Delivery Technologies

for Biopharmaceuticals

Delivery Technologies for Biopharmaceuticals: Peptides, Proteins, Nucleic Acids and Vaccines
Edited by Lene Jorgensen and Hanne Mrck Nielsen
2009 John Wiley & Sons, Ltd. ISBN: 978-0-470-72338-8

Delivery Technologies
for Biopharmaceuticals
Peptides, Proteins, Nucleic Acids
and Vaccines

Editors
Lene Jorgensen
Faculty of Pharmaceutical Sciences
University of Copenhagen, Denmark
Hanne Mrck Nielsen
Faculty of Pharmaceutical Sciences
University of Copenhagen, Denmark

A John Wiley & Sons, Ltd., Publication

This edition first published 2009

2009 John Wiley & Sons Ltd
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Library of Congress Cataloging-in-Publication Data
Delivery technologies for biopharmaceuticals : peptides, proteins, nucleic acids, and vaccines / editors, Lene Jorgensen,
Hanne Mrck Nielsen.
p. ; cm.
Includes bibliographical references and index.
ISBN 978-0-470-72338-8 (cloth : alk. paper) 1. Drug delivery systems. 2. Drug carriers (Pharmacy) I. Jorgensen,
Lene. II. Nielsen, Hanne Mrck.
[DNLM: 1. Drug Delivery Systemsmethods. 2. Biological Products. QV 785 D355 2009]
RS199.5.D45 2009
615.6dc22
2009025904
A catalogue record for this book is available from the British Library.
ISBN 9780470723388 (H/B)
Set in 10 on 12 pt Times by Toppan Best-set Premedia Limited
Printed and bound in Great Britain by CPI Antony Rowe Ltd, Chippenham, Wiltshire

Contents

Preface
List of Contributors

page xv
xvii

INTRODUCTION

1. Challenges in Delivery of Biopharmaceuticals; the Need for Advanced

Delivery Systems
Hanne Mrck Nielsen and Lene Jorgensen
1.1
1.2

1.3
1.4
1.5

Introduction
Overcoming Delivery Barriers
1.2.1 Stabilization
1.2.2 Enhancing Delivery
Drug Delivery Technologies and Excipients
Risks
Conclusion
References

DELIVERY OF BIOPHARMACEUTICALS
2.

Novel Formulation Approaches for Peptide and Protein Injectables

Mingshi Yang and Sven Frokjaer
2.1
2.2
2.3

2.4

Introduction
Formulation Challenges
Chemical Modification of Proteins and Peptides
2.3.1 PEGylation
2.3.2 Glycoengineering
2.3.3 Acylation
2.3.4 Amino Acid Substitution
2.3.5 Protein Fusion
Depot Delivery Systems
2.4.1 Micro- and Nanoparticulate Systems
2.4.2 In Situ Depot-Forming Systems
2.4.3 Implant Systems

3
3
4
4
5
5
6
7
7

9
11
11
12
13
13
15
16
17
17
18
18
21
21

vi

Contents

2.5

2.6

3.

Novel Non-Injectable Formulation Approaches of Peptides

and Proteins
Shirui Mao, Dongmei Cun and Yoshiaki Kawashima
3.1
3.2

3.3

3.4

3.5

3.6

3.7

4.

Other Delivery Systems

2.5.1 Protein Crystallization and/or Precipitation
2.5.2 Injection Devices
Conclusions
References

Introduction
Intranasal Delivery of Peptide/Protein Drugs
3.2.1 Properties of Intranasal Delivery
3.2.2 Strategies to Enhance Intranasal Absorption
Pulmonary Delivery of Peptide/Protein Drugs
3.3.1 Properties of Pulmonary Delivery
3.3.2 Strategies to Enhance Pulmonary Delivery
Buccal Administration of Peptide/Protein Drugs
3.4.1 Properties of Buccal Delivery
3.4.2 Strategies to Improve Buccal Absorption
Oral Delivery of Peptide/Protein Drugs
3.5.1 Challenges of Oral Delivery
3.5.2 Strategies to Enhance Oral Absorption
Transdermal Delivery of Peptide/Protein Drugs
3.6.1 Properties of Transdermal Delivery
3.6.2 Strategies to Improve Transdermal Absorption of
Proteins/Peptides
Conclusions
References

Chemical Vectors for Delivery of Nucleic Acid-Based Drugs

Elizabeth A. Vasievich and Leaf Huang
4.1
4.2

Introduction
Barriers to Delivery of Nucleic Acids
4.2.1 Anatomical
4.2.2 Cellular
4.3 Major Classes of Delivery Vectors
4.3.1 Introduction
4.3.2 Cationic Lipids
4.3.3 Cationic Polymers
4.3.4 Cationic Dendrimers
4.3.5 Cell Penetrating Peptides (CPP) and DNA Mimics
4.3.6 Naked DNA
4.4 Targeted Delivery
4.4.1 Introduction
4.4.2 Targeting Solid Tumours and/or Metastasis

22
22
22
23
23

29
29
30
30
31
36
36
36
42
42
44
47
47
48
53
53
53
56
57
69
69
70
70
70
71
71
71
73
74
75
76
76
76
77

Contents

4.5 Triggered Release

4.5.1 Introduction
4.5.2 Proton Sponge Effect
4.5.3 Magnetofection (Magnetic Delivery)
4.5.4 Hyperthermic Delivery
4.5.5 Reductive Agents
4.5.6 BiotinAvidin
4.5.7 Electrochemical
4.6 Recent Clinical Trials Using Naked and Chemically Complexed
Nucleic Acids
4.6.1 Introduction
4.6.2 Naked Nucleic Acids in Clinical Trials
4.6.3 Lipid-Based Nucleic Acid Carriers in Clinical Trials
4.6.4 Polymer-Based Nucleic Acid Carriers in Clinical Trials
4.7 Conclusion
References
5.

Viral Technology for Delivery of Nucleic Acids

Shervin Bahrami and Finn Skou Pedersen
5.1
5.2

5.3
5.4

5.5

5.6

6.

Introduction
Barriers and Topological Requirements to Cellular Entry
5.2.1 Enveloped Viruses and Membrane Fusion
5.2.2 Non-Enveloped Viruses
Routes of Administration
Delivery Vector Requirements
5.4.1 Targeting
5.4.2 Expression
5.4.3 Safety
Examples of Viral Delivery Technology
5.5.1 Retroviral and Lentiviral Vectors
5.5.2 Adenovirus Vectors
5.5.3 Adeno-Associated Virus Vectors
5.5.4 Mixed Delivery Systems
5.5.5 In Vitro Assembled Delivery Vehicles
Conclusion
References

The Innate Immune Responses, Adjuvants and Delivery Systems

S. Moein Moghimi
6.1 Introduction
6.2 Dendritic Cell Immunobiology
6.3 Antigen Capture
6.4 Particulate Antigen Delivery Systems
6.5 Signalling Receptors and the Role of Adjuvants
6.6 Conclusions
References

vii

79
79
80
80
81
82
82
83
83
83
83
85
86
86
87
93
93
95
95
97
98
98
98
100
101
103
103
105
106
107
108
108
109
113
113
117
119
120
121
123
124

viii

Contents

DELIVERY TECHNOLOGIES FOR BIOPHARMACEUTICALS

7.

Lipid Nanoparticle-Based Systems for Delivery

of Biomacromolecule Therapeutics
Susana Martins, Domingos C. Ferreira and Eliana B. Souto
7.1 Introduction
7.2 Definitions and Properties of Solid Lipid Nanoparticles (SLNs)
7.3 Definitions and Properties of Lipid-Drug Conjugates (LDCs)
7.4 Administration, Delivery and Targeting
7.5 Toxicity and Safety
7.6 Applications for Biopharmaceuticals
7.6.1 Peptides and Proteins
7.6.2 Nucleic Acids
7.6.3 Vaccines
7.7 Conclusions
References

8.

Dendrimers in Delivery of Biopharmaceuticals

Chandan Thomas and Fakhrul Ahsan
8.1

Introduction
8.1.1 Properties of Dendrimers as a Carrier System
8.2 Case I Application of Dendrimers in Delivery of Large Molecular
Weight Drugs
8.3 Case II Application of Dendrimers in Gene Delivery
8.3.1 Case IIA Application of PAMAM-PLGA Microparticle
Conjugates in Gene Delivery
8.3.2 Case IIB Application of Dendrimers in siRNA Delivery
8.4 Case III Application of Dendrimers in Vaccine Delivery
8.5 Concluding Remarks
References

PEPTIDES AND PROTEINS

9.

Modification of Peptides and Proteins

Susanne Hostrup, Kasper Huus and Henrik Parshad
9.1
9.2

9.3

Introduction
PEGylated Peptides and Proteins
9.2.1 Methods
9.2.2 Conceptual Considerations on PEGylation
9.2.3 Pharmacokinetic Aspects
9.2.4 Pharmaceutical Aspects
9.2.5 PEGylated Proteins in Advanced Delivery Systems
Lipidization of Peptides and Proteins
9.3.1 Methods of Lipidization
9.3.2 Conceptual Considerations on Lipidization

129
131
131
133
135
136
138
139
139
139
142
142
142
149
149
150
153
155
156
158
162
165
166

169
171
171
171
172
173
174
175
177
177
177
178

Contents

9.4

9.5
9.6
10.

178
179
180
181
181
182
183
183
185
185
186
186
187

Nanocarriers for the Delivery of Peptides and Proteins

Kenneth Lundstrom

193

10.1
10.2

193
194
194
195
195
196
196
197
198
199
199
199
200
201
202
202

10.3

10.4
11.

9.3.3 Improved Pharmacokinetics

9.3.4 Improved Delivery via Non-Parenteral Delivery Routes
9.3.5 Pharmaceutical Aspects
9.3.6 Lipidization in Advanced Delivery Systems
Modification of the Primary Structure of Peptides and Proteins
9.4.1 Strategies for Designing Protein Analogues
9.4.2 Advantages and Disadvantages
9.4.3 Pharmaceutical Aspects General Principles for Improving
Protein Stability
9.4.4 Pharmacokinetic Aspects
9.4.5 Manipulating the Isoelectrical Point (pI) by Molecular
Engineering
General Considerations on Processing and Characterization
Conclusions
References

ix

Introduction
Polymeric Nanoparticles
10.2.1 Lactic/Glycotide Polymers
10.2.2 Polycaprolactones
10.2.3 Polyphosphoesters
10.2.4 Polyanhydrides
10.2.5 Polyorthoesters
10.2.6 Block Copolymers with a Specific Emphasis on Medusa
10.2.7 Cross-Linked Dextran
In Situ Depot Forming Systems
10.3.1 Precipitation Systems
10.3.2 Thermal Gelling Systems
10.3.3 Cross-Linked Systems
10.3.4 Thermoplastic Semisolids
Conclusions
References

Polymer-Based Delivery Systems for Oral Delivery of Peptides

and Proteins
Bruno Sarmento, Domingos Ferreira and Tefilo Vasconcelos

207

11.1 Introduction
11.2 Advances in Oral Protein Delivery
11.3 Intestinal Considerations for Protein Absorption
11.4 Polymer-Based Delivery Systems for Oral Delivery of Proteins
11.4.1 Hydrogels
11.4.2 Patches and Tablets
11.4.3 Microparticles
11.4.4 Nanoparticles

207
208
209
211
211
212
213
215

Contents

11.5

12.

Conclusions
References

Advanced Pulmonary Delivery of Peptides or Proteins Using

Polymeric Particles
Yu Seok Youn, Kang Choon Lee, You Han Bae, Kun Na and
Eun Seong Lee
12.1
12.2

12.3
12.4
12.5
12.6
12.7
12.8
12.9

Introduction
Practical Issues in the Pulmonary Delivery of Peptides and Proteins
12.2.1 Physiological Features of the Lungs
12.2.2 Barriers to the Pulmonary Delivery of Peptides and Proteins
12.2.3 Factors Affecting the Pulmonary Delivery of Peptide and
Protein Particles
Polymeric Microparticles for Delivering Peptides and Proteins
Porous Microparticles
Polymeric Nanoparticles
Sustained or Controlled Release Issues in Polymeric Particle
Formulations
Stability Issues of Peptides and Proteins in Particles
Toxicity Issues of Inhaled Particles
Conclusions
References

219
220

227

227
228
228
228
230
231
232
237
239
240
240
240
241

NUCLEIC ACIDS

245

13.

Polymer Microparticles for Nucleic Acid Delivery

Tim Pearce, Jared Hierman and Chun Wang

247

13.1
13.2

247
248
248
248
251
253
254
256
257
257
258
259
260
260
261
262
263
263
264

Introduction
Microparticles Based on Poly(Lactic-co-Glycolic Acids) (PLGA)
13.2.1 Overview
13.2.2 Design Parameters
13.2.3 General Strategies and Fabrication of Microparticles
13.2.4 Optimization of Microparticle Formulations
13.2.5 Combining Polycations with PLGA Microparticles
13.2.6 Current Applications
13.3 Other Types of Polymer Microparticles
13.3.1 Alginate
13.3.2 Chitosan
13.3.3 Hyaluronan (HA)
13.3.4 Macro-Albumin Aggregates (MAA)
13.3.5 Poly(D,L-Lactide-co-4-Hydroxy-L-Proline) (PLHP)
13.3.6 The Pluronics
13.3.7 Poly(Ortho Esters) (POEs)
13.3.8 Polyacrylamide
13.4 Conclusions
References

Contents

14.

15.

Pulmonary Delivery of Small Interfering RNA for Novel

Therapeutics
Qing Ge, David Evans, John J. Xu, Harry H. Yang and
Patrick Y. Lu

269

14.1 Introduction
14.2 RNA Interference: Mechanism of Action
14.3 Lung Physiology
14.4 Animal Models
14.5 siRNA Delivery to Treat Pulmonary Diseases
14.5.1 Challenges
14.5.2 Viral Versus Non-Viral Vectors
14.5.3 Airway siRNA Delivery
14.5.4 Intravenous siRNA Delivery
14.5.5 Other Delivery Methods
14.6 siRNA-Induced Inflammatory Response
14.7 Case Studies
14.7.1 siRNA Therapeutics Against RSV and PIV Infection in
Mouse
14.7.2 siRNA Therapeutics for Treatment of SCV Infection in
Monkey
14.8 Faster Drug for Unknown Bugs
14.9 Conclusions
References

269
270
270
272
273
273
274
275
278
279
279
280

Lipid-Based Formulations for siRNA Delivery

Camilla Foged, Pieter Vader and Raymond M. Schiffelers

291

15.1
15.2

291

15.3
15.4
15.5
15.6

16.

xi

Introduction
Cationic Lipid-Based Delivery Systems for Nucleic
Acid-Based Drugs
15.2.1 Helper Lipids
15.2.2 Lipopolymers
15.2.3 Active Targeting
Neutral and Anionic Lipid-Based Drug Delivery Systems
Mechanisms of Internalization
Immune Activation
Conclusions
References

280
281
284
284
285

292
293
294
294
296
296
297
299
300

Cellular Bioavailability of Peptide Nucleic Acids (PNAs) Conjugated

to Cell Penetrating Peptides
Takehiko Shiraishi and Peter E. Nielsen

305

16.1
16.2
16.3
16.4

305
306
306
307

Introduction
Peptide Nucleic Acids (PNAs)
Cell Penetrating Peptides (CPPs)
Cellular Uptake Versus Bioavailability

xii

17.

Contents

16.5 PNA Conjugates

16.6 Efficacy Versus Efficiency
16.7 Auxiliary Agents
16.8 In Vivo Activity of PNA Conjugates
16.9 Conclusions
References

307
307
330
331
331
336

DEAE-dextran-MMA Graft Copolymer for Non-Viral Delivery of

DNA
Yasuhiko Onishi, Yuki Eshita and Masaaki Mizuno

339

17.1 Introduction
17.2 Theoretical
17.3 Materials and Methods
17.3.1 Preparation of DDMC
17.3.2 Characterization of DDMC
17.3.3 Reaction Between DDMC and DNA
17.3.4 The Structure of the Complex Between DNA and DDMC
17.3.5 Transfection
17.3.6 Protection from DNase Degradation
17.3.7 DNA Delivery Pathways by DDMC
17.4 Conclusions
References

339
340
341
341
341
341
343
345
350
352
353
354

VACCINES

357

18.

Liposomes in Adjuvant Systems for Parenteral Delivery of Vaccines

Dennis Christensen, Karen Korsholm, Grith Kryer Wood,
Afzal Mohammed, Vincent Bramwell, Peter Andersen, Else Marie Agger
and Yvonne Perrie

359

18.1 The Structural Basis of Liposomes

18.2 Key Characteristics Which Make Liposomes Advantagous as Vaccine
Delivery Systems
18.3 Formulation Approaches to Improve Adjuvanticity and Enhance
Immunological Targeting
18.3.1 Immunomodulators
18.3.2 Physicochemical Properties
18.4 Instability Considerations of Liposomes
18.5 Techniques to Formulate Long-Term Stable Liposome Formulations
18.5.1 Protection During Freeze-Drying
18.6 Conclusion
References

359

19.

Influenza Virosomes as Delivery Systems for Antigens

Mario Amacker, Stefan Moese, Andreas R. Kammer, Ari Helenius
and Rinaldo Zurbriggen

360
363
364
365
366
368
369
371
372
377

Contents

xiii

19.1

Introduction
377
19.1.1 Immune Stimulating Reconstituted Influenza Virosomes (IRIVs)
as Adjuvant and Delivery System
377
19.1.2 Mechanisms of Action
379
19.2 A Case Study Antigen-Virosome Drug Delivery System
382
19.2.1 Particle Characterization
382
19.2.2 Virosome Uptake into HeLa Cells
382
19.2.3 Humoral Immune Response to Antigen Virosomes
385
19.2.4 Cellular Immune Response to Antigen Virosomes
386
19.2.5 Efficiency of the Antigen Virosome Drug Delivery System
388
19.3 Methods
389
19.3.1 Virosome Formulation
389
19.3.2 Size Determination
389
19.3.3 FreezeFracture Electron Microscopy
389
19.3.4 Transmission Electron Microscopy
390
19.3.5 TIRF Microscopy
390
19.3.6 Mice and Immunizations
390
19.3.7 Intracellular IFN Staining
390
19.3.8 ELISA
391
References
391

20.

GRAZAX: An Oromucosal Vaccine for Treating Grass Pollen

Allergy with Immunotherapy
Annette Rmmelmayer Lundegaard, Lise Lund and
Jrgen Nedergaard Larsen
20.1
20.2
20.3
20.4
20.5
20.6
20.7

Introduction
Specific Immunotherapy
Description of Grass Pollen Extract
Technologies of Fast Dissolving Tablets
Formulation of GRAZAX
Production of GRAZAX
Specifications and Analytical Procedures
20.7.1 Physical and Chemical Tests
20.7.2 Immunochemical Tests
20.7.3 Microbial Tests
20.8 Feasibility Study: Additional Analyses
20.9 Conclusions
References

FINAL COMMENTS
21.

395

395
396
397
398
399
400
401
401
402
403
403
403
404

405

Delivery Technologies for Biopharmaceuticals: A Critical

Assessment
Marco van de Weert

407

21.1

407

Introduction

xiv

Contents

21.2 Large Scale Production and Reproducibility

21.3 Safety Concerns
21.4 CostBenefit Ratio
21.5 Conclusion
References

Index

408
408
410
410
411

413

Preface

The present book, entitled Delivery Technologies for Biopharmaceuticals: Peptides,

Proteins, Nucleic Acids and Vaccines, reviews the challenges for delivery of biopharmaceuticals, focusing on how to overcome the main delivery barriers by means of pharmaceutical formulation. The overview chapters go into depth with strategies to overcome
the main barriers for successful delivery of therapeutic peptides, proteins and nucleic
acid-based drugs or vaccines related to the site of administration and the target site. For
these categories of biopharmaceuticals, selected types of delivery technologies and delivery routes are described with focus on the state-of-the-art concepts for successful delivery.
Thus, many of the approaches described herein are reported in formulations in current
clinical trials as well as in marketed products. Supplementary to this are the more specific
chapters describing in depth some of the novel approaches, which address the challenges
of successful delivery of biopharmaceuticals.
Compiling research knowledge in the fields of peptide and protein therapeutics, nucleic
acid-based therapeutics and vaccines, respectively, is a challenging task in itself. Therefore,
the issues dealt with in the present book are by no means complete in covering the vast
number of different materials and approaches under investigation and development for
novel formulation approaches. Yet, it is the editors hope that the substantial contributions
of the authors all-in-all will contribute to a generic understanding of how challenges in
the delivery of biopharmaceuticals might be overcome. Finally, we would like to sincerely
acknowledge the contributions of all the authors.
Lene Jorgensen and Hanne Mrck Nielsen
University of Copenhagen, Denmark

List of Contributors

Else M. Agger, Statens Serum Institute, Department of Infectious Disease Immunology,

Copenhagen, Denmark
Fakhrul Ahsan, School of Pharmacy, Department of Pharmaceutical Sciences, Texas
Tech University Health Sciences Center, Amarillo, TX, USA
Mario Amacker, Pevion Biotech, Ittigen/Bern, Switzerland
Peter Andersen, Statens Serum Institute, Department of Infectious Disease Immunology,
Copenhagen, Denmark
You Han Bae, University of Utah, Department of Pharmaceutics and Pharmaceutical
Chemistry, Salt Lake City, UT, USA
Shervin Bahrami, Aarhus University, Department of Molecular Biology, Aarhus,
Denmark
Vincent Bramwell, Aston University, School of Life and Health Sciences, Birmingham,
UK
Dennis Christensen, Statens Serum Institute, Department of Infectious Disease
Immunology, Copenhagen, Denmark
Dongmei Cun, University of Copenhagen, Department of Pharmaceutics and Analytical
Chemistry, Faculty of Pharmaceutical Sciences, Copenhagen, Denmark
Yuki Eshita, Oita University, Department of Infectious Diseases, Faculty of Medicine,
Oita, Japan
David Evans, Sirnaomics, Inc., Gaithersburg, MD, USA
Domingos C. Ferreira, University of Porto, Department of Pharmaceutical Technology,
Faculty of Pharmacy, Porto, Portugal
Camilla Foged, University of Copenhagen, Department of Pharmaceutics and Analytical
Chemistry, Faculty of Pharmaceutical Sciences, Copenhagen, Denmark

xviii

List of Contributors

Sven Frokjaer, University of Copenhagen, Department of Pharmaceutics and Analytical

Chemistry, Faculty of Pharmaceutical Sciences, Copenhagen, Denmark
Qing Ge, Sirnaomics, Inc., Gaithersburg, MD, USA
Ari Helenius, Institute of Biochemistry, ETH Hoenggerberg, Zurich, Switzerland
Jared Hierman, University of Minnesota, Department of Biomedical Engineering,
Minneapolis, MN, USA
Susanne Hostrup, Novo Nordisk A/S, Bagsvrd, Denmark
Leaf Huang, University of North Carolina, Eshelman School of Pharmacy, Chapel Hill,
NC, USA
Kasper Huus, Novo Nordisk A/S, Mlv, Denmark
Lene Jorgensen, University of Copenhagen, Department of Pharmaceutics and Analytical
Chemistry, Faculty of Pharmaceutical Sciences, Copenhagen, Denmark
Andreas R. Kammer, Pevion Biotech Ltd., Ittigen/Bern, Switzerland
Yoshiaki Kawashima, Aichi Gakuin University, School of Pharmaceutical Science,
Nagoya, Japan
Karen Korsholm, Statens Serum Institute, Department of Infectious Disease Immunology,
Copenhagen, Denmark
Jrgen Nedergaard Larsen, ALK-Abell A/S, Hrsholm, Denmark
Kang Choon Lee, SungKyunKwan University, College of Pharmacy, Jangan-ku, Suwon
City, Republic of Korea
Eun Seong Lee, The Catholic University of Korea, Division of Biotechnology, Bucheonsi, Republic of Korea
Patrick Y. Lu, Sirnaomics, Inc., Gaithersburg, MD, USA
Lise Lund, ALK-Abell A/S, Hrsholm, Denmark
Annette Rmmelmayer Lundegaard, ALK-Abell A/S, Hrsholm, Denmark
Kenneth Lundstrom, Pan Therapeutics, Lutry, Switzerland
Shirui Mao, Shenyang Pharmaceutical University, School of Pharmacy, Shenyang,
China

List of Contributors

xix

Susana Martins, University of Porto, Department of Pharmaceutical Technology,

Faculty of Pharmacy, Porto, Portugal
Masaaki Mizuno, Nagoya University, Graduate School of Medicine Program in Cell
Information Medicine, Nagoya, Japan
Stefan Moese, 3-V Biosciences, Schlieren, Switzerland
S. Moein Moghimi, University of Copenhagen, Department of Pharmaceutics and
Analytical Chemistry, Faculty of Pharmaceutical Sciences, Copenhagen, Denmark
Afzal Mohammed, Aston University, School of Life and Health Sciences, Birmingham,
UK
Kun Na, The Catholic University of Korea, Division of Biotechnology, Bucheon-si,
Republic of Korea
Hanne Mrck Nielsen, University of Copenhagen, Department of Pharmaceutics and
Analytical Chemistry, Faculty of Pharmaceutical Sciences, Copenhagen, Denmark
Peter E. Nielsen, University of Copenhagen, Department of Cellular and Molecular
Medicine, The Panum Institute, Faculty of Health Sciences, Copenhagen, Denmark
Yasuhiko Onishi, Ryuju Science Co. Ltd., Seto, Japan
Henrik Parshad, Novo Nordisk A/S, Bagsvrd, Denmark
Tim Pearce, University of Minnesota, Department of Biomedical Engineering,
Minneapolis, MN, USA
Finn Skou Pedersen, Aarhus University, Department of Molecular Biology, Aarhus,
Denmark
Yvonne Perrie, Aston University, School of Life and Health Sciences, Birmingham,
UK
Bruno Sarmento, University of Porto, Department of Pharmaceutical Technology,
Faculty of Pharmacy, Porto, Portugal
Raymond M. Schiffelers, Utrecht University, Department of Pharmaceutical Sciences,
Faculty of Science, Utrecht, The Netherlands
Takehiko Shiraishi, Utrecht University, Department of Pharmaceutical Sciences, Faculty
of Science, Utrecht, The Netherlands, and University of Copenhagen, Department of
Cellular and Molecular Medicine, The Panum Institute, Faculty of Health Sciences,
Copenhagen, Denmark

xx

List of Contributors

Eliana B. Souto, Fernando Pessoa University, Department of Pharmaceutical Technology, Faculty of Health Sciences, Porto, Portugal, and Institute of Biotechnology and
Bioengineering, Centre of Genetics and Biotechnology, University of Trs-os-Montes
and Alto Douro, Vila Real, Portugal
Chandan Thomas, Texas Tech University Health Sciences Center, Department of
Pharmaceutical Sciences, School of Pharmacy, Amarillo, TX, USA
Pieter Vader, Utrecht University, Department of Pharmaceutical Sciences, Faculty of
Science, Utrecht, The Netherlands
Tefilo Vasconcelos, University of Porto, Department of Pharmaceutical Technology,
Faculty of Pharmacy, Porto, Portugal
Elizabeth A. Vasievich, University of North Carolina, Eshelman School of Pharmacy,
Chapel Hill, NC, USA
Chun Wang, University of Minnesota, Department of Biomedical Engineering,
Minneapolis, MN, USA
Marco van de Weert, University of Copenhagen, Department of Pharmaceutics and
Analytical Chemistry, Faculty of Pharmaceutical Sciences, Copenhagen, Denmark
Grith Kryer Wood, Statens Serum Institute, Department of Infectious Disease
Immunology, Copenhagen, Denmark
John J. Xu, Sirnaomics, Inc., Gaithersburg, MD, USA
Harry H. Yang, Sirnaomics, Inc., Gaithersburg, MD, USA
Mingshi Yang, University of Copenhagen, Department of Pharmaceutics and Analytical
Chemistry, Faculty of Pharmaceutical Sciences, Copenhagen, Denmark
Yu Seok Youn, Pusan National University, College of Pharmacy, Geumjeong-gu, Busan,
Republic of Korea
Rinaldo Zurbriggen, Pevion Biotech Ltd., Ittigen/Bern, Switzerland