Pharmaceutical Applications of Dendrimers [1 ed.] 0128145277, 9780128145272

Table of contents :
Cover
Pharmaceutical Applications
of Dendrimers
Copyright
Contributors
Preface
Section A: Dendrimers and their properties
1
Engineering critical nanoscale design parameters (CNDPs): A strategy for developing effective nanomedicine the ...
Introduction
Historical
Dendrimer-based nanoscale atom mimicry: A window to nanoperiodicity
Dendrimer-based protein mimicry
Dendrimer-based dendritic effects as a window to nanoperiodic property patterns
Application of nanoperiodic property patterns and CNDP engineering principles to nanomedicine
Developing an optimum nanotherapeutic platform based on intrinsic structure controlled, tunable nanoparticle CNDPs
Unique dendrimer features that facilitate systematic CNDP engineering for optimized nanomedicine applications
Nanoparticle structural control and engineering CNDPs: Implications for nanomedicine
Size
Recent progress in dendrimer based CNDP-engineering
Size/shape
Surface/interior chemistry
Flexibility/Rigidity
Optimizing dendrimer properties for nanomedicine applications
Early examples of engineering dendrimer CNDPs for nanomedicine applications
An early example of engineering dendrimer CNDPs related to targeted therapy for cancer
Quality assurance of dendrimer quality/CNDP engineering for clinical trials
Quality assurance of dendrimers for experimental literature: Minimum information reporting in bio-nano experimental ...
Progress in dendrimer-based CNDP engineering related to quantitative nanostructure-activity relationships (QNSARs) and ...
Quantitative nanostructure-activity relationships (QNSARs)
CNDP-based QNSARs to determine most effective antiviral dendrimers against influenza A (H1N1)
Conclusions
Acknowledgment
References
Further reading
2
Dendrimers in drug delivery and the role of ``critical nanoscale design parameters´´ (CNDPs)
Introduction
Formulation approach
Nanoconstruct approach
Dendrimer as drug delivery?
Critical nanoscale design parameters (CNDPs) in dendrimers for drug delivery
References
Section B: Dendrimers for pharmaceutical applications
3
Dendrimers for drug solubilization, dissolution and bioavailability
Introduction
Molecular features of dendrimers
Concept of solubility enhancement with dendrimer
Non-covalent interaction
Covalent interaction
Dendrimer-based solubility enhancement of various classes of drugs
NSAIDs
Anticancer drug
Anti-microbial drugs
Antihyperlipidemic/hypolipidemic drugs
Anti-hypertensive drugs
Diuretics
Antipsychotic drug
Anti-glaucoma drug
Phytoconstituents
Anthelmintic/benzimidazole carbamate albendazole (ABZ)
Miscellaneous
Dendrimer-based drug-delivery through various routes
Intravenous delivery
Intranasal delivery
Oral delivery
Ocular delivery
Pulmonary delivery
Transdermal delivery
Conclusion
Acknowledgments
References
4
Pharmacokinetic considerations in design of dendrimer-based nanomedicines
Introduction
Effect of size, molecular weight, hydrophobicity and charge on pharmacokinetic profile of dendrimers
Effect of surface functionalization on pharmacokinetic profile of dendrimers
Conclusion
Acknowledgment
References
5
Dendrimer-based targeted drug delivery
Introduction
Designing of dendrimers for drug targeting
Types of surface modification of dendrimers
PEGylation
For cancer
For bone diseases
Folic acid conjugation
For cancer
For rheumatoid arthritis
Amino acids/peptides/proteins conjugation
For cancer
For human immunodeficiency virus HIV
For dental applications
N-acetyl-cysteine (NAC) conjugation
For cerebral palsy (CP)
For Rett syndrome (RTT)
Carbohydrate functionalization
For cancer
For infections
For human immunodeficiency virus (HIV)
For scar prevention
For antimalarial drug delivery
For lung disorders
Biotinylation
For cancer
Lauroyl chain conjugation
For cancer
Tuftsin grafting
For HIV
Antibody grafting
For cancer
Phosphonate derivatization
Sulfate derivatization
For human immunodeficiency virus (HIV) and herpes simplex virus (HSV)
P-hydroxyl benzoic acid (pHBA) functionalization
Hyaluronic acid functionalization
Conclusion
References
6
Dendrimers for anticancer drug delivery
Introduction
Brief about dendrimers and application for cancer drug delivery
PAMAM dendrimer for cancer therapy
PPI dendrimers for cancer therapy
PPL dendrimers for cancer therapy
Conclusion
Acknowledgment
References
7
Cancer-targeted chemotherapy: Emerging role of the folate anchored dendrimer as drug delivery nanocarrier
Cancer world ``the making of modern disease´´
What is cancer?
Causes of cancer
Origin of the word cancer
The oldest description of cancer
Biology of cancer
Tumor vasculature
Physiology of tumor structure
Stages of tumor development
Classification of cancer
Prevention of cancer
Tobacco
Alcohol
Physical inactivity, dietary factors, and obesity
Infections
Environmental pollution
Occupational carcinogens
Radiation
World cancer day
Diagnosis of cancer
How cancer is treated?
Surgery
Radiation therapy
Immunotherapy
Hormone therapy
Gene therapy
Chemotherapy
Current approaches to cancer therapy
Barriers in cancer therapy
Physiological barriers
Cellular barriers
Pharmacokinetic barriers
Limitations of conventional cancer therapy
Tumor targeting via a novel drug delivery system
Passive targeting
Active targeting
Nanotechnology
Properties of carrier systems used for drug delivery
Nanovision for targeting of cancer
Carbon nanotubes (CNTs)
Gold nanocarriers
Mesoporous silica nanoparticles (MSNs)
Quantum dots
Liposomes
Polymeric micelles
Dendrimers
Dendrimer in nanotechnology
What are dendrimers
Properties of dendrimeric domain
Potential applications of dendrimers
Inkjet inks and toners
In vitro diagnostics
In vitro gene transfection
Controlled drug delivery
Solubilization of drug
Drug delivery units
Catalyst support
Delivery agents for vaccines
Protein mimicry
Patterning and templating
A tribute to dendrimer workers
The rationale for selecting dendrimers as drug delivery carrier
The rationale for opting dendrimer-based drug targeting
Cancer and dendrimer
Drug-loaded dendrimers as nano-vehicles
Target specific dendritic scaffolds
The rationale for selecting folic acid as a ligand
Folate-targeted dendritic nanocarriers
Folate-PEG dendrimer in cancer targeting
DNA assembled dendrimer-folate conjugate
Folate-DTPA dendrimer in cancer targeting
Multimodality dendrimers based diagnostic agents
Conclusion
References
Further reading
8
Design of dendrimer based prodrugs
Introduction
Advantages of dendrimer over other polymers for prodrug preparation
Advantages of dendrimer prodrugs
Requirements for dendrimer prodrugs
Strategies for design and synthesis of dendrimer prodrugs
Dendrimer prodrugs without spacer
Dicyclohexylcarbodiimide (DCC)
1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, EDCI)
Coupling reactions involving N-hydroxysuccinimide (NHS) ester derivative
Dendrimer-prodrugs with spacers
Conclusion
References
9
Dendrimers in gene delivery
Introduction
Mechanism of gene delivery using dendriplexes
Synthesis of dendriplexes
Targeted dendriplexes
Intracellular trafficking
Endolysosomal escape
Proton-sponge hypothesis
Transport across the cytoplasm
Nuclear localization
Release of genetic material from dendriplexes
Factors affecting dendrimer-based gene delivery
Structure of dendrimers
Size and molecular weight
Surface charge and charge density
Polyvalency
Hydrophilicity
Polymer-DNA charge ratio
Design criteria for formulating dendrimer vectors for gene delivery
Problems in dendrimer-based gene delivery
Surface modification in dendrimer for efficient gene delivery
Amino acid-modified dendrimers
Protein- or peptide-modified dendrimers
Carbohydrate-modified dendrimers
Polymer- or lipid-modified dendrimers
Other ligand-modified dendrimers
Modification of dendrimer core for efficient gene delivery
For flexibility of dendrimers
For hydrophobicity of dendrimers
For functional versatility of dendrimers
Conclusion
Acknowledgments
References
Further reading
10
Dendrimers in immunotherapy and hormone therapy
Dendrimers in immunotherapy
Effect of dendrimers on innate immune system
Effect of dendrimers on adaptive immune system
Role of dendrimers as vaccine delivery systems
Dendrimers in hormone therapy
Limitations of dendrimers
Future prospects
Acknowledgments
References
11
Toxicity and biocompatibility aspects of dendrimers
Introduction
Dendrimer architecture
Dendrimer types
Polyether dendrimers
Polyester dendrimers
Tecto/core-shell dendrimers
Triazine dendrimers
Citric acid dendrimers
Phosphate dendrimers
Melamine dendrimers
Polyether imine (PEI) dendrimers
Polyether-polyester (PPE) dendrimers
Other dendrimers
PAMAM dendrimers
PPI dendrimers
Glycodendrimers
Chiral dendrimers
Poly-l-lysine dendrimers
Hybrid dendrimers
PAMAM-organosilicon (PAMAMOS) dendrimers
Liquid crystalline (LC) dendrimers
Polyurea dendrimers
Peptide dendrimers
Mechanism of dendrimer biocompatibility and toxicity
Why are dendrimers toxic?
Cationic charge
Core associated toxicity
Free amine toxicity
Dendrimer toxicity and biocompatible strategies
Cytotoxicity
Hematological toxicity
Hemolytic toxicity
Immunogenic toxicity
In-vivo toxicity
Surface modified dendrimer for compatibility
PEGylation
Folic acid surface tagging
Amino acid/peptide surface modification
Antibody tagging
Miscellaneous
Future prospectus
References
12
Therapeutic dendrimers
Introduction
Classification of therapeutic dendrimer
Antimicrobial therapy
Anti-inflammatory/inflammation
Tumors
Angiogenesis inhibitor
Antiviral
Conclusion
Acknowledgments
References
Section C: Dendrimers for non-pharmaceutical applications
13
Dendrimers for diagnostic applications
Introduction
Problem with carrier free in-vivo delivery of diagnostic agents
Advantage of dendrimer-based imaging systems
Design criteria for formulating dendrimer vectors as contrasting agents
Low polydispersity index
Enhanced permeability and retention effect
High permeability
Sustained/extended effect
Higher solubilization potential
High uniformity and purity
Multifunctional platform
High loading capacity
High stability
Low toxicity
Low immunogenicity
Dendrimers can be modified as stimuli responsive to release drug
Dendrimers microvascular extravasation properties
Dendrimer for encapsulating contrasting agents within the cavity
Dendrimer-based magnetic resonance imaging agents
Dendrimer-based positron emission tomography agents
Dendrimers-based computed tomography agents
Dendrimer-based fluorescence imaging agents
Dendrimer-based PDT, PTT and neutron capture therapy agents
Dendrimer for conjugating contrasting agents on the surface
Dendrimer-based magnetic resonance imaging agents
Dendrimer-based positron emission tomography (PET) agents
Dendrimer-conjugated computed tomography agents
Dendrimer-conjugated florescence imaging agents
Dendrimer-conjugated PDT, PTT and neutron capture therapy agents
Major challenges of dendrimer-based diagnostic systems
Toxicity
Immunogenicity
Conclusion
References
14
Dendrimer-based marketed formulations and miscellaneous applications in cosmetics, veterinary, and agriculture
Current scenario: Translation of dendrimer-based product from research to market
VivaGel
Alert ticket
Stratus CS
SuperFect
Priofect
Gadomer-17
Dendrimer-based formulations under clinical trial
Miscellaneous applications of dendrimers
Dendrimers in cosmetics
Dendrimers in veterinary
Dendrimers in agricultural application
Conclusion
References
Author Index
Subject Index
Back Cover

Citation preview

Pharmaceutical Applications of Dendrimers

Micro and Nano Technologies

Pharmaceutical Applications of Dendrimers Edited by Abhay Chauhan Hitesh Kulhari

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States © 2020 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-814527-2 For information on all Elsevier publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Matthew Deans Acquisition Editor: Simon Holt Editorial Project Manager: Sam W. Young Production Project Manager: Anitha Sivaraj Cover Designer: Greg Harris Typeset by SPi Global, India

Contributors Becka Anton School of Pharmacy, Medical College of Wisconsin, Milwaukee, WI, United States Aparna Areti Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER)-Hyderabad, Balanagar, India Suresh K. Bhargava Centre for Advanced Materials & Industrial Chemistry (CAMIC), School of Science, RMIT University, Melbourne, VIC, Australia Srinivasa Reddy Bonam Vaccine Immunology Laboratory, Natural Products Chemistry Division, CSIR-Indian Institute of Chemical Technology; Academy of Scientific and Innovative Research (AcSIR), CSIR-IICT Campus, Hyderabad, India; CNRS, Biotechnology and Cell Signaling, University of Strasbourg, Illkirch; Laboratory of Excellence Medalis, Strasbourg, France Yogeshwari Borade National Institute of Pharmaceutical Education and Research (NIPER) Ahmedabad, An Institute of National Importance, Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Government of India, Gandhinagar, India Pooja Borisa National Institute of Pharmaceutical Education and Research (NIPER) Ahmedabad, An Institute of National Importance, Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Government of India, Gandhinagar, India Abhay Chauhan School of Pharmacy, Medical College of Wisconsin, Milwaukee, WI, United States Avinash Gothwal Department of Pharmacy, School of Chemical Sciences and Pharmacy, Central University of Rajasthan, Ajmer, India Umesh Gupta Department of Pharmacy, School of Chemical Sciences and Pharmacy, Central University of Rajasthan, Ajmer, India Nitin Gupta School of Nano Sciences, Central University of Gujarat, Gandhinagar, India David M. Hedstrand National Dendrimer & Nanotechnology Center, NanoSynthons LLC, Mt. Pleasant, MI, United States Narenda Kumar Jain Rajiv Gandhi Technological University, Bhopal, India Poonam Jain School of Nano Sciences, Central University of Gujarat, Gandhinagar, India xi

xii

Contributors

Tukaram Karanwad National Institute of Pharmaceutical Education and Research (NIPER) Ahmedabad, An Institute of National Importance, Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Government of India, Gandhinagar, India Prashanth Komirishetty Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER)-Hyderabad, Balanagar, India; Division of Neurology & Neuroscience and Mental Health Institute, Department of Medicine, University of Alberta, Edmonton, AB, Canada Dominika Krynicka School of Pharmacy, Medical College of Wisconsin, Milwaukee, WI, United States Hitesh Kulhari School of Nano Sciences, Central University of Gujarat, Gandhinagar, India Sarita Malik Department of Pharmacy, School of Chemical Sciences and Pharmacy, Central University of Rajasthan, Ajmer, India Sylviane Muller CNRS, Biotechnology and Cell Signaling, University of Strasbourg, Illkirch; Laboratory of Excellence Medalis; University of Strasbourg Institute for Advanced d e ration Hospitalo-Universitaire OMICARE, Fe  de ration de Me decine Study; Fe Translationnelle de Strasbourg, Strasbourg University, Strasbourg, France Linda S. Nixon National Dendrimer & Nanotechnology Center, NanoSynthons LLC, Mt. Pleasant, MI, United States Vruti Patel National Institute of Pharmaceutical Education and Research (NIPER) Ahmedabad, An Institute of National Importance, Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Government of India, Gandhinagar, India Chaitrali Patil School of Pharmacy, Medical College of Wisconsin, Milwaukee, WI, United States; School of Nano Sciences, Central University of Gujarat, Gandhinagar, India Deep Pooja Centre for Advanced Materials & Industrial Chemistry (CAMIC), School of Science, RMIT University, Melbourne, VIC, Australia; Applied Biology Division, Pharmacology and Toxicology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad, India Divya Bharti Rai School of Nano Sciences, Central University of Gujarat, Gandhinagar, India Chitra Rajani National Institute of Pharmaceutical Education and Research (NIPER) Ahmedabad, An Institute of National Importance, Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Government of India, Gandhinagar, India Kuldeep Rajpoot Pharmaceutical Research Project Laboratory, Institute of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya (A Central University), Bilaspur, India

Contributors

xiii

T. Srinivasa Reddy Centre for Advanced Materials & Industrial Chemistry (CAMIC), School of Science, RMIT University, Melbourne, VIC, Australia Mayank K. Singh Department of Applied Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad, India Ramakrishna Sistla Applied Biology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad, India Rakesh K. Tekade Department of Materials Engineering, Indian Institute of Technology-Jammu, India Donald A. Tomalia National Dendrimer & Nanotechnology Center, NanoSynthons LLC, Mt. Pleasant, MI; Department of Chemistry, University of Pennsylvania, Philadelphia, PA; Department of Physics, University of Virginia Commonwealth, Richmond, VA, United States Pushpendra Kumar Tripathi Department of Pharmacy, RITM, Dr APJ Abdual Kalam Technical University, Lucknow, India Shalini Tripathi Department of Pharmacy, RITM, Dr APJ Abdual Kalam Technical University, Lucknow, India Lakshmi Tunki Centre for Advanced Materials & Industrial Chemistry (CAMIC), School of Science, RMIT University, Melbourne, VIC, Australia; Applied Biology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad, India

Preface Dendrimers are engineered, three-dimensional, nanostructures with a high degree of molecular uniformity, tunable size, multivalency, high surface functionality and high aqueous solubility. Because of these important and attractive properties, dendrimers are already being harnessed to address many of the key problems of formulation development especially for water insoluble drugs and for drug targeting. Further, it is expected that the applicability of dendrimers would increase in coming years in different fields like biomedical, cosmeceuticals, veterinary and agriculture. Pharmaceutical Applications of Dendrimers has three sections. First section describes the Critical Nanoscale Design Parameters of dendrimers for its applications as nanomedicine. Second section is focused on the applications of dendrimers in pharmaceutical sciences and medicines. This section illustrates the utilization of dendrimers in designing of pharmaceutical formulations. Initial few chapters of this section are on the role of dendrimers in preformulation stage while later chapters provide the understanding for development of dendrimer-based advanced formulations. This section describes the potential of dendrimers in designing of targeted formulations for cancer treatment, prodrugs, gene delivery, immunotherapy and hormone therapy. A chapter has been included on the biocompatibility and toxicity of the dendrimers which is an important aspect in the development of a safe and an effective formulation. Considering the significance of dendrimers in fields other than pharmaceutical sciences and medicines, Third section includes the chapters describing the applications of dendrimers in diagnosis, cosmeceuticals, veterinary and agriculture. Finally, the editors thank to the generous support and contributions of all the authors of the chapters included in this book.

—Abhay Chauhan, Hitesh Kulhari

xv

1 Engineering critical nanoscale design parameters (CNDPs): A strategy for developing effective nanomedicine therapies and assessing quantitative nanoscale structure-activity relationships (QNSARs) Donald A. Tomaliaa,b,c, Linda S. Nixona, David M. Hedstranda a

NA TIONAL DE NDRIMER & NANOTECHNOLOGY CENTER, NANOSYNTHONS LLC, MT . PL E AS AN T , M I , U N IT ED ST A TE S b DE PART MENT OF CHEMIS TRY , UNIV ER SIT Y O F PENNSYLVANI A, P HI LADELP HIA, PA, UNITED STATES c DE PARTMENT OF PHYSICS, UNIVERSITY OF VI RGINI A COMMONWEALT H, RI CHMOND, VA, UNITED STATES

1 Introduction Essentially all traditional structure activity relationships (QSARs) and successful pharmaceutical/medical therapies during the past century have been based on a simple premise. Simply stated, it was based on the ability to precisely control/engineer critical molecular level design parameters (CMDPs) of small pharmaceutically active molecules. These CMDPs included molecular level size, shape, surface chemistry, flexibility/rigidity, architecture and elemental composition. Mechanistically, CMDPs are largely controlled by fundamental electronic/steric dynamics involving atomic/molecular level bond formation or self-assembly principles. In retrospect, a minimum of human engineering was required for the controlled synthesis of literally millions of precisely defined active structures now recognized as traditional pharmaceuticals. That withstanding, the emergence of unprecedented benefits/advantages associated with active nanostructures and nano-targeting therapies involving nanoparticles has presented revolutionary new possibilities, as well as fundamental new challenges. The revolutionary benefits offered by the emergence of nanomedicine are widely recognized and clearly articulated. However, less apparent is the role and importance of “engineering CNDP controlled nanoparticle

Pharmaceutical Applications of Dendrimers. https://doi.org/10.1016/B978-0-12-814527-2.00001-9 © 2020 Elsevier Inc. All rights reserved.

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4

Pharmaceutical Applications of Dendrimers

syntheses” suitable for use in the life sciences and nanomedicine. This is perhaps one of the highest priority challenges facing the future progress of nanomedicine and is the topic of this chapter.

2 Historical 2.1 Dendrimer-based nanoscale atom mimicry: A window to nanoperiodicity Development of a unified framework for systematically organizing structure-function properties of nanomaterials, much like the Mendeleev Periodic Table for atoms, was proposed to provide a paradigm for understanding important physico-chemical relationships. Defining well-defined nanoperiodic property patterns for nanomaterials would be expected to enable the prediction of new properties without synthesis and facilitate the translation of nanomaterials into clinical applications. It is from this perspective, that a new nanoperiodic concept involving structure-controlled, hard or soft nanoparticles possessing tailorable critical nanoscale design parameters (CNDPs) has been introduced recently [1,2]. These CNDPs include: size, shape, surface chemistry, flexibility/rigidity, architecture and elemental composition. Many of these discrete, well-defined nanoparticles are being considered for a wide variety of nanomedicine applications. This concept teaches that systematic engineering of these CNDPs provides a powerful strategy for optimizing, as well as a priori prediction of ideal function and property designs required for many nanomedicine applications. This paradigm may be used as a perspective from which to appraise the progress/failure of traditional polymer/liposome-based nanomedicine platforms, as well as several contemporary more successful nanoparticle platforms. The ability to control and engineer specific CNDP features in nanostructures was first observed in the 1980–90s, while investigating dendrons and dendrimers and reported in an article entitled: Starburst Dendrimers: Molecular Level Control of Size, Shape, Surface Chemistry, Topology, and Flexibility From Atoms to Macroscopic Matter [3]. This work inspired the evolution of a systematic nanoperiodic concept [1,3–6] for defining and understanding the properties of many discrete hard and soft nanoparticles now currently used in nanomedicine. Within a decade after the discovery of dendrimers [3] and at least a decade before the formal emergence of nanotechnology (i.e., early 2000s), these nanoparticles were formally recognized to represent the first mathematically defined, synthetic entities to exhibit essential structural control of size, shape, surface chemistry and flexibility (Fig. 1.1). This dendrimer-based structural control was proposed to be involved in the transfer of critical hierarchical information from the atomic to macroscale levels. Dendrimers were subsequently used as a model system to demonstrate the critical role that they could in defining new emerging properties [5] and unprecedented nanoperiodic property patterns [4,6,7] as a function of six critical nanoscale design parameters (CNDPs); namely: size, shape, surface chemistry, flexibility/rigidity, architecture and elemental composition.

Chapter 1 • Engineering critical nanoscale design parameters (CNDPs)

5

FIG. 1.1 Front cover of Angew Chem Int Ed Engl 1990;29:138–75 first describing structural control of critical hierarchical design parameters (CHDP) from atoms to macroscopic matter observed during the divergent syntheses of all dendrimers. Adapted with permission. Copyright 1990 Wiley-VCH Verlag GmbH & KGaA.

2.2 Dendrimer-based protein mimicry Furthermore, this unprecedented structural control was recognized to rival the structural control observed in iconic biological macromolecules such as proteins [8]. These unique features, especially their precisely defined sizes, shapes and surface chemistries led to frequent reference to dendrimers as artificial proteins [9,10]. In many respects, certain discrete dendrimer sizes, shapes and surface chemistries were found to be essentially comparable to those found in histones, ubiquitous protein scaffoldings in biological systems used for storing and presenting nucleic acids (Fig. 1.2). In fact, the presumed histone mimicry inspired some of the first examples using PAMAM dendrimers as synthetic DNA/RNA vectors and currently reported in a wide variety of gene transfection applications (https://www.qiagen.com/us/shop/transfection/ superfect-transfection-reagent) [11–14]. Extensive work by Cheng et al. [15] describes the engineering of certain dendrimer CNDPs (i.e., size, surface chemistry, etc.) for optimizing transfection properties of dendritic vectors.

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Pharmaceutical Applications of Dendrimers

FIG. 1.2 A comparison of dimensional length scales (nm) for poly(amidoamine) (PAMAM) dendrimers (Gen. 4–7), Nc ¼ 3, Nb ¼ 2 (NH3 core) and various biological entities (e.g., proteins, DNA and lipid bilayers). Reproduced with chet JMJ. Discovery of dendrimers and dendritic polymers: a brief historical permission from Tomalia DA, Fre perspective. J Polym Sci Part A Polym Chem. 2002;40(16):2719–28. Copyright 2002 John Wiley & Sons.

2.3 Dendrimer-based dendritic effects as a window to nanoperiodic property patterns Unexpected dendrimer guest-host encapsulation properties now routinely associated with many important drug delivery applications were reported as early as 1987 for PAMAM dendrimers [16]. This encapsulation property is now known to be dependent upon branch cell symmetry (i.e., architecture/shape) described in Fig. 1.3. This subtle branch cell symmetry feature found in PAMAM dendrimers accounts for why that dendrimer family exhibit dramatic encapsulation properties; whereas, poly(lysine) dendrimers possess no interior guest-host properties. The unique interplay of specific CNDPs (i.e., size, shape, surface chemistry, flexibility/ rigidity) involved in the tethered dendritic growth of dendrimers very nicely defines specific generational levels that are optimum for encapsulation as illustrated in Fig. 1.4. The importance of dendrimer generation level (i.e., size) [3] for effective encapsulation and dendrimer container properties is illustrated in Fig. 1.5 and has been reviewed extensively elsewhere [15].

.64 .60 .56 .52

Denkewalter (unsymmetrical) branch cell dendrimers

.48 AMU 3

Å

.44

(A)

.40 .36 .32 .28 .24 .20

Tomalia (PAMAM) (symmetrical) branch cell dendrimers

.16 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 Generation

(B)

FIG. 1.3 Comparison of densities as a function of generation for (A) asymmetrical branch cell in Denkewalter-type dendrimers, (B) symmetrical branch cell in Tomalia-type dendrimers (densities calculated from experimental and theoretical hydrodynamic diameters) [16]. Reproduced with permission of Tomalia DA. Dendritic effects: dependency of dendritic nano-periodic property patterns on critical nanoscale design parameters (CNDPs). New J Chem 2012;36:264–81. Copyright 2012 The Royal Society of Chemistry.

10

Surface Chemistry

Congestion

9 8

Amplification (# of terminal groups (Z)

Surface area/ terminal groups (Z)

Size

7 6 5

Shape

Dendrimer Diameters (nm)

Gc

(aspect ratio)

4

Flexibility

3

Container Properties

2 1

1

2

3

4

5

6

7

8

9

10

Dendrimer Generations FIG. 1.4 A comparison of emerging nanoperiodic property patterns (i.e., endo- and exo-dendritic effects) as a function of dendrimer generation level related to congestion, surface chemistry, size, flexibility, nanocontainer properties and shape. A critical generation level (Gc) is defined by the three CNDP parameters, namely: (1) size, (2) surface chemistry amplification and (3) congestion/flexibility. Reproduced with permission of Tomalia DA. Dendritic effects: dependency of dendritic nano-periodic property patterns on critical nanoscale design parameters (CNDPs). New J Chem 2012;36:264–81. Copyright 2012 The Royal Society of Chemistry.

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Pharmaceutical Applications of Dendrimers

(I) Flexible Scaffolding Z–Z Distances

10.71A

Z

Z Z

Z

Z

Z

(II) Container Properties

10.71A

10.25A

9.52A

8.46A

(III) Rigid Surface Scaffolding 7.12A

5.62A

--

--

ZZ

ZZ

Z Z

ZZ

Z Z

Z Z

Z Z

4

5

6

7

8

Z

Z

Z

De Gennes Dense Packing G=0

1

2

3

9

10

Flexible, Open Dendritic Structures No interior Accessible Interior

Inaccessible Interior

FIG. 1.5 Congestion induced dendrimer shape changes (I, II, III) with development of nanocontainer properties for a family of [core:1,2-diaminoethane];(4 ! 2); dendri-{poly(amidoamine)–(NH2)Z}; (G ¼ 0–10) (PAMAM) dendrimers: Nc ¼ 4; Nb ¼ 2, where: Z-Z ¼ distance between surface groups as a function of generation. Reproduced with permission from Tomalia DA. Dendrons/dendrimer: quantized, nano-element like building blocks for soft-soft and soft-hard nano-compound synthesis. Soft Matter 2010;6:456–74. Copyright 2010 The Royal Society of Chemistry.

More recently, new emerging properties related to architectural confinement of electron rich moieties (i.e., amine, carboxylic acids, amido, hydroxyl groups) were observed to exhibit unusual fluorescence emission properties in dendrimers [17,18] and other related hyperbranched systems. These luminescence properties referred to as non-traditional intrinsic luminescence (NTIL) have been examined extensively [19–21] and found to involve a significant paradigm shift from traditional luminescence principles. This new phenomena has been observed in a wide range of structures/compositions and explained mechanistically as a function of chemical or physical spatial confinement of high electron density hetero-atomic moieties induced by CNDPs such as architecture (i.e., dendritic) or by CNDPs associated with chemical/physical flexibility/rigidity [21]. Many early reports of unexpected or unprecedented properties observed for dendrimers were categorically referred to as so-called dendritic effects. It is now recognized that these dendritic effects are actually related to the six well-defined/controlled CNDPs. It is these six dendrimer CNDPs that exquisitely define a wide range of important dendrimerbased nanoperiodic property patterns that heuristically mimic traditional elemental property patterns reported by Mendeleev in the 19th century [7] (Fig. 1.6).

3 Application of nanoperiodic property patterns and CNDP engineering principles to nanomedicine 3.1 Developing an optimum nanotherapeutic platform based on intrinsic structure controlled, tunable nanoparticle CNDPs The primary objectives of the FDA regulatory approval process for all nanomedicine applications include the need to deliver patient compliant nano-platforms with good safety

Chapter 1 • Engineering critical nanoscale design parameters (CNDPs)

9

FIG. 1.6 The relationship of dendritic effects to critical nanoscale design parameters (CNDPs) that include: (1) size, (2) shape, (3) surface chemistry, (4) flexibility/rigidity, (5) architecture and (6) elemental composition and the emergence of nanoperiodic property patterns based on these relationships. Reproduced with permission of Tomalia DA. Dendritic effects: dependency of dendritic nano-periodic property patterns on critical nanoscale design parameters (CNDPs). New J Chem 2012;36:264–81. Copyright 2012 The Royal Society of Chemistry.

margins, high therapeutic efficacy at an acceptable cost. A typical FDA regulatory pathway roadmap through the preclinical, investigational new drug (IND) and Phase I–III clinical evaluation stages is described in Scheme 1.1. As such, the nanomedicine approval process is both complex and challenging. Much as for traditional small pharmaceutical medicine, it is important to develop viable strategies and optimize all critical parameters required for selecting highest probability nanoscale candidates [26–28]. Usually the first step invoked by traditional pharmaceutical scientists was to use quantitative structure-activity relationship (QSAR) analyses for defining and optimizing best small molecule structures as part of the preclinical evaluation process. This strategy has been highly successful for developing predictive patterns and identifying many optimal traditional pharmaceutical candidates. For example, CMDPs such as molecular size, surface chemistry and flexibility were found to determine valuable and predictable patterns for optimizing transport of small molecule pharmaceuticals across blood-brain barriers [29]. As such, many of these QSAR-like principles based on traditional pharmaceuticals are currently being applied to nanoparticle CNDPs and their role in various nanomedicine applications [26–28]. For example, dendrimer CNDPs are currently being examined as a function of their intrinsic interaction relationships with drugs (Fig. 1.7A), as well as their specific mode of delivery as described in Fig. 1.7B. For example, interactions with lipid membranes, proteins [30] and genetic material [31] are actively under investigation in an effort to define specific behavior patterns for their use as delivery vectors in nanomedicine [32]. An extensive QNSAR investigation has been reported recently concerning the use of PAMAM dendrimers as polyvalent scaffolding for presenting sialic acid (i.e., sugars) as decoy moieties for disrupting the adhesion properties of influenza A. This topic is discussed extensively in Section 8.2.

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Pharmaceutical Applications of Dendrimers

SCHEME 1.1 Roadmap for translation of a typical nanomedicine platform application from the discovery stage through Phase I–III human clinical trials to FDA approval and commercialization [22–25]. Reproduced with permission from Kannan RM, Nance E, Kannan S, Tomalia DA. Emerging concepts in dendrimer-based nanomedicine: from design principles to clinical applications. J Intern Med 2014;276:579–617. Copyright 2014 John Wiley & Sons.

FIG. 1.7 Dendrimer-drug relationships based on structure controlled CNDPs that may be readily engineered and various nanomedicine drug delivery strategies. (A) Dendrimers have exhibited at least six unique relationships with traditional drugs including: (a) dendrimer drug conjugates, (b) encapsulation of drugs in dendrimers, (c) dendrimers as nano-excipients for drugs (i.e., oral delivery), (d) dendrimer drug complexes, (e) dendrimer imaging/ablation agent conjugates, and (f ) dendrimers as intrinsically active nanopharmaceuticals. (B) The diversity of well-defined dendrimer-drug relationships provides at least six demonstrated drug targeting strategies; namely: passive, active (receptor mediated), active (angiogenic endothelial cell mediated), magnetically guided, image guided and intrinsic (i.e., polyvalent mediated). Reproduced with permission from Kannan RM, Nance E, Kannan S, Tomalia DA. Emerging concepts in dendrimer-based nanomedicine: from design principles to clinical applications. J Intern Med 2014;276:579–617. Copyright 2014 John Wiley & Sons.

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It is notable that Nel et al. [33] have defined nanoparticle “size” followed by “shape” [34] as perhaps the two most important physical properties (i.e., CNDPs) of nanoparticles in nanomedicine applications. These two CNDPs determine many cellular uptake, transport and accumulation events that occur in vivo. All biological organisms depend on highly tuned and controlled uptake/transport activities that are strictly size and shape regulated. For example, all cellular membrane bilayers exhibit a dimension of 4–10 nm. On the other hand, vertebrate nuclear pore complexes are approximately 80–120 nm in diameter [35]. As such, these size-regulated biostructures exercise very important barrier functions as nanoparticles enter and exit. These size/shape controlled sites determine nanoparticle interactions with biological membranes, organelles, etc. that in turn directly influence cellular uptake, bioavailability, cellular excretion, tissue accumulation, cellular metabolism and organ toxicity to mention a few. Unarguably, other CNDPs such as surface chemistry, flexibility/rigidity, elemental composition and architecture will also exert their own intrinsic influences on many of these events and dynamics [36]. Finally, it is also well known that nanoparticle interactions with functional barrier/porosity parameters related to disease pathology, such as blood brain barrier (BBB) impairment, enhanced permeation and retention (EPR) effect, inflammation and infection may have to be engineered based CNDPs. With this in mind, it is important to consider a nanotherapy (i.e., nanoparticle) roadmap (see Fig. 1.4) describing possible pathways for observed excretion, extravasation and certain biodistribution modes much as used for traditional drugs. More specifically, this flowchart allows one to examine the dependency of therapeutic size on intrinsic in vivo behavior patterns for traditional small molecule CMDPs compared to larger nanoparticle CNDPs. Irrespective of the administration mode (i.e., oral, parenteral, inhalation, etc.), in vivo clearance will be expected to follow certain pathways that we now know are largely directed by either CMDP or CNDP features. As such, these CNDP dependent in vivo barriers must be considered in the total design and engineering of all nanoscale imaging, therapies and diagnostic strategies and devices. Nevertheless, after systemic administration an immediate consequence is that subtle nanoscale size differences will determine: (a) the rate of extravasation from the circulatory system, (b) the mode of excretion (i.e., kidney versus liver or spleen), (c) the blood circulation time, (d) propensity for passive targeting via the enhanced permeation and retention (EPR effect) [37], (e) organ selectivity, (f ) disease site (i.e., tumor) penetration or organ penetration and (g) receptor mediated recognition space or cell uptake behavior, etc. For example, systemic administration of small molecule drug sizes (4 nm) are observed to exhibit longer circulatory residency times, size selective excretion modes and permeability patterns associated with their nanoscale sizes and respective surface chemistries as shown in Fig. 1.8 [38–41]. These remarkable nano-size dependent excretion modes are some of the first examples of CNDP dependent nanoperiodic property patterns observed in vivo [38].

Chapter 1 • Engineering critical nanoscale design parameters (CNDPs) 13

FIG. 1.8 A schematic roadmap depicting in vivo barrier and processing differences (i.e., I–VI) experienced by small molecule drugs (2630 proteins [53] found in the cerebrospinal fluid (CSF). These in vivo nanoparticle-protein interactions lead to specific NP-protein coatings (i.e., coronas) that are largely determined by the CNDPs of the NPs and proteins respectively. These NP-protein coronas define many important pharmacological behavior properties including: (a) absorption, (b) biodistribution, (c) biocompatibility, (d) excretion and (e) biological activity of the nanoparticles.

5.1 Size Nanoparticle dimensions play a dominant role in essentially all nanobiological interface studies and applications. Appropriate nano-size scaling and control of monodispersity are critical nanoparticle features required to successfully negotiate the wide range of in vivo barriers as described in Fig. 1.4. Nanoscale particle dimensions have been shown to dramatically affect essentially all major dynamic in vivo physiology patterns. For example, as a function of increasing nanometric size, nanoparticles may be engineered/tuned to exhibit important properties such as: (a) extravasation from circulation, (b) retention as

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blood pool agents, (c) kidney excretion, (d) lymph node affinity, (e) liver excretion, (f ) enhanced circulation times (Fig. 1.10). Furthermore, nanoparticle size and specific surface chemistry combinations can dramatically influence certain cytotoxicity patterns. In general, soft nanoparticle cytotoxicity (i.e., dendrimers) has been shown to be enhanced as a function of size according to the following surface chemistry order: anionic < neutral-hydrophilic < neutral-hydrophobic < cationic as illustrated in Fig. 1.9. On the other hand, it is notable that certain hard nanoparticles (i.e., gold nanoclusters) have been reported to exhibit inverse cytotoxicity size patterns compared to soft nanoparticles such as dendrimers as described above. In the case of gold nanoclusters particles, sizes 15 nm) appear to present little risk [54]. It is generally recognized that many important in vivo physiological phenomena including: (a) complement activation, (b) protein optimization, (c) bilayer disruption and (d) platelet lysis, (e) G protein coupled receptor (GPCR) communication and (f ) inflammatory/allergenic cascade processes are strongly influenced by both nanoscale particle sizes and surface chemistry. Understanding the role of these two CNDPs is also

FIG. 1.9 Dependency of biodistribution, toxicity, biopermeability, immunogenicity and membrane/platelet disruption on dendrimer size (i.e., generation) and surface chemistry. Reproduced with permission from Kannan RM, Nance E, Kannan S, Tomalia DA. Emerging concepts in dendrimer-based nanomedicine: from design principles to clinical applications. J Intern Med 2014;276:579–617. Copyright 2014 John Wiley & Sons.

Chapter 1 • Engineering critical nanoscale design parameters (CNDPs)

17

FIG. 1.10 Nanoperiodic drug encapsulation and organ/tumor targeting patterns observed for poly(amidoamine) (PAMAM) dendrimers based on critical nanoscale design parameters such as: size, interior/surface chemistry and flexibility/rigidity [38, 49]. Adapted with permission from Tomalia DA, Reyna LA, Svenson S. Dendrimers as multipurpose nanodevices for oncology drug delivery and diagnostic imaging. Biochem Soc Trans 2007;35(1):61–67, http:// www.biochemsoctrans.org/content/35/1/61, Copyright 2007 Portland Press.

critical in the treatment of certain diseases. For example, cancer tumor penetration and intra-tumoral distribution by nanoparticle delivery vectors is highly size dependent relative to tumor porosity. As such, the use of smaller nanoparticle dimensions as nano-therapy vectors is observed to be more effective against most tumors than larger nanoparticles [43,55–60]. Although specific nanometric sizes are important for optimizing certain nanobiological interface functions/applications, it is also very important to control nanoparticle dispersity. Therefore, it is very critical to understand nanoparticle aggregation properties as a function of environment (i.e., pH, solvent polarity, temperature, etc.). Undoubtedly, polydispersed particle sizes associated with supramolecular liposome assemblies account for many spurious, as well as unexpected in vivo biodistribution patterns and targeting results [61]. In fact, it has been shown that nanometric size differences as small as 1–3 nm may completely alter the overall excretion mode of a nanoparticle. It has been reported by Tomalia et al. [1,4,5,38], Kobayashi et al. [41] and others that in vivo administered PAMAM dendrimerbased MRI contrast agents exhibit very selective blood pool, organ selectivity and renal/liver excretion properties which are very sensitive to their monodispersed, nanoscale dimensions (Fig. 1.10). Similarly, Bawendi et al. [39] and Wiesner et al. [40] observed analogous are corroborating kidney and liver size excretion patterns for a wide range of soft and hard nanoparticles including: proteins, quantum dots and silica nanoparticles, respectively.

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Very important trans-epidermal penetration properties involving dendrimers were found to be dependent not only on size, but also surface chemistry as shown in Fig. 1.11. Combining low PAMAM dendrimer generations (i.e., G2) with cationic, neutral or anionic polar surface moieties (i.e., amino-/amido-/amido-carboxylate groups) produced complete penetration through the stratum corneum and well into the viable epidermis and dermis domains. Replacing the dendrimer surface polar amide moieties with hydrophobic functionality (i.e., oleate) reduced penetration; whereas, increasing the generation level from 2 to 4 for cationic amine terminated dendrimers completely suppressed dermal penetration (Fig. 1.11).

6 Recent progress in dendrimer based CNDP-engineering As described above, a versatile concept has now emerged for relating critical nanoscale design parameters (CNDPs) to nanoperiodic property patterns [4,5,7] and engineering

FIG. 1.11 A comparison of critical nanoscale design parameters (i.e., size and surface chemistry, etc.) directed patterns associated with epidermal penetration by poly(amidoamine) (PAMAM) dendrimers. These quantized epidermal penetration patterns are useful for designing and engineering optimized dendrimer prototypes for use as topical microbicides or transdermal drug delivery vectors. Reproduced and adapted with permission from Yang Y, Sunoqrot S, Stowell C, Ji J, Lee C-W, Kim JW, et al. Effect of size, surface charge, and hydrophobicity of poly(amidoamine) dendrimers on their skin penetration. Biomacromolecules 2012;13:2154–62. Copyright 2012 American Chemical Society.

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FIG. 1.12 A schematic illustration of dendrimer-based critical nanoscale design parameter control and engineering for optimizing prototypes suitable for various nanomedical applications. (i) Size control (approximately 1 nm per generation) with mathematically defined polyvalent surface functionality, (ii) polyvalent dendrimer surface chemistry can be chemically portioned into imaging moieties (A), therapy with cleavable linkers (B), targeting groups (C) and biocompatibility or circulatory enhancement groups for stealth properties (Z) [38,49]. Adapted with permission from Tomalia DA, Reyna LA, Svenson S. Dendrimers as multi-purpose nanodevices for oncology drug delivery and diagnostic imaging. Biochem Soc Trans 2007;35(1):61–67, http://www.biochemsoctrans.org/content/ 35/1/61, Copyright 2007 Portland Press.

NP-CNDPs as a strategy for optimizing NPs to specific applications has now been demonstrated [2,5,7,49]. In this regard, recent progress describing CNDP directed biological activity will be overviewed briefly (Fig. 1.12). The importance of engineering CNDPs (Fig. 1.13) of nanoparticles used in nanomedicine applications may be noted by the explosion of literature citations appearing in the last decade. A small sampling of these citations is documented in Table 1.1.

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FIG. 1.13 Engineering critical nanoscale design parameters using dendritic architecture, (I) size, (II) shape, (III) surface chemistry, (IV) flexibility/rigidity and (V) elemental composition.

Table 1.1

Dendrimer-based CNDP engineered applications and properties

CNDPs

Applications

Functions/properties

References

(I) Size

Infectious diseases Neuro-therapeutics Drug delivery Cancer therapy Gene therapy Wound healing Photodynamic therapy Catalysis Toxicology Surface patterning Antiviral MRI imaging

Anti-inflammatory Anti-inflammatory Encapsulation Targeted scaffolding Scaffolding Scaffolding Scaffolding Encapsulation – Scaffolding Scaffolding Scaffolding

[62–67] [49,68] [49,63,69–82] [49,63,72,74,77,78,83–87] [49,63,81,83,88,89] [90] [91] [92,93] [73,94,95] [96] [63,97,98] [99,100]

Chapter 1 • Engineering critical nanoscale design parameters (CNDPs)

Table 1.1 CNDPs

(II) Shape

(III) Surface chemistry

(IV) Flexibility/ rigidity

21

Dendrimer-based CNDP engineered applications and properties—cont’d Applications

Functions/properties

References

Anti-bacterial Nano-assemblies Ocular diseases Bioadhesives Bioimaging Nano-separations Immuno-conjugates Wound healing Cancer therapy Surface patterning Drug delivery MRI imaging Nano-assemblies Ocular diseases Infectious diseases Neuro-therapeutics Drug delivery Cancer therapy Gene therapy Anti-prion Targeted delivery Diagnostics Immuno-conjugates Imaging Photothermal therapy Wound healing Protein delivery Anti-protein fouling Photodynamic therapy Transdermal delivery Catalysis Toxicology Antiviral Anti-bacterial Nano-assemblies Ocular diseases Bioadhesives

Scaffolding Biodistribution Nanofibers UV curable/low toxicity Intrinsic fluorescence Templates Scaffolding Scaffolding Scaffolding Scaffolding Scaffolding Scaffolding Biodistribution Nanofibers Anti-inflammatory Anti-inflammatory Encapsulation Targeted scaffolding Scaffolding Scaffolding Scaffolding Scaffolding Scaffolding Scaffolding Scaffolding Scaffolding Scaffolding Scaffolding Scaffolding Scaffolding – – Scaffolding Scaffolding Biodistribution Nanofibers UV-curable, reduced toxicity PABA based Intrinsic fluorescence

[101,102] [103] [76] [104] [105] [106] [107] [90] [72] [96] [69] [100] [103] [76] [49,62,64–67,108] [49,68,83] [49,69–72,75–77,80,82,87] [49,72,73,77–79,84,85,89,109] [13–15,49,81,83,87–89,108–110] [111] [49,74,83] [83,84] [107] [99,100,105,107] [109] [90] [72,112] [113] [91] [114] [92,93] [73,94,95] [97,98,101] [101,102] [103] [76] [104]

Templates Stealth features Self-assembly

[106] [111] [69,75,76,81,116]

Antioxidants Non-traditional intrinsic fluorescent Nano-separation Anti-prion agents Drug delivery

[115] [105]

Continued

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Pharmaceutical Applications of Dendrimers

Table 1.1

Dendrimer-based CNDP engineered applications and properties—cont’d

CNDPs

(V) Composition

Applications

Functions/properties

References

Anti-bacterial Ocular diseases Neuro-therapeutics Drug delivery Cancer therapy Gene therapy Anti-prion Ocular diseases Immuno-conjugates Photodynamic therapy Transdermal delivery Catalysis Toxicity Surface patterning Antiviral Anti-bacterial Bioadhesives Antioxidants Fluorescence imaging

Adhesion Nanofibers Anti-inflammatory Encapsulation Targeted scaffolding – Scaffolding – Scaffolding Scaffolding Scaffolding Encapsulation Scaffolding Scaffolding Scaffolding Scaffolding UV curable, low toxicity PABA based Intrinsic fluorescence

[101] [76] [49,62,63,67,68,108] [63,69,71,72,75,79,80,83,116] [49,72,84,89] [49,63,89,108] [111] [49,76,83] [107] [91] [114] [92] [94,95,108] [96] [97] [101,102] [104] [115] [105]

6.1 Size/shape Control of persistent 3-D size and shape of soft therapeutic nanoparticles is important for predicting in vivo biodistribution [117], cellular uptake, tumor uptake [118], as well as excretion modes. It is known, that traditional random coil, linear polymers and certain rod-like nanostructures may reptate and undergo extravasation through very small vascular pores even when G5 (37.30%) [35% survival] (high K); >G4 (31.45% survival) [moderate K]; >G5 (40.35%) [moderate K]; >G4 (40.30%) [weak K, no survivors]; >G5 (55.45%) [weak K, no survivors]; >G5 (74.55%) [weak K, no survivors]. Adapted from Kwon S-J, Na DH, Kwak JH, Douaisi M, Zhang F, Park EJ, et al. Nanostructured glycan architecture is important in the inhibition of influenza A virus infection. Nat Nanotechnol 2017;12:48–54. Copyright 2017 Springer Nature.

when tested with animal models. Therefore, as shown in Fig. 1.21, binding constants (K) were determined for sialic acid (i.e., sugar), surface modified PAMAM dendrimers (i.e., G2-G5) as a function of the sugar moiety surface density (density %) for each dendrimer generation (i.e., size). Firstly, essentially no sugar-HA interaction was noted for G2 and G3 conjugates. This QNSAR study demonstrated the viability of an unprecedented non-vaccine/nonantibody therapy strategy based on the interaction of CNDP engineered PAMAM dendrimer conjugates (i.e., possessing sialic acid, surface functionality) with critical trimeric HA moieties found on the surfaces of most virulent viruses. These dendrimer sialic acid-viral HA interactions were shown to dramatically disrupt critical viral adhesion events with healthy cells. These interactions were observed to quantitatively reduce viral infectivity as a function of the strongest dendrimer-HA interaction constant K. Corroborating

Chapter 1 • Engineering critical nanoscale design parameters (CNDPs)

37

FIG. 1.22 Systematic CNDP engineering a library of PAMAM-sialic acid conjugates produced the best antiviral candidate, namely G4 (sialic acid saturation level; 20.55%). This selection was based on observing the highest complexation constant (K) for the influenza A virus-dendrimer conjugate interaction compared to all the library candidates. This selection was corroborated by observing that it protected the highest level of mice (i.e., 75%) when exposed to a lethal dose of influenza A. Adapted from Kwon S-J, Na DH, Kwak JH, Douaisi M, Zhang F, Park EJ, et al. Nanostructured glycan architecture is important in the inhibition of influenza A virus infection. Nat Nanotechnol 2017;12:48–54. Copyright 2017 Springer Nature.

evidence for this hypothesis was obtained by observing that the CNDP engineered PAMAM dendrimer conjugates exhibiting the highest dendrimer-HA interaction constants (K), produced the greatest in vivo reduction in mortality (i.e., >75%) of mice infected with fatal amounts of influenza A as shown in Fig. 1.22. More specifically, the PAMAM dendrimer conjugate; G4 (20.55%) exhibited the highest K value and produced the highest in vivo survival level in mice infected with a lethal dose of influenza A. Systematic CNDP engineering of PAMAM sialic acid conjugates as a function of G (size) and sialic acid surface density showed that inter-ligand spacing rather than number of ligands (i.e., dendrimer valency) was found to be critical. This critical spacing (i.e., 3 nm) was associated with the scaffolding sizes (i.e., G4/G5) and surface saturation levels of 20–38%. Higher surface saturation levels and smaller dendrimer generation sizes reduced the binding constants between the PAMAM dendrimer conjugates and the viral HA trimeric spikes. On the other hand, strong K values were observed when the inter-ligand spacing was 3 nm as illustrated in Fig. 1.23.

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FIG. 1.23 An illustration of the optimal interactions between multivalent PAMAM; G4 (density level ¼ 20.55%) conjugate (i.e., inter-ligand spacing ¼ 3.1 nm) and the receptor binding sites on the HA trimers of influenza A to produce a strong interaction; whereas, the PAMAM; G4 (density level ¼ 40.36) conjugate with an inter-ligand spacing of 1.6 nm yields a weak interaction. Reproduced with permission from Kwon S-J, Na DH, Kwak JH, Douaisi M, Zhang F, Park EJ, et al. Nanostructured glycan architecture is important in the inhibition of influenza A virus infection. Nat Nanotechnol 2017;12:48–54. Copyright 2017 Springer Nature.

As virulent virus strains similar to pandemic influenza A continue to evolve and diversify in birds and mammals, genetic mutations can alter their infectivity, virulence or ability to spread among humans [162]. This work demonstrates that the use of multivalent dendrimer-based inhibitors with specific CNDP engineered nanostructures offers an unprecedented alternative to vaccine/antibody based strategies that are sensitive to even minor viral mutations. That withstanding, the articulate CNDP engineering required to obtain optimum inhibitor candidates depends entirely on the homogeneity and high purity of the multivalent dendrimer particles used for these viral inhibitor conjugates. Although this seminal investigation demonstrated the validity of a very powerful new strategy for controlling a pandemic-like virus with an unprecedented survival level of 75%. Unfortunately, the PAMAM dendrimer sialic acid conjugates used in this investigation [98] were optimized using the same commercial source of heterogeneous PAMAM dendrimers as described in Section 7.1 and Figs. 1.17 and 1.19 of this account. Just imagine how much higher the in vivo survival level and more dramatic the reported results might have been if the optimized dendrimer conjugates in this work had been derived from a proven homogenous PAMAM dendrimer source.

9 Conclusions The historical evolution of dendrimer-based atom mimicry and the dendritic effect leading to the current concept of dendrimer-based critical nanoscale design parameters (CNDPs) and nanoperiodicity [7] was briefly reviewed. This review provided an introduction to dendrimer based nanoperiodic property patterns and CNDP engineering principles which may be used to develop quantitative nanostructure-activity relationships (QNSARs) useful for optimizing prospective nanostructure candidates for specific nanomedicine applications and clinical trials. Several critical examples were presented showing the importance

Chapter 1 • Engineering critical nanoscale design parameters (CNDPs)

39

of using high purity, monodisperse, low defect level dendrimer samples especially for all life science applications. This chapter was concluded by focusing on an actual study reporting the use of dendrimer-based CNDP engineering principles as a strategy to select the best CNDP optimized dendrimer-sialic acid conjugate candidate for in vivo protection against a recognized pandemic pathogen; namely, influenza A (i.e., the Spanish flu).

Acknowledgment We gratefully acknowledge Prof. A. Sharma (Central Michigan University) for helpful discussions and certain analytical results described in this account.

References [1] Tomalia DA, Christensen JB, Boas U. Dendrimers, dendrons, and dendritic polymers: discovery, applications and the future. New York, NY: Cambridge University Press; 2012. [2] Tomalia DA. Dendritic effects: dependency of dendritic nano-periodic property patterns on critical nanoscale design parameters (CNDPs). New J Chem 2012;36:264–81. [3] Tomalia DA, Naylor AM, Goddard III WA. Starburst dendrimers: molecular level control of size, shape, surface chemistry, topology and flexibility from atoms to macroscopic matter. Angew Chem Int Ed Engl 1990;29(2):138–75. [4] Tomalia DA. In quest of a systematic framework for unifying and defining nanoscience. J Nanopart Res 2009;11:1251–310. [5] Tomalia DA. Dendrons/dendrimer: quantized, nano-element like building blocks for soft-soft and soft-hard nano-compound synthesis. Soft Matter 2010;6:456–74. [6] Tomalia DA, Khanna SN. In quest of a systematic framework for unifying and defining nanoscience. Mod Phys Lett B 2014;28(3):1–48. [7] Tomalia DA, Khanna SN. A systematic framework and nanoparticle concept for unifying nanoscience: hard/soft nanoelements, superatoms, meta-atoms, new emerging properties, periodic patterns, and predictive Mendeleev-like nanoperiodic tables. Chem Rev 2016;116:2705–74. [8] Goodsell DS. Biomolecules and nanotechnology. Am Sci 2000;88:230–7. chet JMJ. Dendritic encapsulation of function: applying nature’s site isolation principle [9] Hecht S, Fre from biomimetics to materials science. Angew Chem Int Ed Engl 2001;40(1):74–91. [10] Tomalia DA, Huang B, Swanson DR, Brothers II HM, Klimash JW. Structure control within poly(amidoamine) dendrimers: size, shape and regio-chemical mimicry of globular proteins. Tetrahedron 2003;59:3799–813. [11] Reebye V, Sætrom PI, Mintz PJ, Huang K-W, Swiderski P, Peng L, et al. A novel RNA oligonucleotide improves liver function and inhibits liver carcinogenesis in vivo. Hepatology 2014;59(1):216–27. [12] Tomalia DA, Baker JR, Cheng R, Bielinska AU, Fazio MJ, Hedstrand D, et al. Bioactive and/or targeted dendrimer conjugates. US Patent 5,714,166, 1998. [13] Liu X, Zhou J, Yu T, Chen CZ, Ching Q, Sengupta K, et al. Adaptive amphiphilic dendrimer-based nanoassemblies as robust and versatile siRNA delivery system. Angew Chem Int Ed Engl 2014;53:11822–7. [14] Posocco P, Liu X, Cheng Q, Laurini E, Zhou J, Liu C, et al. Mastering dendrimer self-assembly for efficient siRNA delivery: from conceptual design to in vivo efficient gene silencing. Small 2016;12:3667–76.

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chet JMJ, Tomalia DA. Dendrimers and other dendritic polymers. Chichester: Wiley; 2001. [151] Fre [152] Dvornic PR, Tomalia DA. Genealogically directed syntheses (polymerizations): direct evidence by electrospray mass spectroscopy. Macromol Symp 1995;98:403–28. [153] Tomalia DA. Some examples of dendrimer synthesis. In: Davis FJ, editor. Polymer chemistry: a practical approach. New York: Oxford University Press; 2004. p. 188–200. [154] Tomalia DA, Christensen JB, Boas U. Characterization methodologies. In: Dendrimers, dendrons, and dendritic polymers: discovery, applications and the future. New York, NY: Cambridge University Press; 2012. p. 162–86. [155] Kobayashi H, Kawamoto S, Sato N, Choyke PL, Knopp MV, Star RA, et al. Comparison of dendrimerbased macromolecular contrast agents for dynamic micro-magnetic resonance lymphangiography. Magn Reson Med 2003;50:758–66. [156] Rupp R, Rosenthal SL, Stanberry LR. VivaGelTM (SPL7013 Gel): a candidate dendrimer-microbicide for the prevention of HIV and HSV infection. Int J Nanomedicine 2007;2:561–6. [157] Bjornmalm M, Faria M, Caruso F. Increasing the impact of materials in and beyond bio-nano science. J Am Chem Soc 2016;138:13449–56. [158] Mulvaney P, Parak WJ, Caruso F, Weiss PS. Standardizing nanomaterials. ACS Nano 2016;10:9763–4. [159] Fourches D, Pu D, Tassa C, Weissleder R, Shaw SY, Mumper RJ. Quantitative nanostructure-activity relationship modeling. ACS Nano 2010;4:5703–12. [160] Toropova AP, Toropov AA, Rallo R, Leszcynska D, Leszczynski J. Nano-QSAR: genotoxicity of multiwalled carbon nanotubes. Int J Environ Res 2016;10(1):59–64. [161] Reuter JD, Myc A, Hayes MM, Gan Z, Roy R, Qin D, et al. Inhibition of viral adhesion and infection by sialic-acid-conjugated dendritic polymers. Bioconjug Chem 1999;10:271–8. [162] Lam TT-Y, Zhou B, Wang J, Chai Y, Shen Y, Chen X, et al. Dissemination, divergence and establishment of H7N9 influenza viruses in China. Nature 2015;522:102–5.

Further reading [163] Yang Y, Sunoqrot S, Stowell C, Ji J, Lee C-W, Kim JW, et al. Effect of size, surface charge, and hydrophobicity of poly(amidoamine) dendrimers on their skin penetration. Biomacromolecules 2012;13:2154–62.

2

Dendrimers in drug delivery and the role of “critical nanoscale design parameters” (CNDPs) Abhay Chauhan SCHOOL OF PHARMACY, MEDICAL COLLEGE OF WISCONSIN, MILWAUKEE, WI, UNITED STATES

1 Introduction PAMAM dendrimers are one of the most investigated dendrimers among the various dendrimer families for drug delivery [1–10]. In general dendrimer architecture is comprised of three salient features (i) core (ii) branching points, and (iii) outside surface groups (Fig. 2.1). Each part helps in drug entrapment by (i) molecular entrapment in void spaces; (ii) hydrogen bonding and van der Waals force with branching points and (iii) chargecharge interactions with the outside surface groups (Fig. 2.2). We have proposed two approaches for drugs (or any guest molecules) association with dendrimers: (i) formulation approach and (ii) nanoconstruct approach (Fig. 2.3) [12].

1.1 Formulation approach In this approach drugs are entrapped in the dendrimers by non-covalent approach. The entrapment of drug (or any guest molecules) in dendrimer can be explained by two mechanisms: (i) inward and (ii) outward. The inward entrapment happens due to the spherical architecture of dendrimers at the core and branches, whereas outward entrapment happens at the outer surface of dendrimers via polyvalency (Fig. 2.4). In this approach drugs remain in the free form and in constant equilibrium with the ambience. Drug release is mainly governed by diffusion and interaction force between drug and dendrimer.

1.2 Nanoconstruct approach It is a classical prodrug kind of approach with polyvalency involved. Guest molecules are covalently coupled on the polyvalent surface of dendrimers. Drug remains with dendrimer until it is cleaved by chemical or biological processes.

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Pharmaceutical Applications of Dendrimers

DIAMETERS

Engineerable sizes (1 to 15 nm) (1 nm/generation)

INTERIOR VOID SPACE Nano-container encapsulation properties

CORES

Small molecules Nanoparticles, metals Polymers, latexes Dendrimers

INTERIOR BRANCHING Robust covalent framework Derived from traditional monomer Architecture may be engineered to include functionality

SURFACE GROUPS (Z)n Hydrophilic: cationic, anionic & neutral Hydrophobic: aliphatic & aromatic Combinations of surface groups FIG. 2.1 Schematic presentation of a G4 dendrimer containing four generations [11]. Reproduced with permission.

FIG. 2.2 Possible drug entrapment mechanisms in a dendrimer by the formulation approach.

2 Dendrimer as drug delivery? Drug is a pharmacological moiety which provides therapeutic effect. Historically, herbal formulations were used in early civilizations and these herbs were then collected and dried to prepare powder dosage form. The conventional dosage forms (which includes

Chapter 2 • Dendrimers in drug delivery and the role of CNDPs

Formulation Approach

51

NanoConstruct

FIG. 2.3 A drug may be either (i) physically entrapped in a dendrimer by the formulation approach or (ii) chemically conjugated to a dendrimer by the nanoconstruct approach [12].

FIG. 2.4 Role of dendrimer architecture in “outward” and “inward” entrapment of drug molecules [12].

tablet, capsule, powder, solution, suspension, emulsion, ointment) were aimed to “dose” the drug inside the body and their target was systemic circulation (Fig. 2.5). In last 30 years, the target was changed from systemic delivery to the disease specific areas creating novel drug delivery systems. Liposomes and micelles are vesicular systems, which works by encapsulating drug inside the vesicles. Liposomes are lipid based, whereas micelles are amphiphilic

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Pharmaceutical Applications of Dendrimers

Solid

Semi-Solid

Unit Solid: •Tablets •Capsules

Gas

Monophasic liquid Cream

Bulk dosage forms •Powders •Dusting powder

Liquid

•Solution

Inhaler

Biphasic liquid Paste

•Emulsion •Suspension

Aerosols

Gel

Suppositories

FIG. 2.5 Conventional dosage forms.

surfactant-based vesicles. Nanoparticles and microparticles are particle based small size drug delivery systems. Drug is embedded onto to the surface of these particles. Dendrimers are three-dimensional polymeric structure with core, branches (generation) and surface. The drug entrapment occurs throughout the dendrimer architecture. Drug entrapment in the vesicular and particulate systems occurs during the synthesis of these delivery structures, whereas in case of dendrimers it happens after synthesis of the drug delivery system. Dendrimers can also serve as a multifunctional delivery system by assisting in preformulation, formulation and advanced formulation applications (Fig. 2.6).

3 Critical nanoscale design parameters (CNDPs) in dendrimers for drug delivery Dendrimers are an architecture and it depends on (1) sizes, (2) shapes, (3) surface chemistries, (4) flexibility/rigidity, (5) architecture or (6) interior elemental compositions. These are called “critical nanoscale design parameters” (CNDPs). It is very important to understand the CNDPs to exploit the potential of dendrimers for drug delivery. The effect of generations (size) and surface of dendrimer was studies using indomethacin (Fig. 2.7). The anionic indomethacin shown more entrapment for cationic

Chapter 2 • Dendrimers in drug delivery and the role of CNDPs

Preformulation

Formulation Advanced formulation

53

• solubility • stability- storage • transport • dissolution • stability • enzymatic and hydrolytic degradation • permeability • multiple drug entrapment • bioavailability • targeting • toxicity

FIG. 2.6 Multifunctional dendrimer application in preformulation, formulation and advanced formulation [12].

100

I/D

80

G3

60

G4

40

G5 G6

20 0

Surface Functionality FIG. 2.7 Effect of dendrimer surface chemistry and generation (i.e., size) on entrapment potential of dendrimer with indomethacin. I/D: indomethacin/dendrimer mole ratio [13].

dendrimers compared to anionic dendrimer. Similarly, entrapment of drug increases with increase in dendrimer generation (size). The drug release from dendrimer-drug complex depends upon the intensity of interactions and kind of interactions (Fig. 2.8). Indomethacin release very fast from G4.5COOH surface anionic dendrimer due to their weak interactions, whereas very slow from G4-NH2 dendrimer due to strong interactions. The transdermal application of dendrimer-indomethacin formulation too showed vivid trend justifying CNDPs [15]. The drug plasma concentration was significantly higher for G4-NH2 and G4-OH formulations compared to the pure drug. G4.5-COOH dendrimer formulation showed only marginal improvement. These dendrimers can be stitched together with other nanostructures to create nanocompounds [16]. We have developed a hybrid dendrimer concept using two different dendrimers, whereas specific property of each dendrimer can be exploited (PCT/US2007/ 014402) [17]. Amine dendrimers depicted sustained release of the drug and PEG

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Pharmaceutical Applications of Dendrimers

% indomethacin released

100 80 60 G4-NH2

40 G4.5-COOH

20 0 0

20

40

60

Time (h) FIG. 2.8 In-vitro release profiles of dendrimer-drug complexes [14].

FIG. 2.9 In-vitro drug release profile of the G4-NH2 + G4-PEG hybrid dendrimer, G4-NH2 dendrimer and G4-PEG dendrimer [16].

dendrimers showed fast drug release. The hybrid dendrimer of amine dendrimer and PEG dendrimer showed intermediate drug release profile (Fig. 2.9). Self-assembly of dendrimer and albumin nanoparticles was evaluated for effective and safe delivery of paclitaxel (Fig. 2.10) [18]. Fullerene C60 was conjugated on PAMAM dendrimers and its effect on complex formation was examined [19] (Fig. 2.11). Lower generation dendrimers showed very linear increase in C60/dendrimer mole ratio and the mole ratio increased exponentially at G4 due to the dendritic effect. At G5 and G6, C60/dendrimer mole ratio plateaued. The dis˚ for G2 dendrimers whereas it tance between the two adjacent surface groups is 10.72 A ˚ narrowed down significantly to 8.46 A for G6 dendrimers. This behavior can be explained by the nanoscale sterically induced stoichiometry (NSIS) [2,4].

PTXL (p)

PTXL loaded dendrimer (pD)

Free amine

(3) Surface amine crosslinking (Glutaraldehyde addition) NH2

H N

(2) Dissolvation (Ethanol addition)

H N

HN

4.0G PAMAM Dendrimer (D) (1) Complexation

NH

H2 N

NH2

HN

NH HN

pD loaded ANP (pD-ANP)

NH2

N H

(4) Folate anchoring Activated

Aq. BSA solution

folic acid H N

HN

H N

HN NH HN NH HN

NH

HN NH

N H

Folate conjugated pD loaded ANP (pD-ANP-f) FIG. 2.10 Nano-assembly for delivery of paclitaxel in PAMAM dendrimer-albumin nanoparticle [17].

Steric Effect

40

C 60/ Dendrimer mole ratio

35 30 25 20

Dendritic Effect

15 10 5 0 G0-C60

G1-C60

G2-C60

G3-C60

G4-C60

G5-C60

G6-C60

PAMAM Dendimer- C60 Conjugate FIG. 2.11 Effect of PAMAM dendrimer architecture on the C60-dendrimer conjugate mole ratio [19].

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Pharmaceutical Applications of Dendrimers

References [1] Tomalia DA. Dendrimer research. Science 1991;252(5010):1231. [2] Tomalia DA. In quest of a systematic framework for unifying and defining nanoscience. J Nanopart Res 2009;11(6):1251–310. [3] Tomalia DA. Interview: an architectural journey: from trees, dendrons/dendrimers to nanomedicine. Interview by Hannah Stanwix. Nanomedicine (Lond) 2012;7(7):953–6. [4] Tomalia DA. Dendritic effects: dependency of dendritic nano-periodic property patterns on critical nanoscale design parameters (CNDPs). New J Chem 2012;36(2):264–81. [5] Tomalia DA. Special issue: “functional dendrimers” Molecules 2016;21(8):E1035. [6] Tomalia DA. A serendipitous journey leading to my love of dendritic patterns and chemistry. Molecules 2018;23(4):824. [7] Tomalia DA, et al. Partial shell-filled core-shell tecto(dendrimers): a strategy to surface differentiated nano-clefts and cusps. Proc Natl Acad Sci USA 2002;99(8):5081–7. [8] Tomalia DA, Christensen JB, Boas U. Dendrimers, dendrons, and dendritic polymers: discovery, applications, and the future. Cambridge University Press; 2012. p. 1–412. [9] Tomalia DA, Khanna SN. A systematic framework and nanoperiodic concept for unifying nanoscience: hard/soft nanoelements, superatoms, meta-atoms, new emerging properties, periodic property patterns, and predictive Mendeleev-like nanoperiodic tables. Chem Rev 2016;116(4): 2705–74. [10] Tomalia DA, Reyna LA, Svenson S. Dendrimers as multi-purpose nanodevices for oncology drug delivery and diagnostic imaging. Biochem Soc Trans 2007;35(Pt. 1):61–7. [11] Pentek T, et al. Development of a topical resveratrol formulation for commercial applications using dendrimer nanotechnology. Molecules 2017;22(1):137. [12] Chauhan A, Kaul M. Engineering of “critical nanoscale design parameters” (CNDPs) in PAMAM dendrimer nanoparticles for drug delivery applications. J Nanopart Res 2018;20(226):1–11. [13] Chauhan AS. Dendrimer nanotechnology for enhanced formulation and controlled delivery of resveratrol. Ann NY Acad Sci 2015;1348(1):134–40. [14] Chauhan A, et al. Solubility enhancement propensity of PAMAM nanoconstructs. Mater Matters Nanomater 2007;2:24–6. [15] Chauhan AS, et al. Dendrimer-mediated transdermal delivery: enhanced bioavailability of indomethacin. J Control Release 2003;90(3):335–43. [16] Tomalia DA, Nixon LS, Christensen JB, Cachau R, Turro N, flere M. “Periodic patterns, relationships and categories of well-defined nanoscale building blocks”: report on a National Science Foundation workshop, September 24–25, 2007. 1st ed; 2008. Mount Pleasant, USA. [17] Chauhan A, Svenson S. Formulations containing hybrid dendrimers. WTO; 2007. [18] Tekade RK, et al. Dendrimer-stabilized smart-nanoparticle (DSSN) platform for targeted delivery of hydrophobic antitumor therapeutics. Pharm Res 2015;32(3):910–28. [19] Kujawski M, et al. Molecular dynamics simulation of polyamidoamine dendrimer-fullerene conjugates: generations zero through four. J Nanosci Nanotechnol 2007;7(4–5):1670–4.

3 Dendrimers for drug solubilization, dissolution and bioavailability Abhay Chauhana, Becka Antona, Mayank K. Singhb a

SCHOOL OF PHARMACY, MEDICAL COLLEGE OF WI SCONSIN, MILWAUKEE, WI, UNITED STATES b DEPART MENT OF AP PLI ED B IO LOGY , C SIR- I NDIAN INSTITUTE O F CHEMI CAL T ECHNOLOGY, HY DERABAD, INDIA

1 Introduction Solubility (solute dissolved in a solvent to give a homogenous system) is one of the major concerns that possess enormous hindrance in the formulation development of pharmacologically active new chemical entities and generic dosage forms. The development of a new drug is estimated to take 12–15 years, at a cost of over $800 million [1]. By controlling the time and location of a drug, abnormal cells can be targeted while avoiding healthy cells (i.e., chemotherapy drugs), side effects will be reduced, and drug efficacy can be maximized, leading to a more precise and effective dosage for patients. The therapeutic effectiveness of any drug is often diminished by its inability to gain site of action in an appropriate dose. The oral bioavailability of drugs is dependent upon two main parameters, solubility and permeability. Solubility of a drug influences the behavior of compounds in biomedical applications. Poor solubility of drugs and hydrophobicity limit their possible applications in drug formation and drug delivery. Approximately 40% of newly developed drugs are rejected by the pharmaceutical industry and will unable to benefit patients due to low water solubility. With the growing impact and need for improving drug delivery, a thorough understanding of delivery technologies to enhance the bioavailability of drugs is essential [2]. The human body is composed of 55–75% water; therefore, a drug must have a certain polarity or hydrophilicity to function effectively inside the body. These drugs must also contain certain lipophilicity to be able to cross lipophilic (“fat liking”) cell membranes; which would allow it to dissolve in fats, oils and other non-polar solvents. Thus, lipophilic substances will tend to dissolve in other lipophilic molecules, while hydrophilic molecules tend to dissolve in water and other hydrophilic molecules. Solubility becomes a major challenge for formulation scientists using only water as the solvent of choice for their pharmaceutical formulations. Majority of drugs have application issues due to their poor aqueous solubility and often require high doses in order to reach therapeutic plasma concentrations [3–5]. The therapeutic effectiveness of any drug

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is dependent upon its ability to permeate the body and reach its targeted site of action. To achieve this, the drug must have a certain degree of both solubility and permeability. Solubility depends on the form of the solid molecule, the composition of the solvent medium, pressure, temperature, particle size, the nature of the solute and solvent, molecular size polarity and the rate of the solution. There are various techniques to enhance water solubility of drugs. Hydrotropy increases the mass of an aqueous solvent to dissolve by adding organic salts or non-electrolytes. The use of salt form has improved solubility, simply because a drug that is converted into salt will be more vulnerable to dissolve. A poorly water-soluble drug with parts of a molecule that can be protonated or deprotonated may potentially be dissolved by employing a pH change. One can also improve the solubility of a drug through solid dispersion, and selective absorption on insoluble molecules. Another method of aiding solubilization is to encapsulate the drug within the hydrophobic domain of a colloidal or surfactant-based system (i.e., liquid crystals, emulsions or micelles) [6]. Drug solubility can be increased by decreasing the particle size. Various techniques have been anticipated to overcome the solubility problems of drugs. In many cases, β-cyclodextrin and surfactant-based systems have been utilized to improve drug solubility, dissolution and bioavailability. However, these systems have problems regarding leakage of drugs in physiological environment, due to their thermodynamic instability. On the other hand, the conventional approaches of solubility enhancement have certain limitations, such as choice of co-solvents, pH dependence solubility etc. [7,8]. To improve solubility and bioavailability, advances are being made with innovations in nanoscience. Dendrimers can now provide an alternate route to create well-defined nanostructures that are now suitable for drug-solubilization applications. Dendrimers have been shown to exhibit micelle-like behavior and to have container like properties in solution [2,8]. The ability to tailor dendrimers properties for a particular application makes them ideal carriers for small-molecule drugs and biomolecules. Dendrimers are being considered as additives in several routes of administration, including intravenous, transdermal, oral and ocular [9,10]. We studied the role of dendrimers for solubility enhancers with focus on showcasing the dendrimer molecular features, mechanism, entrapment of different classes of drugs, and its site-specific delivery through various route of administration.

2 Molecular features of dendrimers Dendrimers are precisely engineered nanostructures which are characterized by its extensively branched 3D structure which provides a high degree of surface functionality. Dendrimers consist of a core unit upon which branches are build outward, in a generation-by-generation fashion, with the final addition of the outermost units, or surface groups. The architecture of dendrimers can be tailored at various levels, making them

Chapter 3 • Dendrimers for drug solubilization, dissolution and bioavailability

61

ideal carriers for drug delivery. One is able to customize the size, branching density, and surface functionality of dendrimers to suit both the drug of choice and its target site of delivery. Its unique properties associated in the form of its polyvalency, uniform nanosize and shape, well-defined molecular weight and existing internal cavities make it attractive for biological, chemical and pharmaceutical applications [9,11–23]. Unlike a buckyball (another 3-D molecule that is—unless filled with a payload—an empty spherical shell of carbon atoms), a dendrimer is made of bonded atoms branching in a layer-by-layer fashion (generation) from a central (hydrophobic) core unit, with final addition of functional groups like Amine, Hydroxyl and Carboxylate on its periphery. A wide variety of dendrimer structures have been classified and used in research to showcase solubility enhancement. One of the most widely studied class of dendrimer is the polyamidoamine (PAMAM) dendrimer. PAMAM dendrimers are comprised of an amidoamine repeat branching structure built around an ethylenediamine core. This dendritic nano-material composed of a huge number of smaller vacant spaces.

3 Concept of solubility enhancement with dendrimer There are two methods by which drugs can be delivered via dendrimers: entrapment or covalent attachment. The entrapment method works well for lipophilic drugs, as they can be hidden within the hydrophobic center of the dendrimer to increase their solubility [24]. Alternatively, the covalent attachment method, also called conjugation, links the drug molecule to the outer surface of the dendrimer. This is commonly done with prodrugs to increase the loading capacity [24]. The exterior of the dendrimer can be conjugated to the drug through various linkages, such as amides, esters, or disulfides, depending upon what surface groups are present for interaction. The biggest obstacle of the conjugation method is multistep drug release process [24]. Solubility enhancement with host-guest interaction (non-covalent) is a significant aspect of dendrimer and this is a synergy with its site-specific drug transportation [11,14,25–28]. Due to the presence of hydrophobic core and solubilizing functional groups at periphery, it resembles micellar property [29]. The core of dendrimer imbibes the drug with the capability to encapsulate and carry water insoluble drugs within their interior core. Outer hydrophilic functional groups of dendrimers make it soluble in aqueous media. The solubilization process is reversible and the drug will be free to diffuse out of the micellar architecture under pH-responsive conditions.

3.1 Non-covalent interaction The physical entrapment of guest molecule takes place by depending upon dendrimer and drug type, involving the mechanism of electrostatic interaction, hydrophobic interactions, hydrogen bonding, either alone or all in combination (Fig. 3.1).

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Pharmaceutical Applications of Dendrimers

Z Z

Z

Z Z

Z

Z

Z Z

Z

Z

Z

Z

Z

Z

Z

Z Z

Z

Z Z Z

Z

Z

Z

Z

Entrapment by van der Waals Forces Z

Z

Z Z

Z

Z

Z

Z Z

Z

Z

Z

Z

Z

Z

Z

Z

Z

Z

Z Z

Z

Z Z

Z

Z

Z Z

Z Z

Z Z Z

Z

Z

Z

Z

Molecular Entrapment

Z Z

Z

Entrapment by Charge Association FIG. 3.1 Possible non-covalent interaction for drug entrapment with dendrimer [30].

• •

•

Electrostatic interaction: It is one of the major interactions taking part between the surface charges of dendrimer and guest molecules. Hydrophobic interaction: This interaction happens in the core of dendrimer with hydrophobic guest molecule. A hydrophobic drug would be expected to associate with dendrimer core to provide maximum contact with its hydrophobic domain. Hydrogen bonding: Hydrogen bonding can be possible due to the presence of hydrogen atom on both components.

3.2 Covalent interaction The chemical bond that involves the coupling of guest molecule to the outer surface of the dendrimer through various molecular interactions, such as amides, esters, or disulfides (Fig. 3.2).

Z

Z

Z Z

Z

Z

Z

Z

Z

Z

Z

Z Z

Z Z

Z Z

Z Z

Z

Z Z

Z

Z

FIG. 3.2 Covalent interaction for drug conjugation with dendrimer [30].

Chapter 3 • Dendrimers for drug solubilization, dissolution and bioavailability

63

Thus, physical entrapment of guest molecules into dendrimer seems to be straight forward approach as compared to the chemical conjugation method, which requires several steps [24,31–34]. A wide variety of drugs have been solubilized using dendrimer by healthcare industries and research laboratories and delivered via various routes like intravenous intranasal, oral, ocular, transdermal and pulmonary. Fig. 3.2 highlights the structure of drugs using dendrimer nanotechnology for solubility enhancement. D’Emanuele and Chauhan groups have first reported the application of dendrimer as solubility enhancers for drug molecules [35,36].

4 Dendrimer-based solubility enhancement of various classes of drugs PAMAM dendrimers have been extensively used for solubility enhancement for various classes of drugs as demonstrated below: •

• • • • • • • • •

Anti-inflammatory/nonsteroidal anti-inflammatory drugs (NSAIDs): Diclofenac, Diflunisal, Ibuprofen, Indomethacin, Ketoprofen, Mefenamic acid, Methylprednisolone, Naproxen and Dexamethasone Anticancer: Camptothecin, Cisplatin, Dimethoxycurcumin, Doxorubicin, Etoposide, 5-Fluorouracil, Methotrexate, Paclitaxel and Docetaxel Anti-microbial: Sulfamethoxazole, Quinolones, Artemether, Niclosamide and Antichagasics Antihyperlipidemic/Hypolipidemic: Simvastatin Anti-hypertensive: Nifedipine, Ramipril Diuretics: Furosemide, Hydrochlorothiazide Antipsychotic: Haloperidol Anti-glaucoma: Brimonidine, Timolol maleate, Pilocarpine Phytoconstituents/flavonoids: Resveratrol, Tetramethlscutellarein Anthelmintic: Benzimidazole Carbamate Albendazole

4.1 NSAIDs NSAIDs represent an important class of drugs, frequently prescribed in modern medicine (Diclofenac, Diflunisal, Ibuprofen, Indomethacin, Ketoprofen, Mefenamic acid, Methylprednisolone, Naproxen and Dexamethasone) which is very effective in the alleviation of pain, fever and inflammation and almost available over the counter in most countries [37]. Since 1971, Vane and coworkers discovered their therapeutic mechanism. But it lacks behind due to their poor aqueous solubility, oral bioavailability and gastrointestinal toxicities during long term therapy. Thus, NSAIDs has been solubilized with dendrimer to improve its physicochemical properties and toxicity (Fig. 3.3).

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Pharmaceutical Applications of Dendrimers

FIG. 3.3 Solubility enhancement of Diclofenac, Diflunisal, Ibuprofen, Indomethacin, Ketoprofen, Mefenamic acid, Methylprednisolone, Naproxen and Dexamethasone (NSAIDs) using dendrimer technology.

Diclofenac is often used to treat chronic pain associated with cancer, in particular with presence of inflammation. Biocompatible dendrimers of G1.0, G2.0, and G3.0 were synthesized using PEG 600 as the core and citric acid as a branching unit. These dendritic triblock have been successfully explored for the solubilization of various NSAIDs such as 5-amino salicylic acid (5-ASA), pyridine, mefenamic acid, and diclofenac [38]. Diflunisal is a salicylic acid derivative with analgesic and anti-inflammatory activity. It was developed by Merck Sharp & Dohme in 1971 [39]. Increased risk of stomach ulcers, and their complications, with long-term use, has limited its therapeutic claim parallel to its aqueous solubility profile. Polyamidoamine (PAMAM) dendrimers have been utilized to facilitate enhanced solubility of Diflunisal [40] and in a parallel study, PAMAM G2–G4 dendrimers have been used to enhance the solubility profile of Ketoprofen [41]. As, Ketoprofen is generally prescribed in the treatment of a variety of acute and chronic inflammatory diseases and including rheumatoid arthritis, osteoarthritis, ankylosing spondylitis or severe toothaches that result in the inflammation of the gums as well as menstrual abdominal cramps [42]. Ibuprofen was discovered in 1961 by Stewart Adams and initially marketed as Brufen [40] and is a medication is used for treating fever (including post-vaccination fever), inflammation, painful menstrual periods, migraines, and rheumatoid arthritis [43].

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PAMAM G4 dendrimer was able to encapsulate and enhance the solubility of Ibuprofen from 0.10 to 7.45 mg/mL. The solubility of ibuprofen in G4 dendrimer solutions in the range 2–4% weight/volume was determined and compared to that in a 2% w/v solution of sodium dodecyl sulfate (SDS) [35]. The influence of dendrimer concentration and temperature on the solubility of ibuprofen and the effect of pH on the solubility of ibuprofen in PAMAM G4 was investigated. Enhancement of solubility is due to the electrostatic interactions of the amine groups of dendrimers with carboxyl groups of ibuprofen. It was found that the solubility of ibuprofen was directly proportional to dendrimer concentration and inversely proportional to temperature. Indomethacin is a hydrophobic drug which is commonly used as a remedy medication to reduce fever, pain, stiffness, and swelling from inflammation. It works by inhibiting the production of prostaglandins, endogenous signaling molecules known to cause these symptoms. It does this by inhibiting cyclooxygenase, an enzyme that catalyzes the production of prostaglandins [44]. PAMAM Dendrimers (dNH2, dOH and dCOOH) were used to enhance the solubility of Indomethacin. Interestingly, this dendrimer complex reported the enhanced solubility by 29-folds with dNH2 followed by 26-folds with dOH and 10-folds with dCOOH functionality [36,45]. Mefenamic acid was discovered and brought to market by Parke-Davis in the 1960s as a member of the anthranilic acid derivatives class of NSAIDs and is used for the treatment of mild to moderate pain, including menstrual pain, and is sometimes used to prevent migraines associated with menstruation [46]. Mefenamic acid has been encapsulated into dendrimers G1–G3 suggesting dendrimers as a potential drug carrier for its solubility enhancement [37]. Methylprednisolone was approved for medical use in 1955 and is, the most effective and safe medicines needed in a health system. It is a corticosteroid (glucocorticoid family) medication used to suppress the immune system and decrease inflammation [47]. PAMAM dendrimer-based methylprednisolone-loaded carboxymethyl-chitosan nanoparticles (NPs) were developed and evaluated for cell uptake, retention into secretory vesicles of rat cultured astrocytes [48]. Naproxen (brand names: Aleve, Naprosyn, and many others) is a NSAID of the propionic acid class (the same class as ibuprofen) that relieves pain, fever, swelling, and stiffness. As NSAID, naproxen exerts its anti-inflammatory action by reducing the production of inflammatory mediators called prostaglandins [49]. Solubility of Naproxen was significantly enhanced (0.02–31.41 mg/mL) in association with PAMAM G4 Dendrimer prodrug approach [43]. Dexamethasone was first made in 1957 and is a type of corticosteroid medication used in the treatment of many conditions, including rheumatic problems, several skin diseases, severe allergies, asthma, chronic obstructive lung disease, croup, brain swelling, and along with antibiotics in tuberculosis [50]. Dexamethasone was complexed with PAMAM G4 and PAMAM G3.5 dendrimer for Transcorneal iontophoresis and increase in dexamethasone solubility was reported to 3.9-fold with G4, and 10.3-fold with G3.5 dendrimer [51]. The ex-vivo studies showed that iontophoresis increased

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Pharmaceutical Applications of Dendrimers

the amount of Dexamethasone that penetrated into the cornea by 5.6- and 3.0-fold for PAMAM G4 and PAMAM G3.5, respectively. Ketoprofen was introduced as a nonsteroidal anti-inflammatory drug that contains both properties of analgesic and antipyretic effects; it also helps treat moderate pain and dysmenorrhea. Since ketoprofen is not freely soluble in water it can create gastrointestinal problems, its poor solubility in distilled water restricts its parenteral applications. Different generations of PAMAM dendrimers (G3–G5) were investigated to increase the solubility of hydrophobic drugs such as ketoprofen [52]. The results were shown that of a Generation 4 dendrimer, which the solubility of ketoprofen increased in a linear correlation with an increase in dendrimer concentration. This was due to increase in the number of surface amines that can interact with the ketoprofen molecules. It can be concluded that the solubility enhancement can be due to the electrostatic interactions between the surface amine groups of molecules and the carboxyl group of ketoprofen. At low pH, there is a lower increase of solubility compared to that of a high pH. This was due to a weak acid ketoprofen molecule that is not completely ionized at low pH conditions and cannot freely interact with the dendrimer molecule. Solubility of ketoprofen was increased with increasing dendrimer concentration. The solubility of aceclofenac in PAMAM dendrimer solutions in the range 0–10 mg/mL was determined in phosphate buffers [53]. Solubility was carried out using the Higuchi rotating bottle method. Various trials of solubility of aceclofenac were carried out using G0 and G3 PAMAM dendrimers and they both showed a positive correlation with the concentration increasing with an increase in the concentration of PAMAM at three different pH values (4, 7 and 10). In the presence of PAMAM dendrimers at a fixed pH condition, the solubility of aceclofenac in the solutions increased in an almost linear pattern with an increase in the concentration of the PAMAM dendrimer. The solubility of aceclofenac was affected by the generation of the PAMAM generation; the solubility of aceclofenac in G3 was higher than G0 dendrimer solution. It can be determined that the solubility of a hydrophobic compound in dendrimer solution most likely depends on the dendrimer generation. The solubility of aceclofenac in PAMAM solutions at a given pH condition depends on the surface area and amino groups on the dendrimer particles. Solubility of aceclofenac at various temperatures was studied as well. The amount of aceclofenac of both G0 and G3 dendrimers was inversely proportional to temperature.

4.2 Anticancer drug Anticancer drug also called as antineoplastic drugs, are effective in the treatment of malignant, or cancerous, disease. There are several major anticancer drugs; these include Camptothecin, Cisplatin, Dimethoxycurcumin, Doxorubicin, Etoposide, 5-Fluorouracil, Methotrexate, Paclitaxel and Docetaxel, natural products, as well as a variety of other chemicals that do not fall within these discrete classes but can prevent the replication of cancer cells and thus are used in the treatment of cancer (Fig. 3.4).

Chapter 3 • Dendrimers for drug solubilization, dissolution and bioavailability

67

FIG. 3.4 Solubility enhancement of Camptothecin, Cisplatin, Dimethoxycurcumin, Doxorubicin, Etoposide, 5-Fluorouracil, Methotrexate, Paclitaxel and Docetaxel (Anticancer drugs) using dendrimer technology.

Camptothecin is a topoisomerase inhibitor. It was discovered in 1966 by M. E. Wall and M. C. Wani in systematic screening of natural products for anticancer drugs. It was isolated from the bark and stem of Camptothecaacuminata (Camptotheca, Happy tree). It has shown remarkable anticancer activity in preliminary clinical trials. However, its application may be limited due to poor aqueous solubility [54]. Thus, a biocompatible polyester dendrimer composed of natural metabolites, glycerol and succinic acid has been utilized to entrap camptothecin for toxicity evaluation in various human cell lines (breast adenocarcinoma-MCF7, colorectal adenocarcinoma-HCT29, non-small cell lung carcinoma NCI-H460 and glioblastoma SF-268) at low IC50 (nmol/L) values and demonstrated significant higher cellular uptake in MCF7 cell lines [55]. Cisplatin was discovered in 1845 and licensed for medical use in 1978–79. Cisplatin is a chemotherapy medication used to treat a number of cancers such as testicular, ovarian, cervical, breast, bladder, head and neck, esophagal, lung, mesothelioma, brain tumors and neuroblastoma. It is in the platinum-based antineoplastic family of medications which works in part by binding to DNA and inhibiting its replication [56]. Carboxylateterminated PAMAM dendrimer cisplatin complex was prepared by an efficient method and entrapment of cisplatin in dendrimers was found to be independent on the dendrimer surfaces and generations (Table 3.1). The effect of the mole ratios on the percentage of Pt

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Pharmaceutical Applications of Dendrimers

Table 3.1 Effect of core and generation of dendrimer on cisplatin loading Gen

Pt content (%w/v) Mean
SD

EDAa

3.5

27.27
0.67

EDA DABb DODc

4.5 4.5 3.5

27.6
1.21 27.7
0.53 26.7
1.4

DOD

4.5

25.02
0.39

Core

a b c

Dendrimer

Diaminoethane. Diaminobutane. Diaminododecane.

Table 3.2 The effect of drug/dendrimer molar ratio on the percentage of Pt content (STARBURST dendrimer G3.5 with EDA core and COONa surface) Drug/dendrimer molar ratio

Pt content (%w/v)

9.4 19 28 53 78 106 304

10.92 18.65 26.19 35.8 42.8 50.1 58.05

content was studied using the G3.5 dendrimer with diaminoethane core and sodium carboxylate surface groups. It was observed that Pt content enhanced with increase in drug/ dendrimer molar ratio (Table 3.2). The drug release rate was related to the amount of drug that was loaded into the dendrimer with slow release (at retention time) at low loading and high release at high loading [10,57]. Release of the drug from the dendrimer matrix mainly depends on the nature of the drug and also the interactions with the various dendrimers. Dendrimers help to increase the solubility of the poorly soluble drugs and that would enhance water solubility and oral bioavailability of the drugs. Dexamethasone complexation with PAMAM G4 increased the aqueous solubility of dexamethasone to 4-fold, while complex with PAMAM G3.5 resulted in 10-fold increase [58]. Doxorubicin is DNA interacting anthracycline type of chemotherapy that is used alone or with other medications to treat several different types of cancer. Doxorubicin works by slowing or stopping the growth of cancer cells. Doxorubicin has been encapsulated into PAMAM G3 and G4 dendrimers, which had polyethylene glycol (PEG) monomethyl ether chains of molecular weights 550 and 2000 Da conjugated to their surface by urethane

Chapter 3 • Dendrimers for drug solubilization, dissolution and bioavailability

69

bonds. The encapsulation efficiency was dependent on PEG chain length and size of the dendrimer, with the highest encapsulation efficiency of 6.5 Doxorubicin molecules per dendrimer found for G4 PAMAM-PEG 2000 [59]. Etoposide is a chemotherapy medication also used for the treatment of several types of cancer. This includes testicular cancer, lung cancer, lymphoma, leukemia, neuroblastoma, and ovarian cancer. Etoposide was first synthesized in 1966 and US Food and Drug Administration approval was granted in 1983 [60]. Etoposide has been formulated in micelles composed of block copolymers, lipophilic poly(caprolactone) (PCL) and hydrophilic PEG5000, conjugated to PAMAM G2 dOH dendrimer as the core. The loading capacity of these micelles achieved with etoposide was up to 22% (w/w) with significant cytotoxic activity [61]. 5-Fluorouracil is in the antimetabolite and pyrimidine analogue families of medications. Fluorouracil was patented in 1956 and came into medical use in 1962 [62,63]. To improve solubility of 5-Fluorouracil, G4 PAMAM dendrimer has been utilized with PEG 5000. Drug loading was enhanced to 12-fold compared to non-PEGylated dendrimer, whereas in-vitro drug release rates were enhanced 6-fold, allowing sustained release over a period of 6 days from G4 PEGylated system [64]. Methotrexate is analogue of folic acid developed in 1947 (then known as amethopterin) proposed in treatment for leukemia as it was less toxic than the current treatments. In 1956 it provided the first cures of a metastatic cancer. Methotrexate is an antimetabolite and antifolate drug used in the treatment of many cancers, which acts by inhibiting the metabolism of folic acid [65]. Methotrexate was encapsulated in PAMAM G3 and G4 dendrimer, having PEG 550 and PEG 2000 monoethyl ether chains conjugated to their surfaces to modify bioavailability and toxicity. The highest encapsulation of 26 Methotrexate molecules per dendrimer was found with G4-PEG2000 dendrimer [66]. Paclitaxel and Docetaxel are a class of diterpenes, commonly known as Taxanes. The high lipophilicity and the high lattice energy of paclitaxel and docetaxel reflect their bulky and fused ring structure with several lipophilic substituents; resulting in very limited aqueous solubility. The low water solubility of paclitaxel has been reported as 0.35–0.7 μg/mL, in contrast to docetaxel, which has a much greater aqueous solubility of 3–25 μg/mL. Paclitaxel and Docetaxel are both widely used as chemotherapy agents but present difficulties in formulation due to their poor solubility in water [67–71]. Dendrimer-D-α-tocopherol polyethylene glycol succinate (TPGS) mixed micelles were prepared for solubility enhancement of two taxanes, Docetaxel and Paclitaxel. The maximum solubility of both drugs was found at a ratio of 1:2 (dendrimer to TPGS). Formulations were found to be biocompatible in hemolytic toxicity. Cytotoxicity studies from both drugs showed enhanced toxicity after encapsulation in micelles against cancer cells [72]. Micelles of a linear-dendritic copolymer (BE-PAMAM), formed by conjugating the poly(butylene oxide) (B)-poly(ethylene oxide) (E) block copolymer B16E42 (BE) with a G2 dendrimer, that have been compared with diblock copolymer B16E42 for the anticancer drug paclitaxel [73]. Solubility capacity is influenced by the solubilization technique; in which the drug and copolymer were brought into direct contact before the formation

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Pharmaceutical Applications of Dendrimers

of micelles. There was a significantly higher drug encapsulation result compared to the more conventional method in which drug was added to a solution containing preformed micelles. The BE-PAMAM copolymer showed a greater solubility enhancement than BE. The release profiles from the micellar solution showed that the release of paclitaxel was showed in both BE and BE-PAMAM copolymers. Jain et al. compared the various solubilizing agents for their solubilization propensity as well as effect on concentration, pH and time on solubility of a hydrophobic drug [74]. The solubility of paclitaxel was increased with an increase in concentration of all the solubilizing agents that were under investigation with dendrimers. The solubility was the highest at a pH of 5.0 than at 7.4 followed by pH 9.2. From the results of this study, the solubilization efficiency of the solubilizers toward paclitaxel is listed in the following order: G5 PPI dendrimers > β-CD > Tween 80 > PEG 6000. It can be concluded that the G5 PPI dendrimer was found to be the best solubilizer among all the selected solubilizing agents for safe and effective formulation.

4.3 Anti-microbial drugs Anti-microbial drugs are agents that kill microorganisms or halt their growth. Antimicrobial agents can be classified according to the microorganisms they primarily act against. In the 19th century, Louis Pasteur and Jules Francois Joubert observed antagonism between some bacteria and discussed the merits of controlling these interactions in medicine. Later, in 1928, Alexander Fleming became the first to discover a natural anti-microbial fungus known as Penicillium rubens and named the extracted substance penicillin which in 1942 was successfully used to treat a Streptococcus infection [75,76]. There are various antimicrobial agents which have been entrapped in dendrimers such as Sulfamethoxazole, Quinolones, Artemether, Niclosamide and Antichagasics (Fig. 3.5). Sulfamethoxazole is one of the highly prescribed drugs in the class of anti-microbials and used for various bacterial infections such as urinary tract infections, bronchitis, and prostatitis and is effective against both Gram-negative and Gram-positive bacteria such as Listeria monocytogenes and E. coli. Due to its low aqueous solubility and poor therapeutic potential, it has been physically associated with PAMAM G2–G4 dendrimers and reported to enhance the aqueous solubility by 40-fold with increased potential of antibacterial activity 4- to 8-fold when compared to pure Sulfamethoxazole [77]. Quinolones are large group of broad-spectrum bactericides that share a bicyclic core structure related to the compound 4-quinolone, with an attached fluorine atom in their chemical structure. They are effective against both Gram-negative and Gram-positive bacteria [78,79]. Nadifloxacin and Prulifloxacin are the most widely used quinolones but their poor aqueous solubility hinders their antibacterial property and thus their solubility was significantly enhanced with PAMAM G3–G5 amine dendrimers for excellent anti-microbial activity [80]. Artemether is a methyl ether derivative of artemisinin, which is a peroxide-containing lactone isolated from the antimalarial plant Artemisia annua. It is a relatively lipophilic

Chapter 3 • Dendrimers for drug solubilization, dissolution and bioavailability

71

FIG. 3.5 Solubility enhancement of Sulfamethoxazole, Quinolones, Artemether and Niclosamide (Anti-microbial drugs) using dendrimer technology.

and unstable drug, which acts by creating reactive free radicals in addition to affecting the membrane transport system of the Plasmodium organism [81–83]. Dendritic micelles have been developed for solubility and stability enhancement of artemether and found to escalate the solubility depending upon concentration and size of these micelles by 3- to 15-fold and were stable up to 5 h [84]. Niclosamide was discovered in 1958 as anthelmintic family to treat tapeworm infestations. Niclosamide is having poor aqueous solubility at physiological pH and sparingly soluble at pH 8–10. Complexation of Niclosamide with PAMAM carboxylate dendrimer had shown insignificant effect on the solubility while, complexation with PAMAM dendrimer having amine functionality had significantly enhanced the aqueous solubility of Niclosamide by 372- to 6176-fold with G0–G3 amine dendrimers [33]. Synthesis of surface-modified TREN-cored PAMAM dendrimers and their effects on the solubility of sulfamethoxazole (SMZ) as an analogue antibiotic drug was investigated [85]. Results showed that the solubility of SMZ improved with an increasing generation size and PAMAM dendrimer concentration. The role of PAMAMs in the solubility enhancement of SMZ was in the order of G4.NH2 > G4.COOH > G3.NH2 > G4. TRIS > G2.NH2 > G3.COOH > G3.TRIS > G2.COOH > G2.TRIS.

4.4 Antihyperlipidemic/hypolipidemic drugs Antihyperlipidemic/hypolipidemic drugs, also called lipid-lowering drugs, reduce the level of lipids and lipoproteins in the blood. Lipoproteins bind cholesterol and can accumulate in blood vessels. High levels of specific lipoproteins, namely, low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL), have been associated with an elevated risk of certain forms of cardiovascular disease, including coronary artery disease,

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Pharmaceutical Applications of Dendrimers

heart attack, and stroke. Statins are hypolipidemic drugs that block the enzyme HMG-CoA (5-hydroxy-3-methylglutaryl-coenzyme A) reductase, which is required for the synthesis of cholesterol. Simvastatin, which was developed by Merck and came into medical use in 1992, is generally quite safe but lacks behind in the formulation development due to its solubility effects [86]. There have been several attempts to deliver simvastatin (SMV) with Self-emulsifying drug delivery systems (SEDDS). SEDDS are thermodynamically unstable, it is diluted prior to drug administration that is only stable for less than 6 h. PAMAM dendrimer-SMV formulations were developed and effects of dendrimer concentration on solubility, pH effect, effects of surface groups were studies (Fig. 3.6). The solubility profile of Simvastatin was enhanced with PEGylated dendrimer (33 times) followed by dNH2 (23 times) and dOH (17.5 times) dendrimers at various pHs and concentrations of dendrimers [87]. The solubility profile graph showed a linear correlation between solubility and dendrimer concentration (% w/v). Also, the curve of the graph showed that positive and linear relationship in lower dendrimer concentrations (up to 0.1%) and the curve became non-linear due to nanosize induced stoichiometry (N-SIS). The effect of pH on the solubility was also tested in the presence of amine dendrimers. At a higher pH (that being 10) the increase in solubility of SMV could have contributed to electrostatic interaction between the deprotonated dendrimers and ionized drug HO

O O

O H

O

Solubility (µg/ml)

Simvastatin 500 450 400 350 300 250 200 150 100 50 0

DN DO DP

0

0.1

0.2

0.3

0.4

0.5

Dendrimer concentration (%w/v) FIG. 3.6 Solubility enhancement of Simvastatin (Antihyperlipidemic drug) using dendrimers [87].

Chapter 3 • Dendrimers for drug solubilization, dissolution and bioavailability

73

molecules. pH could also be dependent on solubility enhancement on weak acidic drugs at a higher pH as well. So, dendrimer would improve solubility and disintegration of simvastatin, but it would depend on the concentration of dendrimers, the functional group type that is found of the dendrimer surface or the pH of the solution.

4.5 Anti-hypertensive drugs Anti-hypertensive is a class of drugs that are used to treat hypertension (high blood pressure). Nifedipine was discovered and developed by the German pharmaceutical company Bayer, with most initial studies being performed in the early 1970s and approved for human use in 1981. The effect of dendritic size and surface functionality in the aqueous system with Nifedipine has been studied utilizing G0–G3 PAMAM dNH2 and G0.5–G2.5 with ester surface at various pH. The higher solubility was reported with ester group than amine group dendrimers [34]. Ramipril is an angiotensin-converting enzyme (ACE) inhibitor, used to treat high blood pressure (hypertension) and congestive heart failure. By inhibiting an enzyme, ACE inhibitors relax the muscles around small arteries (arterioles). The arterioles expand and allow blood to flow through more easily. Thus, in this way, it reduces blood pressure. But its low aqueous solubility and dissolution make it difficult to use it on a regular basis. The solubility of ramipril was improved by employing dendrimer nanotechnology and 4.91-folds of solubility was enhanced by G4.0 PAMAM dNH2, 4.01-folds with G4.0 PAMAM dOH and 2.95-folds with G3.5 PAMAM dCOONa Dendrimers followed by enhanced dissolution of ramipril in simulated gastric fluid (pH 1.2) and USP dissolution medium (pH 7.0) [88] (Fig. 3.7). Hyperbranched polyglycerol dendrimers were evaluated for the solubilization of hydrophobic drugs [89]. Biphenyl groups were synthesized in three- or four-step procedure by employing Suzuki-coupling reactions. They were then used to solubilize nimodipine and pyrene. It was concluded that the transport properties of the polyglycerol derivatives which are based on hydrophobic interactions that depend solely on the degree and type of core on the polyglycerol dendrimer structure. The enhancement of solubilization increased 300-fold for nimodipine and was 6000-fold for pyrene at a polymer concentration that is 10% weight/volume. The effect of variables such as concentration, generation size (G2, G3 and G4), and surface groups (NH2, COOH and TRIS) of PAMAM on the aqueous solubility of candesartan cilexetil (CC) were studied [90]. The results showed that the aqueous solubility of CC in the presence of carboxyl and TRIS-terminated PAMAMs was higher than those aminePAMAMS, and the effect of surface functional group of the PAMAMS in the aqueous solubility of CC was dependent on the generation size. The observed solubility fold enhancement due to PAMAMs was G4.COOH > G3.COOH > G4.TRIS > G2. COOH > G3.TRIS > G2.TRIS > G4.NH2 > G3.NH2 > G2.NH2. Improvement in the solubility of CC is expected primarily through the intermolecular hydrogen bonding between the drug and the surface functional groups on the studied PAMAMs.

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Pharmaceutical Applications of Dendrimers

RDC

800

RDO

RDN

700

Solubility (µg/ml)

600 500 400 300 200 100 0 0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Dendrimer Concentration (% w/v) FIG. 3.7 Solubility enhancement of Ramipril using dendrimer technology [88].

4.6 Diuretics Diuretics are any substance that promotes increased production of urine. Diuretics are used to treat heart failure, liver cirrhosis, hypertension, influenza, water poisoning, and certain kidney diseases. Diuretic also helps to make the urine more alkaline and are helpful in increasing excretion of substances in cases of overdose or poisoning. Furosemide is primarily used for the treatment of edema, but also in some cases of hypertension (where there is also kidney or heart impairment). It is also used in adjunct therapy for swelling of the brain or lungs where rapid diuresis is required (IV injection), and in the management of severe hypercalcemia in combination with adequate

Chapter 3 • Dendrimers for drug solubilization, dissolution and bioavailability

75

rehydration [91]. The solubility of the Furosemide with (PAMAM) dendrimers G4 was achieved to be 1000-fold higher by varying the pH from 2.0 to 7.0. Further, significantly higher stability constants were observed with the furosemide-dendrimers complexes compared to native Furosemide [92]. Hydrochlorothiazide (HCTZ) belongs to thiazide class of diuretics. It reduces blood volume by acting on the kidneys to reduce sodium (Na+) reabsorption in the distal convoluted tubule. The major site of action in the nephron appears on an electroneutral NaCl co-transporter by competing for the chloride site on the transporter. By impairing Na+ transport in the distal convoluted tubule, hydrochlorothiazide induces natriuresis and concomitant water loss. Thiazides increase the reabsorption of calcium in this segment in a manner unrelated to sodium transport. Additionally, by other mechanisms, HCTZ is believed to lower peripheral vascular resistance [93]. HCTZ solubility was enhanced by 2.83-, 3.22- and 3.72-folds with G4.0 PAMAM dNH2, G4.0 PAMAM dOH and G3.5 PAMAM dCOONa Dendrimers [88] (Fig. 3.8).

4.7 Antipsychotic drug Antipsychotic (neuroleptics or major tranquilizers) are a class of medication primarily used to manage psychosis principally in schizophrenia and bipolar disorder. They are increasingly being used in the management of non-psychotic disorders. Antipsychotics are usually effective in relieving symptoms of psychosis in the short term [94]. Haloperidol is a classic Antipsychotic drug having limited aqueous solubility and hence formulated with dendrimer for brain targeting via intranasal delivery with improved aqueous solubility by more than 100-folds than native Haloperidol [95] (Fig. 3.9).

4.8 Anti-glaucoma drug Anti-glaucoma drug is those drugs, which are used to prevent or alleviate glaucoma, a disease in which the optic nerve is damaged, resulting in progressive, irreversible loss of vision (Fig. 3.10). Thus, ideally one should use the lowest dose of a drug that will produce the greatest therapeutic response with the least number of side effects. Indeed, many of the currently available topical medications are associated with adverse effects, such as dry eye, burning, stinging sensations, tearing and allergic reactions followed by solubility and stability issues [96]. Pilocarpine is a medication used to treat increased pressure inside the eye for glaucoma (before surgery), ocular hypertension, and to bring about constriction of the pupil following its dilation. Pilocarpine was successfully formulated with PAMAM amine, carboxylate and hydroxyl dendrimers for controlled ocular delivery in New Zealand albino rabbit [97,98]. Brimonidine is an α2 adrenergic agonist and indicated for the lowering of intraocular pressure in patients with open-angle glaucoma or ocular hypertension while Timolol maleate was the first β blocker approved for topical use in the treatment of glaucoma in 1978. The mechanism of action of timolol is probably the reduction of the formation of aqueous humor in the ciliary body in the eye. The combination of these two drugs plays

76

Pharmaceutical Applications of Dendrimers

HDN

3000

HDO

HDC

Solubility (µg/ml)

2500 2000 1500 1000 500 0 0

0.2

0.4

0.6

0.8

Dendrimer Concentration (w/v)

FIG. 3.8 Solubility enhancement of Hydrochlorothiazide using dendrimer technology [88].

a significant role in the treatment of glaucoma [99]. Dendrimer hydrogel was formulated for the delivery of brimonidine (0.1% w/v) and timolol maleate (0.5% w/v) to human corneal epithelial cells with enhanced solubility of brimonidine by 77.6% and sustained the in vitro release of both drugs over 56–72 h [98].

4.9 Phytoconstituents Phytoconstituents are chemical compounds that occur naturally in plants. Some are responsible for color and other organoleptic properties. The term is generally referred

Chapter 3 • Dendrimers for drug solubilization, dissolution and bioavailability

77

FIG. 3.9 Solubility enhancement of Haloperidol (Antipsychotic drug) using dendrimer technology.

FIG. 3.10 Solubility enhancement of Anti-glaucoma drug (Pilocarpine, Brimonidine and Timolol maleate) using dendrimer technology.

to biologically significant chemicals, but not established as essential nutrients. Resveratrol is insoluble in water and has stability issues. Dendrimer-resveratrol complex showed solubility enhancement with 1% dendrimer concentration and successfully enhanced solubility up to 0.216 μg/mL, followed by improvement of stability in solution and cream dosage forms [100] (Fig. 3.11). PAMAM dendrimer containing ethylenediamine tetra acetic acid core and ethylenediamine as repeating units was synthesized by divergent approach and then used to encapsulate a flavonoid tetramethylcutellarein (TMScu, 1) to study its solubility and potential bioactivity enhancement. Results showed encapsulation of (TMScu, 1) into dendrimer was achieved by a co-precipitation method with the encapsulation efficiency of 77.8%
0.69% and loading capacity of 6.2%
0.06% as a function of dendrimer concentration at pH 4.07 and 7.2. The amine-terminated PAMAM G4 enhanced the solubility of the studied compound as a function of dendrimer concentration. pH also had an influence on the solubility with solubility being higher at pH 4 than at pH 7. The in vitro release studies indicated that PAMAM G4-MScu complex was more stable in neutral medium at pH 7. Overall, PAMAM dendrimer-G4 was capable of encapsulating, increasing its solubility that could enhance its bioactivity [101].

78

Pharmaceutical Applications of Dendrimers

FIG. 3.11 Solubility enhancement of Resveratrol, Quercetin and Tetramethylscutellarein using dendrimer technology.

In another study, the potential of PAMAM dendrimers for quercetin was investigated with dendrimers of G0, G1, G2 and G3 using concentrations of 0.1, 0.5, 1, 2 and 4 μm. The results indicated an increased positive correlation significantly as a function of PAMAM dendrimer over the whole concentration range [102].

4.10 Anthelmintic/benzimidazole carbamate albendazole (ABZ) Anthelmintic/benzimidazole carbamate albendazole (ABZ) are those drugs which are used to treat infections with parasitic worms. This includes both flat worms, tapeworms and round worms. PAMAM dendrimer was used to improve the aqueous solubility of albendazole (Fig. 3.12). One of the main problems with this drug is that of its poor aqueous solubility (0.61 μg/mL), and its low irregular bioavailability in humans. The G3, G2.5 and G3.5 and G3OH PAMAM dendrimers were used to enhance the solubility of albendazole that the increase in solubility depends on both generation and surface of dendrimers (amine or hydroxyl group). Both hydrophobic interior and specific hydrogen bond interactions can contribute to the albendazole-dendrimer association, enhancing the solubility [103].

FIG. 3.12 Solubility enhancement of Albendazole using dendrimer technology.

Chapter 3 • Dendrimers for drug solubilization, dissolution and bioavailability

79

4.11 Miscellaneous The effect of pH with G5 dendrimers using hydrophobes that differ in functional groups such as famotidine, indomethacin and amphotericin was carried out at pH 4.0, 7.4 and 10.0. It was shown that the solubility enhancement was driven by both pH and functional groups present in all the hydrophobic drugs [104]. The dendrimers were useful for not only stabilizing weak acidic and basic drugs (such as indomethacin and famotidine) but also amphotericin which is amphoteric in nature. It was observed that solubility depends on the protonated/deprotonated state of the dendrimer. PAMAM dendrimers were used to improve the solubility of phenylbutazone, it was found to be more effective than other carriers (i.e., cyclodextrins and micelles) [105]. Solubilization ability depends upon dendrimer generations, dendrimer concentration, and pH condition and carboxyl groups that are attached on the surface. 2D-NOESY spectra showed that the presence of phenylbutazone localizes in the interior cavities as well as on the surface of PAMAM dendrimers or both G3 and G6 dendrimers. Lastly, the solubility of 2D-NOESY results and the analysis suggests that the electrostatic and encapsulation interaction together caused the solubility enhancement of phenylbutazone. A series of dendrimers with different cores were synthesized and characterized by NMR and MS techniques [106]. L-Histidine, Naproxen and Methotrexate were mixed in the dendrimer at room temperature in pH buffers of 6, 7 and 8. The results showed that the solubility enhancement was due to the presence of dendrimers at different pH compared to their corresponding aqueous solubility at different pH values. The enhancement of drug solubility was owed to electrostatic interactions and hydrogen bonding between anionic dendrimers. It also indicated that aspartate-based dendrimers could be considered an effective supplement of PAMAM dendrimers in solubility enhancement and also drug delivery. The properties of CO2 fluorescent dendritic polymers, poly(amidoamine)/Pluronic F 127 (PAMAM/F127) are reported, by dynamic light scattering (DLS) and transmission electron microscopy (TEM) [107]. The polymers showed unimolecular micelle morphologies at low concentrations and changed to multimolecular micelles at higher concentrations. The results indicated that PAMAM/F127 can be used to improve the solubility of curcumin and the drug released faster in the presence of CO2.

5 Dendrimer-based drug-delivery through various routes This section focuses on the performance of dendrimers for drug-delivery through various routes which alter the pharmacokinetic behavior of drug after intravenous, intranasal, oral, ocular, pulmonary and transdermal administration. It has been proved that due to polyvalent structure and nanosized dendrimers alters the pharmacokinetic behavior of covalently and non-covalently associated drugs and affects their circulation in the blood; enhance cellular uptake and absorption across membrane with controlled drug release pattern. Thus, this section will be focused especially on describing the dendrimer-based drug-delivery through various routes.

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Pharmaceutical Applications of Dendrimers

5.1 Intravenous delivery To improve the delivery of docetaxel (DTX) and reduce systemic toxicity, Dendrimer was conjugated with trastuzumab (TZ), a monoclonal antibody for breast cancer and TZ-conjugated dendrimers displayed higher cellular internalization and induction of apoptosis against MDA-MB-453 cells. Binding of TZ with the dendrimer helps the site-specific delivery of DTX resulting from its lack of specificity. In addition, in vivo studies revealed that the pharmacokinetic profile of DTX was significantly improved by the conjugated TZ. Results suggest that the pharmacokinetic profile of dendrimer conjugates (TZ-DendDTX and Dend-DTX) differed from marketed formulation Taxotere (Fig. 3.13). The areaunder-the-curve (AUC0∞) of TZ-Dend-DTX and Dend-DTX was approximately 4.7 and 3.5 times higher, respectively than Taxotere. This pharmacokinetic pattern was due to the release of DTX from dendrimer-based formulations. The observed clearance values for Taxotere, Dend-DTX and TZ-Dend-DTX were 5.5 times less and 7.3 times less, respectively. Mean residence time was enhanced by 3.4 times and 2.5 times for TZ-Dend-DTX and Dend-DTX, respectively. Compared with TZ-unconjugated dendrimers TZ-DendDTX induced a significantly higher AUC0∞ (lower clearance and longer MRT) [108].

5.2 Intranasal delivery For efficacy studies in the brain, PAMAM dendrimer-based Haloperidol complex was formulated and aqueous solubility of the drug in dendrimer formulation increased up to 100fold. Dendrimer-haloperidol complexes were administrated to Sprague-Dawley rats through intranasal, intraperitoneal and oral route for evaluating its potential for brain

FIG. 3.13 Plasma profiles of docetaxel (DTX) concentration after intravenous administration of Taxotere, DTX-loaded G4 PAMAM amine dendrimers (Dend-DTX) and trastuzumab-conjugated Dend-DTX (TZ-Dend-DTX) [108].

Chapter 3 • Dendrimers for drug solubilization, dissolution and bioavailability

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FIG. 3.14 Distribution of haloperidol in the olfactory bulb, striatum, cerebellum, and plasma of adult rats (n ¼ 3) at 60 min following administration of dendrimer-haloperidol formulation through the IP (D-HP IP), IN (D-HP IN), and oral (D-HP Oral) routes and control haloperidol formulation through the IP route (HP-A IP) [95].

delivery. Results showed that the developed dendrimer system showed higher drug distribution in plasma and brain, compared to the control formulation given via intraperitoneal injection. Also, when administered intranasally, the drug-loaded dendrimer yielded a behavioral response similar to intraperitoneal injection, but at 6.7 times lower dose (Fig. 3.14) [95]. PAMAM-G4 was conjugated to drug methylprednisolone (MP) to be delivered intranasal route [109]. A lower concentration of drug was needed to reduce the inflammatory reaction. There was also slightly longer residence time within the lung tissue. No undesired side effects were observed. Dendrimer conjugate with anti-inflammatory drug MP delivered trans-nasal application. MP covalently bound to dendrimer achieved high solubility and increase the length of drug half-life. Delivery of this dendrimer solution via trans-nasal administration was found to be retained within the lung with a half-life of approximately 5 days. An observed decrease in baseline endogenous levels of marginated and interstitial inflammatory cells on treatment with both MP and MP-dendrimer conjugate treatments were consistent with

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the anti-inflammatory effect of methylprednisolone. Observations support the use of dendrimer-drug conjugates to reduce the amount of drug used and perhaps reduce the frequency of administration for controlling chronic inflammatory lung disorders, such as asthma.

5.3 Oral delivery Pharmacokinetic and Pharmacodynamic Studies of Poly(amidoamine) dendrimer Based simvastatin oral for the treatment of Hypercholesterolemia was performed in Male albino Sprague-Dawley rats (Fig. 3.15). Dendrimer-SMV formulations showed better pharmacokinetic performances than pure SMV suspension. The area under the plasma concentration-time profile (AUC0!t) with PAMAM-amine-SMV (DN2), PAMAM-hydroxylSMV (DO2), and PEGylated PAMAM-SMV (DP2) formulations were found to be 19.03, 11.14, and 25.43 μg/mLh, respectively, which was significantly higher than the pure drug (3.4 μg/mLh). Furthermore, SMV absorption and elimination rates were decreased significantly, showing controlled release of SMV from the dendrimer formulations. While in a pharmacodynamic study, the percent increase in cholesterol was less with PAMAM dendrimer formulations as compared to pure drug. The cholesterol level was increased to 20.92% with pure SMV, whereas it was 11.66% with amine dendrimer, 11.49% with PEGylated dendrimer, and 10.86% with hydroxyl dendrimer formulations. Reduction in the increase in triglyceride and low-density lipoprotein level was also more prominent with the drug-dendrimer formulations. The order of increase in high density lipoprotein level was DP2 (4.04%) > DN2 (2.57%) > DO2 (1.48%) > pure SMV (1.09%). D’Emanuele et al., have reported that the mechanism by which dendrimer enhances apical to basal propranolol transport to involve endocytosis-mediated transepithelial transport. The therapeutic use of polymers for reducing the effects of P-glycoprotein

Plasma concentration of SVM (µg/ml)

4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 0

2

SMV

4

DN2

6 8 Time (h) DO2 DP2

10

12

FIG. 3.15 Plasma concentration profile of SMV and dendrimer-SMV formulations [110].

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83

on drug absorption may prove that the bioavailability of propranolol. Results suggest that dendrimer-drug prodrugs may be used as a way to increase drug solubility and bypass drug efflux transporters that would then increase bioavailability [111]. In another study, PEGylated G5 PAMAM dendrimer modified nanoliposome was investigated to increase water solubility, oral absorption of Probucol (PB) and transepithelial transport. All four functionalities of the G5 dendrimers improved the solubility of PB. The positive charged G5-NH2 and G5-PEG improved the water solubility of PB more than negatively charged G4.5-COOH or neutral G5-OH. The solubilization of PB by G5-PEG was concentration-dependent since more dendrimer molecules would be available to encapsulate more drugs at higher concentrations. The results demonstrated that PB-liposome/ G5-PEG significantly increase the oral absorption that would significantly improve its pharmacodynamic effects [112]. Naproxen was conjugated to G0 PAMAM dendrimers with an amide bond or ester bond [113]. The G0 dendrimer was evaluated in 80% human plasma and 50% rat liver homogenate. The ester linkage in prodrug provides high stability in plasma with slow hydrolysis in liver homogenate. When using diethylene glycol as a linker, it yielded an ester conjugate that showed high chemical stability. Permeability studies showed a significant enhancement in the transport of naproxen when conjugated with dendrimers. Dendrimer-based drugs with the appropriate linkers have the potential as carriers for the oral delivery of low solubility drugs such as naproxen. Conjugation of naproxen to G0 dendrimer increased its permeability in both directions. Thus, suggested that the dendrimers potential as nanocarriers for the enhancement of oral bioavailability [113].

5.4 Ocular delivery The rate of penetration of a drug is influenced by multiple factors including solubility, lipophilicity, size of the molecule, charge, molecular shape, and degree of ionization. In the case of ocular drug delivery, these dendrimers need to have enough biocompatibility and low immunogenicity to lower the risk of possible side effects within the ocular tissue [24]. Thus, dendrimer drug delivery through ocular route has many benefits like: • • • •

Topically administered drugs delivered via dendrimers had longer corneal residence time Increased receipt of drug by retina following systemic application In retinal degeneration, dendrimer provided sustained protection of neuroinflammation within the retina Use of dendrimer as corneal glues to provide an alternative option to sutures, following corneal surgeries

Iontophoresis of dendrimer in the eye has been proposed to sustain drug delivery and maintain therapeutic concentrations. Dexamethasone (DEX) is a lipophilic drug and has rapid clearance from eye tissue, thus corneal delivery via dendrimer was developed

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FIG. 3.16 Dex recovered from aqueous humor after 2 h of passive and 4 min iontophoretic in vivo application of Dex, Dex-PAMAM G4 and Dex-PAMAM G3.5 [53].

with increased solubility from 4- to 10-folds with PAMAM G4 and PAMAM G3.5 with better cellular uptake, and drug bioavailability. Stronger interactions were occurred between the G3.5 PAMAM and dexamethasone compared to G4 PAMAM. In vivo experiments, however, have evidenced that iontophoresis of Dex-PAMAM-G3.5 improved Dex concentration in the aqueous humor by 6.6-fold, while iontophoresis of DEX-PAMAM G4 and Dex enhanced it 2.5- and 2-fold, respectively (Fig. 3.16). Therefore, iontophoresis targeted dendrimer to the cornea but it is the sustained delivery of the Dex from dendrimer that prevents its rapid elimination from the aqueous humor. In conclusion, iontophoresis of DEX-dendrimer complexes represent a promising strategy for targeted and sustained topical drug delivery to the eye [53].

5.5 Pulmonary delivery The coadministration of G0–G3 PAMAM dendrimers to the lungs improved the absorption of insulin and calcitonin in rats by disrupting gap junctions and increasing the permeability of the pulmonary epithelium [114]. For instance, an early study showed that G3 PAMAM-PEG micelles enhanced the bioavailability of entrapped low molecular weight heparin from the lungs (60%) and the pulmonary delivery of the dendrimer formulation resulted in improved anticoagulant activity in a rat model of thrombosis when compared to the subcutaneous delivery of heparin alone [115]. Conversely, a G4 PAMAM-OH dendrimer with methylprednisolone had prolonged its retention in the lungs when administration pulmonary, compared to administration of the drug alone and improved the reduction in experimental airway inflammation when compared to the drug in a rat model of lung inflammation [109]. More recently, the pulmonary administration of a PEGylated G5 polylysine dendrimer conjugated with the doxorubicin displayed 13% absorption from the lungs of rats and extended retention of drug in the lungs. When administered to the lungs of rats with experimentally induced lung metastases of breast cancer, anticancer activity was significantly improved when compared to the intravenous administration of the dendrimer or doxorubicin alone [116].

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5.6 Transdermal delivery Hydrophobic drug indomethacin has been solubilized with G4-NH2, G4-OH and G4.5COOH-PAMAM dendrimers. Interestingly, the steady-state flux of indomethacin increased linearly with an increase in dendrimer concentration in the formulations vs the plain drug, despite the non-linear increase in solubility. Dendrimer-indomethacin complexes showed enhanced indomethacin skin penetration in-vitro in rat skin membrane model. Interestingly, In vivo pharmacokinetic and pharmacodynamic assays post transdermal application in rats showed higher blood concentration of indomethacin with PAMAM dendrimers vs pure indomethacin suspension (Fig. 3.17). The [AUC]0–24h of G4-NH2 and G4-OH formulations are higher than pure drug with a ratio of 2.27 times and 1.95-folds respectively. The G4-PAMAM dendrimers maintained the effective indomethacin concentration in the blood for 24 h [45]. Dendrimer-resveratrol formulation was demonstrated to have better transdermal permeation in the in-vitro studies [100]. Application of the drug-dendrimer complex increased the flux of 5-fluorouracil (5-FU) in isopropyl myristate (IPM) and mineral oil [117]. The increased skin partitioning of dendrimer from lipophilic vehicles increased the drug solubility in skin. The dendrimer also increased permeability coefficient of 5-FU by 4-folds in the mineral oil and 2.5-folds in IPM, while there was a decrease by half in the phosphate buffer. The data indicated a linear correlation with increase in pretreatment time; dendrimer pretreatment increased transepidermal water loss and a decrease in skin resistance. The decrease in skin resistance can be correlated to the enhancement in skin permeation of 5-FU. Overall the dendrimer interacts with the drug’s permeability coefficient by decreasing its solubility in the vehicle.

FIG. 3.17 Plasma concentration vs time profile of various dendrimer formulations of Indomethacin and free drug after transdermal application in male Wistar rats [45].

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In other research, deposition and permeation of 5-fluorouracil across human epidermis assisted by appropriately charged peptide dendrimers were investigated [118]. The effect of peptide dendrimers on the solubility and partition coefficient of 5-FU, degrading of drug in skin as well as deposition and permeation of 5-FU in and through the skin was studied. The dendrimers that were tested (R4: 4+ charge, C-to-N sequence Gly-Lys-(Arg)2, [M + H]+ of 515.8; R8: 8+ charge, C-to-N sequence Gly-Lys-(Lys)2-(Arg)4, [M + H]+ of 1084.4; R16: 16+ charge, C-to-N sequence Gly-Lys-(Lys)2-(Lys)4-(Arg)8, [M + H]+ of 2222.5) increased the solubility and partition coefficient of 5-FU with each also significantly enhancing the deposition across human epidermis in a concentration-dependent manner. The peptide dendrimers (R4, R8 and R16) increased the permeation of 5-FU via human epidermis, with R8 showing the maximum skin deposition enhancing effects for 5-FU; the application of peptide dendrimers in skin delivery of other therapeutically relevant such as anti-nociceptive and anti-inflammatory agents. Additionally, PAMAM dendrimers have been utilized to facilitate transdermal delivery of diflunisal [40]. In vitro permeation (excised rat skins) studies indicated that PAMAM dendrimers based Diflunisal system has significantly enhanced the permeability after 24 h as compared to pure drug with 2.48 times higher concentration of diflunisal in blood. Pharmacokinetic studies revealed that the bioavailability was 2.73 times higher for the Ketoprofen-PAMAM dendrimer complex than the pure drug [41].

6 Conclusion Dendrimer for solubility enhancement of the drugs came a long way in last 15 years. The complexation of water-insoluble molecules with dendrimer for increased solubility, high drug loading, enhanced dissolution and increased physicochemical stability closely correlates the dendrimer potential as excipients. Dendrimer as solubility enhancers can be used through all possible drug delivery routes of administration. This chapter aims to construct a bridge between the formulation scientist and the pharmaceutical industry to accept dendrimers as an excipient cum multitasking drug delivery system.

Acknowledgments M.K.S. acknowledges Council of Scientific and Industrial Research, India (CSIR-INDIA) for Senior Research Fellowship. A.C. and B.A. acknowledge Dean of School of Pharmacy, Medical College of Wisconsin, USA. We would like to acknowledge Enica Saffold’s help in collecting information for this chapter under Clinical & Translational Science Institute (CTSI), Star 500 program at Medical College of Wisconsin, Milwaukee, USA.

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4

Pharmacokinetic considerations in design of dendrimer-based nanomedicines

Lakshmi Tunkia, Hitesh Kulharib, Suresh K. Bhargavaa, Deep Poojaa a CENTRE FOR ADVANCED MATERIALS & I NDUST RIAL CHEMIS TRY ( CAMIC), SCHOOL O F SC IE NC E, RMIT UNIVERSI TY, ME LBOURNE, VI C, AUST RALIA b SCHOOL OF NANO SCIENCES, CENTRAL UNIVERSITY OF GUJARAT, GANDHINAGAR, INDIA

1 Introduction A class of synthetic polymers with central core, repetitive layers and an ample number of terminal functional groups constitute to form dendrimers which have unique architecture, monodispersity and uniform size. Design of dendrimer can be controlled at every step starting from core to the addition of functional groups or ligands at the periphery. The inbuilt physicochemical properties of the dendrimers like size, surface charge, molecular weight, type of the core, surface functionalities and their association with drugs or proteins decide their pharmacokinetic behavior. For the development of the delivery system, pharmacokinetic profiling is very much required as it indicates the fate of the delivery system in the body and displays about the potential toxic or beneficial events [1].

2 Effect of size, molecular weight, hydrophobicity and charge on pharmacokinetic profile of dendrimers Pharmacokinetic profile includes parameters like absorption, distribution, metabolism and excretion. In specific, pharmacokinetic properties indicate the behavior of the delivery system inside the body after administration. The properties of dendrimers like size, play a crucial role in elimination through kidneys. With the increase in size or hydrodynamic radius beyond the threshold of kidneys, their plasma residence time gets extended. Increase in generation or molecular weight expands the metabolic stability but at the same time it may also increases toxicity concerns. Surface charges on the dendrimers affect the biodistribution and association with cells or tissues. In general, cationic charged dendrimers like amine dendrimers rapid associates with the endothelial cells and thus are rapid eliminate from plasma.

Pharmaceutical Applications of Dendrimers. https://doi.org/10.1016/B978-0-12-814527-2.00004-4 © 2020 Elsevier Inc. All rights reserved.

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Kobayashi et al. prepared novel, macromolecular MRI contrast agents using different molecular weight PAMAM dendrimers to check the pharmacokinetic profiles of these G3, G4, G5 and G6 dendrimers. It was found that with an increase in molecular weight of dendrimers, the blood to organ retention ratio was increased. Pharmacokinetic studies were also performed for higher generations of PAMAM dendrimers like G7, G8 and G9 and observed that all the generations of PAMAM dendrimers loaded with MRI contrast agents have shown generation dependent renal accumulation as the generation of dendrimer increases the renal accumulation decreases (Fig. 4.1). G8 and G9 dendrimers were accumulated more in liver in comparison to G6 and G7. The highest blood retention was shown by G7 among all the generations [2,3]. In another study, two different types of G6 PAMAM dendrimers were loaded with MRI contrast agents and were evaluated for biodistribution and clearance patterns. The two PAMAM dendrimers differed in their molecular weight where the internal core was either ammonia or ethylenediamine with 192 and 256 peripheral amino groups, respectively. Though the two dendrimers had similar molecular size and structure, a significant difference in pharmacokinetic properties was observed, depending upon the dendrimer core. Ammonia core PAMAM dendrimer showed higher renal clearance because of more efficient renal uptake than the PAMAM dendrimer with ethylenediamine core. In terms of serving as contrasting agent, PAMAM with ethylenediamine core was better because of its extended plasma retention time and ability to conjugate more number of contrasting agents per molecule than the PAMAM dendrimer with ammonia core [4]. *

50

40

G6 G7

*

*

G8 G9 *

*: p dextran > galactose [25]. Singh et al. prepared PAMAM dendrimers up to four generations, characterized and conjugated them with folic acid and PEGylated folic acid. The targeted dendrimers were

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checked for the targeting potential in tumor bearing mice induced with KB cells which are human epidermoid carcinoma with overexpression of folate receptors, using 5-fluorouracil (5-FU) as anticancer drug. Results showed promising anticancer, targeting and prolonged retention time which are attributed to PEGylation as well as folic acid conjugation to the dendritic formulations [26]. Amreddy et al. worked on co-delivery of two different agents, siRNA and chemotherapeutic drug cis-diaminodichlorido platinum using folate conjugated dendrimers targeting to lung cancer cells. RNA binding protein human antigen R (HuR) which is overexpressed in lung cancer is silenced using siRNA. Combining the drug with siRNA is proposed to give additive effect. In vitro studies like cellular uptake, cytotoxicity and molecular studies were carried out. Results revealed that efficient targeting of the dendritic formulations was observed in case of folate conjugated dendrimers and conclude that the delivery system has potential to take in to in vivo studies on orthotopic induced lung cancer models [27]. Sunoqrot et al. designed hybrid nanoparticles encapsulated with folate conjugated dendrimers and grafted with poly(ethylene glycol)-b-poly(D,L-lactide) (PEG-PLA) on surface. Biodistribution studies were carried out in healthy mice indicated that PEGylation improved circulation half-life. In vivo studies on tumor induced mice showed the targeting potential of folate conjugated dendrimers. Authors showed the advantages of hybrid nanoparticles in terms of increased circulation time attributed to PEG PLA nanoparticles and controlled release with good penetration and targeting potential attributed to dendrimers [28]. PAMAM dendrimer were conjugated with folic acid for targeting of anticancer drugs to glioma cells [29]. Borneol (BO), a Chinese medicinal herb, was used for crossing bloodbrain barrier (BBB) and decreasing the toxicity of PAMAM. Authors showed the dual role of delivery system where BO helps in crossing BBB and folate helps in endocytosis of the dendrimer into tumor cells and finally DOX release by performing both in vitro and detailed in vivo studies [29]. Folate conjugated PAMAM dendrimers are loaded with flavonoid 3,4difluorobenzylidene diferuloylmethane (CDF) for targeting human cervical cancer cells (HeLa) and ovarian cancer cells (SKOV3) (Fig. 5.1). Authors performed profound in vitro studies and show cased that the folate-conjugated PAMAM dendrimers loaded with CDF showed many advantages over unconjugated ones. Western blot analysis of conjugated formulation showed higher inhibition of NFκB as compared to non-targeted formulation. Similar trend is observed in all other assays like cellular uptake, apoptosis, cytotoxicity, etc., which indicate that the delivery system has potential to take it for further in vivo studies [30].

3.2.2 For rheumatoid arthritis Thomas et al. explored that PAMAM dendrimers loaded with MTX and conjugated to folic acid for targeting to the macrophages which overexpress folate receptors in inflammatory

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FA-PAMAM-CDF

FA-PAMAM CDF loading

Intravenous administration

CDF release Tumor

Folate receptor

Accumulation of nano-formulation (FA-PAMAM-CDF) by EPR effect

Folate receptor mediated endocytosis of targeted formulation (FA-PAMAM-CDF)

FIG. 5.1 The pictorial representation of accumulation of targeted formulation (FA-PAMAM-CDF) at tumor site by EPReffect, followed by folate receptor mediated endocytosis of the formulation due to the specific binding of FA to folate receptors overexpressed on cancer cells are shown [30].

conditions. Both in vitro and in vivo studies were carried out for establishing the targeting potential. Results revealed that folic acid conjugated dendrimers showed enhanced internalization in macrophages in vitro and anti-inflammatory by reducing arthritis induced inflammatory parameters in vivo [31]. Qi et al. carried out a similar kind of work for evaluating binding affinities on RAW264.7, NR8383 and primary rat peritoneal macrophages using MTX loaded FA-conjugated PAMAM dendrimers and plain dendrimers. Results showed that binding of conjugates was concentration and temperature dependent and can be inhibited by adding free folic acid. The preventive effects on the developed arthritis model revealed the importance of folic acid conjugated dendrimers in the adjuvantinduced arthritis model [32]. In a study, folate conjugated PAMAM dendrimers, loaded with indomethacin, were checked for the targeting efficiency of the conjugate to inflammatory regions. Results showed that active targeting by folate conjugated dendrimers is very much better than the passive targeting of plain dendrimer [33]. Benchaala et al. worked on inflammatory arthritis caused by Chlamydia trachomatis known as Chlamydia-induced reactive arthritis. They used folate conjugated PAMAM dendrimers to target the dendrimer to folate receptors which are known to overexpress in

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inflammatory sites. Results showed increased concentration of folate conjugated dendritic formulations by three- to fourfolds in infected and inflammatory sites, i.e. paws and genital tracts compared to plain dendrimers [34].

3.3 Amino acids/peptides/proteins conjugation Ongoing research in many fields is utilizing proteins, peptides and even amino acids as a ligand for targeting a specific organ or tissue. Dendrimers can be conjugated with wide variety of amino acids, peptides and proteins. Here are few examples where dendrimer surfaces have been modified with these biomolecules for drug delivery purposes in cancer and HIV treatments.

3.3.1 For cancer Shukla et al. conjugated PAMAM dendrimer with Arg-Gly-Asp (RGD) peptide and evaluated its tumor targeting efficiency in cell lines like human umbilical vein endothelial cells (HUVEC) and Jurkat cell lines which are known to have a high number of integrin receptors. Results showed that conjugate was readily taken up by the cell lines indicating binding affinity of conjugate to cancer cells [35]. Chittasupho et al. designed chemokine receptor CXCR4 targeting dendrimers. CXCR4 receptors are reported to be involved in metastasis of breast cancer. A linear peptide LFC131 (Tyr-Arg-Arg-Nal-Gly) was used as ligand and conjugated to DOX loaded dendrimers. In vitro cell uptake studies were performed on BT-549-Luc and T47D cells (human breast cancer cell lines) and showed that peptide-conjugated dendrimers showed enhanced binding and uptake, and improved cytotoxicity [36]. Kong et al. developed multifunctional dendrimers with C12 alkyl chains, poly(ethylene glycol) chains and c(RGDfK) on their surface encapsulated with 10-hydroxycamptothecin and evaluated for cytotoxicity on 22RV1 cells (human prostate carcinoma cell line) and MCF 7 (breast cancer cell line). Results showed higher cytotoxicity on 22RV1 cells which have higher expression of integrin receptors whereas lower cytotoxicity on MCF 7 is attributed to lower expression of integrin receptors. It was demonstrated that the delivery system is selectively targeted toward cancer without harming normal cells [37]. Zhang et al. worked on RGD grafted PEGylated PAMAM dendrimers conjugated to DOX via two types of bonds-acid sensitive cis aconityl (PPCD) and acid insensitive succinic linkage (PPSD), evaluated in vitro for cytotoxicity, cellular uptake studies and in vivo pharmacokinetic, pharmacodynamic studies. Results revealed that both the conjugates accumulated more in brain in comparison to other organs and PPCD conjugate was better targeting tool than PPSD [38]. Liu et al. worked on T7 peptide (His-Ala-Ile-Tyr-Pro-Arg-His), a ligand for transferrin receptor conjugated dendrimers loaded with anti-tumor gene (pORF-hTRAIL) and DOX. DOX was loaded on to surface of dendritic poly L lysine using acid sensitive spacer with

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glutamic acid. The designed polyfunctional dendrimer is subjected to both in vivo and in vitro studies. Results revealed the synergistic effect of gene and chemotherapy drugs in terms of apoptosis assay performed in both in vitro and in vivo conditions. Pharmacodynamic studies showed the potential of targeted co-delivery of gene and drug with pH-triggered drug release at tumor site. The dose of DOX was reduced substantially with multifunctional dendrimers at which the side effects of DOX like weight loss and cardiotoxicity were negligible in mice [39]. Lempens et al. investigated dendrimers grafted with two tumor homing peptides CREKA, which is linear five amino acid peptide whose receptor is yet to be identified and the other peptide is cyclic nonapeptide, LyP-1 (CGNKRTRGC) which recognizes tumor cells. By attaching fluorescent probe to dendrimers the tumor targeting potential has been elucidated. They concluded that the dendrimers grafted with tumor homing peptides showed superiority in reaching the areas of tumor tissues which are otherwise difficult to target via passive diffusion [40]. He et al. developed dual targeted dendritic formulations consisting of transferrin and wheat germ agglutinin (WGA) acting like ligands conjugated to PEGylated PAMAM carrying DOX inside it. In this research, authors checked for the ability of the formulation to cross blood-brain barrier (BBB). Results revealed that dual ligand conjugated dendrimers showed enhanced crossing of BBB by 13.5% of DOX in 2 h whereas plain DOX showed only 5%. In vitro cytotoxicity studies carried out in murine C6 glioma cells and murine brain microvascular endothelial cells (BMVECs) which serve as normal cells showed that dendrimers are non-toxic to normal cells whereas they are toxic to glioma cells. BBB in vitro model developed using BMVECs showed synergistic transport of dendrimers attributed to dual targeting property of the developed nanodevices [41]. The same group worked on similar dual targeting dendrimers where WGA is replaced with estrogen receptor modulator tamoxifen, which is known to improve the transportation across BBB and inhibit multidrug resistance (MDR). Tamoxifen loaded in interior core and PEG, Transferrin was conjugated exterior along with DOX. In vitro studies like cytotoxicity, cellular uptake and transportation assay in BBB model revealed the potential of the dual targeting in overcoming BBB as well as MDR [42].

3.3.2 For human immunodeficiency virus HIV Rivero-Buceta et al. used 9–18 tryptophan moieties to conjugate on PAMAM dendrimers and checked against HIV replication. Authors found that the conjugated dendrimers inhibited viral attachment to host surface by interacting with the glycoproteins gp120 and gp41of HIV envelop. They stated that nine tryptophan residues are sufficient for efficient binding of dendrimer and thus anti-HIV activity [43]. Ciepluch et al. worked on conjugation of HIV peptides Gp160 (NH-EIDNYTNTIYTLLEECOOH) P24 (NH-DTINEEAAEW-COOH) and Nef (NHGMDDPEREVLEWRFDSRLAFCOOH) to two different types of phosphorus-containing dendrimers and checked for their stability. Authors conclude that prepared dendriplexes can help in designing a valuable tool for immunotherapy against HIV [44].

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3.3.3 For dental applications Hill et al. stated that the use of cyclic RGD-PAMAM for dental implants. They have synthesized the conjugate of c(RGDyK) labeled with fluorescein with PAMAM and evaluated for their binding and uptake properties in MDPC-23 cells which are odontoblast-like cells. Selective binding of the conjugate to integrin receptors was shown in in vitro cellular uptake studies conducted on human dermal microvessel endothelial cells (HDMEC), human vascular endothelial cells (HUVEC), or MDPC-23 cells. It has been demonstrated that PAMAM dendrimers can be used to deliver RGD to tooth that can help in odontoblast regeneration [45]. The same group also hypothesized that binding of cyclic RGD-PAMAM conjugate binds the dental pulp and modulate the differentiation of them through activation of c-Jun N-terminal kinase (JNK) signaling pathway. Western blot analysis showed the increased level of expression of dentin matrix protein, dentin sialoprotein, vascular endothelial growth factor and matrix extracellular phosphoglycoprotein [46].

3.4 N-acetyl-cysteine (NAC) conjugation NAC is known to have clinical application by scavenging the ROS through conversion to intracellular glutathione. NAC is having wider applications in treatment of cancer, HIV, heart disorders, etc. [47].

3.4.1 For cerebral palsy (CP) Activation of microglia and astrocytes in new born leads to neuroinflammation which further leads to CP. Kannan et al. used the advantage of dendritic platforms crossing BBB in CP patients whereas this doesn’t occur in healthy control groups. They used NAC tagged PAMAM dendrimers and showed the improvement in motor activity when tested in newborn rabbit neuroinflammation and motor shortfall [48]. Mishra et al. evaluated for the first time the potential of hydroxyl terminated PAMAM dendrimers in reaching the damaged brain cells of domesticated dogs (canine model hypothermic circulatory arrest induced brain injury). Authors claim that the high dose of valproic acid and NAC required for neuroprotection can be reduced with the use of dendrimers. In vivo efficacy studies with reduced side effects showed the importance of drug dendrimers conjugates, i.e. NAC-dendrimer and dendrimer-PEG-valproic acid conjugate in improving the neurological protection activity [49].

3.4.2 For Rett syndrome (RTT) RTT is a rare genetic brain disorder affecting girls. NAC conjugated with anionic PAMAM dendrimer was used in the treatment of RTT. Nance et al. performed in vitro studies on glial cells from Methyl-CpG-binding protein 2 mouse gene (Mecp2)-null and wild type mice and in vivo onMecp2-nullmice showed improved immune regulation and effective down phasing of RTT phenotype after treatment with the targeted dendritic formulations [5].

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3.5 Carbohydrate functionalization Receptors like lectin, asialoglycoprotein are overexpressed in many cancer cells, bacteria and virus cell membranes. Many carbohydrate molecules like lactobionic acid, fucose, mannose, galactose, lactose, sialic acid, etc., can be used as ligands to target various receptors that are overexpressed in various cancers and infectious conditions. Thus, they can be exploited for conjugation as ligands to target particular cancer or infection [50].

3.5.1 For cancer Lagnoux et al. synthesized glycodendrimers where dendrimers are surface decorated with four or more glycans like β-glucose, α-galactose, N-acetyl-galactose, or lactose and their branches were incorporated with amino acids like Ser, Thr, His, Asp, Glu, Leu, Val, Phe with cysteine moiety in the core which is used for attaching colchicine through disulfide linkage. These glycodendrimers were then evaluated for their cytotoxicity against HeLa cells (human cervical cancer cells) and non-transformed mouse embryonic fibroblasts. Results displayed that the glycoconjugated dendrimers showed more selectivity toward HeLa than mouse fibroblasts. This selectivity in terms of cell death was 163-fold more in HeLa cells than fibroblasts [51]. Lactobionic acid (LA), a disaccharide consisted of gluconic acid and galactose, was conjugated to dendrimers for targeting hepatic cancer overexpressing asialoglycoprotein receptors. They have made surface conjugation sequentially using FITC (fluorescein isothiocyanate) and lactobionic acid or PEGylated LA and loaded with DOX. Remaining amine functional groups have been masked by acetylation. Authors concluded that use of PEG spacer has improved the encapsulation, release, targeting power and therapeutic efficacy of the DOX [52]. Liu et al. demonstrated that glycans can be conjugated on to PAMAM dendrimers and showed their binding activity in vitro on HepG2 (human hepatic cancer cells) cell line. To study the glycodendrimers targeting efficiency, a small library of dendrimers surface modified with different glycans like galactose, lactose and N-acetyl galactosamine (NAG) were synthesized and tagged with fluorescein dye and checked for avidity on HepG2 cells. Results showed that dendrimers conjugated with NAG have shown significant specificity and binding efficacy [53]. Similar study was performed by Medina et al. where they have used generation 5 PAMAM dendrimer conjugated with NAG and checked for its targeting potential in HepG2 cells (Fig. 5.2). They evaluated the effect of ligand selectivity, concentration, time of incubation and number of NAG molecules anchored on the uptake studies using HepG2. Results showed the role of asialoglycoprotein receptors (ASGPR) in endocytosing the dendritic conjugated formulations and also observed that conjugation of 12 NAG molecules is sufficient for efficient uptake by 100% cells within short period of time [54].

3.5.2 For infections For prevention of influenza infection, sialic acids (SAs) are used to prevent viral attachment but they are prone to enzymatic cleavage which demands for higher concentration

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NAcGal Targeting Ligand Covalently-Conjugated Drug (e.g. Chemotherapeutic Agent)

Dendrimer Carrier

ASGPR

Receptor-Mediated Endocytosis Endosomal Escape

Receptor Recycling Drug Release

Hepatic Cancer Cell FIG. 5.2 A schematic drawing showing the composition of a drug-loaded G5-NAcGal conjugate binding to the ASGPR expressed on the surface of hepatic cancer cells (e.g. HepG2), which triggers receptor-mediated endocytosis of these G5-NAcGal conjugates followed by endosomal escape and release of the therapeutic cargo into the cytoplasm while the ASGPR recycles back to the cell surface [54].

of SAs. At higher concentrations SAs show toxicity. Therefore, as an alternative PAMAM dendrimer has been used to conjugate sialic acid that can inhibit influenza hemagglutinin protein binding at non-toxic concentrations. Landers et al. adapted three approaches—in vitro, in vivo and computational modeling to show the inhibition of viral protein binding and results have shown that the conjugate can prevent pulmonary infection caused by influenza A virus [55].

3.5.3 For human immunodeficiency virus (HIV) Shane et al. showed the conjugation and characterization of Manα1-2Man (disaccharide found in the higher oligomannose structures), to generation 3 and 4 PAMAM dendrimers. Precipitation assays were carried out to check the binding efficiency of conjugates with Cyanovirin-N (CV-N), a protein with virucidal activity against HIV. Results showed that the higher affinity of conjugate with CV-N could be attributed to the presence of two mannose moieties on it [56]. Dutta et al. worked extensively on t-Boc–glycine and mannosylated poly (propyleneimine) dendrimer loaded with efavirenz for targeting the lectin receptors on macrophages,

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which act as main reservoirs for spreading of the virus in HIV infected persons. Toxicity of the PPI dendrimers was also decreased after conjugating with t-Boc–glycine and mannose. Cellular uptake studies on macrophages/monocytes showed increased uptake of conjugated dendrimers in comparison to plain dendrimers [57]. The same group worked on the mannosylated PPI dendrimers loaded with lamivudine and evaluated for anti-retroviral activity by estimating p24 antigen using MT2 cell lines along with cellular uptake studies where mannosylated dendrimers showed 21 folds higher uptake in comparison to free drug [58].

3.5.4 For scar prevention Shaunak et al. used anionic PAMAM dendrimers to make water-soluble glucosamine and glucosamine 6 sulfate conjugates and showed that conjugates are having immuno modulatory and antiangiogenic properties respectively which are helpful in prevention of scar tissue formation after surgery [59].

3.5.5 For antimalarial drug delivery Galactose, sugar-coated PPI dendrimers have been synthesized for improving the delivery of primaquine phosphate. Results revealed that galactosylation has increased not only the encapsulation efficiency but also prolonged the drug release from 1 to 2 days of uncoated dendrimers to 5–6 days for galactose coated dendrimer. Plasma studies showed that galactosylated formulations are targeted to liver with prolonged period of circulation time and decreased hemolytic toxicity [60]. In a separate study, galactose was conjugated to poly-L-lysine dendrimers having polyethylene glycol (PEG-1000) as core up for delivering chloroquine phosphate. The effect of galactose coating on safety, stability and efficacy was evaluated both in vitro and in vivo. There were fivefolds decrease in uptake of galactose coated dendrimers by macrophages when compared to uncoated dendrimers, which is helpful to safeguard the dendrimers from macrophage action and target to liver. Further galactose coating decreased the toxicity of native dendrimers sustained drug release property and enhanced drug targeting efficiency [61].

3.5.6 For lung disorders Blattes et al. designed dendrimers decorated with mannose and termed as mannodendrimers. Precisely they used generation 3 and generation 4 poly(phosphorhydrazone) dendrimers and modified their surfaces with 48 and 96 mannose groups and named them as 3T and 4D, respectively. They have checked their binding efficacy to dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC SIGN), a C-type lectin receptor present on the surface of dendritic cells. In vitro studies showed that 3T molecule was more active therefore it was taken to in vivo acute lung inflammation mouse model, results showed that 3T reduced neutrophil recruitment in mice exposed to LPS which showcases its anti-inflammatory activity [62].

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3.6 Biotinylation Essential micronutrient like biotin is required for certain functions of cells like gluconeogenesis, fatty acids, amino acids metabolism, growth and development. Biotin levels are extremely high in rapidly proliferating cells like cancer. Thus, researchers have used the strategy of conjugating the nanocarriers with biotin for targeting them to cancerous cells via sodium-dependent multivitamin transporter (SMVT) uptake [63].

3.6.1 For cancer The carboxylic group of biotin was reacted with amine group of PAMAM dendrimers and then cisplatin was physically encapsulated in dendrimer cavities. Anticancer activity of the conjugate was evaluated against ovarian cancer cell lines like OVCAR-3, SKOV-3 and cisplatin-resistant cells A2780 and CP70. Results showed that biotinylated dendrimers had reduced IC50 values in comparison to free cisplatin in OVCAR-3, SKOV-3. IC50 values were also lesser for biotinylated dendrimer formulation in case of CP70 than free cisplatin which shows the potential of delivery system in cisplatin-resistant cells. Further, biotinylation of dendrimers reduced the hemolysis induced by native dendrimers [63]. In another study, the same group worked on biotinylation of different generation of PAMAM dendrimers and evaluated the effect of generation on the cytotoxicity and cellular uptake. Increase in generation led to increase in cytotoxicity as well as cellular uptake of dendrimers. However, the cellular uptake of dendrimers was more in OVCAR-3 ovarian cancer cells compared to HEK 293 human embryonic kidney cells [64].

3.7 Lauroyl chain conjugation The properties of dendrimers can be modified using ligands made of fatty acids like lauryl chains which help in decreasing the toxicity profile and improves the permeability of dendrimers across the biological membranes [65].

3.7.1 For cancer Teow et al. worked on generation 3 PAMAM dendrimer where they have conjugated with lauryl chains which are further grafted with paclitaxel (PTX) and FITC to check the increase in permeability using Caco-2 (human colon adenocarcinoma cell line) and PBECs (primary cultured porcine brain endothelial cells). Results showed an increase of permeability by 12% of lauryl dendrimer conjugate in comparison to plain PTX alone [66].

3.8 Tuftsin grafting Tuftsin, a tetrapeptide (Thr-Lys-Pro-Arg) increase natural killer activity of macrophages, monocytes and leukocytes by exhibiting specific binding to them. Tuftsin is an integral part of Fc portion of immunoglobulin IgG having dual advantage as targeting ligand and phagocytosis inducer. Therefore, dendrimers with this peptide help in targeting and improving the therapeutic efficacy of the payload employed.

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3.8.1 For HIV Dutta et al. worked on in vitro characterization of tuftsin conjugated poly (propyleneimine) dendrimers to show the targeting potential of tuftsin in treatment of HIV using efavirenz, anti-HIV drug. It was demonstrated in 1 h, cellular uptake was increased by 34.5 times in case of conjugate in comparison to pure drug. Accumulation of the conjugate is higher in infected macrophages when compared with normal cells. Authors claim the increase in activity is due to inherent anti-HIV property of tuftsin [67].

3.9 Antibody grafting Use of monoclonal antibodies (mAb) is a type of targeted immunotherapy with tumors overexpressing antigens or proteins known as tumor associated antigens. By employing a mAb on the surface of carrier, it can be directed toward tumor and avoiding their access to other sites. It is advantageous over other targeting ligands because other ligands are also inhibited competitively by presence of endogenous moieties [68].

3.9.1 For cancer PAMAM dendrimers were conjugated to trastuzumab (TZ) monoclonal antibody, which binds to human epidermal growth factor receptor type 2 (HER2) overexpressing cancer cells. Two human breast cancer cells, i.e. MDA-MB-453 and MDA-MB-231 cells which are HER2 positive and negative respectively, were used for checking the uptake of the unconjugated and conjugated formulations. The antibody conjugated dendrimers showed enhanced uptake in comparison to unconjugated dendrimers in HER2 positive cells, on the contrary, no difference in uptake was observed in case of HER2 negative cells which explains the targeting efficiency of TZ. The cytotoxicity results also followed the same trend in which antibody conjugated formulations showed 3.6 folds higher than unconjugated dendrimers in HER2 positive cells [69]. Jain et al. used carboxylic acid terminated G3.5 PAMAM dendrimers, loaded with paclitaxel and conjugated with monoclonal antibody mAbK1, mesothelin protein which overexpress in few cancers like ovarian. In vitro cytotoxicity studies were performed on OVCAR-3 ovarian cell line which showed increased greater cytotoxicity when compared to plain dendrimers and free drug. In vivo studies showed better survival rate and good tumor inhibition in case of antibody conjugated dendrimers when compared to plain dendrimer and drug alone. Authors claim that targeted dendritic formulations not only improved the efficacy but also decreased the toxicity profile of dendrimers [70]. Xie et al. conjugated multiple Sialyl Lewis X antibodies to PAMAM dendrimers for targeting colon cancer. These antibodies are known to bind saliva acidifying Lewis oligosaccharides X antigens which mediate colorectal cancer metastatic process. In this research,

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they have characterized for conjugate formation using atom force microscope, UV, fluorescence measurements and showed capturing efficiency of the HT29 colon cancer cells. The captured circulating tumor cells lost the ability of survival and proliferation as well which will aid in booming prevention of cancer metastasis [71]. Yoon et al. addressed the limitation of systematically administered naked adenovirus, which causes hepatotoxicity and immunotoxicity that was overcome by use of epidermal growth factor receptor (EGFR) precise antibody (ErbB) and PEGylated conjugated poly(amidoamine) (PAMAM) dendrimer. It was demonstrated that exposure of adenovirus was avoided by the steric hindrance of PEGylated antibody conjugated dendrimers which specifically targeted the EGFR positive tumor cells and reduced immunotoxicity [72].

3.10 Phosphonate derivatization Research by Hayder et al. studied surface modified dendrimers with phosphorous containing moieties. These modified dendrimers showed their ability in modulating innate immunity by exhibiting their effects on monocytes. These dendrimers are posed to have dual action both in inflammation and osteoclastogenesis which paves the opportunity for dendrimers to be used in rheumatoid arthritis [73]. Dendrimers with araldehyde on their surface are conjugated with phosphoric acid to give phosphonate modified dendrimers. Poupot/Caminade group from France has adapted this approach and claimed that polyvalency of phosphorous dendrimers with 16 phosphonate groups is important for biological activity with in vitro results, showing the decreased monocyte activation and increased number of natural killer cells [74, 75]. In rheumatoid arthritis mouse model, the fully capped phosphonate dendrimers with 24 phosphonate groups showed dose-dependent decrease in pro-inflammatory cytokine levels from splenocytes, increase in anti-inflammatory cytokine levels and the antiosteoclastic activity which all will make phosphonate dendrimers as a valuable tool in the treatment of rheumatoid arthritis. This fully capped bisphosphonate dendrimers were also found to be nontoxic in non-human primates up to 10 mg/kg for 4 weeks with intervals weekly when injected intravenously [76].

3.11 Sulfate derivatization Anionic surface created by sulfonation helps in increasing the binding efficiency of dendrimers to virus and also inhibits virus from invading the host cells thus avoiding infection [77–79].

3.11.1 For human immunodeficiency virus (HIV) and herpes simplex virus (HSV) The surface amine groups of dendrimers were sulfated to block the chemokine receptors CCR5 and CXCR4 which are crucial for viral way into CD4 + cells after binding of HIV-1 envelope gp120 to human cell surface CD4 [77]. Starpharma developed completely

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sulfated form of dendrimer SPL7013 which is formulated as aqueous gel into a mucoadhesive polymer Carbopol® that can be applied as a topical agent. The anionic surface charge of the SPL7013 is known to block the viral attachment to gp120 of human cells. The first clinical investigation performed by Price et al. on HIV-uninfected women using VivaGel showed the retention of strong HIV-1 and HSV-2 inhibitory activity in humans. They performed ex vivo anti-viral activity, local retention of gel for a period of 24 h following a single application of gel and in vivo estimation of microbicide levels in cervicovaginal fluid (CVF) samples. Results of these clinical studies showed that gel was well recovered and high levels of virus inhibitory activity for both HIV and HSV was observed in CVF samples for all the women participants after 3 h of administration [78]. O’loughlin et al. evaluated SPL7013 gel safety and tolerability and pharmacokinetics through Phase I clinical studies on healthy women. Results showed that gel applied to women over a period of 7 days at a concentration of 0.5–3% was safe with good toleration and no indication of toxicities [79].

3.12 P-hydroxyl benzoic acid (pHBA) functionalization pHBA is a small molecule with an affinity toward sigma receptors that overexpresses in brain tumor. Swami et al. conjugated pHBA on to G4 PAMAM dendrimers loaded with docetaxel for targeting to brain. The prepared conjugates were characterized and preceded for evaluation of in-vitro studies on U87MG brain tumor cells and in vivo studies on mice for determining pharmacokinetic and biodistribution in comparison to marketed formulation taxotere. Results demonstrated that pHBA conjugated dendrimers showed superior activity in terms of cytotoxicity and cellular uptake. In vivo studies for conjugated formulations showed two-fold increase in drug concentration in brain when compared to unconjugated dendrimers [80].

3.13 Hyaluronic acid functionalization Hyaluronic acid (HA) is a linear mucopolysaccharide with inherent tumor targeting potential through exhibiting binding efficiency to CD44, cell-surface glycoprotein known to overexpress in many tumors like ovarian, colon, lung, breast, etc. [81]. Kesharwani et al. synthesized hyaluronic acid conjugated dendrimers for targeting of curcumin toward pancreatic cancer (MiaPaCa-2 cell line) (Fig. 5.3). They have used the derivative of curcumin, 3,4-difluorobenzylidene curcumin (CDF) which was reported to have superior anticancer activity than curcumin. HA-conjugated dendritic formulations showed better cytotoxicity and cellular uptake. Receptor blockade assay show cased that endocytosis occurred because of binding of HA to CD44. It was concluded that even the cationic charge of PAMAM dendrimer is reduced because of conjugation with HA that will help in reduction of toxicity and thus the potential of dendrimer’s usage into clinical translation [82]. Table 5.1 summarizes various dendrimer-based targeted drug delivery systems designed for delivering of different therapeutic molecules.

Chapter 5 • Dendrimer-based targeted drug delivery

PAMAM

HA

HA-PAMAM

CDF

123

HA-PAMAM-CDF

Blood pH 7.4 EPR effect

Nucleus CD44 receptor mediated endocytosis CDF released at acidic Endolysosomes

Acidic tumor Extracellular PHl

FIG. 5.3 Schematic depicting the method used to synthesize CDF-loaded HA conjugated PAMAM dendrimer nanosystem and their uptake in pancreatic cancer cells over-expressing CD44 receptors. The HA-PAMAM-CDF targeted nano-systems are selectively taken up by tumor cells over-expressing CD44 via receptor-mediated endocytosis. The CDF is released in acidic endolysosomes, followed by its release into the cytoplasm for therapeutic action [82].

Table 5.1 diseases Targeting ligand PEG

Dendrimer-based targeted delivery of therapeutic molecules in various

Dendrimers

Drug delivered

Target site/disease

Reference

PAMAM PAMAM PAMAM

Doxorubicin Adriamycin Adriamycin, methotrexate Camptothecin Doxorubicin Doxorubicin Doxorubicin 5-fluorouracil siRNA, doxorubicin – Kartogenin

Gastric cancer HeLa cells –

[9] [10] [11]

Colon cancer Rectum carcinoma Breast carcinoma Breast cancer MCF-7 cancer cells Multidrug resistant cancers Bone targeting Bone targeting

[12] [13] [14,15] [16] [17] [18] [19] [20]

Poly lysine Poly lysine Poly lysine Poly lysine PAMAM PAMAM PAMAM PAMAM

Continued

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Table 5.1 Dendrimer-based targeted delivery of therapeutic molecules in various diseases—cont’d Targeting ligand Folic acid

Dendrimers

Drug delivered

Target site/disease

Reference

PAMAM PAMAM PPI (Poly(propylene imine)) PAMAM PAMAM

Methotrexate – Paclitaxel

KB cells Ovarian cancer HeLa, SiHa cells

[22,23] [24] [25]

5-fluorouracil siRNA, cisdiaminodichlorido platinum – Doxorubicin 3,4-difluorobenzylidene diferuloylmethane Methotrexate Methotrexate Indomethacin – – Doxorubicin 10hydroxycamptothecin Doxorubicin Doxorubicin –

human epidermoid carcinoma Lung cancer

[26] [27]

human epidermoid carcinoma Brain tumor HeLa, SKOV3 cells

[28] [29] [30]

Arthritis RAW264.7, NR8383 Inflammation Arthritis HUVEC, Jurkat cell lines. Breast cancer Prostate and breast cancer cells

[31] [32] [33] [34] [35] [36] [37] [38] [39] [40]

Doxorubicin

Brain tumor Glioma Prostate cancer, MDA-MB-435 cancer cells. Brain tumor

[41]

PAMAM

Doxorubicin

Brain tumor

[42]

PAMAM Phosphorous dendrimers PAMAM

– –

HIV HIV

[43] [44]

–

[45,46]

– Valproic Acid – Colchicine

HDMEC, HUVEC, Odontoblast like cells Neuroinflammation cerebral palsy Rett syndrome HeLa cells

[48] [49] [83] [51]

Doxorubicin – – Efavirenz Lamivudine –

Hepatic cancer HepG2 cells HIV HIV Scar tissue prevention

[52] [53,54] [56] [57,58] [59]

PAMAM PAMAM PAMAM

RGD LFC131 c(RGDfK) RGD T7 CREKA, LyP-1 Transferrin, WGA Transferrin, tamoxifen Tryptophan HIV peptides c(RGDyK) Nacetyl-cysteine Glycodendrimers

PAMAM PAMAM PAMAM PAMAM PAMAM PAMAM PAMAM PAMAM Poly lysine AB5 dendritic wedges PAMAM

PAMAM PAMAM PAMAM Glycopeptide dendrimers PAMAM PAMAM PAMAM PPI PAMAM

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Table 5.1 Dendrimer-based targeted delivery of therapeutic molecules in various diseases—cont’d Targeting ligand

Dendrimers

Drug delivered

Target site/disease

Reference

Primaquine phosphate Chloroquine phosphate –

Malaria Malaria Lung disorders

[60] [61] [62]

Biotin Lauroyl

PPI Poly lysine Poly (phosphorhydrazone) PAMAM PAMAM

Cisplatin Paclitaxel

[63,64] [66]

PPI PAMAM PAMAM PAMAM PAMAM ABP SPL7013 PAMAM PAMAM

Efavirenz Docetaxel Paclitaxel – – – – Docetaxel Curcumin

Ovarian cancer cells Caco-2, primary cultured porcine brain endothelial cells. HIV Breast cancer cells Ovarian cancer Colon cancer Lung tumor Rheumatoid arthritis HIV Brain tumor Pancreatic cancer

Tuftsin Antibody

Phosphonates Sulfate pHBA Hyaluronic acid

[67] [69] [70] [71] [72] [73–76] [77–79] [80] [82]

4 Conclusion Presently the use of dendrimers in targeted drug delivery is on wheels. The in-depth in vitro and in vivo studies of many research groups have enlightened that targeted dendrimers formulations have performed better than non-targeted formulations in the treatment of many serious diseases and disorders.

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Dendrimers for anticancer drug delivery

Pushpendra Kumar Tripathi, Shalini Tripathi DEPARTMENT OF PHAR MACY, RITM, DR AP J ABDUAL KALAM T ECHNICAL UNI VERSITY, LUCKNOW, INDIA

1 Introduction Cancer cells have potential to proliferate abnormally and spread to the whole body which makes it a terrible disease that affects and kills millions of people every year [1]. Defective apoptosis and complexity in genetic and phenotypic level leads to therapeutic resistance and unproductive treatment outcomes. Various approaches such as surgical removal, chemotherapy, radiation, and hormone therapy are common methods to treat the cancer but they have all have significant limitations and side effects [2]. During Chemotherapy, anticancer drugs are delivered systemically to patients for quenching the uncontrolled multiplication of cancerous cells [3]. But, due to nonspecific targeting of anticancer agents, various side effects may occur, and poor drug delivery of those agents cannot show up the desired effect in most of the cases. Drug development comprises a very complex procedure which is allied with advanced polymer chemistry and modification. The major challenge of cancer therapeutics is to identify and differentiate the cancerous cells from the normal body cells, therefore the main objective is to alter drugs in such a way so as it can target the cancer cells to diminish their growth and proliferation. Normal chemotherapy fails to target the cancerous cells only without interacting with the normal body cells and so this may cause serious side effects including organ damage which results in compromised treatment with lower dose and eventually low survival rates [1]. Various therapeutic tactics to fight cancer have been explored using small drug molecules, which showed low therapeutic index and often with harmful side effects. Hence, to attain maximum drug activity with insignificant side effects, various efforts have been made to deliver anticancer drugs using macromolecular formulations. Nanotechnology usually deals with the size range from a few nanometers (nm) to several hundred nm, based on their proposed use [4]. Developing specific drug delivery systems has become the area of attention over the last decade as it offers many advantages to overcome the limitations of conventional formulations [5,6]. Various studies are being carried out in order to find more accurate nanotechnology based cancer treatment and minimizing the side effects of the conventional ones [4]. Pharmaceutical Applications of Dendrimers. https://doi.org/10.1016/B978-0-12-814527-2.00006-8 © 2020 Elsevier Inc. All rights reserved.

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Chemotherapy was given the name “magic bullet” by Paul Ehrlich [7]. Various types of nanoparticles that are used in medical treatment are Fe4 [Fe (CN) 6] [8], nanotubes [9], liposomes, polymer-drug conjugates and others to assist therapeutic agents to pass through biological barriers to mediate molecular interactions, and to identify molecular changes. They are with larger surface area and modifiable properties such as optical, electronic, magnetic, and biologic as compared to macroparticles. The liposomes and polymer-drug conjugates that were developed in the 1960s and 1970s [10]; are now playing the significant role in the area of nanomedicine [11]. Latest drug delivery systems based on nanotechnology for cancer treatment, which are already marketed and under research and evaluation, include liposomes, polymeric micelles, dendrimers, nanospheres, nano capsules, and nanotubes [12,13] DOXIL (liposomal doxorubicin) and Abraxane (albumin bound paclitaxel) are example of such nanoformulations [14]. Recent advances in nanomedicine has significantly improved the life expectancy and quality of life for the cancer patients [15].

2 Brief about dendrimers and application for cancer drug delivery Development of nanomedicines for targeted cancer therapies is one of the most important application of nanotechnology. Recently Dendrimer that is a globular, nanosized (1–100 nm) synthetic macromolecule has been explored as drug delivery vehicle for anticancer drugs. Dendrimer term originates from ‘dendron’ meaning a tree in Greek and in arborols the Latin word ‘arbor’ also meaning a tree. Dendrimers can be characterized by a central core; branches, called ‘generations’, having repeated units and many terminal functional groups [16]. Traditional synthetic polymers have been classified into three major macromolecular structural designs (i) linear (class I), (ii) cross-linked (bridged; class II), and (iii) branched types (class III). Structures or architectures of these classes are produced by largely statistical polymerization processes, rather than exact distribution processes. Dendrimers with monodispersity represent the fourth class of polymers mostly synthesized by convergent or divergent synthesis method by step-by-step controlled synthesis based on chemical reactions [17]. Dendrimer structure consists of inner space and multiple terminal groups for drug encapsulation and drug conjugation. A multifunctional nanocarrier can be prepared by attaching imaging probes and targeting ligands to the dendrimers. The exact morphology of a dendrimer depends both on its chemical composition as well as on the generation number (Fig. 6.1). Dendrimers structure (e.g., G0 and G1) possess open structures and have highly asymmetric shapes compared with higher-generation structures that first appear to be disk-like and then gradually progress to spherical geometries and assume globular structures with a significant reduction in hydrodynamic volume [16,18].

Chapter 6 • Dendrimers for anticancer drug delivery

133

FIG. 6.1 Formation of dendrimer skeleton with synthesis approaches.

In addition to sphere-like dendrimers which based on a dot-like core, cylindrical dendrimers are based on rod-like cores. These macromolecules can be related with spaghetti because they can be rigid or flexible like the uncooked or cooked form of the pasta. These properties can be changed based on the density and chemical composition of packing of the dendritic branches [19]. Tumor-targeting antibody can be effective for killing cancer cells. But tumor-targeting antibody needs to be modified with multiple radioisotopes, toxins, or even with many small molecules to increase its efficacy. This may lead to complex synthesis steps and may also alter its inherent specific antigen-binding affinity. To maximize drug loading while minimizing the toxic effects on the biological integrity of the host, a better approach is to use a carrier molecule such as a dendrimers. Dendrimers can be used for attaching targeting ligands, imaging molecules, whereas drugs can be loaded in their free form. Dendrimeric conjugates of anticancer drug have shown ability to bypass efflux transporter, to deliver the drug intracellularly, and to improve bioavailability of loaded molecular cargo. Cisplatin which is a widely used anticancer drug for example requires internalization of at least 50 cisplatin molecules upon superficial analysis to be effective as an anticancer drug. Complexes of cisplatin with dendrimers have showed reduced cytotoxicity with significant anti-proliferative activity. Dendrimers have also shown promise as a next generation imaging molecules for diagnostic applications and theranostic applications [20] in cancer treatment (Table 6.1) aside from targeted delivery of chemotherapeutic drugs [26]. Mechanisms for cancer therapies also include blockage of signal transduction inhibitor activities, modulation of gene expression, induction of apoptosis, inhibition of

134

Pharmaceutical Applications of Dendrimers

Table 6.1

Dendrimers for cancer diagnosis.

Type of dendrimer

Type of cancer

Use

References

Gadolinium (III) (Gd(III))-complexed dendrimer-gold nanoparticles Ferrocene-cored poly(amidoamine) dendrimers Dendrimers complexed with copper

Lung cancer metastasis

MRI and CT scan

[21]

Breast cancer

electrochemical DNA detection strategy MR imaging

[22]

Multimode imaging CT imaging positron emission tomography (PET) imaging

[23a, 24] [24] [25]

Dendrimer–gold nano flowers PAMAM coated magnetite nanoparticles Amphiphilic dendrimer

Different cancer types and metastases Tumors Liver cancer Tumors

[23]

angiogenesis and utility of cancer vaccines. Anticancer drug molecules may be covalently conjugated to the end groups of a dendrimer or entrapped inside the core via hydrogen bonding, hydrophobic linkage, or electro–static interactions [27]. The number of generations influences the drug loading capacity: a relatively high generation number provides more space for guest drugs and has a larger number of functional groups on the surface for drug conjugation. PPI, PAMAM, PLL, polypeptide, polyesters, polyether dendrimers, and dendrimers based on PEG, or carbohydrates have been mainly investigated for delivery anticancer drugs [23,28]. Drug release from dendrimers depends on the type of interactions between a drug and a dendrimer [29]. A cancer-targeting strategy is reported that is reminiscent of the antibody– toxin/immunoconjugate strategy where distinct, but linked, entities are used to first recognize, bind and then subsequently modify a cancer cell [30]. Their strategy, however, has great potential to improve on both the “targeting” and “payload” aspects of cancer therapy. The key to this approach was to include a DNA “zipper” on each dendrimer that allows the targeting cluster, composed of folate-derivatized PAMAM in proof-ofconcept experiments [31], to be readily combined with the imaging or drug-carrying dendrimer by way of the complementary DNA strand [30]. New technologies like dendrimers that provide a platform to physiologically related display of carbohydrates, are now exploiting these molecules to interfere in malignant disease. Promising efforts in this way include the presentation of oligosaccharides found only in cancer cells [32–35] on a dendritic scaffold for vaccine development. An area of recent exploration is the abnormal glycosylation allied with the cancer cells; specifically dendrimeric scaffolds give a distinctive platform to control the multimeric carbohydrate presentation necessary to endorse the “cluster glycoside effect” [36–38], which is important for targeting. One more approach to exploit glycosylation in the treatment of cancer is the capability to express non-natural sialic acids on the cell surface with the use of N-acetyl-mannosamine analogs [33,39]. Dendrimers offer assistance at several steps

Chapter 6 • Dendrimers for anticancer drug delivery

135

in this process of chemo selective targeting of drug loaded dendrimers to the cell surface [40,41]. PAMAM dendrimers also show anti-inflammatory activities via different mechanisms determined by surface groups [42,43]. Neutral dendrimers have been found to target inflammatory cells within the brain. They can localize in activated microglia and astrocytes in the presence of neuroinflammation. Activated (i.e., SuperFect) and non-activated (i.e., Polyfect) PAMAM dendrimers can differentially modulate EGFR signaling pathway [44,45]. Such intrinsic therapeutic functions endowed by dendrimers themselves may be integrated into drug delivery system design and offer additional therapeutic benefits for cancer therapy. Dendrimers can be easily eliminated from the body and have the advantages of being biocompatible. PAMAM dendrimer- drug complexes circulate for longer time in blood than small-molecule drugs. Dendrimer nanoparticles are eventually cleared from the human body through the kidneys along with used up by metabolic pathways such as growth factors [46], folate [31,47] peptides [48,49] and antibodies [50]. However, cationic dendrimers also have the disadvantages of being cytotoxic to normal cells due to the end groups present on their peripheries [51] such as cationic PAMAM, PLL, and PPI dendrimers. High-quality dendrimer products in high quantities have been made available for fundamental research and translational studies owing to commercialization of PAMAM dendrimers by several companies such as Dendritech (Midland, Michigan), Dendritech Nanotechnologies (Mt. Pleasant, Michigan) and NanoSynthons (Mt. Pleasant, Michigan). The commercially available dendrimers serve as a reliable source of building blocks for development of dendrimer nanomedicine products on a large scale. Nonetheless, manufacturing of functionalized dendrimers with uniform loading of drugs, ligands, and other moieties on the dendrimer surface is challenging and remains to be solved for consistent therapeutic effects [52]. Efficient and robust dendrimer surface chemistries such as click chemistry [53] could help to tackle the heterogeneity of dendrimer surface functionalization and enable scaling up of dendrimer nanomedicine products.

3 PAMAM dendrimer for cancer therapy The well-known poly (amidoamine) PAMAM “starburst” dendrimers and “arborols” are the first dendritic systems that have been exhaustively characterized and studied during the period of 1970–90 (Fig. 6.2A) They are favorably used in drug delivery due to their hydrophilic, biocompatible, and non-immunogenic nature. Ethylenediamine, diaminododecane, diaminoexane, and diaminobutane are most common cores used for PAMAM dendrimers [18,54,55]. Their branching units which are based on methyl acrylate and ethylenediamine, have amine (in full generations) and carboxyl (in half generations) terminated groups (Fig. 6.3) [56]. DOX which is the most widely used antitumor drugs. Despite of its good efficacy shows well known systemic side effects, mainly cardiomyopathy [57]. Many researchers have worked on the development of dendrimer-based delivery system for optimizing the therapy and hence increasing the efficacy and reducing the

136

O

HN O

H3N

NH

H N

N N

O

O

HN

O HN

O

H3N

N H

O

NH O

(A)

NH2 H2N

HN NH O NH3+

H3N

NH

O

H2N

O

H N

NH O

HN

NH2

N H

HN O NH

H2N

NH2

O

O

H2N

O

NH O

H2N

H N

H2N

NH2

N

N

O

O

H N

O O

H2N

NH NH

O

O

O NH

NH NH2

H2N

NH O

H2N

O

NH2

NH2

H2N HN

O

H2N

(B)

H2N

Poly(propylene imine)

(C)

NH2

HN

NH2

O HN

N

NH2

O

H2N

NH2

N

NH2

NH HN

NH2

N

NH2

O

O

O

H2N

N

NH2

HN

O

HN

O

NH2

N H

NH

NH2

O

NH2

H N

HN O

HN H2N

NH2

O N H

PAMAM

+

O HN HN

NH2

O O

H3N

O

O

HN

NH HN

H2N

HN NH

O

O

NH2

HN

NH2

H2N N

O

+

H2N

NH O

H2N

N HN

H2N

O

O

NH

NH

NH3+

O +

O

NH2

N

N

NH2

NH2

NH

H2N

NH2

Poly-I-Iysine

FIG. 6.2 Most studied dendrimers: (A) PAMAM 2.0 generation, (B) PPI dendrimer 2.0 generation, and (C) PLL dendrimer 4.0 generation.

Pharmaceutical Applications of Dendrimers

+

H2N

NH3+

NH3+

NH2

NH2

PAMAM

CO

O

H

COOH

NH2

Full Generation (Amine terminated)

COOH

PAMAM

H

2

2

H

O

CO

CO O

NH

NH

137

COOH

O CO

H

NH2

2 NH

COOH

2 NH

Chapter 6 • Dendrimers for anticancer drug delivery

Half Generation (Carboxyl terminated)

FIG. 6.3 PAMAM dendrimer, amine terminate full generation and half generation.

toxic side effects of DOX [56,58–60]. For improving the drug accumulation in lung tumors, Zhong et al. [60a] worked on DOX-dendrimer conjugates and examined their ability to reduce metastatic lung burden through local administration. PAMAM dendrimers of generation 4 was used and DOX was conjugated to their surface through acid-sensitive hydrazone bonds. In a study when mouse-bearing melanoma B16-F10 cells were used as a model for lung metastasis, the pulmonary administration of DOX-dendrimer conjugate revealed that tumor burden was decreased, accumulation in the lungs was increased, and distribution to the cardiac tissue was found to be diminished. Conjugation of acidsensitive hydrazone bonds to DOX has been used as a method to develop stimuli-sensitive carriers to release their load in the tumor microenvironment or endosomal vesicles [61] when exposed to low pH and thus show improved tumor specificity. Some other such stimuli-sensitive approaches can also be used to increase the specificity of the drug delivery systems to discover microenvironment characteristics of tumor [62,63]. Applying one of these strategies [64], PAMAM dendrimers of fourth generation were conjugated to PTX through a peptide linker that is cleavable by an enzyme named cathepsin B showed upregulation in metastatic breast cancer. This PTX-dendrimer conjugate (PGD) exhibited higher cytotoxicity than free PTX in cells having greater cathepsin B activity. PGD thus revealed better efficacy when compared to free PTX in terms of inhibit tumor growth in mice embedded with high expression of cathepsin B xenografts such as MDA-MB-231 [64]. The PAMAM dendrimers provide the platform for surface modification with variety of ligand moieties which help in active targeting to the cancerous cells and hence, leads to improved tumor specificity with minimal systemic toxicity [65]. One of the most profound examples of active targeting is by the means of antibodies as targeting molecules. Kulhari et al. [66] exploited trastuzumab (TZ) as an targeting moiety and linked it on the surface of

138

Pharmaceutical Applications of Dendrimers

docetaxel (DTX) bearing G4 dendrimers, using PEG as a linker. Herein, TZ selectively attaches to the human epidermal growth factor receptor type 2 (HER2) which are overexpressed on the tumors cells and stop downstream signaling [67]. Moreover, the in vitro efficacy of TZ-DTX-dendrimers was compared with DTX-dendrimers and free DTX in MDA-MB-453 (HER2-positive) and MDA-MB-231(HER2-negative) cells. Seventy percentage higher uptake by HER-positive cells was observed for TZ-DTX-dendrimer as compared to DTX-dendrimer after 4 h of incubation, while no differential uptake pattern was observed for the same in HER-negative cells. TZ modified dendrimer showed higher toxicity in MDA-MB-453 as compared to plain dendrimer. Supportively, the IC50 of TZ-DTX-dendrimer was 3.6-fold greater than DTX-dendrimer, while no difference was detected between any of them and free drug in MDA-MB-123 [66]. Generally, dendritic structure with higher generations (4 and up) of PAMAM show better efficacy and higher drug loading as compared to the structures with lower generations. It may be due to greater physical or chemical interactions as higher generation dendrimers offer more space within the dendritic cavities where the tertiary amines can easily interact with guest molecules [55,68]. The same condition is well-applied in the case of chemical conjugation as the number of changeable surface groups which conjugate with the drugs are increased with increase in generations [55]. Pan and co-workers [69] investigated that multidrug resistance-related protein, P-gp, using its corresponding siMDR-1 is a promising gene and chemo combination approach. Based on G4 PAMAM, a co-delivery system for siRNA and DOX has been developed. PAMAM dendrimer plays important roles in complexing siRNA, facilitating cellular interaction and assisting endosomal escape. PEG moieties shield the cationic charge and homogenize the structure of the nano-preparation. A balance between cellular interaction and cytotoxicity is a crucial factor in terms of maximizing the therapeutic efficiency. They have reported that MDM 1:10 is the most promising platform for co-delivery of nucleic acid with hydrophobic DOX. When complexed with siMDR-1, MDM 1:10 downregulated the amount of membrane bound P-gp as well as the function of P-gp, resulting in reversal of the MDR effect. In addition to successful delivery of siMDR1 into MDR cell lines, a synergistic anti-cancer effect has been achieved by the co-delivery of DOX base and siMDR-1 with dendrimer-based nano-preparation. PAMAM dendrimers were used for combination chemotherapy approach. Guo and coworkers [70] investigated that the synergistic effects of drugs and minimal drug dose for cancer therapy, combination chemotherapy is frequently used in the clinic. In this study, hyaluronic acid-modified amine-terminated fourth-generation polyamidoamine dendrimer nanoparticles were synthesized for systemic co-delivery of cisplatin and doxorubicin (HA@PAMAM-Pt-Dox). Results suggested that HA@PAMAM-Pt-Dox has great potential to improve the chemotherapeutic efficacy of cisplatin and doxorubicin in breast cancer. Huge difficulties are including the antagonistic nature of drugs, variations in drugs in terms of solubility, and limited tumor targeting. Considering the advantages of dendrimers such as control of size and molecular weight, bioavailability, and biosafety, fourthgeneration PAMAM dendrimers were modified by HA as drug vectors by covalently

Chapter 6 • Dendrimers for anticancer drug delivery

139

conjugating with anticancer drugs (cisplatin and doxorubicin) to form a nanodrug delivery system, named HA@PAMAM-Pt-Dox. It was observed that the HA@PAMAM-Pt-Dox system can effectively kill breast cancer cells both in vitro and in vivo, which showed a favorable synergistic effect. Zhang and co-workers [71] investigated that the preparation of multifunctional doxorubicin (DOX)-conjugated poly(amidoamine) (PAMAM) dendrimers for pH-responsive drug release and targeted chemotherapy of cancer cells. DOX was conjugated onto the periphery of partially acetylated and folic acid (FA)-modified generation 5 (G5) PAMAM dendrimers through a pH-sensitive cis-aconityl linkage to form the G5.NHAc-FA-DOX conjugates. FA conjugation onto the dendrimers allowed a specific targeting to cancer cells overexpressing FA receptors. The G5.NHAc-FA-DOX conjugates showed effectiveness for targeted cancer therapy. Yao and Ma [71a] investigated the enhancement of the therapeutic effect of Paclitaxel (a potent anticancer drug) by increasing its cellular uptake in the cancerous cells with subsequent reduction in its cytotoxic effects. To accomplish these aims the paclitaxel (PTX)biotinylated PAMAM dendrimer complexes were prepared using biotinylation method. The primary parameter of Biotinylated PAMAM with a terminal HN2 group - the degree of biotinylation - was evaluated using HABA assay. The basic integrity of the complex was studied using DSC. The Drug Release (DR) and Drug Loading (DL) parameters of Biotinylated PAMAM dendrimer-PTX complexes were also observed. Cellular uptake study was performed in OVCAR-3 and HEK293T cells using fluorescence technique. The statistical analysis was also performed to support the experimental data. The results achieved from HABA assay disclosed the complete biotinylation of PAMAM dendrimer. DSC study established the integrity of the complex as compared with pure drug, biotinylated complex and their physical mixture. Result showed the highest DL (12.09%) and DR (70%) for 72 h as compared to different concentrations of drug and biotinylated complex. The OVCAR-3 (cancerous) cells were characterized by more intensive cellular uptake of the complexes than HEK293T (normal) cells. Both experimental and statistical evaluation confirmed that the biotinylated PAMAM NH2 dendrimer-PTX complex not only increases cellular uptake but also enhances release up to 72 h with the reduction in cytotoxicity. Li and co-workers studied [60] Gastric cancer (GC) which is the second most common leading cause of cancer-related death. Cancer stem cell (CSC) with the mark of CD44 played a significant role in GC. rMETase was wildly exploited as chemotherapeutic option for GC Polymers based nanoparticle drug delivery have been commonly used for cancer therapy. 5G PAMAM-Au-METase with the surface decoration of Hyaluronic acid (HA), a receptor of CD44, nanoparticles exhibit with good biocompatibility and aqueous solubility. The number of CD44(+) GC cells in nude mice injected with generation 5G PAMAMAu-METase decorated by HA was declined causing in the inhibition of tumor growth. Pishavar and Co-workers investigated [72] that Alkyl-PEG and cholesteryl formate modified PAMAM 5G and 4G with tumor necrosis factor-related apoptosis-inducing ligand for gene therapy for colon cancer treatment. Prepared dendrimer system enhances transfection efficiency through overcoming extracellular and intracellular barriers while

140

Pharmaceutical Applications of Dendrimers

reducing PAMAM cytotoxicity. Besides, in vivo study in C26 tumor-bearing mice suggested that the prepared non-toxic safe vector could inhibit the tumor growth. It is, though, important to balance the efficacy of higher generations with their toxicity and considering this, many studies focus on 4G PAMAM as drug and gene carriers.

4 PPI dendrimers for cancer therapy Poly(propylene imine) (PPI) dendrimers were the first ones to be reported by Buhleier et al. [72a] in 1978 (Fig. 6.2B). They referred to them as a cascade of molecules. Along with PAMAM, they were also widely studied. PPI dendrimers having 1,4-diaminobutane (DAB) as a core, but can also be synthesized from an ethylenediamine core and other core molecules by a double Michael addition reaction. Propylene imine monomers are used as branching units. Hence their interior contains various tertiary tris-propylene amines, and they form full generations with primary amines as surface ends [73]. The presence of alkyl chains in their branching units imparts them a more hydrophobic interior than PAMAM dendrimers (which contain amide groups in addition to the alkyl chains) of equivalent generations [74]. Kesharwani and co-workers investigated the PPI dendrimers of different generations and their surface modifications [75,76]. Melphalan is encapsulated in PPI of generations 3, 4, and 5, it showed improved tumor growth inhibition and increased survival rates in BALB/c mice inoculated with MCF-7 cells, especially for G4 and G5. Hemolytic toxicity of these dendrimers increased with increase in generations [77]. When dendrimers were surface-modified with folic acid to increase cancer targeting ability, the biocompatibility increased, probably due to shielding of some cationic groups by folate. Still, lower biocompatibility was seen for G5 compared to G3 and G4. Also, folate-modified dendrimers showed even better tumor growth inhibition in MCF-7 tumor-bearing BALB/c mice [76]. In an attempt to increase tumor specificity, and consequently treatment efficacy, Jain and co-workers [78] reported carboxylic acid terminated G4.5 PPI dendrimers conjugated to monoclonal antibody mAbK1 and to encapsulate PTX (mAbK1-PPI-PTX). mAbK1 targets mesothelin protein, overexpressed in some cancers, but not in normal cells. In vitro evaluation in the OVCAR-3 ovarian cell line (which expresses high amount of mesothelin) showed higher cytotoxicity when immunodendrimers were used compared to free drug and PPI-PTX dendrimers. On the ground of in vitro cell cytotoxicity assay and efficient pharmacodynamic as well as pharmacokinetic profile, establish the concept of anticancer bioactive loaded 4.5G modified PPI immunodendrimers mediated targeting to mesothelin receptors, over-expressed in ovarian cancer. Tietz and Co-worker [79] reported small interfering RNAs (siRNAs) offer a great potential to treat so far incurable diseases or metastatic cancer. This study reports a new modular polyplex carrier system for targeted delivery of siRNA, which is based on transfectiondisabled maltose-modified poly(propyleneimine)-dendrimers (mal-PPI) bioconjugated to single chain fragment variables (scFvs). Results suggest that mal-PPI-based polyplexes is a promising avenue to improve siRNA therapy of cancer and a novel strategy for modular bioconjugation of protein ligands to nanoparticles.

Chapter 6 • Dendrimers for anticancer drug delivery

141

Szulc and coworkers [79a] in 2016 reported a promising strategy for leukemia treatment. Cytarabine (Ara-C) has limited efficacy due to drug resistance, inefficient uptake and accumulation of the drug inside cancer cells where it has to be transformed into the active triphosphate congener. PPI dendrimers makes complexes with nucleotide Ara-C triphosphate forms (Ara-CTP). In this work PPI glycodendrimers used as drug delivery devices in order to facilitate the delivery of activated cytarabine to cancer cells. In vitro study has been carried out using 1301 and HL-60 leukemic cell lines as well as peripheral blood mononuclear cells. Results suggested that this combination might be a versatile candidate for chemotherapy. Jain et al. [78] investigated the drug targeting potential of glycyrrhizin (GL) conjugated dendrimers (GL-PPI) and multi walled carbon nanotubes (GL-MWCNTs) toward liver targeting of a model anti-cancer agent, doxorubicin (DOX). Elevated DOX loading was observed in case of GL-PPI-DOX as compare to GL-MWCNT-DOX (43.02  0.64% and 87.26 0.57%, respectively) and parent nanocarriers. GL attachment reduced the haemolytic toxicity of DOX by 12.38  1.05 and 7.30  0.63% by GL-PPI-DOX and GL-MWCNTDOX, respectively. MTT cytotoxicity studies were carried out in HepG2 cell. The IC50 of DOX was reduced from 4.19  0.05 μM to 2.0  0.01 and 2.7  0.03 μM of GL-PPI-DOX and GL-MWCNT-DOX respectively.

5 PPL dendrimers for cancer therapy Poly-L-lysine (PLL) dendrimers are a type of peptide dendrimer used mostly as gene carriers due to their excellent condensation with oligonucleotides (Fig. 6.2C) [80]. PLL dendrimers have good biocompatibility, water solubility, biodegradability, and flexibility, similar to other dendrimers. With peptide bonds in their structures, both their core and branching units are commonly based on the amino acid lysine [81]. PLL dendrimers be different from the general concept of PAMAM and PPI dendrimers since they are mostly asymmetrical. However, they are still precise molecules, with a controlled number of lysine groups branching out from the core, and terminal amine residues [81]. The lysine in the terminal group of PLL contains two primary amines that are frequently modified for better biological performance [82,83]. PLL dendrimers, also have good potential in the drug delivery field and cancer therapy. These are found to be greatly associated with DOX and improve the anticancer activity, while showing fewer adverse events than the free drug [75,83]. Cationic PLL dendrimers of generation six had good antiangiogenic activity in an in vivo B16F10 xenograft model even in the absence of a therapeutic molecule [84]. These dendrimers showed deeper penetration in 3D tumor spheroid models of DU145 prostate cancer cells and B16F10 melanoma xenograft mouse model, and it has been attributed to their small size (usually below 10 nm) and positive charge [26,85,86]. Li and co-workers (Li et al. [87]) reported another study utilizing sixth generation PLL dendrimers carrying DOX. Niidome et al. [87a] found better tumor accumulation of the dendrimers and enhanced tumor inhibition with no apparent toxicity in a mouse model of BALB/cN mice implanted with Colon-26 mouse

142

Pharmaceutical Applications of Dendrimers

rectum carcinoma cells. PEGylation improved tumor accumulation through the EPR effect, while the oligopeptide link created a hydrophobic cavity to increase DOX encapsulation. In a more advanced research stage, a formulation of PEGylated PLL dendrimer containing docetaxel conjugated to their surface is currently in Phase I clinical trials. The formulation developed by Starpharma (Melbourne, Australia), named DEP® docetaxel, presented improved tumor targeting and effectiveness against different solid tumor including breast, ovarian, prostate, and lung compared to Taxotere, the docetaxel formulation used in the conventional chemotherapy [88]. Some other studies are presented in Table 6.2. Jain and co-workers in 2014 reported poly-L-lysine (PLL) dendrimers as antiangiogenic agent for therapy of cancer. In this study folate conjugated poly-L-lysine dendrimers (FPLL) as an efficient carrier for model anticancer drug, doxorubicin hydrochloride

Table 6.2 Type of cancer Brain cancer

Dendrimers based delivery of anticancer drugs in different types of cancers. Type of dendrimer

Drug/gene

Purpose

Reference

PAMAM-Chitosan conjugate Sialic acid/ glucosamine/ concanavalin anchored poly(propyleneimine, PPI) FA and borneol (Bo) modified PAMAM dendrimer Anti- EGFR dendriworms

Temozolomide (Tmz)

For the treatment of brain glioblastoma by TMZ Sialic acid is better than glucosamine can be used as potential ligands with PPI dendrimers. Enhanced uptake of paclitaxel to the brain

[89]

Doxorubicin

Effective delivery to brain glioma

[26, 90a]

SiRNA

SiRNA delivery in vivo 2.5-fold more efficiently than commercial cationic lipids Boron neutron capture therapy (BNCT) for glioma

[91]

Dendrimer lipid hybrid system development for theranostic applications By conjugates s better anti-tumor agents targeting using ligands for ovarian cancer

[93]

Boronated-PAMAM dendrimer

Ovarian cancer

Lipid-dendrimer

Herceptin and diglycolamic acid functionalized (PAMAM) dendrimer PPI immunodendrimers PAMAM peptide dendrimer

Paclitaxel

Anti-epidermal growth Factor monoclonal antibody (Mab) cetuximab Lipid-dendrimer

Cisplatin

Paclitaxel FSHR

Antibody anchored targeting for ovarian cancer FSH33 targeting gynecological malignancy

[90]

[92]

[78, 93a]

[78, 94] [78, 94a]

Chapter 6 • Dendrimers for anticancer drug delivery

143

Table 6.2 Dendrimers based delivery of anticancer drugs in different types of cancers—cont’d Type of cancer Lymphatic cancer

Skin cancer

Type of dendrimer

Drug/gene

Purpose

Reference

Polyamidoamine-akali blue dendrimer Fatty acid graft of PAMAM PAMAM dendrimer

Paclitexel

Lymphatic targeting for treatment and diagnosis Lymphatic absorption increase bioavailability Significant apoptosis in skin tumor

[50]

Phosphorous dendrimer

Lung cancer

Breast cancer

Biotinylated–PAMAM dendrimer PEG-biotin- PAMAM dendrimer Peptide-labeled dendrimer

5-FU antisense oligonucleotide (ASO) Photosensitizers, (methylene blue and rose) bengal cRGD peptides Paclitaxel DNA-plasmid complex

Dendrimer-mediated

Enzyme-glycoprotein

FA-PAMAM dendrimer nanoparticles

siRNA and cis-diamine platinum

Alkyl modified dendrimer Gold nanoparticle coated dendrimer

SiRNA

Phosphorous and PAMAM dendrimer

Curcumin

Polo-like kinase1siRNA

[16] [95]

Evaluation of skin cancer activity effective therapy

[96]

alphavbeta3 targeting nano system development Biotin receptor active targeting of PTX efficiently achieved to biotin receptor Gene therapy found in transfected lung cancer cells growing in RAG1KO mice Enzyme-based circulating tumor cell capture strategy novel method for cancer cell detection Receptor targeted delivery for lung cancer combination therapy of chemo and siRNA Gene therapy

[49]

Smart carrier for curcumin delivery can be used as a potent anticancer agent in targeted for breast cancer Gene delivery for triple negative breast cancer

[97] [98]

[99]

[100]

[101] [102]

[103]

(Dox) for pH sensitive drug release, selective targeting to cancer cells, anticancer activity and antiangiogenic activity. Patil and coworkers in 2011 investigated triblock PAMAM-PEG-PLL dendrimer for the delivery of siRNA. Each component of this triblock nanocarrier showed a noteworthy role and performs various functions like tertiary amine groups in the PAMAM dendrimer work as a proton sponge and plays a vibrant role in the endosomal escape and cytoplasmic delivery of siRNA; likewise PEG, a connecting linker to PLL and PAMAM dendrimers gives nuclease stability and protects siRNA in human plasma. PLL also acts as penetration enhancer has and with primary amines form polyplexes with siRNA through electrostatic interaction. Toxicity of PLL and the entire triblock nanocarrier PAMAM-PEG-PLL was reduced due to conjugation of PEG and PAMAM. This outcome suggests that triblock

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nanocarrier system is well taken up by the cancer cells and exhibited significant plasma stability in human. Rahimi and co-workers in 2016 reported that poly(L-lysine) (PLL) dendrimer are amino acid-based macromolecules and its application as drug delivery agents. Their branched structure allows them to be functionalized by various groups to encapsulate drug agents into their structure. Molecular simulation was done on PLL dendrimers of different generations. By calculating the moment of inertia and the aspect ratio, the formation of spherical structure for PLL dendrimer was confirmed. The calculated radial probability and radial distribution functions confirm that at pH 7, the PLL dendrimer has more cavities and as a result it can encapsulate more water molecules into its inner structure. Ryan et al. [103a] formulated doxorubicin loaded a PEGylated poly-lysine dendrimer (size 12 nm), a PEGylated liposome (100 nm) and different pluronic micellar formulations (5 nm) and studied against lymph node metastases in rats. Plasma and lymph pharmacokinetics were analyzed by compartmental pharmacokinetic modeling in S-ADAPT, and Berkeley Madonna software. Dendrimer formulation significantly increased the recovery of doxorubicin in the thoracic lymph (both intravenous and subcutaneous dosing) when compared to the micellar system.

6 Conclusion Unlike linear polymers, dendrimers show a precisely controllable architecture with tailormade surface groups. The branches of dendrimers can be adorned with a wide variety of molecules that can be utilized for passive entrapment and eventual release of drugs or other cargoes. The molecular structure of dendrimers can be fine-tuned, due to symmetrical shape and many peripheral functional groups, controlled molecular weight, and nanometer size. Dendrimers are excellent nanocarriers with good fluid mechanic performance, versatility, and strong adsorption ability. In cancer chemotherapy, these desirable size-based features are strengthened by the enhanced permeability and retention (EPR) effect that advances the delivery of macromolecules to tumors. The ability to append more than one type of functionality to a dendrimer allows the inclusion of ligands intended to bind specifically to cancer cells in the design of a multi-functional drug-delivery nanodevice.

Acknowledgment Authors Acknowledge to Hon’ble Vice Chancellor, Dr. APJ Abdul Kalam Technical University, Dr. Vinay Kumar Pathak for support & encouragement.

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7

Cancer-targeted chemotherapy: Emerging role of the folate anchored dendrimer as drug delivery nanocarrier

Chitra Rajania,*, Pooja Borisaa,*, Tukaram Karanwada,*, Yogeshwari Boradea,*, Vruti Patela,*, Kuldeep Rajpootb, Rakesh K. Tekadec a

NATIONAL INSTITUTE OF PHARMACEUTICAL EDUCATION AND RESEARCH (NIPER) A H ME D A B A D, AN IN S T I T UT E O F N A T I ONAL IMPORTANCE, DEPARTMENT OF PHARMACEUTICALS, MINISTRY OF CHEMICALS AND FERTILIZERS, GOVERNME NT OF INDI A, GANDHINAGAR, INDIA b PHARMACEUTICAL RESEARCH PROJE CT LABOR AT OR Y, INST IT UT E O F PHARMACEUTICAL SCIENC ES, GURU G HASIDAS VISHWAVIDYALAYA (A CENT RAL UNIVERSITY), BILASPUR, INDIA c DEPARTMENT OF MATERIALS ENGINEERING, INDIAN INSTITUT E O F T ECHNOLOGY-J AMMU, INDIA

1 Cancer world “the making of modern disease” I know a person named “DD” who lived in a small town near Bhopal, India. He frequently complains of a feeling of tiredness, persistent fatigue which has been occurring with him for a week. He was also losing his weight day by day. He kept ignoring these symptoms, as he thought it would get alright by its own. But 1 day, as he was getting ready for his job, he encountered sudden fever and chills. He was completely unaware of the fact, what was wrong with him, and what kind of disease he was suffering from. After observing his condition, his elder brother took him to a community hospital. The doctor diagnosed and gave him some medications including some nutritional supplements and told him to visit again after 8 days. But, after taking the prescribed medicine, his condition did not improve at all, even got worse. The doctor was unable to find an exact reason for the condition of the patient. Therefore, advised him to undergo some diagnostic tests and referred him to a cancer specialist. DD radially went to the nearest cancer hospital for the examination. His blood sample was taken and examined by a cancer specialist, which revealed that he was suffering from leukemia.

*Authors having equal contribution and can be interchangeably regarded as the first author. Pharmaceutical Applications of Dendrimers. https://doi.org/10.1016/B978-0-12-814527-2.00007-X © 2020 Elsevier Inc. All rights reserved.

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He was then recommended to immediately get admitted to the hospital and undergo chemotherapy. In this span of few days, DD not only entered the hospital ward but in a new world called “world of cancer”. And it was not DD, but his whole family entered a dark phase of life, both mentally and economically. The thought of cancer has always been terrible, but, that fear was not centrally situated in our mind, as it is now. This is the prime reason why most newspapers have started calling cancer as “the modern disease per excellence”. Cancer has become the price of modern life and it was recently estimated by American Cancer Society that by 2019 about 42% of all men and about 50% of all women will start dealing with a different type of cancer once in their lifetime (American Cancer Society, Cancer Facts and Figures 2019). This is the reason most statisticians ranked cancer as the second leading cause of death globally.

1.1 What is cancer? Cancer is a group of disease in which abnormal cells grow without any control as well as its cell division is also faster than normal cells. In addition, these abnormal cells destroy body tissue. According to the prevailing theory of cancer, the normal cells convert into cancerous, which leads to abnormal cellular, molecular, and biochemical networking of normal cells. These cancerous cells also spread or metastasize from the primary location to a secondary location in the body [1]. However, the group of cancerous cells that forms an abnormal mass of tissue is called solid tumor whereas the cancerous cells which do not form the masses of tissue are termed as leukemia or cancer of the blood. The tumor is classified into two types; first is a malignant tumor, which gets spread in the body or nearby tissue. It occurs when a section of malignant tumor sloughs off and travels from primary location to a secondary location through the lymphatic system or blood and grows into a new tumor [2]. Another type of tumor is a benign tumor which does not spread and its size is also not as big as that of a malignant tumor [3]. A malignant tumor is a cancerous and harmful tumor, but the benign tumor is not cancerous. The benign tumor becomes harmful if they exert pressure on vital organ [4]. Gene mutation which occurs due to heritance or non-heritance factors is also responsible for the proliferation as well as the growth of abnormal cells [5].

1.2 Causes of cancer In the 1800s, three fundamental theories highlighting the causes of cancer were proposed, which include; (a) cancer caused due to chronic irritation proposed by Virchow, (b) displaced embryonal tissue leads to cancer hypothesized by Cohnheim, and (c) Infectious agent (e.g., viruses, bacteria, etc.) causes cancer summarized by others. IARC (International Agency for Research on Cancer) was introduced in 1965 to examine various causes of cancer. There are two main causes that lead to cancers: (a) environmental factor and (b) hereditary causes. The main environmental factors responsible for cancers are infections, tobacco, drinking alcohol, excessive exposure to certain chemicals, diet and obesity, and radiation. Inheritance in several genes, like oncogenes, tumor suppresser gene also responsible for developing cancer [6].

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1.3 Origin of the word cancer The Greek physician Hippocrates (460–370 BC) studied most of the diseases which produce mass (onkos). They first described word karkinos for non-healing and ulcerative lumps and karkinomas for non-ulcerative lumps. Therefore, the origin of the word cancer is credited to Hippocrates, who is also referred to as the “father of medicine” [7]. In the Greek language, this word implies crab which can be literally related to this disease because it spreads, and the crab’s finger looks like spreading projection. The Roman physician Aulus Cornelius Celsus (28–50 BC) later translated this Greek word crab into Latin word cancer [8].

1.3.1 The oldest description of cancer The history of cancer has been mentioned in very earlier, in 1500–1600 BC. Ancient Egypt papyri wrote about cancerous cells in dinosaur fossil and human bone. In the 19th century, Georg Ebers and Smith papyri (ancient Egyptian) described the pharmacological, surgical, and magical treatment. Imhotep the physician architect written, “Smith papyrus” in which they described breast cancer that was first referenced in the 30th century BC and this type of cancer used to spread over a breast, which was cool in touch, bulging, and incurable. Further, around 1000 BC, the growth of abnormal cells (cancerous) were also found in Peruvian and Egyptian mummies. In steppes of southern Siberia, nearly 2700 year ago, a disseminated cancer was noticed in Scythian king, who was of 40–50-year-old. In addition, the first case of cancer that was documented observed in the 2700 year ago. Moreover, the discoveries of cancer started from the Hippocrates and ended with radiation and medical oncology [7]. Eventually, the first case of occupational cancer was reported in chimney sweeper (Percivall Pott) in 1775 [9,10].

1.4 Biology of cancer Cancer is a group of disease in which a cell’s inherent ability to divide, grow, multiply and mature normally, is due to a mutation in somatic cells thereby interrupting the homeostasis. Gene which suppresses tumor and prevents cancer is known as anti-oncogenes. Oncogenes sometimes referred to as proto-oncogenes undergo activation in cancer cells, which is responsible for the uncontrolled division of cells. Owing to the activation of oncogenes, the development of cancer occurs by: (a) Amplification of gene resulting in the enhancement of normal protein formation, (b) Fusion protein with high biological activity will form by translocation of a segment of DNA from the location of one chromosome to another. Gene which suppresses tumor undergo inactivation in cancer cells responsible for apoptosis programming and cell cycle progression control. The major cancer cells features (Hallmarks) are the development of limitless replicative potential, independence of growth factor, upregulation of gene associated with metastasis and tissue invasion, evasion of programmed cells death, capacity for sustained angiogenesis, insensitivity of antigrowth signal (Fig. 7.1) [11].

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Independence of Growth factor

Insensitivity to antigrowth signal

Evading apoptosis

Tissue invasion and metastasis

Tumour vasculature

Sustained angiogenesis Limitless replicative potential Tumour cell

Stroma cell

Invading immune cell

FIG. 7.1 Alteration in the physiology of the tumor.

Cancerous cells have a different cellular environment than normal tissue: •

Rate of aerobic glycolysis is higher in tumor cells than the normal cells and thus increase the lactic acid concentration leading to a decrease in pH up to 6.8. In case of a solid tumor, extracellular pH is lesser than intracellular while in case of normal tissue it is reversed where the extracellular pH of normal tissue is higher than intracellular [12].

1.5 Tumor vasculature Tumor vasculature varies from normal vasculature in significant degrees which can take advantage of while designing targeted delivery (Fig. 7.2). Normal vasculature consists of well organized, evenly distributed blood vessels that are well regulated, along with wellorganized lymphatic vessels. It allows the (ample) amount of oxygen as well as other nutrients to be perfused throughout all cells. While in tumor vasculature, not only the growth of cancerous cells is uncontrolled but the overexpression of pro-angiogenic factor is also observed where it causes the development of a network of the irregular unorganized blood vessel. Although, the identification of venules and arterioles in these blood vessels is difficult. However, tumor vasculature exhibit abnormal structure dynamics and it has highly permeable, immature, and tortuous vessels. Further, blood vessels not only exist as an

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Normal cells which line the surface of the airways/gut/milk duct of breast/pancreatic duct/uterus/cervix/skin /mouth/bladder/etc.

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Primary tumour Cancer cells (looks abnormally)

Nucleus inside cells contain genes made from DNA

Layer of heathy tissue

One cell acquires enough mutation to become cancerous

Cancerous cell will divide at accelerated weight and displace healthy tissue Cancer cells invade into local tissue Lymph channel

Dipper tissues such Some cells as muscle layer in Tiny blood vessel break from the gut or bladder/fat (capillaries) primary tumour beneath the skin /etc.

New blood vessel simulated to grow Supply cancer cells to blood supply

Cancer cells spread to other areas of the body via blood vessels or lymph channels

Eventually, normal cells will be eliminated and organ function is compromised

Tumour cells will continue to grow and die as steady state size exists until new blood vessels forms

FIG. 7.2 Schematic showing tumor vasculature.

irregulated shape but also shows uneven diameters with abnormal bulges. The function of tumor vasculature to remove waste product through the lymphatic system and transport of nutrients is lost Immature tumor vessels leads to inadequate supply of oxygen due to the abnormal bulges and irregular geometry, thereby, generating a zone of microregional hypoxia. Moreover, it can show resistance against chemotherapy and radiotherapy which means it narrows down the anticancer treatment strategies. Hence, using the unique properties exhibited by tumor vasculature, some targeted treatment-based tactics can be utilized to combat these associated hurdles [13]. The tumor microenvironment is important for new strategies regarding cancer treatments. Tumor extracellular environment has more acidic pH than the normal cells [12]. For the formation of new blood vessels in a solid tumor, it uses the previously well-established vessel bed process, also known as angiogenesis. However, angiogenesis is different than the vasculogenesis (i.e., the formation of vessels during embryonic stages) [2,14]. Neovascularization in tumor cells imparts excessive permeability to its vessels, while the absence of smooth muscles in blood vessels of tumor thickens the basal membrane thus, resisting the flow of blood. Lack of vascularization and hyperpermeability results in hypoxia which further promotes angiogenesis [15]. This resulting hypoxia not only contributes to immunosuppression but also another parameter like fibrosis, tumor invasion, inflammation, metastasis, and treatment resistance [16]. Thus, limiting the drug delivery and making it tedious. Physical scientist and Engineers studied on a physical, cellular and molecular mechanism to overcome these abnormalities and barriers. Moreover, scientists have re-engineered tumor microenvironment by rectifying the tumor vessels to overcome these barriers, so that the efficacy and delivery of treatment can be improved [17].

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1.6 Physiology of tumor structure The cancer is a progressive generation of tumor which involves an increase in the population of the abnormal cell due to uncontrolled growth of cancerous neoplastic cells. Depending on the location of cells, the type of tumor is defined. The tumor grows in minimum three tissue compartments i.e., the original site, mesenchyme of primary site, and distant mesenchyme. In addition, tumor growth occurs in different formats such as cell suspension and solid tumor. All tumors require stroma for removal of wastage while nutrition requirement of the stromal component is interposed between the malignant cell and normal host tissue. It is composed of fibroblasts, inflammatory cells, plasma, blood vessels, and may represent as much as 90% of the mass of a tumor. Stroma plays a critical role in angiogenesis in the growth of solid tumors. The angiogenesis response is essential for tumor growth. The tumor formation is mediated by various responses like the immune system and growth factor. However, this kind of abnormality mostly occurred at the vessel geometry as well as in the wall. Moreover, the abnormality information and geometry of the vessel and venule is responsible for the incomplete formation of the basement membrane of the vessel. However, the formation of the tumor shows some possibilities like changes in vessel tortuosity, elongation, as well as abnormal and heterogeneous capillary density. Although, the tumor nature depends on its stages and cell type of tumor [18].

1.7 Stages of tumor development At the cellular level, development of cancer involves many steps in which the major cause is the mutation of cells ultimately leading to an increase in their capacity for proliferation, survival, and invasion. The process starts with the alteration in the genetic material which leads to cell proliferation, and thus increase in the mass of the abnormal cells. This process is known as tumor initiation and tumor progression. There may be a generation of further mutations in the tumor cell which may provide the cell with certain advantages making them more cancerous. The daughter cells of cells bearing such mutations will become dominant in the population (Fig. 7.3). This is known as the clonal selection and new clone is produced. This process of clonal selection continues and thus the tumor growth continuously increases [19].

1.8 Classification of cancer Cancer can be classified on the basis of the type of tumor cell as well as on the basis of the organ where the tumor is present. There are major five types of cancer on the basis of the tumor cell and it can be classified as carcinomas, sarcomas, lymphomas, myeloma, and leukemias (Fig. 7.4). (i) Carcinomas are cancer originating in the epithelial tissue like skin or tissues lining the internal organs. They are the most common types of cancer consisting of about 85% of cases. Depending upon the type of epithelial cell it affects it can be classified as squamous cell carcinoma, transitional cell carcinoma, and basal cell carcinoma.

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FIG. 7.3 Stages of tumor development.

FIG. 7.4 Prevalence of different types of cancer.

(ii) Sarcomas are the tumors which start in connective tissues like bone, cartilages, tendons and other fibrous tissues. Sarcomas constitute about 1% of cases. They are generally classified into two main types osteosarcoma which is sarcoma of bone and soft tissues sarcomas. Osteosarcoma starts in the bone cell osteocyte. Soft tissue sarcoma occurring in the cartilage is known as chondrosarcoma and in the muscle, it is known as rhabdomyosarcoma. (iii) Leukemia is characterized by excessive production of white blood cells by the bone marrow. Such white blood cells are defective in structure and lack proper function. They constitute about 3% of cases.

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(iv) Lymphomas are the cancer of lymphatic system characterized by the abnormal growth of the lymphocytes, these abnormal lymphocytes get accumulated in the lymph nodes, bone marrow, spleen, and other places and lead to the formation of a tumor. They constitute about 5% of cases of cancer. (v) Myelomas are cancer that starts in the plasma cells, a type of white blood cells produced in the bone marrow responsible for the production of the immunoglobulins. They constitute about 1% of the cases of cancer. Cancer can also be classified on the basis of the organ it attacks as shown in Fig. 7.5.

1.9 Prevention of cancer As per the World Health Organization (WHO), about 30–50% of cases of cancer can be prevented. For cancer prevention WHO has highlighted several points:

FIG. 7.5 Different types of cancer-based on the organ where it occurs.

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1.9.1 Tobacco Tobacco is responsible for approximately 6 million deaths worldwide due to cancer and several other diseases. Out of 7000 chemicals present in the tobacco smoke, 250 have been reported to be harmful to human health and 50 are known carcinogenic. It can cause several types of cancer such as lung, esophagus, larynx, mouth, throat, kidney, bladder, pancreas, stomach, etc. Second-hand smoke is also known as environmental tobacco smoke can also cause lung cancer in non-smoking adults. Oral tobacco has also been found to cause oral and esophageal cancer. Hence it is necessary to avoid the use of tobacco for the prevention of cancer.

1.9.2 Alcohol Use of alcohol is a risk factor for cancer such as oral, pharynx, larynx, esophagus, liver and colorectum. Alcohol needs to be avoided for the prevention of cancer.

1.9.3 Physical inactivity, dietary factors, and obesity There is an established link between obesity and cancer such as the esophagus, colorectum, breast, endometrium, and kidney. Diet rich in fruits and vegetables may have protective effects against such cancers. Regular exercise, proper diet, and weight control can help in the prevention of cancer.

1.9.4 Infections As per the WHO 2012 report, about 15% of cancers were due to infection by microorganisms such as Helicobacter pylori, human papillomavirus, hepatitis B and C and Epstein Barr virus. Such infections can be prevented by vaccines. Vaccines for Hepatitis B and HPV have been found to reduce the risk of liver and cervical cancer respectively.

1.9.5 Environmental pollution Pollution of air, water, and soil with the carcinogens may lead to cancer. Hence it is necessary to control the pollution for the prevention of cancer.

1.9.6 Occupational carcinogens Exposure to the carcinogens in the working environment may lead to the development of cancer like mesothelioma which is cancer of the outer lining of the lung and the chest cavity is caused by the exposure to the asbestos. For prevention of occupational cancer, it is necessary to provide the working zone that consists of suitable gowning and prevent their direct contact with the harmful chemicals.

1.9.7 Radiation Exposure to all the types of the ionizing radiation may lead to the development of solid tumor as well as leukemia. Ultraviolet radiation particularly solar radiation is harmful to human beings causing basal cell and squamous cell carcinoma and melanoma. Radiation is used in medicine as well as in the diagnosis, however, its inappropriate use may

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cause the development of cancer. Hence for the prevention of cancer, it is necessary to check the exposure of radiation to the various tissues [20].

1.10 World cancer day World cancer day is observed by UN members annually on 4th February to spread awareness of cancer and encourage its prevention, detection, and treatment. It was founded by the Union for International Cancer Control (UICC) to support the goals of the World Cancer Declaration which was written in 2008. For the year 2016 to 2018, the theme was “WE can. I can” and the theme for the year 2019 to 2021 is “I Am, and I Will”.

1.11 Diagnosis of cancer The primary challenge in the management of cancer is an accurate diagnosis of cancer. Early detection of cancer is important for successful elimination otherwise it becomes difficult to treat the late stage cancer. The currently used methods for diagnosis of cancer are endoscopy, blood tests, imaging techniques like MRI, pap test. Nowadays, advances and new technologies are also employed such as immunofluorescence, DNA/RNA analysis, immunohistochemistry and fluorescent in situ hybridization (FISH). But the non-specific nature of symptoms of cancer produces difficulty in diagnosis. In some cases, the patient is asymptomatic as well as in many cases the early signs and symptoms are ignored by patients which causes cancer to spread. Hence the early diagnosis of cancer with the advanced techniques is essential to in successful cancer treatment [21,22].

1.12 How cancer is treated? Various treatment strategies and technologies are employed for cancer management. The type of treatment strategy depends on the stage of cancer, health condition, age. Mostly the combination of therapies is used in treatment. Some of the treatment strategies are as follows:

1.12.1 Surgery Surgery involves the surgical removal of the tumor tissue with the help of various techniques. Surgery is the oldest approach to the treatment of cancer. Developments in the surgical techniques non-invasive imaging, anesthesia, and postoperative care have significantly reduced the operative death rates. The various techniques used in this treatment consists of (a) cryosurgery of the cancers such as esophageal, prostate, oral cancers, (b) laser surgery mainly for oral cancers, (c) photodynamic therapy for oral and laryngeal cancers, and (d) hyperthermia therapy for breast, head, and neck lymph node metastases, cervical cancer, etc. [23–25].

1.12.2 Radiation therapy Radiation therapy is an important treatment strategy along with the surgery and chemotherapy. Radiotherapy involves the use of high energy radiation also known as ionizing

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radiation (X-rays or gamma rays or particle radiation) that damages the genetic material (DNA) of the cell so that they become unable to divide and multiply further. Even though radiotherapy affects both normal and the tumor cells, the radiation therapy aims at maximizing the radiation dose to the tumor cell and minimizing the exposure to normal cells. Normal cells have the ability to recover faster than the tumor cells. As the cancer cell is not that much efficient in repairing the damage, the radiation therapy results in the differential killing of tumor cells. The radiation is delivered to the target tissue in two ways; (a) External beam of radiation in which the high energy rays are exposed from outside of body to the tissue and (b) Internal radiation is also known as brachytherapy in which the radiation is delivered inside the body with the help of radioactive sources, catheters to the tissue. Radiotherapy is usually used along with the surgery, immunotherapy or chemotherapy. Radiation shrinks the tumor when used before the surgery and it destructs the microscopic tumor cells that could have remained after surgery. The various techniques of radiation therapy used are fractionation, 3D conformal radiotherapy, intensity modulated radiation therapy (IMRT), image-guided radiotherapy, stereotactic body radiation therapy (SBRT), etc. [26,27].

1.12.3 Immunotherapy The scientist has been trying from more than half a century to use the immune system of the body against cancer. But the tumors have the ability to escape, beat the normal immune system of the body which is the major barrier in this treatment. Recent immunotherapy aims at activating the body’s immune system in order to recognize and attack tumor cells. The therapy includes the administration of monoclonal antibodies against antigens expressed on tumor cells such as HER2 receptor in HER2-positive breast cancer, vascular endothelial growth factor, epidermal growth factor receptor in colorectal, breast, head and neck cancer, etc. Apart from the antibodies, another immunological reagent such as recombinant interleukin-2 (denileukin diftitox), recombinant interferon is also used for immunotherapy of cancer. The various advancements in immunotherapy are immune checkpoint therapy, adoptive immunotherapy, neoantigen for immunotherapy, cancer vaccine therapy [22].

1.12.4 Hormone therapy Hormone therapy is important and major therapy for the treatment of hormonesensitive cancers such as breast, endometrium, and prostate cancer. This therapy comprises of external administration of the particular hormone or the drugs that are antagonists of such hormones. As the steroid hormones are involved in the gene expression in certain tumor cells, altering the levels of such hormones or by inhibiting them can inhibit the growth of cancer cell and even lead to cell death. The major strategies used in this therapy are; (a) Estrogen receptor-targeted therapeutics in which either selective estrogen receptor modulators (SERMs) or estrogen receptor down-regulators (ERDs) are used to treat various cancers like breast, prostate, colorectal, endometrial, ovarian cancers as estrogen is involved in development as well as progression of this

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diseases. The drugs used in this approach are tamoxifen, raloxifene, idoxifen, fulvestrant, etc. (b) Progesterone targeted therapy for endometrium cancer in which progestin therapy is given [28]. Androgen deprivation therapy (ADT) is used to treat prostate cancer which includes the luteinizing hormone-releasing hormone agonist (LHRa) or the newer LHRH [29].

1.12.5 Gene therapy Gene therapy comprises of delivery of genetic material into the target cells or tissues with the purpose of getting a therapeutic outcome. Thus, gene therapy includes the cancer vaccines, targeting viruses to tumor cell for lysis and cell death, decreasing the blood circulation to cancer cells, as well as introducing the genes into the tumor cell which results in death or correction of the abnormal phenotype. The introduction of genetic material into the target cells or tissues is done by the methods such as (a) physical methods e.g., electroporation, gene gun, ultrasound method, (b) viral method which uses the virus as a vector to deliver the gene into the cells, (c) non-viral method uses synthetic carriers like nanoparticles, liposomes, etc., and (d) bacterial/yeast method that uses different bacteria and yeast to deliver the genetic material [30,31].

1.12.6 Chemotherapy Chemotherapy includes the use of drugs or chemical agents in order to eradicate the tumor cells. This agent acts against the dividing cells and hence have a major effect on the rapidly proliferating tumor cell rather than the normal cells. But also due to such action against dividing cells, these agents have major side effects on the GIT mucosa and bones as their cell are also rapidly dividing [28,32]. Paul Ehrlich (German scientist) coined the term “chemotherapy”. Chemotherapy had a history since the early 20th century but its actual use to treat cancer began in the 1930s. It was noticed that decreased in leukocyte count in soldiers were observed during first and second world war as they exposed to mustard gas. After this, in 1943 nitrogen mustard was used by Gilman as a chemotherapeutic agent to treat lymphoma [33]. The various classes of chemotherapeutic agents used in cancer treatment are: (i) Platinum-based compound for e.g., cisplatin, carboplatin, and oxaliplatin. (ii) Alkylating agents for e.g., cyclophosphamide, nitrogen mustard, carmustine, and busulfan. (iii) Antimetabolites for e.g., cytarabine, mercaptopurine, methotrexate, and gemcitabine. (iv) Natural agent for e.g., vinca alkaloids and taxanes. (v) Antitumor antibiotics for e.g., actinomycin D, mitomycin C, and bleomycin [28,34,35]. Currently, the first line treatment in chemotherapy comprises of the cisplatin compounds along with the third degeneration drugs like gemcitabine, taxanes, and vinca alkaloids. But there is severe short term as well as long term side-effects of the chemotherapeutic agent

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which of major concern in cancer treatment. This side effect includes nausea, vomiting, ulceration, diarrhea, central and peripheral neurotoxicity, bone marrow depression, hair loss, etc. Hence along with this chemotherapeutic drug combination of symptomatic treatment is given to reduce these side effects [35,36].

1.13 Current approaches to cancer therapy (i) In addition to the conventional surgical, radiation and chemotherapeutic approach, the immunotherapy in combination with this conventional model has gained significant use nowadays. For example, the combination of cancer vaccines with the chemotherapeutic agents leads to an increase in the efficacy of the chemotherapy. Also, the checkpoint blockade therapies are the advancement in immunotherapy that further improved the therapeutic effects of treatment. (ii) Use of nanotechnology for the targeted delivery the drugs have resulted in great clinical outcomes in the cancer treatment and thus it has now wide application in treatment. (iii) Antimicrobial proteins (AMP) having anti-tumor activity also have application in cancer therapy as this protein interacts with the supramolecules such as DNA, proteins, membrane phospholipids, ribosomes thereby killing the tumor cells. (iv) Various ligands are now used to specifically target the tumor cells, for example, folic acid as a ligand to target the solid tumors, hyaluronic acid for targeting breast cancer, etc. (v) Enhanced permeability and retention (EPR) effect is also widely used for delivery of the drug to the tumor tissue. [37–40].

1.14 Barriers in cancer therapy 1.14.1 Physiological barriers The various physiological barriers affect the efficient targeting of the drug to tumor tissue. Most of the drug delivery systems such as nanocarriers use passive targeting by utilizing the EPR effect which is due to the abnormal leaky vasculature. But the therapeutic effectiveness is affected by the heterogeneity of EPR effect within as well as between the tumors. The variable gaps between the endothelial cells of blood vessels lead to nonuniform extravasation of nanocarriers into tumors. As a consequence of such abnormal endothelium of vessels, there is leakage of fluid and osmotic proteins into the interstitial space. This leads to the increased osmotic pressure in interstitium whereas reduced hydrostatic pressure in a vessel that further impact the transport of drug to the tumor tissue. The second barrier after the endothelium of vessels in the tumor extracellular matrix which is denser than the normal tissue causing high intra-tumoral fluidic pressure. This creates a barrier for the drug to penetrate into tumor tissue. Apart from this, the abnormal nutrient metabolism results in hypoxia and oxidosis which are responsible for the resistance to chemotherapy. Also, the opsonization and subsequent identification of the

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nanocarriers and other drug delivery system by the phagocytic cell in the blood circulation causes rapid clearance of the drug from circulation that limits the efficacy of the drug. Thus, these physiological barriers like tumor heterogeneity, abnormal pressure in interstitium and vessels extracellular matrix, opsonization are responsible for poor drug penetration into the tumor tissue [41–43].

1.14.2 Cellular barriers The various cellular barriers affect the targeting of drug to the tumor cells. The primary obstacle to the delivery of the drug is the plasma membrane which limits the passage of drug into the cell and the drug delivery system must interact with the membrane in order to get internalized into the cell which depends on the properties of drugs like surface charge, hydrophobicity, and size. The charged particles usually have greater tendency to interact with membrane than an uncharged particle, for example, uncharged PEGylated nanoparticles have decreased membrane interaction due to the steric hindrance which leads to clustering of nanoparticles around the membrane inhibiting the entry of nanoparticle into the cell. After internalization of the drug into the cell, the further barrier is endosome vesicular barrier. Many drugs undergo internalization by endocytosis pathway that leads to the accumulation of drug to nontarget cell organelles and lysosomes in which the drug get degraded especially the gene and peptide-based drugs. Also, the acidic pH inside the tumor reduces the intracellular concentration of drug that is weakly basic. Efflux transporters are also the major barrier in drug internalization in tumor cells. Solid tumors have highly expressed P-glycoproteins (efflux transporter) that efflux out the drugs leading reduced drug concentration inside the cell. This multidrug transporter that efflux out many drugs is one of the reasons behind multidrug resistance in chemotherapy. Efficacy of drug is also affected by the alteration of molecular targets, for example, the alteration in topoisomerase enzyme results in resistance to drugs that act on this enzyme. Increase in DNA repairing enzyme in tumor cells also leads to drug resistance and hence, decrease in drug efficacy [41,43–46].

1.14.3 Pharmacokinetic barriers Many anticancer drugs show inter-individual variation in pharmacokinetics behavior and also the narrow therapeutic index. Hence, the knowledge of the change in pharmacokinetics in cancer is of great concern in treatment. Because of such high variation in pharmacokinetic behavior, it is challenging to understand the responses of the patient to drug and may lead to increased risk of toxicity or sub-therapeutic effect in patients. Most of the anticancer drugs are administered through the i.v. route and hence shows less pharmacokinetic variation due to absorption. But in the case of orally administered drugs, absorption is the first barrier that impacts the bioavailability of the drug. Also, many agents undergo the first-pass metabolism that further reduces the bioavailability. The intestinal P-gp efflux transporter and intestinal metabolizing enzyme are also the hurdles to the oral drugs.

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The next hurdle to the drug is variation in distribution because of the protein binding variability. Some patients with liver dysfunction may lead to hypoalbuminemia which needs to be considered for drugs with high protein binding. Cancer patients show variability in metabolism too. It has been observed that the levels of cytochrome P450 enzymes are reduced in cancer patients as compared to normal individual hence the drugs that are substrate for such enzymes shows pharmacokinetic variation and may lead to toxicity of drugs with narrow therapeutic index or may result in reduced efficacy of drugs which get activated after metabolism by this enzymes. The variation in biliary and renal excretion is also observed in a cancer patient that results in decreased excretion of some drugs [47,48].

1.15 Limitations of conventional cancer therapy The conventional treatment strategies for cancer have a clear limitation that is responsible for the failure of the therapy and hence needs to be overcome for effective treatment. The conventional therapies are successful in treatment if given in the early stage but the asymptomatic nature of most cancer, as well as insufficiency of diagnosis, leads to metastasis of the tumor to other tissues without medical treatment. The conventional anticancer drugs act by killing the rapidly proliferating cells and the main problem with these agents is that they are unable to differentiate between the normal and tumor cells. Because of this non-specificity, the chemotherapeutic agent also acts on the healthy rapidly dividing cell of bone marrow, hair follicles, GIT, macrophages and thus results in serious side effects of this drug like myelosuppression, alopecia, inflammation of GIT mucosa, immunosuppression, etc. Some drugs remain in blood circulation for a short time because of the clearance by macrophages. Also, most of the anti-cancer have poor solubility thus very less penetration of such drugs through the membrane is the limitation of conventional therapy. The conventional treatment strategies are unsuccessful in eliminating cancer stem cells that lead to the recurrence of cancer in most cases. Hence, new treatment strategies with the aim of selectively targeting the tumor tissues are of highest concern to overcome the limitations of conventional therapy [21,27,49].

1.16 Tumor targeting via a novel drug delivery system 1.16.1 Passive targeting Passive targeting to the cancer cells is done by the enhanced permeability and retention (EPR) effect exhibited by tumor cells. With the increase in the stage of cancer, the tumor size also tends to increase. Increase in the size beyond 2 mm, the tumor will require independent blood supply which leads to the process of angiogenesis. Also, with the increase in the tumor size the solid tumors tend to compress the blood vasculature which decreases the blood supply to the tumor leading to the generation of the hypoxic state in the tumor. As a consequence of this, the tumor cells tend to increase the production of the angiogenic factors like vascular endothelial growth factor (VEGF) which will lead to the process of angiogenesis. These new blood vessels which are formed possess several defects in their

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structure like they lack smooth muscle layer and pericytes also they possess larger fenestrations compared to the normal cells. There is also an abnormal production of vascular growth factors and factors which enhance vascular permeability like nitric oxide, prostaglandins, and bradykinin [50]. Nanocarriers can travel through this gap and reach the tumor site. This phenomenon is known as enhanced permeation. It has also been observed for the tumor tissue that lacks optimum lymphatic drainage. This happens due to the elevation in interstitial fluid pressure, dense stromal compartment, fibroblast, smooth muscle cell, and macrophages. This process leads to the retention of the nanocarriers inside the tumor mass. This entire phenomenon of accumulation of nanocarriers at the tumor site and their subsequent retention is known as the EPR effect and is one of the major mechanisms reported for the targeted therapy of cancer. The most important factor which governs the occurrence of the EPR effect is the size of the nanocarrier. Nanocarrier with the size range of 10–200 nm can only accumulate by EPR effect [51].

1.16.2 Active targeting Active targeting means guiding nanocarrier specifically to the cancer cells. Cancer cells differ from normal cells due to the overexpression of certain receptors. The compound specific to these receptors can be attached to the nanocarrier for active targeting to the cancer cells. These ligands will identify the targets on the cancer cell surface will release the drug at that site only. The active targeting process can be distinguished in two main types, targeting cancer cells and targeting tumor endothelium [52]. Targeting to the cancer cell directly is generally preferred for delivery of siRNA, DNA, and proteins inside the cell. The receptors which are overexpressed in case of cancer are transferrin receptors, folate receptors and epidermal growth factor receptor (EGFR). Targeting these receptors can pave the way for effective tumor targeting. Certain glycoproteins are also overexpressed in case of cancer which can also be used for targeting. The transferrin receptor is a type of serum glycoprotein responsible transport of iron through the blood and is involved in iron homeostasis. Transferrin receptor is almost 100 times more overexpressed on cancer cells as compared to normal cells and hence can act as an effective target in cancer treatment [53]. The folate receptors are the most studied targets for cancer targeted therapy. It allows binding of folic acid, conjugates of drug with folic acid and folic acid conjugated nanocarriers. As the tumor cells are actively dividing, there is also an increase in the process of DNA replication which will ultimately require nucleotides. Folic acid plays a major role in the synthesis of the nucleotides, thus, due to increased consumption of folic acid inside the cancer cells, it will express a number of folate receptors which can be used as targets for cancer targeting [54]. EGRF is a type of tyrosine kinase receptors involved in the process of angiogenesis invasion and metastasis. It is generally overexpressed in breast cancer. Another receptor which is overexpressed in breast cancer is human epidermal receptor 2 (HER 2). Lectins are the non-immunological proteins which can bind to the carbohydrate portion of the glycoprotein. Nanocarriers can be conjugated with the lectins

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for targeted therapy known as direct lectin targeting. In reverse lectin targeting the carbohydrates are conjugated with the nanocarriers and targeted towards lectin [53]. The tumor endothelium targeting involves the destruction of the vascular endothelium thereby the oxygen and nutrient supply to the tumor is cut-off ultimately leading to tumor cell death. Also, by cutting off the blood supply the chances of invasion to other tissues and metastasis are reduced. Compared to the tumor cell targeting this approach offers several advantages like extravasation of a nanocarrier are not required and the nanocarrier can directly bind to the endothelial receptors after intravenous administration. The genetic stability of the endothelial cells is comparatively more than the tumor cells hence there are fewer chances of development of drug resistance. The receptors mainly targeted in this approach are vascular endothelial growth factor receptor (VEGFR), αvβ3 integrin receptor, vascular cell adhesion molecule-1 (VCAM-1) which is a transmembrane glycoprotein and matrix metalloproteinases (MMPs) (the type of endopeptidases) [53]. A comparison between passive and active targeting has been shown in Fig. 7.6.

2 Nanotechnology Nanotechnology is science, technology, and engineering conducted at the nanoscale, about 1 to 100 nm. The basic idea about nanotechnology originated in a lecture given by Richard Feynman at an American physical society meeting entitled as “There’s Plenty of Room at the Bottom”. Nanotechnology is nowadays used in many fields and has revolutionized drug delivery methods (Fig. 7.7). Nanomedicine is the application of nanotechnology for the treatment and prevention of disease. A well-designed nano formulation may help in increasing the concentration of the drug at the tumor site by active or passive targeting, thus improving the pharmacokinetic and pharmacodynamic profile, decreases

Tumor

Nucleus

Nucleus

Tumor

Drug loaded nanocarrier

(A)

Drug loaded nanocarrier with targeting ligand receptor

(B)

FIG. 7.6 A comparison between passive and active targeting. (A) passive targeting and (B) active targeting.

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FIG. 7.7 Applications of nanotechnology.

the drug concentration in the healthy tissue leading to reduced side effects. It also helps to improve the stability, solubility and improve biocompatibility [55].

2.1 Properties of carrier systems used for drug delivery Any substrate which is used to transport a drug to the target site is known as a drug carrier, primarily employed for improving the safety, efficacy, selectivity or altering the release of the drug. They can sequester, transport or retain the drug en-route while eluting to or near the vicinity of the target. An ideal drug carrier formulated to deliver an anti-cancer drug to the tumor site should possess the following properties: (i) (ii) (iii) (iv)

Should have enhanced drug loading capacity. Biocompatible with body fluids. Should be less toxic or non-toxic, non-immunogenic and biodegradable. Should be able to reach the tumor site with efficient targetability by overcoming the barriers posed by the vasculature and capillaries of tumorous tissue. (v) The drug-carrier complex formed should be stable in plasma, interstitial and surrounding biofluids. (vi) The carrier should be selective for tumorous cells and should have less accessibility to normal cells. (vii) Should be able to easily release the active moiety after getting internalized inside the tumor tissues.

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There are various types of drug carriers based on different ways of attaching to tumor tissues that can be used for delivery of anti-cancer drugs. These could either be in the form of soluble polymeric carriers, polymeric micelles, cyclodextrins, dendrimers, proteins, and amino acids, microspheres, liposomes, or nanoparticles as discussed above. These carriers have the ability to improve solubility, bioavailability, half-life, and reducing the adverse effects produced by unbound drugs [56]. After the development of reliable delivery devices, Dendrimers came into existence as the advantages offered by dendrimers are numerous like the ease of fabrication, low immunogenicity, targetability [57], polyvalence; it has become a multi-utility carrier system. Due to these constant efforts and continual research in this area, a perspective towards a large dendrimeric family of more than 100 constitutively different dendrimers having great potential for drug delivery has opened up [58, 59].

2.2 Nanovision for targeting of cancer Nanomaterials due to their nanosize range have a larger surface area with unique mechanical, electronic, magnetic, and photonic properties. Hence, special classes of nanocarriers have been designed with unique properties for the targeted therapy of cancer and to avoid the limitation of the conventional drug delivery systems (Fig. 7.8) [55].

2.2.1 Carbon nanotubes (CNTs) CNTs are made of an allotrope of carbon known as fullerene which may be in the form of hollow spheres, hollow tubes or ellipsoidal form. CNTs are formed when the graphene

FIG. 7.8 Different nanocarriers for cancer targeting.

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sheet is rolled upon a seamless cylinder. CNTs are the emerging technologies for the targeted therapy of cancer because of the several advantages offered by it like larger surface area, conjugation as well as encapsulation of drugs it’s permeability to the cell membrane and nucleus membrane as well as its inert nature [52]. CNTs can be categorized into three major categories on the basis of the number of layers and their wrapping as single wall carbon nanotubes (SWCNTs), double-wall carbon nanotubes (DWCNTs) and multi-wall carbon nanotubes (MWCNTs). CNTs also offer versatility in their synthesis as it can be synthesized by both physical as well as chemical method. Physical methods include laser ablation technique and arc discharge technique and chemical methods include vapor deposition method and reaction between carbon monoxide and cobalt molybdenum. Apart from these several other methods are also reported for the preparation of CNTs like electrolysis, helium arc discharge and flame synthesis method [60]. Apart from several advantages, the two major disadvantages which limit the use of CNTs is its highly hydrophobic nature and its cytotoxicity, however, these issues can be resolved by their proper functionalization. For the improvement in solubility and decrease in toxicity, CNTs are functionalized. As the carbon is present is sp2 form there is a generation of strain in its structure which helps in their easy functionalization. The functionalization can be performed by covalent and non-covalent methods. They are conjugated with biomolecules like proteins, nucleic acids, or polysaccharides to make them biocompatible. CNTs are more preferred for cancer because of their tumor accumulation potential. Also, it helps in the prevention of degradation of drug by the enzymes thus imparting stability to the drug. For the treatment of colon cancer, gemcitabine drug was conjugated with MWCNTs containing hyaluronic acid and polyethylene glycol (Fig. 7.9) [61].

FIG. 7.9 TEM images of (A) purify MWCNTs (B) Gemcitabine loaded hyaluronic acid-MWCNTs (C) Gemcitabine loaded hyaluronic acid-polyethylene glycol MWCNTs. Adapted with permission from Prajapati SK, Jain A, Shrivastava C, Jain AK. Hyaluronic acid conjugated multi-walled carbon nanotubes for colon cancer targeting. Int J Biol Macromol 2019;123:691–703.

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2.2.2 Gold nanocarriers Photothermal therapy in which the light energy i.e., the energy of photons is converted to heat energy which leads to increase in the temperature at the tumor site and thus killing the tumor cell, is gaining much more importance in combination with the chemotherapy. A metallic carrier such as gold nanoparticles by the effect of surface plasmon resonance (SPR) can convert the light energy to heat and can also scatter it to the surrounding tumor tissue leading to tumor cell death. Gold nanocarriers are the preferred option for the targeted therapy of cancer due to its properties like tunability of size and can be used to incorporate different types of drug, DNA, etc. Gold nanoparticles as mentioned earlier can be used for photothermal therapy as well as the drug can be conjugated with it thus providing chemotherapy, thus it can function as a carrier as well as a therapeutic agent. For the photothermal therapy, the gold nanoparticles can also be activated by NIR laser. There are several ways by which the drug can be loaded in the gold nanoparticles; for the core-shell nanoparticles the drug can be loaded in the mesoporous silica core; gold nanoshells can be covered with a thin layer of hydrogel formed by the polymer containing drug and the drug can also be intercalated on the gold nanoshells. Control of the size of the nanoparticles is very much important in case of targeted therapy of cancer because the passive targeting, EPR effect and intracellular accumulation of the nanocarrier all are dependent on the size of the nanocarrier [52]. It has been reported that gold nanoparticles (GNPs) have the ability to down-regulate VEGF which is highly expressed in tumor cells. It has been found to bind with the heparin binding domain of the VEGF and inactivate it [62].

2.2.3 Mesoporous silica nanoparticles (MSNs) MSNs contain the porous honeycomb-like structure of silica (SiO2). They offer advantages of the porous structure, tunable size, biocompatibility, and larger surface area. The high surface area of pores allows the attachment of the functional groups on the MSNs [52]. There are mainly three types of MSNs i.e., ordered MSNs, hollow or rattle type of MSNs and core/shell type MSNs. Ordered MSNs possess uniform pore size and pore structure arranged in an orderly manner (Fig. 7.10). The hollow type MSNs are more preferred over ordered ones because of increased drug loading and easy surface functionalization. To improve the functionalization of MSNs it can be attached to the solid cores like gold, platinum, etc. also the surface of the MSNs can be functionalized with the gold for various application [63]. The drug can be loaded in the core or onto the surface of MSNs either by electrostatic adsorption or hydrophobic interactions and covalent binding. These silica surfaces are negatively charged, if unmodified, due to the presence of hydroxyl group of tetraethyl orthosilicate (TEOS). Hence the water-soluble hydrophilic drugs tend to adsorb on MSNs with negatively charged pores and surface. While the hydrophobic anticancer drugs generally attach to the MSNs by the means of hydrophobic interaction. The hydrophobic drug can be dissolved in the organic solvent which is then mixed with MSNs solution followed by vacuum drying to remove the solvent. Apart from this, the functional group present on

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FIG. 7.10 TEM images of mesoporous silica nanoparticles. Adapted under common creative license from Zhou Y, Quan G, Wu Q, Zhang X, Niu B, Wu B, Huang Y, Pan X, Wu C. Mesoporous silica nanoparticles for drug and gene delivery. Acta Pharm Sin B 2018;8:165–177, under common creative license (Elsevier).

the silica surface can be easily functionalized to make it available for chemical conjugation [51]. PEGylation is required to improve the circulation half-life of the MSNs which is limited for conventional MSNs. Polymers can be grafted on the porous structure of the MSN. These polymers act as gatekeepers and control the release of the drug embedded in the porous structure. MSNs can be used for passive targeting by the control of the size as well as for the active targeting by conjugation with certain ligands like folate, mannose, transferrin, and certain peptides [52].

2.2.4 Quantum dots Quantum dots are a type of fluorescent semiconducting devices which can be used for diagnosis as well as therapeutic purposes. They are the inorganic nanomaterials which possess inherent fluorescent properties along with several optical and electronic properties. They are water soluble with suitable modifications and can be designed to a size of about 2–4 nm [64]. The drug incorporated in the carrier can be released at the tumor site while the carrier can be used for diagnosis purpose. Quantum dots are generally made up of three main parts: a core, a shell, and outer capping material. Both the core and shell are made up of semiconductor material which is covered by capping material [52]. The core material is responsible for imparting the fluorescence to the system and the outer layer protects the core and prevents its leaking and photo-bleaching. Different types of QDs include carbon-based QDs, graphene-based QDs, and cadmium based QDs [64].

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They possess several unique photophysical properties which can help in real-time monitoring of the drug release. Quantum dots can be prepared by both top-down and bottomup approaches. The top-down approach includes techniques like molecular beam epitaxy, X-ray lithography, e-beam lithography, and ion implantation, while the bottom-up technique involves chemical reduction after self-assembly in solution [52]. Recently the use of QDs has increased for the diagnostic purpose because it offers certain advantages over the conventional fluorophores like it avoids photo-bleaching, they can be functionalized by the polymeric materials and can be used for the targeting. It possesses photoluminescence property which can be used to diagnose the tumor cells in the body [64]. Graphene quantum dots have been synthesized for the drug doxorubicin for the targeted therapy of cancer. They were prepared from the carbon nanotubes by acidic oxidation and exfoliation. As biotin receptors are overexpressed in case of cancer the QDs were attached to biotin for active targeting [65].

2.2.5 Liposomes Liposomes are a type of vesicular drug delivery system containing phospholipids. Phospholipids which is a major component of the biological membrane consists of the hydrophobic tail made up of fatty acid and a hydrophilic head based on phosphate group. On contact with water, phospholipids self-assemble to form vesicles. The core formed inside the vesicle is of hydrophilic nature and thus can be used for the incorporation of water-soluble drugs. The region between the two layers of phospholipid is hydrophobic in nature and thus can be used for incorporation of lipid soluble drugs. On the basis of a number of bilayers, liposomes can be classified as unilamellar vesicles and multilamellar vesicles. The unilamellar vesicles further depending on their size can be categorized as small unilamellar vesicles and larger unilamellar vesicles. The conventional methods for liposome preparation include film hydration method (which is mostly used) solvent injection method, detergent dialysis and reversed phase evaporation. However, they offer certain drawbacks as well. To overcome that, newer techniques based on supercritical fluid technology like supercritical anti-solvent method and supercritical reverse phase evaporation are used [52]. Conventional liposomes have many issues related to instability, minimum drug loading, entrapment by the reticuloendothelial system (RES), thus, shorter circulation time in blood and lack of ability to control the drug release. Therefore, like other nanocarriers liposomes also require functionalization. PEG is conjugated to liposomes to provide stealthiness and thus decrease its uptake by RES system.

2.2.6 Polymeric micelles Amphiphilic polymers which contain both the hydrophilic and hydrophobic portion when attaining a suitable concentration above a certain critical micelle concentration (CMC) have a tendency to self-assemble themselves to form a circular structure known as polymeric micelles. When these amphiphilic polymers are exposed to the hydrophilic solvent the hydrophilic part of the micelle will get attached to the solvent while the

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hydrophobic part will tend to move away from the solvent this will lead to the formation of the micelle containing hydrophobic core and the outer hydrophilic surface. The opposite case will happen when the amphiphilic polymers are exposed to the hydrophobic solvents leading to the formation of the reverse micelle [52]. There are three ways to incorporate the drugs in the polymeric micelle i.e., by physical entrapment, by chemical conjugation or by emulsification. The physical entrapment method is preferred over chemical conjugation because chemical conjugation leads to the formation of the chemical bond which may cause steric hindrance and thus, offers resistance to the cleavage by enzymes. Physical entrapment of the drug can be carried out by either dialysis method or by preparation of oil in water emulsion. The core can be modified to control the release of the drug. The drug generally releases from the micelle by either of the two ways, by diffusion from the micelles or by breaking of the micelle [66]. Micelles offer several advantages of drug loading, because of a large hydrophobic core, hydrophobic drugs can be easily entrapped. Also, the charged species can be loaded on the micelles by the help of ionic interaction between the polymer and the species. Similar to other nanocarriers it is also possible to design the micelle containing various ligand required for the targeted therapy of cancer. In the polymeric micelle system, it is also possible to design a micelle in which the drug release will be based on certain stimuli. The pH-sensitive micelle is one of the important strategies for cancer treatment due to the acidic pH of the tumor microenvironment, polymers which are sensitive to a particular pH can be incorporated in the preparation of such micelles. Other strategies include temperature sensitive, ultrasound sensitive, enzyme sensitive, oxidation-sensitive, etc. One of the major issues faced by the micelle is early drug release in the blood before reaching the tumor site because of the imbalance in the polymer structure. To overcome this disadvantage some researchers have reported a method of cross-linking the polymer either in the core part or in the shell part to improve the stability of the micelle [67].

2.2.7 Dendrimers Dendrimers are the hyperbranched nanostructure with low polydispersity, high surface activity and uniformity in size. A particular dendrimer structure can be divided into three major parts: a core, a branching system, and peripheral functional groups. Depending upon the moiety present in the core and the peripheral group, the dendrimers can be classified into several classes such as polyamido amine (PAMAM), polypropylene imine (PPI), glycodendrimer, liquid crystalline, peptide, chiral, hybrid, core-shell (tecto), PAMAM organosilicon (PAMAMOS) triazine, melamine, poly-lysine, etc. [52]. Two main methods have been reported for the synthesis of which one is convergent method in which first the branches are synthesized which are then added to the core molecule and the other is divergent method in which the core is first synthesized and then branches are added to it. Some recent techniques like click chemistry and Lego chemistry have also been introduced for the synthesis of the dendrimer.

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Amongst all the classes of dendrimers, the PAMAM dendrimers have positively charged groups on its surface and is, therefore, used to conjugate DNA for gene delivery to the cells. Due to a large number of peripheral groups available on the surface multifunctional dendrimers can also be prepared to contain drug molecule, along with the targeting moiety and/or imaging agent. An example of such a system is the functionalization of biotin on the PEGylated PAMAM dendrimer to deliver paclitaxel to the cancer site. Here biotin is the targeting moiety, lung cancer cells show overexpression of biotin, hence biotin functionalized dendrimer can be actively targeted to the lung cancer [68].

3 Dendrimer in nanotechnology 3.1 What are dendrimers The term dendrimer is adapted from the Greek word “δενδρον” or dendron, implying “tree” and can be defined as highly branched, synthetic, spherical and monodisperse macromolecules with 3D nanometric structure. It can be imagined as a tree-like structure where the trunk of a tree can be assumed as its core, from which all the branches are originating; leading to other branching units (interior shells). These units further terminate as leaves which can be related to surface functional moieties in case of dendrimers. In literature, they are also frequently mentioned as, “cascade molecules”, “arboreal”, “dendritic molecules” [69,70] or because of their nanoscopic size and monodispersity they are often referred to as “nanoscopic compounds”. These dendrimeric structures can also be visualized around us in the form of branches of trees, dendritic cells, roots, vasculatory systems, etc. These all architectural domains smartly teach us one common feature of providing a maximum interface for optimum energy. The drug to be encapsulated gets attached to the dendrimers by a well-known “click in” mechanism [71] and it is believed that an acid-base reaction occurs between the host dendrimer and the guest drug molecule. Here, the hydrogen bonding keeps the guest bound to the host [72] and the host encapsulating the drug molecules by non-covalent (i.e., hydrophobic, physical entrapment, or ionic) interactions. This weak interaction property can be utilized for releasing the drugs at tumor sites, where a pH less than 7 leads to protonation of amide groups, directing to its separation from the host molecule, hence transporting the drug at the tumor site [73, 74]. A schematic representation of a 4.0G dendrimer with 64 surface amino groups has been presented in Fig. 7.11.

3.2 Properties of dendrimeric domain (i) Properties of a dendrimer are strongly associated with the functional groups present on its surface [75,76]. These surface groups can be modified to produce antibody dendrimer, glycodendrimer, peptide dendrimer, PEGylated dendrimer or dendritic boxes in which guest molecule can be encapsulated.

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FIG. 7.11 Schematic representation of a 4.0G dendrimer with 64 surface amino groups. The core of this dendrimer begins from ethylenediamine; while the branches or arms were attached by exhaustive Michael addition to methyl acrylate followed by complete aminolysis of the resulting methyl ester by ethylene diamine.

(ii) It has polyvalent surface groups which are responsible for its manifold interactions with different biological receptor sites, as in case of antiviral therapy. (iii) They have the ability to assemble themselves spontaneously i.e., Self-assembly is possible in the presence of intermolecular forces. (iv) Huge numbers of surface end groups are present on the dendrimers; developing charge on these surface groups leads to polyelectrolyte generation that can attract oppositely charged groups. For example, the formation of aggregates of methylene blue over dendrimer surface occurs by electrostatic attraction [77]. General properties of dendrimers are presented in Fig. 7.12.

3.3 Potential applications of dendrimers Dendrimers have wide applications in biomedical, diagnostics, cancer therapy, and tissue engineering; due to their unique and modifiable physicochemical properties [69] (Fig. 7.13). Some of the major applications are enlisted here:

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FIG. 7.12 Generalized properties of dendrimer.

FIG. 7.13 Representation of the applications of the dendrimer.

3.3.1 Inkjet inks and toners Addition of PAMAM dendrimers to the formulation of inks can help in improving their adhesion to various substrates like paper, glass, plastic or metal and water resistance. Their Newtonian flow behavior makes them suitable for shear stability of these formulations. They are known to impart good admix and flow characteristics, and highquality image.

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3.3.2 In vitro diagnostics Conjugation of dendrimers with diagnostic agents can help to improve their diagnosis through various techniques like MRI, computed tomography (CT) Scan, etc. PAMAM dendrimers labeled with Gd have various advantages over Gd i.e., enhanced clearance, targetability, and contrasting features, therefore, they have been in research for quite a long time [78]. Gd (III)-DTPA-based PAMAM dendrimers, linked via thiourea linkage were developed by Wiener et al. These complexes, when used as MRI imaging agents, showed excellent imaging and longer circulation time, due to the high molecular relaxivities offered by dendrimers. Increasing the generation of dendrimers further increases the relaxivities up to 6th generation [79].

3.3.3 In vitro gene transfection A gene can be delivered to its target site with the help of a dendrimer, using it as a vector protects the drug from the degrading enzymes in the blood and also allows endosomal escape due to the “proton sponge” effect. These advantages lead to the emergence of dendriplexes i.e., conjugates having both dendrimer and nucleic acids [80].

3.3.4 Controlled drug delivery A large number of hydrophilic drugs are available for which the release needs to be controlled. These drugs or other therapeutic agents can be loaded both in the interior void space and on the surface of PAMAM dendrimers via covalent or non-covalent conjugation. This interaction between the drug and the dendrimer makes it release the drug in a controlled manner, for instance, when an aqueous insoluble drug flurbiprofen (FB), an NSAID, was encapsulated inside 3.0G dendrimer, then the terminal half-life and mean residence time was reported to be increased by 3 folds along with escalated distribution as compared to the free drug [81].

3.3.5 Solubilization of drug Dendrimers have the ability to interact with a drug and increase its solubility through physical encapsulation or chemical conjugation. Solubility enhancement can be clearly seen in some drugs which have been experimented upon drugs like aceclofenac, amphotericin, and albendazole, show an increase in solubility with an increase in the generation of the dendrimer. The reason behind this increase in solubility could be the interaction between the functional groups of drugs and the surface amine group of dendrimers. Nevertheless, there are many different factors other than generation of dendrimers that affects its solubility, like the concentration of dendrimers, pH of the solution, temperature, etc. [58] have been enlisted in Fig. 7.14.

3.3.6 Drug delivery units Dendrimers have the ability to deliver the drugs orally, which is the most preferred route of administration. They are of much significance due to the ability of their structures to be altered or managed easily as well as high reactivity of terminal groups than any other polymer [80]. Apart from this, they have a large number of branches along with surface amine

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FIG. 7.14 Factors affecting solubilization of dug by the dendrimer.

groups, in which various antibodies, enzymes, drugs, and other bioactive agents can be immobilized via covalent or non-covalent interactions [82]. Nevertheless, they have the capability to cross the epithelial layer via transcellular as well as paracellular routes and can momentarily open the tight junctions [79]. Their application in the administration of drugs can be clearly portrayed as: (i) Increase in bioavailability was shown by D’Emanuele et al., by combining propranolol with lauroyl-3.0G PAMAM dendrimer which leads to an increase in solubility of the drug as well as bypassing the efflux transporters. (ii) The prolonged release was seen in case of ketoprofen when delivered along with PAMAM dendrimers. Ketoprofen is an NSAID with low aqueous solubility, which can also be countered with the help of dendrimers. (iii) The solubility of camptothecin (which is a plant alkaloid having anticancer properties) also increased significantly when combined with PAMAM dendrimers investigated by Cheng et al. (iv) Targetability can also be achieved by conjugating 3.5G PAMAM dendrimer with SN38 (7-ethyl-10-hydroxycampothecin) and using Glycine or β-alanine as a spacer. This conjugate has the ability to be stable at gastric as well as intestinal pH due to which the premature release of the drug can be avoided. Therefore, targeted treatment in case of colorectal carcinoma is achieved when the drug is released in the wraith of carboxylesterase, by the 3.5G-Gly-SN38 conjugates [79]. (v) Not only this but dendrimers can also be used to increase permeation efficiency of transdermal dendrimers as was seen in case of resveratrol-dendrimer complexes in which the permeation efficiency of this complex was 2.5 times more than resveratrol [82].

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3.3.7 Catalyst support Catalyst supports are insoluble organic, inorganic, or hybrid supports, needed for the separation of catalysts from the products. For this Silica and other inorganic materials have been used previously but due to the partially unknown structures of the catalysts, their slower diffusion (which leads to mass transport limitations) and metal leaching, their use is quite controversial. Using soluble support like dendrimer resolves these issues related to mass transfer of the catalyst systems. Even the catalysts can be recycled using two-phase catalysis, precipitation, and immobilization on insoluble supports. The selectivity, activity or the stability of the system depends upon the architecture of the dendrimer, like whether the catalysts are present on the surface or in the core (Fig. 7.15) [83].

3.3.8 Delivery agents for vaccines For the delivery of vaccines i.e., nonpathogenic antigens, a certain adjuvant is required to enhance the immune responses produced by the antigen. For enhancement of immune response either delivery systems or immune-stimulators that can sensitize the immune system are required, while delivery agents cumulate or agglutinize antigens leading to their easier detection by the antigen presenting cells (APCs). Various adjuvants present lack the ability to induce Th1 responses that can boost immunity at mucosal surfaces. Therefore, in the dearth of a stable, safe system that can induce immune responses dendrimers were used. Peptides can be conjugated onto the surface of PAMAM or PPI dendrimers popularly known as multiple antigenic peptide (MAP) system, that can develop antigenic responses against them [84].

3.3.9 Protein mimicry Due to the high molecular weight and branching in the structure of dendrimer, they have the ability to mimic proteins. Ammonia core PAMAM dendrimers of 3.0G has

(B)

(A)

Catalyst located at the periphery

Catalyst located at the core

FIG. 7.15 Architecture of the dendrimer showing the ways catalyst can be attached to the dendrimer (A) catalysts are present on the surface and (B) in the core.

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approximately the same size and shape as that of insulin (3 nm), 4.0G as that of cytochrome C (4 nm), and hemoglobin (5.5 nm) as 5.0 G. There are a lot of similarities between proteins and dendrimers, but some dissimilarities also exist, like vulnerable nature of proteins towards pH, light, and temperature which is not the case in dendrimers. They can have different applications while mimicking proteins: (i) In angiogenesis: Arginine-loaded dendrimers which were designed by Shimamura et al. can mimic the exterior architecture of endostatin. Endostatin is an endogenously found angiogenic protein which binds to heparin or HSPG and inhibits the process mediated by basic fibroblast growth factor (b-FGF) and vascular endothelial growth factor (VEGF). The inhibition of these factors can lead to the anti-angiogenic effect and can suppress or prevent the escalation and metastasis of solid tumors. (ii) Hydroxyapatite regeneration: PAMAM dendrimers surface functionalized with carboxylic acid groups can mimic amelogenin, an extracellular matrix (ECM) protein. Amelogenin is responsible for the biomineralization of the degraded or matured enamel. PAMAM-COOH can thus persuade the development of hydroxyapatite (HAP) crystals over the demineralized enamel surface by acting as a template. (iii) Collagen mimics: Collagen that is obtained from bovine sources and human cadavers can lead to transmission of disease and anaphylactic reactions. Therefore, another source of collagen is a necessity. Kinberger et al. synthesized collagen mimicking dendrimers having trimesic acid (TMA) as a core and Gly-NleuPro sequence in a tris-based scaffold [79].

3.3.10 Patterning and templating Higher generation dendrimers have enough space inside their architecture to hold nanoparticles, this has been beneficial in the synthesis and stabilization of nanoparticles with the help of tertiary amine groups present inside the dendrimer. (i) Encapsulation of Pt nanoparticles inside 4.0G-OH PAMAM dendrimers was done with the help of coordination of K2PtCl4 with the internal amine groups of dendrimer; following reduction by NaBH4, which reduces Pt from (+2) to (0) [85]. (ii) 2.0G-10G of poly(amidoamine) (PAMAM) dendrimers can be used as templates for the preparation of organic-inorganic hybrid colloids in water. This concept is based on the attraction between the negatively charged dendrimer and positively charged metal ions [86].

3.4 A tribute to dendrimer workers The idea of dendrimers was first put forward by Flory in 1941. He had a good grasp over the processes that occur during the self-polymerization of AB2 type of monomers. He explained this process as increasing the generation of dendrimer by a single level leads to an increase in surface area by a factor of (generation)2 whereas a factorial increase in volume is of (generation)3. But he could not provide any evidence for that idea due to

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lack of man-made and analytical methods available at that time. In 1978, Vogtle published his first paper for the synthesis of low molecular weight oligoamines by repetitive synthesis technique (RST). In 1981, the divergent synthesis of poly(lysine) dendrimers up to 10 generations were patented by Denkewalter [87]. Due to the number of benefits offered by dendrimers, continued research efforts in this area have flourished, that circles around its uses and applications as therapeutic agents. It was way back in the 1980s when D. A. Tomalia who had a hobby of growing trees got insight from the tree-like structures, that can be molded into molecular form and named them “Dendrimers”. In 1985, that was a major breakthrough in the history if dendrimers when Tomalia along with Dow Chemical company published his first article regarding the synthesis of PAMAM dendrimers [88]. E. W. Meijer, a chemistry professor at the Eindhoven University of Technology in Netherlands along with his coworkers Johan F. G. A. Jansen and Ellen M. De Brabander-van den Berg, demonstrated the possibility of dendrimers to be used as containers or hosts, by describing dendrimers as “dendritic box” [89]. Later, Steven C. Zimmerman, who is a professor of chemistry at the University of Illinois, got inspired by nature that even a viral capsid is not made by a whole polypeptide in one go, instead, small subunits are first made. With this insight, he and his colleagues decided to self-assemble small dendrons in order to make a larger dendrimer. They prepared a wedge-like a molecule with dendritic tail and allowed six of these wedges to assemble themselves into a tart like circular hydrogen-bonded aggregate. They were the first to introduce hydrogen bond mediated assembly of benzyl ether dendrons as well as reported its delivery application [90]. These works were “stimulating”, said chemistry professor Jean M. J. Frechet of Cornell University who has been a key player in the field of a dendrimer, since its introduction by Donald Tomalia.

3.5 The rationale for selecting dendrimers as drug delivery carrier There are various types of nanocarriers as discussed above which can serve the purpose of drug delivery but still, dendrimers are more preferred because of several advantages offered by it (Table. 7.1).

3.6 The rationale for opting dendrimer-based drug targeting Less specificity of the prevalent chemotherapeutics makes them non-efficacious for the treatment of tumors, as the side effects produced in this case will be quite large. To avoid these side effects and resistance towards drugs, there is a necessity for an efficient drug delivery carrier that can deliver the drug at its site of action, thereby producing a therapeutic response which is most desirable. (i) As far as the tumor is concerned, there is a phenomenon known as Enhanced Permeability and Retention (EPR), which is observed in the case of tumor endothelial cells. By taking advantage of this EPR effect, many drugs can be delivered to the tumorous sites.

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Table 7.1

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Advantages of dendrimer

Advantages

References

Its nanoscopic size Its mono-dispersity i.e., it possesses a high degree of molecular uniformity Huge drug loading Can reduce the dose and hence, the dose-limiting toxicity Can decrease the secondary effects and toxicity related to the administration of anticancer drugs such as doxorubicin, taxanes in organs (e.g., heart) Help in preventing the enzymatic degradation of the drug Rapid and simple pathway for its synthesis Easy availability of reagents/facilities for synthesis Able to control the release of the drug/therapeutics as it slows and sustains the drug release capability An ample number of amendable surface end groups are available that imparts targetability

[91] [92] [93] [94] [57] [95] [96] [97] [98] [99]

(ii) Another advantage is offered by dendrimers, as they have the majority of modifiable surface groups where various targeting moieties like peptides, antibodies, folic acid can be attached. Hence, efficient target specificity can be achieved: (a) By peptides: ανβ3 integrin is an extracellular matrix protein which is overexpressed in case of tumor cells for the initiation of angiogenesis. Arg-Gly-Asp (RGD) peptide has a high affinity for ανβ3 integrin. Therefore, this peptide series when attached to the surface of PAMAM dendrimers provides targetability towards tumor microvessels [100]. (b) By folic acid: It is the most widely used ligand for targeting cancer cells, as folic acid receptors are overexpressed in case of every tumor type. Therefore, Folic acid conjugated dendrimers have shown high affinity towards these receptors leading to specificity towards cancer cells. (c) By monoclonal antibody: Tumor cells have a characteristic of expressing certain surface antigens which can be targeted to achieve specificity. Monoclonal antibodies bind to these antigens specifically. Conjugating them to the surface of dendrimers can help to serve the purpose of specificity towards tumor cells. (d) By glycosylating dendrimers: Asialoglycoprotein receptors (ASGPR) are receptors present on hepatocytes and have a strong affinity for galactose. The high density of these receptors is observed in the case of tumor cells. So, to achieve target activity towards hepatic tumor cells polypropyleneimine (PPI) dendrimers are glycosylated to achieve targetability [100].

3.7 Cancer and dendrimer Cancer is regarded as the most vulnerable type of disease. Though there are various novel approaches to treat cancer, still there is a great need for efficient targeting treatment methodologies for curing malignant growth of tumors. In the recent scenario, drug delivery strategies such as liposomes, polymeric nanoparticles, microspheres, etc. are being utilized for treating cancer. Some are FDA approved and many are under clinical trials.

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To attain the reliability of such treatment approaches, there is an emerging area of dendrimers. They are mostly being worked upon as drug delivery vehicle for anticancer drugs. Dendrimers are nano-carrier, they contain a branched structure which resembles a tree. They have an interior core and terminal branches possessing functional groups attached at the periphery. Thus, this type of structure imparts it the property of flexibility to be modulated into desired moiety and function as a carrier for drugs. Dendrimers are also biocompatible in nature and possess eminent functions for developing dendrimerbased drugs. Thus, there are a lot of publications related to dendrimers and their use in diagnosis and treatment of various types of cancer Sharma et al. [101]. Dendrimers have immense utility in cancer treatment and diagnosis. They are monodispersing in nature and they contain terminal functional groups which can be easily modified and conjugated with different molecules thus, making them compatible for functioning as carriers for various drugs and diagnostic agents. They also have a high degree of uptake by cells and high drug loading capacity, because they contain functional groups such as carboxylic acid, amine, and hydroxyl which have a high density that imparts them the tendency to entrap various types of drugs and moieties possessing high molecular weight. Therefore they provide a means of treating cancer through passive targeting approach, in which the size of the dendrimer facilitates the entry of the drug into the tumor vasculature through enhanced permeability and retention (EPR) effect, in which the tumor blood vessels become leaky and increase the permeability of such type of drug-loaded nanocarriers (Fig. 7.16). Dendrimers can also be used to develop active targeting therapy, by allowing the drug to target tumor site. This type of targeting involves the conjugation of a dendrimer with certain targeting ligands such as folic acid which can bind to receptors present at the target site and permit the release of drug at that site (Fig. 7.17).

Ligand mediated targeting

Tumor cell Nucleus Receptor

Dendrimer with ligand FIG. 7.16 Ligand-mediated active targeting through dendrimer.

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Endothelium

Leaky vasculature

Dendrimer Tumor tissue

FIG. 7.17 Passive targeting mediated through dendrimer.

A class of dendrimer that is mostly being investigated in this field of cancer research is PAMAM. They are multifunctional and also have the capacity to specifically target cancer cells. They are in literature and thus a lot of information is being available for designing such type of dendrimers to achieve site-specific delivery of drugs and treat cancer. Thus, many other classes of dendrimers have also emerged as therapeutic agents for the treatment of cancer. The structure of PAMAM dendrimers offers it the advantage to control its interaction with drugs. Through generating modifications in its structure, it can be used to encapsulate various drugs. Other attributes such as spherical structure, biocompatibility, non-immunogenicity, reduced non-specific blood-protein binding and its solubility in water, makes it capable of conducting delivery of drugs and genes. The drugs can either form conjugates or complexes with PAMAM dendrimers for obtaining controlled release profile. It is reported that the drug release rate is faster if the pH is low. The microenvironment of the tumor is acidic, therefore, it will cause the protonation of amine groups present in PAMAM which will change its conformation and further lead to the release of the drug. Thus, drug release from PAMAM is pH dependent and there will be a high rate of drug release in acidic pH [102]. Dendrimers have diverse applications ranging from medical, biomedical, diagnostics and various non-medical purposes. They are employed for solubility enhancement, drug delivery, tissue engineering, bone imaging, cancer therapy, oligonucleotide delivery, and siRNA delivery. They are also used as imaging agents and MRI contrast agents. Dendrimers can be employed in boron neutron capture therapy and photodynamic therapy for efficient diagnosis and treatment of cancer. There are some dendrimer-based products also available in the market e.g., Vivagel, Profect, Superfect, etc. The main role of dendrimers is mostly in the area of cancer therapy due to their selective targeting features and modulator structure. Thus extensive research on dendrimers has paved a path for developing novel drug delivery approaches for treating various types of cancer [101].

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3.7.1 Drug-loaded dendrimers as nano-vehicles Dendrimers have the property of containment. From the time of their emergence in the market, they are being extensively worked upon to study drug-carrier chemistry. Dendrimers are conjugated with drugs through physical adsorption, covalent interaction and electrostatic interaction. Therefore, these are referred to as the drug dendrimer binding mechanisms. For developing targeted drug delivery platform dendrimers are being used as nano-vehicles or nanocarrier for different drug moieties [103]. The drug molecules are retained in the branches of dendrimers through the development of electrostatic interaction with the protons of amide groups present in the interior. Therefore, at less than 7 pH value, the inclusion complexes can be separated due to protonation. This important principle can be utilized to demonstrate the intrinsic capability of dendrimers, that in alternate ways can be used to reduce the release of drugs in higher concentration at the site of the tumor, where the microenvironment has acidic pH [104]. Most of the anticancer drugs are lipophilic in nature and this attribute is considered as the major problem in the formulation of such drugs. To overcome such problems these drugs can be encapsulated in dendrimers. This is called dendritic scaffolding and it leads to an increase in solubility of the drug because these scaffolds have the ability to take part in hydrogen bonding with water. Thus, hydrophobic interactions are considered to play an important role in the modification of the structure of dendrimers. Polyglycerol dendrimers of 4.0G and 5.0G were being reported by Ooya et al. They were used for increasing the solubility of paclitaxel, an anticancer drug which is poorly soluble in water. Thus increase in the generation of dendrimer lead to an increase in solubility of the drug [105]. The drug undergoes interaction with the anionic functional groups on the surface of a dendrimer, this mediates the uptake of drug in the interior portion of the dendritic scaffold. Therefore, dendritic scaffolds offer superior properties such as low toxicity, high drug loading efficiency, increase in water solubility as well as imparts stability. Another mechanism of delivering the drug through dendrimers is by utilizing their property of creating small holes. It has been reported that polycationic dendrimers create transient nano-sized holes in the living cells, and these holes allow increased exchange through the cell membrane [106].

3.7.2 Target specific dendritic scaffolds Tumor cells have various differences in comparison to normal cells. The tumorigenic cells show overexpression of certain receptors and biomarkers, these can serve as important targets for facilitating the delivery of various anticancer drugs into the tumor. The drug is being conjugated with a tumor recognition moiety. It is attached either directly or through a linker, this forms a delivery device for targeting tumor. Such targeting strategies improve the therapeutic efficacy of anticancer drugs, therefore, providing an efficient means of treating cancer with manageable therapy. Thus, through dendrimers, complete targeting treatment strategies can be developed for minimization of side effects. Dendrimers possess certain characteristics such as non-immunogenic effect, increased water solubility, low polydispersity index. With terminal amine groups,

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imparting it the efficiency to formulate dendrimer-based targeting drug delivery systems [107]. The size and structure of dendrimers provide them with the ability to enter the pores and gain access into the tumor vasculature and thus, increase the permeability in tumor cells. This type of drug delivery approach paves a path for prevention of multidrug resistance. Many targeting ligands are being used for developing targeted drug dendrimer conjugates. Example of such targeting agents is folic acid, dextran, glycodendrimer and monoclonal antibodies [108].

3.7.3 The rationale for selecting folic acid as a ligand The folate receptors are overexpressed in case of cancer thus they have more affinity for folic acid specifically bind to it providing a means for entry of drug conjugate to folic acid. Thus, these receptors can function as efficient targets for treating tumors. Various research studies have been conducted to prove the potential application of folic acid as a targeting ligand and also there are many reports which confirm that folic acid can easily undergo conjugation with dendrimer. Therefore, many studies are conducted on dendrimers conjugated with folic acid to support their efficient use in the treatment of cancer [109]. Folic acid has various properties such as [110]: (i) It is selectively uptaken by the folate receptors that are overexpressed in cancerous cells. (ii) Its small size reduces the chances of immunogenicity. (iii) Its effective uptake by receptors imparts easy delivery of drugs in the cytosol. (iv) The binding chemistry between drug and folic acid is not complicated hence provide ease of conjugation. (v) Selectively release the drug at cancer site and therefore increase anticancer activity and decrease toxic effects. (vi) It is stable and cost-effective. Folate-targeted dendritic nanocarriers The conjugation of folic acid is being done widely for developing many chemotherapeutic formulations that show targeted effect. Folic acid is also known as vitamin B9. It plays a pivotal role in the production of new cells because it is the main component that is being taken up by most of the cells for its utilization in the synthesis of various amino acids such as serine and methionine. It is also used in the synthesis of purines, ultimately governs the synthesis of deoxyribonucleic acid (DNA). The tumors that originate at epithelial cells have overexpressed folate receptors which have high affinity to bind with folic acid. Such types of tumors are formed in breast, brain, prostate, kidney, and certain choriocarcinomas also resemble them. To develop an antitumor therapy, a culmination of various functions is required which involves targeting the tumor site then conduct its effective imaging and ultimately deliver the drug to the targeted tumor site without affecting major portions of nearby tissues or cells. Hence eliminate the risk of side effects and increases antitumor activity [111].

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Dendrimers can be regarded as appropriate candidates for entrapping various high molecular weight structures to produce the desired specific functions. The size of the dendrimer is similar to that of serum proteins which imparts them the ability to enter the tumor vasculature [112]. Dendrimers can be further utilized to achieve drug release in a pH-dependent manner. Therefore, there is a burst release of drug in the acidic environment of the tumor site and in normal conditions, there is a slow release of drug at physiological pH. In spite of all such advantages, still, drug-loaded formulations of dendrimers produce toxic effects due to the release of drug at off-target sites around the tumor. To overcome such complication, drug conjugation is being studied and modified according to the desired specifications for drug delivery. The excess of free amine groups on the peripheral surface of dendrimers enhances the covalent binding of drugs. But the open structure of dendrimers still leads to certain issues related to biocompatibility and hemolytic toxicity. To improve the biocompatibility of dendrimers, PEGylated dendrimers were developed which showed the sustained release of the drug [113]. In order to resolve toxicity problems “locked in” type of dendrimers were being reported and they further provided along with the circulating dendritic system [114]. Hence the dendrimer structure is such that it has the flexible characteristics to be played around and modified for conjugating various moieties essential for tumor targeting. The conjugation of folic acid to dendrimers involves easy mechanism, and there are many reports available which support the successful use of folate conjugated dendrimers in both imaging as well as targeting tumors in various types of cancers [115]. Folate–PEG dendrimer in cancer targeting Conjugated dendrimers are being developed to reduce the toxicity associated with them. It has been reported that polyethylene glycol (PEG) was grafted on the dendrimer surface that leads to the formation of large interior surface which increased the loading efficiency of drugs such as chloroquine phosphate and 5-fluorouracil. An anticancer drug like Adriamycin and methotrexate have also been encapsulated in PEGylated dendrimer. The highest ability of encapsulation was attained by the fourth generation of dendrimer containing PEG graft with an approximate weight of 2000 Da. This graft was capable of retaining around 6.5 molecules of adriamycin and 26 molecules of methotrexate, per molecule of the dendrimer. The release rate of methotrexate loaded PEGylated dendrimer showed a slow release in the less ionic concentration of the aqueous solution. Thus, these type of dendrimers modulates drug release as well as produce fewer side effects when further conjugated with targeting ligands such as folic acid [116]. Dendrimers if unconjugated may also attack the surrounding normal cells around tumor so, in order to avoid such scenario, they are being conjugated with ligands having a high affinity of uptake at the targeted tumor site, this type of approach is called active targeting. Folic acid conjugates of dendrimer attach on the tumor site as they have increased affinity towards the overexpressed folate receptors at the tumor site. Thus, folate conjugation provides effective drug carrier for delivering the drug at the targeted site. But

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folic acid dendrimers possess a limitation of low solubility in water, this limitation can be removed by the addition of PEG to the folic acid conjugated dendrimer. Thus, along with an increase in water solubility, PEG will also increase the circulation time in blood, enhance kinetic stability, lowers toxicity, imparts high drug loading efficiency, controls drug release and slow down degradation by enzymes [117]. PEGylation also changes the lipophilic character of a molecule into hydrophilic. As the PEG chain length increases, the degree of hydrogen bonding decreases and thus molecular mobility and stability increases. Thus, this, in turn, affects the binding affinity of the conjugating ligand towards the receptor. Folic acid conjugated to dendrimer with a PEG linker has an application as a biodegradable nanoparticle. A Folic acid-PEG amphiphilic dendrimer has been synthesized, its core is made up of poly(L-lactide) and on the exterior part, it is surrounded by six dendrons of polyester. They were being used for constructing nanocarriers to mediate targeted delivery of drugs in cancer cells. PEG-conjugated folic acid dendrimers have demonstrated a lower risk of hemolytic toxicity and an increase in the retention time at the tumor areas [116]. For the treatment of cancer, gene therapy can be employed and to conduct efficient delivery of siRNA into the tumor site the PEG-folic acid dendrimers have been modified and in one such studies, it is reported that they are further conjugated with α-cyclodextrin. This increases the targeting capacity of siRNA; it increases the permeability and mediates easier transfection and further enhance endosomal escape. The PEGylated dendrimers can also be used to load Gadolinium which would then altogether function as an imaging agent used in magnetic resonance imaging (MRI). This type of imaging allows for easy detection and diagnosis of cancer. Thus, PEG-folic acid conjugated dendrimer can be designed to develop improved treatment strategies for cancer [118]. DNA assembled dendrimer–folate conjugate PAMAM dendrimers have been widely explored due to their multifunctional properties. Their structure serves as a great source for modification. They are less toxic, biocompatible, non-immunogenic and have a solubility in water. These features make them capable of designing a therapeutic strategy for treatment as well as diagnosis of cancer. Conjugating such dendrimers with different ligands might alter their properties but in order to improve their targeting potential, conjugation is required. Recently self-assembled PAMAM dendrimers have been reported. These types of dendrimers are being developed using complementary short sequences of DNA i.e., oligonucleotides (ODN’s). These sequences have the property to self-assemble, which can be utilized to construct sitespecific drug delivery approaches for treatment as well as diagnosis of cancer [119]. The dendrimers can be conjugated with ODN through simple conjugating procedures. Thus, the ability of this unique self-assembled dendritic model was being evaluated to target folate receptors by conjugating it with folic acid and fluorescein isothiocyanate (FITC). In a study, these two different ligands were reported to be linked with complementary ODN. The ODN was 34 base pair long with a modified 50 -phosphate group. The folic acid (FA) would function as targeting ligand and FITC as an imaging agent. Therefore, both

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aspects of targeting, as well as imaging, can be culminated with the development of these type of unique and multifunctional DNA assembled dendrimers. In vitro tests of these dendrimers demonstrated that their binding affinity was specific to KB cells of the folate receptor. The confocal microscopy images also indicated that the dendrimer was effectively taken up in the interior of the tumor cell [120]. These types of dendrimers were synthesized through a procedure containing different steps. In the first step, the G5 PAMAM dendrimer was partially acetylated using acetic anhydride, this was done to increase the solubility and specificity of the dendrimer. To prevent undesired interaction with the cell membrane. In the second step, FITC was conjugated with the partially acetylated G5 PAMAM dendrimers by dissolving it in dimethyl sulfoxide (DMSO). Then in the third step, folic acid was conjugated to the partially acetylated dendrimer using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), which acts as a cross-linking agent and activates the carboxylic group of folic acid. In the fourth step, the oligonucleotide was conjugated with folic acid or FITC conjugated G5 PAMAM dendrimers. Then, finally, the DNA conjugated FA-G5 PAMAM and FITC-G5 PAMAM were hybridized to develop DNA assembled G5 PAMAM dendrimer containing FA and FITC, for cancer cell-specific targeting [120]. Folate–DTPA dendrimer in cancer targeting For the effective diagnosis and detection of tumors, the contrasting or imaging agent being used must be delivered to the tumor site. Hence, to develop an imaging agent that reaches the desired target site, dendrimers have been used to incorporate them and function as a carrier to conduct their delivery into tumor sites. For example, gadolinium (Gd) is an imaging agent which is being complexed with folic acid conjugated dendrimer. This dendrimeric complex lead to the increase in relaxation time of tumor cells that overexpressed the folate receptors. It also has a high affinity for the folate binding proteins that are present at the surface of the cancer cells and also in the serum proteins of patients suffering from cancer [109]. PAMAM dendrimers are most widely used for incorporation of different contrasting agents. They have high solubility and adjustable size which renders them useful to function as scaffolds for the conjugation of Gd along with its chelators such as diethylenetriamine penta-acetic acid (DTPA) and tetraazacyclododecane1,4,7,10-tetraacetic acid (DOTA) (Fig. 7.18) [121]. For each 5 nm particle, the modified form of G3 PAMAM dendrimer has the affinity for binding to 32 ions of gadolinium. These types of conjugated chelator imaging agents can eliminate the problem of less retention and inappropriate targeting in tumors. Thus, after conjugation with dendritic scaffolds and targeting ligand such as folic acid, it can be employed as active targeting moiety in MRI for detecting tumors. It has been reported that folic acid was attached to the core of G3 PAMAM dendrimer and then conjugated with Gd contrast agent consisting of a chelator such as DTPA. This type of dendrimer also showed accumulation at the tumor surface possessing overexpressed folate receptors [121].

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Gd

Gd Gd

Gd

Gd

FIG. 7.18 Dendrimer linked with gadolinium.

3.7.4 Multimodality dendrimers based diagnostic agents The early diagnosis of cancer is based on molecular imaging, but certain contrasting agents have less imaging time, therefore, nanotechnology is being explored to develop various approaches for improving the properties of diagnostic agents. Computed tomography (CT) and magnetic resonance imaging (MRI) are mostly being used for cancer diagnosis. The tumor imaging potential of nanomaterials is attributed to the enhanced permeability and retention (EPR) effect which is the basis of passive targeting [54]. Therefore, multifunctional dendrimers can be synthesized and modified via conjugation with different targeting ligands for performing dual mode imaging of tumors. The dendritic structure can be utilized to entrap gold nanoparticles into the internal core and Gd-chelator system conjugates at its peripheral branches. It has been reported that arginine-glycine-aspartic acid peptide acts as a ligand that targets the tumor cells which have overexpressed αv β3 integrin receptors. Therefore, this strategy can be utilized to develop dendrimer containing RGD peptide, which functions as a vector in gene delivery and targeting ligand for drug delivery in anticancer therapeutics. They also specifically target the αv β3 integrin receptors present in cancer cells [122]. The gadolinium entrapped gold nanoparticles (Gd-Au DENP’s) were modified with RGD peptide for imaging and diagnosis of cancer cells overexpressing αv β3 integrin receptors, thus, the imaging can be conducted in dual mode i.e., CT and MRI. These dendrimers were synthesized in a series of steps. Firstly, G5 PAMAM possessing terminal amine groups was modified by conjugation with Gd-chelator, polyethylene glycol (PEG) was used as a linker for attachment of RGD peptide, and further linked to polyethylene glycol monomethyl ether. Then finally this multi-modulated dendrimer was employed to serve as a base for entrapment of gold nanoparticles, favor chelation of gadolinium ions and the remaining peripheral amines were acetylated to form RGDmodified Gd-Au DENP’s. This type of multimodality dendrimer exhibited various

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advantages such as noncytotoxicity, stability at various pH values and was found to be hemocompatible. Therefore, different generations of dendrimers can be modified with different conjugating ligands and functional molecules to develop active targeting therapy and diagnostic aid in cancer [122].

4 Conclusion The field of cancer has an emerging potential for research. The different treatment, as well as diagnostic techniques involved in cancer therapy, have turned out to be of great importance in improving cancer therapeutics. But still, these approaches have certain shortcomings which can be overcome by utilizing the field of nanotechnology. Cancer has led to an increase in the mortality rate in recent times. Thus, effective formulations need to be developed for treating this disease as well as preventing the metastasis process which makes the tumors more malignant. The situation thus becomes more fatal. Therefore, in order to combat such worse conditions, there should be an optimum targeted drug delivery approach. To devise such a therapy, nanocarriers are being developed and studied to evaluate their targeting abilities as well as intrinsic properties which makes them a suitable drug carrier. The different nanocarriers have different use in delivery of different agents to the targeted sites. The area of nanocarriers which has a wide utility in cancer is that of dendrimers. These multifunctional, tree-structured molecules have a highly branched structure, consisting of interior core and terminally placed amino groups. This type of structure imparts the ability to entrap a large amount of drug, hence increase drug loading efficiency. To enhance the targeting ability of dendrimer they are modified and conjugated with ligands such as folic acid. This has good targeting approach in cancer because it has a high affinity for folate receptors overexpressed on tumor cells. The folate–PEG dendrimer was found to exhibit a decrease in toxicity, and it controlled the release of drugs and demonstrated fewer side effects. DNA assembled folate dendrimer also provide better targeting as well as detection of tumors when conjugated with folic acid and FITC. Further, the gadolinium linked folate dendrimer and multimodality dendrimer provide efficient targeting and dual imaging attributes in cancer therapy. Thus, such nano-vehicles can function as the source of building a new transformation in cancer diagnosis and treatment.

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[75] D’emanuele A, Attwood D. Dendrimer–drug interactions. Adv Drug Deliv Rev 2005;57:2147–62. [76] Frechet JM. Functional polymers and dendrimers: reactivity, molecular architecture, and interfacial energy. Science 1994;263:1710–5. [77] Abbasi E, Aval SF, Akbarzadeh A, Milani M, Nasrabadi HT, Joo SW, Hanifehpour Y, Nejati-Koshki K, Pashaei-Asl R. Dendrimers: synthesis, applications, and properties. Nanoscale Res Lett 2014;9:247. [78] Wolinsky JB, Grinstaff MW. Therapeutic and diagnostic applications of dendrimers for cancer treatment. Adv Drug Deliv Rev 2008;60:1037–55. [79] Noriega-Luna B, Godı´nez LA, Rodrı´guez FJ, Rodrı´guez A, Zaldı´var-Lelo De Larrea G, Sosa-Ferreyra CF, Mercado-Curiel RF, Manrı´quez J, Bustos E. Applications of dendrimers in drug delivery agents, diagnosis, therapy, and detection. J Nanomater 2014;2014:1–19. [80] Hu J, Hu K, Cheng Y. Tailoring the dendrimer core for efficient gene delivery. Acta Biomater 2016;35:1–11. [81] Lin Q, Jiang G, Tong K. Dendrimers in drug-delivery applications. Des Monomers Polym 2010;13:301–24. [82] Chauhan AS. Dendrimers for drug delivery. Molecules 2018;23(4). [83] Van Heerbeek R, Kamer P, Van Leeuwen P, Reek J. Dendrimers as support for recoverable catalysts and reagents. Chem Rev 2002;102:3717–56. [84] Heegaard PM, Boas U, Sorensen NS. Dendrimers for vaccine and immunostimulatory uses. A review. Bioconjug Chem 2010;21:405–18. [85] Wang Q, Zhang Y, Zhou Y, Zhang Z, Xu Y, Zhang C, Sheng X. Synthesis of dendrimer-templated Pt nanoparticles immobilized on mesoporous alumina for p-nitrophenol reduction. New J Chem 2015;39:9942–50. € hn F, Bauer BJ, Akpalu YA, Jackson CL, Amis EJ. Dendrimer templates for the formation of gold [86] Gro nanoclusters. Macromolecules 2000;33:6042–50. [87] Sowinska M, Urbanczyk-Lipkowska Z. Advances in the chemistry of dendrimers. New J Chem 2014;38:2168–203. [88] Tomalia D, Fr echet MJ. Discovery of dendrimers and dendritic polymers: a brief historical perspective. J Polym Sci A 2002;40:2719–28. [89] Jansen JF, Meijer E, De Brabander-Van Den Berg EM. The dendritic box: shape-selective liberation of encapsulated guests. J Am Chem Soc 1995;117:4417–8. [90] Zimmerman SC, Zeng F, Reichert DE, Kolotuchin SV. Self-assembling dendrimers. Science 1996;271:1095–8. [91] Tomalia DA. Starburst/cascade dendrimers: fundamental building blocks for a new nanoscopic chemistry set. Adv Mater 1994;6:529–39. [92] Mody N, Tekade RK, Mehra NK, Chopdey P, Jain NK. Dendrimer, liposomes, carbon nanotubes and PLGA nanoparticles: one platform assessment of drug delivery potential. AAPS PharmSciTech 2014;15:388–99. [93] Tran NQ, Nguyen CK, Nguyen TP. Dendrimer-based nanocarriers demonstrating a high efficiency for loading and releasing anticancer drugs against cancer cells in vitro and in vivo. Adv Nat Sci Nanosci Nanotechnol 2013;4, 045013. [94] Sadekar S, Ghandehari H. Transepithelial transport and toxicity of pamam dendrimers: implications for oral drug delivery. Adv Drug Deliv Rev 2012;64:571–88. [95] Caminade A-M, Turrin C-O. Dendrimers for drug delivery. J Mater Chem B 2014;2:4,055–66. chet JM. Water-soluble dendrimer–poly (ethylene glycol) starlike conjugates as [96] Liu M, Kono K, Fre potential drug carriers. J Polym Sci A Polym Chem 1999;37:3492–503.

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[97] Kwok A, Eggimann GA, Reymond JL, Darbre T, Hollfelder F. Peptide dendrimer/lipid hybrid systems are efficient DNA transfection reagents: structure–activity relationships highlight the role of charge distribution across dendrimer generations. ACS Nano 2013;7:4668–82. [98] Lo S-T, Kumar A, Sun X. Delivery and controlled release of therapeutics via dendrimer scaffolds. Future Science Ltd; 2015. [99] Tekade RK, Dutta T, Tyagi A, Bharti AC, Das BC, Jain NK. Surface-engineered dendrimers for dual drug delivery: a receptor up-regulation and enhanced cancer targeting strategy. J Drug Target 2008;16:758–72. [100] Zhu J, Shi X. Dendrimer-based nanodevices for targeted drug delivery applications. J Mater Chem B 2013;1:4199–211. [101] Sharma AK, Gothwal A, Kesharwani P, Alsaab H, Iyer AK, Gupta U. Dendrimer nanoarchitectures for cancer diagnosis and anticancer drug delivery. Drug Discov Today 2017;22:314–26. [102] Gillies ER, Frechet JM. Dendrimers and dendritic polymers in drug delivery. Drug Discov Today 2005;10:35–43. [103] Dufes C, Uchegbu IF, Schatzlein AG. Dendrimers in gene delivery. Adv Drug Deliv Rev 2005;57:2177–202. [104] Carnahan MA, Grinstaff MW. Synthesis and characterization of poly (glycerol  succinic acid) dendrimers. Macromolecules 2001;34:7648–55. [105] Ooya T, Lee J, Park K. Hydrotropic dendrimers of generations 4 and 5: synthesis, characterization, and hydrotropic solubilization of paclitaxel. Bioconjug Chem 2004;15:1221–9. [106] Bayele HK, Ramaswamy C, Wilderspin AF, Srai KS, Toth I, Florence AT. Protein transduction by lipidic peptide dendrimers. J Pharm Sci 2006;95:1227–37. [107] Kolhe P, Khandare J, Pillai O, Kannan S, Lieh-Lai M, Kannan RM. Preparation, cellular transport, and activity of polyamidoamine-based dendritic nanodevices with a high drug payload. Biomaterials 2006;27:660–9. [108] Tomalia DA, Reyna L, Svenson S. Dendrimers as multi-purpose nanodevices for oncology drug delivery and diagnostic imaging. Portland Press Limited; 2007. [109] Konda SD, Aref M, Wang S, Brechbiel M, Wiener EC. Specific targeting of folate–dendrimer MRI contrast agents to the high affinity folate receptor expressed in ovarian tumor xenografts. MAGMA 2001;12:104–13. [110] Hilgenbrink AR, Low PS. Folate receptor-mediated drug targeting: from therapeutics to diagnostics. J Pharm Sci 2005;94:2135–46. [111] Lu Y, Sega E, Leamon CP, Low PS. Folate receptor-targeted immunotherapy of cancer: mechanism and therapeutic potential. Adv Drug Deliv Rev 2004;56:1161–76. [112] Patri AK, Majoros IJ, Baker JR. Dendritic polymer macromolecular carriers for drug delivery. Curr Opin Chem Biol 2002;6:466–71. [113] Guillaudeu SJ, Fox ME, Haidar YM, Dy EE, Szoka FC, Frechet JM. Pegylated dendrimers with core functionality for biological applications. Bioconjug Chem 2008;19:461–9. [114] Gardikis K, Hatziantoniou S, Bucos M, Fessas D, Signorelli M, Felekis T, Zervou M, Screttas CG, Steele BR, Ionov M, Micha-Screttas M, Klajnert B, Bryszewska M, Demetzos C. New drug delivery nanosystem combining liposomal and dendrimeric technology (liposomal locked-in dendrimers) for cancer therapy. J Pharm Sci 2010;99:3561–71. [115] Singh P, Gupta U, Asthana A, Jain NK. Folate and folate-PEG-pamam dendrimers: synthesis, characterization, and targeted anticancer drug delivery potential in tumor bearing mice. Bioconjug Chem 2008;19:2239–52. [116] Luong D, Kesharwani P, Deshmukh R, Mohd Amin MCI, Gupta U, Greish K, Iyer AK. pegylated pamam dendrimers: enhancing efficacy and mitigating toxicity for effective anticancer drug and gene delivery. Acta Biomater 2016;43:14–29.

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[117] Sampogna-Mireles D, Araya-Dura´n ID, Ma´rquez-Miranda V, Valencia-Gallegos JA, Gonza´lezNilo FD. structural analysis of binding functionality of folic acid-PEG dendrimers against folate receptor. J Mol Graph Model 2017;72:201–8. [118] Ohyama A, Higashi T, Motoyama K, Arima H. In vitro and in vivo tumor-targeting siRNA delivery using folate-PEG-appended dendrimer (G4)/α-cyclodextrin conjugates. Bioconjug Chem 2016;27:521–32. [119] Choi Y, Baker Jr JR. Targeting cancer cells with DNA-assembled dendrimers: a mix and match strategy for cancer. Cell Cycle 2005;4:669–71. [120] Choi Y, Thomas T, Kotlyar A, Islam MT, Baker Jr JR. Synthesis and functional evaluation of DNAassembled polyamidoamine dendrimer clusters for cancer cell-specific targeting. Chem Biol 2005;12:35–43. [121] Huang WY, Davis JJ. Multimodality and nanoparticles in medical imaging. Dalton Trans 2011;40:6087–103. [122] Chen Q, Wang H, Liu H, Wen S, Peng C, Shen M, Zhang G, Shi X. Multifunctional dendrimerentrapped gold nanoparticles modified with RGD peptide for targeted computed tomography/magnetic resonance dual-modal imaging of tumors. Anal Chem 2015;87:3949–56.

Further reading [123] Zhou Y, Quan G, Wu Q, Zhang X, Niu B, Wu B, Huang Y, Pan X, Wu C. Mesoporous silica nanoparticles for drug and gene delivery. Acta Pharm Sin B 2018;8:165–77.

8

Design of dendrimer based prodrugs T. Srinivasa Reddy, Suresh K. Bhargava CENTRE FOR ADVANCED MATERIALS & I NDUSTRIA L CHEMI STRY (CAMIC), SC HOOL OF SCI ENCE, RMIT UNIVERSITY, MELBOUR NE , VIC, AUSTRALIA

1 Introduction The term prodrug was introduced in 1958 by Adrien Albert and is defined as “derivatives of drug molecules that are activated or metabolised in the body to generated active parent drug molecule which can produce the desired pharmacological properties” [1]. The prodrug approach was devised to increase the poor drug solubility, bio stability, biocompatibility and bio permeability of drugs. Recently, prodrug approach has become an attractive strategy to improve physicochemical, pharmacokinetic parameters of active drug molecules. About 5–7% of drugs approved worldwide can be classified as prodrugs [2,3]. Dendrimers represent a new class of highly branched polymers with globular structures that have diameters below 10 nm. Highly regular branching pattern in dendrimers allows precise control of size and shape. The diameter of the dendrimers can be tuned through use of different generations to match the sizes and shapes of proteins and biomolecules there by renders them as perfect bio mimics [4]. Generally, dendrimers have an inner core from which branches emerge with terminal surface functional groups at the end. Because of the inner core and terminal function groups, dendrimers display excellent host-guest interactions [5]. The multiple functional groups at the periphery of the dendrimers can be used for conjugation/grafting drug molecules, while inner space can be used for the encapsulation of small molecules. Dendrimers can, therefore, be loaded with drug molecules by encapsulation and/or conjugation (Fig. 8.1). Furthermore, a variety of different drug molecules, imaging agents and targeting compounds can be added to a single dendrimer molecule to produce multifunctional dendrimer prodrugs [6–8]. The encapsulation of hydrophobic drug molecules within inner or outer space of the dendrimers can be achieved by simple mixing of dendrimer and drug solutions, facilitating enhanced aqueous solubility. This physical encapsulation or adsorption of small molecule drugs with dendrimers via non-covalent interactions such as hydrophobic interaction, electrostatic attraction or hydrogen bonding interaction could result in uncontrolled or burst release of drug molecules which results in limited therapeutic

Pharmaceutical Applications of Dendrimers. https://doi.org/10.1016/B978-0-12-814527-2.00008-1 © 2020 Elsevier Inc. All rights reserved.

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Dendrimer-drug networks

Encavitated guest

(A)

(B)

(C)

Covalently bound prodrug

Non-covalently bound prodrug Current Opinion in Chemical Biology

FIG. 8.1 Schematic representation of dendrimer drug-delivery systems. The darkened oval represents an active substance [9].

efficacy and systemic toxicity. For instance, anti-cancer drugs encapsulated in dendrimers via non-covalent interactions show a burst release profile before the accumulation at the tumor site [9]. Alternatively, covalent conjugation of drug molecules with the large numbers of reactive peripheral functional groups on the surface of dendrimers provide an opportunity of high drug payload and controlled drug release. The drug loading in dendrimer can also be tuned by varying the number of reactive functional groups on the outer space of the dendrimer. The release of free drugs from dendrimer conjugates can be precisely controlled via chemical or enzymatic cleavage of hydrolytically labile linkers between the dendrimer peripheral groups and drug molecules thus allowing better control of the intracellular drug release than hydrophobic encapsulation [10–12]. The dendrimers-drug conjugates (prodrugs) as a drug delivery system to increase the drug solubility and permeability has received a considerable attention in the recent years for various drugs including anti-inflammatory, antimicrobial and anti-cancer drugs [10,13]. The control of molecular weight and low polydispersity of dendrimers which are considered to be the main factors in design of polymer-drug conjugates allows the reproducible bio distribution of dendrimer-prodrugs thus making them scaffolds for efficient drug delivery vehicles. These dendrimer based prodrugs offer sustained drug blood concentrations which can lead to simplifying dosing schedules, and improved patient compliance with a potential for safe administration of drugs. Overall, dendrimers are highly promising platforms for the development of prodrugs because of their reproducible syntheses, drug-loading capabilities, multi-functionality and their potential for being passively targeted toward cancer tissues.

2 Advantages of dendrimer over other polymers for prodrug preparation •

Availability of large number of suitable reactive functional groups dCOOH, dOH, or dNH2 on the surface of dendrimers for covalent coupling with drugs provide an opportunity for high drug payload

Chapter 8 • Design of dendrimer based prodrugs

• • • • • • •

201

Passive targeting can be achieved by controlling the molecular weight (size) of the polymer prodrug Biocompatible, nontoxic and non-immunogenic Reproducible bio distribution Low polydispersity Commercially available, reproducibility in manufacturing Dendrimer prodrugs are superior to polymer prodrugs, because their molecular weight and structures are well defined Polyethylene glycol (PEG), targeting ligands and imaging probes can also be incorporated into dendrimer prodrugs.

3 Advantages of dendrimer prodrugs Dendrimer prodrugs may offer the following advantages • • • • • •

Enhanced aqueous solubility of hydrophobic drugs Reduced premature release of the drug from the dendrimers Improved pharmacokinetics of conjugated drug due to decrease in clearance Prolonged blood circulation time and altered bio distribution Improved tumor accumulation through enhanced permeability and retention effect Reduced side effects and improve efficacy compared with the parent drug.

4 Requirements for dendrimer prodrugs • • • •

The linkage between drug and dendrimer usually is covalent bond and the prodrug should be inactive or less active The linkage should be bio-reversible The carrier released after the in vivo cleavage should be nontoxic and biocompatible It should possess suitable number of functional groups to attach drug molecules.

5 Strategies for design and synthesis of dendrimer prodrugs The dendrimer prodrugs are generally prepared by direct conjugation of drug molecules with the surface functional groups of dendrimers or using spacer molecules to form covalent bridges between dendrimer and drug molecules. The dendrimer prodrugs synthesized by both these methods can be absorbed in local injured sites, thus allowing controlled and tissue targeted release which leads to reduced systemic toxicity [7,8]. The general design of a dendrimer prodrug is illustrated in Fig. 8.2. The dendrimerprodrug may contain as many as four of the following components: (i) The drug molecules that exhibits the pharmacological activity; (ii) Dendrimer with suitable functional groups (dCOOH, dNH2, dOH);

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FIG. 8.2 Dendrimer prodrugs design strategies.

(iii) A linker which links the peripheral functional groups of dendrimers to the functional group of drug molecules; (iv) Spacer which separates the drug and dendrimer through linker bonds. In few cases, a targeting or imaging moiety is also conjugated to the surface of the dendrimer for specific delivery and imaging of the disease areas.

6 Dendrimer prodrugs without spacer Since dendrimers have large number of multiple functional groups like dNH2, dOH, dCOOH at the periphery, drugs with carboxyl, aldehyde, amine and ketone groups can be easily conjugated by different synthetic procedures. The resulting linker bond between the dendrimer and drug should be stable to prevent premature release of drug during the transport before the cellular localization. Generally, drugs are chemically conjugated directly to dendrimers through ester (carboxyl, carbonate, carbamate), amide, or coordinate bonds, some of which are hydrolytically cleavable in physiological conditions (Table 8.1). Among these, ester linkages are most commonly used linkages in the dendrimer prodrugs design because carboxylic acid and hydroxyl functional groups that forms the ester linkages are widely available in most parent drugs. Moreover, enzymatic hydrolysis of ester linkages is mediated by esterases which are ubiquitously found in vertebrate tissue and blood leading to the controlled release of the parent drug [14]. The rate of ester hydrolysis is influenced by type of ester bond, steric crowding, hydrophilicity and molecular weight of the conjugate. For example, carbamate ester is stable toward hydrolysis in comparison to carboxyl, phosphate ester and carbonate esters [2]. Amide bond (dCONH2) is another commonly used linkage in the design of dendrimer prodrugs. It is the derivative of carboxyl and amine group. The difference between amideand ester-bond linked prodrugs is their stability in physiological conditions. The amidebond linked prodrugs are stable toward hydrolysis even in extremely acidic conditions with only little drug release from the prodrug matrices after their administration, while

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Table 8.1 Commonly used linkers for direct conjugation of dendrimer to drug molecules Linker

Linkage bond

Ester

Carboxyl ester

Structure

Carbamate ester

Carbonate ester

Phosphate ester Amide Peptide bond

the ester-linkage prodrugs are more susceptible to acid hydrolysis [15]. The rate of hydrolysis will generally in the order carboxyl ester > carbonate ester > carbamate ester > amide. The utilization of these ester or amide linkers to conjugate the dendrimers and drug molecules has been resulted in prodrugs showing distinct properties, such as, solubility, stability, and cytotoxicity (Table 8.2). Several strategies were proposed to directly conjugate drug molecules to the surface of dendrimers using different coupling reagents. Coupling agents link the dendrimer and drug molecules by forming a linkage bond without additional spacer atom. If, the drug contains more than one functional group, then the synthetic method to conjugate drug to dendrimer involves protection or deprotection of one of the functional group of the drug. The most regularly used strategies for conjugating dendrimer directly to the drug molecules involve use of coupling agents such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, EDCI) and dicyclohexyl carbodiimide (DCC) or use of N-hydroxy succinimide esters. Most of these bioconjugation strategies involve coupling reactive nucleophiles with the following order of reactivity: amino, carboxyl and hydroxyl groups. The pH in the reaction and presence of steric hindrance on the coupling moiety controls this order of reactivity [16].

6.1 Dicyclohexylcarbodiimide (DCC) It is majorly used to couple amino acids during synthesis of artificial peptides. It is highly soluble in various organic solvents such as acetonitrile, tetrahydrofuran, dichloromethane

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N C N

FIG. 8.3 Structure of DCC.

and dimethylformamide and is insoluble in water which makes it easy to separate after the coupling reaction. A wide range of alcohols, even some tertiary alcohols, can be esterified by reacting with carboxylic acid derivatives in presence of DCC and catalytic amounts of dimethyl amino pyridine (DMAP) (Fig. 8.3).

6.2 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, EDCI) It is the most widely used coupling agent in the synthesis of amide and phosphoramide bonds where it activates carboxyl and phosphate groups which then react with primary amines. EDC is also used in combination with N-hydroxysuccinimide (NHS) or sulfoNHS to increase coupling efficiency or create a stable amine-reactive product. EDC can be used in esterification reactions (coupling of carboxylic acid to alcohol) with catalytic amounts of DMAP (Fig. 8.4).

6.3 Coupling reactions involving N-hydroxysuccinimide (NHS) ester derivative As NHS is highly reactive at physiological pH, it is used for amine coupling reactions in bioconjugation. As shown in Fig. 8.5, the NHS ester compounds react with nucleophiles to form an acylated product with NHS as a leaving group. Carboxyl groups activated with NHS esters are highly reactive with amine nucleophiles. Carboxyl groups are easily reacted with amine nucleophiles after their activation by NHS esters (Table 8.2). Apart from using coupling reagents, dendrimer prodrugs can be prepared through formation of coordinate covalent bond between the functional groups of dendrimers and metal-containing drugs [26,27].

FIG. 8.4 EDC structure.

FIG. 8.5 NHS structure.

Chapter 8 • Design of dendrimer based prodrugs

Table. 8.2

205

Summary of linkers used in the design of dendrimer prodrugs

Dendrimer

Linker

Drug

Ref

Polyaryl ether and poly ester-OH G4 PAMAM-COOH G4 PAMAM-COOH G2.5 PAMAM-COOH G4 PAMAM-OH G2–6 PAMAM-OH G4 PAMAM-NH2 G0 PAMAM-NH2 G3 PAMAM-NH2 G2.5 PAMAM COOH G1 PAMAM-NH2 G4.5 PAMAM-COOH G4.5 PAMAM-COOH

Carbonate ester Carboxylate ester Carboxylate ester Carboxylate ester Carboxylate ester Phosphate ester Amide Amide Amide Amide Thiourea Coordination bond Coordination bond

Cholesterol-OH Glucosamine 6-sulfate-NH2 Glucosamine-NH2 Venlafaxine-OH Ibuprofen-COOH ( )-Beta-D-(2R, 4R)-dioxolane-thymine (DOT)-OH Ibuprofen-COOH Naproxen-COOH Methotrexate-COOH Methotrexate-NH2 N-Acetyl glucosamine-NCS Cisplatin Diaquo(1,2-diaminocyclohexane)platinum(II)

[17] [18] [18] [19] [20] [21] [22] [23] [24] [24] [25] [26] [27]

7 Dendrimer-prodrugs with spacers The large or bulkier size (molecular weight) of dendrimers causes steric hindrances for direct covalent conjugation with the drug molecules where the reactive functional groups may not be at suitable distance to react each other or other atoms may be blocking any reaction. Further, direct covalent linkage of drugs to dendrimer could lead to resistance toward hydrolysis under mild conditions due to steric crowding around the linkage. Bifunctional spacers may be incorporated to separate drug from dendrimer during the conjugation to avoid the steric hindrance and crowding effect and control the site and the rate of release of free drug from the conjugate. Therefore, spacer plays an important role in the design of dendrimer prodrugs and has a significant influence on properties of the prodrugs, such as drug loading ability, water solubility, site and rate of drug release. The combination of appropriate spacers, as well as conjugation strategies, leads to distinct pharmacokinetic behaviors of the dendrimer prodrugs. For this reason, a wide variety of different spacer units including glycidol, succinic acid, lactic acid, glutaric acid, p-amino benzoic acid etc. have been investigated in terms of their suitability in the design of dendrimer prodrugs [8,9] (Table 8.3).

8 Conclusion Dendrimers offer several advantages such as low polydispersity, regular branching, predictable size, shape, three-dimensional globular architecture, multi valency and well-defined molecular weight. The large number of reactive functional groups on the periphery of dendrimers provide an opportunity for high drug loading ability in the dendrimer-based prodrugs. Further, the drug loading ability can be altered by selecting suitable dendrimer generations, carriers, linkers and model drugs. The release of drug

Summary of spacers used in the design of dendrimer-prodrugs Spacer

G5 PAMAM-NH2

Spacer structure

Drug

Ref

Glycidol

Methotrexate

[28]

G4 PAMAM-NH2

Succinic anhydride

Dexamethasone

[29]

G1 PAMAM-NH2

Succinic acid

Terfenadine

[30]

G4 PAMAM-OH

Succinic acid

Paclitaxel

[31]

G1 PAMAM-NH2

Diethylene glycol

Terfenadine

[30]

G0 PAMAM-NH2

Lactic acid

Naproxen

[32]

1,4,7,10-Tetraazacyclododecane cored PAMAM-NH2

1-Bromoacetyl

5-Fluorouracil

[33]

G4 PAMAM-OH

Glutaric acid

MethylprednisoloneOH

[34]

Polyaryl ether-COOH

Hydrazine

[35]

PEGylated G4 PAMAM-NH2

cis-Acotinyl

MethotrexateCOOH Doxorubicin-NH2

[36]

G3 PAMAM-NH2

p-Amino benzoic acid

Salicylic acid

[37]

Pharmaceutical Applications of Dendrimers

Dendrimer

206

Table 8.3

p-Amino hippuric acid

Salicylic acid

[37]

G4 PAMAM-NH2

Glycine-PhenylalanineLeucine-Glycine (GFLG)

Paclitaxel

[38]

PEGylated peptide dendrimer

Glycine-PhenylalanineLeucine-Glycine (GFLG)

Doxorubicin

[39]

Dendrimer-COOH

Glycine-PhenylalanineLeucine-Glycine (GFLG)

Doxorubicin

[40]

Lysine peptide Dendrimer

Glycine-PhenylalanineLeucine-Glycine (GFLG)

Gemcitabine

[41]

G5 PEGylated Poly L-Lysine dendrimer

PVGLIG

Methotrexate

[42]

Chapter 8 • Design of dendrimer based prodrugs

G3 PAMAM-NH2

207

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Pharmaceutical Applications of Dendrimers

molecules from the dendrimer prodrugs can be controlled by choosing suitable degradable linkers thus allowing targeted accumulation of drug molecules at specified site. Taken together, these attractive properties make dendrimers a perfect scaffold in the design of polymeric prodrugs. Dendrimer prodrugs have demonstrated their applications in increasing the aqueous solubility of hydrophobic drugs, controlled and target specific release and improving the pharmacokinetics. In spite of these advantages, most of them of dendrimer prodrugs that are reported in the literature are limited to proof-of-concept. The major limitation that prevents these prodrugs from pre-clinical translation is their biocompatibility and cytotoxicity. Future efforts should be directed toward the development of dendrimer prodrugs with long blood circulation times, and perfect site-specific drug release systems which allows the drug molecules selectively to accumulate at the targeted sites for the efficient therapy.

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chet JM. Water-soluble dendrimer–poly (ethylene glycol) starlike conjugates as [17] Liu M, Kono K, Fre potential drug carriers. J Polym Sci A 1999;37(17):3492–503. [18] Shaunak S, Thomas S, Gianasi E, Godwin A, Jones E, Teo I, Mireskandari K, Luthert P, Duncan R, Patterson S, Khaw P. Polyvalent dendrimer glucosamine conjugates prevent scar tissue formation. Nat Biotechnol 2004;22(8):977. [19] Yang H, Lopina ST. Extended release of a novel antidepressant, venlafaxine, based on anionic polyamidoamine dendrimers and poly (ethylene glycol)-containing semi-interpenetrating networks. J Biomed Mater Res A 2005;72(1):107–14. [20] Kolhe P, Khandare J, Pillai O, Kannan S, Lieh-Lai M, Kannan RM. Preparation, cellular transport, and activity of polyamidoamine-based dendritic nanodevices with a high drug payload. Biomaterials 2006;27(4):660–9. [21] Liang Y, Narayanasamy J, Rapp KL, Schinazi RF, Chu CK. PAMAM dendrimers and branched polyethyleneglycol (nanoparticles) prodrugs of (-)-β-D-(2R, 4R)-dioxolane-thymine (DOT) and their anti-HIV activity. Antivir Chem Chemother 2006;17(6):321–9. chet JM, Szoka FC. Polyester dendritic systems for drug [22] Padilla De Jesu´s OL, Ihre HR, Gagne L, Fre delivery applications: in vitro and in vivo evaluation. Bioconjug Chem 2002;13(3):453–61. [23] Najlah M, Freeman S, Attwood D, D’Emanuele A. Synthesis, characterization and stability of dendrimer prodrugs. Int J Pharm 2006;308(1–2):175–82. [24] Gurdag S, Khandare J, Stapels S, Matherly LH, Kannan RM. Activity of dendrimer-methotrexate conjugates on methotrexate-sensitive and -resistant cell lines. Bioconjug Chem 2006;17(2):275–83. [25] Vannucci L, Fiserova´ A, Sadalapure K, Lindhorst TK, Kuldova´ M, Rossmann P, Horva´th O, Kren V, Krist P, Bezouska K, Luptovcova´ M. Effects of N-acetyl-glucosamine-coated glycodendrimers as biological modulators in the B16F10 melanoma model in vivo. Erratum in Int J Oncol 2014;44(4):1410. Int J Oncol 2003;23(2):285–96. [26] Howell BA, Fan D. Poly (amidoamine) dendrimer-supported organoplatinum antitumour agents. Proc Math Phys Eng Sci 2009;466(2117):1515–26. [27] Howell B, Fan D, Rakesh L. Thermal decomposition of a generation 4.5 PAMAM dendrimer platinum drug conjugate. J Therm Anal Calorim 2006;85(1):17–20. [28] Zhang Y, Thomas TP, Desai A, Zong H, Leroueil PR, Majoros IJ, Baker Jr JR. Targeted dendrimeric anticancer prodrug: a methotrexate-folic acid-poly (amidoamine) conjugate and a novel, rapid, “one pot” synthetic approach. Bioconjug Chem 2010;21(3):489–95. [29] Choksi A, Sarojini KVL, Vadnal P, Dias C, Suresh PK, Khandare J. Comparative anti-inflammatory activity of poly(amidoamine) (PAMAM) dendrimer-dexamethasone conjugates with dexamethasone-liposomes. Int J Pharm 2013;449(1–2):28–36. [30] Najlah M, Freeman S, Attwood D, D’Emanuele A. Synthesis and assessment of first-generation polyamidoamine dendrimer prodrugs to enhance the cellular permeability of P-gp substrates. Bioconjug Chem 2007;18(3):937–46. [31] Khandare JJ, Jayant S, Singh A, Chandna P, Wang Y, Vorsa N, Minko T. Dendrimer versus linear conjugate: influence of polymeric architecture on the delivery and anticancer effect of paclitaxel. Bioconjug Chem 2006;17(6):1464–72. [32] Najlah M, Freeman S, Attwood D, D’Emanuele A. In vitro evaluation of dendrimer prodrugs for oral drug delivery. Int J Pharm 2007;336(1):183–90. [33] Zhuo RX, Du B, Lu ZR. In vitro release of 5-fluorouracil with cyclic core dendritic polymer. J Control Release 1999;57(3):249–57. [34] Khandare J, Kolhe P, Pillai O, Kannan S, Lieh-Lai M, Kannan RM. Synthesis, cellular transport, and activity of polyamidoamine dendrimer-methylprednisolone conjugates. Bioconjug Chem 2005;16(2):330–7.

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[35] Danieli E, Shabat D. Molecular probe for enzymatic activity with dual output. Bioorg Med Chem 2007;15(23):7318–24. [36] Zhu S, Hong M, Zhang L, Tang G, Jiang Y, Pei Y. PEGylated PAMAM dendrimer-doxorubicin conjugates: in vitro evaluation and in vivo tumor accumulation. Pharm Res 2010;27(1):161–74. [37] Wiwattanapatapee R, Lomlim L, Saramunee K. Dendrimers conjugates for colonic delivery of 5-aminosalicylic acid. J Control Release 2003;88(1):1–9. [38] Satsangi A, Roy SS, Satsangi RK, Vadlamudi RK, Ong JL. Design of a paclitaxel prodrug conjugate for active targeting of an enzyme upregulated in breast cancer cells. Mol Pharm 2014;11(6):1906–18. [39] Zhang C, Pan D, Luo K, Li N, Guo C, Zheng X, Gu Z. Dendrimer–doxorubicin conjugate as enzymesensitive and polymeric nanoscale drug delivery vehicle for ovarian cancer therapy. Polym Chem 2014;5(18):5227–35. [40] Lee SJ, Jeong YI, Park HK, Kang DH, Oh JS, Lee SG, Lee HC. Enzyme-responsive doxorubicin release from dendrimer nanoparticles for anticancer drug delivery. Int J Nanomedicine 2015;10:5489–503. [41] Zhang C, Pan D, Li J, Hu J, Bains A, Guys N, Zhu H, Li X, Luo K, Gong Q, Gu Z. Enzyme-responsive peptide dendrimer-gemcitabine conjugate as a controlled-release drug delivery vehicle with enhanced antitumor efficacy. Acta Biomater 2017;55:153–62. [42] Kaminskas LM, Kelly BD, McLeod VM, Sberna G, Boyd BJ, Owen DJ, Porter CJ. Capping methotrexate α-carboxyl groups enhances systemic exposure and retains the cytotoxicity of drug conjugated PEGylated polylysine dendrimers. Mol Pharm 2011;8(2):338–49.

Dendrimers in gene delivery

9

Divya Bharti Raia, Deep Poojab, Hitesh Kulharia a

SCHOOL OF NANO SCIENCES, C ENTRAL UNIVERSITY OF GUJARAT, GANDHINAGAR, INDIA b PHARMACOLOGY AND T OX ICOLOGY DIVISIO N , C S I R - I NDI A N I N S T I T U T E O F C H E MI C A L TECHNO LOGY , HYDERABAD, INDIA

1 Introduction Gene therapy is a recombinant technique that employs genetic material in advance treatment strategies. This technique may allow treating a disorder by inserting a functional gene into a patient’s cells rather than using drugs or surgery [1]. This therapy involves transfer of the identified gene with therapeutic function, specifically and efficiently to the targeted cells, through a carrier termed as vector. Gene vector may be viral or non-viral gene vectors. The viral vectors are used for transfection based on their intrinsic properties such as carrying owns genome from one host cell to the other, navigating a new target cell, entering to the cell nucleus and initiate expression of its genome and replicate [2]. Viruses such as retrovirus, poxvirus, alphavirus, lentivirus, herpes simplex virus, adenovirus, and adeno-associated virus are modified into gene-delivery means by substituting some of the fragment of a viral genome with a functional gene [3]. During initial years of gene therapy development, viral vectors became popular due to their high efficacies in gene delivery both in vitro and in vivo. Later on, certain concerns such as immunotoxicity and genotoxicity, large-scale production problems, and high manufacturing cost were observed [4]. Because of these disadvantages, researchers are coming up with a variety of non-viral alternatives including polymers, liposomes and exosomes, peptides and proteins and nanoparticles [5–9]. Non-viral vectors interact with deoxyribose or ribonucleic acid through electrostatic forces, condense the genetic material into units of a few tens to hundred nanometers thereby protecting the gene fragment and facilitates cellular entry (Fig. 9.1). Such complexes of gene fragment with cationic lipids/polymers are known as lipoplex and polyplex, respectively [10]. Dendrimers are suitable polymer for gene delivery operations attributing to formation of more compact complexes with DNA, oligonucleotides, genes, aptamers, siRNA etc. through a non-viral mechanism [11]. Spherical architecture and polyvalency makes dendrimer a suitable platform for gene delivery. Flexible chemistry of dendrimers offers numerous properties required for efficient gene delivery such as biological compatibility, manufacturing feasibility, stable formulation and controlled delivery of gene. They can efficiently condense nucleic acids into small nanoparticles by ionic interactions and protect them from endosomal or lysosomal degradation [10]. Pharmaceutical Applications of Dendrimers. https://doi.org/10.1016/B978-0-12-814527-2.00009-3 © 2020 Elsevier Inc. All rights reserved.

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FIG. 9.1 Polyplex formation. (A) Polycations and DNA interacting electrostatically and form polyplexes. (B) Micrograph of polyplexes obtained from TEM, scale bar 200 nm [10].

However, the efficiency of transfection depends on properties of carrier, i.e., dendrimers as well as on extracellular and intracellular parameters. The conformation, surface functionality, generation number, size and other physiochemical properties of carrier are crucial of optimum delivery [12]. There are many in vivo factors that create barriers to gene entry into the cell. Hence there is a need to design next generation delivery systems such as dendrimer for obtaining maximum efficiency and easy in gene transport by using best features of both viral and non-viral vectors. This section depicts the mechanism of non-viral gene delivery through dendrimer and the factors influencing the pathway in detail. The barriers for efficient gene delivery and the strategies to overcome the problems such as designing suitable type of dendrimer molecules with the surface or core modifications has also been discussed.

2 Mechanism of gene delivery using dendriplexes Gene delivery is a sophisticated process comprising a sequence of complex events. The process begins with incorporation of a therapeutic gene within the dendrimers, hence forming the dendriplex. This dendriplex adheres to the cellular surface, enters by endocytosis, escapes intracellular endolysosomal vesicles into the cytoplasm, passes across the cytoplasm toward the nucleus and crosses through the nuclear membrane (Fig. 9.2). Alternative pathways exist for several of these steps. Finally, the dendriplex releases the encapsulated or cross-linked gene in the target area [10].

2.1 Synthesis of dendriplexes Dendrimers are cationic polymers bearing a diversity of DNA-binding functional entities, among are primary, secondary, tertiary and quaternary amine groups, as well as other positively charged groups such as amidines groups which lies in the backbone of the polymer [10]. These polymer binds to DNA through electrostatic interactions between the

Chapter 9 • Dendrimers in gene delivery

+

+

+

+

+

Dendrimer

213

+ +

+

+

+

+

Condensation +

+

+

+ +

+

+

+

+

Nucleic acid

Core effect Flexibility Hydrophobicity Functionality

+

Serum stability

+ +

+ +

+ +

+ +

Cellular uptake End

oso me

+ +

+

Nucleus

+

+ +

+ +

Nuclear entry

Endosome escape

+ +

+

intracellular release +

+

+

+

+

+ + +

+ +

+

+

+

+

+

+ +

Proton sponge

Dendrimer

FIG. 9.2 Mechanism of gene transfection through a cationic dendrimer [13].

negatively-charged phosphates along the DNA backbone and positive charges displayed on the dendrimer vector and condense it into tiny, compact particles [14]. The genespolymer condensation is energetically favorable and dendriplexes form readily upon mixing of dendrimer with the genetic sample [15,16]. The resulting particles possess spherical or toroidal structures of diameter ranging from 30 to several hundred nanometers approximately. Each dendriplex comprises several DNA molecules along with dendrimers. The shape and conformation of dendriplexes seems to be altered by kinetic parameters and often depends on the steps of mixing (for example, dendrimer is added to DNA solution or DNA to dendrimer solution) and stoichiometric ratio. For instance, in gene delivery excess of polycationic dendrimer is mixed, which generates particles with a positivelycharged surface. An overall net positive charge of the dendrimer-nucleic acid complex (dendriplex), is thereby essential for attachment to the negatively charged cell membrane and thus facilitating the cellular uptake.

2.2 Targeted dendriplexes Dendrimers lack capacity to target cells specifically itself but it can be modified by attaching target specific ligand on its surface for selective targeting. Modification of dendrimer

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with targeting ligands can be utilized for receptor-mediated endocytosis into the cell. Glycosidic moieties and other low-weight receptor molecules such as folate and retinoic acid enables specific targeting of cell types which display the complementary receptor protein [17–20]. A variety of surface crosslinking interactions have been used to attach targeting ligands to the polymers [21].

2.3 Intracellular trafficking Polyplexes are generally internalized by endocytic pathway. Untargeted polyplex particles bind electrostatically to the surface of cells and enters the cell via adsorptive pinocytosis [22], whereas polyplexes conjugated with targeting ligands attaches to surface receptors on specific cell, and internalized through the process of receptor-mediated endocytosis. In either case, the polyplexes is enclosed inside the endocytic vesicles—the early endosome [23]. Then it may fuse with sorting endosomes and subsequently can be transported back toward the cell membrane and out of the cell in exocytosis or transported to the late endosome vesicles that maintains acidic pH 5–6 due to the action of an ATPase enzyme which constantly pump protons inside the vesicle membrane. Later, polyplexes can be trafficked into lysosomes containing various degradative enzymes and further suspended to lower pH of about 4.5.

2.4 Endolysosomal escape DNA and vector across the cytoplasm, must elude out through these compartments, to reach the nucleus; the event is called endolysosomal escape. Certain materials that are capable of mediating their own endosomal escape are known as “proton-sponge” polymers through a unique mechanism based on proton-sponge hypothesis.

2.4.1 Proton-sponge hypothesis “Proton-sponge” polymers have a large number of secondary and tertiary amines, for example, polyethylenimine (PEI) and polyamidoamine (PAMAM) dendrimers and hence exhibit pKa values within the range of physiological and lysosomal pH [24–26]. An ATPase enzyme is present to actively transport protons from the cytoplasm into the endolysosomal vesicle and creates acidic environment (Fig. 9.3). As the vector polymers internalize, it becomes protonated during endocytic trafficking. In case of proton-sponge polymers, ATPase requires to pump protons in excess number to reach the pH required for degradation of polymer. To balance excess of protons in the vesicle, the counter ions are fluxed inside the vesicle. This increases the ionic concentration and ultimately causes rupture of the endosome membrane due to osmotic swelling, releasing polyplexes into the cytosol [27].

2.5 Transport across the cytoplasm After releasing from endosomal compartments, polyplexes must migrate across the cytoplasm toward the nucleus where the encapsulated gene could be transcribed [28].

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H+ ATPase

B

CI–

H+

CI– BH+

ATPase

+

BH

+

BH

H

CI–

–

CI B

B

BH+

CI–

H+

CI–

+

BH+

H+

B

H+ B

+

BH

CI–

BH+

CI–

CI– CI–

CI–

CI–

FIG. 9.3 Schematic diagram of the proton-sponge mechanism. Protonation of the proton-sponge polymer (green (dark gray)) causes increased influx of protons (and counter-ions) into endocytic vesicles. Increasing osmotic pressure causes the vesicle to swell and rupture [10].

2.6 Nuclear localization It is crucial for polyplex crossing across the double-layered nuclear membrane. Polyplex invasion is mediated by certain nuclear localization signals (NLS) on the nuclear membrane. NLS are short stretch of positively-charged amino acid sequences that can be processed by specific protein involve in import across the nuclear membrane known as importins. Polyplex also being cationic in nature may be recognized by importing the same way as the NLS [29–34]. Moreover, the therapeutic gene may also incorporate within some sequences encoding for nuclear translocation [25].

2.7 Release of genetic material from dendriplexes As described above, complexation within the polymer prevents any enzymes or nucleases such as binding of proteins required for gene expression. At the final step of the gene delivery, the vector must release the genes to be transcribed in the nucleus. Polymer degradation and unbounding of gene from polyplex may be done by the decreasing DNA-carrier binding affinity [35], by reducing the net positive charges [36], PEGylation or reducing the molecular weight of the vector polymer [36] ultimately increasing the gene expression. Polymers must be designed for controlled discharge of the genes, preferably within the nuclear space.

3 Factors affecting dendrimer-based gene delivery Dendrimer-based gene delivery depends on the nature of the polymeric vector and the subsequent environment of the transportation route as well as the localized site. Several factors such as functional group, charge, hydrophilicity, concentration, molecular weight, generation, conformation, and valency affect the success and fate of gene delivery. Furthermore, environmental and physiological factors such as pH, serum resistance and ionic concentration at the biologically targeted area must also be considered.

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3.1 Structure of dendrimers Higher generation dendrimers are more suitable for gene delivery because it provides a higher surface density of functional groups for binding of genes.

3.2 Size and molecular weight Molecular size and weight of the polymeric carrier effects transfection process drastically. Since internalization of polyplex into the cell via endocytosis is a size limiting process [37,38], less bulky polymer would easily be enclosed in endocytic vesicle. However, very small-sized may not load a large-size transgene fragment completely. Considering the two factors, higher generation dendrimers (>G4) with an average size and molecular weight apparently are the best suited as gene carrier.

3.3 Surface charge and charge density As explained earlier, the cationic dendrimer possessing an excess of positive charge on dendriplex facilitates the interaction with the negatively charged cell membrane and consequently the internalization [10]. Additionally, the charge-neutralizing effect of negatively charged macromolecules present in serum is countered by an excess of positive charge, hence conferring some serum stability. The positive charge density increases to twofold with successive generation number with doubling of surface groups. Also, smaller particle size obtained with G5 dendrimers with higher condensation capacity compared with G4 dendrimer because of higher number of amino groups in the surface.

3.4 Polyvalency Dendrimers exhibit two unique architecture features: (i) spherical architecture and (ii) polyvalency. Multiple surface groups on a defined spherical architecture provides polyvalency. Dendrimers with a higher number of terminal groups enables multiple interactions simultaneously with serum solvent, cellular boundaries, or molecular groups and consequently show high affinity, solubility and reactivity. In a similar manner, numerous interactions between phosphate backbone of nucleic acid and polymeric amines are important for proficient binding in dendrimer-DNA polyplex. Additionally, various surface moieties can be conjugated synergically to make interactions with targeting or hydrophilic ligand, for example, PEG for enhancing stability by reducing steric hinderance [39,40].

3.5 Hydrophilicity Hydrophilic nature of the dendrimer surface increases solubility and hence aggregation of the delivery system is forbidden.

3.6 Polymer-DNA charge ratio The stability and cellular uptake of gene vector usually requires a high polymer/DNA charge ratios or nitrogen/phosphorus ratios (N/P ratios) to form polyplex.

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DNA-dendrimer structures with increased solubility and lower density can be formed at higher dendrimer-DNA charge ratio, enhancing transfection levels and generation of more reactive functional complexes. However, such high polymer/DNA charge ratio raises positive charge excessively and causes cytotoxicity [41].

4 Design criteria for formulating dendrimer vectors for gene delivery •

•

• • •

•

•

•

•

•

Protection of therapeutic gene: An efficient condensation and encapsulation of nucleic acid are essential for preventing its degradation in various in-vivo compartments and from various nucleases. Encapsulation of large DNA plasmids: As dendrimers have definite size, a right generation of dendrimers should be selected to provide sufficiently large size and interbranching voids to package longer fragments carrying the whole of gene encoding sequences. Easy administration: Various types of formulation that can be introduced via different routes, could flexibly be manufactured using the polymer. Ease of fabrication: The polymer should have possibility to manufacture at large scale and low cost, easily modified and designed as per requirement. Robustness/stability: Serum resistance and delayed systemic clearance lead to increase half-life of the DNA-dendrimer complex, thus contribute to increase bioavailability. Internalization: Interaction to the cell membrane and permeation through the membrane are the events crucial in both receptor-mediated and non-targeted endocytosis. Endolysosomal escape: The vector particle may be susceptible to degradation while transportation across various extra and intracellular compartments. Thus, it must possess some intrinsic pH buffering capacity to withstand a wide range of pH. Nuclear transport: Nuclear localization signal—the polymeric carrier or the transgene itself—have incorporated sequences recognizable to the importin protein which is responsible for the intranuclear entry. Efficient unpackaging: Dendrimers can be tailored with a system for regulated and stimulus-dependent release of the genes, optimally in the nucleus such as auto degrading properties, tunable integrating forces, binding affinity. Partially heatdegraded or disulfide-containing polymers can easily be fragmented in a reducing environment such as the cytosol and nucleus. Safety: Gene delivery expected to be a vector which is nontoxic, non-immunogenic and biodegradable in nature can be considered as for preventing any adverse effects. As soon as after releasing the gene at its target site, the dendrimers should be degraded, and sub polymeric particles have to be cleared out of the system [10].

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5 Problems in dendrimer-based gene delivery Despite having great advantage of dendrimer-mediated gene delivery over other viral and non-viral vectors, this approach has major challenges of low transfection efficiency and cytotoxicity. Also delivering gene with untailored dendrimer vectors has only a moderate cellular specificity. Certain factors such as polyplex condensation, circulation half-life, cellular entry, bypassing endolysosomal degradation, nuclear localization, unpackaging of the dendriplexes can be accounted for the successful transfection of gene. In vivo toxicity is also triggered by a few factors in dendrimer-based gene delivery such as low biodegradability. Delivery efficacy and cytotoxicity of dendrimer-based delivery system are apparently correlated. The transfection efficacy increases along with the higher generation number so as the toxicity. On the contrary, low-generation dendrimers have minimal toxicity but extremely low transfection efficacy. Therefore, the actual need is to balance optimum efficacy and toxicity [42]. While employing such vector system for clinical purpose, the terminal amine groups on the dendrimer surface (responsible for both efficacy and cytotoxicity) can be modified to attain the optimum efficacy while managing the toxicity. For providing specifically targeted gene delivery, specific ligands could be anchored on dendrimer for targeting the signature molecules of the target cells and guide the controlled transfection in certain cells. Multiple approaches can be used to achieve the above goals which includes: • • •

Designing a novel dendrimer structure by improvising a new core unit. Modification of already designed dendrimer molecules, such as fluorinateddendrimers or photoreactive-dendrimers. Incorporating other biodegradable or bioactive polymer such as PEG to prepare hybrid polymers with diverse properties for gene delivery [10].

6 Surface modification in dendrimer for efficient gene delivery Despite numerous advantages, dendrimer-based gene delivery systems are compromised due to less efficiency and toxicity. Recent studies over this issue have proposed surface-engineered dendrimer vector to design more efficient and biocompatible polymeric gene vector (Table 9.1). Following functional ligands and modifying molecules have been proved successful in increasing potential of dendrimer in gene delivery platforms.

6.1 Amino acid-modified dendrimers Various amino acids with different functional groups have distinct properties. Amino acids hydrophobic in nature such as phenylalanine and leucine often used to improve the cellular uptake of polyplexes and increases their transfection efficiency by balancing the charge and hydrophobic contents in the polymer [43,50].

Chapter 9 • Dendrimers in gene delivery

Table 9.1

219

Dendrimer surface-modifying ligands and their significance

S. no.

Dendrimer

Modifying ligand

Property of ligand

Significance

Ref.

1.

G4 PAMAM

Amino acid

Positively charged groups, buffering capacity, hydrophobicity

[43]

2.

G2, G3 PAMAM

Carbohydrates

3.

G5 PAMAM

Protein or peptide

Targetability, affinity for lipid molecules located on cell membranes Targetability

4.

G4, G5, G6 PAMAM

Polymer or Lipid

Raise molecular weight, act as protective barrier to prevent transgene from nucleases. Fusogenic, lipophilic, hydrophobic, Balance cationic charge and lipid content of polymeric vector

5.

G1, G5 PAMAM, PPI

Others such as Nanoparticles, Cationic moiety, Fluorine

Unique properties, such as photothermal, magnetic, and fluorescent properties. Increases the charge density on the dendrimer. Fluorine are both hydrophobic and lipophobic, low surface energy due to high-affinity fluorous-fluorous interactions, hydrogen bond formation with water, sensitive probe in nuclear magnetic resonance

Improves DNA- and membrane-binding affinity, endosomal escape and cellular uptake, serum resistance Improves affinity for cell membrane, Receptormediated endocytosis Receptor-mediated endocytosis in cancer cells and brain cells Increase the serum stability and blood circulation time of the dendriplexes, which is essential for in vivo applications. Facilitates internalization of dendriplex either by passive transport due to membrane destabilization or by caveolae-mediated endocytosis. Excellent serum-resistance, low cytotoxicity. Generate micelles in aqueous solutions, which allows the co-delivery of anticancer drugs and therapeutic genes Widely used in the diagnosis and treatment of many diseases. Improves DNA binding capacity and pH buffering capacity, reduces cytotoxicity, improved cellular uptake, serum resistance, aqueous solubility, endosomal escape, and intracellular DNA release, transfection at extremely low N/P ratios, in vitro and in vivo monitoring of gene delivery by magnetic resonance imaging (MRI)

[44]

[43]

[45,46]

[47–49]

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Pharmaceutical Applications of Dendrimers

In a different approach, conjugation of cationic amino acids such as lysine and arginine on the terminal amines of dendrimers provides more positive charge, thus improve the transfection process. Arginine and lysine have two positively charged groups which significantly increase the positive charge density at polymer surface and aids to DNA condensation [51–53]. Also, a guanidinium group is present in which positive charge is delocalized on three nitrogen atoms; hence guanidinium strongly interacts with phosphates of DNA backbone [54]. The guanidinium group in arginine strongly binds the cell membranes by ionic and hydrogen bonding [55,56]. The increase of efficacy through arginine- and lysine modification of dendrimers is quite specific for a particular dendrimer species. For example, PAMAM dendrimers with arginine are more efficient than lysine-modified ones [51,52], while in case of lysine-modification, PPI dendrimers are more efficient than those with arginine-modification [57,58], linkage between an amino acid and dendrimer also influence gene delivery; for example, when amino acid conjugate dendrimer with degradable ester bonds, creates more efficient and biocompatible gene vector than those having non-degradable amide bonds [56]. Histidine is another amino acid which improves the transfection efficacy in cationic dendrimers. It has a protonable imidazole group with a pKa value around 6.0. So, histidine improves the pH buffering capacity of cationic dendrimers and facilitates the escape from endolysosomal disruption. The histidine-modified dendrimers also acquires serumresistant the same way as with arginine due to the presence of an inert imidazole group [43]. Multiple functionalizations of dendrimers with several amino acids can be done in a combination of arginine, histidine, and phenylalanine on PAMAM dendrimer surface produces additive results in gene delivery [59,60]. In addition, the amino acid conjugation with cationic polymers can add on some unique physicochemical features of the polymers, such as solubility and mucoadhesiveness [61].

6.2 Protein- or peptide-modified dendrimers Besides saccharide, proteins and peptides also may be used as targeting ligands in cancertargeted or site-specific gene delivery. Epidermal growth factor receptors (EGFR) which are highly expressed in cancer cells, can enhance internalization of EGF conjugated dendrimers by the cancer cells [62]. Another protein, transferrin is used to penetrate highly selective blood-brain barrier (BBB) through receptor-mediated endocytosis. The endothelial cell surface of brain capillaries has an abundance of transferrin receptors. Therefore, conjugation of transferrin to gene carriers may improve the expressions of a target gene in the brain [63]. Several peptides may also help the dendrimer/DNA polyplexes to penetrate the BBB such as 29 residues long amino acid sequences isolated from rabies virus glycoprotein specifically binds to nicotinic acetylcholine receptor on neuronal cells [64]. Peptides conjugation on dendrimers maintain binding affinity with the receptors, reduce spatial hindrance on the exterior of dendrimer, and improve the stability of vectors during gene

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delivery up to more extent as compared to protein. The above examples of protein/peptide-targeted gene delivery are active targeting on the basis of receptor-mediated endocytosis. Similarly, a polymeric vector with a cell-penetrating peptide or a nuclear localization signal (NLS) peptide may improve intracellular trafficking of the polyplex [44,65,66].

6.3 Carbohydrate-modified dendrimers Dendrimers can be functionalized by maltotriose or maltose protected oligonucleotides from nuclear degradation at neutral pH [67]. Saccharide carbohydrates such as cyclodextrin and mannose are another group of molecules potentially enhance the efficacy of gene delivery vectors. Internal cavities in cyclodextrin can encapsulate and bind hydrophobic molecules such as cholesterol, cholic acid, and other lipid molecules located on cell membranes and improve polymer uptake at cell membranes [68–72]. PAMAM dendrimer modified with three β-CD molecules to encapsulate retinoic acid which forms an inclusion structure with the polymeric vector. The small interfering-RNA loaded in such cyclodextrin conjugated dendrimer enhance stem cell differentiation, growth, and survival [73]. Certain sugars, for example, mannose, galactose, and lactose can be internalized by receptor-mediated endocytosis. Mannose receptors are present in a high number of macrophages and dendritic cells. Dendrimers modified with these saccharides may perform cell-specific gene delivery [44,74].

6.4 Polymer- or lipid-modified dendrimers Polyethylene glycol (PEG) is extensively used polymer for synthesizing dendrimer conjugates. Such polymer modification found to increase the transfection efficacy while reducing cytotoxicity [75–77]. PEGylation of dendrimers can increase the serum stability and blood circulation time of the polyplexes, which otherwise rapidly cleared by the reticuloendothelial systems when administrated via the intravenous route [78–80]. For efficient gene delivery to the mucosal tissues, the polyplex should be capable of penetrating across the mucosa layer and be inert to the mucus constituents. A recent study shows that conjugating a cationic PAMAM dendrimer with a high density of PEG chains enable penetration across the mucus layer and potentially can be applied for the treatment of mucosal diseases [81]. Lipid functionalization improves the cellular uptake and membrane-disrupting activity of cationic dendrimers due to the hydrophobic effect and fusogenic property of the lipids [82]. Lipids are amphipathic molecules having a hydrophilic head motif and hydrophobic tail end. A positively as well as a negatively charged region interconnected by a disulfide bond is present at the hydrophobic head at physiological pH. Also, the positive region of the head is attached to the hydrophobic tail at another side. As the lipids contact with cellular membranes or taken up by cells, the disulfide bond is cleaved by the membrane-bound and intracellular factors which removes the region bearing negative charge from the head group. Thus, lipid becomes cationic and therefore fusogenic, i.e., facilitate fusion with negatively charged cell membranes [83]. Lipids with longer hydrophobic chain enhance cellular uptake much more, for example, lauric acid < myristic

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acid < palmitic acid [82]. However, the strong bonding made by longer lipids while modifying dendrimer hinders dendrimer breakdown and intranuclear DNA release. Lipid-modified dendrimers in aqueous environment aggregates to form micelles which may be used to deliver anticancer drugs along with the gene. High-generation dendrimers with more efficacy in gene transport but confers severe cytotoxicity, and on the contrary, low-generation dendrimers are least toxic but then transfection efficacy is also low [84]. Therefore, lipid-association with the dendrimer polymer provides a solution since lipid-modified low-generation dendrimer acts like a lipid gene vector with comparatively more efficacy rather than a polymeric vector.

6.5 Other ligand-modified dendrimers Dendrimers have been modified with several other moieties like hormones, folic acid, cationic molecules for different purposes. Dendrimers have also been conjugated to nanoparticles and photosensitizers for incorporating additional therapeutic or diagnostic properties. •

•

•

•

•

Hormone-modified dendrimers: dendrimer modified with triamcinolone acetonide, a glucocorticoid with nuclear targeting property, significantly improves the transfection efficacy of dendrimers [85]. Folic Acid-modified dendrimers: Folic acid receptors are overexpressed on a variety of cancer cells allows efficient DNA and siRNA delivery in folic acid receptor overexpressing cells [86,87]. Nanoparticle-modified dendrimers: Nanoparticles such as carbon nanotubes, graphene, quantum dots, gold nanoparticles, metal nanoparticles and silicon nanomaterials are widely used in diagnosis and treatment of many diseases due to certain properties such as photothermal, magnetic and fluorescent properties [45,47,88–94]. A dendrimer may be grafted with these nanostructures to prepare multifunctional gene vectors with reduced cytotoxicity [95]. For example, PAMAM dendrimers modified with gold nanorods used to deliver short hairpin RNA and enhance gene transfection efficacy via near-infrared light irradiation. Such materials can kill cancer cells by gene therapy along with photothermal ablation [96]. Dendrimers are modified with magnetic Fe3O4 nanoparticles or bacterial magnetic nanoparticles in siRNA delivery [95–97]. Photosensitizer-modified dendrimers: Photosensitizing compounds such as porphyrin degrade endosomal and lysosomal membranes upon photoactivation [98]. In grafted dendrimer, it improves transfection efficacy upon laser exposure. Also, photosensitizing compounds also be employed to monitor the in-vivo location of gene vector in cells due to fluorescent property [99–101]. Cationic moiety-modified dendrimers: Cationic dendrimers usually have primary amine groups on their surface. Such dendrimers can be further conjugated with other cationic moieties, such as oligoamine, tertiary amine, quaternary ammonium, imidazolium, guanidium, and phosphonium to increases the charge density on the

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dendrimer. There is an odd-even effect on gene transfection efficacy of oligoamines grafted on the polymers. The oligoamines with even-numbered repeating aminoethylene units show high buffering capacity at acidic pH compared to those with odd-numbered aminoethylene units [102]. Fluorinated dendrimers: The cellular uptake and stability of gene vector is maintained by high polymer-to-DNA charge ratios or nitrogen/phosphorus ratios, N/P ratios in polyplexes. However, at such high polymer/DNA charge ratio, excess positive charge leads cytotoxicity [41]. Alternatively, fluorinated dendrimer has a low surface energy due to high-affinity fluorous-fluorous interactions. Hence it assembles into nano- or microaggregates and fluorinated dendrimer bind nucleic acid in the form of polymeric aggregates rather a single polymer [103]. Fluorinated dendrimers such as G5-F768 achieve optimal gene transfection at extremely low positive-to-negative charge ratios or N/P ratios (1.5:1 to 2:1) without neglecting biocompatibility [104]. Perfluoroalkyls are both hydrophobic and lipophobic, hence the fluorinated dendrimers remain inert in serum proteins and exhibit excellent serum-resistance in gene delivery. Fluorine is conventionally used as a highly sensitive probe in nuclear magnetic resonance and this application in association with dendrimers may be used to track in vivo and in vitro gene delivery by magnetic resonance imaging (MRI) [103,105–108]. Fluorophore-modified dendrimers: Dendrimers modified with a fluorescent dye such as Oregon Green enables visualization of the vector during gene transfection. Also, the hydrophobic effect of the fluorescent dye attributes high delivery efficiency [13].

7 Modification of dendrimer core for efficient gene delivery Dendrimer core also plays important roles in gene delivery. It influences the overall dendrimer conformation, number of surface groups and size, and physicochemical properties of the dendrimers, such as flexibility, hydrophobicity, and functionality [108].

7.1 For flexibility of dendrimers The flexibility of dendrimers can be efficiently improved by introducing one or more flexible linkers to the core, modulating branching multiplicity, or increasing the core mobility. Better flexibility of the dendrimers is beneficial for efficient DNA condensation and gene transfection. Partially degraded PAMAM dendrimers (fractured dendrimers) with more open and flexible structures have improved transfection efficacy (>50-fold) as compared to intact dendrimers having a globular shape with a relatively rigid and densely packed structure [109]. Conjugating three flexible ethylene glycol units in the TEA-core dendrimers expand the distance between PAMAM dendrons and hence binds to DNA homogeneously. Also, flexible core creates void spaces within the dendrimer structure, facilitating the interaction of tertiary amine groups in dendrimers to water molecule for tertiary amine protonation and subsequent endosomal escape of the

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dendriplexes [110–112]. Dendrimer flexibility can also be enhanced by tailoring the core branching multiplicity.

7.2 For hydrophobicity of dendrimers Modification of dendrimer core with lipids such as fatty acids and cholesterol can efficiently enhance its transfection efficacy by improving serum stability, membrane fusion, cellular uptake and intracellular DNA unpacking of the dendriplexes [113]. These lipid-conjugated at the dendrimer core facilitate endolysosomal escape due to fusogenic activity of lipid. Moreover, the lipid-cored dendrimers increase efficiency of gene delivery because of lowest nuclease activation. The lipid-cored dendrimer also has minimal toxicity on the transfected cells [114–116]. This is attributed to only one lipid chain in the lipid-cored dendrimer buried in the dendrimer interior, which decreases the damage of lipid moiety to the cell membranes [46].

7.3 For functional versatility of dendrimers Modifying dendrimer cores can introduce new properties and functions in the dendrimers. Similar to conjugation at surface, porphyrins in the dendrimer core is used as photosensitizer to convert light to energy and heat and efficiently improve the cytoplasmic delivery of DNA by photochemical disruption of endosomal membrane [46,98].

8 Conclusion With a growing understanding of human genome and dendrimer chemistry, various types of dendrimer have been designed mediating efficient non-viral gene delivery. The high level of control over size, shape, charge and surface functionality of dendrimers allow to design a nucleic acid or cargo-specific for a particular disease and for desired therapeutic potential. As a non-viral gene delivery platform, dendrimers have already proven to enhance DNA complexation, serum stability, controlled release of encapsulated genes and to increase the tissue and cellular uptake. However, the challenge of safety with efficient delivery of genetic material is still not solved and concerned limitations of dendrimers such as cytotoxicity, low targetability and the release of the nucleic acid from the dendrimer complex needs emphasis. For addressing the mentioned issues, high density of surface groups can allow attachment of a variety of targeting groups, which could enhance transfection efficacy and reduce cytotoxicity. Thus, designing multifunctional dendrimers may be visualized as a promising and safe option of non-viral vectors for gene therapy in the future perspective.

Acknowledgments H.K. acknowledges Department of Science and Technology, New Delhi for INSPIRE Faculty Award. D.B.R. acknowledges University Grant Commission, New Delhi for M. Phil Fellowship. Figures have been incorporated after reprint permissions under licence number 4597581045819 (Fig. 9.1 and 9.3) and 4597581176674 (Fig. 9.2).

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[98] Nishiyama N, Iriyama A, Jang WD, Miyata K, Itaka K, Inoue Y, Takahashi H, Yanagi Y, Tamaki Y, Koyama H, Kataoka K. Light-induced gene transfer from packaged DNA enveloped in a dendrimeric photosensitizer. Nat Mater 2005;4(12):934–41. [99] Nomoto T, Fukushima S, Kumagai M, Machitani K, Arnida, Matsumoto Y, Oba M, Miyata K, Osada K, Nishiyama N, Kataoka K. Three-layered polyplex micelle as a multifunctional nanocarrier platform for light-induced systemic gene transfer. Nat Commun 2014;5(3545):1–10. [100] Ma D, Zhao Y, Zhou XY, Lin QM, Zhang Y, Lin JT, Xue W. Photoenhanced gene transfection by a starshaped polymer consisting of a porphyrin core and poly(L-lysine) dendron arms. Macromol Biosci 2013;13(9):1221–7. [101] Uchida H, Miyata K, Oba M, Ishii T, Suma T, Itaka K, Nishiyama N, Kataoka K. Odd–even effect of repeating aminoethylene units in the side chain of N-substituted polyaspartamides on gene transfection profiles. J Am Chem Soc 2011;133(39):15524–32. [102] Criscione JM, Le BL, Stern E, Brennan M, Rahner C, Papademetris X, Fahmy TM. Self-assembly of pH-responsive fluorinated dendrimer-based particulates for drug delivery and noninvasive imaging. Biomaterials 2009;30(23–24):3946–55. [103] Wang M, Liu H, Li L, Cheng Y. A fluorinated dendrimer achieves excellent gene transfection efficacy at extremely low nitrogen to phosphorus ratios. Nat Commun 2014;5(3053):1–8. [104] Thurecht KJ, Blakey I, Peng H, Squires O, Hsu S, Alexander C, Whittaker AK. Functional hyperbranched polymers: toward targeted in vivo 19F magnetic resonance imaging using designed macromolecules. J Am Chem Soc 2010;132(15):5336–7. [105] Jiang ZX, Liu X, Jeong EK, Yu YB. Symmetry-guided design and fluorous synthesis of a stable and rapidly excreted imaging tracer for 19F MRI. Angew Chem Int Ed Engl 2009;48(26):4755–8. [106] Takaoka Y, Sakamoto T, Tsukiji S, Narazaki M, Matsuda T, Tochio H, Shirakawa M, Hamachi I. Selfassembling nanoprobes that display off/on 19F nuclear magnetic resonance signals for protein detection and imaging. Nat Chem 2009;1(7):557–61. [107] Dı´az-Lo´pez R, Tsapis N, Fattal E. Liquid perfluorocarbons as contrast agents for ultrasonography and 19F-MRI. Pharm Res 2010;27(1):1–16. [108] Tang MX, Redemann CT, Szoka FC. In vitro gene delivery by degraded polyamidoamine dendrimers. Bioconjug Chem 1996;7(6):703–14. [109] Liu X, Liu C, Catapano CV, Peng L, Zhou J, Rocchi P. Structurally flexible triethanolamine-core poly(amidoamine) dendrimers as effective nanovectors to deliver RNAi-based therapeutics. Biotechnol Adv 2014;32(4):844–52. [110] Zhou Z, Lu Z. Dendritic nanoglobules with polyhedral oligomeric silsesquioxane core and their biomedical applications. Nanomedicine 2014;9(15):2387–401. [111] Liu X, Wu J, Yammine M, Zhou J, Posocco P, Viel S, Liu C, Ziarelli F, Fermeglia M, Pricl S. Structurally flexible triethanolamine core PAMAM dendrimers are effective nanovectors for DNA transfection in vitro and in vivo to the mouse thymus. Bioconjug Chem 2011;22(12):2461–73. [112] Liu Z, Zhang Z, Zhou C, Jiao Y. Hydrophobic modifications of cationic polymers for gene delivery. Prog Polym Sci 2010;35(9):1144–62. [113] Yuba E, Nakajima Y, Tsukamoto K, Iwashita S, Kojima C, Harada A, Kono K. Effect of unsaturated alkyl chains on transfection activity of poly(amidoamine) dendron-bearing lipids. J Control Release 2012;160(3):552–60. [114] Khan OF, Zaia EW, Yin H, Bogorad RL, Pelet JM, Webber MJ, Zhuang I, Dahlman JE, Langer R, Anderson DG. Ionizable amphiphilic dendrimer-based nanomaterials with alkyl-chain-substituted amines for tunable siRNA delivery to the liver endothelium in vivo. Angew Chem Int Ed Engl 2014;53(52):14397–401.

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[115] Yu T, Liu X, Bolcato-Bellemin A-L, Wang Y, Liu C, Erbacher P, Qu F, Rocchi P, Behr JP, Peng L. An amphiphilic dendrimer for effective delivery of small interfering RNA and gene silencing in vitro and in vivo. Angew Chem Int Ed Engl 2012;51(34):8478–84. [116] Cheng Y, Zhao L, Li T. Dendrimer-surfactant interactions. Soft Matter 2014;10(16):2714–27.

Further reading [117] Banaszczyk MG, Lollo CP, Kwoh DY, Phillips AT, Amini A, Wu DP, Mullen PM, Coffin CC, Brostoff SW, Carlo DJ. Poly-L-lysine-graft-PEG-comb-type polycation copolymers for gene delivery. J Macromol Sci A 1999;36(7–8):1061–84. [118] Nishikawa M, Huang L. Non-viral vectors in the new millennium: delivery barriers in gene transfer. Hum Gene Ther 2001;12(8):861–70. [119] Wood KC, Little SR, Langer R, Hammond PT. A family of hierarchically self-assembling lineardendritic hybrid polymers for highly efficient targeted gene delivery. Angew Chem Int Ed Engl 2005;44(41):6704–8. [120] Peng F, Su Y, Zhong Y, Fan C, Lee ST, He Y. Silicon nanomaterials platform for bioimaging, biosensing, and cancer therapy. Acc Chem Res 2014;47(2):612–23. [121] Cheng Y, Morshed RA, Auffinger B, Tobias AL, Lesniak MS. Multifunctional nanoparticles for brain tumor imaging and therapy. Adv Drug Deliv Rev 2014;66:42–57.

10

Dendrimers in immunotherapy and hormone therapy

Srinivasa Reddy Bonama,b,c,d,*, Aparna Aretie, Prashanth Komirishettye,f, Sylviane Mullerc,d,g,h a

VACCINE IMMUNOLOGY LABORATORY, NATURAL P ROD U CT S CHEMI STR Y DI VI SIO N , CS IRINDIAN INSTITUTE O F CHEMICAL T ECHNOLOGY, HYDERABAD, INDI A b AC A DE MY O F SCIENTIFIC AND INNOVATIVE RE SEARCH (AcSIR ), CSI R-II CT C AMPUS, HY DERABAD, INDIA c CNRS, B IOTECHN OLOGY AND CELL SIGNALING, UNIVERSITY OF STRA SBOU RG, ILLKIRCH, FRANCE d LAB ORAT ORY OF EXCE LLENCE MEDALIS, S TRASBOURG, FRANCE e DEPARTMENT OF PHARMACOLOGY AND T OX ICOLOGY, NATIONAL INSTITUTE OF PHARMACEUTICAL EDUC AT ION AND RESEAR CH (NIPER)-HYDERABAD, BALANAGAR, INDIA f DIVISION OF NEUROLOG Y & NEUR OSCIENCE AND MENT AL HEALT H I NS TI TU TE , DE PART MENT OF MEDICINE, UNIVERSITY OF ALBERTA, EDMONTON , A B, CANADA g UNI VERSI TY OF STRASBOUR G INSTI TUTE  ERA  FOR ADVANCED STUDY, STRASBOUR G, FRANCE h F ED T IO N HO S P IT AL O - U NI VE RSI TA IR E  ER  AT IO N D E M EDECINE  OMI CA RE, F ED T RANSLATIONNELLE DE STRASBOURG, STRASBOURG UNIVERSI TY, S TRASBOURG, F RANC E *C O R R E S P O NDI NG AUT HOR. E-MAI L ADDR ESS: BSR PH A RMACY9 0@ G MAI L.C OM

1 Dendrimers in immunotherapy 1.1 Effect of dendrimers on innate immune system Innate immune system is the primary defense mechanism and it provides non-specific protection against various invading pathogens. The innate immune system is composed of diversified cells such as monocytes, macrophages, dendritic cells (DCs), granulocytes, natural killer cells, and complement proteins. Many of the aforementioned cells recognize pathogen or its components (pathogen-associated molecular patterns (PAMPs) or damage-associated molecular pattern (DAMPs)) through pattern recognition receptors (PRRs) [1]. Dendrimers use in vaccines and immunostimulatory are reviewed by Peter M. H. Heegaard et al. [2]. Natural killer (NK) cells, robust innate immune cells, play an important role in the infectious and malignant diseases, non-specifically. Dendrimers capped with azabisphosphonate showed increase NK cell activity and its activity is mediating via CD4+ T cells [3]. Nanosized dendrimers tagged with phosphonate have shown increased proliferation of NK cells, thereby killing the tumor cells, in-vitro [4]. The same groupalso developed the anti-inflammatory dendrimers. Phosphorous containing dendrimers capped with acid azabisphosphonate stimulated the monocytes and CD4+ T cells to secrete Th2 mediated cytokines such as IL-4 and IL-10 respectively [5]. Pharmaceutical Applications of Dendrimers. https://doi.org/10.1016/B978-0-12-814527-2.00010-X © 2020 Elsevier Inc. All rights reserved.

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Azadiphosphonate-capped dendrimers containing nitrogen with various chemical substituent’s have generated a logical structure-activity relationship (SAR), smallest hydrophobic groups (methyl, allyl, and butyl) are enhancing the CD14 expression and longer hydrophobic substituent’s decreasing the same expression, which represents monocytes [6]. Recently, different dendrimers with carbohydrates complexation have been reported. Glycopeptide modified dendrimers with different linker substituent’s have come up with successive immunomodulatory dendrimers, which are inducers of antigen cross-presentation [7]. Interestingly, few glycopeptide dendrimers acting against disease-causing organisms (e.g., Pseudomonas aeruginosa) have emerged by the use of combinatorial chemistry technique [8] and these glycopeptides (FD2 (C-Fuc-LysProLeu)4 (LysPheLysIle)2LysHisIleNH2 and PA8 (OFuc-LysAlaAsp)4(LysSerGlyAla)2LysHisIleNH2) have shown potent inhibition and dispersion of biofilms (P. aeruginosa) by forming ligands with lectin LecB [9]. Angela Berzi et al. developed a novel pseudo mannosylated dendrimer (Polyman26), in which mannose moieties are ligands to C-type lectins and exhibits immunomodulating properties. Studies on monocytes derived dendritic cells (MoDCs) with Polyman26 either alone or in combination with lipopolysaccharide (LPS) has proved that Polyman26 is effectively binding to dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) and toll-like receptor (TLR)s 9. It clearly induced the secretion of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and chemokines (CCL3, CCL4, and CCL5) and also an expression of co-stimulatory markers (CD40L) [10]. Interestingly, functional modification of the former said dendrimer with different hydrophobic groups have shown highest competitive binding to LPS and it also inhibited the LPS induced nitric oxide production, which is involved in many inflammatory diseases. Later, the modified dendrimer also is shown its effectiveness in-vivo by protecting mice from LPS induced septic shock [11]. Polyamidoamine (PAMAM) conjugated with amino sugar (glucosamine) also inhibited LPS induced cytokines and chemokines production from both macrophages and DCs but it induced the increased response of co-stimulatory molecules. In the same study, authors conjugated the glucosamine 6-sulfate to the PAMAM dendrimer, which was shown the antiangiogenic property. Consequently, a combination of glucosamine and glucosamine 6-sulfate conjugated to dendrimer inhibited the formation of scar tissue (Table 10.1) [12, 13]. Most of the dendrimer-antibody conjugates were targeting focused receptors without fail, but many of them are not as effective as non-conjugated substances [14,15]. The untold story behind this might be that dendrimers sustained release of the component. Oligomannose decorated PAMAM dendrimers conjugated to a carrier protein (CRM197; non-toxic mutant of diphtheria toxin) have generated high antibody responses than conjugates without dendrimer [16]. G3-carbohydrate dendrimers exhibited potent competitive inhibition of DC-SIGN over the Ebola pseudotyped viral particles [17]. Abnormal glycosylation is used as a biomarker for cancer cells identification. Glycopeptide cancer antigens, especially T-antigen disaccharides, were conjugated to many types of dendrimers (PAMAM, polypropylene imine (PPI), N,N0 -bisacrylamide acetic acid, and hyperbranched L-lysine) were used as screening purposes [18]. It has been proven that Man2, Man4,

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Man9 (mannose), and other fucose/mannan derivatives are standard ligands of DC-SIGN. Juan J. Garcı´a-Vallejo et al. conducted a study on targeted delivery of antigens to DCs, which reveals the effect of polyvalent glycopeptide dendrimers. In this study, they found that polyvalent dendrimers (16–32 glycans) induced the antigen uptake, processing, and crosspresentation [19]. Plant-derived dendrimer like particles composed of α-D-glucan has induced DCs stimulation, secretion of IL-1β and IL-12. It also acted as vehicle system for vaccine delivery, upon injection, it recruited the monocytes at the site of injection [20]. Dendrimers linked with peptides decorated with mannose have shown effective uptake by DCs and macrophages, which is central property required by vaccine delivery systems [21]. Cationic carbosilane dendrimer (G2-NN16) were used to deliver the human immunodeficiency virus (HIV) peptides (p24/gp160 or p24/gp160/NEF) to DCs effectively [22]. Ingrid S. Ganda et al. designed a peptide conjugated dendrimer vaccine against Chlamydia trachomatis. G4-PAMAM dendrimer hydroxyl terminated (G4OH) was conjugated to the similar to chlamydial glycolipid antigen (Peptide 4; AFPQFRSATLLL). This conjugation was made by a cleavable ester linkage, by which authors wants to enhance the antigen presentation. As hypothesized, in in-vivo vaccine adjuvant studies, it has shown increased antibody (IgG) titer, than gold standard, alum. Further, these formulation has shown a reduced infectious load of C. trachomatis [23]. Few nanosized PAMAM dendrimers are used to maintain homeostasis, mainly in the inflammatory diseases involved with prostaglandins [24]. Remy Poupot’s group also developed a novel dendrimer construct [25, 26], which originated monocytes activation [27], either to induce pro-inflammatory cytokines with co-stimulation [28], or to induce anti-inflammatory effect [29] target inflammation and osteoclastogenesis [30], uveitis [31], neuroinflammation [32], immunotherapy against multiple melanoma [33]. PAMAM dendrimers shown generation induced toxicity on macrophages, higher the generation and higher the toxicity (G6 > G5 > G4). Further, this toxicity is mediated by generation induced reactive oxygen spices (ROS) production and thereby activation of inflammatory mediators (MIP-2, TNF-α, and IL-6) [34]. Autophagy, a self-destructive process against unwanted cells, type II programmed cell death and brings homeostasis to the entire biological system, especially immune system. Factors agitating this process have been implicated in the many autoimmune diseases. Thus, developing novel molecules or delivery systems, which reverse the alterations in the immune defense process is useful [35]. Shaofei Wang et al. reported that the cationic G5-PAMAM dendrimers induced neuronal/glioblastoma cell death by regulating autophagy. In this study, they found reduced cell viability upon treatment of PAMAM dendrimer. Their downstream signaling study revealed that dendrimer induced autophagic flux by inhibiting negative regulator of autophagic markers (Akt/mTOR/p70S6K). In addition, they found, the dendrimer is generating ROS production, intracellularly on glioblastoma cells [36]. The same group confirmed the anti-cancer activity/cytotoxicity of said dendrimers on hepatocellular carcinoma, which is also mediated through autophagy pathway. Interestingly, the authors found that PAMAM dendrimer enhances inflammasome

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formation, which activates the inflammatory caspases [37–39]. The antibacterial drug, cysteamine conjugated to the G4-PAMAM dendrimer was also shown to regulate autophagy pathway with increased bioavailability [40].

1.2 Effect of dendrimers on adaptive immune system Immunotherapy is an emerging strategy for all types of diseases. Among the various diseases or ailments, targeted drug delivery is essential for the treatment of cancer. Many monoclonal antibodies (MoAbs) have been directed against tumors and tested in in-vivo models. Notwithstanding, their efficacy is still limited. Efficacy of MoAbs is increased in conjunction with dendrimers in comparison with alone treated. MoAbs linked to boronated starburst dendrimer have shown targeted delivery against murine B16 melanoma [41]. Dendrimers are widely used in the radiolabeling for targeted therapy in cancers. In contrast, Hisataka Kobayashi et al. conjugated a monoclonal antibody IgG1 (OST7) to a polyamine dendrimer (G4-(1B4M)43), which is obtained by the combination of fourth generation PAMAM (G4) and (1B4M 2-(p-isothiocyanatobenzyl)-6-methyl-diethylene triaminepenta-acetic acid). Its radiolabeled (indium (In) and gadolinium (Gd)) products have shown labeling specific immunoreactivity, biodistribution, and tumor targeting in mice. Further, they identified that gadolinium (153Gd-OST7-G4-(1B4M)43/G4-(1B4M)64) has exhibited specificity toward human osteosarcoma xenograft tumor (KT005) compared to indium (111In-OST7-G4-(1B4M)43/G4-(1B4M)64). They found that with this method, they are able to bind 49 times metal atoms to the antibody (Table 10.1) [42]. Antibody conjugated therapies to target tumors is one of the successful strategies in cancer therapeutics. It showed no toxicity to normal tissues. Like other conventional strategies, it also suffers from few difficulties, i.e., solubility and targeted delivery. Modified dendrimers offer a unique property, targeted delivery; G5-PAMAM dendrimer conjugated with anti-PSMA (prostate specific membrane antigen) antibody (J591) has shown binding and internalization into cells overexpressing PSMA [43, 44]. Tumor cells circulated in the bloodstream enable many biomarkers in the patients, which are often hard for the early diagnosis [45]. Dendrimers usage was also considered in early diagnosis applications because of their high sensitivity and capture efficiency. A study was done by James B. Otis et al. provides a stable platform for the detection of HER-2 (human epidermal growth factor receptor-2) positive cancers. In this study, the authors constructed a nano-imaging agent, Au-G5-Gd-Herceptin, which is a combination of modified PAMAM dendrimer and gold nanoparticles (AuNPs) with Gd. These modified dendrimers are obtained by the conjugation of polyethylene glycol (PEG) and DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to the surface of PAMAM dendrimer. Finally, dendrimer encapsulated AuNPs were conjugated to humanized anti-HER-2 (human epidermal growth factor receptor 2) antibody (Herceptin) (with fluorescently tagged it is conjugate 7), which is selective for cancer cells. Their in-vitro studies on lung (A549) and breast (SKBR-3) cancer cells proved that final nanoconjugate (Fig. 10.1) has selectively internalized in the HER-2 overexpressing cells with affecting their survival (Table 10.1) [46].

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FIG. 10.1 PAMAM dendrimer-antibody conjugate against HER-2receptor.

Rameshwar Shukla et al. targeted the former said receptor, i.e., HER-2. In this study, authors wisely used the chemotherapeutic agent, methotrexate, encapsulated with fifth generation PAMAM dendrimer and conjugated to the MoAb against HER-2 (G5-FI-HNMTX). As anticipated, it exhibited the same kind of dihydrofolate reductase inhibitory activity but relatively it has shown less cytotoxicity on HER-2 overexpressing MCA 207 (3-methylcholanthrene (MCA)) cells as compared to methotrexate alone [14]. As dendrimers are enormously used for the different distinct biomarkers targeting, Jingjing Xie et al. explored their views on colorectal metastatic cancer, in which authors targeted against Sialyl Lewis X (saliva acidifying Louis oligosaccharides X (Slex), a type II carbohydrate antigen) biomarker. In this study, authors designed and synthesized two fluorescently labeled dendrimer-antibody conjugates namely G6-3aSlex and G6-5aSlex (Sialyl Lewis X antibodies (aSlex)-conjugated PAMAM dendrimers) and evaluated their binding and restraining abilities against HT29 cells. Interestingly, their results revealed that the conjugates have effectively bound to the HT29 cells in a time and dose-dependent manner, where increasing the time and doses were found to enhance the binding efficiency. Further, these dendrimers have shown the significant antiproliferative effect on HT29 cells. The mechanism behind this antiproliferation was confirmed by cell cycle arrest at S phase and loss of mitochondrial membrane potential [45]. Conjugating DOX (doxorubicin) with dendrimer (Pol(Star)-DOX) alone or dendrimer-antibody conjugate (RTX-Pol-DOX; Rituximab (RTX)), star-shaped polymer conjugates was done by Ondrej Lidicky´ et al. In their findings, they observed that both conjugates are shown effective in-vitro release, cytotoxicity, targeted binding (RTX-Pol-DOX), and antitumor effects. However, the in-vivo antitumor effect of RTX-Pol-DOX has shown equal to that of Pol(Star)-DOX. Interestingly, authors found that combination of Pol(Star)-DOX with RTX has shown highest survival when compared to others (Table 10.1) [15]. Dendrimer-antibody conjugate, cystamine core dendrimer and panitumumab (Ab-(G4S15)4, Ab-(G5S29)4) were designed for the MRI imaging purpose [47]. Dendrimers tagged with targeted ligand have shown high biodistribution studies in tumor xenografts models compared to monoclonal antibodies. Konda S. D. et al. designed a Gd-folate-G4-PAMAM dendrimer, which is targeted to the highly expressed human folate receptors (hFR). Studies on in-vivo xenograft tumor models found that dendrimer tagged with folate has shown better tumor-blood ratio distribution [48].

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Among several companies, Starpharma Pvt. Ltd (Abbotsford, Australia) has come up with dendrimer product called VivaGel™ (SPL7013 Gel). VivaGel™ is composed of SPL7013 and polymer Carbopol®. SPL7013 contains benzhydrylamineamides of L-lysine with four generations of L-lysine, to create amine groups on the surface (32 amine groups). For each amine, sodium 1-(carboxymethoxy)naphthalene-3,6-disulfonate group is attached via amide linker. SPL7013 (3%) admixed to buffered Carbopol® gel with a physiological pH (compatible with the normal human vagina) [49, 50]. Clinical trials (phase I and II (NCT00740584)) have shown potent antiviral activity against HIV-1 and HSV-2 (herpes simplex virus) [51]. Moreover, SPL7013 binds to targets on the pathogens (viruses) thereby blocking the viral attachment to host cells, to prevent infection. In contrast to HIV, SPL7013 targets gp120 viral proteins, which helps in the viral entry into CD4+ T cells [50]. Recently, phase III clinical trials (NCT01577537) concluded that VivaGel™ is effective in the treatment of bacterial vaginosis (Table 10.1).

1.3 Role of dendrimers as vaccine delivery systems Currently, vaccines are abundantly formulated with delivery systems with or without adjuvants to improve its efficacy against targeted antigens [1]. Dendrimers are widely used as delivery systems against drugs, antigens, and in many applications like ocular, transdermal, oral, and others [52]. In modern immunology manipulating materials those can modulate immunity is an important approach, especially, development of vaccines against specific diseases. Vaccine antigen delivery to a specific target with nanoformulations/materials is limitedly available in the clinics, without long-term toxicity [1]. Constructing advanced materials, which could initiate the adaptive immunity through innate immune signals without causing reactogenicity and autoreactivity is a foremost step in novel delivery systems. Dendrimers are one such composite, with well-characterized architecture [53]. In general, vaccine antigens/foreign matter enters through receptor-mediated endocytosis, particularly via mannose receptors like Dectin-1, MR (CD206), DC-SIGN (CD209). The others have shown considerable antigen presentation and T-cell stimulation than non-receptor mediated endocytosis. Several dendrimers were developed and succeeded pre-clinically and cationic charged PAMAM dendrimers have been used as adjuvants in an influenza vaccine [54]. There was a low rate of success in development of DNA vaccines until the formulation delivery systems emerge. PAMAM dendrimers were used for delivery of Chlamydophila psittaci, DNA vaccination [55]. Sheng et al. confirmed the above-statement both in- vitro and in- vivo with modified dendrimer. Mannosylated dendrimer OVA (MDO), synthesized by the authors have shown increased T-cell proliferation (OTI (CD8+) or OTII T (CD4+)) via both MHC I and II antigen presentation. High concentration of mannose has shown early endosomal uptake, with enhanced co-stimulatory markers, antigen-specific antibody titer, cross-presentation in lymph nodes and effective cell-mediated immunity (IFN-γ) with no observed significant toxicity. Further, prophylactic efficiency was summarized against B16-OVA melanoma tumor model. Mice treated with MDO have shown delayed tumor growth or 6 months

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Table 10.1

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List of dendrimer conjugates and uses in immunological disorders

Dendrimer

Type

Use

Reference

Boronated starburst dendrimer 153 Gd-OST7-G4(1B4M)43/G4-(1B4M)64 Au-G5-Gd-Herceptin

Dendrimer-Antibody conjugate Dendrimer-Antibody conjugate Dendrimer-Antibody conjugate Dendrimer-Antibody conjugate Dendrimer-Antibody conjugate Dendrimer-Antibody conjugate Polymer conjugate DendrimerAntibody-Drug conjugate Dendrimer conjugate

Effective against murine B16 melanoma

[41]

Detection of osteosarcoma

[42]

Detection of HER-2 positive cancers

[46]

Detection and inhibition of saliva acidifying Louis oligosaccharides X (Slex) antigen positive cancer cells MRI imaging

[45]

Delivery of methotrexate to HER-2 overexpressing MCA 207 cells Targeted delivery Targeted delivery to the CD20 expressed mature B cells

[14]

G6-3aSlex and G6-5aSlex Ab-(G4S15)4, Ab-(G5S29)4 G5-FI-HN-MTX Pol(Star)-DOX RTX-Pol-DOX

Gd-folate-G4-PAMAM Pseudo mannosylated dendrimer (Polyman26) PAMAM glucosamine PAMAM glucosamine 6-sulfate PAMAM-DENCYS

[47]

[15] [15]

Polymer conjugate

Biodistribution of Gd-folate dendrimer against hFR positive tumors Immunomodulatory

[48] [10]

Polymer conjugate Polymer conjugate

Immunomodulatory Antiangiogenic

[12] [12]

Polymer-drug conjugate

Cystic Fibrosis

[40]

Abbreviations: Ab, antibody; CD, cluster of differentiation; DEN, dendrimer; HuIg, human immunoglobulin; MTX, methotrexate; Slex, saliva acidifying Louis oligosaccharides X/Sialyl Lewis X.

increased survival capacity compared to untreated mice [56]. Istvan Toth and Mariusz Skwarczynski laboratories are continuously working on the development of novel vaccine delivery systems especially polymer-peptide conjugated delivery systems. In contrast, they have developed dendrimer, a 4-arm polymer-peptide conjugate system to deliver human papillomavirus (HPV), type 16 (HPV-16) E7 oncoprotein, which is highly tumorigenic. Among the polymers they have studied, linear poly(t-butyl acrylate) polymer (1), branched linear polymer (2), 4-arm star polymer (3), and 4-arm dendritic polymer (4), the 4-arm dendritic polymer have shown increased uptake on macrophages and DCs. Moreover, the treated groups showed more activation of both CD4+ and CD8+ T cells than untreated group; the survival rate (90%) on mice (C57BL/6 mice bearing myeloma (TC-1) cells) was significantly higher as compared to gold standard, 8Qmin + ISA51 (8Qmin emulsified in Montanide ISA51) [57]. Many peptide-based vaccines efficacy were limited by a deficit of selective adjuvant or delivery systems. In one of the studies by Istvan Toth group on vaccine development against group A Streptococcus (GAS), with dendrimers

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as delivery systems have delivered favorable results. C-terminus of the M protein (p145), is one of the conserved epitopes from the 180 serotypes, it is attached to the chemically synthesized polyacrylate dendritic polymer. In- vivo studies on murine models reveal that p145 with dendrimers have shown increased p145 specific antibody titer, which was found from the blood, withdrawn on 50 days after intranasal immunization, and also observed increased levels of subtype antibodies (IgG1, IgG2a, IgG2b, and IgG3) especially IgG1(Th2) subtype. However, no significant differences in IgA levels from both salivary and fecal titers were observed in the dendrimer treated group. Moreover, this delivery system also induced the high levels of opsonization. Interestingly, delivery system co-administered with cholera toxin B-unit (CTB) has delivered increased vaccine efficacy in all aspects [58]. Multiple epitope copies of J14 were conjugated to the polyacrylate dendrimer has shown higher levels of antigen-specific antibody (IgG) titers along with subtypes (IgG1, IgG2a, IgG2b, and IgG3) and efficacy has been increased, when it was co-administered with complete Freund’s adjuvant, bleed at 37 days, after subcutaneous immunization. However, IgG1 (Th2) titer was superior to IgG2a (Th1) [59]. Xiaoting Wang et al. from China have recently used PAMAM-Lys as DNA vaccine delivery systems against Schistosoma japonicum Infection, one of the major infectious diseases in China. PAMAM conjugated with L-lysine has shown reduced toxicity on HEK293T (human embryonic kidneys) as compared to poly-L-lysine (PLL) and it has also increased the efficiency of gene delivery (75%) than free polymer (unconjugated) one (60%). PAMAM-Lys with PJW4303-SjC23 (DNA vaccine) antigen given intramuscularly has shown higher antigen antibody titers with Th1 (IgG2a) response, i.e., IL-2 and IFN-γ. Being a Th1 mediated vaccine delivery system; it has enhanced the protective immunity against S. japonicum challenge [60]. Dendrimers are eliciting antigen-specific antibody response without hemolysis effect [61]. Of the other formulations, amino terminated 5th generation dendrimer has produced IgG1 (Th2) response, which is essential for B cell-mediated immune responses [62]. Dendrimers also used in the development of vaccine adjuvant systems in the sexually transmitted infections (STI). C. trachomatis infection is one of the serious STI. Many drug conjugated dendrimers and nanodevices were developed against C. trachomatis [63, 64]. The same group was consistently working on the design and development of novel dendrimers; such as dendrimers in loading high amount of drugs [65], targeted delivery of drugs (ocular delivery) [66–69], as sensor for cytokines and other biomarkers [70, 71], anti-inflammatory including but not limited to neuroinflammation [72–74], lung inflammation [75], brain inflammation [76], and anti-oxidant [77], anti-cancer [78], antimicrobial activities [79], and many other outcomes [80].

2 Dendrimers in hormone therapy Hormone therapies exert therapeutic effectiveness by preventing the production of hormones or hampering hormone activities [81]. Hormonal therapy in oncology is one of the major modalities of medical oncology have superiority over the chemotherapy [82]. It involves the maneuvering of the endocrine system through the exogenous delivery of

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specific hormones, particularly steroid hormones, or drugs which inhibit the production or activity of such hormones. Hormonal therapy is used for quite a lot of types of cancers originated from hormonally reactive tissues, together with the breast, prostate, endometrium, and adrenal cortex [81]. Hormonal drugs are steroid in structure and definitely, there is a need to control the delivery of the drug because when they are delivered conventionally, causes serious side effects [83]. In this context, various researchers have investigated the delivery of hormonal drugs as nanomedicines for targeted cancer therapies [84]. Remarkable success in targeted therapies has been achieved with the use of dendrimer-based nanomedicines [85, 86]. Dendrimers offer a high degree of suppleness in drug loading. Drugs can be either covalently conjugated to the dendrimer surface or entrapped inside dendrimers by non-covalent interactions. For example, drugs with gene plasmids and nucleic acids components are complexed with dendrimers through electrostatic interactions [87, 88]. Antiinflammatory and anti-microbial agents with chemical structures can be conjugated by covalent structures to the dendrimers [89]. There is extensive interest in diverse topics ranging from synthesis and functionalization of dendrimers, design principles and considerations for the utility of dendrimer features, to dendrimer-based drug delivery systems for treatment of various diseases [90]. But the construction of suitable dendrimer for hormonal drug delivery presented some problems. This is because hormones are steroidal structures having rather apolar or few functional polar groups available (for example, hydroxyl groups). These structural issues affect the synthesis of suitable dendrimers for hormonal drug therapy [91]. For this purpose, a variety of dendrimers have been developed and used since the 1980s, but the dendrimers which have been derived from PAMAM are unquestionably the most employed. They are hydrophilic, biocompatible, and non-immunogenic systems, most commonly the core is made of ethylenediamine which helps their use in drug delivery [92]. PPI dendrimers are another type of dendrimers synthesized as a cascade of molecules where the core is based on a 1,4-diaminobutane (DAB). PLL dendrimers are a type of peptide dendrimer used mostly as gene carriers due to their excellent condensation with oligonucleotides. Among these types of dendrimers, PAMAM is the one widely used to conjugate the hormonal drugs [93, 94]. Researchers like Kannan and co-workers explored two different PAMAMdendrimers, a G3.5 dendrimer with 32 dCOOH groups and G5-PAMAM-OH dendrimers with 64 dOH groups. These modifications made easy conjugation of hormonal drugs like methylprednisolone. In-vitro experimentations found that these conjugates were rapidly taken up by the cells mainly accumulating in the cytosol and has shown significant improvement in the activity when compared to the conventional delivery of methylprednisolone [95]. In the other example, ovarian cancer can be targeted by designing drug delivery systems carrying follicle stimulating hormone (FSH) ligand. In ovarian cancers, it has been proved that there is overexpression of follicle stimulating hormone receptor (FSHR) [96]. Hence, targeted ovarian cancer therapy can be achieved by designing drug

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delivery systems carrying FSH ligand. In recent times, Modi et al. designed a dendrimer-based therapy to target FSHR. This dendrimer was constructed by coupling the binding peptide domain of FSH (FSH33) to PAMAM dendrimer G5 [97]. The high selectivity of FSH33-dendrimer conjugates was established. Furthermore, the conjugates displayed receptor antagonistic effect by down-regulating the survivin, an antiapoptotic protein [98]. Other major hormones widely used in the treatment of various diseases, including prostate and breast cancer, and reproductive disorders, such as infertility and precocious puberty is Luteinizing hormone-releasing hormone (LHRH) analogues. LHRH is the important regulator of the hypothalamic-pituitary-gonadal (HPG) axis that is accountable for the development and functioning of the reproductive system. Delivery of a continuous supply of LHRH agonists causes down-regulation of the LHRH receptors, resulting in a striking decrease in androgens in males and estrogens in females [99]. However, they require parenteral route of administration, and no oral formulations are presently available. Amirreza Rafiee and co-workers synthesized two types of LHRH mini-dendrimers using thioether ligation, aiming to enhance the stability and bioavailability of the peptide drug while maintaining its biologically active conformation. Because the biodegradable PLL dendrimers were found to exhibit lower toxicity than PAMAM based dendrimers, which accumulate in the liver and spleen after intravenous injection [100]. On the whole, the commercially available dendrimers serve as a trustworthy source of building blocks for the development of dendrimer nanomedicine products on a large scale. Especially, cancer therapy strategy can be definitely successful with the dendrimer-based delivery. Nanostructured carriers like dendrimers are expected to continue to play an important role in delivering the hormonal drugs to the right target at the tissue, cellular, or subcellular levels. Certain intrinsic therapeutic functions performed by dendrimers themselves may be integrated into drug delivery system design and offer additional therapeutic benefits for cancer therapy. The overall summary of dendrimers uses or its effects on the biological system is illustrated in Fig. 10.2.

3 Limitations of dendrimers As usual, as there is no ideal drug delivery system and dendrimers also have few limitations for their usage. Among them the main drawback of cationic dendrimers is they cause cytotoxicity because of their positively charged surface groups, which destabilize the cell membrane and causes cell lysis. Though the higher generation of dendrimers has the advantages of target drug delivery they were proved to be toxic. Sometimes the primary amine terminal used to tag the immune drugs or hormones were found to show cytotoxic and hemolytic activities. Hence the primary amine can be replaced by the secondary or tertiary amines to avoid the unwanted effects. Moreover, non-cationic dendrimers like pyrrolidinone are proven to be safe for future use. Dendrimers are less tested in clinical studies compared to the linear polymers.

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Immune stimulation

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DNA repair Immunogenicity M

Anti-infective

G2

G1 S

Cell cycle arrest Apoptosis

Anti-angiogenesis Autophagy

Endocrine Hormones

Migration

Differentiation

FIG. 10.2 Schematic representation of applications of functional dendrimers in pre-clinical and clinical trials.

4 Future prospects Due to their unique structure compared to the other polymers, dendrimers have improved physical and chemical properties allowing enhancing drug bioavailability. Dendrimers size, shape, density, and surface functionality make them ideal carriers for various applications of drug delivery like therapeutic, diagnostic and many other fields of pharmaceutical applications. Last decade has witnessed the amplified growth of usage of dendrimers in biological systems. They have been also often referred to as “polymers of the 21st century.” The current chapter emphasized the considerable opportunities that dendrimers offer in immunotherapy and hormone therapy. Being a multivalent delivery system, it could deliver multivalent antigens or multiple epitopes, which are highly required against multivalent specific vaccines, e.g., Dengue, HIV etc. [101]. As highlighted in the present work, many attempts have been made to formulate various immune drugs and hormones as dendrimers to improve their efficacy and more research is still going on to develop the functionalized moieties pertaining to its high potential as nano-carriers. With improved synthesis, further understandings of their unique structural characteristics

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and recognition of new applications, dendrimers will become promising candidates for further exploitation in drug discovery and clinical applications. Development of targeted dendrimer delivery system is one of the urgent and challenging medical interventions.

Acknowledgments S.R.B. thanks the Department of Science and Technology (DST), Government of India and the Centre e (CEFIPRA) for the award of a Raman-Charpak Franco-Indien pour la Promotion de la Recherche Avance fellowship. S.M. thanks the French Centre National de la Recherche Scientifique (CNRS), the Laboratory of Excellence Medalis (ANR-10-LABX-0034), Initiative of Excellence (IdEx), Strasbourg University, and ImmuPharma France. The support of the TRANSAUTOPHAGY COST Action, CA15138 is also acknowledged.

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[86] Sharma AK, Gothwal A, Kesharwani P, Alsaab H, Iyer AK, Gupta U. Dendrimer nanoarchitectures for cancer diagnosis and anticancer drug delivery. Drug Discov Today 2017;22(2):314–26. [87] Nanjwade BK, Bechra HM, Derkar GK, Manvi FV, Nanjwade VK. Dendrimers: emerging polymers for drug-delivery systems. Eur J Pharm Sci 2009;38(3):185–96. [88] Luong D, Kesharwani P, Deshmukh R, Amin MCIM, Gupta U, Greish K, et al. PEGylated PAMAM dendrimers: enhancing efficacy and mitigating toxicity for effective anticancer drug and gene delivery. Acta Biomater 2016;43:14–29. [89] Yang H, Lopina ST. Penicillin V-conjugated PEG-PAMAM star polymers. J Biomater Sci Polym Ed 2003;14(10):1043–56. [90] Yuan Q, Yeudall WA, Yang H. PEGylated polyamidoamine dendrimers with bis-aryl hydrazone linkages for enhanced gene delivery. Biomacromolecules 2010;11(8):1940–7. [91] Bledsoe RK, Stewart EL, Pearce KH. Structure and function of the glucocorticoid receptor ligand binding domain. Vitam Horm 2004;68:49–91. [92] Guan L, Huang S, Chen Z, Li Y, Liu K, Liu Y, et al. Low cytotoxicity fluorescent PAMAM dendrimer as gene carriers for monitoring the delivery of siRNA. J Nanopart Res 2015;17(9):385. [93] Palmerston Mendes L, Pan J, Torchilin VP. Dendrimers as nanocarriers for nucleic acid and drug delivery in cancer therapy. Molecules 2017;22(9):1401. [94] Yang H, Kao WJ. Dendrimers for pharmaceutical and biomedical applications. J Biomater Sci Polym Ed 2006;17(1–2):3–19. [95] Hlabathe T. Synthesis, characterization and immobilization of Pd and Pt dendrimer-encapsulated nanoparticles and their application in homogeneous and heterogeneous catalysis. University of Johannesburg; 2015. [96] Bose CK. Follicle stimulating hormone receptor in ovarian surface epithelium and epithelial ovarian cancer. Oncol Res 2008;17(5):231–8. [97] Modi DA, Sunoqrot S, Bugno J, Lantvit DD, Hong S, Burdette JE. Targeting of follicle stimulating hormone peptide-conjugated dendrimers to ovarian cancer cells. Nanoscale 2014;6(5):2812–20. [98] Yang H. Targeted nanosystems: advances in targeted dendrimers for cancer therapy. Nanomedicine 2016;12(2):309–16. [99] Pinski J, Lamharzi N, Halmos G, Groot K, Jungwirth A, Vadillo-Buenfil M, et al. Chronic administration of the luteinizing hormone-releasing hormone (LHRH) antagonist cetrorelix decreases gonadotrope responsiveness and pituitary LHRH receptor messenger ribonucleic acid levels in rats. Endocrinology 1996;137(8):3430–6. [100] Rafiee A, Mansfeld FM, Moyle PM, Toth I. Synthesis and characterization of luteinizing hormonereleasing hormone (LHRH)-functionalized mini-dendrimers. Int J Org Chem 2013;3(01):51. [101] Patravale V, Prabhu P. Potential of nanocarriers in antigen delivery: the path to successful vaccine delivery. Nanocarriers 2014;1(1):10–45.

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Toxicity and biocompatibility aspects of dendrimers Avinash Gothwala, Sarita Malika, Umesh Guptaa, Narenda Kumar Jainb a

DEP A RTME NT OF PHARMACY, SC HOOL OF CHEM ICAL SCIENCES AND PHARMACY, CENTRAL UNIVERSITY O F RAJASTHAN, AJMER, INDIA b RAJI V GANDHI T ECHNOL OGI CAL UNI V ERS I TY , BHOPAL, INDIA

1 Introduction In the current era, nano-drug delivery systems are gaining attention from the research fraternity due to their versatile characteristics, potential and unmatched properties. Nanosystems including polymeric micelles, nanoparticles, microspheres, and dendrimers have proven their excellence in the nano-therapeutical approaches. Based on the well-defined properties, i.e., high drug payload, tailor made-architecture, low polydispersity index, non-immunogenicity, hyperbranched, and high solubilization propensity in aqueous media dendrimers lead the nano-therapeutic systems. These tree-like synthetic macromolecules were first introduced by Tomalia et al. [1] and Newkome et al. [2]. The term “dendrimer” was originally taken from Greek word “dendron,” which literally means tree/branch because of resemblance with tree, and “meros” means part. Dendrimers are highly water soluble due to plenty of terminal functional groups and hence can be used as solubilizer for poorly aqueous soluble drug molecules [3]. It has been reported that dendrimers improve pharmacokinetic parameters of the loaded as well as conjugated drug molecules [4] in terms of improved bioavailability [5]. Dendrimers also prolong the overall cumulative percent drug release from the scaffold [6, 7]. Improved pharmacokinetic parameters with dendrimers can be explained by its hydrophobic interior, which holds insoluble drugs and hydrophilic surface that mimics a container. Dendrimer mediated solubility, in terms of mechanistic aspect, is due to hydrophobic and ionic interaction between drug and dendrimer interior [8]. It is well accepted that dendritic scaffold increases stability as well as protects encapsulated moieties from biological environment. For example, Khopade and Jain [9] developed dendrimer-methotrexate complex for brain delivery. The complex was stable at physiological pH and able to deliver drug cargo to central nervous system (brain and bone marrow). Further, the dendrimer was also explored for gene delivery as one of the established non-viral vectors. Earlier, viruses were the only vector for gene delivery, but the janus face of virus vector led to immune and provoked responses in the patient [10]. To overrule this immunogenicity, scientists opted for

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different non-viral vectors [11]. Several polycationic nanocarriers were explored for genetic material delivery including cationic liposomes, PAMAM and PPI dendrimers [12,13]. Despite so many extensive preliminary biological applications and meritorious properties, dendrimers have a negative aspect of associated toxicity such as hemotoxicity, cytotoxicity, in-vivo toxicity etc. Dendrimer generation, charge and terminal functional groups define its toxicity toward the biological membranes. Several approaches have been documented to develop dendrimers compatible, such as by surface modification with different biodegradable molecules and even entirely new biocompatible dendrimers were also synthesized. Here in this chapter, we will focus on dendrimer associated toxicity and its biocompatibility aspects.

2 Dendrimer architecture Dendrimers are globular in shape and are monodispersed in nature. Dendrimers are synthetic macromolecules with thousands of Dalton molecular weight. Basically, there are three structural components in dendrimers, i.e., core, interior and terminal exterior (Fig. 11.1). Core is the central atoms with multiple functionalities, from the core dendrons emanate by several iterative steps of reaction. Number of reactions depends on generation of the dendrimers. Generation of dendrimers is the focal point from where each branching initiates, moving inside out from the core numbers of focal point to be considered as generation number. Interior of a typical dendrimer is composed of generations where branching elements are attached radially to the core, at the periphery, terminal functionalities are attached with the second last outermost generation. Lower generation dendrimers (i.e., 0, 1, 2) have open and unorganized structure in comparison to higher generation dendrimers. As the dendrimer generation increases, dendrimers turn into a globular shape [14]. When dendrimeric structure reaches to the critical branching point when no more branching is possible due to lack of space it is known as “starburst effect,” for example, PAMAM dendrimers Surface functionality Cavity/Void space

Core

Focal points

FIG. 11.1 This is how a dendrimer looks like.

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cannot be grown beyond 10th generation [15]. With increased generation, a void space develops in the interior of the dendrimer, which is mostly hydrophobic. This hydrophobic interior creates a hydrophobic drag, which may suitably drag hydrophobic moiety and accommodate it within.

3 Dendrimer types In recent years, the unique hyperbranched structure of dendrimers, higher surface functional groups, globular shape and mono-dispersity with different functionalities have shown great potential and interest in biomedical applications. Herein, we have attempted to accumulate the information based on types of dendrimers and their role; especially the incorporated functionality groups. Some of the dendrimers having different functionalities were described with their applications [16]. This description of dendrimer types is oriented to their toxicity and biocompatibility.

3.1 Polyether dendrimers Hawker and Frechet in 1990 prepared polyether dendrimers by convergent route using 1,1,1-tris(4-hydroxyphenyl) ethane as the core material and benzylic bromide and 3,5dihydroxybenzyl alcohol as branching material to form dendrons. Later, MALDI-TOF (matrix-assisted laser desorption ionization) mass spectroscopy was used for the characterization of polyether dendrimers. These types of dendrimers lyse over a period. Malik and coworkers reported a study in which the dendrimers with carboxylate and malonate surface groups were not lysed up to 1 h but underwent lysis after 24 h [17].

3.2 Polyester dendrimers Polyester dendrimers are simple to prepare and have attracted the attention of researchers continuously due to their marvelous properties in drug delivery. Polyester dendrimers are devoid of toxicity, unlike many other dendrimers that is why they are gaining much attention toward drug delivery. Hawker and Frechet [18] synthesized polyester dendrimers first time in 1991 via self-condensation of 3,5-bis(trimethylsilyloxy) benzoylchloride. Then, Haddleton et al. [19] synthesized polyester dendrimers via divergent method using phloroglucinol, hydroquinone and naphthalene-2,6 diol as monomer and used 1,3-dicyclohexylcarbodiimide (DCC) or its acid chloride for activation of reaction [20]. Transesterification reaction and cleavage of functional groups is the main strategy for the formation of ester bond during the synthesis of polyester dendrimers. Aliphatic polyester dendrimers were synthesized by Hult and Soderlind from first to fourth generation [21].

3.3 Tecto/core-shell dendrimers The core-shell or tecto dendrimers are highly organized and composed of a core with branches. The core generation number is more significant than the surrounding shell

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dendrimer, called the core-shell tecto dendrimer [22]. Core-shell/tecto dendrimers represent a polymeric structure obtained as a product of controlled covalent attachment of dendrimer building block. Tecto dendrimers are composed of a core dendrimer, which may or may not contain the therapeutic agents surrounded by the dendrimers. The synthesis of tecto dendrimers is quite simple compared to other types of dendrimers [22]. Initially, FITC (fluorescein isothiocyanate) was used as an identifying agent but encountered some solubility issues. Nowadays, fluorescein can be used as an initiator for recognition and folate as targeting molecule during the synthesis of core-shell dendrimers in place of FITC. Fluorescein conjugates utilization encounters the solubility problems on the dendritic surface. These conjugates are found to be advanced and suitable in comparison to FITC conjugates. The surrounded dendrimers are of several types to perform a function that is necessary for a smart therapeutic nanodevice [16,23]. The core-shell/tecto dendrimers inhibit the growth of melanoma cells at a concentration, which is safe for the keratinocytes epithelial cells. Shirreffs et al. [24] reported cytotoxicity of the core-shell dendrimers to investigate their application in drug delivery. The 5.0 G amine-terminated PAMAM was utilized as core with 2.5 G PAMAM on the peripheral surface having COOH group. The prepared conjugates were examined on SK-MEL-28 human melanoma cancer cell lines for cytotoxic effects. The formulated core-shell dendrimers exhibited cytotoxicity effects on melanoma in a concentration range that was safe for normal keratinocytes epithelial cells in human [24]. These dendrimers can be better explored for biomedical applications and nano-drug delivery.

3.4 Triazine dendrimers The triazine dendrimers used in a drug delivery system have the payback from their synthetic versatility and well-defined structure. They can be synthesized and designed to display orthogonally working surface that will help in the attachment of drug, ligands. Triazine dendrimers have biomedical applications such as drug delivery, non-viral DNA and RNA delivery. The synthesis of triazine is based on the substitution of trichlorotriazine with amine nucleophiles [25] and triazine is used as central core during synthesis. Triazine dendrimers do not show any toxic effects in animal models at preliminary stage. They are prepared by divergent and iterative method at laboratory levels. Many other studies based on application of triazine dendrimers are under process.

3.5 Citric acid dendrimers Citric acid (CA) dendrimers are the most attractive class of dendrimers because of their properties of improving water solubility of insoluble biomolecules. This is one of the hastily growing classes among dendrimers as they are giving potential results in enhancing the therapeutic efficacy and bioavailability of entrapped drugs. Dendrimers are extensively explored for transdermal drug delivery. Our skin is negatively charged, and the cationic dendrimer formulations can interact with skin as maximum dendritic formulation

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concentration reaching the targeted sites. The dendritic formulations can penetrate inside the skin and act efficiently with enhanced permeability in case of dermal problems. Therefore, dendrimers act as carrier for transdermal drug delivery. Rao et al. [26] prepared antifungal hydrogel formulation of econazole based on CA dendrimers. Econazole showed excellent anti-fungal activity but its use is constrained by the less solubility and bioavailability issues. So, the researcher prepared hydrogels of this anti-fungal drug by incorporating it inside the core of CA dendrimer that solubilizes the drug with improved bioavailability as well. Namazi and coworkers synthesized PEGylated CA dendrimers that act as potential drug delivery system. They prepared a triblock of citric acid-based dendrimers; CA-PEG-CA and then attached the imidazole group on the surface of dendrimers to study the type of interaction between anti-inflammatory drug naproxen and prepared dendrimers triblock conjugate. The amount of drug released was studied at different pH such as 1.0, 7.4 and 10. Results suggested that amount of encapsulated drug in the dendritic core increased with increment of dendrimers generations. It was concluded from the study that the prepared CA-PEG-CA triblock may be used for gene/drug delivery system [27]. Interestingly, solubility of mefenamic acid, diclofenac, and 5-amino salicylic acid was also improved using CA dendrimers by researchers in the past. The CA-based dendrimer complexes are found to be stable at pH 10.

3.6 Phosphate dendrimers Phosphate dendrimers are synthesized by introduction of phosphate group on to the PAMAM dendrimers via Mannich-reaction and characterized by SEM, TEM, and XRD. These types of dendrimers along with an amorphous calcium phosphate stabilizing agent, polyacrylic acid (PAA), are utilized in dermal therapy. Domanski et al. [28] reported cytotoxicity and hemotoxicity of phosphate dendrimers and studied their effect on human erythrocytes including blood cells integrity, structure and observed this type of dendrimer biocompatibility with Chinese hamster ovary (CHO-K1) cell line. Results revealed that the membrane stability of blood cells increased upon interaction of phosphate dendrimers with human erythrocytes [28].

3.7 Melamine dendrimers Melamine dendrimers were synthesized by both convergent and divergent methods. These dendrimers reduced the cytotoxicity of some anti-cancer agents such as methotrexate, 6-mercaptopurine along with increased solubility. Zhang group [29] synthesized 3.0G dendrimers using melamine with attachment of 16 carbamate group and characterized the prepared dendrimers via 1H NMR and 13C NMR spectroscopy. These dendrimers are explored as a drug candidate and examined for in vitro and in vivo toxicity. Neerman et al. [30] observed that melamine dendrimers at doses up to 10 mg/kg showed no hepatic toxicity at sub-chronic doses although doses up to 40 mg/kg showed liver necrosis in mice.

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3.8 Polyether imine (PEI) dendrimers Krishna and coworkers synthesized 3-amino-propan-1-ol based polyether imine dendrimers and evaluated them for biological applications. Later this group synthesized another alcohol-based imine dendrimers and tested for cytotoxicity against different cancer cell lines including breast cancer cell line such as T47D and monkey kidney cell line by MTT assays. They concluded from the results that the polyether imine dendrimers did not cause cell cytotoxicity as 98% cells were viable after treating with 100 mg/mL of dendrimer formulation [31].

3.9 Polyether-polyester (PPE) dendrimers In one of the studies, Carnahan and Grinstaff prepared PPE dendrimers from lactic acid and glycerol. PPE dendrimers can be synthesized using PEG as core and characterized by AFM and X-ray photoelectron spectroscopy (XPS). Dhanikula and Hildgen [32] used this type of dendrimers for encapsulation of anti-cancer drug methotrexate and studied the dendrimers for gliomas treatment. Authors noticed enhanced encapsulation and better intra-tumoral efficacy of dendrimer formulation. The cytotoxicity of dendritic solution was examined on RAW 264.7 cell lines. PPE dendrimers were found to be safe up to a concentration of 250 μg/mL without any cell death, therefore, can be used in drug delivery due to their biocompatibility [33].

3.10 Other dendrimers In addition to all the important classes of dendrimers discussed above, some others type of dendrimers are also synthesized and characterized by the scientists. The classification of dendrimers is not specific. Various other types are extensively utilized for nanodevice based anti-cancer agents, transdermal drug delivery and ocular drug delivery. These types include some PAMAM based multifunctional dendrimers, micellar form and biodegradable polymers, pH sensitive (poly-lysine) based dendrimers formulations. Micellar dendrimers are highly branched and aqueous soluble dendrimers can be used as therapeutics. These dendrimers with their large unique properties such as small size, highly constructed structure and non-toxicity may be explored extensively further for gene therapy, imaging agents etc.

3.11 PAMAM dendrimers Tomalia in 1980s for the first time developed and discussed the family of dendrimers that were synthesized, characterized and commercialized for the drug delivery. Later, he named PAMAM dendrimers a “starburst dendritic molecule” and introduced the synthesis from first to sixth generations. These may be synthesized by a divergent method using ethylenediamine (EDA) as the core. The synthesis of PAMAM dendrimers involves two steps (i) Michael addition of a suitable amine initiator core with methyl acrylate, and (ii) amidation of the esters with the large excess of ethylenediamine (EDA). For each

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generation, PAMAM molecular weight, number of atoms, number of terminal amine group and radius increase exponentially. Lower generations PAMAM dendrimers exhibited linear structure while higher generations showed globule-like shapes. In the globular structures, the central core plays pivotal role in encapsulating the biomolecules inside their cavities. NMR, size exclusion chromatography and IR spectroscopy [1] were used to characterize both alkylation and amidation steps. The internal structure of PAMAM dendrimers with their distinctive functional groups such as amines, amide linkages can be used to hold contrast agents, and for adsorbing control release agents and used in gene transfection because of the presence of positive charge on its surface. The terminal amines of PAMAM going to attach with the targeting molecule and many surface modified PAMAM are non-immunogenic in nature with significant water solubility. PAMAM toxicity and hemolysis depend on concentration. From the literature, it was concluded that the PAMAM from 5.0G to 10.0G showed polydispersity index less than 1.08 that suggested that the particle size distribution is uniform [16]. PAMAM dendrimers are most explored class of dendrimers in drug delivery.

3.12 PPI dendrimers PPI dendrimers contain poly-alkyl amines with primary amines as end groups and tertiary tris-propylene amines are present in the internal structure. These are commercially available to fifth generations with a trade name of Astramol™ [34]. These can be synthesized by the divergent method, which is obtained by repetition of double Michael addition of acrylonitrile to primary amines followed by heterogeneously catalyzed hydrogenation of nitrile to primary amines [35]. In the synthesis of PPI dendrimers, 1,4-diaminobutane was used as dendrimer core. The poly(propylene imine) was introduced commercially by DSM (Dutch State Mines), Netherlands and Aldrich Chemical Company (Milwaukee, WI) [23]. Two different types of amine are available in PPI dendrimer; primary and tertiary. The primary amines are basic in nature with a pKa 10 and tertiary amines are acidic having pKa 6–9. Different molecules can be used as core with primary and tertiary amine in PPI dendrimer synthesis. These classes of dendrimers are explored much and possess remarkable drug delivery potential like PAMAM dendrimers. The only structural difference between PAMAM and PPI is the core; PPI has much hydrophobic core as compared to PAMAM.

3.13 Glycodendrimers The word “glycodendrimer” is used to describe dendrimers that are composed of carbohydrates such as glucose, mannose, galactose. The primary groups of glycodendrimers have saccharide residue on their surface but containing a sugar moiety as a central core. Glycodendrimers are classified into three types: (i) carbohydrate coated, (ii) carbohydrate centered, and (iii) carbohydrate based. Glycodendrimers with surface carbohydrate units have been used in the study of protein-carbohydrate interactions [16,23]. They have been widely utilized for a variety of biologically relevant applications such as in analytical

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devices, gene-drug delivery, MRI contrasting agents and in gel formulations. An anticipated application of these dendrimers is their site-specific delivery to lectin-rich organs.

3.14 Chiral dendrimers Chiral dendrimers offer the possibility to investigate the impact of chirality in the macromolecular system. They are constructed constitutionally in a different way with identical branching to chiral core. Their properties are based on their well-defined highly ordered structure with nanoscopic dimensions. Chiral dendrimers differ from achiral dendrimers by following features: (i) the functional group’s arrangement on the surface could be chiral, (ii) chiral structures in chiral dendrimers should be detected by optical measurement; (iii) their shape could be chiral, not spherical [36]. The chirality in the dendrimers is based on the construction of constitutionally different but chemically similar branches to a chiral core [23,37]. These are suitable for addition of useful functionalities aimed at an application as drug delivery system and light-harvesting materials. Chiral dendrimers have applications in identifying the chiral molecular and asymmetric catalytic reactions [16]. Ghorai et al. [38] studied for the first time the anthracene molecules enclosed in chiral dendrimers that were derived from 1,3,5-trisubstituted aromatic core and carbohydrate units. They reported that chiral dendrimers have potential to encapsulate the anthracene molecules and widely utilized in drug delivery system and in light-harvesting molecules. The research evidence toward this claim is still awaited at exploring chiral dendrimers applications in the biomedical field.

3.15 Poly-L-lysine dendrimers Poly-L-lysine is a cationic polypeptide with amino acids as repetition units. The polypeptide chain of PLL is ideal for in vivo gene delivery. These are the first polymer developed as substitute viral vector for gene delivery to avoid the immunogenicity and oncogenicity [39]. Poly-L-lysine dendrimers show systemic antiangiogenic activity, which may be used for solid tumor therapy.

3.16 Hybrid dendrimers Hybrid dendrimers are linear polymers composed of mono-functionalized zero generation PAMAM dendrimers. The structures are lamellar, cubic, centered self-organized lattices. Hybrid dendrimers consist of dendritic and linear polymeric grafted structures. Javier et al. [20] prepared and characterized gold nanoparticles of hybrid dendrimers of PVV-PAMAM and explained that these nanoparticles can be used as biomarkers in living cells. The prepared hybrid dendrimers are able to bind with siRNA (small interfering ribonucleic acid) [20].

3.17 PAMAM-organosilicon (PAMAMOS) dendrimers PAMAMOS dendrimers are radially layered dendrimers having hydrophobic organosilicon (OS) on periphery and water-soluble, nucleophilic PAMAM present interiorly that

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comprises an inverted unimicellar molecule. They have a wide range of applications in electronics, chemical catalysis, nano-lithography and photonics etc., due to their exclusive properties such as constancy of structure and ability to form complex. PAMAMOS dendrimers can be used in nano drug delivery because they can encapsulate various biomolecules in their compact structure [40]. PAMAMOS dendrimers’ use in drug delivery is continuously under exploration by researchers.

3.18 Liquid crystalline (LC) dendrimers LC dendrimers are multi-splitted oligomers or polymers consist of mesogenic LC monomers, e.g., mesogen functionalized carbosilane dendrimers. Mesogens are comprised of rod-shaped (calamitic) or disk-like (discotic) molecules. In 1995, Percec and group first reported AB type mesogens. Later Frey et al. [41] attached carbosilane group such as cyanobiphenyl [42] and cholesteryl [41] to the mesogen units. These dendrimers were studied for delivery of short-chain siRNA and anti-HIV oligodeoxynucleotide to HIVinfected blood cells. Their potential is limited for long-chain double-stranded nucleic acid delivery but the dendrimer complexes of carbosilane group have shown stability with antiHIV nucleic acid and less cytotoxicity for human erythrocytes.

3.19 Polyurea dendrimers Polyurea dendrimers (PUD) are green dendrimers with two functional groups. They have remarkable characteristics such as aqueous solubility, blue photo-luminescence, biocompatibility, biodegradability and efficient transfection ability. PUD can be synthesized by divergent method under supercritical fluid such as CO2 in one pot reaction. The mode of application or use of polyurea dendrimers in various cells is still under investigation. Some efforts toward the exploration of applications are keenly awaited for PUD dendrimer uses in the medical and material sciences. PUDs have been used in cytosol siRNA delivery at molecular level [43,44].

3.20 Peptide dendrimers Peptide dendrimers are the dendrimers containing peptide bonds. These are radically branched macromolecules that include peptidyl branching core and peripheral peptides chains. These can be classified into three subclasses (i) grafted peptide dendrimer, these contain unnatural amino acids or organic groups as the branching core and peptide or proteins are attached as functional groups on the surface, (ii) peptide dendrimer, consist of mostly peptides, and (iii) contains amino acids in the branching core and surface functional groups and having non-peptide branching units [16,23]. Divergent and convergent methods are used for the synthesis of peptide dendrimers. Peptide dendrimers are used as contrast agents for magnetic resonance imaging (MRI), magnetic resonance angiography (MRA), fluorogenic imaging [45], proteins, liposomal mimetics, and vehicles for drug and gene delivery and also as a biomaterial in life science and biomedical applications. Darbre

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and Reymond [46] reported that peptide dendrimers can be utilized as esterase catalysts in some reactions. The solid phase combinatorial methods can be applied to screen out the desired properties of peptide dendrimers from the large libraries. With this background of dendrimer types now we can examine their biocompatibility and toxicity aspects.

4 Mechanism of dendrimer biocompatibility and toxicity “Toxicity” is defined as the measure of harmful effects on the tissues, cells, and different organs of the body. Testing of toxicity profile is considered as most important parameter for drug before commercialization for the safety reasons of patient. Many novel polymers, polymeric conjugates, dendritic formulation and other biomedical devices, and other therapeutics are examined for toxicity screening in the preclinical and clinical phases in the pharmaceutical industries. The term “biocompatibility” means the compatibility of materials or formulation with the living tissues. Biocompatible materials are devoid of any toxic or immunological response when exposed to the host and can perform a suitable response with the immune system in a specific condition. These definitions are meaningful in testing of a formulation in terms of safety and response toward the body. The exact meaning of biocompatible material is uncertain. Physical, chemical and biological properties of a compound have direct relationship with biocompatibility. The small size, shape, stability and structural functionalities and biological uptake and immune response of particles are important consideration for biocompatible material. A dendritic therapeutic is said to be biocompatible for cells if it produces a desirable response with no or very less toxicity in the system. Dendrimer is a new class of macromolecules with peripheral surface functionality that determines its physical properties. Dendrimers can be attached with drug by physical (encapsulation) and chemical (covalent bonding) forces and via spacer conjugates strategies. Dendrimers are advantageous over other nanotherapeutics in drug delivery system due to their small size, biocompatibility toward cellular system in cancer diagnosis and therapy. They have ability to accumulate within the tumor cells specifically by enhanced permeation effect (EPR) ascribed to their nano size via passive targeting [47]. Scientists have examined the toxicity of dendrimers in vitro and in vivo over last decades. Dendrimers have strong capacity to bind with body’s vitamins, heavy metals, ions, lipids, proteins after administration as formulation. One conclusive finding is that the higher generation dendrimers are more toxic than lower generation. MTT assays were performed on many cell lines to find out the cytotoxicity of dendrimers. Another conclusive finding is that cationic dendrimers produce more toxicity than anionic dendrimers. This causes hemolysis at a greater extent but with biocompatible dendrimers such as melamine exhibited very acute or no toxicity in the animal models [48]. The fate of dendrimer is important to be considered and it must be excreted out from the body. The long stay of undegradable dendrimers in the body may cause toxicity over a period. Still, critical efforts are required in the designing of biodegradable dendrimers for the safety concern of patients in drug delivery system.

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5 Why are dendrimers toxic? Despite unmatched characteristics of dendrimers, its undesirable associated toxicity restricts its application in therapeutics. The possible reasons behind the associated toxicity of dendrimers are discussed in this section briefly.

5.1 Cationic charge Dendrimers are cationic in nature due to tertiary amines in the inner branching and primary amines at the surface, this may damage the cellular membranes such as erythrocytes and other biological membranes. It has been believed that cationic polymers lead to morphological changes in cellular milieu. For example, cationic PAMAM dendrimers create a pore in the membrane fluid phase while plasma membrane is not affected due to gel phase [49,50]. L929 cells were incubated with polyethylene imine (PEI) with varying concentration and it was observed that at higher PEI concentration and prolonged incubation time, cell lysis occurred with development of distorted cells followed by loss of adherent capacity. Similarly, the cationic charge also leads to hemotoxicity, in case of PAMAM dendrimers it is generation dependent and interestingly increases gradually when the incubation time increases [17]. Gothwal et al. [51] demonstrated that G4.0 PAMAM dendrimers were hemotoxic due to surface amine and cationic charge. However, bendamustine conjugation reduced overall toxicity of dendrimers by several folds due to masking of surface amine functionality and cationic charge. Further, surface modification with human origin lactoferrin modification on the G3.0 PAMAM dendrimer also reduced the hemotoxicity by several folds [52]. Plethora of literature is available to describe the cationic charge of dendrimers for its toxicity to the biological membranes.

5.2 Core associated toxicity Core mediated toxicity is not evident in higher generation dendrimers because surface functionality and charge play critical role in the toxicity. But in case of lower generation dendrimers core comes into the scene of toxicity due to open structure and its interactive possibility with biological membranes. Core of higher generation dendrimers are sterically hindered due to multiple numbers of surface functionalities while in lower generation surface is more accessible to end groups [53].

5.3 Free amine toxicity Toxicity of amine terminated PAMAM dendrimer (G3.0, G5.0 and G7.0) was investigated in Swiss-Webster mice and severe biological complications in G7.0 dendrimer treated animals were observed. The authors concluded that PAMAM associated toxicity increased as the generation numbers of amine and cationic charge increased [48]. It was also claimed that maximum tolerable i.v. dose of G4.0 and G7.0 amine-terminated PAMAM dendrimers was less than 10 mg/kg. Not only i.v. but also oral administration caused

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similar toxicity. The half generation dendrimers with dCOOH and dOH terminal functionalities were observed to be safer than the amine-terminated PAMAM dendrimers [54]. A very interesting study was conducted by Kitchens et al. [55] to investigate the PAMAM dendrimer-based toxicity in Caco-2 cells. The authors incubated Caco-2 cells with different generation PAMAM dendrimers. Therefore, authors selected a combo of PAMAM dendrimer and Caco-2 cells for demonstration of toxicity. The authors observed that lower generation dendrimer G2.0NH2 or G1.5COOH were not able to change the cellular morphology even after 2 h of incubation, while G4.0NH2 dendrimer caused microvilli disruption in the cellular membrane (Figs. 11.2 and 11.3). Authors presumed that dendrimers with higher number of cationic groups on surface interacts with negatively charged cell membrane and causes more toxicity over the anionic dendrimers. It has been also claimed that cationic dendrimers create holes in the lipid bilayer followed by internalization [56–58]. A comparative study of G4.0 PAMAM-NH2 and G4.0 PAMAM-OH on E. coli infected guinea pig revealed that amine-terminated dendrimers was more cytotoxic over the hydroxyl terminated dendrimers. Outer and inner membrane permeation studies revealed that amine-terminated dendrimers disrupt both outer and inner membrane which led to bacterial cell lysis [59]. It was concluded that number of cationic functionalities on the dendrimer surface plays a critical role in the associated toxicity.

FIG. 11.2 Transmission electron microscopy (TEM) images of Caco-2 cell monolayers after treatment with PAMAM dendrimers (1 mM) for 2 h; (A) control cells; (B) G2NH2; (C) G1.5COOH; (D) G4NH2; (E) G3.5COOH. The images display a generation-dependent effect of PAMAM dendrimers on Caco-2 microvilli (magnification ¼ 12,500 ). Scale bars ¼ 1 μm. Figure reused from Kitchens KM, Foraker AB, Kolhatkar RB, Swaan PW, Ghandehari H. Endocytosis and interaction of poly (amidoamine) dendrimers with Caco-2 cells. Pharm Res 2007;24:2138–45 with copyright permission.

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FIG. 11.3 TEM images of Caco-2 cell monolayers after treatment with G4NH2 dendrimers for 2 h: (A) control cells; (B) 0.01 mM G4NH2; (C) 0.1 mM G4NH2; (D) 1.0 mM G4NH2. The images display a concentration-dependent effect of G4NH2 on Caco-2 microvilli (magnification ¼ 12,500 ). Scale bars ¼ 1 μm. Figure reused from Kitchens KM, Foraker AB, Kolhatkar RB, Swaan PW, Ghandehari H. Endocytosis and interaction of poly (amidoamine) dendrimers with Caco-2 cells. Pharm Res 2007;24:2138–45 with copyright permission.

6 Dendrimer toxicity and biocompatible strategies Unmatched drug delivery capacity of dendrimers gained the attention of drug delivery research fraternity over nearly past four decades. Dendrimers have been explored for the effective delivery of drug candidates in different ailments preliminarily including cancers [51], cardiac disorders, and brain ailments [52] etc. However, the nanometric size (1–100 nm) facilitates the interaction with cell membranes effectively and especially with the cell organelles, proteins [60,61]. As of now, most of the developed nanocarrier systems are non-selective in terms of delivery potentials until and unless these are tagged with any ligand molecules, which drives the developed nanoarchitecture to the desired site. However, passive targeting is possible with the enhanced permeation and retention effect (EPR). EPR is a characteristic feature for higher drug accumulation in tumors only, according to EPR concept biocompatible macromolecules gets deposited at the tumor site comparatively in the higher than the normal concentration in plasma [62–64]. This EPR aspect is desired for better pharmacokinetic parameters. Interestingly, the non-selectivity was also an aspect of use for gene delivery due to better cell transfection efficiency [60,61]. This non-selectivity also has a janus face of toxicity associated with dendrimers. In this section, we will focus on the toxic behavior of dendrimers. Despite the worthy applications in biomedical and pharmaceutical field the toxic nature limits its candidature for clinical applications [17,65,66].

6.1 Cytotoxicity One of the very first cytotoxicity reports were published by Malik et al. [17], they explored cytotoxicity of PPI and PAMAM dendrimers against the B16F10, CCRF and HepG2 cancer cell lines. Later, Jevprasesphant et al. [67] also observed cytotoxicity of PAMAM dendrimers in Caco-2 cancer cells. Similarly, Agashe et al. [40] observed that PPI G5.0 dendrimers were cytotoxic to HepG2 cells over COS-7 cells. Toxicity depends on time of incubation and concentration; authors concluded that free surface

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amines and cationic charge were responsible for the toxicity. Interestingly, Stasko et al. [68] demonstrated toxicity of surface modified G5.0 PPI dendrimer against HUVEC and found that unmodified dendrimer showed higher plasma membrane permeability over the modified one.

6.2 Hematological toxicity Cationic nature of dendrimer leads to hematological parameter disturbance, Bhadra et al. [6] observed that primaquine phosphate loaded PPI dendrimers alleviated RBC while leukocyte was boosted than the normal level in Sprague-Dawley male rat. Agashe et al. [40] experienced the very same results when G5.0 PPI dendrimer decreased RBC, Hb, HCT and MCH levels while a change was observed in WBCs level. Later Agrawal et al. [69] also observed quite similar changes in the hematological parameters with G4.0 PLL dendrimers, increased WBC level and decreased RBC level. Considering these results, it may be concluded that cationic dendrimers are needed to be altered in a better biocompatible mode.

6.3 Hemolytic toxicity Terminal functionality of dendrimer plays a crucial role in the hemolytic toxicity, cationic amine terminal functionality causes hemolysis [70,71]. Malik et al. [17] observed that PPI and PAMAM dendrimers of G3.0 were hemotoxic at 1 mg/mL concentration. Similarly, Bhadra et al. [71] documented that G4.0 PAMAM dendrimers caused around 15% hemolysis. Almost similar hemolysis was documented by Asthana et al. [70] with same generation of PAMAM dendrimers. With higher generation of dendrimers and higher incubation time, boosted hemotoxicity due to increased density of terminal cationic functionality and prolonged interaction time with RBCs was observed that was in line with the previous studies. For example, Agashe et al. [40] observed that G5.0 PPI dendrimer caused up to 80% hemolysis when incubated for 4 h at same concentration over 1 h of incubation (around 34% hemolysis). Agrawal et al. [69] claimed that G4.0 PLL dendrimers was around 14% hemolytic and suggested that higher generations may be more toxic because of higher cationic charges.

6.4 Immunogenic toxicity It is still unclear whether dendrimers cause immunogenicity or not. There is not much literature available, but some reports were documented. Roberts et al. [48] investigated immunogenic responses of PAMAM dendrimers, but there was no immunogenicity with a dose range of 0.1–0.0001 μM observed. Later, Agashe et al. [40] examined immunogenicity of PPI G5.0 dendrimers in Balb/C mice and claimed no immunogenic provoked responses. These studies are positive sign for utilizing these excellent nanocarriers as a drug delivery scaffold. It may be concluded that host immune system treats dendrimer as intrinsic molecules however more studies are needed to reach any clear decision.

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6.5 In-vivo toxicity It is necessary to check the safety profile of any drug-delivery system before marching to clinical studies. In case of dendrimer, researchers demonstrated in-vivo toxic studies to establish safety parameters of dendrimers in animal systems. Roberts et al. [48] examined toxicity of PAMAM G3.0, 5.0 and 7.0 in Swiss-Webster mice. The researchers claimed that G7.0 PAMAM caused severe biological complications, additionally concluded that dendrimers are safe for biological applications. Malik et al. [17] explained that PAMAM dendrimers eliminated from the body regardless of i.p. or i.v. administration route. Neerman et al. [30] performed behavioral and acute toxicity studies of melamine dendrimers. The authors observed that higher dose 160 mg/kg was lethal for the mice with 100% mortality rate and hepatic toxicity was noted at 40 mg/kg dose. Lower dose up to 10 mg/kg was safe for the mice as neither toxicity nor mortality was observed.

7 Surface modified dendrimer for compatibility Surface modification of dendrimers ends up with suppressed toxicity and improved signature characteristics of dendrimers. Although the toxicity of dendrimers is the janus face of its applicability in the drug delivery yet surface modification is the most explored and best strategy for masking this toxicity. Several approaches have been used to tag some biodegradable on the surface of dendrimer scaffold including poly(ethylene glycol), peptides, antibodies etc. These molecules are generally known as ligands, these moieties play multiple roles despite of toxicity masking, for example, active targeting of dendrimer to the target site. Most of the used ligands are tending to bind to specific receptors which improve the overall accumulation of the scaffold in the target sites. In this section, we will focus on the surface modification to overrule the dendrimer associated toxicity. Apart from the reduced cytotoxicity and targeting surface modification it also improves drug loading efficiency, entrapment efficiency of the scaffold, pharmacokinetic, prolonged release of drug and transfection efficiency. In other words, overall therapeutics gets improved when drug is delivered through dendrimers [6,7,69,71,72].

7.1 PEGylation PEG is basically a polyether and it is the extensively used ligand moiety to overcome the dendrimers toxicity. It will not be wrong if we say PEG a “ligand of choice” to reduce the toxicity. Mainly PEG masks the free amine/cationic functionality which leads to restriction in the electrostatic interaction with biological membranes. Liu et al. [73] introduced a new water-soluble dendrimer-PEG scaffold for drug delivery and improved solubility. The authors used cholesterol and amino acid derivatives as model drug molecules to tether with dendrimeric scaffold. Further, conjugates were characterized using MALDI-TOF. Later on, a milestone study was done by Bhadra et al. [71] who reported that when PAMAM G4.0 dendrimer was decorated with PEG on the surface and observed that hemotoxicity

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and hematological toxicity were minimized over pure dendrimer. Interestingly, drug loading was also increased, and leakage was minimized after PEGylation. Luo et al. [74] PEGylated PAMAM G5.0 for effective DNA delivery. The nano-constructed architecture was highly biocompatible and overall transfection efficiency of 20 times improved. The dendrimer-based cytotoxicity was also significantly minimized. Further, PAMAM-PEG conjugation approach was explored by other researchers, for example, PAMAM G5.0 and G6.0 were conjugated with PEG and investigated intramuscular gene delivery potency of both scaffolds. The overall transfection efficiency was several folds increased in another hand the PAMAM-PEG conjugates were very efficient transfecting vector and biocompatible [75]. Sabri et al. [76] synthesized G4.0 PAMAM-PEG conjugate and further conjugated with methotrexate (MTX) to establish a drug delivery system with higher potentials. PEGylation was used as a linker to conjugate MTX and minimize the toxicity. Later in 2016, Xu et al. [77] demonstrated that higher generation PAMAM G5.0 dendrimers were efficient nano-vector for siRNA delivery in neovascularization model. Prior to this work Qi et al. [75] reported that PEGylation reduces hemotoxicity and cytotoxicity of G5 and G6 PAMAM dendrimers. Recently, Urbiola et al. [78] constructed a dendriplex composed of PAMAMPEG conjugate. The authors claimed that PAMAM-PEG has an unmatched complex ability with siRNA, higher transfection efficiency against HeLa and LS174T with less toxicity and viability. So, PEGylation is extensively dig out for two decades and still, it is the best strategy to minimize toxicity of nanocarrier or to improve the drug loading as well as pharmacokinetics of the drug. This concept is yet to be explored as all the studies are in preliminary stage.

7.2 Folic acid surface tagging Folic acid (FA) is basically one of the vitamins B complex, mainly used as a targeting ligand as folate receptors are overexpressed in various tumor cells [79,80]. Receptor-ligand mediated mechanism facilitate internalization of drug-loaded nanocarriers and increases overall deposition of drug in the tumor. Even folate tagging also used utilized for genetic material delivery, diagnostic agents, imaging agents as well and results were encouraging [80]. This phenomenal concept was also used for cancer targeting with dendrimers. It has been observed that FA modification suppress dendrimer associated toxicity. In 1999, Kono et al. [81] introduced polyether dendrimer-FA conjugate and characterized. Further Quintana et al. [82] developed a PAMAM dendrimer-FA conjugate for the improved delivery of methotrexate. Choi et al. [83] developed a nano-conjugate of PAMAM G5.0 dendrimer decorated with FA and methotrexate on the surface and investigated drug delivery potency, the conjugate showed a selective binding toward the KB cells and kills. Further, Zong et al. [84] synthesized PAMAM G5.0 dendrimer-FA conjugate via triazene scaffold and explained the FR mediated mechanism of internalization which causes toxicity in the KB cells. Despite dendrimer-FA conjugation, PEG was also introduced as a ligand to develop dendrimer-PEG-folate conjugates. Developed nanocarrier system was least hemotoxic over the dendrimer and the potency of drug delivery was also improved

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with sustained drug release [85]. This concept is still being explored extensively, even after two decades. Recently, Zhang et al. [86] developed a nanocarrier system composed of PAMAM G5.0 dendrimer tagged with FA and further conjugated with doxorubicin. The cytotoxicity of the surface modified PAMAM dendrimer was significantly alleviated and cellular uptake was improved. Conclusively, folate modification on the surface of dendrimers improves the drug loading, release and most importantly, alleviates the dendrimer associated toxicity. Folate tagging can be phrased as “killing two birds with one stone” because it felicitates two different causes one is as a ligand and second is reducing the dendrimeric toxicity.

7.3 Amino acid/peptide surface modification Amino acid or peptides are molecules of intrinsic origin, which means these are biocompatible and safe for human applications. Phenyl alanine and glycine tagged dendrimers have shown significant reduced dendrimeric toxicity [40]. Higher generation PPI dendrimer (G5.0) tailored with these amino acids which led to improved biocompatibility, reduced hemotoxicity even time-dependent hemotoxicity was not observed in the developed conjugates. Similarly, Kono et al. [87] decorated PAMAM (G4.0) dendrimers with leucine or phenyl alanine for effective gene delivery. The authors claimed, transfection potency was improved while cytotoxicity was suppressed. Similarly, arginine tagging also improved transfection potency for gene delivery [88,89]. Apart from the amino acid, some peptides were also conjugated with peptides. For example, Yang and Kao [90] decorated starburst PAMAM G3.5 and G4.0 with arginine-glycine-aspartate (RGD) peptides and evaluated the cellular uptake in fibroblast cells and observed that G3.5 conjugates do not affect the cell viability. The authors suggested that peptide conjugation may be used in drug delivery aspect as it has great potential for the same. Recently, Kojima et al. [91] developed peptide-dendrimer for tumor targeting against the acute myelogenous leukemia (AML). They used CPP44 peptide as tumor targeting ligand further cathepsin B was also linked to the scaffold. Evaluation revealed that the scaffold has higher penetration and antitumor activity. Dendrimer-peptide conjugation is not much explored till date however it may be the ray of hope in the safety profiling of the dendrimer.

7.4 Antibody tagging Antibodies (Ab) are also known as immunoglobins (Ig), Igs are basically large “Y” shape proteins, synthesized by plasma cells. Ab can be used as a homing device which drives the nanocarriers to targeting site especially in cancer. Certain antigens are overexpressed in malignancy and Ab has a selective binding tendency toward those antigens which means therapeutic agents could be delivered directly to the tumor sites via an active targeting approach [92]. Ab acts through different modes, i.e., antiangiogenic effect means it targets blood vessels of tumor supply [93] or may suppress the growth factors responsible for the growth of tumor cells.

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Dendrimer-Ab conjugates have been explored for the targeted drug delivery which also led to retarded dendritic toxicity. Thomas et al. [94] conjugated PAMAM (G5.0) dendrimer with two different antibodies, 60bca and J591 for selective targeting to CD14-expressed HL-60 cells and PSMA express LNCaP cells. In-vitro studies showed significantly higher uptake of antibodies tethered scaffold. Shukla et al. [95] developed G5.0 PAMAMAlexafluor conjugate for targeting overexpressed HER-2 ovarian and cancer cells. The authors got higher accumulation of the scaffold in HER-2 overexpressed in-vivo tumor models. Similarly, Patri et al. [96] tailored J59 monoclonal Ab on PAMAM dendrimer surface to target overexpressed PSMA. Similarly, Otis et al. [97] encapsulated gold NPs in PAMAM dendrimers, further tailored with Herceptin Ab and observed that the nanoassembly selectively binds to the HER-2 overexpressed cells, interestingly the unmodified nano-assembly did not show any binding affinity toward the HER-2 expressed cells. Another monoclonal Ab Cetuximab, which targets EGFR was tethered on PAMAM G5.0 dendrimers covalently, further loaded with methotrexate antineoplastic drug. In-vitro evaluation revealed that the developed conjugate was quite cytotoxic to the F98 cell lines with high binding affinity [93]. Not so earlier, Xie et al. [98] conjugated a Slex Ab on PAMAM dendrimer and characterized the same. The authors claimed that cellular uptake of the modified dendrimers in HT29 cells was higher and concentration dependent. Most recently, Marcinkowska et al. [99] developed a PAMAM-trastuzumab conjugate further loaded with doxorubicin. The authors claimed that the developed conjugate holds a great potential of selective binding toward the HER-2 overexpressed cells. Cytotoxicity of the developed conjugate against the cancer cells was higher over the free drug. Conclusively, Ab-based approaches are very effective for the drug delivery against the cancerous cells. Intrinsic origin of Ab provides several advantages over the synthetic polymers. Additionally, the approach improves the biocompatibility factors when tagged with dendrimers.

8 Miscellaneous Apart from the above surface modification techniques, several other approaches have also been explored by the researchers. For example, acetylation of full generation dendrimers leads to neutralization of the surface amine cationic charge which is the main cause of toxicity to biological membranes [68]. It was observed that full generation PPI dendrimer causes toxicity to the HUVEC cells while 0.5G with acetylation showed significantly less toxicity toward HUVEC cells. Similarly, half-generation and anionic dendrimers have shown less toxicity over the cationic and full generation dendrimers. The half generation dendrimers do not have amine functionality on the surface carboxylic groups instead. These half generation dendrimers have shown least hemotoxicity and cytotoxicity [17,48,71]. Interestingly, drug conjugation on dendrimer surface also masks the cationic charge, and dendriplex formation also masks the cationic charge. For example, Bendamustine tagging on the PAMAM G4.0 dendrimer reduces the hemotoxicity by several folds [51]. Lactoferrin surface modification on PAMAM G3.0 dendrimers also alleviate the hemotoxicity [52].

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9 Future prospectus Dendrimers are the nanocarrier of new era with great potential for nanotechnological based applications including therapeutics, imaging and diagnostics. Dendrimers have already proven their capability in drug delivery systems very well. On ground reality, several dendrimeric marketed formulations are available such as Vivagel® (manufactured by Starpharma, Australia) application as a topical microbicide, Superfect® (manufactured by Qiagen Inc., USA) application as a gene transfecting agent, Alert Ticket™ (manufactured by US army research Laboratory) application as anthrax detector etc. These products also proved that dendrimers have clinical potential and in near future, many more clinically approved products will be there in the market as several formulations are under clinical trial process. Despite so many unmatched properties, cationic dendrimers do have the undesired toxicity as discussed in this chapter. But, as we have discussed there are so many strategies to overrule this risk factor, which improves the overall applicability of dendrimers. Still, researchers should explore new techniques to make dendrimers more biocompatible for biomedical applications. In fact, the newer research outcomes are majorly focusing exclusively on the surface engineered dendrimers. Additionally, the option of hydroxyl, carboxylic and other biocompatible surface functionality is always open for exploration. In future, it should be possible to rank the biocompatibility and toxicity propensity of different dendrimers in a predictable manner so that the research efforts on their therapeutic and clinical aspects could be selectively focused.

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[50] Wolinsky JB, Grinstaff MW. Therapeutic and diagnostic applications of dendrimers for cancer treatment. Adv Drug Deliv Rev 2008;60:1037–55. [51] Gothwal A, Khan I, Kumar P, Raza K, Kaul A, Mishra AK, Gupta U. Bendamustine-PAMAM conjugates for the improved apoptosis, efficacy and in vivo pharmacokinetics: a sustainable delivery tactic. Mol Pharm 2017;5:203–14. [52] Gothwal A, Nakhate KT, Alexander A, Ajazuddin, Gupta U. Boosted memory and improved brain bioavailability of rivastigmine: targeting effort to the brain using covalently tethered lower generation PAMAM dendrimers with lactoferrin. Mol Pharm 2018;15:4538–49. [53] Castro RI, Forero-Doria O, Guzma´n L. Perspectives of dendrimer-based nanoparticles in cancer therapy. An Acad Bras Cienc 2018;90:2331–46. [54] Greish K, Thiagarajan G, Herd H, Price R, Bauer H, Hubbard D, Burckle A, Sadekar S, Yu T, Anwar A, Ray A, Ghandehari H. Size and surface charge significantly influence toxicity of silica and dendritic nanoparticles. Nanotoxicology 2012;6:713–23. [55] Kitchens KM, Foraker AB, Kolhatkar RB, Swaan PW, Ghandehari H. Endocytosis and interaction of poly (amidoamine) dendrimers with Caco-2 cells. Pharm Res 2007;24:2138–45. [56] Hong S, Bielinska AU, Mecke A, Keszler B, Beals JL, Shi X, Balogh L, Orr BG, Baker Jr JR, Holl MMB. Interaction of poly(amidoamine) dendrimers with supported lipid bilayers and cells: hole formation and the relation to transport. Bioconjug Chem 2004;15:774–82. [57] Hong S, Leroueil PR, Janus EK, Peters JL, Kober MM, Islam MT, Orr BG, Baker Jr JR, Holl MMB. Interaction of polycationic polymers with supported lipid bilayers and cells: nanoscale hole formation and enhanced membrane permeability. Bioconjug Chem 2006;17:728–34. [58] Mecke A, Uppuluri S, Sassanella TM, Lee DK, Ramamoorthy A, Baker Jr JR, Orr BG, Holl MMB. Direct observation of lipid bilayer disruption by poly (amidoamine) dendrimers. Chem Phys Lipids 2004;132:3–14. [59] Wang B, Navath RS, Menjoge AR, Balakrishnan B, Bellair R, Dai H, Romero R, Kannan S, Kannan RM. Inhibition of bacterial growth and intramniotic infection in a guinea pig model of chorioamnionitis using PAMAM dendrimers. Int J Pharm 2010;395:298–308. [60] Choi SH, Lee SH, Park TG. Temperature-sensitive pluronic/poly(ethylenimine) nanocapsules for thermally triggered disruption of intracellular endosomal compartment. Biomacromolecules 2006;7:1864–70. [61] Lee H, Larson RG. Lipid bilayer curvature and pore formation induced by charged linear polymers and dendrimers: the effect of molecular shape. J Phys Chem B 2008;112:12279–85. [62] Iwai K, Maeda H, Konno T. Use of oily contrast medium for selective drug targeting to tumor: enhanced therapeutic effect and X-ray image. Cancer Res 1984;44:2115–21. [63] Maeda H, Matsumoto T, Konno T, Iwai K, Ueda M. Tailor-making of protein drugs by polymer conjugation for tumor targeting: a brief review on Smancs. J Protein Chem 1984;3:181–93. [64] Matsumura Y, Maeda H. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent Smancs. Cancer Res 1986;46:6387–92. [65] Brazeau GA, Attia S, Poxon S, Hughes JA. In vitro myotoxicity of selected cationic macromolecules used in non-viral gene delivery. Pharm Res 1998;15:680–4. [66] Wilbur D, Pathare P, Hamlin D, Bhular K, Vessella R. Biotin reagents for antibody pretargeting: synthesis, radioiodination, and evaluation of biotinylated starburst dendrimers. Bioconjug Chem 1998;9:813–25. [67] Jevprasesphant R, Penny J, Jalal R, Attwood D, McKeown N, D’Emanuele A. The influence of surface modification on the cytotoxicity of PAMAM dendrimers. Int J Pharm 2003;252:263–6.

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Therapeutic dendrimers

12

Abhay Chauhana, Dominika Krynickaa, Mayank K. Singhb a

SCHOOL OF PHARMACY, MEDICAL COLLEGE OF WI SCONSIN, MILWAUKEE, WI, UNITED STATES b DEPART MENT OF AP PLI ED B IO LOGY , C SIR- I NDIAN INSTITUTE O F CHEMI CAL T ECHNOLOGY, HY DERABAD, INDIA

PA MA

M

De

nd

rim

er

Arachidonic Acid

Arachidonic Acid Inflammation

Inflammation absent

COX Prostaglandins

Prostaglandins

1 Introduction Important biological building blocks such as DNA, proteins, and lipid-bilayers have nanometer dimensions and therefore nano-moieties can interact easily and effectively with these biological moieties. Dendrimers of different sizes (2–10 nm) have dimensions like the proteins found in the body. This opens a window for nanometer range platforms like dendrimers. Dendrimers can interact with biological entities and produce therapeutic effects. These dendrimers are called Therapeutic Dendrimers. Approval of dendrimer products that have been commercially, as well as clinically important, has led to exponential interest in research and development of Therapeutic Dendrimers (TD). Therapeutic Dendrimers are closely entwined in the diagnosis, prevention and treatment of diseases. TD has the potential to be immensely beneficial to human health due to their multitasking-polyvalent architecture [1]. As evident through interdisciplinary research, Pharmaceutical Applications of Dendrimers. https://doi.org/10.1016/B978-0-12-814527-2.00012-3 © 2020 Elsevier Inc. All rights reserved.

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Table 12.1 List of TD with their applications in pharmaceutical, medical and agricultural research Products (Pharmaceutical and medical) In-vitro and In-vivo Cardiac Diagnostics Stratus CS® Anthrax-detecting agent Alert Ticket™ Gene Transfection Reagent SuperFect® siRNA Transfection Reagents PrioFect™ HIV and STDs VivaGel™ Multiple cancer types DEP™ docetaxel Colon Cancer Dendrimer-Oxaliplatin Agrochemicals NYSE: AGU, TSE: AGU

Dendrimer

Corporation

Status

PAMAM

Dade Behring

Marketed

PAMAM

US Army Lab

Marketed

PAMAM

Qiagen

Marketed

PAMAM

Starpharma

Marketed

Poly-L-lysine

Starpharma

Clinical trial (Phase-III)

#

Starpharma

Clinical trial (Phase-I)

#

Starpharma

Clinical trial (Phase-I)

Priostar

Starpharma-Agrium

Marketed

PAMAM, poly-amidoamine; HIV, human immunodeficiency virus; STDs, sexually transmitted diseases; #, not disclosed.

dendrimers have shown potential to be designed as therapeutically active molecules for human use. There are many dendrimer-based inventions that are currently in preclinical and clinical trials. Besides the diagnosis and/or in-vitro dendrimer-based technology already in the market, dendrimer systems have reached the clinical evaluation as antimicrobicides (VivaGel™) and drug carriers for solid tumors (DEP™ docetaxel). The key features of few TD are shown in Table 12.1. In this chapter, we carried out an analysis of TD and their application as platforms in preclinical and clinical trials with a special focus on their active role as dendrimers in nanomedicine in preclinical, clinical and FDA-Approved development trials [2–21]. The field of pharmaceutical application has exhibited rapid growth in the past decade. Among them, polyamidoamine (PAMAM) dendrimers have emerged as a promising therapeutic system to be used in diverse areas, which will be discussed in the following sections as Antimicrobial therapy, Anti-inflammatory activity, tumors, angiogenesis inhibitors and antivirals.

2 Classification of therapeutic dendrimer 2.1 Antimicrobial therapy The Centers for Disease Control and Prevention claims that antimicrobial resistance is among the top three threats to the survival of mankind [22]. Since the 1950s, and most drastically the 1990s, organisms such as VRE (Vancomycin Resistant Enterococci), ESBL

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(Extended Spectrum Beta-Lactamase), KPC (Klebsiella pneumoniae Carbapenemase), and many more have developed resistance to the most powerful drugs on the market (i.e., vancomycin and carbapenems) [23]. As use of these drugs continues, more bacteria will fall under the category of “superbug,” like those mentioned above. In order to combat bacterial resistance, dendrimers have been studied in search for new antimicrobial therapy. Dendrimers with amphiphilic and cationic assets may demonstrate antimicrobial activity due to their membrane disrupting properties, which has consequences in solubilization of the microbial membrane and pore formation, followed by death. Mechanistically, the dendrimer utilizes an innovative cytotoxic method as compared to other antibiotics, thus overcoming the antimicrobial resistance crisis [24]. Poly(lysine) dendrimer modified with sulfonated naphthyl groups (VivaGel™) is a well-developed waterbased vaginal product of 3% (w/w) SPL7013 mixed in carbopol gel to provide relief and prevent recurrence of bacterial vaginosis. It also helps prevent genital herpes, HIV, and other sexually transmitted infections [25–27]. VivaGel is currently in clinical trial for human applications. Dendrimer properties have been explored by Gholami et al. [28], who studied a seventh-generation poly(amidoamine) dendrimer, also known as PAMAM-G7, against Gram-negative and Gram-positive strains and isolates, such as Pseudomonas aeruginosa ATCC 27853, E. coli ATCC 25922, Acinetobacter baumannii ATCC 17957, Shigella dysenteriae ATCC 13313, K. pneumoniae ATCC 1705, Proteus mirabilis ATCC 29906, Staphylococcus aureus ATCC 25923, and Bacillus subtilis ATCC 23857 in-vitro. These are among the primary strains of bacteria that are classified as multi-drug resistant bacteria that contribute to healthcare-associated infections [29]. It was demonstrated that PAMAM-G7 can inhibit the growth of majority of the strains listed above. Antibacterial activity was determined by utilizing the disc diffusion method. Cytotoxic assay was administered in order to analyze the dendrimer’s cytotoxic effects on human cells. Overall, PAMAM-G7 inhibited growth in both isolated Gram-positive and Gram-negative bacteria and standard strains but had stronger antimicrobial effects on the standard strains. The highest sensitivity was found in P. mirabilis ATCC 29906, S. dysenteriae ATCC 13313, and S. aureus ATCC 25923 strains based on zones of inhibition. Based on MIC values, the most resilient bacterial isolates, or those with the highest MIC50 and MIC90 in regard to PAMAM-G7, were E. coli, A. baumannii, P. aeruginosa and S. aureus isolates. The smallest MIC50 and MIC90 were identified for organisms S. dysenteries and P. mirabilis. The highest MBC50 and MBC90 strain values were similar in rank to the highest MIC50 and MIC90 isolate values, minus A. baumannii. S. dysenteriae was found to have the smallest MBC50 and MBC90. MIC and MBC values were similar for the isolates and strains. It was demonstrated that the cytotoxic effect was increased with increase in concentration of PAMAM-G7 dendrimer. Also, cytotoxicity was relatively low at the MIC values used in this study (i.e., 2–4 and 4–8 μg/mL). Cytotoxicity occurs when the dendrimer creates nanoscale holes in the cell’s membrane. In a similar way, the mechanism of action of

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the PAMAM-G7 against bacteria also includes the penetration of their cell walls. It is thought to involve the dendrimer’s terminal amine functional groups; this dendrimer has 512 amine groups. The functional groups disrupt outer and inner membranes by diffusing through the bacterial cell wall, allowing for the release of electrolytes, DNA and RNA. Pen˜a-Gonza´lez et al. have synthesized water-soluble silver nanoparticles (AgNPs) that were capped with cationic carbosilane dendrons, which are lipophilic in nature. Both water-soluble AgNPs (silver nanoparticles) and AuNPs (gold nanoparticles) capped with cationic dendrons were studied against Gram-positive, Gram-negative bacterial strains, and yeast. These strains included methicillin-susceptible S. aureus (MSSA), methicillinresistant S. aureus (MRSA), Enterobacter faecalis, E. coli, P. aeruginosa, multi-drug resistant Staphylococcus haemolyticus, and yeast strains Candida albicans and Candida glabrata. Without the AgNp, the carbosaline dendron was inherently inactive, while the AgNP is bactericidal against many resistant organisms, other than P. aeruginosa and fungi like C. glabrata. In order for the capped nanoparticle to interact well with the bacterial and fungal cell, the lipophilic dendron is necessary to surpass the cell membrane of the organisms [30]. Bin Sun et al. looked at the bactericidal activity of nitric oxide (NO)-releasing poly-propylene-imine (PPI) dendrimers against Gram-positive and negative organisms such as P. aeruginosa, methicillin-susceptible S. aureus, and methicillin-resistant S. aureus. NO-releasing PPI dendrimers were compared to non-NO releasing PPI dendrimers, and it was determined that NO-releasing dendrimers have superior bactericidal properties and less cytotoxic activity against mammalian fibroblast cells, as opposed to quaternary ammonium-functionalized dendrimer scaffolds. NO functions as a reactive free-radical that produces a multitude of toxic byproducts such as dinitrogen trioxide and peroxynitrite; this function allows the molecule to have broad bactericidal effects. Among all the NO-releasing PPI dendrimers tested, the dendrimer modified with styrene oxide (SO) demonstrated the most biocidal activity, but minimal cytotoxicity against in vitro mammalian cells at bactericidal doses. It is hypothesized that SO-modified dendrimers allow for increased electrostatic interactions between the bacteria and the dendrimer, which ultimately helps increase localized NO release. In addition, Bin Sun et al. synthesized N-diazeniumdiolate functionalities, which also increase the local release of NO to the bacterial cell, primarily due to the functionality’s dense structure [31]. Choline-binding proteins (CBPs) are common pneumococcal virulence factors common to all serotypes of the bacteria and have been found to be promising targets for pneumococcal infections. Ribes et al. studied a choline dendrimer with specific affinity for CBPs on microglia-induced pneumococcal phagocytosis. Choline dendrimers were incubated with the bacteria, leading to the uptake of the dendrimers, which ultimately replaced the bacteria’s CBPs. This allowed for continued growth of the pneumococci that was readily phagocytosed by microglial cells. It has been demonstrated that multivalent dendrimers with choline end groups are a promising antimicrobial agent for the control of pneumococcal disease [32].

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2.2 Anti-inflammatory/inflammation Inflammation is a reaction of the body in response to injury. Upon the introduction of injury or harmful stimuli, the body attempts to protect itself. Chemicals from white blood cells are released into the blood to remove harmful substances and start the healing process. Increased blood flow to the infected area will cause the redness and warmth of inflammation. Inflammation is not an infection, which is caused by bacteria, fungi, or a virus. Inflammation is merely the body’s reaction to infection. Some people turn to home remedies such as fish oil, ice packs, or green tea to treat inflammation along with drug therapy available for the treatment of inflammation [33]. A group of anti-inflammatory drugs called nonsteroidal anti-inflammatory drugs (NSAIDs) are popularly used for this indication. NSAIDs work by blocking the enzyme cyclooxygenase (COX). COX is responsible for the release of prostaglandins, which cause the inflammation. Prostaglandins are chemicals responsible for body reactions that cause pain and inflammation; therefore, if COX enzymes are blocked, prostaglandin release is blocked, thus resulting in reduced pain and inflammation. NSAIDs can be used in the treatment of arthritis, fever, headaches, and even menstrual cramps. Long-term use of NSAIDs, however, can have some severe side effects. As mentioned before, NSAIDs block the cyclooxygenase enzymes, COX-1 and COX-2. COX-1 is responsible for maintaining stomach lining. Inhibiting this enzyme can cause stomach ulcers and gastrointestinal upset. COX-2 is usually specific to just sites of inflammation, so inhibiting this enzyme does not cause as much GI upset. Renal failure, heart attack, and stroke, however, are all associated with the inhibition of COX-2. With the evolving world of medicine, there has been researching into alternative drug therapy for the treatment of inflammation. One of which has been the use of dendrimers as a potential inflammation reliever. Dendrimers were used as a delivery mechanism for an NSAID to relieve inflammation in a specific area. This is beneficial because the drug can be delivered directly to the site of inflammation, decreasing side effects that arise from the drug’s effect on non-inflamed areas. As research continued, it was discovered that the naked dendrimer could produce inhibitory properties of its own [34]. In the seminal paper of “the unexpected in-vivo anti-inflammatory activity observed for simple, surface functionalized poly(amidoamine) dendrimers” experiments Chauhan et al. [35] compared the inhibition rates of inflammation between naked dendrimer, indomethacin and dendrimer-indomethacin complex. Following a group of studies including the carrageenan-induced paw edema in rats, the cotton pellet test, adjuvant-induced arthritis in rats, and the cyclooxygenase assay. Indomethacin is a well-known anti-inflammatory drug, but it does have some undesirable side effects. The effect of dendrimer generation and surface was also investigated. It was discovered that “naked” dendrimers were more effective in inhibiting inflammation than the NSAID indomethacin (Fig. 12.1). For in-vivo testing, a group of rats were given saline and a group was given dendrimer with different surface groups (amine, hydroxyl and carboxylate). The rats were then dosed with carrageenan, a food additive, which caused inflammation. The swelling was

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FIG. 12.1 (A) A polyvalent; [core; 1,2-diaminoethane]; (G4.5); {dendri-poly(amidoamine)-(CO2H)55-(glucosamine)9} dendrimer conjugate and (B) various simple, terminal functionalized [core; 1,2-diaminoethane]; (G4.0); {dendripoly(amidoamine)-(Z)n}; wherein: Z ¼ dNH2, dOH, and other surface groups. From Chauhan AS, Diwan PV, Jain NK, Tomalia DA. Unexpected in vivo anti-inflammatory activity observed for simple, surface functionalized poly (amidoamine) dendrimers. Biomacromolecules 2009;10(5):1195–202.

measured at constant intervals. Finally, the level of swelling in the drug-treated group of rats was compared to that in the group treated with just saline. It was discovered that the dendrimer showed higher inhibition of inflammation than indomethacin. However, as time elapsed and dose levels were altered, percent inhibition changed. The cotton pellet test is a sub-chronic model for inflammation activity testing. In this test, a cotton pellet was surgically inserted into each rat followed by saline and drug administration. After day 8, the cotton pellets were removed and then surrounded by inflamed tissue, then dried out by heat. The results were compared to that of the rats given saline. Overall, the dendrimers were more effective at inhibiting inflammation than indomethacin. However, dendrimers did not show significant difference among themselves in inhibiting inflammation (Fig. 12.2). Adjuvant-induced arthritis in rats is a chronic test for inflammation. This test involves a group of rats that are treated with dendrimers, one treated with indomethacin, and the last group treated with saline. The rats were then injected with the inactivated, dried mycobacteria, Freund’s adjuvant. The rats were then dosed daily with the naked dendrimer and the indomethacin. The results again showed a higher percent inhibition with the dendrimers than that of the NSAID. Ultimately, inhibition rates were similar between dendrimers. Cyclooxygenase Assay was done to study the in-vitro mechanism. Cyclooxygenase 1 (COX-1) is responsible for kidney and platelet function in the body. It also maintains the lining of the stomach. Cyclooxygenase 2 (COX-2) is primarily at the site of inflammation. Some NSAIDs inhibit both COX-1 and COX-2. Low levels of COX-1 can lead to stomach ulcers and low levels of COX-2 in non-inflamed regions can also be dangerous. Ideally, researchers are looking to find an anti-inflammatory agent that inhibits only COX-2 that are specific to areas that are inflamed. The cyclooxygenase assay was conducted to

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Mean %Inhibition

60 50 40 30 20 10 0 Indomethacin

G4-NH2

G4-NH2-Indo

Formulation FIG. 12.2 Mean percentage inhibition exhibited by naked Indomethacin (Indo), (G4-NH2) and (G4-NH2-Indo complex), respectively, after i.p. administration in a cotton pellet granuloma assay using male Wistar rats (n ¼ 6); P < 0.05. From Chauhan AS, Diwan PV, Jain NK, Tomalia DA. Unexpected in vivo anti-inflammatory activity observed for simple, surface functionalized poly (amidoamine) dendrimers. Biomacromolecules 2009;10(5):1195–202.

determine the cyclooxygenase inhibitory properties of multiple dendrimers. It was shown that carboxylate and succinamic acid surfaced dendrimers did not have effects on either COX-1 or COX-2. The amine and cationic surfaced dendrimers had substantial effects on the inhibition of both COX-1 and 2, with amine dendrimer having the greater effect on COX-2. It was also proven that the increase in generation of dendrimers leads to increased COX-2 inhibition rates (Fig. 12.3). Xavier Bosch and team examined the effects that an azabisphosphonate capped dendrimer would have on the processes involved in the pathology of rheumatoid arthritis. 80

COX1 COX2

% COX inhibition

70 60 50 40 30 20 10

4PE 4CO G O Na G 4SU C G 4AE EA SC -5 6 N 0 S39 8 G

G

TR IS 4-

G

4-

PY

AE G

2

4G

4NH G

R

0

PAMAM Dendrimer FIG. 12.3 Effect of G ¼ 4/4.5; PAMAM dendrimer surface functionalization on cyclooxygenase (COX-1 and COX-2) inhibition as determined by COX inhibitor screening assay, (n ¼ 4)*. *Concentration of dendrimers: 0.174% w/v; SC 560 (specific COX-1 inhibitor): 18 nM; NSC 398 (specific COX-2 inhibitor): 1.74 μM. From Chauhan AS, Diwan PV, Jain NK, Tomalia DA. Unexpected in vivo anti-inflammatory activity observed for simple, surface functionalized poly (amidoamine) dendrimers. Biomacromolecules 2009;10(5):1195–202.

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Two mouse models containing rheumatoid arthritis were used in this study. The mice were dosed weekly with the dendrimer capped with ABP and it showed the following results: serum levels of pro-inflammatory cytokines were normal with a weekly dose of ABP, inflammation was decreased, there was zero joint damage, no monocytes turned into osteoclasts, and no osteoclasts in the bones of the mice. This study proved that the dendrimer capped with ABP can work on a cellular level and inhibit the processes thought to cause rheumatoid arthritis [36]. Rheumatoid arthritis (RA) is an auto-immune disorder, meaning that the immune system attacks the body that it itself originated. Two main processes associated with rheumatoid arthritis are the production of osteoclasts and the secretion of pro-inflammatory cytokines [37]. Osteoclasts are large multinuclear cells associated with the dissolution of bone. Cytokines are inflammatory signaling molecules excreted from immune cells. Drug therapy does exist for the treatment of RA, but these treatments require a very large dose and have a frequent demand. The dendrimer that was used in this study was capped with azabisphosphonate (ABP). ABP was originally sought out in the scientific community as a possible element used to turn the immune system against cancerous cells. Severine Fruchon et al. studied the effects of injecting cynomolgus macaques, a non-human primate, with ABP. Biochemical substances, blood levels, and clotting all stayed within a normal range throughout the duration of the experiment. The tissue of the primates was studied. This histopathological analysis and the study of serum cytokines showed no lesions or non-physiological occurrence. It was proven that ABP can be a therapeutic candidate for the treatment of inflammation [38]. ABP capped dendrimers were also studied in the treatment of uveitis, an inflammation of the middle layer of tissue in the eye. Mice models were given lipopolysaccharide (LPS) to induce uveitis. LPS is found on the outer membrane of Gram-negative bacteria and causes a strong immune response in animals. In this study, ABP was compared to dexamethasone, a popular steroid used to treat inflammation. Researchers found that ABP was most effective when given immediately following LPS injection. At this time, ABP was stronger than dexamethasone at its lowest dose. ABP was no different at inhibiting inflammation than dexamethasone at a higher dose. In addition, both ABP capped dendrimer and dexamethasone caused an increase in Interleukin-10, an anti-inflammatory cytokine. In this study, ABP capped dendrimer successfully inhibited inflammation in the eye of the mice models and worked systemically to greatly increase interleukin [39]. Cerebral palsy is a term that includes many disorders and it is an immune disorder. These disorders affect one’s ability to move and control muscle movement. Scientists do not have a definite cause of these disorders, but it is thought that cerebral palsy is caused by damage to the underdeveloped brain of the child during pregnancy or shortly after birth. These groups of disorders are permanent and have no cure. The study conducted by Fan Zhang et al. [40] unearthed the inhibitory properties of dendrimer in relation to microglial cells, which are present in inflammatory brain disorders such as cerebral palsy. Microglial cells are located throughout the brain and spinal cord and are the first and main form of active immune defense in the central nervous system. Microglial cells

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are seen in neuro-inflammation on the brain in regard to cerebral palsy and autism. An activation of microglial cells results in an increase in major histocompatibility complex (MHC). MHC is on the surface of cells to help the immune system recognize foreign substances. An upregulation of MHC makes microglia act as antigen-presenting cells to T-cells. These antigen-presenting cells can then enter the brain during active infection, causing inflammation. To analyze the dynamics of microglial migration and its interaction with dendrimers, researchers created an organotypic interaction between two or more cell types from complex tissue or organ and whole-hemisphere brain slice culture from rats. One group of rats was exposed to inflammation while in the womb the other was not exposed. Rats that were exposed to cerebral palsy had cells with amoeboid, ability to alter own shape, morphology and impaired microglial migration. In comparison to the healthy rats, the group with inflammation had decreased levels of cell migration and velocity. It was concluded that organisms exposed to inflammation in the womb can be expected to have impaired microglial function and movement. Leukocytes are white blood cells that circulate in the blood and body fluids to counteract disease. When the body recognizes an antigen or disease-causing substance, white blood cells attack. One immune response the body produces in response to antigens is inflammation. Endothelial cells line the entire vascular system and control the transit of white blood cells. Binding interactions between E-, L-, and P-selectin proteins play a role in leukocyte migration. E-selectin proteins are only expressed on the endothelial cells after activation. These proteins bind neutrophils, monocytes, and T-cell subsets to inflamed tissue. L-selectin proteins contribute to the lymphocyte and neutrophil entry into inflamed areas [41]. P-selectin proteins are transmembrane proteins that are specific to the alpha granules of platelets and Weibel-Palade bodies of endothelial cells [42]. Weibel-Palade bodies are storage granules of endothelial cells. They store and release von Willebrand and P-selectin protein. When certain cells are activated, P-selectin protein is moved to the plasma membrane where it acts as a monocyte and neutrophil receptor. Being that the E-, L-, and P-proteins play a key role in white blood cell migration, scientists were anticipating the use of a dendrimer to inhibit the binding of these proteins, thus inhibiting inflammation. The dendrimer used in this experiment is synthetic dendritic polyglycerol sulfate (DPG). This dendrimer is macromolecular, meaning it is a very large molecule with smaller structural units linked together. It also operates via a binding system with several sites to attach an antigen and mimics the naturally occurring ligands in the body. SPR based-binding assay was used to determine the IC50 values of DPG over a large concentration. IC50 is a measure of effectiveness of a substance in inhibiting specific biological function. The results showed that L- and P-protein bindings were successfully blocked with DPG, however, E-selectin was not affected by DPG [43]. Effects of the core size of dendrimer in blocking protein binding were evaluated. Unfractionated heparin with 63 sulfate groups was used as a control. Unfractionated heparin has more sulfate groups than the dendrimer that was utilized, although it still produced a lower IC50 value

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than the dendrimer. This result is thought to be because of the backbone and spaced out ligand structure that the unfractionated heparin has. In addition, the degree of sulfur modification of DPG was studied. As DPG sulfate levels increased, L-selectin binding was reduced. With the results of these studies, it was determined that inhibitory properties depended on many characteristics such as core size, number of sulfurs, and ligand spacing. Leukocyte extravasation to inflamed areas with DPG was measured. An allergic reaction was aroused in mice models with trimellitic anhydride. This reaction caused the typical effects of dermatitis such as redness, ear swelling, edema, and cellular infiltration. DPG was compared to glucocorticoid prednisolone, an anti-inflammatory drug. Glucocorticoid drugs are man-made versions of glucocorticoids which interrupt inflammation in the body by moving into cells and suppressing proteins. It was found that DPG was just as effective at reducing inflammation as the prednisolone. Neutrophil elastase activity in the mouse ear was measured to determine the amount of extravasation occurring in response to administration of DPG. Neutrophil elastase is secreted by neutrophils and macrophages during inflammation. DPG showed a decrease in white blood cell extravasation to inflamed tissue. DPG effectively blocked the binding of E-, L-, and P-selectin proteins. This means that the dendrimer is capable of controlling leukocyte migration, which can lead to a decrease in inflammation. In conclusion, drug therapy indicated for inflammation is evolving. Nonsteroidal antiinflammatory drugs are effective in treating inflammation, but the lasting side effects are a concern. As studies are conducted on this subject, researchers are finding potential in the use of dendrimers in treating inflammation. Scientific experiments are showing that dendrimers can have better anti-inflammatory rates than some NSAIDs. The use of dendrimers an effective way to inhibit inflammation is still being studied.

2.3 Tumors Zhang et al. synthesized a tryptophan-rich peptide dendrimer (TRPD) as a new type of effective tumor therapy. This entity is highly effective due to its excellent water solubility, precise structure that is highly branched with multiple terminal groups, and overall protein-like shape. The TRPD can interact with intracellular DNA by interaction of the indole ring connected to tryptophan residues, creating effective supramolecular aggregates. In addition, TRPDs have high membrane permeability and can exert highly cytotoxic effects on tumor cells. Overall, the TRPs were able to obstruct the proliferation of tumor cells in vivo and aid in tumor cell apoptosis [44].

2.4 Angiogenesis inhibitor Dendrimers have been conjugated with glucosamine and glucosamine-6-sulfate and claimed to exhibit immunomodulatory and antiangiogenic properties which prevent scar tissue formation during glaucoma surgery [10]. In another study it was assumed that dendrimers mimic heparin or heparin sulfate, sequestering fibroblast growth factor, and possibly other growth factors, thereby disrupting the formation of new blood vessels [45].

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2.5 Antiviral Dendrimers with sialic acid functional groups have been utilized to enhance anti-viral activity and claim to be therapeutically effective inhibitors of influenza virus when binding to a host cell receptor in in-vitro and in-vivo models [46–48].

3 Conclusion Dendrimers have been showing promise as Therapeutic Dendrimers. The successful demonstration of such biomimetic dendrimers has broadened the potential to use as macromolecular vectors in novel drug delivery and biomedical applications.

Acknowledgments D.K. acknowledges Dean, School of Pharmacy, Medical College of Wisconsin, USA. M.K.S. acknowledges Council of Scientific and Industrial Research, India (CSIR-INDIA) for Senior Research Fellowship. We would like to acknowledge Alexis Robinson-Cooper’s help in collecting information for this chapter under CTSI, Star 500 program at Medical College of Wisconsin, Milwaukee, USA.

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13 Dendrimers for diagnostic applications Divya Bharti Raia, Nitin Guptaa, Deep Poojab, Hitesh Kulharia a

SCHOOL OF NANO SCIENCES, C ENTRAL UN IVERSITY OF GUJARAT, GANDHINAGAR, INDIA b PHARMACOLOGY AND T OX ICOLOGY DIVISIO N , C S I R - I ND I A N I N S T I T U T E O F C H E MI C A L TECHNO LOGY , HYDERABAD, INDIA

1 Introduction Medical diagnosis is the process of determining the disease or condition by seeing the patient’s symptoms and signs. It is usually called diagnosis when the medical context is unexpressed. The information required for diagnosis is typically collected from a history and physical examination of the patient seeking medical hospitality. Several kinds of diagnostic imaging are also done during the process to conclude the result [1,2]. Currently, in-vivo diagnostic imaging comprises an important focus area of medical research. The rapidly evolving field of molecular imaging improves early disease detection and progression of disease and enables image-guided therapy and treatment personalization. Furthermore, it provides essential information on the therapy efficacy. However, molecular imaging requires the use of molecular imaging probes to visualize and characterize biological processes at the cellular and molecular level [3–7]. In this section, a brief overview of frequently used non-invasive imaging modalities with respect to the application of nanoparticles will be provided. Medical imaging comprises the non-invasive assessment of anatomical (or morphological), functional and molecular information, which enables the diagnosis of pathophysiological abnormalities. Current imaging modalities that are routinely used in preclinical research and clinical practice include Magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET) as well as theranostic modalities such as photothermal therapy (PTT), photodynamic therapy (PDT) and neutron capture therapy (NCT). These modalities are based on different underlying physical principles and, therefore, possess specific disadvantages with respect to sensitivity and specificity to contrast agents, tissue contrast, spatial resolution, quantitative estimation and tissue penetration [8–10]. Recent advances in nanotechnology have led to the development of various nanoparticle formulations for diagnostic and therapeutic applications. Diagnostic nanoparticles aim to visualize pathologies and to improve the understanding of important pathological and physiological principles of various diseases and disease treatments [5,11]. The use of Pharmaceutical Applications of Dendrimers. https://doi.org/10.1016/B978-0-12-814527-2.00013-5 © 2020 Elsevier Inc. All rights reserved.

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nanocarriers as diagnostic systems for therapeutic or imaging agents can improve the pharmacological properties of commonly used compounds in cancer non-invasive imaging techniques, diagnosis and treatment [12]. Nano- and microparticles based on polymers, proteins, lipids, lipoproteins, metals, and silica, as well as fullerenes, carbon nanotubes, selenium-cadmium nanocrystals (i.e., quantum dots), and microbubbles, are frequently suggested as diagnostic and theranostic agents [7]. However, nanodiagnostics are only useful in a limited number of situations, due to the complex demands on their pharmacokinetic properties and elimination [13]. Therefore, the intrinsic characteristics of nanoparticles hold great promise for integrating diagnostic and therapeutic agents into a single nanoparticle formulation, enabling their application for theranostic purposes, such as monitoring the biodistribution and target site accumulation, visualizing and quantifying drug release and longitudinally assessing the therapeutic efficacy [14–18]. For better and more specific imaging, several requirements are proposed for contrast agents, which generally include a long half-decay time, a relatively low renal toxicity, and a high specificity. With the development of nanotechnology, researchers are trying to apply nanoparticles (NPs) as contrast agents [19]. These NP systems have many advantages over the conventional contrast agents, such as a prolonged blood circulation time, facile surface functionalization, an extended imaging time and the desired biocompatibility [20]. Dendrimers, as a new nanomaterial, are broadly involved in the design and exploitation of contrast agents. Specifically, atom-by-atom modifications on the cores, interiors, and surface groups of dendrimers permit rational manipulation of dendrimer-based agents to optimize the physical characteristics, biodistribution, receptor-mediated targeting and controlled release of payload contrast agents. As a function of their size, shape, surface chemistry and interior void space, dendrimers are routinely synthesized as tunable nanostructures that may be designed and regulated. Critical Nanoscale Design Parameters (CNDPs) such as: (a) size, (b) shape, (c) surface chemistry, (d) flexibility and (e) architecture should be controlled to obtain a wide range of synthetic nanostructures [21]. Key research priorities for targeted delivery and in vivo imaging should address: (i) design of nanostructures with stealth properties that prevent them from being opsonized or cleared before reaching the target cells, (ii) ability to penetrate into cells and crossover biological barriers like the BBB, uptake and recycling of nanostructures, (iii) nanocarriers or strategies that selectively targets diseased cells, tissues and organs, (iv) trans-endocytosis of nanostructures, (v) safety evaluation (in vitro/in vivo cytotoxicity, hematological compatibility and immunogenicity), in vivo carrier biodistribution, and (vi) compatibility with external activation by magnetic field, ultrasound, X-ray, or optics to generate contrast [18]. This chapter illustrates the basic features of hyperbranched dendrimer as probes, carriers, solubilizers and protective agents for imaging in vivo and in vitro and various advantages of using dendrimer for imaging purpose in various diagnostic and theranostic modalities.

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2 Problem with carrier free in-vivo delivery of diagnostic agents • • • • • •

Hydrophobicity and consequently low bioavailability Short retention time-rapidly cleared from the blood by glomerular filtration and subsequently excreted by the kidneys without significant retention Non-specific accumulations/non-specific distribution Biological barriers and cellular impermeability Toxicity or immunogenicity Poor diagnostic properties such as contrast, resolution, X-ray attenuation etc.

3 Advantage of dendrimer-based imaging systems Various contrasting compounds conjugated with dendrimers have been widely synthesized and evaluated for bioimaging because of following advantages: • • • • •

reduced premature release of contrasting agents from the dendrimers and improved tissue-specific accumulation through enhanced permeability and retention effect high payload enhanced aqueous solubility of hydrophobic agents; improved pharmacokinetics of conjugated imaging compounds due to decrease in clearance prolonged blood circulation time and altered biodistribution compatibility with external activation by magnetic field, ultrasound, X-ray, or optics to provide contrast.

4 Design criteria for formulating dendrimer vectors as contrasting agents The following properties of the dendrimers make them as an ideal carrier for drug delivery, therapy and diagnosis.

4.1 Low polydispersity index They have lower polydispersity index, due to stringent control during synthesis. As the density of branches increases the outer most branches arrange themselves surrounding a lower density core in the form of spheres and outer surface density is more and most of the space remains hollow toward core. This region can be utilized for entrapment of variety of drugs [22].

4.2 Enhanced permeability and retention effect Size of dendrimers (i.e., Generation 4–4.4 nm) is in nano range. Cancer cells have leaky membranes leading to enhanced permeation of drugs. Dendrimers demonstrate

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enhanced permeability and retention effect (depending on their M.W.) that allows dendrimer-drug complex to target tumor cells more effectively than small entities such as typical drug molecules. Lymphatic system is one way drainage system and drug loaded dendrimers may get retained inside the cancer area [23].

4.3 High permeability This property improves intracellular trafficking of drugs. Dendrimers can cross biobarriers like blood brain barrier, cell membrane. Nanometer range and uniformity in size enhance their ability to cross cell membranes and diminishes the risk of undesired clearance from the body through the liver or spleen [24,25].

4.4 Sustained/extended effect Dendrimers can release drug in a sustained manner. PAMAM dendrimers exhibited slower release, higher accumulation in solid tumors, and lower toxicity. Conjugation with Polyethylene glycol on the surface of these nanocarriers avoids non-specific interaction with plasma proteins or engulfment. Increase in blood circulation time is essential to achieve desired clinical effect [26].

4.5 Higher solubilization potential Ionic interaction, hydrogen bonding and hydrophobic interactions are probable mechanism by which dendrimers show its solubility enhancing property. Most anticancer drugs have poor solubility and can be loaded into dendrimers to improve solubility [27, 28]. Dendrimers can improve the solubility, biodistribution, and efficacy of a number of therapeutics as well as being used as imaging and diagnostic molecules in animal models bearing brain tumors.

4.6 High uniformity and purity The synthetic process used produces dendrimers with uniform sizes range, well-defined surface functionality, and negligible impurity. Monodispersed dendrimers would facilitate to attain effective and targeted drug delivery [29,30].

4.7 Multifunctional platform Multiple functional groups are present on outer surface of dendrimers, which can be used to attach vector devices for targeting to particular site in the body. Terminal groups may also be modified to reorganize specific receptors. The surface modification may allow designing dendrimers mimicking biological exo-receptors, substrates, inhibitors or cofactors. Free surface groups can form complex or conjugates with drug excellent molecules or ligands by using cross linking agents. The surface of dendrimers may be conjugated with ligands, solubility modifiers, and stealth molecules [31].

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4.8 High loading capacity Dendrimers structures can be used to load a wide range of organic or inorganic molecules by encapsulation and absorption on surface. Drug can get entrapped inside the internal cavities as well as electrostatically in the surface of dendrimers [32].

4.9 High stability Dendrimer-drug complex or conjugate shows better colloidal, biological and shelfstability. Dendrimers have nanoscopic particle size range from 1 to 100 nm, which makes them less susceptible for reticuloendothelial uptake.

4.10 Low toxicity Most dendrimers systems display very low cytotoxicity levels but have good biodegradability. PEGylation of the dendrimer surface can prolong its circulation time and reduce its toxicity [33].

4.11 Low immunogenicity Dendrimers shows low or negligible immunogenic response when injected or used topically. The problems in vesicular systems like chemical instability, drug leakage, aggregation and fusion during storage, solubility in physiological environment, lysis of phospholipids, purity of natural phospholipids lack in dendritic system [34].

4.12 Dendrimers can be modified as stimuli responsive to release drug The similarity of dendrimers structure with IgM antibodies (pentamers radially distributed) suggest that they may be used to simulate as antibodies, e.g., activation of macrophages, recognition, and high affinity to antigen.

4.13 Dendrimers microvascular extravasation properties An ideal polymeric carrier suitable for parenteral administration should be essentially non-toxic, nonimmunogenic and biodegradable. The carrier should display suitable tissue distribution attributes that confine the therapy to the targeted disease site and, ideally, avoid exposure to collateral healthy cells and/or tissue. Extravasation is the movement of smaller, lower molecular weight molecules from the blood circulatory system across the endothelial lining of capillary walls into the neighboring interstitial tissues. Efficacious drug delivery systems must extravasate from the systemic circulation across the microvascular endothelium into the interstitial tissue to reach a targeted site of therapeutic action. The influence of size and molecular weight within a series of PAMAM–NH2 dendrimers on extravasation across the microvascular endothelium was investigated [21]. Extravasation time(s) increased exponentially with an increase in molecular weight and size of the PAMAM dendrimers. The order of extravasation time for PAMAM–NH2 dendrimers was

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G0 < G1 < G2 < G3 < G4, ranging from 143.9 to 422.7 s. This size-dependent selectivity is due to the increased exclusion of PAMAM–NH2 dendrimers from the endothelial pores, 4–5 nm in radius, as the dendrimer size was increased. Based on the reported molecular sizes of PAMAM–NH2 dendrimers, ranging 1.5–4.5 nm, it seems that dendrimers can cross the microvascular endothelium through the endothelial pores of diameter 4–5 nm if their dimensions are small enough. The observed extravasation of PAMAM–NH2 dendrimers might be a function of the electrostatic interactions between the dendrimer and the negatively charged endothelium glycocalyx lining. The study demonstrated that an increase in molecular weight and size of polymers results in increased extravasation time across the microvascular endothelium. Molecular geometry and surface charge also influence the microvascular extravasation of water-soluble polymers across the endothelial barrier. As such, spherical, positively charged PAMAM dendrimers usually exhibit shorter extravasation times than linear macromolecules such as PEGs [35].

5 Dendrimer for encapsulating contrasting agents within the cavity Bioimaging agents can be either encapsulated in the dendrimer core or attached to polymer surface via covalent linkage (amide, ester etc.). Incarceration in this void space overcome hydrophobicity, reduces drug toxicity and allows controlled release. The interior is defined by the size and nature of the core (i.e., hydrophobic compared with hydrophilic), as well as surface congestion, which increases with generation level. Generally, contrasting agents are encapsulated in the interior cavities of dendrimers by physical encapsulation or via non-covalent interactions between imaging molecules and interior functional groups of dendrimers. The non-covalent interaction between dendrimers and imagining molecules may be an electrostatic attraction, hydrophobic interaction, or hydrogen bonding interaction [36]. These dendrimer cavities act as a typical container to provide controlled release of diagnostic agents, specifically for hydrophobic imaging molecules. However, as diagnostic molecules can cause severe unwanted toxicities due to accumulation, the prerequisite for the development of a nanocarrier-based bioimaging system for is targeting and controlled elimination of imaging agents from the body [37].

5.1 Dendrimer-based magnetic resonance imaging agents MRI is an important non-invasive medical tool that helps physicians to diagnose and treat medical conditions. It provides high-quality three-dimensional images without the use of harmful ionizing radiation. MRI uses a powerful magnetic field, radio frequency pulses and a computer to produce detailed images of organs, soft tissues, bone and virtually all other internal body structures. Magnetic resonance imaging (MRI) is a technique that uses a magnetic field and radio waves to create detailed images of the organs and tissues within your body. Most MRI machines are large, tube-shaped magnets. Inside an MRI machine, the magnetic field temporarily realigns hydrogen atoms in the body. The signal

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intensity in MRI stems mainly from the relaxation rate of in vivo water protons and is enhanced by the administration of a contrast agent prior to the scan. Such agents include a paramagnetic metal ion that decreases the relaxation times of nearby water protons. Different groups of contrast agents are established for clinical application such as gadolinium chelates, superparamagnetic iron oxide particles, and hepatobiliary contrast agents. However, the gadolinium chelates constitute the largest group of MRI contrast agents and are safe. The crucial properties of MRI contrast agents include good biocompatibility, low toxicity, and high relaxivity. Low molecular weight MRI contrast agents diffuse rapidly from blood vessels into the interstitial space and are excreted from the body very rapidly [38]. Paramagnetic metal chelates such as Gd(III)-N,N0 ,N00 ,N000 -tetracarboxymethyl-1,4,7,10tetraazacyclododecane (Gd(III)-DOTA), Gd(III)-diethylenetriamine pentaacetic acid (Gd(III)-DTPA), and their derivatives are used as contrast agents for magnetic resonance imaging (MRI), because these metal chelates increase the relaxation rate of surrounding water protons [39]. Every tissue of the body is intrinsically different, and the water protons of each tissue act in a distinct, detectable way. Radio frequency pulses disrupt the magnetization of the protons in tissue. The time it takes for the magnetization to be restored is measured as relaxation rates. Relaxation rates are measured by changes in the electromagnetic signal strength over very short periods of time (milliseconds). Mathematical analysis transforms differences in relaxation times into visual contrast between different tissue types. Magnetic gradients are applied so that the spatial coordinates of relaxation measurements can be assigned. Together, a very high-resolution, two- or threedimensional image of body tissue can be produced and analyzed for diagnostic purposes. Introduction of contrast agents to bodily tissues can enhance the images obtained by MRI and other imaging techniques. These contrast agents operate by increasing the relaxivity of the protons in their near vicinity [40]. However, the shortcomings of these low molecular weight contrast agents include short circulation times within the body and inefficient discrimination between diseased and normal tissues. Subsequently, macromolecular Gd(III) complexes have been developed by conjugating Gd(III) chelates to biomedical polymers, including poly(amino acids), polysaccharides, and proteins to improve image contrast enhancement. These macromolecular agents have demonstrated superior contrast enhancement for blood pool imaging and cancer imaging in animal models. Unfortunately, the clinical application of macromolecular agents in general is limited by their slow excretion rate which results in their accumulation within the body, that is, the liver. In addition, the long residence time of MRI agents enhances the risk of potential toxicity by Gd(III) ions released during the metabolism of these agents [41,42]. Therefore, medical researchers have tested dendrimers in preclinical studies as contrast agents for magnetic resonance as well as macromolecular carrier for conjugating contrasting agent. Addition of contrast agents (paramagnetic metal cations) improves sensitivity and specificity of the method. Gadolinium (Gd) paramagnetic contrast agents for MRI have been complexed with dendrimer molecules over the last two decades for contrast enhancement, improved clearance characteristics, and

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potential targeting [43,44]. Gadolinium salt of diethylenetriaminepentaacetic acid (DTPA) is used clinically but it diffuses into the extravenous area due to its low molecular mass [45]. Dendrimers due to their properties are highly suited for use as image contrast media. Several groups have prepared dendrimers containing gadolinium ions chelated on the surface [46,47]. Preliminary tests show that such dendrimers are stronger contrast agents than conventional ones. They also improve visualization of vascular structures in magnetic resonance angiography (MRA) of the body. It is a consequence of excellent signal-to-noise ratio [48]. Wiener et al. developed a new class of magnetic resonance imaging contrast agents, Gd(III)-DTPA-based PAMAM dendrimers, with large proton relaxation enhancements and high molecular relaxivities. These sixth generation PAMAM dendrimers possess 192 reactive terminal amines, which can be conjugated to the chelating ligand 2-(4-isothiocyanato-benzyl)-6methyl-(DTPA) through a thiourea linkage. This dendrimer has a relaxivity a six times higher than that of free Gd(III)-DTPA complex. In vivo experiments on rabbits show excellent MRI images of blood vessels and long blood circulation times (>100 min) upon intravenous injection [49]. In addition, Kobayashi et al. synthesized small dendrimer-based MRI contrast agents and investigated the relationship between relaxivity and dendrimer generations using Gd(III)-DTPA based PAMAM dendrimers. The results of this study revealed that relaxivities increased as the dendrimer generation increased, but there was no significant increase in relaxivities beyond seventh generation [50–52] (Table 13.1). Table 13.1

Dendrimer-encapsulated MRI agents

Dendrimer

Imaging/contrasting agent

Target

Remarks

Ref.

G6-PAMAM

(1B4M-Gd) 256

Breast cancer

• Distinguishes between the differ-

[52]

ent types of angiogenesis Dual magnetic resonance and fluorescence imaging

[53]

G6-PAMAM

G8 PAMAM

G6-Cy(5.5)1.25(1B4MGd)145 (Dual modality: MRI and FI) (1B4M-Gd)1024

G6-PAMAM

(1B4M-Gd)256

G5-G7 PMPA

99mTc

Sentinel (mammary) lymph nodes

•

Sentinel (mammary) lymph nodes Sentinel (mammary) lymph nodes Kidney/Bladder

• Enhanced resolution

[54]

• Study of tumor metastasis

[55]

• Rapid elimination from blood via

[56]

• A 4 PAMAM

(1B4M-Gd)64

Kidney

G2-PAMAMCystamine

Gd(III)-1B4M-DTPA & Rhodamine green (Dual modality: MRI and FI)

Ovarian tumors

• • • •

kidneys Negligible non-specific binding to organs or tissues Enhanced contrast Dynamic imaging High-resolution micro-MRI images Dual-modality MRI and fluorescence imaging

[57]

[58]

Chapter 13 • Dendrimers for diagnostic applications

Table 13.1 Dendrimer G1–5 Poly (propyleneimine) Dendrimers G6 PAMAM

G3 PAMAM

299

Dendrimer-encapsulated MRI agents—cont’d Imaging/contrasting agent

Target

Remarks

Ref.

Gd-DTPA

–

• Tunable molecular relaxivities

[59]

111In and Cy5, Alex (660, 680, 700, 750) (Dual Modality: radionuclide and 5NIR) Gd(III) and Alexa Fluor 594 Dual Modality (MRI and FI)

Optical lymphatic imaging and sentinel lymph nodes

• Dual-modality MRI and five-color

[60]

Tumors

• αvβ3-targeted delivery of contrast

near-infrared optical imaging

• G4 PAMAM

Gd(III)-1B4M-DTPA

Angiography

•

G6 PAMAM

(1B4M-Gd)192

•

DAB-Am64 (G6) DAB-Am64 (G4) G4 PAMAM

(1B4M-Gd)64

Intertumoral vasculature Liver micrometastasis Liver micrometastasis

G3–6 PAMAM

(1B4MGd)64

Blood pool

•

(1B4MGd)64

• •

agents Dual-modality MRI and five-color near-infrared optical imaging Higher yields and efficiency of dendrimer-based MRI agents with versatility 3D MR angiography (MRA) providing more clear visualization Visualization of micrometastatic tumors with better contrast Rapid accumulation of hydrophobic contrasting agent in liver for better MRI imaging Increased blood retention of contrast agent with increasing generation number (molecular weight) of PAMAM

[61]

[62]

[63] [64] [65]

[66]

5.2 Dendrimer-based positron emission tomography agents Positron emission tomography (PET) is a functional imaging technique that produces a three-dimensional image of metabolic processes in the body. The system detects pairs of gamma rays emitted indirectly by a positron-emitting radionuclide (tracer), which is introduced into the body on a biologically active molecule. A positron emission tomography (PET) scan is an imaging test that helps reveal how tissues and organs are functioning, hence offering physiological and biological information through the in vivo distribution of PET agents for disease diagnosis, therapy monitoring and prognosis evaluation. A PET scan uses a radioactive drug (tracer) to show this activity. The tracer may be injected, swallowed or inhaled, depending on which organ or tissue is being studied by the PET scan [67]. Due to the unique structural characteristics allowing for facile modification of targeting ligands and radionuclides, dendrimers can be served as a versatile scaffold to build up various PET imaging agents, and significant breakthroughs have been made in this field over the past decades [68]. Recent advances in dendrimer-based contrast agents for PET has improved imaging of cancer, cardiovascular and other diseases. For instance, the researchers recently applied

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Pharmaceutical Applications of Dendrimers

Table 13.2

Dendrimer-encapsulated PET agents

Dendrimer

Imaging/contrasting agent

(LyP-1) 4-dendrimer TrifluoroboroarylPAMAM-biotin

64

Polyethylene oxide-Dendrimer

76

G0-PAMAM

64

Cu

18

F

Br

Cu and Cy5.5

Target

Remarks

Ref.

Atherosclerotic plaque Breast tumor cells

• • • •

[71,72]

Tissues overexpressing αvβ3 receptors Ovarian cancer

• • • •

Enhanced uptake Specific accumulation Targeted delivery Increased anti-tumor activity of 18 F-dendron-biotin with decreasing dendron size Enhanced bioavailability In vivo radiostability Increased specificity Dual modality near-infrared florescent and PET imaging

[73]

[74]

[75]

PET for live imaging of the macrophage accumulation and ingestion of lipids into foam cells to monitor the progression of atherosclerosis (AS) which are substantially important hallmarks in the pathogenesis of AS and can facilitate the diagnosis of AS and improve the management of patients [69, 70]. Thus, imaging systems detecting, and quantifying macrophage accumulation will provide diagnostic and prognostic information for atherosclerotic plaque. Further, to improve the accumulation efficacy of LyP-1 in case of AS, Seo et al. designed and synthesized a dendrimer with multiple LyP-1 ligands using lysine as a core structural element, which was named as (LyP-1)4-dendrimer-64Cu. AS is a chronic inflammatory vascular disease, is a high-risk factor for myocardial infarction and cerebrovascular events. Macrophages piled in an AS lesion always express many, biomarkers, such as p32 protein which could be a potential target for the non-invasive identification of AS progression [71]. The (LyP-1)4-dendrimer-64Cu was intravenously injected into ApoEatherosclerotic mice. After 2 h of circulation, PET-CT co-registered images demonstrated a greater uptake of the (LyP-1) 4-dendrimer-64Cu than the (ARAL) 4-dendrimer-64Cu, where ARAL is another type of peptide like LyP-1 except for the ability to bind to p32 in the aortic root and descending aorta. Ex vivo images and biodistribution acquired at 3 h after injection also demonstrate a significantly higher uptake of the (LyP-1) 4-dendrimer-64Cu in the aorta. Similarly, the subcutaneous injection of LyP-1dendrimeric carriers resulted in preferential accumulation in plaque-containing regions over 24 h. Taken together, these results suggested that the (LyP-1) 4-dendrimer can be applied as a nanocarrier of contrast media for the in vivo PET imaging [72] (Table 13.2).

5.3 Dendrimers-based computed tomography agents Computed tomography (CT) is one of the routine medical imaging tools for disease diagnosis in hospital, which offers high-resolution three-dimensional (3D) tomography information of the anatomic structure of organ systems based on their different X-ray absorption characteristics. CT contrast agents with a high X-ray attenuation coefficient

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are generally required for contrast enhancement and for desirable imaging quality, especially in the imaging and differentiating malignancies among normal tissues [76]. To obtain high sensitivity and specificity, contrast agents are indispensable for CT imaging. Commercially available iodine-based small molecular CT contrast agents (e.g., Omnipaque) are usually able to be rapidly cleared from blood after injection, making CT imaging at a reasonably long time impossible. In addition, they often cause significant side-effects, such as renal toxicity at high concentrations. Therefore, much effort has been made toward the development of novel and efficient CT contrast agents with improved contrast quality and prolonged circulation time CT imaging technologies have undergone a very fast development in the last years. Recently, the development of CT contrast agents has brought new hope for CT molecular imaging [77]. Recently, nanoparticle (NP)-based contrast agents, such as bismuth sulfide, gold NPs (AuNPs), and ytterbium based-NPs, have been extensively investigated as CT imaging contrast agents because of their higher X-ray attenuation coefficient than that of conventional iodine-based small molecular agents (e.g., Omnipaque), which display several disadvantages such as short half-decay time, renal toxicity at a relatively high concentration, and non-specificity [78–80]. Development of new types of CT imaging contrast agents with high X-ray attenuation intensity, specificity, and excellent biocompatibility remains a great challenge. The unique dendrimer chemistry allows the use of dendrimers as stabilizers to spontaneously conjugate CT agents with DSNPs with a spherical shape, good water solubility and colloidal stability, and excellent cytocompatibility. Dendrimer-based CT agents with radiodense metals (gold or silver) size usually larger than 5 nm formed by entrapping one or more metal NPs within one single dendrimer molecule, or one metal NP is surrounded by multiple dendrimer molecules [81–83]. The dendrimer templating or stabilization approaches have been used for CT imaging applications with enhanced or comparable X-ray attenuation property (Table 13.3). Table 13.3 Dendrimer

Dendrimer-encapsulated CT agents Imaging/contrasting agent

Target

Remarks

Ref.

– Lung adenocarcinoma Mouth adenocarcinoma

• Good X-ray attenuation • Biocompatible

[84] [78]

• • • • •

[85]

G5-PAMAM Acetylated PAMAM FA-G2 PAMAM

Au Au

Lactobionic acidPAMAM 131 I-PAMAM G5-PAMAM

Au

Hepatic cancer

Au Ag

Gliomas Blood pool

Au

• • •

Simple synthesis approach Easy modification Good cytocompatibility High X-ray attenuation Higher X-ray attenuation compared to iodine-mediated CT Improved stability of CT agents Prolonged stability Enhanced imaging

[86] [87] [88]

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Pharmaceutical Applications of Dendrimers

5.4 Dendrimer-based fluorescence imaging agents In fluorescence imaging, an external light of appropriate wavelength is used to excite a target fluorescent molecule, followed almost immediately by release of longerwavelength, lower-energy light for imaging. Fluorescence imaging at depths greater than a few millimeters requires NIRF probes and dyes. There is an ever-growing list of fluorophores for NIRF imaging in small animals, including Cy5.5, the Alexa dye series, indocyanine green, and quantum dots. Whereas potential toxicity may limit applications of Cy5.5 or quantum dots in patients, gaining Food and Drug Administration approval for clinical use of a NIRF dye will be a key step toward using fluorescence imaging for routine clinical applications [89–91]. Of the many fluorescent probes, there are quantum dots, fluorescent proteins, fluorescent nanostructures such as metal complexes, semiconductor nanocrystals, upconversion nanophosphors, and other nanoparticles. Ideally a probe should be easily yet specifically internalized by cells, yet non-toxic and not affecting metabolism [92, 93]. Moreover, some specific probes are expected to being taken up by specific cell compartments. However, several imperfections limit their application. Organic dyes are not readily internalized by cells, and most of them do not combine specifically with cellular compartments or biochemical compounds. Moreover, they often leak uncontrollably from the cells and quickly lose their photochemical properties [89]. Knowing these problems, the idea of engaging dendrimers in bioimaging has recently been developed. For bioimaging based on fluorescence, dendritic polymers were extensively studied as conventional probe carriers. Dendrimers (dendrites) can be both hydrophilic and hydrophobic and are easily internalized by cells. Unique fluorescent properties of PAMAM dendrimers have been observed, i.e., they are able to emit weak fluorescent signal when excited at proper wavelength. The fluorescence lifetime grew with increase of generation suggesting that a specific fluorescent component was located in a more protected or

FIG. 13.1 Confocal microscopy photography of mHippo-E18 cell line (A) treated with autofluorescent PAMAM dendrimer for 24 h and (B) treated with autofluorescent PAMAM dendrimer for 24 h and stained with fluorescent stains: RedDot2 (nucleus) and NeuroDiO (cytoplasm). Blue emission is the PAMAM dendrimer fluorescence signal [94].

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constrained microenvironment. It was postulated that fluorescence came from n ! π transition of interior amid groups of the particles. Since PAMAM dendrimers did not carry any traditional fluorescent moieties, this phenomenon was named non-traditional intrinsic fluorescence (NTIF) or intrinsic or autofluorescence [95]. NTIF was later reported also for PEI and PPI [96, 97]. The fluorescence of autofluorescent dendrimers in cell line in vitro is presented in Fig. 13.1 [94]. A product showing strong blue photoluminescence was obtained by oxidation of OH-terminated PAMAM dendrimers, such as G4-OH, G2-OH, and G0-OH, with HAuCl4 or (NH4)2S2O8. The fluorescence emission spectrum peaked at 450 nm, while the excitation maximum was at 380 nm, independent of the generation of dendrimer. The product also shows two weak electrogenerated chemiluminescence (ECL) signals upon cycling the potential between about 1.2 and 1.7 V. Since PAMAMNH2 of corresponding generations showed significantly weaker signal, the authors postulate that the backbone of dendrimer is less important for its autofluorescence than the surface. Moreover they emphasized that oxidation of the termini was necessary to obtain fluorescence [98]. Although PAMAM and PPI dendrimers have so-called non-traditional intrinsic fluorescence (NTIF), the fluorescence of dendrimer usually originates from a label that must be attached to the hyperbranched dendrimer skeleton such that no self-quenching does occur. Dendrimer-based systems for fluorescence bioimaging in most cases are composed of fluorophore conjugated or coated with the dendrimer [99]. Complexing of organic dyes with these macromolecules improves their solubility and enhances cellular uptake. Moreover, it helps overcome other limitations in using them for photobleaching, and their lack of specificity or cytotoxicity. Protective properties of dendrimers are especially valuable for use with quantum dots, which have great optical potential but contain heavy metals that can adversely affect biological objects. Dendrimer based fluorescent probes have been widely applied in vitro and in vivo bioimaging. The brightness of dendrimeric NPs can be controlled by size, and color by the terminal fluorophore. Their size exceeds 2 nm in very few cases. They have been applied to plain imaging, to targeted imaging, and to sensing/imaging. Features of fluorescent dendrimers include very large molar absorbances. In a work by Dougherty et al., G5-NH2-TAMRA were prepared with mixture of dendrimers with 5 or more dye per dendrimer. The absorption intensity increased in non-linear fashion whereas the fluorescence emission and lifetime decreased with an increasing number of dyes per dendrimer [100]. Some of the fluorophore show pH- and oxygen-dependent fluorescence. For example, Albertazzi et al. found that PAMAM dendrimers conjugated simultaneously with two different dyes could be made for ratiometric analysis: green fluorescent dye—a pH-sensitive one—and red, pH-insensitive (rhodamine). Since these dyes have different excitation and emission spectra of their fluorescence, the ratio of green:red indicated the pH. The dendrimers were used for HeLa cell line in vitro bioimaging. Based on physiological pH differences in particular cell compartments (e.g., acidic for lysosome and neutral for plasma membrane) a pixel-by-pixel map was generated from a confocal microscope image. Further development of this approach led to conjugating a third particle on the surface of

304

Pharmaceutical Applications of Dendrimers

Fluo

0

Rhod

255

Ratio

0

255 0

4

FIG. 13.2 Targeted ratiometric pH imaging in plasma membrane and lysosomes with dendrimer-based G6-NH3+Fluo-RhRed sensor which specifically targets organelles owing to its surface cationic charge. Scale Bar ¼ 5 μm [101].

Table 13.4

Dendrimer-encapsulated florescence imaging agents

Dendrimer

Imaging/contrasting agent

Target

Remarks

Ref.

OH-PAMAM G5-PAMAM

– TAMRA

– Kidney

• Exhibit autofluorescence • Increased absorption intensity with increasing

[98] [100]

Lauroyl/ propranololG3 PAMAM PAMAM FA-G5 PAMAM-Taxol DHA-G1 PAMAM

number of dyes per dendrimer whereas fluorescence emission and lifetime decrease Enhanced cellular internalization; mode of internalization influenced by surface properties of dendrimer pH-dependent ratiometric analysis using two different dyes simultaneously Simultaneous drug delivery and labeling

FITC

Colon cancer

•

Florescence and rhodamine red FITC

Breast cancer

•

Oral cancer

•

SNARF conjugated CdSe/ZnS quantum dots (QDs)

Mesenchymal stem cells

• pH sensing with high sensitivity detecting up • •

Gly-Lys-G6 dendrimer

–

Colorectal cancer

• •

APS-G4 PAMAM

–

Glioma

•

to 0.2 pH change Fluorescence of PAMAM-QDs was 70–80 times stronger compared with free QDs Endosomal escape observed in PAMAM-QDs rather than in free QDs Exhibit autofluorescence Enhanced membrane interaction and cellular uptake being cationic in nature Intensified autofluorescence due to oxidation of APS

[102]

[101] [103] [104]

[105]

[106]

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305

dendrimer—PEG. PEGylation helped set a stable distance between dye molecules and limited their ability to move, thereby enhancing the intensity of fluorescence of both dyes [101] (Fig. 13.2; Table 13.4).

5.5 Dendrimer-based PDT, PTT and neutron capture therapy agents Neutron capture therapy is concerned with the treatment of cancers that is based on Boron capture reaction [107]. The radiation energy generated from the capture reaction of low-energy thermal neutrons by 10B atoms has been used successfully for the selective destruction of tissue. Due to their well-defined structure and multivalency, dendrimers are a very fascinating compound for use as boron carriers [108]. The applicability of PAMAM dendrimers in investigating intratumoral delivery of agents for neutron capture therapy is remarkable in biomedical science. In a study by Wu et al. functionalized G5 PAMAM dendrimers and conjugated cetuximab specific to EGF receptor to starburst dendrimers that carried around 1100 boron atoms. The in vivo results revealed that accumulation of conjugates were 10 times higher in brain tumor tissues in comparison to healthy brain tissues. This study was first to demonstrate the efficacy of a boronated monoclonal antibody for boron neutron capture therapy of an intracerebral glioma [109]. Phototherapy has attracted considerable attention as a non-invasive therapeutic technique for cancer treatment due to its unique advantages such as remote controllability, improved selectivity, and low systemic toxicity [110]. Phototherapies induced by visible or near infrared (NIR) light usually involve phototherapeutic agents that have little toxicity in the dark, but are able to selectively kill cancer cells when exposed to light irradiation without causing much damage to normal tissues. Photodynamic therapy (PDT) and photothermal therapy (PTT), two different types of phototherapy that are commonly used for therapeutic purposes, require the absorption of incoming light by a photosensitizer (PS) or a photothermal agent (PTA) to generate reactive oxygen species (ROS) or heat to kill cancer cells, respectively [111]. Photodynamic therapy (PDT) uses a photosensitizer or photosensitizing agent, and a particular type of light to treat various types of cancers and other non-neoplastic conditions, including psoriasis, muscular degeneration of the retina and also some autoimmune diseases like rheumatoid arthritis. On exposure to light of a specific wavelength, these photosensitizers produces singlet oxygen which kills the nearby cells [112, 113]. Each photosensitizer gets activated by a distinct range of wavelength of light and that particular wavelength determines how deep the light penetrates into the body [114, 115]. Thus, PDT requires particular photosensitizers and wavelengths of light which are specific to treat various types of cancers. Briefly, in PDT, a photosensitizing agent is injected into the bloodstream, which is absorbed by all the cells of the body. However, this PS is eliminated at a faster rate from the normal cells and PS retained for a longer time in cancer cells. This is because cell membranes of tumor cells distinctly possess high numbers of low-density lipoprotein receptors, prompting to excessive accumulation of

306

Pharmaceutical Applications of Dendrimers

photosensitizer molecules at extracellular surface of tumor cells. The photosensitizers may also accumulate in tumor cells due to disorders in the local microvasculature, abnormal blood supply and aggravated vascular permeability [116–118]. Targeted delivery of PSs is one of the main challenges in PDT. The fact that most effective PSs tend to be insoluble, hydrophobic molecules with a high propensity to aggregate means that encapsulation in nano-drug carrier may make a big difference to their performance. Dendrimers have sites appropriate for binding various classes of functional groups and are a promising nanomaterial for promoting experimentations. Their specific advantages include the control and consistency of size and number of functional moieties accessible for designing. In this way, it offers reproducible pharmacokinetics, which makes dendrimers a fascinating framework for PDT drug delivery [119]. In a trial, dendrimer with dual functionality were synthesized for efficient near infraredsensitive photosensitizers. Narsireddy et al. modified dendrimer with nitrilotriacetic corrosive (NTA) and 5,10,15,20-tetrakis(4-hydroxyphenyl)-21H,23H-porphine (PS) groups. After that, a peptide with specificity for human epidermal development factor 2 was added to the NTA of the dendrimer. The peptide was used for targeting tumor, assessing photodynamic therapy in-vitro and in-vivo. The examinations demonstrated reduction of tumors in a xenograft animal tumor model administered with PS-dendrimers. Thus, PS incorporated with dendrimer proved to be productive in PDT-assisted apoptosis measures in human epidermal development factor receptor 2 (HER2) positive SKOV-3 cells [120]. Dendrimer can be used for treating both superficial cancers as well as for vascular PDT for atherosclerosis. Rodriguez et al. carried out a study to administer 5-aminolevulinic acid (ALA) for activating protoporphyrin-IX already present within the body, then used the activated porphyrin for many applications in photodynamic therapy (PDT). It was already shown that porphyrin synthesis was enhanced by conjugating with ALA dendrimers. In this work, ALA dendrimers were evaluated for encapsulating 9 and 6 ALA groups (9m-ALA and 6m-ALA) to photosensitize LM3 mammary tumor cell lines. It was observed that the porphyrin synthesis was higher with even low concentrations of dendrimer in comparison to that of ALA, while there is no significant difference among the production level of porphyrin at high concentrations at from the two compounds. But ALA dendrimers showed a negative result on topical application showing no diffusion via subcutaneous route into LM3 cells. So, later vascular PDT was proposed and focused on higher affinity of ALA-dendrimer for macrophages (RAW264.7 macrophages) than affinity for endothelial cells (line). The results showed that ALA dendrimers induced increased production of protoporphyrin-IX in macrophages, where 9m-ALA and 6m-ALA residues correspondingly induced 4.6- and 6-times higher production compared to the HMEC-1 microvasculature cell, indicating higher selectivity of ALA dendrimers for macrophages. So the following study paves a way for studying PDT for vascular applications rather than superficial therapy [121]. PTT is a promising paradigm for cancer therapy. To conduct efficient PTT, diverse photothermal agents (PAs) including metal nanoparticles, carbon-based materials, semiconductor nanoparticles and polymeric nanostructures have been intensively developed in the past decade [122–125]. All of these PAs represent distinguished photothermal effect

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307

and complete tumor ablation, but few of them are translated into clinical trials due to several issues related to the particle size and multiple functionalization [126, 127]. Dendrimers have well-defined size and shape, hollow interior, and high density of surface functional groups which are widely used as templates to synthesize ultra-small nanoparticles (typically 93.0% at pH 4.0, incubation time of 30.0 min and reaction temperature ranging between 90 and 100 °C. The decay corrected radiochemical yield was found to be 79.4  0.01%. The radiolabeled preparation [(68)Ga]-DOTA-PAMAM-D remained stable (radiolabeling efficiency of 96.0%) at room temperature and in serum for up to 4-h. The plasma protein binding was observed to be 21.0%. After intravenous administration, 50.0% of the tracer cleared from the blood circulation by 30-min and