1.1. Synthetic Hydrogels

Chapter 1: Introduction

1.1. Synthetic Hydrogels

Hydrogels by definition are three-dimensional swollen networked structures. Certain

materials, when placed in a compatible aqueous medium, are able to swell and retain the

volume of the adsorbed aqueous medium in their three-dimensional swollen network.

Such aqueous gel networks are known as hydrogels or aquagels [1]. Included in this

definition are a wide variety of natural materials of both, plant and animal origins,

chemically modified, naturally occurring materials and synthetic polymeric materials [2].

Synthetic polymeric hydrogels are generally three-dimensional swollen networks of

hydrophilic homopolymers or copolymers covalently or ionically crosslinked [1-8]. The

original polymeric hydrogel network was developed by Wichterle and Lim in

Czechoslovakia in 1954 [9]. It was a copolymer of 2-hydroxyethyl methacrylate (HEMA)

and ethylene dimethacrylate (EDMA) for use as contact lenses. Due to lack of interest

and support from the appropriate authorities, no success was achieved. Wichterle and Lim

however, continued to work on their development and it was not until the 1960s when the

versatility of synthetic polymeric hydrogels was visualised from a commercial point of

view.

Polymeric hydrogel networks may be formed by various techniques, however the most

common synthetic route is the free radical polymerisation of vinyl monomers in the

presence of a bifunctional crosslinking agent and a swelling agent. The resulting polymer

is interesting in the sense that it exhibits both liquid-like and solid like properties [3]. The

liquid-like properties result from the fact that the major constituent (>80%) is water.

However, the polymer also exhibits solid-like properties due to the network formed by the

crosslinking reaction, or more like elastic solids in the sense that there exists a

remembered reference configuration to which the hydrogel returns after being deformed

for a long time [3, 10].

The classification of hydrogels depends on their physical structure and chemical

composition. A common classification, especially useful in biomedical applications

includes neutral hydrogels, ionic hydrogels and swollen interpenetrating networks (IPNs)

[4] is described in detail in Section 1.1.1. The most characteristic property of a hydrogel

1

Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies

is that it swells in the presence of an aqueous media and shrinks in its absence [7]. In

general hydrogels swell to an equilibrium value of 10 – 98 % at physiologic temperature,

pH and ionic strength [2,8, 11,]. A dried hydrogel imbibing at least 20 times its own

weight of the aqueous media while retaining its original shape could be referred to as

superabsorbent. The capacity of hydrogels to absorb the aqueous media, however, could

be enormous and can be as much as 1000 times the weight of the polymer [3,12]. Figure 1

depicts a hydrogel network upon placement in water [13].

Figure 1. A schematic representation of a single chain in a hydrogel network upon

placement in water.

Swelling in hydrogels when in aqueous media occurs in a similar manner as that of an

analogous linear polymer dissolving in the media to form an ordinary solution [14].

Mainly the nature, predominantly the hydrophilicity/hydrophobicity of polymer chains

and the crosslinking density determine the extent of swelling. A hydrogel from a linear

polymer upon dissolving in the aqueous media becomes a hydrosol, which is a dispersion

of colloidal particles or simply an aqueous solution from a physical point of view [1,15].

A number of polymer systems may undergo a reversible transformation between hydrogel

and hydrosol but through chemical crosslinking of dispersed particles in hydrosol result in

an irreversible hydrogel [1].

A gel is typically a large macromolecule, which forms network extending from one end to

the other and occupying the whole reaction vessel [16]. Microgels, which are crosslinked

polymer networks as that of macromolecules but composed of small particles with

diameters smaller then 1 µm are water soluble as that of linear polymers due to their

molecular nature [17,18]. The term hydrogel is referred to a material currently in swollen

state but upon drying, the swollen network of the hydrogel collapses due to the high

2


surface tension of water rendering a xerogel or a dry gel [1,7]. The overall shape of the

hydrogel is preserved during the swelling and shrinking process.

Synthetic hydrogels have been a field of extensive research for the past four decades and

it still remains a very active area of research today. Hydrogels can be designed with

controllable responses as to shrink or expand with changes in external environmental

conditions [1]. The extent of swelling or de-swelling in response to the changes in the

external environment of the hydrogel could be so drastic that the phenomenon is referred

to as volume collapse or phase transition [19-21].

Hydrogels may respond uniquely to changes in external environmental conditions such as

ionic strength [22], electromagnetic radiation [23], pH [24-28], and temperature

[24,26,29-31]. These conditions could be introduced individually or in combinations and

altered as desired. Other important factors such as the type of salt used for the preparation

of buffer [32,33], solvent used as the medium [34], photoelectric stimulus [35] and

external stress [36,37] are also influential on the hydrogel’s performance. These unique

properties make hydrogels excellent candidates in numerous biomedical, pharmaceutical,

agricultural and consumer-oriented fields [1].

1.1.1. Classification of Hydrogels

Polymeric hydrogels are classified in accordance to their monomeric composition based

on the method of preparation giving some important class of hydrogels namely

homopolymeric hydrogels, copolymeric hydrogels and interpenetrating polymeric

hydrogels. Furthermore, the chemical constituent of monomers used in the preparation of

hydrogels plays an important role in classifying the hydrogels. The hydrogels are classed

as either neutral, anionic, cationic or ampholytic based on the presence of ionic charges

on the monomer. Hydrogels are also classed as amorphous or semi-crystalline materials

based on their physical nature.

1.1.1.1. Homopolymeric Hydrogels

Homopolymers are formally referred to as a polymer network derived from a single

species of the monomer, which is the basic structural unit comprising of any polymer

3


network [38-41]. Homopolymers could have crosslinked or uncrosslinked skeletal

structure depending on the nature of the monomer and polymerisation technique.

Homopolymers, which are generally crosslinked, find important applications such as slow

drug delivery devices and contact lenses. An important category of crosslinked

homopolymeric hydrogels of poly(hydroxyalkyl methacrylates) include poly(3-

hydroxypropyl methacrylate (PHPMA), poly(glyceryl methacrylate), (PGMA) and

poly(2-hydroxyethyl methacrylate) (PHEMA) [2,42]. PHEMA hydrogels is among the

most widely studied and used of all synthetic hydrogel materials [23,42-52].

There are some uncrosslinked homopolymers, which have been of interest to a number of

researchers [53-57]. Poly(N-vinyl-2-pyrrolidinone) (PNVP), poly(acrylamide) (PAM),

poly(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA) are classed as uncrosslinked

water-soluble homopolymers. PNVP has found useful applications in biomedicine due to

its extreme solubility in water and adequate solubility in many other polar and non-polar

solvents [2,53-55]. PVA is another important class of uncrosslinked homopolymeric

material with numerous potential biomedical and agricultural applications when

crosslinked [56,57]. PEG and PAM have been widely used in agricultural applications

[58].

1.1.1.2. Copolymeric Hydrogels

Copolymeric hydrogel networks are comprised of two or more different monomer species

with at least one hydrophilic component, arranged in a random, block or alternating

configuration along the chain of the polymer network [38-41]. The copolymeric hydrogel

networks are generally covalently or ionically crosslinked structures, which are not water

-soluble [1-8].

A wide range of important copolymeric hydrogels with vast combinations of compatible

monomers, some of which include poly(NVP-co-HEMA), poly(HEMA-co-MMA) and

poly(HEMA-co-AA) have been studied by a number of researchers [59-61]. Copolymers

designed to function as hydrogels are confined to the combination of compatible

4


monomers, which will give the hydrogels the desirable properties for their intended

potential applications.

1.1.1.3. Interpenetrating Polymer Network (IPN) Hydrogels

IPN, an important class of hydrogel materials, are defined as two independent crosslinked

synthetic and/or natural polymer components contained in a network form as shown in

Figure 2 [62]. A semi-IPN is an IPN where one of the components is a crosslinked

polymer while the other component is a non-crosslinked polymer [1,4,41,63-67].

Figure 2. Structure of an IPN

The two basic synthetic routes to form IPNs are sequential and simultaneous

polymerisation methods [63]. The formation of an IPN increases the compatibility of the

polymer components thus preventing phase separation and allows access to properties

that may be hybrids of those of the component macromolecules [1,66-68]. Park et al [1]

have described the IPN formation between a pH sensitive hydrogel and temperature

sensitive second polymer as an example of such behaviour. The IPN formed will be both,

pH and temperature sensitive. Since there is no chemical bonding between the two

polymeric components, each component may retain its own property while the proportion

5


of each network can be varied independently thus obtaining the desired combinations of

the properties of the two macromolecule components [63,69,70].

The mechanical strength of the hydrogel can be improved by using relatively hydrophobic

second polymer in the IPN. Furthermore, one or both of the macromolecular networks of

the IPN could be made biodegradable [1,71]. A number of IPNs and semi-IPNs based on

polysaccharides such as chitosan and its derivatives, PNVP, PVA, poly(ethylene oxide)

(PEO), poly(N-isopropyl acrylamide) (PNIPAM), PEG and poly (methacrylic acid)

(PMAA) with potential bioapplications as hydrogel materials have been reported by

numerous researchers [66,67,69,72-83].

1.1.1.4. Non-Ionic Hydrogels

Non-ionic hydrogels, often referred to as neutral hydrogels, are homopolymeric or

copolymeric networks, which do not bear any charged groups in their structure. Neutral

hydrogels may be prepared by various polymerisation techniques or by conversion of

existing polymers. Although generalizations can be made about hydrogels, the wide range

of chemical compositions of the monomers used, give them different properties with

regards to biocompatibility, physical and chemical properties of the resultant polymer [2].

Neutral hydrogels swell to equilibrium when the osmotic pressure of the solvent is

balanced with the sub-chain stretching energy. The collapse and swelling of neutral

hydrogel networks occur normally as a result of change in the environmental temperature

[84]. Some neutral monomers commonly utilized to form hydrogels are shown in Figures

3 - 8 [2].

CH2

CH3

O

O

R

H2C C

H2

OH H2C C

HOH

CH3

H2C C

HCH

2

OH

OH

R = ,

Figure 3. Hydroxyalkyl methacrylates

6


CH3

CH

CH

R'

R

O R''

CH3

CH3 C

2H

5

CH2CHOHCH

3

R =

R', R'' =

H ,

H , ,

Figure 4. Acrylamide derivatives

NCH

2

CH2 C

H2

CH

CH2

O

CH2

CH

CH

CH

CH

2

OH

CH2

C

H2

C

H2

CH2

CH2

N O

CHCH

2

Figure 5 Figure 6 Figure 7

N-Vinyl pyrrolidinone N-Vinyl caprolactam 2, 4 Pentadiene-1-ol

CH2

R

O

O

R'

CH3

CH3 C

4H

9OMe

CNOCH2CH

2OCH

3

R = H ,

, ,

,

R' =

Figure 8. Hydrophobic acrylics

1.1.1.5. Ionic Hydrogels

Ionic hydrogels also known as polyelectrolytes are prepared from monomer/s

accompanying ionic charges. The charges could be positive or negative thus classing the

hydrogels as cationic or anionic hydrogel respectively and furthermore, a combination of

both positive and negative charges gives an ampholytic macromolecule [2,26,85,86]. The

phase transition phenomenon of the polyelectrolytes was theorized by Dusek and

Patterson (1968) [87]. Inclusion of charged species in the polymer backbone enhances the

stimuli responsive properties, which could be controlled, depending on the nature of the

pendent group thus widening its scope of bioapplications as hydrogels [26,84-89].

7


1.1.1.5.1. Anionic Hydrogels

Anionic hydrogel networks are usually referred to as either homopolymers of negatively

charged acidic or anionic monomers or copolymers of an anionic monomer and a neutral

monomer. However, anionic hydrogels could also be prepared through modification of

existing polymeric non-ionic hydrogels such as by the partial hydrolysis of poly(hydroxy

alkyl methacrylates) or by the addition of excess polyanions in the case of polyelectrolyte

complexes to form anionic hydrogels [2]. Anionic monomers commonly utilized to form

anionic hydrogels are shown in Figures 9-11 [2]. Anionic hydrogels are known to exhibit

a marked increase in the swelling ratio with increase in the environmental pH [88,89].

CH2

OH

R

O

CH3R = H ,

Figure 9. Acrylic acid derivatives

CH

CH

OH

O

CH3

CH

CH

CH

CH

SO3 NaC

H

CH2

Figure 10 Figure 11

Crotonic acid Sodium Styrene sulfonate

1.1.1.5.2. Cationic Hydrogels

Homopolymers of positively charged basic or cationic monomers or copolymers of

cationic and neutral monomers are commonly referred to as cationic hydrogel networks.

Cationic monomers commonly utilized to prepare cationic-based hydrogel networks are

shown in Figures 12-13 [2]. As described in section 1.1.1.5.1, cationic polymeric

networks could also be derived through modifications such as partial hydrolysis of the

existing non-ionic pre-formed polymer networks. It is also possible to synthesize cationic

hydrogels through polyelectrolyte complexation reactions by addition of excess

polycations [2].

8


Cationic pendant groups in polymer network in the contrary behaviour to anionic pendant

give rise to hydrogels, which remain collapsed in the basic environment and swollen in

the acidic environment due to the electrostatic repulsion between the positively charged

groups [88,89].

CH2

R

O

O

CH

2

CH

2

HN

R'

R''

CH3 C

4H

9H , ,R, R', R'' =

Figure 12. Aminoethyl methacrylate derivatives

NCH

CH2

Figure 13. 4-Vinyl pyridine

1.1.1.5.3. Polyampholytic Hydrogels

Polyampholytic hydrogel networks are referred to as macromolecules capable of

possessing both positively and negatively charged moieties in the polymer network

[26,90,81]. The presence of ionic species along the polymer chain has a distinct effect on

the solution and solid-state properties of the polyampholytes [90]. The columbic

attractions between the oppositely charged sides afford inter- and intramolecular ionic

interactions that are stronger than Van der Waals forces, yet weaker than covalent bonds

[90].

The net charges on these materials can be changed to achieve the desired functional

property by changing the monomeric composition of the feed mixture [36,91]. Some

common acidic and basic monomer combinations used to prepare polyampholytes are

illustrated in Figures 14 - 16 [90]. Preparation of numerous polyampholytic networks

with a wide range of important biomedical applications including sustained drug delivery

systems have been reported [26, 85,86,91-93].

9


CH2

CH

COOHN

CH

CH2

Acrylic acid 2-Vinylpyridine

Figure 14

OO O

N

CH

CH2

OO

Figure 15

Maleic anhydride N- vinylsuccinimide

CH2

CH

COOH

CH2

ON

CH3

O

CH3

CH3

Acrylic acid

Figure 16

2-(Diethylamino)ethyl 2-methylacrylate

1.1.1.6. Hydrogel Network Structures

Flory (1953) [14] states that the polymeric hydrogel network structure may have several

roles. In an aqueous medium the network may dissociate and take the role of the solute as

in the case of some water-soluble hydrogel networks described in Section 1.1.1.1 or swell

to equilibrium by imbibing the medium in its structure. As the network expands in an

aqueous medium (Figure 1) [13], a force resisting the expansion occurs due to the

elongation of the chain into a lesser entropically desirable conformation. When the

osmotic pressure driving the medium into the hydrogel network is matched by the exerted

expansion resistance force, equilibrium degree of swelling is achieved [8].

10


The physical and other properties of the hydrogels depend on the structures of the

polymeric networks [4]. To maintain the three-dimensional structures, polymer chains of

hydrogels are usually crosslinked chemically or physically as described earlier in the sub-

sections of Section 1.1.1. In chemically crosslinked hydrogels, polymer chains are

connected by covalent bonds and thus it is difficult to change the shape of such networks.

Polymer chains of physical gels are connected through non-covalent bonds, such as van

der Waals interactions, ionic interactions, hydrogen bonding, or hydrophobic interactions

[7]. Physical and ionotropic forms represent secondary valence networks while the

covalent form indicates primary valence networks (Figure 17) [3].

Physical CovalentIonotropic

Figure 17. Schematic representation of hydrogel structures

The extent of crosslinking in the hydrogel network is referred to as crosslinking density.

Increased crosslinking density will increase the resistive force to chain elongation

consequently reducing the degree of equilibrium swelling in contrast to hydrogels with

low crosslinking density [8].

The polymeric hydrogel networks can be classified as hydrogen bonded, amorphous, or

semi-crystalline based on the structural analysis of the polymer network using a number

of physiochemical techniques such as small angle X-ray scattering (SAXS), wide angle

X-ray Scattering (WAXS), electron microscopy, differential scanning calorimetry (DSC),

along with electron and neutron diffraction [4,38,39,94].

11


1.1.1.6.1. Amorphous Hydrogel Structures

The term ‘amorphous’ also known as non-crystalline is usually attributed to optically

transparent isotropic polymeric networks that contain randomly arranged macromolecular

chains as suggested by Flory [14]. The amorphous hydrogel network often contains

localized ordered structures or non-homogeneous structures that are not suggested by the

common Flory definition of amorphous polymers. Thus the most acceptable definition of

such networks is a collection of Gaussian chains between crosslinks [4].

The temperature at which the polymeric network undergoes the transformation from a

glassy to a rubbery state is referred to as the glass transition temperature (Tg) [4,38,41].

The characteristic feature of amorphous polymeric networks is that when exposed to

temperature conditions below its Tg value, they pass successfully through the

transformation from a rubbery to glassy state without any clear demarcation between the

two phases [41]. The glassy, transparent nature, an important characteristic of amorphous

hydrogel networks, has widened their scope as bioapplicable materials requiring optical

transmittance [95].

1.1.1.6.2. Semicrystalline Hydrogel Structures

Semicrystalline hydrogel networks are complex mixtures of amorphous and crystalline

phases, which contain dense regions of ordered macromolecular chains (crystallites)

[96,97]. The lack of mechanical strength in some conventional crosslinked hydrogel

network structures for certain biomedical applications has led the development of

anisotropic semicrystalline polymeric networks which are characterized by the presence

of strong covalent bonds along the polymer chain [96,98].

Semicrystalline hydrogel networks are produced by heat treatment of noncrystalline

hydrogels above their Tg [4,98]. Crystallization of polymers in polymer-diluent systems is

the typical method of preparing semicrystalline hydrogel networks [4,99]. In the

crystallization process the short chains that are not able to fold are rejected from the

crystalline phase and thus they participate in the amorphous phase hence the resultant

polymer network contains continuous composition of amorphous and crystalline regions

[4,100]. The tendency of the polymers to crystallize is enhanced by its regularity and

12


polarity [4]. Peppas (1977) [101] suggests that when semicrystalline polymer networks

are placed in aqueous medium, only the amorphous regions swell and the crystalline

regions are not affected by the medium thus they play the role of crosslinks in the

polymer network.

1.1.1.6.3. Hydrogen Bonded Hydrogel Structures

Hydrogen bonding is referred to as an electrostatic interaction between electronegative

atoms such as oxygen, nitrogen, fluorine and chlorine and hydrogen atoms that are

covalently bound to similar electronegative atoms [1,102]. The strength of the hydrogen

bonding (< 10 Kcal/mol), however, is far weaker than covalent bonding (> 100 Kcal/mol)

but still stronger than the van der Walls interactions (~ 1 Kcal/mol) [102].

The formation of multiple hydrogen bonds between two water-soluble macromolecules

may result in strong intermolecular structures [1], which are physically crosslinked three-

dimensional polymeric networks such as IPNs and semi-IPNs described in Section

1.1.1.3. The driving force behind the formation of the multiple simultaneous hydrogen

bonds between the macromolecules is the co-operative interaction between the

macromolecules, which is restricted to the chain length of the macromolecule [103].

1.2. Synthesis of Polymeric Hydrogels

Polymerisation reactions based on kinetics can be divided into chain or step

polymerisation reactions [41,64,65]. Step polymerisation reactions generally occur

between functionally substituted monomers and are characterized by a rapid

disappearance of the monomer at an early stage of the reaction and the existence of broad

molecular weight distribution in the later stages of the reaction [41,64].

Chain polymerisation, however, involves a three-step process namely: initiation,

propagation and termination thus allowing the monomer concentration to decrease

steadily with time thus ideally the reaction mixture at any stage of the polymerisation

reaction contains the monomer and the converted high polymer [41,64]. In contrary to

step polymerisation reactions, longer reaction times in chain polymerisation produces

high yield polymer but the molecular weight of the polymer is not affected [64].

13


The saturated monomers described in Section 1.1.1 for hydrogel synthesis typically react

through a chain polymerisation process [64]. The characteristic polymerisation of

hydrogel network begins from the reactive centre initiated by the polymerisation source

and terminates upon the loss of the radical reactivity of the radicals as described in

Sections 1.2.1–1.2.3. The reactive centres at the initiation stage could be of free radical

nature or ionic nature [64,104] thus promoting free radical or ionic polymerisation as

described in Section 1.2.4.

1.2.1. Chain Initiation

A trace quantity of an initiator is normally required in the case of photopolymerisation

and thermal polymerisation processes to create free radicals for chain initiation [64]. The

initiators for specific curing processes readily fragment into radicals under the influence

of the applied source, which could be either heat or UV light as described in Scheme 1.

∆R R

/ hv

Initiator Free radical

Scheme 1. Formation of radicals

However, in addition to heat and light some high-energy ionization radiation sources,

which do not require initiators, can also generate radicals through electrochemical means

[64]. The radicals created according to Scheme 1 [104] react with an unsaturated

monomer also referred to as vinyl monomers to create a new species, which is still a

radical as shown in Scheme 2 [104] thus initiating the chain polymerisation process.

R CH

2

CH2

RH2C CH

2

Unsaturated

monomer

Propagating

species

+

Free Radical

Scheme 2. Chain initiation

14


The efficiency of the initiator is a measure of the extent to which the number of radicals

formed reflects the number of polymer chains formed [64]. For thermal and photo-curable

systems the percentage of the initiator may vary with the weight of total resin. However,

high amounts of initiator may not necessarily enhance polymerisation, instead it may

have adverse effect since increased free radicals may undergo recombination and inhibit

the polymerisation process.

1.2.2. Chain Propagation

The propagation step involves growth of the polymer chain by rapid sequential addition

of monomer to the active center as shown in Scheme 3 [104]. The reactivity of the

propagating radicals is assumed to be independent of the size or degree of polymerisation

[41].

CH

2

CH2

R CH2

CH2

n

CH

2

CH2

CH

2

CH2

n

Propagating

species

+ R

Chain addition Propagating species

Scheme 3. Chain propagation

1.2.3. Chain Termination

The chain polymerisation does not continue until all the participating monomers are used

up because the free radicals involved are so reactive that they find a variety of ways of

losing their radical activity [64]. Thus the polymer chain terminates by disproportionation

or combination reactions.

CH

2

CH2

CH

2

CH3

n

CH

2

CH2

CH

2

CH2

n

CH

2

CH2

CH

CH2

n

R

R

Propagating species

2

Reaction products

Disproportionation

R

+

Scheme 4. Chain termination through disproportionation reaction

15


The disproportionation reaction is characterized by the interaction of two reactive radial

species via hydrogen abstraction process leading to the formation of a saturated and an

unsaturated compound as shown in Scheme 4 [104].

The combination reaction in the termination stage of the polymer chain occurs by the

combination of two reactive radical species to form a single bond and one reaction

compound as shown in Scheme 5 [64,104].

CH

2

CH2

CH

2

CH

2n

CH

2

CH2

CH

2

CH2

n

RR

Propagating species

2

2Single reaction product

Combination

Scheme 5. Chain termination through combination reaction

1.2.4. Nature of the Reactive Radical Species

The nature of the radical species characterizes the type of chain polymerisation. The

categories of chain polymerisation reactions based on the nature of the reactive species

are referred to as free radical for non-ionic radical species, cationic and anionic for

cationic and anionic reactive radical species respectively. The presence of reactive ionic

centres makes ionic chain polymerisations more monomer specific than free radical

polymerisation reactions [64]. Furthermore, the propagating ionic centre is accompanied

by a counter-ion of opposite charge and termination does not occur by the reaction of two

ionic centres since they are of similar charge and thus repel each other [64].

The polarity of the polymerisation solvent and the ability to solvate the counter ion are

significant factors in ionic polymerisation [64]. Cationic active centres are created by the

reaction of an electrophilic monomer in the presence of protonic acids, which serve as

initiators. The propagation mechanism is the same as that of free radical polymerisation

described in Section 1.2.2. The termination step in cationic polymerisation is achieved by

either unimolecular arrangement of the ion pair or through chain transfer [64].

16


Anionic chain polymerisation begins with active centres created by the reaction of a

nucleophilic monomer and propagates as described in Section 1.2.2 but there is no

inherent termination process, which is a unique property of such polymers [38,64].

Termination by ion pair arrangement in contrary to cationic polymerisation does not

occur in anionic polymerisation due its unfavourable requirement to eliminate the hydride

ion and furthermore, the alkaline earth metal counter ions used do not have the tendency

to combine with the active carbanion thus rendering the polymer molecule active, also

referred to as living polymers [38,64].

1.2.5. Curing Processes

The polymerisation techniques described in Section 1.2.1 could be carried out using a

variety of curing processes such as thermal, redox and radiation methods. Thermal

polymerisation technique involves the use of heat in the presence of a suitable initiator

while the redox method simply involves a reduction-oxidation reaction between the

participating species. Radiations sources commonly utilized by researchers [105,106] to

synthesize polymeric hydrogels include low energy ultraviolet (UV) radiation technique

and high-energy ionisation techniques such as gamma radiation and electron beam

radiation.

1.2.5.1. Ionizing Radiation Sources

Ionization radiation covers a wide range of different radiations some of which are of

primary source or secondary source. Ionization radiation is a high-energy process

involving electronic radiation of moving particles, which carry enough energy to ionize

simple molecules either in air or water and therefore more penetrative [4]. It involves the

use of either electron beams from an electron accelerator or gamma radiation from a 60

Co

source [4].

1.2.5.1.1. Electron Beam (EB) Radiation Process

Electron beam radiation is a high-energy process, which involves artificially accelerated

electron beams delivered from several systems with energy ranging from 0.5 to 20 MeV

[4]. Electron accelerators, such as commonly used Van de Graaff accelerator, substitute

for isotopes emitting β-rays, which are not utilized in radiation chemistry due to technical

17


problems. The EB process is an efficient process, which does not require initiators in the

reactive mixture, however the penetration of fast electron is lower than that of gamma

radiation [4]. Radical as well as ionic species are capable of participating in the

fundamental process.

1.2.5.1.2. Gamma Radiation Process

Gamma radiation involves emission of γ-rays by radioactive isotopes and they cover a

wide range of energies [4]. The ease of its preparation and fairly long half-life of 5.3

years compared to other present isotopes makes 60

Co, which sources two monochromatic

γ beams with energies of 1.17 and 1.33 MeV, the most widely used isotope for this

purpose [4]. 60

Co is produced by neutron irradiation of normal 59

Co in a nuclear reactor.

Gamma radiation technique does not require the inclusion of chemical initiators of any

sort, however, they can be used to remove any residual initiator that is present after other

conventional polymerisation processes such as thermal or UV curing. Residual initiator

may act as an undesirable contaminant [105-107]. The gamma rays in contrary to electron

beam radiation have very high penetrative power and the dose of radiation could be

varied from 5 to 100 rad/sec [4].

1.2.5.2. Ultra Violet (UV) Radiation Process

UV radiation curing technique involves the use of UV rays from a special light source of

desired intensity normally in the presence of a photosensitive chemical. This chemical

serves as an initiator in the photopolymerisation process to form radicals at a wavelength

of 360nm at which monomers are not affected. The curing process could be achieved by

the use of an electrode type or electrodeless lamp.

A medium pressure mercury lamp is an electrode type quartz tube filled with an inert gas

such as argon or xenon along with small amount of mercury installed with an electrode at

either end. The lamp when connected to an appropriate power source, an electrical arc

passes between the electrodes vaporizing mercury resulting in the energy emission, which

is primarily a white light, infrared and ultraviolet.

18


Electrodeless lamp displays a similar spectral emission to that of an electrode type lamp,

however the operation of energization of the lamp is different between the two. The lamp

is energized by microwaves generated by magnetrons rather than an electrical arc. Even

ambient lighting contains UV rays, which could initiate some highly photosensitive

monomers in the presence of a photoinitiator.

The use of UV curing is a field of growing interest. UV curing has unique advantages

over thermal systems, which include cost effectiveness, minimal space requirements,

reduction in energy consumption, and rapid polymeric network formation on heat

sensitive substrates [108,109]. Energy and pollution constraints represent the major

contribution factors to the growth of this technology [109]. However, the only drawback

in this curing method is the use of chemical initiators. The photoinitiators are seldom

consumed fully during the polymerisation process. These materials trapped in the

polymer matrix tend to leach out when the polymer is in contact with an aqueous

medium. The in vivo leaching of additives used during the fabrication of polymers has

been cited as the cause of inflammation and eventual rejection of the implanted

biomaterial [2,110].

1.2.6. Charge-Transfer (CT) Complex Polymerisation

Scott et al [111] and Ellinger [112] were the pioneers in the field who initially reported

the spontaneous polymerisation of N-vinylcarbazole (VCZ) through the formation of

charge-transfer (CT) complexes in the presence of a variety of electron acceptor

monomers in 1963. The CT complex formation phenomenon has been widely recognized

since then, and has been an area of great research interest [113-123]. The complex

formations occur between electron-rich (donor) and electron-poor (acceptor) olefins and

are often spontaneous, that is, no chemical initiator is required in the process [124].

Chemical initiators used in the curing process to speed up the reactions are never fully

consumed in the reaction. Thus the resultant polymer is usually fairly unstable and

susceptible to non-desirable degradation. Chemical initiators besides being one of the

most costly components in the polymerisable compositions are also a significant

contributor to the toxicity of such formulations [122]. CT complex reactions, however,

19


may require heat or light depending on the nature of the participating monomers for the

reaction to commence. Thermal and photochemically induced CT complex

polymerisation reactions studied in recent years have been thoroughly revised by Shirota

and Mikawa [115].

1.2.6.1. Charge-Transfer (CT) Interactions

The CT complex system involves two components, an acceptor and a donor. The donor

molecule contains unshared pair of electrons and the acceptor molecule has vacant

molecular orbitals. The interaction between the donor and the acceptor counterparts in CT

polymerisation reactions is based on Mulliken’s charge-transfer theory, which states that

charge-transfer complexes are formed as a result of partial electron transfer from the

donor to the acceptor [125]. The formation of a CT complex depends on the nonbonding

interactions between the pi bonds of a donor and acceptor that create a species lower in

energy [126].

A A D

A A D

A D

A D

A D+ + D**

A D+ + D**

*

A D+*hv

*Scheme 6. Formation of an AD from a ground state CT

Scheme 7. Formation of an exciplex type AD

hv

hv

The degree of charge transfer depends on the compatibility of the donor and acceptor

monomers. For weak electron donor-acceptor pairs, the no-bond structure contributes

greatly to the ground state charge-transfer complex, whereas the electron transferred

dative structure contributes greatly to the excited state complex [121]. Scheme 6 [127]

outlines the formation of an excited state CT complex (AD*) by direct excitation of the

ground state CT complex.

20


The formation of CT complexes not only occurs in the ground state but also in the excited

state of an electron donor or acceptor [121]. Scheme 7 [127] outlines the formation of an

AD* from excited donor or acceptor. The excited donor (D

*) or acceptor (A

*) associates

with the ground state acceptor (A) or donor (D) respectively. Such excited state

complexes are termed exciplexes [121]. Exciplexes are stable in the excited state but

dissociative in the ground state. The enhanced donor and acceptor properties of the

molecules in their excited states contribute to the formation of exciplexes [121]. It has

been suggested in the literature [127,128] that both the mechanisms of AD* formation as

illustrated in Schemes 6 and 7 could lead to the generation of photochemically identical

AD* species.

EHOMO (D) ELUMO (A)

Hückel calculations

Formation of ground state CTC: EHOMO (D) ≈ELUMO (A)

Scheme 8. Relative energy differences between electron donors (D) and acceptors (A)

21


The Mulliken theory [125] predicts a maximum charge transfer stabilization for the cases

where a maximum overlap exists between the highest occupied molecular orbital

(HOMO) of the donor and the lowest unoccupied molecular orbital (LUMO) of the

acceptor, thus producing coloured species of high ionic character. Scheme 8, adopted

from Jönsson et al [127], indicates the relative energy differences between typical donor

and acceptor monomers. In the case of a strong overlap between the HOMO and LUMO

of the monomers, a thermodynamically favoured separated radical ion pair may be

formed. In such cases the thermal stability and the photo latency are completely lost and a

spontaneous polymerisation occurs [127,120].

1.2.6.1.1. Decay Mechanisms of Excited State CT Complexes

Excited state CT complexes besides decaying directly back to ground state could also go

through a photochemically allowed [2+2] cycloaddition. Such interaction between the

donor and the acceptor olefins involved in the CT complexes is based on Huisgen’s

seminal work on the cycloaddition reactions between electron rich and electron deficient

olefins [129,130]. Huisgen’s work suggests the involvement of tetramethylene

intermediates in the process of cycloaddition.

The tetramethylene intermediates resulting from the decomposition of excited state CT

complexes could be predominantly of 1, 4 biradical in nature or predominantly

zwitterionic in nature [127,124,120]. Influence of ionic/biradical ratio of the short-lived

excited CT complex, depending on the polarity of the olefins involved, is also noticeable

among the intermediates [127,120]. The formation of excited state complex and the decay

mechanism via the formation of possible tetramethylene intermediates is illustrated in

Scheme 9 [120].

Hall and Padias [124,131] suggest that the terminal substituents on the donor and the

acceptor olefins direct the nature of the intermediates formed. The zwitterions are

predominantly in the cis or syn conformation due to the coulombic attraction, while the

biradicals exist in extended trans-conformation [124]. An extensive study on the

formation of these tetramethylene intermediates has been reported by Hall and Padias

[131]. All the possible resultant intermediates from the decomposition of the excited CT

22


complexes proceed to form a thermodynamically stable cyclobutane ring. However, the

relative yield of ring closure via each of these intermediates may vary depending on the

nature of the intermediate [120]. The strength of the CT complex could be controlled to

favour the existence of a free radical process over zwitterionic. Jonssön et al [120] have

also proposed the existence of fused radical ion pair as a possible intermediate upon

acceptor donor interaction. If the fused radical/ion pair dissociates into radical ions, they

will be capable of inducing both, the free radical and the ionic process [120]. The

dissociative mechanism of the fused radical/ion pair is illustrated in Scheme 10.

AD

*

*

D A

D A

D A

AD

AD

D A

- e-

Excited state complex

(AD )

-

1, 4 biradical

Zwitter ion

+

- +

+-

Fused radical/ionic pair

Cyclobutane

Tetramethylene intermediates

+

Scheme 9. Mechanism of [2+2] cycloaddition of the donor (D) and the acceptor (A) via

the possible tetramethylene intermediates.

23


AD

AD

AD

AD

- +

+-

Fused radical/ionic pair

- +

+-

Separated radical ionsDissociation

+

+

Scheme 10. Dissociation of the radical/ionic pair

To achieve the formation of 1, 4 biradical intermediate and subsequent free radical

polymerisation, it is crucial to generate an excited state complex with minimum ionic

character as possible [127,120].

1.2.6.2. Inducement of CT Complex Polymerisation

CT complex formulations could be induced by heat or light depending on the sensitivity

of the donor/acceptor monomers involved. Thermal induced reactions normally include

various initiation mechanisms whereas photo-induced reactions generally involve the

intermediacy of ions or radicals [115]. Photo-induced CT complex formations are by far

the predominant area of current research with numerous publications available to date

[120-122,126,127].

The use of ultra-violet light (UV) to cure unsaturated monomers in the absence of

photosensitizers has been known since the introduction of CT complexes. However, it

was restricted to very short wavelength UV lights and thickness of the polymerisable film

[122]. Such systems were not commercially viable. Jönsson et al (1995) [122] developed

UV polymerisable CT complex formulations, which were photoinitiator free and not

restricted to short wavelengths. Furthermore, the process was found to be more efficient

in the sense that the thickness of the polymerisable film was not an interfering factor in

the degree of polymerisation.

Such formulations often referred to as photoinitiator-free UV curable systems involve the

use of ‘charge-transfer complexes’ as a substitute to chemical photoinitiators [132] to

24


accomplish photopolymerisation reactions. Copolymerisation reactions via CT complexes

although being slower in comparison to photoinitiator-initiated systems but are vastly

superior in terms of stability is a field of growing interest [127].

1.2.6.3. Proposed Mechanisms of CT Complex Polymerisation

Polymerisation mechanisms always have to take at least a two-step process into account,

namely initiation and propagation. A number of researchers have proposed various

mechanisms to account for the CT complex polymerisation phenomenon. It has been

suggested in the literature that spontaneous CT polymerisation could be initiated by a

partial electron transfer (ET) from the donor to the acceptor molecule as illustrated in

Scheme 11 [124].

D+

A- e-

D A-

Scheme 11. Radical ions formation in ET

However, this is only true for the CT complex reactions, which involve extremely

electron rich and extremely electron deficient olefins [133]. The initiation of CT complex

polymerisation through the formation of tetramethylene intermediates proposed by Hall

and Padias [124] has been the most conclusive and widely accepted concept to date. In an

ideal system without chain transfer, the polymerisation may start from any of the decayed

species illustrated in Scheme 9 [120]. Depending on the character of the monomers

present in the system, the polymerisation will occur via a free radical or ionic process

[124].

An alternating copolymer has often been the observed as the phenomenal result of CT

complex polymerisation. A number of theories have been proposed in the past to account

for this phenomenon [124]. The CT complex acting as the co-monomer in the reaction as

illustrated in Scheme 12 has been a very attractive theory. This theory by Bartlett and

Nozaki [134] states that the colour observed as the result of CT complex formation fades

as the polymerisation via cycloaddition reaction proceeds.

25


D

A

DAD

ADA

D A D A D A

A

D A D A D A

D

AD

+

+

Scheme 12. CT complex acts as the monomer

Scheme 13. Matrix polymerisation

The other theory based on the difference of the polarities between the reacting molecules

was that of the matrix polymerisation, illustrated in Scheme 13. The theory of matrix

polymerisation suggests that the monomers align themselves in an alternating fashion in

the polymerisable mixture. The theories illustrated in Schemes 12 and 13 however, do not

provide sufficient evidence on the CT complex polymerisation [124]. In thermal initiated

CT complex polymerisations, Coote and Davies [135] concluded that the CT complex

was not involved in the propagation step. Hall and Padias suggest that the alternating

propagation is ascribed to the polarity difference of the monomers involved in the free

radical polymer chain.

1.2.6.4. Influential Factors in CT Complex Polymerisation

There are certain factors such as the choice of donor/acceptor monomer pairs involved,

inclusion of catalytic additives such as hydrogen donors or Lewis acids which govern the

efficiency and nature of the CT complex polymerisation. Hall and Padias [124] have shed

light on further advancement on their present theory of CT complex initiation. They

hypothesise that any force that brings the donor or acceptor together will enhance the rate

of tetramethylene intermediate formation and consequently the rate of spontaneous

polymerisation. The duo suggests possible modes of interactions such as protic acid/base,

Coulombic attractions, hydrophobic and hydrophilic interactions, Lewis acid/base, or the

use of templates or tethering beyond CT interaction.

26


1.2.6.4.1. Effect of Monomers

The efficiency and the nature of CT complex polymerisation is heavily dependent on the

choice the acceptor and the donor monomer pairs. The strength of electron donating and

electron withdrawing capabilities of the donor and acceptor respectively is the most

crucial factor, which is taken in consideration for the selection of desirable donor/acceptor

pairs. A strong donor/strong acceptor pair is known to give a CT complex of very ionic

character. Strong donor/weak acceptor or weak donor/strong donor pairs, however, give

CT complexes of absolutely minimum ionic character thus promoting free radical

polymerisation [120,127,136,137]. Table 1, adopted from Jönsson et al [120], summarises

a list of some commonly used acceptor and donor monomers with indicative strength of

electron acceptation and donation.

Table 1. Commonly utilized acceptor/donor monomers

Acceptors (A) Donors (D)

Incr

easi

ng

acc

epto

r st

ren

gth

‡‡

Maleic anhydride

Maleimide

N-methylmaleimide

p-Carbomethoxyphenylmaleimide

N-phenylmaleimide

4, 4- Dimaleimidobisphenol

Dimethyl fumarate

Diallyl maleate

Dimethyl maleate

Tetrahydrofurfuryl vinyl ether

Triethyleneglycol divinyl ether

Paraglycidyloxystyrene

Paramethoxystyrene

4-Propenyloxymethyl-1, 3, 2-dioxolanone

IsoEugenol (IEU)

N-Vinyl pyrrolidinone

††In

crea

sin

g d

on

or

stre

ng

th

Increasing donor or acceptor strength in the direction of the arrows

The donor/acceptor monomer feed ratio also critically affects the efficiency of CT

complex polymerisation. According to Decker et al [138], their study on N-substituted

alkyl maleimide (MI) and vinyl ether (VE) has indicated that the monomer feed

composition plays a decisive role on the polymerisation kinetics. They have reported

similar disappearance rate of both the monomers when VE is in excess yielding an

alternating copolymer. However, when the MI is in excess, the copolymerisation and the

27


MI homopolymerisation occur simultaneously to yield a copolymer with isolated VE

units. Based on this observation the authors suggest that the VE radicals act as the main

propagating species.

1.2.6.4.2. Effect of Lewis Acids

Lewis acids enhance the efficiency of spontaneous copolymerisation. The role of Lewis

acids in such reactions is that they make the acceptor olefin more electron deficient [124].

A literature example of alternating copolymerisation of styrene and acrylonitrile in the

presence of Lewis acids is illustrated in Scheme 14 [124].

Ph CN

ZnCl2

+ Alternating copolymer

Scheme 14. Role of Lewis acids in copolymerisation

Inorganic salts such as ZnCl2 or SbCl2 complex with the lone electron pairs on the

electron withdrawing substituent on the acceptor olefin. Cole et al [139], Garnett and

Zilic [140] in their recent studies on CT complexes have reported a significant increment

in the rate of CT complex polymerisation in the presence of SbCl2.

1.2.6.4.2. Effect of Hydrogen Donors

Certain N-substituted maleimides are known to homopolymerise in the absence of

chemical sensitizers [127,138,141] but the process is fairly inefficient. According to

Jönsson et al [127] however, in the presence of strong H-donors, which contain labile

hydrogens, the H-abstraction process will be quite efficient. The hydrogen abstraction

process increases the number of initiating radicals thus increasing the rate of

polymerisation.

Jönsson et al [127] and Clark et al [142] have reported that mixtures of N-

alkylmaleimides and acrylates polymerise rapidly, provided that the mixture contains

easily abstractable hydrogens. Hydrogens could be sourced either by added hydrogen

donors such as tertiary amines, secondary alcohols or by ethyleneglycol/ propyleneglycol

backbones in acrylate monomer/oligomer. The mechanisms of the intramolecular and

28


intermolecular H-abstraction from the excited state N-substituted maleimide, proposed by

Jönsson et al [127] are illustrated in Scheme 15.

N

O

O

N

OH

O

RH

N

O

O

R

R 1

NR

2

R 3

HH

OOR'

H H

R"

HS-R N

OH

O

R

R1 NR

2

R 3

H

OOR'

H

R"

S-R

N

OH

O

R N

OH

O

R N

OH

O

R

RHH

hv

Mechanism of intramolecular H abstraction

+ +

Mechanism of intermolecular H abstraction

hv

Mechanism of radical rearrangement

Scheme 15. Hydrogen abstraction from the excited state N-alkyl maleimide

1.2.6.5. Polymeric Hydrogel Synthesis via CT Complex Formation

Synthesis of polymeric hydrogels through a PI-free method is indeed a novel technique,

which was introduced recently in the course of this study. Although the concept of CT

complex polymerisation has been known for decades and is now well established, to the

29


best knowledge of the author, it has never been used to synthesize polymeric hydrogels.

The use of conventional PIs to assist the photopolymerisation of biomedical polymers has

always been a concern due to the possible leach of PIs from the polymer matrix. As

previously mentioned the PIs besides being expensive are fairly toxic materials.

Successful synthesis of polymeric hydrogels formed through the initiation of CT

complexes formed between a series of N-hydroxyalkyl maleimides and NVP, has been

published by the author in the duration of this course [143-148]. The N-hydroxyalkyl

maleimides are strong electron acceptors while NVP is a weak electron donor, thus

making them a compatible donor/acceptor pair for free radical polymerisation. The

hydrogels formed through this novel process has been reported to be very stable, quite

resilient, non-toxic and very promising materials for drug delivery applications.

1.3. Applications of Hydrogels

Polymeric hydrogels, owing to their dynamic structural properties have been commonly

utilized in numerous biomedical and agricultural applications. The biomedical

applications of hydrogels could be classified into three distinct categories namely,

coatings such as catheters, homogeneous materials such as contact lenses and devices

such as sustained drug delivery systems [2,149]. Biomedical applications of hydrogels are

discussed in detail in Section 1.4.

1.3.1. Agricultural Applications

Hydrogels have been commonly utilized in agricultural field mainly as water storage

granules [150,151]. The need for improving the physical properties of soil to increase

productivity in the agricultural sector was visualized in 1950s [152]. This led to the

development of water-soluble polymers such as PVA, PEG and PAM to function as soil

conditioners [58,152] followed by the introduction of water-swellable polymeric

hydrogels in the early 1980s [153,154]. Water-swellable hydrogels from crosslinked

PAM, crosslinked polyacrylates and copolymers of acrylamide and acrylates for such

applications have been reported [153, 154].

30


Soils with moist hydrogels acting as conditioners, increase water-holding capacity of the

potting media by 50 to 100 %. The increased water supply enhances the germination

process by reducing the relative amount of water loss via evaporation and drainage.

Swellable hydrogel delivery systems are also commonly utilized for controlled release of

agrochemicals and nutrients of importance in agricultural applications to enhance plant

growth with reduced environmental pollution. A number of researchers have reported the

versatility of polymeric hydrogels in agricultural applications [150-155].

1.3.2. Other Applications

Hydrogels have been commonly utilized commercially in other important industrial areas

such as cosmetics, food industry, photography and instrumentation. A list of some

important industrial applications of polymeric hydrogel systems are summarised in Table

2 [3].

Table 2. Additional important applications of hydrogels

¬ Hydrogels are used as thickening agents (e.g., starch and gelatin) in foods.

¬ The addition of hydrogel-forming agents to incontinence products increases

the fluid uptake and ensures improved retention capacity.

¬ Technical and electronic instruments can be protected from corrosion and

short-circuit exposure of, or sheathing with highly absorbent hydrogel-forming

agents.

¬ Hydrogels are used in photographic technology because they are light

permeable and can also store light sensitive substances.

¬ In electrophoresis and chromatography, the separation and diffusion

characteristics of the gel structure are exploited. Hydrogels, thus applied,

operate within only a very limited range of swelling.

31


1.4. Hydrogels as Biomaterials

The field of biomedical research has advanced rapidly in the past several years, mainly as

a result of attempts to replace body tissues with natural or synthetic biomaterials [1-

8,149]. Success in the application of biomaterials is strictly confined to their

biocompatibility. The term biocompatibility is referred to as the appropriate biological

performance, both local and systematic of a given biomaterial in a specific application

[1]. An appropriate response of the biomaterial for its particular application would be

referred to as an inert or positive interaction with the host [156]. One promising class of

biomaterials in this field of research is that of polymeric hydrogels.

The landmark paper by Wichterle and Lim [9] on the biomedical usage of PHEMA

hydrogel as contact lenses captivated the interest of biomaterial researchers around the

globe. Since then preparation and study of numerous hydrogel systems with various

properties have been reported. Hydrogels resemble in the physical properties of living

tissue more than any other class of synthetic biomaterial [2]. The high water content and

the soft rubbery consistency of hydrogels contribute to their superficial resemblance of

human tissues and may also contribute to their biocompatibility by minimising

mechanical irritation to surrounding tissue [1-8]. The highly hydrated and water-

plasticised polymer network of the hydrogel often has a low mechanical strength, but is

still finding ever-increasing use as biomaterials [157-161].

The wide range of biomedical applications of hydrogels can be attributed to both their

satisfactory performance upon in vivo implantation in either blood contacting or tissue

contacting situations and to their ability to be fabricated into a wide range of

morphologies [1-8,149]. It should be emphasized that a particular hydrogel composition

for one biomedical application may have to be significantly modified for a different

application as described in the earlier sections of this chapter. The various applications of

hydrogels in the biomedical field are summarised in Table 3 [2].

32


Table 3. Biomedical Applications of Synthetic Hydrogels

Coatings

‘Homogeneous”

Materials

Devices

Sutures

Catheters

IUD’s

Blood detoxicants

Sensors

Vascular grafts

Electrophoresis cells

Cell structure substrates

Electrophoresis gels

Contact lenses

Artificial corneas

Vitreous-humour

replacements

Oestrous –Induces

Soft tissue substitutes

Burn dressings

Bone ingrowth sponges

Dentures

Ear drum plugs

Synthetic cartilage’s

Hemodialysis membranes

Particulate carriers of

tumour antibodies

Enzyme therapeutic

systems

Artificial organs

Sustained drug

delivery systems

The area of interest presented in this work is that of controlled drug release. Sustained

delivery of drugs has been one very important application, where hydrogels have been

extensively used [1-8]. Delivery of bioactive agents through sustained release devices has

been a major field of research over the last three decades [60].

1.4.1. Sustained Drug Delivery Devices

The principle of slow release has been utilized since 1950 in the pharmaceutical industry

but it was not until the mid 1960’s that polymers were used for slow release of molecules

[162]. Folkman and Long [163] first reported sustained drug release from polymers in

1964. However, it was not until the 1970’s when polymeric hydrogels were considered as

drug delivery devices [162]. In recent years major emphasis has been put on the studying

of polymeric hydrogels in biomedical research related to drug delivery [1-8,164] due to

their dynamic properties described in Section 1.1 of this chapter.

33


Until the early 1970’s, drugs were delivered to the human body exclusively via oral and

intravenous means [6]. Though these conventional means of drug administration are still

common to date due to their relative availability and easy excess, there are various

disadvantages associated with them. Firstly, high doses of drug cannot be injected into the

body at one time. Secondly, intravenous delivery leads to high concentration of drug into

the blood stream leading to toxic side effects. Furthermore, only a very small percentage

of the injected drug reaches the affected area in the body and hence multiple injections are

often required for an effective treatment. A typical graphical representation of the drug

dose level in blood when administered through conventional methods is depicted in

Figure 18 [165].

Figure 18. Drug levels in blood with traditional drug dosing

Supplying the appropriate amount of medicine to the body is essential to the success of a

treatment. This concept serves as the foundation for sustained drug release systems [162].

In the early stages of the research on controlled drug delivery devices the major challenge

faced was in obtaining zero-order sustained release of the drug over a prolonged period

[1,164]. The premise of zero-order drug release is to maintain a constant drug

concentration in blood for an extended period of time [1]. Current technology on

sustained drug release devices has improved to such a level that delivery of drugs at a

constant rate could be achieved for a certain period of time ranging from days to years

[1,166,167].

34


A variety of methods have been used to target biologically active molecules to the

specific site and extend their therapeutic lifetimes once inside the body [60,164]. The use

of swellable materials for drug delivery applications has followed experimental and

theoretical investigations of drug transport in polymeric delivery systems [168]. A

graphical representation of drug dosing through a sustained release device is depicted in

Figure 19 [165]. Controlled drug delivery occurs when the polymer, whether natural or

synthetic is judiciously combined with a drug, and the drug is then released over a desired

period into the appropriate biological environment [6,165]. The release of the active drug

may be constant over a long period, cyclic over a long period or triggered by the

environment or other external factors [6,165].

Advantages of controlled drug release devices thus possibly include delivery to the

required site, delivery at required rate, fewer applications, reduced dangers of overdose

and economic advantages by the virtue of more efficient dosage, at the expense of

possibly more complicated fabrication [6,165,169].

Figure 19. Drug levels in blood with controlled drug delivery dosing

Hydrogels have to be biocompatible and biodegradable to be ideal for drug delivery

applications. The degradation products should be non-toxic and should not cause an

inflammatory response. The degradation should also occur within a reasonable period as

required by the application [1-8].

35


1.4.2. Mechanisms of Controlled Drug Delivery

A convenient classification of controlled-release systems is based on the mechanism that

triggers the release of the incorporated drug. A drug delivery mechanism is ideal for a

device, which exhibits a zero order drug release [1,6,164].

Table 4. Classification of Controlled Release Systems

Type Controlling step Drug release mechanism

Diffusion-controlled devices

Reservoir (membrane)

devices

Matrix (monolithic)

devices

Concentration difference

Concentration difference

Diffusion

Diffusion

Chemically-controlled devices

Biodegradable

(bioerodible) devices

Pendant chain devices

Degradation

Hydrolysis

Reaction-dependent

diffusion

Reaction and Diffusion

Solvent-activated systems

Osmotically controlled

devices

Swellable systems

Swelling-controlled

systems

Osmosis

Swelling

Swelling front

Osmotic flow

Diffusion

Relaxation-dependent

diffusion

External force-induced release

Magnetically controlled

devices

Magnetic field

Diffusion

36


Depending on the mode of delivery, the mechanisms involved in the controlled release of

the drug from the device may vary. The three primary mechanisms by which the

therapeutic drugs can be released from a delivery system are diffusion, polymeric

degradation and swelling followed by diffusion [6,165,168,170]. Any or all of these

mechanisms may occur for a given drug release system. Table 4, adopted from Peppas

and Korsmeyer [171], summarizes the various types of controlled release systems.

1.4.2.1. Diffusion-Controlled Release

The most common mechanism of release is that of diffusion. In diffusion systems the

therapeutic drug, which may be either encapsulated in the polymer membrane or

suspended within the polymer matrix passes through the polymer that forms the

controlled release device when placed in an aqueous media [6,165]. The medium diffuses

into the matrix, dissolves the incorporated drug, which then diffuses out of its carrier. The

diffusion can occur on a macroscopic scale as through pores in the polymer matrix,

depicted in Figure 20 [165] or on a molecular level by passing between chains as that of

reservoir devices depicted in Figure 21 [165]. In the matrix system the drug release rate

depends upon the amount of drug present at a particular time, thus the rate of release is

time dependent. [6,165,170,172].

Figure 20. Drug delivery from a typical matrix drug delivery system

37


Figure 21. Drug delivery from typical reservoir devices

(a) transdermal systems (b) implantable or oral systems

In reservoir devices the active ingredient within the polymer matrix forms a core

surrounded by an inert polymeric film or membrane, which acts as the diffusion barrier.

This membrane surrounding the reservoir is the only structure, which effectively limits

the release of the drug molecule in such systems [1,165]. The diffusion of the aqueous

medium through the polymeric membrane surrounding the reservoir device is the rate-

determining step. Furthermore, since the polymeric membrane systems surrounding the

reservoir is essentially uniform and of a non-changing nature, the rate of drug release is

fairly constant and is proportional to the concentration of the incorporated drug initially

present [6,165,170,172].

1.4.2.2. Drug Release Through Biodegradation

Polymer degradation is another interesting phenomenon in which the drug is released

from the carrier device. Biodegradable polymers are designed to degrade into biologically

acceptable and progressively smaller molecules and as the polymer degrades the

imbedded drug is freed into the host [6,165,170,172]. The drug release through polymer

degradation is depicted in Figure 22 [165].

38


Figure 22. Drug delivery through polymer degradation

(a) Bulk erosion (b) Surface erosion

The biodegradation may occur through bulk hydrolysis where the polymer randomly

degrades throughout the matrix. The rate of erosion is dependent on the volume of the

matrix rather than the thickness thus the rate of drug release in this case is unpredictable

and dumping effect of the dose is commonly observed [165,173,174]. This could be

solved by the use of polymer systems that are highly hydrophobic yet contain water labile

linkages [174]. These systems undergo surface erosion with minimum internal

degradation, thus the release rate is proportional to the polymer degradation rate with

proper surface geometry [165,173,174]. The most common formulations of biodegradable

materials are that of microparticles used in oral delivery and injectable delivery systems

[165].

1.4.2.3. Swelling-Controlled Release Systems

The release of the drug could occur from matrix or reservoir devices. The coupling of

diffusion and the macromolecular relaxation of the carrier control the release mechanism

of the incorporated drug providing conditions for zero-order release. A typical polymeric

swelling-controlled release system is depicted in Figure 23 [165]. The dry polymer slab

39


when placed in an aqueous medium swells thus increasing its aqueous solvent content

within the formulation as well as the polymer mesh size as a result, allowing the

incorporated drug to diffuse out into the host environment [6,60,165].

Figure 23. Drug delivery from (a) reservoir and (b) matrix

swelling-controlled release systems

1.4.2.3.1. Solute Transport in Swelling-Controlled Release Systems

The incorporated drug is essentially immobile in the glassy region of the polymer but

begins to diffuse out as the polymer swells in the compatible penetrant medium. The

release of drug thus depends on two simultaneous rates processes, medium migration into

the polymer network and the solute diffusion out of the network. The solubility of the

drug for a given medium is also essential.

In swelling-controlled release systems, the polymer has to swell to some extent before the

drug can diffuse out, thus the initial burst effect of the drug is often observed [6,171,175].

The continued swelling of the polymer eases the diffusion of the drug, ameliorating the

40


slow tailing off of the release curve [171,176,177]. The net effect of the swelling process

is to prolong and linearize the release profile of the drug. A schematic representation of

the swelling-controlled release action is illustrated in Figure 24 [171].

P G

M

D

υ

Figure 24. Schematic representation of a swelling controlled release system

The penetrant medium (M) enters the initially glassy polymer (P) with velocity (υ). The

incorporated (D) drug diffuses through the swollen gel layer (G).

1.4.2.3.1.1. Fick’s Laws of Diffusion

Understanding the mechanisms of the penetrant medium diffusion into the swellable

polymer is crucial to define the release profile of the incorporated solute. Fick [178]

developed two differential equations referred to as Fick's First and second laws to

describe the diffusion phenomenon in thin membranes in one dimension. Fick’s first law

is described by Equation 1 where J is the flux, j is the flux per unit area, A is the area

across the diffusional field, D is the diffusional coefficient, c is the concentration of

solute, z is the distance and /∂ is the concentration gradient across the z axis. c∂ z

41


c

J Aj ADz

∂= − = − ∂ Equation 1

The law states that the flux of a component of concentration across a membrane of unit

area, in a predefined plane, is proportional to the concentration differential across that

plane. In the case of diffusion without convection and unitary area, Equation 1 could be

re-written as described by Equation 2, which is the starting point for numerous

descriptions of diffusion behaviour in swellable polymers [171,179,180].

c

J Dz

∂= − ∂ Equation 2

Fick’s second law with constant boundary conditions can successfully describe much of

the observed solute transport in polymers. It is frequently successful in describing

transport both above and below Tg [175]. It states that the rate of change of concentration

in a volume element of a membrane, within the diffusional field, is proportional to the

rate of change of concentration gradient at that point in the field, as given by Equation 3

[175,179].

2

2

CD

t x

∂ ∂=∂ ∂C

Equation 3

The boundary conditions are:

t = 0 -1/2 < x < 1/2 C = C1

t > 0 x = + 1/2 C = C0

1.4.2.3.1.2. Fickian and Non-Fickian Diffusion

Diffusion in polymers is known to be associated with the physical properties of the gel

network and the interaction between the polymer and the penetrant medium. Based on

Fick’s law of diffusion, Alfrey et al [181] proposed a classification of diffusional

behaviours namely Fickian (Case I) and non-Fickian (Case II and anomalous) in

swellable polymers. The propositions were made in accordance to the penetrant diffusion

rate and the polymer relaxation rate.

42


In the Fickian (Case I) diffusion the penetrant mobility rate is much lower than the

segmental relaxation rate. The reduced driving concentration gradient slows down the

diffusion rate in the polymer slab geometry. In non-Fickian (Case II) diffusion, however,

the mobility rate of the penetrant is much higher than the segmental relaxation rate. The

sharp boundary between the gel phase formed by the penetrant and the glassy portion of

the polymer becomes the rate-determining step [171,179]. Anomalous diffusion

behaviour is characterized by the intermediate properties between the Fickian and Case II

behaviour [179].

Time dependent swelling behaviour in swellable polymers has been generally described

in the literature [27,171,175,176,182] according to Equation 4, normally termed as the

power-law model, with n being the diffusional exponent.

t nM

KtM ∞

= Equation 4

Mt/M∞ represents the fractional uptake of the penetrant medium or release of the

incorporated solute at time (t) normalized with respect to equilibrium conditions. The k

value is a constant, which incorporates the characteristics of the macromolecular

network/drug system and the dissolution medium [175]. The parameter n determines the

dependence of the medium uptake or release rate on time.

Table 5. Transport mechanisms of penetrant through a polymer slab

Exponent n Type of transport Time dependence

0.5 Fickian diffusion f (t –0.5

)

0.5 < n < 1.0 Non-Fickian diffusion (anomalous) f (t n-1

)

1.0 Case II transport Time-independent

n > 1.0 Super Case II transport f (t n-1

)

Table 5 [171,175] summarizes a list of possible transport mechanisms with their

characteristic n values and time dependence.

43


Figure 25. The Fickian (A) and Case-II transport (B) penetrant uptake isotherm into a

sphere of radius a, with diffusion coefficient D (for Fickian diffusion) or relaxation

constant ka (for Case-II transport). Mt and M∞ represent mass of the swollen gel at time (t)

and infinity respectively.

A graphical representation of the two extreme solute transport diffusion mechanisms,

Fickian and non-Fickian diffusion behaviour in swellable polymers is shown in Figure 25

[171]. The power-law model despite being widely used in the literature and its importance

in defining Fickian and non-Fickian diffusion pattern has limitations [171,175,176]. The

power-law model generally accommodates only for Mt/M∞ ≤ 0.7. Thus in situations

where it is important to model the entire swelling or release curve, more sophisticated

models must be used [171].

1.4.2.3.1.3. Dimensionless Analytical Parameters

Brazel and Peppas [176] suggest that penetrant uptake and drug delivery from swelling-

controlled release systems can be described sufficiently by the two dimensionless

parameters for most polymer/solvent systems. These parameters are referred to as the

diffusional Deborah number (De), and the swelling interface number (Sw).

Vrentas et al [183] attempted to establish the regions of Fickian and non-Fickian

diffusion in the swellable polymer behaviour by introducing a dimensionless parameter,

the De. The ratio of the characteristic relaxation time to the characteristic diffusion time is

referred to as the De (Equation 5) where λ is the characteristic relaxation time for

polymer when subjected to swelling and θ is the characteristic diffusion time into the

44


swelling sample [176,177,184]. The parameter θ is defined as the square of the

diffusional distance divided by the diffusion coefficient of the penetrant in the polymer.

De = λθ Equation 5

When a glassy polymer is placed in contact with a swelling agent, the swelling process is

accompanied by the transition of glassy to rubbery state as a result of lowering the glass

transition temperature (Tg) of the polymer [171,180]. This is attributed to the increased

mobility of the macromolecular chains in the presence of the swelling agent as shown in

Figure 26 [177].

Figure 26. Effect of penetrant concentration on the glass transition temperature

In Figure 26, the glassy to rubbery state transmission is represented by a dashed curve.

Zone III, defined arbitrarily around this transition, is the region where the De is

approximately equal to one. Non-Fickian (anomalous) transport is observed in this region.

In zone II, which is the rubbery state, the De is much smaller than one and thus Fickian

diffusion is observed. In zone I (glassy state) the De is much larger than one and Fickian

diffusion is again observed.

45


Fickian diffusion is observed when the time scale of the macromolecular relaxation is

either effectively infinite or zero compared to the time required to establish a

concentration profile in the polymer [175,179,180]. Non-Fickian diffusion behaviour is

normally observed in glassy polymers when the Tg of the polymer is higher than the

environmental temperature [180]. At a specific temperature below the Tg, the polymer

chains are not sufficiently mobile to permit immediate penetration of the medium in the

polymer core [185].

The swelling interface number (Sw) is important in describing the balance between the

solvent penetration and the release of the incorporated drug according to Equation 6

[184].

Sw = s,s

u

D

δ Equation 6

The Sw value (Equation 6), expresses the ratio between the solvent motion and solute

diffusion where u is the velocity of the penetrant front, δ is the thickness of the swollen

region through which the solute diffuses out and Ds,s is the diffusion coefficient of the

incorporated solute [176,177,182,184]. In the case where Sw value smaller or greater than

1 the release pattern has been suggested to be controlled by the solvent penetration or

drug diffusion and the time dependence is Fickian. However, in the case where the Sw

value is in the order of one, anomalous behaviour is observed [176].

1.4.2.3.2. Influential Factors in Swelling-Controlled Release Systems

One of the most remarkable, and useful, features of a polymer's swelling ability manifests

itself when the swelling can be triggered by a change in the environment surrounding the

delivery system [26,165,186]. The external environmental conditions could involve pH,

temperature, magnetic field or ionic strength. The gels may either shrink or swell in

response to such environmental changes as illustrated in Figure 27 [[6,7,165,186-189].

46


Figure 27. Environmental sensitive swelling-controlled release system

The effect of the environmental conditions on the polymer’s performance, however, is

dependent on the nature of the polymer, which could be ionic or neutral. The swelling-

release action in neutral hydrogels is driven by the thermodynamic mixing contribution of

the penetrant medium and the polymer to the overall free energy, which is coupled with

an elastic polymer contribution [175,190]. In ionic hydrogels the driving forces are the

same as that of neutral gels along with some additional contributors such as the ionic

interactions between the charged polymer and the free ions [191]. For most of these

polymers, the structural changes are reversible and repeatable upon additional changes in

the external environment [165].

Hydrogels could be synthesized appropriately to achieve the desired response from a

given environmental condition. Parameters such as the polymer composition, degree of

crosslinking density and the size and nature of the incorporated drug molecule play an

important role in determining the drug release behaviour and thus must be considered

during the design of swelling-controlled release devices [6,8,175].

47


1.4.2.3.2.1. Effect of the Polymer Composition

The composition of the polymer defines its nature as a neutral or ionic network and

furthermore, its hydrophilic/hydrophobic characteristics. Neutral and ionic polymeric

hydrogel networks have been discussed in Section 1.1.1. Presence of hydrophilic

components in the polymer network enhances the swelling characteristics of the polymer.

Hydrophobic components on the other hand reduce the swelling efficiency [175,192].

1.4.2.3.2.2. Effect of the Crosslinking Density

Increase in crosslinking density through addition of crosslinking agents such as divinyl

glycol (DVG), divinyl benzene (DVB) or tripropyleneglycol diacrylate (TPGDA) are

known to reduce the equilibrium swelling [6,8]. Reduced swelling is often marked with

reduced diffusion coefficient. Lee et al [193] in their study on the diffusion coefficients in

crosslinked PHEMA hydrogels found a reduction in the diffusion coefficient values with

increased crosslinking density.

1.4.2.3.2.3. Effect of the Environmental pH

Ionic hydrogels, which could be cationic, containing basic functional groups or anionic,

containing acidic functional groups, have been reported to be very sensitive to changes in

the environmental pH [2,26,27,88,175,186]. The swelling properties of the ionic

hydrogels are unique due to the ionization of their pendent functional groups [175]. The

equilibrium swelling behaviour of ionic hydrogels containing acidic and/or basic

functional groups is illustrated in Figure 28 [175].

Ng et al [26] in their studies on cationic rich polyampholytes found increased swelling

activity in acidic conditions and reduced swelling in basic conditions. Brannon-Peppas

and Peppas [27] on the other hand studied the swelling behaviour of pH sensitive anionic

hydrogels based on HEMA, methacrylic acid and maleic anhydride in varied pH

environments. They reported low swelling activity of the hydrogels in acidic medium but

very high swelling activity in basic medium.

48


Figure 27. Equilibrium degree of swelling in response to pH

Most useful pH-sensitive polymers swell at high pH values and collapse at low pH

values, the triggered drug delivery occurs upon an increase in the pH of the environment.

Such materials are ideal for systems such as oral delivery, in which the drug is not

released at low pH values in the stomach but rather at high pH values in the upper small

intestine [26,165].

1.4.2.3.2.4. Effect of the Environmental Temperature

Changes in the environmental temperature may either enhance the swelling ability of the

hydrogel or in contrary could cause the hydrogel to collapse. Physical gels that contain

hydrophilic components exhibit enhanced swelling behaviour at elevated temperatures

and are referred to as thermo-swelling gel [194]. However, gel networks composed of

relatively hydrophobic components shrink at elevated temperatures. These networks are

referred to as thermoshrinking networks [195]. Thermoshrinking gels undergo reversible

swelling and de-swelling in response to changes in environmental temperature [1,195].

Hoffman et al [196] in their studies on thermo-responsive hydrogels based on N-

isopropyl acrylamide and methacrylic acid found that the gels shrunk at elevated

temperatures but swelled to equilibrium at low temperatures. According to their study the

process was reversible and they suggested the existence of a low critical solution

temperature (LCST). The temperature, which induces the polymer to collapse, is referred

to as the LCST [164,175,197]. Tanaka [197] has used the thermodynamic approach to

49


explain this behaviour. An increased swelling activity is observed at temperatures lower

than the LCST and the collapse of the hydrogel is observed above the LCST. Thermal

responsive hydrogels and membranes have been extensively evaluated as platforms for

pulsatile delivery of drugs [25,164,188]

1.4.2.3.2.5. Effect of the Ionic Strength

According to the concept of Donnan equilibrium, an increase in the ionic strength of the

swelling agent increases the ionization of a weakly polyelectrolytes system thus leading

to high swelling activity [26,175]. However, once the ionic hydrogel has been fully

ionized, further increase in the ionic content of the swelling agent will cause the hydrogel

to de-swell due to the screening effect of the counterions [187]. Anionic gels are normally

unionized at a pH lower than the gel pKa while cationic gels display the opposite

behaviour and the pH is dependent on the pKb of the gel [175].

Khare and Peppas [187] in their study on anionic hydrogels observed a decrease in the

swelling activity upon a further increase in the ionic strength of the swelling agent at a

constant pH higher than the pKa of the gel. According to a recent study by Ostroha et al

[84], the dependence of the swelling behaviour on the ionic strength is significant when

the operational pH is close to the transitional value of the degree of swelling.

1.4.2.3.2.6. Effect of the Nature and Size of the Drug

The size and the nature of the incorporated drug play a very important role in determining

the efficiency of its release from the carrier. Yasuda et al [198,199] found a linear

dependence of the solute diffusion coefficient in the swollen polymer system on the

molecular size of the solute and the reciprocal of the degree of swelling. An increase in

the molecular size of the drug reduces the drug release rate [6,168].

Ng et al [26] in their recent study on the release rate of the model drugs vitamin B1 and

vitamin B in ionic hydrogels showed that the nature of the drug also affects the release

properties of the carrier. They suggest that the coulombic interactions between the

charges borne by the vitamin and the hydrogel matrices are also influential in the release

pattern of the drugs.

50


1.5. Testing of Biomedical Polymeric Hydrogels

Biomaterials should perform with appropriate host response in a specific application

without toxic, inflammatory, carcinogenic and immunogenic responses [200,201]. An

appropriate response of the biomaterial for its particular application would be referred to

as an inert or positive interaction with the host [156]. Toxicology testing of biomaterials

generally includes examination of the local tissue response, systematic toxicological

response, and allergic, pyrogenic, carcinogenic and teratogenic responses [202].

The local tissue cells are usually tested for toxicity activity of the given biomaterial. A

cell proliferation assay is normally conducted to determine the cell viability, which

indicates whether or not the material for intended biomedical use is biocompatible.

Mosmann [203] developed a quantitative colorimetric assay for mammalian cell survival

and proliferation using a tetrazolium salt, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl

tetrazolium bromide (MTT). The assay has been widely accepted as a better alternative to

the previously used radioactive assays, which made use of hazardous radioactive isotopes.

Furthermore, the technique using the tetrazolium salt reflects metabolic activity rather

than cell division thus providing a new approach for the study of cell function [204].

NN

+

NN

S

N N

N NH

N

S

N

e-

MTT (yellow crystals) Formazan (purple crystals)

Scheme 16. Reductive mechanism in the formazan formation

The MTT assay is based on the conversion of yellow water-soluble MTT salt to water-

insoluble purple formazan crystals in the presence of live cells by the reductive cleavage

51


of the tetrazolium ring [203,204] as illustrated in Scheme 16. The exact cellular reactions

involved in the reduction of the salt are not clearly understood to date. However, a

number of reaction models have been proposed. The widely accepted assumption of the

reduction process is based on the study carried out by Slater et al [205]. They suggest that

the mitochondrial succinate dehydrogenase in living cells reduces the MTT to formazan.

The amount of the formazan generated is directly proportional to the live cell numbers

over a wide range, using a homogeneous population. Mosmann [203] also found that the

activated cells produce more formazan than the resting cells, which allowed the

measurement of activation even in the absence of cell proliferation. Gerlier and

Thomasset [204] in their study on cell activation have also reported concurrent findings.

Furthermore, the MTT assay could be used for cell viability assessment of all cell types.

1.6. Research Insight

Polymeric hydrogels, since their discovery and introduction into the biomedical arena in

the early 1960s have been of great research interest [1,2]. Numerous researchers around

the globe have carried out extensive research on intelligent polymeric hydrogels and this

has resulted in some very classical and important developments in such materials.

However, there still seems to be an infinite range of possible applications of these

versatile materials. Thus there remains an everlasting quest to achieve superiority over

present hydrogel systems in terms of biocompatibility, mechanical strength, response to

environment and economy to meet the requirements of such applications. These versatile

materials have found numerous important medical and pharmaceutical applications.

One such area of application of great interest has been that of swelling-controlled drug

release systems. Much of the research on hydrogels has been focussed on application in

controlled drug delivery systems, as these are more effective, more patient friendly and

more cost effective towards a particular treatment in comparison to pills and injections,

which are the conventional drug administration methods [1-8]. However, the need for

cheaper, more responsive and more biocompatible substitute drug delivery systems

continues.

52


1.6.1. Research Direction

The direction of the research in this Ph.D. project will be geared towards obtaining

hydrogels for slow drug delivery applications through an economical and efficient

polymerisation process. These hydrogels would possess enhanced properties such as

mechanical strength, swelling-drug release properties and biocompatibility. Hydrogels

will be prepared from a range of monomers, some with specific functional groups, which

contribute to responses to changes in environmental conditions such as pH, temperature

and ionic strength. The use of UV radiation to achieve polymerisation of hydrogels is a

widely used radiation technique today, which is largely due to its ease of operation,

economy and environmental friendliness.

However, in order to proceed efficiently with the polymerisation process, UV curable

systems normally require the presence of a photoinitiator in the reacting monomer

mixture. A photoinitiator is a photosensitive chemical, which is converted into reactive

radicals upon exposure to the UV light. Photoinitiators besides being costly if not

completely utilized in the polymerisation process can lead to undesirable toxic impurities

trapped in the polymer matrix that may leach out of the matrix in a biomedical

application. This has been a major issue pointed by a number of research publications

[110,122] and this will be addressed in this research project through the formation of

charge-transfer complexes. This process is photoinitiator-free.

This project will involve the study of the swelling-controlled release systems synthesized

via UV radiation in the presence and absence of photoinitiators. NVP and HEMA are the

monomers of interest in this study for the synthesis of hydrogels. PHEMA has been

widely used as controlled release devices [51] due to its high level of biocompatibility. A

good reason for combining NVP, a highly hydrophilic monomer and HEMA is to give

high water absorbing hydrogels [52], which makes them suitable for rapid drug delivery

systems. In this study, photoinitiator-free UV curable systems, which involve electron

donor/acceptor type monomers, will include acrylic acid, HEMA and N-hydroxyalkyl

maleimides as acceptors and NVP as a donor used to form charge-transfer complexes,

which generate initiating radicals essential for the polymerisation process.

53


A series of N-hydroxyalkyl maleimides namely, HMMI, HEMI, HPrMI and HPMI will

be used as acceptor type monomers and will be photopolymerised in conjunction with

NVP, a donor monomer in the absence of a photoinitiator. IPNs involving chitosan and its

derivative carboxymethyl chitosan will also be prepared through the PI-free

polymerisation technique. The resultant polymers will be investigated in vitro for their

biocompatibility.

The Differential Photocalorimetric technique will be used to evaluate the suitability of the

donor/acceptor pairs for the formation of CT complexes. The effect of hydrogen donors

such as glucose and glucosamine hydrochloride on the efficiency of CT complex

formation will also be investigated.

Hydrogels synthesized through the conventional photopolymerisation process in presence

of a photoinitiator and through the CT complex polymerisation will be tested for their

water uptake and drug release capabilities. The swelling and drug release tests on these

hydrogels will be conducted in varied pH environments to evaluate the effect of pH

changes on the swelling and drug release behaviour. A number of model drugs with

varying molecular weights will be utilized for the drug release experiments. The kinetics

of drug release from the various hydrogel networks and the effect of the molecular weight

of the incorporated drug on the release kinetics will be investigated.

The hydrogels prepared are intended for biomedical applications as implants or

transdermal controlled drug release devices thus there is a need for these materials to

satisfy the definition of being biocompatible. Hydrogels are known to be biocompatible

owing their dynamic structures. However, these materials are prepared via UV radiation

where the conversion of monomers to polymer networks is never 100 %. Thus the

possibility of some unreacted monomers contained in polymer network is inevitable.

These unreacted components could be highly toxic thus unsuitable for biomedical

applications. The hydrogels prepared will be cleaned thoroughly and subjected to 3-[4,5-

dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) cell proliferation assay

using human keratinocyte (HaCaT) cells to confirm the inertness or positive response of

the hydrogels to the host cells.

54


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1.1. Synthetic Hydrogels 1

1.1.1. Classification of Hydrogels 3

1.1.1.1. Homopolymeric Hydrogels 3

1.1.1.2. Copolymeric Hydrogels 4

1.1.1.3. Interpenetrating Polymer Network (IPN) Hydrogels 5

1.1.1.4. Non-Ionic Hydrogels 6

1.1.1.5. Ionic Hydrogels 7

1.1.1.5.1. Anionic Hydrogels 8

1.1.1.5.2. Cationic Hydrogels 8

1.1.1.5.3. Polyampholytic Hydrogels 9

1.1.1.6. Hydrogel Network Structures 10

1.1.1.6.1. Amorphous Hydrogel Structures 12

1.1.1.6.2. Semicrystalline Hydrogel Structures 12

1.1.1.6.3. Hydrogen Bonded Hydrogel Structures 13

1.2. Synthesis of Polymeric Hydrogels 13

1.2.1. Chain Initiation 14

1.2.2. Chain Propagation 15

1.2.3. Chain Termination 15

1.2.4. Nature of the Reactive Radical Species 16

1.2.5. Curing Processes 17

1.2.5.1. Ionizing Radiation Sources 17

1.2.5.1.1. Electron Beam (EB) Radiation Process 17

1.2.5.1.2. Gamma Radiation Process 18

1.2.5.2. Ultra Violet (UV) Radiation Process 18

1.2.6. Charge-Transfer (CT) Complex Polymerisation 19

1.2.6.1. Charge-Transfer (CT) Interactions 20

1.2.6.1.1. Decay Mechanisms of Excited State CT Complexes 22

1.2.6.2. Inducement of CT Complex Polymerisation 24

1.2.6.3. Proposed Mechanisms of CT Complex Polymerisation 25

1.2.6.4. Influential Factors in CT Complex Polymerisation 26

1.2.6.4.1. Effect of Monomers 27

1.2.6.4.2. Effect of Lewis Acids 28

1.2.6.4.2. Effect of Hydrogen Donors 28

1.2.6.5. Polymeric Hydrogel Synthesis via CT Complex Formation 29

1.3. Applications of Hydrogels 30

1.3.1. Agricultural Applications 30

1.3.2. Other Applications 31

1.4. Hydrogels as Biomaterials 32

1.4.1. Sustained Drug Delivery Devices 33

1.4.2. Mechanisms of Controlled Drug Delivery 36

1.4.2.1. Diffusion-Controlled Release 37

1.4.2.2. Drug Release Through Biodegradation 38

1.4.2.3. Swelling-Controlled Release Systems 39

1.4.2.3.1. Solute Transport in Swelling-Controlled Release Systems 40

1.4.2.3.1.1. Fick’s Laws of Diffusion 41

66


1.4.2.3.1.2. Fickian and Non-Fickian Diffusion 42

1.4.2.3.1.3. Dimensionless Analytical Parameters 44

1.4.2.3.2. Influential Factors in Swelling-Controlled Release Systems 46

1.4.2.3.2.1. Effect of the Polymer Composition 48

1.4.2.3.2.2. Effect of the Crosslinking Density 48

1.4.2.3.2.3. Effect of the Environmental pH 48

1.4.2.3.2.4. Effect of the Environmental Temperature 49

1.4.2.3.2.5. Effect of the Ionic Strength 50

1.4.2.3.2.6. Effect of the Nature and Size of the Drug 50

1.5. Testing of Biomedical Polymeric Hydrogels 51

1.6. Research Insight 52

1.6.1. Research Direction 53

1.7. References 55

67

Chapter 2: Experimental

2.1. Materials

In this research work, chemicals of high purity were utilized as received from the

suppliers with the exception of two monomers, which were further purified. These were

N-vinyl-2-pyrrolidinone (NVP) and 2-hydroxyethyl methacrylate (HEMA). HEMA was

purified by passing through an inhibitor remover column supplied by Aldrich to remove

the stabilizer hydroquinone while NVP was distilled off at 95 oC

under vacuum of 7 mm

Hg. The materials used in the experimental work and their suppliers are listed in Table 1.

The chemicals required for the synthesis of a series of N-hydroxyalkyl maleimides,

carboxymethyl chitosan and a model drug, manganese-5, 10, 15, 20-tetrakis(4-

hydroxyphenyl) porphyrin are also included in Table 1.

Table 1. Materials list and respective suppliers

Monomers

N-vinyl caprolactam (98%) Aldrich

N-vinyl-2-pyrrolidinone (99%) Sigma

2-hydroxyethyl methacrylate (98%) Sigma

Acrylic acid (99%) Sigma

Polysaccharide

Chitosan (85 % deacetylation) Sigma

Hydrogen Donors

D(+)-Glucose Sigma

D-Glucosamine hydrochloride Sigma

Photoinitiator

Bis(2,4,6-trimethylbenzoyl)-phenylphosphineoxide (Irgacure® 819) Ciba-Geigy

66


Chemical Reagents- Buffer preparation

Disodium hydrogen orthophosphate (Na2HPO4) (A.R. grade) Ajax Chemicals

Sodium dihydrogen orthophosphate (NaH2PO4) (A.R. grade) BDH

Phosphoric acid (A.R grade) BDH

Chemical Reagents - Synthetic work

Maleic anhydride (95%) Sigma

Maleimide (99%) Aldrich

Formaldehyde 37 wt.% Aldrich

3-Amino-1-propanol (99%) Aldrich

5-Amino-1-pentanol (95%) Aldrich

Ethanolamine (98%) Aldrich

Furan (98%) Sigma

Manganese chloride (A.R grade) Ajax chemicals

Monochloro acetic acid (A.R. grade) BDH

Sodium hydroxide (97%) Aldrich

5, 10, 15, 20 tetrakis(4-hydroxyphenyl)-21H, 23H-porphine (99%) Aldrich

67


Solvents

Dichloromethane (A.R grade) BDH

Chloroform (A.R grade) BDH

Chloroform-d (CDCl3) (A.R grade) Aldrich

Deuterium oxide (D2O) (A.R grade) Aldrich

Acetone (A.R grade) APS Chemicals

Methanol (A.R grade) BDH

Ethanol (A.R grade) BDH

Ethyl acetate (99.5 + %) Aldrich

Levulinic acid (98%) Sigma

Cremophor EL (CRM) Sigma

1,2-propanediol (Prg) Sigma

Toluene (L.R grade) BDH

Petroleum ether (A.R grade) Ajax chemicals

Model Drugs

Theophylline Sigma

Thiamine hydrochloride (vitamin B1) Aldrich

Uranyl Actinometer – Lamp calibration reagents

Uranyl nitrate [UO2 (NO3)2. 6H2O] (A.R grade) Ajax chemicals

Oxalic acid (COOH)2 (99 + %) Aldrich

Potassium permanganate (KMnO4) (A.R grade) BDH

Sulphuric acid (A.R grade) BDH

68


Biological Reagents- Cytotoxicity test

HaCaT cells Skin Tech, UWS

Fetal calf (bovine) serum JRH Biosciences

Dulbecco's modified Eagle's medium (DMEM) - Ca2+

free Thermo Trace

DMEM – w/o phenol red Thermo Trace

L-Glutamine Sigma

Penicillin/streptomycin (5000U/ml, 5000ug/ml) Thermo Trace

Anhydrous isopropanol Sigma

Hydrochloric acid (HCl) BDH

Ethylenediamine tetraacetic acid Sigma

Dulbecco's phosphate buffered saline Thermo Trace

Trypsin Sigma

D-Glucose Sigma

EDTA Disodium Sigma

Potassium chloride (KCl) Sigma

Sodium bicarbonate (NaHCO3) Sigma

Sodium chloride (NaCl) Sigma

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) Sigma

N-[2-hydroxyethyl] piperazine-N-[2-ethane sulfonic acid] (HEPES) Sigma

69


2.2. Equipment

2.2.1. Radiation Source

A 90 W high-pressure mercury vapour filled lamp, manufactured by Phillips (Holland),

was used as the ultra violet (UV) light source. The lamp was mounted at the centre of an

enclosed drum, which contained a circular rack fitted with a rotor to hold six Pyrex

sample tubes, each with a diameter of 2 cm and a height of 10 cm at a distance of 10 cm

from the lamp. The drum had air vents at the bottom and was also fitted with a fan to

control excessive heat. The energy absorption per sample was determined by an uranyl-

oxalate actinometer.

The calculations were made on the assumption that the energy of all the wavelengths

between 254 nm and 435 nm was absorbed completely. An average wavelength of 350

nm and an average quantum yield value of 0.57 were used in the calculations to determine

the dose rate [1]. The UV lamp calibration procedure is outlined in detail in Section 2.4.4.

The calculated energy flux value from the calibration was 9.65 x 10-2

J s-1

. This dose rate

value was used to determine the radiation dose applied to the monomer mixture samples

to achieve the formation of polymers which function as hydrogels.

2.2.2. Analytical Instruments

2.2.2.1. Ultra Violet - Visible (UV-Vis) Spectrophotometer

A Shimadzu UV-1601 PC spectrophotometer was used as the quantitative analytical tool

in the drug release experiments. The samples were measured in quartz cuvettes with a cell

length of 1 cm. The spectroscopic measurements were carried out in the range of 800 nm

to 200 nm with a slit width value of 2.0 nm. Calibration curves of the standards of each of

the drugs analysed were plotted and used for drug release calculations.

2.2.2.2. Gas Chromatograph Mass Spectrometer (GC-MS)

Low-resolution mass spectra were recorded on a Shimadzu QP5000/GC17A equipped

with an MS ion trap detector. Solid samples were dissolved in CH2Cl2 and subsequently

admitted to the ion source using a direct insertion probe. The probe had at its tip a small

container in which a small amount of sample solution was placed. The probe was inserted

through a vacuum lock to within a few millimetres of the ion beam, where precise and

70


controllable internal heating was used to gently evaporate the sample with minimum

decomposition.

2.2.2.3. Nuclear Magnetic Resonance (NMR) Spectrometer

2.2.2.3.1. Carbon-13 (13

C) and Proton (1H) NMR

NMR spectroscopic analysis of liquid samples was performed on Varian unity-plus 300

MHz and Mercury 400 MHz spectrometers. Solid samples were either dissolved in CDCl3

or D2O. Chemical shifts were recorded in ppm. 1H and

13C{

1H} spectra were referenced,

internally to the residual solvent peak. The 13

C and 1H spectra in D2O were referenced

with respect to TSP dissolved in D2O.

2.2.2.3.2. Proton NMR Relaxation (T1 and T2) Measurements

Proton NMR relaxation (T1 and T2) measurements were conducted on Varian unity-plus

300 MHz NMR spectrometer equipped with micro-imaging accessory. A micro-imaging

probe was employed for these measurements. The swollen hydrogel samples were

inserted into the micro-imaging probe with 30 mm insert. T1 measurements were

performed using inversion recovery technique while T2 measurements were performed

employing spin-echo technique with CPMG pulse sequence.

2.2.2.4. Fourier Transform Infrared (FT-IR) Spectrometer

Fourier Transform Infrared spectroscopic analysis was conducted on a BIO-RAD FTS

3000 MX FT-IR spectrometer. Potassium bromide (KBr) discs containing samples to be

analysed were prepared by mixing 0.5 – 1.0 mg of the sample with approximately 100 mg

of powdered KBr. The mixture was ground before being compressed in a special metal

KBr die under pressure of 15 - 30 tonnes to produce transparent KBr discs. Merlin,

Version 1.2 was the analytical software used in conjunction with the FT-IR to process the

data. The sampling range was between 4000 cm-1

and 400 cm-1

with an average of 16

scans per sample.

2.2.2.5. Differential Photocalorimeter (DPC)

A Differential Photocalorimeter/DSC 2910 system by TA Instruments was used for

kinetic studies on CT complex formation of various donor/acceptor systems. Samples

71


were placed in a pre-weighed DSC aluminium pan using a micro-syringe, accurately

weighed to an approximate sample size of 2 mg. The aluminium pans were specially

crimped to maintain uniform sample size. The DSC 2910 system is equipped with a 200

W mercury arc lamp with a variable light intensity of 1 - 56 mW cm-2

. Light intensity was

measured with an IL 1440-A Radiometer supplied by International Light. Samples were

degassed in the DSC for 2 minutes prior to irradiation under a measured light intensity of

55.8 mW cm-2

in the presence of N2. Thermal Solutions software was used to operate the

instrument while Universal Analysis software, Version 2.5 was used to process the

acquired data.

2.2.2.6. Texture Analyser (TA)

A TA.XT2 texture analyser from Stable Micro Systems was used to evaluate the stiffness

and the viscoelasticity of the swollen hydrogel networks. A 1/2" ebonite cylinder was

used as the probe. The hydrated samples were in a cylindrical shape. The height and the

diameter of the samples were recorded prior to placement under the probe. The probe was

set to approach the sample at 1.0 mm s-1

with a trigger force of 0.01 N. Once the probe

was in contact with the sample, the test duration was 30 seconds with increase in

compression distance from 0-1.0 mm at a rate of 0.1000 + 0.0001 mm s-1

in the first 10

seconds and maintained compression distance of 1.0 mm in the later 20 seconds. A

compression stress-strain graph was obtained for each sample.

2.2.2.7. Atomic Absorption Spectrometer (AAS)

A GBC 902 double beam AAS was used to carry out the metal analysis. The samples

were dissolved in de-ionized water and aspirated into the air/acetylene flame. The

measurements were carried out using a hollow cathode lamp with a wavelength and a slit

width of 279.50 nm and 0.2 nm respectively. A range of standard solutions (1 ppm –10

ppm) of the metal was analysed to construct a calibration curve from which the metal

content in the sample was determined.

2.2.2.8. Microplate Reader

A BMG Labtechnologies FLUOstar OPTIMA microplate reader was used for the cell

proliferation assay. The reader was equipped with a high-energy xenon flash lamp as the

72


light source and a side window, current type photomultiplier tube as the detector.

Absorbance filters A560 and A650 were used as excitation and background filters

respectively. The absorbance measurements were recorded at 560 nm with background

absorbance of 650 nm in a plate mode over a single cycle with 20 flashes per well. The

FLUOstar OPTIMA v1.30-0 software was used to operate the instrument and process the

acquired raw data.

2.2.2.9. Microscope

A Nikon inverted microscope (TMS) equipped with a Nikon digital sight camera unit

(DS-5M-L1) was used for the image analysis of the cell cultures in the cytotoxicity

experiments. Microscopic examinations were carried out at 200 x magnification and the

images were captured on the digital camera.

2.3. Synthesis of Chemical Compounds

A range of chemical compounds used in this work was synthesized using the chemical

reagents described in Table 1. The syntheses and characterization of these compounds are

described in Sections 2.3.1-2.3.3. Detailed reaction mechanisms of the syntheses of these

compounds are illustrated in Appendix V.

2.3.1. Synthesis of a Model Drug

Manganese-5, 10, 15, 20-tetrakis(4-hydroxyphenyl) porphyrin (Mn-TPP-OH), a cancer

tumour-tracing agent was obtained through insertion of Mn into 5, 10, 15, 20 tetrakis(4-

hydroxyphenyl)-21H, 23H-porphine (TPP-OH). The detailed synthesis of Mn-TPP-OH is

described in Section 2.3.1.1. Mn-TPP-OH was used for the drug release studies.

2.3.1.1. Synthesis of Mn-TPP-OH.

TPP-OH (0.30 g, 0.055 mmol) was added to a 50 ml, three-neck, round-bottomed flask

fitted with a reflux condenser under N2 atmosphere, containing distilled CH2Cl2 (20 ml).

The solution was stirred magnetically for 10 minutes. To this, a solution of a manganese

(II) chloride (0.18 g, 0.053 mmol) in methanol (5 ml) was added and the reaction vessel

was shielded from ambient lighting. The flask was immersed in a water bath and the

solution was refluxed overnight. Insertion of the metal into the porphyrin was monitored

73


by UV-vis spectroscopy for completion. De-ionized water (50 ml) was added to the

precipitated crude product. This product was filtered, dissolved in CH2Cl2 and purified

via open column chromatography by passing through a silica column and eluting the

product fraction with CH2Cl2 : MeOH mixture (100:1) as the eluent. The solvents in the

eluted product fraction were removed under reduced pressure yielding 0.19 g (59 %) of

green crystalline solid Mn-TPP-OH (Figure 1).

NN

N N

OH

OH

OH

OH Mn

Figure 1. Mn-TPP-OH

2.3.1.1.1. UV-Vis Spectroscopic Analysis

UV-vis (CH2Cl2) λmax, ε (L mol-1

cm-1

): 610 nm, 5209; 571 nm, 4768; 520 nm, 4842.

2.3.1.1.2. AAS Analysis

Observed Mn composition: 7.40 % w/w

Calculated Mn composition (based on C44H30MnN4O4.H2O): 7.31 % w/w

2.3.2. Synthesis of N-Hydroxyalkyl Maleimides

A series of N-hydroxyalkyl maleimides, which were used as monomers were synthesized

through reverse Diels-Alder reaction of N-hydroxyalkyl maleimide adducts of furan. The

detailed syntheses of the starting materials and the N-hydroxyalkyl maleimides are

described in Sections 2.3.2.1-2.3.2.8.

74


2.3.2.1. Synthesis of HMMI

The N-hydroxymethyl maleimide (HMMI) (Figure 2) was synthesized according to the

method described by Tawney et al [2]. Maleimide (10 g, 0.103 mol) was added to 10 ml

of a 37 % solution of formaldehyde and heated to approximately 35 oC and 0.31 ml of a 5

% solution of NaOH was added. Within 10 minutes all of the maleimide had dissolved

and an exothermic reaction proceeded. The solution was stirred for 2 hours where white

crystals were observed after cooling to room temperature.

N

O

O

OH

ab c

Figure 2. HMMI

The solution was placed in a freezer overnight and the resulting crystals were filtered and

washed with ice-cold ethanol and diethyl ether. The crude product was purified twice by

sublimation yielding HMMI (9.77 g, 74.6 %) as white crystals, m.p. 104 oC (lit. 104-106

oC). The purity of the product was further evaluated using NMR and mass spectroscopy.

2.3.2.1.1. Mass Spectroscopic Analysis

M.S. (molecular ion, m/z (peak intensity)

Observed: M+ and (M+1)

+ were not observed.

Calculated (C5H5NO3): 127.10

MS fragmentation, m/z (peak intensity): 99.15 (23.87, (M – 28); 80.15 (10.93, (M – 47));

55.10 (25.11, (M – 66)); 54.10 (100.00, (M – 73)); 53.10 (49.11, (M – 74)).

2.3.2.1.2. NMR Spectroscopic Analysis

1H NMR (D2O): δ ppm 6.79 (s, 2H, Ha); 4.88 (s, 2H, Hc)

13C NMR (D2O): δ ppm 172.26 (Cb); 135.31 (Ca); 59.73 (Cc).

75


2.3.2.2. Synthesis of Furan-A

The 3, 6-endoxo-1, 2, 3, 6-tetrahydrophthalic anhydride (Furan-A) (Figure 3) was

synthesized according to the method described by Narita et al [3]. Maleic anhydride (20

g, 20.4 mmol) and ethyl acetate (25.5 ml) were mixed in a 100 ml round-bottomed flask.

Furan (17.35 g, 25.5 mmol) was added and the reaction mixture was stirred overnight at

room temperature. The precipitate was filtered and washed with ethyl acetate producing

18.15 g (89 %) of a white solid as the final product, m.p. 107 oC. NMR and mass

spectroscopy were used to confirm the purity of the product.

O

O

O

O

ab

c d

Figure 3. Furan-A





Calculated (C8H6O4): 166.03

MS fragmentation, m/z (peak intensity): 149.10 (0.68, (M – 17); 94.05 (4.14, (M – 72));

69.05 (5.59, (M – 80)), 68.00 (100.00, (M – 98)).


1H NMR (CDCl3): δ ppm 6.56 (s, 2H, Ha); 5.44 (s, 2H, Hc); 3.15 (s, 2H, (Hb).

13C NMR (CDCl3): δ ppm 172.9 (Cd); 137.0 (Ca); 82.2 (Cb); 48.7 (Cc).

2.3.2.3. Synthesis of HEMI-A

The synthesis of 2-hydroxy-N-ethyl-3, 6-endoxo-1, 2, 3, 6-tetrahydrophthalimide (HEMI-

A) (Figure 4) was carried out in accordance to the method described by Narita et al [3].

Ethanolamine (4.2 g, 6.17 mmol) in ethanol (5 ml) was added drop-wise to a slurry of

Furan-A (10 g, 6 mmol) in ethanol (15 ml) in a 50 ml round-bottomed flask. This mixture

76


was then refluxed for 4 hours. After cooling to room temperature overnight, a white solid

had formed. The crude product was filtered and washed with ethanol followed by

petroleum ether yielding 7.27 g (58 %) of a white crystalline solid as the final product,

m.p. 132 oC (lit. 132

oC). NMR and mass spectroscopy were used to further evaluate the

purity of the product.

N

O

O

O

OH

a

bc d

e

f

Figure 4. HEMI-A






MS fragmentation, m/z (peak intensity): 110.10 (5.56, (M – 99)); 82.10 (3.60, (M – 127)),

68.00 (100.00, (M – 141)).


1H NMR (CDCl3): δ ppm 6.50 (s, 2H, Hb); 5.26 (s, 2H, Ha); 3.75, 3.68 (m, 2H, Hf, He);

2.87 (s, 2H, Hc); 2.20 (br s, 1H, OH).

13C NMR (CDCl3): δ ppm 176.8 (Cd); 136.5 (Ca); 81.0 (Cb); 60.4 (Cf); 47.5 (Cc); 41.8

(Ce).

2.3.2.4. Synthesis of HEMI

The 2-hydroxy-N-ethyl maleimide (HEMI) (Figure 5) was synthesized according to the

method described by Shigeyoshi [4]. HEMI-A (5.8 g, 2.78 mmol) was refluxed in toluene

(40 ml) under a N2 atmosphere overnight in a 100 ml round-bottomed flask. After cooling

the reaction mixture overnight, the precipitate was filtered and washed with petroleum

ether. The crude product was purified twice by sublimation yielding HEMI (3.25 g, 83 %)

77


as white crystals, m.p. 72 oC (lit. 72

oC). The purity of the product was further evaluated

using NMR and mass spectroscopy.

N

O

O

OH

a bc

d

Figure 5. HEMI



Observed: 141.15 (1.11); 142.15 (0.68).

Calculated (C6H7NO3): 141.04 (1.11); 141.12 (0.81).

MS fragmentation, m/z (peak intensity): 110.10 (85.04, (M – 31)); 82.05 (88.08, (M –

59)), 54.10 (100.00, (M – 87)).


1H NMR (D2O): δ ppm 6.73 (s, 2H, (Ha)); 3.76, 3.72 (m, 2H, Hc, Hd); 2.13 (t, 1H, J(HH)

5.4 Hz, OH).

13C NMR (D2O): δ ppm 171.1 (Cb); 134.2 (Ca); 60.8 (Cd); 40.6 (Cc).

2.3.2.5. Synthesis of HPrMI-A

Synthesis of 3-hydroxy-N-propyl-3, 6-endoxo-1, 2, 3, 6-tetrahydrophthalimide (HPrMI-

A) (Figure 6) was carried out in accordance to the method described by Narita et al. [3]

with the exception of using ethanolamine. Instead, 3-amino-1-propanol (7.0 g, 93 mmol)

in ethanol (10 ml) was added drop-wise to a slurry of Furan-A (15.0 g, 90 mmol) in

ethanol (25 ml) in a 50 ml round-bottomed flask. This mixture was then refluxed for 4

hours. After cooling the reaction mixture overnight, a white precipitate had formed. The

crude product was filtered and washed with ethanol followed by petroleum ether yielding

10.27 g (51 %) of a white crystalline solid as the final product, m.p. 117 oC. Mass

spectroscopy and NMR were used to confirm the purity of product.

78


N

O

O

O

OH

ab

c de

f

g

Figure 6. HPrMI-A






MS fragmentation, m/z (peak intensity): 110.10 (85.04, (M – 31)); 125.20 (2.21, (M –

80)), 82.10 (3.08, (M – 123)), 68.05 (100.00, (M – 155)).


1H NMR (CDCl3): δ ppm 6.51 (s, 2H, Ha); 5.26 (s, 2H, Hb); 3.64 (t, 2H, J(HH) 6.2 Hz, Hg);

3.50 (t, 2H, J(HH) 5.7 Hz, He); 2.86 (s, 2H, (Hc)); 2.48 (br s, 1H, OH); 1.75 (quintet, 2H,

J(HH) 6.2 Hz, Hf).

13C NMR (CDCl3): δ ppm 179.7 (Cd); 136.5 (Ca); 81.1 (Cb); 58.9 (Cg); 47.5 (Cc); 35.9

(Ce); 29.4 (Cf).

2.3.2.6. Synthesis of HPrMI

The 3-hydroxy-N-propyl maleimide (HPrMI) (Figure 7) was synthesized according to the

method described by Shigeyoshi [4]. HPrMI-A (5.0 g, 22.4 mmol) was refluxed in

toluene (40 ml) under a N2 atmosphere for 96 hours in a 100 ml round-bottomed flask.

The reaction mixture was allowed to cool overnight and then the crude product was

filtered and washed with petroleum ether, which gave the final product as a white

crystalline solid with a yield of 70 % (2.43 g). The purity of the product was evaluated

using NMR and mass spectroscopy.

79


N

O

O OH

a bc

de

Figure 7. HPrMI



Observed: 155.10 (0.87); (M+1)+ was not observed.


MS fragmentation, m/z (peak intensity): 137.10 (22.44, (M – 18)); 110.00 (91.35, M –

45)), 82.05 (65.30, (M – 73)), 54.05 (100.00, (M – 101)).


1H NMR (D2O): δ ppm 6.74 (s, 2H, Ha); 3.48 (m, 4H, Hc, He); 1.69 (quintet, 2H, J(HH) 6.6

Hz, Hd).

13C NMR (D2O): δ ppm 173.4 (Cb); 134.5 (Ca); 59.1 (Ce); 34.7 (Cc); 30.3 (Cd).

2.3.2.7. Synthesis of HPMI-A

Synthesis of 5-hydroxy-N-pentyl-3, 6-endoxo-1, 2, 3, 6-tetrahydrophthalimide (HPMI-A)

(Figure 8) was carried out in accordance to the method described by Narita et al [3] with

the exception of using ethanolamine. Instead, 5-Amino-1-pentanol (7.6 g, 7.35 mmol) in

ethanol (15 ml) was added drop-wise to a slurry of Furan-A (5 g, 7.13 mmol) in ethanol

(15 ml) in a 50 ml round-bottomed flask. This mixture was then refluxed for 4 hours.

After cooling the reaction mixture overnight, a white precipitate had formed. The crude

product was filtered and washed with ethanol followed by petroleum ether yielding 8.98 g

(50 %) of a white crystalline solid as the final product. The purity of the product was

evaluated using NMR and mass spectroscopy.

80


N

O

O

O

OH

ab

c de

f

g

h

i

Figure 8. HPMI-A






MS fragmentation, m/z (peak intensity): 183.15 (4.30, (M – 68); 165.10 (1.85, (M – 18)),

121.15 (1.17, (M – 62)), 82.05 (68.10, (M – 169)); 68.05 (100.00, (M – 183)).


1H NMR (CDCl3): δ ppm 6.48 (s, 2H, Ha); 5.24 (s, 2H, Hb); 3.58 (t, 2H, J(HH) 6.4 Hz, Hi);

3.46 (t, 2H, J(HH) 7.2 Hz, He); 2.81 (s, 2H, (Hc); 1.48-1.54 (m, 4H, (Hf, Hh)); 1.31 (m, 2H,

Hg).

13C NMR (CDCl3): δ ppm 176.32 (Cd); 136.48 (Ca); 80.90 (Cb); 62.45 (Ci); 47.33 (Cc);

38.73 (Ce); 32.01 (Ch); 27.18 (Cf); 22.64 (Cg).

2.3.2.8. Synthesis of HPMI

The 5-hydroxy-N-pentyl maleimide (HPMI) (Figure 9) was synthesized according to the

method described by Shigeyoshi [4]. HPMI-A (5.0 g, 1.99 mmol) was refluxed in toluene

(40 ml) under a N2 atmosphere for 48 hours in a 50 ml round-bottomed flask. The

reaction mixture was allowed to cool overnight and then precipitate was filtered and

washed with petroleum ether. The crude product was purified twice by sublimation

yielding HPMI (2.73 g, 75 %) as white crystals, m.p. 51 oC. NMR and mass spectroscopy

were used to confirm the purity of the product.

81


N

O

O

OH

a bc

d

e

f

g

Figure 9. HPMI



Observed: 183.15 (3.23); (M+1)+ was not observed.


MS fragmentation, m/z (peak intensity): 153.15 (3.83, (M – 30)), 110.05 (100.00, (M –

73)), 82.05 (4.20, (M – 101)).


1H NMR (D2O): δ ppm 6.71 (s, 2H, Ha); 3.42 (t, 2H, J(HH) 6.6 Hz, Hg); 3.36 (t, 2H, J(HH)

7.0 Hz, Hc); 1.50-1.30 (m, 4H, Hd, Hf); 1.17 (quintet, 2H, J(HH) 7.1 Hz, He).

13C NMR (D2O): δ ppm 176.3 (Cb); 137.1 (Ca); 64.4 (Cg); 40.4 (Cc); 33.6 (Cd); 30.2 (Cf);

25.2 (Ce).

2.3.3. Water-Soluble Derivative of Chitosan

A water-soluble derivative of chitosan, carboxymethyl (CM) chitosan was prepared

through partial deacetylation of chitosan at a specific temperature. The detailed synthesis

of the CM chitosan is described in Section 2.3.3.1.

2.3.3.1. Synthesis of CM Chitosan

CM chitosan was synthesized according to the method described by Liu et al [5].

Chitosan (10 g) was added to a 500 ml flask containing sodium hydroxide (13.5 g), and

solvent (100 ml, 1/9: water/isopropanol) to swell and alkalise at ~ 10 oC for 1 hour. The

temperature was maintained in a water bath. Monochloroacetic acid (15 g) dissolved in

82


isopropanol (20 ml), was then added to the reaction mixture drop-wise for 30 minutes and

allowed to react for 4 hours at the same temperature. The reaction was quenched by

adding 70 % ethyl alcohol (200 ml). The solid was filtered and rinsed with 70-90 % ethyl

alcohol to desalt and dewater followed by vacuum drying at room temperature yielding

sodium salt CM chitosan (6.21 g) as the product.

O

CH2OH

OH

NH2

O

CH2OH

OH

NR2

O

O

CH2OH

OH

NHR

O

CH2OH

OH

NHAc

OO O

(R= CH2COOH)

Figure 10. CM Chitosan

Na salt CM chitosan (1 g) was suspended in 80 % ethyl alcohol aqueous solution (100

ml), and hydrochloric acid (10 ml, 37 %) was added with stirring for 30 minutes. The

solid was filtered, rinsed in 70-90 % ethyl alcohol to neutral pH and dried at room

temperature under vacuum. The final product obtained was the N-form of CM chitosan

(Figure 10) [6] as pale brown flakes. The final product was characterized using FT-IR

spectroscopy.

2.3.3.1.1. FT-IR Analysis

3466 cm-1

(broad -OH stretch), 1747 cm-1

(-COOH peak), 1660 and 1540 cm-1

(-NH3+

peak), and 1070-1136 cm-1

(-C-O- stretch).

The FT-IR spectrum is attached in Section 5.3.2.

2.4. UV Lamp calibration

The UV lamps were calibrated on the basis of the amount of energy, (energy flux), given

out per second. The energy flux given out per second was determined using a chemical

actinometer, which involved the decomposition of oxalic acid in the presence of uranyl

nitrate upon exposure to the UV light. Uranyl nitrate and oxalic acid solution samples

83


were exposed to the lamp for varying time intervals. The excited UO22+

species formed

according to Equation 1 decomposes the oxalic acid as shown in Equation 2. The

irradiated samples were then titrated against standardized KMnO4 to determine the

unreacted oxalate ion concentration according to Equation 3.

UO22+

hv (UO22+

)* Equation 1

(UO22+

)* + (COOH) 2 UO2

2+ + H2O + CO2 + CO Equation 2

2 MnO4- + 5(COOH) 2 + 6H

+10CO2 + 8H2O + 2Mn

2+ Equation 3

2.4.1. Preparation of Solutions

2.4.1.1. Oxalic Acid Solution (COOH) 2

Oxalic acid solution (0.02 M) was prepared in a 100 ml volumetric flask by dissolving

accurately weighed oxalic acid (0.180 g, MW. 90.04 g mol-1

) in milli-Q- water. The

volumetric flask was shaken gently until the solid was entirely dissolved and then the

flask was filled up to the mark with milli-Q-water.

2.4.1.2. Uranyl Nitrate Solution (UO2 (NO3) 2. 6H2O)

Accurately weighed hydrated uranyl nitrate (1.005 g, MW. 502.13 g mol-1

) was dissolved

in a 100 ml volumetric flask with milli-Q-water to give approximately a 0.02 M solution

of uranyl nitrate.

2.4.1.3. Potassium Permanganate Solution (KMnO4)

Potassium permanganate is widely used as a titrant in inorganic and organic analysis.

Potassium permanganate solution (0.001 M) was prepared in a 100 ml volumetric flask by

dissolving an accurately weighed KMnO4 (0.016 g, MW. 158.03 g mol-1

) with milli-Q-

water. The KMnO4 solution was standardized by titration with hot acidified oxalic acid at

60-90 oC until an endpoint was detected upon a colour change from a clear colourless

solution to a pink solution.

84


2.4.1.4. Preparation of Sample Solutions

Sample solutions for UV irradiation were prepared in several test tubes, each containing

20 ml of uranyl nitrate and 20 ml of oxalic acid solution. A reference sample solution was

prepared in the same manner.

2.4.2. Irradiation of Samples

The ultraviolet lamp was turned on and equilibrated for 5 minutes prior to placing the test

tubes containing sample solutions prepared as described in Section 2.4.1.1, in the fitted

rotating circular rack around the lamp. Each sample was irradiated for a designated period

of time. The reference sample solution was not exposed to the ultraviolet light.

2.4.3. Analysis of the Irradiated Samples

Aliquots (5 ml) were removed from the irradiated samples along with the reference

sample (described in Section 2.4.2), heated to 60-90 oC, acidified with 2 M sulphuric acid

and titrated against the standardized solution of KMnO4. Oxalic acid that was not

decomposed by UO22+

ion reacted with KMnO4 according to Equation 3. The titration was

that of a redox reaction. Potassium permanganate and oxalic acid were the oxidising and

reducing agents respectively. The titrations were done in duplicates for each irradiated

sample with UV exposure period ranging from 10-90 minutes.

2.4.4. UV Dose Calculation

The UV lamp calibration was carried over 90 minutes to observe the decomposition rate

of oxalic acid at varying exposure times. A plot illustrating the amount of oxalic acid

decomposed as a function of UV exposure time is attached in Appendix IV. Titration

results are listed below in Table 2.

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Table 2. Titration results – KMnO4 consumption

UV exposure

time (mins)

Titre 1 values

(KMnO4) (ml)

Titre 2 values

(KMnO4) (ml)

Average titre values

(KMnO4) (ml)

0 14.43 14.45 14.44

10 10.68 10.72 10.70

20 6.98 7.04 7.01

30 3.99 4.03 4.01

60 0.71 0.73 0.72

90 0.23 0.25 0.24

The UV radiation dose rate calculations however, were based on the sample exposed to

the lamp for 30 minutes. The extent of oxalic acid decomposition was obtained by

comparing the moles of acid in the irradiated sample at time (tx = 30 mins) to that in the

non-irradiated (reference) sample. An energy flux or dose rate value of 9.63 x 10-2

J s-1

was calculated as illustrated in Sections 2.4.4.2 - 2.4.4.5.

2.4.4.1. Decomposition of Oxalic Acid

Mol (COOH)2 tx = mol (COOH)2 to x titre vol [(KMnO4 (to) - KMnO4 (tx)) / KMnO4 (to)]

= (0.02 mol L-1

x 0.02 L) x [(14.44 – 4.01 ml) /14.44 ml]

= 2.89 x 10-4

mol of oxalic acid decomposed

Concentration of oxalic acid: 0.02 mol L-1

Volume of oxalic acid in sample: 0.02 L

Where to = non-irradiated sample; tx = irradiated sample for time x.

2.4.4.2. Number of Einstein’s (s-1

) Required

No: Einstein’s = (Moles of decomposed oxalic acid) / [(time of exposure (s)) x Φ] = (2.89 x 10

-4 mol) / [(30 x 60 s) (0.57)]

= 2.82 x 10-7

Einstein’s s-1

Where Φ = 0.57 (Quantum yield of oxalic acid)

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2.4.4.3. Energy (J)

E = hc/λ E = [(6.626 x 10

-34 J s

-1) (2.998 x 10

8 m s

-1)] / 3.5 x 10

-7 m

E = 5.68 x 10-19

J

Where λ = 350 nm (average energy quanta between 254-435 nm); h = 6.626 x 10-34

J s

(Planks constant) and c = 2.998 x 108 m s

-1 (speed of light).

2.4.4.4. Dose Rate (J s-1

)

Dose rate = (Einsteins (s-1

)) x (avagadro’s number) x (energy J)

= [(2.82 x 10-7

s-1

) (6.022 x 1023

mol-1

) (5.68 x 10-19

J)

= 9.63 x 10-2

J s-1

2.5. Preparation of Hydrogels

An ultra violet radiation source, described in Section 2.2.1 was used for the curing of the

monomer solutions. A wide range of monomers with exceptional chemical properties was

used to synthesize polymeric hydrogels. The monomers used were HEMA, NVP, acrylic

acid (AA), N-vinyl caprolactam (NVC) and a series of N-hydroxyalkyl maleimides,

synthesized as described in Section 2.3.2. Polysaccharides namely, chitosan and its

derivative CM chitosan were also utilized. In this work, majority of the polymerisation

processes were initiated by the donor/acceptor pairs under the influence of the UV source.

Polymerisation of certain monomer systems was also achieved in the presence of a

photoinitiator under the influence of a UV source.

2.5.1. Hydrogels Synthesis Initiated By Photoinitiator (PI)

The monomers used for the synthesis of copolymers with PI included were HEMA and

NVP. Irgacure 819 was the photoinitiator used. The following ratios by volume: 0:5, 1:4,

2.5:2.5, 4:1 and 5:0 of the respective monomers were added to give a total of 5 ml

solution in each test tube to which was added Irgacure 819 (0.005 g, 0.1 % w/v). Two

formulations (5 ml), each containing 10 % v/v of water were prepared in the following

ratios by volume of HEMA: NVP: H2O; 3.6: 0.9: 0.5 and 0.9: 3.6: 0.5 with added PI

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(0.005 g, 0.1 % w/v). Approximately 1ml of each monomer mixture was transferred into

non-pigmented polypropylene straws cut to lengths of ~8 cm with a bore diameter of 0.5

cm and were sealed at one end. Straws were used instead of micro test tubes to avoid

problems associated with having to break glass test tubes to retrieve the polymer gels for

testing. The straws containing the monomer samples were placed in test tubes and then

subjected to radiation as described in Section 2.5.3.

2.5.2. Hydrogels Synthesis via Photoinitiator-Free Process

A number of PI free systems were studied in which the formations of hydrogels were

initiated by the monomers, which functioned as electron donor/acceptor pairs. HEMA,

AA and a series of N-hydroxyalkyl maleimides: HMMI, HEMI, HPrMI and HPMI were

used as electron acceptor monomers combined with NVP, which served as an electron

donor monomer. Interpenetrating polymer networks (IPNs) involving chitosan and its

derivative CM chitosan in conjunction with HMMI, NVP and HEMA were also prepared

using the PI- free technique.

2.5.2.1. Preparation of the N-Hydroxyalkyl Maleimide and NVP Systems

The N-hydroxyalkyl maleimides, HMMI, HEMI, HPrMI and HPMI were each combined

with NVP to form donor/acceptor pairs respectively. All these systems were prepared in

the presence of hydrogen donors, glucose and glucosamine HCl. The monomer solutions

each of an approximate total mass of 2 g containing the respective maleimide (MI) were

prepared in accordance to the following composition: NVP (71.45 % w/w); MI (4.68 %

w/w); H2O (23.37 % w/w); H-donor (0.5 % w/w). H-donor and the respective maleimide

were first dissolved in water followed by the addition of NVP. Each solution was briefly

sonicated to ensure homogeneity in the mixture and was then transferred into clear

polypropylene straws, which were then subjected to UV radiation.

2.5.2.2. Preparation of the HPMI- NVP-HEMA System

A stock solution of HEMA (50 % v/v) and NVP (50 % v/v) was prepared. The HPMI-

NVP-HEMA system was prepared in accordance to the procedure described in Section

2.5.2.1 with the exception of substituting NVP (71.45 % w/w) with the HEMA-NVP

stock solution (71.45 % w/w).

88


2.5.2.3. Preparation of the HPMI- NVP-NVC System

Stock solutions of NVP and NVC were prepared according to the following composition

in % v/v, (NVP:NVC; 20:80; 50:50; 80:20). The HPMI-NVP-NVC systems were

prepared in accordance to the procedure described in Section 2.5.2.1 with the exception of

substituting NVP (71.45 % w/w) with the NVP-NVC stock solutions (71.45 % w/w).

2.5.2.4. Preparation of the IPN, HMMI-NVP-Chitosan System

Chitosan (85 % de-acetylated) derived from crab shells was used for this work. A

chitosan stock solution A was prepared by dissolving chitosan (0.4249 g) in levulinic acid

(0.4238 g, 0.09 M) to which was added 40 ml of milli-Q water. The mixture was stirred

overnight to allow chitosan to dissolve fully in the acidic medium. The same method of

preparation was repeated for a stock solution B containing chitosan dissolved in acrylic

acid. The stock solutions were slightly viscous upon full dissolution of chitosan. Both the

stock solutions, A and B were used in separate similar formulations with the exception of

excluding HMMI in formulations containing stock solution B to form IPNs. The

monomer solutions of an approximate total mass of 2 g were prepared with varied

compositions of NVP and chitosan solution. Mixture A contained HMMI (4.0 % w/w);

NVP (62.4 % w/w) and chitosan stock solution (33.6 % w/w). Mixture B contained

HMMI (4.0 % w/w); NVP (33.6 % w/w) and chitosan stock solution (62.4 % w/w).

Mixture C contained HMMI (4.0 % w/w); NVP (48 % w/w) and chitosan stock solution

(48 % w/w). The solutions were sonicated for homogeneity, transferred into clear

polypropylene straws and subjected to UV radiation as described in Section 2.5.3.

2.5.2.5. Preparation of the IPN, HMMI-NVP-CM Chitosan System

CM chitosan synthesized under controlled physical conditions (Section 2.3.3) is a water-

soluble derivative of chitosan. CM chitosan (0.2 g) was accurately weighed in a beaker to

which was added 20 ml of milli-Q-water. The mixture was stirred overnight to allow

complete dissolution of CM chitosan. The CM chitosan solution, which was slightly

viscous, was used as the stock solution in the preparation of the IPN, HMMI-NVP-CM

chitosan system. The monomer solutions of an approximate total mass of 2 g were

prepared with varied compositions of NVP and CM chitosan solution. Mixture A

contained HMMI (4.0 % w/w); NVP (62.4 % w/w) and CM chitosan stock solution (33.6

89


% w/w). Mixture B contained HMMI (4.0 % w/w); NVP (33.6 % w/w) and CM chitosan

stock solution (62.4 % w/w). Mixture C contained HMMI (4.0 % w/w); NVP (48 % w/w)

and CM chitosan stock solution (48 % w/w). The final monomer mixtures were sonicated,

transferred into clear polypropylene straws and subjected to UV radiation.

2.5.2.6. Preparation of the IPN, HEMA-NVP-Chitosan System

Chitosan stock solutions A and B prepared as described in Section 2.5.2.2 were utilized to

prepare the HEMA-NVP-chitosan IPNs. The monomer mixture was prepared in

accordance to the following composition: HEMA (25 % w/w): NVP (25 % w/w):

chitosan solution (50 % w/w) to give a final approximate mass of 2 g. A 2 g mixture

containing HEMA (50 % w/w): chitosan solution (50 % w/w) was also prepared. HMMI

was excluded from these formulations. The mixtures were placed in an ultra sonic bath

for a brief moment, transferred into clear polypropylene straws and subjected to UV

radiation.

2.5.2.7. Preparation of the Hydrogels Based on HEMA-NVP-AA System

The hydrogels based on HEMA-NVP-AA system were prepared in a wide range of

variable compositions of the three monomers. The monomer mixtures, each of a total

volume of 5 ml were prepared in accordance to the following composition in % v/v of

(HEMA:NVP:AA): (50:40:10); (50:10:40); (50:25:25); (50:50:0); (40:50:10); (10:50:40);

(25:50:25); (0:50:50); (50:0:50); (25:25:50); (10:40:50); (10:10:50). Each solution was

mixed thoroughly before being transferred into clear polypropylene straws. The samples

were subjected to UV radiation.

2.5.3. Polymerisation Procedure

The clear polypropylene straws filled with the monomer sample solutions as described in

Sections 2.5.1 and 2.5.2 were placed in test tubes and sealed with a rubber stopper. The

tubes were placed around the mercury lamp in the fitted circular rack drum for exposure

to the UV source. The specifications of the UV drum are described in Section 2.2. The

time taken for each sample solution to polymerise was recorded. The polymeric gel

samples were cut open from the straws and subjected to washing for several days in de-

ionized water to remove uncured monomers present in the gel matrix. The washing media

90


was changed regularly with fresh de-ionized water during this period. The washing

process was repeated until the concentration of the leached materials in the wash solution

was less than 1 ppm, estimated using an UV- Vis spectrophotometer. The gel samples

after being washed and dried were subjected to tests described in Sections 2.6, 2.7 and

2.9.

2.6. Equilibrium Water Content (EWC) Evaluation

The polymers synthesized were subjected to swelling test to evaluate the EWC in the

polymeric hydrogels. The swollen hydrogel networks were then subjected to texture

analysis and proton NMR experiments.

2.6.1. Equilibrium Swelling

Equilibrium swelling experiments on the hydrogel samples were conducted at 37 + 0.5 oC

mainly in neutral media with the exception of the use of basic and acidic media for some

hydrogel systems. The experiments were carried out at 37 oC to simulate the body

temperature for the purpose of indicating the gels potential application at such

temperature. Isotonic solution at pH 7.4 was also used as the swelling media for selected

hydrogel systems. Preparations for the buffers at different pH values are described in

Section 2.6.1.1.

The hydrogels prior to EWC evaluation were washed and dried as described in Section

2.5.3. Duplicate hydrogel samples for each system prepared as described in Section 2.5

were accurately weighed prior to immersion into the swelling media. The hydrogel

samples were removed periodically from the swelling media, blotted dry with an

absorbent tissue and weighed. The EWC evaluation experiment was carried out over a

period of 170 hours, which allowed the water content in the hydrogels to reach

equilibrium. A Mettler digital weighing balance was used to carry out the measurements.

The EWC calculations were carried out according to Equation 4 where Wt is the weight of

the swollen hydrogel at time t and Wo is the weight of the dry polymer [7].

EWC (%) = t

t

W W

W

− o x 100 Equation 4

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2.6.1.1. Preparation of Media

The EWC evaluation experiments were conducted in a range of swelling media. Many

biological reactions in body occur in the pH range of 2 to 8 [8] thus the media at certain

pH values were selected to simulate different pH environments in the body for this work.

Neutral media (milli-Q-water) was commonly used along with phosphate buffers (pH 2

and pH 8) for selected systems. A physiological phosphate-buffered isotonic solution (pH

7.4), which simulates the pH of blood, was also used for selected systems.

2.6.1.1.1. Preparation of Phosphate Buffer (pH 2)

The pH 2 buffer preparation method was adopted from Christian [8]. Monobasic sodium

phosphate, NaH2PO4.2H2O (7.80 g, MW. 156.01 g/mol) was combined with phosphoric

acid (3.4 ml, 85 %, 14.7 M) and prepared to a total volume of 1 L with milli-Q-water to

give a 100 mM solution. The final pH of the solution was adjusted to 2.0 by addition of

diluted phosphoric acid drop-wise to this solution while being monitored by a pH meter.

2.6.1.1.2. Preparation of Phosphate Buffer (pH 8)

The pH 8 buffer preparation method was adopted from Christian [8]. Monobasic sodium

phosphate, NaH2PO4.2H2O (3.12 g, MW. 156.01 g/mol) was added to a 100 ml flask and

made up to the mark with milli-Q-water to give a 0.2 M stock solution A. Stock solution

B (0.2 M) was prepared in a 500 ml flask containing anhydrous dibasic sodium

phosphate, Na2HPO4 (14.20 g, MW. 141.96 g/mol). Stock solution A (21.2 ml) was

combined with stock solution B (378.8 ml) and milli-Q-water (400 ml) to give a pH 8

buffered solution.

2.6.1.1.3. Preparation of Phosphate-Buffered Isotonic Solution (pH 7.4)

The phosphate-buffered isotonic solution preparation method was adopted from Christian

[8]. Two stock solutions, A and B were prepared as described in Section 2.6.1.2. Stock

solution A (76 ml) was combined with stock solution B (324 ml) and milli-Q-water (400

ml) to give a pH 7.4 buffered solution.

92


2.6.2. Texture Analysis

The hydrogels in their swollen state were tested for viscoelasticity and stiffness/elasticity

using a TA.XT2 texture analyser. The specifications of the texture analyser and

experimental conditions are described in Section 2.2.2.6. The samples were subjected to

load for a brief period and let to relax back to their original physical state. A stress-strain

graph was obtained (Appendix III). The samples were checked for any deformation after

the test. The linear portion of the compression-strain curve was used to compute the

Young’s modulus, which defines the flexibility/stiffness of the sample. The Young’s

moduli were calculated according to Equation 5 where stress is the measured compression

force F (N) divided by the contact surface area (m2) of sample and the strain is the ratio of

the deformed length and the undeformed length of the sample.

Young’s Modulus (MPa) = Stress

Strain Equation 5

The relative stress relaxation (SR) values, which are a direct measure of viscoelasticity in

the samples, were calculated according to Equation 6 where F1 is the measured

compression force at t = 10 seconds and F2 is the measured relaxation force exerted by

the sample post the stress period.

Relative1

1

-F F

F= 2

SR Equation 6

2.6.3. Proton NMR Relaxation (T1 and T2) Measurements

The proton NMR relaxations times, T1 and T2 were measured for selected swollen

hydrogel samples to investigate the dynamics of the water molecules in the swollen

networks. The specifications of the instrument and T1 and T2 measurements are described

in Section 2.2.2.3.2.

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2.7. Equilibrium Drug Release (EDR) Evaluation

Equilibrium drug release experiments were conducted at 37 + 0.5 oC in neutral, acidic and

basic media. Thiamine hydrochloride (HCl), theophylline, and Mn-TPP-OH were the

model drugs used for this work to evaluate the drug release behaviour of the various

hydrogels synthesized.

2.7.1. Preparation of the Model Drug Solutions

The model drugs listed in Section 2.7 are water-soluble with the exception of Mn-TPP-

OH, which is only water soluble upon prior dissolution in 2/3:1/3 cremophor EL: 2-

propan-diol mixture. Concentrated drug solutions (8000 ppm) were prepared to allow

maximum drug loading into the gel matrix. Details of the drug loading technique are

described in Section 2.7.2.

2.7.1.1. Preparation of Theophylline Solution

Theophylline (1,3 dimethylxanthine) is an oral bronchodilator used to treat asthma,

emphysema, and bronchitis [9]. It is a widely used model drug for drug release studies by

a number of researchers [10-20]. The high stability under the experimental conditions

described in Section 2.7, ease of detection under UV-Vis spectroscopy and ready

availability made theophylline (Figure 11) a suitable candidate as one of the model drugs

used for the controlled release studies in this research. An accurately weighed mass of

about 0.8 g of theophylline (MW. 180.16 g/mol) was dissolved in a 100 ml volumetric

flask with milli-Q-water to give a 8000 ppm solution of theophylline. The mixture was

warmed up gently and sonicated to give a uniform solution.

N

NN

NH

O

O

Figure 11. Theophylline

94


2.7.1.2. Preparation of Thiamine HCl Solution

Thiamine HCl (vitamin B1) plays a key role in the body’s metabolic cycle for generating

energy. The molecular weight of the incorporated model drug has been reported to be a

crucial factor in the drug release experiments [10,13]. Thiamine HCl (Figure 12) was

considered as a model drug for this work due its stability at the required experimental

conditions described in Section 2.7 and its relatively higher molecular weight in

comparison to theophylline to allow comparative studies on release rate of the model

drugs based on molecular weight.

Accurately weighed mass of approximately 0.8 g of thiamine HCl (MW. 337.26 g/mol)

was dissolved in a 100 ml volumetric flask with milli-Q-water to give a 8000 ppm

solution. The solution was warmed gently and sonicated briefly to allow complete

dissolution of the drug.

N

SO N

N

NH H

H

Cl+

-

Figure 12. Thiamine HCl

2.7.1.3. Preparation of Mn-TPP-OH Solution

Mn-TPP-OH (Figure 1) is a cancer tumor-tracing agent used in conjunction with the

magnetic resonance imaging (MRI) technique. Interest in this material lies in the fact that

it has a relatively larger molecular weight thus making it a good candidate as a model

drug for observing the effect of large molecular weight on the drug release rate.

Furthermore, the use of Mn-TPP-OH would indicate the possibility of using the hydrogels

under study as carriers of such drugs in cancer therapy.

The Mn-TPP-OH is not readily soluble in water thus had to be pre-dissolved in specially

prepared cremophor EL (CRM) and 1, 2-propanediol mixture. Mn-TPP-OH (0.1531 g,

MW. 733.69 g/mol) was dissolved in CRM (3 ml) and 1, 2-propanediol (1.5 ml) mixture

95


prior to addition of 25 ml of milli-Q-water. The solution was gently warmed and briefly

sonicated to make the solution uniform with a concentration of 8000 ppm.

2.7.2. Drug Loading Technique

The model drug could be loaded into the hydrogel in several ways. In crosslinked

polymer systems it is possible to have the drug incorporated during the formation step but

this could lead to problems associated with other unreacted components leaching out of

the gel matrix along with the incorporated drug. The drug could also be sensitive to the

gelation conditions and may undergo chemical change or possibly interfere with the

polymerisation process. Thus in such instances the drug must be loaded from solution

[21]. In this experiment the drug was loaded from a concentrated drug solution for each

drug prepared as described in Section 2.7.1.

All gel samples were washed and dried as described in Section 2.5.2 prior to drug

incorporation. Duplicate samples of each hydrogel were weighed accurately and each

immersed into 25 ml of concentrated drug solutions. The gel samples were left immersed

in the concentrated drug solution for a period of seven days to allow maximum drug

loading into the hydrogel matrix.

2.7.3. Controlled Drug Release Studies

Controlled drug release experiments were conducted in duplicate samples for each

hydrogel system. The duplicate hydrogel samples were removed from the concentrated

drug solution after seven days of loading and each swollen gel loaded with the model

drug was placed in approximately 100 ml of milli-Q-water for 10 minutes to allow any

excess drug on the surface of the gel to be washed off. The samples were removed from

these wash solutions, blotted dry with a tissue paper and weighed. Each of these samples

was then placed into a test-tube containing accurately measured, 50 ml of release

medium.

Milli-Q-water was mainly used as the release medium with the exceptional use of pH 2

and pH 8 phosphate buffered solutions, prepared as described in Section 2.6.1.1, for

selected hydrogels. The release medium temperature was equilibrated to 37 oC prior to

96


placement of the samples. In order to obtain a homogeneous solution, each test-tube was

equipped with a stirrer bar. Aliquots (1 ml) of the release medium were removed

periodically, diluted to 21 ml and analysed.

The periodic removal of the release medium was carried out until the change in the

concentration of the released drug was negligible or had reached equilibrium. At this

stage the release medium was changed to an accurately measured 50 ml of fresh medium

and any further drug released was analysed for several days. This was repeated until

negligible amount of drug released (< 0.1 ppm) into the medium was detected.

The controlled drug release experiments on the polymeric hydrogel samples were

conducted at 37 oC. Calculations on fractional drug release were based on the amount of

drug release analysed at each time interval divided by the total amount of drug released

from each sample. The same procedure was repeated for all the hydrogels synthesized

along with all the model drugs used in conjunction with these gels.

2.7.3.1. The Analytical Technique (Ultraviolet - Visible Spectroscopy)

A Shimadzu UV-1601 PC instrument was used for this work. The specifications of the

instrument are described in Section 2.2.2.1. The working wavelength range for the

analysis of the three model drugs was from 800 nm to 200 nm. Mn-TPP-OH was detected

at 424 nm while theophylline and thiamine HCl were detected at 271 nm and 266 nm

respectively. Five standard solutions of varying known concentrations ranging from 3

ppm to 20 ppm of each model drug was prepared and analysed to construct standard

calibration curve of absorbance against concentration. This curve was used to determine

the concentration of the drug released into the media with the fact that absorbance is

directly proportional to concentration. Fractional drug released (FDR) from each hydrogel

sample was calculated according to Equation 7 where Mt is the mass of the drug released

at time t and M∞ is the total mass of the incorporated drug.

FDR = tM

M ∞ Equation 7

97


The total amount of drug released from each hydrogel system as observed from the UV

analysis was compared with the calculated amount of incorporated drug from weight

measurements made prior and post drug loading to confirm equilibrium drug release.

2.8. Kinetic Studies on Electron Donor/Acceptor Systems

Kinetic studies were conducted on a range of donor/acceptor pairs using a Differential

Photocalorimeter/DSC 2910 system by TA Instruments. These donor/acceptor systems

were utilized to form hydrogels as described previously in Section 2.5.1.2. The

specifications of the DPC, experimental conditions and analytical procedure are described

in Section 2.2.2.5. The electron acceptor monomers, which were tested for this work,

were AA, HEMA and a series of N-hydroxyalkyl maleimides, HMMI, HEMI, HPrMI and

HPMI, synthesized as described in Section 2.3.2. NVP was the electron donor monomer

utilized. Glucosamine HCl and glucose were used as hydrogen donors in the N-

hydroxyalkyl maleimide-NVP systems.

2.8.1. Preparation of NVP-HEMA and AA-NVP Systems

The donor/acceptor monomer combinations in each of the following systems

HEMA:NVP and AA:NVP were prepared in three different mol ratios. For each system,

the monomer pairs were weighed in sample tubes to give (1:2; 1:1; 2:1) mol ratios. The

sample tubes were wrapped in aluminium foil to protect samples from being exposed to

ambient light. Samples were analysed using a DPC unit as described in Section 2.2.2.5.

2.8.2. Preparation of N-Hydroxyalkyl Maleimides-NVP Systems

The four maleimides synthesized as described in Section 2.3 namely HMMI, HEMI,

HPrMI, and HPMI were each combined with NVP in a 1:1 mol ratio and tested for their

relative reactivity in the absence as well as in the presence of hydrogen donors, glucose

and glucosamine HCl. Details of preparation of the systems with and without the

hydrogen donors are described in Sections 2.8.2.1 and 2.8.2.2.

2.8.2.1. Preparation in the Presence of H-Donor

Accurately weighed amounts of NVP, N-hydroxyalkyl maleimide and H2O were

combined to form stock solutions. The final mixture contained NVP (1.42 M); N-

98


hydroxyalkyl maleimide (1.42 M) and the H-donor (0.15 M). The mass of the different

maleimides varied in each case due to the difference in the molecular weight and thus the

final mass was adjusted with water. However, the mol ratio of NVP and maleimide was

maintained as 1:1. Two sample mixtures, glucose solution (0.15 g; 0.56 M) and

glucosamine HCl solution (0.15 g; 0.46 M), each combined with stock solution (0.33 g)

were prepared in sample tubes wrapped with aluminium foil to prevent unwanted

polymerisation initiated by light in the laboratory. Each sample was dispensed into an

aluminium DSC pan as described in Section 2.2.2.5 and its exotherm was subsequently

obtained using the DPC technique.

2.8.2.2. Preparation in the Absence of H-Donor

Systems without a hydrogen donor were prepared and analysed in accordance to the

procedure described in Section 2.8.2.1 with the hydrogen donor being excluded. The

concentrations of the acceptor and the donor in the final sample mixture were maintained

by substituting the mass of the hydrogen donor solution with milli-Q-water to maintain

the same total mass of the final mixture as that of the systems with hydrogen donor.

2.9. Cytotoxicity Tests on Mammalian Cells

The study of hydrogels in this work is oriented towards its biomedical application as

controlled drug delivery devices. It is a known fact that the complete conversion of

monomers to polymers may not be achieved in the polymerisation process thus there is

always a certain component of unreacted toxic monomers still present in the polymer

matrix. These monomers have the tendency to leach out of the polymer matrices when the

polymers are in contact with an aqueous medium thus rendering the hydrogel to be non-

biocompatible.

The hydrogel systems prepared as described in Section 2.5 were tested in vitro for their

biocompatibility with human epidermal keratinocyte (HaCaT) cells, provided by the Skin

Technologies Research Centre at the University of Western Sydney. In the panorama of

numerous established cell lines, HaCaT has a very interesting feature, having a close

similarity in the functional competence to that of normal keratinocytes [22]. This cell line

has been used in numerous studies as a paradigm for epidermal cells and furthermore, is

99


readily available, highly sensitive and easily regenerated, making it an ideal candidate as

the cell model for this work.

A 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, which is a

colorimetric based technique for non-radioactive quantification of cellular proliferation,

viability and cytotoxicity was used for this work. The assay is based on the cleavage of

the yellow tetrazolium salt, (MTT) to form purple formazan crystals by dehydrogenase

activity in active mitochondria, which indicate the presence of live cells [23,24].

2.9.1. Sustaining HaCaT Cells

The HaCaT cells were handled with great care to avoid any contaminants thus aseptic

handling techniques were enforced at all times. A specially prepared growth medium was

used to sustain the HaCaT cells during the course of the cytotoxicity experiments.

2.9.1.1. Preparation of HaCaT Growth Medium

The HaCaT medium was prepared in milli-Q-water by adding Dulbecco's modified

Eagle's medium (DMEM)- calcium-free (12.7 g/L) powdered media containing glucose

(4.5 g/L) and sodium pyruvate (0.11 g/L) to which was added sodium bicarbonate (2.2

g/L), penicillin/streptomycin (100 U/ml, 0.1 g/L), N- [2-hydroxyethyl] piperazine-N- [2-

ethane sulfonic acid] (HEPES) (2.38 g/L) and L-glutamine (0.29 g/L).

The mixture was gently stirred and then accurately diluted to a total volume of 900 mL

with milli-Q-water. The medium was filter sterilized through a 0.2 µm membrane filter,

aseptically dispensed into sterile bottles and stored in the dark at 4 oC. For cell growth,

the HaCaT medium was supplemented with sterile heat inactivated fetal calf (bovine)

serum (FCS) (10 % v/v). The media was warmed to 37 oC and filter sterilized each time

prior to feeding cells.

2.9.1.2. Aseptic Techniques

It was absolutely crucial that at no point in time during the cytotoxicity experiments,

contaminants of any kind were introduced to the cells. Aseptic handling procedures were

enforced at all times. The biohazard hood was sterilized with ultra violet radiation for 10

100


minutes prior to commencement of every experiment. The working bench under the hood

and surrounding atmosphere was sterilized with 70 % ethanol spray. All biological

reagents including the media and test samples were filter sterilized through a 0.2 µm

membrane filter at all times prior to introduction to the cells.

2.9.1.3. Generation of HaCaT Cells

The supplied HaCaT cell suspension was stored in growth medium supplemented with

FCS (30 % v/v) and DMSO (10 % v/v) in a cryo vial (1 ml) under liquid nitrogen. A

specially prepared growth medium as described in Section 2.9.1.1 was used to culture the

HaCaT cells. The HaCaT medium (5 ml) supplemented with 10 % v/v of fetal calf

(bovine) serum FCS was warmed to 37 oC, filter sterilized and aseptically dispensed into

a centrifuge tube to which was added the thawed HaCaT cell suspension.

The mixture was centrifuged for 5 minutes at 2000 rpm separating the cells from the

suspension. The liquid content of the tube was discarded and the cells were re-suspended

in fresh media (1ml). Two 75 cm2 culture flasks each containing 0.5 ml of the cell

suspension and 10 ml of media supplemented with 10 % v/v FCS were prepared and

incubated at 37 oC in the presence of CO2 (5 %). The culture flasks were replenished with

fresh medium every third day until the cells were fully confluent.

2.9.1.4. Trypsinizing HaCaT Cells

The HaCaT cells after becoming fully confluent were trypsinized using ethylenediamine

tetraacetic acid (EDTA) and trypsin solution. The exhausted media in the culture flask

was discarded and replaced with sterile EDTA solution (10 ml, 0.02 %) prepared in

Dulbecco's phosphate buffered saline (DPBS). The flask was then incubated at 37 oC for

20 minutes. At the end of the incubation period the EDTA solution was discarded and

replaced with trypsin solution (5 ml) containing EDTA (0.02 % w/v), D-Glucose (0.1 %

w/v), KCl (0.04 % w/v), NaCl (0.8 % w/v) and trypsin (0.1 % w/v) followed by a further

5 minutes incubation period at 37 oC. The process involved detachment of the cells from

each other by EDTA treatment, and detachment from the surface of the culture flask by

trypsin treatment.

101


The culture flask was tapped gently on the sides to assist in the separation of the cells and

observed under the microscope to determine whether the cell had dissociated from the

flask and each other. The contents of the flask were emptied into a sterile centrifuge tube

containing fresh filtered media (5 ml). The tube was centrifuged for 5 minutes at 2000

rpm. The supernatant was discarded and the cell pellet re-suspended in fresh media (1ml).

The cell suspension was gently swirled before transferring 20 µL into the counting

chamber of a hemacytometer to determine cell density.

A new culture flask containing fresh media (10 ml) was seeded with an aliquot of the cell

suspension (50 µL) to maintain the cell line. The cells were fed as described in Section

2.9.1.3 and trypsinized upon becoming confluent. The process of subculturing and

sustaining the HaCaT cells was repeated throughout the course of the cytotoxicity

experiments.

2.9.2. Preparation of Test Samples

The hydrogel samples used for this work were cleaned thoroughly as described in Section

2.5.2 to eliminate the leaching of any toxic unreacted monomers from the hydrogel

matrix. The pre-weighed gel samples were further cleaned with milli-Q-water in the

biohazard hood and exposed to ultra violet light for 20 minutes for sterilization. Aseptic

techniques were employed in handling the hydrogel samples post this process to avoid the

growth of micro-organisms on the samples, which could affect the validity of this

experiment.

The gel samples after sterilization were immersed in accurately measured volumes of

milli-Q-water with gel mass to water volume ratio of 20 g/ml and incubated at 37 oC for a

period of 14 days. The water conditioned by the hydrogels (samples) was then filter

sterilized using a 0.2 µm syringe filter and used for cytotoxicity tests described below.

Standard solutions of the monomers, AA, HEMA, NVP and the N-hydroxyalkyl

maleimides were prepared in HaCaT medium without FCS in varying concentrations (125

ppm, 250 ppm, 500 ppm and 1000 ppm) and also tested for cytotoxicity.

102


2.9.2.1. Preparation of Culture Plates

Greiner 96 well tissue culture plates were utilized for this work. The HaCaT cell

suspension prepared as in Section 2.9.1.4 was used to make cell suspensions of varying

cell concentrations ranging from 1 x 106 – 5 x 10

4 cells/mL. A standard culture plate was

prepared for a standard curve by transferring 100 µL aliquots of each cell suspension per

well in five replicate wells in a 96 well plate. Fresh HaCaT media (100 µL) served as the

blank for background reference.

The experimental culture plate was prepared by adding 100 µL of a cell suspension of 1 x

105 cell/mL into each well in the plate, followed by transferring 20 µL aliquots of each

sample medium prepared as described in Section 2.9.2 making a total volume of 120

µL/well. Fresh HaCaT medium (120 µL) served as the blank. The experimental control,

which was the untreated cell suspension, was prepared by adding 20 µL of FCS-free

medium to 100 µL of 1 x 105 cells/ml cell suspension. The response of the HaCaT cells to

the sample medium at 24 hours and at 48 hours of exposure was evaluated. The

cytotoxicity experiment on each sample medium was repeated in triplicate.

2.9.2.2. MTT Cell Proliferation Assay

Sterile MTT solution (5 mg/ml) was prepared by dissolving MTT (100 mg) in DMEM-

phenol red free medium (20 ml). The solution was filter sterilized and stored in the dark

at minus 20 oC. Anhydrous isopropanol with HCl (0.1 M) was used as the MTT solvent

solution. The standard culture plates prepared as described in Section 2.9.2.1 were

incubated at 37 oC in the CO2 (5 %) atmosphere for 10 minutes to allow the cells to settle.

MTT solution (10 µL) was then added to the wells in the culture plates and incubated at

37 oC in the presence of CO2 (5 %) for 3 hours.

The experimental culture plates were incubated at 37 oC in the CO2 atmosphere for 24

hours and 48 hours prior to the addition of the MTT solution (10 µL). The cell cultures

were examined under a microscope after the designated treatment time. MTT was then

subsequently added to the culture plates followed by a further 3 hour incubation period at

37 oC in the presence of CO2 (5 %). The medium from the wells was replaced with MTT

103


solvent solution (100 µL) after the 3 hour incubation period. Each plate was placed on a

gyratory shaker to dissolve the purple MTT formazan crystals.

A BMG Labtechnologies FLUOstar OPTIMA microplate reader was used to carry out the

absorbance measurements. The readings were carried out at a wavelength of 560 nm with

background absorbance of 650 nm. The FLUOstar OPTIMA v1.30-0 software was used

to analyse the acquired raw data. A standard curve was constructed from the standard

culture plate measurements of known cell densities to estimate the cell density of the

unknown cell cultures treated with the monomers and the hydrogel sample media.

2.9.2.2.1. Statistical Analysis

The MTT assay data were expressed as means + SEM. The MTT assay data obtained for

the hydrogels were compared with that of the monomers and also with the experimental

untreated controls. A one-way analysis of variance (ANOVA) was performed using the

MINITAB 7.2 statistical software. A p value of < 0.05 was regarded as statistically

significant.

104


2.10. References

1. Leighton, W. G., Forbes, G. S., J. Am. Chem. Soc., 52, 3139-3152, (1930).

2. Tawney, P. O., Snyder, R. H., Conger, R. P., Leibbrand, K. A., Stiteler, C. H.,

Williams, A. R., J. Org. Chem., 26, 15-21, (1961).

3. Narita, M., Teramoto, T., Okawara, M., Bull. Chem. Soc. Jap., 44, 1084-1089,

(1971).

4. Shigeyoshi, H., Chem Abs., 114, 185261z, (1990).

5. Liu, X. F., Guan, Y. L., Yang, D. Z., Li, Z., Yao, K. D., J. Appl. Polymer Sci., 79,

1324-1335, (2001).

6. Muzzarelli, R. A. A., Ilari, P., Petrarulo, M., Int. J. Biol. Macromol., 16, 177-180,

(1994).

7. Aggarwal, S. L., “COMPREHENSIVE POLYMER SCIENCE” – The Synthesis,

Characterisation, Reactions & Applications of Polymers, vol 7, Pergamon Press,

Oxford, pp. 221, (1989).

8. Christian, G. D., “Analytical Chemistry”, 5th

edition, John Wiley & Sons, New

York, pp. 207-209, (1994).

9. Evans, D., Taylor, D., Zetterstrom, O., and Chung, K., New Eng. J. Med., 337,

1412-1418, (1997).

10. Brazel, C. S., Peppas, N. A., Polymer, 40, 3383-3398, (1999).

11. Grassi, M., Colombo, I., Lapasin, R., J. Controlled Release, 76, 93-105, (2001).

12. Ward, J. H., Peppas, N. A., J. Controlled Release, 71, 183-192, (2001).


14. Inoue, T., Chen, G., Nakamae, K., and Hoffman, A. S., J. Controlled Release, 49,

167-176, (1997).

15. Katime, I., Novoa, R., Díaz de Apodaca, E., Mendizábal, E., Puig, J., Polymer

Testing, 18, 559-566, (1999).


17. Shantha, K. L., Harding, D. R. K., Int. J. Pharm., 207, 65-70, (2000).

18. Shah, S. S., Kulkarni, M. G., Mashelkar. R. A., J. Controlled Release, 15, 121-

131, (1991).

19. Harland, R. S., Peppas, N. A., J. Controlled Release, 26, 157-174, (1993).

105


20. Bettini, R., Colombo, P., Peppas, N. A., J. Controlled Release, 37, 105-111,

(1995).


22. Pessina, A., Raimondi, A., Cerri, A., Piccirillo, M., Neri, M. G., Croera, C., Foti.

P., Berti, E., Cell Prolif., 34, 243-252, (2001).



106


2.1. Materials 66

2.2. Equipment 70

2.2.1. Radiation Source 70

2.2.2. Analytical Instruments 70

2.2.2.1. Ultra Violet - Visible (UV-Vis) Spectrophotometer 70

2.2.2.2. Gas Chromatograph Mass Spectrometer (GC-MS) 70

2.2.2.3. Nuclear Magnetic Resonance (NMR) Spectrometer 71

2.2.2.3.1. Carbon-13 (13

C) and Proton (1H) NMR 71

2.2.2.3.2. Proton NMR Relaxation (T1 and T2) Measurements 71

2.2.2.4. Fourier Transform Infrared (FT-IR) Spectrometer 71

2.2.2.5. Differential Photocalorimeter (DPC) 71

2.2.2.6. Texture Analyser (TA) 72

2.2.2.7. Atomic Absorption Spectrometer (AAS) 72

2.2.2.8. Microplate Reader 72

2.2.2.9. Microscope 73

2.3. Synthesis of Chemical Compounds 73

2.3.1. Synthesis of a Model Drug 73

2.3.1.1. Synthesis of Mn-TPP-OH. 73

2.3.1.1.1. UV-Vis Spectroscopic Analysis 74

2.3.1.1.2. AAS Analysis 74

2.3.2. Synthesis of N-Hydroxyalkyl Maleimides 74

2.3.2.1. Synthesis of HMMI 75

2.3.2.1.1. Mass Spectroscopic Analysis 75

2.3.2.1.2. NMR Spectroscopic Analysis 75

2.3.2.2. Synthesis of Furan-A 76



2.3.2.3. Synthesis of HEMI-A 76



2.3.2.4. Synthesis of HEMI 77



2.3.2.5. Synthesis of HPrMI-A 78



2.3.2.6. Synthesis of HPrMI 79



2.3.2.7. Synthesis of HPMI-A 80



2.3.2.8. Synthesis of HPMI 81



2.3.3. Water-Soluble Derivative of Chitosan 82

107


2.3.3.1. Synthesis of CM Chitosan 82

2.3.3.1.1. FT-IR Analysis 83

2.4. UV Lamp calibration 83

2.4.1. Preparation of Solutions 84

2.4.1.1. Oxalic Acid Solution (COOH) 2 84

2.4.1.2. Uranyl Nitrate Solution (UO2 (NO3) 2. 6H2O) 84

2.4.1.3. Potassium Permanganate Solution (KMnO4) 84

2.4.1.4. Preparation of Sample Solutions 85

2.4.2. Irradiation of Samples 85

2.4.3. Analysis of the Irradiated Samples 85

2.4.4. UV Dose Calculation 85

2.4.4.1. Decomposition of Oxalic Acid 86

2.4.4.2. Number of Einstein’s (s-1

) Required 86

2.4.4.3. Energy (J) 87

2.4.4.4. Dose Rate (J s-1

) 87

2.5. Preparation of Hydrogels 87

2.5.1. Hydrogels Synthesis Initiated By Photoinitiator (PI) 87

2.5.2. Hydrogels Synthesis via Photoinitiator-Free Process 88

2.5.2.1. Preparation of the N-Hydroxyalkyl Maleimide and NVP Systems 88

2.5.2.2. Preparation of the HPMI- NVP-HEMA System 88

2.5.2.3. Preparation of the HPMI- NVP-NVC System 89

2.5.2.4. Preparation of the IPN, HMMI-NVP-Chitosan System 89

2.5.2.5. Preparation of the IPN, HMMI-NVP-CM Chitosan System 89

2.5.2.6. Preparation of the IPN, HEMA-NVP-Chitosan System 90

2.5.2.7. Preparation of the Hydrogels Based on HEMA-NVP-AA Systems 90

2.5.3. Polymerisation Procedure 90

2.6. Equilibrium Water Content (EWC) Evaluation 91

2.6.1. Equilibrium Swelling 91

2.6.1.1. Preparation of Media 92

2.6.1.1.1. Preparation of Phosphate Buffer (pH 2) 92

2.6.1.1.2. Preparation of Phosphate Buffer (pH 8) 92

2.6.1.1.3. Preparation of Phosphate-Buffered Isotonic Solution (pH 7.4) 92

2.6.2. Texture Analysis 93

2.6.3. Proton NMR Relaxation (T1 and T2) Measurements 93

2.7. Equilibrium Drug Release (EDR) Evaluation 94

2.7.1. Preparation of the Model Drug Solutions 94

2.7.1.1. Preparation of Theophylline Solution 94

2.7.1.2. Preparation of Thiamine HCl Solution 95

2.7.1.3. Preparation of Mn-TPP-OH Solution 95

2.7.2. Drug Loading Technique 96

2.7.3. Controlled Drug Release Studies 96

2.7.3.1. The Analytical Technique (Ultraviolet - Visible Spectroscopy) 97

2.8. Kinetic Studies on Electron Donor/Acceptor Systems 98

108


2.8.1. Preparation of NVP-HEMA and AA-NVP Systems 98

2.8.2. Preparation of N-Hydroxyalkyl Maleimides-NVP Systems 98

2.8.2.1. Preparation in the Presence of H-Donor 98

2.8.2.2. Preparation in the Absence of H-Donor 99

2.9. Cytotoxicity Tests on Mammalian Cells 99

2.9.1. Sustaining HaCaT Cells 100

2.9.1.1. Preparation of HaCaT Growth Medium 100

2.9.1.2. Aseptic Techniques 100

2.9.1.3. Generation of HaCaT Cells 101

2.9.1.4. Trypsinizing HaCaT Cells 101

2.9.2. Preparation of Test Samples 102

2.9.2.1. Preparation of Culture Plates 103

2.9.2.2. MTT Cell Proliferation Assay 103

2.9.2.2.1. Statistical Analysis 104

2.10. References 105

109

Chapter 3: Photo-cured Hydrogels Containing 2-Hydroxyethyl Methacrylate and N-Vinyl-2-Pyrrolidinone

3.1. Introduction

Wichterle and Lim [1] first developed hydrogels based on 2-hydroxyethyl methacrylate

(HEMA) for their potential application as contact lenses. Polymeric hydrogels have been

heavily utilized in the biomedical arena since then as versatile and commercially viable

materials [2-7]. HEMA is by far the most extensively used monomer for hydrogel

preparation. Numerous publications are available to date on poly(HEMA) (PHEMA)

hydrogels [8-18]. HEMA besides being a proven versatile homopolymeric biomaterial

could be combined with a pronounced hydrophilic co-monomer such as N-vinyl

pyrrolidinone (NVP) to produce copolymers with enhanced structural properties.

Polymers of HEMA and NVP have been investigated in recent years as potential hydrogel

materials for biomedical applications such as sustained drug delivery systems and contact

lenses [19-24].

The attractive bioapplications of HEMA-co-NVP hydrogels is attributed to their

remarkable water absorption ability and durability in harsh environments [9,12,20,22].

The high water content in hydrogels is believed to be intrinsically related to their high

biocompatibility [25,26]. Factors favourable to swelling include high osmotic potential,

high free volume, high chain flexibility, low crosslinking density and strong interaction

with water [27]. The water adsorption ability of a polymer is dependent on the nature and

the composition of the monomers in the polymer [22].

Inclusion of additives such as crosslinkers has been reported to govern the swelling in

polymers. Lai [19], Perera and Shanks [20] have reported a marked reduction in the

swelling of HEMA-co-NVP hydrogels upon inclusion of crosslinking agents, ethylene

glycol dimethacrylate and methylene diacrylamide. The rate of the drug delivery is

controlled by the macromolecular structure of the carrier as defined by the degree of

swelling [28,29].

The swelling and drug release kinetics in hydrogels could range from Fickian diffusion to

high order diffusion such as non-Fickian (anomalous and case II). Fickian diffusion is

characterized by a solute mass uptake that is directly proportional to the square root of

time. High order non-Fickian diffusion however, is characterized by linear mass uptake as

107


a function of time [28-30]. Ideal release kinetics of a drug would be a zero-order process

where the diffusion of the solute is time independent as that in the case of case II

diffusion. Bhardwaj et al [31] studied the diffusion behaviour in a range of copolymers of

HEMA and NVP and observed Fickian diffusion kinetics.

In contrary, Korsmeyer and Peppas [21] from their swelling and drug release experiments

on HEMA-co-NVP copolymers observed non-Fickian swelling and drug release kinetics.

However, they also further suggest that the swelling and drug release kinetics is

dependent on the relative composition of HEMA to NVP. Franson and Peppas [32] in an

earlier study on the effect of HEMA and NVP composition on the swelling behaviour of

their copolymers have also suggested the dependence of diffusion kinetics on the

monomeric composition.

Non-fickian hydrogels display a phenomenal abrupt volume change as a result of rapid

swelling in contrary to Fickian gels, which have a gradual swelling process. The diffused

water contained in hydrogel is evidenced to vary in molecular form. Khare and Peppas

[33] suggest the existence of water molecules in the polymer in three states, bound water,

interfacial water and bulk water. The bound water is suggested to be associated with the

polymer chains by means of hydrogen bonding. Hydrophobic interactions between the

functional groups on the polymer chain and water result in interfacial water. Free or bulk

water has the same physical properties as normal water and is not attached to the polymer

matrix [33,34].

Researchers have commonly resorted to Nuclear Magnetic Resonance (NMR)

experiments to investigate the state of water [30,34,35]. The T1 and T2 relaxation times

obtained from NMR experiments have been shown to indicate the relative mobility of

water in the polymer network. Furthermore, it is indicative of the diffusion kinetics of the

macromolecular network.

The stress relaxation phenomenon in polymers is another interesting feature, which

describes the texture of polymers. Polymeric hydrogels are often defined as two phase

systems where one phase is the water insoluble macromolecular network while the other

108


is water [2]. Polymers could behave like solids or liquids depending on the relative water

content [36]. The stress relaxation in polymers could also be termed as the viscoelasticity

of the polymer. Hong et al [37] from their studies on the texture analysis of hydrated

copolymers of HEMA and NVP reported the dependence of viscoelasticity on the

monomer composition.

Hydrogels of HEMA and NVP have been synthesized via a number of techniques such as

thermal and radiation methods [19-24]. Thermal and low energy UV radiation techniques

require additional chemical initiators while high-energy radiation processes such as

gamma and electron beam do not. Furthermore, some researchers have made use of

chemical crosslinkers in the formulations [19,20,24].

Bhardwaj et al [31] reported similar diffusion kinetics in HEMA-co-NVP hydrogels

prepared through chemical initiation and gamma radiation. In a recent study by Malak et

al [22,23], similar observations were made. However, they do suggest that these different

polymerisation protocols could lead to subtly different network structures. They state that

the efficiency of polymerisation would vary in each curing protocol thus the copolymers

could be inhomogeneous with varied crosslinking density.

In the present study HEMA and NVP were photopolymerised in a wide range of varied

compositions in the presence of a photoinitiator, Irgacure 819. Irgacure 819, also

chemically known as bis(2,4,6-trimethylbenzoyl)-phenylphosphineoxide, is an efficient

photoinitiator, which was newly introduced in the market at the time of this research. The

hydrogels formed were evaluated for their equilibrium swelling and drug release ability at

310 K. The effect of water, as an added plasticiser in the polymer formulation, on the

swelling and drug release kinetics of the polymer was investigated.

Hydrogels were also swollen in an isotonic medium to evaluate the effect of ionic

strength on the diffusion characteristic of the hydrogels. Texture analysis experiments

were conducted on the hydrogels to investigate their relative stress relaxation phenomena

after an applied stress. Furthermore, proton NMR experiments were conducted to

109


measure the relaxation times, T1 and T2 in the hydrogel samples in their fully hydrated

state.

3.2. Experimental Procedure

HEMA and NVP were combined in varying ratios by volume and subjected to UV

radiation in the presence of an external photoinitiator, Irgacure 819. A fraction of water

was included in some formulations to act as a plasticiser. The detailed synthesis

procedure of HEMA-NVP hydrogels in the presence of a photoinitiator is described in

Section 2.5.1. The hydrogels formed were subjected to swelling drug release experiments

in neutral pH environment at 37 oC. The effect of ionic strength on the swelling ability of

the hydrogels in an isotonic (pH 7.4) environment was also evaluated. Theophylline was

used as the model drug for drug release experiments.

Swelling and drug release experimental specifications and procedure have been described

in detail in Sections 2.6.1 and 2.7. UV-vis spectroscopy was employed as the analytical

tool for quantitative drug release measurements. A TA instrument was used carry out

texture analysis experiments on swollen hydrogel samples to investigate their relative

stiffness and viscoelasticity. NMR spectroscopic technique was used to measure the

relaxation times, T1 and T2 in selected swollen hydrogel samples. Detailed experimental

specifications on NMR and TA analysis are described in Sections 2.2.2.3.2 and 2.2.2.6

respectively.

3.3. Results

3.3.1. Hydrogel Formation

HEMA and NVP formulated in varying ratios by volume in the presence of Irgacure 819

were subjected to UV radiation. Certain formulations were inclusive of 10 % v/v of water,

which served as a plasticiser. The observations on the status of polymerisation upon

applying approximately 174 J of radiation dose are described in Table 1.

110


Table 1. Polymerisation status of HEMA : NVP : H2O formulations in the presence

of Irgacure 819 (0.1 % w/v)

Hydrogel formulation

HEMA: NVP: H2O (% v/v)

Polymerisation status upon application

of curing dose (~ 174 J)

100: 00: 00 Hard, clear pale yellow gel







The radiation dose was calculated according to Equation 1 where t is the total radiation

time in seconds. The monomer formulations were exposed to UV radiation for a period of

approximately 30 mins at a dose rate of 9.6 x 10-2

J s-1

. The dose rate was calculated as

described in Section 2.4.4.4.

Radiation dose (J) = dose rate (J s-1

) x t (s) Equation 1

3.3.2. Experimental Swelling Results

The swelling experimental data of HEMA-NVP hydrogels formed is presented in Tables

2-5. The data reflects the effect polymer composition on the swelling behaviour of the

gels and also the effect on the swelling behaviour upon introduction of a plasticiser,

water. Furthermore, the data also reflects the effect of ions in the swelling medium on the

swelling behaviour of the gels. Poly(NVP) (PNVP) hydrogel disintegrated upon a short

exposure to aqueous swelling medium thus further experiments on this system was not

conducted. Graphical representations of the swelling test data are illustrated in Figures 1-

13.

111


Table 2. Swelling test on hydrogels in neutral pH environment at 37 oC

Average % water content values at time (t)

Time (h) Gel A Gel B Gel C Gel D Gel E Gel F

0.00 0.0 0.0 0.0 0.0 0.0 0.0

0.17 8.2 9.2 15.9 36.1 13.1 44.5

0.33 9.8 11.8 21.8 45.8 15.5 55.4

0.50 10.9 13.8 26.0 52.6 17.4 61.4

0.67 12.1 15.6 28.9 57.1 19.6 65.8

0.83 13.2 17.0 31.5 60.3 21.0 68.9

1.00 14.2 18.5 34.0 62.9 23.2 71.5

2.00 17.5 23.1 42.4 73.4 27.6 79.5

3.00 21.1 26.2 47.6 78.3 30.9 83.3

4.00 22.1 28.4 51.6 81.3 33.7 85.7

5.00 23.9 30.4 55.0 83.3 35.7 87.3

7.00 26.6 33.3 59.5 86.0 39.0 89.4

9.00 28.6 35.4 62.4 87.5 41.3 90.5

12.00 30.4 37.3 65.4 89.1 43.6 91.7

24.00 33.5 40.8 69.7 91.3 47.2 93.5

48.00 35.2 41.8 71.1 92.3 48.6 94.6

72.00 35.8 42.0 71.2 92.3 48.8 94.9

96.00 36.0 41.9 71.1 92.2 48.9 95.0

120.00 36.4 41.9 71.1 92.0 49.1 95.0

144.00 36.7 42.0 70.9 91.8 49.1 95.0

170.00 36.6 41.9 70.9 91.6 49.3 95.0

Hydrogel compositions: (NVP : HEMA : H2O (% v/ v)); 00: 100: 00 (Gel A); 20: 80: 00

(Gel B); 50: 50: 00 (Gel C); 80: 20: 00 (Gel D); 15: 75: 10 (Gel E) and 75: 15: 10 (Gel F).

112


The swelling data are expressed as percentage water content at respective time intervals.

The percentage water content values were calculated as described in Section 2.6.1.

Graphical representations of the swelling behaviour observed in the NVP-HEMA

hydrogels in neutral pH environment are illustrated in Figures 1 -6. The comparative plots

illustrate the effect of added water to gel formulation E and F, on the swelling efficiency.

0

20

40

60

80

100

0 50 100 150 200

Time (h)

% W

ate

r C

on

ten

t

Gel A Gel B Gel C Gel D

Figure 1. Plot of % water content in Gels A-D at 37 oC in neutral pH environment as a

function of time.

0

20

40

60

80

100

0 50 100 150 200

Time (h)

% W

ate

r C

on

ten

t

Gel E Gel B Gel F Gel D

Figure 2. Comparative plot of % water content in Gels B, D, E and F at 37 oC in neutral

pH environment as a function of time.

113


0.0

0.4

0.8

1.2

0 5 10 15

t1/2

(h1/2

)

Mt/

Min

ifin

ity


Figure 3. Plot of fractional swelling in Gels A-D at 37 oC in neutral pH environment as a

function of the square root of time.

0.0

0.4

0.8

1.2

0 5 10 15t1/2

(h1/2

)

Mt/

Min

fin

ity

Gel B Gel D Gel E Gel F

Figure 4. Comparative plot of fractional swelling in Gels B, D, E and F at 37 oC in

neutral pH environment as a function of the square root of time.

114


-1.5

-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G M

t/ M

infi

nit

y

Gel A Gel B Gel E

Figure 5. Plot of the LOG of fractional swelling as a function of the LOG of time, in the

initial stages of the swelling in Gels A, B and E at 37 oC in neutral pH environment.

-1.5

-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G M

t/ M

infi

nit

y

Gel C Gel D Gel F


initial stages of the swelling in Gels C, D and F at 37 oC in neutral pH environment.

The kinetics of diffusion, namely, Fickian and non-Fickian diffusion mechanisms in

swellable polymers have been discussed in Section 1.4.2.3.1.2. The diffusion mechanisms

in polymers can be observed by plotting the fractional swelling data as a function of the

square root of time. A LOG plot derived from this information gives quantitative

information on the diffusion kinetics.

115


Equation 2, a mathematical expression where y and x are the values on the y and x-axis

respectively, c is the intercept on y-axis and m is the slope, derived from the LOG graphs

illustrated in Figures 5 and 6.

Equation 2 y mx c= ±

The value of the slope represents the parameter n in the power law expression (Equation

3) where Mt is the swollen mass at time t and M∞ is the swollen mass at equilibrium. The

k value is a constant specific to the polymer/solvent system and the parameter n defines

the diffusion mechanism in operation in the polymer matrix.

Mt/M∞ = k t n Equation 3

The slope (n) values calculated from Figures 5 and 6 for the hydrogels A-F are presented

Table 3.

Table 3. Characteristic exponential n values for diffusion in hydrogels in neutral

medium

Hydrogels n values

Gel A 0.39 + 0.05

Gel B 0.47 + 0.02

Gel C 0.53 + 0.01

Gel D 0.62 + 0.01

Gel E 0.41 + 0.02

Gel F 0.63 + 0.01

116


Table 4. Swelling test on hydrogels in isotonic (pH 7.4) environment at 37 oC



0.00 0.0 0.0 0.0 0.0 0.0 0.0

0.17 6.9 9.0 14.3 28.6 9.5 43.3

0.33 8.6 11.9 18.8 36.7 12.6 49.3

0.50 9.8 13.5 22.2 42.2 15.5 56.4

0.67 11.1 14.9 24.5 46.3 15.8 60.7

0.83 11.9 16.2 27.4 49.6 17.1 64.4

1.00 12.7 17.5 29.2 52.1 18.2 66.1

2.00 16.8 21.7 36.7 63.4 23.6 73.8

3.00 20.2 24.4 41.8 69.3 25.5 78.6

4.00 21.8 26.5 45.5 74.0 27.2 80.6

5.00 23.8 28.2 48.3 75.9 28.8 82.6

7.00 26.7 30.8 52.5 79.7 31.2 84.9

9.00 27.5 32.5 55.8 81.8 33.2 86.4

12.00 31.2 34.2 58.6 83.9 34.5 88.0

24.03 33.8 37.1 62.8 87.1 36.7 90.4

48.03 36.2 38.3 64.0 88.3 37.8 91.7

72.02 35.0 39.0 63.7 88.3 38.1 92.0

96.00 35.2 39.3 63.5 88.1 38.2 91.9

120.00 35.0 39.8 62.7 87.7 38.4 91.8

144.00 34.9 39.7 61.4 86.8 39.1 91.5

170.00 35.0 39.7 61.5 87.1 39.9 91.2



117


Graphical representations of the swelling behaviour observed in the HEMA-NVP

hydrogels in isotonic (pH 7.4) environment are illustrated in Figures 7 -13.

0

20

40

60

80

100

0 50 100 150 200Time (h)

% W

ate

r C

on

ten

t


Figure 7. Plot of % water content in Gels A-D at 37 oC in isotonic (pH 7.4) environment

as a function of time.

0

20

40

60

80

100

0 50 100 150 200

Time (h)

% W

ate

r C

on

ten

t


Figure 8. Comparative plot of % water content in Gels B, D, E and F at 37 oC in isotonic

(pH 7.4) environment as a function of time

118


0

20

40

60

80

100

0 50 100 150 200

Time (h)

% W

ate

r C

on

ten

t

neutral isotonic

Figure 9. Comparative plot of % water content in 50 HEMA: 50 NVP hydrogel at 37 oC

in isotonic (pH 7.4) and neutral environments as a function of time.

0.0

0.4

0.8

1.2

0 5 10 15t1/2

(h1/2

)

Mt/

Min

fin

ity


Figure 10. Plot of fractional swelling in Gels A-D at 37 oC in isotonic (pH 7.4)

environment as a function of the square root of time.

119


0.0

0.4

0.8

1.2

0 5 10 15

t1/2

(h1/2

)

Mt/

Min

fin

ity


Figure 11. Comparative plot of fractional swelling in Gels B, D, E and F at 37 oC in

isotonic (pH 7.4) environment as a function of the square root of time

-1.5

-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G M

t/ M

infi

nit

y

Gel A Gel B Gel E


initial stages of the swelling in Gels A, B and E at 37 oC in isotonic (pH 7.4)

environment.

120


-1.5

-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G M

t/ M

infi

nit

y

Gel C Gel D Gel F


initial stages of the swelling in Gels C, D and F at 37 oC in isotonic (pH 7.4) environment

The slope (n) values for hydrogels A-F calculated from Figures 12 and 13 are presented

in Table 5.

Table 5. Characteristic exponential n values for diffusion in hydrogels in isotonic

(pH 7.4) medium

Hydrogels n values

Gel A 0.39 + 0.01

Gel B 0.41 + 0.01

Gel C 0.50 + 0.01

Gel D 0.57 + 0.01

Gel E 0.42 + 0.01

Gel F 0.56 + 0.01

121



The T1 and T2 relaxation measurements were performed on fully hydrated hydrogel

samples. Table 6 presents the NMR relaxation time data for restricted water in the

hydrated hydrogel samples of varying HEMA and NVP content.

Table 6. 1H NMR relaxation T1 and T2 values

Relaxation times (s)

Hydrogels T1 T2

A 0.45830 + 0.06687 0.00321 + 0.00001

C 0.66080 + 0.03113 0.03678 + 0.00077

D 1.86500 + 0.00917 1.01300 + 0.00385

3.3.4. Texture Analysis

Stress relaxation (SR) evaluation was also conducted on the hydrated hydrogel samples of

varying HEMA and NVP content. The hydrogel samples were subjected to a load of

certain magnitude for a period of 30 seconds and then allowed to relax. The stress

relaxation phenomenon is related to the viscoelasticity of the polymer. The Young’s

modulus (E) values of the samples were computed from the linear portion of the stress-

strain curves. The relative SR and E values were calculated as described in Section 2.6.2.

The SR and E values are summarised below in Table 7.

Table 7. Relative SR and E values of the hydrogel samples

Hydrogel samples Relative SR values E (MPa)

Gel A 0.1302 + 0.0291 0.3572 + 0.0218

Gel B 0.1231 + 0.0182 0.0837 + 0.0030

Gel C 0.1020 + 0.0070 0.0468 + 0.0071

Gel D 0.0347 + 0.0109 0.0135 + 0.0003

Gel E 0.1447 + 0.0095 0.0844 + 0.0026

Gel F 0.0424 + 0.0085 0.0096 + 0.0001

122


3.3.5. Experimental Drug Release Results

The experimental data expressed as fractional drug release at specific time intervals are

presented in Table 8. The fractional drug release values were calculated as described in

Section 2.7. Graphical representations of the drug release behaviour in the HEMA-NVP

hydrogels are illustrated in Figures 14 – 19.

Table 8. Theophylline release from HEMA - NVP hydrogels at 37 oC in neutral pH

environment

Average fractional theophylline release values at time (t)


0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.17 0.07 0.09 0.17 0.20 0.10 0.22

0.33 0.10 0.15 0.25 0.31 0.15 0.32

0.50 0.12 0.18 0.33 0.39 0.19 0.41

0.67 0.14 0.21 0.38 0.44 0.22 0.47

0.83 0.16 0.24 0.44 0.50 0.25 0.52

1.00 0.18 0.25 0.48 0.55 0.28 0.56

2.00 0.24 0.35 0.65 0.76 0.40 0.74

3.00 0.29 0.44 0.76 0.83 0.48 0.84

4.00 0.35 0.50 0.83 0.88 0.55 0.89

5.00 0.40 0.54 0.87 0.88 0.61 0.92

7.00 0.44 0.62 0.88 0.89 0.71 0.94

9.00 0.49 0.69 0.90 0.90 0.77 0.94

12.00 0.56 0.74 0.92 0.92 0.83 0.96

24.00 0.73 0.83 0.94 0.93 0.95 0.94

48.00 0.83 0.88 0.93 0.94 0.96 0.95



123


0.0

0.4

0.8

1.2

0 10 20 30 40 50 6

Time (h)

Fra

ctio

nal

Dru

g R

elea

se

0


Figure 14. Plot of fractional release of theophylline from Gels A-D at 37 oC in neutral pH

environment as a function of time.

0.0

0.4

0.8

1.2

0 10 20 30 40 50 6

Time (h)

Fra

ctio

nal

Dru

g R

elea

se

0


Figure 15. Comparative plot of fractional release of theophylline from Gels B, D, E and F

at 37 oC in neutral pH environment as a function of time.

124


0.0

0.4

0.8

1.2

0 2 4 6 8

t1/2

(h1/2

)

Fra

ctio

nal

dru

g r

elea

se


Figure 16. Plot of fractional release of theophylline from Gels A-D at 37 oC in neutral pH


0.0

0.4

0.8

1.2

0 2 4 6 8

t1/2

(h1/2

)

Fra

ctio

nal

Dru

g R

elea

se


Figure 17. Comparative plot of fractional release of theophylline from Gels B, D, E and F

at 37 oC in neutral pH environment as a function of the square root of time

125


-1.5

-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G F

DR

Gel A Gel B Gel E

Figure 18. Plot of the LOG of fractional drug released (FDR) as a function of the LOG of

time, in the initial stages of the theophylline release from Gels A, B and E at 37 oC in

neutral pH environment

-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G F

DR

Gel C Gel D Gel F


time, in the initial stages of the theophylline release from Gels C, D and F at 37 oC in


126


Table 9 presents the slope (n) values calculated from Figures 18 and 19.

Table 9. Characteristic exponential n values for theophylline release from hydrogels

in neutral medium

Hydrogels n values

Gel A 0.51 + 0.03

Gel B 0.52 + 0.02

Gel C 0.61 + 0.01

Gel D 0.56 + 0.02

Gel E 0.55 + 0.01

Gel F 0.55 + 0.02

127


3.4. Discussion


HEMA and NVP based hydrogels of varying monomer compositions were prepared using

a photoinitiator, Irgacure 819, which was found to be a very efficient photoinitiator. The

photolytic decomposition of Irgacure 819 into subsequent free radicals is illustrated in

Scheme 1. Hydrogel formulations upon subjection to UV radiation were successfully

polymerised in ~ 30 minutes at a low dose rate of 9.65 x 10-2

J s-1

. The monomer

formulations containing a higher percentage of HEMA polymerised faster than those

containing high percentage of NVP.

OO

P

O

O

P

O

O

O

P

O+

+

hv

hv

Irgacure 819

Scheme 1. Photolysis of Irgacure 819 under the influence of UV light

128


The results on the rate of polymerisation of the hydrogels shed light on the relative

reactivity of the monomers. The free radical formations of the monomers under the

influence of UV light are shown below in Schemes 2 and 3.

N

CH2

C

H2

CH2

OCH

CH2 CHC

H2

R N

CH2

C

H2

CH2

O

R+hv

NVP 2o Free Radical

Scheme 2. Free radical formation of NVP

O

CH3

CH2

O

C

H2

CH

2

OHR O

CH3

O

C

H2

CH

2

OHCH2

R

+hv

HEMA 3o Free Radical

Scheme 3. Free radical formation of HEMA

Lai [19,38], Perera and Shanks [20] have reported the efficient reactivity of HEMA over

NVP. They further suggest that NVP and HEMA do not polymerise well based on

difference of reactivity of the two monomers. According to them, HEMA being a more

reactive monomer tends to enter the polymer more quickly by homopolymerising then

NVP. As a result, NVP is not fully consumed by HEMA and thus it may remain in

partially homopolymerised or unreacted state where it is susceptible to leaching. The

secondary free radical of NVP is an electron donor while the tertiary free radical of

HEMA is electron deficient, thus an acceptor (Schemes 2 and 3). HEMA and NVP can

possibly form a donor/acceptor pair. Scheme 4 illustrates a possible donor/acceptor

interaction between HEMA and NVP.

129


N

O

Donor (D) Acceptor (A)

hv

DA interaction

(NVP) (HEMA)

O

OOH

O

OOH

N

O+

Scheme 4. Interaction of HEMA and NVP

However, the copolymerisation reaction between of NVP and HEMA tends to favour the

homopolymerisation of HEMA over the copolymerisation reaction. The relative reactivity

of HEMA could be related to the number of α-hydrogens available for abstraction in

comparison to NVP.

O

CH3

O

OHCH2

R H

HH

H

CH

2

R N

CH2

C

H2

O

H

H

H

HEMA NVP

Figure 20. Structures of HEMA and NVP radicals with available abstractable hydrogens

Figure 20 illustrates the number of α-hydrogens available on the structure on HEMA

radical and NVP radical. HEMA has four abstractable hydrogens over NVP, which has

only three abstractable hydrogens. Thus HEMA tends to preferably undergo

intramolecular hydrogen abstraction leading to favourable homopolymerisation reaction.

However, the presence of an efficient photoinitiator such as Irgacure 819 enhances the

rate of polymerisation providing effective co-polymerisation of HEMA and NVP. All the

hydrogel samples formed from the photopolymerisation process were clear, transparent

and yellowish in colour. The yellow colour is a characteristic of the photoinitiator.

130


3.4.2. Swelling and Drug Release Investigations

Fickian or non-Fickian diffusion kinetics was used to characterize the swelling and drug

release phenomena in the HEMA-NVP hydrogels. Fickian and non-Fickian diffusion

behaviour have been discussed in detail in Section 1.4.2.3.1.2. The swelling action in

polymers is generally time dependent, however, a time independent diffusion is desirable

in drug release applications which gives a zero order release. Diffusion kinetics could be

determined according to a power law expression (Equation 3).

As described in Section 1.4.2.3.1.2, a n value of 0.5 is indicative of Fickian diffusion

while a value of n higher than 0.5 represents non-Fickian diffusion behaviour which could

be further described as anomalous (0.5 < n < 1), case II (n = 1) and super case II (n > 1).

The process of the medium diffusing into the polymer matrix and the incorporated solute

diffusing out of the polymer is simultaneous. The drug release in swellable

macromolecular networks is dependent on the degree of swelling of the network.

Influential factors such as the nature and composition of monomers used in the synthesis

govern the degree of swelling in the hydrogels. Furthermore, the nature of swelling agent

is also an influential contributing factor to swelling efficiency of the hydrogel.

3.4.2.1. Hydrogel Swelling Behaviour

3.4.2.1.1. Effect of the Monomeric Composition on Swelling

Most of these polymer samples when immersed in water produced clear colourless

hydrogels. PNVP hydrogel was found to be water soluble due to insufficient crosslinking

of the macromolecular network. Uncrosslinked PNVP has been reported in the literature

to be water-soluble [2,6,7]. NVP is known to be an extremely hydrophilic monomer. For

polymers, which are highly hydrophilic, and of relatively low molecular weight, the

equilibrium state is an aqueous solution of the polymer.

A gradual increase in water uptake was observed in PHEMA (Gel A), 80 HEMA-20 NVP

(Gel B) and 50 HEMA-50 NVP (Gel C) hydrogels in neutral pH environment at 37 oC

yielding equilibrium water content (EWC) values of 36.6 %, 41.9 % and 70.9 %

respectively (Table 2, Figure 1). The hydrogels showed a linear mass increase upon

131


swelling as a function of the square root of time (Figure 3) indicating typical Fickian

diffusion kinetics. This was confirmed by the LOG plots (Figure 5 and 6, Table 3) from

which average n values (Equation 2, 3) of 0.39 for hydrogel A, 0.47 for hydrogel B and

0.53 for hydrogel C were calculated. In the later stages of the swelling process, Case II

diffusion behaviour prevailed, which was indicated by a gradual decrease in the rate of

water uptake by the hydrogels. At this stage the swelling process becomes time

independent where the parameter, n = 1. The rate of polymer chain relaxation at this point

is equal to the rate of solute transport [29].

The 80 NVP-20 HEMA (Gel D) hydrogels however, showed an exponential increase in

water uptake in the first hour followed by a gradual decrease in the swelling around 48

hours yielding an EWC value of 91.6 % (Table 2, Figure 1). The diffusion kinetics in this

hydrogel system was a characteristic of a high order non-Fickian (anomalous) diffusion in

the initial stages followed by case II diffusion in the later stages (Figure 3). Non-Fickian

diffusion behaviour was confirmed by a LOG plot (Figure 6, Table 3), which yielded an

average n value of 0.62. The high water content of the hydrogels containing high

percentage of NVP is due the high hydrophilicity/polarity nature of NVP. Korsmeyer and

Peppas [21] have reported non-Fickian swelling kinetics in copolymers of HEMA and

NVP whilst Bhardwaj et al [31] reported Fickian diffusion kinetics in copolymers of

HEMA and NVP. These researchers have made use of copolymers of varying ratios of

HEMA and NVP.

Bhardwaj et al [31] used high HEMA containing copolymers while Korsmeyer and

Peppas used high NVP containing copolymers. Franson and Peppas [32] have suggested

the dependence of diffusion kinetics on the copolymer composition of HEMA and NVP.

Thus the present results obtained for diffusion kinetics of the hydrogels under study

ranging from Fickian to non-Fickian with increasing NVP content is in agreement with

these researchers.

Water was added to selected formulations in 10 % v/v to enhance the porosity of the

network. The 80 HEMA-20 NVP hydrogel and 20 HEMA-80 NVP hydrogel were the

chosen systems. Thus upon addition of 10 % v/v of H2O, the final formulations of 75

132


HEMA-15 NVP-10 H2O (Gel E) and 15 HEMA-75 NVP-10 H2O (Gel F) were obtained.

Hydrogels E and F upon swelling displayed Fickian and non-Fickian (anomalous)

behaviour with EWC values of 49.3 % and 94.3 % respectively (Tables 2 and 3, Figures

2, 4 and 6). A gradual water uptake was observed in Gel E, however, the efficiency of

swelling was higher than that of 80 HEMA-20 NVP hydrogel. Gel F displayed

exponential swelling in the initial stages with enhanced swelling efficiency in comparison

to 80 NVP-20 HEMA hydrogel. Water acts as a plasticiser for many hydrophilic

polymers and if they can exhibit hydrogel character, the backbone and the side chains

exhibit relatively unrestricted rotational mobility [39].

3.4.2.1.2. Effect of the Ionic Strength on Swelling

PHEMA, copolymers of HEMA-co-NVP and copolymers of HEMA-co-NVP with 10 %

v/v H2O were subjected to swelling in isotonic, pH 7.4 environment at 37 oC. The

hydrogels displayed reduced swelling behaviour in the isotonic environment. Hydrogels

A-F displayed EWC values of 35.0 %, 39.7 %, 61.5 %, 87.1 %, 39.9 % and 91.2 %

respectively (Table 4, Figures 7 and 9). The diffusion kinetics was also influenced by the

presence of ions in the environment. A typical Fickian diffusions kinetics was observed to

be in operation with the exception of hydrogels D and F, which adhered to slight non-

Fickian (anomalous) behaviour with n values of 0.57 and 0.56 respectively (Table 5,

Figures 10-13]. Reduction in the swelling efficiency of these non-ionic hydrogels in

isotonic environment could be attributed to the increase in the ionic strength in the

environment, which effectively reduces the polymer mesh thus reducing the swelling

ratio. Another notable behaviour observed particularly in high swelling hydrogels was

that upon reaching EWC value they began to de-swell upon continual swelling

experimentation.

This observation could be attributed to the concentration gradient established between the

restricted ions in the hydrogels and the free ions in the medium. Hence once the

concentration of the restricted ions is higher than the free ions the diffusion direction is

reversed. The absorbed medium with the ions starts diffusing out of the polymer matrix.

This phenomenal diffusion process is repeatedly reversed in order to maintain an

equilibrium between the ionic concentration of restricted and free ions while the hydrogel

133


maintains its maximum swelling capacity for the given environment. This behaviour was

not apparent in low swelling hydrogels due to low swelling activity as that in the case of

high HEMA content hydrogels.

3.4.2.2. Drug Release Studies

A significant variation in the drug release behaviour was observed with varying

monomeric compositions. Hydrogels A-F were tested for theophylline release in neutral

medium at 37 oC. Hydrogels A-F yielded equilibrium drug release (EDR) values of 0.83,

0.88, 0.93, 0.94, 0.96 and 0.95 respectively (Table 8, Figures 14 and 15). A characteristic

rapid release of theophylline was observed in the initial 20 minutes of the experiment.

This rapid release could be described as the burst effect release [29].

PHEMA hydrogel baring a slight initial burst displayed the slowest drug release. The

release kinetics in Gels A and B was that of typical Fickian diffusion behaviour with n

values of 0.51 and 0.52 (Table 9, (16-19). The release profile in the initial stages of

experiment was characterized by a linear increase in the release rate of theophylline as a

function of the square root of time. Hydrogels C-F on the other hand displayed non-

Fickian (anomalous) diffusions kinetics with n value of 0.61, 0.56, 0.55 and 0.55

respectively (Table 9).

The diffusivity of theophylline was observed to increase with increasing water content in

the hydrogel. Hydrogels with high NVP content displayed high theophylline diffusivity.

Thus it could be suggested that the theophylline release was dependent on the swelling

ratio of the polymer. NVP being the more hydrophilic component in the copolymer

contributed to the increased swelling efficiency of the hydrogel leading to an increase in

the polymer mesh size thus allowing rapid diffusion of the incorporated theophylline from

the network. The dependence of solute diffusion on the polymer mesh is well documented

[40-43].

Peppas and Korsmeyer [21] in their study on the swelling-drug release kinetics in

poly(HEMA-co-NVP) hydrogels observed increased diffusivity of theophylline from

networks with increasing NVP content. Teijón et al [13] and Trigo et al [18] from their

134


study on the release kinetics in PHEMA hydrogels observed Fickian release mechanism

characterized by a slow linear release of the drug as a function of the square root of time.

The release kinetics observed for PHEMA and poly(HEMA-co-NVP) hydrogels in the

present study is in agreement with these researchers.


The relaxation times T1 and T2 reflect the dynamics of water molecules in the polymer

matrix. The copolymers of HEMA and NVP displayed longer relaxation times with

longer T1 and T2 values with increasing NVP content in the copolymer (Table 6).

PHEMA on the other hand displayed significantly shorter relaxation times. The

experimental data on the relaxation time measurements indicated that the T2 values were

more sensitive to the variation in the polymer composition and their relative water content

than the T1 values.

A short T2 indicates high mobility of water in the polymer matrix, thus indicating that the

water present is interfacial water, which is repelled by the relative hydrophobicity of the

polymer and is effectively mobile. The increased mobility of water in PHEMA sample

could also be attributed to a relatively short T1 value. Shorter T1 values result from rapid

tumbling of free water molecules in the polymer matrix. Due to minimum hydrogen

bonding or hydrophilic attraction between polymer and the water molecules, a faster

rotational motion (tumbling) of the water molecules is observed in the matrix of PHEMA.

The HEMA-co-NVP copolymers have a relatively longer T2 times. This is indicative of

the fact that these copolymers have increased hydrophilicity in comparison to PHEMA.

Furthermore, longer T2 values in the copolymers with higher NVP content could be

attributed to the highly hydrophilic nature of NVP. The study suggested that the

proportion of restricted water, which is due to high hydrogen bonding, is significantly

higher in HEMA-co-NVP hydrogels in comparison to PHEMA hydrogel.

The T1 and T2 relaxation data also correlate to the swelling data and are in agreement with

the deduced diffusion kinetics of the hydrogels under study. Shorter relaxation times are

indicative of Fickian diffusion kinetics while longer relaxation times are indicative of

135


non-Fickian diffusion kinetics. Barbieri et al [34], Ghi and Hill [30] in their study on the

diffusion kinetics of HEMA based hydrogels have reported similar results. They also

suggest a direct relevance of relaxation times to the swelling kinetics in polymers.

3.4.4. Texture Analysis (Stress Relaxation)

Texture analysis experiments revealed that the hydrogels under study were viscoelastic.

However, the hydrogels displayed varying degrees of viscoelasticity. The SR values

(Table 7) of the hydrogels decreased with increasing NVP content of the hydrogels.

PHEMA showed a SR value of 0.1302. In comparison, hydrogels D and F displayed

relatively low SR values of 0.0347 and 0.0424 respectively. The viscoelasticity of a

polymer can be described in terms of local frictional forces encountered by a short

segment of a moving chain, together with additional entanglement coupling to other

chains. The entanglements profoundly inhibit long-range conformational rearrangements

and separation of chains from each other.

The SR data suggested that the stress relaxation process in high HEMA content hydrogels

(Gels A, B, C and E) was relatively slower in comparison to high NVP content hydrogels

(Gels E and F). The longer stress relaxation time in high HEMA content hydrogels could

be attributed to the compact nature of the networks. The compactness of the networks

gives rise to increased local frictional force restricting the conformational rearrangement

and separation of the chains.

Hong et al [37], Chirila and Hong [44] recently reported stress relaxation behaviour in

HEMA and NVP based hydrogels. They observed a decrease in the SR value with

increasing NVP content in the hydrogels. The SR data from the present study is in

agreement these researchers. The study suggests that high HEMA content hydrogel

networks (Gels A, B, C and E) are more elastic whilst the hydrogels (Gel D and F) with

high NVP content hydrogel networks have characteristics similar to that of a viscous

fluid.

PHEMA displayed the highest Young’s modulus value of 0.3572 MPa followed by

hydrogels B (0.0837 MPa), E (0.0844 MPa), C (0.0468 MPa), D (0.0135 MPa) and F

136


(0.0096 MPa). The Young’s modulus values decreased in the hydrogels with increasing

NVP content suggesting a decrease in the stiffness of the hydrogels. Davis and Huglin

[45] studied the mechanical properties of copolymers of HEMA and NVP of varying

compositions and found increasing stiffness with increasing HEMA content of the

copolymer. The trend of stiffness in relation to HEMA-NVP composition observed in the

present study is in agreement with Davis and Huglin. Furthermore, it is indicative from

this study that there exists a trend between Fickian and non-Fickian diffusion to the SR

values. Hydrogels, which displayed non-Fickian diffusion kinetics, were observed to

behave more like viscous fluid whilst the opposite was observed for hydrogels displaying

Fickian diffusion kinetics. However, the data obtained in the present study does not

sufficiently confirm this trend.

3.5. Conclusions

Irgacure 819 was found to efficiently polymerise NVP and HEMA. High HEMA content

polymers exhibited shorter time of polymerisation in comparison to high NVP content

polymers. Experimental swelling data revealed that the diffusion kinetics in HEMA-NVP

hydrogels range from Fickian to high order non-Fickian (anomalous) diffusion behaviour

in the earlier stages of the experiment followed by case II diffusion in the later stages.

Inclusion of water into the hydrogel formulation resulted in an increased swelling activity

of hydrogels. Presence of ionic environment was found to reduce the swelling efficiency

of the hydrogels.

The proton NMR relaxation times, T1 and T2 were found to correlate with the diffusion

kinetics in polymers. Longer relaxation times were observed in hydrogels, which

displayed non-Fickian diffusion behaviour while hydrogels, which adhered to Fickian

diffusion kinetics displayed shorter relaxation times. Longer relaxations times resulted

from large proportion of restricted water due to enhanced hydrophilic nature of the

hydrogels. Hydrophobic interactions between the polymer and water molecules resulted

in interfacial water, which was increasingly mobile leading to shorter relaxation times.

The texture analysis indicated that hydrogels understudy were viscoelastic with longer

stress relaxation time in high HEMA content hydrogels. The Young’s modulus values

137


indicated that hydrogels with high HEMA content were more rigid. The present study

suggests a possible trend between the SR data and the diffusion kinetics exhibited by the

hydrogels, however the present study does not sufficiently confirm this trend.

The drug release experiments revealed that only PHEMA hydrogel adhered to Fickian

transport mechanism in releasing theophylline into the neutral pH environment at 37 oC.

The copolymers of HEMA-co-NVP and copolymers with added plasticiser, water

displayed non-Fickian diffusion behaviour. The burst effect release of the drug was

observed in the initial stages of the release experiment followed by linear release profile.

The rate of theophylline release gradually increased with increase in the concentration of

the hydrophilic monomer, NVP suggesting direct dependence of release kinetics on the

swelling ratio of the polymers.

138


3.6. References

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Fundamentals, Peppas, N. A., ed., vol I, CRC Press, Inc., Florida, pp. 1-25,

(1986).

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5. Park, H., Park, K., “Hydrogels and Biodegradable Polymers for Bioapplications”,

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Washington, D.C., pp. 2-10, (1996).


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(1976).


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8. Brannon-Peppas, L., Peppas, N. A., Biomaterials, 11, 635-644, (1990).

9. Hsieh, K.-H., Young, T.-H., ”Polymer Materials Encyclopedia”, Salamone, J. C.,

ed., vol 5, CRC Press, New York, pp. 3087-3091, (1996).

10. Clayton. A. B., Chirila, T. V., Dalton, P. D., Polymer. Int., 42, 45-56, (1997).

11. Hill, D. J. T., Lim, M. C. H., Whittaker, A. K., Polymer Int., 48, 1046-1052,

(1999).


13. Teijón, J. M., Trigo, R. M., García, O., Blanco, M. D., Biomaterials, 18, 383-388,

(1997).

14. Hoch, G., Chauhan, A., Radke, C. J., J. Membrane Sci., 214, 199-209, (2003).

15. Jeyanthi, R., Rao, K.P., J. Controlled Release, 13, 91-98, (1990).

16. Yıldırmaz, G., Akgöl, S., Arıca, M. Y., Sönmez, H., Denizli, A., React. Func.

Polym., 56, 103-110, (2003).

17. Duncan, A. C., Boughner, D., Campbell, G., Wan, W. K., Eur. Polym. J., 37,

1821-1826, (2001).

139


18. Trigo, R. M., Blanco, M. D., Teijón, J. M., Sastre, R., Biomaterials, 15, 1181-

1186, (1994).

19. Lai, Y.-C., J. Appl. Polym. Sci., 66, 1475-1484, (1997).

20. Perera, D. I., Shanks, R. A., Polymer Int., 39, 121-127, (1996).


22. Malak, M., Hill, D. J. T., Whittaker, A. K., Polymer. Int., 52, 1740-1748, (2003).

23. Malak, M., Hill, D. J. T., Whittaker, A. K., Polymer. Int., 53, 235, (2004).

24. El-Din, H. M. M. N., Maziad, N. A., El-Naggar, A. W. M., J. Appl. Polym. Sci.,

91, 3274-3280, (2004).

25. Corkhill, P. H., Jolly, A. M., Ng, C. O., Tighe, B., Polymer, 28, 1758-1766,

(1987).

26. Bruck, S. D., J. Biomed. Mat. Res., 7, 387-404, (1973).

27. Ratner, B. D., “Comprehensive Polymer Science” – The Synthesis,

Characterisation, Reactions & Applications of Polymers, Aggarwal, S. K., ed., vol

7, Pergamon Press, Oxford, pp. 201-247, (1989).

28. Peppas, N. A., Khare, A. R., Adv. Drug Deliv. Rev., 11, 1-35, (1993).



pp. 109-135, (1987).

30. Ghi, P. Y., Hill, D. J. T., Maillet, D., Whittaker, A. K., Polymer Commun., 38,

3985-3989, (1997).

31. Bhardwaj, Y. K., Sabharwal, A., Majali, A. B., J. Polym. Mater., 11, 29-34,

(1994).

32. Franson, N. M., Peppas, N. A., J. Appl. Polym. Sci., 28, 1299-1310, (1983).

33. Khare, A., Peppas, N. A., Polymer, 34, 4736-4739, (1993).

34. Barbieri, R., Quagila, M., Delfini, M., Brosio, E., Polymer, 39, 1059-1066,

(1998).

35. Yung, K.-T., Magnetic Resonance Imaging, 21, 135-144, (2003).

36. Kulicke, W. M., Nottelmann, H., “Polymers in Aqueous Media” – Performance

Through Association, Glass, J. E., ed., American Chemical Society, Washington,

D.C., pp.15-44, (1989).

140


37. Hong, Y., Chirila, T. V., Cuypers M. J. H., Constable, I. J., J. Biomater. Appl., 11,

135-181, (1996).

38. Lai, Y.-C., J. Polym. Sci. Part A: Polym. Chem., 35, 1039-1046, (1997).

39. Ratner, B. D., “Hydrogels in Medicine and Pharmacy”- Fundamentals, Peppas, N.

A., ed., vol I, CRC Press, Inc., Florida, pp. 85-94, (1986).


(1969).

41. Wood, J. M., Attwood, D., Collet, J. H., J. Pharm. Pharmacol., 34, 1-4, (1982).


(1995).


(1995).

44. Chirila, T. V., Hong, Y., Polym. Int., 46, 183-195, (1998).

45. Davis, T. P., Huglin, M. B., Macromolecules, 22, 2824-2829, (1989).

141


3.1. Introduction 107

3.2. Experimental Procedure 110

3.3. Results 110

3.3.1. Hydrogel Formation 110

3.3.2. Experimental Swelling Results 111


3.3.4. Texture Analysis 122

3.3.5. Experimental Drug Release Results 123

3.4. Discussion 128


3.4.2. Swelling and Drug Release Investigations 131

3.4.2.1. Hydrogel Swelling Behaviour 131

3.4.2.1.1. Effect of the Monomeric Composition on Swelling 131

3.4.2.1.2. Effect of the Ionic Strength on Swelling 133

3.4.2.2. Drug Release Studies 134


3.4.4. Texture Analysis (Stress Relaxation) 136

3.5. Conclusions 137

3.6. References 139

142

Chapter 4: Photoinitiator-Free Photopolymerisation of N-Substituted Maleimides and N-Vinylpyrrolidinone Hydrogels

4.1. Introduction

Rapidly polymerisable systems, which respond to light without the necessity of adding a

photosensitiser has been one of the most extensively explored frontiers in photocurable

free radical polymerisation process in recent years [1-17]. The widespread popularity of

such systems is attributed the disadvantages associated with photoinitiators (PI).

The most critical concern in the use of PI is that they are only partially consumed in the

polymerisation process since relatively high concentrations are utilized to ensure adequate

light absorption. This unused PI leads to considerable leachable undesirable small

molecule toxic contaminants in the polymer matrix [1,7]. The PI in the polymer matrix

may cause undesirable degradation leading to an early mechanical failure of the polymer

network thus limiting its possible applications. Generation of coloured and harmful side

products in addition to the primary radical photoproducts of the initiation of the

polymerisation is also a drawback factor in the use of PI [1,7].

The development of these photoinitiator-free photocurable systems has led to the

development of more versatile polymers with enhanced stability and superior

performance. A range of olefins, which function as acceptors and donors could be utilized

to achieve polymerisation in the absence of added photoinitiator. In recent years efficient

photoinduced free radical polymerisation by excited state acceptor monomers, such as N-

substituted maleimides has been shown in numerous donor/acceptor pair combinations.

Jönsson et al [2,8,11,14], Morel et al [13], and Decker et al [5,16] in their respective

study on donor/acceptor pair systems have reported successful use of a range of vinyl

ethers and N-vinyl pyrrolidinone (NVP) as donors combined with a range of N-

substituted maleimides as acceptors. Yamada et al [18] initially reported that maleimides

(MI) and their N-substituted derivatives could homopolymerise in the absence of added

initiator when photo-radiated.

Maleimides undergo a transition from ground to an excited triplet state when subjected to

ultraviolet radiation [17,19]. The excited N-substituted maleimides, which have molar

absorptivity of ~700 M-1

cm-1

have been reported to readily abstract available hydrogens

142


from either a vinyl ether backbone or undergo an intra-molecular hydrogen abstraction

process [1,11,12,16]. The resulting free radicals initiate a rapid alternating

copolymerisation [1,2,5,9,10-17]. The excitation and hydrogen abstraction processes of

the maleimides have been described in detail in Section 1.2.6.

MI*

DH

MI

MI*

VE

MI

D

P

P

P

P DH PH D

P

DH

+ +

- Growing polymer chain

- Hydrogen donor

Scheme 1. Interaction of a typical MI with a vinyl ether (VE)

The excited state maleimides besides abstracting available hydrogens from the present co-

monomer such as vinyl ether could also abstract hydrogens from an external additive.

Introducing additives such as hydrogen donors to the donor/acceptor formulation could

enhance the process of donor/acceptor polymerisation initiation [1,2,4,5,9-17,20-22].

Hydrogen donors contain abstractable labile hydrogens that are located adjacent to

heteroatoms such as oxygen, nitrogen and sulfur. An excited state maleimide interacting

with a vinyl ether in the presence of a hydrogen donor is illustrated in Scheme 1 [5].

The unique feature of such systems is their ability to function as the initiator and as well

as the co-monomer in the formulation [9,21,22]. Although the photoinitiator-free

polymerisation process is not as rapid as formulations with added external photoinitiator,

143


they are rapid enough to be potentially useful in various applications. Formulations

containing N-substituted maleimides are transparent at wavelengths above 300 nm [7].

This is due to the replacement of N-substituted maleimides by succinimide

chromophores, which have virtually no ultraviolet absorbance at wavelengths greater than

300 nm [1,7]. This ability contributes to a greater stability of such systems in prolonged

ultraviolet exposed environments.

Despite the availability of this photoinitiator- free technology for quite some time, it has

never been utilized to synthesize potential biopolymers. Photoinitiators are also known to

be significant contributors towards the toxicity of finished polymers, thus make

photoinitiator-free formulations ideal candidate for biomedical applications [7]. In this

present work the author has made use of donor/acceptor pairs involving a range of water-

soluble N-hydroxyalkyl maleimides as moderately strong acceptors and NVP as a strong

donor monomer to synthesize polymers, which could function as hydrogels for drug

delivery.

This part of the study firstly involved the evaluation of the efficiency of copolymerisation

of NVP with a series of N-hydroxyalkyl maleimides namely, N-hydroxymethyl

maleimide (HMMI), 2-hydroxy-N-ethyl maleimide (HEMI), 3-hydroxy-N-propyl

maleimide (HPrMI) and 5-hydroxy-N-pentyl maleimide (HPMI). A Differential

Photocalorimetric (DPC) technique was used to investigate the relative kinetics of these

donor/acceptor pairs. Glucose and glucosamine hydrochloride (HCl) were utilized as

added hydrogen donors to enhance the rate of polymerisation.

Suitable donor/acceptor pairs for the initiation of polymerisation were evaluated based on

kinetics data obtained from the DPC measurements. In the synthesis of hydrogels,

comparatively less hydrophilic monomers such as 2-hydroxyethyl methacrylate (HEMA)

and N-vinyl caprolactam (NVC) were also utilized as additional monomers to vary the

swelling capacity of the polymers. Results on photopolymerisation of hydrogels for

sustained drug delivery applications via photoinitiator-free process involving

donor/acceptor interaction have been published by the author in the duration of this

course [20-25].

144



The DPC technique was used to evaluate the efficiency of complex formation between

the donor/acceptor pairs. Experimental specifications and detailed procedure of DPC

measurements are outlined in Sections 2.2.2.5 and 2.8. The hydrogels formed through this

photoinitiator-free process were subjected to swelling drug release experiments in neutral

pH environment at 37 oC. The effect of ionic strength on the swelling ability of hydrogels

in an isotonic (pH 7.4) environment was also evaluated. Three model drugs, theophylline,

thiamine hydrochloride (vitamin B1) and Mn-tetrahydroxyphenyl porphyrin (Mn-TPP-

OH) were utilized for drug release investigations on the hydrogels.

Experimental procedures for the synthesis of photoinitiator-free hydrogels based on N-

hydroxyalkyl maleimides, NVP, HEMA and NVC are outlined in detail in Sections

2.5.2.1-2.5.2.2. Experimental specifications and procedure of swelling-drug release

experiments have been described in Sections 2.6.1 and 2.7. UV-vis spectroscopy was

utilized for the quantitative drug release measurements.

4.3. Results

4.3.1. DPC Measurements

The DPC measurements were performed in N2 atmosphere at an isothermally controlled

temperature of 15 oC under a light intensity of 55 mW cm

-2. The reaction temperature of

15 oC was chosen to eliminate undesirable evaporation of volatile components in the

formulation, which would contribute to inconsistent results. The presence of nitrogen

eliminates atmospheric oxygen, which is known to inhibit the polymerisation reaction.

The maleimides, HMMI, HEMI, HPrMI were each formulated with NVP. The

formulations were exposed to the UV light in the DPC equipment with and without the

presence of hydrogen donors, glucose and glucosamine HCl. The DPC results are

expressed in Figures 1- 4. The calculations on the rate of charge-transfer (CT) complex

polymerisation of the hydroxyalkyl maleimides and NVP are presented in Table 1.

145


HMMI-NVP

HPrMI-NVP

HPMI-NVP

-2

8

18H

eat

Flo

w (

W/g

)

0 200 400 600Time (sec)Exo Up

HEMI-NVP

Figure 1. Photo-exotherms of the N-hydroxyalkyl maleimides with NVP in the absence

of an external H-donor

146


HPrMI-NVP-Glucose

HPMI-NVP-Glucose

HMMI-NVP-Glucose

-2

8

18H

eat

Flo

w (

W/g

)


HEMI-NVP-Glucose

Figure 2. Photo-exotherms of the N-hydroxyalkyl maleimides with NVP in the presence

of glucose as the external H-donor

147


HPrMI-NVP-Glucosamine HCl

HPMI-NVP-Glucosamine HCl HMMI-NVP-Glucosamine HCl

-2

8

18H

eat

Flo

w (

W/g

)


HEMI-NVP-Glucosamine HCl

Figure 3. Photo-exotherms of the N-hydroxyalkyl maleimides with NVP in the presence

of glucosamine HCl as the external H-donor

148


No H-donor

Glucosamine HCl

-2

8

18H

eat

Flo

w (

W/g

)


Glucose

Figure 4. Photo-exotherms illustrating the effect of H-donors on the rate of acceptor/

donor interaction between NVP and the N-hydroxyalkyl maleimides.

Table 1. Comparison of the rate of polymerisation of the N-hydroxyalkyl maleimides

and NVP with and without the presence of external H-donors

Rate (J g-1

s-1

) at 15 oC, N2 Systems

1:1 mol No H-donor Glucose Glucosamine HCl

HPMI:NVP 1.18 1.57 2.01

HMMI:NVP 0.73 1.15 1.63

HPrMI:NVP 0.42 0.61 1.39

HEMI:NVP 0.31 0.54 1.08

149


The polymerisation rates between the donor/acceptor pairs were calculated according to

Equation 1 where t is the time taken to reach peak max.

Rate of polymerisation (J g-1

s-1

) = -1Peak max (J g )

t (s) Equation 1


The N-hydroxyalkyl maleimides were formulated with NVP in the presence of glucose

and glucosamine HCl and subjected to UV-radiation. Other monomers, HEMA and NVC

were also included in selected formulations. The observations on the extent of

polymerisation upon applying approximately 9 KJ of radiation dose are described in

Tables 2- 4.

Table 2. Polymerisation status of formulations in the presence of glucosamine HCl

Hydrogel formulation Polymerisation status upon application

of curing dose (~9 KJ)

NVP-HMMI DNP

NVP-HEMI DNP

NVP-HPrMI DNP

NVP-HPMI DNP

Table 3. Polymerisation status of formulations in the presence of glucose



NVP-HMMI Hard, clear gel

NVP-HEMI Hard, clear gel

NVP-HPrMI Hard, clear gel

NVP-HPMI Hard, clear gel

150


Table 4. Polymerisation status of HPMI-NVP formulations with additional

monomers, HEMA and NVC in the presence of glucose



NVP-HPMI-HEMA Hard, clear gel

NVP-HPMI-NVC Hard, clear pale yellow gel

The details of the monomer compositions are described in Section 2.5.2. DNP indicates

that the formulation did not polymerise. The radiation dose was calculated according to

Equation 2 where t is the total radiation time in seconds. The samples were exposed to

UV radiation for approximately 25 hours at a dose rate of 9.6 x 10-2

J s-1

. The dose rate

was calculated as described in Section 2.4.4.4.




The swelling experimental data of the N-hydroxyalkyl maleimide-NVP hydrogels formed

are presented in Tables 5-10. The data reflect the effect on the swelling behaviour of the

hydrogels upon introduction of additional monomers and changes in the nature of the

swelling environment such as presence of an ionic environment. HEMI-NVP hydrogels

disintegrated upon a short exposure to aqueous swelling medium thus further experiments

on this system were not conducted.

151


Table 5. Swelling test on N-hydroxyalkyl maleimide -NVP hydrogels at 37 oC in



Time (hr) Gel A Gel B Gel C Gel D Gel E

0.00 0.0 0.0 0.0 0.0 0.0

0.17 44.0 44.1 47.4 25.9 16.0

0.33 54.2 56.0 58.2 36.0 20.0

0.50 60.6 63.1 64.2 44.4 24.4

0.67 64.9 67.3 68.6 48.8 27.2

0.83 68.1 70.3 71.7 53.3 30.2

1.00 70.6 73.6 73.8 56.3 32.5

2.00 79.5 82.0 81.4 67.4 42.4

3.00 83.6 85.8 85.1 73.0 48.4

4.00 85.9 88.1 87.1 76.8 52.8

5.00 87.4 89.3 88.5 79.3 56.2

7.00 89.3 91.2 90.2 82.3 60.9

9.00 90.5 92.3 91.2 84.4 63.9

12.00 91.6 93.4 92.2 86.3 66.8

24.00 93.5 95.2 93.8 89.9 71.6

48.00 94.4 96.1 94.5 92.1 73.4

72.00 94.7 96.4 94.7 92.5 73.8

96.00 94.8 96.4 94.7 92.7 73.6

120.00 94.8 96.4 94.7 92.8 73.3

144.00 94.8 96.4 94.7 92.8 73.1

170.00 94.8 96.3 94.7 92.7 73.0

Hydrogel compositions: HPMI-NVP (Gel A), HPrMI-NVP (Gel B), HMMI-NVP (Gel

C), HPMI-NVP-NVC (Gel D), HPMI-NVP-HEMA (Gel E)

152




Graphical representations of the swelling behaviour observed in the NVP-maleimide

hydrogels in neutral pH environment are illustrated in Figures 5 -10.

0

20

40

60

80

100

0 50 100 150 200

Time (h)

% W

ate

r C

on

ten

t

Gel A Gel B Gel C

Figure 5. Plot of % water content in hydrogels A - C at 37 oC in neutral pH environment

as a function of time.

0

20

40

60

80

100

0 50 100 150 200

Time (h)

% W

ate

r C

on

ten

t

Gel A Gel D Gel E

Figure 6. Plot of % water content in hydrogels A, D and E at 37 oC in neutral pH


153


0.0

0.4

0.8

1.2

0 5 10 15

t1/2

(h1/2

)

Mt/

Min

fin

ity

Gel A Gel B Gel C

Figure 7. Plot of fractional swelling in hydrogels A - C at 37 oC in neutral pH


0.0

0.4

0.8

1.2

0 5 10 15

t1/2

(h1/2

)

Mt/

Min

fin

ity

Gel A Gel D Gel E

Figure 8. Plot of fractional swelling in hydrogels A, D and E at 37 oC in neutral pH


154


-2.0

-1.5

-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G M

t/ M

infi

nit

y

Gel A Gel B Gel C

Figure 9. Plot of the LOG of fractional swelling in hydrogels A - C in the initial stages of

the swelling experiment at 37 oC in neutral pH environment as a function of the LOG of

time.

-2.0

-1.5

-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G M

t/ M

infi

nit

y

Gel A Gel D Gel E

Figure 10. Plot of the LOG of fractional swelling in the hydrogels A, D and E in the

initial stages of the swelling experiment at 37 oC in neutral pH environment as a function

of the LOG of time

155


As previously stated the value of the slope represents the parameter n in the power-law

expression (Equation 3) where Mt is the swollen mass at time t and M∞ is the swollen

mass at equilibrium. The k value is a constant specific to the polymer/solvent system and

the parameter n determines the dependence of the medium uptake or release rate on time.


Table 6 presents the slope (n) values for hydrogel A - E calculated from Figure 9 and 10.

Table 6. Characteristic exponential n values for diffusion in hydrogels A - E in

neutral medium

Hydrogels n values

Gel A 0.60 + 0.01

Gel B 0.64 + 0.02

Gel C 0.60 + 0.01

Gel D 0.65 + 0.04

Gel E 0.60 + 0.03

156


Table 7. Swelling test on HPMI -NVP, HPMI-NVP-HEMA and HPMI-NVP-NVC

hydrogels at 37 oC in pH 7.4, isotonic environment


Time (h) Gel A Gel D Gel E

0.00 0.0 0.0 0.0

0.17 54.1 23.7 10.8

0.33 63.2 34.8 14.8

0.50 68.1 42.7 18.2

0.67 71.3 48.9 20.8

0.83 73.9 53.7 23.2

1.00 76.1 57.7 25.4

2.00 82.3 70.5 33.9

3.00 85.1 76.0 39.2

4.00 86.8 79.2 43.4

5.00 88.0 81.4 46.3

7.00 89.4 84.3 50.8

9.00 90.3 86.1 53.7

12.00 91.1 87.9 56.6

24.00 92.3 91.0 61.3

48.00 92.6 92.7 63.5

72.00 92.4 93.3 63.7

97.00 92.2 93.5 64.1

120.00 91.9 93.6 63.5

144.00 91.6 93.6 62.5

170.00 91.2 93.6 61.9

157


Graphical representations of the effect of isotonic environments on the swelling

behaviour of the NVP-HPMI, NVP-HPMI-HEMA and NVP-HPMI-NVC hydrogels are

illustrated in Figures 11 - 14.

0

20

40

60

80

100

0 50 100 150 200

Time (h)

% W

ate

r C

on

ten

t

Gel A Gel D Gel E

Figure 11. Plot of % water content in hydrogels A, D and E at 37 oC in isotonic, pH 7.4


0.0

0.4

0.8

1.2

0 5 10 15

t1/2

(h1/2

)

Mt/

Min

fin

ity

Gel A Gel D Gel E

Figure 12. Plot of fractional swelling in the hydrogels A, D and E at 37 oC in isotonic, pH

7.4 environment as a function of the square root of time.

158


0.0

0.4

0.8

1.2

0 5 10 15t1/2

(h1/2

)

Mt/

Min

fin

ity

Neutral pH 7.4

Figure 13. Comparative plot of fractional swelling in hydrogel E in isotonic and neutral

pH environments at 37 oC as a function of the square root of time

-2.0

-1.5

-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G M

t/M

infi

nit

y

Gel A Gel D Gel E

Figure 14. Plot of the LOG of fractional swelling in gels A, D and E in the initial stages

of the swelling experiment at 37 oC in pH 7.4 environment as a function of the LOG of

time.

159


Table 8 presents the slope (n) values for hydrogels A, D and E calculated from Figure 14.

Table 8. Characteristic exponential n values for diffusion in hydrogels A, D and E in

isotonic medium at 37 oC

Hydrogels n values

Gel A 0.57 + 0.02

Gel D 0.83 + 0.07

Gel E 0.57 + 0.02

160



The drug release experiments on the N-hydroxyalkyl maleimide–NVP hydrogels were

conducted at 37 oC in neutral pH environment. Theophylline, thiamine HCl and Mn-TPP-

OH were used as the model drugs. The experimental data expressed as fractional drug

release at specific time intervals are presented in Tables 9-12.

Table 9. Theophylline release from N-hydroxyalkyl maleimide -NVP hydrogels at 37

oC in neutral pH environment

Average fractional theophylline release values at

time (t)

Time (h) Gel A Gel B Gel C Gel D Gel E

0.00 0.00 0.00 0.00 0.00 0.00

0.17 0.22 0.24 0.24 0.26 0.20

0.33 0.35 0.37 0.34 0.37 0.30

0.50 0.43 0.45 0.43 0.46 0.38

0.67 0.49 0.52 0.49 0.52 0.46

0.83 0.55 0.58 0.52 0.58 0.53

1.00 0.60 0.63 0.57 0.64 0.55

2.00 0.76 0.80 0.74 0.80 0.72

3.00 0.85 0.88 0.82 0.87 0.85

4.00 0.92 0.93 0.88 0.93 0.88

5.00 0.92 0.95 0.91 0.92 0.93

7.00 0.94 0.97 0.92 0.96 0.98

9.00 0.94 0.97 0.92 0.94 0.99

12.00 0.95 0.97 0.91 0.97 0.96

24.00 0.96 0.97 0.95 0.96 0.95

48.00 0.95 0.97 0.95 0.95 0.97

Hydrogel compositions: HPMI-NVP (Gel A), HPrMI-NVP (Gel B), HMMI-NVP (Gel

C), HPMI-NVP-NVC (Gel D), HPMI-NVP-HEMA (Gel E)

161


The fractional drug release values were calculated as described in Section 2.7. Graphical

representations of the drug release behaviour in the N-hydroxyalkyl maleimide-NVP

hydrogels are illustrated in Figures 15 – 20.

0.0

0.4

0.8

1.2

0 10 20 30 40 50 6

Time (h)

Fra

ctio

nal

Dru

g R

elea

se

0

Gel A Gel B Gel C

Figure 15. Plot of the fractional release of theophylline from hydrogels A-C at 37 oC in

neutral pH environment as a function of time.

0.0

0.4

0.8

1.2

0 20 40

Time (h)

Fra

ctio

nal

Dru

g R

elea

se

60

Gel A Gel D Gel E

Figure 16. Plot of the fractional release of theophylline from hydrogels A, D and E at 37

oC in neutral pH environment as a function of time.

162


0.0

0.4

0.8

1.2

0 2 4 6 8

t1/2

(h1/2

)

Fra

ctio

nal

Dru

g R

elea

sed

Gel A Gel B Gel C

Figure 17. Plot of the fractional release of theophylline from hydrogels A-C at 37 oC in


0.0

0.4

0.8

1.2

0 2 4 6 8

t1/2

(h1/2

)

Fra

ctio

nal

Dru

g R

elea

sed

Gel A Gel D Gel E

Figure 18. Plot of the fractional release of theophylline hydrogels A, D and E at 37 oC in


163


-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G F

DR

Gel A Gel C Gel B


time in the initial stages of theophylline release from hydrogels A-C in neutral

environment at 37 oC.

-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G F

DR

Gel A Gel D Gel E


time in the initial stages of theophylline release from hydrogels A, D and E in neutral

environment at 37 oC.

164


Table 10 presents the slope (n) values for theophylline release from NVP-HPMI, NVP-

HPrMI and NVP-HMMI hydrogel systems calculated from Figures 19 and 20.

Table 10. Characteristic exponential n values for theophylline release kinetics of

NVP - N-hydroxyalkyl maleimide hydrogels in neutral medium at 37 oC

Hydrogels n values

Gel A 0.53 + 0.01

Gel B 0.52 + 0.02

Gel C 0.51 + 0.02

Gel D 0.50 + 0.04

Gel E 0.60 + 0.02

165


Table 11. Theophylline, thiamine HCl and Mn-TPP-OH release from HMMI -NVP

hydrogels at 37 oC in neutral pH environment

Average fractional release values for the

model drugs at time (t)

Time (h) Mn-TPP-OH Thiamine HCl Theophylline

0.00 0.00 0.00 0.00

0.17 0.08 0.28 0.24

0.33 0.16 0.42 0.34

0.50 0.20 0.52 0.43

0.67 0.28 0.59 0.49

0.83 0.28 0.67 0.52

1.00 0.32 0.69 0.57

2.00 0.46 0.85 0.74

3.00 0.57 0.90 0.82

4.00 0.60 0.91 0.88

5.00 0.60 0.91 0.91

7.00 0.62 0.93 0.92

9.00 0.63 0.92 0.92

12.00 0.70 0.93 0.91

24.00 0.82 0.95 0.95

48.00 0.85 0.95 0.93

Graphical representations of the effect of varying molecular weight and nature of the

model drugs on the release behaviour in NVP-HMMI hydrogels are illustrated in Figures

21 – 23.

166


0.0

0.4

0.8

1.2

0 20 40Time (h)

Fra

ctio

nal

Dru

g R

elea

sed

60

Mn-TPP-OH Thiamine HCl Theophylline

Figure 21. Plot of the fractional release of theophylline, thiamine HCl and Mn-TPP-OH

from NVP-HPMI hydrogels at 37 oC in neutral pH environment as a function of time.

0.0

0.4

0.8

1.2

0 2 4 6

t1/2

(h1/2

)

Fra

ctio

nal

Dru

g R

elea

sed

8


Figure 22. Plot of the fractional release of theophylline, thiamine HCl and Mn-TPP-OH

from NVP-HPMI hydrogels at 37 oC in neutral pH environment as a function of the

square root of time.

167


-1.5

-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G F

DR



time in the initial stages of Mn-TPP-OH, thiamine HCl and theophylline release from

NVP-HMMI hydrogels in neutral pH environment at 37 oC.

Table 12 presents the slope (n) values calculated from Figure 23 for release kinetics of

varying molecular weight drugs from the NVP-HMMI hydrogel systems.

Table 12. Characteristic exponential n values for release kinetics of theophylline,

thiamine HCl and Mn-TPP-OH from HMMI-NVP hydrogel in neutral medium

Model drugs n values

Mn-TPP-OH 0.74 + 0.08

Thiamine HCl 0.52 + 0.01

Theophylline 0.52 + 0.02

168


4.4. Discussion


The DPC measurements (Table 1) revealed that the HPMI-NVP system was the most

efficient donor/acceptor pair system followed by HMMI-NVP and then HPrMI-NVP. The

rate calculations were carried out according to Equation 1. Efficient systems were

characterized by a sharp peak in the photo-exotherms (Figures 1-4). The HEMI-NVP

system was found to be the least efficient donor/acceptor pair combination. Furthermore,

the rates of polymerisation of these systems were observed to have enhanced upon

addition of hydrogen donors, including glucose and glucosamine HCl. Glucosamine HCl

was found to be a more superior hydrogen donor over glucose as suggested by the

polymerisation rate calculations in Table 1.

The polymerisation reaction of the N-hydroxyalkyl maleimides and NVP in the absence

of external hydrogen donors could be explained as an electron/proton transfer process

between NVP and the N-hydroxyalkyl maleimides. Scheme 2 [9] illustrates a typical

electron/proton transfer between a N-hydroxyalkyl maleimide and NVP leading to the

formation of radical initiators. The relative efficiency of the polymerisation of N-

hydroxyalkyl maleimides with NVP could be related to the strength of the CT complex

formed between the donor/acceptor pair. The strength of the CT complex relies on the

nature of its donor/acceptor pair in terms of the chemical structure, which governs their

compatibility and reactivity.

4.4.1.1. Effect of the Monomer Structure on Polymerisation

The chemical structures of the acceptor and the donor monomers play a very crucial role

in determining the rate of polymerisation. Ng et al [26] in their study on CT complex

polymerisation reactions have emphasised the importance of donor/acceptor monomer

structures on the strength of CT complex formed. The only difference between the four

maleimides under study is the chain length of the hydroxyalkyl N-substituent. The

presence of α hydrogens, which are self abstracted by the maleimides is a very influential

structural characteristic in determining their relative efficiencies. HPMI, HPrMI and

HEMI have four readily abstractable α-hydrogens in comparison to HMMI, which has

only two α abstractable hydrogens as illustrated in Figure 24.

169


N OH

O

O

H

H

H

HOH

H

HN

O

O

H

H

N OH

O

O

H

H

H

H

N

O

O

H

H

OH

(1)

(2)

(3)

(4)

(1)

(2)

(3)

(4)

HPMI

HMMI

HPrMI

HEMI

(1)

(2)

(1)

(2)

(3)

(4)

Figure 24. Structures of the N-substituted maleimides illustrating the number of available

abstractable hydrogens located adjacent to heteroatoms, N and O

Based on the fact on the number of abstractable hydrogens contained by the maleimides,

the order of expected reactivity would be as such: HPMI = HEMI = HPrMI > HMMI.

However this was only true for HPMI, which showed the highest reactivity (1.18 J g-1

s-1

).

HMMI despite containing only two abstractable hydrogens was found to be more reactive

(0.73 J g-1

s-1

) than HEMI (0.31 J g-1

s-1

) and HPrMI (0.42 J g-1

s-1

). The present study

thus suggests the involvement of further influential factors such as the chain length of the

N-substituent hydroxyalkyl group. The relative inertness of HEMI paired with NVP in

comparison to HMMI, HPrMI and HPMI paired with NVP could be explained along this

line.

Jönsson et al [9] from their studies on the role of N-hydroxyalkyl maleimides as initiators

and acceptors in donor/acceptor formulations have suggested a predominant intra-

molecular H-abstraction of HEMI over the inter-molecular abstraction from the donor.

They further suggest a phenomenal thermodynamically favoured seven membered ring

formation of HEMI upon excitation as illustrated in Scheme 2.

170


N R

O

O

N R

O

O

N R

O

O

N R

O

O

N R

O

O

N+

O

HH

N R

O

O

N R

O

O

N+

OH

H

N

O

H

N R

OH

O

N R

O

O

e- H+

Transfer/

-CH2CH

2CH

2CH

2CH

2OH

-CH2CH

2CH

2OH

-CH2CH

2OH

-CH2OH

hv

*

(RMI)

NVP

+

+

R:

Scheme 2. Direct excitation of a typical N-substituted maleimide by electron/proton

transfer from NVP

171


N

O

OH

OH

H

N

O

OH

OH

H

N

O

OHH

OHN

O

OH H

OH

N

O

OH

H

OH

N

O

OH

OHH

NH

O

O

OHH

H+ - Transfer +

hv

Scheme 3. Intramolecular H-abstraction for HEMI leading to a thermodynamically stable

ring formation

172


However, the ring formation inevitably reduces the number of initiating radicals, thus

reducing the rate of polymerisation. The extent of the predominance of intra-molecular H-

abstraction over inter-molecular H-abstraction in HEMI however has not been established

to date. The ring formation of the hydroxyalkyl group with the carbonyl centre as

suggested in Scheme 3 would not be thermodynamically favourable at all for HMMI and

HPMI. The hydroxymethyl N-substituent of HMMI is very short in chain length thus the

possibility of ring formation would be a three membered ring, which is highly strained

and thermodynamically unstable.

The hydroxypentyl N-substituent of HPMI has a fairly long chain length, which would

inhibit the ring formation. Firstly, being a longer chain there will be a considerable

amount of steric hindrance for it to form a ring with the carbonyl centre. Secondly, a

possible the ring formation would be a 10 membered ring, which would also be

thermodynamically unstable due to a relatively high angle strain.

The slow reactivity of HPrMI compared to HMMI and HPMI could also be attributed to

some extent to the ring formation phenomenon displayed by HEMI. However, a faster

reactivity of HPrMI as opposed to HEMI suggests that the ring formation in HPrMI is not

as thermodynamically favourable as that in HEMI. The availability of four readily

abstractable α-hydrogens on HPMI in comparison to two readily abstractable α-

hydrogens on HMMI explains the polymerisation efficiency of HPMI over HMMI.

4.4.1.2. Hydrogen Abstraction

NVP has three readily abstractable α hydrogens, which are abstracted by the excited state

maleimide via an inter-molecular process. However, the introduction of glucose and

glucosamine HCl, which have several abstractable α hydrogens, caused a dramatic

increase in the rate of polymerisation reaction by increasing the number of initiating free

radicals. Hydrogens available for inter-molecular hydrogen abstractions are illustrated in

Figure 25.

173


O

OH OH

OH

OH

H

HH

H

OH

H

HH

O

OH NH3

+

OH

OH

H

HH

H

OH

H

HH

N

O

H

HH Cl

(1)

(2)(3)

(4)

(5)

(6)(7)

(1)

(2)(3)

(4)

(5)

(6)(7)

Glucose Glucosamine HClNVP

(1)

(2)(3)

Figure 25. Structures of the NVP, glucose and glucosamine HCl illustrating the number

of available hydrogens located adjacent to heteroatoms, N and O for inter-molecular

hydrogen abstraction.

Scheme 4 [21] illustrates the inter-molecular H-abstraction from a glucose molecule by an

excited state N-substituted maleimide. Glucose and glucosamine HCl have the same

number of abstractable α-hydrogens, however, glucosamine HCl was found to be a more

superior H-donor and reaction rate enhancer. This observation could be attributed to the

fact that glucosamine HCl (pKa = 7.75) is more acidic than glucose (pKa = 12.34) due to

the presence of a positive charge on the nitrogen making it a superior hydrogen donor.

Hence a rapid CT complex polymerisation reaction was observed from the DPC

measurements


Hydrogel formations via ultraviolet radiation were attempted using the donor/acceptor

formulations of N-hydroxyalkyl maleimides and NVP (Tables 2-4). Formulations

containing glucosamine HCl did not polymerise despite its enhancement of reaction

activity shown in the DPC measurements. A clear colourless formulation containing

glucosamine HCl turned into an intense orange coloured solution after being exposed to

UV light. UV light could not penetrate through the intense coloured formulation hence a

complete polymerisation was not achieved.

174


N

O

O

R

N

O

O

R

T

N

OH

O

R

O

OH OH

OH

OH

H

HH

H

OH

H

H

O

OH OH

OH

OH

H

HH

H

OH

H

HH

O

OH OH

OH

OH

HH

H

OH

H

H

OH OH

OH

OH

HH

H

OH

O

H

N

O

O

R

H

H

H

(1)

(2)(3)

(4)

(5)

*

1

hv

H- abstraction

Rearrangement

+

+

+

+

(1)

(2)(3)

(4)

(5)

(6)

(1)

(2)(3)

(4)

(5)

(2)(3)

(4)(5)

(7)

(7) (6)

(7) (6)

(7) (6)

Scheme 4. Direct intermolecular H-abstraction of an excited state N-substituted

maleimide from a glucose molecule

175


However, the colouration of the formulation can be due to several factors. Glucosamine

HCl could also react with the carbonyl group on N-hydroxyalkyl maleimide via a

nucleophilic attack on the carbonyl followed a proton transfer from nitrogen to oxygen

leading to the formation of an imine. The colour could also be due to the formation of

short polymer chains. The formation of the coloured compound was not investigated in

sufficient detail thus no definite conclusions could drawn on nature of the coloured

compound from this experimental observations. The photocurable sample size should also

be taken into consideration with respect to the light intensity. The sample sizes for DPC

measurements were much smaller than the sample formulation for hydrogel formation.

Furthermore, the UV light source used to cure the hydrogel samples has a relatively lower

intensity thus requiring longer polymerisation time. Thus it could be suggested from this

observation that a more intense light source over a short span of time would be required

to achieve complete polymerisation of such systems.

Hydrogel formulations containing glucose as the hydrogen donor on the other hand were

successfully synthesized. HEMA and NVC, monomers less hydrophilic in nature

compared to NVP, were also included in some formulations, and resulted in successfully

synthesized polymers. Glucose was also observed to enhance the mechanical strength of

the polymeric hydrogels, allowing them to be more resilient upon swelling.

4.4.3. Swelling and Drug Release Investigations

The swelling and drug release behaviour in the hydroxyalkyl maleimide-NVP hydrogels

were characterized on the basis of Fickian or non-Fickian diffusion behaviour. Fickian

and non-Fickian diffusion have been discussed in detail in Section 1.4.2.3.1.2. As

previously described, the swelling action in polymers is generally time dependent and

could be described according Equation 3. The parameter n in the equation characterizes

the diffusion kinetics in the polymer. The characteristic n values signifying Fickian and

non-Fickian diffusion have been mentioned in Section 1.4.2.3.1.2.

176


4.4.3.1. Hydrogel Swelling Behaviour


The HEMI-NVP hydrogel network disintegrated after a short exposure to the aqueous

swelling medium. This was indicative of the fact that the hydrogel network was not

suitably crosslinked. The bulk of the composition of the hydrogel formulation is NVP

(71.45% w/w), which gives a water-soluble polymer upon homopolymerisation. The

water solubility of the HEMI-NVP hydrogel network thus suggests that HEMI did not

actively crosslink with NVP. The final polymer was simply a homopolymer of NVP,

which disintegrated upon placement in the swelling medium. This can be explained by the

ring formation mechanism (Scheme 3) of HEMI, which suggests that upon preferable ring

formation, HEMI became inert and did not react with NVP.

An exponential increase in the water uptake by the HMMI-NVP, HPrMI-NVP and

HPMI-NVP hydrogels in neutral pH environment at 37 oC was observed in the first hour

(Figure 7) indicating non-Fickian (anomalous) transport behaviour. This was confirmed

by a LOG plot (Figure 9, Table 6) from which average n values (Equation 3) of 0.64 for

HPrMI-NVP and 0.60 for HMMI-NVP and HPMI-NVP were calculated. In the later

stages of the swelling process, Case II diffusion behaviour prevailed which was indicated

by a gradual decrease in the rate of water uptake by the hydrogels.

The gradual decrease in the rate of water uptake was observed around 7 hours of swelling

which became constant upon 48 hours of constant swelling, thus indicating equilibrium

water content was achieved (Table 5, Figure 5). At this stage the swelling process became

time independent where the parameter n = 1. The rate of polymer chain relaxation at this

point is equal to the rate of diffusion [27]. Equilibrium water content (EWC) values of

94.8 %, 96.3 % and 94.7 % were observed for the HPMI-NVP, HPrMI-NVP and HMMI-

NVP hydrogels respectively.

The HPMI-NVP-HEMA and HPMI-NVP-NVC hydrogels also adhered to non-Fickian

(anomalous) diffusion in the initial stages of the swelling experiment followed by case II

transport in the later stages (Figures 8 and 10). However, a reduced swelling activity was

observed from these systems in comparison to HPMI-NVP system. Equilibrium swelling

177


was achieved after 48 hours of constant experimental swelling which yielded EWC values

of 73.4 % and 92.1 % for HPMI-NVP-HEMA and HPMI-NVP-NVC hydrogels

respectively (Table 5, Figure 6). HPMI-NVP-HEMA hydrogel displayed significant

suppressed swelling in comparison to HPMI-NVP hydrogel throughout the swelling

process with the percentage water content values ranging from 16.0 % at 10 minutes to an

EWC value of 73.4 % at 48 hours.

HPMI-NVP-NVC gel showed suppressed swelling in the initial stages of the swelling

experiment with % water content values ranging from 25.9 % at 10 minutes to an EWC

value of 92.1 % at 48 hours. Inclusion of relatively hydrophobic monomers such as

HEMA and NVC has led to a reduction in the equilibrium water content uptake with

HEMA proving to be a more effective monomer in reducing water uptake. HEMA is

slightly acidic and also a weak acceptor monomer while NVC is a slightly basic and

could function as a donor. Thus it could be stated that HEMA and NVC were effectively

consumed in the formulation upon UV curing, as their presence was clearly demonstrated

by the swelling process.

4.4.3.1.2. Effect of the Ionic Strength on Swelling

The HPMI-NVP, HPMI-NVP-NVC and HPMI-NVP-HEMA hydrogels were subjected to

swelling in isotonic, pH 7.4 environment at 37 oC. The hydrogels were observed to de-

swell in the isotonic environment (Table 7, Figures 11 - 13). The isotonic environment

had a more pronounced de-swelling effect on HPMI-NVP-HEMA hydrogels in

comparison to HPMI-NVP and HPMI-NVP-NVC hydrogels. The diffusion order was

however influenced by the presence of ions in the environment. A slight reduction in the

swelling efficiency of the hydrogels was observed.

HPMI-NVP, HPMI-NVP-NVC and HPMI-NVP-HEMA hydrogels displayed non-Fickian

(anomalous) behaviour in the initial stages of the swelling experiment in isotonic

environment. This was confirmed by a LOG plot (Figure 14, Table 8) from which average

n values of 0.57, 0.83 and 0.57 were obtained for hydrogels A, D and E respectively.

EWC values of 93.6 %, 91.3 % and 61.9 % for HPMI-NVP-NVC, HPMI-NVP and

HPMI-NVP-HEMA respectively were observed after 48 hours of constant swelling.

178


Another notable behaviour observed particularly in HPMI-NVP and HPMI-NVP-HEMA

was that upon reaching EWC value these two hydrogels began to de-swell upon continual

swelling process. This observation could be attributed to the concentration gradient

established between the restricted ions in the hydrogels and the free ions in the medium.

Hence once the concentration of the restricted ions is higher than the free ions, the

diffusion direction is reversed. The absorbed medium with the ions starts diffusing out of

the polymer matrix. This phenomenal diffusion process is repeatedly reversed in order to

maintain an equilibrium between the ionic concentration of restricted and free ions while

the hydrogel maintains its maximum swelling capacity for the given environment.


4.4.3.2.1. Effect of the Monomeric Composition on Drug Release

The drug release experiments conducted on HPMI-NVP, HPrMI-NVP and HMMI-NVP

hydrogels (Table 9, Figures 15 and 17) in neutral pH environment at 37 oC using

theophylline as the model drug yielded equilibrium drug release (EDR) values of 0.95,

0.97 and 0.93 respectively in 48 hours. The drug release rate increased rapidly in the first

2 hours of the experiment after which the release rate gradually slowed down and

eventually became constant around 7 hours. The quick release of the drug from the carrier

in the early stages of the experiment is attributed to the burst effect release [27]. As

described previously the polymer containing the incorporated solute has to swell to a

certain extent before it can release its contents. The resultant effect is the fast release of

the solute in a short span of time. The LOG plot (Figure 19, Table 10) indicated Fickian

diffusion kinetics of theophylline release with an average n value of 0.53, 0.52 and 0.51

obtained for hydrogels A, B and C respectively.

The drug release experiment on HPMI-NVP-NVC and HPMI-NVP-HEMA using

theophylline at 37 oC in neutral pH environment yielded EDR values of 0.95 and 0.97

respectively in 48 hours (Table 9, Figures 16, 18 and 20). The theophylline release trends

in HPMI-NVP-NVC and HPMI-NVP-HEMA were similar to the HPMI-NVP hydrogel

with a burst effect characterized by a rapid release of theophylline in the first 2 hours. The

release rate in the hydrogels gradually slowed down post this period and became constant

around 7 hours.

179


The drug release rate however in HPMI-NVP-HEMA hydrogel was slightly suppressed in

the initial stages of the experiment confining to non-Fickian solute transport behaviour

(Figure 18). This was confirmed by a LOG plot (Figure 20, Table 10) which yielded an

average n value of 0.6. The reduction in release of theophylline in HPMI-NVP-HEMA

hydrogel could be explained with relation to the reduced hydrophilic nature of the gel.

HPMI-NVP-NVC hydrogel adhered to Fickian transport with very similar drug release

rate to that of HPMI-NVP hydrogel despite the fact that HPMI-NVP-NVC had a

relatively lower swelling activity. This observation could be attributed to the relatively

low molecular weight of theophylline, which diffused with ease through the pores of

these two hydrogel networks at a similar rate.

4.4.3.2.2. Effect of the Nature and the Molecule Size of the Drug

The release experiment conducted on HMMI-NVP hydrogel using three model drugs,

theophylline, thiamine HCl and Mn-TPP-OH yielded EDR values of 0.93, 0.95 and 0.85

respectively in 48 hours (Table 11, Figures 21-23). A rapid release of theophylline and

thiamine HCl was observed in the first 2 hours followed by a gradual reduction in the

drug release activity post this period. The release rate became constant around 7 hours.

Mn-TPP-OH was observed to release with an initial burst followed by a gradual increase

in the release rate in the first two hours of the experiment and then a gradual reduction in

the release rate post this period.

The release of Mn-TPP-OH (MW. 733.69 g mol-1

) was the slowest among the model

drugs. The release profile adhered to typical non-Fickian solute transport behaviour

(Figure 22). The slow release could be attributed to its relatively large molecular mass.

Brazel and Peppas [28] have reported similar observation from their study on release rates

of drugs with varying molecular weights. However, theophylline (MW. 180.16 g mol-1

)

release was observed to be slightly slower than thiamine HCl (MW. 337.26 g mol-1

) in the

initial stages of the drug release experiment but followed by similar release rates in the

later stages of the release experiment. An average n value of 0.52 obtained from the LOG

graph (Figure 23, Table 12) for thiamine HCl and theophylline suggested that Fickian

transport mechanism was in operation. Furthermore, the study suggested that the release

rate of the drug was not only affected by the molecular mass but also the nature of the

180


drug in terms of solubility. Based on the molecular weights of the drugs, the order of

expected release rate would be: theophylline > thiamine HCl > Mn-TPP-OH. This only

holds true for Mn-TPP-OH. The faster release of thiamine HCl over theophylline despite

the fact that thiamine HCl has a larger molecular mass could be explained in terms of the

relative solubility of the drugs. Colombo et al [29] in their drug release studies observed

an increased release rate with increased solubility of the drug. Theophylline has a limited

solubility of 8.33 x 10-3

g ml-1

in water at 25 oC compared to thiamine HCl, which is a salt

that readily dissolves in water. Thus, despite thiamine HCl having a larger molecular

weight, its ease of dissolutions allows it to be released more efficiently than theophylline.

4.5. Conclusions

The kinetic studies on the N-hydroxyalkyl maleimides and NVP as donor/acceptor pairs

revealed that HPMI was the most efficient acceptor monomer followed by HMMI and

then HPrMI. HEMI-NVP was found to be the least efficient donor/acceptor pair.

Inclusion of hydrogen donors, glucose and glucosamine HCl were found to enhance the

rate of polymerisation with glucosamine HCl being a more efficient donor based on DPC

measurements.

Polymeric hydrogels based on N-hydroxyalkyl maleimides and NVP were successfully

synthesized via photoinitiator-free UV curing technique. Inclusion of more hydrophobic

monomers, HEMA and NVC resulted in successfully synthesized hydrogels. The

hydrogels were found to be resilient and competent drug delivery devices.

Experimental swelling data revealed that the HPMI-NVP, HPrMI-NVP and HMMI-NVP

hydrogels adhered to non-Fickian (anomalous) diffusion behaviour in the early stages of

the experiment followed by case II diffusion in the later stages. Inclusion of HEMA and

NVC into the hydrogel network resulted in a reduced swelling activity of hydrogels.

Presence of ionic environment was found to reduce the swelling efficiency of the

hydrogels with a more pronounced effect on HPMI-HEMA-NVP hydrogels. A time

independent, case II swelling was observed in HPMI-NVP and HPMI-NVP-HEMA

hydrogels while HPMI-NVP-NVC hydrogel displayed a super case II swelling behaviour

in isotonic environment

181


The drug release experiments revealed that HPMI-NVP, HPrMI-NVP and HMMI-NVP

hydrogels adhered to Fickian transport mechanism in releasing theophylline into the

neutral pH environment at 37 oC. The burst effect on the release of drugs was observed in

the initial stages of the release experiment followed by a linear release profile. The rate of

theophylline release from HPMI-NVP, HPrMI-NVP and HMMI-NVP hydrogels were

found to be similar. Inclusion of NVC, despite showing reduced swelling, had a very

similar release rate to that observed in the HPMI-NVP hydrogel without NVC indicating

that theophylline being a low molecular weight drug could diffuse through the hydrogels

at a similar rate.

The drug release experiments involving the various model drugs in neutral pH

environment indicated a faster release of thiamine HCl followed by theophylline and then

Mn-TPP-OH. The study suggested that increase in the molecular weight of the drug

reduces the release rate. Additionally the nature of the drug such as the solubility

parameter is also crucial and could predominantly govern the kinetics of release as

indicated by the experimental data on a faster release of thiamine HCl over theophylline.

182


4.6. References

1. Hoyle, C. E., Clark, S. C., Jönsson, S., Shimose, M., Polymer, 38, 5695-5697,

(1997).

2. Jönsson, S., Sundell, P.-E., Hultgren, J., Sheng, D., Hoyle, C. E., Prog. Org.

Coat., 27, 107-122, (1996).

3. Lee, C.-W., Kim, J.-M., Han, D. K., Ahn, K.-D., J.M.S.-Pure Appl. Chem., A36,

1387-1399, (1999).

4. Jönsson, S., Viswanathan, K., Hoyle, C. E., Clark, S. C., Miller, C., Morel, F.,

Decker, C., Nuclear Instruments Methods Phys. Res. B, 151, 268-278, (1999).

5. Decker, C., Morel, F., Jönsson, S., Clark, S., Hoyle, C., Macromol. Chem. Phys.,

200, 1005-1013, (1999).

6. Clark, S. C., Jönsson, S., Hoyle, C. E., Polym. Prep., 37, 348-349, (1996).

7. Jönsson, S., Sundell, P.-E. G., Schaeffer, W. R., United States Patent., 5446073,

(1995).

8. Jönsson, S., Ericsson, J. E., Sundell, P.-E., Shimose, M., Clark, S. C., Miller, C.,

Owens, J., Hoyle, C., Proc. RadTech North America’96, Nashville, USA, pp. 377-

385, (1996).


Hoyle, C. E, Proc. RadTech Asia’01, Kunming, China, pp. 182-195, (2001).


Hoyle, C. E., Proc. RadTech Asia’95, Bangkok, Thailand, pp.118-125, (1995).

11. Jönsson, S., Sundell, P.-E., Shimose, M., Owens, J., Miller, C., Clark, S. C.,

Hoyle, C. E., ACS Polymeric Mater. Sci. Eng., 74, 319-320, (1996).

12. Miller, C. W., Hoyle, C. E., Howard, C., Polym. Prep., 37, 346-347, (1996).

13. Morel, F., Decker, C., Jönsson, S., Clark, S. C., Hoyle, C. E., Polymer, 40, 2447-

2454, (1999).

14. Jönsson, S., Sundell, P.-E., Shimose, M., Clark, S., Miller, C., Morel, F., Decker,

C., Hoyle, C. E., Nuclear Instruments Methods Phys. Res. B, 131, 276-290,

(1997).

15. Clark, S. C., Hoyle, C. E., Jönsson, S., Morel, F., Decker, C., Polymer, 40, 5063-

5072, (1999).

183


16. Decker, C., Morel, F., Jönsson, S., Clark, S. C., Hoyle, C. E., ACS Polymeric

Mater. Sci. Eng., 75, 198-199, (1996).

17. Viswanathan, K., Clark, S., Miller, C., Hoyle, C. E., Jönsson, S., Shao, L., Polym.

Prep., 39, 644-645, (1998).

18. Yamada, M., Takase, I., Koutou, N., Polym. Lett., 6, 883-888, (1968).

19. Aida, H., Takase, I., Nozi, T., Makromol. Chem., 190, 2821-2831, (1989).

20. Ng, L.-T., Jönsson, S., Lindgren. K., Swami, S., Hoyle, C. E., Clark, S., Proc.

RadTech Europe’01, Basel, Switzerland, 609-613, (2001).


22. Swami, S., Ng, L.-T., Jönsson, S., Proc. RadTech Asia ’03, Yokohama, Japan, pp.

677-680, (2003).

23. Ng, L.-T., Jönsson, S., Swami, S., Lindgren, K., Polym. Int., 51, 1398-1403,

(2002).

24. Jönsson, S., Viswanathan, K., Lindgren, K., Swami, S., Ng, L.-T., Polym. Prep.,

44, 7-8, (2003).

25. Garnett, J. L., Ng, L.-T., Nguyen, D., Swami, S., Zilic, E., Radiation Phys. Chem.,

63, 459-463, (2002).

26. Ng, L.-T., Garnett, J. L., Zilic, E., Ngyuen, D., Radiation Phys. Chem., 62, 89-98,

(2001).



pp. 109-135, (1987).

28. Brazel, C. S., Peppas, N. A., S.T.P. Pharm. Sci., 9, 473-485, (1999).

29. Colombo, P., Bettini, R., Santi, P., De Ascentiis, A., Peppas, N. A., J. Controlled

Release, 39, 231-237, (1996).

184




4.3. Results 145

4.3.1. DPC Measurements 145




4.4. Discussion 169


4.4.1.1. Effect of the Monomer Structure on Polymerisation 169

4.4.1.2. Hydrogen Abstraction 173


4.4.3. Swelling and Drug Release Investigations 176

4.4.3.1. Hydrogel Swelling Behaviour 177


4.4.3.1.2. Effect of the Ionic Strength on Swelling 178


4.4.3.2.1. Effect of the Monomeric Composition on Drug Release 179

4.4.3.2.2. Effect of the Nature and the Molecule Size of the Drug 180


4.6. References 183

185

Chapter 5: IPN Hydrogels Synthesized Through Photoinitiator-Free Polymerisation Technique

5.1. Introduction

Interpenetrating network (IPN) hydrogels are an important class of materials, which are

defined as two independent synthetic and/or natural polymer components contained in a

network form [1-6]. Figure 1 illustrates an IPN where at least one component is

crosslinked in the immediate presence of another [1,2].

Figure 1. Interpenetrating polymer network.

Two separate polymer networks interpenetrating each other.

IPN formation is an excellent way to enhance the compatibility of the polymeric

components. An IPN displays a more superior performance for its particular application

in comparison to its component macromolecules [1]. Various synthetic routes of

obtaining such materials have been discussed in detail in Section 1.1.1.3. Numerous

researchers have reported the use of a variety of combination of synthetic and natural

polymers to form IPNs [4-11). The use of polysaccharides such as chitosan and its

derivatives in conjunction with other compatible synthetic or natural polymers to form

IPNs has been very common in recent years as indicated by the publications [4-11].

Chitosan also chemically known as (1-4)-[2-amino-2-deoxy-β-D-glucan] is a natural

derivative of chitin, obtained through its partial deacetylation [11,12]. Chitin, extracted

from crustacean shells, is the most abundant mucopolysaccharide after cellulose and is

known to consist of β-[(1-4)-2-acetamido-2-deoxy-D-glucose] units [11-15]. Amine

185


functional groups on the chitosan structure contribute to its cationic character and it is

well known to have the ability to form intermolecular complexes with carboxylic and

polycarboxylic acids [11]. Furthermore, chitosan is biodegradable and highly

biocompatible, which contributes to its versatility as an extremely useful material in

biomedical applications [16-20]. Figure 2 [15] illustrates chemical structures of chitin and

its derivatives.

H

NHCOCH3

H

OH

H

CH2OH

HOH H

H OO H

O

H

CH2OH

H

H NHCOCH3

n

H

NH2

H

OH

H

CH2OH

HOH H

H OO H

O

H

CH2OH

H

H NHCOCH3

n

H

NHCOCH3

H

OH

H

CH2OH

H

OH

OH

NHCH2COOHH

OH

H

CH2OH

HOH H

H OO H

O

H

CH2OH

H

H NH2

n

Chitin

Chitosan

Carboxymethyl chitosanN-

Figure 2. Structures of chitin, chitosan and CM chitosan

186


Chitosan, however, has certain limitations to its reactivity and processability [21,22]. In

recent years several researchers have attempted to obtain functional derivatives of

chitosan with enhanced properties such as biocompatibility and water-solubility through

partial N-acetylation or chemical modification [23-27]. It has been suggested in the

literature that the degree of deacetylation of the starting chitosan governs the solubility of

the derivatives [15,24,27]. Experimental conditions such as reaction temperature and

solvent used for deacetylation have also been suggested to affect the nature of the

derivative [26].

Derivatives of chitosan with enhanced reactivity and processability have been utilized to

form IPN hydrogels for bioapplications. Chen et al [7] have reported successful synthesis

of IPN based on a water-soluble chitosan derivative, carboxymethyl (CM) chitosan for

drug delivery application. Chen et al [10] in their study of IPN based on CM chitosan

obtained through varying degree of deacetylation of chitosan found phenomenal

sensitivity of the gels to changes in environmental pH. They further suggest the

dependence of the nature of the derivative on the degree of deacetylation.

In this present work the author has made use of chitosan and its water-soluble derivative,

CM chitosan in conjunction with NVP and its copolymer with HEMA to synthesize IPNs

for swelling-drug release applications. The IPNs were synthesized by allowing NVP to

polymerise within the matrix of the polysaccharides through the formation of charge-

transfer (CT) complexes with HEMA and HMMI as the acceptors. The CT complex

formation reactions have been discussed in Sections 1.2.6 and 4.4.1.


The IPNs formed through this photoinitiator-free process were subjected to swelling drug

release experiments at 37 oC in neutral pH environment. The effect of acidic, basic and

isotonic environments on the swelling behaviour of the IPNs was also evaluated. A model

drug, theophylline was used for drug release investigations. Detailed experimental

procedure of IPN formation is outlined in Section 2.5.2.3-2.5.2.5. Spectroscopic

techniques such as FT-IR and UV-vis were employed in this work for IPN

characterization and the quantitative measurements of drug release respectively.

187


Experimental specifications and procedure of swelling-drug release experiments have

been described in Sections 2.6.1 and 2.7.

5.3. Results

5.3.1. Polymerisation of IPNs

The IPNs of chitosan and its derivative CM chitosan were prepared using the NVP and

HMMI via UV curing in the absence of a photoinitiator. Due to restricted solubility of

chitosan, it had to be pre-dissolved in a carboxylic acid prior to usage in the IPN

formulation. CM chitosan on the other hand is water-soluble thus it did not require any

additive for assistance in dissolving.

5.3.1.1. Chitosan Based IPNs

Two carboxylic acids namely, acrylic and levulinic acid were used to pre-dissolve

chitosan prior to use. The viscous mixtures of the chitosan in acid were combined with

NVP, HEMA and HMMI in varied proportions. The observations on the extent of


Tables 1 and 2.

Table 1. Polymerisation status of formulations with chitosan pre-dissolved in acrylic

acid

IPN formulation (% w/w) Polymerisation status upon application


NVP (65 %)-chitosan (35 %) DNP



HEMA (50 %)-chitosan (50 %) DNP

NVP (25 %)-HEMA (25 %)-chitosan (50 %) DNP

188


Table 2. Polymerisation status of formulations with chitosan pre-dissolved in

levulinic acid

IPN formulation (% w/w) Polymerisation status upon

application of curing dose (~9 KJ)

NVP (62.4 %)-chitosan (33.6 %)-HMMI (4.0 %) Soft, rubbery and partially opaque gel

NVP (48 %)-chitosan (48 %)-HMMI (4.0 %) Soft, rubbery and partially opaque gel

NVP (33.6 %)-chitosan (62.4 %)-HMMI (4.0 %) DNP

HEMA (50 %)-chitosan (50 %) DNP

NVP (25 %)-HEMA (25 %)-chitosan (50 %) Hard, clear and pale yellow gel

The monomer compositions are expressed as percentage w/w. DNP indicates that the

formulation did not polymerise. The radiation dose was calculated according to Equation

1 where t is the total radiation time in seconds. The samples were exposed to UV

radiation for approximately 25 hours at a dose rate of 9.6 x 10-2

J s-1

. The dose rate was

calculated as described in Section 2.4.4.4.



5.3.1.2. CM Chitosan Based IPNs

CM chitosan, a water-soluble derivative of chitosan was dissolved in milli-Q-water. The

viscous mixture of the CM chitosan was then added to the mixture of NVP and HMMI.

The observations on the extent of polymerisation upon applying approximately 9 KJ of

radiation dose are described in Table 3.

Table 3. Polymerisation status of formulations with CM chitosan

IPN formulation (% w/w)

(Presence of HMMI (4.0 %)



NVP (62.4 %)-CM chitosan (33.6 %) Soft, rubbery and partially opaque gel

NVP (48 %)-CM chitosan (48 %) Soft, rubbery and partially opaque gel

NVP (33.6 %)-CM chitosan (62.4 %) DNP

189


The monomer compositions are expressed as percentage w/w of the components. DNP

indicates that the formulation did not polymerise. The radiation dose was calculated

according to Equation 1.

5.3.2. Characterization of the IPNs

The IPNs were thoroughly cleaned in milli-Q-water to remove any unreacted component

and then characterized using the FT-IR spectroscopic technique. The 48 % chitosan- 48 %

NVP- 4 % HMMI and 48 % CM chitosan- 48 % NVP- 4 % HMMI IPNs, though

successfully synthesized, were found to be water-soluble. These IPNs disintegrated

during the washing process. The presence of characteristic bands of the monomers on the

FT-IR spectra of IPN gel samples confirmed the IPN formations. The spectra of chitosan,

CM chitosan and the IPNs are illustrated in Figures 3 and 4.

5.3.2.1. FT-IR Data Analysis

The characteristics of the FT-IR spectra corresponding to the chemical compositions of

the polysaccharides and the successfully synthesized water swellable IPNs are described

in Sections 5.3.2.1.1 – 5.3.2.1.5. Figures 3 and 4 illustrate the characteristic peaks of

chitosan, CM chitosan and the IPNs: 33.6 % CM chitosan- 62.4 % NVP- 4 % HMMI (Gel

A), 33.6 % chitosan- 62.4 % NVP- 4 % HMMI (Gel B) and 50 % chitosan- 25 % NVP-

25 % HEMA (Gel C) observed in the FT-IR spectra.

5.3.2.1.1. Chitosan

Figure 4 shows the characteristic peaks of chitosan observed at 3479 cm-1

(-O-H stretch),

2956 cm-1

(-CH stretch), 1720 cm-1

(-NH2 deformation), 1581 cm-1

(-NH bend), 1170 cm-1

(bridge-O-stretch) and 1099 cm-1

(-C-O- stretch).

5.3.2.2.2. CM Chitosan

Figure 3 shows the characteristic peaks of CM chitosan observed at 3466 cm-1

(broad -OH

stretch), 1747 cm-1

(-COOH peak), 1660 and 1540 cm-1

(-NH3+

peaks), and 1070-1136

cm-1

(-C-O- stretch).

190


5.3.2.2.3. IPN Gel A

Figure 3 shows the characteristic peaks of 33.6 % CM Chitosan- 62.4 % NVP- 4 %

HMMI IPN observed at 3479 cm-1


(-COO stretch (acid)),

1662 cm-1

(-COO stretch (amide)), 1384-1467 cm-1

(-CH bend (saturated)) and 1298 cm-1

(-C-O stretch).

5.3.2.1.4. IPN Gel B

Figure 4 shows the characteristic peaks of 33.6 % Chitosan- 62.4 % NVP- 4 % HMMI

IPN observed at 3500 cm-1


(-NR2 peak), 1683 cm-1

(-COO

peak (amide)), 1392-1487 cm-1

(-CH bend (saturated)) and 1296 cm-1

(-C-O -stretch).

5.3.2.1.5. IPN Gel C

Figure 4 shows the characteristic peaks of 50 % Chitosan- 25 % NVP- 25 % HEMA IPN

observed at 3500 cm-1


(-NR2 peak), 1740 cm-1

(-COO

stretch (ester)), 1683 cm-1

(-COO stretch (amide)), 1392-1487 cm-1

(-CH bend

(saturated)), 1296 cm-1

(-C-O stretch) and 1188 cm-1

(-COOR peak).

191


34

54

400140024003400

Wavenumber (cm-1

)

% T

ran

smit

tan

ce

CM Chitosan Gel A

Figure 3. FTIR spectrum illustrating the characteristics of CM chitosan and the IPN

formed of CM chitosan-NVP-HMMI (Gel A).

40

70

100

400140024003400

Wavenumber (cm-1

)

% T

ran

smit

tan

ce

Gel B Gel C Chitosan

Figure 4. FTIR spectrum illustrating the characteristics of chitosan and the IPNs formed

of chitosan-NVP-HMMI (Gel B) and chitosan-NVP-HEMA (Gel C).

192



The IPNs formed were subjected to swelling test in varying pH environments at 37 oC.

The IPNs were found to be resilient with high swelling efficiency and also slightly

sensitive to pH variations. The data on the degree of swelling of the IPNs: Gel A, Gel B

and Gel C in varying pH conditions are presented in Tables 4-11.

Table 4. Swelling test on IPN hydrogels at 37 oC in neutral pH environment


Time (h) Gel A Gel B Gel C

0.00 0.0 0.0 0.0

0.17 44.3 48.0 20.5

0.33 54.3 60.1 26.7

0.50 60.1 66.9 31.6

0.67 64.9 70.3 35.1

0.83 67.5 73.6 37.4

1.00 70.0 75.9 40.0

2.00 78.5 83.1 49.2

3.00 82.2 86.1 54.7

4.00 84.3 88.3 59.9

5.00 85.6 89.1 61.2

7.00 87.4 90.3 65.1

9.00 88.4 91.1 67.8

12.00 89.4 91.9 70.4

24.00 90.9 93.0 75.0

48.00 91.3 93.3 77.4

72.00 91.3 93.4 78.4

96.00 91.5 93.5 79.2

120.00 91.5 93.6 79.8

144.00 91.5 93.6 79.9

170.00 91.4 93.6 79.6

193


The swelling data is expressed as percentage water content at the respective time

intervals. The percentage water content values were calculated as described in Section

2.6.1. The chitosan-NVP-HMMI formulation containing 48 % w/w of NVP and the CM

chitosan-NVP-HMMI formulation containing 48 % w/w of NVP disintegrated after a

short exposure to milli-Q-water during the washing process thus no further experiments

were conducted on these samples. Graphical representations of the swelling behaviour

observed in the IPN hydrogels in neutral pH environment at 37 oC are illustrated in

Figures 5 - 7.

0

20

40

60

80

100

0 50 100 150 200

Time (h)

% W

ate

r C

on

ten

t

Gel A Gel B Gel C

Figure 5. Plot of % water content in the IPNs: Gel A, Gel B and Gel C at 37 oC in neutral

pH environment as a function of time.

0.0

0.4

0.8

1.2

0 5 10 15

t1/2

(h1/2

)

Mt/

Min

fin

ity

Gel A Gel B Gel C

Figure 6. Plot of fractional swelling in the IPNs: Gel A, Gel B and Gel C at 37 oC in


194


-1.5

-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G M

t/ M

infi

nit

y

Gel A Gel B Gel C

Figure 7. Plot of the LOG of fractional swelling in the IPNs: Gel A, Gel B and Gel C in

the initial stages of the swelling experiment at 37 oC in neutral pH environment as a

function of the LOG of time.

As previously discussed, the value of n in the power-law equation defines the kinetics of

the diffusion in the polymer, which in turn governs the solute release. The value of the

slope from the LOG graph fractional swelling against time represents the n value.

Table 5 presents the slope (n) values for the IPNs: Gel A, Gel B and Gel C calculated

from Figure 7.

Table 5. Characteristic exponential n values for diffusion in IPN hydrogels in

neutral medium

IPN hydrogels n values

Gel A 0.61 + 0.01

Gel B 0.66 + 0.02

Gel C 0.52 + 0.02

195


5.3.3.1. Swelling Test in Varying pH Environments

The data on the swelling behaviour of the IPN hydrogels observed at 37 oC in pH 2,

isotonic (pH 7.4) and pH 8 environments is presented in Tables 6-10.

Table 6. Swelling test on IPN hydrogels in acidic (pH 2) environment at 37 oC



0.00 0.0 0.0 0.0

0.17 47.2 47.9 20.6

0.33 56.1 57.6 26.1

0.50 63.9 64.3 31.0

0.67 66.4 69.1 34.6

0.83 69.5 71.3 36.3

1.00 72.6 74.4 38.9

2.00 79.7 82.2 46.7

3.00 83.1 85.1 52.0

4.00 85.0 86.7 55.6

5.00 86.2 88.0 58.5

7.00 87.8 89.7 62.2

9.00 87.8 90.4 65.3

12.00 89.0 91.3 67.7

24.00 90.1 92.7 70.3

48.00 90.8 93.3 71.3

72.00 90.9 93.6 71.4

96.00 91.0 93.6 71.5

120.00 91.2 93.5 71.6

144.00 91.3 93.4 71.9

170.00 91.3 93.3 71.8

Graphical representations of the swelling behaviour observed in the IPN hydrogels in

acidic (pH 2) environment at 37 oC are illustrated in Figures 8 - 10.

196


0

20

40

60

80

100

0 50 100 150 200

Time (h)

% W

ate

r C

on

ten

t

Gel A Gel B Gel C

Figure 8. Plot of % water content in the IPNs: Gel A, Gel B and Gel C at 37 oC in acidic

(pH 2) environment as a function of time.

0.0

0.4

0.8

1.2

0 5 10 15

t1/2

(h1/2

)

Mt/

Min

fin

ity

Gel A Gel B Gel C


acidic (pH 2) environment as a function of the square root of time.

197


-1.5

-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G M

t/ M

inif

init

y

Gel A Gel B Gel C


the initial stages of the swelling experiment at 37 oC in acidic (pH 2) environment as a


Table 7 presents the slope (n) values for IPNs: Gel A, Gel B and Gel C calculated from

Figure 10.

Table 7. Characteristic exponential n values for diffusion in IPN hydrogels in acidic

medium


Gel A 0.68 + 0.01

Gel B 0.74 + 0.04

Gel C 0.48 + 0.03

198


Table 8. Swelling test on IPN hydrogels in isotonic (pH 7.4) environment at 37 oC



0.00 0.0 0.0 0.0

0.17 51.3 49.0 20.0

0.33 61.5 58.1 27.4

0.50 69.6 67.2 30.9

0.67 73.7 70.7 33.5

0.83 76.2 74.0 36.8

1.00 78.2 76.8 39.0

2.00 83.9 83.0 47.8

3.00 86.4 85.4 53.2

4.00 88.0 86.8 57.1

5.00 88.8 87.5 60.0

7.00 90.1 88.7 63.5

9.00 91.1 89.5 65.6

12.00 91.6 89.6 68.5

24.00 92.2 90.8 71.2

48.00 92.8 91.6 72.3

72.00 92.8 91.7 72.1

96.00 92.7 91.7 71.7

120.00 92.7 91.8 71.7

144.00 92.8 91.9 70.9

170.00 92.8 91.9 70.8


isotonic (pH 7.4) environment at 37 oC are illustrated in Figures 11 - 13.

199


0

20

40

60

80

100

0 50 100 150 200

Time (h)

% W

ate

r C

on

ten

t

Gel A Gel B Gel C

Figure 11. Plot of % water content in the IPNs: Gel A, Gel B and Gel C at 37 oC in

isotonic (pH 7.4) environment as a function of time.

0.0

0.4

0.8

1.2

0 5 10 15

t1/2

(h1/2

)

Mt/

Min

fin

ity

Gel A Gel B Gel C


isotonic (pH 7.4) environment as a function of the square root of time.

200


-1.5

-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G M

t/ M

infi

nit

y

Gel A Gel B Gel C


the initial stages of the swelling experiment at 37 oC in isotonic (pH 7.4) environment as a



from Figure 13.

Table 9. Characteristic exponential n values for diffusion in IPN hydrogels in

isotonic medium


Gel A 0.70 + 0.03

Gel B 0.70 + 0.04

Gel C 0.51 + 0.02

201


Table 10. Swelling test on IPN hydrogels in basic (pH 8) environment at 37 oC



0.00 0.0 0.0 0.0

0.17 42.6 40.2 19.1

0.33 52.8 53.5 25.4

0.50 62.9 63.0 28.3

0.67 64.8 66.3 31.4

0.83 68.5 68.8 34.0

1.00 71.1 71.7 36.5

2.00 78.7 79.7 45.2

3.00 82.4 83.6 49.7

4.00 84.7 85.5 53.3

5.00 86.1 87.0 56.2

7.00 87.5 88.8 59.1

9.00 88.6 90.0 61.7

12.00 89.6 90.8 64.3

24.00 90.3 92.4 67.1

48.00 91.0 92.9 68.5

72.00 91.0 93.0 68.5

96.00 91.1 93.0 68.5

120.00 91.3 93.0 68.2

144.00 91.4 93.0 67.9

170.00 91.4 92.8 67.5


basic (pH 8) environment at 37 oC are illustrated in Figures 14 -16.

202


0

20

40

60

80

100

0 50 100 150 200

Time (h)

% W

ate

r C

on

ten

t

Gel A Gel B Gel C

Figure 14. Plot of % water content in the IPNs: Gel A, Gel B and Gel C at 37 oC in basic

(pH 8) environment as a function of time.

0.0

0.4

0.8

1.2

0 5 10 15

t1/2

(h1/2

)

Mt/ M

infi

nit

y

Gel A Gel B Gel C


basic (pH 8) environment as a function of the square root of time.

203


-1.5

-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G M

t/ M

inif

inty

Gel A Gel B Gel C


the initial stages of the swelling experiment at 37 oC in basic (pH 8) environment as a



from Figure 16.

Table 11. Characteristic exponential n values for diffusion in IPN hydrogels in basic

medium


Gel A 0.60 + 0.02

Gel B 0.64 + 0.02

Gel C 0.50 + 0.02

204


Figures 17-19 illustrate the comparative swelling behaviour observed for the IPNs: Gel A,

Gel B and Gel C in varied pH environments as a function of time.

0

20

40

60

80

100

0 50 100 150 200

Time (h)

% W

ate

r C

on

ten

t

pH 2 neutral isotonic (pH 7.4) pH 8

Figure 17. Comparative plot of % water content in Gel A in varied pH environments at

37 oC as a function of time.

0

20

40

60

80

100

0 50 100 150 200

Time (h)

% W

ate

r C

on

ten

t


Figure 18. Comparative plot of % water content in Gel B in varied pH environments at


205


0

20

40

60

80

100

0 50 100 150 200Time (h)

% W

ate

r C

on

ten

t


Figure 19. Comparative plot of % water content in Gel C in varied pH environments at


206



The drug release experiments on the IPNs were conducted at 37 oC in neutral pH

environment using theophylline as the model drug. The experimental data expressed as

fractional drug release at specific time intervals is presented in Table 12.

Table 12. Drug release test on IPN hydrogels at 37 oC in neutral pH condition

Average fractional theophylline released

values at time (t)


0.00 0.00 0.00 0.00

0.17 0.28 0.28 0.27

0.33 0.40 0.44 0.41

0.50 0.49 0.54 0.49

0.67 0.55 0.63 0.58

0.83 0.61 0.69 0.63

1.00 0.65 0.73 0.67

2.00 0.82 0.89 0.86

3.00 0.89 0.93 0.93

4.00 0.91 0.95 0.96

5.00 0.93 0.96 0.98

7.00 0.94 0.97 0.99

9.00 0.94 0.96 0.97

12.00 0.95 0.97 0.99

24.00 0.95 0.96 0.98

48.00 0.97 0.96 0.99


representations of the drug release behaviour in the IPN hydrogels are illustrated in

Figures 20 - 22.

207


0.0

0.4

0.8

1.2

0 20 40

Time (h)

Fra

ctio

nal

Dru

g R

elea

sed

60

Gel A Gel B Gel C

Figure 20. Plot of the fractional release of theophylline from the IPNs: Gel A, Gel B and

Gel C at 37 oC in neutral pH environment as a function of time.

0.0

0.4

0.8

1.2

0 2 4 6 8

t1/2

(h1/2

)

Fra

ctio

nal

dru

g r

elea

se

Gel A Gel B Gel C

Figure 21. Plot of the fractional release of theophylline from the IPNs: Gel A, Gel B and

Gel C at 37 oC in neutral pH environment as a function of the square root of time.

208


-1.5

-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G F

DR

Gel A Gel B Gel C

Figure 22. Plot of the LOG of fractional drug released (FDR) from the IPNs: Gel A, Gel

B and Gel C in the initial stages of release experiment at 37 oC in neutral environment as

a function of the LOG of time.

Table 13 presents the slope (n) values for IPNs: Gel A, Gel B and Gel C calculated from

Figure 22.

Table 13. Characteristic exponential n values for theophylline release from IPN

hydrogels in neutral medium


Gel A 0.50 + 0.01

Gel B 0.50 + 0.02

Gel C 0.50 + 0.01

209


5.4. Discussion

Attempts to synthesize several IPN formulations via photoinitiator-free UV curing

technique resulted in a number of successfully synthesized IPNs (Tables 1-3). These

successfully synthesized IPNs were characterized using FT-IR (Figures 3 and 4) to

confirm their chemical composition. They were then subjected to a number of

experiments to evaluate their ability to function as biocompatible slow drug release

devices. The FT-IR characterizations of these IPN hydrogels are specified in Section

5.3.2.1.

The first part of this discussion is focussed on the formation of the IPNs using various

formulations, some of which were unsuccessful. A number of factors, which are

influential towards the fate of the polymerisation, have been proposed. The composition

of the formulation could be regarded as the utmost important factor. The relative

reactivity of the components and secondly the compatibility of the components are the

key issues outlined by this factor. Furthermore, reaction conditions such as the solvent

type and radiation dose are also influential towards the fate of the polymerisation. These

factors may either lower the reaction kinetics as to minimise the rate of polymerisation or

hinder the polymerisation path by initiating undesirable side reactions.

5.4.1. Influential Factors on Polymerisation

The chemical composition and the type of solvent used for the preparation of IPNs were

found to be critically influential factors towards the efficiency and the nature of the

polymerisation process. These factors are discussed in Sections 5.4.1.1 - 5.4.1.2.

5.4.1.1. Effect of Solvent on Polymerisation

Chitosan is insoluble in neutral and basic medium due to its cationic nature, however it is

soluble in acidic medium. The solubility of chitosan is essentially governed by the

possibility of formation of inter and intramolecular hydrogen bonds [20]. The acidic

solvents used to achieve this were also evaluated for their role in the polymerisation

process. IPN formulations, which involved chitosan pre-dissolved in acrylic acid and

NVP resulted in a dark reddish coloured complex, which did not solidify upon the applied

radiation dose of approximately 9 KJ. However, formulations containing chitosan pre-

210


dissolved in levulinic acid resulted in three successfully polymerised IPN out of the four

formulations.

The solvents, which are carboxylic acids in this instance, are known to form an

intermolecular complex with chitosan, seem to play an important role in the nature of the

polymerisation reaction. Acrylic acid (AA) was the first choice of solvent for this work

due to the fact that it is a moderately strong electron withdrawing monomer. Since this

work was based on CT complex polymerisation, it was proposed that AA would make an

excellent acceptor monomer in the donor/acceptor pair with NVP as the donor and at the

same time, AA would also serve as a solvent. Thus, inclusion of another solvent or an

acceptor monomer could be avoided.

The formulation, which contained pre-dissolved chitosan in acrylic acid and NVP and

NVP/HEMA formed a strong coloured complex observed after a short exposure to the

UV light. This indicated the formation of a CT complex of very high ionic character [28].

Chitosan has a number of labile abstractable hydrogens, which would effectively enhance

the rate of CT complex formation between AA and NVP. Ng et al [29,30] in their studies

on CT complex systems have described a significant positive effect of hydrogen donors

on the efficiency of CT complex formation.

Due to the formation of a strong coloured complex, the UV light could not penetrate

through the sample thus a complete polymerisation reaction was not achieved. In the case

with levulinic acid as the solvent, it can be concluded that the acid did not affect the

nature of polymerisation. However, its participation in the reaction must be accounted for

as an acid base reaction with the chitosan, which is a weak base with a pKa value between

6.2-7 [31]. Scheme 1 illustrates the formation of an intermolecular complex via an

interaction between weakly basic chitosan and carboxylic acids [32,33].

211


O

OH N

OH

OO

H

H

O

OH N+

OH

OO

H

H H

R O

O

HRO

O

H+

+

Chitosan Carboxylic acid Intermolecular complex

+

Scheme 1. Acid base interaction between a carboxylic acid and chitosan

.

5.4.1.2. Effect of Monomer Composition on Polymerisation

IPN formulations containing pre-dissolved chitosan in levulinic acid showed positive

signs of forming IPN with NVP. However, formulations containing high amount of

chitosan/acid mixture showed very low reactivity of IPN formation upon exposure to the

UV light. This could be attributed to the fact that sufficient amount of the NVP and

HMMI complex was not present to initiate the polymerisation of NVP leading to the

formation of an IPN with chitosan. A similar observation was made in formulations

containing CM chitosan. It was experimentally shown that the formulations with low

NVP-HMMI mixture or the formulation containing 50 % HEMA and 50 % chitosan/acid

solution did not successfully yield an IPN when exposed to UV radiation.

HEMA is an acceptor monomer, which has been previously described in Sections 3 and 4

to effectively homopolymerise through the intramolecular hydrogen abstraction process.

However, the cause of unsuccessful polymerisation in the case of 50 % HEMA and 50 %

chitosan/acid solution formulation could be attributed to the fact that a higher

composition of HEMA was required to initiate the polymerisation process. The 25 %

HEMA -25 % NVP -50 % chitosan/levulinic acid solution formulation however resulted

in a successfully synthesized IPN. The successful IPN formation between HEMA-NVP

and chitosan could be attributed to the donor/acceptor interaction between HEMA and

NVP, which initiated the polymerisation leading to the IPN formation.

The IPNs formed from chitosan and CM chitosan were opaque upon curing with the

exception of chitosan-HEMA-NVP IPN, which was clear. It could be stated from this

212


observation that the IPNs containing just the polysaccharides and NVP in the presence of

HMMI were crystalline in nature. The optical clarity in the chitosan-HEMA-NVP is

indicative of an amorphous network.

5.4.2. Swelling and Drug Release Evaluation

The IPN hydrogels formed were subjected to the swelling and drug release experiments.

The 48 % chitosan- 48 % NVP- 4 % HMMI and 48 % CM chitosan- 48 % NVP- 4 %

HMMI, though successfully synthesized were found to be water-soluble. The solubility of

these IPNs could be attributed to the lack of sufficient crosslinking in the structure. The

swelling and drug release behaviour in the IPN hydrogels were characterized on the basis

of Fickian or non-Fickian diffusion behaviour. The swelling action in polymers is

generally time dependent and could be described according to the power-law equation

described in previous sections. The value of n in the power-law equation indicates the

diffusion kinetics. The characteristic n values for Fickian and non-Fickian diffusion

kinetics have been previously mentioned in Section 1.4.2.3.1.2.

5.4.2.1. IPN Swelling Behaviour


An exponential increase in the water uptake by Gel A and Gel B in neutral pH

environment at 37 oC was observed in the first hour (Figure 6) indicating non-Fickian

anomalous transport behaviour. This was confirmed by a LOG plot (Figure 7, Table 5)

from which average n values of 0.61 and 0.66 were calculated for gels A and B

respectively. In the later stages of the swelling process, Case II diffusion behaviour

prevailed which was indicated by a gradual decrease in the rate of water uptake by the

IPNs.

The gradual decrease in the rate of water uptake was observed around 7 hours of swelling

which became constant upon 24 hours of constant swelling thus indicating that

equilibrium water content uptake was achieved (Table 4, Figure 5). At this stage the

swelling process becomes time independent where the parameter n = 1. Equilibrium water

content (EWC) values of 91.4 % and 93.6 % were observed for the Gel A and Gel B

respectively. The slight reduction in the swelling efficiency of Gel A could be attributed

213


to the formulation composition. A slight reduction in NVP content of the IPN

composition could have been the cause of slight variation in the swelling efficiencies

between the IPNs.

Gel C however adhered to Fickian diffusion behaviour in the initial stages of the swelling

experiment followed by an anomalous transport in the later stages (Figures 6 and 7, Table

5). Equilibrium swelling was achieved after 48 hours of constant experimental swelling,

which yielded an EWC value of 79.6 % (Table 4, Figure 5). Inclusion of a relatively

hydrophobic monomer such as HEMA has led to a reduction in the equilibrium water

content. As previously described, HEMA will form a donor/acceptor pair with NVP. Thus

it could be stated that HEMA was effectively consumed in formulation upon UV curing,

as its presence was effective on the swelling process. Presence of HEMA in the IPN was

also evidenced by the FT-IR spectroscopic data as described Section 5.3.2.1.

5.4.2.1.2. Effect of the Environmental pH on Swelling

Risbud et al [34] have reported highly pH sensitive IPNs based on chitosan and NVP.

Chitosan is polycationic thus it will respond to pH changes below its pKa value. Gupta

and Kumar [35] in their study on the diffusion behaviour in chitosan beads over a wide

pH range have also reported similar observations. Chen et al [10] have reported the

amphoteric nature of CM chitosan based IPNs. This could be attributed to the fact that

CM chitosan has basic amine groups as well as acidic acetyl groups. CM chitosan based

IPNs have a certain isoelectric point (IEP) at which these IPNs shrink the most. The IEP

of polyampholytic hydrogels is described as a point where equal amount of anionic and

cationic units exist on their backbones and thus at near the IEP, the Coulombic attraction

between the oppositely charged units within the hydrogel matrix causes the collapse of

the network. However, upon deviating the environmental pH from the IEP these gels

display an increased swelling behaviour.

The results of the swelling experiments on the IPNs at varied pH environments showed

slight variations in the swelling efficiency. The chitosan based IPNs, Gel B and Gel C

showed a slight reduction in the swelling efficiency in pH 8 environment in comparison

to neutral, isotonic and pH 2 environments (Tables 6,8 and 10, Figures 8-10, 11-13,14-

214


16). Gel C displayed EWC values of 71.8 %, 70.8 % and 67.5 % in pH 2, pH 7.4 and pH

8 environments respectively. EWC values of 93.3 %, 91.9 % and 92.8 % were observed

in pH 2, pH 7.4 and pH 8 environments respectively for Gel B. These results on chitosan

based IPNs are in agreement with other researchers [34,35]. However, a drastic variation

in the swelling behaviour was not observed as reported by Risbud et al [34], Gupta and

Kumar [35]. The low pH sensitivity of the chitosan based IPNs could be attributed to the

fact that a low amount of chitosan was used in the formulation thus the pH sensitivity of

the polysaccharide was suppressed.

The results on Gel B in different pH environments indicated non-Fickian diffusion

kinetics in the early stages of swelling, with n values (Tables 7,9, 11) of 0.74, 0.70 and

0.64 in pH 2, pH 7.4 and pH 8 environments respectively. The diffusion mechanism in

Gel C is reflective of Fickian diffusion kinetics in the early stages of swelling, with n

values of 0.48, 0.51 and 0.50 in pH 2, pH 7.4 and pH 8 environments. The chitosan based

IPNs showed a rapid swelling up to 7 hours after which a gradual decrease in the swelling

efficiency was observed with equilibrium swelling achieved around 48 hours.

The swelling experiments on Gel A in different pH environments are indicative of

ampholytic behaviour. However, as in the case of chitosan based IPNs, the sensitivity to

varied pH environments were not pronounced. The IPNs yielded EWC values of 91.3 %,

91.8 % and 91.4 % in pH 2, pH 7.4 and pH 8 environments respectively. Despite similar

EWC values in varied pH environments, a slight reduction in the swelling efficiency was

observed in pH 8 environment in the initial stages of swelling in comparison to pH 2,

neutral and pH 7.4 environments. The swelling efficiency was observed to be highest in

pH 7.4 environment followed by pH 2, neutral and pH 8 environment. The % water

content values of 44.3 %, 47.2 %, 51.3 % and 42.6 % in neutral, pH 2, pH 7.4 and pH 8

environments respectively were observed at the initial 10 minutes of swelling.

The sharp increase in swelling activity from neutral to pH 7.4 environment could be

attributed to the increase in the ionic strength of the swelling agent. Ng et al [36] in their

studies on polyampholytic hydrogels have described a similar observation in relation to

the concept of Donnan equilibrium. The pH 7.4 buffer has an ionic strength of 0.2 in

215


comparison to neutral medium which is just milli-Q-water with an assumed ionic strength

of zero.

According to the concept of Donnan equilibrium, more electrolytes will penetrate into the

hydrogel matrices with increase in ionic strength of the swelling agent and the mobile

counterions surrounding the charged groups in the hydrogel matrices will affect the net

osmotic pressure within the hydrogel network. Chen et al [7] have also observed

increased swelling behaviour in CM chitosan based IPN in pH 7.4 environment. They

suggest that the carboxylic acid groups become progressively ionized at pH 7.4, thus the

significant electrostatic repulsion between the ionized acid groups cause an increase in

swelling.

However, the experimental swelling data indicated reduced swelling activity in pH 8

environment. The data thus suggest the presence of IEP in the vicinity of pH 8, which has

caused the slight reduction in the swelling activity of the IPN. Gel A displayed non-

Fickian swelling kinetics in the early stages of swelling, with n values of 0.68, 0.70 and

0.60 in pH 2, pH 7.4 and pH 8 environments respectively (Tables 7, 9 and 11).


The drug release experiments conducted on the chitosan-NVP-HEMA, chitosan-NVP and

CM chitosan-NVP IPNs (Table 12, Figures 20-22) in neutral pH environment using

theophylline as the model drug yielded equilibrium drug release (EDR) values of 0.99,

0.96 and 0.97 respectively in 48 hours. The drug release rate increased rapidly in the first

2 hours of the experiment after which the release rate gradually slowed down and

eventually became constant around 7 hours. The quick release of the drug from the carrier

in the early stages of the experiment is attributed to the burst effect release [37]. As

previously described, the polymer containing the incorporated solute has to swell to a

certain extent before it can release its contents. The resultant effect is the fast release of

the solute in a short span of time.

The drug release profile observed in the IPNs could be described as Fickian transport

behaviour as illustrated in Figure 21 where the fractional drug released at time t is directly

216


proportional to the square root of time. The LOG plot (Figure 22, Table 13) of the

fractional drug released from the IPNs yielded an average n value of 0.50, which

indicated that Fickian release mechanism was in operation. A very similar release rate

was observed in all the IPNs even though the chitosan-NVP-HEMA had a relatively low

swelling activity. This could be attributed to the relatively low molecular weight of

theophylline, which diffuses with ease through the pores of all the IPNs at a similar rate.

5.5. Conclusions

IPN hydrogels based on the polysaccharides, chitosan and CM chitosan in conjunction

with NVP and HEMA were successfully synthesized through a photoinitiator-free curing

technique. Levulinic acid was found to be a better solvent over AA in the formulation

resulting in successfully synthesized IPNs. However, it was necessary to have NVP-

HMMI and NVP-HEMA in sufficient quantity for efficient curing and adequate

crosslinkage in the IPN matrix. The hydrogels were found to be resilient and competent

drug delivery devices.

Experimental swelling data revealed that the CM chitosan-NVP and chitosan-NVP IPNs

adhere to non-Fickian anomalous diffusion behaviour in the earlier stages of the

experiment followed by case II diffusion in the later stages. Inclusion of HEMA into the

IPN resulted in reducing the swelling activity in the IPN, which adhered to a typical

Fickian behaviour. The IPNs under study were found to be sensitive to variations in the

environmental pH with chitosan based IPNs displaying cationic behaviour while CM

chitosan based IPN displayed ampholytic behaviour. However, a pronounce pH

sensitivity of the IPNs based on polysaccharides under study as reported by other

researchers was not observed.

The drug release experiments revealed that all the IPNs understudy adhered to Fickian

transport mechanism in releasing theophylline into the neutral pH environment at 37 oC.

The burst effect release of the drug was observed in the initial stages of the release

experiment followed by linear release profile. The rate of release of theophylline in all the

IPNs under study were found to be similar indicating that theophylline being a low

molecular weight drug could diffuse through the IPN membranes with ease.

217


5.6. References

1. Sperling, L. H., “Interpenetrating Polymer Networks and Related Materials”,

Plenum Press, New York, pp. 1-10, (1981).



3. Sperling, L. H., Mishra, V., Polym. Adv. Technol., 7, 197-208, (1996).

4. Maolin, Z., Jun, L., Min, Y., Hongfei, H., Radiation Phys. Chem., 58, 397-400,

(2000).

5. Xuequan, L., Maolin, Z., Jiuqiang, L., Hongfei, H., Radiation Phys. Chem., 57,

477-480, (2000).

6. Bartolotta, A., Di Marco, G., Lanza, M., Carini, G., D’Angelo, G., Tripodo, G.,

Fainleib, A., Danilenko, I., Grytsenko, V., Sergeeva, L., Materials Sci. Eng. A,

370, 288-292, (2003).

7. Chen, S.-C., Wu, Y.-C., Mi, F.-L., Lin, Y.-H., Yu, L.-C., Sung, H.-W., J.

Controlled Release, 96, 285-300, (2004).

8. Lee, J. W., Kim, S. Y., Kim, S. S., Lee, Y. M., Lee, K. H., Kim, S. J., J. Appl.

Polym. Sci., 73, 113-120, (1999).

9. Risbud, M. V., Bhat, S. V., J. Mater. Sci. Mater. Med., 12, 75-79, (2001).

10. Chen, L., Tian, Z., Du, Y., Biomaterials, 25, 3725-3732, (2004).

11. Peniche, C., Argüelles-Monal, W., Davidenko, N., Sastre, R., Gallardo, A.,

Román, J. S., Biomaterials, 20, 1869-1878, (1999).

12. Yao, K.-D., Liu, J., Cheng, G.-X., Zhao, R.-Z., Wen, H. W., Wei, L., Polym. Int.,

45, 191-194, (1998).

13. Furlan, L., De Fàvere, V. T., Laranjeira, M. C. M., Polymer, 37, 843-846, (1996).

14. Smirnova, L, A., Semchikov, Y. D., Tikhobaeva, Y. G., Pastukhova, N. V.,

Polym. Sci. Series B, 43, 33-36, (2001).

15. Kumar, M. N. V. R., React. Func. Polym., 46, 1-27, (2000).

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308, (2000).

17. Mi, F.-L., Tan, Y.-C., Liang, H.-F., Sung, H.-W., Biomaterials, 23, 181-191,

(2002).

18. Chandy, T., Sharma, P., Biomater. Artif. Cells Artif. Organs, 18, 1-24, (1990).

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19. Singh, D. K., Ray, A. R., J. Appl. Polym. Sci., 53, 1115-1121, (1994).

20. Domard, A., Domard, M., “Polymeric Biomaterials”, Dumitriu, S, ed., 2nd

ed.,

Marcel & Decker, Inc., New York, pp.187-212, (2002).

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22. Illum, L., Pharm. Res., 15, 1326-1331, (1998).

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214, (1994).

26. Chen, X.-G., Park, H.-J., Carbohydr. Polym., 53, 355-359, (2003).

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Hoyle, C. E., Proc. Radtech Asia’95, Bangkok, Thailand, pp. 118-125, (1995).

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(2002).

30. Ng, L.-T., Jönsson, S., Lindgren. K., Swami, S., Hoyle, C., Clark, S., Proc.

Radtech Europe’01, Basel, Switzerland, pp. 609-613, (2001).

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ed., Marcel

& Decker, Inc., New York, pp. 213-237, (2002).

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pp. 109-135, (1987).

219




5.3. Results 188

5.3.1. Polymerisation of IPNs 188

5.3.1.1. Chitosan Based IPNs 188

5.3.1.2. CM Chitosan Based IPNs 189

5.3.2. Characterization of the IPNs 190

5.3.2.1. FT-IR Data Analysis 190

5.3.2.1.1. Chitosan 190

5.3.2.2.2. CM Chitosan 190

5.3.2.2.3. IPN Gel A 191

5.3.2.1.4. IPN Gel B 191

5.3.2.1.5. IPN Gel C 191


5.3.3.1. Swelling Test in Varying pH Environments 196


5.4. Discussion 210

5.4.1. Influential Factors on Polymerisation 210

5.4.1.1. Effect of Solvent on Polymerisation 210

5.4.1.2. Effect of Monomer Composition on Polymerisation 212

5.4.2. Swelling and Drug Release Evaluation 213

5.4.2.1. IPN Swelling Behaviour 213


5.4.2.1.2. Effect of the Environmental pH on Swelling 214



5.6. References 218

220

Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique

6.1. Introduction

Variation in pH is the most important signal in the human body, which occurs naturally in

different parts of the body. The pH of the gastric condition is much lower than that of the

enteric condition [1]. A pH sensitive hydrogel is an excellent candidate for an intelligent

delivery device, which will respond to the variation in the environmental pH [1-6].

Numerous pH sensitive hydrogel based drug delivery systems for varied pH domains of

the human body such as periodontal, oral, gastric and intestinal applications have been

developed in recent years [3,7-11].

Polymeric hydrogels, which exhibit pH sensitivity contain either acidic or basic pendent

groups on it structure. In appropriate pH environment with adequate ionic strength, these

pendent groups ionize and develop fixed charges on the hydrogel [4]. The degree of the

ionization determines the swelling efficiency of the ionic hydrogel network [12-14].

These polymers with large number of ionizable groups are also referred to as

polyelectrolytes [2,5,15-18].

Polyelectrolytes could be anionic, cationic or ampholytic in nature depending on the

nature of pendent groups. Anionic hydrogels containing acidic pendent groups exhibit a

marked increase in the degree of swelling at high pH whilst the opposite response is

observed for cationic hydrogels containing basic pendent groups [15-19]. Ampholytic

behaviour in hydrogels is referred to as having both acidic and basic pendent groups on

the polymer structure [20].

Figure 1. Swelling of an ionic hydrogel network due to ionization of pendent groups at

specific pH values.

220


Figure 1 illustrates a typical swelling and de-swelling behaviour of an ionic hydrogel in

response to environmental pH variations [4]. Ionic hydrogels have been described in

detail in Section 1.1.1.5.

A wide range of acidic, neutral and basic monomers has been used by the researchers in

numerous varied compositions to achieve desirable environmental sensitivity in specific

applications [11-27]. Use of AA, HEMA and NVP has been commonly reported in the

literature. Kaczmarek et al [26], Devine and Higginbotham [11] reported the use of NVP

and AA where the monomers were individually polymerised and then let to form an inter-

macromolecular complex. Khare et al [14], Am Ende et al [19,24], Khare and Peppas

[12] made use of AA and HEMA to synthesize anionic hydrogels. Sahoo et al [25]

copolymerised NVP and AA in the presence of a crosslinking agent, NN’ methylene bis-

acrylamide to function as hydrogel nanoparticles.

Chapiro and Trung [28] studied interactions between AA derivatives and NVP, and they

described the formation of a complex due to an donor/acceptor interaction. Garnet and

Zilic [29] in a recent study on charge transfer (CT) complexes have also commented on

the suitability of AA as an electron acceptor monomer. However, to date, the concept of

CT complex formation has not been applied to the synthesis of ionic hydrogels.

Researchers [11-26] have either used high-energy source such as gamma radiation or

conventional photo and thermal curing methods in the presence of initiators to prepare

this particular class of hydrogels.

In the present work the author has made use of HEMA, NVP and AA to form negatively

charged anionic polymeric networks via a photoinitiator-free process. These networks

were intended to function as pH sensitive hydrogels for slow drug delivery applications.

The hydrogel formation is based on the concept of donor/acceptor pair interaction

discussed previously in Section 4. In this study HEMA and AA were utilized as electron

acceptor monomers combined with an electron donor monomer, NVP.

The first part of the work involved kinetic studies using the Differential Photocalorimetric

(DPC) technique on the donor/acceptor pair interactions with AA and HEMA as

221


acceptors and NVP as the donor. The donor/acceptor pair were formulated in varying

mole ratios of the acceptor and donor and subjected to DPC measurements. Following the

kinetics study, which indicated the suitability of the donor/acceptor pairs, the anionic

hydrogels were synthesized through the photoinitiator-free process. They were

subsequently tested for their swelling and drug release behaviours in acidic, neutral and

basic pH environments.


DPC measurements were carried out to evaluate the efficiency of complex formation

between the donor/ acceptor pairs. Experimental specifications and detailed procedure of

DPC measurements have been outlined in Sections 2.2.2.5 and 2.8. The NVP-HEMA-AA

hydrogels formed through this photoinitiator-free process were subjected to swelling drug

release experiments at 37 oC. The effect of acidic and basic environment on the swelling

and drug release behaviours of NVP-HEMA-AA hydrogels was also investigated. A

model drug, theophylline was used for drug release investigations. Detailed experimental

procedure for the synthesis of NVP-HEMA-AA hydrogels is outlined in Section 2.5.2.7.

Specifications and procedure of swelling-drug release experiments including the

preparation of the swelling and drug release media have been described in Sections 2.6.1

and 2.7. Quantitative drug release measurements were carried out on a UV-vis

spectrophotometer.

222


6.3. Results

6.3.1. DPC Measurements on AA/NVP and HEMA/NVP Systems

AA and HEMA were considered as potential acceptors, which were combined with NVP,

a donor monomer in varying mole ratios. The photo-exotherms (Figures 2 and 3) and

rates of polymerisation (Tables 1 and 2) indicate the relative efficiency of the

donor/acceptor pair.

AA : NVP 1mol : 1mol


-2

8

18

Hea

t F

low

(W

/g)

0 100 200 300 400 500Time (sec)Exo Up


Figure 2. Photo-exotherms of AA: NVP in varying mole ratios

Table 1. Rate of polymerisation of AA: NVP in varying ratios

Molar composition

AA: NVP

Polymerisation rate (J g-1

s-1

)

at 15 oC, N2

1 mol: 1 mol 0.53

1 mol: 2 mol 0.23

2 mol: 1 mol 0.37

223


1 mol HEMA : 2 mol NVP


-1

0

1

Hea

t F

low

(W

/g)



Table 2. Rate of polymerisation of HEMA: NVP in varying ratios

Figure 3. Photo-exotherms of HEMA: NVP in varying mole ratios

Molar composition

HEMA: NVP

Polymerisation rate (J g-1

s-1

)

at 15 oC, N2

1 mol: 1 mol DNP

1 mol: 2 mol DNP

2 mol: 1 mol DNP

DNP indicates that the sample did not polymerise within the duration of DPC

measurement. The polymerisation rates between the donor/acceptor pairs were calculated

according to equation 1 where t, is the time taken to reach peak max.

Rate of polymerisation (J g-1

s-1

) = -1Peak max (J g )

t (s) Equation 1

224


6.3.2. Photopolymerisation of Hydrogels Containing NVP, AA and HEMA

NVP, AA and HEMA were combined in varying ratios by volume and subjected to UV

radiation in the absence of photoinitiator. The observations on the extent of


Table 3.

Table 3. Polymerisation status of formulations of NVP, HEMA and AA in varying

% v/v ratios

HEMA-NVP-AA hydrogel formulation

NVP : HEMA : AA (% v/v)



50 : 00: 50 Hard, clear, pale yellow gel

50 : 50 : 00 Hard, clear, colourless gel




00 : 50 : 50 Hard, clear, pale yellow gel

10 : 50: 40 Hard, clear, colourless gel






The monomer compositions are expressed as percentage v/v. The radiation dose was

calculated according to Equation 2 where t is the total radiation time in seconds. The

samples were exposed to UV radiation for approximately 25 hours at a dose rate of 9.6 x

10-2

J s-1

. The dose rate was calculated as described in Section 2.4.4.4.



225



The anionic hydrogels based on HEMA, AA and NVP in varying compositions were

subjected to swelling test in varying pH environments at 37 oC. The hydrogels were in

general found to respond to the pH variations with varying extent of swelling with

varying hydrogel compositions and the environmental pH. The swelling test results on the

hydrogels at varied pH environments expressed as % water content values at designated

time intervals are presented in Tables 4-21. The 50 NVP: 50 AA hydrogel though

successfully polymerised was found to be water-soluble, thus no further tests were

conducted on this hydrogel. Graphical representations of the swelling data are illustrated

in Figures 4-32.

226


Table 4. Swelling test on the formulations of HEMA (50 % v/v) with varied ratios (

% v/v) of NVP and AA in neutral pH environment at 37 oC


Time (h) Gel A Gel B Gel C Gel D

0.00 0.0 0.0 0.0 0.0

0.17 10.6 9.7 9.2 7.8

0.33 14.1 13.5 12.4 11.0

0.50 16.5 16.5 15.4 13.2

0.67 18.2 18.2 16.4 18.2

0.83 19.7 20.2 17.7 17.4

1.00 20.5 21.4 19.1 18.3

2.00 27.9 28.3 25.3 24.3

3.00 30.5 31.6 27.0 26.2

4.00 33.3 33.4 29.9 29.3

5.00 35.6 35.8 32.4 30.8

7.00 39.0 39.0 36.0 34.8

9.00 41.1 41.3 37.5 36.7

12.00 43.3 43.4 40.2 38.8

24.00 47.5 46.8 45.2 42.0

48.00 49.2 48.1 45.9 43.4

72.00 49.5 48.2 46.2 42.7

96.00 48.9 47.9 46.2 42.3

120.00 49.2 47.5 46.2 42.4

144.00 48.8 47.8 47.2 42.5

170.00 49.1 48.4 46.6 42.6

Hydrogel compositions: (NVP : HEMA : AA) (% v/ v)); 00 : 50 : 50 (Gel A); 10 : 50 : 40

(Gel B); 40 : 50 : 10 (Gel C) and 25 : 50 : 25 (Gel D)

227




Graphical representations of the swelling behaviour observed in the NVP-HEMA-AA

hydrogels in neutral pH environment at 37 oC are illustrated in Figures 4 - 6.

0

20

40

60

80

100

0 50 100 150 200

Time (h)

% W

ate

r C

on

ten

t


Figure 4. Plot of % water content in Gels A - D at 37 oC in neutral pH environment as a

function of time.

0.0

0.4

0.8

1.2

0 5 10 15

t1/2

(h1/2

)

Mt/

Min

fin

ity


Figure 5. Plot of fractional swelling in Gels A - D at 37 oC in neutral pH environment as

a function of the square root of time.

228


-1.5

-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G M

t/ M

inif

inty



initial stages of swelling in Gels A - D at 37 oC in neutral pH environment.

As previously described in Sections 1 and 3, the parameter n is the kinetic exponential

value in the power law equation (equation 3), which indicates the time dependence

swelling and solute release kinetics in a swellable polymeric material.


The n values were calculated as the slope of the LOG graph. Table 5 presents the slope

(n) values calculated from Figure 6.

Table 5. Characteristic exponential n values for diffusion in NVP-HEMA-AA


Hydrogels n values

Gel A 0.44 + 0.03

Gel B 0.52 + 0.01

Gel C 0.47 + 0.02

Gel D 0.53 + 0.01

229


Table 6. Swelling test on the formulations of AA (50 % v/v) with varied ratios ( %

v/v) of NVP and HEMA in neutral pH environment at 37 oC


Time (h) Gel E Gel F Gel G

0.00 0.0 0.0 0.0

0.17 34.5 12.0 15.4

0.33 42.7 16.1 20.8

0.50 48.7 19.7 24.3

0.67 53.7 21.3 28.0

0.83 57.8 23.4 30.3

1.00 60.0 25.1 32.4

2.00 69.3 32.6 41.1

3.00 72.4 36.7 45.7

4.00 75.0 40.1 50.0

5.00 76.9 43.3 52.0

7.00 79.0 46.3 55.7

9.00 80.0 48.5 57.4

12.00 81.3 50.7 59.5

24.00 82.9 54.5 62.1

48.00 84.1 55.0 63.0

72.00 84.6 54.6 63.0

96.00 84.7 54.3 63.3

120.00 85.0 54.4 63.1

144.00 85.1 54.4 63.4

170.00 85.6 55.1 63.5

Hydrogel compositions: (NVP: HEMA: AA (% v/ v)); 40 : 10 : 50 (Gel E); 10 : 40 : 50

(Gel F) and 25 : 25 : 50 (Gel G)

230


0

20

40

60

80

100

0 50 100 150 200

Time (h)

% W

ate

r C

on

ten

t

Gel E Gel F Gel G

Figure 7. Plot of % water content in Gels E - G at 37 oC in neutral pH environment as a

function of time.

0.0

0.4

0.8

1.2

0 5 10 15

t1/2

(h1/2

)

Mt/ M

infi

nit

y

Gel E Gel F Gel G

Figure 8. Plot of fractional swelling in Gels E - G at 37 oC in neutral pH environment as a


231


-1.5

-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G M

t/ M

infi

nit

y

Gel E Gel F Gel G


initial stages of swelling in Gels E - G at 37 oC in neutral pH environment.

Table 7 presents the slope (n) values calculated from Figure 9.



Hydrogels n values

Gel E 0.60 + 0.02

Gel F 0.50 + 0.02

Gel G 0.52 + 0.01

232


Table 8. Swelling test on the formulations of NVP (50 % v/v) with varied ratios (%

v/v) of AA and HEMA in neutral pH environment at 37 oC


Time (h) Gel H Gel I Gel J Gel K

0.00 0.0 0.0 0.0 0.0

0.17 13.8 11.6 18.3 8.7

0.33 18.3 15.9 22.0 11.8

0.50 21.6 19.1 24.9 14.4

0.67 24.8 21.8 28.3 15.6

0.83 27.0 24.4 30.8 17.4

1.00 29.4 25.9 32.5 19.0

2.00 38.0 34.4 39.8 25.2

3.00 42.3 36.7 43.8 26.8

4.00 46.5 39.8 47.5 29.0

5.00 49.4 42.6 49.7 31.5

7.00 53.6 46.6 52.6 34.4

9.00 55.8 49.2 53.4 36.8

12.00 58.2 52.3 55.0 39.1

24.00 62.5 57.8 56.5 42.9

48.00 64.4 60.1 57.4 44.2

72.00 64.5 60.8 57.8 43.7

96.00 64.5 60.9 57.4 43.9

120.00 64.6 61.5 57.2 43.0

144.00 64.8 61.8 57.1 43.4

170.00 65.2 62.1 59.2 43.2

Hydrogel compositions: (NVP : HEMA : AA (% v/ v)); 50 : 50 : 00 (Gel H); 50 : 40 : 10

(Gel I); 50 : 10 : 40 (Gel J) and 50 : 25 : 25 (Gel K)

233


0

20

40

60

80

100

0 50 100 150 200

Time (h)

% W

ate

r C

on

ten

t

Gel H Gel I Gel J Gel K

Figure 10. Plot of % water content in Gels H - K at 37 oC in neutral pH environment as a

function of time.

0.0

0.4

0.8

1.2

0 5 10 15

t1/2

(h1/2

)

Mt/

Min

fin

ity


Figure 11. Plot of fractional swelling in Gels H - K at 37 oC in neutral pH environment as

a function of the square root of time.

234


-1.5

-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G M

t/ M

inif

init

y



initial stages of swelling in Gels H - K at 37 oC in neutral pH environment.




Hydrogels n values

Gel H 0.53 + 0.01

Gel I 0.52 + 0.01

Gel J 0.44 + 0.04

Gel K 0.49 + 0.03

235


Table 10. Swelling test on the formulations of HEMA (50 % v/v) with varied ratios

(% v/v) of NVP and AA in acidic (pH 2) environment at 37 oC



0.00 0.0 0.0 0.0 0.0

0.17 10.7 12.1 8.3 8.3

0.33 12.7 13.1 10.7 9.8

0.50 15.0 16.0 12.6 12.7

0.67 17.4 18.4 14.4 13.3

0.83 19.7 21.0 15.1 15.7

1.00 20.3 21.3 16.6 17.6

2.00 26.4 29.2 22.6 23.1

3.00 29.3 31.3 24.5 26.3

4.00 31.6 33.4 26.9 27.8

5.00 32.8 35.8 28.8 29.3

7.00 35.9 40.0 31.9 32.0

9.00 37.6 40.4 34.1 34.4

12.00 40.2 42.8 36.9 36.9

24.00 43.3 45.3 41.5 39.9

48.00 45.4 45.6 43.2 40.7

72.00 44.6 46.5 43.1 40.5

96.00 44.5 45.9 43.1 40.5

120.00 43.9 45.5 42.8 40.3

144.00 44.2 45.3 42.7 39.8

170.00 44.7 45.4 42.8 39.7



236


0

20

40

60

80

100

0 50 100 150 200

Time (h)

% W

ate

r C

on

ten

t


Figure 13. Plot of % water content in Gels A - D at 37 oC in pH 2 environment as a

function of time.

0.0

0.4

0.8

1.2

0 5 10 15

t1/2

(h1/2

)

Mt/

Min

fin

ity


Figure 14. Plot of fractional swelling in Gels A - D at 37 oC in pH 2 environment at 37

oC in pH 2 environment as a function of the square root of time.

237


-1.5

-1.0

-0.5

0.0

-1.0 -0.5 0.0

LOG time

LO

G M

t/ M

inif

inty



initial stages of swelling in Gels A - D at 37 oC in pH 2 environment.



hydrogels in acidic medium

Hydrogels n values

Gel A 0.45 + 0.04

Gel B 0.42 + 0.05

Gel C 0.44 + 0.03

Gel D 0.47 + 0.02

238



v/v) of NVP and HEMA in acidic (pH 2) environment at 37 oC



0.00 0.0 0.0 0.0

0.17 28.7 14.5 15.3

0.33 37.4 17.5 20.1

0.50 43.0 20.9 23.4

0.67 46.7 22.6 26.8

0.83 49.6 25.0 28.6

1.00 52.3 26.5 30.2

2.00 58.7 32.3 38.9

3.00 60.8 36.9 43.8

4.00 62.7 38.9 46.9

5.00 63.0 41.3 49.6

7.00 63.4 44.7 52.6

9.00 63.8 46.2 54.5

12.00 64.6 48.2 56.2

24.00 64.0 50.7 58.1

48.00 64.1 51.5 59.3

72.00 64.2 51.5 58.9

96.00 64.2 51.3 58.8

120.00 64.1 51.1 58.8

144.00 64.4 51.1 58.7

170.00 63.6 50.8 59.0


(Gel F) and 25 : 25 : 50 (Gel G)

239


0

20

40

60

80

100

0 50 100 150 200

Time (h)

% W

ate

r C

on

ten

t

Gel E Gel F Fel G

Figure 16. Plot of % water content in Gels E - G at 37 oC in pH 2 environment as a

function of time.

0.0

0.4

0.8

1.2

0 5 10 15

t1/2

(h1/2

)

Mt/

Min

fin

ity

Gel E Gel F Gel G

Figure 17. Plot of fractional swelling in Gels E - G at 37 oC in pH 2 environment as a


240


-1.5

-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G M

t/ M

infi

nit

y

Gel E Gel F Gel G


initial stages of swelling in Gels E - G at 37 oC in pH 2 environment




Hydrogels n values

Gel E 0.53 + 0.01

Gel F 0.43 + 0.03

Gel G 0.50 + 0.02

241


Table 14. Swelling test on the formulations of NVP (50 % v/v) with varied ratios ( %

v/v) of AA and HEMA in acidic (pH 2) environment at 37 oC



0.00 0.0 0.0 0.0 0.0

0.17 13.5 11.8 18.3 7.4

0.33 17.2 15.0 22.8 11.0

0.50 20.1 16.8 26.7 14.4

0.67 22.4 19.1 29.5 15.5

0.83 24.2 20.7 30.4 16.0

1.00 27.0 22.0 32.6 17.1

2.00 34.2 30.2 39.4 22.8

3.00 38.8 32.1 42.1 25.1

4.00 43.2 35.3 44.1 27.3

5.00 45.2 37.2 46.3 29.1

7.00 49.1 40.9 48.2 32.1

9.00 51.9 43.3 49.9 34.2

12.00 54.0 46.0 49.8 36.8

24.00 58.0 50.5 50.8 40.1

48.00 59.8 51.8 51.1 41.3

72.00 60.6 52.2 51.5 41.2

96.00 60.5 52.2 51.1 40.5

120.00 60.3 52.3 50.9 40.3

144.00 59.7 52.4 50.7 40.4

170.00 59.9 52.7 50.2 40.5



242


0

20

40

60

80

100

0 50 100 150 200

Time (h)

% W

ate

r C

on

ten

t


Figure 19. Plot of % water content in Gels H - K at 37 oC in pH 2 environment as a

function of time.

0.0

0.4

0.8

1.2

0 5 10 15

t1/2

(h1/2

)

Mt/

Min

fin

ity


Figure 20. Plot of fractional swelling in Gels H - K at 37 oC in pH 2 environment at 37


243


-1.5

-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G M

t/ M

inif

init

y



initial stages of swelling in Gels H - K at 37 oC in pH 2 environment.




Hydrogels n values

Gel H 0.47 + 0.02

Gel I 0.42 + 0.03

Gel J 0.43 + 0.04

Gel K 0.53 + 0.01

244


Table 16. Swelling test on the formulations of HEMA (50 % v/v) with varied ratios (

% v/v) of AA and NVP in basic (pH 8) environment at 37 oC



0.00 0.0 0.0 0.0 0.0

0.17 25.2 25.6 24.2 25.8

0.33 36.7 34.5 31.5 35.1

0.50 41.8 40.9 36.6 41.8

0.67 42.5 46.6 42.6 46.6

0.83 44.9 51.3 47.0 51.1

1.00 46.5 55.4 50.0 54.4

2.00 60.7 68.7 65.1 69.5

3.00 64.9 72.6 69.6 73.4

4.00 69.7 74.9 72.9 77.7

5.00 72.3 77.8 76.1 80.7

7.00 76.9 81.1 80.8 84.6

9.00 79.9 82.7 83.9 86.8

12.00 82.7 86.3 88.0 89.5

24.00 87.4 89.4 91.1 93.1

48.00 88.8 90.1 91.6 93.2

72.00 89.3 90.4 91.6 93.0

96.00 89.2 89.8 91.6 92.8

120.00 89.0 89.6 91.6 92.8

144.00 89.1 89.6 91.7 92.9

170.00 89.1 89.8 91.6 93.0



245


0

20

40

60

80

100

0 50 100 150 200

Time (h)

% W

ate

r C

on

ten

t


Figure 22. Plot of % water content in Gels A - D at 37 oC in pH 8 environment as a

function of time.

0.0

0.4

0.8

1.2

0 5 10 15

t1/2

(h1/2

)

Mt/

Min

fin

ity


Figure 23. Plot of fractional swelling in Gels A - D at 37 oC in pH 8 environment at 37


246


-2.0

-1.5

-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G M

t/ M

infi

nit

y



initial stages of swelling in Gels A - D at 37 oC in pH 8 environment.



hydrogels in basic medium

NVP : HEMA : AA n values

Gel A 0.51 + 0.03

Gel B 0.71 + 0.04

Gel C 0.65 + 0.02

Gel D 0.69 + 0.02

247



v/v) of NVP and HEMA in basic (pH 8) environment at 37 oC



0.00 0.0 0.0 0.0

0.17 92.8 29.0 34.6

0.33 94.1 38.6 46.2

0.50 95.3 45.3 53.6

0.67 95.3 50.1 58.7

0.83 95.5 53.5 62.6

1.00 95.7 56.3 66.0

2.00 96.0 68.3 76.9

3.00 96.0 73.1 81.5

4.00 96.0 77.5 84.8

5.00 96.0 79.6 86.9

7.00 96.1 81.9 89.6

9.00 96.0 84.6 91.2

12.00 96.0 84.9 92.4

24.00 95.9 87.7 94.1

48.00 95.9 88.6 94.4

72.00 96.0 89.3 94.3

96.00 96.1 89.3 94.4

120.00 96.2 89.2 94.4

144.00 96.1 89.0 94.4

170.00 96.0 89.0 94.4


(Gel F) and 25 : 25 : 50 (Gel G)

248


0

20

40

60

80

100

0 50 100 150 200

Time (h)

% W

ate

r C

on

ten

t

Gel E Gel F Gel G

Figure 25. Plot of % water content in Gels E - G at 37 oC in pH 8 environment as a

function of time.

0.0

0.4

0.8

1.2

0 5 10 15

t1/2

(h1/2

)

Mt/

Min

fin

ity

Gel E Gel F Gel G

Figure 26. Plot of fractional swelling in Gels E - G at 37 oC in pH 8 environment as a


249


-2.0

-1.5

-1.0

-0.5

0.0

-2.0 -1.5 -1.0 -0.5 0.0

LOG time

LO

G M

t/ M

inif

init

y

Gel E Gel F Gel G


initial stages of swelling in Gels E - G at 37 oC in pH 8 environment




Hydrogels n values

Gel E 2.13 + 0.10

Gel F 0.65 + 0.04

Gel G 0.72 + 0.02

250


Table 20. Swelling test on the formulations of NVP (50 % v/v) with varied ratios ( %

v/v) of AA and HEMA in basic (pH 8) environment at 37 oC



0.00 0.0 0.0 0.0 0.0

0.17 15.0 31.2 51.0 37.4

0.33 20.5 38.3 62.8 47.4

0.50 23.5 44.4 68.7 54.8

0.67 27.6 48.7 73.3 59.6

0.83 28.9 53.1 76.0 63.5

1.00 30.8 55.3 78.1 66.3

2.00 37.5 69.3 86.0 78.8

3.00 44.0 73.1 90.1 81.7

4.00 47.9 79.9 92.1 84.8

5.00 50.9 83.2 93.4 87.4

7.00 54.6 86.4 95.0 90.6

9.00 57.5 88.6 95.9 92.3

12.00 59.9 90.2 96.6 93.9

24.00 65.7 92.2 97.3 96.3

48.00 67.3 92.3 97.3 96.5

72.00 67.3 92.6 97.3 96.6

96.00 67.1 92.3 97.3 96.5

120.00 67.5 92.2 97.3 96.5

144.00 67.1 92.3 97.4 96.5

170.00 67.1 92.4 97.4 96.5



251


0

20

40

60

80

100

0 50 100 150 200

Time (h)

% W

ate

r C

on

ten

t


Figure 28. Plot of % water content in Gels H - K at 37 oC in pH 8 environment as a

function of time.

0.0

0.4

0.8

1.2

0 5 10 15

t1/2

(h1/2

)

Mt/

Min

fin

ity


Figure 29. Plot of fractional swelling in Gels H - K at 37 oC in pH 8 environment as a


252


-2.0

-1.5

-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G M

t/ M

infi

nit

y



initial stages of swelling in Gels H - K at 37 oC in pH 8 environment.




Hydrogels n values

Gel H 0.52 + 0.02

Gel I 0.57 + 0.02

Gel J 0.69 + 0.03

Gel K 0.72 + 0.02

253


Figure 31 illustrates comparative effect of varying pH environments on the swelling

behaviour of 40 NVP: 10 HEMA: 50 AA hydrogel. Figure 32 illustrates the comparative

effect of the variations in the environmental pH in relation to the hydrogel composition on

the swelling behaviour of the anionic hydrogels.

0

20

40

60

80

100

0 50 100 150 200

Time (h)

% W

ate

r C

on

ten

t

pH 8 neutral pH 2

Figure 31. Comparative plot of % water content in the 40 NVP: 10 HEMA: 50 AA

hydrogel in varying pH environments at 37 oC as a function of time.

0

20

40

60

80

100

0 50 100 150 200

Time (h)

% W

ate

r C

on

ten

t

Gel H (pH 2) Gel H (pH 8) Gel H (neutral)Gel K (pH 2) Gel K (pH 8) Gel K (neutral)

Figure 32. Comparative plot of % water content in Gel H and Gel K in varying pH

environments at 37 oC as a function of time.

254



The drug release experiments on NVP-AA-HEMA hydrogels H - J were conducted at 37

oC in varied pH environments using theophylline as the model drug. The experimental

data expressed as fractional drug release at specific time intervals are presented in Tables

22, 24 and 26. The hydrogels tested were of the following compositions: (NVP : HEMA

: AA (% v/ v)); 50 : 50 : 00 (Gel H); 50 : 10 : 40 (Gel J) and 50 : 40 : 10 (Gel I)

Table 22. Drug release test on formulations of NVP, AA and HEMA in varied

ratios (% v/v) in neutral environment at 37 oC

Fractional theophylline released

values at time t

Time (h) Gel H Gel I Gel J

0.00 0.00 0.00 0.00

0.17 0.24 0.19 0.28

0.33 0.33 0.28 0.39

0.50 0.41 0.34 0.54

0.67 0.46 0.39 0.64

0.83 0.51 0.43 0.70

1.00 0.55 0.46 0.75

2.00 0.73 0.63 0.92

3.00 0.82 0.72 0.97

4.00 0.88 0.79 0.98

5.00 0.92 0.84 0.97

7.00 0.96 0.90 0.99

9.00 0.95 0.94 0.99

12.00 0.97 0.95 0.98

24.00 0.98 0.97 0.99

48.00 0.98 0.98 0.99

255



representations of the drug release behaviour in the IPN hydrogels are illustrated in

Figures 33 - 35.

0.0

0.4

0.8

1.2

0 10 20 30 40 50 6

Time (h)

Fra

ctio

na

l D

rug

Rel

ease

d

0

Gel H Gel I Gel J

Figure 33. Plot of the fractional release of theophylline from Gels H - J at 37 oC in

neutral pH environment as a function of time.

0.00

0.40

0.80

1.20

0 2 4 6

t1/2

(h1/2

)

Fra

ctio

na

l D

rug

Rel

ease

d

8

Gel H Gel I Gel J

Figure 34. Plot of the fractional release of theophylline from Gels H - J at 37 oC in


256


-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G F

DR

Gel H Gel I Gel J


time, in the initial stages of theophylline release from Gels H - J at 37 oC in neutral pH

environment.


Table 23. Characteristic exponential n values for drug release from NVP-HEMA-

AA hydrogels in neutral medium

Hydrogels n values

Gel H 0.49 + 0.01

Gel I 0.47 + 0.04

Gel J 0.58 + 0.03

257



ratios (% v/v) in acidic (pH 2) environment at 37 oC


values at time t


0.00 0.00 0.00 0.00

0.17 0.20 0.15 0.17

0.33 0.30 0.22 0.27

0.50 0.39 0.28 0.33

0.67 0.44 0.32 0.40

0.83 0.49 0.36 0.44

1.00 0.53 0.39 0.47

2.00 0.70 0.53 0.64

3.00 0.80 0.63 0.76

4.00 0.87 0.70 0.82

5.00 0.91 0.76 0.87

7.00 0.95 0.83 0.94

9.00 0.97 0.88 0.95

12.00 0.97 0.93 0.96

24.00 0.99 0.98 0.99

48.00 0.97 0.98 0.98

258


0.0

0.4

0.8

1.2

0 10 20 30 40 50 6

Time (h)

Fra

ctio

na

l D

rug

Rel

ease

d

0

Gel H Gel I Gel J

Figure 36. Plot of the fractional release of theophylline from Gels H - J at 37 oC in pH 2


0.0

0.4

0.8

1.2

0.0 2.0 4.0 6.0 8.0

t1/2

(h1/2

)

Fra

ctio

na

l D

rug

Rel

ease

d

Gel H Gel I Gel J



259


-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G F

DR

Gel H Gel I Gel J


time, in the initial stages of theophylline release from Gels H - J at 37 oC in pH 2

environment.



AA hydrogels in acidic medium

Hydrogels n values

Gel H 0.53 + 0.01

Gel I 0.52 + 0.01

Gel J 0.56 + 0.01

260



ratios (% v/v) in basic (pH 8) environment at 37 oC


values at time t


0.00 0.00 0.00 0.00

0.17 0.31 0.35 0.32

0.33 0.41 0.47 0.43

0.50 0.49 0.56 0.51

0.67 0.54 0.62 0.57

0.83 0.59 0.69 0.63

1.00 0.63 0.72 0.67

2.00 0.79 0.87 0.82

3.00 0.87 0.93 0.88

4.00 0.91 0.95 0.91

5.00 0.93 0.97 0.93

7.00 0.95 0.97 0.94

9.00 0.96 0.97 0.93

12.00 0.96 0.96 0.93

24.00 0.97 0.97 0.95

48.00 0.98 0.99 0.95

261


0.0

0.4

0.8

1.2

0 10 20 30 40 50 6

Time (h)

Fra

ctio

na

l D

rug

Rel

ease

d

0

Gel H Gel I Gel J



0.0

0.4

0.8

1.2

0 2 4 6

t1/2

(h1/2

)

Fra

ctio

na

l D

rug

Rel

ease

d

8

Gel H Gel I Gel J



262


-1.0

-0.5

0.0

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

LOG time

LO

G F

DR

Gel H Gel I Gel J


time, in the initial stages of theophylline release from Gels H - J at 37 oC in pH 8

environment.



AA hydrogels in basic medium

NVP : HEMA : AA n values

Gel H 0.40 + 0.02

Gel I 0.42 + 0.02

Gel J 0.41 + 0.03

263


6.4. Discussion


The DPC measurements on AA: NVP systems at varied mol ratios revealed that the 1:1

mol ratio of AA:NVP was the most optimum donor/acceptor pair yielding the highest

polymerisation rate of 0.53 J g-1

s-1

(Table 1). The AA: NVP formulations with either

excess of AA or excess of NVP showed lower reactivity. The formulation containing

excess NVP displayed the lowest reactivity of 0.23 J g-1

s-1

indicating its relative

inertness. The rate calculations were carried out according to Equation 1. Efficient

systems were characterized by an intense peak in the photo-exotherms (Figure 2). The

optimum ratio of 1mol:1mol of AA:NVP could be described in terms of the number of

double bonds present in the monomers, which effectively take part in the complex

formation. Scheme 1 illustrates the 1:1mol interaction between AA and NVP.

N

O

OH

O

N

O

OH

O

NVP AA

Donor Acceptor

+ hv

NVP-AA complex

Scheme 1. 1:1 mol of NVP:AA interaction

AA and NVP each have 1 pair of double bonds, which form the ring closure (cyclobutane

ring) as illustrated in Scheme 1. Thus when excess NVP or AA is present, they are not

consumed due to insufficient quantity of the other. The resultant possibly is the

homopolymerisation of the excess monomer, which is relatively less efficient than the

copolymerisation reaction. The formulation containing excess AA showed a slightly

higher rate over the formulation containing excess NVP.

Chapiro and Trung [28] studied the reactivity of AA and NVP under the influence of

gamma radiation. They observed a relatively low reactivity with increasing NVP content

264


in the polymer. Khodzhaev and Mushraipov [30] made use of NMR spectroscopy to study

the interaction between AA and NVP. The also suggest low reactivity of high NVP

content formulation. This observation could be attributed to the fact that AA being an

acrylate has a relatively higher reactivity than NVP, thus AA homopolymerises more

efficiently than NVP.

The kinetic studies using the DPC technique on HEMA-NVP system revealed that it was

not an efficient donor/acceptor system as compared to AA-NVP as it did not completely

polymerise when exposed to the UV light (Table 2). The non-existence of sharp

exotherms (Figure 3) as in the case of AA-NVP illustrates the incomplete polymerisation

of the HEMA-NVP system. This could be attributed to the fact HEMA did not efficiently

react with NVP probably due to insufficient exposure to the UV light under the DPC

measurement condition. The relative reactivity of HEMA and NVP has been discussed in

Section 3.


All the AA-NVP-HEMA formulations in varied compositions resulted in successful

hydrogel synthesis (Table 3). The formulations were cured upon an applied UV dose of 9

KJ. HEMA-NVP system despite showing low reactivity in DPC measurements resulted in

successful synthesis of a hydrogel in the absence of photoinitiator. This observation could

be related to the total dose of UV radiation applied to the monomer mixture. The radiation

time in DPC was only several minutes in comparison to curing time of approximately 25

hours for hydrogel synthesis. Thus the study suggests that HEMA and NVP could be

polymerised in the absence of a photoinitiator provided a longer radiation time is allowed.

HEMA and AA when exposed to the UV source also resulted in a successfully

polymerised hydrogel in the absence of a photoinitiator. Synthesis of anionic hydrogels

based on HEMA and AA has been reported by Khare and Peppas [12] who utilized redox

polymerisation technique in the presence of initiators to achieve copolymerisation of

these two monomers. Am Ende et al [19,24] have utilized thermal polymerisation

technique in the presence of initiators to obtain HEMA-co-AA hydrogel networks. The

successful polymerisation of AA-HEMA system as observed in the present study could be

265


attributed to the possible role of HEMA as a donor to AA. HEMA contains a slightly

electron rich methyl group adjacent to the carbon-carbon double bond on its structure.

Thus AA being a relatively strong electron acceptor could form a complex with HEMA,

which changes its role from a weak electron acceptor to an electron donor monomer as

illustrated in Scheme 2.

CH3

O

OOH

O

OH

O

OH

CH3

O

OOH

HEMA AA

Possible donor Acceptor

+hv

HEMA-AA complex

Scheme 2. Possible donor/acceptor interaction between HEMA and AA

6.4.3. Swelling and Drug Release Evaluation

The AA-NVP-HEMA hydrogels were subjected to swelling and drug release experiments

in pH 2, neutral and pH 8 environments. The phenomena of Fickian and non-Fickian

diffusion kinetics were used to characterize the swelling and drug release behaviours in

these anionic hydrogel networks. Equation 3 describes time dependent swelling action of

hydrogel networks. The parameter n in the power-law equation as described previously,

effectively indicates the type of diffusion mechanism in the hydrogel network. As

previously mentioned, Fickian diffusion is characterized by a n value of 0.5. High order

non-Fickian diffusion could be described in various degrees of non-Fickian diffusion

behaviour. A non-Fickian (anomalous) behaviour is characterized by 0.5 < n < 1. Case II

diffusion is characterized by a n value of 1 while super case II behaviour is characterized

by n > 1.

266


6.4.3.1. Swelling Behaviour of AA-NVP-HEMA Hydrogels

The hydrogels were thoroughly washed in milli-Q-water to remove any unreacted

component in order to avoid any variations in the degree of swelling. The uncrosslinked

copolymer of AA-co-NVP disintegrated during the washing process. The early

disintegration of AA-co-NVP network could be the cause of insufficient crosslinkage in

the polymer network. The hydrogels with HEMA however were reasonably resilient. The

swelling experiments revealed that the degree of swelling in the anionic AA-NVP-HEMA

hydrogels was significantly dependent on pH.

6.4.3.1.1. pH Dependent Swelling Behaviour

The anionic AA-NVP-HEMA hydrogels were found to be significantly dependent on the

environmental pH. Hydrogels swelled in varied pH environments showed an increase in

the degree of swelling with increase in the environmental pH. The extent of pH

sensitivity, however, varied with the monomeric composition.

The 50 % HEMA hydrogels with varied ratios of NVP and AA hydrogels displayed very

low degree of swelling in pH 2 buffer solution with hydrogels A-D yielding average

EWC values of 44.7 %, 45.4 %, 42.8 % and 39.7 % respectively (Table 10, Figure13).

The hydrogels remained collapsed in this acidic environment with a very gradual water

uptake adhering to Fickian diffusion kinetics (Table 11, Figures 14 and 15). The swelling

curve (Figure 13) showed a gradual reduction in the water uptake rate around 12 hours

reaching equilibrium swelling in 48 hours.

In neutral pH environment a very slight increase in the swelling behaviour was observed

with hydrogels A-D yielding EWC values of 49.1 %, 48.4 %, 46.6 % and 42.6 %

respectively (Table 4, Figure 4). Hydrogels A-D displayed a gradual increase in water

uptake in the initial stages of swelling followed by a gradual reduction in the water

absorption rate leading to equilibrium swelling around 48 hours. The swelling data of

hydrogels A-D in neutral pH environment indicate Fickian diffusion mechanism was in

operation (Figures 5 and 6, Table 5).

267


However, in pH 8 buffer solution, a significant increase in the swelling behaviour was

observed with hydrogels A-D yielding average EWC values of 89.1 %, 89.8 %, 91.6 %

and 93.0 % respectively (Table 16, Figure 22). A rapid increase in water content was

observed in the initial stages with gradual reduction in swelling efficiency around 9 hours

and equilibrium swelling was reached in 48 hours. The swelling experimental data on the

hydrogel samples suggested that non-Fickian diffusion kinetics was in operation with the

exception of hydrogel A, which adhered to Fickian diffusion kinetics yielding a n value of

0.51 (Table 17, Figures 23 and 24).

Hydrogels composed of 50 % AA with varying ratios of NVP and HEMA behaved in a

similar fashion in swelling to that of hydrogels A-D. The degree of swelling observed in

pH 2 environment was slightly lower than that in neutral and basic environments.

Hydrogels E-G displayed EWC values of 63.6 %, 50.8 % and 59.0 % respectively (Table

12, Figure 16). A gradual reduction in the swelling efficiency was observed around 7

hours with equilibrium swelling achieved in 48 hours. Hydrogels E-G adhered to Fickian

diffusion kinetics in the initial stages of swelling (Table 13, Figures17 and 18). An

increase in EWC was observed in neutral environment with hydrogels E-G yielding EWC

values of 85.6 %, 55.1 % and 63.5 % (Table 6, Figure 7). A rapid diffusion was observed

in Gel E, which adhered to anomalous diffusion kinetics (Table 7, Figures 8 and 9)

reaching equilibrium swelling in 48 hours. The diffusion process in hydrogels F and G

were indicative of Fickian diffusion kinetics characterized by gradual swelling leading to

equilibrium swelling in 48 hours.

Hydrogels E-G showed rapid water uptake behaviour in pH 8 environment yielding EWC

values of 96.0 %, 89.0 % and 94.4 % respectively (Table 18, Figure 25). Hydrogel E

showed a phenomenal increase in water uptake reaching near equilibrium swelling within

the initial 10 minutes of swelling yielding a water content value of 92.8 %. Hydrogels F

and G displayed a gradual reduction in the swelling efficiency around 7 hours reaching

equilibrium swelling in 48 hours. Hydrogels adhered to non-Fickian (anomalous)

diffusion kinetics in the initial stages of swelling experiment with the exception of

Hydrogel E, which displayed extreme non-Fickian diffusion behaviour described as super

case II diffusion kinetics with a n value of 2.13 (Table 19, Figures 26 and 27).

268


Formulations containing 50 % NVP with varied ratios of HEMA and NVP also showed

significant pH dependent swelling behaviour. In pH 2 environment hydrogels H-K

displayed a low degree of swelling yielding EWC values of 59.9 %, 52.7 %, 50.2 % and

40.5 % respectively (Table 14, Figure19). A gradual increase in water uptake was

observed in the initial stages of the swelling experiment followed by a reduction in the

water uptake rate around 9 hours, reaching equilibrium swelling in 48 hours. The

diffusion kinetics observed in the hydrogels is typical of Fickian diffusion behaviour

(Table 15, Figures 20 and 21). An increase in the EWC was observed in neutral pH

environment with hydrogels H-K yielding EWC values of 65.2 %, 62.1 %, 59.2 % and

43.2 % respectively (Table 8, Figure 10). However, a gradual water uptake rate in

hydrogels H-K was observed indicating that the Fickian diffusion mechanism was in

operation (Table 9, Figures 11 and 12). Equilibrium swelling in hydrogels H-K was

observed around 48 hours of constant swelling.

In pH 8 buffer solution, a rapid increase in the swelling efficiency was observed in

hydrogels I-K, which yielded EWC values of 92.4 %, 97.4 % and 96.5 % respectively

(Table 20, Figure 28). Hydrogel H showed a slight increase in swelling kinetics with an

EWC value of 67.1 %. Hydrogels showed increasing water uptake rate in the initial stages

of the experiment followed by a gradual decrease in the water uptake rate around 9 hours

reaching equilibrium swelling in 48 hours. Hydrogels adhered to non-Fickian diffusion

(anomalous) kinetics in the initial stages of the experiment with the exception of hydrogel

H, which displayed Fickian diffusion kinetics with a n value of 0.52 (Table 21, Figures 29

and 30).

The experimental swelling data could be explained in terms of the monomer composition

of the hydrogels and their relative responses to variations in the environmental pH. The

swelling behaviour of the hydrogels in neutral pH revealed that the formulations

containing high amounts of AA and NVP showed high water absorption ability. The

hydrogel formulations containing 50 % AA (hydrogels E-G) and 50 % NVP (hydrogels

H-K) displayed higher degree of swelling in neutral pH environments in comparison to

hydrogel formulations with 50 % HEMA (hydrogels A-D).

269


Hydrogels E-K showed a reduction in the degree of swelling with increasing HEMA

content. This effect was more significant in hydrogels E-G, which displayed the highest

equilibrium swelling in neutral environment. The high swelling behaviour in hydrogels

with high NVP and AA contents could be attributed to their higher hydrophilic nature as

compared to HEMA. Poor water uptake ability of high HEMA content hydrogels has

been discussed in Section 3. The relatively higher swelling activity of hydrogels E-G in

neutral medium in comparison to H-K suggests superior hydrophilicity of AA over NVP.

Am Ende et al [19] in their studies on the swelling activity in HEMA-co-AA hydrogels

observed reduced swelling with increasing HEMA content. They suggested that increase

in AA content in the copolymer led to a significant reduction in the molecular weight

between crosslinks, which led to high degree of swelling.

However, deviation in the environmental pH from neutral to acidic and basic conditions

resulted in variations in the degree of swelling of the AA-NVP-HEMA hydrogels. The

extent of the variations in the swelling behaviour varied with sample compositions. A

swelling and de-swelling phenomenon was observed in basic and acidic environments

respectively. Furthermore, swelling experiments in varying pH environments indicated

increasing sensitivity of hydrogels with high AA content to variations in the

environmental pH. Hydrogels displayed higher EWC values with increase in the

environmental pH with a more pronounced increase with high AA content hydrogels. .

Significant increase in EWC values with increasing in environmental pH could be

attributed to the significantly acidic nature of AA, which has a pKa value of 4.25. As the

environmental pH is increased from acidic to basic condition, the H+ ions from the

carboxyl groups in AA react with the OH- in alkali leading to an increase in the

dissociation of AA to form carboxyl anions. The degree of dissociation of AA in varying

pH environments was calculated according to Equation 4 [20,22] and the results are

summarised in Table 28.

( )

1

10 1apK pHα −= + Equation 4

270


Table 28. Degree of dissociation of AA with respect to pH variation

pH Degree of dissociation ( ) α2 0.0056

7 0.9982

8 0.9998

The enhanced swelling action in the hydrogels with increase in the environmental pH

could be described as an increase in the electrostatic repulsion within the polymer matrix,

which led to significant swelling. The anionic pendent groups became increasingly

ionized with increase in pH leading to enhanced electrostatic repulsion within the

polymer matrix. This resulted in a disruption of hydrogen bondings between the acid

groups, leading to an increase in the mesh size, thus increasing the swelling ratio. In

acidic condition (pH 2 buffer solution) the anionic hydrogels are non-ionized where they

are in a compact conformation with relatively smaller mesh size thus causing a reduction

in the swelling fraction. The high EWC values observed for the hydrogels under study

suggested high ionization activity of AA in pH 8 buffer solution. However, in milli-Q-

water (neutral pH) environment only a slight increase in swelling was observed despite a

high degree of dissociation of AA as described in Table 28.

This could be attributed to the fact that the nature of the swelling agent, which is milli-Q-

water in this instance, has an ionic strength of zero. Thus the sharp increase in swelling

efficiency in the pH 8 buffer solution in comparison to milli-Q-water (neutral pH) could

also be explained in terms of the ionic strength of the immediate environment. Khare and

Peppas [12] reported an increase in the swelling fraction of HEMA-co-AA in buffered

solutions in comparison to unbuffered milli-Q-water and they described this behaviour

according to the Donnan equilibrium. The Donnan equilibrium, previously discussed in

Section 5.4.2, suggests that an increase in the ionic strength of the swelling medium leads

to a higher equilibrium water uptake by the ionic hydrogels. Thus in milli-Q-water, due to

limited ionization of the hydrogel, the swelling fraction was lower than that in the pH 8

buffered solution where the degree of ionization was relatively higher.

271


The 50 HEMA-50 NVP hydrogel was found to be slightly sensitive to the variations in

the environmental pH. Copolymers of HEMA and NVP have always been reported as

neutral networks. In a recent study, Firreira et al [31] evaluated PHEMA hydrogels as

drug delivery devices in varying pH environments and observed a slight increase in the

degree of swelling with increase in pH. This is in agreement with Svecik et al [32] who

reported partial hydrolysis of PHEMA in basic environment to be the cause of increase in

the degree of swelling. Brannon-Peppas and Peppas [33] also observed a slight increase in

the swelling ratio of PHEMA between pH 6 and pH 8. They explained the slight pH

dependent swelling to be due to the delocalisation of the electron density on the OH

group of HEMA to the electron attracting carbonyl group in basic environment. This

phenomenon explains the slight variations observed in the swelling ratio of the 50

HEMA-50 NVP hydrogel in varying pH environments. The slight pH dependant swelling

behaviour of this network thus could be attributed to the presence of HEMA in the

network.


Hydrogels tested were 50 NVP-50 HEMA (Gel H), 50 NVP-40 AA-10 HEMA (Gel J)

and 50 NVP-10 AA-40 HEMA (Gel I). In neutral pH, hydrogels H and I displayed an

initial burst effect release followed by gradual increase in the release rate of theophylline

in the initial stages of the experiment adhering to Fickian diffusion kinetics with n values

of 0.49 and 0.47 respectively (Tables 22 and 23, Figures 33-35). The rate of release

gradually decreased around 7 hours leading to equilibrium drug release around 9 hours.

Hydrogel J displayed a burst effect release followed by a rapid release of the theophylline

in the initial stages adhering to non-Fickian diffusion (anomalous) kinetics with a n value

of 0.58. A gradual reduction in the release rate was observed in hydrogel J around 3 hours

leading to case II, time independent release around 5 hours.

In acidic environment, lower drug release rates were observed for hydrogels H-J. A

gradual increase in release rate of theophylline was observed in initial stages of the

experiments baring an initial burst followed by a gradual decrease in the release rate

around 7 hours (Tables 24 and 25, Figures 36-38). Hydrogels H and I adhered to Fickian

release kinetics with n values of 0.53 and 0.52 respectively while hydrogel J showed a

272


slight non-Fickian anomalous behaviour with a n value of 0.56. Equilibrium drug release

was observed around 12 hours, which was characterized by case II time independent

diffusion indicated by a constant release rate as function of time.

A relatively high diffusivity of theophylline from hydrogel H-J was observed in pH 8

environment with slightly higher diffusivity of theophylline observed in hydrogel I. An

initial burst effect release of theophylline was observed in the initial stages followed by a

constant linear increase leading to time independent release around 7 hours (Table 26,

Figure 39). The release kinetics in the hydrogels was characteristic of Fickian release

behaviour as illustrated by a linear increase in the release rate as a function of the square

root of time with characteristic n values of 0.40, 0.41 and 0.42 for hydrogels H, J and I

respectively (Table 27, Figures 40 and 41).

In general an increased amount of theophylline release was observed with increase in the

environmental pH. As described previously, anionic hydrogels become increasingly

ionized with increase in environmental pH thus allowing a rapid release of theophylline

from the hydrogels. In neutral environment the hydrogel J displayed the highest

diffusivity of theophylline followed by hydrogels H and I. This behaviour could be

explained in terms the relative hydrophilicity/hydrophobicity of the hydrogel network and

their degree of ionization at the particular pH. The high diffusivity of theophylline from

hydrogel J in neutral environment indicates that the network is highly hydrophilic in

nature, which, as described previously, could be attributed to high AA and NVP content.

Thus a higher swelling activity in hydrogel J led to higher theophylline diffusivity.

Hydrogels H and I on the other hand contained higher amount of HEMA in the

formulation with respect to hydrogel J. As previously described, HEMA is relatively less

hydrophilic than AA and NVP. Thus the hydrogel networks H and I were less favourable

to swelling in neutral environment, which subsequently retarded the release rate of

theophylline.

In pH 2 environment lower release rates were observed but in contrast to neutral

environment, hydrogel H displayed slightly higher release rate in comparison to

hydrogels I and J. This behaviour could be explained in terms of high pH sensitivity of

273


AA containing hydrogels J and I, which remain collapsed in acidic environment due to

nonionization of the carboxyl groups. Thus mesh size of the polymer is significantly

smaller, which restricts the diffusion of theophylline. In pH 8 environment, higher

theophylline diffusivity was observed. This could be explained in terms of increase in the

ionization of carboxyl groups, which lead to an increase in the mesh size thus allowing

high diffusivity of theophylline.

It has been well established in the literature that the diffusion of an entrapped solute from

a swollen polymer increases with the degree of swelling [13,19,27,34-36]. Bettini et al

[13] studied the release kinetics of theophylline from pH sensitive anionic HEMA-co-AA

hydrogels in isotonic environment and reported that the release rate of theophylline was

dependent on the swelling ratio of the hydrogels. Am Ende et al [19] also reported a

similar trend of theophylline release from HEMA-co-AA hydrogels. Shah et al [36]

studied theophylline release behaviour in pH sensitive HEMA-co-4-carboxy styrene

hydrogels and have reported similar observations. Theophylline is a non-ionizable drug

hence it does not interact with the anionic polymer chains. Thus as reported by other

researchers, the rate of solute release is typically dependent on the swelling ratio of the

hydrogels.

The high diffusivity of theophylline from hydrogels H-J in basic medium (Figures 37 and

38) is in agreement with other researchers. A slight increase in the diffusivity of

theophylline was observed in hydrogel I in comparison to hydrogel J despite a higher

EWC value was observed for hydrogel J in pH 8 environment. This observation could be

explained in terms of the extent of ionization achieved by the network. As previously

described, according to the Donnan equilibrium, upon full ionization of the polymer

chain, a further increase in the ionic strength would cause the anionic polymer network to

de-swell thus reducing its mesh size. Thus the slightly lower diffusivity of theophylline

from hydrogel J in comparison to hydrogel I could be attributed to the fact that hydrogel

J, which contained high amounts of AA reached maximum ionization in the presence of a

basic drug, theophylline (pKb = 13.5) within its structure in pH 8 environment. A slightly

lower diffusivity could be due to a reduction of the polymer mesh size as a result of the

shrinking of the network. However, this behaviour was not highly significant.

274


6.5. Conclusions

The DPC measurements revealed that the donor/acceptor pair of AA and NVP was

significantly more efficient than that of HEMA and NVP in CT complex formation. The

kinetics data of AA-NVP system indicate an optimum reactive ratio at 1:1 mol of AA:

NVP. HEMA and NVP on the hand were found to be an inefficient donor/acceptor pair,

which did not completely polymerise when exposed to the UV light in the differential

photocalorimeter.

Anionic hydrogels were successfully synthesized via the photoinitiator-free process.

Hydrogels were found to be reasonably resilient and competent drug delivery devices.

Hydrogels with high AA and NVP content were found to be highly water-swellable in

comparison to high HEMA content networks, which exhibited relatively low swelling

behaviour. The swelling experiments at varied pH environments revealed that the pH

sensitivity in the hydrogel networks was contributed by the presence of AA and HEMA.

However, the pH sensitivity was more pronounced in high AA containing networks.

The drug release experiments were also indicative of pH dependence of the hydrogel

networks in releasing theophylline. The release of theophylline from the anionic networks

increased with increase in the environmental pH, suggesting that the release rate was

controlled by the swelling ratio of the hydrogels. Increase in the mesh size of the anionic

hydrogels with increase in pH led to high amount of drug released. The study on these pH

sensitive photoinitiator-free anionic hydrogels indicate that these promising materials

would be ideal as drug delivery systems for pH sensitive bioapplications such as delivery

devices for the intestinal tract where varied pH environments exist.

275


6.6. References

1. Miyata, T., Uragami, T., “Polymeric Biomaterials”, Dumitriu, S, ed., 2nd

ed.,


2. Qiu, Y., Park, K., Adv. Drug Deliv. Rev., 53, 321-339, (2001).

3. Risbud, M. V, Hardikar, A. A., Bhat, S. V., Bhonde, R. R., J. Controlled Release,

68, 23-30, (2000).

4. Peppas, N. A., Curr. Opin. Colloid Interf. Sci., 2, 531-537, (1997).

5. Ostroha, J., Pong, M., Lowman, A., Dan, N., Biomaterials, 25, 4345-4353, (2004).

6. Kost, J., Langer, R., Adv. Drug. Deliv. Rev., 46, 125-148, (2001).

7. Needleman, I. G., Smales, F. C., Martin, G. P., J. Clin. Periodontol., 24, 394-400,

(1997).

8. Dong, L.-C., Hoffman, A. S., J. Controlled release, 15, 141-152, (1991.

9. Ravichandran, P., Shanta, K. L., Rao, K. P., Int. J. Pharm., 154, 89-94, (1997).

10. Park, K., Robinson, J R., Int. J. Pharm., 19, 107-127, (1984).

11. Devine, D. M., Higginbotham, C. L., Polymer, 44, 7851-7860, (2003).

12. Khare, A. R., Peppas, N. A., Biomaterials, 16, 559-567, (1995).


(1995).

14. Khare, A. R., Peppas, N. A., Massimo, G., Colombo, P., J. Controlled Release,

22, 239-244, (1992).

15. Schwarte, L. M., Peppas, N. A., Polymer Prep., 38, 596-597, (1997).

16. Inoue, T., Chen, G., Nakamae, K., Hoffman, A. S., J. Controlled Release, 49, 167-

176, (1997).

17. Şen, M., Güven, O., Radiation Phys. Chem., 55, 113-120, (1999).

18. Rosso, F., Barbarisi, A., Barbarisi, M., Petillo, O., Margarucci, S., Calarco, A.,

Peluso, G., Mater. Sci., Eng., 23, 371-376, (2003).


(1995).


669-672, (2003).

21. Alvarez-Lorenzo, C., Concheiro, A., J. Controlled Release, 80, 247-257, (2002).

276


22. Sutani, K., Kaetsu, I., Uchida, K., Matsubara, Y., Radiation Phys. Chem., 64, 331-

336, (2002).

23. Kusonwiriyawong, C., Van de Wetering, P., Hubbell, J. A., Merkle, H. P., Walter,

E., Eur. J. Pharm. Biopharm., 56, 237-246, (2003).


25. Sahoo, S. K., De, T. K., Gosh, P. K., Maitra, A., J. Colloid Interf. Sci., 206, 361-

368, (1998).

26. Kaczmarek, H., Szalla, A., Kamińska, A., Polymer, 42, 6057-6069, (2001).

27. Vyavahare, N. R., Kulkarni, M. G., Mashelkar, R. A., J. Membrane Sci., 54, 221-

228, (1990).

28. Chapiro, A., Trung, L. D., Eur. Polym. J., 10, 1103-1106, (1974).

29. Garnett, J. L., Zilic, E., Proc. RadTech Europe’01, Basel, Switzerland, pp.233-

238, (2001).

30. Khodzhaev, S. G., Mushraipov, R., Polym. Sci. U.S.S.R., 32, 1254-1263, (1990).


32. Sveick, S., Vacik, J., Chmelikova, D., Smetana, Jr, K., J. Mater. Sci. Mater. Med.,

4, 505-509, (1995).

33. Brannon-Peppas, L., Peppas, N. A., J. Controlled Release, 16, 319-330, (1991).


(1969).

35. Wood, J. M., Attwood, D., Collet, J. H., J. Pharm. Pharmacol., 34, 1-4, (1982).

36. Shah, S. S., Kulkarni, M. G., Mashelkar, R. A., J. Controlled Release, 15, 121-

132, (1991).

277




6.3. Results 223

6.3.1. DPC Measurements on AA/NVP and HEMA/NVP Systems 223

6.3.2. Photopolymerisation of Hydrogels Containing NVP, AA and HEMA 225



6.4. Discussion 264



6.4.3. Swelling and Drug Release Evaluation 266

6.4.3.1. Swelling Behaviour of AA-NVP-HEMA Hydrogels 267

6.4.3.1.1. pH Dependent Swelling Behaviour 267



6.6. References 276

278

Chapter 7: Biocompatibility of Hydrogels – In Vitro Cytotoxicity Investigations on Mammalian (HaCaT) Cells

7.1. Introduction

Polymeric hydrogels as sustained drug delivery devices have been discussed in detail in

Sections 1-6. However, hydrogels must be biocompatible in order to be considered for

biomedical applications. Biocompatibility is the ability of a material to perform with an

appropriate host response in a specific application without toxic, inflammatory,

carcinogenic and immunogenic responses [1-7]. An appropriate response of the

biomaterial for its particular application would be referred to as an inert or positive

interaction with the host [8]. The biocompatibility of a biomaterial is directly related to its

chemical and biochemical characteristics [1,6].

Cytotoxicity of a biomaterial can be evaluated in vitro by incubating the biomaterial

samples for prolonged periods in the direct presence of suitable host environmental cells

or indirectly through biomaterial leachates [6,7,9-14]. However, for eventual regulatory

approval of a biomaterial, in vitro and in vivo tests are necessary [15]. In vitro testing is

generally less costly, and a non-invasive means of primary cytotoxicity testing, preferably

used by researchers to avoid extensive testing on animals [9,15].

In vitro investigations into possible cytotoxicity of biomedical devices, their component

materials and leachates evaluate lysis, growth inhibition and other impacts on cell

viability using morphological, biochemical and metabolic criteria. [7]. Determination of

cell viability and proliferation are common assays in cytocompatibility testing of

biomaterials in vitro. The ability of the cells to survive and proliferate is used as the

measure of functional status of the cells [10,16].

A tetrazolium-based colorimetric assay is often used for quantitative measurements of

mammalian cell survival and proliferation using the tetrazolium salt, 3-[4,5-

dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) [7]. The assay developed

by Mosmann [17] is based on the reduction of a yellow tetrazolium salt, MTT, by the

mitochondrial dehydrogenase in living cells to insoluble purple formazan crystals. The

MTT assay has been widely accepted as a better alternative to the previously used

radioactive assays, which made use of hazardous radioactive isotopes [7,18-20]. The

purple formazan crystals upon dissolution in an appropriate solvent can be measured

278


spectrophotometrically [20]. The amount of the formazan generated is directly

proportional to the live cell numbers over a wide range, using a homogeneous population

[18,21].

In the present study, the hydrogel networks described in Sections 3-6 were evaluated for

their cytocompatibility in vitro using an indirect contact methodology. A complete

conversion of monomers to polymers may not be achieved in the polymerisation process,

thus there is always a certain component of unreacted monomers, which may be toxic,

still present in the polymer matrix. These monomers have the tendency to leach out of the

polymer matrices when the polymer is in contact with an aqueous environment causing

discomfort to the host environment. However, through thorough cleaning of the finished

polymer, the unreacted toxic components present in the polymer matrix could be

eliminated [7,11-13].

In the study, hydrogel leachates were introduced to human keratinocyte (HaCaT) cells

and incubated for 48 hours. HaCaT cells are epidermal cells, which closely resemble

normal keratinocytes [22]. Furthermore, HaCaT cells are readily available, highly

sensitive and easily regenerated making them an ideal candidate as the human cell model

for in vitro studies. The effect of the hydrogel leachates on the HaCaT cells after 48 hours

of incubation was evaluated using the MTT cell proliferation assay.


Sterile hydrogel pieces (see Section 2.9.2) were immersed in milli-Q-water, which served

as the sample media, at 37 oC for a period of 14 days. These sample media were subjected

to in vitro cytotoxicity experimentation on HaCaT cells using the MTT cell proliferation

assay. The monomeric components in the hydrogels were also tested individually in their

free form. N-vinyl-2-pyrrolidinone (NVP), acrylic acid (AA), 2-hydroxethyl methacrylate

(HEMA), N-hydroxymethyl maleimide (HMMI), 2-hydroxy-N-propyl maleimide

(HPrMI) and 5-hydroxy-N-pentyl maleimide (HPMI) were the monomers tested. Detailed

experimental specifications and procedure of the cytotoxicity experiments have been

described in Section 2.9. A microplate reader was used to carry out the absorbance

measurements. The MTT assay data obtained for the hydrogels were compared with the

279


monomers and also with the untreated experimental control. The MTT assay data were

expressed as means + SEM of the replicate percentage cell numbers with respect to the

experimental control. A one-way analysis of variance (ANOVA) was performed using

MINITAB 7.2 statistical software. A p value of < 0.05 was regarded as statistically

significant.

7.3. Results

7.3.1. HaCaT Cell Proliferation in the Presence of Monomers

The monomers utilized to synthesize the hydrogels were tested for their effect on HaCaT

cell growth using the MTT cell proliferation assay. The HaCaT cells were treated with the

N-hydroxyalkyl maleimides, AA, NVP or HEMA at varying concentrations for 24 and 48

hours. However, results for the 24 hr treatment did not indicate significant growth or

inhibitory activity, thus suggesting that the treatment time was not sufficient for the cells

to grow and show any inhibitory effect on growth imposed by the samples. The results

after 48 hr treatment on the other hand clearly showed the effect of the samples on HaCaT

cell growth and the treatment time was considered adequate as the untreated experimental

control showed that the cells were fully confluent.

The MTT assay data revealed that HPrMI and HPMI did not have any adverse effects on

the HaCaT cells. The assay data indicated that the cells had proliferated in the presence of

HPrMI or HPMI. HMMI on the other hand, had a negative effect on cell growth. All the

cells died when treated with HMMI at 250 ppm and 500 ppm. A very small percentage (~

15 %) of cells were viable in the presence of HMMI at 125 ppm. Microscopic

examinations on the cells cultures treated with 500 ppm of HMMI, HPrMI or HPMI

revealed that cells in HPMI and HPrMI environment had become confluent and were still

attached (Figure 2 C & D). The cells treated with HMMI for 48 hours, died and had

detached from the surface of the culture dish (Figure 2 B).

The MTT assay data on HaCaT cell growth observed in the presence of the N-

hydroxyalkyl maleimides after 48 hours of treatment is presented in Table 1. The cell

numbers observed in the treated cultures are expressed as a percentage of the cell density

280


measured in the untreated control culture. A graphical representation of the percentage

number of HaCaT cells after the treatment is illustrated in Figure 1.

Table 1. Percentage HaCaT cell numbers after treatment with the N-hydroxyalkyl

maleimides at varying concentrations for 48 hours

N-hydroxyalkyl

maleimides

Concentration

(ppm) % Live cell numbers + SEM

125 14.76 + 7.42

250 5.35 + 5.35 HMMI

500 5.05 + 5.05

125 124.47 + 7.48

250 121.54 + 6.26 HPrMI

500 116.08 + 6.32

125 106.61 + 6.77

250 114.66 + 6.84 HPMI

500 102.06 + 9.21

0

50

100

150

200

125 250 500Monomer Concentration (ppm)

% L

ive

Cel

l N

um

ber

s

HMMI HPrMI HPMI

******* ***

*** ******

****

Figure 1. Percentage number (density) of HaCaT cells after 48 hr treatment with HMMI,

HPrMI or HPMI at varying concentrations. Significant cell growth in comparison to

untreated control is represented by ∗ where p < 0.05. Highly significant growth compared

to the control is represented by ∗ ∗ ∗ where p < 0.001.

281


Untreated cells

(A)

Cells treated with HMMI (500 ppm)

(B)

Cells treated with HPrMI (500 ppm)

(C)

Cells treated with HPMI (500 ppm)

(D)

Figure 2. HaCaT cells cultured for 48 hours in the presence of HMMI (B) (500 ppm),

HPrMI (C) (500 ppm) or HPMI (D) (500 ppm). Micrograph A is a representation of the

untreated control. The cell cultures were observed at a magnification of 200 x. The

micrographs illustrate live, attached cells in the HPrMI and HPMI environments. Where

as the HMMI environment caused the cells to round up and die, and detach from the

culture dish surface.

282


The MTT assay data revealed that the monomers AA and HEMA did not have adverse

effect on the HaCaT cells at 250 ppm. However, at 500 and 1000 ppm, a slight growth

inhibitory effect imposed by the presence of HEMA and AA was observed. NVP on the

other hand did not have any adverse effect on the cell growth. The MTT assay data

indicated that cells proliferated in the presence of NVP at 250 ppm, 500 ppm and 1000

ppm. The MTT assay data on AA, HEMA and NVP are presented in Table 2 and

illustrated in Figure 3. Microscopic examinations were carried out on the cell cultures

treated with 1000 ppm of HEMA, AA or NVP for 48 hours. The cells were found to be to

be fully confluent and attached to the culture dish surface in NVP environment after 48

hours of treatment. HaCaT cell cultures in HEMA and AA showed slow cell growth. The

number of attached cells in AA and HEMA environment after 48 hours of treatment was

relatively lower in comparison to that in the NVP environment. The micrographs of the

cell cultures in AA, HEMA or NVP environment are illustrated in Figure 4 (A-D).

Table 2. Percentage HaCaT cell numbers after treatment with HEMA, NVP and AA

at varying concentrations for 48 hours

Monomers Concentration

(ppm) % Live cell numbers + SEM

250 105.00 + 7.86

500 84.07 + 6.77 HEMA

1000 64.17 + 4.41

250 98.71 + 9.13

500 101.17 + 8.52 NVP

1000 105.25 + 7.91

250 97.82 + 8.99

500 91.30 + 7.70 AA

1000 63.97 + 7.29

283


0

50

100

150

200

250 500 1000Monomer Concentration (ppm)

% L

ive

Cel

l N

um

ber

s

HEMA NVP AA

*** ***

*

Figure 3. Percentage number of HaCaT cells after 48 hr treatment with HEMA, NVP or

AA at varying concentrations. Significant cell growth in comparison to untreated control

is represented by where p < 0.05 while a highly significant growth compared to the

control is represented by ∗ ∗ ∗ where p < 0.001. Absence of ∗ indicates insignificant

cellular proliferation in comparison to the untreated control where p > 0.05.

∗

284


Untreated cells

(A)

Cells treated with AA (1000 ppm)

(B)

Cells treated with NVP (1000 ppm)

(D)

Cells treated with HEMA (1000 ppm)

(C)

Figure 4. HaCaT cells cultured for 48 hours in the presence of AA (B) (1000 ppm),

HEMA (C) (1000 ppm) or NVP (D) (1000 ppm). The untreated experimental control is

illustrated in micrograph A. The cell cultures were observed at a magnification of 200 x.

The cells were attached to the culture dish and had become confluent in the presence of

NVP environment after 48 hours of treatment. The presence of HEMA and AA seemed to

have inhibited cell growth to some extent as the HaCaT cells were not fully confluent

after 48 hours of treatment.

285


7.3.2. HaCaT Cell Proliferation in the Presence of Hydrogels

The polymeric hydrogels described in Sections 3-5 were tested for cytotoxicity on HaCaT

cells. The cells were treated with the hydrogel leachates for 48 hours at 37 oC. The effect

of the hydrogel leachates on the HaCaT cell growth was observed and compared with the

effects imposed by the monomeric components of the hydrogels on the cells. The results

of the MTT assay on the effect of hydrogel leachates samples on the cells are described in

Sections 7.3.2.1-7.3.2.4. The cell numbers observed in the treated cultures are expressed

as a percentage of the cell density measured in the untreated control cell culture.

7.3.2.1. Effect of N-Hydroxyalkyl Maleimide-NVP Hydrogels

The MTT assay data on the HaCaT cells treated with HMMI-NVP, HPrMI-NVP, HPMI-

NVP, HPMI-NVP-HEMA or HPMI-NVP-NVC (Table 3 and Figure 5) revealed that the

hydrogel leachates did not contain cytotoxins. The assay indicated that the cells had

proliferated after 48 hours of treatment. A significant (p < 0.05) increase in cell growth

was observed in the presence of the N-hydroxyalkyl maleimide-NVP hydrogel leachates.

The percentage cell numbers observed after the 48 hr treatment are tabulated in Tables 3

and illustrated graphically in Figure 5. Microscopic examinations revealed that the cells

were fully confluent and were still attached to the culture dish after treatment. A

micrograph representing the status of the HaCaT cells after 48 hours of treatment with

hydrogels based on N-hydroxyalkyl maleimides is illustrated in Figure 6.

Table 3. Percentage HaCaT cell numbers after 48 hr treatment with HMMI-NVP,

HPrMI-NVP, HPMI-NVP, HPMI-NVP-HEMA and HPMI-NVP-NVC hydrogels

Hydrogel samples % Live cell numbers + SEM

Gel A HMMI-NVP 141.59 + 13.57

Gel B HPrMI-NVP 165.02 + 16.80

Gel C HPMI-NVP 170.27 + 15.43

Gel D HPMI-NVP-HEMA 143.36 + 9.05

Gel E HPMI-NVP-NVC 151.48 + 24.44

286


0

50

100

150

200

Gel A Gel B Gel C Gel D Gel E

Hydrogel Samples

% L

ive

Cel

l N

um

ber

s

***

*** ***

******

Figure 5. Percentage number of HaCaT cells after 48 hr treatment with leachates of

HMMI-NVP (Gel A), HPrMI-NVP (Gel B), HPMI-NVP (Gel C), HPMI-NVP-HEMA

(Gel D) or HPMI-NVP-NVC (Gel E). The ∗ ∗ ∗ represent highly significant cell growth

in comparison to the untreated control where p < 0.001.

Figure 6. HaCaT cells treated with the N-hydroxyalkyl maleimides–NVP hydrogel

sample leachates after 48 hours of treatment. The cells were mostly attached to the culture

dish and were confluent. The micrograph (magnification 200 x) is a representation of the

cell cultures observed in all the hydrogel samples in this category.

7.3.2.2. Effect of IPN Hydrogels

The MTT assay data (Table 4 and Figure 7) revealed that the IPN hydrogel leachates did

not have any adverse effects on the HaCaT cell growth. The cells were observed to

proliferate after 48 hours of treatment with the IPN hydrogel leachates, thus indicating

that the leachates were free of any cytotoxins. Furthermore, the leaches appeared to

287


significantly (p < 0.05) stimulate cell growth. Microscopic examinations also indicated

that the cells were fully confluent and were still attached after 48 hours of treatment with

the leachates. A micrograph representing the HaCaT cell culture treated with the IPN

hydrogel leachate is illustrated in Figure 8.

Table 4. Percentage HaCaT cell numbers after 48 hr treatment with IPN leachates

IPN hydrogel samples % Live cell numbers + SEM

Gel A CM chitosan-NVP-HMMI 154.37 + 27.72

Gel B HEMA-NVP-chitosan 168.24 + 26.56

Gel C Chitosan-NVP-HMMI 136.52 + 27.92

0

50

100

150

200

Gel A Gel B Gel C

IPN Hydrogel Samples

% L

ive

Cel

l N

um

ber

s

******

***

Figure 7. Percentage number of HaCaT cells after 48 hr treatment with leachates of CM

chitosan-NVP (Gel A), chitosan-HEMA-NVP (Gel B) or chitosan-NVP (gel C) IPN

hydrogel. The ∗ represent highly significant cell growth in comparison to the

untreated control where p < 0.001.

∗ ∗

288


Figure 8. HaCaT cells treated with the IPN hydrogel sample leachates for 48 hours.

HaCaT cells were observed to have become confluent with the majority of the cells

attached to the culture plate. The micrograph (magnification 200 x) is a representation of

the cell cultures treated with all the hydrogel samples in this category.

7.3.2.3. Effect of HEMA-NVP-AA Anionic Hydrogels

The MTT assay data (Tables 3-5 and Figures 9-11) indicated that the HEMA-NVP-AA

ionic hydrogels did not have any significant adverse effect on the HaCaT cell viability.

However, the percentage cell numbers indicated by the assay were relatively lower than

that observed for the cells treated with N-hydroxyalkyl maleimide-NVP and IPN

hydrogel leachates. The treated cultures observed under microscope revealed that the cells

were attached to the culture dish thus indicating that the cells were alive after the 48 hr

treatment. A micrograph illustrating the status of the cell culture treated with the HEMA-

AA-NVP anionic hydrogels is illustrated in Figure12.

289


Table 5. Percentage HaCaT cell numbers after 48 hr treatment with HEMA-NVP-

AA anionic hydrogel leachates

Hydrogel samples

HEMA:NVP:AA % Live cell numbers + SEM

Gel A 50:00:50 122.70 + 13.64

Gel B 50:40:10 111.45 + 7.60

Gel C 50:25:25 124.55 + 15.45

Gel D 50:10:40 111.68 + 11.76

Gel E 10:40:50 98.06 + 9.03

Gel F 25:25:50 113.54 + 9.13

Gel G 40:10:50 98.89 + 15.43

Gel H 10:50:40 94.96 + 14.93

Gel I 25:50:25 113.93 + 11.73

Gel J 40:50:10 124.29 + 15.15

0

50

100

150

200


Hydrogel Samples

% L

ive

Cel

l N

um

ber

s

*** **** *

Figure 9. Percentage number of HaCaT cells after 48 hr treatment with leachates of 50

HEMA-50 AA (Gel A), 50 HEMA-40 NVP-10 AA (Gel B), 50 HEMA-25 NVP-25 AA

(Gel C) or 50 HEMA-10 NVP-40 AA (Gel D). Significant cell growth in comparison to

the untreated control is represented by where p < 0.05 while a highly significant growth

compared to the control is represented by where p < 0.001.

∗∗ ∗ ∗

290


0

50

100

150

200

Gel E Gel F Gel G

Hydrogel Samples

Per

cen

tag

e C

ell

Nu

mb

ers

*


AA- 40 NVP-10 HEMA (Gel E), 50 AA-25 NVP-25 HEMA (Gel F) or 50 AA-10 NVP-

40 HEMA (Gel G). The ∗ represents significant cell growth in comparison to the

untreated control where p < 0.001. The absence of ∗ indicates insignificant cell growth in

comparison to the untreated control where p > 0.05.

0

50

100

150

200

Gel H Gel I Gel J

Hydrogel Samples

Per

cen

tag

e C

ell

Nu

mb

ers

***


NVP- 40 AA-10 HEMA (Gel H), 50 NVP-25 AA-25 HEMA (Gel I) or 50 NVP-10 AA-

40 HEMA (Gel J). The ∗ ∗ ∗ represent highly significant cell growth in comparison to

the untreated control where p < 0.001. Insignificant cell growth in comparison to the

untreated control is indicated by the absence of ∗ where p > 0.05.

291


Figure 12. HaCaT cells treated with the AA-NVP-HEMA hydrogel sample leachates for

48 hours. HaCaT cells were attached to the culture dish indicating cellular viability. The

micrograph (magnification 200 x) is a representation of the cell cultures observed in all

the hydrogel samples in this category.

7.3.2.4. Effect of HEMA-NVP Hydrogels

The MTT assay data (Table 6 and Figure 13) revealed that the hydrogel leachates from

HEMA-NVP hydrogels were free of any cytotoxins. The assay data indicated that the

HaCaT cells were viable after 48 hours of treatment. Furthermore, a significant (p < 0.05)

cellular proliferation was observed. Microscopic examination revealed that the cells were

fully confluent and attached to the culture plate indicating cellular viability (Figure 14).

Table 6. Percentage HaCaT cell numbers after 48 hr treatment with HEMA-NVP

hydrogel leachates

Hydrogel samples % Live cell numbers + SEM

Gel A PHEMA 150.50 + 42.76

Gel B 80 HEMA-20 NVP 148.76 + 25.61

Gel C 50 HEMA-50 NVP 142.41 + 15.33

Gel D 20 HEMA-80 NVP 116.37 + 5.79

Gel E 75 HEMA-15NVP-10H2O 111.17 + 19.59

Gel F 15 HEMA-75NVP-10H2O 156.57 + 40.90

292


0

50

100

150

200

Gel A Gel B Gel C Gel D Gel E Gel F

Hydrogel Samples

% L

ive

Cel

l N

um

ber

s

******

******

******

Figure 13. Percentage number of HaCaT cells after 48 hr treatment with the leachates of

PHEMA (Gel A), 80 HEMA-20 NVP (Gel B), 50 HEMA-50 NVP (Gel C), 20 HEMA-80

NVP (Gel D), 75 HEMA-15NVP-10H2O (Gel E) or 15 HEMA-75 NVP-10H2O (Gel F).

The represent highly significant cell growth in comparison to the untreated control

where p < 0.001.

∗ ∗ ∗

Figure 14. HaCaT cells treated with the poly(HEMA-co-NVP) hydrogel leachates for 48

hours. The cells were attached to the culture dish indicating cellular viability. The

micrograph (magnification 200 x) is a representation of the cell cultures observed in all

the hydrogel samples in this category.

293


7.4. Discussion

7.4.1. Effect of the Monomers on HaCaT Cells

The monomers HMMI, HPrMI, HPMI, NVP, HEMA and NVP were utilized to make

hydrogels in various combinations. HEMA, NVP and AA are widely used monomers for

polymeric hydrogel synthesis. Numerous researchers have made use of HEMA and NVP

in varying compositions for specific biomedical applications [2,12,13,23-29]. AA has

been commonly used to form pH sensitive hydrogels for stimuli response drug delivery

applications [2,30-32]. The N-hydroxyalkyl maleimides, HMMI, HPrMI and HPMI have

been used as suitable acceptor monomers in charge-transfer complex polymerisation [33].

However, the use of photoinitiator-free polymers composed of N-hydroxyalkyl

maleimides as bioapplicable hydrogels has been recently reported by the author [34,35].

In vitro cytotoxicity investigations using the MTT assay revealed that these monomers

bring about a characteristic response in the HaCaT cells. The monomers were therefore

classified in accordance to the level of cytotoxicity exhibited towards the HaCaT cells.

The N-hydroxyalkyl maleimides were found to be fairly inert with the exception of

HMMI. The HaCaT cells were exposed to the maleimides at varying concentrations (125

ppm, 250 ppm and 500 ppm). The MTT assay data revealed that HPrMI and HPMI did

not have a negative impact on cellular viability and proliferation after 48 hours of

exposure at both, high and low concentrations (Table 1, Figure 1). Microscopic

examinations revealed that the cells were attached to the culture dish and the cell cultures

had become confluent indicating cellular growth (Figure 2 (C & D)).

HMMI on the other hand had adverse effects on the HaCaT cells. The cells treated with

HMMI at 125 ppm showed low survival rate (~15%) (Table 1, Figure 1). At higher

concentrations all the cells died. Microscopic observation indicated that all the cells had

detached or had died after 48 hours of exposure thus suggesting that HMMI is toxic to

HaCaT cells (Figure 2 (B)).

The relative toxicity of HMMI in comparison to HPrMI and HPMI could be attributed to

the structural features of these N-hydroxyalkyl maleimides. HMMI, owing to it low

molecular weight and enhanced solubility due to the presence of hydroxyl (-OH) group,

294


will easily diffuse across the cell membrane in comparison to HPrMI and HPMI. Cooney

et al [36] from their studies on biochemical and toxicological properties on N-substituted

maleimides suggested that the addition of alkyl substitutents diminishes the cytotoxicity

of maleimides. The cytotoxicity results of the N-hydroxyalkyl maleimides in this study

are in agreement with Cooney et al. HPMI and HPrMI being larger molecules with longer

N-alkyl substitutents did not have apparent toxic effect at a high concentration of 500

ppm.

NVP did not have an adverse effect on cell viability. The cells were viable as indicated by

the MTT assay data after 48 hours of treatment with NVP at 250 ppm, 500 ppm and 1000

ppm (Table 2, Figure 3). Microscopic examination on the HaCaT cells cultured in NVP

(1000 ppm) revealed that the cells were attached to the culture dish (Figure 4 (H)).

Furthermore, the cultures were almost confluent after 48 hours of treatment, indicating

cellular growth and the inertness of the monomer. NVP is an extremely hydrophilic

neutral monomer. The absence of charged groups on its structure makes it relatively inert.

Van de Wetering et al [37,38] observed a marked reduction in the cytotoxicity of

polymers with increasing NVP content. They described this phenomenon as a result of the

reduction in the charge density on the polymer due to increasing content of a non-charged

monomer, NVP.

HEMA and AA did not show cytotoxic effect on the cell cultures at 250 ppm after the 48

hr treatment (Table 2, Figure 3). The cells appeared viable and appeared to have

proliferated in this environment. However, when the concentration of AA and HEMA

was increased to 500 ppm and 1000 ppm, a toxic effect was observed. A slight growth

inhibitory effect was observed in the 500 ppm environment followed by a pronounced

effect in the 1000 ppm environment. Microscopic examinations revealed that the number

of viable cells had increased with low concentration of AA and HEMA. The cells

remained attached to the culture dish after 48 hours of treatment. However, at 1000 ppm,

the live cell numbers were reduced as indicated by a lower number of attached cells

(Figure 4 (B & C)).

295


The study suggests that HEMA and AA at high concentrations inhibit cell proliferation

and have a toxic effect. The relative cytotoxicity of HEMA and AA in comparison to

NVP at high concentration could be attributed to their slightly acidic nature in

comparison to NVP. HEMA and AA are both of low molecular weight. Furthermore, they

are water-soluble owing to the -OH group on their structure, which would dissociate in

the culture medium. The dissociation of the -OH group will alter the pH of the immediate

cellular environment. As suggested by the experimental data, at low concentrations of the

monomer, change in the pH of the environment was negligible thus the effect on the

cellular growth was not pronounced. However, at high concentration when the pH change

was significant, the cell growth was inhibited in the acidic environment, thus a lower

number of attached cells were observed (Figure10).

Yoshii [39] studied the cytotoxic effect of a range of acrylates and methacrylates and

found the dependence of the cytotoxicity of the monomers on their chemical structures.

Yoshii observed enhanced cytotoxicity in acrylates in comparison to methacrylates.

Furthermore, a more pronounced cytotoxicity was observed in the monomers containing

OH groups. The enhanced cytotoxicity of acrylates in comparison to the methacrylates

could be attributed to the presence of the methyl group on the methacrylates, which

stabilize the -OH bonds thus reducing the degree of dissociation. Bouillaguet et al [40,41]

studied the cytotoxic effect of HEMA, and suggested that HEMA is cytotoxic at high

concentrations.

The results obtained on the cytotoxicity of AA and HEMA in the present work are in

agreement with other researchers. However, as suggested by Yoshii, AA being an

acrylate should be more cytotoxic due to a higher dissociation. In the present study, a

marked cytotoxic effect of AA in comparison to HEMA was not observed. This could be

due to the fact the monomer concentrations used for this work were not sufficiently high

for AA to show a more pronounced effect.

296


7.4.2. Cytocompatibility of the Polymeric Hydrogels

7.4.2.1. Hydrogels Based on N-Hydroxyalkyl Maleimides and NVP

The data from the MTT assay (Table 3, Figure 5) revealed that the N-hydroxyalkyl

maleimide-NVP hydrogels synthesized via a photoinitiator-free technique were free of

any toxins. The leachates from the hydrogels had a significant positive effect on HaCaT

cell growth. Cells were observed to proliferate at a higher growth rate than the untreated

cell cultures. Microscopic observation showed the cells were still attached to the culture

dish and the cultures were almost confluent after 48 hours of exposure thus indicating that

the hydrogels did not have any adverse effect on HaCaT cell growth (Figure 6).

Despite a significant cytotoxicity effect of HMMI in its free form on HaCaT cells, the

HMMI-NVP hydrogels not only sustained the HaCaT cells but also stimulated positive

growth. The concentration of HMMI in the hydrogel was approximately 1000 ppm,

determined from the formulation described in Section 2.5.2 and the sample preparation

for cytotoxicity (Section 2.9.2). The study suggests that the traces of the unreacted

monomers that may have been present in the gel matrix were successfully washed off as

no apparent cytotoxic effect was observed. Furthermore, the inertness of the polymeric

hydrogels to the cells also suggests that the networks are very stable under the

experimental conditions similar to the physiological environment of pH 7.4 and a

temperature of 37 oC. Thus the hydrogels based on the N-hydroxyalkyl maleimides and

NVP could be successfully applied in these specified conditions without any adverse

effects. Hydrogels for sustained drug delivery applications based on N-hydroxyalkyl

maleimides and NVP have been discussed in Section 4.

7.4.2.2. IPN Hydrogels Based on Polysaccharides and NVP

The IPN hydrogels discussed in Section 5 also revealed cytocompatibility with the

HaCaT cells. The MTT assay data (Table 4, Figure 7) showed significant cell

proliferation activity (> 100 %) in the presence of the IPN hydrogel leachates. The IPNs

were synthesized via the photoinitiator-free technique using HMMI in the presence of

NVP and two polysaccharides, chitosan and CM chitosan. As observed previously in the

case of the HMMI-NVP hydrogels, despite apparent cytotoxicity of HMMI, the IPNs

sustained and significantly stimulated HaCaT cell growth.

297


Microscopic investigation revealed that the cells were still attached to the culture dish and

the cultures were almost confluent after 48 hours of exposure thus indicating that the

hydrogels did not have any adverse effect on HaCaT cell growth (Figure 8). The

enhanced cell proliferation (> 100 %) in the presence IPNs could be attributed to the

presence of the polysaccharides in the structure.

Domard and Domard [42] have described polysaccharides to be highly cytocompatible

towards a number of cell types including keratinocytes. Chitosan, although considered

non-toxic, has been suggested to be a strong elicitor of biological activity. They have

further suggested enhanced cell proliferation of keratinocytes in the presence of chitosan

and its derivatives. Risbud et al [11] from their studies on indirect in vitro cytotoxicity

evaluation of chitosan and poly(N-vinyl-2-pyrrolidinone) IPNs on epithelial cells have

reported similar results of enhanced cellular proliferation. The results on the

cytocompatibility of the IPNs based on chitosan and its water-soluble derivative are in

agreement with other researchers. Furthermore, CM chitosan has been reported as an

extremely useful derivative of chitosan owing to its solubility in water and high

biocompatibility [43,44]. Thus the enhanced growth effect by the CM-chitosan based

IPN, as that of chitosan based IPNs could be explained in terms of its biocompatibility.

7.4.2.3. Hydrogels Composed of AA, NVP and HEMA

AA-NVP-HEMA hydrogels discussed in detail in Section 6 are polyanionic in nature.

Results of the MTT assay using these gels indicated that the AA-NVP-HEMA hydrogel

leachates did not have an adverse effect on the HaCaT cells (Table 5, Figures 9-11).

However, the cell proliferation in some of these gels was considerably lower than the

previously discussed samples in Sections 7.4.2.1 and 7.4.2.2. Thus it could be suggested

that these hydrogel networks do not significantly stimulate HaCaT cell growth. Cell

numbers expressed as a percentage of the control were between ~ 90-120% for most of

these hydrogels.

Microscopic investigations revealed that the cells were attached to the culture dish after

48 hours of treatment indicating that the cells were still viable (Figure 12). The hydrogels

298


containing large amounts of AA were found to display slightly more growth inhibitory

behaviour than those systems containing lesser amounts of AA.

As previously described, AA is acidic in nature with a pKa value of 4.25, which readily

dissociates in the culture medium altering the pH of the surrounding environment. The

hydrogels were thoroughly washed prior to testing which eliminates the presence of any

unreacted monomers such as free AA. It could be suggested that the relative growth

inhibitory effect of the hydrogels with high AA content on the HaCaT cell growth is an

intrinsic property of the hydrogel and was not caused by the free monomers. Furthermore,

the data from the MTT assay and microscopic observations also revealed that the

hydrogels tested did not have an adverse effect on the cells. The cells were still alive after

48 hours of exposure to the hydrogel leachates. Therefore the hydrogels can be

considered to be cytocompatible.

Recently Foss and Peppas [45] studied cytotoxic effects of AA based copolymers

containing varying ratios of AA and observed a reduction in the cellular growth with

increase in AA content of the copolymer. They suggested that an increase in AA made the

copolymer increasingly ionizable. Thus increasing ionization of the AA units would

reduce the pH of the immediate environment making it acidic. As a result, the cellular

growth will be reduced in the acidic environment. Hydrogels responsive to pH variations

such as anionic networks based on AA are useful in stimuli responsive release

applications.

7.4.2.4. Poly(HEMA-co-NVP) Hydrogels

Investigations on the cytocompatibility of the hydrogel networks based on HEMA and

NVP revealed that the systems were cytocompatible with the HaCaT cells. Furthermore,

the hydrogels seemed to stimulate cell proliferation. Leachates from the hydrogels were

observed to significantly enhance HaCaT cell growth (Table 6, Figure 13). Cells were

observed to proliferate (>100%) after 48 hours of treatment. Microscopic observations

revealed that the cells were still attached to the culture dish and the cultures were almost

confluent after 48 hours of exposure indicating that the hydrogels did not have any

adverse effect on HaCaT cell proliferation and viability (Figure 14).

299


The biocompatibility of HEMA and NVP based hydrogels has been well established in

recent years [2,12,13,23-29,46-50]. The cytotoxicity evaluation of HEMA-NVP

hydrogels in the present study confirms the biocompatibility of these gels as reported in

the literature. HEMA despite showing some cytotoxicity in its free monomeric form at

high concentration does not show any adverse effect on the HaCaT cells, once

polymerised. Polymeric hydrogel networks based on HEMA and NVP for sustained drug

delivery applications have been discussed in detail in Section 3.

7.5. Conclusions

The monomers were found to have no effect on HaCaT cell growth with the exception of

HEMA, AA and HMMI, which inhibited cell growth at high concentrations with HMMI

being the most toxic monomer. The relative cytotoxicity of the monomers has been

suggested to be dependent on the chemical structure of the monomers. However,

structure-cytotoxicity relationships were not clearly established in this work.

HaCaT cells were observed to be viable and to proliferate in the presence of all the

hydrogel samples tested. The positive cell growth is indicative of the fact that the cells

were not affected by the presence of the hydrogel leachates suggesting that the hydrogels

were free of cytotoxins. This study provides in vitro evidence of the biocompatibility of

the polymeric hydrogels synthesized via UV radiation. These hydrogels may therefore be

suitable for biomedical applications.

300


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303




7.3. Results 280

7.3.1. HaCaT Cell Proliferation in the Presence of Monomers 280

7.3.2. HaCaT Cell Proliferation in the Presence of Hydrogels 286

7.3.2.1. Effect of N-Hydroxyalkyl Maleimide-NVP Hydrogels 286

7.3.2.2. Effect of IPN Hydrogels 287

7.3.2.3. Effect of HEMA-NVP-AA Anionic Hydrogels 289

7.3.2.4. Effect of HEMA-NVP Hydrogels 292

7.4. Discussion 294

7.4.1. Effect of the Monomers on HaCaT Cells 294

7.4.2. Cytocompatibility of the Polymeric Hydrogels 297

7.4.2.1. Hydrogels Based on N-Hydroxyalkyl Maleimides and NVP 297

7.4.2.2. IPN Hydrogels Based on Polysaccharides and NVP 297

7.4.2.3. Hydrogels Composed of AA, NVP and HEMA 298

7.4.2.4. Poly(HEMA-co-NVP) Hydrogels 299


7.6. References 301

304

Chapter 8: Research Outcomes and Future Recommendations

8.1. Research Outcomes

The research work carried out on polymeric hydrogels resulted in a number of versatile

materials with potential applications on sustained drug delivery. A range of experiments

including swelling, drug release and cytotoxicity tests were conducted on these materials

to evaluate their potentials as controlled drug delivery biomedical devices.

A novel photoinitiator-free curing method was proposed to synthesize hydrogels using

monomers, which functioned as donor/acceptor pairs. Kinetics studies were conducted on

these donor/acceptor monomers to evaluate their suitability as donor/acceptor pairs. A

range of N-hydroxyalkyl maleimides and acrylic acid were found to be competent

acceptor monomers in combination with N-vinyl-2-pyrrolidinone (NVP), which served as

an excellent donor monomer. Hydrogels based on these donor/acceptor pairs were

successfully synthesized. Interpenetrating polymer networks were also successfully

formed using chitosan and its water-soluble derivative, carboxymethyl chitosan with NVP

via the photoinitiator free method.

Swelling and drug release experiments conducted on the hydrogel systems prepared

revealed the versatility of these materials as potential drug delivery systems. The swelling

and drug release behaviour observed in the hydrogel networks was described in terms of

Fickian and non-Fickian diffusion kinetics. A wide range of swelling-drug release

kinetics was observed in the hydrogels, which varied from Fickian diffusion to non-

Fickian diffusion kinetics. Furthermore, the polyelectrolyte hydrogel networks displayed

pH dependent swelling and drug release behaviour suggesting their potential application

as stimuli response delivery systems. Finally it could be added that these hydrogel

systems studied could serve a wide range drug delivery applications ranging from slow to

fast releasing systems.

In vitro cytotoxicity experiments on human keratinocyte (HaCaT) cells revealed that

hydrogels did not have any adverse effect on the HaCaT cells suggesting that they were

satisfactorily biocompatible with the host HaCaT cells. A biomaterial must be

satisfactorily biocompatible to be considered for bioapplications. Thus the in vitro

304


biocompatibility studies further indicate the suitability of these hydrogel networks as

biomaterials.

8.2. Future Recommendations

Hydrogels as more effective, more patient friendly and more cost effective substitutes to

the conventional pills and injections for delivery of bioactive agents has been investigated

for several decades. However, the need for cheaper, more stimuli responsive and more

biocompatible substitute drug delivery systems continues. In the present work, hydrogels

were cured via a novel photoinitiator-free curing technique, which were found to be cost

effective, free of toxins and competent drug delivery systems.

Future work could involve exploration of a wider range of donor/acceptor pairs, which

would lead to more efficient polymerisable systems. Furthermore, these monomers may

possess characteristics such as ionic, hydrophilic or hydrophobic nature, which could be

effectively used to design hydrogels with desirable swelling-drug release properties for

specific applications.

As emphasized previously, the ability of the hydrogel to be biocompatible governs its

possible use in biomedical applications. Hydrogels synthesized in the present work were

tested in vitro, which provides sufficient preliminary evidence on the biocompatibility of

the hydrogels. However, in vivo tests are more important as they provide a better image of

the biocompatibility nature of a biomaterial. Thus as a suggestion for future work, in vivo

studies involving clinical trials on the hydrogels prepared in the present work would

provide a better understanding of their biocompatibility. Furthermore, in vivo studies will

confirm the findings from this work on the suitability of these materials for biomedical

use.

305

Appendix I: NMR Spectra

1H NMR spectrum of HMMI in D2O

306


13

C NMR spectrum of HMMI in D2O

307


1H NMR spectrum of HEMI in D2O

308


13

C NMR spectrum of HEMI in D2O

309


1H NMR spectrum of HPrMI in D2O

310


13

C NMR spectrum of HPrMI in D2O

311


1H NMR spectrum of HPMI in D2O

312


13

C NMR spectrum of HPMI in D2O

313


Inversion recovery pulse response of a hydrated gel showing typical T1 relaxation profile

314


CPMG pulse sequence of a hydrated gel displaying typical T2 relaxation profile

315

Appendix II: UV-visible Spectra

316

UV-vis spectrum of theophylline


317

UV-vis spectrum of thiamine hydrochloride

Appendix II: UV-visible Spectra

318

UV-vis spectrum of Mn-TPP-OH


319

UV-vis spectra of TPP-OH and Mn-TPP-OH

Appendix III: Texture Analysis

Appendix III. Texture Analysis

The swollen hydrogels were compressed for 30 seconds with increasing compression

distance (0.1 mm/s) in the first 10 seconds and maintained compression distance in the

later 20 seconds of the experiment. The load was withdrawn from the sample after 30

seconds and the sample was let to relax back to its original physical state. The

compression stress-strain graph (Figure AIII.1) bellow illustrates the behaviour of a

swollen hydrogel under load where F is the compression force measured in the linear

portion of the stress-strain curve, used to calculate the Young’s modulus with the

corresponding strain parameter. F1 is the maximum compression force measured at 10

seconds with a compression distance of 1.0 mm. F2 is the measured maximum relaxation

force exerted by the sample once the load was removed.

Figure AIII.1. A typical compression stress-strain graph of a swollen hydrogel under

load

320

Appendix IV: UV Lamp Calibration

Appendix IV. UV Lamp Calibration

The UV lamp calibration was done using a uranyl-oxalate actinometer. As described in

Section 2.4, 0.02 L of 0.02 M oxalic acid was mixed with 0.02 L of 0.02 M uranyl nitrate

solution and subjected ultra-violet radiation. The radiation dose rate calculations were

carried out according to the decomposition rate of oxalic acid at varying exposure time

intervals. The rate of decomposition of oxalic acid as a function of exposure time is

illustrated in Figure AIV.1.

0.0

1.0

2.0

3.0

4.0

5.0

0 20 40 60 80 100

Time of exposure (mins)

Oxali

c aci

d d

ecom

pose

d

(mol

x 1

0-4

)

Figure AIV.1. Plot of the amount of oxalic acid decomposed as a function of exposure

time to the UV light

A linear decomposition rate of oxalic acid was observed in first 30 minutes of UV

exposure with ~ 70 % oxalic acid decomposed followed by a gradual reduction in

decomposition rate until complete decomposition of the acid. The UV radiation dose rate

calculations as described in Section 2.4.4 were carried out for the uranyl nitrate –oxalic

acid mixture exposed to the UV lamp for 30 minutes on the basis of maximum exposure

time with a linear decomposition rate.

321

Appendix V: Organic Syntheses - Reaction Mechanisms

Appendix V. Organic Syntheses - Reaction Mechanisms

A cancer tumour-tracing agent, Mn-TPP-OH, a series of N-hydroxyalkyl maleimides and

a water-soluble derivative of chitosan were synthesized in accordance to methods

published in the literature. The detailed syntheses and characterization of the compounds

are described in Section 2.3. The reaction mechanisms of the compounds are illustrated in

Schemes 1-10.

NN

N N

R

R

MnR R

NNH

N NH

R

R R

R

OHR

Mn2+

H+

=

TPP-OH Mn-TPP-OH

-2

Scheme 1. Insertion of Mn into the TPP-OH cavity

N

O

O

OH

NH

O

O

H

H

OOH-

Maleimide HMMI

+

Formaldehyde

Scheme 2. Reaction of maleimide with formaldehyde to yield HMMI

322


O

O

O

OO

O

O

O +

Furan Maleic anhydride Furan-A

Scheme 3. Preparation of the maleic anhydride adduct of furan (Furan-A)

N

O

O

O

OH

O

O

O

O

NH2

OH

Furan-A HEMI-A

+

Ethanolamine

Scheme 4. Preparation of the HEMI adduct of furan (HEMI-A)

N

O

O

OH

N

O

O

O

OH

O

HEMI-A HEMI

+

Furan

Scheme 5. Decomposition of the HEMI adduct of furan to yield HEMI

N

O

O

O

OH

O

O

O

O

NH2

OH

Furan-A HPrMI-A3-Amino-1-propanol

+

Scheme 6. Preparation of the HPrMI adduct of furan (HPrMI-A)

323

Appendix V: Organic Syntheses - Reaction Mechanisms

N

O

O OH

N

O

O

O

OH

O

HPrMIHPrMI-A

+

Furan

Scheme 7. Decomposition of the HPrMI adduct of furan to yield HPrMI

N

O

O

O

OH

O

O

O

O

NH2

OH

Furan-A HPMI-A5-Amino-1-pentanol

+

Scheme 8. Preparation of the HPMI adduct of furan (HPMI-A)

N

O

O

OH

N

O

O

O

OH

O

HPMI-A HPMI

+

Furan

Scheme 9. Decomposition of the HPMI adduct of furan to yield HPMI

324


O

CH2OH

OH

O

NH2

O

CH2OH

OH

O

NHAc

O

CH2OH

OH

O

NHR

O

CH2OH

OH

O

NH2

O

CH2OH

OH

O

NR2

O

CH2OH

OH

O

NHAc

ClOH

O

NaOH

O

CH2OH

OH

O

NHR

O

CH2OH

OH

O

NH2

O

CH2OH

OH

O

NR2

O

CH2OH

OH

O

NHAc

H+

O

O

Na+

OH

O

Chitosan

Na Salt CM Chitosan

R =

CM Chitosan

R =

Scheme 10. Partial deacetylation of chitosan to yield carboxymethyl chitosan

325

STATEMENT OF AUTHENTICATION

I, Salesh Narayan Swami, hereby declare that the content of this thesis submitted under

the Doctor of Philosophy program is solely my own work carried out at the School of

Science, Food and Horticulture research laboratory at the University of Western Sydney.

No part of this work has been previously submitted to an educational institution for an

award or postgraduate degree. Information obtained from published or unpublished work

of other researchers has been appropriately acknowledged.

Salesh N. Swami

Doctor of Philosophy Candidate

25 / 07 / 2004

ACKNOWLEDGEMENTS

The author wishes to express his gratitude to several people whose help and suggestions

have been so valuable towards the completion of his doctoral research. Foremost is his

principal supervisor, Dr. Loo-Teck Ng, who has tirelessly supported and guided him

throughout the course of this work with great patience. This work would not have

completed without her invaluable assistance. He wishes to acknowledge with many

thanks, Dr. Clare-Gordon Thompson and Prof. Phil Moore for their help and guidance in

the cytotoxicity studies, Dr. Narshima Reddy for being so helpful in processing the NMR

data, Dr. Michael G. Stevens for his helpful suggestions, advice and support and Dr. Janet

Patterson for her assistance and advice on the texture analysis. The author is grateful to

the technical staff, Mr. Paul Roddy, Mr. Bert Aarts and Ms. Sylvia DeNetto, who have

been very helpful in taking care of the technical needs of the project. He also thanks his

fellow students for their helpful suggestions and encouragement. The author would like to

extend his appreciation to the APA scholarship for the financial support and also the

scholarships selection committee at UWS for such an opportunity. Above all, the author

is very much indebted to his beloved mum and uncle, Mr. Laurie Reilly for their

unconditional love, support and guidance at every step of the way.

i

AWARD

Recipient of the Australian Postgraduate Award (APA) 2001

PUBLICATIONS

Loo-Teck Ng, Salesh Swami and Sonny Jönsson, 2004. “Kinetics study of the

photopolymerisation of donor/acceptor pairs using the differential photocalorimetric

technique and the relation of the kinetics data to hydrogels formation,” Radiation Physics

and Chemistry, 69: pp. 321-328.

Salesh Swami, Loo-Teck Ng and Sonny Jönsson, 2003. “Kinetics studies of

photopolymerisation initiated by donor/acceptor pair systems based on NVP with a series

of N-hydroxyalkyl maleimides, and hydrogel formations via these systems,” Conference

Proceedings of RadTech Asia’03, Yokohama, Japan, pp. 677-680.

Sonny Jönsson, Viswanathan Kalyanaraman, Karin Lindgren, Salesh Swami and Loo-

Teck Ng, 2003. Photopolymerisation of maleimide/N-vinylpyrrolidinone hydrogels,”

Polymer Preprints, 44 (1): pp. 7-8.

Loo-Teck Ng, Sonny Jönsson, Salesh Swami and Karin Lindgren, 2002. “Synthesis of a

hydrogel for drug delivery studies utilising photoinitiator-free photopolymerisation

process based on donor/acceptor pair, N-vinylpyrrolidinone and hydroxypentyl

maleimide,” Polymer International, 51: pp. 1398-1403.

John L. Garnett, Loo-Teck Ng, Duc Nguyen, Salesh Swami and Elvis Zilic, 2002. “CT

complexes in radiation polymerisation processes including grafting, curing and hydrogel

formation,’’ Radiation Physics and Chemistry, 63: pp. 459-463.

Loo-Teck Ng, Sonny Jönsson, Karin Lindgren, Salesh Swami, Charles Hoyle and Shan

Clark, 2001. “Efficiency of hydrogen donors in photo-induced copolymerisation of NVP

and water soluble N-alkyl maleimide,” Conference Proceedings of RadTech Europe’01,

Basel, Switzerland, pp. 609-613.

ii

CONFERENCE PRESENTATIONS

Salesh Swami, Loo-Teck Ng and Clare-Gordon Thompson (poster presentation),

“Hydrogels prepared by photoinitiator-free UV curing technique and their effect on

human keratinocyte (HaCat) cell viability,” 7th

World Biomaterials Congress, 17th

-21st

May 2004, Sydney, Australia.

Loo-Teck Ng and Salesh Swami (poster presentation), “Chitosan-NVP IPN hydrogels

synthesized through photoinitiator-free photopolymerisation technique for drug delivery,”

31st Annual Meeting & Exposition of the Controlled Release Society, 12

th-16

th June 2004,

Hawaii, USA.

Salesh Swami, Loo-Teck Ng and Michael G. Stevens (oral presentation), “Radiation

synthesis of polymeric hydrogels for swelling-controlled drug release studies,” 1st Annual

CSTE Innovation Conference, 8th

-10th

June 2004, UWS, Sydney, Australia.

Salesh Swami, Loo-Teck Ng and Sonny Jönsson (oral presentation), “Kinetics studies of

photopolymerisation initiated by donor/acceptor pair systems based on NVP with a series

of N-hydroxyalkyl maleimides, and hydrogel formations via these systems,” RadTech

Asia’03 Conference, 9th

-12th

December 2003, Yokohama, Japan.

Salesh Swami, Andjelka Arsenin, Vera El-Khoury, Loo-Teck Ng and Sonny Jönsson

(poster presentation), “Hydrogels for controlled-release of drugs prepared through

photoinitiator-free photopolymerisation,” RACI QLD Polymer Group Symposium-

Polymers in Dentistry, Medicine and Surgery, 6th

-8th

February 2002, Brisbane, Australia.

Loo-Teck Ng, Sonny Jönsson, Karin Lindgren, Salesh Swami and Charles Hoyle (oral

presentation), “ Efficiency of hydrogen donors in photo-induced copolymerisation of

NVP and water soluble N-alkyl maleimide,” RadTech Europe’01 Conference, 8th

-10th

October 2001, Basel, Switzerland.

iii

ABSTRACT

Hydrogels are three-dimensional networks of hydrophilic homopolymers or copolymers

generally covalently or ionically crosslinked. They interact with aqueous media by

swelling to some equilibrium value by retaining the aqueous media in their structures.

This study concerns the investigation of the swelling and the controlled drug release

behaviour of hydrogels synthesized via the photopolymerisation process.

Copolymers of varying compositions of 2-hydroxyethyl methacrylate (HEMA) and N-

vinyl-2-pyrrolidinone (NVP) were prepared by allowing the monomers to be exposed to

ultra violet (UV) radiation in the presence of a photoinitiator (PI), Irgacure 819.

However, the use of photoinitiators is a growing concern with regards to their incomplete

usage in the polymerisation process leading to undesirable residual toxic impurities in the

polymer matrix. Hence NVP and HEMA based polymers were synthesized via the

“photoinitiator-free” (PI-free) photopolymerisation process, which was initiated by

charge transfer (CT) complexes formed when electron donor/acceptor pairs were under

the influence of the UV source.

A series of N-hydroxyalkyl maleimides, namely N-hydroxymethyl maleimide (HMMI),

2-hydroxy-N-ethyl maleimide (HEMI), 3-hydroxy-N-propyl maleimide (HPrMI) and 5-

hydroxy-N-pentyl maleimide (HPMI) were synthesized and used as acceptor monomers.

The acceptor monomers were combined with NVP, which was the electron donor

monomer to form the donor/acceptor pairs. Kinetic studies were conducted on these

donor/acceptor pairs in the CT complex formation by using the Differential

Photocalorimeter (DPC) technique. This technique evaluated their efficiency in

polymerisation with respect to the heat released. The kinetics data revealed HPMI-NVP

system as the most efficient followed by HMMI-NVP system, then HPrMI-NVP and

HEMI-NVP being the least efficient. Glucosamine hydrochloride (HCl) and glucose were

use as hydrogen donors. Glucosamine HCl was found to be a more superior hydrogen

donor than glucose with its relative efficiency in enhancing the polymerisation rate.

iv

Neutral hydrogels were prepared based on these N-hydroxyalkyl maleimides as the

acceptors and NVP as the donor.

Prior to the synthesis of anionic hydrogels using the PI-free polymerisation technique

using acrylic acid (AA) as the acceptor and NVP as the donor, kinetics studies using

various mole ratios of these monomers were performed using the DPC technique. The

kinetics data reflected that the 1:1 mole ratio of AA:NVP was the most efficient system

with the highest exotherm. Hydrogels based on AA and NVP were subsequently

synthesized.

Interpenetrating polymer networks (IPNs) involving chitosan and its derivative

carboxymethyl chitosan were synthesized by allowing NVP and NVP/HEMA to

polymerise within the matrices of these polysaccharides via the PI-free technique. The

IPNs were characterized using the Fourier Transform Infrared (FT-IR) spectroscopic

technique.

The polymeric hydrogels synthesized were evaluated for their potential applications as

hydrogels through swelling and drug release experiments conducted at 37 oC in aqueous

media. Polyelectrolyte hydrogels were evaluated for their swelling and drug release

behaviour in varying pH environments. The effect of polymer composition on the drug

release and swelling behaviour of the hydrogels were evaluated.

The drug release studies were conducted using Mn-tetrahydroxyphenyl porphyrin (Mn-

TPP-OH), thiamine hydrochloride and theophylline as the model drugs. The effect of

varying molecular weights of the model drugs on the equilibrium drug release was also

evaluated. The equilibrium water content (EWC) and the equilibrium drug release (EDR)

values from swelling and drug release experiments respectively were reflective of varying

polymer compositions. The differences in these properties of the hydrogels were

explained in terms of heterogeneous crosslinkage and the hydrophilicity/hydrophobicity

of the components. Hydrogels showed a decrease drug release rate with increase in the

molecular weight of the incorporated drug.

v

The study of hydrogels in this work was oriented towards their biomedical applications

as controlled drug delivery devices. It is a known fact that the complete conversion of

monomers to polymers may not be achieved in the polymerisation process thus there is

always a certain component of unreacted toxic monomers still remained in the polymer

matrix. These monomers have the tendency to leach out of the polymer matrices when the

polymers are in contact with an aqueous medium thus rendering the hydrogel to be non-

biocompatible. The polymers synthesized in this work were washed thoroughly in milli-

Q-water and then evaluated in vitro for any possible toxic effect on human keratinocyte

(HaCaT) cells using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide

(MTT) cell proliferation assay. The cytotoxicity results indicated that the hydrogels

understudy sustained and allowed a positive growth of the HaCaT cells in the duration of

the cytotoxicity experiment, thus proving to be satisfactorily biocompatible.

vi

LIST OF ABBREVIATIONS

AA Acrylic acid

AAS Atomic Absorption Spectrometer

ANOVA Analysis of variance

CM Carboxymethyl

CPMG Carr-Purcell-Meiboom-Gill

CT Charge transfer

DMEM Dulbecco's modified Eagle's medium

DMSO Dimethyl sulfoxide

DNP Did not polymerise

DPBS Dulbecco's phosphate buffered saline

DPC Differential Photocalorimeter

DSC Differential Scanning Calorimeter

EDMA Ethylene dimethacrylate

EDR Equilibrium drug release

EDTA Ethylenediamine tetraacetic acid

ER Electron transfer

EWC Equilibrium water content

FCS Fetal calf (bovine) serum

FDR Fractional drug released

FT-IR Fourier transform infrared

Furan-A 3,6-Endoxo-1, 2,3,6-tetrahydrophthalic anhydride

GC-MS Gas Chromatograph Mass Spectrometer

HaCaT Human keratinocyte

HEMA 2-Hydroxyethyl methacrylate

HEMI 2-Hydroxy-N-ethyl maleimide

HEMI-A 2-Hydroxy-N-ethyl-3, 6-endoxo-1, 2, 3, 6-tetrahydrophthalimide

HEPES N-2-Hydroxyethyl piperazine-N-2-ethane sulfonic acid

HMMI N-Hydroxypropyl maleimide

vii

HOMO Highest occupied molecular orbital

HPMI 5-Hydroxy-N-pentyl maleimide

HPMI-A 5-Hydroxy-N-ethyl-3, 6-endoxo-1, 2, 3, 6-tetrahydrophthalimide

HPrMI 3-Hydroxy-N-propyl maleimide

HPrMI-A 3-Hydroxy-N-ethyl-3, 6-endoxo-1, 2, 3, 6-tetrahydrophthalimide

IPNs Interpenetrating polymer networks

LCST Low critical solution temperature

LUMO Lowest unoccupied molecular orbital

MA Maleic anhydride

Mn-TPP-OH Manganese-5, 10, 15, 20-tetrakis(4-hydroxyphenyl) porphyrin

MRI Magnetic Resonance Imaging

MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

MW Molecular weight

NMR Nuclear Magnetic Resonance

NVC N-Vinyl caprolactam

NVP N-Vinyl-2-pyrrolidinone

PI Photoinitiator

PAM Poly(acrylamide)

PEG Poly(ethylene glycol)

PEO Poly(ethylene oxide)

PGMA Poly(glyceryl methacrylate)

PHEMA Poly(2-hydroxyethyl methacrylate)

PHPMA Poly(3-hydroxypropyl methacrylate)

PMMA Poly(methacrylic acid)

PNIPAM Poly(N-isopropyl acrylamide)

PNVP Poly(N-vinyl-2-pyrrolidinone)

PVA Poly(vinyl alcohol)

SAXS Small angle X-ray scattering

SEM Standard error mean

SR Stress relaxation

viii

TA Texture analysis

TPGDA Tripropylene glycol diacrylate

TPP-OH 5, 10, 15, 20-Tetrakis(4-hydroxyphenyl)-21H, 23H-porphine

TSP 2, 2, 3, 3-d(4)-3-(Trimethylsilyl)propionic acid sodium salt

UV Ultra violet

VCZ N-Vinylcarbazole

VE Vinyl ether

WAXS Wide angle X-ray scattering

ix

1.1. Synthetic Hydrogels

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