Nanomaterials for Advanced Biological Applications [1st ed.] 978-3-030-10833-5, 978-3-030-10834-2

Table of contents :
Front Matter ....Pages i-xii
Nanoparticles and Biological Environment Interactions (Armin Azadkhah Shalmani, Pouria Sarihi, Mohammad Raoufi)....Pages 1-17
Nanotopographical Control of Cell Assembly into Supracellular Structures (Francesco Gentile)....Pages 19-53
Polymeric Nanoparticulates as Efficient Anticancer Drugs Delivery Systems (Shima Asfia, Mahsa Mohammadian, Hasan Kouchakzadeh)....Pages 55-84
Hydroxyapatite for Biomedicine and Drug Delivery (Behrad Ghiasi, Yahya Sefidbakht, Maryam Rezaei)....Pages 85-120
Nanoparticles for Biosensing (Pouria Sarihi, Armin Azadkhah Shalmani, Vida Araban, Mohammad Raoufi)....Pages 121-143
Carbon Quantum Dots in Nanobiotechnology (Hamidreza Behboudi, Golnaz Mehdipour, Nooshin Safari, Mehrab Pourmadadi, Arezoo Saei, Meisam Omidi et al.)....Pages 145-179
Size-Dependent Nonlinear Mechanics of Biological Nanoporous Microbeams (Saeid Sahmani, Mohammad M. Aghdam)....Pages 181-207
Mechanical Behaviour of PMMA Bio-polymer Loaded by Nano-scale Additives (Hadi Asgharzadeh Shirazi, Majid R. Ayatollahi, Mahdi Navidbakhsh, Alireza Asnafi)....Pages 209-224

Citation preview

Advanced Structured Materials

Moones Rahmandoust Majid R. Ayatollahi Editors

Nanomaterials for Advanced Biological Applications

Advanced Structured Materials Volume 104

Series editors Andreas Öchsner, Faculty of Mechanical Engineering, Esslingen University of Applied Sciences, Esslingen, Germany Lucas F. M. da Silva, Department of Mechanical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal Holm Altenbach, Otto-von-Guericke University, Magdeburg, Sachsen-Anhalt, Germany

Common engineering materials reach in many applications their limits and new developments are required to fulfil increasing demands on engineering materials. The performance of materials can be increased by combining different materials to achieve better properties than a single constituent or by shaping the material or constituents in a specific structure. The interaction between material and structure may arise on different length scales, such as micro-, meso- or macroscale, and offers possible applications in quite diverse fields. This book series addresses the fundamental relationship between materials and their structure on the overall properties (e.g. mechanical, thermal, chemical or magnetic etc.) and applications. The topics of Advanced Structured Materials include but are not limited to • classical fibre-reinforced composites (e.g. class, carbon or Aramid reinforced plastics) • metal matrix composites (MMCs) • micro porous composites • micro channel materials • multilayered materials • cellular materials (e.g. metallic or polymer foams, sponges, hollow sphere structures) • porous materials • truss structures • nanocomposite materials • biomaterials • nano porous metals • concrete • coated materials • smart materials Advanced Structures Material is indexed in Google Scholar and Scopus.

More information about this series at http://www.springer.com/series/8611

Moones Rahmandoust Majid R. Ayatollahi

•

Editors

Nanomaterials for Advanced Biological Applications

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Editors Moones Rahmandoust Protein Research Center Shahid Beheshti University G.C. Tehran, Iran

Majid R. Ayatollahi Fatigue and Fracture Research Laboratory School of Mechanical Engineering Iran University of Science and Technology Tehran, Iran

ISSN 1869-8433 ISSN 1869-8441 (electronic) Advanced Structured Materials ISBN 978-3-030-10833-5 ISBN 978-3-030-10834-2 (eBook) https://doi.org/10.1007/978-3-030-10834-2 Library of Congress Control Number: 2018965881 © Springer Nature Switzerland AG 2019 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface

Experimental and theoretical nanotechnology is serving medicine, biotechnology and biomaterials summit. The employment of nanomaterial is not only beneficial in enhancing the efficiency of common therapeutics and lowering their side effects, but also in providing many emerging capabilities, due to the specific properties they can introduce, including advancements in tissue engineering, biosensing, bioimaging, benefiting from targeted and smart therapies, as well as unifying diagnostic and therapeutic approaches together. This volume strives to represent an overview of recent advances on how various nanomaterials have been employed for advanced biological applications. Nanomedicine is currently in its infancy. The fate of a nanomedicine after its administration into the body is actually determined on the nano–bio interface. Even though a large body of studies has been carried out in this field, so many aspects of its entity and behaviour are still obscure. This complexity is the result of diverse environments that comprise thousands of different types of proteins, different flow rates and so many other variants that cannot be fully replicated in an in vitro model. That being said, Chapter “Nanoparticles and Biological Environment Interactions” of this book provides an in-depth knowledge about the nano–bio interface. The horizon for the forthcoming progress in nanomedicine is very promising and a fully fledged insight into its complicated interaction with the body can lead to unprecedented improvements in medical interventions. In the first chapter of this book, the extent of knowledge on the interaction of the nanoparticles with the biological environment is reviewed and discussed. Along with the interaction of the nanoparticles with their surrounding biological medium, the term cell adhesion becomes very significant. In this interaction, the complex interplay of cell signalling, physical and chemical properties of materials, as well as the biological functions of cells, depends on the competition of forces exchanged between cells and their biological medium. Thus, adhesion to the extracellular matrix or to neighbouring cells and subsequent multiple cellular processes, such as cell migration, morphogenesis, proliferation, gene expression and survival, depends on the balance/unbalance between the spatial and temporal distribution of cell adhesion molecules, and cell shaping through actin–microtubule crosstalk. v

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Chapter “Nanotopographical Control of Cell Assembly into Supracellular Structures” looks at the nanotopographical control over the assembly of the cell adhesion molecules into supracellular structures. After taking a look at the interface of nano–bio mediums, the impact of nanomaterial on promoting the efficiency of common cancer treatment techniques is introduced. Although conventional chemotherapy is considered as the first-line cancer therapy for many years, it has not been successful enough due to the problems such as non-specificity and toxicity. Thus, development of an effective treatment method in order to be selective and less toxic is necessary. Smart delivery of the drug to targeted tissues has been therefore an important aspect of how nanoparticles can significantly serve towards improving human health over recent decades. In Chapter “Polymeric Nanoparticulates as Efficient Anticancer Drugs Delivery Systems”, polymeric nanoparticles which are used as efficient anticancer drugs delivery systems are introduced. These nanoparticles can be engineered to accumulate specifically at diseased cells, which helps to improve curing, increasing treatment efficacy, reducing side effects, and facilitating co-delivery of two or more drugs or therapeutic modality for combination therapy, and visualization of sites of action by combining therapeutic agents with imaging modalities. In addition, therapeutic nanomaterials admit the potency to overcome biological barriers and effectively transport hydrophobic, poorly water-soluble drugs, and biologics. Other than polymeric nanoparticles, hydroxyapatite is also considered for biomedicine and drug delivery recently. Hydroxyapatite is, in fact, a bioceramic member of the calcium phosphates family, known for its specific chemical similarity to the mammalian inorganic structures, with high thermodynamic stability and solubility in physiological conditions. Furthermore, its similarity with the structure and function of bones and teeth makes it a considerable particle for treatments of the skeleton and dental defects. In Chapter “Hydroxyapatite for Biomedicine and Drug Delivery”, hydroxyapatite is introduced and its applications in biomedicine and drug delivery are elaborated. The next chapter focuses on “Nanoparticles for Biosensing”. Biosensors are analytical devices that use a biological recognition element which incorporates a transduction system to generate a measurable signal output proportional to the concentration of the analyte of interest. In spite of all advances made in medical intervention over the past few decades, efficient curing of many diseases has remained a challenge, due to incompetent means of the diagnosis result in the late recognition of the malicious situation. Some strategies that have been taken into consideration in order to achieve early diagnosis include screening of disease-related biomarkers, and disease marker detection, making biosensors alluring devices which can be utilized for the diagnosis and monitoring of diseases. Biosensor technology has been greatly influenced by recent progress in nanotechnology science, providing rapid, real-time and accurate results in a comparatively easy way. This led not only to an improvement in the sensitivity and performance of biosensors by using nanomaterials, but also allowed the use of new signal transduction technologies in these devices. Chapter “Carbon Quantum Dots in Nanobiotechnology” looks at carbon quantum dots as a unique fluorescent nanomaterial that is employed in many

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bio-related applications, including advanced biosensing applications. Due to low toxicity, biocompatibility and the unique physical, chemical and photochemical properties of these materials, they are capable to increase the efficiency of fluorescent bioimaging (in vitro and in vivo), and biosensing. Along with the advantages of employing carbon quantum dots in various biosensing applications, focus on their use in theranostics applications has also been a recent matter of interest. Unifying diagnostic and therapeutic approaches together, using the superior properties of nanoparticles like carbon quantum dots can lead to the design of efficient systems that simultaneously recognize, deliver therapeutic cargo and evaluate the response to treatment. This approach can overcome the challenges of cancer heterogeneity and adaptation. As another important concern, the influence of nanomaterial in tissue engineering has also been introduced. Tissues in the human body may be degenerated due to different causes, e.g. diseases, trauma, congenital defects. Recent developments in the tissue engineering have enabled material scientists and biomedical engineers to regenerate a damaged tissue, instead of its replacement. For instance, biomaterials of the porous structure have received much attention in a variety of orthopaedic applications. The porous structure of scaffolds enhances tissue regeneration by accelerating cell migration and growth. Technological advancements have made porous biomaterials with nanoscale pore sizes available to biomedical engineers. Therefore, it is important to determine the mechanical properties of nanoporous biomaterials using experimental techniques or theoretical models and to study how pore size may influence the mechanical behaviour of a porous biomaterial. Chapter “Size-Dependent Nonlinear Mechanics of Biological Nanoporous Microbeams” deals with some theoretical models developed for exploring the size-dependent mechanics of a sample biological nanoporous structure. Bone cement is another synthetic biomaterial which has been used extensively in orthopaedic diseases and traumas with the aim to fill the space between a bone and its prosthesis. In addition to possessing very good biocompatibility characteristics, the bone cement is expected to transfer the in vivo loads from implants to the bone and to increase the load-bearing capacity of the prosthesis–cement–bone system. However, the classical bone cement has displayed some disadvantages such as poor mechanical properties and insufficient biocompatibility. Using bone cement of inadequate tensile/compressive strength may lead to prosthesis failure, which in some cases requires subsequent surgical procedures. There have recently been attempts to improve the mechanical and biocompatibility properties of traditional biopolymers, such as bone cement, by loading additive particles of nanometre sizes. Among various types of additive particles proposed by researchers, the addition of hydroxyapatite, particularly its nanosized particles, into the cement matrix has been shown to be able to provide significant improvements in both the mechanical and the biocompatibility properties of the cement. It is, therefore, necessary to study in more detail the bone cement composites and nanocomposites loaded by microscale or nanoscale particles from the experimental and theoretical points of view. This will be elaborated and discussed in the final chapter of the book, “Mechanical Behaviour of PMMA Bio-Polymer Loaded by Nano-Scale Additives”.

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The editors would like to thank all authors who accepted the challenges of preparing valuable chapters and invested their time to achieve this high-quality book. They are also thankful to Springer and specifically to the distinguished editors of the “Advanced Structured Materials” series for their professional support. Tehran, Iran November 2018

Moones Rahmandoust Majid R. Ayatollahi

Contents

Nanoparticles and Biological Environment Interactions . . . . . . . . . . . . Armin Azadkhah Shalmani, Pouria Sarihi and Mohammad Raoufi

1

Nanotopographical Control of Cell Assembly into Supracellular Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Francesco Gentile

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Polymeric Nanoparticulates as Efficient Anticancer Drugs Delivery Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Shima Asfia, Mahsa Mohammadian and Hasan Kouchakzadeh

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Hydroxyapatite for Biomedicine and Drug Delivery . . . . . . . . . . . . . . . Behrad Ghiasi, Yahya Sefidbakht and Maryam Rezaei

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Nanoparticles for Biosensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pouria Sarihi, Armin Azadkhah Shalmani, Vida Araban and Mohammad Raoufi

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Carbon Quantum Dots in Nanobiotechnology . . . . . . . . . . . . . . . . . . . Hamidreza Behboudi, Golnaz Mehdipour, Nooshin Safari, Mehrab Pourmadadi, Arezoo Saei, Meisam Omidi, Lobat Tayebi and Moones Rahmandoust

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Size-Dependent Nonlinear Mechanics of Biological Nanoporous Microbeams . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Saeid Sahmani and Mohammad M. Aghdam Mechanical Behaviour of PMMA Bio-polymer Loaded by Nano-scale Additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hadi Asgharzadeh Shirazi, Majid R. Ayatollahi, Mahdi Navidbakhsh and Alireza Asnafi

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Contributors

Mohammad M. Aghdam Department of Mechanical Engineering, Amirkabir University of Technology, Tehran, Iran Vida Araban Faculty of Pharmacy, Nanotechnology Research Center, Tehran University of Medical Sciences, Tehran, Iran Shima Asfia Protein Research Center, Shahid Beheshti University, Velenjak, Tehran, Iran Alireza Asnafi Hydro-Aeronautical Research Center, Shiraz University, Shiraz, Iran Majid R. Ayatollahi Fatigue and Fracture Research Laboratory, Center of Excellence in Experimental Solid Mechanics and Dynamics, School of Mechanical Engineering, Iran University of Science and Technology, Tehran, Iran Hamidreza Behboudi Protein Research Center, Shahid Beheshti University, Tehran, Iran Francesco Gentile Department of Electrical Engineering and Information Technology, University Federico II, Naples, Italy Behrad Ghiasi Protein Research Center, Shahid Beheshti University, G.C, Tehran, Iran Hasan Kouchakzadeh Protein Research Center, Shahid Beheshti University, Velenjak, Tehran, Iran Golnaz Mehdipour Protein Research Center, Shahid Beheshti University, Tehran, Iran Mahsa Mohammadian Protein Research Center, Shahid Beheshti University, Velenjak, Tehran, Iran

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Contributors

Mahdi Navidbakhsh Tissue Engineering and Biological Systems Research Laboratory, School of Mechanical Engineering, Iran University of Science and Technology, Tehran, Iran Meisam Omidi Protein Research Center, Shahid Beheshti University, Tehran, Iran Mehrab Pourmadadi Protein Research Center, Shahid Beheshti University, Tehran, Iran Moones Rahmandoust Protein Research Center, Shahid Beheshti University, Tehran, Iran Mohammad Raoufi Faculty of Pharmacy, Nanotechnology Research Center, Tehran University of Medical Sciences, Tehran, Iran Maryam Rezaei Institute of Biochemistry and Biophysics (IBB), Tehran University, Tehran, Iran Arezoo Saei Protein Research Center, Shahid Beheshti University, Tehran, Iran Nooshin Safari Protein Research Center, Shahid Beheshti University, Tehran, Iran Saeid Sahmani Department of Mechanical Engineering, Amirkabir University of Technology, Tehran, Iran Pouria Sarihi Faculty of Pharmacy, Nanotechnology Research Center, Tehran University of Medical Sciences, Tehran, Iran Yahya Sefidbakht Protein Research Center, Shahid Beheshti University, G.C, Tehran, Iran; Nanobiotechnology Laboratory, The Faculty of New Technologies Engineering (NTE), Shahid Beheshti University, G.C, Tehran, Iran Armin Azadkhah Shalmani Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Hadi Asgharzadeh Shirazi Fatigue and Fracture Research Laboratory, Center of Excellence in Experimental Solid Mechanics and Dynamics, School of Mechanical Engineering, Iran University of Science and Technology, Tehran, Iran; Tissue Engineering and Biological Systems Research Laboratory, School of Mechanical Engineering, Iran University of Science and Technology, Tehran, Iran Lobat Tayebi School of Dentistry, Marquette University, Milwaukee, WI, USA

Nanoparticles and Biological Environment Interactions Armin Azadkhah Shalmani, Pouria Sarihi and Mohammad Raoufi

1 Introduction Deployment of nanoparticles (NPs) as diagnostic and therapeutic agents has been an intriguing approach over the past recent years. Downscaling to nano-dimension can drastically change the properties of a given substance. The term ‘Nanomedicine’ first appeared in research publications in the year 2000. Nanomedicine could be defined as the engineering and employment of nanoscale or nanostructured materials for the purpose of prevention, diagnosis, and treatment of diseases. These nanostructures confer distinctive medical advantages to our medical intervention compared to their non-nanostructured counterparts. Unlike the general consensus in physical definition of nanotechnology that confines the eligibility of the name ‘nanoparticle’ to particles with dimensions in the range of 1–100 nm, particles up to 1000 nm are usually considered as nanomedicines since particles with this dimension are still small enough to exert distinctive medical effects and functionalities compared to conventional medicines. Also noteworthy, the traditional small-molecule drugs that had not been advertently designed to achieve new therapeutic effects by nano-downscaling do not comply with the term nanomedicine (Wagner et al. 2006). NP candidates for medical purposes comprise a diverse group consisting of quantum dots, carbon nanotubes (CNT), polymeric NPs, metallic NPs (silver, silica, gold etc.), biological NPs (e.g. bovine serum albumin) and so on. Implementation of these small medicines A. Azadkhah Shalmani Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 1417614411, Iran e-mail: [email protected] P. Sarihi · M. Raoufi (B) Faculty of Pharmacy, Nanotechnology Research Center, Tehran University of Medical Sciences, Tehran 1417614411, Iran e-mail: [email protected] P. Sarihi e-mail: [email protected] © Springer Nature Switzerland AG 2019 M. Rahmandoust and M. R. Ayatollahi (eds.), Nanomaterials for Advanced Biological Applications, Advanced Structured Materials 104, https://doi.org/10.1007/978-3-030-10834-2_1

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with new properties could lead to improvement in drug delivery via different mechanisms, including improving site-specific targeting, enhancement of the permeability of a drug through a sophisticated barrier, modification of the pharmacokinetic of a drug and mitigating its toxicity. Even though the application of NPs for drug delivery seems tempting, the results of studies on these particles deviate from our expectations in many cases. One major reason for this discrepancy stems from the fact that NPs are coated by biomolecules after being exposed to a biological fluid (Monopoli et al. 2012). This coat is a protein-based layer called ‘protein corona’ (PC) which is drawn to NPs by adsorption. Adsorption of biomolecules is the consequence of immense surface energy of the NPs due to their high surface area to volume ratio (Zanganeh et al. 2016). Cloaking the NP with the biological layer minimizes its surface energy. Some of the major biomolecules that contribute to the formation of PCs include immunoglobulins, apolipoproteins, complement factors, coagulation factors and albumin (Walkey and Chan 2012). Introduction of PC into the equation of biointeractions dramatically changes the fate of the NP. Corona-coated NPs differ from their pristine counterparts in terms of their physiochemical properties. As a matter of fact, the biomolecular coat that covers the NP gives it a brand-new biological identity which consequently influences the physiological response to it. Thorough apprehension of the characteristics of this new entity and its interactions with its surrounding bio-environment are greatly demanded for a predictable, safe and efficient medical intervention, when dealing with nanomedicines. Figure 1 depicts the formation of PC and its biological aftermath.

2 Formation and Composition It was in the late 1990s when scientists figured out that the properties of an NP alter after it is being injected into the bloodstream. The term ‘corona’ was coined by Dawson, Linse, and co-workers, referring to the corona (Latin for the word “crown”) that enshrouds the NP after immersion into the biological environment (Cedervall et al. 2007). The crown comprises biological molecules present in the biological fluid, with proteins being the predominant component of it, While, other biomolecules such as lipids and carbohydrate contributing to its formation to a lesser extent. Crowning NPs with PCs bestows a new biological identity on them. Formation of NP-PC complex is a dynamic process that is harnessed by not only the affinity between NP surface and proteins but also protein-protein interactions. The interactions contributing to the adsorption of PC include van der Waals (vdW) interactions, hydrogen bonds, hydrophobic forces and electro-static and π–π stacking interactions (Yang et al. 2013). The composition of the corona changes over time and its components dissociate and reassociate continuously. The so-called ‘Vroman effect’ suggests that this ongoing desorption and adsorption of proteins results in the abundant proteins of the fluid to be the predominant component of the corona in short exposure times,

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Fig. 1 When nanoparticles enter the biological milieu, a layer of biomolecules enshrouds them. This new layer known as protein corona gives new biological identity to the particles. The new identity determines not only how these particles interact with one another, but also how the body responds to them. Reproduced with permission (Walkey and Chan 2012)

while in long exposure times, resulting in proteins with the highest affinity being the main constituent of the PC (Vroman 1962). Although this model was in line with some studies conducted later, some further studies failed to corroborate its conclusiveness which left compatibility of Vroman effect to the nano-scale settings to be controversial (Jansch et al. 2012). Some studies showed that the abundance of a particular protein in the corona does not follow a simple increasing or decreasing pattern and there is a fluctuation of increase and decrease in its abundance. For instance, a low-abundance protein in short exposure can show enhancing trend in intermediate exposure, while in the long exposure, a declining trend might be observed from the

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same protein. On the other hand, the study concluded that the formation of PC is quantitative rather than qualitative, meaning that during different exposure times, the components of a PC remain the same, while the amount and proportion of each component differ over further incubation (Tenzer et al. 2013). This highlights the fact that the formation of PC is a multifaceted and intricate process and a comprehensive multifactorial model is needed to predict the composition of PC during different exposure times. Based on the affinity and exchange time of PC components, it is classified as the hard corona and the soft corona. PC is a multi-layered coat. Hard corona is the first layer of proteins which is tightly bound to NP surface. The hard corona has a long exchange time and can maintain adsorbed to the NP for hours. On the contrary, the soft corona is the second layer of biomolecules which is not directly bound to the NP. This layer is bound to hard corona via protein-protein interactions. Soft corona has low affinity and undergoes rapid exchange (from seconds to minutes) during incubation (Milani et al. 2012). Figure 2 demonstrates a schematic illustration of hard and soft corona. From a thermodynamical standpoint, the rigidity of PC-NP interaction can be explained by Gibbs free energy of adsorption (GADS  HADS − TSADS). In this equation, HADS and SADS are changes in enthalpy and entropy respectively and T indicates the temperature. GADS is the net change of Gibbs free energy and in a thermodynamically favourable reaction, it is negative, indicating the simultaneous process of adsorption. The quality of the adsorption depends on GADS. Hard corona is the layer which is adsorbed onto the surface of the NP with a large net binding energy of adsorption (Gads). On the other hand, the soft corona is the result of protein adsorptions with small Gads (Norde 1994). Hard corona can remain adsorbed onto the NP during different biological processes. This is not the

Fig. 2 Schematic illustration of protein corona. Hard corona is directly adsorbed to the nanoparticle. The proteins in this layer are firmly bound to the nanoparticle, thus possess long residence time. Soft corona is the layer that is weakly attached to the first layer. Proteins in this layer have short residence time. Each layer is composed of multiple proteins

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case for soft corona that undergoes rapid alteration after the PC-NP complex enters new biological environments (in case of hard corona this alteration happens at a considerably lower extent). Events like endocytosis and traveling from one biological milieu to another one can lead to rapid dissociation and association of proteins in soft corona layer. As a result of the aforementioned circumstances, the composition of hard corona has a pivotal role in the physiochemical properties of the nano-bio interface.

3 Evaluating Protein Corona In order to understand different properties of PCs, these biomolecular layers should be rigorously examined. PC analysis can be performed via in situ and ex situ approaches. The in situ approach attempts to evaluate the PCs within the biological environment, while ex situ evaluation is performed on the isolated NP-PC complexes. Some of the most common methods for the isolation of such complexes are Centrifugation (the most commonly used method), Size exclusion chromatography (SEC) and Magnetic separation/magnetic flow field fractionation (MgFFF). PCs can be analysed from different aspects. These aspects include the composition of PC, its thickness, and binding affinity, the amount of proteins, the relative abundance of each protein and their conformations. After isolating NP-PC complex, its composition can be examined by techniques like sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), twodimensional gel electrophoresis (2-DE) and mass spectrometry (MS). For evaluating the thickness of PC techniques such as transmission electron microscopy (TEM), dynamic light scattering (DLS) and size exclusion chromatography (SEC) can be implemented. Assessing the binding affinity and stoichiometry of PCs is of paramount importance. Measuring these parameters can be performed using different techniques. These techniques can be categorized into two groups based on their approaches:

3.1 Experimental Approach Different experimental methods can be employed to evaluate PCs in terms of their binding affinity and stoichiometry. Some of which are as below: Isothermal Titration Calorimetry (ITC) ITC can directly measure enthalpy, binding affinity and binding stoichiometry of the protein-NP interactions. In this technique, protein is inserted into an NP suspension in the sample cell and the difference in heat that is required to be added to the

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sample and reference cells to keep both cells at equal temperature is calculated. ITC can provide us with the number of protein molecules attached to each particle, the apparent affinity, and the enthalpy change if the concentrations of both the NP and inserted protein are available. ITC is a non-destructive method that can be employed to quantitatively assess the above-mentioned parameters in in situ settings. Size Exclusion Chromatography (SEC) As mentioned above, this method separates particles based on their size. SEC can also be employed to explore the affinity and lifetime of the NP-PC complex. Surface Plasmon Resonance (SPR) SPR is another technique that can be used to explore biomolecular binding interactions in real time. This technique is performed by immobilizing one binding partner to the surface of a metallic film. The metallic film is then struck by polarized light at a specific condition which leads to the excitation of surface plasmons. Excitation of surface plasmons in the metal can be altered by the adsorption of proteins on the surface of NPs. Such alteration can be detected and utilized to provide information about the kinetics of adsorption. Measuring Zeta Potential Zeta potential demonstrates the electrostatic potential value at the plane of shear. Adsorption of proteins to NP surface alters its zeta potential. Zeta potential can be correlated with the amount of the adsorbed protein on the surface of the NPs. Other experimental techniques that can be used to probe binding affinity and stoichiometry of PCs are Quartz crystal microbalance (QCM) and Fluorescence correlation spectroscopy (FCS). Quantification of proteins in NP-PC can be performed by conventional protein concentration methods such as Bicinchoninic acid (BCA) assay, Bradford assay and also Thermogravimetric analysis (TGA). For evaluating Protein conformation in PCs, techniques such as Circular dichroism (CD) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, Raman spectroscopy (RS), Nuclear magnetic resonance (NMR) spectroscopy, Differential scanning calorimetry (DSC) spectroscopy and Fluorescence correlation spectrometry (FCS) can be employed.

3.2 Computational Approach This approach utilizes computational modelling to simulate protein-NP interactions. Modelling the protein-NP interactions can simulate the binding process that can broaden our understanding of such phenomenon (Pederzoli et al. 2017) (Table 1).

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Table 1 Common experimental techniques employed to examine protein corona properties. Reproduced with permission (Pederzoli et al. 2017) with small modification Protein corona parameter

Technique

Isolation

Centrifugation, size exclusion chromatography (SEC), magnetic separation/magnetic flow field fractionation (MgFFF)

Composition

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), 2D-PAGE, liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Thickness

Dynamic light scattering (DLS), differential centrifugal sedimentation (DCS), transmission electron microscopy (TEM), size exclusion chromatography (SEC)

Affinity

Surface plasmon resonance (SPR), fluorescence quenching titration (FQT), fluorescence correlation spectroscopy (FCS), zeta potential (Z-pot), Size exclusion chromatography (SEC), isothermal titration calorimetry (ITC), quartz crystal microbalance (QCM)

Quantification

Bicinchoninic acid (BCA) assay, bradford assay, thermogravimetric analysis (TGA)

Conformation

Differential scanning calorimetry (DSC), circular dichroism (CD), Fourier-transform infrared (FTIR), fluorescence correlation spectrometry (FCS), Raman spectroscopy (RS), nuclear magnetic resonance (NMR)

4 Factors Affecting Protein Corona The properties of a PC which are the main determiners of nanomedicine behavioural characteristics in bio-environments depend on a myriad of factors. These factors can be divided into three groups: • Factors regarding the physiochemical properties of the NP including the type of NP and its chemical composition, its size, shape (e.g. surface roughness, porosity etc.), surface charge and the type of functional groups located on its surface. • Factors regarding the biological environment encompassing the NP such as the type of the biological fluid, diseases affecting sample donor’s health conditions and inter-individual variations. • conditions under which the NPs have been immersed in the biological milieu e.g. temperature and incubation time Properties of the NP greatly impact PC characteristics. Studies revealed that NPs with different compositions produce distinct PCs when incubated in identical milieus. For instance, in a study, probing PC variations in NPs with different compositions, it was evidenced that some proteins such as Transferrin and Haptoglobin that were part of the PC composition in zinc oxide NPs were absent from the composition of PCs of silicon dioxide NPs. Similarly, some PC constituents of silicon dioxide were missing from PCs of zinc oxide NPs (Deng et al. 2009). NPs can be found in a variety of shapes including cubic, spherical, rod shape, star etc. NPs with similar composition but different shapes adsorb proteins differ-

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ently (Cox et al. 1999; García-Álvarez et al. 2018). Moreover, the size of the NP can also affect its adsorbed PC; same NPs of different sizes have different curvatures and surface areas. NP surface charge is another crucial factor determining PC composition. Studies showed that positively charged NPs tend to adsorb proteins with isoelectric points (PI) lower than 5.5 like albumin. On the other hand, NPs with negative surface charge entice proteins with PI above 5.5 such as IgG (Aggarwal et al. 2009). Besides, positively charged NPs are more inclined to penetrate into cell membrane (Harush-Frenkel et al. 2007). Charged NPs can impose greater conformational changes on proteins as compared to neutral NPs (Lynch and Dawson 2008). Additionally, hydrophilicity/hydrophobicity of the NP affects its PC. NPs with hydrophobic surfaces demonstrate affinity to different proteins compared to hydrophilic ones (e.g. hydrophobic NPs have high affinity to apolipoproteins while hydrophilic NPs are more prone to bind to proteins like albumin and IgG). Hydrophobic NPs also have a higher tendency to induce conformational changes in proteins compared to hydrophilic ones (Roach et al. 2005). The properties of the biological environment in which NPs are immersed can greatly impact their compositions. Biological fluids comprise a plethora of different biomolecules. Type and quantity of these molecules vary abundantly between different types of biological fluids and individuals. This could not only influence the composition of the PC, but can also effect the overall size of the PC-NP complex (Monopoli et al. 2011). Differences in the biological environments are not just confined to the composition of their biomolecules (e.g. difference in parameters like PH can effectively influence PCs). Different routes of administration also result in various PCs since NPs encounter a different environment in each one of them (Foroozandeh and Aziz 2015). Another factor revealed by studies to influence PC composition is the temperature (Mahmoudi et al. 2013a, b, c). Body temperature differs between different individuals based on their age, sex, reproductive status (e.g. Being pregnant or not), etc. Besides, body temperature can be altered during different activities like sleeping and exercising. A condition such as fever can also considerably affect body temperature. Moreover, different organs of an individual vary thermally based on factors such as metabolism rate and proximity to ambient. All these circumstances provide PCs with different temperatures which ultimately affect their properties. PC is a dynamic interface and its composition alters over time. As a result, the time points (after NP incubation in the biological fluid) at which PCs are being investigated in studies should be taken into consideration (Barrán-Berdón et al. 2013). Properties of a PC are the result of concomitant effects of all these factors. When evaluating PCs, all these different parameters should be taken into consideration.

5 Impacts of Protein Corona on Nanoparticle Behaviour As mentioned above, when NPs are coated by PCs, they obtain new biological identity. Contemplating the differences between coated NPs and naked ones are indispensable for an effective treatment since their performances might be quite dissimilar

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and even contradicting at times. Hence, when different aspects of PC impacts on NP are adequately elucidated, its smart manipulation and application can pave the way to excel in nanomedicine. • Bio-distribution and immunogenicity Of most principal parameters when evaluating a substance as a pharmacotherapeutic agent, is how it distributes throughout the body after administration. Formation of NP-PC complex can profoundly influence the absorption, distribution, metabolism, and excretion (ADME) of a substance. This formation could account for the compromised effectiveness of nanomedicines. Nanomedicine circulation time has a key role in its effectiveness. As soon as an alien substance enters the body, eliminatory means commence counteractions. Mononuclear phagocyte system (MPS) is one of the mechanisms through which body eliminates exogenous materials. The system consists of phagocytic cells and its fulfilment is largely dependent on molecules called opsonins, which include molecules like antibodies and complement proteins that expedite and encourage phagocytosis. Adsorption of immunoglobulins to the NP is shown to facilitate phagocytosis by promoting opsonisation (Chen et al. 2017). Fibrinogen and complement factors have shown similar phagocytosispromoting effect thus diminish blood circulation time for the NPs. On the other hand, proteins like albumin and apolipoproteins are observed to increase the circulation time of NPs (Monopoli et al. 2011). Therefore, PC-coated NPs can demonstrate shorter or longer circulation time as compared to pristine NPs depending on their constituent proteins. Cellular internalization, uptake and endocytic pathway of an NP can also be manipulated by PC (Caracciolo et al. 2010). Coating NPs with PCs can hinder its adhesion to cell surface, thus affecting its uptake (Lesniak et al. 2012). Furthermore, adsorption of proteins onto NP surface increases its size. This size change can influence its internalization and bio-distribution (Gaumet et al. 2008). PC-coated NPs can trigger immunological responses. Cloaked NPs with PCs can be regarded by the body as nanomaterial-associated molecular patterns (NAMPs) (Farrera and Fadeel 2015). NAMPs can be recognized by pattern recognition receptors (PRR) such as toll-like receptors (Neagu et al. 2017). Such recognition can initiate cascades that invoke innate immune system. Importantly, alteration in the conformation of proteins induced by nano-bio interaction might propagate inflammatory reactions. For instance, a study demonstrated that introducing negatively charged gold NPs into biological milieu results in unfolding and adsorption of fibrinogen protein. such PC can interact with some integrin receptors that ultimately promotes NF-κB signalling pathway (Deng et al. 2011). NF-κB is a transcription factor that has a pivotal role in immune responses. Additionally, colloidal NPs constantly collide with one another in a liquid due to Brownian motion. Stability of such an environment is achieved by repulsive electrostatic and steric forces between NPs (Segets et al. 2011). The sustainability of these inter-particle repulsive forces is influenced by the electrolytic and thermal changes of the suspending solution. Debilitating these repulsive forces could eventually lead

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to agglomeration. Studies show that the presence of a biological layer around these NPs can minimize agglomeration through steric stabilization (Gebauer et al. 2012). Adsorption of PCs to NPs can also alter their interactions with biological barriers. Embodiment of NPs can be considered as some kind of metamorphosis. Therefore, these protein-coated particles may cross barriers that seemed impermeable when there were naked. For instance, NPs coated with Apolipoprotein (Apo) A-I and ApoA-II can cross the blood-brain barrier. This alteration in drug direction can impose devastating CNS complications on the patient (Mahmoudi et al. 2015). • Toxicity Toxicological tests are one of the first experiments to conduct when exploring a potential drug candidate for clinical application. Enveloping NPs with PCs can affect their toxicological properties (Hu et al. 2011a, b). This effect is not always the same in different NPs. For instance, is it shown that positively charged NPs can disturb the integrity of cell membrane (Fröhlich 2012). This could be the result of the interactions between the particle and negatively charged cell surface. Coating the positively charged NPs with PCs hampers this interaction and therefore alleviates the toxicity. On the other hand, some particles can induce the formation of reactive oxygen species (ROS) (Carlson et al. 2008). Overproduction of these species can result in DNA damage, cytotoxicity, apoptosis and cell death. Concealing NP surface with PC can prohibit the production of such species. Additionally, NPs can cause disruption to red blood cells (RBC). PCs can protect RBCs from haemolysis induced by NPs (Saha et al. 2014). Contradictorily, PC can sometimes dictate de novo toxicity to our NP. Some studies showed that PC-coated NPs possess higher cytotoxicity compared to naked ones (Gheshlaghi et al. 2008). This unexpected toxicity might arise from an undesired targetability conferred by the new biological suit (PC). An example of this pitfall is when superparamagnetic iron oxide nanoparticles (SPIONs) are administered into the body. Envelopment of SPIONs by biomolecules enhances their permeability through BBB and brings about CNS complications (Mahmoudi et al. 2015). • Protein fibrillation Protein fibrillation is the process through which misfolded proteins cluster together and form bigger linear aggregates (Dobson 2003). These highly insoluble aggregates are called amyloids fibrils. The formation of amyloids has been solidly linked to many pathophysiological conditions like dialysis-related amyloidosis (DRA) and neurodegenerative abnormalities such as Alzheimer’s disease and Parkinson’s disease. NPs can influence protein fibrillation in different manners. Naked NPs like CNTs and silica can accelerate amyloid formation while when cloaked with PCs, they decelerate protein fibrillation (Mahmoudi et al. 2013a, b). By contrast, naked gold NPs have inhibitory effects on protein fibrillation while their protein-coated counterparts foster amyloid formation (Mirsadeghi et al. 2015). It has been suggested that surface interactions play a pivotal role in fibrillation and introducing a nano-bio interface can remarkably influence the equilibrium of these interactions (Mahmoudi et al.

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2013a, b). For example, the particle can induce conformational changes to the proteins, exposing their core-hidden hydrophobic residues leading to attractive forces between hydrophobic residues of neighbouring protein which ultimately makes them more susceptible to aggregation and fibrillation. On the other hand, such interactions might produce fibrillation-incompetent protein conformations that diminish fibrillation. • Targeting Selectivity of our treatment is one of the foremost parameters in drug delivery. Delivering the substance exclusively to our targeted cells is tremendously favourable. Precluding the interaction of our medicine with cells other than the intended target could excessively minimize side-effects. One of the techniques employed by scientists to enhance the selectivity of a given substance is to functionalize its surface with a targeting ligand. This would allow the payload to highly accumulate in the targeted area. An example of this approach is in neoplastic circumstances where some receptors are overwhelmingly expressed in the affected cells. Implementing selective targeting in such setting can result in a more effective treatment while ameliorating its adverse reactions. However, some studies demonstrated that employing this approach in nanomedicine does not result in a satisfactory outcome in in vivo settings since PCs can veil the ligand and hinder its interaction with the target (Lynch and Dawson 2008). NP enshroudment can affect its targetability in various ways. Examples of such effects are provided later in this chapter.

6 Opportunities and Challenges When it comes to actual clinical settings, PC can be deemed as a double-edged sword. The layer acts in our favour in some conditions, while in others, it imposes more adverse effects, complication, and unpredictability to our treatment. Harnessing PC to act in our favour can significantly improve our medical intervention. One strategy called “stealth NP” attempts to disguise the NP form immune system. Studies demonstrated that coating NPs with poly ethylene glycol (PEG), a hydrophilic polymer hampers protein adsorption to the particle. Decline in adsorbed protein translates into a weaker opsonisation that eventually dampens MPS activation and elimination of our NP (Pelaz et al. 2015). Stealth NP can also be achieved by employing biomimetic compounds on NP surface (Hu et al. 2011a, b). For instance, scientists showed that leukosomes, that are engineered leukocyte-resembling liposomes, have prolonged circulation times in the bloodstream and are highly accumulated in inflammatory environments (Molinaro et al. 2016). Coating NPs with such leukosomes might enhance the efficacy of our treatment in contexts where we are trying to mitigate vigorous inflammatory reactions. Decorating NPs with specific ligands might enhance the targetability of our medicine but some studies reported that such functionalization has failed to accomplish the desired outcome (Maiolo et al.

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2015). The effectiveness of a functionalized NP can be greatly jeopardized upon its entrance into the body by the formation of PC (Salvati et al. 2013). PCs can interfere with our targeting moieties by affecting its orientation, inducing conformational disruption or obscuring it from its intended target (Zanganeh et al. 2016). One approach proposed to overcome the drawbacks of PC formation is to produce corona-free NPs. Zwitterionic NPs are resilient to adsorption thus immersing them in physiological serum only facilitates weak corona formation (Moyano et al. 2014). Another suggested approach is to take advantage of such interaction between PCs and our engineered ligand. For instance, engineered gamma globulin-coated silica particles can recruit immune-inflammatory proteins which makes them ideal agents for aiming immunological targets (Mirshafiee et al. 2016). In some cases, selectivity can be achieved without the need to employ a targeting ligand. So, in these conditions, instead of such functionalization, using a meticulously optimized NP that can adsorb a specific protein to its surface might be a better solution for selective targeting. Such adsorbed protein can direct our NP to distribute and accumulate in the organ of our interest. As mentioned before, some constituents of PC can enhance NP ability to cross sophisticated biological barriers. Even though such infiltration can cause unwanted side-effects, it can also be employed to deliver drugs to some inaccessible locations in the body. Employing NPs in amyloidogenic complications especially neurodegenerative diseases can be extremely beneficial. This has particularly drawn considerable attention not only because these agents are shown to manipulate protein fibrillation but also their small size allows them to easily trespass the stringent BBB. PCs can also be used for diagnostic purposes. For instance, one study demonstrated that immersing designed lipid NPs in the blood of pancreatic cancer patients and patients without malignancy, produces PCs with distinct compositions and PCs of the pancreatic cancer patient are more enriched as compared to that of healthy individuals (Caputo et al. 2017). Furthermore, it has been suggested that the evaluation of PC can be an inexpensive and rapid method to identify and monitor neurodegenerative diseases viz. Alzheimer’s Disease and Multiple Sclerosis (Hajipour et al. 2017). Different pathological conditions can alter the composition of the plasma and these variations can make a significant impact on the composition of the PC formed around the NP. Such findings underline the opportunity of interrogating the so-called “disease-specific protein corona” (DSPC) as diagnostic markers, after rigorouslyengineered NPs are enshrouded with the biological environment of our interest. This method could be extremely beneficial in cases such as malignancies and neurodegenerative diseases where early diagnosis can profoundly improve our interventions. One of the main challenges encountered regarding NPs, is the compromised reliability and accuracy of their evaluation in preclinical studies. As mentioned above, the composition of PC which is the de facto determining factor for the fate of the administered NP, is highly dependent on a plethora of parameters. These parameters are highly variable in different studies; therefore, debates have been raised about the transferability of the preclinical studies to clinical settings. For instance, in ordinary preclinical studies, employing murine models for the evaluation of a medicine is

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eminently ubiquitous, but when investigating NPs, this strategy becomes perplexing (Müller et al. 2018). Interspecies variations in plasma composition results in forging different PCs after NPs are administered. These different PCs influence the result of such studies and menace their reliability. Moreover, ex vivo study of NPs cannot fully simulate the complexity of the dynamics of a biological environment and the interactions of the NPs with different biological components including different cells and immune factors (Hadjidemetriou and Kostarelos 2017). Preclinical evaluation of a given medical substance is an inevitable necessity for the commencement of its clinical implementation. Such evaluation cannot be concluded unless a strong body of convergent studies (that use different established in vitro and in vivo models) point out similar results. Figure 3 shows differences between ex vivo and in vivo formation of PCs.

Fig. 3 Differences between the formation of protein coronas in ex vivo and in vivo. Corona formed in in vivo is determined by the dynamic exchange of biomolecules and diverse components of the body. The dynamics and rich biological environment of in vivo settings cannot be thoroughly replicated through ex vivo studies. Reproduced with permission (Hadjidemetriou and Kostarelos 2017)

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Fig. 4 General concept for personalized protein corona. Various genetical, epigenetical, environmental and habitual factors determine the composition of plasma. All these differences can affect the properties of the protein corona after a nanoparticle is administered into an individual’s body. Reproduced with permissions (Corbo et al. 2017)

7 Personalized Protein Corona Tailoring PCs for an individual is an ideal approach leading to a successful drug delivery. As mentioned above, the biological milieu has a colossal impact on PC specifications; therefore, administering the same nanomedicine to different patients can bring about different consequences. Studies revealed that administering the same nanomedicine to patients with different underlying pathophysiological conditions provides different PCs (Hajipour et al. 2014). Additionally, it has been revealed that PC features can even be affected by differences in healthy individuals’ plasma proteomes (Colapicchioni et al. 2016). The concept ‘Personalized protein corona’ pointed out by scientists further underlines the necessity of an extensive and conclusive understanding of the impact of the biological environment on corona characteristics. When such deep understanding is procured, one could imagine the nanomedicine to be utilized in a patient-specific and disease-specific manner. Figure 4 demonstrates some inter-individual variations that can affect PCs.

8 Conclusion Nanomedicine is currently in its infancy. The fate of a nanomedicine after its administration into the body is determined by the nano-bio interface. Even though a large body of studies has been carried out in this field, so many aspects of its entity and

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behaviour are still obscure. Most of the studies conducted hitherto have explored PCs in in vitro settings even though the complexity of the nano-bio interface makes extrapolating in vitro observations to the actual clinical situations less reliable. This complexity is the result of diverse environments that comprise thousands of different types of proteins, different flow rates and so many other variants that cannot be fully replicated in an in vitro model. That being said, an in-depth knowledge about the nano-bio interface is overwhelmingly demanded. The horizon for the forthcoming progress in nanomedicine is very promising and a fully-fledged insight into its complicated interaction with the body can lead to unprecedented improvements in medical interventions.

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Nanotopographical Control of Cell Assembly into Supracellular Structures Francesco Gentile

1 Cell Adhesion Molecules (CAMs) Control the Organization of Cells Within the ECM Cell adhesion is a result of a complex interplay of cell signalling, physical and chemical properties of materials, and biological functions of cells (Armstrong et al. 2006; Decuzzi and Ferrari 2010; Sackmann and Smith 2014). It depends on the competition of forces exchanged between cells and their surrounding medium (i.e. short-range attractive forces, medium range repellant forces), and the elastic forces associated with the deformation of the cell membrane. Short-range attraction forces are caused by interaction among cell adhesion molecules (CAMs); long-range repulsion forces are mediated by the glycoproteins that surround cell membrane; elastic forces at the cell surface interface emerge because of the deformation of the lipid protein bilayer (Armstrong et al. 2006; Bell 1978; Coombs et al. 2004; Decuzzi and Ferrari 2010; Evans and Calderwood 2007; Sackmann and Smith 2014). Thus, fine adjustment of (i) CAMs number, density and topography; (ii) types and properties of cell covering and (iii) ECM type, stiffness, compliance, and geometry, whereby cells differently and selectively morph shapes during capture, rolling, slow rolling, firm adhesion, and migration (that is the cascade of events at the basis of full adhesion), can control cell behaviour and ultimately cell fate. These mechanisms can be modelled using the classical theory of elasticity (Decuzzi and Ferrari 2010), and recently nano-mechanics that are perhaps more appropriate in analyzing nano-scale, non-continuum domains (Bruno et al. 2015) (i.e. deformation of cell membrane), statistical physics, thermodynamics and bio-

F. Gentile (B) Department of Electrical Engineering and Information Technology, University Federico II, 80125 Naples, Italy e-mail: [email protected] © Springer Nature Switzerland AG 2019 M. Rahmandoust and M. R. Ayatollahi (eds.), Nanomaterials for Advanced Biological Applications, Advanced Structured Materials 104, https://doi.org/10.1007/978-3-030-10834-2_2

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chemistry (Bell 1978; Coombs et al. 2004; Evans and Calderwood 2007; Farhadifar et al. 2007) (CAMs recruitment, diffusion and migration; cell shell description), and a combination of these. On cell surface, adhesion molecules and trans-membrane receptors cluster together to form isolated groups of molecules. Similar supra-molecular assemblies are called focal adhesions and focal complexes (i.e. small focal adhesions developed at the early stages of adhesion) (Wozniak et al. 2004), and have a characteristic length scale comprised in the low nanometer interval (30–100 nm) (Geiger et al. 2001, 2009). Focal adhesions serve as the mechanical linkages to the ECM, and as a biochemical signaling hub to concentrate and direct numerous signaling proteins at sites of integrin binding and clustering (Geiger et al. 2001, 2009). Formation of adhesion domains enables activation of strong cohesive forces just with a relatively small number of attractive CAMs (~104 ) (Sackmann and Smith 2014). Thus adhesion and adhesion strength would depend on the CAMs within focal adhesions, and varies with time as their number, density and localization change following exocytosis, endocytosis, or segregation (Decuzzi and Ferrari 2010). Thus, adhesion to the extracellular matrix (ECM) or to neighbouring cells and subsequent multiple cellular processes such as cell migration, morphogenesis, proliferation, gene expression and survival, depends on the balance/unbalance between the spatial and temporal distribution of CAMs, and cell shaping through actin–microtubule cross-talk. Activation and regulation of CAMs distribution depend, in turn, on multiple environmental cues. In the following, we will examine whether and to which extent one can control adhesion molecules through nano-topography to guide cell assembly and organization. This chapter is dedicated to the articulation of this topic. It is a review of some important contributions that have joined nanotechnology, cell biology and cell assembly, presented from the perspective of the author.

2 Nanotechnology, Scaffolds and Artificial Analogues of the ECM Cells respond to environmental features at length scales ranging from the submillimetric scale to the molecular level (Stevens and George 2005). Conventional biomaterial scaffolds (with the function of supporting cell growth, tissue growth, generation or regeneration, and improving or replacing the biological functions of organs and tissues), present features at the macroscale but lack the resolution necessary to reproduce the nanoscale detail of real tissues and organs. While the search for tissue engineered materials and designs has been limited for a long time to properties like biocompatibility, biodegradability, porosity, chemical and mechanical properties (Subramanian et al. 2009; Battista et al. 2015), only recently advances in nanotechnology have enabled the design, fabrication and characterization of biomaterials with a controlled topography at the nanoscale (Arnold et al. 2004; Kim et al. 2014). Nanotechnology may be used to generate controlled nano-scaled geometries to examine

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how the cellular sensory machinery interacts with extremely small cues to regulate cell behaviour (Kanchanawong et al. 2010). As the natural extracellular matrix (ECM) provides a natural environment of intricate nanofibers to support cells and present an instructive background to guide their behaviour (Stevens and George 2005), surfaces with a controlled nano-geometry may represent ECM analogues for the cell adhesion and proliferation (Geiger et al. 2001, 2009; Kanchanawong et al. 2010; Kim et al. 2014). The mechanisms of cell-surface interaction and effects thereof have been heretofore examined in a number of studies. Exploring a variety of geometries, including anisotropic gratings, islands of carbon nanotubes, ridges and pillars, and randomly rough surfaces, researchers demonstrated that a nano-scale architecture may direct, control and, in some cases, improve cell polarity (Ferrari et al. 2011), adhesion (Sorkin et al. 2009; Xie et al. 2010), growth (Ankam et al. 2013; Baranes et al. 2012), differentiation (Migliorini et al. 2013, 2011; Moe et al. 2012), organization or self-organization into simple or complex networks (Huang and Jiang 2013; Limongi et al. 2013), electrical signaling (Tang et al. 2013). In De Vitis et al. (2015), Limongi et al. (2013), super-hydrophobic 2 + 1 dimensional surfaces have been used for guiding neuronal growth, activity, and analyzing the relative expression of transmembrane receptor molecules. In other reported experiments (Gentile et al. 2010, 2013), researchers have demonstrated that the adhesion and proliferation of various cell lineages are maximized on surfaces with moderate roughness and large fractal dimension. Recently, the adhesive behaviour of neuroblastoma N2A cells was verified over porous silicon with a fixed (Gentile et al. 2012c) or smoothly variable pore size (Khung et al. 2008). In Accardo et al. (2017), Accardo and collaborators realized three-dimensional, self-standing scaffolds using multiphoton direct laser writing. Because of the accuracy of the fabrication process, they attained tight control over the features of the scaffolds at the nano-scales. Then, they examined N2A cells seeded within the scaffolds using light sheet fluorescence microscopy and multiphoton confocal imaging to explore cell morphology and cell networks characteristics.

3 Generation of Nano-patterned Surfaces In what follows, I expose some of the methods that have been used to generate nano-textured surfaces. Notice that these may be decorated by irregular rather than periodic motives, or a combination of the two.

3.1 Generating Rough Surfaces Using Wet Etching Procedures Here, we recapitulate a method used, for instance, in Gentile et al. (2010, 2013), Onesto et al. (2017). For this configuration, (111)—oriented Si wafers are used as

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Fig. 1 Examples of rough silicon surfaces generated by KOH wet etching at different etching times

substrates. The superficial layer of SiO2 is removed by immersion in a hydrofluoric acid solution (HF : H2 O  1 : 5 v/v) for 30 s. Fresh silicon samples are then immersed in a corrosive bath of a solution of potassium hydroxide (KOH : H2 O  1 : 4 v/v) at different times and at the constant temperature (T  70 °C). Since strong alkaline substances (pH > 12) such as aqueous KOH etch silicon via the reaction Si + 4OH− → Si(OH)4 + 4e− , the method yields surfaces with different roughness as a function of exposure time to the corrosive solution. At 60 min of time of exposition, one can obtain surfaces with values of average surface roughness Ra and root mean square roughness Rrms up to Ra ~ 100 nm and Rrms ∼ 120 nm (Fig. 1), where     1/2 . In the above, l is the sampling Ra  l z(r )dr l and Rrms  l z 2 (r )dr l length and z(r ) is the profile of the surface along the r direction. Vertical sample profile can be measured using atomic force microscopy. Examples of rough surfaces obtained by a similar method are reported in Fig. 1.

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3.2 Generating Nanoporous Surfaces Using Electrochemical Wet Etching Porous silicon is a material where arrays of vertically aligned pores penetrate through its structure. Size, shape, the density of the holes, and their penetration depth in the porous matrix can be finely adjusted on changing the parameters of the fabrication process, including etching time, current intensity, active etchant concentration, the temperature of the process (Gentile et al. 2011, b). Porous silicon has peculiar characteristics that include, but are not restricted to, biodegradability under physiological conditions (Foll et al. 2002), biocompatibility (Hu et al. 2010; Yiu and Wright 2005), hydrophobicity (Gentile et al. 2011), photoluminescence (Godefroo et al. 2008). Depending on pore size (s), porous silicon is classified into nanoporous silicon (s < 2 nm), mesoporous silicon (2 < s < 50 nm), macroporous silicon (s > 50 nm) (Marinaro et al. 2015). Porous silicon is prepared via a porosification process thoroughly described in (Foll et al. 2002). Porous silicon substrates are generated from bulk boron-doped p-type (100) silicon wafer via anodization. Silicon samples are placed within an electrolytic cell where a platinum cathode and the silicon wafer (anode) are immersed in a solution of hydrofluoric acid (HF) (Fig. 2a). Upon removal of silicon from the pores (Fig. 2a) the substrate exhibits clear luminescence under UV radiation (Fig. 2c). Substrates with different pore sizes can be obtained by tailoring the etching conditions being: (i) the intensity of etching current, (ii) the concentration of HF, (iii) the length of the process. Before electrochemical etching, samples are cleaned with acetone and ethanol to remove possible contaminants; then they are treated with a 4% HF solution to remove the superficial layer of silicon dioxide. The method has been exploited to generate macroporous (MaP), mesoporous (MeP), nanoporous (NP) silicon substrates. In the case of MaP silicon with a characteristic pore size greater than ~50 nm, the etching electrolyte mixture is composed of HF, DI water and DMF (Dimethylformamide)  (9 : 1 : 115, v/v/v). The external applied current is set as I  4 mA cm2 and is maintained for 5 min at 25 °C. In the case of MeP silicon, the etching electrolyte mixture is composed of HF, DI water and methanol (5 : 3 : 2, v/v/v). MeP substrates with a characteristic pore size comprised in the2–50 nm range are obtained by maintaining a constant current density of I  4 mA cm2 for 5 min at 25 °C. In the case of NP silicon, the etching electrolyte mixture is composed of HF, DI water and ethanol (1 : 1 : 2, v/v/v). MeP substrates with a characteristic pore size comprised in the 2–50 nm range are obtained by maintaining a constant current density of I  20 mA cm2 for 5 min at 25 °C. In all cases, the porous layer extends vertically within the bilk silicon for few tens of micrometres. Samples are finally rinsed in DI water, ethanol, and pentane. Since porous silicon is super-hydrophobic, substrates are treated to assure hydrophilicity by oxidation in oven at 200 °C for 2 h. Examples of porous surfaces obtained by electrochemical methods are reported in Fig. 2d.

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Fig. 2 a Porous silicon surfaces can be generated by electrochemical etching; b in the technique, portions of silicon material are removed from the surface; c resulting porous silicon substrates are photoluminescence under UV irradiation; d one can prepare substrates with a mean pore varying from few nm to few tens of nm

3.3 Silver and Gold Nanoparticle Clusters Obtained by Electroless Deposition Sometimes it may be convenient obtaining nanostructured surfaces by incrementally depositing smaller building blocks on a flat substrate. This bottom-up approach deviates from the etching techniques described above and is more akin in nature to additive manufacturing. Electroless deposition (sometimes called electroless growth) is a nanotechnology method for nanoparticle synthesis that attains tight control over the size and shape of nanostructures (Coluccio et al. 2014). During electroless deposition, metal ions in solution are reduced, transported and deposited as metals on a substrate (Fig. 3a–b). The reduction is mediated by specific classes of reducing agents and may be accelerated using catalyzing agents. The process implies absorption of metal ions and reducing agents by a catalytic surface. While electroless deposition is a general process, here we describe the methods in relation to the deposition of silicon because silicon behaves simultaneously like a catalytic and reducing agent. This implies that metal ions can be reduced as atoms without the need of an external

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reducing agent. Deep-centre defects on the silicon surface (dangling bonds) with a bound state that lies below the conduction band of silicon are responsible for its auto-catalytic properties (Schofield et al. 2013). On the active site of a silicon surface exposed to growth, metal ions and associated dangling bonds react through a direct redox reaction to form the intended structures (Qiu and Chu 2008; Tao et al. 2008). Silicon oxidizes and reduces metal ions into metals, with a law that reads mMen+ + nRed  mMe0 + nOx

(1)

where Men+ are metal ions and Red represents the reducing agent—the reaction uses n electrons necessary to produce the atomic metal Me0 . The driving force of the process is the potential differences of the two redox half reactions: thus the reaction is spontaneous and does not need externally applied electric sources or potentials to evolve, with understandable advantages over traditional techniques of particle growth. In the case of silver ions in solution (Fig. 3c), the reaction takes the form: at the anode (silicon oxidation): Si + 2H2 O → SiO2 + 4H+ + 4e−

(2)

At the cathode (silver reduction): Ag+ + e− → Ag0

(3)

where redox potentials are E01  −0.86 V, E02  0.8 V (Goia and Matijevic 1998). In the case of gold ions in solution, the reaction takes the form: at the anode (silicon oxidation): Si + 2H2 O → SiO2 + 4H+ + 4e−

(4)

At the cathode (gold reduction): Au3+ + 3e− → Au0

(5)

where redox potentials are E03  −0.86 V, E04  1.52 V (Yae et al. 2007). Particle growth involves an initial nucleation phase with the formation of metallic nuclei and a steady state phase of growth. Reaction kinetics, shape and size of the final structures, and the time that it takes to reach a steady state value, depend on the concentration of reducing agents, temperature, time of reaction and type of metals. Typical values of solute concentrations in solution are 0.15 M for hydrofluoric acid (HF), 0.1–5 mM for auric chloride (AuCl3 ), 0.1–5 mM for silver nitrate (AgNO3 ). The function of hydrofluoric acid is to solve silicon oxide resulting from the reaction. Electroless deposition enables to produce nanoparticles, nanoparticle clusters and nanoparticle arrays where the mean size of the particles may be finely adjusted in the 5–100 nm

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Fig. 3 a Nano-patterned substrates can be generated by electroless deposition. In the technique, silicon samples are placed in contact with a solution of metal salts and hydrofluoric acid; b metal nanoparticles are then reduced on the surface with time; c the chemical reaction of reduction at the silicon interface is reported for the specific case of silver. d Pre-patterning of silicon by optical or electron beam lithography techniques enables tight control over the shape and size of the resulting metal nanomaterials

range, the standard deviation is maintained low, and particles are repeated over large areas.

3.4 Lithography Techniques for the Definition of Periodic Nanostructures Electroless deposition enables the fabrication of small and ultra-small nanoparticles on a surface. To harness the potentialities of the method and achieve ultra-high resolution, surfaces may have been previously patterned using optical or electron beam lithography techniques. In doing so, the region of the surface exposed to the solution is restricted and growth takes place at predefined sites of the silicon substrate.

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Patterns on a silicon surface can be obtained using optical lithography, electron beam lithography (EBL) or focused ion beam (FIB) techniques. Where the (i) first optical lithography technique allows the formation of large, millimetre patterns with a micrometric resolution in few seconds. The (ii) second EBL technique enables the formation of patterns at the low nanometer scale in a time that may vary from few minutes for micrometric areas to several hours for millimetre areas. The (iii) third FIB technique enables to create, modify, and characterize complex structures below 100 nm either removing or depositing material on a substrate. Patterns on silicon are made of polymeric materials that do not interact with solutions prepared for electroless deposition, enabling spatial selectivity and precise positioning or metal particles on a surface: silicon substrate decorated with specific patterns may be placed in contact with an electroless solution, starting the formation of metal nanograins in the patterned portions of the substrate. If patterns are larger than some tens of nanometers, more particles may growth in the same pattern, creating a random distribution of nanoparticles. When pattern size decreases to a value that is approximately 50 nm, patterns may accommodate single isolated particles: this configuration yields maximum resolution and control of particle shape and particle size. Intermediate values of pattern size enable to modulate the size of deposited particles (Fig. 3d). The fabrication of nanoparticles with a tight control over their geometry and the topography of the substrate itself necessitates an interweaving combination of top down (lithography techniques) and bottom up (electroless deposition) approaches.

3.5 Reactive Ion Etching Techniques for the Fabrication of Super-Hydrophobic, 2 + 1 Dimensional Surfaces Heretofore we have described bidimensional structures where their complexity is lumped within their superficial texture. Extensive use of nanofabrication techniques allows including an additional dimension in the devices. Arrays of gold or silver nanoparticle clusters precedently described can be further processed using Deep Reactive Ion Etching (DRIE) techniques. The DRIE process uses a time varying electromagnetic field to accelerate ions towards the sample and modify it through interaction with these ions. In certain configurations (Limongi et al. 2013), the DRIE process realizes a pulsed, time-multiplexed etching that alternated repeatedly between three modes: (i) deposition of a chemically inert passivation layer of C4 F8 , (ii) isotropic plasma etch of SF6 and (iii) phase for sample and chamber cleaning. The process results in arrays of micrometric cylinders, or pillars, constituted of silicon and covered with silver or gold nanoparticle clusters, which serve as a mask in the etching phase of the DRIE process, as in the examples shown in Fig. 4a–d. Since resulting structures develop preferentially in the vertical direction, they are 2 + 1 dimensional surfaces (Manhattan-like structures). Nonetheless, claiming that the details of the device are limited to the sole 2D surface is a misnomer: based on DRIE alternate process, pillars are fabricated with nano-indentations at the sidewalls, which may be

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Fig. 4 a One can use the RIE technique to obtain super-hydrophobic 2 + 1 dimensional surfaces for cell culture growth, super-hydrophobic surfaces are typically constituted by arrays of micropillars; b larger magnification factors reveal that individual pillars are covered by silver; c or gold; d nanoparticle clusters; e super-hydrophobic has vanishingly small friction coefficients whereby solutions deposited on similar surfaces maintain a quasi-spherical shape

responsible for the peculiar behaviour of cells that are incubated on these structures. The geometrical parameters of the pillars, i.e. diameter d, distance l, height h, are chosen in accordance to a criterion of rational design and set as d  10 µm, l = 20 µm, h > 15 µm (Gentile et al. 2012a). The pillars form a hexagonal packing arrangement that is the highest-density lattice arrangement of circles in a plane. The structures are finally covered by a thin (~5 nm) film of C4 F8 , that assure super-hydrophobicity. Super-hydrophobic surfaces are non-wetting surfaces that prevent firm adhesion of water solutions deposited on them. Instead, solutions assume a spherical shape as it were floating in air (Fig. 4e) that in turn translates into an increased ability to manipulate, transport and control solutions and cells.

4 Characterization of Nano-Patterned Surfaces 4.1 Fractal Dimension of a Surface Surfaces can be described by mathematical variables. While roughness represents the gold standard for describing a surface, still in many practical instances it lacks the resolution necessary to capture the miniature details of nanoscale surfaces. Moreover, roughness is not a complete and consistent metric for a surface, meaning that surfaces with different topological characteristics may have the same value of average surface roughness Ra and root mean square roughness Rrms. While effective in the realm of mechanical and other classical engineers, roughness may have limited use and lead to flawed conjectures in biomedical nanotechnology. In opposition to roughness, the

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fractal dimension D f is a mathematical measure of a surface that coagulates different scales in a single parameter. Fractals are mathematical objects that are too irregular to be described by conventional geometry. They exhibit properties that may be reviewed as follows: (i) details on arbitrarily small scales (fine structure); (ii) fractals can be generated by short algorithms (perhaps recursively); (iii) fractals exhibit a fractal dimension D f strictly greater than the classical topological dimension (Gentile et al. 2011). D f quantifies the complexity of patterns as the ratio of a change in detail to a change in scale (Gentile et al. 2011). Assume to have the Atomic Force Microscopy (AFM) topographical measure z(x · y) of a surface S, acquired using the methods reported, for instance, in (Gentile et al. 2010), where x and y are spatial variables in an orthonormal basis. Then, one can derive the power spectrum density function C2D (qx , q y ) associated to S (Gentile et al. 2011):    1 C2D (qx · q y )  z(x · y) z(o) e−iq(x · y) d x 2 dy 2 (6) 2 (2π) S  where q  2π λ is the wave number, and λ the characteristic wave-length. The symbol · · · stands for ensemble averaging over a collection of different surfaces with identical statistical properties. Since Eq. (6) is bi-dimensional, it may be more convenient deriving its mono-dimensional version C(q), that is obtained through averaging C2D over every circumference  of radius q and origin (0, 0) [the method is called Fractal Analysis by Circular Averaging—FACA (Gentile et al. 2010) as: C(q) 

1 



  1 C2D qx · q y dγ  2π 



2π

C2D (q cosψ · q sinψ)dψ.

(7)

0

1/2    and ψ  arctan q y /qx are polar variables. The In Eq. (7), q  qx + q y power spectrum density function C(q) delivers the information content of a surface at different scales q. In the case of self -affine surfaces, for which a rescale in the planar coordinates x → bx, y → by is accompanied by a rescaling in z: z(b(x · y)) → bHz(x · y), C(q) takes the simplified form

  H h o 2 q −2(H +1) C(q)  2π qo qo

(8)

Equation (8) is accurate ∀q > q0 , q0 is the lower cut-off wavenumber correspond√ ing to an upper cut-off wavelength λ0  2π/q0 , and h o  2 Rrms. A self-affine fractal surface can be univocally identified by specifying the surface roughness (Rrms), the cut-off wavenumber q0 and the Hurst coefficient H . In a loglog plot, in a region of the plot C(q) is linear with slope β. If β is small, the information content of a surface spans across different scales, and D f is large. If β is large, the information content of a surface is limited to specific scales, and D f is small. β and the fractal dimension D f are not independent and from the measure of β one may obtain D f as D f  (8 − β)/2. The fractal dimension D f for a surface ranges

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from 2, representing a perfectly flat surface (Euclidean dimension of a surface), to 3, representing an extremely rough surface. For D f  2.5, the so-called Brownian surfaces are identified which have totally random and uncorrelated profiles.

4.2 Diffusion Limited Aggregation (DLA) of Atoms into Supramolecular Structures Some of the methods for the bottom-up formation of nanoparticles on a substrate imply electroless deposition, i.e. the self-assembly of metal ions into metals on an autocatalytic surface. Particle migration and coalescence into supramolecular structure may take place with or without the influence of external fields, i.e. convective flows, electromagnetic fields, gravitational force fields, or the alike (Glotzer 2012; Glotzer and Anderson 2010; Glotzer and Solomon 2007; Jackson et al. 2004). In the case that the external fields are absent, then particle growth is governed by pure diffusion. Since diffusion and the mathematics of diffusion are very well understood, mathematical models and simulations can be used to duplicate the results of aggregation. A perfect match between experiments and simulations is achieved through molecular dynamics, where the trajectories of atoms are determined by solving Newton’s equations of motion for a system of interacting particles - where forces between the particles and potential energy are defined by molecular mechanics force fields (Gentile et al. 2014b). Nevertheless, molecular dynamics methods may be unpractical in many instances. In contrast to molecular dynamics, Diffusion Limited Aggregation (DLA) is an approximate method of simulation of particle formation and growth on a surface (Coluccio et al. 2014; Gentile et al. 2012c, 2014a). In DLA, randomly displaced particles stick together to form the intended structures. The assumption that random walk is an accurate replica of ions dynamics is correct if chemical reactions at the interface are instantaneous. In the model, the displacement of a metal ion obeys to a uniform distribution, i.e. there are no preferential directions of motion (Fig. 5a–c): the trajectory of a particle can be correctly described by a random walk as in Brownian dynamics. This walk, in turn, can be reproduced in a regular lattice, where particles are dislodged by the finite distance x in the time interval τ . The root mean square distance walked by a particle over time yields a measure of the variation of an ensemble as of particles (Saltzmann 2001)  2  2  2 r  x + y  4Dt.

(9)

Where x, y, r are the Cartesian coordinates in a plane, D is the diffusion coefficient. In the following, we consider bidimensional geometries. The scheme in Fig. 5(d) describes a practical implementation of the method. Sufficiently far from the adhesion surface (at a distance l), n particles are released in the system. At any iteration i, particles move within a regular pattern of cells by one lattice unit (1 l.u.), thus x  1 l.u.—a lattice unit is the smallest block of the lattice and represents

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the resolution of the system. We impose periodic boundary conditions at the lateral boundaries of the lattice, and a bouncing condition at the upper boundary, that means that any particle colliding with the wall rebounds, maintaining direction and velocity intensity but reversing velocity versus. At the lower border, the seed  represents the portion of the silicon substrate exposed to growth in an electroless deposition: it is a line of nucleation sites. During motion, when a particle comes in contact with  it is incorporated by  and the size of the aggregate increases. This process proceeds iteratively and is interrupted by a stop condition (i.e. the aggregates reach a maximum size or i exceeds a maximum iterations number). The result of the simulation is an aggregate where the multi branched arrangement of particles recalls the fractal nature of real electroless deposits (Fig. 5e). The structure of clusters of occupied lattice sites exhibit geometric scaling relationships which are characteristic of fractals and can be used to estimate an effective fractal dimensionality of the aggregates as D f  5/3 ∼ 1.667. The fractal dimension, in turn, can be used to derive the mean cluster size Sc of the aggregates as a function of the total number N of deposited particles (Racz and Vicsek 1983; Meakin 1984): Sc ∼ N D f /( D f −1) .

(10)

Equation (10) is a quantitative estimate of cluster characteristics. As such, it can be used to analyse aggregation results as a function of internal (i.e. temperature, concentration, PH) and external (i.e. pattern size, pattern distance) factors, and compare them to experiments, as for instance in (Coluccio et al. 2014; Gentile et al. 2012b, 2014a).

4.3 Surface Wettability, Cassie Baxter and Recursive Cassie Baxter Surface hydrophilicity or hydrophobicity of nano-patterned surfaces can be determined by measuring the water contact angle with one drop of deionized water using an automatic contact angle meter at room temperature. Measurements permit to evaluate the average contact angle θ developed at the solid/liquid interface. Following the Young-Dupre equation, the energy of adhesion ν per unit area at the solid/liquid interface is determined as v  vlg (1 + cos θ), where vlg  72.8 mJ/m2 is the air/water surface tension. If a surface has a superficial roughness different from zero, then a liquid develops an angle of contact θb that deviates from θ. The extent of the deviation depends on the ratio of asperities of a profile to the profile length, that is the solid fraction φ of a surface. With Cassie and Baxter (Lafuma and Quéré 2003), one can write: cos θb  −1 + (cos θ + 1)φ

(11)

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Fig. 5 a–b DLA representation of electroless growth; c in the walk, the most probable position after a certain number of steps is the initial position; d using numerical schemes, one can reproduce the pattern of metal ions transported in a medium through pure diffusion as in a random walk; e more sophisticated evolution of the scheme enables the simulation of numerical aggregates, where the structure of aggregates is fractal

In the limiting cases (i) φ → 1, then θb  θ (flat surface with no textures); (ii) φ → 0, then θb  180◦ (the surface is composed of an array of vertical, ideally mono-dimensional segments whereby water is suspended in air—this is the Fakir state). Equation (11) has a multi-scale analogue (Herminghaus 2000):   b cosθn+1  −1 + cosθnb + 1 φ

(12)

where the recursive nature of the equation reflects the hierarchical nature of fractal surfaces. In Eq. (12), n is the recursion step that can be interpreted as the level of detail of the real physical prototype at which a nanoscale structure has macroscale effects (i.e. the scale factor). Increasing n increases sample hydrophobicity. To identify the correct value of n one can use the area magnification factor σ written using the formalism of fractals (Gentile et al. 2011):

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(D f −2) η σ , η

(13)

and its definition: σ

 l

(14)

and comparing the terms. In doing so, one obtains σ(n) 

a 2 n

+

A n 2−D f

  A 2 2  n−1 n 2−D f ·  2  g 2 a A a/n + 2−D f n

(15)

n

for n > 1. In the above,  is the real profile length, l is a length of base, η and A are the upper and lower limits of fractal behavior in a power spectrum density function, D f is the fractal dimension of the substrate, a and A are geometrical constants, g is the elliptic integral of the second kind. Equation (15) attains a steady state value for large n. The value of n for which σ reaches the 90% of its final value, is the correct magnification factor (scale) that characterizes the system.

5 Characterization of Cell Networks 5.1 Network Analysis and Cell Network Topology Cell nuclei on a substrate can take specific configurations. That is, their spatial coordinates may follow statistical distributions that are uniform or deviate from uniformity to an extent that depends on the characteristics of the substrate, the cells, or an interplay thereof. The spatial organization of cells, in turn, may be indicative of the augmented ability that cohorts of cells have in contrast to individual cells taken in isolation. Network analysis provides means to describe collections of cells on planar surfaces and gives hints on how cell network topology may influence their functions. One way to examine the topology of a group of elements in a plane is using the mathematical variables: (i) clustering coefficient and (ii) characteristic path length. The clustering coefficient (Cc) describes the propensity of the nodes of a graph to form few groups in which the elements of the groups are inter-connected by an elevated number of edges (Onesto et al. 2017). The characteristic path length (C pl) indicates the number of passages that on average separates two nodes randomly picked in a network. Cc ranges between 0 and 1, C pl is greater than 1. Cc and C pl are used to describe and assess the efficiency of complex systems and dynamical systems (Watts and Strogatz 1998; Strogatz 2001). Networks with high Cc and low C pl are named small world networks. Small world networks typically feature overabundance of hubs with a high number of connections. Thus networks with a small

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world architecture may possibly mediate information between nodes of the network and function more efficiently than equivalent random, periodic or regular graphs (Watts and Strogatz 1998; Strogatz 2001). Small world networks have SW > 1 as described below. To examine cell networks, it is necessary the knowledge of network connectivity, i.e. to know whether pairs of nuclei (nodes) are connected or not. The Waxman model makes a decision on whether two nodes in a grid (u · v) are connected or not (Gunduz et al. 2004). The model makes use of a probability function P(u · v) which decays exponentially with the Euclidean distance d(u · v) between u and v: P(u · v) ∝ e−d(u · v)/ β L , , where L is the largest possible Euclidean distance in the grid and β is a parameter. If a randomly generated number between 0 and 1 is smaller than P, then nodes shall be connected. Notice that if β is large then it is very likely that two nodes in the grid may be connected. If d(u · v)  β L, then P  e−1 , and P < e−1 for every d(u · v) > β L. Thus β L  d p has the significance of a probabilistic cut off distance, which determines with which probability (P) nodes are joined, in contrast to the classical concept of deterministic cut off distance dc , whereby after network conditioning, maximum edge length in the network is set as dc (Onesto et al. 2017). The information about the connections among the nodes (cells) in a graph is then stored in the adjacency matrix A  ai j , where the indices i and j run through the number of nodes n in the graph; ai j  1, if there exists a connection between i and j, ai j  0 otherwise. Since we assume reciprocity, A is also symmetric, being ai j  a ji . Moreover, ai j  0 (Marinaro et al. 2015). With these premises, we show now how to calculate the parameters Cc, C pl and SW of a network. The clustering coefficient is defined as Ci 

2E i k(k − 1)

(16)

where k is the number of neighbors of a generic node i, E i is the number of existing connections between those, k(k − 1)/2 being the maximum number of connections that can exist among k nodes. Notice that the clustering coefficient Ci is defined locally, and a global value, Cc , is derived upon averaging Ci over all the nodes that compose the graph. The characteristic path length (C pl) is defined as the average number of steps along the shortest paths for all possible pairs of network nodes. We shall call the minimum distance between a generic couple of nodes n l and n m the shortest path length, Spl, which is expressed as an integer number of steps. In A, al · i and ai · m account for all the pairs of nodes which are connected to n l and n m respectively. The  sum of al ·i and ai · m over all the nodes in A, is stored in a new al · i ai,m for all the l and m and A2 has the same dimension of A. matrix A2  Now multiplicate A2 and A repeatedly A2  A2 A, until all the terms of A2 are non-zero and those terms in position i j will be the Spl between node i and node j. Finally, the characteristic path length C pl is calculated like the average of Spl over A2 . Once obtained the Cc and C pl values, we can derive a precise measure of ‘small-world-ness’, the ‘small-world-ness’ coefficient (SW), based on the trade off between high local clustering and short path length.

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A network G with n nodes and m edges is a small-world network if it has a similar path length but greater clustering of nodes than an equivalent Erdos–Rényi (E–R) random graph with the same m and n (an E–R graph is constructed by uniquely assigning each edge to a node pair with uniform probability). Let C pln and Ccu be the mean shortest path length and the mean clustering coefficient for the E–R random graphs, and C plgraph and Ccgraph the corresponding quantities for the graphs derived using the methods described above. We can calculate: γ

Ccgraph C plgraph γ ·λ · SW  Ccu C plu λ

(17)

the categorical definition of small-world network above implies λ ≥ 1, γ 1 which, in turn, gives SW > 1.

6 Shannon Information Entropy in Cell Networks A small world configuration of a network of cells may lead to a maximum of information in that network. We can use generalized leaky integrate and fire models (de la Rocha and Parga 2005; FitzHugh 1955) to estimate signal propagation in networks of cells and neuronal cells (Onesto et al. 2016). Here, we assume that the nodes of the grid represent the nuclei of the cells and are extracted from confocal images of cultured neural networks. Nodes of the grid are therefore connected using the Waxman model as described previously and in Gentile et al. (2012c, Marinaro et al. (2015). In doing so, we obtain undirected graphs where the edges of the graphs represent cell-cell connections. Then, nodes of the network are randomly picked and excited with (i) random and (ii) periodic signals of time. Spikes propagate in cascade in the grid. We use generalized leaky integrate and fire model (de la Rocha and Parga 2005; FitzHugh 1955) to analyze the corresponding signal flow. In individual neurons, electric pulses excite the neuron until the response (potential) at the postsynaptic sites reaches and surpasses a limiting value (a threshold), then, the target neuron produces an impulse (an action potential) that propagates in turn to another neuron. This process is described by the following Equation, in which the membrane potential V obeys to a function of time (Fig. 6a): Cm

dV  −gl (V − Vo ) + Istim dt

(18)

where Cm is the capacitance of the membrane, gl is the conductance, Vo is the resting potential of the neuron. The current Istim is the stimulus that excites the neuron until the membrane potential reaches a threshold Vth and an action potential is released from the system. Neurons in a grid (Fig. 6b) are described by a set of differential equations that generalizes the model described by Eq. (18). Each node in the network sends and receives information and this process is mediated through the integrate

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Fig. 6 a The response of individual neurons is described by the celebrated leaky fire and integrate model, in which a pattern of impulses excite a neuron until its membrane voltage V reaches a threshold Vth; b this event triggers the release of an action potential that propagates in turn in the network; c the propagation model is applied to a subset of nodes of the network in which each node both receives and sends information to the other nodes of the set; d the output of individual neurons in the grid is a train of action potentials; e that can be represented in a binary chart in which each entry of the chart is a bit; f bits indicate whether a neuron discharges (1) or not (0) an action potential at a specific time. Bits of information can be divided into groups or words, and words can be sorted in order of decreasing frequency in the train; g from the analysis and comparison of these frequency distributions upon application of a time locked and h a random signal of time, it can be derived i how information propagates in the network. Reproduced from (Onesto et al. 2018)

and fire model and Eq. (18). Assuming linearity, Istim is given by the superposition of current pulses J generated by the neurons i that fire on a neuron j B r el       Istim (t)  ζ di j J δ t − tik i

k

(19)

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where r el is the number of neurotransmitter release events, δ is the Dirac delta function, tik is the timing of individual pulses, ζ is a damping term which accounts for the inter-nodal distance di j (Fig. 6c). Pulses repeatedly excite a neuron until V  Vth and an action potential is discharged from the target neuron (Fig. 6d). The action potential generates in turn an impulse that propagates through the network. The generation of an action potential at a node of the grid at a specific time is an event. The temporal sequence of events encodes the information transmitted over that grid (Fig. 6e). Resulting patterns of multiple spike trains are interpreted using information theory approaches (Onesto et al. 2018). Time spikes are grouped in sets of words, in which a word is an array of on (presence of a spike)/off (absence of a spike) events in a binary representation. By sorting words in order of decreasing occurrence in the train (Fig. 6f) we derived the associated Shannon entropy H : H (S)  −



P(s)log2 P(s)

(20)

S

that quantifies the average amount of information gained with each stimulus presentation. Entropy is measured in bits if the logarithm is taken with base 2. In the equation, P(s) represents the probability with which a stimulus s is presented in the set S. H is the variability of individual neurons in response to a long random sample of stimuli (total entropy) (Fig. 6h), N is the variability of the spike train in response to a sample of repeated stimuli (noise entropy) (Fig. 6g), then information that the spike train provide about the input is the difference between entropies I  H − N . This permits to derive information over all the nodes of the graph (Fig. 6i). In deriving information, we assume that networks are unidirectional. The process of information propagation is irreversible and reciprocity does not generally hold in the grid. This accounts for the fact that, upon the generation of an action potential, a neuron experiences short-time synaptic depression and remains inactive for a refractory time.

7 Cell Adhesion and Proliferation on Nano-patterned Surfaces 7.1 Cell Adhesion on Nano-scale Rough Surfaces Nano-patterned surfaces have been used as a platform to guide cell adhesion and proliferation. In Gentile et al. (2010) Gentile and collaborators cultivated four different cell lines (i.e. A549 human lung carcinoma, human HeLa, human umbilical vein endothelial cells and mouse 3T3 fibroblasts) on silicon substrates with variable roughness ranging in the 0–100 nm interval and a fractal dimension varying from about 2–2.6. For all cell types, the number of adhering cells and their proliferation

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Fig. 7 a The relative density of stably adhering cells as a function of the silicon substrate average roughness Ra at different time points, for mouse 3T3 fibroblasts; b Human HeLa cells; c Human A549 lung carcinoma; and d HUVEC-C. Partially reproduced from (Gentile et al. 2010)

rates exhibited a maximum on moderately rough (Ra  45 nm) nearly Brownian (D f ∼ 2.5) substrates at the time intervals t  24, 36, 48, 60 h (Fig. 7a–d). The increased proliferation rate observed on moderately rough Brownian substrates could be attributed to several mechanisms among which (i) the increased effective surface energy γo , typically associated with moderately rough substrates and (ii) the nonuniform surface adsorption and preferential conformation of proteins over non planar substrates. The effective surface energy γ of a cell adhering to a substrate is defined as the total work per unit area required for full detachment. In general, this work would depend on the surface roughness, and it would be ν for perfectly planar surfaces. Cell adhesion is mediated by the formation of discrete ligand-receptor bonds (specific interactions) and short ranged interfacial forces as van der Waals, double layer electrostatic and steric (non-specific interactions), which together contribute to the surface energy of specific and non-specific adhesion γadh . This grows with the density of the ligands adsorbing on the silicon substrate, the density of the receptors expressed on the cell membrane and the affinity of the ligand-receptor bonds. Under the simplistic assumption that cells can be represented as thin elastic layers of modulus E sitting over a rigid wavy surface with a fixed wavenumber q and an amplitude h; the ratio γ/ν is larger than unity for h smaller than h cr (Decuzzi and Ferrari 2010). In other words, cell stable adhesion and, consequently, proliferation would be energetically favorable on rough surfaces as long as h < h cr , which would then identify the subset of moderately rough substrates for a given q. Also, γ/ν and

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39

h cr have been predicted to grow with the normalized parameter qγadh /E (Decuzzi and Ferrari 2010). For h < h cr , the free energy reduction associated with the increase in the size of the adhesion spot would be larger, in modulus, than the free energy increase associated with the stretching of the cell membrane and the recruitment of new cell receptors at the adhesion site. Conversely, for h > h cr the stretching and ‘recruitment’ energies would outgrow the contribution of γadh , thus impairing stable cell adhesion and proliferation. Similar conclusions have been drawn in the more general context of adhesion on randomly rough substrates (Decuzzi and Ferrari 2010), where γ/ν and hcr have been shown to depend also on the fractal dimension D f , in addition to the surface roughness and the material parameter qγadh /E. In particular, γ/ν and h cr have been predicted to grow whit D f in that for a given surface roughness, the surface area available for adhesion would grow with the fractal dimension D f . For a given roughness interval (moderately rough substrate), the effective energy γ would steadily grow with the fractal dimension D f .

7.2 Cell Adhesion on Porous Silicon Surfaces Similar results have been obtained on porous silicon surfaces. In cultivating different cell lines (primary human endothelial, mouse mesenchymal normal, mouse neuroblastoma and human cortical neuron cell line) on porous surface with varying pore size, Gentile and colleagues (Gentile et al. 2012c) found that cell adhesion is maximized on porous silicon substrates with small average pore size (~5 nm) and large fractal dimension (~2.8) (Fig. 8a–c). It is speculated that the higher adhesion on similar mesoporous MeP1 substrates could be attributed to a preferential matching of the substrate topography with the recently observed multiscale molecular architecture of focal adhesions. In a recent evolution of the research, we have studied the combined effect of surface nano-topography and delivery of therapeutics on the adhesion of tumour cells on porous silicon substrates (De Vitis et al. 2016). We verified the adhesion of MCF-7 breast cancer cells on mesoporous silicon substrates (Fig. 9a) with and without the administration of the DMSO antitumor drug (Fig. 9b). We found that large pore sizes vehicle elevated drug dosages, while large fractal dimensions enhance cell adhesion (Fig. 9c–d). Substrates with a small fractal dimension and large pore size hamper tumour cell growth. Thus, a controlled balance between nano-topography and drug delivery may modulate cell adhesion on porous silicon surfaces.

7.3 Controlling Cell Adhesion and Proliferation on Fractal Surfaces In Gentile et al. (2013), a set of randomly rough, self-affine fractal silicon substrates for cell culture was produced using KOH etching (Fig. 10a). The surface roughness

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Ra (Fig. 10b) and fractal dimension D f (Fig. 10c) were controlled independently by modulating the etchant concentration and processing time. The adhesion, proliferation and morphology of 3T3 fibroblasts were assessed on these different substrates. Adhesion and proliferation rates are selectively enhanced on specific substrates. Proliferation rates are maximized on substrates with moderate roughness (Ra  40nm) and sufficiently large fractal dimension (D f  2.4) (Fig. 10d). Differently, from proliferation, adhesion is optimized on substrates with high roughness (Ra  50nm) and low fractal dimension (D f  2.2) (Fig. 10e–f). Although the regulating mechanisms remains unclear, cell adhesion and proliferation can be selectively singled out with this set of randomly nanorough silicon substrates.

8 Measuring CAMs on Nano-patterned Surfaces Recently, techniques have been developed to measure the biochemical activity of cells at the interface with a surface during adhesion.

Fig. 8 a Number of adhering mouse 3T3 fibroblasts and b HUVEC cells on mesoporous silicon substrate with varying pore size at different time frames. Cells adhere more firmly to MeP1 substrate with an intermediate 5–15 nm pore size; c fluorescence confocal images of cells permit to reveal cell morphology on different substrates. Partially reproduced from (Gentile et al. 2012c)

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Fig. 9 a Porous silicon substrates can be loaded with anti-tumour drugs. Balance between surface nano-topography and the drug release dynamics b enables the modulation of viability c and adhesion d of cells on substrates with different pore sizes. Reproduced from (De Vitis et al. 2016)

In De Vitis et al. (2015), researchers fabricated periodic arrays of silicon micropillars covered with gold nanoparticles. Then, they used these devices as culturing substrates for MCF-7 breast cancer cells. Multiscale nature of the device enables cell manipulation at the microscales and SERS analysis of cells at the nanoscales. Using SEM microscopy, they observed that cells modify their morphology to adapt to the external microenvironment, producing invadopodia. Using Raman spectroscopy and SERS spectroscopy, they demonstrated that cell phenotype is influenced by the substrate preparation. Heterogeneous microstructured surfaces, differently from planar smooth surfaces, induce morphological mutations in cells. In Coluccio et al. (2016) the group led by Francesco Gentile fabricated mesoporous silicon substrates and incorporated gold nanoparticles clusters within the pores. The device combines the capability of porous surfaces to support cell adhesion with the ability of gold nanoparticles to enhance the EM radiation in a SERS effect. The device can, therefore, interrogate cell response during adhesion. Analysis of MFC7 breast cancers demonstrated that the method can uncover the expression of adhesion molecules on rough substrates.

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Fig. 10 a Rough surfaces were generated using wet chemical etching; b the roughness; c and the fractal dimension of the surfaces were independently controlled. d Proliferation rates are maximized on substrates with moderate roughness and sufficiently large fractal dimension; e–f adhesion is optimized on substrates with high roughness and low fractal dimension. Partially reproduced from (Gentile et al. 2013)

9 Cell Networking on Nano-patterned Surfaces 9.1 The Topology of Neuronal Networks on Nano-patterned Surfaces Nano-patterned surfaces were also used to guide the spontaneous self-assembly of the neuronal cell into complex structures with some level of order. In Marinaro et al. (2015), Gentile and colleagues produced silicon porous substrates with a pore size in the low 5–20 nm range. Then, the team grew neuroblastoma N2A brain cancer

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Fig. 11 a–b N2A Neuroblastoma cells cultured on porous silicon form few aggregates with many cells per aggregate compared to the same cells cultured on flat silicon; c the clustering coefficient and the characteristic path length of neuronal cell networks are reported one as a function of the other in a scatter plot: scatter plot representation of neuronal cell networks topology reveal that cells cultured on nanostructured surfaces have high clustering coefficients and small characteristic path lengths; d similar values of Cc and cpl are peculiar of small world networks. Reproduced from (Marinaro et al. 2015)

cells on smooth silicon and silicon etched with nanoscale pores. Neuroblastoma cells display many of the same properties as ordinary neurons but are easier to grow in culture. The cells grew much more quickly on the etched silicon than on the smooth surface. Furthermore, the porous, etched silicon induced the cells to form a more clustered network with a small-world topology in contrast to flat silicon (Fig. 11a–b). In the scatter plots in Fig. 11c, the clustering coefficient and the characteristic path length of cell networks are reported in an individual diagram at diverse time frames to highlight differences between substrates. In the Cc − C pl plane, cells on porous substrates with high fractal dimension are confined in the upper left region of the diagram, while cells on flat silicon with low fractal dimension are clustered in the

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lower right side of the diagram. The diagrams in Fig. 11c indicate that N2A cells on a nanoscale surface have an increased ability to create patterns in which the nodes of the patterns form highly clustered groups and the elements of the groups are connected by a finite, and generally low, number of steps, in contrast to a nominally flat surface. Diagrams indicate that it is possible to categorize classes of networks on the basis of the clustering coefficient and characteristic path length. While regular ordered networks have elevated clustering coefficients and characteristic path lengths, and random graphs possess low clustering and short paths, small world networks lie between the extremes of order and randomness (Crutchfield 2012; Strogatz 2001). Small world networks exhibit short paths and high clustering (Fig. 11d), similar networks are named small world in analogy with the concept of small world phenomenon developed in social psychology (Latora and Marchiori 2001) (a more rigorous definition of small-world-ness is provided above). Dynamical systems with short paths and high clustering may feature enhanced signal propagation speed and computational capabilities compared to regular grids of the same size.

9.2 Nano-scale Cues Influence Information Flow in Planar Neuronal Networks Research reported by Marinaro et al. (2015) analysed the topology of neuronal networks on etched substrates but did not examine how information fluxes within those networks. In Onesto et al. (2017), we studied the organization and signalling of primary neural cells on planar rough silicon surfaces. Using conventional wet etching procedures, we produced surfaces with a controlled roughness in the 0–33 nm interval. We observed that cultured neural networks exhibit topological properties that depend on the nano-topography of the substrate. Large roughness values trigger cell assembly into small world networks. Using functional calcium imaging techniques, computer simulation and mathematical modelling, we demonstrated that small world networks on rough substrates vehicle information ~4 folds more efficiently compared to random networks on flat surfaces. Computer simulation and mathematical modelling have been described in the previous sections of the chapter. Functional calcium imaging techniques allow to determine spikes of spontaneously active neurons as somatic Ca2+ transients as explained in Onesto et al. (2017). Figure 12a–b reports confocal images and associated neural activity for neurons over smooth (a) and moderately corrugated substrates with Ra ∼ 22 nm (b). Spikes were registered throughout a time interval of 40 s. Closely spaced spikes in small world networks over corrugated surfaces suggest that neural small world networks are topologically biased to enhance local connectivity. We found that the ratio of the density of spikes measured on rough surfaces to that measured on flat ones ~3 is consistent with values predicted by simulations. Data imply that excitatory neurons are specifically wired to ensure enhanced neural activity in small world networks over corrugated surfaces.

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Fig. 12 a Functional multi calcium imaging (fMCI) techniques indicate that neuronal cell networks cultured on rough surfaces are topologically biased to transmit information more efficiently compared to flat silicon substrates; b experimental results match with the predictions of computer simulations. Partially reproduced from (Onesto et al. 2017)

9.3 3D Neuronal Networking Reported studies investigated neuronal networks formation on bi-dimensional, planar surfaces. Nevertheless, the generation of 3D networks of primary neurons is a big challenge in neuroscience. In Limongi et al. (2013), researchers presented a method for a 3D neuronal culture on super-hydrophobic substrates. Scanning electron microscopy and confocal imaging show that soon after plating neurites adhere to the nano-patterned pillar sidewalls and they are subsequently pulled between pillars in a suspended position. These neurons display an enhanced survival rate compared to standard cultures and develop mature networks with physiological excitability. In the device, the microscale of pillars is responsible for the super-hydrophobicity, while the nanoscale at the sidewalls of the pillars introduces the third topological dimension. The combination of these two characteristic length scales is fundamental for the docking of neuronal processes on pillars sidewalls and for the ‘suspended’ 3D assembly of the neuronal network. The 3D character of the culture virtually disappeared in the presence of smooth pillar sidewalls, with neurons growing in a 2D fashion on the bed of the device. Results of the research underlined the importance of using nanostructured super-hydrophobic surfaces for directing 3D neuronal growth and designing biomaterials for neuronal regeneration.

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In Accardo et al. (2017), researchers realized 3D architectures for the study of neuronal cell growth and proliferation. They fabricated 3D freestanding scaffolds by multiphoton direct laser writing and seeded them with N2A cells. Then, characterized cell growth within the biomimetic scaffolds using advanced 3D fluorescence imaging approaches. The high accuracy of the fabrication process allows a much finer control of the micro- and nanoscale features compared to other 3D printing technologies. Thus, cells reached elevated penetration depths and formed complex three-dimensional networks that were examined using scanning electron microscopy, light sheet fluorescence microscopy and multiphoton confocal imaging. Results indicate an optimal cell colonization both around and within the 3D scaffold as well as the formation of long neuritic extensions.

10 Possible Physical Reasons for Cell Clustering: Minimization of Energy Density Results presented in the previous sections reinforce the view that cells on a substrate tend to form highly clustered groups of elements. This configuration is termed small world topology and neuronal cells in a small world network transmit information efficiently. Here, we speculate that the main driver of cell organization on a substrate is a criterion of energy optimization. Then, we hypothesize that the nano-scale architecture of surfaces helps cells to overcome frictional adhesion forces to collapse into clusters.

10.1 Free Energy Landscape of Small World Neuronal Networks Figure 13 describes the density of energy change u as a function of small-worldness associated to a system of 200 cells on a planar surface (Onesto et al. 2017). The energy density was derived by summing the potential energy of interaction between cells over all the possible cell pairs in a network: 

 2 k δ /2 i  j s i j (21) U n where ks δi2j /2 is the harmonic potential associated to each of the cell pairs in the ensemble and n is the total number of cells. Notice that the number of connections is lower or equal to n(n − 1)/2. ks is the effective spring constant of the structural linkages between cells, δi j is their separation. The potential describes the chemical energy of interaction between cells due to specific (cell adhesion molecules, CAM, mediated adhesion) and not specific (electrostatics, electrodynamics, van der Waals)

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Fig. 13 Free energy landscape of small world networks. Reproduced from (Onesto et al. 2017)

adhesion forces (Bell 1978; Evans and Calderwood 2007; Sackmann and Smith 2014). We generated networks with a small-world-ness ranging from SW ∼ 0.7 (random graphs) to SW ∼ 2.9 (small world graphs) and calculated for each the associated energy density U : the energy landscape in the diagram associates each conformation of the system to its energy levels. The total free energy of the system decreases as 0.78 + 0.189 SW2 − 0.147 SW2 —thus the higher the SW the higher the energy decrease associated to a conformational change of the system. Since physical systems evolve to maintain their free energy to a minimum, Fig. 13 indicates that cells would tend to assemble into clustered groups with low energy levels. Any system of a sufficiently large number of elements would spontaneously form aggregates if the potential energy of interaction between elements obeys to relation (21). Thus, arguments based on energy optimization would explain why we observe small world networks on rough nano-patterned surfaces. The reason why small world attributes do not emerge on smooth surfaces is presented below.

10.2 Out of Equilibrium Self-assembly The time evolution of neural cell density u in a mono-dimensional domain (Fig. 14a) is described by the partial differential equation (Armstrong et al. 2006) ∂u ∂u K (u) ∂2u −  + ϒη(x)α2 (t)(u) 2 ∂t ∂x ∂x

(22)

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Fig. 14 a One can use physical modelling to obtain the b steady-state cell density distribution on rough surfaces; c steady-state values of cell density are given as a function of the substrate instability force to the cell-cell adhesion force (ρ). Reproduced from (Onesto et al. 2017)

where x and t are the space and time coordinates, u K (u) is the cell-cell adhesive force proportional to the parameter ξ. The term ϒη(x)α2 (t)(u) represents the perturbation force that is related to surface roughness. In the term , it is lumped the dependence on the position on the substrate and cell density, α2 describes how rapidly the cell-substrate force component decays with time, η reflects the random nature of the substrate, ϒ is the intensity of the substrate instability force (Onesto et al. 2017). Thus   ϒ/ξ is the relative intensity of the substrate perturbation force to the cellcell adhesion force. Steady state solution of cell density is reported in Fig. 14b for  ranging from 0 to 6. We observe that while for small values of  ( < 2) cells density is uniform, when  > 2 cells cluster together to form isolated peaks. Asymptotic cell behavior as a function of  is reported in Fig. 14c. Value  in the ordinates is the average ratio of maximum (peaks) to minimum (valleys) values of cell density at the regime, thus large ’s are suggestive of cells clustering. We can distinguish between the two regions in the diagram. When cell-cell adhesion forces dominate over substrate perturbation forces ( < 2) we have isolated cells in the domain, when substrate perturbation forces dominate over cell-cell adhesion forces ( > 2) cells create clusters. Similarly to what happens for undercooled liquids (Debenedetti and Stillinger 2001), cells may maintain a state of unstable equilibrium and remain uniformly distributed on a surface contravening a principle of energy minimization. Surface roughness, lumped here in the sole ϒ parameter, breaks equilibrium and leads to an assemblage of cells into isolated groups. Thus, surfaces with a nano-scale architecture induce cells to form into small world networks that (i) minimize energy and (ii) maximize information density:

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rough surfaces → SW neural networks

(23)

low energy density ↔ high information flows.

(24)

11 The Equivalence Principle in the Self-assembly of Cells Described results indicate that, depending on surface topography, cells on a substrate can adhere more firmly and form small world clusters. If cells are neurons, cell clusters with a small world topology elaborate information more efficiently than the same cells in random, periodic or ordered graphs. This thesis is condensed in Eqs. (23) and (24). Let’s assume now that the main drivers for neuronal cells organization in the plane are the following: (i) energy optimization, whereby (neuronal) cells create networks which minimize energy consumption; (ii) information propagation, and thus cells on a substrate form networks through which the information propagation is maximized; (iii) neural morphogenesis, the positions of the cells on the substrate is dictated by the morphology of neurons and synaptic connections on that substrate. Thus, we may have 3 different criteria which regulate cells networking, and namely the energy criterion, the information criterion, the biology (or evolution) criterion. Equations (23) and (24) demonstrate that the above criteria are not independent, and that cell fate is dictated by a (perhaps non-linear) balance of energy, information, biology. This equivalence principle represents a new tool of analysis in tissue engineering, tissue regeneration, biomedicine, in the study of neurodegenerative disorders. In establishing the correspondence between energy, biology and information, we suggest that these criteria can be used interchangeably. That is to say that the same problem can be addressed from different perspectives. Thus, making experiments or studies in one of those domains, one would obtain the information necessary to design strategies in the other. Similarly, in concept to the Fourier transform that, transforming a signal from the time to the frequency domain, allows to solve a problem in the more convenient conditions and then to transfer the solution to the original domain. This new concept may promote the development of scaffolds with exceptional efficiency. Researchers in the area of cell biology such as tissue engineering, tissue repair and regeneration, neuroscience, bio-computing, may be inspired to implement similar equivalence principle in their studies and reap the rewards.

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Polymeric Nanoparticulates as Efficient Anticancer Drugs Delivery Systems Shima Asfia, Mahsa Mohammadian and Hasan Kouchakzadeh

1 Introduction Over recent decades, improving human health is undergoing an eruption of consideration led by the use of nanoparticles (NPs) as platforms for delivering drugs to cells. These NPs can be engineered to accumulate specifically at diseased cells, which allows direct delivering of drugs to target tissue. Such a delivering approach helps to improve curing, increasing treatment efficacy, reducing side effects, facilitating co-delivery of two or more drugs or therapeutic modality for combination therapy, in order to visualize the sites of action by combining therapeutic agents with imaging modalities, which all of them recuperating human health (Jahangirian et al. 2017; Ferrari 2005). In addition, therapeutic NPs admit the potency to overcome biological barriers and effectively transport hydrophobic, poorly water-soluble drugs, and biologics (Kouchakzadeh et al. 2015). Cellular targeting is an important strategy to improve therapeutic efficacy besides reduction of drug toxicity in healthy tissues. Therefore, various ranges of drug delivery vehicles have been expanded to target chemotherapeutic drugs to solid tumours while struggling with its complexity of dose-limiting toxicity is still controversial (McDaniel et al. 2013). Since the first application of nanocarriers as drug delivery systems (DDSs) in the 1980s, wide researches have been done to introduce them as a promising platform for multiple pharmaceutical applications (Kazunori et al. 1993; Movassaghian et al. 2015). The first US Food and Drug Administration (FDA) approval of DDSs was Liposomal amphotericin B, in 1990. Nowadays, more than ten DDSs are commercially available with different applications such as cancer treatment, muscular degeneration, and treatment of fungal infections (Zhang et al. 2013). S. Asfia and M. Mohammadian—Equal contributors. S. Asfia · M. Mohammadian · H. Kouchakzadeh (B) Protein Research Center, Shahid Beheshti University, G.C., Velenjak, Tehran, Iran e-mail: [email protected] © Springer Nature Switzerland AG 2019 M. Rahmandoust and M. R. Ayatollahi (eds.), Nanomaterials for Advanced Biological Applications, Advanced Structured Materials 104, https://doi.org/10.1007/978-3-030-10834-2_3

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This kind of therapy utilizing nanocarriers offers a set of advantages including the nano-size range with a narrow size distribution that enables them to be injected intravenously without the risk of blood vessel blockage; besides increased longterm circulation time in the bloodstream due to their micelle-like shape and high static and dynamic structural stability which is essential for their in vivo applications (Movassaghian et al. 2015). Nanometer size of the carrier influences its extravasation to a tumour and diffusion in the interstitium. Faster accumulating and dipper diffusion in a tumour can be accrued by the smaller carriers than the larger ones. Generally, the size of the particle effects on the circulation kinetics of the particles, their functions, and cell targeting capabilities (Bertrand et al. 2014). In order to have an efficient delivery of therapeutics into the intracellular sites of action, a smart design and construction of the nanocarrier is needed for the effective cellular uptake (Farokhzad and Langer 2009). In this regard, nanocarriers can target the diseased tissue actively or passively. Active targeting, also called ligand-mediated targeting, involves utilizing affinity ligands which are conjugated to the surface of NPs that facilitate their specific retention and uptake by targeting molecules or receptors overexpressed in diseased organs, tissues, cells or subcellular domains. In this approach, therapeutics are released specifically in their site of action from the NPs which are accumulated in tumours through the sensitivity to temperature or pH or through the ligand-receptor attachment mechanism. On the other hand, passive targeting is known for preferential distribution to the tumours through the presence of fenestrations in the imperfect tumour blood vessels and to the poor lymphatic drainage in the tissue. These two phenomena combined together and have coined as the enhanced permeation and retention (EPR) effect. This targeting mechanism that utilizes the properties of the tumours and NPs, has become the desirable issue of many scientists for the efficient delivery of anticancer drugs to tumours without using any kind of ligands (Bertrand et al. 2014). The rapid development of nanoparticle-based DDSs introduces various types of materials such as polymers, lipids, and polysaccharides that can be used for the construction of carriers (Bhatia 2016). Nowadays, natural- and synthetic-based polymeric NPs are the most commonly used drug delivery vectors (Irache and Espuelas 2006). Herein, recent advances in the field of polymeric nanoparticulate drug delivery systems are presented. In addition, their targeting strategies to improve the therapeutic efficacy for the cancer treatment are discussed. Finally, the current polymeric NPs marketed and under clinical trials are summarized in order to exhibit the bright perspective of this novel technology.

2 Polymeric Nanoparticulate Systems as Platforms for Drug and Gene Delivery Besides common carriers recognized as drug delivery systems such as liposomes, dendrimers, carbon nanotubes, and metal-based NPs, the focus of many researchers who are working on the development of several innovative delivery systems is

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on polymer conjugates and polymeric NPs. These polymeric particles are mostly biodegradable, biocompatible and provide a proper condition for controlled drug delivery and improved release kinetics (Kouchakzadeh and Shojaosadati 2016b; Ajazuddin 2010; Bonifácio et al. 2014). Active pharmaceutical ingredients (API) can be incorporated chemically or physically to polymers or polymeric matrix using different techniques. From the drug release point of view, polymer-based devices can be categorized as diffusion-controlled (monolithic devices), solvent-activated (swelling- or osmotically-controlled devices), chemically controlled (biodegradable), or externally-triggered systems (e.g., pH, temperature) (Liechty et al. 2010). Currently, natural and synthetic polymers can be employed as effective vectors for drug and gene delivery. Protein NPs are one of the widely used natural-based polymeric carriers and most of them are derived from gelatine, albumin, and gliadin. Recently, it was reported that soy and milk proteins can effectively be used for the formulation of NPs (Lohcharoenkal et al. 2014). The other natural carriers such as amphiphilic particles can be classified into missiles and liposomal vectors, and also polysaccharides have known as other opportunities for targeting applications. Chitosan (cationic) and low methoxy pectins (anionic) are two well-known examples of polysaccharides that have been considered as promising delivery systems. Polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), Polyethyleneimine (PEI) and dendrimers are the examples of synthetic-based polymers for the decoration of nanoparticulate delivery system (Kouchakzadeh and Shojaosadati 2016b; Amreddy et al. 2017; Kim et al. 2014). Natural-based polymeric NPs have been more attracted due to their biocompatibility, non-toxicity, and non-immunogenicity. Furthermore, these polymers can be produced by several living organisms and are easy to access (Labhasetwar 2005). In addition, the surface of polymeric NPs can be functionalized in order to conjugate to biomolecules that plays an important role in therapeutic targeting into cancerous cells (Kim et al. 2015; Jahanshahi 2004).

2.1 Natural-Based Polymers as Building Blocks of NPs Natural molecules such as proteins, lipids, and polysaccharides demonstrate unique functionalities and have potential applications specifically in biomedical sciences. Because of their amphiphilicity property which enables them to interact and conjugate appropriately with both drug and solvent under the preparation procedure, they have been considered as ideal materials for the formulation of nanoparticulate delivery systems (Kouchakzadeh et al. 2015; Jahanshahi 2004; Marty et al. 1978). Among the natural-based polymeric carriers, protein NPs are known for being biodegradable, metabolisable, and easily amenable for surface modifications in order to allow attachment of drugs and targeting ligands. In addition, these nanocarriers have been successfully synthesized from various sources including water-soluble proteins (such as bovine serum albumin (BSA) and human serum albumin (HSA)) and insoluble proteins (such as zein and gliadin) (Weber et al. 2000; Ezpeleta et al. 1996). The most

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widely used natural-based polymeric NPs that have been employed for targeting and delivering purposes are discussed in the following. • Albumin Albumin can be obtained from a variety of sources, including bovine serum, human serum and egg white (ovalbumin). Albumin is the major soluble protein in the bloodstream (35–50 g/L in human serum) and plays an important role in the maintenance of osmotic pressure, and binding to and transportation of nutrients to the cells. High water solubility of albumin (up to 40% w/v) at pH 7.4 makes it a noteworthy macromolecular carrier capable of conjugating with a wide variety of drugs. This protein can be stable at pH range of 4–9 and resist to heat at 60 °C up to 10 h without any destructive effects (Peters 1985; Larsen et al. 2016). Albumin is commonly used in the preparation of nanospheres and nanocapsules (Kratz 2008). Albumin nanocarriers contain reactive functional groups (thiol, amino, and carboxyl) on their surface that makes it possible ligand binding or other surface modifications. Therapeutics can be released from the polymeric matrix of albumin NPs after protease degradation under a controlled and sustained release profile (Kouchakzadeh et al. 2015; Kratz et al. 1997). In order to surface modification of NPs, chemical coupling of polyethylene glycol (PEG) to BSA NPs was performed by SPA activated mPEG through the superficial free amino groups. PEGylation of proteins or particles can prolong their circulation half-life, reduce their immunogenicity, and helps to promote their accumulation in tumours through the EPR effect. Reduction of the negative surface charge to −31.7 mV obtained after PEGylation of BSA NPs. Drug release from the PEGylated NPs is slower than the bare ones, probably due to the presence of a PEG layer around the modified particles (Kouchakzadeh et al. 2010). The poorly water-soluble drug, Tamoxifen (Tmx), loaded successfully under an optimized condition in the amphipathic matrix of HSA NPs by a modified desolvation method. In order to enhance the long-term circulation and additional control over the drug release, Tmx loaded albumin NPs were PEGylated (Kouchakzadeh et al. 2014). In addition, an optimized BSA nanoparticulate delivery system dual loaded with anticancer drug 5-fluorouracil (5-FU) and magnetic NPs was prepared, characterized and its anticancer efficiency evaluated. These protein-based NPs had shown rapid cancer diagnostics and can be considered as theranostic tools for efficient cancer treatment (Kouchakzadeh and Shojaosadati 2016a). Gopinath et al. encapsulated niclosamide (Nic) into albumin NPs and showed its high therapeutic efficacy against human lung and breast cancer cell lines. Enhanced solubilisation and stability of Nic in an aqueous environment were reported by preparing a protein-drug nanoformulation via desolvation technique. The average hydrodynamic size for BSA-Nic NPs was found to be 199.9 nm plays a crucial role in drug delivery, as the NPs up to 400 nm get preferentially accrue in the tumour microenvironment via EPR effect. In addition, they reported the successful induction of apoptosis in BSA-Nic NPs treated cancerous cells (Gopinath et al. 2015).

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A green method for blood–brain barrier (BBB)-penetration of albumin NPs developed with the capacity for simultaneous encapsulation of different drugs without needing any crosslinkers. The prepared albumin NPs have the ability to penetrate the BBB and target glioma cells via the mechanisms of protein acidic and rich in cysteine (SPARC) and glycoprotein 60 (gp60) mediated biomimetic transport, two kinds of albumin-binding proteins that overexpress in many tumours. Results demonstrated that, this strategy was potent in arresting tumour growth in both subcutaneous and orthotropic glioma models through multiple mechanisms, such as antiangiogenesis, apoptosis, and tumour immune microenvironment regulation (Lin et al. 2016). Availability of a single free thiol at Cys34 offers the potential for site-selective covalent drug attachment. Recent improvements indicate that recombinant albumin has the ability to make chemically conjugations with nearly all type of drugs (Larsen et al. 2016; Caspersen et al. 2017). In a study by Kushwah et al. a novel conjugate of gemcitabine (GEM)-BSA prepared and thereof these NPs (GEM-BSA NPs) showed a potential therapeutic efficacy by altering physicochemical properties, improving cellular uptake and stability of GEM. In this case, GEM-BSA conjugate was transformed into NPs via high-pressure homogenization technique with a particle size of 147.2 ± 7.3, PDI of 0.16 ± 0.06 and zeta potential of −19.2 ± 1.4 that were morphologically analysed by scanning electron microscopy (SEM) and Atomic force microscopy (AFM) (Kushwah et al. 2017). Generally, albumin NPs have been employed as an appropriate carrier for delivery of drugs such as GEM (Dubey et al. 2015), teniposide (VM-26) (He et al. 2015), lapatinib (LPT) (Wan et al. 2015), scutellarin (STA) (Wei et al. 2014), paclitaxel (PTX) (Li et al. 2014), tamoxifen (TMX) (Kouchakzadeh et al. 2014), doxorubicin (DOX) (Dreis et al. 2007), tacrolimus (FK506) (Gao et al. 2012), 5-FU (Wilson et al. 2012), curcumin (CUR) (Kim et al. 2011), 10-hydroxycamptotecin (HCPT) (Yang et al. 2007), and methotrexate (MTX) (Taheri et al. 2012a). • Gelatine Gelatine is one of the most widely used animal proteins that can be obtained from controlled hydrolysis of collagen which is a significant component of the skin, bones, and connective tissues (Coester et al. 2006). Gelatin has two different types, A and B, that can be produced by both acid or base hydrolysis, that leads to creating proteins with different features including isoelectric point, molecular weight, amino acid composition, and viscosity. Since this protein is inexpensive, can be sterilized and is non-pyrogenic, and also possesses low antigenicity, have got attention of researchers for delivery goals (Kouchakzadeh et al. 2015; Schwick and Heide 1969). Gelatine contains functional groups such as carboxyl, amino, phenol, guanidine, and imidazole, which are potential sites for bonding or chemical conjugations (Kouchakzadeh et al. 2015). Gelatine NPs can be prepared with predictable size, it ranges between 168 and 460 nm by two desolvation method that can be used as drug delivery vehicles (Babaei et al. 2008). Another application of gelatine micro- and NPs is to hold all the needed properties that pose successful topical ophthalmic delivery that can enrich the different delivery formats. These biodegradable polymers can be used as carrier

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systems and hold significant promise for ocular drug delivery since they are suitable for several drugs and would allow drop-wise administration while maintaining the drug activity at the site of action (Hathout and Omran 2015). Furthermore, in other studies to improve the performance of gelatine-based castedfilms for controlled delivery of hydrophobic drugs, Piperine (a model drug) was loaded into gelatine (type A) casted-films by a solvent evaporation method. Then, glutaraldehyde was used as a cross-linker for the gelatine films to modulate the drug release behaviour and to prolong the rate of degradation of the film. Gelatine castedfilm based drug delivery systems can be effectively used for a wide range of release requirements i.e. from fast release to delayed release (Laha et al. 2015). • Liposome Lipid-based delivery systems have been developed as an alternative to traditional treatments, opened a new pathway for targeting due to their excellent tolerability. Liposomes, the first lipid-based nanocarriers introduced in 1965, are composed of one or more phospholipid layers separated by an aqueous internal compartment that confer liposomes amphiphilic features and allows encapsulation of water-soluble therapeutics whereas the phospholipid membrane can solubilize lipophilic molecules. Liposomes are able to improve drug performance by extending the plasma half-life. Despite these properties of liposomes, they may suffer from slow and incomplete drug release in some cases. Recently, in order to solve this problem, thermally sensitive liposomes are well developed and more applied (Gainzaa et al. 2015; McDaniel et al. 2013). Lipoproteins (high-density lipoprotein; HDL and low-density lipoprotein; LDL) were isolated from human plasma, characterized and tethered to G5.0 PPI dendrimers to construct LDL- and HDL-conjugated dendrimeric nanoconstructs for target and delivery of docetaxel (DCX) to cancer cells. Developed formulations showed a sustained release in both in vitro and in vivo studies. Thus, these precisely synthesized dendrimeric nanoconstructs could serve as a promising drug carrier for fighting with the fatal disease like cancer and increase the rate of survival (Jain et al. 2013). A new lipid-based drug delivery system was developed by using a recently approved second-generation type 5 phosphodiesterase inhibitor used for erectile dysfunction. Avanafil (AVA) was formulated as self-nanoemulsifying drug delivery system (SNEDDS) utilizing various oils, surfactants, and cosurfactants. This development increased half-life by about 45% and its bioavailability by more than 1.4-fold in comparison with the pure drug (Fahmy et al. 2015). In addition, Shen et al., utilized cationic lipid-assisted polymeric NPs for systemic delivery of siRNA targeting GATA2, a specific transcription factor that plays a critical role in KRAS mutant in order to treat non-small-cell lung carcinoma (NSCLC) patients (Shen et al. 2014). A liposome-based co-delivery system composed of a fusogenic liposome encapsulating ATP-responsive elements with chemotherapeutics developed by Mo et al. for ATP-mediated drug release. Actually, this liposome contained a protein-DNA complex core including ATP-responsive DNA scaffold with DOX that could release

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DOX through a conformational change from the duplex to the aptamer/ATP complex in the presence of ATP. Directly delivering extrinsic liposomal ATP promoted a pHsensitive drug release and anticancer efficacy was enhanced both in vitro and in vivo (Mo et al. 2014). Moreover, scientists designed a dual-mediated (receptor-mediated and adsorption-mediated) liposome, named transferrin–cell penetrating peptide–sterically stabilized liposome (TF-CPP-SSL), in order to improve therapy of glioma cells. This method operates by combining molecular recognition of transferrin receptors (TF-Rs) on the BBB and gliomas with the highest targeting efficacy for brain microvascular endothelial cell and C6 cell uptake but avoided capturing by normal cells. TF-CPP-SSL offered advantages for crossing the BBB and entering into glioma C6 cells. Thus, TF-CPP-SSL was entrapped in endosomes of glioma C6 cells and then escaped from lysosomes successfully to release the liposomal contents into the cytosol, so the entrapped DOX could enter the nucleus to exert pharmacological effects (Liu et al. 2017). Recently, liposomes were prepared and modified with folic acid on their surface for targeted delivery of 5-FU to cancer cells. Folate-targeted liposomes showed better tumour inhibition than free drug and no tissue abnormalities were found in histological examination. Therefore, these targeted particles with average size of 114.00 ± 4.58 nm provide an effective and safe strategy for colon cancer targeted chemotherapy (Moghimipour et al. 2018a). Generally, the most loaded drugs in liposomal delivery systems are Amphotericin B (Hann and Prentice 2001), DOX (Krauze et al. 2007), Daunorubicin (Feldman et al. 2011), Verteporphin (Bressler et al. 2001), Morphine sulphate (Gambling et al. 2005), Cytosine Arabinoside (Kobayashi et al. 1975), Propofol (Patrick et al. 1985), Estrogen (Heger et al. 2014), Vincristine (Johnston et al. 2006), Cisplatin (Cis) (Feng et al. 2016), Topotecan (Jain and Jain 2016), Vinorelbine (Wang et al. 2016a, b), Annamycin (Booser et al. 2002), Cationic liposomal PTX (Patel et al. 2011), Bupivacaine (Richard et al. 2012) and siRNA (Jayaraman et al. 2012). Liposomes have been used for applications as diverse as imaging tumours and sites of infection, for vaccine and gene medicine delivery, for treatment of infections and for cancer treatment, for lung disease and for skin conditions. One of the most important feature of liposomal delivery systems is to be useful for their ability to realizing drugs “passively” at sites of increased vasculature permeability, when their average diameter is in the ultra-filterable range (less than 200 nm in diameter), and for their ability to reduce the side effects of the encapsulated drugs relative to free drugs (Allen and Cullis 2013). • Chitosan Chitin (β-(1-4)-poly-N-acetyl-D-glucosamine) is the second most abundant polysaccharide after cellulose in polysaccharide groups. Chitin is available as the major structural component in the exoskeleton of crab and shrimp shells and the cell wall of fungi and yeast. As chitin is not readily dissolved in common solvents, it is often converted to its more deacetylated derivative, chitosan (CS), they both known for their poor solubility. CS is the only polysaccharide that possesses a high density of

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positive charges due to the protonation of amino groups on its backbone. In addition, non-toxicity, biocompatibility, and biodegradability of CS introduced it as a potential carrier for drug delivery applications (Luo and Wang 2014; Azuma et al. 2015). Wang et al. prepared boronic acid-rich CS-poly(N-3-acrylamidophenylboronic acid) NPs (CS-PAPBA NPs) by polymerizing N-3-acrylamidophenylboronic acid in the presence of CS in an aqueous solution. The cellular uptake, tumour penetration, and antitumour activity were evaluated by using three-dimensional (3D) multicellular spheroids (MCs) as the in vitro model and H22 tumour-bearing mice as the in vivo model. The in vitro examinations had shown efficient cell killing through iRGDconjugated NPs significantly improved the efficiency of DOX penetration in MCs, compared with free DOX and non-conjugated NPs. On the other hand, In vivo tests indicated that iRGD-conjugated CS-PAPBA NPs promoted the accumulation of NPs in tumour tissue and enhanced their penetration in tumour areas, thus the efficiency of DOX-loaded NPs has been improved and led to restraining tumour growth and prolonging the lifetime of H22 tumour-bearing mice (Wang et al. 2013). In another study, researchers developed a freeze-dried formulation of CS nanocapsules containing DCX for the evaluation of its efficacy in the NCI-H460 cancer cell line. The nanocapsules maintained the anti-proliferative effect of the drug that it was not affected by the freeze-drying process and exhibited high encapsulation efficiencies of DCX (78%). In this experiment, the ability of DCX intracellularly delivering of CS nanocapsules were tested and the results showed that CS nanocapsules still had the antiproliferative effect of the drug so it was not affected by the freeze-drying process. Also, it was found that this cytostatic effect of the DCX was linked to its intracellular delivery in the cancer cells (Lozano et al. 2013). Maya et al. evaluated in vitro the cytotoxicity effects and targeting ability of CS cross-linked γ-poly (glutamic acid) (γ-PGA) NPs loaded with an anti-cancer drug, DCX and decorated with Cetuximab (CET), on EGFR over-expressing NSCLC cells. The cytotoxicity of NPs quantified by using cell-based assays (MTT/LDH) and flow cytometry demonstrated excellent anti-proliferative activity of CET-DTXL-γ-PGA NPs over DTXL-γPGA NPs and which is promising for targeted therapy of NSCLC (Maya et al. 2014). Soares et al. found that DOX encapsulation efficiency is about 70% and 50% in CS and O-HTCC (ammonium-quaternary derivative of CS) NPs, respectively. In the second system, DOX release is under influence of pH and so, in this drug delivery system they could reduce DOX side effects and improve its effectiveness in treating several tumours, so they led to a higher amount of DOX released than CS NPs (Soares et al. 2016). SELEX method employed for the isolation of DNA aptamer (Ap) that could specifically bind to a human epidermal growth factor receptor-2 (HER-2) overexpressing SK-BR-3 human breast cancer cell line. They developed a novel multifunctional composite micelle with surface modification of Ap for PTX delivery to cancer cells. This mixed system containing Ap-modified pluronic® F127 and CS could enhance PTX loading capacity, increased micelle stability and showed higher cytotoxicity of drug (Nguyen et al. 2016).

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CS NPs can be a platform for loading drugs such as DOX (Mitra et al. 2001), chlorhexidine digluconate (Senel et al. 2000), 5-FU (Ohya et al. 1994), mitoxantrone (Jameela and Jayakrisnan 1995), cytarabine (Blanco et al. 2000), PTX (Kim et al. 2006) for improvement in cancer treatment and insulin (Jintapattanakit et al. 2009) for diabetics (Elgadir et al. 2014).

2.2 Synthetic-Based Polymers as Building Blocks of NPs Synthetic polymeric nanoparticle should have some properties for gaining the ability for drug delivery in body, such as hydrophilicity in aqueous solutions, biocompatibility to offer a safe passage of the system, biodegradability to break due to cleavage of covalent bonds between them and have the ability to erosion of the polymer due to dissolution of linking chains without bringing about any change in chemical structure of the molecule. In addition, these polymeric drug carriers have to be water soluble, nontoxic and non-immunogenic in order to minimize drug degradation and improve blood circulation time (Srivastava et al. 2016; Liechty et al. 2010). More applied synthetically produced polymers that have been used as excellent nanocarriers for drug delivering are discussed in the following. • Polylactic-co-glycolic acid (PLGA) PLGA is one of the extensively used synthetic biodegradable polymers due to its favourable properties. It has been among the most attractive polymeric candidates for drug delivery and tissue engineering applications. PLGA is known as a ‘Smart Polymer’ due to its stimuli sensitive behaviour and is approved by the FDA for several therapeutic applications because of its biodegradability, biocompatibility and sustained-release properties (Srivastava et al. 2016; Kapoor et al. 2015; Biondi et al. 2008). PLGA undergoes hydrolytic degradation in an aqueous environment where ester linkages present along the polymer backbone are randomly hydrolysed and then each of ester group forms one hydroxyl and one carboxylic acid group (Makadia and Siegel 2011). The PLGA molecular weight is directly related to the polymeric chain size and it is proven that polymers with high molecular weight mostly exhibits low degradation rate (Park 1994). In a study by Wang et al., PLGA NPs were modified with CS through physical adsorption and chemical binding methods that changed the surface charge of the NPs. After the introduction of CS, the surface charge of the PLGA NPs measured as the zeta potential altered from the negative amount to positive, making the drug carrier more affinity to cancer cells. The presence of functional amino groups at the surface of CS-modified PLGA NPs confirmed by Fourier Transform Infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy. As a result, the positive charge and hydrophilic property of PLGA NPs induced a moderate and prolonged drug release (Wang et al. 2013a). A multimodal bioimaging and anticancer drug delivery system was fabricated by encapsulating inorganic imaging agents of superparamagnetic iron oxide NPs (SPI-

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ONs), manganese-doped zinc sulphide (Mn:ZnS) quantum dots (QDs) in combination with the anticancer drug busulfan into PLGA NPs via an emulsion-evaporation method. The magnetic resonance imaging (MRI) of PLGA vesicles indicated high r*2 relaxivity and greatly enhanced T*2 -weighted MRI contrast. On the other hand, macrophage cells showed high uptake of PLGA vesicles labelled with quantum dots, and the uptake was improved over time. Moreover, histological studies showed that the presence of PLGA vesicles in organs was moved from the lungs to the liver and spleen over time (Ye et al. 2014). The amphiphilic graft copolymer poly(lactic-co-glycolic acid)-g-dextran (DexPLGA) synthesized to fabricate micelles with the size below 100 nm with a relatively narrow size distribution for the delivery of PTX with low critical micelle concentration (CMC). This strategy could overcome the drug resistance of multi-drug resistant human breast carcinoma cells (MCF-7/Adr cells). The antitumour activity results indicated that treatments with Dex-PLGA/PTX micelles effectively suppressed the tumour growth and highly reduced the toxicity (Liua et al. 2015). Metformin (Met) and CUR were co-encapsulated in PEGylated PLGA NPs. Dialysis method and MTT assay were employed for the evaluation of drug release and the cytotoxicity effect, respectively. In addition, the inhibitory effect of individual and combined drugs on the expression level of hTERT in T47D breast cancer cells was measured by qPCR technique. A dose-dependent cytotoxicity against cancer cells observed for both free drugs and formulations. More synergistic antiproliferative effect that significantly arrested the growth of cancer cells was observed for Met–CUR–PLGA/PEG NPs than the other groups. Also, real-time PCR results revealed that CUR, Met, and combination of Met–CUR in free and encapsulated forms inhibited hTERT gene expression. Met–CUR–PLGA/PEG NPs in comparison to free combination could further decline hTERT expression in all concentration so holds promising potential towards the treatment of breast cancer (Farajzadeh et al. 2017). DCX (Chaudhari et al. 2012), DOX (Niu et al. 2013), PTX (Steele et al. 2011), clonidine (Dubey et al. 2012), tobramycin (Lucia et al. 2012) and dexamethasone (Chang-Lin et al. 2011) are the samples that had been loaded in PLGA-based polymeric NPs which demonstrated mostly higher efficacy in comparison with their free form (Farokhzad and Langer 2009). • Polyglycolic acid (PGA) and poly-l-glutamic acid Polyglycolic acid (PGA) is a biodegradable, thermoplastic polymer and the simplest linear, aliphatic semicrystalline polyester that can be degraded in the body over a period of several weeks. On the other hand, Poly-l-glutamic acid or Gamma PGA is a polymer of the amino acid glutamic acid (GA). PGA is able to be prepared starting from glycolic acid by means of polycondensation or ring-opening polymerization. PGA has been known since 1954 as a tough fibre-forming polymer. Whereas, Gamma PGA is formed by bacterial fermentation. Gamma PGA has a wide number of potential applications ranging from food and medicine to water treatment and the most important of them as a drug delivery system in cancer treatment (Liechty et al. 2010; Dickers et al. 2003).

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• Polylactic acid (PLA) Polylactic acid (PLA) is a biodegradable thermoplastic aliphatic polyester made from two possible monomers or building blocks: lactic acid, and lactide. Lactic acid can be produced by the bacterial fermentation of a carbohydrate source under controlled conditions. In the industrial scale, production of lactic acid is from renewable and sustainable resources, such as corn starch (mostly in the United States and Canada), tapioca roots, chips or starch (mostly in Asia), or sugarcane (in the rest of the world) (Liechty et al. 2010). The utilization of both nano- and microparticles based on PLA and related polymers and copolymers in the field of controlled release of drugs is become an interesting topic due to its excellent biocompatibility and biodegradability. PLA-based nano- and microparticles are able to incorporate with a wide variety of compounds, for instance, PEG, CS, and PVA (Lassalle and Ferreira 2007). Rancan et al. had investigated the suitability of biodegradable PLA particles loaded with fluorescent dyes as carriers for transepidermal drug delivery. PLA particles with the size of 228 and 365 nm penetrated into 50% of the vellus hair follicles, reaching a maximal depth corresponding to the entry of the sebaceous gland in 12–15% of all observed follicles. Therefore, PLA NPs may be ideal carriers for hair follicle and sebaceous gland targeting due to their stability in aqueous solution, destabilization of the particles and significant release of incorporated dye occurred upon contact with organic solvents and the skin surface (Rancan et al. 2009). Recently, DOX is covalently attached to a polylactide building block (Mw  2600, n  36) and then the particle surface is decorated with an HCC specific peptide moiety (i.e., SP94) to achieve preferential tumour accumulation for improving the therapeutic outcome of hepatocellular carcinomas (HCCs). The resulting HCC-targetable DOX-encapsulating NPs (termed tNP-PLA-DOX) exhibited several unique characteristics, including the feasible fabrication of sub-100 nm NPs, substantially delayed drug release for several weeks, HCC cell-specific uptake and tumour accumulation in an in vivo mouse model, as well as alleviated drug toxicity in animals and eventually. It is suggested that the prepared DOX-based nanomedicines have potential for enhancing the therapeutic index in patients (Xu et al. 2018). • Polyethylenimine (PEI) PEI is known for its high effectivity for its complexation with nucleic acid materials which could make it possible to accelerate the process of polyplex endocytosis. The branched PEI of 25 kDa (PEI25) has shown a superior transfection activity and has become a gold standard for gene therapy by polymeric carriers, however, one of the major factors limiting its use is the relatively high toxicity of PEI, which has been attributed to both necrotic and apoptotic mechanisms resulting from cell membrane damage. Therefore, an effective strategy to reduce the toxicity of PEI has been demonstrated that is the attachment of PEG to PEI polymers (Wang et al. 2016a, b). One of the novel types of drug delivery systems is termed NanoGel™ that represents particles of a hydrophilic polymer network that were synthesized by

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cross-linking of PEI and carbonyldiimidazole-activated PEG through emulsification/solvent evaporation technique with the average particle size of 120 nm. Antisense phosphorothioate oligonucleotides (SODN) specific to the human mdr1 gene were incorporated in these NanoGel™ particles, therefore loaded NanoGel™ particles with SODN reduced the particle diameter to 80 nm and decreased zeta-potential due to neutralization of the charge of PEI chains by SODN. Accumulation of SODN incorporated in NanoGel™ particles in multidrug-resistant (MDR) human oral epidermoid carcinoma cells (KBv) was obviously increased compared to the free SODN, thus these particles were found to effectively inhibit expression of P-glycoprotein (P-gp) efflux pump in MDR cell lines (Vinogradov et al. 1999). Wang et al. designed PEI segments to integrate a sufficient nucleic acid condensation as well as only a mild cytotoxic behaviour by a hydrophobic oligoester core with glycolide units resulting in fast degradation after cellular internalization in combination with PEG moieties acting as shielding agents. By interconnecting branched 25 kDa PEI and PEG on poly((ε-caprolactone)-co-glycolide) (CG), amphiphilic PEICG-PEI and PEG-CG block copolymers were used to form a series of micelles via self-assembly of PEI-CG-PEI or co-assembly of both copolymers for DNA and siRNA delivery. Their properties and transfection activity could be controlled by the length of the hydrophobic part in PEI-CG-PEI, the incorporation of PEG-CG in mixed micelles, the polymer/nucleic acid ratio and the type of nucleic acid. These micelles showed several advantages in comparison with homo-PEI25 such as lower toxicity, higher DNA transfection activity according to reporter geneexpression and an exceptionally high knockdown in siRNA delivery experiments (Wang et al. 2016a, b).

3 Active Targeting Utilizing Ligand Decorated NPs Various studies have shown that nanocarriers are able to accumulate in the tumour site by a mechanism called enhanced permeability and retention effect (so-called passive targeting). This preferential distribution to the tumours was ascribed to the presence of fenestrations in the imperfect tumour blood vessels that makes them highly permeable, and to the poor lymphatic drainage that leads to the accumulation of NPs in their site of action. Besides passive targeting mechanism, active targeting (so-called ligand mediated targeting) involves using affinity ligands on the surface of NPs for specific retention and uptake by the targeted disease cells. Ligands such as antibody, peptide, aptamer, folic acid, and transferrin are the most selected ones that associated with their specific receptors and molecules on the surface of diseased tissues, organs or cells which is schematically illustrated in Fig. 1. Targeted delivery utilizing ligands that are discussed in continue, is expected to enhance the interactions between NPs and cells and increase drug internalization without overall biodistribution (Bertrand et al. 2014; Zhong et al. 2014; Wang and Thanou 2010).

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Fig. 1 Modified NPs are able to pass through the blood vessels and reach their target tissues due to the ligands available on the surface of NPs and on the other hand, their specific receptors on the surface of tissues and cells. Each modified drug delivery system attach specifically to its desired site of action due to the interaction between the ligand and its exact receptor resulting reduced side effect and enhanced therapeutic effect of the drug

3.1 Monoclonal Antibodies (mAbs) Over the last decades, mAbs have received wide attention for cancer therapy because of their remarkable activity against tumour cells. They have been used as therapeutic agents or as targeting ligands due to their high specificity (Kouchakzadeh et al. 2017). Ligand more specifically interacts with the overexpressed receptors on the surface of damaged cells, and not with the receptors on the surface of normal tissues, and thus reduces the side effects of chemotherapeutic agents (Carter et al. 2016). Most of the cancer cells express a high number of HER-2 receptors on their surface and mAbs targeted against HER-2 have shown a promising heading to these cells (Kouchakzadeh et al. 2013). In a study by Li et al., a novel theranostics agent made up of bismuth sulphide@ mesoporous silica (Bi2 S3 @mps) core-shell NPs, DOX and Trastuzumab was produced in order to target HER-2-positive tumours. Based on the findings, good biocompatibility, high drug loading ability, and active tumour targeting observed for the functional system. Trastuzumab-modified NPs can be served as a great contrast and therapeutic enhancement system due to the presence of diagnosis and therapy elements which are directly targeted against HER-2-positive tumours. 16 times more accumulation obtained for mAb modified system than the non-targeted group (Li et al. 2018). Protein-based albumin NPs have significant properties such as biodegradability, lack of toxicity, and nonantigenicity (Kouchakzadeh et al. 2012; Fadaeian et al. 2015;

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Kirpotin et al. 2006). In addition, their production is easy and their targeting is possible because of the functional groups on their surface (Elzoghby et al. 2012; Wagner et al. 2010). A novel mAb (1F2) conjugated to the surface of HSA NPs and cellular uptake of these particles was evaluated. mAb thiolation with a 1-fold molar excess of 2-imminothiolane and the ratio of 10:1 for the thiolated 1F2 (μg) to PEGylated NPs (mg) were optimum for the attachment process. Under this condition, almost 24% of 1F2 was conjugated to NPs, Targeted NPs interacted with almost all HER-2 receptors on the surface of BT474 cells, and MCF-7 cells that are HER-2 negative had no cellular uptake. To consider all into account, it was found that targeted albumin NPs are a promising approach in drug delivery system toward HER-2-positive tumour cells (Kouchakzadeh et al. 2013). In another related study, PR81, an anti-MUC1 mAb, attached to the surface of BSA NPs through a PEG-based crosslinker and loaded with anticancer medicine 5-FU to lead the drug toward a specific cancerous tissue. The results showed that these targeted drug-loaded NPs work more efficiently than free drug. In addition, stability studies demonstrated an acceptable immunoreactivity of conjugated NPs even after 11 days of storage at room temperature that verified by an enzyme-linked immunosorbent assay (ELISA) (Kouchakzadeh et al. 2012). In another study performed by Dinarvand et al., the surface of albumin NPs containing MTX was decorated with trastuzumab mAb. The cytotoxicity tests showed that targeted drug conjugated NPs have a much higher effect than non-targeted ones or free drug on HER-2-positive cells. In addition, all kind of MTX formulations showed similar cytotoxicity on HER-2 negative cells approximately (Taheri et al. 2012b). Pang et al. conjugated (BSA)-branched polyethylenimine layer by layer NPs with PSMA antibody to deliver DCX and p44/42 mitogen-activated protein kinase (MAPK) siRNA at the same time to CSR22R cells. It was obtained that the median survival of mice which received targeted drug delivery system prolonged from 18 days to more than 45 days in comparison with the mice that received free drug (Pang et al. 2017). In two different studies, long-circulating immunoliposomes (one of them containing Mellitin) were targeted with anti-HER2 mAbs, and their cytotoxicity and selectivity abilities were investigated. One group of researchers observed that targeted and non-targeted liposomes had a nearly similar rate of accumulation in HER-2positive breast cancer xenografts (BT474) by the means of EPR effect but the results of flow cytometry showed that targeted liposomes had 6-fold greater internalization in tumour cells (Bhatia 2016). The other group of researchers found a decrease in viability of HER-2 overexpression SKBR-3 breast cancer cells in a dose-response manner. Liposomes with selective internalization could be considered as efficient agents for cancer treatment, provide new opportunities for drug delivery and have the potential to be developed for early phase I-II clinical trials (Barrajón-Catalán et al. 2010). In a study by Nguyen et al., rapamycin and a photosensitizer polypyrrole were conjugated to liposomes and modified with trastuzumab for combined chemophotothermal therapy. Targeted liposomes showed higher accumulation in BT-474 cells and higher therapeutic influence on HER-2-positive cells compared to HER-2 negative ones (Nguyena et al. 2017).

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CD147 antibody considered as a targeting ligand and attached to the surface of Doxil (PEGylated liposomal DOX) to improve its specificity against hepatocellular cells. The results showed an enhancement in the accumulation of Doxil in hepatocellular cells and xenograft models in vitro and in vivo, respectively, after antibody conjugation. Long circulation time and efficient accumulation in tumours were investigated and proved by pharmacokinetic and biodistribution studies (Wang et al. 2017). It was also reported that treatment with Doxil causes a mucocutaneous reaction in mice identified as auricular erythema (AE). Therefore, a study was done to target Doxil toward tumours for decreasing its adverse side effects. In this regard, Doxil was coupled to an anticancer mAb called 2C5. This modification diminished the accumulation of DOX in normal skin and symptoms of AE 3-4 fold as a result. Thus, targeting of DOX-loaded long-circulating liposomes with 2C5 not only increased the accumulation of drug in tumours, but also decreased the side effects of original Doxil (Elbayoumi and Torchilin 2008). Haibu et al. produced GEM-loaded glycol CS NPs (GC complex) and GEMloaded glycol CS NPs conjugated with anti-EGFR antibody (Abc complex). Both complexes showed inhibitory effects on SW1990, but the inhibitory effect of Abc complex was much higher than GC complex, so they suggested a promising potential for pancreatic cancer treatment (Xiao and Yu 2017). PLGA NPs employed for targeted delivery of DCX by their conjugation to cetuximab as the targeting ligand to improve cytotoxicity and specificity of drug to NSCLC cells. In vitro studies demonstrated higher accumulation and more anti-proliferative activity of targeted NPs compared to non-targeted ones. In vivo evaluations showed a higher reduction in tumour growth by conjugated NPs (Patel et al. 2018). Lee et al. encapsulated SPIONs to Herceptin-conjugated PLGA NPs for targeted delivery to HER-2-positive cancer cells. The results showed that the amount of accumulation of Herceptin-conjugated PLGA in the target tissue is higher than non-conjugated ones and the signal intensity of in vivo MRI reduced as the amount of Herceptin increased (Lee et al. 2018).

3.2 Folic Acid Folic acid has been used in a large number of studies as a targeting ligand because it is inexpensive, easy to reach, stable and can be attached to drug carriers easily. Folate receptors are overexpressed in many cancers such as ovary, kidney, brain, breast, myeloid cells and lung (Lu et al. 2004). Folate can also be considered as a receptor for targeted gene delivery. In a study by Zhang et al. folate\PEG\PEI was combined with mini circle DNA as a new tumour gene delivery system. Gene expression increased about 2-8 fold in cells which have folate receptors on their surface by folate modified vectors in comparison with conventional plasmids. In addition, it was found that PEG reduced the toxicity of PEI (Zhang et al. 2010). Shi et al. prepared gefitinib-loaded folate-decorated BSA conjugated Carboxymethyl-β-cyclo dextrin (FA-BSA-CM-β-CD) NPs to improve drug delivery

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to folate receptor-positive tumour cells. The produced system showed enhancement in gefitinib uptake and toxicity in folate receptor-positive Hela cells. The results expressed that FA-BSA-CM-β-CD NPs provide a new efficient strategy for the treatment of overexpressed folate receptor cancer cells (Shi et al. 2014). Recombinant viral vectors are widely used for transferring genes into body, but this approach has some side effects such as immunogenicity and oncogenicity. In order to overcome these problems, synthetic delivery systems for gene therapy can be developed. Folate-based complexes were prepared by Mornet et al. and were compared together in order to reach an efficient carrier for gene therapy. The results indicated that folate-modified lipids (or polymers) could enhance non-toxicity and cell-specific gene delivery in folate receptor expressing cells in vitro (Mornet et al. 2013). FA also can be considered for dual-targeting. For example, folate was attached to the surface of HSA NPs loaded with GEM as a targeting ligand. Folate dual-targeted HSA NPs showed a higher rate of cytotoxicity, cellular uptake and apoptosis induction on a folate receptor overexpressing tissue in comparison to non-targeted NPs. Therefore, dual-targeted HSA NPs can be considered as potential carriers for cancer therapy with improved stability, efficacy and selectivity of GEM (Norouzi et al. 2017). Poor bioavailability and retention time of CUR improved through its encapsulation to PEGylated PLGA NPs. Folate-conjugated CUR-loaded PLGA-PEG NPs resulted in maximum cytotoxicity on cancer cells among other formulations. Folic acid conjugation could improve the bioavailability and chemosensitizing efficacy of CUR-encapsulated PLGA-PEG NPs towards paclitaxel chemotherapy in vitro and in vivo and introduced a promising system for CUR delivery (Thulasidasan et al. 2017).

3.3 Transferrin Transferrin (Tf) receptor is a type II membrane glycoprotein that is expressed at low levels on the surface of most normal tissues and supports the transport of iron to the cells that grow rapidly. Tumour’s increasing demand of iron causes the overexpression of Tf receptors on the surface of cancerous cells. Thus, Tf can lead the anticancer drugs toward overexpressed tumour cells and enhance the accumulation of drug there (Widera et al. 2003; Kouchakzadeh et al. 2017). The potent anticancer drug, DCX, was conjugated to Tf-targeted NPs of poly(lactide)-d-α-tocopheryl polyethylene glycol succinate (PLA-TPGS) diblock co-polymer and its efficacy was compared to unmodified PLA-TPGS NPs and the commercial free DCX, Taxotere. The results demonstrated great advantages of the targeted system as IC50 tests showed its cytotoxicity could be 23.4%, 16.9%, and 229% higher than PLGA NPs, PLA-TPGS NPs and Taxotere, respectively. They also proved that Tf-conjugated PLA-TPGS NPs could pass BBB and has the potential to deliver the drugs across BBB (Gan and Feng 2010). In a similar study, the surface of PLGA NPs and PEG synthesized co-polymer was modified by Tf ligands for

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receptor-mediated targeting to the brain. In vitro cytotoxicity assays performed on human cancer cell lines such as PC3, DU145, colo-205, HCT15, and MCF-7. Findings showed enhanced uptake of Tf-targeted PEGylated NPs and their accumulation in brain tissue. Thus, the proposed system could be used as a potential carrier for drug delivery to the brain (Jain et al. 2011). PTX loaded CS NPs were covered with polyethylene glycol and targeted with Tf to promote drug delivery to tumours. The uptake of Tf-targeted NPs into cancer cells was found to be higher in comparison to non-targeted ones. PTX loaded targeted NPs demonstrated higher cytotoxicity to human NSCLC cell lines (Hop-62), higher intracellular uptake, lower haemolytic toxicity than free drug, PTX loaded NPs and PEGylated PTX NPs. Pharmacokinetic studies showed that Tf targeted NPs also enhanced retention time in lungs and blood (Nag et al. 2016). A group of researchers in Japan screened the toxic effect of 11 isoquinoline derivatives and α-methylene-γ-butyrolactones by MTT assay to identify their potential anticancer agent. Then, they attached the drug to Tf-targeted liposomes and demonstrated that the targeted system has significant antitumour activity and selectivity to tumour cells in comparison with free drug and non-targeted system (Yang et al. 2015). In another study, liposomal cisplatin (Cis) was modified with Tf to evaluate its selectivity potential towards glioma. The in vitro tests on bEnd3/C6 co-culture indicated that drug-loaded liposomes modified with Tf cause higher transport efficacy through BBB and cytotoxicity in C6 other than Cis-Lipo and Cis-solution (Lv et al. 2013). Tf also conjugated to 5-FU loaded liposomes and their in vitro cytotoxicity was evaluated by MTT assay on HT-29 cancer cells and fibroblast normal cells. The results indicated that the cytotoxic effect of targeted liposomes was much higher than free drug and non-targeted liposomes. They also did not show any cytotoxicity on normal cells. This strategy was introduced as a potential strategy for colon cancer treatment due to increasing apoptosis, reducing the dose of the drug and decreasing side effects (Moghimipour et al. 2018b). Transferrin can also be used in combination with another ligand to produce dualtargeted carriers for achieving more efficiency. In this regard, Tf and TAT (cationic cell penetrating peptide) were attached to liposomes and their in vitro cellular uptake was assessed on three different Tf receptor-positive cells. In vitro findings showed that dual-targeted liposomes enhanced cellular uptake and selectivity in comparison to single-ligand Tf or TAT modified liposomes. The in vivo studies also verified a higher delivery efficiency for dual-modified liposomes than the other ones (Tang et al. 2013).

3.4 Peptide Peptide conjugation is a precise and easy controlled process. On the other hand, the physicochemical features of NPs are hardly changed after conjugation due to the very small size of the peptides. The mostly used peptide in cancer treatment research is

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arginine-glycine-aspartate or “RGD” that is able to target αv β3 integrin overexpress on the surface of endothelial cells and many types of solid tumours (Zhong et al. 2014; Ruoslahti 2012; Danhier et al. 2012). Shikonin is an anticancer drug with a great antitumour effect. However, poor water solubility and low bioavailability limit its usage. To solve these problems, RGDmodified shikonin-loaded liposomes were prepared and characterized. The system revealed excellent physicochemical properties together with greater cytotoxicity and apoptosis in vitro than the free drug. Targeted liposomal system could also inhibit cell proliferation, migration, and invasion. Findings recommend a new efficient strategy for targeted delivery of shikonin to breast cancer cells (Wen et al. 2018). In a study by Minura et al. micelles incorporated with platinium anticancer drug were modified with cyclic Arg-Gly-Asp (cRGD) and cyclic Arg-Ala-Asp (cRAD). Although two different targeted systems demonstrated similar physicochemical characteristics such as size and surface change, the cRGD-linked polymeric micelles shoed more rapid accumulation and higher permeability in U87MG cancer cells rather than the non-targeted or cRAD-targeted ones. These findings indicated that cRGD-modified micelles reached into tumours by an active internalization pathway more effectively (Miura et al. 2013). Poly(D,L-lactic-co-glycolic acid)-block-polyethylene glycol (PLGA-PEG) NPs loaded with Cis prodrug conjugated to cyclic pentapeptide (RGDfk). This system was evaluated for prostate and breast cancer epithelial cells in vitro, and orthotropic human breast cancer xenograft model in vivo. The results displayed enhanced cytotoxicity as compared to original free drug both in vitro and in vivo and provided new opportunities in Cis delivery (Graf et al. 2012). Yang et al. modified the surface of gold NPs simultaneously with two different peptides. One of them increased the cellular uptake while the other one increased nuclear delivery. The results showed that targeted NPs cause four-time enhancement in therapeutic response in comparison with non-targeted ones. However, there was a slightly increase in DNA damage of cells which treated with targeted NPs (Yang et al. 2014). A breast cancer targeting peptide (P18-4) was added to the surface of two micelles (poly(ethyleneoxide)-poly(ester)) (PEO) with poly(2-caprolactone) (PCL) and poly(α-benzyl carboxylate-2-caprolactone) (PBLC) cores. Although a better uptake of PEO-PCL by MDA-MB-231 cells observed in vitro, PEO-PBCL demonstrated higher stability and accumulation in MDA-MB-231 tumours in vivo. Using PEO-PBCL instead of PEO-PCL more efficiently increased the accumulation of carriers in tumour cells than the conjugation of micelles and targeting peptides (Garg et al. 2017). Yeh et al. produced peptide (SP204)-PEGylated lipids and then added to liposomal DOX and vinorelbine to target it against PC3 human prostate carcinoma cell line in vivo and in vitro. Cellular uptake and MTT assays showed augmented accumulation and cytotoxicity of targeted drugs compared to free drugs. In addition, the conjugation of this peptide to imaging agents indicated more precise delivery of the agents to tumour sites (Yeh et al. 2016).

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3.5 Saccharides Cancerous tissues often express higher amounts of glycans in comparison to normal tissues. Therefore, saccharides can be considered as ligands for targeted drug delivery (Zhong et al. 2014). Polymeric micelles modified with galactose are promising agents for hepatomatargeted drug delivery. DOX-loaded micelles were targeted with gal and showed an exhibited stability at physiological pH. An enhanced DOX release without burst effect observed in response to acidic pH environment. Cellular uptake and in vitro cytotoxicity results indicated effective transformation of DOX to hepatoma cells with promoted proliferation inhibition (Feng et al. 2014). In another study by Wang et al. DOX was loaded in galactose-decorated cross-linked micelles with ionic cores. In vitro studies showed rapid drug release into the cell nucleus of HepG2 cells compared to non-targeted micelles (Wang et al. 2013b). The combination of therapeutic nucleic acids and chemotherapeutic drugs has shown great promise for cancer therapy. Hea Ry Oh et al. co-delivered vimentin siRNA and DOX to hepatocellular carcinoma. In this regard, they developed galactose-DOX/vimentin siRNA liposomes (gal-DOX/siRNA-L). This agent showed stronger affinity to human hepatocellular carcinoma cells than other cells and inhibited tumour growth more efficiently than single DOX or vimentin siRNA. These findings demonstrated great potential for synergistic anti-tumour therapy (Oh et al. 2016). Lapatinib is an orally administered drug for the treatment of metastatic breast cancer. For the improvement of its properties, nanocrystals (NCs) of LPT were produced and then coated with hyaluronic acid (HA). The in vitro and in vivo investigations of LPT-HA-NCs demonstrated a significant anticancer activity in comparison with LPTNCs or free LPT. This agent also increased the residence time of LPT in triple negative 4T1 cells and overall survival (Agrawal et al. 2017). HA was also employed for targeting of daunorubicin plus honokiol cationic liposomes to human breast cancer cells. In vitro results showed an enhanced cellular uptake and destroyed vasculogenic mimicry (VM) channel. In addition, in vivo results revealed a prolonged circulation time in blood, enhanced accumulation in tumour cells and elevated anticancer effects of drugs (Ju et al. 2018).

3.6 Aptamers Aptamers are single-stranded DNA or RNA that can bind to various molecular ligands with high affinity and specificity. With comparison to antibodies, they have smaller size, lower immunogenicity, lower production cost and higher stability (Zhong et al. 2014; Ladju et al. 2017). In a study by Xu et al. unimolecular micelles were conjugated to aptamer and loaded with DOX. Then the produced system was tested in prostate-specific mem-

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brane antigen (PSMA)-positive prostate carcinoma cells. The results indicated much higher cellular uptake and cytotoxicity of the targeted unimolecular micelles. DOXloaded targeted unimolecular micelles also demonstrated a higher level of DOX accumulation in tumour cells than DOX-loaded non-targeted ones (Xu et al. 2013). DOX was also targeted with GMT-3 and single-stranded DNA (ssDNA) aptamer to A-172 glioblastoma cells. Findings demonstrated a potential in GMT-3 aptamer-mediated therapeutic drug transportation to glioblastoma cells and the authors claimed that the system can be used for clinical purposes (Bayrac et al. 2018). Min et al. developed a dual-aptamer conjugated superparamagnetic iron oxide NPs to target PSMA-positive and PSMA negative prostate cancer cells. They also loaded DOX into the complex and observed apoptosis in both types of prostate cancer, selective cell uptakes and effective drug delivery (Min et al. 2011). DOX also was loaded to a DNA nanostructure for heading to resistant cancer cells. This nanostructure consisted of two constituents: a DNA aptamer to target cancer cells by binding with nucleolin, and a double-stranded DNA (dsDNA) as a carrier for DOX due to its GC rich base pairs. The intercalation of dsDNA and DOX did not have any effect on the structure of ssDNA and this agent enhanced tumour growth inhibition while reduced cardiotoxicity (Liu et al. 2016).

4 Polymeric Nanoparticles Marketed and Under Clinical Trials Abraxane, Doxil, Marqibo, Depocite, Ambisome, Paclical, Diprivan, and Visudyne are the examples of marketed nano-based drugs as an alternative for traditional drugs for treatment (Ragelle et al. 2016). In addition, there are nano-formulations that are under different stages of clinical trials. Albumin-bound rapamycin (ABI-009) is based on albumin NPs and has been passed through phase I and II of clinical trials for advanced non-hematologic malignancies (phase I completed), pulmonary arterial hypertension (phase I ongoing), advanced malignant perivascular epithelioid cell tumour (phase II ongoing), nonmuscle invasive bladder cancer (phase I and II ongoing) and advanced cancer with mTOR mutation (phase II ongoing) (Tan and Ho 2018). Livatag, a polymer-based drug delivery system that is consists of DOX and poly(alkyl cyanoacrylate) (PACA) NPs, is planned to be used for hepatocellular carcinoma treatment and currently is in phase III of clinical trials (Ragelle et al. 2016). CRLX301 (Cerulean) is another example of polymer-based drug delivery system which is produced by conjugation of cyclodextrin NPs and camptothecin and has the potential to be used for ovarian, renal cell, NSCL or rectal cancers. It is currently under phase I and II of clinical evaluations (Wagner et al. 2018). BIND-014 is a PSMA targeted polymeric nanoparticle loaded with DCX that is under clinical studies for treatment of prostate, NSCL, cervical and head and neck cancers. Promising results

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for the treatment of NSCL cancer with BIND-014 obtained in late 2014 (Wagner et al. 2018). There are a number of NPs based on polymeric micelles for drug delivery to solid tumours. NC-6004, containing Cis and CriPec, containing DCX are polymeric micelles which are under phase I, II and phase I of clinical trials, respectively (Ragelle et al. 2016; Wagner et al. 2018). For the treatment of pancreatic cancer, a lipid-based NPs modified with PKN3 siRNA (Atu-027) developed and is currently under phase IIb of clinical trials (Ragelle et al. 2016). A liposomal delivery system containing CEBPA siRNA (MTL-CEBPA) developed for liver cancer therapy and is in phase I of clinical studies (Ragelle et al. 2016). CPT-11 is a camptothecin-11-containing liposomal drug carrier that targets high-grade glioma with finished phase I of clinical trials (Mazur et al. 2017). Genexol is a PTX conjugated PEG-poly(D,L-lactic acid) that was approved for the treatment of breast and non-small cell lung cancers but is still in phase III of trials for ovarian cancer treatment (Movassaghian et al. 2015). Besides the polymeric NPs, Auroshell is a gold-coated silica nanoshelles for the treatment of prostate and head and neck cancers through the laser irradiation which is currently under phase I of clinical studies (Ragelle et al. 2016; Manisekaran et al. 2018).

5 Conclusion Although conventional chemotherapy is considered as the first line cancer therapy for many years, it has not been successful enough due to the problems such as nonspecificity and toxicity. Thus, development of an efficient treatment method in order to be selective and less toxic is necessary. In addition, the poor water solubility of anticancer drugs has a major impact on the survival time of cancer patients and much efforts have been made to overcome the challenges pertaining to conventional therapy. Targeted therapy by the use of nanoparticulate delivery systems, can be a solution for achieving an efficient treatment and natural and synthetic polymer-based carriers can be employed for this purpose. Despite the challenges over the development of targeted drug delivery systems such as limitations of ligand conjugation and difficulty of process scale up, but the future of using these systems is promising due to their advantages over the free drug.

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Hydroxyapatite for Biomedicine and Drug Delivery Behrad Ghiasi, Yahya Sefidbakht and Maryam Rezaei

1 Introduction Hydroxyapatite (HA) is a member of the calcium phosphates family (Table 1) and like the other ones is known as a bioceramic with specific advantages raise from chemical similarity to the mammalian inorganic structure. In comparison to other CaPs, HA has highest thermodynamic stability and solubility (after Fluorapatite) in physiological conditions. Besides this, HA has the highest similarity with the structure and function to the biominerals like bone and teeth which makes it considerable particle for researchers to treat bone and dental defects. However, there are also some differences between biological HA and synthetic HA such as the substitution of ion in biological HA’ network. HA’ crystal orientation also has a difference and exact axle growth have not achieved in the laboratory. HA is a major constitution of bone as Bone contains 65–70% HA in addition to 5–8% water and 20–25% organic materials (Batchelar et al. 2006; Sato 2007; Malmberg and Nygren 2008). HA nanoparticles have also used for other applications, such as remediation of the environment, removal of metals and absorbance of organic molecules on the surface (Piccirillo and L Castro 2017). Hydroxyapatite has the capability to adsorb oxytetracycline from the aqueous medium (Harja and Ciobanu 2018). The 1960’s was an initiative time to start the study about the potentiality of ceramics as a bone substitute and other biomedical applications. The potency of this material B. Ghiasi · Y. Sefidbakht (B) Protein Research Center, Shahid Beheshti University, G.C, Tehran, Iran e-mail: [email protected] Y. Sefidbakht Nanobiotechnology Laboratory, The Faculty of New Technologies Engineering (NTE), Shahid Beheshti University, G.C, Tehran, Iran M. Rezaei Institute of Biochemistry and Biophysics (IBB), Tehran University, Tehran, Iran © Springer Nature Switzerland AG 2019 M. Rahmandoust and M. R. Ayatollahi (eds.), Nanomaterials for Advanced Biological Applications, Advanced Structured Materials 104, https://doi.org/10.1007/978-3-030-10834-2_4

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Symbol

HA

ACP

(β-TCP)

(α-TCP)

OCP

DCPA

DCPD

MCPA

MCPM

Name

Hydroxyapatite

Amorphous calcium phosphate

β-Tricalcium phosphate

α-tricalcium phosphate

Octacalcium phosphate

Dicalcium phosphate anhydrous (monetite)

Dicalcium phosphate dehydrate (Brushite)

Monocalcium phosphate anhydrous

Monocalcium phosphate monohydrate

0.5

0.5

1.0

1.0

1.33

1.5

1.5

1.2–2.2

1.67

Ca/P

Table 1 Characteristics of CaP family

Ca(H2 PO4 )2 , H2 O

Ca(H2 PO4)2

CaHPO4 · 2H2 O

CaHPO4

Ca8 (HPO4 )2 (PO4 )4 · 5H2 O

Ca3 (PO4 )2

Ca3 (PO4 )2

Cax (PO4 )y nH2 O

Ca10 (PO4)6 (OH)2

Formulas

1.14

1.14

6.59

6.90

96.6

25.5

28.9

116.8

pKs at 25 °C

0.0–2.0

2.0–6.0

5.5–7.0

~5–12

9.5–12

pH stability range in aqueous

~18

~17

~0.088

~0.048

~0.0081

~0.0025

~0.0005

~0.0003

Solubility at 25 °C, g/L

Triclinic p 1

Triclinic p 1

Monoclinic la

Triclinic p 1

Triclinic p 1

Monoclinic P21/a

Rhombohedral R3cH

Monoclinic P21/b or hexagonal P63/m

Space group

2.23

2.58

2.32

2.89

2.61

2.86

3.08

3.16

Density g/cm3

Soluble phosphate fertilizer (Nasri et al. 2015)

Orthopedic (Zhang et al. 2017)

Bone regeneration (Torres et al. 2015)

Bone defect (Tadashi et al. 2014)

Orthopedic and dentistry (Carrodeguas and De Aza 2011)

Repair bone defects (Agarwalla et al. 2018)

Dentistry (Zhao et al. 2011)

Application

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Fig. 1 The growing number of published papers per year with specific subjects

makes it interesting so that many studies have been indexed by PubMed in recent decades (Fig. 1). As it is shown in the number of publications related to this subject, tissue engineering and implants are the most related topics, however, the applications in drug delivery and combination with antibacterial agents are also growing fields. Bioactivity, biocompatibility, osteoconductivity and absence of immune response are predictable and tested features of HA due to the chemical similarity to biological form. Facile fabrication methods and proper cost alongside intrinsic feature of HA makes it as an appropriate agent for implants, scaffolds and also targeted drug carrier for a verity of bone disease. Indeed, constructing a suitable scaffold which is able to form bone, needs exact porosity and special morphology to provide cell connectivity, cell attachment, cell migration, and finally osteoformation. Several investigations indicate that morphology and porosity are tuneable in HA nanoparticles that help to induce vascularization and bone formation. In addition, there are studies which confirm the improvement of implants by HA either as filler or coat (Kim et al. 2004a, b). Same as other nanoparticles HA has a high surface to volume ratio and can be loaded by a wide variety of drugs like antibiotics, hormones, growth factors, RNA and DNA to enhance treatment efficiency and decrease duration. For example, studies have shown Combination of HA with other polymers like chitosan and collagen to improve bone mineralization (Li et al. 2002; Chen et al. 2012). Targeted drug delivery is approachable and significantly for reducing the duration of treatment.

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2 Different Synthesis Methods Variety of methods are published to synthesise HA or extract it from natural resources (Hu et al. 2001; Akram et al. 2014) generally, preparation methods are divided into three groups and twelve subgroups: (a) Dry methods: (1) solid-state and (2) mechanochemical. (b) Wet methods: (1) chemical precipitation, (2) hydrolysis, (3) sol-gel (4) hydrothermal, (5) emulsion and 6) sonochemical. (c) High-temperature processes: (1) combustion, (2) pyrolysis, (3) synthesis from biogenic sources and (4) combination procedures. Other methods have also been reported, such as sol-gel, chemical methods, extract from biosource (Fig. 2), and synthesis by alkoxides. The coating on other substrates is also desirable and are achievable by utilizing plasma spray and electrochemical deposition. Dry methods are mostly used to produce a large amount of product; due to the simplicity in controlling processing conditions and smooth influence of processing parameters on the achieved product. In the solid-state synthesis, typical precursors such as chemicals that contain calcium and phosphate or prepared CaP salts are first milled and then gone under high temperature (e.g. 1000 C) to be calcined. Hydroxyapatite with good crystallinity is achieved by this method because of being calcined under high temperature. Calcination under high temperature provides a well-crystallized structure of HA. The size is large and the shapes are irregular in the HA production by solid state method. Unlike dry methods, in the wet methods, the solvent is used (an organic solvent is also possible). Many efforts have been applied to prepare HA particles with nanosized structure besides regular morphology under wet methods. From the fundamental point of view, growing HA crystals in the aqueous phase can clarify the biomineralization pathway which occurs in vivo. These options turn wet methods to the promising method. For Instance, until 2013, 60% of articles discussed this method (Sadat-Shojai et al. 2013). The low preparation temperature leads to low crystallinity and production of other CaP phases moreover the impurity in the HA crystals increase when other ions Present in aqueous solution. Chemical precipitation is the simplest pathway to synthesize nanoHA among wet chemistry methods. The reaction temperature is a range from room temperature to the boiling point of the solvent, and the pH of the reaction should be higher than 4.2. Calcium and phosphate salt are procedures of synthesis, (Tripathi and Basu 2012) such as calcium nitrate, calcium hydroxide and diammonium hydrogen phosphate or orthophosphoric acid (Dhand et al. 2014). The typical procedures are shown in Fig. 3. Hydrothermal method is another common process to prepare HA, the reaction applied inside a chamber which is called pressure vessel or autoclave, which provide high pressure to conduct ageing step at a higher temperature than the boiling point. The product is highly crystalline with homogeneous composition and good stoichiometric, also phase purity improved in this method due to the high temperature.

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Fig. 2 Co-precipitation process, and schematic setup

Hydrolysis also occurs under aqueous phase, for example, hydrolysis of brushite and TCP at a temperature ranged from 40 to 60 and pH 8.

3 Applications HA has been under attention for decades due to the excellent biocompatibility (Rabiei et al. 2007; Liang et al. 2011), affinity to biopolymers (Chen et al. 2007; Pelin et al. 2009), and high osteogenic potency (Gu et al. 2004; O’Hare et al. 2010). Several investigations have demonstrated the promoting effect of HA on bone growth through osteoconduction mechanism without local or systematic toxicity, inflammation, or immune response to the HA particles (Fig. 4) (Marini et al. 2004; Kokubo and Takadama 2006; Habibovic et al. 2008). These properties make HA a suitable candidate for orthopaedic and orthodontic applications including regeneration, replacement, and reconstruction. The main achievement of the material containing HA or coated by HA, is providing an appropriate surface for cell adhesion which remains for a long time.

Fig. 3 The typical procedures of HA synthesis

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Advantages

Disadvantages

Bioactive(hydration shell)

Strong hydration shell, Ionic surface, Fragile Surface corona formation, aggregation Precipitation and turbid solution dispersity in chemical composition , size and shape polymorphism, High pH sensitivity and solubility, low stability

Well attachment to polymers Ease of modification and surface functionalization Ease of composite formulation Biodegradable Biocompatible Self-assembly

Biomedical Applications Tissue engineering drug delivery implants

Fig. 4 HA advantages and disadvantages and biomedical applications

Furthermore, investigators suggest that HA or HA Derivatives can be considered as a model to study mineralization in the human body (Vallet-Regí and GonzálezCalbet 2004; Jee et al. 2011) Lately, research has shown HA particles can inhibit the growth of a wide Variety of cancer cells (Li et al. 2008b; Hou et al. 2009). Hydroxyapatite is an accepted material for medical applications, especially in hard tissue treatment, for example, improving and eliminating bone and tooth defects (Furukawa et al. 2000; Trombelli et al. 2010); Lateral alveolar ridge augmentation (Strietzel et al. 2007), middle ear implants (Ye et al. 2001), tissue engineering systems (Lv et al. 2009; Seol et al. 2009), drug delivery agent (Itokazu et al. 1998b), teeth material, and bioactive coat on metallic bone implants. HA and its derivatives have led to a number of industrial and technological applications such as chemical reactions catalyser, for example methane oxidation (Sugiyama et al. 1996), host material for leaser and fluorescent materials (Li et al. 2008c), ion conductivity and gas sensor (Mahabole et al. 2005). Deploying HA to chromatography columns provide simple and fast separation of biomolecules such as nucleic acid and proteins (Jungbauer et al. 2004). Moreover, it has been shown

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that HA might increase quality of water (Lin et al. 2009) refiner and also purification of soil from heavy metals (Hashimoto et al. 2009). Nowadays, compatible implant and substitution are needed. Biocompatibility, bioactivity, mechanical properties and lack of immune response are great challenges for scientists. In the individual treatment, implants must require flexibility to achieve controlling details in micrometry level, similarity, non-toxicity, and biocompatibility to provide a suitable implant for the patient. Targeted drug delivery system with the authorization of controlling rate and duration of drug release is a popular field of research. Due to the high amount of hydroxyl on the surface and polar charge, hydroxyapatite has appropriate properties to attract and preserve DNA and peptides in along of HA osteoconductivity feature, make it a suitable drug carrier (Sadat-Shojai et al. 2010). The Biohydroxyapatite which is found in the hard biologic organ is plate or cone shape like nanocrystals that have few thicknesses but a length equal to 10 nm. There is a belief that due to the similarity of hydroxyapatite with natural mineral in mammals, HA is one of the best material for substitution and bone regeneration (Cai et al. 2007). HA nanocrystal demonstrates enhanced densification and improved sinterability due to the higher surface to volume ratio which might increase fracture toughness. The comparison with micro hydroxyapatite has shown that Nano hydroxyapatite has higher reabsorbance capability and bioactivity (Dong et al. 2009; Wang et al. 2010). Moreover, calcium phosphate clusters can Couse forming and catalysed synthesis path (Dorozhkin 2010). Release calcium ion from biological hydroxyapatite is more similar to what Happened to nanohydroxyapatite in comparison with micro apatite. Furthermore, demineralization stops when the particle size reaches the critical point of nanoscale (Wang and Nancollas 2009). Nanohydroxyapatite shows higher density (Eriksson et al. 2011) and improved sinterability (Bianco et al. 2009; Bose et al. 2009, 2010), due to high surface energy. In general, nanostructured HA has enhanced mechanical properties because of reduction in flaw sizes (Ahn et al. 2001), the ability of HA in decreasing apoptotic death, improving cell activity and division reported in studied (Li et al. 2008a). In hydroxilation process, calcium and phosphate preserve DNA until the appropriate time. Cell proliferation and cellular differentiation can come from the excellent functional surface of nanoHA, which has more surface and rigidity, that leads to better cell attachment and better interaction in cellular matrix. HA nanoparticles are under attentions in dentistry and oral care issues due to the statistical data that demonstrate the ability of HA to decline hypersensitivity and inhibit early lesions. So in the last decade, bioceramics and biocomposite base on Nanohydroxyapatite (nHA) turned to promising material for the different medical application. In one hand many advantages come with HA particles which makes HA a promising agent for biomedical applications. On the other hand, there are also disadvantages such as low mechanical strength and slow release of the drug. So more research is provided to enhance HA properties and fit features to applications.

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4 Tissue Engineering Likely, more bone defects are consequences of Accident, osteoporosis, neoplasms, osteoarthritis, tissue infection or bone tumours. Bone defects might lead to bone loss or surgical removal in some cases. To regenerate these defects and bring the functionality of the injured parts back, bone substitution is required. In general, bone substitute origin is divided to three groups involving autologous (the most desirable), allograft and bone graft. Bone tissue engineering is an effective and risk-free alternative offer to autografts and allografts treatment (replacement). Bone graft has more advantages over other substitute, merits such as low cost, availability, lack of probability of pathogen transmission (like HIV and hepatitis virus) and also immune rejection are reported. The bioengineered biomaterials are typically in the form of prostheses, scaffolds and hydrogels. In fact, slow resorption of an implant which is used in bone healing is desirable in certain circumstances, while resorption rate has to be set to tissue regeneration rate. If the solubility rate of implants is higher than the tissue regeneration rate, the application will be limited to use in the bone cavity and defect filling (Best et al. 2008). HA usually used as a bone substitute in granular structure and pore form (with tuneability), this structure and form is an ideal for attachment of cell and their migration until bone formation. Since 1981 HA has known as an ideal bone substitute for bone regeneration due to its advantages like the superior biocompatibility, similarity and bioactivity, affinity to host hard and soft tissues, slow biodegradability in situ, osteoconductivity, osteoinductivity, osteoblastic differentiation and growth-promoting function are also recognized as advantages. HA applications in the tissue engineering is a broad territory from acting as a filler for bone scaffolds (Khanna et al. 2017; Antony et al. 2018; Raucci et al. 2018) to metallic implants coating (Chakraborty et al. 2017; Ke et al. 2017; Yan et al. 2017; Furko et al. 2018) and self-setting bone cements (Sato et al. 2017; Dorozhkin 2018). Physically, HA is accessible in many forms like particles, dense blocks, porous scaffolds, granules, powders, implanting coating and composite component in the specific shapes mostly for the orthopaedic applications. HA is prepared both from natural sources and synthetic methods. Tissue engineering is going to develop bone substitute with biocomposites’ help which has the power to restore and improve tissue functions. To achieve this goal there are several requirements such as porous scaffold which has enough space for cell growth and mimics the biological function beside the specific structure of the host extracellular matrix. Not only in the physical structure but also in the chemical composition, also bioactive surface for cell adhesion, proliferation and differentiation of osteoblast cell have to be provided to achieve an efficient treatment. As far as bone tissue engineering develop, weaknesses and defects of HA particles be more obvious such as slow degradation rate, low mechanical strength, weak intensity, brittleness, fatigue failure and disability to induce vascularization.

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However, the insufficient mechanical strength of pure HA restricts its application to those which only require low-bearing applications, researchers suggest to fabricate composition to solve this problem and match all needs in a system, so there are challenges to match mechanical properties to biological features. Therefore, toughness, poor tensile strength and weak wear resistance become major obstacles for potential clinical applications. Exploiting graphene and its derivative as reinforcement to hydroxyapatite composites has been studied recently (Li et al. 2018). nHA/polymer composite is a promising agent for overcome these shortages. Polymers are high elastic modulus and able to fill lack of stress shielding in ceramic biomaterials that are easy to fabricate and control on mechanical properties and degradation rate is possible so the characteristics properties are tailor-made. Facility of fabricating polymers made researchers able to construct materials in the exact shape and size. Natural polymers which commonly use in bone healing regeneration are included polysaccharides, proteins and polynucleotides and lots of investigations have proceeded to examine their combination in the multiplex system consists of polymers and bioceramics like HA to developed bone substitute (Fig. 5). For decades, synthetic polymers have attracted much attention as they longevity in term of shelf life although the synthetic polymer biocompatibility has to be under attention. There is a well cellular affinity for the natural polymer, on the other hand, synthetic polymer exhibit better mechanical strength (Sanjay et al. 2018) and tuneable degradation rate. Although in the many cases natural polymers exhibit admissible biocompatibility and bioactivity, the probability of being denatured under scaffold preparation is always there and necessary care must be kept as well. The porous structure is required to provide enough space to promote bone ingrowth, mesoporous structure facilitate mineralization and osteoid formation by expansion osteoprogenitors and osteoblast migration inside the scaffolds, enhance-

Fig. 5 Schematic of HA application in bone regeneration

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ment of nutrient diffusion and vascularization can be obtained by interconnected microporous and it has been shown that porosity is adjustable by polymers. The pure form of HA has a brittle nature and makes desirable fabrication in the size and shapes hard. HA-chitosan combination is well studied, chitosan obtained from chitin under alkaline conditions. Chitosan like the other derivates of chitin has inherent antibacterial activity in addition to biocompatibility and biodegradability. Chitosan uses in many applications such as tissue regeneration and wound healing. It’s known as an excellent composition for bone tissue engineering because of having pivotal factors in bone regeneration applications. HA conjugate to chitosan through the interaction of calcium ions and the amine group of CS residue which, provide nucleate sites for HA crystals that initiate growth and make mechanical fixation. HA-chitosan exhibits more biocompatibility than pure chitosan. Collagen is the major constituent of the bone and is familiar to have degradation characteristics, cells attachment, and excellent biocompatibility. col-HA composite matches the HA’s inherent bioactivity with cell migration and binding feature of collagen to elevate osteogenic differentiation. By adding collagen to porous HA mechanical properties can enhance due to a reduction in porosity. Fracture energy is increased through the formation of H-bond between HA and collagen. Hyaluronic acid is suggested to be added to HA composition, to provoke cell differentiation and proliferation since hyaluronic acid has a significant role in the cell signalling pathway in addition of properties such as elasticity and antimicrobial activity. HA-hyaluronic acid interaction has studied for years. Polylactic acid (PLL) is a synthetic polymer and generally exhibits high Young’s modulus and tensile strength. There is 4 form of PLAs base on the chirality of lactic acid and each one has a different amount of tensile strength and degradation rate which application can be determined by these properties. For example, poly L-lactic acid (PLLA) used in an orthopaedic fixation device, 4.8 GPa tensile strength and degradation rate up to 5 years in vivo. On the other hand, poly D, L-lactic acid (PDLLA) has lower tensile stress (1.9 GPa), but a faster degradation rate which makes them suitable for drug delivery applications. PLA/HA nanocomposites have been used for both scaffold material and drug carrier to deliver drug and protein to target that mainly is hard tissue in bone regeneration. Furthermore, PLA/HA rate in this composition can moderate mechanical properties. The degradation rate significantly depends on the process temperature. For example, Young’s modulus of composites would gradually decrease when the process developed at 220c for 5 min. The composite which consists of 70% HA shows more similar bending strength and fracture toughness to the bone than composite with 80–85% HA. Biocompatibility of PLA/HA has been confirmed via different cell lines such as MG-63 osteosarcoma cells, MC3T3-E1 and L929 fibroblasts. Cell adhesion, cell numbers, osteocalcin expression and osteoblast differentiation increased significantly in each line. Collagen also examined as a promising addition to enhance biomaterial properties as it can act as a template that HA form on it. Collagen negative charge that came

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from carboxylate group play an important role and has a high effect on the nucleation of HA crystals on the membrane of collagen. Collagen-HA interaction can promote the mechanical properties like elasticity. Gelatine which is derived from collagen and has many usages in pharmaceutical, cosmetic and food applications. HA-gelatine composite also has been well studied. Polylactic acid (PLA), polyglycolic acid (PGA) and poly (lactic-co-glycolic) acid (PLGA) are aliphatic polyesters with biodegradability feature which have been severally studied as a member of bone substitute composite. In addition of biodegradability PLA has shown thermal plasticity and in combination to HA would possess mechanical strength. Adding a second phase as a reinforcement suggest as a solution to proper adequate fracture toughness and wear resistance. Carbon nano-tubes were introduced as an effective reinforcement. Several methods are reported to coat HA and HA composite on implant surface like electrophoretic deposition, plasma spraying, spark plasma sintering (sps), hot isostatic pressing, and aerosol deposition and laser surface alloying Biocompatibility of HA also can be enhanced by the use of some reinforcement like carbon nano-tube, gelatine, carboxymethyl cellulose, pectin, hyaluronic acid graphene oxide and montmorillonite which indicate an important role to improve bioactivity. HA coating on the other implants is an alternative solution to provide tough biocompatible implants by coating metal implants with HA to improve bioactivity and biocompatibility. Plasma sprayed HA coatings have known as major innovations in the last decades. The results show HA coatings enhanced lifetime implantation device in compare to uncoated this advantage is durable due to the expanse of lifetime and high demand in a young patient. De Groot et al., Furlong and Osborn are the very first developer of plasma sprayed HA coating, who published their work about 20 years ago (De Groot et al. 1987; Furlong and Osborn 1991). Osteosarcoma is a highly aggressive and lethal cancer. Osteosarcoma cells are sustainable in the presence of nano-HA due to the activation of caspase-9- pathway and suppression, and apoptosis is depending on nanoparticles’ sizes, where larger ones are more effective. The scaffolds also can be loaded with a wide range of drugs to boost functionality of biomaterials, since the bonding time in relatively long (Oonishi et al. 1999), biomolecule entities were suggested to shortage bonding time, for example, loading of vancomycin with a short release rate has studied by Martinez-Vazquez and exhibit inhibiting of bacterial growth around scaffold. Controlled release of growth factored also checked out in several investigations. There are many regulating factors, which control bone regeneration process like vascular endothelial growth factor (VEGF), bone morphogenetic proteins (BMP), fibroblast growth factor (FGF), insulin-like growth factor (IGF-1) and plateletderived growth factor (PDFG-BB) and all are well studied in the bone regeneration process. Addition of growth factors like VEGF and BMPs to induce vascularization but they are high cost and has low stability. Deepak Kumar Khajuria et al. synthesized a nitrogen doped carbon dots (NCDs) conjugated with HA, and demonstrated the

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osteogenic potential of NCDs-HA by a zebrafish jaw bone regeneration model and MC3T3-E1. The nanoparticles significantly indicate an enhancement in alkaline phosphatase activity, and the expression of genes that are involved in osteogenic formation, in addition, bone regeneration in zebrafish enhanced (Khajuria et al. 2018).

5 Antibacterial Activity As Stamm et al. reported, implant surgery is associated with about 50% of infections that occur in the hospital (Stamm 1978). The use of implant increase daily and adding an antibacterial feature to them is a desirable approach because Materials and tissues integration have to start without any bacterial adhesion or biofilms formation (Zimmerli et al. 1984). On the other hand, antibacterial resistance made it more complicated to remove infections so there are few alternatives to overwhelm antibacterial resistance. A comparative study is brought in Table 2. Incorporating of metal ions are known as a preventive method, for example, silver ions which inhibited the enzymatic uptake of phosphate that lead to the structural changes of DNA (Schreurs and Rosenberg 1982; Yang et al. 2009) or releasing reactive oxygen species (Kim et al. 2007). Placing an implant into the body can raise the oxidative stress in the environment of implants, the oxidative stress causing delayed healing, apoptosis, and implant failure that the last one leads to the bacterial infection. Panday et al. investigated a matrix based on HA that contains ceria (IV) and Ag NPs and reported the matrix effect on reducing ROS levels, and antibacterial efficiency. ROS scavenging is provided by Ce3+ , and the Ag NPs provide antibacterial properties (Pandey et al. 2018). BS Gholizadeh et al. fabricated a nanocomposite that contains HA and sodium alginate with different amount of HA (1 up to 5%). they examined the HA effect on physical and mechanical properties and antibacterial activity. Nanocomposite consist 5% HA demonstrated the highest antibacterial activity in counter to the foodborne pathogen (3 CFU/mg reduction). Addition of HA improved tensile strength and elongation but also reduced water solubility and vapour permeability by 50% (Gholizadeh et al. 2018). D. Nancy et al. modified pure titanium with two layers that first contains TiO2SrHA (TH) and second contains Chitosan/Gelatine with the incorporation of vancomycin (THV). THV samples showed enhancement in cell attachment and decrease in bacterial adhesion. The nanocomposite indicates higher antibacterial activity than free drug, in the concentration of 2.74 μg vancomycin (Nancy and Rajendran 2018). Vuk Uskokovi´c et al. construct a Hydroxyapatite-Gelatine-Silica nanocomposite to overwhelm defects such as the speedy release of drug and inappropriate pore size that are not large enough to provide space for cell growth. The gelatine and silica causing more pore formation on the surface in the physiological fluid that leads to a sustained release of the drug. The composite has promoted antibacterial activity without harmful effect on the host cell (Uskokovic et al. 2017). Mónica Cicuéndez et al. prepare a three-dimensional scaffold with a multifunctional-therapeutic feature that is able to eradicate biofilm while enhancing

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Table 2 Antibacterial loaded HA, drug and purpose HA system used

Drug (Antibacterial agent)

Bacteria

Reason/advantage References

Hydroxyapatite nanowires

Silver nanoparticles and ciprofloxacin (CIP)

Escherichia coli., Staphylococcus aureus

High flexibility, high drug loading capacity (447.4 mg/g) sustained and pH-responsive drug release

Xiong et al. (2017)

Silver-doped hydroxyapatite

Silver doped

Staphylococcus aureus

Addition of silver increased the antibacterial activity

Riaz et al. (2018)

Antioxidant ceria (CeO2 ) and antibacterial silver (Ag) reinforced hydroxyapatite (HA) composite

Silver (Ag) reinforced hydroxyapatite (HA) composite

Escherichia coli., Staphylococcus aureus

Combat Postimplantation oxidative stress and bacterial infections

Pandey et al. (2018)

Antibacterial ion substituted calcium deficient hydroxyapatite (CDHA) nanoparticles

Zinc, silver, and strontium doxycycline

Staphylococcus aureus and Escherichia coli.

Nanoparticles of length 40–50 nm and width of 5–6 nm The release of antibacterial ions was studied over a period of 21 days A long-term antibacterial activity

Sampath Kumar et al. (2015)

Calcium-deficient hydroxyapatite (CDHA, Ca/P  1.61) and tricalcium phosphate (beta-TCP)

Tetracycline and ibuprofen

Biocompatible with significant antibacterial and antiinflammatory activity

Madhumathi et al. (2018)

NanoAntibacterial gallium/hydroxyapatite nanospheres of nanocomposite elemental gallium

Pseudomonas aeruginosa

Enhance biocompatibility and alternative for cytotoxic

Kurtjak et al. (2016)

Dense circular HA disks

Streptococcus sanguinis Actinomyces viscosus Streptococcus mutants

Improved retention and antibacterial efficacy for oral pathogen control

Huang et al. (2016)

(HA)-binding antimicrobial peptide (HBAMP)

(continued)

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Table 2 (continued) HA system used

Drug (Antibacterial agent)

Bacteria

Reason/advantage References

HA, nanorods

Monocyclic Nthio-substituted β-lactams (monocyclic Nthio-substituted β-lactams)

Staphylococcus aureus Escherichia coli.

Strongly inhibited the bacterial growth of both methicillin resistant and methicillin susceptible clinical isolates of S. aureus from surgical bone biopsies

Giacomini et al. (2017)

nHA with a diameter of 200–700 nm nHA/PCL (electrospun polycaprolactone)

Tetracycline hydrochloride (TCH)

E. coli. and B. cereus

Higher surface roughness and lower mechanical properties than PCL nanofibers TCH/nHA/PCL membrane exhibited increased drug release behaviour than TCH/PCL membrane

Hassan and Sultana (2017)

bone regeneration. HA embedded into the scaffold and mesoporous loaded with levofloxacin as an antibiotic. The drug release significantly increases under pH infection (6.7 and 5.5) which demonstrate pH sensitive reaction.

6 Ions Substitutions There are several methods to incorporate metal ions into HA such as precipitation (Ma et al. 1994; Kim et al. 1998; Stani´c et al. 2010), coating methods (Zhang et al. 2018), and spraying (Ke et al. 2017). Different methods have different effects on metal ion distribution so the biological response and antibacterial properties are different. Pure HA particles consist calcium (Ca2+ ), phosphate (PO4 3− ) and hydroxyl (OH− ) groups, the ions can be substituted by other ions which have affected crystallinity, solubility, thermal stability, lattice parameters and crystal morphology. Chlorapatite (Fahami et al. 2013; Nasiri-Tabrizi and Fahami 2014) and fluorapatite (Wei et al.

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2003; Jarlbring et al. 2006; Shanmugam and Gopal 2014) are well-studied examples of ions substitution. Despite providing structural support and biocompatible material for healing bone, the presence of therapeutic agents in scaffolds are necessary to control local infections or post-implantation treatment for improved bone regeneration and inhibit systemic infection are required. Due to the insufficiency of systemic drug delivery to deliver planned concentrations of drugs, local drug delivery introduced as an alternative by having advantages like the limited risk of overdose, and high efficiency and control release rate and period. Covalent attachment and self-assembly are conventional methods to immobilize drugs on scaffolds, whereas the capacity of loading drug is related to the surface volume ratio and pore size. Immobilization of drugs can limit the drug activity. Berit Mueller et al. examine an open-porous Hydroxyapatite/lysozyme scaffold, scaffold obtained by a one-pot freeze gelation and different amounts of loaded lysozyme were tested. One-pot freeze gelation method is reported as a one-step method to produce protein loaded scaffold without deploying damage to biomolecules. Their composition implanted into domestic pigs without inflammation, resorption of material was over 50% and new bone formation of 21% after eight weeks.

7 Dental Treatment The alveolar bone loss is a consequence of the severe form of periodontitis which is a kind of oral disease (Mombelli 2003). In general treatment, high dosage of antibiotic administers systematically for a long duration (Slots and Ting 2002). To treat kinds of infections which cause vascular damage, finding a suitable alternative for delivering the antibiotic is necessary and the novel drug-delivery system has to be able to transmit intended dosage of the antibiotic because the parenteral administration of the effective concentration of antibiotics locally is difficult (Etienne 2003; Hanes and Purvis 2003; Sundararaj et al. 2013; Bansal et al. 2018). The similarity in the chemical composition of HA and biological hard tissue such as bone and tooth causing the bio-conduction of HA particles to hard tissues for remineralization (Otsuka et al. 1994; Krishnan et al. 2015; Wu et al. 2015; Kolanthai et al. 2016). Coupling the drug loading capability with controlled release rate and the possibility of inducing osteointegration, is desirable for treating bone defects and periodontal disease. The particle size of HA is a determining parameter for cell activity and injectability (Gauthier et al. 1999). The proper hydroxyapatite for biomedical applications has to have features like the uniformity in particle size at the nano range, slow agglomerated rate, and phase homogeneity. The pathogen that is supposed to be eliminate in the periodontal disease, is the Indicating factor for choosing appropriate antibiotic (Durgesh et al. 2015) Since there

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is a wide range of microorganisms in oral microflora, about 400 different species, and only a few of them are the pathogen that has to be eliminated (Ferraz et al. 2007). M.P. Ferraz et al. report the release kinetics and antimicrobial activity of antibioticloaded HA with different porosity. The HA particle which has higher porosity also has more stable release rate than the particle with lower porosity (Ferraz et al. 2007). The control data in this paper indicate that pure HA microparticle has no antibiotic activity. Preventing dental decay and surgical free treatment of incipient lesion enamel caries are a novel approach in contemporary dental research. So many efforts have done to introduce an agent that has an ability to remineralize subsurface enamel lesion, but additional investigations are required to achieve the agent (Azarpazhooh and Limeback 2008). A vast number of investigation suggest delivery of bioavailable calcium and phosphate ions into the lesion to induce remineralization (Huang et al. 2011; Elkassas and Arafa 2014; Cai et al. 2018). An obstacle to deliver remineralizing ions to the subsurface of the lesion is the presence of a dense mineral layer on the top of the carious lesion that blocks transportation of incoming ions to the core of the lesion (Larsen and Fejerkov 1989). A significant number of the investigation evaluated the effect of HA particle on remineralization and reconstruction (Mahdi et al. 2018; Roveri et al. 2008; Jayasree et al. 2017) and also HA potential to act as an preventing agent in dentistry (Hannig et al. 2013). HA particles were used as an additive to toothpaste and mouthwash (Tschoppe et al. 2011; Hiller et al. 2018; Vano et al. 2018). Andrej M. Kielbassa et al. studied showed the similar remineralization capacities for different HA contained toothpaste (Tschoppe et al. 2011). S. Huang et al. by cross-sectional microhardness (CSMH) tests and polarized light microscopy (PLM) indicate that nano-HA has higher remineralizing potential than micro-HA and mineral deposition occur mostly on the top layer of the lesion and has a low potency to decrease lesion depth. A detailed investigation using pH cycling conditions demonstrate that remineralization effect gets higher when pH is less than 7.0 (Huang et al. 2011). Nano-HA properties have a relation to environmental pH change (Olsson et al. 2000). Bacterial adhesion on tooth surface and acid associated demineralization are two of major reason for dental problems and decay, an alternative strategies to prevent adhesion suggested to affect the thermodynamic, physical, and electrostatic interactions that provoke microbial adhesion to interrupt their interaction (Besinis et al. 2015; Kensche et al. 2016). Kensch A et al. investigated antibiofilm activity and anti bioadhesion effect of HA in situ on bovine enamel and bacterial adhesion was measured by DAPI staining. Result show significant reduction of adhesion in presence of HA but no observable effect on streptococcus mutants viability (Kensche et al. 2017). Shahmoradi M et al. design a method to achieve stable HA nanosuspensions for remineralization of enamel caries. HA prepared by wet chemistry than transfer to a high-pressure homogenizer. The obtained HA particles size were about 20–40 nm. The nanosuspensions indicate higher remineralizing efficiency than the control group (Shahmoradi et al. 2018).

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The remineralizing of enamel depends on physiochemical mechanism since there is no cellular mechanism to repair enamel caries. For years, fluoride has known as a beneficial agent to repair early lesions. Kulkarni VK et al. examined the efficiency of nano-HA as an alternative to fluoride. The result demonstrates that 10% of nanoHA has a remineralization effect similar to 1000 ppm fluoride, so nano-HA can be considered as an alternative to fluoride. Fluoride has used for widely for remineralizing of early carious lesion, Vyavhare et al. design an experiment to study (remineralizing enamel and make artificial lesion, and treat with HA solution at pH cycle) the effect of HA as an alternative for fluoride, the experiment shows the effect of HA on remineralizing but they conclude that it has to use as an additive to enhance the fluoride therapy (Vyavhare et al. 2015). In another study, Juntavee et al. examined the potency of HA gel on remineralizing of enamel cementum by computer-aided design and manufacturing. Analysing of surface micro-hardness (SMH) date indicate an increase in the demineralized enamel after treating with HA gel (Juntavee et al. 2018). Vicker test is a common test for asses SMH. Ali Nozari et al. compared the ability of three remineralizing agents include NaF varnish, Nano Silver fluoride (NSF) and n-HA on remineralize enamel. The results show a significant superiority for NSF effect, and a similarity in the effect of n-HA and NaF. A protective layer was formed in all cases as AFM images showed (Nozari et al. 2017).

8 Implant Hydroxyapatite implants made from sea coral are treated so that their structure and the chemical composition becomes nearly identical to natural bone which is used in plastic surgery. The advantage of these implants is osteoconduction, osteointegration, and porous nature that allow tissue integration. In addition, these implants are usually intensely heated and therefore are no immunogenic reactions. There are several methods to coat HA on metallic implants to improve biofeatures include laser pulse deposition, electrochemical and electrophoretic deposition, etching associated with sandblasting by aluminium dioxide or titanium dioxide, and plasma spraying. The mentioned methods are complex and expensive so more research is needed to introduce modification on these methods or develop new facile and cheap methods which be universal. Coating by phytic acid-metal complex multilayer was developed by Wang Q et al., the formed crystal by this group improved the osteogenic ability and biocompatibility of MG63 cells, they suggest this method for bone implants and orthopaedic applications (Predoi et al. 2016). In the other hand, Joo L.ong and Daniel C.N Chan discussed the important issues for in vivo, and their clinical development is briefly summarized. Although there are benefits in using HA, disadvantages are also seen, for example, risk of dissolution might increase while HA used as coating and the susceptibility of bacterial infection

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in HA coating increase in the camper to titanium implants, beside this fracture can occur on the surface. To provide proper HA for clinical usage attention have to payed on some properties such as chemical composition, coating thickness, the effect of crystallinity or crystallite size and porosity, adjusting these properties lead to the HA which is optimum in biological and physical properties (Ong and Chan 2000). Biological performance of chemical hydroxyapatite coating associated with implant surface modification by a laser beam (Faeda et al. 2009). To overwhelm deficiencies of pure HA coatings, investigators suggested substitution of a dopant into the HA structure. Sahar Vahabzadeh et al. doped strontium (Sr) into plasma sprayed HA to and checked out the changes on the protein release kinetics (new generation of HA coating are supposed to deliver biomolecules), dissolution behaviour and crystallinity. The Sr-HA has a lower crystallinity and higher dissolution rate than pure HA, this result illustrate that the HA properties are able to be tailor through ion substitution and ion dopant (Vahabzadeh et al. 2015). Zhou-Shan et al. prepared four groups of Sr-HA with different amount of Sr (0, 5, 10, 20%), 20% Sr coating displayed best osseointegration property (Tao et al. 2016). Sr-substituted HA also exhibits higher osseointegration in compare to zinc and magnesium substitute (Tao et al. 2016). Apart from ions substitution, drug and biomolecule have loaded on HA coating to explore the effect on healing, for example, parathyroid hormone was loaded on Sr-HA coating and significantly increased the osteointegration ability on rat samples (Tao et al. 2016). As were mentioned on preparation part HA particles can be achieved both with chemical synthesis and extraction from biosources. A comparative study is done by Karthik Alagarsmy and revealed the differences. HA nanoparticles which provided from co-precipitation had higher crystallinity than goat femur bone extracted HA(cHA) and control of morphology was available in n-HA. Both particles were active in vivo but n-HA showed better performance (Karthik et al. 2018). β-tricalcium phosphate is another member of CaP family that is used for bone repair, it shows interfacial compatibility and osteoinductive characteristic, but osteointegration have to be added to its features. Chen Q et al. suggest a coating of HA on it, the collected data demonstrate improvement in the osteointegration and Osteogenesis (Chen et al. 2018). HA is also used to stabilize β-TCP blocks in the defected part and make β-TCP blocks able to be used in reconstructing surgery (Sakamoto et al. 2018).

9 Drug Delivery Among several components of calcium-based bioceramics, hydroxyapatite (HA) is recognized as a proper analogue to apatite which exists in the natural bone structure. In term of the advantages of HA (reaching third generation of drug carriers Fig. 6 and Table 3), it can target the damaged area of bone and deliver intended drug such as stem cell containing biomaterials (Suchanek and Yoshimura 1998; Son et al. 2011;

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Kang et al. 2013). This bioceramic can associate the drug molecules physically and chemically and release them under control in a favourite time scale (Yunoki et al. 2011; Uskokovi´c and Desai 2014). Since numerous elderlies all over the world suffer from a variety of bone injuries, therefore, bone tissue engineering is applied tremendously to help the patients to improve the quality of life (Wei and Ma 2004; Wahl and Czernuszka 2006; SadatShojai et al. 2013; Dorozhkin 2015). There are numerous drugs which are not proper clinically because of insolubility, separation of phases and toxicity (Yih and Al-Fandi 2006; Sun et al. 2018). Several kinds of biodegradable Nanocarriers have been reported for drug delivery. For instance, organic silica, hydroxyapatite (HA) nanoparticles and polymeric nanoparticles. Among them, HA has been recognized as the most favourite material to design drug carriers (Kong et al. 2016; Xiong et al. 2016). HA with a hexagonal structure (Cui et al. 2014), can be extracted easily in animal bones, eggshells and codfish bones (Ha et al. 2015; Hamdy et al. 2016). Indeed, a pure HA surface bioactivity causes the lack of biofunctionality of HA which is considered as a disadvantage in HA-mediated drug delivery. To combat this negative function, the loading of the anticancer drug into hydroxyapatite structure may intensify the interaction force between drug and HA in order to reduce the drug leakage and several composites such as chitosan (Zhao et al. 2002), agarose (Khanarian et al. 2012), collagen (Meagher et al. 2016), polyesters (Zhang

Fig. 6 Three generations of nanoparticles engineered for biomedical applications Table 3 Different types of HA used for drug delivery Porous HA types

Anti-cancer drug carriers

Nanoparticles

Dietylenediamineplatinum medronate (Palazzo et al. 2007) Biphosphonate alendronate (Palazzo et al. 2007) Paclitaxel (Venkatasubbu et al. 2013)

Block

Cis—diamminedichloreplatinum (II) (Uchida et al. 1992; Netz et al. 2001) Methotrexate (Itokazu et al. 1998b)

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et al. 2009b; Hu et al. 2014), cellulose and its derivatives (Kong et al. 2005; Kwak et al. 2014; Lukasheva and Tolmachev 2015), has been developed. A new version of porous HA scaffold containing PLGA microsphere accompanied with Dexamethasone to deliver inorganic calcium phosphate in order to generate bone tissue in vivo. This model has excellently affected the efficiency in Dexamethasone delivery. Most studied have shown that hydroxyapatite could result in drug leakage during blood circulation (Kundu et al. 2013). Penetration of drug into the blood can cause severe side effects and also reduce its bioavailability. To overcome this problem, hydroxyapatite Nanocarriers with drug loading inside the materials had been applied (Sun et al. 2018). Doxorubicin (Dox)-loaded hydroxyapatite Nanorods consisting of folic acid (FA) modification (DOX@HA-FA) were recognized as a powerful anticancer treatment. This modified drug Nanocarriers, DOX@HA-FA Nanorods, were developed to suggest a new template to load drug in hydroxyapatite materials. In fact, in the microenvironment with folate receptors, endocytosis happened and the nanorods exhibited an increase in cellular uptake (Fig. 7), more degradation. So, the proliferation of targeted cells was inhibited. DOX@HAFA illustrated good stability in neutral solution, but release under low pH conditions with no apparent side effects (Sun et al. 2018). Cancer is categorized in the group of disease which medical world deals with as the second cause of the death that is a really big challenge (Almeida et al. 1998; Barakat et al. 2009; Bian et al. 2010). Recently, using Nano-sized particles to attack cancer cells attracted the attention (Yoo and Park 2004; Sumer and Gao 2008; Bamrungsap et al. 2012; Elsabahy and Wooley 2012; Chan et al. 2017). To enhance the tumour accumulation, Nanocarriers can be activated with a tumourtargeting group which enable them to target cancer cells (Wei et al. 2013; Park et al. 2015; Wei et al. 2015). In an experiment, Macroporous HA block was able to release the drug slowly, which was up to 42 and 18 days only (Liu 1996; Itokazu et al. 1998a). On the other hand, the specific properties of the drugs and the morphology of the HA nanoparticles affected the uptake and release kinetics of the drugs. The negatively charged alendronate was strongly adsorbed, while the neutral DPM complex showed a lower affinity towards the negative surface of the HA nanoparticles (Fig. 8). Different mechanisms of drug loading have applied on HA nanoparticles (Fig. 9). Among them, porous HA nanoparticles are appropriate options for anticancer drugs. Comparison between needle-shaped and plat shaped porous HA nanoparticles show that drugs desorb faster from the needle-shaped HA. In fact, the surface charge of drugs and the morphology of the HA nanoparticles play a key role in the association and dissociation kinetics which is a pH-sensitive process. HA sensitivity to pH acts as a factor degradation into calcium and phosphorous elements under weak acidic condition (Xie et al. 2014, 2016; Munir et al. 2018). In recent years, MgO has been applied as a microcarrier to facilitate the delivery of HA toward the HepG2 cancer cells (Awwad et al. 2017). In a typical experiment, the ability of ZnO–MgO (bimetal) as a powerful drug carrier for cancer cells was observed (Sun et al. 2018).

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Fig. 7 Schematic showing of HA nanoparticles loaded with DOX (drug) delivery process into the tumour cells

Fig. 8 Schematic representation of negative charged drug molecules association with the positive site of the HA and positive charged drug molecules association with the negative site of the HA

Fig. 9 Different loaded agent on HA delivery agents. a Gold dotted Hydroxyapatite for therapeutic and diagnostic application. b The multilayer coated HA, conjugated with alendronate for bone regeneration. c Schematic figure of lactoferrin absorbance on HA, the interaction at pH 9 is not so strong since the electrostatic charge is mix on HA surface so Lf removes more easily after washing

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Fig. 10 Schematic diagram of the porous HA scaffold containing Dex-loaded PLGA

Cellulose/HA-nanocomposites have good cytocompatibility and relatively high protein adsorption ability toward haemoglobin. These data indicate that the cellulose/ HA nanocomposites are promising for using in different biomedical fields such as tissue engineering and protein/drug delivery (Fu et al. 2018). It was believed that in vivo bone regeneration could be jumped with HA scaffolds containing DEX-loaded PLGA poly (lactic-co-glycolic acid) microspheres compared to the use of HA scaffolds alone. With modification in form of PEI coated on PLGA microsphere surfaces, resulting in a net positive charged surface. Of this microsphere surfaces, DEX-loaded PLGA were immobilized on the negatively charged HA scaffold surfaces (Fig. 10). Data illustrated enhanced volume and quality of new bone formation when compared to defects drug with HA scaffolds alone (Son et al. 2011). These days, HA is recognizing a favourite molecule for gene therapy. Activated hydroxyapatite Nanorods with polyethylene mine (PEI) with Varying amounts of EGFP encoding DNA Which were added applied in order to dispersion and the dispersion stability was monitored by dynamic light scattering. In HeLa and MG-63 cells, the surface zeta potential of the cationic HA-PEI delivery system was reduced. The Nanorods accompanied with small amounts of DNA illustrated higher positive zeta potential and better cellular uptake by the negatively charged cell membrane (Klesing et al. 2012). Since the cell membrane is negatively charged, therefore, positively charged nanoparticles lead to the higher degree of attraction due to the ionic interactions between positively charged particles and cell membranes (Gratton et al. 2008; Wang et al. 2015). In addition, an investigation reported that the positively charged nanoparticles might escape from lysosomes and show perinuclear localization, while the negatively and neutrally charged nanoparticles experience fusion with lysosomes (Rabinovich-Guilatt et al. 2004; Vasir and Labhasetwar 2008). As a result, conjugating the nanoparticles with special functional groups can increase the cellular uptake and thus the transfection efficiency of the delivery system (Wang et al. 2015). In an investigation, hydroxyapatite nanoparticles (HANP) which was synthesized by hydrothermal method (Zhang et al. 2009a; Guo et al. 2011; Maia et al. 2016), zeta potential is altered in pH lower than 3.45 which can be defined as the isoelectric point of these nanostructures. The high specific surface area plays a key role in HANP application as bioactive molecules for drug delivery. In this specific study, the high surface area present

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Table 4 Averages obtained water used for sample dilution and VCR incorporation into HANP after modification of pH value (pH 3.4 and 5.0) pH of the purified water used for dilution

Mean concentration (μg/ml) ± SD

VCR incorporated mean concentration (μg/ml) ± SD

Incorporation efficiency (%) ± SD

3.4

25.70 ± 0.92

5.96 ± 3.93

5.92 ± 3.92

5.0

24.25 ± 0.95

1.24 ± 0.95

1.88 ± 5.27

7.4

25.23 ± 0.61

2.61 ± 6.31

3.06 ± 0.36

by HANP may favour the incorporation of VCR (vincristine) high concentration (Table 4). VCR-loaded HANP displayed inhibitory role both tubulin polymerization and mitotic spindle formation in cancer cells. However, anticancer drugs efficacy in a bone metastasis can be studied so as to prove the capacity of VCR-loaded HANP as a drug delivery system (Maia 2018). In another study, well-designed NHA (amine-functionalized hydroxyapatite) nanoparticles were properly prepared for the delivery of p53 and candesartan (CD) (p53/CD/NHA) nanoparticles to treat the breast cancer. The obtained NHA nanoparticles with suitable amine groups supported effective condensation, and the p53/CD/NHA nanoparticles with small particle size, positive charges illustrated the excellent loading of drug and gene in vitro. Also, NHA nanoparticles had almost no cytotoxicity (Zhao et al. 2017). In conclusion, desirable drug delivery systems are expected to open new windows to overcome the challenge of incurable disease. HA can amuse researchers from several fields which is proved that necessity of these kinds of solutions in the medical world. This review contributes to such multidisciplinary aspect to elucidate the HA functions in the field of degradable and compatible biomaterial systems.

10 Conclusion Nanohydroxyapatite is one of the well-studied members of CaP ceramics due to the good characteristics that are appropriate for biomedical applications, such as biocompatibility, nontoxicity, bioactivity and osteoinduction feature. On the other hand, low mechanical strength has limited it’s utilization in bone regenerating to low loaded organs an investigating is provided to achieve HA particles that would be able to tolerate the high load of force to expand HA applications to the sites like backbone and femur. There are several methods to synthesise HA nanoparticle such as dry method and wet method and each method has its own advantages, for example, some methods are simple and are doable with different sources but it is hard to achieve well dispersity. On the other hand, some methods are durable to achieve HA with high homogeneity and narrow dispersity but require high temperature and expensive instruments.

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To fabricate a desirable HA particle, many investigators suggest a combination of methods. Porosity of HA particles is also an important property in both tissue engineering and drug delivery applications, in tissue engineering porous have to proper enough space for cell growth and the pore should be connected to provide cell-cell interactions and initiation of bone formation while biodegradation rate should be math to cell proliferation, choosing pore site must be done with care in these applications because it has a direct effect on decreasing mechanical strength. In the drug delivery issue, porous has to have enough space to achieve highest load capacity of the drug. The Role of HA in preventive and curative dentistry is also studied well, due to the high similarity of HA to teeth HA is known as a promising agent for remineralizing early lesion and cure dentin hypersensitivity. It is used highly as an additive to mouthwashes and toothpaste and promising data have been reported. In this chapter, we reviewed HA nanoparticles, and summarize HA advantages and disadvantages for each application, several investigations and innovative methods are required to achieve the HA nanoparticles that would be utilizable in clinical treatment.

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Nanoparticles for Biosensing Pouria Sarihi, Armin Azadkhah Shalmani, Vida Araban and Mohammad Raoufi

1 Introduction In spite of all advances made in medical intervention over the past few decades, efficient curing of many diseases such as diabetes, Alzheimer’s disease, cardiovascular disease, cancer disease etc. has remained a challenge (Bast 2004). Delayed onset of treatment is one of the main contributors to the failure in treating these diseases to a satisfactory extent. On time diagnosis is an absolute necessity for a triumphant therapy. Incompetent means of the diagnosis result in the late recognition of the malicious situation which leads to an overdue medical intervention (Bast 2004; Gruhl et al. 2013). Some strategies that have been taken into consideration in order to achieve early diagnosis include screening policies in cases like breast cancer. Screening disease-related biomarkers can effectively enhance our ability to accomplish early diagnosis. Disease marker detection methods have been equipped with new technologies such as new gene expression array analysis techniques and advances in proteomics and lipomics (Bast 2004). Over the last decade, biosensors have introduced themselves as alluring devices which can be utilized for the diagnosis and monitoring of diseases that can provide rapid, real-time and accurate results P. Sarihi · V. Araban · M. Raoufi (B) Faculty of Pharmacy, Nanotechnology Research Center, Tehran University of Medical Sciences, 1417614411 Tehran, Iran e-mail: [email protected] P. Sarihi e-mail: [email protected] V. Araban e-mail: [email protected] A. Azadkhah Shalmani Faculty of Pharmacy, Tehran University of Medical Sciences, 1417614411 Tehran, Iran e-mail: [email protected] © Springer Nature Switzerland AG 2019 M. Rahmandoust and M. R. Ayatollahi (eds.), Nanomaterials for Advanced Biological Applications, Advanced Structured Materials 104, https://doi.org/10.1007/978-3-030-10834-2_5

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in a comparatively easy way. Biosensor technology has been greatly influenced by recent progress in nanotechnology science. This led not only to an improvement in the sensitivity and performance of biosensors by using nanomaterials, but also allowed the use of new signal transduction technologies in these devices (Bast 2004; Jianrong et al. 2004; Rasooly and Herold 2008; Gruhl et al. 2013). Biosensors are analytical devices that use a biological recognition element which incorporates a transduction system to generate a measurable signal output proportional to the concentration of the analyte of interest (Arnold and Meyerhoff 1988; Belkin 2003; Wilson 2004; Wilson and Gifford 2005; Eggins 2008). The foundation of biosensors origins from Clark and Lyon’s work on the combination of the electrochemical oxygen sensor (Clark oxygen electrode) and the enzyme glucose oxidase (the first biological recognition element) in 1962. Modification of the structure of this electrode highlighted the need to expand the biological elements. With new studies, other biological molecules like nucleotides, antibodies and even cells proved themselves as suitable bio-recognition elements. These advances led the term enzyme electrode, which just qualified enzyme as a biological element, to be transformed to Biosensor (Kirsch et al. 2013). Nowadays, biosensors are being used in diverse areas such as different fields of medicine, pharmacology, defence, agriculture and food safety, homeland security and environmental monitoring (Kirsch et al. 2013).

2 Biosensor Structure As mentioned earlier biosensors mainly use two systems, consist of biorecognition elements and transduction system, in order to measure the concentration of an analyte of interest. Figure 1 represents a schematic view of a typical biosensor’s structure. The interaction (input signal) between biorecognition elements and sample transforms to an output signal by a transduction system (transducer) with or without the help of an amplifier. In this part, definition, special features and functions of the main parts of biosensors which are biorecognition element and transducer are presented.

Fig. 1 Elements and components of a typical biosensor, with permission from (Chambers et al. 2008)

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• Biorecognition element (ligand) This part of a biosensor is responsible for the selectivity of the biosensor. Such selectivity results from a specific interaction between the ligand and the analyte of interest (Gruhl et al. 2013). Biorecognition Element can be a biologically derived material or biomimetic components (e.g. tissues, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids) that interacts with analyte (Chambers et al. 2008). Most commonly used ligands are Antibodies, Aptamers and peptides (Chambers et al. 2008). It is also notable that in order to use some of the biorecognition elements, physical or chemical techniques should be applied to immobilize these elements on sensor surface (Chambers et al. 2008; Gruhl et al. 2013). • Transducer The transducer is a signal conversion unit which transforms the input signal (resulted from the interactions of the analyte with biorecognition element) to a detectable signal that can be measured and quantified (Rasooly and Herold 2008; Gruhl et al. 2013). The output system is the signal processor, responsible for amplification, processing and producing a user-friendly visualization of the output (Gruhl et al. 2013). Interestingly, the current general trend is toward more complex and integrated multi-analyte biosensors that are capable of more comprehensive analysis. This trend has been utilized by advances in micro/nano electrical and mechanical systems (MEMS/NEMS) (Rasooly and Herold 2008). Biosensors can be classified based on the type of analyte that they interact with or the type of transformation which is happening on their interface.

2.1 Biosensors Categorization Based on the Type of Analyte Direct recognition sensors: In this category, the direct interactions between the target element and ligand are measured. The important difference between direct and indirect biosensors is the labelling process which is not always necessary in direct sensors. Labelling can be used in direct biosensors to amplify the input signal. Some examples of direct biosensors are optical-based systems (surface plasmon resonance) and mechanical systems such as quartz crystal resonators (Rasooly and Herold 2008). Indirect recognition sensors: This type of biosensor’s function is based on the interactions between ligand and a secondary element (label). Secondary element’s concentration is directly influenced by the concentration of the primary element. Interaction signals between label and ligand, are more precise and convenient than the primary element’s interaction. Enzymes such as glucose oxidase (enzyme electrode) and fluorescent labelled antibodies are examples of secondary elements used in biosensors (Rasooly and Herold 2008).

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2.2 Biosensors Categorization Based on Transformations Although biosensor’s transformation systems are diverse, most of them can be categorised as acoustic wave biosensors, electrochemical biosensors, magnetic biosensors, optical biosensors and thermal biosensors.

3 Biorecognition Elements Biorecognition elements can be categorized based on their structure into receptors, enzymes, antibodies, aptamers, peptide nucleic acids (PNA), molecular Imprint and lectins (Chambers et al. 2008).

3.1 Receptors Synthetic receptors are an alluring group of biorecognition elements to mimic the cellular environment in biosensor’s structure. They have interesting properties such as high ligand specificity and affinity (Chambers et al. 2008). Receptors are transmembrane (plasma and intracellular membranes) or soluble proteins in the cytoplasm, which bind to the analyte of interest and cause the desired physiochemical change. The disadvantages of using receptors are their low yield, relative instability and timeconsuming purification process. However, advances in recombinant technology and gene expression systems solved this problem and large-scale production of proteins and modification of their structure is now conceivable (Valdes et al. 1988; Valdes et al. 1990; Subrahmanyam et al. 2002).

3.2 Enzyme-Based Recognition The physiochemical changes by enzymatic reactions can be initiated by protons, electrons, light and heat. The products of these reactions can produce suitable output signal (Barhoumi et al. 2006). As an example, the catalytic turnover of enzyme-fluorescence based techniques can result in amplification of the signal, which increases the sensitivity of the assay (Yanagawa 1999).

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3.3 Antibody-Based Recognition After foundation of the monoclonal antibody technology in 1990 by Kohler and Milstein’s seminal work, antibodies has been used extensively for recognition, identification and quantification processes. The advantage of employing antibodies as biorecognition elements are their suitable sample handling process which does not require a purification step (Jayasena 1999). An interesting example of using antibodies in identification processes is the immuno-PCR method, which utilized numerous PCR steps (Sano et al. 1992).

3.4 Aptamer-Based Recognition Aptasensors are special reagentless ligands which are composed of single-stranded strings of nucleic acids (RNA, ssDNA, modified ssDNA, or modified RNA). They are semisynthetic products that can be easily manufactured. Due to their single-stranded string, they are capable of folding and creating three-dimensional structures. These structures have high affinity and specificity; therefore, they are capable of being used as ligands. Interestingly, Aptamers recognize their target primarily by its shape rather than its sequence. Due to this feature aptamers are very useful for a wide variety of detection purposes ranging from small molecules to proteins and whole cells (Ellington and Szostak 1990; Tuerk and Gold 1990; Luzi et al. 2003; Lim et al. 2005). As mentioned before, Aptamers have great potentials for future studies for multiple reasons. The first reason is their user-friendly feature. They can be engineered unlimitedly and easily (Chambers et al. 2008). Due to their chemical stability and neutrality, they do not require any special conditions like low temperature to maintain their stability (except for aptazymes). They are also low-cost products because of their reusable feature (Chambers et al. 2008). The second reason is their capacity to facilitate the transduction process. They can emit detectable signals with some modifications in their structure (Chambers et al. 2008). In the end, it is valuable to mention that another group of aptamers, called aptazymes, have catalytic activities and can have allosteric properties like enzymes. This property is an advantage which makes both enzymes and aptamers usable as biorecognition element in multi-analyte analysis techniques (Hesselberth et al. 2003).

3.5 Peptide Nucleic Acid (PNA)-Based Recognition Peptide nucleic acid (PNA) are synthetic DNA analogues or mimics which have a polyamide backbone instead of a sugar-phosphate bone (Egholm et al. 1993). This group has considerable thermal stability and permanent shape. Permanent shape of PNAs cannot be easily disrupted by folding. This advantage makes this group

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suitable for detection of cDNAs and RNA sequences (Demidov et al. 1994). PNADNA interactions have great advantages over DNA-DNA interactions due to their thermal stability, their independent interaction with DNA which doesn’t require any salt media and their capability to act in a variety of PH range (Kim et al. 1990; Ersöz et al. 2005; Bertozzi and Kiessling 2001). It is also notable that this group can also exhibit a colour transition in different modes of interaction which can act as a sensitive detection method for discriminating DNA single-base mismatches (Chakrabarti and Klibanov 2003).

3.6 Molecular Imprint Based Recognition Molecular imprints are synthetic polymers that provide selective binding sites for the analyte of interest. Imprints are stable artificial biosensing elements (Chambers et al. 2008). Sample pretreatment is not necessary for Molecular imprint Based Biosensors. Molecular imprint based quartz crystal microbalance detection is an accepted analysing method for detecting a variety of analytes, especially glucose (Ersöz et al. 2005).

3.7 Lectin-Based Recognition Lectins are a class of plant proteins that are chiefly toxic. These proteins bind especially to certain sugars and cause agglutination (Kim et al. 1990). Lectins are excellent biorecognition elements for saccharide moieties (Bertozzi and Kiessling 2001). Concanavalin A (Con A) is one of the widely-used lectins for saccharide detection. Con A has been coupled with specific detection labels (fluorescent dye) as an indirect biosensor for the detection of various analytes of interest. It is also noteworthy that binding of lectin to the target sugar can be used in many biosensors like electrical-oscillation, piezoelectric crystal oligosaccharide and microcalorimetric platforms (Yoshikawa and Omochi 1986; Cardullo et al. 1998; Gemeiner et al. 1998; Wong et al.2002; Nagase et al. 2003).

4 Acoustic Based Biosensors Acoustic based biosensors have been investigated for the detection of a wide range of biological and chemical analytes. Feasibility is the key advantage of these devices which are based on gravimetric sensing of the analyte of interest (Fogel et al. 2016). Acoustic based biosensors utilize acoustic or mechanical waves to cause changes in mass, elasticity, conductivity and dielectric properties (input signal) and then convert variations of these parameters into medical, biochemical, and biophysical data

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(output signal) (Drafts 2001). The majority of these devices use piezoelectric effect to transform acoustic waves into an electrical signal (Lec and Lewin 1998; Drafts 2001; Durmu et al. 2015). New generations of acoustic wave biosensors do not require piezoelectric substrate and they employ capacitive micro-machined ultrasound transducers (CMUTs) (Durmu et al. 2015). Piezoelectricity is a phenomenon which was proposed in the 1880s by Pierre and Paul. They had shown that in certain crystals such as quartz and Rochelle salt, mechanical stress can induce voltage generation and vice versa. Later this phenomenon was used to construct sonar detection systems for submarines (Durmu et al. 2015). Examples of piezoelectric crystals that can be used in acoustic biosensors are quartz, lithium niobate and Lithium tantalate. As can be seen in Fig. 2, two traditional ‘cuts’ for quartz crystal are being used in acoustic sensors (the AT cut and the ST cut). Differences between these two, cause different piezoelectric deformations. Each of these cuts produces a different type of acoustic waves (BAWs for AT and SAWs for ST). Each type propagates efficiently in different directions and has its own pros and cons (Fogel et al. 2016). The transduction process in acoustic-based biosensors is based on the acoustic energy storage or dissipation which happens at the interface. Acoustic resonators exhibit infinite resonant modes of vibration, however the process of storage or dissipation of energy (impact of substrate-ligand interaction) causes frequency shifts and changes in the acoustic oscillation frequency. These variations will play the role of input data for the transduction system (Länge et al. 2008; Durmu et al. 2015).

Fig. 2 Graphical diagram describing processes for the generation of SAWs and BAWs, with permission from (Fogel et al. 2016)

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4.1 Bulk Acoustic Wave (BAW) Devices These kinds of biosensor devices employ either longitudinal or shear waves (Fig. 3). it’s also noteworthy that shear waves are significantly preferred. BAW devices consist of parallel electrodes located on both sides of the thin piece of crystal (Substrate). When an alternating electric field (AC) is applied, the induced potential differences result in shear deformation of the crystal which causes mechanical oscillation of a standing wave across the bulk of substrate. The frequency of the vibrations is dependent on the properties (density, size, and phase) of the piezoelectric crystal and the media which is in contact with the crystal surface (Kaspar et al. 2000; Durmu et al. 2015; Fogel et al. 2016). In this part, a summery on two kinds of BAW devices is presented. • Thickness shear mode (TSM) resonator Thickness shear mode (TSM) resonator also referred to as Quartz crystal microbalance (QCM) is composed of a quartz plate which is inserted between two electrodes on opposite sides. When voltage is applied, electric field crosses through this plate to the electrodes, resulting in a shear mechanical strain or displacement in the quartz (maximum displacement occurs on the surfaces). Oscillation of the voltage frequency can generate mechanical resonance. Variations in the frequency are the inquired data from any interactions with the biorecognition element in acoustic biosensors (Durmu et al. 2015). • Shear-horizontal acoustic plate mode (SH-APM) sensors The difference between SH-APM and TSM (as shown in Fig. 4) is the interdigital transducer which is applied by SH-APM sensors instead of electrode plates, however

Fig. 3 Classification of acoustic waves, SAWs and BAWs with permission from (Lec and Lewin 1998)

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Fig. 4 Elements and components of a typical SH-APM sensor; with permission from (Drafts 2001)

the other parts of their system are the same. Two Interdigital devices are applied in the SH-APM sensor. The first one is the input transducer which converts electric signals to acoustic waves and the second one is the output transducer which converts acoustic waves to electrical signal. The advantages of using interdigital devices are their immunity to corrosion problems (a common problem with electrode biosensors in biological solutions) (Wessa et al. 1998; Drafts 2001; Länge et al. 2008). SH-APM devices are more sensitive compared to TSM devices, because SH-APMs generate greater frequencies but they are also more complex and expensive (Milstein and Das 1977).

4.2 Surface Acoustic Wave Sensors (SAW) This type of biosensor consists of basic components including a piezoelectric substrate (crystals such as quartz, GaAs, or LiNbO3 ), micro metallization patterns (electrodes), interdigital transducers (IDT) and active thin films. The generated acoustic wave propagates along the surface of the solid medium (Drafts 2001; Länge et al. 2008) and its velocity changes due to the mass, temperature, or viscosity variations on its surface. Interestingly these variations are resulted from the interactions which happen on substrate surface (Milstein and Das 1977). We can also apply SAW sensors coated with organic and/or inorganic materials to analyse interactions on the surface of a device (McGill et al. 1998). SAW devices operate at high frequencies, hence they are very sensitive devices. SAW devices can be miniaturized by using photolithographic techniques and applying complex circuits on the substrate surface (Lin et al. 1993; Durmu et al. 2015).

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4.3 Micro/Nano-electromechanical Systems (MEMs/NEMs) Micro/Nanoelectromechanical systems are new technologies that can be applied in biosensors to produce complex devices which have unique properties due to their small or intermediate size (Waggoner and Craighead 2007). Advances like planar fabrication techniques facilitated the use of Micro/Nanoelectromechanical resonators for acoustic biosensing. One of the unique technologies which have been explored a lot is cantilever structure. These devices can be suitably packaged to provide electrical and fluidic access (microfluidics) (Waggoner and Craighead 2007). Biorecognition element molecules may be attached to the surface of MEMs/NEMs with different techniques such as ink-jet printing, micro-contact printing, microfluidic interfacing or other techniques. The Micro/Nano scale of electromechanical resonant sensors have enhanced mass sensitivities of attograms or less (Waggoner and Craighead 2007; Fogel et al. 2016). Advances in NEMs/MEMs have facilitated the detection of living analytes such as living cells, bacteria or viruses even in inappropriate conditions (Waggoner and Craighead 2007).

5 Optical Biosensors Optical biosensors are the most explored type of biosensors. These devices are direct biosensors which provide sensitive, selective and real-time detection of a wide range of biological and chemical substances. The input signal in optical biosensors origins from interactions of the optical field with the biorecognition element (Damborský et al. 2016). Optical biosensors can be categorized into two main groups consisting of direct detectors (label-free) and indirect detectors (label-based). Briefly, in a labelfree mode, the detected signal is generated directly by analysing variations, which are resulted from the interactions of the ligand-analyte complex with optical field. In contrast, in label-based mode, different methods like calorimetric, fluorescent or luminescent techniques are involved in generation of the optical signal (Rasooly and Herold 2008). Different mechanisms are being used in each type of these biosensors.

5.1 Surface Plasmon Resonance (SPR) Biosensors Surface plasmon resonance is the oscillation of conduction electrons at interfaces of two media (usually glass and liquid). This oscillation can be stimulated by polarized light at a specific angle. The main mechanism of these biosensors is based on Kretschmann configuration (Fig. 5). As can be noticed in Fig. 5, a prism is applied for the purpose of light-surface plasmon coupling at the surface of a thin metal film (usually gold or silver). The angle of the incident light should be greater than a certain limiting degree which total internal reflection occurs. Total internal reflection is

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Fig. 5 Kretschmann configuration, with permission from (Rasooly and Herold 2008)

vital for SPA analysing because only in this situation the reflected prism can give us the input signal. By changing the angle of the prism, at a defined SPR angle (also known as resonance angle), an evanescent light field travels through the thin gold film and light-SP coupling happens. This coupling causes variations in the intensities of scattered and transmitted light fields and ultimately provides the input signals for determining the thickness and/or dielectric constant of the coating (Vogt et al. 2004; Damborský et al. 2016). To measure properties of ligand-analyte interaction, biorecognition element must be immobilized on the sensor surface (a sensor chip with a gold surface and a layer enabling ligand immobilization) which is integrated with a fluidics system enabling a flow-through operation and the intensity shift will be used as input data in transduction systems (Rasooly and Herold 2008). One of the limitations in using SPR technology is the lack of resolution. In order to overcome such limitation, researchers have come up with new approaches that are shown in Fig. 6. One approach to improve the resolution is to increase the mass which is the main idea for (a), (b) and (c) method in Fig. 6 (Rasooly and Herold 2008). Another approach is to integrate two mechanisms to attain high resolution as shown by method (d) in Fig. 6.

5.2 SPR Imaging SPR imaging (SPRi) results from the integration of SPR and spatial imaging devices in a microarray format that allows the simultaneous study of multiple different interactions (Fig. 7). A successful application of this method was investigated in a study in which the binding between an immunosuppressive drug (FK506) and its target (FK506-binding protein 12 (FKBP12)) was accomplished and qualified (Damborský et al. 2016).

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Fig. 6 Methods which have applied successfully to improve the resolution of SPR technique, with permission from (Rasooly and Herold 2008)

Fig. 7 Elements and components of a typical SPRi imaging device with permission from (Safina 2012)

5.3 Localized Surface Plasmon Resonance (LSPR) Localized SPR (LSPR) which is considered to be the next generation of plasmonic label-free technique, is based on the integration of surface plasmon in a nanoparticle when its size is comparable or smaller than the polarized probe light. The input signal for the transducer is related to the variations in electron charge oscillations in MNPs which can be observed by colour changes and absorption peak shifts (Harris et al. 1997; Voinova et al. 2002). Electron charge oscillation in MNPs is induced by the electromagnetic field of light and later alterations in the media are related to the subsequent absorbance of light within the Ultraviolet-Visible (UV-VIS) band by MNPs (Fig. 8) (Wang et al. 2013). Variation in oscillation is caused by the surrounding

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Fig. 8 a schematic comparison between SPR and LSPR imaging, with permission from (Estevez et al. 2014)

dielectric environmental changes. These changes produce LSPR spectral shifts; also, referred to as ‘wavelength-shift’ sensing. LSPR-based detection can simply be miniaturized to increase the throughput of detection and reduce operational costs (Özalp 2011), for example, LSPR multiarray biosensors were used for screening antigen-antibody interactions as a multianalyte biosensor (considered analytes were immunoglobulins, C-reactive protein and fibrinogen) (Fant et al. 2000) and also in a study for diagnosis of ovarian cancer, a Nano chip was assembled to detect the analyte of interest (HE-4) by its antibody which was assembled on the LSPR Nano chip’s surface (Antosiewicz et al. 2015).

5.4 Evanescent Wave Fluorescence Biosensors The evanescent wave is an oscillating electric and/or magnetic field that does not propagate and its energy is spatially concentrated in the vicinity of the source (Damborský et al. 2016). When a guided light passes through the optical fibre, it undergoes a total internal reflection as it meets the interface of the optic fibre and surrounding medium (which has a lower index of refraction). As a result of this reflection, an electromagnetic field called an evanescent wave extends out from the interface into the lower index medium (Damborský et al. 2016). The evanescent wave decays exponentially with distance from the interface. Due to near-surface effects of the evanescent wave, its generation can be used to excite and generate a fluorescent signal as an input data for transducer (Damborský et al. 2016). This geometric limitation is deemed as an advantage since it minimizes unwanted background signals and excites near fluorescent molecules. A schematic review of these processes is presented in Fig. 9. The excellent sensitivity of this method for HIV, syphilis and hepatitis C has been demonstrated in recent studies (Davis and Higson 2005).

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Fig. 9 Elements and components of a typical evanescent wave fluorescent biosensor with permission from (Miyajima et al. 2013)

5.5 Bioluminescent Optical Fibre Biosensors Bioluminescent optical fibre biosensors are considered as biosensors to detect genotoxins. These devices use recombinant bioluminescent cells and the bioluminescent signal is transferred from the analyte by an optical fibre. Changes in intensity of these signals are considered as input signals (Damborský et al. 2016).

5.6 Some Other Types of Optical Biosensors Five major devices that are extensively used were presented. Here we give you a brief review about different mechanisms that can be applied to these biosensors. The first group is the Ellipsometric biosensor. This biosensor’s mechanism is based on changes in polarization of light that passes through the sample (Damborský et al. 2016). The other group is Reflectometric interference spectroscopy biosensor which is a multilayer system that measures phase and amplitude of the polarized light in thin films. Changes in phase and intensity of reflected light in its specific wavelength will be measured as an input data in these biosensors (Damborský et al. 2016). At last, surface enhanced Raman scattering biosensors use specific metal nanoparticles on the surface of the biosensor to enhance Raman scattering and Raman shifts will be the input data (Damborský et al. 2016).

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6 Electrochemical Biosensors Electrochemical sensors operate by interacting with the analyte of interest in a catalytic or binding event to produce an electrical signal as an input data for transduction system. Electrochemical biosensors can be classified into three major different groups: (1) Potentiometric biosensors, (2) Amperometric biosensors, (3) Impedometric biosensors. It is noteworthy that other electrochemical systems are being applied in Biosensors but here we focus on these three groups (Hammond et al. 2016). A very good example of electrochemical biosensors is the group of glucose biosensors. The oxidoreductive reactions which happen in the interfaces of the media, generate charge flow that will serve as biosensing element (Hammond et al. 2016). The reactions which happen only in close proximity to the electrode surface can be detected by the electrode, hence properties of the electrode such as surface modifications, materials or its dimensions play a crucial role in the performance of electrochemical biosensors (Hammond et al. 2016). Electrochemical sensing usually requires a reference electrode (to maintain a known and stable potential), a working electrode also known as the sensing or redox electrode (the transduction element) and a counter or auxiliary electrode to maintain current to the working electrode (Hammond et al. 2016). Nanomaterials like metal nanoparticles, nanowires and nanotubes, which had a great influence on the development of electrochemical biosensors, are discussed below (Wang 2005).

6.1 Amperometric Biosensors Amperometric biosensors are a class of electrochemical biosensors that transform the interactions in the sensing surface into a current signal for transducer system. This type of transduction system receives variations in charge transfer as an input signal. The current is measured in these biosensors at a constant potential which is referred to as amperometry (Hammond et al. 2016). Clark’s enzyme electrodes represent the quintessence of simple amperometric biosensor. • The first generation of amperometric biosensors In this generation two main reactions happen: (i) Substrate/Product identification step, which is a chemical oxide-reductive reaction causes changes in concentration of indentifiable mediaters which are ideal substitutes for the analyte of interest. Consumption of O2 and consequent production of H2 O2 or vice versa are the main reactions involved in this step that cause charge flow. The charge flow which is produced by these reactions, will be measured by the transduction system.

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(ii) The main reactions, which happen between the biorecognition element (Glucose Oxidase in the enzyme electrode) and the analyte of interest cause variations in concentration of the substrates and products of type (i) reactions. S + O2 + 2H+ → P + H2 O2 The major issues with the first generation of amperometric biosensors are the fluctuation of substrates (like O2 in enzyme electrode) and interferences which can happen between Products and substrates of the identifying reactions (Clark and Lyons 1962; Guilbault and Lubrano 1973; Shimizu and Morita 1990). • The second generation of amperometric biosensors In order to overcome these issues, a mediator was introduced which acts as both a donor and an acceptor of electrons. This solution can omit the first problem by its reusable feature and the second one by its bifunctional acceptor-donor feature. This technique also facilitates miniaturizing the biosensor which improves its selectivity (Cass et al. 1984). • The third generations of amperometric biosensor Recent advances in nanotechnology and nanomaterial science like self-assembly techniques have facilitated the creation of the third generation of amperometric biosensors by direct electron transfer between an immobilized enzyme and electrode surface (Hammond et al. 2016). Figure 10 will give you a brief view of the differences between these 3 types.

6.2 Impedimetric Sensors The electrochemical impedance spectroscopy is the most common technique used in impedimetric biosensors. This technique measures the impedance of system over a wide range of frequencies. The impedance number is linked to the opposition that a circuit presents to an applied current. The input data in these biosensors is frequencydomain responses which are resulted from storage and dissipation of energy. Variation in the input data is related to the physiochemical changes (storage and dissipation of energy which is related to dielectric properties) that take place when an interaction happens between the analyte of interest and biorecognition element (Hammond et al. 2016).

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Fig. 10 Schematic comparison between three generations of amperometric biosensors, with permission from (Freire et al. 2003)

6.3 Chronocoulometric Sensors Chronocoulometry (CC) refers to the measurement of the charge as a function of time. CC is a controlled-potential technique. In this technique, the potential constant changes in order to induce oxide-reductive reactions (which are reversible) and variations in the potential constants which induce reactions can be used to identify the concentration of the electroactive species on the surface of the electrode by Nernst equation (Hammond et al. 2016). Chronocoulometry sensors are widely used in the quantification of nucleotide molecules (because of their negative charge) (Hammond et al. 2016).

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Fig. 11 Graphical diagram describing probe-coated magnetic bead’s role in electronic DNA hybridization biosensors, with permission from (Wang 2005)

6.4 Nanoparticle, Nanowire and Nanotube’s Roles • Nanoparticle An interesting example of using nanoparticles in the development of electrochemical biosensors is their amplification role in detection of the oligonucleotide interactions within electrical DNA hybridization biosensors. The sensing mechanism of these devices relies on hybridization of probe-coated magnetic beads with the metaltagged targets. The ‘magnetic’ collection of such magnetic-bead/DNA/metal-label assembly onto the electrode leads to direct contact of the metal label and the surface of the electrode. This absorbance results in CC sensing of metal-tags and enables measurement of the target DNAs (Wang 2005; Hammond et al. 2016). The whole process is shown in Fig. 11.

6.5 Nanowires and Nanotubes One-dimensional nanostructures, like carbon nanotubes (SWCNTs and MWCNTS) or semiconducting or conducting nanowires, present amazing properties like their electron conductivity. Because of their small size and high surface to volume ratio, these structures possess special physical, electrical and chemical properties (e.g. special light-emitting properties of quantum dots) (Wang 2005; Hammond et al. 2016). Thermal and electron conductance changes due to perturbation (as a result of binding to the analyte of interest) has high sensitivity (because of their wide electroactive surface) and can be used in biosensors. Carbon nanotubes (CNTs) have a wide range of applicability including in touch screens, solar cells, batteries, supercapacitors, transistors, super-strong materials for structural composites, biosensors, etc. (Wang 2005; Hammond et al. 2016). CNTs can act as molecular wires to allow electrical communication between the underlying electrode and redox proteins (Wang 2005; Hammond et al. 2016).

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6.6 Graphene-Based Electrochemical Biosensors Graphene is an allotrope of carbon which contains a single layer of carbon atoms arranged in a hexagonal lattice. Graphene has contributed to the fabrication of biosensors with high sensitivity due to its exceptional properties such as good sensing ability, excellent mechanical, thermal, excellent conductivity and small band gap, which can be beneficial for electron transfer between the enzyme and electrode surface. High carrier mobility and density, Passive absorption and covalent immobilization of biomolecules are methods for surface loading on the large surface of graphene. This set of features allowed the development of electrode interfaces capable of hosting high amounts of bio-receptors, thereby enhancing the sensitivity of biosensor devices (Hammond et al. 2016; Nikoleli et al. 2016; Justino et al. 2017).

7 Thermal Biosensors Thermometric biosensors are a class of biosensors which their input data is temperature alteration of the media following enzymatic reaction between biorecognition element (immobilized enzyme) and the analyte of interest. Variation in temperature is measured using sensitive thermistors. Since thermal activities exist ubiquitously in biological processes, thermal biosensing is a widely applicable direct biosensing method; However, conventional thermal biosensors are rather complicated. The bioreactions which happen in the media are exothermic and have rapid turnover rates. Thermal biosensors, usually measure enthalpy changes since enthalpy of major reactants and products are constant. These measurements provide unique opportunities for detecting a wide range of biomolecules and serve as an important complementary method to other biosensor detection schemes (Ramanathan et al. 1999; Ramanathan and Danielsson 2001; Marks et al. 2007).

8 Magnetic Nanoparticle Sensors Magnetic nanoparticles have alluring properties which make them a suitable option in biological labelling, hyperthermia and as MRI contrast agents. Many types of biosensors employ magnetic nanoparticles that are functionalized to recognize specific molecular targets.

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8.1 Magnetic Relaxation Switch Assay-Sensors These sensors are based on the effects of magnetic nanoparticles on proton relaxation of water. SPIONs are a group of MRI contrast agent which can be used in pre-clinical targeted molecular imaging with special surface modifications. When these NPs bind to the analyte of interest they produce local inhomogeneity in the applied magnetics. This inhomogeneity results in a decrease in relaxation time which leads to changes in the contrast of MR images (Koh and Josephson 2009).

8.2 Magnetic Particle Relaxation Sensors When the magnetic field is turned off, magnetic nanoparticles in the liquid can go through different kinds of relaxation mechanisms including Neel Relaxation and Brownian Relaxation. The effective relaxation rate is equal to the sum of the Brownian relaxation rate (SQUID or an AC magnetosusceptometer) and the Neel relaxation rate (SQIDs devices). Variation in relaxation rates is the basis for detection in relaxation sensors (Koh and Josephson 2009).

8.3 Magnetoresistive Sensors Binding of magnetic particles (which are attached to the analyte of interest) to the surface of magnetoresistive sensors results in electrical current changes within the sensor due to nanoparticle’s magnetic field. Variation in the current flow is the input signal for the transducer system in these sensors (Koh and Josephson 2009).

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Carbon Quantum Dots in Nanobiotechnology Hamidreza Behboudi, Golnaz Mehdipour, Nooshin Safari, Mehrab Pourmadadi, Arezoo Saei, Meisam Omidi, Lobat Tayebi and Moones Rahmandoust

1 Introduction There has been an outburst of attraction in the use of nanoparticles (NPs) and nanomaterials for bioimaging, biosensing and other chemical and biological applications (Yao et al. 2014). At the leading edge of the new trend (Demchenko and Dekaliuk 2013) of the emerging nanoparticles in the carbonic nanomaterials family, fluorescent carbon quantum dots (CQDs) have drawn considerable attention due to their unique and tunable properties (Zhang et al. 2012). CQDs, first reported in 2004 (Shi et al. 2011), are mentioned to be clusters of carbon atoms with diameters of generally 2–10 nm, which also include a substantial section of hydrogen, oxygen and nitrogen. They did measurably dissolve in the aqueous solution, however, their aggregation was infrequently observed (Baker and Baker 2010). CQDs can produce strong fluorescent emission and usually do need not to be labelled or doped. Their colour of emission can be tuned by differing the conditions of the synthesis (Ding et al. 2015), leading to various sizes. Both the excitation and the emission spectra are reported to be wide. Although the emission spectra generally extend from blue (430 nm) to red (650 nm) wavelengths, strongest fluorescence is limited to blue, and longer excitation wavelengths normally induce green to yellow emissions (Yang et al. 2009a). According to the reported experiences, slight variation in reaction parameters during the synthesis can lead to adjustment of the properties of the obtained CQDs. In addition, the functional groups on CQDs, such as amino, carboxyl, hydroxyl, and carbonyl, facilitate more modification possibilities, which expand the range of H. Behboudi · G. Mehdipour · N. Safari · M. Pourmadadi · A. Saei · M. Omidi · M. Rahmandoust (B) Protein Research Center, Shahid Beheshti University, Tehran, Iran e-mail: [email protected] L. Tayebi School of Dentistry, Marquette University, Milwaukee, WI, USA © Springer Nature Switzerland AG 2019 M. Rahmandoust and M. R. Ayatollahi (eds.), Nanomaterials for Advanced Biological Applications, Advanced Structured Materials 104, https://doi.org/10.1007/978-3-030-10834-2_6

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excitation and emission wavelengths, as well as improve selectivity and sensitivity in bioimaging applications (Hola et al. 2014). Taking advantage from uniqueness of the CQDs in terms of excellent dispersion (Li et al. 2010a), photo- and chemical-stability (da Silva and Gonçalves 2011), excitation-dependent emission, providing multicolor emissions (Fig. 1) (Cao et al. 2007), ease and cost-effectiveness of the synthesis and modification procedures (Bourlinos et al. 2008), good cell permeability (Zheng et al. 2015b), biocompatibility and low toxicity (Yang et al. 2009b), chemically inertness, small size (below than 10 nm), tunable photoluminescence (PL) properties (Zhu et al. 2015), easy functionalizing with biomolecules, and multi-photon excitation that provides up-converted photoluminescence (Zhu et al. 2015), considerable efforts have been made for CQDs to be used for potential applications as promising agents for bioimaging, biosensing, wound-dressing and drug delivery system, as well as in other biological applications with similar aspects (Zhu et al. 2013b). In addition, researchers have discovered that CQDs exhibit strong photoluminescence upon two-photon excitation in the 650 nm (near-infrared region), which further develops their applications in anti-stoke biosensing and bioimaging (Cao et al. 2007). Being pH-dependent (Jia et al. 2012), the fluorescent emissions of CQDs can be divided into the up-conversion and the down-conversion types (Alam et al. 2015) that will be explained later in this chapter. Compared to common heavy-metal quantum dots, generally referred to as quantum dots (QDs), carbon quantum dots are environmentally friendly and show superior biocompatibility and low toxicity (Sun et al. 2006). This chapter is prepared in a way to provide an overview of the fundamental properties of carbon quantum dots and then focus on their in vivo and in vitro bioimaging, biosensing, drug delivery, wound-dressing and photocatalysis applications. We expect that this chapter would offer beneficial insight into the topic and encourage more exploration of carbon quantum dots.

Fig. 1 Excitation-dependent PL in CQDs

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2 Carbon Quantum Dots Structure Carbon is a substance with a low water-solubility and weak fluorescence properties. Instead, the quantum-sized carbon dots have strong luminescence and high watersolubility and biocompatibility, due to the innumerable carboxyl groups on their edges (Lim et al. 2015) and their special structure, which regulates their unique properties at nanoscale (Baker and Baker 2010; Weng et al. 2011; Tao et al. 2016; Namdari et al. 2017). In general, all nanoscale carbon materials are called nanocarbons, in which at least one dimension is below 10 nm in size. However, in this class of nanostructured materials, CQDs have all their three dimensions less than 10 nm. CQDs have a spatial structure of sp2/sp3 with fluorescence emission as their innate feature (Baker and Baker 2010; Yan et al. 2010). The surface of the CQDs encompass modified or connected various chemical groups, such as polymer chains, oxygen-containing group, nitrogen-based aminocontaining groups, etc. (Baker and Baker 2010) and according to the synthesis method, they show diverse chemical structures and fluorescent properties. For instance, graphene quantum dots (GQDs) are a sub-class of CQDs, which possess only a single or a few sp2 graphene layers, whereas another member of the group, known as carbon nanodots (CNDs), are always spherical. However, the obvious crystal lattice is always distinguishable in the structure of GQDs (Zhu et al. 2015). Chemical modification and surface passivation with diverse organic or inorganic, biological and polymeric materials is another possibility in this class of nanomaterial that can be employed to improve their properties. In addition, due to being based on carbon, these structures have photochemical stability, good conductivity, and safe chemical composition (Yang et al. 2013). Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) is a technique that is used to recognize the chemical and functional groups of CQDs, and their physical and chemical structures (Tetsuka et al. 2012; Demchenko and Dekaliuk 2013). Other techniques for obtaining the general structure and the grafting of chemical groups, and direct characterization methods include high-resolution TEM (HRTEM), Raman spectroscopy, and X-ray diffraction (XRD), Fourier transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), nuclear magnetic resonance (NMR), matrix-assisted laser desorption ionization timeof-flight (MALDI-TOF), and element analysis (Tetsuka et al. 2012).

3 Carbon Quantum Dots Synthesis Carbon quantum dots were accidentally discovered by Xu et al. in (2004) during purification of single-walled carbon nanotubes (Xu et al. 2004). Since then, many methods have been reported to generate CQDs, which can be categorized into two major groups, namely, “bottom-up” and “top-down”. The “bottom-up” pathway includes the synthesis of CQDs from smaller precursors, such as carbohydrates, and citrates, whereas in the “top-down” synthetic pathway, larger carbon compounds, for

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instance, graphite sheets, and carbon nanotubes, are decomposed to make CQDs, with possible modification during post-treatment steps (Robertson and O’Reilly 1987; Gong et al. 2014; Chen et al. 2016). In general, in preparation of CQDs, three issues should be considered: (i) controlling size and uniformity that is important for the mechanical perusal and can be optimized through post-treatment methods, such as dialysis, centrifugation, and gel electrophoresis; (ii) carbon capture along carbonization, which can be accomplished through using electrochemical synthesis, pyrolysis or solvent chemotherapy; and (iii) surface properties and functional groups that can be adjusted during production or post-treatment steps (Cao et al. 2007; Baker and Baker 2010; Briscoe et al. 2015). There are also different techniques for dehydration and carbonization of various precursors including hydrothermal carbonization in a micro-reactor, microwave hydrothermal and plasma-hydrothermal methods (He et al. 2011; Zong et al. 2011; Tang et al. 2012). These methods allow the superior control on the properties of the ultimate product. In this part, the most important methods of this group are summarized.

3.1 Hydrothermal/Solvothermal Treatment The hydrothermal/solvothermal reaction is a chemical reaction between the precursors in a solvent environment inside a closed system of high boiling point and under high-pressure condition. Hydrothermal carbonization (HTC) or solvent carbonization is an inexpensive, environmentally friendly and non-toxic way to produce new carbon-based nanomaterials, in which a high precision pre-formulation solution is reacted in a hydrothermal reactor (Fig. 2) (Jana et al. 2016). HTC-made CQDs have been produced by many various precursors, including glucose, chitosan, banana juice, citric acid, diammonium hydrogen citrate and proteins (He et al. 2011). Bhunia et al. synthesized two types of hydrophobic and hydrophilic CQDs, with a diameter of less than 10 nm, by carbonization of carbohydrates (Bhunia et al. 2013). Hydrophobic CQDs were prepared via mixing different amounts of carbohydrates within octadecylamine and octadecene for 30 min at temperatures between 70 and 300 °C, whereas the hydrophilic ones were created by heating a carbohydrate solution in a wide range of pH (Bhunia et al. 2013).

3.2 Microwave Irradiation Microwaves are electromagnetic waves with the frequency range from about 300 MHz to 300 GHz or wavelengths in air ranging from 100 cm to 1 mm. In other words, microwaves are the shortest waves in the wavelength region of the radio spectrum within the electromagnetic spectrum (Zhu et al. 2009; Zhai et al. 2012). In microwave synthesis approach (Fig. 3), there is an opportunity to modify the

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Fig. 2 One step hydrothermal synthesis of CQDs

Fig. 3 Schematic diagram showing the microwave irradiation synthesis of CQDs

combination, morphology and compound structure of materials, exclusively composites by differential heating (Jaiswal et al. 2012). Microwave irradiation of organic compounds is a quick and inexpensive approach to produce CQDs. For instance, using diethylene glycol (DEG) as the carbon source, green luminescent CQDs will be produced (Jaiswal et al. 2012). These DEG-CQDs are well diffused in water with a clear appearance. In addition, they can be subsequently absorbed by glioma C6 cells, making them suitable for bioimaging applications (Liu et al. 2014).

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3.3 Carbonization As introduced initially by Dong et al. in (2012), thermal decomposition of citric acid was proved to be a cheap and facile bottom-up technique to produce a homogenous distribution of pristine blue-emitting CQDs in aqueous solution. They thermally decomposed citric acid by heating it for 30 min at 200 °C. He also reported fabrication of graphene oxide (GO) particles by heating CA at the same temperature for longer duration of 120 min. Since elemental analysis of the products showed higher content of carbon in the so-called GOs, compared to CQDs and citric acid, respectively, and hence, they named the process as “carbonization” (Dong et al. 2012). Later on, various heating temperatures and durations were investigated by various scholars (Dong et al. 2012; Wu et al. 2013; Amjadi et al. 2014; Wang et al. 2015a; Bagheri et al. 2017). In most of the reports employing the mentioned technique, the achieved Quantum Yields (QYs) were not significant yet. In 2015, Wang et al. tried to use a set of various synthesis conditions and compared the achieved QYs. However, since a systematic control of the synthesis conditions was not employed, results did not lead to manifest improvement of the QY (Wang et al. 2015a). Recently, Rahmandoust and Mohammadi employed a two-step systematic optimization approach to enhance the QY of as-prepared CQDs to about three times higher values (Rahmandoust and Mohammadi 2018). Carbonization is not however limited to chemical thermal decomposition. Electrochemical carbonization (Fig. 4) is another powerful method for producing CQDs, using a wide variety of carbon materials as precursors (Zhou et al. 2007). There are a few reports about the electrochemical carbonization of small molecules to CQDs. Deng, Lu et al. produced CQDs by electrochemical carbonization of alcohols using two sheets of Pt as the counter electrodes and a sheet of Ag/AgCl fixed in the freely customizable Lugging capillaries as the reference electrode. Alcohols have been converted to CQD after electrochemical carbonization (Deng et al. 2014). The size of these CQDs is reported to increase with increasing the applied the potential (Sun et al. 2006; Deng et al. 2014).

3.4 Laser Ablation The laser ablation method (Fig. 5) is summarized as the process of combining laser pulses in specific time intervals leading to the synthesis of the targeted semiconductor. This combination can decrease factors that cause thermal effects such as evaporation, explosive melting, and laser-plasma interactions, thereby decreasing problems with delamination and micro-cracks that can affect the yield and reliability of the produced semiconductor (Sun et al. 2006; Hu et al. 2009). Hu, Niu et al. reported the synthesis of fluorescent CQDs by laser ablation method, showing that by using organic solvents, the surface state of fluorescent CQDs can be modified to regulate their emission intensity. According to control experiments, the

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Fig. 4 Electrochemical carbonization synthesis of CQDs

Fig. 5 Laser ablation synthesis of CQDs

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linkers on the surface of the CQDs in relation to surface states are attributed as the source of the luminescence property of the nanomaterial (Hu et al. 2009). Li et al. used nanosize carbon material, as the carbon source in an organic solvent, as the liquid medium, and produced fluorescent CQDs. In this method, 0.02 g of nanosize carbon material was solved in 50 mL of an organic solvent such as water, acetone or ethanol. After ultrasonication, 4 mL of suspension was transferred to a cell for laser radiation using a 532 nm wavelength to irradiate the prepared suspension. Subsequently, to obtain supernatant, which includes CQDs, the solution was centrifuged at 6000 rpm (Li et al. 2010c; Wang and Hu 2014).

3.5 Chemical Ablation Strong oxidizing acids can carbonize small-sized organic molecules to carbonic materials, which can be cut into sheets by controlling the process of oxidation (Baker and Baker 2010; Dong et al. 2010; Qiao et al. 2010; Luo et al. 2013; Zhu et al. 2013a). Although the approach requires hard conditions and severe control over the process, Travas-Sejdic and Peng reported a simple and facile method for producing fluorescent CQDs using this technique. In their proposed technique, carbohydrates were dehydrated in an aqueous media by concentrated sulfuric acid and then the carbonic structures were broken into CQDs through nitric acid (Peng and TravasSejdic 2009). The surface passivation of CQDs was performed by post-treatment of CQDS by amine-terminated compounds as an important step for the photoluminescence enhancement. The emission wavelength of reported CQDs can be adjusted by differing the amount of the precursors and the time assigned for the nitric acid treatment.

4 Photoluminescence Mechanism in CQDs Photoluminescence, as schematically illustrated in Fig. 6, is the phenomenon of the emission of an electromagnetic wave, in the visible light wavelength range, after absorption of photons or electromagnetic radiation, in any material (Gong et al. 2014). It is one of the many forms of luminescence (light emission) and is initiated through photoexcitation. The period between absorption and emission may vary ranging from short femtoseconds to delayed emission of up to minutes or hours, under special conditions. The source of the detected optoelectronic demeanour is an issue of discourse to date, however, in the scope of CQDs, there have been plenty of efforts concentrated on the physicochemical features to explain the source of photoluminescence (Baker and Baker 2010). Various reasons were proposed in literature, including the quantum confinement effect, which leads to recombination of electronhole pairs in small sp2 carbon clusters, or on an sp3 matrix; and the effect of the surface states, which is fueled by different functional groups and defects (Baker and Baker 2010; Wang and Hu 2014), which are explained in more details.

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Fig. 6 Illustrate the PL mechanism in CQDs Fig. 7 CQDs absorption includes a broad UV-IR spectral range because of their conjugated π-domains

4.1 Quantum Confinement Derived PL The first quantum confinement based PL mechanism is related to bandgap transitions through conjugated π-domains (Fig. 7). It’s been well known that the expansion of the new π-conjugated molecules is critical for optoelectronic materials, such as light-emitting materials, photovoltaic materials, and organic semiconductors. This is mostly due to the electronic structure of π-systems, which can cause extensive delocalization of electrons all over the molecules. CQDs absorption includes a broad UV-IR spectral range because of their conjugated π-domains. There may be several absorption peaks attributed to the n–π* transition of C=O bonds, the π–π* transition of the C–C bonds, etc. (Bailey et al. 2005).

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Robertson and O’Reilly proposed that the CQDs PL is lead to the renewal of electron-hole pairs in the strongly localized π and π* electronic levels of the sp2 positions. These positions lie between the bandgap of the σ and σ* situations of the sp3 matrix, leading to intense visible emission (Robertson and O’Reilly 1987). In essence, HOMO/LUMO energies, as to be the highest-occupied- and the lowestunoccupied-molecular orbitals, and their intermolecular interplays of π-conjugated molecules can be tuned by improving their chemical structures (Shen et al. 2014). From the mechanical aspect, the optical absorption and fluorescence emissions in CQDs is not related to the band gap, but rather because of π-plasmon and the radiative recombination of the surface-limited electrons and holes, which differs from semiconductor metalic quantum dots (Hu et al. 2015). Unique size-associated PL property of CQDs leads to the discovery of size-tunable synthesis approaches. The QY and emission wavelength can be adjusted by varying the particle size, excitation wavelength and structure via tuning the reaction factors, such as the reaction time/temperature and precursors composition/ratio (Robertson and O’Reilly 1987). Photoexcited CQDs are demonstrated to be both excellent electron acceptors and electron donors, because either electron acceptor or electron donor molecules disorder the radiative recombination of CQDs and hence impel PL quenching (Wang et al. 2009). Zhang, Abbasi et al. has recently suggested an efficient method to synthesize a tunable size CQDs via varying the percentage or composition of the reactants (Zhang et al. 2015).

4.2 Surface State Derived PL The surface state, which is determined by different hybridized groups with the carbon backbone, leads to the formation of energy traps on the surface of CQDs. It’s been proved that these energy traps can control the PL mechanism. Although size can affect the PL spectrum, the surface states are also believed to play a dominant role in CQDs, so that most of the PL centres in CQDs are originated from the surface state (Hilderbrand et al. 2009). Therefore, by engineering these trapping states, the luminescence of CQDs can be controlled (Wang et al. 2010; Zhu et al. 2015). Quantum yield of a quantum dot is the number of emitted photon relative to the number of absorbed ones. The QY can dramatically increase based on surface chemistry and synthesis method (Shi et al. 2016). CQDs usually show low QY, which is due to the inadequate surface state. Different defects make energy traps on the surface, which cause the electrons to recombine with them in a non-radiative way, thereby the total QY is reduced. However, surface passivation, as shown in Fig. 8, with a certain limit of passivation agent can facilitate the radiative recombination of electron-hole pairs and enhance the PL (Sun et al. 2006; Wang et al. 2009). Surface passivation is also applied for stabilization. Passivation is achieved via different natural proteins, polymers and any organic molecule that is capable of introducing different functional groups to the surface of CQDs. The PL mechanism is also

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Fig. 8 The PL improvement by surface oxidation or reduction

improved by surface oxidation or reduction, which is induced by different synthesis routes (Zheng et al. 2011). Although this strategy is the most often used one, another approach to improve the QY measure in CQDs is doping the nanoparticle by different heteroatoms to which the carriers (electron or holes) deliver their energy. Different atoms such as nitrogen, phosphorous, sulfur, and oxygen are used to enhance the optical properties of CQDs (Zheng et al. 2011; Chandra et al. 2013). To use the synergic effect of different atoms, co-doping is possible by using suitable precursors, which can provide both atoms (Lim et al. 2015). Using an amine molecule as a source for CQDs synthesis can simultaneously serve as a passivation and N-doping agent (Wang and Hu 2014). Therefore surface modifications will improve the physicochemical properties of CQDs and their usability for different bio applications (Namdari et al. 2017).

5 Biocompatibility and Cytotoxicity Although CQDs have attracted a large amount of attention for their special characteristics that make them a potential candidate for application in many fields, still more studies are needed in order to have them used in biological and medical applications. Usually, in these studies, CQDs are examined for biocompatibility and cytotoxicity. Biocompatibility It is a term used to explain the degree of compatibility of a substance with a cell, tissue or living organism. Biocompatible materials do not create a toxic or immunological response inside the body and cytotoxicity is the quality of being toxic to cells. Many studies have concentrated on the biocompatibility studies of CQDs. In a study on strongly fluorescent polyethylene glycol (PEG)-functionalized CQDs in 2007, no significant cytotoxicity was observed. In general, CQDs that were injected into mice did not show any considerable toxicity and cell death. As a result, it was proved that they were suitable for optical in vivo imaging. Even after surface pas-

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sivation of CQDs by Poly-propionylethylenimine-co-ethylenimine (PPEI-EI) agent, no cytotoxicity for living cells was observed (Cao et al. 2007). In 2008, Zhao et al. exert two types of CQDs to evaluate them for cytotoxicity. The bare CQDs did not show a toxic state up to a high dose concentration of 0.4 mg/mL for cells. In addition, for the CQD prepared by the electrochemical treatment method, the cytotoxicity was appraised in human kidney cells, which did not seem to affect the viability of the kidney cells (Zhao et al. 2008). Ray and colleagues synthesized CQDs with a diameter of 2–6 nm, through nitric acid oxidation. The obtained Ndoped CQDs were examined for cytotoxicity and the results indicated that they were not toxic, based on the cell survival rate as measured by MTT and Trypan blue assay. The cells were exposed to 0.1–1 mg/mL of CQDs for 24 h. This dose is about 100–1000 times greater than the amount required for bioimaging. The cell viability in doses less than 0.5 mg/mL was about 90–100% and at higher concentrations, the mortality rate of the cells increased. Therefore, these particles can be used for bioimaging and other medical applications (Ray et al. 2009). The PEGylated CQDs are compatible at the concentrations required for bioimaging and they have no reported cytotoxicity in recent studies. For example, in a research, the PEG1500N passivated CQDs were injected into the mice for 28 days. Blood and organ were sampled for cytotoxic assays and the result was that there were no toxic effect on mouse cells (Yang et al. 2009b).

6 Upconversion PL of CQDs Upconversion PL (UCPL) of CQDs were first observed by Sun’s group in 2007 upon excitation with a femtosecond IR pulsed laser at 800 nm (Hilderbrand et al. 2009). Upconversion is the procedure wherein longer-wavelength light is transformed into the photons of higher energy (Wen et al. 2014). Accordingly, the UCPL property of CQDs, as shown in Fig. 9, can be associated to the multi-photon excitation (Hilderbrand et al. 2009), in which the concurrent absorbency of two or more photons results in the emission of light at shorter wavelengths and higher energy than the excitation wavelength. The UCPL of CQDs creates a great opportunity for cell imaging with two-photon luminescence microscopy, and also a very efficacious catalytic method, for usage in energy technology and bioscience (Kong et al. 2012). Most CQDs may not have traceable UCPL. To evaluate the UCPL it is essential to eliminate the typical fluorescence and measure the amount of excitation intensity of fluorescence. UCPL is actually a typical fluorescence that is excited by flow from the second dispersion in a single-phase spectrophotometer, which can be omitted by adding a passing filter to the excitation path of a commercial fluorescence spectrophotometer. Intensity-related experiments have been clearly confirmed that UCPL is the typical fluorescence with lineal reply against a multiple photon proceeding (Kong et al. 2012). However, under controlled experimental conditions in five different types of synthesized CQDs in a fluorescence spectrophotometer, no observable UCPL was reported (Wen et al. 2014).

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Fig. 9 The UCPL property of CQDs can be assocthe iated to the multi-photon excitation

7 Bioimaging The term imaging can be understood in numerous ways. Imaging may immediately lead to a perception of a sort of photography in people’s minds, however, the scientific meaning of imaging is far beyond this. The so-called images can also be obtained by various methods such as near-infrared imaging (Giavalisco et al. 2004; Luo et al. 2014), Raman spectroscopy (Keren et al. 2008), CT imaging (Bar-Shalom et al. 2003), radio-imaging by respective nuclides (Love et al. 2003), electrochemical imaging by rastering electrode (Barker et al. 2004), positron emissions tomography (Bailey et al. 2005), and even more complicated scanning methods like laser ablation (Geohegan et al. 1998). Most of the imaging methods are toxic and destructive or require expanded sample preparation, but not all of them. Therefore, only biocompatible techniques such as fluorescence-based imaging can be applied to living systems or intact tissues. The term of biological imaging by means of fluorescent labeled nanomaterials is typically referred to as bioimaging. In addition to MRI and conventional light microscopy, fluorescence bioimaging is probably the other most widespread imaging technique in biosciences (Wolfbeis 2015). For instance, the term fluorescence does not imply a single spectroscopic technique but rather contains a variety of approaches. The images not only can be obtained with measurement of decay time (lifetime) (Smith and Ghiggino 2015), polarization (Camacho et al. 2015), and intensity (Das et al. 2014), but also with studying the effects caused by photo-induced electron transfer, quenching (Song et al. 2014), or resonance energy transfer (Stanisavljevic et al. 2015). Fluorescence imaging has become an efficient method to represent biological processes inside the living cells (Luo et al. 2014), due to high benefits including the absence of radioactivity risk, improved high throughput capability, acceptable spatial resolution, higher selectivity, and sensitivity (Zhao et al. 2015). During the past years, there has been a tremendous increase in imaging resolution up to the nanometers scale. There are two different types of fluorescent imaging. The first one involves imaging based on intrinsically fluorescent chemical and biochemical species, such as NADH in tissues, or chlorophyll in most types of plants (Sarder

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Fig. 10 The synthetic and the intrinsic fluorescent probes by their color of emission: a the synthetic fluorescent probes that are used for metal sensing inside living cells; b some of the intrinsic fluorescent probes that exist in normal cells and tissues; c fluorescent proteins, and d CQDs

et al. 2015). The second one covers imaging approaches that display fluorescence by adding chemical synthetic fluorescent labels, probes, nanosensors or nanoparticles. The use of synthetic probes is indispensable to detect species, which are not susceptible to direct fluorescent imaging (Walker et al. 2015). In Fig. 10, the synthetic and the intrinsic fluorescent probes are illustrated and their corresponding emission colours are shown. Among various available probes, fluorescent CQDs have numerous advantages, containing comparable optical properties and adequate chemical and photochemical stability (Lim et al. 2015). More importantly, CQDs are largely non-toxic, biocompatible and environmentally friendly (Namdari et al. 2017). As

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previously mentioned, carbon quantum dots are able to exhibit multicolour emission (Jiang et al. 2015), that is a huge benefit setting them apart from the most of other fluorescent labelling agents and probes. This allows researchers to choose and control the excitation and the emission wavelengths (Ding et al. 2015). These features make CQDs very desirable as alternatives to heavy metal quantum dots to visualize biological systems, processes and mechanism both in vitro and in vivo (Luo et al. 2013). Table 1 shows various nanoparticle fluorescent probes, stating their properties, advantages and disadvantages. More details on in vitro and in vivo bioimaging are provided in upcoming sections.

7.1 In Vitro Bioimaging As mentioned earlier, optical properties are the key factors of CQDs for bioimaging applications (Wang and Hu 2014). CQDs can be used in biological systems as a bioimaging agent, because of their low cytotoxicity and strong fluorescence emission (Wang and Hu 2014). On the first step of in vitro bioimaging, CQDs must be internalized by the cells, in a way that at the specific experimental conditions, cells are incubated with the CQDs. It was proposed that CQDs likely translocate into cells by endocytosis (Zhou et al. 2014). The up-taking ability of CQDs by cells is revealed to be temperature-dependent (Zheng et al. 2015b). Since only at the specific range of temperature CQDs internalize into the cells, it was recommended that CQDs must be translocated into cells by endocytosis (Zheng et al. 2015b). In addition, as illustrated in Fig. 11, the CQDs’ coupling with translocation peptides of membrane leads to enhancement of their cell uptake (Jafari et al. 2015; Wei et al. 2017). Therefore, by overcoming the membrane barrier of cells, translocation procedure can be facilitated. Surface modified CQDs can be used for labelling of biological cells (Sun et al. 2006). Endocytosis is the proposed mechanism for translocating of CQDs into human cells such as breast cancer cells MCF-7 (Hsu and Chang 2012) and HeLa cells but there is some research that indicates this processes can be done through nonendocytic pathways (Yacobi et al. 2011), so internalization mechanism theory still need more investigations. The CQDs are gradually taken up by HeLa cells, for instance, according to obtained images during the experiment (Ding et al. 2013b). The required concentration of CQDs toward HeLa cells for in vitro bioimaging is a way less than the lethal dosage, which is more than 5 mg mL−1 against HeLa cells, whereas in case of in vitro cell bioimaging, the required concentration is a hundred times lower (Lewinski et al. 2008; Zheng et al. 2015b). In addition, to temperature, size, surface composition, and surface charge are the other important factors that influence translocation of CQDs across the cell’s membrane (Zhao et al. 2011; Fröhlich 2012). As previously discussed, it is possible to improve CQDs crossing through cellular membrane barrier by recruitment of the translocation peptides of cells membrane, such as trans-activator proteins (Zuo et al. 2016). Due to the fast and irregular growth of tumour cells, the iron ion requirement of cancerous cells increase (Toyokuni 1996),

Technique

NIRF

NIRF

MRI

PET/SPECT

NPs

CQDs

QDs, dye-doped NPs

Iron oxide NPs

Radioisotopes NPs

Positron-rays

Magnetic fields

Light

Light

Signal type

Table 1 Features and properties of most useable fluorescent probes

No limit

No limit

50 μm

1–2 mm