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This is to certify that the
thesis entitled

DEVELOPMENT AND SYNTHESIS OF NOVEL
POLY(B-AMINO ACID) DRUG DELIVERY SYSTEMS

presented by

PING CAO

has been accepted towards fulfillment
of the requirements for the

Master degree in Chemis

 
   

 

n I 2 ‘2
AA A 2 <2 A
Maéor professor’s'fSViignatulle

UY/vl/odf

Date

 

.UBRARY
Michigan State
University

 

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

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6/01 c:/CIRC/DateDue.p65-p.15

DEVELOPMENT AND SYNTHESIS OF NOVEL
POLY(B-AMINO ACID) DRUG DELIVERY SYSTEMS

By

PING CAO

A THESIS

Submitted to
Michigan State University
in partial firlfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Chemistry

2004

ABSTRACT

DEVELOPMENT AND SYNTHESIS OF NOVEL POLY([3-AMINO ACID) DRUG
DELIVERY SYSTEMS

By

Ping Cao

A new drug delivery system based on a poly(B-amino acid) has been synthesized from
2(5H)—furanone and N, N—dimethylethylene diamine by a simple, one step
polymerization. The resulting polymer was purified by Bio Gelp-P6O size exclusion
chromatography and auto separated into high, medium and low molecular weight
fractions. The molecular weight distribution was controlled by adjusting the reaction
temperate and reaction time. The drug delivery properties of the polymers were evaluated
by studying the transfer of the anticancer drug doxorubicin to mouse embryonic
fibroblasts cells and the transfer of telomerase antisense RNA (with sequence GCG CGG
GGA AAA GCA) and a 4.2 kb plasmid containing the green fluorescent protein gene into
the same cells. Best results were obtained using the high molecular weight polymer
fraction. This result proves that this polymer is a very promising delivery system which
can be used for those anticancer drugs with serious side effects and also successfully

bring gene or plasmid which is hardly enter the cells by themselves before.

ACKNOWLEDGMENT

I would like to express my sincere gratitude to my academic advisor Dr. Howllingsworth
in the first place. It is Dr. Howllingsworth who taught me how to enjoy the research
process no matter failure or success. Also I am very grateful to my committee members:

Dr. Baker and Dr. Watson for their constant encouragement and understanding for me.

I am thankful to all my colleagues fi'om the laboratory of Dr.Hollingsworth. They are not
only my lab-mates but also good fiiends of mine. I can hardly find words to describe my
gratefulness to their consideration, help, and support. They are: Besy, Carol, Chang,
Changyou, Felicia, Gia, Li, Kun, Linjuan, Hanmi, Xuezheng, Xiaoyu, Zhen, Zhiyuan,
and Yuqing. It is my good luck to become a member of this lab during the past two and
half years. Working in such nice and worm environment helps me build a good sense of

team cooperation, which are invaluable for my future career.

Thanks to all my friends both in USA and in China, you always make my life so exciting.

Last but not least, I want to say thanks to my family for their love always makes me full

of confidence and strength.

iii

TABLE OF CONTENTS

LIST OF FIGURES ................................................................................. v
CHAPTER 1
LITERATURE REVIEW .......................................................................... 1
Introduction ....................................................................................... 1
Administration methods ......................................................................... 6
Vehicles of drug elivery ....................................................................... 12
Inorganic vehicles ........................................................................... 12
Organic vehicles ............................................................................ 14
Liposome ................................................................................. l4
Polymer vehicles ....................................................................... 19
Gene therapy .................................................................................. 38
Introduction ............................................................................... 38
Antisense human telomerase RNA .................................................... 39
Bibliography ................................................................................. 43
CHAPTER 2
DESIGN OF CATIONIC POLY B-AMINO ACID DDS .................................... 50
Introduction .................................................................................. 50
Two main elements of design ............................................................. 51
Molecular weight control .............................................................. 51
Ability to traverse membranes ........................................................ 53
The construction of poly B-amino acid DDS ........................................................ 56
Bibliography ................................................................................. 58
CHAPTER 3
EXPERIMENTAL ......................... , ........................................................ 6 0
Synthesis of poly B-amino ................................................................. 6O
Purification and separation ............................................................... 61
Measurement of drug delivery properties ............................................. 62
CHAPTER 4
RESULTS AND DISCUSSION ................................................................. 64
Synthesis and MW control of poly B-amino acids .................................... 64
Separation and MW estimation of polymer fractions ................................ 76
Characterization of high MW fraction of the poly B-amino acids ................. 82
Drug delivery properties of poly B-amino acids ....................................... 85
Anticancer drug delivery (doxorubicin) ........................................... 85
Delivery of antisense human telomerase RNA ................................... 89

iv

Plasmid GFP gene delivery by the poly B-amino acids ........................... 93

Bibliography ................................................................................... 99
CHAPTER 5
PREPARATION OF CATIONIC POLYELECTROLYTES ........................... 100
Introduction ................................................................................. l 00
Experimental section ...................................................................... 105
Results and discussion .................................................................... 108
Bibliography ................................................................................ l 12

LIST OF TABLES

CHAPTER]
Table 1.1 Common routes of drug administration .............................................. 6
Table 1.2 Methods for Hormones Delivery ........................................................ 11
Table 1.3 A summary of the main properties of applications of
natural polymers in DDSs ........................................................................... 20
CHAPTER 2
Table 2.1: Amino acid sequence of characterized protein-
transduction domains .............................................................................. 53

Table 2.2: intracellular delivery of various molecules using PTDs. . . . . . . . . . . . . . . . . ..........54

CHAPTER 4
Table 4.1 Data of standard curve (1 gMW v.s Kay) ............................................. 77
Table 4.2 Relative yield of each fraction (percentage of recovered materials) ........... 78
CHAPTER 5
Table 5.1 stimuli-responsive hydrogels in drug delivery ................................... 103

vi

LIST OF FIGURES

CHAPTER 1

Figure 1.1 drug levels in blood with ......................................................... 4
(a) Traditional drug dosing
(b)controlled-de1ivery dosing

Figure1.2 Drug Delivery Methods ............................................................ 9
Figure 1.3 Plasma IgG following different administration routes in humans ......... 10
Figure 1.4 MicroCHIPS Technology ....................................................... 13
Figure 1.5 Classifications of Liposomes ................................................... 14
Figure 1.7 Drug delivery from typical matrix delivery systems ........................ 35
Figure 1.8 Drug delivery from typical reservoir devices ................................. 35

a. Implantable or oral systems
b. Transdermal systems
Figure 1.9 Drug delivery from ............................................................... 37
a) Bulk degradation controlled system.
b) Surface degradation controlled system
Figure 1.10 Drug delivery from swelling—controlled release system .................. 37
a) Reservoir polymeric DDS
b) Matrix polymeric DDS

Figure1.11 Gene addition technique ...................................................... 38
Figure 1.12 Antisense Technology ......................................................... 38
Figure 1.13 A Comparison of Mitosis of Normal Somatic Cells with
Cancer Somatic cells ........................................................................... 42
Figure 1.14 Cancer Cell Treatment with Antisense Drug ............................... 42
CHAPTER 4
Figure 4.1(a) 1H-NMR spectrum of reaction mixture at 00C after 2 hrs ............... 67
Figure 4.1(b) l3C-NMR spectrum of reaction mixture at 00C afier 2 hours ......... 68
Figure 4.2 lH-NMR spectrum of reaction mixture at 70°C after 1 hour (a)
and after 4 hours (b) ........................................................................... 69
Figure 4.2(c) 1H-NMR spectrum of reaction mixture at 70°C after 8 hours ........... 70
Figure 4.3 IH-NMR spectrum of reaction mixture at 80°C afier 4 hours (a)
and after 10 hours(b) ........................................................................ 71
Figure 4.4 1H—NMR spectrum of reaction mixture at 90°C after 2 hours
(a) and after 4 hours ........................................................................ 72
Figure 4.4 (c) lH-NMR spectrum of reaction mixture at 90°C after 8 hours ......... 73
Figure 4.5 1H-NMR spectrum of reaction mixture at 120°C after 3.5 hours (a),
6.5 hours (b), and 9.5 hours (c) ............................................................. 74

Figure 4.6 (a) IH-NMR spectrum of reaction mixture at 140°C afier 0.5 hour(a),

vii

1 hour (b) 2 hours (0), and 4 hours(d) ...................................................... 75

Figure 4.7 Plot of fraction number vs absorbance ........................................ 76
Figure 4.8 MW v.s. Km, calibration ......................................................... 77
Figure 4.9 (a) GPC results for the high MW fraction isolated by

Bio GelP-60 chromatography ............................................................... 79
Figure 4.9 (b) Mass spectrum of the high MW fraction isolated

by Bio GelP-60 chromatography ........................................................... 79
Figure 4.10 (a) GPC results of the medium MW fraction isolated

by Bio GelP-60 chromatography ............................................................ 80
Figure 4.10 (b) Mass spectrum of the medium MW fraction isolated

by Bio GelP-6O chromatography ............................................................ 80
Figure 4.11 GPC result of high MW fraction isolated by acetone

precipitation method .......................................................................... 81
Figure 4.12 1H-NMR spectrum of the high MW fraction isolated

by Bio GelP-60 chromatography ........................................................... 83

Figure 4.13 FTIR spectrum of the high MW fi'action isolated

by Bio GelP-60 chromatography ............................................................ 84
Figure 4.14 Structure of doxorubicin ....................................................... 86
Figure 4.15 (A-F) Light micrographs showing of doxorubicin

sequestration and delivery to MEF Cells ................................................... 88
Figure 4.16 (AD) The light micrographs of antisense delivery ........................ 89
Figure 4.17 Subcellular drug delivery by poly(B-amino acid)polymers ............... 92

Figure 4. 18 (A—L) fluorescence and transmission light micrographs of MEF
cells illustrating transfection of plasmid DNA encoding GFP mediated by the

drug delivery vehicles described here and a commercial transfection agent .......... 93
CHAPTER 5
Figure 5.1 Scheme of hydrogel formation ................................................ 104

Figure 5.2 1H-NMR spectrum of methylated polymer product before
ion change with chloride anion (a) and after ion change with chloride
anion (b) ...................................................................................... 107

viii

Chapter 1 Literature review

1.1 Introduction

Beginning with the botanical phase of early human civilizations, through the synthetic
chemistry age in the middle of 20th century, and finally to the biotechnology era at the
dawn of the 21 st century, drug research has evolved and matured" 2' 3. In spite of this
progress, it is still common to find someone who has suffered the pain of losing a friend
or relative because of an incurable infection or cancer. Thus, scientists still search for
better therapeutics for human beings. Some may think better medical treatment means
stronger medicine; however the effectiveness of drugs strongly depends on how drugs are
introduced into the human body. For example, many anti-cancer drugs effectively kill
cancer cells, but without an appropriate carrier to sequester them before they are released
to the cancer site, the drugs can be very toxic to human beings. The term “drug delivery
system” (DDS) refers to both the methods of administration and the delivery vehicles,

which are as significant as the pharmacological activities of the drug itself.

Drug delivery is becoming an increasingly important field in the pharmaceutical industry.

It strongly affects patient compliance, cost efficiency and the development of new drugs.

Patient compliance

When people fail to comply with medication regimen, they mainly complain about the
inconvenience of taking drugs and their concern about adverse long-term effects“. DDSs
address three critical issues; the desire of patients to take the lowest number of drug
administrations, the patients’ favorite administration route, and minimizing side effects

(by preventing the exposure of drugs to entire untargeted tissues) with the same efficiency.

These techniques drive the drug delivery market in the most beneficial way, which is

estimated to be more than $50 billion worldwides.

Cost efficiency

The average development cost of a new drug remains $400-650 million and a time
consuming 10-15 yearsz. Facing this risky process, pharmaceutical companies are under
constant pressure to maximize the full potential of a drug candidate at an early stage of its
life cycle. Fortunately, this aim can be accomplished by incorporating the drug into
different kinds of drug delivery systems, which lead to extended drug patent life and more
convenient dosage forms that overcome the administration problems. Specifically, a new
DDS only costs around $80-130 million and requires less than 5 years of development
allowing pharmaceutical companies to make the best use of their investment. DDSs give
old drugs a second life with increased efficiency and patient satisfaction. For example,
Cardizem DDS °, a simple once-daily form of deltiazem had $4 billion in sales after the

native composition of matter patent expired.

Development of new drugs

Developments in genetics, immunology, biochemistry, molecular biology, and
information technology have made it possible to decipher the whole human genome.
Meanwhile, the focus of therapeutics has moved from mainly symptomatic treatment to
curing and preventing the cause of disease. Therefore, gene delivery has become a
promising therapeutic method, as well as a new challenge, since larger molecules such as
antisense DNA/RNA or plasmids pose greater problems in drug absorption and

distribution.

What are ideal DDSs?

1. Target function

Optimal DDSs sequester and carry drugs efficiently to the appropriate part of the body
(some times even to particular cells and/or a special organelle in the cell). Without the
protection afforded by a DDS, the immune system, the reticuloendothelial system (RES)
and a variety of enzymes can easily break, degrade, and clear drugs when they circulate
within the human body. Even if the drugs are not functional during blood circulation,
they still should be enveloped to prevent harmful side effects to normal tissues. The
required DDS must be sufficiently inert, biocompatible, and mechanically strong. Afier
being modified by a specific antibody or biochemical agent, the ideal DDS will only

release drugs at the specific site.

2. Intelligent release
Some polymer materials are called ‘smart’ DDSs because they are self-regulated. They
keep the drug sequestered and release it only when triggered by abnormal /disease

signals (stimuli) from the human body.

3. Programmable release
One kind of DDS carries several different chemical reagents into the human body at one
time and when prompted by either physician or the patient releases them selectively in a

controllable manner.

4. Simple to administer

The methods of administration should be comfortable and convenient for patients.

5. Sustainable and constant release

Both patients and physicians wish medicine could be given the minimum number of

times.

Ideal DDSs not only are capable of high drug loadings, but also release the drug at a
controlled and constant rate to maintain the concentration in the blood within a desired
range for an extended period of time. (See figure 1.1) Depending on the formulation and
the application, this time may be anywhere from 24 hours (Procardia XL) to one month

(Lupron Depot) to five years (Norplant) 7.

~-- .. _...~—- . . .. --._ ..~_- __..--. .~ ......_ . .‘...._-..\- -~—-—-—.‘uw~-fi—O“—Qra ._ - “I- -.._.,..‘..‘_1

 

Maximum desired level (a) l

A x”? 1 [’1 x1

(UK/l
l/ \l \l \

— Minumum desired level

Drug level

 

Time ———‘> '2

Maximum desired level

1 2
2 \ i

i f Minumum desired level (b) a
l

 

/

 

Drug level

 

Time ———§

Figure 1.1 Drug levels in the blood with
(a) Traditional drug dosing
(b) Controlled-delivery dosing

Figure is adapted from Rct‘l7]

 

L..—-.>.-—.—-.___v.__.... - L. _.....--_.._ av... . h.-._._ -sw..._._.~..- . . . h we... -_ - _.._h _._.- _....._..._...--_...._..~- .4

6. Biodegradability
After their delivery, DDSs are broken down into biologically acceptable molecules that

can be absorbed or removed via normal metabolic pathways without adverse effects to

human body.

7. Easy to fabricate and sterilize
In addition to having all these desirable properties, DDSs should be easy to produce and
process. Therefore, if we find a simple platform for DDS synthesis that effortlessly

produces a series of DDSs for different drugs, the goal will be accomplished.

Those issues are not trivial and they are as complex as drug discovery.
In summary, drug delivery is becoming a core technology that always operates alongside

earlier aspects of drug development.

1.2 Administration methods

As early as four thousands years ago pills, ointments, and salves were employed by

Egyptian physicians. In 1665, intravenous injections were first performed in humans.

Wood introduced subcutaneous injections in 1853. Luer developed the modem

hypodermic syringe in 1884. Since then, a large variety of administration methods have

evolved '. Saltzman reviewed the current administration methods shown in Table 1.1'.

 

 

 

 

 

 

 

 

Table 1.1 Common routes of drugadministration
Route of Example Advantages Disadvantages
Administration
Intravenous Antibiotics for 100% bioavailability Discomfort to patient
injection (i.v.) sepsis Requires health care-
provider
Risk of overdose or
toxicity
Risk of infection
Intravenous Heparin for anti- 100% bioavailability Requires
infusion coagulation Continuous control hospitalization
over plasma levels Risk of infection
Subcutaneous Usually high bio- Discomfort to patient
injection (s.c.) availability
Intramuscular Insulin for Usually high Discomfort to patient
injection diabetes bio- availability
(i.m.)
Oral (p.o.) Many Convenient Drug degradation
Self-administered before absorption
Limited absorption of
many drugs
Sublingual or Nitroglycerin Avoids first-pass Limited to lipophilic,
buccal for angina metabolism in liver highly potent
Ophthalmic Pilocarpine for Local delivery Discomfort to some
glaucoma Self-administered patients for frequent

 

 

 

 

administration

 

 

 

 

 

 

 

 

 

Topical Antibiotic Local delivery Limited to agents that
ointments Self-administered are locally active
Intra-arterial Control of vascular High Risk
injection delivery to specific
regions
Intra-arterial Direct delivery to Limited drug
injection brain penetration into
brain tissue
Rectal Avoids first-pass Discomfort leads to
metabolism in liver; poor compliance in
Self-administered some patients
Transdermal Nitroglycerin Continuous, constant Skin irritation
patches for delivery Limited to lipophilic,
angina Self-administered highly potent agents
Vaginal Spermicides Self-administered Discomfort leads to
poor compliance in
some patients
Controlled- Norplant for Long-term release Requires surgical
release contraception procedure
implants

 

 

 

 

 

 

"' Table is taken from Ref [1]

The aim of any drug administration methods is always to combine the tissue absorption
and distribution of the drug with patients’ compliance in a maximum beneficial way.
Among them, oral dosage forms are always preferred since they are painless,
uncomplicated and self —administered. However, DDSs intended for oral administration
require more consideration of gastrointestinal physiology. Otherwise, most drugs will be
easily degraded within the gastrointestinal tract (GIT) or cleared by macrophage (Kupffer
cells) of the liver and spleen, and not absorbed in sufficient quantity to be effective.
Therefore, protein pharmaceuticals are mostly administered by parenteral delivery in

order to quickly achieve the efficient concentration in the blood, while avoiding the “first

pass elimination”. This occurs when drug molecules enter the circulation through a
mucosal surface, and then circulate through the liver, where they can be metabolized,
before distribution throughout the rest of the body’. However, frequent parenteral
injections are poorly accepted by the elderly and children, plus they are not well supplied

to some undeveloped countries and urban areas.

Besides parenteral and oral delivery methods, there are other modes of administration,
such as transderrnal delivery, inhalation delivery, sublingual delivery, rectal delivery,
topical delivery, etc. Each delivery scheme has special attributes. See figure 2.18.
Inhalation delivery is fit for treating diseases of the lungs and respiration system, and it
requires that the DDS be dispersed as well as the drugs. Sublingual delivery is usually
used for acute cardiac dysfunction because it is faster than oral delivery, convenient for
patients, and it requires the DDS to protect drugs from degradation by saliva enzymes.
Topical delivery is more efficient than systemic delivery since a drug can be administered
directly to the target organ or tissue. In all of the various regional deliveries, the design of
the DDS always needs to take into consideration the environmental situations where the
drugs will be released, such as pH, enzymes, and different barriers for further absorption

and distribution in the body.

The drug concentrations in the blood realized by these administrations are different over
time because all need to be absorbed (except in the case of intravenous injections) before
entering the blood. It is therefore useful to know that the rate of absorption by these
administration methods from the fastest to slowest is: inhalation, sublingual, rectal,
intramuscular, subcutaneous, oral, and transdermal. Thus, delivery of identical amounts

of drug to tissue sites with different administration routes

 

 

 
 

l iv
A: °'|_
F A“
- 23‘ . r
t . . ' '

/ b h J‘ ‘ A
Sublingual . Inhalation
Delivery

Oral '
Delivery
Intra-
arterial
injectin
LII- ‘
. 'F

E 9
3 2
a _.
r E
a 5'
fl =
:a
c-
a
'<

_' ' Rectal

. ’T' delivery
I ‘50"
i-'

 

 

 

 

 

 

Figure1.2 Drug Delivery Methods *Figure is adapted from Ref [8]

can result in measurable differences in the drug concentration in the blood within the
same period of time. In addition, the route of drug administration will influence the
kinetics of biodistribution and elimination and thus the effectiveness of the therapy. For
example, human immunoglobulin G (IgG) were administered orally, subcutaneously,
intramuscularly, and intravenously to human patients (without delivery systems) (Figure
1.3)9. These different methods led to different patterns of IgG concentrations in the

plasma over time.

 

 

 

'100
90%
sea a
1 Intramuscular
°\° 7°} '3 Subcutaneous
a 60-1 o Intravenous
g j 0 Oral
a 50-:
.E 3
3. 4°?
sol.
201
i
101
i
o e e :7 a
r'r"r"*r' 'r *“t*** I Y I
o 5 1o 15 20 25 so hrs

Figure 1.3 Plasma IgG following different administration routes in humans

* Figure 1.3 was taken from Ref [1]

Figure 1.3 shows that without the protection of a DDS, the IgG cannot be delivered orally.

Although the intravenous injections give the highest initial drug concentration in the

blood, the concentration decreases sharply during the first 5 hours. With an ideal DDS the

10

delivery could be accomplished in a controlled manner; the initial drug concentration is
not too high (nontoxic), and also the rate of drug release is kept constant. Subcutaneous
and intramuscular injections show smoother changes in drug concentration in the blood,

which is more like controlled delivery. However, DDSs are still needed to help improve

the rate at which the drug concentration in the blood reaches the efficient level.

Table 1.2 Methods for Hormones Delivery (*Adapted from Ref [10])

 

Advantages

Disadvantages

 

Less liver stress

Less steady hormone level. Pain and slight

 

Injection than oral delivery. infection risk from hypodermic needle
Inexpensive usage.
Convenience.
Possrbly more Increased stress on the liver since it has to
beneficral for . .

Oral process the hormones multiple times,

blood cholesterol
levels than other
methods.

resulting in an increase in clotting factors.

 

Transdermal film

Less liver stress

than oral delivery.

Hormone level
more steady than
injections.

Inconvenience and skin irritation. Multiple
simultaneous patches required for pre-op
dosage.

Expensive.

 

Uncoated tablets
can be placed
under the tongue

 

 

Sublingual/ or between the Some is also dissolved in the saliva and
Buccal cheek and gum. swallowed. The taste is not good.
Less liver stress
than oral delivery.
Absorption through a mucosal membrane
Cream, Le . is best; absorption through scrotal skin is
. ss liver stress
Supposrtories, and than oral delivery not as good as mucosal, but better than
Pessaries ' through other skin (need more data about

 

 

typical doses and absorption).

 

 

A medicine commonly has a number of delivery methods, which provides patients more
opportunities to get the most suitable administration method, such as the delivery of
hormones (see table 1.2). Therefore, it becomes more critical for scientists to develop
DDSs that can be used for different administration routes with the lowest side effects and

the same therapeutic efficiency.

11

1.3 Vehicles of drug delivery

1.3.1 Inorganic vehicles

In the beginning of drug delivery development, inorganic materials were unnoticed
because of their lower biocompatibility. However, the excellent adsorption properties of
calcium hydroxyapatite, Ca.o(PO4)6(OH)2' ”2 and activated charcoal'3’” have made them
successful carriers of antitumor agents for the local treatment of metastatic lesions of
lymph nodes afier surgical removal of the main tumor, and for the treatment of solid

tumors by inhibiting cancer sell growth (Dunn osteosarcoma cells).

Another exciting development of inorganic materials for DDSs is the use of microchips,”
as controlled drug—delivery devices. (See figure 1.4) This patented technology is based on
tiny silicon chips containing hundreds of micro—reservoirs capped by noble metal
membranes that open when electronically activated. Each reservoir can be filled with any
combination of bioactive agents and hermetically sealed to protect the contents. Complex
biochemical delivery can be achieved by opening the reservoir caps on demand in
response to a preprogrammed clock, biosensor feedback, or a wireless signal from a
physician or patient. This product is still waiting for the evaluation in vivo. However, the

versatility of the technology offers tremendous potential for future development.

12

 

Drug releasing
Drug reservoir
Gold caps

Figure 1.4: MicroCHIPS Technology (taken from Ref [15])

FeRx Corporation 1° is developing magnetically targeted systems, which make use of
elemental iron magnets with drug adsorbed to the surface of carriers. Carriers that localize
within tumors because of the applied magnetic field are undergoing trials for the
treatment of metastatic liver cancer. Although research on DDSs still focus mainly on
organic material carriers, in the future, it is believed that inorganic materials will get

greater attention from drug developers.

1. 3. 2 Organic vehicles

1.3.2.1 Liposome

Introduction

Liposomes are colloidal, vascular structures based on (phospho) lipid bilayers which can
be unilamellar or multilamellar oriented around an aqueous core. Since Gregoriadis,
Ryrnan, and Bangham invented the liposome drug delivery systems (LDDS) in the early
l970s'7'20, liposomes have been under extensive investigation as DDS for a broad

spectrum of agents including drugs, antibodies & antigens, genetic materials, etc.

 

Figure 1.5 Classifications of Liposomes (adopted from Ref [21])

14

As drug delivery materials, liposomes may solubilize lipophilic drugs that would
otherwise be difficult to administer intravenously. Through interaction with cells in
various ways, liposomes can be successfully used for passive and active target delivery
with additional surface modifications. Liposomes not only protect encapsulated drugs
from degradation by metabolizing enzymes, but also prevent the non-targeted part of the

body from being exposed to the full dose of drugs.

Classification of Liposomes

There are four major liposome types that can be broadly distinguished on the basis of
composition and in vivo application (2" (See Figure 1.5). These four classes include:
conventional liposomes, long-circulating liposomes, immunoliposomes, and cationic

liposomes which will be described below.

Conventional liposomes refer to the liposomes composed of only phospholipids (neutral
and/or negatively charged) and/or cholesterol. Such non-surface modified liposomes are
characterized by a relatively short blood circulation time because they either disintegrate
in the blood stream or circulate and then are picked up predominately by macrophages
(Kupffer cells) in the spleen and liver. According to this behavior, conventional
liposomes are good candidates for the delivery of antimicrobial agents to infected

macrophages.

Long-circulating liposomes or stealth liposomes are produced by covalently attaching a
hydrophilic polymer, polyethylene-glycol (PEG), to the outer surface. PEG-modified
lipids prevent plasma protein adsorption to the liposome surface and thus the subsequent
recognition and uptake by RES”. It also reduces the opportunities for attack of multiple

reactive groups by shielding the membrane surface of lipid323'29. This protection leads to a

15

longer circulation time, which consequently enhances the opportunity for liposomes to
take advantage of “the leaky endothelium effect” attributed to the tumor site and

inflammatory sites and leave the vascular system.

Immunoliposomes have specific antibodies or antibody fragments covalently attached to
their surfaces to enhance target site binding. With the help of PEG coating,
immunoliposomes are given a greater chance to reach targets other than macrophages. In
addition to antibodies, glycolipids, proteins and vitamins have also been used to target
specific cells via cell surface receptors. Moreover, immunoliposomes can be used for
antibody-directed enzyme pro-drug therapy (ADEPT) designed to generate a high

concentration of anticancer molecules only in close proximity to tumor cell membranes.

Cationic liposomes represent the pioneering form of liposome drug delivery systems. The
cationic lipid components interact with, and neutralize, the negatively-charged DNA,
thereby condensing it into a more compact structure. The resulting lipid-DNA complex,
rather than DNA encapsulated within liposomes, offers the protection and improves

cellular internalization and expression of the condensed plasmid.

Requirements of liposome gene delivery system in vivo 3°

1. Liposomes should be targeted to endocytic receptors to increase the rate of endocytosis.

2. Fusion processes should be optimized to enable efficient escape from the endosome

and entry into the cytoplasm.

3. Cytoplasmic stability and nuclear targeting of the plasmids should be ensured.

l6

Mechanism

Liposome encapsulation can alter the spatial and temporal distribution of drugs in the
body, which may significantly reduce unwanted toxic side effects and increase the
efficacy of the treatment. These exciting drug delivery applications result from the
physicochemical and colloidal characteristics as well as their biological interactions with
the cells. Liposomes resemble cell membranes in their structure and composition and

have four major interactions with cells”.

1. Lipid exchange involves the exchange of liposome lipids for the lipids of various cell

membranes and depends on the mechanical stability of the bilayer.

2. Adsorption onto cells occurs when the attractive forces exceed the repulsive forces.

3. Endocytosis delivers the liposome and its contents into the cytoplasm indirectly via a

lysosomal vacuole in which low pH and enzymes may inactivate the encapsulated agent.

4. Fusion is the process in which the liposomes’ contents are delivered directly into cells

as the liposomal lipids merge into the plasma membrane.

Problems of liposomal DDSs

There are still some difficulties 2" 3'

in the development of liposome drug delivery

systems (LDDS). First of all, the qualities of the raw material (phospholipids) such as
phosphatidylcholine (PC), phosphatidyl glycerol (PG), and phosphatidylethanolamine (PE)
are poor. They all have a source-dependent composition of acyl chains. Ester hydrolysis

and peroxidation also make their quality vary considerably. Because liposome behavior in

vivo strongly depends on size, bilayer rigidity, charge and morphology, poor

17

characterization of the physicochemical properties of the liposomes becomes a more
serious problem. Further, low drug-loading capacities and leakage, which leads to poor

stability, are serious problems for DDSs.

18

1.3.2.2 Polymer vehicles

The combination of polymer science with pharmaceutical science has led to a
quantum leap in design and development of novel drug-delivery systems. Bioadhesive
polymers were first used to improve the residence time and efficacy of the DDS
through their intimate contact with the epithelial cell layer. Biodegradable polymers
were also recognized as capable of accomplishing drug delivery functions without
surgical removal of delivery materials. Now ‘smart’ hydrogel break-throughs have
launched a promising field of self —regulated drug delivery in response to an
environmental stimulus. Compared with other materials used for DDSs, polymers
have great advantages for achieving either temporal or spatial control of dmg

delivery32'3°.

Polymeric materials used in DDSs can be naturally occurring, synthetic or a
combination of both. The main classes of natural polymeric materials used in DDSs
are summarized in Table 1.3 33 according to their origin, properties and principal
applications. Although naturally derived polymers are abundant and usually
biodegradable, their structural complexity often makes modification and purification
difficult. In addition, significant batch-to-batch variations occur because of their bio

‘preparation’ in living organisms (plants, crustaceans).

l9

 

Table 1.3 A summary of the main properties of applications of natural polymers in

 

 

 

 

 

DDSs.
Polymer Main applications and comments Refs
Proteins and Absorbable, biocompatible, nontoxic, naturally
protein-based available.
polymers:
Collagen Used as drug delivery micro-spheres. 37
Albumin Used in cell and drug micro-encapsulation. 38
Poly (amino Nontoxic, nonantigenic and biocompatible. 39
acids):
POIY (a, L-lysine) Used as oligomeric drug carriers. 40,41
Poly (0t, L-glutamic
acid)
Poly (aspartic acid)
Polysaccharides
and derivatives:
Carboxymethyl From vegetable sources, pH responsive and water- 39.42
cellulose soluble.
Sodium Widely used in oral and topical pharmaceutical 43,44,
Carboxymethyl formulations because of its viscosity—increasing 45
0611111056 properties. Also used as a tablet binder and to stabilize
emulsions.
Al . From marine sources, algae, excellent gel-formation
grnate . . . . . . .
properties; relative brocompatrbrlrty; microstructure and
viscosity are dependent on the chemical composition 46,47
(batch to batch) variations. Used for controlled release
of bioactive substances, injectable microcapsules for
treating neurodegenerative and hormone-deficiency
diseases.
Carrageenan Excellent thermoreversible properties. Used for 48
microencapsulatio‘n.
Dextran From human and animal sources. Excellent rheological
properties. Widely used as sustainable DDSs,
particularly for injectable and colon—specific DDSs. 49.50
Chitosan Biocompatible, nontoxic, excellent gel-and film-
forrning ability, good absorption-enhancing, natural
polycation, pH-responsive, controlled release as well as 5152

 

bioadhesive properties. The degree of deacetylation and
derivation with various side chains can be a source of
manipulation for specific drug-delivery applications
(e.g., gels, membranes, micro-spheres).

 

 

Table is from Ref. [33]

20

 

Synthetic polymers overcome many of the limitations of natural polymers. They are
available in a wide variety of compositions with readily adjusted properties.
Additionally, processing, copolymerization and blending provide a simultaneous
means of optimizing a polymer’s delivery properties. The following is a
representative list of synthetic polymers used in DDSs. It is mainly divided by

biodegradable polymers and non-biodegradable polymers 33’ 35’ 3° 53.

Non-biodegradable polymers

Silicones

Silicones, mostly referred to as polysiloxanes, are a unique class of non-deformable
polymers possessing good low-temperature flexibility, remarkable biocompatibility,
excellent electrical properties and water repellency features that are not common with
hydrocarbon polymers. They are usually synthesized by hydrolysis of alkylsilicon or
arylsilicon halidess3 . Chemical modification commonly involves introducing constituents

in place of one or both of the pendent methyl groups in the structure shown below:

'3‘ H20 51 F31
Cl-Si-CI -_.-. HO-Si-OH . 4 H‘fO-Si-lBOH + H20
92 HQ 5'32 '

Because of their case of fabrication and high permeability, polydimethyl siloxanes
(PDMSs) are used for water-soluble drugs and for the delivery of steroids through long-
acting DDSs such as subdermal implants and intravaginal systems. In addition,
dimethylsilicones can be copolymerized with methyl methacrylate and ethylene oxide to
create a series of polymers with controlled permeability to hydrophilic or hydrophobic

steroids, enhanced mechanical properties, and improved adhesion to tissuesS4'5°.

Poly [ethylene-co-(vinyl acetate)] (EVAc)

21

The most commonly used copolymer contains 40% vinyl acetate and has the general
structure: . . ‘
-«'- CH2 “CH2 9: CH2 "CHv-r
‘X‘ 0 Y
1' -‘0
CH3

The copolymer is synthesized by free—radical polymerization of vinyl acetate and

ethylene. Since EVAc is one of the most biocompatible polymers that has been tested as
implant materials 57, it has been widely used as a matrix for controlled drug delivery.” 59
As stimuli-responsive hydrogels, EVAc can be used to deliver drugs using different
environmental stimuli such as magnetic fields, ultrasonic radiation and changes in glucose

concentration °°‘°2.

Poly(ethylene terephthalate)
Commonly used linear polyester is poly(ethylene terephthalate) (PETE), which is

synthesized by condensation of ethylene glycol and terephthalic acid:

9 l? f, 9 O 1
Ho-c—Q—c-OH + HO'-H20-CH2-'OH——'> -.—c o-CH2—0H2~o+—

0

Acrylic polymers:
Poly(methyl methacrylate) (PMMA) is the one of the most popular acrylic polymers, and
is prepared in large quantities commercially using free-radical polymerization. PMMA

has exceptional transparency and physical strength (structure as follows).

. CH3 :
._. CH2‘C _ 4
. 0' n

0
CH3

22

Biodegradable synthetic polymers

Aliphatic polyesters

Polylactide (PLA)

Lactic acid exists as two optical isomers, D and L. L-lactide is the cyclic dimmer of
naturally occuning isomer, and DL-lactide or meso lactide is the synthetic blend of D-
lactide and L-lactide. The homopolymer of L-lactide (LPLA) is a semicrystalline
polymer with high tensile strength and slow degradation time. Poly(DL-lactide)
(DLPLA) is an amorphous polymer exhibiting a random distribution of both isomeric
forms of lactic acid, and accordingly low tensile strength and more rapid degradation

time3°' 53 .

0' 0
Me, C r Me O
/ 0 t 1 t 'A . v 2 ‘
C‘, ,' -———°~§-°y§---—» 'iO‘“CH- c-o ~CH c»
‘C/ 2, heat 1 g
.2 Me Me . n
l o
Monomer;
O
T Me\/C~ ' so Me 0'
0 catalyst ; w ; E
O '\ —~— —--—— -- OCH—C-O-CH-“C:
0
D-Lactide Poly(D—Iactide)

Poly(lactide-co-glycolide) (PLGA)

Copolymers of glycolide with both L-lactide and DL-lactide have been developed for
biodegradable drug delivery systems. Adjusting the percentage of monomers can be
used to regulate the degradation time of copolyrners (PLGA). Although PLGA
represents the ‘gold standard’ of biodegradable polymers (exemplified by more than
500 patents), the disadvantage of PLGA is that increased local acidity due to
degradation can lead to irritation at the site of polymer application“. Moreover, the

increased local acidity may be detrimental to the stability of protein drugs”.

23

j 0 Me 0. .j 0 Me ,1 1H2° H20 _

40 -CH c 0 CH cmoa CHv-C 0 CH ci 9:0 c o c c-o-«

Poly(L-Iactide) Poly(D-Iactide) Poly(Glycolide)
Poly(e-caprolactone)

The ring opening polymerization of e-caprolactone yields a semicrystalline polymer with
a melting point of 59-64 °C. The polymer has been regarded as a tissue compatible and
biodegradable DDS. Copolymers of e-caprolactone and DL-lactide have yielded materials

with more rapid degradation rates.

0
,C ~ .\ mtalyst

2. -» AWA

\ ,—
v

Poly(hydroxybutyrate-co-hydroxyvalerate)

The poly(hydroxy butyrate) is crystalline and brittle, whereas the copolymers of poly
(hydroxybutyrate-co-hydroxyvalerate) are less crystalline, more flexible, and easier to
process. These polymers typically require the presence of enzymes for biodegradation but
can degrade in a range of environments and are under consideration for several

biomedical applications”.

. o, , o,
‘TO‘CH- CH2-:C~l-—iO-CH--CH2 -cj-
Me x E! y

PoIy(hydroxybutyrate-eo-hydroxyvalerate)

Poly(dioxanone) (a polyether-ester)
Poly(dioxanone) is synthesized by the ring-opening polymerization ofp-dioxanone. This

material is approximately 55% crystalline, with a glass transition temperature of —10 to 0

24

°C. The polymer should be processed at the lowest possible temperature to prevent
depolymerization back to the monomer. Poly(dioxanone) has demonstrated no acute or

toxic effects on implantation and it is re-absorbable after being broken down by

hydrolysis“.
(0:0 catalyst .’ O;
., j --—-- > +o~CH2-Hzco--CH2--c-—
‘02 heat _ «n
p-dioxanone Poly(dioxanone)
Polyurethanes

Polyurethanes have excellent mechanical properties, making them suitable for many
different biomedical applications. The biocompatibility of polyurethanes appears to be
determined by their purity, i.e., the effectiveness of removing catalyst residues and low
molecular weight oligomers from the polymer“. Polyurethanes can be formed by reacting

a bis-chloroformate with a diamine”,

 

CI-COO-(CH2)2-O-COCI +H2N'(CH2)6‘NH2 -_-_.. -—4—COO-(CH2)2-OCGNH-(CH2)5NH+—

n

or by reacting a diisocyanate with a dihydroxy compound. For example, ethylene glycol

and hexanediisocyanate react as shown53.

HO '(CH2)2 -OH + O=C=N-(CH2)6-N=C=O ‘ ~ ._ - > fCO‘NH-(CHQ)5-NH-COO-(CH2)2'O":1“
. I

As DDSs, the surfaces of polyurethanes can be surface modified to produce materials that

are resistant to thrombosis or that interact with cells and tissues in specific ways”‘ °"'°9.

Polyorthoester

25

Polyorthoesters have been synthesized by the addition of diols to diketene acetals.
The mechanical properties of polyorthoesters can be readily varied by choosing

appropriate diols or a mixture of diols in their synthesis”.

0 ,_ Q ./ °-\ lO-\ ~~O\ /“" \‘
2’ "MI \ l ‘ Y 1
HO R . OH // _-_~-.4. ‘ ’(r:7.~\\ ---~ —-’ 3 J ‘~ ‘\.\ .‘
+ o -./-..-O ‘ “‘1‘0‘ 0'“ “ 0 so mn—
010' Diketene acetal Polyorthoester

The degradation mechanism of polyorthoester is shown below.

I," "\>(IO' ‘-,/’MO.\//“ \1 H20 El"i"‘0 “0“:“El
4.0, *0 Ai-ofl‘ 'g " “"’”"‘" \ ’ + HO-Fl-OH
Form" 140"" \-~70H
Polyorthoester
l
I
l H20
V
HO—'\ ""OH
X + CH3CH2COOH
H0 " \“OH

Degradation rates of polyorthoesters can be controlled by incorporating of esters of
short-chain alpha-hydroxy acids such as esters of glycolic acid, lactic acid or glycolic-
co-lactic acid copolymer into the polymer chain and by variation of the amount of
these esters relative to the polymer as a whole”‘ 53. At room temperature,
polyorthoesters can be made as an ointment, which is appropriate for a variety of
t0pical and periodontal applications. Polyorthoesters can also be obtained as a viscous
liquid at room temperature. Proteins and other labile molecules can be mixed into the

polymer, as well, without using solvents or increasing temperature”.

Phosphorous-based polymers

Polyphosphazenes have the general structure

Polymers with a variety of physical, chemical, and biological properties can be produced

by performing substitution reactions on the base polymer, poly(dichlorophosphazene)”:

R

F? PM I
{—N=P-}- ___> -+N=P—l-
I n l n
R Cl
R NH-R
l RNH2 l
l—N=T+n —'—" +N-T+n
a HN—R
I? RONa Q‘R
Jr~=t+. —> +~=A+A
n o-n

This basic structure provides for considerable flexibility in the design of biomaterials, by
selection of the side groups on the polymer chain". The resulting polymers can be
hydrophilic, hydrophobic or amphiphilic. In addition, a variety of bioactive compounds

can be linked to the backbone to make multifunctional DDS.

Polyanhydrides

Polyanhydrides are characterized by their excellent biocompatibility and fast degradation
through hydrolysis. They can be synthesized via the dehydration of diacid molecules
through melt polycondensation as is the case with poly(sebacic acid)”. By selecting
appropriate monomers with different degrees of hydrophobicity, the rate of drug release
can be controlled from days to weeks. For example, copolymers of sebacic acid, a
hydrophilic monomer, with carboxyphenoxypropane, a hydrophobic monomer can be
made into a controlled drug delivery system. By adjusting the monomer ratio during
copolymerization, the degradation of the resulting polymer can be modulated in a

controlled manner“ 72' 73.

l /:.\
Ho‘y/\~V/A“-./\/’\\/H\OH + HO'C'NE [>— O/\/\O“‘\/§

-o
D.

6:0

0

I

O O -1
Sebacic acid Carboxyphenoxypropane
l
i
V
, O . 5 /~ .':\ r11: 0 ']
—+C ‘(CHzle‘C‘Or -.~c .4 X o cum- 0»./,\ /-—c oi-
l. . l ' i> // \.‘_ _' l
O x O ' “ Y

1, 3-Bis (p-carboxyphenoxy) propane

27

Copolymers of methyl vinyl ether and maleic anhydride

Most materials such as biodegradable polyesters erode in a disorderly pattern: defects,
cracks, and holes that initially appear grow in size with time throughout the material. To
provide better control of polymer matrix erosion, materials that erode homogeneously
have been designed. In particular, for materials that erode from the surface only, the
kinetics of dissolution and the release of incorporated drugs can be precisely controlled.
A copolymer of maleic anhydride and methyl vinyl ether was synthesized for this goal.

The following is obtained after partial esterification”:

9 CH3
,0, o coon
“2‘C “*0 CH3 + :1 ,0 ' ‘ --> 'i--*CH2~CH'~CHCH-l
c , 2 .
O l HOOC ‘n

When the copolymer was placed in an aqueous environment, the carboxylic acid groups
on the polymer-water interface become ionized, thus the erosion of polymer is limited to
the polymer surface, and the rate of erosion strongly depends on pH. (The rate increases
when pH drops). The disadvantage of this DDS is the erosion products are

macromolecules and not easily metabolized or excreted by the body”.

Polyamides

A common strategy in the design of biodegradable polymers for medical applications is to
use naturally occurring monomers, with the hope that these polymers will degrade into
non-toxic components. For example, poly(lactide-co-glycolide) degrades into lactic and
glycolic acid, normally occuning metabolites. Thus, amino acids are an obvious choice as
monomers for the production of polymeric biomaterials. Three conventional synthetic

methods are shown below,

28

HzN-R-NHz + H02C-R'-C02H ——> Ho-g-NH-n-Nnco-Rucoéon + H20
'n

HzN-R-NHz + cuco-nucocr *-—> HO'lNH-R-NHCO-R'COL OH + H20

l'l

HzN'R'CozH —<——> HO l—NH-a-col 60H + H20
Poly(amino acid) have good biocompatibility for the delivery of low molecular weight
compounds. Unfortunately, amino acids polymerized by conventional methods usually
yield materials that are extremely antigenic and exhibit poor mechanical properties which
make them difficult to process". Because of their high crystallinity, the degradation of
pure poly(aminoacids) is relatively slow” 4‘. To circumvent these problems, several
approaches have been developed. A few amino acids, like glutamic acid and lysine, can
be modified through their side chains to produce polymers with different mechanical
properties. Copolymers of L-glutamic acid and y-ethyl L-glutamate have been used to
release a variety of drugs, and the variation of the ratio of monomers in the polypeptide
influences the rate of degradation of the resulting polymers". Due to the stability of the
peptide bond in water, the biodegradation of these implants occurs by dissolution of intact
polymer chains and subsequent enzymatic hydrolysis in the liver. Alternatively, amino
acids can be polymerized by linkages other than the conventional peptide bond, yielding
pseudopoly(amino acids)”. For example, the amino acid serine can be used to produce

poly(serine ester), poly(serine imine), orconventional polyserine”:

NH2

{O-CHz-C-C-f Poly(sen‘ne ester)
H 11
/////’r O n
9
HgN-C'H-C-OH

_——’
CH2 {HN‘9H'CH2‘]—' Poly(sen’ne imine)
OH O=C n

\ OH

O
{HN-C'SH‘C-f Polyserine
n

29

Dendrimers (PAMAM)
Relative newcomers to the collection of biodegradable materials used for DDSs are
dendrimers, a type of highly branched macromolecule. The name "dendrimer" is derived

from the ancient Greek word "dendron" (tree), and from the Greek suffix "-mer"

(segment).

Dendrimers consist of a series of chemical shells built on a small core molecule. The
synthesis begins with a simple seed molecule such as ethylene diamine and ammonia,
which normally has two or three reaction sites. With an excess of the first monomer
molecule reacting with all of the reaction sites of the seed molecule, the first branches are
raised. This first monomer molecule has two distinct reactive groups, one at each end.
After one end reacts, the other end will provide reaction sites for the next layer of the
shell 7°79. For example, polyamidoamine (PAMAM) dendrimers are synthesized from an
ethylenediamine core with branching units containing tertiary amine and amide
functionality. This core is reacted with the double bond in acrylic acid to produce a
tertiary-acid molecule. This tertiary-acid is reacted with ethylene diamine to produce a
tertiary-amine (G0). This tertiary-amine is reacted with acrylic acid to produce an Oct-
acid, doubling the number of acids in this half-generation (G0.5). Next, another round of
ethylene diamine is reacted with the G05 to give a G1 molecule with Oct-amines, twice
the number at G0. This alternation of acrylic acid with ethylene diamine continues until

the desired generation is reached. (See figure 1.6)

Dendrimers are like ordinary organic molecules for the first three generations. By G4 they
are beginning to have a preference of three-dimensional structure and to become
spherical. By G5 they have a consistent and specific three-dimensional structure. Then
they are highly structured spheres“. The spherical surface of a dendrimer acts like a

microsc0pic form of Velcro, and a variety of bioactive agents can bind to the surface.

30

Dendrimers have a high drug-carrying capacity because of their multivalency7°'79. The
consistency of structure makes dendrimers ideal building blocks for creating biologically
active nano-materials, which can target structures less than 5 nm in diameter. This
provides an excellent drug delivery system that can get through vascular pores and into
tissue more efficiently than larger carriers". Another advantage of dendrimers is that their
synthesis results in a single molecular weight rather than a distribution of sizes, which is
critical for controlled DDSs77. So far, all the excellent properties of dendrimers make

them promising materials for controlled drug delivery systems”.

Seed molecule:

H2N_\‘—NH2

0
first step 1 HzcletCOH
P
A.-.
N ..
HO- N xNN
g \AC-OH
11

second step 1 H2N—\_
NH2

HZN _\ '0 jNHZ
tl‘dvx 9
N NC'NH
HN'qN —\‘—N
I _
/~—/ 0 9 NH__\
HZN O NH;

0
1
third step 1 Heep-con

H N
fourth step 1 2 xNHz

Figure 1.6 Synthesis of PAMAM Dendrimer (I)

31

O c—OH C\
HO’CXNJ K ftp
\-- O JN“ ‘OH
\N-ll /
\/\ "
H N'\_ N 'N“
”fl-(EN N
,r" "J O E'NH
N~. \ ,OH
"l/ \\ ’N,/"\‘ v’ C\\
HO c ’ c OH <\ o
O 0 C/
HO “O
i
l
V
1 Repeat the first step and second step
I
l
o . S
,NH .
C HN ,0
\/ \ .
\
H N O, 2 7
2 \4/AxN/‘C/\/N O «N /\ ,9
H ”\ n O _ / \ C“N/\, NHZ
H‘Cm .. f" A
N NC'NH
N —\—N
HN'SE \/\ H
[-4 I O n N\
H N, O \\
H NA /N‘,C--/ ‘ ,AN-fi
2 0' i 2 \_
\C HN" OJ C I NC
CS NH ./ b O \\
'\ H NF “”2
2

Figure 1.6 Synthesis of PAMAM Dendrimer (ll)

32

Factors which affect degradation of polymers

A great deal of attention and research effort is being concentrated on biodegradable

polymers because biodegradable polymers provide a better opportunity for controlled

drug delivery without the concerns of removal of the extraneous delivery system.

However, measuring the degradability of polymers is not a simple question since there are

so many factors to be considered“. These factors include

Chemical composition and structure

1.

2.

3.

4.

Distribution of repeat units in multimers.
Presence of ionic groups.
Presence of unexpected units or chain defects.

Configuration structure.

Physical factors

1.

2.

Molecular weight.

Molecular weight distribution.

Shape and size changes.

Variations of diffusion coefficients.

Mechanical stress, stress- and solvent-induced cracking, etc.
Morphology (amorphous/semicrystalline, microstructures, residue

stressed).

Physicochemical factors (ion exchange, ionic strength, pH).

Sites of implantation

Absorbed compounds (water, lipids, ions, etc.).

Mechanism of hydrolysis (enzymes versus water).

As well as the factors mentioned above, the processing conditions (annealing,

sterilization, etc.) and storage history also affect the degradation properties of polymers.

33

Thus, designing controlled—release drug delivery system using biodegradable polymers is

still time-consuming.

Mechanisms of drug release from polymeric DDSs

There are four primary mechanisms by which active agents can be released from
polymeric drug delivery systems: diffusion, solvent-activated release, degradation, and
swelling followed by diffusion. Any or all of these mechanisms may occur in a given

release system“ 8°.
Diffusion

Diffusion occurs when a drug or other active agent passes through the polymer. There are
two diffusion-controlled release systems, matrix and reservoir, which are shown in Figure

1.7 and Figure 1.8.

In a matrix system (See Fi gure1.7) a polymer and active agent are mixed to form a
homogeneous system. Diffusion occurs when the drug passes from the polymer matrix
into the external environment. With this type of system, the rate of drug release normally
decreases, since the active agent has a progressively longer distance to travel and

therefore requires a longer diffusion time to release.

In a reservoir systems (See Figures 1.8 a and 1.8 b) the drugs (pure or in a dilute or highly
concentrated solution) are surrounded by a polymer film or membrane that controls the
diffusion rate. Since this polymer coating is essentially uniform and of a nonchanging
thickness, the diffusion rate of the active agent can be kept fairly stable throughout the

lifetime of the delivery system.

When talking about diffusion-controlled systems, the drug delivery device is understood
to be fundamentally stable in the biological environment and does not change its size
either through swelling or degradation. In these systems, the combinations of polymer
matrices and bioactive agents chosen must allow for the drug to diffuse through the pores
or macromolecular structure of the polymer upon introduction of the delivery system into

the biological environment without inducing any change in the polymer itself.

   

 

é‘j/ , \‘if.
fall, ’I I " ll?
Time ..
:“7,2'*”’"""\~;;
“\g ,,./31- 1 _'
/"'~'"‘\ ' I
./ .,.',‘ \l :
\\"- J’f/ '
l. '
Figure 1.7: Figure 1.8:
Drug delivery from typical matrix Drug delivery from typical reservoir
delivery systems. devices: a. Implantable or oral systems

b. Transdermal systems

*Figure adapted from Ref [34] *Figure adapted fiom Ref [34]

35

Solvent-activated

Solvent-activated systems usually employ a semi-permeable membrane containing a
small, laser-drilled hole. Within the membrane there is a high concentration of an osmotic
agent, either the drug itself or a salt, which causes water to enter through the membrane.
The drug is then forced out through the hole because of the increased pressure. Drug

release could be kept at a constant rate in solvent-activated systems.

Degradation

There are three types of degradation mechanisms. A) Water—soluble polymers are made
insoluble by cross-linking. When the cross-links are broken at some point in the body, the
polymer will dissolve. B) Water-insoluble polymers are made soluble by hydrolysis or
ionization of side groups. C) Insoluble polymers are broken into smaller soluble
molecules with an environmental stimulus. One or all of these mechanisms can be used in

degradation of controlled release systems.

There are two forms of degradation, bulk degradation and surface degradation. (See
Figure 1.9 a and 1.9 b) Bulk degradation occurs throughout the polymer structure in a
rather random fashion”. The rate of release is unpredictable, and entire dose dumping can
often occur“. Surface degradation delivery systems eliminate the problems of bulk
degradation systems by using hydrophobic polymers, which contain water-labile linkages.
Thus, diffusion of water into the matrix and internal degradation are minimized.8|
Examples include polyanhydrides and polyorthoesters. The degradation occurs only at the
surface of the polymer, resulting in a release rate that is proportional to the surface area of
the drug delivery system. With proper surface geometry design, zero order degradation

kinetics is possible“.

36

 

Figure 1-9= Figure 1.10:

Drug delivery from: Drug delivery from swelling-
a) Bulk degradation controlled system. controlled release system:
b) Surface degradation controlled system. a) RCSGWOII' polymeric DDS

b) Matrix polymeric DDS

*Figure adapted from Ref [341 *F- d3 d fr R f[34]
igurea pte om e

Swelling-controlled release systems

Swelling-controlled release systems are initially dry and, when placed in the body, will
absorb water or other bodily fluids and swell. The swelling increases the aqueous solvent
content within the formulation as well as the polymer mesh size, enabling the drug to
diffuse through the swollen network into the external environment”. Examples of these
types of devices are shown in Figures 1.10 a and 1.10 b for reservoir and matrix systems,

respectively.

37

1.4 Gene therapy

1.4.1 Introduction

Gene therapy is a technique for correcting defective genes responsible for disease

development. There are two main approaches for correcting faulty genes, gene addition

and antisense delivery techniques.

Gene Addition: A normal gene which is lacking or dysfunctional in patients is

inserted into the human body (see figure 1.11).

Therapeutic
Protein

DNA Cell chromosome

Therapeutic
Protein

 

Figurel.ll Gene addition technique (taken from Ref [82])
Images in this thesis are presented in color.

 

DNA mRN A No protein
_ produced
(3)4 ' '
:x I“
‘4'!” .0 translation
\‘;."/ '3»: r.
{5‘ 3; i /T‘
w =-
v 41—1,
A'TTV' V\~A .
Q . . Antisense
' 1 Drug

 

Figure 1.12 Antisense Technology (taken from Ref [82])

38

Antisense technique: A technology of interrupting rnRNA translation by using an
antisense strand to hybridize with a specific messenger to block the expression of

disease-related genetic code (see figure 1.12).

Actually, gene delivery is the introduction of specific polynucleotides into human cells
with the aim of altering the production of a specific protein. The changes in protein
expression result in the reduction or elimination of disease”. In the future, all diseases
including cancer“, infectious disease”, vascular disease“, inflammatory disease”,
neurological disorders 88as well as inheritable genetic abnormalities”90 have the potential

to be cured by this promising strategy.

From the beginning of gene therapy (1990), researchers have mostly used viruses as the
gene delivery system“. In this method, disease-causing genes are removed and
therapeutic genes are inserted into a virus. The virus vector carries and unloads the
therapeutic human gene into the target cell to accomplish therapy. However, in 1999, the
death of lS-year-old Jesse Gelsinger who was participating in a gene therapy trial for
omithine transcarboxylase deficiency (OTCD) caused gene therapy to suffer its first big
setback'o". Since it is believed that the boy’s death was triggered by a severe immune
response to the adenovirus carrier, researchers began to notice the high risk of virus
delivery methods. Viruses present a variety of potential problems to the patient: toxicity,
immune and inflammatory responses, and gene control and targeting issues. Further, there
is unavoidable concern that the viral vector, once inside the patient, may recover its

ability to cause disease.

Achieving success in gene therapy is not easy. It not only depends on the accurate

expression of the therapeutic agent, but also strongly depends on efficient delivery

39

into target cells, successfully overcoming the subcelluar barriers such as crossing
membranes of cells, drugs escaping from lysosomes, and targeting and entering the
nucleus°3' 92’ 93. Now it is crucial to develop an improved delivery system for gene
therapy since compared with conventional small molecules, polynucleotides exhibit

different chemical and physical properties that are not well suited to cell delivery.

40

1.4.2 Antisense human telomerase RNA (GCG CGG GGA AAA GCA)

An average human chromosome contains a single molecule of DNA of about 150
million nucleotide pairs. The DNA molecules in eukaryotic chromosomes are linear
with two ends, which are called telomeres. Telomeres are crucial to the life of the
cell“. They keep the ends of the various chromosomes in the cell from becoming
entangled and sticking to each other, and also assist in the pairing of homologous
chromosomes and crossing over during prophase of m_eio_s§_ 1. Human telomeres lose
about 100 base pairs from their telomeric DNA at each mitosis. With this rate, after
125 mitotic divisions, the telomeres would be completely gone. Therefore, the steady
shrinking of telomeres imposes a finite life span on cells. Most cancers arise from
somatic cells, but one of the specific features is their ability to divide indefinitely”. It
turns out that cancer cells have the ability to synthesize telomeres and, thus, to
compensate the shortening of their telomeres. The reason cancer cells can be
distinguished from normal somatic cells is that they have telomerase, an enzyme that
can add telomere repeat sequences to the end of DNA strands during the DNA

- - - 9 ,97
replication to make cancer cells immortal ° .

Telomerase is a ribonucleoprotein. Its single RNA molecule provides an AAUCCC
template to guide the insertion of TTAGGG. Thus telomerase is a reverse
transcriptasegg, synthesizing DNA from an RNA template. The sequence GCG CGG
GGA AAA GCA is complementary to the sequence between residues 76 and 94 of
human telomerase RNA. Using this antisense telomerase RNA (GCG CGG GGA
AAA GCA) can interrupt the telomerase production with the aim to shorten the life

span of cancer cells”.

41

DNA Molecules

////\

Telomors } ’ } Without T elomerase

A

Mitosis of Normal somatic cells

/ With T elomerase
'l'olom<A ' ' ’ ’

Mitosis of Cancer somatic cells

 

Figure 1.13 A Comparison of Mitosis of Normal Somatic Cells with Cancer Somatic

slit)

Telmerase RNA Antisense Drug

 

Figure 1.14 Cancer Cell Treatment with Antisense Drug (GCG CGG GGA AAA
GCA)

42

10.

ll.

12.

l3.

14.

15.

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49

Chapter 2 Design of Cationic Poly-B-amino acid DDS

2.1 Introduction

In the past 30 years, liposomes have been the most commonly used drug delivery

2'3to

material. However, even with several improvements and diverse modifications“
overcome their shortcomings, liposomes still have many drawbacks. Some are due to
the poor quality of raw materials, low drug-loading capacity and high instability 4' 5.

Therefore, the development of efficient and reliable polymeric drug delivery systems

has become imperative.

Generally, there are a few requirements for designing a prospective polymeric drug
delivery system:
1. Biocompatibility— stable in blood circulation, good for administration and
distribution
2. Drug loading/binding capability
3. Ability to penetrate cell membranes
4. Biodegradability: Drug delivery systems can biodegrades into safe,
physiologically benign fragments.
5. Stimuli release: with different environmental stimuli, drugs can be accordingly
freed from the drug delivery systems, especially within endosomes.
6. Target-ability— with antibody fragments to accomplish target-specific cell or
tissue delivery.

7. To be traceable, with a molecular beacon, reporter group or label.

50

2.2 Two main elements of the design

2.21 Molecular Weight Control

Why do we need to control the molecular weight of polymers?

With oral administration, most macromolecules cannot be absorbed from the gut and
also small molecules may be easily modified or degraded by glucuronyl, sulfate,
acetyl, glutathione, or glycine conjugation 6 upon the first pass or subsequent exposure
in the liver. Further, most animal research studies and early clinical trials are
performed by direct injections. Therefore, the development of injectable dosage forms
of new drugs (anticancer-drugs, proteins and genes) are more likely to succeed than
alternative routes of delivery. The molecular weight control of parenteral drug

delivery systems is the key element for the success of the delivery.

Regarding parenteral delivery, we need to understand several barriers in the human
body. First, at the systemic level, the reticuloendothelial system (RES) and renal
filtration are mainly responsible for the rapid clearance of drug delivery system from
the systemic circulation7. The RES mainly gets rid of larger hydrophobic particles
over smaller, more hydrophilic ones; while the kidney filtration selectively removes
smaller molecules from the circulation in favor of larger molecules. So the
hydrophilic polymeric drug delivery system with appropriate molecular weight will
have a longer circulation time, which helps them to distribute drugs to different

compartments or be retained at the target site.8

51

Macromolecules cross the normal vascular endothelium slowly, as evidenced by the
appearance of serum proteins in the lymph. However, this process is also sensitive to
molecular weight. Higher molecular weight polymers are found at lower
concentration in the extracellular fluid, relative to serum, than lower molecular weight
polymerss. This is true except for the special organs like the liver or spleen where the
endothelium is fenestrated or discontinues, or the tumor site where the enhanced

permeability and retention effect (EPR) 9' '0' ”

can allow high molecular weight
polymers to readily diffuse through. Higher MW polymers do not reach the same
concentration in the extracellular medium of normal tissue as is as found in serum.

Because of this, the size of the drug delivery system needs to be considered.

52

2.22 Ability to traverse membranes

Among the requirements for the design of polymeric drug delivery systems, their
ability to penetrate cell membranes while carrying large molecules such as proteins,
genes, etc. is the most critical element. The significant discovery of protein-

transduction domains (PTDs) opens a promising scene in this field.

Protein-transduction domains (PT Ds) refer to special polyamino acid sequences that
can efficiently translocate a variety of bioactive agents into living cells'2"8. (See table
2.1”, the protein-transduction domains include human immunodeficiency virus
(HIV)-l Tat (48-46), drosophila antennapedia (Antp)-(43-5 8), and herpes simplex

virusl(HSV) VP22.)

 

Table 2.1: Amino acid sequence of characterized protein-transduction domains

 

PT Ds Amino acid sequence

 

HIV -1 TAT Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg

 

HSV VP22 Asp-Ala-Ala-Thr-Ala-Thr-Arg-Gly-Arg-Ser-Ala-Ala-Ser-Arg-Pro-Thr-
Glu-Arg-Pro-Arg-Ala-Pro-Ala-Arg-Ser-Ala-Ser-Arg-Pro-Arg-Arg-Pro-
Val-Glu .

 

 

Antp Arg-Gln-Iso-Lys-Iso-Trp-Phe-G1n-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys

 

 

From table 2.2'3 intracellular delivery of various molecules using PTDs, we can see

there is no limit on the type of molecules that can be transduced into cells with a PTD:

enzymes, antibodies, oligonucleotides, peptides, full-length proteins, etc'g' 20‘ 2'46.

53

 

Table 2.2: intracellular delivery of various molecules using PTDs

 

 

 

 

 

 

 

 

 

Molecules Carrier peptides Conjugation

Synthetic peptides Antp, Tat, Oligoarginine Direct attachment,
disulfide

Protein Tat, Oligoarginine, Antp Chemical cross-link
genetic

Megnet beads Tat Chemical cross-link

liposomes Tat Chemical cross-link

Antisense oligoDNA Tat, Antp Disulfide

Radioisotopes Tat Chelate

Natural products Oligoarginine Chemical cross-link

 

 

 

Currently, the mechanism of PTDs is not known. However, most scientists presume
that the arginines play a more critical role than lysine or other amines, especially
exemplified in the Tat PTD'Z‘ 13' '5 . Further, some scientists deduce that the direct
penetration of the lipid bilayer of the cell membrane is mainly caused by the localized

positive charge of the PTD's.

No matter what the mechanism is, it is not easy to produce or extract PTDs' 2. In
addition, there is still a concern that PTDs from viruses may become reactive again in
vivo. So the focus has been moved to preparing some polymers that have a similar
structure to Tat. Oligomers of lysine, and L/D- argininel2 were synthesized by
Wender et al. A limitation to this approach is that such synthesis requires many steps

and most of the steps need to be repeated several times during the synthesis.

54

 

Structure of Polyarginines:

fie-NH};

'1‘” o
(l::NH2

NH2

55

2.3 The construction of poly(B-amino acid) drug delivery system

Amid linkages and hydroxyl groups are characteristic functionalities in biomolecules.
Ways of preparing polymers containing predominately these functionalities were
explored because they would be expected to have a high degree of biocompatibility.
Simplicity in the synthesis of this material was also very desirable. With this in mind,

the structure shown below was designed and a synthetic route was developed.

on
Me\ 0 i 9 CH2
[N’\’NH2 + do ___.. . N-—C -CH2-CH -(
Me i \
(v3!
HN Me
Me

2.4 Prospective advantages of the novel poly(B-amino acid) drug
delivery system

1. This drug delivery system has a poly(B-amino acid) backbone structure that
resembles the poly a-amino acids (peptide), but should be more stable to

biodegradation compared with peptides.

2. This drug delivery system has dimethylaminoethyl side chains where the positive

charges resemble polyarginine.

3. The molecular weight of this drug delivery system can be controlled by adjusting

the time and temperature of the synthesis.

56

 

4. With the positive charge, the polymer will be able to bind and mediate the uptake
of a variety of drugs such as the anticancer drug—doxorubicin, antisense RNA,

plasmid DNA, etc.

57

 

10.

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12.

13.

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Ennio Tasciotti, Monica Zoppe and Mauro Giacca., Transcellular transfer of
active HSV -l thymidine kinase mediated by an 11- amino-acid peptide from
HIV-1 Tat. Cancer Gene Therapy 2003, 10, 64-74.

TA Khan, F W Sellke, RJ Laham, Gene therapy progress and prospects:
therapeutic angiogenesis for limb and myocardial ischemia. Gene Therapy 2003,
10, 285—291

Hagan Bayley., Protein therapy-delivery guaranteed. Research News and Views.
1999, 17, 1066-1067.

Steven R. Schwarze, Keith A. Hruska and Steven F. Dowdy., Protein
transduction: unrestricted delivery into all cells? Trends in cell Biol. 2000, 10.

Derossi, D., Trojan peptides: the penetratin system for intracellular delivery.
Trends in cell Biol. 1998, 8, 84-87.

Fawell, S. Tat-mediated delivery of heterologous proteins into cells., Proc. Natl.
Acad. Sci. U. S. A. 1994, 91 ,664-668.

M., Maité Lewin, Nadia Carlesso, Tat peptide-derivatized magnetic
nanoparticles allow in vivo tracking and recovery of progenitor cells., Nature
Biotechnology 2000, 18 (4), 410 - 414

Allinquant, B., Downregulation of amyloid precursor protein inhibits neurite
outgrowth in vitro. J. Cell Biol. 1995, 128, 919-927.

Anderson, D.C., Tumor cell retention of antibody Fab fragments is enhanced by
an attached HIV TAT protein-derived peptide. Biochem. Biophys. Res. Commun.
1993, 194,876-884.

Pooga, M., Cell penetrating PNA constructs regulate galanin receptor levels and
modify pain transmission in vivo. Nat. Biotechnol.1998, 16, 857-861.

Ezhevsky, S. A, Hypo-phosphorylation of the retinoblastoma protein (PRB) by
cyclin D: Cdk4/6 complexes results in active pRB. Proc. Natl. Acad. Sci.
U.S.A.1997, 94, 10699-10704.

Nagahara, H., Transduction of full length TAT fusion proteins into mammalian
cells: TAT- p27kip1 induces cell migration. Nat. Med. 1998, 4, 1449-1452.

59

Chapter 3.0 Experimental

3.1. Synthesis of Poly(B-amino acid)
3.1.1. Materials and characterization

All chemical materials used were obtained from Aldrich Company and were
analytical grade unless otherwise noted. NMR measurements were made on a Varian
VXR 500 MHZ Spectrometer with CD3OD as solvent. The FT-IR spectra were
obtained with a Nicolet 710 FT-IR spectrometer. Mass Spectra were taken with a
MALDI Voyager-DETM STR The molecular weight of different polymer fractions
were estimated by means of gel permeation chromatography (GPC) using Shodex
Asahipak GF-310 HQ column (exclusion limit is 40,000 Daltons) and using Mili-Q

water as solvent with a flow rate of 0.7m1/rnin.
3.1.2. Synthesis of Poly(B-amino acid)

N, N- dimethyl ethylene diamine (0.01 mole, 0.9279 g) was dissolved in cold
methanol (4ml) in a 50 ml vial (A). 2(5H) Furanone (0.01 mole, 0.8579g) was
dissolved in cold methanol (4ml) in another 50 ml vial (B). The solution in vial (B)
was quickly added to vial (A) at 0° C. Cold methanol (2 ml) was used to rinse vial (B)
several times, and the rinsate was added to vial (A). The reaction mixture was left at

0° C for 4 hours. And then the reaction temperature was changed to 25° C for another

4 hours. Reaction mixtures were heated to different temperatures (60° C, 70° C, 80° C,

90° C, 120° C, 140° C, etc) and different polymerization time (0.5 hours, 1 hour, 2

60

hour, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, etc) were allow the

production of polymers with a variety of molecular weight.

3.2 Purification and separation

A) Using Bio Gelp-60 Size exclusion chromatography

The purification, characterization and application experiments are described to the
polymer synthesized at 120 °C for 3.5 hours. Polymer (0.2 g) was dissolved in 10 ml
Mili-Q water. The polymer water solution was injected onto the Bio Gelp-60 Size
exclusion column (3X80 cm) with water as the elutant. The fractions of polymer were
collected by the Auto Fraction collector at a flow rate of 0.1 ml/minute with 5 ml in
each test tube. Polymer solutions (200 pl) were taken from each tube of the Auto
Fraction collector and dropped into the corresponding cells of an 8* 12 cm plate. The
UV absorbance values of polymer fractions were measured by u Quant Universal
Microplate Spectrophotometer at a wavelength of 220 nm. High (1), medium (11), low
MW (111) fractions of the polymer were separated and collected according to UV
absorbance results. The solvent was removed with a rotary evaporator and each

polymer fraction was kept in a freeze dryer for 72 hours.

B) Precipitation and extraction

The polymers (0.2 g) were dissolved in 0.5 m1 hot water (60 °C), and then 3.5 m1
acetone (poor solvent) was added into the polymer water solution. The mixture was

left in a 10—ml separation funnel for 24 hours. Then, the first fraction (high molecular

61

weight) was collected. Medium molecular weight fraction and low molecular weight

fractions were collected by repeating this step two times, respectively.

3.3 Measurement of drug delivery properties

I. Anticancer drug—doxorubicin delivery*

Mouse embryonic fibroblasts (MEF) were grown in Dulbecco’s Modification of
Eagles Medium (DMEM) at 37°C, 5% C02. The cells were plated on cover slips. One
cover slip (24 hours later) was taken and flipped over onto a microscope slide which
has a droplet of 5 ul of the stock solution of drug Doxorubicin (0.0002 g) and labeled
polymer (0.0040 g) dissolved in lml phosphate buffered saline (PBS). The samples

were prepared right before visualizing with the Laser Scanning Confocal Microscope.

Labeling polymer with fluorescein isothiocyanate (FTIC)

Fluorescein isothiocyanate (0.004 g) “was dissolved in 40 ul acetonitrile. Polymer
(0.01 g) was added along with 200 pl of water and 0.005 g of sodium bicarbonate
(NaHCO3). The sample was sonicated for 2 minutes and stirred overnight. On the
second day, the sample was diluted with 200 ul of water and the excess FTIC was
separated by passing the solution through a SEP-PAK cartridge C18. The FTIC

labeled polymer solution was then dried using a lyophilizer.

62

11. RNA Antisense (GCG CGG GGA GCA AAA GCA) delivery*

Mouse embryonic fibroblasts (MEF) were grown in DMEM at 37°C, 5% C02.

The cells were plated on cover slips. One cover slip (24 hours later) was taken and
flipped over onto a microscope slide which has a droplet of 5 ul of stock solution of
Cy5 labeled oligonucleotide (GCG CGG GGA GCA AAA GCA) (0.0002 g) and
polymer (0.0040 g) dissolved in 1 ml phosphate buffered saline (PBS). The samples
were prepared immediately before visualizing with the Laser Scanning Confocal

Microscope.

III. Plasmid GFP gene delivery*

Mouse embryonic fibroblasts (MEF) were grown in DMEM at 37°C, 5% C02. The
cells were plated on cover slips. Phosphate buffer saline (PBS) (1 ml) containing 2 pg
of plasmid with the green fluorescence protein gene (GFP) and 0.0040 g of non-
labeled polymer were added to the MEF cells in DMEM and left for 12, 13, 18, and
33 hours before visualizing with the Laser Scanning Confocal Microscope. Phosphate
buffer saline (PBS) (1 ml) containing. 2 pg of plasmid with the green fluorescence

Tm

protein gene (GFP) and 0.0040 g of non-labeled Super Fect were added to the

MEF cells in DMEM and left for 12 and 17 hours before visualizing with the Laser

Scanning Confocal Microscope.

*Method provided by Felicia Codrea.

63

Chapter 4 Results and Discussion

4.1 Synthesis of poly(ll-amino acid) and MW control of Poly(B-amino acid)

Me‘ O M' ha lAdd'ti W 0
[N’\/NH2 + do iii—Lon NNNHZ do

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Self polymerization

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Poly B-amino acids

Scheme 4.1 Synthesis of poly(fl-amino acid)

The route of synthesizing poly(B-amino acid) is outlined in Scheme 4.1. This route
can be used for a series of drug delivery systems. For example, a mixture of different
primary amines can be used such that the total molar amount of these amines is equal
to the molar amount of furanone used. By regulating the relative amounts of polar vs
non-polar amines, charged vs neutral groups, anionic vs cationic groups etc, the
pharmacological and physicochemical properties of the material can be controlled. By
adjusting the temperature and/or reaction time, the molecular weight of the polymers

can be controlled.

The extent of the reaction to produce poly(B-amino acid) was monitored by lH-NMR
spectroscopy. Performing the exothermic Michael addition at low temperatures is
important because it ensures that the addition has taken place completely before
appreciable amide formation begins. After 2 hours at 0 °C, the disappearance of the
signals for furanone at fields higher than 5 ppm indicated that the Michael addition
was complete. The spectra indicated that the dimer product from the Michael addition

was the major product. There was no evidence of polymerization (Figure 4.1 a & b).

Increasing temperature and time aided polymerization as shown in Figure 4.2 A-C
(proton NMR spectra of the mixture after reaction at 70 °C for 1 hour, 4 hours and 8
hours). The signals for the methylene protons at the 4-position of the lactone ring
(4.17 ppm and 4.42 ppm) disappeared indicating complete reaction of the dimer.
However, the narrow linewidths of the signals in the spectra indicated that the product
was composed mainly of oligomeric species. It was also found that increasing the
time at 70 °C slightly increased the degree of polymerization. This is indicated by the

increased width of peaks especially those between 2.0 ppm to 2.6 ppm. Figure 4.3 A-

65

B (proton NMR spectra of polymer reacted at 80°C for 4 hours and 10 hours) and
Figure 4.4 A-C (proton NMR spectra of polymer reacted at 90 °C for 2, 4, and 8 hours
showed the same trend as above. The spectra show that oligomeric species were the
main products at 90°C or lower. Even though increasing the reaction time showed
some benefits, the temperature was still too low for polymerization. If high molecular
weight products were being formed, a broad envelope of signals will be observed
instead of sharp peaks. When the reaction temperature was increased to 120 °C, the
NMR results were very different (Figure 4.5 A-C). The linewidths of signals between
2.0 ppm and 3.4 ppm were significantly broadened. All of the sharp monomer signals
disappeared indicating that polymerization had occurred. The spectra also show that
the longer the reaction time, the better the polymerization. Analyses were done from

3.5 hours to 9.5 hours.

When the reaction was carried out at 140 °C for 0.5, 1, 2 and 4 hours, respectively,

the relationship between the molecular weight (broad NMR lines) and the reaction
time was still the same; however, a few sharp peaks between (3.2-3.4ppm) were found
in the spectra. These were attributed to degradation to low molecular weight species
as shown in Figure 4.6 (A-D). Therefore these results indicate that the polymerization
is best conducted at 120°C is and that lengthening the reaction time will accordingly
increase the molecular weight of the polymers. Therefore, all of the following tests
such as purification, characterization, and application experiments were done on the

polymer synthesized at 120 °C for 3 hours.

66

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after 4 hours (b).

69

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after 10 hours (b).

71

 

 

 

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72

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75

4.2 Separation and estimation of molecular weight (MW) of the polymer fractions:

4.2.1 Biogel P-60 Size exclusion chromatography of the poly(B-amino acid)

During biogel P-60 Size exclusion chromatography, fractions of the poly(B-amino acid)
were collected and monitored at 220 nm. In Figure 4.7, a plot of the fraction number vs

UV absorption is shown.

Bio-Gel p-60 Size exclusion chromatography

 

Absobance at 220 nm

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16111621263136414651566166717681869196

Tubes number

Figure 4.7 Plot of fraction number vs absorbance
Based on the plot, the polymer distribution was preliminarily assigned to a high molecular

weight fraction (tubes 19-47), a medium molecular weight fraction (tubes 48-73) and a

low molecular weight fraction (tubes 74-98).

76

4.2.2 Determination of the MW of poly(B-amino acid) fractions by Waters 1525

HPLC

The molecular weights of three polymer fractions after Biogel P-60 Size exclusion
chromatography were estimated by means of gel permeation chromatography (GPC)
using a column with an exclusion limit of 40,000 Daltons. The retention times (Te) were
obtained of Dextrans standards (MW 5200, 11600, 23800). Table 4.1 shows the data
from the standards. Figure 4.8 shows the curve of log MW v.s. K... [K..,= (Te-Toy (T.-To)].
To (the exclusion limit, 7.05 minutes) was the retention time of Dextran with MW

686,000. T. (12.54 minutes) was the retention time of glucose (MW=180).

Table 4.1 Data of standard curve (IgMW v.s. K")

 

      
  
   

  
   

       
   
 

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Figure 4.8 MW v.s. KIv calibration

77

The linear line fit to the data from the dextran standards is

log MW= -2.72K.v + 4.91

The high MW fraction’s retention time was 8.565 minutes (Fig 4.9 a) corresponding to a
molecular weight of 15,000 Da. The medium MW fraction’s retention time was 9.532
minutes (Fig 4.10 a) indicating that its molecular weight should be 5000 D. The MALDI
Voyager-DETM STR spectrometer was used to obtain more accurate values of 16,986 Da
and 5383 Da for the molecular weights at the peaks of the distributions (Figure 4.9 b and
Figure 4.10 b). The relative yield for each MW fraction (high, medium, and low) is

shown in Table 4.2.

Table 4.2 Relative yield of each fraction (percentage of recovered materials)

 

 

 

 

 

Fractions MW Yield %
High molecular weight l6,000-17,000 53.9%
Medium molecular weight 5,000-6,000 31.4%
Low molecular weight <1000 14.7%

 

 

 

78

 

100.00

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8 565

 

 

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
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Figure 4.9 (a) GPC results for the high MW fraction isolated by Bio GelP-60
chromatography

 

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chromatography

79

K
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chromatography

 

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method

4.2.3 GPC result of high MW fraction isolated by acetone precipitation method

(Figure 4.11)

From figure 4.11, we can see the high MW fraction from acetone precipitation was
noticeably impure. The peak width is broad and the low MW product accounts for

more than 20% of the sample. Compared with the precipitation

method of fractionation, gel filtration using size exclusion chromatography gave a much
better separation. However, the acetone precipitation method allowed the processing of a
much larger amount of material and could be used as an efficient initial fractionation

method before final purification and sizing.

81

4.3. Characterization of high MW fractions of the poly(B-amino acid)

The high MW fractions of the poly(B-amino acid) were characterized by proton NMR and
FT-IR spectroscopy. (Figure 4.12 and Figure 4.13). From the proton NMR spectrum, the
apparently broad envelope indicated high molecular weight poly(B—amino acid). In the
FTIR spectrum, the signal at 3297 cm" indicated the presence of —OH, signals at 2943
cm", 2858 cm" and 2768 cm" indicated the structures of —CH2, —CH;, —CH respectively,

the signal at 1660 cm'I indicated ~C=O, and the signal at 1042 cm'l indicated —C-0.

82

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84

4.4 Drug delivery properties of poly(B-amino acid)

4.4.1 Anticancer drug delivery (doxorubicin)

There are countless drugs that are effective in killing cancer cells in the laboratory, but act
differently in a living system because they do not affect only the tumor, and cause
extremely harmful side-effects. Doxorubicin is one of the most widely used anticancer
drugs (see figure 4.14). It is effective in the treatment of many solid tumors such as
lymphomas, tumors of the breasts, lungs, ovaries, testes, prostate, cervix, head and neck.
It is also a therapy for osteogenic sarcomas, Ewing’s sarcoma, AIDS related Kaposi’s

sarcoma etc."2

Doxorubicin is an anthracycline antineoplastic agent produced by the fungus
streptomyces peucetius3. Doxorubicin damages DNA by intercalation of the anthracycline
portion of the molecule, metal ion chelation, or by generation of free radicals. It also
inhibits DNA topoisomerase 11, an enzyme that is critical to DNA function by making the

reproduction of DNA effective." 3

Doxorubicin also is called “red death" because of its physical appearance (red color) and
its extremely inherent toxicity. From the name, we could imagine how dangerous its side
effects could be. Since doxorubicin is water soluble and diffuses into cells quickly and
freely, it has been associated with a number of toxicities such as hair loss, nausea,
vomiting, diarrhea, allopecia, stomatitis, esophagitis, cardiotoxicity and bone marrow
depression which lead to anemia, greater risk of bleeding, infection etc" 3. Among them,
cardiotoxicity is a major concern during doxorubicin therapy. The heart contains

excessive enzymes that convert doxorubicin to free radicals“, however, unlike most

85

tissues, the heart has poor defense mechanisms against free radicals. The risk of

cardiotoxicity is proportional to the cumulative dose of doxorubicin received'.

0 OH O OH
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Figure 4.14 Structure of doxorubicin

Liposome delivery systems have been used to deliver doxrubicins, however the side
effects are large" 3. Developing an efficient polymeric drug delivery system with good
drug binding ability is the key to solving this problem6’ 7. The polymeric delivery system
we prepared and tested was very effective at sequestering doxorubicin and releasing it
inside mouse embryonic fibroblasts (MEF) cells. This is illustrated in Figure 4.15 A to F.
Figure A shows a florescence micrograph of a solution of doxorubicin in phosphate
buffer saline (PBS) solution. The entire field is evenly covered showing that the drug is
soluble and completely diffused throughout the solution. Figure 4.153 shows a
florescence micrograph of a PBS solution of doxorubicin with the high molecular weight
fraction of poly (IS-amino acid) as the carrier. Figure 4.15 C shows a florescence
micrograph of a solution of doxorubicin with the medium molecular weight fraction of
poly (B-amino acid) as the carrier in phosphate buffered saline solution. These two
figures (B &C) show that for the two different polymers, the brightness of colloidal gel
particles of polymers in PBS solution were different, the higher MW polymers have better

binding ability to doxorubicin. Figure 4.15 D shows the high MW polymer without

86

doxorubicin inside of one cell which is undergoing division. The green color corresponds
to the green fluorescence of poly(B-amino acids). Figure E shows that with the carrier,
doxorubicin molecules were inside the nucleus of one cell. The orange color is due to the
green fluorescence of the polymer plus red fluorescence of doxorubicin. Figure F shows
that with the carrier, doxorubicin molecules inside several cells were trapped in a polymer
colloidal gel particle (green plus red). With such good sequestering properties by the
polymers, the doxorubicin can be delivered to the target in a controllable manner. As a

result, the serious side effects of doxorubicin can be decreased by a large degree.

87

 

(A) (B) (C)
( D) (E) (F)

Figure 4.15 (A-F) Light micrographs showing of doxorubicin sequestration and
delivery to MEF Cells.

(A) Doxrubicin buffer solution without polymer binding

(B) Doxorubicin buffer solution with polymer (MW=16978)

(C) Doxorubicin buffer solution with polymer (MW=5383)

(D) Polymer inside MEF cells without doxorubicin

(E) Polymer in one MEF cell with doxorubicin

(F) Polymer gel particle with doxorubicin inside MEF cells

* Doxorubicin was red fluorescence, polymer was green fluorescence (labeled by

FT IC) Orange is due to red plus green when polymer exists with doxorubicin.

88

4.4.2. Delivery of antisense human telomerase RNA (GCG CGG GGA AAA GCA)

The light micrographs of the poly(B-amino acid)polymers delivery system with the
antisense drug (GCG CGG GGA AAA GCA) are shown as follows: (See Figure 4.16 A-

D)

 

(D)

Figure 4.16 The light micrographs of antisense delivery
(A) MEF cells with Cy5 labeled antisense without polymers (blue fluorescence
image)
(B) MEF cells with Cy5 labeled antisense with polymers (green + blue fluorescence
image)

(C) MEF cells with Cy5 labeled antisense and polymers (transmission image)

(D) MEF cells with Cy5 labeled antisense and polymers (green + blue fluorescence
image)
One of the barriers of antisense delivery is that unlike most small molecule drugs,
antisense oligonuleotides have very high densities of negative charge which inhibit
penetration of cell membranes. Hence in Figure 4.16 (A), the labeled antisense molecules
are not taken up by cells in the absence of carrier. Figure 4.16 (B) shows the efficient
antisense translocation by the carrier. Two channels (blue + green) were monitored. The
antisense molecule fluoresces blue with Cy5 labeled and the carrier was labeled with
fluorescent fluorescein isothiocyanate (FTIC) to green fluorescence. Figure 4.16 (C) is
a bright field image of a cell that was treated with the carrier and antisense
oligonucleotides. The cell is still intact. Figure 4.16 (D) shows that in the presence of
carrier, no lysis of cells was observed and the Cy5 labeled antisense oligonuleotides (blue)

were delivered into the cells and mainly localized in the nucleus.

Barriers to antisense treatment of cancer

1. Target to cancer site-«by passive targeting mechanisms.

Tumors always require a large blood supply and demand highly vascularized tissue to
maintain their rapid rate of growth8 .The vasculature of tumors is extremely‘different from
normal tissues. Unlike normal tissue, tumors have leaky capillaries, high vascular density

9'10. Another characteristic is the dysfunction of the

and permeability—enhanced factors
lymphatic system that is responsible for the drainage of macromolecules from normal
tissues.”' '2 Because of the enhanced permeability and retention effect, polymers can
enter tumor tissues and remain there for a prolonged time, while small molecules are not

retained because of their ability to return to the circulation by diffusion. Rapidly dividing

cancer cells constantly ingest nutrients from their surroundings by macropinocytosis or

90

random gulping of extracellular fluid”. Because of this, the DDS can be efficiently
internalized without modification with some special targeting of cell-surface receptors in

certain types of cancers”.

11. Subcellar barrier—lysosome escape

The major barrier to the subcellular level of drug delivery is whether the drug can
successfully escape from the lysosomes. The lysosomes contain a number of degradation
enzymes and also have a harsh environment that renders drugs ineffective too soon after
the drug delivery system enters the cell. Figure 4.16 (D) proves that the antisense drug is

able to successfully escape from the lysosome and reach the final target—the nucleus.

The following Figure 4.17 is the strategy for subcelluar drug delivery by the cationic
poly(B-amino acid). In the delivery mechanism outlined in Figure 4.17, the positive
charge of the carrier binds the drugs tightly and facilitates their interaction with the cell
membrane which has an overall negative charge. The drugs are taken up into cells by the
process of endocytosis within structures called endosomes. A family of enzymes called
lysosomal thiol-dependent proteases catalyze the cleavage of the polymer-backbone to set
the drug free. Protonation of the carrier also leads to unfolding and this also facilitates
drug release. The drug /carrier complex also passes through the nuclear membrane, which

is the ultimate target for drug processing.

91

 

%
e + % % Drug binding
a 0 q,

Drug molecules

 

Polyamide

Enter cells

 

Figure 4.17 Subcellular drug delivery by poly(ll-amino acid)polymers

92

4.4.3 Plasmid GFP gene delivery by the poly(p-amino acid)polymers

The delivery of an entire plasmid into a cell is the biggest challege in the gene therapy
field. There are a few commercial products for this “transfection” process, but their
efficiency is usually very low. In some cases, a large proportion of cells is damaged.
Super Fect is one of these commercial products. We compared the efficiency of our
carriers to Super Fect (SF) in the delivery of plasmids containing a gene for green
fluorescent protein (GFP) into mouse embryonic fibroblast (MEF) cells. Light
micrographs describing the results of these experiments are shown below in figure 4.18

(A-L):

 

(A)

   

(C) (D)

 

94

Figure 4. l8 (A—L) fluorescence and transmission light micrographs of MEF cells
illustrating transfection of plasmid DNA encoding GFP mediated by the drug

delivery vehicles described here and a commercial transfection agent (Super F ect Tm)

(A) MEF cells 12 hours in incubation (transmission image)

(B) MEF cells 12 hours in incubation (green fluorescent image)

(C) MEF cells with GFP gene plasmid 12 hours in incubation (transmission
image)

(D) MEF cells with GFP gene plasmid 12 hours in incubation (green fluorescent
imge)

(E) MEF cells with SF and GF P gene plasmid 12 hours in incubation
(transmission image)

(F) MEF cells with SF and GF P gene plasmid 12 hours in incubation (green
fluorescent image)

(G) MEF cells with polymer and OF P gene plasmid 12 hours in incubation
(transmission image)

(H) MEF cells with polymer and GFP gene plasmid 12 hours in incubation
(green fluorescent imge)

(I) MEF cells with polymer and GFP gene plasmid 18 hours in incubation
(green fluorescent image) .

(J) MEF cells with polymer and GFP gene plasmid 33 hours in incubation
(green fluorescent image)

(K) MEF cells with SF and GFP gene plasmid 17 hours in incubation
(transmission image)

(L) MEF cells with SF and GFP gene plasmid 17 hours in incubation

(transmission image)

95

Figure 4.18 A is a transmission light micrograph of mouse embryonic fibroblast (MEF)
cells after 12 hours of incubation without the carrier or the plasmid. Figure 4.18 B shows
the same microscope field but this time only monitoring the green channel for the green
fluorescent protein (GFP). From the images, it is known that the cells have no intrinsic

green fluorescence.

Figure 4.18 C is a transmission light micrograph of mouse embryonic fibroblasts (MEF)
cells after they were incubated with a GFP encoding plasmid and no carrier for 12 hours.
Figure 4.18 D shows the same microscope field, but only monitoring the green channel

for GFP. The results show no evidence for transfection.

Figure 4.18 E is a transmission light micrograph of mouse embryonic fibroblast (MEF)
cells after incubation with a plasmid encoding for the OF P gene and the commercial
transfection agent Super F ectT’“ for 12 hours. Figure 4.18 F shows the same microsc0pe
field monitoring the green channel for GFP. The results indicate that most cells show

lysis. The cells were destroyed by Super Fect during the transfection.

Figure 4.18 G is the transmission light micrograph of mouse embryonic fibroblast (MEF)
cells after they were incubated with the GFP encoding plasmid and the carrier—Poly(B-
amino acid) polymers for 12 hours. Figure 4.18 H shows the same microscope field
monitoring the green channel for GFP. The carrier not only efficiently mediates

transfection, but also does not affect the structure of the cells.

Figure 4.18 I is a green fluorescent light micrograph of mouse embryonic fibroblast
(MEF) cells after they were incubated with the GF P encoding plasmid and the carrier—

Poly(B-amino acid) polymers for 18 hours. From the image, a larger number of cells show

96

green fluorescence after 18 hours treated with the polymer. All of the cells are intact.

None of cells were destroyed by the carrier.

Figure 4.18 J is a green fluoresce light micrograph of mouse embryonic fibroblast (MEF)
cells after they were incubated with the GFP encoding plasmid and the carrier—poly(B-
amino acid) polymers for 33 hours. Many more cells show green fluorescence after 33
hours with the carrier and still the cells are intact (none of them showed lysis). After 33
hours, the delivery ability of the poly(B-amino acid) is still very good, which gives a
strong indication that this polymer has the potential to be used as a long term controlled

release delivery system.

Figure 4.18 K and L are two images of transmission light micrographs of mouse
embryonic fibroblast (MEF) cells after they were incubated with the GFP encoding
plasmid and the commercial transfection agent Super F ectTm for 17 hours. The images

show that the cells were destroyed more seriously by Super Feet after 17 hours.

97

Conclusion:

The experimental results show that the poly(B-amino acid) polymers are a promising
drug delivery system. They successfiilly sequester and mediate the delivery of the
anticancer drug doxorubicin into mouse embryonic fibroblast (MEF) cells. This
carrier might be useful in reducing or eliminating the serious side effects of this drug.
The delivery of RNA, gene or gene sequences into cells is an especially challenging
task. Poly(B-amino acid) carriers described here efficiently delivered the RNA
antisense (GCG CGG GGA GCA AAA GCA) drug into MEF cells without any cell
lysis occurring. Thus, they provided another exciting prospect for curing cancer.
Poly(B- amino acid) carriers also showed excellent suitability for the transfection of
plasmids. Compared with the commercial transfection agent Super FectTm , they also
showed superb non-cell lysis properties, which will be the most significant property as a

drug delivery system.

98

10.

ll.

12.

13.

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Joshua Mecham, Preston Maxwell and Will Davids, Doxil liposomal delivery
system of doxorubicin. Pharmaceuticals III.

Hardman JG, Limbird LE, Molinoff PB, Ruddon RW, Gilman AG., The
Pharmacological Basis of Therapeutics, Goodman & Gilman 's, 9thEd. 1264-
1266.

Http: www. aegis.com/factshts/network/access/drugs/doxo.html

Martee L. Hensley, The cost and efliciency of liposomal Doxorubicin in
Platinum -Refractory Ovarian Cancer in Heavily Pretreated Patients.

Igor V. Zhigaltsev, Triggered release of doxorubicin following mixing of
cationic and anionic liposomes. Biochimica et biophysica Acta 2002, 1 5 65 ,
129-135.

B. Rihova, Doxorubicin bound to a HPMA copolymer carrier through
hydrazone bond is effective also in a cancer cell line with a limited content of
lysosomes. Journal of Controlled Release 2001 , 74, 225-232.

T. Nakanishi, Development of the polymer micelle carrier system for
doxorubicin. Journal of Controlled Release 2001, 74, 295-302.

Dean K. Pettit and Wayne R. Gombotz, The development of site —specific
drug-delivery systems for protein and peptide biopharmaceuticals. Tibtech
August 1998 16.

D. F. Bahan, L.W. Seymore, Control of tumor vascular permeability, Adv.
Drug Deliv. Rev. 1998, 34, 109-119.

H. Maeda, Tumor vascular permeability and the EPR effect in macromolecular
therapeutics: a review. J. Control. Release 2000, 65, 271-278.

Vladimir P. Torchilin and Anatoly N. Lukyanov, Peptide and protein drug
delivery to and into tumors: challenges and solutions. DDT 2003, 8(6).

Maeda, H. Mechanism of tumor—targeted delivery of macromolecular drugs,
including the EPR effect in solid tumor and clinical overview f the prototype
polymeric drugs SMANCS. J. Control. Release 2001, 74, 47-61.

Maeda. H. SMANCS and polymer—conjugated macromolecular drugs:
advantages in cancer chemotherapy. Adv. Drug Deliv. Rev.2001, 46, 169-185.

99

Chapter 5.0 preparation of cationic polyelectrolytes

Introduction

Polyelectrolytes are polymers containing charged groups at regular intervals along the
length of the chain. The preparation of such materials is another important application
for the poly(B-amino acid) we described in the earlier chapter 3. At low pH, protonation
of the dimethylaminoethyl side chain would result in the formation of a polyelectrolyte.
A permanent charge can be obtained by alkylation leading to quaternization of the side
chain nitrogen and the formation of a cationic polyelectrolyte. Polyelectrolytes can be
used to stabilize or destabilize interactions between charged particles in numerous
applications. There are several examples showing when polyelectrolytes can be used to
inhibit the aggregation of particles. For instance, printing inks are suspensions of colloidal
particles and the aggregation of these particles leads to loss of resolution and blockage of
the ink dispenser. This can be reversed or inhibited by coating the pigment (color)
particles with a polyelectrolyte so they do not associate with each other. Further, bacteria
and other microorganisms are invariably negatively charged and tend to disperse in
aqueous media. Polyelectrolytes are often used as flocculants"3 to produce aggregates
from these dispersions in wastewater treatment. Polyeletrolytes are also frequently
applied directly to soil as conditioning agents to maintain soil structural conditions by
stabilizing the colloidal nature of the soil particles. They are also used as carrier gels for
fluid drilling in the placement of pre-germinated seeds in agricultural and horticultural
practice‘. Besides other uses as thickeners, detergents5 and coagulants, one important and
exciting use of polyelectrolyte gels is in the fabrication of solid electrolyte batteries that

have greater safety because they are less likely to leak toxic liquidsG. Last, but not least,

100

as a biosensor, polyelectrolytes can be used for the controlled drug delivery7'9 triggered
by different bio-stimuli such as change in pH, the concentrations of enzymes, sugars,
antigens, etc. In this study, cationic polyelectrolytes were prepared by protonation and by
methylation of poly(B-arnino acid) with dimethylaminoethyl side chains. Polyelectrolytes
often form hydrogels, which make them significant materials for use in controlled drug

delivery systems.

Smart polymer material—Hydrogels

Hydrogels are polymers that will absorb at least 10-20% amount of fluid and swell
when placed in water or other biological liquids to form a gel. Water absorbance of
hydrogels, depending on hydrophilic structure of polymers, can vary from 20% to
many times their dry weight. One of the most remarkable properties of hydrogels is
that the swelling or shrinking can be triggered by a change in the environment
surrounding the delivery system. The swelling or shrinking of a hydrogel is reversible
and repeatable after additional changes from the external environment. Depending on
the different compositions of hydro gels, the environmental change can involve pH,
temperature, ionic strength etc. A number of these environmentally sensitive or
"intelligent" hydrogel materials are listed in Table 5.1”). Because variations of pH are
known at several body sitesl 1, the pH sensitive hydrogel drug delivery system has
received more attention by scientists. Hydrogels are elastic in nature because of the
presence of a memorized reference configuration to which they return even afier
being deformed for a very long time. In addition, they consist of polymers combined
with water and as such have dual characteristics. Hydrogels show a solid character
due to the polymer, which make them available in a variety of structures for different

drug delivery functions. They also display certain water—like properties, such as

101

permeability, for many water-soluble substances. When hydro gels are loaded with
drugs, they can be implanted into the human body and establish the controlled drug
release at specific pH values. Through further modifications, hydrogels can release
drugs under different stimulations as we mentioned in table 5.1: such as different pHs,

temperatures, magnetic fields, ultrasonic pulses, electric fields, etc.

102

 

Table 5.1 stimuli-responsive hydrogels in drug delivery

 

Stimuli

Magnetic
field

Ultrasonic
radiation

Electric field

Glucose

Urea

Morphine

Antibody

pH

Temperature

pH and
temperature

Polymer

Ethylene-co-vinyl acetate (EVAC)

(EVAC);
Ethylene-co-vinyl alcohol

Poly(2-hydroxyethyl methacrylate)

EVAC

Methyl vinyl ether-co-maleic
anhydride

Methyl vinyl ether-co-maleic
anhydride

Poly(ethylene-co-vinyl acetate)

Chitosan-Poly(ethylene oxide)

Poly(acrylic acid) : PEO

Gelatin-PEO
Poly(Z-hydroxyethyl methacrylate)
Poly(acrylamide-co-maleic acid)

N-vinyl pyrrolidone, polyethylene
glycol diacrylate, chitosan

Poly(N-isopropyl acrylamide)

Poly(N-isopropyl acrylamide-co-
butyl methacrylate-co-acrylic acid)

*Table is adapted from Ref [10]

103

Drug

Insulin

Zinc bovine insulin
Insulin

Propranolol
Hydrochloride

Insulin

Hydrocortisone

Naltrexone

Naltrexone
Ethinyl estradiol
Amoxicillin
Metronidazole

Salicylamide
Nicotiamide
Clonidine
Hydrochloride
Prednisolone
Riboflavin

Salicylic acid

Terbinafine
Hydrochloride

Theophylline
S-fluorouracil

Heparin

Calcitonin

Refs

12

14

15

16

17

18

19

20

21

22

23

24

25

26

H(‘l , H30

. _
( Drugsa. )‘

 

 

 

Figure 5.1 Scheme of hydrogel formation

Preparation of polyelectrolytes hydrogles by protonation

We prepared polyelectrolytes by protonating the dimethylaminoethyl side chain. A
scheme illustrating hydrogel formation by the poly(B-amino acid) polymer leading to
capture of drugs is shown in Figure 5.1. (* Means drug could be added into hydrogel.)
The hydrogels are formed because protonated poly(B-amino acid) molecules repelled
each other and then water molecules inserted into the space between the charged

groups to form the hydrogels.

104

Experimental section

1. Polyelectrolytes by protonation

The high, medium, and low fraction polymers (0.01 g) were each added to a
preweighed watch glass. One drop of concentrated HCl followed by a few drops of
water were added to each watch glass. The watch glass was turned over to cover the
top of a 50-mL flask containing 10 mL concentrated HCl for 24 hours. (The amount
of water added was controlled to prevent the polymer solution from dripping when the
watch glass was turned over.) After 24 hours, each watch glass was weighed and then
the three gels were dried in a vacuum oven for 4 hours to determine the amount of
water absorbed by the polymer gels. (* Diameter of watch glass = 2.5 cm.) In addition,
one drop of hexane and one drop of iodine were added to the gels as a stain to aid

visualization, and then photographs of gels were taken.

105

2. Polyelectrolyte hydrogels by methylation

Materials and characterization

All chemical materials used were obtained from the Aldrich Company and were
analytical grade unless otherwise noted. NMR measurements were made on a Varian

VXR 500 MHZ Spectrometer.

Synthesis

Na2C03 (0.030 g) dissolved in 2 ml H20 and 0.030 g of the poly(B-amino acid) dissolved
in lml of methanol, were mixed in a 50 ml vial. Dimethly Sulfate (3011.1) was then quickly
added to the vial at room temperature. The reaction mixture was heated at 60 °C degree
for 1 hour. The solvents were then removed by rotary evaporator. The crude polymer
(0.01 g) was dissolved in 1 ml of H20 and the polymer solution was passed over an anion
exchange resin (chloride form) to remove the methyl sulfate anion. The solvent (H20)
was removed again by rotary evaporator. The final polymer solutions were lyophilized for

72 hours.

 

OH
O OH
’1 k n 1.1120 '
H 2.N82CO3 N n
N
M

3. CH ) SO
( a 2 4 5 e H
I \ Hac-O— '0 eNMe

6
Me Me’ Me

Scheme of methylation of the poly(B-amino acid)

106

 

 

Walks -. Amy/L

#71 I I-I.I. ‘,_I I, I 1" I .I 1 1' r_ r ' Y.Tfr I "f

5.0 4.5 4.0 3.5 3.0

 

 

2.5 ppm

0))

 

5.0 4.5 4.0 3.5 3,0

 

2.5 ppm

Figure 5.2 lH-NMR spectrum of methylated polymer product before ion change with
chloride anion (a) and after ion change with chloride anion (b). * Solvent: D20

107

Results and discussion:

NMR spectra

Figure 5.2 A is the NMR spectrum of the polymer product before ion exchange and
Figure 5.2 B is the NMR spectrum of the polymer product after ion exchange with
chloride anion. In Figure 5.2 A, the sharp peak at 3.65 ppm is the signal for the
H3C0803' anion. In Figure B, disappearance of the peak indicated that the CH30803'

anion was exchanged successfully.

Photographs of hydrogels of poly([l amino acid) polymer:

Iodine-stained hydrogel formed from
0.01g poly(fl-amino acid) and 82% (w)
water.

 

Figure 5.4 The hydrogel of the high MW fraction

Iodine-stained hydrogel formed from
0.01g poly(fl-amino acid) and 50% (w)
water.

 

Figure 5.5 The hydrogel of the medium MW fraction.

108

Table 5.2 Water absorbance of different molecular weight polymer fractions

 

 

 

 

 

Fractions Water Absorbance %
High molecular weight 82%,
Medium molecular weight 50%
Low molecular weight 33%

 

 

 

Table 5.2 shows water absorbance for the different molecular weight fractions of poly(B-
amino acid) polymers. Figure 4.15 and Figure 4.16 are the photographs of hydrogels of
high MW and medium MW polymer. The photographs show that the hydrogel volume
from the high MW polymer is noticeably larger than that from the medium MW polymer.
Experimental results demonstrate that the water absorbance is increased with the

molecular weight of the poly(B-amino acid) polymers.

Cationic hydrogels are relatively rare compared to anionic and neutral hydrogels. Cationic
polyelectrolytes with amide backbone are uncommon. Polylysine is one example of a
cationic polyelectrolyte with a polyamide backbone, but its gel-forming ability is not well
documented. The materials we described here are therefore important new contributions.
Polyethyleneimine is another example of a cationic polyelectrolyte. It can be prepared

from ethyleneimine as shown below”. It cannot be readily degraded in biological systems.

H2?\NH CH2 N ~CH2 CH2 NH— CH2 fie»
’ l H2C ' CH2 NH‘ CH2 CH2 NH’r‘

H2O n
Chitosan (poly B-l ,4-D-glucosamine) is the only biogenic cationic polyelectrolyte. Unlike
polyethyleneimines, chitosan has good water-absorbing properties. Chitosan forms gels at
high pH (>6.3)28. These contain as much as 98% water”. In contrast to the systems

developed here, gel structure is maintained by a hydrogen-bonding network and not by

109

charge. At low pH (pH < 6), chitosan is protonated and soluble. When the pH is raised
above about 6.3, the amino groups become deprotonated and this polysaccharide can form

an insoluble hydrogel network 29‘ 30.

OH O
15% “w
0
O
HO QNH; OH n

Sdume

OH
HO NH
1 O . 2 + 2nH“
H

NH2 OH 11

Insoluble

Materials that can form hydrogels under all pH conditions are desirable. This can be
facilitated by converting groups that are charged in a fashion depending on pH into
permanently charged groups. This was successfully accomplished in this study by

methylation of the poly(B-amino acid).

Since they were first synthesized in 1960, pHEMA (poly-hydroxyethyl methacrylate)
gels have been utilized for biomedical applications. Extensive studies have been
carried out on the structural, chemical properties, and applications of pHEMA.
pHEMA is one of the most popular neutral hydrogels with water content of
approximately 40%3 '. The water content can be regulated by copolymerization with
hydrophobic or hydrophilic monomers. Currently, much research is being carried out
on biomedical applications for pHEMA. This cannot be degraded enzymatically or
hydrolyzed by acids or bases. One approach is to copolymerize pHEMA with maleic

acid and maleic anhydride to improve their degradation, but this results in poorer

110

water absorption compared to pure pHEMA32. Compare with pHEMA hydrogels, the
polymer we described here has much better water absorption properties. In addition,
this polymer is biodegradable, and its poly B-peptide backbone makes it a potentially

excellent biocompatible drug delivery system.

The poly(B-amino acid) system we describe also offer significant advantages over
those containing polyacrylic acid (PAA) backbones, which are among the most
intensely studied hydrogel systems.33 Polyacrylic acids hydrogels are known for their
ability to form extended polymer networks through hydrogen bonding. The water
content of smart poly (acrylic acid) hydrogels (37.27-47.71%) prepared by Kim34 is
considerably lower than our poly(B-amino acid) system. This limits their capacity to
carry drugs. Another advantage of our system is that the cationic charges on the side
chains make this polymer more efficient at transporting drugs through cell membranes

which have negative charges.

Me

1 1 ‘1 l

1 CH2 C “1.. 1 CH2 9H1“
c=o otc ‘ "
o--CH2 CH2 OH OH

Poly-hydroxyethyl methacrylate Polyacrylic acid

111

10.

ll.

12.

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