Nanotoxicity: Prevention and Antibacterial Applications of Nanomaterials (Micro and Nano Technologies) [1 ed.] 0128199431, 9780128199435

Nanotoxicity: Prevention, and Antibacterial Applications of Nanomaterials focuses on the fundamental concepts for cytoto

1,755 141 16MB

English Pages 504 [465] Year 2020

Report DMCA / Copyright

DOWNLOAD FILE

Polecaj historie

Nanotoxicity: Prevention and Antibacterial Applications of Nanomaterials (Micro and Nano Technologies) [1 ed.]
 0128199431, 9780128199435

Table of contents :
Nanotoxicity
Copyright
Contents
List of Contributors
Foreword
References
1 Nanoparticle–physiological media interactions
1.1 Introduction
1.1.1 Particle–cell interactions in physiological media
1.1.2 Engineered nanoparticles in many commercial products
1.1.3 Nanoparticle-mediated therapies
1.1.4 Protein corona
1.1.5 Quantification of particle uptake
1.1.6 Flow cytometry
1.1.7 Use of plasmonic properties
1.1.8 Other methods used to quantify the nanoparticle uptake
1.1.9 Limitations of the above methods
1.1.10 Use of atomic force microscopy
1.2 Recent advances on the interaction of nanoparticles with biological media
1.2.1 Dynamical modeling of manipulation process in trolling-mode atomic force microscopy
1.2.2 Limits of the effective medium theory in particle amplified surface plasmon resonance spectroscopy biosensors
1.2.3 Aromatic nitrogen mustard-based autofluorescent amphiphilic brush copolymer as pH-responsive drug delivery vehicle
1.2.4 Dynamic changes of protein corona compositions on the surface of zinc oxide nanoparticle in cell culture media
1.2.5 In vitro methods for assessing nanoparticle toxicity
1.2.6 Nanoparticles targeting retinal and choroidal capillaries in vivo
1.2.7 Distribution of superparamagnetic Au/Fe nanoparticles in an isolated guinea pig brain with an intact blood–brain barrier
1.2.8 Long-term real-time tracking live stem cells/cancer cells in vitro/in vivo through highly biocompatible photoluminesc...
1.2.9 The effect of silica nanoparticles stability in biological media
1.2.10 Experimental challenges regarding the in vitro investigation of the nanoparticle-biocorona in disease states
1.2.11 Effect of ionic strength on shear-thinning nanoclay–polymer composite hydrogels
1.2.12 The effect of surface charge and pH on the physiological behavior of cobalt, copper, manganese, antimony, zinc, and ...
1.2.13 Sweet strategies in prostate cancer biomarker research: focus on a prostate-specific antigen
1.2.14 Iron oxide colloidal nanoclusters as theranostic vehicles and their interactions at the cellular level
1.2.15 Assembly of carboxylated zinc phthalocyanine with gold nanoparticle for colorimetric detection of calcium ion
1.2.16 Developing the next generation of graphene-based platforms for cancer therapeutics
1.2.17 pH-Responsive morphology-controlled redox behavior and cellular uptake of nanoceria in fibrosarcoma
1.2.18 pH- and thermo-sensitive MTX-loaded magnetic nanocomposites: synthesis, characterization, and in vitro studies on A5...
1.2.19 Monitoring the dynamics of cell-derived extracellular vesicles at the nanoscale by liquid-cell transmission electron...
1.2.20 In vivo formation of protein corona on gold nanoparticles—the effect of their size and shape
1.2.21 Fabrication of folic acid magnetic nanotheranostics: an insight on the formation mechanism, physicochemical properti...
1.2.22 Multifunctional pH sensitive three-dimensional scaffolds for treatment and prevention of bone infection
1.2.23 Antibody-pHPMA functionalized fluorescent silica nanoparticles for colorectal carcinoma targeting
1.2.24 Size-controlled, colloidally stable, and functional nanoparticles based on the molecular assembly of green tea polyp...
1.2.25 Influence of interaction between α-Fe2O3 nanoparticles and dissolved fulvic acid on the physiological responses in S...
References
2 In vitro methods to assess the cellular toxicity of nanoparticles
2.1 Introduction
2.2 Materials and methods
2.2.1 Cytotoxicity
2.2.1.1 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay
2.2.1.1.1 Materials
2.2.1.1.2 Medium, buffer, and solutions
2.2.1.1.2.1 In vitro components
2.2.1.1.2.1.1 Preparation of culture medium
2.2.1.1.2.1.2 Phosphate buffered saline (Ca2+, Mg2+ free)
2.2.1.1.2.1.3 Trypsin-EDTA (0.25%)
2.2.1.1.3 Method
2.2.1.1.4 Limitations
2.2.2 Live/dead assessment
2.2.2.1 Propidium iodide uptake assay
2.2.2.1.1 Materials
2.2.2.1.2 Medium, buffer, and solutions
2.2.2.1.2.1 In vitro components
2.2.2.1.2.1.1 Preparation of culture medium
2.2.2.1.2.1.2 Phosphate buffered saline (Ca2+, Mg2+ free)
2.2.2.1.2.1.3 Trypsin-EDTA (0.25%)
2.2.2.1.3 Methods
2.2.2.1.3.1 In vitro cell preparation and treatment
2.2.2.1.3.2 In vitro method for propidium iodide uptake assay
2.2.2.1.4 Limitations
2.2.2.2 Trypan blue exclusion test
2.2.2.2.1 Materials
2.2.2.2.2 Medium, buffer, and solutions
2.2.2.2.2.1 In vitro components
2.2.2.2.2.1.1 Preparation of culture medium
2.2.2.2.2.1.2 Phosphate buffered saline (Ca2+, Mg2+ free)
2.2.2.2.2.1.3 Trypsin-EDTA (0.25%)
2.2.2.2.3 Methods
2.2.2.2.3.1 In vitro cell preparation and treatment
2.2.2.2.3.2 In vitro method for trypan blue exclusion test
2.2.2.2.4 Limitations
2.2.3 Genotoxicity
2.2.3.1 Single cell gel electrophoresis (Comet) assay
2.2.3.1.1 Materials
2.2.3.1.2 Medium, buffer, and solutions
2.2.3.1.2.1 Normal melting agarose
2.2.3.1.2.2 Low-melting-point agarose
2.2.3.1.2.3 Stock lysing solution
2.2.3.1.2.4 Working lysing solution
2.2.3.1.2.5 Electrophoresis buffer
2.2.3.1.2.6 Neutralization buffer
2.2.3.1.2.7 Staining solution
2.2.3.1.2.8 5,6-Carboxyflourescein dye
2.2.3.1.2.9 In vitro components
2.2.3.1.2.9.1 Preparation of culture medium
2.2.3.1.2.9.2 Phosphate buffered saline (Ca2+, Mg2+ free)
2.2.3.1.2.9.3 Trypsin-EDTA (0.25%)
2.2.3.1.3 Methods
2.2.3.1.3.1 In vitro cell preparation
2.2.3.1.3.1.1 Viability test
2.2.3.1.3.1.2 Preparation of base slides
2.2.3.1.3.1.3 Cell lysis
2.2.3.1.3.1.4 Electrophoresis of microgel slides
2.2.3.1.3.1.5 Neutralization of microgel slides
2.2.3.1.3.1.6 Staining of microgel slides
2.2.3.1.3.1.7 Scoring of the microgel slides
2.2.3.1.4 Limitations
2.2.3.2 Cytokinesis block micronucleus assay
2.2.3.2.1 Materials
2.2.3.2.2 Medium, buffer, and solutions
2.2.3.2.2.1 Cytochalasin B
2.2.3.2.2.2 Geimsa stock solution
2.2.3.2.2.3 Working geimsa solution
2.2.3.2.2.4 In vitro components
2.2.3.2.2.4.1 Preparation of culture medium
2.2.3.2.2.4.2 Phosphate buffered saline (Ca2+, Mg2+ free)
2.2.3.2.2.4.3 Trypsin-EDTA (0.25%)
2.2.3.2.2.4.4 Hypotonic solution (potassium chloride)
2.2.3.2.2.4.5 Carnoy’s fixative
2.2.3.2.2.4.6 Sorenson’s buffer
2.2.3.2.2.5 In vivo components
2.2.3.2.2.5.1 Anticoagulant
2.2.3.2.2.5.2 Buffer
2.2.3.2.3 Methods
2.2.3.2.3.1 In vitro cell preparation and treatment
2.2.3.2.4 Limitations
2.2.4 Oxidative stress
2.2.4.1 Reactive oxygen species generation
2.2.4.1.1 Materials
2.2.4.1.2 Medium, buffer, and solutions
2.2.4.1.2.1 In vitro components
2.2.4.1.2.1.1 Preparation of culture medium
2.2.4.1.2.1.2 Phosphate buffered saline (Ca2+, Mg2+ free)
2.2.4.1.2.1.3 Trypsin-EDTA (0.25%)
2.2.4.1.3 Methods
2.2.4.1.3.1 Stock 2,7-dichlorofluorescein diacetate
2.2.4.1.3.2 Working 2,7-dichlorofluorescein diacetate
2.2.4.1.3.3 tert-Butyl hydrogen peroxide
2.2.4.1.3.4 In vitro cell preparation
2.2.4.1.3.5 Method for in vitro reactive oxygen species detection
2.2.4.1.4 Limitations
2.3 Conclusion
References
3 In vivo studies: toxicity and biodistribution of nanocarriers in organisms
List of abbreviations
3.1 General overview
3.2 Types of nanocarriers
3.2.1 Carbon nanotubes
3.2.2 Nanoparticles
3.2.2.1 Inorganic nanoparticles
3.2.2.2 Polymeric nanoparticles
3.3 Polymeric micelles
3.4 Dendrimers
3.4.1 Poly(amidoamine) dendrimers
3.4.2 Poly(amidoamine) dendriplexes (complex with nucleic acid)
3.4.3 Polypropyleneimine dendrimers
3.4.4 Melamine dendrimers
3.5 Liposomes
3.6 Conclusion
3.7 Future directions
References
4 Standard biological assays to estimate nanoparticle toxicity and biodistribution
4.1 Introduction
4.2 In vitro methods for determination of nanoparticle toxicity
4.2.1 Methods for cytotoxicity assessment
4.2.1.1 Dye exclusion assays
4.2.1.1.1 Trypan blue exclusion assay
Protocol
4.2.1.1.2 Erythrosin B dye exclusion assay
4.2.1.2 Colorimetric assays
4.2.1.2.1 MTT assay
4.2.1.2.1.1 Protocol
4.2.1.2.2 WST-1 assay
4.2.1.2.3 Lactate dehydrogenase assay
4.2.1.2.3.1 Protocol
4.2.1.2.4 Neutral red uptake
4.2.1.2.4.1 Protocol
4.2.1.3 Fluorescence-based assays
4.2.1.3.1 Alamar blue assay
4.2.1.3.1.1 Protocol
4.2.1.3.2 Protease-based viability assay
4.2.1.3.2.1 Protocol
4.2.1.4 Luminometric methods for cell viability assessment
4.2.1.4.1 Adenosine triphosphate based method
4.2.1.4.1.1 Protocol
4.2.1.5 Cell viability test in real-time
4.2.1.5.1 Protocol
4.2.1.5.2 Estimation of oxidative stress
4.2.1.5.3 Reactive oxygen species level measurement
4.2.1.5.3.1 Protocol
4.2.1.5.4 Lipid peroxidation
4.2.1.5.4.1 Protocol
4.2.1.5.5 Glutathione estimation
4.2.1.5.5.1 Protocol
4.2.1.6 Apoptosis based assays
4.2.1.6.1 Annexin-V FITC/propidium Iodide assay
4.2.1.6.2 TUNEL assay
4.2.2 Methods for determining the genotoxicity potential of nanoparticles
4.2.2.1 Micronucleus formation
4.2.2.2 Cytokinesis block micronucleus assay
4.2.2.3 Flow micronucleus assay
4.2.2.4 Comet assay
4.3 In vivo bio-distribution and toxicity of nanoparticles
4.3.1 Quantitation and bio-distribution of nanoparticles from tissues
4.3.2 Electron microscopy
4.3.3 Liquid scintillation counting
4.3.4 Quantification of nanoparticles by drug loading and release
4.3.5 Whole body imaging-based methods for assessment of nanoparticle toxicity and bio-distribution
4.3.5.1 In vivo optical imaging
4.3.5.2 Computed tomography
4.3.5.3 Magnetic resonance imaging
4.3.5.4 Nuclear medicine imaging
4.4 Conclusion and future aspects
Conflict of interest
References
5 Toxicity of metal oxide nanoparticles
5.1 Introduction
5.2 Metal oxide nanoparticles
5.3 Zinc oxide nanoparticles
5.3.1 In vitro toxicity studies of ZnO nanoparticles
5.3.2 In vivo toxicity studies of ZnO nanoparticles
5.4 Iron Oxide-based magnetic nanoparticles
5.4.1 In vitro toxicity studies of IO nanoparticles
5.4.2 In vivo toxicity studies of IO nanoparticles
5.5 Titanium dioxide Nanoparticles
5.5.1 In vitro toxicity studies of TiO2 nanoparticles
5.5.2 In vivo toxicity studies of TiO2 nanoparticles
5.6 Copper oxide nanoparticles
5.6.1 In vitro toxicity studies of CuO nanoparticles
5.6.2 In vivo toxicity studies of CuO nanoparticles
5.7 Toxicity mechanism of metal oxide nanoparticles
5.7.1 Oxidative stress
5.7.2 Metal ion toxicity
5.8 Conclusion
Conflicts of interest
References
Further reading
6 Toxicity of silver and other metallic nanoparticles
6.1 Introduction
6.2 Toxicity of silver nanoparticles
6.2.1 Biocorona formation contributes to silver nanoparticle induced endoplasmic reticulum stress
6.2.2 Silver nanoparticles to prepare scaffolds for the regeneration of infected full-thickness skin defects
6.2.3 Responses of flocculated activated sludge to bimetallic Ag-Fe nanoparticles toxicity
6.2.4 Influence of addition of L-cysteine into inositol hexaphosphate (IP6) modified Ag nanoparticles
6.2.5 Silver accumulation in adults and abnormal embryo development in their offspring
6.2.6 Toxicity of silver nanoparticles released by Hancornia speciosa (Mangabeira) biomembrane
6.3 Toxicity of gold nanoparticles
6.3.1 Toxicity of gold nanoparticles on human health and environment
6.3.2 Gold nanoparticles and quantification of individual mycotoxin concentrations
6.3.3 Interaction of gold nanoparticles with nanoparticles of silver and titanium dioxide
6.3.4 Differential cytotoxicity effect of dragon fruit extract capped gold nanoparticles on breast cancer cells
6.3.5 Anticancer activity of gold nanoparticles prepared by using fruit extract of Lycium chinense
6.3.6 Mechanism of intracellular uptake and localization and the subsequent toxicity of nanoparticles
6.3.7 The interaction strategy of diosmin functionalized gold nanoparticles with calf thymus DNA
6.3.8 Effects and bioaccumulation of gold nanoparticles in the gilthead seabream (Sparus aurata)
6.3.9 Application of gold nanoparticles in drug uptake and induction of cell death on breast cancer cell line
6.3.10 Gold nanoparticles in ophthalmology
6.4 Toxicity of copper nanoparticles
6.4.1 Rapid and selective detection of trace Cu2+ in sea water
6.4.2 Quaternized chitosan-stabilized copper sulfide nanoparticles for cancer therapy
6.4.3 Copper nanoparticles and osteoblast response
6.4.4 Copper nanoparticles and immune response in the liver of juvenile Takifugu fasciatus
6.4.5 Copper sulfide nanoparticles for T1-weighted magnetic resonance imaging guided photothermal cancer therapy
6.4.6 Removal of Cu2+ from water and wastewaters
6.4.7 Toxicity of copper oxide nanoparticles on spring barley (Hordeum sativum distichum)
6.4.8 Toxicity of copper oxide nanoparticles to neotropical species
6.5 Toxicity of iron nanoparticles
6.5.1 Responses of flocculated activated sludge to bimetallic Ag-Fe nanoparticles toxicity
6.5.2 Biogenic magnetite nanoparticles for efficient removal of azo dyes and phenolic contaminants from water
6.5.3 Functionalization of T lymphocytes for magnetically controlled immune therapy
6.6 Toxicity of zinc nanoparticles
6.6.1 Comparative toxicity of organic, inorganic, and nanoparticulate zinc
6.6.2 Nano zinc oxide hydrogels as wound healing materials
6.6.3 Evaluation of toxicity of phycocyanin-ZnO nanorod composites
6.6.4 DNA damages and offspring quality in sea urchin Paracentrotus lividus sperms exposed to ZnO nanoparticles
6.6.5 Reclaimable La: ZnO/PAN nanofiber catalyst for toxicological evaluation utilizing early life stages of zebra fish (Da...
6.7 Conclusion
References
7 Recent advances in the study of toxicity of polymer-based nanomaterials
7.1 Introduction
7.2 Recent advances in the study of toxicity of polymeric nanomaterials
7.2.1 Nanodiamonds: emerging face of future nanotechnology
7.2.2 Nanomaterials as versatile adsorbents for heavy metal ions in water
7.2.3 Toxicological responses of Chlorella autotrophica and Dunaliella salina to Ag and CeO2 nanoparticles
7.2.4 Nanoparticles as a solution for eliminating the risk of mycotoxins
7.2.5 Nanoparticles modulate membrane interactions of human Islet amyloid polypeptide
7.2.6 Self-assembled antimicrobial nanomaterials
7.2.7 Construction of dual-functional polymer nanomaterials with near-infrared fluorescence imaging and polymer prodrug by ...
7.2.8 General overview of lipid–polymer hybrid nanoparticles, dendrimers, micelles, liposomes, spongosomes, and cubosomes
7.2.9 Nanomaterials for water cleaning and desalination, energy production, disinfection, agriculture, and green chemistry
7.2.10 Nanomaterials as protein, peptide, and gene delivery agents
7.2.11 Development of cholate conjugated hybrid polymeric micelles for farnesoid X receptor receptor mediated effective sit...
7.2.12 Antibacterial properties of electrospun Ti3C2Tz (MXene)/chitosan nanofibers
7.2.13 Effects of surface charge of hyperbranched polymers on cytotoxicity, dynamic cellular uptake and localization, hemot...
7.2.14 Nanomaterials for skin care
7.2.15 Evaluation of the effects of polymeric chitosan/tripolyphosphate and solid lipid nanoparticles on germination of Zea...
7.2.16 Synthesis, characterization, and cytotoxicity of S-nitroso-mercaptosuccinic acid-containing alginate/chitosan nanopa...
7.2.17 Polymeric nanomaterials as nanomembrane entities for biomolecule and drug delivery
7.2.18 Fabrication and application areas of mixed matrix flat-sheet
7.2.19 CO2-based amphiphilic polycarbonate micelles enable a reliable and efficient platform for tumor
7.2.20 Electrospun polyacrylonitrile templated 2D nanofibrous mats: a platform toward practical applications for dye remova...
7.2.21 Nanomaterials for neurology: state-of-the-art
7.2.22 Effect of different nanomaterials on the metabolic activity and bacterial flora of activated sludge medium
7.2.23 Fluorescent polymeric nanovehicles for neural stem cell modulation
7.2.24 Graphene oxide–enriched double network hydrogel with tunable physico-mechanical properties and performance
7.2.25 Advances in nanobiomaterials for topical administrations: new galenic and cosmetic formulations
7.2.26 Fluorescent polymer nanoparticles based on dyes: seeking brighter tools for bioimaging
7.2.27 Nanostructured materials functionalized with metal complexes: in search of alternatives for administering anticancer...
7.2.28 Sequestration of nanoparticles by an extracellular polymeric substance matrix reduces the particle-specific bacteric...
7.2.29 In vitro cytotoxicity evaluation of functional polyethylene glycol-PDMA block copolymer in liver human hepatocellula...
7.2.30 Polyethylene glycolylated boron nitride nanotube-reinforced poly(propylene fumarate) nanocomposite biomaterials
7.2.31 Evaluation of potential acute cardiotoxicity of biodegradable nanocapsules in rats by intravenous administration
7.2.32 A study of the catalytic ability of in situ prepared AgNPs-PMAA-PVP electrospun nanofibers
7.2.33 Caenorhabditis elegans as an alternative in vivo model to determine oral uptake, nanotoxicity, and efficacy of melat...
7.2.34 Evaluation of the effects of nitric oxide-releasing nanoparticles on plants
7.2.35 Planktonic and biofilm-grown nitrogen-cycling bacteria exhibit different susceptibilities to copper nanoparticles
7.2.36 Inorganic nanocarriers for platinum drug delivery
7.2.37 Low-bandgap biophotonic nanoblend: a platform for systemic disease targeting and functional imaging
7.2.38 Unique roles of nanotechnology in medicine and cancer-II
7.2.39 Nanotechnology and vaccine development
7.2.40 Environmental interactions of geo-and bio-macromolecules with nanomaterials
7.3 Concluding remarks
References
8 Toxicity of polymeric nanomaterials
8.1 Introduction
8.2 Classification of polymeric nanomaterials
8.2.1 Natural polymeric nanomaterials
8.2.1.1 Chitosan
8.2.1.2 Cellulose
8.2.1.3 Starch
8.2.1.4 Alginate
8.2.2 Biosynthesized polymeric nanomaterials
8.2.2.1 Poly β-hydroxybutyrate
8.2.2.2 Xanthan gum
8.2.2.3 Polylactic acid
8.2.2.4 Polysialic acid
8.2.3 Chemical-synthesized polymeric nanomaterials
8.2.3.1 Dendrimers
8.2.3.2 Poly(lactic-co-glycolic acid)
8.2.3.3 Polyethylenimines
8.2.3.4 Poly(l-lysine)
8.2.3.5 Dextran
8.2.3.6 Polymeric micelles
8.3 In vitro toxicity of polymeric nanomaterials
8.3.1 Chitosans
8.3.2 Poly(lactic-co-glycolic acid)
8.3.3 Poly(amidoamine) dendrimers
8.3.4 Polyethylenimines
8.3.5 Polystyrene
8.4 In vivo toxicity of polymeric nanomaterials
8.4.1 Respiratory system
8.4.2 Cardiovascular system
8.4.3 Neurological system
8.4.4 Immune system
8.4.5 Skin
8.4.6 Gastrointestinal system
8.4.7 Reproduction and development
8.5 Mechanisms of polymeric nanomaterials-induced toxicity
8.5.1 Physicochemical mechanisms of polymeric nanomaterials-induced toxicity
8.5.1.1 Size
8.5.1.2 Charge
8.5.1.3 Shape
8.5.1.4 Surface chemistry
8.5.2 Biochemical mechanisms of polymeric nanomaterials-induced toxicity
8.5.2.1 Apoptosis and necrosis
8.5.2.2 Ferroptosis
8.5.2.3 Autophagy
8.5.2.4 Pyroptosis
8.5.2.5 Cell cycle arrest
8.5.2.6 Oxidative stress
Conflict of interest
References
9 General methods for detection and evaluation of nanotoxicity
9.1 Introduction
9.2 General nanotoxicity methods
9.3 Mechanism of antibacterial activities
9.4 Methods for detection and evaluation of nanotoxicity
9.4.1 Plate counting, colony counting, or colony forming efficiency assay
9.4.2 Disk diffusion test: zone of inhibition
9.4.3 Gradient method (Etest)
9.4.4 Optical density
9.4.5 Dilution methods
9.4.6 Broth dilution
9.4.7 Time-kill test (time-kill curve)
9.4.8 Adenosine triphosphate cell viability assay
9.4.9 Reactive oxygen species
9.4.10 Lipid peroxidation measurement
9.4.11 Omics methods: transcriptomic, metabolomics, genomics, and proteomic profiles
9.4.12 Microscope
9.4.12.1 Fluorescence microscope
9.4.12.2 Flow cytometry
9.4.12.3 Electron microscopy
9.4.13 Other methods
9.5 Conclusion and outlooks
References
10 Safer-by-design for nanomaterials
10.1 Introduction
10.2 Hazard and release reduction for engineered nanomaterials in production and products
10.3 Reducing releases to the environment from nanomaterial production and processing facilities
10.4 Safer-by-design hazard reduction of engineered inorganic and carbonaceous nanomaterials for organisms
10.4.1 Evaluating safer-by-design regarding inorganic and carbonaceous nanoparticles
10.4.2 Safer-by-design strategies to reduce (aspects of) nanomaterial hazards
10.5 Reducing releases to the environment of nanomaterials from relatively large nanocomposites and products
10.5.1 Safer-by-design for relatively large nanocomposites: reduction of nanomaterial releases
10.5.1.1 Nanomaterials in relatively large nanocomposites
10.5.1.2 Functional releases from and unintended releases of nanomaterials from relatively large nanocomposites
10.5.1.3 Releases from relatively large nanocomposites during their use stage and strategies to reduce those releases
10.5.1.4 Releases from relatively large nanocomposites to the environment after the use stage
10.5.2 Controlling releases from nanomaterials present as components in products
10.6 Reducing hazards of fragments released from nanocomposites
10.7 Conclusions
References
11 Antibacterial activity of metal oxide nanoparticles
11.1 Introduction
11.2 Effective physicochemical properties of MO-NPs on antibacterial activity
11.2.1 Chemical composition of metal oxide nanoparticles
11.2.2 Particle size and surfaces properties of metal oxide nanoparticles
11.2.3 Concentration of metal oxide nanoparticles
11.2.4 The shape-dependent antimicrobial properties of metal oxide nanoparticles
11.2.5 Solubility of metal oxide nanoparticles
11.2.6 The generation of reactive oxygen species
11.2.7 Mechanisms of antimicrobial activity metal oxide nanoparticles
11.3 Antibacterial activity of magnesium oxide and calcium oxide nanoparticles
11.4 Antibacterial activity of aluminum oxide nanoparticles
11.5 Antibacterial activity of silver oxide nanoparticles
11.6 Antibacterial activity of copper oxide nanoparticles
11.7 Antibacterial activity of zinc oxide nanoparticles
11.8 Antibacterial activity of iron oxide nanoparticles
11.9 Antibacterial activity of titanium oxide nanoparticles
References
12 Antibacterial activity of platinum nanoparticles
12.1 Platinum nanoparticles
12.2 Antibacterial activity
12.3 Antibiotics and antimicrobial compounds
12.4 Determination of the microbial activity
12.5 Recent trends in the antibacterial activity of platinum nanoparticles
References
13 Antibacterial property of metal oxide-based nanomaterials
13.1 Introduction
13.2 Mechanism of antimicrobial resistance
13.3 Methods to evaluate MO-NPs antibacterial efficiency
13.3.1 In vitro methods for antimicrobial evaluation of nanoparticles-based metal oxide
13.3.2 Regulatory testing
13.3.3 Antimicrobial activity tests
13.4 Antimicrobial effect of metal and metal oxide nanoparticles
13.5 Mode of antimicrobial action by metal and metal oxides nanoparticles
13.5.1 Formation of reactive oxygen species
13.5.2 Damage to the cell-wall membrane due to electrostatic interaction and accumulation
13.5.3 Loss of homeostasis by metal ions
13.5.4 Dysfunction of proteins and enzymes-binding to cytosolic proteins
13.5.5 Inhibition of the transduction signal
13.5.6 Interactions with phospholipid bilayer
13.6 Nanoparticle characteristics and their influence on antimicrobial activity
13.6.1 Size and shape
13.6.2 Surface and zeta potential
13.6.3 Chemical doping
13.7 Metal oxide-based antibacterial membrane
13.8 Antibacterial functions of multi-metal oxide nanoparticles
13.9 Magnetic bio-metal oxide-magnetosome
13.10 Toxicity concerns of MO-NPs as antimicrobial agents
13.11 Conclusions, challenges, and future perspectives
References
14 Antimicrobial properties of carbon quantum dots
14.1 Introduction
14.2 Antibacterial properties of carbon nanodots
14.2.1 Non-functionalized carbon nanodots with inherent antibacterial properties
14.2.2 Functionalized carbon nanodots with inherent antibacterial properties
14.2.3 Nonfunctionalized carbon nanodots exhibiting antibacterial properties after irradiation
14.2.4 Functionalized carbon nanodots exhibiting antibacterial properties after irradiation
14.3 Conclusion
References
15 Applications of nanotechnology in agry-food productions
15.1 Introduction
15.2 Nanoencapsulation techniques applied to food and agriculture
15.2.1 Nanoencapsulation for food applications
15.2.2 Nanoencapsulation for agriculture applications
15.3 Nanosensors in food and agriculture
15.4 Nanotechnology applied to environmental remediation
15.5 Manufacture of protective clothes for farm workers
15.6 Conclusion and outlooks
References
Further reading
16 Nanoparticle applications in sustainable agriculture, poultry, and food: trends and perspective
16.1 Introduction
16.2 Nanoparticle applications in agriculture
16.2.1 Seed germination and growth
16.2.2 Plant protection and production
16.2.2.1 Nanopesticides
16.2.2.2 Nanofungicides
16.2.2.3 Nanoherbicides
16.2.2.4 Nanofertilizers
16.2.2.5 Nutrient management
16.3 Nanoparticle applications in poultry
16.4 Nanoparticle applications in food
16.5 Nano-biosensors sustainable agriculture, poultry, and food
16.6 Regulatory aspects of nanotechnology in agriculture, poultry, and food
16.6.1 Regulatory aspects of nanomaterials in agriculture/food/poultry feed in the European Union
16.6.2 Risk management of pesticides
16.6.3 Risk assessment of food and feed
16.6.4 Regulatory aspects of nanomaterials in agriculture/food/poultry feed in non-European Union countries
16.7 Conclusion and future perspectives
Conflicts of interest
References
17 Antibacterial nanocomposite coatings
17.1 Introduction
17.2 Inorganic nanocomposite coating
17.3 Organic nanocomposite coating
17.4 Environmental benefits and impacts of antibacterial nanocomposite coatings
References
18 Antimicrobial nanomaterials for water disinfection
18.1 Introduction
18.2 Significance of nanotechnology
18.3 Antibacterial metal oxides and metal nanoparticles
18.3.1 Copper nanoparticles
18.3.2 Zinc oxide nanoparticles
18.3.3 Silver nanoparticles
18.3.4 Silica nanoparticles
18.3.5 Cerium nanoparticles
18.3.6 Nickel and magnesium nanoparticles
18.3.7 Carbon-based nanomaterials
18.3.7.1 Carbon nanotube
18.4 Mechanisms for nanoparticle-mediated microbial disinfection
18.5 Advanced technologies for nanoparticle-based water disinfection
18.5.1 Membrane-based nanotechnology for microbial water disinfection
18.5.2 Nanoparticle embedded column induced microbial disinfection
18.5.3 Phtocatalysis-based microbial destruction disinfection
18.5.3.1 Titanium oxide nanoparticles
18.5.3.2 Zinc oxide nanoparticles
18.5.4 Carbon nanotubes
18.5.5 Magnetic nanoparticles
18.5.6 Nano-zero valent iron
18.6 Some commercialized products and their information
18.7 Current status of technology transfer, scale up, and challenges
References
19 Nanomaterials for antifungal applications
19.1 Introduction
19.1.1 Oils having antifungal properties
19.1.2 Rosemary having antifungal properties
19.1.3 Best medicine for antifungal
19.1.4 Side effects of antifungal creams
19.1.5 Antifungal properties of olive oil
19.1.6 Natural treatments for fungal infections, such as ringworm
19.1.7 Antifungal properties of coconut oil
19.1.8 Antifungal nature of honey
19.1.9 Killing of fungus by antifungals
19.1.10 Killing of fungus by baking soda
19.1.11 Antifungal nature of onion
19.1.12 Antifungal activity and mode of action of silver nanoparticles on Candida albicans
19.2 Recent trends in the study of antifungal activities of nanoparticles
19.2.1 Biological activity of disulfide- and triazole-linked peptides
19.2.2 Attractive properties nanodiamonds
19.2.3 Nano-based antifungals mediated by biomolecule coronas
19.2.4 Gray mold of table grapes controlled by eco-friendly nanomaterials
19.2.5 Rapid killing of microbes by polymer-silver nanocomposites
19.2.6 Antimicrobial activity of essential oil components
19.2.7 Utility of noble metal-based nanoparticles in medicine and pharmacology
19.2.8 Antimicrobial activity of cellulose acetate—essential oil nanocapsules
19.2.9 Use of nanoparticles in veterinary medicine
19.2.10 Use of propolis in animal nutrition and animal health
19.2.11 Toxicological studies of surfactant-functionalized praseodymium oxide nanoparticles
19.2.12 Antifungal mechanisms for chemical fungicides, biological agents, and nanoparticles
19.2.13 Biomedical applications of selenium nanomaterials
19.2.14 Ag(I) ions monitored by fluorescent probe
19.2.15 Antimicrobial activity, cytotoxicity, and DNA-binding studies of carbon dots
19.2.16 Synergistic effect of silver nanoparticles, chitosan, and fungicide zineb
19.2.17 ZnO-based nanoplatforms in drug delivery and theranostic
19.2.18 Antimicrobial activity gold nanoparticles
19.2.19 Toxicity of zinc oxide and silver nanoparticles in Saccharomyces cerevisiae
19.2.20 Applications of fungi-assisted silver nanoparticle
19.2.21 Antibacterial, antiviral, antiangiogenic, and anticancer activity mechanism of AgNPs
19.2.22 Antifungal activity of zinc oxide produced by green synthesis
19.2.23 Controlling growth of plant-pathogenic fungi by metallic nanoparticles
19.2.24 Ag2O nanostructures with strontium for structural changes in DNA
19.2.25 Antimicrobial applications of nanoparticles of inorganic, organic, and hybrid materials
References
20 Antibacterial nanocoatings
20.1 Introduction
20.2 Novel and smart antibacterial nanocoating approaches
20.3 Applications of antibacterial nanocoatings
20.3.1 Marine systems
20.3.2 Medical and healthcare related applications
20.3.3 Construction
20.3.4 Food and packaging
20.3.5 Textile
20.4 Safety and toxicological issues
20.5 Conclusion
References
Further reading
21 Emerging antibacterial and antifungal applications of nanomaterials on food products
21.1 Introduction
21.2 Organic nanomaterial applications
21.2.1 Nanofibers
21.2.1.1 Electrospun nanofibers
21.2.2 Polymeric nanoparticles
21.2.2.1 Polymeric nanoparticles as Pickering emulsions
21.2.2.2 Application of polymeric nanoparticles in functional coatings and films
21.2.2.3 Electrosprayed nanoparticles
21.2.3 Nanoemulsions
21.2.4 Solid lipid nanoparticles
21.2.5 Nanoliposomes
21.2.6 Cyclodextrins/molecular inclusions
21.2.6.1 Cyclodextrins-based nanosponges
21.2.7 Contact surface
21.3 Inorganic Nanomaterial Applications
21.3.1 Metal and metal oxide nanoparticles
21.3.1.1 Food packaging applications of metal and metal oxide nanoparticles
21.4 Conclusion
References
Further reading
Index

Citation preview

NANOTOXICITY

NANOTOXICITY PREVENTION AND ANTIBACTERIAL APPLICATIONS OF NANOMATERIALS Edited by

Susai Rajendran PSNA College of Engineering and Technology, Dindigul, India

Anita Mukherjee Department of Botany, Centre of Advanced Study, University of Calcutta, Kolkata, India

Tuan Anh Nguyen Institute for Tropical Technology, Vietnam Academy of Science and Technology, Hanoi, Vietnam

Chandraiah Godugu Department of Regulatory Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Balanagar, India

Ritesh K. Shukla School of Arts & Sciences, Ahmedabad University, Gujarat, India

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States Copyright © 2020 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-819943-5 For Information on all Elsevier publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Matthew Deans Acquisitions Editor: Simon Holt Editorial Project Manager: Mariana Kuhl Production Project Manager: Debasish Ghosh Cover Designer: Victoria Pearson Typeset by MPS Limited, Chennai, India

Contents 3.5 Liposomes 62 3.6 Conclusion 63 3.7 Future directions References 64

List of Contributors ix Foreword xiii

PART 1 Basic principles

4. Standard biological assays to estimate nanoparticle toxicity and biodistribution 71

1. Nanoparticle physiological media interactions 3

Juhi Shah, Stuti Bhagat and Sanjay Singh

4.1 Introduction 71 4.2 In vitro methods for determination of nanoparticle toxicity 72 4.3 In vivo bio-distribution and toxicity of nanoparticles 85 4.4 Conclusion and future aspects 96 Acknowledgments 96 Conflict of interest 97 References 97

R. Dorothy, N. Karthiga, S. Senthil Kumaran, R. Joseph Rathish, Susai Rajendran and Gurmeet Singh

1.1 Introduction 3 1.2 Recent advances on the interaction of nanoparticles with biological media 5 References 18

2. In vitro methods to assess the cellular toxicity of nanoparticles 21 Krupa Kansara and Ashutosh Kumar

2.1 Introduction 21 2.2 Materials and methods 2.3 Conclusion 38 Acknowledgments 38 References 38

PART 2 Toxicity of nanomaterials

23

5. Toxicity of metal oxide nanoparticles 107 Thodhal Yoganandham Suman, Wei-Guo Li and De-Sheng Pei

3. In vivo studies: toxicity and biodistribution of nanocarriers in organisms 41

5.1 5.2 5.3 5.4 5.5 5.6 5.7

Introduction 107 Metal oxide nanoparticles 107 Zinc oxide nanoparticles 108 Iron Oxide-based magnetic nanoparticles Titanium dioxide Nanoparticles 112 Copper oxide nanoparticles 114 Toxicity mechanism of metal oxide nanoparticles 115 5.8 Conclusion 118 Acknowledgments 118

Nivya Sharma, Mohd Aslam Saifi, Shashi Bala Singh and Chandraiah Godugu

List 3.1 3.2 3.3 3.4

64

of abbreviations 41 General overview 43 Types of nanocarriers 44 Polymeric micelles 56 Dendrimers 57

v

110

vi

CONTENTS

PART 3 Prevention of nanotoxicity

Conflicts of interest 118 References 119 Further reading 122

6. Toxicity of silver and other metallic nanoparticles 125

9. General methods for detection and evaluation of nanotoxicity 195

T. Umasankareswari, Gurmeet Singh, S. Santhana Prabha, Abdulhameed Al-Hashem, S. Senthil Kumaran and Susai Rajendran

Hani Nasser Abdelhamid

6.1 Introduction 125 6.2 Toxicity of silver nanoparticles 126 6.3 Toxicity of gold nanoparticles 128 6.4 Toxicity of copper nanoparticles 132 6.5 Toxicity of iron nanoparticles 136 6.6 Toxicity of zinc nanoparticles 137 6.7 Conclusion 139 Acknowledgment 140 References 140

9.1 9.2 9.3 9.4

Introduction 195 General nanotoxicity methods 196 Mechanism of antibacterial activities 198 Methods for detection and evaluation of nanotoxicity 198 9.5 Conclusion and outlooks 208 Acknowledgment 209 References 209

10. Safer-by-design for nanomaterials

215

L. Reijnders

7. Recent advances in the study of toxicity of polymer-based nanomaterials 143 A. Suriya Prabha, R. Dorothy, S. Jancirani, Susai Rajendran, Gurmeet Singh and S. Senthil Kumaran

7.1 Introduction 143 7.2 Recent advances in the study of toxicity of polymeric nanomaterials 144 7.3 Concluding remarks 163 References 163

8. Toxicity of polymeric nanomaterials 167 Yubin Li, Shaofei Wang and Dianwen Ju

8.1 Introduction 167 8.2 Classification of polymeric nanomaterials 168 8.3 In vitro toxicity of polymeric nanomaterials 172 8.4 In vivo toxicity of polymeric nanomaterials 174 8.5 Mechanisms of polymeric nanomaterialsinduced toxicity 179 Acknowledgments 185 Conflict of interest 186 References 186

10.1 Introduction 215 10.2 Hazard and release reduction for engineered nanomaterials in production and products 217 10.3 Reducing releases to the environment from nanomaterial production and processing facilities 217 10.4 Safer-by-design hazard reduction of engineered inorganic and carbonaceous nanomaterials for organisms 218 10.5 Reducing releases to the environment of nanomaterials from relatively large nanocomposites and products 224 10.6 Reducing hazards of fragments released from nanocomposites 227 10.7 Conclusions 228 References 228

PART 4 Antibacterial activity of nanomaterials 11. Antibacterial activity of metal oxide nanoparticles 241 Vojislav Stani´c and Sladjana B. Tanaskovi´c

11.1 Introduction

241

vii

CONTENTS

11.2 Effective physicochemical properties of MONPs on antibacterial activity 242 11.3 Antibacterial activity of magnesium oxide and calcium oxide nanoparticles 250 11.4 Antibacterial activity of aluminum oxide nanoparticles 253 11.5 Antibacterial activity of silver oxide nanoparticles 254 11.6 Antibacterial activity of copper oxide nanoparticles 255 11.7 Antibacterial activity of zinc oxide nanoparticles 258 11.8 Antibacterial activity of iron oxide nanoparticles 261 11.9 Antibacterial activity of titanium oxide nanoparticles 263 Acknowledgements 266 References 266

12. Antibacterial activity of platinum nanoparticles 275 Susai Rajendran, S. Santhana Prabha, R. Joseph Rathish, Gurmeet Singh and Abdulhameed Al-Hashem

12.1 Platinum nanoparticles 275 12.2 Antibacterial activity 275 12.3 Antibiotics and antimicrobial compounds 276 12.4 Determination of the microbial activity 276 12.5 Recent trends in the antibacterial activity of platinum nanoparticles 276 References 280

13. Antibacterial property of metal oxide-based nanomaterials 283 Md Abdus Subhan

13.1 Introduction 283 13.2 Mechanism of antimicrobial resistance 285 13.3 Methods to evaluate MO-NPs antibacterial efficiency 285 13.4 Antimicrobial effect of metal and metal oxide nanoparticles 287 13.5 Mode of antimicrobial action by metal and metal oxides nanoparticles 288

13.6 Nanoparticle characteristics and their influence on antimicrobial activity 292 13.7 Metal oxide-based antibacterial membrane 293 13.8 Antibacterial functions of multi-metal oxide nanoparticles 294 13.9 Magnetic bio-metal oxidemagnetosome 296 13.10 Toxicity concerns of MO-NPs as antimicrobial agents 297 13.11 Conclusions, challenges, and future perspectives 298 References 299

14. Antimicrobial properties of carbon quantum dots 301 Theodoros Chatzimitakos and Constantine Stalikas

14.1 Introduction 301 14.2 Antibacterial properties of carbon nanodots 302 14.3 Conclusion 313 References 313

PART 5 Emerging antibacterial and antifungal applications 15. Applications of nanotechnology in agry-food productions 319 J.L. Castro-Mayorga, L. Cabrera-Villamizar, J. Balcucho-Escalante, M.J. Fabra and A. Lo´pez-Rubio

15.1 Introduction 319 15.2 Nanoencapsulation techniques applied to food and agriculture 320 15.3 Nanosensors in food and agriculture 328 15.4 Nanotechnology applied to environmental remediation 331 15.5 Manufacture of protective clothes for farm workers 332 15.6 Conclusion and outlooks 333 References 333 Further reading 340

viii

CONTENTS

16. Nanoparticle applications in sustainable agriculture, poultry, and food: trends and perspective 341 N. Chandra Mohana, P.R. Mithun, H.C. Yashavantha Rao, C. Mahendra and S. Satish

16.1 16.2 16.3 16.4 16.5

Introduction 341 Nanoparticle applications in agriculture 342 Nanoparticle applications in poultry 347 Nanoparticle applications in food 347 Nano-biosensors sustainable agriculture, poultry, and food 348 16.6 Regulatory aspects of nanotechnology in agriculture, poultry, and food 348 16.7 Conclusion and future perspectives 350 Conflicts of interest 351 References 351

17. Antibacterial nanocomposite coatings 355 Tien Viet Vu, Van Thang Nguyen, Phuong Nguyen-Tri, The Huu Nguyen, Thien Vuong Nguyen and Tuan Anh Nguyen

17.1 17.2 17.3 17.4

Introduction 355 Inorganic nanocomposite coating 356 Organic nanocomposite coating 357 Environmental benefits and impacts of antibacterial nanocomposite coatings 360 References 360

18. Antimicrobial nanomaterials for water disinfection 365 Nidhi Verma, Sachin Vaidh, Gajendra Singh Vishwakarma and Alok Pandya

18.1 Introduction 365 18.2 Significance of nanotechnology 366 18.3 Antibacterial metal oxides and metal nanoparticles 367 18.4 Mechanisms for nanoparticle-mediated microbial disinfection 373 18.5 Advanced technologies for nanoparticle-based water disinfection 375

18.6 Some commercialized products and their information 377 18.7 Current status of technology transfer, scale up, and challenges 378 Acknowledgment 379 References 379

19. Nanomaterials for antifungal applications 385 K. Kavitha, N. Vijaya, A. Krishnaveni, M. Arthanareeswari, Susai Rajendran, Abdulhameed Al-Hashem and A. Subramania

19.1 Introduction 385 19.2 Recent trends in the study of antifungal activities of nanoparticles 387 References 397

20. Antibacterial nanocoatings 399 Majid Montazer and Tina Harifi

20.1 Introduction 399 20.2 Novel and smart antibacterial nanocoating approaches 400 20.3 Applications of antibacterial nanocoatings 403 20.4 Safety and toxicological issues 409 20.5 Conclusion 410 References 411 Further reading 413

21. Emerging antibacterial and antifungal applications of nanomaterials on food products 415 Dılhun Keriman Arserim-Uc¸ar and Burcu C ¸ abuk

21.1 Introduction 415 21.2 Organic nanomaterial applications 417 21.3 Inorganic Nanomaterial Applications 435 21.4 Conclusion 439 References 440 Further reading 453

Index 455

List of Contributors Hani Nasser Abdelhamid Advanced Multifunctional Materials Laboratory, Department of Chemistry, Assiut University, Assiut, Egypt Abdulhameed Al-Hashem Petroleum Research Centre, Kuwait Institute for Scientific Research, Safat, Kuwait Dılhun Keriman Arserim-Uc¸ar Food Engineering Department, Faculty of Engineering and Architecture, Bingo¨l University, Bingo¨l, Turkey M. Arthanareeswari PG and Research Department of Chemistry, SRM University, Chennai, India J. Balcucho-Escalante Nanobiotechnology and Applied Microbiology Research Group (NANOBIOT), University of the Andes, Bogota´, Colombia Stuti Bhagat Division of Biological and Life Sciences, School of Arts and Sciences, Ahmedabad University, Ahmedabad, Gujarat, India L. Cabrera-Villamizar Nanobiotechnology and Applied Microbiology Research Group (NANOBIOT), University of the Andes, Bogota´, Colombia Burcu C ¸ abuk Gastronomy and Culinary Arts Department, Arts and Design Faculty, Alanya Hamdullah Emin Pa¸sa University, Antalya, Turkey J.L. Castro-Mayorga Nanobiotechnology and Applied Microbiology Research Group (NANOBIOT), University of the Andes, Bogota´, Colombia Theodoros Chatzimitakos Laboratory of Analytical Chemistry, Department of Chemistry, University of Ioannina, Ioannina 451 10, Greece R. Dorothy Department of EEE, AMET University, Chennai, India M.J. Fabra Food Safety and Preservation Department, Institute of Agrochemistry and Food Technology (IATA-CSIC), Valencia, Spain Chandraiah Godugu Department of Regulatory Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, India Tina Harifi Department of Textile Engineering, Functional Fibrous Structures & Environmental Enhancement (FFSEE), Amirkabir University of Technology, Tehran, Iran S. Jancirani PG and Research Department of Chemistry, MVM Government College for Women, Dindigul, India Dianwen Ju Department of Microbiological and Biochemical Pharmacy; The Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai, P. R. China Krupa Kansara Division of Biological and Life Sciences, School of Arts and Sciences, Ahmedabad University, Ahmedabad, India

ix

x

LIST OF CONTRIBUTORS

N. Karthiga Department of Chemistry, SBM College of Engineering, Dindigul, India K. Kavitha PG and Research Department of Chemistry, National College, Trichy, India A. Krishnaveni Department of Chemistry, Yadava College, Madurai, India Ashutosh Kumar Division of Biological and Life Sciences, School of Arts and Sciences, Ahmedabad University, Ahmedabad, India S. Senthil Kumaran

School of Mechanical Engineering, VIT University, Vellore, India

Wei-Guo Li College of Life Science, Henan Normal University, Xinxiang, P.R. China Yubin Li Department of Neurology, Xinqiao Hospital, Third Military Medical University, Chongqing, P. R. China; Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States; Corporal Michael J. Crescenz VA Medical Center, Philadelphia, PA, United States A. Lo´pez-Rubio Food Safety and Preservation Department, Institute of Agrochemistry and Food Technology (IATA-CSIC), Valencia, Spain C. Mahendra

Department of Studies in Botany, University of Mysore, Mysore, India

P.R. Mithun Elexes Medical Consulting Pvt Ltd., Bengaluru, India N. Chandra Mohana Microbial Drugs Laboratory, Department Microbiology, University of Mysore, Mysore, India

of

Studies

in

Majid Montazer Department of Textile Engineering, Functional Fibrous Structures & Environmental Enhancement (FFSEE), Amirkabir University of Technology, Tehran, Iran The Huu Nguyen Faculty of Chemical Technology, Hanoi University of Industry, Hanoi, Vietnam Thien Vuong Nguyen Institute for Tropical Technology, Vietnam Academy of Science and Technology, Hanoi, Vietnam Tuan Anh Nguyen Institute for Tropical Technology, Vietnam Academy of Science and Technology, Hanoi, Vietnam Van Thang Nguyen Faculty of Chemical Technology, Hanoi University of Industry, Hanoi, Vietnam Phuong Nguyen-Tri Department of Chemistry, University of Montreal, Montreal, QC, Canada Alok Pandya Department of Physical Science, Institute of Advanced Research, Gandhinagar, India De-Sheng Pei College of Life Science, Henan Normal University, Xinxiang, P.R. China; Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing, P.R. China S. Santhana Prabha PSNA College of Engineering and Technology, Dindigul, India Susai Rajendran Corrosion Research Centre, St Antony’s College of Arts and Sciences for Women, Amala Annai Nagar, Dindigul, India; PSNA College of Engineering and Technology, Dindigul, India; Corrosion Research Centre, Department of Chemistry, St Antony’s College of Arts and Sciences for Women, Dindigul, India; Department of Chemistry, St. Antony’s College of Arts and Sciences for Women, Dindigul, India

xi

LIST OF CONTRIBUTORS

H.C. Yashavantha Rao Department of Biochemistry, Indian Institute of Science, Bengaluru, India R. Joseph Rathish PSNA College of Engineering and Technology, Dindigul, India L. Reijnders IBED, University of Amsterdam, Amsterdam, The Netherlands Mohd Aslam Saifi Department of Regulatory Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, India S. Satish Department of Studies in Microbiology, Manasagangotri, University of Mysore, Karnataka, India Juhi Shah Division of Biological and Life Sciences, School of Arts and Sciences, Ahmedabad University, Ahmedabad, Gujarat, India Nivya Sharma Department of Regulatory Toxicology, National Pharmaceutical Education and Research (NIPER), Hyderabad, India Gurmeet Singh Pondicherry University, Puducherry, India

Institute

of

Sanjay Singh Division of Biological and Life Sciences, School of Arts and Sciences, Ahmedabad University, Ahmedabad, Gujarat, India Shashi Bala Singh Department of Regulatory Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, India Constantine Stalikas Laboratory of Analytical Chemistry, Department of Chemistry, University of Ioannina, Ioannina 451 10, Greece Vojislav Stani´c Vinˇca Institute of Nuclear Sciences, Laboratory of Radiation and Environmental Protection, University of Belgrade, Belgrade, Serbia Md Abdus Subhan Department of Chemistry, Shah Jalal University of Science and Technology, Sylhet, Bangladesh A. Subramania Centre for Nano Sciences & Technology, Madanjeet School of Green Energy Technologies, Pondicherry University, Puthucherry, India Thodhal Yoganandham Suman College of Life Science, Henan Normal University, Xinxiang, P.R. China; Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing, P.R. China; Ecotoxicology Division, Centre for Ocean Research, Sathyabama Institute of Science and Technology, Chennai, Tamil Nadu, India A. Suriya Prabha Department of Chemistry, Mount Zion College of Engineering and Technology, Pudukkottai, India S. Santhana Prabha PSNA College of Engineering and Technology, Dindigul, India Sladjana B. Tanaskovi´c Faculty of Pharmacy, Department of General and Inorganic Chemistry, University of Belgrade, Belgrade, Serbia T.

Umasankareswari Rajapalayam, India

Department

of

Chemistry,

Rajapalayam

Rajus

College,

Sachin Vaidh Department of Biological Science and Biotechnology, Institute of Advanced Research, Gandhinagar, India Nidhi Verma Department of Physical Science, Institute of Advanced Research, Gandhinagar, India

xii

LIST OF CONTRIBUTORS

N. Vijaya Department of Chemistry, Vellalar College for Women, Thindal, India Gajendra Singh Vishwakarma Department of Biological Science and Biotechnology, Institute of Advanced Research, Gandhinagar, India Tien Viet Vu Faculty of Chemical Technology, Hanoi University of Industry, Hanoi, Vietnam Shaofei Wang Department of Cellular and Genetic Medicine, School of Basic Medical Sciences, Fudan University, Shanghai, P.R. China

Foreword I have always enjoyed multidisciplinary scientific meetings as they are the melting pot for ideas. As most know, the progress in science nowadays is very dependent upon having an input from different skill sets. Nowhere is this more important at this point in time than the field of nanotechnology. The Royal Microscopical Society (RMS) is one such multidisciplinary group—physicists, chemists, biologists, engineers, medics, etc. However, in the 1990s RMS meetings often resulted in parallel sessions with groups of specialists only talking amongst themselves. During my presidency of the RMS I was charged with finding a topic to which all strands of the society could contribute. The topic that quickly suggested itself was the health effects of fine particles—it needs high resolution microscopy, sophisticated chemical, and physical analysis of minute amounts of material, biological experimentation, and observance of health outcomes. The meeting led to a multiauthor book, Particulate Matter: Properties and Effects Upon Health [1]. This appeared just prior to the emergence of societal knowledge of the existence of the nanotechnology industry and was of a certain amount of influence in framing the subsequent nanotoxicology debate. This book addresses a topic of crucial importance. It is widely known that the profligate use of antibiotics in human medicine and animal husbandry over the past 50 1 years has led and continues to lead to the emergence of strains of bacteria that are resistant to all known antibiotics. Society has had less than a century’s benefit from this technology and during that time we have come to expect that the wonders of modern surgery and the perinatal survival of most of our offspring will continue. But now the bugs are fighting back via Darwinian evolution and we stand to lose much of the benefit. Nanotechnology holds out the prospect of being able to target medicines more precisely within the body. With that comes the tempting idea that we may be able to devise mechanisms of delivering antimicrobial therapies more directly to foci of infection or indeed to specific types of bacteria directly. There is a world of difference between accepting a therapy under informed consent and having something thrust upon you, without consent, via the environment. If you have a serious enough illness, cancer for example, you may consider accepting quite potentially dangerous or even totally experimental therapies. And yet we know that some medicines or their metabolites can be passed on to the environment in bodily effluvia and cause subsequent problems. The secret with nanomedicines is going to be to achieve the former and avoid the latter. Nanoparticles have toxicological properties associated with their enhanced surface chemistry and ability to move easily through the body and the environment [2]. There have also been some indications that they may cause ecological problems [3,4]. The nanotechnology industry has so far been an example of collaboration between society, industry, and science to apply the precautionary principle to the development of nanomedicines. It is not clear to what extent this was due to the juxtaposition of emerging knowledge of the

xiii

xiv

FOREWORD

negative effects of particles on health with the arrival of a brand new industry or, on the other hand, to the application of ethical self-governance. It is probably a bit of both. The outcome, however, is good because it appears to be making product developers perform thought experiments to look at what might go wrong and test for that before going into full production. History is littered with examples where this approach has not been heeded (simply look at the costs currently being incurred by the chemical industry because of PCBs in the environment for a good example). A comprehensive list of such poor outcomes is described in the European Environment Agency’s late lessons from early warnings series [5]. Society really does need some sort of thought experiment think tank to be applied in many areas of current technological development, particularly where the technology is both powerful and has the ability to be pervasive in the environment. My current reading of the situation is that the nanomedicine industry may provide a good template for such a collaboration between society and industry—and from that respect it is a very welcome development. This book mirrors that approach in that it covers many of the areas of concern in the realms of toxicology and ecotoxicology while also demonstrating the ingenuity and technological skill that is being applied to the field of nanomedicine. I commend it to you. C. Vyvyan Howard Nano Systems Biology, Centre for Molecular Bioscience, University of Ulster, Coleraine, United Kingdom

References [1] R.L. Maynard, C.V. Howard (Eds.), Particulate Matter: Properties and Effects Upon Health, Bios Scientific Publishers, Oxford UK, 1999. ISBN 1-85996-172-X. [2] A. Elsaesser, C.V. Howard, Toxicology of nanoparticles, Adv. Drug Deliv. Rev. 64 (2012) 129 137. [3] K. Van Hoecke, J.K. Quik, J. Mankiewicz-Boczek, K.C. Deschamphelaere, A. Elsaesser, P. Vandermeeren, et al., Fate and effects of CeO2 nanoparticles in aquatic ecotoxicity tests, Environ. Sci. Technol. 43 (2009) 4537 4546. [4] K. Van Hoecke, K.A.C. De Schamphelaere, Z. Ali, F. Zhang, A. Elsaesser, P. Rivera Gil, et al., Ecotoxicity and uptake of polymer coated gold nanoparticles, Nanotoxicology 7 (1) (2013) 37 47. [5] European Environment Agency Report 22/2001, Late lessons from early warnings: the precautionary principle 1896 2000, ISBN: 92-9167-323-4. https://www.eea.europa.eu/publications/environmental_issue_report_ 2001_22.

C H A P T E R

1 Nanoparticle physiological media interactions R. Dorothy1, N. Karthiga2, S. Senthil Kumaran3, R. Joseph Rathish4, Susai Rajendran4 and Gurmeet Singh5 1

Department of EEE, AMET University, Chennai, India 2Department of Chemistry, SBM College of Engineering, Dindigul, India 3School of Mechanical Engineering, VIT University, Vellore, India 4PSNA College of Engineering and Technology, Dindigul, India 5 Pondicherry University, Puthucherry, India

1.1 Introduction Nanoparticles are surrounded by proteins called the corona, which has been investigated by many techniques. The corona is used in drug delivery and diagnosis. When nanoparticles (NPs) enter into a biological system many interesting incidents can occur. Some occurrences are known, but the majority are unknown.

1.1.1 Particle cell interactions in physiological media Particle cell interactions in physiological media are significant in determining the fate and transport of NPs and the biological responses to them. These interactions are assessed in real time using many techniques including atomic force microscopy -(AFM) based platform.

1.1.2 Engineered nanoparticles in many commercial products Engineered nanoparticles (ENPs) are involved in many industrial processes. Hence environmental [1], occupational [2,3], and consumer exposure are inevitable [4,5]. Nano-enabled technologies are presently used in various biomedical applications. They are used in preventing the transmission of infectious diseases [6,7] and theranostic applications [8].

Nanotoxicity DOI: https://doi.org/10.1016/B978-0-12-819943-5.00001-4

3

Copyright © 2021 Elsevier Inc. All rights reserved.

4

1. Nanoparticle physiological media interactions

1.1.3 Nanoparticle-mediated therapies Nanoparticle-mediated therapies have been introduced in many fields. They can either enhance current diagnostic methods like magnetic resonance imaging (MRI) [9] and X-rays [10], or introduce new methods such as photo-acoustic tomography [11].

1.1.4 Protein corona The potential adverse health effects and the efficacy of theranostics depend on the nanoparticle cell interactions and particle uptake from cells [12]. There is a plethora of published literature documenting ENPs and their ability to penetrate biological barriers and initiate a cascade of events, which probably lead to adverse health effects [13]. When NPs enter physiological media, there is an instantaneous formation of a protein coating, generally known as the protein corona (PC) [14] (Fig. 1.1). The PC is responsible for undesirable biological aspects, tunable drug delivery systems, and novel medical applications. The behavior and the fate of the NPs in biological systems are governed by the PC [15]. The PC has influence of their agglomeration potential [16], the NP adhesion to the cell membrane [17], and potential cell uptake and possible toxicity [18]. Because of the importance of the corona in the nanoparticle cell interactions, numerous studies have focused on the identification of (1) parameters influencing the adsorption of proteins on the surface of NPs in various physiological fluids [19] and (2) the role of the corona on the NP cell uptake [20].

1.1.5 Quantification of particle uptake Even though these studies have investigated the nanoparticle cell interactions, they are done so indirectly by observing secondary features such as the cell adhesion/viability,

FIGURE 1.1 Nanoparticle with protein corona.

1. Basic principles

1.2 Recent advances on the interaction of nanoparticles with biological media

5

morphology, metabolic activity, oxidative stress, and particle uptake. They are later related to NP properties such as size, shape, and surface chemistry/modifications [21]. The most frequently used metric is the quantification of particle uptake [22,23].

1.1.6 Flow cytometry Flow cytometry is the most significant method used for the NP uptake quantification. This requires fluorescence ENPs [24]. Nevertheless the fluorescent dyes may alter the chemistry and affect the nanoparticle cell interactions [25].

1.1.7 Use of plasmonic properties Wang et al. used the plasmonic properties of gold nanoparticles (AuNPs) to study the intracellular localization of NPs to recreate a three-dimensional (3D) mapping of their distribution [26]. However, this approach is limited to a small number of ENPs with intrinsic particle properties.

1.1.8 Other methods used to quantify the nanoparticle uptake Conventional methods like inductively coupled mass spectrometry [27] have been used to quantify the NP uptake. James et al. employed X-ray fluorescence microscopy to map ZnO particles distribution in THP-1 cells [28]. This is considered a very sophisticated method. Recently, molecular dynamic simulations have been used to investigate these interactions [12].

1.1.9 Limitations of the above methods The abovementioned methods have the following limitations. • They do not provide a direct quantification of the nanoparticle cell interactions. • They depend on intrinsic particle properties (e.g., fluorescence, plasmonic resonance, etc.). This limits their applicability to only a few particle systems. • They require highly specialized equipment.

1.1.10 Use of atomic force microscopy Recently AFM has been used to investigate nanoparticle nanoparticle interactions [29]. AFM has been widely used in material science for surface imaging [30] and corrosion inhibition study [31].

1.2 Recent advances on the interaction of nanoparticles with biological media Recent advances on the interaction of NPs with biological media are discussed in this section.

1. Basic principles

6

1. Nanoparticle physiological media interactions

1.2.1 Dynamical modeling of manipulation process in trolling-mode atomic force microscopy Dynamical bulged modeling of trolling-mode AFM in manipulation of bio-samples is presented. The combination of high accuracy and compatibility with physiological conditions makes AFM a unique tool for studying biological materials in liquid medium. However, AFM microcantilever undergoes rigorous sensitivity degradation and noise amplification while operating in liquid; the large hydrodynamic pull between the cantilever and the surrounding liquid overwhelms the tip-sample interface forces that are significant in controlling the process. Consequently, a suitable nanoneedle should be long enough to maintain the cantilever out of liquid medium and short enough to be able to transmit the required force to push NP. Nevertheless, a long nanoneedle may deflect under the approaching force; therefore, its bending deflection should be accounted for in governing equations. Moreover, analytical and finite element stress analysis of nanoneedle and cantilever is carried out to assure about their selected material and geometry. Johnson Kendall Roberts theory is used to model contact mechanics between the needle/surface and the particle. Pull and meniscus forces are utilized to model the liquid media. Governing equations are solved using ODE45 and the system behavior is simulated. Critical conditions of descending including critical time and force are produced, and changes of pushing force, needle deflection, and indentation depths are illustrated. Also, effects of velocity variations are observed. Then, diverse heights for nanoneedle are tested and an appropriate one is preferred for our purpose (to keep the needle out of liquid and transmit the force appropriately). The simulation is repeated for a variety of biological particles and their behaviors are studied. At the end, the present simulation is validated through comparing the results with an earlier work. This comparison shows that the simulation is reliable for the proposed purpose [32].

1.2.2 Limits of the effective medium theory in particle amplified surface plasmon resonance spectroscopy biosensors The resonant wave modes in monomodal and multimodal planar surface plasmon resonance (SPR) sensors and their response to a bidimensional array of AuNPs are investigated both theoretically and experimentally, to examine the parameters that rule the correct NP counting in the emerging metal nanoparticle-amplified surface plasmon resonance (PA-SPR) spectroscopy. With numerical simulations based on the finite element method, we calculate the error executed in the determination of the surface density of NPs σ when the MaxwellGarnett effective medium theory is used for fast data processing of the SPR reflectivity curves upon NP detection. The variation increases directly with the demonstrations of nonnegligible scattering cross-section of the single NP, dipole dipole interactions between adjacent AuNPs and dipolar interactions with the metal substrate. Near field simulations show clearly the set-up of dipolar interactions when the dielectric thickness is smaller than 10 nm and confirm that the strange dispersion usually observed experimentally is due to the failure of the effective medium theories. Using citrate stabilized AuNPs with a nominal diameter of about 15 nm, we express experimentally that dielectric loaded waveguides can be used as correct nanocounters in the range of surface density between 20 and 200 NP/μm2,

1. Basic principles

1.2 Recent advances on the interaction of nanoparticles with biological media

7

opening the way to the use of PA-SPR spectroscopy on systems mimicking the physiological cell membranes on SiO2 supports [33].

1.2.3 Aromatic nitrogen mustard-based autofluorescent amphiphilic brush copolymer as pH-responsive drug delivery vehicle The delivery of clinically accepted nonfluorescent drugs is challenged due to how hard it is to monitor the intracellular drug delivery without incorporating any integrated fluorescence moiety into the drug carrier. The present investigation reports the synthesis of a pH-responsive autofluorescent polymeric nanoscaffold for the administration of nonfluorescent aromatic nitrogen mustard chlorambucil (CBL) drug into the cancer cells. Copolymerization of poly(ethylene glycol) (PEG) attached styrene and CBL conjugated N-substituted maleimide monomers allows the formation of well-defined luminescent alternating copolymer. These amphiphilic brush copolymers self-organized in aqueous medium into 25 68 nm NPs, where the CBL drug is enclosed into the core of the self-assembled NPs. In vitro studies exposed B70% drug was retained under physiological conditions at pH 7.4 and 37 C. At endolysosomal pH 5.0, 90% of the CBL was released by the pH-induced cleavage of the aliphatic ester linkages connecting CBL to the maleimide unit. Although the nascent NP (without drug conjugation) is nonhazardous, the drug conjugated NP confirmed higher toxicity and better cell killing capability in cervical cancer (Henrietta Lacks) cells rather than in normal cells. Interestingly, the copolymer without any predictable chromophore exhibited photoluminescence under ultraviolet (UV) light irradiation due to the presence of “through-space” π π interaction between the C 5 O group of maleimide unit and the adjacent benzene ring of the styrenic monomer. This property used intracellular tracking of CBL conjugated autofluorescent nanocarriers through fluorescence microscope imaging. Finally, the 4-(4-nitrobenzyl)pyridine colorimetric assay was executed to examine the ability of CBL-based polymeric nanomaterials toward alkylation of DNA [34].

1.2.4 Dynamic changes of protein corona compositions on the surface of zinc oxide nanoparticle in cell culture media The potential functions of nanomaterials used in nanomedicine as constituents in drug delivery systems and in other products continue to expand. When nanomaterials are introduced into physiological environments and driven by energetics, they readily associate proteins forming a PC on their surface. This PC could result in a modification of the nanomaterial’s surface characteristics, disturbing their interaction with cells due to conformational changes in adsorbed protein molecules. However, our current understanding of nanobiological interactions is still very limited. Utilizing a liquid chromatography mass spectroscopy/mass spectroscopy technology and a Cytoscape plugin (ClueGO) approach, we studied the composition of the PC for a set of zinc oxide nanoparticles (ZnONP) from cell culture media characteristically and further analyzed the biological interaction of recognized proteins, respectively. In total, 36 and 33 common proteins were examined as being bound to ZnONP at 5 and 60 min, respectively. These proteins were further studied with ClueGO, which provided gene ontology and the biological interaction processes of identified proteins. Proteins bound to the surface of NPs that may change the structure,

1. Basic principles

8

1. Nanoparticle physiological media interactions

therefore the function of the adsorbed protein could accordingly affect the difficult biological processes [35].

1.2.5 In vitro methods for assessing nanoparticle toxicity As a result of their increase in annual production and widespread distribution in the environment, NPs potentially cause an important public health risk. The sought-after catalytic activity approved by their physiochemical properties doubles as a hazard to physiological processes following exposure through inhalation, oral, transdermal, subcutaneous, and intravenous uptake. Upon uptake into the body, their size, morphology, surface charge, coating, and chemical composition supplement the response of biological systems to the materials and increase their toxicity. Recognition of each property is essential to predict the harm imposed by foreign nanomaterials in the body. Assay methods ranging from endotoxin and lactate dehydrogenase signaling to apoptosis and oxidative stress detection supply valuable techniques for exposing biomarkers of NP-induced cellular damage. Spectroscopic investigation of epithelial barrier penetration and distribution within living cells reveals the proclivity of NPs to enter the body’s natural protective boundaries and deposit themselves in cytotoxic locations. Combination of the various characterization methodologies and assays is required for every new nanoparticulate system despite preexisting data for similar systems due to the lack of deterministic trends among investigated NPs. The propensity of nanomaterials to denature proteins and oxidize substrates in their local environment produces significant concern for the applicability of several traditional in vitro assays, and the alteration of susceptible approaches into novel methods suitable for the evaluation of NPs comprises the focus of future work centered on NP toxicity analysis [36].

1.2.6 Nanoparticles targeting retinal and choroidal capillaries in vivo The functionalization of NPs with exact receptor ligands enables their accumulation in targeted tissues and can be used therapeutically to transport drugs or for diagnostic purposes (Parveen et al., Nanomedicine 8:147 166, 2012). Targeting endothelial cells in retinal and choroidal capillaries can be realized even under physiological conditions using quantum dots as model NPs functionalized with an integrin-binding peptide (Pollinger et al., Proc. Natl. Acad. Sci. 110:6115 6120, 2013). Even though the chemistry is standard and was well-explained in the literature, that we used was well-explained in the literature, there are a number of preparation steps that are delicate and deserve special notice. It is, therefore, our goal to describe step by step the significant methods of ligand immobilization on quantum dot surfaces to assist the reader to reproduce our work. Here we describe the chemical alteration of quantum dots with c as a targeting peptide that allows the resulting modified NPs to adhere to endothelial cells also in the retinal tissue. We demonstrate the properties of the resulting particles by showing some of the in vitro results from our previous studies. Doing so, we concurrently encourage the reader to check particles intended for targeting cells in vivo first by extensive in vitro analysis of particle interaction with cells by the means of flow cytometry and confocal microscopy to prove the successful functionalization. Only then the application of functionalized quantum dots into the

1. Basic principles

1.2 Recent advances on the interaction of nanoparticles with biological media

9

systemic circulation of mice led to the preferred localization of NPs in the retinal and choroidal blood vessels [37].

1.2.7 Distribution of superparamagnetic Au/Fe nanoparticles in an isolated guinea pig brain with an intact blood brain barrier Diagnosis and treatment of brain disorders, such as epilepsy, neurodegenerative diseases, and tumors, promote from innovative approaches to deliver therapeutic or diagnostic compounds into the brain parenchyma, with either a homogeneous or a targeted localized distribution pattern. To evaluate the mechanistic feature of diffusion of NPs into the brain parenchyma, a complex, yet controlled and facilitated environment was used: the isolated guinea pig brain maintained in vitro by arterial perfusion. In this unique preparation the blood brain barrier and the interactions between vascular and neuronal sections are morphologically and functionally conserved. In this study, superparamagnetic Au/Fe nanoparticles (MUS: OT Au/Fe NPs), recently studied as a promising magnetic resonance T2 contrast agent with high cellular penetration, were arterially perfused into the in vitro isolated brain and showed high and homogeneous penetration through transcytosis into the brain parenchyma. Ultramicroscopy investigation of the in vitro isolated brain sections by transmission electron microscope (TEM) analysis of the electron-dense center of the MUS: OT Au/Fe NPs was conducted to understand NPs’ brain penetration through the blood brain barrier after in vitro arterial perfusion and their distribution in the parenchyma. The data shows that MUS: OT Au/Fe NPs enter the brain using a physiological route and therefore can be developed as brain penetrating nanomaterials with potential contrast agent and theranostics capabilities [38].

1.2.8 Long-term real-time tracking live stem cells/cancer cells in vitro/in vivo through highly biocompatible photoluminescent poly(citrate-siloxane) nanoparticles Long-term live cell tracking is desirable and essential to understand the dynamics and complexity of biological interactions in stem cells and cancer cells. Conventional live cells fluorescence trackers are generally nondegradable and show increased toxicity concerns during the long-standing application. Previously we developed ecofriendly fluorescent poly(citrate)-based hybrid elastomers for bone regeneration applications. Here, we fabricated the photoluminescent poly(citrate-siloxane) nanoparticles (PCSNPs) through an oil/ water emulsion method and confirmed their long-term live stem cells/cancer cells imaging applications. PCSNPs showed a uniform size distribution (mean diameter 120 nm) and highly stable dispersability (above 30 days) in different physiological medium, as well as outstanding fluorescent properties and photostability. PCSNPs possess excellent cellular biocompatibility, which could be efficiently internalized by cells and selectively image the cell lysosome with a high photostability. Compared with commercial Cell Tracker Green and Cell Tracker Red, the adipose-derived mesenchymal stem cells or human hepatoma cells were stably labeled by PCSNPs for over 14 days as they grew and developed (seven passages). Additionally, PCSNPs capably tracked cells up to 7 days in vivo through a noninvasive way compared with 1 day of commercial tracker. This study demonstrates an

1. Basic principles

10

1. Nanoparticle physiological media interactions

important approach to design biodegradable multifunctional delivery platforms for biomedical applications such as long-term bioimaging [39].

1.2.9 The effect of silica nanoparticles stability in biological media The stability and level of aggregation of NPs in physiological conditions or different media are significant for biomedical applications. The interaction of NPs in different media could change the physicochemical properties of NPs. In this study, two dissimilar sizes of amorphous silica nanoparticles (SiNPs) encapsulated dye were synthesized using the micelle entrapment method. The SiNPs encapsulated dyes suspension was blended with a different concentration of salt solution, NaCl and mouse serum and protected at 37 C to mimic the human body environment in order to study the interaction of SiNPs encapsulated dyes in physiological conditions. Particles agglomeration or aggregation of SiNPs encapsulated dyes in NaCl solution and mouse serum were examined and analyzed. The absorbance spectra and the stability efficiency were recorded and calculated using ultraviolet visible (UV Vis) spectrometer, while the particle size was measured using Zetasizer particle analysis and TEM. The results showed that 53 nm of SiNPs was more stable compared to 30 nm both in NaCl solution and in mouse serum [40].

1.2.10 Experimental challenges regarding the in vitro investigation of the nanoparticle-biocorona in disease states Toxicological evaluation of NPs requires the utilization of in vitro techniques due to their number and diverse properties. Cell culture systems are often deficient in their aptitude to carry out comparative toxicity evaluation due to dosimetry issues and capability to simulate in vivo environments. Upon encountering a physiological environment, NPs become coated with biomolecules forming a biocorona (BC), influencing function, biodistribution, and toxicity. Disease-induced alterations in the biological milieu can alter BC formation. This study evaluates the role of low-density lipoprotein (LDL) in changing macrophage responses to iron oxide (Fe3O4) NPs. BCs were formed by incubating Fe3O4NPs in serum-free media, or 10% fetal bovine serum with or without LDL present. Following exposures to a normalized dose (25 μg/mL), macrophage association of Fe3O4NPs with a LDL-BC was enhanced. TNFα mRNA expression and protein levels were differentially stimulated due to BCs. Cell surface expression of SR-B1 was condensed following all Fe3O4NPs exposures, while only NPs with an LDL-BC enhanced mitochondrial membrane potential. These findings propose that elevations in LDL may give to distinct BC formation thereby influencing NP-cellular interactions and response. Further, our study highlights challenges that may arise during the in vitro evaluation of disease-related variations in the NP-BC [41].

1.2.11 Effect of ionic strength on shear-thinning nanoclay polymer composite hydrogels Nanoclay polymer shear-thinning composites are designed for a broad range of biomedical applications, including tissue engineering, drug delivery, and additive biomanufacturing.

1. Basic principles

1.2 Recent advances on the interaction of nanoparticles with biological media

11

Despite the advances in clay-polymer injectable nanocomposites, colloidal properties of layered silicates are not fully considered in evaluating the in vitro performance of shearthinning biomaterials (STBs). Here, as a model system, we investigate the effect of ions on the rheological properties and injectability of nanoclay gelatin hydrogels to know their behavior when prepared in physiological media. In particular, we learn the effect of sodium chloride (NaCl) and calcium chloride (CaCl2), common salts in phosphate buffered saline (PBS) and cell culture media (e.g., Dulbecco’s Modified Eagle’s Medium), on the structural organization of nanoclay (LAPONITE XLG-XR, a hydrous lithium magnesium sodium silicate)-polymer composites, responsible for the shear-thinning properties and inject ability of STBs. The formation of nanoclay polymer aggregates due to the ion-induced shrinkage of the dispersed double layer and finally the liquid-solid phase separation decreases the resistance of STB against elastic deformation, decreasing the yield strain. Accordingly, the strain corresponding to the onset of structural breakdown (yield zone) is regulated by the ion type and concentration. These results are independent of the STB composition and can directly be converted into the physiological conditions. The exfoliated nanoclay undergoes visually undetectable aggregation upon mixing with gelatin in physiological media, resulting in heterogeneous hydrogels that phase divide under stress. This work gives fundamental insights into nanoclay polymer interactions in physiological environments, paving the way for designing clay-based injectable biomaterials [42].

1.2.12 The effect of surface charge and pH on the physiological behavior of cobalt, copper, manganese, antimony, zinc, and titanium oxide nanoparticles in vitro There is inadequate knowledge regarding various interactions of metal NPs in a living organism. Assumingly, metals can connect to nucleic acids, peptides, and proteins (e.g., enzymes), and change the functioning of vital cellular sections after entering the organism. The predictive factors for quantitative nanostructure activity relationship analysis could enhance efficient and harmless usage of NPs in the industry as well in the medicine. The studies value the composition of the NP corona determined by time, temperature, and source of protein which has been found to implicate the physiological behavior of NPs. One has largely been ignored: the NPs specific isoelectric point (IEP) and pH at the state of measurement. Herein, this study investigates the effect of pH and surface charge of six metal oxide (MeOx) NPs on time dependency of cytotoxicity. Several aspects of the characterization of ultrafine particles in the actual test system, which is the most relevant for the explanation of the toxicological data, are referred: (1) the difference of pH in the room temperature and in the incubation conditions; (2) the difference of dispersions in MilliQ and complete cell media; (3) the need to demonstrate the pH and IEP when the hydrodynamic size is measured; (4) the significance of time due to the time-dependent equilibration and changes of NPs corona. The surface charge determines the formation of corona and could be modified by pH. MeOx NPs without fully charge equilibrated corona might play the main role of MeOx NPs entering into the cell and accordingly the time-dependent materialization of the cellular effect [43].

1. Basic principles

12

1. Nanoparticle physiological media interactions

1.2.13 Sweet strategies in prostate cancer biomarker research: focus on a prostate-specific antigen A clarion call for early diagnosis of prostate cancer (PCa) can be addressed using new approaches such as abnormal protein glycosylation. Proteins are naturally affected by numerous posttranslational modifications, mainly by glycosylation which is associated with physiological and pathological transformation participating in the growth of diseases such as various types of cancer, but also neurodegenerative disorders, endocrine abnormalities, AIDS, etc. Therefore, glycoproteins play a vital role in cancer biomarker research, and determination of glycosylation is nowadays one of the key analytical tasks. The predominantly used approach based on affinity assays using lectins as glycorecognition elements has become an essential part in the biomedical research as it shows great prospects in the clinical diagnostics. Due to their ability to understand saccharide structures, lectins can be applied for binding to different molecules and substrates such as proteins, lipids, cell walls as well as in biological materials, including stem cells and microorganisms. In order to improve the diagnostic potential of well-known cancer biomarkers, lectin-based biosensors and biochips are being widely used for the finding of glycoproteins. In this review, we will focus on various bioassay strategies for glycoprofiling of a prostatespecific antigen (PSA) with an emphasis on modern and potential techniques suitable for the analysis of PSA glycan patterns biosensors, biochips, and mass spectrometry methods. All mentioned methods are suitable for applications in research, diagnosis, and therapy of PCa [44].

1.2.14 Iron oxide colloidal nanoclusters as theranostic vehicles and their interactions at the cellular level Advances in surfactant-assisted chemical approaches have led the way for the utilization of nanoscale inorganic particles in medical diagnosis and treatment. In this field, magnetically-driven multimodal nanotools that perform both detection and therapy, welldesigned in size, shape, and composition, are highly advantageous. Such a theranostic material—which entails the controlled assembly of smaller (maghemite) nanocrystals in a secondary motif that is highly dispersible in aqueous media—is discussed here. These surface functionalized, pomegranate-like ferrimagnetic nanoclusters (40 85 nm) are made of nanocrystal subunits that show a remarkable MRI contrast efficiency, which is better than that of the superparamagnetic contrast agent Endorem. Going beyond this feature and with their demonstrated low cytotoxicity in hand, we study the critical interaction of such nanoprobes with cells at different physiological environments. The time-dependent in vivo scintigraphic imaging of mice experimental models, combined with a biodistribution study, revealed the accretion of nanoclusters in the spleen and liver. Moreover, the in vitro production of spleen cells and cytokine production witnessed a size-selective regulation of immune system cells, inferring that smaller clusters induce mainly inflammatory activities, while larger ones stimulate anti-inflammatory actions. The preliminary findings corroborate that the modular chemistry of magnetic Fe3O4 nanoclusters stimulates unknown pathways that could be determined to modify their function in favor of healthcare [45].

1. Basic principles

1.2 Recent advances on the interaction of nanoparticles with biological media

13

1.2.15 Assembly of carboxylated zinc phthalocyanine with gold nanoparticle for colorimetric detection of calcium ion A series of water-soluble carboxylated zinc phthalocyanine (ZnPc-COOH) were obtained from a facile hydrolyzation of terminating nitriles groups of zinc phthalocyanine synthesized via bisphthalonitrile based precursor. After the AuNPs with positively charged surfactant cetrimonium bromide were added to as-prepared ZnPc-COOH solution, the electronic interaction between them would contribute to the tunable conjugate of AuNPs/ZnPc-COOH and direct to a red-shifted absorption peak in UV Vis spectrum. Particularly, both the amount of phthalocyanine rings and concentrations of ZnPc-COOH would create a large difference in the interaction with AuNPs. In the presence of different metal ions, the ZnPc-COOH/AuNPs aqueous solution revealed a selective response to Ca21, leading to an increased aggregation extent, while the naked eye visualized color change. The further experiment exposed that the red-shift was available in a wide concentration range of Ca21, and the red-shift degree was proportional to the concentration of Ca21 in the range of 2 8 μM with a limit of detection defined as 1 μM. Combing the photosensitivity of ZnPc-COOH and localized surface resonance plasmon of AuNPs, this label-free search would give a potential application in colorimetric detection and photosensitization under a physiological environment [46].

1.2.16 Developing the next generation of graphene-based platforms for cancer therapeutics Graphene has a hopeful future in applications such as disease identification, cancer therapy, drug/gene delivery, bioimaging, and antibacterial approaches due to graphene’s distinctive physical, chemical, and mechanical properties alongside minimal toxicity to normal cells, and photostability. However, these unique features and bioavailability of graphene are fraught with uncertainties and concerns for environmental and occupational exposure. Changes in the physicochemical properties of graphene influence biological responses including reactive oxygen species (ROS) production. Less production of ROS by currently available theranostic agents, for example, magnetic nanoparticles (MNP), carbon nanotubes, gold nanostructures or polymeric NPs, controls their clinical application in cancer therapy. Oxidative stress made by graphene accumulated in living organs is owing to acellular factors, which may affect physiological interactions between graphene and target tissues and cells. Acellular factors include particle size, shape, surface charge, surface containing functional groups, and light activation. Cellular responses such as mitochondrial respiration, graphene cell interactions and pH of the medium are also determinants of ROS production. The mechanisms of ROS production by graphene and the role of ROS for cancer treatment are inadequately understood. The aim of this study is to set the theoretical basis for further research in growing graphene-based theranostic platforms [47].

1.2.17 pH-Responsive morphology-controlled redox behavior and cellular uptake of nanoceria in fibrosarcoma Mehmood et al. reported on structural/microstructural associations with biological performance for three nanoceria morphologies, aiming to explain the major factors in their

1. Basic principles

14

1. Nanoparticle physiological media interactions

interactions with fibrosarcoma [48]. These include the pH of the in vitro medium and the crystallinities, stoichiometries, surface areas and chemistries, and maximal oxygen vacancy concentrations ([VO••]Max). Although the [VO••]Max is dominant in the redox behavior, the role of the morphology was marked in the order of usefulness of the redox regulation, which was nanocubes (NC) , nanorods (NR) , nanooctahedra (NO). The proposed mechanism illustrates the role of VO•• in explaining antioxidant behavior at physiological pH 7.4 and prooxidant behavior in the tumor microenvironment pH 6.4. Cellular uptake at pH 7.4 was dominated by the morphology of the NP, demonstrating the order NO , NC , NR. Control of the [VO••]Max, morphology, and dependent structural and microstructural parameters can be used to optimize the uptake and redox performance of nanoceria [48].

1.2.18 pH- and thermo-sensitive MTX-loaded magnetic nanocomposites: synthesis, characterization, and in vitro studies on A549 lung cancer cell and MR imaging Farshbaf et al. have proposed [49] a simplistic method for fabrication of multifunctional pH- and thermo-sensitive magnetic nanocomposites (MNCs) as a theranostic agent for use in targeted drug delivery and MRI. To this end, the investigators decorated Fe3O4 MNPs with N,N-dimethylaminoethyl methacrylate and N-isopropylacrylamide, best known for their pH- and thermo-sensitive properties, respectively. The investigators also conjugated mesoporous silica nanoparticles (MSNs) to polymer matrix acting as a drug container to increase the drug encapsulation efficiency. Methotroxate (MTX), as a model drug, was effectively loaded in MNCs (M-MNCs) via surface adsorption onto MSNs and electrostatic interaction between drug and carrier. The pH- and temperature-triggered liberate of MTX was concluded through the estimation of in vitro release at both physiological and simulated tumor tissue conditions. Based on in vitro cytotoxicity assay results, M-MNCs significantly exposed higher antitumor activity compared to free MTX. In vitro MR susceptibility experiment showed that M-MNCs relatively possessed high transverse relaxivity (r2) of about 0.15/mM/ms and a linear relationship between the transverse relaxation rate (R2), and the Fe concentration in the M-MNCs was also demonstrated. Therefore, the designed MNCs can potentially become an elegant drug transporter, while they also can be a promising MRI negative contrast agent [49].

1.2.19 Monitoring the dynamics of cell-derived extracellular vesicles at the nanoscale by liquid-cell transmission electron microscopy Cell-derived extracellular vesicles (EVs) circulating in body fluids hold assures as bioactive therapeutic agents and as biomarkers to detect an extensive range of diseases. However, nano-imaging methods are required to characterize these complex and heterogeneous soft materials in their native wet environment. The investigators exploit liquid-cell transmission electron microscopy (LCTEM) to characterize the morphology and dynamic behavior of EVs in physiological media with nanometer resolution. The beam-induced controlled growth of AuNPs on bilayer membranes is used as an original in situ staining method to advance the contrast of EVs and artificial liposomes. LCTEM provides

1. Basic principles

1.2 Recent advances on the interaction of nanoparticles with biological media

15

information about the size distribution and concentration of EVs that are consistent with Cryo-TEM and NP tracking analysis measurements. Moreover, LCTEM offers a distinctive insight into the dynamics of EVs depending on their liquid environment. The sizedependent morphology of EVs is sensitive to osmotic stress which tends to transform their spherical shape to ellipsoidal, stomatocyte, or discocyte morphologies. In the liquid-cell, EVs show a subdiffusive motion due to strong interactions between the AuNPs and the liquid-cell windows. Finally, the high-resolution monitoring of EV aggregation and fusion illustrate that LCTEM opens up a new way to learn cell-membrane dynamics [50].

1.2.20 In vivo formation of protein corona on gold nanoparticles—the effect of their size and shape The efficiency of drug delivery and other nanomedicine-related therapies largely relies on the ability of NPs to attain the target organ. However, when NPs are injected into the bloodstream, their surface is instantly modified upon interaction with blood components, chiefly with proteins. It is well known that a dynamic and multilayered protein structure is formed spontaneously on the NP upon contact with physiological media, which has been expressed PC. Although several determinant factors involved in PC formation have been recognized from in vitro studies, definite relationships between the nanomaterial synthetic identity and its resulting biological identity under realistic in vivo conditions ´ lvarez et al. [51] have presented a complete study of in vivo remain mysterious. Garcı´a-A PC formation after blood circulation of anisotropic AuNPs (NR and nanostars). Plasmonic AuNPs of different shapes and sizes were coated with PEG, intravenously administered in CD-1 mice and consequently recovered. The results from gel electrophoresis and mass spectrometry analysis revealed the formation of complex PCs, as early as 10 min postinjection. The total amount of protein adsorbed onto the particle surface and the PC composition were found to be affected by both the particle size and shape [51].

1.2.21 Fabrication of folic acid magnetic nanotheranostics: an insight on the formation mechanism, physicochemical properties, and stability in simulated physiological media Nanodevices based on magnetite functionalized with folic acid (FA) with enhanced properties to be employed as theranostics in various types of cancer are proposed in the following. Two methodologies for FA incorporation were explored aiming to attain appropriate loading efficiency as well as sufficient stability of nanosystems in physiological media. To this end, easy adsorption and covalent binding of FA and some experimental conditions derived from both procedures were studied. A systematic physicochemical characterization was performed using all the formulations. The mechanism of the interaction between FA and MNPs was elucidated from characterization results supported by theoretical studies using spin-polarized density functional theory. Both data coincide in that the selective functional group of FA (pteridine group) remained available after FA binding MNPs. Such studies also demonstrated that any of FA carboxylate groups could be available to potentially connect other molecules (i.e., therapeutic agents). Additionally, other issues that are not normally accomplished in

1. Basic principles

16

1. Nanoparticle physiological media interactions

reported articles were included; that is, the stability according to two different criteria: size evolution (expressed as hydrodynamic diameter) as a function of time in aqueous media; and the capacity FA retention in PBS, pH 5 7.4. Recovered data showed that the samples are stable at least 15 days in water and 4 h in buffer without significant modifications of their properties. The possibility of these formulations interacting with simulated physiological fluid was also assayed. The results revealed that PC was formed around all the tested formulations leading to more stable nanodevices in terms of their hydrodynamic sizes and size evolution along the time. To complete the theranostic characteristic, Doxorubicin was added to the MNPs@FA by physical adsorption, to offer the therapeutic function. The suitable absorption was verified by Fourier transform infrared (FTIR) spectroscopy [52].

1.2.22 Multifunctional pH sensitive three-dimensional scaffolds for treatment and prevention of bone infection Multifunctional-therapeutic 3D scaffolds have been prepared. These biomaterials are capable of destroying the Staphylococcus aureus bacterial biofilm and allowing bone regeneration at the same time. The present study is focused on the design of pH sensitive 3D hierarchical meso-macroporous 3D scaffolds based on mesoporous glass/hydroxyapatite (MGHA) nanocomposite formed by a mesostructured glassy network with implanted hydroxyapatite NPs, whose mesopores have been loaded with levofloxacin (Levo) as an antibacterial agent. These 3D platforms exhibit controlled and pH-dependent Levo release, sustained over time at physiological pH (7.4) and notably increased at infection pH (6.7 and 5.5), which is owing to the different interaction rate between diverse Levo species and the silica matrix. These 3D systems are able to hinder the S. aureus growth and destroy the bacterial biofilm without cytotoxic effects on human osteoblasts, allowing a sufficient colonization and differentiation of preosteoblastic cells on their surface. These findings propose promising applications of these hierarchical MGHA nanocomposite 3D scaffolds for the treatment and prevention of bone infection. Statement of significance multifunctional 3D nanocomposite scaffolds with the ability for loading and continued delivery of an antimicrobial agent, to eliminate and prevent bone infection and at the same time to contribute to bone regeneration process without cytotoxic effects on the surrounding tissue has been proposed. These 3D scaffolds exhibit a sustained Levo delivery at physiological pH (pH 7.4), which increases particularly when pH decreases to characteristic values of bone infection process (pH 6.7 and 5.5). In vitro competitive assays between preosteoblastic and bacteria onto the 3D scaffold surface demonstrated an adequate osteoblast colonization in whole scaffold surface together with the ability to eliminate bacterial contamination [53].

1.2.23 Antibody-pHPMA functionalized fluorescent silica nanoparticles for colorectal carcinoma targeting The general application of highly effective drugs such as cytostatics poses the risks of side effects, which could be reduced by using a carrier system able to specifically deliver the encapsulated drug to the target tissue. Essential components of a NP-based drug delivery system include the drug carrier itself, a targeting moiety, and a surface coating that

1. Basic principles

1.2 Recent advances on the interaction of nanoparticles with biological media

17

ˇ ´ et al. [54] have reported on the prepminimizes recognition by the immune system. Lizonova aration, in vitro characterization and in vivo testing of a new delivery system consisting of fluorescent silica NPs functionalized with a nonimmunogenic stealth polymer poly(N-(2hydroxypropyl)methacrylamide) (pHPMA) and a monoclonal antibody immunoglobulin (IgG) M75 that specifically joins to carbonic anhydrase IX (CA IX). CA IX is a promising therapeutic target, as it is a hallmark of several hypoxic tumors including colorectal carcinoma. Exclusively in this work, the monoclonal antibody was covalently attached to the surface of fluorescently labeled silica NPs via a multivalent amino-reactive copolymer rather than a traditional bivalent linker. The pHPMA-M75 functionalized SiO2 NPs exhibited excellent colloidal stability in physiological media. Their in vitro characterization by flow cytometry confirmed a highly specific interaction with colorectal carcinoma cells HT-29. In vivo study on athymic NU/NU nude mice exposed that the SiO2-pHPMA-M75 NPs are capable of circulating in the blood after intravenous administration, and they accumulate in the tumor at tenfold higher concentration than NPs without exact targeting, with a significantly longer retention time. Additionally, it was found that by reducing the dose administered in vivo, the selectivity of the NP biodistribution could be further improved in favor of the tumor [54].

1.2.24 Size-controlled, colloidally stable, and functional nanoparticles based on the molecular assembly of green tea polyphenols and keratins for cancer therapy While intellectual NPs with therapeutic effects offer a resolving strategy for low drug loading efficiency, poor metabolism and elimination of current nanoparticulate drug delivery systems, accurate preparation of colloidally stable but stimuli-responsive nanocarriers with size tunability is still a challenging task. Yi et al. [55] have extended a simplistic and sustainable method through the use of naturally reproducible green tea polyphenols and hair keratins to prepare biocompatible, colloidally stable, stimuli-responsive NPs with therapeutic effects. The present strategy simply involves covalent interactions of tea catechins and keratins, giving rise to the molecular assembly of size-controlled NPs (30 230 nm) which are long-term colloidally stable at physiological media but are disassembled under pathological conditions, ideally for targeted delivery of anticancer drugs. The cell experiments established that these NPs are bio-safe, have the inherent bioactivity of tea catechins, and that the drug-loaded NPs yield a higher cancer cell inhibition rate than free drugs. In addition, the NPs are found to improve the bioavailability of tea polyphenols, according to animal studies, which further demonstrates that the use of NPs as drug carriers results in enhanced anticancer efficiency with insignificant systemic toxicity. Given that large-scale preparation of size-controlled NPs could already be easily achieved, Yi et al. [55] have provided an innovative nanotechnological approach to make good use of green tea polyphenols with beneficial health effects, potentially for therapeutic and protective purposes [55].

1.2.25 Influence of interaction between α-Fe2O3 nanoparticles and dissolved fulvic acid on the physiological responses in Synechococcus sp. PCC7942 The ecotoxicity of α-Fe2O3 NPs and its interaction with a typical natural organic matter, fulvic acid (FA) on the physiological responses of Synechococcus sp. PCC7942 was

1. Basic principles

18

1. Nanoparticle physiological media interactions

calculated. α-Fe2O3 NPs reduced the algae growth at a concentration higher than 10 mg/L and induced oxidative stress, indicated by improved antioxidant enzymes activities, elevated protein, and sugar content. FA could efficiently recover cell growth and decrease antioxidant enzyme actions stimulated by α-Fe2O3 NPs, indicating that the toxicity of NPs was alleviated in the presence of FA. α-Fe2O3 NPs could form large aggregates coating on the cell surface and inhibit cell development. FTIR spectra confirmed FA interacted with α-Fe2O3 NPs through carboxyl groups, partly replaced the binding sites of α-Fe2O3 NPs on algal cell walls, which thus reduced NP aggregates coating on the cell surface. This favors reducing the oxidative strain caused by direct contact and increasing light availability, thus diminishes NPs toxicity [56].

References [1] W. Gao, J. Wang, The environmental impact of micro/nanomachines: a review, ACS Nano 8 (2014) 3170 3180. [2] B. Dhimiter, J. Martin, C. Santeufemio, Q. Sun, K. Lee Bunker, et al., Physicochemical and morphological characterisation of nanoparticles from photocopiers: implications for environmental health, Nanotoxicology 7 (2012) 989 1003. [3] S. Pirela, G. Pyrgiotakis, B. Dhimiter, T. Treye, V. Castranova, P. Demokritou, Development and characterization of an exposure platform suitable for physico-chemical, morphological and toxicological characterization of Printer Emitted Particles (PEPs), Inhal. Toxicol. 26 (7) (2014) 400 408. [4] M.A. Philbert, G.V. Alexeeff, T. Bahadori, J.M. Balbus, M.G. Bawendi, P. Biswas, et al., Review of Federal Strategy for Nanotechnology-Related Enviromental, Health, and Safety Research, The National Academic Press, Washington, DC, 2008, pp. 1 131. [5] G. Sotiriou, C. Watson, K. Murdaugh, T.H. Darrah, G. Pyrgriotakis, A. Elder, et al., Engineering safer-bydesign, transparent, silica-coated ZnO nanorods with reduced DNA damage potential, Environ. Sci. Nano 1 (2014) 144 153. [6] G. Pyrgiotakis, J. McDevitt, A. Bordini, E. Diaz, R. Molina, C. Watson, et al., A chemical free, nanotechnologybased method for airborne bacterial inactivation using engineered water nanostructures, Environ. Sci. Nano 1 (2014) 15 26. [7] G. Pyrgiotakis, J. McDevitt, T. Yamauchi, P. Demokritou, A novel method for bacteria inactivation using engineered water nanostructures, J. Nanopart. Res. 14 (2012) 1027 1038. [8] S.R. Grobmyer, D.L. Morse, B. Fletcher, L.G. Gutwein, P. Sharma, V. Krishna, et al., The promise of nanotechnology for solving clinical problems in breast cancer, J. Surg. Oncol. 103 (2011) 317 325. [9] L. Bu, J. Xie, K. Chen, J. Huang, Z.P. Aguilar, A. Wang, et al., Assessment and comparison of magnetic nanoparticles as MRI contrast agents in a rodent model of human hepatocellular carcinoma, Contrast Media Mol. Imaging 7 (2012) 363 372. [10] K.K. Jain, Advances in the field of nanooncology, BMC Med. 8 (2010) 83. [11] L. Xi, S.R. Grobmyer, G. Zhou, W. Qian, L. Yang, H. Jiang, Molecular photoacoustic tomography of breast cancer using receptor targeted magnetic iron oxide nanoparticles as contrast agents, J. Biophotonics 7 (2014) 401 409. [12] H.-M. Ding, Y.-Q. Ma, Controlling cellular uptake of nanoparticles with pH-sensitive polymers, Sci. Rep. 3 (2013) 2804. [13] J.M. Cohen, R. Derk, L. Wang, J. Godleski, L. Kobzik, J. Brain, et al., Tracking translocation of industrially relevant engineered nanomaterials (ENMs) across alveolar epithelial monolayers in vitro, Nanotoxicology 8 (2014) 216 225. [14] T.A. Faunce, J. White, K.I. Matthaei, Integrated research into the nanoparticle protein corona: a new focus for safe, sustainable and equitable development of nanomedicines, Nanomedicine 3 (2008) 859 866. [15] I. Lynch, A. Salvati, K.A. Dawson, Protein-nanoparticle interactions: what does the cell see? Nat. Nanotechnol. 4 (2009) 546 547. [16] J. Cohen, G. DeLoid, G. Pyrgiotakis, P. Demokritou, Interactions of engineered nanomaterials in physiological media and implications for in vitrodosimetry, Nanotoxicology 7 (2012) 417 431.

1. Basic principles

References

19

˚ berg, Nanoparticle adhesion [17] A. Lesniak, A. Salvati, M.J. Santos-Martinez, M.W. Radomski, K.A. Dawson, C. A to the cell membrane and its effect on nanoparticle uptake efficiency, J. Am. Chem. Soc. 135 (2013) 1438 1444. [18] Y. Yan, K.T. Gause, M.M.J. Kamphuis, C.-S. Ang, N.M. O’Brien-Simpson, J.C. Lenzo, et al., Differential roles of the protein corona in the cellular uptake of nanoporous polymer particles by monocyte and macrophage cell lines, ACS Nano 7 (2013) 10960 10970. [19] S.C. Wasdo, J. Juntunen, H. Devarajan, K.B. Sloan, A comparison of the fit of flux through hairless mouse skin from water data to three model equations, Int. J. Pharm. 366 (2009) 65 73. [20] P. Nativo, I.A. Prior, M. Brust, Uptake and intracellular fate of surface-modified gold nanoparticles, ACS Nano 2 (2008) 1639 1644. [21] P.V. AshaRani, G. Low Kah Mun, M.P. Hande, S. Valiyaveettil, Cytotoxicity and genotoxicity of silver nanoparticles in human cells, ACS Nano 3 (2009) 279 290. [22] L. Treuel, G.U. Nienhaus, Toward a molecular understanding of nanoparticle protein interactions, Biophys. Rev. 4 (2012) 137 147. [23] L. Treuel, X. Jiang, G.U. Nienhaus, New views on cellular uptake and trafficking of manufactured nanoparticles, J. R. Soc. Interface 10 (2013) 20120939. [24] Y. Ibuki, T. Toyooka, Nanoparticle uptake measured by flow cytometry, Methods in Molecular Biology, Vol. 926, Humana Press, Totowa, NJ, 2012, pp. 157 166. [25] A. Salvati, A.S. Pitek, M.P. Monopoli, K. Prapainop, F.B. Bombelli, D.R. Hristov, et al., Transferrinfunctionalized nanoparticles lose their targeting capabilities when a biomolecule corona adsorbs on the surface, Nat. Nanotechnol. 8 (2013) 137 143. [26] S.-H. Wang, C.-W. Lee, A. Chiou, P.-K. Wei, Size-dependent endocytosis of gold nanoparticles studied by three-dimensional mapping of plasmonic scattering images, J. Nanobiotechnol. 8 (2010) 33. [27] B.D. Chithrani, A.A. Ghazani, W.C.W. Chan, Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells, Nano Lett. 6 (2006) 662 668. [28] S.A. James, B.N. Feltis, M.D. de Jonge, M. Sridhar, J.A. Kimpton, M. Altissimo, et al., Quantification of ZnO nanoparticle uptake, distribution, and dissolution within individual human macrophages, ACS Nano 7 (2013) 10621 10635. [29] G. Pyrgiotakis, C.O. Blattmann, S. Pratsinis, P. Demokritou, Nanoparticle-nanoparticle interactions in biological media by atomic force microscopy, Langmuir 29 (2013) 11385 11395. [30] J. Li, A. Cassell, H. Dai, Carbon nanotubes as AFM tips: measuring DNA molecules at the liquid/solid interface, Surf. Interface Anal. 28 (1999) 8 11. [31] V.R. Nazeera Banu, S. Rajendran, S. Senthil Kumaran, Investigation of the inhibitive effect of Tween 20 self assembling nanofilms on corrosion of carbon steel, J. Alloys Compd. 675 (2016) 139 148. [32] S.Z. Mohammadi, M. Moghaddam, H.N. Pishkenari, Dynamical modeling of manipulation process in TrollingMode AFM, Ultramicroscopy 197 (2019) 83 94. [33] J.S. Costa, Q. Zaman, K.Q. da Costa, V. Dmitriev, O. Pandoli, G. Fontes, et al., Limits of the effective medium theory in particle amplified surface plasmon resonance spectroscopy biosensors, Sensors (Basel) 19 (3) (2019) 584. [34] B. Saha, N. Choudhury, S. Seal, B. Ruidas, P. De, Aromatic nitrogen mustard-based autofluorescent amphiphilic brush copolymer as pH-responsive drug delivery vehicle, Biomacromolecules 20 (1) (2019) 546 557. [35] V.-V. Giau, Y.-H. Park, K.-H. Shim, S.-W. Son, S.-S.A. An, Dynamic changes of protein corona compositions on the surface of zinc oxide nanoparticle in cell culture media, Front. Chem. Sci. Eng. 13 (2019) 90 97. [36] D.T. Savage, J.Z. Hilt, T.D. Dziubla, In vitro methods for assessing nanoparticle toxicity, Methods Mol. Biol. 1894 (2019) 1 29. [37] A. Haunberger, A. Goepferich, Nanoparticles targeting retinal and choroidal capillaries in vivo, Methods Mol. Biol. 1834 (2019) 391 404. [38] B. Sanavio, L. Librizzi, P. Pennacchio, G.V. Beznoussenko, F. Sousa, P.J. Silva, et al., Distribution of superparamagnetic Au/Fe nanoparticles in an isolated guinea pig brain with an intact blood brain barrier, Nanoscale 10 (47) (2018) 22420 22428. [39] F. Li, Y. Du, G. Pi, B. Lei, Long-term real-time tracking live stem cells/cancer cells in vitro/in vivo through highly biocompatible photoluminescent poly(citrate-siloxane) nanoparticles, Mater. Sci. Eng. 93 (2018) 380 389. [40] A. Ahmad, N.D. Zakaria, Z. Lockman, K.A. Razak, The effect of silica nanoparticles stability in biological media, J. Phys. Conf. Ser. 1082 (1) (2018) 012047. [41] S.X.-F. Adamson, Z. Lin, R. Chen, L. Kobos, J. Shannahan, Experimental challenges regarding the in vitro investigation of the nanoparticle-biocorona in disease states, Toxicol. Vitro 51 (2018) 40 49.

1. Basic principles

20

1. Nanoparticle physiological media interactions

[42] A. Sheikhi, S. Afewerki, R. Oklu, A.K. Gaharwar, A. Khademhosseini, Effect of ionic strength on shearthinning nanoclay-polymer composite hydrogels, Biomater. Sci. 6 (8) (2018) 2073 2083. [43] T. Titma, The effect of surface charge and pH on the physiological behaviour of cobalt, copper, manganese, antimony, zinc and titanium oxide nanoparticles in vitro, Toxicol. Vitro 50 (2018) 11 21. ˇ Belicky´, J. Tka´cˇ , J. Katrlı´k, Sweet strategies in prostate cancer biomarker [44] P. Damborsky´, D. Damborska´, S. research: focus on a prostate specific antigen, BioNanoScience 8 (2) (2018) 690 700. [45] A. Kostopoulou, K. Brintakis, E. Fragogeorgi, A. Anthousi, L. Manna, S. Begin-Colin, et al., Iron oxide colloidal nanoclusters as theranostic vehicles and their interactions at the cellular level, Nanomaterials 8 (5) (2018) 315. [46] X. Zhou, K. Jia, X. He, S. Wei, P. Wang, X. Liu, Assembly of carboxylated zinc phthalocyanine with gold nanoparticle for colorimetric detection of calcium ion, J. Mater. Sci. Mater. Electron. 29 (10) (2018) 8380 8389. [47] T.A. Tabish, S. Zhang, P.G. Winyard, Developing the next generation of graphene-based platforms for cancer therapeutics: the potential role of reactive oxygen species, Redox Biol. 15 (2018) 34 40. [48] R. Mehmood, N. Ariotti, J.L. Yang, P. Koshy, C.C. Sorrell, pH-responsive morphology-controlled redox behavior and cellular uptake of nanoceria in fibrosarcoma, ACS Biomater. Sci. Eng. 4 (3) (2018) 1064 1072. [49] M. Farshbaf, R. Salehi, N. Annabi, R. Khalilov, A. Akbarzadeh, S. Davaran, pH- and thermo-sensitive MTXloaded magnetic nanocomposites: synthesis, characterization, and in vitro studies on A549 lung cancer cell and MR imaging, Drug. Dev. Ind. Pharm. 44 (3) (2018) 452 462. [50] M. Piffoux, N. Ahmad, J. Nelayah, C. Wilhelm, A. Silva, F. Gazeau, et al., Monitoring the dynamics of cellderived extracellular vesicles at the nanoscale by liquid-cell transmission electron microscopy, Nanoscale 10 (3) (2018) 1234 1244. ´ lvarez, M. Hadjidemetriou, A. Sa´nchez-Iglesias, L.M. Liz-Marza´n, K. Kostarelos, In vivo formation [51] R. Garcı´a-A of protein corona on gold nanoparticles. The effect of their size and shape, Nanoscale 10 (3) (2018) 1256 1264. [52] P. Azcona, I. Lo´pez-Corral, V. Lassalle, Fabrication of folic acid magnetic nanotheranostics: an insight on the formation mechanism, physicochemical properties and stability in simulated physiological media, Colloids Surf. A Physicochem. Eng. Asp. 537 (2018) 185 196. [53] M. Cicue´ndez, J.C. Doadrio, A. Herna´ndez, M.T. Portole´s, I. Izquierdo-Barba, M. Vallet-Regı´, Multifunctional pH sensitive 3D scaffolds for treatment and prevention of bone infection, Acta Biomater. 65 (2018) 450 461. ˇ ´ , M. Majerska´, V. Kra´l, M. Pechar, R. Pola, M. Kova´rˇ, et al., Antibody-pHPMA functionalised [54] D. Lizonova fluorescent silica nanoparticles for colorectal carcinoma targeting, RSC Adv. 8 (39) (2018) 21679 21689. [55] Z. Yi, Z. Sun, G. Chen, H. Zhang, X. Ma, W. Su, et al., Size-controlled, colloidally stable and functional nanoparticles based on the molecular assembly of green tea polyphenols and keratins for cancer therapy, J. Mater. Chem. B 6 (9) (2018) 1373 1386. [56] M. He, Y. Chen, Y. Yan, S. Zhou, C. Wang, Influence of interaction between A-Fe2O3 nanoparticles and dissolved fulvic acid on the physiological responses in Synechococcus sp. PCC7942, Bull. Environ. Contam. Toxicol. 99 (6) (2017) 719 727.

1. Basic principles

C H A P T E R

2 In vitro methods to assess the cellular toxicity of nanoparticles Krupa Kansara and Ashutosh Kumar Division of Biological and Life Sciences, School of Arts and Sciences, Ahmedabad University, Ahmedabad, India

2.1 Introduction The series of nanotechnology breakthroughs have been the source of numerous novel possibilities for new products and technological interventions. One of the most salient examples in this regard is the widespread employment of nanoparticles across a range of products such as photo-detectors, catalysts, sorbents, biosensors, and semiconductors [1,2]. Metal oxide nanoparticles are considered as the most versatile platform for applications in biomedical and therapeutic areas [35]. Zinc oxide and titanium oxide nanoparticles are used in consumer products including paints and coatings due to their small particle size. These particles have seen an explosion of interest, with its property of being resistant to ultraviolet light being utilized in everything from sunscreen and other cosmetics, to paints, paper, fibers, and a variety of other products [68]. It has genuinely begun to proliferate in our daily environment. However, there is a danger involved with this breakthrough. Such technological innovations often flood the market before every aspect of their use has been thoroughly researched, and this can have adverse consequences. Human beings are in constant exposure to nanoparticles (production, usage, and disposal) either through the usage of nano-based products, inadvertent release of engineered nanoparticles (ENPs) in environment, or by deliberate administration through nanomedicines. Since nanoparticles are small, increased surface area and increased number of atoms on the boundary, the possibility for their reactivity to the biological system is also high. The particle size, surface properties, shape, and composition of the ENPs influence their uptake, distribution, transport, and interaction with plasma proteins in a human system which further induces the adverse effects [6,913]. A variety of nanoparticles with different chemical compositions, synthesized through different methods, differing in size, shape, surface coatings, etc. have been reported to be genotoxic and cytotoxic in different models

Nanotoxicity DOI: https://doi.org/10.1016/B978-0-12-819943-5.00002-6

21

Copyright © 2020 Elsevier Inc. All rights reserved.

22

2. In vitro methods to assess the cellular toxicity of nanoparticles

such as prokaryotes, plants, human cell lines, primary human cells, in vivo, and aquatic models [1419]. There are several in vitro reports that have demonstrated the genotoxic, carcinogenic and apoptotic properties of nanoparticles to human [2022]. Studies have shown that nanoparticles also adversely affect the microbes (Escherichia coli, Pseudomonas aeruginosa, and Streptococcus aureus) which are responsible for maintaining environmental health [21]. This also raises the possibility that the release of nanoparticles into the environment may be detrimental to important bio-geochemical processes in soil such as carbon or nitrogen cycling. Therefore organisms, especially those that interact strongly with their immediate environment, are expected to be affected as a result of their exposure to nanoparticles. It is also likely that the nanoparticles can directly interact with the food web at different trophic levels and affect the ecological sustenance [12,23]. The bio-magnification of engineered nanomaterials across the genera is also a big concern. Different analytical techniques such as microscopy, flow cytometry, and spectroscopy among others are being used to provide an insight toward understanding the adverse effects induced by nanoparticles [24,25]. Cytotoxicity assays determine the number of metabolically active viable cells/or the dead cells, therefore determining the extent of toxicity of nanoparticles [26]. The various properties of nanoparticles such as size, surface properties, internalization, reactivity, aggregation and agglomeration, interaction with the proteins and natural organic matters, and others exhibit a different extent of cytotoxicity in different model organisms (Fig. 2.1) [9,2733]. It has also been postulated that oxidative stress is one of the key mechanisms for nanoparticles-induced toxicity [25]. An increase in the reactive oxygen species (ROS) level and a decrease in the glutathione level play a major role in nanoparticlesinduced toxicity [34]. An increase in the intracellular ROS in different organ specific cell lines has shown to induce cell death by activating multiple signaling pathways [35]. The extent of DNA damage can be quantified by comet and cytokinesis block micronucleus (CBMN) assays. Several studies have shown that metal oxide nanoparticles cause concentration dependent DNA damage as assessed by comet and chromosomal aberrations [36,37]. Nanoparticles have also been reported to modulate the human immune system. Immunotoxicity comprises of the imbalance in cytokines levels, changes in activity of macrophages, and antigen presenting cells [38]. The induction in the release of inflammatory cytokine in human blood cells by zinc oxide ENPs and in the levels of interleukin-1, tumor FIGURE 2.1 Schematics for challenges faced in nanomaterial toxicology.

Coating during synthesis Exposure assessment

Polydispersity

Characterization Challenges in nanomaterial toxicity

Control

Visualization

Dose metric Agglomeration

1. Basic principles

2.2 Materials and methods

23

necrosis factor-α, and IL-6 by iron oxide ENPs in Institute of Cancer Research mice have been reported [9,39]. In vitro studies are often performed to assess the hazard associated with the nanoparticles, as it is rapid, low cost, less uncontrolled variables, and minimum ethical concerns. However, in vitro studies suffer from the accurate prediction of the in vivo toxicity due to unavailability of cell to cell communication and the lack of metabolic activities. The lack of regulatory guidelines, reference standards, and certification processes for nanoparticles (from manufacture to product development) is a major stumbling block in hazard identification through risk and exposure assessment. This is compounded by the lack of equipment for accurate and sensitive measurement of nanoparticles with respect to their number, mass, and surface area in the environment. Hence, it is prudent to address the issues of risks associated with nanoparticles and develop ethical, legal, and regulatory framework to mitigate their exposure. The major thrust of the nanotoxicology research is to assess the hazardous property of the nanoparticles and the risk associated with it. Therefore this chapter describes the different in vitro methods to assess the ENPs induced toxicity.

2.2 Materials and methods 2.2.1 Cytotoxicity Cytotoxicity is the degree to which an agent, that is, nanoparticles, has specific destructive ability on cells. Cytotoxicity assessment is usually determined by the cellular reduction of tetrazolium salts to produce insoluble formazan dyes. 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay is a frequently used assay to assess ENP induced cytotoxicity in vitro. 2.2.1.1 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay MTT is a yellow colored dye that reduces to purple formazan crystals within the mitochondria of a cell by means of dehydrogenase enzymes. The crystals formed are dissolved by dimethysulphoxide (DMSO) and measured spectrophotometrically at 530 nm in a microplate reader. The increase in cytotoxicity corresponds to the increase in the percentage of MTT reduction (Fig. 2.2). 2.2.1.1.1 Materials

1. 5 3 105 cells/mL cell suspension 2. Culture medium, bovine serum albumin (BSA), phosphate buffered saline (PBS; Ca21, Mg21 free) and fetal bovine serum (FBS), trypsin-EDTA, L-glutamine, antibioticantimycotic solution (10,000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) 3. ENPs, 10% Triton X-100 4. MTT stock solution—5 mg/mL MTT in PBS (can be stored for 23 weeks at 4 C or longer at 220 C) 5. MTT working solution—0.5 mg/mL MTT in incomplete media/PBS 6. DMSO

1. Basic principles

24

2. In vitro methods to assess the cellular toxicity of nanoparticles

Grow the cells in 96 well culture plate

Control and ENPs treated cells Aspirate the media with ENPs

Incubate the cells with MTT at 37°C for 3 h

Remove MTT

Add MTT

Add 200 µL DMSO

Record absorbance at 550 nm

FIGURE 2.2 Schematics for cytotoxicity assessment by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

7. Tissue culture flasks (25 cm2), 96-well tissue culture plates, syringe filters (0.22 μm), filter holders 8. Micropipettor (101000 μL) with sterile micropipettor tips 2.2.1.1.2 Medium, buffer, and solutions 2.2.1.1.2.1 In vitro components

2.2.1.1.2.1.1 Preparation of culture medium Dissolve the powdered medium in autoclaved Milli-Q water. Add sodium bicarbonate (2 g/L) and L-glutamine (300 mg/L) to maintain the pH to 7.2. Further add antibiotic/antimycotic solution (10 mL/L) to prevent contamination. Make up the volume to 1 L. Filter the medium with 0.22 μm membrane filters. Check for contamination by keeping the medium at 37 C overnight and if not contaminated then store at 4 C. 2.2.1.1.2.1.2 Phosphate buffered saline (Ca21, Mg21 free) Dissolve the powdered PBS in 990 mL autoclaved Milli-Q water. Maintain the pH to 7.4 and make up the volume to 1000 mL. To inhibit the endonuclease activity of the cells, the PBS used should be calcium and magnesium free. 2.2.1.1.2.1.3 Trypsin-EDTA (0.25%) The ready-to-use solution of trypsin-EDTA is to be thawed, aliquoted into 15 mL tubes, and stored at 220 C. 2.2.1.1.3 Method

1. Seed 1 3 104 cells/well in 96-well tissue culture plate for 24 h at 37 C and 5% CO2. 2. Also in parallel maintain a cell free system with medium without cells.

1. Basic principles

2.2 Materials and methods

25

3. Treat the cells with nanoparticles and positive control (10% Triton X-100) and repeat the experiment thrice. 4. After the treatment, remove the medium and add 100 μL MTT working solution. 5. Incubate for 3 h at 37 C and 5% CO2. 6. Aspirate the MTT solution. 7. Add 200 μL DMSO. Dissolve the formazan crystals by slow pipetting. 8. Read the absorbance of the plate on microtiter plate reader at 550 nm. 9. Calculate the percent MTT reduction using the following formula:   F 550sample 2 F 550sample blank Percent MTT reduction 5 3 100 F 550control 2 F 550control blank 10. Calculate the mean standard deviation (SD) and standard error (SE) for control, positive control, and unknown samples. 11. Compare the mean values of percent MTT reduction in treated cells among various concentrations with the control and analyze the results using analysis of variance (ANOVA) [40]. 2.2.1.1.4 Limitations

1. The MTT assay does not provide the cell counts as the dye is extracted from the cells using DMSO. 2. Does not determine the cellular death mechanism.

2.2.2 Live/dead assessment The number of viable cells in a cell population can be determined by using different dyes based on the changes in membrane permeability. The various assays used for live/dead assessment are explained below. 2.2.2.1 Propidium iodide uptake assay Propidium iodide (PI) is a fluorescent dye that binds to DNA and is commonly used in evaluation of cell viability by flow cytometry, as described by Crowley et al. [41]. The loss in integrity of plasma membrane allows uptake of PI, while intact plasma membranes exclude it. When PI is bound to nucleic acids, the fluorescence excitation maximum shifts to 535 nm and the emission maximum is 617 nm. 2.2.2.1.1 Materials

1. 5 3 105 cells/mL cell suspension 2. Culture medium, BSA, PBS (Ca21, Mg21 free), FBS, trypsin-EDTA, L-glutamine, antibiotic-antimycotic solution (10,000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) 3. ENPs, 10% Triton X-100 4. PI Stock solution at concentration 0.5 mg/mL 5. Ethanol

1. Basic principles

26

2. In vitro methods to assess the cellular toxicity of nanoparticles

6. Tissue culture flasks (25 cm2), syringe filters (0.22 μm), filter holders 7. Micropipettor (101000 μL) with sterile micropipettor tips 2.2.2.1.2 Medium, buffer, and solutions 2.2.2.1.2.1 In vitro components

2.2.2.1.2.1.1 Preparation of culture medium Dissolve the powdered medium in autoclaved Milli-Q water. Add sodium bicarbonate (2 g/L) and L-glutamine (300 mg/L) to maintain pH to 7.2. Further add antibiotic/antimycotic solution (10 mL/L) to prevent contamination. Make up the volume to 1 L. Filter the medium with 0.22 μm membrane filters. Check for contamination by keeping the medium at 37 C overnight and if not contaminated then store at 4 C. 2.2.2.1.2.1.2 Phosphate buffered saline (Ca21, Mg21 free) Dissolve the powdered PBS in 990 mL autoclaved Milli-Q water. Maintain the pH to 7.4 and make up the volume to 1000 mL. The PBS used should be calcium and magnesium free to inhibit the endonuclease activity of the cells. 2.2.2.1.2.1.3 Trypsin-EDTA (0.25%) The ready-to-use solution is thawed, aliquoted into 15 mL tubes, and stored at 220 C. 2.2.2.1.3 Methods 2.2.2.1.3.1 In vitro cell preparation and treatment

1. Seed 1 3 105 cells/well in a 12 well tissue culture plate for 24 h at 37 C and 5% CO2. 2. Treat the cells with ENPs and positive control (10% Triton X-100) and repeat the experiment thrice. 3. Aspirate medium from the wells and detach the cells from the surface by adding 0.25% trypsin-EDTA. 4. Quench the trypsin by adding an equal amount of complete medium. 5. Centrifuge the cells at 250 g for 5 min, discard the supernatant and resuspend the pellet in 200 μL PBS. 2.2.2.1.3.2 In vitro method for propidium iodide uptake assay

To 100 μL of cell suspension, add PI (stock: 0.5 mg/mL; working: 2 μL/100 μL). Incubate the sample at 4 C in dark for 1015 min. After incubation, add 200 μL PBS. Acquire the samples using flow cytometer. Analyze the results by comparing the increased fluorescence intensity as in terms of percentage cell death as compared to control. 6. Calculate the mean SD and SE for control, positive control and unknown samples. 7. Compare the mean values of percent viable cells among various concentrations with the control and analyze the results using ANOVA.

1. 2. 3. 4. 5.

2.2.2.1.4 Limitations

1. PI is carcinogenic and needs to be handled with care. 2. Detects only necrotic cells.

1. Basic principles

2.2 Materials and methods

27

2.2.2.2 Trypan blue exclusion test The trypan blue exclusion test is used to measure the number of viable cells present in a cell suspension. It is based on the principle that viable cells have intact cell membranes that exclude trypan blue whereas dead cells have damaged membrane, hence they take in the trypan blue dye [42]. Therefore nonviable cells will have a blue cytoplasm and viable cells will have a clear cytoplasm (Fig. 2.3). 2.2.2.2.1 Materials

1. Cell suspension (B5 3 105 cells/mL) 2. Culture medium, BSA, PBS (Ca21, Mg21 free), FBS, trypsin-EDTA, L-glutamine, antibiotic-antimycotic solution (10,000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) 3. ENPs, 10% Triton X-100 4. Trypan blue—0.4% 5. Ethanol 6. Tissue culture flasks (25 cm2), syringe filters (0.22 μm), filter holders 7. Micropipettor (101000 μL) with sterile micropipettor tips 8. Fine scissors, forceps, and scalpel 9. Ice buckets 2.2.2.2.2 Medium, buffer, and solutions 2.2.2.2.2.1 In vitro components

2.2.2.2.2.1.1 Preparation of culture medium Dissolve the powdered medium in autoclaved Milli-Q water. Add sodium bicarbonate (2 g/L) and L-glutamine (300 mg/L) to maintain pH to 7.2. Further add antibiotic/antimycotic solution (10 mL/L) to prevent contamination. Make up the volume to 1 L. Filter the medium with 0.22 μm membrane filters. Check for contamination by keeping the medium at 37 C overnight and if not contaminated then store at 4 C. 2.2.2.2.2.1.2 Phosphate buffered saline (Ca21, Mg21 free) Dissolve the powdered PBS in 990 mL autoclaved Milli-Q water. Maintain the pH to 7.4 and make up the volume to 1000 mL. To inhibit the endonuclease activity of the cells, PBS used should be calcium and magnesium free. 2.2.2.2.2.1.3 Trypsin-EDTA (0.25%) The ready-to-use solution is thawed, aliquot into 15 mL tubes, and stored at 220 C. 2.2.2.2.3 Methods 2.2.2.2.3.1 In vitro cell preparation and treatment

1. Seed 2 3 104 cells/well in a 24 well tissue culture plate for 24 h at 37 C and 5% CO2. 2. Treat the cells with ENPs and positive control (10% Triton X-100) and repeat the experiment thrice. 3. Aspirate medium from the wells and detach the cells from the surface by adding 0.25% trypsin-EDTA. 4. Quench the trypsin by adding equal amount of complete medium. 5. Centrifuge the cells at 250 g for 5 min, discard the supernatant and resuspend the pellet in 1000 μL PBS.

1. Basic principles

28

2. In vitro methods to assess the cellular toxicity of nanoparticles

Neutral red uptake assay 10,000 cells seeded in a 96 well plate Cells exposed to different concentration of TiO2 ENPs Medium discarded

Cells incubated with 50 µg/mL of NR dye for 3 h at 37 °C Cells washed with 0.5% formaldehyde and 1% calcium chloride Dye taken up by cells dissolved in solution (50% ethanol + 1% acetic acid + 49% water)

Absorption at 540 nm

FIGURE 2.3 Schematics for cytotoxicity assessment by neural red uptake assay.

1. Basic principles

2.2 Materials and methods

29

2.2.2.2.3.2 In vitro method for trypan blue exclusion test

1. Dilute the cell samples in 0.4% trypan blue dye by preparing a 1:1 dilution of the cell suspension and dye. 2. Add 10 μL trypan blue dye to 10 μL of cell suspension in a microfuge tube. 3. Mix and incubate the same for at least 2 min. 4. Load 10 μL of mixture (cell suspension and trypan blue) onto a hemocytometer and count the average number of cells per square under light microscope. 5. Record the percentage of viable cells using the following formula: 

 Total no: of viable cells per mL of aliquot Viable cells ð%Þ 5 3 100 Total no: of cells per mL of aliquot 2.2.2.2.4 Limitations

1. This assay is tedious as each sample has to be counted manually using hemocytometer. 2. Detects only necrotic cells.

2.2.3 Genotoxicity Genotoxicity tests include the in vitro and in vivo tests, designed to identify the compounds that can induce damage to the genetic makeup. Genotoxicity determines the DNA or chromosomal damage. 2.2.3.1 Single cell gel electrophoresis (Comet) assay Comet assay detects the single and double stranded DNA damage in individual cells (in vitro and in vivo). The comet assay has been frequently used to determine the genotoxicity of nanoparticles and chemical compounds in multiple organ specific cell lines. The protocol for the Comet assay was first described by Singh et al. [43]. 2.2.3.1.1 Materials

1. 5 3 105 cells/mL cell suspension 2. Culture medium, BSA, PBS (Ca21, Mg21 free), FBS, trypsin-EDTA, L-glutamine, antibiotic-antimycotic solution (10,000 U/mL penicillin, 10 mg/mL streptomycin, and 25 μg/mL amphotericin-B) 3. ENPs, ethyl methane sulfonate (EMS)—1 μM 4. Low-melting-point agarose (LMPA), normal melting agarose (NMA), ethidium bromide (EtBr), ethylene diamine tetra acetic acid disodium salt [(EDTA)Na2] 5. Sodium hydroxide and DMSO 6. Ethanol and methanol 7. Tissue culture flasks (25 cm2), syringe filters (0.22 μm), and filter holders 8. End-frosted conventional glass slides (75 mm 3 25 mm, with 19 mm frosted end), cover slips (No. 1, 24 3 60 mm), and screw cap bottles (1002000 mL) 9. Fine scissors, forceps, and scalpel 10. Petri dishes 11. 1 mL syringes with 21-gauge needles

1. Basic principles

30

2. In vitro methods to assess the cellular toxicity of nanoparticles

12. Ice buckets 13. Micropipettor (101000 μL) with sterile micropipettor tips 14. Electrophoresis unit 2.2.3.1.2 Medium, buffer, and solutions 2.2.3.1.2.1 Normal melting agarose Prepare 1% NMA by adding 1 g of NMA in 90 mL of dH2O. Mix and dissolve the agarose by boiling. Make up the volume to 100 mL with dH2O. Keep the NMA at 60 C in dry bath, while preparing the base slides. 2.2.3.1.2.2 Low-melting-point agarose To make 1% LMPA, add 500 mg LMPA in 50 mL of PBS. Mix and dissolve the agarose by boiling. Aliquot and store at 4 C for a week. Prepare 0.5% LMPA by diluting 1% LMPA with equal volume of PBS. 2.2.3.1.2.3 Stock lysing solution Add 146.1 g NaCl, 37.2 g EDTA and 1.2 g Trizma base in 700 mL dH2O. Add 8 g NaOH into it and dissolve by stirring. Maintain the pH of solution to 10 using either HCl or NaOH. Make up the volume to 1 L and store at room temperature. 2.2.3.1.2.4 Working lysing solution The working lysing solution is prepared by adding 1% Triton X-100 and 10% DMSO (for in vivo samples only) to lysing solution. Keep the final lysing solution in a refrigerator for at least 30 min, prior to use. 2.2.3.1.2.5 Electrophoresis buffer

1. Stock solution of 10 N NaOH—Dissolve 200 g of NaOH in 500 mL dH2O. 2. Stock solution of 200 mM EDTA—Dissolve 14.89 g of EDTA in 200 mL dH2O, pH 10. The stock solutions can be stored at room temperature for 2 weeks. 3. 1X working electrophoresis buffer—Add 30 mL of 10 N NaOH stock solution to 5 mL of 200 mM EDTA stock solution, and make the volume to 1000 mL. It should be freshly prepared before each experiment. 2.2.3.1.2.6 Neutralization buffer Add 48.5 g of Tris base in 800 mL dH2O and adjust the pH to 7.5 with concentrated ( . 10 M) HCl. Make up the volume to 1000 mL with dH2O. Keep it refrigerated for 30 min before use. It can be stored at room temperature till 2 weeks. 2.2.3.1.2.7 Staining solution

1. 10 3 Stock solution of 20 μg/mL EtBr  Add 10 mg of EtBr in 50 mL dH2O. Store at room temperature. 2. 1 3 working solution—Mix 1 mL of stock solution with 9 mL of dH2O. 2.2.3.1.2.8 5,6-Carboxyflourescein dye

Ready-to-use solution of 5,6-carboxyflourescein dye

is used. 2.2.3.1.2.9 In vitro components

2.2.3.1.2.9.1 Preparation of culture medium Dissolve the powdered medium in autoclaved Milli-Q water. Add sodium bicarbonate (2 g/L) and L-glutamine (300 mg/L) to maintain pH to 7.2. Further add antibiotic/antimycotic solution (10 mL/L) to prevent the

1. Basic principles

2.2 Materials and methods

31

growth of bacteria and fungus. Make up the volume to 1 L. Filter the medium with 0.22 μm membrane filters. Check for contamination by keeping the medium at 37 C overnight and if not contaminated then store at 4 C. 2.2.3.1.2.9.2 Phosphate buffered saline (Ca21, Mg21 free) Dissolve the powdered PBS in 990 mL autoclaved dH2O. Maintain the pH to 7.4 and make up the volume to 1000 mL. To inhibit the endonuclease activity of the cells, the PBS used should be calcium and magnesium free. 2.2.3.1.2.9.3 Trypsin-EDTA (0.25%) The ready-to-use solution is thawed, aliquoted into 15 mL tubes and stored at 220 C. 2.2.3.1.3 Methods 2.2.3.1.3.1 In vitro cell preparation

1. Seed 5 3 104 cells/well in a 24 well tissue culture plate for 24 h at 37 C and 5% CO2. 2. Treat the cells with ENPs and positive control (EMS; 1 μM) and repeat the experiment thrice. 3. Aspirate medium from the wells and detach the cells from the surface by adding 0.25% trypsin-EDTA. 4. Quench the trypsin by adding an equal amount of complete medium. 5. Centrifuge the cells at 250 g for 5 min, discard the supernatant and resuspend the pellet in 1000 μL PBS. 2.2.3.1.3.1.1 Viability test 1. To 10 μL of cell suspension in a microfuge tube, add 10 μL of carboxyfluorescein (for cells from different organs). 2. Incubate the mixture for 2 min. 3. Apply 10 μL of cells to hemocytometer and count the number of cells under a fluorescent microscope. 4. Record the number of viable cells. 2.2.3.1.3.1.2 Preparation of base slides 1. The base slides should be prepared one day before the experiment. 2. Clean the end-frosted slides, dip this in methanol, and burn over the flame. 3. Dip the slides into 1% NMA up to one-third of the frosted area and remove gently. 4. Wipe the lower side of the slide to remove agarose and air-dry the slide. 5. Mark the upper/coated layer of the slide and store in slide boxes at room temperature until used. 2.2.3.1.3.1.3 Cell lysis 1. To the resuspended pellet add 100 μL of 1% LMPA. 2. Pour 80 μL of the cell suspension on NMA coated slide and place a coverslip over it for even spreading. 3. Allow to solidify the LMPA layer by keeping the slides on an ice pack. 4. Remove the coverslip slowly and add a third agarose layer (80 μL, 0.5% LMPA) to the slide. 5. Allow to solidify the third layer by keeping the slides on an ice pack. 6. Remove coverslip and put the slides in freshly prepared cold lysing solution in a coupling jar. 7. Keep the slides in refrigerator for a minimum of 3 h to overnight for the lysis of the cells.

1. Basic principles

32

2. In vitro methods to assess the cellular toxicity of nanoparticles

2.2.3.1.3.1.4 Electrophoresis of microgel slides 1. After the lysis, remove the slides and keep it side by side on the horizontal electrophoretic chamber. 2. Pour the electrophoresis buffer on the slides and also remove any bubbles over the agarose. 3. The slides should be equilibrated in the alkaline electrophoretic buffer for 20 min to allow for unwinding of the DNA. 4. Perform electrophoresis for 30 min at 24 V (B0.74 V/cm) and 300 mA which can be reached by adjusting the level of the buffer. 2.2.3.1.3.1.5 Neutralization of microgel slides 1. Switch off the power and remove the slides from the buffer and keep on a drain tray. 2. Pour neutralization buffer on the slides; cover it with buffer for at least 5 min. 3. Drain the buffer from the slides and repeat the process for at least two times. 2.2.3.1.3.1.6 Staining of microgel slides 1. Stain the slides with 80 μL of 1 3 working solution of EtBr for 5 min and then remove excess stain by dipping the slides in chilled distilled H2O. 2. Wipe the water from the underneath of the slide and cover with a coverslip. 3. Store the slides in a humidified slide box at 4 C. 2.2.3.1.3.1.7 Scoring of the microgel slides 1. After 2 h the slides can to be scored in minimum light to prevent the additional DNA damage from bright white light. 2. For visualization of DNA damage, observe the EtBr-stained DNA using a 40 3 objective on a fluorescent microscope. 3. Score 25 random cells from each replicate slide per sample using Komet 5 image analysis software developed by Andor Technology (Belfast, United Kingdom). 4. The percentage tail DNA, the length of DNA migration, and Olive tail moment determines the DNA damage (Fig. 2.4). 2.2.3.1.4 Limitations

It reveals only strand breaks and alkali labile sites; effects on cell-cycle checkpoints cannot be determined with this assay. 2.2.3.2 Cytokinesis block micronucleus assay Micronucleus is a fragment of damaged chromosomes or whole chromosomes that fail to attach onto the spindle during the anaphase stage of cell division (Fig. 2.3). These are much smaller than the principle nuclei and hence referred to as micronucleus [44]. The CBMN assay determines the chromosome loss, chromosome breakage, nondisjunction, necrosis, and apoptosis [45]. 2.2.3.2.1 Materials

1. 5 3 105 cells/mL cell suspension 2. Culture medium, BSA, PBS (Ca21, Mg21 free), FBS, trypsin-EDTA, L-glutamine, antibiotic-antimycotic solution (10,000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B)

1. Basic principles

33

2.2 Materials and methods

Protocol for Comet assay Seeded human cells in a 12 well plate

FIGURE 2.4 Schematics for genotoxicity assessment by Comet assay.

24 h at 37°C

TiO 2 ENPs exposure to the cells for 3 and 6 h at 37°C Trypsinize Neutralize with complete media

Single cell suspension

1% LMPA + cells

Preparation of slides 10 min. on Ice

Alkaline lysis 0.5% LMPA

2.5 M NaCl, 100 mMEDTA,

10 min. on Ice

10 mM Tris,1% Triton ×–100 pH 10

Alkaline unwinding 300 mMNaOH, 1 mM EDTA pH >13

20 min

Alkaline electrophoresis 30 min

~0.7–1.0 V/cm, 300 mA

Neutralizing 5 min, thrice

0.4 M Tris pH 7.5

Staining slides 75 µL EtBr, 20 µg/mL 5 min

Image processed using Komet5.1 software

3. 4. 5. 6. 7. 8. 9. 10.

ENPs, EMS—6 mM, Giemsa, and DPX mountant DMSO Ethanol, methanol, glacial acetic acid, and glycerol Tissue culture flasks (25 cm2), syringe filters (0.22 μm), and filter holders End-frosted conventional glass slides (75 mm 3 25 mm, with 19 mm frosted end) Syringe with 26-gauge needle Micropipettor (101000 μL) with sterile micropipettor tips Cytofunnel

2.2.3.2.2 Medium, buffer, and solutions 2.2.3.2.2.1 Cytochalasin B Dissolve 0.5 mg cytochalasin B in 1 mL DMSO. Prepare aliquots of 500 μL and store at 220 C till use. The final working concentration of cytochalasin B is 3 μg/mL.

1. Basic principles

34

2. In vitro methods to assess the cellular toxicity of nanoparticles

2.2.3.2.2.2 Geimsa stock solution To a mixture of 20 mL methanol and 30 mL glycerol, add 500 mg of Geimsa. Keep the solution for maturation for 23 days. 2.2.3.2.2.3 Working geimsa solution Add 10 mL of Giemsa stock in 90 mL of Sorenson’s buffer (as described in Section 2.2.3.2.2.4.6). Keep the solution stored at room temperature. 2.2.3.2.2.4 In vitro components

2.2.3.2.2.4.1 Preparation of culture medium Dissolve the powdered medium in autoclaved Milli-Q water. Add sodium bicarbonate (2 g/L) and K-glutamine (300 mg/L) to maintain the pH to 7.2. Further add antibiotic/antimycotic solution (10 mL/L) to prevent contamination. Make up the volume to 1 L. Filter the medium with 0.22 μm membrane filters. Check for contamination by keeping the medium at 37 C overnight and if it is not contaminated then store at 4 C. 2.2.3.2.2.4.2 Phosphate buffered saline (Ca21, Mg21 free) Dissolve the powdered PBS in 990 mL autoclaved dH2O. Maintain the pH to 7.4 and make up the volume to 1000 mL. To inhibit the endonuclease activity of the cells, the PBS used should be calcium and magnesium free. 2.2.3.2.2.4.3 Trypsin-EDTA (0.25%) The ready-to-use solution is thawed, aliquoted into 15 mL tubes, and stored at 220 C. 2.2.3.2.2.4.4 Hypotonic solution (potassium chloride) Dissolve 0.56 g of potassium chloride (KCl) in 100 mL dH2O and store at room temperature. 2.2.3.2.2.4.5 Carnoy’s fixative Mix methanol and glacial acetic acid in the ratio of 3:1 v/v. Prepare fresh and chilled before use. 2.2.3.2.2.4.6 Sorenson’s buffer Add 0.445 g of disodium hydrogen phosphate (Na2HPO4) and 0.34 g of potassium dihydrogen phosphate (KH2PO4) in 200 mL of dH2O. Maintain the pH to 6.8. 2.2.3.2.2.5 In vivo components

2.2.3.2.2.5.1 Anticoagulant From the stock of 1000 U/mL heparin sodium salt, use 10 μL of heparin to coat a microfuge tube for collection of 50100 μL blood. 2.2.3.2.2.5.2 Buffer The buffer consists of 0.5% FBS and 2 mM EDTA in PBS. 2.2.3.2.3 Methods 2.2.3.2.3.1 In vitro cell preparation and treatment

1. Seed 2 3 105 cells/well in a 6 well tissue culture plate for 24 h at 37 C and 5% CO2. 2. Treat the cells with ENPs and positive control (EMS; 6 mM) and repeat experiment thrice. 3. Aspirate the medium from the wells and wash the cells with PBS. 4. Add cytochalasin B (final concentration; 3 μg/mL) in medium till the doubling time of the cells is completed. It inhibits the cytokinesis. 5. Aspirate the medium from the wells and detach the cells from the surface by adding 0.25% trypsin-EDTA. 6. Quench the trypsin by adding equal amount of complete medium. 7. Centrifuge the cells at 250 g for 5 min, discard the supernatant. 8. Add 5 mL of chilled Carnoy’s fixative to the cells for 15 min at 4 C.

1. Basic principles

2.2 Materials and methods

35

9. Again centrifuge the cells at 250 g for 10 min, remove the supernatant and add 600 μL of chilled Carnoy’s fixative. 10. In Shandon double cytofunnel pour the cells and centrifuge at 880 g for 10 min using a cytospin (Thermo Shandon, Hampshire, United Kingdom). 11. Dip the slides in 90% chilled methanol for 5 min, air-dry and store until staining. 12. Stain the slides with 10% Giemsa working solution for 10 min. 13. Air-dry the slides and mount with DPX. 14. Check for the presence of micronuclei in the bi-nucleated cells using a bright field microscope. 15. Count the number of micronuclei in a minimum of 1000 bi-nucleate cells from each slide of the sample (500 bi-nucleate cells from each dots; Fig. 2.5) [45]. 2.2.3.2.4 Limitations

More than 2000 cells have to be scored in cultures with low micronucleus frequency.

FIGURE 2.5

Schematics for genotoxicity assessment by micronucleus assay.

Protocol for micronucleus assay Seeded human cells in a 6 well plate 24 h at 37ºC

TiO2 ENPs exposure to the cells for 6 h at 37º C CytochalasinB (3 µg/mL)

Harvested after 20 h Cell suspension was centrifuged and washed Pellet resuspendedin 500 µL serum free medium Loaded 250 µL of cell suspension in cytofunnel and centrifuged

Slides were air dried and fixed in MeOH Slides dried over night

Stained with 10% Giemsadried and mounted with DPX 1000 binucleate cells scored

1. Basic principles

36

2. In vitro methods to assess the cellular toxicity of nanoparticles

2.2.4 Oxidative stress Oxidative stress can be defined as a shift in the balance between oxidants and antioxidants in favor of oxidants [46]. Free radicals such as ROS and nitrogen species are generated in our body by various endogenous systems and exposed to different physiochemical conditions. Reactive oxygen species contain one or more unpaired electrons which can oxidize protein amino acids and unsaturated fatty acids of cell membranes leading to lipid peroxidation. 2.2.4.1 Reactive oxygen species generation 2,7-Dichlorofluorescein diacetate (DCFDA) dye is a nonfluorescent dye that is used to measure ROS activity within the cell [47]. DCFDA enters into the cell through diffusion. Cellular esterases deacetylate converts DCFDA to a nonfluorescent compound (2,7-dichlorofluorescein, DCFH). Further DCFH is converted into fluorescent dichlorofluorescein on oxidation by ROS (Fig. 2.6). It is detected by fluorescence spectroscopy at excitation of 485 nm and emission of 528 nm. 2.2.4.1.1 Materials

1. 5 3 105 cells/mL cell suspension 2. Culture medium, BSA, PBS (Ca21, Mg21 free), FBS, trypsin-EDTA, L-glutamine, antibiotic-antimycotic solution (10,000 U/mL penicillin, 10 mg/mL streptomycin and amphotericin-B) Protocol for reactive oxygen species (ROS)

FIGURE 2.6 Schematic representing the protocol for reactive oxygen species (ROS) detection.

10,000 cells seeded in a 96 well black bottom plate

Cells exposed to different concentration of TiO2 ENPs Medium discarded and cells incubated with 20 µM of DCFDA dye for 30 min at 37°C Cells washed with PBS Added 200 µL of PBS

Fluorescence at 485/528 nm

1. Basic principles

2.2 Materials and methods

3. 4. 5. 6. 7. 8. 9. 10.

37

ENPs, tert-butyl hydrogen peroxide DCFDA Ethanol Tissue culture flasks (25 cm2), black bottom 96-well tissue culture plate, syringe filters (0.22 μm), and filter holders Fine scissors, forceps, and scalpel 1 mL syringes with 21-gauge needles Ice buckets Micropipettor (101000 μL) with sterile micropipettor tips

2.2.4.1.2 Medium, buffer, and solutions 2.2.4.1.2.1 In vitro components

2.2.4.1.2.1.1 Preparation of culture medium Dissolve the powdered medium in autoclaved Milli-Q water. Add sodium bicarbonate (2 g/L) and L-glutamine (300 mg/L) to maintain pH to 7.2. Further add antibiotic/antimycotic solution (10 mL/L) to prevent contamination. Make up the volume to 1 L. Filter the medium with 0.22 μm membrane filters. Check for contamination by keeping the medium at 37 C overnight and if not contaminated then store at 4 C. 2.2.4.1.2.1.2 Phosphate buffered saline (Ca21, Mg21 free) Dissolve the powdered PBS in 990 mL autoclaved dH2O. Maintain the pH to 7.4 and make up the volume to 1000 mL. To inhibit the endonuclease activity of the cells, the PBS used should be calcium and magnesium free. 2.2.4.1.2.1.3 Trypsin-EDTA (0.25%) The ready-to-use solution is thawed, aliquoted into 15 mL tubes, and stored at 220 C. 2.2.4.1.3 Methods 2.2.4.1.3.1 Stock 2,7-dichlorofluorescein diacetate Dissolve 5 mg of DCFDA in 1 mL of DMSO to prepare stock solution of 10 mM DCFDA. 2.2.4.1.3.2 Working 2,7-dichlorofluorescein diacetate Dissolve 20 μL of 10 mM stock DCFDA to 10 mL PBS buffer to prepare 20 μM DCFDA working solution. The dye should not be exposed to light. The working solution should be prepared fresh before use. 2.2.4.1.3.3 tert-Butyl hydrogen peroxide Prepare 55 mM of tert-butyl hydrogen peroxide

solution in the culture medium. 2.2.4.1.3.4 In vitro cell preparation

1. 2. 3. 4.

Seed (1 3 104 cells/well) in a 96-well tissue culture plate for 24 h at 37 C and 5% CO2. In parallel maintain a cell free system with medium but without cells. Treat the cells with ENPs and positive control (tert-butyl hydrogen peroxide; 55 mM). After the completion of treatment time, aspirate the medium and wash the cells twice with PBS. 2.2.4.1.3.5 Method for in vitro reactive oxygen species detection

1. Add 100 μL of DCFDA dye (20 μM) prepared in PBS to each well and incubate it for 30 min at 37 C. 2. Aspirate the PBS containing DCFDA after incubation and add 200 μL of PBS to each well.

1. Basic principles

38

2. In vitro methods to assess the cellular toxicity of nanoparticles

3. Measure the fluorescence intensity in a multi-well plate reader at an excitation and emission wavelengths of 485 and 528 nm respectively. 4. Measure the percent ROS generation using the following formula:   F 485=528sample 2 F 485=528sample blank Percent ROS generation 5 3 100 F 485=528control 2 F 485=528control blank 5. Calculate the mean SD and SE for all the samples. 6. Compare the mean values of percent ROS generation using ANOVA. 2.2.4.1.4 Limitations

• DCFDA measures the concentration of hydrogen peroxide in cells along with the superoxide and nitric oxide generation which are also capable of oxidizing DCFH.

2.3 Conclusion The increase in applications of ENPs in various consumer and therapeutic products has enhanced the interaction of ENPs with biological systems. ENPs have profound advantages, however its toxicity to different in vitro and in vivo model organisms are well documented. Different types of ENPs with different size, shape, composition, and surface properties are known to exhibit different levels of toxicity. Therefore there is a need for proper assessment of the cytotoxic, genotoxic, and immunotoxic effects of ENPs. To evaluate the type of cell injuries, ENPs can be evaluated by the techniques discussed above. A few particles have the potential to damage cells through different mechanical action; so more than one assay is required to determine uncharacterized material. An integrated approach investigating multiple parameters is also required to assess the toxicity of ENPs. Apart from above described traditional assays, few companies are marketing more rapid, precise, and commercially available assays for cellular viability. However, these kits need to validate with a range of ENPs as the toxic potential of these ENPs are mainly depending on their composition, size, and shape. Regardless, complete characterization of ENPs is required to advocate some conclusion.

Acknowledgments Funding received from the Department of Biotechnology, Government of India under the project “NanoToF: Toxicological evaluation and risk assessment on Nanomaterials in Food” (Grant Number BT/PR10414/PFN/20/ 961/2014) and DST SERB Project “Nanosensors for the Detection of Food Adulterants and Contaminants” (Grant Number EMR/2016/005286) is gratefully acknowledged. We would also like to acknowledge the financial assistance from The Gujarat Institute for Chemical Technology (GICT) for the establishment of a facility for environmental risk assessment of chemicals and nanomaterials.

References [1] C. Petrarca, et al., Engineered metal based nanoparticles and innate immunity, Clin. Mol. Allergy 13 (1) (2015) 13. [2] V. Sharma, A. Kumar, A. Dhawan, Nanomaterials: exposure, effects and toxicity assessment, Proc. Natl Acad. Sci. India Sect. B Biol. Sci. 82 (1) (2012) 311.

1. Basic principles

References

39

[3] J.W. Rasmussen, et al., Zinc oxide nanoparticles for selective destruction of tumor cells and potential for drug delivery applications, Expert Opin. Drug. Deliv. 7 (9) (2010) 10631077. [4] P. Patel, et al., Nanotherapeutics for the treatment of cancer and arthritis, Curr. Drug. Metab. (2019). [5] K. Kansara, et al., Synthesis of biocompatible iron oxide nanoparticles as a drug delivery vehicle, Int. J. Nanomed. 13 (2018) 79 (T-NANO 2014 Abstracts). [6] A. Kumar, et al., Engineered ZnO and TiO2 nanoparticles induce oxidative stress and DNA damage leading to reduced viability of Escherichia coli, Free. Radic. Biol. Med. 51 (10) (2011) 18721881. [7] R.K. Shukla, et al., TiO2 nanoparticles induce oxidative DNA damage and apoptosis in human liver cells, Nanotoxicology 7 (1) (2013) 4860. [8] A. Kumar, et al., Zinc oxide nanoparticles affect the expression of p53, Ras p21 and JNKs: an ex vivo/ in vitro exposure study in respiratory disease patients, Mutagenesis 30 (2) (2014) 237245. [9] V.A. Senapati, et al., ZnO nanoparticles induced inflammatory response and genotoxicity in human blood cells: a mechanistic approach, Food Chem. Toxicol. 85 (2015) 6170. [10] V. Sharma, D. Anderson, A. Dhawan, Zinc oxide nanoparticles induce oxidative DNA damage and ROStriggered mitochondria mediated apoptosis in human liver cells (HepG2), Apoptosis 17 (8) (2012) 852870. [11] R.K. Shukla, et al., Titanium dioxide nanoparticles induce oxidative stress-mediated apoptosis in human keratinocyte cells, J. Biomed. Nanotechnol. 7 (1) (2011) 100101. [12] A. Kumar, A. Dhawan, R. Shanker, The need for novel approaches in ecotoxicity of engineered nanomaterials, J. Biomed. Nanotechnol. 7 (1) (2011) 7980. [13] P. Patel, et al., Cytotoxicity assessment of ZnO nanoparticles on human epidermal cells, Mol. Cytogenet. 7 (1) (2014) P81. [14] K. Kansara, et al., TiO2 nanoparticles induce DNA double strand breaks and cell cycle arrest in human alveolar cells, Environ. Mol. Mutagen. 56 (2) (2015) 204217. [15] P. Patel, et al., Cell cycle dependent cellular uptake of zinc oxide nanoparticles in human epidermal cells, Mutagenesis 31 (4) (2016) 481490. [16] G.S. Gupta, et al., Impact of humic acid on the fate and toxicity of titanium dioxide nanoparticles in Tetrahymena pyriformis and zebrafish embryos, Nanoscale Adv. 1 (1) (2019) 219227. [17] V.A. Senapati, et al., Monitoring characteristics and genotoxic effects of engineered nanoparticleprotein corona, Mutagenesis 32 (5) (2017) 479490. [18] K. Kansara, et al., Montmorillonite clay and humic acid modulate the behavior of copper oxide nanoparticles in aqueous environment and induces developmental defects in zebrafish embryo, Environ. Pollut. 255 (Pt 2) (2019) 113313. [19] K. Kansara, A. Kumar, A.S. Karakoti, Combination of humic acid and clay reduce the ecotoxic effect of TiO2 NPs: a combined physiochemical and genetic study using zebrafish embryo, Sci. Total Environ. 698 (2019) 134133. [20] Z. Magdolenova, et al., Mechanisms of genotoxicity. A review of in vitro and in vivo studies with engineered nanoparticles, Nanotoxicology 8 (3) (2014) 233278. [21] A. Kumar, et al., Cellular uptake and mutagenic potential of metal oxide nanoparticles in bacterial cells, Chemosphere 83 (8) (2011) 11241132. [22] A. Kumar, A. Dhawan, Genotoxic and carcinogenic potential of engineered nanoparticles: an update, Arch. Toxicol. 87 (11) (2013) 18831900. [23] G.S. Gupta, et al., Assessment of agglomeration, co-sedimentation and trophic transfer of titanium dioxide nanoparticles in a laboratory-scale predator-prey model system, Sci. Rep. 6 (2016) 31422. [24] A. Kumar, et al., A flow cytometric method to assess nanoparticle uptake in bacteria, Cytometry Part A 79 (9) (2011) 707712. [25] N.S. Vallabani, et al., TiO2 nanoparticles induced micronucleus formation in human liver (HepG2) cells: comparison of conventional and flow cytometry based methods, Mol. Cytogenetics 7 (S1) (2014) P79. [26] T.L. Riss, R.A. Moravec, A.L. Niles, Cytotoxicity testing: measuring viable cells, dead cells, and detecting mechanism of cell death, Mammalian Cell Viability, Springer, 2011, pp. 103114. [27] D. Sahu, G. Kannan, R. Vijayaraghavan, Size-dependent effect of zinc oxide on toxicity and inflammatory potential of human monocytes, J. Toxicol. Environ. Health Part A 77 (4) (2014) 177191. [28] M.V. Park, et al., The effect of particle size on the cytotoxicity, inflammation, developmental toxicity and genotoxicity of silver nanoparticles, Biomaterials 32 (36) (2011) 98109817.

1. Basic principles

40

2. In vitro methods to assess the cellular toxicity of nanoparticles

[29] M. Bajpayee, A. Kumar, A. Dhawan, The Comet assay: assessment of in vitro and in vivo DNA damage, Genotoxicity Assessment, Springer, 2019, pp. 237257. [30] A. Kumar, et al., Cellular response to metal oxide nanoparticles in bacteria, J. Biomed. Nanotechnol. 7 (1) (2011) 102103. [31] V.A. Senapati, et al., Zinc oxide nanoparticle induced age dependent immunotoxicity in BALB/c mice, Toxicol. Res. 6 (3) (2017) 342352. [32] K. Kansara, et al., TiO2 nanoparticles induce cytotoxicity and genotoxicity in human alveolar cells, Mol. Cytogenet. 7 (1) (2014) P77. [33] A. Kumar, et al., Microorganisms: a versatile model for toxicity assessment of engineered nanoparticles, Nano-Antimicrobials, Springer, 2012, pp. 497524. [34] J.J. Wang, B.J. Sanderson, H. Wang, Cyto-and genotoxicity of ultrafine TiO2 particles in cultured human lymphoblastoid cells, Mutat. Res. Genet. Toxicol. Environ. Mutagenesis 628 (2) (2007) 99106. [35] H.C. Bae, et al., Oxidative stress and apoptosis induced by ZnO nanoparticles in HaCaT cells, Mol. Cell. Toxicol. 7 (4) (2011) 333337. [36] V. Sharma, et al., DNA damaging potential of zinc oxide nanoparticles in human epidermal cells, Toxicol. Lett. 185 (3) (2009) 211218. [37] E.K. Dufour, et al., Clastogenicity, photo-clastogenicity or pseudo-photo-clastogenicity: genotoxic effects of zinc oxide in the dark, in pre-irradiated or simultaneously irradiated Chinese hamster ovary cells, Mutat. Res. Genet. Toxicol. Environ. Mutagenesis 607 (2) (2006) 215224. [38] V. Kodali, et al., Dysregulation of macrophage activation profiles by engineered nanoparticles, ACS Nano 7 (8) (2013) 69977010. [39] E.-J. Park, et al., Inflammatory responses may be induced by a single intratracheal instillation of iron nanoparticles in mice, Toxicology 275 (13) (2010) 6571. [40] A. Kumar, V.A. Senapati, A. Dhawan, Protocols for in vitro and in vivo toxicity assessment of engineered nanoparticles, Nanotoxicology. (2017) 94132. [41] L.C. Crowley, et al., Measuring cell death by propidium iodide uptake and flow cytometry, Cold Spring Harb. Protoc. 2016 (7) (2016). 10.1101/pdb. prot087163. [42] W. Strober, Trypan blue exclusion test of cell viability, Curr. Protoc. Immunol. 111 (1) (2015) A3. B. 1-A3.B.3. [43] N.P. Singh, et al., A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell Res. 175 (1) (1988) 184191. ˙ [44] K. Zelazna, K. Rudnicka, S. Tejs, In vitro micronucleus test assessment of polycyclic aromatic hydrocarbons, Environ. Biotechnol. 7 (2011) 7080. [45] A. Kumar, V. Sharma, A. Dhawan, Methods for detection of oxidative stress and genotoxicity of engineered nanoparticles, Oxidative Stress and Nanotechnology, Springer, 2013, pp. 231246. [46] D.J. Betteridge, What is oxidative stress? Metabolism 49 (2) (2000) 38. [47] C. Wan, et al., Content audit of POISINDEX, Vet. Hum. Toxicol. 35 (2) (1993) 168169.

1. Basic principles

C H A P T E R

3 In vivo studies: toxicity and biodistribution of nanocarriers in organisms Nivya Sharma, Mohd Aslam Saifi, Shashi Bala Singh and Chandraiah Godugu Department of Regulatory Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, India

List of abbreviations Au AST APA APD ALT ABC APTES BSA BBB BUN CuO CNT CD-1 cDNA CD-44 CNS DNA DSHemsPC DPK DTPA EPR EDA

gold aspartate aminotransferase action potential amplitude action potential duration alanine aminotransferase accelerated blood clearance 3-aminopropyltriethoxysilane bovine serum albumin blood brain barrier blood urea nitrogen copper oxide carbon nanotube cluster of diefferentiation-1 complementary DNA cluster of differentiation-44 central nervous system deoxyribonucleic acid 1,2-distigmasterylhemisuccinoyl-sn-glycero-3-phosphocholine dendritic poly(L-lysine) diethylenetreiaminepentaacetic enhanced permeation and retention effect ethylenediamine

Nanotoxicity DOI: https://doi.org/10.1016/B978-0-12-819943-5.00003-8

41

Copyright © 2020 Elsevier Inc. All rights reserved.

42 GPER HR ILs IV IP IM IONPs LHRH LCN-2 LDH MCP-1 MWCNTs miRNA MRI NO NOS NFKB NAC NP PLA PLGA PDGF PEG PAMAM pORF-hTRAIL PEI PPI QDs RES ROS RNA RGD RP RBCs SD SWCNTs SiNPs SPIO SC SMA-PAEEPEG siRNA SGOT TGF-β TNF-α TUNEL TBIL TiO2 UCNP WBC ZnO

3. In vivo studies: toxicity and biodistribution of nanocarriers in organisms

G-protein coupled estrogen receptors heart rate interleukins intravenous intraperitoneal intramuscular iron oxide nanoparticles luteinizing hormone-releasing hormone lipocalin-2 lactate dehydrogenase monocyte chemoattractant protein-1 multiwalled carbon nanotubes micro RNA magnetic resonance imaging nitric oxide nitric oxide synthase nuclear factor kappa light chain enhancer of activated B cells N-acetyl cysteine nanoparticles polylactic acid poly(lactic-co-glycolic acid) platelet derived growth factor polyethylene glycol poly(amidoamine) tumor necrosis factor-related apoptosis-inducing ligand (hTRAIL)-encoding plasmid open reading frame polyethyleneimine polypropyleneimine quantum dots reticuloendothelial system reactive oxygen species ribonucleic acid arginine-glycine-aspartic acid resting potential red blood cells Sprague Dawley single walled carbon nanotubes silica nanoparticles superparamagnetic iron oxide subcutaneous poly(styrene maleic acid)-poly(amide-ether-ester-imide)-poly(ethylene glycol) small interference RNA serum glutamic oxaloacetic transaminase tumor growth factor-β tumor necrosis factor-α terminal deoxynucleotidyl transferase dUTP nick end labeling total bilirubin titanium dioxide upconversion nanoparticles white blood cell zinc oxide

1. Basic principles

3.1 General overview

43

3.1 General overview Science and technology advancements have ignited the field of drug delivery and developmental research. However, despite having potential pharmacodynamic properties, a number of new chemical moieties and drugs show compromised activity primarily due to poor pharmacokinetic properties, which is one of the major hurdles for the failure of clinical translation of these molecules. Hence, modifying pharmacokinetic properties to ultimately improve the pharmacodynamic effects of these potential molecules is very challenging in drug development [1]. Nanotechnology is a potential solution to address these hurdles with a primary aim to improve the pharmacokinetic properties and ultimately improve the drug delivery and targeting aspects. Nanotechnology has been on a boom leading to immense employment of nanotechnology-based production for various applications including imaging, gene and drug delivery, and diagnostics. As novel drug delivery systems, nanocarriers are potential candidates with numerous advantages including specific tissue and organ targeting, improving the stability of hydrophobic drugs rendering them suitable for administration; enhancing biodistribution and pharmacokinetics resulting in improved efficacy, etc. [2]. Nanocarriers are colloidal-sized particles, owning diameters ranging between 1 and 1000 nm which transport drug molecules encapsulated, adsorbed or dispersed in them. The commonly used nanocarriers include carbon nanotubes (CNTs), micelles, liposomes, polymers, inorganic materials, metal nanoparticles and others. Due to exceptionally small size, nanocarriers behave quite differently as compared to their bulk counterparts in the form of dominant optical, electric and magnetic properties which provide them characteristic attributes suitable for different applications such as drug delivery, diagnosis, theranostic etc. Taking leverage of their high surface area to volume ratio, basic properties and bioactivity of drug can be altered to achieve an improved pharmacokinetic properties with reduced toxic effects unlike conventional therapies [3]. Technological advantages of nanocarriers encompass long shelf-life leading to high stability, high carrier capacity pertaining to incorporation of single or multiple drug molecules, feasibility of incorporating both hydrophilic and hydrophobic substances, and the flexibility to enable controlled drug release [4]. In addition, nanocarriers can also deliver drugs to otherwise, inaccessible sites around the body and are therefore, extra favored when there is a need for controlled drug delivery to a specific organ [5]. Further, encapsulation of therapeutic/diagnostic agents in the nanocarriers provide improved pharmacokinetic properties by protecting them from early degradation and provide long circulation time. Nanocarriers are known to display decreased clearance rate and thus, a prolonged circulation time owing to their nano size. Small diameter of the particles blended with the extended circulation time lead to increased accumulation of the entrapped drug in tissues with augmented vascular permeability and impaired lymphatic drainage such as inflamed tissues and tumors. This phenomenon is called the enhanced permeability and retention (EPR) effect which can be leveraged for passive targeting of the encapsulated drug to the tumor site, thus abating its accumulation in healthy tissues and subsequent side effects [3]. A motley of nanocarriers composed of lipids, polymers, and inorganic materials have been evolved, resulting in the development of delivery systems which are unique in their physicochemical properties and hence their application.

1. Basic principles

44

3. In vivo studies: toxicity and biodistribution of nanocarriers in organisms

The popularity of these systems is due, in part, to the numerous advantages they provide for delivering their payloads to a particular site [6]. However, despite being a promising solution to effectively target the tissue with more bioavailability and specificity, nanocarriers are no less in eliciting organ toxicities. This book chapter provides detailed insights into the biodistribution and pharmacokinetic profiling and subsequent in vivo organ toxicities of various nanocarriers like CNTs, polymeric and inorganic nanoparticles (NPs), polymeric micelles, dendrimers, dendriplexes, and liposomes with varying size, surface chemistry, route of administration, etc. In addition, a brief overview about the factors affecting biodistribution and toxicity has also been provided.

3.2 Types of nanocarriers 3.2.1 Carbon nanotubes CNTs are large molecules made up of carbon which are formed by rolling the sheet(s) of graphene. CNTs can be single walled carbon nanotubes (SWCNTs) or multiwalled carbon nanotubes (MWCNTs) which are immensely employed in several biomedical applications, majorly diagnosis, bioimaging and drug delivery attributing to their high surface functionalization potential, huge payload carrying capacity, and high specificity. They are relatively biocompatible, stable, and have the finesse to escape the body’s immune system. In addition, they are known to show high entrapment efficacy when compared to their other nanocarrier counterparts such as nanoparticles, liposomes, and dendrimers [7]. To date, no CNT based nanocarrier has entered into clinics, making them novice with huge underlying potentials yet to be explored. But the major challenge lies in their biodistribution profile leading to reported organ toxicities which cannot be overlooked. Following administration, CNTs accumulate in the local tissue and get distributed to other organs via lymphatic circulation keeping in view their size. They travel through lymphatic vessels and get lodged into lymph nodes making them pivotal in cancer involving lymph nodes. However, irrespective of their electronic properties and surface charge, all CNTs show almost similar biodistribution profile. They enter lymphatic circulation and reach gastrointestinal tract, showing highest assimilation there after intravenous (IV) administration. However, when given through inhalational route, CNTs accumulate in lungs and distribute to lymph, blood, and central nervous system (CNS). The circulating CNTs in the blood reaches to organs like spleen, kidney, bone marrow, and heart. Owing to their nanoscale dimensions, these have shown rapid distribution, assimilation, and retention for longer times. CNTs with high aspect ratio raise concerns about changes in lung pathology when inhaled or instilled intratracheally. Despite showing some attractive properties, the toxicity of CNTs is a prime concern. The toxicity of CNTs depends on certain physicochemical properties including size, shape, aspect ratio, degree of aggregation, surface charge, purity, dose, and concentration. The toxicity profile of CNTs ranges from cellular to reproductive toxicity covering different organ toxicity. On the cellular level, CNTs have been shown to penetrate the cell membrane of macrophages consequently leading to alteration of cellular and physiological function of macrophages and simultaneously showing dose-dependent increase in oxidative stress and alteration in mitochondrial membrane potential [8]. Further, the authors also demonstrated

1. Basic principles

3.2 Types of nanocarriers

45

FIGURE 3.1 A schematic representation of inhalational toxicity of carbon nanotubes (CNTs). The CNTs enter into lung interstitium and activate a number of singling events. CNTs can damage the DNA either by directly interacting with it, or indirectly, by activating a generation of reactive oxygen species which are highly damaging to the cellular components including proteins, lipids, and DNA. In addition, the exposure of CNTs can also activate inflammatory pathways. CNTs are known to activate the synthesis of proinflammatory cytokines which also reflect in the bronchoalveolar lavage fluid. Further, the persistent injury to lungs by CNTs leads to granuloma formation in the lungs.

that degree of agglomeration affects the toxicity potential of CNTs as shown by less toxicity of the surfactant dispersed CNTs as compared to agglomerated CNTs. In addition, the genotoxicity of CNTs is also available in the literature. As demonstrated by Paulsen et al., intratracheal administration of MWCNTs was associated with DNA damage in the lungs exposed [9]. However, there are contradictory reports available on the genotoxicity potential of CNTs wherein the CNTs did not produce genotoxicity [10,11]. The pulmonary toxicity of CNTs is well studied and reported in literature and there is a lack of literature on other organ toxicity. The CNTs exposure has been found to be associated with granuloma formation and induction of fibrosis in lungs of animals [12] (Fig. 3.1). Further, Warheit et al. also focused on the pulmonary toxicity profile of pristine SWCNTs and they observed that about 15% of rats administered with 5 mg/kg of SWCNTs died within 24 h which was suspected to be due to blockage of airway by SWCNTs [13]. Furthermore, Shvedova et al. reported that pharyngeal administration of SWCNTs induced inflammation, fibrosis, and granuloma in the mice [14] (Fig. 3.1). Along the similar lines, after 2 months of lung exposure, the MWCNTs produced pulmonary lesion and collagen rich granuloma in the mice exposed. CNTs of various types are also reported to produce pneumonia in guinea pigs as demonstrated by Grubek-Jaworska et al. [15]. A study by Ryman-Rasmussen and colleagues has shown the ravaging effects of MWCNTs at different lung sites in rodents. They thoroughly studied the outreach of MWCNTs at various lungs sites including macrophages, mesenchymal cells, and collagen matrix of sub pleural region, and peritoneum for different day

1. Basic principles

46

3. In vivo studies: toxicity and biodistribution of nanocarriers in organisms

interval and concluded the migratory nature of MWCNTs causing inflammatory response [16] (Fig. 3.1). Further, Wang et al. studied the biodistribution of water-soluble and biocompatible radioisotope I125 labeled multiple hydroxylated SWCNTs. They relied on radiotracing to determine the quantity of SWCNTs congregated in various organs of male mice. They commented that multiple hydroxylated SWCNTs behaving as small molecules rapidly distribute throughout the body, readily accumulate in organs, and are retained for longer times. The conclusion of the experiment was that major accumulation was seen in stomach, kidney, and bones followed by skin, blood, lungs, and intestine in decreasing order, irrespective of the route of administration [17]. On the other hand, cardiovascular effects of SWCNTs were evident by activation of heme oxygenase-1 in the aorta and cardiac tissue along with aortic DNA damage [18]. A closer look at the literature also suggests potential reproductive and developmental toxicity of CNTs. The fetal and developmental abnormalities were evident in the animals exposed to CNTs [19,20]. In addition, CNTs were also reported to assimilate in the testes after single IV administration without causing any harm to sex hormones and sperm [21]. The functionalization of CNTs is cardinal to their biocompatibility, stability, safety, and encapsulating plethora of payloads to make it an effective delivery system with an array of characteristics to choose from [22]. However, surface functionalization has been reported to accelerate their excretion through urine and feces, substantially reducing their contribution to chronic organ toxicities and making them an apt nanocarrier for various applications. To investigate the effects of functionalization on biodistribution profile of CNTs, Singh et al. used 1,3-dipolar cycloaddition method to covalently attach diethylenetriaminepentaacetic (DTPA) dianhydride, a chelating agent that allows rapid complexation with radionuclide required to undergo biodistribution studies. They found that after functionalization of SWCNTs, the biodistribution profile changed significantly and showed the highest accumulation in the kidney followed by muscle, skin, femur, and blood after 30 min of IV injection. However, they did not observe the effect of surface charge on biodistribution profiling of SWCNTs [23]. Similarly, Xiang et al. tested the impact of electronic properties of SWCNTs on lung inflammation. They used differential gradient ultracentrifugation to purify two types of SWCNTs namely metallic SWCNT and semiconductor SWCNTs and tested both separately using oropharyngeal aspiration in C57BL/6 mice species. They evaluated the bronchoalveolar lavage (BAL) fluid for the presence of interleukin-6 (IL-6), platelet derived growth factor with two polypeptide A chains and tumor growth factor-β1 levels and tested collagen in lungs. Interestingly, they did not see any significant deviation in the parameters observed for both types and thus, concluded that electronic properties of SWCNTs do not make a major independent impact on lung toxicity profile [24]. On the other hand, the study conducted by Guo et al. underscored on the biodistribution of glucosamine functionalized MWCNTs in vivo. The movement of water-soluble MWCNTs with radiolabeled 99mTc was traced in the body of female mice after intraperitoneal (IP) injection. The presence of MWCNTs in various body fluids and tissues was looked for at different time points. This study acknowledged the release of more than 70% of active MWCNTs through urine and feces, in contrast with nonfunctionalized MWCNTs which show a general trend of accumulation in the lymph organs. Following the radioactivity measurement by scintillor, the highest concentration was found in enterogastric area after 6 h [25]. On the other hand, the study by Kafa et al. investigated the brain penetration potential of CNTs in an animal model. Gamma counting was

1. Basic principles

3.2 Types of nanocarriers

47

employed to study the gathered [111In] DTPA-MWNTs in mouse brain following systemic administration. The highest accumulation was found in lungs and liver after 5 min and 24 h of injection. Capillary depletion confirmed their presence in brain capillaries and parenchyma fractions. However, the levels soon nosedived, stressing upon the fact that CNTs were rapidly cleared from the brain within few hours [26]. The biodistribution study for combination approach using two drugs doxorubicin and metformin inside MWCNTs was done for orthotopic model of triple negative breast cancer. Consistent with the other studies, the highest concentration was found in the lungs and liver [27]. IV administration of SWCNT has shown highest concentration in the liver where it was localized in Kupffer cells, followed by lung and spleen [28]. Another study conducted in mice reflected notably high concentration in the lung, liver, and spleen 90 days after administration and thus, supporting the previous finding [29]. IV injection of SWCNT in rabbits reflected liver as major depot for its assimilation [30]. Polyethylene glycol (PEG) coated SWCNT showed prominent uptake by liver but concentration was less, owing to PEG properties [31]. Another study using water-soluble SWCNT showed the kidney as the major site of assimilation followed by the liver and spleen [32]. Conversely, MWCNT has also shown almost similar biodistribution profile to SWCNT. In a study following IV exposure, they showed highest congregation in the liver followed by the spleen and lung [28]. Even functionalized MWCNT also showed significant distribution to the liver with smaller amounts detected in the spleen and lungs [33]. Surface modified thick and thin MWCNT of the same length after administration through IV route reflected the assimilation of thicker tubes in the liver and spleen and that of narrow tubes in the lungs [34]. Hence, the CNTs, despite being one of the important nanocarriers also raise the concerns of their toxicity primarily due to their accumulation in the stomach and lymph organs; however, their functionalization could afford to reduce their toxicity potential.

3.2.2 Nanoparticles Ps, as the name itself defines, are the solid particles or particulate dispersions with size commonly ranging from 1 to 100 nm. NPs are broadly classified into two types, organic and inorganic. Organic NPs are generally fabricated using biodegradable proteins, synthetic polymers, such as polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyacrylates or natural polymers, along with albumin, gelatin, alginate, collagen, and chitosan etc. Conversely, inorganic NPs are made up of metals and their oxides. NPs have been employed in different fields including cosmetics, industry, therapy, diagnostics, molecular imaging, and theranostics. Apart from this, NPs have been under the limelight for the past few years for their use as effective gene and drug delivery vehicles for specific tissue targeting, both actively and passively. Albeit, offering a wide spectrum of advantages over conventional therapeutics, NPs face a drawback in terms of eliciting tissue and organ toxicity because of their wide array of biodistribution profiles. Several chemical and physical properties like size, charge, and surface chemistry decide the pharmacokinetic profile and toxicity of NPs [35]. Majorly, the metal NPs have put forth the toxicity concerns because of their rapid clearance from systemic circulation and prolonged retention in reticuloendothelial system (RES) organs like the liver and spleen. Due to their small size, NPs can be

1. Basic principles

48

3. In vivo studies: toxicity and biodistribution of nanocarriers in organisms

FIGURE 3.2 Major types of inorganic nanoparticles taking part in toxicity.

readily cleared from systemic circulation and eventually accumulate in the nontargeted tissues. Once they are taken up by RES organs, they are readily cleared by the body leading to short circulation time and failure to elicit the required pharmacological response [36]. However, the rapid uptake and retention of NPs in the RES organs also raise toxicity concerns for these organs due to undesirably higher levels of NPs. 3.2.2.1 Inorganic nanoparticles Inorganic nanoparticles are made up of metals (Au, Ag, Cu, and Pt), semiconductor nanocrystals [quantum dots (QDs)], metal oxides, rare earth metals (cerium and selenium), and a class of lanthanide-doped upconversion nanoparticles (UCNPs) (Fig. 3.2). They have various favorable properties like ease of fabrication, tunable size, X-ray absorption, generating heat or reactive oxygen species (ROS), and energy transfer properties. These are of mammoth importance in image guided therapy, photodynamic therapy, X-ray computed tomography imaging, magnetic resonance imaging (MRI), and positron emission tomography scanning (PET) [37]. Metal oxide NPs like iron oxide nanoparticles (IONPs) are widely used either as bioimaging contrast agents or for delivery of a variety of biomolecules by exploiting their tunable magnetic properties. Further, these are extensively utilized in cell separation, as contrasting agents, cell labeling, drug delivery, food additive, and magnetic hyperthermia. The synthesis process, their surface chemistry, use and administration methods greatly determine the biodistribution and pharmacokinetic profile of these NPs [38] (Fig. 3.3). Due to involvement of different factors, the biodistribution profiles of these NPs vary in different conditions. One study evaluated the influence of orally administered IONPs on young adult male Wistar rats. They reported an increase in biochemical parameters like alanine transferase (ALT), urea, hemoglobin, hematocrit, and eosinophil levels. On the contrary, a decrease was observed in iron and neutrophil levels when compared to the control group. The histopathological analysis reflected liver inflammation and necrosis. A decrease in the relative weight of certain organs such as brain, liver, kidneys, lungs, and stomach was seen attributed to hypotrophy of cells [39]. Elbialy et al. studied the

1. Basic principles

3.2 Types of nanocarriers

49

FIGURE 3.3 Summarizing the organ accumulation of different inorganic nanoparticles (NPs) taken via different routes.

biodistribution and toxicity profile of curcumin capped IONPs after single dose IV administration in BALB/c mice and suggested that significant amount of IONPs assimilated in the liver and kidney causing changes in their biochemical parameters [40]. They reported the assimilation of iron majorly in the liver, spleen, kidney, and brain with the highest concentration in the liver and decreasing sequentially. Notably, IONPs crossed blood brain barrier (BBB) and showed time dependent accumulation in the brain which may be due to the presence of transferrin and ferritin binding to brain receptors. Further, they also observed a drastic increment in liver enzymes post 0.5 h of injection. The increase was

1. Basic principles

50

3. In vivo studies: toxicity and biodistribution of nanocarriers in organisms

correlated to the augmented uptake and congregation of NPs by Kupffer cells and hepatocytes. On thorough study of serum biochemical parameters of kidney, they observed an increase in serum urea, creatinine, and uric acid but they all decreased and returned to normal within 1 day of administration. This shows that the IONPs have evoked toxicity majorly in liver as can be confirmed from changes in its biochemical parameters. Yang and colleagues explored the impact of surface modification on IONPs’ biodistribution and toxicity in adult male Sprague Dawley (SD) rats. They prepared three IONPs with PEG, carboxyl group (COOH) and bovine serum albumin (BSA) capping. The results have shown the elevation in the iron contents in spleen and blood both in the PEG IOMNs and COOH IOMNs treatment groups. Post 1 day of injection, biochemical analysis reflected augmented levels of liver injury markers in both former groups, whereas a few additional hepatic markers were observed in COOH IOMNs group. In the later treatment group, the hepatic injury markers elevated on day 9 and 16 postinjection. In addition to this, the PEG coated treatment group encountered individual neutrophils and cytoplasmic vacuolization [41]. Another study involving evaluation of biodistribution and toxicity of dendrimer coated IONPs was conducted in BALB/c mice. The results reported a significant increase in blood urea nitrogen (BUN) and hyperuricaemia in acute toxicity analysis, whereas chronic toxicity studies showed augmented direct bilirubin levels. In line with other studies, the histopathological investigation showed edema and losing cytoplasm in the liver cells. They concluded that most of the IONPs are excreted by kidney pertaining to their minute size and absorption by lungs [42]. However, chronic toxicity studies reflected an interesting finding in terms of enhanced apoptosis of cardiac tissue with an elevation in lactate dehydrogenase (LDH) and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. IP administration of superparamagnetic maghemite iron oxide NPs in mice showed maximum amassing of NPs in peritoneal cavity away from administration site after 4 h of administration and it got cleared after 48 h. Majority of NPs dispelled from tissues after 7 days of IP administration [43]. Owing to their vast catalytic, electrocatalytic, and plasmonic effects causing high biocompatibility, gold nanoparticles (Au NPs) are extensively employed these days in therapeutics and biomedical applications. However, the Au NPs are widely reported to be toxic in different scenarios. Chen et al. demonstrated the in vivo toxicity of bare Au NPs ranging from 3 to 100 nm in BALB/C mice [44]. The authors reported lethality of Au NPs within a period of 21 days. Further, pathological examination revealed toxicity of major organs including the liver, lung, and spleen. However, the study conducted by LasagnaReeves et al. addressed bioaccumulation and toxicity aspects of the Au NPs after repeated IP administration [45]. Although, the authors reported efficient brain uptake, there was no evidence of significant toxicity. On the other hand, Bailly et al. studied the toxic effects of dextran-coated Au NPs prepared by laser ablation method and given IV to athymic nude female mice. Pharmacokinetic analysis of gold concentration in central compartment reflected its short half-life and rapid clearance. In peripheral compartments, the highest concentration was observed in the liver which maintained till day 14 followed by spleen where concentration plummeted after day 7. Agglomerated Au NPs were observed to be taken up by macrophages of spleen and liver. There was no observed functional impact on liver and kidney [46]. Conversely, PEGylated gold nanoprisms were tested for in vivo toxicity following IV administration and they showed no significant changes in

1. Basic principles

3.2 Types of nanocarriers

51

biochemical parameter. After 4 months, histopathological analysis reflected enlarged vacuoled hepatocytes and Kupffer cells with enlarged cytoplasm. Macrophages of spleen reported a black pigment in cytoplasm. Liver and spleen showed accumulation of nanoprisms after 72 h of injection. After 4 months, the accumulation in liver reduced whereas it was the same in the spleen suggesting its degradation by liver [47]. Subcutaneous (SC) injection of glutathione coated Au NPs has shown that it is cleared majorly through the kidney without imparting any renal stress. Also, highest congregation was seen in the spleen after 24 h and a slight accumulation in lung tissue was reported pertaining to macrophage uptake and phagocytic engulfment [48]. Biodistribution and toxicity profiling of micellar platinum NPs was studied in BALB/c mice following IV administration. The study revealed the maximum sequestration of NPs in the liver and spleen with the spleen showing decreased white pulp [49]. In another experiment, platinum nanoparticles have been proven to show acute toxic effect on heart rhythm. The study was conducted in neonatal mice cardiomyocytes using patch clamp technique. Depolarized resting potential, suppressed depolarization and delayed repolarization of action potential was observed which was concentration dependent. A decrease in the current densities of sodium and potassium channels was seen. At higher dose, they observed resting potential depolarization, action potential amplitude reduction and action potential duration prolongation. In adult mice, dose-dependent decrease in the heart rate, prolonged P-R intervals and finally complete A-V conduction block were reported [50]. Another study investigated the nephrotoxic and hepatotoxic potential of platinum nanoparticles (diameter 1 and 8 nm), biochemically and histologically in mice through IV route. For platinum nanoparticles of diameter 1 nm, they noticed hepatic and renal damage as evident by dose-dependent increase in hepato-renal biomarkers with increase in IL formation. However, no physiological change was noted for 8 nm diameter Pt NPs [51]. Dey et al. reported elevation in LDH, serum glutamic oxaloacetic transaminase (SGOT) and creatinine levels in plasma indicating liver and kidney dysfunction by copper oxide nanoparticles (CuO NPs). In line with other studies, the highest assimilation was observed in the liver and spleen. Histopathological analysis revealed infiltration by lymphocytes, hypertrophy, vacuolization, and disorganization of hepatocytes and also, loosened parenchyma at higher doses. In the kidney, swollen and dilated Bowman’s capsule with deposition of hyaline-like materials in proximal tubules, degenerated epithelium of the proximal tubules as well as ruptured Malpighian corpuscles, and Bowman’s capsule were reported [52]. CuO NPs were also tested for their environmental toxicity profiles. In one of the studies, skeletal muscle toxicity in adult zebrafish was evaluated following chronic exposure to CuO NPs. The results indicated an increase in functional markers like creatine kinase-MB, acetylcholinesterase, and LDH, oxidative stress markers, degradation and atrophy, all pointing towards muscle damage [53]. In another study, toxicity profiling of CuO NPs in different life stage of Artemia salina was performed. Concentration dependent decrease in catalase activity and GSH levels was reported with evident accumulation of NPs in gut causing toxicity [54]. These studies on aquatic organisms conclude that CuO NPs augments oxidative stress and creates an imbalance in antioxidant levels. Wide application of nanosized titanium oxide nanoparticles (TiO2 NPs) in cosmetics, medicine, and food industry raises concerns about its potential health risk. Following oral route, most of them are reported to be excreted out by feces but long-term consumption

1. Basic principles

52

3. In vivo studies: toxicity and biodistribution of nanocarriers in organisms

causing accumulation in intestine presents an entirely different story. A study conducted by Wei et al. reflected the impact of long-term oral dietary intake of TiO2 NPs on intestine inflammation in mice. They reported altered gut microbiota composition, splenomegaly, an increase in colon length and lipocalin-2, intestinal inflammatory markers augmenting with increase in NPs size [55]. Oral toxicity of TiO2 NPs was also examined in adult and young rats. They reported high susceptibility in young rats as evidenced by heart injury and liver injury, demonstrated by increased total bilirubin (TBIL) content, ALT/AST ratio and decreased activities of AST, edema at higher doses along with histological changes such as hepatic cord disarray, vacuolization, perilobular cell swelling, or hydropic degeneration but no kidney injury unlike the adult rats. Adult rats only exhibited decrease in TBIL levels and an increase in blood urea nitrogen levels along with inflammatory cells infiltration indicating towards a slight liver and kidney injury. Low intestinal permeability along with hyper responsiveness and allergic reaction due to mast cell activation was also seen in adult rats affecting nutrient absorption and thus, causing significant body weight changes. Biodistribution profiling asserted that particles with diameter 60 200 nm majorly present in stomach and small intestine with less absorption and no systemic distribution leading to intestinal inflammation, in line with the previous study. However, a decrease in size will enhance absorption and thus, increasing translocation to other organs [56]. Intragastric administration of nanoparticulate anatase TiO2 NPs was evaluated for its effects on hematological and hepatic biochemical parameters. Histopathological analysis of liver tissue in high dose group reflected blurred hepatocytes and congested interstitial vessels. Analysis of liver biochemical parameters for 30 days with high dose group showed an increase in liver injury markers and a reduction in certain hematological parameters. Significant reduction in immunologically competent cells, nitric oxide (NO) and Interleukin-2 was observed [57]. The same route of administration has shown kidney injury in mice with renal inflammation, cell necrosis, and dysfunction. After digging up the molecular mechanism, an increase in nuclear factor-κB (NF-κB) signaling resulting in the production of inflammatory cytokines and reduction in heat shock proteins having role in detoxification of TiO2 NPs was noted [58]. Exposure of female mice to intragastric administration of TiO2 NPs for consecutive 90 days resulted in deposition of particles in the ovary leading to alterations of hematological and serum parameters and sex hormone levels, reduction of body weight, relative weight of ovary and fertility and augmented atretic follicle, inflammation, and necrosis [59]. TiO2 NPs were also investigated for their fetal toxicity in pregnant mice where their ability to cross placental barrier was revealed. After IP administration, anomalous liver and brain anatomy was observed in fetus along with reduced weight and diameter of placenta [60]. A study to unveil the ability of TiO2 NPs to cause developmental toxicity was conducted in mice. It was noteworthy that TiO2 NPs at higher doses decreased crown rump length, head length, number of body sections. and increased malformation rate in embryo [61]. These observations are further strengthened by a study from Hong et al., where they evaluated toxicity of TiO2 NPs following oral administration on embryo development in mice. This study pointed towards effective crossing of fetal blood and placental barrier by nanosized TiO2 particles. Reduction in fetal weight, placental weight, number of live fetuses, and fetal cauda as well as crown rump length and increased number of both dead fetuses and resorptions were noted alongside skeletal malformations like a significant absence of cartilage, reduced or

1. Basic principles

3.2 Types of nanocarriers

53

absent ossification, and an augmented number of fetuses with dysplasia [62]. In another study, biodistribution profiling of TiO2 NPs following oral exposure in rats showed high titanium content in placenta at higher doses [63]. Due to their large-scale use owing to their appealing physicochemical properties such as size and surface chemistry, stability, and tunable biocompatibility, silica nanoparticles (SiNPs) are heavily deployed in agriculture, food, cosmetics, molecular imaging, drug and gene delivery. Compared to their amorphous forms which are less pathogenic due to rapid clearance by lungs, crystalline forms are associated with several lethal diseases like silicosis, emphysema, lung cancer, so on and so forth [64]. Single exposure study by Lee et al. highlighted that the smaller the size of SiNPs, the higher the excretion from the body is due to rapid decomposition. They on looked high silicon levels in kidney, liver, lung, and spleen post 6 h of oral administration, persisting in the lungs and spleen for 2 days [65]. Tissue distribution and toxicity profiling of mesoporous SiNPs following oral instillation shed light on the impact of aspect ratio or shape on clearance profile of SiNPs. An increased aspect ratio was reported to be linked with reduced urinary excretion, intestinal absorption, and organ distribution of SiNPs and augmented renal damage such as hemorrhage, vascular congestion, and renal tubular necrosis [66]. A study by Kim et al. in C57BL/6 mice using SiNPs administered by oral route stressed upon immunotoxicity being altered by surface charge. They noted a size-dependent decrease in white blood cell count and decreased proinflammatory cytokines, and reduction in proliferation of B and T cells only for uncoated SiNPs. The most potent ones in showing immunotoxicity were negatively charged and small sized SiNPs [67]. Inhalational exposure of C57BL/6 mice to nonporous SiNPs elicited inflammatory response in a dose-dependent fashion resulting in an increase in infiltration of neutrophils and macrophages in BAL fluid. However, functionalization of SiNPs surface with amine functional group like 3-aminopropyltriethoxysilane has been noted to significantly reduce lung inflammation following intratracheal instillation [68]. Cardiovascular toxicity following intratracheal instillation of SiNPs was evaluated in male Wistar rats using variable doses and sizes. The reports highlighted an increase in oxidative stress and inflammatory cytokines at cardiovascular site and serum for small size NPs compared to bigger one at a particular dose. A significant decrease in antioxidant activity, NO and nitric oxide synthase levels combined with an elevation in levels of reactive oxygen species (ROS), intercellular adhesion molecule-l and vascular cell adhesion molecule-1 points towards endothelial dysfunction. This experimental data stresses upon the leakage of SiNPs through alveoli capillary barrier undergoing systemic distribution [69]. Intranasal instillation repeatedly for 30 days in Wistar rats suggested impaired antioxidant mechanism and size-dependent elevation in lipid peroxidation in various regions of brain along with enhanced mRNA and protein expression levels of nuclear factor kappa light chain enhancer of activated B cells, interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) in different regions of rat brain via redox sensitive signaling cascade [70]. Talking about parenteral exposure to SiNPs in vivo, IV administration in SD rats reflected an increase in inflammatory cell infiltration in the portal area of the liver along with activation of Kupffer cells. The imbalance in antioxidant mechanism was noted. Serum biochemical analysis highlighted an augmented differential leukocyte count, hepatic and renal markers, etc. [71]. A 24 h study discerning acute IP administration of amorphous SiNPs in mice to

1. Basic principles

54

3. In vivo studies: toxicity and biodistribution of nanocarriers in organisms

evaluate oxidative stress, DNA damage and inflammation in multiple organs was conducted. Lipid peroxidation was significantly augmented in the lung, liver, kidney, and brain, but was unchanged in the heart. Antioxidant imbalance and DNA damage were reported in all organs studied along with leucocytosis and enhanced plasma activities of LDH, creatine kinase, alanine aminotransferase, and aspartate aminotransferase. Levels of TNF-α were statistically significant in the lung, heart, and kidney. In the lung, liver, and brain, a significant elevation in pulmonary and renal IL-6 and IL-1β was noted [72]. A strong connection between inflammation and nephrotoxicity by high dose of SiNPs was established by Chen et al., unraveling the role of NF-κB pathway in causing renal interstitial fibrosis. BALB/c mice were injected intraperitoneally with variable doses of SiNPs. In a dose-dependent manner, high expression of fibrosis markers, the nuclear translocation of NF-κB p65 in the kidney combined with elevated biochemical parameters like aspartate and alanine aminotransferase, BUN levels were noted following exposure. Histopathological analysis of kidney showed renal interstitial fibrosis, lymphocytic infiltration, tubular necrosis, glomerular basement membrane thickening, and renal tubular regeneration in higher dose groups [73]. Zinc nanoparticles, mainly zinc oxide nanoparticles (ZnO NPs) are widely used in cosmetics and paints owing to their efficient ultraviolet blocking, low toxicity, high skin tolerance, and antibacterial properties. They are also employed in electronic devices due to their super hydrophobic and photocatalytic properties. Selective apoptosis in tumor cells reveals their huge potential as anticancer drugs [74]. However, this immense use is associated with potential health and environmental risks as proven by different studies across the world. IV administration of ZnO NPs was evaluated in rats for developmental toxicity during gestation period which highlighted the dose-dependent toxicity in dams with an increased number of dead fetuses. Hematological analysis reflected enhanced multifocal mixed cell infiltration with histocytes, neutrophils, and cellular debris in lungs along with tubular dilation with basophilic change in kidneys, thrombosis in lung and extramedullary hemopoiesis in liver in dams whereas, only liver showed significant zinc concentration in fetuses which can be attributed to placental transfer [75]. Intermittent intraperitoneal exposure of ZnO NPs to male Wistar rats was evaluated for its effect on splenocytes. Hematological parameters like neutrophils, platelets, and eosinophils significantly increased in a time dependent fashion. An increase in oxidative stress and apoptosis was observed along with altered antioxidant levels and reduced mitochondria membrane potential was noted in splenic tissue. It was noteworthy that ZnO NPs caused DNA damage at high dose, as analyzed by COMET assay [76]. Further, study by Li et al. using ZnO NPs with IP and oral administration was done which reflected broad tissue spectrum of zinc distribution with major depot as liver followed by spleen and lung in IP administered group. However, serum biochemical analysis in orally administered group showed hepatic injury marker along with moderate to severe hepatic swelling and vacuolization, especially in hepatocytes around the terminal hepatic vein region as evident by histopathological profiling [77]. Long-term toxicity testing of orally exposed ZnO NPs pointed to an increased zinc concentration in serum, liver, and kidney along with augmented activity of glutamic-pyruvic transaminase activity indicating liver damage [78]. Inhalational exposure to ZnO NPs in rats showed alveolar epithelial injury as evident by decreased mitochondrial activity, increased lactate dehydrogenase (LDH) release and intracellular ROS and translocation of ZnO NPs from apical to basolateral fluid across

1. Basic principles

3.2 Types of nanocarriers

55

damaged paracellular pathways [79]. As concluded by various aforementioned studies, different metal nanoparticles evoke toxicities to different organs which are influenced by their size, surface charge, and dose to some extent. Thus, serious evaluation of these parameters on toxicological profiling of inorganic NPs will prove to be apt in designing more such nanocarriers to tackle issues with traditional therapies. QDs are spherical or quasi spherical semiconductor nanocrystals having a size between 1 and 10 nm. They contain several elements belonging to groups II VI (CdSe, CdS, CdTe, ZnS, etc.) or group III-V of periodic table. Owing to their outstanding optical properties, QDs are heavily deployed in electronics in solar battery and light emitting devices, pharmaceutical, medical sector, as powerful fluorescent probes for long-term, multiplexed imaging, drug delivery, cancer diagnosis, and treatment [80]. However, their growing use comes with a worry of their biosafety. QDs to date have been shown to cause various toxicities of liver, kidney, etc. In one study, tail vein administration of CdSe/ZnS QDs in male BALB/c mice has shown cardiotoxicity as evident by an increase in cardiac parameters along with long-term cadmium accumulation in the heart, although cumulative accumulation was low. Significant increase in oxidative stress markers in the heart at high dose was observed [81]. The reproductive toxicity was evaluated for the same aforementioned class of QDs in female mice by Xu and team. They noted the dose-dependent accumulation of cadmium in major organs like liver, spleen, lungs, ovary, and heart following IV exposure. RES organs showed accumulation for QDs with size greater than 5.5 nm. However, SC administration at a higher dose showed accumulation in ovary with reduced oocyte maturation and mRNA levels of female hormones like follicle stimulating hormone and luteinizing hormone (LH) [82]. Cd/Se-ZnS QDs were evaluated for their biodistribution and lung toxicity after pulmonary instillation in rats. They reported dosedependent augmentation in lung injury and inflammation, as suspected in BAL fluid along with chronic persistence of cadmium in lungs for 28 days. No cadmium was analyzed in the liver, brain, heart, blood or spleen at any time point, maybe due to targeted exposure to lungs [83]. Oral administration of hydroxylated graphene QDs in C57BL/6J mice has shed light on its tendency to induce intestinal toxicity. They reported intestinal injuries like shortened villi, crypt loss, and enhanced intestinal permeability, in dose-dependent paradigm along with a decrease in intestinal proliferative progenitor cells and stem cells and enhanced apoptosis of intestinal cells [84] (Fig. 3.3). 3.2.2.2 Polymeric nanoparticles Polymeric nanoparticles with variable surface coatings of polymers like PLGA, PLA, chitosan, etc. have attracted immense attention as drug delivery vehicle pertaining to their low toxicity. Oral administration of PLGA was given for 21 days in rats to evaluate biodistribution profile and suspected toxicity of PLGA NPs. The concentration profile was reported as: spleen . kidney . intestine . liver . lungs . brain . heart after daily oral gavage but following distribution in 7 and 14 days, the highest concentration was reported in the intestine and liver respectively. Hepatic enzymes levels were reported to be significantly elevated along with mild lymphoplasmacytic inflammatory infiltration, as noted by histopathological analysis. Lamina propria of intestine also showed histiocyte infiltration. However, all the aberrant deviations returned to normal within a few days, thus, highlighting no chronic toxicity by PLGA NPs [85]. Another study to track biodistribution of suberoylanilide hydroxamic acid loaded PLGA NPs following oral exposure by Sankar

1. Basic principles

56

3. In vivo studies: toxicity and biodistribution of nanocarriers in organisms

et al. pointed out the retention of PLGA in the liver, kidney, lung, and heart tissue after 3 days of administration. Red blood cells hemolysis was reported as minor toxicity [86]. All these reports highlight that polymeric nanoparticles evoke minimal toxicity and thus, are used as effective drug delivery vehicles compared to their counterparts.

3.3 Polymeric micelles Polymeric micelles or amphiphilic block copolymers have come under the limelight in lieu of their ability to solubilize poorly water-soluble drugs or prodrugs in a hydrophobic reservoir or core. The reservoir or core area encapsulates poorly water-soluble drugs in a dissolved state and is often stabilized by surfactants or polymeric shell to abate rapid diffusion of encapsulated drug. Micelles are classified as association or amphiphilic colloids based on their nanoscopic size and the nature by which they are formed, but should not be considered solid particles. They have diameters ranging from 10 to 100 nm and are characterized by core shell architecture whereas the inner core consists of the hydrophobic regions of the amphiphiles for the solubilization of lipophilic drugs. The benefits of polymeric micelles as delivery agent are twofold: first, the hydrophobic core serves as a cargo space for drugs with poor aqueous solubility; second, the hydrophilic shell provides shielding against opsonin adsorption, which contributes toward better blood stability and subsequently, longer blood circulation time. Micelle’s ability to solubilize a poorly watersoluble drug is determined by its hydrophobic core. PEG is invariably preferred as the shell forming polymer due to certain reasons. First, it is one of the few synthetic polymers approved by Food and Drug Administration to be used in the drug products and is nontoxic. Second, PEG undergoes hydration moves rapidly to sweep out a large exclusion volume in aqueous environment. Third, functionalization of PEG to tether ligands for targeted drug delivery is comparatively easy [6]. Additionally, the small size of polymeric micelles contributes toward longer blood circulation time via escaping scavenging by the mononuclear phagocytic system in the liver and bypassing the filtration of interendothelial cells in the spleen. Ultimately, longer circulation time results in improved accumulation at tissue sites with vascular abnormalities. This unique characteristic provides one of the most powerful arguments for using polymeric micelles for delivering drugs, most of which have very low formulation oriented aqueous solubility [87]. Polymeric micelles offer numerous advantages as drug delivery vehicle, like improvement in drug solubility and augmented bioavailability by solubilizing the drug in hydrophobic core of the micelle [87], inhibition of drug degradation by reducing the nonspecific interactions, including RES (due to steric repulsion by the hydrophilic polymers surrounding the drug- encapsulated hydrophobic core) resulting in improved stability [87], passive targeting of drug through EPR effect [6], utilizing receptor-mediated endocytosis for active drug targeting to increase the selectivity for tumor cells. Compared to their counterparts, polymeric micelles owing to their large hydrophobic core can incorporate a large amount of hydrophobic drugs. They are easy to go and flexible carriers for loading charged macromolecules like nucleic acids and proteins due to ionic interactions with their inner core. In the last few years, high stability and intactness of hydrophobic low molecular weight drugs have remained a major reason for popularity of this nanocarrier system. The easy

1. Basic principles

3.4 Dendrimers

57

and precise control over its size had been advantageous in escaping renal clearance and RES capture. Another advantage is that size is controlled majorly by surface polymers and not through a preparation process unlike liposomes which maintain uniformity. Disassembly of polymeric chains accelerates its renal and hepatic clearance through the body making it less contributing to chronic toxicities unlike other nanocarriers [88]. A study involving comprehension of biodistribution and toxicity of polymeric micelles containing doxorubicin and superparamagnetic iron oxide (SPIO) with glucose as ligand was done. They studied micelles at a different concentration of SPIO and observed that the one with lowest concentration undergone rapid opsonization, quick accumulation and metabolism in liver with shorter circulation time [89]. When used as delivery vehicles, polymeric micelles with PEG coating undergo an immunogenic accelerated blood clearance reaction whereby it has been observed that they are rapidly excreted from the blood on second injection at a particular dose and interval compared to the first one where they persist for a longer time ensuring good bioavailability. This is due to the formation of antiPEG antibodies after first exposure which evokes an immunological response. This peculiar phenomena is not observed with anticancer drugs loaded polymers [88]. Systemic toxicity evaluation of raloxifene loaded polymeric micelle for targeting breast cancer in mice was done along with tissue distribution study. The polymer used composed of poly(styrene maleic acid)-poly(amide-ether-ester-imide)-poly(ethylene glycol). Results revealed a significant uptake of micelles by G-protein coupled estrogen receptors [90]. Polymeric micelles coated with methoxy poly(ethylene glycol)-b-poly(D,L-lactide) and loaded with alpinumisoflavone were tested for their pharmacokinetic profile in rats following IV administration. It was observed that micelles exhibited longer circulation time with sustained release drug profile and lower plasma clearance. Biodistribution studies indicated higher assimilation in RES organ liver along with lung, kidney, spleen, heart, and muscle with congregation decreasing in same sequence. In vivo toxicity assay reported no toxicity by drug loaded polymeric micelles [91].

3.4 Dendrimers Dendrimers are nano molecules with symmetrical branching units constructed around a well-defined linear polymer core or a small molecule. Besides having a compact molecular structure, they have end groups which can be easily functionalized to tune their physicochemical or biological characteristics. They have been widely employed in the supramolecular chemistry field majorly in host guest reaction and self-assembly processes and are gaining immense limelight in anticancer therapies as drug delivery agent, gene delivery, and diagnostic imaging techniques [92].

3.4.1 Poly(amidoamine) dendrimers They were first biologically evaluated in mice by Roberts et al. in 1996 where they acknowledged the extremely high assimilation in pancreas for groups with 96 and 384 terminal amines after 24 h. Conversely, 24 terminal amine group showed the highest

1. Basic principles

58

3. In vivo studies: toxicity and biodistribution of nanocarriers in organisms

accumulation in the kidney after 48 h. This reflected that cationic dendrimers are rapidly cleared out from the blood [93]. After IV and IP injection of radiolabeled poly(amidoamine) (PAMAM) dendrimers, cationic and anionic both showed the highest congregation in the liver whereas blood concentration for anionic outnumbered cationic ones [94]. CNS penetration of cationic PAMAM dendrimers was studied. Confocal imaging reflected the time dependent movement of fourth generation dendrimers starting with plasma membrane to nucleus to perinuclear region after 24 h. Intraventricular injection of fourth generation dendrimer in mice showed their free diffusion in the cerebrospinal fluid and penetration into the brain parenchyma after crossing ependymal cell border. They showed accumulation in perinuclear region, ependymal barrier, and distribution in brain parenchyma. They tried injecting G4 (generation 4) and G4-C12 to brain via intraparenchymal route and concluded that G4-C12 showed high assimilation in brain parenchyma compared to its counterpart pertaining to its high hydrophobicity [95]. Biotinylated PAMAM dendrimers, owing to their huge application in pretargeting approach in cancer cells targeting, are used pertaining to their small tunable spherical sizes. Different generations of radioiodinated starburst dendrimers were tested in athymic mice for their toxicity and biodistribution profiling post 4 h of IV injection. This study acknowledged their rapid clearance by hepatobiliary and renal routes and the kidney retention was augmented with increasing size and charge [93]. Hemmer et al. studied the biodistribution and toxicity of biotinylated G4 PAMAM dendrimers in rat’s brain and discern after 24 h of systemic injection that dorsal striatum was found to contain highest fluorescence intensity since it has thin BBB lining with enhanced permeability [96]. For their application as contrast agents, conjugation of dendrimers with gadolinium, indium or yttrium salts are highly employed which leads to their lodging in various tissues and subsequent toxicity. Unsaturated conjugates are known for their higher assimilation rates compared to saturated ones in the liver, kidney, spleen, and bones. Quick excretion is on looked with lower generation dendrimers. Dendrimers with more size and hence higher generation are taken up by RES and sequester in liver and spleen. Higher generations of anionic MRI contrast agent, Gd labeled DTPA dendrimer have shown the inability to cross BBB following IV infusion pertaining to their large size [97]. PEG has attracted a lot of attention in past few years to show protracted circulation half-life of nanocarriers by preventing their RES uptake, renal uptake, and blood clearance [98]. Ethylenediamine (EDA) and PEG cored PAMAM were studied for their biodistribution fate in Swiss albino female mice following IP route. They showed high retention at the site of injection, peritoneal cavity, liver, and kidney which happened after they entered bloodstream [99]. Multifunctional PAMAM G3 dendrimer-based conjugates encompassing covalently decorated arginine-glycine-aspartic acid-cyclopeptides for targeting monoclonal antibodies, fluorescent dyes for optical imaging, and chelates for gadolinium-based MRI were created to serve as a multimodal nanocarrier. The biodistribution studies were carried out in M21 tumor bearing mice model which acknowledged the highest uptake by the kidney followed by the liver and spleen post 2 h of IV injection. However, the levels fell after 4 h indicating rapid blood clearance [100]. PAMAM dendrimers have not been reported to be immunogenic as such. However, higher generation like G7 dendrimer at high dose has shown some complications following parenteral administration. Liver vacuolation has been reported to be the prominent feature after G3, G5, and G7 administration [93]. IV administration of radiolabeled amino acid dendrimers

1. Basic principles

3.4 Dendrimers

59

have shown rapid blood clearance and congregation in the liver and kidney with hepatic assimilation being generation dependent. But this notion does not commensurate with renal assimilation. Moreover, no significant hepatotoxicity was reported. However, PEGylation dispelled the hepatic and renal accumulation to a greater extent [101]. Clearance of anionic dendrimers commensurates with their generation as the higher generation shows quick clearance [102]. Radiolabeled anionic G6.5 dendrimer in female cluster of diefferentiation-1 mice following oral route has shown 22% of bioavailability and a major chunk got excreted through the kidneys in 4 h. Mild accumulation was reported in the liver, kidney, lungs, and blood. Owing to their big size, they took some time for degradation and excreted eventually [103]. Kannan et al. studied the in vivo toxicity of PAMAM-OH dendrimer carrier employed to deliver antiinflammatory drug N-acetyl cysteine (NAC) for neuroinflammation and cerebral palsy in the rabbit model. He noted no remarkable changes in hepatic, renal, and neuronal functions but less weight gain was recorded for animals with dendrimer alone, which pertains to high catabolic rate due to ongoing induced inflammation [104].

3.4.2 Poly(amidoamine) dendriplexes (complex with nucleic acid) Nucleic acid complexes with dendrimers, known as dendriplexes are utilized in tumor targeted delivery of nucleic acid, be it siRNA, miRNA, etc. for silencing or knocking down the cancer causing genes [105]. The biodistribution and pharmacokinetics of these dendriplexes were studied for the first time by Latallo and team where they intravascularly and endobronchially delivered DNA complexed with G9 PAMAM dendrimers to murine lung tissue for evaluating intrapulmonary transgene expression. Significant concentration of transgenic was found in lung tissue when administered intravascularly at 6 h and reaching peak at 12 h, and a decline was seen on day 5 and day 7 [106]. Mamede et al. experimented targeting hepatocytes with radiolabeled oligo DNA complexed with cationic PAMAM G4 dendrimers and avidin-biotin system via IV route. In the former case, the highest uptake was observed I the kidney and spleen followed by the liver and lungs whereas in the latter case, major assimilation happened in the liver followed by the kidney and spleen. However, when the carrier was embellished with both G4 dendrimer and avidinbiotin, the highest uptake was observed for the lungs pertaining to rapid entrapment of large molecular weight aggregates by lungs RES followed by kidney, liver, and spleen. This study underscored the impact of carrier on organ uptake and resulting bioavailability [107]. Conjugation of pLNC-FVIII vector (which regulates expression of human factor VIII gene) with PAMAM dendrimers was evaluated for biodistribution in vivo in the mice model following IV injection. After successfully recording the required expression of gene in plasma, they discerned using syngene imaging technique that the human FVIII BD cDNA transcription occurred mainly in the spleen and lung and secondarily in the liver and kidney. No prominent side effects were recorded [108]. Another study involving complex of green fluorescent ES312 gene with starburst PAMAM dendrimers was conducted for biodistribution profiling via intramuscular (IM) route in BALB/c mice. The expression was recorded in the heart, liver, spleen, lung, kidney, brain and injected muscle from 2 h to 7 days with highest level in kidneys and endothelial cells [109]. Compared to unmodified dendrimer carriers, dendriplexes are preferred

1. Basic principles

60

3. In vivo studies: toxicity and biodistribution of nanocarriers in organisms

because they are less toxic due to neutralization of charges [102]. Combination cancer therapy using peptide HAIYPRH (T7) conjugated PAMAM dendrimer for codelivery of anticancer drug Doxorubicin and Plasmid pORF-hTRAIL was studied in vivo. And they reported no apparent toxicity due to carrier [110]. Polyplex of G4 and G5 PAMAM starburst dendrimers with luciferase plasmid was employed to target neuroblastoma tumor in vivo following IV route. PAMAM G4 polyplex reported significant hepatic toxicity with elevated enzyme levels at a charge ratio of 6 only at a dose of 10 mg/kg along with remarkable areas of liver necrosis. Besides tumor, the lung showed noteworthy luciferase expression but negligible accumulation was seen in other organs [111].

3.4.3 Polypropyleneimine dendrimers With a variety of polymeric dendrimers in hand and PAMAM being the most popular and widely used one, focus will be shifted to other types of polymeric dendrimers and study their biodistribution and in vivo toxicity in detail. PPI dendrimers are the next in terms of use as the drug and nucleic acid delivery vehicle after PAMAM. Their core nucleus is synthesized by a double Michael addition reaction with propylene imine monomers as branching units and primary amines decorating surface ends. They have a comparatively more hydrophobic interior attributing to the presence of surface alkyl chains [112]. The presence of ample surface primary amines makes them efficient nucleic acid carriers with a generation dependent paradigm. However, their use is restricted owing to their exposed cationic charges accounting for toxicity. Conjugation of a PPI dendrimer with a bifunctional DTPA derivative complexing Gd (III) was studied for its biodistribution and pharmacokinetic profile in the first of its kind study to target intrahepatic micrometastasis in mice. The MRI reported the high signal intensity in the liver post 25 min of injection, in blood after 3 min which decreased gradually over time [113]. A generation 4 PPI Dab dendrimer was used as a liver MRI contrast agent owing to its rapid accumulation in the liver. When given in vivo, it showed highest accumulation in the kidney followed by liver and blood. It has also been reported to show twofold urinary excretion compared to its counterpart PAMAM dendrimer. This quick albumin binding causing more circulation time and hepatic uptake could be due to more hydrophobicity of this dendrimer than PAMAM [114]. Different conjugates bestow varied molecular sizes to these carriers and thus vary their biodistribution fate. Scientists also tried their hands at combination therapy by delivering an anticancer drug along with SiRNA entrapped within these PPI dendrimers. Shah and team targeted ovarian cancer with the aforementioned method with paclitaxel and siRNA directed to CD-44 mRNA. They studied biodistribution of this cationic PPI dendrimer in mice and found significant assimilation in the liver, kidneys, lungs, spleen, and heart. They also showed that surface modification of dendrimer by synthetic analogue of luteinizing hormone-releasing hormone (LHRH) peptide dramatically impeded its accumulation in healthy organs to a great extent and made targeting more efficient [115]. Carbohydrate (mannose and lactose) coated G5 cationic PPI dendrimers were investigated in New Zealand rabbits for their body distribution. The rapid blood clearance was shown along with more hepatic accumulation and retention and less cardiac detention compared to

1. Basic principles

3.4 Dendrimers

61

their unmodified PPI G5 counterparts. Mannose PPI showed peak kidney concentration after 1 h which fell drastically after 6 h indicating rapid urinary excretion. Lactose and mannose modification also succeed in decreasing liver and spleen assimilation comparatively. However, lactose PPI deviated a bit and showed higher accumulation in spleen [116]. Another carbohydrate modified fourth generation PPI dendrimer with maltotriose sugar was evaluated in Wistar rats for their ability to cross BBB. It was observed that the partially modified cationic open dendrimers readily entered the BBB acting as a great therapeutic agent for CNS drug delivery compared to the neutral dense sugar shell with complete glycodendrimer on surface. The body distribution accounted for the highest amount in the liver followed by kidney, spleen, heart, brain, lung, and plasma [117]. Concentrating on biodistribution of PPI dendriplexes, Taratula et al. tried formulating siRNA NPs combined with PPI dendrimer inside PEG polymer for enhanced plasma stability and coated with tumor specific LHRH moiety for efficient tumor uptake. They reported that the tumor nontargeted ones showed major accumulation in the liver and kidney after 72 h of injection [118]. With the PEG polymer becoming popular day by day owing to an increase in circulation half-life of nanocarriers, avid scientists experimented with PEG coatings to discern the effect of its molecular weight and chain length on its body distribution and apparent toxicity, if any. Tritium labeled poly-L-lysine dendrimers with PEG coating were prepared and tested in male SD rats for biodistribution profiling through IV infusion. PEG polymers with lower molecular weights (smaller chain length) quickly cleared off from plasma with increased renal excretion indicating that the higher the molecular weight, the more the terminal half-life of dendrimer will be. PEG also enhanced their accumulation by RES system in the liver and spleen [119].

3.4.4 Melamine dendrimers A library of PEGylated melamine dendrimers with different surface modifications were evaluated for their acute in vivo toxicity in mice through IV route where no conclusion was drawn on any significant liver and kidney toxicity paving way for safe use of PEG in the future [99]. Neerman and colleagues conducted the in vivo acute and subchronic toxicity studies of melamine dendrimers to evaluate their potential as drug carrier. Following IP injection in acute toxicity studies, 160 mg/kg was reported as a lethal dose with visible changes in BUN levels and ALT in serum. However, subchronic toxicity studies ruled out that 40 mg/kg dose is lethal based on a significant increase in ALT activity which is in line with studies conducted with cationic PAMAM dendrimers, stressing upon the impact of the choice of surface groups on toxicity and biodistribution profile of a drug [120]. With viral vectors being in limelight for years as an efficient gene delivery tool, their limitations motivated scientists to come up with nonviral alternatives to assist in gene delivery with more efficacy and safety. Consistent with this idea, dendriplexes of triazine poly ethylene imine (PEI) dendrimer were employed as nonviral gene delivery vectors and tested for the impact of peripheral groups and core structure on their biological activity. In vivo studies were conducted in BALB/c mice with G201g and G2-5 variants owing to their success in vitro. Their biodistribution was compared with PEI polyplexes where they acknowledged the release of siRNA and deposition of polyplex in liver upon hepatic

1. Basic principles

62

3. In vivo studies: toxicity and biodistribution of nanocarriers in organisms

passage indicating faster first pass effect. However, the two former dendriplexes showed longer retention time and less hepatic accumulation which is a beacon of their immense stability. Amphiphilic modification of G2-5 dendrimer enhanced its lungs intracellular uptake [121]. Poly-L-lysine and poly-L-ornithine coated dendrimers were tested following IV administration along with PEGylated sixth generation of DPK (KG6). Consistent with other studies, PEGylated KG6 showed enhanced blood retention. They also showed scant accumulation in the spleen, heart, lung, and urine with no renal and hepatic assimilation. Conversely, amino acid dendrimers showed rapid blood clearance and major congregation in the liver and kidney in generation dependent and independent paradigms respectively [101]. In last few years, a huge amount of data has been generated worldwide on biodistribution and in vivo toxicity of dendrimers with varied size, inner core, and surface modifications as reflected by the studies delineated above. Although we can see dichotomy in some results, one thing remains the same which is dendrimers out of all nanocarriers discovered and synthesized to date are the least toxic and provide a plethora of advantages with flexibility in surface tuning.

3.5 Liposomes Liposomes as nanocarriers have won widespread interest in the recent past due to their attractive biological properties, inclusive of general biocompatibility, biodegradability, isolation of hydrophobic and hydrophilic drugs from the encircling surroundings by encapsulation inside hydrophobic tail region and hydrophilic head respectively and the capacity to entrap both hydrophilic and hydrophobic drugs [122]. Liposomes are essentially the self-assembled artificial vesicles developed from amphiphilic phospholipids and their size can vary from 50 nm to numerous micrometers through the addition of agents to the lipid membrane. They can be unilamellar or multilamellar. The liposomes characteristics including size, surface charge, and capability, can be easily tuned by altering their surface chemistry. Their efficacy has been proven in reducing systemic effects and toxicity, as well as in attenuating drug clearance [122]. Modification of liposomes which includes temperature sensitive liposomes prepared using lipids, transferrin-modified liposomes geared up with a pH sensitive fusogenic peptide, lectin-modified insulin liposomes [123], etc. have top notch pharmacokinetic profiles for the shipping of anticancer agents at nano level. Surface modification of liposomes confers stability and structural integrity in opposition to a harsh bio-environment and it can be executed by attaching PEG devices, or by means of attaching other polymers, inclusive of poly(methacrylic acid-co-cholesteryl methacrylate), poly (acetylic acid), and many others [122]. Liposomes unlike other nanocarriers elicit a comparatively negligible toxic response in the body which can be easily eschewed with varying surface chemistry. Liposomes encapsulating sirolimus were evaluated for their in vivo toxicity in New Zealand albino rabbits intravitreally. Histopathological and immunohistochemistry analysis showed no signs of toxicity to eyes [124]. In another study, 1,2-distigmasterylhemisuccinoyl-sn-glycero-3-phosphocholine liposome encapsulating Amphotericin B employed for anti-leishmanial and antifungal activity was tested for in vivo toxicity and biodistribution profile in vivo in

1. Basic principles

3.6 Conclusion

63

BALB/c mice following IV administration. The drug accumulation was found in the liver and spleen with a low amount in kidneys. Serum concentration was found the highest at 6 h after treatment and that too was insignificant [125]. To extract a better understanding of the effect of protein corona imparted on liposomal surface once they reach blood circulation, Corbo and team conducted a study to understand changes in surface characteristics of liposome and subsequently, liposome-liposome interaction and liposome-cell interaction once they disperse in plasma using a combination of confocal microscopy and flow cytometry techniques. After 1 h of incubation, the results were confounding. They reported that protein corona enhanced uptake of liposomes, be it by cancer cells or macrophages of the liver and spleen leading to their assimilation and reduced their clearance [126]. Size tunable unilamellar cationic liposomes based on novel microfluidic approach were evaluated for their biodistribution and cellular uptake. In vivo studies in mice stressed upon using small diameter liposomes to increase their congregation in draining lymphatics and quick clearance from site of injection which will ultimately enhance biodistribution of liposomes to target site [127]. Biodistribution analysis of 188 Re-liposome in patients with metastatic tumors in phase 0 clinical trial studies revealed that they were majorly uptaken by RES of the liver and spleen and also showed great assimilation in tumors due to EPR effect. After 1 h of injection, the radioactivity was observed in normal organs in the order: Lungs . spleen . kidneys . heart [128].

3.6 Conclusion The inability of drug candidates with huge pharmacodynamic potential to reach the market pertains to their high number of formulation related issues leading to less specificity to diseased area and killing of healthy cells causing undesirable side effects and thus, reduced patient compliance. The overall objective of nanotechnology is providing targeted delivery with minimum side effects and broad safety margin. There are different types of nanocarriers, all with a similar objective but each with its own benefits and limitations. A few of them are like CNTs, NPs, liposomes, polymeric micelles, dendrimers, dendriplexes, etc. Many of them have successfully proved their finesse and have reached clinical trials but some of them like CNTs are novice and underlie huge potential to reach market. The ease of making a variety of NPs with flexible surface tuning has attracted a lot of attention. The ability to play with the surface chemistry, size, and electronic attributes of nanocarriers to deliver payload to desirable sites is bringing limelight to them. As evident from the reported studies, the use of nanocarriers is associated with potential toxicological concerns. Although, the rapid pace of nanotechnology research has raised our hopes for better and improved therapeutic options for certain difficult-to-treat diseases. However, due to their wide applications in various fields ranging from molecular imaging, diagnostics, theranostics to highly precise drug and gene delivery, it is warranted to screen the nanocarriers for their suspected toxicological potential. There is a need to perform comprehensive toxicity analysis for different nanocarriers to rule out their toxicity concerns. Moreover, the case-to-case variations in the physicochemical and in turn toxicological properties of nanocarriers point toward the dire need of toxicity testing in different experimental conditions.

1. Basic principles

64

3. In vivo studies: toxicity and biodistribution of nanocarriers in organisms

3.7 Future directions Although, there are enough reports on toxicological aspects of nanomaterials, there is still a lack of concrete evidences to reach a consensus. There are multiple reasons behind the observed discrepancies in the toxicological studies of the nanomaterials. The first one being the use of exceptionally high doses for carrying out toxicological studies. As everything could be toxic above threshold levels; nanocarriers are no different from it. Further, the use of in vitro/in vivo models for toxicity testing also raises the concerns for their suitability as these models do not accurately recapitulate the clinical conditions. Moreover, there is a need to validate and develop the new models which can effectively and accurately mimic the ideal conditions required for toxicity testing. However, the available literature puts forth a strong case for the toxicity potential of the nanocarriers highlighting the importance of toxicity testing procedures for nanocarriers. In addition, the lack of strict regulatory guidelines also adds to it. Nevertheless, the use of high throughput techniques, organ culture, microfluidics, omics techniques etc. could help to predict and evaluate the toxicity potential of nanocarriers in a fast and reliable manner which would ultimately translate to safe and efficacious use of these glaring moieties. Research is also required for the development of various analytical tools and methods to analyze different types of NPs in various samples including bioanalysis.

References [1] G.A. Silva, Introduction to nanotechnology and its applications to medicine, Surg. Neurol. 61 (3) (2004) 216 220. [2] A.Z. Wilczewska, K. Niemirowicz, K.H. Markiewicz, H. Car, Nanoparticles as drug delivery systems, Pharmacol. Rep. (PR) 64 (5) (2012) 1020 1037. [3] F.U. Din, W. Aman, I. Ullah, O.S. Qureshi, O. Mustapha, S. Shafique, et al., Effective use of nanocarriers as drug delivery systems for the treatment of selected tumors, Int. J. Nanomed. 12 (2017) 7291 7309. [4] S. Gelperina, K. Kisich, M.D. Iseman, L. Heifets, The potential advantages of nanoparticle drug delivery systems in chemotherapy of tuberculosis, Am. J. Respir. Crit. Care Med. 172 (12) (2005) 1487 1490. [5] G. Sharma, A.R. Sharma, S.-S. Lee, M. Bhattacharya, J.-S. Nam, C. Chakraborty, Advances in nanocarriers enabled brain targeted drug delivery across blood brain barrier, Int. J. Pharm. 559 (2019) 360 372. [6] K. Letchford, H. Burt, A review of the formation and classification of amphiphilic block copolymer nanoparticulate structures: micelles, nanospheres, nanocapsules and polymersomes, Eur. J. Pharm. Biopharm. 65 (3) (2007) 259 269. [7] N. Mody, R.K. Tekade, N.K. Mehra, P. Chopdey, N.K. Jain, Dendrimer, liposomes, carbon nanotubes and PLGA nanoparticles: one platform assessment of drug delivery potential, AAPS PharmSciTech 15 (2) (2014) 388 399. [8] K. Pulskamp, S. Diabate´, H.F. Krug, Carbon nanotubes show no sign of acute toxicity but induce intracellular reactive oxygen species in dependence on contaminants, Toxicol. Lett. 168 (1) (2007) 58 74. [9] S.S. Poulsen, P. Jackson, K. Kling, K.B. Knudsen, V. Skaug, Z.O. Kyjovska, et al., Multi-walled carbon nanotube physicochemical properties predict pulmonary inflammation and genotoxicity, Nanotoxicology 10 (9) (2016) 1263 1275. [10] M. Ema, S. Masumori, N. Kobayashi, M. Naya, S. Endoh, J. Maru, et al., In vivo comet assay of multi-walled carbon nanotubes using lung cells of rats intratracheally instilled, J. Appl. Toxicol. (JAT) 33 (10) (2013) 1053 1060. [11] J. Catala´n, K.M. Siivola, P. Nymark, H. Lindberg, S. Suhonen, H. Ja¨rventaus, et al., In vitro and in vivo genotoxic effects of straight versus tangled multi-walled carbon nanotubes, Nanotoxicology 10 (6) (2016) 794 806. [12] C.-W. Lam, J.T. James, R. McCluskey, R.L. Hunter, Pulmonary toxicity of single-wall carbon nanotubes in mice 7 and 90 days after intratracheal instillation, Toxicol. Sci. 77 (1) (2004) 126 134.

1. Basic principles

References

65

[13] D.B. Warheit, B.R. Laurence, K.L. Reed, D.H. Roach, G.A.M. Reynolds, T.R. Webb, Comparative pulmonary toxicity assessment of single-wall carbon nanotubes in rats, Toxicol. Sci. 77 (1) (2004) 117 125. [14] A.A. Shvedova, E.R. Kisin, R. Mercer, A.R. Murray, V.J. Johnson, A.I. Potapovich, et al., Unusual inflammatory and fibrogenic pulmonary responses to single-walled carbon nanotubes in mice, Am J. Physiol. Lung Cell Mol. Physiol. 289 (5) (2005) L698 L708. ´ [15] H. Grubek-Jaworska, P. Nejman, K. Czuminska, T. Przybyłowski, A. Huczko, H. Lange, et al., Preliminary results on the pathogenic effects of intratracheal exposure to one-dimensional nanocarbons, Carbon 44 (6) (2006) 1057 1063. [16] J.P. Ryman-Rasmussen, M.F. Cesta, A.R. Brody, J.K. Shipley-Phillips, J.I. Everitt, E.W. Tewksbury, et al., Inhaled carbon nanotubes reach the subpleural tissue in mice, Nat. Nanotechnol. 4 (11) (2009) 747 751. [17] H. Wang, J. Wang, X. Deng, H. Sun, Z. Shi, Z. Gu, et al., Biodistribution of carbon single-wall carbon nanotubes in mice, J. Nanosci. Nanotechnol. 4 (8) (2004) 1019 1024. [18] Z. Li, T. Hulderman, R. Salmen, R. Chapman, S.S. Leonard, S.-H. Young, et al., Cardiovascular effects of pulmonary exposure to single-wall carbon nanotubes, Environ. Health Perspect. 115 (3) (2007) 377 382. [19] N.A. Philbrook, V.K. Walker, A.R.M.N. Afrooz, N.B. Saleh, L.M. Winn, Investigating the effects of functionalized carbon nanotubes on reproduction and development in Drosophila melanogaster and CD-1 mice, Reprod. Toxicol. 32 (4) (2011) 442 448. [20] A. Pietroiusti, M. Massimiani, I. Fenoglio, M. Colonna, F. Valentini, G. Palleschi, et al., Low doses of pristine and oxidized single-wall carbon nanotubes affect mammalian embryonic development, ACS Nano 5 (6) (2011) 4624 4633. [21] Y. Bai, Y. Zhang, J. Zhang, Q. Mu, W. Zhang, E.R. Butch, et al., Repeated carbon nanotube administrations in male mice cause reversible testis damage without affecting fertility, Nat. Nanotechnol. 5 (9) (2010) 683 689. [22] M.I. Sajid, U. Jamshaid, T. Jamshaid, N. Zafar, H. Fessi, A. Elaissari, Carbon nanotubes from synthesis to in vivo biomedical applications, Int. J. Pharm. 501 (1 2) (2016) 278 299. [23] R. Singh, D. Pantarotto, L. Lacerda, G. Pastorin, C. Klumpp, M. Prato, et al., Tissue biodistribution and blood clearance rates of intravenously administered carbon nanotube radiotracers, Proc. Natl Acad. Sci. U.S.A. 103 (9) (2006) 3357 3362. [24] X. Wang, N.D. Mansukhani, L.M. Guiney, J.H. Lee, R. Li, B. Sun, et al., Toxicological profiling of highly purified metallic and semiconducting single-walled carbon nanotubes in the rodent lung and E. coli, ACS Nano 10 (6) (2006) 6008 6019. Available from: https://doi.org/10.1021/acsnano.6b01560. [25] J. Guo, X. Zhang, Q. Li, W. Li, Biodistribution of functionalized multiwall carbon nanotubes in mice, Nucl. Med. Biol. 34 (5) (2007) 579 583. [26] H. Kafa, J.T.-W. Wang, N. Rubio, K. Venner, G. Anderson, E. Pach, et al., The interaction of carbon nanotubes with an in vitro blood-brain barrier model and mouse brain in vivo, Biomaterials 53 (2015) 437 452. [27] G. Biagiotti, F. Pisaneschi, S.T. Gammon, F. Machetti, M.C. Ligi, G. Giambastiani, et al., Multiwalled carbon nanotubes for combination therapy: a biodistribution and efficacy pilot study, J. Mater. Chem. B. 7 (16) (2019) 2678 2687. [28] S. Yang, W. Guo, Y. Lin, X. Deng, H. Wang, H. Sun, et al., Biodistribution of pristine single-walled carbon nanotubes in vivo, J. Phys. Chem. 111 (48) (2007) 17761 17764. Available from: https://doi.org/10.1021/jp070712c. [29] S.T. Yang, X. Wang, G. Jia, et al., Long-term accumulation and low toxicity of single-walled carbon nanotubes in intravenously exposed mice, Toxicol. Lett. 181 (3) (2008) 182 189. [30] P. Cherukuri, C.J. Gannon, T.K. Leeuw, H.K. Schmidt, R.E. Smalley, S.A. Curley, et al., Mammalian pharmacokinetics of carbon nanotubes using intrinsic near-infrared fluorescence, Proc. Natl Acad. Sci. U.S.A. 103 (50) (2006) 18882 18886. [31] Z. Liu, W. Cai, L. He, N. Nakayama, K. Chen, X. Sun, et al., In vivo biodistribution and highly efficient tumour targeting of carbon nanotubes in mice, Nat. Nanotechnol. 2 (1) (2007) 20 21. [32] M.R. Mcdevitt, D. Chattopadhyay, J.S. Jaggi, R.D. Finn, P.B. Zanzonico, C. Villa, et al., PET imaging of soluble yttrium-86-labeled carbon nanotubes in mice, PLoS ONE 2 (9) (2007) e907. Available from: https://doi. org/10.1371/journal.pone.0000907. [33] X. Deng, S. Yang, H. Nie, H. Wang, Y. Liu, A generally adoptable radiotracing method for tracking carbon nanotubes in animals, Nanotechnology. 19 (7) (2008) 075101. Available from: https://doi.org/10.1088/09574484/19/7/075101. [34] J.T. Wang, C. Fabbro, E. Venturelli, C. Me´nard-Moyon, O. Chaloin, T. Da Ros, et al., The relationship between the diameter of chemically-functionalized multi-walled carbon, Biomaterials 35 (35) (2014) 9517 9528.

1. Basic principles

66

3. In vivo studies: toxicity and biodistribution of nanocarriers in organisms

[35] S.-D. Li, L. Huang, Pharmacokinetics and biodistribution of nanoparticles, Mol. Pharm. 5 (4) (2008) 496 504. [36] J.P.M. Almeida, A.L. Chen, A. Foster, R. Drezek, In vivo biodistribution of nanoparticles, Nanomed. 6 (5) (2011) 815 835. [37] H.Y. Yoon, S. Jeon, D.G. You, J.H. Park, I.C. Kwon, H. Koo, et al., Inorganic nanoparticles for image-guided therapy, Bioconjug. Chem. 28 (1) (2017) 124 134. [38] H. Arami, A. Khandhar, D. Liggitt, K.M. Krishnan, In vivo delivery, pharmacokinetics, biodistribution and toxicity of iron oxide nanoparticles, Chem. Soc. Rev. 44 (23) (2015) 8576 8607. [39] D. Askri, S. Ouni, S. Galai, B. Chovelon, J. Arnaud, N. Sturm, et al., Nanoparticles in foods? A multiscale physiopathological investigation of iron oxide nanoparticle effects on rats after an acute oral exposure: Trace element biodistribution and cognitive capacities, Food Chem. Toxicol. 127 (2019) 173 181. [40] N.S. Elbialy, S.F. Aboushoushah, W.W. Alshammari, Long-term biodistribution and toxicity of curcumin capped iron oxide nanoparticles after single-dose administration in mice, Life Sci. 230 (2019) 76 83. [41] P. Yang, H. Xu, Z. Zhang, L. Yang, H. Kuang, Z.P. Aguilar, Surface modification affect the biodistribution and toxicity characteristics of iron oxide magnetic nanoparticles in rats, IET Nanobiotechnol. 12 (5) (2018) 562 568. [42] M. Salimi, S. Sarkar, S. Fathi, A.M. Alizadeh, R. Saber, F. Moradi, et al., Biodistribution, pharmacokinetics, and toxicity of dendrimer-coated iron oxide nanoparticles in BALB/c mice, Int. J. Nanomed. 13 (2018) 1483 1493. [43] B.T.T. Pham, E.K. Colvin, N.T.H. Pham, B.J. Kim, E.S. Fuller, E.A. Moon, et al., Biodistribution and clearance of stable superparamagnetic maghemite iron oxide nanoparticles in mice following intraperitoneal administration, Int. J. Mol. Sci. 19 (1) (2018) E205. [44] Y.S. Chen, Y.C. Hung, I. Liau, G.S. Huang, Assessment of the in vivo toxicity of gold nanoparticles, Nanoscale Res. Lett. 4 (8) (2009) 858 864. Available from: https://doi.org/10.1007/s11671-009-9334-6. [45] C. Lasagna-Reeves, D. Gonzalez-Romero, M.A. Barria, I. Olmedo, A. Clos, V.M. Sadagopa Ramanujam, et al., Bioaccumulation and toxicity of gold nanoparticles after repeated administration in mice, Biochem. Biophys. Res. Commun. 393 (4) (2010) 649 655. [46] A.L. Bailly, F. Correard, A. Popov, G. Tselikov, F. Chaspoul, R. Appay, et al., In vivo evaluation of safety, biodistribution and pharmacokinetics of laser-synthesized gold nanoparticles, Sci. Rep. 9 (1) (2019) 12890. [47] M. Pe´rez-Herna´ndez, M. Moros, G. Stepien, P. Del Pino, S. Menao, M. de Las Heras, Multiparametric analysis of anti-proliferative and apoptotic effects of gold nanoprisms on mouse and human primary and transformed cells, biodistribution and toxicity in vivo, Part Fibre Toxicol 14 (1) (2017) 41. [48] C.A. Simpson, K.J. Salleng, D.E. Cliffel, D.L. Feldheim, In vivo toxicity, biodistribution, and clearance of glutathione-coated gold nanoparticles, Nanomedicine 9 (2) (2013) 257 263. [49] A.L. Brown, M.P. Kai, A.N. DuRoss, G. Sahay, C. Sun, Biodistribution and toxicity of micellar platinum nanoparticles in mice via intravenous administration, Nanomaterials (Basel) 8 (6) (2018) E410. [50] C.-X. Lin, J.-L. Gu, J.-M. Cao, The acute toxic effects of platinum nanoparticles on ion channels, transmembrane potentials of cardiomyocytes in vitro and heart rhythm in vivo in mice, Int. J. Nanomed. 14 (2019) 5595 5609. [51] K. Isoda, T. Daibo, K. Yushina, Y. Yoshioka, Y. Tsutsumi, Y. Akimoto, et al., Hepatotoxicity, nephrotoxicity, and drug/chemical interaction toxicity of platinum nanoparticles in mice, Pharm. 72 (1) (2017) 10 16. [52] A. Dey, S. Manna, J. Adhikary, S. Chattopadhyay, S. De, D. Chattopadhyay, et al., Biodistribution and toxickinetic variances of chemical and green copper oxide nanoparticles in vitro and in vivo, J Trace Elem. Med. Biol. 55 (2019) 154 169. [53] R. Mani, S. Balasubramanian, A. Raghunath, E. Perumal, Chronic exposure to copper oxide nanoparticles causes muscle toxicity in adult zebrafish, Environ. Sci. Pollut. Res. Int. (2019). Available from: https://doi. org/10.1007/s11356-019-06095-w. [54] M.R. Madhav, S.E.M. David, R.S.S. Kumar, J.S. Swathy, M. Bhuvaneshwari, A. Mukherjee, et al., Toxicity and accumulation of copper oxide (CuO) nanoparticles in different life stages of Artemia salina, Environ. Toxicol. Pharmacol. 52 (2017) 227 238. [55] W. Mu, Y. Wang, C. Huang, Y. Fu, J. Li, H. Wang, et al., Effect of long-term intake of dietary titanium dioxide nanoparticles on intestine inflammation in mice, J. Agric. Food Chem. 67 (33) (2019) 9382 9389. [56] Y. Wang, Z. Chen, T. Ba, J. Pu, T. Chen, Y. Song, et al., Susceptibility of young and adult rats to the oral toxicity of titanium dioxide nanoparticles, Small Weinh. Bergstr. Ger. 9 (9 10) (2013) 1742 1752.

1. Basic principles

References

67

[57] Y. Duan, J. Liu, L. Ma, N. Li, H. Liu, J. Wang, et al., Toxicological characteristics of nanoparticulate anatase titanium dioxide in mice, Biomaterials 31 (5) (2010) 894 899. [58] S. Gui, Z. Zhang, L. Zheng, Y. Cui, X. Liu, N. Li, et al., Molecular mechanism of kidney injury of mice caused by exposure to titanium dioxide nanoparticles, J. Hazard Mater. 195 (2011) 365 370. [59] X. Zhao, Y. Ze, G. Gao, X. Sang, B. Li, S. Gui, et al., Nanosized TiO2-induced reproductive system dysfunction and its mechanism in female mice, PLoS One 8 (4) (2013) e59378. [60] P. Naserzadeh, F. Ghanbary, P. Ashtari, E. Seydi, K. Ashtari, M. Akbari, Biocompatibility assessment of titanium dioxide nanoparticles in mice fetoplacental unit, J. Biomed. Mater. Res. A 106 (2) (2018) 580 589. [61] X. Jia, S. Wang, L. Zhou, L. Sun, The potential liver, brain, and embryo toxicity of titanium dioxide nanoparticles on mice, Nanoscale Res. Lett. 12 (1) (2017) 478. [62] F. Hong, Y. Zhou, X. Zhao, L. Sheng, L. Wang, Maternal exposure to nanosized titanium dioxide suppresses embryonic development in mice, Int. J. Nanomed. 12 (2017) 6197 6204. [63] J. Lee, J.S. Jeong, S.Y. Kim, M.K. Park, S.D. Choi, U.J. Kim, et al., Titanium dioxide nanoparticles oral exposure to pregnant rats and its distribution, Part. Fibre Toxicol. 16 (1) (2019) 31. [64] S. Murugadoss, D. Lison, L. Godderis, S. Van Den Brule, J. Mast, F. Brassinne, et al., Toxicology of silica nanoparticles: an update, Arch. Toxicol. 91 (9) (2017) 2967 3010. [65] J.-A. Lee, M.-K. Kim, H.-J. Paek, Y.-R. Kim, M.-K. Kim, J.-K. Lee, et al., Tissue distribution and excretion kinetics of orally administered silica nanoparticles in rats, Int. J. Nanomed. 9 (Suppl. 2) (2014) 251 260. [66] L. Li, T. Liu, C. Fu, L. Tan, X. Meng, H. Liu, Biodistribution, excretion, and toxicity of mesoporous silica nanoparticles after oral administration depend on their shape, Nanomed. Nanotechnol. Biol. Med. 11 (8) (2015) 1915 1924. [67] J.-H. Kim, C.-S. Kim, R.M.C. Ignacio, D.-H. Kim, M.E.J. Sajo, E.H. Maeng, et al., Immunotoxicity of silicon dioxide nanoparticles with different sizes and electrostatic charge, Int. J. Nanomed. 9 (Suppl. 2) (2014) 183 193. [68] A.S. Morris, A. Adamcakova-Dodd, S.E. Lehman, A. Wongrakpanich, P.S. Thorne, S.C. Larsen, et al., Amine modification of nonporous silica nanoparticles reduces inflammatory response following intratracheal instillation in murine lungs, Toxicol. Lett. 241 (2016) 207 215. [69] Z. Du, D. Zhao, L. Jing, G. Cui, M. Jin, Y. Li, et al., Cardiovascular toxicity of different sizes amorphous silica nanoparticles in rats after intratracheal instillation, Cardiovasc. Toxicol. 13 (3) (2013) 194 207. [70] A. Parveen, S.H.M. Rizvi, Sushma, F. Mahdi, I. Ahmad, P.P. Singh, et al., Intranasal exposure to silica nanoparticles induces alterations in pro-inflammatory environment of rat brain, Toxicol. Ind. Health. 33 (2) (2017) 119 132. [71] Q. Chen, Y. Xue, J. Sun, Kupffer cell-mediated hepatic injury induced by silica nanoparticles in vitro and in vivo, Int. J. Nanomed. 8 (2013) 1129 1140. [72] A. Nemmar, P. Yuvaraju, S. Beegam, J. Yasin, E.E. Kazzam, B.H. Ali, Oxidative stress, inflammation, and DNA damage in multiple organs of mice acutely exposed to amorphous silica nanoparticles, Int. J. Nanomed. 11 (2016) 919 928. [73] X. Chen, W. Zhouhua, Z. Jie, F. Xinlu, L. Jinqiang, Q. Yuwen, et al., Renal interstitial fibrosis induced by high-dose mesoporous silica nanoparticles via the NF-κB signaling pathway, Int. J. Nanomedicine 10 (2015) 1 22. [74] E.-J. Park, U. Jeong, C. Yoon, Y. Kim, Comparison of distribution and toxicity of different types of zinc-based nanoparticles, Environ. Toxicol. 32 (4) (2017) 1363 1374. [75] J. Lee, W.-J. Yu, J. Song, C. Sung, E.J. Jeong, J.-S. Han, et al., Developmental toxicity of intravenously injected zinc oxide nanoparticles in rats, Arch. Pharm. Res. 39 (12) (2016) 1682 1692. [76] N. Singh, M.K. Das, R. Gautam, A. Ramteke, P. Rajamani, Assessment of intermittent exposure of zinc oxide nanoparticle (ZNP)-mediated toxicity and biochemical alterations in the splenocytes of male Wistar rat, Environ. Sci. Pollut. Res. Int. 26 (32) (2019) 33642 33653. [77] C.-H. Li, C.-C. Shen, Y.-W. Cheng, S.-H. Huang, C.-C. Wu, C.-C. Kao, et al., Organ biodistribution, clearance, and genotoxicity of orally administered zinc oxide nanoparticles in mice, Nanotoxicology 6 (7) (2012) 746 756. [78] C. Wang, K. Cheng, L. Zhou, J. He, X. Zheng, L. Zhang, et al., Evaluation of long-term toxicity of oral zinc oxide nanoparticles and zinc sulfate in mice, Biol. Trace Elem. Res. 178 (2) (2017) 276 282. [79] Y.H. Kim, F. Fazlollahi, I.M. Kennedy, N.R. Yacobi, S.F. Hamm-Alvarez, Z. Borok, et al., Alveolar epithelial cell injury due to zinc oxide nanoparticle exposure, Am J. Respir. Crit. Care Med. 182 (11) (2010) 1398 1409.

1. Basic principles

68

3. In vivo studies: toxicity and biodistribution of nanocarriers in organisms

[80] Y. Wang, L. Chen, Quantum dots, lighting up the research and development of nanomedicine, Nanomedicine 7 (4) (2011) 385 402. [81] L. Li, J. Tian, X. Wang, G. Xu, W. Jiang, Z. Yang, et al., Cardiotoxicity of intravenously administered CdSe/ ZnS quantum dots in BALB/c mice, Front. Pharmacol. 10 (2019) 1179. [82] G. Xu, G. Lin, S. Lin, N. Wu, Y. Deng, G. Feng, et al., The reproductive toxicity of CdSe/ZnS quantum dots on the in vivo ovarian function and in vitro fertilization, Sci. Rep. 6 (2016) 37677. [83] J.R. Roberts, J.M. Antonini, D.W. Porter, R.S. Chapman, J.F. Scabilloni, S.H. Young, et al., Lung toxicity and biodistribution of Cd/Se-ZnS quantum dots with different surface functional groups after pulmonary exposure in rats, Part. Fibre Toxicol. 10 (2013) 5. [84] L. Yu, X. Tian, D. Gao, Y. Lang, X.X. Zhang, C. Yang, et al., Oral administration of hydroxylated-graphene quantum dots induces intestinal injury accompanying the loss of intestinal stem cells and proliferative progenitor cells, Nanotoxicology 13 (10) (2019) 1409 1421. [85] S.M. Navarro, T.W. Morgan, C.E. Astete, R.W. Stout, D. Coulon, P. Mottram, et al., Biodistribution and toxicity of orally administered poly (lactic-co-glycolic) acid nanoparticles to F344 rats for 21 days, Nanomedicine (Lond) 11 (13) (2016) 1653 1669. [86] R. Sankar, V. Ravikumar, Biocompatibility and biodistribution of suberoylanilide hydroxamic acid loaded poly (DL-lactide-co-glycolide) nanoparticles for targeted drug delivery in cancer, Biomed. Pharmacother 68 (7) (2014) 865 871. [87] Y. Lu, K. Park, Polymeric micelles and alternative nanonized delivery vehicles for poorly soluble drugs, Int. J. Pharm. 453 (1) (2013) 198 214. [88] M. Yokoyama, Polymeric micelles as drug carriers: their lights and shadows, J. Drug Target 22 (7) (2014) 576 583. [89] N. Thitichai, C. Thanapongpibul, M. Theerasilp, W. Sungkarat, N. Nasongkla, Study of biodistribution and systemic toxicity of glucose functionalized SPIO/DOX micelles, Pharm. Dev. Technol. 24 (8) (2019) 935 946. [90] J. Varshosaz, F. Hassanzadeh, B. Hashemi-Beni, M. Minaiyan, S. Enteshari, Tissue distribution and systemic toxicity evaluation of raloxifene targeted polymeric micelles of poly (styrene-maleic acid)-poly (amide-etherester-imide)-poly (ethylene glycol) loaded with docetaxel in breast cancer bearing mice, Recent Pat. Anticancer Drug Discov. 14 (3) (2019) 280 291. [91] M.J. Jo, Y.H. Jo, Y.J. Lee, C.-W. Park, J.-S. Kim, J.T. Hong, et al., Physicochemical, pharmacokinetic, and toxicity evaluation of methoxy poly(ethylene glycol)-b-poly(d,l-lactide) polymeric micelles encapsulating alpinumisoflavone extracted from unripe Cudrania tricuspidata fruit, Pharmaceutics. 11 (8) (2019) E366. [92] E. Abbasi, S.F. Aval, A. Akbarzadeh, M. Milani, H.T. Nasrabadi, S.W. Joo, et al., Dendrimers: synthesis, applications, and properties, Nanoscale Res. Lett. 9 (1) (2014) 247. [93] J.C. Roberts, M.K. Bhalgat, R.T. Zera, Preliminary biological evaluation of polyamidoamine (PAMAM) Starburst dendrimers, J. Biomed. Mater. Res. 30 (1) (1996) 53 65. [94] N. Malik, R. Wiwattanapatapee, R. Klopsch, K. Lorenz, H. Frey, J.W. Weener, et al., Dendrimers: relationship between structure and biocompatibility in vitro, and preliminary studies on the biodistribution of 125Ilabelled polyamidoamine dendrimers in vivo, J. Control Release 65 (1 2) (2000) 133 148. [95] L. Albertazzi, L. Gherardini, M. Brondi, S. Sulis Sato, A. Bifone, T. Pizzorusso, et al., In vivo distribution and toxicity of PAMAM dendrimers in the central nervous system depend on their surface chemistry, Mol. Pharm. 10 (1) (2013) 249 260. [96] R. Hemmer, A. Hall, R. Spaulding, B. Rossow, M. Hester, M. Caroway, et al., Analysis of biotinylated generation 4 poly(amidoamine) (PAMAM) dendrimer distribution in the rat brain and toxicity in a cellular model of the blood-brain barrier, Molecules 18 (9) (2013) 11537 11552. [97] H. Sarin, A.S. Kanevsky, H. Wu, A.A. Sousa, C.M. Wilson, M.A. Aronova, et al., Physiologic upper limit of pore size in the blood-tumor barrier of malignant solid tumors, J. Transl. Med. 7 (2009) 51. [98] H. Kobayashi, S. Kawamoto, T. Saga, N. Sato, A. Hiraga, T. Ishimori, et al., Positive effects of polyethylene glycol conjugation to generation-4 polyamidoamine dendrimers as macromolecular MR contrast agents, Magn. Reson. Med. 46 (4) (2001) 781 788. ¨ ztu¨rk, A.S. Ertu¨rk, D. Yoyen-Ermis, G. Esendagli, S. C [99] M.U. Gu¨rbu¨z, K. O ¸ alı¸s, et al., Cytotoxicity and biodistribution studies on PEGylated EDA and PEG cored PAMAM dendrimers, J. Biomater Sci. Polym. Ed. 27 (16) (2016) 1645 1658.

1. Basic principles

References

69

[100] C.A. Boswell, P.K. Eck, C.A.S. Regino, M. Bernardo, K.J. Wong, D.E. Milenic, et al., Synthesis, characterization, and biological evaluation of integrin alphavbeta3-targeted PAMAM dendrimers, Mol. Pharm. 5 (4) (2008) 527 539. [101] T. Okuda, S. Kawakami, T. Maeie, T. Niidome, F. Yamashita, M. Hashida, Biodistribution characteristics of amino acid dendrimers and their PEGylated derivatives after intravenous administration, J. Control Release 114 (1) (2006) 69 77. [102] D. Shcharbin, A. Janaszewska, B. Klajnert-Maculewicz, B. Ziemba, V. Dzmitruk, I. Halets, et al., How to study dendrimers and dendriplexes III. Biodistribution, pharmacokinetics and toxicity in vivo, J. Control Release 181 (2014) 40 52. [103] G. Thiagarajan, S. Sadekar, K. Greish, A. Ray, H. Ghandehari, Evidence of oral translocation of anionic G6.5 dendrimers in mice, Mol. Pharm. 10 (3) (2013) 988 998. [104] S. Kannan, H. Dai, R.S. Navath, B. Balakrishnan, A. Jyoti, J. Janisse, et al., Dendrimer-based postnatal therapy for neuroinflammation and cerebral palsy in a rabbit model, Sci. Transl. Med. 4 (130) (2012) 130ra46. [105] J. Li, L. Chen, N. Liu, S. Li, Y. Hao, X. Zhang, EGF-coated nano-dendriplexes for tumor-targeted nucleic acid delivery in vivo, Drug Deliv. 23 (5) (2016) 1718 1725. [106] J.F. Kukowska-Latallo, E. Raczka, A. Quintana, C. Chen, M. Rymaszewski, J.R. Baker, Intravascular and endobronchial DNA delivery to murine lung tissue using a novel, nonviral vector, Hum. Gene Ther. 11 (10) (2000) 1385 1395. [107] M. Mamede, T. Saga, T. Ishimori, T. Higashi, N. Sato, H. Kobayashi, et al., Hepatocyte targeting of 111Inlabeled oligo-DNA with avidin or avidin-dendrimer complex, J. Control Release 95 (1) (2004) 133 141. [108] W.-Y. Kang, H.-L. Wang, H. Wang, X.-F. Wang, C.-Z. Wang, Q.-H. Fu, et al., In vivo transfection and expression of human coagulant factor VIII cDNA in mice, Zhongguo Shi Yan Xue Ye Xue Za Zhi 12 (2) (2004) 188 193. [109] J.-J. Ding, C.-Y. Guo, Q.-L. Cai, Y.-H. Lin, H. Wang, In vivo expression of green fluorescent protein gene and immunogenicity of ES312 vaccine both mediated by starburst polyamidoamine dendrimers, Zhongguo Yi Xue Ke Xue Yuan Xue Bao 27 (4) (2005) 499 503. [110] L. Han, R. Huang, J. Li, S. Liu, S. Huang, C. Jiang, Plasmid pORF-hTRAIL and doxorubicin co-delivery targeting to tumor using peptide-conjugated polyamidoamine dendrimer, Biomaterials 32 (4) (2011) 1242 1252. [111] G. Navarro, G. Maiwald, R. Haase, A.L. Rogach, E. Wagner, C.T. de Ilarduya, et al., Low generation PAMAM dendrimer and CpG free plasmids allow targeted and extended transgene expression in tumors after systemic delivery, J. Control Release 146 (1) (2010) 99 105. [112] L. Palmerston Mendes, J. Pan, V.P. Torchilin, Dendrimers as nanocarriers for nucleic acid and drug delivery in cancer therapy, Mol. J. Synth. Chem. Nat. Prod. Chem. 22 (9) (2017) 1401. [113] H. Kobayashi, T. Saga, S. Kawamoto, N. Sato, A. Hiraga, T. Ishimori, et al., Dynamic micro-MRI of liver micrometastasis with a novel liver macromolecular MR contrast agent DAB-Am64-(1B4M-Gd)64, Acad. Radiol. 9 (Suppl. 2) (2002) S452 S454. [114] H. Kobayashi, S. Kawamoto, T. Saga, N. Sato, A. Hiraga, T. Ishimori, et al., Novel liver macromolecular MR contrast agent with a polypropylenimine diaminobutyl dendrimer core: comparison to the vascular MR contrast agent with the polyamidoamine dendrimer core, Magn. Reson. Med. 46 (4) (2001) 795 802. [115] V. Shah, O. Taratula, O.B. Garbuzenko, O.R. Taratula, L. Rodriguez-Rodriguez, T. Minko, Targeted nanomedicine for suppression of CD44 and simultaneous cell death induction in ovarian cancer: an optimal delivery of siRNA and anticancer drug, Clin. Cancer Res. 19 (22) (2013) 6193 6204. [116] H.B. Agashe, A.K. Babbar, S. Jain, R.K. Sharma, A.K. Mishra, A. Asthana, et al., Investigations on biodistribution of technetium-99m-labeled carbohydrate-coated poly(propylene imine) dendrimers, Nanomed. Nanotechnol. Biol. Med. 3 (2) (2007) 120 127. [117] K. Ciepluch, B. Ziemba, A. Janaszewska, D. Appelhans, B. Klajnert, M. Bryszewska, et al., Modulation of biogenic amines content by poly(propylene imine) dendrimers in rats, J. Physiol. Biochem. 68 (3) (2012) 447 454. [118] O. Taratula, O.B. Garbuzenko, P. Kirkpatrick, I. Pandya, R. Savla, V.P. Pozharov, et al., Surface-engineered targeted PPI dendrimer for efficient intracellular and intratumoral siRNA delivery, J. Control Release 140 (3) (2009) 284 293.

1. Basic principles

70

3. In vivo studies: toxicity and biodistribution of nanocarriers in organisms

[119] L.M. Kaminskas, B.J. Boyd, P. Karellas, G.Y. Krippner, R. Lessene, B. Kelly, et al., The impact of molecular weight and PEG chain length on the systemic pharmacokinetics of PEGylated poly l-lysine dendrimers, Mol. Pharm. 5 (3) (2008) 449 463. [120] M.F. Neerman, W. Zhang, A.R. Parrish, E.E. Simanek, In vitro and in vivo evaluation of a melamine dendrimer as a vehicle for drug delivery, Int. J. Pharm. 281 (1 2) (2004) 129 132. [121] O.M. Merkel, M.A. Mintzer, D. Librizzi, O. Samsonova, T. Dicke, B. Sproat, et al., Triazine dendrimers as non-viral vectors for in vitro and in vivo RNAi: the effects of peripheral groups and core structure on biological activity, Mol. Pharm. 7 (4) (2010) 969 983. [122] S. Bamrungsap, Z. Zhao, T. Chen, L. Wang, C. Li, T. Fu, et al., Nanoparticles as a drug delivery system, Nanomedicine 7 (8) (2012) 1253 1271. [123] N. Zhang, Q.N. Ping, G.H. Huang, W.F. Xu, Investigation of lectin-modified insulin liposomes as carriers for oral administration, Int. J. Pharm. 294 (1 2) (2005) 247 259. [124] M.B. Abud, R.N. Louzada, D.L.C. Isaac, L.G. Souza, R.G. dos Reis, E.M. Lima, et al., In vivo and in vitro toxicity evaluation of liposome-encapsulated sirolimus, Int. J. Retina Vitr. 5 (2019) 35. [125] M. Iman, Z. Huang, S.H. Alavizadeh, F.C. Szoka, M.R. Jaafari, Biodistribution and in vivo antileishmanial activity of 1,2-distigmasterylhemisuccinoyl-sn-glycero-3-phosphocholine liposome-intercalated amphotericin B, Antimicrob. Agents Chemother. 61 (9) (2017) e02525 16. [126] C. Corbo, R. Molinaro, F. Taraballi, N.E. Toledano Furman, M.B. Sherman, A. Parodi, et al., Effects of the protein corona on liposome-liposome and liposome-cell interactions, Int. J. Nanomed. 11 (2016) 3049 3063. [127] G. Lou, G. Anderluzzi, S. Woods, C.W. Roberts, Y. Perrie, A novel microfluidic-based approach to formulate size-tuneable large unilamellar cationic liposomes: formulation, cellular uptake and biodistribution investigations, Eur. J. Pharm. Biopharm. 143 (2019) 51 60. [128] S.-J. Wang, W.-S. Huang, C.-M. Chuang, C.-H. Chang, T.-W. Lee, G. Ting, et al., A phase 0 study of the pharmacokinetics, biodistribution, and dosimetry of 188Re-liposome in patients with metastatic tumors, EJNMMI Res. 9 (1) (2019) 46.

1. Basic principles

C H A P T E R

4 Standard biological assays to estimate nanoparticle toxicity and biodistribution Juhi Shah, Stuti Bhagat and Sanjay Singh Division of Biological and Life Sciences, School of Arts and Sciences, Ahmedabad University, Ahmedabad, Gujarat, India

4.1 Introduction Nanotechnology describes the study of objects that consist at least of one dimension in the size regime of 1100 nm [1,2]. Owing to their small size, nanomaterials display unique physicochemical and optoelectronic properties and large surface area to volume ratio, which makes them significantly different from their corresponding bulk counterparts. The potential applications of nanotechnology in various industries and consumer products has led to the exponential growth in methods and types of nanomaterials. Applications of nanomaterials have been explored in various fields such as medicine [35], industry [69], public health [10,11], and agriculture [1214]. Exposure of nanomaterials is unavoidable in our day to day life as they have been used in many products of direct human use such as cosmetic materials [15], food packaging [16], food additives [17], drug delivery [18], and therapeutics [19]. Therefore the utilization of nanomaterials is expected to expand, which is associated with the exponential rise in a myriad of applications. Considering the increase in the rate of synthesis and the usage of nanomaterials for a variety of purposes, it is imperative to study their potential influence on the biological system. The small size of nanoparticles (NPs) leads to the accumulation or deposition in important organs and subsequently interactions with surrounding biological tissues. NPs can easily cross the plasma membrane of the cells and react with intracellular biomolecules and thus affect the cellular metabolism [20]. It has been suggested that interaction of NPs with cells stimulate pro-oxidant effects which leads to the formation of reactive oxygen species (ROS) and oxidative stress. Workers of the industries are expected to experience a comparatively high amount of NPs exposure

Nanotoxicity DOI: https://doi.org/10.1016/B978-0-12-819943-5.00004-X

71

Copyright © 2020 Elsevier Inc. All rights reserved.

72

4. Standard biological assays to estimate nanoparticle toxicity and biodistribution

during the production of NPs incorporating products [21]. Prevalent usage of NPs and their products could lead to the serious health issues, therefore, extensive assessment of the toxicity of NPs must be performed prior to their routine use. Thus as soon as any novel NPs have developed it is essential to investigate the biocompatibility and possible harmful effects on the host or consumer. Such investigations can be achieved by certain in vitro and in vivo methods of testing. In vitro tests are quick lab scale methods utilizing specific cell culture models, and provide preliminary screening of the nanomaterials tested. However, in vivo methods involve live animal models and provide accurate information about nanomaterials accumulation in different organs as well as possible effects on the various biochemical pathways of tested animals. This chapters discusses about various in vitro and in vivo methods for testing nanomaterials toxicity.

4.2 In vitro methods for determination of nanoparticle toxicity Assessment of NPs toxicity by in vitro methods is an imperative technique because they provide quick results. Additionally, they offer several benefits such as: (1) In vitro experiments are comparatively convenient, fast, and a large number of NPs can be tested simultaneously. Several concentrations of NPs as well as many cell line types can be studied quickly. NP treatment on the cultivated cells can be performed within 23 days. (2) A few factors can be changed in an in vitro study such as exposure of NPs to various cell types, which is also easy to handle [22]. (3) Simple quantification of data is a major benefit in in vitro studies. Particularly, this aspect provides a way for screening studies of NPs of different types, compositions, and sizes [23].

4.2.1 Methods for cytotoxicity assessment The methods of cytotoxicity assessment are based on the functions of cells such as permeability of cell membrane, activity of certain enzymes, production of cellular adenosine triphosphate (ATP), enzyme and nucleotide uptake activity which ultimately could lead to the death due to cell damage [24]. Cytotoxicity assays can be divided into four major categories of assays: (1) dye exclusion assays, (2) colorimetric assays, (3) fluorometric assays, and (4) luminometric assays. 4.2.1.1 Dye exclusion assays This assay provides evaluation of membrane integrity in terms of influx or efflux of certain dyes after NP exposure. In this method, live cells containing intact cellular membrane excludes the dye of interest. Conversely, dye can be easily taken up by the dead cells. A set of such dyes are easily available such as trypan blue, Congo red, eosin, and erythrosine B. 4.2.1.1.1 Trypan blue exclusion assay

This assay is useful for the estimation of viable/nonviable cells present in the suspension. Trypan blue is an anionic dye, therefore, it can only stain the cells with compromised cell

1. Basic principles

4.2 In vitro methods for determination of nanoparticle toxicity

73

membrane, which specifies cell death. Generally, trypan blue is impermeable to the cell membrane in the case of viable cells. Conversely, it is readily up taken by nonviable cells because they have compromised cell membrane. Therefore during light microscopic imaging, only dead cells appears blue because they have taken up the dye. A percentage of viable cells showing clear cytoplasm are calculated against the nonviable cells with blue cytoplasm [25]. Protocol

1. Seed the cells at a density of 5000 cells/well in 96 well plate and allow to adhere for 24 h at 37 C in a 5% CO2 and 95% humidity. Treat cells with different concentrations of NPs and incubate at appropriate culture conditions. 2. Centrifuge the suspension of cells for 5 min at 100 g and decant supernatant followed by addition of 1 mL of PBS in the cell pellet. 3. Mix trypan blue (0.4%) and cell suspension in 1:1 ratio followed by incubation at ambient temperature for 3 min. 4. Cast a drop of mixture containing trypan blue/cells in a hemocytometer and observe under microscopy. Count the stained and unstained cells and estimate viable cells (%) as mentioned below: Viable cells 5

total no of viable cells per mL of aliquote 3 100 total no of cells per mL of aliquote

4.2.1.1.2 Erythrosin B dye exclusion assay

Erythrosin B is also known as a red no. 3 and frequently used as a food additive. It is considered as a safe and vital dye used for measuring live cells. The cell viability assessment follows the similar principle of the trypan blue exclusion assay. Erythrosin B is considered as a substitute of trypan blue dye but with lesser toxicity, however, less popular than trypan blue for staining viable and nonviable cells [26]. 4.2.1.2 Colorimetric assays Colorimetric assays for cell viability assessment depend on the presence of certain functional biochemical markers, and their activity is correlated with the metabolic activity of the cells. With the use of suitable reagents in such assays, color is developed as a result of viable cells that provide the color based determination of cell viability. These assays are extremely easy to perform and the obtained results are quantified by the absorbance intensity recorded by spectrophotometer. Colorimetric assays are suitable for both adherent or suspension cells, easy to handle, and comparably low cost [27]. Although there are several colorimetric tests for determination of cell viability, we will discuss the important and most common types of assays. 4.2.1.2.1 MTT assay

3-(4,5-Dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) assay is a widely used colorimetric cell viability assessment method for the determination of biocompatibility of NPs exposed to various cell lines [28]. MTT dye contains a lipophilic side group and

1. Basic principles

74

4. Standard biological assays to estimate nanoparticle toxicity and biodistribution

net positive charge which allows dye to penetrate readily into viable cells. This assay evaluates the viability of cells via estimation of the function of mitochondria through assessing the activity of enzymes present in mitochondria such as succinate dehydrogenase [29]. Viable cells convert colorless MTT dye into purple color formazan crystals which are soluble in dimethyl sulfoxide (DMSO). The solubilized formazan crystals show absorbance at 570 nm. Dead cells do not convert MTT dye into formazan crystals because of the lack of viable mitochondria. Thus development of color acts as a tool for indication of only viable cells. MTT assay was used to evaluate the viability of Leishmania donovani Ag83 upon liposome encapsulated halofantrine exposure as shown in Fig. 4.3. It was found that cell survival was decreased in time and in concentration dependent manner [30]. It has been suggested that nicotinamide adenine dinucleotide phosphate (NADPH) plays a significant role in the reduction of MTT dye into purple formazan [31]. Although MTT dye-based cell viability estimation is rapid and simple, it still suffers with major limitations such as formation of water insoluble formazan [32]. 4.2.1.2.1.1 Protocol

1. Seed cells at a density of 100010,000 cells/well in a 96 well plate and allow to adhere for 24 h at 37 C in a 5% CO2 and 95% humidity. 2. Treat the cells with desired concentration of NPs and discard the treatment after incubation. 3. Add 100 μL of serum free media and 10 μL MTT (5 mg/mL) in PBS to each well and reincubate it for another 34 h at 37 C. 4. Discard the medium and add 200 μL of DMSO solution to all the well and ensure proper mixing to solubilize purple formazan crystals followed by measuring the absorbance at 570 nm using UVVisible spectrophotometer. 4.2.1.2.2 WST-1 assay

In order to overcome the drawbacks of water insoluble formazan crystals certain water soluble tetrazolium derivatives have been developed. For example, 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium [33], 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide [34,35], and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST) [36] are some of the most commonly used dyes. WST-1 is an assay that helps in determining the relative rate of cell proliferation. The principle of this assay includes the conversion of tetrazolium salt into water soluble formazan crystals through mitochondria dehydrogenase enzymes in the presence of intermediate electron acceptor, such as 1-methoxy-5-methyl-phenazinium methyl sulfate. The biggest advantage of this assay is, it eliminates the interference of phenol red indicator in cell culture medium as the colored dye produce at the end of the reaction is water soluble. 4.2.1.2.3 Lactate dehydrogenase assay

Lactate dehydrogenase (LDH) assay determines the integrity of the cell membrane. Upon rupture of the cell membrane the cytosolic components, including certain important enzymes, are released in the media. One of such key enzymes is LDH. NPs exposure causes the damage of plasma membrane which results in the release of LDH enzyme in

1. Basic principles

4.2 In vitro methods for determination of nanoparticle toxicity

75

FIGURE 4.1 Schematic representation showing principle and working of lactate dehydrogenase (LDH) assay in cells damaged by the exposure of nanoparticles (NPs).

the supernatant of cells [37]. LDH activity is estimated with a coupled enzymatic reaction that involves the conversion of tetrazolium salt, iodonitrotetrazolium, into red color formazan. As represented in Fig. 4.1, LDH enzyme catalyze the conversion of lactate into pyruvate with concomitant interconversion of NAD to NADH/H1. In the subsequent step, the catalyst (diaphorase) transfers H/H1 from NADH/H1 to the tetrazolium salt, which is reduced into red color formazan [38,39]. The obtained red formazan shows maximum absorbance at 492 nm. The amount of red formazan is directly correlated with the amount of LDH enzyme released in cell culture thus indirectly suggests the number of damaged cells. To assess the total release of LDH enzyme from cells, Triton X-100 is utilized as a positive control [40]. One of the key concerns of this method is the interference of serum with LDH enzyme activity which gives high background signal. Therefore, the applicability of this method is limited to the low serum or serum-free conditions [41]. LDH assay was performed on normal human keratinocytes (HaCat) cells to evaluate that exposure of buthionine sulfoximine (BSO) caused the damage to cell membrane integrity. Therefore, it showed increase in LDH release (B1.8 fold) whereas cells pre-exposed to PEGylated cerium oxide nanoparticles (CeNPs) did not show increase in LDH release observed from Fig. 4.3 [42]. Various kits are available to conduct this assay for example, pierce LDH cytotoxicity assay kit from Thermofisher Scientific, LDH assay kit from Abcam, LDH cytotoxicity assay kit from Cayman Chemical. 4.2.1.2.3.1 Protocol

1. Seed cells at the density of 200020,000 cells/well in 96 well plate and incubate at 37 C in 5% CO2 and 95% humidity. 2. Incubate the cells with different concentration of NPs. For positive control, treat cells with lysis buffer provided in kit which demonstrates the maximum LDH release. 3. After incubation, centrifuge the cells at 250g for 4 min and transfer 50 μL of each cell supernatant to a fresh 96 well plate in triplicate wells. 4. Add 50 μL of LDH substrate to the medium and incubate it at room temperature for 1530 min in dark.

1. Basic principles

76

4. Standard biological assays to estimate nanoparticle toxicity and biodistribution

5. Add 50 μL of stop solution to each well and mix properly and record absorbance at 490 nm using UVVisible spectrophotometer. Cytotoxicity ð%Þ 5

Experimental LDH release ðOD490 Þ 3 100 Maximum LDH release ðOD490 Þ

4.2.1.2.4 Neutral red uptake

Neutral red uptake (NRU) is a widely used colorimetric test for determining biocompatibility of nanomaterials. This assay was first established by Borenfruend and Purner [43]. Depending on the viability, cells internalize this supervital dye, which contains a weak positive charge. Due to this charge, the dye penetrates in the cells through the nonpassive diffusion and subsequently accumulates in the lysosomes. Further, the extraction of the dye from the viable cells can be achieved through the washing of cells with acidified ethanol solution followed by measuring the absorbance at 540 nm using a spectrophotometer. Uptake of neutral red dye relies on the ability of cells to maintain the pH of cytoplasm by modulating the ATP synthesis mechanism. At physiological pH, the dye carries a net zero charge, which facilitates it to penetration through the cell membrane. Presence of a proton gradient inside the lysosomes assists to maintain the pH lesser than the cytoplasm. In this case, the dye becomes charged and therefore, lies within the lysosomes [44]. Upon cell death or decrease in pH gradient, the dye cannot be retained in the lysosomes but released in the cytoplasm. Subsequently, absorbance can be recorded at 540 nm using UVVisible spectrophotometer to quantify cell viability. The internalization of neutral red dye by live cells can also be altered by varying the cell surface or lysosomal membranes [45]. Thus this method is a promising approach to discriminate the viable cells from damaged or dead cells [46]. 4.2.1.2.4.1 Protocol

1. Seed cells at a density of 5000 cells/well in a 96 well plate and incubate at 37 C in 5% CO2 and 95% humidity. 2. Add the NP of different concentrations and incubate at appropriate conditions generally for 24 h. 3. Prepare the neutral red working solution (40 μg/mL) by diluting the neutral red stock solution (4 mg/mL) with cell culture medium in 1:100 ratio (12 mL of medium and 0.12 mL of stock solution) and incubate overnight at 37 C. 4. Centrifuge the neutral red medium for B10 min at 600 g in order to remove any precipitated crystals of dye and add 100 μL of neutral red to each of the well following by incubation of plate for 2 h at 37 C. Discard the neutral red medium and wash the cells with PBS. 5. Add 150 μL neutral red destain solution containing (1% acetic acid and 50% ethanol) into each well and shake the plate on shaker for 10 min for the extraction of dye from cells followed by recording the absorbance of neutral red at 540 nm using UVVisible spectrophotometer. 4.2.1.3 Fluorescence-based assays Fluorescence-based methods for cytotoxicity assessment can be performed using a variety of instruments such as fluorescence microscope, fluorescence spectrophotometer, and flow cytometer. Fluorescence-based cytotoxicity assays are suitable for adherent as well as

1. Basic principles

4.2 In vitro methods for determination of nanoparticle toxicity

77

suspension cell lines and also considered to be more sensitive with respect to colorimetric methods [47]. Some commercial kits are also available for these assays and experimental methods are described in the kit itself. Some examples are  in vitro toxicology assay kitresazurin based from Sigma-Aldrich, alamarBlue—rapid and accurate cell health indicator from Life Technologies, CellTiter-Blue cell viability assay from Promega Corporation. In the subsequent section, we will discuss the common methods for cytotoxicity determination utilizing fluorescence method. 4.2.1.3.1 Alamar blue assay

This method is also known as resazurin reduction assay because it involves conversion of nonfluorescent resazurin to the fluorescent dye resorufin with the help of mitochondrial and other enzymes such as diaphorases. Resazurin dye is easily internalized by cells and acts as a redox indicator, which is used for the measurement of cytotoxicity following a protocol similar to tetrazolium dyes-based methods [48]. Viable cells readily reduce the blue colored nonfluorescent resazurin into the intense red fluorescent resorufin, which leads to the overall enhancement of the cell fluorescence [49]. Quantity of fluorescence can be directly proportional to the number of viable cells. Using a fluorimeter (Ex./Em. 560/590 nm) the viability of the cells can be easily estimated. The assay may require a variable incubation period (14 h) which is imperative to achieve the appropriate fluorescent signal corroborating the metabolic activity, density, and the type of medium used for cell cultivation. 4.2.1.3.1.1 Protocol

1. Seed cells at a density of 104 cells/well in a 96 well plate and incubate at 37 C in 5% CO2 and 95% humidity. Treat the cells with different concentrations of NPs and incubate at appropriate conditions. 2. Add 150 μg/mL Alamar blue solution at 10% volume of cell culture medium in each well and incubate for 2 h followed by recording the fluorescence intensity with 590 nm (Ex. 560 nm). 4.2.1.3.2 Protease-based viability assay

Estimation of the activity of conserved and constitutive protease enzymes of live cells is a good tool for estimating the cytotoxicity of nanomaterials. Glycylphenylalanyl-aminofluorocoumarin (GF-AFC) is a cell permeable fluorogenic substrate that specifically investigates the protease activity of live cells [50]. The GF-AFC substrate can easily permeate in viable cells where cytoplasmic aminopeptidase enzyme eliminates the glycine and phenylalanine amino acids to release aminofluorocoumarin (AFC). This biochemical reaction results in the production of fluorescence signal which is proportional to the index of live cells. Due to cell death, protease activity will quickly disappear, which makes this method one of the important biomarkers for the estimation of viable cell population. This approach is offered as a commercial kit CellTiter-Fluor cell viability assay obtained from Promega Corporation [51]. 4.2.1.3.2.1 Protocol

1. Seed cells in 96 well black bottom plate and give treatment with different concentrations of NPs to receive a total volume of 100 μL/well.

1. Basic principles

78

4. Standard biological assays to estimate nanoparticle toxicity and biodistribution

2. Preparation of GF-AFC reagent: Transfer 10 μL of GF-AFC substrate into assay buffer to get 2X reagent. Vortex it for complete solubilization of GF-AFC substrate. 3. Add CellTiter-Fluor reagent in an equal volume (100 μL/well) to all wells, mix it, and then incubate for 30 min at 37 C and measure resulting fluorescence intensity using a fluorimeter at 505 nm (Ex. 390 nm). 4.2.1.4 Luminometric methods for cell viability assessment Luminometric methods are rapid and easy to perform for estimation of cell proliferation and cellular toxicity. These methods are based on the presence of certain key biomolecules in cells exposed to nanomaterials. The changes in the concentration of these key biomolecules are directly proportional to the cell viability. Several commercial kits are available to assess the cell viability, however, one of the most common methods has been described here. 4.2.1.4.1 Adenosine triphosphate based method

ATP is one of the key biomolecules required for the viability of mammalian cells, therefore, it has been considered for developing novel assays to determine cell viability. ATP is present in cells as an energy currency needed for several important processes including synthesis of biomolecules, cell signaling, transport and movement of essential ions. Hence, cellular ATP acts as one of the sensitive end points during the evaluation of biocompatibility of nanomaterials [52,53]. Damage to cells frequently leads to the loss of membrane integrity which results in the drop in ATP synthesis. Subsequently, concomitant effect of endogenous ATPase leads to quick depletion of remaining ATP molecules in cytoplasm. Therefore any drop in cytoplasmic concentration of ATP is correlated with the cytotoxicity of NPs. ATP is dependent on the fluorescence method based on the conversion of luciferin into oxyluciferin by luciferase enzyme. This enzyme catalyzes the reaction in the presence of Mg21 ions and ATP and thus produces luminescence signals. Thus there is a direct correlation between the intensity of luminescence signal and amount of ATP [54] or number of cells [55]. This assay is very sensitive and can be used to detect about 10 cells/well. This method has been used as a high throughput method (in a 1536 well format) for a quick estimation of biocompatibility of variety of nanomaterials. Commercial kits are available to perform this assay. CellTiter-Glo Luminescent Cell Viability Assay from Promega Corporation, ATPLite 1 step, Perkin Elmer and ATP bioluminescent somatic cell assay kit from Sigma-Aldrich. The protocol for this method has been provided below [56]. 4.2.1.4.1.1 Protocol

1. Seed cells in 96 well black bottom plate and give treatment with different concentrations of NPs to receive a total volume of 100 μL/well. 2. Preparation of GF-AFC reagent: Transfer 10 μL of GF-AFC substrate into assay buffer to get 2 3 reagent. Vortex it for complete solubilization of GF-AFC substrate. 3. Add CellTiter-Fluor reagent in an equal volume (100 μL/well) to all wells, mix it, and then incubate for 30 min at 37 C and measure resulting fluorescence intensity using a fluorimeter 390 nm (Ex.) and 505 nm (Em.).

1. Basic principles

79

4.2 In vitro methods for determination of nanoparticle toxicity

4.2.1.5 Cell viability test in real-time Currently, a new method has been established for monitoring the number of live cells in real-time [57]. For this pursuit, an engineered luciferase was acquired from a marine shrimp along with a small molecule pro-substrate. The pro-substrate and luciferase act as reagent, which are added into the cell culture medium. Subsequently, the pro-substrate is converted into a substrate by intracellular reduction in live cells (Fig. 4.2). The substrate utilizes luciferase to produce luminescent signals. However, dead cells are unable to convert the pro-substrate into the substrate, thus no luminescence signal is produced. There are two ways to carry out this assay, continuous read and endpoint measurement. There are assay kits available commercially for these assays from Promega Corporation (RealTime-Glo reagent). 4.2.1.5.1 Protocol

1. Seed 10,000 cells/well in a 96 well plate and give exposure of NPs as per the experimental requirement. 2. Prepare the 2 3 RealTime-GLO reagent by dilution of MT cell viability substrate and NanoLuc enzyme in cell culture media to get a 2X concentration of each reagent. For instance, prepare 1 mL of 2 3 RealTime-Glo reagent, add 2 μL of MT cell viability substrate, (1000 3 ), and 2 μL of NanoLuc enzyme, (1000 3 ) to 996 μL of cell culture media to form the 2 3 RealTime-Glo reagent. Mix well with a vortex mixer. 3. Add equal amount of RealTime-Glo reagent to the cells and incubate the cells for 1060 min in an incubator. Measure luminescence using luminometer. 4.2.1.5.2 Estimation of oxidative stress

It is well documented that exposure of NPs to cells leads to the production of ROS and reactive nitrogen species (RNS) which results in the alteration of cellular redox state [5862]. Generation of cellular ROS due to NPs are considered as the primary outcome of nanotoxicity and it has been ascribed to the existence of pro-oxidant functional groups on the reactive surfaces of NPs or due to NPs cell interaction [63]. The widespread methods for determination of ROS includes 20 ,70 -dichlorodihydrofluorescein-diacetate (H2DCF-DA) assay, lipid peroxidation, and glutathione peroxidase assay. Each of these methods are comprehensively summarized in the following section. FIGURE 4.2 Schematic representation of the principle of luminescence-based assay for detection of live and dead cells utilizing luciferase enzyme.

1. Basic principles

80

4. Standard biological assays to estimate nanoparticle toxicity and biodistribution

4.2.1.5.3 Reactive oxygen species level measurement

20 -70 -Dichlorodihydrofluorescein diacetate (DCFH-DA) is one of the commonly utilized methods for assessing the overall level of ROS and RNS in cells. DCFH-DA is nonfluorescent but is also a highly permeable dye to cells. Within cells, DCFH-DA molecules are cleaved by intracellular esterase at the two ester (diacetate) bonds which results in a polar cell membrane impermeable to the product (H2DCF). This nonfluorescent product accumulates intracellularly and subsequent oxidation by ROS yields the highly fluorescent product DCF [64]. Fluorescence of DCF can be measured using fluorescent spectrophotometer using excitation and emission wavelengths at 485 and 528 nm, respectively [65]. ROS can also observed by confocal microscopic analysis as seen in Fig. 4.3. HaCaT cells exposed to BSO and stained with DCFH-DA show green fluorescence from their cytoplasm indicating the high amount of ROS generation compared to control [42]. PEGylated CeNPs scavenge the ROS generated by BSO as cells pre-exposed to CeNPs did not exhibit any green fluorescence. 4.2.1.5.3.1 Protocol

1. Seed cells at 1.02.0 3 104 cells/well in a 96 well plate and incubate at 37 C in 5% CO2 and in 95% humidity. 2. Treat the cells with NPs for a defined period of time. Decant the medium and wash cells with PBS. 3. Prepare DCFDA stock, 20 mM in DMSO. Stain cells with 20 μM DCFDA prepared in PBS for 30 min in the dark at 37 C and wash cells with PBS. 4. Add 200 μL PBS in each well of assay and record fluorescence intensity of sample at 528 nm (Ex. 485 nm) in fluorescence spectrophotometer. 4.2.1.5.4 Lipid peroxidation

Peroxidation of cellular lipids elucidate the oxidative stress induced by a variety of NPs expose to cells. Lipid peroxidation demonstrates the oxidative degradation of cell membranes instigated through the ROS, which can be quantitatively estimated by evaluating the presence of malondialdehyde (MDA) or other thiobarbituric acid reactive substances (TBARS) [68]. TBARS are represented as the MDA equivalents and normalized to total cellular protein [69]. This assay is carried out in cell or tissue lysates, MDA then forms an adduct with thiobarbituric acid (TBA) which is fluorescent in nature and can be identified with an excitation and emission wavelength at 530 and 550 nm, respectively. 4.2.1.5.4.1 Protocol

1. Seed cells at a density of 1.5 3 106 in a 6 well plate and incubate at 37 C in 5% CO2 and in 95% humidity. 2. Treat the cells with NPs for defined period of time and scrape the cells by adding 1 mL 2.5% trichloroacetic acid. Centrifuge the cells at 11,000 rpm for 35 min for removal of solids. 3. Centrifuge again for 20 min at 11,000 rpm to ensure the removal of cells, NPs and precipitated proteins, and add 3 mL freshly prepared 0.67% (w/v) TBA solution to supernatant. Keep the samples in boiling water bath for 10 min followed by cooling. Measure the fluorescence intensity at 550 nm (Ex.530 nm) in fluorescence spectrophotometer.

1. Basic principles

4.2 In vitro methods for determination of nanoparticle toxicity

81

FIGURE 4.3 Various in vitro assays used for evaluation of nanoparticle (NP) toxicity. MTT assay: Source: Reprinted from S. Bhagat, Y. Parikh, S. Singh, S. Sengupta, A novel nanoliposomal formulation of the FDA approved drug halofantrine causes cell death of Leishmania donovani promastigotes in vitro. Colloids Surf. A Physicochem. Eng. Asp. 582 (2019) 123852 with permission from Elsevier, Copyright r 2019. LDH assay: Source: Reprinted from R. Singh, A.S. Karakoti, W. Self, S. Seal, S. Singh, Redox-sensitive cerium oxide nanoparticles protect human keratinocytes from oxidative stress induced by glutathione depletion. Langmuir 32 (46) (2016) 1220212211 with permission from American Chemical Society, Copyright r 2016. Apoptosis assay: Source: Reprinted from R. Singh, S. Singh, Redox-dependent catalase mimetic cerium oxide-based nanozyme protect human hepatic cells from 3-AT induced acatalasemia. Colloids Surf. B Biointerfaces 175 (2019) 625635 with permission from Elsevier, Copyright r 2019. Micronucleus assay: Source: Reprinted from R. Singh, A.S. Karakoti, W. Self, S. Seal, S. Singh, Redox-sensitive cerium oxide nanoparticles protect human keratinocytes from oxidative stress induced by glutathione depletion. Langmuir 32 (46) (2016) 1220212211 with permission from American Chemical Society, Copyright r 2016. Comet assay: Source: Reprinted from S. Singh, V. D’Britto, A. Prabhune, C. Ramana, A. Dhawan, B. Prasad, Cytotoxic and genotoxic assessment of glycolipid-reduced andcapped gold and silver nanoparticles. N. J. Chem. 34 (2) (2010) 294301 with permission from Royal Society of Chemistry, Copyright r 2010. ROS estimation assay: Source: Reprinted from R. Singh, A.S. Karakoti, W. Self, S. Seal, S. Singh, Redox-sensitive cerium oxide nanoparticles protect human keratinocytes from oxidative stress induced by glutathione depletion. Langmuir 32 (46) (2016) 1220212211 with permission from American Chemical Society, Copyright r 2016. 4.2.1.5.5 Glutathione estimation

Glutathione (GSH) is a natural antioxidant molecule present in cells. GSH presents in two forms: the reduced sulfhydryl from (GSH) and the oxidized glutathione disulfide

1. Basic principles

82

4. Standard biological assays to estimate nanoparticle toxicity and biodistribution

(GSSG) form. Oxidative stress leads to a deep impact on the thiol balance of cells, which can result in a lower GSH/GSSG ratio. GSH assay is dependent on a reaction between GSH with 5,50 -dithiobis (2-nitro-benzoic acid) (DTNB) (also known as an Ellman’s reagent) that generates a TNB chromophore, which gives absorbance maxima at 412 nm due to oxidized glutathioneTNB adduct (GS-TNB). This adduct is proportional to the quantity of GSH present in the sample. Thus the amount of TNB formation estimated at 412 nm is concomitant to the amount of GSH present in the given sample. The disulfide product (GS-TNB) is reduced through glutathione reductase (GR) enzyme in the presence of NADPH in GSH, which is recycled back into the cycle. GR leads to the reduction of GSSG (oxidized) form into 2GSH (reduced). The amount of glutathione estimated indicates the sum of oxidized and reduced glutathione in sample. Therefore GSH total 5 [GSH] 1 2 3 [GSSG]. The rate of change in absorbance (Δ412 nm/min) is made to be linear for the convenience and consistency of measurement. Standard curve using GSH standards has been performed that helps in the determination of unknown sample concentration [70]. 4.2.1.5.5.1 Protocol

1. Seed cells 0.20.4 3 106 cells/well in 6 well plate and incubate it for 37 C in 5% CO2 and in 95% humidity. Treat the cells with NPs for desire period of time and harvest the cells and resuspend in 1 mL PBS. 2. Centrifuge lysate at 1000 g for 30 min at 4 C. Remove the supernatant and add 1 mL of PBS to the cell pellet. Centrifuge again and decant the supernatant. 3. Resuspend the cells in 1 mL of extraction buffer (0.1% Triton-X a 0.6% sulfosalicyclic acid in potassium phosphate EDTA buffer (KPE). Sonicate the suspension and add 20 μL of KPE, 20 μL of samples and standards in each well of 96 well plate. 4. Mix equal volumes of freshly prepared DTNB and GR solution together and add 120 μL to each well. 5. Allow reaction to stand for 30 s for the conversion of GSSG to GSH and then add 60 μL of β-NADPH. Measure the absorbance at 412 nm in UVVisible spectrophotometer. 6. Assess the actual total GSH concentration in the samples by using linear regression to estimate the values gained via a standard curve. Indicate the total GSH concentration in μM or nM/mg protein or nM per 106 cells. 4.2.1.6 Apoptosis based assays Apoptosis and necrosis are two different forms of cell death that occurs during normal and diseased conditions [71]. Apoptosis is a programmed cell death that takes place in the normal or pathological conditions including embryogenesis and immune system regulation. It can be caused by different chemical and physical stimuli [72]. Apoptosis is one of the crucial markers studied during the in vitro safety assessment of NPs. Necrosis is a type of cell death that can be considered as a swelling and rupture of intracellular organelles, ultimately resulting in the plasma membrane disruption. It is a passive and accidental form of cell death [73]. Oxidative stress generated by NPs that leads to cell death is associated with the intrinsic apoptotic network. Many studies indicate that apoptosis is induced by NPs [74]. Studies demonstrate that silver NPs coated with fruit extract Rubus fairholmianus induce the apoptosis in MCF-7 breast cancer cells through mitochondrial mediated intrinsic apoptotic

1. Basic principles

4.2 In vitro methods for determination of nanoparticle toxicity

83

pathway [75]. Polyethylene glycol modified titanium oxide NPs (TiO2 NPs) induce apoptosis in epithelial cell line (NCL-H292) which is evident by elevated activity of caspase 3 [76]. NPs stimulate intrinsic and extrinsic apoptosis pathways. Intrinsic pathways are regulated by the mitochondria and proteins present in the mitochondria. The overall pathway is regulated by the B cell lymphoma 2 (BCL-2) family proteins [77]. In the case of the extrinsic pathway, it includes the activation of death receptors on the cell surface, activation of caspase 8 and activation of caspase 3, ultimately resulting in cell death. When TiO2 NPs is exposed in the human bronchiole cells (BEAS-2B) it activates the extrinsic apoptotic pathways. It is evident by the change in the activity of BCL-2 protein, BCL-2 associated X protein, cytochrome c, and p53 protein [78]. There are several assays for the assessment of apoptosis including Annexin-V fluorescein isothiocyanate/propidium iodide (Annexin-V FITC/PI) assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. 4.2.1.6.1 Annexin-V FITC/propidium Iodide assay

Annexin-V and propidium Iodide (PI) both are cell death markers utilized for the measurement of toxicity induced due to NPs exposure. The appearance of phosphatidylserine (PS) (normally present within the plasma membrane) on the surface of cells is an early event of apoptosis. When cells undergo apoptosis, phospholipid PS is flipped from the cytoplasmic face of plasma membrane to the cell surface, thus revealing PS to the outer environment of the cell. Annexin-V protein has a strong affinity for PS in the presence of calcium ions [79]. Exposed PS has been detected by Annexin-V FITC/PI which is a useful probe for the discrimination between healthy and dead cells [80]. As early apoptotic and healthy cells have undamaged plasma membrane, it excludes PI stain. In contrast, late apoptotic and necrotic cells have compromised integrity of plasma and nuclear membrane; their plasma membrane is permeable to PI that intercalates with nucleic acid to give red fluorescence [8183]. Therefore, early apoptotic cells are Annexin-V positive and PI negative whereas late apoptotic cells and necrotic cells are Annexin-V/PI double positive [84]. Annexin-V/PI assay was performed in normal human liver (WRL-68) cells as shown in Fig. 4.3. Exposure of a catalase inhibitor 3-amino-1,2,4-triazole (3-AT) on WRL-68 cells indicates that cells undergo early apoptosis which is evident by the increase in green fluorescence of fluorescein isothiocyanate (FITC) (5.5 fold increase) [66]. Pre-incubation of cells with CeNPs with a concentration of 100 and 150 μM results in significantly lower populations of apoptotic cells exposed to 3-AT. 4.2.1.6.2 TUNEL assay

Morphological changes has occurred as an outcome of apoptosis. Change in nucleus during apoptosis is based on the DNA cleavage into specific DNA fragments which can be detectable through TUNEL assay [85]. DNA damage is not distinct only in apoptosis but it can be part of necrosis also. Therefore, TUNEL assay can give false positive staining of necrotic cells and this limitation is eliminated in Annexin-V FITC/PI assay [72]. It is a widely used assay for the detection of DNA damage [86]. TUNEL assay has a unique feature as it tags the ends of DNA which are fragmented through endonucleases due to apoptosis, causing into biotinylated dUTP at the 30 -OH group. Identification of these groups is done using the streptavidin-horseradish peroxidase and a diaminobenzidine chromogen

1. Basic principles

84

4. Standard biological assays to estimate nanoparticle toxicity and biodistribution

through light microscopy. A fluorescent dye can be utilized for the labeling of incorporated dUTP nucleotides and observed under fluorescence microscopy [87].

4.2.2 Methods for determining the genotoxicity potential of nanoparticles Nanomaterials exhibit distinct physiochemical characteristics due to their nanometer dimension [88], vast surface area [89], tunable chemical composition, surface modification [90], and shape [91,92]. These characteristics of NPs may assist them to directly interact with biomolecules and other key components, which may lead to the change in cell signaling pattern and thus cell function. Although complex, there is limited clarity about the interaction of NPs with the lipid component of the cell membrane and their consequent intracellular transport. It has shown that NPs can easily pass the cell membrane and internalize into the cells through different endocytosis pathways [91,93]. These characteristics are also reported to be dependent on the surface properties of NPs. Once internalized, NPs could interact with the components of the cytoplasm and thus cause damage to the genetic material. Therefore, it is essential to study the direct effects of NPs on the genetic materials which may provide primary information about the genotoxic traits of nanomaterials. There have been several sensitive methods that are being used to study the genotoxic potential of NPs. The following section discusses about some of the common methods practiced in laboratories to determine the genotoxicity caused by NPs. 4.2.2.1 Micronucleus formation Micronuclei (MNi) originates from the chromosomal fragments or whole chromosomes that lag behind the anaphase during the cell division [94]. In vitro micronucleus assay formation in cells occurs due to the gross chromosome damage induced by the NPs exposure. In this method, scoring of MNi is performed. MNi are considered as miniature nuclei (as a result from lagging acentric chromosomal fragments or lagging whole chromosomes), which are unable to get integrated in the daughter nuclei during cell division. Therefore, these MNi are left in the cytoplasm as a broken piece of nuclei [95]. MNi produced from dividing cells either acquire chromosome breaks without centromeres and/or whole chromosome which are incapable to move towards the spindle poles during mitosis. At the telophase stage, a nuclear envelope develops around the lagging chromosome and fragments, which then uncoil and gradually mimic the morphology of interphase nuclei with the exception that they are smaller than main nuclei in the cells, henceforth termed as MNi. There are two common methods to score MNi: 4.2.2.2 Cytokinesis block micronucleus assay As mentioned, MNi assay is the indicator for the evaluation of genomic instability, DNA damage and mammalian chromosome abnormalities. Cytokinesis block micronucleus (CBMN) assay is dependent upon the principle of cytokinesis interfaces by cytochalasin B which works as a microtubule inhibitor [96]. The advantage of this method includes the scoring of MNi in the cells which have completed only one cell division. Another benefit includes estimation of rate and progression of nuclear division in a dividing cell population. This can be obtained by measuring the frequency of mononucleate, binucleate, and multinucleate cells

1. Basic principles

4.3 In vivo bio-distribution and toxicity of nanoparticles

85

after a defined time point, following the addition of cytochalasin B. The drawbacks of CBMN include the labor intensive process, slow, assay completion that requires 12 weeks, and only a small number of cells can be measured B1000 cells and two dimensional visualization which may lead to false negative results. 4.2.2.3 Flow micronucleus assay MNi formation occurs during the metaphase/anaphase transition of mitosis and can be analyzed using a flow cytometer [97]. Dissimilar from CBMN cells are analyzed after anaphase of cell division in flow cytometer. The discrimination between the formed MNi and main nucleus is dependent upon the size variation of MNi and main nucleus [98]. A log scale plot can be used to register the DNA and side scatter signal. G1 phase nuclei can be sorted around 20,000 cells and MNi can be calculated in the 5% to 40% region of the DNA content of G1 phase nuclei [99]. As indicated in Fig. 4.3 BSO exposed in HaCaT cells exhibited the MNi formation. In contrast, cells pre-exposed to CeNPs escape the MNi formation [42]. 4.2.2.4 Comet assay The term, comet assay, was introduced by Ostling and Johnson in 1984. It is a modified version of one of the earlier methods used for identification of DNA damage [100] however, subsequently revised by Singh et al. in 1988 [101]. This assay is also known as single cell gel electrophoresis assay and facilitates the identification of double as well as single DNA strand breaks and alkaline labile sites. The aforementioned method relies on the norm that DNA with strand breaks tend to migrate more quickly in agarose gel with respect to intact DNA under the influence of electric field which gives a comet-like structure. Length of comet tail indicates the index of DNA damage which can be visualized if NPs cause genetic material damage [102]. In comet assay, the intact DNA nucleoid part is also known as “head,” whereas the trailing DNA damage streak is known as the “tail” [103]. Olive tail moment can be also be calculated from the quantity of DNA present in the tail and the mean distance of migration in the tail [102]. Comet assay was performed to study the effect of sophorolipid (OA-SL) molecules on the DNA of HepG2 cells. Cells treated with 0. 60, 6, 0.6, and 0.0006 mg/mL OA-SL did not display any increase in the tail length with respect to control as demonstrated in Fig. 4.3 [67]. Table 4.1 enlists some of the common in vitro assays for evaluating the toxicity of NPs.

4.3 In vivo bio-distribution and toxicity of nanoparticles According to the National Institute of Health (NIH), “Nanomedicine is clinical application to nanotechnology, which refers to highly specific medical intervention of nano scale material for screening, diagnosis, and treatment of biological systems” [113]. The distinctive properties of NPs make them strong agents for various applications in medicines such as in vivo targeted imaging, in vitro diagnostic assays, drug delivery, and therapy. The administered NPs are reported to distribute themselves in various organs of the body. Biodistribution of NPs is influenced by several factors such as surface properties, the physical environment where NPs are introduced, and the route of administration [114]. There are many routes for administering NPs in in vivo animal models including intravenous (IV)

1. Basic principles

86

4. Standard biological assays to estimate nanoparticle toxicity and biodistribution

TABLE 4.1 List of in vitro methods for assessment of nanoparticle (NP) toxicity. Nanoparticle type

Assay

Dosage

Results

References

Albumin coated copper NPs

MTT

400 μM

NPs inhibited the proliferation of breast cancer MDA-MB-231 cells while the same concentration did not inhibit the normal breast MCF-10A cells proliferation

Ferromagnetic NPs

NRU

0.322 mg/mL Growth of mouse fibroblast L929 cells was inhibited by a very low amount of NPs

[105]

Cerium oxide LDH nanoparticles (CeNPs) (14)

100 μM

WRL-68 treated with 3-AT (100 mM) displayed high toxicity. Cells pre-treated with CeNPs showed protection upon 3-AT exposure.

[66]

Titanium oxide nanoparticles (TiO2 NPs)

GSH

50 μg/mL

Concentration of GSH was found significantly lower in NPs treated gill cell line from wallago attu (WAG) in a dose dependent manner. NPs exposure (50 μg/ mL) in cells leads to GSH depletion (B176.16%)

[106]

Zinc oxide nanoparticles (ZnO-NPs)

DCFDA

50 μg/mL

ROS level was found to be elevated in human alveolar fibroblast cells (MRC-5) and it was associated with increased expression of endoplasmic reticulum related stress response gene

[107]

Multiwalled carbon nanotubes (MWCNT)

Comet

400 μg/mL

This dose induced significant enhancement of tail DNA [16.39%] compared to control [2.02%] after 48 h of exposure which indicates genotoxicity.

[108]

Nickel oxide NPs (NiO NPs)

Annexin-V FITC/PI

300 μg/mL

Kidney epithelial cells (NRK-52E) exposed to NiO NPs at a highest concentration of 300 μg/mL leads to 75% of total cell death in which apoptosis contributes 62.5% whereas necrosis was 14.4%.

[109]

OA-SL reduced gold and silver nanoparticles (AuNPs and AgNPs)

MTT

51.4 μg/mL (AuNPs)

OA-SL-AuNPs exposed in cancerous liver cells (HepG2) at a concentration of 0.0005, 0.51, and 5.14 μg/mL did not demonstrate any toxicity, at a high dose (51.4 μg/mL) cell viability decreased up to B72%. In case of OA-SL-AgNPs, lower concentration of (0.0001, 0.12, and 1.27 mg/mL) B90% cell survival was observed whereas in high dose of NPs (12.75 mg/mL) 58% cell survival was found.

[67]

Ironplatinum (FePt) alloy NPs

MTT

150 μM

FePt alloy NPs exposed in WRL-68 were found to biocompatible up to 150 μM concentration

[110]

[104]

(Continued)

1. Basic principles

87

4.3 In vivo bio-distribution and toxicity of nanoparticles

TABLE 4.1 (Continued) Nanoparticle type

Assay

Dosage

Results

Multifunctional Micronucleus 1.5 μg/mL nanoliposomes (MN) containing CeNPs and PTEN plasmid Polyoxyethylene cholesteryl ether (ChEO5) coated copper (Cu) platinum (Pt) nanoalloys

DCFH-DA

100 μM

References

MN consists of PTEN plasmid (1.5 μg/mL) exposed to prostate cancer cells which demonstrates the increase in micronuclei formation

[111]

CuPt nanoalloys exposed to normal WRL68 did not show green fluorescent upto 100 μM concentration indicating that they are safe and can utilized for biomedical application

[112]

NRU, Neutral red uptake; LDH, lactate dehydrogenase; GSH, glutathione, DCFDA, dichlorodihydrofluorescein-diacetate; Annexin-V FITC/PI, Annexin-V fluorescein isothiocyanate/propidium iodide; DCFH-DA, 20 -70 -dichlorodihydrofluorescein diacetate; OA-SL, sophorolipid; PTEN, phosphatase and tensin homolog.

FIGURE 4.4 Diagrammatic representation of the different techniques to measure bio-distribution of nanoparticles (NPs) under in vivo experimental models.

injection, oral, pulmonary, sub cutaneous, and dermal administration. Among these, IV is the most commonly used method because nanomedicines are directly injected into the blood of the tested animal model [115]. In vivo bio-distribution of certain NPs can be easily identified by imaging the whole animal in computed tomography (CT), nuclear medicine imaging (NMI), magnetic resonance imaging (MRI) or by harvested tissues (Fig. 4.4). Bio-distribution of NPs from harvested tissue can be visualized by electron microscopy, liquid scintillation counting, optical imaging, and histology, etc.

4.3.1 Quantitation and bio-distribution of nanoparticles from tissues Histology is qualitative, invasive, and a very common technique to measure the biodistribution of nanomaterials in in vivo animal models. This technique predominantly

1. Basic principles

88

4. Standard biological assays to estimate nanoparticle toxicity and biodistribution

involves microscopy including light and fluorescence microscopy. In a typical method, tissues or organs of the nanomaterials exposed animals are harvested at desired time points. Subsequently, tissues are fixed by either conventional paraffin embedded processing or cryostat processing method. In conventional paraffin method the tissue is preserved or fixed by using neutral buffer formalin which is equivalent to 4% paraformaldehyde. Formalinbased fixation takes about 48 h for dehydration. Due to fixation, cells do not degrade and maintain the structural integrity (autolysis and putrefaction). Thus, fixation is a necessary step prior to embedding and sectioning of tissues. Sometimes alcohol-based dehydration is also used such as in the case of BrdU staining. After fixation, tissues can be stored at 4 C in 70% ethanol. Subsequently, a gradient of alcohol wash is given to remove water and xylene is used as a clearing agent to remove ethanol and lipids from the samples. The clearing agent allows infiltration of paraffin wax in the tissues [116]. The paraffin wax solidifies at room temperature however, melts at a higher temperature (65 C to 70 C). Tissue embedded in paraffin is sectioned by microtome. Paraffin embedding process cannot be used for micelles, solid lipid NPs, and liposome because of the use of lipid solubilizing solvents such as ethanol and xylene. Therefore, these NPs containing tissues are processed by cryostat freezing method [116]. In this method, microtome mounted in refrigeration device (cryostat) is used. In cryostat, unfixed or fixed frozen tissues can be sectioned and mounted on glass slides. These samples can be further stained to develop visualization of the cells’ anatomy and NP’s bio-distribution. Various histological stains are used to distinguish between NPs and biological structures such as immuno-histochemicals, hematoxylin eosin (H&E) stain, Mallory’s stain, toluidine blue, Masson’s stain, etc. Among these, H&E stain is the most commonly used [116] which stains nuclei (purple) and cytoplasmic organelles (pink) within cells. It is very important to choose the stain which only stains biological moieties and does not affect the NPs. Conventional staining methods are used to measure bio-distribution of certain types of NPs. For an example, NPs .200 nm in size are visualized based on resolution limitations of light microscope [117]. It has been reported that SPIO and USPIO (small and ultra-small superparamagnetic iron oxide, respectively) can be visualized by light microscope [118,119]. Iron positive area can be measured by paraffin-embedded method whereas organs can be differentiated by Prussian blue dye [120]. Cationic stain, Alcian blue, have been used to stain negatively charged sulfates in organic dendritic polyglycerol sulfte NPs [121]. The bio-distribution of different NPs such as polymeric micelles, [122] and silica NPs [123] which are tagged with fluorophore or NPs could be visualized in tissue sections by fluorescence microscopy.

4.3.2 Electron microscopy Electron microscopic analysis is a semi-quantitative method that provides detailed information of NPs and their cellular association in tissues under very high magnification. For electron microscopy analysis, tissue samples are harvested at determined time points and fixed with formalin. Afterwards, samples undergo serial dehydration by using gradients of alcohol and propylene oxide to remove water prior to embed in resin and sectioned by microtome followed by 4% uranyl acetate or lead citrate staining [124]. Uranyl acetate binds with proteins and lipids with their sialic acid (carboxyl) groups such as ganglioside and glycoproteins which produce contrast image. Lead citrate enhances the contrast of the image for cytoskeleton, lipid membranes, ribosomes, and other compartments of the cytoplasm. For lead citrate

1. Basic principles

4.3 In vivo bio-distribution and toxicity of nanoparticles

89

staining, samples are fixed with osmium tetroxide. The enhancement of contrast depends on interaction with reduced osmium. Osmium allows lead ions to attach with polar molecule groups [124]. Electron microscopy is mostly used to determine the NP’s size, morphology, and dispersity. However, limited studies have used electron microscopy to show in vivo biodistribution of NPs [125]. Jong et al. [126] reported 10 and 250 nm AuNPs bio-distribution in rat tissues by using transmission electron microscope (TEM). Post treatment tissue exposed to AuNPs was harvested and ultrathin sections (5070 nm) were prepared by microtome followed by staining with uranyl acetate and lead citrate. AuNPs of 10 nm size were found at reticuloendothelial system (RES) of phagocytic cells however, AuNPs of 250 nm were not detected in the investigated organs. One of the major drawbacks of this technique is that electron microscopy cannot image large tissue sections compared to standard histology method. A single microtome section covers about 110 μm3 area of the tissue. Additionally, only 50150 nm thickness samples can be used for analysis, which may affect the results as all the sections cannot be examined. Recently, researchers have used scanning transmission electron microscope (STEM) for analysis of bio-distribution of NPs in tissues. STEM works on the combined principle of TEM and scanning electron microscope (SEM) [127]. In STEM, very thin sections of tissue is needed because a very fine tuned electron beam scans the sample in a raster pattern. STEM can also analyze scattered beam electrons, characteristic X-rays, secondary electrons, and electron energy loss spectra, which is not possible with simple electron microscopy. Also, spatial resolution is also improved than SEM. For an example, Kempen et al. [128] used STEM to analyze bio-distribution of polyethylene glycol coated Raman-active-silicagold-NPs (PEG-RSi AuNPs) in mouse liver tissue. Tissue samples were fixed and stained by osmium tetroxide and subsequently with uranyl acetate followed by sectioning (150 nm) by an ultra-microtome. This study showed that intravenously injected NPs accumulated in liver tissues whereas orally administered NPs remained within colon.

4.3.3 Liquid scintillation counting The principle of liquid scintillation counting (LSC) is based on the quantification of radioactivity of low energy radioisotopes which are majorly released by β or α-emitters. LSC detection is a standard laboratory method where a specific cocktail is used as scintillator [129]. LSC cocktail contains aromatic solvents and scintillators such as oxazole, 2,5-diphenyloxazole (PPO), polyphenyl hydrocarbons, ρ-terphenyl, butyl-PBD(2-(4-tertbutylphenyl)5-(4-phenylphenyl)-1,3,4-oxadiazole), etc. [130]. Scintillators are also known as “fluorophores” or “phosphors.” LSC depends on the released energy from radioactive decay (from β or α particle). This energy further excites the aromatic molecule which is present in LSC cocktail and transfers it to the scintillator. Subsequently, the excited scintillator produces electrons, which further decay to the ground state and emit light detected by photomultiplier tube (PMT) of the LSC. This technique is very sensitive, specific, and quantitatively measures the bio-distribution of NPs in different tissues/organs [131]. In vivo bio-distribution of NPs can be measured quantitatively by LSC using tagged or labeled NPs with isotopic marker prior to administration in test animals. For an example,

1. Basic principles

90

4. Standard biological assays to estimate nanoparticle toxicity and biodistribution

Bauhinia purpurea agglutinin and polyethylene coated liposome (BPA-PEG-LP) encapsulating doxorubicin (BPA-PEG-LPDOX) NPs were radiolabeled with cholesteryl hexadecyl ether (3H-CHE) to investigate the bio-distribution when administered intravenously in mice [132]. 3H-CHE is a commonly used fluorophore to investigate the bio-distribution of NPs. 3H-CHE has resistance to degradation once attached with liposomes or lipid. BPAPEG-LPDOX showed suppression of the growth of prostate cancer cells whereas BPAPEG-LP did not show any toxicity to cells even after internalization due to the absence of ThomsenFriedenreich antigen. Subsequently, organs and tissues were harvested at different time points and LSC samples were prepared. Liquid samples with less amount of proteinaceous material such as plasma, serum, and urine etc. can be directly mixed with scintillator. However, samples with higher protein concentration must be first solubilized followed by mixing with scintillator. There are different types of solubilizers such as Soluene-350, SOLVABLE, Hyamine, and Hydroxide 10-X etc. which are used for the estimation of NPs in a harvested organ. Radioactivity in LSC is measured in form of disintegrations per min (DPM) of radiotracer material. The total radioactivity can be converted from DPM to the number of Becquerel (Bq, SI derived unit for radioactivity) per gram of tissue using following conversation: 1 Bq 5 60 DPM [131].

4.3.4 Quantification of nanoparticles by drug loading and release This is an indirect technique to quantify the bio-distribution of NPs loaded with drugs. NPs accumulate in specific tissues (depending on route of administration) where NPs release their cargo (drug). NPs localization in specific tissue depends on various factors such as RES, blood clearance and renal filtration, penetration through dense stroma to the microenvironment of specific tissue, or tumor. As a result of these effects, NPs may remain in specific tissue for extended time and release the drug to induce pharmacological effect [133] (Fig. 4.5). Post NPs administration blood samples can be drawn from tested animal models and analyzed for different parameters such as total blood count, lipid profile (cholesterol and triglycerides fats), etc. To check the bio-distribution of NPs, blood can also be collected from retro orbital site or by cardiac puncture. Subsequently, the blood sample is centrifuged at 5000 rpm for 10 min to obtain plasma protein which undergo precipitation by using 1:1 ratio of methanol and acetonitrile followed by centrifugation at 5000 rpm for 10 min. The supernatant is then processed through high performance liquid chromatography (HPLC) or mass spectroscopy (MS) to quantify the NPs concentration [133,134]. Organs/tissues are harvested at specific time points and grinded by different procedures such as blending, sonication, homogenization, or sieving. Afterwards, particulates are removed from the biological sample by using centrifugation, filtration, or solid phase extraction method. The supernatant contains drug and other biological compounds which are further purified and then processed through HPLC or MS to measure the drug concentration [135,136]. The extraction of drugs from samples majorly depends on physicochemical property of NPs as well the nature of drugs such as hydrophobicity/hydrophilicity, ionization, solubility molecular weight, and partition coefficient [136]. This technique is widely used for quantitative determination of NP concentration in in vivo model systems. Xu and Meng [137] assessed the bio-distribution of paclitaxel loaded stealth liposomes in SpragueDawley rats. Animals were euthanized at 6, 12, 24,

1. Basic principles

4.3 In vivo bio-distribution and toxicity of nanoparticles

91

FIGURE 4.5 Schematic diagram showing systemic and intra-tumoral biodistribution of nanoparticles (NPs).

and 48 h of post administration of NPs followed by collection of tumor, liver, spleen, and blood plasma for HPLC analysis. Results indicated that compared to free paclitaxel, paclitaxel loaded liposome had long circulation half time. A major benefit of this technique is that it provides a quantitative measurement of accumulation of NPs. If the drug is prematurely dissociated from NPs, false results would be obtained. The accurate measurement of drug concentration also depends on the efficient purification and extraction of drug from the tissue or organs. This technique is laborious and time consuming and realtime bio-distribution monitoring is not possible. This technique can be used to support bio-distribution results attained from qualitative technique [138].

4.3.5 Whole body imaging-based methods for assessment of nanoparticle toxicity and bio-distribution 4.3.5.1 In vivo optical imaging Optical imaging technique provides qualitative information about the bio-distribution of NPs by measuring the bioluminescence or fluorescence intensity of NPs from the organs/ tissues or whole animal imaging. In vivo Imaging System (IVIS) and Kodak in-vivo FX imaging station are some of the common tools that are being used to analyze bio-distribution of NPs. These tools have the potential to study the bio-distribution of NPs in real-time and within live animals. The in vivo optical imaging system is noninvasive and relatively simple to operate. Mostly, fluorescence-based detection method is used to evaluate the bio-distribution of NPs. Therefore, NPs must have the intrinsic fluorescence property or be conjugated with a fluorescent protein or dye [such as green fluorescence protein (GFP), red fluorescence protein (RFP), and Cy5], and conjugated polymers [139]. These fluorophores are tagged or encapsulated in NPs which can be excited at a particular wavelength so that the emission is obtained B 600 to 1000 nm [red or near infrared (IR)], which helps to optimize the fluorescence sensitivity in the in vivo model system. In a typical imaging system,

1. Basic principles

92

4. Standard biological assays to estimate nanoparticle toxicity and biodistribution

the sample (whole animal or harvested organ/tissue) is placed in an imaging chamber, subsequently excited by suitable light source. The emitted fluorescence is recorded by a charge coupled device camera which converts the light signals into electrical signals. The collected signals will give a three dimensional (3D) tomographic image of bio-distribution of the fluorescence or bioluminescence exhibiting NPs in animals or tissues. IVIS is commonly used in investigation of tumor dynamic, morphology, and biodistribution of NPs at different time points. There are several fluorescently labeled NPs such as carbon nanotubes [140], lipid-based NPs [141], polymer-based NPs [142], silicon NPs [143], quantum dots (QDs) [144], metal NPs [145], etc used for imaging of in vivo models through IVIS. Li et al. [146] have synthesized arginine-glycine-aspartic acid peptide labeled CdSe/ZnS quantum dots (RGD-QDs) and used them for photodynamic therapy of pancreatic neoplasm. QDs are formulated with core-shell structure which allows the imaging in the near IR region. The bio-distribution of RGD-QDs was studied at a different time point (1, 3, 5, and 24 h) in BALB/C female mice using IVIS Lumina XRMS III imaging system (PerkinElmer). It was found that the accumulation of RGD-QDs was significantly higher in tumor region. Optical imaging of harvested organs such as liver, heart, lungs, and kidneys did not show any accumulation of RGD-QDs. The results attained from IVIS were also comparable with the quantitative data of inductively-coupled mass spectrometry. In vivo optical imaging is a direct and easy technique to conduct, and also does not require exposure of ionizing radiations. Further, imaging of same group of animals can be performed at different time points to assess the real-time bio-distribution of NPs, thus reducing the number of animals used for the experiment [138]. Since this is a qualitative technique, it is not necessary that the intensity of fluorescence or bioluminescence can be co-related with the number of NPs present in the organs/tissues. This technique can provide qualitative information about the accumulated NPs in an organ or tissue. Further, this method does not provide any information about the distribution of NPs in animal tissues. Some tissues show auto fluorescence which causes interference during the analysis of data. Another issue could be the photo-bleaching of fluorophore which can also lead to negative results. Photo-bleaching issues can be avoided by using fluorophores or NPs having a high signal to background ratio [147]. 4.3.5.2 Computed tomography CT is a radiological technique in which a 3D and cross sectional (tomographic) image of animal or tissue is produced by using X-rays. In this technique, NP exposed tissues form ionizing radiation by absorbing irradiated X-rays with the wavelength of B0.01 to 10 nm. The CT scan consists of a rotating X-ray unit, detector, collimators (to protect detector from scattered X-rays), image building system, and filters. The X-ray’s tube of CT scan is composed of tungsten alloy (anode) whereas cathode is placed within a vacuum which accelerates electron at high voltage. These electrons interact with the electron of tungsten nuclei (anode) and thus cause emission of X-rays. The emitted X-rays are then passed through the animal or tissue which leads to the reduction in the intensity of X-rays due to absorption or scattering. These signals are collected by the detector that is located directly opposite to the X-ray source. As the X-rays leave the animal or tissues, they are identified by the detectors as a series of projections. In CT scanner, the X-ray tube and detectors move together on a circular axis around the sample which allows a complete dataset of

1. Basic principles

4.3 In vivo bio-distribution and toxicity of nanoparticles

93

projections to be acquired over 360 degrees. CT data analyzing computer uses sophisticated mathematical techniques to produce a 3D reconstruction of the scanned data. Thickness and density play a major role to obtain the final contrast image. Bones are highly dense in the body therefore, they are easily imaged through CT scan however, the soft tissues are challenging to obtain a contrast image from. Therefore, the contrast imaging agent is used to increase CT sensitivity and enhance the difference between different tissues. As the contrast agent iodinated or barium-based contrast media are commonly used because they contain a high atomic number (higher number of electrons) and therefore absorb external X-rays efficiently. Iodine based media are commonly used for vascular imaging whereas barium based media used for gastrointestinal tract [138]. CT scanner can be used for in vivo investigation of NP bio-distribution in real-time. The bio-distribution of different NPs such as liposomes [148], micelles [149], nano-emulsions, [150] metal NPs (gold, platinum, palladium, tungsten, gadolinium) [151], iron oxide NPs [152], and dendrimers [153] have been studied by several groups. To visualize the sample contrast agent (iodine and bismuth based agents) are incorporated in the core or on the surface of the NPs. Engudar et al. [154] have synthesized PEGlyated liposome with a remote loading of 124I (radioiodine) for evaluation of bio-distribution in murine tumor model. The radiolabeling efficiency of this liposome was 77 6 1% used for imaging of CT26 murine colon cancer model. Interestingly, significantly lower accumulation of these liposomes was observed in the spleen, kidney, and liver. Recently, AuNPs have gained a lot of interest as a contrast agent as well as for their clinical ability and cost effectiveness. AuNPs have found successful in vivo experiments in a variety of cell types, including stem cells and immune cells, without affecting their therapeutic efficacy [155]. Noninvasive, quantitative, and longitudinal cell tracking with high sensitivity have also been reported with the use of AuNPs. Utilizing CT, bio-distribution of NPs can be visualized in real time at different time points with the same sample. This reduces the usage of the number of animals for the experiments. CT imaging give qualitative measurement of bio-distribution of NPs and only detect NPs at organ or tissue level. Additionally, CT requires exposure of contrasting agent to increase sensitivity. Information regarding cellular accumulation of NPs cannot be analyzed by a CT scan [138]. 4.3.5.3 Magnetic resonance imaging MRI is another scanning technique to produce 3D anatomical images with fine details but without using any ionizing radiation. This imaging technique uses powerful magnets to produce strong magnetic field (B2 to 3 tesla) and radio waves to produce images of the parts of animals that cannot be seen through X-rays, ultrasound, or CT scanning techniques [156]. An animal body mostly consists of water molecules (H2O) which contain hydrogen nuclei as a proton. These protons align along with the magnetic field and absorb the energy and subsequently flip their spin. When the magnetic field is turned off, the protons return to their original spin; this process is called precession. During precession, radio signals are emitted from proton which can be detected by the receiver present in the scanner and converted into an image [156]. Different body tissue display different time rates for protons to come back to their normal spin therefore the scanner can distinguish between different tissues of the body. MRI can produce a high resolution image by determining the spin magnetization of polarized proton and their respective longitudinal (T1)

1. Basic principles

94

4. Standard biological assays to estimate nanoparticle toxicity and biodistribution

and transverse (T2) rates in the body. Sometimes to speed up the proton realignment, contrast agents may be administered to patients intravenously before MRI. This agent can shorten the T1 and T2 relaxation timing. When contrast agents shorten the T1 relaxation the image will be bright, however, contrary to this, shorten T2 relaxation leads to dark image. As contrast agent SPIO crystals (Fe21 or Fe31) and USPIO are used for shorting the T2 relaxation to produce dark images [157]. For a brighter image (shorting T1 relaxation) paramagnetic gadolinium ions are used [158]. Currently, MRI is being used to determine the bio-distribution of NPs containing a core of SPIO. Additionally, PEG, alginate, dextran, chitosan, and albumin-coated SPIOs can also be used for in vivo imaging through MRI [152]. Several formulations of iron oxide are clinically approved such as ferumoxide and ferucarbotran and are used to enhance the contrast image of liver [159]. Gadolinium NPs are reported as a theranostic agent for neutron capture therapy [160]. The in vivo bio-distribution of NPs such as dendrimers, [161] liposome, [158] AuNPs [162], and micelles [163] are reported which consist of gadolinium or SPIO as their core or at the surface. MRI has a major advantage of producing high spatial resolution images and thus provides a better contrast image and differentiates between soft tissue, muscles, water, and fat. Although MRI is a noninvasive technique that provides 3D imaging in real time, it is cost intensive because of the high amount of contrast agent required to obtain a better contrast image. 4.3.5.4 Nuclear medicine imaging In NMI, radioactive material (radioisotopes or radiotracers) are introduced to animals before imaging. These agents emit γ-rays (gamma rays), which can be detected from the specific tissue [164]. There are two commonly used NMI techniques: single photon emission computed tomography (SPECT) and positron emission tomography (PET). Both techniques are noninvasive and detect γ-rays to produce a 3D image of the specific tissue. In this technique, γ-rays are detected by gamma scintillation camera which is placed around the sample. The scintillation crystal of detectors convert γ-rays energy into a lower energy photon which is further converted in electrical signals by PMT. Afterwards, radionuclide captured from several angles to construct 3D tomographic image concomitant with the bio-distribution of the radiotracer. PET and SPECT use gamma radiation for detections but use a different radiotracer. SPECT directly detects the γ-rays emitted from radioactive decay whereas PET detects positrons produced from the breakdown of radionuclei. The frequently used radiotracers in SPECT are 111In, 99mTc, and radioiodine (131I) and in PET are 15O, 64Cu, 11C, 18F, and 13N [164]. NPs are coated or encapsulated with gamma emitting radionuclides or positron emitters facilitate the information about the bio-distribution in tissues. PET imaging has been used to detect bio-distribution of different NPs such as iron oxide NPs, [165] liposomes [166], QDs [167], solid lipid [166], micelles [168], carbon-based [167], and polymers [169]. Likewise, SPECT imaging is used for the detection of a range of NPs such as dendrimers [170], AuNPs [170], polymeric [171], and carbon-based NPs [172]. Sometimes, due to the dissociation of radiolabels from the NPs, misleading results from which do not reflect the actual bio-distribution. Thus radiolabels remain attached to the NPs throughout the process. PET and SPECT are highly sensitive compared to other imaging techniques because a small amount of radiotracer can also be used for detection in this technique. NMI is

1. Basic principles

95

4.3 In vivo bio-distribution and toxicity of nanoparticles

TABLE 4.2 List of in vivo methods for assessment of nanoparticle (NP) bio-distribution and toxicity. Nanoparticle type

In vivo model

Dosage and route of administration Method

Results

References

Silver nanoparticles (AuNPs)

Wistar rats

15.1 μg/mL (intravenous, IV)

Inductivelycoupled mass spectrometry (ICP-MS)

Accumulation of AuNPs [173] was found in liver (72.2 ng 6 40.5) and spleen (8.9 ng 6 4.9) after single dose throughout two months which leads to significant effects on different genes

Zirconium labeled cerium oxide nanoparticles (ZrCeNPs)

C57BL/6 mice

3.711.1 MBq (IV)

LSC and positron emission tomographycomputed tomography (PET-CT) imaging

Compared to uncoated ZrCeNPs, poly acrylic acid (PAA) coated ZrCeNPs showed B1% ID/g increase in biodistribution. NPs did not cause toxicity and retain their catalytic activity

Iron oxide NPs (IO-NPs) coated with poly ethylene glycol (PEG) and glucose (Glc)

Swiss mice (Mus musculus)

0.1 mmol of NPs/kg (intravenous, IV)

Transmission electron microscope (TEM) and fluorescence microscopy

0.016 and 0.08 mg/kg [175] PEG@IONPs accumulation was found in liver and spleen, respectively. 0.06 and 0.13 mg/kg, Glc@IONPs was found in liver and spleen, respectively

Chitosan coated paclitaxel encapsulated liposome (PCL)

Mice

11.97 μg/mL (IV)

LSC

PCL (0.6% ID/g) showed maximum tumor efficiency compared to free paclitaxel. Also, PCL showed low clearance rate, lower RES, and high blood concentration

Copper nanoparticles (CuNPs)

SpragueDawley rat

200 mg/kg Histopathology (oral [hematoxylin administration) eosin (H&E) staining]

[174]

[176]

CuNPs (80 nm) found in [177] kidney (0.97 mg/kg 6 0.04), liver (4.88 mg/kg 6 0.13), spleen (0.22 mg/kg 6 0.08) (Continued)

1. Basic principles

96

4. Standard biological assays to estimate nanoparticle toxicity and biodistribution

TABLE 4.2 (Continued) Nanoparticle type

In vivo model

Naproxen loaded SpragueDawley magnetic rats polymeric micelle NPs

Dosage and route of administration Method 10 mg/kg (IV)

High performance liquid chromatography (HPLC)

Results

References

NPs showed prolonged [178] drug circulation time and high drug accumulation found in spleen (12 μg/mL), liver (11.5 μg/mL), and lung (10 μg/mL) compare to free drug (4 μg/mL)

LSC, Liquid scintillation counting.

a qualitative technique and its capability is not limited by tissue depth. PET is more sensitive than SPECT however, SPECT is less expensive than PET. Also, PET requires generator or cyclotron to process the image [138]. Examples of some in vivo methods for the determination of bio-distribution and toxicity of NPs are provided in Table 4.2.

4.4 Conclusion and future aspects NPs are rapidly capturing the market of nanotechnology for various applications. Health concerns are associated with profound usage of NPs. In this pursuit, appropriate toxicity assessment of NPs is necessary to make their full safe usage in recent advances. A myriad of in vitro cytotoxicity and genotoxicity assays are established to detect the individual markers of toxicity but none of them provide direct pass or fail quantification for their usage. Along with traditional assays, a number of kits are available from companies for toxicity evaluation which give endpoints for types of damage caused by nanomaterials. In the future, it is imperative to design an assay that helps in investigations of multiple parameters for toxicity assessment of NPs that can give clear insight for their beneficial use. Further, a validation of in vitro assays for NPs can be fulfilled by performing in vivo analysis. There are different techniques to evaluate in vivo bio-distribution NPs. The choice of technique depends on physicochemical property of NPs, type of analysis (quantitative or qualitative), type of animal examine (organ/tissue or whole animal or secession of tissue), etc. The in vivo real-time bio-distribution technique allows qualitative analysis of NPs by using optical imaging, PET, SPET, MRI, and CT. Whereas the quantitative biodistribution of NPs can be analyzed by LSC, electron microscopy imaging of sectioned tissue, histology, etc. Also, different procedures to determine the NP’s bio-distribution are currently being explored by researchers such as NPs consisting of an ultrasound contrast agent (perfluorocarbons and sulfur hexafluoride) to enhance the imaging.

Acknowledgments J. Shah thanks Department of Science and Technology, New Delhi, for providing INSPIRE Senior Research Fellowship (INSPIRE-SRF). The financial assistance for the Center for Nanotechnology Research and Applications

1. Basic principles

References

97

(CENTRA) by The Gujarat Institute for Chemical Technology (GICT) is acknowledged. The funding from the Department of Science and Technology - Science and Engineering Research Board (SERB) (Grant No.: ILS/SERB/ 2015-16/01) to Dr. Sanjay Singh under the scheme of a Start-Up Research Grant (Young Scientists) in Life Sciences is also gratefully acknowledged. The Seed Grant (AU/SG/SAS/DBLS/17-18/03) support from Ahmedabad University provided to S. Singh is also acknowledged.

Conflict of interest Authors declare no conflict of interest.

References [1] [2] [3] [4] [5] [6]

[7]

[8] [9] [10] [11]

[12] [13] [14] [15] [16] [17] [18] [19]

O.C. Farokhzad, R. Langer, Impact of nanotechnology on drug delivery, ACS Nano. 3 (1) (2009) 1620. M. Ferrari, Cancer nanotechnology: opportunities and challenges, Nat. Rev. Cancer 5 (2005) 161. Y. Wang, S. Sun, Z. Zhang, D. Shi, Nanomaterials for cancer precision medicine, Adv. Mater. 30 (17) (2018) e1705660. J. Yao, H. Wang, M. Chen, M. Yang, Recent advances in graphene-based nanomaterials: properties, toxicity and applications in chemistry, biology and medicine, Mikrochim. Acta 186 (6) (2019) 395. B. Formicola, A. Cox, R. Dal Magro, M. Masserini, F. Re, Nanomedicine for the treatment of Alzheimer’s Disease, J. Biomed. Nanotechnol. 15 (10) (2019) 19972024. E. Semenzin, V. Subramanian, L. Pizzol, A. Zabeo, W. Fransman, C. Oksel, et al., Controlling the risks of nano-enabled products through the life cycle: the case of nano copper oxide paint for wood protection and nano-pigments used in the automotive industry, Environ. Int. 131 (2019) 104901. Z. Wang, Q. Wu, J. Zhang, H. Zhang, J. Feng, S. Dong, et al., In situ polymerization of magnetic graphene oxide-diaminopyridine composite for the effective adsorption of Pb(II) and application in battery industry wastewater treatment, Environ. Sci. Pollut. Res. Int. 26 (32) (2019) 3342733439. B. Shen, D. Zhang, Y. Wei, Z. Zhao, X. Ma, X. Zhao, et al., Preparation of Ag doped keratin/PA6 nanofiber membrane with enhanced air filtration and antimicrobial properties, Polymers (Basel) 11 (9) (2019) E1511. M. Hoseinnejad, S.M. Jafari, I. Katouzian, Inorganic and metal nanoparticles and their antimicrobial activity in food packaging applications, Crit. Rev. Microbiol. 44 (2) (2018) 161181. S.L. Lim, C.T. Ng, L. Zou, Y. Lu, J. Chen, B.H. Bay, et al., Targeted metabolomics reveals differential biological effects of nanoplastics and nanoZnO in human lung cells, Nanotoxicology. 13 (8) (2019) 11171132. V. Marassi, L. Di Cristo, S.G.J. Smith, S. Ortelli, M. Blosi, A.L. Costa, et al., Silver nanoparticles as a medical device in healthcare settings: a five-step approach for candidate screening of coating agents, R. Soc. Open. Sci. 5 (1) (2018) 171113. R. Prasad, A. Bhattacharyya, Q.D. Nguyen, Nanotechnology in sustainable agriculture: recent developments, challenges, and perspectives, Front. Microbiol. 8 (2017) 1014. ˇ ca´k, M. Ghorbanpour, et al., Application of silicon A. Rastogi, D.K. Tripathi, S. Yadav, D.K. Chauhan, M. Zivˇ nanoparticles in agriculture, 3 Biotech. 9 (3) (2019) 90. I. Iavicoli, V. Leso, D.H. Beezhold, A.A. Shvedova, Nanotechnology in agriculture: opportunities, toxicological implications, and occupational risks, Toxicol. Appl. Pharmacol. 329 (2017) 96111. H. Pastrana, A. Avila, C.S.J. Tsai, Nanomaterials in cosmetic products: the challenges with regard to current legal frameworks and consumer exposure, NanoEthics 12 (2) (2018) 123137. N. Bumbudsanpharoke, J. Choi, S. Ko, Applications of nanomaterials in food packaging, J. Nanosci. Nanotechnol. 15 (9) (2015) 63576372. M.E. Abd El-Hack, M. Alagawany, M.R. Farag, M. Arif, M. Emam, K. Dhama, et al., Nutritional and pharmaceutical applications of nanotechnology: trends and advances, Int. J. Pharmacol. 13 (4) (2017) 340350. J.K. Patra, G. Das, L.F. Fraceto, E.V.R. Campos, Md.P. Rodriguez-Torres, L.S. Acosta-Torres, et al., Nano based drug delivery systems: recent developments and future prospects, J. Nanobiotechnol. 16 (1) (2018) 71. V. Steffes, Z. Zhang, J. Crowe, S. MacDonald, K.K. Ewert, B. Carragher, et al., Lipid Nanomaterials for paclitaxel delivery in cancer therapeutics: effect of pegylation and charge on the morphology and efficacy, Biophys. J. 116 (3) (2019) 507a.

1. Basic principles

98

4. Standard biological assays to estimate nanoparticle toxicity and biodistribution

[20] C. Hanley, A. Thurber, C. Hanna, A. Punnoose, J. Zhang, D.G. Wingett, The Influences of cell type and ZnO nanoparticle size on immune cell cytotoxicity and cytokine induction, Nanoscale Res. Lett. 4 (12) (2009) 14091420. [21] A.J. Koivisto, J. Lyyranen, A. Auvinen, E. Vanhala, K. Hameri, T. Tuomi, et al., Industrial worker exposure to airborne particles during the packing of pigment and nanoscale titanium dioxide, Inhal. Toxicol. 24 (12) (2012) 839849. [22] S. Mullick Chowdhury, G. Lalwani, K. Zhang, J.Y. Yang, K. Neville, B. Sitharaman, Cell specific cytotoxicity and uptake of graphene nanoribbons, Biomaterials. 34 (1) (2013) 283293. [23] R.R. Arvizo, S. Saha, E. Wang, J.D. Robertson, R. Bhattacharya, P. Mukherjee, Inhibition of tumor growth and metastasis by a self-therapeutic nanoparticle, Proc. Natl Acad. Sci. U.S.A. 110 (17) (2013) 67006705. [24] M. Ishiyama, H. Tominaga, M. Shiga, K. Sasamoto, Y. Ohkura, K. Ueno, A combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet, Biol. Pharm. Bull. 19 (11) (1996) 15181520. [25] W. Strober, Trypan blue exclusion test of cell viability, Curr. Protoc. Immunol. 21 (1) (2001). Appendix 3: Appendix 3B. [26] S.I. Kim, H.J. Kim, H.J. Lee, K. Lee, D. Hong, H. Lim, et al., Application of a non-hazardous vital dye for cell counting with automated cell counters, Anal. Biochem. 492 (2016) 812. [27] K. Prabst, H. Engelhardt, S. Ringgeler, H. Hubner, Basic colorimetric proliferation assays: MTT, WST, and resazurin, Methods Mol. Biol. 1601 (2017) 117. [28] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods. 65 (1-2) (1983) 5563. [29] V. Stone, H. Johnston, R.P.F. Schins, Development of in vitro systems for nanotoxicology: methodological considerations, Crit. Rev. Toxicol. 39 (7) (2009) 613626. [30] S. Bhagat, Y. Parikh, S. Singh, S. Sengupta, A novel nanoliposomal formulation of the FDA approved drug halofantrine causes cell death of Leishmania donovani promastigotes in vitro, Colloids Surf. A: Physicochem. Eng. Asp. 582 (2019) 123852. [31] N.J. Marshall, C.J. Goodwin, S.J. Holt, A critical assessment of the use of microculture tetrazolium assays to measure cell growth and function, Growth Regul. 5 (2) (1995) 6984. [32] M.V. Berridge, P.M. Herst, A.S. Tan, Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction, Biotechnol. Annu. Rev. 11 (2005) 127152. [33] A.H. Cory, T.C. Owen, J.A. Barltrop, J.G. Cory, Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture, Cancer Commun. 3 (7) (1991) 207212. [34] K.D. Paull, R.H. Shoemaker, M.R. Boyd, J.L. Parsons, P.A. Risbood, W.A. Barbera, et al., The synthesis of XTT: A new tetrazolium reagent that is bioreducible to a water-soluble formazan, J. Heterocycl. Chem. 25 (3) (1988) 911914. [35] D.A. Scudiero, R.H. Shoemaker, K.D. Paull, A. Monks, S. Tierney, T.H. Nofziger, et al., Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines, Cancer Res. 48 (17) (1988) 48274833. [36] M. Ishiyama, M. Shiga, K. Sasamoto, M. Mizoguchi, P.-g He, A new sulfonated tetrazolium salt that produces a highly water-soluble formazan dye, Chem. Pharm. Bull. 41 (6) (1993) 11181122. [37] P. Kumar, A. Nagarajan, P.D. Uchil, Analysis of cell viability by the lactate dehydrogenase assay, Cold Spring Harb. Protoc. 6 (2018) 465467. [38] S. Kaja, A.J. Payne, T. Singh, J.K. Ghuman, E.G. Sieck, P. Koulen, An optimized lactate dehydrogenase release assay for screening of drug candidates in neuroscience, J. Pharmacol. Toxicol. Methods. 73 (2015) 16. [39] T. Decker, M.L. Lohmann-Matthes, A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity, J. Immunol. Methods 115 (1) (1988) 6169. [40] G. Fotakis, J.A. Timbrell, In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride, Toxicol. Lett. 160 (2) (2006) 171177. ¨ .S. Aslantu¨rk, In vitro cytotoxicity and cell viability assays: principles, advantages, and disadvantages, [41] O Genotoxicity: A Predictable Risk Our Actual World, IntechOpen, Rijeka, 2018, p. 1. [42] R. Singh, A.S. Karakoti, W. Self, S. Seal, S. Singh, Redox-sensitive cerium oxide nanoparticles protect human keratinocytes from oxidative stress induced by glutathione depletion, Langmuir. 32 (46) (2016) 1220212211.

1. Basic principles

References

99

[43] E. Borenfreund, J.A. Puerner, A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR-90), J. Tissue Cult. Methods 9 (1) (1985) 79. [44] D.J. Filman, R.J. Brawn, W.B. Dandliker, Intracellular supravital stain delocalization as an assay for antibodydependent complement-mediated cell damage, J. Immunol. Methods 6 (3) (1975) 189207. [45] L. Bitensky (Ed.), The reversible activation of lysosomes in normal cells and the effects of pathological conditions, in: Ciba Foundation Symposium-Anterior Pituitary Secretion (Book I of Colloquia on Endocrinology), 1963, Wiley Online Library. [46] G. Repetto, A. del Peso, J.L. Zurita, Neutral red uptake assay for the estimation of cell viability/cytotoxicity, Nat. Protoc. 3 (7) (2008) 11251131. [47] B. Page, M. Page, C. Noel, A new fluorometric assay for cytotoxicity measurements in-vitro, Int. J. Oncol. 3 (3) (1993) 473476. [48] E.M. Czekanska, Assessment of cell proliferation with resazurin-based fluorescent dye, Methods Mol. Biol. 740 (2011) 2732. [49] J. O’Brien, I. Wilson, T. Orton, F. Pognan, Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity, Eur. J. Biochem. 267 (17) (2000) 54215426. [50] A.L. Niles, R.A. Moravec, P. Eric Hesselberth, M.A. Scurria, W.J. Daily, T.L. Riss, A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers, Anal. Biochem. 366 (2) (2007) 197206. [51] CellTiter-Fluor Technical Bulletin. Online. ,http://www.promegacom/B/media/Files/Resources/Protocols/ Technical%20Bulletins/101/CellTiter-Fluor%20Cell%20Viability%20Assay%20Protocolpdf.. [52] Y. Maehara, H. Anai, R. Tamada, K. Sugimachi, The ATP assay is more sensitive than the succinate dehydrogenase inhibition test for predicting cell viability, Eur. J. Cancer Clin. Oncol. 23 (3) (1987) 273276. [53] O. Garcia, L. Massieu, Glutamate uptake inhibitor L-trans-pyrrolidine 2,4-dicarboxylate becomes neurotoxic in the presence of subthreshold concentrations of mitochondrial toxin 3-nitropropionate: involvement of mitochondrial reducing activity and ATP production, J. Neurosci. Res. 74 (6) (2003) 956966. [54] H. Mueller, M.U. Kassack, M. Wiese, Comparison of the usefulness of the MTT, ATP, and calcein assays to predict the potency of cytotoxic agents in various human cancer cell lines, J. Biomol. Screen. 9 (6) (2004) 506515. [55] P.E. Andreotti, I.A. Cree, C.M. Kurbacher, D.M. Hartmann, D. Linder, G. Harel, et al., Chemosensitivity testing of human tumors using a microplate adenosine triphosphate luminescence assay: clinical correlation for cisplatin resistance of ovarian carcinoma, Cancer Res. 55 (22) (1995) 52765282. [56] CellTiter-Glo Technical Bulletin #288, Online. ,http://www.promegacom/B/media/Files/Resources/Protocols/ Technical%20Bulletins/101/Caspase-Glo%203%207%20Assay%20Protocolpdf.. [57] S.J. Duellman, W. Zhou, P. Meisenheimer, G. Vidugiris, J.J. Cali, P. Gautam, et al., Bioluminescent, nonlytic, real-time cell viability assay and use in inhibitor screening, Assay Drug. Dev. Technol. 13 (8) (2015) 456465. [58] P.P. Fu, Q. Xia, H.M. Hwang, P.C. Ray, H. Yu, Mechanisms of nanotoxicity: generation of reactive oxygen species, J. Food Drug Anal. 22 (1) (2014) 6475. [59] S.E. Lehman, A.S. Morris, P.S. Mueller, A.K. Salem, V.H. Grassian, S.C. Larsen, Silica nanoparticle-generated ROS as a predictor of cellular toxicity: mechanistic insights and safety by design, Environ. Sci. Nano 3 (1) (2016) 5666. [60] R. Ludwig, F.J. Teran, U. Teichgraeber, I. Hilger, Nanoparticle-based hyperthermia distinctly impacts production of ROS, expression of Ki-67, TOP2A, and TPX2, and induction of apoptosis in pancreatic cancer, Int. J. Nanomed. 12 (2017) 10091018. [61] M. Soh, D.W. Kang, H.G. Jeong, D. Kim, D.Y. Kim, W. Yang, et al., Ceria-zirconia nanoparticles as an enhanced multi-antioxidant for sepsis treatment, Angew. Chem. Int. Ed. Engl. 56 (38) (2017) 1139911403. [62] P. Ma, H. Xiao, C. Yu, J. Liu, Z. Cheng, H. Song, et al., Enhanced cisplatin chemotherapy by iron oxide nanocarrier-mediated generation of highly toxic reactive oxygen species, Nano Lett. 17 (2) (2017) 928937. [63] P. Khanna, C. Ong, B.H. Bay, G.H. Baeg, Nanotoxicity: an interplay of oxidative stress, inflammation and cell death, Nanomaterials. 5 (3) (2015) 11631180. [64] E. Eruslanov, S. Kusmartsev, Identification of ROS using oxidized DCFDA and flow-cytometry, Methods Mol. Biol. 594 (2010) 5772. [65] A. Wojtala, M. Bonora, D. Malinska, P. Pinton, J. Duszynski, M.R. Wieckowski, Methods to monitor ROS production by fluorescence microscopy and fluorometry, Methods Enzymol. 542 (2014) 243262.

1. Basic principles

100

4. Standard biological assays to estimate nanoparticle toxicity and biodistribution

[66] R. Singh, S. Singh, Redox-dependent catalase mimetic cerium oxide-based nanozyme protect human hepatic cells from 3-AT induced acatalasemia, Colloids Surf. B Biointerfaces 175 (2019) 625635. [67] S. Singh, V. D’Britto, A. Prabhune, C. Ramana, A. Dhawan, B. Prasad, Cytotoxic and genotoxic assessment of glycolipid-reduced and-capped gold and silver nanoparticles, N. J. Chem. 34 (2) (2010) 294301. [68] T.M. Potter, B.W. Neun, S.T. Stern, Assay to detect lipid peroxidation upon exposure to nanoparticles, Methods Mol. Biol. 697 (2011) 181189. [69] J.A. Buege, S.D. Aust, Microsomal lipid peroxidation, Methods Enzymol. 52 (1978) 302310. [70] I. Rahman, A. Kode, S.K. Biswas, Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method, Nat. Protoc. 1 (6) (2006) 31593165. [71] A. Lekshmi, S.N. Varadarajan, S.S. Lupitha, D. Indira, K.A. Mathew, A. Chandrasekharan Nair, et al., A quantitative real-time approach for discriminating apoptosis and necrosis, Cell Death Discov. 3 (1) (2017) 16101. [72] K. Kyrylkova, S. Kyryachenko, M. Leid, C. Kioussi, Detection of apoptosis by TUNEL assay, Methods Mol. Biol. 887 (2012) 4147. [73] F.K. Chan, K. Moriwaki, M.J. De Rosa, Detection of necrosis by release of lactate dehydrogenase activity, Methods Mol. Biol. 979 (2013) 6570. [74] S. Chakraborty, V. Castranova, M.K. Perez, G. Piedimonte, Nanoparticles-induced apoptosis of human airway epithelium is mediated by proNGF/p75(NTR) signaling, J. Toxicol. Environ. Health A 80 (1) (2017) 5368. [75] B. Plackal Adimuriyil George, N. Kumar, H. Abrahamse, S.S. Ray, Apoptotic efficacy of multifaceted biosynthesized silver nanoparticles on human adenocarcinoma cells, Sci. Rep. 8 (1) (2018) 14368. [76] Q. Sun, T. Ishii, K. Kanehira, T. Sato, A. Taniguchi, Uniform TiO2 nanoparticles induce apoptosis in epithelial cell lines in a size-dependent manner, Biomater. Sci. 5 (5) (2017) 10141021. [77] C.M. Pfeffer, A.T.K. Singh, Apoptosis: a target for anticancer therapy, Int. J. Mol. Sci. 19 (2) (2018) E448. [78] H. Lujan, C.M. Sayes, Cytotoxicological pathways induced after nanoparticle exposure: studies of oxidative stress at the ‘nano-bio’ interface, Toxicol. Res. (Camb.). 6 (5) (2017) 580594. [79] M. Eray, M. Matto, M. Kaartinen, L. Andersson, J. Pelkonen, Flow cytometric analysis of apoptotic subpopulations with a combination of annexin V-FITC, propidium iodide, and SYTO 17, Cytometry 43 (2) (2001) 134142. [80] I. Lakshmanan, S.K. Batra, Protocol for apoptosis assay by flow cytometry using Annexin V staining method, Bio Protoc. 3 (6) (2013). [81] Y. Shounan, X. Feng, P.J. O’Connell, Apoptosis detection by annexin V binding: a novel method for the quantitation of cell-mediated cytotoxicity, J. Immunol. Methods 217 (1-2) (1998) 6170. [82] I. Vermes, C. Haanen, C. Reutelingsperger, Flow cytometry of apoptotic cell death, J. Immunol. Methods. 243 (1-2) (2000) 167190. [83] A.M. Rieger, K.L. Nelson, J.D. Konowalchuk, D.R. Barreda, Modified annexin V/propidium iodide apoptosis assay for accurate assessment of cell death, J. Vis. Exp. (50))(2011). [84] E. Brauchle, S. Thude, S.Y. Brucker, K. Schenke-Layland, Cell death stages in single apoptotic and necrotic cells monitored by Raman microspectroscopy, Sci. Rep. 4 (2014) 4698. [85] D. Kylarova, J. Prochazkova, J. Mad’arova, J. Bartos, V. Lichnovsky, Comparison of the TUNEL, lamin B and annexin V methods for the detection of apoptosis by flow cytometry, Acta Histochem. 104 (4) (2002) 367370. [86] Z. Darzynkiewicz, D. Galkowski, H. Zhao, Analysis of apoptosis by cytometry using TUNEL assay, Methods 44 (3) (2008) 250254. [87] V. Kumar, N. Sharma, S.S. Maitra, In vitro and in vivo toxicity assessment of nanoparticles, Int. Nano Lett. 7 (4) (2017) 243256. [88] J.W. Hickey, J.L. Santos, J.M. Williford, H.Q. Mao, Control of polymeric nanoparticle size to improve therapeutic delivery, J. Control. Release 219 (2015) 536547. [89] M.A. Gatoo, S. Naseem, M.Y. Arfat, A.M. Dar, K. Qasim, S. Zubair, Physicochemical properties of nanomaterials: implication in associated toxic manifestations, Biomed. Res. Int. 2014 (2014) 498420. [90] R.A. Sperling, W.J. Parak, Surface modification, functionalization and bioconjugation of colloidal inorganic nanoparticles, Philos. Trans. A Math. Phys. Eng. Sci. 368 (1915) (2010) 13331383. [91] S. Lanone, J. Boczkowski, Biomedical applications and potential health risks of nanomaterials: molecular mechanisms, Curr. Mol. Med. 6 (6) (2006) 651663. [92] Y. Liu, J. Tan, A. Thomas, D. Ou-Yang, V.R. Muzykantov, The shape of things to come: importance of design in nanotechnology for drug delivery, Ther. Deliv. 3 (2) (2012) 181194. [93] A.V. Kabanov, Polymer genomics: an insight into pharmacology and toxicology of nanomedicines, Adv. Drug. Deliv. Rev. 58 (15) (2006) 15971621.

1. Basic principles

References

101

[94] F. Maffei, J.M. Zolezzi Moraga, S. Angelini, C. Zenesini, M. Musti, D. Festi, et al., Micronucleus frequency in human peripheral blood lymphocytes as a biomarker for the early detection of colorectal cancer risk, Mutagenesis 29 (3) (2014) 221225. [95] B.C. Nelson, C.W. Wright, Y. Ibuki, M. Moreno-Villanueva, H.L. Karlsson, G. Hendriks, et al., Emerging metrology for high-throughput nanomaterial genotoxicology, Mutagenesis 32 (1) (2017) 215232. [96] M. Fenech, The in vitro micronucleus technique, Mutat. Res. 455 (1-2) (2000) 8195. [97] K. Williams, R.W. Sobol, Mutation research/fundamental and molecular mechanisms of mutagenesis: special issue: DNA repair and genetic instability, Mutat. Res. 743-744 (2013) 13. [98] M. Nusse, J. Kramer, Flow cytometric analysis of micronuclei found in cells after irradiation, Cytometry 5 (1) (1984) 2025. [99] G.A. Schreiber, W. Beisker, M. Bauchinger, M. Nusse, Multiparametric flow cytometric analysis of radiationinduced micronuclei in mammalian cell cultures, Cytometry 13 (1) (1992) 90102. [100] P.L. Olive, Cell proliferation as a requirement for development of the contact effect in Chinese hamster V79 spheroids, Radiat. Res. 117 (1) (1989) 7992. [101] N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell Res. 175 (1) (1988) 184191. [102] T.S. Kumaravel, A.N. Jha, Reliable Comet assay measurements for detecting DNA damage induced by ionising radiation and chemicals, Mutat. Res. 605 (1-2) (2006) 716. [103] E. Clonfero, G.M. Ferri, S. Pavanello, [Molecular epidemiology in occupational medicine: methodological features and impact of individual genetic susceptibility], G. Ital. Med. Lav. Ergon. 25 (3) (2003) 279284. [104] M. Azizi, H. Ghourchian, F. Yazdian, F. Dashtestani, H. AlizadehZeinabad, Cytotoxic effect of albumin coated copper nanoparticle on human breast cancer cells of MDA-MB 231, PLoS One 12 (11) (2017) e0188639. [105] O. Minaeva, E. Brodovskaya, M. Pyataev, M. Gerasimov, M. Zharkov, I. Yurlov (Eds.), Comparative study of cytotoxicity of ferromagnetic nanoparticles and magnetitecontaining polyelectrolyte microcapsules, J. Phy. Conf. Ser. 784 (2017) 012038. [106] A. Dubey, M. Goswami, K. Yadav, D. Chaudhary, Oxidative stress and nano-toxicity induced by TiO2 and ZnO on WAG cell line, PLoS One 10 (5) (2015) e0127493. [107] C.T. Ng, L.Q. Yong, M.P. Hande, C.N. Ong, L.E. Yu, B.H. Bay, et al., Zinc oxide nanoparticles exhibit cytotoxicity and genotoxicity through oxidative stress responses in human lung fibroblasts and Drosophila melanogaster, Int. J. Nanomed. 12 (2017) 16211637. [108] A. Patlolla, B. Knighten, P. Tchounwou, Multi-walled carbon nanotubes induce cytotoxicity, genotoxicity and apoptosis in normal human dermal fibroblast cells, Ethn. Dis. 20 (1 Suppl 1) (2010) S1-65S1-72. [109] M. Abudayyak, E. Guzel, G. Ozhan, Nickel oxide nanoparticles induce oxidative DNA damage and apoptosis in kidney cell line (NRK-52E), Biol. Trace Elem. Res. 178 (1) (2017) 98104. [110] K. Shah, S. Bhagat, D. Varade, S. Singh, Novel synthesis of polyoxyethylene cholesteryl ether coated Fe-Pt nanoalloys: a multifunctional and cytocompatible bimetallic alloy exhibiting intrinsic chemical catalysis and biological enzyme-like activities, Colloids Surf. A Physicochem. Eng. Asp. 553 (2018) 5057. [111] S. Singh, R. Asal, S. Bhagat, Multifunctional antioxidant nanoliposome-mediated delivery of PTEN plasmids restore the expression of tumor suppressor protein and induce apoptosis in prostate cancer cells, J. Biomed. Mater. Res. A. 106 (12) (2018) 31523164. [112] M. Shah, J. Shah, H. Arya, A. Vyas, A. Vijapura, A. Gajipara, et al., Biological oxidase enzyme mimetic Cu-Pt nanoalloys: a multifunctional nanozyme for colorimetric detection of ascorbic acid and identification of mammalian cells, Chem. Sel. 4 (21) (2019) 65376546. [113] T.J. Webster, Nanomedicine: what’s in a definition? Int. J. Nanomed. 1 (2) (2006) 115116. [114] S. Harper, C. Usenko, J.E. Hutchison, B.L.S. Maddux, R.L. Tanguay, In vivo biodistribution and toxicity depends on nanomaterial composition, size, surface functionalisation and route of exposure, J. Exp. Nanosci. 3 (3) (2008) 195206. [115] T. Lammers, F. Kiessling, W.E. Hennink, G. Storm, Drug targeting to tumors: principles, pitfalls and (pre-) clinical progress, J. Control. Release 161 (2) (2012) 175187. [116] H.A. Alturkistani, F.M. Tashkandi, Z.M. Mohammedsaleh, Histological stains: a literature review and case study, Glob. J. Health Sci. 8 (3) (2015) 7279. [117] A.L. Robson, P.C. Dastoor, J. Flynn, W. Palmer, A. Martin, D.W. Smith, et al., Advantages and limitations of current imaging techniques for characterizing liposome morphology, Front. Pharmacol. 9 (2018) 80.

1. Basic principles

102

4. Standard biological assays to estimate nanoparticle toxicity and biodistribution

[118] J. Zhao, J. Vykoukal, M. Abdelsalam, A. Recio-Boiles, Q. Huang, Y. Qiao, et al., Stem cell-mediated delivery of SPIO-loaded gold nanoparticles for the theranosis of liver injury and hepatocellular carcinoma, Nanotechnology 25 (40) (2014) 405101. [119] T. Xia, F. Yu, K. Zhang, Z. Wu, D. Shi, H. Teng, et al., The effectiveness of allogeneic mesenchymal stem cells therapy for knee osteoarthritis in pigs, Ann. Transl. Med. 6 (20) (2018). 404-. [120] B.T.T. Pham, E.K. Colvin, N.T.H. Pham, B.J. Kim, E.S. Fuller, E.A. Moon, et al., Biodistribution and clearance of stable superparamagnetic maghemite iron oxide nanoparticles in mice following intraperitoneal administration, Int. J. Mol. Sci. 19 (1) (2018). [121] C. Holzhausen, D. Groger, L. Mundhenk, P. Welker, R. Haag, A.D. Gruber, Tissue and cellular localization of nanoparticles using (3)(5)S labeling and light microscopic autoradiography, Nanomedicine 9 (4) (2013) 465468. [122] H. Asem, Y. Zhao, F. Ye, A. Barrefelt, M. Abedi-Valugerdi, R. El-Sayed, et al., Biodistribution of biodegradable polymeric nano-carriers loaded with busulphan and designed for multimodal imaging, J. Nanobiotechnol. 14 (1) (2016) 82. [123] M. Cho, W.S. Cho, M. Choi, S.J. Kim, B.S. Han, S.H. Kim, et al., The impact of size on tissue distribution and elimination by single intravenous injection of silica nanoparticles, Toxicol. Lett. 189 (3) (2009) 177183. [124] J. Kuo, Electron Microscopy: Methods and Protocols, Springer Science & Business Media, 2007. [125] J. Zhang, X. He, M.T. Tseng, Application of Electron microscopes in nanotoxicity assessment, Methods Mol. Biol. 1894 (2019) 247269. [126] W.H. Jong, M.C. Burger, M.A. Verheijen, R.E. Geertsma, Detection of the presence of gold nanoparticles in organs by transmission electron microscopy, Materials (Basel) 3 (9) (2010) 46814694. [127] C.A. Garcia-Negrete, M.C. Jimenez de Haro, J. Blasco, M. Soto, A. Fernandez, STEM-in-SEM high resolution imaging of gold nanoparticles and bivalve tissues in bioaccumulation experiments, Analyst 140 (9) (2015) 30823089. [128] P.J. Kempen, A.S. Thakor, C. Zavaleta, S.S. Gambhir, R. Sinclair, A scanning transmission electron microscopy approach to analyzing large volumes of tissue to detect nanoparticles, Microsc. Microanal. 19 (5) (2013) 12901297. [129] I. Stojkovi´c, N. Todorovi´c, J. Nikolov, I.K. Broni´c, J. Bareˇsi´c, U.K. Luburi´c, Methodology of tritium determination in aqueous samples by liquid scintillation counting techniques, Advances in Research and Applications, Nova Science Publishers, 2018, p. 99. [130] D. Horrocks, Organic Scintillators and Scintillation Counting, Elsevier, 2012. [131] PerkinElmer, LSC in Practice: LSC Sample Preparation by Solubilization. Application Note  PerkinElmer, 2008. [132] K. Ikemoto, K. Shimizu, K. Ohashi, Y. Takeuchi, M. Shimizu, N. Oku, Bauhinia purprea agglutinin-modified liposomes for human prostate cancer treatment, Cancer Sci. 107 (1) (2016) 5359. [133] M.J. Ernsting, M. Murakami, A. Roy, S.-D. Li, Factors controlling the pharmacokinetics, biodistribution and intratumoral penetration of nanoparticles, J. Control. Release 172 (3) (2013) 782794. [134] P. Ebrahimnejad, R. Dinarvand, M.R. Jafari, S.A.S. Tabasi, F. Atyabi, Characterization, blood profile and biodistribution properties of surface modified PLGA nanoparticles of SN-38, Int. J. Pharm. 406 (1) (2011) 122127. [135] C.W. Chumbley, M.L. Reyzer, J.L. Allen, G.A. Marriner, L.E. Via, C.E. Barry 3rd, et al., Correction to absolute quantitative MALDI imaging mass spectrometry: a case of rifampicin in liver tissues, Anal. Chem. 88 (17) (2016) 8920. [136] J.B. Arsand, L. Jank, M.T. Martins, R.B. Hoff, F. Barreto, T.M. Pizzolato, et al., Determination of aminoglycoside residues in milk and muscle based on a simple and fast extraction procedure followed by liquid chromatography coupled to tandem mass spectrometry and time of flight mass spectrometry, Talanta 154 (2016) 3845. [137] Y. Xu, H. Meng, Paclitaxel-loaded stealth liposomes: development, characterization, pharmacokinetics, and biodistribution, Artif. Cells Nanomed. Biotechnol. 44 (1) (2016) 350355. [138] L. Arms, D.W. Smith, J. Flynn, W. Palmer, A. Martin, A. Woldu, et al., Advantages and limitations of current techniques for analyzing the biodistribution of nanoparticles, Front. Pharmacol. 9 (2018) 802. [139] B. Priem, C. Tian, J. Tang, Y. Zhao, W.J. Mulder, Fluorescent nanoparticles for the accurate detection of drug delivery, Expert. Opin. Drug. Deliv. 12 (12) (2015) 18811894. [140] P.W. Barone, R.S. Parker, M.S. Strano, In vivo fluorescence detection of glucose using a single-walled carbon nanotube optical sensor: design, fluorophore properties, advantages, and disadvantages, Anal. Chem. 77 (23) (2005) 75567562.

1. Basic principles

References

103

[141] J. Merian, R. Boisgard, P.A. Bayle, M. Bardet, B. Tavitian, I. Texier, Comparative biodistribution in mice of cyanine dyes loaded in lipid nanoparticles, Eur. J. Pharm. Biopharm. 93 (2015) 110. [142] A. Wagh, S.Y. Qian, B. Law, Development of biocompatible polymeric nanoparticles for in vivo NIR and FRET imaging, Bioconjug. Chem. 23 (5) (2012) 981992. [143] E. Rampazzo, D. Genovese, F. Palomba, L. Prodi, N. Zaccheroni, NIR-fluorescent dye doped silica nanoparticles for in vivo imaging, sensing and theranostic, Methods Appl. Fluoresc. 6 (2) (2018) 022002. [144] S. Chinnathambi, N. Shirahata, Recent advances on fluorescent biomarkers of near-infrared quantum dots for in vitro and in vivo imaging, Sci. Technol. Adv. Mater. 20 (1) (2019) 337355. [145] Z. Ma, D. Dosev, M. Nichkova, R.K. Dumas, S.J. Gee, B.D. Hammock, et al., Synthesis and characterization of multifunctional silica core-shell nanocomposites with magnetic and fluorescent functionalities, J. Magn. Magn Mater. 321 (10) (2009) 13681371. [146] M.M. Li, J. Cao, J.C. Yang, Y.J. Shen, X.L. Cai, Y.W. Chen, et al., Biodistribution and toxicity assessment of intratumorally injected arginine-glycine-aspartic acid peptide conjugated to CdSe/ZnS quantum dots in mice bearing pancreatic neoplasm, Chem. Biol. Interact. 291 (2018) 103110. [147] M. Vats, S.K. Mishra, M.S. Baghini, D.S. Chauhan, R. Srivastava, A. De, Near Infrared fluorescence imaging in nano-therapeutics and photo-thermal evaluation, Int. J. Mol. Sci. 18 (5) (2017). [148] S. Dimchevska, N. Geskovski, R. Koliqi, N. Matevska-Geskovska, V. Gomez Vallejo, B. Szczupak, et al., Efficacy assessment of self-assembled PLGA-PEG-PLGA nanoparticles: correlation of nano-bio interface interactions, biodistribution, internalization and gene expression studies, Int. J. Pharm. 533 (2) (2017) 389401. [149] L. Jennings, O. Ivashchenko, I.J. Marsman, A.C. Laan, A.G. Denkova, G. Waton, et al., In vivo biodistribution of stable spherical and filamentous micelles probed by high-sensitivity SPECT, Biomater. Sci. 4 (8) (2016) 12021211. [150] N. Anton, A. Parlog, G. Bou About, M.F. Attia, M. Wattenhofer-Donze, H. Jacobs, et al., Non-invasive quantitative imaging of hepatocellular carcinoma growth in mice by micro-CT using liver-targeted iodinated nano-emulsions, Sci. Rep. 7 (1) (2017) 13935. [151] J.R. Ashton, J.L. West, C.T. Badea, In vivo small animal micro-CT using nanoparticle contrast agents, Front. Pharmacol. 6 (2015) 256. [152] N.V.S. Vallabani, S. Singh, A.S. Karakoti, Magnetic nanoparticles: current trends and future aspects in diagnostics and nanomedicine, Curr. Drug Metab. 20 (6) (2019) 457472. [153] P. Mohammadzadeh, R.A. Cohan, S.M. Ghoreishi, A. Bitarafan-Rajabi, M.S. Ardestani, AS1411 aptameranionic linear globular dendrimer G2-iohexol selective nano-theranostics, Sci. Rep. 7 (1) (2017) 11832. [154] G. Engudar, H. Schaarup-Jensen, F.P. Fliedner, A.E. Hansen, P. Kempen, R.I. Jolck, et al., Remote loading of liposomes with a (124)I-radioiodinated compound and their in vivo evaluation by PET/CT in a murine tumor model, Theranostics 8 (21) (2018) 58285841. [155] R. Meir, K. Shamalov, O. Betzer, M. Motiei, M. Horovitz-Fried, R. Yehuda, et al., Nanomedicine for cancer immunotherapy: tracking cancer-specific T-cells in vivo with gold nanoparticles and CT imaging, ACS Nano 9 (6) (2015) 63636372. [156] V.P. Grover, J.M. Tognarelli, M.M. Crossey, I.J. Cox, S.D. Taylor-Robinson, M.J. McPhail, Magnetic resonance imaging: principles and techniques: lessons for clinicians, J. Clin. Exp. Hepatol. 5 (3) (2015) 246255. [157] N.V.S. Vallabani, S. Singh, Recent advances and future prospects of iron oxide nanoparticles in biomedicine and diagnostics, 3 Biotech. 8 (6) (2018) 279. [158] Y. Jin, N. Zhang, C. Li, K. Pu, C. Ding, Y. Zhu, Nanosystem composed with MSNs, gadolinium, liposome and cytotoxic peptides for tumor theranostics, Colloids Surf. B Biointerfaces 151 (2017) 240248. [159] A.S. Thakor, J.V. Jokerst, P. Ghanouni, J.L. Campbell, E. Mittra, S.S. Gambhir, Clinically approved nanoparticle imaging agents, J. Nucl. Med. 57 (12) (2016) 18331837. [160] A. Narmani, B. Farhood, H. Haghi-Aminjan, T. Mortezazadeh, A. Aliasgharzadeh, M. Mohseni, et al., Gadolinium nanoparticles as diagnostic and therapeutic agents: Their delivery systems in magnetic resonance imaging and neutron capture therapy, J. Drug. Deliv. Sci. Technol. 44 (2018) 457466. Mater. Chem. Phys. 181 2016. [161] W.G. Lesniak, N. Oskolkov, X. Song, B. Lal, X. Yang, M. Pomper, et al., Salicylic acid conjugated dendrimers are a tunable, high performance CEST MRI nanoplatform, Nano Lett. 16 (4) (2016) 22482253. [162] D. Maniglio, F. Benetti, L. Minati, J. Jovicich, A. Valentini, G. Speranza, et al., Theranostic gold-magnetite hybrid nanoparticles for MRI-guided radiosensitization, Nanotechnology. 29 (31) (2018) 315101. [163] A. Babic, V. Vorobiev, G. Trefalt, L.A. Crowe, L. Helm, J.P. Vallee, et al., MRI micelles self-assembled from synthetic gadolinium-based nano building blocks, Chem. Commun. (Camb.). 55 (7) (2019) 945948.

1. Basic principles

104

4. Standard biological assays to estimate nanoparticle toxicity and biodistribution

[164] P. Suetens, Fundamentals of Medical Imaging, Cambridge University Press, 2017. [165] J. Pellico, J. Ruiz-Cabello, M. Saiz-Alia, G. Del Rosario, S. Caja, M. Montoya, et al., Fast synthesis and bioconjugation of (68) Ga core-doped extremely small iron oxide nanoparticles for PET/MR imaging, Contrast Media Mol. Imaging 11 (3) (2016) 203210. [166] F. Man, P.J. Gawne, R. TMdR, Nuclear imaging of liposomal drug delivery systems: a critical review of radiolabelling methods and applications in nanomedicine, Adv. Drug. Deliv. Rev. (2019). [167] Q. Wang, H. Yang, Q. Zhang, H. Ge, S. Zhang, Z. Wang, et al., Strong acid-assisted preparation of greenemissive carbon dots for fluorometric imaging of pH variation in living cells, Mikrochim. Acta 186 (7) (2019) 468. [168] X. Xue, A. Lindstrom, Y. Li, Porphyrin-based nanomedicines for cancer treatment, Bioconjug. Chem. 30 (6) (2019) 15851603. [169] G. Huang, T. Zhao, C. Wang, K. Nham, Y. Xiong, X. Gao, et al., PET imaging of occult tumours by temporal integration of tumour-acidosis signals from pH-sensitive (64)Cu-labelled polymers, Nat. Biomed. Eng. (2019) 111. [170] Y. Li, L. Zhao, X. Xu, N. Sun, W. Qiao, Y. Xing, et al., Design of (99m)Tc-labeled low generation dendrimerentrapped gold nanoparticles for targeted single photon emission computed tomography/computed tomography imaging of gliomas, J. Biomed. Nanotechnol. 15 (6) (2019) 12011212. [171] D.P. Feldmann, S. Jones, K. Douglas, A.F. Shields, O.M. Merkel, Microfluidic assembly of siRNA-loaded micelleplexes for tumor targeting in an orthotopic model of ovarian cancer, Methods Mol. Biol. 1974 (2019) 355369. [172] Z. Dong, L. Feng, Y. Chao, Y. Hao, M. Chen, F. Gong, et al., Amplification of tumor oxidative stresses with liposomal fenton catalyst and glutathione inhibitor for enhanced cancer chemotherapy and radiotherapy, Nano Lett. 19 (2) (2019) 805815. [173] S.K. Balasubramanian, J. Jittiwat, J. Manikandan, C.-N. Ong, L.E. Yu, W.-Y. Ong, Biodistribution of gold nanoparticles and gene expression changes in the liver and spleen after intravenous administration in rats, Biomaterials 31 (8) (2010) 20342042. [174] P.R. McDonagh, G. Sundaresan, L. Yang, M. Sun, R. Mikkelsen, J. Zweit, Biodistribution and PET imaging of 89-zirconium labeled cerium oxide nanoparticles synthesized with several surface coatings, Nanomed. Nanotechnol. Biol. Med. 14 (4) (2018) 14291440. [175] G. Stepien, M. Moros, M. Pe´rez-Herna´ndez, M. Monge, L. Gutie´rrez, R.M. Fratila, et al., Effect of surface chemistry and associated protein corona on the long-term biodegradation of iron oxide nanoparticles in vivo, ACS Appl. Mater. Interfaces 10 (5) (2018) 45484560. [176] B. Nanda, A.S. Manjappa, K. Chuttani, N.H. Balasinor, A.K. Mishra, R.S. Ramachandra Murthy, Acylated chitosan anchored paclitaxel loaded liposomes: Pharmacokinetic and biodistribution study in Ehrlich ascites tumor bearing mice, Int. J. Biol. Macromol. 122 (2019) 367379. [177] H. Tang, M. Xu, X. Zhou, Y. Zhang, L. Zhao, G. Ye, et al., Acute toxicity and biodistribution of different sized copper nano-particles in rats after oral administration, Mater. Sci. Eng. C. Mater Biol. Appl. 93 (2018) 649663. [178] Z. Karami, S. Sadighian, K. Rostamizadeh, S.H. Hosseini, S. Rezaee, M. Hamidi, Magnetic brain targeting of naproxen-loaded polymeric micelles: pharmacokinetics and biodistribution study, Mater. Sci. Eng. C 100 (2019) 771780.

1. Basic principles

C H A P T E R

5 Toxicity of metal oxide nanoparticles Thodhal Yoganandham Suman1,2, 3, Wei-Guo Li1 and De-Sheng Pei1,2 1

College of Life Science, Henan Normal University, Xinxiang, P.R. China Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing, P.R. China 3Ecotoxicology Division, Centre for Ocean Research, Sathyabama Institute of Science and Technology, Chennai, Tamil Nadu, India

2

5.1 Introduction Nanotechnology is a rising field that gives sustainable development at an atomic level and nanoscale, and nanoparticles (NPs) are the foremost vital structural masses of nanotechnology [1]. NPs are clusters ranging from 1 to 100 nm in size. Their high volume-tosurface ratio gives NPs different electrical, mechanical, optical, magnetic, chemical, magneto-optical, and electro-optical properties from their bulk properties Recently, the fast growth of nanotechnology has increased the usage of engineered NPs in marketable products, making engineered NPs one of the significant causes for particle introduction in the cutting-edge society [2,3]. Current research has shown that a total of 1814 commercial goods are being marketed globally, containing some NPs that can be released to the environment increasingly [4]. The discharge of NPs into the environment and the exposure of ecosystems to nanomaterials are also anticipated to increase with the increased use of nanomaterials [5]. NPs bioaccumulation can lead to potential hazards to humans, aquatic life, plants, and animals.

5.2 Metal oxide nanoparticles A metal oxide nanoparticle (MO-NPs) is the largest proportion of manufacture and application among various nanomaterials [6]. MO-NPs have special characteristics like physical, chemical, and extensive structural and functional among the most versatile groups of semiconductors. Due to these MO-NPs unique and tunable properties, making

Nanotoxicity DOI: https://doi.org/10.1016/B978-0-12-819943-5.00005-1

107

Copyright © 2020 Elsevier Inc. All rights reserved.

108

5. Toxicity of metal oxide nanoparticles

them excellent candidates for different high-tech applications such as medical system, catalytic, thermal, mechanical, electrical, magnetic, optoelectronic, and optical [7]. MO-NPs seem to have no toxic effects. However, these particles have larger volume-tosurface ratio which creates their higher chemical reactivity, resulting in enhanced production of reactive oxygen species (ROS). Indeed, due to the interaction of their surfaces with the biological system, the surface of NPs is a key factor for their intrinsic toxicity [8]. Different metal oxides are generally available in nature, but some of them have a wide variety of technological uses. Transition metal oxides, such as zinc oxide nanoparticles (ZnO NPs), iron oxide nanoparticles (IO-NPs), copper oxide nanoparticles (CuO NPs), and titanium dioxide nanoparticles (TiO2 NPs), are highlighted. In the following sections, we discuss the toxicity of MO-NPs.

5.3 Zinc oxide nanoparticles Zinc oxide (ZnO) exists as a mineral zincite in the earth’s crust, while most of it is produced by synthetic methods that are used commercially [9]. ZnO has spot wide gap (3.37 eV) and excessive exaction binding energy (60 meV) that could be the chemical compound with an enormous range of enticing properties and the most popular multitasking metal [10]. Due to its exceptional electrical and optical properties, optoelectronic applications, consider it a possible material to function in visible and near ultraviolet (UV) spectral regions. Nanoscale zinc is used to absorb UV light mainly in the rubber industry, white pigment, and as a heat conductor [11]. ZnO NPs have also been used in several goods such as catalysts, paints, and semiconductors. Furthermore, due to the resilient UV absorption properties of ZnO, these particles are found even more in consumer products, such as anti-dandruff shampoos, fabric treatments for UV shielding, and baby powders [12 14]. The US Food and Drug Administration (USFDA) consider ZnO to be a commonly accepted safe substance [15]. However, they are usually recognized as a secure category of these substances which can develop new features and can produce toxic effects.

5.3.1 In vitro toxicity studies of ZnO nanoparticles Currently, many in vitro cytotoxicity studies of ZnO NPs have been reported. Suman et al. [16] investigated the toxicity of ZnO NPs (40 48 nm) following the exposure of Chlorella vulgaris (C. vulgaris) in a dose- and time-dependent manner. C. vulgaris was treated with 50 300 mg/L ZnO NPs for 24 and 72 h. The viability of C. vulgaris has declined in 50 mg/L reduced by 90.4% in at 24 h and while exposure for 72 h decreased substantially in 300 mg/L to 23.8%. After exposure to ZnO NPs, C. vulgaris showed decreased viability, increased lactate dehydrogenase (LDH), and decreased superoxide dismutase (SOD) and glutathione (GSH). These outcomes show that oxidative stress is a key factor for cytotoxicity induction. The concentration of ZnO NPs increases the significant morphological

2. Toxicity of nanomaterials

5.3 Zinc oxide nanoparticles

109

FIGURE 5.1 Scanning electron micrographs (SEM) morphological analysis of algal cells. (A) Control cell shows no damage; (B, C) comprehensive membrane destruction after 72 h treatment with zinc oxide nanoparticles (ZnO NPs) of up to the 50 mg/L and 100 mg/L; (D) 300 mg/L ZnO NPs displays morphological distortions after 72 h treatment.

changes, and the damage to the cell wall in C. vulgaris has been confirmed by microscopic analyses (Fig. 5.1). The surface charge of the NPs is also a significant consideration of toxicity. The positively charged NPs have greater toxicity, and the variance surface charge is recognized to cellular uptake and intracellular location [17,18]. Kim et al. [19] draw the same conclusion about the importance of positively charged ZnO NPs in RAW264.7 cells. The positively charged ZnO NPs induced higher cytotoxicity, compared to the negatively charged one. Ng et al. [20] reported a substantial release of extracellular LDH and reduced cell viability in MRC5 lung cells treated with ZnO NP, showing genotoxicity and DNA damage. Some studies reported DNA damage and cytotoxicity in Caco-2 cells [21]. The release of Zn21 ions through dissolution of ZnO NP could influence the ZnO NP-induced cytotoxicity and the development of free radicals from the ZnO surface, leads to metabolic imbalance and cellular ionic related with an inhibition of ion transport and a defect of ionic homeostasis [16,22].

2. Toxicity of nanomaterials

110

5. Toxicity of metal oxide nanoparticles

5.3.2 In vivo toxicity studies of ZnO nanoparticles Sharma et al. [23] exposed mice to 30 nm ZnO NPs with 300 mg/kg dose. The result showed major accumulation of NPs in the liver, resulting in cell injury following 14 consecutive days of subacute oral exposure to ZnO NPs. Fpg-modified comet assay was used to evaluate the DNA damage in mice’s liver and kidney cells, and the result showed a significant DNA lesion in the liver, indicating that oxidative stress caused DNA damage compared to control. Cardozo et al. [24] exposed Drosophila melanogaster to 27 nm ZnO NPs. The NPs showed the genotoxicity, and homologous recombination is the main mechanism for the loss of heterozygosis in the somatic cells of D. melanogaster. After exposure of ZnO NP in D. melanogaster, nuclear factor κB (NF-κB) inhibitory protein in the cell decreases and consequently increased NF-κB dimers in the nucleus; this induced DNA double-strand breaks. Abbasalipourkabir et al. [25] investigated the oxidative stress analysis after injected intraperitoneally with a dose of 50 200 mg of ZnO NPs/kg body. In a dose-dependent manner, the NPs induced significant oxidative stress. The results showed a major increase in liver enzymes at the concentration of 100 mg/kg animal body weight of ZnO NPs. ZnO NPs at the concentrations above 50 mg/kg were also found to result in considerably increased SOD but insignificant decreased glutathione peroxidase, indicating oxidative stress occurred in treated groups. The liver tissue of animals exposed to ZnO NPs displayed inflammation, increased congestion, chromatin condensation, and apoptosis. Effects following the oral exposure dermal exposure of ZnO NP (20 nm) have been studied in rats for 90 days. Tissue distribution of ZnO NP showed the increasing doses of zinc in the liver, large intestine, small intestine, and feces. Hyperkeratosis and papillomatosis were induced in the skin, compared to the normal [26]. ZnO NP has shown distinctly toxic effects both in vitro and in vivo, including the cytotoxicity, oxidative stress, and genotoxicity. Thus, exposure to ZnO NPs should be avoided.

5.4 Iron Oxide-based magnetic nanoparticles Iron oxide nanoparticles (IO-NPs) have drawn considerable attention in various applications, due to their unique physicochemical characteristics in the nanoscale. Due to their superparamagnetic properties, IO-NPs have a particular interest among different types of magnetic nanoparticles. Together with their elevated colloidal stabilization, this characteristic makes them very appealing for a broad spectrum of applications [27]. IO-NPs are often used from an industrial point of view as the pigment in building materials, which are capable of imparting different colors, colorfast, low cost, and food additives [28]. However, the most significant usage of IO-NPs is in the medicinal field. For example, they are used in hyperthermia, magnetic resonance imaging, drug delivery, tissue repair, immunoassays, and cell separation. Iron oxides occur in different types in nature, such as magnetite, maghemite, and hematite [29]. Hematite is commonly found in soil and rock, and is also called as ferric oxide. It is highly stable under ambient conditions, and transforms the end product of other iron oxides [29]. Magnetite is also regarded as black iron oxide, or magnetic iron ore, ferrite.

2. Toxicity of nanomaterials

5.4 Iron Oxide-based magnetic nanoparticles

111

It displays the most powerful magnetism of any metal oxide [30]. Oxidation of magnetite can lead to the formation of maghemite [31]. IO-NPs are manufactured for the biomedical application used for both diagnostic and therapeutic purposes, which are characteristically developed by a core of maghemite or magnetite.

5.4.1 In vitro toxicity studies of IO nanoparticles Most findings analyzing the cytotoxicity of IO-NPs focus on assessing reductions in the viability of these nanoparticles on cell cultures. The NIH3T3 cells were incubated with superparamagnetic IO-NPs (10 50 nm) for 3, 24, and 48 h for assessing the cell viability. For 3 and 24 h, no cytotoxicity was observed. However, after 48 h of incubation, a small reduction in cell viability was detected. During 48 h of incubation, the cells internalize nanoparticles, and the particles were accumulated in the perinuclear region toward the entire cytosol and large vesicles. After 48 h exposure, NIH3T3 treated with superparamagnetic IO-NPs decreased the activity rate of mitochondria [32]. In contrast, Feng et al. [33] used IO-NPs with PEGylated coated and polyethylenimine coated to study the in vitro cell uptake and cytotoxicity activity in SKOV-3 cells and RAW264.7 macrophages. In test concentrations (3.125 100 μg/mL) on SKOV-3 cancer cells and RAW264.7 macrophages, IO-NPs (10 nm) with polyethylenimine caused dose-dependent cytotoxicity. Most nuclei in SKOV-3 cells showed positive propidium iodide staining, after 16 h exposure to 5 μg/ mL IO-NPs (10 nm) with polyethylenimine, a sign of cell death. In both macrophages and cancer cells, the intake of polyethylenimine-coated IO-NPs was considerably greater than the level of PEGylated, and produced cytotoxicity by multiple methods, such as apoptosis and ROS production. Numerous in vitro studies evaluated the impact of IO-NPs; however, no genotoxicity was found in various studies [34,35]. Recently, a study showed activity in genotoxicity. Sonmez et al. [36] evaluated the toxicity of magnetite nanoparticles on cytotoxicity, genotoxicity, and oxidative damage using various magnetite concentrations (0 1000 mg/L) to treat whole blood cultures for 72 h. Cell viability was assessed by LDH release and MTT assay. The higher concentration of magnetite decreases cell viability. The magnetite concentration higher than 10 mg/L induces total oxidation level and decreased total antioxidant capacity level in human blood cells. The increased concentration of magnetite NPs showed significant increases in sister chromatid exchange, micronuclei, and chromosome aberration. Given these studies, the genotoxic potential of IO-NPs seems to be strongly induced by the concentrations and size. However, due to the lack of consistency in the outcomes obtained, further research is necessary in order to identify the particular processes of genotoxicity caused by these nanoparticles.

5.4.2 In vivo toxicity studies of IO nanoparticles Feng et al. [33] used PEGylated-coated IO-NPs (10 and 30 nm) and polyethyleniminecoated IO-NPs (10 nm) to study the in vivo toxicity, including distribution, biodegradation, and clearance. Biodistribution studies have shown that all IO-NPs tend to be distributed in the liver and spleen. The clearance and biodegradation of PEGylated IO-NPs were comparatively slow in the tissues ( . 2 weeks). They included the maximum tumor uptake

2. Toxicity of nanomaterials

112

5. Toxicity of metal oxide nanoparticles

was achieved by 10 nm PEGylated IO-NPs. No apparent toxicity for PEGylated IO-NPs was found in BALB/c mice for while the dose-dependent lethal toxicity of polyethylenimine-coated IO-NPs was found. The toxicity analysis in rats exposed to bulk and nanostructured iron oxide (30 1000 mg/kg body weight) in a 28 day period and investigated their effects on biomarkers of histopathology, biodistribution, and oxidative stress. Research on oxidative stress biomarkers has shown that lipid peroxidation has been significantly increased and decreased in the dose-dependent content of glutathione in the liver, brain, and the kidney of the treated groups. In addition, the activity of antioxidant enzymes, glutathione reductase, glutathione peroxidase, catalase, and glutathione S transferase were significantly elevated, but SOD effect prominently decreased in treated rat organs. Inductively coupled plasma-optical emission spectrometric (ICP-OES) analysis showed an accumulation of IO-NPs in the liver followed by kidney and brain based on the size and dose accumulation of IO-NPs [37]. Sundarraj et al. investigated the toxicity of IO-NPs (25 and 50 mg/kg) [38]. The histopathology finding showed that sloughing of germ cells, lesions like detachment and vacuolization were also noted in response to IONPs treatment. In addition, 25 and 50 mg/kg iron(III) oxide nanoparticles administration increased glutathione peroxidase activity, protein carbonyl content, lipid peroxidation, nitric oxide, and ROS with a concomitant reduction antioxidant levels vitamin C, catalase, glutathione, and SOD. The increased expression levels of cleaved-PARP cleaved-caspase-3 and Bax confirm the occurrence of apoptosis. In vivo studies with IO-NPs in mice and rats may conclude that the administration dose plays a critical role in oxidative stress and accumulation of iron.

5.5 Titanium dioxide Nanoparticles TiO2 is the oxide form of titanium and occurs naturally. It may be sourced from minerals such as anatase, rutile, and ilmenite. Anatase and brookite are rare polymorphs, whereas rutile is a natural form of TiO2 [39]. The most popular technique for producing TiO2 from the mineral limonite is sulfate and chloride. The manufacturing includes the transformation of ilmenite into aqueous titanium, sulfate solution, or titanium tetrachloride, which is converted into a crystalline form, characterized by size specific anatase or rutile kinds [40]. TiO2 NPs have attracted a considerable amount of attention in the industrial and scientific fields. Because of their unique properties (chemical composition, small size, solubility, surface structure, aggregation, and shape), TiO2 NPs are used in many applications, such as laminates, food wraps, lacquers, paints, foils, plastics, textiles, food products, cosmetics, transparent papers, medicines, toothpaste, and pharmaceuticals. They are also used in anti-fogging car mirrors, windows, self-cleaning tiles, sewage, purification of water, and combustion gases [41]. TiO2 NPs are the maximum manufactured nanomaterial with yearly manufacture maximum of 10,000 tons. Large production, extensive use, and uncontrolled disposal of TiO2 NPs will unavoidably result in environmental releases, in which individual organisms and ecosystems may be affected. In order to assess the health risk, it is important to know the impact of TiO2 NPs on organisms in vitro and in vivo.

2. Toxicity of nanomaterials

5.5 Titanium dioxide Nanoparticles

113

5.5.1 In vitro toxicity studies of TiO2 nanoparticles A wide range of studies came to a consensus that the anatase form of TiO2 NPs showed more toxicity compared to other forms of TiO2 NPs. In A549 cells and MCF-7 cells, De Matteis et al. [42] investigated the toxicity of rutile and anatase crystalline forms of TiO2 NPs. They found that in comparison to the rutile type, titanium ions were released more in anatase form. The A549 cell lines displayed considerable defiance to anatase and rutile NPs related to MCF-7. The TiO2 anatase induced higher ROS production in MCF-7 cell line, affected the mitochondrial membrane, and activated the apoptosis pathway. In cellular uptake study, MCF-7 cell line showed significant endocytosis to the anatase form. The crystal phase appeared to influence cell toxicity. Pedata et al. [43] examined the toxicity mechanism of TiO2 NPs using Caco-2 cell to 42 or 84 μg/mL TiO2 NPs for 4, 24, and 48 h exposure, and found that the intestinal epithelium layer was affected after 24 h exposure with 42 or 84 μg/mL TiO2 NPs and reduced about 13% cell viability. Transmission electron microscope analysis showed that the particles of TiO2 were trapped preferentially and internalized by monolayers of Caco-2. Analysis of ICP-OES showed titanium ions within the cells could trigger the production of pro-inflammatory cytokines. TiO2 NPs exert toxic effects on the intestinal epithelium layer. Stoccoro et al. studied the effect of citrate-coated TiO2 NPs exposure on human lung epithelial cell line A549. The citrate-coated nanoparticles exposures induced cytotoxic effects that were statistically significant. Investigation of DNA damage by comet assay showed that the genotoxic effects were increased by citratecoated nanoparticles. Changing the surface of coating nanoparticles influences the nanoparticle toxicity activity by changing their physicochemical properties [44]. Gea et al. showed the toxicity of TiO2 NPs in BEAS-2B cells using various forms (bipyramids, rods, and platelets). The cytotoxicity of the rod was higher than that of bipyramids or platelets. Platelets could cause direct genotoxicity and oxidative DNA damage. Bipyramids and rods did not induce oxidative stress or genotoxicity damage. The accumulation of platelets was higher than that of rod or bipyramids, which is correlated with genotoxicity [45].

5.5.2 In vivo toxicity studies of TiO2 nanoparticles So far, data on human absorption of TiO2 NPs through inhalation have not been available. However, there are more studies on rats and mice. Intravenous administration of TiO2 NPs (,100 nm) in male Wistar rats at a low dose (5 mg/kg body weight) for 1, 14, and 28 days were performed. Bioaccumulation studies showed that, in blood cells, plasma, brain, and lymph nodes, there was no detectable level of TiO2. But the bioaccumulation of titanium was found in kidney, lung, and spleen in a lower amount, compared to the liver. No modification in the cytokines and enzymes observed in male Wistar rats blood samples indicate that organ toxicity has not been detectable [46]. Younes et al. [47] intraperitoneally injected TiO2 NPs to a rat at a moderate dose (20 mg/kg) for 20 days. TiO2 NPs have been observed to induce liver changes, including congestion, prominent vasodilatation, and vacuolization, resulting in damage to liver function. Similar pathologies were found after the instillation of dissolved titanium ion into rat lungs. After 20 days of treatment, the enzyme ratio of aspartate aminotransferase/alanine aminotransferase and LDH activity increases, indicating the organ damage. High doses of TiO2 NPs (1387 mg/kg body

2. Toxicity of nanomaterials

114

5. Toxicity of metal oxide nanoparticles

weight) were introduced into mice intravenously, but the second day of injection resulted in mortality. While reduced doses (10 mg/kg body weight) triggered acute toxicity symptoms, such as decreased water and food consumption, it also enhanced the number of white blood cells and increased white blood cells. TiO2-treated mice spleen showed greater coefficients of body weight/tissue weight, compared to the kidney and liver. Interestingly, TiO2 NPs treatment caused damage to the kidney, lung, brain, spleen, and liver; however, no pathological effects were found in the heart [48]. Abdelgied et al. examined whether the crystalline form of TiO2 NPs induces pleural toxicity and pulmonary toxicity in rats. After treatment, the rutile TiO2 NPs treated rats showed normal external lung morphology, while the crystalline form of TiO2 NPs (80% anatase and 20% rutile) induced pulmonary toxicity in rats [49]. Mixed TiO2 NPs provide the most potent inflammatory response in the short term with the mixed sample [50]. In summary, a higher dose of TiO2 NPs showed toxicity. Dose and crystal phase influenced the toxicity. In vitro and in vivo anatase and a mixture of anatase phase showed higher toxicity.

5.6 Copper oxide nanoparticles Copper is the starting material for copper oxide (CuO) synthesis. Nanoparticles are abundant in nature as they are presented in different salts (chlorides, sulfates, and etc.). Copper oxide nanoparticles (CuO NPs) have attracted particular attention among different MO-NPs, because of its useful physical properties, such as spin dynamics, hightemperature superconductivity, and electron correlation effects [51,52]. In addition, CuO NPs are commonly added as materials to lubricants, plastics, ink, skin cosmetics, and metallurgical coatings [53]. Copper is available in two Cu11 and Cu21 states of oxidation. This enables copper to function in biochemical reactions as a reducing or oxidizing agent. However, this property also makes copper potentially toxic, because copper ions can induce oxidative stress [54], free radical production [55,56], and genotoxic substances [57].

5.6.1 In vitro toxicity studies of CuO nanoparticles With the widespread applications of CuO NPs, the biological consequences of exposure to CuO NPs need to be clearly understood. Numerous studies have recently examined the toxic effects of CuO NPs on in vitro cell lines and animals. Laha et al. [58] evaluated the toxic effects of CuO NPs on cellular viability, apoptosis, and cell cycle analysis in the MCF-7 cells. Morphological changes were observed using transmission electron microscopic, compared to control. CuO treated cells showed autophagic vacuoles and cell cycle arrested in G0, indicating the induction of apoptosis. Jing et al. [59] investigated the toxic effects on lung epithelial cells treated with CuO NPs 9.2 nm at different concentrations. The CuO NPs decreased cell cytotoxicity and increased the level of oxidative stress in dose-dependent levels. The toxicity of CuO NPs is related to different forms and particle sizes. CuO particles with two different sizes (6 nm CuO NPs and ,100 nm larger polydispersed CuO NPs), microparticles, and Cu ions were examined in epithelial kidney cell. Polydispersed CuO NPs was more toxic than any other Cu treatment tested.

2. Toxicity of nanomaterials

5.7 Toxicity mechanism of metal oxide nanoparticles

115

Polydispersed CuO NPs showed a substantial increase in intracellular ROS generation, DNA damage, and cell death [60].

5.6.2 In vivo toxicity studies of CuO nanoparticles CuO NPs instillation leads to a studied increase in the discharge of reliable pulmonary injury markers, LDH. After exposure to CuO NPs, the methylation status in the mouse lung was observed. In the mouse model, copper nanoparticles induced obviously epigenetic changes [61]. Lee et al. [62] used ICP-MS to detect copper from CuO NPs or copper microparticles in tissues samples taken in rats after 14 days oral exposure. Nanoparticles were observed at spleen, liver, kidney, brain, blood, lung, heart, urine, and feces samples in the CuO treated groups. A high level of copper was found in feces for CuO microparticles treated rat, while a lower level of copper was detected by CuO NPs treated rat. A high level of copper was found in kidney and liver for CuO microparticles treated rats. The rat treated with CuO NPs showed extensive damage to liver, thymus, red blood cells, kidney, and spleen, but the high-dose CuO microparticles did not cause adverse effects on rats. For five consecutive days, De Jong et al. [63] treated the rats with CuO NPs and tested the toxicity at the 6th and 26th day. Nanoparticles of CuO increased ALT, which is an indicator of liver damage. In the initial dose, no alteration of histopathology was observed in liver, bone marrow, and stomach at the highest dose 512 mg/kg. The copper ion released from copper nanoparticles severally affected the immune system by severe lymphoid cell depletion in thymus and spleen. It might be assumed that the dissolution and biodistribution act as a determining factor in the toxic responses of CuO particles in vivo.

5.7 Toxicity mechanism of metal oxide nanoparticles As we can see from the above, various nanoparticles of metal oxide have different toxicity. Scientists have explained some factors for nanoparticles toxicity based on the outcomes of nano size, the chemical composition of nanoparticles, concentration, dissolution, aggregation, and oxidative stress [64 66]. Nanoparticles might induce local and systemic toxicity dependent on the method of exposure, possibly due to increased oxidative stress causing vascular injury, fibrogenesis and tissue remodeling, blood clotting, immune response, inflammation, genotoxicity, and neurotoxicity [67 69]. The toxic effects of MONPs were summarized both in vitro (Fig. 5.2) and in vivo (Fig. 5.3). There are consensus nanoparticles frequently taken up via endocytosis. Internalization of nanoparticles through the entry pathway of endocytosis causes severe damage to the cell. Compared to the entry pathway of nonendocytosis. The endocytosis vesicle is possibly transformed into lysosomes by autophagosomes [70]. The lysosomes that carry the MONPs result in the liberation of ions, due to the lower pH acting as a Trojan horse. Nanoparticles facilitate the generation of free radicals, especially ROS that may cause oxidative stress [71].

2. Toxicity of nanomaterials

116

5. Toxicity of metal oxide nanoparticles

Mitochondrial impairment Reactive oxygen species

Cell death

DNA damage

Inflammation

In vitro study

Loss of membrane integrity

Lipid peroxidation

FIGURE 5.2 Toxicity mechanism of metal oxide nanoparticles (MO-NPs) in vitro.

Obstruction in intestine Genotoxicity

Delay in growth and reproduction

Accumulation in liver, kidney, and brain

Reactive oxygen species In vivo study

Mortality

Lung inflammation

FIGURE 5.3 Toxicity mechanism of metal oxide nanoparticles (MO-NPs) in vivo.

5.7.1 Oxidative stress ROS are chemically reactive chemical species, such as peroxides, superoxide, hydroxyl radical, alpha oxygen, and singlet oxygen. ROS is extremely reactive because of the appearance of unpaired valence shell electrons. ROS are formed as a byproduct of the normal metabolism of oxygen and plays a major role in apoptosis, cell signaling, and homeostasis. ROS levels are increased during exposure to environmental stress, which causes

2. Toxicity of nanomaterials

5.7 Toxicity mechanism of metal oxide nanoparticles

117

significant damage in cell structures. The situation is called oxidative stress. Excessive ROS causes polyunsaturated fatty acid peroxidation and damages the permeability of the cell membrane. Once the cell membrane is damaged, the MO-NPs penetrate the cell and attach to the organelles, causing effusion of cytoplasm and denaturation of protein [72]. Damaged cells produced a number of toxicants and affected organism systems. Recent studies have exposed that nanoparticles caused oxidative stress by increasing ROS and lipid peroxidation, but decreasing intracellular GSH [57,73]. The toxicity of nanoparticles strongly related to the ROS. For nanoparticles, there are other indirect pathways to promote redox cell imbalance. Nanoparticles may play a direct role in the formation of ROS. The oxidative stress created by MO-NPs was studied by Yu et al. He tested the effect of ZnO NPs against normal skin cells and explained the molecular mechanism in a normal skin cell model. He found that ZnO NPs accumulated in autophagic vacuoles lead to cell death and induce ROS which damages mitochondria in normal skin cells. The nanoparticles induce the production of ROS depending on the type of antioxidant response [74]. By exposing J774 cells, the oxidative stress posed by IO-NPs was explored. The amounts of OH in IO-NPs suspension were more, compared to control [75]. OH is regarded as one of the most toxic ROS, and can oxidize virtually all cellular components. The MO-NPs generate the extracellular OH by inducing oxidative damage, which can cause a toxic effect on the organism. A nanoparticle causes ROS to rise above the cells oxidative stress level and ultimately leads to apoptosis [76]. Apoptosis is activated by two major signaling pathways: the death receptor-mediated exogenous and the mitochondria-mediated endogenous pathways [77]. The mitochondria-mediated endogenous apoptotic pathway plays a significant role in MO-NPs induced cell death, which is the major target organelle for nanoparticles inducing oxidative stress. From mitochondrial chain, a small proportion of electron escapes and interacts with molecular oxygen to form O2, which later reduce to the damaging OH or give rise to H2O2. The generation of O2 is catalyzed by nanoparticles either by blocking the electron transport chain or by accelerating the electron to molecular oxygen [78,79]. Toxicity of magnetic IO-NPs tested against RAW264.7 cells. Cell viability decreases in a dose-dependent manner with the formation of autophagosome [80]. Roy et al. [81] investigated the enhancement of autophagy through P13K/mTOR/Akt inhibition. In ZnO NPs exposed to macrophages, the phosphorylated levels of Akt/mTOR/PI3K were significantly reduced. p53 is identified as unique of the key pathways triggered by nanoparticles. The apoptosis in RKO cell treated with ZnO NPs was observed by Moos et al. Cell toxicity was considered to be nanoparticles-dependent rather than soluble zinc salt exposure [82].

5.7.2 Metal ion toxicity Metal ions are often attributed to the toxicity of partially soluble MO-NPs. This assumption is usually evaluated by comparing the toxicity of MO-NPs with the soluble metal salt. Metal ion toxicity for CuO, ZnO, and antimony trioxide nanoparticles (Sb2O3 NPs) was reported and the highest dissolution was observed in CuO followed by Sb2O3 and ZnO [83]. The bioaccumulation of both MO-NPs and metal ions within bacterial cells is reported [84].

2. Toxicity of nanomaterials

118

5. Toxicity of metal oxide nanoparticles

Metal ion dissolution is associated with bulk and nanoparticles. Dimkpa et al. [85] confirmed the discharge of ions from ZnO NPs, CuO NPs, and bulk by centrifugation pellet from suspension. Cu ions release was higher in nanoparticles, when compared to the bulk. But, the bulk and ZnO NPs level were similar. The biological function of Cu ions release from CuO NPs can be eliminated by the bathocuproine and chelator specific to Cu ions. Another important factor of toxicity is metal ion dissolution. Compared to bulk ones, nanosized particles have a big surface area. ZnO NPs dissolve rapidly and inhibited the C. vulgaris growth rate, which indicated that ZnO NPs were likely a continuous source of Zn21 ions toxic to C. vulgaris [16]. The solubility of MO-NPs is related to the pH. Studer et al. (2010) studied the solubility of CuO NPs in two different pH (5.5 and 7.0). Copper ions were not found after 3 days at neutral pH. The particles of CuO dissolve rapidly to a pH 5.5 and even stable inside the lysosomes. Copper ions generated toxicity in Chinese hamster oocytes and Henrietta Lacks cells. The cellular membrane is an evolutionary obstacle for most ions. However, the membrane is easily penetrated by the soluble nanoparticles. The heavy metal ions released inside the cell can rupture lysosomes, therefore, harm the cell [86] or generate radicals within the cells [34,87]. It is understood that both dissolved ions and MO-NPs will influence the toxicity.

5.8 Conclusion Recent nanotechnology developments have facilitated the synthesis of novel nanoparticles with new and different physicochemical properties. In numerous applications, the use of MO-NPs is continually increasing, which also raises their chance of interacting with different ecosystem factors. The widespread use of MO-NPs in various consumer goods without following safety guidelines and regulations has become a growing global concern. MO-NPs simply enter into the cell because of their smaller size and are further dispersed to various organs causing cytotoxicity. Many studies have reported that the mechanism is involved in nanomaterial toxicity, such as oxidative stress, mitochondrial dysfunction, DNA damage, and cytomembrane. Thus, the toxicological aspects of MO-NPs need to be further explored, which can guarantee their secure applications for human advantage.

Acknowledgments D.S. Pei (corresponding author) and T.Y. Suman contributed equally to this work. The authors are thankful for the funding from the CAS Team Project of the Belt and Road (to D.S.P.), the Chongqing Key Program of Basic Research and Advanced Exploration Project (No. cstc2019jcyj-zdxmX0035), the Three Hundred Leading Talents in Scientific and Technological Innovation Program of Chongqing (No. CSTCCXLJRC201714), the postdoctoral scholarship program of Henan Normal University, Xinxiang, Henan (to T.Y. Suman), and the program of China-Sri Lanka Joint Research and Demonstration Center for Water Technology and China-Sir Lanka Joint Center for Education and Research by Chinese Academy of Sciences, China.

Conflicts of interest The authors declare no conflict of interest.

2. Toxicity of nanomaterials

References

119

References [1] M.A. Albrecht, C.W. Evans, C.L. Raston, Green chemistry and the health implications of nanoparticles, Green. Chem. 8 (5) (2006) 417 432. [2] Y. Liu, G. Wang, C. Li, Q. Zhou, M. Wang, L. Yang, A novel acetylcholinesterase biosensor based on carboxylic graphene coated with silver nanoparticles for pesticide detection, Mater. Sci. Eng. C 35 (2014) 253 258. [3] J. Sripriya, S. Anandhakumar, S. Achiraman, J.J. Antony, D. Siva, A.M. Raichur, Laser receptive polyelectrolyte thin films doped with biosynthesized silver nanoparticles for antibacterial coatings and drug delivery applications, Int. J. Pharm. 457 (1) (2013) 206 213. [4] M.E. Vance, T. Kuiken, E.P. Vejerano, S.P. McGinnis, M.F. Hochella Jr, D. Rejeski, et al., Nanotechnology in the real world: redeveloping the nanomaterial consumer products inventory, Beilstein J. Nanotechnol. 6 (1) (2015) 1769 1780. [5] F. Gottschalk, C. Ort, R.W. Scholz, B. Nowack, Engineered nanomaterials in rivers—exposure scenarios for Switzerland at high spatial and temporal resolution, Environ. Pollut. 159 (12) (2011) 3439 3445. [6] R.J. Aitken, M.Q. Chaudhry, A.B.A. Boxall, M. Hull, Manufacture and use of nanomaterials: current status in the UK and global trends, Occup. Med. 56 (5) (2006) 300 306. [7] Z. Sun, T. Liao, L. Kou, Strategies for designing metal oxide nanostructures, Sci. China Mater. 60 (1) (2017) 1 24. [8] O. Choi, Z. Hu, Size dependent and reactive oxygen species related nanosilver toxicity to nitrifying bacteria, Environ. Sci. Technol. 42 (12) (2008) 4583 4588. [9] H. Mirzaei, M. Darroudi, Zinc oxide nanoparticles: biological synthesis and biomedical applications, Ceram. Int. 43 (1) (2017) 907 914. [10] M.H. Huang, S. Mao, H. Feick, H. Yan, Y. Wu, H. Kind, et al., Room-temperature ultraviolet nanowire nanolasers, Science 292 (5523) (2001) 1897 1899. [11] L. Vayssieres, K. Keis, A. Hagfeldt, S.E. Lindquist, Three-dimensional array of highly oriented crystalline ZnO microtubes, Chem. Mater. 13 (12) (2001) 4395 4398. [12] A. Becheri, M. Du¨rr, P.L. Nostro, P. Baglioni, Synthesis and characterization of zinc oxide nanoparticles: application to textiles as UV-absorbers, J. Nanopart. Res. 10 (4) (2008) 679 689. [13] B. Ludi, M. Niederberger, Zinc oxide nanoparticles: chemical mechanisms and classical and non-classical crystallization, Dalton Trans. 42 (35) (2013) 12554 12568. [14] H. Ma, P.L. Williams, S.A. Diamond, Ecotoxicity of manufactured ZnO nanoparticles—a review, Environ. Pollut. 172 (2013) 76 85. [15] USFDA, Code of Federal Regulations, Title 21, Food and drug. ,http://www.accessdata.fda.gov/scripts/ cdrh/cfdocs/cfCFR/CFRSearch.cfm?fr 5 114.3,2., 2011. [16] T.Y. Suman, S.R. Rajasree, R. Kirubagaran, Evaluation of zinc oxide nanoparticles toxicity on marine algae Chlorella vulgaris through flow cytometric, cytotoxicity and oxidative stress analysis, Ecotoxicol. Environ. Saf. 113 (2015) 23 30. [17] A. Asati, S. Santra, C. Kaittanis, J.M. Perez, Surface-charge-dependent cell localization and cytotoxicity of cerium oxide nanoparticles, ACS Nano 4 (9) (2010) 5321 5331. [18] I. De Angelis, F. Barone, A. Zijno, L. Bizzarri, M.T. Russo, R. Pozzi, et al., Comparative study of ZnO and TiO2 nanoparticles: physicochemical characterisation and toxicological effects on human colon carcinoma cells, Nanotoxicology 7 (8) (2013) 1361 1372. [19] C.S. Kim, H.D. Nguyen, R.M. Ignacio, J.H. Kim, H.C. Cho, E.H. Maeng, et al., Immunotoxicity of zinc oxide nanoparticles with different size and electrostatic charge, Int. J. Nanomed. 9 (Suppl. 2) (2014) 195. [20] C.T. Ng, L.Q. Yong, M.P. Hande, C.N. Ong, L.E. Yu, B.H. Bay, et al., Zinc oxide nanoparticles exhibit cytotoxicity and genotoxicity through oxidative stress responses in human lung fibroblasts and Drosophila melanogaster, Int. J. Nanomed. 12 (2017) 1621. [21] K. Gerloff, C. Albrecht, A.W. Boots, I. Fo¨rster, R.P. Schins, Cytotoxicity and oxidative DNA damage by nanoparticles in human intestinal Caco-2 cells, Nanotoxicology 3 (4) (2009) 355 364. [22] F. Namvar, H.S. Rahman, R. Mohamad, S. Azizi, P.M. Tahir, M.S. Chartrand, et al., Cytotoxic effects of biosynthesized zinc oxide nanoparticles on murine cell lines, Evid. Based Complem. Alternat. Med. 2015 (2015) 593014. [23] V. Sharma, P. Singh, A.K. Pandey, A. Dhawan, Induction of oxidative stress, DNA damage and apoptosis in mouse liver after sub-acute oral exposure to zinc oxide nanoparticles, Mutat. Res. Genet. Toxicol. Environ. Mutagenesis 745 (1 2) (2012) 84 91.

2. Toxicity of nanomaterials

120

5. Toxicity of metal oxide nanoparticles

[24] T.R. Cardozo, R.F. De Carli, A. Seeber, W.H. Flores, J.A. da Rosa, Q.S. Kotzal, et al., Genotoxicity of zinc oxide nanoparticles: an in vivo and in silico study, Toxicol. Res. 8 (2) (2019) 277 286. [25] R. Abbasalipourkabir, H. Moradi, S. Zarei, S. Asadi, A. Salehzadeh, A. Ghafourikhosroshahi, et al., Toxicity of zinc oxide nanoparticles on adult male Wistar rats, Food Chem. Toxicol. 84 (2015) 154 160. [26] H.J. Ryu, M.Y. Seo, S.K. Jung, E.H. Maeng, S.Y. Lee, D.H. Jang, et al., Zinc oxide nanoparticles: a 90-day repeated-dose dermal toxicity study in rats, Int. J. Nanomed. 9 (Suppl. 2) (2014) 137. [27] A. Prina-Mello, K. Crosbie-Staunton, G. Salas, M. del Puerto Morales, Y. Volkov, Multiparametric toxicity evaluation of SPIONs by high content screening technique: identification of biocompatible multifunctional nanoparticles for nanomedicine, IEEE Trans. Magnet. 49 (1) (2013) 377 382. [28] N. Dissanayake, K. Current, S. Obare, Mutagenic effects of iron oxide nanoparticles on biological cells, Int. J. Mol. Sci. 16 (10) (2015) 23482 23516. [29] R.M. Cornell, U. Schwertmann, The Iron Oxides: Structure, Properties, Reactions, Occurrences and Uses, John Wiley & Sons, 2003. [30] P. Majewski, B. Thierry, Functionalized magnetite nanoparticles—synthesis, properties, and bio-applications, Crit. Rev. Solid. State Mater. Sci. 32 (3-4) (2007) 203 215. [31] H. Fischer, J. Luster, A.U. Gehring, EPR evidence for maghemitization of magnetite in a tropical soil, Geophys, J. Int. 169 (2007) 909 916. [32] G. Jarockyte, E. Daugelaite, M. Stasys, U. Statkute, V. Poderys, T.C. Tseng, et al., Accumulation and toxicity of superparamagnetic iron oxide nanoparticles in cells and experimental animals, Int. J. Mol. Sci. 17 (8) (2016) 1193. [33] Q. Feng, Y. Liu, J. Huang, K. Chen, J. Huang, K. Xiao, Uptake, distribution, clearance, and toxicity of iron oxide nanoparticles with different sizes and coatings, Sci. Rep. 8 (1) (2018) 2082. [34] H.L. Karlsson, J. Gustafsson, P. Cronholm, L. Mo¨ller, Size-dependent toxicity of metal oxide particles—a comparison between nano-and micrometer size, Toxicol. Lett. 188 (2) (2009) 112 118. [35] H.L. Karlsson, P. Cronholm, J. Gustafsson, L. Moller, Copper oxide nanoparticles are highly toxic: a comparison between metal oxide nanoparticles and carbon nanotubes, Chem. Res. Toxicol. 21 (9) (2008) 1726 1732. ¨ zbek, B. Togar, K. Meral, et al., Cytotoxicity and genotoxicity of iron [36] E. Sonmez, E. Aydin, H. Turkez, E. O oxide nanoparticles: an in vitro biosafety study, Arch. Biol. Sci. 68 (1) (2016) 41 50. [37] U.A. Reddy, P.V. Prabhakar, M. Mahboob, Biomarkers of oxidative stress for in vivo assessment of toxicological effects of iron oxide nanoparticles, Saudi J. Biol. Sci. 24 (6) (2017) 1172 1180. [38] K. Sundarraj, V. Manickam, A. Raghunath, M. Periyasamy, M.P. Viswanathan, E. Perumal, Repeated exposure to iron oxide nanoparticles causes testicular toxicity in mice, Environ. Toxicol. 32 (2) (2017) 594 608. [39] L.S. Dubrovinsky, N.A. Dubrovinskaia, V. Swamy, J. Muscat, N.M. Harrison, R. Ahuja, et al., Materials science: the hardest known oxide, Nature 410 (6829) (2001) 653. [40] L.L. Chang, Industrial Mineralogy: Materials, Processes, and Uses, Prentice Hall, 2002. [41] H. Shi, R. Magaye, V. Castranova, J. Zhao, Titanium dioxide nanoparticles: a review of current toxicological data, Part. Fiber Toxicol. 10 (1) (2013) 15. [42] V. De Matteis, M. Cascione, V. Brunetti, C.C. Toma, R. Rinaldi, Toxicity assessment of anatase and rutile titanium dioxide nanoparticles: the role of degradation in different pH conditions and light exposure, Toxicol. In Vitro 37 (2016) 201 210. [43] P. Pedata, G. Ricci, L. Malorni, A. Venezia, M. Cammarota, M.G. Volpe, et al., In vitro intestinal epithelium responses to titanium dioxide nanoparticles, Food Res. Int. 119 (2019) 634 642. [44] A. Stoccoro, S. Di Bucchianico, F. Coppede`, J. Ponti, C. Uboldi, M. Blosi, et al., Multiple endpoints to evaluate pristine and remediated titanium dioxide nanoparticles genotoxicity in lung epithelial A549 cells, Toxicol. Lett. 276 (2017) 48 61. [45] M. Gea, S. Bonetta, L. Iannarelli, A.M. Giovannozzi, V. Maurino, S. Bonetta, et al., Shape-engineered titanium dioxide nanoparticles (TiO2-NPs): cytotoxicity and genotoxicity in bronchial epithelial cells, Food Chem. Toxicol. 127 (2019) 89 100. [46] E. Fabian, R. Landsiedel, L. Ma-Hock, K. Wiench, W. Wohlleben, B. Van Ravenzwaay, Tissue distribution and toxicity of intravenously administered titanium dioxide nanoparticles in rats, Arch. Toxicol. 82 (3) (2008) 151 157. [47] N.R.B. Younes, S. Amara, I. Mrad, I. Ben-Slama, M. Jeljeli, K. Omri, et al., Subacute toxicity of titanium dioxide (TiO2) nanoparticles in male rats: emotional behavior and pathophysiological examination, Environ. Sci. Pollut. Res. 22 (11) (2015) 8728 8737.

2. Toxicity of nanomaterials

References

121

[48] J. Xu, H. Shi, M. Ruth, H. Yu, L. Lazar, B. Zou, et al., Acute toxicity of intravenously administered titanium dioxide nanoparticles in mice, PLoS One 8 (8) (2013) e70618. [49] M. Abdelgied, A.M. El-Gazzar, D.B. Alexander, W.T. Alexander, T. Numano, M. Iigou, et al., Pulmonary and pleural toxicity of potassium octatitanate fibres, rutile titanium dioxide nanoparticles, and MWCNT-7 in male Fischer 344 rats, Arch. Toxicol. 93 (4) (2019) 909 920. [50] D.B. Warheit, R.A. Hoke, C. Finlay, E.M. Donner, K.L. Reed, C.M. Sayes, Development of a base set of toxicity tests using ultrafine TiO2 particles as a component of nanoparticle risk management, Toxicol. Lett. 171 (3) (2007) 99 110. [51] F. Marabelli, G.B. Parravicini, F. Salghetti-Drioli, Optical gap of CuO, Phys. Rev. B 52 (3) (1995) 1433. [52] A. El-Trass, H. ElShamy, I. El-Mehasseb, M. El-Kemary, CuO nanoparticles: synthesis, characterization, optical properties and interaction with amino acids, Appl. Surf. Sci. 258 (7) (2012) 2997 3001. [53] N. Cioffi, N. Ditaranto, L. Torsi, R.A. Picca, L. Sabbatini, A. Valentini, et al., Analytical characterization of bioactive fluoropolymer ultra-thin coatings modified by copper nanoparticles, Anal. Bioanal. Chem. 381 (3) (2005) 607 616. [54] M.M.H.C.M. Valko, H. Morris, M.T.D. Cronin, Metals, toxicity and oxidative stress, Curr. Med. Chem. 12 (10) (2005) 1161 1208. [55] R. Ha¨nsch, R.R. Mendel, Physiological functions of mineral micronutrients (Cu, Zn, Mn, Fe, Ni, Mo, B, Cl), Curr. Opin. Plant. Biol. 12 (3) (2009) 259 266. [56] A. Ivask, O. Bondarenko, N. Jepihhina, A. Kahru, Profiling of the reactive oxygen species-related ecotoxicity of CuO, ZnO, TiO2, silver and fullerene nanoparticles using a set of recombinant luminescent Escherichia coli strains: differentiating the impact of particles and solubilised metals, Anal. Bioanal. Chem. 398 (2) (2010) 701 716. [57] M. Ahamed, R. Posgai, T.J. Gorey, M. Nielsen, S.M. Hussain, J.J. Rowe, Silver nanoparticles induced heat shock protein 70, oxidative stress and apoptosis in Drosophila melanogaster, Toxicol. Appl. Pharmacol. 242 (3) (2010) 263 269. [58] D. Laha, A. Pramanik, J. Maity, A. Mukherjee, P. Pramanik, A. Laskar, et al., Interplay between autophagy and apoptosis mediated by copper oxide nanoparticles in human breast cancer cells MCF7, Biochim. Biophys. Acta (BBA) Gen. Subj. 1840 (1) (2014) 1 9. [59] X. Jing, J.H. Park, T.M. Peters, P.S. Thorne, Toxicity of copper oxide nanoparticles in lung epithelial cells exposed at the air liquid interface compared with in vivo assessment, Toxicol. In Vitro 29 (3) (2015) 502 511. [60] A. Thit, H. Selck, H.F. Bjerregaard, Toxic mechanisms of copper oxide nanoparticles in epithelial kidney cells, Toxicol. In Vitro 29 (5) (2015) 1053 1059. [61] X. Lu, I.R. Miousse, S.V. Pirela, J.K. Moore, S. Melnyk, I. Koturbash, et al., In vivo epigenetic effects induced by engineered nanomaterials: a case study of copper oxide and laser printer-emitted engineered nanoparticles, Nanotoxicology 10 (5) (2016) 629 639. [62] I.C. Lee, J.W. Ko, S.H. Park, N.R. Shin, I.S. Shin, C. Moon, et al., Comparative toxicity and biodistribution assessments in rats following subchronic oral exposure to copper nanoparticles and microparticles, Part. Fiber Toxicol. 13 (1) (2016) 56. [63] W.H. De Jong, E. De Rijk, A. Bonetto, W. Wohlleben, V. Stone, A. Brunelli, et al., Toxicity of copper oxide and basic copper carbonate nanoparticles after short-term oral exposure in rats, Nanotoxicology 13 (1) (2018) 50 72. [64] S. Tedesco, H. Doyle, J. Blasco, G. Redmond, D. Sheehan, Oxidative stress and toxicity of gold nanoparticles in Mytilus edulis, Aquat. Toxicol. 100 (2) (2010) 178 186. [65] L. Yan, F. Zhao, S. Li, Z. Hu, Y. Zhao, Low-toxic and safe nanomaterials by surface-chemical design, carbon nanotubes, fullerenes, metallofullerenes, and graphenes, Nanoscale 3 (2) (2011) 362 382. [66] W. Zhuang, X. Gao, Methods, mechanisms and typical bioindicators of engineered nanoparticle ecotoxicology: an overview, Clean Soil Air Water 42 (4) (2014) 377 385. [67] M. Gasser, P. Wick, M.J. Clift, F. Blank, L. Diener, B. Yan, et al., Pulmonary surfactant coating of multiwalled carbon nanotubes (MWCNTs) influences their oxidative and pro-inflammatory potential in vitro, Part. Fibre Toxicol. 9 (1) (2012) 17. [68] F.A. Murphy, A. Schinwald, C.A. Poland, K. Donaldson, The mechanism of pleural inflammation by long carbon nanotubes: interaction of long fibers with macrophages stimulates them to amplify pro-inflammatory responses in mesothelial cells, Part. Fiber Toxicol. 9 (1) (2012) 8.

2. Toxicity of nanomaterials

122

5. Toxicity of metal oxide nanoparticles

[69] A.A. Shvedova, A. Pietroiusti, B. Fadeel, V.E. Kagan, Mechanisms of carbon nanotube-induced toxicity: focus on oxidative stress, Toxicol. Appl. Pharmacology 261 (2) (2012) 121 133. [70] Z. Wang, N. Li, J. Zhao, J.C. White, P. Qu, B. Xing, CuO nanoparticle interaction with human epithelial cells: cellular uptake, location, export, and genotoxicity, Chem. Res. Toxicol. 25 (7) (2012) 1512 1521. [71] O.S. Adeyemi, Y. Murata, T. Sugi, Y. Han, K. Kato, Modulation of host HIF-1α activity and the tryptophan pathway contributes to the anti-Toxoplasma gondii potential of nanoparticles, Biochem. Biophysics Rep. 11 (2017) 84 92. [72] G. Applerot, A. Lipovsky, R. Dror, N. Perkas, Y. Nitzan, R. Lubart, et al., Enhanced antibacterial activity of nanocrystalline ZnO due to increased ROS-mediated cell injury, Adv. Funct. Mater. 19 (6) (2009) 842 852. [73] M.J. Akhtar, S. Kumar, R.C. Murthy, M. Ashquin, M.I. Khan, G. Patil, et al., The primary role of ironmediated lipid peroxidation in the differential cytotoxicity caused by two varieties of talc nanoparticles on A549 cells and lipid peroxidation inhibitory effect exerted by ascorbic acid, Toxicol. In Vitro 24 (4) (2010) 1139 1147. [74] T. Xia, M. Kovochich, M. Liong, L. Madler, B. Gilbert, H. Shi, et al., Comparison of the mechanism of toxicity of zinc oxide and cerium oxide nanoparticles based on dissolution and oxidative stress properties, ACS Nano 2 (10) (2008) 2121 2134. [75] S. Naqvi, M. Samim, M.Z. Abdin, F.J. Ahmed, A.N. Maitra, C.K. Prashant, et al., Concentration-dependent toxicity of iron oxide nanoparticles mediated by increased oxidative stress, Int. J. Nanomed. 5 (2010) 983. [76] C. Stewart, K. Konstantinov, M. McDonald, K. Bogusz, D. Cardillo, S. Oktaria, et al., Engineering of bismuth oxide nanoparticles to induce differential biochemical activity in malignant and nonmalignant cells, Part. Part. Syst. Charact. 31 (9) (2014) 960 964. [77] S.A.A. Azim, H.A. Darwish, M.Z. Rizk, S.A. Ali, M.O. Kadry, Amelioration of titanium dioxide nanoparticles-induced liver injury in mice: possible role of some antioxidants, Exp. Toxicol. Pathol. 67 (4) (2015) 305 314. [78] J. Boonstra, J.A. Post, Molecular events associated with reactive oxygen species and cell cycle progression in mammalian cells, Gene 337 (2004) 1 13. [79] J.F. Turrens, Mitochondrial formation of reactive oxygen species, J. Physiol. 552 (2) (2003) 335 344. [80] E.J. Park, D.H. Choi, Y. Kim, E.W. Lee, J. Song, M.H. Cho, et al., Magnetic iron oxide nanoparticles induce autophagy preceding apoptosis through mitochondrial damage and ER stress in RAW264. 7 cells, Toxicol. In Vitro 28 (8) (2014) 1402 1412. [81] R. Roy, S.K. Singh, L.K.S. Chauhan, M. Das, A. Tripathi, P.D. Dwivedi, Zinc oxide nanoparticles induce apoptosis by enhancement of autophagy via PI3K/Akt/mTOR inhibition, Toxicol. Lett. 227 (1) (2014) 29 40. [82] P.J. Moos, K. Chung, D. Woessner, M. Honeggar, N.S. Cutler, J.M. Veranth, ZnO particulate matter requires cell contact for toxicity in human colon cancer cells, Chem. Res. Toxicol. 23 (4) (2010) 733 739. [83] A. Ivask, T. Titma, M. Visnapuu, H. Vija, A. Kakinen, M. Sihtmae, et al., Toxicity of 11 metal oxide nanoparticles to three mammalian cell types in vitro, Curr. Top. Med. Chem. 15 (18) (2015) 1914 1929. [84] B. Wu, Y. Wang, Y.H. Lee, A. Horst, Z. Wang, D.R. Chen, et al., Comparative eco-toxicities of nano-ZnO particles under aquatic and aerosol exposure modes, Environ. Sci. Technol. 44 (4) (2010) 1484 1489. [85] C.O. Dimkpa, A. Calder, D.W. Britt, J.E. McLean, A.J. Anderson, Responses of a soil bacterium, Pseudomonas chlororaphis O6 to commercial metal oxide nanoparticles compared with responses to metal ions, Environ. Pollut. 159 (7) (2011) 1749 1756. [86] T.J. Brunner, P. Wick, P. Manser, P. Spohn, R.N. Grass, L.K. Limbach, et al., In vitro cytotoxicity of oxide nanoparticles: comparison to asbestos, silica, and the effect of particle solubility, Environ. Sci. Technol. 40 (14) (2006) 4374 4381. [87] K. Midander, P. Cronholm, H.L. Karlsson, K. Elihn, L. Mo¨ller, C. Leygraf, et al., Surface characteristics, copper release, and toxicity of nano-and micrometer-sized copper and copper (II) oxide particles: a crossdisciplinary study, Small 5 (3) (2009) 389 399.

Further reading M. Amde, J.F. Liu, Z.Q. Tan, D. Bekana, Transformation and bioavailability of metal oxide nanoparticles in aquatic and terrestrial environments. A review, Environ. Pollut. 230 (2017) 250 267.

2. Toxicity of nanomaterials

Further reading

123

X. Jia, S. Wang, L. Zhou, L. Sun, The potential liver, brain, and embryo toxicity of titanium dioxide nanoparticles on mice, Nanoscale Res. Lett. 12 (1) (2017) 478. R.J. Miller, H.S. Lenihan, E.B. Muller, N. Tseng, S.K. Hanna, A.A. Keller, Impacts of metal oxide nanoparticles on marine phytoplankton, Environ. Sci. Technol. 44 (19) (2010) 7329 7334. V.V. Mody, R. Siwale, A. Singh, H.R. Mody, Introduction to metallic nanoparticles, J. Pharm. Bioallied Sci. 2 (4) (2010) 282. K.W. Ng, S.P. Khoo, B.C. Heng, M.I. Setyawati, E.C. Tan, X. Zhao, et al., The role of the tumor suppressor p53 pathway in the cellular DNA damage response to zinc oxide nanoparticles, Biomaterials 32 (32) (2011) 8218 8225. H.G. Vo¨lz, J. Kischkewitz, P. Woditsch, A. Westerhaus, W.D. Griebler, M. De Liedekerke, et al., Pigments, inorganic, Ullmann’s Encyclopaedia of Industrial Chemistry, Wiley-VCH Verlag GmbH, 2006.

2. Toxicity of nanomaterials

C H A P T E R

6 Toxicity of silver and other metallic nanoparticles T. Umasankareswari1, Gurmeet Singh2, S. Santhana Prabha3, Abdulhameed Al-Hashem4, S. Senthil Kumaran5 and Susai Rajendran6 1

Department of Chemistry, Rajapalayam Rajus College, Rajapalayam, India 2Pondicherry University, Puthucherry, India 3PSNA College of Engineering and Technology, Dindigul, India 4 Petroleum Research Centre, Kuwait Institute for Scientific Research, Safat, Kuwait 5 School of Mechanical Engineering, VIT University, Vellore, India 6Corrosion Research Centre, Department of Chemistry, St Antony’s College of Arts and Sciences for Women, Dindigul, India

6.1 Introduction In recent years, research on the synthesis of nanoparticles (NPs) has been growing exponentially. NPs are small in size and have extraordinary physicochemical properties. A diversity of NPs, that is, metallic, carbon-based, fluorescent, and polymer-based have been exploited in different fields such as tissue engineering, drug delivery, and various other therapeutic applications. Instead of multidisciplinary applications of NPs, research dealing with the toxicity concerns and the influence of such materials, on the public health, plants, and environment is still in its infancy. NPs can cause injury at the cellular, subcellular, molecular, and protein levels owing to their smaller size, large surface area to volume ratio, shape, and surface functionality. Extensive information related to NPs toxicology, the mechanisms of action, routes of their entry into the body and probable impacts on human health are the need of the hour [1]. The toxicity of various metallic NPs are discussed in this section.

Nanotoxicity DOI: https://doi.org/10.1016/B978-0-12-819943-5.00006-3

125

Copyright © 2020 Elsevier Inc. All rights reserved.

126

6. Toxicity of silver and other metallic nanoparticles

6.2 Toxicity of silver nanoparticles Silver nanoparticles (AgNPs) have become favorable as antibiotic agents in textiles and wound dressings, medical strategy, and appliances such as refrigerators and washing machines. The number of AgNP-containing products has grown from less than 30 in 2006 to over 300 at the beginning of 2011. They are most often employed as bacteriostatic coatings for preventing infections or as deodorants. About 280 tons of AgNPs were shaped for use in commercial or industrial products and that number is expected to quadruple by 2015 [2]. The number of applications for AgNPs will continue to grow. However, there is still much that needs to be understood with respect to their fate and buildup in the environment and their potential long-term effects on humans and other organisms. Recent research shows that the release of AgNPs into the environment is growing. There are large gaps in our considerate of how these particles are transported through ecosystems and migrate into the food chain and their cost on human health. At the cellular level, many mechanisms of AgNPs toxicity have been reported. This includes reactive oxidative species (ROS) generation, DNA damage, and cytokine induction during in vitro studies. The few in vivo studies reveal the potential for unpleasant effects at the organismic level, with vulnerabilities in the circulatory, respiratory, central nervous, hepatic, and dermal systems. More studies are needed to determine the biodistribution and subsequent toxicity of AgNPs using in vivo systems. These future studies should include modeling of the toxicological impact of AgNPs leached from textiles, a major anthropogenic silver source. The physicochemical properties of AgNPs are also significant factors and should be monitored during the course of a toxicological study to assess the effects of any physical changes on NP uptake and bioavailability. While AgNPs present some challenges for traditional toxicological assays, they also have exclusive qualities that enable entirely new approaches to examine their toxicological impact on cells and organisms. This includes the use of self-referencing microsensors for real-time physiological sensing and novel imaging modalities that take advantage of the strong plasmon resonances produced by AgNPs, permitting their tracking in a label-free manner. These newly developed tools can easily be incorporated into experimental designs that will enhance the superiority of risk assessment of AgNPs.

6.2.1 Biocorona formation contributes to silver nanoparticle induced endoplasmic reticulum stress Persaud et al. [3] have investigated rat aortic endothelial cells (RAEC) exposed to 20 or 100 nm AgNPs with or without a biocorona (BC) consisting of bovine serum albumin (BSA), high-density lipoprotein or fetal bovine serum (FBS) to form a complex BC. The presence of a BC consisting of BSA or FBS proteins significantly reduced uptake of 20 and 100 nm AgNPs in RAEC. Western blot analysis indicated robust activation of the IREα and PERK pathways in RAEC exposed to 20 nm despite the reduction in uptake by the presence of a BC. This was not noticed for the 100 nm AgNPs. Hyperspectral darkfield microscopy established that the preformed BC was maintained following uptake by RAEC. Transmission electron microscopy demonstrated a size dependent effect on the subcellular localization of AgNPs. Overall, these results put forward that AgNP size,

2. Toxicity of nanomaterials

6.2 Toxicity of silver nanoparticles

127

surface area, and BC formation govern the induction of ER stress and alterations in intracellular trafficking.

6.2.2 Silver nanoparticles to prepare scaffolds for the regeneration of infected full-thickness skin defects AgNPs have been widely used in antibacterial applications to solve the antibiotic resistance problems. Sanghuangporus sanghuang polysaccharides (FSHPs) were used as a green reducing agent to prepare AgNPs with a size of 3 35 nm. The FSHPs-silver nanoparticles (FSHPs-Ag) composite with chitosan solution were then freeze-dried to obtain a porous sponge dressing of chitosan-FSHPs-Ag (CS-FSHPs-Ag). This investigation reveals that the material showed almost no toxicity in L929 cells. To conclude, this material was used for dressing animal wounds [4].

6.2.3 Responses of flocculated activated sludge to bimetallic Ag-Fe nanoparticles toxicity Ever-increasing production and use of NPs have aroused overarching concerns for their toxic effects on the environment and humans. Yazdanbakhsh et al. have investigated the toxic effects of silver (Ag) and iron (Fe) NPs on the function of activated sludge, under continuous aerobic/anoxic/anaerobic conditions in laboratory-scale sequencing batch reactors (SBRs). The metagenome analysis confirmed a marked shift in the microbial community structure signifying that both heterotrophic and autotrophic communities were affected by the presence of Ag-Fe NPs. This study offers proof for compounded effects of NPs in their simultaneous presence, and generates new leads for future research activities [5].

6.2.4 Influence of addition of L-cysteine into inositol hexaphosphate (IP6) modified Ag nanoparticles Cu21 ions threaten human health by way of cellular toxicity, liver damage, and neurodegenerative diseases. Hence, developing detection method for trace copper ions (Cu21) has become a primary concern. Addition of L-cysteine (L-Cys) into inositol hexaphosphate (IP6) modified AgNPs (designated as L-Cys/IP6@Ag) induces a certain aggregation of NPs, resulting in an increase in the surface enhanced Raman scattering (SERS) signal of rhodamine 6G (R6 G). The presence of Cu21 ions are adsorbed by IP6 and the concentrated Cu21 ions further selectively oxide Cys molecules under a specific pH value, which disperses the IP6@Ag again and decreases the SERS signal of R6 G. The L-Cys/IP6@Ag-R6 G-based SERS sensor has been fruitfully used to detect trace Cu21 in river water [6].

6.2.5 Silver accumulation in adults and abnormal embryo development in their offspring Toxicity of AgNPs to aquatic organisms has been widely investigated. The cytotoxicity of poly-N-vinyl-2-pirrolidone/polyethyleneimine (PVP/PEI) coated 5 nm AgNPs in hemocytes

2. Toxicity of nanomaterials

128

6. Toxicity of silver and other metallic nanoparticles

exposed in vitro has been screened and the effects of dietary exposure to AgNPs on mussels growth, immune status, gonad condition, reproductive success, and offspring embryo development have been assessed by Duroudier et al. [7]. AgNPs taken up through the diet can appreciably affect ecologically relevant endpoints such as reproduction success and embryo development in marine mussels.

6.2.6 Toxicity of silver nanoparticles released by Hancornia speciosa (Mangabeira) biomembrane Almeida et al. have reported [8] that latex from diverse species is able to create tissue replacement and regeneration. Particularly, biomembranes obtained from Hancornia speciosa latex (HSB) have shown high angiogenic and osteogenic activity. Considering novel materials for wound healing, it would be attractive to develop a product combining antibacterial and antifungal properties. AgNPs have been usually used for this purpose in medicinal products and strategy for decades. In order to unite angiogenic, antibacterial, and antifungal properties on the same platform, Almeida et al. have developed an HSB containing three concentrations of AgNP. It was observed that the HSB successfully accommodated the AgNP in the matrix and released them in a controlled manner. The release dynamics of AgNP by HSB was described by UV vis absorption spectroscopy. The cytotoxic and genotoxic effects were determined using the Allium cepa assay. The results revealed no cytotoxic effect of HSB-AgNP in all studied concentrations [8].

6.3 Toxicity of gold nanoparticles 6.3.1 Toxicity of gold nanoparticles on human health and environment Gold nanoparticles (AuNPs) have extensive uses. However, their toxicity on human health and the environment has been established beyond doubt. Hence there is pressing need to remove AuNPs from environment. The hierarchically porous poly(ethylenimine) modified poly (styrene-co-divinylbenzene) microsphere (PEI-PS-DVB) was organized and characterized by techniques such as scanning electron microscopy, X-ray diffraction (XRD), transform infrared spectrometry, and energy dispersive X-ray spectrometry. The developed hierarchically porous PEI-PS-DVB was a gifted adsorbent for AuNPs with high adsorption capacity, and recycling usage of waste AuNPs conformed to the green and sustainable concept [9].

6.3.2 Gold nanoparticles and quantification of individual mycotoxin concentrations Due to the broadly occurring co-contamination of mycotoxins in raw food materials, simultaneous monitoring of multiple mycotoxins is needed. Lu and Gunasekaran have reported [10] the design and fabrication of an electrochemical immunosensor for simultaneous detection of two mycotoxins, fumonisin B1 (FB1) and deoxynivalenol (DON), in a single test. A dual-channel three-electrode electrochemical sensor pattern was etched on a transparent indium tin oxide coated glass via photolithography and was integrated with

2. Toxicity of nanomaterials

6.3 Toxicity of gold nanoparticles

129

capillary-driven polydimethylsiloxane (PDMS) microfluidic channel. The two working electrodes were functionalized with gold nanoparticles and anti-FB1 and anti-DON antibodies. Tests were conducted by incubating the working electrodes in a sample solution introduced in the PDMS channel. The creation of toxin-antibody immunocomplexes on the working electrode surface created electrochemical signal responses, which were recorded and compared with control signal to quantify individual mycotoxin concentrations [10].

6.3.3 Interaction of gold nanoparticles with nanoparticles of silver and titanium dioxide Global manufacturer of engineered nanoparticles (ENPs) continues to increase due to the demand of enabling properties in consumer products and industrial uses. Release of individual or aggregates of ENPs have been shown to interact with one another consequently resulting in adverse biological effects. Sharma et al. [11] have discussed AgNPs, which are currently used in numerous applications, including but not limited to antibacterial action. As a result, the release of AgNPs into the aquatic environment, the dissociation into ions, the binding to organic matter, reactions with other metal-based materials, and disruption of normal biological and ecological processes at the cellular level are all potential negative effects of AgNPs usage. The probable sources of AgNPs include leaching of intact particles from consumer products, disposal of waste from industrial processes, intentional release into contaminated waters, and the natural formation of AgNPs in surface and ground water. Further, Sharma et al. have presented current knowledge on the interactions of AgNPs with gold nanoparticles (AuNPs) and titanium dioxide nanoparticles (TiO2 NPs). The interaction between AgNPs and AuNPs result in stable bimetallic Ag-Au alloy NPs. Whereas the interaction of AgNPs with TiO2 NPs under dark and light conditions results in the release of Ag 1 ions, which may be subsequently converted back into AgNPs and adsorb on TiO2 NPs. The possible chemical mechanisms and toxic effects of AgNPs with AuNPs and TiO2 NPs have been discussed.

6.3.4 Differential cytotoxicity effect of dragon fruit extract capped gold nanoparticles on breast cancer cells Biosynthesis of gold nanoparticles from dragon fruit (DF) extract can be considered as an ecofriendly alternative for synthesis of gold nanoparticles. The ability of this fruit to synthesize gold nanoparticles and its anticancer activity has been investigated by Divakaran et al. [12]. The synthesized gold nanoparticles have been characterized by UV vis spectrophotometry, XRD, Fourier transform infrared spectroscopy, and transmission electron microscopy. The DF extract and DF-AuNPs induced important growth inhibition of MCF-7 breast cancer cells however, no significant toxicity effect was observed on MDA-MB-231 cells.

6.3.5 Anticancer activity of gold nanoparticles prepared by using fruit extract of Lycium chinense Chokkalingam et al. [13] have proposed a simple green approach for the synthesis of gold (Au) and silver (Ag) nanoparticles which were produced by using Lycium chinense

2. Toxicity of nanomaterials

130

6. Toxicity of silver and other metallic nanoparticles

(LC) fruit extract as a reducing and stabilizing agent. The synthesized NPs were characterized by relevant surface plasmon resonance (SPR) peaks for gold and silver NPs at 536 and 480 nm, respectively, with an UV vis spectrophotometer. LC-prepared silver NPs (LCAgNPs) antimicrobial activity demonstrated inhibitory activity against pathogenic microorganisms such as Escherichia coli and Staphylococcus aureus. The synthesized LC-prepared gold NPs (LC-AuNPs) did not show inhibitory activity. The LC-AgNPs exhibited major cytotoxicity to the human breast cancer MCF7 cell line and less cytotoxicity to nondiseased RAW264.7 (murine macrophage) cells, whereas LC-AuNPs showed minimal toxicity to both cell lines. The quickly synthesized NPs could play a role in the field of nanotechnology and in biomedical uses.

6.3.6 Mechanism of intracellular uptake and localization and the subsequent toxicity of nanoparticles Physicochemical characteristics of NPs have been shown to modify the uptake and toxicity of NPs. Vetten and Gulumian [14] have investigated the uptake of six gold nanoparticles (AuNPs) into the human bronchial epithelial cell line BEAS-2B. The AuNPs studies included colloidal citrate-stabilized AuNPs of 14 nm in diameter; and 14 nm AuNPs conjugated to functional groups via polyethylene glycol (PEG), namely hydroxyl-PEG (POH), carboxyl-PEG (PCOOH), biotin-PEG (PBtn), nitrilotriacetic acid-PEG (PNTA), and azidePEG (PAZ). An examination into the energy dependence of uptake of the citrate-stabilized and PCOOH AuNPs revealed that uptake was an active process. Cells pretreated with either chlorpromazine or genistein as endocytosis inhibitors for clathrin- and caveolae-mediated pathways, respectively, prior to addition of AuNPs, recommended a caveolae-dependent mechanism of endocytosis. These results further sustain recent findings on the mechanism of intracellular uptake and localization and the following toxicity of NPs.

6.3.7 The interaction strategy of diosmin functionalized gold nanoparticles with calf thymus DNA The interactions of natural drug functionalized metal NPs with DNA play a pivotal role in developing effective therapeutic agents that have a wide range of potential biomedical applications. Thomas et al. [15] have undertaken a study to decipher the binding mechanism of diosmin-capped gold nanoparticles (DM-AuNPs) with calf thymus DNA (ctDNA) through a combination series of spectroscopic and calorimetric studies. The gold nanoparticles were synthesized by the facile one-pot synthesis using DSM as a capping and reducing agent. The DM-AuNPs were characterized using UV visible spectroscopy, XRD, FTIR, DLS, and HRTEM analysis confirming the formation of stable AuNPs with an average size of 30 6 3 nm. The MTT assay revealed a moderate antiproliferative and toxicity effects of DM-AuNPs on MCF-7 and normal human cell lines. In this context the entire results based on ctDNA-DM-AuNPs binding mechanism may facilitate sensible synthesis and design of various synthetic/natural drug functionalized NPs possessing better therapeutic and sensing efficacy with minimal toxicity.

2. Toxicity of nanomaterials

6.3 Toxicity of gold nanoparticles

131

6.3.8 Effects and bioaccumulation of gold nanoparticles in the gilthead seabream (Sparus aurata) Gold nanoparticles (AuNPs) are found in a wide range of applications and therefore expected to present increasing levels in the environment. There is however limited knowledge concerning the potential toxicity of AuNPs as well as their combined effects with other pollutants. Hence, Barreto et al. have undertaken a study to investigate the effects of AuNPs alone and combined with the pharmaceutical gemfibrozil (GEM) on different biological responses (behavior, neurotransmission, biotransformation, and oxidative stress) in one of the most consumed fish in southern Europe, the seabream Sparus aurata. Fish were exposed for 96 h to waterborne 40 nm AuNPs with two coatings—citrate and polyvinylpyrrolidone (PVP), alone or combined with GEM. Antioxidant defences were induced in liver and gills upon both AuNPs exposure. Decreased swimming performance (1600 μg/L) and oxidative damage in gills (4 and 80 μg/L) were observed following exposure to polyvinylpyrrolidone-coated gold nanoparticles (PVP-AuNPs). Generally, accumulation of gold in fish tissues and deleterious effects in S. aurata were higher for PVP-AuNPs than for cAuNPs exposures. Although AuNPs and GEM combined effects in gills were generally low, in liver, they were higher than the predicted. The accumulation and effects of AuNPs showed to be dependent on the size, coating, surface charge, and aggregation/ agglomeration state of NPs. Additionally, it was tissue specific and dependent on the presence of other contaminants. Although, gold intake by humans is expected to not exceed the estimated tolerable daily intake, it is highly recommended to keep it on track due to the increasing use of AuNPs [16].

6.3.9 Application of gold nanoparticles in drug uptake and induction of cell death on breast cancer cell line Linalool is a monoterpene alcohol that occurs naturally in several aromatic plants. Jabir et al. have undertaken a study to load Linalool on gold nanoparticles, conjugate the complex with CALNN peptide, and investigate them for in-vitro anticancer activities against breast cancer (MCF-7) cell line. Linalool was obtained with 98% purity while gold nanoparticles and CALNN peptide were chemically synthesized. The formation of LIN-AuNPs and LIN-AuNPs-CALNN was observed through a color change. These compounds were confirmed and characterized using SEM, DLS, AFM, UV vis spectrophotometer, XRD, and FTIR. The free radical scavenging potential of each compound was confirmed based on its stable antioxidant effects using different parameters. Blood compatibility on red blood cells was confirmed by hemolytic and in vitro cytotoxicity assays. The in vitro anticancer activity of each compound toward MCF-7 cell line was investigated using various parameters. The study revealed that Linalool, AuNPs, LIN-AuNPs, and LIN-AuNPsCALNN were found to exert cell growth arrest against MCF-7 cell line. The antiproliferative effect of these compounds was due to cell death and induction of apoptosis confirmed using acridine orange-Ethidium bromide dual staining, DAPI staining, and electrophoresis analysis of DNA fragmentation. High fluorescent signals specific for the cellular uptake of LIN-AuNPs and LIN-AuNPs-CALNN into the cytoplasm of the cell line were confirmed. To study the toxicity of LIN-AuNPs-CALNN in animal models, the hematological,

2. Toxicity of nanomaterials

132

6. Toxicity of silver and other metallic nanoparticles

histopathological, and body weight changes were estimated after 4 weeks of intraperitoneal injection of the compounds into the animal models. The investigation reveals that Linalool, AuNPs, Linalool-AuNPs, and Linalool-AuNPs-CALNN peptide had no side effects and could be clinically used for future therapeutic purposes [17].

6.3.10 Gold nanoparticles in ophthalmology Numerous research projects are underway to improve the diagnosis and therapy in ophthalmology. In fact, visual acuity deficits affect 285 million people worldwide and different strategies are being developed to strengthen patient care. One of these strategies is the use of gold nanoparticles (AuNP) for their multiple properties and their ability to be used as both diagnosis and therapy tools. The exploration of Masse et al. [18] details research developing AuNPs for use in ophthalmology. The toxicity of AuNPs and their distribution in the eye are described through in vitro and in vivo studies. All publications addressing the pharmacokinetics of AuNPs administered in the eye have been analyzed critically. Besides, their use as biosensors or for imaging with optical coherence tomography is illustrated. The future of AuNPs for ophthalmic therapy is also discussed. AuNPs can be used to deliver genes or drugs through different administration routes. Their antiangiogenic and antiinflammatory properties are of great interest for different ocular pathologies. In conclusion, AuNPs can be used to improve stereotactic radiosurgery, brachytherapy, and photothermal therapy (PTT) because of their lots of properties.

6.4 Toxicity of copper nanoparticles 6.4.1 Rapid and selective detection of trace Cu21 in sea water Developing detection method for trace copper ions (Cu21) has attracted great attention since Cu21 ions threaten human health, in terms of cellular toxicity, liver damage, and neurodegenerative diseases. Liu et al. [6] have reported that adding L-cysteine (L-Cys) into inositol hexaphosphate (IP6) tailored AgNPs (designated as L-Cys/IP6@Ag) induces a certain aggregation of nanoparticles, resulting in an increase in the SERS signal of rhodamine 6G (R6 G). The presence of Cu21 ions are adsorbed by IP6 and the concentrated Cu21 ions further selectively oxide Cys molecules under a certain pH value, which disperses the IP6@Ag again and decreases the SERS signal of R6 G. The linear range of determination of Cu21 ions is from 1025 to 1021 M as well as a low detection concentration is of 10 pM. Furthermore, the L-Cys/ IP6@Ag-R6 G-based SERS sensor has been fruitfully applied to detect trace Cu21 in river water.

6.4.2 Quaternized chitosan-stabilized copper sulfide nanoparticles for cancer therapy Huang et al. [19] have reported a smart and green strategy to synthesize copper sulfide nanoparticles (CuS-NPs) for clinically translatable cancer treatment. For the first time, the preparation of CuS-NPs was developed by taking advantage of the copper-amine complex

2. Toxicity of nanomaterials

6.4 Toxicity of copper nanoparticles

133

as the copper source and sodium sulfide as the sulfide source, in which the quaternized chitosan (QCS) was used as a biotemplate and stabilizing agent. The obtained QCS/CuSNPs composites (CuS@QCS-NPs) were spherical and stable with an average diameter of 5.6 nm, and showed strong NIR absorbance for photothermal conversion. Moreover, in vitro and in vivo cancer theranostic capability of CuS@QCS-NPs without any biomodification was evaluated. The study revealed that after intratumoral (i.t.) injection of CuS@QCS-NPs with NIR laser irradiation (808 nm, 1.5 W/cm2, 5 min), the 4T1 mammary tumor growth could be effectively suppressed comparing with the other control groups, and there was no obvious lethal toxicity regarding liver function, kidney function, and vital organs. Such QCS-stabilized CuS-NPs may offer an alternative for clinical application of CuS-based PTT.

6.4.3 Copper nanoparticles and osteoblast response The development of Cu-incorporated micro/nano-topographical bioceramic coatings for enhanced osteoblast response has been investigated by Huang et al. [20]. In order to take recompense of the structural complexity of hierarchical topography and benefits of Cu, novel Cu-incorporated micro/nano-topographical coatings were developed on titanium (Ti) substrates using microarc oxidation (MAO) and hydrothermal treatment. A postheat treatment was employed to alter the morphology of the nanostructures and modulate the release of Cu21. The viability of cells was significantly inhibited on MAO-HT surface [a thin layer of nanoparticles (20 nm in diameter) composed of CuO and calcium silicate hydrate formed on MAO surface] due to the cytotoxicity of high concentration Cu21 release. However, the attachment, proliferation, and differentiation of SaOS-2 cells were enhanced on MAO-HT2 surface compared to MAO surface. The results show that modifying Ti surface with hierarchical micro/nanotopography could lead to enhanced osteoblast response.

6.4.4 Copper nanoparticles and immune response in the liver of juvenile Takifugu fasciatus Cu NPs are a new pollutant in aquaculture. They present a hazard to aquatic organisms. Wang et al. have investigated the effects of Cu NPs exposure on oxidative stress, apoptosis, and immune response in an economically important model species, Takifugu fasciatus. The juvenile fish were exposed to control, 20 or 100 μg Cu NPs/L for 30 days. The growth of T. fasciatus was inhibited after Cu NPs exposure. Copper buildup in liver increased with increasing Cu NPs dose. Oxidative stress indicators [malondialdehyde (MDA), total superoxide dismutase (T-SOD), catalase (CAT), and glutathione], apoptosis index and activities of caspases (caspase-3, caspase-9) all increased with the increase of Cu NPs concentration in the liver. With an increase in Cu NPs dose, the activities of succinate dehydrogenase and Na 1 -K 1 -ATPase as well as cytochrome c (Cyt-c) concentration in mitochondria decreased, accompanied by increased Cyt-c concentration in cytosol. Apoptosis-related gene expressions of p53, caspase-3, caspase-9, and Bax increased with the increase of Cu NPs dose. However, the opposite result was found in Bcl2 expression.

2. Toxicity of nanomaterials

134

6. Toxicity of silver and other metallic nanoparticles

The physiological indicators of immune response [heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), immunoglobulin M (IgM), and lysozyme (LZM)] as well as the mRNA levels of HSP70, HSP90, IgM, and C-LZM all increased after Cu NPs exposure. These results are helpful in understanding the mechanism of Cu NPs toxicity in T. fasciatus [21].

6.4.5 Copper sulfide nanoparticles for T1-weighted magnetic resonance imaging guided photothermal cancer therapy Copper sulfide nanoparticles (CuS NPs) have attracted considerable interest in the PTT field due to its low cost, easy preparation and favorable photothermal effect. However, lack of reliable visualization and relatively poor biocompatibility restrict its further bioapplication. To overcome these limitations, polydopamine (PDA, a melanin-like biopolymer) stabilized CuS NPs and further chelated with iron ions (denoted as CuPDF) were designed as a versatile nanoplatform for T1-weighted MR imaging-guided PTT. In this system, PDA served as both biotemplate to synthesis CuS NPs and an active platform to give MRI diagnostic capability. The as-prepared CuPDF NPs demonstrated strong absorption at NIR region, nearly three times higher than that of pure PDA NPs at 808 nm. Moreover, toxicity studies and histology evaluation verified that CuPDF NPs possess excellent biocompatibility. In addition, CuPDF NPs showed significant MRI signal enhancement with high longitudinal relaxivity (r1 5 4.59 per mM/s). In vivo MRI and biodistribution test confirmed the efficient accumulation of CuPDF NPs in the tumor region. After intravenous injection of CuPDF, irreversible tumor ablation was successfully achieved without inducing any obvious side effects by using 808-nm laser irradiation. All in all, these results revealed that the developed CuPDF NPs hold great potential as an effective theranostic agent for MR imaging guided PTT in vivo [22].

6.4.6 Removal of Cu21 from water and wastewaters One of the techniques for the removal of Cu21 from water and wastewaters is adsorption, with special attention on biosorption. Compared to the conventional method, today adsorption is the most appropriate method for removing pollutants from wastewater. The adsorption of Cu21 on natural materials such as zeolites and clays is considered, and modified natural materials are also in use because they possess the ion-exchange ability. For industrial applications, the use of biopolymers and hydrogels is projected because they can reduce the initial concentrations of metal ions to the billions of original concentrations, and are widely available and environmentally safe. The modified biopolymer adsorbents on the basis of polysaccharides (derived from chitin, chitosan, and starch) have been proposed as new materials for the removal of Cu21 ions from wastewater. NPs based on carbon nanotubes CNTs, as well as new nano-adsorbents based on graphene and its composites, show a very high efficiency of removal of Cu21 from wastewater. The adsorption process on natural adsorbents (biosorption) is a relatively recent process that has found great application in the removal of heavy metals, phenols, paints, and other organic pollutants from wastewater. Adsorption on biosorption has become a potential alternative to existing technologies for the effective removal of low concentrations of Cu21 and other

2. Toxicity of nanomaterials

6.4 Toxicity of copper nanoparticles

135

metals from aqueous solutions and wastewaters because of the simplicity, cost-effectiveness, and high capacity of removing both organic and inorganic water pollutants. The results of bibliographic searches show that many agricultural byproducts, as well as waste materials from the food and wood industry, which have low or almost no economic value, can be used as adsorbents for the adsorption of Cu21 ions from the solution [23].

6.4.7 Toxicity of copper oxide nanoparticles on spring barley (Hordeum sativum distichum) The rapid growth of CuO NPs manufacture and its abundant uses in many industries, and increasing release into an environment from both intentional and unintentional sources, create risks to spring barley (Hordeum sativum distichum), one of the most important stable food crop. Thereby, the aim of this study was to investigate the phytotoxicity of CuO NPs on H. sativum growth in hydroponic system. The CuO NPs inhibited H. sativum growth by affecting the germination rate, root and shoot lengths, maximal quantum yield of photosystem II, and transpiration rate. Structural and ultrastructural examination of H. sativum tissues using light, transmission, and scanning electron microscopy showed effects on stomatal aperture and root morphology, metaxylem size and changes in cellular organelles (plastids, mitochondria), as well as in plastoglobules, starch granules, protoplasm, and membranes. The formation of electron-dense materials was noted in the intercellular space of cells of CuO NPs-treated plants. Additionally, relative root length was one-third (35%) that of the control, and relative shoot length (10%) was also reduced. Further, the Cu content of roots and leaves of CuO NPs-treated plants was 5.7 and 6.4-folds higher than the control (without CuO NPs), respectively. Presented data were significant at P # 0.05 compared to control. Conclusively, the results provide insights into our understanding of CuO NPs toxicity on H. sativum, and findings could be used for developing strategies for secure disposal of NPs [24].

6.4.8 Toxicity of copper oxide nanoparticles to neotropical species The increase of production and consumption of copper oxide nanostructures in several areas contributes to their release into aquatic ecosystems. Toxic effects of CuO NPs, in particular, on tropical aquatic organisms are still unknown, representing a risk for biota. In this study, the effects of rod-shaped CuO NPs on the neotropical species Ceriodaphnia silvestrii and Hyphessobrycon eques were investigated. We also compared the toxicity of CuO NPs and CuCl2 on these species to investigate the contribution of particles and copper ions to the CuO NPs toxicity. Considering the low copper ions release from CuO NPs (,1%), our results revealed that the toxicity of CuO NPs to C. silvestrii and H. eques was mainly induced by the NPs. The 48 h EC50 for C. silvestrii was 12.6 6 0.7 μg Cu/L and for H. eques the 96 h LC50 was 211.4 6 57.5 μg Cu/L of CuO NPs. There was a significant decrease in reproduction, feeding inhibition and increase in ROS generation in C. silvestrii exposed to CuO NPs. In fish H. eques, sublethal exposure to CuO NPs caused an increase in ROS generation in gill cells and an increase in cell numbers that were in early apoptotic and necrotic stages. Our results showed that CuO NPs caused toxic effects to C. silvestrii

2. Toxicity of nanomaterials

136

6. Toxicity of silver and other metallic nanoparticles

and H. eques and ROS play an important role in the toxicity pathway observed. Data also indicated that C. silvestrii was among the most sensitive species for CuO NPs. Based on predicted environmental concentration in water bodies, CuO NPs pose potential ecological risks for C. silvestrii and H. eques and other tropical freshwater organisms. CuO NPs cause toxic effects to C. silvestrii and H. eques in environmentally relevant concentrations, posing potential risks for these species and the Neotropical biota [25].

6.5 Toxicity of iron nanoparticles 6.5.1 Responses of flocculated activated sludge to bimetallic Ag-Fe nanoparticles toxicity Escalating production and use of NPs have aroused overarching concerns for their toxic effects on the environment and human. In the present study, the toxic effects of silver (Ag) and iron (Fe) NPs on the performance of activated sludge were investigated under continuous aerobic/anoxic/anaerobic conditions in laboratory-scale SBRs. Activated sludge was exposed to various concentrations (5 100 mg/L) of Ag-Fe NPs for 60 days and its response was assessed through the enzymatic activity, COD, nitrogen (TN) and phosphorus (TP) removal, toxicity tests, as well as variations in bacterial community. Compared with the pristine control sample, the exposure to NPs suppressed TN and TP removal efficiencies. Indeed, the respiration rate and biomass concentration were significantly affected by the NPs. Although the simultaneous exposure to Ag-Fe NPs did affect the integrity of cell membrane (LDH) and key enzymes activities, the higher concentration induced an increased generation of ROS. The metagenome analysis revealed a marked shift in the microbial community structure suggesting that both heterotrophic and autotrophic communities were affected by the presence of Ag-Fe NPs. The present study provides some proof for compounded effects of NPs in their simultaneous presence, and generates new leads for future research efforts [5].

6.5.2 Biogenic magnetite nanoparticles for efficient removal of azo dyes and phenolic contaminants from water Abbasi Kajani and Bordbar [26] have conducted a study toward the green and facile synthesis of biocompatible magnetite NPs for the proficient removal of organic contaminants from water. The NPs were synthesized using a modified coprecipitation method and functionalized by the taxane diterpenoids extracted from Taxus baccata L., and fully characterized using UV vis spectroscopy, SEM, FTIR, VSM, and XRD. The synthesized monodisperse magnetite NPs, with a narrow size distribution of less than 50 nm, displayed significant and stable magnetic activity without surface oxidation after several months. The batch experiments clearly revealed the efficient iron-catalyzed removal of Nile blue, methylene blue, methylene orange, and 4-nitrophenol for several cycles without significant loss of catalytic activity. The relevant kinetic data of the dye removal reactions were fitted to a pseudo-first order model. Furthermore, in vitro MTT assay revealed high biocompatibility of the NPs with no significant toxicity on different human cell lines.

2. Toxicity of nanomaterials

6.6 Toxicity of zinc nanoparticles

137

The overall results showed a high potential of green synthesized, biocompatible magnetite NPs for the environmental applications, especially wastewater remediation.

6.5.3 Functionalization of T lymphocytes for magnetically controlled immune therapy The World Health Organization feels that cancer is the second most important cause of death in Europe. Due to its manifold manifestations, it is not possible to treat all patients according to a uniform scheme. Nevertheless, all solid tumors have one thing in common: independent of the tumor’s molecular subgroup and the treatment protocol, the immune status of the tumor, especially the amount of tumor infiltrating lymphocytes (TILs), is important for the patient’s clinical outcome—the higher the number of TILs, the better the outcome. For this reason it seems desirable to increase the number of TILs. One way to build up T cells in the tumor area is to make them magnetizable and attract them with an external magnetic field. Magnetization can be achieved by superparamagnetic iron oxide nanoparticles (SPIONs) which can be bound to the cells’ surface or internalized into the cells. For this study, SPIONs with different coatings were synthesized and incubated with immortalized mouse T lymphocytes. SPIONs only stabilized with lauric acid (LA) coated in situ or afterwards showed high toxicity. Addition of an albumin layer increased the biocompatibility but reduced cellular uptake. To increase the cellular uptake the albumin coated particles were aminated, leading to both higher uptake and toxicity, dependent on the degree of amination. In the presence of an externally applied magnetic field, T cells loaded with selected types and amounts of SPIONs were guidable. With this gifted pilot study Mu¨hlberger et al. [27] have demonstrated that it is possible to attract SPION bearing T cells by an external magnet. Biocompatibility and uptake of SPIONs by T cells are opposing events. Thus, for the functionalization of T cells with SPIONs the balance between uptake and toxicity must be evaluated carefully.

6.6 Toxicity of zinc nanoparticles 6.6.1 Comparative toxicity of organic, inorganic, and nanoparticulate zinc Dekani et al. [28] have carried out a study to contrast the dietary toxicity of organic zinc (Zn-proteinate, Bioplex Zn), mineral zinc (ZnSO4), and nanoparticulate zinc (ZnO-NPs) on the basis of some biological responses including growth performance and whole-body proximate composition, and antioxidant enzymes, as well as their accumulative affinity to target organs. These Zn sources with the nominal concentrations of 0, 30, 100, and 500 mg/kg diet were added to a basal diet. Juvenile common carp (n 5 400; weight of 25.3 6 2.7 g) were fed with the diets for 56 days. ZnSO4 significantly reduced condition factor (CF) at 500 mg/kg diet. The highest activity of SOD and alkaline phosphatase was observed in the plasma of the animals received 500 mg/kg diet of all experimental Zn sources. However, this concentration of ZnONPs significantly increased the activity of SOD when compared to the respective amount of ZnSO4 and Zn-proteinate. Catalase (CAT) showed a zinc-concentration decreasing activity; the minimum activity was observed in the fish group treated with the diet containing 500 mg/kg ZnSO4. Digestive, muscular, and integumentary systems demonstrated the following tissue

2. Toxicity of nanomaterials

138

6. Toxicity of silver and other metallic nanoparticles

zinc burden: liver . muscle . bone . posterior intestine  skin . anterior intestine, for ZnONPs; liver . muscle  bone  posterior intestine  skin . anterior intestine, for Zn-proteinate; and liver . muscle  bone  skin . posterior intestine  anterior intestine, for ZnSO4. Based on accumulative affinity, taken together, ZnO-NPs displayed the highest affinity to all of the analyzed target organs, and also intestinal Zn accumulation suggested that the gut tissue has the lowest rendering ability against ZnO-NPs in comparison to ZnSO4 and Zn-proteinate.

6.6.2 Nano zinc oxide hydrogels as wound healing materials Composite hydrogels as wound dressings feature healing properties in treating wounds. Khorasani et al. have carried out a study wherein polyvinyl (alcohol)/chitosan/nano zinc oxide nanocomposite hydrogels were formed using the freeze-thaw method and essential process parameters including thawing time, thawing temperature, and the number of freeze-thaw cycles was investigated to model nanocomposites employing response surface methodology. Critical properties including water vapor transmission rate, porosity, wound fluid absorption, and gel content were modeled using process parameters. Analysis of morphology, mechanical properties, toxicity, protein absorption, antibacterial activity, and invitro wound healing were also performed. Results showed that increased freeze-thaw cycles caused reduced pore size and increased porosity and wound fluid absorption. Besides, increased freeze-thaw cycles and reduced thawing temperature resulted in increased elastic modulus and tensile strength, while elongation at break point decreased. Antibacterial properties, biocompatibility, and in vitro wound healing tests demonstrated that the designed system showed no toxicity and it was able to treat the wounds adequately [29].

6.6.3 Evaluation of toxicity of phycocyanin-ZnO nanorod composites C-Phycocyanin pigment was purified from a native cyanobacterial strain using a novel consecutive multistep procedure and utilized for the first time for the green synthesis of phycocyanin-zinc oxide nanorods (PHY-ZnO NRs) by a simple, low-cost, and ecofriendly approach. The PHY-ZnO NRs were characterized using UV vis spectroscopy, XRD, zeta potential measurement, FTIR, SEM, TEM, differential scanning calorimetry, thermogravimetric, and EDX spectroscopy analysis. The UV vis spectra showed an absorption band at 364 nm which is characteristic of ZnO nanoparticles (ZnONPs). The rod-shaped PHYZnO NRs observed in the TEM and SEM images had an average diameter size of 33 nm, which was in good agreement with the size calculated by XRD. The elemental analysis of PHY-ZnO NRs composition showed that three emission peaks of zinc metal and one emission peak of oxygen comprised 33.88% and 42.50%, respectively. The thermogram of PHY-ZnO NRs sample exhibited the weight loss of biosynthesized NPs registered to be 3%, emphasizing the purity and heat stability of zinc oxide nanorods coated with phycocyanin pigment-protein. MTT assay indicated that PHY-ZnO NRs had less cytotoxicity on fibroblast L929 compared to the ZnONRs-treated cells. A remarkable increase in ROS level was measured in cells treated with ZnO at final concentrations of 100, 200, and 500 μg/mL (78 6 7, 99 6 8, and 116 6 11, respectively). When it comes to PHY-ZnO NRs, a protective effect for phycocyanin was detected which declined the level of ROS content

2. Toxicity of nanomaterials

6.7 Conclusion

139

as established by fluorescent microscopy. The characteristic features of phycocyanin for surface functionalization of ZnO NPs deserve to be deemed as a nano-drug candidate for further researches [30].

6.6.4 DNA damages and offspring quality in sea urchin Paracentrotus lividus sperms exposed to ZnO nanoparticles The current advances in nanotechnology lead to a potential increase of the release of NPs into the sea environment through different routes, with possible toxic effects upon the living part of this ecosystem. NP marine contamination is gaining more concern due to the widespread use of cosmetics containing ZnO NPs as UV-filter. Although the possible adverse effects on marine organisms have been already ascertained, the information about the possible genotoxicity of ZnO NPs is still scant. In this work the spermiotoxicity of ZnO particles of different sizes (ZnO Bulk . 200 nm, ZnO NPs 100 nm, and ZnO NPs 14 nm) was assessed, using Paracentrotus lividus spermatozoa, by evaluating the DNA damage of the exposed sperm, fertilization capability, and DNA damage transmission to progeny. Our results showed that ZnO NPs induced DNA damages in spermatozoa after 30 min of exposure. While the sperm fertilization capability was not affected, morphological alterations (skeletal alterations) in offspring were observed and a positive correlation between sperm DNA damage and offspring quality was reported. This study underlines that a possible spermiotoxic action of ZnO NPs at concentration close to those reported in marine coastal water could occur [31].

6.6.5 Reclaimable La: ZnO/PAN nanofiber catalyst for toxicological evaluation utilizing early life stages of zebra fish (Danio rerio) Lakshmi et al. [32] have designed and evaluated La: ZnO/PAN and ZnO/PAN nanofibers as a catalyst for the photodegradation of diethyl (4-nitrophenyl) phosphate (MP). The solid catalyst was characterized by various analytical techniques. Hexagonal and wurtzite crystal structure of the fabricated rod-shaped La-ZnO NPs were substantiated from the XRD pattern. The catalytic activity of ZnO nanorods improved in the presence of La due to their SPR effect. Real-time experiment carried out in the wastewater sample spiked with MP showed 100% degradation efficiency at 150 min as quantified by HPLC. The product of degradation (4-nitrophenol) was directly transformed to tertiary compound (p-amino phenol) eschewing the intermediate as attested by GC MS analysis. Acute toxicity of MP was evaluated utilizing early stages of zebra fish and the degraded product was found to be less toxic for the aquatic living system. They have antibacterial activity also. Some NPs are toxic in nature. The toxicity of NPs such as silver, gold, iron, copper, and zinc are discussed.

6.7 Conclusion Metallic NPs have antibacterial activity. Some NPs are toxic in nature. The toxicity of NPs such as silver, gold, iron, copper, and zinc have been recognized.

2. Toxicity of nanomaterials

140

6. Toxicity of silver and other metallic nanoparticles

Acknowledgment The authors are thankful to their respective managements for their help and support.

References [1] R. Singla, C. Sharma, A.K. Shukla, A. Acharya, Toxicity concerns of therapeutic nanomaterials, J. Nanosci. Nanotechnol. 19 (4) (2019) 1889 1907. [2] M.C. Stensberg, Q. Wei, E.S. McLamore, D.M. Porterfield, A. Wei, M.S. Sepu´lveda, Toxicological studies on silver nanoparticles: challenges and opportunities in assessment, monitoring and imaging, Nanomed. (Lond.) 6 (5) (2011) 879 898. Available from: https://doi.org/10.2217/nnm.11.78. [3] I. Persaud, J.H. Shannahan, A.J. Raghavendra, N.B. Alsaleh, R. Podila, J.M. Brown, Biocorona formation contributes to silver nanoparticle induced endoplasmic reticulum stress, Ecotoxicol. Environ. Saf. 170 (2019) 77 86. [4] L. Ran, Y. Zou, J. Cheng, F. Lu, Silver nanoparticles in situ synthesized by polysaccharides from Sanghuangporus sanghuang and composites with chitosan to prepare scaffolds for the regeneration of infected full-thickness skin defects, Int. J. Biol. Macromolec. 125 (2019) 392 403. [5] A.R. Yazdanbakhsh, M. Rafiee, H. Daraei, M.A. Amoozegar, Responses of flocculated activated sludge to bimetallic Ag-Fe nanoparticles toxicity: performance, activity enzymatic, and bacterial community shift, J. Hazard. Mater. 366 (2019) 114 123. [6] Y. Liu, Y. Wu, X. Guo, Y. Wen, H. Yang, Rapid and selective detection of trace Cu21 by accumulationreaction-based Raman spectroscopy, Sens. Actuat. B: Chem. 283 (2019) 278 283. [7] N. Duroudier, A. Katsumiti, M. Mikolaczyk, J. Scha¨fer, E. Bilbao, M.P. Cajaraville, Dietary exposure of mussels to PVP/PEI coated Ag nanoparticles causes Ag accumulation in adults and abnormal embryo development in their offspring, Sci. Total Environ. 655 (2019) 48 60. [8] L.M. Almeida, L.N. Magno, A.C. Pereira, E´.J. Guidelli, O.B. Filho, A. Kinoshita, et al., Toxicity of silver nanoparticles released by Hancornia speciosa (Mangabeira) biomembrane, Spectrochim. Acta Part A: Mol. Biomolec. Spectrosc. 210 (2019) 329 334. [9] S. Qin, L.-Y. Ma, X. Sun, X. Mao, L. Xu, Hierarchically porous poly(ethylenimine) modified poly(styrene-codivinylbenzene) microspheres for the adsorption of gold nanoparticles and simultaneously being transformed as the nanoparticles immobilized catalyst, J. Hazard. Mater. 366 (2019) 529 537. [10] L. Lu, S. Gunasekaran, Dual-channel ITO-microfluidic electrochemical immunosensor for simultaneous detection of two mycotoxins, Talanta 194 (2019) 709 716. [11] V.K. Sharma, C.M. Sayes, B. Guo, S. Pillai, J.G. Parsons, C. Wang, et al., Interactions between silver nanoparticles and other metal nanoparticles under environmentally relevant conditions: a review, Sci. Total. Environ. 653 (2019) 1042 1051. [12] D. Divakaran, J.R. Lakkakula, M. Thakur, M.K. Kumawat, R. Srivastava, Dragon fruit extract capped gold nanoparticles: synthesis and their differential cytotoxicity effect on breast cancer cells, Mater. Lett. 236 (2019) 498 502. [13] M. Chokkalingam, P. Singh, Y. Huo, V. Soshnikova, S. Ahn, J. Kang, et al., Facile synthesis of Au and Ag nanoparticles using fruit extract of Lycium chinense and their anticancer activity, J. Drug Deliv. Sci. Technol. 49 (2019) 308 315. [14] M. Vetten, M. Gulumian, Differences in uptake of 14 nm PEG-liganded gold nanoparticles into BEAS-2B cells is dependent on their functional groups, Toxicol. Appl. Pharmacol. 363 (2019) 131 141. [15] R.K. Thomas, S. Sukumaran, S. Prasanth, C. Sudarsanakumar, Revealing the interaction strategy of Diosmin functionalized gold nanoparticles with ctDNA: multi-spectroscopic, calorimetric and thermodynamic approach, J. Lumin. 205 (2019) 265 276. [16] A. Barreto, L.G. Luis, E. Pinto, A. Almeida, P. Paı´ga, L.H.M.L.M. Santos, et al., Effects and bioaccumulation of gold nanoparticles in the gilthead seabream (Sparus aurata) single and combined exposures with gemfibrozil, Chemosphere (2019) 248 260. [17] M.S. Jabir, A.A. Taha, U.I. Sahib, Z.J. Taqi, A.M. Al-Shammari, A.S. Salman, Novel of nano delivery system for Linalool loaded on gold nanoparticles conjugated with CALNN peptide for application in drug uptake and induction of cell death on breast cancer cell line, Mater. Sci. Eng. C 94 (2019) 949 964.

2. Toxicity of nanomaterials

References

141

[18] F. Masse, M. Ouellette, G. Lamoureux, E. Boisselier, Gold nanoparticles in ophthalmology, Medicinal Res. Rev. 39 (1) (2019) 302 327. [19] X. Huang, C. Xu, Y. Li, H. Cheng, X. Wang, R. Sun, Quaternized chitosan-stabilized copper sulfide nanoparticles for cancer therapy, Mater. Sci. Eng. C 96 (2019) 129 137. [20] Q. Huang, X. Liu, R. Zhang, X. Yang, C. Lan, Q. Feng, et al., The development of Cu-incorporated micro/ nano-topographical bio-ceramic coatings for enhanced osteoblast response, Appl. Surf. Sci. 465 (2019) 575 583. [21] T. Wang, X. Wen, Y. Hu, X. Zhang, D. Wang, S. Yin, Copper nanoparticles induced oxidation stress, cell apoptosis and immune response in the liver of juvenile Takifugu fasciatus, Fish. Shellfish. Immunol. 84 (2019) 648 655. [22] Y. Xiong, F. Sun, Y. Zhang, Z. Yang, P. Liu, Y. Zou, et al., Polydopamine-mediated bio-inspired synthesis of copper sulfide nanoparticles for T1-weighted magnetic resonance imaging guided photothermal cancer therapy, Colloids Surf. B: Biointerf. 173 (2019) 607 615. [23] V. Krsti´c, T. Uroˇsevi´c, B. Peˇsovski, A review on adsorbents for treatment of water and wastewaters containing copper ions, Chem. Eng. Sci. 192 (2018) 273 287. [24] V. Rajput, T. Minkina, A. Fedorenko, S. Sushkova, S. Mandzhieva, V. Lysenko, et al., Toxicity of copper oxide nanoparticles on spring barley (Hordeum sativum distichum), Sci. Total Environ. 645 (2018) 1103 1113. [25] A.S. Mansano, J.P. Souza, J. Cancino-Bernardi, F.P. Venturini, V.S. Marangoni, V. Zucolotto, Toxicity of copper oxide nanoparticles to Neotropical species Ceriodaphnia silvestrii and Hyphessobrycon eques, Environ. Pollut. 243 (2018) 723 733. [26] A. Abbasi Kajani, A.-K. Bordbar, Biogenic magnetite nanoparticles: a potent and environmentally benign agent for efficient removal of azo dyes and phenolic contaminants from water, J. Hazard. Mater. 366 (2019) 268 274. [27] M. Mu¨hlberger, C. Janko, H. Unterweger, E. Schreiber, J. Band, C.H.K. Lehmann, et al., Functionalization of T lymphocytes for magnetically controlled immune therapy: selection of suitable superparamagnetic iron oxide nanoparticles, J. Magnetism Magnetic Mater. 473 (2019) 61 67. [28] L. Dekani, S.A. Johari, H.S. Joo, Comparative toxicity of organic, inorganic and nanoparticulate zinc following dietary exposure to common carp (Cyprinus carpio), Sci. Total. Environ. 656 (2019) 1191 1198. [29] M.T. Khorasani, A. Joorabloo, H. Adeli, Z. Mansoori-Moghadam, A. Moghaddam, Design and optimization of process parameters of polyvinyl (alcohol)/chitosan/nano zinc oxide hydrogels as wound healing materials, Carbohydr. Polym. 207 (2019) 542 554. [30] S. Davaeifar, M.-H. Modarresi, M. Mohammadi, E. Hashemi, M. Shafiei, H. Maleki, et al., Synthesizing, characterizing, and toxicity evaluating of Phycocyanin-ZnO nanorod composites: a back to nature approaches, Colloids Surf. B: Biointerf. 175 (2019) 221 230. [31] M. Oliviero, S. Schiavo, S. Dumontet, S. Manzo, DNA damages and offspring quality in sea urchin Paracentrotus lividus sperms exposed to ZnO nanoparticles, Sci. Total. Environ. 651 (2019) 756 765. [32] K. Lakshmi, K. Kadirvelu, P.S. Mohan, Reclaimable La: ZnO/PAN nanofiber catalyst for photodegradation of methyl paraoxon and its toxicological evaluation utilizing early life stages of zebra fish (Danio rerio), Chem. Eng. J. 357 (2019) 724 736.

2. Toxicity of nanomaterials

C H A P T E R

7 Recent advances in the study of toxicity of polymer-based nanomaterials A. Suriya Prabha1, R. Dorothy2, S. Jancirani3, Susai Rajendran4, Gurmeet Singh5 and S. Senthil Kumaran6 1

Department of Chemistry, Mount Zion College of Engineering and Technology, Pudukkottai, India 2Department of EEE, AMET University, Chennai, India 3PG and Research Department of Chemistry, MVM Government College for Women, Dindigul, India 4Corrosion Research Center, St Antony’s College of Arts and Sciences for Women, Dindigul, India 5Pondicherry University, Puthucherry, India 6School of Mechanical Engineering, VIT University, Vellore, India

7.1 Introduction Nanotoxicology is the study of the toxicity of nanomaterials (NMs). Because of quantum size effects and large surface area to volume ratio, NMs have unique properties compared to their larger counterparts that affect their toxicity. Mechanisms of toxicity are attributed to oxidative stress, cytotoxicity, and genotoxicity. Characterization of a NM’s physical and chemical properties is important for ensuring the reproducibility of toxicology studies, and is also vital for studying how the properties of NMs determine their biological effects. The properties of a NM, such as size distribution and agglomeration state, can change as a material is prepared and be used in toxicology studies, making it important to measure them at different points in the experiment Inhalation exposure is the most common route of exposure to airborne particles in the workplace. The deposition of nanoparticles (NPs) in the respiratory tract is determined by the shape and size of particles or their agglomerates, and they are deposited in the lungs to a greater extent than larger respirable particles. Based on animal studies, NPs may enter the bloodstream from the lungs and translocate to other organs, including the brain.

Nanotoxicity DOI: https://doi.org/10.1016/B978-0-12-819943-5.00007-5

143

Copyright © 2021 Elsevier Inc. All rights reserved.

144

7. Recent advances in the study of toxicity of polymer-based nanomaterials

The inhalation risk is affected by the dustiness of the material, the tendency of particles to become airborne in response to a stimulus. Dust generation is affected by the particle shape, size, bulk density, and inherent electrostatic forces, and whether the NM is a dry powder or incorporated into a slurry or liquid suspension.

7.2 Recent advances in the study of toxicity of polymeric nanomaterials Many research works have been undertaken in the field of toxicity of NMs. Recent advancements in the field of toxicity of nanopolymeric materials are presented.

7.2.1 Nanodiamonds: emerging face of future nanotechnology Amazing advancements in nanostructured materials have been achieved through their integration into recent nanotechnology in almost each phase of society. Amongst NMs, nanodiamonds (NDs) have become a subject of lively research due to their attractive properties (e.g., large diameter, elevated thermal conductivity, hardness, resistance to friction, nontoxicity, small and tunable surface structure, high surface area, chemical dullness, and excellent optical/mechanical properties). These exceptional properties have expanded their applications which extend to quantum optics, electrochemical coatings, antifriction coatings, antibacterial/antifungal coatings, polymer strengthening, bioimaging probes, implants, polishing, lubricants and fuels, drug delivery, catalyst supports, water cleaning processes, nanomagnetometry, and nano-electrometry. This research of Kumar et al. [1] is controlled to critically review the varied commercial applications of NDs, including their use in thin-film electronics, photovoltaic devices, energy storage devices, water treatment, nanofluids, and electrochemical sensors. Kumar et al. have explained the developmental history of carbon NMs with a major emphasis on the structure and chemical nature of NDs, different synthesis techniques of NDs, and their associated properties. Recent market challenges and future guidelines for the commercial application of NDs have been explained by Kumar et al. [1].

7.2.2 Nanomaterials as versatile adsorbents for heavy metal ions in water Over the years, heavy metal pollution has become a very grave environmental difficulty worldwide. Even though anthropogenic sources are supposed to be the major cause of heavy metal pollution, they can also be introduced into the environment from natural geogenic sources. Heavy metals, because of their toxicity and carcinogenicity, are considered to be nearly all destructive contaminants from groundwater as well as surface water, which pose a serious threat to both human and aquatic life. NMs due to their size and higher surface area to volume ratio have some unique properties compared to their bulk counterpart and have drawn significant consideration of the scientific community in the last few decades. This large surface area can make these materials valuable adsorbents in pollution remediation studies. In this review, an attempt has been made to focus on the applicability of different types of NMs, such as clay-nanocomposites, metal oxide-based NMs, carbon nanotubes, and various polymeric nanocomposites as adsorbents for the removal of variety

2. Toxicity of nanomaterials

7.2 Recent advances in the study of toxicity of polymeric nanomaterials

145

of heavy metals, such as As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sn, U, V, and Zn, from water as reported during the last few years. This work tries to analyze the metalNM interactions, the mechanism of adsorption, the adsorption capacities of the NMs, and the kinetics of adsorption under a mixture of experimental circumstances. The relation between the physicochemical properties of the NMs and deep metal adsorption on them have been explained by Sarma et al. [2].

7.2.3 Toxicological responses of Chlorella autotrophica and Dunaliella salina to Ag and CeO2 nanoparticles The application of NPs, such as Ag and CeO2 NPs, has increased considerably in the last decade due to their significance for the production of engineered nanomaterials (ENMs) functional to new consumer products. This generalized use in everyday products has made the presence of NPs in aquatic systems more recurrent and makes them potential environmental disturbers. Marine phytoplankton is at the bottom of the food web and, therefore, microalgae are potentially susceptible to NPs at different levels: reproductive (population growth), structural and metabolic. One of the first mechanisms of toxicity is caused by the adsorption of NPs onto the cell wall later leading to their internalization. As the cell wall may be a barrier against the intake of NPs, species missing a cell wall would be expected to show a higher sensitivity. In the present study, two microalgae species with a marked structural difference, Dunaliella salina, lacking a cell wall, and Chlorella autotrophica, with a typical cellulosic cell wall, were uncovered to ionic and NP forms of the metals Ag and Ce for 72 h. The biomarkers employed as indicative of toxicity were: cell density, cell practicability, cell size, cell complexity, autofluorescence of chlorophyll a, active chlorophyll, effective quantum yield of PII (photosystem II), and reactive oxygen species (ROS). Exposure to Ag and Ce in both ion and NP forms affected the reproductive, structural, and physiological mechanisms of D. salina and C. autotrophica. In common, toxicity resulted in a reduction of active chlorophyll, efficient quantum yield of PII, and cell density and an increase in cell complexity and ROS. For both species, treatments with Ag were more toxic than those of Ce and, for both metals, the ionic form was more toxic than the NPs. Although D. salina lacks a cell wall, it was more forbearing than C. autotrophica, indicating that the absence of a cell wall does not make it a more sensitive species. The higher tolerance of D. salina might be related to different processes to manage metal exposure that prevents toxicity such as biosorption, by producing extracellular polymeric substances (EPS), and the elimination of the compounds to the peripheral surroundings [3].

7.2.4 Nanoparticles as a solution for eliminating the risk of mycotoxins Mycotoxins are toxic secondary metabolites produced by certain filamentous fungi. The occurrence of mycotoxins in food and feed causes negative health impacts on both humans and animals. Clay binders, yeast cell walls, or antioxidant additives are the most extensively used products for mycotoxin elimination to decrease their impact. Although conservative methods are continuously improving, present research trends are looking for novel solutions. Nanotechnology approaches seem to be a promising, efficient, and low-cost way

2. Toxicity of nanomaterials

146

7. Recent advances in the study of toxicity of polymer-based nanomaterials

to decrease the physical condition effects of mycotoxins. The objective of the work is to shed light on the critical knowledge gap in mycotoxin elimination by nanotechnology. There are three major strategies: mold inhibition, mycotoxin adsorption, and tumbling the toxic effect via NPs. One of the most gifted methods is the use of carbon-based NMs. Graphene has been shown to have an enormous surface and high binding capacity for mycotoxins. Polymeric NPs have also drawn attention due to the fact that they could replace adsorbents or enclose any substance, which would progress the health status of the organism [4].

7.2.5 Nanoparticles modulate membrane interactions of human Islet amyloid polypeptide The spectacular expansion of nanotechnology applications, particularly the dawn of NMs and NPs into the consumer financial system, have led to heightened responsiveness of their budding health risks. This study of Peretz et al. [5] examines the impact of several NPs upon membrane-induced aggregation and bilayer interactions of the human Islet amyloid polypeptide (hIAPP). The authors have reported that several NPs—polymeric NPs, TiO2NPs, and Au NPs displaying coating layers exhibiting different electrostatic charges— did not significantly interfere with the fibrillation procedure and fibril morphology of hIAPP, both in buffer or in biomimetic DMPC:DMPG vesicle solutions. Spectroscopic and microscopic analyses propose, in fact, that the NPs promoted membrane-induced fibrillation. Importantly, we find that all the NPs examined, regardless of work of art or surface properties, gave rise to more distinct, synergistic bilayer interactions when co-incubated with hIAPP. NP-enhanced bilayer interactions of hIAPP might point to probable toxicity and pathogenicity risks of amyloidogenic peptides in the attendance of NPs [5].

7.2.6 Self-assembled antimicrobial nanomaterials Nanotechnology aimed to continue improving human life by dropping environmental pollution of earth and water with pathogens. This review discusses how self-assembled antimicrobial NMs can contribute to humans, their water, and their environment surrounded by safe boundaries for human life, even though some of these NMs show an overt toxicity. At the core of their strategic use, the self-assembled antimicrobial NMs exhibit optimal and biomimetic organization leading to movement at low doses of their toxic components. Antimicrobial bilayer fragments, bilayer-covered or multilayered NPs, functionalized inorganic or organic polymeric materials, coatings and hydrogels reveal their potential for environmental and communal health applications [6].

7.2.7 Construction of dual-functional polymer nanomaterials with near-infrared fluorescence imaging and polymer prodrug by reversible addition-fragmentation chain transfermediated aqueous dispersion polymerization The presentation of functional polymer NMs is an energetically discussed topic in polymer science. The authors are dedicated in investigating polymer NMs based on

2. Toxicity of nanomaterials

7.2 Recent advances in the study of toxicity of polymeric nanomaterials

147

near-infrared (NIR) fluorescence imaging and polymer prodrug. Aza-boron dipyrromethene (BODIPY) is an important organic dye, having characteristics such as ecological confrontation, light resistance, high molar extinction coefficient, and fluorescence quantum yield. The authors have incorporated it into target monomer, which can be polymerized without shifting its parent structure in a polar solvent and copolymerized with water-soluble monomer to improve the solubility of the dye in an aqueous solution. At the same time, the hydrophobic drug camptothecin was designed as a prodrug monomer, and the polymeric NPs with NIR fluorescence imaging and prodrug were synthesized in situ in reversible additionfragmentation chain transfer (RAFT)-mediated aqueous dispersion polymerization. The dynamic light scattering (DLS) and transmission electron microscopy (TEM) exposed the final uniform size of the dual-functional polymeric NPs morphology. The dual-functional polymeric NPs had a strong absorption and emission signal in the NIR region ( . 650 nm) base on the fluorescence tests. In consideration of the long-term biological toxicity, confocal laser scanning microscopy results indicated that the dual-functional NPs with controlled drug content exhibited successful capability of killing HeLa cells. In addition, in vivo imaging of the dual-functional NPs was observed in real time, and the fluorescent signals clearly established the dynamic process of prodrug transfer [7].

7.2.8 General overview of lipidpolymer hybrid nanoparticles, dendrimers, micelles, liposomes, spongosomes, and cubosomes In recent years, the wider use of nanotechnology has involved better concentration from scientists in multidisciplinary fields. Nanotechnological research has come a long way in the past decade, with chief advances being made, both in terms of diagnostic and therapeutic potential of NPs. Some of the prominently discussed NPs in this day and age are polymeric micelles, liposomes, lipidpolymer hybrid NPs, dendrimers, spongosomes, and cubosomes. This review attempts to place a focal point on the conformist advantages and exemplary features that these particles possess, thus making them some of the most ideal vehicles for drug release. Specialist opinion: Particulate systems, which have been lengthily studied in this article, have been employed to enhance the pharmacokinetic and pharmacodynamic characteristics of a range of hydrophobic and hydrophilic drug moieties, thus attempting to prolong the blood circulation times and augment their usefulness over unchanged drug molecules. These adaptation techniques have enabled these drug molecules to be delivered to the pharmacological sites of action at an optimized controlled rate, thus trying to minimize the potential for any toxicity resulting from the nonspecific delivery of drug to different organs [8].

7.2.9 Nanomaterials for water cleaning and desalination, energy production, disinfection, agriculture, and green chemistry NMs may assist to solve issues such as water accessibility, clean energy generation, manage of drug-resistant microorganisms, and food safety. The authors have reviewed innovative approaches to unravel these issues using nanotechnology. The major topics discussed are wastewater treatment using carbon-based, metal-based, and polymeric nanoadsorbents for

2. Toxicity of nanomaterials

148

7. Recent advances in the study of toxicity of polymer-based nanomaterials

removing organic and metal contaminants; nanophotocatalysis for microbial control; desalination of seawater using nanomembranes; energy conversion and storage using solar cells and hydrogen-sorbents nanostructures; antimicrobial properties of NMs; smart release systems; biocompatible NMs such as nanolignocellulosis and starches-based materials, and methods to diminish the toxicity of NMs. Significantly, here it is reviewed two ways to palliate NMs toxicity: (1) controlling physicochemical factors moving this toxicity in order to arrange more safe NMs, and (2) harnessing greener synthesis of them to bring down the environmental impact of toxic reagents, wastes and byproducts. All of these contemporary challenges are reviewed at the present article in an effort to evaluate environmental implications of NMs technology by means of a complete, dependable, and dangerous vision [9].

7.2.10 Nanomaterials as protein, peptide, and gene delivery agents NMs propose noteworthy reward in delivery of different biomolecules which suffer from drawbacks like poor bioavailability, low stability and retention time, degradation in biological systems, etc. Nanotechnological approach has shown promising results for the continuous release of these biomolecules with minimal toxicity concerns. The present review describes an inclusive outlook of the different NMs used for the delivery of these biomolecules. Current literature information related to protein, peptide, and gene delivery agents have been reviewed and classified according to their applications. Studies reveal that the NM based delivery agents can be generally classified into five categories which include metallic NPs, polymeric NPs, magnetic NPs, liposomes, and micelles. All these materials provided major improvement in the under attack delivery of biomolecules. Concerns regarding the bioavailability, stability and delivery of proteins, peptides, and genes need to be investigated to improve their therapeutic potential in the biological milieu. The use of NPs as drug delivery vehicles may avoid uninvited hazards and may amplify their pharmaceutical usefulness [10].

7.2.11 Development of cholate conjugated hybrid polymeric micelles for farnesoid X receptor receptor mediated effective site-specific delivery of paclitaxel The intent of the present study was to explore the tumor targeting potential of a cholic acid (CA) conjugated polymeric micelle system for the effective delivery of paclitaxel (PTX). CA has a high binding similarity to the farnesoid X receptor (FXR) which is overexpressed in a good number of breast cancer cells. CA grafted poly(bis(carboxyphenoxy) phosphazene)-poly(diallyldimethylammonium chloride) micelles were prepared by nanoprecipitation in the presence of a surfactant. The polymeric nanomicelles (PNMs) exhibited well-defined spherical morphology with a hydrodynamic diameter of around 218 nm. CA incorporated hybrid polymeric micelles form a physically powerful gel at body temperature and exhibited prolonged drug discharge. In addition, the surface charge of the micelles undergoes positive to negative exchange based on the environmental pH. Positively charged NMs promote faster cellular uptake in the cancer acidic milieu. CA formulation specifically targeted the FXR overexpressed in breast cancer for significant

2. Toxicity of nanomaterials

7.2 Recent advances in the study of toxicity of polymeric nanomaterials

149

progression of the cytotoxicity of PNM with a 2.69-fold raise in comparison to free PTX. γH2AX and comet assays demonstrate proficient cellular uptake, preferential tumor buildup, and drug release in the cytoplasmic region. This work suggests that the prepared micelle system has a good tumor-targeting facility with reduced systemic toxicity, proving it to be a capable formulation for breast cancer therapy [11].

7.2.12 Antibacterial properties of electrospun Ti3C2Tz (MXene)/chitosan nanofibers Electrospun natural polymeric bandages are extremely attractive due to their low-cost, biodegradability, nontoxicity, and antimicrobial properties. Functionalization of these nanofibrous mats with two-dimensional NMs is a striking strategy to augment the antibacterial effects. Herein, the authors have demonstrated an electrospinning process to manufacture encapsulated delaminated Ti3C2Tz (MXene) flakes within chitosan nanofibers for passive antibacterial wound dressing applications. In vitro antibacterial studies were performed on crosslinked Ti3C2Tz/chitosan composite fibers touching Gram-negative Escherichia coli and Gram-positive Staphylococcus aureusdemonstrating a 95% and 62% reduction in colony forming units, respectively, following 4 h of action with the 0.75 wt% Ti3C2Tz-loaded nanofibers. Cytotoxicity studies to establish biocompatibility of the nanofibers indicated the antibacterial MXene/chitosan nanofibers are nontoxic. The incorporation of Ti3C2Tz single flakes on fiber morphology was analyzed by scanning electron microscopy (SEM) and TEM set with an energy-dispersive detector. The results put forward that the electrospun Ti3C2Tz/chitosan nanofibers are a hopeful candidate material in wound healing applications [12].

7.2.13 Effects of surface charge of hyperbranched polymers on cytotoxicity, dynamic cellular uptake and localization, hemotoxicity, and pharmacokinetics in mice Nanoscaled polymeric materials are gradually being investigated more as pharmaceutical products, drug/gene delivery vectors, or health-monitoring devices. Surface charge is one of the dominant parameters that regulates NM behavior in vivo. The authors have demonstrated how control over chemical synthesis allowed manipulation of NP surface charge, which in turn greatly influenced the in vivo behavior. Three methacrylate/methacrylamide-based monomers were used to synthesize well-defined hyperbranched polymers (HBP) by RAFT polymerization. Each HBP had a hydrodynamic diameter of approximately 5 nm as determined by DLS and TEM. Incorporation of a fluorescent moiety within the polymeric NPs allowed determination of how charge exaggerated the in vivo pharmacokinetic behavior of the NMs and the biological response to them. A direct correlation between surface charge, cellular uptake, and cytotoxicity was experimental, with cationic HBPs exhibiting higher cellular uptake and cytotoxicity than their neutral and anionic counterparts. Evaluation of the distribution of the differently charged HBPs within macrophages showed that all HBPs accumulated in the cytoplasm, but cationic HBPs also trafficked to, and accumulated within, the nucleus. Although cationic HBPs

2. Toxicity of nanomaterials

150

7. Recent advances in the study of toxicity of polymer-based nanomaterials

caused slight hemolysis, this was generally below accepted levels for in vivo protection. Analysis of pharmacokinetic activities showed that cationic and anionic HBPs had short blood half-lives of 1.82 6 0.51 and 2.34 6 0.93 h respectively, compared with 5.99 6 2.30 h for neutral HBPs. This was qualified to the fact that positively charged surfaces are more readily covered with opsonin proteins and thus more visible to phagocytic cells. This was supported by in vitro flow cytometric and qualitative live cell imaging studies, which showed that cationic HBPs tended to be used up by macrophages more successfully and quickly than neutral and anionic particles [13].

7.2.14 Nanomaterials for skin care There are a lot of cosmetic products on the market based on nanotechnology. Those products have NMs since they have many advantages such as better delivery of active ingredients, strength, and photostability of potentially unbalanced cosmetic ingredients, increased effectiveness, and tolerance of the skin for different UV filters. NMs contribute to ease of application and aesthetic appearance of final products. Although they offer several possibilities, their use demands caution. Nanoparticles have a large surface to volume ratio leading to their reactivity and alteration in biological activity compared to the parent bulk materials. The shape and size of the particles are the cause of their toxic effects, rather than their chemical properties. There are different nanosystems at present in use, in pharmaceutical and cosmetic industry, and a lot of new ones, waiting to be applied. In this article we are starting to introduce those who find an application in personal care products such as: liposomes, niosomes, transfersomes, nanoemulsions, solid lipid nanoparticles (SLN), polymeric systems, nanocrystals, fullerenes and ending with metal oxide NPs [14].

7.2.15 Evaluation of the effects of polymeric chitosan/tripolyphosphate and solid lipid nanoparticles on germination of Zea mays, Brassica rapa, and Pisum sativum Even though the potential toxicity of many metallic and carbon NPs to plants has been reported, few studies have evaluated the phytotoxic effects of polymeric and SLN. The present work described the research and characterization of chitosan/tripolyphosphate (CS/TPP) NPs and SLN and evaluated the effects of different concentrations of these NPs on germination of Zea mays, Brassica rapa, and Pisum sativum. CS/TPP NPs presented a usual size of 233.6 6 12.1 nm, polydispersity index (PDI) of 0.30 6 0.02, and zeta potential of 121.4 6 1.7 mV. SLN showed an average size of 323.25 6 41.4 nm, PDI of 0.23 6 0.103, and zeta potential of 213.25 6 3.2 mV. Nanotracking analysis enabled resolve of concentrations of 1.33 3 1010 (CS/TPP) and 3.64 3 1012 (SLN) NPs per mL. At high concentrations, CS/TPP NPs caused complete inhibition of germination, and thus negatively affected the initial growth of all experienced species. Differently, SLN presented no phytotoxic effects. The dissimilar size and composition and the opposite charges of SLN and CS/TPP NPs could be related with the differential phytotoxicity of these NMs. The present study reports the phytotoxic potential of polymeric CS/TPP NPs towards plants, indicating that further

2. Toxicity of nanomaterials

7.2 Recent advances in the study of toxicity of polymeric nanomaterials

151

study is needed on the effects of such formulations intended for future use in agricultural systems, in order to shun injury to the surroundings [15].

7.2.16 Synthesis, characterization, and cytotoxicity of S-nitroso-mercaptosuccinic acid-containing alginate/chitosan nanoparticles Nitric oxide (NO) is an endogenous free radical, which plays a key role in quite a lot of biological processes including vasodilation, neurotransmission, inhibition of platelet adhesion, cytotoxicity beside pathogens, wound healing, and defense against cancer. Due to the relative unsteadiness of NO in vivo (half-life of c.0.5 s), there is increasing attention on the growth of low molecular weight NO donors, such as S-nitrosothiols (RSNOs), which are able to prolong and preserve the biological activities of NO in vivo. In order to enhance the sustained NO release in a number of biomedical applications, RSNOs have been effectively allied to NMs. In this context, this work describes the synthesis and characterization of the NO donor S-nitrosomercaptosuccinic acid (S-nitroso-MSA), which belongs to the class of RSNOs, and its incorporation in polymeric biodegradable NPs composed by alginate/chitosan. First, chitosan NPs were obtained by gelation process with sodium TPP, followed by the addition of the alginate layer, to enhance the NP guard. The obtained NPs presented a hydrodynamic diameter of 343 6 38 nm, PDI of 0.36 6 0.1, and zeta potential of 30.3 6 0.4 mV, indicating their thermal stability in aqueous deferment. The negative zeta potential value was assigned to the presence of alginate chains on the surface of chitosan/TPP NPs. The encapsulation efficiency of the NO donor into the polymeric NPs was found to be 98% 6 0.2%. The high encapsulation efficiency value was accredited to the positive interactions between the NO donor and the polymeric content of the NPs. Kinetics of NO release from the NPs exposed a spontaneous and sustained release of therapeutic amounts of NO, for several hours under physiological temperature. The incubation of NO-releasing alginate/chitosan NPs with human hepatocellular carcinoma (HepG2) cell line revealed a concentration-dependent toxicity. This outcome points to the hopeful uses of NO-releasing alginate/chitosan NPs for anti-cancer chemotherapy [16].

7.2.17 Polymeric nanomaterials as nanomembrane entities for biomolecule and drug delivery Bionanomaterials assembled into nanomembrane entities are actively considered to circumvent the uncontrollable list of shortcomings of conventional delivery systems: low water solubility, hostile stability, short circulation time in plasma, quick clearance from the human body, poor bioavailability, nonspecific toxicity against normal tissue and cells, low cellular uptake, and receptiveness to enzyme degradation. Basically, these nanoentities enable the exploitation of the therapeutic value of many gifted biomolecules and drugs (B&D), controlling the group transport of B&D at a certain rate or even on demand if an incentive is applied. The bulky surface-to-volume ratio of bio-nanomaterials as well as their tunable properties enable to increase the biocompatibility, bioavailability, solubility and permeability of many unique B&D which are otherwise tricky to deliver. Albisa et al. have reported on the last advances of bio-nanomaterials applied as nanomembranes in biomolecule and drug delivery, as well as their more extraordinary properties and applications in biomedicine. New advances have

2. Toxicity of nanomaterials

152

7. Recent advances in the study of toxicity of polymer-based nanomaterials

been drastically established in the production of smart nanomembranes that alter their own structure and function in response to the surroundings. These new insights have been used for the production of smart drug delivery nanomembranes. These nanomembranes entities have the potential to revolutionize biomedicine but there are shortcomings needing to be addressed in order to convert the laboratory production to the clinic [17].

7.2.18 Fabrication and application areas of mixed matrix flat-sheet Polymer-NP composite materials have exclusive characteristics, such as high mechanical strength, good electrical conductivity, optical and thermal properties. Nanoparticles can gain high functionality and therefore they raise the overall performance of conventional materials, such as membranes, used in environmental applications. Membrane partition properties can be controlled for each specific application by the proper choice of fabrication components (main polymer, solvent, additives like NPs, pore forming agents, etc.) and parameters (evaporation time and temperature, coagulation bath temperature, etc.). Furthermore, especially in recent years, the membrane fouling problem can be avoided with dissimilar membrane fabrication components. Membrane fouling can be defined as the uncontrolled deposition of particles, colloids, macromolecules, or salt ions from feed solution at the membrane surface or inside the membrane pores. It is a strict problem for membrane materials used in pressure-driven processes such as invalidate osmosis (RO), nanofiltration (NF), ultrafiltration (UF), and microfiltration (MF), and also for other membrane processes. Different polymers (polyethersulfone, polysulfone, cellulose acetate, polyvinylidene fluoride, etc.) are possible preferred membrane materials for these membrane systems due to their physical and chemical characteristics, such as good chemical, heat, mechanical and cleaning resistance, and environmental endurance, as well as easy processing and manufacturing. For instance, the intrinsic hydrophobicities of some polymers are high and so this results in hydrophobic membrane materials and leads to a low membrane flux, poor anti-fouling properties and low application and useful life. The fouling causes a decrease in membrane performance, either temporarily or permanently. The fouling mechanism includes the interaction between the membrane surface and the foulants (inorganic, organic, and biological substances in many different forms). The foulant molecules not only physically interact with the membrane surface but also chemically degrade the membrane material. Most of the latest membrane fouling studies focused on the physical or chemical modifications of membrane material for low fouling properties. These studies can be summarized in three main areas: (1) the modification of the membrane surface with in-situ physical and chemical treatments, (2) the coating of the membrane with special materials that have low fouling properties, and (3) the preparation of the membrane by adding nanomaterials (mixed matrix membranes, MMMs). MMMs are formed by the addition of inorganic or metal oxide particles, having micrometer or nanometer sizes, to the polymeric casting solution or by in-situ generation [18].

7.2.19 CO2-based amphiphilic polycarbonate micelles enable a reliable and efficient platform for tumor Biodegradable polymeric NMs can be straight broken down by intracellular processes, offering a desirable way to solve toxicity issues for cancer diagnosis and action. Among

2. Toxicity of nanomaterials

7.2 Recent advances in the study of toxicity of polymeric nanomaterials

153

them, aliphatic polycarbonates are approved for application in biological fields by the United States Food and Drug Administration (FDA), however, high hydrophobicity, lacking functionality and improper degradation offer major room for development in these materials. To achieve progress in this direction, herein, we demonstrate that CO2-based amphiphilic polycarbonates (APC) with improved hydrophilicity and processability can be used as a reliable and efficient platform for tumor imaging. To better investigate their potential, we devised a convenient strategy through conjugation of APC with gadolinium (Gd). Results: The consequential polymeric micelles (APC-DTPA/Gd) exhibit excellent magnetic resonance imaging performance, simultaneously enabling real-time visualization of bioaccumulation and decomposition of polymeric micelles in vivo. Outstandingly, these micelles can be degraded to renally cleared products within a sensible timescale without confirmation of toxicity. The findings may help the development of CO2-based APC for cancer diagnosis and treatment, accompanied by their low-toxicity degradation pathway [19].

7.2.20 Electrospun polyacrylonitrile templated 2D nanofibrous mats: a platform toward practical applications for dye removal and bacterial disinfection The fabrication of polymeric nanofibers and its potential adaptability instigated to promote smart hybrid nanomaterials for the deletion of environmental pollutants. In this pursuit, in this research work, polyacrylonitrile (PAN)-based two-dimensional (2D) nanofibrous mats with polyethyleneimine (PEI)/Fe and quaternary ammonium (QA)/Fe as hybrid fillers were prepared by the electrospinning development for the effective dye removal and bacterial disinfection. The characteristics of the fabricated nanomaterials were extensively explored by quite a lot of analytical techniques such as field emission-SEM, TEM, X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and Brunauer, Emmett, Teller analysis. Magnetic and thermal properties were investigated by superconducting quantum interference device and thermogravimetric measurements. The kinetic and isothermal models confirmed the adsorption performance of the PAN. PEI/Fe nanofibers, and further regenerative studies substantiated the sustainability of the mats for the elimination of industrial dye effluents. Subsequently, the magnetic-QA-loaded PAN nanofiber mats exhibited bactericidal killing efficacy of 99% and 89.5% in both S. aureus and green fluorescence protein expressing E. coli bacterial models evaluated from the conventional quantitative bacterial colony-counting assay. Disk diffusion technique and microscopic investigations corroborated the disinfection effectiveness with a zone of inhibitions of B23 and 33 mm, respectively. Fascinatingly, in vitro cell culture studies conducted in BHK-21 and NIH 3T3 cell lines demonstrated the cytocompatibility, and the in vivo toxicity investigations using the zebrafish models necessitated the realtime submission of these nanofibrous mats. Consequently, the comprehensive study of the fabricated PANlated functionalized 2D nanofibrous mats confirmed to be competent for the remediation of industrial dye effluents and bacteria in water bodies [20].

7.2.21 Nanomaterials for neurology: state-of-the-art Despite the plentiful challenges associated with the application of nanotechnology in neuroscience, it should have a noteworthy impact on our understanding of how the

2. Toxicity of nanomaterials

154

7. Recent advances in the study of toxicity of polymer-based nanomaterials

nervous system works, how it fails in disease, and the progress of earlier and less-invasive diagnostic procedures so we can intervene in the preclinical stage of neurological disease before extensive neurological damage has taken place. Eventually, both the challenges and opportunities that nanotechnology presents stem from the fact that this technology provides a way to interact with neural cells at the molecular level. This review has provided a neurobiological overview of key neurological disorders, explaining the special types of nanomaterials in use and discussing their recent and potential uses in neuroscience. The authors also discuss the issue of toxicity in these nanomaterials. Veloz-Castillo et al. [21]. have reported many of the diverse applications that advances in nanotechnology are having in the field of neurological sciences, especially the high impact they are having in the progress of new treatment modalities for neurological disorders that will induce the expected physiological response while minimizing unwanted secondary effects. In conclusion, the authors weigh in on what the promises and challenges are for future development in this innovative field [21].

7.2.22 Effect of different nanomaterials on the metabolic activity and bacterial flora of activated sludge medium In this study, the central and delegate bacteria of activated mud before and after the adding up of different nanomaterials (NMs) (multiwalled carbon nanotube, MWCNT, silicon dioxide NP, nSiO2, titanium oxide NP, nTiO2, aluminum oxide-NP, nAl2O3, and silver NP, nAg) were discretely isolated, enumerated, and recognized. In total, 17 different representative isolates were identified by using 16S rDNA gene sequence analysis. The effects of NMs on the growth (viable counts), biological oxygen demand (BOD) removal performances, respiration mechanism, and EPS construction of bacteria were also investigated. Both BOD removal capacity and respiration rate of bacterial medium decreased later than NMs exposure. Most toxic NMs were MWCNT (46.15%) and nAg (42.30%) related with BOD removal capacity and nAg (62%) and nTiO2 (56%) for respiration mechanism. Consequently, the protein satisfied of soluble EPS (sEPSp) increased with addition of nAg, nTiO2, and nAl2O3. NMs also have a negative effect on the structure of community of activated sludge in both diversity and density [22].

7.2.23 Fluorescent polymeric nanovehicles for neural stem cell modulation Nanomaterials are emerging as physically powerful candidates for applications in drug release and offer an alternative platform to modulate the differentiation and activity of neural stem cells. Herein the authors have reported the synthesis and characterization of two different classes of polymeric NPs: N-isopropylacrylamide-based thermoresponsive nanogels RM1 and P(TEGA)-b-P(d, lLA)2nano-micelles RM2. We covalently linked the NPs with fluorescent tags and display their capacity to be internalized and tracked in neural stem cells from the postnatal subventricular zone, without moving their proliferation, multipotency, and differentiation characteristics up to 150 μg/mL. The distinction in chemical structure of RM1 and RM2 does not appear to impact toxicity nevertheless it influences the loading capacity. Nanogels RM1 loaded with retinoic acid improve solubility of

2. Toxicity of nanomaterials

7.2 Recent advances in the study of toxicity of polymeric nanomaterials

155

the drug which is on the loose at 37 C, resulting in an augment in the number of neurons, similar to what can be obtained with a solution of the free drug solubilized with a tiny percentage of DMSO [23].

7.2.24 Graphene oxideenriched double network hydrogel with tunable physico-mechanical properties and performance An emerging move toward gaining polymeric hydrogels with superior properties is integration of carbon-based nanomaterials within their network structure. Alternatively, hydrogels with modified physical and mechanical characteristics are an attractive class of materials which have extensive application in drug delivery and tissue engineering. This study presents a strategy to achieve graphene oxide (GO)-enriched hydrogels with modulated physico-mechanical properties and performance. GO/poly acrylic acid/gelatin hydrogels are fabricated via in situ polymerization technique followed by chemical crosslinking of gelatin molecules. N,N0 -Methylenebisacrylamide in a range of concentrations is used within the prepolymer composition as a cross-linking agent to organize a set of nanocomposite hydrogels. FTIR, XRD, and atomic force microscopy are used to characterize the fabricated hydrogel samples. The microstructure of samples is analyzed with SEM. The mechanical properties of the specimens are evaluated by rheometry. The swelling performance, degradation kinetic, and porosity of the hydrogels as well as their in vitro cytotoxicity are also assessed. The results show successful synthesis of nano-GO sheets and polymer composites. The augment of cross-linker concentration decreased the swelling ratio and increased the porosity of hydrogel samples. A wide range of pore diameters (70300 μm) and mechanical stiffness (storage modulus of 200025,000 Pa) is obtained. Through manipulation of cross-linking density, the degradation rate of nanocomposite hydrogels is controlled. Lastly, no toxicity is detected by contact of the hydrogel extracts to osteoblast osteosarcoma cells. The optimized hydrogel samples having appropriate range of physical characteristics and functionality suggest the application of the 3D structures as scaffold material for rigid tissue construction [24].

7.2.25 Advances in nanobiomaterials for topical administrations: new galenic and cosmetic formulations Nanomaterials are systems with at least one outside dimension in the size range of approximately 1100 nm (or 10291027 m). Nanoscale changes the chemical, physical, and biological properties of the raw material, ensuing in a wide multiplicity of applications, for example, drug delivery, diagnostics, implants, biosensors, medical imaging, and tissue engineering. Among the cosmetic and galenic formulations for topical administration, nanomaterials are being employed with the purpose of protection of the active pharmaceutical ingredient (API) from degradation, to improve the limited action and to avoid the API penetration into the bloodstream, thus reducing the danger of systemic effects and toxicity. The most popular nanomaterials for topical application are based on lipid or polymeric NPs, while others containing metals are chiefly used for diagnostic purposes and imaging analysis. Of particular relevance are those composed of biodegradable and

2. Toxicity of nanomaterials

156

7. Recent advances in the study of toxicity of polymer-based nanomaterials

biocompatible raw materials (nanobiomaterials), making them striking for the formulation of topical pharmaceuticals and cosmetics. The new advances in nanobiomaterials and their applications in galenic and superficial formulations have been discussed [25].

7.2.26 Fluorescent polymer nanoparticles based on dyes: seeking brighter tools for bioimaging Speed, resolution, and sensitivity of today’s fluorescence bioimaging can be radically enhanced by fluorescent NPs which are many-fold brighter than organic dyes and fluorescent proteins. While the field is currently subjugated by inorganic NPs, notably quantum dots (QDs), fluorescent polymer NPs encapsulating large quantities of dyes (dye-loaded NPs) have emerged newly as an attractive substitute. These new nanomaterials, inspired from the fields of polymeric drug delivery vehicles and advanced fluorophores, can combine superior brightness with biodegradability and low toxicity. The strategies for synthesis of dye-loaded polymer NPs by emulsion polymerization and assembly of preformed polymers have been reported. Superior intensity requires strong dye loading without aggregation-caused quenching (ACQ). Only recently a number of strategies of dye design were proposed to overcome ACQ in polymer NPs: aggregation induced emission, dye change with bulky side groups and use of bulky hydrophobic counterions. The resulting NPs now surpass the brightness of QDs by  10-fold for a comparable size, and have started reaching the level of the brightest conjugated polymer NPs. Other property, remarkably photostability, color, blinking, as well as particle size and surface chemistry are also systematically analyzed. Finally, main and emerging applications of dye-loaded NPs for in vitro and in vivo imaging are reviewed. Dye-loaded polymer NPs have emerged newly as a nice-looking alternative to QDs and conjugated polymer NPs in fluorescence bioimaging. Design concepts of bright and small NPs with minimized dye self-quenching are summarized, their properties are analyzed scientifically, and their major and rising applications in cells and small animals are explained [26].

7.2.27 Nanostructured materials functionalized with metal complexes: in search of alternatives for administering anticancer metallodrugs Nanotechnology has unraveled several important issues of conventional anticancer chemotherapy. Expectations project that a new generation of effective cancer therapies will be residential with enormous potential to overcome the biological, biophysical, and biomedical obstacles that the human body enacts against standard chemotherapeutic treatments. Generally, nanostructures protect the entombed drug molecules from poverty in blood, allowing their safe and unimpaired delivery to precise goal sites in the body. Nanostructured macromolecular systems such as curcubit[n]urils, cyclodextrins, liposomes, lipid nanocapsules, proteins, polynuclear organometallic compounds, carbon nanotubes, polymeric NPs, and ceramic materials have shown great potential in facilitating the administration of effective anticancer metallodrugs. The uniqueness of these nanostructured materials lies in their suitability for functionalization with small molecule drugs. Recently, there has been a great deal of interest among oncologists and therapeutic chemists in functionalizing nanostructured

2. Toxicity of nanomaterials

7.2 Recent advances in the study of toxicity of polymeric nanomaterials

157

materials with anticancer metallodrugs to ensure better administration. In light of these facts, the state-of-art nanostructured materials functionalized with metallodrugs as anticancer agents are described in this review. The functionalization of numerous classes of metal complexes including platinum and nonplatinum compounds is also addressed. Particular focus is given to the co-delivery of metallodrugs within nanostructures. Toxicity of nanomaterials is discussed and the linked concerns are also highlighted. Finally, the recent challenges and the future perspectives of metallodrug functionalization have been commented upon [27].

7.2.28 Sequestration of nanoparticles by an extracellular polymeric substance matrix reduces the particle-specific bactericidal activity Most artificial nanomaterials are known to display broad-spectrum bactericidal activity; however, the defense mechanisms that bacteria use based on EPS to detoxify NPs are not well known. The authors have ruled out the likelihood of ion-specific bactericidal activity by showing the need of equivalent dissolved zinc and silicon toxicity and determined the particle-specific toxicity of ZnO and SiO2 NPs (ZnONPs/SiO2NPs) through dialysis isolation experiments. Astoundingly, the manipulation of the E. coli EPS (i.e., no EPS manipulation or EPS removal by sonication/centrifugation) showed that their particle-specific bactericidal movement could be antagonized by NP-EPS sequestration. The survival charge of pristine E. coli (no EPS manipulation) reached 65% (ZnONPs, 500 mg/L) and 79% (SiO2NPs, 500 mg/L), whereas continued existence rates following EPS removal by sonication/centrifugation were 11% and 63%, respectively. TEM joint with fluorescence micro-titration analysis and FTIR showed that protein-like substances (N-H and C-N in amide II) and secondary carbonyl groups (C 5 O) in the carboxylic acids of EPS acted as significant binding sites that were involved in NP sequestration. So, the amount and composition of EPS produced by bacteria have vital implications for the bactericidal usefulness and potential environmental issues of NPs [28].

7.2.29 In vitro cytotoxicity evaluation of functional polyethylene glycol-PDMA block copolymer in liver human hepatocellular carcinoma cells The development of matrices to manage the release of drugs into specific sites in the human body is a standpoint biomedical application of polymeric materials. The purpose of this work was to estimate the cytotoxicity of a recently synthesized functional block copolymer of composition poly(ethylene glycol-b-2-N,N-dimethylaminoethyl methacrylate) PEO-b-PDMA for application in nanosized drug release systems. The toxicological effect of the copolymer was studied by in vitro exposure of human liver HepG2 cell line. Toxicity was examined by two methods—MTT test and neutral red examination following the exposure to the copolymer in the concentration range from 1 to 1000 μg/mL for 24 and 48 h. It was revealed that no toxic outcome was experimental in the concentration range from 1 to 1000 μg/mL, even after 48 h of incubation. The results from the revise demonstrated a good safety profile for the investigated hydrophilic PEO-PDMA block copolymer [29].

2. Toxicity of nanomaterials

158

7. Recent advances in the study of toxicity of polymer-based nanomaterials

7.2.30 Polyethylene glycolylated boron nitride nanotube-reinforced poly (propylene fumarate) nanocomposite biomaterials Boron nitride nanotubes (BNNTs) are the most promising inorganic nanomaterials explored so far. Aiming to reach polymeric biomaterials with tailored physical properties for tissue engineering, polyethylene glycol (PEG)-grafted BNNTs were used as reinforcements for biocompatible poly(propylene fumarate) (PPF). The nanocomposites were synthesized via sonication and thermal curing, and their morphology, hydrophilicity, biodegradability, cytotoxicity, thermal, mechanical, tribological, and antibacterial properties were examined. Morphological observations exposed that the PEG-g-BNNTs were randomly and uniformly detached within PPF, and suggested good compatibility between the covalently functionalized nanotubes and the matrix. The thermal stability, hydrophilicity, water uptake, biodegradation rate, and protein absorption capability steadily increased upon increasing PEG-g-BNNTs loading. The nanocomposites displayed better stiffness and strength compared to PPF, and maintained enough inflexibility under physiological conditions to be used for bone tissue regeneration. The friction coefficient and wear rate decreased upon addition of the PEG-g-BNNTs, which is desirable for biomedical applications. The nanocomposites exhibited antibacterial activity against Gram-positive S. aureus and Gram-negative E. coli bacteria, the biocide result being stronger with increasing nanotube loading, which did not cause toxicity on human dermal fibroblasts [30].

7.2.31 Evaluation of potential acute cardiotoxicity of biodegradable nanocapsules in rats by intravenous administration Nanotoxicology aims to study the protection of nanomaterials, especially towards human exposure. Biodegradable polymeric nanocapsules have been indicated as potential drug carriers applicable for treating several pathologies. Therefore the objective of this investigation was to assess the potential cardiotoxicity of biodegradable lipid-core nanocapsules (LNC) containing poly(ε-caprolactone). Nanocapsules were characterized and the acute toxicity evaluation was conducted in Wistar rats. Two control groups (saline and tween/glycerol) were utilized, and three treated groups were chosen for low, midway and high doses: 28.7 3 1012 (LNC-1), 57.5 3 1012 (LNC-2), and 115 3 1012 (LNC-3), expressed as a number of nanocapsules per milliliter per kg. Blood pressure measurements were conducted in nonanesthetized animals by caudal plethysmography. The electrocardiographic (ECG) and echocardiographic analyses were carried out after anesthesia by isoflurane at two points, prior to treatment and behind 14 days. Blood was collected 24 h and 14 days after treatment. Biochemical and histopathological analyses were performed. During the evaluation period, no deaths, weight loss, or clinical signs were observed. Posttreatment systolic pressures (24 h and 14 days) were appreciably increased in association to pretreatment in both control groups and treated groups, which is recommended as a possible consequence of the infused volume. Serum sodium, potassium, aspartate aminotransferase, and alkaline phosphatase, as well as, hematological parameters were within reference values reputable for rats. ECG showed no indications of cardiotoxicity. Despite the echocardiograms, no alterations in the ejection fraction were found as indicators of cardiotoxicity. Cardiac histopathology also demonstrated no alterations. Therefore the present

2. Toxicity of nanomaterials

7.2 Recent advances in the study of toxicity of polymeric nanomaterials

159

results on acute assessment after i.v. administration, by unhurried infusion, showed potential safety since no cardiotoxic effects by ECG, echocardiographic, arterial pressure, biochemical, and histopathological analyses were found [31].

7.2.32 A study of the catalytic ability of in situ prepared AgNPs-PMAA-PVP electrospun nanofibers The catalysis of nanomaterials is attractive and striking. Herein, electrospinning was engaged to afford poly(methyl acrylate) (PMAA)-poly(vinyl pyrrolidone) (PVP) electrospun nanofibers. Next, in situ photosynthesis resulted in the formation of a uniform congregation of silver NPs (AgNPs) over the electrospun fibers, generating AgNPs-PMAA-PVP electrospun nanofibers. The as-formed AgNPs-PMAA-PVP electrospun nanofibers were utilized for investigating the catalytic fall of 4-nitrophenol (4-NP) in the presence of NaBH4. For the in situ synthesis, the available light sources that were used included sunlight, table lamps, and 365 nm UV lamps. The outcome showed that the AgNPs-PMAA-PVP electrospun nanofibers could catalyze the reduction of 4-NP in the presence of NaBH4, generating 4-aminophenol, therefore reducing the toxicity of 4-NP. After the use of the AgNPs-PMAA-PVP electrospun nanofibers for up to four cycles, the catalytic competence remained as high as 90%, signifying that the noble metal/polymeric electrospun nanofibrous catalyst is extremely efficient and reusable [32].

7.2.33 Caenorhabditis elegans as an alternative in vivo model to determine oral uptake, nanotoxicity, and efficacy of melatonin-loaded lipid-core nanocapsules on paraquat damage Caenorhabditis elegans is a choice in vivo model that is being effectively used to assess the pharmacological and toxic effects of drugs. The exponential growth of nanotechnology requires the use of substitute in vivo models to charge the toxic effects of theses nanomaterials. The use of polymeric nanocapsules has shown gifted results for drug delivery. Moreover, these formulations have not been used in cases of intoxication, such as in treatment of paraquat (PQ) poisoning. Thus using drugs with properties superior by nanotechnology is a hopeful approach to conquer the toxic effects of PQ. This study aimed to evaluate the absorption of rhodamine B-labeled melatonin (Mel)-loaded LNC by C. elegans, the application of this model in nanotoxicology, and the protection of Mel-LNC against PQ damage. The formulations were prepared by self-assembly and characterized by particle sizing, zeta potential, drug content, and encapsulation efficiency. The results demonstrated that the formulations had fine size distributions. Rhodamine B-labeled Mel-LNC were orally absorbed and distributed in the worms. The toxicity assessment of LNC showed a lethal dose 50% near the highest dose tested, indicating low toxicity of the nanocapsules. Moreover, pretreatment with Mel-LNC considerably increased the survival rate, abridged the ROS, and maintained the development in C. elegans exposed to PQ compared to those worms that were either untreated or pretreated with free Mel. These results demonstrated for the first time the uptake and delivery of Mel-LNC by a nematode, and indicated that while LNC is not toxic, Mel-LNC prevents the effects of PQ poisoning. Thus C. elegans may be an interesting substitute model to test the nanocapsules toxicity and efficiency [33].

2. Toxicity of nanomaterials

160

7. Recent advances in the study of toxicity of polymer-based nanomaterials

7.2.34 Evaluation of the effects of nitric oxide-releasing nanoparticles on plants These days, there are numerous commercially available products containing nanostructured materials. Meanwhile, despite the many benefits that can be obtained from nanotechnology, it is still essential to understand the mechanisms in which nanomaterials interact with the surroundings, and to obtain information concerning their achievable toxic effects. In agriculture, nanotechnology has been used in different applications, such as nanosensors to detect pathogens, NPs as controlled release systems for pesticides, and biofilms to deliver nutrients to plants and to protect food products against degradation. Moreover, plants can be used as models to study the toxicity of NPs. Indeed, phytotoxicity assays are required to identify possible negative effects of nanostructured systems, prior to their implementation in agriculture. Nitric oxide (NO) plays a key role in plant growth and protection, and recently, several papers described the beneficial effects due to application of exogenous NO donors in plants. The tripeptide glutathione (GSH) is an important antioxidant molecule and is the precursor of the NO donor, S-nitrosoglutathione (GSNO). In this context, the present work investigates the property of different concentrations of alginate/chitosan NPs, containing either GSH or GSNO, on the development of two experiment species (Z. mays and Glycine sp.). The results showed that the alginate/chitosan NPs present a size with an average range from 300 to 550 nm, a PDI of 0.35, and encapsulation efficiency of GSH between 45% and 56%. The NO release kinetics from the alginate/chitosan NPs containing GSNO showed sustained and controlled NO release over several hours. Plant assays showed that at the concentrations tested (1, 5, and 10 mM of GSH or GSNO), polymeric NPs showed no significant inhibitory effects on the development of the species Z. mays and Glycine sp., considering the variables shoot height, root length, and dry mass. Consequently, these NPs seem to have promising uses in agriculture, and might be potentially used as forbidden release systems useful by the foliar route [34].

7.2.35 Planktonic and biofilm-grown nitrogen-cycling bacteria exhibit different susceptibilities to copper nanoparticles Proper characterization of NP interactions with environmentally applicable bacteria under representative conditions is required to enable their sustainable manufacture, use, and disposal. Earlier nanotoxicology research based on planktonic growth has not adequately explored biofilms, which serve as the predominant mode of bacterial increase in natural and engineered environments. Copper nanoparticle (Cu-NP) impacts on biofilms were compared with respective planktonic cultures of the ammonium-oxidizing Nitrosomonas europaea, nitrogen-fixing Azotobacter vinelandii, and denitrifying Paracoccus denitrificans using a suite of independent toxicity diagnostics. Median inhibitory concentration (IC50) values derived from adenosine triphosphate for Cu-NPs were lower in N. europaea biofilms (19.6 6 15.3 mg/L) than in planktonic cells (49.0 6 8.0 mg/L). Nevertheless, in absorbancebased growth assays, compared with unexposed controls, N. europaea growth rates in biofilms were twice as resilient to inhibition than those in planktonic cultures. Likewise, relative to unexposed controls, growth rates and yields of P. denitrificans in biofilms exposed to CuNPs were 40-fold to 50-fold less inhibited than those in planktonic cells. Physiological evaluation of ammonium oxidation and nitrate decrease suggested that biofilms were also less

2. Toxicity of nanomaterials

7.2 Recent advances in the study of toxicity of polymeric nanomaterials

161

inhibited by Cu-NPs than planktonic cells. Furthermore, functional gene expression for ammonium oxidation (amoA) and nitrite reduction (nirK) showed lower reserve by NPs in biofilms relative to planktonic-grown cells. These results suggest that biofilms mitigate NP impacts, and that nitrogen-cycling bacteria in wastewater, wetlands, and soils might be more flexible to NPs than planktonic-based assessments advocate [35].

7.2.36 Inorganic nanocarriers for platinum drug delivery These days platinum drugs take up approximately 50% of all the clinically used anticancer drugs. Besides cisplatin, novel platinum agents including sterically hindered platinum (II) drugs, chemically reductive platinum (IV) drugs, photosensitive platinum (IV) drugs, and multinuclear platinum drugs have been developed recently, with a few ingoing clinic trials. Rapid development of nanobiotechnology makes targeted delivery of anticancer platinum agents to the tumor site probable, while simultaneously minimizing toxicity and maximizing the drug efficacy. Being versatile drug carriers to deliver platinum drugs, inorganic nanovehicles such as gold NPs, iron oxide nanomaterials, carbon nanotubes, mesoporous nanosilica, metal-organic frameworks, have been widely studied over the past decades. In contrast to conventional polymeric and lipid NPs, inorganic NP-based drug carriers are strange as they have shown outstanding theranostic effects, placing themselves as an indispensable part of future nanomedicine. The authors have elaborate current research advances on fabrication of inorganic NPs for platinum drug release [36].

7.2.37 Low-bandgap biophotonic nanoblend: a platform for systemic disease targeting and functional imaging Photonic nanomaterials have found extensive applications in theranostics. The authors have introduced a design of all-organic photonic NPs, different from traditional ones, in which they have employed nanoblend of a low-bandgap π-conjugated polymer (LB-CP) and polystyrene as the photonic core, surrounded by an FDA-approved polymeric surfactant. This design provides capability for efficient deep tissue imaging using highly penetrating NIR excitation and emission of LB-CP and also allows us to incorporate a NIR phosphorescent oxygen-sensitive dye in the core to serve as a dual-emissive probe for hypoxia imaging. These biophotonic nanoblend particles (B20 nm in diameter) show superficial blood circulation, proficient disease targeting, and negligible liver filtration as well as sustained renal excretion in the intravenously administered mouse models, as noninvasively visualized by the NIR emission signals. In diseased mouse models, pathological tissue deoxygenation at hypoxic sites was effectively detected with ratiometric spectral information. The authors have shown that the nanoformulation exhibits no apparent toxicity, thus serving as a versatile biophotonics stage for diagnostic imaging [37].

7.2.38 Unique roles of nanotechnology in medicine and cancer-II Applications of nanotechnology in medicine and cancer are attractive increasingly popular. Common nanomaterials and devices applicable in cancer medicine are classifiable as liposomes,

2. Toxicity of nanomaterials

162

7. Recent advances in the study of toxicity of polymer-based nanomaterials

polymeric-micelles, dendrimers, nano-cantilevers, carbon nanotubes, QDs, magnetic-NPs, gold nanoparticles (AuNPs), and certain miscellaneous NPs. The authors have presented assessment of the structure, function, and utilities of the various accepted, under trial, and pretrial nanodevices applicable in the cancer care and medicine. The liposomes are phospholipid-vesicles made useful in transport drugs to the target site minimizing the biodistribution toxicity, and a number of such theranostics have been approved for clinical practice. Newly worked out liposomes and polymeric micelles are under the trail phases for nano-therapeutic utility. A multifunctional dendrimer conjugate with imaging, targeting, and drug molecules of PTX has been recently synthesized for cancer theranostic applications. Nano-cantilever based assays are likely going to replace the conventions methods of chemical pathological investigations. Carbon nanotubes are emerging for utility in regenerative and cancer medicine. Quantum dots hold great promise for the micrometastasis and intraoperative tumor imaging. Important applications of magnetic NPs are in the cardiac stents, photodynamic therapy, and liver metastasis imaging. The AuNPs have been employed for cell imaging, computed tomography, and cancer therapy. Moreover these categories, miscellaneous NPs are being discovered for efficacy in the cancer analysis and disease management. However, the use of NPs should be cautious since the toxic effects of NPs are not well-known. The use of NPs in the clinical practice and their toxicity profile need further widespread research [38].

7.2.39 Nanotechnology and vaccine development Regardless of the development of conventional vaccines, improvements are clearly required due to concerns about the weak immunogenicity of these vaccines, intrinsic instability in vivo, toxicity, and the need for multiple administrations. To overcome such problems, nanotechnology platforms have newly been incorporated into vaccine development. Nanocarrier-based delivery systems offer an opportunity to enhance the humoral and cellular protected responses. This advantage is attributable to the nanoscale particle size, which facilitates uptake by phagocytic cells, the gut-associated lymphoid tissue, and the mucosa-associated lymphoid tissue, most importantly to proficient antigen recognition and presentation. Modifying the surfaces of nanocarriers with a multiplicity of targeting moieties permits the delivery of antigens to specific cell surface receptors, thereby stimulating specific immune responses. In this review, we introduce recent advances in nanocarrier-based vaccine delivery systems, with a focus on the types of carriers, including liposomes, emulsions, polymer-based particles, and carbon-based nanomaterials. The authors have leftover challenges and possible breakthroughs, including the development of needle-free nanotechnologies and a fundamental understanding of the in vivo behavior and stability of the nanocarriers in nanotechnology-based rescue systems [39].

7.2.40 Environmental interactions of geo-and bio-macromolecules with nanomaterials ENMs are typically synthesized with tailored surfaces using various surfactants, polymeric, or biomolecule coatings to achieve desired functionality. When exposed to the surroundings, coatings on the ENMs will undergo the first set of interactions with natural geo-and bio-macromolecules preexisting in aqueous and/or soil matrices. Such interfacial

2. Toxicity of nanomaterials

References

163

interaction will probably change the conformation and extent of coverage of the synthetic ENM surface coatings via exchange, displacement, and/or overcoating by environmental macromolecules. The exchange kinetics and extent of replacement of the synthetic coatings will intensely crash environmental fate, transport, transformation, and toxicity of the ENMs. Saleh et al. have discussed the state-of-the-art literature to make out key synthetic coating types, their interaction with the environmental and biological macromolecules, and exemplify the obtainable challenges to determine covering exchange kinetics and its environmental implications on ENMs [40].

7.3 Concluding remarks Polymeric NPs are submicron (11000 nm) colloidal particles. They are comprised of APIs that are within/adsorbed macromolecular substances (polymer) [41]. Nanotoxicology is the study of the toxicity of nanomaterials. Because of quantum size effects and large surface area to volume ratio, nanomaterials have unique properties compared to their larger counterparts that influence their toxicity. Mechanisms of toxicity are ascribed to oxidative stress, cytotoxicity, and genotoxicity [42].

References [1] S. Kumar, M. Nehra, D. Kedia, N. Dilbaghi, K. Tankeshwar, K.-H. Kim, Nanodiamonds: emerging face of future nanotechnology, Carbon 143 (2019) 678699. [2] G.K. Sarma, S. Sen Gupta, K.G. Bhattacharyya, Nanomaterials as versatile adsorbents for heavy metal ions in water: a review, Environ. Sci. Pollut. Res. 26 (2019) 62456278. Available from: https://doi.org/10.1007/ s11356-018-04093-y. [3] M. Sendra, J. Blasco, C.V.M. Arau´jo, Is the cell wall of marine phytoplankton a protective barrier or a nanoparticle interaction site? Toxicological responses of Chlorella autotrophica and Dunaliella salina to Ag and CeO2 nanoparticles, Ecol. Indic. 95 (2018) 10531067. [4] P. Horky, S. Skalickova, D. Baholet, J. Skladanka, Nanoparticles as a solution for eliminating the risk of mycotoxinsOpen Access Nanomaterials 8 (9) (2018) 727. [5] Y. Peretz, R. Malishev, S. Kolusheva, R. Jelinek, Nanoparticles modulate membrane interactions of human Islet amyloid polypeptide (hIAPP), Biochimica et. Biophysica Acta  Biomembranes 1860 (9) (2018) 18101817. [6] A.M. Carmona-Ribeiro, Self-assembled antimicrobial nanomaterialsOpen Access Int. J. Environ. Res. Public. Health 15 (7) (2018) 1408. [7] C. Tian, J. Niu, X. Wei, Y.-J. Xu, L. Zhang, Z. Cheng, et al., Construction of dual-functional polymer nanomaterials with near-infrared fluorescence imaging and polymer prodrug by RAFT-mediated aqueous dispersion polymerization, Nanoscale 10 (21) (2018) 1027710287. [8] R.R. Wakaskar, General overview of lipidpolymer hybrid nanoparticles, dendrimers, micelles, liposomes, spongosomes and cubosomes, J. Drug. Target. 26 (4) (2018) 311318. ´ . Rı´os, Nanomaterials for water cleaning and desalination, energy production, disinfection, [9] M.J. Villasen˜or, A agriculture and green chemistry, Environ. Chem. Lett. 16 (1) (2018) 1134. [10] A. Guliani, A. Acharya, Nanomaterials as protein, peptide and gene delivery agents, Open Biotechnol. J. 12 (1) (2018) 154165. [11] S. Mehnath, M. Arjama, M. Rajan, M. Jeyaraj, Development of cholate conjugated hybrid polymeric micelles for FXR receptor mediated effective site-specific delivery of paclitaxel, N. J. Chem. 42 (20) (2018) 1702117032. [12] E.A. Mayerberger, R.M. Street, R.M. McDaniel, M.W. Barsoum, C.L. Schauer, Antibacterial properties of electrospun Ti3 C2 Tz (MXene)/chitosan nanofibers, RSC Adv. 8 (62) (2018) 3538635394.

2. Toxicity of nanomaterials

164

7. Recent advances in the study of toxicity of polymer-based nanomaterials

[13] L. Chen, J.D. Simpson, A.V. Fuchs, B.E. Rolfe, K.J. Thurecht, Effects of surface charge of hyperbranched polymers on cytotoxicity, dynamic cellular uptake and localization, hemotoxicity, and pharmacokinetics in mice, Mol. Pharmaceut. 14 (12) (2017) 44854497. [14] S. Miljkovi´c, M. Tomi´c, I. Hut, S. Pelemis, Nanomaterials for skin care (Book Chapter 9), in: Commercialization of Nanotechnologies—A Case Study Approach, Editors: D. Brabazon, E. Pellicer, F. Zivic, J. Sort, M. Baro´, N. Grujovic, K.-L. Choy, 2018, pp. 205226. Available from: https://doi.org/10.1007/978-3319-56979-6. ISBN: 978-3-319-56978-9. [15] D.Y. Nakasato, A.E.S. Pereira, J.L. Oliveira, H.C. Oliveira, L.F. Fraceto, Evaluation of the effects of polymeric chitosan/tripolyphosphate and solid lipid nanoparticles on germination of Zea mays, Brassica rapa and Pisum sativum, Ecotoxicol. Environ. Saf. 142 (2017) 369374. [16] A.B. Seabra, G.K. Fabbri, M.T. Pelegrino, L.C. Silva, T. Rodrigues, Synthesis, characterization and cytotoxicity of S-nitroso-mercaptosuccinic acid-containing alginate/chitosan nanoparticles, J. Phys. Conf. Ser. 838 (1) (2017) 012032. [17] A. Albisa, L. Espan˜ol, M. Prieto, V. Sebastian, Polymeric nanomaterials as nanomembrane entities for biomolecule and drug delivery, Curr. Pharm. Des. 23 (2) (2017) 263280. [18] D.Y. Koseoglu-Imer, I. Koyuncu, Chapter 18: Fabrication and application areas of mixed matrix flat-sheet membranes (Book Chapter), in: Application of Nanotechnology in Membranes for Water Treatment, in: A. Figoli, J. Hoinkis, S.A. Altinkaya, J. Bundschuh (Eds.), Application of Nanotechnology in Membranes for Water Treatment, 2017, pp. 4965. CRC Press, Available from: https://doi.org/10.1201/9781315179070. ISBN 9781315179070. [19] Y. Li, S. Liu, X. Zhao, Y. Wang, J. Liu, X. Wang, et al., CO2-based amphiphilic polycarbonate micelles enable a reliable and efficient platform for tumor imaging, Theranostics 7 (19) (2017) 46894698. [20] R.K. Sadasivam, S. Mohiyuddin, G. Packirisamy, Electrospun polyacrylonitrile (PAN) Templated 2D Nanofibrous Mats: a platform toward practical applications for dye removal and bacterial disinfection, ACS Omega 2 (10) (2017) 65566569. [21] M.F. Veloz-Castillo, R.M. West, J. Cordero-Arreola, O. Arias-Carrion, M.A. Me´ndez-Rojas, Nanomaterials for neurology: state-of-the-art, CNS Neurol. Disord. Drug. Targets 15 (10) (2016) 13061324. ˙ ˙ Koyuncu, Effect of different nanomaterials on the metabolic ¨ .F. Algur, I. ˘ Imer, [22] T. Ergo¨n-Can, D.Y. Ko¨seogluO activity and bacterial flora of activated sludge medium, Clean. Soil Air Water 44 (11) (2016) 15081515. [23] S.A. Papadimitriou, M.P. Robin, D. Ceric, R.K. O’Reilly, S. Marino, M. Resmini, Fluorescent polymeric nanovehicles for neural stem cell modulation, Nanoscale 8 (39) (2016) 1734017349. [24] S. Mohammadi, H. Keshvari, M. Eskandari, S. Faghihi, Graphene oxideenriched double network hydrogel with tunable physico-mechanical properties and performance, React. Funct. Polym. 106 (2016) 120131. [25] P. Severino, J.F. Fangueiro, M.V. Chaud, J.C. Cardoso, A.M. Silva, E.B. Souto, Advances in nanobiomaterials for topical administrations: new galenic and cosmetic formulations (Book Chapter 1), in: Nanobiomaterials in Galenic Formulations and Cosmetics: Applications of Nanobiomaterials, Editor: A.M. Grumezescu, 2016, pp. 123. ISBN: 978-0-323-42868-2, Imprint William Andrew, Elsevier. [26] A. Reisch, A.S. Klymchenko, Fluorescent polymer nanoparticles based on dyes: seeking brighter tools for bioimaging, Small 12 (15) (2016) 19681992. [27] W.A. Wani, S. Prashar, S. Shreaz, S. Go´mez-Ruiz, Nanostructured materials functionalized with metal complexes: in search of alternatives for administering anticancer metallodrugs, Coord. Chem. Rev. 312 (2016) 6798. [28] Q. Wang, F. Kang, Y. Gao, X. Mao, X. Hu, Sequestration of nanoparticles by an EPS matrix reduces the particle-specific bactericidal activity, Sci. Rep. 6 (2016) 21379. [29] V. Tzankova, N. Doneva, M. Frosini, M. Valoti, B. Kostova, D. Rachev, et al., In vitro cytotoxicity evaluation of functional PEG-PDMA block copolymer in liver HEPG2 cells, Pharmacia 63 (1) (2016) 913. [30] A.M. Dı´ez-Pascual, A.L. Dı´ez-Vicente, PEGylated boron nitride nanotube-reinforced poly(propylene fumarate) nanocomposite biomaterials, RSC Adv. 6 (83) (2016) 7950779519. [31] R. Fracasso, M. Baierle, G. Goe¨thel, A. Barth, F. Freitas, S. Nascimento, et al., Evaluation of potential acute cardiotoxicity of biodegradable nanocapsules in rats by intravenous administration, Toxicol. Res. 5 (1) (2015) 168179. [32] L. Zhong, T. Yang, J. Wang, C.Z. Huang, A study of the catalytic ability of in situ prepared AgNPs-PMAAPVP electrospun nanofibers, N. J. Chem. 39 (12) (2015) 95189524. [33] M.F. Chara˜o, C. Souto, N. Brucker, A. Barth, D.S. Jornada, D. Fagundez, et al., Caenorhabditis elegans as an alternative in vivo model to determine oral uptake, nanotoxicity, and efficacy of melatonin-loaded lipid-core nanocapsules on paraquat damage, Int. J. Nanomed. 10 (2015) 50935106.

2. Toxicity of nanomaterials

References

165

[34] A.E.S. Pereira, A.M. Narciso, A.B. Seabra, L.F. Fraceto, Evaluation of the effects of nitric oxide-releasing nanoparticles on plants, J. Phys.: Conf. Ser. 617 (1) (2015) 012025. [35] V.C. Reyes, S.O. Opot, S. Mahendra, Planktonic and biofilm-grown nitrogen-cycling bacteria exhibit different susceptibilities to copper nanoparticles, Environ. Toxicol. Chem. 34 (4) (2015) 887897. [36] P. Ma, H. Xiao, C. Li, Y. Dai, Z. Cheng, Z. Hou, et al., Inorganic nanocarriers for platinum drug delivery, Mater. Today 18 (10) (2015) 554564. [37] Y.H. Seo, M.J. Cho, O.J. Cheong, W.-D. Jang, T.Y. Ohulchanskyy, S. Lee, et al., Low-bandgap biophotonic nanoblend: a platform for systemic disease targeting and functional imaging, Biomaterials 39 (2015) 225233. [38] F. Alam, M. Naim, M. Aziz, N. Yadav, Unique roles of nanotechnology in medicine and cancer-II, Indian J. Cancer 52 (1) (2015) 19. [39] M.-G. Kim, J.Y. Park, Y. Shon, G. Kim, G. Shim, Y.-K. Oh, Nanotechnology and vaccine development, Asian J. Pharm. Sci. 9 (5) (2014) 227235. [40] N.B. Saleh, J.R. Lead, N. Aich, D. Das, I.A. Khan, Environmental interactions of geo-and bio-macromolecules with nanomaterials (Book Chapter 9), in: M.R. Knecht, T.R. Walsh, (Eds.), Bio-Inspired Nanotechnology: From Surface Analysis to Applications ISBN: 9781461494461, 2014, pp. 257290. Available from: https://doi. org/10.1007/978-1-4614-9446-1. [41] , https://www.sciencedirect.com/topics/materials-science/polymer-nanopart . . [42] , https://en.wikipedia.org/wiki/Nanotoxicology . .

2. Toxicity of nanomaterials

C H A P T E R

8 Toxicity of polymeric nanomaterials Yubin Li1,2,3, Shaofei Wang4 and Dianwen Ju5 1

Department of Neurology, Xinqiao Hospital, Third Military Medical University, Chongqing, P. R. China 2Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States 3Corporal Michael J. Crescenz VA Medical Center, Philadelphia, PA, United States 4Department of Cellular and Genetic Medicine, School of Basic Medical Sciences, Fudan University, Shanghai, P.R. China 5Department of Microbiological and Biochemical Pharmacy; The Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai, P. R. China

8.1 Introduction Nanomaterials are a series of engineered structures with a diameter of less than 100 nm which could be manufactured by different physiochemical processes based on different types of properties and applications [1]. The exclusive characteristics of these nanomaterials including unique structures, monodispersity, and high surface to mass ratio make them existing effective cellular property to uptake genes, peptides, proteins, and chemical drugs for biomedical applications [2,3]. Among them, polymeric nanomaterials especially cationic polymers are widely used for therapeutic and diagnostic vectors, and also developed as antipathogen or antitumor agents [4 6]. But the toxicity of polymeric nanomaterials has been raised to challenge their nanomedicinal applications [7]. A series of studies have suggested that polymeric nanomaterials exposure could trigger global toxicity including respiratory system, cardiovascular system, neurological system, immune system, skin, gastrointestinal system, reproduction, and development dysfunction; meanwhile their related mechanisms of polymeric nanomaterials-induced toxicity have also been widely investigated [8,9]. This chapter introduces toxicity of polymeric nanomaterials. It starts by presenting classification of polymeric nanomaterials based on different classification standard (including nanocapsules and nanospheres or natural polymeric nanomaterials and synthetic polymeric nanomaterials); It then shows the toxicity of polymeric nanomaterials in vitro (different cell types) and in vivo (different tissues, organs, and systems); Continually, it explains the related mechanisms of polymeric nanomaterials-induced toxicity, which mainly including biophysical mechanisms,

Nanotoxicity DOI: https://doi.org/10.1016/B978-0-12-819943-5.00008-7

167

Copyright © 2020 Elsevier Inc. All rights reserved.

168

8. Toxicity of polymeric nanomaterials

biochemical mechanisms, and cellular and molecular biological mechanisms. Size, shape, charge, surface chemistry, and dispersity of polymeric nanomaterials are involved in the biophysical mechanisms section; Cellular biological mechanisms mainly focus on apoptosis, necrosis, necroptosis, autophagy, pyroptosis, and cell cycle arrest; while molecular biological mechanisms are mainly including DNA damage and reactive oxygen species (ROS), although there still exist potential factors involved in polymeric nanomaterials-induced toxicity need to be explored. Finally, the chapter briefly discusses current perspective usage of polymeric nanomaterials-triggered toxicity and the strategies to overcome their toxicity. The chapter highlighted toxicity of polymeric nanomaterials and related mechanisms, which might contribute to the evaluation of polymeric nanomaterials-induced toxicity.

8.2 Classification of polymeric nanomaterials Polymeric nanomaterials are defined as solid colloidal materials of sub-micron (1 1000 nm) [10]. Based on the different preparation methods, the most common form of polymeric nanomaterials are nanocapsules and nanospheres [11]. Nanospheres are formed by a polymeric matrix, where the molecules are adsorbed on the surface or encapsulated inside the matrix. Nanocapsules constitute a vesicular system, forming an interior reservoir where the molecules are entrapped, the core is of a liquid form surrounded by a shell of solid material [12]. According to the origin of polymeric nanomaterials, they can be divided into two main categories: natural polymeric nanomaterials and synthetic polymeric nanomaterials. Natural polymeric nanomaterials are generally hydrophilic while synthetic polymeric nanomaterials are hydrophilic in nature. Natural polymeric nanomaterials can be divided into proteins, like gelatin and albumin, and polysaccharides, like alginate and chitosan [13]. Synthetic polymeric nanomaterials used in synthesis are either in prepolymerized form [like polystyrene and poly (ε-caprolactone) (PCL)] or polymerized during a process [like poly(methyl methacrylate) and poly(isobutyl cyanoacrylate)] [14]. Among synthetic polymeric nanomaterials, they can be mainly divided into biosynthesized polymeric nanomaterials and chemical-synthesized polymeric nanomaterials (Fig. 8.1).

8.2.1 Natural polymeric nanomaterials 8.2.1.1 Chitosan Chitosan is a polysaccharide composed of 2-amino-2-deoxy-beta-D-glucan combined with glycosidic linkages, and is the most important derivative of chitin, produced by removing the acetate moiety from chitin [15]. Being a cationic polysaccharide in neutral or basic pH conditions, chitosan contains free amino groups, and is insoluble in water. The primary amine groups present special properties making chitosan very useful in pharmaceutical applications [16]. 8.2.1.2 Cellulose Cellulose polymeric nanomaterials are one of the most abundant and inexhaustible natural polymers, which includes cellulose nanofibers and cellulose nanocrystals [17].

2. Toxicity of nanomaterials

8.2 Classification of polymeric nanomaterials

169

FIGURE 8.1 Classification of Polymeric Nanomaterials.

Cellulose nanocrystals are extracted from never dried cellulose nanofibers using sulfuric acid under controlled hydrolysis time [18]. Cellulose polymeric nanomaterials have remarkable properties such as light weight, low cost, and availability of the raw material, renewability, nanoscale dimension, and unique morphology [19,20]. Cellulose nanomaterials have been widely used as reinforcing fillers in nanocomposite materials [21]. Cellulose and its derivatives are broadly used in the drug delivery systems to modifying drug solubility and gelation which could control the same release profile [22]. 8.2.1.3 Starch Starch is a natural, renewable, and biodegradable polymer produced by many plants as a source of stored energy. It is the second most abundant biomass material in nature [23]. Starch polymeric nanomaterials are defined as particles that have at least one dimension smaller than 1000 nm, but larger than a single molecule or atom. It can be easily synthesized by acid or enzymatic hydrolysis of native starch [24]. As an abundant, inexpensive, renewable, biodegradable, and biocompatible natural polymeric nanomaterials, starch nanomaterials have been suggested as one of the promising biomaterials for novel utilization in foods, pharmaceuticals, cosmetics, and various nanocomposites [25]. 8.2.1.4 Alginate Alginate is one of the most used polymers in formation of (micro)particles [26]. As natural derived polysaccharides existing biocompatibility, biodegradability, stability, sustainability, and alginate based polymeric nanomaterials have raised considerable attentions in the drug-delivery systems [27]. This biopolymer with final carboxyl groups has been classified as anionic polymer and exists much better mucoadhesive strength when compared with cationic and neutral biopolymers [28].

2. Toxicity of nanomaterials

170

8. Toxicity of polymeric nanomaterials

8.2.2 Biosynthesized polymeric nanomaterials 8.2.2.1 Poly β-hydroxybutyrate Polyhydroxyalkanoates (PHAs) are a family of polyesters that are synthesized and intracellularly accumulated as a carbon and energy storage material by various microorganisms [29]. In 1926, PHAs were first discovered as Poly β-hydroxybutyrate (PHB) in Bacillus megaterium by Lemoigne. To date, it has been demonstrated that many bacteria can synthesize various types of PHAs. PHA-producing microorganisms such as Ralstonia eutropha which have been used for the commercial production of PHAs. PHAs are widely used for various biomedical applications, including drug delivery and tissue engineering scaffolds, due to their excellent biocompatibility and biodegradability [30]. PHB can be synthesized by many bacteria. It exists as a carbon reserve, in cytoplastic fluid in the form of crystalline granules about 0.5 μm in diameter. PHB exhibits high stiffness and crystallinity [31]. Increasing PHB content in PHB also reduces the stiffness, melting point, and crystallinity of PHB. Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a copolymer of PHB with randomly arranged 3-hydroxybutyrate (HB) groups and 3-hydroxyvalarate groups. In order to increase its flexibility and processing capabilities, PHB is often copolymerized with polyhydroxyvalerate to form PHBV. 8.2.2.2 Xanthan gum Xanthan gum is a high molecular weight heteropolysaccharide sourced from Xanthomonas campestris. It is a polyanionic polysaccharide and exists great bioadhesive properties. Because it is considered nontoxic and nonirritating, xanthan gum is widely used as a pharmaceutical excipient such as supporting hydrogels for drug release applications, particularly due to its acid resistant property [32]. 8.2.2.3 Polylactic acid Polylactic acids (PLAs) are produced from renewable biomass through a chemobioprocess consisting of fermentative production of lactic acid (LA) and chemical polymerization. PLA, a polymer produced from renewable resources has emerged as a potential substitute to the conventional synthetic petrochemical-derived plastics owing to their biodegradeable and bioresorbable nature [33]. Bacteria have been genetically manipulated to synthesize lactate-based polymers in a single-step metal-free system by using a lactatepolymerizing enzyme. Production of LA-enriched and PLA-like polymers in bacteria from glucose and xylose, metabolic/genetic engineering for polymer yields enhancement, polymer properties [34]. There are several methods for synthesizing high-molecular-weight PLAs: condensation, chain elongation, and ring-opening polymerization of cyclic lactides [35]. In 2008, Taguchi et al. developed a recombinant Escherichia coli strain allowing the synthesis of LA-based polyesters by introducing the gene encoding engineered PHA synthase with acquired LA-polymerizing activity. They thus achieved the one-step biosynthesis of a copolymer with 6 mol% of lactate and 94 mol% of HB units. This extremely important result represents a milestone toward the biological synthesis of PLA and confirms that the work is moving in the right direction [36]. At present, the LA fraction in the copolyesters has been enriched up to 99.3 mol%, so the synthesis of homopolymers of LA has been almost accomplished.

2. Toxicity of nanomaterials

8.2 Classification of polymeric nanomaterials

171

8.2.2.4 Polysialic acid Polysialic acid is a collective name for liner polymers of sialic acid that are covalently bound to proteins, a post-translational modification. It is widely expressed in nature in bacteria capsules, fish, sea urchin eggs, embryonic tissues, amphibians, animal and human brains [37]. Sialic acid and polysialic acid serve as endogenous substances, which are nonimmunogenic and biodegradeable. Prostate-specific antigen modification can reduce the immunity of the proteins or polypeptides and increase circulation time of the modified drugs in the blood, thus achieving active targeting effect [38].

8.2.3 Chemical-synthesized polymeric nanomaterials 8.2.3.1 Dendrimers First reported by Tomalia et al. in the 1980s, poly(amidoamine) (PAMAM) dendrimers were the first complete family of dendrimers to be synthesized and commercialized and were one of the most studied [39]. Because of their highly bifurcated, monodisperse, welldefined and three-dimensional structures, dendrimers are globular-shaped and their surface could be functionalized and easily controlled, which makes dendrimers excellent candidates as drug delivery systems [40]. There are two main approaches for dendrimers synthesis: (1) starting from their core and then extending outwards; (2) starting from outside and then eaching their core [41]. 8.2.3.2 Poly(lactic-co-glycolic acid) Poly(lactic-co-glycolic acid) (PLGA) is a copolymer combined with PLA and poly glycolic acid. PLA contains an asymmetric alpha-carbon that is typically described as the D or L form in classical stereochemical terms [42]. PLGA is normally an acronym for poly(D,L-lacticco-glycolic acid) where D- and L-lactic acid forms are in equal ratio. As PLGA polymeric nanomaterials exhibits excellent biocompatibility, long-standing track record, and properties for continuous drug release, it is widely used in biomedical field [43]. 8.2.3.3 Polyethylenimines Polyethylenimines (PEI) are aliphatic polyamines characterized by a (C2H5N) repeating unit and synthesized by acid-catalyzed polymerization of aziridine. PEIs yield a highly branched network with a high cationic charge-density potential. As one of the most typical polycationic nanomaterials, PEIs can be divided into linear polyethylenimines (LPEIs) and branched PEI based on different synthesized structures [44]. Besides shapes and sizes, the difference between LPEIs and branched PEIs is also shown by different amino groups: LPEIs process primary and secondary amino groups while branched PEIs also process tertiary amino groups. However, different shaped or sized PEIs present different transfection efficiency and cytotoxicity [45]. 8.2.3.4 Poly(L-lysine) Poly(L-lysine) (PLL) polymeric nanoparticles are linear polypeptides with the amino acid lysine for the repeat unit. PLL polymeric nanoparticles and their derivatives exist well-defined architecture, excellent biocompatibility, and biodegradable properties as they

2. Toxicity of nanomaterials

172

8. Toxicity of polymeric nanomaterials

consist of amino acid lysine [46]. Although PLL polymeric nanoparticles were one of the first cationic polymers used for gene transfer, high molecular weight PLL polymers still showed potential cytotoxicity [47]. 8.2.3.5 Dextran Dextrans are glucose polymers characterized by highly content of α-1,6-gluocopyranosidic linkages (95%) compared to 1,3-linkages (5%). Dextrans have been broadly used for various therapeutic applications: dextrans with 40 70 kDa molecular weights have been used for drug delivering agents because of their well-defined repetitive chemical motifs and functional groups for derivatization, soluble property in water, and resistant acidic and basic conditions, which could protect drugs from degradation [48]. 8.2.3.6 Polymeric micelles Polymeric micelles are amphiphilic block copolymers consisting of nanoparticles which could self-gather to form a core shell structure in the aqueous solution. The hydrophobic core of polymeric micelles can be loaded with hydrophobic drugs, and the hydrophilic shell will make the whole system soluble in water and stabilize the core [49]. Polymeric micelles under 100 nm are normally having a narrow distribution to avoid fast renal excretion. Besides, their polymeric shell restrains nonspecific interactions with biological components. These nanoparticles have a strong prospective for hydrophobic drug delivery since their interior core structure permits the assimilation of drugs resulting in enhancement of stability and bioavailability [22].

8.3 In vitro toxicity of polymeric nanomaterials 8.3.1 Chitosans Most in vitro studies of polymeric nanomaterials have focused on nanoparticle-assisted absorption and systemic drug delivery of orally administered therapeutics. The majority of these studies have used the Caco-2 cell monolayer model [50]. Dale et al. described the effects of chitosans with differing molecular weights and degree of deacetylation on CaCo-2 cells, HT29-H and in situ rat jejunum. The toxicity of chitosans was dependent in deacetylation degree and molecular weight. At high deacetylation degree the toxicity is related to the molecular weight and the concentration while at lower deacetylation degree, toxicity is less pronounced and less related to the molecular weight [51]. Interestingly, chitosan and its derivatives seem to be toxic to several bacteria, fungi, and parasites. Emulsions containing chitosans were tested and confirmed could against both Pseudomonas aeruginosa and Staphylococcus aureus. A lipid emulsion of the same chitosans was found to have antimycotic effect against Candida albicans and Aspergillus niger. In tests of meglumine antimoniate against Leishmania infantum it was found that the chitosan excipient had anti-parasitic properties [16].

2. Toxicity of nanomaterials

8.3 In vitro toxicity of polymeric nanomaterials

173

8.3.2 Poly(lactic-co-glycolic acid) Size-dependent cytotoxic effect was explored in both RAW264.7 cells and BEAS-2B cells after cells were incubated with three different sizes of PLGA for 24 h. Although PLGA nanoparticles did not trigger significantly lethal toxicity up to a concentration of 300 μg/mL, the proinflammatory cytokine tumor necrosis factor-α (TNF-α) release after PLGA nanoparticles exposure should raise our concern especially in clinic. Platel et al., evaluated effects of the surface charge of PLGA nanoparticles on their in vitro genotoxicity, cytotoxicity, ROS production, and endocytosis by using PLGA nanoparticles with different positive, neutral, or negative surface charges. In vitro genotoxicity assays (including microneus and comet assays) together with an examination of cytotoxicity, were performed in different cell lines. Induction of ROS and endocytosis were also explored. PLGA nanoparticles are endocytosed by the clathrin pathway and induced ROS in all the experimental cell lines. Besides, PLGA nanoparticles led to chromosomal aberrations without primary DNA damage in human bronchial epithelial cells [52]. PLGA polymeric nanoparticles coated with cell-penetrating peptides was also assessed to explore their toxicity on two lung epithetial cell lines (A549 and Beas-2B cells). Cells exposed to the PLGA-DNA-CPP presented low inflammatory effect, low apoptotic level and no cleavage of caspase-3. Increase of necrotic cells (around 10% 15%) after 24 h of exposure and increase in autophagy, triggered by PLGA-DNA-CPP, are likely to be related to the lysosomal escape mechanism [53]. Besides, PLGA-poly(ethylene oxide) nanoparticles could be untaken and localized in human brain derived endothelial cells, and induced oxidative stress generation and DNA-damaging effects in exposed human brain-derived endothelial cells [54].

8.3.3 Poly(amidoamine) dendrimers The immunotoxicity of three generations of PAMAM dendrimers was evaluated in mouse macrophage cells (J774A.1 cells) in vitro. Using the Alamar blue and 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide assays, a generation dependent cytotoxicity of the PAMAM dendrimers was found whereby G-6 . G-5 . G-4. The toxic response of the PAMAM dendrimers correlated well with the number of surface primary amino groups, with increasing number resulting in an increase in toxic response. A generation dependent ROS and cytokine production was found, which correlated well with the cytotoxicological response and therefore number of surface amino groups. A clear time sequence of increased ROS generation, TNF-α and interleukin-6 secretion, MIP-2 levels and cell death was observed. The intracellular ROS generation and cytokine production induced cytotoxicity point toward the mechanistic pathway of cell death upon exposure to PAMAM dendrimers [55].

8.3.4 Polyethylenimines PEI polymeric nanoparticles could reduce intracellular adenosine triphosphate (ATP) activity, change cell membrane integrity, and trigger direct necrosis and apoptosis during inhibiting cell viability in primary astrocytes. Flow cytometry assessments revealed that the branched PEI can result in greater internalization than the liner PEI, which also induced

2. Toxicity of nanomaterials

174

8. Toxicity of polymeric nanomaterials

greater cytotoxicity. Annexin V assay confirmed early and late apoptosis by branched PEI, imposing somewhat DNA damage detected by comet assay. Western blot analysis resulted in induction of Akt-kinase which is possibly one of biomolecules affected by PEI [56]. Florea et al., explored transfection efficiency and toxicity of polyethyleneimine in differentiated Calu-3 and nondifferentiated COS-1 cell cultures, and found that transfection efficiency was strongly correlated to PEI cytotoxicity. Also, some evidence for PEI-induced apoptosis in both cell lines was found [57]. The cytotoxicity results showed that linear 250 kDa PEI was nontoxic whereas branched PEIs with lower molecular weights showed toxicity effects in a concentration dependent manner. Also, the cytotoxicity effects of branched PEIs were proportional with carrier/plasmid ratio and were more for the polyplexes prepared in HBG buffer compared to HEPES-buffered saline buffer after 24 h incubation. Flow cytometry results confirmed that apoptosis is the main mechanism of cell toxicity produced by polyplexes [58].

8.3.5 Polystyrene Cationic polystyrene nanoparticles induced apoptosis followed by a secondary necrotic response in 1321N1 human brain astrocytoma cells. Immunoblot showed that apoptosisrelated proteins such as caspase-3, caspase-7, caspase-9, and poly(ADP-ribose) polymerase-1 (PARP-1) were cleaved after cationic polystyrene nanoparticles incubation. Morphological changes of lysosomes and mitochondria, which were consistent with quickly increase of ROS formation, were also confirmed by transmission electron microscopy analysis [59].

8.4 In vivo toxicity of polymeric nanomaterials 8.4.1 Respiratory system Cullen et al. ever explored the inhalation toxicity of cellulose fiber. The experimental rats were exposed to an aerosol of cellulose fibers for 7 h per day, 5 days per week for periods from 1 day to 3 weeks. After 1 day exposure, early inflammatory response was found from lavaging the lungs, but subsequently the numbers and percentages of inflammatory granulocytes declined. Histopathology results showed that aggregations of macrophages and adjacent epithelial cells were around at the bifurcations of the terminal and respiratory bronchioles (toxicity of cellulose fibers) [60]. Huang et al. examined effects of lung tissues to chitosan microparticles exposure: inhaled chitosan microparticles triggered proinflammatory effects in rat lungs in a dose-dependent manner, which including increases in bronchoalveolar lavage fluid protein and lactate dehydrogenase activity and increases in lung tissue myeloperoxidase activity and leukocyte migration. Moreover, a large of polymorphonuclear neutrophils has been found infiltrated in lung tissues. Taken together, these results indicated that inhaled chitosan microparticles could have significant proinflammatory effects on lung tissues [61] (Fig. 8.2). Different delivery methods of nanoparticles might cause different effects. Here the inflammatory effects of respire cellulose fibers were studied in two short-term animal models: intraperitoneal injection in mice, and inhalation in rats. The mouse peritoneal cavity is particularly sensitive to fibrous compared to nonfibrous particles. Cellulose caused

2. Toxicity of nanomaterials

8.4 In vivo toxicity of polymeric nanomaterials

175

FIGURE 8.2 Systemic Toxicity of Nanomaterials.

marked, dose-dependent recruitment of inflammatory cells to the mouse peritoneal cavity. For the inhalation study, the inhalation exposure induced an early inflammatory response in rat lung, as determined by bronchoalveolar lavage [60]. Also different genders might also generate different responses to the same nanoparticles. Anna et al., explored the gender differences in murine pulmonary responses elicited by cellulose nanocrystals, and

2. Toxicity of nanomaterials

176

8. Toxicity of polymeric nanomaterials

found that cellulose nanocrystals exposure could induce oxidative stress, tumor growth factor -β and collagen levels elevation, and pulmonary functions damage [62]. Apart from cellulose nanomaterials, hydroxyethyl starch could also trigger respiratory system toxicity through mediating pulmonary cytokines release and edema formation [63]. In addition, PLA nanoparticles were reported to induce biological functions modification of A549 lung epithetial cells [64]. Cationic starburst PAMAM dendrimers could induce acute lung injury in vivo. PAMAM dendrimers triggered autophagic cell death via deregulating the Akt-TSC2-mTOR signaling pathway. The autophagy inhibitor 3-methyladenine rescued PAMAM dendrimer-induced cell death and ameliorated acute lung injury caused by PAMAM dendrimers in mice [65].

8.4.2 Cardiovascular system Zebrafish are becoming a model organism for investigating cardiovascular toxicity because of their short reproductive cycles, numerous transparent productions, and low cost [66]. Kim et al., studied acute cardiovascular toxicity of sterilizers, polyhexamethylene guanidine phosphate (PHMG), and oligo-[2-(2-ethoxy)-ethoxyethyl]-guanidinium-chloride (PGH) in human cells and heart failure in zebrafish models: The PGH and PHMG at normal dosages caused severe atherogenic process in human macrophages, cytotoxic effect, and aging in human dermal cell. Zebrafish embryos exposed to the sterilizer displayed early death with acute inflammation and attenuated developmental speed. All zebrafish treated by different working concentration of PHMG and PGH died within 70 min and showed serum triacylglycerol level acute increases and fatty liver induction. The dead zebrafish showed severe accumulation of fibrous collagen in the bulbous artery of the heart with elevation of ROS, which confirmed that the sterilizers showed acute toxic effect in blood circulation system, caused by severe inflammation, atherogenesis, and aging, with embryo toxicity [67]. High molecular weight of PEIs such as PEI 25 kD are promising for their drug carrying capacity. The systemic evaluation of branched PEI 25 kD and PEI-CyD (PC) composed of low molecular PEI (Mw 600) and beta-cyclodextrin was performed in zebrafish model and endothelial cells. Exposure of PC and PEI 25 kD can induce high mortality rate, shorten hatching time, promote malformations and cell apoptosis of zebrafish embryos in a dose-dependent manner. Most signify, the cationic polymer PC and PEI 25 kD can decrease heart rate of zebrafish embryos and downregulate the expression of heart development-related genes, which demonstrate their cardiovascular toxicity. PC can induce endothelial cells dysfunction, including oxidative stress and apoptosis which are involved in cardiovascular diseases [68]. Micro/nanoparticles could induce serious effects on cardiovascular system and increase cardiovascular disease-related risks. poly(ethylene glycol) (PEG)-b-poly(ε-caprolactone) nanomicelle caused dose-dependent embryo mortality and embryonic and larval malformations. PEG-b-PLC nano-micelle also dose-dependently decreased growth of intersegmental vessels and caudal vessels, as well as reduced fetal liver kinase 1 expression. In addition, PEG-b-PCL nano-micelle exposure upregulated p53 pro-apoptotic pathway and induced cellular apoptosis. Suppressing p53 activity by pharmacological inhibitor or siRNA could abrogate the apoptosis and angiogenic effects triggered by PEG-b-PCL nano-micelles, indicating that PEGb-PCL nano-micelle inhibits angiogenesis through activating p53-mediated apoptosis [69].

2. Toxicity of nanomaterials

8.4 In vivo toxicity of polymeric nanomaterials

177

8.4.3 Neurological system Chitosan nanoparticles could enter into the brain and specially accumulate in the frontal cortex and cerebellum after systemic injection. Exposure of chitosan nanoparticles for 7 days could significantly decrease the body weight of rats in a dose-dependent manner [70]. Apoptosis and necrosis of neurons, slight inflammatory response in frontal cortex, and decrease of glial fibrillary acidic protein expression in cerebellum also confirmed the neurotoxicity of chitosan nanoparticles. PEIs could also induce acute brain toxicity in adult Sprague Dawley rats through detecting the size of gross tissue loss and measuring reactive astrocytes and microglial cell recruitment. Compared with negative control, PEIs loosed gross tissue volume in brain tissues and triggered astrocytes and microglia accumulation during their induced neurotoxicity [71]. In addition, researchers used human brain-derived endothelial cells, human brain astrocytoma cells, and glioma cells as in vitro cell models for neurotoxicity study. PLGA-poly(ethylene oxide) nanomaterials were reported to be localized and accumulated in human brain-derived endothelial cells, caused oxidative stress, and triggered DNA-damaging effects [54]; polystyrene nanoparticles exposed to human brain astrocytoma cells could induce caspase and PARP-1 cleavage followed by neurosis [72]. Cationic PAMAM dendrimers could obviously induce morphology changes of glioma cells and concentration-dependently decrease cell viability [73]. Moreover, PAMAM dendrimers mediated toxicity was tightly associated with DNA damage, apoptosis, and ROS production [74]. Our previous publications also showed that cationic PAMAM dendrimers caused mitochondrial oxidative fluorescence and autophagic fluorescence accumulation in neuronal cells [75]; Meanwhile, other studies also suggested that the interaction between PAMAM dendrimers and plasma membranes induced neurons and astroglial cells activation, led to mitochondrial depolarization, and oxidation impaired in brain tissues [76].

8.4.4 Immune system To explore in vitro blood cell viability profiling of polymers used in molecular assembly, Jeong et al., investigated the red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs) cytotoxic effects of polymers frequently used in the layer-by-layer assembly technique; and implemented three types of assays: a hemolysis assay using RBCs, cell viability and Annexin V-FITC/propidium iodide (PI) staining assays using PBMCs. The polymers used in the study were divided into three groups based on their surface charge: cationic polymeric nanoparticles; neutral polymeric nanoparticles; and anionic polymeric nanoparticles. Cytotoxicity is indicated at over 10% hemolysis, and only LPEI at its highest dose showed mild toxicity. After 24 h, toxicity was indicated at the higher concentrations of several polymers in the order of LPEI (62.1%) . tannic acid (TA) (21.5%) . poly(diallyl dimethyl ammonium chloride) (PDAC) (19.2%) . chitosan low molecular weight (CHI) (16.5%) . branched polyethylenimines (BPEI) (13.6%) at 50 μg/mL. After 48 h, the toxic effects of the above polymers continuously increased; In addition, the higher concentrations of polyacrylamide (PAAM) and PAH showed toxicity. Most of the toxic effects were limited to higher concentration of PAAM and poly(allylamine hydrochloride) (PAH) showed toxicity. However, most of the toxic effects were limited to higher concentrations, longer exposure times, and cationic polymers. After 9 h, a large proportion of cells exposed to PDAC

2. Toxicity of nanomaterials

178

8. Toxicity of polymeric nanomaterials

(90.5%), LPEI (82.4%), and PLL (77.3%) at 10 μg/mL were altered to apoptotic forms compared to PEG (45.6%). These results correspond to those from the hemolysis and cell viability assays, and confirmed that polycations directly damage the cell membrane. Although PEG is nontoxic and nonimmunogenic, the early apoptotic cell phase could be caused by the high amounts of polymers on the cell surface [77].

8.4.5 Skin The dermal toxicity of cationic PAMAM dendrimers were evaluated, 10 days topical application of various concentrations of PAMAM-NH2 resulted in signify morphological changes of epidermal cells including cytoplasmic vacuolization of keratinocytes in the basal and spinouts layers, hyperplasia of connective tissue fibers and leukocyte infiltration. Furthermore, visible granulocyte infiltration in the upper dermis and sockets formed by necrotic, cornified cells in the hyperplastic foci of epithelium were also mentioned [78]. The increased nuclear immunity to proliferating cell nuclear antigen extended throughout the skin layers might suggest abnormal cell proliferation, which might lead to neoplastic changes. The anaphylactoid reaction caused by dextran in rats has been studied by several investigators. Edlund et al., found that after intradermal injections of dextran, menkin’s intravenous dye test shows seepage of dye into the wheals when the concentration of commercial dextran is at least 10 μg/mL. Dextran preparations with highly branched molecular chains are more toxic than those with relatively unbranched chains [79]. Flexner reported that continuous intravenous injection of dextran sulfate was toxic, producing profound but reversible thrombocytopenia in all subjects who received drug for more than 3 days and extensive but reversible alopecia in most subjects. As its toxicity and lack of beneficial effect on surrogate markers, dextran sulfate is unlikely to have a practical role in the treatment of symptomatic HIV infection [80]. The influence of two chitosans with a similar degree of deacetylation but different molecular weight, chitosan 1130 (120 kDa) and chitosan oligosaccharide (5 kDa), on the human keratinocyte cell line HaCaT was analyzed. Chitosans exhibit a molecular-weight-dependent negative effect on HaCaT cell viability and proliferation in vitro. The chitosans tested also stimulated the release of inflammatory cytokines by HaCaT cells depending on incubation time and concentration. Chitosan 1130 and chitosan oligosaccharide induced apoptotic cell death, which was mediated by activation of the effector caspase 3/7 [81].

8.4.6 Gastrointestinal system The safety of orally-administered polymeric nanoparticles was mainly based on the physical properties such as surface charge and surface chemistry. Wiwattanapatapee et al., ever explored the effects of different PAMAM dendrimer size, charge, and concentration on the uptake and transport across rat intestine by using everted rat intestinal sac system. Cationic PAMAM dendrimers have lower absorption due to adherence to negatively charged cell membranes of the gut epithelium, while anionic PAMAM dendrimers showed serosal transfer rate which were faster than natural macromolecules in the everted sac system [82]. Similarly, rat gut explant study using lipid core nanoparticles for delivery of indomethacin showed that the nanoparticle was degraded and the core exposed in the gut lumen, indicated by metabolic conversion of

2. Toxicity of nanomaterials

8.5 Mechanisms of polymeric nanomaterials-induced toxicity

179

indomethacin in the lumen prior to absorption into blood [83]. Dextran sulfate sodium has been used to destroy intestinal epithelium structure and impair barrier function to mimic clinic intestine damages, which causes the intestinal inflammation and further tissue destruction [84]. Navarro et al., quantified the biodistribution and assessed the toxicity of PLGA and surfacemodified PLGA chitosan nanoparticles orally administered for different days to rats, and found that biodistribution of PLGA and PLGA chitosan nanoparticles were similar, with highest amounts found in the intestine and liver. Alkaline phosphatase increased significantly in treated rats. Mild histological differences were detected in the intestine and liver [85].

8.4.7 Reproduction and development Embryo exposure to chitosan nanoparticles resulted in a concentration dependently decreased hatching rate and increased mortality. The exposure of chitosan nanoparticles to embryo showed an increased rate of cell death, high expression of ROS, and overexpression of heat shock protein 70, indicating that chitosan nanoparticles caused reproduction and development toxicity in zebrafish [86]. Wick et al., explored whether polystyrene nanoparticles could cross the placental barrier and affected the fetus: They chose fluorescently labeled polystyrene nanoparticles with diameters of 50, 80, 240, and 500 nm to investigate whether these effects was in size-dependent manner. The polystyrene nanoparticles with diameter up to 240 nm could be taken up by the placenta and were able to cross the placental barrier without affecting the viability of the placental explant [87]. Scsukova et al., examined the effect of polymeric nanoparticles PEG-b-PLA on in vitro luteinizing hormone release from anterior pituitary cells of infantile and adult female rats, and found that neonatal treatment with PEG-b-PLA signify increased basal and luteinizing hormone releasing hormone-induced luteinizing hormone release from pituitary cells in both infantile and adult rats [88]. They also studied delayed effects of neonatal exposure to polymeric nanoparticle PEG-b-PLA on the endpoints related to pubertyl development and reproductive function in female rats. Intraperitoneally injection of 20 or 40 mg/kg of PEG-b-PLA could accelerate the onset of vaginal opening, while 20 mg/kg of PEG-b-PLA intraperitoneal injection significantly reduced regular estrous cycles numbers, increased pituitary because of hyperemia, vascular dilatation and congestion, altered hypothalamic gonadotropin-releasing hormone-stimulated luteinizing hormone secretion course, and increased progesterone serum level, which indicated that PEG-b-PLA could affect hypothalamic-pituitary-ovarian axis development and function in adult female rats [89].

8.5 Mechanisms of polymeric nanomaterials-induced toxicity 8.5.1 Physicochemical mechanisms of polymeric nanomaterials-induced toxicity The toxicity of polymeric nanomaterials could be caused by their unique physicochemical characteristics, which includes size, charge, shape, dispersity, surface chemistry, crystal types, and so on. Besides, dosage, administrated types, and exposure routes of nanomaterials are all common factors affecting toxicity [90] (Fig. 8.3).

2. Toxicity of nanomaterials

180

8. Toxicity of polymeric nanomaterials

FIGURE 8.3 Toxicity of Polymeric Nanomaterials.

8.5.1.1 Size The size-dependent cytotoxicity of nanomaterials could be smaller nanomaterials with larger specific surface existing more biomolecules absorbing ability, and these abilities might be important paradigm to predict the in vitro toxicity of nanomaterials. PEIs including LPEI and BPEI showed a strong cytotoxic effect due to their large molecular mass and high charge density resulting from a large number of secondary amine groups. High molecular weight PEI (800 kDa) induced massive necrosis within 30 min and low molecular weight PEI (25 kDa) had low cytotoxicity. Higher molecular weight (39,800 kDa) PLL showed a higher toxicity than low molecular weight PLL (8000 kDa). Although biomedical used PLGA polymeric nanomaterials exhibit no obvious cytotoxic effects, 60 nm PLGA nanomaterials could induce proinflammatory cytokines release, while 200 nm PLGA nanomaterials could not induce any negative response from the cells. Different with PLGA, when compared with the cytotoxicity of PLL polymeric nanomaterials of low molecular weight and high molecular weight by exploring mitochondrial membrane potential, cytochrome C release, and caspase activation, low-molecular-weight PLL polymeric nanomaterials did not trigger cytochrome C release or mitochondrial dysfunction, while high-molecular-weight PLL polymeric nanomaterials caused more cytochrome C release, mitochondrial membrane potential loss, and caspase activation. Another independent study also confirmed that PLL polymeric nanomaterials-triggered cytotoxicity was tightly related with their molecular weight: 29.8 kDa PLL polymeric nanomaterials were much more toxic than 4.0 kDa and 7.5 kDa PLL polymeric nanomaterials. So although toxicity of polymeric nanomaterials was depends on their size, it does not mean smaller sized polymeric nanomaterials were less toxic than the larger ones, it still depends on the different types of nanomaterials [91].

2. Toxicity of nanomaterials

8.5 Mechanisms of polymeric nanomaterials-induced toxicity

181

8.5.1.2 Charge The charge of polymeric nanomaterials could significantly affect their cellular uptake in vitro [92]. Because of electrostatic attraction between cell membranes and positively charged cationic polymeric nanomaterials, cationic polymeric nanomaterials with positively charged surfaces could be more easily attached on the cell membranes and accumulated inside the cells [93]. A previous study has suggested that the toxicity of PAMAM dendrimers was mainly influenced by size and charge. Researchers reported that surface charge and functional group-dependent toxicity with amine-terminated dendrimers (both G4 and G7) were safely only at doses lower than 10 mg/kg, while carboxyl (G3.5 and G6.5) and hydroxyl (G4 and G7) terminated dendrimers were safely administered at 50fold or higher doses. Employment of amine-terminated dendrimers in human applications could be fatal unless amine-terminated surface properties were modified to reduce their toxic effects [39]. Besides, hundred nanomolar of G4-C12 PAMAM dendrimers caused obvious apoptotic cell death of neurons in vitro, while G4 PAMAM dendrimers did not trigger apoptotic effect even in the submicromolar range of concentration [94]. The oral toxicity of PAMAM dendrimers also depended on charges of polymeric nanoparticles: 300 mg/kg of G7-OH PAMAM dendrimers treatment proved to be toxic, whereas G7-NH2 PAMAM dendrimers with a dose of 30 mg/kg was at least 10 times less tolerated than G7-OH PAMAM dendrimers [95]. 8.5.1.3 Shape Toxicity of polymeric nanomaterials is also affected by polymeric architecture. Polymeric nanomaterials with higher toxicity usually exist within a larger size (molecular weight) and much more branching of the macromolecule (shape) [96]. Kafil and Omidi ever compared cytotoxic effects of LPEIs and branched PEIs, and reported that branched PEIs could result in greater internalization and trigger more serious cytotoxicity than LPEIs. Branched PEIs could also induce early and late apoptosis, which has been confirmed by Annexin V assay and DNA fragmentation analysis [56]. The effects of chain length on cytotoxicity and endocytosis of Rhodamine B (RhB) end-labeled cationic linear poly(2-(N,N-dimethylamino)ethyl methacrylate) (RhB-PDMAEMA) polymeric nanomaterials were also investigated: for the short chain PDMAEMA, its triggered cytotoxicity, membrane disruption, and apoptotic effects were low and independent of the chain length; while for the medium-length chain PDMAEMA, its caused cytotoxicity was increased with chain length and polymer concentration, and mainly due to the cooperative effect of membrane disruption and apoptosis; for long-length chain PDMAEMA, they were very disruptive to the cellular membrane, proapoptotic, and able to enter cytoplasm and nucleus faster than short chains, and their cytotoxicity is independent of PDMAEMA chain length [97]. Vaine et al., compared the role of polymers surface texturing and found that textured particles could not only influenced uptake efficiency but also on the subsequent immune cell activation of the inflammasome: textured polymers could recruit neutrophil to the injection site much more quickly and could be more readily phagocytosed and recruit lipid raft to the phagosome than smooth particles. Moreover, textured polymers could also induce more IL-1β secretion than smooth particles via NLRP3 inflammasome activation [98]. PLGA-PEG nanoparticles could induce cytotoxicity via

2. Toxicity of nanomaterials

182

8. Toxicity of polymeric nanomaterials

lysosome damage-mediated cell apoptotic signaling pathway, and finally triggered DNA fragmentation and cell death, while needle-shaped, but not sphere-shaped PLGA-PEG nanoparticles could trigger obvious cytotoxicity [99]. 8.5.1.4 Surface chemistry Typical dispersities vary based on the mechanism of polymerization and can be affected by a variety of reaction conditions. In synthetic polymeric nanomaterials, it can be vary greatly due to reactant ratio, how close the polymerization went to completion [100]. The toxic properties of dendrimers also relied on their structural components including the core, interior monomers, and surface groups, of which amino surface groups are known to be the most toxic [40]. Due to existing amine groups, dendrimers are limited in their clinical applications. These groups are positively charged or cationic which makes them toxic, hence dendrimers are usually modified to reduce their toxicity or to eliminate it [101]. Janaszewska et al., compared neurotoxicity of cationic amino-terminated PAMAM dendrimers and pyrrolidone modified PAMAM dendrimers in mouse embryonic hippocampal mHippoE-18 cells by using lactate dehydrogenase release and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide assay, and found that cationic amino-terminated PAMAM dendrimers triggered significant release of LDH and inhibited cell growth. Shrinkage of cells, shortening of axonal branches, and cell death mediated by cationic amino-terminated PAMAM dendrimers could be obviously diminished by pyrrolidone modification [102]. To explore the genotoxic effect of polymeric nanomaterials, Choi et al., evaluated cell death induced by polyethyleneimine and PAMAM dendrimers via comet assay and cytokinesis-block micronucleus assay, and found that polyethyleneimine and PAMAM dendrimers treatment induced significant increases in DNA damage [103]. The toxicity of unmodified generation 4 poly propylene imine (PPI) dendrimers and generation 4 PPI dendrimers existing 25% and 100% of the surface amino groups substituted with maltotriose residues. A higher dose of unmodified PPI dendrimers triggered significant toxicity, but surface modification nearly abolished these toxic effects [104].

8.5.2 Biochemical mechanisms of polymeric nanomaterials-induced toxicity 8.5.2.1 Apoptosis and necrosis Apoptosis is one of programmed cell death (PCD) types which usually occur in multicellular organisms [105]. Apoptosis could be triggered by different extrinsic death receptors or intrinsic mitochondrial pathway, as mitochondrion is one of the major regulators of cell death, especially for nanomaterials-induced apoptotic cell death. Mitochondrial dysfunction related apoptosis is characterized as mitochondrial membrane potential loss, cytochrome C release, and caspases activation [106]. Amine-modified polystyrene nanoparticles could increase cell membrane permeability of PI, induce caspase 3, caspase 7, and caspase 9 activation, trigger PARP-1 cleavage, and induce an apoptotic response in human brain astrocytoma cell lines [107]. PEI polymeric nanomaterials mediated cytotoxicity could be divided into necrosis in the early stage and apoptosis in the late stage [96]. Necrosis is unprogrammed death of cells characterized by the rapid breakdown of cell membranes, which resulting massive intracellular components are released into the

2. Toxicity of nanomaterials

8.5 Mechanisms of polymeric nanomaterials-induced toxicity

183

extracellular space. In contrast to apoptosis, where caspases are the key death proteases, calpains and lysosomal proteases are major players in necrosis. As a passive and uncontrolled process, necrosis could usually be triggered by series of factors such as infections, cellular stress, and toxins, and necrosis cannot be reversed [108]. Exposure of cationic polymeric nanoparticles, including cationic liposomes, PEI, and chitosan, led to the rapid appearance of necrotic cells. Cell necrosis induced by cationic polymeric nanoparticles dependent on their positive surface charges [109,110]. Instead, intracellular Na 1 overload was found to accompany the cell death. Cell necrosis induced by cationic polymeric nanoparticles and the resulting leakage of mitochondrial DNA could trigger severe inflammation in vivo, which confirmed that cationic polymeric nanoparticles induced acute cell necrosis through the interaction with Na 1 /K 1 -ATPase, with the subsequent exposure of mitochondrial damage-associated molecular patterns as a key event that mediates the inflammatory responses [111]. 8.5.2.2 Ferroptosis Ferroptosis is characterized morphologically by the presence of smaller than normal mitochondria with condensed mitochondrial membrane densities, reduction or vanishing of mitochondria crista, and outer mitochondrial membrane rupture; and is recognized as one of the regulated cell death types [112]. Activation of mitochondrial voltage-dependent anion channels and mitogen-activated protein kinases, upregulation of endoplasmic reticulum stress, and inhibition of cysteine/glutamate antiporter is involved in the activating process of ferroptosis, and is characterized by the accumulation of lipid peroxidation products and lethal ROS derived from iron metabolism [113]. Kim et al., presented that ultra small PEG-coated silica nanoparticles, functionalized with melanoma-targeting peptides can trigger ferroptosis in starved cancer cells and cancerbearing mice. Tumor xenografts in mice intravenously injected with nanoparticles using a high-dose multiple injection scheme exhibit reduced growth or regression, in a manner that is reversed by the pharmacological inhibitor of ferroptosis, liproxstatin-1 [114]. Szwed et al., recently reported that poly(alkylcyanoacrylate) with butyl alkyl side chains (PBCA) triggered cytotoxicity could be almost totally reversed by the ferroptosis inhibitors and the iron chelater deferiprone could also abolish PBCA-induced cytotoxicity, while pan-caspase inhibitor z-VAD had no significant effect. PBCA particles treatment could induce lipid peroxidation in a liproxstatin-sensitive manner. The cellular import of cysteine is identified as one of the key regulators of ferroptosis. When MDA-MB-231 cells were incubated in medium lacking only cysteine, potent cell death was induced by significantly lower PBCA concentrations than those required to induce cytotoxicity in full medium. The cell death induced by PBCA particles in cysteine-free medium could be fully inhibited by ferroptosis inhibitors ferrostatin, liproxstatin or by re-addition of cysteine [115]. Although currently limited reports showed ferroptosis triggered by polymeric nanoparticles during their induced toxicity, it might be one of the critical mechanisms during polymeric nanoparticles-mediated toxicity and deserve further explorations. 8.5.2.3 Autophagy Autophagy is the evolutionarily conserved process of degradation cytoplasmic components such as dysfunctional organelles and misfolded protein within lysosomes under

2. Toxicity of nanomaterials

184

8. Toxicity of polymeric nanomaterials

stress conditions [116]. This process in different with endocytosis-mediated degradation and recycle of cytoplasmic components. Autophagy triggered by polymeric nanoparticles during toxicity might be an adaptive cellular response aiming to clearance nanomaterials or could be harmful during cellular dysfunction [117]. Our previous review has presented the different role and mechanisms of autophagy in nanoparticles-induced toxicity [118], and also introduced cationic polymers mediated autophagy in their neurotoxicity [73]. Here we mainly focused on recent studies regarding polymeric nanoparticles-triggered autophagy. Lin et al., reported that pH-sensitive polymeric nanomaterials could modulate autophagic effect through lysosome impairment. They evaluated four types of polymeric nanomaterials with different physical properties and explored their triggering autophagy capabilities: pH-sensitivity is one of the most critical factors for autophagy, lower dose of polymeric nanomaterials triggered autophagy in an mTOR dependent manner while higher dose of polymeric nanomaterials led to autophagic cell death [119]. Sun et al., explored new intracellular trafficking networks of 3-hydroxybutyrate-co-3-hydroxyhexanoate (PHBHHx) and presented the autophagy mechanism of PHBHHx nanomaterials: PHBHHx nanomaterials could induce intracellular autophagy and be degraded through endolysosomal pathways; autophagy inhibitors 3-methyladenine and chloroquine could impair nanomaterials degradation in lysosomes by blocking endolysosomal pathways [120]. 8.5.2.4 Pyroptosis Pyroptosis is an inflammatory form of PCD that occurs most frequently upon infection with intracellular pathogens [121]. In contrast to apoptosis and necrosis, pyroptosis requires the function of the enzyme caspase-1. Caspase-1 is activated during pyroptosis by a large super molecular complex termed pyroptosome (inflammasome), and subsequently mediates the maturation and secretion of interleukin-1 beta (IL-1β) and interleukin-18 [122]. Lower size of chitosan, but not larger size of chitosan, provoked greater activity on NLRP3 inflammasome activation, and mediated interleukin-1β secretion. Intraperitoneal poly(ethylenimine) injection could activate NLRP3 inflammasome, cleave caspase-1, and mediate pyroptosis in vivo [123]. Amino-functionalized polystyrene nanomaterials could activate NRLP3 inflammasome and trigger the release of interleukin-1β much more effectively than carboxyl- and nonfunctionalized nanomaterials in human macrophages [124]. Cationic PAMAM dendrimers could induce inflammasome-signaling activation as demonstrated by NLRP3 activation, caspase-1 cleavage, and interleukin-1β maturation during their triggered hepatotoxicity. Suppression of NLRP3 inflammasomes by belnacasan could significantly protect cationic PAMAM dendrimers-induced hepatotoxicity [125]. Hu et al., presented that administration of PEI 25 kD could obviously promote breast cancer metastasis in liver and lung tissues, and these effects were correlated with its ability to induce high oxidative stress and NLRP3 inflammasome activation, which suggested that polymeric nanoparticles have potential to trigger immune-stimulation and cancer metastasis [68]. 8.5.2.5 Cell cycle arrest Cell cycle arrest is a stopping point in the cell cycle, where it is no longer involved in the processes surrounding duplication and division. Although low dose of amino-modified

2. Toxicity of nanomaterials

8.5 Mechanisms of polymeric nanomaterials-induced toxicity

185

polystyrene nanoparticles induced lower cell death, cell cycle was significantly arrested, and further studies suggested that nanoparticle uptake in the cells was affected by cell cycle arrest. Wang et al., reported that polyvinylpyrrolidone (PVP) K-30 treatment could induce toxicity to HeLa cells in a dose-dependent and time-dependent manner: cells exposed to PVP K-30 showed obviously morphological apoptotic features. Flow cytometric analysis confirmed that PVP K-30 induced HeLa cell cycle arrest at G2/M phase and the subsequent appearance of sub-G1 population [126]. Salehi et al., showed that exposure of MDA-MB-231 cells to chitosan led to depolarization of the mitochondrial membrane, increase in ROS, DNA oxidation, and S phase cell cycle arrest [127]. Furthermore, Annexin-PI staining, Terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and altered expression of caspase 3 in MDA-MB-231 cells indicated that cell progressively became apoptotic upon chitosan exposure. S phase arrest in MDA-MB-231 cells suggested possible chitosan DNA interaction. The effects of resistant starch treatment on tumor cells and colonic mucosal cells were also been explored: When compared with ordinary starch, resistant starch treatment triggered significant upregulation of cell cycle regulatory genes cyclin-dependent kinase 4 and growth arrest and DNA damage-inducible alpha [128]. Legaz et al., studied the cytotoxicity of PLA nanomaterials in Drosophila model, and found that oxidative stress as well as P53 and ATP pathways led to cell cycle arrest at G1, and ultimately to cell death when using near lethal nanoparticle doses [129]. Researchers also explored the cell cycle distribution of fibroblasts and keratinocytes treated by different doses of second and third generation PAMAM dendrimers for 24 h: a decrease in the proportion of cells in the G1 phase while increase in the S phase; and the increase in the S phase was directly proportional to the increase in dendrimer concentration and generation [130]. 8.5.2.6 Oxidative stress Overproduction of ROS can induce oxidative stress, resulting in an imbalance between prooxidant and antioxidant levels in cells [131]. A series of literature have reported that oxidative stress could lead to apoptosis, DNA damage, ion channels changes, and has also become the critical factor for nanotoxicity mediated by polymeric nanomaterials [132]. PLGA nanoparticles could be endocytosed by the cells, and triggeroxidative stress by inducing ROS production, which in turn cause DNA damage without influencing cell viability [133]. Besides, cationic polystyrene nanomaterials could induce mitochondria morphological changes and rapidly increase ROS accumulation in human brain astrocytoma cells [72]. PAMAM dendrimers could also evoke neuronal depolarization and induce cell death: PAMAM dendrimers could activate neurons and astroglial cells, resulting in mitochondrial depolarization and also impair oxidative metabolism of neurons [75]. Moreover, different surface modified PAMAM dendrimers showed different influence on the mitochondrial membrane potential and intracellular ROS levels: unmodified PAMAM dendrimers caused a toxic response via ROS generation and mitochondria damage, while pyrrolidonemodified dendrimers lacked the abilities on ROS formation [101].

Acknowledgments This work was supported by grants from the National Key Basic Research Program of China (2015CB931800), and the National Natural Science Foundation of China (81901232, 81773620, 81573332).

2. Toxicity of nanomaterials

186

8. Toxicity of polymeric nanomaterials

Conflict of interest All the figures in “Toxicity of Polymeric Nanomaterials” are original, unpublished materials designed and prepared by Yubin Li, Shaofei Wang, and Dianwen Ju. The authors declared no conflict of interests.

References [1] J. Jeevanandam, A. Barhoum, Y.S. Chan, A. Dufresne, M.K. Danquah, Review on nanoparticles and nanostructured materials: history, sources, toxicity and regulations, Beilstein J. Nanotechnol. 9 (2018) 1050 1074. [2] L. Sun, C. Zheng, T.J. Webster, Self-assembled peptide nanomaterials for biomedical applications: promises and pitfalls, Int. J. Nanomed. 12 (2017) 73 86. [3] M. Goldberg, R. Langer, X. Jia, Nanostructured materials for applications in drug delivery and tissue engineering, J. Biomater. Sci. Polym. Ed. 18 (2007) 241 268. [4] S. Barua, J. Ramos, T. Potta, D. Taylor, H.C. Huang, G. Montanez, et al., Discovery of cationic polymers for non-viral gene delivery using combinatorial approaches, Combin. Chem. High. Throughput Screen. 14 (2011) 908 924. [5] G. Lin, H. Zhang, L. Huang, Smart polymeric nanoparticles for cancer gene delivery, Mol. Pharm. 12 (2015) 314 321. [6] M.R.E. Santos, A.C. Fonseca, P.V. Mendonca, R. Branco, A.C. Serra, P.V. Morais, et al., Recent developments in antimicrobial polymers: a review, Materials 9 (2016). [7] S.K. Murthy, Nanoparticles in modern medicine: state of the art and future challenges, Int. J. Nanomed. 2 (2007) 129 141. [8] R.D. Brohi, L. Wang, H.S. Talpur, D. Wu, F.A. Khan, D. Bhattarai, et al., Toxicity of nanoparticles on the reproductive system in animal models: a review, Front. Pharmacol. 8 (2017) 606. [9] R. Wang, B. Song, J. Wu, Y. Zhang, A. Chen, L. Shao, Potential adverse effects of nanoparticles on the reproductive system, Int. J. Nanomed. 13 (2018) 8487 8506. [10] J.W. Hickey, J.L. Santos, J.M. Williford, H.Q. Mao, Control of polymeric nanoparticle size to improve therapeutic delivery, J. Control. Release 219 (2015) 536 547. [11] S.S. Guterres, M.P. Alves, A.R. Pohlmann, Polymeric nanoparticles, nanospheres and nanocapsules, for cutaneous applications, Drug. Target. Insights 2 (2007) 147 157. [12] F.S. Poletto, R.C.R. Beck, S.S. Guterres, A.R. Pohlmann, Polymeric nanocapsules: concepts and applications, in: R. Beck, S. Guterres, A. Pohlmann (Eds.), Nanocosmetics and Nanomedicines, Springer, Berlin, 2011. [13] K. DeFrates, T. Markiewicz, P. Gallo, A. Rack, A. Weyhmiller, B. Jarmusik, et al., Protein polymer-based nanoparticles: fabrication and medical applications, Int. J. Mol. Sci. 19 (2018) E1717. [14] J. Han, D. Zhao, D. Li, X. Wang, Z. Jin, K. Zhao, Polymer-based nanomaterials and applications for vaccines and drugs, Polymers 10 (2018) E31. [15] M.A. Mohammed, J.T.M. Syeda, K.M. Wasan, E.K. Wasan, An overview of chitosan nanoparticles and its application in non-parenteral drug delivery, Pharmaceutics 9 (2017) E53. [16] R.C. Cheung, T.B. Ng, J.H. Wong, W.Y. Chan, Chitosan: an update on potential biomedical and pharmaceutical applications, Mar. Drugs 13 (2015) 5156 5186. [17] J. George, S.N. Sabapathi, Cellulose nanocrystals: synthesis, functional properties, and applications, Nanotechnol. Sci. Appl. 8 (2015) 45 54. [18] M. Li, Q. Wu, K. Song, S. Lee, Y. Qing, Y. Wu, Cellulose nanoparticles: structure-morphology-rheology relationships, ACS Sustain. Chem. Eng. 3 (5) (2015) 821 832. [19] A.H. Tayeb, E. Amini, S. Ghasemi, M. Tajvidi, Cellulose nanomaterials-binding properties and applications: a review, Molecules 23 (2018). [20] A. Dufresne, Cellulose nanomaterials as green nanoreinforcements for polymer nanocomposites, Philos. Trans. Ser. A Math. Phys. Eng. Sci. 376 (2018). [21] E.C. Ramires, A. Dufresne, Cellulose nanoparticles as reinforcement in polymer nanoposites, in: F. Gao (Ed.), Advances in Polymer Nanocomposites, Woodhead Publishing, 2012, pp. 131 163.

2. Toxicity of nanomaterials

References

187

[22] J.K. Patra, G. Das, L.F. Fraceto, E.V.R. Campos, M.D.P. Rodriguez-Torres, L.S. Acosta-Torres, et al., Nano based drug delivery systems: recent developments and future prospects, J. Nanobiotechnol. 16 (2018) 71. [23] D. Le Corre, J. Bras, A. Dufresne, Starch nanoparticles: a review, Biomacromolecules 11 (2010) 1139 1153. [24] H.Y. Kim, S.S. Park, S.T. Lim, Preparation, characterization and utilization of starch nanoparticles, Colloids Surf. B Biointerfaces 126 (2015) 607 620. [25] P. Raigond, R. Ezekiel, B. Raigond, Resistant starch in food: a review, J. Sci. Food Agric. 95 (2015) 1968 1978. [26] J.P. Paques, E. van der Linden, C.J. van Rijn, L.M. Sagis, Preparation methods of alginate nanoparticles, Adv. Colloid Interface Sci. 209 (2014) 163 171. [27] M. Lopes, B. Abrahim, F. Veiga, R. Seica, L.M. Cabral, P. Arnaud, et al., Preparation methods and applications behind alginate-based particles, Expert Opin. Drug. Deliv. 14 (2017) 769 782. [28] G.S. Asane, S.A. Nirmal, K.B. Rasal, A.A. Naik, M.S. Mahadik, Y.M. Rao, Polymers for mucoadhesive drug delivery system: a current status, Drug. Dev. Ind. Pharm. 34 (2008) 1246 1266. [29] S.Y. Lee, Bacterial polyhydroxyalkanoates, Biotechnol. Bioeng. 49 (1996) 1 14. [30] S. Mohapatra, S. Maity, H.R. Dash, S. Das, S. Pattnaik, C.C. Rath, et al., Bacillus and biopolymer: prospects and challenges, Biochem. Biophys. Rep. 12 (2017) 206 213. [31] K. Uchino, T. Saito, B. Gebauer, D. Jendrossek, Isolated poly(3-hydroxybutyrate) (PHB) granules are complex bacterial organelles catalyzing formation of PHB from acetyl coenzyme A (CoA) and degradation of PHB to acetyl-CoA, J. Bacteriol. 189 (2007) 8250 8256. [32] R.A. Hassler, D.H. Doherty, Genetic engineering of polysaccharide structure: production of variants of xanthan gum in Xanthomonas campestris, Biotechnol. Prog. 6 (1990) 182 187. [33] R. Song, M. Murphy, C. Li, K. Ting, C. Soo, Z. Zheng, Current development of biodegradable polymeric materials for biomedical applications, Drug Des. Dev. Ther. 12 (2018) 3117 3145. [34] J. Cappello, J. Crissman, M. Dorman, M. Mikolajczak, G. Textor, M. Marquet, et al., Genetic engineering of structural protein polymers, Biotechnol. Prog. 6 (1990) 198 202. [35] J.E. Yang, S.Y. Choi, J.H. Shin, S.J. Park, S.Y. Lee, Microbial production of lactate-containing polyesters, Microb. Biotechnol. 6 (2013) 621 636. [36] S. Taguchi, M. Yamada, K. Matsumoto, K. Tajima, Y. Satoh, M. Munekata, et al., A microbial factory for lactate-based polyesters using a lactate-polymerizing enzyme, Proc. Natl Acad. Sci. U.S.A. 105 (2008) 17323 17327. [37] K. Bork, W. Reutter, R. Gerardy-Schahn, R. Horstkorte, The intracellular concentration of sialic acid regulates the polysialylation of the neural cell adhesion molecule, FEBS Lett. 579 (2005) 5079 5083. [38] M.S. Ravindran, L.B. Tanner, M.R. Wenk, Sialic acid linkage in glycosphingolipids is a molecular correlate for trafficking and delivery of extracellular cargo, Traffic 14 (2013) 1182 1191. [39] K. Madaan, S. Kumar, N. Poonia, V. Lather, D. Pandita, Dendrimers in drug delivery and targeting: drugdendrimer interactions and toxicity issues, J. Pharm. Bioallied Sci. 6 (2014) 139 150. [40] E. Abbasi, S.F. Aval, A. Akbarzadeh, M. Milani, H.T. Nasrabadi, S.W. Joo, et al., Dendrimers: synthesis, applications, and properties, Nanoscale Res. Lett. 9 (2014) 247. [41] T. Barrett, G. Ravizzini, P.L. Choyke, H. Kobayashi, Dendrimers in medical nanotechnology, IEEE Eng. Med. Biol. Mag. Q. Mag. Eng. Med. Biol. Soc. 28 (2009) 12 22. [42] H.K. Makadia, S.J. Siegel, Poly lactic-co-glycolic acid (PLGA) as biodegradable controlled drug delivery carrier, Polymers 3 (2011) 1377 1397. [43] P. Gentile, V. Chiono, I. Carmagnola, P.V. Hatton, An overview of poly(lactic-co-glycolic) acid (PLGA)-based biomaterials for bone tissue engineering, Int. J. Mol. Sci. 15 (2014) 3640 3659. [44] M. Jager, S. Schubert, S. Ochrimenko, D. Fischer, U.S. Schubert, Branched and linear poly(ethylene imine)-based conjugates: synthetic modification, characterization, and application, Chem. Soc. Rev. 41 (2012) 4755 4767. [45] A. Zakeri, M.A.J. Kouhbanani, N. Beheshtkhoo, V. Beigi, S.M. Mousavi, S.A.R. Hashemi, et al., Polyethyleniminebased nanocarriers in co-delivery of drug and gene: a developing horizon, Nano Rev. Exp. 9 (2018) 1488497. [46] D.P. Walsh, R.D. Murphy, A. Panarella, R.M. Raftery, B. Cavanagh, J.C. Simpson, et al., Bioinspired starshaped poly(l-lysine) polypeptides: efficient polymeric nanocarriers for the delivery of DNA to mesenchymal stem cells, Mol. Pharm. 15 (2018) 1878 1891. [47] R. Rai, S. Alwani, I. Badea, Polymeric nanoparticles in gene therapy: new avenues of design and optimization for delivery applications, Polymers 11 (2019) E745. [48] J. Varshosaz, Dextran conjugates in drug delivery, Expert Opin. Drug Deliv. 9 (2012) 509 523.

2. Toxicity of nanomaterials

188

8. Toxicity of polymeric nanomaterials

[49] N.A.N. Hanafy, M. El-Kemary, S. Leporatti, Micelles structure development as a strategy to improve smart cancer therapy, Cancers 10 (2018) E238. [50] Y. Yun, Y.W. Cho, K. Park, Nanoparticles for oral delivery: targeted nanoparticles with peptidic ligands for oral protein delivery, Adv. Drug. Deliv. Rev. 65 (2013) 822 832. [51] O. Dale, T. Nilsen, G. Olaussen, K.E. Tvedt, F. Skorpen, O. Smidsrod, et al., Transepithelial transport of morphine and mannitol in Caco-2 cells: the influence of chitosans of different molecular weights and degrees of acetylation, J. Pharm. Pharmacol. 58 (2006) 909 915. [52] A. Platel, R. Carpentier, E. Becart, G. Mordacq, D. Betbeder, F. Nesslany, Influence of the surface charge of PLGA nanoparticles on their in vitro genotoxicity, cytotoxicity, ROS production and endocytosis, J. Appl. Toxicol.: (JAT) 36 (2016) 434 444. [53] L. Gomes Dos Reis, W.H. Lee, M. Svolos, L.M. Moir, R. Jaber, N. Windhab, et al., Nanotoxicologic effects of PLGA nanoparticles formulated with a cell-penetrating peptide: searching for a safe pDNA delivery system for the lungs, Pharmaceutics 11 (2019) E12. [54] B. Halamoda Kenzaoui, C. Chapuis Bernasconi, S. Guney-Ayra, L. Juillerat-Jeanneret, Induction of oxidative stress, lysosome activation and autophagy by nanoparticles in human brain-derived endothelial cells, Biochem. J. 441 (2012) 813 821. [55] P.C. Naha, M. Davoren, F.M. Lyng, H.J. Byrne, Reactive oxygen species (ROS) induced cytokine production and cytotoxicity of PAMAM dendrimers in J774A.1 cells, Toxicol. Appl. Pharmacol. 246 (2010) 91 99. [56] V. Kafil, Y. Omidi, Cytotoxic impacts of linear and branched polyethylenimine nanostructures in a431 cells, BioImpacts (BI) 1 (2011) 23 30. [57] B.I. Florea, C. Meaney, H.E. Junginger, G. Borchard, Transfection efficiency and toxicity of polyethylenimine in differentiated Calu-3 and nondifferentiated COS-1 cell cultures, AAPS PharmSciTech 4 (2002) E12. [58] R. Kazemi Oskuee, M. Dabbaghi, L. Gholami, S. Taheri-Bojd, M. Balali-Mood, S.H. Mousavi, et al., Investigating the influence of polyplex size on toxicity properties of polyethylenimine mediated gene delivery, Life Sci. 197 (2018) 101 108. [59] F. Wang, M.G. Bexiga, S. Anguissola, P. Boya, J.C. Simpson, A. Salvati, et al., Time resolved study of cell death mechanisms induced by amine-modified polystyrene nanoparticles, Nanoscale 5 (2013) 10868 10876. [60] R.T. Cullen, A. Searl, B.G. Miller, J.M. Davis, A.D. Jones, Pulmonary and intraperitoneal inflammation induced by cellulose fibres, J. Appl. Toxicol.: JAT. 20 (2000) 49 60. [61] Y.C. Huang, A. Vieira, K.L. Huang, M.K. Yeh, C.H. Chiang, Pulmonary inflammation caused by chitosan microparticles, J. Biomed. Mater. Res. Part A 75 (2005) 283 287. [62] A.A. Shvedova, E.R. Kisin, N. Yanamala, M.T. Farcas, A.L. Menas, A. Williams, et al., Gender differences in murine pulmonary responses elicited by cellulose nanocrystals, Part. Fibre Toxicol. 13 (2016) 28. [63] J. Krabbe, N. Ruske, T. Braunschweig, S. Kintsler, J.W. Spillner, T. Schroder, et al., The effects of hydroxyethyl starch and gelatine on pulmonary cytokine production and oedema formation, Sci. Rep. 8 (2018) 5123. [64] C.M. da Luz, M.S. Boyles, P. Falagan-Lotsch, M.R. Pereira, H.R. Tutumi, E. de Oliveira Santos, et al., Poly-lactic acid nanoparticles (PLA-NP) promote physiological modifications in lung epithelial cells and are internalized by clathrin-coated pits and lipid rafts, J. Nanobiotechnol. 15 (2017) 11. [65] C. Li, H. Liu, Y. Sun, H. Wang, F. Guo, S. Rao, et al., PAMAM nanoparticles promote acute lung injury by inducing autophagic cell death through the Akt-TSC2-mTOR signaling pathway, J. Mol. Cell Biol. 1 (2009) 37 45. [66] W. Heideman, D.S. Antkiewicz, S.A. Carney, R.E. Peterson, Zebrafish and cardiac toxicology, Cardiovasc. Toxicol. 5 (2005) 203 214. [67] J.Y. Kim, H.H. Kim, K.H. Cho, Acute cardiovascular toxicity of sterilizers, PHMG, and PGH: severe inflammation in human cells and heart failure in zebrafish, Cardiovasc. Toxicol. 13 (2013) 148 160. [68] Q. Hu, F. Zhao, F. Guo, C. Wang, Z. Fu, Polymeric nanoparticles induce NLRP3 inflammasome activation and promote breast cancer metastasis, Macromol. Biosci. 17 (2017). [69] T. Zhou, Q. Dong, Y. Shen, W. Wu, H. Wu, X. Luo, et al., PEG-b-PCL polymeric nano-micelle inhibits vascular angiogenesis by activating p53-dependent apoptosis in zebrafish, Int. J. Nanomed. 11 (2016) 6517 6531. [70] Z.Y. Yuan, Y.L. Hu, J.Q. Gao, Brain localization and neurotoxicity evaluation of polysorbate 80-modified chitosan nanoparticles in rats, PLoS One 10 (2015) e0134722.

2. Toxicity of nanomaterials

References

189

[71] B. Newland, T.C. Moloney, G. Fontana, S. Browne, M.T. Abu-Rub, E. Dowd, et al., The neurotoxicity of gene vectors and its amelioration by packaging with collagen hollow spheres, Biomaterials 34 (2013) 2130 2141. [72] M.G. Bexiga, J.A. Varela, F. Wang, F. Fenaroli, A. Salvati, I. Lynch, et al., Cationic nanoparticles induce caspase 3-, 7- and 9-mediated cytotoxicity in a human astrocytoma cell line, Nanotoxicology 5 (2011) 557 567. [73] S. Wang, Y. Li, J. Fan, Z. Wang, X. Zeng, Y. Sun, et al., The role of autophagy in the neurotoxicity of cationic PAMAM dendrimers, Biomaterials 35 (2014) 7588 7597. [74] Y. Li, S. Wang, Z. Wang, X. Qian, J. Fan, X. Zeng, et al., Cationic poly(amidoamine) dendrimers induced cyto-protective autophagy in hepatocellular carcinoma cells, Nanotechnology 25 (2014) 365101. [75] Y. Li, H. Zhu, S. Wang, X. Qian, J. Fan, Z. Wang, et al., Interplay of oxidative stress and autophagy in PAMAM dendrimers-induced neuronal cell death, Theranostics 5 (2015) 1363 1377. [76] G. Nyitrai, L. Heja, I. Jablonkai, I. Pal, J. Visy, J. Kardos, Polyamidoamine dendrimer impairs mitochondrial oxidation in brain tissue, J. Nanobiotechnol. 11 (2013) 9. [77] H. Jeong, J. Hwang, H. Lee, P.T. Hammond, J. Choi, J. Hong, In vitro blood cell viability profiling of polymers used in molecular assembly, Sci. Rep. 7 (2017) 9481. [78] K. Winnicka, M. Wroblewska, K. Sosnowska, H. Car, I. Kasacka, Evaluation of cationic polyamidoamine dendrimers’ dermal toxicity in the rat skin model, Drug. Design, Dev. Ther. 9 (2015) 1367 1377. [79] T. Edlund, B. Lofgren, L. Vali, Toxicity of dextran in rats, Nature 170 (1952) 125. [80] C. Flexner, P.A. Barditch-Crovo, D.M. Kornhauser, H. Farzadegan, L.J. Nerhood, R.E. Chaisson, et al., Pharmacokinetics, toxicity, and activity of intravenous dextran sulfate in human immunodeficiency virus infection, Antimicrob. Agents Chemother. 35 (1991) 2544 2550. [81] C. Wiegand, D. Winter, U.C. Hipler, Molecular-weight-dependent toxic effects of chitosans on the human keratinocyte cell line HaCaT, Skin. Pharmacol. Physiol. 23 (2010) 164 170. [82] R. Wiwattanapatapee, B. Carreno-Gomez, N. Malik, R. Duncan, Anionic PAMAM dendrimers rapidly cross adult rat intestine in vitro: a potential oral delivery system? Pharm. Res. 17 (2000) 991 998. [83] I.L. Bergin, F.A. Witzmann, Nanoparticle toxicity by the gastrointestinal route: evidence and knowledge gaps, Int. J. Biomed. Nanosci. Nanotechnol. 3 (2013) 1 2. [84] A.M. Westbrook, A. Szakmary, R.H. Schiestl, Mechanisms of intestinal inflammation and development of associated cancers: lessons learned from mouse models, Mutat. Res. 705 (2010) 40 59. [85] S.M. Navarro, C. Darensbourg, L. Cross, R. Stout, D. Coulon, C.E. Astete, et al., Biodistribution of PLGA and PLGA/chitosan nanoparticles after repeat-dose oral delivery in F344 rats for 7 days, Therap. Deliv. 5 (2014) 1191 1201. [86] Y.L. Hu, W. Qi, F. Han, J.Z. Shao, J.Q. Gao, Toxicity evaluation of biodegradable chitosan nanoparticles using a zebrafish embryo model, Int. J. Nanomed. 6 (2011) 3351 3359. [87] P. Wick, A. Malek, P. Manser, D. Meili, X. Maeder-Althaus, L. Diener, et al., Barrier capacity of human placenta for nanosized materials, Environ. Health Perspect. 118 (2010) 432 436. [88] S. Scsukova, A. Mlynarcikova, A. Kiss, E. Rollerova, Effect of polymeric nanoparticle poly(ethylene glycol)block-poly(lactic acid) (PEG-b-PLA) on in vitro luteinizing hormone release from anterior pituitary cells of infantile and adult female rats, Neuro Endocrinol. Lett. 36 (Suppl 1) (2015) 88 94. [89] E. Rollerova, J. Jurcovicova, A. Mlynarcikova, I. Sadlonova, D. Bilanicova, L. Wsolova, et al., Delayed adverse effects of neonatal exposure to polymeric nanoparticle poly(ethylene glycol)-block-polylactide methyl ether on hypothalamic-pituitary-ovarian axis development and function in Wistar rats, Reprod. Toxicol. 57 (2015) 165 175. [90] Y. Li, D. Ju, The Application, Neurotoxicity, and Related Mechanism of Cationic Polymers, Neurotox. Nanomater. Nanomed. (2017) 285 329. [91] Y. Li, J. Fan, D. Ju, Neurotoxicity concern about the brain targeting delivery systems, Brain Targeted Drug Delivery System (2019) 377 408. [92] E.W. Hsu, S. Liu, A.R. Shrivats, A.C. Watt, S. McBride, S.E. Averick, et al., Cationic nanostructured polymers for siRNA delivery in murine calvarial pre-osteoblasts, J. Biomed. Nanotechnol. 10 (2014) 1130 1136. [93] X.N. Yang, D.D. Xue, J.Y. Li, M. Liu, S.R. Jia, L.Q. Chu, et al., Improvement of antimicrobial activity of graphene oxide/bacterial cellulose nanocomposites through the electrostatic modification, Carbohydr. Polym. 136 (2016) 1152 1160.

2. Toxicity of nanomaterials

190

8. Toxicity of polymeric nanomaterials

[94] L. Albertazzi, L. Gherardini, M. Brondi, S. Sulis Sato, A. Bifone, T. Pizzorusso, et al., In vivo distribution and toxicity of PAMAM dendrimers in the central nervous system depend on their surface chemistry, Mol. Pharm. 10 (2013) 249 260. [95] G. Thiagarajan, K. Greish, H. Ghandehari, Charge affects the oral toxicity of poly(amidoamine) dendrimers, Eur. J. Pharm. Biopharm. 84 (2013) 330 334. [96] A.C. Hunter, Molecular hurdles in polyfectin design and mechanistic background to polycation induced cytotoxicity, Adv. Drug. Deliv. Rev. 58 (2006) 1523 1531. [97] B. Mendrek, A. Fus, K. Klarzynska, A.L. Sieron, M. Smet, A. Kowalczuk, et al., Synthesis, characterization and cytotoxicity of novel thermoresponsive star copolymers of N,N’-dimethylaminoethyl methacrylate and hydroxyl-bearing oligo(ethylene glycol) methacrylate, Polymers 10 (2018) 1255. [98] C.A. Vaine, M.K. Patel, J. Zhu, E. Lee, R.W. Finberg, R.C. Hayward, et al., Tuning innate immune activation by surface texturing of polymer microparticles: the role of shape in inflammasome activation, J. Immunol. 190 (2013) 3525 3532. [99] B. Zhang, P. Sai Lung, S. Zhao, Z. Chu, W. Chrzanowski, Q. Li, Shape dependent cytotoxicity of PLGA-PEG nanoparticles on human cells, Sci. Rep. 7 (2017) 7315. [100] K.E.B. Doncom, L.D. Blackman, D.B. Wright, M.I. Gibson, R.K. O’Reilly, Dispersity effects in polymer selfassemblies: a matter of hierarchical control, Chem. Soc. Rev. 46 (2017) 4119 4134. [101] A. Janaszewska, J. Lazniewska, P. Trzepinski, M. Marcinkowska, B. Klajnert-Maculewicz, Cytotoxicity of dendrimers, Biomolecules 9 (2019) E330. [102] A. Janaszewska, M. Studzian, J.F. Petersen, M. Ficker, J.B. Christensen, B. Klajnert-Maculewicz, PAMAM dendrimer with 4-carbomethoxypyrrolidone--in vitro assessment of neurotoxicity, Nanomed. Nanotechnol. Biol. Med. 11 (2015) 409 411. [103] Y.J. Choi, S.J. Kang, Y.J. Kim, Y.B. Lim, H.W. Chung, Comparative studies on the genotoxicity and cytotoxicity of polymeric gene carriers polyethylenimine (PEI) and polyamidoamine (PAMAM) dendrimer in Jurkat T-cells, Drug. Chem. Toxicol. 33 (2010) 357 366. [104] B. Ziemba, A. Janaszewska, K. Ciepluch, M. Krotewicz, W.A. Fogel, D. Appelhans, et al., In vivo toxicity of poly(propyleneimine) dendrimers, J. Biomed. Mater. Res. Part A 99 (2011) 261 268. [105] S. Elmore, Apoptosis: a review of programmed cell death, Toxicol. Pathol. 35 (2007) 495 516. [106] C.P. Baines, Role of the mitochondrion in programmed necrosis, Front. Physiol. 1 (2010) 156. [107] T. Xia, M. Kovochich, M. Liong, J.I. Zink, A.E. Nel, Cationic polystyrene nanosphere toxicity depends on cell-specific endocytic and mitochondrial injury pathways, ACS Nano 2 (2008) 85 96. [108] A. Negroni, S. Cucchiara, L. Stronati, Apoptosis, necrosis, and necroptosis in the gut and intestinal homeostasis, Mediators Inflamm. 2015 (2015) 250762. [109] S. Di Gioia, M. Conese, Polyethylenimine-mediated gene delivery to the lung and therapeutic applications, Drug Des. Dev. Ther. 2 (2009) 163 188. [110] S.M. Moghimi, P. Symonds, J.C. Murray, A.C. Hunter, G. Debska, A. Szewczyk, A two-stage poly(ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy, Mol. Ther. J. Am. Soc. Gene Ther. 11 (2005) 990 995. [111] X. Wei, B. Shao, Z. He, T. Ye, M. Luo, Y. Sang, et al., Cationic nanocarriers induce cell necrosis through impairment of Na(1)/K(1)-ATPase and cause subsequent inflammatory response, Cell Res. 25 (2015) 237 253. [112] S.J. Dixon, K.M. Lemberg, M.R. Lamprecht, R. Skouta, E.M. Zaitsev, C.E. Gleason, et al., Ferroptosis: an iron-dependent form of nonapoptotic cell death, Cell 149 (2012) 1060 1072. [113] Y. Xie, W. Hou, X. Song, Y. Yu, J. Huang, X. Sun, et al., Ferroptosis: process and function, Cell Death Differ. 23 (2016) 369 379. [114] S.E. Kim, L. Zhang, K. Ma, M. Riegman, F. Chen, I. Ingold, et al., Ultrasmall nanoparticles induce ferroptosis in nutrient-deprived cancer cells and suppress tumour growth, Nat. Nanotechnol. 11 (2016) 977 985. [115] M. Szwed, T. Sonstevold, A. Overbye, N. Engedal, B. Grallert, Y. Morch, et al., Small variations in nanoparticle structure dictate differential cellular stress responses and mode of cell death, Nanotoxicology 13 (2019) 761 782. [116] Y. Chun, J. Kim, Autophagy: an essential degradation program for cellular homeostasis and life, Cells 7 (2018) E278.

2. Toxicity of nanomaterials

References

191

[117] E. Tasdemir, L. Galluzzi, M.C. Maiuri, A. Criollo, I. Vitale, E. Hangen, et al., Methods for assessing autophagy and autophagic cell death, Methods Mol. Biol. 445 (2008) 29 76. [118] Y. Li, D. Ju, The role of autophagy in nanoparticles-induced toxicity and its related cellular and molecular mechanisms, Adv. Exp. Med. Biol. 1048 (2018) 71 84. [119] Y.X. Lin, Y. Wang, S.L. Qiao, H.W. An, R.X. Zhang, Z.Y. Qiao, et al., pH-Sensitive polymeric nanoparticles modulate autophagic effect via lysosome impairment, Small 12 (2016) 2921 2931. [120] X. Sun, C. Cheng, J. Zhang, X. Jin, S. Sun, L. Mei, et al., Intracellular trafficking network and autophagy of PHBHHx nanoparticles and their implications for drug delivery, Sci. Rep. 9 (2019) 9585. [121] T. Bergsbaken, S.L. Fink, B.T. Cookson, Pyroptosis: host cell death and inflammation, Nat. Rev. Microbiol. 7 (2009) 99 109. [122] S.M. Man, R. Karki, T.D. Kanneganti, Molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases, Immunol. Rev. 277 (2017) 61 75. [123] F. Wegmann, K.H. Gartlan, A.M. Harandi, S.A. Brinckmann, M. Coccia, W.R. Hillson, et al., Polyethyleneimine is a potent mucosal adjuvant for viral glycoprotein antigens, Nat. Biotechnol. 30 (2012) 883 888. [124] O. Lunov, T. Syrovets, C. Loos, G.U. Nienhaus, V. Mailander, K. Landfester, et al., Amino-functionalized polystyrene nanoparticles activate the NLRP3 inflammasome in human macrophages, ACS Nano 5 (2011) 9648 9657. [125] Y. Li, S. Wang, J. Fan, X. Zhang, X. Qian, X. Zhang, et al., Targeting TNFα Ameliorated cationic PAMAM dendrimer-induced hepatotoxicity via regulating NLRP3 inflammasomes pathway, ACS Biomater. Sci. Eng. 3 (5) (2017) 843 853. [126] Y.B. Wang, Y. Lou, Z.F. Luo, D.F. Zhang, Y.Z. Wang, Induction of apoptosis and cell cycle arrest by polyvinylpyrrolidone K-30 and protective effect of alpha-tocopherol, Biochem. Biophys. Res. Commun. 308 (2003) 878 884. [127] F. Salehi, H. Behboudi, G. Kavoosi, S.K. Ardestani, Chitosan promotes ROS-mediated apoptosis and S phase cell cycle arrest in triple-negative breast cancer cells: evidence for intercalative interaction with genomic DNA, RSC Adv. 7 (2017) 43141 43150. [128] S.S. Dronamraju, J.M. Coxhead, S.B. Kelly, J. Burn, J.C. Mathers, Cell kinetics and gene expression changes in colorectal cancer patients given resistant starch: a randomised controlled trial, Gut 58 (2009) 413 420. [129] S. Legaz, J.Y. Exposito, C. Lethias, B. Viginier, C. Terzian, B. Verrier, Evaluation of polylactic acid nanoparticles safety using Drosophila model, Nanotoxicology 10 (2016) 1136 1143. [130] R. Czarnomysy, A. Bielawska, K. Bielawski, Effect of 2nd and 3rd generation PAMAM dendrimers on proliferation, differentiation, and pro-inflammatory cytokines in human keratinocytes and fibroblasts, Int. J. Nanomed. 14 (2019) 7123 7139. [131] A. Rahal, A. Kumar, V. Singh, B. Yadav, R. Tiwari, S. Chakraborty, et al., Oxidative stress, prooxidants, and antioxidants: the interplay, Biomed. Res. Int. 2014 (2014) 761264. [132] P. Khanna, C. Ong, B.H. Bay, G.H. Baeg, Nanotoxicity: an interplay of oxidative stress, inflammation and cell death, Nanomaterials 5 (2015) 1163 1180. [133] Calarco A., Bosetti M., Margarucci S., Fusaro L., Nicoli E., Petillo O., et al. The genotoxicity of PEI-based nanoparticles is reduced by acetylation of polyethylenimine amines in human primary cells. Toxicol. Lett. 218:10 17, 2013.

2. Toxicity of nanomaterials

C H A P T E R

9 General methods for detection and evaluation of nanotoxicity Hani Nasser Abdelhamid Advanced Multifunctional Materials Laboratory, Department of Chemistry, Assiut University, Assiut, Egypt

9.1 Introduction Nanotechnology has advanced several fields and has become a growing area for scientific research and industry [1]. The global nanotechnology market involved hundreds of billions of US dollars and has a reported expected growth of 17% for the 2018 24 market [2]. Currently, there are thousands of nanomaterials-based products including sporting and food goods, cosmetics, electronics, and devices; which goes without saying that humans are in contact with products containing nanoparticles. Due to the small size of nanoparticles, they have a high risk to humans [3]. Thus it is required to follow the safety procedures for the usage of nanotechnologies in medicine and medical devices [3]. Characterization of nanomaterials is very important in order to understand nanoparticles properties and decide their potential applications. Several analytical techniques were developed to characterize the physicochemical properties of nanoparticles. Composition and element percentages of nanoparticles can be determined using elemental analysis, inductively coupled mass spectrometry (ICP-MS), spectroscopy techniques (absorption, emission, or scattering of either wavelength or frequency), atomic absorption spectrometry (AAS), atomic emission spectroscopy (AES), synchrotron radiation-induced X-ray fluorescence, and X-ray photoelectron spectroscopy. Chemical structures’ connectivity can be determined using diffraction (X-ray, neutron, and electron), and nuclear magnetic resonance (NMR). Nanoparticle size can be determined using several techniques including dynamic light scattering techniques, electron microscopes [transmission electron microscope (TEM) and scanning electron microscope (SEM)], and ultraviolet visible (UV Vis) spectroscopy. Morphology of nanomaterials can be evaluated using electron microscopy (TEM and SEM). Surface properties can be determined using zeta potential measurements for relative particle charge, nitrogen adsorption-desorption for surface area (Brunauer Emmett Teller, and Langmuir), and pore size distribution. Analyze cell or

Nanotoxicity DOI: https://doi.org/10.1016/B978-0-12-819943-5.00009-9

195

Copyright © 2020 Elsevier Inc. All rights reserved.

196

9. General methods for detection and evaluation of nanotoxicity

tissue uptake and biodistribution of nanoparticles can be achieved using ICP-MS, AAS, and AES after digestion [4]. The nanoparticle products are very broad and widely spread overall the world so human exposure to products containing nanoparticles is inevitable. The term “nanotoxicology” was coined in 2004 [5]. Nanotoxicology has been defined as “the study of the adverse effects of engineered nanomaterials on living organisms and the ecosystems, including the prevention and amelioration of such adverse effects” [6]. Cell upon interactions with nanoparticles undergo cell death via necrosis or apoptosis. Cell death via rupture of the cell membrane and spilling of cellular contents is defined as necrosis. Necrosis is a pathological process involving in response to external factors induced toxicity, including cellular processes related to DNA damage, inflammation, or disrupting the oxidant-antioxidant balance creating; (1) reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), superoxide radical (O2*), and hydroxyl radical (OH*), and (2) reactive nitrogen species (RNS), such as nitric oxide (NO*), peroxynitrate (ONOO2), and peroxynitrous acid (ONOOH). It involves breakdown of the cell constituents into precursor amino acids and nucleotides that can be reused by neighboring cells. Apoptosis is “programmed cell death” or “controlled cell death” whereby the cell maintains the cell membrane integrity. Apoptosis can be characterized using cell membrane blabbing, mitochondrial DNA damage, nuclear and cytoplasmic shrinkage, fragmentation into apoptotic bodies, and chromatin condensation. Nanotoxicology can be evaluated via in vitro and in vivo assessments [7]. Pathogenic bacteria present a serious threat to humans [8 13]. Traditional antibiotics suffer from low efficiency, and antibiotic resistant. Thus advanced materials including nanoparticles are highly attractive due to their advantages including high efficiency, simple processing, and easy for conjugation with conventional antibiotics. Antibiotic-conjugated nanoparticles offer a higher antibacterial activity compared to the antibiotic or nanoparticles alone. This is due to a synergistic effect of the antibiotic and nanoparticles [14]. Analytical methods are highly required to characterize the activity of nanoparticles as antibacterial agents. This chapter aims to summarize analytical methods used for evaluation of nanotoxicology. Analytical methods for in vitro and in vivo analysis will be covered. Principles, requirements, and examples are reviewed for each technique. Because of the numerous kinds of these methods, widely used methods will be only covered.

9.2 General nanotoxicity methods Toxicity of nanoparticles is often reported as indexes such as minimal inhibitory concentration (MIC), half maximal effective concentration (EC50), half inhibitory concentration (IC50), half lethal concentration (LC50), half minimal cell toxicity concentration (CC50), toxic concentration (TC50), minimal bactericidal concentration (MBC) [15], and half lethal dose (LD50). Definition and differences of these terms are tabulated in Table 9.1. These indexes may be evaluated using analytical and biochemical-based methodologies, including: 1. Viability and cytotoxic; cellular processes like proliferation, necrosis, and apoptosis. 2. Studies of biochemical pathways and mechanism, cell responses, and pathological changes to understand the effects of NPs exposure on processes in biological systems. 3. Strategies for general toxicity assessment in cells or whole organisms.

3. Prevention of nanotoxicity

197

9.2 General nanotoxicity methods

TABLE 9.1 Therapeutic indexes, definition, and comments. Terms

Definition

Comments

EC50

Concentration of a toxicant which induces a response halfway between the baseline and maximum after a specified exposure time

It is comparable to an EC50 for agonist drugs

IC50

Measure of the potency of nanoparticles in inhibiting 50% of the cells

The values are typically expressed as molar concentration

LD50

The value of LD50 for a substance is the dose required to kill half the members of a tested population after a specified test duration

LD50 is usually determined by tests on animals such as laboratory mice

CC50

The cytotoxicity concentration of a nanoparticle that will kill 50% of the cells in an uninfected cell culture

EC50, Half maximal effective concentration; IC50, half maximal inhibitory concentration; LD50, median lethal dose; CC50, half minimal cell toxicity concentration.

TABLE 9.2 Advantages, and disadvantages of in vitro and in vivo assays. Assay

Advantages

Disadvantages

In vitro

• • • • • •

• Lacks entire set of stimuli • Cells don’t represent in vivo phenotype • Lack of standard methods for cell line

In vivo

No need to follow ethical rules Cheap and simple Homogenous and control Easy for mechanism study Realistic Absorption, metabolism, biodistribution, and excretion can be studied

• High variation among different species • Require ethical rules • Requires high experience

It is important to keep in mind that these indexes depend on several parameters including the method of administration and type of assessments (in vitro or in vivo) (Table 9.2). Nanoparticles are less toxic when administered orally than when intravenously administered. The exposure time is also important. The time of incubation should be considered such as LD50/10 or LD50/50 referring 10 or 50 days, respectively. The values of these indexes are somewhat unreliable, and results may vary greatly between testing facilities due to factors such as the genetic characteristics of the sample population, animal species tested, environmental factors, and mode of administration [16]. Thus it is more useful to use therapeutic indexes, which are simply the ratio of LD50 to ED50. Nanoparticles may cause several effects at the same time. In vitro assays using HEK 293T cells revealed that the small and large sizes of graphene (G), and graphene oxide (GO) reduced the cell viability, increased DNA damage, generated ROS, and induced various expressions of associated critical genetic markers [17]. Thus several tests are required in parallel in order to understand the materials nanotoxicity. Two points are required to understand the materials activity: bacteria cell wall structure and mechanism of nanoparticles action. Bacteria can be classified into gram-positive, and gram-negative. Gram-positive bacteria contain a thick layer of peptidoglycan in their cell walls; whereas gram-negative bacteria have a thin

3. Prevention of nanotoxicity

198

9. General methods for detection and evaluation of nanotoxicity

peptidoglycan layer with an additional layer of lipopolysaccharide. Lipopolysaccharide layer has a negative charge and ensures strong interaction with the positive charge of nanoparticles. Thus gram-positive bacteria are more resistant to nanoparticles compared to gram-negative bacteria. The interactions of nanoparticles are increased also in species such as Salmonella typhimurium, gram-negative bacteria, due to the presence of a mosaic of anionic surfaces domains rather than a continuous layer. Electrophoretic mobility, and mathematical calculations revealed that Escherichia coli is more negatively charged and rigid than Staphylococcus aureus [18]. These differences show different responses of the same nanoparticles.

9.3 Mechanism of antibacterial activities Antibacterial activities of nanoparticles can be explained in several mechanisms including generation of ROS, cation release, damages of cell components, disrupt the function of enzymes, break necessary bonds that maintain the integrity of cells proteins, and adenosine triphosphate (ATP) depletion [19]. Clear mechanism of the nanoparticle toxicity is under debate. Because it is difficult to track nanoparticles inside the cells, the large variation between samples, strains dependent, and the unclear characterization of nanoparticles (surface capping agents and their structures).

9.4 Methods for detection and evaluation of nanotoxicity 9.4.1 Plate counting, colony counting, or colony forming efficiency assay Counting the number of cells before and after treatment with nanoparticles is the simplest method to evaluate biological activity. The method involves the measuring of the ability of surviving cells to form colonies. It is a bulk analysis, label-free method, and each microbial strain form different colonies that can differentiate clearly from each other. The cell counting can take place using naked-eyes, or using a plate reader. For simple counting, the cells were diluted first before counting. Cell numbers can be presented as a number or as a relative ratio for the cell before and after treatments. The cell numbers reveal cytotoxic effects (reduction of the number of colonies formed), enhance bacteria growth (increase of the number of colonies formed), and cytostatic effects (reduction in colony size). The cells number can be determined using naked-eyes or a microscope via the pour plate method, the spread plate method, the membrane filter method, the Miles and Misra methods, or drop-plate method. Colony-forming units require only viable cells counts. The results can be presented as a colony-forming unit (CFU, cfu, and Cfu), or as a colony forming efficiency (CFE). It is important to take into account that single cell rises to a colony through replication. In most cases, the number of CFU is an undercount of the real number of living cells using a simple counting method because bacteria cells can grow in chains or clumps. However, using microscope the numbers may be higher because it counts living and death cells. Plate counting was used to evaluate antibacterial activity of silver nanoparticles (Au NPs) [20], copper nanoparticles (Cu NPs) [21], zinc oxide nanoparticles (ZnO NPs) [22], polyurethanes (PUs) and its composite with Ag, Au, ZnO, TiO2, carbon nanotubes (CNTs), and chitosan (CTS)

3. Prevention of nanotoxicity

199

9.4 Methods for detection and evaluation of nanotoxicity

[23], CST-CdS [24], SrTiO3 nanotubes embedded with Ag2O nanoparticles [25], SnO@graphene oxide [26], polydopamine-sodium alginate-capsaicin@CST, and CST [27], two-dimensional SnO@graphene oxide nanosheets [28]. ZnO NPs displayed potent inhibition on Bacillus subtilis, and S. aureus with MIC at 128 and 256 µg/mL, respectively. Authors also observed no inhibition against gram-negative bacteria [22]. Flat colony counting method (standard norm ASTM E218007) was used to evaluate the nanotoxicity for CTS, Ag doped TiO2-chitosan, and TiO2-chitosan composites under visible-light and dark conditions against E. coli, S. aureus, and Pseudomonas aeruginosa [29]. Data showed antibacterial activity of these nanoparticles. The CFE assay, is a clonogenic assay. It measures the ability of surviving single cells to form colonies. Cell survival is expressed as the number of colonies formed from surviving cells being exposed to a nanoparticle to the number of colonies observed for control cells (untreated cells). This assay were reported to assess the cytotoxicity of several nanoparticles including AuNPs [30], AgNPs [31], TiO2 NPs [32], ZnO NPs [33], ZnLaFe2O4-NiTiO3 [34], silica nanoparticles [35], CeO2 NPs [36], titanium embedded with AgNPs (Ag-NPs@Ti) [37], multiwalled carbon nanotubes (MWCNT) [38], and antibiotic loaded nanoparticles [39]. Gallic acid modified AgNPs showed effective antibacterial activity with a MIC of 10 µg/mL for E. coli and S. aureus as model bacteria [40].

9.4.2 Disk diffusion test: zone of inhibition Antibacterial activity of a nanoparticle can be evaluated using the disk diffusion test, agar diffusion test, or Kirby Bauer test (Fig. 9.1) [41]. In this test, disk containing the nanoparticles are placed on an agar plate where a pure bacterial culture swabbed uniformly across a culture plate that is left to incubate resulting an area around the wafer without any bacteria growth. This area has been defined as zone of inhibition (ZOI). ZOI, depends on several factors including bacteria strains, cells number, cell media, incubation time and temperature, and nanoparticles activity. Thus most of these aspects are

Growth time

24 h

Agar media, spread on Petri dish

Zone of inhibition A, B, C, and D are nanoparticles

FIGURE 9.1 Zone of inhibition (ZOI) for nanoparticles.

3. Prevention of nanotoxicity

200

9. General methods for detection and evaluation of nanotoxicity

standardized via following the Kirby Bauer procedure that may ensure consistent and reliable results. The cell media should be Mueller Hinton Broth (MHB, pH value of 7.2 7.4) at only 4 mm deep, poured into either 100 mm or 150 mm petri dishes. Concentrated broth culture is usually diluted to match a 0.5 McFarland turbidity standard, which is approximately equivalent to 1.5 3 108 cells/mL. In agar diffusion method [42], bacteria colonies were transferred to Mueller Hinton Broth medium and placed in an incubator for 2 4 h at 37 C to achieve 0.5 McFarland. After dilution, 500 mL suspension 1.5 3 108 cfu/mL (equal to 0.5 McFarland standard turbidity) was transferred to Mueller Hinton agar and cultured using a sterile loop. Nanoparticles are carefully placed on the agar surface. Finally, the plates were incubated at 37 C and the ZOI was measured after 24 h. ZOI is presented as diameter (millimeter, mm). The zone diameter should be compared to a database of zone standards to relatively sort the nanoparticles activity. Disk diffusion test has been used to evaluate AgNPs [43], AgNPs-decorated reduced graphene nanocomposites [44], Ag-cellulose [45], Pd Ag-cellulose paper [46], copper sulfide nanoparticles (CuS NPs), and ZnO-Zn(OH)2 [47], chitosan-lysozyme nanoparticles (CS-Lys-NPs) [48], and ZnO [49]. Tragacanth gum-ZnO NPs coated cotton fabric showed 100% antimicrobial properties with inhibition zone of 3.3 6 0.1, 3.1 6 0.1, and 3.0 6 0.1 mm against S. aureus, E. coli, and Candida albicans, respectively [49]. Green synthesized ZnO NPs using Cuminum cyminum showed ZOI of 18 25 6 1 mm [50]. Inhibition zone diameters using Aspergillus oryzae for CuS NPs, and ZnO-Zn(OH)2 NPs are 12.38 and 10.50 mm for, respectively [47]. Disk-diffusion assay offers many advantages including simplicity, low cost, and the ability to test enormous numbers of microorganisms and nanoparticles. However, the ZOI is not easy to interpret as efficiency. The distance number has no meaning for user. Furthermore, there is no reference to refers these distance.

9.4.3 Gradient method (Etest) The gradient method, or Etest, combines the principle of dilution methods with that of diffusion methods. It has been commercialized under the name such as Etest (BioMe´rieux). It is based on the possibility of creating a concentration gradient in the agar medium. In the procedure, a strip impregnated with an increasing concentration gradient of the antimicrobial agent from one end to the other is deposited on the agar surface, previously inoculated with the microorganism tested. MIC value is determined at the intersection of the strip, and the growth inhibition ellipse. Several previous studies have shown a good correlation between the MIC values determined by Etest and those obtained using broth dilution or agar dilution method. Etest strips are routinely used. However, Etest strips cost about $2 3 each. Therefore this approach becomes costly if numerous nanomedicines are tested [51].

9.4.4 Optical density Absorbance (scattered, optical density) using a UV Vis spectrophotometer at 600 nm (OD600, or simplified as OD) can be used to evaluate antibacterial activity of a

3. Prevention of nanotoxicity

9.4 Methods for detection and evaluation of nanotoxicity

201

nanoparticle. The measurements are based on the amount of light scattered via cells rather than true absorbance. General procedure of OD measurements involves the data acquisition of the cultured bacteria in the broth medium after incubation with nanoparticles. Control experiments using a mixture of sterile water and bacterial solution are usually carried out under the same conditions. The bacterial growth rates were determined via measuring OD at 600 nm using a UV Vis spectrophotometer. Several nanoparticles, including AgNPs [52,53], Ag-cellulose [45], AgNPs-decorated porous reduced GO [54], Au@AgNPs [55], hydroxyapatite-AgNPs-calcium phosphate [56], metal complexes [57], BaTiO3 NPs [58], and ZnO-graphene quantum dot nanocomposites [59] have been evaluated using OD. According to the decrease in the OD, ZnO NPs have a marked inhibitory effect on the growth of E. coli colonies [59].

9.4.5 Dilution methods Dilution methods are the most appropriate methods for the determination of MIC values. They can be used to estimate the concentration of the tested nanoparticles in the agar (agar dilution), or broth medium (macrodilution, or microdilution). The Clinical and Laboratory Standards Institute (CLSI), and the European Committee on Antimicrobial Susceptibility Testing provided a uniform procedure for testing which is practical to perform in most clinical microbiology laboratories.

9.4.6 Broth dilution Nanoparticles antibacterial activity can be measured using broth dilution. In this method, a suspension of bacteria is prepared from fresh culture as stock. Then, the suspension is diluted with sterile media and used as inoculums. The broth dilution can be performed using macrodilution (uses broth volume of 1 mL in standard test tubes), or microdilution (uses about 0.05 0.1 mL total broth volume in a microtiter plate) [60]. Microdilution method is simple compared to macrodilution method and offers minimum errors in the preparation of antimicrobial solutions. It can be used for the comparatively large amount of reagents. It provides high reproducibility with low costs due to the miniaturization of the test. The suspensions were measured using a reader. Different concentrations of the nanoparticles were prepared in broth using test tubes. The changes of a control sample and the cells incubated nanoparticles can be followed photometrically (wavelength 625 nm and a 1 cm path), and nephelometers. Readers can find more details in a book chapter ref. [60]. Several colorimetric methods were used for nanoparticle evaluation using dye molecules as a probe such as Alamar blue dye (resazurin, an effective growth indicator) [61], Live/ Dead BacLight Bacterial Viability Kit [62], PrestoBlue assay [63], and BacLight LIVE/DEAD membrane [64]. In general, these methods are based on that live and dead cells exhibit two different colors. For examples, live cells display green fluorescence (SYTO 9), whereas nonviable bacterial cells display red fluorescence (propidium iodide, PI). Trypan blue dye stains only dead cells, and excluded via viable cells. Dihydrorhodamine-123 is converted to the fluorescent form upon reaction with peroxide, singlet oxygen, and other reactive species.

3. Prevention of nanotoxicity

202

9. General methods for detection and evaluation of nanotoxicity

Nanoparticles

P

N

FIGURE 9.2 96-well plate assay incorporating a blue dye as an indicator of cell growth. Conventional antibiotic (ref.) is used as control, with positive (P, dye changes to pink color), and negative (N) controls.

Increase concentration

Ref.

Nanotoxicity assessment based on optical-based methods use of a dye molecule as probe (Fig. 9.2). Typically, a tiny volume (microlitters) of a nanoparticle solution (prepared in sterile water) was pipette into a sterile 96-well plate (Fig. 9.2). Serial dilutions were performed to all other wells (Fig. 9.2). Then, a dye indicator solution was added. The volume of all wells was uniformed using a pipette of broth (Fig. 9.2). Finally, bacterial suspension (5 3 106 cfu/mL) was added to each well to achieve a concentration of 5 3 105 cfu/mL. A set of controls using a broad-spectrum antibiotic as positive control (Ref.) are used (Fig. 9.2). The plates were prepared in triplicate (Fig. 9.2, NPs), and incubated at 37 C for 48 h. The color change is assessed visually; any color change, for example from blue to pink is recorded as positive (Fig. 9.2). The lowest concentration at which color change occurred was taken as the MIC value (Fig. 9.2). The incubation was performed in the dark for 15 30 min at room temperature. SYTO 9 excitation and emission were recorded at 488 and 500 575 nm, respectively. In other side, red fluorescence (PI, dead cells) displays excitation and emission wavelengths at 561 and 570 620 nm, respectively (Fig. 9.2). Broth microdilution was used to evaluate antibacterial activity of Ag, GO, and their composite against E. coli [65]. It was also used for metal-organic framework nanocubes against P. aeruginosa strains (with MBC 3.13 mg/mL) [15]. Results showed that plate count method is more accurate than dilution method which showed false estimation of 4% viability percentage [15]. MgO enhanced ultrasound-induced lipid peroxidation (LPO) in the liposomal membrane [61]. Measuring of viable cells using a dye molecule conveys the actual number of viable cells. The changes of the dye color represent the changes of the cell number including increasing cell numbers (cell proliferation), or decreasing cell numbers (cytotoxicity). It is an accurate method

3. Prevention of nanotoxicity

9.4 Methods for detection and evaluation of nanotoxicity

203

because the results can be compared to control, or untreated cells. However, care must be taken because nanoparticles can interfere with the spectrophotometric readout signals [66].

9.4.7 Time-kill test (time-kill curve) Time-kill test is used to evaluate the dynamic interaction between nanoparticles and the microbial strain. It can be also used to determine synergism or antagonism between nanomedicines (two or more) in combinations. It is a time-dependent or a concentration-dependent method. According to CLSI, the method is performed in broth culture medium using three tubes containing a bacterial suspension of 5 3 105 cfu/mL. One of these three tubes is considered as the growth control, while the other two tubes contain tested nanoparticles usually at final concentrations of 0.25 3 MIC, and 1 3 MIC are used for the test. Under the normal conditions, the tubes are incubated for varied time intervals (0, 4, 6, 8, 10, 12, and 24 h). The percentage of dead cells is determined relatively to the growth control (cfu/mL) of each tube using the agar plate count method.

9.4.8 Adenosine triphosphate cell viability assay ATP, is the chemical form of energy for all living cells. Measuring ATP produced by bacteria or fungi after incubation with nanoparticles solution is useful method for nanotoxicity evaluation. The ATP amount is almost constant in a cell. Therefore changes of ATP amount can be used quantitively to estimate the microbial population in a sample. The measurement of ATP is based on the conversion of D-luciferin in the presence of the ATP; via luciferase, to oxyluciferin that generates light. The produced light is measured using a luminometer and expressed as relative light unit (RLU) or as RLU/mol of ATP. There is a linear relationship between cell viability and the measured signals of luminescence. Thus it can be used to assess the nanotoxicity of nanoparticles.

9.4.9 Reactive oxygen species Nanoparticles can create reactive species such as ROS or RNS which kill cells. These species may be scavenged in the cell via redox reaction of glutathione (GSH) to oxidize glutathione (GSSG) [67]. Nanoparticles cause oxidative stress leading to increased depletion of GSH [68]. The intracellular ROS can be measured by detecting the ratio of GSH to GSSG inside the cells. Exposure of bacteria to AgNPs [67], ZnO NPs [69], and TiO2 NPs [70] causes a GSH depletion and an increase in the formation of GSSG. In general, bacterial cells were cultured in cell medium at 37 C for 12 h. Then, they were incubated with a certain concentration of the nanoparticles at 37 C for 6 h. ROS generation in bacteria was measured using a dye molecule such as dichlorofluorescein diacetate (DCFHDA, λDCE 5 530 nm, λex 5 480 nm). Finally, ROS emissions were observed using fluorescence equipment. ROS can be also quantified using electron paramagnetic resonance, or electron spin resonance spectroscopy using 5,5-dimethyl-1-pyrroline N-oxide as probe. The generated ROS depends on the nanoparticles properties. For example, physical deformations or defects of ZnO NPs; via activation using UV and visible-light, increase defects in ZnO crystals [71]. The defects of ZnO NPs creates electron hole pairs resulting in the splitting of suspended H2O molecules into OH2 and H1. The dissolved molecules

3. Prevention of nanotoxicity

204

9. General methods for detection and evaluation of nanotoxicity

eventually react to form H2O2, a ROS that is able to penetrate the cell membrane and kill bacteria. Thus ROS measurements can be used to evaluate the effect of different crystal structures of crystalline nanoparticles such as TiO2 [72]. Results show no effect of crystal forms rutile and anatase in their antibacterial activity [72]. The method is sensitive to study impurities effects in nanoparticles such as single- and MWCNT [72].

9.4.10 Lipid peroxidation measurement Antibacterial activity of nanoparticle can be measured via LPO measurements. LPO assay determines the free radical production [73]. Briefly, LPO was evaluated using the thiobarbituric acid reactive substances assay (TBARS, OxiSelect TBARS Assay Kit). Bacteria culture at mid-log phase grown under aerobic conditions are pelleted and resuspended in NPs dispersions. After 30 min, TBARS reagent is added and the solution is incubated at 95 C for 50 min. Cells, and nanoparticles are centrifuged and the supernatant is collected for fluorescent spectroscopy measurements using excitation and emission wavelengths at 540 and 590 nm, respectively. Standard curve using malondialdehyde (MDA) is used to convert the fluorescence signals into MDA equivalent concentrations. LPO measurements are used to evaluate antibacterial activity of several nanoparticles including ZnO NPs [74], MgO [61], Ag-ZnO nanocomposite [75], and AgNPs [76]. Data reveals disruption of cell membrane and subsequent cell death due to the oxidation chain reaction of fatty acids.

9.4.11 Omics methods: transcriptomic, metabolomics, genomics, and proteomic profiles Omics technologies (transcriptomics, proteomics, metabolomics, genomics, and fluxomics) provide insights into cellular dynamics [77]. They provide useful knowledge that can be conjugated with traditional methods, for example, cell counting to identify novel therapeutic targets against bacterial pathogens. Schematic representation of metabolomics is shown in Fig. 9.3. It can be used for in vivo and in vitro studies (Fig. 9.3). Animals or cells are treated with Top-down

Statistical analysis using known metabolites Map to metabolic pathways

NMR

Nanoparticles treatment Animal

Identify metabolite and detect difference in peak area

LC-MS

Tissue or biofluid

FIGURE 9.3 General flow of a metabolomics study of nanotoxicity.

3. Prevention of nanotoxicity

Bottom-up

Statistical analysis of spectra Discover an identify metabolites responsible for nanotoxicity

9.4 Methods for detection and evaluation of nanotoxicity

205

nanoparticles with and without extraction before analysis using NMR, or liquid chromatography mass spectrometry (Fig. 9.3). The signals were analyzed for identification and quantitative analysis using peak area under the curve and a standard calibration curve (Fig. 9.3). The analysis can be achieved using top-down, or bottom-up approaches (Fig. 9.3). Transcriptional analysis indicated that AgNPs restrained the expression of key genes related to denitrification [78]. Specifically, the genes involved in denitrifying catalytic reduction and electron transfer were significantly downregulated. Proteomic profiling revealed that the syntheses of the proteins involved in catalytic process, electron transfer, and metabolic process were inhibited by AgNPs [78]. Proteomics analysis of P. aeruginosa identified 59 silver-regulated proteins (27 upregulated, and 32 downregulated proteins), and 5 silver-binding proteins after treatment [79]. Proteomics analysis of E. coli upon incubation with AgNPs showed that approximately 65% of E. coli proteins bound to AgNPs are enzymes (e.g., tryptophanase, alcohol dehydrogenase, and cytochrome C), and nonenzymatic proteins [e.g., membrane porins (OmpA and OmpB), chaperonins, and periplasmic peptide binding proteins] [80]. Proteomics analysis showed also that periplasmic peptide has a high binding affinity towards AgNPs [81]. The toxicity of nanoparticle doesn’t require membrane rupture for the protein upregulation [82,83]. Translational ribosomal proteins S2 and L9 are involved in translational regulation and also have functions in structure and stress regulation [84]. Transcriptome analysis of AgNPs treated S. aureus reveals differential regulation of 21% (i.e., 629 genes), and 28.5% (i.e., 830 genes) of the total functional coding genes [85]. While, 1884 and 5834 were differentially expressed genes after 1 and 12 h, respectively for Euplotes vannus [86]. CuO NPs show metal-resistance genes in P. aeruginosa PAO1 [87]. E. coli treated with MgO NPs differentially regulated 109 proteins with 83 being downregulated [88]. Genomics analysis of bacteria cells upon incubation with AgNPs and Ag1 revealed an upregulation of a shared 161 genes and downregulation of 27 genes in E. coli with exclusively regulated 309 and 70 genes, respectively [89]. Another study reported that E. coli treated with AgNPs upregulated many genes covering a wide range of functions such as membrane structure and biofilm formation (bolA), the citric acid cycle (sdhC), electron transfer (sdhC), cellular transport (mdfA), protein efflux (fsr, yajR, and emrE), and DNA repair (recN, uvrA, ybfE, yebG, ssb, sbmc, and nfo) [82]. CeO2 NPs tested against E. coli, B. subtilis, and S. oneidensis showed differential expression of genes rnt, thiS, cysI, cysN, cysW, yciW, ilvG, and pyrB [90]. TiO2 NPs showed downregulated genes dnaX and holB, both involved in DNA replication for E. coli [91].

9.4.12 Microscope 9.4.12.1 Fluorescence microscope Nanotoxicity of a nanoparticle can be evaluated using fluorescence microscope. Antibacterial activity can be evaluated via imaging using confocal laser scanning microscopy (CLSM), and quantified using fluorescence-activated cell sorting (FACSTM) analysis. Biological activity of metal oxide nanoparticles is evaluated against zebrafish HTS using bright-field, and fluorescence images [92]. Analysis can be used to evaluate defects, such as reduction in hatching rate, vertebral malformation, viability, and other physical deformations [92]. Analysis of the levels of intra-bacterial free Zn21 ions using two-photon

3. Prevention of nanotoxicity

206

9. General methods for detection and evaluation of nanotoxicity

FIGURE 9.4 Two-photon fluorescence microscopy (collected at 500 620 nm upon 780 nm excitation with femtosecond pulses) and bright-field images of AZn2-labeled (A J) Staphylococcus aureus and (K F) Klebsiella pneumoniae after incubation with ZnO nanoplates (NPs), nano-assemblies (NAs), and conventional nanoparticles (CNs), and ZnCl2 of 0.35 mM. Source: Reprinted with permission from A. Joe, S.-H. Park, K.-D. Shim, et al., Antibacterial mechanism of ZnO nanoparticles under dark conditions. J. Ind. Eng. Chem. 45 (2017) 430 439, Available from: https://doi.org/ 10.1016/j.jiec.2016.10.013.

microscopy shows direct visualization of Zn21 ions in the living bacteria (Fig. 9.4) [69]. Upon excitation at 780 nm with femtosecond pulses, the images of AZn2-labeled bacteria showed weak two-photon excited fluorescence (TPEF, Fig. 9.4). Data shows mean TPEF intensities of B12 times higher for S. aureus, and six times higher for Klebsiella pneumoniae than that of the control group (Fig. 9.4). The observed intra-bacterial free Zn21 ions is due to the local dissolution of the attached or transfected ZnO NPs (Fig. 9.4) [69]. CLSM was used to evaluate the antibacterial of AgNPs against S. aureus biofilms [52]. CLSM was used to evaluate the nanotoxicity of TiO2 NPs [62], AgNPs [93], (PDA-AlgCAP@CS-n)m [27], and Au@AgNPs [55]. The change in the contrast becomes blurry, of the bacterial cell wall is an indication of the cell wall degradation [93]. AgNPs cause destruction of the cell wall after subsequent penetration of NPs.

3. Prevention of nanotoxicity

9.4 Methods for detection and evaluation of nanotoxicity

207

The antibacterial activity of the prepared nanoparticles is evaluated using a CLMS using a dye such as Live/dead BacLight Bacterial Viability Kit. Briefly, few microlitters of the tested nanoparticles were added to bacterial cells, and the mixture was incubated at 37 C for 24 h. Then, the solutions were mixed with a dye solution containing SYTO9 and PI, for 20 min at room temperature. Finally, the bacteria cells were imaged using a CLMS instrument. The rapid detection of damaged cells depends on the use of appropriate dyes staining. 9.4.12.2 Flow cytometry Fluorescence-activated cell sorting (FACS), or flow cytometry (FCM) are used for sorting a heterogeneous mixture of biological cells into two or more containers; one cell per a time [94]. It is based on the specific light scattering, and fluorescent characteristics of each cell. Upon light scattering, excitation, and emission of a fluorochrome molecule, a specific multiparameter data from particles and cells (1 40 µm diameter) is recorded. It provides fast, and quantitative recording of fluorescent signals from individual cells. It is a useful method for physical separation of cells of particular interest, evaluating nanotoxicity of a nanoparticle, pathogen host interactions, and the mechanisms governing pathways. The cells of a mixture are modified with an antibody associated to a fluorescent molecule (Fig. 9.5). The specific cells pass through a laser beam for monitoring. The cells of the mixture were charged (positive or negative) or noncharged, based on whether the cell has limited the fluorescently tagged antibody or not. The charged cells were separated using an electric field into a collection tube based on their charges (Fig. 9.5). Scattered light can be classified to the forward scatter (FS, typically up to 20 degrees offset from the laser beams axis), or side-scatter (SS) light (measured approximately at a 90 degrees angle to the excitation line). The FS intensity equates to the cell’s and particle’s size, while the SS intensity provides information about the granular content within a cell and a particle. Both FS and SS are unique for each cell. Thus they can be used to differentiate among cells of a heterogeneous sample. 9.4.12.3 Electron microscopy Imaging bacteria cell using TEM can be used to evaluate a nanoparticle cytotoxicity. TEM images provide useful information about the cell integrity. Electron microscopy has been used to evaluate the nanotoxicity of CS-Lys-NPs [48], MgO NPs [96], and TiO2 [97]. TEM images showed that the different types of TiO2 NPs (A12, R9, and A140) behaved in a different way [97]. For example, TiO2 A12, which was synthesized using laser pyrolysis localized in the periplasm of both strains, whereas TiO2 R9 (rutile from Sigma-Aldrich, Cat # 637262), and A140 (anatase from Sigma-Aldrich, Cat # T-8141) did not, suggesting a specific mechanism of internalization [97]. Data reveals that the adsorption of the NPs onto bacterial cell wall is a prerequisite for the internalization. TEM images show a disruption in the cell wall when B. subtilis cells were exposed to MgO NPs [96]. The damaged cells can be imaged using TEM that physically separated walls from the internal cellular environment and that electron dense aggregation of compounds were surrounding the lysed cell [98]. SEM can be also used to evaluate the nanotoxicity of AuNPs layer (GNPL), and a phase-transitioned lysozyme film for E. coli and S. aureus upon treatment with near-infrared laser irradiation [99].

3. Prevention of nanotoxicity

208

9. General methods for detection and evaluation of nanotoxicity

Cell suspension

Ultrasonic nozzle vibrator (forms droplets)

Detectors Analyser

Laser

Charged (–, +) and noncharged small drops –20000 V

_ _ _

+ + +

+20000 V

Cell collector FIGURE 9.5 Flow cytometry (FCM) can be used to characterize bacterial physiological responses [95].

9.4.13 Other methods In vitro cell-based characterizations for nanotoxicity respect to biodistribution, metabolism, hematology, immunology, and neurological ramifications [100]. Thus it can be accomplished using newer methods such as microfluidics, microelectrochemistry, gas chromatography coupled to mass spectrometry, thin-layer chromatography coupled to mass spectrometry, matrixassisted laser desorption ionization mass spectrometry, Fourier transform mass spectrometry, and solid-state nuclear magnetic resonance studies using magic angle spinning.

9.5 Conclusion and outlooks Antibacterial properties of nanoparticles can be measured using different techniques including liquid culture tests, spectrophotometric method, linear cultivation tests, disk diffusion method, 3. Prevention of nanotoxicity

References

209

CFU, and counting method [101]. Most of these techniques were used for small molecules that could have different modeling of the metabolism compared to nanoparticles. Thus special techniques for nanoparticles are highly required (Table 9.2). For accurate evaluation of the nanoparticle toxicity, it is critical to ensure the purity of nanomaterials and absence of impurities [102]. In general, a stabilizer reagent is used during the synthesis of nanoparticles so that purification of nanoparticles should be carried out using chromatographic techniques such as high pressure liquid chromatography, or size exclusion chromatography. Thus any nonspecific toxicity attributed to material impurities or contaminations can be excluded. The bacteria cells media are heterogeneous, thus the overall absorbance should be evaluated carefully. Therefore a better alternative to bulk-cell assays such as single-cell analysis is accurate, and easy for interpretation. The single-cell assays can be used to acquire dynamic information from individual cells, such as differences in gene and protein expression, proliferation, cell cycle, and drug response.

Acknowledgment Author would thank the Ministry of Higher Education and Scientific Research (MHESR) and Institutional Review Board (IRB) of the Faculty of Science in Assiut University, Egypt for the support.

References [1] V. Prakash Sharma, U. Sharma, M. Chattopadhyay, V.N. Shukla, Advance applications of nanomaterials: a review, Mater. Today Proc. 5 (2018) 6376 6380. Available from: https://doi.org/10.1016/j.matpr.2017.12.248. [2] Global Nanotechnology Market Outlook 2018 2024. ,https://www.researchandmarkets.com/reports/4536705/ global-nanotechnology-market-outlook-2018-2024. (accessed 24.05.19). [3] The Lancet, The risks of nanotechnology for human health, Lancet 369 (2007) 1142. Available from: https:// doi.org/10.1016/S0140-6736(07)60538-8. [4] Z.-J. Zhu, R. Carboni, M.J. Quercio, et al., Surface properties dictate uptake, distribution, excretion, and toxicity of nanoparticles in fish, Small 6 (2010) 2261 2265. Available from: https://doi.org/10.1002/smll.201000989. [5] K. Donaldson, Nanotoxicology, Occup. Environ. Med. 61 (2004) 727 728. Available from: https://doi.org/10.1136/ oem.2004.013243. [6] G. Oberdo¨rster, Safety assessment for nanotechnology and nanomedicine: concepts of nanotoxicology, J. Intern. Med. 267 (2010) 89 105. Available from: https://doi.org/10.1111/j.1365-2796.2009.02187.x. [7] D.B. Warheit, T.R. Webb, K.L. Reed, et al., Pulmonary toxicity study in rats with three forms of ultrafineTiO2 particles: differential responses related to surface properties, Toxicology 230 (2007) 90 104. Available from: https://doi.org/10.1016/j.tox.2006.11.002. [8] H.N. Abdelhamid, H.F. Wu, Multifunctional graphene magnetic nanosheet decorated with chitosan for highly sensitive detection of pathogenic bacteria, J. Mater. Chem. B 1 (2013) 3950 3961. Available from: https://doi.org/10.1039/C3TB20413H. [9] H.F. Wu, J. Gopal, H.N. Abdelhamid, N. Hasan, Proteomics 12 (2012) 2949 2961. Available from: https:// doi.org/10.1002/pmic.201200295. [10] J. Gopal, H.N. Abdelhamid, P.Y. Hua, H.F. Wu, Chitosan nanomagnets for effective extraction and sensitive mass spectrometric detection of pathogenic bacterial endotoxin from human urine, J. Mater. Chem. B, 1 (2013) 2463 2475. Available from: https://doi.org/10.1039/C3TB20079E. [11] H.N. Abdelhamid, J. Gopal, H.F. Wu, Synthesis and application of ionic liquid matrices (ILMs) for effective pathogenic bacteria analysis in matrix assisted laser desorption/ionization (MALDI-MS), Analytica chimica acta 767 (2013) 104 111. Available from: https://doi.org/10.1016/j.aca.2012.12.054.

3. Prevention of nanotoxicity

210

9. General methods for detection and evaluation of nanotoxicity

[12] M.L. Bhaisare, H.N. Abdelhamid, B.S. Wu, H.F. Wu, Rapid and direct MALDI-MS identification of pathogenic bacteria from blood using ionic liquid-modified magnetic nanoparticles (Fe3O4@SiO2), J. Mater. Chem. B, 2 (2014) 4671 4683. [13] HN Abdelhamid, ML Bhaisare, HF Wu, Ceria nanocubic-ultrasonication assisted dispersive liquid–liquid microextraction coupled with matrix assisted laser desorption/ionization mass spectrometry for pathogenic bacteria analysis, Talanta 120, 2014, 208-217. Available from: https://doi.org/10.1016/j.talanta.2013.11.078. [14] H.N. Abdelhamid, H.F. Wu, Proteomics analysis of the mode of antibacterial action of nanoparticles and their interactions with proteins, TrAC Trends in Analytical Chemistry 65 (2015) 30 46. Available from: https://doi.org/10.1016/j.trac.2014.09.010. [15] V. Pezeshkpour, S.A. Khosravani, M. Ghaedi, et al., Ultrasound assisted extraction of phenolic acids from broccoli vegetable and using sonochemistry for preparation of MOF-5 nanocubes: comparative study based on micro-dilution broth and plate count method for synergism antibacterial effect, Ultrason. Sonochem. 40 (2018) 1031 1038. Available from: https://doi.org/10.1016/j.ultsonch.2017.09.001. [16] E. Hodgson, A Textbook of Modern Toxicology, John Wiley & Sons, Inc, Hoboken, NJ, 2004. [17] P.-P. Jia, T. Sun, M. Junaid, et al., Nanotoxicity of different sizes of graphene (G) and graphene oxide (GO) in vitro and in vivo, Environ. Pollut. 247 (2019) 595 606. Available from: https://doi.org/10.1016/j.envpol.2019.01.072. [18] R. Sonohara, N. Muramatsu, H. Ohshima, T. Kondo, Difference in surface properties between Escherichia coli and Staphylococcus aureus as revealed by electrophoretic mobility measurements, Biophys. Chem. 55 (1995) 273 277. Available from: https://doi.org/10.1016/0301-4622(95)00004-H. [19] Y.N. Slavin, J. Asnis, U.O. Ha¨feli, H. Bach, Metal nanoparticles: understanding the mechanisms behind antibacterial activity, J. Nanobiotechnology 15 (2017) 65. Available from: https://doi.org/10.1186/s12951-017-0308-z. [20] M.M. Mohamed, S.A. Fouad, H.A. Elshoky, et al., Antibacterial effect of gold nanoparticles against Corynebacterium pseudotuberculosis, Int. J. Vet. Sci. Med. 5 (2017) 23 29. Available from: https://doi.org/10.1016/j.ijvsm.2017.02.003. [21] Q. Lv, B. Zhang, X. Xing, et al., Biosynthesis of copper nanoparticles using Shewanella loihica PV-4 with antibacterial activity: novel approach and mechanisms investigation, J. Hazard. Mater. 347 (2018) 141 149. Available from: https://doi.org/10.1016/j.jhazmat.2017.12.070. [22] S. Fanny Chiat Orou, K.J. Hang, M. Thuya Thien, et al., Antibacterial activity by ZnO nanorods and ZnO nanodisks: a model used to illustrate “nanotoxicity threshold”, J. Ind. Eng. Chem. 62 (2018) 333 340. Available from: https://doi.org/10.1016/j.jiec.2018.01.013. [23] Z. Farrokhi, A. Ayati, M. Kanvisi, M. Sillanpa¨a¨, Recent advance in antibacterial activity of nanoparticles contained polyurethane, J. Appl. Polym. Sci. 136 (2019) 46997. Available from: https://doi.org/10.1002/ app.46997. [24] H.N. Abdelhamid, H.F. Wu, Probing the interactions of chitosan capped CdS quantum dots with pathogenic bacteria and their biosensing application, J. Mater. Chem. B 1 (2013) 6094 6106. Available from: https://doi. org/10.1039/C3TB21020K. [25] Y. Chen, A. Gao, L. Bai, et al., Antibacterial, osteogenic, and angiogenic activities of SrTiO3 nanotubes embedded with Ag2O nanoparticles, Mater. Sci. Eng. C 75 (2017) 1049 1058. Available from: https://doi.org/ 10.1016/j.msec.2017.03.014. [26] B.S. Wu, H.N. Abdelhamid, H.F. Wu, Synthesis and antibacterial activities of graphene decorated with stannous dioxide, RSC Adv. 4 (2014) 3722 3731. Available from: https://doi.org/10.1039/C3RA43992E. [27] X. Hao, W. Wang, Z. Yang, et al., pH responsive antifouling and antibacterial multilayer films with self-healing performance, Chem. Eng. J. 356 (2019) 130 141. Available from: https://doi.org/10.1016/j.cej.2018.08.181. [28] M.S. Khan, H.N. Abdelhamid, H.F. Wu, Near infrared (NIR) laser mediated surface activation of graphene oxide nanoflakes for efficient antibacterial, antifungal and wound healing treatment, Colloids and Surfaces B: Biointerfaces 127 (2015) 281 291. Available from: https://doi.org/10.1016/j.colsurfb.2014.12.049. [29] J. Li, B. Xie, K. Xia, et al., Enhanced antibacterial activity of silver doped titanium dioxide-chitosan composites under visible light, Materials (Basel) 11 (2018) 1403. Available from: https://doi.org/10.3390/ma11081403. [30] R. Coradeghini, S. Gioria, C.P. Garcı´a, et al., Size-dependent toxicity and cell interaction mechanisms of gold nanoparticles on mouse fibroblasts, Toxicol. Lett. 217 (2013) 205 216. Available from: https://doi.org/10.1016/ j.toxlet.2012.11.022. [31] E. Locatelli, F. Broggi, J. Ponti, et al., Lipophilic silver nanoparticles and their polymeric entrapment into targeted-PEG-based micelles for the treatment of glioblastoma, Adv. Healthc. Mater. 1 (2012) 342 347. Available from: https://doi.org/10.1002/adhm.201100047.

3. Prevention of nanotoxicity

References

211

[32] U.L.N.H. Senarathna, S.S.N. Fernando, T.D.C.P. Gunasekara, et al., Enhanced antibacterial activity of TiO2 nanoparticle surface modified with Garcinia zeylanica extract, Chem. Cent. J. 11 (2017) 7. Available from: https:// doi.org/10.1186/s13065-017-0236-x. [33] I. De Angelis, F. Barone, A. Zijno, et al., Comparative study of ZnO and TiO2 nanoparticles: physicochemical characterisation and toxicological effects on human colon carcinoma cells, Nanotoxicology 7 (2013) 1361 1372. Available from: https://doi.org/10.3109/17435390.2012.741724. [34] A. Sobhani-Nasab, Z. Zahraei, M. Akbari, et al., Synthesis, characterization, and antibacterial activities of ZnLaFe2O4/NiTiO3 nanocomposite, J. Mol. Struct. 1139 (2017) 430 435. Available from: https://doi.org/10.1016/ j.molstruc.2017.03.069. [35] C. Uboldi, G. Giudetti, F. Broggi, et al., Amorphous silica nanoparticles do not induce cytotoxicity, cell transformation or genotoxicity in Balb/3T3 mouse fibroblasts, Mutat. Res. Toxicol. Environ. Mutagen. 745 (2012) 11 20. Available from: https://doi.org/10.1016/j.mrgentox.2011.10.010. [36] N. El Yamani, A.R. Collins, E. Runde´n-Pran, et al., In vitro genotoxicity testing of four reference metal nanomaterials, titanium dioxide, zinc oxide, cerium oxide and silver: towards reliable hazard assessment, Mutagenesis 32 (2017) 117 126. Available from: https://doi.org/10.1093/mutage/gew060. [37] G. Wang, W. Jin, A.M. Qasim, et al., Antibacterial effects of titanium embedded with silver nanoparticles based on electron-transfer-induced reactive oxygen species, Biomaterials 124 (2017) 25 34. Available from: https:// doi.org/10.1016/j.biomaterials.2017.01.028. [38] J. Ponti, R. Colognato, H. Rauscher, et al., Colony forming efficiency and microscopy analysis of multi-wall carbon nanotubes cell interaction, Toxicol. Lett. 197 (2010) 29 37. Available from: https://doi.org/10.1016/ j.toxlet.2010.04.018. [39] S. Hussain, J. Joo, J. Kang, et al., Antibiotic-loaded nanoparticles targeted to the site of infection enhance antibacterial efficacy, Nat. Biomed. Eng. 2 (2018) 95 103. Available from: https://doi.org/10.1038/s41551-017-0187-5. [40] G. Liu, G. Haiqi, K. Li, et al., Fabrication of silver nanoparticle sponge leather with durable antibacterial property, J. Colloid Interface Sci. 514 (2018) 338 348. Available from: https://doi.org/10.1016/j.jcis.2017.09.049. [41] M. Balouiri, M. Sadiki, S.K. Ibnsouda, Methods for in vitro evaluating antimicrobial activity: a review, J. Pharm. Anal. 6 (2016) 71 79. Available from: https://doi.org/10.1016/j.jpha.2015.11.005. [42] International Standard ISO 20645, Textile Fabrics—Determination of Antibacterial Activity—Agar Diffusion Plate Test. International Organization for Standardization, Switzerland, 2004. [43] P. Le Thi, Y. Lee, T.T. Hoang Thi, et al., Catechol-rich gelatin hydrogels in situ hybridizations with silver nanoparticle for enhanced antibacterial activity, Mater. Sci. Eng. C 92 (2018) 52 60. Available from: https:// doi.org/10.1016/j.msec.2018.06.037. [44] J. Peng, J. Lin, Z. Chen, et al., Enhanced antimicrobial activities of silver-nanoparticle-decorated reduced graphene nanocomposites against oral pathogens, Mater. Sci. Eng. C 71 (2017) 10 16. Available from: https://doi. org/10.1016/j.msec.2016.09.070. [45] T.G. Volova, A.A. Shumilova, I.P. Shidlovskiy, et al., Antibacterial properties of films of cellulose composites with silver nanoparticles and antibiotics, Polym. Test. 65 (2018) 54 68. Available from: https://doi.org/10.1016/ j.polymertesting.2017.10.023. [46] H. Chen, X. Zhao, Y. Liu, et al., Facile synthesis of elemental silver by the seed nucleus embedding method for antibacterial applications, Cellulose 25 (2018) 5289 5296. Available from: https://doi.org/10.1007/s10570-018-1952-7. [47] H. Zare Khafri, M. Ghaedi, A. Asfaram, et al., Synthesis of CuS and ZnO/Zn(OH)2 nanoparticles and their evaluation for in vitro antibacterial and antifungal activities, Appl. Organomet. Chem. 32 (2018) e4398. Available from: https://doi.org/10.1002/aoc.4398. [48] T. Wu, C. Wu, S. Fu, et al., Integration of lysozyme into chitosan nanoparticles for improving antibacterial activity, Carbohydr. Polym. 155 (2017) 192 200. Available from: https://doi.org/10.1016/j.carbpol.2016.08.076. [49] S. Ghayempour, M. Montazer, Ultrasound irradiation based in-situ synthesis of star-like Tragacanth gum/zinc oxide nanoparticles on cotton fabric, Ultrason. Sonochem. 34 (2017) 458 465. Available from: https://doi.org/ 10.1016/j.ultsonch.2016.06.019. [50] E. Zare, S. Pourseyedi, M. Khatami, E. Darezereshki, Simple biosynthesis of zinc oxide nanoparticles using nature’s source, and it’s in vitro bio-activity, J. Mol. Struct. 1146 (2017) 96 103. Available from: https://doi. org/10.1016/j.molstruc.2017.05.118. [51] J.H. Jorgensen, M.J. Ferraro, Antimicrobial susceptibility testing: a review of general principles and contemporary practices, Clin. Infect. Dis. 49 (2009) 1749 1755. Available from: https://doi.org/10.1086/647952.

3. Prevention of nanotoxicity

212

9. General methods for detection and evaluation of nanotoxicity

[52] Z. Qiao, Y. Yao, S. Song, et al., Silver nanoparticles with pH induced surface charge switchable properties for antibacterial and antibiofilm applications, J. Mater. Chem. B 7 (2019) 830 840. Available from: https://doi.org/ 10.1039/C8TB02917B. [53] A. Alshareef, K. Laird, R.B.M. Cross, Shape-dependent antibacterial activity of silver nanoparticles on Escherichia coli and Enterococcus faecium bacterium, Appl. Surf. Sci. 424 (2017) 310 315. Available from: https://doi.org/ 10.1016/j.apsusc.2017.03.176. [54] L.T. Hoa, N.T.Y. Linh, J.S. Chung, S.H. Hur, Green synthesis of silver nanoparticle-decorated porous reduced graphene oxide for antibacterial non-enzymatic glucose sensors, Ionics (Kiel) 23 (2017) 1525 1532. Available from: https://doi.org/10.1007/s11581-016-1954-0. [55] L. Yang, W. Yan, H. Wang, et al., Shell thickness-dependent antibacterial activity and biocompatibility of gold@silver core shell nanoparticles, RSC Adv. 7 (2017) 11355 11361. Available from: https://doi.org/10.1039/C7RA00485K. [56] M.A. Surmeneva, A.A. Sharonova, S. Chernousova, et al., Incorporation of silver nanoparticles into magnetronsputtered calcium phosphate layers on titanium as an antibacterial coating, Colloids Surf. B Biointerfaces 156 (2017) 104 113. Available from: https://doi.org/10.1016/j.colsurfb.2017.05.016. [57] H.N. Abdelhamid, H.F. Wu, A method to detect metal drug complexes and their interactions with pathogenic bacteria via graphene nanosheet assist laser desorption/ionization mass spectrometry and biosensors, Analytica chimica acta 751, 2012, 94 104. Available from: https://doi.org/10.1016/j.aca.2012.09.012. [58] A.A. Shah, A. Khan, S. Dwivedi, et al., Antibacterial and antibiofilm activity of barium titanate nanoparticles, Mater. Lett. 229 (2018) 130 133. Available from: https://doi.org/10.1016/j.matlet.2018.06.107. [59] J. Liu, M.D. Rojas-Andrade, G. Chata, et al., Photo-enhanced antibacterial activity of ZnO/graphene quantum dot nanocomposites, Nanoscale 10 (2018) 158 166. Available from: https://doi.org/10.1039/C7NR07367D. [60] L.S. Garcia (Ed.), Broth microdilution MIC test. in: Clinical Microbiology Procedures Handbook, third ed., American Society of Microbiology, Orlando, FL, 2010, pp 25 41. [61] K. Krishnamoorthy, G. Manivannan, S.J. Kim, et al., Antibacterial activity of MgO nanoparticles based on lipid peroxidation by oxygen vacancy, J. Nanopart. Res. 14 (2012) 1063. Available from: https://doi.org/10.1007/s11051-0121063-6. [62] B. Jalvo, M. Faraldos, A. Bahamonde, R. Rosal, Antibacterial surfaces prepared by electrospray coating of photocatalytic nanoparticles, Chem. Eng. J. 334 (2018) 1108 1118. Available from: https://doi.org/ 10.1016/j.cej.2017.11.057. [63] S. Jaworski, M. Wierzbicki, E. Sawosz, et al., Graphene oxide-based nanocomposites decorated with silver nanoparticles as an antibacterial agent, Nanoscale Res. Lett. 13 (2018) 116. Available from: https://doi.org/ 10.1186/s11671-018-2533-2. [64] X. Huang, X. Bao, Y. Liu, et al., Catechol-functional chitosan/silver nanoparticle composite as a highly effective antibacterial agent with species-specific mechanisms, Sci. Rep. 7 (2017) 1860. Available from: https:// doi.org/10.1038/s41598-017-02008-4. [65] M. Moghayedi, E.K. Goharshadi, K. Ghazvini, et al., Kinetics and mechanism of antibacterial activity and cytotoxicity of Ag-RGO nanocomposite, Colloids Surf. B Biointerfaces 159 (2017) 366 374. Available from: https://doi.org/10.1016/j.colsurfb.2017.08.001. [66] A. Kroll, M.H. Pillukat, D. Hahn, J. Schnekenburger, Interference of engineered nanoparticles with in vitro toxicity assays, Arch. Toxicol. 86 (2012) 1123 1136. Available from: https://doi.org/10.1007/s00204-012-0837-z. [67] B. Ramalingam, T. Parandhaman, S.K. Das, Antibacterial effects of biosynthesized silver nanoparticles on surface ultrastructure and nanomechanical properties of gram-negative bacteria viz. Escherichia coli and Pseudomonas aeruginosa, ACS Appl. Mater. Interfaces 8 (2016) 4963 4976. Available from: https://doi.org/ 10.1021/acsami.6b00161. [68] A.K. Madl, L.E. Plummer, C. Carosino, K.E. Pinkerton, Nanoparticles, lung injury, and the role of oxidant stress, Annu. Rev. Physiol. 76 (2014) 447 465. Available from: https://doi.org/10.1146/annurev-physiol-030212-183735. [69] A. Joe, S.-H. Park, K.-D. Shim, et al., Antibacterial mechanism of ZnO nanoparticles under dark conditions, J. Ind. Eng. Chem. 45 (2017) 430 439. Available from: https://doi.org/10.1016/j.jiec.2016.10.013. [70] A. Kumar, A.K. Pandey, S.S. Singh, et al., Engineered ZnO and TiO2 nanoparticles induce oxidative stress and DNA damage leading to reduced viability of Escherichia coli, Free. Radic. Biol. Med. 51 (2011) 1872 1881. Available from: https://doi.org/10.1016/j.freeradbiomed.2011.08.025. [71] N. Padmavathy, R. Vijayaraghavan, Enhanced bioactivity of ZnO nanoparticles—an antimicrobial study, Sci. Technol. Adv. Mater. 9 (2008) 035004. Available from: https://doi.org/10.1088/1468-6996/9/3/035004.

3. Prevention of nanotoxicity

References

213

[72] A. Simon-Deckers, S. Loo, M. Mayne-L’hermite, et al., Size-, composition- and shape-dependent toxicological impact of metal oxide nanoparticles and carbon nanotubes toward bacteria, Environ. Sci. Technol. 43 (2009) 8423 8429. Available from: https://doi.org/10.1021/es9016975. [73] A.C. Gasparovic, M. Jaganjac, B. Mihaljevic, et al., Assays for the measurement of lipid peroxidation, in: L. Galluzzi, I. Vitale, O. Kepp, G. Kroemer (Eds.), Cell Senescence. Methods in Molecular Biology (Methods and Protocols), vol. 965, Humana Press, Totowa, NJ, 2013, pp. 283 296. [74] U. Kadiyala, E.S. Turali-Emre, J.H. Bahng, et al., Unexpected insights into antibacterial activity of zinc oxide nanoparticles against methicillin resistant Staphylococcus aureus (MRSA), Nanoscale 10 (2018) 4927 4939. Available from: https://doi.org/10.1039/C7NR08499D. [75] S. Wang, J. Wu, H. Yang, et al., Antibacterial activity and mechanism of Ag/ZnO nanocomposite against anaerobic oral pathogen Streptococcus mutans, J. Mater. Sci. Mater. Med. 28 (2017) 23. Available from: https://doi. org/10.1007/s10856-016-5837-8. [76] Y.-M. Long, L.-G. Hu, X.-T. Yan, et al., Surface ligand controls silver ion release of nanosilver and its antibacterial activity against Escherichia coli, Int. J. Nanomed. 12 (2017) 3193 3206. Available from: https://doi.org/ 10.2147/IJN.S132327. [77] V.M. Chernov, O.A. Chernova, A.A. Mouzykantov, et al., Omics of antimicrobials and antimicrobial resistance, Expert. Opin. Drug. Discov. 14 (2019) 455 468. Available from: https://doi.org/10.1080/17460441.2019.1588880. [78] X. Zheng, J. Wang, Y. Chen, Y. Wei, Comprehensive analysis of transcriptional and proteomic profiling reveals silver nanoparticles-induced toxicity to bacterial denitrification, J. Hazard. Mater. 344 (2018) 291 298. Available from: https://doi.org/10.1016/j.jhazmat.2017.10.028. [79] X. Yan, B. He, L. Liu, et al., Antibacterial mechanism of silver nanoparticles in Pseudomonas aeruginosa: proteomics approach, Metallomics 10 (2018) 557 564. Available from: https://doi.org/10.1039/C7MT00328E. [80] N.S. Wigginton, A. de Titta, F. Piccapietra, et al., Binding of silver nanoparticles to bacterial proteins depends on surface modifications and inhibits enzymatic activity, Environ. Sci. Technol. 44 (2010) 2163 2168. Available from: https://doi.org/10.1021/es903187s. [81] O. Choi, Z. Hu, Size dependent and reactive oxygen species related nanosilver toxicity to nitrifying bacteria, Environ. Sci. Technol. 42 (2008) 4583 4588. Available from: https://doi.org/10.1021/es703238h. [82] N. Gou, A. Onnis-Hayden, A.Z. Gu, Mechanistic toxicity assessment of nanomaterials by whole-cell-array stress genes expression analysis, Environ. Sci. Technol. 44 (2010) 5964 5970. Available from: https://doi.org/10.1021/ es100679f. [83] A. Ivask, A. ElBadawy, C. Kaweeteerawat, et al., Toxicity mechanisms in Escherichia coli vary for silver nanoparticles and differ from ionic silver, ACS Nano 8 (2014) 374 386. Available from: https://doi.org/10.1021/nn4044047. [84] L.V. Aseev, A.A. Levandovskaya, L.S. Tchufistova, et al., A new regulatory circuit in ribosomal protein operons: S2-mediated control of the rpsB-tsf expression in vivo, RNA 14 (2008) 1882 1894. Available from: https://doi. org/10.1261/rna.1099108. [85] N. Singh, J. Rajwade, K.M. Paknikar, Transcriptome analysis of silver nanoparticles treated Staphylococcus aureus reveals potential targets for biofilm inhibition, Colloids Surf. B Biointerfaces 175 (2019) 487 497. Available from: https://doi.org/10.1016/j.colsurfb.2018.12.032. [86] Y. Pan, W. Zhang, S. Lin, Transcriptomic and microRNAomic profiling reveals molecular mechanisms to cope with silver nanoparticle exposure in the ciliate Euplotes vannus, Environ. Sci. Nano 5 (2018) 2921 2935. Available from: https://doi.org/10.1039/C8EN00924D. [87] J. Guo, S.-H. Gao, J. Lu, et al., Copper oxide nanoparticles induce lysogenic bacteriophage and metal-resistance genes in Pseudomonas aeruginosa PAO1, ACS Appl. Mater. Interfaces 9 (2017) 22298 22307. Available from: https:// doi.org/10.1021/acsami.7b06433. [88] Y.H. Leung, A.M.C. Ng, X. Xu, et al., Mechanisms of antibacterial activity of MgO: non-ROS mediated toxicity of MgO nanoparticles towards Escherichia coli, Small 10 (2014) 1171 1183. Available from: https://doi.org/10.1002/ smll.201302434. [89] J.S. McQuillan, A.M. Shaw, Differential gene regulation in the Ag nanoparticle and Ag1-induced silver stress response in Escherichia coli: a full transcriptomic profile, Nanotoxicology 8 (2014) 177 184. Available from: https://doi.org/10.3109/17435390.2013.870243. [90] D.A. Pelletier, A.K. Suresh, G.A. Holton, et al., Effects of engineered cerium oxide nanoparticles on bacterial growth and viability, Appl. Environ. Microbiol. 76 (2010) 7981 7989. Available from: https://doi.org/10.1128/ AEM.00650-10.

3. Prevention of nanotoxicity

214

9. General methods for detection and evaluation of nanotoxicity

[91] B. Sohm, F. Immel, P. Bauda, C. Pagnout, Insight into the primary mode of action of TiO 2 nanoparticles on Escherichia coli in the dark, Proteomics 15 (2015) 98 113. Available from: https://doi.org/10.1002/ pmic.201400101. [92] S. Lin, Y. Zhao, T. Xia, et al., High content screening in zebrafish speeds up hazard ranking of transition metal oxide nanoparticles, ACS Nano 5 (2011) 7284 7295. Available from: https://doi.org/10.1021/nn202116p. [93] I.P. Mukha, A.M. Eremenko, N.P. Smirnova, et al., Antimicrobial activity of stable silver nanoparticles of a certain size, Appl. Biochem. Microbiol. 49 (2013) 199 206. Available from: https://doi.org/10.1134/ S0003683813020117. [94] J. V. Watson, Introduction to Flow Cytometry, First Paperback Edition. Cambridge University Press, 2004. [95] V. Ambriz-Avin˜a, J.A. Contreras-Gardun˜o, M. Pedraza-Reyes, Applications of flow cytometry to characterize bacterial physiological responses, Biomed. Res. Int. 2014 (2014) 1 14. Available from: https://doi.org/10.1155/ 2014/461941. [96] P.K. Stoimenov, R.L. Klinger, G.L. Marchin, K.J. Klabunde, Metal oxide nanoparticles as bactericidal agents, Langmuir 18 (2002) 6679 6686. Available from: https://doi.org/10.1021/la0202374. [97] B. Pignon, H. Maskrot, V. Guyot Ferreol, et al., Versatility of laser pyrolysis applied to the synthesis of TiO2 nanoparticles—application to UV attenuation, Eur. J. Inorg. Chem. 2008 (2008) 883 889. Available from: https://doi.org/10.1002/ejic.200700990. [98] H.N. Abdelhamid, M.S. Khan, H.F. Wu, Graphene oxide as a nanocarrier for gramicidin (GOGD) for high antibacterial performance, RSC Adv. 4 (2014) 50035 50046. Available from: https://doi.org/10.1039/ C4RA07250B. [99] Y. Qu, T. Wei, J. Zhao, et al., Regenerable smart antibacterial surfaces: full removal of killed bacteria via a sequential degradable layer, J. Mater. Chem. B 6 (2018) 3946 3955. Available from: https://doi.org/ 10.1039/C8TB01122B. [100] K. Greish, G. Thiagarajan, H. Ghandehari, In vivo methods of nanotoxicology, Mol. Biol. 926 (2012) 235 253. [101] H.N. Abdelhamid, A. Talib, H.F. Wu, Facile synthesis of water soluble silver ferrite (AgFeO2) nanoparticles and their biological application as antibacterial agents, RSC advances 5 (44) (2015) 34594 34602. Available from: https://doi.org/10.1039/C4RA14461A. [102] V. Kumar, N. Dasgupta, S. Ranjan, Nanotoxicology, CRC Press, Taylor & Francis Group, Boca Raton, FL, 2018.

3. Prevention of nanotoxicity

C H A P T E R

10 Safer-by-design for nanomaterials L. Reijnders IBED, University of Amsterdam, Amsterdam, The Netherlands

10.1 Introduction Nanomaterials are defined here as materials that are in at least one dimension ,100 nm. The focus of this chapter will be on engineered inorganic and carbonaceous nanomaterials and will include engineered materials that are partly nanosized [1 4]. Maynard et al. [5] called safe-by-design “a grand challenge of safe nanotechnology.” In Ref. [6] 2016 Maynard and Aitken reviewed the progress in the field of safe-by-design and noted “some progress most notably in pharmaceutical applications.” The progress of safeby-design strategies regarding pharmaceutical applications of nanomaterials (also: nanopharmaceuticals or nanomedicines) has been reviewed by Yan et al. [7]. The focus of the empirical studies reviewed by Yan et al. [7] is on reducing negative side effects of nanomedicines, often while increasing intended positive effects on consumers. Safety after excretion is not considered in the studies reviewed by Yan et al. [7], which limits the scope for safety claims. Consumer safety in the context of pharmaceuticals is furthermore an ambiguous concept. Pharmaceuticals admitted to the market as “safe” are commonly characterized by negative side effects on parts of the consumer population [8]. In view of the negative side effects of medicines, the second part of the title used by Yan et al. [7] “A safe-by-design strategy: towards safer nanomaterials in nanomedicines,” which refers to safer rather than safe, seems more fitting than just safe. In recent years safe-by-design for nanomaterials is also the subject of rapidly increasing interest in scientific publications outside the field of nanopharmaceuticals [9]. Such safe-bydesign studies are commonly based on toxicological research that would be assigned to the preclinical stage in the development of medicines [10]. This might well be an important limitation as unintended negative effects of pharmaceuticals often emerge after preclinical testing [8]. Also, outside the field of nanopharmaceuticals “safe” can be an ambiguous concept. An illustration thereof regards food-grade TiO2 that is partially nanosized [1]. In 1969 the Joint Expert Committee on Food Additives of WHO and FAO (JECFA) [11] concluded that “studies regarding food-grade TiO2 in several species including man did show neither significant absorption

Nanotoxicity DOI: https://doi.org/10.1016/B978-0-12-819943-5.00010-5

215

Copyright © 2020 Elsevier Inc. All rights reserved.

216

10. Safer-by-design for nanomaterials

nor tissue storage following ingestion. Establishment of an acceptable daily intake for man was therefore considered unnecessary.” However, recent studies have shown that there is significant absorption of nanoparticulate food-grade TiO2 after ingestion and that commonly ingested TiO2 nanoparticles constitute a potential risk to the liver, ovaries, and testes of humans [12]. Also there are strong indications that TiO2 nanoparticles may impair gut homeostasis in mice and lead to inflammation of the mammalian intestine [13 15]. Furthermore, in US policy, amorphous nanosilica is considered as generally recognized as safe, whereas there are strong indications that amorphous nanosilica can have inflammatory and fibrinogenic effects when inhaled, and may cause corneal injury in the case of eye exposure [16 18]. In the context of safe-by-design studies outside the field of nanopharmaceuticals it might be argued that the exposure to nanomaterials remains below the lowest no-adverse effect level derived from toxicological studies and that therefore such exposure is safe [19]. However, this argument does not take into account the simultaneous exposure to particles in the same size range from other sources but with similar impacts on organisms (e.g., the generation of reactive oxygen species). Exposure to current “background” nanosized inorganic and carbonaceous materials may be associated with risk to which additional nanomaterial exposures may add [20 22]. In view of the preceding considerations it would seem proper to use safer-by-design instead of safe-by-design. Safer-by-design is a concept also used in recent nanotechnology studies [23 28]. It should be noted that there are constraints regarding safer-by-design for engineered nanomaterials. A very important constraint originates in limited toxicological knowledge about the negative impacts of nanomaterials on organisms and the differences between materials used in nanotoxicity testing and nanomaterials to which organisms or cells are exposed in the real world [28,29]. This limits the scope for safer design and weakens the support for claims that a specific design makes nanomaterials safer. A further constraint follows from trade-offs between functionality and safety. It might, for example, be argued that negative impacts linked to the use of Ag nanoparticles in textiles can be reduced by decreasing the release of Ag ions. However, the antibacterial effect of Ag ions released from nanoparticles is intended to be functional by decreasing bacterial activity, for example, to suppress odors [30]. The application of nanomaterials can also be a constraint for safer-by-design. For instance, Truffier-Boutry et al. [31] suggested grafting TiO2 nanoparticles on larger TiO2 particles for the application in photocatalytic paints. This would not an option for sunscreens as the white color of such grafted particles would be considered unacceptable in sunscreens, whereas photocatalytic activity is a negative property in sunscreens. Safer-by-design strategies may aim at [24,31 33]: • reducing hazard (potential to harm organisms) of nanomaterials, and • reducing the release of nanomaterials to the environment, including indoor environments. Section 10.2 will briefly present hazard and release reduction for nanomaterial and nanocomposite production and processing, and for products. Section 10.2 will also introduce Sections 10.3 10.5. It has been argued that to the extent that engineered nanomaterials are released from nanocomposites (also: nano-enabled materials), these nanocomposites should be designed

3. Prevention of nanotoxicity

10.3 Reducing releases to the environment from nanomaterial production and processing facilities

217

in such a way that nanocomposite fragments released in the use stage are less hazardous than the engineered nanomaterials used in nanocomposite production [34]. This safer-bydesign proposal will be discussed in Section 10.6. Section 10.7 will present the conclusions of this chapter.

10.2 Hazard and release reduction for engineered nanomaterials in production and products Safer-by-design for nanomaterial and nanocomposite production and processing largely relies on reducing the release of nanomaterials, which will be discussed in Section 10.3. To the extent that nanomaterials are released, safer-by-design hazard reduction, discussed in Section 10.4 is relevant. Engineered nanomaterials are widely applied in products. In part such applications regard nanomaterials that are not linked to other materials and rather often such applications are inherently dispersive: they inevitably imply releases to the environment and may also inevitably lead to human exposure. Examples thereof are inorganic and carbonaceous nanomaterials applied in medicines, cosmetics, and food [7,14,18,35]. For inherently dispersive applications of nanomaterials safer-by-design relies on hazard reduction discussed in Section 10.4. It may also be that applications of engineered nanomaterials that are not linked to other materials are not inherently dispersive and that nanomaterials are contained in the use stage. Examples thereof are nanomaterials applied in reactors [2] and applications of nanomaterials in drilling fluid [36,37]. Regarding such applications, highly efficient recycling is conducive to limiting releases [2,38]. To the extent that releases occur, safer-by-design approaches discussed in Section 10.4 may be conducive to limiting risk. Other noninherently dispersive applications regard the use of relatively large nanocomposites in which nanomaterials are linked to other materials. Safer-by-design for such materials will be discussed in Section 10.5.1. A further case of applying noninherently dispersive nanomaterials regards products such as electronics and batteries, and includes the use of inorganic and carbonaceous nanomaterials for components such as wiring, sheets, thin film transistors, electrodes and sensors, generated by printing or by depositing nanosized materials from precursors in the vapor phase [39 41]. Releases from these applications and control thereof will be discussed in Section 10.5.2.

10.3 Reducing releases to the environment from nanomaterial production and processing facilities Nanomaterial production and processing, including the machining of (relatively large) nanocomposites, may lead to substantial releases of nanomaterials [42 45]. Geraci et al. [46] have considered the design of safer production processes for nanomaterials. Isolation of production processes by containment, including the use of high-efficiency particulate air filters removing nanomaterials, for treating gaseous flows to the environment, is central to such design. Regarding carbonaceous and metallic nanomaterials, there is furthermore a case to design processes for preventing dust explosions (avoiding sparks and dust clouds in contained areas) [46,47].

3. Prevention of nanotoxicity

218

10. Safer-by-design for nanomaterials

Containment as proposed by Geraci et al. [46] is also likely to be conducive to limiting releases in the case of processing, including the machining, of relatively large nanocomposites for the production of parts [45]. In situ synthesis of nanofibers on substrates may ceteris paribus be safer than first synthesizing nanofibers followed by further processing [33,48]. A further matter relevant to reduction of releases to the environment is the handling of nanosized production residues which may be solid, present in sludges, or suspended in water [39,49 52]. Nanosized materials present in solid wastes and sludges can be landfilled as hazardous wastes. A problem with this option is that to remain safe such stored nanosized wastes should remain contained for an indefinite period of time, which cannot be guaranteed. Well-contained recycling of such nanosized wastes [49,51,52] may in this context be considered a safer-by-design option. When nanomaterials are suspended in wastewater, it should be noted that standard urban wastewater treatment is not well adapted to prevent the release of nanomaterials [23,53,54]. The fate of specific nanomaterials in urban wastewater treatment plants may vary [53]. However, nanomaterials are likely to be largely adsorbed by sludge, whereas up to 10% of engineered nanosized inorganic and carbonaceous materials entering aerobic wastewater treatment and up to 30% of engineered inorganic and carbonaceous nanomaterials entering anaerobic wastewater treatment may be released to surface water [54]. Sludges are often subject to land surface spreading that corresponds with emission to soil, or disposal by landfill, which cannot be guaranteed to allow for indefinite containment of nanomaterials. Settable biomass, sludge and solid wastes containing nanomaterials may also be incinerated [50], which, in the case of nanomaterials containing metals might give rise to particulate residues, which cannot be guaranteed to be indefinitely contained [2,55]. Exposure to such residues containing metals may lead to an elevated generation of reactive oxygen species [56]. Removal of nanomaterials from wastewater flows at the source by suitable filtration technology seems like a necessary step in reducing releases of nanomaterials and hazardous substances derived thereof to the environment [57,58].

10.4 Safer-by-design hazard reduction of engineered inorganic and carbonaceous nanomaterials for organisms Size, structure, shape (e.g., sheet, fiber, tube, sphere, cube, and sharp edges), composition, crystallinity, surface characteristics (e.g., area, impurities, charge, defects, and presence of coating), aggregation state, persistence, and the corona of adsorbed biogenic molecules are determinants of inorganic and carbonaceous nanomaterial hazard [7,10,27,46,57,59 64]. The nanomaterial, dissolved substances originating in nanomaterials (e.g., Pb ions leached from PbS nanocrystals) and biogenic substances present in coronas such as endotoxins and allergens may be hazardous [30,65 69]. Also nanomaterials may adsorb hazardous synthetic organic compounds and inorganic substances and may transport them into organisms [70 75]. Endpoints of toxicological studies used in safe(r)-by-design strategies are often generation of reactive oxygen species/oxidative stress (that may lead to inflammation and genotoxicity), immune responses, inflammation, cytotoxicity, viability and hatching of zebrafish embryos, [76 77]. Usually acute effects following single large exposures are considered, whereas in

3. Prevention of nanotoxicity

10.4 Safer-by-design hazard reduction of engineered inorganic and carbonaceous nanomaterials for organisms

219

the real world chronic low exposures are more relevant [77]. The cell-based tests used in toxicological studies may vary in their predictive value depending on the nature of mechanisms underlying toxicity. For instance, regarding the inflammatory response to metal oxide nanoparticles, cell-based tests did relatively well as to the prediction of negative effects on mammals when the underlying mechanism is based on dissolved metal ions, but cell-based tests did poorly regarding the prediction of negative effects on mammals when the underlying mechanism is based on surface reactions [78]. Nanomaterial hazards may be specific for applications. This is illustrated by the following two examples: • Nanosized Ag may be applied in textiles (for antibacterial impact) or in electronics (e.g., for wiring and contacts). Ag ions released from Ag nanoparticles in the use stage of textiles may negatively impact benign bacteria on the human skin [33] and may lead to the emergence of microbial resistance to silver ions [79,80]. The negative impact on benign skin bacteria can be hazardous as benign skin bacteria may, for example, protect against wound infections [81]. Microbial resistance to silver may be hazardous because such resistance may reduce the benefit of applying nanosized Ag in wound dressings. Such hazards are not linked to nanosilver in the use stage of electronics. Conversely, electronic devices might contain Ag nanowires which may be released after the use stage (e.g., due to shredding). Ag nanowires .20 µm may give rise to a relatively prolonged inflammatory response of murine macrophages [82]). This is a kind of hazard that would not apply to Ag nanoparticles used in textiles for antibacterial impact. • A further example regards the application of photocatalytic nano-TiO2 in paints. A rationale for this application of TiO2 nanoparticles is their potential for reducing concentrations of airborne pollutants such as volatile organic compounds and nitrogen oxides. Truffier-Boutry et al. [31] and Gandolfo et al. [83] found that the photocatalytic effect of TiO2 nanoparticles caused degradation of the paint matrix and an unexpectedly high release of hazardous volatile organic compounds such as formaldehyde, originating in paint degradation. When, for example, applying photocatalytic nano-TiO2 in ceramic products this type of hazard would not occur. In view thereof safer-by-design should aim at reducing hazard taking account of nanomaterial applications.

10.4.1 Evaluating safer-by-design regarding inorganic and carbonaceous nanoparticles Several safe(r)-by-design studies have focused on Fe doping of CuO or ZnO nanoparticles [84 86]. The rationale behind this is that Fe doping should decrease the dissolution of metal ions (of Cu or Zn) that are held to be responsible for cytotoxicity [27,60]. A decrease of metal ion dissolution by Fe doping of ZnO and CuO nanoparticles in several media was shown in the studies of George et al. [86], Xia et al. [85], and Naatz et al. [84]. A reduction of negative effects by doping metal oxide nanoparticles with Fe was found in several tests. Cytotoxicity studies for Fe-doped ZnO nanoparticles with a duration of 3 h

3. Prevention of nanotoxicity

220

10. Safer-by-design for nanomaterials

were performed by George et al. [86]. Xia et al. [85] tested Fe-doped ZnO nanoparticles up to 30 days in rodent lungs and 5 days on hatching zebrafish embryos. Naatz et al. [84] used a 3-day test for Fe-doped and nondoped CuO nanoparticles involving the inhibition of hatching by zebrafish embryos. Cytotoxicity tests for Fe-doped CuO nanoparticles of 24 h were performed by Naatz et al. [84]. George et al. [86] concluded that Fe doping of ZnO nanoparticles “improves nanosafety.” Xia et al. [85] stated that Fe doping of ZnO nanoparticles is a “possible safe design strategy for preventing toxicity in animals and the environment.” Naatz et al. [84] concluded that their study “demonstrated the safe use of Fe-doped CuO nanoparticles in the environment.” Do the studies of George et al. [86], Xia et al. [85], and Naatz et al. [84] show that Fedoped CuO and ZnO particles are safe, or safer than nondoped CuO and ZnO nanoparticles? The rationale behind Fe doping is, as noted before, that negative impacts are determined by the dissolution of metal ions. But is this correct? First, Adeleye et al. [87] tested Fe-doped CuO nanoparticles generated in a way similar to those studied by Naatz et al. [84] and found an increased dissolution of Cu ions from Fe-doped CuO in natural waters, if compared with nondoped CuO. It has been suggested that Cu ions are relatively toxic to selected crustaceans, algae, and fish present in natural waters [88]. Second, there is evidence that dissolved metal ions are not the only cause of negative impacts of ZnO and CuO nanoparticles on cells [89,90,91,92]. For instance, Fairbairn et al. [93] tested the toxicity of ZnO and Fe-doped ZnO nanoparticles to sea urchin development. They concluded that Fe doping reduced the dissolution of Zn ions but did not reduce toxicity and suggested that this was due to surface reactions of Fe-doped ZnO nanoparticles. Bai et al. [94] reported that both (surfaces of) ZnO nanoparticles and dissolved Zn ions contributed to negative impacts in the zebrafish embryo hatching test used by Xia et al. [85]. There may be mechanical damage to membranes and intracellular structures caused by, and inflammatory responses to, (surfaces of) CuO and ZnO nanoparticles and it is likely that both dissolved ions and (surface reactions of) nanoparticles contribute to the generation of reactive oxygen species [90]. Reduced metal ion dissolution might furthermore lead to metal oxide nanoparticles becoming more persistent in cells. This might, for example, in the longer run lead to a larger negative impact of Fe-doped CuO and ZnO nanoparticles on cells, if compared with nondoped nanoparticles [90,95,96]. Such an effect would not have been picked up by the relatively short-term tests in the papers of George et al. [86] and Naatz et al. [84]. Mendes et al. [97] stressed the importance of long-term testing for adequately predicting the effects of modified CuO nanomaterials on organisms. Also, both CuO and ZnO nanoparticles show photocatalytic activity under solar irradiation [98 100]. This may lead to the degradation of biogenic materials which in turn may negatively affect organisms [95,101]. There are also reports that photocatalytic activity of CuO and ZnO nanoparticles may be enhanced by Fe doping [99,100,102], which in turn might increase negative impacts of ZnO and CuO nanoparticles. As to the cytotoxicity tests used by George et al. [86] and Naatz et al. [84], it may be noted that Fe doping may change nanoparticle surface characteristics, which in the real world might lead to differences in the corona of adsorbed biogenic molecules between Fe-doped and nondoped nanoparticles [103]. Differences in corona in might lead to differences in real-world cytotoxicity and inflammatory and immune responses [103,104], which may not be picked up by the cytotoxicity tests used by George et al. [86] and

3. Prevention of nanotoxicity

10.4 Safer-by-design hazard reduction of engineered inorganic and carbonaceous nanomaterials for organisms

221

Naatz et al. [84]. The rodent lung- and zebrafish embryo-based toxicity tests used by Xia et al. [85] and Naatz et al. [84] are furthermore not validated as fully predictive for effects that might occur during human exposure [77,78,105]. All in all, it would appear that the studies of George et al. [86], Xia et al. [85], and Naatz et al. [84] do not provide strong support for claims that doping of CuO and ZnO by Fe makes CuO and ZnO nanoparticles safe or safer. Another example of safe(r)-by-design studies regards the coating of ZnO and TiO2 nanoparticles that are applied in sunscreens. As noted before, ZnO nanoparticles are photocatalytic, and so are TiO2 nanoparticles [101], which makes their application in sunscreens at least remarkable. A commonly applied safer-by-design strategy aims at reducing the generation of reactive oxygen species (or radicals) on exposure to sunlight by silica or alumina coatings (combined with silicones) [101,106 109]. However, this reduction is limited or may in practice even be absent [109 113]. As a possible explanation for the disappointing reduction in the generation of reactive oxygen species by silica-coating of TiO2 nanoparticles, it has been suggested that silica may inhibit the recombination of radicals and may increase photocatalytic activity [111]. There are also other safety-related matters to consider. First, leaching of Zn from ZnO nanoparticles considered in the studies of George et al. [86] and Xia et al. [85] has not been explicitly considered as the focus of safer-by-design coatings has been on reducing the generation of reactive oxygen species linked to solar irradiation. Second, Rowenczyk et al. [108] noted that aging of sunscreen emulsions with TiO2 nanoparticles coated with silica or alumina (combined with silicones) favored the development of potential pathogenic bacteria. Furthermore, it has been suggested that alumina coatings of TiO2 nanoparticles might degrade when released from the human skin while swimming and that TiO2 nanoparticles with degraded coatings might subsequently enter the digestive tract of water organisms [114]. Also the coating of ZnO and TiO2 nanoparticles with the antioxidant lignin has been advocated for the application in sunscreens [115]. One might, however, expect that lignin coatings of nanoparticles can degrade. Photocatalytically enhanced lignin degradation on ZnO and TiO2 nanoparticles has been reported [116], and so has the degradation of lignin after releasing it to the environment [117]. The studies discussed here suggest that the hazards of ZnO and TiO2-based-sunscreens for consumers specifically linked to the generation of reactive oxygen species may be reduced by silica, alumina, and lignin coatings. On the other hand, limitations to safer-bydesign by coating TiO2 and ZnO would seem to provide a strong case to search for (nanosized) ingredients of sunscreens that are not photocatalytically active (see Section 10.4.2). The term safer-by-design suggests that the design effort regards the overall hazard of engineered nanomaterials. However, the two previous evaluations suggest that the design effort rather regards an aspect of the overall hazard or a specific hazard (respectively linked to dissolved metal ions and the photocatalytic generation of reactive oxygen species). A specific hazard is also central to the safer-by-design strategy for a class of rigid long-fiber ( . 4 10 µm) multiwalled carbon nanotubes (also called: high aspect-ratio multiwalled carbon nanotubes). This class of carbon nanotubes resembles fibrous asbestos and there are strong indications that in mammals these nanotubes may cause mesothelioma in a way similar to fibrous asbestos [78,118 120]. The presence of transition metals used in the synthesis of multiwalled carbon nanotubes and poor degradability of long rigid

3. Prevention of nanotoxicity

222

10. Safer-by-design for nanomaterials

multiwalled nanotubes is thought to contribute to this hazard [121 123]. As such, a number of safer-by-design options for reducing mesothelioma hazard for mammals have been suggested. A first option is strongly reducing fiber length and transition metal contamination of carbon nanotubes originating in catalyst use [121]. As to the latter, for example, the use of fullerenes as a catalyst for the synthesis of multiwalled carbon nanotubes [124] might be considered. Also the switch to flexible carbon nanotubes that are better degradable [123,125] has been recommended to reduce this specific hazard. It would seem that safer-by-design strategies targeting aspects of the overall nanomaterial hazard or specific hazards are common in the scientific literature.

10.4.2 Safer-by-design strategies to reduce (aspects of) nanomaterial hazards A variety of strategies that may reduce (aspects of) nanomaterial hazards have been suggested. These are the following, illustrated by examples of their application. 1. Coating Coating inorganic nanoparticles with silica and polymers can reduce nonspecific interactions with biomolecules and improve aqueous stability, which may improve the performance of nanopharmaceuticals [7,10]. Coating of rare earth-oxide nanoparticles with phosphonates or rare earth phosphates has been suggested as a safer-by-design technology to reduce damage caused by phosphate stripping of cellular components [126 128]. Coating with phosphonates has also been suggested as a safer-by-design strategy for metal oxides because such a coating can reduce the dissolution of metal ions and the generation of reactive oxygen species at the nanomaterial surface [94]. Coating multiwalled carbon nanotubes with Pluronic F 108 (a nonionic triblock copolymer) may reduce the lung fibrosis hazard of such nanotubes [129]. 2. Control of size Regarding hazards linked to human skin permeation of inorganic and carbonaceous nanoparticles, the size of nanoparticles is important. Guidance for control of size to minimize penetration of the human skin may be based on a paper by Filon et al. [130]. Filon et al. [130] reviewed the available scientific literature and concluded that nanoparticles with a diameter ,20 nm can permeate both the intact and damaged skin, that nanoparticles with a diameter between 20 and 45 nm can only permeate the damaged skin and that nanoparticles with a diameter .45 nm cannot permeate the skin. Furthermore, small graphene sheets have been suggested as a safer-by-design option for reducing graphene hazard by more efficient removal of deposited graphene and reduced shielding of cells [57,131]. Relatively short lengths have been proposed to reduce the fibrinogenic effect of Ag nanowires [82]. Increased size of nanosilica is linked to a reduction of surface area per unit of mass. Reducing the surface area has been proposed as a safe(r)-by-design strategy for amorphous silica nanoparticles in products that may be ingested [132]. In this way the perturbation of cholesterol homeostasis might be minimized [132]. Increasing the size of amorphous nanosilica would be in line with the design strategy proposed by Chatterjee et al. [132]. Hazards linked to the dissolution of substances from inorganic nanomaterials may be reduced when nanoparticulate size increases [2].

3. Prevention of nanotoxicity

10.4 Safer-by-design hazard reduction of engineered inorganic and carbonaceous nanomaterials for organisms

223

3. Doping Doping may change the energy bandgap (the difference in energy between the valence band and the conduction band) of nanomaterials and thereby reduce the generation of reactive oxygen species and oxidative stress [7]. For instance, Fe doping of TiO2 nanoparticles has been suggested by Ghiazza et al. [133] to change the bandgap in order to reduce the generation of reactive oxygen species under solar irradiation. However, it should be noted that the impact on the formation of reactive oxygen species under solar illumination is dependent on Fe concentration and synthetic routes. For instance, George et al. [134] have shown that Fe doping of titania nanoparticles may lead to the opposite effect under solar irradiation: an increase in the generation of reactive oxygen species. Fe and Ti doping of amorphous nanosilica have been suggested as safer-by-design strategies to reduce inhalation hazards [16,17]. 4. Grafting Grafting regards the covalent binding of nanomaterials to other substances. One example thereof is the grafting of TiO2 nanoparticles to larger TiO2 particles, proposed by Truffier-Boutry et al. [31] for safer-by-design paints. Grafting small organic molecules to carbon nanotubes may reduce cytotoxicity and persistence of nanotubes in cells [135]. 5. Loading Loading regards the noncovalent binding of molecules to nanomaterials. Loading nanomaterials with organic molecules can be conducive to improved performance in drug delivery, medical imaging, and tissue repair and regeneration [7]. 6. Managing shape and crystallinity Bottero et al. [23] compared different shapes of rutile and anatase, varieties of photocatalytic nano-TiO2, and concluded that small compact cubes of anatase TiO2 were the best compromise to minimize negative impacts by reactive oxygen species while retaining photocatalytic activity. 7. Reducing the presence of substances at the nanomaterial surface that contribute to hazard Sun et al. [16] suggested reduction of silanol display at the surface of silica nanomaterials to reduce acute inflammation of the lung. Fe doping and calcination were used for this purpose. 8. Reduction of persistence Chemical modification of graphene compounds to enhance degradability has been suggested as a safer-by-design option [57]. 9. Substitution Substitution regards the functional replacement of specific nanomaterials by other substances. The latter may be other nanomaterials. For instance, Tarantini et al. [136] investigated quantum dots based on InZnS or InZnPS nanoparticles, whether or not capped with a shell of ZnSeS and coated with glutathione, as a potentially safer alternative for CdSe quantum dots. Cytotoxicity was tested for 24 h using primary human keratinocytes. Pristine nanoparticles with shells did show the lowest cytotoxicity (much lower than CdSe quantum dots) and no significant oxidative stress [136]. However, on aging cytotoxicity of capped nanoparticles increased, showing the need for a more robust shell [136]. As noted in Section 10.4.1, coating of TiO2 and ZnO nanoparticles applied in sunscreens tends to provide only a limited reduction of

3. Prevention of nanotoxicity

224

10. Safer-by-design for nanomaterials

hazard. In view thereof Hayden et al. [137] have suggested a potentially safer substitute: transparent nanoparticles composed of ethyl cellulose and zein (a cornderivative consisting mainly of protein) with for example encapsulated quercetin or retinol. These nanoparticles absorb ultraviolet (UV) radiation [137]. The stability of the nanoparticles proposed by Hayden et al. [137] in commercial sunscreens merits further investigation. Zhang et al. [138] have suggested SiO2, HfO, and Gd2O3 as potentially safer substitutes for CoO, Cr2O3, and MnO2 nanoparticles.

10.5 Reducing releases to the environment of nanomaterials from relatively large nanocomposites and products As pointed out in Section 10.1 reducing releases to the environment is an important aspect of safer-by-design. Section 10.5.1 will deal with releases from nanocomposites and Section 10.5.2 deals with releases from products such as batteries and electronics with nanomaterials as components.

10.5.1 Safer-by-design for relatively large nanocomposites: reduction of nanomaterial releases In this section the application of nanoparticles linked to relatively large nanocomposites will be considered. Section 10.5.1.1 briefly outlines the application of nanomaterials in relatively large nanocomposites. Section 10.5.1.2 briefly discusses functional releases originating in nanomaterials and unintended releases of nanomaterials from relatively large nano-enabled materials. Releases of nanomaterials from relatively large nanocomposites during the use stage and strategies to reduce those releases are discussed in Section 10.5.1.3. Section 10.5.1.4 deals with releases from relatively large nano-enabled materials after the use stage. 10.5.1.1 Nanomaterials in relatively large nanocomposites In relatively large nano-enabled materials engineered inorganic and carbonaceous nanomaterials are linked to other materials. Examples are a variety of paints, stains and sealants [139,83,140,141], nanoparticle-coated textiles [19], nanocomposites of nanoparticles and polymers [34,142], nanocomposite ceramics and cement-based products [42,84,143], and nanoparticle-coated glass [42]. 10.5.1.2 Functional releases from and unintended releases of nanomaterials from relatively large nanocomposites In some cases the release of constituents of nanomaterials from relatively nanocomposites is functional; a case in point is textile coated with Ag nanoparticles. The function of such coatings is their antibacterial effect, associated with the release of Ag ions. There is also the potential for unintended effects of nano-enabled materials relevant to nanomaterial release that may merit consideration. For instance, the use of nanoparticles for conferring reinforcement to polymers may increase the release of nanoparticulate material, if compared with the neat material, in the case of mechanical treatments such as

3. Prevention of nanotoxicity

10.5 Reducing releases to the environment of nanomaterials from relatively large nanocomposites and products

225

drilling [142]. Also, photocatalytically active TiO2 nanoparticles generate nitrous acid on converting NOx [23] and this might reduce the fixation of TiO2 nanoparticles (applied to reduce air pollution) in cement matrixes. 10.5.1.3 Releases from relatively large nanocomposites during their use stage and strategies to reduce those releases In this section the release of nanomaterials and/or its constituents from relatively large nano-enabled materials during their use stage (including the reuse stage) is considered First, release mechanisms are discussed for textiles coated with ZnO and CuO nanoparticles. Thereafter strategies to reduce releases from nanocomposites will be outlined. Mantecca et al. [19] proposed metal oxide (ZnO and CuO) nanoparticle-coated textiles (using an ethanol-based coating process) as a ‘safe’ alternative to a coating with Ag nanoparticles. The intended functionality of the nanoparticle, as studied by Mantecca et al. [19], is its antibacterial effect. Mantecca et al. [19] performed abrasion tests on nanoparticle-coated textile (nano-textile) and found the release of safe amounts of nanoparticles to air. However, release of nanomaterials by abrasion is only one of the processes leading to (potential) releases of nanomaterials and/or constituents thereof during the use stage that should be considered. Another process than abrasion conducive to release of nanoparticles from textiles is washing [144,145]. Furthermore, an increased release of nanoparticles might occur on aging of the coated textile, for example, due to exposure to UV radiation [145,146]. The latter is all the more important as the preparation of the nanoparticles studied by Mantecca et al. [19] suggests that the nanoparticles used for coating are likely to be photocatalytically active when exposed to UV radiation, which in turn may lead to accelerated aging of textiles on exposure to solar radiation. Also, the absorption of UV radiation by ZnO and CuO nanoparticles might increase the temperature of synthetics such as polyamides, which may lead to warming that can be conducive to accelerated aging [147]. Dissolution of Zn an Cu ions from the metal oxides, central to the studies of George et al. [86], Xia et al. [85], and Naatz et al. [84], discussed in Section 10.4.1, was not considered by Mantecca et al. [19] whereas contact with sweat and water may be conducive to such dissolution. So, there are several ways in which nanomaterials or constituents thereof may be released during the use stage of textiles coated with CuO or ZnO nanomaterials that were not considered by Mantecca et al. [19]. Several strategies conducive to enhanced integrity and durability of nanocomposites in their use stage have been proposed. Enhanced integrity is linked to reduced releases of nanomaterials. Durability can extend the time that nanomaterials remain in use, thereby indirectly reducing nanomaterial releases linked to the production of (new) nanomaterials. • Ko¨hler and Som [148] suggested a number of factors that may be conducive to low releases of nanoparticles from nano-textiles during their use stage. These include: a polar nanoparticle surface, covalent links between nanoparticle and textile and embedding nanoparticles in abrasion-resistant fibers. One might view this as a strategy aiming at enhancing the integrity of a nanocomposite. • Weathering and abrasion of nano-enabled paints and coatings containing nanoparticulate SiO2, and TiO2 leading to the release of nanoparticulate material have been studied by Kaegi et al. [149], Al-Kattan et al. [150,151], and Zhang et al. [152]. Such

3. Prevention of nanotoxicity

226

10. Safer-by-design for nanomaterials

releases are nonfunctional and may be reduced. For instance, Truffier-Boutry et al. [31] suggested a safer-by-design strategy for photocatalytic paints with TiO2 nanoparticles aiming at enhancing paint integrity and durability, including the use of binders more resistant to photocatalytic degradation and better dispersion of nanoparticles. Fiorentino et al. [139] studied ways to reduce the release of nano-SiO2 present in paints due to abrasion and weathering. They suggested that the addition of relatively large-sized TiO2 pigments and the substitution of acrylic copolymer binder by styrene acrylic copolymer binder could strongly decrease the release of nano-SiO2. • Specific kinds of nanocomposite production and composition may be selected to reduce the release of nanomaterials from nanocomposites during their use stage. For instance, to enhance integrity and durability, Reijnders [32,33] suggested the use of ceramic or glass supports and high temperature fixation of photocatalytically active TiO2 nanoparticles in environmental technology, instead of low temperature fixation and the use of supports made from steel or organic polymers. • Bottero et al. [23] investigated leaching of photocatalytic nano-TiO2 from cement products. They found that degradation and aging significantly increased leaching of nanomaterial by impacting pore networks connected to the cement surface, and suggested that nanoparticle leaching can be reduced by improved design of pore networks. • Several authors have suggested exploiting the retro Diels Alder reaction to restore the integrity and enhance the durability nanocomposites. The retro Diels Alder reaction gives these nanocomposites self-healing properties that can be exploited by infrared irradiation generating a temperature of about 130 C in the nanocomposite. Engel and Kickelbick [153] and Scha¨fer and Kickelbick [154] studied this option for functionalized silica nanoparticles and methacrylates. Li et al. [155] investigated the retro Diels Alder reaction for a nanocomposite consisting of epoxy resin and amino-functionalized carbon nanotubes. Clark et al. [156], Cai et al. [157,158], and Pilate et al. [159] focused on the retro Diels Alder reaction to restore the integrity and enhance the durability of thermoset polymer-graphene nanocomposites. 10.5.1.4 Releases from relatively large nanocomposites to the environment after the use stage Preferentially safer-by-design strategies should target complete life cycles, including the fate of the nanocomposite after the use (including reuse) stage [48]. First the case of textiles will be considered. After the use stage nanocomposite textile materials may be recycled [160]. Recycling practices may give rise to the release of nanoparticles from nano-enabled textile materials to the environment [160]. Also discarded textiles may be incinerated. Inorganic nanoparticles may be persistent in waste incineration [2,161 163]. They can be efficiently removed from flue gas by best available flue gas treatment technology, but, if so, end up in waste incineration residues [161,162]. Such residues can include particulate materials collected from filtration units such as baghouses. When nanomaterials containing metals are incinerated, particulate materials collected from filtration units might on exposure of organisms give rise to elevated generation of reactive oxygen species [56]. Solid waste incineration residues may be landfilled or used, for instance in the production of building and geotechnical materials [55]. Such fates for waste incineration residues in turn may lead

3. Prevention of nanotoxicity

10.6 Reducing hazards of fragments released from nanocomposites

227

to releases of nanoparticles to the environment [161]. Alternatively, textiles with nanomaterials may be landfilled [164]. Materials used in textiles, such as polyester and cotton, may degrade under landfill conditions [165,166], which might in turn lead to the release and mobility of nanomaterials [167]. More in general, one may note that only limited research is available regarding the stability of nanocomposite products under landfill conditions. Releases of Ag, Cu, and Zn ions from respectively Ag, CuO, and ZnO nanoparticles are, however, likely to occur in landfills [168 170]. Landfills would moreover not guarantee the indefinite containment of nanomaterials. What holds for the incineration of nano-enabled textiles is also likely to hold for nanocomposite plastics. Recycling nanocomposite plastics may release more nanomaterials than recycling neat plastics. Size reduction by practices such as grinding used in nanocomposite plastic recycling have been found to generate relatively large releases of particles, especially after aging [45,171]. As recycling of nanocomposite cement products also implies size reduction, relatively large releases of nanomaterials from nano-enabled cement products may be also expected in this case [147]. Reducing releases of nanomaterials after the use stage does not appear to be a focus in safer-by-design strategies. Still, it would seem that containment of recycling activities and high-efficiency capture of materials released from nanocomposites in recycling plants could be conducive to reduced releases of nanomaterials to the environment.

10.5.2 Controlling releases from nanomaterials present as components in products Releases of nanomaterials from products such as electronics and batteries during normal use would seem unlikely, but releases may occur in the case of mishaps such as fires or faulty operations [48]. Releases from end-of-life products are a matter of concern. This regards both disposal (landfilling and incineration) and processing for the recovery of valuable materials [48]. The latter holds for informal recycling with primitive technology (such as open burning) and also for sophisticated recycling [48]. Unit processes applied in sophisticated recycling such as shredding and milling can lead to the release of nanosized materials to the environment [48]. Design for disassembly as an alternative to shredding might reduce the release of nanosized materials to the environment. Feeding end-of-life printed circuit boards or small electronic devices such as mobile phones containing nanosized components to smelters that can recover a wide range of relatively rare metals would seem preferable to feeding shredded end-of-life printed circuit boards or mobile phones to such smelters, as in shredding high percentages of rare earths, noble metals, and copper tend to be lost from recycling by ending up in the wrong shredder output [172 174].

10.6 Reducing hazards of fragments released from nanocomposites It has been argued that to the extent that engineered nanomaterials are released from nano-enabled materials, nanocomposites should be designed in such a way that released

3. Prevention of nanotoxicity

228

10. Safer-by-design for nanomaterials

nanocomposite fragments are less hazardous than the engineered nanomaterials used in nanocomposite production [34]. In practice, design of nanocomposites may well lead to releases of materials that exceed nanosize [34,175 177]. There is suggestive evidence that relatively large fragments released from nanocomposites may be less hazardous than engineered nanoparticles. Amorim et al. [177] studied fragments released from nano-enabled cement and three types of nanocomposites with organic polymers in a variety of tests including sewage treatment, zebrafish embryo hatching, trout cell lines and soil worms, and found toxicities that were not different from the matrix material (cement or organic polymer). A similar result was found for fragments released from nanocomposite thermoplastics and cement as tested by in vivo installation in rats [178]. There is also evidence that when TiO2 nanoparticles embedded in paint matrix are released, the hazard thereof following rodent lung exposure is reduced if compared with pristine TiO2 nanoparticles as tested for toxicity in the mouse lung [179,180] and for systemic blood toxicity after inhalation by mice [180]. However, the fate of relatively large fragments of nanocomposites released to the environment can give rise to increased hazards. Fragments released from nanocomposites with organic polymers might adsorb hazardous substances when released to the environment [74,181] and may be further degraded in the environment to smaller sized materials [182,183], including the engineered nanoparticles used in nanocomposite production and/or constituents thereof. Relatively large fragments released from vehicle catalytic converters containing nanosized platinum group elements degrade in the environment and give rise to the release of nanomaterials [184].

10.7 Conclusions Safer-by-design strategies for engineered inorganic and carbonaceous nanomaterials mainly aim at reducing hazard (potential to harm organisms) and the release of nanomaterials to the environment. In practice, the focus as to hazard is often on aspects of the overall hazard or specific hazards. Important constraints for safer-by-design are limited toxicological knowledge, differences between tested nanomaterials and the nanomaterials that organisms or cells are exposed to in the real world, and trade-offs between functionality and safety. Proposed strategies to reduce (aspects of) hazard for nanomaterials include: coating, control of size, doping, grafting, loading, managing shape and crystallinity, reducing the presence of substances at the surface of nanomaterials that contribute to hazard, reduced persistence and substitution. Isolation of nanomaterials production and processing, in situ synthesis of nanomaterials, enhanced integrity and durability of nanocomposites, design for disassembly of products and efficient recycling of materials may contribute to reducing nanomaterial releases. In designing nanocomposites one might aim at the release in the use stage of fragments to the environment that are less hazardous than nanomaterials. However, such fragments released from nanocomposites can become increasingly hazardous once in the environment.

References [1] Y. Yang, K. Doudrick, X. Bi, K. Hristovski, P. Herckes, P. Westerhoff, et al., Characterization of food-grade titanium dioxide: the presence of nanosized particles, Environ. Sci. Technol. 48 (2014) 6391 6400.

3. Prevention of nanotoxicity

References

229

[2] L. Reijnders, Safe recycling of materials containing persistent inorganic and carbon nanoparticles, in: J. Njuguna, K. Pielichowski (Eds.), Health and Environmental Safety of Nanomaterials: Polymer Nanocomposites and Other Materials Containing Nanoparticles. Composites Science & Engineering No. 49, Woodhead Publishing, Cambridge, 2014, pp. 222 250. Chapter 11. [3] M. Dorier, D. Beal, C. Tisseyre, C. Narie-Desvergne, M. Dubosson, F. Barreau, et al., The food additive E171 and titanium dioxide nanoparticles indirectly alter the homeostatsis of human intestinal epithelial cells in vitro, Environ. Sci. Nano 6 (2019) 1549 1561. [4] R.S. Lankone, K. Challis, L. Pourzahedi, D.F. Durkin, Y. Bi, Y. Wang, et al., Copper release and transformation following natural weathering of nano-enabled pressure treated lumber, Sci. Total Environ. 668 (2019) 234 244. [5] A. Maynard, R.J. Aitken, T. Butz, V. Colvin, K. Donaldson, G. Oberdo¨rster, et al., Safe handling of nanotechnology, Nature 444 (2006) 267 269. [6] A. Maynard, R. Aitken, ‘Safe handling of nanotechnology’ ten years on, Nat. Nanotechnol. 11 (2016) 998 1000. [7] L. Yan, F. Zhao, J. Wang, Y. Zu, Z. Gu, Y. Zhao, A safe-by-design strategy towards safer nanomaterials in nanomedicines, Adv. Mater. (2019) 1805391 (33 pp.). [8] R. Hjorth, L. van Hove, F. Wickson, What can nanosafety learn from drug development? The feasibility of ‘safety by design’, Nanotoxicology 11 (2017) 2305 2312. [9] A. Kraegeloh, B. Suarez-Merino, T. Sluijters, C. Micheletti, Implementation of safe-by-design for nanomaterial development and safe innovation: why we need a comprehensive approach, Nanomaterials 8 (4) (2018) 239 (12 pp.). [10] S. Siegrist, E. Co¨rek, P. Detampel, J. Sandstro¨m, P. Wick, J. Huwyler, Preclinical hazard evaluation strategy for nanomedicines, Nanotoxicology 13 (2018) 73 99. Available from: https://doi.org/10.80/17435390. 2018.1505000 (27 pp.). [11] JECFA, Thirtieth report of the joint FAO/WHO expert committee on food additives. FAO Nutrition Meetings Report Series/WHO Technical Report Series. PAS 70.36/NMRS 46A-JECFA 13/55 titanium dioxide (INS171). Geneva, 1969. [12] M.B. Heringa, L. Geraets, J.C.H. van Eijkeren, R.J. Vandebriel, W.H. de Jong, A.G. Oomen, Risk assessment of titanium dioxide nanoparticles via oral exposure, including toxicokinetic considerations, Nanotoxicology 10 (2016) 1515 1525. [13] C.M. Nogueira, W.M. de Azevedo, M.L.Z. Dagli, S.H. Toma, A.Z. de Arruda Leite, M.L. Lordello, et al., Titanium dioxide induced inflammation of the small intestine, World. J. Gastroenterol. 18 (2012) 4729 4735. [14] G. Pinget, J. Tan, B. Janac, N.O. Kaakoush, A.S. Angelatos, J.O. Sullivan, et al., Impact of food additive titanium dioxide (E 171) on gut microbiota-host interaction, Front. Nutr. 6 (2019) 57 (13 pp.). [15] B. Jovanovic, Critical review of public health regulation of titanium dioxide, a human food additive, Integr. Environ. Assess. Manag. 11 (2014) 10 20. [16] B. Sun, S. Pokhref, D.R. Dunphy, H. Zhang, Z. Ji, X. Wang, et al., Reduction of acute inflammatory effects of fumed silica nanoparticles in the lung by adjusting silanol display through calcination and metal doping, ACS Nano 9 (2015) 9357 9372. [17] B. Sun, X. Wang, Y. Liao, Z. Ji, C.H. Chang, S. Pokhrel, et al., Repetitive dosing of fumed silica leads to profibrinogenic effects through unique structure-activity relationships and biopersistence in the lung, ACS Nano 10 (2016) 8054 8066. [18] D. Sun, L. Gong, J. Xie, X. Gu, Y. Li, Q. Cao, et al., Toxicity of silicondioxide nanoparticles with varying sizes and protein corona as a strategy for therapy, Sci. Bull. 63 (2018) 907 916. [19] P. Mantecca, K. Kasemets, A. Deokar, I. Perelshtein, A. Gedanken, Y.K. Bakh, et al., Airborne nanoparticle release and toxicological risk from metal-oxide coated textiles: toward a multiscale safe-by-design approach, Environ. Sci. Technol. 51 (2017) 9305 9317. [20] K. Jantzen, P. Moller, D.G. Karottki, Y. Olsen, G. Beko¨, G. Xlausen, et al., Exposure to ultrafine particles, production of reactive oxygen species in leukocytes and altered levels of endothelial progenitor cells, Toxicology 359 360 (2016) 11 18. [21] N. Li, S. Georas, N. Alexis, P. Fritz, T. Xia, M.A. Williams, et al., A workgroup report on ultrafine particles. Why ambient ultrafine and engineered nanoparticles should receive special attention for possible adverse

3. Prevention of nanotoxicity

230

[22] [23]

[24] [25] [26] [27] [28]

[29] [30]

[31]

[32] [33] [34] [35] [36]

[37]

[38]

[39]

[40] [41] [42] [43]

10. Safer-by-design for nanomaterials

health outcomes in human subjects, J. Allergy Clin. Immunol. 138 (2016) 386 396. Available from: https:// doi.org/10.106/j.jaci.2016.02.023. T. Gonet, B.A. Maher, Airborne vehicle-derived Fe-bearing nanoparticles in the urban environment: a review, Environ. Sci. Technol. 53 (2019) 9970 9991. J.Y. Bottero, J. Rose, C. de Garidel, A. Masion, T. Deutsch, G. Brochard, et al., SERENADE: safer and ecodesign research and education applied to nanomaterial development, the new generation of materials safer by design, Environ. Sci. Nano 4 (2017) 526 538. M. Cobaleda-Siles, A.P. Guillamon, C. Delpivo, S. Va´zquez- Campos, V.F. Puntes, Safer by design strategies, J. Phys. Conf. Ser. 838 (2017) 012016 (9 pp.). C. Schwarz-Plaschg, A. Kallhof, I. Eisenberger, Making nanomaterials safer by design? Nanoethics 11 (2017) 277 281. R. Hwang, V. Mirshafiee, Y. Zhu, T. Xia, Current approaches for safer design of engineered nanomaterials, Ecotoxicol. Environ. Saf. 166 (2018) 294 300. S. Lin, T. Yu, X. Hu, D. Yin, Nanomaterials safer-by-design: an environmental safety perspective, Adv. Mater. 30 (2018) e1705691 (5 pp.). A.V. Singh, P. Laux, A. Luch, C. Sudrik, S. Wiehr, A. Wild, et al., Review of emerging concepts in nanotoxicology: opportunities and challenges for safer materials design, Toxicol. Mech. Methods 29 (5) (2019) 378 387. G.V. Lowry, K.B. Gregory, S.C. Apte, J.R. Lead, Transformations of nanomaterials in the environment, Environ. Sci. Technol. 46 (2012) 6893 6899. S. Wijnhoven, W. Peijnenburg, C. Herberts, W. Hagens, A. Oomen, E. Heugens, et al., Nano-silver a review of available data and knowledge gaps in human and environmental risk assessment, Nanotoxicology 3 (2009) 109 138. D. Truffier-Boutry, B. Fiorentino, V. Bartolomei, R. Souklas, O. Sicardi, A. Benayad, et al., Characterization of photocatalytic paints: a relationship between the photocatalytic properties- release of nanoparticles and volatile organic compound, Environ. Sci. Nano 4 (2017) 1998 2009. L. Reijnders, Hazard reduction for the application of titania nanoparticles in environmental technology, J. Hazard. Mater. 162 (2008) 440 445. L. Reijnders, Hazard reduction in nanotechnology, J. Ind. Ecol. 12 (3) (2008) 297 306. L. Reijnders, The release of TiO2 and SiO2 nanoparticles from nanocomposites, Polym. Degrad. Stabil. 94 (2009) 873 876. J. Jeevanandam, A. Barhoum, Y.S. Chan, A. Dufresne, M.K. Danquah, Review on nanoparticles and nanostructured materials: history, sources, toxicity and regulations, Beilstein J. Nannotechnol. 9 (2019) 1050 1074. V. Kosynkin, G. Cerotti, K.C. Wilson, J.R. Lomeda, J.T. Scorsone, A.D. Patel, et al., Graphene oxide as a highperformance fluid-loss-control additive in water based drilling fluids, ACS Appl. Mater. Interfaces 4 (2011) 222 227. A.R. Ismail, A. Aftab, Z.H. Ibupoto, N. Zolkifile, The novel approach for the enhancement of rheological properties of water-based drilling fluids by using multi-walled carbon nanotubes, nanosilica and glass beads, J. Pet. Sci. Eng. 139 (2016) 264 275. N.D. Kno¨fel, H. Rothfuss, J. Willenbacher, C. Barner-Kowollik, P. Roesky, Platinum (II)-crosslinked singlechain nanoparticles: an approach towards recyclable homogeneous catalysts, Angew. Chem. Int. Ed. 56 (2017) 4950 4954. N.J. Mohr, A. Meijer, M.A.J. Huibregts, L. Reijnders, Environmental life cycle assessment of roof integrated flexible amorphous silicon/nanocrystalline silicon solar cell laminate, Prog. Photovolt. Res. Appl. 21 (2013) 802 815. W. Wu, Inorganic nanomaterials for printed electronics, Nanoscale 9 (2017) 7342 7372. G. Pallas, M.G. Vijver, W.J.G.M. Peijnenburg, J. Guinee, Life cycle assessment of emerging technologies at the lab scale, J. Ind. Ecol. (2019). Available from: https://doi.org/10.1111/jie.12855 (12 pp.). P. Van Broekhuizen, F. van Broekhuizen, R. Cornelissen, L. Reijnders, Use of nanomaterials in the European construction industry and some occupational health aspects thereof, J. Nanopart. Res. 13 (2011) 447 462. A.A. Keller, S. McFerran, A. Lazareva, S. Suh, Global life cycle of engineered nanomaterials, J. Nanopart. Res. 15 (2013) 1692 (17 pp.).

3. Prevention of nanotoxicity

References

231

[44] Y. Ding, T.A.J. Kuhlbusch, M. van Tongeren, A.S. Jime´nez, I. Tuinman, R. Chen, et al., Airborne engineered nanomaterials in the workplace a review of release and worker exposure during nanomaterial production and handling processes, J. Hazard. Mater. 322 (2017) 17 28. [45] Gendre, L., A Study of Emission of Nanoparticles During Physical Processing of Aged Polymer-Matrix Nanocomposites (Ph.D. thesis), Cranfield University, 196 pp., 2016. [46] C. Geraci, D. Heidel, C. Sayes, L. Hodson, P. Schulte, A. Eastlake, et al., Perspectives on the design of safer nanomaterials and manufacturing processes, J. Nanopart. Res. 17 (2015) 366 (13 pp.). [47] H. Sun, Y. Pan, J. Guan, Y. Jiang, J. Yao, J. Jiang, et al., Thermal decomposition behaviors and dust explosion characteristics of nano-polystyrene, J. Therm. Anal. Calorim. 135 (2019) 2359 2366. [48] A. Ko¨hler, C. Som, A. Helland, F. Gottschalk, Studying the release of carbon nanotubes throughout the application life cycle, J. Clean. Prod. 16 (2008) 927 937. [49] Z. Zhuang, X. Xu, Y. Wang, Y. Wang, F. Huang, Z. Lin, Treatment of nanowaste via fast crystal growth: with recycling of nano-SnO2 from electroplating sludge as a study case, J. Hazard. Mater. 211 212 (2012) 414 419. [50] G.P. Nichols, Exploring the need for creating standardize approach to managing nanowaste based on similar experiences from other wastes, Environ. Sci. Nano 3 (2016) 946 952. [51] P. Pati, S.M. McGinnis, P.J. Vikesland, Waste not want not: life cycle implications of gold recovery and recycling from nanowaste, Environ. Sci. Nano 3 (2016) 1133 1143. [52] W. Liu, C. Weng, J. Zheng, X. Peng, J. Zhang, Z. Lin, Treatment and recycling of heavy metals from nanosludge, Environ. Sci. Nano 6 (2019) 1657 1673. [53] O. Suarez-Iglesias, S. Collado, P. Oulego, M. Diaz, Graphene-family nanomaterials in wastewater treatment plants, Chem. Eng. J. 313 (2017) 121 125. [54] P.K. Westerhoff, M.A. Kiser, K. Hristovski, Nanomaterial removal and transformation during biological wastewater treatment, Environ. Eng. Sci. 30 (2013) 109 117. [55] L. Reijnders, Disposal uses and treatments of combustion ashes- a review, Resour. Conserv. Recycl. 43 (2005) 313 336. [56] E.P. Vejerano, Y. Ma, A.L. Holder, A. Pruden, S. Elankumaran, L.C. Marr, Toxicity particulate matter from incineration nanowaste, Environ. Sci. Nano 2 (2015) 143 154. [57] M.V.D.Z. Park, E.A.J. Bleker, W. Brand, F.R. Cassee, M. van Elk, I. Gosens, et al., Considerations for safe innovation: the case of graphene, ACS Nano 11 (2017) 9574 9593. [58] H. Lee, S. Kang, S.C. Kim, D.Y.H. Pui, Deposition and reentrainment of colloidal particles in disordered fibrous filters under chemically and physically unfavorable conditions, J. Membr. Sci. 582 (2019) 322 334. [59] D.M. Brown, H. Johnston, E. Gubbins, V. Stone, Serum enhanced cytokine responses of macrophages to silica and iron oxide particles and nanomaterials: a comparison of serum to lung fluid and albumin dispersions, J. Appl. Toxicol. 34 (2014) 1177 1187. [60] H.L. Karlsson, P. Cronholm, Y. Hedberg, M. Tornberg, L. De Battice, S. Svedhem, et al., Cell membrane damage and protein interaction induced by copper containing nanoparticles importance of metal release process, Toxicology 313 (2013) 59 69. [61] A.E. Nel, W.J. Parak, W.C.W. Chan, T. Xia, M.C. Hersam, C.J. Brinker, et al., Where are we heading in nanotechnology environmental health and safety and materials characterization, ACS Nano 9 (2015) 5627 5630. [62] C.C. Le, H. Yin, R. Chen, L. Zhao, P.S. Casey, C. Chen, et al., An experimental and computational approach to the development of ZnO particles that are safe by design, Small 12 (2016) 3568 3577. [63] S. Halappavavar, U. Vogel, H. Wallin, C.L. Yauk, Promise and peril in nanomedicine: the needs and challenges for integrated systems biology approaches to define health risk, WIREs Nanomed. Nanotechnol. 10 (2018) e1465 (7 pp.). [64] S. Zhu, L. Gong, Y. Li, H. Xu, Z. Gu, Y. Zhao, Safety assessment of nanomaterials to eyes: an important but neglected issue, Adv. Sci. 6 (2019) 1802289 (16 pp.). [65] D. Boraschi, S.M. Moghimi, A. Duschl, Interaction between the immune system and nanomaterials: safety and medical exploitation, Curr. Bionanotechnol. 2 (2016) 3 5. [66] E. Oh, R. Liu, A. Nel, K.B. Gemill, M. Bilal, Y. Chen, et al., Meta-analysis of cellular toxicity for cadmiumcontaining quantum dots, Nat. Nanotechnol. 11 (2016) 479 486. [67] S. Smulders, J. Kaiser, S. Zuin, K.L. van Landuyt, L. Golanski, J. Vanoirbeek, et al., Contamination of nanoparticles by endotoxin: evaluation of different test methods, Part. Fibre Toxicol. 9 (2012) 41 (11 pp.).

3. Prevention of nanotoxicity

232

10. Safer-by-design for nanomaterials

[68] Y. Chang, K. li, Y. Feng, N. Liu, Y. Chen, X. Sun, et al., Crystallographic facet-dependent stress responses by polyhedral lead sufide nanocrystals and potential safe-by-design approach, Nano Res. 9 (2016) 3812 3827. [69] M. Westerka, K. Dziendzikowska, J. Gromadzka-Ostrowska, J. Dudek, H. Polkowska-Motrenko, J.N. Audinot, et al., Silver ions are responsible for memory impairment induced by oral administration of silver nanoparticles, Toxicol. Lett. 290 (2018) 133 144. [70] T. Tuutija¨rvi, J. Lu, G. Chee, As(V) adsorption on maghemite nanoparticles, J. Hazard. Mater. 166 (2009) 1415 1420. [71] G.K. Ramesha, A.V. Kumara, H.B. Muralidhara, S. Sampath, Graphene and graphene oxide as effective adsorbents toward anionic and cationic dyes, J. Colloid Interface Sci. 361 (2011) 270 277. [72] Y. Tu, C. You, C. Chang, Kinetics and thermodynamics of adsorption for Cd on green manufactured nanoparticles, J. Hazard. Mater. 235 236 (2012) 116 122. [73] C.J.A.F. Kwadijk, J. Velzeboer, A.A. Koelmans, Sorption of perfluorooctane sulfonate to carbon nanotubes in aquatic sediment, Chemosphere 90 (2013) 1631 1636. [74] I. Velzeboer, C.,J.A.F. Kwadijk, A.A. Koelmans, Strong sorption of PCBs to nanoplastics, microplastics, carbon nanotubes and fullerenes, Environ. Sci. Technol. 48 (2014) 4869 4876. [75] Q. Zhao, Y. Li, X. Chai, L. Zhang, L. Xu, J. Huang, et al., Interaction of nanocarbon particles and anthracene with pulmonary surfactant: the potential hazards of inhaled nanoparticles, Chermosphere 215 (2019) 746 751. [76] J.A. Shatkin, K.J. Ong, C. Beaudric, A.J. Clippinger, C.O. Hendren, L.T. Haber, et al., Advancing risk analysis for nanoscale materials: report from an international workshop on the role of alternative testing strategies for advancement, Risk Anal. 36 (2016) 1520 1537. [77] J.A. Shatkin, K.J. Ong, Alternative testing strategies for nanomaterials: state of the science and considerations for risk analysis, Risk Anal. 36 (2016) 1564 1579. [78] W. Cho, R. Duffin, M. Bradley, I.L. Megson, W. MacNee, J.K. Lee, Y. Jeong, K. Donaldson, Predictive value of in vitro assays depends on the mechanism of toxicity of metal oxide nanoparticles, Particle Fibre Toxicol. 10 (2013) 55 (15pp). [79] S. Silver, L.T. Phung, G. Silver, Silver as biocides in burn and wound dressings and bacterial resistance to silver compounds, J. Ind. Microbiol. Biotechnol. 33 (2006) 627 634. [80] A. Pana´cek, L. Kvitek, M. Sme´kalova, R. Vecerova´, M. Kola´r, M. Roderova, et al., Bacterial resistance to silver nanoparticles and how to overcome it, Nat. Nanotechnol. 13 (2018) 65 71. [81] A. Han, J.M. Zenilman, J.H. Melendez, M.E. Shirtliff, A. Agostinho, G. James, et al., The importance of a multifaceted approach to characterizing the microbial flora of chronic wounds, Wound Rep. Reg. 19 (2011) 523 541. [82] D. Toybou, C. Celle, C. Aude-Garcia, T. Rabilloud, J. Simonato, A toxicology-informed safer by design approach for the fabrication of transparent electrodes based on silver nanowires, Environ. Sci. Nano 6 (2019) 684 694. [83] A. Gandolfo, S. Marque, B. Temine-Roussel, R. Gemayel, H. Wortham, D. Truffier-Boutry, et al., Unexpectedly high levels of organic compounds released by indoor photocatalytic paints, Environ. Sci. Technol. 52 (2018) 11328 11337. [84] H. Naatz, S. Lin, R. Li, W. Jiang, Z. Ji, C.H. Chang, et al., Safe-by-design CuO nanoparticles via Fe-doping Cu-O bond length variation and biological assessment in cells and zebrafish embryos, ACS Nano 11 (2017) 501 515. [85] T. Xia, Y. Zhao, T. Sager, S. George, S. Pokhrel, J. Li, et al., Decreased dissolution of ZnO by iron doping yields nanoparticles with reduced toxicity to the rodent lung and zebrafish embryos, ACS Nano 5 (2011) 1223 1235. [86] S. George, S. Pokhrel, T. Xia, B. Gilbert, Z. Ji, M. Schowalter, et al., Use of a rapid cytotoxicity screening approach to engineer a safer zinc oxide particle through iron doping, ACS Nano 4 (2010) 15 29. [87] A.S. Adeleye, S. Pokhrel, L. Ma¨dler, A.A. Keller, Influence of nanoparticle doping on colloidal stability and toxicity of copper oxide nanoparticles in synthetic natural waters, Water Res. 132 (2018) 12 22. [88] O. Bondarenko, K. Juganson, A. Ivask, K. Kasemets, M. Mortimer, A. Kahru, Toxicity of Ag, CuO and ZnO nanoparticles to selected environmentally relevant test organisms and mammalian cells in vitro: a critical review, Arch. Toxicol. 87 (2013) 1181 1200.

3. Prevention of nanotoxicity

References

233

[89] S.L. Chia, D.T. Leong, Reducing ZnO nanoparticles toxicity through silica coating, Heliyon 2 (2016) e00117 (18 pp.). [90] A. Ivask, K. Juganson, O. Bondarenko, M. Mortimer, V. Aruaoja, K. Kasemets, et al., Mechanisms of toxic action of Ag, ZnO and CuO nanoparticles to selected ecotoxicological test organisms and mammalian cells in vitro: a comparative review, Nanotoxicology 8 (S1) (2014) 57 71. [91] J.W. Ko, J.W. Park, N.R. Shin, J.H. Kim, Y.K. Cho, D.H. Shin, et al., Copper oxide nanoparticle induces inflammatory response and mucus production via MAPK signaling in bronchial epithelial cells, Environ. Toxicol. Pharmacol. 43 (2016) 21 26. [92] A.K. Sharma, V. Singh, R. Gerra, M.P. Purohit, D. Ghosh, Zinc oxide nanoparticle induces microglial death by NADPH-oxidase-independent reactive oxygen species as well as energy depletion, Mol. Neurobiol. 54 (2017) 6273 6286. [93] E.A. Fairbairn, A.A. Keller, L. Ma¨dler, D. Zhou, S. Pokhrel, G.N. Cherr, Metal oxide nanomaterials in seawater; linking physicochemical characteristics with biological response in sea urchin development, J. Hazard. Mater. 192 (2011) 1565 1571. [94] W. Bai, Z. Zhang, W. Tian, X. He, Y. Ma, Y. Zhao, Z. Chai, Toxicity of zinc oxide Nanoparticles to zebrafish embryo: a physicochemical study of toxicity mechanism, J. Nanopart. Res. 12 (2010) 1645 1654. [95] H. Ma, P.L. Williams, S.A. Diamond, Ecotoxicity of manufactured ZnO nanoparticles - a review, Environ. Pollut. 172 (2013) 76 85. [96] E. Burello, A. Worth, A rule for designing safer nanomaterials: do not interfere with the cellular redox equilibrium, Nanotoxicology 9 (S1) (2015) 116 117. [97] L.A. Mendes, M.J.B. Amorim, J.J. Scott-Fordsmand, Assessing the toxicity of safer by design CuO surfacemodifications using terrestrial multispecies assays, Sci. Total Environ. 678 (2019) 457 465. [98] L. Huang, F. Peng, H. Yu, H. Wang, Preparation of cuprous oxides with different sizes and their behaviors of adsorption, visible light driven photocatalysis and photocorrosion, Solid. State Sci. 11 (2009) 129 138. [99] R. Saleh, N.F. Djaja, UV light photocatalytic degradation of organic dyes with Fe-doped ZnO nanoparticles, Superlattice Microstruct. 74 (2014) 217 233. [100] A.M. El Sayed, M. Shaban, Structural, optical and photocatalytic properties of Fe and (Co, Fe) co-doped copper oxide spin coated films, Spectrochim. Acta A 149 (2015) 638 646. [101] E. Gilbert, F. Pirot, V. Bertholle, J. Roussel, F. Falson, K. Padois, Commonly used UV filter toxicity on biological functions: review of last decade studies, Int. J. Cosmet. Sci. 35 (2013) 208 219. [102] W. Bousslama, H. Elhouichet, M. Ferid, Enhanced photocatalytic activity of Fe doped ZnO nanocrystals under sunlight irradiation, Optik 134 (2017) 88 98. [103] I. Lynch, A. Ahluwalia, D. Boraschi, H.J. Byrne, B. Fadeel, P. Gehr, et al., The bio-nano-interface in predicting nanoparticle fate and behavior in living organisms: towards grouping and categorizing nanomaterials and ensuring nanosafety by design, BioNanoMaterials 14 (3 4) (2013) 195 216. [104] B. Pelaz, G. Charron, C. Pfeiffer, Y. Zhao, J.M. de la Fuente, X. Liang, et al., Interfacing engineering nanoparticles with biological systems: anticipating adverse nano-bio interactions, Small 9 (2013) 1573 1584. [105] M. Hofmann-Amtenbrink, D.W. Grainger, H. Hofmann, Nanoparticles in medicine: current challenges facing inorganic nanoparticle toxicity assessments and standardizations, Nanomed. Nanotechnol. Biol. Med. 11 (2015) 1689 1694. [106] T.G. Smijs, S. Pavel, Titanium dioxide and zinc oxide nanoparticles in sunscreens: focus on their safety and effectiveness, Nanotechnol. Sci. Appl. 4 (2011) 95 112. [107] M. Auffan, M. Pedeutour, J. Rose, A. Masion, F. Ziarelli, C. Borschnek, et al., Structural degradation at the surface of an TiO2 based nanomaterial used in cosmetics, Environ. Sci. Technol. 44 (2010) 2689 2694. [108] L. Rowenczyk, C. Duclairoi-Poc, M. Barreau, C. Picard, N. Hucher, N. Orange, et al., Impact of coated TiO2nanoparticles in sunscreens on two representative strains of the human microbiota: effect of the particle surface nature and aging, Colloids Surf. B 158 (2017) 339 348. [109] Y. Tang, R. Cai, D. Cao, X. Kong, Y. Lu, Photocatalytic production of hydroxyl radicals by commercial TiO2 nanoparticles and phototoxic hazard identification, Toxicology 406 407 (2018) 1 8. [110] M.J. Osmond, M.J. Mccall, Zinc oxide nanoparticles in modern sunscreens: an analysis of potential exposure and hazard, Nanotoxicology 4 (2010) 15 41.

3. Prevention of nanotoxicity

234

10. Safer-by-design for nanomaterials

[111] S. Ortelli, A.L. Costa, P. Matteucci, M.R. Miller, M. Blosi, D. Gardini, et al., Silica modification of titania nanoparticle enhances photocatalytic production of reactive oxygen species without increasing toxicity potential in vivo, RSC Adv. 8 (2018) 40369 40377. [112] L. Reijnders, Cleaner technology and hazard reduction of manufactured nanoparticles, J. Clean. Prod. 14 (2006) 124 133. [113] R. Dunford, A. Salinaro, L. Cai, N. Serpone, S. Horiskoshi, H. Hidaka, et al., Chemical oxidation and DNA damage catalyzed by inorganic sunscreen ingredients, FEBS Lett. 418 (1997) 87 90. [114] J. Labille, J. Feng, C. Botta, D. Borschnek, M. Sammut, M. Cabie, et al., Aging of TiO2 nanocomposites used in sunscreen. Dispersion and fate of degradation products in aqueous environment, Environ. Pollut. 158 (2010) 3482 3489. [115] M. Morsella, N. d’Alessandro, A.G.F. Lanterna, J.C. Sciaiano, Improving sunscreen properties of TiO2 through and understanding of its catalytic properties, ACS Omega 1 (2016) 464 469. [116] S.K. Kansal, M. Singh, D. Dud, Studies in TiO2/ZnO photocatalysed degradation of lignin, J. Hazard. Mater. 153 (2008) 412 417. [117] T.K. Kirk, T. Higuchi, H. Chang (Eds.), Lignin Biodegradation: Microbiology, Chemistry and Potential Applications, CRC Press, Boca Raton, FL, 2018. [118] J. Port, D.J. Murphy, Mesothelioma; identical routes to malignancy from asbestos and carbon nanotubes, Curr. Biol. 27 (2017) R1173 R1176. [119] A. Schinwald, F.A. Murphy, A. Prina-Mello, C.A. Poland, F. Byrne, D. Movia, et al., The threshold length for fiber-induced acute pleural inflammation: shedding light on the early events in asbestos-induced mesothelioma, Toxicol. Sci. 128 (2012) 461 470. [120] F. Ito, H. Hisashi, S. Toyokuni, Polymer coating on carbon coatings into Durobeads is a novel strategy for human environmental safety, Ngoya J. Med. Sci. 80 (2018) 597 604. [121] G. Morose, The 5 principles of design for safer nanotechnology, J. Clean. Prod. 18 (2010) 285 289. [122] S. Tsuruoka, F.R. Cassee, V. Castranova, A new approach to design safe CNTs with an understanding of redox potential, Part. Fibre Toxicol. 10 (2013) 44 (2 pp.). [123] K. Bhattacharya, S.P. Mukherjee, A. Gallud, S.C. Burkert, S. Bistarelli, S. Belluci, et al., Biological interactions of carbon-based nanomaterials: from coronation to degradation, Nanomed. Nanotechnol. Biol. Med. 12 (2016) 333 351. [124] Q. Zhang, J. Huang, W. Qian, Y. Zhang, F. Wei, The road for nanomaterials industry: a review of carbon nanotube production, post treatment, and bulk applications for composites and energy storage, Small 8 (2013) 1237 1265. [125] K. Donaldson, G.A. Poland, F.A. Murphy, M. MacFarlane, T. Chernova, A. Schinwald, Pulmonary toxicity of carbon nanotubes and asbestos - similarities and differences, Adv. Drug. Deliv. Rev. 65 (2013) 2078 2086. [126] V. Mirshafiee, B. Sun, C.H. Chang, Y. Liao, W. Jiang, J. Jiang, et al., Toxicological profiling of metal oxide nanoparticles in liver context reveals pyroptosis in Kupfer cells and macrophages versus apoptosis in hepatocytes, ACS Nano 12 (2018) 3836 3852. [127] C. Gao, Y. Jin, G. Jia, X. Suo, H. Liu, D. Liu, et al., Y2O3 nanoparticles caused bone tissue damage by breaking the intracellular phosphate balance in bone marrow stromal cells, ACS Nano 13 (2019) 313 323. [128] R. Li, Z. Ji, C.H. Chang, D.R. Dunphry, X. Cai, H. Meng, et al., Surface interactions with compartimentalized cellular phosphates explain rare earth oxide nanoparticle hazard and provide opportunities for safer design, ACS Nano 8 (2014) 1771 1783. [129] X. Wang, T. Xia, M.C. Duch, Z. Ji, H. Zhang, R. Li, et al., Pluronic F 108 coating decreases lung fibrosis potential of multiwall carbon nanotubes by reducing lysosomal injury, Nano Lett. 12 (2012) 3050 3061. [130] L.F. Filon, M. Mauro, G. Adami, M. Bovenzizi, M. Crosera, Nanoparticle skin absorption: new aspects for a safety profile, Regul. Toxicol. Pharmacol. 72 (2015) 310 322. [131] C. Bussy, H. Al-Boucetta, K. Kostarelos, Safety considerations for graphene: lessons learnt from carbon nanotubes, Acc. Chem. Res. 46 (2013) 692 701. [132] N. Chatterjee, J. Yang, R. Atluri, W. Lee, J. Hong, J. Choi, Amorphous silica nanoparticle-induced perturbation of cholesterol homeostasis as a function of surface area highlights safe-by-design implementation: an integrated multi-omics analysis, RSC Adv. 6 (2016) 68606 68614.

3. Prevention of nanotoxicity

References

235

[133] M. Ghiazza, E. Alloa, S. Oliaro-Bosso, F. Viola, S. Livraghi, D. Rembges, et al., Inhibition of the ROS mediated cytotoxicity and genotoxicity of nano-TiO toward human keratinocyte cells by iron doping, J. Nanopart. Res. 16 (2014) 2263 (17 pp.). [134] S. George, S. Pokhrel, Z. Ji, B.L. Henderson, T. Xia, L. Li, et al., Role of Fe doping in tuning the bandgap of TiO2 for the photo-oxidation-induced cytotoxicity paradigm, J. Am. Chem. Soc. 133 (2011) 11270 11278. [135] D. Fourches, D. Pu, L. Li, H. Zhou, Q. Mu, G. Su, et al., Computer-added design of carbon nanotubes with the desired bioactivity and safety profiles, Nanotoxicology 10 (2016) 374 383. [136] A. Tarantini, K.D. Wegner, F. Dussert, G. Sarret, D. Beal, L. Mattera, et al., Physicochemical alterations of InP alloyed quantum dots aged in environmental conditions: a safer by design evaluation, NanoImpact 14 (2019) 100168 (13 pp.). [137] D.B. Hayden, H.V.M. Kibbelaar, A. Imhof, K.P. Velikov, Fully-biobased UV-absorbing nanoparticles from ethyl cellulose and zein for environmentally friendly photoprotection, RSC Adv. 8 (2018) 25104 25111. [138] H. Zhang, Z. Ji, T. Xia, H. Meng, C. Low-Kam, R. Liu, et al., Use of metal oxide nanoparticle band gap to develop a predictive paradigm for oxidative stress and acute pulmonary inflammation, ACS Nano 6 (2012) 4349 4368. [139] B. Fiorentino, L. Golanski, A. Guiot, J. Damlencourt, D. Boutry, Influence of paints formulation on nanoparticle release during their life cycle, J. Nanopart. Res. 17 (2015) 149 (13 pp.). [140] I. Hincapie´, A. Caballero-Guzman, D. Hiltbrunner, B. Nowack, Use of engineered nanomaterials in the construction industry with specific emphasis on paints and their flows in construction and demolition waste in Switzerland, Waste. Manag. 43 (2015) 398 406. [141] J.G. Clar, W.E. Platten, E. Baumann, A. Remsen, S.M. Harmon, K. Rodgers, et al., Release and transformation of ZnO nanoparticles used in outdoor surface coatings for UV protection, Sci. Total Env. 670 (2019) 78 86. [142] K. Starost, E. Frijns, J. van Laer, N. Faisal, A. Egizabal, E. Elizetxea, et al., The effect of nanosilica (SiO2) and nanoalumina (Al2O3) reinforced polyester nanocomposites on aerosol emissions into the environment during automated drilling, Aerosol Sci. Technol. 51 (2017) 1035 1046. [143] J. Silvestre, N. Silvestre, J. de Brito, Review on concrete nanotechnology, Eur. J. Environ. Civ. Eng. 20 (2016) 455 485. [144] H.J. Lee, S.Y. Yeo, S.H. Jeong, Antibacterial effect of nanosized silver colloidal solution on textile fabrics, J. Mater. Sci. 38 (2003) 2199 2204. [145] S.J. Froggett, S.F. Clancy, D.R. Boverhof, R.A. Canady, A review and perspective of existing research on the release of nanomaterials from solid nanocomposites, Part. Fibre Toxicol. 11 (2014) 17 (28 pp.). [146] D. Mitrano, S. Motellier, S. Clavaguera, B. Nowack, Review of nanomaterial aging and transformations through the life cycle of nano-enhanced products, Environ. Int. 77 (2015) 132 147. [147] W. Wohlleben, N. Neubauer, Quantitative rates of release from weathered nanocomposites are determined across 5 orders of magnitude by the matrix, modulated by the embedded nanomaterial, NanoImpact 1 (2016) 30 45. [148] A. Ko¨hler, C. Som, Risk preventive innovation strategies for emerging technologies: the cases of nanotextiles and smart textiles, Technovation 34 (2014) 420 4230. [149] R. Kaegi, B. Sinner, S. Zuleeg, H. Hagendorfer, E. Mueller, R. Vonbank, et al., Release of silver nanoparticles from outdoor facades, Environ. Pollut. 158 (2010) 2900 2905. [150] A. Al-Kattan, A. Wichser, R. Vonbank, S. Brunner, A. Ulrich, S. Zuin, et al., Release of TiO2 from paints obtaining pigment-TiO2 or nano-TiO2 by weathering, Environ. Sci. Process. Impacts 15 (2013) 2186 2193. [151] A. Al-Kattan, A. Wichser, R. Vonbank, S. Brunner, A. Ulrich, S. Zuin, et al., Characterization of materials released into water from paint containing nano SiO2, Chemosphere 1119 (2015) 1314 1321. [152] X. Zhang, M. Wang, S. Guo, Z. Zhang, H. Li, Effects of weathering and rainfall conditions on the release of SiO2, Ag and TiO2 engineered nanoparticles from paints, J. Nanopart. Res. 19 (2017) 19338 (13 pp.). [153] T. Engel, G. Kickelbick, Self-healing nanocomposites from silica-polymer core-shell nanoparticles, Polym. Int. G3 (2014) 915 923. [154] S. Scha¨fer, G. Kickelbick, Self-healing polymer nanocomposites based on Diels-Alder reactions with silica nanoparticles: the role of the polymer matrix, Polymer 69 (2015) 357 368.

3. Prevention of nanotoxicity

236

10. Safer-by-design for nanomaterials

[155] Q. Li, M. Jiang, G. Wu, L. Chen, S. Chen, Y. Cao, et al., Photothermal conversion triggered precisely targeted healing of epoxy resin based on thermoreversible Diels-Alder network and amino-functionalized carbon nanotubes, Appl. Mater. (2017) 20797 20807. [156] J.H. Clark, T.J. Farmer, L. Herrero-Davila, J. Sherwood, Circular economy design considerations for research and process development in the chemical science, Green Chem. 18 (2016) 3914 3934. [157] C. Cai, Y. Zhang, X. Zou, R. Zhang, X. Wang, Q. Wu, et al., Rapid self-healing and recycling of multipleresponsive mechanically enhanced epoxy resin/graphene nanocomposites, RSC Adv. 7 (2017) 46336 46343. [158] C. Cai, Y. Zhang, M. Lei, Y. Chen, R. Zhang, X. Wang, et al., Multiple-response shape memory polyacrylonitrile/graphene nanocomposites with rapid self-healing and recycling properties, RSC Adv. 8 (2018) 1225 1231. [159] F. Pilate, Z. Wen, F. Khelifa, Y. Hui, S. Delpierre, L. Dan, et al., Design of melt-recyclable polycaprolactonebased supramolecular shape-memory nanocomposites, RCS Adv. 8 (2018) 27119 27130. [160] H. Wigger, S. Hackman, T. Zimmermann, J. Ko¨ser, J. Tho¨ming, A. von Gleich, Influences of use activities and waste management on environmental releases of engineered nanomaterials, Sci. Total Environ. 535 (2015) 160 171. [161] T. Walser, L.K. Limbach, R. Brogioli, E. Erismann, L. Flamigni, B. Hattendorf, et al., Persistence of engineered nanoparticles in a municipal solid-waste incineration plant, Nat. Nanotechnol. 7 (2012) 520 524. [162] H. Fo¨rster, T. Thajudeen, C. Funk, W. Peukert, Separation of nanoparticles: filtration and scavenging from waste incineration plants, Waste Manag. 52 (2016) 346 352. [163] G. Ounoughene, C. Chivas-Joly, C. Longuet, G. Le Bihan, J.M. Lopez-Cuesta, L. Le Coq, Evaluation of nanosilica emission in polydimethylsiloxane composite during incineration, J. Hazard. Mater. 371 (2019) 415 422. [164] A.L. Hicks, T.L. Theis, A comparative life cycle assessment of commercially available household silverenabled polyester textiles, Int. J. Life Cycle Assess. 22 (2017) 256 265. [165] M.J. Kay, R.W. McCabe, L.H.G. Morton, Chemical and physical changes occurring in polyester polyurethane during biodegradation, Int. Biodeterior. Biodegrad. 31 (1993) 209 225. [166] J.E. McDonald, J.N.I. Houghton, D.J. Rooks, H.E. Allison, A.J. McCarthy, The microbial ecology of anaerobic cellulose degradation in municipal waste landfill sites: evidence of a role for fibrobacters, Environ. Microbiol. 14 (2012) 1077 1087. [167] E. Mendes de Oliveira, D.A. Nogueira, L.C.R. Lopes, J.F. Silveira Feiteira, J.A. de Castro, Analysis of percolation of the stabilized suspensions of TiO2 and SiO2 nanoparticles in soil columns simulating landfill layers, J. Mech. Eng. Autom. 6 (2016) 47 52. [168] R. Kaegi, A. Englert, A. Gondikas, B. Sinnet, F. von der Kammer, M. Burkhardt, Release of TiO2 nanoparticles from construction and demolition landfills, NanoImpact 8 (2017) 73 79. [169] S. Bolyard, D. Reinhart, S. Santra, Behavior of engineered nanoparticles in landfill leachate, Environ. Sci. Technol. 47 (2013) 8114 8122. [170] E. Mendes de Oliveira, J.A. de Castro, I.L. Feirera, Study of the interaction of copper nanoparticles with titanium in landfill soils, Mater. Sci. Forum 869 (2016) 778 783. [171] J. Zhang, A. Panwar, D. Bello, T. Jozokos, J.A. Isaacs, C. Barry, et al., The effects of recycling on the properties of carbon nanotube filled polypropylene composites and worker exposure, Environ. Sci. Nano 3 (2016) 409 417. [172] M. Bigum, L. Brogaard, T.H. Chistensen, Metal recovery from high grade WEEE: a life cycle assessment, J. Hazard. Mater. 207-208 (2012) 8 14. [173] A. Marra, A. Cesaro, V. Belgiorno, Separation efficiency of valuable and critical metals in WEEE mechanical treatments, J. Clean. Prod. 186 (2018) 490 498. [174] G. Zhang, Y. He, Y. Feng, T. Zhang, H. Wang, X. Zhu, Recovery of residual metals from fine nonmetallic fractions of waste printed circuit boards using a vibrated gas-solid fluidized bed, Sep. Purif. Technol. 207 (2018) 312 328. [175] A. Hellmann, K. Schmidt, S. Rippenberger, M. Berges, Freisetzung ultafeiner Staube bei der machanische Bearbeitung von Nanokompositen (Release of ultrafine dusts during the machining of nanocomposites), Gefahrst. Reinhalt. Luft. 72 (11/12) (2012) 473 476.

3. Prevention of nanotoxicity

References

237

[176] S. Harper, W. Wohlleben, M. Doa, B. Nowack, S. Clancy, R. Canady, et al., Measuring nanomaterial release from carbon nanotube composites: review of the state of the science, J. Phys. Conf. Ser. 617 (2015) 012026 (19 pp.). [177] M.J.B. Amorim, S. Lin, K. Schlich, J.M. Navas, A. Brunelli, N. Neubauer, et al., Environmental impacts by fragments released from nanoenabled products: a multiassay, multimaterial exploration by the SUN approach, Environ. Sci. Technol. 52 (2018) 1514 1524. [178] W. Wohlleben, S. Brill, M.W. Meier, M. Mertler, G. Cox, S. Hirth, et al., On the life cycle of nanocomposites: comparing released fragments and their in-vivo hazards from three release mechanisms and four nanocomposites, Small 16 (2011) 2384 2395. [179] A.T. Saber, N.R. Jacobsen, A. Mortensen, J. Szarek, P. Jackson, A. Madsen, et al., Nanotitanium dioxide toxicity in mouse lung is reduced in sanding dust from paint, Part. Fibre Toxicol. 9 (2012) 4 (15 pp.). [180] S. Smulders, K. Luyts, G. Brabants, K. van Landuyt, C. Kirschhock, E. Smolders, et al., Toxicity of nanoparticles embedded in paints compared with pristine nanoparticles in mice, Toxicol. Sci. 141 (2014) 132 140. [181] Y.K. Song, S.H. Hong, M. Jang, J.H. Kang, O.Y. Kwon, G.M. Han, et al., Large accumulation of micro-sized polymer particles in the sea surface microlayer, Environ. Sci. Technol. 48 (2014) 9014 9021. [182] J. Pinto da Costa, P.S.M. Santos, A.C. Duarte, T. Rocha-Santos, (Nano)plastics in the environment sources, fates and effects, Sci. Total Environ. 566 567 (2016) 15 26. [183] S. Lambert, M. Wagner, Characterization of nanoplastics during degradation of polystyrene, Chemosphere 145 (2016) 265 268. [184] H.M. Prichard, P.C. Fisher, Identification of platinum and palladium particles emitted from vehicles and dispersed into the surface environment, Environ. Sci. Technol. 46 (2012) 3149 3154.

3. Prevention of nanotoxicity

C H A P T E R

11 Antibacterial activity of metal oxide nanoparticles Vojislav Stani´c1 and Sladjana B. Tanaskovi´c2 1

Vinˇca Institute of Nuclear Sciences, Laboratory of Radiation and Environmental Protection, University of Belgrade, Belgrade, Serbia 2Faculty of Pharmacy, Department of General and Inorganic Chemistry, University of Belgrade, Belgrade, Serbia

11.1 Introduction Bacterial infections present a large problem due to the fact that they are the cause of chronic infections which lead to a huge mortality rate. Antibiotics are known to have the best effect in treating infections caused by bacterias. The main problem arises from inappropriate use, overuse and misuse of the antibiotics for prophylactic and corrective purpose without adequate medical indications, thus leading to bacterial resistance to antibiotics. There are types of bacteria called super-bacterias which are resistant to almost all antibiotics known to us and own a gene for super-resistance New Delhi metalloβ-lactamase-1 [1]. There are several mechanisms of antibiotics affects. Most antibiotics known today operate in one of the three following ways: based on synthesis of the cell wall, by translatory mechanism, or by DNA replication mechanism. Bacterial resistance can show toward any of these mechanisms. This is the main reason for continuous research for new antimicrobial agents. Nanoparticles (NPs) are now being used more and more for antibacterial purpose as an alternative for antibiotics. This implies the use of NPs in implants plates and medical materials. The following thesis has been accepted: “Nanomaterials as antibacterial complements to antibiotics are highly promising and are gaining large interest as they might fill the gaps where antibiotics frequently fail” [2]. The advantages of nanoparticles as antibacterial agents compared to antibiotics are following: (1) the overcoming of the existing mechanisms of resistance including prevention of biofilm creation, (2) battling against microbes with combined mechanisms, and (3) working as good carriers of antibiotics [3]. Generally, biofilm-growing bacteria are highly resistant to antibacterial drugs and the host immune system, although the exact mechanisms underlying such resistance are still

Nanotoxicity DOI: https://doi.org/10.1016/B978-0-12-819943-5.00011-7

241

Copyright © 2020 Elsevier Inc. All rights reserved.

242

11. Antibacterial activity of metal oxide nanoparticles

not fully understood [4,5]. Biofilm is a structured community of bacteria embedded in a self-produced extracellular matrix of proteins, polysaccharides, and DNA. Consequently, infections involving biofilm formation are chronic and difficult to treat. Therefore there is an urgent need to find alternative therapeutic approaches for overcoming the increasing resistance of bacteria to current antibiotics [6]. Several metal oxides are widely used in medicine, agriculture, and environmental protection as antibacterial agents. Metal oxides, especially Ag2O, CaO, MgO, ZnO, NiO, CoO, CuO, Cu2O, TiO2, SiO2, and FexOy in their nano-forms, have been proposed as potential antibacterial agents. They may show bacteriostatic or/and bactericidal effect. The antibacterial activity depends on its physicochemical properties: chemical composition, particle size, surface properties, shape, aggregation, solubility, crystal phase, and crystallinity. The activity also depends on nanoparticle concentration, experimental conditions, and type and number of bacteria.

11.2 Effective physicochemical properties of MO-NPs on antibacterial activity 11.2.1 Chemical composition of metal oxide nanoparticles The antibacterial activity of metal oxides depends very much on the chemical composition, respectively, of the type of metal ions. Metal ions like Ca21, Mg21, Cu21, Zn21, Co21, Fe21; 31, and Ni21 in the small quantities are essential for various metabolic processes in most of the living bacterial strains, while in the higher amounts are potentially toxic [7]. Nonessential metal ions such as Ag1 and Hg21 showed much stronger antibacterial effect at exceptionally low concentrations. Mercury compounds have less and less importance in medicine because they show high toxicity and have a negative effect on the environment. The antibacterial activity of metal ions may be partially related to their binding affinity to certain ligand atoms from biomolecules and cellular components. The interaction between metal ions and ligand atoms is based on hardsoft acid base theory (HSAB principle) [8]. According to HSAB principle, hard acids prefer to coordinate to hard bases and soft acids to soft bases. Soft acids, such as: Ag1, Hg1, Hg21, and Cd21 make very stable complex compounds with soft bases as sulfur from thiol group that are found in proteins (Table 11.1). Some metal ions such as: Cu21, Zn21, Co21, Fe21, and Ni21, have medium TABLE 11.1 Some hard-soft acids and bases [7,8]. Hard Acids H1, Li1, Na1, and K1 21

Mg Fe

31

21

and Ca

Borderline

Soft

Fe21, Co21, Ni21, Cu21, and Zn21

Ag1, Au1, Hg1, Hg21, Cd21, Pt21, and Pd21

C6H5NH2, imidazole, and pyridine

R-SH, R-C6H5, ethylene, and R-CN

and Co31

La31 and Ce41 Bases R-COO2, R-NH2, R-OH, R-O-R, OH2, CO322, PO432, NH3, and H2O

4. Antibacterial activity of nanomaterials

11.2 Effective physicochemical properties of MO-NPs on antibacterial activity

243

hard-soft properties and are classified in a special borderline acid group (Table 11.1). They can form stable complexes with hard (R-NH2) and soft bases (R-SH). Biomolecules from bacteria behave like multidentate ligands, except OH and NH2 groups, and other groups particularly COOH (COO2), OH; participate in interaction with metal ions. The antibacterial activity of metals ions is approximately proportional to their affinity for thiol groups. Silver has the strongest antimicrobial effect in the following sequence [9]: Ag . Hg . Cu . Cd . Cr . Pb . Co . Au . Zn . Fe . Mn . Mo . Sn: The IrvingWilliams series of ligand affinity for the first-row transition essential divalent metal ions clearly demonstrates the affinity of biological molecules is as follows: Ca21 , Mg21 , Mn21 , Fe21 , Co21 , Ni21 , Cu21 . Zn21 and shows that divalent copper has a strong affinity for biological molecules, suggesting it can have the most toxic effect in higher concentrations.

11.2.2 Particle size and surfaces properties of metal oxide nanoparticles Particle size plays a major role in interaction of MO-NPs oxides with bacterial cells and other biological systems. Nanoparticles exhibited stronger antimicrobial activity than microscaled particles (bulk particles), due to their larger surface to volume ratios and the number of particles per given mass is significantly higher. Decreasing the size of the particles to nano size, leads to significant increase of total number of atoms on the surface and therefore can have a large influence on the overall properties of the crystal. Metal ions on the surface of nanoparticles are coordinated unsaturated, that is, “active centers” and are very reactive and can be easily released into the surrounding environment when in contact with biomolecules. Size-dependent interaction of metal oxide nanostructures with bacteria has been widely reported. The zinc oxide nanoparticles (ZnONPs) show a significant inhibitory effect against both gram-negative and gram-positive bacteria under normal lighting conditions, as compared to the bulk particles [1012]. Similarly, antibacterial studies showed that tested bacteria is highly affected by the size of the CuO nanoparticles [13,14]. The nanoparticles due to larger specific surface area have better contact with bacterial cells than bulk particles. The zeta potential of a metal oxide is commonly used to characterize the surface charge property of nanoparticles. The point of zero charge (PZC) is the pH at which the total number of positive and negative charges on its surface becomes neutral or zero. The knowledge of the PZC of the MO nanomaterials can be crucial for predicting antimicrobial activity at certain pH values of the environment. The MO-NPs may have a positive, negative or neutral charge on their surface. The MO-NPs with positive charge had the strongest antimicrobial activity, neutral charge had intermediate activity, and the nanoparticles with negative charge were the least effective. At pH value below the PZC, the oxide surface is positively charged. At pH value above the PZC, the oxide surface is negatively charged. The value of the PZC of a metal oxide depends on several factors such as: crystal structure, particle morphology, presence of impurities and the type of electrolyte present in the aqueous solution.

4. Antibacterial activity of nanomaterials

244

11. Antibacterial activity of metal oxide nanoparticles

11.2.3 Concentration of metal oxide nanoparticles Antibacterial activity of MO-NPs increases with its concentration in the medium [15]. It may be related that higher MO-NP concentrations imply the higher surface area, which results in intensive contact with bacterial cells, leading to higher antimicrobial activity. The minimum inhibitory concentration (MIC) is the most common indicator in microbiology for quantitative evaluation in vitro antibacterial activity of MO-NP [12,16]. MIC is the lowest concentration of an antimicrobial agent that inhibits the visible growth of microorganisms after incubation overnight [17].

11.2.4 The shape-dependent antimicrobial properties of metal oxide nanoparticles The shape of MO-NPs may also affect the antibacterial activity, although the exact method of action is not entirely clear. Different shape of MO-NPs can be controlling by the synthesizing method, type and concentration of precursors, solvents, the presence of various molecules: surfacants, polymers, biomolecules, and small organic molecules; as well as preparation condition: temperature, pH synthesis and time [18,19]. Each morphology of nanoparticles has specific physicochemical properties, such as: specific surfaces, crystallinity, solubility and for some metal oxides there is a possibility of creating reactive oxygen species (ROS); which are reflected in antibacterial activity. Several studies suggest that different shape of MO-NP have different degrees of antibacterial activities. ZnO nanopyramids showed much greater antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) compared with of nanoplates and nanospheres [20]. Another study showed that rod-shaped ZnONP being less antibacterial than spherical NP [10].

11.2.5 Solubility of metal oxide nanoparticles Solubility of MO-NPs can have a major impact on their antimicrobial activity. MO-NPs solubility depend on their physico-chemical properties of particles: size, shape, specific surface, crystal phase, crystallinity, types, and amounts of foreign ions present in its structure, as well as conditions in the medium: the type of solution, pH, temperature and etc. Particle size is a key property affecting the solubility of MO-NPs. Solubility tends to increase with decreasing particle size and increasing specific surface. Nanoparticle aggregation can reduce their solubility [21]. The pH of the medium is a very important factor that affects the solubility of metal oxides. The solubility of metal oxides can be affected by other molecules and a metal ions present in the solution. Periclase [MgO(s)] recrystallizes to the mineral brucite [Mg(OH)2(s)] in pure water or converts to magnesite, MgCO3(s), or a hydromagnesite, Mg(OH)2(MgCO3)4(H2O)3(s), if CO2(aq) is present [22].

11.2.6 The generation of reactive oxygen species All aerobic organisms can generate ROS as intermediates under various physiological states and play important roles in modulation of cell survival, cell death, differentiation, cell signaling, and inflammation-related factor production. Their cellular levels are strongly regulated by various antioxidant enzymes, such as superoxide dismutase, catalase, and

4. Antibacterial activity of nanomaterials

11.2 Effective physicochemical properties of MO-NPs on antibacterial activity

245

peroxidases or bound to different types of antioxidants, including flavonoids, ascorbic acids, vitamin E, and glutathione. Some metal oxides: TiO2, ZnO, CuO, SiO2, MgO, and Fe2O3 are photocatalytic semiconductors and can produce ROS on the surface of the particles under light irradiation. ROS important effectiveness factors for the antibacterial activity of MO-NPs. Photocatalytic semiconductors are characterized by the filled valence level and empty conductive levels. After irradiation/exposure to light photons that have energy equal to or greater than the energy-band energy (Eg) of the semiconductor, electrons are excited from the valence band (VB) to the conduction band (CB), to give a photoexcited electron (e2) and an electronic hole (h1) in the CB and VB, respectively (Fig. 11.2). In this way, the charge carrier is generated in the semiconductors, the electron/electronic hole pair (e2/h1). The value of bandgap energies of some antibacterial oxides was shown in Table 11.1. Reducing particle size leads to an increase of the bandgap energy and can enhance the redox potential of photogenerated electrons and holes, that is, smaller particles will be more photoactive than larger ones [23]. The lifespan of e2/h1 pairs is only a few nanoseconds, but it is long enough to initiate redox reactions with semiconductor material in solutions or gaseous phases. Electrons and holes can also react to each other, which is undesirable because recombination occurs thus resulting into heat. Nano particles exhibit high photocatalytic activity. Namely, the smaller the particle size the larger the catalyst surface, so the shorter the path of the transition of e2/h1 pairs from the interior to the surface of the particles. This leads to a decrease in the recombination potential of pairs, and consequently to an increase in photocatalytic activities. Generated electrons and holes are highly reactive species that can migrate to the surface of the metal oxide where they can participate in oxidation-reduction reactions with chemisorbed molecules (Fig. 11.1). In order to obtain the oxidation of ions or molecules on the surface of the semiconductor, the potential of these species must be less positive than the potential of the photogenerated electronic holes of the VB. Electronic holes can react with electron donor species such as adsorbed water or hydroxyl ions, in which strong oxidizing agent, hydroxyl free FIGURE 11.1 Processes involved in semiconductor particles upon band gape excitation. CB, Conduction band; VB, valence band.

4. Antibacterial activity of nanomaterials

246

11. Antibacterial activity of metal oxide nanoparticles

radical (•OH) and hydrogen peroxide are formed. The electrons in the CB can reduce adsorbed molecules when their potential is less negative. The electron also can react with adsorbed molecular oxygen to produce superoxide (O2•2) anion. This processes are described by Eqs. (11.1) and (11.12) [24].  (11.1) TiO2 1 hν-TiO2 e2 1 h1 Surface reactions involving holes:  TiO2 h1 1 2H2 O-TiO2 1  OH 1 H1  TiO2 h1 1 2H2 O-TiO2 1 2H1 1 H2 O2  TiO2 h1 1 OH2 -TiO2 1  OH2

(11.2) (11.3) (11.4)

Surface reactions involving electrons: TiO2 ðe2 Þ 1 O2 -TiO2 1  O2 2 2

TiO2 ðe Þ 1



O2 2 -TiO2

1 H2 O2

TiO2 ðe2 Þ 1 H2 O2 -  OH 1 OH2 1 O2



O2 2

1 H2 O2 -  OH 1 OH 1 O2 2

 O22 1 H1 -  HO2 TiO2 ðe2 Þ 1  HO2 -TiO2 1  HO2  HO2 1 H1-H2 O2 2  HO2 -O2 1 H2 O2

(11.5) (11.6) (11.7) (11.8) (11.9) (11.10) (11.11) (11.12)

The antibacterial activity of MO-NPs depends on the type and quantity of the generated ROS species. ROS such as •OH, •O2a, and H2O2 are highly toxic to bacterial cells. Hydroxyl radicals have a strongest oxidation potential of 2.8 V and can damage different types of biomolecules, including lipids, proteins, nucleic acids, and carbohydrates. The hydroxyl radical and hydrogen peroxide can penetrate the bacterial cell walls while superoxide anion cannot penetrate due to its negative charge [24]. MO-NPs shows more power to create ROS types of their bulk materials. Structural, electronic, and surface features of nanoparticles play an important role in light absorption [25]. Nanoparticles create larger quantities of ROS compared to their bulk counterpart, due to their larger surface areas with more reaction sites for ultraviolet (UV) absorption. Along with the particle size, a number of other factors affect the amount of ROS created, such as: concentration of nanoparticles, intensity of UV radiation, pH environment, temperature and various organic and inorganic ions, and molecules which can be adsorbed at the surface of nanoparticles.

11.2.7 Mechanisms of antimicrobial activity metal oxide nanoparticles Metallic oxide nanoparticles can exhibit a bacteriostatic or bactericidal effect. Bacteriostatic activity means that the treated bacteria cease to grow or multiply. When

4. Antibacterial activity of nanomaterials

11.2 Effective physicochemical properties of MO-NPs on antibacterial activity

247

treated bacterial cells are removed from a solution containing NP, they begin to grow again. In case of a bactericidal effect, the death of a bacterial colony comes as a consequence of MO-NPs action. Metal oxides are highly effective inhibitors against a wide range of bacteria when administered in well-defined physical-chemical properties of NPs and environmental conditions. The exact mechanism of bacterial toxicity of metal oxide nanoparticles is still undefined in full, even though there are several suggested: (1) Liberation of antibacterial metal ions from oxide particles (2); (3) direct mechanical destruction of the cell wall of the bacteria and/or its membrane, and (4) oxidative stress via generation of ROS on surfaces of the nanoparticles, generation of ROS from MO-NPs. Each of the proposed mechanisms can be independent, although in most cases it is simultaneous and depends on the type of metal oxide. The antibacterial activities of MO-NPs depend on the type of bacterial organisms and they are mainly related to the structure of the bacterial cell envelope. Many studies have shown that MO-NPs have larger activity towards gram-positive than gram-negative bacterias due to the difference in their membrane structure. Gram-negative bacterias have a wall made of lipoproteins and phospholipids which makes the wall penetrable for macromolecules only. Contrary to this, the cell wall of the gram-positive bacterias is made of thin peptidoglycan layer and teichoic acid which enables penetration of the unknown molecules through the pores, causing the damage of the membrane and leading to the effective cell death. Beside all the above, these bacterias have high negative charge at the membrane surface which also attracts NPs. Liberation of antibacterial metal ions from MO-NPs in a liquid medium containing bacteria is an important factor in increasing bactericidal effects. Some of the most noted and researched MO-NPs include, Ag2O, CuO, and ZnO can kill bacteria most likely via the release of metal ions. The amount of released metal ions depends on the physico-chemical properties of the MO-NPs and on the properties of the bacterial medium. The antibacterial mechanism of the action of a metal ions on bacterial cells is very complex. One possible explanation for the action of the metal ion on the cell envelope of bacteria is the reaction with thiol (SH), amino (NH) and carboxylic (COOH) groups of proteins present in the cell wall of bacteria, which leads to their denaturation [26]. All interactions of membrane proteins and metal ion lead to a drastic change in permeability of the membrane by degradation of the lipopolysaccharide and denaturation of proteins, where as the result of the quenching the proton gradient [27]. Destruction of lipids in cell membranes is the cause of intracellular leakage and it is believed that this leads to the death of a bacterial cell. Intracellular ions of metals can bind to respiratory chain proteins resulting in a separation of the breathing process, or the transport of electrons through membrane proteins, from the pathway of oxidative phosphorylation. Metal ions can inhibit various proteins present in the cytoplasm and ribosomes, and interacts with nucleic acids by preventing replication and translation processes, causing cell death. Similar to metal ions, MO-NPs may disrupt the integrity of the bacterial envelope and the cell membrane potential by electrostatic interactions. MO-NPs with a positive charged surface electrostatic force interact with the negative surface of the bacterial cell. The most of gram-positive and gram-negative bacteria under physiological conditions has a negative charge of the cell surface due to the presence of carboxyl, phosphate, and amino groups. The opposite charge of the bacteria and nanoparticles reveals one reason for the

4. Antibacterial activity of nanomaterials

248

11. Antibacterial activity of metal oxide nanoparticles

antibacterial activity of MO-NPs. The same charge (negative) on the bacterial cell and the surface of the nanoparticles leads to repulsion and thus interferes mutual contact. Metal ions on the surface of MO-NPs are coordinated unsaturated and responsible for antibacterial activity. They are very reactive and can be easily released in contact with cell envelope and thus contribute to a stronger bactericidal effect. Due to breaking of the cell barrier, abundant amount of cytosol is released from cell leading to the death of the bacteria. The adhesion of MO-NPs structures on bacteria can induce mechanical damage and osmotic rupture their cells [2830]. Degree of the cell damage depends on physical properties and quantities of adsorbed MO-NPs as well as the bacteria type. Contact with highly crystalline MO may cause more mechanical damage of the bacterial cells [31]. Gramnegative cells are generally more sensitive to mechanical lysis and osmotic rupture than gram-positive cells due to their peptidoglycan cell wall being B45 times thinner than those of the gram-positive bacteria [29,32]. Bacteriocidal effects of some MO can also occur due to the formation of ROS on the surface of nanoparticles. ROS can attack vital cell components like polyunsaturated fatty acids, proteins, nucleic acids and to a lesser extent carbohydrates [33]. The antibacterial effect depends on the quantity of the created species. These reactions lead to severe oxidative stress and can alter intrinsic membrane properties like fluidity and lysis of the cells. ROS can attack directly polyunsaturated fatty acids in membranes and initiate oxidative damage to lipids (lipid peroxidation). Phospholipids play an important role in the structure and function of all biological membranes. Unsaturated phospholipids from bacterial cell envelope are susceptible to peroxidation as they contain multiple double bonds and the methylene group that lies within is prone to abstraction of hydrogen atom. The lipid peroxidation consists of three phases: initiation, propagation, and termination (Fig. 11.2). The initiation phase of lipid peroxidation starts by abstraction of a hydrogen atom from a methylene carbon in the lipid substrate (LH) by ROS like hydroxyl radical, forming the carbon-centered lipid radical (L). In the propagation phase, lipid radical (L) rapidly reacts with oxygen to form a lipid peroxy radical (LOO). The peroxyl radical can abstract a hydrogen atom from another lipid molecule generating a new L (that continues the chain reaction) or abstract a hydrogen atom from other biomolecules, such as DNA and proteins, to form the primary oxidation product, a lipid hydroperoxide (LOOH). The lipid hydroperoxide (ROOH) is unstable in the presence of metal ions like Fe21 leading to the formation of reactive alkoxy radicals. In the termination reaction, antioxidants In the termination phase, antioxidants like enzymes (superoxide dismutase, catalase, and peroxidases), donate a hydrogen atom to the lipid peroxy radical species resulting in the formation of nonradical products. The primary consequence of lipid oxidation are an increase membrane fluidity and disrupt the cell integrity Aldehydes are a secondary product of lipid oxidation. Some of them, such as 4-hydroxy2-nonenal (HNE) and malondialdehyde (MDA), are very reactive and can diffuse from the site of their origin to the inside of the bacterial cell. They can covalently binds to lysine residues of proteins, enzymes, nucleic acids, and phospholipids, which results in loss of their function [34]. The amount of MDA created is considered a reliable indicator of lipid peroxidation and oxidative damage to cell. Proteins are crucial in all cell functions, including versatile cellular processes, structural role in bacterial cell membranes, catalytic function, a number of metabolic functions and so on. The oxidative damage of cellular proteins caused by ROS is manifested oxidation of

4. Antibacterial activity of nanomaterials

11.2 Effective physicochemical properties of MO-NPs on antibacterial activity

249

FIGURE 11.2 Lipid peroxidation process takes place in three phases: Initiation 1, propagation 2, and termination phase 3. In the termination reaction, various aldehydes may occur [malondialdehyde (MDA) or 4-hydroxy-2-nonenal (HNE)] or antioxidants can neutralize the lipid peroxy radical species to neutral products.

the side chains of almost type of amino acids, chain fragmentation, cross-linked protein aggregates and disorder of the secondary and tertiary structure [34,35]. These modifications lead to functional changes that disturb cellular metabolism and impair in the permeability of cell membranes and can lead to cell death. The oxidation of some amino acid side chains leads to the generation of aldehyde and ketone groups, referred to as carbonyl derivatives (Table 11.2). The carbonyl groups may also be introduced into the protein by α,β-unsaturated alkenals such as MDA and 4-hydroxynonenal produced during the lipid peroxidation. The levels of carbonylation within the protein molecule serve as a marker for oxidative protein damage. Carbonyl derivatives are harmful because they change the conformation of the protein, which inevitably reflects on their function. The consequences of amino acid side chain modifications include the formation of new species bound and protein-protein crosslinkages which may give be inert end products. Functional groups of proteins can react with oxidation products of polyunsaturated fatty acids and with carbohydrate derivatives to produce inactive derivatives. ROS can cause oxidative damages to nucleic acids in bacteria, acting on their bases and sugar moieties. Oxidative damage can result in multiple effects, including (1) single and double-strand breaking in the backbone, (2) adduction of the base and sugar groups, (3) cross-linking between DNA and proteins, and (4) causing base lesions [37,38]. Attack of ROS on sugar ultimately leads to sugar fragmentation, base loss, and a strand break with a terminal fragmented sugar residue [39]. ROS can react with nucleic bases through the abstraction of hydrogen atom from methyl groups, resulting in allyl radicals or by creating

4. Antibacterial activity of nanomaterials

250

11. Antibacterial activity of metal oxide nanoparticles

TABLE 11.2 Amino acid residues of proteins and oxidized products formed by reactive oxygen species (ROS) [35,36]. Amino acid

Oxidation products

References

Arginine

Glutamic semialdehyde

[35,36]

Cysteine

Disulfides and cysteic acid

Histidine

2-Oxohistidine, asparagine, and aspartic acid

Lysine

2-Aminoadipic semialdehyde

Proline

2-Pyrrolidone, 4- and 5-hydroxyproline pyroglutamic acid, and glutamic semialdehyde

Threonine

2-Amino-3-ketobutyric acid

Leucine

3-,4-,5-hydroxyleucine

[36]

Glutamyl

Oxalic acid and pyruvic acid

[35]

Methionine

Methionine sulfoxide and methionine sulfone

Phenylalanine 2,3-Dihydroxyphenylalanine and 2-, 3-, and 4-hydroxyphenylalanine Tryptophan

2-, 4-, 5-, 6-, and 7-hydroxytryptophan, nitrotryptophan, kynurenine, 3hydroxykynurenine, and formylkynurenine

Tyrosine

3,4-Dihidroxyphenylalanine, Tyr-Tyr cross-linkages, Tyr-O-Tyr, and cross-linked nitrotyrosine

nucleic base-OH adduct radicals. More than 20 different oxidized and ring-fragmented nitrogen bases formed by ROS. Damage to the bacterial nucleic acid can disrupt the normal functioning of the cell and lead to its death.

11.3 Antibacterial activity of magnesium oxide and calcium oxide nanoparticles Magnesium and calcium are essential elements needed for a broad variety of physiological functions. Calcium in the form of biological hydroxyapatite is the main inorganic constituent of bone tissue. MgO(s) and CaO(s) are not stable in the presence of water and recrystallize to a hydroxide forms Mg(OH)2(s) and Ca(OH)2(s). Both metallic hydroxides have considerable use in the repair of damaged dental tissue and as antibacterial agents in dental care [40] The antimicrobial mechanism of MgO and CaO, were researched and were found to indicate strong activity related to alkalinity, creation of ROS and attachment of the particles on a bacterial cell envelope. High pH value of the environment can slow the growth of bacteria or completely kill them. As a suspension in water at room temperature, Mg(OH)2 has pH value of 10.4, while Ca(OH)2 has pH of 12.5. Dong et al. [41] described that the treatment of Escherichia coli in NaOH, Luria Broth (LB) culture solution at pH of 10 did not damage the cell, its growth rate is much slower than neutral LB solution. In contrast, all E. coli cells were killed at the same pH, but with Mg(OH)2 nanoparticles suspension. Several studies reported that bactericidal effect of Ca(OH)2 is related

4. Antibacterial activity of nanomaterials

11.3 Antibacterial activity of magnesium oxide and calcium oxide nanoparticles

251

to its high pH value [42]. High alkalinity of Ca(OH)2 can disrupt the structural protein in the bacterial cell envelope [43]. The alkalinization of the cell caused by Ca(OH)2 induces the breakdown of ionic bonds that maintain the structure of proteins. As a consequence, these changes frequently result in the loss of biological activity of the enzyme and disruption of the cellular metabolism. Mg21 and Ca21 ions did not result in any significant toxicity [41,44,45]. Antimicrobial activity of MgO and CaO related to generation of ROS such as superoxide anions has been observed from the powder slurry [46]. Hydroxyl ions are highly oxidant free radicals that show extreme reactivity according to biomolecules. MgO has a high band gap energy of 7.8 eV, making it an insulator. Generation of ROS on the surface of alkaline earth oxides is the presence of defects or oxygen vacancy at the surface of the particle [47]. This effect is especially noticeable in nanoparticles due to its larger specific surface area and larger number of defects (oxygen vacancies). Hewitt et al. [48] reported that MgO initiated the sensitivity changes in E. coli induced by ROS. Strong antibacterial activity of the MgO-NPs could be observed in absence of any ROS production. Sawai et al. [45] examined antibacterial activity of MgO against E. coli or S. aureus. They suggested that the presence of ROS, such as superoxide, on the surfaces of MgO nanoparticles was one of the primary factors that affect their antibacterial activity. The authors suggested that MgO nanoparticles could be utilized as an effective antibacterial agent to enhance food safety. The antimicrobial effect of MgO-NPs on the morphology of E. coli studied by Castillo et al. [49] by transmission electron microscopy (TEM) is presented on Fig. 11.3. The high pH 9, generated by suspending MgO-NPs, caused damage of individual cells (Fig. 11.3B). The dominant effect of the action of MgO-NPs on bacterial cells is the loss of normal healthy form and condensation of the cytoplasmic material (Fig. 11.3C). Higher concentrations of MgO-NPs cause increased permeability of the bacterial envelope and leakage of cytoplasmic material (Fig. 11.3D). Doping another metal ion into the MgO structure, produces lattice defects such as vacancies, which play an important role in modifying the physical and chemical properties of the oxide to a certain extent. Copper doping promotes the generation of oxygen vacancies and increases the zeta potential and antibacterial activity, while causing the size of the particles to decrease [50]. Rao et al. [51] reported that Li-doped MgO exhibits better antibacterial activity, Zn-doped and Ti-doped MgO display poorer antibacterial activity than pure MgO. In contrast, Ohira et al. [52] reported that the antibacterial activity enhanced with increasing Zn content in MgO. MgO nanoparticles showed strong bactericidal activity against both pathogens, grampositive and gram-negative bacteria, such as E. coli, B. cereus, B. subtilis, and S. aureus [45,53]. The antibacterial activity of MgO-NPs increased as the concentrations of nanoparticles increased [54]. The mechanism of antimicrobial activity of MgO-NPs might occur as a result of the cell membrane damage. MgO-NPs attached to the bacterial membranes by electrostatic interactions, causing an increase in membrane permeability and efflux of intracellular contents which in turn lead to death of the bacterial cells [54]. The antibacterial activity of MgO-NPs was found to be dependent upon the size of the nanoparticles. Smaller MgO-NPs particles showed greater activity than larger particles. Sellik et al. [55] compared the effect of the particle size of MgO-NPs on bactericidal activity. MgO (11 nm) exhibited an increased activity against E. coli and S. aureus, whereas MgO (25 nm) showed

4. Antibacterial activity of nanomaterials

252

11. Antibacterial activity of metal oxide nanoparticles

FIGURE

11.3 Transmission electron microscopy (TEM) micrographs of Escherichia coli, (A) control cells without any treatment; (B) cells growth in the medium at pH 9; (C) cells incubated with 0.5 mg/mL MgONPs, and (D) cells incubated with 1 mg/mL MgO-NPs [49].

a weaker effect on E. coli but had no activity against S. aureus. In another study, MgO-NPs sintered at lower temperature had a smaller particle size and showed greater antibacterial activity toward both gram-positive (S. aureus) and gram-negative bacteria (E. coli) [56]. The studies done by Makhluf et al. [57] also demonstrated that the bactericidal effect of MgO-NPs is better when the particle size is smaller. The penetration of smaller particles into bacterial cells was most pronounced. CaONPs possess excellent antimicrobial potential and capability to inactivate microbial endotoxin [58,59]. Due to CaONPs unique structural and optical properties they can be used as a potential drug delivery agent, such as delivery of chemotherapeutic agents— CaONPs are safe material to human beings and animals [60]. The reason of antibacterial effect of CaONPs is owing to the active oxygen and the alkaline pH when dissolved in water, which in turn cause destruction to the bacterial cell membrane and consequently death [61]. The antibacterial activity of CaONPs studied to against E. coli and S. aureus using growth curve and disc diffusion methods under physiological conditions (pH 5 7.4). In the presence of CaONPs optical density of bacterial cell suspension was decreased with the increase of CaONPs with time. The MIC of CaONPs was found to be 10 μg/mL for both bacteria. It has been verified that the antibacterial mechanism of CaO and MgO nanoparticles is brought about by the generation of ROS which is higher in alkaline pH due to dehydration of CaONPs. The alkaline pH in CaO leads to effective killing of E. coli, Listeria monocytogenes, and Salmonella typhimurium strains [42]. The ROS interact with carbonyl group present in bacteria cell wall peptide linkages/polyunsaturated phospholipids and influence on proteins

4. Antibacterial activity of nanomaterials

11.4 Antibacterial activity of aluminum oxide nanoparticles

253

degradation consequently, leading to the destruction of bacteria cell wall [48]. The existence of active oxygen, such as O22, on the surface of MgO and CaONPs, has been observed in mechanistic studies [45]. The results indicated that, MgO and CaONPs alone or in combination [62] with other disinfectants showed excellent antibacterial effect. These nanoparticles also are low cost, biocompatible, and available materials. These properties make them promising antibacterial agent [63] which can be utilized in environmental preservations, in food processing and as a safe new therapeutic for bacterial infections [64].

11.4 Antibacterial activity of aluminum oxide nanoparticles Aluminum oxide nanoparticles (Al2O3 NPs) are suitable nanomaterials and were found to be applicable in different aspects of biomedical science and biotechnology such as: an abrasive material, an absorbent in heterogeneous catalysis, a biomaterial and in ceramic industry [65]. Some observed that biotoxicity may hinder their use as vehicles for intracellular delivery of therapeutic nucleic acids and proteins. They are thermodynamically stable particles over a wide temperature range and corundum structure with oxygen atoms adopting hexagonal close packing with Al31 ions filling two-thirds of the octahedral sites in the lattice [66]. The co-precipitation method is the most commonly used for the synthesis of Al2O3 NPs in antimicrobial activity testing. The X-ray diffraction result confirmed aluminum oxide has the crystallite size of 35 nm. The antibacterial activity of Al2O3 NPs was tested against various bacterial strains Escherichia coli, Proteus vulgaris, Staphylococcus aureus, and Streptococcus mutans by using agar well diffusion method. The results indicated that Al2O3 NPs synthesized by co-precipitation method showed effective antibacterial activity against pathogenic bacteria. The inhibitory effect of Al2O3 NPs increased with the increase in concentration. The diameter of inhibitory zone shows the degree of susceptibility of microorganisms. The strain susceptible to Al2O3 NPs exhibited a larger zone of inhibition (E. coli), while resistant strain exhibited a smaller zone of inhibition (P. vulgaris) [67]. Al2O3 NPs show strong antimicrobial activities owing it to the large surface area. Mentioned particles (179 nm sized) over a wide concentration range (101000 μg/mL) were incubated with bacterium E. coli in a study by Sadiq et al. [68] and a mild antigrowth effect has been observed only at very high concentrations. This is due to surface charge interactions between positively charged particles at near-neutral pH and negatively charged E. coli cells. Electrostatic interaction has led to the adhesion of nanoparticles on the surface of bacteria [69]. The antimicrobial property of these metal oxides has been attributed to the generation of ROS, which causes cell wall disruption and subsequent cell death [70]. AlNPs can act as free radical scavengers because they are able to rescue cells from oxidative stress-induced cell death in a manner that appears to be dependent on the structure of the particle but independent of its size [71]. Also, a small decrease was reported in extracellular protein content of the bacterium. In a similar study, AlNPs showed antigrowth effects of 57% on B. subtilis, 36% on E. coli and 70% on Pseudomonas fluorescens, originating from direct attachment of the NPs to cell walls of these bacteria [72]. In addition to the application in the form of pure NPs, aluminum oxide nanomaterials have shown potential antimicrobial properties against E. coli and Staphylococcus epidermidis when used in the form of aluminum oxidesilver

4. Antibacterial activity of nanomaterials

254

11. Antibacterial activity of metal oxide nanoparticles

nanocomposite, indicating the potential biomedical applications of nano-aluminum oxide as composite structures [73]. In another study, aluminum oxide nano-coatings, in the form of Fe3O4/aluminum oxide core/shell magnetic NPs, showed an incredibly magneticallyderived photothermal killing effects on a range of gram-negative, gram-positive, and drug-resistant bacterial isolates. In this intelligently designed nanocomposite, aluminum oxide shell functions as a recognizer of bacterial cells that subsequently are killed photothermally through the Fe3O4 core [74].

11.5 Antibacterial activity of silver oxide nanoparticles Silver and its inorganic compounds such as: AgNO3, Ag2O, and silver halides, are the most commonly used as antimicrobials and additives in consumer, health-related and industrial products. The antibacterial activity of Ag2O originates from the released Ag1 ions, direct interaction nanoparticles with the cell envelope and through the generated ROS. Silver ions have expressed an oligodynamic effect with a minimal development of microorganism’s resistance. Low concentrations of silver ions are not toxic, but high concentrations can cause cytotoxicity. The antibacterial actions of silver ions are very complex, including physicochemical interactions with biomolecules and ROS generation. Silver ions dominant binding to SH but also with NH2, COO2 groups of amino acid residues of proteins and other biomolecules from bacterial cell envelope, leading to disruption of these structures. The silver ions penetrate into the cell, inactivates various proteins and formation of sparingly soluble chloride causing inhibition of cell respiration. Their interaction with nucleic acids can affect replication and translation processes. It was found that the Ag1 ions could induce the ROS generation and further accelerate the killing of bacteria [75]. Several studies have reported the Ag2O NPs are more active against gram-negative species than gram-positive species [76,77]. This difference in activity is due to the difference in cell envelope composition of both types of bacterial species. Antibacterial activity of Ag2O NPs increases with increasing particle concentrations [76,78,79]. Several studies have reported that the morphology of Ag2O particles affects antibacterial activity. The antibacterial effect of the cubic Ag2O particles is better in comparison to that of the octahedral particles [78]. The studies done by Kim et al. [80] demonstrated that the antibacterial activities of Ag2O microparticles were in the following order: cubes . cubes with small voids . cubes with large voids . octapods. Silver compounds have been used in medicine for prevention and treatment of infections, burns and wounds. Orthopedic and dental implant infections are significant because of their morbidity and usually require the removal or replacement of installed materials. Incorporation of silver antimicrobial agents in a bone implant material alone proved to be very successful in the prophylaxis [81,82]. The Ag2O NPs-decorated Ta2O5 and TiO2 nanotubes on Ti6Al4V substrates and showed bactericide effect against E. coli, promoted the formation of bone-like apatite layer and improved the osseointegration of these implant materials [83,84]. Similar results were reported by Lv et al. [85] that the addition of an increased amount of Ag2O NPs enhances antibacterial capability of the resultant Ag-incorporated TiO2 coating. Ag2O NPs showed excellent antibacterial activities against the two dental bacteria S. mutans and Lactobacilli sp. [79]. Materials with silver compounds

4. Antibacterial activity of nanomaterials

255

11.6 Antibacterial activity of copper oxide nanoparticles

TABLE 11.3 oxides.

Crystal structure, space group, values of bandgap and point of zero charge (PZC) of metal

Oxide

Crystal structure

Space group

Bandgap (eV)

PZC

References

Cu2O

Cubic

Pn3m

2.22.5

CuO

Monoclinic

C2/c

1.211.55

8.6

[90,91]

ZnO

Hexagonal

P63mc; C6v

3.20

9.3; 8.9

[92,93]

TiO2 and Rutile

Tetragonal

P42/mnm

3.02 and 3.00

3.8

[94,95]

TiO2 (anatase)

Tetragonal

I41/amd

3.2 and 3.21

[94,95]

TiO2 (brookite)

Orthorhombic

Pbca

2.96 and 3.13

[94,95]

[89]

are widely used for wound treatments with respect to antimicrobial activity. The methylcellulose hydrogel with Ag2O NPs showed excellent antimicrobial activity against: S. aureus, Klebsiella pneumoniae and E. coli; and burn wound healing [86]. Babu et al. [87] developed silk fibroin (Ag2O-SF) spuns which showed synergistic wound healing and antibacterial property when compared to individual Ag2O and SF. Ag2O-SF spuns showed excellent antibacterial activity against both pathogenic (S. aureus and Mycobacterium tuberculosis) and non-pathogenic (E. coli) microorganisms. In vitro wound healing assay indicates the migration of cells in the scratch, treated with extract of Ag2O-SF spuns is complete within 24 h. Chitosan is a natural biopolymer and has attracted considerable interest due to its biological properties, such as antimicrobial activity, antitumor activity, and immune enhancing effect. Due to antimicrobial property, chitosan film may be used in medicine and food packaging. The antimicrobial effect of chitosan occurred without migration of active agents. The incorporation of Ag2O NPs into chitosan matrix significantly improving antimicrobial activity of chitosan film [88]. Ag2O NPs as a semiconductor with a band gap of 1.46 eV (Table 11.3) can generate ROS. They can interact with cell envelope, penetrate inside the cell and disturb the cytoplasmic functions and prove fatal for the bacteria. Chen and Liu [96] fabricated photocatalytic paper by incorporating cellulose fibers with graphite fibers which were pre-loaded with Ag2O NPs, that is, Ag2O@graphite fibers. The synthesized photocatalytic paper showed strong antibacterial properties towards E. coli in both dark and office lighting conditions. Radical-trapping experiments showed that Ag2O NPs under UV, visible and nearinfrared light irradiation generate O22 and OH radical species.

11.6 Antibacterial activity of copper oxide nanoparticles Copper is an essential trace element for living organisms due to its role in many enzymes important for metabolism. High concentrations of copper ions are toxic to a bacteria cells and can cause cell death [97101]. Copper has long been known to have antimicrobial activity and is used in agriculture, medicine, healthcare, and industry, drinking water treatment and transportation. With antibiotic resistance, research on copper as an antimicrobial agent has again became very attractive. Copper oxides exist in two different

4. Antibacterial activity of nanomaterials

256

11. Antibacterial activity of metal oxide nanoparticles

forms: cupric oxide (CuO) and cuprous oxide (Cu2O). Both oxides are p-type semiconductors with a band gap of 2.22.5 eV for Cu2O and 1.211.55 eV for CuO (Table 11.3). The CuO is thermodynamically more stable than Cu2O. CuONPs exhibit a broad spectrum of antibacterial activity against gram (1) and gram (2) bacteria (Table 11.4). The physicochemical characteristics of copper oxide nanoparticles, such as size, shape, crystal structure and concentration of particles, are directly associated with enhanced antibacterial effects. Some study suggests that Cu2O have better antibacterial activity then CuO because it can release more toxic Cu1 [110,115]. The bactericidal action of Cu2O must be based on the binding nature of released Cu1 ions with thiol groups from amino acid residua from proteins [116]. Antibacterial activity of copper oxides are highly dependent on the concentration of nanoparticles in the test medium. Several studies have presented

TABLE 11.4 Antibacterial oxides (Cu2ONPs and CuONPs): particle size, shape, and tested bacteria. Oxide

Size (nm)

Shape

Bacteria

References

CuO

1820



Streptococcus mutans

[102]

CuO

B92

Plate, grain, and Streptococcus iniae, Streptococcus parauberis, Escherichia needle coli, and Vibrio anguillarum

CuO

33

Spherical and irregular

Staphylococcus, Salmonella, Pseudomonas, and E. coli

[104]

CuO

5080

Spherical

E. coli, Proteus mirabilis, and Klebsiella pneumoniae

[105]

CuO

7 and 14

Spherical

Staphylococcus aureus and E. coli

[106]

Cu2O

10, 50, 100, and 200

Irregular

E. coli

[107]

CuOcotton

1015

Irregular

S. aureus and E. coli

[108]

Cu2O and CuO

B40 and B30

Irregular

E. coli

[109]

Cu2O and CuO

60 and 50150

Spherical

S. aureus and Klebsiella

[110]

CuO

510

Spherical

S. aureus, Klebsiella aerogenes, E. coli and Pseudomonas desmolyticum

[111]

CuO

2095

Equi-axes

S. aureus, MRSA, E. coli, S. epidermidis, Pseudomonas aeruginosa, and Proteus spp.

[112]

CuO

20

Spherical

Aeromonas hydrophila, Pseudomonas fluorescens, and Flavobacterium branchiophilum

[113]

CuO

B35

Spherical and sheet

E. coli, Proteus vulgaris, Bacillus subtilis, and Micrococcus luteus

[114]

B257 3 42

4. Antibacterial activity of nanomaterials

[103]

11.6 Antibacterial activity of copper oxide nanoparticles

257

that CuONPs showed stronger antibacterial activity than bulks [117120]. The smaller NPs have more efficient antibacterial activity and higher affinity to the bacterial cells, that is, can easily penetrate into bacterial cells and may release copper ions faster upon dissolution. While studying the effect of particle size on antibacterial activity, Xiong et al. [107] reported that the decrease in size of the Cu2ONPs resulted in the increase in antibacterial activity. Shape of particles is another crucial factor that determines the antibacterial activity of the copper oxides. Ananth et al. [103] and they found that the plate-like CuONPs had more potent antibacterial activity than grain- or needle-shaped ones. Laha et al. [114] reported, according scanning electronic microscopic (SEM) studies, that antibacterial effect of CuONPs depends on size, specific morphology and nature of the bacterial strain (Fig. 11.4). From the SEM images can see that spherical shaped CuONPs produced more membrane damage on E. coli compared to CuONPs and sheet shaped induced more membrane damage on B. subtilis. From the SEM images, it can be seen that spherical CuONPs created more membrane damage on E. coli compared to sheet shaped CuONPs. Sheet shaped NPs caused more membrane damage on B. subtilis. The mechanism of antibacterial action of copper oxides nanoparticles can be associated with: the release of copper ions (Cu1 or Cu21) from oxides; direct contact of CuONPs nanoparticles with bacterial cell envelope and the production of ROS. Copper ions released from nanoparticles can react with negatively charged cell envelope components through electrostatic attraction and rupture it, thereby leading to protein denaturation and cell death. In addition, copper ions can penetrate in a bacterial cell and disrupt biochemical processes [121].

FIGURE 11.4 Scanning electronic microscopic (SEM) micrographs of different shaped CuONPs treated or mock-treated gram (2) E. coli and gram positive Bacillus subtilis bacterial cells [114].

4. Antibacterial activity of nanomaterials

258

11. Antibacterial activity of metal oxide nanoparticles

Copper ions may bind with DNA molecules and lead to disorder of the helical structure by crosslinking within and between the nucleic acid strands [122]. Xiong et al. [107] suggested that mechanisms of inactivation of E. coli by Cu2ONPs in the dark are result of adsorption of the nanoparticles on the bacterial cell envelope. This interaction may cause denaturation or degradation of bacteria, resulting in their inactivation. The antibacterial activity of copper oxides nanoparticles could be geenerated by ROS originated through either an oxidative or a reductive process. The generation of ROS can be related to the presence of copper ions (Cu1 and Cu21) in the liquid medium as well as on the surface of the copper oxide nanoparticles. Copper oxides (ions) react with adsorbed O2 or endogenous H2O2 to generate hydroxyl radicals in a process analogous to the Fenton reaction to generate O22 or hydroxyl radicals OH (Eqs. 11.1311.16) [109]. The ROS production from copper oxides generally originates from their electrondonating nature. It is assumed that bacterial cells, Cu21 ions are reduced by thiol groups from cysteine amino acid residua from protein; to Cu1 ions (Eq. 11.13). The Fenton-like reaction generates O22 and hydroxyl radicals from H2O2, Eqs. (11.14)(11.16) [123].  Cu21 1 Protein 2 CysðSÞ-Cu1 1 protein Cys S1 (11.13) Cu1 1 O2 -Cu21 1  O2 2

(11.14)

21 1 1 H2 O2 Cu1 1  O2 2 1 2H -Cu

(11.15)

Cu1 1 H2 O2 -  OH 1 OH2 ðFenton-like reactionÞ

The level of O2 decreased fast because it reacts with Cu Cu1 (Eq. 11.17) [109]. 2

21

(11.16)

ions from CuO to form

1 Cu21 1  O2 2 -Cu 1 O2

(11.17)

The amount generated of OH2 radicals is higher for CuO than Cu2O [109]. Recycling redox reactions between Cu21 and Cu1 ions can occur at the bacterial cell surface and inside the cell [102]. The production of more amount of ROS can lead to lipid peroxidation, oxidation of proteins and damage to nucleic acids. That is, lead to oxidative stress of the bacterial cell. Xiong et al. [107] reported that H2O2 and OH species generate through a reductive process on surface of Cu2ONPs under visible light. These species were partially responsible for better bacterial inactivation under irradiation.

11.7 Antibacterial activity of zinc oxide nanoparticles Zinc is an essential trace metal for various metabolic processes in most of the living organisms, while in the higher amounts is potentially toxic. ZnO-NPs exhibit a broad spectrum of antibacterial activity and are widely used in cosmetics products, in medicine for wound healing, treatment of acne or fungal infection, because its less toxic to humans than CuONPs and AgONPs and low-cost synthesis [99,124127]. ZnO can exist in three crystalline forms: hexagonal Wurtzite, cubic zinc blende, and cubic rock salt. Wurtzite structure is the most stable and the most represented and is described by the space group P63mc or C6v (Table 11.3). The other two crystalline forms of ZnO are less stable. ZnO is a

4. Antibacterial activity of nanomaterials

11.7 Antibacterial activity of zinc oxide nanoparticles

259

semiconductor with a wide band gap (3.3 eV) and has high UV absorption efficiency and good transparency to visible light (Table 11.3). Several comparative studies showed that ZnO has better antibacterial properties than various metal oxides. Dasari et al. [128] investigated the toxicity of MNPs (CuO, ZnO, TiO2, and Co2O3) to E. coli. The ranking of antibacterial activity according to lethal concentrations (LC50) for the E. coli, for studied MNPs was ZnO , CuO , TiO2 , Co3O4 and ZnO , CuO , Co3O4 , TiO2 under light and dark conditions, respectively. Release of metal ions observed in the E. coli cells treated with ZnO and CuONPs, while at Co3O4 and TiO2 were not significant. Similarly, the study by Jones et al. [12] reported that nanoparticles of ZnO have significantly higher antibacterial effects on S. aureus than nanoparticles of: TiO2, Al2O3, CuO, CeO2, and ZnO, under normal laboratory lighting condition. The mechanism of antibacterial action of ZnO-NPs is complex and encompassing: the release of Zn21 ions from ZnO-NPs; direct contact of ZnO-NPs with bacterial cell envelope resulting in destruction of cell integrity and the production of ROS, resulting in oxidative stress and cell wall damage, enhanced membrane permeability and uptake of toxic dissolved Zn21 ions [129131]. The released Zn21 from ZnO-NPs ions can have significant effect on the permeability of the bacterial membrane and can also inactivate of various biomolecules, especially enzymes and lead to cell death [132]. Li et al. [133] reported that the antibacterial activity of ZnO-NPs is mainly associated with the release of Zn21 ions. The antibacterial activity of ZnO-NPs may result from electrostatic interaction between nanoparticles and bacterial cell envelope. The antibacterial activity of ZnO-NPs depends on the size of NPs. ZnONPs showed excellent bactericidal potential which increased with an increase in surface-to-volume ratio due to a decrease in particle size of nanoparticles [13,120]. Namely, NPs of smaller sizes and large surface area penetrate easily into bacterial membranes, which results in higher antibacterial activity [130]. The size-dependent antimicrobial response of ZnO-NPs was confirmed in gram (1) bacteria as B. subtilis, S. epidermidis, Streptococcus pyogenes, S. aureus and MRSA and gram (2) bacteria: E. coli, Salmonella paratyphi B, and K. pneumoniae [11,12,133135]. On the contrary, Adams et al. [136] reported that size of ZnO-NPs has no greater significance on antibacterial activity. Important factor responsible for antibacterial activity of ZnO-NPs is concentration [134]. Some studies have indicated that ZnO-NPs at a concentration of between 3 and 10 mM caused 100% inhibition of bacterial growth due to disorganization of E. coli membranes, which increases membrane permeability leading to accumulation of nanoparticles in the bacterial membrane and cytoplasmic regions [131]. Synthesized ZnONPs nanoparticles with diameter of 12 nm in concentration of 3 mM are able to slow down 100% the growth of E. coli. It is a result of disorganization of the membranes, which increases the membrane permeability leading to the accumulation of nanoparticles in the bacterial membrane and cytoplasm regions of the cells. An alternative hypothesis suggested that the binding of ZnO-NPs to the bacterial surface is due to electrostatic forces that directly kill bacteria [28]. A different protective mechanism of ZnO has been suggested in that ZnO may protect intestinal cells from E. coli infection by inhibiting the adhesion and internalization of bacteria by preventing the increase of tight junction permeability and modulating cytokine gene expression The shape of ZnO-NPs was found to affect the mechanism of their internalization into bacteria, and it was observed that rods and wires penetrate into cell walls of bacteria more easily than spherical ZnO-NPs [137] as well as flower-shaped ZnO-NPs show higher

4. Antibacterial activity of nanomaterials

260

11. Antibacterial activity of metal oxide nanoparticles

antimicrobial activity against S. aureus and E. coli than the spherical ones [138]. The effect of particle morphology on antimicrobial activity was studied by Cai et al. [139] against E. coli and S. aureus bacteria. Zinc oxide nanoflowers showed antibacterial activity in an order of petal flowers . fusiform flowers . rod flowers. Differences in morphological characteristics of particles such as, specific surface area, pore size, and Zn-polar plane; play a decisive role in antimicrobial activity. Numerous studies indicated the occurrence of ROS (superoxide radical, hydroxyl radical, and singlet oxygen) on surface of ZnO-NPs is an important factor in antibacterial activity FIGURE 11.5 (A) Transmission electron microscopy (TEM) micrograph showing adherence of bare zinc oxide nanoparticles (ZnONPs) on the surface of Escherichia coli, (B) TEM micrograph of Escherichia coli exposed to thioglycerol capped ZnO-NPs, (C) Scanning electronic microscopic (SEM) micrograph showing deformed E. coli engulfed by bare ZnONPs, supported by energydispersive X-ray spectroscopy spectrum showing Zn L-X-ray, and (D) TEM micrograph shows damage of E. coli cell due to interaction with ZnO-NPs [140].

4. Antibacterial activity of nanomaterials

11.8 Antibacterial activity of iron oxide nanoparticles

261

[132]. ZnO has been found to possess high photocatalytic efficiency among metal oxides. The average concentration of total ROS produced within a specified irradiation period (48 h) followed the order for nanoparticles TiO2 . ZnO . Al2O3 . SiO2 . Fe2O3 . CeO2 . CuO and ZnO . TiO2 for bulk oxide [132]. The amount of ROS generated from the surface of ZnO should increase proportionally with increasing concentrations of nanoparticles. The bacterial survival rate decreases with an increasing average concentration of ROS. Dutta et al. [140] reported that larger bare ZnO-NPs has higher ROS production and better antibacterial activity of smaller capped ZnO-NPs with thioglycerol (TG). The lesser antibacterial effect of ZnO-NPs was attributed to the capping agent. TEM studies showed adherence of bare ZnO-NPs (Fig. 11.5A) and TG capped ZnO-NPs (Fig. 11.5B) on the surface of E. coli. Deformation of cellular morphology, cell swelling and membrane ruptures were observed in interaction with bar ZnO-NPs as a consequence of the greater release of ROS (Fig. 11.5C and D). In order to improve photocatalytic properties and antibacterial activity of ZnO-NPs, metal ions have been added into its crystal structure to narrow or split the band gap. Some metal ions, such as: Ag1, Cu21, Mg21, Co21, and rare earth metal ions; have been doped into ZnO-NPs [141145]. The consequences of doping of ZnO-NPs are reflected in an enhancement of crystal defects, reduced particle size, increases solubility, and also affects the optical properties by shifting the optical absorption towards the solar region. Antibacterial studies showed that doped ZnO-NPs have a higher activity and production of ROS.

11.8 Antibacterial activity of iron oxide nanoparticles Iron oxides were frequently used in a large variety of biomedical and biotechnological applications. Nanoparticles synthesized from these oxides have also been employed for diagnostic imaging (magnetic resonance imaging), cancer treatment (magnetic hyperthermia and thermal ablation), scale-up bioseparation processes and biosensing-based applications [146]. Iron oxide magnetic nanoparticles are physically and chemically stable, biocompatible, and environmentally safe [147,148], thus presenting unique characteristics for clinical application. Iron oxide (III) is very stable oxide, it crystallizes in hexagonal form and is found in nature as the mineral hematite α-Fe2O3. The nanostructures of this oxide take different forms as they are nanowires, nanotubes, nanospheres, etc. [149]. Methods of synthesis are directly related to size, shape, coating and stability of iron oxide nanoparticles (FeO NPs) [150,151]. When FeO NPs [Fe3O4 (magnetite) or -Fe2O3 (maghemite)] reach smaller sizes (about 1020 nm for iron oxide), superparamagnetic properties become evident, so that the particles reach a better performance for most of the aforementioned applications [152]. Particles smaller than 10 nm are easily excreted from the body [153], what reduces their blood-circulating time. Further, hydrophobic and negatively charged nanoparticles tend to suffer proteic opsonization and are quickly recognized by phagocytic cells [154], also resulting in faster clearance. These and other Fe2O3 NPs limitations can be overcome by an adequate surface-coating. Different organic and inorganic coatings have been examined in studies showing that shape, spatial configuration, and nature of the coating play an important role on the nanosystem’s performance.

4. Antibacterial activity of nanomaterials

262

11. Antibacterial activity of metal oxide nanoparticles

The Fe2O3 NPs bactericidal effect against E. coli and S. aureus has been reported, where an increase of this effect is observed, as the concentration of Fe2O3 NPs increases [155]. A bactericidal effect has also been noticed on Pseudomonas aeruginosa with a MIC of 0.06 mg/L [156]. Another study reports on the bactericidal activity of nanostructured hematite against a variety of Gram-positive and Gram-negative bacteria: P. aeruginosa, S. aureus, K. pneumoniae, Lysinibacillus sphaericus, and Bacillus safensis [157], proposing some mechanisms of action depending on the activity observed in each stage of the growth of the bacteria in question. A bactericidal effect of NPs of Fe2O3 against S. epidermidis has also been determined [158]. Studies suggest that the potential of magnetic nanoparticles to generate microbial toxicity is due to a series of interactions, including membrane depolarization with consequent impairment of cell integrity [3], production of ROS with lipid peroxidation and DNA damage [159], and release of metal ions that affect cellular homeostasis and protein coordination [160]. Iron oxide and free iron ions can induce the formation of hydroxyl radical in biological systems via Fenton (Eq. 11.19) and HaberWeiss reaction (Eq. 11.20) [161]. Fe31 can reduced to Fe21 in the presence of superoxide radicals (O22), which are mainly produced in the mitochondria through respiration. Fe21 1 H2 O2 -Fe31 1 OH2 1  OH

(11.18)

1 OH 1  OH

(11.19)

21

Fe

1 H2 O2 -Fe

31

2

 O22 1 H2 O2 -O2 1 OH2 1  OH

(11.20)

It is believed that nanoparticles have the ability to adsorb and penetrate into biofilms due to their physicochemical characteristics, such as surface charge, hydrophobicity and high surface area ratio by volume [26,162]. Positively charged and neutral Fe2O3 NPs promoted higher reduction of cells of Streptococus mutans biofilms with respect to negatively charged counterparts [163], highlighting the influence of the surface properties of magnetic nanoparticles on their antibiofilm activity [160]. Li et al. [30] used the sensitive bacterial strain E. coli K12 to investigate the antibacterial and growth-inhibiting effects of ferric oxide nanoparticles and to elucidate the process of internalization of ferric oxide nanoparticles. It was found that internalization of the nanoparticles into the cell damaged the cell membranes. The nanoparticles first approached the cell membrane and began to invade, and then destroyed the membrane and embedded into it. After completely penetrating through the cell membrane, the ferric oxide nanoparticles entered the cytoplasm, causing vacuoles to form around these nanoparticles in the cytoplasm. The process whereby the nanoparticles destroyed the membranes was as follows: the ferric oxide nanoparticles approached the cell and adhered to the cell surface; adhered to the cell’s outer membrane and tore the outer membrane; uplifted the membranes; adhered closely to the outer membrane and made it separate from the cell membrane; and finally, broke the cell membrane, causing the release of cytoplasm. The elemental analysis results showed that there were high levels of Fe in the regions around the highly dense particles in bacteria, indicating that the highly dense particles were ferric oxide nanoparticles. Vasantharaj et al. [164] reported that FeO NP alone and FeO NPs incorporated cotton fabrics exhibited effective bactericidal activity against E. coli and

4. Antibacterial activity of nanomaterials

11.9 Antibacterial activity of titanium oxide nanoparticles

263

FIGURE 11.6

Scanning electronic microscopic (SEM) micrographs of iron oxide nanoparticles (FeO NPs) untreated and treated pathogens (AC) E. coli and (BD) Staphylococcus aureus [164].

weaker against K. pneumonia and S. aureus. The SEM study showed bacterial cell shrinkage and that the cells had deformities when treated with FeO NPs, whereas the untreated cells were in good condition and morphology (Fig. 11.6).

11.9 Antibacterial activity of titanium oxide nanoparticles Nanoparticulate of TiO2 is applied in various fields: medicine, fabrics, water treatment, purification, and removal of pollutants from air and water. TiO2 NPs has received a great deal of attention as an antibacterial agent due to its broad effect on gram negative and gram positive bacteria, chemical stability, low toxicity and cost. Antibacterial activity of TiO2 is related to generation of ROS, such as: OH and O22 under UV radiation, that are directly involved in the oxidation processes leading to the degradation of microorganisms. TiO2 NPs can lead to mechanical damage to bacterial cells. The study done by PigeotRe´mya et al. [165] suggested that the contact between TiO2 NPs and E. coli K-12 in the dark leads to damages to the outer membrane integrity. Matusunga [166] first reported that photoactivated TiO2 showed the bactericidal activity. Foster et al. [167] reviewed that TiO2 materials and substrates showed a wide range of antimicrobial activity including Gram-negative and Gram-positive bacteria, filamentous and unicellular fungi, algae, protozoa, mammalian viruses and bacteriophage.

4. Antibacterial activity of nanomaterials

264

11. Antibacterial activity of metal oxide nanoparticles

FIGURE 11.7 Scanning electronic microscopic (SEM) micrographs of Pseudomonas putida (A, B, E, and F) and Staphylococcus aureus (C, D, G, and H) biofilms on TiO2-coated glass slides (AD) and TiO2-coated filters (EH) without ultraviolet (UV) treatment (A, C, E, and G) and after UV irradiation (B, D, F, and H) [169].

There are several studies focusing on the effect of ROS created on TiO2 NPs surfaces by UV irradiation on bacterial cell morphology [165,168,169]. SEM studies done by Jalvo et al. [169] showed that in UV non-irradiated bacteria on surface of TiO2 coating, the morphology of Pseudomonas putida cells (rod shaped) (Fig. 11.7A and E) and of S. aureus (roundshaped) were retained (Fig. 11.7C and G). After 2 h of UV irradiation all bacterial cells were damaged (Fig. 11.7B, D, F, and H). Significant amounts of ROS were detected in bacterial cells in all TiO2 irradiated specimens. The photocatalytic production of ROS on TiO2 surfaces greatly depends on the properties of the nanoparticles: crystal structure, particle size, surface area and porosity. TiO2 in nature generally exists in three crystalline structures: anatase, rutile and brookite, although monoclinic and orthorhombic are found in negligible quantities. The structures of the main three forms are shown in Table 11.1. Under ambient conditions macrocrystalline anatase and brookite are metastable and are readily transformed to stable form rutile when heated. The anataserutile phase transformation temperature depends on the particle size as well as the specific TiO2 surfaces. Thermodynamic stability of TiO2 nanocrystalline phases are particlesize dependent. If nanoparticle sizes of the three phases are equal, anatase is most thermodynamically stable at sizes less than 11 nm, brookite is most stable for crystal sizes between 11 and 35 nm, and rutile is most stable at sizes greater than 35 nm [170]. Compared to rutile and brookite, anatase is most represented in photocatalytic tests because it shows the best photocatalytic activity. TiO2 with a higher amorphous phase content exhibits weaker photocatalytic activity [171]. Li et al. [172] reported that antibacterial activity of anatase (antibacterial rate B20%) is superior to that of rutile (B10%) and amorphous TiO2 (B3%). Several studies have reported that the highest photocatalytic efficiency and the best antibacterial activity is

4. Antibacterial activity of nanomaterials

11.9 Antibacterial activity of titanium oxide nanoparticles

265

achieved when TiO2 contains a mixture of anatase and rutile [173,174]. Pantaroto et al. [173] evaluated the antibacterial activity of TiO2 films with different crystalline phases (anatase, rutile and a mixture of both) on the oral multispecies biofilm composed of Streptococcus sanguinis, Actinomyces naeslundii, and Fusobacterium nucleatum. After 1 h of UV-A light activation anatase and mixture of TiO2 films showed significant antibacterial effect on multispecies biofilm with a reduction about 99% and 99.9% of bacterial counts, respectively. Rutile films had no antibacterial effect on multispecies biofilm. The main limitation of practical primary TiO2 NPs is that their band gape lies in the UV part of the spectrum, which makes up only a small fraction of the solar radiation energy (B5%), as well as a large degree of electron and hole recombination. Therefore great attention has been paid to the modification of TiO2 in order to improve its photocatalytic efficiency, i. antibacterial activities. Improving the efficiency of TiO2 can be achieved modification of surfaces of photocatalysts by metals, doping with transition metal ions, formation of nanocomposites, and more [94]. Modifying the surface of TiO2 NPs with metals such as Ag, Pt, Pd, and Au aims at reducing the recombination of e2/h1 pairs, and therefore improving the efficiency of photocatalysis. By depositing silver on the TiO2 NPs (Degusa P25), the absorption of light is shifted to the visible part of the spectrum, and induces an increase in the photocatalytic activity under both UV and visible ligh [175]. Due to its antimicrobial properties, silver nanoparticles are very often used in the manufacture of composite materials with TiO2 NPs. Ag/TiO2 nanocomposite particles exhibit very good antibacterial properties without the effects of UV light, that is, in daylight and in the dark [74,176,177]. Chen et al. [178] reported that Ag/TiO2 the presence of the TiO2 matrix enhances the bactericidal action effect of silver nanoparticles. The antibacterial activity of the Au/TiO2 composite was weaker than the TiO2 sample. Similar results was reported by Armelao et al. [179] that Au NPs on the TiO2 matrix increases photoactivity, whereas the antimicrobial activity of Au/TiO2 films is retarded with respect to pure TiO2. A wide range of transition metal ions, such as: Ag1, Cu21, Co21, Ni21, and Fe31, have been used as dopants for TiO2 to improve antibacterial efficacy [180184]. Antibacterial studies showed that doped TiO2 materials exhibit better activity against the bacteria tested, compared to results with transition metal compounds or TiO2 NPs efficacy. The better antibacterial effect of a metal ions doped TiO2 NPs, can be attributed to the release of dopant ions and production of larger quantities of ROS. Co21 and Ni21 ions are bioelements while at higher concentrations they may show antibacterial activity [185187]. The role of the metal ion as a dopant can be twofold, it can improve adsorption of photons into visible spectral regions and/or act as electron and electron hole traps, reduces the degree of recombination to the electron/hole [94]. Doping of TiO2 with transition metal ions can cause surface modifications as well as crystal defects. Modifications and defects lead to changes in photocatalytic properties. It is necessary to determine the optimum amount of dopant to achieve the desired improving the photocatalytic efficiency of TiO2. Apatite biomaterials are often used for coating titanium or other metals implants to ensure their osseointegration. The mechanical stability of the interface between the apatite coating and metal substrate could be a problem either during surgical operation or after implantation for a period of time. Another problem may be the occurrence of infection after implant placement, because bacteria on the apatite surface attachment easily [97,188,189]. Composite biomaterials of apatite and TiO2 NPs have advantageous

4. Antibacterial activity of nanomaterials

266

11. Antibacterial activity of metal oxide nanoparticles

characteristics: better mechanical properties after calcination or sintering and show antibacterial activity [190192]. Ti41 doped calcium hydroxyapatite crystals showed the bactericidal effect even in the dark although TiO2 shows it only under UV irradiation [193].

Acknowledgements This chapter was supported by the Ministry of Education, Science and Technological Development, Republic of Serbia (Grant No. 172014 and No III43009). The authors gratefully acknowledge Danijela Nikoli´c.

References [1] P.R. Hsueh, New Delhi metallo-β-lactamase-1 (NDM-1): an emerging threat among enterobacteriaceae, J. Formos. Med. Assoc. 109 (2010) 685687. [2] N. Beyth, Y. Houri-Haddad, A. Domb, W. Khan, R. Hazan, Alternative antimicrobial approach: nanoantimicrobial materials, Evid. Based Complement. Altern. Med. 2015 (2015) 246012. [3] R.Y. Pelgrift, A.J. Friedman, Nanotechnology as a therapeutic tool to combat microbial resistance, Adv. Drug. Deliv. Rev. 65 (2013) 18031815. [4] Y. Shen, J. Zhao, C. de la Fuente-Nu´n˜ez, Experimental and theoretical investigation of multispecies oral biofilm resistance to chlorhexidine treatment, Sci. Rep. 6 (2016) 27537. [5] T. Bjarnsholt, The role of bacterial biofilms in chronic infections, APMIS Suppl. 121 (Suppl s136) (2013) 158. [6] K. Alzahrani, A. Niazy, A. Alswieleh, R. Wahab, A. El-Toni, H. Alghamdi, Antibacterial activity of trimetal (CuZnFe) oxide nanoparticles, Int. J. Nanomed. 13 (2018) 7787. [7] J.A. Lemire, J.J. Harrison, R.J. Turner, Antimicrobial activity of metals: mechanisms, molecular targets and applications, Nat. Rev. Microbiol. 11 (2013) 371384. [8] R.G. Pearson, Hard and soft acids and bases, J. Am. Chem. Soc. 85 (1963) 35333539. [9] G. Zhao, S.E. Stevens, Multiple parameters for the comprehensive evaluation of the susceptibility of Escherichia coli to the silver ion, BioMetals 11 (1998) 2732. [10] S. Nair, A. Sasidharan, V.V.D. Rani, D. Menon, S. Nair, K. Manzoor, et al., Role of size scale of ZnO nanoparticles and microparticles on toxicity toward bacteria and osteoblast cancer cells, J. Mater. Sci. Mater Med. 20 (2009) S235S241. [11] K.R. Raghupathi, R.T. Koodali, A.C. Manna, Size-dependent bacterial growth inhibition and mechanism of antibacterial activity of zinc oxide nanoparticles, Langmuir 27 (2011) 40204028. [12] N. Jones, B. Ray, K.T. Ranjit, A.C. Manna, Antibacterial activity of ZnO nanoparticle suspensions on a broad spectrum of microorganisms, FEMS Microbiol. Lett. 279 (2008) 7176. [13] A. Azam, A.S. Ahmed, M. Oves, M.S. Khan, S.S. Habib, A. Memi´c, Antimicrobial activity of metal oxide nanoparticles against gram-positive and gram-negative bacteria: a comparative study, Int. J. Nanomed. 7 (2012) 60036009. [14] O. Mahapatra, M. Bhagat, C. Gopalakrishnan, K.D. Arunachalam, Ultrafine dispersed CuO nanoparticles and their antibacterial activity, J. Exp. Nanosci. 3 (2008) 185193. [15] T. Pandiyarajan, R. Udayabhaskar, S. Vignesh, R.A. James, B. Karthikeyan, Synthesis and concentration dependent antibacterial activities of CuO nanoflakes, Mater. Sci. Eng. C 33 (2013) 20202024. [16] E.A.S. Dimapilis, C.-S. Hsu, R.M.O. Mendoza, M.-C. Lu, Zinc oxide nanoparticles for water disinfection, Sustain. Environ. Res. 28 (2018) 4756. [17] J.M. Andrews, Determination of minimum inhibitory concentrations, J. Antimicrob. Chemother. 48 (2001) 516. [18] P.J.P. Espitia, N.-F.F. Soares, J.S.R. Coimbra, N.J. Andrade, R.S. Cruz, E.A.A. Medeiros, Zinc oxide nanoparticles: synthesis, antimicrobial activity and food packaging applications, Food Bioprocess. Technol. 5 (2012) 14471464. [19] R.S. Liu, Controlled Nanofabrication: Advances and Applications, CRC Press, Boca Raton, FL, 2013 (Chapter 10). [20] S.H. Cha, J. Hong, M. McGuffie, B. Yeom, J.S. VanEpps, N.A. Kotov, Shape-dependent biomimetic inhibition of enzyme by nanoparticles and their antibacterial activity, ACS Nano 9 (2015) 90979105.

4. Antibacterial activity of nanomaterials

References

267

[21] L. Zhong, Y. Yu, H. Lian, X. Hu, H. Fu, Y. Chen, Solubility of nano-sized metal oxides evaluated by using in vitro simulated lung and gastrointestinal fluids: implication for health risks, J. Nanopart. Res. 19 (2017) 375. [22] G.E. Brown, V. Henrich, W. Casey, D. Clark, C. Eggleston, A.F.A. Felmy, et al., Metal oxide surfaces and their interactions with aqueous solutions and microbial organisms, Chem. Rev. 99 (1999) 77174. ´ [23] M. Kopaczynska, M. Vargova´, K. Wysocka-Kro´l, G. Plesch, H. Podbielska, Photocatalytic effects in doped and undoped titania, in: S.A.M. Tofail (Ed.), Biological Interactions with Surface Charge in Biomaterials, The Royal Society of Chemistry, Thomas Graham House, Cambridge, 2012. [24] S. Banerjee, J. Gopal, P. Muraleedharan, A.K. Tyagi, B. Raj, Physics and chemistry of photocatalytic titanium dioxide: visualization of bactericidal activity using atomic force microscopy, Curr. Sci. 90 (2006) 13781383. [25] G. Colo´n-Iba´n˜ez, C. Belver-Coldeira, M. Ferna´ndez-Garcı´a, Nanostructured oxides in photo-catalysis, in: J.A. Rodrı´guez, M. Ferna´ndez-Garcı´a (Eds.), Synthesis, Properties, and Applications of Oxide Nanomaterials, John Wiley & Sons, Inc., Hoboken, NJ, 2007. [26] J.R. Morones, J.L. Elechiguerra, A. Camacho, K. Holt, J.B. Kouri, J.T. Ramı´rez, et al., The bactericidal effect of silver nanoparticles, Nanotechnology 16 (2005) 23462353. [27] I. Sondi, B. Salopek-Sondi, Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria, J. Colloid Interface Sci. 275 (2004) 177182. [28] P.K. Stoimenov, R.L. Klinger, G.L. Marchin, K.J. Klabunde, Metal oxide nanoparticles as bactericidal agents, Langmuir 18 (2002) 66796686. [29] L.C. Ann, S. Mahmud, S.K.M. Bakhori, A. Sirelkhatim, D. Mohamad, H. Hasan, et al., Antibacterial responses of zinc oxide structures against Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pyogenes, Ceram. Int. 40 (2014) 29933001. [30] Y. Li, D. Yang, S. Wang, C. Li, B. Xue, L. Yang, et al., The detailed bactericidal process of ferric oxide nanoparticles on E. coli, Molecules 23 (2018) 606618. [31] E.E. Roden, J.M. Zachara, Microbial reduction of crystalline Iron (III) oxides: Influence of oxide surface area and potential for cell growth, Environ. Sci. Technol. 30 (1996) 16181628. [32] E.P. Ivanova, J. Hasan, H.K. Webb, G. Gervinskas, S. Juodkazis, V.K. Truong, et al., Bactericidal activity of black silicon, Nat. Commun. 4 (2013) 2838. [33] U. Bandyopadhyay, D. Das, R.K. Banerjee, Reactive oxygen species: oxidative damage and pathogenesis, Curr. Sci. 77 (1999) 658666. [34] E. Cabiscol, J. Tamarit, J. Ros, Oxidative stress in bacteria and protein damage by reactive oxygen species, Int. Microbiol. 3 (2000) 38. [35] E.R. Stadtman, R.L. Levine, Free radical-mediated oxidation of free amino acids and amino acid residues in proteins, Amino Acids 25 (2003) 207218. [36] B.S. Berlett, E.R. Stadtman, Protein oxidation in aging, disease, and oxidative stress, J. Biol. Chem. 272 (1997) 2031320316. [37] J. Cadet, J.R. Wagner, DNA base damage by reactive oxygen species, oxidizing agents, and UV radiation, Cold Spring Harb. Perspect. Biol. 5 (2013) a012559. [38] S. Kim, K. Ghafoor, J. Lee, M. Feng, J. Hong, D. Lee, et al., Bacterial inactivation in water, DNA strand breaking, and membrane damage induced by ultraviolet-assisted titanium dioxide photocatalysis, Water Res. 47 (2013) 44034411. [39] J.A. Imlay, S. Linn, DNA damage and oxygen radical toxicity, Science 240 (1988) 13021309. [40] A. Milosevic, Calcium hydroxide in restorative dentistry, J. Dent. 19 (1991) 313. [41] C. Dong, J. Cairney, Q. Sun, O.L. Maddan, G. He, Y. Deng, Investigation of Mg(OH)2 nanoparticles as an antibacterial agent, J. Nanopart. Res. 12 (2010) 21012109. [42] D.H. Bae, J.H. Yeon, S.Y. Park, Bactericidal effect of CaO (Scallop-Shell powder) on foodborne pathogenic bacteria, Arch. Pharm. Res. 29 (2006) 298301. [43] J.F. Siqueira, H.P. Lopes, Mechanisms of antimicrobial activity of calcium hydroxide: a critical review, Int. Endod. J. 32 (1999) 361369. [44] L. Matevosyan, I. Bazukyan, A. Trchounian, Comparative analysis of the efect of Ca and Mg ions on antibacterial activity of lactic acid bacteria isolates and their associations depending on cultivation conditions, AMB Express. 9 (2019) 32. [45] J. Sawai, H. Kojima, H. Igarashi, A. Hashimoto, S. Shoji, T. Sawaki, et al., Antibacterial characteristics of magnesium oxide powder, World J. Microbiol. Biotechnol. 16 (2000) 187194.

4. Antibacterial activity of nanomaterials

268

11. Antibacterial activity of metal oxide nanoparticles

[46] J. Sawai, E. Kawada, F. Kanou, H. Igarashi, A. Hashimoto, T. Kokugan, et al., Detection of active oxygen generated from ceramic powders having antibacterial activity, J. Chem. Eng. Jpn. 29 (1996) 627633. [47] K. Krishnamoorthy, G. Manivannan, S.J. Kim, K. Jeyasubramanian, M. Premanathan, Antibacterial activity of MgO nanoparticles based on lipid peroxidation by oxygen vacancy, J. Nanopart. Res. 14 (2012) 1063. [48] C.J. Hewitt, S.R. Bellara, A. Andreani, G. Nebe-von-Caron, C.M. McFarlane, An evaluation of the antibacterial action of ceramic powder slurries using multiparameter flow cytometry, Biotechnol. Lett. 23 (2001) 667675. [49] I.F. Castillo, L. De Matteisb, C. Marquina, E.G. Guille´n, J.M. de la Fuente, S.G. Mitchell, Protection of 18th century paper using antimicrobial nano-magnesium oxide, Int. Biodeterior. Biodegrad. 141 (2019) 7986. [50] H. Cui, X. Wu, Y. Chen, J. Zhang, R.I. Boughton, Influence of copper doping on chlorine adsorption and antibacterial behavior of MgO prepared by co-precipitation method, Mater. Res. Bull. 61 (2014) 511518. [51] Y. Rao, W. Wang, F. Tan, Y. Cai, J. Lu, X. Qiao, Influence of different ions doping on the antibacterial properties of MgO nanopowders, Appl. Surf. Sci. 284 (2013) 726731. ˝ [52] T. Ohira, M. Kawamura, M. Fukuda, K. Alvarez, B. Ozkal, O. Yamamoto, Extension of the optical absorption range in Zn-doped MgO powders and its effect on antibacterial activity, JMEPEG 19 (2010) 374379. [53] O.B. Koper, J.S. Klabunde, G.L. Marchin, K.J. Klabunde, P. Stoimenov, L. Bohra, Nanoscale powders and formulations with biocidal activity toward spores and vegetative cells of Bacillus species, viruses, and toxins, Curr. Microbiol. 44 (2002) 4955. [54] T. Jin, Y. He, Antibacterial activities of magnesium oxide (MgO) nanoparticles against foodborne pathogens, J. Nanopart. Res. 13 (2011) 68776885. [55] A. Sellik, T. Pollet, L. Ouvry, S. Brianc¸on, H. Fessi, D.J. Hartmann, et al., Degradation of paraoxon (VX chemical agent simulant) and bacteria by magnesium oxide depends on the crystalline structure of magnesium oxide, Chem. Biol. Interact. 267 (2017) 6773. [56] M. Sundrarajan, J. Suresh, R.R. Gandhi, A comparative study on antibacterial properties of MgO nanoparticles prepared under different calcination temperature, Dig. J. Nanomater. Biostruct. 7 (2012) 983989. [57] S. Makhluf, R. Dror, Y. Nitzan, Y. Abramovich, R. Jelinek, A. Gedanken, Microwave-assisted synthesis of nanocrystalline MgO and its use as a bacteriocide, Adv. Funct. Mater. 15 (2005) 17081715. [58] L. Wang, C. Hu, L. Shao, The antimicrobial activity of nanoparticles: present situation and prospects for future, Int. J. Nanomed. 12 (2017) 12271249. [59] J. Sawai, Quantitative evaluation of antibacterial activities of metallic oxide powders (ZnO, MgO and CaO) by conductimetric assay, J. Microbiol. Methods 54 (2003) 177182. [60] A.R. Butt, S. Ejaz, J.C. Baron, M. Ikram, S. Ali, CaO nanoparticles as a potential drug delivery agent for biomedical applications, Dig. J. Nanomater. Biostruct. 10 (2015) 799809. [61] S.M. Dizaj, F. Lotfipour, M. Barzegar-Jalali, M.H. Zarrintan, K. Adibkia, Antimicrobial activity of the metals and metal oxide nanoparticles, Mater. Sci. Eng. C 44 (2014) 278284. [62] J. Vidic, S. Stankic, F. Haque, D. Ciric, R. Le Goffic, A. Vidy, et al., Selective antibacterial effects of mixed ZnMgO nanoparticles, J. Nanopart. Res. 15 (2013) 110. [63] L.H. Leung, A. Ng, X. Xu, Z. Shen, L.A. Gethings, M.T. Wong, et al., Mechanisms of antibacterial activity of MgO: non-ROS mediated toxicity of MgO nanoparticles towards Escherichia coli, Small 10 (2014) 11711183. [64] J. Sawai, H. Igarashi, Evaluation of antibacterial activity of inorganic materials and application of natural inorganic materials to controlling microorganisms, food ingredients, J. Jpn. 203 (2002) 4757. [65] D. Manyasree, P. Kiranmayi, R.V.S.S.N. Ravi Kumar, Synthesis, characterization and antibacterial activity of aluminum oxide nanoparticles, Int. J. Pharm. Pharm. Sci. 10 (2018) 3235. [66] N. Varghese, M. Hariharan, B. Cherian, P.V. Sreenivasan, J. Paul, PVA-Assisted synthesis and characterization of nano αalumina, Int. J. Sci. Res. 4 (2014) 14. [67] G. Geoprincy, N. Nagendhra, S. Gandhi, S. Renganathan, Novel antibacterial effects of alumina nanoparticles on Bacillus cereus and Bacillus subtilis in comparison with antibiotics, Int. J. Pharm. Pharm. Sci. 4 (2012) 544548. [68] I.M. Sadiq, B. Chowdhury, N. Chandrasekaran, A. Mukherjee, Antimicrobial sensitivity of Escherichia coli to alumina nanoparticles, Nanomed. Nanotechnol. Biol. Med. 5 (2009) 282286. [69] B. Li, B.E. Logan, Bacterial adhesion to glass and metal oxide surfaces, Colloids Surf. B 36 (2004) 8190. [70] J.P. Rupareli, A.K. Chatterjee, S.P. Duttagupta, S. Mukherji, Strain specificity in antimicrobial activity of silver and copper nanoparticles, Acta Biomater. 4 (2008) 707771.

4. Antibacterial activity of nanomaterials

References

269

[71] G. Mohammad, V.K. Mishra, H.P. Pandey, Antioxidant properties of some nanoparticles may enhance wound healing in T2DM patient, Dig. J. Nanomater. Biostruct 3 (2008) 159162. [72] W. Jiang, H. Mashayekhi, B. Xing, Bacterial toxicity comparison between nano- and micro-scaled oxide particles, Environ. Pollut. 157 (2009) 16191625. [73] T. Bala, G. Armstrong, F. Laffir, R. Thornton, Titaniasilver and alumina silver composite nanoparticles: novel, versatile synthesis, reaction mechanism and potential antimicrobial application, J. Colloid Interface Sci. 356 (2011) 395403. [74] T.J. Yu, P.H. Li, T.W. Tseng, Y.C. Chen, Multifunctional Fe3O4 / alumina core/shell MNPs as photothermal agents for targeted hyperthermia of nosocomial and antibiotic-resistant bacteria, Nanomedicine 6 (2011) 13531363. [75] K. Zheng, M.I. Setyawati, D.T. Leong, J. Xie, Antimicrobial silver nanomaterials, Coord. Chem. Rev. 357 (2018) 117. [76] A. Shah, S. Haq, W. Rehman, M. Waseem, S. Shoukat, M. Rehman, Photocatalytic and antibacterial activities of paeonia emodi mediated silver oxide nanoparticles, Mater. Res. Express. 6 (2019) 045045. [77] S. Haq, W. Rehman, M. Waseem, V. Meynen, S.U. Awan, S. Saeed, et al., Fabrication of pure and moxifloxacin functionalized silver oxide nanoparticles for photocatalytic and antimicrobial activity, J. Photochem. Photobiol. B 186 (2018) 116124. [78] X. Wang, H.F. Wu, Q. Kuang, R.B. Huang, Z.X. Xie, L.S. Zheng, Shape-dependent antibacterial activities of Ag2O polyhedral particles, Langmuir 26 (2010) 27742778. [79] V. Manikandan, P. Velmurugan, J.H. Park, W.S. Chang, Y.J. Park, P. Jayanthi, et al., Green synthesis of silver oxide nanoparticles and its antibacterial activity against dental pathogens, 3 Biotech. 7 (2017) 72. [80] M.J. Kim, S. Kim, H. Park, Y.D. Huh, Morphological evolution of Ag2O microstructures from cubes to octapods and their antibacterial activities, Bull. Korean Chem. Soc. 32 (2011) 37933795. [81] V. Stani´c, D. Jana´ckovi´c, S. Dimitrijevi´c, S.B. Tanaskovi´c, M. Mitri´c, M.S. Pavlovi´c, et al., Synthesis of antimicrobial monophase silver-doped hydroxyapatite nanopowders for bone tissue engineering, Appl. Surf. Sci. 257 (2011) 45104518. ˇ [82] V. Stani´c, A.S. Radosavljevi´c-Mihajlovi´c, V. Zivkovi´ c-Radovanovi´c, B. Nastasijevi´c, M. Marinovi´c-Cincovi´c, J.P. Markovi´c, et al., Synthesis, structural characterisation and antibacterial activity of Ag1-doped fluorapatite nanomaterials prepared by neutralization method, Appl. Surf. Sci. 337 (2015) 7280. [83] M. Sarraf, A.D. Dabbagh, B.A. Razak, R. Mahmoodian, B. Nasiri-Tabrizi, H.R.M. Hosseini, et al., Highlyordered TiO2 nanotubes decorated with Ag2O nanoparticles for improved biofunctionality of Ti6Al4V, Surf. Coat. Technol. 349 (2018) 10081017. [84] M. Sarraf, A. Dabbagh, B.A. Razak, B. Nasiri-Tabrizi, H.R.M. Hosseini, S. Saber-Samandari, et al., Silver oxide nanoparticles-decorated tantala nanotubes for enhanced antibacterial activity and osseointegration of Ti6Al4V, Mater. Des. 154 (2018) 2840. [85] Y. Lv, Y. Wu, X. Lu, Y. Yu, S. Fu, L. Yang, et al., Microstructure, bio-corrosion and biological property of Ag-incorporated TiO2 coatings: influence of Ag2O contents, Ceram. Int. 45 (2019) 2235722367. Available from: https://doi.org/10.1016/j.ceramint.2019.07.265. [86] M.H. Kim, H. Park, H.C. Nam, S.R. Park, J.Y. Jung, W.H. Park, Injectable methylcellulose hydrogel containing silver oxide nanoparticles for burn wound healing, Carbohydr. Polym. 181 (2018) 579586. [87] P.J. Babu, M. Doble, A.M. Raichur, Silver oxide nanoparticles embedded silk fibroin spuns: microwave mediated preparation, characterization and their synergistic wound healing and anti-bacterial activity, J. Colloid Interface Sci. 513 (2018) 6271. [88] S. Tripathi, G.K. Mehrotra, P.K. Dutta, Chitosansilver oxide nanocomposite film: preparation and antimicrobial activity, Bull. Mater. Sci. 34 (2011) 2935. [89] Y. Yang, D. Xu, Q. Wu, P. Diao, Cu2O/CuO bilayered composite as a high-efficiency photocathode for photoelectrochemical hydrogen evolution reaction, Sci. Rep. 6 (2016) 35158. [90] L. Miao, C. Wang, J. Hou, P. Wang, Y. Ao, S. Dai, et al., Effects of pH and natural organic matter (NOM) on the adsorptive removal of CuO nanoparticles by periphyton, Environ. Sci. Pollut. Res. Int. 22 (2015) 76967704. [91] M.Z. Sahdan, M.F. Nurfazliana, S.A. Kamaruddin, Z. Embong, Z. Ahmad, H. Saim, Fabrication and characterization of crystalline cupric oxide (CuO) films by simple immersion method, Procedia Manuf. 2 (2015) 379384.

4. Antibacterial activity of nanomaterials

270

11. Antibacterial activity of metal oxide nanoparticles

[92] E. Topoglidis, A.E.G. Cass, B. O’Regan, J.R. Durrant, Immobilisation and Bioelectrochemistry of proteins on nanoporous TiO2 and ZnO Films, J. Electroanal. Chem. 517 (2001) 2027. [93] S.R. Kanel, S.R. Al-Abed, Influence of pH on the transport of nanoscale zinc oxide in saturated porous media, J. Nanopart. Res. 13 (2011) 40354047. [94] S.M. Gupta, M. Tripathi, A review of TiO2 nanoparticles, Chin. Sci. Bull. 56 (2011) 16391657. [95] D. Reyes-Coronado, G. Rodrı´guez-Gattorno, M.E. Espinosa-Pesqueira, C. Cab, R. Coss, G. Oskam, Phasepure TiO2 nanoparticles: anatase, brookite and rutile, Nanotechnology 19 (2008) 145605. [96] H. Chen, W. Liu, Cellulose-based photocatalytic paper with Ag2O nanoparticles loaded on graphite fibers, J. Bioresour. Bioprod. 1 (2016) 192198. [97] V. Stani´c, S. Dimitrijevi´c, J. Anti´c-Stankovi´c, M. Mitri´c, B. Joki´c, I.B. Ple´caˇs, et al., Synthesis, characterization and antimicrobial activity of copper and zinc-doped hydroxyapatite nanopowders, Appl. Surf. Sci. 256 (2010) 60836089. ˇ [98] G. Vuˇckovi´c, M. Antonijevi´c-Nikoli´c, S.B. Tanaskovi´c, V. Zivkovi´ c-Radovanovi´c, New Cu (II) and Co(II) octaazamacrocyclic complexes with 2-amino-3-phenyl-propanoic acid, J. Serb. Chem. Soc. 76 (2011) 719731. [99] M. Antonijevi´c Nikoli´c, B. Draˇzi´c, J. Anti´c Stankovi´c, S. Tanaskovi´c, New mixed-ligand Ni(II) and Zn(II) macrocyclic complexes with bridged bicyclo-[2,2,1]-hept-5-en-endo-2,3-cis-dicarboxylate: synthesis, characterization, antimicrobial and cytotoxic activity, J. Serb. Chem. Soc. 84 (2019) 28. Available from: https://doi. org/10.2298/JSC181216028A. [100] M. Antonijevi´c-Nikoli´c, J. Anti´c-Stankovi´c, S.B. Tanaskovi´c, Synthesis, characterization, and in vitro antiproliferative and antibacterial studies of tetraazamacrocyclic complexes of Co(II) and Cu(II) with pyromellitic acid, J. Coord. Chem. 71 (2018) 15421559. [101] M. Antonijevi´c-Nikoli´c, J. Anti´c-Stankovi´c, S.B. Tanaskovi´c, M.J. Korabik, G. Gojgi´c-Cvijovi´c, G. Vuˇckovi´c, Preparation, characterisation and study of in vitro biologically active azamacrocyclic Cu(II) dicarboxylate complexes, J. Mol. Struct. 10541055 (2013) 297306. [102] M. Eshed, J. Lellouche, S. Matalon, A. Gedanken, E. Banin, Sonochemical coatings of ZnO and CuO nanoparticles inhibit Streptococcus mutans biofilm formation on teeth model, Langmuir 28 (2012) 1228812295. [103] A. Ananth, S. Dharaneedharan, M.S. Heo, Y.S. Mok, Copper oxide nanomaterials: synthesis, characterization and structure-specific antibacterial performance, Chem. Eng. J. 262 (2015) 179188. [104] R. Chandrasekaran, S.A. Yadav, S. Sivaperumal, Phytosynthesis and characterization of copper oxide nanoparticles using the aqueous extract of Beta vulgaris L and evaluation of their antibacterial and anticancer activities, J. Clust. Sci. (2019). Available from: https://doi.org/10.1007/s10876-019-01640-6. [105] A.B. Devi, D.S. Moirangthem, N.C. Talukdar, M.D. Devi, N.R. Singh, M.N. Luwang, Novel synthesis and characterization of CuO nanomaterials: biological applications, Chin. Chem. Lett. 25 (2014) 16151619. [106] S. Moniri Javadhesari, S. Alipour, S. Mohammadnejad, M.R. Akbarpour, Antibacterial activity of ultra-small copper oxide (II) nanoparticles synthesized by mechanochemical processing against S. aureus and E. coli, Mater. Sci. Eng. C 105 (2019) 110011. [107] L. Xiong, H. Yu, C. Nie, Y. Xiao, Q. Zeng, G. Wang, et al., Size-controlled synthesis of Cu2O nanoparticles: size effect on antibacterial activity and application as a photocatalyst for highly efficient H2O2 evolution, RSC Adv. 7 (2017) 5182251830. [108] I. Perelshtein, G. Applerot, N. Perkas, E. Wehrschuetz-Sigl, A. Hasmann, G. Guebitz, et al., CuOcotton nanocomposite: formation, morphology, and antibacterial activity, Surf. Coat. Technol. 204 (2009) 5457. [109] S. Meghana, P. Kabra, S. Chakraborty, N. Padmavathy, Understanding the pathway of antibacterial activity of copper oxide nanoparticles, RSC Adv. 5 (2015) 12293. [110] S. Kumar, A.K. Ojha, D. Bhorolua, J. Das, A. Kumar, A. Hazarika, Facile synthesis of CuO nanowires and Cu2O nanospheres grown on rGO surface and exploiting its photocatalytic, antibacterial and supercapacitive properties, Physica B 558 (2019) 7481. [111] H.R. Naika, K. Lingaraju, K. Manjunath, D. Kumar, G. Nagaraju, D. Suresh, et al., Green synthesis of CuO nanoparticles using Gloriosa superba L. extract and their antibacterial activity, J. Taibah Univ. Sci. 9 (2015) 712. [112] G. Ren, D. Hu, E.W.C. Cheng, M.A. Vargas-Reus, P. Reip, R.P. Allaker, Characterisation of copper oxide nanoparticles for antimicrobial applications, Int. J. Antimicrob. Agents 33 (2009) 587590. [113] A. Kumar, A.K. Pandey, S.S. Singh, R. Shanker, A. Dhawan, Engineered ZnO and TiO2 nanoparticles induce oxidative stress and DNA damage leading to reduced viability of Escherichia coli, Free. Radic Biol. Med. 51 (2011) 18721881.

4. Antibacterial activity of nanomaterials

References

271

[114] D. Laha, A. Pramanik, A. Laskar, M. Jana, P. Pramanik, P. Karmakar, Shape-dependent bactericidal activity of copper oxide nanoparticle mediated by DNA and membrane damage, Mater. Res. Bull. 59 (2014) 185191. [115] M. Hans, A. Erbe, S. Mathews, Y. Chen, M. Solioz, F. Mu¨cklich, Role of copper oxides in contact killing of bacteria, Langmuir 29 (2013) 1616016166. [116] Z. Wu, F.A. Fernandez-Lima, D.H. Russell, Amino acid influence on copper binding to peptides: cysteine versus arginine, J. Am. Soc. Mass Spectrom. 21 (2010) 522533. [117] Y.-W. Baek, Y.-J. An, Microbial toxicity of metal oxide nanoparticles (CuO, NiO, ZnO, and Sb2O3) to Escherichia coli, Bacillus subtilis, and Streptococcus aureus, Sci. Total Environ. 409 (2011) 16031608. [118] V. Vellora, T. Padil, M. Cernı´k, Green synthesis of copper oxide nanoparticles using gum karaya as a biotemplate and their antibacterial application, Int. J. Nanomed. 8 (2013) 889898. [119] S. Jadhav, S. Gaikwad, M. Nimse, A. Rajbhoj, Copper oxide nanoparticles: synthesis, characterization and their antibacterial activity, J. Clust. Sci. 22 (2011) 121129. [120] A. Azam, A.S. Ahmed, M. Oves, M.S. Khan, A. Memic, Size-dependent antimicrobial properties of CuO nanoparticles against Gram-positive and -negative bacterial strains, Int. J. Nanomed. 7 (2012) 35273535. [121] J.H. Kim, H. Cho, S.E. Ryu, M.U. Choi, Effects of metal ions on the activity of protein tyrosine phosphatase VHR: highly potent and reversible oxidative inactivation by Cu21 ion, Arch. Biochem. Biophys. 382 (2000) 7280. [122] R.B. Thurman, C.P. Gerba, G. Bitton, The molecular mechanisms of copper and silver ion disinfection of bacteria and viruses, Crit. Rev. Environ. Sci. Technol. 18 (1989) 295315. [123] P.P. Fu, Q. Xia, H.M. Hwang, P.C. Ray, H. Yu, Mechanisms of nanotoxicity: generation of reactive oxygen species, J. Food Drug. Anal. 22 (2014) 6475. [124] A. Moezzi, A.M. McDonagh, M.B. Cortie, Zinc oxide particles: synthesis, properties and applications, Chem. Eng. J. 185186 (2012) 122. [125] F. Piccinno, F. Gottschalk, S. Seeger, B. Nowack, Industrial production quantities and uses of 10 engineered nanomaterials for Europe and the world, J. Nanopart. Res. 14 (2012) 11091120. [126] O. Bondarenko, K. Juganson, A. Ivask, K. Kasemets, M. Mortimer, A. Kahru, Toxicity of Ag, CuO and ZnO nanoparticles to selected environmentally relevant test organisms and mammalian cells in vitro: a critical review, Arch. Toxicol. 87 (2013) 11811200. [127] A.A. Keller, S. McFerran, A. Lazareva, S. Suh, Global life cycle releases of engineered nanomaterials, J. Nanopart. Res. 15 (2013) 1692. [128] T.P. Dasari, K. Pathakoti, H.M. Hwang, Determination of the mechanism of photoinduced toxicity of selected metal oxide nanoparticles (ZnO, CuO, Co3O4 and TiO2) to E. coli bacteria, J. Environ. Sci. 25 (2013) 882888. [129] A. Sirelkhatim, S. Mahmud, A. Seeni, N.H.M. Kaus, L.C. Ann, S.K.M. Bakhori, et al., Review on zinc oxide nanoparticles: antibacterial activity and toxicity mechanism, Nano-Micro Lett. 7 (2015) 219242. [130] L. Zhang, Y. Jiang, Y. Ding, M. Povey, D. York, Investigation into the antibacterial behaviour of suspensions of ZnO nanoparticles (ZnO nanofluids), J. Nanopart. Res. 9 (2007) 479489. [131] R. Brayner, R. Ferrari-Iliou, N. Brivois, S. Djediat, M.F. Benedetti, F. Fie´vet, Toxicological impact studies based on Escherichia coli bacteria in ultrafine ZnO nanoparticles colloidal medium, Nano Lett. 6 (2006) 866870. [132] Y. Li, W. Zhang, J. Niu, Y. Chen, Mechanism of photogenerated reactive oxygen species and correlation with the antibacterial properties of engineered metal-oxide nanoparticles, ACS Nano 6 (2012) 51645173. [133] M. Li, L. Zhu, D. Lin, Toxicity of ZnO nanoparticles to Escherichia coli: mechanism and the influence of medium components, Environ. Sci. Technol. 45 (2011) 19771983. [134] L. Palanikumar, S.N. Ramasamy, C. Balachandran, Size-dependent antimicrobial response of zinc oxide nanoparticles, IET Nanobiotechnol. 8 (2014) 111117. [135] I. Rago, C.R. Chandraiahgari, M.P. Bracciale, G. De Bellis, E. Zanni, M.C. Guidi, et al., Zinc oxide microrods and nanorods: different antibacterial activity and their mode of action against Gram-positive bacteria, RSC Adv. 4 (2014) 5603156040. [136] L.K. Adams, D.Y. Lyon, P.J. Alvarez, Comparative eco-toxicity of nanoscale TiO2, SiO2, and ZnO. Water suspensions, Water Res. 40 (2006) 35273532. [137] H. Yang, C. Liu, D. Yang, H. Zhang, Z. Xi, Comparative study of cytotoxicity, oxidative stress and genotoxicity induced by four typical nanomaterials: the role of particle size, shape and composition, J. Appl. Toxicol. 29 (2009) 6978.

4. Antibacterial activity of nanomaterials

272

11. Antibacterial activity of metal oxide nanoparticles

[138] N. Talebian, S.M. Amininezhad, M. Doudi, Controllable synthesis of ZnO nanoparticles and their morphology-dependent antibacterial and optical properties, J. Photochem. Photobiol. 120 (2013) 6673. [139] Q. Cai, Y. Gao, T. Gao, S. Lan, O. Simalou, X. Zhou, et al., Insight into biological effects of zinc oxide nanoflowers on bacteria: why morphology matters, ACS Appl. Mater. Interfaces 816 (2016) 1010910120. [140] R.K. Dutta, B.P. Nenavathu, M.K. Gangishetty, A.V.R. Reddy, Studies on antibacterial activity of ZnO nanoparticles by ROS induced lipid peroxidation, Colloids Surf. B 94 (2012) 143150. [141] S.B. Rana, R.P.P. Singh, S. Arya, Structural, optical, magnetic and antibacterial study of pure and cobalt doped ZnO nanoparticles, J. Mater. Sci. Mater Electron. 28 (2017) 26602672. [142] A.T. Ravichandran, R. Karthick, A. Robert Xavier, R. Chandramohan, S. Mantha, Influence of Sm doped ZnO nanoparticles with enhanced photoluminescence and antibacterial efficiency, J. Mater. Sci. Mater Electron. 28 (2017) 66436648. [143] C. Selvaraju, R. Karthick, R. Veerasubam, The modification of structural, optical and antibacterial activity properties of rare earth gadolinium-doped ZnO nanoparticles prepared by co-precipitation method, J. Inorg. Organomet. Polym. Mater. 29 (2019) 776782. [144] R. Guan, H. Zhai, D. Sun, J. Zhang, Y. Wang, J. Li, Effects of Ag doping content and dispersion on the photocatalytic and antibacterial properties in ZnO nanoparticles, Chem. Res. Chin. Univ. 35 (2019) 271276. [145] P. Maddahi, N. Shahtahmasebi, A. Kompany, M. Mashreghi, S. Safaee, F. Roozban, Effect of doping on structural and optical properties of ZnO nanoparticles: study of antibacterial properties, Mater. Sci. Pol. 32 (2014) 130135. [146] M. Mohapatra, S. Anand, Synthesis and applications of nanostructured iron oxides/hydroxides—a review, Int. J. Eng. Sci. Technol. 2 (2010) 127146. [147] L.S. Arias, J.P. Pessan, A.P.M. Vieira, T.M.T. Lima, A.C.B. Delbem, D.R. Monteiro, Iron oxide nanoparticles for biomedical applications: a perspective on synthesis, drugs, antimicrobial activity, and toxicity, Antibiotics 7 (2018) 4678. [148] A.H. Lu, E.L. Salabas, F. Schu¨th, Magnetic nanoparticles: synthesis, protection, functionalization, and application, Angew. Chem. Int. Ed. Engl. 46 (2007) 12221244. [149] M. Tadic, L. Kopanja, M. Panjan, S. Kralj, J. Nikodinovic-Runic, Z. Stojanovic, Synthesis of coreshell hematite (α-Fe2O3) nanoplates: quantitative analysis of the particle structure and shape, high coercivity and low cytotoxicity, Appl. Surf. Sci. 403 (2017) 628634. [150] A. Raghunath, E. Perumal, Metal oxide nanoparticles as antimicrobial agents: a promise for the future, Int. J. Antimicrob. Agents 49 (2017) 137152. [151] M. Mahmoudi, S. Sant, B. Wang, S. Laurent, T. Sen, Superparamagetic iron oxide nanoparticles (SPIONs): development, surface modification and applications in chemotherapy, Adv. Drug. Deliv. Rev. 63 (2011) 2446. [152] C. Ru¨menapp, B. Gleich, A. Haase, Magnetic nanoparticles in magnetic resonance imaging and diagnostics, Pharm. Res. 29 (2012) 11651179. [153] T. Banerjee, S. Mitra, A. Kumar Singh, R. Kumar Sharma, A. Maitra, Preparation, characterization and biodistribution of ultrafine chitosan nanoparticles, Int. J. Pharm. 243 (2002) 93105. [154] S.M. Moghimi, A.C. Hunter, J.C. Murray, Long-circulating and target-specific nanoparticles: theory to practice, Pharm. Rev. 52 (2001) 283318. [155] A. Rufus, N. Sreeju, D. Philip, Synthesis of biogenic hematite (α-Fe2O3) nanoparticles for antibacterial and nanofluid applications, RSC Adv. 6 (2016) 9420694217. [156] R. Irshad, K. Tahir, B. Li, A. Ahmad, A.R. Siddiqui, S. Nazir, Antibacterial activity of biochemically capped iron oxide nanoparticles: a view towards green chemistry, J. Photochem. Photobiol. B 170 (2017) 241246. [157] H. Muthukumar, N.I. Chandrasekaran, S.N. Mohammed, S. Pichiah, M. Manickam, Iron oxide nano-material: physicochemical traits and in vitro antibacterial propensity against multidrug resistant bacteria, J. Ind. Eng. Chem. 45 (2017) 121130. [158] S. Groiss, R. Selvaraj, T. Varadavenkatesan, R. Vinayagam, Structural characterization, antibacterial and catalytic effect of iron oxide nanoparticles synthesised using the leaf extract of Cynometra ramiflora, J. Mol. Struct. 1128 (2017) 572578. [159] X. Pan, J.E. Redding, P.A. Wiley, L. Wen, J.S. McConnell, B. Zhang, Mutagenicity evaluation of metal oxide nanoparticles by the bacterial reverse mutation assay, Chemosphere 79 (2010) 113116. [160] N.B. Saleh, B. Chambers, N. Aich, J. Plazas-Tuttle, H.N. Phung-Ngoc, M.J. Kirisits, Mechanistic lessons learned from studies of planktonic bacteria with metallic nanomaterials: implications for interactions between nanomaterials and biofilm bacteria, Front. Microbiol. 6 (2015) 677.

4. Antibacterial activity of nanomaterials

References

273

[161] T. Nakamura, I. Naguro, H. Ichijo, Iron homeostasis and iron-regulated ROS in cell death, senescence and human diseases, Biochim. Biophys. Acta Gen. Subj. 1863 (2019) 13981409. [162] A.E. Nel, L. Ma¨dler, D. Velegol, T. Xia, E.M. Hoek, P. Somasundaran, et al., Understanding biophysicochemical interactions at the nano-bio interface, Nat. Mater. 8 (2009) 543557. [163] T. Javanbakht, S. Laurent, D. Stanicki, K.J. Wilkinson, Relating the surface properties of superparamagnetic iron oxide nanoparticles (SPIONs) to their bactericidal effect towards a biofilm of Streptococcus mutans, PLoS One 11 (2016) e0154445. [164] S. Vasantharaj, S. Sathiyavimal, P. Senthilkumar, F.L. Oscar, A. Pugazhendhi, Biosynthesis of iron oxide nanoparticles using leaf extract of Ruellia tuberosa: antimicrobial properties and their applications in photocatalytic degradation, J. Photochem. Photobiol. B 192 (2019) 7482. [165] S. Pigeot-Re´mya, F. Simonet, E. Errazuriz-Cerda, J.C. Lazzaroni, D. Atlan, C. Guillard, Photocatalysis and disinfection of water: identification of potential bacterial targets, Appl. Catal. B 104 (2011) 390398. [166] T. Matusunga, Sterilization with particulate photosemiconductor, J. Antibact. Antifung. Agents 13 (1985) 211220. [167] H.A. Foster, I.B. Ditta, S. Varghese, A. Steele, Photocatalytic disinfection using titanium dioxide: spectrum and mechanism of antimicrobial activity, Appl. Microbiol. Biotechnol. 90 (2011) 18471868. [168] S. Pigeot-Re´mya, F. Simonet, D. Atlan, J.C. Lazzaroni, C. Guillard, Bactericidal efficiency and mode of action: a comparative study of photochemistry and photocatalysis, Water Res. 46 (2012) 32083218. [169] B. Jalvo, M. Faraldos, A. Bahamonde, R. Rosal, Antimicrobial and antibiofilm efficacy of self-cleaning surfacesfunctionalized by TiO2 photocatalytic nanoparticles against Staphylococcus aureus and Pseudomonas putida, J. Hazard. Mater. 340 (2017) 160170. [170] H. Zhang, J.F. Banfield, Understanding polymorphic phase transformation behavior during growth of nanocrystalline aggregates: insights from TiO2, J. Phys. Chem. B 104 (2000) 34813487. [171] L. Gao, Q. Zhang, Effects of amorphous contents and particle size on the photocatalytic properties of TiO2 nanoparticles, Scr. Mater. 44 (2001) 11951198. [172] H. Li, Q. Cui, B. Feng, J. Wang, X. Lu, J. Weng, Antibacterial activity of TiO2 nanotubes: influence of crystal phase, morphology and Ag deposition, Appl. Surf. Sci. 284 (2013) 179183. [173] H.N. Pantaroto, A.P. Ricomini-Filho, M.M. Bertolini, J.H.D. da Silvad, N.F.A. Neto, C. Sukotjo, et al., Antibacterial photocatalytic activity of differentcrystalline TiO2 phases in oral multispecies biofilm, Dent. Mater. 34 (2018) e182e195. [174] R. Su, R. Bechstein, L. Sø, R.T. Vang, M. Sillassen, B. Esbjo¨rnsson, et al., How the anatase-to-rutile ratio influences the photoreactivity of TiO2, J. Phys. Chem. C 115 (2011) 2428724292. [175] M.G. Me´ndez-Medrano, E. Kowalska, A. Lehoux, A. Herissan, B. Ohtani, D. Bahena, et al., Surface modification of TiO2 with Ag nanoparticles and CuO nanoclusters for application in photocatalysis, J. Phys. Chem. C 120 (2016) 51435154. [176] B. Yu, K.M. Leung, Q. Guo, W.M. Lau, J. Yang, Synthesis of AgTiO2 composite nano thin film for antimicrobial application, Nanotechnology 22 (2011) 115603. [177] H. Zhang, G. Chen, Potent antibacterial activities of Ag/TiO2 nanocomposite powders synthesized by a one-pot sol 2 gel method, Environ. Sci. Technol. 438 (2009) 29052910. [178] S.F. Chen, J.P. Li, K. Qian, W.P. Xu, Y. Lu, W.X. Huang, et al., Large scale photochemical synthesis of M@TiO2 nanocomposites (M 5 Ag, Pd, Au, Pt) and their optical properties, CO oxidation performance, and antibacterial effect, Nano Res. 3 (2010) 244255. [179] L. Armelao, D. Barreca, G. Bottaro, A. Gasparotto, C. Maccato, C. Maragno, et al., Photocatalytic and antibacterial activity of TiO2 and Au/TiO2 nanosystems, Nanotechnology 18 (2007) 375709. [180] C.C. Trapalis, P. Keivanidis, G. Kordas, M. Zaharescu, M. Crisan, A. Szatvanyi, et al., TiO2 (Fe31) nanostructured thin films with antibacterial properties, Thin Solid Films 433 (2003) 186190. [181] H.M. Yadav, S.V. Otari, R.A. Bohara, S.S. Mali, S.H. Pawar, S.D. Delekar, Synthesis and visible light photocatalytic antibacterial activity of nickeldoped TiO2 nanoparticles against grampositive and gramnegative bacteria, J. Photochem. Photobiol. A 294 (2014) 130136. [182] T. Amna, M.S. Hassan, M. Pandurangan, M.S. Khil, H.K. Lee, I.H. Hwang, Characterization and potent bactericidal effect of cobalt doped titanium dioxide nanofibers, Ceram. Int. 39 (2013) 31893193. [183] Q. Cheng, C. Li, V. Pavlinek, P. Saha, H. Wang, Surface-modified antibacterial TiO2/Ag1 nanoparticles: preparation and properties, Appl. Surf. Sci. 252 (2006) 41544160.

4. Antibacterial activity of nanomaterials

274

11. Antibacterial activity of metal oxide nanoparticles

[184] C. Karunakaran, G. Abiramasundari, P. Gomathisankar, G. Manikandan, V. Anand, Cu-doped TiO2 nanoparticles for photocatalytic disinfection of bacteria under visible light, J. Colloid Interface Sci. 352 (2010) 6874. [185] S.B. Tanaskovi´c, G. Vuˇckovi´c, M. Antonijevi´c-Nikoli´c, T. Stanojkovi´c, G. Gojgi´c-Cvijovi´c, Binuclear biologically active Co (II) complexes with octazamacrocycle and aliphatic dicarboxylates, J. Mol. Struct. 1029 (2012) 17. ´ [186] G. Vuˇckovi´c, V. Stani´c, S.P. Sovilj, M. Antonijevi´c-Nikoli´c, J. Mrozinski, Cobalt (II) complexes with aromatic carboxylates and N-functionalized cyclam bearing 2-pyridylmethyl pendant arms, J. Serb. Chem. Soc. 70 (2005) 11211129. [187] G. Vuˇckovi´c, S.B. Tanaskovi´c, Z.M. Miodragovi´c, V. Stani´c, High-spin binuclear Co (II) complexes with a pendant octaazamaclocycle and carboxylates, J. Serb. Chem. Soc. 72 (2007) 12951308. [188] S. Raiˇcevi´c, V. Stani´c, T. Kaludjerovi´c-Radoiˇci´c, Theoretical assessment of calcium arsenates stability: application in the treatment of arsenic contaminated waste, Mater. Sci. Forum 555 (2007) 131136. [189] V. Stani´c, S. Dimitrijevi´c, D.G. Antonovi´c, B.M. Joki´c, S.P. Zec, S.T. Tanaskovi´c, et al., Synthesis of fluorine substituted hydroxyapatite nanopowders and application of the central composite design for determination of its antimicrobial effects, Appl. Surf. Sci. 290 (2014) 346352. [190] X.B. Zheng, C.X. Ding, Characterization of plasma-sprayed hydroxyapatite/TiO2 composite coatings, J. Therm. Spray. Technol. 9 (2000) 520525. ´ . Dea´k, S.P. Tallo´sy, D. Seb˝ok, E. Csapo´, K. Bohinc, et al., Hydroxyapatite-enhanced structural, [191] L. Janova´k, A photocatalytic and antibacterial properties of photoreactive TiO2/HAp/polyacrylate hybrid thin films, Surf. Coat. Technol. 326 (2017) 316326. [192] K. Kaviyarasu, A. Mariappan, K. Neyvasagam, A. Ayeshamariam, P. Pandi, R.R. Palanichamy, et al., Photocatalytic performance and antimicrobial activities of HAp-TiO2 nanocomposite thin films by sol-gel method, Surf. Interfaces 6 (2017) 247255. [193] M. Wakamura, K. Hashimoto, T. Watanabe, Photocatalysis by calcium hydroxyapatite modified with Ti(IV): albumin decomposition and bactericidal effect, Langmuir 198 (2003) 34283431.

4. Antibacterial activity of nanomaterials

C H A P T E R

12 Antibacterial activity of platinum nanoparticles Susai Rajendran1, S. Santhana Prabha2, R. Joseph Rathish2, Gurmeet Singh3 and Abdulhameed Al-Hashem4 1

Department of Chemistry, St. Antony’s College of Arts and Sciences for Women, Dindigul, India 2PSNA College of Engineering and Technology, Dindigul, India 3Pondicherry University, Puducherry, India 4Petroleum Research Centre, Kuwait Institute for Scientific Research, Safat, Kuwait

12.1 Platinum nanoparticles Platinum nanoparticles (PtNPs) are sometimes within the sort of a suspension or colloid of nanoparticles (NPs) of platinum in a very fluid, sometimes water. A colloid is technically outlined as a stable dispersion of particles in a very fluid medium (liquid or gas). Spherical noble metal NPs will be created with sizes between concerning 2 and 100 nm, looking on reaction conditions. PtNPs are suspended within the colloid of maroon or black color. NPs are available in a large choice of shapes together with spheres, rods, cubes, and tetrahedra. PtNPs are the topic of considerable analysis, with potential applications in a very large choice of areas. These embrace chemical change, medicine, and therefore the synthesis of novel materials with distinctive properties.

12.2 Antibacterial activity Anything that destroys microorganism or suppresses their growth or their ability to breed is taken into account as antibacterial. Heat, chemicals like gas, and antibiotic medication all have medicament properties. Several medicament merchandise for cleanup and hand washing are oversubscribed nowadays.

Nanotoxicity DOI: https://doi.org/10.1016/B978-0-12-819943-5.00012-9

275

Copyright © 2020 Elsevier Inc. All rights reserved.

276

12. Antibacterial activity of platinum nanoparticles

12.3 Antibiotics and antimicrobial compounds Antibiotics are a broader variation of antimicrobial compounds that may act on fungi, bacteria, and different compounds. Although antibacterials come back underneath antibiotics, antibacterials will solely kill microorganisms. Antibiotic was the primary discovered by Sir Alexander Fleming.

12.4 Determination of the microbial activity The determination of the medicament activity (microbicide) of surfaces is delineated within the following norms: ISO 22196 and JIS Z 2801 (the Japanese norm JIS Z 2801). Within the take, each a surface system coated with sporicide associate degreed the same surface system while the medicament coating is charged with designated microorganisms. A once-only assessment of the reduction issue is allotted once 24 h by crucial colony counts on the reference surface and on the medicament surface.

12.5 Recent trends in the antibacterial activity of platinum nanoparticles The recent trends in the antibacterial activity of PtNPs are discussed. Nishanthi et al. reported the green synthesis and characterization of bioinspired silver, gold and PtNPs and evaluation of their synergistic antibacterial activity after combining with different classes of antibiotics [1] A facile one-step green synthesis of silver nanoparticle (AgNP), gold nanoparticle (AuNP), and PtNP has been tried exploitation the rind extract of the fruit of Garcinia mangostana L. The authors evaluated the antibacterial activity of the metal NPs before and when combining with commercially used antibiotics in addition as free antibiotics against human pathogenic bacteria. Their findings open up windows for the treatment of antibiotic resistant bacteria when combining with totally different NPs under clinical set up. Endo et al. reported the bactericidal properties of plasmonic photocatalysts composed of noble metal nanoparticles (NPs) on faceted anatase titania [2]. In their work, Octahedral anatase particles (OAP) with eight equivalent sides and decahedral anatase particles (DAP) with two extra facets were changed with NPs of noble metals (silver, copper, gold, and platinum) by photodeposition, and applied for inactivation of Escherichia coli K12. The authors found that DAP beneath UV light irradiation showed very high bactericidal activity, which may well be attributed to economical generation of reactive oxygen species (ROS), thanks to intrinsic properties of DAP, that is, charge carriers’ separation (migration of electrons and holes to and sides, respectively). However, an sudden decrease in activity when DAP modification with AuNPs and PtNPs (mainly deposited on facets) instructed that bacteria cells were directly rotten on DAP surface.

4. Antibacterial activity of nanomaterials

12.5 Recent trends in the antibacterial activity of platinum nanoparticles

277

Jiang et al. reported the light-induced assembly of metal NPs on ZnO enhances the generation of charge carriers, ROS, and antibacterial activity [3]. The authors showed a facile way to build ZnO/metal heteronanoparticles by the mixing/irradiation method of ZnO and metal NPs. Their findings indicated that smaller-sized PtNPs are economical in promoting charge carrier generation and ROS production. At 5 nm, silver NPs promoted charge carrier generation with more efficiency than Pt and AuNPs, however, PtNPs promoted ROS generation with more efficiency than Au and AgNPs. Subramaniyan et al. reported the preparation of self-assembled platinum nanoclusters to combat Salmonella typhi infection and inhibit biofilm formation [4]. In their work, phytoprotein functionalized platinum nanoclusters (PtNCs) have been synthesized using the proteins from fresh green spinach leaves. These PtNCs inhibits the expansion of the foodborne pathogen, S. typhi with minimum restrictive concentration (MIC) of 12.5 µM. Zou et al. reported the functionalization of silk with in-situ synthesized PtNPs [5]. In their study, after PtNPs were in-situ synthesized on silk fabrics through heat treatment, it absolutely was determined that the treatment of the silk materials with PtNPs imparted multiple functions, as well as coloring, catalysis, and bactericide activity. These PtNP-treated silk material exhibited vital catalytic perform and a notable antibacterial impact against E. coli. Dobrucka and Dlugaszewska reported the antimicrobial activity of the biogenically synthesized core-shell Cu@Pt nanoparticles [6] The authors presented the biological strategies of synthesizing bimetallic core-shell Cu@PtNPs , using Agrimoniae herba extract. As reported the synthesized NPs exhibited most activity against gram-negative bacterium E. coli ATCC 25922, S. aureus ATCC 25923, and P. aeruginosa NCTC 6749. The core-shell Cu@PtNPs additionally exhibited activity against the yeast C. albicans ATCC 10231 and dermatophytes T. mentagrophytes ATCC 9533. Peana et al. reported the intriguing potential of “minor” noble metals: emerging trends and new applications [7] The authors indicated that platinum compounds have clearly been the foremost studied derivatives because of the acknowledged biological properties of this part. Additionally, palladium, ruthenium, gold, silver, and copper species, below the shape of salts, complexes, or NPs formulations, found fascinating applications within the medical specialty fields as anticancer, medication, antiparasitic, and antifungal agents. Jeyapaul et al. reported an eco-friendly approach for synthesis of PtNPs using leaf extracts of Jatropa gossypifolia and Jatropa glandulifera and their antibacterial activity [8]. The authors showed anenvironmental-friendly approach is bestowed for the formation of PtNPs using liquid leaf extracts of Indian medicative herbs like J. gossypifolia and J. glandulifera as economical reducing and capping agents. These biologically synthesized PtNPs of leaf extracts exhibit a huge potent of medicinal drug activity against pathogenic bacteria. Jeyapaul et al. reported the green synthesis of PtNPs using Saudi’s dates extract and their usage on the cancer cell treatment [9] The authors evaluated PtNPs’ anticancer activities against various cancer cells together with the colon cancer cells (HCT-116), breast cells (MCF-7), and hepatocarcinoma (HePG-2). To get the antibacterial result, antibacterial agents SK-Ampicillin and antibiotic drug are used.

4. Antibacterial activity of nanomaterials

278

12. Antibacterial activity of platinum nanoparticles

Lastly, the gram-negative bacteria: E. coli (RCMB 010052) and gram-positive bacteria: grass bacillus (RCMB 010067) were accustomed to verify the bactericide application of PtNPs. Chelli and Golder presented the pot green synthesis of Pt, Co, and Pt@Co core-shell nanoparticles using Sechium edule [10]. The authors reported a simple ecofriendly (green) method to engineer the structure of cobalt (Co), platinum (Pt) and PtCo coreshell nanoparticles using plant-based analytes present in the extract of S. edule, the fruit of a perennial climber plant. The synergistic effects such as ligand interaction between core and shell and the geometric effect improved the antibacterial activity against Bacillus subtilis and E. coli bacteria in comparison to Pt nanospheres and Co nanoprisms, and these results were comparable with ciprofloxacin and cefprozil as the antibiotic controls. Ismail and Al-Radadi reported an eco-friendly synthesis of PtNPs and their applications on the cancer cell treatments [11]. In their work, a green synthesis of platinum nanosize is investigated as using Ajwa aqueous extract solution that is an ecofriendly reducing and capping agents by the biodegradation of the plant-based surfactants for cancer cell treatments. In vitro antitumor activity analysis of the freshly synthesized PtNPs are disbursed against human neoplastic cell lines colon malignant neoplastic disease cells (HCT-116), malignant hepatoma (HePG-2) and breast cells (MCF-7). Antibacterial application of these PtNPs is disbursed against gram-positive bacteria: Bacillus subtilis (RCMB 010067) and gram-negative bacteria: E. coli (RCMB 010052). SK-Ampicillin and gentamicin are the foremost effective medicament agents, several that are used as a reference in these antibacterial activity studies. Sankarganesh et al. reported new pyrimidine-based ligand capped gold and platinum NPs: synthesis, characterization, antimicrobial, antioxidant, DNA interaction, and in vitro anticancer activities [12]. In their study, new pyrimidine-based Schiff base ligand, 2-((4,6-dimethoxypyrimidine-2-yl)methyleneenamino)-6-methoxyphenol (DPMM) capped gold (Au) and platinum (Pt) NPs have been synthesized by modified Brust Schiffrin method [12]. The authors indicated that DPMM-AuNPs and DPMMPtNPs have potent antimicrobial against E. coli, Klebsiella pneumonia, Pseudomonas fluorescens, Shigella sonnei, Staphylococcus aureus and Aspergillus niger, Candida albicans, Candida tropicalis, Mucor indicus, and Rhizopus strains. The DPMM-AuNPs and DPMMPtNPs have good antioxidant activities rather than the free ligand (DPMM). Moreover, the in vitro anticancer activity of DPMM, DPMM-AuNPs, and DPMM-PtNPs against cancer (MCF-7, HeLa, and HEp2) and normal (NHDF) cell lines have performed using 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Their results reveal that, DPMM-AuNPs and DPMM-PtNPs have significant cytotoxic activity against the cancer cell lines and the least toxic effect on normal cell line as compared to standard drug cisplatin. Ramkumr et al. reported the synthesis of PtNPs using seaweed Padina gymnospora and their catalytic activity as PVP/PtNPs nanocomposite toward biological applications [13] The authors represented one-step synthesis of PtNPs using binary compound extract of Indian brown algae P. gymnospora and their chemical action activity with a compound polyvinylpyrrolidone (PVP) as PVP/PtNPs nanocomposite toward antimicrobial, hemolytic, cytotoxic (A. salina), and antioxidant properties. The authors indicted that the chemical action behavior of PtNPs as polymer/metal nanocomposite (PVP/PtNPs)

4. Antibacterial activity of nanomaterials

12.5 Recent trends in the antibacterial activity of platinum nanoparticles

279

preparation for antibacterial activity against seven sicknesses inflicting infective bacterial strains with the utmost activity against E. coli (15.6 mm) followed by Lactococcus lactis (14.8 mm) and E. pneumoniae (14.4 mm). However, no hemolytic activity was seen at their effective germicidal concentration, whereas increase within the hemolytic activity was seen solely in higher concentrations (600, 900, and 1200 µg/mL). On the opposite hand, PVP/PtNPs nanocomposite has shown cytotoxic activity at 100 6 4 µg/mL (LC50) against A. salina nauplii. Moreover, PVP/PtNPs nanocomposite showed increased scavenging activity against DPPH, superoxide, nitric oxide, and hydroxyl radicals. Cai et al. presented the porous Pt/Ag nanoparticles with excellent multifunctional enzyme-mimic activities and antibacterial effects [14]. In their work, a series of porous Pt/Ag NPs were fictitious from regular Pt 3 Ag100 x (x 5 25, 50, and 75) octahedra by a facile and economical dealloying method. Exceptional improvement in multiple enzyme-mimic activities associated with ORR was ascertained for the dealloyed Pt50Ag50 (D-Pt50Ag50) NPs. This impact is attributed to the ensuing Pt-rich surface structure, increased extent, and a synergistic impact of Pt and silver atoms within the D-Pt50Ag50 NPs. Moreover, the D-Pt50Ag50 NPs exerted glorious medicine effects on two model bacterium (gram-negative Escherichia and gram-positive S. aureus). Tahir et al. presented the facile and green synthesis of phytochemicals capped PtNPs and in vitro their superior antibacterial activity [15]. In their study, plant extract of extremely active medicative plant, asterid dicot genus laevigatum, was used for the synthesis of PtNPs to boost its bio-activities. The authors found that zone of inhibition of PtNPs against P. aeruginosa was 15 ( 6 0.5) metric linear unit and B. subtilis was 18 ( 6 0.8) mm. The foremost vital outcome of this examination is that PtNPs exhibited sturdy medicament activity against P. aeruginosa and B. subtilis that have sturdy defensive system against many antibiotics. Ayaz Ahmed et al. reported the PtNPs inhibit bacteria proliferation and rescue zebrafish from bacterial infection [16]. The authors indicated that for the primary time, the in vivo antibacterial activity of PtNPs was incontestable using adult zebrafish because the animal model. As a signal of idea, zebrafish infected with a model pathogen, E. coli and a fish-specific pathogen, Aeromonas hydrophila, were subjected to treatment with PtNPs. A microorganism colony count assay discovered that the PtNPs exhibit dose-dependent inhibition of microorganism proliferation and saved zebrafish fully from bacteria infection. Pharmacology studies revealed that the antibacterial concentration of PtNPs employed in their study is nontoxic to zebrafish. Being nontoxic to zebrafish, these PtNPs may open up new avenues in antimicrobial medical care for future medicine applications. Rajathi and Nambaru reported the phytofabrication of nanocrystalline platinum particles by leaves of Cerbera manghas and its antibacterial efficacy [17]. The authors indicated that biologically synthesized PtNPs were found to be economical against designated microorganism pathogens. A study by Nam [18] has characterized the synthesis of a changed PMMA dental appliance acrylic loading noble metal nanoparticles (PtN) and assessed its microorganism repressing effectuality to provide novel antimicrobial denture base material. Polymerized PMMA dental appliance acrylic disc (20 3 2 mm) specimens

4. Antibacterial activity of nanomaterials

280

12. Antibacterial activity of platinum nanoparticles

containing 0 (control), 10, 50, 100, and 200 mg/L of PtN were fictitious severally. In antimicrobial assay, specimens were placed on the cell culture plate, and 100 µL of microbic suspensions of Streptococcus mutans (S. mutans) and Streptococcus sobrinus (S. sobrinus) were inoculated then incubated at 37 C for 24 h. His as-prepared platinumPMMA nanocomposite (PtNC) expressed vital microorganism antiadherent impact instead of germicidal effect on top of 50 mg/L PtNPs loaded when put next to pristine PMMA (P 5 .01) with no or very little amounts of atomic number 78 particle eluted. This PtNC can be a doable intrinsic antimicrobial denture material with correct mechanical characteristics, meeting those given for dental appliance bases. Konieczny et al. reported the effects triggered by PtNPs on primary keratinocytes [19]. Since the human skin is often exposed to toxic particles; thus, within the gift study we have a tendency to self-addressed the question of whether or not PVP -coated PtNPs could have any negative effects on skin cells, together with preponderantly epidermal keratinocytes. In their study, PtNPs of two sizes were used: 5.8 nm and fifty seven nm, in concentrations of 6.25, 12.5, and 25 µg/mL. each kinds of NPs were protected with PVP. Primary keratinocytes were treated for 24 and 48 h, then toxicity, genotoxicity, morphology, metabolic activity, and changes within the activation of signal pathways were investigated in PtNP-treated cells. The authors have a tendency to found that PtNPs trigger toxic effects on primary keratinocytes, decreasing cell metabolism, however these changes don’t have any effects on cell viability or migration. Moreover, smaller NPs exhibited a lot of injurious impact on DNA stability than the massive ones. Analyzing activation of caspases, they found changes in activity of proteolytic enzyme nine and caspase 3/7 triggered principally by smaller NPs. Elhusseiny and Hassan reported the antimicrobial and antitumor activity of platinum and palladium complexes of novel spherical aramides nanoparticles containing flexibilizing linkages: Structure-property relationship [20] In their study, a square planar palladium (II) and octahedral Pt (IV) complexes with novel spherical aramides nanoparticles containing versatile linkages ligands are synthesized and characterized using analytical and spectral techniques. These Pd complexes of polyamides containing sulfones showed the best efficiency as medication and antifungal agents. In addition, the platinum complexes containing sulfone and ether versatile linkages and chloro teams exhibited high efficiency as anticancer and antimicrobial agents.

References [1] R. Nishanthi, S. Malathi, S. John Paul, P. Palani, Green synthesis and characterization of bioinspired silver, gold and platinum nanoparticles and evaluation of their synergistic antibacterial activity after combining with different classes of antibiotics, Mater. Sci. Eng. C. 96 (2019) 693 707. [2] M. Endo, M. Janczarek, Z. Wei, K. Wang, A. Markowska-Szczupak, B. Ohtani, et al., Bactericidal properties of plasmonic photocatalysts composed of noble metal nanoparticles on faceted anatase titania, J. Nanosci. Nanotechnol. 19 (1) (2019) 442 452. [3] X. Jiang, W. He, X. Zhang, Y. Wu, Q. Zhang, G. Cao, et al., Light-induced assembly of metal nanoparticles on ZnO enhances the generation of charge carriers, reactive oxygen species, and antibacterial activity, J. Phys. Chem. C. 122 (51) (2018) 29414 29425.

4. Antibacterial activity of nanomaterials

References

281

[4] S.B. Subramaniyan, A. Ramani, V. Ganapathy, V. Anbazhagan, Preparation of self-assembled platinum nanoclusters to combat Salmonella typhi infection and inhibit biofilm formation, Colloids Surf. B Biointerfaces 171 (2018) 75 84. [5] F. Zou, J. Zhou, J. Zhang, J. Li, B. Tang, W. Chen, et al., Functionalization of silk with in-situ synthesized platinum nanoparticles, Materials 11 (10) (2018) 1929. [6] R. Dobrucka, J. Dlugaszewska, Antimicrobial activity of the biogenically synthesized core-shell Cu@Pt nanoparticles, Saudi Pharm. J. 26 (5) (2018) 643 650. [7] M.F. Peana, S. Medici, M.A. Zoroddu, The intriguing potential of “minor” noble metals: emerging trends and new applications (Book Chapter), in: Biomedical Applications of Metals, 2018, pp. 49 72. [8] U. Jeyapaul, M.J. Kala, A.J. Bosco, P. Piruthiviraj, M. Easuraja, An eco-friendly approach for synthesis of platinum nanoparticles using leaf extracts of jatropa gossypifolia and jatropa glandulifera and their antibacterial activity, Orient. J. Chem. 34 (2) (2018) 783 790. [9] N.S. Al-Radadi, Green synthesis of platinum nanoparticles using Saudi’s Dates extract and their usage on the cancer cell treatment, Arab. J. Chem. 12 (3) (2019) 330 334. [10] V.R. Chelli, A.K. Golder, One pot green synthesis of Pt, Co and Pt@Co core-shell nanoparticles using Sechium edule, J. Chem. Technol. Biotechnol. 94 (3) (2019) 911 918. [11] E.H. Ismail, N.S. Al-Radadi, An eco-friendly synthesis of platinum nanoparticles and their applications on the cancer cell treatments, J. Comput. Theor. Nanosci. 14 (12) (2017) 6044 6052. [12] M. Sankarganesh, P. Adwin Jose, J. Dhaveethu Raja, M.P. Kesavan, M. Vadivel, J. Rajesh, et al., New pyrimidine based ligand capped gold and platinum nano particles: synthesis, characterization, antimicrobial, antioxidant, DNA interaction and in vitro anticancer activities, J. Photochem. Photobiol. B 176 (2017) 44 53. [13] V.S. Ramkumar, A. Pugazhendhi, S. Prakash, N.K. Ahila, G. Vinoj, S. Selvam, et al., Synthesis of platinum nanoparticles using seaweed Padina gymnospora and their catalytic activity as PVP/PtNPs nanocomposite towards biological applications, Biomed. Pharmacother. 92 (2017) 479 490. [14] S. Cai, X. Jia, Q. Han, X. Yan, R. Yang, C. Wang, Porous Pt/Ag nanoparticles with excellent multifunctional enzyme mimic activities and antibacterial effects, Nano Res. 10 (6) (2017) 2056 2069. [15] K. Tahir, S. Nazir, A. Ahmad, B. Li, A.U. Khan, Z.U.H. Khan, et al., Facile and green synthesis of phytochemicals capped platinum nanoparticles and in vitro their superior antibacterial activity, J. Photochem. Photobiol. B 166 (2017) 246 251. [16] K.B. Ayaz Ahmed, T. Raman, V. Anbazhagan, Platinum nanoparticles inhibit bacteria proliferation and rescue zebrafish from bacterial infection, RSC Adv. 6 (50) (2016) 44415 44424. [17] F.A.A. Rajathi, V.R.M.S. Nambaru, Phytofabrication of nano-crystalline platinum particles by leaves of Cerbera manghas and its antibacterial efficacy, Int. J. Pharma Biol. Sci. 5 (1) (2014) P619 P628. [18] K.-Y. Nam, Characterization and bacterial anti-adherent effect on modified PMMA denture acrylic resin containing platinum nanoparticles, J. Adv. Prosthodont. 6 (3) (2014) 207 214. [19] P. Konieczny, A.G. Goralczyk, R. Szmyd, L. Skalniak, J. Koziel, F.L. Filon, et al., Effects triggered by platinum nanoparticles on primary keratinocytes, Int. J. Nanomed. 8 (2013) 3963 3975. Available from: https://doi. org/10.2147/IJN.S49612. [20] A.F. Elhusseiny, H.H.A.M. Hassan, Antimicrobial and antitumor activity of platinum and palladium complexes of novel spherical aramides nanoparticles containing flexibilizing linkages: structure-property relationship, Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 103 (2013) 232 245.

4. Antibacterial activity of nanomaterials

C H A P T E R

13 Antibacterial property of metal oxide-based nanomaterials Md Abdus Subhan Department of Chemistry, Shah Jalal University of Science and Technology, Sylhet, Bangladesh

13.1 Introduction Microbial infectious diseases are a global threat to humans. Improper and excess use of antibiotics has created antimicrobial-resistant microbes that can resist clinical treatment. The hunt for safe and alternate antimicrobial agents in order to overcome such resistant microorganisms offers promise to combat infectious organisms. Over the past two decades, metal oxide nanoparticles (MO-NPs) have become an attractive alternative to combat microbes that are highly resistant to various classes of antibiotics. Their vast range of physicochemical properties enables MO-NPs to act as antimicrobial agents through various mechanisms. Due to various properties, potencies, and spectra of activity, some metals such as silver, copper, gold, titanium, zinc and their oxides have been used as antibacterial agents [1]. Antibacterial agents are also very important in the textile industry, water disinfection, medicine, and food packaging. Usually, organic compounds used for disinfection have some disadvantages including toxicity to the human body. For this reason, the interest in inorganic disinfectants, such as MO-NPs, is increasing rapidly. Apart from exhibiting antimicrobial properties, MONPs also serve as drug carriers. These immense multiple properties exhibited by MO-NPs will have an impact on the treatment of deadly infectious diseases. This book chapter discusses the mechanisms of action of MO-NPs against microorganisms, safety concerns, challenges, and future perspectives. With an increase in antibiotic resistance, an increasing interest in developing new antimicrobial agents has gained popularity. Metal and metal oxide-based nanoparticles (NPs) are able to distinguish bacterial cells from mammalian cells and can provide long-term antibacterial and biofilm prevention. These NPs elicit bactericidal properties through the generation of reactive oxygen species (ROS) that are able to target physical structures, metabolic pathways, and DNA synthesis of prokaryotic cells leading to cell death. In this chapter, the critical analysis of current literature on antimicrobial effect of metal and MO-NPs is examined.

Nanotoxicity DOI: https://doi.org/10.1016/B978-0-12-819943-5.00013-0

283

Copyright © 2020 Elsevier Inc. All rights reserved.

284

13. Antibacterial property of metal oxide-based nanomaterials

The antimicrobial mechanisms of metal ions and metal nanomaterials (NMs) are discussed. Antimicrobial efficiency of NMs is correlated with the structural and physical properties, such as size, shape, and/or zeta potential (ZP). A critical analysis of the current state of metal and metal oxide nanomaterial research may advance our understanding to overcome antibiotic resistance and provide alternatives to combat bacterial infections. Finally, emerging approaches to identify and minimize metallic toxicity for biomedical applications are addressed [2]. Nanotechnology is a scientific and engineering technology conducted at the nanoscale, such as in the fields of compound fabric manufacturing, food processing, agricultural processing, and engineering, as well as in medical and medicinal application. In the recent decade, many researchers have been interested in applications of NMs for antimicrobial use. Recently available reports show that some of the MO-NPs including; AgO, Ag2O, Al2O3, TiO2, ZnO, CuO, Co3O4, In2O3, MgO, SiO2, ZrO2, Cr2O3, Ni2O3, Mn2O3, CoO, NiO, and CeO2 have toxicity toward several microorganisms and they could successfully kill numerous bacteria [3,4]. There are some effective factors that can influence the ability of NM in reducing or killing the cells, and there are mechanisms for NM against bacteria, which are briefly listed as follows: surface charge of the metal NM, shape, type and material, concentration of NM, dispersion and contact of NM to the bacterial cell, presence of active oxygen, liberation of antimicrobial ions, medium components and pH, physicochemical properties, specific surface-area-to-volume ratios, size, role of growth rate, role of biofilm formation, cell wall of bacteria, and effect of ultraviolet (UV) illumination. In the use of NM as antimicrobial agents, consideration of the many factors is highly important. Antibacterial resistance to common chemical antibacterial agents can be due to long production consumption cycle, thereby reducing their efficiency, and use of poor quality or fake medicines in undeveloped and developing countries. NPs as antimicrobial agents have become an emerging approach against this challenge, which can establish an effective nanostructure to deliver the antimicrobial agent for targeting the bacterial community efficiently; in addition, they are so potent that microbial pathogens cannot develop resistance to them. On the other hand, most of the MO-NPs have no toxicity toward humans at low and effective concentrations used to kill bacterial cells, which thus becomes an advantage for using them in a full scale [1]. Over the present decade, several studies have suggested that NPs are excellent antibacterial agents [4]. MO-NPs are known to effectively inhibit the growth of a wide range of gram-positive and gram-negative bacteria. They have emerged as promising candidates to challenge the rising global issue of antimicrobial resistance (AMR). However, a comprehensive understanding of their mechanism of action and identifying the most promising NP materials for future clinical translation remains a major challenge due to variations in NP preparation and testing methods. With various types of MO-NPs being rapidly developed, a robust, standardized, in vitro assessment protocol for evaluating the antibacterial potency and efficiency of these NPs is needed. Calculating the number of NPs that actively interact with each bacterial cell is critical for assessing the dose response for toxicity. Methods to evaluate MO-NPs antibacterial efficiency with focus on issues related to NPs in these assays with highlight on sources of experimental variability including NP preparation, initial bacterial concentration, bacterial strains tested, culture microenvironment, and dose have been reported [5].

4. Antibacterial activity of nanomaterials

13.3 Methods to evaluate MO-NPs antibacterial efficiency

285

In recent years, infectious diseases, specifically those are caused by pathogens, have seen a dramatic proliferation due to resistance to multiple antibiotics, opening the colony by opportunistic pathogens. Nanotechnology and tissue engineering have been applied in the development of new antimicrobial therapies, capable of fighting opportunistic infections. In the medical field, research on antimicrobial properties of MO-NPs has emerged to find new antimicrobial agents as an alternative against resistant bacteria. The metal oxides, particularly those formed by transition metals are compounds with electronic properties, and most magnetic phenomena involve this type of oxides. NP-based metal oxide properties such as shape, size, roughness, ZP, and their large surface area, make oxides ideal candidates to interact with bacteria and able to have an antimicrobial effectiveness. However, investigating the interaction patterns at nano-bio interface is a key challenge for safe use of NPs to any biological system [6]. A recent study, for example, explored the role of interaction pattern at the iron oxide nanoparticle (IONP) bacteria interface affecting antimicrobial propensity of IONP. The aim of this chapter is to offer an updated landscape about the relationships between the use of MO-NPs in the medical field, with an emphasis on their role as antimicrobial agents and the properties that influence their antimicrobial response. In addition, the mechanism of nano-antimicrobial action is described and the importance of using in vitro test methods, adopted by leading international regulatory agencies, which can be used to determine the antimicrobial activity of the MO-NPs.

13.2 Mechanism of antimicrobial resistance AMR includes two levels of resistance, the cellular level resistance and the community level resistance [7]. The development of cellular resistance occurs due to endogenous gene mutations as well as via HGT [8,9] of resistance determinants from other microorganisms. Also, a group of bacteria can be tolerant to the environmental stress that individual cells can not, which is called the community level resistance. Such tolerance can cause an increased resistance to antimicrobials. For example, the resistance obtained by microorganisms in biofilm can be up to 1000 times higher than that gained by their planktonic counterparts, which impairs the treatment of biofilm-associated infections in clinical treatment. The main mechanism currently proposed to explain such tolerance is the presence of persister cells. The persisters can escape the lethal action of antimicrobials by entering a physiological state in which the antimicrobials do not kill them, a phenomenon known as bacterial persistence. Moreover, the cellular and community levels of resistance can be synergistic, thereby greatly enhancing the overall AMR of the microbial community [7].

13.3 Methods to evaluate MO-NPs antibacterial efficiency 13.3.1 In vitro methods for antimicrobial evaluation of nanoparticles-based metal oxide Bacteria exposed to antimicrobials are under selective pressure to evolve and adapt, this natural process leads to AMR [5]. Humans are facing the growing threat of rapid evolution

4. Antibacterial activity of nanomaterials

286

13. Antibacterial property of metal oxide-based nanomaterials

and dissemination of bacteria resistant to multiple antibiotics. Therefore there is an urgent need to develop new antimicrobials. Antimicrobial agents include disinfectants, antiseptics, and antibiotics. New agents must be exhaustively tested for efficacy and safety. Evidencebased selection of the microorganisms and the evaluation system are of paramount importance for adequate interpretation of the test results, and for extrapolating from in vitro to real-life scenarios. The use of NPs especially based in metal oxides emerge as new antimicrobial agents, therefore it is necessary to test the efficacy of nano-antimicrobials against representative bacterial species. One known limitation of the testing systems currently in use, is that formulations are often challenged in vitro with one microbial species at the time, and rarely against multi-species biofilms.

13.3.2 Regulatory testing Regulatory agencies require adherence to well-established evaluation systems. Regulatory tests applicable to disinfectants, antiseptics, or therapeutic antimicrobials vary greatly and could include application to NPs with potential use as antimicrobials.

13.3.3 Antimicrobial activity tests A relevant test microorganism is chosen: preferably a strain from the American Type Culture Collection (ATCC) or a similar repository; although, wild-type bacteria from clinical samples also has been used. All necessary controls must be included to assess test reliability and reproducibility. Also, it is important to differentiate between kill and inhibition of growth. The antimicrobial capability of NPs has been explored by these techniques; due studies have suggested that NPs are excellent microbicidal agents. The in vitro tests described below are the ones that have been the most used, and the regulatory agencies recommend to determine antimicrobial activity of chemical formulations and can be used in the studies of nano-antimicrobials. The use of such tests depends on the objectives and the type of information it wants to obtain. If NP has antimicrobial activity the first approach is to conduct an antimicrobial activity test, such as a disc diffusion test. The currently used methods are: disk-diffusion method, agar dilution method, broth dilution method, and time-kill method [5]. Disk-diffusion method: Mueller-Hinton agar (pH 7.2 7.4) is the culture medium of choice. To standardize disc diffusion, the agar is poured into either Petri dish to only 4 mm in depth, as indicated in the Clinical and Laboratory Standards Institute method. The bacteria are suspended to a 0.5 McFarland turbidity standard equivalent to 50 3 106 cfu/mL. From this suspension, 100 μL are uniformly spread onto the agar. Filter-paper discs of 6 mm diameter, containing the test, nano-antimicrobial, will be placed over the seeded agar (alternatively, a 50 100 μL well, punched into the agar, will contain the test antimicrobial). After overnight incubation at 37 C, the plates will be examined to assess inhibition rings around the disc. The size of the NP, its rate of diffusion, the agar’s porosity, and possible charge interactions between the antimicrobial and the agar may affect diffusion and the final size of the inhibition zone. In theory, the highest concentrations will be near the antimicrobial-containing disc and will be diluted away from the center.

4. Antibacterial activity of nanomaterials

13.4 Antimicrobial effect of metal and metal oxide nanoparticles

287

Agar dilution method: this method is the gold standard for assessing the minimal inhibitory concentration (MIC). In this method, the melted agar is mixed to contain serial dilutions of the nano-antimicrobial. The resulting antimicrobial containing medium is plated into Petri dishes. An aliquot containing 104 cfu of the test microorganism is placed onto the agar’s surface, and incubated overnight. Then, the plates will be examined for growth to determine the last effective concentration to inhibit growth. Broth dilution method: this method is often used because it is more versatile and less laborious than the agar dilution method. Its microtiter plate version (broth microdilution), allows for testing more microorganisms against diverse concentrations of nano-antimicrobials, and can be automated. Test tubes or wells in a microtiter plate, are prepared with bacteriological broth containing serial dilutions of the test nano-antimicrobial, and seeded with bacteria. After, overnight incubation, the tubes or wells are inspected for growth. The lowest concentration of NPs that results in no-growth is the MIC. Time-kill method: after adding the test formulation to a broth culture, antimicrobial activity can be assessed in vitro by collecting sequential samples to count survivors. Time-kill allows the assessment of in vitro synergy or antagonism between nano-antimicrobials. For the timekill experiments, Mueller Hinton broth is prepared with serial dilutions of the test antimicrobial, alone or in combination. The nano-antimicrobial concentrations may span a range above and below the formulation’s MIC, previously obtained from agar dilution tests. Broths are then inoculated with 106 cfu/mL and incubated overnight at 37 C. From time 0 when bacteria are first exposed to the test antimicrobial, samples are obtained at 30 min intervals for up to 6 h. The samples are then plated on nutrient agar. After incubation overnight at 37 C, survivor counts are plotted to obtain a time-kill curve.

13.4 Antimicrobial effect of metal and metal oxide nanoparticles Similar to antibiotics, metal-based NPs are able to differentiate prokaryotic (bacterial cells) from eukaryotic (mammalian cells) through bacteria’s metal transport system and metalloproteins. However, unlike antibiotics, metal-based NPs prompt bactericidal efficiency via multiple mechanisms. Given this distinction, multiple gene mutations within the same bacterial cell are needed to elicit any form of resistance. Metal NPs physically interact with bacterial cells through three major pathways to kill resistant bacteria [2]. These are (1) destabilization of phospholipid bilayer of the cell (2) binding to cytosolic proteins and (3) oxidative stress through formation of ROS. Although not well-understood, bacteria precipitate metal compounds as oxides, sulfides, protein aggregates, or elemental crystals. These precipitates form particulates that meticulously interact with the membrane, sequestering these materials into the cell. Metal compounds have been shown to disrupt biofilm production and synergistically exert antimicrobial effects by inhibiting enzyme activity, altering membrane stability and function, damaging DNA, and overall inhibiting planktonic growth. Due to the mutating nature of bacteria, studying the antimicrobial effects of metal and metal ions has been slow and difficult. However, metal NPs, specifically silver, gold, and gallium, have demonstrated unique antimicrobial effects that have been extensively investigated.

4. Antibacterial activity of nanomaterials

288

13. Antibacterial property of metal oxide-based nanomaterials

The emergence of antibiotic- and/or multidrug-resistant bacteria is recognized as a crucial challenge for public health. Killing of antibiotic-resistant bacteria requires multiple expensive drugs that may have side effects. As a result, treatments are costly and require more time. NPs can offer a new strategy to tackle multidrug-resistant bacteria. Several types of silver carbon complexes with different formulations including micelles and NPs have efficient toxicity against medically important pathogens such as targeting bactericidal NPs to specific bacteria or specific infected tissue, which is an efficient prospect in treating infection because this phenomenon minimizes side effects and enhances antibacterial activity. In this case, multifunctional NPs can be very useful; for instance, multifunctional IgG Fe3O4@TiO2 magnetic NPs are able to target several pathogenic bacteria and have efficient antibacterial activity under UV irradiation [10]. The IgG and TiO2 play a critical role in the targeting and killing properties of these NPs respectively. These NPs are toxic to Streptococcus pyogenes M9022434 and M9141204. Nitric-oxide-releasing NPs (NO NPs) are broad spectrum antibacterial agents that are able to inhibit the growth of many antibiotic-resistant and sensitive clinically isolated bacteria such as Klebsiella pneumoniae, Enterococcus faecalis (1), Str. pyogenes, E. coli, and P. aeruginosa ( ). The toxicity of these NPs depends on the delivery of NO to the target. These NPs are able to change the structure of the bacterial membrane and produce reactive nitrogen species, which lead to modification of essential proteins of bacteria. Beside NO NPs, ZnO NPs are toxic to antibiotic (methicillin)-resistant bacteria such as Streptococcus agalactiae (1) and S. aureus. These NPs are able to disorganize and damage the cell membrane and increase the permeability, which leads to cell death. The polyvinyl alcohol -coated ZnO NPs are able to internalize the bacteria and induce oxidative stress. The toxicity of ZnO NPs is concentration-dependent and these NPs are mildly toxic at low concentration [3].

13.5 Mode of antimicrobial action by metal and metal oxides nanoparticles Some of these include the action of ROS, the electrostatic interaction, accumulation, ions delivered and contact by itself of NPs, which introduce several effects from outside and into the bacteria [5] (Fig. 13.1), and are described below:

13.5.1 Formation of reactive oxygen species They are a group of reactive molecules produced in some metabolic processes in which oxygen participates: the superoxide anion O22 which is a powerful oxidizing agent very reactive with water. Hydrogen peroxide H2O2 and the hydroxyl radical (•OH) which is the most reactive, since accepting one more electron, gives rise to a water molecule (Fig. 13.2). MO-NPs are capable of producing different ROS that may participate in different types of reactions in which they can undergo oxidation or reduction processes. The alteration of the balance in the mechanisms of production and elimination of ROS, in favor of production, originates the state of oxidative stress in the bacteria cell. In the case of O2 and H2O2 cause less acute stress reactions and can be neutralized by endogenous antioxidants, such as superoxide and catalase enzymes, while OH2 and O2 can lead to acute microbial death.

4. Antibacterial activity of nanomaterials

13.5 Mode of antimicrobial action by metal and metal oxides nanoparticles

289

FIGURE 13.1 Mechanisms of action of the bactericidal effect from metal oxide nanoparticles (MO-NPs). Reproduced with permission from A.L. Vega-Jime´nez, A.R. Va´zquez-Olmos, E. Acosta-Gı´o, et al., In vitro antimicrobial activity evaluation of metal oxide nanoparticles, Nanoemulsions—Properties, Fabrications and Applications, 2019, IntechOpen, 1 13. Available from: https://doi.org/10.1039/C9NJ01760G [5]

ROS lead to severe oxidative stress and damage to the cell’s macromolecules which overall cause lipid peroxidation, alteration of proteins, inhibition of enzymes, and RNA/DNA damage. ROS produce disruption of DNA, damage by oxidation of polyunsaturated fatty acids and amino acids. This severe oxidative stress can also form holes or pits within the bacterial membrane, causing cell lysis. Hydroxyl radical (•OH) formation has been observed with silver. Gold, zinc oxide, and magnesium oxide demonstrate ROS formation through increased catalytic activity generating H2O2 from glucose oxidase. Titanium dioxide, upon exposure to light, has elicited ROS formation from both OH and H2O2. Gallium, a unique case, has induced ROS production when mistaken for iron.

4. Antibacterial activity of nanomaterials

290

13. Antibacterial property of metal oxide-based nanomaterials

FIGURE 13.2 Metal nanomaterials are able to physically interact with prokaryotic cells compromising cellular functions. (A) Metal nanomaterials are able to destabilize the phospholipid bilayer of the cell, causing cell lysis. (B) Metal nanoparticles are able to bind to cytosolic proteins, such as DNA, triggering cell death. (C) Metal nanomaterials produce reactive oxygen species (ROS), leading to increased oxidative stress and cell instability. Reproduced with permission from A.L. Vega-Jime´nez, A.R. Va´zquez-Olmos, E. Acosta-Gı´o, et al., In vitro antimicrobial activity evaluation of metal oxide nanoparticles, Nanoemulsions—Properties, Fabrications and Applications, 2019, IntechOpen, 1 13. Available from: https://doi.org/10.5772/intechopen.84369 [2] with permission from Copyright WileyVCH Verlag GmbH & Co. KGaA

13.5.2 Damage to the cell-wall membrane due to electrostatic interaction and accumulation The electronegative groups of the polysaccharides in the bacterial membrane have attraction sites by metal cations. The difference in charge between bacterial membranes and the NPs of metal oxides leads to electrostatic attraction and thus accumulates on the bacteria surface, altering the structure and permeability of the cell membrane. Gram-negative

4. Antibacterial activity of nanomaterials

13.5 Mode of antimicrobial action by metal and metal oxides nanoparticles

291

bacteria have a higher negative charge than gram-positive bacteria and therefore the electrostatic interaction will be stronger in gram-negative strains. The pores of the membranes are in the order of nanometers, therefore the smaller the particle size and the greater the surface area, the greater the efficiency of the MO-NPs. In the same way, the cations extracted from the NPs of the metal oxides and their accumulation in the cell wall, create pits in it, leading to a change in permeability due to the sustained release of lipopolysaccharides(LPSs), membrane proteins, and intracellular factors. In addition, this mechanism has been linked to the interruption of the replication of adenosinetriphosphate (ATP) and the DNA of the bacterium, leading to its death. One study indicates that the action of NPs depends on the components and structure of the bacterial cell. The unique components of gram-negative bacteria, such as LPS, can prevent the adhesion of metal oxide NPs to the barrier of bacterial cells and regulate the flow of ions in and out of the bacterial cell membrane.

13.5.3 Loss of homeostasis by metal ions The balance of metallic elements is essential for microbial survival, since it regulates metabolic functions by helping coenzymes, cofactors, and catalysts. When the bacteria have an excess of metals or metal ions, there will be a disorder in the metabolic functions. Metal ions bind with DNA and alter the helical nature by cross-linking between and within the DNA strands. The metal ions neutralize the charges in LPS and increase the permeabilization of the outer membrane. The ions of metal oxides might also cause the decomposition of bacterial cells due to the diffusion of metal ions by generating large amounts of hydroxyl radicals and diffusion in bacterial cells. Other studies indicate that NPs of metal oxides slowly release metal ions through adsorption, dissolution, and hydrolysis; they are toxic and abrasive to bacteria and, therefore, lyse the cells.

13.5.4 Dysfunction of proteins and enzymes-binding to cytosolic proteins Protein dysfunction is another mode of antibacterial activity exhibited by NPs of metal oxides. The metal ions catalyze the oxidation of the side chains of amino acids resulting in carbonyls bound to proteins. The carboxylation levels within the protein molecule serve as a marker for the oxidative damage of the protein. This carboxylation of proteins will lead to the loss of catalytic activity in the case of enzymes, which finally triggers the degradation of proteins. The main mechanism in which metallic-based NPs induce an antimicrobial response is through binding to cytosolic proteins, such as enzymes and DNA. This interaction leads to decreased function, inhibiting respiratory and metabolic pathways and ATP production. For example, silver binds to enzymes within the respiratory chain and DNA, inhibiting replication and division. Gold, on the other hand, interacts with DNA by upregulating genes within the cell. This results in decreased membrane integrity and a buildup of ROS within the cytosol of the cell.

4. Antibacterial activity of nanomaterials

292

13. Antibacterial property of metal oxide-based nanomaterials

13.5.5 Inhibition of the transduction signal Electrical properties of metal oxide NPs interact with nucleic acids inducing a suppression of cell division by altering processes of replication of the chromosomal DNA and the plasmid in microorganism. It is known that signal transduction in bacteria is affected by NPs of metal oxide. Phosphotyrosine is an essential component of mechanism of signal transduction in bacteria. NPs dephosphorylate the phosphotyrosine residues, which inhibits signal transduction and, ultimately, obstructs the growth of bacteria.

13.5.6 Interactions with phospholipid bilayer Metal-based NPs can disrupt the cell membrane potential and integrity by binding electrostatically to the bacterial cell wall and/or releasing metallic ions. Given the positive charge of the NPs and the negative charge of cellular components, the two interact at the surface through electrostatic communication. These interactions disrupt the membrane and produce increased oxidative stress that damages bacterial proteins. Due to breaking of the cell barrier, an abundant amount of water from the cytosol is released. Cells try to compensate for this loss through the bacteria’s proton efflux pumps and electron transport. However, the high demand of these ions causes severe damage to these transmembrane systems. Overall, this imbalance of ions and membrane stability results in impaired respiration, interruption of energy transduction, and eventually cell death. This effect has been demonstrated through the interaction of silver, gold, zinc oxide, magnesium oxide, and titanium oxide NPs. Silver NPs specifically interact with sulfur-containing constituents within the cell membrane, and the ions produced impede cell wall synthesis. Yet another mechanism found to be effective against E. coli occurs when Ag-TiO2 acts as a photocatalyst under solar light illumination [12]. The surface plasmon resonance effect under solar light sustains the generation of photoinduced electrons, eventually leading to the production of ROS within bacterial cells, thus killing them. Biofilms are the most difficult to tackle with antibiotics. MeO-NPs offer a great advantage against biofilm formation.

13.6 Nanoparticle characteristics and their influence on antimicrobial activity The factors of the antimicrobial activity have been sought to analyze what characteristics influence the microbial response to the action of the MO-NPs. It is known of existing reports concerning the chemical-physics properties from the MO-NPs, but certain factors must be taken into consideration like the shape, size, roughness, ZP and coatings, etc., which influence the resultant antimicrobial effectiveness. These results could have a mainly therapeutic application in medicine, but it can also be extended to the food industry, to water purification and to the textile industry.

13.6.1 Size and shape Several reports mention that size and shape are the most important factors to the antimicrobial activity. With respect to size there are findings where this is a crucial factor to damage the bacterial systems for many reasons. The sizes as ,30 nm are factors that allow accumulation

4. Antibacterial activity of nanomaterials

13.7 Metal oxide-based antibacterial membrane

293

and penetration into the bacteria causing damage and consequently leading to bacteria death (,10 nm). MO-NPs with a size greater than 10 nm can promote the permeability when coming into contact with bacteria. In relation with this, the specific surface area by the NP size affects the surface to mass ratio affecting surface reactivity. For this reason, they can also have influence in many direct mechanisms of toxicity against the bacteria and the subsequent loss of viability. With respect to shape, it is by knowing that depending on the synthesis method, it will obtain the form of the NP. Numerous studies exhibited various forms obtained like spherical, rod-shaped, truncated triangular, nanotubes, nanorods, nanowires, nanosphere, nanoneedles, nanorings, and nanocubic. Evidence reports that needle-shaped MO-NPs present higher antibacterial activity than cubic shaped, based on the optical and fluorescence intensity.

13.6.2 Surface and zeta potential The relation between the surface NP/NM and bacterial adhesion has not been fully studied and there are few reports about it. Some studies report that the adsorption of bacterial proteins is promoting by the surface area to mass ratio carry out the reduction in bacterial adhesion. Surface of NM has a high degree of roughness; therefore, bacteria cell membranes can not adhere to the surface NM; so, the bacteria adhesion is reduced. The surface charge or ZP could be another property of the NP related with bacteria adhesion since it is important to mention that if the surfaces with negative charge are capable to decrease the interaction with bacteria charged negatively, the surface of NM with negative charge could obtain the same effect, compromising bacterial adhesion. On the other hand, the electrostatic attraction occurs when the NPs are positively charged promoting the accumulation in bacterial cell membrane, which is negatively charged and then they penetrate inside the bacteria triggering other mechanisms. ZP of the NP with slightly positive or slightly negative is suitable for interaction with bacterial cell membrane.

13.6.3 Chemical doping Nanoparticle chemical doping is a modification and functionalization around the surface of NPs to regulate and control the interaction with bacteria and enhance their antimicrobial effect. Studies have shown this method as a factor to improve the presence of surface oxygen atoms that promote the production of ROS. Similarly, the chemical functionalization and increases of the surface-area-to-volume ratio result in increasing the antimicrobial potential activity. Also, this procedure has prevented the agglomeration and the solubility in different solutions [5].

13.7 Metal oxide-based antibacterial membrane Antibacterial activity on the membrane surface shows that the addition of metal oxide can decrease microorganism’s attachment on the membrane surfaces. The metal oxide on the polysulfone membrane surface can remove almost all bacteria colonies by using ZnO-GO and AgGO antibacterial agents. Other than that, the addition of Ag-SiO2 and Ag@TiO2-CNTs make the

4. Antibacterial activity of nanomaterials

294

13. Antibacterial property of metal oxide-based nanomaterials

bacteria colonies expire. The antibacterial activity of metal oxide will vary depending on its antibacterial mechanism, the number of bacterial colonies on the membrane surface due to the amount or type of different metal oxides used. To improve membrane performance, many modifications have been made to the membrane preparation. Membrane preparation with the addition of metal oxide can increase membrane antibacterial, antifouling, and membrane filtration properties. Antibacterial mechanisms in the metal oxide involve the release of metal ions into the water which causes the amount of metal oxide in the membrane surface to decrease. The improvement of interaction between metal oxide and membrane surface is required to reduce the rate of metal ions released from the membrane surface so that maintaining the antibacterial activity can go for a long time, moreover so it can reduce soluble metal ions concentration in water. By combining two metal oxides or with another metal NP can increase membrane performance. There have been studies, reported that optimized metal NPs exhibited better antibacterial properties than a single metal oxide [1]. The other challenge is to combine the metal oxide with another natural resource, to give better antibacterial activity and be more friendly to the environment. Some research has shown the combination from ZnO and Eugenol to give antibacterial activities in polysulfone membrane. The next challenge to improve the antibacterial property of membrane is to combine various polymeric membranes with NPs to find the most appropriate combinations and applications of the membrane fabricated. For example, in drinking water application, metal oxide must be handled carefully due to potentially its toxicity toward the human. Moreover, the antibacterial agent activity only prevents microorganism growth on membrane surface. Beside of that, the polymeric materials and NPs researches need to be optimized so that cost of production becomes more competitive, gives a high performance and long time antibacterial activity of membranes. Membrane is an advanced treatment technology that has many benefits such as cheap, highly selective, using less chemicals and energy, simple design, and easy to maintenance. Membrane also shows great performance for wastewater treatment application in many industries by using physical filtration processes or membrane bioreactor. On another hand, biofouling causes the blockage of the membrane’s pores and reduces the membrane performance. But it can be removed by modifying the membrane with antibacterial agent like MO-NPs. The proposed antibacterial mechanism of MO-NPs is free metal ion toxicity from dissolution of MO-NPs and oxidative stress via the generation of ROS on the surface of NPs. The antibacterial properties of MO-NPs were affected by morphological and physicochemical properties of NMs such as crystal structure, shape, size, concentration, and pH. The modified membrane using MO-NPs has better antifouling properties and also improved other properties such as mechanical strength, water flux, hydrophilicity, permeability, porosity, and rejection tendency. It is expected that further research into the development of MO-NPs in membrane technology will enable many applications [13].

13.8 Antibacterial functions of multi-metal oxide nanoparticles Multidrug-resistant (MDR) bacterial strains have emerged because of the extensive misuse of antibiotics. For this reason, the development of new antibacterial materials and therapeutics to kill bacteria effectively has become essential. Metal oxide and sulfide nanocomposites exhibit excellent antibacterial activity [14]. Nano silver containing composite microspheres also revealed superior bacteriostatic and bactericidal activities. Bifunctional Fe3O4@AgNPs

4. Antibacterial activity of nanomaterials

13.8 Antibacterial functions of multi-metal oxide nanoparticles

295

with both superparamagnetic and antibacterial properties have been reported. Magnetooptical Fe3O4/Au/AgNPs have great potential for NP-based diagnostic and therapeutic applications. In the recent study, a bioassay test of Fe3O4@Ag@Ni was performed using the well diffusion method against Staphylococcus aureus, Proteus mirabilis, K. pneumoniae, Escherichia coli, and Pseudomonas aeruginosa bacteria [14]. Bacterial culture was carried out with agar nutrients where a 6 mm well was created to introduce the sample, and a reference was used named imipenem (ipm 10). The result showed that all the synthesized particles have very good antibacterial properties, both for gram-positive and gram-negative bacteria. S. aureus, which is a gram-positive bacterium, was destroyed with 2.83 ratio. Similarly, P. mirabilis, K. pneumoniae, E. coli, and P. aeruginosa which are gram-negative bacteria were also destroyed with 2.5, 2.83, 2.83, and 3.5 ratio. These activities confirmed that the synthesized particles are biologically active. Silver has biological properties, due to its localized surface electrons. Magnetite and Ni particles are also active, which caused mechanical damage of the bacterial cell and enhanced inhibition. Fe3O4@Ag@Ni may be a magnetically recyclable brilliant antibacterial agent as recently Ag@Fe3O4 PEI NPs showed excellent photothermal stability, high magnetic recyclability, and low cytotoxicity. This multifunctional core shell nanocomposite is expected to be very promising for biomedical sterilization applications [14]. Inorganic nanostructured materials and their surface modifications exhibit good antimicrobial functions. These improved antibacterial agents in low concentrations locally destroy bacteria without being toxic to the surrounding healthy tissues/cells keeping them unharmed [15]. The antibacterial activity of the Ag2O3 SnO2 Cr2O3 NPs was investigated using the well diffusion method against gram-positive bacteria (S. aureus) and gram-negative bacteria (Escherichia coli) bacteria. Agar nutrients were used for the bacterial culture where the sample was introduced, creating a 6 mm well; Gentamicin10 (GEN 10) was used as a reference. The different inhibition zones were measured after overnight incubation at 37 C. The zone of inhibition confirmed the antibacterial effect of Ag2O3 SnO2 Cr2O3 NPs, which killed both gram-positive and gram-negative bacteria; this indicated the considerable effectiveness against gram-negative bacteria than that against gram-positive bacteria [1]. Coated and noncoated Ag ZnO Fe3O4, curcumin and Ag ZnO Fe3O4-APTMS NPs were subjected to antibacterial activity for five pathogenic bacteria strains [15]. The antibacterial activity was investigated by modified Kirby Bauer disk diffusion method. Well diffusion test was carried out on nutrient agar plates to observe the antimicrobial activity against pathogenic organisms. For bacteria, 250 mL nutrient agar medium (NAM) was prepared by maintaining the value of pH between 6.8 and 7.0. Nutrient agar medium was poured in the sterile petriplates and it was solidified bacterial suspension spreading on petriplates. The pure cultures of organisms were subcultured in Muller Hinton broth at 37 C. For bacterial growth, a lawn of culture was prepared by spreading the 30 μL fresh culture having 106/mL colony-forming units (cfu) of each test organism on nutrient agar plates with the help of L-shaped glass-rod spreader. Plates were left standing for 30 min to let the culture get absorbed. Then around 6 mm wells were punched into the nutrient agar plates for testing antimicrobial activity. Wells were sealed with one drop of molten agar (0.8% agar) to prevent leakage of NMs from the bottom of the wells. Using a micropipette, 100 μL (50 μg) of the sample of NP suspension was poured onto each of two wells on all plates. After overnight incubation at 37 C, the different levels of zone of inhibition were measured. The inhibition zones appeared when the plates were









4. Antibacterial activity of nanomaterials

296

13. Antibacterial property of metal oxide-based nanomaterials

FIGURE 13.3 Inhibition zone produced by antidose (a), Ag ZnO Fe3O4-APTMS (b), Ag ZnO Fe3O4 (c), Ag ZnO Fe3O4-curcumin (d) and curcumin (e) nanocomposite for (i) Escherichia coli, (ii) Staphylococcus aureus, (iii) Klebsiella pneumonia, (iv) Pseudomonas aeruginosa and (v) Proteus mirabilis [15].

allowed to incubate for 24 h. Ag ZnO Fe3O4-APTMS and curcumin show minimum inhibition effect against pathogens but Ag ZnO Fe3O4 and Ag ZnO Fe3O4-curcumin showed better activity like antidotes. Among them, Ag ZnO Fe3O4-curcumin shows maximum range of effect against pathogenic bacteria. Fig. 13.3 illustrates the results of the antimicrobial test performed by Ag ZnO Fe3O4, Ag ZnO Fe3O4-APTMS, curcumin and Ag ZnO Fe3O4-curcumin. Hence, Ag ZnO Fe3O4 and coated Ag ZnO Fe3O4-Curcumin NPs may be used in pharmaceuticals to prepare best antibacterial drugs.

13.9 Magnetic bio-metal oxide-magnetosome Magnetosomes are intracellular structures produced by magnetotactic bacteria, which comprise magnetic nanoparticles (Fe3O4) surrounded by lipid bilayer membrane. They have attracted attention due to their very promising biotechnological applications including antibacterial and drug delivery [16,17]. This is due to a series of appealing properties summarized below which are not usually found in chemically synthesized nanoparticles, Fe3O4: • The magnetosomes are magnetic nanoparticles, which possess a narrow size distribution and uniform morphology when the magnetotactic bacteria are cultivated in optimum conditions, that is, essentially using a low oxygen concentration (varied between 0.25 and 10 mbar) during the growth. In these conditions, the magnetosome size distribution can be as small as B10 nm with magnetosome sizes typically lying between 45 and 55 nm for the most commonly studied species of magnetotactic bacteria (AMB-1 and MSR-1). • The core of the magnetosomes is usually composed of magnetite (Fe3O4) which can oxidize into maghemite (γ-Fe2O3). The magnetosome core is also usually of high levels of purity and crystallinity. • The magnetosomes are usually large single magnetic domain nanoparticles. This leads to a magnetic moment that is thermally stable at physiological temperature. Therefore it produces better magnetic properties than those found in chemically synthesized iron oxide nanoparticles which are usually superparamagnetic and possess a thermally unstable magnetic moment. It also yields high values of the coercivity (HcB20 40 mT) and ratio between the remanent and saturation magnetization (Mr/MsB0.4 0.5).

4. Antibacterial activity of nanomaterials

13.10 Toxicity concerns of MO-NPs as antimicrobial agents





• • • •

297

In specific conditions, these magnetic properties result in higher heating capacities and better magnetic resonance imaging contrast agents for the magnetosomes than for chemically synthesized nanoparticles. The magnetosomes are usually arranged in chains inside the bacteria. This arrangement is stable enough to be preserved even after disrupting the bacteria to isolate the magnetosomes. Such arrangement is appealing since it prevents aggregation and yields a high rate of internalization within human cells, two properties that are usually desired for medical applications. The magnetosomes are covered by biological material made of a majority of lipids and a minority of proteins. This biological coating results in negatively charged magnetosomes with a good dispersion in water. By contrast, chemically synthesized nanoparticles are not naturally coated and need to be stabilized, for example, by being covered with dextran or poly(ethylene glycol) molecules. This usually makes their synthesis more complicated than that of the magnetosomes. The magnetosomes can easily be functionalized, due to the presence of various chemical groups at their surface, which iare suitable for drug delivery applications [17]. Methods have been published that enable to produce a large quantity of magnetosomes up to 170 mg/L/day of magnetosomes. When they are prepared in specific conditions, the magnetosomes possess a high biocompatibility and a low toxicity. Finally, magnetosomes are obtained by cultivating magnetotactic bacteria in a growth medium that is not toxic (e.g., ATCC medium 1653 for the AMB-1 species). This contrasts with the use of toxic products often used during the preparation of chemically synthesized nanoparticles [16].

13.10 Toxicity concerns of MO-NPs as antimicrobial agents Researchers around the world are putting their concerted efforts into unraveling the interaction of MO-NPs with organisms. However, the mechanisms of toxicity in humans still remain largely unexplored. Some MO-NPs exhibited varying degrees of toxicity, which was noticed less in their bulk counterparts; Most of the MO-NPs and their respective bulk materials do not exhibit a similar toxic effect. The following features pose