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POLYMERIC

BIOMATERIALS
Second Edition, Revised and Expanded

Severian Dumitriu
University of Sherbrooke
Sherbrooke, Quebec, Canada

Marcel Dekker, Inc. New York Basel


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Copyright 2001 by Marcel Dekker, Inc. All Rights Reserved.

Copyright 2002 Marcel Dekker, Inc.


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Dedicated with affection

In memory of my parents

To my wife, Maria,
and my children, Daniela and Cristina,
who make everything worthwhile

Copyright 2002 Marcel Dekker, Inc.


Preface

The need for spare parts for the human body has never been greater than at present. More people than ever are living longer, but the
human body, shaped by millions of years of evolution, isnt keeping up. Spare parts must be available to replace organs and tissues
that are worn out from operating beyond their expiration dates, as well as those damaged by injury, disease, or developmental mishaps.
The biomaterials eld has come a long way from its empirical beginnings, with researchers taking whatever materials were available
and attempting to integrate them into the human body, sometimes with disastrous results. Now, the bodys response to foreign materials
is better understood than ever before. Furthermore, the past 15 years have seen great strides in tissue engineering. Spare parts consisting
of living tissues are poised for signicant clinical application. Tissue engineering, especially of tissues derived from the patients own
cells, offers total acceptance by and integration with the patients body, unlike nonliving materials or living tissues from other humans
or species.
The active ingredient in a medicine is only part of the arsenal against disease. The drug must somehow get to the right place at the
right time. Thats where drug delivery comes in. Drug delivery companies work to devise new dosage forms for medications. The main
challenge is to create the technologies for the easier and most convenient systematic delivery systems. For proteins and other macromole-
cules, however, the oral route is by far the hardest to accomplish.
From a practical perspective, medical applications of polymers fall into three broad categories: (1) extracorporeal uses (catheters,
tubing, and uid lines; dialysis membranes/articial kidney; ocular devices; wound dressings and articial skin), (2) permanently im-
planted devices (sensory devices; cardiovascular devices; orthopedic devices; dental devices), and (3) temporary implants (degradable
sutures; implantable drug delivery systems; polymeric scaffolds for cell or tissue transplants; temporary vascular grafts and arterial
stents; temporary small bone xation devices).
Today, biomaterial research has developed into a major interdisciplinary effort involving chemists, biologists, engineers, and physi-
cians. Biomaterials research has provided the clinician with a large number of new materials and new medical devices. As the biomaterials
device industry continues to grow, degradable polymers will increase at the expense of traditional biomaterials such as metals and
conventional, biostable polymers.
This volume consists of two parts: Part I: Polymers as Biomaterials and Part II: Medical and Pharmaceutical Applications of Polymers.
The fundamental questions of polymer synthesis, the types of polymers used for medical purposes, and modication of polymers to
increase their biocompatibility, are presented in the rst part. The applications of the two major groups of biomaterialsnatural biomate-
rials (polysaccharides, cellulose, chitosan, proteins, etc.) and synthetic biomaterials (polyesters, silicones, elastomers, etc.)are also
reviewed. Part II deals with concrete utilization of polymeric biomaterials in the domains of tissue engineering, ophthalmic delivery,
vascular prostheses (grafts), dental and maxillofacial surgery, blood contacting, skin graft polymers, sensors in biomedical applications,
medical adhesives, medical textiles, and topical hemostat biomaterials.
The uses of polymers in the pharmaceutical domain fall into two areas: drug polymers and drug carrier polymers for controlled
release. Part II provides the groundwork for understanding the fundamentals of drug delivery including conventional, nonconventional,
and modulated systems; structureproperty relations of selected supports and their role in drug delivery; delivery of drugs to sites such
as the gastrointestinal tract, lung, skin, tumors, and blood vessels; and marketing considerations in new drug delivery systems.
This book is truly international, with authors from Austria, Canada, Finland, France, Germany, India, Israel, Italy, Japan, the Nether-
lands, Portugal, Slovenia, Spain, Switzerland, Thailand, the United Kingdom, and the United States. I am grateful to all the contributors.

Severian Dumitriu

Copyright 2002 Marcel Dekker, Inc.


Contents

Preface
Contributors

Part I. Polymers as Biomaterials

1. Polysaccharides as Biomaterials
Severian Dumitriu

2. Biomimetics
Weiyuan John Kao

3. Silicones for Pharmaceutical and Biomedical Applications


Haissam S. El-Zaim and John P. Heggers

4. Biodegradable Polymers as Drug Carrier Systems


Abraham J. Domb, Neeraj Kumar, Tzviel Sheskin, Alfonso Bentolila, Joram Slager, and
Doron Teomim

5. Biodegradable Biomaterials Having Nitric Oxide Biological Activity


C. C. Chu

6. Hydrogels for Biomedical and Pharmaceutical Applications


Akio Kishida and Yoshito Ikada

7. Mucoadhesive Polymers
Andreas Bernkop-Schnurch

8. Polymers for Tissue Engineering Scaffolds


Howard W. T. Matthew

9. Chitosan: StructureProperties Relationship and Biomedical Applications


Alain Domard and Monique Domard

10. Chitosan-Based Delivery Systems: Physicochemical Properties and Pharmaceutical Applications


Radi Hejazi and Monsoor Amiji

Copyright 2002 Marcel Dekker, Inc.


11. Immobilization of Active Biopolymers from Cold PlasmaFunctionalized Surfaces for the Creation
of Molecular Recognition and Molecular Manufacturing Systems
Ferencz Denes and Sorin Manolache

12. Advances in Designed Multivalent Bioconjugates with Dendritic Structure


Bogdan Comanita

13. Biocompatibility of Elastomers


D. J. Chauvel-Lebret, P. Auroy, and M. Bonnaure-Mallet

14. Control of CellBiomaterial Interactions


Danielle C. Giliberti, Kyle K. White, and Kay C Dee

Part II. Medical and Pharmaceutical Applications of Polymers

15. Polymeric Systems for Ophthalmic Drug Delivery


O. Felt, S. Einmahl, P. Furrer, V. Baeyens, and R. Gurny

16. Dental and Maxillofacial Surgery Applications of Polymers


A. Bascones, J. M. Vega, N. Olmo, J. Turnay, J. G. Gavilanes, and M. A. Lizarbe

17. Biomaterials in Burn and Wound Dressings


Robert L. Sheridan, Jeffrey R. Morgan, and Rashid Mohammad

18. Dermocosmetic Applications of Polymeric Biomaterials


P. Corvi Mora and P. G. Baraldi

19. Textile-Based Biomaterials for Surgical Applications


C. C. Chu

20. Bioabsorbable Polymers for Medical Applications with an Emphasis on Orthopedic Surgery
Pentti U. Rokkanen

21. Polymers for Articial Joints


Naohide Tomita, Kazuya Nagata, and Hiroshi Fujita

22. Polymeric Occluders in Tilting Disc Heart Valve Prostheses


G. S. Bhuvaneshwar, A. V. Ramani, and K. B. Chandran

23. Blood-Contacting Polymers


T. Avramoglou, J. Jozefonvicz, and M. Jozefowicz

24. Surface Modication of Dacron Vascular Grafts: Incorporation of Antithrombin and


Mitogenic Properties
Matthew D. Phaneuf, Martin J. Bide, William C. Quist, and Frank W. LoGerfo

25. AntithrombinHeparin Complexes


Leslie R. Berry, Maureen Andrew, and Anthony K. C. Chan

26. Adhesives for Medical Applications


Iain Webster and Peter J. West

27. Glucose-Sensitive Hydrogel Membranes


Jin Ho Lee, Jung Ju Kim, and Kinam Park

Copyright 2002 Marcel Dekker, Inc.


28. Polymeric Micro- and Nanoparticles as Drug Carriers
G. Barratt, G. Couarraze, P. Couvreur, C. Dubernet, E. Fattal, R. Gref, D. Labarre, P. Legrand,
G. Ponchel, and C. Vauthier

29. Liposomes in Drug Delivery


Yuan-Peng Zhang, Boris Ceh, and Danilo D. Lasic

30. Liposomes for Cancer Therapy Applications


Lawrence D. Mayer, Rajesh Krishna, and Marcel B. Bally

31. Systemic Cancer Therapy Using Polymer-Based Prodrugs and Progenes


Leonard W. Seymour

32. Anticancer Drug Conjugates with Macromolecular Carriers


F. Kratz, A. Warnecke, K. Riebeseel, and P. C. A. Rodrigues

33. EnzymeProdrug Therapies of Cancer


Richard J. Knox, Roger G. Melton, and Ronit Satchi

34. New Lipid/DNA Complexes for Gene Delivery


Kenneth W. Liang and Leaf Huang

35. Gene Delivery by Cationic LiposomeDNA Complexes


Nejat Duzgunes, Sergio Simoes, Pedro Pires, and Maria C. Pedroso de Lima

36. Biological Stimulus-Responsive Hydrogels


Takashi Miyata and Tadashi Uragami

37. Biocompatible Polymers in Liver-Targeted Gene Delivery Systems


Edwin C. Ouyang, George Y. Wu, and Catherine H. Wu

38. Bioarticial Pancreas


Riccardo Calaore

39. Transdermal Delivery of Drugs


B. B. Michniak and A. El-Kattan

40. Drug Delivery via Mucosal Routes


Nimit Worakul and Joseph R. Robinson

41. Bioadhesive Drug Delivery Systems


A. David Woolfson, R. Karl Malcolm, Paul A. McCarron, and David S. Jones

42. Recent Developments in Drug Delivery to the Nervous System


Dusica Maysinger, Radoslav Savic, Joseph Tam, Christine Allen, and Adi Eisenberg

43. Glucose-Mediated Insulin Delivery from Implantable Polymers


Larry R. Brown

44. Drug Targeting to the Kidney: The Low-Molecular-Weight Protein Approach


R. F. G. Haverdings, R. J. Kok, M. Haas, F. Moolenaar, D. de Zeeuw, and D. K. F. Meijer

Copyright 2002 Marcel Dekker, Inc.


Contributors

Christine Allen McGill University, Montreal, Quebec, Canada


Mansoor Amiji Northeastern University, Boston, Massachusetts
Maureen Andrew Hamilton Civic Hospitals Research Centre, Hamilton, Ontario, Canada
P. Auroy Universite de Rennes, Rennes, France
T. Avramoglou Universite de Paris, Villetaneuse, France
V. Baeyens Centre Interuniversitaire de Recherche et dEnseignement, Archamps, France
Marcel B. Bally BC Cancer Agency, Vancouver, British Columbia, Canada, and University of British Columbia, Vancouver, British
Columbia, Canada
P. G. Baraldi Ferrara University, Ferrara, Italy
G. Barratt UMR CNRS, Chatenay-Malabry, France
A. Bascones Universidad Complutense, Madrid, Spain
Alfonso Bentolila The Hebrew University of Jerusalem, Jerusalem, Israel
Andreas Bernkop-Schnurch University of Vienna, Vienna, Austria
Leslie R. Berry Hamilton Civic Hospitals Research Centre, Hamilton, Ontario, Canada
G. S. Bhuvaneshwar Sree Chitra Tirunal Institute of Medical Sciences and Technology, Thiruvananthapuram, India
Martin J. Bide University of Rhode Island, Kingston, Rhode Island
M. Bonnaure-Mallet Universite de Rennes, Rennes, France
Larry R. Brown HarvardMassachusetts Institute of Technology, Cambridge, Massachusetts
Riccardo Calaore University of Perugia School of Medicine, Perugia, Italy
Boris Ceh University of Ljubljana, Ljubljana, Slovenia
Anthony K. C. Chan Hamilton Civic Hospitals Research Centre, Hamilton, Ontario, Canada
K. B. Chandran University of Iowa, Iowa City, Iowa
D. J. Chauvel-Lebret Universite de Rennes, Rennes, France
C. C. Chu Cornell University, Ithaca, New York
Bogdan Comanita Institute for Chemical Process and Environmental Technology, Ottawa, Ontario, Canada

Copyright 2002 Marcel Dekker, Inc.


P. Corvi Mora EUPHAR Group, Piacenza, Italy
G. Couarraze UMR CNRS, Chatenay-Malabry, France
P. Couvreur UMR CNRS, Chatenay-Malabry, France
Kay C Dee Tulane University, New Orleans, Louisiana
Ferencz Denes University of Wisconsin, Madison, Wisconsin
D. de Zeeuw University of Groningen, Groningen, The Netherlands
Alain Domard University Claude Bernard Lyon I, Villeurbanne, France
Monique Domard University Claude Bernard Lyon I, Villeurbanne, France
Abraham J. Domb The Hebrew University of Jerusalem, Jerusalem, Israel
C. Dubernet UMR CNRS, Chatenay-Malabry, France
Severian Dumitriu University of Sherbrooke, Sherbrooke, Quebec, Canada
Nejat Duzgunes University of the Pacic, San Francisco, California
S. Einmahl University of Geneva, Geneva, Switzerland
Adi Eisenberg McGill University, Montreal, Quebec, Canada
A. El-Kattan Pzer Inc., Ann Arbor, Michigan
Haissam S. El-Zaim University of Texas Medical Branch, Galveston, Texas
E. Fattal UMR CNRS, Chatenay-Malabry, France
O. Felt University of Geneva, Geneva, Switzerland
Hiroshi Fujita Kyoto University, Kyoto, Japan
P. Furrer University of Lausanne, Lausanne, Switzerland
J. G. Gavilanes Universidad Complutense, Madrid, Spain
Danielle C. Giliberti Tulane University, New Orleans, Louisiana
R. Gref UMR CNRS, Chatenay-Malabry, France
R. Gurny University of Geneva, Geneva, Switzerland
M. Haas University of Groningen, Groningen, The Netherlands
John P. Heggers University of Texas Medical Branch, Galveston, Texas
R. F. G. Haverdings University of Groningen, Groningen, The Netherlands
Radi Hejazi Northeastern University, Boston, Massachusetts
Leaf Huang University of Pittsburgh, Pittsburgh, Pennsylvania
Yoshito Ikada Suzuka University of Medical Science, Mie, Japan
David S. Jones The Queens University of Belfast, Belfast, Northern Ireland
J. Jozefonvicz Universite de Paris, Villetaneuse, France
M. Jozefowicz Universite de Paris, Villetaneuse, France
Weiyuan John Kao University of Wisconsin, Madison, Wisconsin
Jung Ju Kim Purdue University, West Lafayette, Indiana
Akio Kishida National Cardiovascular Center Research Institute, Osaka, Japan
Richard J. Knox Gnact Pharma Plc, Salisbury, England
R. J. Kok University of Groningen, Groningen, The Netherlands

Copyright 2002 Marcel Dekker, Inc.


F. Kratz Tumor Biology Center, Freiburg, Germany
Rajesh Krishna* BC Cancer Agency, Vancouver, British Columbia, Canada, and University of British Columbia, Vancouver, British
Columbia, Canada
Neeraj Kumar The Hebrew University of Jerusalem, Jerusalem, Israel
D. Labarre UMR CNRS, Chatenay-Malabry, France
Danilo D. Lasic Liposome Consultations, Newark, California
Jin Ho Lee Purdue University, West Lafayette, Indiana
P. Legrand UMR CNRS, Chatenay-Malabry, France
Kenneth W. Liang University of Pittsburgh, Pittsburgh, Pennsylvania
M. A. Lizarbe Universidad Complutense, Madrid, Spain
Frank W. LoGerfo Beth Israel Deaconess Medical Center, Boston, Massachusetts
R. Karl Malcolm The Queens University of Belfast, Belfast, Northern Ireland
Sorin Manolache University of Wisconsin, Madison, Wisconsin
Howard W. T. Matthew Wayne State University, Detroit, Michigan
Lawrence D. Mayer BC Cancer Agency, Vancouver, British Columbia, Canada, and University of British Columbia, Vancouver,
British Columbia, Canada
Dusica Maysinger McGill University, Montreal, Quebec, Canada
Paul A. McCarron The Queens University of Belfast, Belfast, Northern Ireland
D. K. F. Meijer University of Groningen, Groningen, The Netherlands
Roger G. Melton Gnact Pharma Plc, Salisbury, England
B. B. Michniak University of Medicine and Dentistry of New Jersey, Newark, New Jersey
Takashi Miyata Kansai University, Osaka, Japan
Rashid Mohammad Shriners Burns Hospital, Massachusetts General Hospital, Boston, Massachusetts, and Harvard Medical School,
Cambridge, Massachusetts
Jeffrey R. Morgan Shriners Burns Hospital, Massachusetts General Hospital, Boston, Massachusetts, and Harvard Medical School,
Cambridge, Massachusetts
F. Moolenaar University of Groningen, Groningen, The Netherlands
Kazuya Nagata Industrial Technology Center of Okayama Prefecture, Okayama, Japan
N. Olmo Universidad Complutense, Madrid, Spain
Edwin C. Ouyang University of Connecticut Health Center, Farmington, Connecticut
Kinam Park Purdue University, West Lafayette, Indiana
Maria C. Pedroso de Lima University of Coimbra, Coimbra, Portugal
Matthew D. Phaneuf Beth Israel Deaconess Medical Center, Boston, Massachusetts
Pedro Pires University of Coimbra, Coimbra, Portugal
G. Ponchel UMR CNRS, Chatenay-Malabry, France
William C. Quist Beth Israel Deaconess Medical Center, Boston, Massachusetts
A. V. Ramani T.T.K. Pharma, Ltd., Bangalore, India

* Current afliation: Bristol-Meyers Squibb, Princeton, New Jersey

Copyright 2002 Marcel Dekker, Inc.


K. Riebeseel Tumor Biology Center, Freiburg, Germany
Joseph R. Robinson University of Wisconsin, Madison, Wisconsin
P. C. A. Rodrigues Tumor Biology Center, Freiburg, Germany
Pentti U. Rokkanen Helsinki University Central Hospital and University of Helsinki, Helsinki, Finland
Ronit Satchi Center for Polymer Therapeutics, London, England
Radoslav Savic McGill University, Montreal, Quebec, Canada
Leonard W. Seymour University of Birmingham, Birmingham, England
Robert L. Sheridan Shriners Burns Hospital, Massachusetts General Hospital, Boston, Massachusetts, and Harvard Medical School,
Cambridge, Massachusetts
Tzviel Sheskin The Hebrew University of Jerusalem, Jerusalem, Israel
Sergio Simoes University of Coimbra, Coimbra, Portugal
Joram Slager The Hebrew University of Jerusalem, Jerusalem, Israel
Joseph Tam McGill University, Montreal, Quebec, Canada
Doron Teomim The Hebrew University of Jerusalem, Jerusalem, Israel
Naohide Tomita Kyoto University, Kyoto, Japan
J. Turnay Universidad Complutense, Madrid, Spain
Tadashi Uragami Kansai University, Osaka, Japan
C. Vauthier UMR CNRS, Chatenay-Malabry, France
J. M. Vega Universidad Complutense, Madrid, Spain
A. Warnecke Tumor Biology Center, Freiburg, Germany
Iain Webster Smith & Nephew Group Research Centre, York, England
Peter J. West Smith & Nephew Group Research Centre, York, England
Kyle K. White Tulane University, New Orleans, Louisiana
Nimit Worakul University of Wisconsin, Madison, Wisconsin, and Prince of Songkla University, Haad Yai, Thailand
A. David Woolfson The Queens University of Belfast, Belfast, Northern Ireland
Catherine H. Wu University of Connecticut Health Center, Farmington, Connecticut
George Y. Wu University of Connecticut Health Center, Farmington, Connecticut
Yuan-Peng Zhang ALZA Corp., Mountain View, California

Copyright 2002 Marcel Dekker, Inc.


1

Polysaccharides as Biomaterials

Severian Dumitriu
University of Sherbrooke, Sherbrooke, Quebec, Canada

I. INTRODUCTION Applications, and Polysaccharides: Structural Diversity


and Functional Versatility, all edited by S. Dumitriu and
Polymers have found applications in virtually every disci- published by Marcel Dekker, Inc.
pline of medicine, ranging from simple extracorporeal de-
vices to intricately designed implants. Since each medical
application has its own highly specialized requirements, a II. BACTERIAL POLYSACCHARIDES
range of diverse materials with good biocompatibility but
different chemical and physicomechanical properties must Encapsulated bacterial diseases are still the most prevalent
be available. and serious infections in humans. There is increasing re-
Polysaccharides constitute an important component of search of the chemical basis of immunogenicity to capsular
life matter. Polysaccharides display a perfect biocompati- polysaccharides and prevention of bacterial infections
bility and biodegradability, which are the basic characteris- through immunization.
tics for polymers used as biomaterials. They have several The capsular polysaccharides of bacteria are attractive
characteristics not found in other natural polymers. Re- vaccine candidates because they constitute the most highly
cently, specic properties of antivirals, antitumorals, gene conserved and most exposed bacterial surface antigens.
modulators, etc., have been discovered for various classes They can be readily isolated and puried; they are non-
of polysaccharides. toxic; and they are immunogenic in humans (with few ex-
This chapter presents all polysaccharide classes with ceptions) (1). Another important feature is that capsular
applications as biomaterials and as pharmaceutical sys- polysaccharides can be chemically and physically dened,
tems. For the rst grouppolysaccharides used as bioma- which is necessary for control over their efcacy as human
terialsa special emphasis is given to the presentation of biologicals. In fact, the use of capsular polysaccharides as
membranes, implants, skin replacements, matrices for cel- immunoprophylactic agents against human disease caused
lular engineering (scaffolds), and hemostatic agents. The by encapsulated bacteria is now rmly established (2,3).
second grouppolysaccharides used as pharmaceutical Efcacious vaccines composed of the capsular polysaccha-
systemsdescribes mainly the use of polysaccharides as rides of Nisseria meningitidis, Streptococcus pneumoniae,
components for drug conditioning and drug delivery sys- Haemophilus inuenzae, and Salmonella typhi have been
tems fabrication. This chapter presents a review of the lit- developed as commercial products (Table 1).
erature based on the three books previously published: A new generation of highly successful semisynthetic
Polymeric Biomaterials, Polysaccharides in Medicinal glycoconjugate vaccines in which the polysaccharides were

Copyright 2002 Marcel Dekker, Inc.


Table 1 Bacterial Polysaccharides droxyamino acid units or asparagine units of proteins. In
proteoglycans, polysaccharides are bound to polypeptides.
Pathogenic organism Reference
The crystal structure of the complex of an antiId Fab
1. Neisseria meningitidis with a Fab specic for a Brucella polysaccharide antigen
Group A 4 has previously been reported (33). To complement this
Group B 5 study, the binding characteristics and immunological prop-
Group C 5 erties of this Ab2 and two others raised with a second anti-
2. Streptococcus pneumoniae
Brucella antibody were investigated, including quantita-
Type 19F 6
Type 14 7
tive kinetic measurements by surface plasmon resonance
3. Hemophilus inuenzae (34). Protective immunity to encapsulated bacteria is re-
Type b 8 lated to antibody response to polysaccharide (PS) antigen,
interactions with T and B cells, and host defense mecha-
nisms (35).
Klebsiella pneumoniae is one of the most frequently
conjugated (covalently linked) to protein carriers were de- isolated gram-negative pathogens in severe nosocomial in-
veloped (1). During the last 15 years, comprehensive fections. Alvarez et al. (36) have demonstrated the exis-
studies specially directed to the development of glycocon- tence of K. pneumoniae clinical isolates decient in the
jugate vaccines as potential human vaccines have been re- lipopolysaccharide O side chain, the major factor for resis-
ported (Table 2) (911). tance to complement-mediated killing in this bacterial spe-
Complex carbohydrates are essential cell surface com- cies. These isolates are complement resistant, and their
ponents. Generally, the surface carbohydrates of bacteria mechanisms to resist complement were investigated by
are polysaccharides, either capsular polysaccharides or a selecting transposon-generated complement-sensitive mu-
part of cell wall lipopolysaccharides (LPS) (2224). Bacte- tants. One mutant with a drastically reduced capacity to
rial cells are protected by capsules in inadvertent surround- grow in nonimmune human serum carried the transposon
ings (2529). inserted in an open reading frame of a gene cluster in-
Capsular polysaccharides (CPS) are generally nega- volved in capsule synthesis. Four additional clinical iso-
tively charged (30). All structures of bacterial polysaccha- lates representing four different K serotypes were studied,
rides published until early 1982 have been recorded in a and results showed that capsular polysaccharide is a ma-
review (31). jor complement resistance factor in these O side chain
Generally, bacterial polysaccharides have repetitive decient isolates.
units that may consist of monosaccharides and octasac- Barnett et al. (37) compared responses to pneumococcal
charides and they may be linear or branched. conjugate and polysaccharide vaccines in 48 otitis-free and
Cell wall lipopolysaccharides of gram-negative bacteria 64 otitis-prone children. Pre- and postimmunization con-
consist of a lipid moiety, a core oligosaccharide, and a centrations of antibodies to pneumococcal serotypes 6B,
polysaccharide moiety which is made up from repeating 14, 19F, and 23F were measured by enzyme-linked immu-
units (32). Mammalian glycoproteins contain oligosaccha- nosorbent assay. Postimmunization mean concentrations
rides with different complexities, which are bound to hy- of antibodies to all four serotypes were signicantly higher
for children receiving conjugate vaccine than for those
receiving polysaccharide vaccine; the difference in re-
Table 2 Synthetic Glycoconjugate Vaccines sponses was primarily due to a better response to conjugate
vaccine in the otitis-prone group. Signicantly higher post-
Polysaccharide source Protein carrier Reference immunization concentrations of antibodies to all four sero-
Haemophilus inuenzae Diphtheria toxoid 12,13 types and to one of the four serotypes were found in otitis-
Tetanus toxoid 14 prone children and otitis-free children who received conju-
Streptococcus pneumonia Tetanus toxoid 15 gate vaccine, respectively. Pneumococcal conjugate vac-
Cholera toxin 16,17 cine has the potential to reduce the incidence of disease
Pertussis toxin 16 due to vaccine serotypes, even among children with recur-
Neisseria meningitidis Tetanus toxoid 18 rent otitis media.
Bovine serum albumin 19 Protein antigens induce signicant mucosal immuno-
Salmonella typhi Bovine serum albumin 20 globulin A (IgA) and systemic IgG responses when admin-
Cholera toxin 20
istered intranasally (i.n.) with the glyceride-polysorbate
Tetanus toxoid 21
based adjuvant RhinoVax (RV) both in experimental

Copyright 2002 Marcel Dekker, Inc.


animals and humans (38). The pneumococcal polysaccha- ysis is regenerated from cuproammonium solution of re-
ride conjugates (PNCs) induced signicant systemic IgG ned cotton linters. Cellulose hollow bers are regenerated
and IgA antibodies after i.n. immunization only when from cellulose acetate by deacetylation (45). Current ex-
given with RV and, for serotype 1, serum IgG titers were tensive efforts to improve hemodialysis membranes are
comparable to titers induced by subcutaneous immuniza- focusing on removal of -microglobulin and improv-
tion. In addition, i.n. immunization with PNC-1 in RV elic- ing blood compatibility. The improvement of hemocom-
ited detectable mucosal IgA. These results demonstrate patibility of cellulose membranes is achievement by sur-
that RV is a potent mucosal adjuvant. face modication through uretanation, acylation/sulfation,
Zhong et al. (39) describe the generation, molecular chloroacetylation/sulfonation, and polyacylation (4648).
structure, and protective efcacy of a human monoclonal Ishihara et al. (49) have synthesized a water-soluble
antibody (MAb) reactive with the capsular polysaccharide graft polymer composed of a cellulose and polymer having
of serotype 8 Streptococcus pneumoniae. In vitro studies a phospholipid polar group, poly(2-methacryloyloxyethyl)
revealed MAb D11dependent complement deposition on phosphoacrylcholine (MGC) as a coating material on the
the capsule of serotype 8 organisms via either the classical cellulose hemodialysis membrane. The MGC could be
or the alternative complement pathway. In vivo, MAb D11 coated on the hollow ber probe and the performance of
prolonged the survival of both normal and C4-decient the probe did not decrease even after the coating. By re-
mice with lethal serotype 8 S. pneumoniae infection. cording rapid changes in the glucose concentration from
100 to 200 mg/dL, the time to reach 90% of the maximum
value was within 7 min. To determine the glucose concen-
III. CELLULOSE AND ITS DERIVATIVES tration in subcutaneous tissue, the hollow ber probe modi-
ed with MGC was applied to human volunteers.
Polysaccharides currently used in medicine on a large scale Both cellulose diacetate and triacetate are used for he-
include cellulose, cellulose derivatives, heparin, dextrin, modialysis. The blood compatibility of the cellulose ace-
pullulan, hyaluronic acid, and alginate. tate membrane seems to be excellent if monolayer cover-
Cellulosics, regenerated cellulose and its methyl, ethyl, age of the surface with serum albumin is employed (50).
amino ethyl, and acetate-phthalate derivatives (40) have Collodion, a cellulose trinitrate derivative, was the rst
been widely investigated as membranes in articial kid- polymer to be used as an articial membrane, and it played
neys, encapsulating materials for controlled drug delivery a central role in further investigations and applications
(41), sutures and bandages (42), and blood-compatible ma- (51,52). Cellophane and Cuprophan (5357) membranes
terials (43), and are used mostly for blood purication, as replaced collodion later because of their better perfor-
anticoagulant, and as plasma expander in aqueous solution. mance and mechanical stability. However, due to their al-
leged lack of hemocompatibility, membranes made from
A. Blood Purication unmodied cellulose lost their market share. They have
been replaced by modied cellulosic and synthetic dialysis
1. Hemodialysis
membranes which show a better hemocompatibility than
Hemodialysis is a therapeutic treatment of chronic renal unmodied cellulose membranes. Most of the new mem-
disease to remove an excess of water and uremic toxins brane materials are also available in high-ux modica-
from the patient blood by dialysis under the extracorporeal tions and for this reason are suitable as well for more ef-
blood circulation. The most important part of the hemodi- fective therapy modes, such as hemodialtration and
alysis system is the semipermeable membrane installed in hemoltration. The success of hemodialysis as a routine
the dialyzer. The rst semipermeable membrane was re- therapy is also the success of membrane development,
ported in 1944 using cellophane. Cellulosic membranes are because both a reproducible membrane production and
still used for the hemodialyzer, largely synthetic semiper- an unlimited availability of dialysis membranes have in-
meable membranes made of polysulfone (PS), polyacrylo- creased the number of dialyzed patients to about 1 million
nitrile (PAN), poly(methyl methacrylate) (PMMA), and worldwide in 1999 (58).
ethylene-vinyl alcohol copolymer (EVAL) (44). Dialysis membranes made from regenerated cellulose
The cellulosic hemodialysis membrane can be divided are under dispute because of their alleged lack of he-
into regenerated cellulose and cellulose acetate (di- and mocompatibility. The introduction of membranes from
triacetate). In the early years of hemodialysis therapy, at-, synthetically modied cellulose, like cellulose acetate or
tube-, hollow bertype membranes were employed. At Hemophan, has proven, however, that hemocompatible
present almost 100% of hemodialyzers appear to be made membranes can be fabricated from cellulose by means of
from hollow bers. The cellulose membrane for hemodial- chemical surface modications (59,60). Diamantoglou et

Copyright 2002 Marcel Dekker, Inc.


al. (59) have synthesized a series of cellulose carbamate lose is as a barrier for the prevention of surgical adhesions
derivatives to prot from the excellent hemocompatibil- (64). The use of oxidized cellulose (Surgicel) was largely
ity pattern of the urethane family. In vitro investigations unsuccessful in animal models in reducing surgical adhe-
on membranes made from these cellulose modications sions in a reproducible fashion.
proved a direct relationship between the degree of modi-
cation and hemocompatibility. This was proven for the fol- B. Drug Delivery Systems
lowing three representative hemocompatibility parame-
ters: complement C5a generation, thrombinantithrombin Hydrophilic matrices based on polysaccharide carriers re-
(TAT) III formation, and platelet count (PC). As already main a highly popular design of sustained release dosage
shown for modications made from cellulose esters, a form. Control of drug release from tablets containing poly-
direct dependency between improved hemocompatibility saccharide excipient system was found to be capable using
and the degree of substitution (DS) in the cellulose mole- a variety of different formulations and process methods to
cule could be found. provide a variety of different release modalities which
were capable of matrix-dimension independence. Control-
2. Plasmapheresis lability of drug release was achieved by manipulation of
the synergistic interactions of the heterodisperse polysac-
Plasmapheresis is a therapeutic treatment of blood to sepa- charides (65).
rate only plasma from the whole blood of a patient through Release data from ethylcellulose (EC) matrix tablets
extracorporeal circulation when the plasma contains patho- were analyzed to determine which release equation pro-
genic substances which are mostly high molecular weight vided the best t to the data and to observe the effect of
proteins. The separated plasma is discarded and substituted drug solubility on the release mechanism(s) (66). Theoph-
with fresh plasma. The plasma separation is based on ultra- ylline, caffeine, and dyphylline were selected as nonelec-
ltration by hollow ber membrane with pore size from trolyte xanthine derivatives, with solubilities from 8.3 to
0.1 to 0.5 m, prepared from cellulose acetate, polyvinyl 330 mg/mL at 25C. At high drug loading, drug was re-
alcohol, polyethylene, and polypropylene. leased by a diffusion mechanism with a rate constant that
increased with an increase in aqueous solubility. At low
3. Virus Removal drug loading, polymer relaxation also became a component
Cellulose is a very good candidate as the lter material of the release mechanism. However, its contribution to
since the cellulose surface exhibits the least protein adsorp- drug release was less pronounced as solubility decreased,
tion of the conventional polymer surfaces. The membrane becoming negligible in the case of theophylline.
is prepared by coagulation of the cuproammonium solu- Microcrystalline cellulose (MCC), sodium carboxy-
tion. It was proved that this lter with an average pore methylcellulose (NaCMC), hydroxypropylmethylcellulose
size of 35 nm could completely remove hepatitis C virus (HPMC), hydroxyethylcellulose (HEC), hydroxypropylcel-
(61,62). lulose (HPC), and ethylcellulose were used for the pro-
duction of time-controlled acetaminophen delivery sys-
tems using a spray-drying technique. The inuence of fac-
4. Hemostasis
tors such as polymer concentration, inlet temperature, and
Oxidized cellulose has been used as hemostat. This is an drug/polymer ratio were investigated (67). Dissolution
acidic polysaccharide produced by oxidation of cellulose studies in pH 1 dilute HCl and pH 6.8 phosphate buffer
with periodic acid or nitrogen oxide. A more effective dissolution media showed that formulations consisting of
method for homeostasis is to use oxidized cellulose gauze 1% polymer with a drug/polymer ratio of 1:1 exhibited
together with a liquid-type hemostat. Wiseman et al. (63) the slowest drug release, with the spheroids coated with
evaluated two adhesion barriers composed of oxidized re- NaCMC and HEC showing the longest T50% values (45
generated cellulose (ORC) in a model of bowel surgery, and 53 min at pH 1 and 49 and 55 min at pH 6.8, respec-
with and without bleeding. They concluded that with he- tively). Slightly better sustained drug release in pH 6.8 dis-
mostasis both absorbable fabrics of ORC reduced adhesion solution medium was reached, showing the following
formation between the injured cecum and abdominal side trend: HEC NaCMC MCC EC HPMC. Concern-
wall. The effectiveness of INTERCEED Barrier, but not ing the additives, the trends in dissolution T50% of drug
nTC7, was reduced but not eliminated in the presence of revealed TA SA CA OA PVP PA DBS
bleeding. This conrms similar observations in models of in acidic pH 1 dissolution medium and PVP OA
gynecologic surgery. TA SA PA CA DBS in phosphate buffer at pH
A particularly interesting application of oxidized cellu- 6.8 (67).

Copyright 2002 Marcel Dekker, Inc.


Hydroxypropylmethylcellulose has been the most side linkages. Very few (1 6)--D-glycoside linkages
widely used hydrophilic drug carrier. Matrix tablets were seem to occur in this polysaccharide by the chemical (83)
prepared by wet granulation (68), slugging (69), or direct and enzymatic (84) methods. Curdlan is insoluble but
compression (70). The drug release was controlled through swells in water.
variations in the molecular weight of the polymer, drug
solubility, the drug/polymer ratio, the particle size of the A. Clinical Application of Curdlan
drug and polymer, and the addition of different additives
(71,78). Maeda and Chihara (85) and Sasaki et al. (86) reported
The available grades of HPMC have varied molecular that curdlan, lentinan, and other similar polysaccharides
weight, degree of substitution, and particle size. The drug inhibited the growth of Sarcoma-180. Moreover, Sasaki et
and HPMC particles sizes also inuence the drug release al. (87) found that a serum factor is involved in the tumor
parameters, although to a lesser extent. The major disad- inhibition by curdlan. When peritoneal macrophages from
vantage of single-unit hydrophilic matrix systems such as normal untreated mice were incubated in vitro with serum
tablets or capsules has been the possibility of uncontrolled of mice treated with curdlan, they become cytotoxic to tu-
erosion as a result of mechanical stress during passage mor cells. This was attributed to two serum factors, a pep-
through the gastrointestinal tract, causing erratic drug re- tide (mw 4500) and probably a peptidoglycan (mw
lease and absorption. 9000) found in serum after administration of the polysac-
A multiple-unit indomethacin delivery system based on charide.
HPMC as the hydrophilic carrier material was developed Morikawa et al. (88) showed that when curdlan is given
by a novel technique using the insolubility of cellulose i.p. to mice, it produces a high and persistent level of both
ether at elevated temperatures and the ionotropic gelation polymorphonuclear (PMN) leukocytes and macrophages.
of the polysaccharide, sodium alginate with calcium ions The activated PMN leukocytes were spontaneously cyto-
(79). toxic to mammary carcinoma cells in vitro.
In order to develop nasal powder preparations with Cellulose sulfate, curdlan sulphate, and sulfopropyl cur-
higher bioavailability for peptide delivery, the effect of a dlan have been found to exhibit strong antihuman immu-
combination of hydroxypropylcellulose and microcrystal- nodeciency virus (anti-HIV) activity (89,90). Yoshida et
line cellulose used as base materials and microenvironment al. (91) showed that curdlan sulfate with a sulfur content
for the drugs in the preparations was examined (80). Sig- of 14.4% at concentrations as low as 3.3 g/mL completely
nicant enhanced absorption of leuprolide, calcitonin, and inhibited infection by HIV.
FITC-dextran was attained by the addition of a small Antitumor active polysaccharide against Sarcoma-180
amount of HPC to MCC. It is suggested that MCC works was isolated by DEAE-Sepharose CL-6B and Sepharose
as an absorption enhancer by causing a locally high con- 4B column chromatography from the hot watersoluble
centration of drugs in the vicinity of the mucus surface. fraction of the mycelium of liquid-cultured Agaricus blazei
On the other hand, HPC works to increase retention of mill (92). This polysaccharide did not react with antibodies
drugs on the nasal mucus due to its gel-forming property of antitumor polysaccharides such as lentinan, gliforan,
(80). and FIII-2-b, which is one of the antitumor polysaccharides
The aqueous interaction of the sodium salt of ibupro- from A. blazei. Moreover, the analyses of 13C-NMR and
fen with the cellulose ethers ethyl hydroxyethylcellulose GC-MS suggested that this polysaccharide was prelimi-
(EHEC) and HPMC has been investigated in the concen- nary glucomannan with a main chain of -1,2linked
tration range 0500 mM ibuprofen and 0.11% (w/w) D-mannopyranosyl residues and -D-glucopyranosyl-3-
polymer by cloud point, capillary viscometry, equilibrium O--D-glucopyranosyl residues as a side chain. This poly-
dialysis, and uorescence probe techniques (81). A combi- saccharide was completely different from the antitumor
nation of time-resolved and static uorescence quenching polysaccharide obtained from fruiting body of A. blazei,
shows that micellelike ibuprofen aggregates are formed in -1,6-glucan.
the solution. The average aggregation number of pure ibu-
profen micelles in water is about 40.
V. GUAR GUM

IV. CURDLAN Targeting of drugs to the colon, following oral administra-


tion, can be accomplished by the use of modied biode-
Harada et al. (82) and Saito et al. (83) showed that more gradable polysaccharides as vehicles. Guar gum (GG) was
than 99% of the linkages in curdlan are (1 3)--D-glyco- crosslinked with increasing amounts of trisodium trimeta-

Copyright 2002 Marcel Dekker, Inc.


phosphate (STMP) to reduce its swelling properties for use microspheres were prepared by crosslinking with glutaral-
as a vehicle in oral delivery formulations, especially drug dehyde. Nifedipine was loaded into these matrices before
delivery systems aimed at localizing drugs in the distal por- and after crosslinking in order to study its release patterns
tions of the small bowel (93). Swelling of GG in articial (97). The mean particle size of the microspheres was found
gastrointestinal uids was reduced from 100- to 120-fold to be around 300 m. The molecular transport phenome-
(native GG) to 10- to 35-fold depending on the amount of non, as studied by the dynamic swelling experiments, indi-
crosslinker used, showing a bell shape dependency. As a cated that an increase in crosslinking affected the transport
result of the crosslinking procedure GG lost its nonionic mechanism from Fickian to non-Fickian. The in vitro re-
nature and became negatively charged. lease study indicated that the release from these micro-
Functionalizing of GG crosslinked products (GGP) spheres is not only dependent upon the extent of crosslink-
as possible colon-specic drug carriers was analyzed by ing, but also on the amount of the drug loaded as well as
studying (1) the release kinetics of preloaded hydrocorti- the method of drug loading (97).
sone from GGP hydrogels into buffer solutions with or Guar gum tablet formulations were prepared and evalu-
without GG-degrading enzymes (-galactosidase and - ated under a variety of in vitro dissolution conditions. The
mannanase) and (2) direct measurements of the polymers formulations, along with Dilacor XR (diltiazem), were ad-
degradation in the cecum of conscious rates (94). The ef- ministered to a group of eight fasted, healthy volunteers in
fect of a GG diet on -galactosidase and -mannanase ac- a four period crossover study (98). Dissolution of diltiazem
tivity in the cecum of the rat and GGP degradation was also from guar gum tablets was essentially independent of stir
measured. It was found that the product GGP-0.1 (loosely speed under normal conditions (USP Apparatus II).
crosslinked with 0.1 equivalents of STMP) was able to pre-
vent the release of 80% of its hydrocortisone load for at
least 6 h in PBS, pH 6.4. In vivo degradation studies in VI. PULLULAN
the rat cecum showed that, despite the chemical modica-
tion of GG, it retained its enzyme-degrading properties in Although many fungi are known to produce exopoly-
a crosslinker concentrationdependent manner. Eight days saccharides with an interesting range of chemical and
of GG diet prior to the study increased -galactosidase physical properties (99), most of the research effort has
activity in the cecum of the rat threefold compared to its been directed to the -glucan pullulan, produced by Aureo-
activity without the diet. basidium pullulans (100). Bernier (101) rst described
A novel tablet formulation for oral administration using the production of two exopolysaccharides by Pullularia
guar gum as the carrier and indomethacin as a model drug (A. pullulans), a heteropolysaccharide and a neutral glu-
has been investigated for colon-specic drug delivery us- can. These contain -(1-4) and -(1-6) glucosidic link-
ing in vitro methods (95). Drug release studies under con- ages.
ditions mimicking mouth-to-colon transit have shown that Pullulan is a polysaccharide with high liver afnity.
guar gum protects the drug from being released completely Considering this property, interferonpullulan conjugation
in the physiological environment of stomach and small in- was promising for interferon (IFN) targeting to the liver
testine (95). Studies in pH 6.8 phosphate-buffered saline with efcient exertion of its antiviral activity therein (102).
(PBS) containing rat cecal contents have demonstrated the The cyanuric chloride method enabled the preparation of
susceptibility of guar gum to the colonic bacterial enzyme an IFNpullulan conjugate that retained approximately 7
action with consequent drug release. 9% of the biological activity of IFN. Pullulan conjugation
Also, it has been shown that the swelling of guar gum enhanced the liver accumulation of IFN and the retention
is affected by concentration of the drug and viscosity grade period with the results being reproducible. When injected
of the polymer. This study examines the mechanism of intravenously to mice, the IFNpullulan conjugate en-
behavior of guar gum in a polymerdrug matrix (96). The hanced the activity of 2-5A synthetase in the liver. The
swelling action of guar gum, in turn, is controlled by the activity could be induced at IFN doses much lower than
rate of water uptake into the matrices. An inverse relation- those of free IFN injection. In addition, the liver 2-5A syn-
ship exists between the drug concentration in the gel and thetase induced by conjugate injection was retained for 3
matrix swelling. This implies that guar gum swelling is days, whereas it was lost within the rst day for the free
one of the factors affecting drug release. The swelling be- IFNinjected mice.
havior of guar gum is therefore useful in predicting drug A chelating residue [diethylenetriamine pentaacetic
release. acid (DTPA)] was introduced to pullulan (103). This
Poly(vinyl alcohol)guar gum interpenetrating network DTPApullulan could conjugate with IFN through Zn2

Copyright 2002 Marcel Dekker, Inc.


coordination on mixing these three components. Intrave- transplanted mice using 3H-liposome, the tumor/serum ra-
nous injection of the IFNDTPApullulan conjugate with dioactivity ratio in mice injected with 1-AL/CHP liposome
Zn2 coordination induced activity in the liver of an antivi- was higher than that of mice injected with other liposomes.
ral enzyme (103). Liver targeting of IFN by this conjuga- These observations suggest that 1-AL is effective as a cell
tion technique based on Zn2 coordination opens a new recognition element. As a result, 1-AL/CHP liposome is
method of IFN therapy. considered to be a good carrier of anticancer drugs for the
Gu et al. (104) and Wang et al. (105) reported a novel active targeting of tumor cells (107).
formula of hydrophobized polysaccharide nanoparticles Insulin spontaneously and easily complexed with the
which can deliver a HER2 oncoprotein containing an epi- hydrogel nanoparticle of CHP in water (108). The com-
tope peptide to the MHC class I pathway. A protein con- plexed nanoparticles (diameter 2030 nm) thus obtained
sisting of the 147 amino-terminal amino acids of oncogene formed a very stable colloid (108). The original physiolog-
erbB-2/neu/HER2 (HER2) was complexed with two kinds ical activity of complexed insulin was preserved in vivo
of hydrophobized polysaccharides, cholesteryl group after i.v. injection.
bearing mannan (CHM) and cholesteryl groupbearing Suzuki and Sunada (109) have investigated the inu-
pullulan (CHP), to form nanoparticles (CHM-HER2 and ence of water-soluble polymers on the dissolution behavior
CHP-HER2). CHM-HER2 and CHP-HER2 were able of nifedipine from solid dispersions with combined carri-
to induce CD3/CD8 CTLs against HER2-transfected ers. All the solid dispersions of nifedipine were prepared
syngeneic brosarcoma cell lines. In addition, vaccina- by the fusion method using nicotinamide and four differ-
tion by CHM-HER2 complexes led to a strongly enhanced ent water-soluble polymers: hydroxypropylmethylcellulose
production of IgG antibodies against HER2, whereas vac- (HPMC), polyvinylpyrrolidone (PVP), partially hydro-
cination with HER2 proteins alone resulted in a produc- lyzed polyvinyl alcohol (PVA), and pullulan. HPMC, PVP,
tion of antibodies at a marginal level. Mice immunized and PVA dissolved in the fused liquid of nicotinamide and
with CHM-HER2 or CHP-HER2 before tumor challenge operated efciently on the amorphous formation of nifedi-
successfully rejected HER2-transfected tumors. The com- pine in solid dispersions. In dissolution studies, the drug
plete rejection of tumors also occurred when CHM-HER2 concentration for these dispersions increased to more than
was applied not later than 3 days after tumor implanta- twice the intrinsic drug solubility. The rank order of the
tion. drug concentration was HPMC PVP PVA. However,
The effect of liposomal adriamycin with tumor recogni- since pullulan did not dissolve in the fused nicotinamide,
tion molecule, 1-aminolactose (1-AL), on AH66 hepatoma nifedipine was present as a crystalline state in the solid
transplanted into nude mice was investigated by Ichinose dispersion; the supersaturation behavior of the drug was
et al. (106). Adriamycin (ADM) was encapsulated in lipo- scarcely observed (109).
some coating with CHP to increase the stability in the
blood stream. 1-Aminolactose (1-AL) was assembled to
the outer layer of CHP-coated liposomal ADM as a tumor VII. DEXTRIN AND CYCLODEXTRIN
recognition molecule. In an in vivo therapeutic study, 1-
AL/CHP-coated liposomal ADM restrained tumor growth Dextrin consist of an -(16)-linked glucan with
more when compared with CHP-coated liposomal ADM. branches attached to O-3 of the backbone chain units. The
Thus, 1-AL/CHP-coated liposome seems to be a carrier of degree of branching is approximately 5%. Recently, enzy-
ADM to tumor cells. matic hydrolysis combined with chemical and nuclear
Yamamoto et al. (107) synthesized CHP bearing 1- magnetic resonance studies have enabled the ratio of single
aminolactose and introduced a saccharide, cholesteryl pul- to multiple branches to be elegantly elucidated (110).
lulan bearing 1-aminolactose (1-AL/CHP), to an outer The molecular size is a pivotal importance for each of
layer of the conventional liposome as a cell recognition the pharmacological properties of dextrin, for example,
element. Lectin recognized the -galactose by aggregation colloid osmotic pressure, viscosity, cell surface adsorption,
of 1-AL/CHPcoated liposome (1-AL/CHP liposome). and steric exclusion principles (111,112).
The uptake of this liposome to AH66 rat hepatoma cells The effects of dextrin on hemostasis have been re-
was greater than in liposomes without 1-aminolactose in viewed (113,114). Although it is generally agreed that the
vitro. Furthermore, 1-AL/CHP liposomal adriamycin coagulation mechanism remains normal after infusion of
showed a stronger antitumor effect in comparison with a standard clinical dose of dextrin (115,116). Dextran ap-
other types of liposomal adriamycin in vitro. When in vivo pears to be adsorbed to the surfaces of the vascular endo-
tumor-targeting efcacy was investigated in AH66 tumor thelium and various cells (117,118).

Copyright 2002 Marcel Dekker, Inc.


Whereas native dextrin is immunogenic in humans Cyclodextrins have the potential to enhance drug re-
(119), lower molecular weight fractions in the clinical range lease by increasing the concentration of diffusible species
were not found to be immunogenic (120,121). The antigenic within matrix. Guo and Cooklock (137) used a range of
determinants on the dextrin chain appear to correspond additives including CDs to increase the solubility of the
to segments of two to seven glucose units (121,122). poorly water-soluble opoid analgesic, buprenorphine,
pH-sensitive dextran hydrogels were prepared by acti- and modify its release from buccal patches composed of
vation of dextrin (T-70) with 4-nitrophenyl chloroformate, poly(acrylic acid), poly(isobutylene), and poly(isoprene).
followed by conjugation of the activated dextran with 4- By inclusion of guest molecule inside the cavity of CDs,
aminobutyric acid and crosslinking with 1,10-diaminode- side effects decreased. Inclusion in CDs can reduce the
cane (123). The crosslinking efciencies determined by bitterness of femoxetine (138), reduce the local irritation
mechanical measurements were in the range of 5263%. of pirprofen (139), and decrease the ulcerous effect of
Incorporation of carboxylpropyl groups in dextran hy- phenylbutazone (140) or indomethacin (141). In the case
drogels led to a higher equilibrium and faster swelling un- of active ingredients exhibiting a poor bioavailability due
der high pH conditions. The swelling reversibility of hy- to water nonsolubility or low solubility, but without ab-
drogels was also observed after repeated changes in buffers sorption problems, the improvement in apparent solubility
between pH 2.0 and 7.4 (123). can improve the bioavailability (142,143). Other examples
Cyclodextrin (CDs) are naturally occurring homochiral of using CDs to promote drug release through dissolution-
oligosaccharides composed of from 6 to 13 -1,4-linked erosion mechanisms are given by Giunchedi et al. (144)
D-glucopyranose units. They possess annular structures and Song et al. (145).
whose wide and narrow hydrophilic ends are delineated by Because of their ability to enhance the stability, solubil-
OH (2) and OH (3) secondary and OH (6) primary hy- ity, or bioavailability of drugs, CDs have been the subject
droxyl groups, respectively, whereas their hydrophobic an- of studies concerning every administration route: oral
nular interiors are lined with methyl and methylene groups (146158); rectal (159163); dermal (164178); ocular
and ether oxygens. Interest in CDs stems from their ability (179182); nasal (183188); pulmonary (189191); par-
to partially or completely include a wide range of guest enteral (192196); intracerebral (197); intrathecal (198),
species within their annuli to form inclusion complexes, and epidural (199,200) administration. Recently, modied
also referred to a hostguest complexes (124130). The CDs (201212) were prepared either to allow the direct
bonding between the CDs and guests is solely secondary in formation of targeting agents (213217) or to enable them
nature; nevertheless, the inclusion complexes can exhibit to be targeting agents.
considerable thermodynamic stability (131).
Cyclodextrins are potentially very interesting as the for-
mulator in pharmaceutical technology. These cyclic oligo-
VIII. STARCH
saccharides have the ability to form noncovalent com-
plexes with a number of drugs and in so doing alter their A. Microspheres and Microcapsules
physicochemical properties. In addition, the primary and
secondary hydroxyl group of the native (, , ) cyclodex- Starch, in its native and modied form, has been subjected
trins are potential sites for chemical modications (132). to extensive study over the past 40 years. Early interest in
Cyclodextrins have remarkable properties in improving starch was associated with the food and paper industry,
stability, solubility, and bioavailability of drugs after oral textile manufacture, and medicine. Crosslinked starch and
administration (133,134). Natural CDs undergo enzymatic starch networks have both the required biodegradability
degradation along the gastrointestinal tract, which proba- and a relatively high mechanical and chemical stability.
bly occurs mainly in the colon (135). No denitive results Starch microspheres were prepared using epichlorohy-
of acute toxicity have been published because the highest drin as a crosslinking agent. Recently the need for three-
doses administered to animals do not result in any mortal- dimensional matrices with controlled release properties for
ity (133). Because CDs are most often used to enhance the pharmaceutical and agrochemical uses has increased inter-
solubility and consequently the bioavailability of poorly est in starch gelation (218223).
water-soluble active ingredients, intravenous administra- The condensation mechanism of epichlorohydrin with
tion is among the most interesting parenteral routes. Inclu- amylose involves epoxide ring opening, mediated by the
sion of a guest molecule in the cavity of CDs constitutes nucleophilic attack of the alkali amylose, and subsequent
a protected state of the included molecule. This protection chlorine displacement and epoxidation (224,225). The
is especially effective in the solid state, with respect to the starchepichlorohydrin reaction follows second order ki-
oxygen from ambient air (136). netics, rst order with respect to epichlorohydrin as well

Copyright 2002 Marcel Dekker, Inc.


as starch. The activation energy for the reaction is 38 able -1,6 bonds related to the presence of amylopectin
kJ/mol and the temperature coefcient K323/K303 is 2 (226). in the raw starch, (2) glycerol diether, and (3) monoether
The crosslinking of starch with epichlorohydrin under groups, all of these being likely to block the activity of -
homogeneous and heterogeneous condition was studied amylase.
with a particular view of measuring the extent of the side A novel silicone polymer-grafted starch micropar-
reaction, where starch was substituted to give a monoether ticlestarch microparticles (MPs) grafted with 3-(trie-
derivative (227). The extent of this reaction was strongly thoxysilyl)-propylterminated polydimethylsiloxane (TS-
dependent on the reaction conditions (temperature, time, PDMS)was developed that is efcacious both orally and
molar ratio of all reactants). Depending on these condi- intranasally (229,230). Unlike most other microparticle
tions, 525% epichlorohydrin was bound as glycerol systems, this novel system does not appear to retard the
monoether substituent by starch. The degree of swelling release of antigen or to protect antigen from degradation.
of the crosslinked starch was linearly dependent on the The results indicate that a unique physiochemical relation-
water/starch molar ratio in the reaction mixture (227). The ship occurs between protein antigen and silicone in a starch
crosslinking of starch proceeded with remarkable efciency matrix that facilitates the mucosal immunogenicity of anti-
when epichlorohydrin was applied in the vapor phase. gen. This leads to predominance of Th2 antibody response
An interfacial crosslinking process was applied to hy- (229,230).
drosoluble starch derivatives: hydroxyethyl starch and car- The efcacy of temporary arterial emmobilization using
boxymethyl starch (223). All crosslinked polysaccharide degradable starch microspheres combined with hyperther-
microcapsules were characterized by a total resistance to mia was investigated in rabbits bearing VX2 tumors (231).
digestive media. Microsphere injection caused a marked decrease of tumor
Shefer et al. (224) characterized the structure and mor- blood ow and pH. During heating, there was a marked
phology of starch networks formed by two distinct meth- increase of the maximum temperature in tumor tissue com-
ods using cross-polarization magic anglespinning 13C- pared with normal muscle. Tumor growth was suppressed
NMR spectroscopy (CP-MAS 13C-NMR) combined with 330% times at 3 weeks after hyperthermia alone and 270%
wide angle x-ray diffraction measurements. The rst step times following combined treatment with microspheres
in the process involves the gelatinization of amylose using and hyperthermia. Damage to normal muscle tissue was
sodium hydroxide. After the addition of NaOH solution mild (231).
(6.6% w/w) the resonance lines of the amylose were broad- Evaluation of reticular endothelial systemspecic
ened. Broadening can be caused by distribution of isotropic magnetic starch microspheres (MSMs) as an i.v. contrast
chemical shifts due to the loss of crystallinity (224). The agent for MR imaging in a model of experimental liver
loss of crystallinity during this process is also observed in metastases has been studied. A loss of liver signal intensity
the wide angle x-ray diffractogram of a sample following was obtained at all MSM dose levels. No metastases were
treatment with NaOH. detected in the precontrast images. The optimal detection
The amorphous nature of the networks formed follow- rate of hepatic metastases was reached with the T1-
ing the reaction of amylose with epichlorohydrin is also weighted spin-echo (SE) sequence at a dose of 1.0 mg
supported by x-ray diffraction analysis. The resonance of Fe/kg b.w. MSM and the diameters of the smallest lesions
the C6 carbon in the network formed is of weaker intensity depicted were 1 mm (232). The use of MSM dramatically
and is shifted downeld by about 1 ppm from about 61.4 increased the detection of experimental hepatic metastases.
ppm in the native amylose molecule to 60.4 ppm in the
crosslinked network. The secondary OH(2) and OH(3) of B. Starch Derivatives
the native amylose at 5.05.4 ppm appear in the cross-
1. Hydroxyethyl Starch
linked network spectrum. This indicates that both crosslink
points as well as glycolic functional group (4.7 ppm) are Hydroxyethyl starches (HES) are high-polymeric com-
formed (224). These observations suggest that epichloro- pounds obtained via hydrolysis and subsequent hydroxy-
hydrin crosslinks and reacts monofunctionally at C2, C3, ethylation from the highly branched amylopectin con-
and C6 (224). Their swelling degree, reecting the number tained in maize. The glucose units can be substituted at
of glycerol dieter bridges in the polymeric network, and carbon 2, 3, and 6 leading to various substitution patterns.
the number of noncrosslinking monoglycerol ether groups This pattern is described by the C2/C6 hydroxyethylation
corresponding to a side reaction of epichlorohydrin with ratio. The higher the degree of substitution and the C2/C6
starch were determined (228). Degradation by -amylase ratio, the less the starch is metabolized. The in vitro molec-
was surface controlled and could be modulated by the in- ular weight, the degree of substitution, and the C2/C6 ratio
troduction in the polymeric network of (1) nonhydrolyz- are the main determinants of the in vivo molecular weight,

Copyright 2002 Marcel Dekker, Inc.


which is clinically relevant. Hemorrhagic complications kinetics of ACS with those of HES in 32 patients (ASA
that occur after infusing larger volumes of HES can be physical status I and II) undergoing elective surgery. In
avoided with a starch of low in vivo molecular weight contrast to hydroxyethyl starch, this new agent undergoes
(233). Furthermore high molecular weight HES macromol- rapid and nearly complete enzymatic degradation.
ecules lead to a distinctive decrease in bronectin concen-
tration that reects saturation of the reticuloendothelial 3. Carboxymethyl Starch
system (RES). Another advantage of low in vivo molecular
weight HES is its rather short half-life. Patients with an Claudius et al. (240) determined effects of sodium carbox-
increased bleeding risk, microcirculatory disturbance, or ymethyl starch (CMS) on the antimicrobial activity of van-
affected RES should receive HES with low in vivo molecu- comycin. In particular, the in vitro activity of vancomycin
lar weight. In the future, HES should be mainly character- against two clinically relevant bacteria, Staphylococcus
ized by the in vivo and not the in vitro molecular weight. aureus and Enterococcus faecalis, was studied in the pres-
Articial colloids affect hemostasis. Particularly, HES ence of varying concentrations of sodium CMS. From two
solutions may have detrimental effects on hemostatic independent studies conducted using an agar dilution
mechanisms (234). method, it appeared that the binding of vancomycin to so-
Hydroxyethyl starch is frequently used as a volume ex- dium carboxymethyl starch had no effect on the in vitro
pander in critically ill patients. Hofbauer et al. (235) in- antimicrobial activity of vancomycin.
vestigated whether HES inuences the chemotaxis of
polymorphonuclear leukocytes (PMNs) through endothe- IX. FUCAN SULFATES
lial cell monolayers by using a test system that allows the
simultaneous treatment of both cell types; HES was shown Marine algal sulfated polysaccharides have been found to
to signicantly reduce the chemotaxis of PMNs through possess various pharmacological activities, i.e., antibacte-
endothelial cell monolayers. rial, antiviral, antitumor (241,242), immunosuppressive,
The effects of HES on blood coagulation were investi- antilipemic, antihemostatic, and anticoagulant (243). Fu-
gated in 20 patients undergoing surgery to determine can sulfates are a type of sulfated polysaccharide occurring
whether its use places recipients at risk of hemorrhage or in brown marine algae.
thrombosis (236). The partial thromboplastin times are sig- The anticoagulant activity of fucan sulfates (244) was
nicantly prolonged; factor VIII activities and brinogen mainly assayed by activated partial thromboplastone time,
levels are decreased. After infusion of HES, no signicant which expresses the intrinsic pathway of blood anticoagu-
differences were detected in platelet count or prothrombin lation; prothrombin time, which explores the extrinsic
time. A decreased platelet aggregation was also found after pathway; thrombin clotting time; and repilase clotting time
the infusion of HES (236). methods (245). The anticoagulant components of brown,
Jamnicki et al. (237,238) compared the effects of pro- red, and green algae are found in fucan sulfates. They all
gressive in vitro hemodilution (30 and 60%) on blood comprise a family of polydisperse heteromolecules based
coagulation in 80 patients receiving one of two different on fucose, xylose, glucuronic acid, galactose, mannose,
6% HES solutions using thrombelastography (TEG). The and half ester sulfate. They differ in sugar composition and
newly developed solution has a mean molecular weight of sulfate content, and thus in structure (246249).
130 kD and a degree of substitution, dened as the average The correlation between the sulfate and uronic acid con-
number of hydroxyethyl groups per glucose moiety, of 0.4 tents and the anticoagulant activity of fucan sulfates was
(HES 130/0.4); the conventional solution has a mean mo- conrmed by a study on fucan sulfate from Ecklenia kur-
lecular weight of 200 kD and a degree of substitution of ome (250). It has also been reported that fucoidans (pure
0.5 (HES 200/0.5). Both HES solutions signicantly com- fucans) from F. vesiculosus (251), Eisenia bicyclis (252),
promised blood coagulation, as seen by an increase in reac- Hizikia fusiforme (253), Laminaria angustata (254), and
tion time and coagulation time and a decrease in angle P. canaliculata (255) showed antithrombin activity.
alpha, maximal amplitude, and coagulation index (all p
0.05).
X. LECTINS
2. Acetyl Starch
Lectins are proteins or glycoproteins of nonimmune origin
Acetyl starch (ACS) is a new synthetic colloid solution for capable of binding to one or more specic sugar residues
plasma volume expansion and is now undergoing phase 2 and mediating a variety of biological processes, such as
clinical trials. Behne et al. (239) compared the pharmaco- cellcell and hostpathogen interactions, serum glycopro-

Copyright 2002 Marcel Dekker, Inc.


tein, and innate immune responses. Currently, over 200 Hyaluronan (sodium hyaluronate) and hyaluronan de-
three-dimensional structures of lectins from plants, ani- rivatives (hylans) have been developed as topical, in-
mals, bacteria, and viruses and their complexes are avail- jectable, and implantable vehicles for the controlled and
able (256262). localized delivery of biologically active molecules (283).
Excellent recent reviews on the structure and interactions Hyaluronan is the original lastoviscous, biocompatible
of lectins are available (263272). Thus, lectins possess polysaccharide developed for use in eye surgery and visco-
various specicities that are associated with their ability surgery, orthopedic surgery, rheumatology, otology, plas-
to interact with acetylaminocarbohydrates, aminocarbohy- tic surgery, and veterinary medicine (284,285). Hyaluronic
drates, sialic acid, hexoses, pentose, and many other carbo- acid, either by itself or mixed with bronectin, may be a
hydrates (258,273). Lectins from plant sources were the potentially optimal bioimplant for the surgical manage-
rst proteins of this class to be studied (274277). Human ment of vocal fold mucosal defects and lamina propria de-
foods of both plant and animal origin contain a variety of ciencies (e.g., scarring) from a biomechanical standpoint
simple and complex carbohydrates as well as lectins. Both (286).
saccharides and lectins have the capacity to interfere with Hyaluronic acid in the range of Mw 1300 kD may prove
bacterial and viral attachment to epithelial cell surfaces benecial in minimizing bacterial contamination of surgi-
within the alimentary canal, as has the major mucosal im- cal wounds when used in guided tissue regeneration sur-
munoglobulin, secretory IgA. It is well known that the lec- gery (287). The 1.0 mg/mL concentration of high molecu-
tin from jack fruits can bind to serum IgA1, but secretory lar weight HA had the greatest overall bacteriostatic effect,
IgA also possesses oligosaccharide receptors for bacterial inhibiting the growth of all six bacterial strains tested.
lectins in mbriae and can agglutinate E. coli by this anti- Among the bacterial strains studied, HA was found to have
gen nonspecic mechanism (264). no bactericidal effects, regardless of concentration or mo-
Membrane lectins of certain cells are capable of inter- lecular weight.
nalization of their ligands, and hence glycoconjugates spe- An animal model study was conducted to compare the
cically recognized by these lectins can be used as carriers efcacy of recurrent topical applications of hyaluronic acid
of metabolite inhibitors and drugs (278). Galactose-termi- and gentamicin ointment for the treatment of noninfected,
nated glycoproteins and neoglycoproteins have been used mechanical corneal erosions (288). Rabbit eyes treated
to carry antiparasitic (279) and antiviral drugs (280,281). with hyaluronic acid showed a signicantly enhanced rate
The potential for using lectins as a means of anchor- of epithelial defect closure compared with untreated eyes
ing a drug delivery system to the mucosal surfaces of the and a similar rate to that achieved with gentamicin oint-
eye has been investigated (282). In this study the acute ment. In the eyes treated with hyaluronic acid a normal,
local dermal irritancy of these lectins, in terms of their po- multilayered epithelium was observed 48 h after complete
tential to cause inammation and tissue necrosis, was healing, whereas the gentamicin-treated eyes showed an
investigated. There was no evidence of tissue necrosis, imperfectly layered epithelium, with irregularity of the cu-
edema, or Evans blue inltration with any of the lectin boidal cells.
solutions administered. The rabbits did not display any Through the esterication of the carboxyl group of the
signs of discomfort such as scratching or continued groom- glucuronic acid moiety, polymeric prodrugs of hyaluronic
ing throughout the experiment. Histological examination acid have been prepared by several groups (289291).
of the injection sites revealed little sign of any inamma- Two drugs made up of HA derivatives have recently be-
tion, such as heterophil migration, edema, or tissue dam- come available for patients in whom simple analgesics and
age. It was concluded that these lectins demonstrate mini- conservative nonpharmacological therapy have failed. Les-
mal acute irritancy, and will therefore be taken forward for lie (292) reviews the epidemiology, pathogenesis, diagno-
formulation and in vivo studies. sis, and medical management of osteoarthritis of the knee,
with an emphasis on the physiologic and pharmacological
mechanisms of HA. Health care providers may administer
XI. HYALURONIC ACID, HYALURONAN, HA via intra-articular injection in primary care and rheu-
AND HYALURONAN DERIVATIVES matologic or orthopedic settings or they may refer their
patients to specialists for consultation.
Hyaluronic acid (HA) is a natural mucopolysaccharide Hyaluronic acid grafted with poly(ethylene glycol)
which consists of alternating residues of D-glucuronic acid (PEG) (PEG-g-HA) were synthesized. The materials char-
and N-acetyl-D-glucosamine. Hyaluronic acid functions as acterization, enzymatic degradability, and peptide (insulin)
the backbone of the proteoglycan aggregates necessary for release from solutions of the copolymers were examined
the functional integrity of articulate cartilage of the knee. (293). Insulin was preferentially partitioned into the PEG

Copyright 2002 Marcel Dekker, Inc.


phase in a PEG/HA solution system. Leakage of insulin that the biomaterial, with or without mesenchymal pro-
from the copolymers was dependent upon the PEG content. genitors, did not elicit any inammatory response and
Leakage rate of insulin from copolymer containing be- was completely degraded within 4 months after implanta-
tween 7 and 39% by weight of PEG were similar. The tion (296).
conformational change of insulin was effectively pre- The new composite biomaterial made from hydroxyap-
vented in PEG-g-HA solutions, although insulin was dena- atite and collagen conjugated with hyaluronic acid has
tured in storage of both phosphate buffered solution and been studied (297). The structure evaluation of the com-
HA solution. Such a heterogeneous-structured polymeric posite showed more dense arrangement due to the forma-
solution may be advantageous as an injectable therapeutic tion of collagen hyaluronic acid conjugate, and particles of
formulation for ophthalmic or arthritis treatment. inorganic component are closely anchored in the structure
To increase the availability of sodium butyrate over a (297). The test of contact cytotoxicity showed a very good
longer period of time. Coradini et al. (294) covalently biocompatibility of the biomaterial.
linked isodium butyrate to HA (a component of the extra- The adsorption of glycosaminoglycans (heparin, he-
cellular matrix). Its major advantages as a drug carrier con- paran sulfate, dermatan sulfate, highly sulfated chondroitin
sist of its high biocompatibility and its ability to bind sulfate, chondroitin sulfate, and hyaluronan) onto coral has
CD44, a specic membrane receptor frequently overex- been investigated (298). Granules of natural coral of spe-
pressed on the tumor cell surface (294). The biological cic diameter, between 100 and 500 m, having high con-
activity of hyaluronic acidbutyric ester derivatives was tent of calcium (98%) and a homogeneous surface ad-
evaluated in terms of the inhibition of the growth of the sorb glycosaminoglycans with different capacity. Heparin
MCF7 cell line and compared with that of sodium butyrate. (maximum adsorption 1.29 0.10 mg/20 mg of coral,
After 6 days of treatment, we observed a progressive im- 6.45% w/w) is adsorbed more than highly sulfated chon-
provement of the antiproliferative activity up to DS 0.20; droitin sulfate species (maximum adsorption 0.90 0.06
thereafter, the antiproliferative effect of the ester deriva- mg/20 mg of coral, 4.50% w/w), chondroitin sulfate (maxi-
tives decreased (294). Fluorescence microscopy showed mum adsorption of 0.72 0.06 mg/20 mg of coral, 3.60%
that after 2 h of treatment uorescein-labelled compounds w/w), dermatan sulfate (maximum adsorption of 0.70
appeared to be almost completely internalized into MCF7 0.06 mg/20 mg of coral, 3.50% w/w), and heparan sulfate
cells, expressing CD44 standard and variant isoforms. (maximum adsorption of 0.72 0.07 mg/20 mg of coral,
Two biomaterials based on hyaluronic acid modied by 3.60% w/w) (298). Hyaluronan is not adsorbed onto gran-
esterication of the carboxyl groups of the glucuronic acid ules of coral. The percentage adsorption of polyanions onto
(HYAFF 11 and ACP sponges) were tested as osteogenic coral depends mainly on their charge density, with sulfate
or chondrogenic delivery vehicles for rabbit mesenchymal groups being more important than carboxyl groups. The
progenitor cells and compared with a well-characterized adsorption of glycosaminoglycans is driven by electro-
porous calcium phosphate ceramic delivery vehicle (295). static interactions with calcium sites of coral that are de-
The hyaluronic acidbased delivery vehicles are superior pendent on pH and blocked in the presence of large
to porous calcium phosphate ceramic with respect to the amounts of salt. Due to these peculiar properties, the com-
number of cells loaded per unit volume of implant, and bination of granules of natural coral with glycosaminogly-
HYAFF 11 sponges are superior to the ceramics with re- cans makes this material potentially useful in osseointegra-
gard to the amount of bone and cartilage formed. Addition- tion in bone metabolism or periodontal therapy (298).
ally, hyaluronic acidbased vehicles have the advantage of Several biomaterials are available for the purpose of
degradation/resorption characteristics that allow complete soft tissue augmentation, but none of them has all the prop-
replacement of the implant with newly formed tissue. erties of the ideal ller material. The recent development
The tolerability and safety of hyaluronan-based three- of HA gels for dermal implantation give the physician new
dimensional scaffolds as a culture vehicle for mesenchy- possibilities of effective treatment in this eld (299). Stabi-
mal progenitor cells was investigated (296). The prolifera- lized, nonanimal hyaluronic acid gel is well tolerated and
tion patterns and extracellular matrix production of rabbit effective in augmentation therapy of soft tissues of the
and human mesenchymal, bone marrowderived progeni- face. This material presents several advantages in compari-
tors rst were characterized in vitro. Subsequently rabbit son to previously used injectable biomaterials and expands
autologous cells were cultured in this hyaluronan-based the arsenal of therapeutic tools in the eld of soft tissue
scaffold and implanted in a full thickness osteochondral augmentation.
lesion. In vitro histologic ndings showed that mesen- With the aim of producing a biomaterial for surgical
chymal progenitor cells adhered and proliferated onto the applications, the alginatehyaluronate association has
hyaluronan-derived scaffold. In vivo data demonstrated been investigated (300). Crossed techniques were used to

Copyright 2002 Marcel Dekker, Inc.


assess the existence of polymer interactions in aqueous so- alginate hydrogels without buffering of stomach acid may
lutions up to 20 mg/mL. result in desegregation of the calcium alginate complex.
Several antacids can buffer gastric uid at pH 4.5, which
is an appropriate pH for pancreatic buffering in the duode-
XII. ALGINATE num. An increase in enteric pH to 6.5 will occur in the
ileum. Buffering of gastric uid with antacids would be
The alginates is a copolymer composed of D-mannuronic necessary in order to facilitate release of drugs from cal-
acid (M) and L-guluronic acid (G) arranged in MM and cium alginate into small intestinethe most appropriate
GG blocks interrupted by regions of more random distribu- application of this system.
tion of M and G units. Due to the presence of carboxylate The sodium alginate from Sarassum fulvellum showed
groups, alginate is a polyelectrolyte at neutral pH, with one a considerable antitumor activity against various murine
charge per repeating unit in the coil conformation (301). tumors, such as Sarcoma-180, Ehrlich ascites carcinoma
and IMC carcinoma (318).
A. Alginate Hydrogel With the aim of producing a biomaterial for surgi-
cal applications, the alginatehyaluronate association has
Interactions of alginate with univalent cations in solution been investigated (319). Crossed techniques were used to
have been investigated by circular dichroism (c.d.) (302) assess the existence of polymer interactions in aqueous so-
and rheological measurements. Poly-L-guluronate chain lutions up to 20 mg/mL. Viscometry measurements using
segments show substantial enhancement (approximately the capillary technique or the Couette ow, together with
50%) of c.d. ellipticity in the presence of excess of K, circular dichroism investigations, evidenced the moderate
with smaller changes for other univalent cations: Li signicance of interactions between the two polysaccha-
Na K Rb Cs NH4 (302). Pass and Hales rides in dilute solutions. In addition, the case of more con-
(303) have investigated the effect of the cation on the en- centrated solutions and containing 20 mg/mL alginate was
thalpy of dilution of alkali metal salts of alginate. approached by rheological measurements in the ow
Calcium alginate has been one of the most extensively mode; the behavior of the polymer associations appeared
investigated biopolymers for binding heavy metals from as a compromise between those of individual polysaccha-
dilute aqueous solutions (304307). Alginate forms gels rides (319).
in the presence of divalent ions at concentration of 0.1%
w/w (308). The gels are not thermoreversible. The ratio of B. AlginatePolyelectrolyte Complexation
calcium to alginate over which thixotropic gels are formed
depends on the alginate type, the pH, and the solids content Irreversible hydrogels are insoluble in water, as well as in
of the system (309). Previous studies of alginate gelation other solvents, in a wide range of temperatures and dilu-
by c.d. and other techniques (310312) have shown that tions. Due to their solubility proles, irreversible hydrogels
the primary event in network formation is dimerization of have found multiple applications as food additives, in cos-
poly-L-guluronate chain sequences, in a regular 2(1) con- metics, in medicine, and in biotechnology (320326).
formation (313,314) with specic chelation of Ca2 ions The complexation of complementary polymers has been
between the participating chains (315). modeled theoretically (327,328). In this analysis the total
For high M alginates (high D-mannuronic acid algi- free energy of complexation was divided into two contri-
nate), thixotropic gels exist at calcium levels that, in an butions: one arising from the specic interactions between
alginate with a high proportion of L-guluronic acid blokcs, complexing functional groups and a second arising from
would be holding the alginate chains in a permanent gel congurational changes of the system upon complexation.
structure. Specic intermolecular cooperative interactions Several authors have modeled the association of biological
occur between calcium and glucuronate blocks owing to polymers (329332).
the buckled ribbon structure of the polyguluronic acid. The interactions of alginates of various compositions
This observation has led to the well-known proposal of with basic polypeptides, namely, poly(L-lysine) and poly
egg-box junction zones (308,316). 13C-NMR spectro- (Lys-Ala-Ala), have been studied by means of circular di-
scopic studies have been made on alginate solutions under- chroism (333). The alginates used differ from each other
going solgel transition induced by four different divalent in the content of L-guluronate and mixed sequences. The
cations: Ca, Cu, Co, and Mn (317). content of D-mannuronate sequences in all alginates is al-
The rigid structure and large pore size of these gels are most identical. The lower complexation efciency with al-
useful for the encapsulation of enzymes, proteins, drugs, ginate II (MM 33%, GG 30%, MG 37%) than with alginate
liposomes, and living cells. Oral administration of calcium I (MM 30%, GG 20%, MG 50%) and the nearly zero ef-

Copyright 2002 Marcel Dekker, Inc.


ciency with alginate III (MM 33%, GG 47%, MG 20%) tion of islets in the absence of immunosuppression. It has
are due to the presence of considerable amounts of non- previously been suggested that alginate rich in mannuronic
complexing L-guluronate sequences in the alginate struc- acid (high M) is more immunogenic than alginate rich in
ture (333). On the basis of the results of complexation guluronic acid (high G). The ability of these alginates to
achieved in this study (333), it may be suggested that the induce an antibody response in the recipient or act as an
L-guluronan chain is more rigid, i.e., less adaptable to the adjuvant to antibody responses against antigens leaked
changes in the surrounding medium than the D-galacturo- from the capsule was investigated (335). High Galginate
nan chain. capsules are less immunogenic than high M capsules. Be-
The inuence of charge density of a polycation on com- cause encapsulation did not protect against the generation
plexation was studied with the sequentially regular poly of antibodies against islet-like cell clusters (ICC), it can
(Lys-Ala-Ala), which is characterized by a charge density be assumed that antigen leakage from the capsules occurs,
one-third that of poly(L-lysine) (333). Alginates interacted as no evidence was found for capsules breaking in vivo.
with poly(Lys-Ala-Ala) rather intensively. The difference Alginate and proteins were also used in polyionic com-
in efciency of interaction of L-guluronan and D-galactur- plexation (336). Freshly prepared gels of gelatine with al-
onan with poly(L-lysine) results from the difference in the ginate or pectate below the isoelectric point of gelatine
conformational exibility of their polyanionic chains in so- (e.g., at pH 3.9) melt over the same temperature range as
lution. L-Guluronan maintains the rigid twofold symmetry gelatine (3040C), but on aging they become thermosta-
in solution, and D-galacturonan is conformationally adapt- ble (336). The gels are also stable in 7M urea, arguing
able in the course of interaction. against hydrogen bonding or hydrophobic interactions, but
Many of the present controlled-release devices for in the enhanced thermal stability can be eliminated by high
vivo delivery of drugs involve elaborate preparations, of- salt (e.g., 0.3M NaCl) or by raising the pH to above the
ten employing either harsh chemicals, such as organic sol- isoelectric point of gelatine, as expected for an ionic net-
vent, or extreme conditions, such as elevated temperatures. work (336).
These conditions have the potential to destroy the activity A systematic study of the alginatepolycation micro-
of sensitive macromolecule drugs, such as proteins or oli- encapsulation process, as applied to encapsulation of bio-
gopeptides. active macromolecules such as protein, was conducted
Drug delivery particulates were prepared using alginate, by Wheatly et al. (337). When protein drugs (myoglobin)
polylysine, and pectin. Theophylline, chlorothiazide, and were suspended in sodium alginate solution and sprayed
indomethacin were used as the model drugs for in vitro into buffered calcium chloride solution to form crosslinked
assessments, and mannitol was the model for assessing microcapsules. The drug-loaded capsules were coated with
paracellular drug absorption across Caco-2 cell mono- a nal layer of poly(L-lysine).
layers. Alginate and pectin served as the core polymers,
and polylysine helped to strengthen the particulates. Use C. Calcium Alginate as a Matrix for Delivery
of pectin specially helped in forming a more robust particu- of Nucleic Acids
late that was more resistant in acidic pH and modulated
the release proles of the encapsulated model drugs in the Advances in the design of genetically targeted agents offer
alkaline pH. Alginate and pectin were also found to en- new opportunities for drug therapy (338340). Accord-
hance the paracellular absorption of mannitol across Caco- ingly, the encapsulation of DNA and its derivatives may be
2 cell monolayers by about three times. The release rate useful for enteric targeting of nucleic acids as gene transfer
could be described as a rst-order or square-root time agents, carriers for DNA intercalaters, and modied oligo-
process depending on the drug load. Use of alginate nucleotides (341).
polylysinepectin particulates is expected to combine the The biodegradable microspheres based on sodium algi-
advantages of bioadhesion, absorption enhancement, and nate were used to encapsulate plasmid DNA containing the
sustained release. This particulate system may have poten- bacterial -galactosidase (LacZ) gene under the control of
tial use as a carrier for drugs that are poorly absorbed after either the cytomegalovirus (CMV) immediate-early pro-
oral administration (334). moter or the Rous sarcoma virus (RSV) early promoter.
The polyionic complex based on alginate were used to Mice inoculated orally with microspheres containing plas-
included the animal cells (335). Microencapsulation of is- mid DNA expressed LacZ in the intestine, spleen, and
lets of Langherhans in alginatepoly(L-lysine) capsules liver. Inoculation of mice with microspheres containing
provides an effective protection against cell-mediated im- both the plasmid DNA and bovine adenovirus type 3
mune destruction, and ideally should allow the transplanta- (BAd3) resulted in a signicant increase in LacZ expres-

Copyright 2002 Marcel Dekker, Inc.


sion compared to those inoculated with microspheres con- tection was found to be comparable to high molecular
taining only the plasmid DNA. Our results suggest that weight poly(L-lysine) membranes (197.1 kDa) (345). Co-
adenoviruses are capable of augmenting transgene expres- guanidine membranes coating alginate result in a molecu-
sion by plasmid DNA both in vitro and in vivo (342). lar weight cutoff sufcient to retain DNA and exclude 31-
Chitosan and poly(L-lysine) membranes, coating algi- kDa DNAse, while providing access to the low molecular
nate beads, were almost totally inert to the enzymatic hy- weight carcinogen, ethidium bromide.
drolysis by lysozyme; chitosanase; and trypsin, chymo- Somatic gene therapy using nonautologous recombi-
trypsin, or proteinase (343). Less than 2% of the membrane nant cells immunologically protected with alginate micro-
weight was hydrolyzed. It appears that either membrane capsules has been successfully used to treat rodent genetic
material would be stable for in vivo application, and in diseases. Ross et al. (346) have reported the delivery of
particular in the protection of DNA during gastrointestinal recombinant gene products to the brain in rodents by im-
transit. At chitosanase concentrations of 1.4 mg/mL and planting microencapsulated cells for the purpose of even-
in the presence of sodium ions, 20% of the total double- tually treating neurodegenerative diseases with this tech-
stranded DNA was released from chitosan coated beads. nology. Alginatepoly(L-lysine)alginate microcapsules
An exchange of calcium for sodium within the bead lique- enclosing mouse C2C12 myoblasts expressing the marker
ed the alginate core releasing DNA. The presence of cal- gene human growth hormone (hGH) at 95 20 ng per
cium stabilized the alginate bead, retaining all the DNA. million cells per hour were implanted into the right lateral
Highly pure DNA was recovered from beads through me- ventricles of mice under stereotaxic guidance. Control
chanical membrane disruption, core liquefaction in citrate, mice were implanted similarly with nontransfected but en-
and use of DNA spin columns to separate DNA/alginate capsulated cells. Delivery of hGH to the different regions
mixtures in a citrate buffer. DNA recovery efciencies as of the brain at various times postimplantation was exam-
high as 94% were achieved when the initial alginate/DNA ined. At 7, 28, 56, and 112 days postimplantation, hGH
weight ratio was 1000 (343). was detected at high levels around the implantation site
Alginate gels produced by an external or internal gela- and also at lower levels in the surrounding regions, while
tion technique were studied so as to determine the optimal control mice showed no signal. Immunohistochemical
bead matrix within which DNA can be immobilized for staining of the implanted brains showed that on days 7,
in vivo application. The encapsulation yield of double- 56, and 112 postimplantation, hGH was localized in the
stranded DNA was over 97 and 80%, respectively, for tissues around the implantation site.
beads formed using external and internal calcium gelation
methods, regardless of the composition of alginate. Homo- D. Calcium Alginate as Microparticles for Drug
geneous gels formed by internal gelation absorbed half and Drug Proteins Delivery Systems
as much DNAse as compared with heterogeneous gels
formed by external gelation. Testing of bead weight Present and future applications of alginates are mainly
changes during formation, storage, and simulated gastroin- linked to the most striking feature of the alginate molecule,
testinal (GI) conditions (pH 1.2 and 7.0) showed that high i.e., a solgel transition in the presence of multivalent ca-
alginate concentration, high G content, and homogeneous tions, e.g., Ca2. The properties of alginate gels suggest
gels (internal gelation) result in the lowest bead shrinkage biomedical and pharmaceutical uses. Calcium alginate has
and alginate leakage. These characteristics appear best been extensively studied and employed in a number of
suited for stabilizing DNA during GI transit (344). pharmaceutical applications (347,348), especially in con-
Co-guanidine membranes were shown to form intact, trolled drug release (349). For many drug candidates a
ionically complexed membranes on alginate beads, serving modied in vivo drug release is desired to improve ef-
as an alternative to the commonly used polymers, poly(L- cacy, sustain effect, or minimize toxicity. Polymeric de-
lysine) and chitosan. DNA was encapsulated (345). The livery systems, such as microspheres, nanospheres, and
level of DNA protection from nuclease diffusion and the polymeric lms, have been extensively researched in an
degree of DNA complexation with co-guanidine mem- attempt to achieve modied drug release (350). Calcium
branes were all shown to be dependent on both polymer alginate offer an alternative approach.
concentration and coating time. The highest level of The release rate of nicardipine HCl from various algi-
DNAse exclusion was possible within beads coated with nate microparticles was investigated (351,352). The effect
a polymer concentration of 5 mg/mL. Recovery of double- of drug/polymer weight ratio, CaCl2 concentration, and
stranded DNA after nuclease exposure for 60 min reached curing time on parameters such as the time for 50% of
90% of that initially encapsulated. The level of DNA pro- the drug to be released (t50%) and the drug entrapment

Copyright 2002 Marcel Dekker, Inc.


efciency were evaluated with analysis of variance. The Ionotropic gelation by divalent metal interaction was
release of drug from alginate microparticles took place by employed of indomethacinsodium alginate dispersion
both diffusion through the swollen matrix and relaxation with calcium ions to induce the spontaneous formation of
of the polymer at pH 1.24.5. However, the release was indomethacincalcium alginate gel discs (357). A three-
due to diffusion and erosion mechanisms at pH 77.5. phase approach was developed to establish the critical cur-
Pellets of calciumalginate, calciumpectinate, and ing parameters. Since curing involved crosslinking of the
calciumalginatepectinate were produced via crosslink- sodium alginate with calcium ions, an optimal concentra-
ing in an aqueous medium for site-specic drug delivery tion of calcium chloride (phase one) and crosslinking reac-
in the gastrointestinal tract (353). In general, texture analy- tion time (phase two) had to be determined. Furthermore,
sis of various pellets indicated that both strength and resil- the third phase involved the optimization of the air drying
ience proles were in the order of calciumalginate time of the gel discs. In phases one and two, stabilization
calciumalginatepectinate calciumpectinate. Calcium of in vitro drug release characteristics was used as the
alginate pellets were found to be viscoelastic, while marker of optimal crosslinking efciency. Phase three was
calciumpectinate was highly brittle. based on achieving fully dried gel discs by drying to con-
Alginate gel beads containing tiaramide (TAM) were stant weight at 21C under an extractor. The study revealed
prepared (354) using a gelation of alginate with calcium that optimal crosslinking efciency was achieved in 1%
cations. Bead performance was evaluated in vitro for dif- w/v calcium chloride solution for 24 h and air dried at 21C
ferent dissolution media, and beads were also subjected under an extractor for 48 h (357).
to coating. Tiaramide release was dependent both on its Growth and progression of malignant brain tumors oc-
solubility in dissolution medium and the guluronate resi- curs in a micromilieu consisting of both tumor and normal
due content of the alginate used. The release rate was in cells. Several proteins have been identied with the poten-
the following order: in pH 1.2 pH 6.8 water. The fast tial of interfering directly with tumor cells or with the neo-
release rate in pH 1.2 is the result of the high solubility of vascularization process, thereby inhibiting tumor growth.
TAM in acidic medium (354). Beads with high guluronate A continuous delivery of such inhibitory proteins to the
content gave the best controlled results. tumor microenvironment by genetically engineered cells
Calcium-induced alginate gel beads containing chitosan could theoretically be of considerable therapeutic impor-
salt (Alg-CS) were prepared using nicotinic acid (NA), a tance. Read et al. (358) have investigated the growth char-
drug for hyperlipidemia, and investigated its two functions acteristics of cells encapsulated in alginate, which repre-
in gastrointestinal tract: (1) NA release from Alg-CS and sents a potential delivery system for recombinant proteins
(2) uptake of bile acids into Alg-CS. The amount of NA that may have antitumor effects. Three different cell lines,
incorporated in Alg-CS increased according to increment NHI 3T3, 293, and BT4C, were encapsulated in alginate,
of CS content. Nicotinic acid was rapidly released from which is an immunoisolating substance extracted from
Alg-CS in diluted HCl solution (pH 1.2) or physiological brown seaweed. Morphological studies showed that encap-
saline without disintegration of the beads. When Alg-CS sulated cells proliferated and formed spheroids within the
was placed in bile acid solution it took bile acid into alginate in the in vitro cultures and after implantation into
itself. About 80% of taurocholic acid dissolved in the me- the brain. Even after 4 months in vivo a substantial amount
dium was taken into Alg-CS. According to increment of of living cells were observed within the alginate beads. A
bile acid concentration the uptake amount increased, and vigorous inltration of mononuclear cells was observed in
an approximately linear relationship existed among them the brain bordering the alginate beads 1 week after implan-
(355). tation (358).
Calcium alginate beads containing ampicillin were pre- The insecticide/nematicide carbofuran was incorpo-
pared (356). Morphological studies and drug contents, in rated in alginate-based granules to obtain controlled re-
vitro release, and erosion tests were carried out for the lease (CR) properties (359). The basic formulation (sodium
characterization of the prepared beads. The dried particles alginate 1.61%, carbofuran 0.59%, water) was modied
were characterized by irregular shape and a smooth or by addition of sorbents. The effect on carbofuran release
rough surface, depending on the viscosity grade of the algi- rate, caused by the incorporation of natural and acid-
nate used. The control of the drug for different time inter- treated bentonite in alginate formulation, was studied by
vals depended on the molecular weight of the polymer immersion of the granules in water under shaking (359).
used; however, the pH change test showed that this capac- The use of alginate-based CR formulations resulted in a
ity was much lower in the case of acid-treated particles. reduction of the leached amount of carbofuran compared
The results obtained show that the ampicillin beads pre- with the total amount of pesticide leached using the techni-
pared are suitable for intramammary therapy. cal product (50 and 75% for CR granules containing natu-

Copyright 2002 Marcel Dekker, Inc.


ral and acid-treated bentonite, respectively). Alginate that the calcium alginate tested may improve some cellular
bentonite CR formulations might be efcient systems for aspects of normal wound healing but not others (361).
reducing carbofuran leaching in clay soils, which would Drug-impregnated polyelectrolyte complex (PEC)
reduce the risk of groundwater pollution. sponge composed of chitosan and sodium alginate was pre-
pared for wound dressing application (362). Equilibrium
water content and release of silver sulfadiazine (AgSD)
E. Calcium Alginate Wound Dressing could be controlled by the number of repeated in situ PEC
reactions between chitosan and sodium alginate. The re-
It is commonly accepted that the ideal wound covering lease of AgSD from AgSD-impregnated PEC wound
should mimic many properties of human skin. It should dressing in PBS buffer (pH 7.4) was dependent on the
be adhesive, elastic, durable, impermeable to bacteria, and number of repeated in situ complex formations for the
occlusive. Alginates are highly absorbent, gel-forming ma- wound dressing (362). In vivo tests showed that granula-
terials with hemostatic properties, and it has long been tion tissue formation and wound contraction for the AgSD
known that more rapid wound healing occurs when a gel is plus dihydroepiandrosterone (DHEA)impregnated PEC
formed at the wound surface and dehydration is prevented wound dressing were faster than any other groups.
(306). Because of the biocompatibility, exudate absorb- Control of hemorrhage during excision and grafting is
ability, and lm-forming properties of calcium alginate difcult and postoperative hematoma may reduce graft
product, they are good candidates for burn and wound take. Kneafsey et al. (363) have found calcium alginate
management uses. dressings can be of immense help in minimizing these
Ueng et al. (360) investigated the calcium alginate technical problems. Calcium alginate dressings following
dressing as a drug delivery system for the treatment of vari- hemorrhoidectomy effectively reduce postoperative pain
ous surgical infections. Cytotoxicity of the calcium algi- compared to more bulky anal packs (364).
nate dressing to broblasts and HeLa cells was evaluated A prospective controlled trial was carried out to assess
by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetra- the healing efcacy of calcium alginate and parafn gauze
zolium bromide colorimetric assay. The calcium alginate on split skin graft donor sites (365). Calcium alginate
dressing was mixed with vancomycin, and lyophilized or dressings provide a signicant improvement in healing
not lyophilized to form two types of antibiotic dressings. split skin graft donor sites.
The results suggested that the antibiotic dressings present
no obvious toxic risk to their use as a drug delivery system. F. Calcium Alginate as Tissue Engineering
All antibiotic dressings released bactericidal concentra-
tions of the antibiotics in vitro for the period of time New cartilage formation has been successfully achieved
needed to treat surgical infections. This study offers a con- by technology referred to as tissue engineering. Recent ad-
venient method to meet the specic antibiotic requirement vances in tissue engineering permit us to focus on produc-
for different infections. tion of larger amounts of cartilaginous tissue, such as
The healing of cutaneous ulcers requires the develop- might be needed for reconstructive surgery of the entire
ment of a vascularized granular tissue bed, lling of large auricle.
tissue defects by dermal regeneration, and the restoration Polymers and hydrogels such as poly(glycolic acid),
of a continuous epidermal keratinocyte layer. These pro- calcium alginate, and poly(ethylene) and poly(propylene)
cesses were modelled in vitro in the present study, utilizing hydrogels have been used as cell carriers to regenerate car-
human dermal broblast, microvascular endothelial cell tilage in the nude mouse model. This study compared the
(HMEC), and keratinocyte cultures to examine the effect suitability of three polymers for generating tissue engi-
of calcium alginate on the proliferation and motility of neered elastic cartilage using autologous cells in an immu-
these cultures, and the formation of capillarylike structures nocompetent porcine animal model (366). When using
by HMEC (361). This study demonstrates that the calcium pluronics as scaffold, histologic features resemble those of
alginate tested increased the proliferation of broblasts but native elastic cartilage, showing a more organized arrange-
decreased the proliferation of HMEC and keratinocytes. In ment of the cells, which seems to correlate to functional
contrast, the calcium alginate decreased broblast motility properties as elastin presence in the tissue.
but had no effect on keratinocyte motility. There was no Transplantation of isolated chondrocytes has long been
signicant effect of calcium alginate on the formation of acknowledged as a potential method for rebuilding small
capillarylike structures by HMEC. The effects of calcium defects in damaged or deformed cartilages. Chalain et al.
alginate on cell proliferation and migration may have been (367) describe modication of the basic techniques that
mediated by released calcium ions. These results suggest lead to production of a large amount of elastic cartilage

Copyright 2002 Marcel Dekker, Inc.


originated from porcine and human isolated chondrocytes. re-expression of their differentiated function prior to im-
Small fragments of auricular cartilage were harvested from plantation. Glicklis et al. (371) have examined the behavior
children undergoing ear reconstruction for microtia or ex- of freshly isolated rat adult hepatocytes seeded within a
tirpation of preauricular tags and from ears of juvenile novel three-dimensional (3-D) scaffold based on alginate.
pigs. Enzymatically isolated elastic chondrocytes were The attractive features of this scaffold include a highly po-
then agitated in suspension to form the chondronlike ag- rous structure (spongelike) with interconnecting pores, and
gregates, which were further embedded in molded hy- pore sizes with diameters of 100150 m. Due to their
drogel constructs made of alginate and type I collagen aug- hydrophilic nature, seeding hepatocytes onto the alginate
mented with -elastin. The constructs were then implanted sponges was efcient. DNA measurements showed that the
in nude mice and harvested 4 and 12 weeks after hetero- total cell number within the sponges did not change over 2
transplantation. The resulting neocartilage closely resem- weeks, indicating that hepatocytes do not proliferate under
bled native auricular cartilage at the gross, microscopic, these culture conditions. More than 90% of the seeded cells
and ultrastructural levels (367). participated in the aggregation; the high efciency is attrib-
In vitro multiplication of isolated autologous chondro- uted to the nonadherent nature of alginate. The 3-D ar-
cytes is required to obtain an adequate number of cells to rangement of hepatocytes within the alginate sponges pro-
generate neocartilage, but is known to induce cell dediffer- moted their functional expression; within a week the cells
entiation. Marijnissen et al. (368) and Demoor-Fossard et secreted the maximal albumin secretion rate of 60 microg
al. (369) investigated whether multiplied chondrocytes can albumin/10(6) cells/day (371).
be used to generate neocartilage in vivo. Adult bovine ar- Tissue engineering, a eld that combines polymer scaf-
ticular chondrocytes were suspended in alginate at den- folds with isolated cell populations to create new tissue,
sities of 10 or 50 million/mL, or after multiplication in may be applied to soft tissue augmentationan area in
monolayer for one (P1) or three passages (P3). Alginate which polymers and cell populations have been injected
with cells was seeded in demineralized bovine bone matrix independently. Marler et al. (372) have developed an in-
(DBM) or a eece of polylactic/polyglycolic acid (E210) bred rat model in which the subcutaneous injection of a
and implanted in nude mice for 8 weeks. The newly hydrogel, a form of polymer, under vacuum permits direct
formed tissue was evaluated. Structural homogeneity of comparison of different materials in terms of both histo-
the tissue, composed of freshly isolated as well as serially logic behavior and their ability to maintain the specic
passaged cells, was found to be enhanced by high-density shape and volume of a construct. Using this model, three
seeding (50 million/mL) and the use of E210 as a carrier. forms of calcium alginate, (a synthetic hydrogel) were
The percentage of collagen type II, positive-staining P3 compared over an 8-week period: a standard alginate that
cells was generally higher when E210 was used as a car- was gelled following injection into animals (alginate post-
rier. Furthermore, seeding P3 chondrocytes at the higher gel); a standard alginate that was gelled before injection
density (50 million/mL) enhanced collagen type II expres- into animals (alginate pregel); and alginate-RGD, to which
sion. the cell adhesion tripeptide RGD was linked covalently
Chondrocytes from 21-day-old rat fetal nasal cartilage (RGD postgel). Parallel groups that included cultured syn-
were cultured in alginate beads for up to 20 days (370). It geneic broblasts suspended within each of these three
was found that chondrocytes retained their spherical shape gels were also evaluated (alginate postgel plus cells, algi-
and typical chondrocytic appearance. During the culture nate pregel plus cells, and RGD postgel plus cells). Histo-
time, chondrocytes underwent differentiation, as demon- logically, the gel remained a uniform sheet surrounded by
strated by the alkaline phosphatasespecic activity and a brous capsule in the alginate postgel groups. In the algi-
rate of proteoglycan synthesis. Morphological data con- nate pregel and RGD postgel groups, there was signicant
rmed chondrocyte differentiation with the appearance of ingrowth of a brovascular stroma into the gel with frag-
hypertrophic chondrocytes scattered in the alginate gel and mentation of the construct. In constructs in which syn-
a dense extracellular matrix containing lamentous struc- geneic broblasts were included, cells were visualized
tures and matrix vesicles (370). These results indicate that throughout the gel but did not extend processes or appear
the alginate system represents a relevant model for studies to contribute to new tissue formation. Material compres-
of chondrogenesis and endochondral ossication. Further- sion testing indicated that the alginate and RGD postgel
more, the encapsulation method could prove useful for constructs became stiffer over a 12-week period, particu-
studies of tissuebiomaterial interactions in an in vitro en- larly in the cell-containing groups (372).
vironment which more closely mirrors the cartilage matrix Bone morphogenetic proteins (BMPs) are unique mole-
than other culture methods. cules with a specic biological activity for inducing ec-
A potential approach to facilitate the performance of topic bone formation when implanted with a suitable
implanted hepatocytes is to enable their aggregation and carrier matrix. A novel BMP-2derived oligopeptide,

Copyright 2002 Marcel Dekker, Inc.


NSVNSKIPKACCVPTELSAI, was coupled covalently to on alternate glucose residues with a charged trisaccharide
alginate. Then NSVNSKIPKACCVPTELSAI-linked al- side chain (377). Xanthans secondary structure has been
ginate hydrogel composites were implanted into the calf studied by x-ray ber diffraction (378) and analyzed be
muscle of rats and harvested 3 or 8 weeks after surgery. molecular modeling (379). Addition of the side chain
Ectopic bone formation was observed in alginate hydrogel causes the backbone to change from a twofold ribbonlike
linked with BMP-2derived peptide. It is suggested that cellulose conformation to a vefold helix (380383). A
alginate hydrogel linked with an oligopeptide derived from single helix stabilized by backbonesidechain bonding has
BMP-2 might provide an alternative system for topical de- been proposed, but double helical models cannot be ex-
livery of the morphogenetic signal of BMP-2 (373). cluded (378,379).
Alginate membrane was proposed as a self-setting bar- The majority of studies of xanthan in the literature have
rier membrane that can be used for guided tissue regenera- been molecular, including optical rotation, NMR, DSC,
tion. The alginate membrane can be prepared and placed and circular dichroism (381385), but the interpretation of
at the bone defect during the surgical procedure. The pro- some of these have been complicated by the nature of the
cedure consists of two simple steps. First, the bone defect macromolecule. The orderdisorder transition has rst or-
is lled with sodium alginate (Na-Alg) aqueous solution. der kinetics, is fully reversible, and shows no thermal hys-
Then calcium chloride aqueous solution is dropped on the teresis. From this evidence Morris et al. (380) suggested
surface of the Na-Alg aqueous solution. An alginate mem- a single helix stabilized intramolecularly by ordered pack-
brane is formed on the bone defect, keeping the inside of ing of side chains along the polymer backbone.
the bone defect lled with unreacted Na-Alg aqueous solu-
tion (374). A. Xanthan Gels
Many materials have been used for articial tubular
prostheses to assist peripheral nerve gap reconstruction. A number of polysaccharides interact with galactoman-
However, the clinical use of these devices has been re- nans resulting in synergistic viscosity increases or gel for-
stricted because a microsurgical procedure requires spe- mation, which have been extensively investigated be Dea
cialized techniques and expensive equipment, such as op- et al. (386,387) and other groups (388398). Synergistic
erating microscope systems. Therefore Suzuki et al., (375) interactions of polysaccharides in binary mixtures have of-
developed a new gluing method, without sutures, that uses ten been considered to be synonymous with intermolecular
freeze-dried alginate gel. A 7-mm gap in the sciatic nerve binding of the two polysaccharides. The synergisms be-
of rats was bridged with freeze-dried alginate gel. Regener- tween plant galactomannans (carob, tara, or enzymatically
ation was evaluated by electrophysiologic testing and his- modied guar gum) and xanthan or certain algal polysac-
tologic study. Eighteen weeks after surgery, functional re- charides (-carrageenan, furcelaran, or agarose) have been
innervation of motor and sensory nerves had occurred, as attributed to intermolecular binding of the backbone of the
demonstrated by recovery of compound muscle action galactomannan and the helix of the order polysaccharide
potentials (CMAPs), compound nerve action potentials (386,390). The evidence for this and for similar (388,389)
(CNAPs), and somatosensory-evoked potentials (SEPs). well-accepted such intermolecular models. The structural
Histologically, many regenerated nerve fasciculi, includ- similarity of galacturonic acid and glucuronic acid blocks
ing myelinated and unmyelinated bers, were observed favors the formation networks between pectin and alginate
and the implanted alginate gel had disappeared. In conclu- (387). The crosslinking of cellulose brils by galactoman-
sion, a gluing technique using alginate gel is a potential nans has indeed been demonstrated by electron microscopy
alternative to the conventional nerve autograph technique. (391). Cairns et al. (392,393) have proposed a different
Advantages include simple application and rapid repair. model for the gelation of xanthan and galactomannans,
Freeze-dried alginate gel is a promising material for arti- based on x-ray ber diffraction studies on stretched gels.
cial nerve guides for peripheral nerves and also could be Mixed junction zones were formed by interaction between
used for repair of disrupted pathways in central nervous the xanthan and galactomannan backbones, with the rela-
tissue that is amorphous and cannot be sutured (375). tive positions of xanthan side chains on either side of a
sandwiched galactomannan molecule being staggered. The
x-ray results indicate a repeat distance of 0.52 nm (393).
XIII. XANTHAN GUM Xanthan is widely used in foodstuffs in the form of syner-
gistic gels with gluco- and galactomannans (394); these
Xanthan gum is a widely used thickening agent in foods two types of polysaccharide per se and xanthan alone will
and is a recent addition to the hydrophilic matrix carrier not form gels, whereas mixtures of xanthan with either of
list (376). The primary structure is a (1 4)-linked -D- the plant polysaccharides, when heated and allowed to
glucan backbone (cellulose) substituted through position 3 cool, form thermoreversible gels (395,396).

Copyright 2002 Marcel Dekker, Inc.


The inuence of the galactomannan characteristic ratios degradation occurred more slowly. It was concluded that
(M/G) on the temperature of gelation (Tg) and the gel xanthan is an appropriate vehicle for antimicrobial pep-
strength of mixtures of galactomannan with xanthan is re- tides in a retention-increasing formulation.
ported (397). Two galactomannans were investigated: one
highly substituted from the seeds of Mimosa scabrella
(M/G 11) and the other, less substituted, from the endo- XIV. PECTIN
sperm of Schizolobium parahybae (M/G 30). The
xanthan/galactomannan systems (4: 2 g l(1), in 5 mM Pectin is a general term for a group of natural polymers
NaCl) showed a Tg of 24C for that of S. parahybae (398) that occur as structural materials in all land-growing plants.
and 20C for the galactomannan of M. scabrella, deter- Polymerized galacturonic acid partially esteried with
mined by viscoelastic measurements and microcalorime- methanol accounts for the major part of any commercial
try. A Tg of 4050C was found by Shatwell et al. (399) pectin. Commercial pectins are divided into low-ester pec-
for locust bean gum (LBG) (M/G 43). Lundin and Her- tin and high-ester pectins. Pectin is not degraded by en-
mansson (400) reported a difference of 13C Tg of two zymes secreted in the upper gastrointestinal tract and
LBG samples, with M/G 3 (40C) and 5 (53C), in mix- passes intact through the small intestine. Pectin has been
tures with xanthan. It appears that the more substituted ga- used as ber source in numerous studies of the effects on
lactomannans have lower temperatures of gelation in the gastric emptying, gastric ulcer, glucose and insulin level,
presence of xanthan. The mechanism of gelation depends bile acid binding, gallstone, binding of divalent and triva-
also on the M/G ratio. For the lower values it involves only lent cations, inuence on the enzymatic activity of the
disordered xanthan chains in contrast to M/G ratios higher upper digestive tract, lowering serum cholesterol level,
than 3. In addition, the presence of the galactomannan from healing intestinal wounds, colon cancer, colonic cell
M. scabrella increased slightly the temperature of the con- proliferation, and as source of volatile fatty acids in the
formational change (Tm) of xanthan, probably due to the colon (404409). Pectin inuences the viscosity of the
ionic strength contribution of proteins (3.9%) present in meal, and its short-term effect on the rate of gastric empty-
the galactomannan. On the other hand, the galactomannans ing may be explained in this way. A sustained effect of
from S. parahybae, with 1.5% of proteins, and M. sca- pectin consumption in the studies by Schwartz et al. (410),
brella, with 2.4% of protein, did not show this effect, the however, cannot be explained by increased viscosity of the
Tm of xanthan alone or in a mixture being practically un- stomach content. Apparently, pectin delays absorption of
changed (397). glucose in two ways: by delaying gastric emptying and by
Examples of current or potential applications of xanthan increasing the intestinal barrier layer (407).
in pharmaceutics (401,402) or biomedical uses are as ex- Kohen et al. (411) demonstrated a protective effect of
cipients in tablets or clear blood uid substitutes. The func- pectin against oxidative damages of the jejunal mucosa in
tion of xanthan in tablets is similar to other polysaccha- rats. Protection against radicals is important because the
rides used in the same way, namely, to erode or dissolve mucosa is exposed to oxidative stress from the diet.
slowly and thereby yield a delayed release of active ingre- The antidiarrhea effect of pectin (in the form of dried
dients compared to formulations not containing hydrocol- citrus peels or waste from potato starch manufacturing) in
loids. Recently reported is that xanthan used in tablets cattle is enhanced by mixing with lecitin from soy oil re-
yields a comparable kinetics in the release of drugs to those ning. The positive effect of lecithin supplementation is
of formulations containing N-CMC or carrageenan (403). explained by increased adhesion of pathogenic bacteria to
Oral candidiasis frequently occurs in individuals with the pectinlecithin complex (412).
dry mouth syndrome (xerostomia), in immunocomprom- The pectin-based raft-forming antireux agent Aurax
ised patients, and in denture wearers. Russien et al. (401) (Idoux) was examined, rst regarding reduction of
developed a formulation which will prolong the retention esophageal acid exposure and also as to its efcacy as
time of antimicrobial agents at the site of application. The maintenance treatment in patients with healed esophagitis
activity against Candida albicans of a synthetic cationic (413). The median (interquartile range) acid exposure
peptide dhvar 1, based on the human fungicidal salivary times in the upright position were 3.1% (1.613.0%) on
peptide histatin 5, was tested in a mixture with the bioadhe- Aurax versus 6.7% (2.514.9%) on placebo (p 0.10).
sive. Coupling caused a reduction of the viscosity and elas- In the supine position no difference was found (Aurax
ticity of the xanthan solution related to the applied concen- 13.7%, placebo 13.2%). The time to recurrence of heart-
tration of the coupling agent. Incubation of the peptide burn with Aurax treatment was prolonged signicantly;
with claried human whole saliva resulted in proteolytic after 6 months the life table estimates were 48% of patients
degradation of the peptide. In the presence of xanthan the in remission on Aurax versus 8% on placebo (p 0.01).

Copyright 2002 Marcel Dekker, Inc.


Following treatment, erosive esophagitis was found in taining 2.5 or 5% apple or orange pectin or without pectin
17/34 on Aurax versus 28/38 on placebo (p 0.05). (control) (420). Cholesterol concentrations were deter-
Aurax signicantly delays recurrence of moderate or se- mined in feces after 1, 2, and 3 weeks of treatment, and
vere heartburn and erosive esophagitis, when used as main- in liver and serum at the end of the experimental trials.
tenance treatment. The acid exposure was not signicantly Cholesterol concentration in feces showed a signicant in-
reduced with pH monitoring (413). crease by week 3 in rats fed 5% orange or apple pectin.
Lowering the serum cholesterol level is, without doubt, Hepatic cholesterol concentration declined signicantly in
the most studied physiological effect of pectin and other all pectin-fed groups. Serum cholesterol only declined sig-
soluble dietary bbers. Numerous studies in hypercholes- nicantly in apple-fed groups. The decrease of cholesterol
terolemic and normolipidemic humans, rats, and other ex- levels in liver and serum and its increase in feces could
perimental animals have proven the serum cholesterol low- explain the benecial effect of including these bbers in
ering effect of pectin (404408,414417). The conclusion the diet to prevent some currently very frequent diseases
from the last 25 years studies is that a pectin dosage of (420).
1015 g/day leads to a decrease in the serum cholesterol Because a high daily consumption of polysaccharide-
level of 10%. containing food is assessed to decrease the risk of cancer
The dietary effect of the water-soluble dietary - of the gastrointestinal system, different types of carbohy-
bers (WSDF) guar gum, partially hydrolyzed guar gum drates were investigated for their antimutagenic activity
(PHGG), glucomannan, and highly methoxylated (HM) against different standard mutagens. Within the screening
pectin on the serum lipid level and immunoglobulin (Ig) pronounced antimutagenic effects were found for xyloglu-
production of SpragueDawley rats was compared with can and different pectins and pectinlike rhamnogalacturo-
that of water-insoluble cellulose (418). Although serum to- nans against 1-nitropyreneinduced mutagenicity. Inhibi-
tal cholesterol and triglyceride levels were signicantly tion rates were dose-dependent and varied between 20 and
lower in the rats fed with WSDF than in those fed with 50%. Concerning the mode of action, a direct interaction
cellulose, a decrease in the level of phospholipids was only of the polymers with the cells is claimed, protecting the
observed in the rats that had been fed on guar gum or glu- organisms from the mutagenic attack (421).
comannan. In addition, all WSDF feeding enhanced IgA Anticarcinogenic and tumor growthinhibiting effects
productivity in the spleen and mesenteric lymph node lym- of nonsoluble bers have been described (422). In a pre-
phocytes, although the increase in serum IgA level was liminary study on methylnitrosourea-induced mammary
only observed in the rats fed on WSDF, and not on PHGG. carcinogenesis in female SpragueDawley rats, 15% oli-
When mesenteric lymph node lymphocytes were cultured gofructose added to the basal diet modulated this carcino-
in the presence of various concentrations of guar gum or genesis in a negative manner (423). There was a lower
glucomannan, no signicant increase in Ig production was number of tumor-bearing rats and a lower total number of
apparent. These data suggest that WSDF indirectly en- mammary tumors in oligofructose-fed rats than in the
hanced the Ig production of lymphocytes, and that serum group fed the basal diet alone.
lipid reduction and IgA productionenhancing activities of The effect of dietary nondigestible carbohydrates (15%
WSDF were dependent on their molecular sizes (418). oligofructose, inulin, or pectin incorporated into the basal
Experimental hypercholesterolemia and its modulation diet) on the growth of intramuscularly transplanted mouse
by some natural dietary supplements (pectin, garlic, and tumors, belonging to two tumor lines (TLT and EMT6),
ginseng) and by the drug gembrozil were studied (419). was also investigated (424). The results were evaluated by
Results of the study demonstrated that feeding the choles- regular tumor measurements with a vernier caliper. The
terol-enriched diet caused a signicant increase in total, mean tumor surface in the experimental groups was com-
LDL, and HDL cholesterol; plasma MDA and post-heparin pared with that in animals of the control group fed the basal
total; and hepatic lipase activities. On the other hand, serum diet containing starch as the only carbohydrate. The growth
Tg and erythrocyte superoxide dismutase were not changed. of both tumor lines was signicantly inhibited by supple-
Histopathological examination revealed marked alteration menting the diet with nondigestible carbohydrates. Such
in the aortic wall with the appearance of large multiple ather- nontoxic dietary treatment appears to be easy and risk free
omatous plaques. Both garlic and pectin were successful in for patients, applicable as an adjuvant factor in the classical
a signicant reduction of the hypercholesterolemia in a way protocols of human cancer therapy (424).
comparable to gembrozil. Garlic was the only treatment Among pectin, apple pectin exerts stronger bacteriostat-
that has antilipid peroxidative property (419). ical action on Staphylococcus aureus, Streptococcus fae-
To investigate the effects of pectin on cholesterol me- calis, Pseudomonas aeruginosa, and Escherichia coli in
tabolism, normal rats were fed for 3 weeks a diet con- comparison with citrus pectin. In this study, we used water-

Copyright 2002 Marcel Dekker, Inc.


soluble methoxylated pectin from apple. The diet, supple- widespread in animal tissue (435). Heparin and chondroi-
mented by 20% apple pectin, signicantly decreased the tin sulfates, which are the most abounded sulfated gly-
number of tumors and the incidence of colon tumors. cosaminoglycans, contribute an enormously important role
PGE2 level in distal colonic mucus in 20% apple pectin in the successful development of open heart surgery. Due
fed rats were lower than those in basal diet fed rats. Fecal to its antithrombotic and anticoagulant properties, heparin
-glucuronidase activities in the apple pectinfed group, is extensively used in the management of cardiovascular
which has been considered a key enzyme for the nal acti- diseases (436438). Long-term treatments with heparin, as
vation of dimethylhydrazine metabolism to carcinogens in well as short-term ones in the case of patients susceptible
the colonic lumen, were signicantly lower than those in to hemorrhage, involve risk of bleeding. Another unwanted
control group at initiation stage of carcinogenesis. The side effect of heparin is its interaction with platelet func-
concentrations of -glucosidase and azoreductase were tion, which may lead to depletion of these blood compo-
also decreased. The effect of apple pectin on the colon car- nents (thrombocytopenia, HIT). During the last decade,
cinogenesis may partially depend on PGE, concentration fundamental aspects of the pathogenesis of HIT have been
decrease in colonic mucus, and on the type of pectin, also resolved. The understanding of some the mechanisms un-
related to fecal enzyme activities. derlying the development of new, paradox thromboem-
The resistance of pectin to degradation in the upper GI bolic complications in HIT led to the concept that thrombin
tract and its complete dissolution in the colon makes pectin generation plays a key role in clinically manifest HIT.
an ideal ingredient for colon-specic delivery. The solubil- Consequently new therapeutic concepts imply the use of
ity in the GI uids is suppressed by crosslinking with cal- drugs with either indirect or direct antithrombin activity,
cium or by chemical means. Coacervate with gelatine per- such as donaparoid sodium and the recombinant hirudin
mits the formation of microglobules suitable for lepirudin. In recent years results of the rst prospective
controlled-release products (425427). studies assessing various treatment regimens in HIT be-
To develop an enzymatically controlled pulsatile drug came available. Although data of randomized trials are still
release system based on an impermeable capsule body, missing, some treatment recommendations can already be
which contains the drug and is enclosed by an erodible drawn from these studies (439).
pectin/pectinase plug have been studied (428). The plug The discovery of new activities of heparin, relating to
was prepared by direct compression of pectin and pec- inhibition of growth of smooth muscle cells (440), angio-
tinase in different ratios. In addition to the disintegration genesis (441), the human immunodeciency virus (HIV)
times of the plugs, the lag times and the release proles (442), and tissue engineering (443), has widened the scope
of the pulsatile system were determined as a function of of potential uses of this GAG. Incorporation of heparin-
pectin/enzyme ratio, the pH of the surrounding medium, binding peptides into brin gels enhances neurite exten-
and the addition of buffering or chelating agents. The drug sion, an example of designer matrices in tissue engineer-
release was controlled by the enzymatic degradation and ing.
dissolution of pectin (429). Kratz et al. (444) have recently shown that heparin in
The use of pectin in tablet formulations have been stud- combination with chitosan stimulates re-epithelialization
ied by Meshali and Gabr (430) using a blend of pectin and in an in vitro model of human wound healing. The chito-
chitosan. Ashford et al. (431,432) protected a core tablet sanheparin membrane stimulated the increased stabiliza-
with a coat of HM pectin. It was shown that the tablets tion and concentration of growth factors in the wound area,
disintegrated in the colon. Coprecipitation of cationic which stimulated healing.
drugs with pectin was shown to be applicable for sustained Some of the biological properties of heparin can be sim-
release of water-soluble drugs (433). Encapsulation of ulated by other GAGs or by chemically sulfated vegetal,
liver cells by coacervation of carboxymethylcellulose, chon- algal, or microbial polysaccharides. However, the type of
droitin sulfate, chitosan, and polygalacturonic acid gave carbohydrate backbone and the sulfation pattern of the
viable cells that could be used for extracorporeal liver sup- polysaccharide are important for the emergence and level
port (434). of specic activities (445). As the prophylactic use of hep-
arin continues to increase, nurses must be aware that hepa-
rin use may cause heparin-induced skin necrosisa rare
XV. HEPARIN but serious complication. Although even more severe com-
plications may occur from heparin use, this discussion will
Heparin is the most biologically reactive member of the focus on skin necrosis caused by subcutaneous heparin.
family of sulfated glycosaminoglycans (GAGs), which are Should heparin-induced skin necrosis develop, heparin

Copyright 2002 Marcel Dekker, Inc.


therapy must be discontinued immediately. Reports of one as in the treatment of stroke and unstable angina. In all
patients reaction to this complication have been presented these situations, LMWH had a similar or even a better
(446,447). risk/benet ratio than unfractionated heparin (UH) (451).
Angiogenesis is a prerequisite for tumor expansion and Acute myocardial infarction and coronary angioplasty are
metastasis. The angiogenic potential of the heparin-bind- among the new targets presently under investigation with
ing growth factors acidic broblast growth factor (FGF) various LMWH. Patients with mechanical heart valves re-
and basic FGF has been demonstrated in various publica- quire life-long anticoagulation. Therefore, the anticoagula-
tions. Zugmaier et al. (448) have studied the inhibitory ef- tion with LMWH after mechanical heart valve replacement
fects of suramin and the polysulfated heparinoids pentosan were studied (451). Anticoagulation activity with LMWH
polysulfate, dextran sulfate, and fucoidan on the action of after mechanical heart valve replacement appears feasible,
FGF. Polysulfated heparinoids exert a selective inhibitory provides adequate biological anticoagulation, and com-
effect on heparin-binding angiogenesis factors at an IC50, pares favorably with UH anticoagulation. Randomized
which is 100 times below the IC50 of suramin. Therefore, studies are now needed to further evaluate this new thera-
the administration of polysulfated heparinoids might be- peutic approach.
come a novel approach to tumor therapy based on blocking Albumin has also been utilized with heparin preadsorp-
angiogenesis. tion of both molecules or by their covalent coupling, which
Aspirin, a potent antiplatelet drug, and heparin, an anti- results in heparinalbumin conjugates. Albumin pread-
coagulant, are commonly used for postimplant complica- sorbed onto surface reduce platelet adhesion, while heparin
tions such as thrombosis and thromboembolism. Aspirin is able to interact with antithrombin III, preventing throm-
and heparin were embedded in chitosanpolyethylene vi- bus formation (452456). Crosslinked gels of albumin as
nylacetate comatrix to develop a prolonged release form well as heparinized albumin gels, potential sealant of pros-
(449). The effect of these drugs toward the bioprosthetic thetic vascular grafts, were studied with regard to in vitro
calcication was investigated by in vitro and in vivo mod- stability, binding of basic broblast growth factor, and cel-
els (449). In vitro and in vivo evaluation suggest that the lular interactions (457). It can be concluded that cross-
released aspirinheparin from the comatrix had a syner- linked gels of albumin to which heparin is immobilized,
gistic effect in inhibiting glutaraldehyde pretreated bovine are candidate sealants for prosthetic vascular grafts and
pericardium calcication. Biochemical, histological, and suitable substrates for endothelial cell seeding.
scanning electron microscopic evaluation of retrieved sam- Inhibitory effects of heparin coupling on calcica-
ples demonstrated a signicant reduction in calcium depo- tion of bioprosthetic vascular grafts of different origin
sition. were studied. Heparin-bonded and 0.625% glutaraldehyde
Many of the acute coronary ischemic syndromes are crosslinked (GA) segments of porcine thoracic aorta (AO),
triggered by spontaneous or mechanical disruption of ath- pulmonary artery (PA), jugular vein (JV) and rabbit aorta
erosclerotic plaques with resultant activation of platelets (RA) were implanted subcutaneously in weanling rats for
and coagulation. Given the central role of platelets and 5 months (458). Heparin bonding was ineffective in pre-
thrombin in arterial thrombosis, current strategies for its vention of calcication of JV and RA. Calcium content of
prevention and treatment focus on both inhibition of plate- heparin-coupled PA and AO was signicantly less when
let aggregation and control of thrombin generation and ac- compared with their GA-treated counterparts. Calcication
tivity. Although aspirin and unfractionated heparin are the inhibition was achieved to a greater extent in heparin-
cornerstones of current treatment strategies, both have lim- bonded PA than in the AO coupled to heparin. Heparin-
itations (450). bonded porcine pulmonary artery seemed to be the best
Among the biomedical applications of synthetic poly- among all vascular bioprostheses in this study.
mers, the development of blood-handling equipment Clinical procedures involving extracorporeal blood cir-
(e.g., the hemodialysis and cardiopulmonary bypass, angi- culation are potentially complicated by the interaction of
ographic catheters, intra-aortic balloon pumps, cardiovas- various blood systems with foreign surfaces. In cardiopul-
cular prostheses, articial heart devices) has been one of monary bypass, exposure of blood to synthetic surfaces
the most investigated areas in the past two decades. Bioma- generally leads to activation of cellular and humoral blood
terial thrombogenicity remains the single most important systems with activation of complement cascade. This reac-
concern preventing even more widespread application. tion can be associated with a variety of postoperation clini-
In recent years, low molecular weight heparins cal complications, such as increased pulmonary capillary
(LMWH) have been tested in the prevention and treatment permeability, anaphylactic reactions, and various degrees
of deep vein thrombosis and pulmonary embolism, as well of organ failure which contribute to mortality in routine

Copyright 2002 Marcel Dekker, Inc.


cardiac operations. Application of biocompatible materials accurate and reproducible these sensors are. Polymer
in an extracorporeal circuit modies the normal pattern membranebased potentiometric sensors were developed
of blood activation, and therefore may potentially reduce earlier to provide a rapid and direct method of analysis
clinical complications in routine cardiac surgery (459). for polyions such as heparin. These heparin sensors are
The use of heparin-coated circuits resulted in reduction of irreversible, requiring a membrane renewal procedure be-
systemic leukocyte activation of cardiopulmonary bypass tween measurements which currently prevents the sensors
reected by reduced leukocyte and neutrophil counts 24 h from being used for continuous monitoring of blood hepa-
after operation (p 0.05). rin. Indeed, recent research has revealed that these sensors
Various sugar-carrying polystyrenes (PS), which con- behave quite differently than classical ion-selective elec-
sist of synthetic styrene and sugar moieties, are glyco- trodes. Mathison and Bakker (463,464) explored ways to
conjugates that are able to attach to polymeric surfaces. improve measurement reproducibility and long-term po-
Heparin-carrying PS (HCPS) is especially able to retain tential stability by considering the unique pseudosteady
the binding of heparin-binding growth factors (GFs) such state response mechanism of the polyion sensors devel-
as vascular endothelial GF 165 or broblast GF 2. Human oped so far. Heparin may be stripped out of the phase
skin broblast cells, human coronary smooth muscle cells, boundary membrane surface with a high sample NaCl con-
and human coronary endothelial cells have good adherence centration, and this characteristic is used to modify the cal-
to the HCPS-coated plate. These results indicate that ibration procedure in order to avoid memory effects. It is
growth of various cells can be controlled by the HCPS also attempted to reduce long-term potential drifts by con-
coating, thereby retaining the bioactivity of molecules such tinuously stripping heparin out of the membrane at the
as heparin-binding GFs. Thus, HCPS-coated surfaces con- membrane inner lling solution side. A theoretical model
trol selective growth of various cells (460). is presented to explain the experimental results (464).
Using polystyrene microspheres coated with heparin or
heparan sulfate, it was shown that coated microspheres
specically bound eukaryotic cells and were endocytosed XVI. CHITOSAN
by nonprofessional phagocytic cells. Coated microspheres
displayed properties of binding to eukaryotic cells that Chitosan is a polysaccharide comprising copolymers of
were similar to those of Chlamydiae, and the microspheres glucosamine and N-acetylglucosamine. Chitosan is usually
were competitively inhibited by chlamydial organisms. prepared from chitin (2-acetamido-2-deoxy -1,4-D-glu-
Endocytosis of heparin-coated beads resulted in the tyro- can), and chitin has been found in a wide range of natu-
sine phosphorylation of a similar set of host proteins, as ral sources (crustaceans, fungi, insects, annelids, mollusks,
did endocytosis of Chlamydiae; however, unlike viable coelenterate, etc.). However chitosan is only manufactured
chlamydial organisms, which prevent phagolysosomal fu- from crustaceans (crab and craysh), primarily because a
sion, endocytosed beads were trafcked to a lysosomal large amount of the crustacean exoskeleton is available as
compartment. These ndings suggest that heparin-coated a byproduct of food processing. It is a natural, nontoxic,
beads and Chlamydia trachomatis enter eukaryotic cells biodegradable polysaccharide available as solution, ake,
by similar pathways (461). ne powder, bead, and ber. Due to the fact that chitosan
Poly(ethylene terephthalate) (PET) lm was exposed to has a large molecular weight, exhibits a positive charge,
oxygen plasma glow discharge to produce peroxides on its and demonstrates lm-forming ability and gelation char-
surfaces. These peroxides were then used as catalysts for acteristics, the material has been extensively used in the
the polymerization of acrylic acid (AA) in order to prepare industry, foremost as a occulant in the clarication of
a carboxylic acid groupintroduced PET (PET-AA). Insu- wastewater (465,466), as a chelating agent for harmful
lin and heparin coimmobilized PET (PET-I-H) was pre- metals for the detoxication of hazardous waste (467,468),
pared by the grafting of poly(ethylene oxide) to PET-AA, for the clarication of beverages, such as fruit juices and
followed by reaction rst with insulin and then heparin beers (469), and for agricultural purposes such as fungi-
(462). The blood compatibility of the surface-modied cides (470). In addition chitosan has been exploited in the
PETs were examined using in vitro thrombus formation, cosmetic industry, in the dental industry, for hair care prod-
plasma recalcication time, activated partial thromboplas- ucts, and for ophthalmic applications, such as for contact
tin time, and platelet adhesion and activation. lens coatings or the contact lens material itself (471473).
The growing importance of polymer membranebased It has been extensively used as a biomaterial (474) due to
potentiometric polyion sensors in biomedical research and its immunostimulatory activities, anticoagulant properties
clinical measurements has brought up the question of how (475), antibacterial and antifungal action (476481), and

Copyright 2002 Marcel Dekker, Inc.


also as a promoter of wound healing in the eld of surgery using biopolymer chitosan and its heparinlike derivative
(482484). Chitosan caused leakage of glucose and lactate as the organic species to be incorporated into the silicon
dehydrogenase from E. coli cells. These data support the alkoxidebased network (493). These hybrid materials dis-
hypothesis that the mechanism of chitosan antibacterial played good blood compatibility in comparison with their
action involves a crosslinkage between the polycations single component systems.
of chitosan and the anions on the bacterial surface that
changes the membrane permeability. A. Biocompatibility and Bioadhesivity
Recently, Artursson et al. (485) and Schipper et al. of Chitosan
(486) reported that the structural properties of chitosans,
such as degree of acetylation and molecular weight, are Chitin and chitosan are substrate for lysozyme, an enzyme
very important for its absorption enhancement of hydro- found in various mammalian tissues (494,495). The enzy-
philic drugs. They found that a low degree of acetylation matic hydrolysis of chitin and chitosan leads to the produc-
and/or a high molecular weight appear to be necessary for tion of N-acetyl-D-glucosamine and D-glucosamine. It is
chitosans to increase the epithelial permeability. believed that the glucosamine plays an important physio-
Chitosan ingestion effectively lowers serum choles- logical role in in vivo biochemical processes. It is known
terol (487489). Oligosaccharides of chain length less than that glucosamine takes part in detoxication functions of
ve residues are ineffective. Orally administered chitosan liver and kidney and possesses anti-inamatory, hepato-
binds fat in the intestine, blocking absorption, and has been protective, antireactive, and antihypoxic qualities (496
shown to lower blood cholesterol in animals and humans. 498).
As a result it has been proposed that dietary supplementa- Chitosan lacks irritant or allergic effect and is biocom-
tion with chitosan may inhibit the formation of atheroscle- patible with both healthy and infected human skin (499).
rotic plaque (490,491). Animals were fed for 20 weeks on When chitosan was administered orally in mice, the LD50
a diet containing 5% chitosan or on a control diet. Blood was found to be in excess of 126 g/kg, which is higher
cholesterol levels were signicantly lower in the chitosan- than that of sucrose (500).
fed animals throughout the study, and at 20 weeks were Chitosan has been proposed for the development of
64% of control levels. When the area of aortic plaque in membranes and bers for hemodialysis and blood oxy-
the two groups was compared, a highly signicant inhibi- genators, skin substitute, and wound dressing materials
tion of atherogenesis in both the whole aorta and the aortic (501503). Chitosan as hemodialysis membranes promotes
arch was observed in the chitosan-fed animals42 and surface-induced thrombosis and embolization (501). When
50%, respectively. Body growth was signicantly greater blood comes in contact with biomaterial surfaces like
in the chitosan-fed animals. This study shows a direct cor- chitosan, there is an initial adsorption of plasma proteins,
relation between lowering of serum cholesterol with chito- followed by adhesion and activation of platelets (504).
san and inhibition of atherogenesis, and suggests that the To improve blood compatibility, chitosan surface was
agent could be used to inhibit the development of athero- modied by the complexationinterpenetration method us-
sclerosis in individuals with hypercholesterolemia. ing an anionic derivative of poly(ethylene glycol), me-
The effects of chitosan have been investigated on 80 thoxypropyl(ethylene glycol) sulfonate (505). The result of
patients with renal failure undergoing long-term stable he- this study shows that chitosan surface can be permanently
modialysis treatment (492). The patients were tested after modied by complexationinterpenetration of anionic
a control treatment period of 1 week. Half were fed 30 poly(ethylene glycol) derivative to improve blood compat-
chitosan tablets (45 mg chitosan per tablet) three times a ibility of chitosan. When in contact with blood, the modi-
day. Ingestion of chitosan effectively reduced total serum ed surface can resist plasma protein adsorption and cell
cholesterol levels (from 10.14 4.40 to 5.82 2.19 mM) adhesion by the steric repulsion mechanism (505).
and increased serum hemoglobin levels (from 58.2 12.1 Mucoadhesive properties of three viscosity grades of
to 68 9.0 g L-1). Signicant reductions in urea and creat- chitosan were investigated to assess the suitability of this
inine levels in serum were observed after 4 weeks of chito- polymer for gastroadhesive formulations. The inuence of
san ingestion (492). During the treatment period, no clini- the pH, ionic strength, and temperature of the hydration
cally problematic symptoms were observed. These data medium on the rheological properties were studied. The
suggest that chitosan might be uneffective treatment for mucoadhesive performance was assessed in vitro by means
renal failure patients, although the mechanism of the effect of rheological synergism and tensile stress testing (506).
should be investigated further. It was found that the interaction of chitosan with mucin
New silicachitosan hybrid biomaterials were produced decreases on increasing molecular weight.

Copyright 2002 Marcel Dekker, Inc.


Toxicity of chitosan also depends on its high charge a wound healing product for human use containing chito-
density but appears to be less affected by the molecular san as an excipient.
weight (507,508). A photocrosslinkable chitosan to which both azide
and lactose moieties were introduced (Az-CH-LA) was
B. Chitosan and Its Derivatives as Biomaterials prepared as a biological adhesive for soft tissues, and its
for Wound Healing effectiveness was compared with that of brin glue. Intro-
duction of the lactose moieties resulted in a much more
It is commonly accepted that the ideal wound covering water-soluble chitosan at neutral pH. Application of ultra-
should mimic many properties of human skin. Because violet light irradiation to photocrosslinkable Az-CH-LA
of the biocompatibility, exudate absorbability, and lm- produced an insoluble hydrogel within 60 s (518). The
forming properties of chitosan products, they are good can- binding strength of the chitosan hydrogel prepared from
didates for burn and wound management uses (509). 3050 mg/mL of Az-CH-LA was similar to that of brin
Chitosan may be used to inhibit broplastia in wound heal- glue. Compared to the brin glue, the chitosan hydrogel
ing and promote tissue growth and differentiation in tissue more effectively sealed air leakage from pinholes on iso-
culture. Chitosan also shows a biological aptitude to stimu- lated small intestine and aorta and from incisions on iso-
late cell proliferation and histoarchitectural tissue organi- lated trachea. Neither Az-CH-LA nor its hydrogel showed
zation (510). any cytotoxicity in cell culture tests of human skin bro-
Earlier, Prudden (511) and Allen and Prudden (512) had blasts, coronar endothelial cells, and smooth muscle cells.
investigated the tricking effect of cartilage preparations in These results suggest that the photocrosslinkable chitosan
accelerating wound healing. The phenomenon was later at- developed here has the potential of serving as a new tissue
tributed to the presence of N-acetyl-D-glucosamine. adhesive in medical use.
Chitosan has been reported as a wound healing acceler- Water-soluble chitin was prepared by controlling de-
ator (513). Cotton ber type chitosan (DA 18%) was gree of deacetylation and molecular weight of chitin
applied on open skin wounds for 15 days, and the process through alkaline and ultrasonic treatment (519). Its acceler-
of wound healing was evaluated histologically and immu- ating effect on wound healing in rats was compared with
nohistochemically. On day 3 postwounding, the chitosan- those of chitin and chitosan. The water-soluble chitin was
treated wounds showed histologically severe inltration found to be more efcient than chitin or chitosan as a
of polymorphonuclear cells. Granulation was more pro- wound-healing accelerator. The wound treated with water-
nounced on day 9 and 15. Immunohistochemical typing of soluble chitin solution was completely re-epithelialized,
collagen I, III, and IV showed increase of the production granulation tissues in the wound were nearly replaced by
of type III collagen in the chitosan group. The appear- brosis, and hair follicles were almost healed at 7 days
ance of mitotic cells occurred numerously in the control after initial wounding.
on postwounding day 3 and in the chitosan group on post-
wounding day 6. These results suggest chitosan has a func- C. Chitosan Use as a Matrix for Drug Delivery
tion in the acceleration of inltration of polymorpho- Systems
nuclear cells at the early stage of wound healing, followed
by the production of collagen by broblasts (513). As a pharmaceutical excipient, chitosan has been added
Chitosan acetate lms, which were tough and protec- for sustained release (520522) and to improve dissolution
tive, had the advantages of good oxygen permeability, high of poorly soluble drugs (523). The applications of chitosan
water absorptivity, and slow lysozyme degradation (514). as an excipient in pharmaceutical products are listed in
Loke et al. (515) have prepared wound dressing con- Table 3.
sisting of two layers: the upper layer a carboxymethyl Kotze et al. (524) showed that chitosan hydrochloride
chitin hydrogel material, while the lower layer is an antimi- and chitosan glutamate are potent absorption enhances in
crobial impregnated biomaterial. The hydrogel layer acts acidic environments. It was concluded that there is a need
as a mechanical and microbial barrier and is capable of for chitosan derivatives with increased solubility, espe-
absorbing wound exudate. From the in vitro release stud- cially at neutral and basic pH values, for use as absorption
ies, the loading concentration was optimized to deliver suf- enhancers aimed at the delivery of therapeutic compounds
cient antimicrobial drug into the wound area to sustain in the more basic environment of the large intestine and
the antimicrobial activity for 24 h. colon.
Chitosan may facilitate wound healing by stimula- The efciency of chitosan in tablets was dependent on
ting granulation tissue formation and re-epithelialization chitosan cristallinity, degree of deacetylation, molecular
(516,517). The 3M company has marketed TEGASORB, weight, and particle size (525,526).

Copyright 2002 Marcel Dekker, Inc.


Table 3 Chitosan as a Pharmaceutical Excipient Table 5 Hydrophobic Counterions for the
Ionotropic Gelation of Chitosan
Conventional formulations:
Gels Polycation Hydrophobic counterions
Films
ChitosanNH2 Octyl sulfate
Emulsions
Lauryl sulfate
Direct compression tablets
Hexadecyl sulfate
Wetting agent
Cetylstearyl sulfate
Coating agent
Controlled release matrix tablets:
Microspheres and microcapsules
Transmucosal drug delivery in globules (Table 4) (528). Using more hydrophobic coun-
Vaccine delivery terions (Table 5) it is possible to prepare hydrophobic car-
DNA delivery
riers (532).

D. Microspheres and Microcapsules


Sabnis et al. (527) studied the potential utility of chito-
san (I) in inhibiting diclofenac sodium (II) release in the Over the past decade, several microsphere drug formula-
gastric environment from a directly compressible tablet tions have matured beyond the stage of promising investi-
formulation. Analyses of variance model was tested using gational agents in preclinical and clinical trials to become
SAS (SAS Institute, Inc., NC) indicated that the degree viable pharmaceutical products approved for widespread
of N-deacetylation of chitosan signicantly affected drug human use. Chitosan microspheres are produced, either
release at pH 1.2 and 6.8 (p 0.0001). An increase in the by an emulsicationcrosslinking process or by use of
pH of the dissolution medium resulted in an increase in complexation between oppositely charged macromole-
drug release (p 0.0001). The ionic strength of the disso- cules. The resulting crosslinked chitosan microspheres
lution medium did not signicantly affect drug release at were characterized by being smooth, spherical, and in the
any of the pHs studied (p 0.198). size range of 45300 m.
Using chitosan solution (pH 6), gel formation will Inuence of chitosan molecular weight on drug load-
occur with a number of different multivalent anionic coun- ing and drug release of drug-loaded chitosan microspheres
terions. By adding a chitosan solution dropwise into a was studied (533). Chitosans of 70,000, 750,000, and
crosslinking solution having pH 6, a real ionotropic gel 2,000,000 molecular weight were employed. Ketoprofen
is formed (528). The NH2 groups of chitosan are proton- (ket) was chosen as the model drug to be encapsulated.
ated and an ionic crosslinking occurs. Prepared chitosan microparticle delivery systems can mod-
Chitosan crosslinked with high molecular weight coun- ulate ket release within 48 h.
terions (529531) results in capsules (Table 4), while A new microparticulate of chitosan controlled-release
crosslinking with low molecular weight counterions results system, consisting of hydrophilic chitosan microcores en-
trapped in a hydrophobic cellulosic polymer, such as cellu-
lose acetate butyrate (CAB) or ethyl cellulose (EC), was
Table 4 Possible Counterions for the Ionotropic Gelation
proposed (534). These microparticles were obtained with
of Chitosan
different types of chitosan and various core/coat ratios,
Polycation Counterions with the particle size in all cases being smaller that 70 m.
ChitosanNH2 Low molecular weight
Using sodium diclofenac (SD) and uorescein isothiocya-
Pyrophosphate nate-labeled bovine serum albumin (FITC-BSA) as model
Tripolyphosphate compounds, the properties of these new microparticles for
Tetrapolyphosphate the entrapment and controlled release of drugs and proteins
Octapolyphosphate were investigated. The microparticles were stable at low
Hexametaphosphate pH and thus suitable for oral delivery without requiring
[Fe(CN)6]4, [Fe(CN)6]3 any harmful crosslinkage treatment.
High molecular weight A new system which combines specic biodegradabil-
Poly(1-hydroxy-1-sulfonate-2-propene) ity and pH-dependent release is presented. The system con-
Poly(aldehydocarbonic acid) sists of chitosan (CS) microcores entrapped within acrylic
Xanthane
microspheres. Sodium diclofenac (SD), used as a model
Pectin
drug, was efciently entrapped within CS microcores us-

Copyright 2002 Marcel Dekker, Inc.


ing spray-drying and then microencapsulated into Eudragit entered the colon, as evaluated by the transit time experi-
L-100 and Eudragit S-100 using an oil-in-oil solvent evap- ments with chitosan capsules. These ndings suggest that
oration method (535). The size of the CS microcores was chitosan capsules may be useful carriers for the colon-
small (1.82.9 m) and they were encapsulated within Eu- specic delivery of peptides including insulin.
dragit microspheres (size between 152 and 233 m) form-
ing a multireservoir system. A combined mechanism of
2. Microspheres and Microcapsules
release is proposed, which considers the dissolution of the
for Cancer Therapy
Eudragit coating, the swelling of the CS microcores and
the dissolution of SD and its further diffusion through the Intravenously administered anticancer drugs are distrib-
CS gel cores. uted throughout the body as a function of the physicochem-
Novel CS and CS/ethylene oxidepropylene oxide ical properties of the molecule. A pharmacologically active
block copolymer (PEO-PPO) nanoparticles and their po- concentration is reached in the tumor tissue at the expense
tential for the association and controlled release of proteins of massive contamination of the rest of the body. The use
and vaccines were studied by Calvo et al. (536). The mech- of microsphere drug carriers could represent a more ratio-
anism of protein association was elucidated using several nal approach to specic cancer therapy.
proteins, bovine serum albumin, and tetanus and diphtheria Kawashima et al. (539) have developed a novel mu-
toxoids, and varying the formulation conditions (different coadhesive DL-lactide/glycolide copolymer (PLGA)
pH values and concentrations of PEO-PPO) and the stage nanosphere system to improve peptide absorption and
of protein incorporation into the nanoparticle formation prolong the physiological activity following oral ad-
medium. ministration. The desired PLGA nanospheres with elca-
tonin were prepared by the emulsion solvent diffusion
method to coat the surface of the resultant nanospheres
1. Microspheres and Microcapsules with Insulin
with a mucoadhesive polymer such as chitosan, poly
As a consequence of poor oral bioavailability and the cur- (acrylic acid), and sodium alginate. The chitosan-coated
rent lack of alternative delivery routes, insulin is presently nanospheres showed higher mucoadhesion to the everted
administered parenterally. The difculties in achieving a intestinal tract in saline than the other polymer-coated
normal physiological prole of insulin by injectable ther- nanospheres.
apy has led to the investigation of alternative, nonparen- Nishioka et al. (540) produced glutaraldehyde cross-
teral routes for the delivery of insulin in an attempt to im- linked chitosan microspheres that contained cisplatin. Sim-
prove glycemic control. An insulin delivery system has ilar microspheres were prepared by Jameela and Jayakrish-
been widely investigated as an alternative to subcutaneous nan (541) containing mitoxantrone. Drug release was
injection for the treatment of diabetes. found to be effectively controlled by the degree of cross-
Bugamelli et al. (537) investigated the production of linking. Wang et al. (542) described an orthogonal experi-
chitosan microparticles containing insulin by interfacial mental design to optimize the formulation of cisplatin
crosslinkage of chitosan solution in the aqueous phase of (CDDP)-loaded chitosan microspheres (namely, CDDP-
a water/oil dispersion in the presence of ascorbil palmitate, DAC-MS), which were produced by an emulsion-chemical
thus permitting covalent bond formation with the amino crosslinking technique. Seven factors and three levels for
group of chitosan when its oxidation to dehydroascorbyl each factor that might affect the formulation of micro-
palmitate takes place during microparticle preparation. spheres were selected and arranged in an L27(3(13)) or-
This preparation method produced microparticles charac- thogonal experimental table.
terized by high loading levels of insulin, completely releas- Chitosan nanoparticles, readily prepared without the
ing the drug in about 80 h at an almost constant release use of organic solvents, are a suitable vehicle for the deliv-
rate. ery of these immunostimulants from Mycobacterium vac-
Tozaki et al. (538) studied the colon-specic insulin de- cae (PS4A); the formulations might nd application as an-
livery with chitosan capsules. A marked absorption of in- titumor agents (543).
sulin and a corresponding decrease in plasma glucose lev- The fate of 166Hochitosan complex, a radiophar-
els was observed following the oral administration of these maceutical drug for cancer therapy, was determined by
capsules that contain 20 IU of insulin and sodium glyco- studying its absorption, distribution, and excretion in
cholate (PA% 3.49%), as compared with the capsules rats and mice (544). To determine the effects of chito-
containing only lactose or only 20 IU of insulin (PA% san in 166Hochitosan complex, 166Ho alone (0.75 mg
1.62%). The hypoglycemic effect started from 8 h after of Ho(NO3)3 5H2O/head) was intrahepatically adminis-
the administration of chitosan capsules when the capsules tered to male rats; and radioactive concentrations in blood,

Copyright 2002 Marcel Dekker, Inc.


urinary and fecal excretion, and radioactive distribution an external or an internal calcium source, and then mem-
were examined. The radioactive concentrations in tissues brane coated with chitosan or poly-L-lysine. Membrane
and the whole-body autoradiography images showed that thickness increased with decreasing polymer molecular
most of the administered radioactivity was localized at the weight and increasing degree of deacetylation of chitosan
administration site, and only slight radioactivity was de- (547,548). Less than 1% of total double-stranded DNA re-
tected from the liver, spleen, lungs, and bones. These re- mained unhydrolyzed within chitosan- or poly-L-lysine
sults strongly suggest that 166Ho is retained at the admin- coated beads, corresponding with an increase in DNA re-
istration site only when it forms a chelate complex with siduals (i.e., double- and single-stranded DNA, poly-
chitosan. Autoradiographs after intratumoral administra- nucleotides, bases). Chitosan membranes did not offer suf-
tion of 166Hochitosan complex showed that radioactivity cient DNA protection from DNase diffusion since all of
was localized at the site of administration without distribu- the double-stranded DNA was hydrolyzed after 40 min of
tion to the other organs and tissues. These results strongly exposure.
suggest that 166Ho is retained at the administration site
only when it forms a chelate complex with chitosan. Auto-
3. Microspheres and Microcapsules
radiographs after intratumoral administration of 166Ho
with Antiinammatory Drugs
chitosan complex showed that radioactivity was localized
at the site of administration without distribution to the An inclusion complex composed of hydrocortisone acetate
other organs and tissues (544). (HC) and hydroxypropyl--cyclodextrin (HPCD) was
The potential of gadolinium neutroncapture therapy prepared by the spray-drying method (549). Hydrocorti-
(Gd-NCT) for cancer was evaluated using chitosan nano- sone acetate alone, HC inclusion complex, or HC with
particles as a novel gadolinium device (545). The nanopar- HPCD as a physical mixture were incorporated into chi-
ticles, incorporating 1200 g of natural gadolinium, were tosan microspheres by spray-drying (549). The HC release
administered intratumorally twice in mice bearing subcuta- rates from chitosan microspheres were inuenced by the
neous B16F10 melanoma. The thermal neutron irradiation drug/polymer ratio in the manner that an increase in the
was performed for the tumor site, with the uence of release rate was observed when the drug loading was de-
6.32 1012 neutrons/cm2 8 h after the second gadolinium creased. However, release data from all samples showed
administration. After the irradiation, the tumor growth in signicant improvement of the dissolution rate for HC,
the nanoparticle-administered group was signicantly sup- with 2540% of the drug being released in the rst hour
pressed compared to that in the gadopentetate solution compared with about 5% for pure HC.
administered group, despite radioresistance of melanoma Mooren et al. (550) investigated the inuence of chito-
and the smaller Gd dose than that administered in past Gd- san microspheres on transport of the hydrophilic, antiin-
NCT trials. This study demonstrated the potential use- ammatory drug prednisolone sodium phosphate (PSP)
fulness of Gd-NCT using gadolinium-loaded nanoparticles across the epithelial barrier. The effect is dependent on the
(545). integrity of the intercellular cell contact zones and the mi-
Hojo et al. (546) have prepared a hybrid of a water- croparticles are able to pass the epithelial layer. Their po-
soluble chitosan and a laminin-related peptide, and have tential benet under inammatory conditions like in in-
examined its inhibitory effect on experimental metasta- ammatory bowel disease, in order to establish high drug
sis in mice. Four methods were tried to achieve a cou- doses at the region of interest, remains to be shown.
pling reaction, the diphenylphosphoryl azide method, the Chitosan microspheres were also used as support for
diisopropylcarbodiimide/1-hydroxybenzotriazole method, the antiinammatory drugs in order to assure the gastroin-
the water-soluble carbodiimide, and the 2-(1H-benzotri- testinal tract protection. In this way the chitosan micro-
azole-1-yl)-1,1,3,3-tetramethyluronium tetrauoroborate spheres covalently linked with citric acid and loaded with
(TBTU) method, but all four methods were unsuccessful. indomethacin are prepared (551). Also, a pharmacokinetic
Therefore, a small spacer, tert-butyloxycarbonyl-Gly, was model of colon-specic drug has been validated by use of
intercalated in chitosan by the TBTU method to facilitate 5-aminosalicylic acid (5-ASA) as a model antiinamma-
its coupling with the peptide. Conjugation of the peptide tory drug (552). The simulation curves obtained from the
with the larger chitosan molecule did not reduce the inhibi- pharmacokinetic model were in good agreement with ex-
tory effect of the peptide on experimental metastasis in perimental data obtained after oral administration of 5-
mice, it actually potentiated the antimetastatic effect, dem- ASAcontaining chitosan capsules. The concentrations of
onstrating that chitosan may be effective as a drug carrier 5-ASA in the large intestinal mucus after drug adminis-
for peptides. tration were higher than after administration of the drug
DNA was immobilized within alginate matrix using in carmellose suspension. These ndings suggest that

Copyright 2002 Marcel Dekker, Inc.


our pharmacokinetic model of colon-specic drug delivery Thin lms of a biopolymer chitosan (CHIT) were cast
can accurately evaluate this colon-specic delivery system on glassy carbon electrodes, modied by grafting Luc-
and that 5-ASAcontaining chitosan capsules are more ef- ifer Yellow VS dye (LYVS) onto chitosan chains, and
fective than other 5-ASA formulations for treatment of co- crosslinked with glutaric dialdehyde (GDI) (568). The ion
litis in rats. transport and ion exchange properties of such polymeric
structures (CHIT, CHIT-LYVS, CHIT-LYVS-GDI) were
4. Microspheres and Microcapsules studied using cyclic voltammetry, rotating disk electrode,
as Local Injection and ow injection analysis. The results showed that the
chitosan matrix supported a fast ion transport as demon-
Local implantation or injection of microspheres containing strated by aqueous-like values of the apparent diffusion
bisphosphonates for site-specic therapy may aid in treat- coefcients of Ru(NH3)6(3) and dopamine in the lms
ing several pathological conditions associated with bone (568). The results indicate that the chemically modied
destruction. Chitosan microspheres containing two antire- chitosan is an attractive new coating for the development
sorption and anticalcication agents, pamidronate and su- of fast, selective, and reversible sensors.
beroylbisphosphonate, were prepared from a water-in-oil Miyazaki et al. (569) and Kristl et al. (565) investigated
emulsion. Various formulation variables were studied for the suitability of dried chitosan gels as vehicles for the
their effect on the release rate prole of these bone-seeking sustained release of the poorly soluble drugs such as indo-
agents (553). Polymer coating of micromatrices yielded methacin, papaverine hydrochloride, and lidocaine. The
microspheres with the most retarded release rate, and the gel showed zero order release into pH 7.4 buffer at 24 h. It
drug delivery system was found biocompatible in endothe- was found that the degree of deacetylation and the chitosan
lial cell culture. Bisphosphonate released from chitosan content were important for the release properties.
microspheres effectively inhibited bioprosthetic tissue cal- The inuence of the acid type used to dissolve chitosan
cication in the rat subdermal model. on the resulting sponge, physical properties, and the conse-
The formation of microcapsules which contain rose- quent effect on the drug liberation were investigated (570).
mary oil is herewith described (554). The process is based Chitosan was dissolved in different acid solutions and
on two steps: (1) formation of oil-in-water emulsions using chitosangelatine sponges were produced by frothing up
lecithin as emulsier, thus imparting negative charges on the polymer solution and then freeze-drying the foam. Pred-
the oil droplets and (2) addition of a cationic biopolymer, nisolone was used as a model drug. Using tartaric or citric
chitosan, in conditions that favor the formation of an insol- acid resulted in unstable, soft, elastic, and disintegrating
uble chitosanlecithin complex. sponges with fast drug release. Elastic but harder sponges
from stable foams were obtained when hydrochloric or lac-
E. Gels tic acid were used. The use of acetic or formic acid enabled
the production of stable foams and soft and elastic sponges,
Hydrogels formed from chitosan are usually covalently allowing low drug release. The rate of drug release was
crosslinked. Crosslinking agents such as glutaraldehyde decreased by crosslinking the polymers with glutaralde-
(555557), glyoxal (558), and diethyl squarate (559) have hyde. Therefore, it is possible to manipulate the mechani-
been employed in the fabrication of chitosan hydrogels. cal properties and the drug liberation rate by using differ-
Chitosan has also been covalently crosslinked with the ent acids to dissolve chitosan (570).
carbohydrate sceleroglucandialdehyde (560). In addition A biodegradable drug delivery system of a gentamicin-
semi-interpenetring polymer network hydrogels have been loaded chitosan bar with sustained antibiotic effect is de-
produced between chitosanpolyethylene oxide diacrylate scribed (571). Combined crosslinking, solvent evapora-
crosslinked by UV irradiation (561), glyoxal crosslinked tion, and a cylinder model cutting technique was used to
chitosanpolyethylene oxide (562), and glutaraldehyde prepare the chitosan bar. Sustained diffusion of gentamicin
crosslinked chitosan combined with silk broin (563) or into the surrounding medium was seen using a release test
polyacrylic acid (564). in vitro. Approximately 11% gentamicin was released
Noncovalent crosslinking has been used to prepare from the bar in the rst 24 h. The gentamicin released from
chitosan-based gels by the O- and N-acetylation of chito- the bar showed signicant antibacterial activity. The bar
san (565) and the attachment of C10-alkyl glycosides to implanted in the proximal portion of the rabbit tibia pro-
chitosan (566). By the attachment of hydrophobic palmi- duced a low blood concentration of gentamicin, but a much
toyl groups to glycol chitosan, a water-soluble chitosan higher concentration was produced in local bone and in
derivative has been produced (567). the hematoma. In all bone tissue around the bar, the genta-

Copyright 2002 Marcel Dekker, Inc.


micin concentration exceeded the minimum inhibitory polymers gelled as the temperature was raised from 4 to
concentration for the common causative organisms of os- 37C and provided signicant extension of drug release.
teomyelitis for approximately 8 weeks (571). Based on
these test results together with the chitosan characteristics F. ChitosanPolyelectrolyte Complexation
of biodegradable, antibiotic, and immunologic activity, the
gentamicin-loaded chitosan bar seems to be a clinically Polymer complexes are formed by the association of two
useful method for the treatment of bone infection. This or more complementary polymers and may arise from elec-
system has an advantage over other systems in that it trostatic forces, hydrophobic interactions, hydrogen bond-
avoids a second operation for removal of the carrier. ing, van der Waals forces, or combinations of these inter-
Chitosanethylene diamine tetracetic acid (EDTA) con- actions (579583). The formation of complexes may
jugate gels have also been reported in which the carboxylic strongly affect the polymer solubility, rheology, conduc-
acid group of EDTA are covalently linked to the amino tivity, and turbidity of polymer solutions (583). Hydrogen
groups of chitosan (572,573). A recently developed chito- bonds are distinctly directional and specic and are more
sanEDTA conjugate, neutralized with sodium hydroxide localized than any other type of weak intermolecular inter-
(NaChito-EDTA), has been tested for possible topical use action (584). Many polymer complexes form as a result of
(571). This antimicrobial activity of NaChito-EDTA can electrostatic forces. Polymeric acids may form complexes
be explained by its highest binding afnity toward mag- by proton transfer to complementary polymeric bases, re-
nesium, which stabilizes the outer membrane of gram- sulting in poly(cation)/poly(anion) pairs. In addition to the
negative bacteria. interpolymer complexes, intrapolymer complexes of poly-
New mucoadhesive polymers, exhibiting a high capac- ampholytes have been studied (585587). In these systems
ity to bind bivalent cations that are essential cofactors for complexes may be formed between oppositely charged
intestinal proteolytic enzymes, were synthesized (574). moieties on the same polymer chain, or even on the same
Under the formation of amide bonds, the complexing agent monomer units. Finally, simple polyelectrolytes in solution
EDTA was covalently bound to the primary amino groups may be linked together by multivalent ions to form gels or
of chitosan (574,575). Whereas proteolytic activity of the coacervates (588,589). The complex stability is dependent
serine proteases trypsin, -chymotrypsin, and elastase upon such variables as charge density, solvent, ionic
could not be inhibited, proteolytic activity of the zinc pro- strength, pH, and temperature. Polyelectrolytes of high
teases carboxypeptidase A and aminopeptidase N was charge density may form relatively insoluble complexes.
strongly inhibited by the chitosanEDTA conjugate. The Characteristics of the macromolecule complexes are
adhesive force of the conjugate was even higher than of largely based on their higher molecular weight, which are
chitosan HCl. called polymer effects, such as cooperative interactions,
Berberine (the main ingredient of Coptis spp.) was in- concerted interactions, steric compatibility, and microenvi-
corporated into chitosan hydrogel to prepare ointments. ronmental effects (590).
The physicochemical properties of the ointments and the Gel technology based on ionic crosslinked polysaccha-
release prole of berberine were investigated. The results rides is currently in use and of considerable interest for
indicated that the viscosity of chitosan hydrogel increased potential application in the extraction with solvents (591),
with an increasing amount of lactic acid or EDTA (576). as controlled release systems (592,593), as mechanochemi-
The release rate of berberine was inversely proportional to cal devices (594), in the oil industry (595597), and also
ointment viscosity. in several other biomedical applications (598,599).
The effect of heparin ionically linked to chitosan on the The binding of chitosan to alginate beads was studied
stimulation of re-epithelialization of full thickness wounds quantitatively by using radioactive-labelled fractions of
in human skin was investigated in an in vitro model (577). chitosan (600). The binding of chitosan was markedly in-
After 7 days of incubation, heparinchitosan gel stimu- creased by reducing the number average molecular weight
lated 9/10 of the full thickness wounds to re-epithelialize of chitosan below 20,000 Da and by increasing the porosity
compared with only 3/10 of the wounds that were covered of the alginate gel. The porosity was increased by produc-
with chitosan gel or membrane; and none of the wounds ing homogeneous gels, and by adding calcium chloride to
incubated without gel or membrane or with heparin solu- the chitosan solution during the membrane-forming stage.
tion alone. The binding of chitosan was also found to increase with
One approach taken by Hoffman et al. (578) was to decreasing fraction of N-acetylations (FA) on chitosan, in
synthesize hybrid copolymers by grafting temperature- the range of FA 0.3 to FA 0, and with increasing pH,
responsive polymers (Pluronic) to chitosan backbones. The in the range of pH 4 to 6. Capsules with a diameter of 500

Copyright 2002 Marcel Dekker, Inc.


m had a higher weight ratio of chitosan to alginate after sules with high mechanical strength were obtained after
24 h of binding than the capsules with the larger diameter shorter reaction times when the number average molecular
of 1500 m. weight of the chitosan was reduced to around 15,000 Da,
To prepare spray-dried composite particles containing when the capsules were made more homogeneous, and
alginatechitosan complex [SD(L/AL-CS)], an aqueous when the capsule diameter was reduced to around 300 m
solution of lactose and sodium alginate and the acetic acid (602).
solution of chitosan were concomitantly fed into the rotary Noncovalent crosslinked chitosan gel mixtures can be
atomizer of a spray-dryer (601). The drug release proles prepared by the use of polyelectrolyte complexes of chito-
of dry-coated tablet with SD(L/AL-CS) indicated a long san and polyanions such as carboxymethylcellulose (603)
induction period followed by a rapid drug release phase in and xanthan (604,605).
the articial intestinal uid. The induction period for drug Gelatine forms polyionic complexes with chitosan at
release to occur was increased with an increase in the de- the suitable pH value (606). These complexes show a
gree of deacetylation of chitosan and in the amount of chi- slower rate of dissolution than chitosan at the correspond-
tosan in the formulation. The prolongation of induction pe- ing pH. Therefore a combination of chitosan and gelatine
riod was attributed to the formation of an insoluble ion in a sponge will rst lead to an absorption of the wound
complex between sodium alginate and chitosan in the com- uid and second slowly release a wound-treating drug
posite particles, which could form a rigid gel structure on (607,608). The release prole and biodegradability of both
the tablet surface. polymers can be effectively controlled by glutaraldehyde
The stability of alginatechitosan capsules was shown crosslinking (609,610).
to depend strongly on the amount of chitosan bound to The complexation reaction between the polyions leads
the capsules. When the capsules were made by dropping to structural changes in both polymers, xanthan and chito-
a solution of sodium alginate into a chitosan solution (one- san. A water-insoluble hydrogel is formed by blocking the
stage procedure), all the chitosan was located in a thin hydrophilic functions (RCOOH in case of polyanions
alginatechitosan membrane on the surface. These cap- and RNH2 for the polycations) and by the interaction
sules were much weaker than the capsules made by re- between the macromolecular chains (Fig. 1). The latter ef-
acting calcium alginate beads in an aqueous solution of fect created an ionic reticular network that immobilized
chitosan and calcium chloride (two-stage procedure). Cap- the polymers. The hydrogel has the property of incorporat-

Figure 1.1 The complexation reaction between xanthan and chitosan.

Copyright 2002 Marcel Dekker, Inc.


ing a signicant amount of structural water as well as the
capacity of stabilizing, in its matrix, biological products
(611615).
Recently, we have studied the complexation reaction
between xanthan and chitosan by determining the yields
and the structure of the complex as well as its swelling
capacity. The complexation efciency readily reaches 90%
of the available chitosan and the less deacetylated chitosan
(DA 34%) forms a stable hydrogel having the highest
chitosan/xanthan ratio. In fact, the lower the DA, the
higher the amount of xanthan needed to form a stable hy-
drogel. Additionally, we have studied the inuence of the
pH of the chitosan solution on the yield of complexation
with xanthan. The results are shown in Table 6. We notice
a poor complexation at low pH due to strong ionization Figure 1.2 Evaluation of chitosanxanthan complex stability
of the amine groups, which impedes its reaction with the at different pH conditions. Time: 48 h; buffer solution concentra-
carboxylic groups present in the xanthan. Similar results tion: 0.2M.
have been reported for the complexation of chitosan
with carboxymethyl cellulose (CMC) and alginic acid
(616,617). sage that the gels are quite porous and that formation of
The decomposition of the chitosanxanthan hydrogel brilar structures takes place. The channels present in the
depends on the pH and the ionic concentration of the buffer gels have a pore size between 107 and 106 m (0.11 m),
solution. In the pH range between 1.2 and 9.2 the hydrogel whereas the brils have a typical dimension of 107 m (100
is stable no matter the ionic concentration present (Fig. 2). nm). The surface of the microsphere has a homogeneous
The swelling characteristics and the water content of porous structure (Fig. 3b), which allows the passage of
the hydrogel are shown in Table 7, from which we can polymeric substrates to the regions where the immobilized
notice that water constitutes, in all cases, the largest weight enzymes are lodged.
concentration of the hydrogel. The large swelling capacity Transmission electron microscope (TEM) images of the
of these hydrogels does not lead to their disintegration hydrogels are shown in Fig. 4. Since the preparation of
when prepared as microspheres, as in the case for the the samples for TEM inevitably leads to modications of
xanthanCMC complex (616). the sample, care has to be exercised in the interpretation
Scanning electron microscope (SEM) images of the hy- of the micrographs. Figure 4 shows the presence of loose
drogels are shown in Fig. 3 for typical gel samples (24.25% aggregates of brils probably resulting from the rupture
chitosan, 75.75% xanthan). The images convey the mes- of the elaborate network, observed via scanning electron
microscopy (SEM), due to the drying and cutting proce-
dures used. The aggregates are formed of ber fragments
Table 6 Inuence of the pH of the
having typical diameters of 50 to 100 nm in agreement
Chitosan Solution on the Yield of Its
Complexation with Xanthan

pH Xanthan complexation yield (%) Table 7 Inuence of the Molecular Weight of Chitosan on
1.5 21 the Absorption of Water by the ChitosanXanthan Hydrogel
2.5 36 Degree of Complexation max
3.5 56 Chitosan Mna acetylation (%) yield (%) (%)
4.5 60
5.5 98 691,930 28.0 98.9 2560
6.3 82 452,875 27.9 97.2 1805
191,325 28.2 84.5 992
Note: Ratio CH/X 0.65 g/g. Mn chitosan 122,350 28.8 81.3 457
691,390, determined by measuring the intrinsic
viscosity. [] 1.81 103 M0.93, determined Note: pH 5.0; buffer acetate 0.2 M.
in 0.1 M acetic acid/0.2 M NaCl solution at a
Determined by intrinsic viscosity [] 1.81 103 M0.93, in 0.1 M
25C. acetic acid/0.2 M NaCl solution at 25C.

Copyright 2002 Marcel Dekker, Inc.


(a)
Figure 1.4 Transmission electron microscope images of typical
gels (24.25% chitosan, 75.75% xanthan) 48,000.

an immune effector molecule has been also demonstrated


in murine models (622). The TNF-, IL-, and NO secre-
tion as markers for macrophages activation were assessed.
The CH-X complex did not show a cytotoxic effect re-
gardless of degradation time periods and concentration
used (Fig. 5) (623).
Production of TNF- by macrophages was stimulated
by CH-X particles for the concentration of 1 mg/mL. This
effect was more evident at higher concentration (10
mg/mL). Production of IL-1 was enhanced after macro-
phage incubation with CH-X particles, while the concen-
tration did not affect the secretion level (Fig. 6). Our study
(b) demonstrated clearly that macrophages incubated with

Figure 1.3 Scanning electron microscope images of typical


gels (24.25% chitosan, 75.75% xanthan). (a) Image of external
surface, 30,000; (b) image of internal section, 60,000.

with the SEM observations. A concentration of material


near the external surface of the sample is also evident. The
open spaces (pores) are quite developed toward the cen-
ter of the sample.
Evaluation of their bicompatibility was carried out with
L929 broblasts cell line to determine the cytotoxicity and
with J774 macrophages cell line to assess the inammatory
response. Cytokines and nitric oxide (NO) are also secreted
by the macrophages as indicators of the inammatory re-
sponse, and they are known to induce other cells such as
T lymphocytes to proliferate and synthesize proteins and
additional factors which, in turn, enhance macrophage acti- Figure 1.5 Effect of CH-X particles on L-929 cell viability par-
vation (618621). Nitric oxide production and efcacy as ticles (direct contact).

Copyright 2002 Marcel Dekker, Inc.


Figure 1.6 Effect of CH-X particles on IL-1 secretion.

CH-X particles were activated to produce IL-1. The size Figure 1.8 TEM micrograph showing broblast adhering along
of the particles could be not phagocytosable. CH-X particles (P) after 8 weeks of implantation in rat subcutane-
Secretion of TNF- and NO (Fig. 7) is inuenced in ous tissue.
the same way by CH-X extract products with a slight sensi-
tivity for TNF- secretion.
Macrophages also play an important role in the phago- model used indicated a phagocytosis of the CH-X particles
cytosis of the material and its degradation in vivo. The by macrophages, which can lead to the entire resorption
inammatory reaction is essential for preparing the wound of the hydrogel.
for the production of a new extracellular matrix. Cytokines Shioya et al. (616) developed the encapsulation method
and growth factors released by the inammatory cells at- using chitosan. The encapsulation is based on the electro-
tract the broblasts into the wound to initiate the recon- static interactions of chitosan as a polycation with sodium
struction process. In vivo studies based on light micros- carboxymethyl cellulose as a polyanion. The objective of
copy observations showed a recruitment of specic cell this investigation was to identify the factors which control
populations responsible for the preparation of wound re- the transmembrane permeability of the capsules prepared
pair and deposition of new matrices. Their presence is with chitosan and CMC (616). The permeability slightly
shown in Fig. 8, and the presence of collagen attests to the decreases with time. Longer reaction times did not improve
healing process (Fig. 9). Degradation study on the animal

Figure 1.9 LM micrograph (156 ) showing dense brosis for-


mation with blood vessels (arrows) and inammatory reaction
Figure 1.7 Effects of CH-X extract products on NO2 secretion around CH-X particles (P) after 2 weeks implantation in rat sub-
by J-774 macrophages. cutaneous tissue.

Copyright 2002 Marcel Dekker, Inc.


the capsule membrane (616). The permeability increases phoric acid gel beads using a polyelectrolyte complexa-
with an increase in the salt concentration. Adding salt may tion method for the sustained release of anticancer agent,
cause a reduction in the effective charge by a shielding 6-mercaptopurine (627). The complexation mechanism of
effect (623) and then prevent the chitosan from reacting chitosan beads gelled in pentasodium tripolyphosphate or
with CMC. In parallel with permeability, the capsule mem- polyphosphoric acid solution was ionotropic crosslinking
branes formed by chitosan with salt also have lower me- or interpolymer complexation, respectively. The drug re-
chanical strengths because of a lower density of interpoly- lease patterns of all chitosan gel beads in pH 6.8 seemed
mer bridge. The lower molecular weight of chitosan may to be diffusionally based, whereas release proles in pH
give a more compact membrane structure, thus lower per- 1.2 medium seemed to be non-Fickian diffusion controlled
meability. due to the swelling or matrix erosion.
The equilibrium adsorption rates of chitosan on oxi- Insulin-loaded chitosan nanoparticles were prepared by
dized cellulose substrates were determined and are plotted ionotropic gelation of chitosan with tripolyphosphate
as a function of carboxylic acid group content (624). The anions (628). The ability of chitosan nanoparticles to en-
increase in absorption with increase in carboxyl group con- hance the nasal absorption of insulin was investigated in
tent may arise from two effects (624): a conscious rabbit model by monitoring the plasma glucose
levels. Chitosan nanoparticles enhanced the nasal absorp-
1. Increased electrostatic interaction between poly-
tion of insulin to a greater extent than an aqueous solution
anion (oxidized cellulose) and polycation (chi-
of chitosan. The amount and molecular weight of chitosan
tosan)
did not have a signicant effect on insulin response. In
2. Increased substrate surface area due to increased
vivo absorption studies in animals have been carried out
swelling on immersion in water because of increase
to evaluate the nasal absorptionpromoting activity of chi-
in the hydrophilic character of the substrate (oxi-
tosan using insulin as a model peptide (629630).
dized cellulose) with increase in the extent of modi-
cation
F. Chitosan as Scaffolds
The addition of NaCl has two opposing effects on the ab-
sorption of chitosan on oxidized cellulose (624). At low It is known that glycosaminoglycans are involved in cell
sodium chloride concentration, the dominant effect is re- cell and cellmatrix interactions, and probably act as
duction of the hydrodynamic volume of the chitosan mole- modulators of cell morphology, differentiation, movement,
cules leading to an increase in the equilibrium adsorption. synthesis, and function (631633). Chitosan, having struc-
At higher sodium chloride concentration, competition be- tural similarity to glucosaminoglycans, was modied using
tween Na ions and cationic groups on the chitosan for several proteins (collagen, albumin, and gelation) to in-
the carboxylic acid groups on the cellulose predominates, crease surface area and improve biocompatibility. In vitro,
leading to a gradual decrease in the equilibrium adsorption collagen-blended chitosan matrices were found to attach
of chitosan (624). more readily to chromafn cells than gelatine- or albumin-
Remunan-Lopez and Bodmeier (625) investigated opti- blended matrices (634). Morphological evidence showed
mal conditions for the complex between chitosan and gela- that the chromafn cells attached to collagen-blended chi-
tine. All of the optimal conditionspH, polymer ratio, tosan substrate integrated well with the hydrogel matrix
polymer concentration, temperature, reaction time, and and survived for at least 2 weeks under in vivo conditions.
ionic strengthwere essentially coincident with chitosan Collagen-blended chitosan substrates produce signicantly
polysaccharide complexes. improved bovine adrenal medullary chromafn cell attach-
Glycol chitosan (GC) and methyl glycol chitosan ment characteristics, which can be considered for neural
(MGC) were, individually, reacted with potassium meta- tissue engineering studies. Morphological examinations re-
phosphate (MPK) to form a series of water-insoluble vealed that the chromafn cells survived for at least 2
macromolecular complexes (MC) in aqueous solution at weeks with collagenchitosan scaffolds.
different hydrogen ion concentration (GC-MPK and MGC- The effectiveness of chitosan as a scaffold of hepatocyte
MPK) systems (626). These coagulation curves indicate attachment was examined (635). Since chitosan gel was
the dependence of hydrogen ion concentration on the mole too fragile to use for cell culture, its free amino groups
ratio of the reacting group of MPK to that of GC, GC were crosslinked by glutaraldehyde to increase its strength.
CaCl2, or MGC in the reaction mixture at the beginning Rat hepatocytes seeded onto glutaraldehyde-crosslinked
and end of the coagulation. chitosan (GAchitosan) gel could attach with stability to
Enzymatic-hydrolyzed chitosan was employed to pre- the surface, retaining its spherical form, the same as in
pare chitosantripolyphosphate and chitosanpolyphos- vivo, and then release a very small amount of lactate dehy-

Copyright 2002 Marcel Dekker, Inc.


drogenase during the 5-day culture period. By contrast, he- Scanning electron microscopy of the cellular lattices
patocytes on a collagen-coated surface spread at, and they showed bers of the collagenchitosan mixture to be the
released much more lactate dehydrogenase than those on thickest and with altered organization. These results show
the GAchitosan gel. Hepatocytes on GAchitosan also that chitosan sulfation markedly enhances broblast adhe-
retained higher urea synthesis activity, a liver-specic sion and promotes contraction of a collagen lattice com-
function, than those on the collagen-coated surface. These pared to the unsulfated material. By analogy to the in vivo
results indicate that chitosan is a promising biopolymer as sequence of hyaluronan followed by sulfated glycosami-
a scaffold of hepatocyte attachment which can be applied noglycans in wounds, the results suggest that glycosami-
to an effective bioarticial liver support system. noglycan sulfation may be a contributing signal for pheno-
The quality of articulate cartilage engineered using a typic transformation during wound healing (638).
cellpolymer construct depends, in part, on the chemical
composition of the biomaterial and whether that biomate- G. Chitosan-Based VectorDNA Complexes
rial can support the chondrocytic phenotype. Acknowledg- for Gene Delivery
ing the supportive inuence of tissue-specic matrix mole-
cules on the chondrocytic phenotype, Sechriest et al. (636) Chitosan is a candidate nonviral vector for gene delivery.
combined chondroitin sulphate-A (CSA) and chitosan, a Therefore, formation of DNA particles with chitosan (639)
glycosaminoglycan (GAG) analog, to develop a novel bio- or chemically modied chitosan (640), such as N,N,N-
material to support chondrogenesis. Chitosan may be com- trimethylchitosan, has been investigated for gene trans-
bined with the polyanionic CSA such that ionic crosslink- fer. Successful chitosan-mediated transfection has been re-
ing results in hydrogel formation. Bovine primary articular ported for a few cell types, such as HEK 293 cells (639).
chondrocytes, when seeded onto a thin layer of CSA Erbacher et al. (641) have found chitosan presents some
chitosan, form discrete, focal adhesions to the material characteristics favorable for gene delivery, such as the abil-
and maintain many characteristics of the differentiated ity to condense DNA and to form homogeneous population
chondrocytic phenotype, including round morphology, of complexes, smaller than 100 nm, in dened conditions.
limited mitosis, and collagen type II and proteoglycan pro- Moreover, chitosan could easily be chemically modied
duction. by coupling ligands, such as lactose, in order to target cells
With an aim of improving bone regeneration, chito- expressing a galactose-binding membrane lectin. With the
san sponge containing platelet-derived growth factor-BB aim of developing a chitosan-based vector system, various
(PDGF-BB) was developed. For fabrication of chitosan biophysical characteristics of chitosan-condensed DNA
sponge, chitosan solution was freeze-dried, crosslinked, complexes were measured, and several transfection experi-
and freeze-dried again. PDGF-BB was incorporated into ments mediated by chitosan and lactosylated chitosan were
the chitosan sponge by soaking chitosan sponge into the performed (642).
PDGF-BB solution. Release kinetics of PDGF-BB, cell at- In order to achieve an efcient gene delivery via
tachment, proliferation capacity, and bony regenerative receptor-mediated endocytosis, the synthesis of a novel
potentials of PDGF-BBloaded chitosan sponge were in- chitosan derivative having recognizable saccharide resi-
vestigated (637). Release rate of PDGF-BB could be dues, N,N,N-trimethylchitosangalactose conjugate, was
controlled by varying initial loading content of PDGF-BB performed (643). The formation of a polyelectrolyte com-
to obtain optimal therapeutic efcacy. PDGF-BBloaded plex with DNA and the cellular recognition of N,N,N-
chitosan sponge induced signicantly high cell attachment trimethylchitosangalactose conjugate were tested, and
and proliferation levels, which indicated good cellular then the possibility of its application as a gene delivery
adaptability. PDGF-BBloaded chitosan sponge demon- tool was investigated.
strated marked increase in new bone formation and rapid Chitosan is a polysaccharide that demonstrates much
calcication. Degradation of the chitosan sponge was pro- potential as a gene delivery system. The ability of a com-
ceeded at defect site and subsequently replaced with new mercially available chitosan and depolymerized chitosan
bone. Histomorphometric analysis conrmed that PDGF- oligomers to condense plasmid was determined using TEM
BBloaded chitosan sponge signicantly induced new and microtitration calorimetry, while the diameter and sta-
bone formation. These results suggested that chitosan bility of the resultant complexes were measured using laser
sponge and PDGF-BBloaded chitosan sponge may be light scattering (644). A selected complex containing a
benecial to enhance periodontal bone regeneration (637). lytic peptide was administered in the upper small intestine
The ability of cultured human foreskin broblasts to and colon of rabbits, and reporter gene expression was
bind and to contract lattices of collagen, collagenchito- measured in dened intestinal tissues. Reporter gene ex-
san, and collagenchitosan sulfate was determined (638). pression was enhanced in dened intestinal tissues, al-

Copyright 2002 Marcel Dekker, Inc.


though levels of expression remained low. The combina- systems. For peroral peptide delivery these tasks focus on
tion of strong complex stability and low in vivo expression overcoming the absorption (I) and enzymatic barrier (II)
levels suggest that uptake and/or decomplexation, but not of the gut. On the one hand, even unmodied chitosan
endosomal release, may be the critical rate-limiting steps proved to display a permeation-enhancing effect for pep-
in the uptake process. tide drugs. On the other hand, a protective effect for poly-
Many cationic polymers (645649) have been explored mer embedded peptides toward degradation by intestinal
as nonviral vectors. Highly puried chitosan fractions of peptidases can be achieved by the immobilization of en-
5000 Da (N1), 500010,000 Da (N2), and 10,000 Da zyme inhibitors on the polymer. Whereas serine proteases
(N3) were prepared and characterized in respect of their are inhibited by the covalent attachment of competitive in-
cytotoxicity, ability to cause hemolysis, ability to complex hibitors such as the Bowman-Birk inhibitor, metallopepti-
DNA, as well as ability to protect DNA from nuclease deg- dases are inhibited by chitosan derivatives displaying com-
radation (650). ChitosanDNA interaction at a charge of plexing properties such as chitosanEDTA conjugates. In
1:1 was much greater than seen for poly(L-lysine), and addition, because of the mucoadhesive properties of chito-
complexation resulted of DNA degradation by DNase II: san and most of its derivatives, a presystemic metabolism
99.9 0.1, 99.1 1.5, and 98.5 2.0% for N1, N2, of peptides on the path between the dosage form and the
and N3, respectively. After intravenous injection, all the absorption membrane can be strongly reduced. Based on
chitosans showed rapid blood clearance. The observations these unique features, the co-administration of chitosan
that the highly puried chitosan fractions used were neither and its derivatives leads to a strongly improved bioavail-
toxic nor hemolytic, that they have ability to complex ability of many perorally given peptide drugs such as insu-
DNA and protect against nuclease degradation and that lin, calcitonin, and buserelin. These polymers are therefore
low molecular weight chitosan can be administered intra- useful excipients for the peroral administration of peptide
venously without liver accumulation suggest there is po- drugs (653).
tential to investigate further low molecular weight chito- To increase chitosans utility, it has been N-derivatized
sans as components of a synthetic gene delivery system by phosphonomethylation (PM-chitosan) or by carboxy-
(650). methylation (CM-chitosan). Both derivatives disolve eas-
Hydrophobically modied chitosan containing 5.1 ily in water at neutral conditions. Moreover, these de-
deoxycholic acid groups per 100 anhydroglucose units was rivatives maintain the benecial cosmetic properties of
synthesized by an EDC-mediated coupling reaction. For- chitosan (654).
mation and characteristics of self-aggregates of hydropho- Chitosan succinate and phthalate matrices showed pH-
bically modied chitosan were studied by uorescence dependent release proles of the entrapped diclofenac so-
spectroscopy and dynamic light scattering method (651). dium (655). Maximum drug release was observed under
Charge complex formation between self-aggregates and pH 7.4, in contrast to pH 2 at which the matrices resisted
plasmid DNA was conrmed by electrophoresis on an dissolution.
agarose gel. A simple carbohydrate polymer glycol chitosan (degree
Mao et al. (652) describe a new immunoprophylactic of polymerization approximately 800) has been investi-
strategy using oral allergen gene immunization to mod- gated for its ability to form polymeric vesicle drug carriers.
ulate peanut antigeninduced murine anaphylactic re- The attachment of hydrophobic groups to glycol chitosan
sponses. Oral administration of DNA nanoparticles synthe- should yield an amphiphilic polymer capable of self-
sized by complexing plasmid DNA with chitosan resulted assembly into vesicles. Chitosan is used because the
in transduced gene expression in the intestinal epithelium. membrane-penetration enhancement of chitosan polymers
Oral allergen gene immunization with chitosanDNA na- offers the possibility of fabricating a drug delivery system
noparticles is effective in modulating murine anaphylactic suitable for the oral and intranasal administration of gut-
responses, and indicates its prophylactic utility in treating labile molecules. Glycol chitosan modied by attachment
food allergy. of a strategic number of fatty acid pendant groups (1116
mol%) assembles into unilamellar polymeric vesicles in
H. Chitosan Derivatives the presence of cholesterol. These polymeric vesicles are
found to be biocompatible and hemocompatible and capa-
In the 1990s chitosan turned out to be a useful excipient ble of entrapping water-soluble drugs (656). These poly-
in various pharmaceutical formulations. By modications meric vesicles efciently entrap water-soluble drugs.
of the primary amino group at the 2 position of this poly Investigations of chitosan citrate as a hydrocolloidal
(1 4-D-glucosamine), the features of chitosan can even matrix material (657659) and as the wall of a mcrocap-
be optimized according to a given task in drug delivery sule (660) have been reported.

Copyright 2002 Marcel Dekker, Inc.


A chitosan with lauryl groups attached to amino groups glu-MMC hardly exhibited any tumor growth inhibition,
to provide the hydrophobic moieties and carboxymethyl but N-suc-chitosan-glu-MMC showed signicant tumor
groups attached to hydroxy groups to provide the hydro- growth suppression. As to the intratumoral administration,
philic moieties [N-lauryl-carboxymethylchitosan (LCC)] the tendency to suppress tumor growth was observed in
was newly synthesized. The solubility of taxol in LCC mi- MMC and both the conjugates.
celles in aqueous solution was examined (661). It was
found that LCC solubilized taxol by forming micelles with J. Sterilization of Chitosan
particle sizes less than 100 nm. This particle size was con-
sidered effective for passive targeting for tumors. The con- Chitosan has potential biomedical applications that may
centration of taxol in the micellar solution was very high, require the nal products to be sterilized before use. The
with a maximum of 2.37 mg/mL. gamma irradiation of puried and highly deacetylated chi-
tosan bers and lms at sterilizing doses (up to 25 KGB)
I. Macromolecular Produgs Based on Chitosan caused main chain scissions. The viscosity average molec-
ular weight of the polymer decreased with increasing irra-
5-Fluorouracil (5FU) has remarkable antitumor activity. In diation dose (666). Irradiation in anoxia did not affect lm
order to provide a polymeric prodrug of 5FU with reduced properties signicantly.
side effects, having afnity for tumor cells, and exhibiting
high antitumor activity, four kinds of chitosan derivatives
were synthesized carrying 5FU through some kinds of
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2

Biomimetics

Weiyuan John Kao


University of Wisconsin, Madison, Wisconsin

I. INTRODUCTION methodologies have been employed as an engineering tool


to develop novel material processing methods such as cal-
Current biomaterial research efforts mainly focus on eluci- cium phosphate coatings on metal alloys (26) and poly(L-
dating the specic interaction among material physico- lactic acid)co-apatite composites (27), to formulate bio-
chemical properties, proteins/enzymes and various biolog- functional materials for biomedicine and bioengineering
ical molecules, observed cellular functions, and such as synthetic peptide amphiphiles (28) and polymers
physiological parameters such as mechanical forces in derived from molecular and microbiology (2931), and to
vitro and in vivo (110). Based on these investigations, continue to be utilized in probing the molecular fundamen-
biomaterial developments have begun to utilize the diver- tals and mechanisms of biology.
sity and uniqueness of biology for potential methods to Although the biomimetic approach has been employed
control biological behavior of a myriad of tissue engi- by various scientic disciplines to develop novel mole-
neering and biomedical applications. As the result of this cules and materials, there is not a consensus in the deni-
novel approach and new technology, the eld of biomi- tion of biomimetics that is accepted by various science
metic materials emerges. However, the root of biomimetics communities, namely, engineering, chemistry, medicine,
can be traced to the early 1970s when functional articial and life sciences. Nonetheless, the semantic denition is
molecules were designed and synthesized by mimicking the ability to camouage a substance with suitable contriv-
the functional structure and synthesis pathway of natural ances similar to the environmental background with the
compounds. For example, biomimetic methodologies were aim to elude a hostile observer. However, such a denition
employed to selectively synthesize antileukemic triptolides is insufcient and narrow. Hence, the author argues that
(11), cyclical polyenes such as racemic 11-hydroxy- in the biological and engineering arena, the application of
progesterone (1214), camptothecin chromophores (15), mimetism should be broad and hence dynamic and encom-
catechol estrogens (16), polyketone-derived phenols (17), passing. The author will attempt to address the wide spec-
D-glucose-derived ()-biotin (18), and much more. In the trum of biomimetics in this chapter by highlighting se-
1980s and early 1990s, the eld of biomimetics expanded lected research investigations in which the knowledge of
dramatically to such areas as antitumor pharmaceutics biological sciences has been adopted as a template in the
(1921), the development of receptor agonists and antago- study and development of novel molecules and materials.
nists (22,23), the study of functional structures and synthe- The author will emphasize the complementary interrela-
ses of proteins and nucleotides (24), and the elucidation tionship between biomimetic research and the pursuit of
of biological pathways such as enzyme catalysis (25). In understanding the complex biological system. Biological
the late 1990s and into the new millennium, biomimetic systems fundamentally function at the angstrom scale of

Copyright 2002 Marcel Dekker, Inc.


molecular atoms, the nano scale of macromolecules, the chrome-c, the low molecular weight metalloporphyrins
micron scale of cells and protein matrices, and the macro catalyzed the dismutation of O 2 with a rate constant of
scale of tissues, organs, and macroorganisms (32). The 10 7 10 8 /M/s. The k cat value for the multifunctional chi-
uniqueness of biological materials and systems is the meta- mera of metalloporphyrins and poly(styrene-co-maleic an-
stable microstructure formed from the molecular to mac- hydride) was found to be 1.1 0.1 10 6 /M/s at 8.1 pH
roscale in order to attain a combination of required proper- in vitro. In vivo studies demonstrated that the chimera con-
ties (i.e., physical, chemical, biological, structural, etc.) for struct of metalloporphyrins and poly(styrene-co-maleic an-
optimal function. Hence, the intricate micro- and macroar- hydride) bound to the warfarin site of albumin and showed
chitecture and multifunctional properties of biological sys- an enhanced half-life circulation time; while the SOD ac-
tems are utilized as foundations in the investigation of bio- tivity was retained.
mimetic materials. By studying the hierarchical structure In the design of biofunctional molecules to study and
of biological systems and biological constituents at all pos- modulate host cell behavior (34), Kao et al. have chosen
sible scales of spatial resolution, the fundamentals of the a family of active proteins, i.e., interleukin-1 (IL1), as a
unique structural designs can be deduced and mimicked model in the design of biofunctional molecules. IL1 is a
by currently available techniques. Alternatively, the mo- potent proinammatory cytokine that upregulates cellular
lecular synthesis and processing mechanism of biological function upon ligation with IL1 receptor type I (IL1RI) on
systems and biological components can be elucidated and the extracellular membrane (35). IL1-activated macro-
applied to develop novel materials (32). Both strategies phages (a primary class of phagocytic leukocytes) may re-
embody the principle and the dynamic nature of biomime- lease high levels of IL1, tumor necrosis factor , and
tism. Investigations adopting these broad research princi- granulocyte/macrophagecolony stimulating factor (GM-
ples will be presented in this chapter as illustrative exam- CSF), which all have important roles in the healing process
ples of biomimetic macromolecules and novel biomaterials (1,4,9,36). Circulating IL1 receptor antagonist (IL1ra) is
with potential biomedical values. the natural antagonist for IL1. IL1 ligates with Domain
1, 2, and 3 of IL1RI and initiates signaling pathways to
upregulate cellular functions (37); whereas, IL1ra only
II. BIOFUNCTIONAL MOLECULAR binds with Domain 1 and 2 of IL1RI, resulting in no postli-
BIOMIMETICS gation signal transduction (38). It has been proposed that
IL1 complexes with Domain 3 of IL1RI by a strong elec-
From the beginning of biomimetic research in the led of trostatic interaction. Kao et al. designed macromolecules
chemistry, pharmaceutical sciences, and biochemistry, based on the functional architecture of IL1 and IL1ra.
bioactive molecules have been synthesized by studying the Antagonists were designed from known IL1 and IL1ra
native functional structure and natural synthesis pathways amino acid residues showing strong avidity toward Do-
of model molecules. This approach continues to this day. main 1 and 2 of IL1RI (35,3739). These residues are R (4)
Of particular interest to biomedicine is the investigation on L(6) F (46) I (56) K (103) E (105) of IL1 and W (17) Q (21) Y (35) Q (37)
superoxide dismutase (SOD) enzymes to catalyze oxygen Y (148) of IL1ra (35,3740). Strong evidence suggests that
radicals for the management of chronic inammation, ex- these amino acids are essential in ligandreceptor interac-
cessive oxidative metabolism, and tumor (1921). For ex- tion. The tertiary structures of the native IL1 and IL1ra
ample, hemodialysis polymer membranes may activate the molecule in solution were utilized as a guide in determin-
phagocyte oxidative metabolism resulting in the highly ox- ing the spatial interrelationship between each residue. The
idized low-density lipoprotein found in uremic patients. minimum distance between each residue was approxi-
Superoxide dismutase eliminates the highly reactive and mated using the structural coordinates archived in the
hence destructive superoxide radicals and participates in SwissProt Database. Based on the measurement, a poly-
the oxygen radical detoxication process. However, the glycine sequence of approximately the same length was
native SOD molecule is highly unstable and has low bio- utilized to link each amino acid at all possible orientations.
availability and high immunogenicity. The biomimetic ap- Kao et al. further hypothesized that the resulting antagonist
proach to develop molecules with SOD activity was rst sequences, which target Domains 1 and 2 of IL1RI, can
pioneered by Yamamoto et al. (21) and continued by Ohse be converted to agonists by coupling a strong electrostatic
and Nagaoka, who demonstrated that the reaction of vari- moiety (i.e., poly-lysine) to the terminal amino acid of the
ous metalloporphyrins and poly(styrene-co-maleic anhy- antagonist sequence, thus allowing the complexation be-
dride) copolymers resulted in the formulation of novel chi- tween the peptide macromolecule and Domains 1, 2, and
meras with pH sensible sites and SOD-like active sites 3 of IL1RI to occur. To formulate agonists, a trimeric gly-
(Fig. 1) (33). Using in vitro assay systems based on cyto- cine linker was utilized to join the antagonist domain with

Copyright 2002 Marcel Dekker, Inc.


Figure 2.1 Structure of poly(styrene-co-maleic anhydride)co-metalloporphyrin derivatives with superoxide dismutase activity.
(Adopted from Ref. 33.)

the poly-lysine domain. The trimeric glycine linker was the presence of autologous serum in the culture medium.
designed to introduce spatial exibility between the poly- After 2 h, nonadherent cells were removed and adherent
lysine sequence and the antagonist oligopeptide domain cells were challenged with free peptides at an optimal con-
since chain mobility may impact the dynamics of ligand centration of 50 pmol/mL that had been determined previ-
receptor association. Consequently, a library of linear oli- ously. Simultaneously, adherent cells were also challenged
gopeptides were formulated and synthesized using solid- with or without recombinant human IL1 or IL1ra at 25
resin methods with standard 9-uorenylmethyloxycarbo- pmol/mL. Peptides with scrambled amino acid sequences
nyl chemistry (41). Peptides thus formulated were desig- were also employed as peptide controls. Cells were cul-
nated as follows. Antagonists included those modeled after tured thereafter in the presence of autologous serum and
the IL1 molecule: (IL1iant) RGGLGGFGGIGKGGEG, supernatants were collected at various time points for
(IL1iiant) FGRGGLGGGIGKGGEG, and (IL1iiiant) assay. At 4 h after the peptide challenge, no differences in
LGGRGFGGIGKGGEG; those modeled after the IL1ra GM-CSF release by adherent macrophages were observed
molecule: (IL1ivant) WGGGQGGYGGQGGGYG and among all test samples and controls. At 18 h after the free
(IL1vant) WGGYGGQGGYGGQG; and that modeled peptide challenge, IL1-treated adherent macrophages on
after antagonists developed via combinatorial chemistry tissue-culture polystyrene (TCPS) showed a higher GM-
(40): (IL1viant) YWQPYALPL. Agonists included those CSF release than controls without treatment (Fig. 2a). Ad-
modeled after the IL1 molecule: (IL1i) KKKGGGRGGL herent macrophages treated with agonist IL1v showed a
GGFGGIGKGGEG, (IL1ii) KKKGGGFGRGGLGGGIG higher GM-CSF release than that treated with IL1, IL1ra,
KGGEG, and (IL1iii) KKKGGGLGGRGFGGIGKGGEG; none, and other agonists. It is known that IL1 upregulates
those modeled after the IL1ra molecule: (IL1iv) KKKGGG GM-CSF release by human macrophages via the induction
WGGGQGGYGGQGGGYG and (IL1v) KKKGGGWGG of AP-1 and NFB gene expression factors. The result sug-
YGGQGGYGGQG; and those modeled after antagonists gests that IL1v activates these gene expression factors re-
developed via combinatorial chemistry (40): (IL1vi) KKK sulting in an increased GM-CSF production. The GM-CSF
GGGYWQPYALPL and (IL1vii) YWQPYALPLGGGK level of cells treated with IL1v remained comparable when
KK. Human blood monocytederived macrophages were IL1ra was added simultaneously as the IL1v peptide indi-
allowed to adhere on tissue culture polystyrene for 2 h in cating that the effect of IL1v in increasing GM-CSF release

Copyright 2002 Marcel Dekker, Inc.


lease of GM-CSF by adherent macrophages. Recombinant
IL1 and IL1ra proteins have been investigated for their
potential therapeutic values. For example, IL1 augments
hematopoiesis and increases wound healing; whereas,
IL1ra attenuates host inammatory reaction. However, the
clinical value of these regimens has yet to be demonstrated
due to high cost, low bioavailability, deleterious side ef-
fects of the high dosage that is often required, and the pres-
ence of natural agonists, antagonists, and serum proteases.
Hence, IL1-derived agonists and antagonists are currently
under extensive investigation by pharmaceutical compa-
nies and basic science research laboratories to improve the
therapeutic value of native IL1 and IL1ra moieties (42
44). IL1-derived agonists have demonstrated potential in
increasing hematopoietic stem cell proliferation, increas-
ing B cell proliferation, enhancing local wound healing,
and modulating collagenase production. IL1-derived an-
tagonists have shown promise in decreasing the extent of
acute and chronic inammation in diseases such as rheu-
matoid arthritis, treating septic shock, suppressing chronic
leukemia, and modulating bone resorption by osteoclasts.
These two examples illustrate the uniqueness of a biomi-
metic approach to develop synthetic molecules derived
from naturally occurring precursors such as SOD and IL1
family proteins. These biomimetic molecules demonstrate
enhanced biochemical and physical properties that can be
employed to study biological systems and potentially im-
prove clinical medicine.

Figure 2.2 The release of GM-CSF by adherent human blood-


derived macrophages (normalized to adherent macrophage den- III. NOVEL POLYMERIC MATERIALS
sity) on tissue-culture polystyrene after 18 h incubated with (a) CONTAINING PEPTIDE/PROTEIN
biomimetic agonists derived from IL1-family proteins with or
BIOMIMETICS
without recombinant human IL1ra (* represents p 0.05 versus
values of none control; # represents p 0.05 versus values
of IL1 control; IL1v values were larger than that of IL1i, IL1ii, Currently, several laboratories have taken the biomimetic
IL1iii, IL1vi, and IL1vii at p 0.05); or (b) biomimetic antago- approach to develop novel biomaterials containing biologi-
nists derived from IL1-family proteins with or without recombi- cally derived functional moieties to modulate specic cel-
nant human IL1 (* represents p 0.05 versus values of none lular behavior in a variety of physiological systems such
control; # represents p 0.05 versus values of IL1 control; no as cardiovascular, orthopedic, and nervous. The overall
differences at p 0.95 were found among each test group indi- goal of these investigations is to obtain fundamental under-
cating that the GM-CSF release mediated by antagonists was not standing of the complex biologicalmaterial interaction;
neutralized by the presence of IL1). All values mean s.e.m., the knowledge is necessary for the construction of tissue
n 4. engineered devices and novel therapeutic biomedical prod-
ucts. Hubbell and West have focused on the study and de-
velopment of enzymatically degradable hydrogels that are
by adherent macrophages was not neutralized in the pres- responsive to the local host environment (45,46). For ex-
ence of the natural IL1 antagonist, namely, IL1ra. No sig- ample, West et al. utilized known protease substrate pep-
nicant differences in the GM-CSF release were observed tides (i.e., sequence LGPA from collagenase, sequence
between macrophages without treatment and those treated AAA from elastase, or sequence NRV from plasmin) co-
with both IL1 and one of the antagonists (Fig. 2b). These polymerized with polyethyleneglycols using dicyclohexyl-
results indicate that the IL1-derived biomimetic antago- carbodiimide-based chemistry to formulate BAB (peptide
nists neutralized the ability of IL1 in increasing the re- polyethyleneglycolpeptide) block copolymers. The BAB

Copyright 2002 Marcel Dekker, Inc.


copolymers were reacted with acryloyl chloride to provide of adherent macrophage activation and fusion to form mul-
photopolymerizable end groups. ABA (polyethylenegly- tinucleated foreign body giant cells (FBGC) is unique to
colpeptidepolyethyleneglycol) block copolymers were the macrophage phenotype. The presence of FBGCs is uti-
formed by reacting the peptide with two amine groups such lized as a histopathology marker for chronic inammation
as lysine residues at the C terminus with polyethylenegly- and the host foreign body reaction. Foreign body giant
colN-hydroxysuccinimide monoacrylate. Cell adhesive cells have been demonstrated on implanted biomaterials
peptides such as RGDs were also reacted with polyethyl- and the rate of material degradation underneath the giant
eneglycolN-hydroxysuccinimide monoacrylate and were cells has been shown to be markedly increased (50). How-
subsequently grafted into the photopolymerized ABA hy- ever, the molecular mechanisms involved in FBGC forma-
drogel network. Hydrogel networks were formulated via tion remain unclear. Activated macrophages may release
photopolymerization with block copolymers, triethanol- a variety of cytokines, growth factors, and other bioactive
amine, and 2-2-dimethoxy-2-phenyl acetophenone photo- agents to modulate the function of other cell types in the
initiator in 1-vinyl-2-pyrrolidone (47). After the BAB inammatory milieu (1). Several macrophage-derived cy-
(peptidepolyethyleneglycolpeptide)-containing hydro- tokines and growth factors are utilized on a limited basis
gels were hydrated to equilibrium, type IV collagenase, to alter the inammatory response and promote the healing
porcine pancreatic elastase, or plasmin at various concen- process. For example, IL1 and GM-CSF are given to in-
trations was added to the hydrogel to commence degra- jured patients and bone marrow transplant recipients to as-
dation. Hydrogels were found to degrade in a protease- sist the recovery of hematopoiesis (4244). Pharmaceutics
specic and dose-dependent fashion that increased with based on the property, function, and structure of anti
increasing treatment time. When the ABA (polyethylene- tumor narcosis factor are administered to patients suffer-
glycolpeptidepolyethyleneglycol)-containing hydrogel ing from chronic inammation in order to enhance the
network grafted with the RGDs cell adhesion mediating wound healing process and attenuate inammation (44).
motif was cultured with human dermal broblasts, cells However, the long-term clinical efcacy of treatments in-
were found to adhere and migrate throughout the hydrogel volving the systemic administration of these bioactive
matrix. Since the polymer construct degrades enzymati- agents has yet to be demonstrated. Hence, the localized
cally in the presence of a specic enzyme substrate, the delivery of selected cytokines and growth factors produced
degradation path within the hydrogel can be controlled by endogenous inammatory cells is desirable and may
spatially and temporally by varying the hydrogel chemical have potential therapeutic value in the fundamental pro-
composition. These bioresponsive materials allow the cesses of tissue healing, growth regulation, and biocompat-
study of cell migration mechanisms and function. Similar ibility.
methodologies are currently been utilized by Hubbell et Before the ability to control specic cellular function
al. to investigate and to partly control the biomolecular via biomaterials can be realized, a more detailed under-
mechanism of neurite extension and migration within a standing of the interplay between material-bound ligands
similar polyethyleneglycol-containing, bioresponsive hy- and receptors on the cell surface must be obtained. Further-
drogel system immobilized with enzymatically degradable more, the redundancies that exist between receptors and
peptides (48,49). target proteins must also be addressed. For instance, the
In the development of biofunctional materials by mim- tripeptide RGD sequence is found in several extracellular
icking ligandreceptor interactions, a clear understanding matrix proteins such as bronectin, vitronectin, and -
of the functionstructure relationship between target pro- brinogen. Several cell membrane receptors on different
teins and cell membrane receptors is crucial. Furthermore, types of cells have been shown to complex with the RGD
the ultimate challenge of eliciting specic cellular function cell adhesion motif of these matrix proteins. For example,
by utilizing biofunctional materials lies within the normal platelet surface glycoprotein IIb/IIIa recognizes the RGD
host foreign body response, which might overcome the sequence of brinogen, and integrin 5 1 and IIb 3 recep-
bioactive functionalities in the material intended to modu- tors on macrophages also recognize the RGD motif of -
late cellular function. The major function of blood-derived bronectin. Other integrin receptors on a variety of cell
phagocytic monocytes and macrophages is to mediate the types that recognize the RGD cell binding motif are
normal host immune and inammatory response against (2,3,4,5,7,8,v) 1 and v (1,3,5,6,8) . Some of the current biomate-
foreign objects such as microorganisms and biomaterials rial development and research utilize the RGD sequence
(1). Several characteristic macrophage functions are identi- to provide bioactive sites for cell adhesion. However, it is
ed as critical events in the foreign body response. Macro- apparent that such a design strategy to elicit a specic cel-
phages recognize adsorbed proteins on the foreign surface lular function is limited by the redundancies that exist be-
via several ligandreceptor superfamilies (4). The process tween receptors and target proteins.

Copyright 2002 Marcel Dekker, Inc.


A clear elucidation of the mechanism involved in the (55) and RGD (56), which are located in the FIII-9 and the
interaction among cells, proteins/enzymes, materials, and FIII-10 modules, respectively, of the central cell binding
various physiological parameters such as mechanical domain (Fig. 4) and the sequence PRRARV of the C termi-
forces is critical for the development of biomimetic materi- nal heparin binding domain of bronectin (53) were cho-
als with specic cellular activity in the new eld of cellular sen for exploration. RGD and PHSRN are present on adja-
engineering (Fig. 3). The author acknowledges the critical cent loops of two connecting FIII modules and bind
role of this type of fundamental research at the molecular synergistically to the same integrin receptor (57). The hep-
level between cells and material surfaces in the develop- arin binding domain of bronectin in which the sequence
ment of novel enabling technologies. The resulting tech- PRRARV resides also binds directly with integrin recep-
nologies and know-how are vital for the future of tissue tors (58,59); however, the precise mechanisms involved in
engineered products, the improvement of clinical biomate- this association remain unclear. The tertiary structure of
rials, and the construction of biofunctional materials. bronectin (60,61) was used as a guide in the design of
Several methodologies were utilized to examine the in- peptides that included RGD and PHSRN. The distance be-
terrelationship between material chemistry and cellular be- tween these sequences was approximated using the struc-
havior at protein and cellular levels under in vitro and in tural coordinates archived in the SwissProt Database
vivo environments (110). Based on these ndings, Kao (sequence FINC HUMAN P02751). Based on the mea-
et al. designed methodologies to probe the molecular surement, a hexamer of glycine (G 6 ) of approximately the
mechanism of receptorligand recognition and postliga- same length was used to link the two bioactive sequences
tion cellular function (51,52). In one of the studies, Kao in both possible orientations. The combination of RGD and
et al. designed and employed biomimetic oligopeptides to PRRARV was studied using a G 6 linker; although in this
probe the effect of primary and tertiary structures of a case the G 6 linker was not selected based on any structural
model protein (i.e., bronectin) in ligandreceptor interac- considerations. A terminal trimeric glycine domain (G 3 )
tion (52). Synthetic peptides were formulated based on the was employed as a spacer in all peptides. Oligopeptides
primary and tertiary structures of human plasma bronec- were synthesized using solid-resin methods with standard
tin (53,54). Specically, the amino acid sequences PHSRN 9-uorenylmethyloxycarbonyl chemistry (41). The follow-
ing oligopeptides were synthesized: G 3 RGDG, G 3 PHSR
NG, G 3 PRRARVG, G 3 RGDG 6 PHSRNG, G 3 PHSRNG 6 R
GDG, G 3 RGDG 6 PRRARVG, and G 3 PRRARVG 6 RGDG.
A cyclic RGD peptide with an amino acid sequence of
LNQEQVSPD(cRGDGRN) was utilized for comparison.
LNQEQVSPD is a leader sequence and the cyclical
RGDGRN sequence has been shown to bind with v 3 in-
tegrin with high specicity and afnity (62). Peptides were
immobilized onto a polymer network terpolymerized with
polyethyleneglycol, acrylic acid, and trimethylolpropane-
triacrylate (51,52). The network had been shown to medi-
ate low levels (30 cells/mm 2 ) of adhesion by human
macrophages, human neonatal foreskin dermal broblasts,
and human umbilical vein endothelial cells in serum-
containing culture medium up to 10 days. Macrophage ad-

Figure 2.3 Current research focuses on elucidating the complex


mechanisms in the interaction among cells, proteins/enzymes,
materials, and various physiological parameters such as mechani-
cal forces. Fundamental research at the molecular level results in
the development of novel enabling technologies that are criti-
cal for the development of biomimetic materials with specic cel-
lular activity in the new eld of cellular engineering. Manipu- Figure 2.4 Amino acid sequences PHSRN and RGD (in ball-
lation of cellular function is vital for the future of tissue- and-stick model) located in the FIII-9 and the FIII-10 modules,
engineered products, the improvement of clinical biomaterials, respectively, of the central cell-binding domain of human plasma
and the pursuit of novel biofunctional materials. bronectin.

Copyright 2002 Marcel Dekker, Inc.


hesion and FBGC formation in vitro assays (63) were per- Table 2.2 Macrophage-Derived FBGC Adherent Density and
formed. Under the FBGC culture condition described, Size on Polymer Networks Grafted with Fibronectin-Derived
FBGCs containing up to 50 nuclei/cell formed consistently Biomimetic Peptides at 240 h of the FBGC Assay
on tissue-culture polystyrene control surfaces. Briey, Average FBGC
freshly isolated human blood-derived monocytes were in- Peptide FBGC/mm 2 Size (100 m 2 )
cubated with samples in RPMI containing 20% autologous
serum. At 96 and 168 h, the medium was changed to RPMI G 3 RGDG 18 9 21 8
G 3 PHSRNG 14 9 27 9
with heat-inactivated autologous serum 10 ng/mL of
G 3 PRRARVG 15 9 17 4
recombinant human interleukin-4 5 ng/mL of recombi-
G 3 RGDG 6 PHSRNG 16 7 19 5
nant human GM-CSF. Each adherent cell with three or G 3 PHSRNG 6 RGDG 88 38 a 32 13
more nuclei per cell was dened as a FBGC. Competitive G 3 RGDG 6 PRRARVG 14 7 20 8
inhibition studies utilizing soluble free peptides and neu- G 3 PRRARVG 6 RGDG 17 9 19 7
tralizing antibodies were performed to ascertain ligand LNQEQVSPD(cRGDGRN) 20 11 18 5
receptor specicity and identication. Kao et al. (52) found Tissue-culture polystyrene 60 20 162 38
that serum bronectin modulated macrophage adhesion
Note: All values expressed in mean s.e.m., n 3 to 6.
and the extent (i.e., size) of FBGC formation on control a
Values comparable ( p 0.95) versus that of tissue-culture polystyrene
surfaces in the presence of serum proteins. Macrophages controls.
adhered to all peptide-grafted polymer networks with rela-
tively subtle differences between adhesion mediated by
RGD, PHSRN, PRRARV, or combinations thereof (Table in the presence of serum proteins (Table 2) (52). This re-
1) (52). 1 integrin subunit was essential in macrophage sponse was highly dependent upon the relative orientation
adhesion to peptide-grafted networks in a receptorpeptide between RGD and PHSRN. PRRARV alone or in tandem
specic manner; whereas, 3 integrin subunit was less im- with RGD in a single peptide formulation did not support
portant. Macrophage adhesion to PRRARV was mediated FBGC formation. Furthermore, neutralizing antibody was
primarily by the direct interaction with integrins. RGD or utilized in the FBGC culture assay to partly determine the
PHSRN alone did not provide an adequate substrate for role of integrins and bronectin-derived oligopeptides in
macrophage fusion to form FBGCs. However, the PHSRN modulating the function of adherent macrophages to fuse
synergistic site and the RGD site in a single oligopeptide and form FBGCs. Antibody isotype negative controls were
provided a substrate for FBGC formation that was statisti- also utilized for conrmation. Both integrin 3 and 1 sub-
cally comparable to that on the positive control material units were found to play an importance role in mediating
FBGC formation by macrophages adhered on peptide-
grafted networks (Table 3) (52). Results showed that
Table 2.1 Adherent Macrophage Density on Polymer no antibody and anti- 3 neutralizing antibodytreated
Networks Grafted with Fibronectin-Derived Biomimetic
groups had a comparable FBGC density on TCPS (Table
Peptides
3). On most of the peptide-grafted network, no FBGC for-
Culture time (h) mation was observed in the anti- 3 antibodytreated
group. One notable exception was the peptide that contains
Peptide 24 168
the PHSRN and the RGD domain in the optimal orienta-
G 3 RGDG 378 59 a
155 83 a tion, namely G 3 PHSRNG 6 RGDG, on which antiintegrin
G 3 PHSRNG 243 30 a,b 121 70 a 3 reduced FBGC formation by about 70%. When anti- 1
G 3 PRRARVG 304 46 a,b 121 48 a neutralizing antibody was utilized, no FBGC formation
G 3 RGDG 6 PHSRNG 271 28 a,b 92 25 a was observed on any sample. Macrophages and FBGCs
G 3 PHSRNG 6 RGDG 309 34 a,b 97 29 a
express 2 , 4 , 1 , and 3 integrin subunits. It is also
G 3 RGDG 6 PRRARVG 311 39 a,b 88 30 a
G 3 PRRARVG 6 RGDG 151 24 a,b 62 24 a,b
known that the rst 160 amino acids of integrin 1 , spe-
LNQEQVSPD(cRGDGRN) 290 83 a,b 220 140 a cically the -helix formed by residues 141 to 160, are
No grafted peptides 69 37 b 24 14 b critical in integrinligand recognition. These ndings sug-
Tissue-culture polystyrene 400 49 a 131 22 a gest that the association between this region of the integrin
1 receptor and G 3 PHSRNG 6 RGDG, but not G 3 RGDG 6 P
Note: All values 10 macrophage/mm 2, mean s.e.m., n 3 to 5.
HSRNG, results in the necessary binding characteristic
a
Value different at 95% condence level (p 0.05) versus no grafted
peptide controls.
that determine the subsequent cellular event leading to
b
Value different at 95% condence level ( p 0.05) versus tissue-culture FBGC formation. Activated integrin 1 or 3 intracellular
polystyrene controls. domains stimulate cell migration, modulate proliferation

Copyright 2002 Marcel Dekker, Inc.


Table 2.3 Macrophage-Derived FBGC Adherent Density on Polymer Networks
Grafted with Fibronectin-Derived Peptides at 240 h of FBGC Culture

No antibody Anti-integrin Anti-integrin


Peptides treatment a 3 treatment 1 treatment
G 3 RGDG 18 9 0 0 0 0
G 3 PHSRNG 14 9 0 0 0 0
G 3 PRRARVG 15 9 0 0 0 0
G 3 RGDG 6 PHSRNG 16 7 0 0 0 0
G 3 PHSRNG 6 RGDG 88 38 30 30 0 0
G 3 RGDG 6 PRRARVG 14 7 0 0 0 0
G 3 PRRARVG 6 RGDG 17 9 0 0 0 0
LNQEQVSPD(cRGDGRN) 20 11 0 0 0 0
Tissue-culture polystyrene 60 20 90 30 0 0

Notes: All values expressed in FBGC/mm 2, mean s.e.m., n 3 to 6. Cells treated with
anti-integrin antibody. To determine the role of integrins in mediating FBGC formation, neu-
tralizing antibody [antihuman integrin 1 (JB1a, puried IgG isotype ascite) or antihuman
integrin 3 (25E11, puried IgG2a isotype ascite) neutralizing antibody at 60 g/mL] was
added to the culture at 96 and 168 h of the FBGC assay.
a
All values were signicantly lower ( p 0.05) than that of tissue-culture polystyrene and
networks grafted with G 6 PHSRNG 6 RGDG.

and gene expression, induce the assembly of F-actin cy- shown to upregulate these selected signaling molecules un-
toskeleton, and localize the activity of focal adhesion ki- der a variety of ligandreceptor associations. For example,
nase pp125 FAK. These cellular events may contribute to the Src is involved in integrin signaling upon ligation with ex-
process of FBGC formation; however, the exact interrela- tracellular matrix proteins, such as bronectin, brinogen,
tionship between ligandreceptor architecture and associa- or vitronectin, leading to macrophage adhesion and focal
tion in activating intracellular signaling events resulting in adhesion kinase formation. Treated cells were incubated
the control of cellular behavior remains unclear. with tissue-culture polystyrene or networks terpolymerized
To yield insights into the mechanisms coordinated by with polyethyleneglycols, acrylic acid, and trimethylolpro-
the interaction between integrins and bronectin in mediat- panetriacrylate. Tissue-culture polystyrene- and polyethyl-
ing macrophage adhesion and FBGC formation, Kao et al. eneglycol-based networks were preadsorbed or immobi-
utilized the aforementioned biomimetic bronectin- lized with recombinant human bronectin or bronectin-
derived oligopeptides to probe the structurefunction char- derived biomimetic oligopeptides containing RGD and/or
acteristic and signaling pathways of bronectinintegrin PHSRN domains. Surfaces without preadsorption nor im-
association in modulating cellular function. Specically, mobilization were employed as negative surface controls.
the key role played by RGD, PHSRN, and the spacing and Inhibitor vehicle was also utilized as additional controls,
orientation between the peptide sequences in modulating and bovine serum albumin was employed as controls for
macrophage and FBGC behavior was investigated. Freshly bronectin and peptides. From cell adhesion assays, the
isolated human blood-derived monocytes were preincu- results showed the following. On networks immobilized
bated with inhibitors of various signaling molecules at sev- with bronectin-derived oligopeptides, macrophage adhe-
eral concentrations to screen candidate signaling cascades sion was found to be independent of protein tyrosine ki-
in regulating macrophage behavior mediated by bronec- nases and protein serine/threonine kinases at 24, 48, and
tin and bronectin-derived biomimetic oligopeptides. The 120 h, except that on surfaces grafted with G 3 PHSRNG
signaling and transcriptional events and the corresponding and G 3 PHSRNG 6 RGDG where cell adhesion was depen-
inhibitor chosen for exploration included activated protein dent upon protein serine/threonine kinases at 48 h (Table
tyrosine kinases inhibitor AG82, lavendustin A, which in- 4). On networks immobilized with bronectin or albumin,
hibits Src-family kinases, activated protein serine/threo- macrophage adhesion was found to be dependent of pro-
nine kinases inhibitor H-7, protein kinase-A inhibitor 14- tein tyrosine kinases but not Src and dependent of protein
22 amide, PI-3K inhibitor wortmannin, PCK inhibitor serine/threonine kinases but not protein kinase-A at 24 and
EGF-R fragment, MAPK inhibitor PD98059, and NFB 48 h. Furthermore, the promotion of protein serine/threo-
inhibitor PSI. Activated integrin receptors have been nine kinases, and specically protein kinase-C, compen-

Copyright 2002 Marcel Dekker, Inc.


Table 2.4 Macrophage Adhesion on Polymer Networks and TCPS Immobilized with Proteins or Fibronectin-Derived
Oligopeptides in Vitro

24 h 48 h 120 h

Networks/TCPS Control AG82 H7 Control AG82 H7 Control AG82 H7


Fibronectin 11 5 4 2a 3 1a 5 3 1 1a 1 1a 8 6 2 0 2 0
Albumin 11 3 5 3a 4 1a 5 3 1 1a 2 1a 6 4 3 1 2 1
G 3 RGDG 6 3 3 1 2 0 3 2 2 1 1 0 7 6 2 2 1 0
G 3 PHSRNG 6 4 3 1 2 1 4 1 1 1 1 0a 7 5 3 0 1 1
G 3 RGDG 6 PHSRNG 5 3 2 1 2 1 6 2 2 1 2 1 5 2 1 1 1 1
G 3 PHSRNG 6 RGDG 6 5 3 1 2 1 3 1 2 1 1 1a 6 4 2 0 0 0
TCPS 10 6 6 3 4 2 7 3 5 3 3 2a 4 1 3 1a 1 1
TCPS bronectin 12 4 7 2a 6 4a 10 3 4 2a 4 3a 5 2 3 1 2 0
TCPS albumin 8 4 7 4 5 2 11 4 3 2a 2 1a 5 1 3 2 1 0

Note: All values expressed in macrophage 100/mm 2, mean sem, n 3 to 6.


a
p 0.1 versus respective values of controls. AG82 inhibits PTK and H7 inhibits PSK. Tissue-culture polystyrene (TCPS).

sated protein tyrosine kinases inhibition in mediating mac- styrene was found to be independent of protein tyrosine
rophage adhesion at 24 h (Table 5). However, macrophage and serine/threonine kinases by 120 h. Assays for FBGC
adhesion on networks immobilized with bronectin or al- demonstrated the following. On networks immobilized
bumin was found to be independent of both protein tyro- with bronectin, FBGC formation was dependent of pro-
sine and serine/threonine kinases at 120 h. On tissue- tein serine/threonine kinases, specically protein kinase-
culture polystyrene immobilized with bronectin, A, but was independent of Src (Table 6). It should be noted
macrophage adhesion was found to be dependent of pro- that macrophage adhesion was independent of protein
tein tyrosine kinases but not Src and dependent of protein kinase-A by 48 h and was independent of both protein tyro-
serine/threonine kinases but not protein kinase-A at 24 h. sine and serine/threonine kinases by 120 h. On tissue-
The promotion of protein serine/threonine kinases, and culture polystyrene immobilized with bronectin, FBGC
specically protein kinase-C, did not compensate protein formation was found to be independent of both protein ty-
tyrosine kinases inhibition in mediating macrophage adhe- rosine and serine/threonine kinases. Macrophage adhesion
sion at 24 and 48 h. These results indicate that the crosstalk on tissue-culture polystyrene immobilized with bronectin
between protein tyrosine and serine/threonine kinases is was independent of protein tyrosine and serine/threonine
different between adherent macrophages on bronectin kinases by 120 h. Taken together, these results support the
that was immobilized onto networks or tissue-culture poly- role of activated protein tyrosine kinases and serine/threo-
styrene. Furthermore, macrophage adhesion on bronec- nine kinases in integrin signalings leading to macrophage
tin-immobilized tissue-culture polystyrene was found to be adhesion mediated by bronectinintegrin association.
dependent of protein tyrosine kinases but not Src and de- Furthermore, RGD and PHSRN appears to be signicant
pendent of protein serine/threonine kinases, specically in mediating this receptorligand association resulting in
protein kinase-C but not protein kinase-A, by 48 h. Cell the necessary signaling characteristic for macrophage ad-
adhesion on bronectin-immobilized tissue-culture poly- hesion and the subsequent development. Specically, pro-

Table 2.5 Macrophage Adhesion on Networks and TCPS Immobilized with Proteins in Vitro

2h 24 h

Networks/TCPS Control AG82 H7 AG82 H7 AG82 PMA Control AG82 H7 AG82 H7 G82 PMA
Fibronectin 16 5 10 7 a
85 a
50 a
21 a
11 5 4 2 3 1 a a
3 1a 51
TCPS 12 8 11 1 13 6 41 62 10 6 6 3 4 2 5 1a 83
TCPS FN 14 6 9 3 11 2 3 1a 4 1a 12 4 7 2 a 6 4 4 0a 5 1a

Note: All values expressed in macrophage 100/mm 2, mean sem, n 3 to 6.


a
p 0.1 versus respective values of controls. AG82 inhibits protein tyrosine kinases, H7 inhibits protein serine/threonine kinases, and PMA promotes
protein kinase-C of protein serine/threonine kinases.

Copyright 2002 Marcel Dekker, Inc.


Table 2.6 FBGC Formation on Networks and TCPS Immobilized
with Fibronectin by 240 h of FBGC Assay in Vitro

Networks/TCPS Control Lavendustin A PKI


Fibronectin 39 16 21 4 26 17 a
TCPS 25 18 23 15 23 16
TCPS bronectin 95 72 17 13

Note: All values expressed in FBGC/mm 2, mean sem, n 3 to 6.


a
p 0.1 versus respective values of controls. Lavendustin A inhibits Src of
protein tyrosine kinases and PKI inhibits protein kinase-A of protein serine/
threonine kinases.

tein kinase-C of tyrosine kinases showed a signicant role IV. PROTEIN MATRIX AND
in modulating FBGC formation mediated by bronectin, SUBCELLULAR BIOMIMETICS
most importantly the RGD and PHSRN domains of bro-
nectin. Kao et al. showed the important role of RGD and In previous sections, examples were given to illustrate the
PHSRN domains of bronectin, and specically the inter- utilization of macromolecules and proteins as templates to
positional spacing between the motifs, in the complexation develop biomimetic molecules and novel materials. How-
with integrin receptors to upregulate tyrosine kinases and ever, biological systems are composed hierarchically;
serine/threonine kinases in mediating macrophage adhe- hence, the impact of biological organization at the submi-
sion and FBGC formation. These ndings represent a cron and subcellular scales needs further elucidation. For
mechanistic correlation between the role of protein func- example, most cells are intimately associated with base-
tional architectures in ligandreceptor recognition and the ment membrane matrices that have a complex three-
postligation signaling events that control cellular behavior. dimensional nanoarchitecture of 100500 nm features.
The fundamental understanding of these complex phenom- Hence, this submicron architecture may play an important
ena provides future researchers with necessary tools in the role in mediating cell adhesion and function. A point of
development of unique biomimetic enabling technologies note, cellular focal adhesion sites are about 250 nm in size.
that are vital for the advancement of cellular engineering Various three-dimensional fabrication methods such as
and tissue engineered devices. inkjet, molding, impregnation, and laser perforation have
The incorporation of biospecic and biofunctional pep- been developed in an attempt to construct or reconstruct
tides into polymeric networks has been adopted in other the unique topographical feature of protein layers, extracel-
systems in the management of various pathological condi- lular membranes, or extracellular matrices with high reso-
tions. Similar to the strategy outlined above by Kao et al., lution and accuracy. Such topographical biomimetics can
Healy et al. grafted RGD and FHRRIKA (putative heparin- be utilized in tandem with current clinically utilized mate-
binding) peptides onto interpenetrating polymer networks rials or novel biofunctional materials as a part of the con-
containing poly(acrylamide) and polyethyleneglycol (64 struction of fully biomimetic material constructs (6769).
66). The resulting polymer network containing both RGD Goodman et al. utilized the inherent three-dimensional
and FHRRIKA peptides mediated extensive adhesion, connement of nonlinear photo-optical processes to de-
spreading, and mineralization of the extracellular matrix velop three-dimensional freeform fabrication of proteins
by human osteoblastlike cells when compared to that of or synthetic polymers by conventional photochemically in-
homogenous peptide surfaces and controls. The cellular re- duced polymerization methods (7073). A laser scanning
sponse was found to rely on the participation of integrin confocal microscope was modied for near-infrared exci-
2 , 1 , and v subunits in a temporally dependent manner. tation to direct protein crosslinking by 2-photon photoacti-
Specically, 2 1 and v 3 integrin receptors mediated the vation. Albumin, brinogen, and alkaline phosphatase
initial cell adhesion; whereas, only v 3 integrin receptors were chosen as model proteins for fabrication in solution.
governed cell adhesion at a longer culture time. The cited Rose bengal was employed as the photoactivator. At the
investigations illustrate the diversity and capability of a focal point of the lens where the photon density is suf-
biomimetic approach in the construction of biofunctional ciently high for 2-photon excitation, crosslinking was in-
materials to study fundamental cell biology and to poten- duced by a mechanism where the photoactivator directly
tially manipulate local host environment for biomedical abstracts hydrogen atoms from protein molecules. Fabrica-
applications. tion was then directed point by point and layer by layer

Copyright 2002 Marcel Dekker, Inc.


within the solution and proceeded to achieve a desired, that tendon has six discrete levels of structure organized in
predetermined geometry and topography. Features of less a hierarchical manner from the molecular to the centimeter
than 200 nm, such as three-dimensional lattices, were syn- scale. Clearly, current composite materials lack the com-
thesized by varying the type of lenses. The bioactivity of plexity and sophistication necessary to achieve highly or-
model proteins was maintained as determined by various dered and multileveled hierarchical structure. Hence, com-
biological functional assays. Goodman et al. demonstrated posite material research has focused on elucidating the
that human platelets extensively adhered to lines fabricated microstructureproperty correlation that includes the cou-
from brinogen molecules in vitro, and the lines were ten- pling between the properties of an individual organic and
fold narrower than the diameter of a platelet cell body. A inorganic component at various scales and in tandem at
minimal level of platelet spreading was observed on sub- different levels of organization. The understanding of the
strates fabricated from the native albumin molecule in structural organization and relationship between each dis-
vitro. Furthermore, micron and submicron structures fabri- crete unit (i.e., molecules, macromolecules, cells, tissues,
cated from alkaline phosphatase molecules demonstrated or extracellular matrix) that makes up a biological system
a high level of enzymatic activity when assayed with en- is one important step in understanding basic structure
zyme linked uorescence imaging in vitro. These exam- function relationships and the construction of complex bio-
ples illustrate the feasibility of mimicking the complex hi- mimetic materials.
erarchical structure of biological systems that expands Due to the specicity of biological synthesis pathways
from the nano scale to subcellular levels in the study and such as biomineralization systems and protein and nucleic
the partial control of biological functions. acid syntheses, microbiological and other biological tech-
niques are currently being adopted and utilized in the con-
struction and processing of novel biomimetic materials
(7783). For example, analysis of a variety of mineralizing
V. BIOMIMETIC-DERIVED MATERIAL biosystems leads to several guiding principles that have
PROCESSING METHODS signicant implications for biology and materials sciences.
Biomineralization occurs within specic subunit compart-
Currently, biomaterial development and synthesis have ments or microenvironments. The process produces spe-
been focused on the utilization of a myriad of synthetic cic minerals with dened crystal size, orientation, and or-
methodologies to obtain a wide range of polymers, ceram- dered macroscopic growth. Many incremental units are
ics, metal alloys, or biologically derived materials with a packaged together to form unique composites (76). These
diverse physicochemical bulk and surface property. How- complex systems are controlled by the structure and chem-
ever, synthetic methodologies often result in heterogeneity istry of the interfaces between the organic substrate, min-
and anisotropy (i.e., polydispersive molecular weight of eral, and medium. The presence of proteins such as osteo-
polymers, large range of size distributions of polymer and pontin and catalysts is also critical for the control of
ceramic particulates, phase separation of chemical and/or biomineralization. Some examples of novel biomaterials
physical properties). Furthermore, current synthetic fabri- synthesized by mimicking biomineralization systems in-
cation methods cannot incorporate or mimic the complex clude monolayer lms, self-assembled monolayer lms,
multilayer microstructure of biological systems to produce self-assembled amphiphilic structures, and supramolecular
materials containing controlled microarchitecture and templates (76). However, low stability, lack of control over
multifunctional properties present in biological materials, local ordering, and inability to direct crystal growth in
such as cellular membranes, extracellular matrices, or tis- three dimensions are some of the existing technological
sues. Several researchers are developing novel material hurdles that have yet to be overcome. One exciting area
synthesis and processing techniques to enhance the limita- of research attempts to utilize microorganisms as a mean
tion of conventional synthesis and fabrication procedures. to synthesize calcium phosphate nanoparticles of a precise
By studying the complex interrelationship of structural size distribution (84). Nanoparticles about 50 nm in diame-
organizations in biological systems, biomaterial research- ter with a uniformed crystal size and structure were synthe-
ers such as Baer and Hiltner pioneered the development sized on the extracellular membrane of bacteria of the Ci-
of complex organic and inorganic composites that incorpo- trobacter genus in the presence of calcium and phosphate
rated some of the ordered microstructure found in nature ions. The Citrobacter genus contains membrane-bound
(32,7476). For example, load-baring tissues such as ten- phosphatase enzymes and has been utilized in the water
don, comprise brous organic components embedded in a purication process through the accumulation of heavy
soft organic matrix that are analogous to ber- or particle- metal ions as cell-bound insoluble metal phosphates. The
reinforced polymeric composites. Baer et al. elucidated nature of the phosphate formed depends on the type of

Copyright 2002 Marcel Dekker, Inc.


ions in the culture media. In the presence of calcium and VI. SUMMARY
phosphate ions in the culture medium, investigators
showed that the crystals formed at the bacteria extracellu- As illustrated by the investigations discussed, the underly-
lar membrane were amorphous calcium phosphates pro- ing basis of biomimetics is the need to better understand
duced by membrane-bound phosphatase enzymes via simi- the structural and functional relationship of biological sys-
lar mechanisms as those involved in the extraction of tems and organizations. Hence, the eld of biomimetics
heavy metals from water. The 50-nm particles within the represents a diverse and interdisciplinary approach to for-
bacteria were of the composition of CaHPO 4 and were pro- mulate functional compounds and materials with enhanced
duced through migration of calcium through the cell mem- physicochemical and biological properties when compared
brane. The exact mechanism of the CaHPO 4 transport to these synthesized via conventional formulation method-
across the extracellular membrane remains unclear. Nano- ologies. Furthermore, the biomimetic approach offers
particles were analyzed and characterized by Fourier trans- unique tools that can, in time, be utilized to probe the fun-
form infrared spectroscopy, x-ray diffraction, and scanning damental questions of the complex biological systems and
and transmission electron microscopy. CaHPO 4 can be fur- mechanisms.
ther processed to hydroxyapatite and then the bacteria rep-
resents a self-constructed soft agglomerate of the particles.
Hence, the utilization of Citrobacter provides a new ap-
proach to produce well-controlled nanoparticulates of opti- ACKNOWLEDGMENTS
mal characteristics that are hydroxyapatite precursor pow-
ders. The use of the natural function of microorganisms in The author thanks the National Institutes of Health (HL
fabricating materials represents a unique ability of novel 63686), the Whitaker Foundation (RG99-0285), the New
material synthesis and processing systems based on bio- Investigator Program Award of AACP, the Burroughs
mimetism. Wellcome Fund and the American Foundation for Pharma-
As another example of utilizing the specicity and accu- ceutical Education, and the American Cancer Society
racy of biological systems to synthesize complex materi- (IRG-58011423). The author also thanks Drs. Y. P. Liu,
als, genes that contain the instruction for the synthesis of D. Lok, J. Li, and D. Lee of the University of Wisconsin
desired macromolecules such as proteins and carbohy- Madison for technical assistance. As the session organizer,
drates were incorporated into vectors that were utilized to the author also thanks the Society for Biomaterials (USA)
transfect candidate cells such as bacteria. The new genetic and the presenters for establishing and participating in the
code becomes a part of the genetic and biochemical ma- Biomimetic Session of the 6th World Biomaterials Con-
chinery of the transfected cell. The marriage of microbiol- gress, May 1520, 2000, in Kamuela, HI.
ogy and biomaterial development was pioneered by Tirrell
et al. and continued in several other laboratories (2931).
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Copyright 2002 Marcel Dekker, Inc.


3

Silicones for Pharmaceutical and Biomedical Applications

Haissam S. El-Zaim and John P. Heggers


University of Texas Medical Branch, Galveston, Texas

I. INTRODUCTION thousands of hydrocephalic shunt procedures have been


performed using silicone elastomer. In other words, sili-
Until the early 1950s, the practice of medicine had not seen cone is responsible for saving the lives of thousands of
major changes for centuries. Before that time surgeons, for children borne with this birth defect (1 in 400600 live
example, still used sutures processed from the intestinal birth), as well as improving the quality of life of survivors
walls of sheep or cows to sew wounds together. Around who otherwise would become mentally retarded. Hydro-
the mid-20th century, Eugene Rochow invented silicone, cephalic shunt is not the only procedure where the use of
which proved to be one of the major inventions of the cen- silicone has made a major contribution. Today, all intra-
tury. Silicone has some unique chemical and physical cranial indwelling tubing is made of silicone. Silicone is
properties. It is inert, which implies low risk for interaction now used in many other surgical and nonsurgical proce-
with the cells and chemicals in the body. It sustains high dures, and its use extends to a variety of medical instru-
temperature, which suggests that silicone-based products ments and indwelling medical devices.
could be easily sterilized. It was these properties that in- Before we go into the details of the different applica-
spired scientists and physicians to consider the use of sili- tions for silicone in medical and pharmaceutical practice,
cone for medical applications. let us briey review the chemistry and physical properties
The rst successful use of silicone in medical practice of silicone.
came about in 1956. At that time Doctors F. E. Nulsen
and E. B. Spitz were cooperating with scientists from Dow
Corning to develop a tube to be used in a hydrocephalic II. CHEMICAL AND PHYSICAL
shunt procedure (68). In this procedure, an elastic tube is PROPERTIES
placed in the head of a child suffering from hydrocephalus
in order to relieve the intracranial pressure caused by the Knowledge of the fundamental chemistry of the element
excess of cerebrospinal uid (CSF) in the cranial cavity. silicon (Si) and understanding how Si can be integrated in
The shunt allows excess CSF to ow in one direction to biosystems are essential to understanding the role of Si in
the drainage site, which can be either intravenous, intratho- health and how it can be responsible for disease. Just like
racic, or intra-abdominal. The tube had to be soft, exible, carbon, Si is capable of forming four bonds and is known
elastomeric, and biocompatible with the human body. All for its ability to polymerize and form network covalent
of these requirements were met by the silicone elastomer structures (9,134). However, unlike carbon, stable SiSi
that was successfully used in the procedure. Since then bonds do not usually form. Silicon dioxide (SiO2), also

Copyright 2002 Marcel Dekker, Inc.


Figure 3.4 Condensation of two molecules of trimethylchloro-
silane into hexamethyldisiloxane. (From Ref. 63b.)
Figure 3.1 The chemical structure of polydimethylsiloxane, the
basic silicone compound used in medicine. (From Ref. 63b.)
misinterpreted as oxygen linked to Si with a double bond,
thus the term silicone. Further reaction with water yields
called silica, is the most abundant mineral in the earths silanolsdimethylsiloxanes with terminal hydroxyls. Poly-
crust and is the natural source of Si used in manufacturing merization usually yields a mixture of silanol oligomers
silicone. with n 3 to 9 and cyclic siloxanes.
Silicone is simply a generic term that refers to siloxane The polysiloxanes then are capped by trimethylsi-
polymers (polysiloxanes). In general, they are compounds loxane groups, a process termed equilibration (50). This
of the structure (R1SiR2O)n , where atoms of silicon are is rst accomplished by condensing two molecules of tri-
linked together by oxygen in a fashion analogous to the methylchlorosilane to hexamethyldisiloxane via the addi-
ether linkage of carbons by oxygen (36,89). The SiO tion of water (Fig. 4) and then reacting hexamethyldisilox-
Si bond is correctly termed a siloxane bond and thus the ane with ailanol oligomers to produce silicones (Fig. 5).
silicones are polysiloxanes (38,48,104,133). In polysilox- This process results in a uid polysiloxane often termed an
anes, the relatively stable SiO bond is further stabilized oil. Consequently, these compounds have widespread
by substitution of alkyl groups as the two side chain groups industrial and consumer uses as dielectric uids, lms, and
pensile from the Si molecule, which were CH3 groups. coatings.
Thus, the most common core structure of medical utility Polydimethylsiloxane, which has a specic gravity
is polydimethylsiloxane (PDMS) (Fig. 1). lower than that of water, is the most commonly used uid
These highly hydrophobic compounds are chemically in medical practice (133). The viscosity of the oil is a pri-
stable under physiologic conditions, can be synthesized mary function of its molecular weight, but also of the abil-
with a variety of physical properties, and can be molded ity of the molecules to slip past one another. The pensile
easily. Dialkylsiloxanes are prepared by the reaction of the methyl groups of silicones have little or no inuence on
dialkyldichlorosilane (generally dimethyldichlorosilane) this slippage. The polymerization reactions described can
with water (Figs. 2 and 3). Originally this structure was also be conducted using other alkyl or aromatic side chain

Figure 3.2 Chemical reaction depicting the formation of dimethylsiloxane from dimethyldichlorosilane in the presence of water. (From
Ref. 63b.)

Figure 3.3 Generation of silanols with terminal hydroxyl groups from dimethylsiloxane with water. (From Ref. 63b.)

Copyright 2002 Marcel Dekker, Inc.


Figure 3.5 Production of silicone in a reaction between hexamthyldisiloxane and ailanol oligomers. (From Ref. 63b.)

modied chlorosilanes such as ethylmethyldichlorosilane imately 400 m2/g. By changing the amount of ller added
or phenylmethyldichlorosilane as the feedstock and equili- to a silicone elastomer, one can change its degree of hard-
brating with the homologous end groups. These will pro- ness. This same property can be modied by changing the
duce polysiloxanes, with increased viscosity because of the type of polymer used. Polydimethylsiloxane is used to pre-
resistance of the more bulky pensile groups to slipping past pare the soft grade silicone elastomers, whereas polysilox-
each other. Silicone gels produced by this procedure con- ane is used to prepare medium and hard grade silicone elas-
sist of covalently crosslinked polysiloxane networks and tomers. Another type of silicone rubber is the room
interpenetrating polymer networks swollen in a silicone temperaturevulcanizing (RTV) silicone rubber. These sil-
uid. Commercial PDMS are not only produced as uids icones have much less tensile and tear strength than the
or oils and gels, but also as gums, or rubber elastomers. high temperaturevulcanized materials. Unlike the other
The strength and elasticity of polysiloxane elastomers is a type of silicone elastomers, RTV silicones are provided as
function of the length of the polymer chain and the degree intermediate components which require mixing before
of crosslinking. Elasticity occurs because polysiloxanes their intended use (7,11).
(like other polymers) exist in highly coiled conformers.
As the material is stretched, the polymer unwinds. When
tension is released the polymer recoils. Elasticity relies on III. BREAST AUGMENTATION IMPLANTS
the ability of adjacent polymer regions to slip past each
other and therefore will be inuenced by the presence of Thomas Cronin and Frank Gerow, two plastic surgeons at
the bulky pendent groups. The physical rigidity of a silox- the University of Texas, were looking for an alternative to
ane polymer is increased by crosslinking the polymeric the spongy breast implants commonly used for reconstruc-
chains. Highly crosslinked polymers will lose elasticity be- tion of breasts among female patients undergoing mastec-
cause uncoiling will be inhibited as a result of the inability tomy. The problem encountered with the spongy implants
of the adjacent regions to slip past each other, but they was that after a period of time they hardened and started
will not deform easily and hence are more rigid. A lightly to look and feel less natural. Around the year 1960, the
crosslinked polymer will deform easily but will have sig- two surgeons developed the rst silicone breast implants
nicant elasticity, with the ability to return to its original in collaboration with Dow Corning (28,93). This prototype
shape when stress is released. of silicone rubber sac lled with silicone gel was called a
The basic ingredient to make silicone elastomers, or si- mammary prosthesis and was later marketed by Dow
licone rubbers, is a clear and highly viscous PDMS with Corning after undergoing the rst human trial. Soon after
minor amounts of other substituents, particularly vinyl Dow Corning introduced this new product to the market,
groups. Other radicals in the silicone rubber determine the and to everybodys surprise, the demand for mammary
physical properties of the resultant elastomer. The most prosthesis was much greater for breast augmentation pro-
common mechanism by which crosslinking occurs is by a cedures for cosmetic purposes (80%) rather than recon-
radical attack on the alkyl groups pensile from the Si initi- struction procedures following mastectomy. It was esti-
ated by heat (heat vulcanization) or benzyl peroxide. The mated that by the year 1992, 12.5 million North
reactive intermediates containing carbon free radicals then American women and 100,000150,000 British women re-
combine to form the carboncarbon covalent crosslinking ceived breast implants (62). During the 1960s and 1970s
bonds. In some cases, crosslinking is facilitated by substi- Dow Corning introduced many improvements to enhance
tuting vinyl groups for occasional methyl groups because the quality of the mammary prosthesis, which became a
the double bond of this group is quite susceptible to free multimillion dollar product. Other companies joined Dow
radical attack. In order to increase its tear and tensile Corning to cash in on this rapidly growing market.
strength, the prevulcanized silicone is usually mixed with Silicone breast implants have been associated with
a ller. A common ller used for this purpose is the submi- complications such as capsular contracture, enlargement
croscopic fumed silica, which has a surface area of approx- of lymph nodes draining the implant site, occasional rup-

Copyright 2002 Marcel Dekker, Inc.


ture of the silicone gel sac, and bacterial contamination issue rose steeply and the Food and Drug Administration,
(26). Capsular contracture, which may be triggered by following the recommendations of two independent advi-
bleed of the silicone gel through the outer bag, is believed sory panels, requested a halt on the use of silicone breast
to be due to the natural attempt by the body to engulf the implants other than within clinical trials (27,94). In 1994,
foreign material (18). The drying effect of silicone in the while the battle between the litigants and the manufactur-
surrounding soft wet tissue enhances the scar formation ers of the implants was raging inside the court and on the
and is another possible contributing factor to the formation media front, a Department of Health advisory group in
of the capsule. This pseudocapsule is composed of brous Great Britain reported that there was no evidence of an
tissue typically seen in the process of wound healing. Phys- increased risk of connective tissue disease in patients with
ical changes and alterations of the nature of the silicone silicone breast implants (43). Analysis of published case
implants, as well as use of wetting agents such as provi- series as well as several casecontrol and cohort studies
done, have reduced the incidence of capsular contracture suggested that the cumulative incidence of connective tis-
(45). sue disorders among recipients of breast implants is no dif-
It was not these complications but rather the possible ferent from that expected in the general population (101).
link of breast implants to a systemic connective tissue Only one retrospective cohort study, by Hennekens et al.,
disease that fueled the debate over the safety of this pro- published in the Journal of the American Medical Associa-
cedure. Several reports appeared in the literature suggest- tion in 1996, pointed to a weak association (52). The au-
ing an association between breast implants and some ill- thors of this study themselves concluded that an associa-
characterized immunological disorders. Most of the reports tion between silicone breast implants and increased risk of
described cases of a poorly dened syndrome referred to major connective tissue disease is unlikely. Further, this
as human adjuvant disease. Some reports claimed an study was criticized for the lack of certain diagnostic valid-
association with systemic, probably autoimmune, connec- ity with potential bias due to differential over-report-
tive tissue diseases (73,77). The rst report of an associa- ing.
tion between silicone breast implant and connective tissue
disease dates back to 1964, and the rst documented cases
were those of three patients in 1982. It is estimated now IV. OPHTHALMOLOGY PROCEDURES
that the number of reported cases exceeds 290 patients AND OPTIC DEVICES
(62). Reported symptoms and signs include fatigue, joint
pain, bone pain, dry eyes, dry mouth, dry skin, cognitive Retinal detachment is a pathological process in which the
dysfunction, myalgia, weakness, hair loss, nail changes, neuronal layers of the retina, including the layer of photo-
skin rashes, paresthesia, dysethesia, freckling, pigment receptors (rods and cones), are separated from the pigment
change, headache, dizziness, nausea, foul taste, weight epithelium. Melanin in the pigment epithelium, located in
gain or weight loss, bruising, increased sensitivity to light, the back of the neuronal retina, captures light that makes
fever, chills, infections, diarrhea, constipation, periodontal it through the photoreceptors layer and therefore prevents
disease, skin papules, muscle twitching, urinary symp- reection of light off the sclera. In addition, the pigment
toms, dysphagia, dysmenorrhea, blurred vision, tinnitus, epithelium is essential for the proper metabolism of the
drug reactions, emotional lability, insomnia, edema, hem- neuronal layer of the retina. It supplies the retina with nu-
angiomas, delayed wound healing, vascular abnormalities, trients and is involved in phagocytizing and clearing out
partial hearing loss, reduced smell, tremor, mouth sores, the debris generated by the constant turnover of the outer
tight skin, shortness of breath, wheezing, palpitations, sei- segments of the photoreceptors containing the photopig-
zures, parotid gland swelling, heat intolerance, and cancer. ments. Retinal detachment can be caused by numerous dis-
The range of symptoms was very large and many of the ease conditions. Cytomegalovirus-induced retinitis is a
cases did not fulll conventional clinical and laboratory major cause of retinal detachment among patients with ac-
criteria for a particular connective tissue disease. Sclero- quired immunodeciency syndrome (AIDS) (56,57). Pa-
derma was the most common specic diagnosis (10,15, tients with retinal detachment are at serious risk of total
16,30,76,99, 100). blindness unless the problem is corrected. Traditionally,
In 1991, an American jury found that silicone breast retinal reapposition was achieved by suturing a sponge or
implants were responsible for the plaintiffs symptoms of a band to the sclera to produce a buckle which closes
a mixed connective tissue disease. The jury found that the the hole by external tamponade. With this procedure the
company was responsible as they misrepresented the safety retina resumes contact with the pigment epithelium. This
of their product. After this trial, public awareness of the procedure works well in uncomplicated cases; however, in

Copyright 2002 Marcel Dekker, Inc.


complex retinal detachment, which requires removal of the Urinary incontinence is a common problem affecting
preretinal membranes by vitrectomy, an external tampon- 1025% of women between the ages of 15 and 60 years
ade must be supplemented with an internal tamponade in (64). Stress urinary incontinence, which is the involuntary
order to successfully bring the retina and the pigment epi- loss of urine during physical activities that increase intra-
thelium together. Investigators felt the need for a substance abdominal pressure, is a common type. This condition
with stable surface properties at aqueous interfaces, that is could result from loss of pelvic support, and hence hyper-
biologically inert, and is amenable to surgical procedures mobility of the vesicourethral junction (anatomic inconti-
to serve this purpose. Gases, such as air, sulfur, hexauo- nence) or intrinsic sphincter deciency (type III stress in-
ride, and peruoropenthrane were tested and proved to be continence). The latter condition may be caused by
effective in the short term. However, for longer-term main- myelodysplasia, sympathetic nerve injury, and surgical in-
tenance of break closure and relief of retinal traction, a jury or trauma (75). Urinary incontinence may also occur
material that is insoluble in tissue uid seemed to be more as a result of spinal cord injury or following prostatectomy
appropriate. Because of its long-term biotolerability and procedure in males (2,25,131).
optical clarity, silicone seemed to be the perfect substance As early as the year 1947, attempts to control urinary
for this purpose. In 1962, Cibis was the rst to report the incontinence by applying a constricting cuff around the
use of liquid silicone (silicone oil) as a vitreous prosthe- urethra via a cutaneous tunnel were reported. In 1973, Jo-
sis in human eyes (23). Since then the use of silicone has seph Kaufman described a procedure in which a silicone
been extended to include providing temporary or perma- balloon is implanted around the crura of the penis to con-
nent internal tamponade, delaminating periretinal mem- trol incontinence (59). Upon distention the balloon would
branes, manipulating the retinal edge in giant tears, closing compress the urethra against the symphysis pubis. Follow-
macular holes, and maintaining the surgeons view of the ing implantation the balloon can be further enlarged by
back of the eye during surgery involving vascularized tis- injecting more uid into it if necessary. That same year,
sue. The use of intraocular silicone is considered a major Brantley Scott and his colleagues developed an articial
breakthrough in the treatment of retinal detachment, which sphincter which consisted of a silicone rubber cuff, a small
when left untreated can lead to blindness. Complex retinal pump, a balloon, and a stainless steel control assembly
detachments are now routinely repaired, and very few are (98). The cuff is surgically implanted around the neck of
deemed to be inoperable. Side effects due to intraocular the bladder, or sometimes around the bulbous urethra of
silicone can be avoided or reduced by proper surgical tech- men. The balloon is positioned in the perivesical space
nique. Foaming or emulsication is responsible for the and, for easy access, the pump is placed under the skin
main long-term complications such as glaucoma and im- either in the scrotum in males or the labia minora in
paired vision. This can be avoided with a more complete women. The inated cuff cuts off the ow of urine in a
silicone ll (21,37,41,74,82,90,97). manner similar to that of the natural sphincter muscle. By
In addition to the previous example, an experimental squeezing the pump several times, forcing the uid out of
procedure to restore accommodation in primate eyes by the cuff and into the balloon, the patient could deate the
relling the lens capsule with injectable silicone com- cuff and allow urine to ow. Within minutes the cuff rells
pounds is currently being investigated (87,88). Last but not automatically restoring continence. An overall success rate
least, polysiloxane lenses coated with povidone are rou- of 70% for an average of 3.5 years was reported with this
tinely used for patients after cataract surgery (58). Silicone device. Drawbacks of this procedure included cost and
is also used in manufacturing soft contact lenses. complications, such as periurethral brosis, tissue erosion,
and infection.
Transurethral or periurethral injection of biomaterials
V. TREATMENT OF URINARY AND FECAL into the intrinsic sphincter was developed as a less invasive
INCONTINENCE AND GENITOURINARY technique for the treatment of incontinence (8). Various
DEVICES forms of bioinjectable materials have been developed for
this purpose. Injection of polytetrauoroethylene (Teon)
Until the introduction of silicone-based prosthesis, at- paste has had relatively good results in the short term (24).
tempts to replace any part of the urinary tract were plagued Long-term follow-up studies on patients treated with
by the problem of phosphatic encrustation. Whereas sili- Teon paste injections revealed a low success rate of only
cone catheters may be left indwelling for 8 to 12 weeks 38% and the occurrence of local side effects, such as -
with minimal phosphatic deposition, latex or plastic cathe- brosis in the urethra and bladder granuloma balls (17).
ters have to be replaced within 3 to 4 weeks (130). More recently, injection of autologous fat was tested and

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the results from this study were not impressive (6,20,95). 107,109125). Niebauer and his colleagues introduced a
Collagen has a good (4677%) long-term (2 years) success hinged silicone joint with a dacron mesh covering for in-
rate and is currently the most widely used injectable (4). growth xation. A hinge spacer originally intended to im-
The major problem encountered with collagen use is the prove the alignment and stability of resection and
potential development of allergic reactions (3). Collagen, arthroplasty was introduced by Swanson. Swanson, who
as well as autologous fat, are absorbed rather rapidly, pioneered the eld of silicone implant arthroplasty, intro-
which calls for repeated injections. Recently, reports on duced other designs used in the treatment of arthritis in the
use of silicone particles for treatment of urinary inconti- wrist, ulnar head, scaphoid, lunate, trapezium, and meta-
nence appeared in the literature (61). According to these carpophalangeal and interphalangeal joints. Designs by
reports, injection of polydimethylsiloxane is a simple, safe, Swanson and Niebauer became very popular soon after
and effective procedure for the treatment of urinary incon- their introductions, but some of the problems encountered
tinence. Another possible application for this procedure is were implant fracture, persistent weakness of grip and
in treating vesicoureteral reux (61). pinch, and increasing deformity. This led to careful recon-
Soiling (fecal incontinence) is another distressing, de- sideration of both prosthesis designs and materials. Today,
moralizing, and costly condition which may result from prostheses based on those original designs continue to be
various causes. Although the pathophysiologic mecha- widely used in arthroplasty. Another use for silicone in
nisms are not fully understood, it is almost always associ- hand orthopedic surgery is in tendon transplantation (19).
ated with anorectal disorders that can deform the contour In this procedure, Hunters Silastic rods are used to form
of the anus and anal canal. The condition is usually cor- a tunnel composed of a thin pseudocapsule in which tendon
rected by medical or surgical means; however, in certain transplants are inserted later on. The purpose of this tunnel
situations reconstruction of the contour deformity of the is to prevent the adhesion of the transplanted tendon to the
anus is the only successful resort (86). Moderate success adjacent structures in the hand, which allows for the glid-
with sublevatorial implantation of a silicone ring was re- ing function of the tendon. Silicone is also used in recon-
ported by Hansen in 1996 (47). Currently, perianal injec- structing the wrist using silicone joint prostheses or bone
tion of PDMS paste is being investigated for treating prostheses made of silicone. Silicone can also be used to
soiling. reconstruct larger joints such as the elbow (71,111,117
Silicone is also used in the making of penile implants 125). This may involve replacement of the head of the ulna
used to treat male impotence. One model of these implants with silicone components. However, reconstruction of
is a solid polysiloxane component that is inserted subcuta- large joints and long bones may require additional rein-
neously in the penis. Another model consists of an inat- forcement.
able silicone bag connected to a reservoir which is used The three major complications following silicone im-
to pump uid to the prosthesis when an erection is desired plant arthroplasty are fracture, subluxation, and synovitis.
(92). Fracture commonly occurs in the wrist and metacarpopha-
langeal implants, whereas subluxation occurs mainly with
the carpal implants (63). Silicone synovitis is an inam-
VI. ORTHOPEDIC AND RECONSTRUCTIVE matory response to small particulate debris of 10100 m
SURGERY in diameter (5). This phenomenon is more commonly seen
in association with high performance (HP) silicone elasto-
Silicone prostheses play a major role in the treatment of mer than with the conventional silicone elastomer (125).
degenerative arthritis and rheumatoid arthritis in the hand Small particles shed from HP elastomer implants are in-
and wrist. Silicone devices are often used in orthopedic gested by phagocytes which fail to clear these foreign par-
surgery, especially in operations to reconstruct the hand ticles away. Frustrated macrophages release proinamma-
(45). Until the late 1950s, success rates were not impres- tory cytokines and attract more immune cells. This results
sive in attempts to achieve pain-free, stable implant in a chronic inammatory response characterized by the
arthroplasty with metacarpal cups or with soft tissue inter- presence of lymphocytes, eosinophils, and giant multinu-
positions (8). The metal hinge implants for the metacarpo- cleated epithelial cells (42). In very few cases, silicone
phalangeal joint introduced in 1959 were not well tolerated lymphadenopathy has been reported following silicone im-
due to bone resorption and metal corrosion (12,35). The plant arthroplasty (22).
search for better techniques and new biocompatible ma- In addition to hand orthopedic surgery, silicone is com-
terials continued until silicone rubber prostheses were in- monly used in maxillofacial reconstructive surgery. Sili-
troduced in the late 1960s and early 1970s (13,84,85, cone products are used to reconstruct the mandible, to aug-

Copyright 2002 Marcel Dekker, Inc.


ment the chin of patients with microgenia and to all the speech restoration methods, the method of shunt
reconstruct the missing ear of patients with microtia esophageal speech by means of silicone tracheoesophageal
(45,69). valve prosthesis have the most consistently high success
rate (102,132).
The major risks associated with the silicone devices are
VII. LARYNGEAL, TRACHEAL, AND bacterial and fungal infections (55,72). The risk of infec-
ESOPHAGEAL PROSTHESES tion can be greatly reduced by proper surgical techniques
and by patient education on how to properly use, maintain,
A variety of devices used in the upper respiratory and di- and clean their prostheses. Also, early intervention to dis-
gestive system are made of silicone. The silicone Safe-T- infect or replace a contaminated device has been very ef-
Tube is designed to maintain adequate tracheal airway and cient in preventing major complications.
to provide support in the stenotic trachea. It can be kept
in place for many years, if necessary. It is commonly used
in cases with acute tracheal injuries, a need to support a VIII. CARDIOVASCULAR PROCEDURES
reconstructed trachea or reconstituted trachea, as well as AND DEVICES
many other procedures (78). In cases such as obstructive
sleep apnea, bilateral vocal cord paralysis, laryngeal carci- With the advent of extracorporeal circulation in 1954 and
noma with glottic insufciency, and neurological disorders the introduction of heartlung machine by Gibbon (40),
with intermittent laryngeal insufciency, a silicone device direct open treatment of valvular diseases became possible.
called the Tracheal Cannula System is usually used (79). Deformed, calcied valves could not be completely xed
By diverting the saliva into the distal esophagus, a silicone- by conservative procedures such as valvuloplasties and
made salivary bypass tube greatly reduces the healing time calcium debridement. Such procedures represented a major
in cases with cervical esophageal and hypopharyngeal challenge to the cardiac surgeon (34). Early attempts to
strictures and stulas resulting from a variety of causes use articial valves were not very successful in the long
including malignancy, surgery, irradiation, trauma, and run due to the loss of exibility of the material and to in-
caustic indigestion (80). Esophageal tube is used to bridge growth of tissue (14). Charles Hufnagel was the rst to
the gap between the pharyngostome and esophagostome implant a valvular prosthesis (46,53). His design consisted
following laryngoesophagectomy and reconstructive sur- of a caged Lucite ball valve which he used to treat aortic
gery of the cervical esophagus (78,80,126,127). Silicone insufciency. Hufnagels attempt set the stage for other
laryngeal stent can be used alone or in combination with investigators to develop more biocompatible and safer
the Safe-T-Tube to prevent and treat laryngeal stenosis, prostheses (33,49,106). Today, there are numerous heart
when the glottic stenosis involves the midglottis, posterior valves available from different manufacturers and many of
glottis, supraglottis, and subglottis (80). Laryngeal keel, an them consist of or contain silicone (34). Valvular endocar-
umbrella-shaped silicone prosthesis, is used to prevent and ditis is the most dreaded complication of valve replace-
treat anterior laryngeal stenosis limited to the anterior com- ment (32). The source of bacteria in early nosocomial in-
missure of the vocal cords (80). Silicone esophageal stents fection is believed to be patients themselves or their
are used as a palliative therapy for tracheoesophageal s- environment. Staphylococci and gram-negative bacilli are
tula, a life-threatening late complication of carcinoma of the leading cause of prosthetic valve infections (103).
the esophagus and lung (83,108). This procedure prevents When the patient presents with symptoms of acute septice-
food and gastric reuxate from entering the respiratory mia secondary to valvular endocarditis, death may ensue
tract and restores the ability to swallow so proper nutrition within few days. The best approach to treat early valvular
intake can be maintained (54). Without treatment, most of endocarditis is the institution of appropriate antibiotics fol-
these patients succumb to respiratory problems and die in lowed by surgery (70). The prognosis for late valvular en-
a matter of few months (31,60). docardtitis is less severe and may be responsive to antibiot-
Since the rst laryngectomy was performed in 1866, ics alone.
several methods to restore speech have been attempted The idea of externally stimulating the heart with an
(1,44). This required an alternative vibratory source to re- electric current was rst conceived in 1952 by Paul Zoll
place the larynx in the reconstructed pharyngoesophageal (135). The rst method of pacing was described in 1958
region. This goal can be achieved by means of an articial by Furman and Robinson (39). According to their method
larynx that has an electric pharyngeal speech vibrator a catheter electrode with a metal guide wire was inserted
which can be externally applied to the neck. However, of into the right ventricle through a peripheral vein. The pace-

Copyright 2002 Marcel Dekker, Inc.


maker itself was kept external to the body. Today, the sive dressing is used in the treatment of hypertrophic scars
transvenous approach is the most commonly applied pro- and keloid. Silicone is also used in manufacturing a variety
cedure (91). In this method, a permanent pacemaker is im- of many other useful devices (65).
planted near the site where the electrode enters the cephalic
vein just beneath the clavicle. In a pacemaker the only
metal exposed to the body uids is the tip of the electrode
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Cendo, D. Hynes, J. N. Wei. Bipolar implant shoulder Elbow, M. Hamalainen, F. W. Hagena (Eds.). Krager,
arthroplasty. Clin. Orthop. Rel. Res. 249:227247 Basel.
(1989). 125. A. B. Swanson. Silicone arthroplasty in the hand, upper
123. A. B. Swanson, G. deGroot Swanson. Elbow joint recon- extremity, and foot25 years experience. In: Silicone in
struction. In: The CIBA Collection of Medical Illustra- Medical Devices. Conference Proceedings, FDA, Center
tions, Vol. 8, Part 2, Musculoskeletal System. CIBA- for Devices and Radiological Health, Baltimore Maryland,
GEIGY, Summit, NJ, 1990, pp. 261262. February 12, 1991, pp. 233259.
124. A. B. Swanson, G. deGroot Swanson, K. Masada, M. Ma- 126. Y. Takimoto, N. Okumura, T. Nakamura, T. Natsume, Y.
kino, P. R. Pires, D. M. Gannon, A. B. Sattel, V. A. Ces- Shimizu. Long-term follow-up of the experimental re-
tari. Constrained total elbow arthroplasty. Rheum. 15: placement of the esophagus with a collagen-silicone com-
113126 (1991). In: Rheumatoid Arthritis Surgery of the posite tube. ASAIO Journal 39:M736M739 (1993).

Copyright 2002 Marcel Dekker, Inc.


4

Biodegradable Polymers as Drug Carrier Systems

Abraham J. Domb, Neeraj Kumar, Tzviel Sheskin, Alfonso Bentolila,


Joram Slager, and Doron Teomim
The Hebrew University of Jerusalem, Jerusalem, Israel

I. INTRODUCTION proteins (19), antibiotics (20), anesthetics (21,22), and


vaccines (23). Injectable formulations containing micro-
Over the past decade the use of polymeric materials for spheres of lactide/glicolide polymers have received the
the administration of pharmaceuticals and as biomedical most attention in recent years.
devices has increased dramatically. The most important
biomedical applications of biodegradable polymers are in a. Synthesis
the areas of controlled drug delivery systems (14) and in
Linear lactide- and glycolide-based polymers are most
the form of implants and devices for fracture repairs (5,6),
commonly synthesized by ring-opening melt polymeriza-
ligament reconstruction (7), surgical dressings (8), dental
tion of lactide and glycolide at 140180C for 210 h us-
repairs, articial heart valves, contact lenses, cardiac pace-
ing a catalyst (24,25). When polymerization temperature
makers, vascular grafts (9), tracheal replacements (10), and
is less than the melting point of polymer (175C), crys-
organ regeneration (11). The purpose of this chapter is to
tallization of the polymerizing polymer occurs, resulting
review the chemistry, properties, and formulation proce-
in solid-state polymerization as with poly(glycolide) (PG).
dures of the different biodegradable polymers actually
Solid-state polymerization has been found useful for very
available and to describe some of their release kinetics,
high molecular weight polymers, around 1000 kD (26).
safety, and biocompatibility considerations.
The polymerization reaction was studied and several
mechanisms were proposed including cationic (2730),
anionic (3134), and coordinationinsertion (35,36). The
II. BIODEGRADABLE POLYMERS
common polymerization catalysts are tin derivatives such
A. Polyesters as tin octoate or tin hexanoate. A hypothetical mechanism
of the ring-opening polymerization of lactide using a tin
1. Lactide and Glycolide Copolymers
catalyst was suggested by Kissel et al. (Scheme 1) (37).
Linear polyesters of lactide and glycolide have been used In this mechanism, Lewis acid character of the tin cata-
for more than three decades for a variety of medical appli- lyst activates the ester carbonyl group in the dilactone (a,
cations (58,1214). Extensive research has been devoted in Scheme 1). The activated species reacts with the alcohol
to the use of these polymers as carriers for controlled drug initiator to form an unstable intermediate, which opens to
delivery of a wide range of bioactive agents for human the ester alcohol (b, in Scheme 1). The propagation reac-
and animal use (15). They have been used for the delivery tion proceeds by tin catalyst activation of another dilactone
of steroids (16,17), anticancer agents (18), peptides and carbonyl group and reaction with the hydroxyl end group.

Copyright 2002 Marcel Dekker, Inc.


Polymerization of L,L- and (L,L D,D)-LA initiated
with Al(O-iPr)3 is claimed to be the rst example of fully
controlled synthesis of high molecular weight PLA (45).
The aluminum trialkoxidegrowing species belong to the
most selective ones, in comparison with other metal (e.g.,
K, Sm, La, Fe, Sn, Ti) alkoxides.
Low molecular weight poly(lactic acid) (3000 kD)
can be synthesized by direct polycondensation of lactic or
glycolic acid using phosphoric acid, p-toluene sulfonic
acid, and antimony triuoride as acid catalysts (46).
An interesting method of polymerization of -hydroxy
acids was offered by Tighe and coworkers (4749), which
did not receive much attention. In this method, the anhy-
drosulte (I) and the anhydrocarboxylate (II) cyclic deriva-
tives of -hydroxy acids are polymerized in reuxing diox-
ane or nitrobenzene for 1852 h with alcohol catalysis
(Scheme 2).
Scheme 1 Hypothetical mechanism of ring-opening polymer- The polymerization was preceded by thermal decompo-
ization using tin octoate as catalyst. (From Ref. 37). sition of (I) to give the sulfur dioxide and -lactone (III).
The -lactone polymerizes by ring-opening polymeriza-
tion using an alcohol catalysis to give exclusively poly(-
Ring-opening rate for cyclic lactone can be increased by hydroxyalkanoic acid). The formation of (III) was rate con-
activation of a Zn- or Sn-based catalyst with carbonyl es- trolling, and the subsequent polymerization was so rapid
ter. Stannous octoate S[CO2CH(Bu)(Et)2]2 is commonly that sulfur dioxide and polymer were formed simulta-
used because it has FDA approval as a food stabilizer (38). neously. Pure polymers in 80% yield and molecular
Alternatively, resorbable Fe(II) salts have been utilized as weights of MN 20,000 were reported (49).
initiators for lactide polymerization above 150C (39).
Zinc powder and CaH2 have also been evaluated as poten-
tial nontoxic catalysts for copolymerization of poly(lactic
acid) (PLA) with poly(ethylene oxide) (PEG) (40). Lauryl
alcohol is generally added to control the molecular weight
of the polymer (24,25). Polymers with a molecular weight
as high as 500,000 can be obtained by the melt process
when high purity monomers (99.9%) are used (37).
Since Sn(OCT)2 promoted polymerizations are hardly
controlled, a variety of organometallic derivatives, particu-
larly metal alkoxides, are continuously tested as initiators
in the polymerization of lactides (41,42). The aluminum
alkoxide initiators are the most versatile and readily avail-
able. Both monoalkoxides (R2-AlOR) and trialkoxides
(Al(OR)3) were applied by several groups (43,44). By us-
ing only one form of the trialkoxide isopropyloxide (Al(O-
iPr)3), namely, its trimer, a perfect control of polymeriza-
tion can be achieved.

Scheme 2 Polymerization of -hydroxy acids using anhydro-


sulte cyclic derivatives of -hydroxy acids.

Copyright 2002 Marcel Dekker, Inc.


Branched lactide polymers prepared from the polymer- the initial concentration of polymer. In concentrations
ization of lactide and pentaerithritol as branching agent up to 10 mg/mL discs were formed either with or without
were reported. These branched polymers are degraded in cavity in the middle (shape of a red blood cell). Polymers
a monophasic character, and their degradation is in general of higher molecular weight formed spheres consisting of
faster than for the linear polymers (50). Various polyol a berlike structure. If polymer was used of 150,000 or
molecules were used to prepare a range of branched struc- higher molecular weight, a three-dimensional network
tures with different properties (37,51). could be noticed in which the previously mentioned
Copolymers of LA/GA 55 :45 branched with 0.21 spheres were absorbed (55).
wt% of small polyols like manitol, cyclodextrine, penta- The PLA stereocomplex appears to be soluble in dichlo-
erithritol, and xylitol had a weight average molecular romethane or hexauoroisopropanol (HFIP). By casting a
weight of 15,000 to 46,000. The copolymers branched with low-concentrated solution on mica crystals PLA stereo-
1 wt% of poly(vinyl alcohol) of 3,000 to 72,000 molecular complexes were obtained. As opposed to isotactic PLA
weight range resulted in a tenfold increase in molecular several reports indicate the formation of triangle-shaped
weight of 182,000 to 676,000, respectively. Star poly(eth- crystals. There is a difference in opinion as to whether the
ylene oxide)-polylactide copolymers in a spherical form PLA stereocomplexes consist of 31-helixes (-form) or
were obtained from the block copolymerization of lactic 103-helixes (-form). Brizollara et al. predicted 31-helix on
acid on the end groups of three or four arm poly(ethylene the basis of computer calculations. The D- and L-helixes
oxide) molecules (51). are intertwined forming double strand helices, which
Blends of low and high molecular weight poly(DL- are packed in a hexagonal cell forming triangle-shaped
lactide) were studied as carriers for drugs. As expected, crystals (57). On the other hand x-ray data suggested no
the addition of low molecular weight polymer accelerated change in the 103-helical conformation. Frustration of
the release of drug from the blend formulation (52). the packing of the L- and D-helixes was suggested to cause
Different interactions are known to be involved in com- the triangular shape (58). According to the latter view, the
plex formation between two polymer molecules. It was helices are not intertwined, as was also suggested in earlier
found that PLA complex was formed in a solution of both reports for stereocomplexes of other polymers (59,60). Re-
enantiomers in chloroform. The reaction proceeds very cently it has been reported that both conformations exist
slowly at room temperature (53). First, gel formation was in the PLA stereocomplex: 103-helix with a minimum of
noticed which turned into a precipitate only after about 30 11 units and 31-helices with a minimum of 7 monomers
days. For high molecular weight PLA the reaction takes since only two helical turns are needed for complexation
even longer, up to 1 year. A quicker procedure was found reaction (61). Also, stereocomplexes of PLA containing
by precipitating D-PLA and L-PLA together. This can be PEG have been synthesized and characterized by means
achieved by pouring a solution of D- and L-PLA in dichlo- of DSC and various microscopic techniques (62).
romethane into a nonsolvent like methanol or ether. When
a lm is casted from a solution of low molecular weight
b. Polymer Properties
isotactic PLA enantiomers in dichloromethane or chloro-
form, preferably stereocomplex crystals are formed. With Polyesters based on poly(lactic acid), poly(glycolic acid),
PLA of higher molecular weight, less stereocomplex for- and poly(lactic-co-glycolic acid) are found as the best bio-
mation was noticed. An easy procedure to obtain the ste- material with regard to design and performance. Among
reocomplex both for low and for height molecular weight them, lactic acid contains an asymmetric -carbon atom
PLA consists of heating a mixture of both enantiomers at with three different isomers as D-, L-, and DL-lactic acid.
60C in acetonitrile. Poly(lactic acid) was soluble in aceto- The physiochemical properties of optically active homo-
nitrile only at elevated temperatures (from 52C upwards). polymers poly(D-lactic acid) (PDLA) and poly(L-lactic
Within several hours the solution became turbid and a pre- acid) (PLLA) are the same, whereas the racimic PLA
cipitate was obtained after 3 days. It is suggested that com- has very different characteristics (63). Racimic PLA and
plex formation takes place during precipitation from a sol- PLLA have Tg of 57 and 56C, respectively, but PLLA is
vent. Neither L-PLA or D-PLA gives a reaction on its own highly crystalline with a Tm of 170C, and racemic PLA
in various solvents under similar conditions (54,55). Dif- is purely amorphous. The polymer characteristics are af-
ferential scanning calorimetry (DSC) shows a shift of the fected by the comonomer composition, the polymer archi-
melting temperature of about 50C (from Tm 180C for tecture, and molecular weight. The crystallinity of the
D- or L-PLA, MW 100,000, to Tm 230C for PLA polymer is an important factor in polymer biodegradation
stereocomplex of similar molecular weight) (5456). and varies with the stereoregularity of the polymer. Steril-
Scanning electron microscope (SEM) images revealed ization using -irradiation decreases the polymer molecular
the formation of particles whose shape was inuenced by weight by 30 to 40% (64). The irradiated polymers con-

Copyright 2002 Marcel Dekker, Inc.


tinue to decrease in molecular weight during storage at has been published (69). The tensile and exural strength
room temperature. This decline in molecular weight affects and modulus, as well as the biodegradation of various
the mechanical properties and the release rate from the lactide/glycolide polymers, polyorthoester, and polycapro-
polymers. Poly(lactic acid) and its copolymers with less lactone have been summarized in a tabular or diagram
than 50% glycolic acid content are soluble in common sol- format.
vents such as chlorinated hydrocarbons, tetrahydrofuran,
and ethyl acetate. Poly(glycolic acid) is insoluble in
c. Biodegradation
common solvents, but soluble in hexauoroisopropanol
and hexauoroacetone sesquihydrate (HFASH). In its Lewis (70), Gopferich (71), Holland et al. (72), and Tracy
highly crystalline form, PGA has a very high tensile (73) reviewed the biodegradation behavior of lactide/
strength (10,00020,000 psi) and modulus of elasticity glycolide polymers. The molecular weight and polydisper-
(1,000,000 psi) (65). sity as well as the crystallinity and morphology of the poly-
The solubility parameters of several polymers were de- mers are important factors in polymer biodegradation.
termined by Siemann (66). The solubility parameters were The factors that may affect the polylactide degradation
in the range of 16.2 and 16.8, which is comparable to those include chemical and congurational structure, molecular
of polystyrene and polyisoprene (67). weight and distribution, fabrication conditions, site of im-
A comparison study on the physicomechanical proper- plantation, physical factors, and degradation conditions
ties of several biodegradable polyesters was reported (68). (74,75). The degradation of semicrystalline polymers pro-
The thermal properties, tensile properties, and the exural ceeds in two phases: in the rst phase the amorphous re-
storage modulus as a function of temperature were deter- gions are hydrolyzed, and then the crystalline regions in
mined. The following polymers were compared: poly(L- the second. The polymers degraded by bulk hydrolysis of
lactic acid), poly(DL-lactic acid), poly(glycolic acid), the ester bonds, which resulted in a decrease in molecular
poly(e-caprolactone), poly(hydroxybutirate) and copoly- weight with no weight loss.
mers with hydroxyvaleric acid, and poly(trimethylene car- A comprehensive investigation on the hydrolysis of lac-
bonate). The thermal and mechanical properties of several tide polymers was described recently by Vert et al. (76).
of the polymers tested are summarized in Table 1 (68). In these studies, a standardized set of experiments was de-
A comprehensive review on the mechanical properties of signed. All specimens (2 10 15 mm) were prepared
several biodegradable materials used in orthopedic devices in a similar way and allowed to hydrolyze at 37C in dis-

Table 4.1 Thermal and Mechanical Properties of Biodegradable Polyestersa

Tensile Tensile Elongation


strength modulus
Polymer MW Tg (C) Tm (C) (MPa) (MPa) Yield (%) Break (%)
Poly(lactic acid)
L-PLA 50,000 54 170 28 1,200 3.7 6.0
L-PLA 100,000 58 159 50 2,700 2.6 3.3
L-PLA 300,000 59 178 48 3,000 1.8 2.0
DL-PLA 107,000 51 29 1,900 4.0 5.0
Poly(glycolic acid)
PGA 50,000 35 210 NA NA NA NA
Poly(-hydroxybutyrate)
PHB 370,000 1 171 36 2,500 2.2 2.5
P(HB-11%HV) 529,000 2 145 20 1,100 5.5 17
Poly(-caprolactone)
PCL 44,000 62 57 16 400 7.0 80
Poly(trimethylene carbonate)
PTC 48,000 15 0.5 3 20 160
Poly(orthoesters)
t-CDM: 1,6-HD 35: 65 99,000 55 20 820 4.1 220
t-CDM: 1,6-HD 70: 30 101,000 84 19 800 4.1 180
Source: Ref. 68.

Copyright 2002 Marcel Dekker, Inc.


tilled water or isotonic phosphate buffer. The changes The polymers were prepared by direct condensation of
in the polymer during hydrolysis were monitored by mandelic acid and lactic acid at 200C under vacuum. The
weighing for water uptake and weight loss, gel permeation copolymers containing 15 to 100% MA were amorphous,
chromatography (GPC) for molecular weight change, and and the degradation rate decreased with the increase in MA
DSC and x-ray scattering for thermal properties and crys- content with no degradation observed for the MA homo-
tallinity change, potentiometry and enzymatic assays for polymer after 15 weeks in buffer at 37C.
pH change and lactic acid release, and dynamic mechanical Low molecular weight -hydroxy acid copolymers
tests for changes in mechanical properties (76). The poly- composed of 70 mole% L-lactic acid and 30 mole% DL-
mer, semicrystalline PLA lost about 50% of its mechanical hydroxy acids of the structure
strength after 18 weeks in buffer, with no weight loss until
about 30 weeks of hydrolysis. The degradation of branched
PLA was characterized as bulk erosion, like the linear
polymers (37).
Vert et al. (77) demonstrated the complexicity of PLA,
PGA, and PLGA degradation, and suggested that size de-
pendency exists for hydrolytic degradation of PLA sys-
tems. Other research efforts suggest that PLA-derived mi-
croparticles will degrade faster than nanoparticles derived
from PLA (78,79) and that this phenomenon is based on
a diffusion mechanism.
The biodegradation of branched PLA with glucose or
macromolecular polyol in rats is determined by weight loss
(37). In vitro degradation was essentially the same, indicat-
ing minimal involvement of enzymatic degradation. The
branched materials degraded much faster than the refer- were synthesized by direct polycondensation in the ab-
ence linear PLA. On the contrary, the linear PLA had a sence of catalysts (82,84). The polymers had a molecular
higher water uptake than the branched polymers. For ex- weight of 5000 and Tg in the range of 20 to 37C. These
ample, after 36 weeks the linear PLA contained about 21 polymers were evaluated in vivo for their capabilities as
wt% water, while the corresponding branched PLA con- biodegradable carriers for drug delivery systems. Small
tained only 2%. No adequate explanation was given for cylinders (2 10 mm) implanted subcutaneously in rats
this phenomenon. were 60 to 90% degraded in 15 weeks postimplantation.
The degradation of several aliphatic polyesters in the The copolymer L-LA/DL-HBA degraded the least with
form of microspheres in phosphate buffer solution at 37 a lag time of no weight loss for 5 weeks. Polymer cylin-
and 85C was reported (80). Lower molecular weight poly- ders containing luteinizing hormone releasing hormone
mers degraded faster than higher molecular weight poly- (LHRH) agonist were implanted in rats and released the
mers. Degradation at 85C resulted in a similar degradation drug for 15 weeks, with poly(L-LA/DL-HICA) being the
prole, but faster. The biodegradation of low molecular most pharmacologically effective.
weight PLA used in tablets for oral delivery of drugs was
also studied (81). 2. Polycaprolactones
The successful use of lactide and glycolide polymers in
d. Other Copolymers of Lactic Acid absorbable drug delivery systems and medical devices and
Low molecular weight homopolymers of D,L-mandelic absorbable sutures encouraged the evaluation of other
acid (MA) and its copolymers with lactic acid were re- polyesters for this purpose. The most studied polymers in
ported (82,83). this category are the polycaprolactones, polyhydroxybuty-
rates, and polymers of other -hydroxy acids. These poly-
mers were developed originally as synthetic plastics to be
degraded by microorganisms in the environment (85,86).

a. Synthesis
Poly(-caprolactone) (PCL) has been synthesized from the
anionic, cationic, and coordination polymerization of -

Copyright 2002 Marcel Dekker, Inc.


caprolactone. The synthesis of polycaprolactones has been merization of -caprolactone (CL) and lactides (LA)
recently reviewed (87,88). A schematic description of cap- allows the permeability of the PCL to be combined with
rolactone polymerization using these three types of initia- the rapid biodegradation of PLA (95).
tors is shown in Scheme 3 (87). Block copolymers of CL and LA were synthesized by
Various initiators and polymerization conditions were ring-opening polymerization of lactides (D,L and L,L) and
reported for each type of catalyst. Effective anionic reac- -caprolactone using aluminum isopropoxide as initiator
tion systems are tertiary amines, alkali metal alkoxides, (96). Block copolymerization of CL and LA was also re-
and carboxylates in tetrahydrofuran, toluene, and benzene ported by Feng and Song using bimetallic (Al, Zn -oxo
(89). The anionic method of polymerization is most use- alkoxides) initiators (97,98). Because of the difference in
ful for the synthesis of low molecular weight hydroxy- reactivity, the sequential polymerization of these two mo-
terminated oligomers and polymers (90). nomers can only be achieved when CL is rst polymerized
Known cationic catalysts in organic synthesis affect followed by the lactide (99). Formation of large amounts
cationic polymerization and include protic acids, Lewis of homo-PLA is observed and has been attributed to the
acids, acylating agents, and alkylating agents. The follow- increase in the mean degree in association of aluminum
ing agents, FeCl3, BF3, Et2O, alkyl sulfonate, and trimethyl- alkoxide in toluene from one to three in the presence of
silyl triate, have been used in 1,2-dichloroethane at 50C CL and LA, respectively. The homopolymer formation can
to yield polymers with a molecular weight range of 15,000 be prevented by the addition of a small amount of an alco-
to 50,000. High molecular weight homopolymers and ran- hol, like 2-propanol, or the use of Al derivative that bears
dom copolymers with lactides and other lactones were ob- only one alkoxide group (99).
tained using coordination catalysts such as di-n-butyl zinc, Degradable block copolymers with polyethylene glycol,
stannous octoate, and alkoxides and halids of Al, Sn, Mg, diglycolide, substituted caprolactones, and -valerolactone
and Ti. Polymerization occurs at 120C under argon to were also reviewed (87).
yield polymers with a narrow molecular weight distribu-
tion (MW/MN 1.1) and molecular weights above 50,000 b. Polymer Properties
(91,92). Polycaprolactone was also obtained from the radi-
Polycaprolactone is soluble in chlorinated and aromatic
cal polymerization of 2-methylene-1,3-dioxepane (93).
hydrocarbons, cyclohexanone, and 2-nitropropane, and it
In contrast to random copolymers, block and graft
is insoluble in aliphatic hydrocarbons, diethyl ether, and
multicomponent systems are most often multiphase mate-
alcohols. The homopolymer melts at 5964C with a Tg
rials with properties that are different from the homopoly-
of 60C. Copolymerization with lactide increases the Tg
mers or random copolymers (94). They are useful in im-
with the increase in the lactide content in the polymer
proving the phase morphology, the interfacial adhesion
(100). The crystallinity of the polymer decreases with the
and, accordingly, the ultimate mechanical properties of im-
increase in polymer molecular weight; polymer of 5000
miscible polymer blends. As an example, block copoly-
is 80% crystalline, whereas the 60,000 polymer is 45%
crystalline (101).

c. Biodegradation
The biodegradation of PCL has been extensively studied
in the past 30 years and several reviews are available
(87,102). Like the lactide polymers, PCL and its copoly-
mers degrade both in vitro and in vivo by bulk hydrolysis
(101), with the degradation rate affected by the size and
shape of the device and additives.
The kinetic equivalency of the degradation of PCL in
buffer and in rabbit was demonstrated by measuring the
polymer intrinsic viscosity for 60 weeks (87). The poly-
mers degrade in two phases. In the rst phase a random
hydrolytic chain scission occurs, which results in a reduc-
tion of the polymer molecular weight. In the second phase
Scheme 3 Polymerization of -caprolactone using (A) anionic, the low molecular fragments and the small polymer parti-
(B) cationic, and (C) coordination catalysts. cles are carried away from the site of implantation by solu-

Copyright 2002 Marcel Dekker, Inc.


bilization in the body uids or by phagocytosis, which The hydrolytic degradation of HB polymers was studied
results in a weight loss. Complete degradation and elimina- (109). Microspheres degraded slowly in phosphate buf-
tion of PCL homopolymers may last for 2 to 4 years. The fer at 85C, and after 5 months 20 to 40% of the poly-
degradation rate is signicantly increased by copolymer- mer eroded under these conditions. Copolymers having a
ization or blending with lactide and glycolide. The rate of higher fraction of 3HV and low molecular weight poly-
degradation can be increased also by the addition of oleic mers were more susceptible to hydrolysis.
acid or tertiary amines to the polymer, which catalyzes the
chain hydrolysis (103,104). 4. Polycarbonates
Poly(ethylene carbonate) and poly(propylene carbonate)
3. Poly(-hydroxybutyrate) have been tested as biodegradable carriers for the delivery
a. Synthesis of 5-uorouracil (5-FU) (110). They are linear thermoplas-
tic polyesters of carbonic acid with aliphatic dihydroxy
Poly(-hydroxybutyrate) (PHB) is made by a controlled compounds.
bacterial fermentation. The producing organism occurs
naturally. An optically active copolymer of 3-hydroxybu- a. Synthesis
tyrate (3HB) and 3-hydroxyvalerate (3HV) has been pro-
duced from propionic acid or pentanoic acid by Alcali- The polymers are synthesized from the reaction of dihy-
genes eutrophus (105): droxy compounds with phosgene or with bischlorofor-
mates of aliphatic dihydroxy compounds by transesteri-
cation and by polymerization of cyclic carbonates:

The copolymer compositions (0 to 95 mole% 3HV con-


tent) can be controlled by the composition of the carbon These polymers have been synthesized from carbon diox-
sources. Random copolymers of 3HB and 4HV were pro- ide and the corresponding epoxides in the presence of or-
duced from 4-hydroxybutyric acid and butyric acids by Al- ganometallic compounds as initiators (111).
caligenes eutrophus (106,107):
b. Biodegradation
Since the carbonate linkage may be labile to hydrolysis,
the biodegradability of polycarbonates has been stud-
ied (110,111). Pellets of poly(ethylene carbonate) and
poly(propylene carbonate) were implanted into the perito-
neal cavity of rats, and the toxicity and weight loss of poly-
b. Properties and Biodegradation.
mer pellets were determined. Poly(ethylene carbonate) was
The polymers are characterized as having a high molecular completely eliminated 15 days postimplantation, while
weight (100,000, [n] 3 dL/g) with a narrow polydis- poly(propylene carbonate) remained intact after 60 days.
persity and a crystallinity of around 50%. The melting When pellets of the polymers were incubated in phosphate
point depends on the polymer composition; P(3HB) homo- buffer pH 7.4 and 37C, both polymers did not degrade
polymer melts at 177C with a Tg at 9C, the 91: 9 copoly- even after 40 days. These data indicate that poly(ethylene
mer with 4HB melts at 159C, and the 1: 1 copolymer with carbonate) was degraded by enzymes. No visible inam-
3HV melts at 91C. The PHB properties in the living cells matory reaction was noted at the implantation sites. In vitro
of Alcaligenes eutrophus were determined using x-ray and release of 5-FU from poly(ethylene carbonate) pellets con-
variable-temperature 13C NMR relaxation studies (108). taining 20% 5-FU was poor, 20% of the drug was released
Polyhydroxybutyrate is an amorphous elastomer with a Tg in 2 h, and only an additional 15% of 5-FU were released
around 40C in its native state within the granules. during the following 60 days. Better release proles were
The biodegradation of these polymers in soil and activated obtained when poly(propylene carbonate) was used.
sludge show the rate of degradation to be in the following Copolymers of aliphatic carbonates and lactide showed
order: P(3HB-co-9% 4HB) P(3HB) P(3HB-co-50% excellent biocompatibility and mechanical properties.
3HV). Block copolymers of trimethylene carbonate (TMC) and

Copyright 2002 Marcel Dekker, Inc.


lactide were synthesized from the reaction of the mono- acid groups in the polymer chains can be used to alter wa-
mers with stannous octoate as catalyst at 160C for 16 h: ter swellability and solubility of the polymers. Biocompati-
bility evaluation in mice indicated that poly (-malic acid)
is nontoxic (115).
A series of polyesters, poly(propylene fumarate) (PPF),
based on the reaction product of fumaric acid (116,117),
and tartaric acid (118,119) with different aliphatic diols
have also been evaluated as drug carriers. Poly(propylene
The polymers were soluble in common organic solvents fumarate) with acrylate and epoxide terminal groups was
and had a weight average molecular weight of 90,000. produced by Domb et al. (120). The polymers were cross-
They completely degraded in vivo in 1 year (112). linked in a ratio of 30 wt% PPF to 70 wt% calcium carbon-
atetricalcium phosphate mixture and were synthesized by
5. Other Polyesters a direct melt condensation of the acids with the diols with
Poly(dihydropyrans) were developed for contraceptive de- acid catalysis.
livery. The in vivo and in vitro release of contraceptive
steroids and antimalarial agents from polymer matrices has B. Poly(amides)
been studied (113). 1. Synthesis and Biodegradation
The utilization of amide-based polymers, especially natu-
ral proteins, in the preparation of biodegradable matrices
has been extensively investigated in recent years (121).
Microcapsules and microspheres of crosslinked collagen,
gelatin, and albumin have been used for drug delivery
(122). The synthetic ability to manipulate amino acid se-
Poly(p-dioxanone) is clinically used as an alternative to quences has seen its maturity over the last two decades,
poly(lactide) in absorbable sutures with similar properties with new techniques and strategies continually being intro-
to poly(lactide) with the advantage of better irradiation sta- duced. Poly(amides) such as poly(glutamic acid) and poly-
bility during sterilization (114): (lysine) and their copolymers with various amino acids
have also been studied as drug carriers (123,124). Re-
cently, Nathan and Kohn (125) have excellently reviewed
the history of amino acidderived polymers.
Pseudopoly(amino acids), a new approach for biomate-
rials based on amino acids, were rst suggested by Kohn
This polymer has not yet been developed as a carrier for and Langer (126,127), who prepared a polyester from N-
controlled drug delivery. protected trans-4-hydroxy-L-proline, and poly(iminocar-
Biodegradable polymers derived from naturally occur- bonate) from tyrosine dipeptide as monomeric starting ma-
ring, multifunctional hydroxy acids and amino acid have terial. The structures of poly(N-acylhydroxyproline esters)
been investigated by Lenz and Guerin (115). The mono- and a homologous series of tyrosine-derived polymers is
mers, malic acid, and aspartic acid were polymerized into described in Scheme 4 (121).
a polyester or polyamide using a ring-opening polymeriza- The properties, biodegradability, drug release, and bio-
tion process as follows: compatibility of this class of polymers have been reviewed
(121,122). Biodegradable polyamides for use in controlled
delivery of drugs were obtained from the reaction of 2,2-
bis[5(4HO-oxazolones] and alkane diamines (128). Drug
release time from these polymers was short, less then 24 h.
Copolymers of glutamic acid and ethylglutamate were
used for the delivery of naltrexone (129). The poly-
The molecular weights of the polymers were highly de- mers were synthesized in three steps: (1) synthesis of N-
pendent on the purity of the cyclic monomers. Polymers carboxyanhydrides of -benzyl-L-glutamate and -ethyl-
of 50,000 were obtained in 93% polymerization yield when L-glutamate; (2) polyamidation of the benzyl blocked
very pure monomers were used. Both polymers are water monomers; and, (3) debenzylation of the intermediate.
soluble; however, crystallinity and controlled number of Poly(amino acids) are generally hydrophilic, and their deg-

Copyright 2002 Marcel Dekker, Inc.


The in vitro enzymatic degradation using pronase E as
protease showed that these polymers degrade from the sur-
face, and the polymers were completely dissolved in from
several hours to several days as a function of polymer
structure and enzyme concentration. Negligible weight
loss was detected without the enzyme.
Scheme 4 Molecular structure of pseudopoly(amino acids). Random copolymers of the -amino acids N-(3-hydro-
(A) Poly(N-acylhydroxyproline esters); (B) Tyrosine-derived
xypropyl)-L-glutamine and L-leucine were synthesized
polyiminocarbonate, poly(CTTE), x 1; poly(CTH), x 4;
poly(CTTP), x 15.
and used as carriers for naltrexone (134). Naltrexone was
covalently bound through the 3-phenolic or the 14-tertiary
hydroxyls to the polymer hydroxyl side chains via a car-
bonate bond. Naltrexone was released from the polymer
in a relatively constant way for 30 days, in both in vitro
radation rates are dependent upon hydrophilicity of the
and in vivo experiments in rats (134). Tyrosine-derived
amino acids.
poly(carbonates) are readily processible polymers that sup-
The degradation rate increases as the glutamic acid con-
port the growth and attachment of the cells and have also
tent increases. For example, copolymer containing 13
shown a high degree of tissue biocompatibility (135,136).
mole% glutamic acid remained intact for more than 79
In vitro degradation of these compounds occurs due to hy-
days, while the 40% copolymer disintegrated in 7 days.
drolysis of the pandent ester bonds and the iminocarbon-
Tubular capsules of 18 :82 glutamic acid/ethylglutamate
ate bonds of the backbone (136). Degradation goes over a
copolymer were used as a reservoir implant. The copoly-
period of months and rates are comparable to the degrada-
mer was biocompatible and completely degraded in 90
tion time of poly(L-lactic acid).
days. The degradation process involves hydrolysis of the
ethyl esters followed by hydrolysis of the peptide bonds
to produce glutamic acid, which enters the metabolic cycle. C. Poly(phosphate Esters)
Amino acids are attractive due to their functionality by
which they provide a polymer. Recently, poly(lactic acid- Poly(phosphoester) has been studied as a potential biode-
co-lysine) (PLAL) was synthesized using a stannous oc- gradable matrix for drug delivery (137,138). The polymers
toate catalyst from lactide and a lysine-containing mono- were synthesized from the reaction of ethyl or phenyl phos-
mer analogous to lactide (130). Inclusion of the amino acid phorodichloridates and various dialcohols including bis-
lysine provides an amino group that allows for further phenol A and poly(ethylene glycol) of various molecular
modication of the PLAL system. In a recent report, Cook weights:
and coworkers (131) have successfully attached a peptide
sequence which promotes cell adhesion to PLAL. The use
of N-carboxyanhydrideactivated amino acids was the rst
efcient method for production of amino acid homopoly-
mers. In a further study, they have exploited the PLAL
system by the reaction with lysine N-carboxyanhydride de- Interfacial condensation using a phase transfer catalyst
rivatives to increase the systems functionality with a poly- and bisphenol A as comonomer yielded polymers with a
(lysine) graft. Poly(lactic acid-co-lysine) has been formu- weight average molecular weight around 36,000. Leong et
lated into microspheres that exhibit deep lung delivery al. have incorporated phosphoester groups into poly(ure-
from porous particles (132). thanes) (139). Poly(urethanes) have been used as blood
Copolymers of n-hydroxyalkyl-L-glutamine and - contacting biomaterials due to having a broad range of
methyl-L-glutamate were synthesized from the reaction of physical properties that can be obtained, from hard and
poly(-methyl-L-glutamate) and 2-amino-1-ethanol or 5- brittle to soft and tacky. Leong et al. designed inert bioma-
amino-1-pentanol as follows (133): terial for controlled release applications by introducing

Copyright 2002 Marcel Dekker, Inc.


terization, and medical applications of polyphosphazenes
was published by Allcock (144).

1. Synthesis and Biodegradation


The polymers are most commonly synthesized by a substi-
tution reaction of the reactive poly(dichlorophosphazene)
with a wide range of reactive nucleophiles such as amines,
alkoxides, and organometallic molecules (Scheme 6). The
reaction is carried out in general at room temperature in
tetrahydrofuran or aromatic hydrocarbone solutions. Poly-
Scheme 5 Synthesis of poly(phosphoester-urethanes). mers containing mixed substituent can be obtained from
the sequential or simultaneous reaction with several nu-
cleophiles (Scheme 6) (145).
The properties of the polymers depend on the nature of
phosphoester linkage in poly(urethane). Introduction of the side groups. Hydrolytically degradable polyphospha-
phosphoester linkage does not change the mechanical zene was obtained when amino acid and imidazole de-
properties inherent in the poly(urethanes) and provides an rivatives were used as substituents (142,146). The rst
excellent biodegradable material. bioerodible polymer was the ethylglycinato derivative
Poly(phosphoester-urethanes) are obtained by the reac- (R C NHCH2COOEt), which hydrolytically degrades to
tion of polydiols and di-isocyanates with phosphates as ethanol, glycine, phosphate, and ammonia (146). In vitro
chain extenders as shown in Scheme 5. degradation studies on poly(phosphazenes) with different
Phosphoester bonds are readily cleaved under physio- amino acid derivatives show that it takes several months to
logical conditions, and hydrolysis of poly(phosphoester- degrade the material depending on the amino acid present
urethanes) leads to phosphates, alcohols, amines, and car- (147). Allcock et al. (148,149) synthesized three differ-
bon dioxide, which makes it an excellent biomaterial for ent poly[(amino acid ester)phosphazenes]: poly[di(ethyl-
drug delivery applications. Leong et al. observed that the glycinato)phosphazene], poly[di(ethylalanato)phophazene],
release kinetics of poly(phosphoester-urethanes) were in- and poly[di(benzylalano)phosphazene]. According to the
uenced by the side chains attached via the phosphoester order of polymers, they show a tendency to decrease the
of the polymer backbone and thus pentavalency of the molecular weight and mass loss with release of small mole-
phosphorus contributes a site for future functionalization cules. The release of small molecules occurred through dif-
(140). The release mechanism was dependent combinedly fusion and decomposition of polymer.
on diffusion, swelling, and degradation of polymer. Biodegradable poly(phosphazenes) that are insoluble in
The polymers based on bisphenol A release drug for a water prior to hydrolysis have been employed in the tem-
long period of time; 8 to 20% cortisone was released after poral controlled release of many drug classes including
75 days in buffer solution. The degradation rate depends nonsteroidal anti-inammatory agents and peptides (150
on the nature of the polymer side chain; polymers with 153). Amino acid estersubstituted polyphosphazenes
phenyl side chains degrade much slower than those con- have been used for controlled release of the covalently
taining ethyl or ethoxyethyl side chains (138). The in vivo
degradation of these polymers in rabbits was faster than
in vitro.

D. Polyphosphazenes

The uniqueness of the polyphosphazenes stems from the


inorganic backbone (N C P), which with certain organic
side groups can be hydrolyzed to phosphate and ammonia.
Several polymer structures have been used as matrix carri-
ers for drugs (141,142) or as a hydrolyzable polymeric
drug, where the drug is covalently bound to the polymer
backbone and released from the polymer by hydrolysis
(143). A comprehensive review on the synthesis, charac- Scheme 6 Synthesis and hydrolysis of polyphospazenes.

Copyright 2002 Marcel Dekker, Inc.


bonded anti-inammatory agent naproxen. Steroids having much faster than in the interior of the device. To exhibit
a hydroxyl group were bound to the polymer chain through such a phenomenon, the polymer has to be extremely hy-
the hydroxyl group (154). drophobic with very labile linkages. Hence, it was envi-
Poly(organophosphazenes) have been synthesized that sioned that polymeric devices with an orthoester linkage
possess amino acid side groups. The mechanical properties in the backbone, which is an acid-sensitive linkage, could
and rates of degradation have been controlled by appro- provide a surface-eroding polymer if the interior of the ma-
priate selection of amino acid side chain groups (155). The trix is buffered with basic salts.
versatility of these polymers has been demonstrated by the
formation of 200-nm diameter poly(organophosphazene) 1. Synthesis
nanoparticles that present covalently coupled poly(ethyl-
ene glycol) at their surface (156). The rst poly(orthoesters) were reported in a series of pat-
Imidazolyl-substituted polyphosphazenes are hydrolyti- ents by Choi and Heller (160163), assigned to Alza Cor-
cally unstable and hydrolyze in room moisture (142). The poration. These proprietary polymers were rst designated
rate of hydrolysis can be slowed by the incorporation of as Chronomer and later as Alzamer. They were prepared
hydrophobic side groups such as phenoxy or methylphe- by a transesterication reaction, as follows (164):
noxy groups (141). The in vivo and in vitro release of
progesterone and bovine serum albumin (BSA) from
imidazole, 4-methylphenoxysubstituted polyphospha-
zene matrices were reported (141). Almost 90% of the
loaded progesterone was released in 30 days with about
60% released in 8 days when placed in phosphate buffer The general synthesis involved the heating of the reaction
pH 7.4 at 37C. An in vivo release study in rats using radio- mixture to 110 to 115C for about 1.5 to 2 h and then
labelled progesterone demonstrated a zero-order release further heated at 180C and 0.01 torr for 24 h. The synthe-
for 30 days. Various hydrophilic and hydrophobic poly- sis of such polymers, with a minimal amount of cross-
mers, hydrogels, water-soluble polymers, bioactive poly- linkage, requires an orthoester starting material in which
mers, and various drugpolyphosphazene conjugates have one alkoxy group has a greatly reduced reactivity. This can
been reviewed (144). be achieved by using a cyclic structure as shown in the
A number of approaches have been proposed to gener- reaction.
ate crosslinked polyphosphazene for temporal controlled Hydrolysis of these polymers regenerates the diol and
release. Andianov and coworkers have synthesized poly v-butyrolactone (165). The -butyrolactone rapidly hydro-
[bis(carboxylatophenoxy)-phosphazene] crosslinked with lyzes to -hydroxybutyric acid. The production of -hydroxy-
Ca2 ions for an ionically stabilized system (157). This butyric acid would further catalyze the breakdown of
polymer allowed drug molecules to be encapsulated into orthoester linkages leading to bulk erosion of the matrix.
poly(phosphazene) microspheres under mild environmen- Thus it was decided to incorporate basic salts into the poly-
tal conditions. Recently, pH-sensitive hydrogels have been mer matrix to neutralize the generated acid and keep the
synthesized by the formation of poly(phosphazenes) with hydrolysis process under control. These Alzamer materials
oxybenzoate and methoxyethoxy side group. Swelling at were investigated as bioerodible implants for the release
different pH values was controlled by varying the ratios of naltrexone and contraceptive steroids. Human clinical
of the two side groups (158). trials of the steroidal implant revealed local tissue irri-
tation, and thus further work was discontinued (166).
E. Poly(orthoesters) Recently the use of these polymers for the release of
indomethacin in the prevention of reossication of experi-
Poly(orthoesters) were invented during the pursuit of de- mental bone defects was reported (167).
veloping a bioerodible polymer, subdermally implantable, Subsequently, another family of poly(orthoesters) was
that would release contraceptive steroids by close to zero- developed not related to Alzamer (168). These polymers
order kinetics for at least 6 months (159). An additional are prepared by the addition of polyols to diketene acetals.
objective was that the polymer erosion and drug release The general reaction can be schematically represented as
should be concomitant so that no polymer remnants are follows:
present in the tissue after all the drug has been released.
These objectives could only be met if the polymer were
truly surface eroding. For a surface-eroding polymer, the
erosion process at the surface of the polymer should be

Copyright 2002 Marcel Dekker, Inc.


Initial work was conducted with the monomer, diketene
acetal, derived form pentaerythritol, and 1,6-hexanediol.
The reaction is exothermic and proceeds to completion vir-
tually instantaneously. The reaction is as follows:

Scheme 7 Synthesis of crosslinked polyorthoesters.

The general method used for the synthesis of cross-


linked polymers was by reacting prepolymer with the triols
or a mixture of diols and triols (Scheme 7) (171).
where R H, for 3,9-bis(methylene 2,4,8,10-tetra ox- The prepolymer is an acetal with a diol and is a viscous
aspiro [5,5] undecane). The diols investigated were 1,6- liquid at room temperature. Thus, the compound of interest
hexanediol trans-1,4-cyclohexane dimethanol, 1,6-cyclo- could be incorporated into the prepolymer along with the
hexanediol, ethylene glycol, and biophenol A. triol, and the mixture can be crosslinked at temperatures as
low as 40C. This can be a good method for incorporating
2. Polymer Properties thermolabile drugs. However, one should be cautious with
using compounds with hydroxyl functionality.
The molecular weight of poly(orthoesters) were signi- In a series of experiments, Heller et al. (172) have re-
cantly dependent on the type of diol and catalyst used for ported the family of poly(orthoesters) which can be pre-
synthesis. A linear, exible diol like 1,6-hexanediol gave pared by reacting a triol with two vicinal hydroxyl groups
molecular weights greater than 200 K, whereas bisphenol and one removed by at least three methylene groups with
A in the presence of catalyst gave molecular weight only an alkyl orthoacetate as shown:
around 10,000 (169).
Mechanical properties of the linear poly(orthesters) can
be varied over a large range by selecting various composi-
tions of diols. It was shown that the glass transition temper-
ature of the polymer prepared from DETOSU can be varied
from 25 to 110C by simply changing the amount of 1,6-
hexanediol in trans-1,4-cyclohexane dimethanol from 100
to 0% (170). There seems to be a linearly decreasing rela-
tionship between the Tg and percentage of 1,6-hexanediol.
One could take advantage of this relationship in selecting
the polymer for in vivo applications because in vivo the
The use of exible triols such as 1,2,6-hexanetriol pro-
Tg of the polymer would drop due to inbibition of water.
duces highly exible polymers that have ointmentlike
This can result in the loss of stiffness and rigidity of the
properties even at relatively high molecular weights. How-
polymer.
ever, properties such as viscosity, and hydrophobicity can
be readily varied by controlling molecular weight and the
3. Crosslinked Poly(orthoesters)
size of the alkyl group R. This polymer has an oint-
To prepare a crosslinked polymer, there should be at least mentlike consistency at room temperature and is applicable
one monomer which has a functionality greater than 2. In where sensitive therapeutic agents such as proteins are in-
case of poly(orthoesters), it is possible for a ketene acetal corporated into the polymer without use of solvent.
or an alcohol to have a functionality greater than 2. Due In another experiment, Heller et al. replaced the exible
to the difculty in preparing trifunctional ketene acetals, triol to a rigid one such as 1,1,4-cyclohexanetrimethanol
triols were used to prepare the crosslinked polymer. to obtain a solid poly(orthoester), as shown:

Copyright 2002 Marcel Dekker, Inc.


Wuthrich et al. (176) proposed the hydrolysis of poly
(orthoesters) derived from 1,2,6-hexanetriol as follows:

4. Polymer Hydrolysis
The primary mechanism for the degradation of poly
(orthoesters) is via hydrolysis. Depending on the reactants This initial hydrolysis is proceeded at a slow rate to pro-
used during the synthesis of the polymer, the hydrolysis duce a carboxylic acid and a triol, thus no autocatalysis is
products are diol, or a mixture of diols, and pentaerythritol observed.
dipropronate or diacetate if 3,9-bis(methylene-2,4,8,10-tet-
raoxaspiro [5,5]undecane) was used (144). The pentaeryth- 5. Polymer Processing
ritol esters hydrolyze at a slower rate to pentaerythritol and
the corresponding acetic or propionic acid. The sequence The orthoester linkage is inherently unstable in the pres-
of reaction is as follows: ence of water. However, because of the polymers highly
hydrophobic nature, they can be stored without careful ex-
clusion of moisture.
Even though the polymer is relatively stable in trace
amounts of moisture, it is unstable to heat and undergoes
disproportionation to an alcohol and ketene acetal. The
combination of moisture and heat can be fatal for the pro-
cessing of poly(orthoesters), which are designed to erode
within days (177). Thus, if injection molding is necessary
to fabricate the device, then moisture must be rigorously
excluded during fabrication. One should also consider the
interaction between the incorporated anhydride as cata-
lysts, the polymer, and the drug during the thermal pro-
The difference in the sensitivity of the hydrolysis of cessing.
orthoester linkages in an acid versus alkaline medium has In one of the studies it was shown that phthalic anhy-
been used to advantage in designing the orthoester-based dride reacted with the free hydroxyl end groups of the
delivery systems. Incorporating acid anhydrides into the polymers. Using high-performance liquid chromatography
matrix to accelerate the rate of hydrolysis uses this prefer- and infrared spectroscopy the formation of half phthalate
ential sensitivity. While, on the other hand, a base is used esters of 1,6-hexanediol and trans-cyclohexane dimethanol
to stabilize the interior of the matrix. was conrmed (178). The reaction led to the decrease in
Acid-catalyzed hydrolysis of these polymers can be concentration of phthalic anhydride, leading to increased
controlled by introducing acidic and basic excipients into time for erosion of the matrix. The preferred method for
the matrices. Rate of hydrolysis can be increased by the fabricating the devices would be under low thermal and
addition of acidic excipients, e.g., suberic acid, as demon- shear stresses (179). This would include solution mixing
strated by the zero-order release of 5-uorouracil over a or powder blending followed by compression molding of
15-day period (173). Alternatively, basic excipients stabi- the devices.
lize the bulk of the matrix but diffuse out of the surface
region. This approach has been used in the temporal con- F. Polyanhydrides
trolled release of tetracycline over a period of weeks in the
treatment of periodontal disease (174). Ng et al. described A large number of biodegradable polymers have been in-
the synthesis of self-catalyzed poly(orthoesters) that con- vestigated as carriers in the design of controlled drug deliv-
tain glycolide sequences and can be degraded hydrolyti- ery systems. It has been generally recognized that the
cally without excipient catalysis (175). matrix should undergo heterogeneous degradation to max-

Copyright 2002 Marcel Dekker, Inc.


imize the control over release process. In the early 1980s, temperature, and an efcient system to remove the byprod-
Rosen et al. (180) envisioned the use of hydrophobic poly- uct, acetic anhydride. The highest molecular weight poly-
anhydrides in designing the surface-eroding matrix for ap- mers were obtained using pure isolated prepolymers and
plications in controlled drug delivery. However, the inven- heating them at 180C for 90 min with a vacuum of 104
tion and development of polyanhydrides dates as far back mmHg, using a dry ice/acetone trap. Molecular weights in
as the early 1900s. excess of 125,000 were obtained.
In 1909, Bucher and Slade (181) reported the develop- Signicantly higher molecular weights were obtained
ment of aromatic polyanhydrides composed of isophthalic in shorter times by using coordination catalysts such as
acid and terephthalic acid. Subsequently, Hill and Caroth- cadmium acetate, earth metal oxides, and ZnEt2H2O (159).
ers (182,183) reported a series of aliphatic polyanhydrides. The weight average molecular weight varied from 90,000
Systematic development of polyanhydrides as substitutes to 240,000 when the concentration of cadmium acetate was
for polyesters in textile applications was undertaken ini- changed from 0.5 to 3 mole%, with the reaction time of
tially by Conix (184,185) and then followed by Yoda and less than 1 h. Other catalysts, such as titanium and iron,
Miyake (186,187). They prepared and studied a number of inhibited the polymerization reaction and the polymers
aromatic and heterocyclic polyanhydrides. These polymers were dark brown in color. Acidic catalysts, such as p-tolu-
were not suitable for textile applications because of the ene sulfonic acid, did not show any effect on polymer mo-
extreme reactivity of anhydride linkage toward water. The lecular weight, while the basic catalyst 4-dimethyl amino
fact that they are extremely hydrophobic and hydrolyti- pyridine caused a decrease in molecular weight.
cally unstable renders them useful in drug delivery applica-
Solution polymerization. Syntheses of polyanhydrides
tions (187189).
using melt polycondensation is useful to obtain high mo-
lecular weight polymers but is not useful if the monomers
a. Synthesis are thermolabile. Hence, methods were developed to syn-
Melt polycondensation. The majority of the polyan- thesize polyanhydrides under ambient conditions for heat-
hydrides are prepared by melt polycondensation. The se- sensitive monomers such as dipeptides and therapeutically
quence of reaction involves rst the conversion of a dicar- active diacids.
boxylic acid monomer into a prepolymer consisting of a The solution polymerization is carried out by the Scot-
mixed anhydride of the diacid with acetic anhydride. This tenBaumann technique. In this method the solution of di-
is achieved by simply reuxing the diacid monomer with acid chloride is added dropwise into an ice-cooled solution
acetic anhydride for a specied length of time. The poly- of a dicarboxylic acid. The reaction is facilitated by using
mer is obtained subsequently by heating the prepolymer an acid acceptor such as triethylamine. Polymerization
under vacuo to eliminate the acetic anhydride (190): takes place instantly on contact of the monomers and is
essentially complete within 1 h. The solvents employed
can be a single solvent or a mixture of solvents like dichlo-
romethane, chloroform, benzene, and ethyl ether. It was
found that the order of addition is very important in ob-
taining relatively high molecular weight polyanhydrides.
Addition of a diacid solution dropwise to the diacid chlo-
ride solution consistently produced high molecular weight
polymers (192):

The drawback of this homogeneous SchottenBau-


mann condensation reaction in solution is that the diacid
This procedure was used by most of the early investigators. chloride monomer should be of very high purity. An alter-
The polyanhydride thus obtained was of low molecular native approach was the conversion of dicarboxylic acid
weight. For most practical applications high molecular monomer into the polyanhydride using a dehydrative cou-
weight polyanhydrides are desirable. Hence, a systematic pling agent under ambient conditions. The dehydrative
study was undertaken to determine the factors that affected coupling agent, NN-bis[2-oxo-3-oxazolidinyl]phosphonic
the polymer molecular weight (191). It was found that the chloride was the most effective in forming polyanhydrides
critical factors were monomer purity, reaction time and with the degree of polymerization around 20 (193). It is

Copyright 2002 Marcel Dekker, Inc.


essential that the catalyst be ground into ne particles be- bility compared to the corresponding homopolymers of ar-
fore use and should be freshly prepared. A disadvantage omatic diacids (199).
of this method is that the nal product contains polymer- The data on mechanical properties of polyanhydrides
ization byproducts which have to be removed by washing are very limited. The bers of poly[1,2-bis(p-carboxyphe-
with protic solvents such as methanol or cold dilute hydro- noxy) ethane anhydride] showed a tensile strength of 40
chloric acid. The washing by protic solvents may evoke kg/mm2 with an elongation of 17.2% and a Youngs mod-
some hydrolysis of the polymer. ulus of 505 kg/mm 2. A systematic study on the tensile
Coupling agents such as phosgene and diphosgene strength of the copolymers of CPP and SA showed that
could also be used for the polyanhydride formation. Poly- increasing the CPP content in the copolymer or the mo-
merization of sebacic acid using either phosgene or diphos- lecular weight of the copolymer increased the tensile
gene as coupling agents with the amine based hetero- strength (177). Unsaturated polyanhydrides of the structure
geneous acid acceptor poly(4-vinyl pyridine) produced [E(OOCECHCCHECO)xE(OOCERECO)y]n were
higher molecular weights in comparison to nonamine het- developed to improve the mechanical properties of the
erogeneous base K2CO3 (194). polymers. The advantage of the unsaturated polyanhy-
drides is that they can undergo secondary polymerization
Ring-opening polymerization. Ring-opening poly-
of the double bonds to create a crosslinked matrix (200)
merization (ROP) takes place in two steps; the rst step
is preparation of the cyclic monomer and the second is
polymerization of the cyclic monomers. Albertsson and c. Polymer Hydrolysis
coworkers prepared adipic acid polyanhydride from cyclic
Anhydride linkage is extremely susceptible to hydrolysis
adipic anhydride (Oxepane-2,7-dione) using cationic [e.g.,
in presence of moisture to generate the dicarboxylic acids
AlCl3 and BF3(C2H5)2O] anionic (e.g., CH3COOK and
(201). Hydrolysis of monomeric anhydrides is catalyzed
NaH) and coordination-type inhibitors such as stannous-
by both acid and base, the hydrolytic degradation rate of
2-ethylhexanoate and dibutyltinoxide (195197).
polyanhydrides increases with increase in pH. It is believed
that the poor solubility of the oligomeric products, under
b. Polymer Properties
low pH conditions, formed at the surface of the matrix im-
Almost all polyanhydrides show some degree of crystallin- pedes the degradation of the core. In general, the hy-
ity as manifested by their crystalline melting points. An in drophobic polymers such as P(CPP) and P(CPH) display
depth x-ray diffraction analysis was conducted with the ho- constant erosion kinetics. The degradation rates of the
mopolymers of sebacic acid (SA); bis(carboxyphenoxy) polyanhydrides can be altered in a number of ways. The
propane (CPP); bis(carboxyphenoxy)hexane (CPH) and degradation rates can be enchanced by incorporating the
fumaric acid; and the copolymers of SA with CPP, CPH, aliphatic monomer, such as sebacic acid, into the polymer.
and fumaric acid. The results indicated that the homopoly- The degradation can be slowed by increasing the methy-
mers were highly crystalline and the crystallinity of the lene groups into the backbone of the polymer. For exam-
copolymers was determined, in most cases, by the mono- ple, in the case of the poly[bis(p-carboxyphenoxy)alkane]
mer of highest concentration. Copolymers with a composi- series, increasing the methylene groups from one to six
tion close to 1:1 were essentially amorphous (198). increased the hydrophobicity of the polymer, and the ero-
The melting point, as determined by differential scan- sion rates underwent a decrease of three orders of magni-
ning calorimetry, of aromatic polyanhydrides are much tude.
higher than the aliphatic polyanhydrides. The melting To achieve a variety of degradation rates, aliphatic aro-
point of the aliphatic aromatic copolyanhydrides is propor- matic homopolyanhydrides of the structure E(OOCE
tional to the aromatic content. For this type of copolymer C6H4EO(CH2)xECOE)n were prepared with x varying
there is characteristically a minimum Tm between 5 to 20 from 1 to 10. Increasing the value of x decreases the ero-
mole% of the lower melting component (191). sion rates (202). Increased erosion rates were also observed
The majority of polyanhydrides dissolves in solvents when poly(sebacic acid) was branched with either 1,3,5-
such as dichloromethane and chloroform. However, the ar- tricarboxylic acid or low molecular weight poly(acrylic
omatic polyanhydrides display much lower solubility than acid) (203).
the aliphatic polyanhydrides. In an attempt to improve the Apart from the reactivity of anhydride linkage toward
solubility and decrease the Tm, copolymers of two different water, aliphatic polyanhydrides and their copolymers are
aromatic monomers were prepared. These copolymers dis- found to undergo self-depolymerization under anhydrous
played a substantial decrease in Tm and an increase in solu- conditions in the solid state and in solution (204). The de-

Copyright 2002 Marcel Dekker, Inc.


polymerization reaction mainly affects the high molecular A typical example of such an aromatic aliphatic mono-
weight fraction of the polymer. Aromatic homopolymers mer is the one obtained by the reaction of trimellitic anhy-
show no sign of depolymerization when stored under anhy- dride with glycine:
drous conditions. The depolymerization rate is found to
follow a rst-order kinetics, accelerate with temperature,
and increase with polarity of the solvent (204). The depoly-
merized polymer can be repolymerized to yield the original
polymer, suggesting that inter- or intramolecular anhydride
interchange takes place during depolymerization.

d. Polymer Processing
Drug-incorporated matrices can be formulated either by
compression or injection molding. The polymer and drug
can be ground in a Micro Mill grinder, sieved into a parti-
cle size range of 90120 m and can be pressed into circu-
lar discs using a Carver press. Alternatively, the drug can
be mixed into the molten polymer to form small chips of
For the synthesis of polyanhydride, the aliphatic aro-
drugpolymer conjugate. These chips are fed into the in-
matic diacid is rst converted to the diacetyl derivative
jection molder to mold the drugpolymer matrix into the
by reuxing the diacid in the presence of excess acetic
desired shaped device. One must consider the thermal sta-
anhydride. The diacetyl derivative is polymerized either by
bility of the polymer and potential chemical interaction be-
melt polycondensation or in solution. The polyanhydride-
tween drug and polymer at the high temperatures of injec-
imides thus obtained are very soluble in polar organic sol-
tion molding.
vents. However, they showed melt transitions at tempera-
The preferred method of drug delivery, in many in-
tures of 245C and above (209). Along with that, insuf-
stances, is by injection. This requires the development of
cient data are available on the hydrolytic stability of these
microcapsules or microspheres of the drug. Several differ-
materials rendering them questionable materials as a car-
ent techniques have been developed for the preparation of
rier in drug delivery systems.
microspheres from polyanhydrides, including hot-melt
On similar lines, another research group has developed
microencapsulation (205) and solvent removal technique
polyanhydrides containing amido groups. The polyanhy-
(206).
drides thus synthesized were of relatively low molecular
weight, low melting points, and a glass transition higher
e. Other Polyanhydrides
than the room temperature (210). The same group also de-
In addition to the previously discussed aliphatic aromatic veloped polyanhydrides containing ester groups (211).
and the copolyanhydrides of the respective diacids, several These polymers are in the early stages of development and
other modications of the backbone of the polyanhydrides have not been characterized with respect to their suitability
have been reported. These new polyanhydrides were devel- in drug delivery systems.
oped to improve their physicochemical, mechanical, ther- Another class of polyanhydrides are based on natural
mal, and hydrolytic properties. fatty acids. The dimers of oleic acid and eurucic acid are
One of these new polyanhydrides is polyanhydride- liquid oils containing two carboxylic acids available for
imides, also referred to as copolyimides (207). They anhydride polymerization. The homopolymers are viscous
showed good thermal resistance but were essentially insol- liquids. Copolymerization with increasing amounts of se-
uble in most organic solvents (208). In an attempt to im- bacic acid forms solid polymers with increasing melting
prove the solubility in more polar solvents, polyanhydrides points as a function of SA content. The polymers are solu-
were synthesized using imide-diacids containing aliphatic ble in chlorinated hydrocarbones, tetrahydrofuran, 2-buta-
aromatic characteristics. A systemic study was reported in none, and acetone (212).
which the starting monomers, imide-diacids, were pre- Polyanhydrides synthesized from nonlinear hydropho-
pared from aromatic acid anhydrides and x-amino acids. bic fatty acid esters based on ricinoleic, maleic, and sebacic
Varying the number of methylenic units in the -amino acid possessed desired physicochemical properties such as
acids provided the variability in the aliphatic character of low melting point, hydrophobicity, and exibility to the
the aromatic aliphatic monomer. polymer formed in addition to biocompatibility and biode-

Copyright 2002 Marcel Dekker, Inc.


gradability. The polymers were synthesized by melt con- The last section of this chapter reviews existing data
densation to yield lm-forming polymers with molecular about the biocompatibility and toxicity of the different
weights exceeding 100,000 (213). polymers actually available for biomedical applications.

A. Polyesters
1. Lactide/Glycolide Copolymers
Biocompatibility of monomer is considered as the founda-
tion for biocompatibility of degradable polymer systems,
not the polymer itself. Thus, PLLA is found as an excellent
biomaterial and safe for in vivo use because its degradation
product L-lactic acid is a natural metabolite of the body.
Even though PLGA is extensively used and represents the
gold standard of degradable polymers, increased local
acidity due to its degradation can lead to irritation at the
site of polymer implant. Agrawal and Athanasiou have in-
troduced a technique in which basic salts are used to con-
trol the pH in local environment of PLGA implant (215).
The properties of polyanhydrides were modied by the The feasibility of lactide/glycolide polymers as excipients
incorporation on long chain fatty acid terminals such as for the controlled release of bioactive agents is well
stearic acid in the polymer composition, which alters its proven, and they are the most widely investigated biode-
hydrophobicity and decreases its degradation rate (214). gradable polymers for drug delivery.
Since natural fatty acids are monofunctional, they would Most of the research work on the use of lactide/
act as polymerization chain terminators and control the glycolide polymers as matrices for delivery systems has
molecular weight. A detailed analysis of the polymeriza- focused on the development of injectable microsphere for-
tion reaction shows that up to about 10 mole% content of mulations, although implantable rod and pellet devices are
stearic acid, the nal product is essentially a stearic acid also being investigated.
terminated polymer. Whereas higher amounts of acetyl The lactide/glycolide copolymers have been subjected
stearate in the reaction mixture resulted in the formation to extensive animal and human trials without any signi-
of increasing amounts of stearic anhydride byproduct with cant harmful side effects (211). No evidence of inamma-
minimal effect on the polymer molecular weight, which tory response, irritation, or other adverse effects have been
remains in the range of 5000. Physical mixtures of polyan- reported upon implantation of lactide/glycolide polymer
hydrides with triglycerides and fatty acids or alcohols did devices. However some limited incompatibility of certain
not form uniform blends. macromolecules with lactide/glycolide was observed.
Lam and coworkers (216,217) studied the particles of
PLA with particles of polytetrauoroethylene (PTFE) as
control. They injected intraperitoneally in mice as well as
III. BIOCOMPATIBILITY AND TOXICITY in rats. After up to 7 days, cells were harvested from the
abdominal cavity. Microscopic examinations of cell mor-
In all the potential uses of polymeric material, a direct con- phology revealed the evidence of cell damage and death
tact between the polymer and biological tissues is evident. caused by phagocytosed PTFE particles. It was observed
Therefore, for the eventual human application of these bio- that there was more pronounced inammatory response
medical implants and devices, an adequate testing for with PLA lms than with PTFE lms in subcutaneous tis-
safety and biocompatibility of the specic polymer matrix sues of rats.
used in each case is essential. Many conventional pharmaceutical agents formulated
Whenever a synthetic polymer material is to be utilized in lactide/glycolide polymer matrices were widely studied
in vivo, the possible tissueimplant interactions must be almost two decades ago, especially as injectable micro-
taken into consideration. In the case of biodegradable ma- sphere dosage forms (1623). One of the most successful
trices, not only the possible toxicity of the polymer have lactide/glycolide drug delivery formulations, in terms of
to be evaluated, but also the potential toxicity of its degra- clinical results obtained, is the steroid-loaded injectable
dation products. microspheres for the controlled release of contraceptives

Copyright 2002 Marcel Dekker, Inc.


(218221). Many animal and clinical trials with these sys- problems in achieving long-term release have been re-
tems were performed showing very good biocompatibility. ported in several cases where the macromolecules lost
For example a 90-day female contraceptive based on nor- bioactivity in vivo after a few days, as in the case of
ethisterone as the active hormone and 85: 15 DL- lactide/glycolide copolymer containing growth hormone.
lactide/glycolide as the excipient has undergone successful A complex interaction apparently occurs in vivo between
Phase I and Phase II clinical trials in various geographic the acidic polymer and the hormone. In vitro release stud-
areas, demonstrating safety and efcacy of the formulation ies have shown that growth hormone can become insoluble
in about 300 subjects (222). when incorporated in poly(lactide) lms. Similar problems
The success of the steroid microsphere system based on in maintaining biological activity for longer than 510
lactide/glycolide matrices is probably due to the combina- days were also observed with interferonlactide/glycolide
tion of several factors: the reproducibility of the microen- polymer formulation (211). On the other hand, promising
capsulation process, the in vivo drug-release performance, results were obtained with luteinizing hormonereleasing
reliability in the treatment procedure, and the safety of the hormone (LHRH) incorporated in lactide/glycolide poly-
polymer (223). mer showing long-term delivery of the macromolecule
Lactide/glycolide implants containing naltrexone and (232,233). In general, hydrophilic polypeptides of low mo-
other narcotic antagonist agents have also been extensively lecular weight (5000) are considered quite stable in the
studied (224227). In one of these studies describing the presence of lactide/glycolide excipients and their acidic bi-
clinical evaluation of a bead preparation containing 70% oerosion byproducts.
naltrexone and 30% of a 90 :10 lactide/glycolide copoly- Lactide/glycolide polymer implants, in the form of mi-
mer, a local inammatory reaction at the site of implanta- crobeads and pellets, containing insulin were reported to
tion was reported in two of three subjects after subcutane- be effective in lowering blood glucose levels in diabetic
ous implantation of the beads containing the drug (226). rats for about 2 weeks, and no adverse effects, including
This nding prevented further clinical testing of that par- inammation at the implant site or deactivation of the mac-
ticular formulation. No similar problems were reported romolecule, were observed (234).
with other lactide/glycolide polymer preparations, and that Toljan and Orthner (235) published details of the clini-
incident was related to some unique aspect of that product cal use of an interference screw made from PGL copoly-
(226). mer. After 6 months, MRI data show the presence of local
Bergshma et al. (228) reported the use of a copolymer reactions at the implant site and a liquidization of the
made of 96% L- and 4% D-lactic acid in subcutaneous screw in 41% of the patients. Clinically, no febrile epi-
tissues of rats for up to 52 weeks. Prior to implantation, sodes or sterile effusions were observed.
discs made from the copolymer and homopolymer PLA Bezwada et al. (13) studied in vitro and in vivo biocom-
were predegraded in vitro (229). The degradation of co- patibility and efcacy of block coplymer of poly(glyco-
polymer was faster than the homopolymer while inam- lide) and PCL in the form of MONOCRYLR sutures. Using
mation was observed with both (230). the size 2/0 monolament sutures implanted subdermally
Good biocompatibility data were also reported with in rats, MONOCRYL sutures retain 50% of their original
lactide/glycolide copolymer matrices containing antineo- strength after about 1 week, whereas homopolymeric PCL
plastic drugs, antibiotics, and anti-inammatory com- monolament size 2/0 sutures retain 50% of their strength
pounds (211). after about 52 weeks.
The delivery of therapeutic molecules to the brain has
been limited in part due to the presence of the bloodbrain
2. Poly(caprolactone)
barrier. The biocompatibility of lactide/glycolide copoly-
mer in the brain was examined regarding the gliotic re- The biocompatibility and toxicity of poly(caprolactone)
sponse following implants of lactide/glycolide copolymer have mostly been tested in conjuction with evaluations of
into the brains of rats. It was found that lactide/glycolide Capronor, which is an implantable 1-year contraceptive
copolymer is well tolerated following implantation into the delivery system composed of a levonorgestrel-ethyl oleate
CNS and that the astrocytic response to lactide/glycolide slurry within a poly(caprolactone) capsule. In a prelimi-
copolymer is largely a consequence of the mechanical nary 90-day toxicology study of Capronor in female rats
trauma that occurs during surgery (231). and guinea pigs, except for a bland response at the implant
Regarding the encapsulation of bioactive macromole- site and a minimal tissue-encapsulating reaction, no toxic
cules as proteins, peptides, and antigens, the studies carried effects were observed (236).
out so far show mixed biocompatibility results. Serious The lack of an inammatory response was conrmed

Copyright 2002 Marcel Dekker, Inc.


by implanting polyvinyl alcohol sponges impregnated with Although many examples on the use of albumin micro-
the powdered polymer in rats. In the Ames mutagenicity spheres were reported in the literature, there are only a few
assay polycaprolactone was negatively tested (87). studies describing gelatin systems. Despite this, compared
The Capronorpolycaprolactone contraceptive delivery with albumin, gelatin offers the advantages of a good his-
system was also tested implanted in rats and monkeys in tory in parenteral formulations and lower antigenicity. A
a 2-year period (87). The results of this second, more ex- biocompatibility study with gelatin microspheres reported
tensive study, based on animal clinical and physical data no untoward effects when injected intravenously into mice
such as blood and urine analysis, ophthalmoscopic tests, over a 12-week period (246).
and histopathology after necropsy, showed no signicant In a second study, when albumin microspheres were
differences between the test and control groups. injected repeatedly into the knee joints of rabbits, pro-
Phase I and II clinical trials with Capronor were re- nounced rapid joint swelling occurred after the second and
cently carried out in different medical centers (87). subsequent injections, compared to no swelling when gela-
tin microspheres were injected in the same way (247).
B. Poly(amides)
1. Natural Polymers 2. Pseudopoly(amino Acids)
The use of natural biodegradable polymers to deliver drugs The pseudopoly(amino acids), belonging to the poly(am-
continues to be an area of active research despite the ad- ides) group, represent one of the latest and therefore less
vent of synthetic biodegradable polymers (237239). Nat- advanced biodegradable polymers for medical use. Only a
ural polymers remain attractive primarily because they are few of them have been synthesized and characterized, and
natural products of living organisms, readily available, rel- no clinical tests have thus far been conducted. However,
atively inexpensive, and capable of a multitude of chemical preliminary promising results were obtained showing no
modications (240). gross toxicity or tissue incompatibility upon subcutaneous
Most of the investigations of natural polymers as matri- implantation for the presently available pseudopoly(amino
ces in drug delivery systems have focused on the use of acids), and they are now being actively investigated in sev-
proteins (polypeptides or polyamides) as gelatin, collagen, eral laboratories for medical applications ranging from bio-
and albumin. Collagen is a major structural protein found degradable bone nails to implantable adjuvants (121).
in animal tissues where it is normally present in the form of In an initial evaluation of hydroxyproline-derived poly-
aligned bers. Because of its unique structural properties, esters, poly(N-palmitoyl hydroxyproline ester) was tested
collagen has been used in many biomedical applications in a seriers of biocompatibility tests (248).
such as absorbable sutures, sponge wound dressings, com- In the rabbit cornea bioassay, implantation of four
posite tissuetendon allografts, injectables for facial re- small pieces of the polymer into four rabbit corneas elic-
constructive surgery, and as drug delivery systems espe- ited a pathological response in three of them and a very
cially in the form of microspheres (241). mild inammatory response in one cornea. Histological
Besides the collagens biocompatibility and nontoxicity examination of the corneas 4 weeks after implantation
for most tissues (242), several factors such as the possible showed no invading blood vessels or migrating inamma-
occurrence of antigenic responses, tissue irritation due to tory cells in the area around the implants (249).
residual aldehyde crosslinking agents, and poor patient tol- In a second biocompatibility study, 10-mg implants of
erance of ocular inserts have adversely inuenced its use poly(N-palmitoyl hydroxyproline ester) were inserted sub-
as a drug delivery vehicle (241). For example, 5-uoro- cutaneously in mice in the dorsal area of the animals be-
uracil and bleomycin crosslinked sponges made from puri- tween the dermis and the adipose tissue layer. The mice
ed bovine skin collagen were implanted in rabbit eyes to responses were examined up to 1 year postimplantation
test their possible use in preventing broblast proliferation (248).
following ophthalmic surgery, resulting in a chronic in- No inammatory reaction at the implantation site was
ammatory reaction elicited by the sponges even in the observed in any of the mice during the rst 7 weeks. A
absence of drug (243). thin but distinct layer of brous connective tissue appeared
Noncollagenous proteins, particularly albumin and to a around the implant by week 14 and, occasionally, a few
lesser extent gelatin, continue to be developed as drug de- multinucleated giant cells were associated with the brous
livery vehicles. The exploitable features of albumin in- connective tissue. Animal necropsies performed at 16 and
clude its reported biodegradation into natural products, its 56 weeks postimplantation showed no histopathological
lack of toxicity, and its nonimmunogenicity (244,245). abnormalities (248).

Copyright 2002 Marcel Dekker, Inc.


These preliminary biocompatibility results from two an- was visualized by UV illumination of sections labeled with
imal models, rabbit and mouse, indicate that the poly(N- uorescent marker, and the degree of calcication around
palmitoyl hydroxyproline ester) elicits a very mild, local the implants was ascertained by backscattered electron mi-
tissue reaction typical of a foreign body response, resulting croscopy. The bone tissue response was characterized by
in encapsulation of the foreign biomaterial without evi- active bond remodeling at the surface of the degrading im-
dence for any signicant pathological abnormalities. How- plant, the lack of brous capsule formation, and an unusu-
ever, additional safety and toxicity tests need to be per- ally low number of inammatory cells at the boneimplant
formed to evaluate possible systemic toxic effects, allergic interface. Poly(DTH-carbonate) exhibited very close bone
reactions, or any mutagenic, teratogenic, and carcinogenic apposition throughout the 26-week period of the initial
activities. study. A roughened interface was observed which was pen-
Pseudopoly(amino acids) containing aromatic side etrated by new bone as early as 2 weeks postimplantation.
chains of tyrosine in their backbone structure were also Bone growth into the periphery of the implant material was
developed recently to investigate whether biodegradable visible at the 26-week time point.
polymers that incorporate aromatic components would
combine a high degree of biocompatibility with a high de- C. Polyphosphazenes
gree of mechanical strength (121).
In order to test the tissue compatibility of tyrosine- Two different types of polyphosphazenes are of interest as
derived poly(iminocarbonates), solvent cast lms of bioinert materials: those with strongly hydrophobic surface
poly(CTTH) were subcutaneously implanted into the back characteristics and those with hydrophilic surfaces. Poly-
of mice. In this study, conventional poly(L-tyrosine) phosphazenes bearing uoroalkoxy side groups are some
served as a control (250). The data obtained from this bio- of the most hydrophobic synthetic polymers known (251,
compatibility assay were very similar to those observed 252). Such polymers are as hydrophobic as poly(tetrauo-
with poly(N-palmitoyl hydroxyproline ester), showing no roethylene) (Teon), but unlike Teon polyphosphazenes
gross pathological changes from visual inspection of the of this type are exible or elastomeric, easy to prepare,
implantation sites over a 1 year period. and can be used as coatings for other materials.
Silver et al. (135) reported a comparative study on bio- Graft polymerization with dimethylaminoethylmetha-
compatibility of solvent cast lms of poly(desaminotyro- crylate (DMAEM) onto the poly(phosphazene) surfaces
syltyrosine hexylesteriminocarbonate), or poly(DTH- highly enhances their biocompatibility. Surface modica-
iminocarbonate), in a subcutaneous rat model. In this tion of poly(triuoroethoxy-phosphazenes) (PTFP) with
study, high-density polyethylene (HDPE) and medical polyethylene glycols was heading to materials with en-
grade poly(D,L-lactic acid) served as controls. Consider- hanced biocompatibility in comparision to nonmodied
ing the signicantly faster degradation rate of poly(DTH- polymers (253). In the next study, the PTFP was grafted
iminocarbonate), one would expect a different response with hydrophilic monomers like dimethylacrylamide
from this material. After 7 days of postimplantation, a (DMAA) and acrylamide (AAm) (254). Large differences
greater cell density and inammatory response was noted in the biocompatibility of these materials were found. Bio-
for poly(DTH-iminocarbonate). However, at a later time compatibility of AAm-grafted samples was greatly en-
point, the biological response observed for poly(DTH- hanced while an opposite behavior for DMAA-grafted
iminocarbonate) was almost similar with the response samples was observed. All studies with these samples were
observed for poly(DTH-iminocarbonate). The tissue re- done by intraperitoneal implantation of thin lms in adult
sponse was characterized by a thin tissue capsule, absence male wistar rats. Tissue around the implantation site was
of gaint cells, and a low inammatory cell count and was evaluated by histological examination after 25 days.
not statistically different from the response observed for Biocompatibility and safety testings of these polymers
polyethylene and poly(lactic acid). by subcutaneous implantation in animals have shown mini-
In vitro attachment and proliferation of broblasts on mal tissue response, similar in fact to the response reported
tyrosine-derived polycarbonates was a function of the pen- with Teon (255). The connection between hydrophobicity
dent chain length. Ertal and Kohn fabricated poly(DTH- and tissue compatibility has been noted for classical or-
carbonate) pins and compared them to commercially avail- ganic polymers (256). Thus, these hydrophobic polyphos-
able orthosorbR pins made of polydioxanone (136). The phazenes have been mentioned as good candidates for use
pins were implanted transcortically in the distal femur and in heart valves, heart pumps, blood vessel prostheses, and
proximal tibia of New Zealand white rabbit for up to 26 as coating materials for pacemakers or other implantable
weeks. In addition to routine histological evaluation of the devices. However, more in vivo testing and clinical trials
implant sites, bone activity at the implanttissue interface are needed (144).

Copyright 2002 Marcel Dekker, Inc.


In their bioerosion reactions polyphosphazenes display of the pocket within about 1 day, despite good adhesive-
a uniqueness that stems from the presence of the inorganic ness.
backbone, which in the presence of appropriate side groups
is capable of undergoing facile hydrolysis to phosphate and E. Polyanhydrides
ammonia. The phosphate can be metabolized and the am-
monia excreted. Theoretically, if side groups attached to Polyanhydrides are a novel class of biodegradable poly-
the polymer are released by the same process being excre- mers under development as vehicles for the release of bio-
table or metabolizable, then the polymer can be eroded active molecules including drug peptides and proteins (1).
under hydrolitic conditions without the danger of a toxic The polyanhydrides constitute so far the only class of
response. Polyphosphazenes of this type are potential can- surface-eroding polymers approved for clinical trials by
didates as erodible biostructural materials for sutures or as the Food and Drug Administration.
matrices for controlled delivery of drugs (144). A series of biocompatibility studies reported on several
Polyphosphazenes containing amino acid ester side polyanhydrides have shown them to be nonmutagenic and
groups were the rst bioerodible polyphosphazenes syn- nontoxic (189). In vitro tests measuring teratogenic poten-
thesized (146). They are solid materials which erode hy- tial were also negative. Growth of two types of mammalian
drolitically to ethanol, glycine, phosphate, and ammonia. cells in tissue culture was also not affected by the polyan-
These polymers were tested in subcutaneous tissue re- hydride polymers (189); both the cellular doubling time
sponse experiments showing no evidence of irritation, cell and cellular morphology were unchanged when either bo-
toxicity, giant cell formation, or tissue inammation vine aorta endothelial cells or smooth muscle cells were
(143,255). grown directly on the polymeric substrate.
Imidazoyl groups linked to polyphosphazene chains Subcutaneous implantation in rats of high doses of the
are also hydrolized very easily, showing good biocom- 20 :80 copolymer of bis-(p-carboxyphenoxy) propane and
patibility tests (141,154). Langone et al. (257) studied sebacic acid for up to 8 weeks indicated relatively minimal
poly[(ethyl-alanate)-co-(imidazole) phosphazene] deriva- tissue irritation with no evidence of local or systemic toxic-
tives for in vivo evaluation. Polymer lms were subcutane- ity (260). Since this polymer was designed to be used clini-
ously implanted in rats. The animals were killed after 30 cally to deliver an anticancer agent directly into the brain
or 60 days. Biopsy samples of the implant zone were histo- for the treatment of brain neoplasms, its biocompatibility
logically examined. In both cases, animals were healthy in rat brain was also studied (261). The tissue reaction of
and biological material surrounding to the polymers was the polymer was compared to the reaction observed with
found to correspond to broplast collagen with only a few two control materials used in surgery, oxidized cellulose
monocytes in the internal site. absorbable hemostat (Surgicel, Johnson and Johnson) and
absorbable gelatin sponge (Gelfoam, Upjohn). The in-
D. Polyorthoesters ammatory reaction of the polymer was intermediate be-
tween the controls (261). A closely related polyanhydride
As mentioned previously, the Chronomer polyorthoester copolymer poly(CPP)SA 50 :50 was also implanted in
material from Alza Corporation, or Alzamer, has been in- rabbit brains and was found to be essentially equivalent to
vestigated as bioerodible inserts for the delivery of the nar- Gelfoam in terms of biocompatibility evaluations (262). In
cotic antagonist naltrexone, and for the delivery of the con- a similar study conducted in monkey brains, no abnormali-
traceptive steroid norethisterone (258,259). The steroidal ties were noted in the CT scans and magnetic resonance
implant was tested in two separate human clinical trials image, nor in the blood chemistry or hematology evalua-
causing local tissue irritation, and therefore further work tions (263). No systemic effects of the implants were ob-
with this formulation was discontinued (166,167). The rea- served on histological examinations of any of the tissues
sons for the local irritation were never properly elucidated. tested (264).
New types of poly(orthoesters) were developed, but no New classes of polyanhydrides have been recently syn-
data on the biocompatibility and safety of these materials thesized and are undergoing extensive preclinical testing,
were reported. including a wide range of biocompatibility studies. Exam-
In vitro studies have shown that good control over re- ples of these new materials are polymers of sebacic acid,
lease of tetracycline could be achieved and very good in poly(SA), and copolymers 1:1 of SA with fatty acid dimer
vitro adhesion to bovine teeth was demonstrated (173). (FAD) [poly(FAD : SA)], fumaric acid (FA) [poly(FA:
However, studies in beagle dogs with naturally occurring SA)], and isophthalic acid [poly(ISO: SA)]. These materi-
periodontitis were not successful because ointmentlike als were implanted in rabbits intramuscularly, subcutane-
polymers with a relatively low viscosity are squeezed out ously, and in the cornea. Ocular and muscle irritation stud-

Copyright 2002 Marcel Dekker, Inc.


ies were performed compared to the material controls were minimal subacute inammation and mild brosis. In
Gelfoam, Surgicel, and Vycryl, a synthetic absorbable comparison, under the same conditions, Vycryl implant in-
suture (Ethicon) (265). Detailed observations of toxicity, duced minimal brosis associated with the presence of
bleeding, swelling, or infection of the implantation site minimal quantity of giant cells and encapsulated foreign
were conducted daily. At the end of the study the animals material.
were sacriced and a gross necropsy examination of the Based on the biocompatibility and safety preclinical
tissues surrounding the implant site was performed. No studies carried out in rats (260,261), rabbits (262,266), and
signicant clinical signs or abnormalities of the incision monkeys (263,264) reviewed here showing acceptability
sites were observed during the study period (4 weeks). No of the polyanhydrides for human use, a Phase I and II clini-
meaningful differences could be seen in reaction between cal protocol was instituted (270). In these clinical trials, a
the various polymer implants tested and the control materi- polyanhydride dosage form (Gliadel), consisting of wa-
als (265). fer polymer implants of poly(CPP-SA) 20 : 80 and con-
In the rabbit cornea bioassay, no evidence of inamma- taining the chemotherapeutic agent Carmustine (BCNU),
tory response was observed with any of the implants at any was used for the treatment of glioblastoma multiforme, a
time. On an average, the bulk of the polymers disappeared universally fatal form of brain cancer. In these studies, up
completely between 7 and 14 days after the implantation to eight of these wafer implants were placed to line the
(265). The cornea is a very sensitive indicator of inam- surgical cavity created during the surgical debulking of the
matory reactions (266,267). The rabbit cornea possesses brain tumor in patients undergoing a second operation for
clear advantages over other implant sites for studying surgical debulking of either a grade III or IV anaplastic
implanthost interactions due to the easy accessibility for astrocytoma. In keeping with the results of the earlier pre-
frequent observations without having to gain surgical ac- clinical studies suggesting a lack of toxicity, no central or
cess to the implantation site. The transparency and avascu- systemic toxicity of the treatment was observed during the
larity of the cornea also enable the observer to distinguish course of treating 21 patients under this protocol. Phase III
among the different inammatory characteristics such as human clinical trials have demonstrated that site-specic
edema, cellular inltration, and ingrowth of blood vessels delivery of BCNU from a poly(CPP-SA) 20 :80 wafer (Gli-
from the perifery of the cornea or neovascularization, adel) in patients with recurring brain cancer (glioblastoma
which are strong indications that the biomaterial under multiforme) signicantly prolongs patient survival (271).
testing is unsuitable for implantation (268). Gliadel has nally won approval from the FDA as therapy
In similar animal experiments in which polyanhydride for the treatment of brain tumors.
matrices containing tumor angiogenic factor (TAF) were
implanted in rabbit cornea, a signicant vascularization re-
sponse was observed without edema or white cells. More-
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1. A. J. Domb, O. Elmalak, V. R. Shastry, Z. Ta-Shma,
showed no adverse vascular response (268,269).
D. M. Masters, I. Ringel, D. Teomim, R. Langer. Polyan-
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Copyright 2002 Marcel Dekker, Inc.


5

Biodegradable Biomaterials Having Nitric Oxide


Biological Activity

C. C. Chu
Cornell University, Ithaca, New York

I. INTRODUCTION biomaterials has two major advantages that nonbiodegrad-


able biomaterials do not have. First, they do not elicit per-
The chemical incorporation of biologically active com- manent chronic foreign body reaction due to the fact that
pounds into synthetic biodegradable biomaterials was suc- they would be gradually absorbed by human body and do
cessfully synthesized by using nitric oxide derivatives not permanently retain trace of residual in the implantation
(e.g., 4-amino-2,2,6,6-tetramethylpiperidine-1-oxy) as the sites. Second, some of them have recently been found to be
biochemical agents and synthetic aliphatic polyesters like able to regenerate tissues, via so-called tissue engineering,
polyglycolide as the biodegradable biomaterials. The re- through the interaction of their biodegradation with immu-
sulting new biomaterial was characterized and its in vitro nologic cells like macrophages. Hence, surgical implants
hydrolytic degradation property was studied to examine made from biodegradable biomaterials could be used as
the release proles of the chemically incorporated nitric temporary scaffold for tissue regeneration. This approach
oxide derivative from polyglycolide. The biological activ- toward the reconstruction of injured, diseased, or aged tis-
ity of this new class of biodegradable biomaterial was sues is one of the most promising elds in the future medi-
tested by examining its ability to retard the proliferation cine.
of human smooth muscle cell in vitro. It was found that Although the earliest and most commercially signicant
this new class of biologically active biodegradable bioma- biodegradable polymeric biomaterials are originated from
terial has indeed one of the well-known biological func- linear aliphatic polyesters like polyglycolide and polylac-
tions of nitric oxide: retardation of the proliferation of tide from poly(-hydroxyacetic acids), recent introduction
smooth muscle cells. The potential biomedical applications of several new synthetic and natural biodegradable poly-
of this new biomaterial include the treatment of hyperpla- meric biomaterials extends the domain beyond this fam-
sia in cardiovascular disorders, promoting wound healing ily of simple polyesters. These new commercially signi-
in healing-impaired patients, and nitric oxiderelated dis- cant biodegradable polymeric biomaterials include poly
eases. (orthoesters), polyanhydrides, polysaccharides, poly(ester-
The interests in biodegradable polymeric biomaterials amides), tyrosine-based polyarylates or polyiminocarbo-
for biomedical engineering use have increased dramati- nates or polycarbonates, poly(D,L-lactide-urethane),
cally during the past decade. This is because this class of poly(-hydroxybutyrate), poly(-caprolactone), poly[bis(car-

Copyright 2002 Marcel Dekker, Inc.


boxylatophenoxy) phosphazene], poly(amino acids), pseu- would describe the new biodegradable biomaterials having
dopoly(amino acids), and copolymers derived from amino nitric oxide function.
acids and nonamino acids.
The earliest and most successful and frequent biomedi-
cal application of biodegradable polymeric biomaterials II. BIODEGRADABLE BIOMATERIALS
has been in wound closure (1). All biodegradable wound HAVING NITRIC OXIDE FUNCTION
closure biomaterials are based upon glycolide and lactide
family. For example, polyglycolide (Dexon from Ameri- Nitric oxide (NO) is a very small but highly reactive and
can Cyanamid), poly(glycolide-L-lactide) random copoly- unstable free radical biomolecule with expanding known
mer with 90-to-10 molar ratio (Vicryl from Ethicon), biological functions. This small biomolecule and its bio-
poly(ester-ether) (PDS from Ethicon), poly(glycolide- logical functions have recently become one of the most
trimethylene carbonate) random block copolymer (Maxon studied and intriguing subjects as recently reviewed by
from American Cyanamid), and poly(glycolide-caprolac- several investigators (818). Nitric oxide is extremely la-
tone) copolymer (Monocryl from Ethicon). Some of these bile and short lived (about 6 to 10 s).
materials like Vicryl have been commercially used as sur- Nitric oxide and its radical derivatives have been known
gical meshes for hernia and body wall repair. Besides to play a very important role in a host of expanding biolog-
wound closure application, biodegradable polymeric bio- ical functions, such as inammation, neurotransmission,
materials that are commercially satisfactory include those blood clotting, blood pressure, cardiovascular disorders,
for drug control/release devices. Some well-known exam- rheumatic and autoimmune diseases, antitumor activity
ples in this application are polyanhydrides and poly with a high therapeutic index, antimicrobial property, sen-
(orthoester). Biodegradable polymeric biomaterials, partic- sitization or protection of cells and tissues against irradia-
ularly totally resorbable composites, have also been experi- tion, oxidative stress, respiratory distress syndrome, and
mentally used in the eld of orthopedics, mainly as compo- cytoprotective property in reperfusion injury, to name a
nents for internal bone fracture xation like PDS pins. few (830). Nitric oxide acts both as an essential regula-
However, their wide acceptance in other parts of orthope- tory agent to normal physiological activities and as cyto-
dic implants may be limited due to their inherent mechani- toxic species in diseases and their treatments. Nathan et
cal properties and their biodegradation rate. Biodegradable al. reported that nitric oxide is a potent antiviral compound
polymeric biomaterials have been experimented as vascu- against two disguring poxvirus and herpes simplex virus
lar grafts, vascular stents, vascular coupler for vessel anas- type 1, which causes cold sores in humans (12). Levi et
tomosis, nerve growth conduits, augmentation of defected al. also found that nitric oxide could protect human heart
bone, ligament/tendon prostheses, intramedullary plug against low oxygen supply, a condition known as myocar-
during total hip replacement, anastomosis ring for intesti- dial ischemia, by widening blood vessels so that more oxy-
nal surgery, and stents in ureteroureterostomies for accu- gen-rich blood reaches the heart (14). Elliott et al. reported
rate suture placement. The details of the biomedical appli- that a new NO-releasing nonsteroidal anti-inammatory
cations of biodegradable polymeric biomaterials and their drug has the benets of accelerating gastric ulcer healing
chemical, physical, mechanical biological, and biodegra- (31,32). It is important to know, however, that excessive
dation properties can be found in other recent reviews (1 introduction of NO into body may have adverse effects
7). like microvascular leakage, tissue damage in cystic bro-
There is one common characteristic among all these sis, septic shock, B cell destruction, and possible muta-
biodegradable biomaterials: they do not actively partic- genic risk, to name a few (18,19,27,3335).
ipate in the process of wound healing, tissue regeneration Nitric oxide and NO-derived radicals are not normal
and engineering. In other words, these biomaterials are not biological messengers whose trafcking depend on spe-
alive and cannot remodel and/or release cytokines upon cic transporters or channels. Instead, nitric oxide radicals
stimulation like normal tissues. These biomaterials, how- released by cells like macrophage and endothelial cells
ever, elicit inammatory and foreign body reactions and would diffuse randomly in all directions from the site of
play a passive role in wound healing. It would be ideal release. Because of this unusual property, the only way to
if these synthetic biomaterials could be engineered so that control the biological functions of nitric oxide is to control
they could become alive after implantation and hence ac- its site of synthesis. This suggests that the only way to
tively participate in the biological functions with the sur- deliver the desirable biological functions of nitric oxide is
rounding tissues, such as the ability to modulate inam- through nature. Existing science and technology are not
matory reactions, to facilitate wound healing, or to mediate able to modulate the release of nitric oxide according to
host defense system to combat diseases. In this chapter, we our wish for a variety of therapeutic purposes.

Copyright 2002 Marcel Dekker, Inc.


A. Synthesis and Characterization

We recently used a patented chemical method to incorpo-


rate nitric oxide derivatives into a series of synthetic biode-
gradable biomaterials (36,37). Upon the hydrolytic degra-
dation of the host biomaterials, nitric oxide derivatives
could be released to the surrounding and the rate of release
could be controlled by the nature of the biodegradable bio-
materials. The amounts of the nitric oxide derivatives that
can be incorporated into biodegradable biomaterials would
depend on the molecular weight of the biomaterials. Figure
1 illustrates the chemical scheme of this patented method
of incorporating 4-amino-2,2,6,6-tetramethylpiperidine-1-
oxy (TAM) as the source of nitroxyl radicals nitric oxide
into synthetic biodegradable polyesters like polyglycolide
or polylactide.
Due to the free radical characteristic of Tempamine ni-
troxyl radicals, the radical incorporated polyglycolide
(PGA) must exhibit an electron paramagnetic resonance
(EPR) spectrum that has the characteristic of nitroxide.
Figure 2 is such an EPR spectrum of TAM-PGA. This EPR
spectrum shows a considerable broadening of linewidth
when compared with an EPR spectrum of free TAM nitric
oxide. An EPR characterization of nitroxyl radicals is
based on the measurement of the signal intensity of an EPR
spectrum. This measurement can provide fundamental in-
formation of free radicals, such as linewidth, which bears
a relationship to the tumbling motion of free radicals; g
value, which largely depends on the immediate environ-
ments of the free radicals; and hyperne splitting con-
stants, which describe the classical multiplicity of EPR
spectram due to the interaction of the unpaired electron Figure 5.2 Electron paramagnetic resonance spectra of TAM
spins with nuclear spins. radical (A) in conjugation with polyglycolide and (B) in free
form.

The considerable broadening of the EPR spectrum of


the TAM-PGA biomaterial (Fig. 2A) when compared with
free TAM nitroxyl radical (Fig. 2B) is attributed to the
viscous macromolecular environment surrounding TAM
nitroxyl radicals that were chemically bound to PGA chain
ends. These nitroxyl radicals cannot move as freely as free
nitric oxide due to the restricted PGA chain segmental mo-
tion. This relationship between free radical motion and the
characteristic of its immediate environment would provide
a useful means to study the release pattern of nitroxyl radi-
cals that were chemically bound to biodegradable sub-
strates.
Because TAM nitroxyl radicals were incorporated only
Figure 5.1 Chemical scheme for incorporating 4-amino- into the carboxylic chain ends of PGA macromolecules,
2,2,6,6-tetramethylpiperidine-1-oxy into the carboxyl chain ends their physical, thermal, and mechanical properties are in-
of linear aliphatic polyesters like polyglycolide. signicantly different from the parent PGA macromole-

Copyright 2002 Marcel Dekker, Inc.


Figure 5.5 The effect of pH of the media on the EPR spectra
intensity of TAM nitroxyl radical at 10 g/mL. The media was
glycolic acid: (a) pH 7.44, (b) pH 4.0, (c) pH 3.5, (d) pH 3.0.

Figure 5.3 The kinetics of in vitro release of TAM nitroxyl


radicals from TAM-PGA biomaterials in buffer media of original leased into the surrounding environment upon hydrolytic
pH 7.44 at 37C. degradation of PGA, the amount and the rate of release of
TAM nitroxyl radicals should have a direct impact on the
applicability of the newly synthesized TAM-PGA bioma-
cules, such as similar melting temperature and heat of fu- terials to medicine. Figure 3 illustrates such an in vitro
sion. This lack of change in fundamental properties release pattern of TAM radicals from PGA. The release of
between TAM-PGA and parent PGA biomaterials should Tempamine nitroxyl radicals upon PGA hydrolysis fol-
be benecial because the similar processing conditions that
have been used to fabricate PGA for a variety of clinical
applications could also be used to fabricate the new TAM-
PGA. In addition, the knowledge of the well-known bio-
degradation properties of PGA could be applied to estimate
the release pattern of TAM nitroxyl radicals from PGA
upon its biodegradation. The main difference between the
parent and TAM-PGA, however, is their degradation prod-
ucts and their subsequent biological properties.

B. In Vitro Hydrolytic Degradation

Since the expected biological functions of the TAM-PGA


must come from the nitroxyl radicals that would be re-

Figure 5.4 The reversible one-electron reduction and oxidation Figure 5.6 The change in pH of the buffer medium used for
reactions of nitroxyl free radicals. the in vitro release study of TAM-PGA at 37C.

Copyright 2002 Marcel Dekker, Inc.


(A)

(C)

Figure 5.7 The hydrolytic release mechanism of TAM radicals


from TAM-PGA biomaterials via alkaline and acid hydrolytic
degradation. (A) Initial alkaline hydrolytic degradation of the es-
ter linkages in polyglycolide backbone during the early stage of
immersion. (B) Acidic hydrolytic degradation of the ester link-