Handbook of Clinical Nanomedicine: Nanoparticles, Imaging, Therapy, and Clinical Applications [1] 9789814669214, 9814669210

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Handbook of Clinical Nanomedicine: Nanoparticles, Imaging, Therapy, and Clinical Applications [1]
 9789814669214, 9814669210

Table of contents :
Front Cover
Dedication
About the Editors
Contents
List of Corresponding Authors
Foreword
Small Is Beautiful: Preface by the Series Editor
Acknowledgements
Section I: General Introduction and Beginnings
Chapter 1: Science at the Nanoscale: Introduction and Historical Perspective
Chapter 2: Nanomedicine: Dynamic Integration of Nanotechnology with Biomedical Science
Chapter 3: A Small Introduction to the World of Nanomedicine
Chapter 4: Top Ten Recent Nanomedical Research Advances
Chapter 5: The Coming Era of Nanomedicine
Chapter 6: What’s in a Name? Defining “Nano” in the Context of Drug Delivery
Section II: Nanoparticles, Nanodevices, and Imaging
Chapter 7: Properties of Nanoparticulate Materials
Chapter 8: Solid Drug Nanoparticles: Methods for Production and Pharmacokinetic Benefits
Chapter 9: Design and Development of Approved Nanopharmaceutical Products
Chapter 10: Nanosizing Approaches in Drug Delivery
Chapter 11: Multilayered Nanoparticles for Personalized Medicine: Translation into Clinical Markets
Chapter 12: Nanomaterials for Pharmaceutical Applications
Chapter 13: Polysaccharides as Nanomaterials for Therapeutics
Chapter 14: The Story of C60 Buckminsterfullerene
Chapter 15: Applications of Nanoparticles in Medical Imaging
Chapter 16: Nanoimaging for Nanomedicine
Chapter 17: Nanoparticles for Multi-Modality Diagnostic Imaging and Drug Delivery
Chapter 18: Magnetic Nanoparticles for Magnetic Resonance Imaging: A Translational Push toward Theranostics
Chapter 19: First-in-Human Molecular Targeting and Cancer Imaging Using Ultrasmall Dual-Modality C Dots
Chapter 20: Atomic Force Microscopy for Nanomedicine
Chapter 21: Atomic Force Microscopy Imaging and Probing of Amyloid Nanoaggregates
Chapter 22: Image-Based High-Content Analysis, Stem Cells and Nanomedicines: A Novel Strategy for Drug Discovery
Chapter 23: Viral Nanoparticles: Tools for Materials Science and Biomedicine
Chapter 24: Bacterial Secretion Systems: Nanomachines for Infection and Genetic Diversity
Chapter 25: The Vascular Cartographic Scanning Nanodevice
Chapter 26: Advancements in Ophthalmic Glucose Nanosensors for Diabetes Management
Section III: Therapy and Clinical Applications
Chapter 27: Towards Nanodiagnostics for Bacterial Infections
Chapter 28: Copaxone® in the Era of Biosimilars and Nanosimilars
Chapter 29: Doxil®: The First FDA-Approved Nanodrug—From an Idea to a Product (January 2015 Update)
Chapter 30: Nanotechnology and the Skin Barrier: Topical and Transdermal Nanocarrier-Based Delivery
Chapter 31: Application of Nanotechnology in Non-Invasive Topical Gene Therapy
Chapter 32: Nanocarriers in the Therapy of Inflammatory Disease
Chapter 33: Advanced 3D Nano/Microfabrication Techniques for Tissue and Organ Regeneration
Chapter 34: Nanomedicine for Acute Lung Injury/Acute Respiratory Distress Syndrome: A Shifting Paradigm?
Chapter 35: Nanoviricides: Targeted Anti-Viral Nanomaterials
Chapter 36: Nanotechnology in Tissue Engineering for Orthopaedics
Chapter 37: Applications of Nanomaterials in Dentistry
Chapter 38: Biomimetic Applications in Regenerative Medicine: Scaffolds, Transplantation Modules, Tissue Homing Devices, and Stem Cells
Chapter 39: Potential Applications of Nanotechnology in the Nutraceutical Sector
Chapter 40: Designing Nanocarriers for the Effective Treatment of Cardiovascular Diseases
Chapter 41: Carbon Nanotubes as Substrates for Neuronal Growth
Chapter 42: Polymeric Nanoparticles for Cancer Therapeutics
Chapter 43: Nanotechnology for Radiation Oncology
Chapter 44: Gold Nanoparticles against Cancer
Chapter 45: Solid Lipid Nanoparticles and Nanostructured Lipid Carriers for Cancer Therapy
Chapter 46: Nanomedicines Targeted to Aberrant Cancer Signaling and Epigenetics
Chapter 47: Biodegradable Nanoparticle-Based Antiretroviral Therapy across the Blood-Brain Barrier
Chapter 48: HIV-Specific Immunotherapy with Synthetic Pathogen-Like Nanoparticles
Chapter 49: Biomedical Engineering and Nanoneurosurgery: From the Laboratory to the Operating Room
Chapter 50: Nanotechnology-Based Systems for Microbicide Development
Chapter 51: Nanotechnology-Based Solutions to Combat the Emerging Threat of Superbugs: Current Scenario and Future Prospects
Chapter 52: Nanolithography and Biochips’ Role in Viral Detection
Chapter 53: Lectins as Nano-Tools in Drug Delivery
Chapter 54: Diagnostics of Ebola Hemorrhagic Fever Virus
Chapter 55: Nanomedicine as a Strategy to Fight Thrombotic Diseases
Back Cover

Citation preview

Handbook of

Clinical Nanomedicine Vol. 1

Pan Stanford Series on Nanomedicine Series Editor

Raj Bawa

Titles in the Series Published

Forthcoming

Vol. 1 Handbook of Clinical Nanomedicine: Nanoparticles, Imaging, Therapy, and Clinical Applications Raj Bawa, Gerald F. Audette, and Israel Rubinstein, eds. 2016 978-981-4669-20-7 (Hardcover) 978-981-4669-21-4 (eBook)

Vol. 3 Immune Effects of Biopharmaceuticals and Nanomedicines Raj Bawa, János Szebeni, Thomas J. Webster, and Gerald F. Audette, eds. 2017

Vol. 2 Handbook of Clinical Nanomedicine: Law, Business, Regulation, Safety, and Risk Raj Bawa, ed., Gerald F. Audette and Brian E. Reese, asst. eds. 2016 978-981-4669-22-1 (Hardcover) 978-981-4669-23-8 (eBook)

Vol. 4 Impact of Nanobiotechnology on the Future of Medicine and Personalized Medicine Shaker A. Mousa and Raj Bawa, eds. 2017

Pan Stanford Series on Renewable Energy — Volume 2 Pan Stanford Series on Nanomedicine Vol. 1

Handbook of

Clinical Nanomedicine Nanoparticles, Imaging, Therapy, and Clinical Applications

edited by

Raj Bawa, MS, PhD

Patent Agent, Bawa Biotech LLC, Ashburn, Virginia, USA Adjunct Professor, Department of Biological Sciences Rensselaer Polytechnic Institute, Troy, New York, USA editors Scientific Advisor, Teva Pharmaceutical Industries, Ltd., Israel

Preben Maegaard Anna Krenz Gerald F. Audette, PhD Wolfgang Palz Department of Chemistry Associate Professor,

Acting Director, Centre for Research on Biomolecular Interactions York University, Toronto, Canada

Israel Rubinstein, MD

Professor, Department of Medicine Wind Energy The Rise of Modern

University of Illinois at Chicago, College of Medicine, Chicago, Illinois, USA Associate Chief of Staff for Research and Development Jesse Brown VA Medical Center, Chicago, Illinois, USA

Wind Power

for the World

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2016 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Version Date: 20160202 International Standard Book Number-13: 978-981-4669-21-4 (eBook - PDF) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www. copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

This book is dedicated with love to my father, Dr. S. R. Bawa, on his 85th birthday, dad, grandfather, inspiration, anatomist, electron microscopist, department founder, professor and head, university dean —the best teacher I ever had. –R.B.

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About the Editors Raj Bawa, MS, PhD, is president of Bawa Biotech LLC, a biotech/pharma consultancy and patent law firm based in Ashburn, VA, USA that he founded in 2002. He is an inventor, entrepreneur, professor and registered patent agent licensed to practice before the U.S. Patent & Trademark Office. Trained as a biochemist and microbiologist, he has been an active researcher for over two decades. He has extensive expertise in pharmaceutical sciences, biotechnology, nanomedicine, drug delivery and biodefense, FDA regulatory issues, and patent law. Since 1999, he has held various adjunct faculty positions at Rensselaer Polytechnic Institute in Troy, NY, where he is currently an adjunct professor of biological sciences and where he received his doctoral degree in three years (biophysics/biochemistry). Since 2004, he has been an adjunct professor of natural and applied sciences at NVCC in Annandale, VA. He is a scientific advisor to Teva Pharmaceutical Industries, Ltd., Israel. He has served as a principal investigator of National Cancer Institute SBIRs and reviewer for both the National Institutes of Health and the National Science Foundation. In the 1990s, Dr. Bawa held various positions at the US Patent & Trademark Office, including primary examiner for 6 years. He is a life member of Sigma Xi, co-chair of the Nanotech Committee of the American Bar Association and serves on the global advisory council of the World Future Society. He has authored over 100 publications, co-edited four texts and serves on the editorial boards of 17 peer-reviewed journals, including serving as a special associate editor of Nanomedicine (Elsevier) and an editor-in-chief of the Journal of Interdisciplinary Nanomedicine (Wiley). Some of Dr. Bawa’s awards include the Innovations Prize from the Institution of Mechanical Engineers, London, UK (2008); Appreciation Award from the Undersecretary of Commerce, Washington, DC (2001); the Key Award from Rensselaer’s Office of Alumni Relations (2005); and Lifetime Achievement Award from the American Society for Nanomedicine (2014).

viii

About the Editors

Gerald F. Audette, PhD, has been a faculty member at York University in Toronto, Canada, since 2006. Currently, he is an associate professor in the Department of Chemistry and acting director of the Centre for Research on Biomolecular Interactions at York University. He received his doctorate in 2002 from the Department of Biochemistry at the University of Saskatchewan in Saskatoon, Canada. Working with Drs. Louis T. J. Delbaere and J. Wilson Quail (1995–2001), Dr. Audette’s research focused on the elucidation of the protein–carbohydrate interactions that occur during blood-group recognition (in particular during the recognition of O blood type) using high-resolution X-ray crystallography. Dr. Audette conducted his postdoctoral research at the University of Alberta (2001–2006) in Edmonton, Canada. Working with Drs. Bart Hazes and Laura Frost; his research again utilized high-resolution protein crystallography to examine the correlation between protein structure and biological activity of type IV pilins that are assembled into pili used by bacteria for multiple purposes, including cellular adhesion during infection. It was during these studies that Dr. Audette identified the generation of protein nanotubes from engineered pilin monomers. Dr. Audette also studied the process of bacterial conjugation (or lateral gene transfer) using the F-plasmid conjugative system of Escherichia coli. Current research directions include: structure/ function studies of proteins involved in bacterial conjugation systems, the structural and functional characterization of several type IV pilins (the monomeric subunit of the pilus), their assembly systems, and adapting these unique protein systems for applications in bionanotechnology. Dr. Audette has previously served as co-editor-in-chief of the Journal of Bionanoscience (2007–2010). Israel Rubinstein, MD, is professor of medicine at the College of Medicine, University of Illinois at Chicago, USA. He is member of the section of pulmonary, critical care, allergy and sleep medicine in the Department of Medicine, University of Illinois at Chicago. He is an attending physician at the University of Illinois Hospital and Health Sciences System and Jesse Brown VA Medical Center in Chicago. Dr. Rubinstein

About the Editors

is also the associate chief of staff for research and development at the Jesse Brown Veterans Administration Medical Center. Prior to his appointment at the University of Illinois at Chicago, he was associate professor of medicine at the University of Nebraska Medical Center in Omaha, Nebraska, USA. Dr. Rubinstein received his medical degree from the Hebrew University-Hadassah School of Medicine in Jerusalem, Israel. He was a medical resident in Israel, fellow in respirology at the University of Toronto and a research fellow at the Cardiovascular Research Institute, University of California at San Francisco. Dr. Rubinstein holds 18 issued and pending patents and has authored close to 200 peer-reviewed papers in scientific journals. Dr. Rubinstein’s funded research endeavors center around nanomedicine and targeted drug delivery with specific focus on lipid-based products and repurposing. Currently, he serves as editor-in-chief of Nanotechnology, Science and Applications, associate editor of the International Journal of Nanomedicine, and editorial board member of several scientific journals. Dr. Rubinstein is member of the scientific advisory board of the International Academy of Cardiology. He is a fellow of the American Heart Association as well as the American College of Physicians and the American College of Chest Physicians. In addition, he is a member of the American Thoracic Society, American Physiological Society, American Society for Pharmacology and Experimental Therapeutics, and American Microbiology Society. Dr. Rubinstein is a board member and director of Advanced Life Sciences, a publicly traded biopharmaceutical company based in Woodridge, Illinois, USA. He is a co-founder of ResQ Pharma, an emerging clinical stage pharmaceutical company focusing on repurposing FDA-approved drugs for cardiopulmonary resuscitation and drug overdoses.

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Contents List of Corresponding Authors xxix Foreword xxxiii Small Is Beautiful: Preface by the Series Editor xxxvii Acknowledgements xlv

Section I: General Introduction and Beginnings 1. Science at the Nanoscale: Introduction and Historical Perspective

Chin Wee Shong, PhD, Sow Chorng Haur, PhD, and Andrew T. S. Wee, PhD



1.1 The Development of Nanoscale Science 1.2 The Nanoscale 1.3 Examples of Interesting Nanoscience Applications



2. Nanomedicine: Dynamic Integration of Nanotechnology with Biomedical Science

3

3 9 12

21

Ki-Bum Lee, PhD, Aniruddh Solanki, PhD, John Dongun Kim, PhD, and Jongjin Jung, PhD

2.1 Introduction 21 2.2 Designing Nanomaterials for Biology and Medicine 22 2.3 Application of Nanomaterials in Biomedical Research 29 2.4 Nanosystematic Approach for Cell Biology 39 2.5 Conclusion 43

3. A Small Introduction to the World of Nanomedicine



61

Rutledge Ellis-Behnke, PhD

3.1 Introduction 3.2 Size Medicine 3.3 Newer Areas of Emerging Therapeutics

61 62 64

xii

Contents

3.4 Safety 3.5 Fad, Promises, or Reality?

4. Top Ten Recent Nanomedical Research Advances



5. The Coming Era of Nanomedicine



Fritz Allhoff, JD, PhD



5.1 Introduction 5.2 The Rise of Nanomedicine 5.3 Diagnostics and Medical Records 5.4 Treatment 5.5 Moving Forward



Raj Bawa, MS, PhD



6.1 Introduction 6.2 Brief Historical Background 6.3 What is “Nano”? 6.4 Physical Scientists versus Pharmaceutical Scientists: Different Views of the Nanoworld 6.5 Is Drug Delivery at the Nanoscale Special? Does Size Matter? 6.6 Summary and Future Prospects

6. What’s in a Name? Defining “Nano” in the Context of Drug Delivery



Section II: Nanoparticles, Nanodevices, and Imaging

7. Properties of Nanoparticulate Materials

73

Melanie Swan, MBA

4.1 Introduction 4.2 Top 10 Areas of Recent Advance in Nanomedicine, Bio-Nanomaterials, and Nanodevices 4.3 Conclusion



66 67 73 76 93

103

103 105 109 114 119

127

128 133 136 146 149 161

171

Takuya Tsuzuki, PhD

7.1 Introduction 7.2 Nanoparticulate Materials 7.3 Common Characteristics of All Types of Nanoparticulate Materials

171 174 175

Contents



7.4 Characteristics of Specific Types of Nanoparticulate Materials 7.5 Summary

8. Solid Drug Nanoparticles: Methods for Production and Pharmacokinetic Benefits



190 202

211

Andrew Owen, PhD, and Steve P. Rannard, DPhil

8.1 Introduction 211 8.2 Physical Properties of Solid Drug Nanoparticles 212 8.3 Approaches for Solid Drug Nanoparticle Manufacture 214 8.4 Pharmacokinetic Advantages 217 8.5 Conclusions 223

9. Design and Development of Approved Nanopharmaceutical Products



Heidi M. Mansour, PhD, RPh, Chun-Woong Park, PhD, and Raj Bawa, MS, PhD

9.1 Introduction 9.2 Approved Nanopharmaceutical Products: Parenteral Drug Delivery 9.3 Approved Nanopharmaceutical Products: Oral Drug Delivery 9.4 Approved Nanopharmaceutical Products: Dermal and Transdermal Drug Delivery 9.5 Approved Nanopharmaceutical Products: Pulmonary Drug Delivery 9.6 Conclusions and Future Prospects

10. Nanosizing Approaches in Drug Delivery

Sandip Chavhan, PhD, Kailash Petkar, PhD, and Krutika Sawant, PhD



10.1 Introduction 10.2 Current Nanonization Strategies 10.3 Conversion to Solid Dosage Forms 10.4 Pharmaceutical Applications and Commercialization Aspects 10.5 Conclusions



233

233 242 246 249 250 252

273

273 277 286 287 291

xiii

xiv

Contents

11. Multilayered Nanoparticles for Personalized Medicine: Translation into Clinical Markets

299



Dania Movia, PhD, Craig Poland, PhD, Lang Tran, PhD, Yuri Volkov, PhD, and Adriele Prina-Mello, PhD



11.1 Introduction 299 11.2 Multilayered Engineered Nanoparticles 300 11.3 The Risk Assessment of Multilayered ENP 302 11.4 The Translation of Multilayered ENP in Clinical Markets 305 11.5 Conclusions 311



12. Nanomaterials for Pharmaceutical Applications

319



Brigitta Loretz, PhD, Ratnesh Jain, PhD, Prajakta Dandekar, PhD, Carolin Thiele, PhD, Yamada Hiroe, PhD, Babak Mostaghaci, PhD, Lian Qiong, MSc, and Claus-Michael Lehr, PhD





12.1 Introduction 319 12.2 Therapeutic Needs and Opportunities for Nanopharmaceuticals 321 12.3 Current Arsenal of Nanocarriers 330 12.4 Translation of Nanopharmaceuticals into Clinics 333 12.5 Challenges for Developing Future Nanopharmaceuticals 343 12.6 Conclusions and Future Perspectives 349



Shoshy Mizrahy, MSc, and Dan Peer, PhD



13.1 Introduction 13.2 Polysaccharides 13.3 Main Mechanisms of Nanoparticle Preparation from Polysaccharides 13.4 Polysaccharide-Based Nanoparticles 13.5 Polysaccharide-Coated Nanoparticles 13.6 Summary



13. Polysaccharides as Nanomaterials for Therapeutics



14. The Story of C60 Buckminsterfullerene

365

365 366 372 376 388 393

409

Harold W. Kroto, PhD



14.1 Carbon Cluster Studies 14.2 Carbon Chains in Space and Stars

414 415

Contents



14.3 Carbon in Space Dust and the Diffuse Interstellar Bands 416 14.4 C60 Pre-discovery Experiments 417 14.5 The Discovery of C60 419 14.6 C60 Theoretical Prehistory 422 14.7 The Circumstantial Supporting Evidence 424 14.8 Skeptical Studies 427 14.9 Spectroscopic Theory 428 14.10 Extraction and Structure Proof 428 14.11 Aftermath Studies 431 14.12 Mechanism 433 14.13 Epilogue: Detection of C60 in Space 434

15. Applications of Nanoparticles in Medical Imaging

443



Jason L. J. Dearling, PhD, and Alan B. Packard, PhD



15.1 Introduction 15.2 Contrast Media for Computed Tomography 15.3 Ultrasound 15.4 Summary



Yuri L. Lyubchenko, PhD, DSc, Yuliang Zhang, Alexey V. Krasnoslobodtsev, PhD, and Jean-Christophe Rochet, PhD



16.1 Introduction 465 16.2 Basic Principles of AFM 466 16.3 AFM: Imaging of Biological Samples 467 16.4 Dynamic Biological Events: Time-lapse AFM 470 16.5 AFM Force-Spectroscopy and Intermolecular Interactions 472 16.6 Concluding Remarks 482



Catherine M. Lockhart, PharmD, and Rodney J. Y. Ho, PhD



17.1 Imaging 17.2 Multi-Modality Imaging 17.3 Nanoparticles

16. Nanoimaging for Nanomedicine

17. Nanoparticles for Multi-Modality Diagnostic Imaging and Drug Delivery

443 445 454 459

465

493

493 498 501

xv

xvi

Contents



17.4 Targeted Nanoparticles for Multi-Modality Molecular Imaging 17.5 Conclusions and Future Prospects



Ryan A. Ortega, Thomas E. Yankeelov, PhD, and Todd D. Giorgio, PhD



18.1 Introduction 18.2 Properties of Iron Oxide Nanoparticles 18.3 Fabrication 18.4 Clinically Available Diagnostic Materials 18.5 Potential Future Clinical Applications 18.6 Conclusions



Michelle S. Bradbury, MD, PhD, and Ulrich Wiesner, PhD





19.1 Introduction 19.2 Pre-Clinical Studies 19.3 Clinical Trial Design 19.4 Particle Tracer PK and Metabolic Analyses 19.5 Clinical Multimodal Imaging with 124I-cRGDY-PEG-C Dots in Melanoma Patients 19.6 Conclusions and Future Prospects

20. Atomic Force Microscopy for Nanomedicine Shivani Sharma, PhD, and James K. Gimzewski, PhD

567



20.1 Introduction 20.2 AFM for Cancer Diagnosis 20.3 Summary and Conclusions

567 572 581



Yuri L. Lyubchenko, PhD, DSc, and Luda S. Shlyakhtenko, PhD

18. Magnetic Nanoparticles for Magnetic Resonance Imaging: A Translational Push toward Theranostics

19. First-in-Human Molecular Targeting and Cancer Imaging Using Ultrasmall Dual-Modality C Dots



21. Atomic Force Microscopy Imaging and Probing of Amyloid Nanoaggregates

21.1 Introduction 21.2 Protein Misfolding and Diseases

505 513

521

521 523 526 530 534 537

549

549 553 554 555

557 559

589

589 590

Contents

21.3 afm Characterization of the Nanoscale Structure of Amyloid Aggregates 592 21.4 AFM Visualization of Dynamic Biological Events 596 21.5 AFM Force Spectroscopy and Intermolecular Interactions 599 21.6 Conclusions 608

22. Image-Based High-Content Analysis, Stem Cells and Nanomedicines: A Novel Strategy for Drug Discovery



Leonardo J. Solmesky, PhD, Yonatan Adalist, MSc, and Miguel Weil, PhD



22.1 Introduction 22.2 Image-Based High-Content Screening 22.3 Stem Cells Used for Lead Discovery 22.4 Personalized Medicine Platforms 22.5 Stem Cells as Tools for Improvement of Safety and Toxicology 22.6 Advances in Nanomedicine for Drug Discovery Using HCS 22.7 Conclusions



Nicole F. Steinmetz, PhD, and Marianne Manchester, PhD



23. Viral Nanoparticles: Tools for Materials Science and Biomedicine

617

617 619 622 627 628 629 631

641

23.1 What Is Nano? 642 23.2 Where Did it all Begin? A History of Vnps: From Pathogens to Building Blocks 643 23.3 Why Vnps? Materials Properties of Vnps 647 23.4 Summary 651

24. Bacterial Secretion Systems: Nanomachines for Infection and Genetic Diversity



Agnesa Shala, PhD, Michele Ferraro, MSc, and Gerald F. Audette, PhD



24.1 Introduction 24.2 Bacterial Secretion Systems

657

657 659

xvii

xviii

Contents



24.3 The Type IV Secretion System 24.4 Conclusions



Frank J. Boehm



25.1 Introduction 25.2 Vascular Cartographic Scanning Nanodevice 25.3 Overview of Envisaged VCSN Capabilities 25.4 Summary of VCSN Components 25.5 Discussion 25.6 VCSN Advantages 25.7 The Gastrointestinal Micro Scanning Device 25.8 Description of Scanning Procedure 25.9 Additional Issues



Angelika Domschke, PhD



26.1 Introduction: Importance of Diabetes Management 707 26.2 Current Continuous Glucose Monitoring Devices 708 26.3 Noninvasive Continuous Glucose Monitoring Devices 709 26.4 Advantages of Noninvasive and Continuous Ophthalmic/Contact Lens Glucose Sensors 710 26.5 Requirements for Ophthalmic/Contact Lens Glucose Sensors 710 26.6 Fluorescence-Based Glucose Sensing 713 26.7 Colorimetric Sensors Based on Periodic Optical Nanostructures 722 26.8 Holographic Glucose Sensors 724 26.9 Photonic Crystal Glucose Sensors 727 26.10 Microelectromechanical-Based Sensors 730 26.11 Progress and Remaining Challenges toward the Development of Ophthalmic Glucose Sensors 736 26.12 Outlook 738

25. The Vascular Cartographic Scanning Nanodevice

26. Advancements in Ophthalmic Glucose Nanosensors for Diabetes Management



665 672

687

687 689 690 692 694 697 698 701 702

707

Contents

Section III: Therapy and Clinical Applications 27. Towards Nanodiagnostics for Bacterial Infections

Georgette B. Salieb-Beugelaar, PhD, and Patrick R. Hunziker, MD



27.1 Introduction 27.2 Diagnostics for Bacterial Infections: From the State of the Art to Nanodiagnostics 27.3 Future Challenges for in vitro Diagnostics Suited for Infectious Diseases 27.4 Conclusion





28. Copaxone® in the Era of Biosimilars and Nanosimilars

749 749 751 767 769

783



Jill B. Conner, MS, PhD, Raj Bawa, MS, PhD, J. Michael Nicholas, PhD, and Vera Weinstein, PhD





28.1 Introduction 784 28.2 Non-Biologic Complex Drugs and Regulatory Pathway for Follow-On Products 791 28.3 Nanomedicines and Regulatory Pathway for Nanosimilars 796 28.4 Colloidal and Nanomedicine Properties of Copaxone® 797 28.5 Immunogenicity 810 28.6 Conclusions and Future Prospects 816



Yechezkel Barenholz, PhD



29.1 Historical Perspective 29.2 First-Generation Liposomal Doxorubicin: Liver-Directed Liposomal Doxorubicin 29.3 OLV-DOX Clinical Trials 29.4 Doxil® Development 29.5 Doxil Performance in Humans 29.6 What Is New about Doxil since 2011 29.7 Conclusions, Take Home Lessons, and What Is Next? 29.8 Doxil Historical Perspectives 29.9 Personal Touch



29. Doxil®: The First FDA-Approved Nanodrug—From an Idea to a Product (January 2015 Update)



827

828

833 846 857 876 883 884 886 886

xix

xx

Contents

30. Nanotechnology and the Skin Barrier: Topical and Transdermal Nanocarrier-Based Delivery

Hagar I. Labouta, PhD, and Marc Schneider, PhD



30.1 Introduction 30.2 Enhancement of Drug Penetration via Encapsulation in Nanocarriers 30.3 Skin Penetration/Permeation of Nanoparticles 30.4 Conclusion



31. Application of Nanotechnology in Non-Invasive Topical Gene Therapy



Mahmoud Elsabahy, PhD, Maria Jimena Loureiro, MSc, and Marianna Foldvari, PhD, DPharmSci





31.1 Introduction 31.2 Topical Delivery as an Alternative Route of Administration 31.3 Biological Barriers toward Topical Gene Therapy 31.4 Nanomedicines for Non-Invasive Topical Gene Delivery Applications 31.5 Toward Needle-Free Future Nanomedicines 31.6 Conclusions



Alf Lamprecht, PhD



32.1 Introduction 32.2 Rheumatoid Arthritis 32.3 Inflammatory Bowel Disease 32.4 Uveitis



Benjamin Holmes, MSc, Thomas J. Webster, PhD, and Lijie Grace Zhang, PhD



33.1 Introduction 33.2 Electrospinning 33.3 3D Printing 33.4 Other Current Methodology 33.5 Summary



32. Nanocarriers in the Therapy of Inflammatory Disease

33. Advanced 3D Nano/Microfabrication Techniques for Tissue and Organ Regeneration

907 907 910 915 926

937

937 939 939 941 960 962

973

973 974 978 982

989

989 991 1002 1011 1013

Contents

34. Nanomedicine for Acute Lung Injury/Acute Respiratory Distress Syndrome: A Shifting Paradigm?

1027



Ruxana T. Sadikot, MD, and Israel Rubinstein, MD



34.1 Introduction 34.2 ALI and ARDS 34.3 Nanomedicine for ALI and ARDS 34.4 Nanomedicine for Drug Delivery to the Lung 34.5 Conclusions



Randall W. Barton, PhD, Jayant G. Tatake, PhD, and Anil R. Diwan, PhD



35.1 Introduction 35.2 Nanoviricides Polymeric Micelle Technology 35.3 The Nanoviricide Antiviral Technology 35.4 Nanoviricides Antiviral Effects



Lesley M. Hamming, PhD, JD, and Mark G. Hamming, MD



36.1 Introduction 36.2 Benefits of Nanometer-Scale Features 36.3 Commercialization 36.4 Tissue Engineering for Bone 36.5 Tissue Engineering for Cartilage 36.6 Conclusion



Karolina Jurczyk, DDS, PhD, and Mieczyslaw Jurczyk, PhD, DSc



37.1 Bulk Nanostructured Titanium 1074 37.2 Bulk Titanium–Bioceramic Nanocomposites 1079 37.3 Dental Implants with Nanosurface 1089 37.4 Nanostructured Materials for Permanent and Bioresorbable Medical Implants 1095 37.5 Nanostructured Dental Composite Restorative Materials 1098

35. Nanoviricides: Targeted Anti-Viral Nanomaterials

36. Nanotechnology in Tissue Engineering for Orthopaedics

37. Applications of Nanomaterials in Dentistry



1027 1029 1030 1032 1034

1039

1039 1040 1043 1046

1053

1053 1056 1057 1060 1063 1065

1073

xxi

xxii

Contents

38. Biomimetic Applications in Regenerative Medicine: Scaffolds, Transplantation Modules, Tissue Homing Devices, and Stem Cells

1109

David W. Green, PhD, and Besim Ben-Nissan, PhD



38.1 Introduction 1109 38.2 Biomimetic Stem-Cell Scaffolds 1111 38.3 Electrospun Polysaccharide Core-Shell Fibres 1123 38.4 Self-Adjusting Bioscaffolds 1125 38.5 Transplantation and Biocargo Modules 1126 38.6 Polysaccharide Chitosan Coated Alginate Nanocapsules 1130 38.7 Native Extracellular Matrix Equivalents 1132 38.8 Tissue Homing Devices 1134 38.9 Summary 1134 38.10 Conclusions 1135

39. Potential Applications of Nanotechnology in the Nutraceutical Sector

Shu Wang, MD, PhD, and Jia Zhang, MS



39.1 Introduction 39.2 Nanoparticles 39.3 Phytochemicals 39.4 Conclusions and Future Prospects

40. Designing Nanocarriers for the Effective Treatment of Cardiovascular Diseases

1141

1141 1142 1144 1156

1167



Bhuvaneshwar Vaidya, MPharm, PhD, and Suresh P. Vyas, MPharm, PhD



40.1 Introduction 1167 40.2 Nanocarriers for the Treatment of Myocardial Infarction 1168 40.3 Novel Carriers for the Treatment of Thromboembotic Diseases 1174 40.4 Nanocarriers for the Treatment of Restenosis 1179 40.5 Concluding Remarks 1185



Contents

41. Carbon Nanotubes as Substrates for Neuronal Growth

1197



Cécilia Ménard-Moyon, PhD



41.1 Introduction 1197 41.2 Effects of Carbon Nanotubes on Neuronal Cells’ Adhesion, Growth, Morphology and Differentiation 1199 41.3 Electrical Stimulation of Neuronal Cells Grown on Carbon Nanotube-Based Substrates 1209 41.4 Investigation of the Mechanisms of the Electrical Interactions Between Cnts and Neurons 1223 41.5 Conclusions and Perspectives 1228



42. Polymeric Nanoparticles for Cancer Therapeutics

1239

Mohit S. Verma, Joshua E. Rosen, Ameena Meerasa, Serge Yoffe, MEng, and Frank X. Gu, PhD





42.1 Introduction 42.2 Biological Interactions of Nanomaterials 42.3 Nanoparticle Targeting 42.4 Classes of Polymeric Nanosystems for Cancer Therapy 42.5 Synthesis Methods 42.6 Conclusions

43. Nanotechnology for Radiation Oncology

1239 1241 1246 1249 1255 1260

1273



Srinivas Sridhar, PhD, Ross Berbeco, PhD, Robert A. Cormack, PhD, and G. M. Makrigiorgos, PhD



43.1 Gold Nanoparticles for Radiation Therapy 1274 43.2 Nanoparticles as Delivery Agents for Radiation Sensitizers 1279 43.3 Nano-Coated Drug-Loaded Implants for Biologically in situ Enhanced, Image-Guided Radio Therapy 1280 43.4 Nanoparticles as Imaging Agents for Radiation Therapy 1284 43.5 Nanotechnology for Radiation Sources 1285 43.6 Conclusions 1286





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44. Gold Nanoparticles against Cancer

1293



Joan Comenge, PhD, Francisco Romero, PhD, Aurora Conill, MS, and Víctor F. Puntes, PhD





44.1 Historical Perspective on the Medical Use of Gold 1294 44.2 Gold in the ERA of Nanotechnology: 1294 New Properties for a Known Material 44.3 Synthesis of Gold Nanoparticles 1295 44.4 Gold Nanoparticles to Target Cancer 1298 44.5 Challenges of Using Gold Nanoparticles in Therapy 1300 44.6 Examples of Gold Nanoparticles in Cancer Therapy 1304 44.7 Conclusions 1307



Melike Üner, PhD



45.1 Introduction 1315 45.2 Principles of Drug Incorporation into SLN and NLC, and Drug Release 1317 45.3 Targeting of SLN and NLC in the Treatment of Cancer 1320 45.4 Incorporation of Anti-Neoplastic Drugs and Genes into SLN and NLC for Cancer Treatment 1328 45.5 NSAIDs as Adjuvant to Cancer Chemotherapy 1333 45.6 Conclusions 1334



45. Solid Lipid Nanoparticles and Nanostructured Lipid Carriers for Cancer Therapy





46. Nanomedicines Targeted to Aberrant Cancer Signaling and Epigenetics



Archana Retnakumari, MTech, Parwathy Chandran, MTech, Ranjith Ramachandran, MSc, Giridharan L. Malarvizhi, MTech, Shantikumar Nair, PhD, and Manzoor Koyakutty, PhD



46.1 Introduction 46.2 Protein Nanomedicine Targeted to Aberrant Kinome Involved in Refractory Cancer 46.3 Protein–Protein Core–Shell Nanomedicine Targeted to Multiple Kinases in Refractory Cancer



1315

1347

1347 1348 1351

Contents



46.4 Polymer–Protein Nanomedicine Targeting Aberrant Kinome in Acute Myeloid Leukemia 1355 46.5 Polymer–Protein Core–Shell Nanomedicine Targeted to Cancer Metastasis 1357 46.6 Protein Nanomedicine Targeted to Aberrant Epigenome 1362 46.7 Conclusions and Future Prospects 1365

47. Biodegradable Nanoparticle-Based Antiretroviral Therapy across the Blood-Brain Barrier

1379



Supriya D. Mahajan, PhD, Yun Yu, PhD, Ravikumar Aalinkeel, PhD, Jessica L. Reynolds, PhD, Bindukumar B. Nair, PhD, Manoj J. Mammen, MD, Tracey A. Ignatowski, PhD, Chong Cheng, PhD, and Stanley A. Schwartz, PhD, MD



47.1 Introduction 47.2 Methods 47.3 Results 47.4 Discussion 47.5 Conclusions and Future Prospects



Orsolya Lorincz, PhD, and Julianna Lisziewicz, PhD



48.1 Introduction 1407 48.2 Nanomedicines for Immunotherapy 1409 48.3 Pathogen-Like DNA-Nanomedicines for Immunotherapy 1411 48.4 DermaVir Immunotherapy for the Cure of HIV/AIDS 1420 48.5 Conclusions 1422

48. HIV-Specific Immunotherapy with Synthetic Pathogen-Like Nanoparticles





49. Biomedical Engineering and Nanoneurosurgery: From the Laboratory to the Operating Room



Mario Ganau, MD, PhD, Roberto I. Foroni, PhD, Andrea Soddu, PhD, and Rossano Ambu, MD



49.1 Neurosurgery at the Crossroads of Nanotechnology, Biomedical Engineering and Neuroscience

1379 1382 1388 1392 1395

1407

1433

1433

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49.2 Applications in Neurovascular Surgery 49.3 Applications in Pediatric Neurosurgery 49.4 Applications in Neuro-Oncology 49.5 Applications in Epilepsy and Functional Surgery 49.6 Applications in Spinal Surgery 49.7 Hemostasis in Brain and Spine Surgery 49.8 New Horizons in Nanoneurosurgery 49.9 Conclusion

50. Nanotechnology-Based Systems for Microbicide Development

Rute Nunes, Carole Sousa, Bruno Sarmento, PhD, and José das Neves, PhD



50.1 Introduction 50.2 Limitations of Microbicide Products Currently under Development 50.3 Why Nanotechnology-Based Microbicides? Potential and Perils 50.4 Nanosystems Presenting Intrinsic Activity against HIV/Competing with the Virus for Host Targets 50.5 Nanosystems Acting as Carriers for Microbicide Agents 50.6 Mucoadhesive or Mucus-Penetrating Microbicide Nanosystems? 50.7 Nanotechnology-Based Rectal Microbicides 50.8 Conclusions and Future Perspectives



51. Nanotechnology-Based Solutions to Combat the Emerging Threat of Superbugs: Current Scenario and Future Prospects

1434 1435 1436 1437 1438 1438 1439 1440

1445

1445 1446 1448 1450 1453 1465 1471 1473

1493



Nisha C. Kalarickal, PhD, and Yashwant R. Mahajan, PhD



51.1 Introduction 1493 51.2 The Discovery and Consequent Development of Antibiotic Resistance 1494 51.3 Nanotechnology-Based Solutions against Superbugs 1498 51.4 Conclusions and Future Perspectives 1509



Contents

52. Nanolithography and Biochips’ Role in Viral Detection

Inbal Tsarfati-BarAd and Levi A. Gheber, PhD



52.1 The Need for Portable Biochips for Viral Detection 52.2 Arrayed Biosensors: Biochips 52.3 The Need for Miniaturization 52.4 Nanolithography 52.5 SPM-Based Nanolithography Methods 52.6 Problems Associated with Miniaturization 52.7 Conclusions



Anita Gupta, MSc, PhD, and G. S. Gupta, MSc, PhD



53.1 Introduction 53.2 Lectins 53.3 Carbohydrate–Lectin Interactions 53.4 Nanotechnology and Drug Targeting Strategies 53.5 Nanocarriers Used in Drug Delivery Systems 53.6 Lectins as Drug Carriers 53.7 Reverse Lectin Targeting 53.8 Carbohydrate-Directed Targeting 53.9 Conclusions



Ariel Sobarzo, PhD, Robert S. Marks, PhD, and Leslie Lobel, MD, PhD



54.1 Ebola Virus 54.2 Etiology and Epidemiology 54.3 Disease Transmission and Clinical Behavior 54.4 Therapy 54.5 The Fear of Ebola 54.6 Current Methods in Ebola Diagnostics 54.7 Nucleic Acid-Based Techniques 54.8 Engineered Recombinant Proteins 54.9 New Trends in Ebola Diagnostics 54.10 Future Diagnostics 54.11 The Effort Continues

53. Lectins as Nano-Tools in Drug Delivery

54. Diagnostics of Ebola Hemorrhagic Fever Virus

1517 1517 1518 1519 1519 1520 1523 1524

1529

1529 1530 1536 1538 1538 1547 1548 1556 1561

1577

1577 1578 1579 1579 1580 1580 1584 1585 1587 1590 1594

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55. Nanomedicine as a Strategy to Fight Thrombotic Diseases

Mariana Varna, PhD, Maya Juenet, MSc, Richard Bayles, PhD, Mikael Mazighi, MD, PhD, Cédric Chauvierre, PhD, and Didier Letourneur, PhD



55.1 Introduction 55.2 Composition of Nano- and Microcarriers Used in Thrombolytic Therapy 55.3 Thrombolytic Therapy Using Nanocarriers and Microbubbles in Preclinical Development 55.4 Microbubbles Associated with Thrombolytic Drugs and/or Ultrasound for Clinical Applications 55.5 Discussion and Conclusion 55.6 Future Perspective



Index

1611

1612 1617 1620 1628 1629 1631 1639

List of Corresponding Authors Fritz Allhoff Department of Philosophy, 3004 Moore Hall, Western Michigan University, 1903 W. Michigan Avenue, Kalamazoo, MI 49008, USA, Email: [email protected] Gerald F. Audette Department of Chemistry and the Centre for Research on Biomolecular Interactions, York University, Life Sciences Building 327C, 4700 Keele Street, Toronto, Ontario M3J 1P3, Canada, Email: [email protected] yorku.ca Yechezkel Barenholz Laboratory of Membrane and Liposome Research, Department of Biochemistry and Molecular Biology, Institute of Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, POB 12272,​​Jerusalem​9112102​,​Israel, Email: [email protected] huji.ac.il Randall Barton NanoViricides, Inc., One Controls Drive, Shelton, CT 06484, USA, Email: [email protected] Raj Bawa Bawa Biotech LLC, 21005 Starflower Way, Ashburn, VA 20147, USA, Email: [email protected] Frank J. Boehm NanoApps Medical, Inc., 141 Empress Avenue South, Thunder Bay, ON, Canada P7B 4N6, Email: [email protected] Michelle S. Bradbury Department of Radiology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA, Email: [email protected] Jill B. Conner Teva Pharmaceutical Industries, Ltd., Specialty Life Cycle Initiatives, 11100 Nall Avenue, Overland Park, KS 66211, USA, Email: [email protected] José das Neves INEB-Instituto de Engenharia Biomédica, University of Porto, Rua do Campo Alegre, 823, 4150-180, Porto, Portugal, Email: [email protected] Jason L. J. Dearling Division of Nuclear Medicine and Molecular Imaging, Department of Radiology, Boston Children’s Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA, Email: [email protected] Angelika Domschke Angelika Domschke Consulting LLC, 3379 Ennfield Way, Duluth, GA 30096, USA, Email: [email protected] Rutledge Ellis-Behnke Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA, Email: [email protected] Marianna Foldvari School of Pharmacy, University of Waterloo, 200 University Avenue West, Waterloo, ON, Canada N2L 3G1, Email: [email protected]

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List of Corresponding Authors

Mario Ganau Department of Surgical Science, Asse Didattico di Medicina, Cittadella Universitaria, S.S. 554. 09042 Monserrato (CA), Italy, Email: [email protected] Howard E. Gendelman Department of Pharmacology and Experimental Neuroscience, College of Medicine, University of Nebraska Medical Center, Omaha, NE 68198, USA, Email: [email protected] Levi A. Gheber Department of Biotechnology Engineering, Ben-Gurion University of the Negev, PO Box 653, Beer-Sheva 84105, Israel, Email: [email protected] James K. Gimzewski Department of Chemistry and Biochemistry, University of California, 607 Charles E. Young Drive East, Los Angeles, CA 900951569, USA, Email: [email protected] Todd Giorgio Department of Biomedical Engineering, Vanderbilt University, VU Station B 351620, Nashville, TN 37235-1620, USA, Email: [email protected] D. W. Green Department of Oral Biosciences, Faculty of Dentistry, University of Hong Kong, The Prince Philip Dental Hospital, 34 Hospital Road, Sai Ying Pun, Hong Kong, Email: [email protected] Frank X. Gu Department of Chemical Engineering, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, N2L 3G1, Canada, Email: [email protected] G. S. Gupta Department of Biophysics, Panjab University, Chandigarh 160014, India, Email: [email protected] Lesley M. Hamming Winston & Strawn LLP, 35 W. Wacker Drive, Chicago, IL 60601-9703, USA, Email: [email protected] Karolina Jurczyk University of Medical Sciences, Conservative Dentistry and Periodontology Department, Bukowska 70 Str., 60-812 Poznan, Poland, Email: [email protected] Manzoor Koyakutty Cancer Nanomedicine, Amrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham University, Ponekkara PO, Kochi, Kerala 682041, India, Email: [email protected] Alexey Krasnoslobodtsev Department of Physics, University of Nebraska at Omaha, 6001 Dodge Street, Omaha, NE 68182, USA, Email: [email protected] Harold W. Kroto Department of Chemistry & Biochemistry, Florida State University, 95 Chieftan Way, Tallahassee, FL 32306-4390, USA, Email: [email protected] Alf Lamprecht Laboratory of Pharmaceutical Engineering, Faculty of Medicine and Pharmacy, University of Franche-Comté Besançon, France, Email: [email protected] Ki-Bum Lee Department of Chemistry and Chemical Biology, Rutgers New Brunswick-Busch Campus, Rutgers, 610 Taylor Road, Piscataway, NJ 08854, USA, Email: [email protected]

List of Corresponding Authors

Claus-Michael Lehr Helmholtz-Institute for Pharmaceutical Research Saarland, Department of Drug Delivery, Saarland University, 66123 Saarbrücken, Universitätscampus E8 1, Germany, Email: Claus-Michael. [email protected] Didier Letourneur INSERM U1148, LVTS, Cardiovascular Bio-Engineering, X. Bichat Hospital, Paris, France, Email: [email protected] Julianna Lisziewicz eMMUNITY Inc., 4400 East West Hwy, Bethesda, MD 20814, USA, Email: [email protected] Catherine M. Lockhart Department of Pharmaceutics, University of Washington, Box 357610, H272 Health Science Building, 1959 NE Pacific Street, Seattle, WA 98195 USA, Email: [email protected] Yuri Lyubchenko Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 601 S. Saddle Creek Road, Omaha, NE 68106, USA, Email: [email protected] Supriya Mahajan Department of Medicine, Division of Allergy, Immunology, and Rheumatology, State University of New York at Buffalo, 6074 UB’s Clinical and Translational Research Center, 875 Ellicott Street, Buffalo, NY 14203, USA, Email: [email protected] Yashwant R. Mahajan Centre for Knowledge Management of Nanoscience and Technology, 12-5-32/8, Vijaypuri Colony, Tarnaka, Secunderabad 500017, Telangana, India, Email: [email protected] Heidi M. Mansour The University of Arizona, College of Pharmacy, 1703 E. Mabel Street, Tucson, AZ 85721-0207, USA, Email: [email protected] arizona.edu Cécilia Ménard-Moyon CNRS, Institut de Biologie Moléculaire et Cellulaire, Laboratoire d’Immunopathologie et Chimie Thérapeutique (UPR 3572), 15 Rue René Descartes, 67084 Strasbourg, Cedex, France, E-mail: [email protected] Andrew Owen Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, 70 Pembroke Place, Liverpool L69 3GF, UK, Email: [email protected] Dan Peer Laboratory of NanoMedicine, Britannia Building, 2nd Floor Room 226, Tel Aviv University, Tel Aviv 69978, Israel, Email: [email protected] ac.il Adriele Prina-Mello School of Medicine and Centre for Research on Adaptive, Nanostructures and Nanodevices, Institute of Molecular Medicine, Trinity College, Dublin D8, Ireland, Email: [email protected] Víctor F. Puntes Inorganic Nanoparticles Group, Catalan Institute of Nanotechnology (ICN-2), 08193 Bellaterra, Barcelona, Spain, Email: [email protected] Steve Rannard Department of Chemistry, University of Liverpool, Liverpool L697ZD, United Kingdom, Email: [email protected] Ruxana T. Sadikot Emory University School of Medicine, Section of Pulmonary and Critical Care Medicine, 201 Dowman Drive, Atlanta, GA 30322, USA, Email: [email protected]

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List of Corresponding Authors

Georgette B. Salieb-Beugelaar Nanomedicine Research Group, Clinic for Intensive Care Medicine, University Hospital Basel, Petersgraben 4, CH-4031 Basel, Switzerland, Email: [email protected] Krutika K. Sawant Pharmacy Department, Faculty of Technology and Engineering, The Maharaja Sayajirao University of Baroda, Kalabhavan, Vadodara 390001, Gujarat, India, Email: [email protected] com Marc Schneider Department of Pharmacy, Biopharmaceutics and Pharmaceutical Technology, Saarland University, Campus A4 1, D-66123 Saarbrucken, Germany, Email: [email protected] Ariel Sobarzo Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, POB 653, Beer-Sheva 84105, Israel, Email: [email protected] Srinivas Sridhar Department of Physics, Northeastern University, 435 Egan Research Center, 120 Forsyth Street, Boston, MA 02115, USA, Email: [email protected] Nicole F. Steinmetz Biomedical Engineering, Case Western Reserve University, School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106 USA, Email: [email protected] Melanie Swan MS Futures Group, PO Box 61258, Palo Alto, CA 94306, USA, Email: [email protected] Takuya Tsuzuki Research School of Engineering, College of Engineering and Computer Science, Australian National University, Ian Ross Building 31, North Road, Canberra, ACT 0200, Australia, Email: [email protected] anu.edu.au Melike Üner Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Technology, Beyazit 34116, Istanbul, Turkey, Email: [email protected] Suresh P. Vyas Drug Delivery Research Laboratory, Department of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar, MP 470003, India, Email: [email protected] Shu Wang Department of Nutritional Sciences, Texas Tech University, 1301 Akron Avenue, Lubbock, TX 79409-1270, USA, Email: [email protected] edu Andrew T. S. Wee Department of Physics, National University of Singapore, 2 Science Drive 3, Singapore 117542, Email: [email protected] Miguel Weil Cell Screening Facility for Personalized Medicine, Laboratory for Neurodegenerative Diseases and Personalized Medicine, The George S. Wise Faculty of Life Sciences, Tel Aviv University, 69978 Ramat Aviv, Tel Aviv, Israel, Email: [email protected] Ulrich Wiesner Department of Materials Science and Engineering, Cornell University, 330 Bard Hall, Ithaca, NY 21047, USA, Email: [email protected] edu Lijie Grace Zhang Department of Mechanical and Aerospace Engineering, The George Washington University, 800 22nd Street NW, 3590 Science and Engineering Hall, Washington, DC 20052, USA, Email: [email protected] gwu.edu

Foreword Major medical advances that emerged during the nineteenth and early twentieth century were based on either “bottom-up” or “topdown” type particle dimensions. In this regard, the importance of early pico/sub-nanoscale (i.e., 0.001–1 nm) elemental/small molecule “bottom-up” therapies is well known. Examples include therapies based on elemental mercury or arsenic compounds (i.e., treatment of syphilis), radium (i.e., cancer therapy) or nineteenth century pharma (i.e., antipyrine, quinine, aspirin, etc.). On the other hand, significant medical insights emerged concurrently with Louis Pasteur’s germ theory of disease (1862) that emphasized “top-down” type particle dimensions. Specifically, this theory emphasized that microbes such as bacteria possessed macro-/ micron scale (i.e., micron to mm) dimensions and caused diseases. Additionally, in 1928, Alexander Fleming was the first to report the therapeutic benefits of controlling infection by the use of antibiotic molds (i.e., small-molecule penicillin). Until recent times however, very little attention has been devoted to the role of nano-sized (i.e., 1–1000 nm) particles and phenomena residing between these two upper and lower hierarchical extremes. Progress toward this new perspective first required critical insights into life sustaining nanoparticles (proteins, DNA, RNA) developed by Linus Pauling, Max Perutz et al. and Francis Crick/James Watson, respectively. Pioneering structural characterization, vaccines and insights developed by Alex Klug, Jonas Salk and others concerning lifethreatening nanoparticles such as viruses clearly demonstrated the important role that “good and bad nanoparticles” could play in defining the human conditions of either health or disease. These pioneering scientists laid the groundwork for understanding life processes and, more importantly, the critical role that nanometric dimensions might play in future medical advancements. In fact, it was Linus Pauling’s deep insights into the nanoscale dimensions and dynamics of proteins (i.e., Hemoglobin) that undoubtedly allowed him to diagnose and propose the first rational therapies for sickle cell anemia as early as 1949. Pauling’s seminal work on sickle cell anemia may represent the very first example of nanomedicine in practice. That withstanding,

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Foreword

it was Richard Feynman’s famous California Institute of Technology lecture in 1959 titled “There is Plenty of Room at the Bottom” that officially launched the present nanotechnology explosion/ revolution. Since that time, the recognized impact of nanoscale complexity on biology and traditional medicine has grown dramatically as witnessed by an exponential increase in publications/patents, establishment of national/global scientific societies and the emergence of several mainstream journals/ publishers—all focusing on the new emerging frontier of nanomedicine and bionanotechnology. My earliest connections to nanomedicine began with industrial/academic activities (1990–2005) which focused on the use of abiotic, nanoscale particles called dendrimers for a variety of biological/medical applications. It was found that, by properly modifying these synthetic nanoparticles as a function of size, shape, surface chemistry, flexibility, architecture or composition, it was possible to selectively mimic, intervene, complex or control the in vivo inter/intracellular dynamics of many important biological nanoparticles such as proteins, DNA, and RNA with minimal cytotoxicity. These strategies provided some of the early working examples of rational in vivo protein mimicry, gene/viral complexation, immuno-diagnostics and targeted drug-delivery by design. Not only did these lead to many successful gene transfection, cardio-diagnostic, antiviral/bacterial and pending targeted drugdelivery products, but they also provided an unbridled personal optimism for the unlimited future possibilities expected in the field of nanomedicine. It is from this broad perspective, that I have had the pleasure to review the Handbook of Clinical Nanomedicine: Nanoparticles, Imaging, Therapy, and Clinical Applications, which is the first in the series on clinical nanomedicine texts edited by Dr. Raj Bawa. The distinguished editors (Raj Bawa, PhD, Gerald F. Audette, PhD, and Israel Rubinstein, MD) have successfully organized critical contributions from an international team of experts into three important sections that constitute this handbook. Throughout all three sections, the editors and authors remain loyal to the handbook theme. They provide a systematic and rational coverage of major issues and challenges encountered in the progression of nanomedical concepts to working prototypes, clinical applications, nanodevices and imaging tools. More specifically, Section I provides a general introduction to nanomedicine, along with historical/

Foreword

background information and ten, recent significant nanomedical advancements. The important chapter on nanotechnology nomenclature and definitional issues of nanotherapeutics highlights the fact that nanomedicines cannot be pigeonholed into any specific numerical range or dimension, especially in the context of drug delivery. Section II consists of a balanced discussion of several nanosystems, nanodevices and imaging tools including magnetic resonance imaging, atomic force microscopy, multi-modal imaging/ diagnostics, in vivo imaging techniques, image-based analysis related to stem cells, and bacterial/viral nanomachines as tools for biomedicine. Of particular interest is the chapter on the first-inhuman molecular targeting and cancer imaging via ultrasmall dualmodality C-dots. The inspirational story of C60 Buckminsterfullerene by Nobel Laureate Dr. Kroto reveals the unique perspectives of many scientists who made major contributions to its discovery. Section III overviews important clinical applications of nanotherapeutics for topical/transdermal delivery; targeted cancer and antiviral therapies; and inflammatory, cardiovascular, respiratory and neuronal diseases. Applications of bionanomaterials for orthopedics and dentistry are reflected in two excellent chapters. This section also provides suitable attention to more specialized topics such as nanolithography and biochips for viral detection, 3D nano/ microfabrication techniques for tissue and organ regeneration, solutions to combat emerging superbugs, diagnostics of Ebola, HIV-specific immunotherapy, and nanoneurosurgery. A superb chapter on the first FDA-approved nanodrug product (Doxil®) by its inventor, Dr. Yechezkel (Chezy) Barenholz, is a fascinating read. The important chapter on the blockbuster MS drug, Copaxone®, by experts from Teva Pharmaceutical Industries, highlights the new era of biosimilars, nanosimilars and NBDCs. The Handbook provides a well-edited, reader-friendly, multidisciplinary roadmap that clearly communicates essential nanomedical information in a style that is attractive to both the novice and expert. It offers the appropriate advice required to advance nanomedical innovations from the concept stage to working prototypes, clinical applications and, finally, to commercial reality. The diversity and expertise of the authors has produced a wellbalanced, comprehensive reference resource which successfully bridges communication and terminology gaps often encountered in such multi-disciplinary efforts. As a result, the Handbook should be of interest to individuals from academia, industry and

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Foreword

government in fields ranging from medicine, biotechnology, drug development, pharmaceutical sciences, engineering, policy, FDA law and commercialization. This authoritative and comprehensive reference bridges the gap between basic biomedical research, engineering, medicine and law while providing a thorough understanding of nano’s potential to address (i) medical problems from both the patient and health provider’s perspective, and (ii) current applications and their potential in a healthcare setting. I view the Handbook of Clinical Nanomedicine: Nanoparticles, Imaging, Therapy, and Clinical Applications as an essential reference asset that should be invaluable to all who are interested in the explosive growth and future promise of this new emerging area.

Donald A. Tomalia, PhD December 2015

Dr. Donald Tomalia received his Bachelor of Arts degree in chemistry from the University of Michigan and his PhD in physical organic chemistry from Michigan State University in 1968 while working at The Dow Chemical Company. In 1990, he moved to Michigan Molecular Institute as professor and director of Nanoscale Chemistry and Architecture. He has subsequently founded three dendrimer-based nanotechnology companies: Dendritech, Inc.; Dendritic Nanotechnologies, Inc.; and NanoSynthons LLC. Dr. Tomalia is currently director of the National Dendrimer & Nanotechnology Center, CEO/founder of NanoSynthons LLC, distinguished visiting professor at Columbia University, adjunct professor in the department of chemistry at the University of Pennsylvania and affiliate professor in the department of physics at Virginia Commonwealth University. He is famous for his discovery of dendrimers and has received several awards for his accomplishments and contributions to science, including the 2012 Wallace H. Carothers Award. He has authored over 250 publications and is the inventor or coinventor of 128 patents. He currently serves as an associate editor of the Journal of Nanoparticle Research (Springer); as member of the Executive Advisory Board of the European Foundation for Clinical Nanomedicine; and as a faculty member of Faculty 1000 Biology. In 2011, Dr. Tomalia was inducted into the Thomas Reuters Hall of Citation Laureates in Chemistry, a listing of the 40 most highly cited international scientists in the field of chemistry.

Small Is Beautiful Preface by the Series Editor Back in 2002, I presented on nanomedicine at the Canadian Space Agency, a few hours’ drive from Montreal. The audience was primarily composed of materials scientists and engineers. After a few excellent questions from the audience, I realized that physical scientists and pharmaceutical scientists view the nanoworld quite differently and that there is tension between the two camps. This was back then, but the state of affairs is the same today, maybe even more profound. Since then, nano has become more sectorized, with nanomedicine at the forefront along with nanoelectronics. Nanomedicine,

an offshoot of nanotechnology, may be defined as the science and technology of diagnosing, treating and preventing disease and improving human health via nano. This exciting and interdisciplinary arena is driven by collaborative research, patenting, commercialization, business development and technology transfer within diverse areas such as biomedical sciences, chemical engineering, biotechnology, physical sciences, and information technology. Optimists see nano as an enabling technology, a sort of next industrial revolution that could enhance the wealth and health of nations. They promise that at least within the medical sector of nano, nanodrugs will be a healthcare game-changer by offering patients access to personalized medicine. Pessimists, on the other hand, take a cautionary position, preaching instead a “go-slow” approach, pointing to a lack of sufficient scientific information on health risks and general failure on the part of regulatory agencies and patent offices. As usual, the reality is somewhere between such extremes. Whatever your stance, nano has already permeated virtually every sector of the global economy, with potential applications consistently inching their way into the marketplace. Nano continues to evolve and play a pivotal role in various industry segments, spurring new directions in research and development and translational efforts. The prefix “nano” in the SI measurement system denotes 10−9 or one-billionth. There is no firm consensus over whether the prefix “nano” is Greek or Latin. While the term “nano” is often linked to the Greek word for “dwarf,” the ancient Greek word for “dwarf” is actually spelled “nanno” (with a double “n”) while the Latin word for dwarf is “nanus” (with a single “n”). While a nanometer refers to one-billionth of a meter in size (10−9 m = 1 nm), a nanosecond refers to one billionth of a second (10−9 s = 1 ns), a nanoliter refers to one billionth of a liter (10−9 l = 1 nl) and a nanogram refers to one billionth of a gram (10−9 g = 1 ng). The diameter of an atom ranges from about 0.1 to 0.5 nm. Some other interesting comparisons: fingernails grow around a nanometer/second; in the time it takes to pick a razor up and bring it to your face, the beard will have grown a nanometer; a single nanometer is how much the Himalayas rise in every 6.3 seconds; a sheet of newspaper is about 100,000 nanometers thick; it takes 20,000,000 nanoseconds (50 times per second) for a hummingbird to flap its wings once; a single drop of water is ~50,000 nanoliters; a grain of table salt weighs ~50,000 nanograms.

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Preface by the Series Editor

However, there is still a clear need for “true” interdisciplinarity because of the different approaches of physical scientists versus pharmaceutical scientists with respect to generation, examination, analysis and discussion of nanomedicine data. I have been extremely cognizant of this fact as I have developed this book series so that everyone finds it relevant and useful. Each stand-alone volume in this series is focused broadly on nanomedicine, pharma and various branches of medicine. It is essential reading for both the novice and the expert in fields ranging from medicine, pharmaceutical sciences, biotechnology, immunology, engineering, FDA law, intellectual property, policy, future studies, ethics, licensing, commercialization, risk analysis and toxicology. Each volume is intended to serve as a useful reference resource for professionals but also to provide supplementary readings for advanced courses for graduate students and medical fellows. Diversity within the broad and evolving arena of nanomedicine is reflected in the expertise of the distinguished contributing authors. All chapters contain key words, figures in full-color and an extensive list of references. As compared to other books on the market, each volume in the series is comprehensive, truly multidisciplinary and intended to be a stand‐alone reference resource—all presented in an easily accessible, user-friendly format. The editors have skillfully curated each chapter to reflect the most relevant and current information possible. The range of topics covered in each volume as well as the interdisciplinary approach of the volumes will attract a global audience and serve as a definitive resource, reference and teaching supplement. My purpose in constructing each volume is to provide chapters that give the reader a better understanding of the subject matter presented therein but also to provoke reflection, discussion and catalyze collaborations between the diverse stakeholders. Each volume is essentially a guided tour of critical topics and issues that should arouse the reader’s curiosity so that they will engage more profoundly with the broader theme reflected. Since 2002, I have been involved in almost all aspects of nanoscience, nanomedicine and nanotechnology—patent prosecution, teaching, research, FDA regulatory filings, conference organizer and speaker. I have intimately seen the evolution of nanomedicine. I have cheered on the cutting-edge discoveries and inventions but also stood up to criticize inept governmental regulatory policies, spotty patent examination at patent offices, hyped-up press

Preface by the Series Editor

releases from eminent university labs and inaccurate depiction of nano by the press and politicians. While the nano die was cast long ago, it is well entrenched now and there is no turning back. Although the air may be thick with news of nano-breakthroughs and a hot topic for discussion in industry, pharma, patent offices and regulatory agencies, the average citizen on the street knows very little about what constitutes a nanoproduct, a nanomaterial or a nanodrug. This was apparent to me early on when the CBS station in Albany, NY, while interviewing me for a story on nanotechnology, approached people on the street and asked them to define nano. To my amazement, not a single person could come up with a description of the term. At the time, I was well aware that there were close to two thousand marketed products that incorporated nano in some fashion. This issue persists. Mind you, these interviews were taking place a few blocks from the massive, world-class education and research complex comprising Albany Nanotech (now SUNY Polytechnic Institute’s Colleges of Nanoscale Science and Engineering), where billions have been invested each year since the early 1990s. Recognizing that the so-called “nanorevolution” will have limited impact unless all stakeholders, especially the public, are fully on board, I immediately initiated an annual nano-conference that continues to this day. The conference is free to all attendees and for the first dozen years was held at Rensselaer Polytechnic Institute in Troy, NY, while the most recent one was held this fall at Albany College of Pharmacy and Health Sciences in Albany, NY. Louis Pasteur famously said: “Science knows no country, because knowledge belongs to humanity, and is the torch which illuminates the world. Science is the highest personification of the nation because that nation will remain the first which carries the furthest the works of thought and intelligence.” Perhaps, nothing is truer in this regard than nanoscience, nanotechnology and nanomedicine. However, we are in a rapidly changing, globalized world—politically, economically, societally, environmentally and technologically. At the present time in our history, national boundaries are rendered artificial or eroding, Too

often though, start-ups, academia and companies exaggerate basic research developments as potentially revolutionary advances and claim these early-stage discoveries as confirmation of downstream novel products and applications to come. Nanotechnology’s potential benefits are frequently overstated or inferred to be very close to application when clear bottlenecks to commercial translation exist. Many have desperately tagged or thrown around the “nano” prefix to suit their purpose, whether it is for federal research funding, patent approval of the supposedly novel technologies, raising venture capital funds, running for office or seeking publication of a journal article.

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there are few true leaders on the global stage, the world power dynamics are shifting and the outdated bureaucratic structures firmly in place are unable to deal effectively with the emerging knowledge economy. These changes are also causing a certain degree of chaos in the world of nano. From my perspective, there is a combination of excitement, potential, confusion, hype, and misinformation in this regard—all of this seen more than in other fields when they were evolving. It is true that in the heady days of any new, emerging technology, definitions tend to abound and are only gradually documented via reports, journals, books and dictionaries. Ultimately, standard-setting organizations like the International Organization for Standardization (ISO) and American Society for Testing and Materials (ASTM) International produce technical specifications. This evolution is typical and essential as the development of terminology is a prerequisite for creating a common language needed for effective communication in any field. Clearly, a similar need for an internationally agreed definition for key terms like nanotechnology, nanoscience, nanomedicine, nanobiotechnology, nanodrug, nanotherapeutic, nanopharmaceutical and nanomaterial, has gained urgency. Nomenclature, technical specifications, standards, guidelines and best practices are critically needed to advance nanotechnologies in a safe and responsible manner. Contrary to some commentators, terminology does matter because it prevents misinterpretation and confusion. It is essential for research activities, harmonized regulatory governance, accurate patent searching and prosecution, standardization of procedures, manufacturing and quality control, assay protocols, decisions by granting agencies, effective review by policymakers, ethical analysis, public dialogue, safety assessment, and more. Also, nomenclature is critical to any translational and commercialization efforts. All definitions of nanotechnology based on size or dimensions must be dismissed, especially in the context of medicine and pharmaceutical science, for the reasons elaborated in chapter 6 of this volume. Similar

disagreements over terminology and nomenclature are seen in other fields as well. For example, the term “super resolution microscopy,” the subject of the 2014 Nobel Prize, is considered an inaccurate description of the technique. Nanoscale therapeutics may have unique properties (nanocharacter) that can be beneficial for drug delivery but there is no specific size or dimensional limit where superior properties reside. Hence, a size limitation below 100 nm cannot be touted as the basis of novel properties of nanotherapeutics. In fact, properties other than size can also have a dramatic effect on the nanocharacter of nanodrugs: shape/ geometry, zeta potential, specific nanomaterial class employed, composition, delivery route, crystallinity, aspect ratio, surface charge, etc. See: Bawa, R. (2016). What’s in

Preface by the Series Editor

Viable sui generis definition of nano having a bright-line size range as applied to nanodrugs blurs with respect to what is truly nanoscale; it is unnecessary, misleading, and in fact, may never be feasible. Since the 100 nm boundary has no solid scientific or legal basis, this series will avoid using the inaccurate and random sub-100 nm definition of nano. The volumes in this series will extensively cover advances in drug delivery as this is leading all developments in nanomedicine. The following points serve to highlight the major impact of nanoscale drug delivery systems:   •  Novel nanodrugs and nanocarriers are being developed that address fundamental problems of traditional drugs because of the ability of these compounds to overcome poor water solubility issues, alter unacceptable toxicity profiles, enhance bioavailability, and improve physical/chemical stability. Additionally, via tagging with targeting ligands, these nanodrug formulations can serve as innovative drug delivery systems for enhanced cellular uptake or localization of therapeutics into tissues of interest (i.e., site-specific targeted delivery).

a name? Defining “nano” in the context of drug delivery. In: Bawa, R., Audette, G. and Rubinstein, I. eds. Handbook of Clinical Nanomedicine: Nanoparticles, Imaging, Therapy, and Clinical Applications, Chapter 6, Pan Stanford Publishing, Singapore. Definitions along these lines will instead be employed in this book series: The design, characterization, production, and application of structures, devices, and systems by controlled manipulation of size and shape at the nanometer scale (atomic, molecular, and macromolecular scale) that produces structures, devices, and systems with at least one novel/superior characteristic or property.” See: Bawa, R. (2007). Patents and nanomedicine. Nanomedicine (London), 2(3), 351–374. Although numerous nanodrugs have been routinely used in medicinal products for decades without any focus or even awareness of their nanocharacter, it is only within the past two decades that they have been highlighted due to their potential of revolutionizing drug delivery. Obviously, the Holy Grail of any drug delivery system is to deliver the correct dose of a particular active agent to a specific disease or tissue site within the body while simultaneously minimizing toxic side effects and optimizing therapeutic benefit. This is often not achievable via conventional drugs. However, the potential to do so may be greater now via engineered nanodrugs. Often, nanodrugs transport active agents to their target binding sites (ligands, receptors, active sites, etc.) to impart maximum therapeutic activity with maximum safety (i.e., protect the body from adverse reactions) while preventing the degradation/denaturation/inactivation of the active agent during delivery/ transit. Targeting can be achieved or enhanced by (i) linking specific ligands or molecules (e.g., antibodies, glycoproteins, etc.) to the carrier, or (ii) by altering the surface characteristics of the carrier. Furthermore, therapeutics that have side effects due to triggering an immune response (e.g., complement activation) or cleared by the reticuloendothelial (RES) system can be entrapped, encapsulated or embedded within a nanoparticle coat or matrix.

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Various FDA-approved liposomal, solid nanoparticle-based, antibody-drug conjugate (ADC) and polymer-drug conjugate delivery platforms overcome associated issues such as (i) low solubility (Abraxane), (ii) extremely high drug toxicity (Brentuximab vedotin and Trastuzumab emtansine), and (iii) side effects related to high doses of free drug (Doxil, Marqibo, DaunoXome).   •  Reformulation of old, shelved therapeutics into nanosized dosage forms could offer the possibility of adding new life to old therapeutic compounds. Classic examples include drugs developed as nanocrystalline products (Rapamune, Emend, Triglide).   •  Coupled with advances in pharmacogenomics, smart nanomachines, information technology and personalized medicine, upcoming innovations in nanomedicine may even generate multifunctional entities enabling simultaneous diagnosis, delivery and monitoring of APIs. Emerging technologies are particularly problematic for governmental regulatory agencies, given their independent nature, slow response rate, significant inertia and a general mistrust of industry. Major global regulatory systems, bodies and regimes regarding nanomedicines are not fully mature, hampered in part by a lack of specific protocols for preclinical development and characterization. Additionally, in spite of numerous harmonization talks and meetings, there is a lack of consensus on the different procedures, assays and protocols to be employed during preclinical development and characterization of nanomedicines. On the other hand, there is a rise of diverse nanospecific regulatory arrangements and systems, contributing to a dense global regulatory landscape, full of gaps and devoid of central coordination. It is often observed that governmental regulatory bodies lack technical and scientific knowledge to support risk-based regulation, thereby leaving a significant regulatory void. In fact, the “baby steps” undertaken by the FDA over the past decade have led to regulatory uncertainty. There are potentially serious and inhibitory consequences if nanomedicine is overregulated. A balanced approach is required here, at least on a case-by-case basis, which addresses the needs of commercialization against mitigation of inadvertent harm to patients or the environment. Obviously, not everything “nanomedical” needs to be regulated. However, more is clearly needed from regulatory agencies like the FDA and EMA

Preface by the Series Editor

than a stream of guidance documents that are generally in draft format, position papers that lack any legal implication, presentations that fail to identify key regulatory issues and policy papers that are often short on specifics. There is a very real need for regulatory guidelines that follow a science-based approach (not policy-based) that are responsive to the associated shifts in knowledge and risks. Other critically important and interrelated regulatory law themes that will permeate this series include transnational regulatory harmonization, FDA law, generic biowaivers, nanosimilars, combination products (nanotheranostics) and non-biologic complex drugs (NBCDs). Let me highlight the translational issues and efforts pertaining to nanomedical products by using the classic example of drug R&D. Creating drugs today is time-consuming, expensive and enormously challenging. De novo drug discovery and development is a 10-17 year process from idea to marketed drug. It may take up to a decade for a drug candidate to enter clinical trials with less than 10% of the tested candidates in trials reaching the market. In fact, more drugs come off patent each year than approved by the FDA. According to a 2014 study by the Tufts Center for the Study of Drug Development, developing a new prescription medicine that gains marketing approval is estimated to cost nearly $2.6 billion. It is clear to everyone that in the past decade, great strides have been made in basic science and research. This is obviously critical to any advanced society. However, the enormous medical advances that should have come from the large public investment in biomedical research are largely absent from a translation pointof-view. All stakeholders—pharma, patients, academia, NIH— have suffered and are to blame for the valley of death. Each needs to re-examine their role and become an active, full partner in the drug development ecosystem so that translation is more robust. Similarly, although great strides have been made in nanomedicine generally at the “science” level, especially with respect to drug delivery and imaging, it continues to be dogged by challenges and bottlenecks at the “translational” level. In pharma, translational research involves the many elements that contribute to the successful conversion of an idea into a drug. Translation of drug products is a challenge faced by pharma at various levels. Bridging the chasm between drug discovery research activities and the successful translation of a drug to the market is a daunting task that requires varying degree of participation from key stakeholders—

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pharma, academia, nonprofit and for-profit institutions, federal agencies and regulatory bodies, diseases foundations and patients. So far, the process of converting basic research in nanomedicine into commercially viable products has been difficult. Securing valid, defensible patent protection from the patent offices along with clearer regulatory/safety guidelines from regulatory agencies is critical to commercialization. This has been a mixed bag at best and much more is warranted. If translation of nanomedicine is to be a stellar success, it is important that some order, central coordination and uniformity be introduced at the transnational level. It is true that this decade has witnessed relatively more advances and product development in nanomedicine than the previous. In this context, many point to the influence of nanomedicine on the pharmaceutical, device and biotechnology industries. One can now say that R&D is in full swing and novel nanomedical products are starting to arrive in the marketplace. It is hoped that nanomedicine will eventually blossom into a robust industry. We will have to see whether there will be giant technological leaps (that can leave giant scientific, ethical and regulatory gaps) or paradigm-shifting advances (that necessitate extraordinary proof and verification). In the meantime, tempered expectations are in order. Key players must come together on a global platform to address some of these crucial issues impacting effective translational efforts. It is important that the public’s desire for novel nanomedical products, venture community’s modest investment, federal infusion of funds and big pharma’s lingering interest in nanomedicine continues. In the end, the long-term prognosis and development of nanomedicine will hinge on effective nanogovernance, issuance of valid patents, clearer safety guidelines, public transparency and full commitment of all the key stakeholders involved—big pharma, academia, governmental regulatory agencies, policy-makers, the venture community and the consumer-patient. Together, these stakeholders can help drive progress and make the future happen faster. Science fiction may become science fact. Not only is this possible, but it will happen. The Times They Are a-Changin’.

Raj Bawa, MS, PhD Ashburn, Virginia, USA December 22, 2015

Acknowledgements Richard J. Apley, Litman Law Offices/Becker & Poliakoff, Washington, DC, USA Lajos P. Balogh, AA Nanomedicine & Nanotechnology Consulting, North Andover, MA, USA Yechezkel (Chezy) Barenholz, Hebrew University-Hadassah Medical School, Israel S. R. Bawa, Bawa Biotech LLC, Schenectady, New York, USA

Sangita Bawa, Novo Nordisk Inc., Princeton, New Jersey, USA

Nancy A. Blair-DeLeon, Institute of Electrical and Electronics Engineers, New York, New York, USA Sara Brenner, SUNY Polytechnic Institute, Albany, New York, USA Stanford Chong, Pan Stanford Publishing Pte. Ltd., Singapore

Jill B. Conner, Teva Pharmaceutical Industries, Ltd., Overland Park, Kansas, USA Jay K. Doshi, Family Dental Care, Spring, Texas, USA

Kamal C. Doshi, Shared Towers VA LLC, McLean, Virginia, USA Eileen S. Ewing, American Bar Association, Chicago, USA

Howard E. Gendelman, University of Nebraska Medical Center, Omaha, Nebraska, USA Harvinder Ghumman, Inova Health System, Reston, Virginia, USA

Susan P. Gilbert, Rensselaer Polytechnic Institute, Troy, New York, USA

Paulette Goldweber, John Wiley & Sons, Inc., Hoboken, New Jersey, USA Neil Gordon, Guanine Inc., Montreal, Quebec, Canada

Drew Harris, Nanotechnology Law & Business, Austin, Texas, USA Jim Hurd, NanoScience Exchange, San Francisco, California, USA

Meghana Kamath Hemphill, John Wiley & Sons, Inc., Hoboken, New Jersey, USA Arvind Kanswal, Pan Stanford Publishing Pte. Ltd., Singapore Francesca Lake, Future Medicine Ltd., London, UK

Gregory Lanza, Washington University Medical School, Saint Louis, Missouri, USA Gregory N. Mandel, Temple University Beasley School of Law, Philadelphia, Pennsylvania, USA

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Acknowledgements

Rajesh Mehra, Chantilly Family Practice Center, Chantilly, Virginia, USA

Lucinda Miller, Northern Virginia Community College, Annandale, Virginia, USA Shaker A. Mousa, Albany College of Pharmacy and Health Sciences, Albany, New York, USA Stefan Muhlenbach, Vifor Pharma, Ltd. and University of Basel, Switzerland

J. Michael Nicholas, Teva Pharmaceutical Industries, Ltd., Overland Park, Kansas, USA

Thurman K. Page, (formerly of) US Patent & Trademark Office, Alexandria, Virginia, USA

Michael Slaughter, CRC Press/Taylor & Francis Group, Boca Raton, Florida, USA Gert Storm, Utrecht University, Utrecht, Netherlands

Robin Taylor, University of Nebraska Medical Center, Omaha, Nebraska, USA Mary A. Vander Maten, Northern Virginia Community College, Annandale, Virginia, USA Thomas J. Webster, Northeastern University, Boston, Massachusetts, USA Vera Weinstein, Teva Pharmaceutical Industries, Ltd., Netanya, Israel Tracy Wold, Morton Publishing, Englewood, Colorado, USA

Section I General Introduction and Beginnings

Copyright © 2016 Raj Bawa. All Rights Reserved.

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Chapter 1

Science at the Nanoscale: Introduction and Historical Perspective Chin Wee Shong, PhD,a Sow Chorng Haur, PhD,b and Andrew T. S. Wee, PhDb aDepartment

bDepartment

of Chemistry, National University of Singapore, Singapore of Physics, National University of Singapore, Singapore

Keywords: nanoscale, carbon nanotubes, bionanotechnology, lithography, molecular electronics, miniaturisation, atomic force microscope, scanning probe microscope, scanning tunnelling microscopy, fullerenes, spintronics

1.1 The Development of Nanoscale Science

The prefix nano comes from the Greek word for dwarf, and hence nanoscience (the commonly used term nowadays for nanoscale science) deals with the study of atoms, molecules and nanoscale particles, in a world that is measured in nanometres (billionths of a metre or 10−9, see Section 1.2). The development of nanoscience can be traced to the time of the Greeks and Democritus in 5th cen­tury B.C., when people thought that matter could be broken down to an indestructible basic component of matter, which scientists now call atoms. Scientists have since discovered the Handbook of Clinical Nanomedicine: Nanoparticles, Imaging, Therapy, and Clinical Applications Edited by Raj Bawa, Gerald F. Audette, and Israel Rubinstein Copyright © 2016 Pan Stanford Publishing Pte. Ltd. ISBN 978-981-4669-20-7 (Hardcover), 978-981-4669-21-4 (eBook) www.panstanford.com

4

Science at the Nanoscale

whole peri­ odic table of different atoms (elements) along with their many isotopes. The 20th century A.D. saw the birth of nuclear and particle physics that brought the discoveries of sub-atomic par­ticles, entities that are even smaller than atoms, including quarks, leptons, etc. But these are well below the nanometre length scale and therefore not included in the history of nanoscale science and technology. The beginnings and developments of nanotechnology, the application of nanoscience, are unclear. The first nanotechnologists may have been medieval glass workers using medieval forges, although the glaziers naturally did not understand why what they did to gold made so many different colours. The process of nanofabrication, specifically in the production of gold nanodots, was used by Victorian and medieval churches which are famed for their beautiful stained glass windows. The same process is used for various glazes found on ancient, antique glazes. The colour in these antiques depends on their nanoscale characteristics that are quite unlike microscale characteristics. The modern origins of nanotechnology are commonly attri­buted to Nobelist Dr. Richard Feynman, who on December 29, 1959, at the annual meeting of the American Physical Society at Caltech, delivered his now classic talk “There’s Plenty of Room at the Bottom” [1]. He described the possibility of putting a tiny “mechanical surgeon” inside the blood vessel that could locate and do corrective localized surgery. He also highlighted a number of interesting problems that arise due to miniaturisation since “all things do not simply scale down in proportion”. Nanoscale mate­rials stick together by molecular van der Waals attractions. Atoms also do not behave like classical objects, for they satisfy the laws of quantum mechanics. He said, “... as we go down and fiddle around with the atoms down there, we are working with different laws, and we can expect to do different things.” Feynman said he was inspired by biological phenomena in which “chemical forces are used in repetitious fashion to produce all kinds of weird effects (one of which is the author)”. He predicted that the principles of physics should allow the possibility of manoeuvring things atom by atom. Feynman described such atomic scale fabrication as a bottom-up approach, as opposed to the top-down approach that is

The Development of Nanoscale Science

commonly used in manufacturing, for example in silicon integrated circuit (IC) fabrication whereby tiny transistors are built up and con­nected in complex circuits starting from a bare silicon wafer. Such top-down methods in wafer fabrication involve processes such as thin film deposition, lithography (patterning by light using masks), etching, and so on. Using such methods, we have been able to fabricate a remarkable variety of electronics devices and machinery. However, even though we can fabricate feature sizes below 100 nanometres using this approach, the ultimate sizes at which we can make these devices are severely limited by the physical laws governing these techniques, such as the wavelength of light and etch reaction chemistry.

Figure 1.1

Moore’s law predicts rapid miniaturization of ICs. Re­printed with permission from Intel Corporation © Copyright Intel Cor­poration.

Figure 1.1 shows that the trend in miniaturisation of ICs will ultimately be limited by quantum mechanics, certainly at scales larger than atoms and molecules. Gordon Moore, co-founder of Intel, made the observation in 1965 (now known as “Moore’s law”) that the number of transistors per square inch on integrated circuits had doubled every year since the integrated circuit was invented. Whilst this trend in IC miniaturisation has more or less been obeyed until now, the current CMOS technology will hit a

5

6

Science at the Nanoscale

“wall” soon as quantum and ballistic electron effects become dom­ inant. The most optimistic proponents of ICs believe that major innovations will be required to reach the ultimate operating limit of the silicon transistor: a length for functional features around 10 nm, or about 30 atoms long. Bottom-up manufacturing, on the other hand, could provide components made of single molecules, which are held together by covalent forces that are far stronger than the forces that hold together macro-scale components. Furthermore, the amount of information that could be stored in devices built from the bottom-up would be enormous.

Figure 1.2

STM image of the Si(111)-7×7 reconstructed surface show­ing atomic scale resolution of the top-most layer of silicon atoms.

Since Feynman’s early visionary ideas on nanotechnology, there was little progress until in 1981 when a new type of microscope, the scanning tunnelling microscope (STM), was invented by a group at IBM Zurich Research Laboratory [2]. The STM uses a sharp tip that moves so close to a conductive surface that the electron wavefunctions of the atoms in the tip overlap with the surface atom wavefunctions. When a voltage is applied, electrons “tun­nel” through the vacuum gap from the foremost atom of the tip into the surface (or vice versa). In 1983, the group published the first STM image of the Si(111)-7×7 reconstructed surface, which nowadays can be routinely imaged as shown in Fig. 1.2 [3]. In 1986, Gerd Binnig and Heinrich Rohrer shared the Nobel Prize in Physics “for their design of the scanning tunneling micro­scope”.

The Development of Nanoscale Science

This invention led to the development of the atomic force microscope (AFM) and a whole range of related scanning probe microscopes (SPM), which are the instruments of choice for nano­technology researchers today. At around the same time in 1985, Dr. Robert Curl, Dr. Harold Kroto and Dr. Richard Smalley made the completely unexpected discov­ery that carbon can also exist in the form of very stable spheres, which they named fullerenes (or buckyballs) [4]. The carbon balls with chemical formulae C60 or C70 are formed when graphite is evaporated in an inert atmosphere (Fig. 1.3). A new carbon chemistry has developed from this discovery, and it is now possible to enclose metal atoms in them, and to create new organic compounds. Not long after in 1991, Iijima et al. reported Transmission Elec­tron Microscopy (TEM) observations of hollow graphitic tubes or carbon nanotubes (Fig. 1.3), which form another member of the fullerene structural family [5]. The strength and flexibility of carbon nano­tubes makes them potentially useful in many nanotechnology applications. Carbon nanotubes are now used as composite fibres in polymers and concrete to improve the mechanical, thermal and electrical properties of the bulk product. They also have potential applications as field emitters, energy storage materials, molecu­lar electronics components, and so on. Some important events in the historical development of nanoscience and nanotechnology are summarised in Table 1.1.

Figure 1.3 Schematic of a C60 buckyball (left) and carbon nanotube (right).

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Table 1.1

Some important events in the historical development of nanoscience and nanotechnology

5th Century Democritus and Leucippus, determined that matter was B.C. made up of tiny, indivisible particles in constant motion. 1803 1897

1911

1914

1959 1974 1977 1981 1985 1986 1989 1990 1991 1993 1997 2000

English chemist and physicist, John Dalton (1766–1844), developed the first useful atomic theory of matter. Cambridge physicist J. J. Thomson (1856–1940), proposed that the mysterious cathode rays were streams of particles (later became known as electrons) much smaller than atoms.

Thomson’s student, Ernest Rutherford, determined there was a centre of the atom, now known as the nucleus, and electrons revolved around the nucleus. Swedish physicist Niels Bohr, advanced atomic theory further in discovering that electrons travelled around the nucleus in fixed energy levels.

Feynman gives after-dinner talk describing molecular machines building with atomic precision. Taniguchi uses term “nano-technology” in paper on ionsputter machining. Drexler originates molecular nanotechnology concepts at MIT.

Scanning tunnelling microscopy (STM) invented by Gerd Binnig and Heinrich Rohrer at IBM Zurich. Buckyball discovered by Robert Curl, Harold Kroto and Richard Smalley.

Atomic Force Microscopy (AFM) invented by Binnig, Quate and Gerber. IBM logo spelled in individual atoms by Don Eigler at IBM Almaden.

Nanotechnology: First nanotechnology journal by Institute of Physics UK. Carbon nanotube discovered by Iijima at NEC, Japan. First Feynman Prize in Nanotechnology awarded. First nanotechnology company founded: Zyvex.

President Clinton announces US National Nanotechnology Initiative.

The Nanoscale

In summary, the key events in the short history of modern nanotechnology may be described as follows: The vision of nanotechnology was first popularised by Feynman in 1959, when he outlined the prospects for atomic-scale engineering. In 1981, Binnig and Rohrer invented the scanning tunnelling microscopy, which enabled scientists to “see” and manipulate atoms for the first time. Corresponding advancements in supramolecular chemistry, particularly the discovery of the buckminsterfullerenes (or buckyballs) by Curl, Kroto and Smalley gave scientists a whole class of nanoscale building blocks with which to construct a whole range of nanostructures.

1.2 The Nanoscale

To start off our discussion on the nanoscale, we first refer to the metric system. The following table gives a summary of the metric system.

Figure 1.4

Schematic showing systematic cutting down of the cross section of a human hair.

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Sometimes it is difficult to appreciate the smallness of the nanoscale. It is thus useful to relate the size scale to items that we commonly find in our home. For example, imagine you take a single strand of human hair. The cross section of a human hair is circular in shape (let us assume to be 100 μm in diameter), and imagine you have a very sharp knife. Use the knife to slice the cross section of the human hair into 100 slices with equal width. After which take out one of the 100 slices and use yet another sharp knife to cut the cross section of that single slice into 1000 slices, again with uniform width. If one takes out one of the 1000 slices, the width of the single strip is equal to 1 nanometre!!! The above hypothetical process is illustrated in Fig. 1.4. This is an extremely small size scale and yet there are a lot of fascinating phe­nomena for us to discover. Table 1.2 shows the metric system units, symbols and prefixes relevant to the nanoscale, as well as representative objects at each size scale. Figure 1.5 shows images representing different size scales from one nanometre to one metre.

Figure 1.5

Scale of things.

The Nanoscale

Table 1.2

Metric system units, symbols and prefix

Yotta- Ym

1024 1,000,000,000,000,000,000,000,000

Zetta- Zm

1021 1,000,000,000,000,000,000,000

Exa-

Em

1018 1,000,000,000,000,000,000

Peta-

Pm

1015 1,000,000,000,000,000

Tera-

Tm

1012 1,000,000,000,000

Giga-

Gm 109

Mega- Mm 106 103

kilo­

km

deka­

dam 101

deci­

dm

milli­

mm 10–3

hecto­ hm

102

metre m

100

10–1

1,000,000,000

1,000,000

Distance to our nearest star is about 4.3 light years ∼40 Pm.

The size of our solar system is about 12 × 1012 m. Diameter of Sun is about 1.4× 109 m.

The total length of The Great Wall of China is about 5 × 106 m.

10

Length of a type of dinosaur (Apatosorus) ∼20 m.

100

1

Height of the Great Pyramid of Giza is about 140 m. Height of a 7-year-old child.

1/10

Size of our palm.

1/1,000

Thickness of ordinary paperclip.

1/1,000,000,000

The diameter of a C60 molecule is about 1 nm.

micro­ µm

10–6

pico­

10–12 1/1,000,000,000,000

pm

The distance from the Sun to the galactic centre is now estimated at 26,000 light-years ∼ 246 Em.

Size of Singapore is about 42 km × 23 km.

10–2

nano­ nm

Mean diameter of the Andromeda Galaxy is 200,000 light years. That is about 2 × 2 Zm.

1,000

centi­ cm

10–9

The Great Wall, a network of galaxies has a length of 4.7 Ym and a width of 1.8 Ym.

1/100

1/1,000,000

Length of a bee.

Size of typical dust particles. Atomic radius of a Hydrogen Atom is about 23 pm.

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Table 1.2 femto­ fm

atto­

am

zepto zm

yocto ym

(Continued)

10–15 1/1,000,000,000,000,000

Size of a typical nucleus of an atom is 10 femtometres.

10–21 1/1,000,000,000,000,000,000,000

Really small.

10–18 1/1,000,000,000,000,000,000

Estimated size of an electron.

10–24 1/1,000,000,000,000,000,000,000,000 Really really small.

If one poses a question, how small is a nanometre? Here are some interesting answers: (1) the diameter of the C60 buckyball molecule (2) half as wide as a DNA molecule (3) 2 times the diameter of a rubidium atom (4) 10 times the diameter of a hydrogen atom (5) The de Broglie wavelength of an electron with an energy of 1.5 eV (6) how much your fingernails grow each second (7) how much the Himalayas rise in every 6.3 seconds (8) the thickness of a drop of water spread over a square metre

1.3 Examples of Interesting Nanoscience Applications

(a) Bionanotechnology One of the most exciting areas of applica­tions of nanotechnology must be in the field of biomedical health­care and disease treatment. The story of tiny “nanobots” acting as miniaturised doctors entering our body to repair damaged cells and to kill foreign bacteria alike is certainly not unheard of to many people. While this remains science fiction in many aspects till today, nobody can say for sure that it will never come to pass in the future. A more promising application of bionanotechnology that has attracted much interest from researchers and industries is the development of nano-drug delivery systems. In our modern busy lifestyle, administration of drugs has progressed from the teaspoon to time-release capsules or implants. Nanotechnology promises delivery mechanisms that can administer drugs at desired rates and at the exact location in the body. This requires the fabrication of precise nanostructures for drug-eluting coatings, membranes,

Examples of Interesting Nanoscience Applications

or even implants. For example, researchers at the University of California, San Francisco have demonstrated how they can use nanotubes made from biocompatible metal oxides to hold therapeutic drugs and deliver these agents in a highly-controlled manner [6]. On the other hand, the dendrimer, a highly branched polymer, has also been investigated by many as a nat­ural form of nanoparticle carrying myriad sites for drug loading. All these developments not only translate to time-saving and bet­ ter treatments, they also help avoid side effects caused by large doses taken orally or by injection. There are also the potential benefits of extension of the bioavailability and economic life-span of proprietary drugs. In 2005, the industry consulting firm NanoMarkets predicted that nanotechnology-enabled drug delivery sys­tems would to generate over US $1.7 billion in 2009 and over $4.8 billion in 2012.

Figure 1.6

The branched capillary structure, feeding adipose tissue in a living mouse, is revealed with multiphoton fluorescence microscopy as nanocrystal quantum dots circulate through the bloodstream. From Lar­son D. T. et al., Science 300, 1434–1436 (2003). Reprinted with permission from AAAS.

Another development in nanoscience that has excited many biomedical researchers is the use of quantum dots (abbreviated QDs) in bio-imaging. These are tiny crystals that give strong fluorescence signals and, when injected into cells,

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allow unprecedented details inside the cells to be imaged. A nice 3D imaging example was demonstrated by Cornell researchers (Fig. 1.6) whereby tiny blood vessels beneath a mouse’s skin were viewed with CdSe/ZnS QDs circulating through the bloodstream. The images appear so bright and vivid in high-resolution that researchers can see the vessel walls ripple at 640 times per minute. (b) Spintronics For many years, scientists and engineers have created a host of electrical devices that rely on electrons in the materials. Such devices include the ubiquitous transistor and the powerful microprocessor. These devices exploit the charge carried by the electrons for their normal function, and they communicate with each other through the flow of electric charges. However, there is another important intrinsic property of electrons that has been neglected in these devices—the spin of the electron. Spin is a purely quantum mechanical property. We normally think of the spin of an electron using the analogy of a spinning top. The spin can be clockwise or counterclockwise in direction. In the case of electrons, the spin could be pointing in the “up” direction or in the “down” direction. The spin in the electron is easily influenced by an externally applied magnetic field. Spin electronics, or spin­tronics, refers to electrical devices that utilise the spin properties of the electrons in addition to their electrical charge in creating useful devices. Scientists and engineers hope to control the spin of electrons within a spintronics device to produce useful devices. As the spintronics device can be influenced by the presence of an electric field, magnetic field or light, the device represents a single device that integrates the multiple functionalities with optoelec­tronics and magnetoelectronics. There are a number of spintronics devices that have been realised. The most widely used spintronics device is the Giant Magnetoresistive (GMR) device commonly used in magnetic harddisk drives. Typically, a simple GMR device consists of two layers of ferromagnetic materials separated by a very thin spacer layer which is nonmagnetic. A simple illustration of such a spin valve device is shown in Fig. 1.7. One of the layers is referred to as the “pinned” layer where its magnetisation direction remains in a fixed direction. The other ferromagnetic layer is known as the “free” layer where its magnetisation direction depends on the externally applied magnetic field. When the two magnetisation

Examples of Interesting Nanoscience Applications

vectors of the ferromagnetic layers are oriented in the same direc­tion, an electrical current will flow freely. On the other hand, if the magnetisation vectors are oriented in the opposite direction, there is a high resistance to the flow of electrons due to spin dependent scattering. The magnitude of the change in the resistance at these two different states is called the Giant Magnetoresistance Ratio. Hence this GMR device is highly sensitive to the external mag­netic field which is capable of switching the relative magnetic ori­ entation of the ferromagnetic layers. Thus it is widely used as the read head for magnetic hard disk drives.

Figure 1.7

Schematic diagram of a simple spin valve.

There are many other spintronics devices that scientists and engineers are working on. These include the spin-based transistor, spin-polarizer, spintronics solar cell, magnetic tunnel junction, and spin-based quantum computer where the spin of a single electron trapped in a quantum dot is used as a qubit. (c) Molecular electronics The emerging field of molecular elec­tronics is now becoming a popular alternative paradigm to current silicon microelectronics. In 1974, Drs. Ari Aviram and Mark Ratner, then at New York University, published a paper in Chemical Physics Letters proposing that individual molecules might exhibit the behaviour of basic electronic devices [7]. Their hypothesis, for­ mulated long before anyone was able to test it, was so radical that it was not pursued for another 15 years. The story continued in

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Science at the Nanoscale

December 1991, when Drs. James Tour and Mark Reed discovered they had a common interest at a small gathering of “moletronics” researchers in the Virgin Islands. The meeting was hosted by Ari Aviram, who was then working at IBM’s Thomas J. Watson Research Center in New York. They started collaborating, but it was not until 1997 when they successfully used the so-called “break-junction” technique to measure the conductance of a sin­gle molecule [8]. In their work, benzene-1,4-dithiol molecules were self-assembled onto two facing gold electrodes of a mechani­ cally controllable break junction to form a stable gold-sulphur-aryl-sulphur-gold system (Fig. 1.8). This allowed the direct observation of charge transport through the molecules for the first time. Their study provided a quantitative measure of the conductance of a junction containing a single molecule, which is a fundamental step towards the realization of the new field of molecular electronics.

Figure 1.8

Schematic of the gold-sulphur-aryl-sulphur-gold system. From Reed et al., Science, 278, 252 (1997). Reprinted with permission from AAAS.

Examples of Interesting Nanoscience Applications

Many papers have since followed demonstrating conductance measurements on single molecules and simple single molecule devices. A useful review of the early days of the field has been written by Drs. Carroll and Gorman [9]. Nanogaps were formed using electromigration whereby a high electric field causes gold atoms to move along the current direction, eventually causing a nanogap. A research team at Hewlett-Packard (HP) Laboratories has proposed the crossbar architecture as the most likely path forward for molecular electronics [10]. A crossbar consists of one set of parallel nanowires less than 100 atoms wide that cross over a second set (Fig. 1.9). A molecule or material that can be stimulated electrically to conduct either more electricity or less is sandwiched between the two sets of wires. The result­ing interwire junctions form a switch at each intersection between crossing wires that can hold its “on” or “off” status over time. Such switches may be able to scale down to nearly single-atom dimensions, and this approach suggests how far the future minia­turisation of ICs might someday go.

Figure 1.9

Left: Schematic showing how the switch is formed at the junction between two crossing nanowires that are separated by a single monolayer of molecules. Right: Picture of fan-out wires that connect the nanoscale circuits to the microscale. Reprinted with permission from P. J. Kuekes, G. S. Snider, R. S. Williams, Crossbar nanocomputers. Sci. Am., 72–80 (2005). Copyright © 2005 by Scientific American, Inc. All rights reserved.

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Disclosures and Conflict of Interest This chapter is a revised version of the author’s chapter that originally appeared in 2010 in Science at the Nanoscale: An Introductory Textbook, Pan Stanford Publishing Pte Ltd., Singapore. The authors declare that no writing assistance was utilized in the production of this manuscript and the authors have received no payment for its preparation. The findings and conclusions here reflect the current views of the authors.

Corresponding Author

Dr. Andrew T. S. Wee Department of Physics, National University of Singapore 2 Science Drive 3, Singapore 117542 Email: [email protected]

About the Authors

Chin Wee Shong is associate professor in the Department of Chemistry at the National University of Singapore. Her research explores the fundamental properties of materials with physical dimension in between individual molecules and the bulk. Currently, her research focuses on the synthesis and characterization of several types of nanomaterials including: (i) semiconducting sulfide and oxide nanocrystals (ii) core/shell and doped functional nanomaterials, (iii) hybridized nanoparticles/polymer composites, (iv) nanomagnets, and (v) surface-derivatized metallic nanoparticles. Dr. Chin and her group aim to optimize the properties of these nanomaterials via chemical preparation and improve their desired functions with various characteristics. Most of their projects involve multidisciplinary collaborative efforts. Sow Chorng Haur is professor in the Department of Physics and group leader of the Nanomaterials Research Lab at the National University of Singapore. His research focuses on the study of a wide variety of nanostructured materials, aiming to understand the physical properties

References

of these nanomaterials and how these properties are influenced by the morphology, stoichiometry and structures and these nanomaterials. His current research focuses on the understanding of the structural assembly and growth mechanism of the nanostructures, in order to achieve greater control in the material composition, physical structure, physical properties and functionality. He is also interested in the synthesis of new classes of nanomaterials and exploring different methods to incorporate two or more different nanostructures together with controlled architecture.

Andrew T. S. Wee is professor in the Department of Physics and vice president (University and Global Relations) at the National University of Singapore. His research interests are in the field of surface and interface science and include scanning tunneling microscopy (STM) and synchrotron radiation studies of the molecule–substrate interface, organic–organic heterojunctions, and graphene and related nanomaterials.

References

1. Feynman, R. P. (1960). There’s plenty of room at the bottom. Eng. Sci., 23(5), 22–36.

2. Binnig, G., Rohrer, H., Gerber, C., Weibel, E. (1982). Tunneling through a controllable vacuum gap. App. Phys. Lett., 40, 178–180.

3. Binnig, G., Rohrer, H., Gerber, C., Weibel, E. (1983). 7 × 7 reconstruction on Si(111) resolved in real space. Phys. Rev. Lett., 50, 120–123.

4. Kroto, H. W., Heath, J. R., O’Brien, S. C., Curl, R. E., Smalley, R. E. (1985). C60: Buckminsterfullerene. Nature, 318, 162–163.

5. Iijima, S. (1991). Helical microtubules of graphitic carbon. Nature, 354, 56–58.

6. Lee, C. C., Gillies, E. R., Fox, M. E., Guillaudeu, S. J., Fréchet, J. M., Dy, E. E., Szoka, F. C. (2006). A single dose of doxorubicin-functionalized bow-tie dendrimer cures mice bearing C-26 colon carcinomas. Proc. Natl. Acad. Sci. U. S. A., 103(9450), 16649–16654. 7. Aviram, A., Ratner, M. A. (1974). Molecular rectifiers. Chem. Phys. Lett., 29(2), 277–283.

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8. Reed, M. A., Zhou, C., Muller, C. J., Burgin, T. P., Tour, J. M. (1997). Conductance of a molecular junction. Science, 278, 252–254. 9. Carroll, R. L., Gorman, C. B. (2002). The genesis of molecular electronics. Angew. Chem. Int. Ed., 41, 4378–4400. 10. Kuekes, P. J., Snider, G. S., Williams, R. S. (2005). Crossbar nanocomputers. Sci. Am., 293, 72–80.

Chapter 2

Nanomedicine: Dynamic Integration of Nanotechnology with Biomedical Science Ki-Bum Lee, PhD,a Aniruddh Solanki, PhD,b John Dongun Kim, PhD,c and Jongjin Jung, PhDd aDepartment of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, New Jersey, USA bBrigham and Women’s Hospital, Harvard Medical School, Cambridge, Massachusetts, USA cDepartment of Biological Sciences, Korea Advanced Institute of Science and Technology, Korea dHannam University, Korea

Keywords: nanopatterning, nanomaterial design, nanocarriers, drug delivery, nanoarrays, microfluidics, biomolecules, molecular imaging

2.1 Introduction

The recent emergence of nanotechnology is setting high expectations in biological science and medicine, and many scientists now pre­dict that nanotechnology will solve many key questions of biologi­cal systems that transpire at the nanoscale. Nanomedicine, broadly defined as the approach of science and engineering at the nanome­ter scale toward biomedical applications, has been drawing con­siderable attention in the area of nanotechnology. Given that the sizes of functional elements in biology are at the nanometer Handbook of Clinical Nanomedicine: Nanoparticles, Imaging, Therapy, and Clinical Applications Edited by Raj Bawa, Gerald F. Audette, and Israel Rubinstein Copyright © 2016 Pan Stanford Publishing Pte. Ltd. ISBN 978-981-4669-20-7 (Hardcover), 978-981-4669-21-4 (eBook) www.panstanford.com

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scale range, it is not surprising for nanomaterials to interact with biolog­ical systems at the molecular level. In addition, nanomaterials have novel electronic, optical, magnetic, and structural properties that cannot be obtained from either individual molecules or bulk materi­als. These unique features can be precisely tuned in order for scien­tists to explore biological phenomena in many ways. For instance, extensive studies have been done with chip-based or solution-based bio-assays, drug delivery, molecular imaging, disease diagnosis, and pharmaceutical screening [1–4]. In order to realize these applications, it is crucial to develop methods that investigate and control the binding properties of individual biomolecules at the fundamental nanometer level. This will require enormous time, effort, and inter­ disciplinary expertise of physical sciences associated with both biol­ogy and engineering. The overall goal of nanomedicine is to develop safer and more effective therapeutics as well as novel diagnostic tools. To date, nanotechnology has revolutionized biomedical sci­ence step by step not only by improving efficiency and accuracy of current diagnostic techniques, but also by extending scopes for the better understanding of diseases at the molecular level [5–8]. In this chapter, nanomaterials and their applications in biomedical research will be discussed.

2.2 Designing Nanomaterials for Biology and Medicine

One of the important technological aspects in nanomedicine lies in the ability to tune materials in a way that their spatial and temporal scales are compatible with biomolecules. That said, materials and devices fabricated at the nanometer scale can investigate and con­trol the interactions between biomolecules and their counterparts at almost the single molecule level. This, in turn, indicates that nano­materials and nanodevices can be fabricated to show high sensitivity, selectivity, and control properties, which usually cannot be achieved in bulk materials. The wide range of the scale of biointeractions is described in Fig. 2.1. Given that one of the major goals of biology is to address the spatial-temporal interactions of biomolecules at the cellular or integrated systems level, the integration of nanotechnology

Designing Nanomaterials for Biology and Medicine

in biomedicine would bring a breakthrough in current biomedical research efforts. In order to apply nanotechnology to biology and medicine, several conditions must be considered: (i) nanomaterials should be designed to interact with proteins and cells without per­turbing their biological activities, (ii) nanomaterials should maintain their physical properties after the surface conjugation chemistry, and (iii) nanomaterials should be biocompatible and non-toxic.

Figure 2.1

Scale of biomolecular interactions.

In general, there are two approaches to build nanostructures or nanomaterials: “top-down” and “bottom-up” methods. Typ­ically, the bottom-up approach utilizes self-assembly of one or more defined molecular building blocks to create higher-ordered functional entities. For the bottom-up approach, the physical and chemical criterion, such as pH, concentration, temperature, and intrinsic properties of building blocks, must be fulfilled. On the other hand, the top-down approach usually involves processes such as lithography, etching, and lift-off techniques to fabricate micro- and nanoscopic structured materials from bulk materials. In many cases, nanomedicine strategies have been derived from what was originally a conventional biomedical application, with a cer­tain degree of modification to address some scientific questions or technical limitations. As far as the applications of nanostructures

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are concerned, we will examine two examples, nanoparticles and nanoarrays/biochips, which are heavily used in biomedical applications.

2.2.1 Inorganic Nanoparticles

Deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptides, and proteins are nanometer scale components that are the best exam­ples of nanomaterials found in nature [9, 10]. For example, DNA has a double-stranded helical structure with a diameter of 2 nm, RNA has a single strand structure with a diameter of 1 nm, and most of protein sizes are less than 15 nm. Likewise, the sizes of functional elements in biology are at the nanoscale level, which inevitably gen­erate significant interests at the intersection between nanotechnol­ogy and biological science. Even though much progress in the life sciences has been achieved over the last few decades, biological and physiological phenomena still remain beyond our understand­ing, because the interactions between elementary biomolecules and other higher components, such as viruses, bacteria, and cells, are complex and delicate. Moreover, the interactions of two biocompo­nents start from the single molecule level, where the recognition sites lie in a nanoscale domain. Thus, studies of these biological compo­nents require not only an ability to handle the biological properties, but also to develop highly advanced tools or techniques to analyze the biological systems [11–14]. Bioconjugated nanomaterials have recently been used as cellular labeling agents to study the biological phenomena at the nanome­ter level. With significant advancements in synthetic and modifica­tion methodologies, nanomaterials can be tuned to desired sizes, shapes, compositions, and properties [15]. Inorganic nanoparticles are one of the most promising examples, since they can be synthesized easily in large quantities from various materials using relatively simple methods. Also, the dimensions of the nanoparticles can be tuned from one to a few hundred nanometers with monodispersed size distribution. Moreover, they can be made up of different met­als, metal oxides, and semiconducting materials, whose composi­tions and sizes are listed in Table 2.1. Given many distinct properties, nanoparticles can be readily tailored

Designing Nanomaterials for Biology and Medicine

with biomolecules via com­bined methodologies from bioorganic, bioinorganic, and surface chemistry. Table 2.1

Selection of available nanoparticle compo­sitions, sizes, and shapes

Particle composition

Particle size (nm)

Metals Au Ag Pt Cu

Semiconductors CdX (X = S, Se, Te) ZnX (X = S, Se, Te) TiO2 PbS ZnO GaAs, InP Ge

Magnetic Fe3O4

Various polymer compositions

2–250 1–80 1–20 1–50

1–20 1–20 2–18 3–50 1–30 1–15 6–30

6–40

20 nm to 500 µm

Despite many significant advances in synthetic and surface mod­ification methods, the fundamental development of bioconjugation methods must first be achieved in order for the nanoparticles to be fully utilized. The bioconjugation strategies involve procedures for coupling biomolecules to nanomaterials, enabling the nanopar­ticles not only to be applied for clinical applications but also to ask and answer fundamental questions in cell biology. For the past few years, many methods have been developed for bio-labeled nanocomposites in various applications in cell biology: cell labeling [19–22], cell tracking, and in vivo imaging [23, 24].

2.2.2 Coupling of Nanoparticles with Biomolecules

Interdisciplinary knowledge from molecular biology, bioorganic chemistry, bioinorganic chemistry, and surface chemistry must

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be employed to functionalize nanostructures with biomolecules. Although nanostructures can be synthesized from various mate­ rials using several methods, the coupling and functionaliza­ tion of nanostructures with biomolecules should be carried out in controlled manners such as a specific salt concentration or pH [9]. Three common methods of functionalizing nanoparticles with biomolecules are: (i) direct interaction between nanoparti­cles and biomolecules via electrostatic interactions or physical adsorptions, (ii) typical conjugation chemistry using organic linker molecules, and (iii) streptavidin-biotin affinity between functional­ized nanoparticles. Typically, solution phase synthesis of nanostructures is carried out in the presence of surfactants such as citrate, phosphates, and alkanethiols. The surfactants not only interact with the atoms of nanostructures by either chemisorption or physisorption at the surface of nanostructures, but also stabilize nanostructures and prevent interparticle aggregation. Using the exchange reactions, sur­factant molecules attached on the nanoparticles can be replaced by biomolecules, making direct biomolecule-nanoparticle covalent bonds. For example, gold nanoparticles can be modified with pro­teins consisting of cysteine residues or with thiol functionalized DNA molecules. There are different types of coupling methods, where the electrostatic forces between proteins and citrate stabilized nanopar­ticles are used for the coupling. For the nanoparticles that are rela­tively unstable, the core-shell strategies can be applied to stabilize the nanoparticles [25, 26]. For example, silver core-shell nanoparticles coated by thin layer of gold can be successfully functionalized with thiol-functionalized DNA [25]. Many semiconductor nanoparticles can also be linked with proteins or DNA by adding a hydrophobic silica shell [16, 27, 28]. Silica surfaces can be tailored with biomolecules, utiliz­ing well known cross-linking methodologies, such as silanization chemisty and self-assembly monolayer (SAM) chemistry [29–32].

2.2.3 Fabrication of Nanoarrays and Biochips

The search for novel ways to explore and understand biomolecu­ lar interactions has been sought in many ways, since interactions between biomolecules are fundamentally intriguing. For example,

Designing Nanomaterials for Biology and Medicine

how proteins such as fibrinogen, fibronectin, and retronectin influ­ence the adhesion of cells and control their morphology and phys­iology has been a central question of cell biology [33, 34]. Several approaches have been examined over the past few years to compre­hend these phenomena, and one of these approaches is the assem­bly of interfacial proteins constructed on micro- or nanoscale [35]. The biomolecular patterned surfaces are not only useful for prob­ing biochemical interactions within whole cells but also crucial for biosensing. However, the realization of these applications is chal­lenging, since the control of the interactions between proteins and surfaces with respect to a binding direction and biomolecular den­sity is technically and biologically not easy to achieve [36]. There­fore, patterning techniques capable of high resolution— molecular to submicron scale—and compatible with biomolecules will be required. Typically, current chip-based biodetection strategies pat­tern molecules with an analyte-capturing ability on the chip surfaces in the micro-/nano scale. This application of chip-based biosensing will allow scientists to detect various analytes at concentrations as low as picomolar in massively parallel ways. The aforementioned features are invaluable for the advancement in genomics and pro­teomics via generating DNA and protein arrays [15]. Yet, the key chal­lenge lies in the fabrication of miniaturized surface structures in the form of nanoarrays that would allow for multi-magnitude orders of complex detection in the same chip and for improved detection sensitivity. There are many techniques available for patterning sur­faces in terms of resolution and compatibility with soft materials (Table 2.2). Among different techniques, one primary distinction is whether the method uses a resist-based process or deposition of materials onto a surface directly. Although the indirect, resist-based patterning methods, such as photolithography, are multi-step pro­cesses that require specialized resists and etching protocols, they are currently by far the most widely used methods in industrial applications. By contrast, direct-printing methods are typically useful for pat­terning soft materials such as small organic or biological molecules with ease. Microcontact printing, developed by Whitesides and coworkers [37–44], is a good example of a directprinting method using elastomer stamps which can be “inked” with molecules and then used to transfer the inking molecules in a form

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Nanomedicine

of desired patterns to various substrates. This technique has been used to generate large area patterns of soft materials on surfaces with pattern resolu­tions approaching 100 nm. However, the technique is limited in its capability to generate multiple, chemically diverse, high-resolution patterns in alignment on a surface. Therefore, in terms of resolu­tion to sub-100 nm, there is a high demand to develop methods of high-resolution lithographic techniques. Since the invention of scan­ning probe microscopes (SPMs), many scientists have realized that it might be possible to manipulate matter, atom-by-atom or molecule-­by-molecule [45, 46]. The early attempts to develop patterning method­ ologies from SPMs were able to demonstrate the high-resolution capabilities of these instruments. Table 2.2

Technique

Features of selected lithography techniques Resolution limit Mode

Comments

Photolithography Currently ~100nm

Parallel only

Resist based, indirect

Indirect SPM methods

Serial only

Resist based, indirect

Electron beam lithography

5–10 nm

Microcontact printing

~100 nm

Ink-jet printing

AFM 5–10 nm STM atomic 6 µm

Dip-pen 5–10 nm nanolithography

Serial only

Parallel only Serial

Resist based, indirect

Direct write or indirect Direct write

Parallel or serial Direct write

The majority of SPM surface patterning methods have focused on either impressive but inherently slow scanning tunneling micro­scope (STM) based methods for moving individual atoms around on a surface in ultra high vacuum (UHV), or on indirect meth­ods using atomic force microscope (AFM) and STM for stepwise etching of organic monolayers on a surface and backfilling with the molecule of interest [37, 47–50]. However, resist-based approaches are inherently restricted to serial processes, and can only be used for a few molecule-substrate combinations. In 1999, Mirkin and his coworkers developed dip-pen nanolithography

Application of Nanomaterials in Biomedical Research

(DPN). DPN uses an AFM tip to transfer “ink” molecules onto a substrate through a water meniscus (Fig. 2.2) [3, 51, 52]. DPN is simple, and its resolution is comparable to electron-beam lithography.

Figure 2.2

Schematic representation of Dip-Pen Nanolithography. “Ink” molecules coated on the AFM tip are transferred to the Au substrate through a water meniscus. Reprinted from Ref. 52 with permission from AAAS.

2.3 Application of Nanomaterials in Biomedical Research

Biomedical scientists have seen a great potential in the nanotechnol­ ogy application. As a result, they have tried to incorporate the intrin­sic properties of nanomaterials with conventional techniques in an attempt to improve detection methods and treatments for greater results [53, 54]. Recently, many studies have reported that innovative nanotechnology has improved biomolecular detection sensitivity, diagnostic accuracy, and treatment efficiency [55–59].

2.3.1 Molecular Imaging for Diagnosis and Detection

Optical imaging methods, such as fluorescence microscopy, differential interference contrast microscopy (DICM), and UV-Vis spectroscopy, are one of the most widely used methods for studying

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biological systems. These methods are simple and highly sensitive, yet tend to have high background noise that is mainly caused by cellular autofluorescence from labeled molecules [60]. Also the lack of quantitative data, requirement for the long observation time, and the loss of signals due to photobleaching in biological systems have initi­ated a need for new imaging agents. In order to complement conven­ tional optical imaging methods, the approach using nanoprobes is one of the major research efforts in nanomedicine mainly due to their ability to recognize and characterize pre-symptomatic diseases [61]. Typically, the nanoprobes comprise of hybrid organic materials such as nanoliposomes and polymeric nanosystems, or inorganic nanoparticles such as quantum dots and magnetic nanomaterials. Depending on the composition and properties of nanomaterials, they can be further utilized in vitro, ex vivo, and in vivo imaging appli­cations. Typical examples include vast arrays of functions such as cell or DNA labeling, molecular imaging, and angiogenesis as an imaging agent, particularly in tumor tissues [15]. Quantum dots (QDs) are fluorescent semiconducting nanocrys­tals that can be used to overcome limitations associated with the more commonly used organic fluorophores. QDs offer many advan­tages such as high quantum yields, high molar extinction coeffi­cients, wide range of absorption spectra from UV to near IR, nar­row emission spectra, resistance to photobleaching and chemical degradation, and long fluorescence lifetimes (>10 ns). Their unique photophysical properties allow time-gated detection for separating their signal from that of the background noise resulting from cell autofluorescence [62–65]. Quantum dots are typically 2–8 nm in diam­eter and their shapes and sizes can be customized by modifying the variables—temperature, duration, and ligand molecules—used in their synthesis. Thus by changing their size and composition, it is possible to precisely tune the absorption and emission spectra to increase or decrease the band gap energy. Due to their narrow emission spectra, they can be used effectively in multiplexing experi­ments where multiple biological units can be labeled simultaneously. One such study was demonstrated by Jain and coworkers [66]. The QDs were applied in vivo to spectrally distinguish multiple species within the tumor tissue [66]. More specifically, they demonstrated that QDs can be customized to concurrently image and differentiate the tumor vessels. The group

Application of Nanomaterials in Biomedical Research

also examined the accessibility to tumor cells depending on the size of QDs. Sizes and compositions of QDs have been extensively studied by many groups [59, 67–70]. Wright and coworkers [71] studied QDs with CdSe core with ZnS shell (CdSe/ZnS) and found that they have potent brightness which is advantageous for optical imaging. The group further studied the core/shell QDs conjugated with an antibody against the respiratory syncytial virus (RSV), a virus which is responsible for causing infections in the lower respiratory tract. It was shown that the use of QDs reduced the detec­tion time from over four days to one hour. In fact, the results were very valuable from a therapeutic point of view as the available anti­viral agent against the RSV is effective only when administered in the initial stages of the infection. Moreover, QDs enable scientists to study live cells and to track down the mechanism of biological processes in a real-time manner due to their resistance to photo-bleaching over long periods of time. The ability to track cells in vivo without having to sacrifice animals signifies a great improvement over the current techniques. Figures 2.3 and 2.4 show two examples of QD imaging: one is the high sensitivity and multicolor capability of QD imaging in live animals and the other is the detection of cancer marker Her2 with QD-streptavidin [21]. (a)

Figure 2.3

(b)

Imaging in live animals using quantum dots (QDs). (a) Molecular targeting and in vivo imaging using antibody-(QD) conjugate. (b) In vivo imag­ing of multicolored QD-encoded microbeads. Reprinted from Ref. 62 with permission from Nature Publishing Group.

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(a)

Figure 2.4

(b)

Detection of cancer marker Her2 in vitro with QD-streptavidin. (a) Her2 detected on surface of free cells using QD 560-streptavidin (yellow). (b) Her2 detected on a section of mouse mammary tumor tissue using QD 630-streptavidin (red). Reprinted from Ref. 21 with permission from Nature Publishing Group.

Cytotoxicity is a primary issue in QD applications [72], because the release of Cd2+ and Se2– ions from QDs could interfere with cell viability or function [73, 74]. While the toxicity may not be critical at low concentrations optimized for labeling, it could be detrimental for the embryo development at higher concentrations. Yet, the problem can be solved by coating the QDs and making them biologically inert or by maintaining a safe concentration range. Several studies have reported that QDs can effectively accomplish their task without adversely affecting cellular processes [75, 76]. Other noble metal nanoparticles, such as Au, Ag, and Cu nanoparticles, are receiving as much attention as QDs, because they exhibit other unique and modifiable optical properties [77]. Typically, when spherical nanoparticles are exposed to electromagnetic field at a certain frequency, the free electrons on a metal surface, known as plasmons, undergo coherent oscillation, resulting in a strong enhancement of absorption and scattering of electromagnetic radi­ation. In case of noble metal nanoparticles, the surface plasmon resonance (SPR) of a metal surface especially yields intense colors and unique optical properties [78]. These noble metals have a greater potential for applications than other materials, because the reso­nance for Au, Ag, and Cu lie at visible frequencies and they have a high stability [77]. Furthermore, Au nanoparticles are insusceptible to photobleaching and can be easily synthesized in a wide range of sizes (4–80 nm) for tunable size-dependent optical properties. They are biocompatible and

Application of Nanomaterials in Biomedical Research

devoid of cytotoxicity, which are big advan­tages over QDs where cytotoxicity can be a limiting factor. The use of biocompatible, nontoxic capping material is critical for medical applications of Au nanoparticles [79] Another reason that makes gold nanoparticles more attractive for optical imaging in biology is the welldefined chemistry between biomolecules with a gold surface. By modifying the surface of Au nanoparticles with an amine or thiol moiety [80], for instance, the nanoparticles can mount antibodies and specifically target tumor cells and biomolecules, such as folic acid [81, 82] and transferrin [83, 84], for imaging and drug/gene delivery. This was well demonstrated in a study by Richards-Kortum and coworkers [85], where the group used 12 nm Au nanoparticles conjugated with anti-EGFR (epithelial growth factor receptor) monoclonal antibodies to image cervical epithelial cancer cell which exhibited an over expression of EGFR as compared to healthy cells. In their study, the conjugation was due to the electrostatic adsorption of the anti­body molecules onto the citrate-capped and negatively charged Au nanoparticles. The use of nanowires and nanotubes in the electrical detection method of analytes at extremely low concentration is one of the hot topics in nanomedicine. Their usage has two major advantages—high sensitivity and fast responses without tedious labeling steps. However, these nanostructures are not as readily functionalized as aforementioned quantum dots or nanoparticles. The unique advan­tages of these nanomaterials come from their one-dimensional mor­phological structures, and many researchers are trying to utilize them as a highly sensitive and selective signal transduction medium. For example, Lieber and coworkers [86] synthesized silicon nanowires with peptide nucleic acid (PNA) functionalization, and demon­strated how the synthetic material could detect DNA without label­ing. A subsequent study modified silicon nanowires with biotin to detect picomolar concentrations of streptavidin and demonstrated high sensitivity to change in conductivity of the nanowires upon the biotin-streptavidin binding. Similar studies have also been carried out by Dai and coworkers [87], where they focused on the application of carbon nanotubes as a material for the sensitive detection. The scheme (Fig. 2.5) [86] illustrates a basic structure of electrical detection of biomolecules with nanowire sensor.

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Figure 2.5

Schematic showing two nanowire devices, 1 and 2, within an array, where nanowires were modified with different (1, green; 2, red) anti­body receptors. Reprinted from Ref. 86 with permission from Nature Publish­ing Group.

Magnetic nanoparticles are emerging as novel contrast reagents for magnetic resonance imaging (MRI) [88–95], revolutionizing current diagnostic tools. Since their unique properties allow precise control of size and composition, magnetic nanocrystals offer great potential for highly specific MRI for biological systems [96–99]. The nanocrystals tend to behave as a single magnetic domain in which all nuclear spins couple to create a single large magnetic domain. At certain temperatures and crystal sizes, these moments wander randomly (superparamagnetic), or become locked in one direction, making the material ferromagnetic [88, 94]. Magnetic nanocrystals of differing com­positions and sizes can be synthesized to generate ultra-sensitive molecular images. Cheon and coworkers [98] developed iron oxide magnetic nanoparticles which were doped with +2 cations such as Fe, Co, Ni, or Mn. It was observed that Mn-doped iron oxide nanoparticles were highly sensitive for detecting cancer cells. The nanoparticles even made it possible to image small tumors in vivo. In the same study, it was noted that 12 nm Mn-doped nanoparticles were bioconjugated with herceptin to have specificity towards the cancer cells. This approach is expected to improve the early diagno­sis of diseases, which is critical for increasing survival rates.

2.3.2 Treatment of Diseases

The motivation to develop nanomedicine stemmed from the limita­tions of current treatments, such as surgery, radiation, or chemother­apy, which often damage healthy cells as well as tumor cells. To address the problem, novel therapeutics which could pas­sively or actively target cancerous cells were developed. To date many nanomedicines are being administered to treat patients. For instance, nanomedicines such as liposomes (DaunoXome)

Application of Nanomaterials in Biomedical Research

[100–102], polymer coated liposomes (Doxil) [100, 103, 104], polymer-protein conju­gates (Oncaspar) [105, 106] antibodies (Herceptin) [107–109], and nanopar­ticles (Abraxane) [110] are already bringing clinical benefits to patients around the world. Most of the nanotherapeutics take two main paths to achieve their goals—passive targeting and active targeting [111]. Rapid vascularization takes place within tumor tissues in order to supply nutrients for fast tumor growth. This inevitably causes the development of a defective, leaky architecture along with dam­aged lymphatic drainage. This leads to the enhanced permeation and retention (EPR) effect, which helps the injected nanoparticles to preferentially permeate and accumulate in the tumor tissue [111, 112]. In order for the passive mechanism to be efficient, the size and sur­face properties of the nanoparticles should be well controlled. The nanoparticle size should be less than 100 nm, and the surface should be hydrophilic to prevent the uptake by the macrophages of the retic­uloendothelial system (RES), which would significantly improve the circulation half-life of the nanoparticles. This is achieved by coating their surfaces with hydrophilic polymers such as polyethylene gly­col (PEG), poloxamers, poloxamines, and polysaccharides [113] since a hydrophilic nanoparticle surface avoids the adsorption of plasma proteins. Another passive targeting method utilizes the unique environ­ment around the tumor, such as cancer-specific enzymes, high or low pH of the tumor tissue, to release the drug/biomolecules within the tumor tissue [114]. Otherwise inactive nanoparticledrug conjugate molecules can specifically be activated by the tumor-specific envi­ronment when they reach the tumor site. The release takes place within the tumor tissue, considerably increasing the drug efficiency. Yet another method to passively target the tumor is by using direct local delivery of anticancer agents into the tumors. This is an effec­tive technique, but can be highly invasive and the access to certain tumors such as lung cancers may be impossible [114]. Active targeting is usually achieved by conjugation of targeting moieties such as proteins, peptides, antibodies, and carbohydrates with nanoparticles. These ligands act as guided missiles which deliver the nanoparticles to the specific cancer tissue and cells. For example, when Doxil (liposomal doxorubicin)

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Nanomedicine

is attached to an anti­body against a growth factor overexpressed in breast cancer (ErbB2), it shows faster and greater tumor regression compared to unmod­ified Doxil [115]. Similarly, several targeting ligands are available for nanocarriers to deliver drugs at specified locations [116].

2.3.3 Nanocarriers for Drug Delivery

Nanocarriers, 2 to 100 nm in diameter, are designed to deliver mul­tiple drugs and/or imaging agents [117]. They have high surface to volume ratios which allow high density functionalization of their surfaces with targeting moieties. Promising nanocarriers summa­rized in Fig. 2.6 include liposomes, polymeric micelles, dendrimers, carbon nanotubes, and inorganic nanoparticles [111].

Figure 2.6

Examples of various drug delivery agents which typically include three components: a nanocarrier, a targeting moiety conjugated to the nanocarrier, and a therapeutic agent. Reprinted from Ref. 111 with permission from the Nature Publishing Group.

Application of Nanomaterials in Biomedical Research

Polymers such as poly lactic acid (PLA) and poly lactic co­ glycolic acid are the most extensively studied and commonly used materials for nanoparticle-based delivery systems [57, 118–122]. Addi­tionally, natural polymers such as chitosan and collagen have already been vigorously studied for encapsulation of drugs with their intrinsic biocompatibility [123–126]. The use of polymers has sig­nificant advantages because the drugs or biomolecules can be encapsulated easily without chemical modification processes, while maintaining a high cost efficiency and yield. Release of the encapsu­lated cargo of the polymeric nanoparticles takes place via diffusion through the polymer matrix, or swelling of the nanoparticles [127–132] due to changes in a local environment such as pH or specific enzyme. Efficiency can be further improved by attaching targeting moieties, and also by passivating the surfaces for longer circulation half-lives. For example, using PEG, Huang and coworkers formulated carbohy­drate conjugated chitosan nanoparticles which effectively targeted the liver cancer cells (HepG2). In the study, the carbohydrate used for targeting was galactose, which specifically bound to the asialo­glycoprotein (ASGP) receptors overexpressed in the HepG2 cells [133]. Lipid-based carriers are attractive due to several properties such as biocompatibility, biodegradability, and ability to protect drugs from harsh environments. These properties came from the unique amphiphilic structure of liposomes, where the spherical amphiphiles consist of closed structures that contain one or more concentric lipid bilayers with an inner aqueous phase. Due to the amphiphilic nature, lipid-based carriers can be easily modified to entrap both hydrophobic and hydrophilic drugs [134–136]. However, Liposomes are rapidly cleared from circulation by the Kupffer cells in the liver. Thus, coating them with PEG could increase their circula­tion half-life [137, 138]. Overall, liposomes have shown reduced toxicity and preferential accumulation in the tumor tissue by EPR effect [127]. They could also be actively targeted by attaching antibodies to their surface. Dendrimers are well defined molecules which are built at a nanoscale with monodispersed systems with sizes ranging from 3 to 10 nm. They have unique molecular architectures and proper­ ties, both of which result in easy conjugation chemistry with tar­geting molecules, making them attractive for the development of nanomedicines [127, 139–142]. Several studies have confirmed that con­jugated dendrimers can be found concentrated in the tumor and

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liver tissue in contrast to non-targeted polymer folates. Increased therapeutic activity and marked reduction in toxicity was also observed [143]. The synthesis of colloidal gold was first reported in 1857 [144]. However, it was about 100 years later when scientists found out that the gold particles could bind proteins without altering their activity, paving a way for their application in biological systems. Several studies confirm that gold nanoparticles are easily taken up by cells [83, 145–147]. The biocompatibility of gold nanoparticles can be further enhanced by PEG coating that prevents uptake by RES. For example, PEG-coated gold nanoparticle formulation, developed by CytImmune Sciences [78], which carries the tumor necrosis fac­tor (TNF), is currently under human trials. These gold nanoparti­cles showed preferential accumulation in MC-38 colon carcinoma tumors and no uptake by liver or spleen. It was further reported that this system is less toxic and more effective in reducing tumor burden than native TNF [148]. Gold nanoparticles are also very useful for delivering nucleic acids. As seen previously, their surface allows for easy functionalization with thiols which makes attachment of oligonucleotides on the surface relatively easy. In a recent study by Mirkin and coworkers, it was reported that the cellular uptake of gold nanoparticles increased with the increase in density of the oligonucleotides on the particle surface [149]. Magnetic nanoparticles are extremely promising as novel drug delivery agents [150]. They show several advantages which are not seen in most nanoparticle-based systems. They can resolve problems related to polydispersity and irregular branching, both of which are common in polymeric nanocarriers. Magnetic nanoparticles, for example, can act as contrasting agents for MRI application [151]. Also, drug loaded magnetic nanoparticles can be guided or held in place with applied external magnetic field using their superb magnetic susceptibility. The magnetic nanoparticles can also be heated to induce hyperthermia of the tissue upon exposure to the external magnetic field. The magnetic nanoparticles can be further function­alized with targeting ligands to improve their uptake efficiency, min­imize their toxicity, and prevent them from aggregating [152, 153]. This is typically done by coating them with hydrophilic to neutral com­pounds such a PEG, dextran, or HSA, which not only stabilizes the particles but also increases their half-life in blood.

Nanosystematic Approach for Cell Biology

With photothermal cancer therapy, which uses optical heating for tumor ablation, doctors can deal with tumors in a noninvasive manner [154–159]. It could be a method of choice for tumors which are inaccessible for surgery or radiotherapy, both of which kill healthy cells along with tumor cells. For this specific application, gold nanoparticles are most widely used, because the gold nanopar­ticles have ability to effectively convert strongly absorbed NIR light to localized heat, and hence are useful in selective photothermal therapy. Using active and passive targeting, these nanoparticles can be localized into the tumor tissue, increasing the effectiveness of the heat produced and at the same time minimizing the non-specificity of treatment. In a recent study, Li and coworkers devel­oped gold nanocages (0.005% are added to chitosan at a ratio of 1:2 and mixed vigorously to generate a stable nano-emulsion whereby ionic complexes of alginate are spontaneously formed within the chitosan solution. In proof-ofconcept studies nanocapsules of mineralized polysaccharides can be produced using this facile method (Fig. 38.12). Surface decoration with important biological molecules using conventional carbodiimide chemistry will significantly enhance cellular responses and more rapidly promote the generation of human tissues [27]. Similar to there macroscopic counterparts these nanotemplates consist of an alginate core coated with a chitosan-CaP shell as confirmed by morphological appearance under TEM and EDAX elemental analysis for presence of calcium phosphate (Figs. 38.12a,b). Work will be carried out to reduce the wide dispersion of capsule sizes cur­rently

842

Biomimetic Applications in Regenerative Medicine

Polysaccharide Chitosan Coated Alginate Nanocapsules

scale cellular niches and (b) to fabricate cellular assembly mimicsprecursors to bone construction. obtained using a less than optimized synthesis. Nanocap­sules have also been shown to retain encapsulates within the alginate core as shown in Fig. 38.12a. In CHITOSAN cell culture nanoparti­ 21.6 POLYSACCHARIDE COATED cles have been demonstrated to readily co-associate with hBMSC within in vitro cell ALGINATE NANOCAPSULES suspensions as shown in Fig. 38.13a. These nanoparticles possess Mineralized polysaccharide capsules have been to that provide a potential capacity to function as intracel­ lular shown vehicles can microenvironments that promote tissue formation and function as deliver genes or proteins into the cell cy­toplasm or nuclear envelope delivery vehicles growth factors and genes. We have been respectively. TEMfor images show mi­crocapsule at theable cell localisation to extend the of our polysaccharide beads for(Fig. intracellular membrane andutility internalisation into the cell cytoplasm 38.13b). Nanocapsule chemistry and size will beofoptimized maximise cell activation and endogenous delivery bioactive to factors by syninternalization, responses (250-900 in vacuoles thethe functional thesizing beads release at the nanoscale nm) and using method effects of encapsulates on12A). targeted stem cells. An understanding of Polysaccharide beads will function of Douglas et al.5 (Figure theintracellular mechanism vehicles (i.e., receptor-mediated?) these as that deliver genesby orwhich proteins intoparticles the cell are endocytosed and envelope. assimilated within the cell will be met by cytoplasm or nuclear deploying of concentrations proteins (clathrin, adaptinareand epsin) In briefinhibitors alginate at of >0.005% added to involved with phagocytosis and endocytosis such as cytochalasin chitosan at a ratio of 1:2 and mixed vigorously to generate a stable be used track B. Fluorescent dyes and con­ focal microscopy nano-emulsion whereby ionic complexes of will alginate are to spontathese particles progresssolution. through In the cell cytoplasm, neously formedduring withintheir the chitosan proof-of-concept alongside antibody marking of Stat-2polysaccharides and AP-2 or presence of studies nanocapsules of mineralized can be proendocyte vesicles. Biochemical ELISA protein assays will be used duced using this facile method (Figure 12). Surface decoration to determine capsule induced over-expression of cytoplasmic and with important biological molecules using conventional carbodimatrixchemistry proteins significant to musculoskeletal tissueresponses regeneration imide will significantly enhance cellular and 27 Similar such as osteonectin andthe os­tgeneration at atissues. subcellular level eocalcin. Imaging more rapidly promote of human will be carried out using TEM using coated gold nanoparticles. (a)

(b)

Figure 38.12 Mineralized polysaccharide (alginate-chitosan) nanocap­ Figure 21.12.sules; Mineralized polysaccharide nanocap(a) TEM of nanocapsules (alginate-chitosan) with alginate stained black/ sules; A) TEM of nanocapsules with alginate stained black/dark greyshell; surdark grey sur­rounded by a lighter grey chitosan outer (b) XRD analysis of a rep­ nanocapsule shows that r esentative rounded by a lighter grey chitosan outer shell; B) XRD analysis of a repcalcium and phosphate ions are con­ c entrated on capsules and resentative nanocapsule shows that calcium and phosphate ions are contherefore indicates the formation of a calcium phosphate outer centrated on capsules and therefore indicates the formation of a calcium shell. This provides evidence that alginate-chitosan cap­sules phosphate outer provides evidence that alginate-chitosan caphaveshell. been This formed. sules have been formed.

ight © 2010 by Pan Stanford Publishing Pte. Ltd.

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38.7 Native Extracellular Matrix Equivalents

844

Decorating the surfaces with important biological molecules will significantly enhance cellular responses and more rapidly pro­mote the formation of human tissues. There is a current need for clinically relevant tissue engineering scaffolds with broad ap­plicability and versatility that can be more specifically tailored to initiate cell responses and fashioned to mimic native ECM thus promote tissue formation that better match with the patient’s own tissues. Our biologically enhanced constructs directly meet this significant need. In this task of the proposal we will prepare cell adhesion peptides/ proprietary functional ligands that stim­ulate stem cell activities Biomimetic Regenerative Medicine and Applications conjugate in them to chitosan (Ca2+ ions 50 mM). Capsules will be synthesized to a range of micron length scales. (a)

(b)

Figure 38.13 (a) High power microscope image showing that polysac­charide Figure 21.13.nanoparticles A) High power microscope image polysacselectively associate withshowing primarythat human bone charide nanoparticles selectively associate with primary (n human marrow stromal cells in a 3D cell suspensions = 12).bone The alginate core is stained with a fluorescent dye marrow stromal cellsnanocapsule in a 3D cell suspensions (n=12). The alginate Red)with which in white transmitted is blue. (b) nanocapsule (Cell coreTracker is stained a fluorescent dye (Celllight Tracker Red) TEM image showing nanocapsules (i) at the surface which in white transmitted light presence is blue. B)ofTEM image showing presence of a primary hBMSC cell membrane and (ii) internalized within of nanocapsules (i) at the a primary hBMSC cellofmembrane and vacuoles. Thesurface yellow of arrows denote position nanocapsules. (ii) internalized within vacuoles. The yellow arrows denote position of nanocapsules. Capsules of this kind were immersed in a primary hBMSC cell

suspension to test the potential of peptide decorated capsules to attach cells to the external surface. It was found that within 7 days metabolically active/viableenhanced primary hBMSC preferentially adhered tissues. Our biologically constructs directly meet this to chitosan-GGRGD complexed capsules (Figs. 38.14b,d). In capsules significant need. In this task of the proposal we will prepare without chitosan conjugated GGRGD ligands onlyligands a few cells were cell adhesion peptides/ proprietary functional that stimseen to be attached (Figs. 38.14a,c). ulate stem cell activities and conjugate them to chitosan (Ca ions This Capsules demonstrated the effectiveness the of RGD coupling to 50 mM). will be synthesized to aofrange micron length chitosan and the effectiveness of RGD presentation to cells in con­ scales. tact Capsules at the surface of kind the chitosan-CaP shell.inThis adaptation will of this were immersed a primary hBMSC cell suspension to test the potential of peptide decorated capsules to attach cells to the external surface. It was found that within 7 days metabolically active/viable primary hBMSC preferentially adhered to chitosan-GGRGD complexed capsules (Figure 14B,D). In capsules without chitosan conjugated GGRGD ligands only a few cells were seen to be attached (Figure 14A,C). This demonstrated the effectiveness of the RGD coupling to

Native Extracellular Matrix Equivalents

1133

be used for (a) extra-cellular activation using a range of other bio­ functional peptide sequences and (b) the potential for exogenous “stem cell-guided” ordered assembly of individual capsules into extra-cellular matrix mimics. Polysaccharide beads can therefore be surface decorated with covalently attached bioactive molecules 21.8. Tissue homing devices (ligands) to enhance cell-to-bead interactions and promote cellu­lar responses that instigate native regeneration processes. (b)

(a)

(c)

(d)

Figure 38.14 Preferential primary human bone marrow stromal cell (2.5 × Figure 21.14. 4 Preferential primary human bone marrow stromal cell (2.5 10 /capsule) attachment to the surface of GGGRGD decorated x 104 /capsule) attachment to the surface of GGGRGD decorated chitosan chitosan coated alginate microcapsules (n = 4). (a) Brightcoated alginate microcapsules (n=4).of A) Brightfield microscope field microscope image chitosan coated alginate image capsule of chitosanwithout coated alginate primary hBMSC B) Brightprimarycapsule hBMSC.without (b) Bright-field microscope image field microscope image coated of chitosan coatedcapsule alginate covered capsule covered with of chitosan alginate with primary primary hBMSC microscope image of of chitosan coated hBMSC.C)(c)Fluorescence Fluorescence microscope image chitosan coated alginate capsule in situ showing absencehBMSC of primary hBMSC. alginate capsule in situ showing absence of primary D) Fluores(d) Fluores­ cence microscope image of chitosan alginate cence microscope image of chitosan coated alginate capsule coated outer surface capsule outer surface showing attachment of primary hBMSC showing attachment of primary hBMSC fluorescing positively with the fluorescing positively with the cytoplasmic stain, Cell Tracker cytoplasmic stain, Cell Tracker Green. Thus attached cells are alive and Green. Thus attached cells are alive and metabolically active. metabolically active. Yellow arrows denote edge of capsule, white arrows Yellow arrows denote edge of capsule, white arrows denote denote primary hBMSC. primary hBMSC.

21.8

TISSUE HOMING DEVICES

Protocols that enhance the accession, processing, function and transplantation of conditioned cells to the patient are imperative. Carrier materials and matrices have been developed extensively to safely and stably transplant sufficiently large numbers of stem

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38.8 Tissue Homing Devices Protocols that enhance the accession, processing, function and transplantation of conditioned cells to the patient are imperative. Carrier materials and matrices have been developed extensively to safely and stably transplant sufficiently large numbers of stem cells and cell types required, into patients and more significantly directly to the site of need. Developments are occuring where transplantation modules are being engineered with homing functions directed to exact tissue sites or regions. A promising example of this technology is described in the invention by Caplan [4]. Protein/lipid compositions were formulated to coat living cells. The lipid component formed strong associations with the cell membrane and by using specific biochemical targeting moieties conjugated with homing peptides (e.g., PWERSL, ASSLNIA, YSGKWGW), antibody fragments and carbohydrates, such treated cells possessed a mobile affinity for specific tis­sue types what has been termed selective direction to the target tissue [4]. Two approaches have been taken. The first is to coat selected progenitor cells with a linker followed by binding of a targeting moiety for a tissue type/character with the linker and administering these treated cells to a safe place where homing can effectively begin. A second approach involves coating the progenitor cell with the linker, targeting moeity in one distinct complex. This may be at the site intended for repair or regeneration or at a distant site where treated cells can be passively transported to the designated location. The invention promises more effective, efficient use of cells and biofactors while reducing invasiveness and use of bulk biomaterials associated with more traditional strategies. We envisage developing polysaccharide modules and patented polysaccharide technology to target specific tissues using highly specific protein, peptide and antibody associations that are positioned on the outer surfaces of these new homing modules.

38.9 Summary

Biomimesis is a concept with growing relevance and impor­tanceto a diverserange of sciences (biology, materials, chem­ istry and

Conclusions

physics), technologies (robotics, nanotechnology), clin­ical edicine and professions (architecture). Learning lessons from nature is pivotal to developing smart materials, structures and processes with self assembling, self-actuating and self stabilizing properties. Tissue engineers are faced with the problem of de­veloping scaffolds with multifarious (often conflicting with each other) functions that must be bio-responsive and evolve in real time to a dynamic host environment. The principles of a bioin­spired approach to tissue engineering scaffold design and synthe­ sis have been described and augmented using BioTriz method­ ology. Further methods focus on nanoscale technologies and building structures and materials using nanoparticles. A num­ ber of worked examples show how biomimesis can generate in­novative functional tissue engineering constructs with complex, intricate structures and morphologies unachievable using conven­tional methods such as vaterite microspheres that mimic plankton shell constructions. We provide an example of a self assembling organic scaffold around which spontaneous mineralization of cal­cium phosphate can be induced—a process analogous to tem­ plate mediated mineralization in nature (e.g., mineralization of eggshell). The internal microenvironments for embedded cells can be modulated to recreate elements of a native extracellular matrix adding a further biomimetic element to this unique system. Bioevaluation provided convincing evidence that these scaf­ folds may provide clinical success, as novel self-assembling scaf­folds or gene/biofactor delivery vehicles for the engineering of mineralized and soft human tissues.

38.10 Conclusions

Regenerative medicine is confronted with a paucity of clinically relevant scaffold designs and biological factors that promote the natural cycle of regeneration. Understanding hierarchical design in nature and harnessing the chemical properties of natural struc­tures at all length scales will be instrumental in re-assembling functional analogues of natural skeletal design. Designing and engineering smart biomaterials and modules with the ability to evolve with and adjust to the local host environment during

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regeneration is a key aim of ours. We believe in using bio-inspired and nanoscale materials chemistry to do this. Such knowledge will enable tissue engineers to synthesize new advanced bio­structures and materials that measure up to the functional de­ mands of regenerating native human tissues that are truly patient ready.

Disclosures and Conflict of Interest

The authors would like to sincerely thank Professor Richard O. C. Oreffo and Professor Stephen Mann for their contribution to the genesis of biomimetics approach and their inspiration, guidance and support given to Dr. D. W. Green while carrying out part of this research at the University of Southampton. They acknowledge the contribution of Dr. Janos Kanclzer, Dr. Xuebin Yang and Dr. Xunhe Xu for provision of fetal derived mesenchymal stem cells and for carrying out in vivo trials and thank Dr. Paul Dal­ton for stimulating discussions on electrospinning for tissue engi­neering applications. Furthermore, the author gratefully acknowledge the financial support of the Australian Research Grant Commission (ARC Discovery), EPSRC (GR/N29860/01) and the Royal Society of UK travel grants. The authors declare that they have no conflict of interest and have no affiliations or financial involvement with any organization or entity discussed in this chapter. Neither any writing assistance was utilized in the production of this chapter nor was there any payment received for its preparation. This is a revised version of the authors’ chapter that appeared in Handbook of Materials for Nanomedicine, 2010 (edited by V. Torchilin and M. M. Amiji), Pan Stanford Publishing Pte. Ltd., Singapore.

Corresponding Author

Dr. D. W. Green Department of Oral Biosciences, Faculty of Dentistry University of Hong Kong The Prince Philip Dental Hospital, 34 Hospital Road Sai Ying Pun, Hong Kong Email: [email protected]

References

About the Authors David W. Green received his PhD from Aston University. He is currently a research assistant professor in the Department of Oral Biosciences, Faculty of Dentistry, University of Hong Kong. His research interests focus on regenerative medicine, bioinspired engineering, and biomimetics materials chemistry.

Besim Ben-Nissan received his PhD from the University of New South Wales in Mechanical and Industrial Engineering/Biomedical Engineering. He is a visiting professor in the Faculty of Science at the University of Technology in Sydney, Australia. His areas of research expertise include nanocoatings, biomaterials, biomimetics, biomechanics, calcium phosphates, implant design, finite element modeling, marine structures, and slow drug delivery. Over the last three decades together with a large numbers of PhD students he has worked on production and analysis of various biomedical implants, hydroxyapatite ceramics, advanced ceramics (alumina, zirconia, silicon nitrides), sol-gel developed nanocoatings for enhanced bioactivity, corrosion and abrasion protections, optical and electronic ceramics. He also has contributed in the areas of mechanical properties of sol-gel developed nanocoatings.

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18. Hollister, S. J. (2005). Porous scaffold design for tissue engineering. Nat. Mater., 4, 518–524. 19. Kang, K. D., Lee, K. H., Ki, C. S., Nahm, J. H., Park, Y. H. (2004). Silk fibroin/chitosan conjugate cross-linked by tyrosinase. Macromol. Res., 12(5), 534–539.

20. Leveque, I., Rhodes, K. H., Mann, S., (2002). Biomineral-inspired fabrication of semi-permeable calcium-phosphate-polysaccharide microcapsules. J. Mat. Chem., 12, 2178–2180. 21. Mann, S. (2005). Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry, Oxford University Press, Oxford, 2001.

22. Mazuka, T., Iwasaki, N., Yamane, S., Funakoshi, T., Majima, T., Minami, A., Ohsuga, N., Ohta, T., Nishimura, S.-I. (2005). Chitosan-RGDSGGC conjugate as a scaffold material for musculoskeletal tissue engineering. Biomaterials, 26(26), 5339–5347. 23. Mohamadnia, Z., Zohuriaan-Mehr, M. J., Kabiri, K., Jamshidi, A., Mobedi, H. (2007). pH-Sensitive IPN hydrogel beads of carrageenan-alginate for controlled drug delivery. J. Bioact. Compat. Polym., 22, 342–356.

24. Nisisako, T., Okushima, S., Torii, T. (2005). Controlled formulation of monodisperse double emulsions in a multi-phase microfluidic sys­tem. Soft Matter, 1, 23–27. 25. Pound, J. C., Green, D. W., Roach, H. I., Mannand S., Oreffo, R. O. C. (2007). An ex vivo model for chondrogenesis and osteogenesis. Biomateri­als, 28(18), 2839–2849. 26. Raeber, G. P., Lutolf, M. P., Hubbell, J. A. (2005). Molecularly engineered PEG hydrogels: A novel model system for proteolytically medi­ated cell migration. Biophys. J., 89, 1374–1388.

27. Rowley, J. A., Mooney, D. J. (2002). Alginate type and RGD density control myoblast phenotype. J. Biomed. Mater. Res., 60(2), 217–223.

28. Silberman, M., Gislason, J., Einarsson, J. M., Martin, P. (2006). Use of chitosan for stimulating bone healing and bone formation. WO/2006/057011. International Patent Application.

29. Wnek, G. E., Carr, M. E., Simpson, D. G., Bowlin, G. L. (2003). Electrospinning of nanofiber fibrinogen structures. Nano Lett., 3, 213–216.

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Chapter 39

Potential Applications of Nanotechnology in the Nutraceutical Sector Shu Wang, MD, PhD, and Jia Zhang, MS Department of Nutritional Sciences, Texas Tech University, Lubbock, Texas, USA

Keywords: biocompatible and biodegradable nanocarriers, phytochemicals, green tea catechins, (-)-epigallocatechin gallate, quercetin, resveratrol, curcumin, bioavailability, bioactivities, cancer, atherosclerosis, stability, sustained release, nanoliposomes, emulsions, micelles, lipid nanoparticles, PLGA nanoparticles

39.1 Introduction The application of nanotechnology in improving bioavailability and bioactivities of nutrients and phytochemicals is a promising and innovative research area. Many natural phytochemicals show promise to remedy diseases, but their low level of solubility, stability, bioavailability and target specificity and the high level of metabolism in the body make administering them in therapeutic doses unrealistic. Green tea catechins and quercetin are wellstudied phytochemicals and have beneficial potentials to human Handbook of Clinical Nanomedicine: Nanoparticles, Imaging, Therapy, and Clinical Applications Edited by Raj Bawa, Gerald F. Audette, and Israel Rubinstein Copyright © 2016 Pan Stanford Publishing Pte. Ltd. ISBN 978-981-4669-20-7 (Hardcover), 978-981-4669-21-4 (eBook) www.panstanford.com

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health. There is a critical need for using nanoparticles to enhance their stability, solubility, absorption, bioavailability, target specificity and bioactivities, protect them from premature degradation in the liver and other tissues, and lower their toxicity to normal tissues.

39.2 Nanoparticles

Although no official universally accepted definition for nanoparticles has been established, most experts consider them to be particles that are in the range of 10–1000 nm. Nanoliposomes, emulsions, micelles, lipid nanocarriers, and poly(lactic-co-glycolic acid) (PLGA) nanoparticles are commonly used biocompatible and biodegradable nanoparticles [1].

39.2.1 Nanoliposomes

Phospholipids and cholesterol are commonly used to synthesize nanoliposomes. The extruder and sonicator are common equipment to make nanoliposomes. After dissolving and drying phosphatidylcholine and cholesterol, saline or aqueous solutions can be added into the dry lipids. After vortexing, the suspension can either pass through the extruder with different sizes of filters or be sonicated to form nanoliposomes. Nanoliposomes are spherical and have lipid bilayered membrane structures. Multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs) and large unilamellar vesicle (LUVs) are major types of nanoliposomes. Hydrophilic phytochemicals can be encapsulated into the central aqueous compartment of nanoliposomes, and the hydrophobic phytochemicals can be embedded into their lipid bilayers [2]. Since all components of nanoliposomes are biocompatible and biodegradable, nanoliposomes have been widely used as delivery carriers in pharmaceuticals and nutraceuticals [2–5].

39.2.2 Emulsions

Emulsions consist of two immiscible liquids. Oil-in-water (O/W) emulsions are made by adding oil into an aqueous phase, and water-in-oil (W/O) emulsions are made by adding an aqueous solution into an oil phase. In order to reduce the interfacial tension, surfactants or co-surfactants are needed. More surfactants or co-

Nanoparticles

surfactants are required to make the smaller size of emulsions. Since our physiological environment is aqueous, O/W emulsions are the common emulsion type. The first FDA approved intravenous fat emulsion was Intralipid®, which was composed of soybean oil, egg phospholipids, and glycerin. Intralipid® can deliver essential fatty acids via intravenous injection to the patients who cannot absorb those nutrients through diet [6]. Many phytochemicals, especially resveratrol, quercetin, and curcumin, are hydrophobic. O/W emulsions can increase their aqueous solubility, stability, bioavailability, and bioactivities.

39.2.3 Micelles

Micelles are composed of many amphiphilic monomers, such as phospholipids and some synthetic polymers. When reaction temperatures reach the critical micellization temperature and amphiphilic monomers concentrations reach the critical micelle concentration (CMC), micelles are formed [7, 8]. Micelles found in human intestines are made by phospholipids and bile acids. Hydrophilic (polar) phospholipid heads form the outer surface of micelles, while hydrophobic (nonpolar) phospholipid fatty acid tails form a hydrophobic core, which can accommodate dietary lipids, lipid-soluble vitamins, phytochemicals, and other hydrophobic compounds. Polymeric micelles are composed of block-copolymers that are made of hydrophobic (nonpolar) and hydrophilic (polar) monomer units in different arrangements [9]. The commonly used core-forming blocks are poly(caprolactone), poly(propylene oxide), poly(D,L-lactic acid) and poly(L-aspartic acid), and polyethylene glycol (PEG) is the widely used hydrophilic monomer [9, 10]. Many hydrophobic compounds can be encapsulated or incorporated into micelles and be used for oral, parenteral, nasal, ocular, and topical administration [9]. Micelles are excellent carriers for phytochemicals.

39.2.4 PLGA Nanoparticles

PLGA is a FDA approved and widely used biocompatible and biodegradable polymer [11]. PLGA is composed of lactic acid and glycolic acid. PLGA can have different numbers of lactic acid  and glycolic acid that give PLGA different molecular weights and properties.

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Many studies have used PLGA nanoparticles as carriers for phytochemicals, including quercetin and curcumin [12–14]. Those phytochemical can be incorporated into PLGA nanoparticles via encapsulation or adsorption on the surface of PLGA nanoparticle [15]. After PLGA nanoparticles deliver those phytochemicals into the animal body, they can be hydrolyzed into lactic acid and glycolic acid, which have minimal toxicity and side effects to human bodies.

39.2.5 Lipid Nanocarriers

Lipid nanocarriers are made of lipids, surfactants, co-surfactants and water [16]. Solid lipid nanoparticles (SLNs) were developed in the early 1990s [17]. The structure of SLNs is similar to lipoproteins, which have a hydrophilic shell and a hydrophobic solid lipid core, which may consist of saturated fatty acids, triglycerides, waxes, or a mixture of the above lipids [16, 18]. SLNs are excellent carriers for hydrophobic compounds, because they can be easily encapsulated into the hydrophobic lipid core. However, low loading capacity is the major limitation of SLNs [18]. The new generation of lipid nanocarriers are called nanostructured lipid carriers (NLCs), whose lipid core is composed of more complex lipid mixtures such as fatty acids with different chain lengths. Such lipid crystal structures are less perfect but they can accommodate more hydrophobic phytochemicals, nutrients, or other compounds, enhancing loading capacity [19–22]. Phase inversion, solvent evaporation/emulsification, cold homogenization, high pressure homogenization, hot homogenization /ultrasonication are common methods to synthesize SLNs or NLCs [22]. Many studies have used NLCs or SLNs as phytochemical carriers to improve their solubility, stability, bioavailability, and bioactivities.

39.3 Phytochemicals

39.3.1 (-)- Epigallocatechin Gallate (EGCG) Nanoparticles Green tea (Camellia sinensis) is a popular beverage worldwide, especially in Asia. Green tea catechins constitute about 10–30% of total dry tea weight [23]. EGCG is the most abundant and bioactive

Phytochemicals

catechin, and accounts for 20–50% of total catechins. Therefore, one tea bag (2 g) contains about 60–300 mg of EGCG [24]. EGCG has anti-inflammatory, anti-oxidant, anti-angiogenic, and antitumorigenic properties [25]. EGCG is not stable in blood and physiological body fluids (neutral pH), but it is stable in an acidic solution (pH < 5.0) [26, 27]. Our group has synthesized EGCG encapsulated nanoliposomes (LIPO-EGCG) and chitosan-coated LIPO-EGCG (CSLIPO-EGCG) using a sonication method [28]. The mean diameter of LIPOEGCG is 56 nm, and the mean diameter of CSLIPO-EGCG is 85 nm. EGCG encapsulation efficiency was more than 90%. As compared to native EGCG, LIPO-EGCG and CSLIPO-EGCG had significantly increased EGCG stability, decreased the viability (50%) of MCF7 breast cancer cells, and induced their apoptosis (27%). Our group has also synthesized EGCG encapsulated NLCs (NLC-EGCG) and chitosan-coated NLC-EGCG (CSNLC-EGCG) using a novel phase inversion method. NLC-EGCG was composed of soy lecithin, glyceryl tripalmitate, glyceryl tridecanoate, polyoxyethylated 12-hydroxystearic acid, a nonionic surfactant, EGCG, NaCl, and deionized water. The mean diameter of NLC-EGCG is 46 nm, and the mean diameter of CSNLC-EGCG is 54 nm. EGCG encapsulation efficiency was about 99%. Compared to native EGCG and NLCEGCG, CSNLC-EGCG increased EGCG stability, increased macrophage cellular EGCG content, decreased macrophage cholesteryl ester accumulation, decreased macrophage mRNA and protein levels of monocyte chemoattractant protein-1 (MCP-1), which indicate their anti-atherogenic potential. The absorption rate of EGCG is extremely low in humans and research animals [29–31]. Many scientists have used rats to conduct pharmacokinetic and bioavailability studies of green tea catechins [32]. They found that the oral bioavailability was about 0.1% [32]. After men and women drinking tea containing 400 mg catechins throughout the day, the EGCG bioavailability is 0.14% and peak plasma EGCG concentrations are less than 2 μM [30, 31]. Many studies have demonstrated that nanoparticles, especially chitosan nanoparticles, significantly increased oral EGCG bioavailability through enhanced transcellular transport process [33, 34]. We summarize the properties and bioactivities of EGCG nanoparticles in Table 39.1.

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Table 39.1

NP type size NLCs 53 nm

The characteristics, findings, and application of EGCG nanoparticles Experiment model/ Findings and Year dose/route potential application [ref.]

THP-1 derived macrophages were treated with 10 µM of free EGCG or nanoEGCG for 16 h.

Nanoliposome MCF7 breast cancer 85 nm cells were treated with 0–10 µM of free EGCG or nano-EGCG.

PLGA 127.2 nm PLGA-PEG 80 nm

Chitosan NPs 440 nm

NLCs 260 nm

7,12-Dimethylbenzanthracene (DMBA)-induced DNA damage in mouse skin PSMA positive prostate cancer (LNCaP) cells were treated with 30 μM of free EGCG or nanoEGCG for 48 and 72 h.

EGCG stability  Sustained release Cellular uptake  Inflammation  Cellular cholesteryl ester content Application in anti-atherosclerosis

2013 [27]

 Sustained release  DNA damage Application in cancer chemoprevention

2013 [35]

EGCG stability 2013 [28]  Sustained release Cellular uptake  Cancer cell viability  Cancer cell apoptosis Application in cancer chemoprevention

 Sustained release  viability of LNCaP cells No effect on viability of normal cells Application in cancer chemoprevention

2011 [36]

EGCG stability

2009 [37]

Excised jejunum from EGCG stability male, Swiss outbred EGCG intestinal mice. absorption Application in Enhancing EGCG bioavailability

N/A

2010 [34]

Phytochemicals

NP type size Polylactic acid– polyethylene glycol (PLA-PEG) NPs 285 nm

Experiment model/ Findings and Year dose/route potential application [ref.] Prostate cancer (PC3) cells were treated with 1–80 μM of free or nano-EGCG for 24 and 48 h. Tumor xenograft mice were given 1 mg and 100 µg of free or nano-EGCG, respectively, via intraperitoneal injection three times per week.

 Apoptosis 2009 [38]  Cell viability  Colony formation  Tumor size  Angiogenesis Nano-EGCG exhibits > 10-fold dose advantage over free EGCG Prostate cancer Application in cancer Chemoprevention and treatment

N/A: not applicable; NP: Nanoparticle; PSMA: Prostate-specific membrane antigen; , increase; , decrease. Reprinted with kind permission of Journal of Nutritional Biochemistry.

39.3.2 Quercetin Nanoparticles Quercetin (3,3,4,5-7-pentahydroxy flavone) is abundant in berries, caper, buckwheat, tea leaves, onion, apple, broccoli, and other leafy green vegetables [39]. Quercetin has many beneficial effects to human health including anti-oxidant, anti-inflammatory, anti-viral and anti-tumorigenic properties [40–42]. Quercetin is a hydrophobic compound and has extremely low aqueous solubility. Quercetin is quickly metabolized in the human body, which results in low circulation time and concentrations [43]. We have successfully synthesized quercetin (Q) encapsulated NLCs (Q-NLCs) and chitosan-coated Q-NLCs (Q-CSNLCs) in our laboratory [44]. The encapsulation efficiency was 95% and the loading capacity of Q-NLCs was 11%. NLCs enhanced aqueous solubility of Q more than 1000 times. Q-NLCs decreased the viability of MCF7 and MDA-MB-231 breast cancer cells in a dosedependent manner (0–50 µM). Q-NLCs had more than two-fold dose advantage than free Q in decreasing the viability of those breast cancer cells. This is partially due to enhanced cellular uptake of Q by Q-NLCs. We also conducted pharmacokinetic studies using male Sprague-Dawley rats. After 1-week acclimation, rats were fasted overnight, weighed and randomly grouped, and received

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the following three treatments via oral gavage or intravenous injection: free Q, Q-NLCs and Q-CSNLCs dissolved in normal saline. Both Q-NLCs and Q-CSNLCs had significantly higher bioavailability than free Q. Q-NLCs and Q-CSNLCs also had significantly higher stability than free Q in blood and gastro-intestinal solutions. As shown in Table 39.2, other published studies also demonstrated that nanoencapsulated Q can increase the solubility, stability, bioavailability and bioactivities of Q, especially anti-cancer activities [45–48]. Table 39.2

The characteristics, findings, and application of quercetin nanoparticles

NP type size NLCs 32 nm

Experiment model/ Findings and dose/route potential application

MCF-7 and MDA-MB231 breast cancer cells were treated with 0–50 µM of free quercetin or nano-quercetin for 24 and 48 h.

NLCs 126.6 nm C57BL/6J mice were given 25 mg/kg body weight of free quercetin or nano-quercetin via oral administration.

PLGA 50 nm

Swiss Albino rats having ischemiareperfusion-induced neuronal damage were given 2.7 mg/kg body weight of free quercetin or nanoquercetin via oral gavage for 3 days.

Year [ref.]

2014  Aqueous solubility of [44] quercetin  Cellular uptake  Sustained release  Apoptosis of cancer cells  Viability of cancer cells Application in the breast cancer chemoprevention and treatment

 Sustained release  Bioavailability Application in oral delivery of quercetin

2014 [49]

 Anti-oxidant status Inducible nitric oxide synthase (iNOS) and caspase-3 activities  Neuronal count in the hippocampal subfields Application in the protection of ischemic neuronal damage

2013 [50]

Phytochemicals

NP type size SLNs & NLCs 282 nm

Experiment model/ Findings and dose/route potential application

Year [ref.]

In vitro permeation  Stability study using full  Sustained release thickness human skin  Quercetin concentrations in skin Application in topical delivery

2012 [51]

NLCs 215.2 nm In vitro skin permeation studies: Franz diffusion cells were treated with 1 mg/mL free quercetin or nanoquercetin for 1, 3, 6, 9 and 12 h. In vivo permeation study: male Kunming mice were given 1.0 mg/ mL of free quercetin or nano-quercetin via topical application for 3, 6, 9 and 12 h.

Nanomicelles 16 nm

Lung tumor mice were given 30 mg/kg body weight of free quercetin or nanoquercetin via oral gavage three times per week for three weeks.

Nanoliposomes C6 glioma cells were 62.3–191.5 nm treated with 0, 50, 100, 200 and 400 µM of free quercetin or nano-quercetin for 12, 24, 36, and 48 h.

PLGA 15 nm

Male Sprague-Dawley rats having gastric ulcer were given 2.5 and 50 mg/kg

 Quercetin concentrations in epidermis and dermis  Anti-oxidant and anti-inflammation Application in topical delivery

2012 [52]

 Stability 2012  Viability of cancer cells [46]  Tumor size Application in the lung cancer chemoprevention and treatment  Viability of C6 glioma 2012 cells [47]  Necrotic cell death Application in the brain cancer chemoprevention and treatment

 Inflammation 2012  Oxidative stress [13] Application in preventing gastric ulcer formation

(Continued)

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Table 39.2  (Continued) NP type size

SLNs 600 min

8.05 ± 5.03

22.70 ± 2.38

14.70 ± 7.15*

*The lower target to non-target ratio of PEG-antimyosin immunoliposomes, relative to anti-myosin-immunoliposomes in the infarct, is due to the higher blood activity of the former at 5 h post intravenous administration of liposome preparation.

Verma et al. [18] have selectively delivered ATP to the ischemic myocardium to protect the myocardial viability. Antimyosin

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antibody (2G4 antibody) was appended at the surface of liposomes as site directing receptor specific ligand. The study was performed in the isolated rat heart model. Results of the study showed that the use of ATP loaded immunoliposomes (ATP-IL) resulted in an effective protection to the myocardium under the ischemia/ reperfusion allowing for the substantial normalization of left ventricle developed pressure (LVDP) as well as left ventricle end diastolic pressure (LVEDP). The lowest LVDP recorded in the case of KH buffer control indicated that the myocardial function was severely compromised in the case of global ischemia. ATP-L also demonstrated significant recovery of both systolic as well as diastolic functions as compared to IL and KH perfusion buffer studies.

40.3 Novel Carriers for the Treatment of Thromboembotic Diseases

Site-targeted drug delivery holds promise in the treatment of vascular injury-associated thrombotic and occlusive events due to CVDs (e.g., atherosclerosis) or interventional procedure (e.g., angioplasty and stenting). On the contrary, conventional techniques are expensive and also they require experienced personnel [35]. Among various drug delivery systems, liposomes have been explored by various research groups for delivery of thrombolytic drugs [36, 37]. Liposomal encapsulation of plasminogen activators might increase efficacy, improve selectivity, possibly reduce or eliminate antigenic complications, retard systemic deactivation and effectively prolong the circulation half-lives of the agents [38]. In an in vitro experiment, Nguyen et al. [36] demonstrated the ability of liposomes to protect streptokinase in plasma without significant loss of activity. Plasminogen activators entrapped in liposomes have shown substantial decrease in the time required to obtain reperfusion and an increased digestion of thrombus as compared to free plasminogen activators perfusion. One of the theories proposed to increase the effectiveness of the liposomal plasminogen activators is the obstruction of channeling by the liposomes that results in a pressure at the leading edge of the thrombus. To prove this theory, Heeremans et al. [39] compared the thrombolytic activity of free t-PA to free t-PA + empty liposomes

Novel Carriers for the Treatment of Thromboembotic Diseases

in a jugular vein model in rabbit. Moreover, the clot lysis activity of free t-PA recorded was nearly equal to that of t-PA + empty liposomes with an equivalent dose. Thus, based on these studies, it was concluded that increased thrombolytic activity of the liposomal plasminogen activator was not due to the pressure created following the blockage of the channels in the thrombus. Moreover, it was suggested that liposomal encapsulation reduces the premature inactivation and prevents degradation of plasminogen activator in the plasma vis-a-vis releases it at the site of thrombus under shear stress. Erdogan et al. [40] developed three types of vesicular systems, (i.e., liposomes, niosomes, and sphingosomes) and evaluated their biodistribution using radiolabeled streptokinase and compared with intravenous injection of free streptokinase. Results demonstrated detectable amounts of streptokinase in the thrombi in all three types of vesicles, with higher amount at 4 h than 1 h. Recently, Baek et al. [41] developed streptokinase (SK) bearing liposomes for the subconjunctival delivery of thrombolytic agents in order to increase the local concentration of drug while decreasing the systemic absorption and/or side effect of the drug. Results of the study demonstrated lower ocular absorption of liposomal SK as compared to non-liposomal SK. Additionally, no detectable systemic and ocular side effects were observed in animals study. Liposomal formulations also enhanced the rate of subconjunctival hemorrhage (SH) absorption.

Figure 40.5 Schematic thrombus targeted drug delivery system.

For optimal drug delivery, the drug should be localized at the site of thrombus. It should avoid its non-specific uptake by or

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delivery to normal non-target tissues. Therefore, a well designed drug delivery system, surface modified with a targeting moiety for targeted delivery of thrombolytic agents may provide an effective alternate (Fig. 40.5). Based on these principles, specific ligandreceptor based approaches have been studied by various research groups [42]. In a recent review [5], we have discussed the role of integrin receptors in the targeted delivery of thrombolytic agents. To target GP IIb/IIIa integrin receptors on the activated platelets, Arg-GlyAsp (RGD) peptides have been explored by various groups as homing devices [43]. Lestini et al. [44] developed RGD-modified liposomes for the targeting of activated platelets. It was found that RGD-liposomes interact more efficiently with the activated platelets as compared to resting platelets. Thus, it was concluded that RGD-liposomes might be useful for the delivery of thrombolytic agents to the platelets embedded in the thrombus. Wang et al. [45] developed RGD peptide conjugated liposomes for the targeted delivery of urokinase to the site of thrombus and observed an increased thrombolytic efficacy. In this study, RGDS conjugated DSPE-PEG3500-COOH was incorporated in to the liposomal bilayer. The hydrophilic PEG imparts long circulation whereas RGD provides selectivity to the carrier system toward activated platelets. It was observed that developed systems could significantly improve the thrombolytic efficacy as compared to plain urokinase liposomes. Suggested mechanism for the improved thrombolysis is the selective release of urokinase at the site of thrombus thereby converting plasminogen in to plasmin and simultaneously reducing the bleeding complications. Later, conformationally constrained cyclic RGD peptide conjugated liposomes were synthesized for improved selectivity towards GP IIb/IIIa integrin receptors expressed on the activated platelets [35]. In a recent study, we have developed cyclic RGD [CNPRGDY(OEt)RC] peptide conjugated long-circulatory liposomes for the delivery of SK to the site of occlusion [46]. In vivo biodistribution study confirmed higher accumulation of modified liposomes to the thrombus area as compared to non-liposomal formulations. RGD-liposomes accumulated 2- and 5-fold higher as compared to non-targeted liposomes and non-liposomal formulation, respectively. In addition, in vivo thrombolysis study revealed higher clot dissolving capacity of RGD-liposomes as compared to plain SK solution. Results showed

Novel Carriers for the Treatment of Thromboembotic Diseases

that 60 min after treatment RGD-liposomes dissolved 34% of initial clot weight whereas non-liposomal SK formulation dissolves only 22% of the clot. Moreover, for the treatment of AMI, a rapid clot lysis is required so that tissue damage can be reduced. For increased and instant clot lysis, drug concentration should be high in the microenvironment of thrombus. Nevertheless, to achieve such concentration, a higher amount of drug is required to be administered, which may cause other circulatory side effects. Based on the hypothesis, target sensitive (TS) liposomes have been developed for the delivery of SK [47]. TS liposomes were prepared using dioleoylphosphatidyl ethanolamine (DOPE) and acylated cyclic RGD peptide. It was found that these liposomes not only specifically target the blood clot but also release drug instantly after interaction with activated platelets embedded in the clot. An in vitro clot dissolving study revealed that RGD conjugated TS liposomes could reduce the     clot lysis time and also could increase the total clot dissolution (Fig. 40.6). (a)

(b)

Figure 40.6 (a) Graphs showing percentage clot lysis with various SK formulations. % clot lysis for RGD liposomes are significantly higher (*p < 0.01, n = 3) than that of SK solution. Percentage clot lysis for RAD liposomes are significantly lower than that of SK solution (p < 0.001, n = 3) and RGD liposomes (**p < 0.001, n = 3). (b) Graphs showing clot lysis time (t50), time to dissolve 50% of clot wt., with various SK formulations. Clot lysis time for RGD liposomes are significantly shorter (*p < 0.01, n = 3) than that of SK solution. From ref. [47] with permission.

Although liposomal incorporation has proven to be an effective method for the delivery of thrombolytic drugs, however such delivery systems suffer a stability problem. To improve the stability, PEGylated (PEG coated) vesicles or polymeric carrier(s) have now being developed and studied for the delivery of various therapeutic

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proteins. Encapsulation in polymeric carriers avoids protein deactivation during systemic circulation and increases circulation in to an acceptable half-life. Leach et al. [48] developed both types of formulations [liposomal (LESK) and polymeric microcapsules (MESK)] and evaluated for various thrombolytic parameters. It was found that both the formulations could reduce reperfusion times, residual clot mass and improved return of flow compared to conventional dosages of free streptokinase in a thrombosed rabbit carotid. The MESK showed comparatively better results. In another experiment, the mechanism for increased thrombolysis by MESK was suggested; MESK resists adsorption to the leading edge of the thrombus, a common limitation for the permeation of free plasminogen activators. Thus, higher thrombolytic activity is due to improved penetration of the MESK to the interior of the clot [49]. Polymeric carriers for the delivery of thrombolytic agents should possess the following properties: (i) They should be small enough to circulate without the risk of vascular occlusion; (ii) they should possess anti-opsonizing properties; (iii) they should be tailored to accumulate at the site of thrombus; and (iv) they must release thrombolytic agents in sufficient concentration to induce and affect clot lysis. To confer these properties, Chiellini et al. [50] synthesized a polymeric material for the preparation of nanoparticles for targeting as well as long circulatory property. Developed carriers could releases the fibrinolytic agents in a controlled manner to fibrin clots [51]. The polymer was synthesized by covalent binding of poly(ethylene glycol) moieties and monoclonal antibody antifibrin Fab fragment to the polymer chain, Poly[(maleic anhydride)alt-(butylvinyl ether)]s. Moreover, size of the particles remains a major concern in their systemic administration and thereby drug delivery. Thus, polymeric nanoparticles were developed for the effective delivery of thrombolytic agents to the site of occlusion and subsequently to the interior of the clot. In the case of microparticles, it was hypothesized that the permeation/penetration of particles into the clot was due to pressure created at the clot. However, in the absence of hydrodynamic pressure, the pores of fibrin clots remain resistant to the permeation of carriers with a size of 1 μm or larger. Therefore, systems should be developed that permeate through the fibrin network into the interior of the clot for intra-clot lysis

Nanocarriers for the Treatment of Restenosis

even in the absence of pressure drop. Surface of the particles should also be modified to increase the interaction of particles with the components of the clot and thus favoring the permeation in to the interior of the clot as well. Chung et al. [52] developed t-PA loaded PLGA nanoparticles coated with chitosan (CS) and CS-GRGD for electrostatic interaction between the positive charge of chitosan and the negative charge of fibrin as well as for ligand-receptor interaction between RGD peptide and GP IIb/IIIa receptors expressed on the activated platelets. A thrombolysis study in a blood clot-occluded tube model revealed that PLGA/CS nanoparticles showed the shortest clot lysis time whereas PLGA/CS-GRGD nanoparticles showed the highest weight percentage of digested clots. The permeation results of the NPs in the blood clots demonstrated that PLGA/CS-GRGD NPs were more adhered to the clot front and aggregated in the interior of the clots compared to other nanoparticles evaluated.

40.4 Nanocarriers for the Treatment of Restenosis

Restenosis may be defined as the process of re-obstruction of an artery following interventional procedures such as angioplasty, atherectomy, or stenting. Restenosis is a complex process that is thought to be possibly triggered by blood vessel wall injury that occurs during an intervention to relieve an arterial obstruction (Fig. 40.7) [53]. Mechanisms contributing to restenosis include elastic recoil, smooth muscle cell migration and proliferation, enhanced extracellular matrix synthesis, vessel wall remodeling, and thrombus formation [54]. A variety of different classes of drugs have been tested for the prevention and treatment of restenosis (Table 40.2). Restenosis may be prevented by delivering drugs in high concentrations for a prolonged period of time. Local drug delivery is the most favorable treatment option for restenosis because it increases localized drug concentration to and above therapeutic levels in the immediate vicinity of vascular injury with minimal or the absence of systemic side effects [55]. However, studies revealed that direct infusion of drug solutions into the arterial wall using infusion catheters fail to offer and maintain adequate therapeutic levels into the arterial wall over time. Most of the drug from the

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  Figure 40.7 Schematic illustration of the processes leading to restenotic lesion development. The figures show a healthy blood vessel (A), formation of atherosclerotic plaque within the blood vessel showing a fatty streak and macrophages encapsulated within a fibrotic tissue (B), insertion of a balloon angioplasty catheter to remove the plaque (C), damage due to stripping of the endothelial cells of the vessel wall after removal of the balloon (D), platelet accumulation and activation as well as rapid growth of smooth muscle cells and fibrous extracellular matrix forming the scaffolding (E), and the late stage restenosis showing neointima protruding into the lumen causing occlusion within the vessel (F).

Table 40.2

Examples of drugs for prevention and treatment of restenosis

Drug action

Examples

Smooth muscle cell growth inhibition

Cytarabine, Doxorubicin, Vincristine, Cyclosporine A, Paclitaxel

PDGF-receptor specific

AG-1295, AGL-2043

Immunosuppressant

Methyl prednisolone, Dexamethasone, Sirolimus, Everolimus, Tacrolimus, Mycophenolic acid

Inflammatory response inhibition Clodronate, Alendronate, Pamidronate, ISA Antiplatelet

Anticoagulant

Cilostazol, Abciximab, Eptifibatide, Argatroban, Clopidogrel, Ticlopidin

Heparin, Hirudin, Iloprost

Nanocarriers for the Treatment of Restenosis

infusion site is washed out within an initial period of 20–30 min with a very small residual amount lasting for a maximum period of 48 h [56, 57]. Thus, colloidal drug carriers including liposomes and nanoparticles have been developed to provide local drug release and sustained retention of drug in the arterial wall [58]. These carriers offer a potentially improved delivery system, since they incorporate the advantages of local drug delivery and, in addition, enable drug targeting to the specific cells [59]. Particle size has been considered to be an important determinant for the effective arterial uptake. It was found that small particles (100 nm) exhibit 3-fold higher accumulation into the artery as compared to larger particles (266 nm). In a different study, it was also observed that small particles penetrate deep in the arterial wall; however, large particles accumulate at the luminal surface of arterial wall. Further, residence time of the particles also depends on the size. The smaller particles (90 nm) showed better ingress into the arterial tissue than the large particles (160 nm), as indicated by a 3.4-fold higher initial drug concentration in the tissue, whereby more than eight-fold higher amount of nanoparticles remained associated with the artery following the rapid washout phase and higher local drug levels were attained as well as maintained over 14 days following administration [60]. It is also well established that the surface charge of the particles influences the accumulation into the arterial wall. For example, it was observed that nanoparticles surface-modified with a cationic compound didodecyldimethylammonium bromide (DMAB), demonstrated 7- to 10-fold greater U-86 arterial levels in comparison to unmodified nanoparticles in different ex vivo and in vivo studies [61]. Balloon-injured coronary arteries also exhibit enhanced permeability analogous to a tumor and it was suggested that the enhanced permeability effect may also be a suitable means for the delivery of antirestenotic drugs to the injured arterial wall. Based on this hypothesis, Uwatoke et al. [62] developed nanoparticles of PEG-based block copolymer (polymeric micelles) for the encapsulation of doxorubicin (NK911). The NK911 accumulated in the vascular lesion; the tissue concentrations of doxorubicin were up to four-fold higher three hours following intravenous injection of NK911 in comparison to free doxorubicin. Low drug levels were recorded in the normal contralateral arteries. The

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intravenous injected NK911 administered immediately, three and six days after the balloon injury, but not doxorubicin alone, significantly inhibited restenosis in the rat carotid artery as recorded at four weeks after injury in both single and double injury models. It was demonstrated by immunostaining that the antirestenotic efficacy of doxorubicin nanoparticles was achieved by the inhibition of SMCs proliferation, rather than due to enhancement of apoptosis or inhibition of inflammatory cell recruitment. Systemic therapy may another approach for the effective treatment of restenosis. Numerous studies revealed the effective use of nanocarriers for the systemic therapy of restenosis [63–65]. Systemic delivery of antirestenotic drugs may be achieved by two methods; passive targeting of the arterial wall and/or active targeting of the carriers to the affected surface. The former might be achieved by controlling the properties of the carriers; however, for the latter approach, the surface of the particles should be appropriately modified using a tissue specific ligand. A systemic approach, however failed to produce satisfactory results for the treatment of restenosis that may be ascribed to the low levels of drug in the injured artery [59]. Recently, better understanding of the involvement of innate immunity in the progression of restenosis evolved a new strategy for the systemic treatment of restenosis [66]. Monocytes/macrophages play a key role in the inflammation cascade and thus the progression of restenosis and comprise up to 60% of neointimal cells after stent-induced arterial injury in various animal models as well as in human autopsy specimens [67]. Following a stimulus, such as inflammation, the amount of circulating monocytes and their migration to the site of inflammation significantly increases [68]. Therefore, systemic inactivation of monocytes and macrophages may lead to the attenuation of neointimal formation. Various nanocarriers including liposomes and polymeric nanoparticles have been used for the targeted delivery of bisphosphonates to the macrophages. Studies demonstrated that systemically administered carriers reduced the level of systemic monocytes and tissue macrophages leading to the low accumulation in the arterial wall. Therefore, SMCs migration and proliferation were reduced as a result. Various nanocarriers used for the delivery of antirestenotic drugs, either locally or systemically, are summarized in Table 40.3.

PLGA NPs

Liposomes

Results of the study

Aminobisphosphonate (ISA)

Alendronate

Sirolimus

Estradiol benzoate (ESB)

Significant attenuation of intimal hyperplasia and stenosis in rat carotid injury model

Inhibited macrophage build up, reducing restenosis in balloon injured hypercholesteremic rabbit carotid artery model

Local delivery of liposomal sirolimus showed a significant reduction in the neointimal formation, the neointimal area to media area ration and the percentage of stenosis as compared to controls

In vivo studies in the balloon-injured rat carotid arteries revealed the potential of ESB-loaded liposomes as efficient local and controlled drug delivery systems to reduce restenosis

10 times more potent inhibitor of cytokine secretion from RAW 264 Bisphosphonates (clodronate, pamidronate, as compared to free drug alendronate) Significant attenuation of intimal hyperplasia and luminal stenosis in balloon injured rabbit model

Drugs encapsulated/ loaded

Examples of nanocarriers used for the delivery of anti-restenotic drugs

Carriers used

Table 40.3

(Continued)

[75]

[74]

[73]

[72]

[69–71, 66]

Refs.

Nanocarriers for the Treatment of Restenosis 1183

Polymeric micelles

Albumin NPs

PVA-g-PLGA

PLA NPs

Carriers used

Doxorubicin

Paclitaxel

Paclitaxel

Sirolimus

Tyrphostins (AG-1295, AGL-2043)

U-86 2-aminochromone

Dexamethasone

Drugs encapsulated/ loaded

Table 40.3 (Continued)

Significant inhibition of SMCs proliferation in balloon injured rat carotid artery model

Reduced neointimal growth in NZ white rabbit model

50% reduction in neointimal area in vessel segments of balloon injured rabbit iliac artery

Results of the study exhibited the potential of sirolimus loaded PLA NPs on promising local and sustained delivery systems administered intraluminary to reduce in-stent restenosis after stent implantation

NP of AGL-2043 exhibited higher antirestenotic efficacy compared to AG-1295 NP

Significant antirestenotic effect was observed in pig model

Significant reduction in neointimal hyperplasia development in balloon injured porcine coronary artery

Significant decrease in the intima/media ratio in rat carotid artery model

Results of the study

[62]

[81]

[57]

[80]

[60, 79]

[78]

[76, 77]

Refs.

1184 Designing Nanocarriers for the Effective Treatment of Cardiovascular Diseases

Abbreviations

40.5 Concluding Remarks Nanocarriers have been widely investigated and commercialized for the delivery of various therapeutic agents for the effective treatment of various disease states. Currently, numerous approved nanoparticle-based therapeutics are in clinical use, validating their ability to improve the therapeutic index of drugs [82–86]. Use of these carriers for the treatment of CVDs is an emerging area of biomedicine. Nanocarriers (e.g., liposomes, polymeric nanoparticles and micelles) have been extensively tested in various preclinical studies with promising results. Thus, based on results of these studies it can be concluded that nanocarrier-based therapeutic delivery could provide significant advantages over conventional approaches for the treatment of CVDs. As discussed in this chapter, various properties of nanocarriers and specific characters of a disease state are used for the designing of a system for delivery of drugs to that diseased area. Thus, in future, the designing of novel nanocarriers with respect to their size, shape and surface properties should offer useful approaches in the clinic for the effective treatment of various CVDs, including myocardial infarction, restenosis, and thromboembolism.

Abbreviations

CVDs: Cardiovascular diseases VTE: Venous thromboembolism DVT: Deep vein thrombosis PE: Pulmonary embolism US FDA: United States Food and Drug Administration AMI: Acute myocardial infarction ATP: Adenosine triphosphate EPR: Enhanced permeability and retention PEG: Polyethylene glycol LVDP: Left ventricle developed pressure LVEDP: Left ventricle end diastolic pressure t-PA: Tissue plasminogen activator SK: Streptokinase RGD: Arg-Gly-Asp DOPE: Dioleoylphosphatidyl ethanolamine PLGA:­ Poly (lactide-co-glycolide)

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Disclosures and Conflict of Interest The authors declare that they have no conflict of interest and have no affiliations or financial involvement with any organization or entity discussed in this chapter. This includes employment, consultancies, honoraria, grants, stock ownership or options, expert testimony, patents (received or pending) or royalties. No writing assistance was utilized in the production of this chapter and the authors have received no payment for its preparation. The findings and conclusions here reflect the current views of the authors. They should not be attributed, in whole or in part, to the organizations with which they are affiliated, nor should they be considered as expressing an opinion with regard to the merits of any particular company or product discussed herein.

Corresponding Author

Prof. Suresh P. Vyas Drug Delivery Research Laboratory Department of Pharmaceutical Sciences Dr. H. S. Gour University, Sagar, MP 470003, India Email: [email protected]

About the Authors

Bhuvaneshwar Vaidya is an associate professor in the Department of Pharmaceutics, ISF College of Pharmacy, Moga, Punjab, India. He obtained his BPharm, MPharm, and PhD from the Department of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar, India. He has previously worked as a research scientist in the Research and Development division of a pharmaceutical company. He has over 7 years of research and teaching experience. He has published more than 30 papers and has presented research papers at both national and international conferences. Dr. Vaidya has also contributed seven chapters in various books. His current area of research is controlled and targeted delivery of therapeutics for the treatment of diseases, including cardiovascular diseases, cancer, and infectious diseases.

References

Suresh P. Vyas is a professor of pharmaceutical biotechnology in the Department of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar, India. He has contributed substantially to research on novel drug delivery systems, drug targeting and nanobiotechnology involving colloidal drug carriers customized for targeted drug and vaccine delivery. He has over 33 years of teaching and research experience in the field of pharmaceutical sciences. His current focus is on the non-invasive, non-conventional delivery of bioactives for targeted therapy of diseases. Prof. Vyas has published over 310 papers, supervised 51 PhD students and 125 MPharm students, authored 16 books and contributed numerous book chapters. He is a Commonwealth Postdoctoral Fellowship awardee and has worked under fellowship at the School of Pharmacy University of London (UK). Prof. Vyas is a recipient of Pandit Lajja Shankar Jha award in the area of science and technology for the excellence in research from Government of Madhya Pradesh, India. In 2001, he received the Best Pharmacy Teacher of the Year award by the Association of Pharmaceutical Teachers of India. He currently serves on the editorial boards of International Journal of Pharmaceutics (Elsevier), Recent Patents on Drug Delivery and Formulation (Bentham), Therapeutic Delivery (Future Science), Journal of Drug Delivery (Hindawi), and Journal of Nanoscience Letters (Simplex publishers).

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78. Humphrey, W. R., Erickson, L. A., Simmons, C. A., Jennifer, L. N., Donn, G. W., et al. (1997). The effect of intramural delivery of polymeric nanoparticles loaded with the antiproliferative 2-aminochromone U-86983 on neointimal hyperplasia development in balloon-injured porcine coronary arteries. Adv. Drug Deliv. Rev., 24, 87–108. 79. Banai, S., Chorny, M., Gertz, S. D., Fishbein, I., Gao, J., et al. (2005). Locally delivered nanoencapsulated tyrphostin (AGL-2043) reduces neointima formation in balloon-injured rat carotid and stented porcine coronary arteries. Biomaterials, 26, 451–461.

80. Luderer, F., Löbler, M., Rohm, H. W., Ocke, C., unna, K., et al. (2011). Biodegradable sirolimus-loaded poly(lactide) nanoparticles as drug delivery system for the prevention of in-stent restenosis in coronary stent application. J. Biomater. Appl., 25, 851–875. 81. Kolodgie, F. D., John, M., Khurana, C., Farb, A., Wilson, P. S., et al. (2002). Sustained reduction of in-stent neointimal growth with the use of a novel systemic nanoparticles paclitaxel. Circulation, 106, 1195–1198. 82. Mansour, H. M., Park, C.-W., Bawa, R. (2015). Design and development of approved nanopharmaceutical products. In: Bawa, R., Audette, G.,

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83. Bawa, R. (2008). Nanoparticle-based therapeutics in humans: A survey. Nanotechnol. Law Bus., 5(2), 135–155.

84. Bawa, R. (2013). FDA and nanotech: Baby steps lead to regulatory uncertainty. In: Bagchi, D., et al., eds. Bionanotechnology: A Revolution in Biomedical Sciences and Human Health. Wiley Blackwell, UK, pp. 720–732. 85. Etheridge, M. L., Campbell, S. A., Erdman, A. G., Haynes, C. L., Wolf, S. M., McCullough, J. (2013). The big picture on nanomedicine: The state of investigational and approved nanomedicine products. Nanomed. Nanotechnol. Biol. Med., 9(1), 1–14.

86. Cheng, Z., Zaki, A., Hui, J. Z., Muzykantov, V. R., Tsourkas, A. (2012). Targeting and imaging capabilities of multifunctional nanoparticles: Cost versus benefit of adding targeting and imaging capabilities. Science, 338, 903–910.

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Chapter 41

Carbon Nanotubes as Substrates for Neuronal Growth Cécilia Ménard-Moyon, PhD CNRS, Institut de Biologie Moléculaire et Cellulaire, Laboratoire d’Immunopathologie et Chimie Thérapeutique (UPR 3572), France

Keywords: adhesion, carbon nanotubes, differentiation, electrical stimulation, electrodes, functionalization, growth, neurites, neurons, substrate

41.1 Introduction Neurons are essential for the processing and transmission of cellular signals. A neuron consists of a central part known as the soma (or cell body) and long processes called neurites, constituted of axons and dendrites, extending over long distances (Fig. 41.1). The soma contains the nucleus of the cell. The growth of neurites and the formation of synapses are controlled by a highly motile structural specialisation at the tips of the neurites called the growth cones. Each neuron has multiple dendrites that carry signals into the soma and a single axon that carries signals away from the soma towards the next neuronal cell. The movement of Handbook of Clinical Nanomedicine: Nanoparticles, Imaging, Therapy, and Clinical Applications Edited by Raj Bawa, Gerald F. Audette, and Israel Rubinstein Copyright © 2016 Pan Stanford Publishing Pte. Ltd. ISBN 978-981-4669-20-7 (Hardcover), 978-981-4669-21-4 (eBook) www.panstanford.com

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ions across the ion channels found on the soma and the axon is driven by electrochemical gradients. It generates electrical signals called action potentials, which normally travel along the axon in one direction, away from the soma and towards the next neuron [1].

Figure 41.1 Structure of a typical myelinated vertebrate motoneuron.

Because of the lack of effective self-repair mechanisms in adults, central nervous system damage results in functional deficits that are often irreversible. The difficult challenge is to find means to cure the disabilities arising from injuries and disorders of nervous systems by stimulating inactive neurons and regulating their growth in a proper way. In order for neural prostheses to augment or restore damaged or lost functions of the nervous system, they need to be able to perform two main functions: stimulate the nervous system and record its activity. For this purpose, nanotechnology offers new perspectives by providing possibilities of repair [2]. Many crucial steps are necessary for a neuron to rebuild a functional network: (i) survival to the injury, (ii) regrowth of neurites (axons and dendrites) and (iii) reconstruction of active synapses that connect neurons [3a]. Therefore, any therapeutic strategy should promote each of these

Effects of Carbon Nanotubes

steps. Nanotechnology and nanomaterials offer exciting promises in neuroscience [3b, 3c]. In particular, the unique combination of physical, chemical, mechanical and electronic properties of carbon nanotubes (CNTs) makes them very attractive in basic and applied neuroscience research because of their small size, electrical conductivity, high flexibility, mechanical strength, inertness, nonbiodegradability and biocompatibility brought about by surface functionalisation [4]. Nevertheless, methods for manipulating the neuronal growth environment at the nanometer scale are still lacking. CNTs have been found to be promising substrates for neuroscience applications. Single-walled carbon nanotube (SWNT) bundles and multi-walled carbon nanotubes (MWNTs) possess diameters that can mimic neuronal processes. In particular, the aspect ratio is similar to that of small nerve fibres, growth cone filopodia and synaptic contacts. Moreover, the high electrical conductivity of CNTs should enable the detection of neuronal electrical activity and allow delivering electrical stimulation to neuronal cells in contact with them. In this chapter, we will present the use of CNTs as substrates for neuronal growth, favouring adhesion of neurons and their survival, growth and differentiation in neurites. We will also detail the use of CNTs to electrically stimulate neuronal cells as well as the influence of CNTs in promoting spontaneous synaptic activity in neuronal networks and in increasing the efficacy of neural transmission. We will then close this chapter by reporting the studies that investigated the mechanisms of the electrical interactions between CNTs and neurons.

41.2 Effects of Carbon Nanotubes on Neuronal Cells’ Adhesion, Growth, Morphology and Differentiation

As it will be described in this part, numerous studies have revealed that CNTs can support and control the growth of neuronal cells as well as their morphology. SWNTs and MWNTs have been found to be permissive substrates for neuronal growth characterised by the presence of growth cones, neurite outgrowth and branching.

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The first use of CNTs as substrates for nerve cell growth was reported by Mattson and coworkers [5]. In this study, embryonic rat hippocampal neurons were grown on MWNTs dispersed on glass coverslips coated with polyethyleneimine (PEI), which is a common substrate for neuronal growth [6]. Neurons require highly permissive substrates for cell attachment and neurite growth, which include positive-charge modification of glass and plastic culture substrates (e.g., polylysine or polyornithine [PLO]) [7] for optimum growth and neurite extension. Two types of MWNTs were utilised to investigate neuronal growth: pristine MWNTs and MWNTs coated with 4-hydroxynonenal (4-HNE), a molecule that effects neurite growth. 4-HNE is known for its ability to regulate neurite outgrowth in cultured embryonic hippocampal neurons [8] via modulation of intracellular Ca2+ levels in cultured hippocampal neurons [9]. Physisorption of 4HNE on the MWNT surface was achieved by sonicating MWNTs in an acidic solution of 4-HNE. Mattson et al. observed that neurons grown on MWNTs survived and continued to grow for at least eight days in culture, indicating that MWNTs support long-term neuronal survival and provide a permissive substrate for neurite outgrowth. It was observed that the direction of growth was not influenced by MWNTs. Scanning electron microscopy (SEM) was used to identify the morphological changes of the neurons grown on the MWNT substrate. Neurons were seen to be attached to the pristine MWNTs while extending one or two neurites. Interestingly, when neurites grew across the MWNTs and then on the glass coverslips coated with PEI, the neurites formed branches on the coverslips but not on the MWNTs. Therefore, the pristine MWNTs did not promote neurite branching, indicating a relatively weak adhesion of growth cones to the surface of non-functionalised nanotubes. This observation suggested that pristine MWNTs were not a suitable support for branch formation. Nevertheless, MWNTs coated with 4-HNE had more and longer neurites (Fig. 41.2). Both neurite outgrowth and branching were enhanced in this case. The authors pointed out that the enhanced adhesion of growth cones to the MWNTs could be favoured by the presence of 4-HNE that could possibly induce changes in intracellular Ca2+ levels. Indeed, Ca2+ influx can regulate growth cone motility and neurite elongation [10].

Effects of Carbon Nanotubes

Figure 41.2 SEM images of neurons grown for three days on CNTs coated with 4-HNE. The right image is a high magnification of the neurite designated by the black arrow in the left image. Scale bars: left image, 5 µm; right image, 100 nm. Reproduced from Mattson et al. [5] with permission.

Further studies were conducted by Haddon et al., and they used MWNTs functionalised with molecules bearing different electrostatic charges to study neuronal growth [11]. The results showed that the neurite outgrowth was controlled by the surface charge of MWNTs. O

O

OH

Oxalyl chloride

Cl

0 °C to reflux

1

EN, DMF 100 °C, 5 days O N H

NH2

2

A

B

C

PABS, DMF 100 °C, 5 days

D

PEG, DMF 100 °C, 5 days

PEI, NMP r.t., 6 h Reflux, 24 h

O

(O

6

3 )

O H

4

n

H2N

O

n

(N

) OH

(N

SO3H

H

N

)

n

5

Scheme 41.1 Functionalisation of CNTs with EN, PABS, PEI and PEG.

Three types of functionalised MWNTs were designed to carry negative, neutral or positive charges at physiological pH. The approach relied on covalent functionalisation of MWNTs rather than on physisorption (used in the previous study) [5] to allow transient retention of grafted molecules on the nanotube surface. MWNTs were first oxidised using nitric acid, and the resulting

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COOH functions introduced on the nanotube ends were then activated and coupled with either ethylenediamine (EN) or polym-aminobenzene sulphonic acid (PABS) [12], as illustrated in Scheme 41.1 (paths A and B).







Scheme 41.2 The effects of pristine and functionalised CNTs on neurite   outgrowth and number of growth cones in comparison with PEI. Reproduced with permission from Hu et al. [11] (a), Hu et al. [14] (b), and Ni et al. [15] (c) with permission.

At physiological pH used to grow neurons, the MWNTs exhibited different surface charges, from negatively charged MWNTCOOH (1), neutral/zwitterionic MWNT-PABS (4), to positively charged MWNT-EN (3). Finally, MWNT films were deposited on glass coverslips coated with PEI, and hippocampal neuronal cells were cultured on these substrates. In addition to SEM analysis, the morphological features of live (rather than fixed) neurons, which directly reflect their potential capability in synaptic

Effects of Carbon Nanotubes

transmission, were characterised by fluorescence spectroscopy. The neurons were labelled with calcein, which is a fluorescent dye that stains only live cells. All neurons that grew on PEI and on MWNT substrates accumulated calcein, which is indicative of their viability. The number of neurites per neuron remained the same for the three substrates (Scheme 41.2a) [11, 15]. However, the average length of neurites was longer on the positively charged MWNT-EN (3), while the number of growth cones was higher on neurons cultured on zwitterionic MWNT-PABS (4) or on positive MWNT-EN (3). Furthermore, the branching of neurites increased as the substrate became more positive. Thus, by varying the surface charge of CNTs it was possible to control the number of growth cones, neurite outgrowth and branching. The use of SWNT composite as scaffold for neuronal growth was also reported by Haddon and coworkers [13]. On the basis of the observations from the previous study showing that neurite outgrowth was enhanced by using positively charged MWNTs [11], a composite constituted of SWNTs functionalised with PEI polymer was prepared. PEI is commonly used as permissive substrate for neuronal growth, and it is positively charged at physiological pH [6]. The precoating of glass coverslips with PEI in the previous studies was necessary because the adhesion of MWNT films on non-coated glass coverslips was low when the films were exposed to aqueous culture medium [5, 11]. Functionalisation of CNTs with PEI should increase their ability to support neurite outgrowth, whereas the precoating of coverslips with PEI could be eliminated. SWNTs were functionalised using a methodology that was comparable to the previous study [11]. PEI (branched PEI, n ≈ 20) was covalently grafted on SWNT-COOH by amidation via oxalyl chloride activation of the carboxylic acid functions (Scheme 41.1, path C). Then, SWNT-PEI films were deposited on glass coverslips, and neuronal growth of hippocampal cells was monitored. The neuronal growth was visualised by SEM, and viability was determined using calcein staining. Neurons were shown to grow on the SWNT-PEI films with neurite outgrowth and branching that are intermediate to those observed for neuronal growth on PEI and as-produced MWNTs (AP-MWNTs). Indeed, the neurite branching on SWNT-PEI was enhanced by comparison with AP-

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MWNTs, while it was comparable to PEI. The number of neurites and growth cones was similar to that of AP-MWNTs, and the neurite lengths were intermediate to those of neurons grown on pristine MWNTs and PEI (Scheme 41.2b). Hence, the growth parameters were found to be sensitive to the nature of the substrate, in particular the surface charge, since the effects of the SWNT-PEI composite on neuronal growth characteristics are intermediate to those observed when using pristine MWNTs and PEI. This behaviour can be explained by the reduced positive charge of PEI, which is proportional to the percentage of SWNTs in the composite. It should be noted that the results obtained in this study with SWNT-PEI were compared with those obtained with pristine MWNTs, not with unmodified SWNTs. Hence, the authors pointed out that the structural parameters of CNTs, in particular the diameter, could also contribute to the observed effects, in addition to the contribution of PEI. In summary, neurite outgrowth and branching could be controlled by varying the ratio of SWNTs and PEI in the graft copolymer. This composite could be implemented in building scaffolds for the formation of neuronal circuits to develop neural prostheses. Haddon et al. also investigated the possibility of using watersoluble SWNTs as substrate for neuronal growth [14]. The SWNTs soluble in water were found to induce an increase of neurite length, while a decrease of the number of neurites and growth cones was observed. SWNTs were functionalised with either PABS [11] or polyethylene glycol (PEG, n ≈ 13) [15] to form the corresponding graft copolymers (Scheme 41.1, paths B and D). Each polymer imparted water solubility to the SWNTs. The methodology used for the functionalisation was based on amidation of the COOH functions located at the nanotube ends. Hippocampal neuronal cells were cultured on the water-soluble SWNT substrates. The neurons accumulated the vital stain calcein, which is indicative of cell viability and of the biocompatibility of the functionalised SWNTs. The copolymers were found to modulate neurite outgrowth by increasing their length, while reducing the number of neurites and growth cones (Scheme 41.2c). Hence, the neurons treated with the water-soluble SWNTs exhibited sparser, but longer neurites.

Effects of Carbon Nanotubes

The authors suggested that water-soluble SWNTs may modulate intracellular Ca2+ homeostasis as Ca2+ influx is known to regulate neurite elongation. Indeed, Ca2+ channel blockers can cause at low concentrations a simultaneous reduction of growth cone filopodia and an increased elongation of neurite [8a]. The same modifications were induced in this study by the water-soluble SWNTs. To confirm this hypothesis, the intracellular Ca2+ levels in cultured individual neurons were monitored. The experiments involved depolarisation of neurons via HiK+-induced intracellular Ca2+ accumulation in neurons. PEG-functionalised SWNTs were found to provoke a reduction of the intracellular Ca2+ accumulation in a dose-dependent manner. Therefore, the SWNT-PEG copolymer could act as blocker of Ca2+ channels by inhibiting the depolarisationdependent influx of Ca2+. Evidence that CNTs can affect the function of ion channels, such as potassium channels, has already been reported [16]. The influence of the electrical conductivity of CNT films on the neuronal growth characteristics was recently studied by Haddon and coworkers [17]. The CNT films were prepared by spraying an aqueous dispersion of SWNT-PEG with an airbrush onto glass coverslips heated at 160°C. The electrical conductivity was controlled by varying the thickness of the nanotube film. Hippocampal rat neurons were then cultured on these substrates. The positive calcein labelling of cells was indicative of cell viability. By comparison with PEI-coated coverslips used as control, the total outgrowth of each neuron (i.e., summed length of all processes and their branches) was superior in neurons grown on the 10 nm thick SWNT-PEG films. However, other parameters of neuronal growth such as the total number of processes and neurites originating from the cell body were unchanged for each neuron. In addition, by increasing the nanotube film thickness to 30 and 60 nm, thus allowing for higher conductivity, these effects disappeared since the neurite outgrowth was unaffected. In summary, Haddon et al. [17] demonstrated that SWNTbased substrates, having a narrow range of electrical conductivity, supported neurite outgrowth, with a decrease in the number of growth cones but an increase in cell body area. Other research groups also reported the use of CNTs as substrate for neuronal growth. Shimizu et al. functionalised MWNTs

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with neurotrophins (a family of proteins that induce survival, development and functions of neurons), in particular with a nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) [18]. The aim of this study was to regulate the differentiation and survival of neurons. Neurotrophins are key proteins for the differential function of neurons. They are endogenous soluble proteins regulating the survival [19], growth and function [20] of neurons. Neurotrophins belong to a class of growth factors, secreted proteins, which are capable of signalling particular cells to survive, differentiate or grow [21]. They may stimulate the synthesis of proteins in either axonal or dendritic compartments, thus allowing synapses to exert local control over the complement of proteins expressed at individual synaptic sites [22]. MWNTs were first oxidised using a mixture of sulphuric acid and nitric acid. Diaminoalkyl compounds were then introduced on the resulting MWNT-COOH via amidation. Neurotrophin was covalently bound to the resulting amino-modified MWNTs using a carbodiimide reagent. The influence of neurotrophin-functionalised MWNTs on the neurite outgrowth of embryonic chick dorsal root ganglion neurons was then examined. These MWNTs were found to promote the neurite outgrowth of dissociated neurons, in a similar manner to soluble NGF and BDNF, while the amino-terminated MWNTs did not promote neurite outgrowth. Romero and coworkers reported the preparation of strong and highly electrically conducting semi-transparent sheets and yarns from pristine MWNTs [23]. The CNT sheets were fabricated by drawing the CNTs from a sidewall of an MWNT forest produced by chemical vapour deposition (CVD). The thickness of the CNT sheet was approximately 50 nm. The CNT yarn was then prepared by spinning the sheet [24]. The authors demonstrated that the CNT sheets were permissive substrates for primary central and peripheral neuronal culture. Indeed, the results indicated that the CNT substrates were able to promote cell attachment and differentiation of a variety of cell types, including cerebellar and sensory dorsal root ganglion neurons, and to support their long-term growth at a similar level to that achieved on PLO-coated glass substrates used in this study for comparison. The neuronal phenotype was demonstrated by

Effects of Carbon Nanotubes

immunofluorescence using β-tubulin [25], which is a specific neuronal marker. In addition, the extension of neuronal axon growth cones was enhanced. Neurons from murine cerebellum and cerebral cortex, which are usually more sensitive to the substrate and the environment for survival and growth, were used too. Also in this case, the neurons were able to dissociate and extend their neurites onto the CNT sheets, as they did similarly onto PLO-coated glass substrates [26]. Thus, contrary to previous studies reporting that axonal growth is limited on pristine CNTs [5, 11], neurons were shown here to grow on pristine CNT sheets and to extend their neurites with similar characteristics (number and length) than those grown on PLO-treated glass substrates. Furthermore, the authors demonstrated that the neuronal growth could be directed by CNT yarns. Neonatal dorsal root ganglion neuron explants were prepared by wrapping a single CNT yarn around the ganglia, where the free end suspended in culture media. The sensory neurons adhered and extended along the CNT yarn by intimately following the surface topography of the CNT substrate. In summary, directionally oriented pristine CNTs configured as sheets or yarns were demonstrated to be viable substrates for neuronal growth. Lu et al. recently reported that thin-film scaffolds constituted of a biocompatible polymer grafted on CNTs can promote neuron differentiation from human embryonic stem cells (hESCs) [27]. The CNT-based composite was prepared by in situ polymerisation of acrylic acid onto oxidised CNTs. The neuron differentiation efficiency on poly(acrylic acid)-grafted CNT (PAAg-CNT) thin films was compared with that of thin films composed of poly(acrylic acid) (PAA) or PLO. The PAA-g-CNT thin films showed enhanced neuron differentiation while maintaining cell viability, as assessed by measuring the metabolic activity of dehydrogenases. SEM images showed that the differentiated neurons on PAA-g-CNT surfaces have more branches, suggesting that they are more mature. In addition, the CNT-based surface exhibited a higher cell adhesion and protein (laminin) adsorption. Laminin is a glycoprotein found in the extracellular matrix, the sheets of protein that form the substrate of all internal organs (i.e., basement membrane). Laminin has been reported to favour neuronal growth

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[28]. Indeed, laminin is a substrate along which nerve axons can grow both in vivo and in vitro. Thus, this work shows for the first time that CNT-based polymer thin films promote the differentiation of hESCs towards neuronal lineage. The growth of somatosensory neurons on functionalised CNT mats was recently reported by Xie et al. [29] MWNTs were functionalised by oxidation using a mixture of sulphuric acid and nitric acid. The CNT mat was then deposited on a tracketch membrane. The CNT substrate was found to be a permissive substrate for the growth of somatosensory neurons from a dorsal root ganglion. Neurite outgrowth and branching were observed by SEM, as well as intertwinement between the neurites and the underlying functionalised CNTs, indicating a strong interaction between both entities at the nanoscale. No obvious neurite growth was observed on neurons plated on a blank polycarbonate membrane. The authors suggested that functional groups on the nanotube surface could act as anchoring seeds which may enhance the adhesion of neurites on the CNT-based substrate. This is consistent with previous studies, such as those reported by Haddon and coworkers, that emphasise the positive impact of the functionalisation of CNTs on permissivity for neuronal attachment and neurite outgrowth [11, 14, 15]. Neurons adhere to substrates via extracellular proteins such as laminin, whose size is about 70 nm [30]. The authors pointed out that the analogous dimension of CNTs and the roughness of the nanotube surface favour contact with neurites by promoting better adhesion. Wick et al. recently investigated the effects of SWNTs with different degrees of agglomeration on primary cells of the nervous system (mixed neuroglial cultures derived from the chicken embryonic spinal cord [SPC] of the central nervous system or dorsal root ganglia [DRG] of the peripheral nervous system) [31]. The cells were exposed to SWNT agglomerates of submicrometre sizes (SWNT-a) and to SWNT bundles (SWNT-b) composed of 10 to 20 tubes. Suspensions of SWNTs-a and SWNTs-b affect glial cells from both tissue types. The level of toxicity was found to be dependent on the agglomeration state of the CNTs. Treatment of mixed neuroglial cultures with up to 30 μg/mL SWNTs significantly decreased the overall DNA content. This effect was more pronounced when

Electrical Stimulation of Neuronal Cells Grown on CNT-Based Substrates

cells were exposed to highly agglomerated SWNTs-a compared with better-dispersed SWNTs-b. Indeed, higher concentrations of SWNTs-a were more toxic than the same amount of SWNTs-b. This result is in agreement with previous studies from Wick et al. in which the aggregation state of SWNTs might be a crucial factor in terms of toxic effects [32]. Additionally, SWNTs reduced the amount of glial cells in both derived cultures as measured by ELISA. The authors observed that neurons were only affected in DRGderived cultures with regard to their ionic conductance (diminished inward conductivity) and resting membrane potential (more positive) according to whole-cell patch recordings. On the contrary, the neurite outgrowth and the electrophysiological properties of neurons derived from SPC cultures were not affected.

41.3 Electrical Stimulation of Neuronal Cells Grown on Carbon Nanotube-Based Substrates

Electrical stimulation of neuronal cells is widely employed in basic neuroscience research, in neural prostheses [33] and in clinical therapy (e.g., treatment of Parkinson’s disease, dystonia and chronic pain) [34]. These applications require an implanted microelectrode array (MEA) with the capacity to stimulate neurons. Neuroprosthetic devices currently face various issues, including (i) long-term inflammatory response of the neuronal tissues, resulting in neuron depletion around the electrodes and their replacement with reactive astrocytes that prevent signal transduction; (ii) delamination and degradation of thin metal electrodes; (iii) miniaturisation of the electrodes and (iv) mechanical compliance with neuronal tissues for long-term performance. Currently, semiconductor devices can only partially solve some of these problems. Nevertheless, CNTs are excellent candidates for MEA applications because of their unique set of properties which offer the possibility of constructing small electrodes with high current density. As will be detailed in this part, microelectrodes coated with CNTs have low impedance and high charge transfer characteristics and provide a rough surface that favours excellent cell–electrode

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coupling, while remaining chemically inert and biocompatible. Different systems have been utilised to guide neuronal cell growth and have been tested for their function in cultured neuronal networks, such as metal electrodes coated with CNTs, patterned CNT surfaces and CNT-polymer composite thin films. For instance, Ozkan et al. demonstrated the formation of directed neurite growth on patterned, vertically aligned MWNTs [35]. Different substrate geometries and nanotube heights were investigated for their ability to induce guided neuronal growth. Vertical MWNT arrays were functionalised with growth adherents that promote neuron adhesion and viability. In particular, the MWNTs were coated with a growth adherent, poly-l-lysine (PLL), which is known to enhance adhesion of neurons by altering surface charges on the culture substrate [36]. Hippocampal rat cells were cultured on patterns of short (500 nm high) and long (10 μm high) vertical MWNTs. Preferential directed growth of neurons was observed over the long MWNT array, but not over the short MWNT array. Indeed, in the latter case, neurons were seen to grow both on the short MWNT pattern and on the silicon chip, with no selection of one of the two types of substrate. On the contrary, in the former case, neurons showed preferential growth along the MWNT array, although the substrate was covered with PLL. The authors pointed out that the difference observed in terms of scaffolding capability between the short and long MWNT arrays could stem from the flexibility of the CNTs, which depends on the nanotube length. Indeed, to allow proliferation of neurites, the long MWNTs were found to undergo deformation, as observed by SEM. This deformation resulted from the extending neurite that interacted with the edges of the MWNT patterns. The neuronal cell networks were found to be viable after they were stained with Fluo-3 calcium. This fluorescent dye binds to the intracellular free calcium ions, which in principle are in high concentration in the cytoplasm of viable neuronal cells. The results of this study highlight the potential applications of long MWNT array substrates for the development of three-dimensional scaffolds suitable for implants. Hanein and coworkers published a review on the development of CNT-based MEAs for neuronal interfacing and network engineering applications [37]. They also described the use of CNT patterns as substrate for neuronal growth [38]. The CNT templates

Electrical Stimulation of Neuronal Cells Grown on CNT-Based Substrates

on which neurons adhered and assembled were fabricated by photolithography and microcontact printing. The deposition of iron nanoparticles on hydrophilic silicon dioxide or quartz substrates was controlled to allow the growth of regular arrays of CNT islands using CVD. During this process, long CNTs (on the order of 100 μm) can thermally fluctuate and may bind to other nanotubes to form a three-dimensional, entangled network. Neurons and glial cells were then deposited on the patterned quartz substrates. After four days’ incubation, neurons were seen to adhere to and grow on the region of the substrate containing CNTs. Interconnections between neighbouring islands were also observed because of the formation of axons and dendrites. The electrical viability of the neuronal networks was then tested and the action potential, generated by electrical stimulations, displayed a normal shape. Thus, this study allowed for the formation of neuronal networks having a normal functionality with pre-defined geometry due to growth on substrates patterned with CNT islands. An original method to pattern cultured neuronal networks was reported by Hanein et al. and was based on the anchoring of neuronal cell clusters constituted of tens of cells [39]. The anchors aimed at stabilising the neuronal cell clusters were CNTs or the strong adhesive substrate poly-d-lysine. The dynamics of the realtime network formation was monitored by placing cultures in an environmental chamber under a microscope. The cell clusters were seen to self-organise by moving away from each other and bridging gaps with concomitant formation of a neurite bundle between neighbouring clusters. Hanein et al. also fabricated CNT-based electrodes by synthesising high-density CNT islands using CVD on a lithographically defined substrate [40]. A mixture of cortical neuron and glial cells from rats were plated and cultured on the surface of the CNT electrode chip. The CNT islands acted as adhesion agents because of the substrate’s roughness, thus favouring the migration and adhesion of cells onto the electrode surface. High-fidelity extracellular recordings from cultured neuronal cells were then performed. A typical extracellular signal was obtained with a shape that reflected the first derivative of the intracellular action potential signal. The signal-to-noise ratio was high compared with that of a conventional TiN electrode.

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It should be noted that one advantage of the CNT electrodes developed by Hanein et al. over standard microelectrodes is the cell-adhesive nature of the nanotubes. Neuronal proliferation can thus be induced without the need for additional adhesion, thereby promoting coating spread between the electrodes. Electrical stimulation of primary neurons was reported by Wang and coworkers, who used vertically aligned MWNTs as microelectrodes [41]. Lithographic patterning of the catalyst allowed the control of the size and location of the MWNT pillars. The MWNTbased microelectrode was constituted of individual MWNTs having a diameter in the 30–50 nm range and separated by a distance of the order of tens of nanometres. The CNT electrode was 40 μm tall and was made more hydrophilic by coating with an amphiphilic PEG–lipid conjugate. Non-covalent binding of the PEG–lipid conjugate allowed for the preservation of the electronic properties of the CNTs. The MWNT MEA was coated with poly-d-lysine to favour cell adhesion to the nanotube substrates. Embryonic rat hippocampal neurons were deposited on the MWNT microelectrode and were shown to grow and differentiate. The viability and neurite outgrowth were comparable to those of cultures on plastic Petri dish controls. The neurons were then electrically stimulated by the MWNT electrode. The calcium indicator Fluo-4 allowed for the detection of action potentials by observation of intracellular Ca2+ level change. Fluo-4 can be loaded into cells and exhibits a large increase in fluorescence intensity on binding free Ca2+. It was also demonstrated that the neurons could be stimulated repeatedly with the MWNT electrode, thus highlighting the longterm endurance of the MWNT electrode. Recently, Hanein and coworkers investigated the neuriteCNT interactions at the nanoscale and elucidated the nature of the interface between neurons and CNTs [42]. Process entanglement was explained as a neuronal anchorage mechanism to CNT-based surfaces. In this study, isolated islands of pristine CNTs were fabricated and plated with cells (mammalian cortex neurons from rats and insect ganglion cells from locusts). The arrangement of neurons and glial cells on CNT-based surfaces was characterised by highresolution scanning electron microscopy (HRSEM), immunostaining and confocal microscopy. The neurons and cells were found to

Electrical Stimulation of Neuronal Cells Grown on CNT-Based Substrates

preferentially adhere to CNT islands and extend towards their periphery. This is in agreement with previous reports that have demonstrated preferential adhesion to rough surfaces [39–41]. The processes at the periphery of the CNT islands were curled and entangled, thus leading to enhanced interactions with the CNT surface that facilitated their anchorage. However, the processes that appeared too thick to interact with CNTs had the tendency to intertwine. Therefore, Hanein et al. pointed out that the roughness of the surface must match the diameter of the neuronal processes to help them anchor. The authors also suggested that adhesion of neuronal cells was in part achieved through an entanglement process. This mechanical effect may thus contribute to the mechanism by which neurons adhere to rough surfaces. Recently, Hanein et al. also investigated the use of CNT MEAs as an interface material for retinal recording and stimulation applications [43]. Electrodes were coated via CVD of CNTs, and electrical stimulation of retinal cells was achieved. The signals obtained with the CNT-based electrodes showed a remarkably high signal-tonoise ratio during recordings in comparison with commercial TiN electrodes. This would be particularly interesting for long-term in vivo implantation of electrodes. In addition, the authors observed an increase of the signal amplitude over several hours of recording. These results were indicative of an improved electrode–tissue coupling of CNT-based electrodes compared with conventional commercial electrodes. This is consistent with previous studies reported by Wang and coworkers, who validated the effectiveness of CNT electrodes for stimulation applications [42]. In summary, this work showed that CNT MEAs provide exceptional electrochemical and adhesive properties. This demonstrates the great potential of CNT-based electrodes for retinal implant applications. The first example of CNT-coated electrodes implanted into different brain areas in rats or monkeys was reported by Keefer et al. [44]. These electrodes showed the ability to enhance the detection of neuronal signals and to stimulate neurons. Indeed, the results indicated that the CNT-coated electrodes improved the electrochemical and functional properties of cultured neurons. CNT coating enhanced both recording and electrical stimulation of

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neurons in culture, rats and monkeys by decreasing the electrode impedance and increasing charge transfer. Electrical stimulation experiments were performed with cultured neuronal networks grown on CNT-based electrodes to determine if the CNT coating could alter the capacity to activate neurons. The CNT coating was found to be permissive for neuronal growth and function. Moreover, the performances of the CNT-coated electrodes were much higher than those of gold-coated control electrodes in evoking neuronal responses. This result highlights the potential of CNTs for the development of electrical brain interfaces. The unique physical, mechanical, chemical and electronic properties of CNTs can be, in part, transferred into CNT composites to combine high electrical conductivity, chemical stability and physical strength with structural flexibility. CNT composite constituted of thin films were prepared by layer-by-layer (LBL) assembly by Kotov and coworkers, as will be detailed later. They demonstrated that CNT composite films were suitable substrates to support growth, proliferation and differentiation, as well as to electrically stimulate neuronal cells. Applications of CNT substrates in medical implementations, such as implanted scaffolding, would require free-standing structure instead of CNTs attached to supporting glass or plastic. For this purpose, Kotov et al. reported the fabrication of free-standing SWNTpolymer thin-film membranes that are biologically compatible with neuronal cell cultures [45]. The membranes constituted of SWNT-polymer composite were prepared by the LBL assembly process. This technique allows for the construction of multilayered composite coatings and free-standing films of SWNTs with versatile architectures, which can be engineered at the nanoscale to attain desirable mechanical, structural, biological and electrical properties. It is based on alternating layers of CNTs and polymers. Coatings made by LBL are generally very mechanically robust [46]. SWNTs were dispersed in an aqueous solution of the amphiphilic poly(Ncetyl-4-vinylpyridinium bromide-co-N-ethyl-4-vinylpyridinium bromide-co-4-vinylpyridine). This polymer bears positively charged groups that favour cell adhesion. The biocompatibility of the SWNT composite thin films towards NG108-15 neuroblastoma × glioma hybrid culture cells was assessed using confocal microscopy and calcein. NG108-15 cells are used as neuronal model system since they

Electrical Stimulation of Neuronal Cells Grown on CNT-Based Substrates

exhibit, after differentiation, many characteristics of mammalian nerve cells. After long-term incubation (up to 10 days) the cells grew and proliferated on the surface of the SWNT films. The neuronal cells were viable, as illustrated by their continuous intracellular esterase activity, which is determined by the enzymatic conversion of the non-fluorescent cell-permeant calcein dye. The neuronal outgrowth of the NG108-15 cells on the surface of the SWNT films was compared with that of the cells on a control culture dish. Neurons were observed by SEM, and their morphology was assessed by cell labelling with the neuronal lipophilic tracer dialkylcarbocyanine. This dye has the capacity to diffuse through the membrane of cells and to trace along their neurites in order to monitor the surface morphology and growth of neurons. On the substrate comprising SWNTs, many elongated neuronal processes were observed with extension of neurites. Many secondary neurites and elaborated branches were grown from these neurites. Interestingly, many processes were seen to start from the neurites and to terminate at the surface of the SWNT film. Free-standing SWNT membranes were then prepared in order to be used as scaffold for neuronal growth and differentiation. The preparation involved the etching of an SWNT film from the surface of a glass substrate using HF and the subsequent rolling into a small elongated thread-like sample. The free-standing films showed a tensile strength that was sufficient for soft neural implants. It is noteworthy that the ionic conductivity of these free-standing films is brought about by its polyelectrolyte nature. This is essential for the interaction with ionic fluxes through neuron membrane. Adhesion and differentiation of NG108-15 cells were seen to be similar to those of SWNT films on solid supports. The neurite extension closely followed the morphological shape and curvature of the surface of the free-standing films. In summary, SWNT composite thin films prepared by LBL and resulting free-standing films can support growth, viability and differentiation of NG108-15 neuroblastoma/glioma hybrid cells. The free-standing SWNT/polymer composite thin film membranes were found to guide the outgrowth of neurites. The combination of different properties, including flexibility, inertness, non-biodegradability and durability, allows us to envisage some applications. For instance, the free-standing films could potentially

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be integrated into extracellular implants for cell stimulation and pain control and into neuroprosthetic devices for neuronal injuries. Kotov and coworkers also reported the stimulation of the neurophysiological activity of NG108-15 cells grown on LBLassembled SWNT-polymer composite films [47]. The LBL SWNT conductive films were prepared from positively charged SWNTs coated with a copolymer, poly(acrylic acid), and were made of 30 multilayers. The NG108-15 cells differentiated and extended long neurites on the surface of the SWNT films. The neurites developed into many secondary processes, branching in many directions on the surface. The SWNT films exhibited a sufficiently high electrical conductivity to electrically stimulate significant ion conductance in excitable neuronal cells that were electrically coupled to the SWNTs. The direction of the electrophysiological response suggested that cells were stimulated by the influx of cations in the cells. Kotov et al. described that mouse cortical neural stem cells (NSCs) can differentiate to neurons, astrocytes and oligodendrocytes with formation of neurites on SWNT-based composite thin films [48]. Note that NSCs are well known for their sensitivity to the environment. The SWNT-polymer composite thin films were prepared by LBL assembly of six bilayers of SWNT-PEI [(PEI/SWNT)6]. SWNTs were first dispersed in a 1 wt% poly(sodium 4-styrene-sulphonate) solution and then assembled using LBL technique with PEI as polyelectrolyte. Mouse embryonic 14-day neurospheres (i.e., spherical clonal structures of NSCs) from the cortex were seen to attach to the SWNT-polyelectrolyte film substrates and to differentiate, in a similar manner to poly-l-ornithine (PLO)-coated substrates used as control. PLO is a standard substrate widely used for NSC cultures as it is known to alter the surface charge, thus providing a more suitable substrate for the negatively charged neurons [27]. The viability of neurospheres determined using the MTT assay was found to be similar on (PEI/SWNT)6- and PLOcoated substrates. In addition, both types of substrates supported differentiation of neurospheres into the three primary neural cell types: neurons, astrocytes and oligodendrocytes, according to immunostaining experiments. Indeed, to analyse the differentiated phenotypes, neurospheres were immunostained with antinestin-,

Electrical Stimulation of Neuronal Cells Grown on CNT-Based Substrates

antimicrotubule-associated protein 2 (anti-MAP2), antiglial fibrillary acidic protein (anti-GFAP) and anti-oligodendrocyte marker O4 (anti-O4). Thus, NSCs grown on LBL-assembled SWNTpolyelectrolyte composite films behaved similarly to NSCs cultured on PLO substrates in terms of biocompatility, neurite outgrowth and expression of neuronal markers. In summary, this study is the first demonstration of the differentiation of environment-sensitive NSCs on a CNT-composite thin film. The results are promising for further development of CNTs for neural interfaces as the CNT-based substrate is able to promote cell viability and to induce the development of neuronal processes, as well as the appearance and progression of neural markers. Further studies were conducted by Kotov and coworkers on the electrical stimulation of NSCs by the SWNT composite constituted of laminin [49]. LBL films (up to 30 bilayers) were prepared from SWNTs wrapped with poly(styrene-4-sulphonate) and laminin, a glycoprotein which is an important protein in the basement membrane. Laminin is commonly used to coat substrates to promote cell adhesion and neurite outgrowth [50]. The SWNT-laminin films were heat-treated to increase their electrical conductivity by enhancing the cross-linking of CNTs among the different layers of the films prepared by LBL. Cell adhesion was clearly seen to depend strongly on the composition of the final layer of the LBL films. Indeed, the film containing SWNTs as top layer was the most suitable for cell adhesion and attachment. Differentiation of NSCs was observed, as indicated by extensive formation of functional neuronal network showing the presence of synaptic connections. By comparison with laminin-coated substrates, longer neuronal outgrowth occurred on the SWNT-laminin substrates that were found to be biocompatible, as indicated by satisfactory cell viability assays. The functionality of the neuronal networks was assessed by immunostaining for the presence of synapsin protein with the above-mentioned neuronal markers. Synapsin was present between the differentiated cells. This indicated that neuronal network was functional. The electrical stimulation of NSCs by the SWNT-laminin films was then investigated. Cell response to chemical and electrical stimuli typically results in a change in the membrane voltage of the cells, referred to as action potential.

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Detection of action potentials can be achieved via the invasive patch-clamp method [51]. The generation of action potentials upon the application of a lateral current through the SWNT film was deduced from the imaging of the NSCs with a Ca2+-dependent dye and was found to be dependent on the status of the surrounding cells. Once one neuronal cell was excited, the excitation sequentially spread from one cell to another. In summary, the combination of different properties of the SWNT/laminin thin film makes them a promising candidate for incorporation in neural electrodes. On the one hand, the films were suitable for NSCs’ growth and proliferation. On the other, as SWNTs exhibit high electrical conductivity, they were able to electrically stimulate NSCs. Chen et al. have been the first to explore the interface between neurons and CNT probes, both extracellularly and intracellularly (i.e., inside the neural membrane) [52]. CNT probes were used to monitor the neuronal activity elicited not only extracellularly but also intracellularly. This work is particularly interesting as it opens the way for intracellular neural probes that minimise damage to the neuron. In this study, two types of CNT probes were fabricated. MWNT bundles were connected to a silver wire. In one case, CNTs were coated with insulating epoxy, while in the other, CNTs were inserted into a sharp glass pipette, whose tip was used to penetrate the neural membrane. Only the tips of the probes were made of CNTs, contrary to the CNT-coated microelectrodes that were also involved in interfacing neurons [41, 42, 53]. The CNT probes were examined with the escape neural circuit of crayfish (Procambarus clarkia) and were found to have comparable performances to conventional Ag/AgCl electrodes. The CNT probes, with the tip pressed against an axon, were able to detect an action potential extracellularly and some small spikes (i.e., potential waveforms) associated with other neurons in the nerve cord. The results obtained from these extracellular experiments were in agreement with those demonstrated with microelectrodes coated with CNTs [41, 42, 54]. The intracellular recording and stimulation with CNT probes has been investigated for the first time. The conduction mechanism was investigated on the basis of impedance

Electrical Stimulation of Neuronal Cells Grown on CNT-Based Substrates

measurements and cyclic voltammetry. CNT probes were shown to transmit electrical signals through not only capacitive coupling but also resistive conduction to a comparable extent, contrary to the suggestion that capacitive impedance is the main mechanism to record and stimulate neural activity [41, 42, 54]. The resistive conduction helps record postsynaptic potentials and equilibrium membrane potentials intracellularly. It also facilitates the delivery of direct-current stimulation. The authors pointed out that the recording capability of the CNTs did not degrade and even improved after delivering direct-current stimuli for a long period of time. This highlights that the CNT probes are suitable for long-term use with a longer endurance compared with conventional Ag/AgCl electrodes, as the latter are inefficient once the silver chloride is reduced to silver. Thus, this work supports the potential use of CNT probes as a promising new neurophysiological tool. Ballerini and coworkers reported that CNT substrates can boost neuronal electrical signalling under chronic growth conditions [54]. They demonstrated that the growth of primary hippocampal neurons on MWNTs increased the frequency of spontaneous postsynaptic currents, but did not affect the general electrophysiological characteristics of the neurons. The substrates coated with CNTs were prepared by deposition of a homogeneous dispersion of pure MWNTs obtained by functionalisation using the 1,3-dipolar cycloaddition of azomethine ylides. Defunctionalisation of the resulting pyrrolidine-MWNTs was then induced at 350°C under nitrogen atmosphere to eliminate the organic functional groups introduced on the nanotube surface. This treatment provided purified non-functionalised MWNTs layered on the glass substrate. Previous attempts to prepare glass coverslips coated with as-produced MWNTs were not satisfactory because of poor reproducibility in terms of neurite growth and elongation. Hippocampal neurons were then seeded on glass coverslips. Attachment and growth of neurons were observed on the substrates covered with purified MWNTs, along with neurite extension. Hence, purified MWNTs layered on glass substrate were found to be permissive substrates to support neuron adhesion, survival and dendrite elongation. Neural network activity was investigated using single-cell patch-clamp recordings. The frequency of spontaneous postsynaptic

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currents (PSCs) of neurons deposited on the glass coverslips covered with MWNTs displayed a sixfold increase in comparison with the control substrate constituted of hippocampal neurons seeded directly on glass coverslips. The generation of PSCs was indicative of functional synapse formation. However, the height and half-width of action potentials of the neurons grown on both substrates were not significantly different. In addition, other electrophysiological characteristics of the neurons grown on the MWNT-coated substrates were nearly similar to those grown on the control substrate, in particular the resting membrane potential, input resistance and capacitance values. In summary, purified MWNTs obtained via a functionalisation/defunctionalisation sequence were demonstrated to be suitable growth surfaces for neurons. They boosted neuronal electrical signalling by promoting an increase in the efficacy of neuronal signal transmission. The authors pointed out that this effect was not attributable to differences in neuronal survival, morphology or passive membrane properties, but that it possibly represented a consequence of the properties of the SWNT substrates. Pappas et al. also examined the use of functionalised SWNTs as substrate for neuronal attachment and growth [55]. They demonstrated that neurons were electrically coupled to SWNTs. Using the diazonium salt approach [56] SWNTs were functionalised to introduce 4-benzoic acid or 4-tert-butylphenyl functional groups on the nanotube surface. Transparent, conductive SWNTpolyethylene terephthalate (PET) films were prepared via predispersion of the SWNTs in water by using 1% SDS. As already reported [46], neuroblastoma × glioma NG108 was used as model of neuronal cell. The highest cell growth and neurite extension of NG108 occurred on unmodified SWNT substrates, with decreasing growth on 4-tert-butylphenyl SWNT and 4-benzoic acid SWNT substrates. The neuronal cells were viable according to the vital dye calcein test. On the basis of acute attachment assays, SWNT substrates were found to display a more dramatic effect on cell attachment than on cell proliferation. It should be noted that cell adhesion is critical for cell survival. In fact, functional groups on the nanotube surface inhibited cell attachment. These results support those obtained by Haddon and coworkers on the influence of the surface charge of CNTs on neurite outgrowth where negatively charged functional groups at physiological pH or pristine MWNTs

Electrical Stimulation of Neuronal Cells Grown on CNT-Based Substrates

induced a reduction of the neurite outgrowth, in comparison with positively charged substrates [11]. The neuronal cells were then electrically stimulated through the SWNT-PET substrates. NG108 and rat primary peripheral neurons showed robust voltage-activated currents. These results suggested that CNTs can be a physiologically compatible substrate to be incorporated into devices for electrical stimulation of neurons. Gabriel et al. reported an alternative and simple method to fabricate CNT-based MEAs [57]. A suspension of purified SWNTs produced by arc discharge was deposited onto platinum electrodes [58]. This method has the advantage to be a post-process procedure that can be used with almost any MEA. The electrical properties of the CNT-based MEAs were found to be superior to those of metal electrodes, as shown by in vitro impedance and electrochemical characterisation. To investigate the biocompatibility of the CNTbased electrodes, in vitro cytotoxicity tests were carried out with different cell types from the nervous system: neurons (PC12 cells), glial cells (42MG-BA cells), fibroblasts (3T3 cells) and endothelial HEK cells (293T cells). The cell activity, estimated by the MTT assay, was not affected by the presence of CNTs, and no significant decrease in proliferation was detected. In addition, the SWNTs did not display any toxicity and remained attached to the surface of the electrode. Extracellular ganglion cell recordings in isolated superfused rabbit retinas were performed to evaluate the capacity of the CNT-based MEAs. It was the first time that CNTs were used to record electrophysiological activity in cultured retinal explants. The results indicated that the SWNT MEA arrays were more efficient in recording the action potential generated by a population of ganglion cells than Pt electrodes. The effect of MWNTs functionalised with cell-adhesion peptides on neuronal functionality was recently investigated by Bianco et al. [59]. In this study, MWNTs were functionalised via 1,3-dipolar cycloaddition of azomethine ylides, and the resulting pyrrolidine ring was derivatised with two peptides, one including the cellbinding domain RGD sequence (GRGDSP) and the other comprising a domain of the laminin protein (IKVAV), as illustrated in Scheme 41.3. GRGDSP is a fibronectin-derived peptide capable of promoting cell adhesion and neurite outgrowth. RGD-containing

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peptides are also involved in the mechanism of integrin regulation of neuronal gene expression [60]. Besides, peptides from different domains of laminin were used to stimulate neuronal growth and   axon regeneration [61].    

1) (CH2O)n, DMF

O

H N

HOOC

 

OH

O

O

O

O

N

NHBoc

NH3 Cl

O

O

120 °C, 3 days

N

O

OH

N O

DIEA, DMF

2) HCl in dioxane

1

7

O O

O O N

O

O

O

O

N H O

 

O N

GRGDSP-C Water

O

O

N

O

N H

N

O

O

S GRGDSP-Cys

OH

 

OH

  8

IKVAV-C Water

9

   

O O N

O

N O

S IKVAV-Cys

10

  Scheme 41.3 Synthesis of peptide–CNT conjugates.  

O

N H

OH

   

O

The activity of the two peptide–nanotube conjugates on   different types of cells, including tumour cells (Jurkat), primary splenocytes and neurons, was examined. Upon incubation of Jurkat cells with increasing doses of the peptide–nanotube conjugates (up to 100 μg/mL), flow cytometry showed no significant loss of cell viability. Similarly, no cytotoxic effect of the peptide–nanotube conjugates on splenocytes isolated from healthy BALB/c mice was observed, thus highlighting the biocompatibility of the functionalised MWNTs. Furthermore, doubly functionalised peptide–nanotube conjugates containing FITC were prepared and found to be internalised into the neuronal cells. The electrophysiological responses of rat dissociated hippocampal neurons in culture incubated with the two peptide– nanotube conjugates were then studied. The spontaneous activity of single neurons in voltage clamp configuration was recorded. The neuronal activity was detected as inward currents, corresponding

Investigation of the Mechanisms of the Electrical Interactions Between Cnts

to synaptic events, of variable amplitude. These events represent a mixed population of inhibitory and excitatory spontaneous postsynaptic currents. Some neuronal passive properties were then examined and measured under voltage clamp. They are indicative of neuronal function such as membrane capacitance and input resistance. By quantifying peak amplitude of inward and outward currents to the membrane capacitance value of each recorded cell, inward and outward current densities were obtained. The values were found to be similar in the case of the RGD-containing peptide conjugated to MWNTs when compared with the peptide alone. Thus, the incubation of neurons in the presence of MWNTs functionalised with peptides did not modify neuronal spontaneous activity. In summary, these data showed that peptide–nanotube conjugates incubated with neuronal cells did not alter the neuronal morphology, viability and basic functions of the neurons. These results are different from those obtained by Haddon and coworkers in which PEG-SWNTs were found to inhibit membrane endocytosis in stimulated neurons, which should lead to a progressive decrease of frequency in spontaneous PSCs, as will be detailed later [62]. Bianco et al. pointed out that this difference may be explained by the different types of treatment carried out on the CNTs (i.e., purification and functionalisation with different chemical groups). Thus, these data indicated that differently functionalised CNTs can have different impacts on neuronal activity, thereby highlighting the importance of determining the influence of the functionalisation of CNTs.

41.4 Investigation of the Mechanisms of the Electrical Interactions Between Cnts and Neurons

Numerous studies have demonstrated that CNTs can sustain and promote neuronal electrical activity in networks of cultured neuronal cells. But the mechanisms by which they affect cellular function are still poorly understood. Little is known about the details of the interactions between CNTs and neurons. A few studies investigating possible hypotheses have been recently reported.

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In vitro models have been developed to investigate the electrophysiological properties of synapses, neurons and networks coupled to CNTs, and to study the electrical interactions between CNTs and membranes. Ballerini and coworkers reviewed various existing experimental in vitro, in vivo and in silico models of neuronal network and compared them by listing their advantages and disadvantages [3a]. The effects of CNT substrates on the electrical behavior of brain networks in vitro were reported for the first time. Studies on the interface between neurons and SWNTs were performed recently by Ballerini and coworkers. They focused on the electrical signal transfer and the synaptic stimulation in cultured brain circuits [63]. Computer simulations were included in the study to model the electrical interactions between neurons connected via SWNTs. The results, strengthened by the modelling of the CNT–neuron junction, showed that SWNTs can directly stimulate brain circuit activity. In this study, rat hippocampal cells were grown on a film of purified SWNTs, via deposition of functionalised SWNTs and subsequent defunctionalisation. Patch-clamp experiments were then performed to determine the neuronal responses to voltage steps delivered via conductive SWNT glass coverslips. The morphology of the neuronal network was determined by SEM and quantified by immunocytochemistry analysis. SEM confirmed the adhesion and growth of hippocampal neurons on SWNT substrates, accompanied by variable degree of neurite extension. The attachment and proliferation processes were similar to those observed for neuronal growth on control glass surfaces. At high magnification, tight interactions between cell membranes and SWNTs could be visualised. The distributed electrical stimulation of cultured hippocampal neurons was then investigated. Hippocampal neurons grown on SWNTs or on control glass substrates exhibited spontaneous electrical activity. In particular, a significant increase in action potentials and frequency of PSCs was observed for neurons grown on SWNT films when compared with control substrates. To assess whether SWNTs could perform local network stimulations, short voltage pulses were delivered to SWNTs to induce the appearance of Na+ fast inward current in the recorded

Investigation of the Mechanisms of the Electrical Interactions Between Cnts

neuron. These stimulations should also induce action potentials in neighbouring neurons that should generate monosynaptic responses to the connected neurons. Indeed, brief SWNT voltage steps effectively delivered PSCs, in 65% of the recorded neurons. To elucidate the electrical interactions occurring in SWNTneuron networks, the modelling of the neuron-SWNT junction was achieved. It was suggested that the coupling between neurons and SWNTs might be partly resistive. But the authors led to the conclusion that because of the non-idealities of the single electrode voltage clamp, eliciting Na+ currents through SWNT stimulation did not conclusively prove a resistive coupling between SWNTs and neurons. Moreover, the authors pointed out that whole-cell patchclamp recordings might yield deceiving results as any resistive coupling between biomembranes and SWNTs is qualitatively undistinguishable from a coupling between SWNTs and the patch pipette. Ballerini and coworkers further investigated the influence of CNTs on the electrical properties of isolated neurons. In particular, the presence of after-depolarisation events in the cell soma was examined [64]. The aim of this study was to determine if the higher efficiency of the signal transmission of neurons grown on conductive nanotube substrates was linked to the interactions between CNTs and neurons at the nanoscale. Purified SWNTs were deposited on glass substrates via the previously described process based on functionalisation and subsequent defunctionalisation. Rat hippocampal cells were cultured on the thin film of purified SWNTs. Electrogenesis was studied in single neurons. The authors injected a brief current pulse into the soma to force the neurons to fire a regular train of six action potentials and measured the presence of membrane depolarisation in the soma at the end of the last action potential. Neurons propagate electrical signals, i.e., action potentials, down an axon. Backpropagation of the action potentials to dendrites can occur occasionally. In this case, the propagation of the electrical signals takes place in the direction opposite to that of the flow. It has been shown that rat hippocampal neurons grown on SWNT substrates exhibited a significantly larger after-potential depolarisation (ADP) by comparison with neurons grown on control glass substrates. ADP was found to be dependent on the degree of

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dendritic branching. Indeed, neurons with minimal dendritic branching grown on the CNT substrate did not display ADP. The backpropagating current induces a voltage change that increases the concentration of Ca2+ in the dendrites and can be measured through the presence of a slow membrane depolarisation following repetitive action potentials. Therefore, the interactions between CNTs and membranes of neurons can affect single-cell activity. These experiments were also conducted on two other types of substrates: indium tin oxide substrate, displaying high conductivity, and a non-conductive nanostructured substrate containing peptides that self-assemble into nanofibres. In both cases, no significant enhancement of ADP was observed, indicating that the ADP enhancement effect is specific to CNT substrate. The authors suggested the electrotonic hypothesis to explain the physical neuron-SWNT interactions and elucidate the mechanisms by which SWNTs might affect the electrical activity of neuronal networks. Electrotonic potential is a non-propagated local potential induced by a local change in ionic conductance. It represents changes to the neuron membrane potential that do not lead to the generation of new current by action potentials. Electrotonic potential is conducted faster than action potential, but it attenuates rapidly and is therefore unsuitable for long-distance signalling. The authors also examined if an electrotonic shortcut could occur between the soma and the dendrite. The results showed the absence of changes brought about by SWNTs in the dendritic passive time constants. Other hypotheses were drawn to tentatively explain the increased effects of ADP, such as (i) potentiation of Ca2+-mediated currents occurring in neurons grown on SWNT thin films and (ii) channels clustering, induced by mechanical interactions between SWNT bundles and the cell cytoskeleton. The presence of the ADPs, their dependence on trains of action potentials, and the detected sensitivity to calcium channel blockers are indicative of the generation of dendritic Ca2+ currents. The discontinuous and tight contacts between CNTs and neuronal membranes were observed by transmission electron microscopy (TEM). The SWNTs were able to modulate the physiology of the neurons. The intimate interactions of the SWNTs with a small area of the neuritic membranes favoured electrical shortcuts between

Investigation of the Mechanisms of the Electrical Interactions Between Cnts

the proximal and distal compartments of the neurons, thus supporting the electrotonic hypothesis. A mathematical model was proposed to simulate how ADP enhancement induced by SWNTs might affect the neuronal activity. More precisely, the aim was to model the membrane voltage input–output relationship between the soma and dendrites. The results indicated that SWNTs may be effectively short-circuiting the dendrites and soma. This shortcut would lead to diverting the electrical activity through the nanotubes, explaining the enhanced ADP effect. In summary, this study reported that SWNTs might affect neuronal information processing. Indeed, the action potential backpropagation was substantially enhanced in neurons grown on SWNT substrates. However, the exact mechanisms at the origin of this effect are not totally clear. One hypothesis relies on the discontinuous and tight interactions between SWNTs and neuronal membranes. Finally, following their study on neuronal growth on CNT substrates, Haddon and coworkers investigated the mechanism by which CNTs induce extension of neurite length [63]. They demonstrated that water-soluble SWNTs are able to inhibit stimulated membrane endocytosis in neurons. Following the observation that water-soluble SWNTs modified Ca2+ dynamics in neurons, thus leading to a decrease of the depolarisation-dependent influx of Ca2+ during cell stimulation, the authors investigated the effect of water-soluble SWNTs on membrane recycling. Indeed, the plasma membrane/vesicular recycling influences the extension of neurites [65] and is regulated by an influx of Ca2+ from the extracellular space induced by the depolarisation of neurons. In this study, hippocampal neuronal cultures were treated with SWNT-PEG. The neurons were exposed to FM1-43 [N-(3triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide] [66], a fluorescent dye that can monitor membrane recycling and that is internalised into cells by endocytosis as it does not passively diffuse across cell membranes. With regard to the constitutive vesicular recycling, taking place in unstimulated cells, the water-soluble graft copolymer SWNT-PEG had no effect on the endocytosis of FM1-43. However, in stimulated neurons (using HIK+), the pegylated SWNTs reduced the endocytotic loading of FM1-43 in a dose-dependent way.

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In summary, the mechanism responsible for the effects of water-soluble SWNTs on enhancement of selected neurite outgrowth was elucidated. The pegylated SWNTs inhibited regulated/ stimulated plasma membrane/vesicular recycling, while the constitutive phenomenon was unaffected.

41.5 Conclusions and Perspectives

CNTs have been demonstrated as a biocompatible substrate that promotes neuronal growth, boosts neural activity and transmits electrical stimulation effectively. Indeed, many studies reported the use of CNTs as substrate to promote neuronal cell adhesion and to induce growth and differentiation of neurons. Functionalisation of CNTs can modulate some processes, such as the outgrowth and branching of neurites. In particular, the surface charge was found to have a strong effect on neuronal growth. Because of their electrical properties, CNTs have also been used as electrodes to stimulate neurons. CNTs were seen to form tight contacts with neuron cell membrane that might favour electrical shortcuts between the proximal and distal compartments of the neurons, thus altering their electrophysiological responses. Therefore, understanding the mechanisms at the origin of the effects of CNTs on neuronal cells is essential for designing functional neuronal circuits. With regard to other materials or devices used for applications in neuroscience, CNTs show great potential as they possess a unique set of physical, chemical, mechanical and electronic properties [67]. A promising substrate candidate suitable for neuronal growth requires several characteristics, such as light weight, tight binding with neurons, controllable branching of neurites and directed neuron network formation. It should also ensure longterm cell viability. Besides, the combination of high electrical conductivity, corrosion resistance, nanoscale size, strength and flexibility is essential for neuroprosthetic devices for electrical recording and stimulation. CNTs can meet these requirements because of their dimension, high electrical conductivity and biocompatibility. Furthermore, CNTs have the exceptional characteristic to be highly flexible, while being very strong. The properties of CNTs can be, in part, transferred into CNT composites.

Disclosures and Conflict of Interest

CNTs are relatively chemically inert, but the possibility of functionalising the nanotube surface offers opportunities to tune their properties. Compared with other devices or materials, CNTs have led to very promising results. Indeed, it has been demonstrated that CNT-based substrates can serve as an extracellular scaffold with a higher capability of directing neurite outgrowth and regulating neurite branching than other types of substrates such as PLL, PEI or PLO, as explained in part 41.2. In terms of recording and stimulating neuronal activity, CNT-based electrodes exhibited better performances because of higher signal-to-noise ratio, longer lifetime and higher tissue–electrode interface in comparison with conventional commercial electrodes. In terms of future potential applications, CNTs are promising candidates for neural prostheses for restoring the function of damaged neuronal circuits. CNTs could serve as an extracellular scaffold to guide neurite outgrowth governed by their tips and growth cones, and also to regulate neurite branching. These processes would lead to the re-establishment of intricate connections between neurons forming synapses. CNTs could be used to selectively enhance neurite elongation directly at the site of nerve injury to aid in nerve regeneration, thus increasing the chance of connecting the injured sites to sustain functional recovery. Several neurological disorders and injuries, such as Parkinson’s disease, epilepsy and stroke, require an implantable device to generate electrical activity in the damaged or diseased tissue.

Disclosures and Conflict of Interest

The author declares that she has no conflict of interest and has no affiliations or financial involvement with any organization or entity discussed in this chapter. No writing assistance was utilized in the production of this chapter and the author has received no payment for its preparation. This is a reformatted version of the authors’ chapter that appeared in Carbon Nanotubes: From Bench Chemistry to Promising Biomedical Applicaons, 2011 (edited by Giorgia Pastorin), Pan Stanford Publishing Pte. Ltd., Singapore.

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Corresponding Author Dr. Cécilia Ménard-Moyon CNRS, Institut de Biologie Moléculaire et Cellulaire Laboratoire d’Immunopathologie et Chimie Thérapeutique (UPR 3572) 15 Rue René Descartes, 67084 Strasbourg, Cedex, France E-mail: [email protected]

About the Author

Cécilia Ménard-Moyon obtained her PhD in 2005 at CEA/Saclay in France on carbon nanotubes, their applications for optical limitation, nanoelectronics, and on the development of novel methods of functionalization. She worked for 1 year as a post-doc fellow at the University of York (U.K.) on the total synthesis of a natural product. Then, she joined the R&D Department of Nanocyl company in Belgium for 18 months to work on the synthesis and functionalization of carbon nanotubes. Since 2008, she has been a researcher at CNRS in the group of Alberto Bianco in Strasbourg. Her research interests focus on the functionalization of carbon-based nanomaterials, mainly carbon nanotubes and graphene, for applications in therapy, imaging, and diagnosis.

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40. Gabay, T., Ben-David, M., Kalifa, I., Sorkin, R., Abrams, Z. R., Ben-Jacob, E., Hanein, Y. (2007). Electro-chemical and biological properties of carbon nanotube based multi-electrode arrays. Nanotechnology, 18, 035201. 41. Wang, K., Fishman, H. A., Dai, H., Harris, J. S. (2006). Neural stimulation with a carbon nanotube microelectrode array. Nano Lett., 6, 2043.

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43. Shoval, A., Adams, C., David-Pur, M., Shein, M., Hanein, Y., Sernagor, E. (2009). Carbon nanotube electrodes for effective interfacing with retinal tissue. Front. Neuroengineering, 2, 4.

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Chapter 42

Polymeric Nanoparticles for Cancer Therapeutics Mohit S. Verma, Joshua E. Rosen, Ameena Meerasa, Serge Yoffe, MEng, and Frank X. Gu, PhD Department of Chemical Engineering and Waterloo Institute for Nanotechnology, University of Waterloo, Canada

Keywords: polymer, drug release, cancer, targeting, biodistribution, therapeutics, immune interaction, drug delivery, nanoprecipitation, copolymer, biocompatibility, micelles, dendrimers

42.1 Introduction

In recent years, the explosion of progress in science and engineering at the nanoscale has paved the way for a new category of healthcare technologies broadly termed nanomedicine. At its core, nanomedicine involves manipulating, engineering, and exploiting different material properties at the nanometer level to design products that can overcome many of the disadvantages found in currently available healthcare technologies. Nanotechnology has been used in a variety of applications: from improved cancer therapeutics with reduced side effects, to more sensitive imaging and diagnostic techniques, to improvements in implantable devices that allow for prolonged device lifetime and superior function; Handbook of Clinical Nanomedicine: Nanoparticles, Imaging, Therapy, and Clinical Applications Edited by Raj Bawa, Gerald F. Audette, and Israel Rubinstein Copyright © 2016 Pan Stanford Publishing Pte. Ltd. ISBN 978-981-4669-20-7 (Hardcover), 978-981-4669-21-4 (eBook) www.panstanford.com

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nanomedicine is clearly at the forefront of a new era of medical technology. One of the main themes employed in nanomedicine is the concept of enhanced control and specificity. While traditional therapeutic agents have allowed for very little control in terms of where they are distributed in the body and how fast they are cleared, engineering at the nanoscale has allowed for significant advances in optimizing the biocompatibility, biodistribution, and pharmacokinetics of various medical technologies. For example, devices can be created that release drugs and other therapeutic agents in a controlled manner, resulting in prolonged and enhanced therapeutic effects. In addition, nanoscale manipulations allow for the development of therapeutic products that target and localize to the area of a particular disease, increasing both the safety and effectiveness of the therapeutic agent, while at the same time minimizing unpleasant side effects. One prominent example of controlled delivery and release is the use of polymeric nanoparticles containing chemotherapeutic agents for the treatment of cancer (Fig. 42.1). In contrast to larger particles, nanoparticles have longer retention times in the body and can penetrate tumors more effectively [1, 2]. Nanoscale drug carriers have shown a remarkable ability to maintain an optimal drug release profile while localizing at the tumor site, minimizing deleterious effects on healthy tissues. Other nanoparticulate structures, such as liposomes, micelles, dendrimers, and metal nanoparticles, have also been evaluated for enhanced drug delivery applications.

Figure 42.1 Commonly used drug delivery platforms for therapeutics in nanomedicine.

Biological Interactions of Nanomaterials

While nanomedical technology has many potential benefits, there are also some significant challenges that must be overcome before these technologies can be put widely to use. One of the major barriers to injecting nanoparticles into the bloodstream is the tendency of these particles to be recognized rapidly by the body’s immune system as foreign entities. This results in their subsequent clearance from the bloodstream and accumulation in the liver and spleen before they have had a chance to exert their therapeutic or diagnostic effects. An equally important challenge is the potentially toxic nature of many types of nanoparticles, which can be a potential barrier towards their implementation in a clinical setting. This chapter serves to provide a general overview of important parameters in nanomedicine, such as biocompatibility and pharmacokinetics, and describes a variety of areas in which nanotechnology is being put to use to design polymer systems capable of delivering therapeutic payloads to tumors.

42.2 Biological Interactions of Nanomaterials

In order to gain an appreciation for the various properties and features of the many polymeric systems that will be discussed in this chapter, it is essential to have a basic understanding of the interactions that occur between nanomaterials and the living body. When designing nanotherapeutic systems, one must remember that the body treats any object administered to it as foreign and attempts to clear it as rapidly as possible. The interactions and physiological mechanisms discussed in this section are the basis of some of the most critical design challenges in the field of nanomedicine as they dictate important parameters such as blood circulation halflife, tissue biodistribution, and excretion pathways. The initial interactions that any nanoparticle encounters are typically dictated by its route of entry into the body; for example, interactions with blood plasma proteins for intravenous administration, the skin for dermal administration, or the upper GI tract for oral administration [3]. However, in all cases, a portion of the particles eventually reach the bloodstream and then accumulate in the liver [3]. This section focuses on the interactions nanoparticles undergo once they are in the bloodstream, as these interactions are generally applicable to a wide range of nanoparticle systems. For detailed reviews of nanoparticle interactions, see [4–10].

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42.2.1 Immune System Response The interaction of nanoparticles with the mononuclear phagocytic system (MPS), also known as the reticuloendothelial system (RES), can result in their degradation or sequestration and removal from circulation. The MPS consists of macrophages and monocytes that have the ability to remove altered cells, senescent cells and foreign particulates [8] including colloidal drug delivery systems, from circulation via non-specific or specific receptor mediated endocytosis. It has been established that the physicochemical properties of nanoparticles, such as particle size, surface charge, hydrophilicity, and polymer conformation, affect immune response and biocompatibility (Table 42.1). Table 42.1

Optimal physicochemical properties of nanoparticles for biocompatibility

Physicochemical Desired property parameter Particle size

200 nm

Surface charge

Neutral

Chain density

High

Hydrophilicity

Conformation

Effect

Target site

Biocompatibility; prolonged circulation

Bloodstream

Hepatic accumulation Liver Splenic accumulation

Spleen

Reduced opsonization; Bloodstream prolonged circulation

Reduced opsonization; Bloodstream Hydrophilic surface polymers, prolonged circulation e.g. PEG Reduced opsonization; Bloodstream prolonged circulation

Bloodstream Steric hindrance; Brush or mushroom-brush reduced opsonization; prolonged circulation (e.g. 5000 MW PEG)

42.2.2 Opsonization

Opsonization is a term used to describe the adsorption or binding of proteins to the surface of a nanoparticle. The specific proteins bound by a given nanoparticle depend on its composition,

Biological Interactions of Nanomaterials

although it has been shown that all particles tend to bind albumin, fibrinogen, IgG, and apolipoproteins [4]. 2D PAGE electrophoresis and Western blot analysis are two methods that are generally used to investigate the constitution of plasma protein adsorption onto a particle surface [4, 7]. Opsonins are defined as blood plasma proteins, such as complement proteins, immunoglobulins, fibronectin, and apolipoproteins, which coat foreign materials and make them more susceptible to phagocytic removal [4, 5, 9, 11]. Opsonization alters the physicochemical properties of the particle, resulting in an increase in particle size or a change in the surface conformation or charge that eventually leads to their recognition by macrophages [9, 10]. The accumulation of proteins on a particle’s surface usually leads to rapid removal of the particle from circulation by cells such as the hepatic Kupffer cells found in the liver, macrophages in the spleen and bone marrow, or dendritic cells [9]. The degree of opsonization experienced by a nanoparticle system is a key parameter in determining its circulation half-life and biodistribution. Nanoparticles that are capable of avoiding opsonization and subsequent clearance are often referred to as “stealthy.”

42.2.3 Complement Activation

The complement system is a component of the innate immune system; it comprises an ensemble of plasma proteins that participate in a biochemical cascade, which is activated upon exposure to foreign antigens. The protein-cleavage products produced throughout the cascade serve to activate the next stage of the cascade, induce an inflammatory response, and act as opsonins to facilitate the recognition of foreign particles by the immune system. The ultimate product of the complement cascade is the formation of a membrane attack complex (MAC), which acts to lyse any invading bacterial species [11]. Since liposomal particles are similar in composition to cell membranes, complement activation can lead to their destruction and the premature release of any payload compounds [10]. The complement system can be activated through three different pathways: classical, alternate, and lectin. The classical pathway is triggered by the binding of IgG and IgM antibodies onto the pathogen surface, which then bind and activate the first protein in the complement cascade. The alternative pathway is

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constantly active at low levels due to the spontaneous cleavage of an intermediate complement protein; however, this activity is amplified by the presence of amine or hydroxyl groups on the surface of a foreign particle leading to full activation of the complement cascade. The lectin pathway is initiated by the binding of mannose-binding lectin to mannose or fructose groups on a pathogen surface, which leads to activation of the complement cascade [6, 10, 11]. Regardless of the mode of activation, the end result of all three pathways is the same: opsonization, inflammation, and MAC formation.

42.2.4 Biocompatibility

In order to be considered biocompatible, a nanoparticle should not elicit an immune or toxic response after administration to the body. Efforts to increase the biocompatibility of nanoparticles typically focus on reducing their susceptibility to opsonization or their ability to activate the complement system. Thus, responses such as macrophage uptake and lysis are reduced and the nanoparticles’ circulation lifetime can be increased, allowing for sustained drug release and improved efficacy. In addition, biocompatible particles also reduce the effective dosage, which reduces possible side effects and toxicity to healthy cells [6]. In order for nanoparticles to be considered non-toxic, they must be both biodegradable and not illicit toxic reactions in the body. Nanoparticle biodegradability can be achieved by choosing biodegradable polymers or materials when synthesizing the particles, while toxic responses need to be evaluated post synthesis. One example of measuring the inherent toxicity of nanoparticles is to assess their hematocompatibility—the ability of nanoparticles to interact with erythrocytes in the bloodstream without causing hemolysis or thrombosis (blood clot formation), conditions that can cause life-threatening pathological conditions such as anemia and jaundice [5]. A percent hemolysis of less than 5% has been established to be acceptable for the use of biomaterials [12].

42.2.5 Effect of Particle Size

Particle size is a key parameter that influences the in vivo interactions of nanoparticles and is thus a major determinant of their pharmacokinetics and biodistribution profiles. Two of the

Biological Interactions of Nanomaterials

primary immune organs involved in nanoparticle clearance, the liver and spleen, have been shown to interact with nanoparticles in a size-dependent manner. Studies have shown that particles around 100 to 150 nm seem to be the most desirable for achieving both biocompatibility and prolonged circulation lifetime [8, 13]. In addition to their ability to avoid size-dependent filtration by both the liver and the spleen, smaller particles have a more rounded surface, which does not allow for strong protein–particle interactions, making opsonization and subsequent immune activation difficult [10]. In comparison, larger particles have increased surface area and a less curved surface, which produces areas of relative flatness that allows for increased complement activity. In addition to its influence upon clearance behavior, size is also a key determinant of a nanoparticle’s ability to extravasate (leave the vasculature) and enter the tissue of organs or tumors to deliver a therapeutic payload.

42.2.6 Importance of Surface Properties

In addition to a nanoparticle’s size, its surface properties are a second major factor in determining the degree of its interactions with various biological systems. It has been shown that hydrophobic and charged surfaces tend to show high degrees of protein binding, while neutral and hydrophilic surfaces show low amounts of opsonization [2, 9]. Studies have shown that the chain density and conformation of a hydrophilic polymer coating both play an important role in immune response. For effective evasion, nanoparticles require a high surface density of hydrophilic polymer chains, thus reducing the ability of proteins to see into the inner hydrophobic core of the particles [14]. In addition, it has been shown that depending on their surface density, polymer chains take on either a mushroom or brush conformation [7]. At lower surface densities, the polymers take on a mushroom-like conformation with flexible chains spread out in solution; at higher surface densities, they extend further from the surface and assume a brush-like conformation. Brush or intermediate mushroom-brush conformations have shown to decrease opsonization the most, as the rigid chains prevent the penetration of proteins into the hydrophobic core and also provide steric repulsive effects [7]. In addition to the general

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physio-chemical properties of a surface, interactions can be further influenced by the presence of specific biomolecules or targeting ligands. As we will discuss now, this effect can be used to great advantage in order to deliver therapeutic materials to specific sites in the body.

42.3 Nanoparticle Targeting

One of the central trends in nanotechnology-based cancer therapeutics and diagnostics is the push to create agents that can distinguish between cancerous and healthy tissues. From a therapeutics standpoint, this is desirable because it will cause drugs and other therapeutic agents to affect cancer cells while reducing the drug’s impact on healthy cells. This will lead to greater therapeutic efficacy, as well as the mitigation of many unpleasant side effects that patients currently face while undergoing chemotherapeutic treatment. Targeting methods can be grouped into two categories that differ in the way in which they distinguish between cancerous and healthy tissues. Passive targeting methods rely on physiological abnormalities in cancer tissues (Table 42.2) such as leaky vasculature and malformed lymphatic drainage systems (collectively known as the enhanced permeability and retention, or EPR, effect) [15–18]. Active targeting methods require that a ligand be attached to the surface of the diagnostic or therapeutic agent, which will then bind to surface markers and receptors that are overexpressed on cancer cells [18]. Table 42.3 lists various surface markers that can be used for active targeting. It should be noted that all active targeting techniques are still subject to the inherent physiological differences between cancerous and normal tissues (Table 42.2), but they have increased specificity compared to purely passive techniques due to the presence of surface ligands. A common problem that arises when employing active targeting ligands is developing a method to conjugate the ligand to the surface of the therapeutic agent without decreasing its affinity for the desired target. The conjugation method that is chosen must take into account both the functional groups available on the particles surface as well as the chemical structure of the ligand. Ligands can be conjugated either directly to the surface (e.g., using a disulfide or peptide bond) if the surface chemistry of the

Nanoparticle Targeting

particle allows for it or by using linker molecules such as NHS or PEG [48]. An interesting strategy that is sometimes used in active targeting is to use avidin–biotin binding to separate the targeting molecule from the nanoparticle. The targeting molecule (coupled to biotin) is administered first and then avidin-functionalized nanoparticles are administered. The particles will then bind to the biotin- tagged cells through the formation of the avidin–biotin complex [49, 50]. This targeting method offers some advantages as it can help reduce the overall size of the particle, which enhances both the particle’s stealth characteristics and its ability to extravasate. Table 42.2

Physiological abnormalities that can be utilized for passive targeting methods

Area of Abnormality body Leaky tumor Many vasculature

Poor lymphatic drainage system

Many

Loss of Liver and phagocytosis spleen

Result

Types of cancer that display this

Ref.

Macromolecules such as polymer encapsulated drugs and diagnostic agents diffuse out of the vasculature and into the tumor interstitium where they can accumulate

Solid tumors

[15– 18]

Solid tumors Once macromole– cules enter the tumor interstitium, they are not cleared out leading to the accumulation of therapeutic and diagnostic agents in clinically relevant levels

[15– 18]

Tumor tissues in the liver and spleen cannot phagocytose macromolecules,

Hepatocellular [19– 22] carcinoma, Liver metastasis, (Contiued)

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Table 42.2

(Contiued)

Area of Abnormality body

Integrin expression

Activated and proliferating endothelial cells

Many Overexpression of surface receptors for nutrients needed for cell proliferation

Result

Types of cancer that display this

Splenic while healthy liver metastasis and spleen tissues can. As a result, diagnostic agents can accumulate in healthy tissues providing added contrast against the cancer tissues.

Solid tumors Integrins such as alphavbeta3 are expressed on growing vasculature. Tumors have a need to recruit and form large amounts of vasculature in order to supply nutrients to their rapidly growing cell mass. As a result, the vasculature in a tumor tends to express high numbers of integrin receptors, which can be targeted by various peptide agents.

Ref.

[23– 25]

Many types of [26– Due to their rapidly cancers 30] proliferating nature, cancer cells tend to overexpress surface receptors for molecules such as folate and transferrin, which are required for cell proliferation. These receptors can be targeted with various molecules.

Classes of Polymeric Nanosystems for Cancer Therapy

Table 42.3

Active targeting ligands and surface receptors that are overexpressed on cancer cells

Targeting ligand

Surface receptor

References

Folic acid

Folate receptor

[26–30]

Monoclonal antibodies

Many

Transferrin

RGD peptides Nanobodies Affibodies Aptamers

Transferrin receptor

[29, 30]

Many

[35–37]

Alphavbeta3 integrin Many Many

[23–25] [31–34]

[32, 38–41] [42–47]

Another advantage of active targeting is that once the agents bind to cell surface receptors, the particles are often internalized via receptor-mediated endocytosis [23, 48]. This allows the therapeutic agent to accumulate in the cytoplasm at clinically relevant levels. Since many receptor-mediated internalization pathways are fairly well characterized, it becomes possible to design “smart” agents that respond to changes in their external environment such as pH or salinity changes. These smart particles can be tailored to only release drugs once inside a cell; for example, a change in pH inside a lysosome can cause the particles to swell and release the drugs they contain.

42.4 Classes of Polymeric Nanosystems for Cancer Therapy

In chemotherapy, high concentrations of therapeutic agents are often required to achieve the desired anticancer effects. Unfortunately, when administered freely, these drugs tend to harm healthy cells in addition to cancerous tissue. Furthermore, many of the promising chemotherapeutic agents are hydrophobic compounds, making it challenging to administer them via intravenous routes in the hydrophilic internal environment of the body. The application of nanotechnology to drug delivery has allowed researchers to begin to address some of these drawbacks. Nanoscale drug delivery technologies allow for smaller doses of drug to be used for treatment, reduced toxicity to the patient, a higher localized buildup of the drug in the affected area and greater temporal stability

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of drug concentrations in the tumor environment. These benefits can be realized by incorporating a desired chemotherapeutic agent in a nanoparticle delivery system. The drug–carrier complex can then be administered into the body where, with the aid of passive and active targeting methods, it will accumulate at the tumor site and begin to release the therapeutic agent while causing minimal damage to healthy tissue. The controlled release of the drug is dependent on the type of nanocarrier used, the materials it is constructed from, the procedures used to encapsulate the drug, and local environmental factors at the drug delivery site. While each type of nanoparticle delivery system has its own advantages and disadvantages, direct comparisons between them are often not feasible or even possible. This is due to the wide variation in study conditions used to evaluate nanoparticles, which are rarely consistent. Consequently, the various classes of therapeutic carriers will be discussed with regard to their individual advantages and drawbacks, and any recent clinical implementations will be emphasized.

42.4.1 Liposomes

Liposomes are spherical vesicles composed of self-assembling amphiphilic phospholipids that can associate themselves into bilayers surrounding an aqueous core. While not strictly a polymerbased system, we have included a discussion of liposomes due to their prominence in the field of nanomedicine. Liposomes have achieved a considerable degree of clinical success as drug carriers [51] due to their versatility, ability to encapsulate a wide variety of drugs, low-toxicity, and biocompatibility, especially following surface modification with a hydrophilic polymer [52]. Drug loading into the liposomal core can be done through various methods, including performing the synthesis in drug-saturated aqueous solution and pH gradient methods [53]. Controlled drug release to the target site can be achieved by two methods. Targeted drug release involves outfitting a liposome with a ligand that upon reaching the target site will be endocytosed by the cell; following endocytosis, the increased acidity experienced by the liposome will result in the loss of its structural integrity and release of the drug. Alternatively, triggered drug release can be based upon the incorporation of materials into the liposome structure that

Classes of Polymeric Nanosystems for Cancer Therapy

can respond to changes in temperature, pH, ionic strength, or irradiation in order to promote drug release [54]. Due to their aqueous core, liposomes are well suited for the containment of water-soluble molecules. FDA-approved Doxil®, which is a PEG-modified liposome product containing the watersoluble drug doxorubicin, has been used to demonstrate anti-tumor efficacy in numerous works [55], including a recent study with a doxorubicin-resistant colon cancer mouse model [56]. Poorly water-soluble drugs can also be incorporated into the lipid bilayer of the liposome, but the loading capacity may be insufficient for this purpose [57–59]. Nonetheless, a paclitaxel-carrying liposome (known by the trade name Lipusu®) has recently been approved for clinical use in China [60]. Studies on paclitaxel-containing liposomes have shown a significant increase in the tolerated dose of paclitaxel, as well as higher blood circulation times and improved anticancer efficacy for drug-resistant tumor models, when compared to free drug administration [60]. The active targeting of “stealth” liposomes that can evade the MPS and stay in the bloodstream for increased periods of time has been a focal point in modern liposome research. Polymers incorporated into the liposome bilayer, such as polyethylene glycol (PEG) and polyvinyl alcohol (PVA), increase blood circulation time but can hinder tumor interaction due to steric effects [53]. To alleviate this problem, targeting ligands such as transferrin have been conjugated to liposomes. Li et al. [53] have recently studied the biodistribution and pharmacokinetics of transferrin-targeted PEG-liposomes in vivo. It was found that the targeted liposomes increased drug uptake by transferrin receptor expressing cancer cells compared to non-targeted liposomes. Despite their clinical success, the efficacy of liposomes as drugcarrying entities has been challenged due to problems arising in vivo with respect to their stability and controlled drug release [58, 61, 62]. Blood circulation times also remain limited as even targeted liposomes accumulate most readily in the liver and spleen [51, 53]. In addition, problems with batch-to-batch reproducibility, effective scale-up and sterilization have been brought to light [54]. In recent years, novel liposome-based nanocarriers have been developed. For instance, hybrid liposome-block copolymer nanocarrier platforms consisting of a hydrophobic core, a hydrophilic

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shell and a lipid monolayer at the interface have been explored [52]. Additionally, lipid nanocapsules, which are composed of a triglyceride core and a shell made from polymers and surfactants, may have several important advantages over liposomes such as improved stability and a higher capacity for drug encapsulation [58].

42.4.2 Polymer–Drug Conjugates

Drugs can be conjugated to the backbones of water-soluble polymers to increase their blood circulation time and prolong their therapeutic effects. The most commonly used polymer for drug conjugation is N-(2-hydroxypropyl) methacrylamide (HPMA) [63]; it has a long history of clinical success [57]. Other polymers such as PEG, polyglutamate, cyclodextrin, dextran, and carboxymethyldextran have been conjugated with drugs such as doxorubicin, camptothecin, and paclitaxel [64]. Many of these conjugates, which are in various stages of clinical or pre-clinical trials [63, 65], have shown improved therapeutic efficacy over free drug administration. For instance, a PEG-camptothecin conjugate has been shown to improve the in vivo stability of the drug, and enhance cell uptake and apoptosis in a nude mouse model of human colon cancer xenografts when compared to the freeacting drug [66]. Drug release rates of polymer–drug conjugates can also be controlled in various ways, such as the use of spacer groups that respond to certain changes in pH or temperature by undergoing hydrolytic degradation [67]. Recently, the potential to use polymer–drug conjugates for combination drug therapy (multiple drugs conjugated to each polymer chain) has been reviewed [68]. Biodegradable polymeric nanoparticles can also be used to encapsulate drugs without covalent bonding. Drug release in this case is typically achieved by controlling diffusion through the polymer matrix or the surface, or by bulk erosion of the nanoparticle [32, 64]. The most common polymers used for this purpose are PEG, poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic) acid (PLGA) and carbohydrates such as chitosan and dextran [64, 69, 70]. Some of the current challenges of polymer–drug conjugate development include molecular weight heterogeneity, difficulties in site-specific conjugation and low drug loading [63].

Classes of Polymeric Nanosystems for Cancer Therapy

42.4.3 Polymeric Micelles Polymeric micelles are core–shell particles in the nanoscale size range formed from amphiphilic block copolymers. They have been investigated extensively for drug delivery applications [59, 64, 70–72]. Polymeric micelles are a very promising technology and have achieved a degree of clinical success. While their small size can provide many advantages, it is also somewhat of a disadvantage because it constrains the amount of drug that can be incorporated into the core and can lead to difficulties with controlling drug release [59]. This example is typical of a design trade-off encountered in nanoparticle formulation, as small size enhances the biodistribution and pharmacokinetic properties while limiting the amount of therapeutic agent that can be encapsulated. The core of a typical polymeric micelle consists of a hydrophobic polymer, such as PLA, PLGA, poly(e-caprolactone) (PCL) or poly(Laspartic acid) (PLAA), which allows for the incorporation of poorly water-soluble drugs. Drugs are typically loaded into the core via physical entrapment [73], although covalent bonding of drug to polymer can also be done in this context [74]. The core significantly increases the water solubility of hydrophobic drugs while protecting them from enzymatic degradation [59]. The outside layer of the micelle is hydrophilic, most often made from PEG or poly(N-vinyl2-pyrrolidone) (PVP), and its main role is to improve the micelle’s biocompatibility and bioavailability. Despite the various polymer and drug combinations that have been explored for their potential as polymeric micelle carriers, relatively few are undergoing clinical trials [71, 75]. Two of these, NK911 and NK105, are doxorubicin and paclitaxel carriers respectively, and are composed of PEGPLAA with strong antitumor activities compared to the free drug [76, 77]. Another formulation of Paclitaxel with mPEG-poly(D,Llactide) micelles has recently completed Phase II clinical trials and has shown tolerance at doses of 300 mg/m2 over 3 weeks [78]. In addition, a PEG-Poly(glutamic acid) block copolymer has been used for forming Cisplatin complexes (NC 6004) and for encapsulating SN-38 (7-ethyl-10-hydroxy-camptothecin) (NK012), both of which have completed Phase I clinical trials [79]. Poloxamers (known by the trade name Pluronics®) are triblock copolymers composed of polyethylene oxide (PEO) and

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propylene oxide (PPO). In this configuration, the hydrophobic PPO is sandwiched between two units of the hydrophilic PEO in the sequence PEO-PPO-PEO. Poloxamers have been used to form polymeric micelles for anticancer drug delivery [80]. Interestingly, poloxamers have been shown to have bioactive capabilities independent of the drug used, including the preferential targeting of cancer cells and suppression of surface proteins that cause cancer drug resistance [81].

42.4.4 Dendrimers

The class of synthetic, highly branched, spherical macromolecules known as dendrimers has gained prominence as an effective type of drug nanocarrier. Typically 1.5–14.5 nm in size, dendrimer molecules contain a central core surrounded by repeating units of highly branched layers with active terminal groups (Fig. 42.1) [32]. The most commonly used dendrimer backbone is polyamidoamine (PAMAM) [82], largely because it is biocompatible with a welldefined structure and can be easily conjugated with other molecules including drugs, targeting ligands and fluorophores for visualization. PAMAM dendrimers are also very small (1-year survival of mice after treatment of subcutaneous EMT-6 tumors. Circles: no treatment (n = 17), and gold only (n = 4), indistinguishable; triangles: irradiation only (26 Gy, 250 kVp) (n = 15); squares: irradiation plus 1.35 g Au/kg AuNP (n = 4); diamonds: irradiation plus 2.7 g Au/kg AuNP (n = 7). From Hainfeld et al. (2004) ref. 14 with permission.

The results of the early theoretical and experimental studies have shown great potential for gold nanoparticle aided radiation therapy to substantially improve cancer treatments. The use of gold NPs to enhance radiation therapy can be broken up into two steps. The first is the preferential targeting of the NPs to the appropriate biological components. The targeting is dependent on conjugation efficiency, molecular affinities, and the biophysical kinetics (extravasation, diffusion, endocytosis, etc.) of delivering the nanoparticle to the correct locations. Individual cells, stem cells, and tumor vasculature have all been proposed as potential targets, each with its own benefits and limitations. The second step is the irradiation either by internal or external radiation sources. In order to ensure safe coverage of the disease, the application of gold nanoparticle radiation therapy must be competitive with

Nanoparticles as Delivery Agents for Radiation Sensitizers

conventional therapeutic irradiation. To this end, employing gold NPs as an adjuvant to accepted safe irradiation procedures is particularly attractive [7].

43.2 Nanoparticles as Delivery Agents for Radiation Sensitizers

Radiation therapy is often delivered in combination with other modalities, such as chemotherapy or hyperthermia. Several anticancer agents have been shown to act as radiosensitizers, enhancing the efficacy of RT when they are accumulated at the tumor site. A variety of nanoparticle formulations have been shown to be highly useful as delivery agents of radiation sensitizers. NK105, a micellar-formulation delivering paclitaxel (PTX) to tumors, has been shown to have superior radiosensitizing activity in Lewislung carcinoma bearing mice, attributable to the more severe cell cycle arrest at the G2/M phase induced by NK105 as compared to that induced by free PTX [15]. The potential of radiation for improving gene delivery by a virus-mimicking nanoparticle, transferrin (Tf)-cationic liposomeDNA complex (Tf-lipoplex), has been explored. Radiation (10–20 Gy) markedly induced transgene (LacZ) expression in Lewis Lung Carcinoma xenografts, correlating with increased plasmid content and TfR expression in irradiated tumors. The results show that radiation improves Tf-lipoplex gene delivery selectively to tumor cells both in vitro and in vivo [16]. It has been proposed that quantum dots (QDs) can be used to excite conjugated photosensitizers and produce cytotoxic singlet oxygen. The photon emission efficiency of CdSe/ZnS QDs on excitation by 6-MV X-rays was measured using dose rates of 100–600 cGy/min. A QD-Photofrin conjugate was synthesized by formation of an amide bond. The number of visible photons generated from QDs excited by 6-MV X-rays was linearly proportional to the radiation dose rate. The Förster resonance energy transfer efficiency approached 100% as the number of Photofrin molecules conjugated to the QDs increased. The combination of the conjugate with radiation resulted in significantly lower H460 cell survival in clonogenic assays compared with radiation alone. The novel QD-

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Photofrin conjugate shows promise as a mediator for enhanced cell killing through a linear and highly efficient energy transfer from X-rays to Photofrin [17].

43.3 Nano-Coated Drug-Loaded Implants for Biologically in situ Enhanced, Image-Guided Radio Therapy

Radiation therapy is delivered either using a LINAC to focus beams of radiation on a target (external beam radiation therapy), or by placing radioactive material in the target to irradiate the target with radiation from the nuclear decay process (brachytherapy). Normal cells have a greater repair capability than cancerous cells so radiation generally kills a greater fraction of cancerous cells than normal cells. The radiation that can be delivered to the target is limited by complications that are associated with radiation damage to normal tissues in the vicinity of the high-dose region. Attempts to increase the therapeutic ratio have focused on decreasing the dose to surrounding normal tissues in order to decrease the normal tissue complication probability. Image-guided radiation therapy (IGRT) uses imaging during radiation therapy procedures to localize the target. Improved target localization allows the use of more conformal dose distributions, which irradiate less normal tissue and lead to fewer toxicities associated with treatments. Two IGRT techniques implant small objects in the tumor as an essential part of the therapy. One to three radio-opaque gold markers, or fiducials, may be implanted in the target to make it visible with X-rays. X-ray imaging mounted on the treatment LINAC can then be used to localize the target immediately before treatment allowing the use of smaller radiation fields by reducing the uncertainty in target position. Permanent 125I prostate brachytherapy uses imaging [18, 19] to guide the insertion of 30 to 100 radioactive sources within the prostate gland. In addition to the radioactive sources, inert spacers are used to maintain a planned geometry between sources. In both these instances, implanted objects essential to reducing normal tissue irradiation offer an opportunity to further increase the therapeutic ratio by providing localized radiosensitization.

Nano-Coated Drug-Loaded Implants for BIS–IGRT

The therapeutic efficiency of IGRT can be further enhanced by biological in situ dose painting (BIS-IGRT) of radiosensitizers through localized delivery within the tumor using gold fiducial markers that have been coated with NPs and nanoporous polymer matrices. Although use of NPs for drug delivery is widely studied, the use of implantable nanoparticle eluters in radiation therapy is a nascent field. Studies of nanoparticle biodistribution involve analysis of nanoparticle accumulation after intravenous delivery. Given the large variety of nanoparticle properties, simulating the biologic effect of NPs offers insight on the particle diffusion and residency properties that are necessary for effective radiosensitization. Computational studies build upon calculations of radiation dose distributions and the biologic effect of radiation. Radiation dose distributions can be calculated by treatment planning systems following the guidelines of the American Association of Physicists in Medicine. Biologic effect can be calculated from the linear quadratic model that is used to describe the results of cell irradiation experiments in terms of the surviving cell fraction SF(d)exp(–ad – bd2), where d is dose, and the parameters a and b are related to the sensitivity of cells to radiation damage. Radiosensitizers enable a given survival fraction to be achieved with less dose, and the sensitization is expressed as s = dsens/dref, where dsens is the dose required for a given SF of sensitized cells and dref is the dose needed to achieve the same SF for unsensitized cells. The biologic effect of a localized distribution of radiosensitizing NPs can be calculated from the resulting sensitization distribution combined with the underlying radiation dose distribution as shown in Fig. 43.3. The concept of the equivalent uniform dose (EUD) [20], derived from the average survival fraction of a region of interest, is a useful construct to provide relative figure of merit to compare the effect of different distributions of radiation dose or nanoparticle sensitization. Simulations of the distribution of particles in the vicinity of implanted eluters can guide the development of the physical characteristics of the eluters and drug carriers. Analytic solutions to the diffusion equation offer a means of understanding how potential radiosensitization depends on both eluter size and the diffusion elimination modulus fb. Cormack et al. [21] have shown that four implanted objects, releasing drug carriers with a low fb, could sensitize a substantial portion of a tumor of the size typical

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of early stage lung cancer often treated with stereotactic body radiation therapy (SBRT). In prostate brachytherapy, avoiding the sensitization of nearby radiosensitive structures adds a competing advantage for higher values of fb. (a)

(b)

(c)

(d)

Figure 43.3 An example of the effect of in situ release of radiation sensitizer within a permanent 125I implant. (a) An axial ultrasound image of the prostate used for treatment planning and image guidance of the implant. (b) The planned radiation dose distribution. (c) A sensitized region achieved by a distribution of implanted radiosensitizer eluters on a 5 mm grid around a point of interest while avoiding the urethra. (d) The biologic effective dose arising from the radiation dose and the planned sensitization.

Radiation therapy may be delivered in a single treatment, many treatments over the course of an extended period of time or with dose being delivered continuously. Implants deliver radiation continuously with the variation of dose with time determined by the half-life of the implanted isotope. The temporal release of NPs from an implanted eluter can be controlled by the chemical composition of the eluter, and the release schedule of radiosensitizers will affect the biologic effect. Integrating the effect of radiation cell kill over the course of a permanent implant shows that the optimal release schedule depends on the concentration

Nano-Coated Drug-Loaded Implants for BIS–IGRT

of intracellular radiosensitizer that can be sustained by the distribution configuration of implanted eluters. Accurate radiation dose calculations have been pursued throughout the history of radiation oncology. Modeling of in vivo distributions of radiation sensitizers has provided guidance to develop the concept of biologic in situ IGRT. Researchers in radiation oncology are investigating the use of biologic effect as a metric to optimize the radiation fields used in intensity modulated radiation therapy (IMRT) [22]. BIS-IGRT offers an additional level of complexity in the treatment planning process by allowing both radiation and drug distributions to be planned and using the biologic effect as a means to optimize the composite effect of both distributions.

43.3.1 Development of Nano-Coated Implants and Sensitizer Release Strategies

The feasibility of nano-coated implants for radiation therapy has been exploited by development of prototype radiosensitizer eluting nano-coated implants. Two approaches have been studied, a free drug release system and a dual-drug release system. The free drug release system comprised Doxorubicin (Dox), a hydrophilic drug, from a non-degradable polymer Poly(methyl methacrylate) (PMMA) coating. In the experiments, an initial release within the first few hours was followed by a sustained release over the course of next 3 months [23]. The dual-release system comprises Poly(D,L-lactic-co-glycolic acid) (PLGA) NPs loaded with fluorescent Coumarin-6, serving as a model for a hydrophobic drug, in a biodegradable chitosan matrix. In this platform, release of NPs and free drug were controlled by degradation rate of chitosan matrix and PLGA. Temporal release kinetics measurements in buffer were carried out using fluorescence spectroscopy. Considering the stability of chitosan film containing PLGA NPs on gold surfaces, this work demonstrates the use of this platform as a coating agent for gold implants. Furthermore, chitosan films could be loaded with hydrophilic model drug (Carboxyfluorescein) and FPTX-loaded PLGA NPs. The release profile of CF and PLGA from chitosan film as well as the release of FPTX from PLGA NPs demonstrated the promising

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application of this platform for localized dual-drug release [22]. The results from these experiments (see Fig. 43.4) show that dosage and rate of release of these radiosensitizers coated on gold fiducials for IGRT can be precisely tailored to achieve the desired release profile for radiation therapy of cancer [24].

Figure 43.4 (Left) Coated fiducial. (Right) Release profile of Doxorubicin from a coated fiducial. From Nagesha, et al. (2010) ref. 24 with permission.

43.4 Nanoparticles as Imaging Agents for Radiation Therapy

Nanoplatforms containing NPs and other functional moieties find many applications as image-enhancing agents using a variety of imaging modalities that are relevant to radiation therapy. Aydogan et al. have studied the feasibility of using 2-deoxy-D-glucose (2-DG)labeled gold nanoparticle (AuNP-DG) as a computed tomography (CT) contrast agent with tumor targeting capability through in vitro experiments [25, 26]. Significant contrast enhancement in the cell samples incubated with the AuNP-DG with respect to the cell samples incubated with the unlabeled AuNP was observed in multiple CT slices. Cinnamon-coated AuNP have been shown to be non-toxic and serve as excellent CT/ photoacoustic contrast enhancement agents [27]. Polymer-coated Bi(2)S(3) NPs have been shown to be attractive platforms for enhanced in vivo imaging of the vasculature, the liver, and lymph nodes in mice. These NPs and their bioconjugates show promise as an important adjunct to

Nanotechnology for Radiation Sources

in vivo imaging of molecular targets and pathological conditions [28]. A long circulating liposomal, nanoscale blood pool agent encapsulating traditional iodinated contrast agent has been used for micro-computed tomography (CT) imaging of rats implanted with R3230AC mammary carcinoma. Three-dimensional vascular architecture of tumors was imaged at 100-micron isotropic resolution [29]. Radio-opaque iodinated polymeric NPs in rats and mice (including those with a liver cancer model) CT-imaging have revealed a significant enhanced visibility of the blood pool for 30 min after injection, followed later by lymph nodes, liver and spleen due to strongly enhanced nanoparticle uptake by the reticuloendothelial system [30]. A novel X-ray luminescence computed tomography (XLCT) has been proposed as a new dual molecular/anatomical imaging modality. XLCT is based on the selective excitation and optical detection of X-ray-excitable NPs. As a proof of concept, a prototype XLCT system and imaged near-IR-emitting Gd(2)O(2)S:Eu phosphors was constructed and imaged in various phantoms. The linear response of the reconstructed images suggests that XLCT is capable of quantitative imaging [31]. Ultra-small paramagnetic iron oxide NPs have been shown to have greater accuracy as compared with conventional techniques and have been instrumental in delineating the lymphatic drainage of the prostate gland [32]. These NPs provide better target definition and delineation for fractionated radiation therapy. The use of multi-modal nanoparticle imaging agents, particularly in integrated clinical environments, such as the AMIGO suite at Brigham and Women’s Hospital, will enable more precise definition of tumor margins and enable more accurate treatment planning for radiation therapy.

43.5 Nanotechnology for Radiation Sources

The ability to control nanostructures surfaces has led to several approaches for use as X-ray emission sources. Electrochemically grown nanotubes on TiO2 surfaces have been explored as X-ray radiation sources [33]. Carbon nanotubes assembled in the TiO2 nanotubes [34] are expected to enhance the emission properties

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potentially leading to a new generation of controllable, portable sources. A prototype cellular irradiator utilizing a carbon nanotube (CNT)–based field emission electron source has been developed for microscopic image-guided cellular region irradiation. The CNT cellular irradiation system has shown great potential to be a high temporal and spatial resolution research tool to enable researchers to gain a better understanding of the intricate cellular and intercellular processes occurring following radiation deposition, which is critical to improving radiotherapy cancer treatment outcomes [35].

43.6 Conclusions

Several novel applications of nanotechnology and nanomedicines to radiation oncology have emerged in recent years. Radiation therapy is frequently used in conjunction with chemotherapy as multi-pronged therapies are needed to treat most cancers. The drug-releasing implants described above can be designed to utilize tailored-release profiles of biologics that are synchronous with the radiation treatment schedule. This chemo radiation therapeutic approach can utilize a wide variety of biologics such as chemotherapeutics like docetaxel, doxorubicin, and others, PARP inhibitors and siRNA, with the advantages of minimum systemic toxicity and high local drug concentrations. These nanoplatforms will provide oncologists with new weapons in the fight against a variety of cancers, including prostate, breast, lung, cervical and other carcinomas, and also sarcomas. Further development of these initial concepts over the next few years is being pursued to achieve successful clinical implementation.

Disclosures and Conflict of Interest

We thank D. Nagesha for helpful comments pertaining to this chapter. This work was supported by the Nanomedicine Science and Technology Center under NSF-DGE-096843 and the Electronic Materials Research Institute at Northeastern University, and by the Division of Medical Physics and Biophysics, Department of Radiation Oncology, Brigham and Women’s Hospital.

About the Authors

The authors declare that they have no conflict of interest and have no affiliations or financial involvement with any organization or entity discussed in this chapter. This includes employment, consultancies, honoraria, grants, stock ownership or options, expert testimony, patents (received or pending) or royalties. No writing assistance was utilized in the production of this chapter and the authors received no payment for its preparation. The findings and conclusions here reflect the current views of the authors. They should not be attributed, in whole or in part, to the organizations with which they are affiliated, nor should they be considered as expressing an opinion with regard to the merits of any particular company or product discussed herein.

Corresponding Author

Prof. Srinivas Sridhar Department of Physics, Northeastern University 435 Egan Research Center, 120 Forsyth Street, Boston, MA 02115, USA Email: [email protected]

About the Authors

Srinivas Sridhar is Arts and Sciences Distinguished Professor of Physics, Biomedical Engineering and Chemical Engineering at Northeastern University, and Lecturer on Radiation Oncology at Harvard Medical School. He is the Director and Principal Investigator of Nanomedicine Science and Technology, an IGERT (Integrative Graduate Education and Research Training) program funded by the National Cancer Institute and the National Science Foundation, and the Director of CaNCURE. He is the founding director of the Electronic Materials Research Institute, an interdisciplinary center with research and education thrusts in nanomedicine, nanomaterials and neurotechnology. From 2004 to 2008, he served as Vice Provost for Research at Northeastern University, overseeing the University’s research portfolio. An elected Fellow of the American Physical Society, Sridhar’s current areas of research are nanomedicine and neurotechnology. His paper in Nature in 2003 was listed among “Breakthroughs of 2003” by the journal Science.

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He has published more than 190 articles on his work in nanomedicine, neurotechnology, nanophotonics, metamaterials, quantum chaos, superconductivity and collective excitations in materials.

Ross Berbeco is a board-certified medical physicist at the Dana-Farber Cancer Institute and Brigham and Women’s Hospital and associate professor of radiation oncology at Harvard Medical School. Dr. Berbeco received undergraduate degrees in Physics and Astrophysics, with a minor in Philosophy, from the University of CaliforniaBerkeley. He earned his doctorate from the University of Michigan, based on high-energy experimental particle physics research performed at CERN in Geneva, Switzerland. His postdoctoral training in radiation oncology physics was completed at the Massachusetts General Hospital and Harvard Medical School. Dr. Berbeco’s pre-clinical research is focused mainly on goldnanoparticle aided radiotherapy. He has published numerous peer-reviewed articles and received federal and private funding to conduct this research. Robert Cormack is a physicist at the Dana-Farber Cancer Institute and Brigham and Women’s Hospital, and an associate professor of radiation oncology at Harvard Medical School. Dr. Cormack received a BA in Physics from Harvard College where he was first exposed to the field of medical physics at the Harvard Cyclotron Laboratory. He received his doctorate from Boston University for research in high-energy particle physics performed under the Gran Sasso at the INFN physics laboratory in L’Aquila, Italy. He completed his postdoctoral training at the Joint Center for Radiation Therapy and the Harvard Medical School. Dr. Cormack mentors students, fellows and residents in the Harvard Medical Physics Residency Program. His research interests include image-guided medical interventions, nano-therapeutics to increase the biologic effectiveness of radiation and adaptive radiation therapy.

References

G. M. Makrigiorgos is a professor of radiation oncology and director of the Medical Physics & Biophysics division at Dana-Farber Cancer Institute and Brigham and Women’s Hospitals, Harvard Medical School. He also directs the DNA technology laboratory and the radiation pre-clinical facility. His research interests include dosimetry of radiation at the microscopic (molecular) level; the development of DNA technologies for molecular diagnostics in oncology and radiosensitization of tissues using nanotechnology. He has developed molecular probes for detecting radiation at the molecular level (Coumarin), and PCR-based techniques for molecular diagnostics and mutation detection, including COLD-PCR. He is a member of the editorial board of Clinical Chemistry and has published over 120 articles, reviews and book chapters.

References

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11. Herold, D. M., Das, I. J., Stobbe, C. C., Iyer, R. V., Chapman, J. D. (2000). Gold microspheres: A selective technique for producing biologically effective dose enhancement. Int. J. Radiat. Biol., 76, 1357–1364. 12. Chithrani, D. B., Dunne, M., Stewart, J., Allen, C., Jaffray, D. A. (2010). Cellular uptake and transport of gold nanoparticles incorporated in a liposomal carrier. Nanomedicine, 6, 161–169. 13. Berbeco, R. I., Korideck, H., Ngwa, W., Johnson, S., Makrigiorgos, G. M., et al. (2011). An in vitro study of dose enhancement from gold nanoparticles with a flattening filter free delivery. Int. J. Radiat. Oncol. Biol. Phys., 81, S150–S151.

14. Hainfeld, J. F., Slatkin, D. N., Smilowitz, H. M. (2004). The use of gold nanoparticles to enhance radiotherapy in mice. Phys. Med. Biol., 49, N309–N315. 15. Negishi, T., Koizumi, F., Uchino, H., Kuroda, J., Naito, S., Matsumura, Y., et al. (2006). NK105, a paclitaxel-incorporating micellar nanoparticle, is a more potent radiosensitising agent compared to free paclitaxel. Br. J. Cancer 95(5), 601–606. 16. Abela, R. A., Qian, J., Xu, L., Lawrence, T. S., Zhang, M. (2008). Radiation improves gene delivery by a novel transferrin-lipoplex nanoparticle selectively in cancer cells. Cancer Gene Ther., 15, 496–507.

17. Yang, W., Read, P. W., Mi, J., Helmke, B. P., Sheng, K., et al. (2008). Semiconductor nanoparticles as energy mediators for photosensitizerenhanced radiotherapy. Int. J. Radiat. Oncol. Biol. Phys., 72, 633–635. 18. Blasko, J. C., Radge, H., Schumacher, D. (1987). Transperineal percutaneous iodine-125 implantation of prostatic carcinoma

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20. Niemierko, A. (1998). Radiobiological models of tissue response to radiation treatment planning systems. Tumori 82, 140–3.

21. Cormack, R. A., Sridhar, S., Suh, W. W., D’Amico, A. V., Makrigiorgos, G. M. (2010). Biological in-situ dose painting for image-guided radiation therapy using drug-loaded implantable devices, Int. J. Radiat. Oncol. Biol. Phys., 76, 615–623. 22. Ling, C. C., Humm, J., Larson, S., Leibel, S., Koutcher, J. A., et al. (2000). Towards multidimensional radiotherapy (MD-CRT): Biological imaging and biological conformality. Int. J. Radiat. Oncol. Biol. Phys., 47, 551–560.

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27. Chanda, N., Shukla, R., Zambre, A., Mekapothula, S., Katti, K. V., et al. (2011). An effective strategy for the synthesis of biocompatible gold nanoparticles using cinnamon phytochemicals for phantom CT imaging and photoacoustic detection of cancerous cells. Pharm. Res., 28(2), 279–291.

28. Rabin, O., Manuel, Perez, J., Grimm, J., Wojtkiewicz, G., Weissleder, R. (2006). An X-ray computed tomography imaging agent based on long-circulating bismuth sulphide nanoparticles. Nat. Mater., 5(2), 118–122.

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30. Aviv, H., Bartling, S., Kieslling, F., Margel, S. (2009). Radiopaque iodinated copolymeric nanoparticles for X-ray imaging applications. Biomaterials, 30, 5610–5616.

31. Pratx, G., Carpenter, C. M., Sun, C., Rao, R. P., Xing, L. (2010). Tomographic molecular imaging of x-ray-excitable nanoparticles. Opt. Lett., 35, 3345–3347.

32. John, S. S., Zietman, A. L., Shipley, W. U., Harisinghani, M. G. (2008). Newer imaging modalities to assist with target localization in the radiation treatment of prostate cancer and possible lymph node metastases. Int. J. Radiat. Oncol. Biol. Phys., 71(1 Suppl), S43–S47. 33. Alivov, Y., Klopfer, M., Molloi, S. (2010). Effect of TiO2 nanotube parameters on field emission properties. Nanotechnology, 21, 505706. 34. Gultepe, E., Nagesha, D. K., Selvarasah, S., Busnaina, A., Sridhar, S. (2008). Large scale 3D vertical assembly of single-wall carbon nanotubes at ambient temperatures. Nanotechnology, 19, 455309.

35. Bordelon, D. E., Zhang, J., Graboski, S., Zhou, O. Z., Chang, S., et al. (2008). A nanotube based electron microbeam cellular irradiator for radiobiology research. Rev. Sci. Instrum., 79, 125102.

Chapter 44

Gold Nanoparticles against Cancer Joan Comenge, PhD,a Francisco Romero, PhD,b Aurora Conill, MS,c and Víctor F. Puntes, PhDa aInorganic

Nanoparticles Group, Catalan Institute of Nanotechnology, Barcelona, Spain Science Institute, University of Valencia, Valencia, Spain cNanotargeting SL, Barcelona, Spain bMolecular

Keywords: nanotechnology, nanopharmaceutical, nanomedicine, targeting, gold, nanoparticles, cancer, drug delivery, enhanced permeability and retention effect, surface plasmon resonance, nanoparticle biodistribution, radiosensitizer, radiotherapy, photothermal therapy

Many of the conventional therapies can be improved using drug delivery systems (DDS). They are designed mainly to modify the pharmacokinetics and biodistribution of small molecular drugs. This is of special importance in the case of anticancer therapies in which a widespread distribution of small molecular chemotherapeutic drugs is often limiting treatments. In this context, nanotechnology emerges as a disruptive technology to design carriers that improve the delivery of drugs to their target organs. The composition of these nanocarriers comprises a wide range of materials such as polymeric nanocapsules, lipidic liposomes, or metallic nanoparticles. Gold nanoparticles (Au NPs) are of special interest due to its demonstrated biocompatibility, their tunable surface chemistry, and their special optical and electronic Handbook of Clinical Nanomedicine: Nanoparticles, Imaging, Therapy, and Clinical Applications Edited by Raj Bawa, Gerald F. Audette, and Israel Rubinstein Copyright © 2016 Pan Stanford Publishing Pte. Ltd. ISBN 978-981-4669-20-7 (Hardcover), 978-981-4669-21-4 (eBook) www.panstanford.com

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properties that allow its use not only as carriers but also as effectors. Hence, Au NPs are perfect candidates to be used for treatment of cancer in the clinics thanks to the capacity to be used as scaffolds to attach drugs or targeting molecules, as imaging agents, and as effectors themselves.

44.1 Historical Perspective on the Medical Use of Gold

Gold in different forms has been used for health in humans since ancient times. The synthesis of Au NPs has been in the spotlight since Faraday discovered in 1857 the mechanism of formation of pure gold colloids. This synthesis has been the keystone of a large amount of chemical routes to obtain Au NPs with controlled size, shape, and surface chemistry. Today, scientists have a wide catalog of Au NPs available, which can be used as excellent model systems to investigate the nano–bio interface. In the 1950s, the first use as contrast agents for radiotherapy was reported. Since the 1970s, Au NPs have been used in combination with antibodies (Abs) or other proteins to visualize specific cellular compartments, proteins, and receptors. Another well-known application of Au NPs is their use as probes of biomarkers in the pregnancy test (e.g., First Response®, marketed in the 1990s). This test is based on the specific recognition of human chorionic gonadotropin (hCG), a hormone produced during pregnancy, by Au NPs conjugated with an antihCG Ab. Nowadays, advanced NP bioconjugate chemistries allow scientists to tailor NPs for much higher sophisticated purposes, such as orchestrating chemical reactions inside cells and manipulating cell response. The successful development of these challenging tasks relies on intelligent surface structure design and the ability to synthesize NP bioconjugates with the desired architecture, which is also paramount in obtaining a well-defined and reproducible behavior.

44.2 Gold in the ERA of Nanotechnology: New Properties for a Known Material

Personalized health care, rational drug design, and targeted drug delivery are some of the proposed benefits of a nanomedicine-

Synthesis of Gold Nanoparticles

based approach to therapy. The progress of the drug development is nowadays limited since most of the delivery methods are based mainly on oral or injection delivery routes, which strongly determines the formulation of the drugs. Precise drug release into highly specified targets involves miniaturizing the delivery systems to become much smaller than their targets. Nanoparticle DDS, due to their small size, can penetrate across the barriers through capillaries into individual cells to allow efficient accumulation at the targeted locations in the body. A wide variety of engineered NPs has been extensively used or is currently under investigation for drug delivery, imaging, biomedical diagnostics, and therapeutic applications. Among those, Au appears as one of the most use and most promising medical nanoparticles for diagnosis, therapy, and theranostics. The employment of NPs for the delivery of pharmaceuticals can result in higher concentrations than possible with other drug delivery methods, which could enhance the drug bioavailability or dosing at the targeted site as well as the overall efficiency of the used drug. For example, the involvement of stable conjugates of Au NPs coated with antibiotic molecules for therapy increases the efficiency of drug delivery to target cells in some studied cases. Au is a biocompatible inert material that can be produced with extreme size control and monodispersity; its functionalization and derivatization are very well developed; and it has a very high electron density, which can be exploited as contrast agents for X-ray imaging or X-ray radiotherapy, or as contrast agents for imaging in the near infrared (a much weaker highly penetrating photon wavelength) and as photoablation (hyperthermia) agents.

44.3 Synthesis of Gold Nanoparticles

When using Au NPs for biological applications, special care has to be taken in the synthesis step since the size and size distribution play an important role in some biological responses such as biodistribution, tumor accumulation, and penetration, time of circulation, and immune response among others. Moreover, the nanoparticles surface should be ready for further chemical modifications in order to link the drug of interest. The synthesis of colloidal gold was introduced by Michael Faraday in the 1850s [1], but it was not until 1951 when the most

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usual synthetic methodology to obtain gold nanoparticles was exhaustively described by Turkevich [2]. This approach, based on the reduction of a gold salt by citrate, results in the production of 20 nm Au NPs with a relatively narrow size distribution. This methodology, as well as subsequent modifications [3], is based on supersaturation of the monomer in solution that induces homogenous nucleation followed by growth of these nuclei without additional nucleation events. This “burst nucleation” is a necessary condition to obtain highly uniform nanoparticles [4]. Nevertheless, the range of Au NP sizes available to obtain using these methods is small and goes from 7 to 25 nm. Frens proposed to decrease the citrate/HAuCl4 ratio in order to increase the nanoparticle size up to 150 nm [5]. However, it has been recently demonstrated that this approach do not follow the “burst nucleation” mechanism and consequently monodispersity becomes poor, and also the spherical shape of the Au NPs is partially lost [6]. To overcome these limitations, a great number of synthetic protocols to achieve better control over the size and shape of nanoparticles have been developed during the past years [4]. Among them, the seeding-mediated strategies based on the temporal separation of nucleation and growth processes are considered very efficient methods to obtain monodisperse NPs [7–9]. In this strategy, small particles are synthesized first and later used as seeds (nucleation centers) to grow larger NPs. For example, Murphy and coworkers proposed the use of a weak reducing agent (ascorbic acid), which reduces Au3+ to Au+ and prevented the total reduction to Au0 unless seeds are present [10]. By using this methodology, additional nucleation that would lead to polydispersity is avoided. However, the use of CTAB as capping agent restricts the possibilities of further functionalization since their replacement by thiols is difficult to achieve [11]. This condition is especially important in nanomedicine, where the ability to render a biological functionality to inorganic nanostructures is one of the cornerstones of this emerging field [12]. In this context, citrate-stabilized Au NPs appear as unique candidates since the loosely bound capping layer provided by the sodium citrate can easily be exchanged by thiolated molecules that pseudo-covalently bind (~45 kcal/mol) to the gold surface [13]. However, the control of the size is complicated since the energy needed to form Au nuclei is not much higher than the needed to grow the NPs and therefore to separate nucleation

Synthesis of Gold Nanoparticles

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Figure 44.1 Representative TEM images of Au NPs after every growth step in the kinetically seeded growth approach. Monodispersity and absence of secondary populations are maintained all over the process. The morphology of the Au NPs is quasi-spherical in all cases avoiding the formation of elongated Au NPs.

from growth is not trivial. Obviously, the formation of new nuclei during growing stages would lead to a broadening of the size distribution and should be avoided in any seed growth approach. Recently, our group proposed a kinetically controlled seeded growth approach that allows obtaining citrate-capped Au NPs with a perfect control of the size from 8 to 200 nm [8]. This methodology, which avoids the use of any surfactants, is based on the reduction of HaAuCl4 by sodium citrate (as in the classic synthesis of Au NPs). Here, the separation of nucleation and growth stages is achieved by manipulating the reaction conditions: pH, temperature, and ratio seed/monomer are key factors to avoid

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secondary nucleation that would have led to a broadening of size distribution (Fig. 44.1). Although spherical Au NPs are the most used in biomedical applications, nanoparticle synthesis is an extremely active field of research. In recent years, protocols allowing the synthesis of Au NPs of exotic shapes have been described. The special physicochemical properties of anisotropic Au NPs, hollow structures or core–shell Au NPs make them appealing to be used in biomedicine as contrast agents, for cell labeling or as effectors for photothermal therapy. Anisotropic gold nanorods (Au NRs) can be synthesized following also a seeding growth approach proposed by El Sayed’s and Murphy’s groups [14, 15]. Au seeds (4 nm) are synthesized with a strong reducing agent (NaBH4) in the presence of cetyltrimethylammonium bromide (CTAB). They are immediately used as nucleation centers for growing Au NRs. It is in this growth stage that the control of the size, and therefore the tuning of the Au NRs aspect ratio, is possible. The growth stage is based on the use of a weak reducing agent (ascorbic acid) to avoid secondary nucleations, and the presence of a micelle-forming surfactant (CTAB and/or benzyldimethylhexadecylammonium chloride, BDAC) and Ag ions to allow the growth in only one direction.

44.4 Gold Nanoparticles to Target Cancer

In cancer, the rapid growth of tumor results in leaky vessels. Macromolecules and NPs are able to permeate through the fenestrations in tumor vessels. In addition, they are retained due to the lack of a functional lymphatic system. This effect (Enhanced Permeability and Retention effect, EPR) is widely reported in the literature [16, 17] and has been exploited to passively accumulate nanocarriers in tumors [18]. This passive accumulation of NPs into the tumor may be even further improved if a ligand that is recognized by a receptor overexpressed in tumors is also attached to the NP. Examples include epidermal growth factor (EGF) [19], folate [20], and transferrin [21]. However, this point is still controversial and it is not clear if the addition of a ligand may change the final biodistribution of the NPs. It has been proposed that NPs with tumor-specific ligands are more rapidly taken up

Gold Nanoparticles to Target Cancer

by tumoral cells rather than affect the biodistribution which is mainly influenced by the physicochemical properties of the NP (size and surface charge) and composition (e.g., presence of stealthing agents) [22]. Long-time circulating NPs maximize the EPR effect since the changes to permeate through the tumor vessels are greater. Related to this, the size of NPs plays an important role in determine tumor accumulation and distribution: 60 nm Au NPs showed a greater accumulation than 20 nm Au NPs likely due to a slower clearance. However, larger Au NPs are accumulated in the perivascular region of the tumor failing to penetrate deeper, while 20 nm Au NPs were still found significantly at 50 μm from the blood vessel [23].

Figure 44.2 Diagram representing EPR effect.

Once the NPs are localized in the tumor, different strategies have been used to release the drug. Normally, the strategies are based on the different physicochemical properties found in the cytoplasm compared to the extracellular environment (e.g., blood). For example, NPs are known to be internalized via endocytic pathways, in which a pH drop is produced [24]. Thus, a pH-sensitive link between drug and NPs ensures non-specific release of drug during the circulation through the body [25]. A controlled release can be also achieved by the reduction of an oxidized inactive prodrug in the cytoplasm, releasing the drug in its active form

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[26]. Also, one can take advantage of specific enzymes to cleave the link between NP and drug [27]. Thus, the use of Au NPs as carriers might provide several advantages, including the following:

• Increased blood half-life of the small drug. Renal filtration is avoided if the NP is larger than 6 nm. • Accumulation in the tumor via the EPR effect. • Reduced toxicity in normal tissue. The drug attached to Au NPs is not able to permeate through the continuous capillaries that irrigate the most part of organs. • Controlled release of the drug, delivering high payloads directly to the site of action. • Possibility of multiple functionalization, which allows combined therapy and/or tuning blood half-life, immune responses toward the vehicle, tumor uptake, etc. • Increase in the solubility of poorly soluble drugs in plasma. • Protection of the drug from immune system. Addition of stealthing agents (e.g., polyethylene glycol) may help to protect the drug from the immune system. • Protection from agents present in the plasma that might deactivate the drug. Active site of the drug or the entire drug can be hindered during the journey through the organism until the moment of action. • Promotion of a double effect by acting as carriers and as effectors by themselves. Physicochemical properties of Au NPs make possible their role as agents of photothermal therapy or as antennas to sensitize cells to radiotherapy.

44.5 Challenges of Using Gold Nanoparticles in Therapy

The use of Au NPs as new therapeutic tools open up a world of potential applications in the biomedical field, and specifically in cancer treatment. However, the use of any new technology needs to be seriously evaluated to minimize risks. Therefore, the use of Au NPs is concurrent to the appearance of challenges that should be considered.

Challenges of Using Gold Nanoparticles in Therapy

Classical syntheses of Au NPs render concentrations of Au NPs in the range of 1012–1013 NP/mL [28]; thus, Au NPs are typically synthesized at the nanomolar scale. Even though high densities of loading have been achieved [29, 30], concentrations of drugs onto NPs as synthesized will rarely be higher than 10 micromolar. Taking into account that there is a limitation of volume that can be injected into the body (e.g., 10 mL/Kg for intravenous injection in mice), the therapeutic dose might not be reached unless NPs had been previously concentrated. This concentration step is not always straightforward because concentration of Au NPs could lead to aggregation if special care is not taken (e.g., concentration of Au NPs without any surfactant increase the chances of aggregation). Note that colloids are intrinsically out of equilibrium and they tend to minimize surface energy when possible via either functionalization or aggregation. The need of concentration of Au NPs to reach therapeutic doses implies that a single lab-scale synthesis might not be enough to achieve the amount of NPs needed to perform in vivo experiments. Thus, not only monodispersity but also reproducibility batch to batch should be guaranteed to control size effects. In addition, robust synthesis protocols will also facilitate the scale-up of the production, which is essential for the transference of the technology to the industry [31] (Fig. 44.3). Obviously, the special properties of Au NPs are lost if they aggregate. In too many cases, the loss of colloidal stability is behind the lack of (or unexpected) biological behaviors [32]. Therefore, one has to ensure the maintenance of colloidal stability along the process (synthesis–storage–exposure/treatment). It is common to assay the stability of Au NPs in the storage conditions (e.g., in aqueous media) even if they are different from the working conditions. However, biological fluids such as cell culture media or blood are complex mixtures of salts, proteins, sugars, etc., that may promote destabilization of Au NPs, and therefore stability should be assayed in this media. The same consideration is valid for the stability of the link with the drug. For example, cisplatin adsorbed on Au NPs is not released in aqueous media, while it is unspecifically and quickly released in cell culture media. On the other hand, cisplatin linked via coordination bond is stable in both media and only released after a pH drop [29].

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Figure 44.3 Example of classical synthesis of Au NPs (inset left) and concentrated Au NPs to reach therapeutic doses (inset right). The monodispersity of the concentrated solution is clearly observed in the TEM image. Scale bar is 200 nm.

The interactions of Au NPs and their environment need also to be evaluated. It is well known that proteins present in biological media interact with Au NPs forming a corona [33]. This corona can influence processes such as cell uptake or immune recognition [34]. Interestingly, the composition of the corona depends on the surfaces properties. Hydrophilic surfaces will likely induce protein corona in which albumin is the main component (note that albumin is the most abundant protein in blood) [33], while more hydrophobic surfaces will induce an increase of proteins such as fibrinogen, or lipoproteins [35]. It is worth noting also that protein corona formation is a dynamic process in which an initial protein corona is progressively hardened: Proteins interact strongly with NPs making the corona less labile. Indeed, the composition of the corona is modified with time from a corona formed by the most abundant proteins in serum to a progressively increase in the proteins that show more affinity to the surface [36]. The choice of appropriate models to determine the potential therapeutic benefits is also not trivial, especially for new technologies in which the amount of literature is limited. In vitro simple tests such as those assessed in monolayer cell cultures do not consider

Challenges of Using Gold Nanoparticles in Therapy

important factors such as tumor vascularity, penetration and other differential properties given by the tumor microenvironment [37]. Consequently, in vitro results do not necessarily reflect the behavior of the assayed DDS in a real case. In this context, 3D cell cultures have been proposed as models to study the behavior of drugs in the particular environment of solid tumors due to the possibility to have an extracellular matrix and different regions of tumor (e.g., hypoxic cells in the intern areas). Since the advantages of using DDS are achieved mainly by modification on the pharmacokinetic properties of the drug, in vivo models are preferred. However, there is a great variety of tumor models including xenografted, orthotopic, and spontaneously and induced autochthonous tumor models [38]. This models show variations between them such as different size of porous in vessels or different immunological responses that may result in different efficiency of the same DDS depending on the model that has been used. Finally, treatments with Au NPs are still very few and not beyond clinical trials. Thus, there is a lack of knowledge about regulatory aspects. Possible adverse effects of NPs such as acute toxicity and longtime accumulation should not be underestimated and should be studied case by case. There is a controversy regarding the in vivo toxicity of NPs and the parameters that play a role in the NPs-induced toxicity [39]. Some works have found no toxic signals after Au NP administration [40] or small alterations in the biochemical markers due to metabolization of the Au NPs or indicating a temporal inflammation [41]. On the other hand, it is believed that the dysfunction of major organs can be related to the presence of NPs at the site of abnormalities. For that reason, there are many studies regarding toxicity in spleen and liver, which are generally accepted to be the organs with the highest accumulation of NPs. The liver toxicity, when found, seems to be associated to a hyperplasia of Kupffer cells that induces an acute inflammation with neutrophils influx [42]. This acute inflammation is a transient response due to the insult of Au NPs; however, apoptosis and necrosis of hepatocytes as well as accumulation of Au NPs could be related to toxic effects [43]. Regarding the spleen, macrophages of the periphery seem to be involved in the uptake of NPs by this organ, thus leading to a temporal inflammation of the spleen. However, in other cases, a loss of weight has been observed after

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intravenous administration [44]. White pulp aberration has been also observed [45]. Any general trend cannot be extracted from the current literature, but it is clear that there are some parameters that have to be taken into account before using Au NPs as a vehicle for medical applications: (i) size of the vehicle, since it will determine the clearance rate and the biodistribution [45]; (ii) surface composition; it has been seen that by changing the surface composition of Au NPs the toxicological profile might be different [46]; (iii) dose and administration route, since they will obviously play a role in the potential toxicity. Therefore, for every Au NP conjugate, a toxicology study must be done to prove their usability in medical applications.

44.6 Examples of Gold Nanoparticles in Cancer Therapy 44.6.1 Drug Delivery

The use of Au NPs as drug delivery agents has been largely reported and is attracting many efforts of both academia and industry. The control over the synthesis, the possibility of doing surface chemistry and consequently attach the molecules of interest (ligands and/or drugs), and the demonstrated biocompatibility of Au NPs are behind the interest of using them as platforms for drug delivery. The rational of using a drug delivery system is to increase the concentration of the drug of interest on the site of action respect the concentration of the drug on healthy organs. This implies that the total dose can be lower. In addition, the drug can be diverted away from organs in which a drug-associated damage is described (e.g., kidneys for cisplatin). Thus, systemic toxicity and/or specific side effects are generally minimized (Fig. 44.4). Paciotti and coworkers demonstrated that TNF attached to 26 nm pegylated Au NPs showed a greater accumulation in tumors than free TNF [47]. Consequently, tumor volume was lower when mice received the Au NPs treatment than the free TNF. Interestingly, TNF plays here a double effect as targeting ligand and therapeutic agent. The therapeutic benefit was even greater when a second anticancer agent (paclitaxel) was added.

Examples of Gold Nanoparticles in Cancer Therapy

(a)

(b)

(c)

Figure 44.4 Toxicity of cisplatin in the kidneys. The appearance of histopathological changes in the proximal tubuli is evidence of nephrotoxicity. (a) Normal appearance of kidney section of control animals and (b) mice treated with Au NP-cisplatin. (c) Proximal tubular degeneration of animals taking high-dose free cisplatin. (Adapted from ref. 25).

In another example, Au NPs were used as a platform to minimize the side effects of one of the most used chemotherapeutic agents, cisplatin. This drug is associated to systemic toxicity and other side effects including nausea, renal toxicity, gastrointestinal toxicity, and peripheral neuropathy among others. Such side effects, especially nephrotoxicity, make it impossible to achieve the full benefit of the treatment in a large number of patients [48]. It is demonstrated in this work that by conjugating cisplatin to Au NPs via a pH-sensitive link, the drug is diverted away from organs irrigated by continuous capillaries such as those found in the kidneys, brain, and lungs in which cisplatin is known to induce toxicity. This is translated to a much lower levels of Pt in those organs. On the contrary, Au NP-cisplatin is accumulated in tumors and phagocytic organs like liver and spleen. Thus, while the toxicity is clearly reduced, the therapeutic benefits of the drug are maintained. The field of drug delivery with Au NPs is very active and many examples can be found in reviews of the topic elsewhere [49, 50].

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44.6.2 Radiosensitizers Radiotherapy is a major modality to treat cancer. Its aim is to deliver a therapeutic radiation dose to the tumor while sparing the damage to the surrounding tissue [51]. However, radiotherapy is still limited by tumor cells that are radioresistant, under irradiated or outside the target region [52]. Moreover, tumor and surrounding tissue normally present similar X-ray absorption characteristics leading to side effects. To overcome these limitations, beam delivery methods are in constant improvement. The use of heavy elements that can be accumulated on the tumors has been also proposed due to their higher mass and energy absorption coefficients [53]. Among these elements, Au NPs are of special interest due to their strong photoelectric absorption coefficient and the ability to be differentially accumulated into tumors due to EPR effect. Therefore, the contrast between tumors and normal tissue would be improved if Au NPs accumulated into the tumor. At KeV energies, X-ray primarily interacts with gold through the photoelectric effect, finally leading to a cascade of Auger electrons. The Auger electrons are weakly bound electrons released as result of electronic shell rearrangements. These electrons have low energies (99.5 atom% D).

47.2.4 In vitro BBB Model

The in vitro BBB model [33–35] uses primary cultures of both BMVECs (Cat# ACBRI-376) and normal human astrocytes (NHAs, Cat# ACBRI-371), which were obtained from Applied Cell Biology Research Institute (ACBRI, Kirkland, WA). Characterization of BMVECs demonstrated that >95% cells were positive for cytoplasmic von Willibrand’s factor/Factor VIII. BMVECs were cultured in CS-C complete serum-free medium (ABCRI, Cat # SF-4Z0-

Methods

500) with attachment factors (ABCRI, Cat # 4Z0-210) and Passage Reagent GroupTM (ABCRI, Cat # 4Z0-800). NHAs were cultured in the CS-C medium, supplemented with 10 µg/mL human epidermal growth factor, 10 mg/mL insulin, 25 µg/mL progesterone, 50 mg/mL transferrin, 50 mg/mL gentamicin, 50 µg/mL amphotericin-B, and 10% FBS. NHAs were characterized on the basis of >99% of these cells being positive for glial fibrillary acidic protein (GFAP). Both BMVECs and NHAs were obtained at passage 2 for each experiment and were used for all experiments between 2–8 passages—within the 6 to 27 cumulative population doublings. The BBB model used consists of two-compartment wells in a sixwell culture plate, with the upper compartment separated from the lower by a 3 µΜ polyethylene terephthalate (PET) insert (surface area = 4.67 cm2). The BMVECs were grown to confluency on the upper side of the insert, while a confluent layer of NHAs were grown on the underside. The formation of a functional and intact BBB takes a minimum of 5 days, which can be confirmed by determining the transendothelial electrical resistance (TEER) value, as described below [35, 36]. Using the in vitro BBB model, we examined BBB permeability and the efficacy of drug delivery of the nanoformulations.

47.2.5 Measurement of TEER

TEER across the in vitro BBB was measured using an Ohm meter Millicell ERS system (Millipore, Bedford, MA, Cat # MERS 000-01). Electrodes were sterilized using 95% alcohol and rinsed in distilled water prior to measurement. A constant distance of 0.6 cm was maintained between the electrodes at all times during TEER measurement.

47.2.6 HIV-1 Infection of THP-1

Monocytic THP-1 cells (American Type Culture Collection [ATCC], Manassas, VA); were maintained at 37°C, under 5% CO2 in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Sigma, St. Louis, MO), penicillin (100 units/mL), streptomycin (100 mg/mL), and L-glutamine (2 mM). THP-1 cells are cultured as a single-cell suspension cultures and were split

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at a ratio of 1:4 once a week. Undifferentiated THP-1 cells are susceptible to infection by T-tropic human immunodeficiency virus type 1 (HIV-1) isolates that use the co-receptor CXCR4 (X4 strains). Undifferentiated THP-1 (1 × 106 cells/mL) were infected for 3 h with HIV-1 IIIB(NIH AIDS Research and Reference Reagent Program) at a concentration of 10 3.0TCID 50/mL cells, equivalent to 10 ng viral isolate/mL of culture media. Following that, the infected cells were washed with Hanks buffered saline, reconstituted in RPMI media (fortified with 10% FBS) and incubated at 37°C/5% CO2 for 7 days. Levels of p24 in the culture supernatants were measured using a commercially available p24 ELISA kit (Zeptometrix, Buffalo, NY) 7 days post-infection. These infected THP-1 cells were then washed and reconstituted in fresh culture medium and used for evaluating the anti HIV-1 efficacy of the nanoplexes.

47.2.7 p24 ELISA

HIV-1 infection was monitored by the viral p24 level in harvested cell culture supernatants, using enzyme-linked immunosorbent assay (ELISA) plates obtained from a commercial vendor (Zeptometrix Buffalo, NY). The results are expressed in pg/mL as the mean and standard deviation (SD) of duplicate determinations from two wells.

47.2.8 Cell Viability Measurement Using an MTT Assay

The MTT assay is a quantitative, sensitive detection of cell proliferation. Cell viability was tested in BMVEC, NHA and THP-1 cells before and after treatment with the nanoplexes. Typically, 10,000 cells suspended in 100 µL of media were incubated for 24 h with various concentration of nanoformulation in a 96-well plate. At the end of the incubation period, 10 µL of MTT reagent (Cat # 30 –1010 K; ATCC) was added to the cells followed by incubation for approximately 3 h, which was followed by addition of a detergent solution to lyse the cells and solubilize the colored crystals. The amount of color produced was directly proportional to the number of viable cells. Colorimetric detection was done at a wavelength of 570 nm. MTT cell proliferation assay measures the reduction

Methods

of a tetrazolium component (MTT-3-(4,5-Dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide, a tetrazole) into an insoluble formazan product by the mitochondria of viable cells.

47.2.9 Evaluation of the Ability of the Nanoformulation to Transverse the BBB and Antiviral Efficacy of the PLGA-AS-Angiopep-2 Nanoformulation

A hundred thousand HIV-1 infected THP-1 cells that were suspended in 1 mL of RPMI complete media were plated in the lower chamber (basolateral end) of the in vitro BBB model. The following formulations (Atazavanir alone, PLGA alone, PLGA-AS alone, PLGA-AS-Angiopep-2) were reconstituted in 500 µL of media and added separate to chambers (apical end) of the in vitro BBB and incubated at 37°C and 5% CO2 for a period of 24–48 h. At the end of this incubation, the THP-1 cells were harvested from the lower chamber, washed, and then analyzed for antiviral efficacy of the nanoformulation by measuring p24 antigen levels using a p24 ELISA assay. Triplicate wells were used for each condition tested.

47.2.10 Statistical Analysis

The primary analysis consists of evaluating the transversing ability of the PLGA-AS-Angiopep-2 nanoformulation, additionally the efficacy of the PLGA-AS-Angiopep-2 nanoformulation is evaluated based on its ability to decrease viral replication as determined by HIV p24 measurements in the HIV-1 infected monocytes/ macrophages harvested from the basolateral end, 48 h posttreatment of the BBB with the nanoformulation. Statistical analysis was done between PLGA-AS-Angiopep-2 treated BBB and other controls namely Atazavanir alone, PLGA alone, PLGA-AS alone, respectively. All experiments are done in triplicate, including the BBB experiments where each condition was done in triplicate. Data was analyzed using ANOVA. A p value of less than 0.05 was considered significant. Analyses were performed using the Prism (Graph Pad Software, Inc., San Diego, CA) software.

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47.3 Results 47.3.1 Characterization of the Nanoplexes The physical properties of the nanoparticle formulations are established using the following techniques: (a) DLS to estimate the hydrodynamic diameter and surface charge of the aqueous dispersed nanoparticles, (b) Transmission Electron Microscopy (TEM) to determine particle size and size distribution, (c) 1H NMR and (d) evaluation of the spectral properties of the fluorescent nanomaterials using simple spectrophotometry and spectrofluorometry. Figure 47.3 shows the correlation of the hydrodynamic diameters with the concentrations and feed ratios of polymer to surfactant. Nile Red–loaded NPs allowed tracking intracellular uptake of the NPs. We synthesized Atazanavir or Nile Red–loaded NPs by the addition of an acetonitrile solution of PLGA and AS or Nile Red into the 4% ethanol–water solution of DSPEPEG-MAL, followed by removal of organic solvents by using centrifugal filter. Our results showed considerable drug loading

Figure 47.3 Correlation between the hydrodynamic diameters with the concentration and feed ratios of polymer to surfactant.

Results

(2–5 wt% relative to PLGA) and effective Nile Red loading (~1 wt%) (For NP uptake tracking) could be obtained (Fig. 47.4). Because of the presence of MAL group at the PEG terminal of the surfactant, the Angiopep-2 peptide was readily conjugated with NPs by using a cysteine-modified Angiopep-2 to react with MAL group via Michael addition mechanism. The optimal concentration of PLGA used in the nanoformulation was 3 mg/mL and that of Atazanavir was 75 µM, respectively. The conjugation efficiency of Angiopep-2 to the PLGA was ~70% as measured by 1H NMR analysis.

Figure 47.4 Drug loading capacity and size of the nanoformulation. Our nanoformulation had a 2–5 wt% Atazanavir loading capacity relative to PLGA and a ~1 wt% Nile Red loading capacity relative to PLGA.

47.3.2 Effect of the Nanoformulations on Cell Viability

Cell viability was tested in BMVEC, NHA and THP-1 cells before and after treatment with the nanoformulations. Our results (Fig. 47.5) indicate greater than 90% cell viability in all three cell lines tested confirming that the nanoformulation are non-toxic. The Atazanavir and PLGA concentrations in the nanoformulation are 75 µΜ and 3 mg/mL, respectively.

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Figure 47.5 Cell toxicity studies using the PLGA-AS-Angiopep-2 nanoformulation.

47.3.3 Determination of the Integrity of in vitro BBB during Transversing of the Nanoplex across the BBB

Our in vitro BBB model is a well-validated co-culture model and reflects several characteristics of the in vivo BBB. We have validated the BBB model of Persidsky et al. [36], which is composed of a co-culture of primary BMVEC and astrocytes, the astrocytic endfeet are in close apposition to the abluminal surface of the brain endothelium and allow formation of a tight BBB. Formation of an intact BBB was measured by determining TEER using MillicellERS microelectrodes (Millipore, Bedford, MA). A mean TEER value of ~250 Ohm/cm2 is consistent with the formation of the BBB. No significant change was observed in the TEER values of the BBB, before and after treatment with the nanoformulations for 24 h (Table 47.1) indicating that the integrity of the BBB is maintained, and that the nanoformulations PLGA alone, Atazanavir-loaded PLGA, PLGA-Angiopep-2 and PLGA-Atazanivir-Angiopep-2 did not cause any damage to the BBB while traversing through this barrier.

Results

Table 47.1

Measurement of transendothelial electrical resistance across the BBB pre- and post-treatment with the nanoformulations

Nanoformulation

Pre-treatment

Post-treatment

PLGA

267.3 ± 13.5

279.6 ± 15.9

PLGA-AS-Angiopep-2

277.4 ± 11.3

282.9 ± 13.8

PLGA-AS

PLGA-Angiopep-2

255.8 ± 16.1

262.2 ± 14.7

258.7 ± 10.2

270.14 ± 16.7

p value NS NS NS NS

47.3.4 Testing the Efficacy of the Nanoformulation Using the in vitro BBB Model

HIV-1 infected THP-1 cells (1 × 105 cells per mL) were added to the lower chamber (Basolateral or CNS end) of the in vitro BBB model. Five hundred microliters of the following solutions were added to the upper chamber (apical end) of the in vitro BBB followed by a 48 h incubation period: Atazavanir alone, PLGA alone, PLGAAS alone, and PLGA-AS-Angiopep-2. We then harvested 500 μL of the media (i.e., volume of media equivalent to the formulations added in the apical chamber) from the lower basolateral chamber and measured the HIV-1 p24 antigen levels in the media using a commercially available p24 ELISA kit. Our results (n = 3) show a >90% decrease in HIV-1 p24 levels in the HIV-1 infected

Figure 47.6 PLGA-AS-Angiopep-2 nanoformulation significantly decreased HIV-1 viral replication as evident from the decreased HIV p24 levels in HIV-1 infected THP-1 cells.

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THP-1 cells treated with PLGA-AS-Angiopep-2 as compared to Atazavanir alone (91.2% decrease; p < 0.001) or PLGA-AS alone (94% decrease; p < 0.001) (Fig. 47.6). Comparison of HIV-1 p24 levels between PLGA-AS-Angiopep-2 and basal levels such as media alone or PLGA-NP alone showed a >97% decrease (p < 0.001).

47.4 Discussion

Nanocarriers provide a means to overcome cellular and anatomical barriers to drug delivery. Nanocarrier-mediated ARV delivery may allow modulation of drug delivery profiles thereby allowing sustained release of the drug at a specific site where the virus is sequestered such as the brain. The therapeutic efficacy and safety of drugs can be significantly improved by targeted delivery using nanocarriers. Nanocarriers enable ARV transport by passive or active transport mechanisms. It is their size that allows them to penetrate through cells and deliver drugs intracellularly without risking extracellular degradation. Biopolymers have attracted great attention for nanotechnology-based drug delivery due to their stability and sustained drug release characteristics and also their reproducibility during synthesis [23, 24]. The important determinants of the type of biopolymers to be used for nanoformulation depends on (1) drug loading capacity needed (2) the requisite drug release pattern and (3) the biodegradability of core polymers, all of which are key issues for a nanoformulation that will be used in vivo. Most antiretroviral drugs are ineffective in reducing the HIV viral load in the brain due to the poor transport of many antiretroviral drugs, particularly the protease inhibitors, across the BBB [16–18]. In the current study we used PLGA, which is been approved by the Food and Drug Administration (FDA). The advantage of using the PLGA biopolymer is that a wide range of drugs of different hydrophilicity or hydrophobicity can be incorporated into these polymers, and their release characteristics can modified by controlling the molecular weight or copolymer composition which influences properties such as degradation rate, thermal sensitivity, and pH sensitivity. These polymers have the capability to sustain drug release for several weeks. Further PLGA undergoes hydrolysis in the body to produce biodegradable metabolite monomers such as lactic acid and glycolic acid that are typically found

Discussion

in the body and therefore, it is an ideal nanocarrier for the drug delivery applications because of minimal systemic toxicity associated with it. We used the PLGA biopolymer as a nanocarrier for an HIV antiretroviral drug called Atazanavir. The rationale for choosing Atazanavir was that this HIV protease inhibitor has limited passage across the BBB, due to its interaction with efflux P-gp and MRPs, which prevent its passage across the BBB and thus its entry into the CNS [31]. A nanoformulation of PLGA and Atazanavir would facilitate the entry of this ARV drug into the brain. In addition to evaluating the transcytosis efficacy of the PLGA nanoformulation using the an in vitro BBB model we tested the anti-HIV efficacy of this nanoformulation in HIV-1 infected THP1 cells cultured on the bottom of the lower chamber (basolateral end or CNS end) of the in vitro BBB model. This study forms the basis for development of a biocompatible nanoformulation that can target HIV-1 in sequestered site such as the brain and can potentially be used as a neurotherapeutic in Neuro-AIDS. Our PLGA-based nanoformulation (Fig. 47.2) is prepared via the nanoprecipitation technique followed by peptide conjugation [37], which avoids tedious synthesis procedure and results in biodegradable NPs with minimal long-term toxicity. TEM imaging was utilized to visualize the morphology and uniformity of these NPs and they showed uniform spherical morphology with average diameter of around 100 nm. Nanoprecipitation readily allows the formation of drug-encapsulated polymer-based NPs via the assembly process of water-insoluble polymers in drugcontaining solutions. Using this technique, we were able to prepare a nanoformulation of Atazanavir-PLGA and were able to effectively control its size through modulation of the concentrations and feed ratios of polymer to the PEG-based surfactants (Fig. 47.3). DLS analysis shows the effects of biopolymer drug conjugate and the surfactant concentrations on the size and size distribution of PLGA-AS-Angiopep-2 NPs. The biopolymer drug conjugate concentrations in THF were 1, 3, and 5 mg/mL and the mass concentration ratio of surfactant to biopolymer drug conjugate was 1, 0.5, and 0.25. Figure 47.3 shows that under these experimental conditions the volume-average hydrodynamic diameter (Dh) of the NPs, as measured by TEM ranged from 60–163 nm, and significantly increased with polymer concentration. Besides size of NPs, size

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distribution of NPs is also an important criterion to assess the quality of formulation. Our optimal PLGA nanoformulation that we used in the study used NPs that had a hydrodynamic diameter (Dh) of 100 nm as measured by TEM and a polydispersity index (PDI) of 0.19, which had an optimal PLGA concentration of 3 mg/mL and a surfactant to biopolymer drug conjugate mass ratio of 0.5. The ~100 nm hydrodynamic size of these NPs helps avoid fast systemic clearance. In addition to size and homogeneity, colloidal stability of PLGA NPs is also crucial in drug delivery. The presence of charged surfactant molecules on surface, allows these PLGA NPs to be readily suspended in water and culture media without noticeable aggregation and they maintain their colloidal stability over extended time periods. A suspension of the PLGA NPs in deionized water (pH = 7.0) was monitored by DLS and showed that the hydrodynamic diameter (Dh) and scattering intensity of the NPs were essentially consistent during 60 days at room temperature, indicating relatively slow degradation of the PLA-based polymer backbones and significant colloidal stability of the NPs. PEGylation also helps avoid recognition by the reticulo-endothelial system. Presence of a MAL group at the w-terminals of PEG chains allowed functionalization with Angiopep-2, which facilitate the penetration of NPs across the BBB. Angiopep-2 was readily conjugated with NPs by using cysteine-modified Angiopep-2 to react with MAL via Michael addition mechanism. The Angiopep2 peptide, binds to low-density lipoprotein receptor-related protein-1 (LRP-1), a receptor expressed on the endothelial cells that constitute the BBB. Figure 47.4 showed considerable Atazanavir loading (2–5 wt% relative to PLGA) and Nile Red loading (~1 wt%) could be obtained and that the size of the biopolymer drug conjugate was ~100 nm. Figure 47.5 shows that our nanoformulation is nontoxic, and greater than 90% cell viability was observed for all the three cell lines (BMVEC, NHA and THP-1) tested, indicating that it will be safe to use this nanoformulation in vivo studies. We observed no appreciable change in the TEER measurements (Table 47.1) when the BBB was treated with PLGA-AS-Angiopep-2, PLGA alone, PLGA-AS and PLGA-Angiopep-2, indicating the absence of any functional damage to the BBB and therefore indicates that

Conclusions and Future Prospects

this PLGA-based nanoformulation may be extremely effective in carrying therapeutic payload across the BBB. The efficacy of this nanoformulation was determined by evaluating the level of HIV-1 p24 released from THP-1 monocytic cells cultured at the basolateral end of the BBB model. We observed a significant decrease (>90%) in HIV-1 p24 production from the HIV-1 infected THP-1, harvested from the lower chamber of the in vitro BBB model (Figs. 47.6). Our results suggest that our PLGA nanoformulation is a great nanocarrier for the antiretroviral drug Atazanavir and significantly enhances the antiviral efficacy of Atazanavir and this may be attributed to the presence of the Angiopep-2 peptide, which binds to low-density lipoprotein receptor-related protein-1 (LRP-1), receptor expressed on the endothelial cells that constitute the BBB [32]. The mechanism of the PLGA-AS-Angiopep-2-based antiretroviral drug transport across the BBB appears to be receptor-mediated endocytosis followed by transcytosis into the brain. Modification of the PLGA surface with covalently attached targeting ligands or by coating with certain surfactants such as PEG is necessary for this receptormediated uptake.

47.5 Conclusions and Future Prospects

In conclusion, we have demonstrated the ability to synthesize a biodegradable biopolymeric nanoformulation that contains an antiretroviral drug and a cell-penetrating peptide on its surface that is able transverse the BBB and significantly inhibit HIV-1 replication in the infected monocytic cells, demonstrating the anti-HIV-1 efficacy of this nanoformulation and its applicability as a neurotherapeutic. Our data encourage further in vivo investigation of these PLGA NPs for nanotherapy within the CNS as the biomedical functions of these PLGA NPs can be further enhanced by multifunctionalization via conjugation with specific targeting ligands and various other diagnostic moieties providing an effective nanotherapeutic strategy in treating Neuro-AIDS and other neurological disorders. The latent HIV reservoir in the CNS serves as a major depot for HIV persistence, and therefore, effective nanotechnology-based approaches to eradicate HIV could provide valuable steps toward

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HIV eradication. Majority of small and large pharmaceutical drugs are unable to cross the BBB, and drug delivery to directly access the brain via either the nasal cavity or inhibition of P-gp transporters to deliver ARV drugs across BBB has had limited success. Therefore, a nanotherapeutic approach that would (1) allow sustained ARV drug delivery, (2) allow accumulation of higher concentration of drug in the CNS, and (3) specifically target CNS cells that harbor the virus can potentially eliminate the virus effectively from the CNS. The important aspect of using PLGA-based nanoformulation is that PLGA has been approved by the United States Food and Drug Administration for human use; therefore, a potentially efficacious PLGA-based antiretroviral formulation could be quickly transitioned from the laboratory to the clinic. The PLGAbased drugs illicit minimal immune response and are biodegradable and are degraded into glycolic acid and lactic acid, via the tricarboxylic acid (TCA) cycle into bi-products like water and CO2 and eliminated from the body. Furthermore, the PLGA nanoparticles allow encapsulation of both hydrophilic and hydrophobic drugs and permit surface modifications that not only allow it to escape immune surveillance but also allow the attachment of various molecules such as antibodies that provide targeting specificity and covalent binding to chemically modified ligands that permit tracking of the nanoparticle within the CNS, thus making it a theranostic (molecules that has both a therapeutic and diagnostic ability). The non-HIV drugs that have used modified PLGA as a delivery system include the 28-amino-acid polypeptide, vasoactive intestinal peptide (VIP), the naturally occurring antioxidant enzyme, superoxide dismutase (SOD) and the glucocorticoid dexamethasone [38–40]. Recent studies by Destache et al. showed that three antiretroviral drugs, namely ritonavir, lopinavir, and efavirenz, could be conjugated to PLGA (cART-NPs) and that sustained release of these antiretrovirals was observed from PLGA NP for 28 days without any noticeable toxicity [41]. The same group demonstrated that in HIV-1-infected H9 monocytic cells treated with cART NPs subcellular fractionation studies demonstrated that these cells contained significantly higher nuclear, cytoskeleton, and membrane antiretroviral drug levels compared to cells treated with ARV drug alone. cART-NPs efficiently inhibited HIV-1 infection and transduction thus demonstrating

Abbreviations

the efficacy of the PLGA NPs formulation in inhibiting HIV-1 replication [42]. Chaowanachan et al. showed that antiretroviral drugs with different physicochemical properties such as Saquinavir, Efavirenz, and Tenofovir can be encapsulated individually into PLGA nanoparticles to potently inhibit HIV [43]. Kuo et al. demonstrated that a nanoparticles synthesized using a combination of three biodegradable components and the antiretroviral drug saquinavir (SQV) is encapsulated within the particle core which composed of PLGA to form SQV-PLGA NPs, and the surface of SQV-PLGA NPs was grafted successively with hydrophilic polyethyleneimine (PEI), poly-(g-glutamic acid) (g-PGA). They showed that this nanoparticulate carrier system proved efficacious in delivering antiretroviral drug across the BBB [44]. Based on our studies and data from other investigators, although antiretroviral drug-based nanotherapeutics show a promise for clinical translation, due to their ability to prolong systemic circulation, cross the BBB, provide specific cell targeting, enhance intracellular drug levels in the CNS and reduce the inherent toxicity of many antiretroviral drugs, no successful clinical trials that use nanoformulations of antiretroviral drugs for CNS delivery have been reported. Issues related to HIV latency, longterm accumulation of nanoparticles and antiretroviral drugs in the CNS thereby contributing to neurotoxicity have hindered their clinical utility. Extensive pharmacokinetic studies of these ARVnanoformulations, their CNS biodistribution, and their toxicity need to be done. With the advent of innovations in personalized medicine, materials science, biomedical engineering, and interdisciplinary research collaborations between clinicians, chemists, and basic scientists, there is no doubt that nanotherapy will become mainstream clinical care within the next couple of decades.

Abbreviations

AIDS: Acquired immunodeficiency syndrome ARV: Antiretroviral AS: Atazanavir BBB: Blood–brain barrier BMVEC: Brain microvascular endothelial cells CNS: Central nervous system DLS: Dynamic light scattering

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DSPE-PEG-MAL: 1,2-Distearoyl-sn-glycero-3 phosphoethanolamine-N-[maleimide PEG-2000] GFAP: Glial fibrillary acidic protein HAART: Highly active anti-retroviral therapy HAND: HIV-associated neurological disorders HIV: Human immunodeficiency virus LRP-1: Lipoprotein receptor-related protein-1 MAL: Maleimide MRPs: Multidrug resistance-associated proteins MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide Neuro-AIDS: Neuro-Acquired immune deficiency syndrome NHA: Normal human astrocytes NMR: Nuclear magnetic resonance NPs: Nanoparticles NR: Nile Red PEG: Polyethylene glycol P-gp: P-glycoprotein PLGA: Poly(lactide-co-glycolide) PET: Polyethylene terephthalate PEI: Polyethyleneimine g-PGA: Poly-g-glutamic acid SOD: Superoxide dismutase TCA: Tricarboxylic acid cycle TEER: Transendothelial electrical resistance TEM: Transmission electron microscopy VIP: Vasoactive intestinal peptide

Disclosures and Conflict of Interest

The authors declare that they have no conflict of interest and have no affiliations or financial involvement with any organization cited in this manuscript. No writing assistance was utilized in the production of this chapter and the authors have received no payment for its preparation. Atazanavir was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, Bethesda, Maryland, USA. This study was supported by the following grants from the National Institutes of Health: 1R21DA03010801 (S. Mahajan), K01DA024577 (J. Reynolds), and Kaleida Health Foundations, Buffalo, New York, USA.

About the Authors

Corresponding Author Dr. Supriya Mahajan Department of Medicine Division of Allergy, Immunology, and Rheumatology State University of New York at Buffalo 6074 UB’s Clinical and Translational Research Center 875 Ellicott St, Buffalo, NY 14203, USA Email: [email protected]

About the Authors

Supriya D. Mahajan is an associate professor in the Department of Medicine, State University of New York at Buffalo. Her research interest is in the area of NeuroAIDS in the context of drug abuse and the current focus of her research work is nanotherapeutics for NeuroAIDS and other neurological diseases. Prof. Mahajan has developed and validated an in vitro blood–brain barrier (BBB) model and has used it to evaluate transendothelial transport of several drugs across the BBB. Yun Yu recently graduated from the Department of Chemical and Biological Engineering, State University of New York at Buffalo. She has expertise in the synthesis of biopolymeric nanoparticles and was involved in the preparation of the PLGA nanoparticles used in this study.

Ravikumar Aalinkeel is a research assistant professor in the Department of Medicine, State University of New York at Buffalo. He has expertise in drug delivery systems in various disease states using nanotechnology and has done extensive work in delivery of siRNA therapeutics in the context of cancer biology and neuro-AIDS.

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Jessica L. Reynolds is an assistant professor in the Department of Medicine, State University of New York at Buffalo. She has expertise in drug delivery using exosomes and has encapsulated antiretroviral (ARV) drugs within exosomes, which are then targeted to HIV-1-infected, CNS macrophage/microglial cells. Exosome-mediated delivery of ARV across the BBB provides increased concentrations of ARV within the CNS and elimination of viral reservoirs. Bindukumar B. Nair is a research assistant professor in the Department of Medicine, State University of New York at Buffalo. He has expertise in cell transfections using various nanomaterials.

Manoj J. Mammen is a clinical assistant professor in the Division of Pulmonology in the Department of Medicine, State University of New York at Buffalo. His research focus is nanotherapy in asthma and COPD, and he has experience with intranasal nanodrug delivery systems. Tracey A. Ignatowski is a research associate professor in the Department of Pathology and Anatomical Sciences at the State University of New York at Buffalo and is actively engaged in research investigating mechanisms involved in cytokine (TNF, IL-1b) regulation of neurotransmitter release in the brain, determining cytokine localization in the brain, and demonstrating brainassociated TNF regulation of peripheral macrophage function. She is involved in investigating biomedical application of nanoparticle formulations, specifically in the field of targeted RNA/DNA/ therapeutic agent delivery.

References

Chong Cheng is a associate professor in the Department of Chemical and Biological Engineering, State University of New York at Buffalo. His research work focuses on the development of general methodologies for the preparation of welldefined biodegradable functional polymers and nanostructures, which can further serve as scaffolds for biomedical and bioengineering applications, especially drug and gene delivery.

References

Stanley A. Schwartz is a distinguished professor of medicine, pediatrics, and microbiology and Chief of the Division of Allergy, Immunology, and Rheumatology in the Department of Medicine at the State University of New York at Buffalo. He is director of the Nanomedicine center at UB’s Clinical and Translational Research Center.

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24. Tosi, G., Bortot, B., Ruozi, B., Dolcetta, D., Vandelli, M. A., et al. (2013). Potential use of polymeric nanoparticles for drug delivery across the blood–brain barrier. Curr. Med. Chem., 20, 2212–2225. 25. Costantino, L., Gandolfi, F., Tosi, G., Rivasi, F., Vandelli, M. A., et al. (2005). Peptide-derivatized biodegradable nanoparticles able to cross the blood–brain barrier. J. Control. Release, 108, 84–96. 26. Destache, C. J., Belgum, T., Christensen, K., Shibata, A., Sharma, A., et al. (2009). Combination antiretroviral drugs in PLGA nanoparticle for HIV-1. BMC Infect. Dis., 9, 198.

27. Shibata, A., McMullen, E., Pham, A., Belshan, M., Sanford, B., et al. (2013). Polymeric nanoparticles containing combination antiretroviral drugs for HIV type 1 treatment. AIDS Res. Hum. Retroviruses, 29, 746–754. 28. Kanmogne, G. D., Singh, S., Roy, U., Liu, X., McMillan, J., et al. (2012). Mononuclear phagocyte intercellular crosstalk facilitates transmission of cell-targeted nanoformulated antiretroviral drugs to human brain endothelial cells. Int. J. Nanomed., 7, 2373–2388.

29. Kuo, Y. C., Lee, C. L. (2012). Methylmethacrylate-sulfopropylmethacrylate nanoparticles with surface RMP-7 for targeting delivery of antiretroviral drugs across the blood–brain barrier. Colloids Surf. B Biointerfaces, 90, 75–82. 30. Kuo, Y. C., Chung, C. Y. (2012). Transcytosis of CRM197-grafted polybutylcyanoacrylate nanoparticles for delivering zidovudine across human brain-microvascular endothelial cells. Colloids Surf. B Biointerfaces, 91, 242–249.

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33. Cucullo, L., Aumayr, B., Rapp, E., Janigro, D. (2005). Drug delivery and in-vitro models of the blood–brain barrier. Curr. Opin. Drug Discov. Dev., 8, 89–99.

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35. Mahajan, S. D., Aalinkeel, R., Sykes, D. E., Reynolds, J. L., Nair, B. B., et al. (2008). Tight junction regulation by Morphine and HIV-1 tat modulates blood brain barrier permeability. J. Clin. Immunol., 28, 528–5541.

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Chapter 48

HIV-Specific Immunotherapy with Synthetic Pathogen-Like Nanoparticles Orsolya Lorincz, PhD, and Julianna Lisziewicz, PhD eMMUNITY Inc., Bethesda, Maryland, USA

Keywords: DNA-nanomedicine vaccine, immunotherapy, Langerhans celltargeted DNA delivery, HIV eradication, polyethylenimine (PEI), DermaVir, therapeutic vaccine, plasmid DNA, intracellular trafficking, gene delivery, polyplex

48.1 Introduction

HIV/AIDS is the leading infectious killer that affects more than 33 million people worldwide [1]. HIV/AIDS is treated with the combination of three or more antiretroviral drugs (ARVs) (highly active antiretroviral therapy, HAART), because using any single drug has not been efficient in controlling infection due to the development of resistant strains of the virus. Currently available HAART is potent in suppressing HIV replication and effective in decreasing HIV RNA levels below the limit of detection (50 copies/ mL) with only minimum side effects. Long-term HAART decreased morbidity and mortality associated with HIV infection [2]. However, even optimal HAART, characterized by suppression of viral Handbook of Clinical Nanomedicine: Nanoparticles, Imaging, Therapy, and Clinical Applications Edited by Raj Bawa, Gerald F. Audette, and Israel Rubinstein Copyright © 2016 Pan Stanford Publishing Pte. Ltd. ISBN 978-981-4669-20-7 (Hardcover), 978-981-4669-21-4 (eBook) www.panstanford.com

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load to undetectable levels for years, has not provided a cure to the disease. Patients on optimal HAART have 12 years shorter life expectancy than HIV-negative people [3–4]. In addition, increased AIDS-related and non-AIDS-related morbidity and mortality has been described in a significant proportion of individuals on optimal HAART due to the lack of normalization of their CD4+ T cell counts [5]. Optimal HAART also failed to decrease the viral reservoirs, especially in the gut mucosa, where the residual low-level viral replication may be the cause of persistent immune activation that facilitates the progression to AIDS and death [6]. One barrier of cure is the stable latent reservoirs of HIV-infected resting memory T cells that are able to produce HIV after cellular activation. HIV producing cells in the reservoirs that are not eliminated by ARVs would be susceptible to immune clearance, but long-term optimal HAART diminishes HIV-specific T cell responses [7]. Therefore, the immune system of successfully treated HIV-infected people is unprepared to kill infected cells, decrease viral reservoirs, and control the virus replication. Immunotherapy improves the reactivity of the immune system to guard the body from internal and external intruders as infections (e.g., HIV), allergens, and malignancies. The innate arm, composed of the phagocytic cells, circulating macrophages and the complement system, is responsible for the immediate defense. Later, the adaptive arm responds in a highly specific manner against molecular determinants of the intruders. Nanoparticles (NPs) made from different types of materials, having various sizes, shapes and surface charges activate the innate and adaptive immune system [8–10]. Therefore, nanotechnology is suitable for improving efficacy and increasing specificity of products indicated as vaccine and immunotherapy. The immunotherapeutic efficacy of NPs is achieved by specific activation (e.g., cancer, infectious diseases) or suppression (e.g., allergy, autoimmune diseases) of the immune system. Efficacy of immune responses is improved by targeted delivery of antigens to professional immune cells specialized for either stimulation or suppression of immune reactivity. This is achieved by using NPs as antigen (virus-like particles; VLPs), NP formulation of soluble antigens (DNA, peptides, proteins) and new administration routes (transdermal, nasal, intratracheal). Increasing the specificity of immunotherapy is achieved by personalization of the antigens in the NP taking into account the

Nanomedicines for Immunotherapy

diversity of the disease (e.g., tumor cells) and genomic background of the individual (e.g., HLA type). More than 30 ARVs and drug combinations are currently available to achieve long-term suppression of HIV RNA to 100 nm nanomedicines target the peripheral immature DCs, and the smaller size ~50 nm nanomedicines drain to the lymph node resident DCs [29]. Modification of the surface of the nanomedicine with DC-specific receptor ligands has been shown to increase the targeting specificity [30]. However, several challenges including crossing physical barriers like the cell and nuclear membranes or adhesion to non-target tissues still need to be overcome during the development of a synthetic delivery system.

48.3 Pathogen-Like DNA-Nanomedicines for Immunotherapy

DermaVir is the first synthetic pathogen-like nanomedicine that has been developed for HIV-specific immunotherapy. A condensed pDNA constitutes the core and a mannobiosylated polymer, polyethylenimine-mannose (PEIm) represents the envelop of this synthetic virus [31, 32]. We have shown that nanomedicine not only looks like a virus but also functions as a replication defective virus: enters cells via receptor-mediated endocytosis, escapes from endosome, and delivers the pDNA to the nucleus. The pDNA expresses proteins that self-assemble to “complex virus-like particles” (VLP+). The huge advantage of this synthetic pathogen-

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like nanomedicine compared to biologic nanomedicines (VLPs, virus vectors, exosomes) is the reproducible and scalable manufacturing method that is independent of the antigens since these are encoded in the pDNA. Therefore, such pDNA-based pathogen-like nanomedicines are not only highly immunogenic but also cost effective to make and therefore suitable for the development of personalized HIV-specific immunotherapy.

48.3.1 Nanomedicine Formulation of Plasmid DNA for Vaccine Delivery

The immunologic objective of a pathogen-like pDNA/PEIm nanomedicine formulation is to target antigen-presenting cells (APCs) and efficiently express the antigens encoded in the pDNA. Targeted delivery is a complex challenge involving binding, nanomedicine uptake, pDNA expression, processing and antigen presentation to naïve T cells [33, 34]. The objectives of the pDNA-based antigen design are efficacy and safety. pDNA does not duplicate in human cells, it expresses protein antigens to induce specific immune responses. Efficacy is achieved by preserving the structure and the epitope content of the disease-causing virus, and safety is achieved by molecular modifications. The first pathogen-like NP, DermaVir, contains a single pDNA (Fig. 48.1a) driving the expression of fifteen HIV proteins. These proteins self-assemble to replication-, reverse transcription–, and integration-defective VLP+. The pDNA is inherently safe because irreversible molecular modifications were introduced into essential viral proteins to prevent the replication and integration of the VLP+. The expressed VLP+ is structurally authentic to the wild-type HIV (Fig. 48.1b). The expression of fifteen HIV proteins from the single pDNA supports the presentation of the highest number of HLA-restricted HIV epitopes and the induction of HIVspecific T cell responses with the broadest specificity. DermaVirinduced T cell responses can kill HIV-infected cells. Beyond inducing T cell responses, this antigen might be suitable for inducing neutralizing antibodies against viral and cellular antigens naturally occurring on the surface of HIV or structures present only during budding or entry [36]. Such a multi-faceted immune response is a unique feature of pDNA vaccines expressing an authentic-looking HIV [35].

Pathogen-Like DNA-Nanomedicines for Immunotherapy

(b)

(a)

Figure 48.1 Key features of DermaVir nanomedicine developed for HIVspecific immunotherapy. (a) One single pDNA encoding 15 HIV antigens serves as the active pharmaceutical ingredient (API) [35]. Red: HIV sequences expressing the antigens in human cells. Gray: Bacterial sequences required for plasmid manufacturing in E. coli. (b) Expression of complex virus-like particles (VLP+) visualized by transmission electron microscopy. Table 48.1

Pathogen-like features of DermaVir nanomedicine compared to a DNA virus

DermaVir Features

pDNA/PEIm nanomedicine DNA virus

Pathogen look

Surface

Sugar residues

Glycoproteins

Pathogen features

Structure

PEIm envelop, condensed pDNA core

Glycoprotein envelop, condensed DNA core

Size, shape 70–300 nm, spherical NPs

Delivery of PEIm-mediated endosomal DNA escape and cytoplasmic trafficking Antigen HIV VLP+ expression

50–500 nm, mostly spherical NPs

Viral protein-mediated endosomal escape and cytoplasmic trafficking HIV

PEIm component of DermaVir is responsible to form a athogenlike nanomedicine with the pDNA (Fig. 48.2a). The pathogen-like features are essential for binding and cellular uptake, and later for the pDNA processing and antigen expression (Table 48.1) [32]. Targeting of the APC population is ensured by the mannobiosylation of the polymer. We modified a transfection agent, the linear polyethylenimine, with mannobiose residues resulting in a mannobiosylated polyethylenimine (PEIm) having grafted sugar

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molecules to resemble pathogens that usually bear similar residues (glycoproteins) on their surface [37–40]. This modified polymer spontaneously forms 70–300 nm NPs with pDNA, the optimal size range for receptor-mediated entry, as this is the size range of viruses [41, 42]. The formed NPs have positive surface charge, indicating that the polymer is on the outside covering the condensed pDNA in the core.

(a)

(c)

(b)

(d)

Figure 48.2 Biophysical characterization of DermaVir nanomedicine. (a) Atomic force microscopic image of naked pDNA and DermaVir pDNA/PEIm NPs. (b) Absorbance spectra of DermaVir nanomedicine (green) and its components; pDNA (blue) and PEIm (red). The shift of the nanomedicine is called hyperchromicity and is attributed to the forming interactions between the pDNA and the PEIm. (c) Inverse correlation between the NaCl concentration of the pDNA solution and the measured Hc%. Hc% is the increase of absorbance measured at 260 nm for DermaVir compared to the sum absorbance at 260 nm of its components expressed in percentage. Molar equivalent on the x-axis stands for molar equivalents calculated on the phosphate concentration of the pDNA ( y = –5.14x + 29.8; r2 = .95) (d) Linear correlation between the pH of the formulation and the Hc% ( y = 1.94x + 6.3; r2 = .98). Adapted from Nanomed. Nanotechnol. Biol. Med., 8, Lorincz, O., Toke, E. R., Somogyi, E., Horkay, F., Chandran, P. L., et al., Structure and biological activity of pathogen-like synthetic nanomedicines, 497–506, Copyright (2012), with permission from Elsevier.

Pathogen-Like DNA-Nanomedicines for Immunotherapy

Once the pathogen-like nanoparticles are taken up by endocytosis the nanomedicine continues its role and function as a pathogen through the intracellular processing. The NPs translocate to a cellular compartment, called endosomes. The polymer envelop buffers the low pH of the endosome that would otherwise degrade the pDNA [43]. The proton-sponge effect of the PEIm supports the buffering of the endosome mimicking the features of viruses that evolved to survive this environment and deliver their genetic material to the host cell’s nucleus. When the NPs reach the cytosol the polymer envelop is loosened enough to release the pDNA at the nucleus. This way the pDNA can translocate to the nucleus where the pDNA-encoded antigens are transcribed. DermaVir’s pDNA expresses a VLP+, and these antigens stimulate the naïve T cells to become HIV-specific memory and effector T cells capable to kill infected cells. The unique pathogen-like feature of the pDNA/PEIm DermaVir nanomedicine is its DNA delivery attributes. As the targeting and cellular uptake of the NPs are controlled by the surface and the size of the NPs, the NPs processing inside the cell is controlled by the PEIm that ensures that the pDNA reaches the nucleus [32, 44]. No other synthetic DNA delivery agents have similar properties, because the DNA either remains in the endosome and is degraded or cannot reach the nucleus where gene expression occurs [38]. The release dynamics of a pDNA-NP inside the cell is a process characterized with a very sensitive equilibrium. This balance is determined by the ionic properties and environment of the components. Since both pDNA and PEIm are polyionic macromolecules and their interaction is driven by electrostatic forces we explored the effect of ionic strength and pH on their biological activity and also on their fine structure. We found that the condensation of the pDNA by PEIm could be followed by UV spectrophotometry, because the structural changes occurring during complexation cause a shift in the UV-spectrum of DermaVir compared to its components (Figs. 48.2a,b). We called this shift hyperchromicity (Hc) and connected it to the forming interactions of the components during NP formation [32]. Our results showed that this phenomenon can be influenced by changing the ionic properties of the components; both ionic strength and pH of the nanomedicine formulation have high impact on Hc. By increasing the ionic strength of the pDNA solution, Hc decreases, and by increasing the pH of the formulation, Hc increases (Figs. 48.2c,d).

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We also found that Hc was associated with the biological activity (Fig. 48.3). The differences in Hc and in biological activity are explained with the fine structure of the NPs. A DermaVir formulation with low Hc and low biological activity shows a loose NP structure where the pDNA protrudes from the polymer envelop [32]. The DermaVir formulation with optimal Hc and high biological activity has a smooth NP surface, where the pDNA is fully protected by a coherent polymer envelop (Fig. 48.2a).

Figure 48.3 Structure and activity relationship of DermaVir nanomedicine. Biological activity of low, optimal, and high hyperchromicity (Hc%) DermaVir formulations.

The equilibrium of pDNA release dynamics inside the cells is controlled by the “compactness.” Compactness is the degree of association between the components of the synthetic NPs that can be controlled by changing the ionic properties of the components. Very high ionic strength causes aggregation of the NPs [45]. When the ionic strength is slightly high or the pH is low, the pDNA and the PEIm will form fewer bonds because the pDNA will have less unprotonated phosphate groups. Low biophysical compactness represents a loose structure. The biological consequence of a loose structure is that the NPs do not survive the endosome and the pDNA is degraded (Fig. 48.4a). When the ionic strength of the pDNA solution is low or the pH is high, more bonds will be formed between the pDNA and the PEIm because the DNA will have many anionic phosphate groups. High compactness represents a tight structure; consequently the NPs reach the proximity of the nucleus but do not release the pDNA (Fig. 48.4b). Optimal NP that survives the endosome and releases the pDNA at the nucleus can be characterized by optimal compactness representing optimal ionic

Pathogen-Like DNA-Nanomedicines for Immunotherapy

strength of the pDNA solution and the optimal pH (6–7.5) of the formulation (Fig. 48.4c). DermaVir is one of the first clinically used nanomedicines in which the structure and biophysical activity have correlated to its biological activity.

Figure 48.4 Compactness is a biophysical property of the pDNA/PEIm NP that determines the release dynamics of the pDNA inside the cell. (a) At low compactness, the NP disintegrates in the endosome. (b) At high compactness, the NP survives the endosome but cannot release the DNA in the cytosol. (c) At optimal compactness, the NP escapes from endosomal degradation, the pDNA is released in the cytosol and reaches the nucleus, where the DNA-encoded antigens are expressed.

48.3.2 Topical Administration of the Langerhans Cell–Targeted Nanomedicine Vaccines

Since Pasteur, the needle injection is the state of art for the administration of antigens. However, injection of soluble antigens (peptides, proteins) does not mimic the natural entry of pathogens in the body creating a challenge to induce potent immune responses. To improve potency, antigens are formulated with different adjuvants, and alternative needle injection techniques are introduced in the form of microneedles and electroporation. Virus vectors and pathogen-like NPs are very potent to deliver the

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DNA into DCs if they are placed in an environment of activated DCs ready to pick up pathogens. Such environment is not created after needle injection for at least two reasons: (i) Injection is not activating DCs to pick up antigens and migrate to lymph nodes and (ii) injection is targeting dermis or muscle, organs that are not prepared for pathogen entry and therefore do not contain many DCs. We developed DermaPrep, the first Langerhans-cell (LC)targeting administration device for pathogen-like nanomedicines. DermaPrep employs a skin preparation method that interrupts the stratum corneum facilitating nanomedicine penetration and providing the essential “danger” signal to the LCs residing just below this protective layer [46, 47]. Once activated, LCs are naturally looking for pathogens and capturing the pathogenlike DermaVir nanomedicine applied to the prepared skin surface under a semi-occlusive patch. The main advantage of DermaPrep is the natural targeting of a large number of LCs (8 million) that form a horizontal 900 to 1800 cells/mm2 network under the skin surface [48].

Figure 48.5 Langerhans cell-targeted delivery of DermaVir NPs after administration with DermaPrep. (1) Skin preparation using body sponge to generate “danger signal.” (2) Nanomedicine application under the patch. (3) Uptake of the NPs by LCs. (4) Migration of LCs to the draining lymph nodes. (5) Antigen presentation in the lymph nodes and priming of naïve CD4+ and CD8+ T cells. (6) HIV-specific central memory T cells with high proliferative capacity circulating in the body [50, 51].

Pathogen-Like DNA-Nanomedicines for Immunotherapy

After DermaVir has been captured, activated LCs migrate to the local lymph nodes [49]. Here DCs express pDNA-encoded antigens and present most HIV epitopes to the passing naïve T cells. HIVspecific precursor/memory T cells primed by DCs further differentiate into HIV-specific effector T cells circulating out of the lymph node to seek virus-infected targets. Each killer effector cell can destroy several HIV-infected cells (Fig. 48.5). During DermaPrep administration one million NPs could be picked up by each LC, because there are 8 × 1012 DermaVir NPs in 0.8 mL solution that is applied under each patch. In contrast, other popular vaccine administration systems such as electroporation and microneedles are needle in a haystack by means of targeting compared to DermaPrep; electroporation delivers naked pDNA usually to muscle and can potentially target APCs only at the site of injection, which usually does not reach the 1 mm3 (which is only 900–1,800 LCs in the epidermal layer) [50–52]. In addition, the intra- and extracellular degradation of the pDNA after electroporation further decreases the success of antigen uptake and expression in the APC. Table 48.2

Comparison of different vaccine delivery devices

Mechanism of action

Targeting

Number of targeted LCs

DermaPrep

Electroporation

Microneedles

Th1 memory T cells

Mainly antibodies

Antibodies and T cells

~8 million

1–2 thousand

Therapeutic vaccines

Prophylactic & Prophylactic and therapeutic vaccines therapeutic vaccines

Epidermal No Langerhans cells

No

A few thousand

DNA expression Lymph node DCs Skin or muscle cells Skin

Potential indications

Formulation

Developmental status

NPs

CE marked

Naked DNA

Safety testing in human ongoing

Several formulations are being tested

Testing in human ongoing

Microneedles that deliver the vaccines into the epidermis are more promising than electroporation, but their small size (few mm2) allows the antigens to target only a few thousand DCs [53]. Regarding the immune responses electroporation is mainly capable

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of inducing Th2-type while DermaPrep elicits Th1-type T cellmediated immunity, microneedles are described to do everything [54, 55]. NP formulations can be administered to specifically target APCs using DermaPrep, electroporation uses naked pDNA with or without adjuvants. All formulations are applicable with microneedles; naked antigens or formulated molecules, coated devices and separate products [56–58]. Table 48.2 summarizes the main features of the pDNA delivery devices compared to DermaPrep.

48.4 DermaVir Immunotherapy for the Cure of HIV/AIDS

DermaVir is the most advanced nanomedicine developed for HIV-specific immunotherapy (therapeutic vaccine). It is the bestcharacterized nanomedicine and has successfully completed Phase II clinical development. Here we summarize the results of the main preclinical and clinical studies. Preclinical studies in chronically infected macaques demonstrated that DermaVir immunotherapy improves the median survival time from 18 to 38 weeks compared to no treatment [50]. In other macaque trial, DermaVir in combination with HAART suppressed virus replication after HAART interruption. DermaVir treatment benefits were associated with the boosting of memory T cell responses. These primate experiments provided the rationale to investigate DermaVir immunotherapy in HIV-infected subjects both as monotherapy and in combination with HAART. The Phase I dose escalation study conducted in Hungary was designed to evaluate the safety and immunogenicity of a single DermaVir immunization in HIV-infected subjects on fully suppressive HAART [51]. Increasing DermaVir doses up to 0.8 mg pDNA were administered by DermaPrep simultaneously on two, four and eight skin sites. DermaVir-associated side effects were limited to the skin, mild, transient and not dose-dependent. Boosting of HIV-specific effector CD4+ and CD8+ T cells expressing IFN-gamma and IL2 was detected against several HIV antigens in every subject of the medium dose cohort. The striking result was the dose-dependent expansion of HIV-specific central memory T cells with high proliferation capacity [59–60]. These findings demonstrated the first time that an immunotherapy (therapeutic

DermaVir Immunotherapy for the Cure of HIV/AIDS

vaccine) is capable to induce potent antigen-specific memory T cell responses in infected subjects. We found that the frequency of DermaVir-boosted memory T cells decreases within a year suggesting that repeated DermaVir immunizations are required for durable high immune reactivity. A Phase I/II clinical trial conducted in several USA clinical centers was designed to investigate repeated administrations of escalating DermaVir doses or placebo on HIV-infected adults receiving fully suppressive HAART. The incidence of adverse events was similar across groups suggesting that DermaVir was as safe as placebo [61, 62]. Immunogenicity data demonstrated the boosting of HIV-specific central memory T cells with high proliferative capacity. The highest frequency of HIV-specific memory T cells was induced in the 0.4 mg DNA dose group. The results of this trial were consistent with the macaque and Phase I studies and confirmed the excellent safety and immunogenicity features of DermaVir immunizations in patients on HAART [61]. The Phase II randomized, placebo controlled multicenter clinical trial conducted in Germany was designed to evaluate the safety and preliminary efficacy of repeated DermaVir immunizations in the absence of HAART. Thirty-six HIV-infected, treatment naïve adults were randomized to receive one of three different DermaVir doses or placebo at Study Weeks 0, 6, 12, and 18. The primary endpoint was safety at Week 24 and secondary endpoints were HIV RNA [63]. The Phase II trial confirmed the safety and the immunogenicity features of DermaVir. Consistently with previous trials, the 0.4 mg DermaVir dose was superior to the others. In this dose group the medium HIV RNA significantly decreased by 70% compared to placebo suggesting the killing of HIV infected cells. Viral load suppression occurred slowly, as predicted by DermaVir mechanism of action, similarly to cancer vaccines [64]. These results were consistent with the macaque and previous clinical studies and confirmed the safety and immunogenicity and antiviral activity of repeated DermaVir immunizations. DermaVir immunotherapy might overcome the following limitations of present ARVs (Table 48.3): (i) reconstitution of HIVspecific immune responses that decreased during optimal HAART (ii) depletion of HIV-infected cells in contrast to current ARVs that inhibit only one step in HIV life cycle. Compared to HAART the antiviral activity of DermaVir is delayed, slow, and less potent,

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because recognizing and killing of millions of infected cells with CTL in the presence of millions of new infections take more time to decrease viral load than blocking HIV replication with drugs. The effectiveness of killing infected cells is revealed by their capacity to manage the infection for ca. 15 years in the absence of any treatment. Therefore, 0.5 log reduction of HIV RNA in 24 weeks, demonstrated with DermaVir, should be sufficient to decrease the amount of HIV-infected cells that are not eliminated by HAART. Table 48.3

Features of DermaVir nanomedicine developed toward the cure of HIV compared with the current state–of-the-art treatment of HIV/AIDS

Features/approach

DermaVir

HAART

Therapeutic target

HIV-expressing cells

HIV life cycle

Adverse events

Transient, skin

Cumulative, systemic

Boosting

Decreasing

Time needed for effectiveness

Slow and durable

Administration schedule

Yearly

HIV-infected cells

Eliminated

Viral load

HIV immunity Cure

48.5 Conclusions

Slow decrease Remission

Rapid and transient Daily

Rapid decrease No effect No

HAART successfully suppresses HIV replication but cannot cure the disease because infected cells remain in the reservoirs. These resting latently infected cells will not be efficiently killed even after HIV reactivation; thus, boosting of T cell responses through HIV-specific immunotherapy will be essential for the cure of HIV [20]. DermaVir is the first synthetic pathogen-like nanomedicine developed to treat HIV. In the clinic, DermaVir is administered topically with DermaPrep targeting ~8 million LCs with ~1013 NPs containing 0.1 mg pDNA. One single dose and two repeated dose randomized, placebo controlled studies demonstrated that DermaVir induce potent and broadly directed HIV-specific central memory T cells in HIV-infected subjects. Such responses have been correlated with low viremia in untreated individuals, protection

Conclusions

in non-human primates and containment of viremia in elite controllers [19, 59, 65].

Figure 48.6 Nanomedicine applications toward the cure of HIV/AIDS: (a) untreated HIV infection: high viral load, high amount of HIV-infected cells in the reservoirs and the disease is partially controlled by the immune system alone for ca. 15 years. (b) HAART is effective in potent and durable suppression of HIV RNA to