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Chapter 4
Chitosan-g-poly(2-Hydroxyethyl methacrylate)
Scope of the Chapter
This chapter concerns the synthesis of chitosan-g-Poly(HEMA) and their
characterization. The preliminary biocompatibility evaluation has been performed. The
chitosan-g-poly(HEMA) films are explored for their suitability as haemodialysis
membranes. Evaluation of low-molecular weight solute (creatinine, urea and glucose)
permeability of these films is covered in this chapter. The ability of the films to isolate
albumin- like permeants are also explored.
The results of the first section of this chapter have been published in Journal of Macromolecular Science Part A- Pure and Applied chemistry, Vol A40, No7, 715-730, (2003), ‘Graft copolymerisation of 2-Hydroxy ethyl methacrylate onto chitosan with cerium (IV) Ion.1.Synthesis and characterization.’ The result of the second section is published in Journal of Applied Polymer Science, Journal of Applied Polymer Science, Vol. 101, 2960–2966, (2006), ‘HEMA grafted chitosan for dialysis membrane applications’.
Chapter Chapter Chapter Chapter 4444
150
4.1. Synthesis and Characterisation
4.1.1. Background
As evident from chapter 3, chitosan-g-PMMA could be synthesized using the
redox initiator ceric ammonium nitrate. The copolymers showed tunable
hydrophilic/hydrophobic properties, pH responsive swellability, blood compatibility,
cytocompatibility and biodegradability. The mechanical properties of the chitosan-g-
PMMA are very poor compared to virgin chitosan films and hence their membrane
applicability could not be explored. However, it is proved that chitosan-g-PMMA could
be used for the synthesis of porous microspheres which could be used for sustained
release of pharmaceuticals. A more hydrophilic synthetic monomer 2-hydroxyethyl
methacrylate (HEMA) was chosen for the modification of chitosan and it is of interest to
examine the physico-chemical properties and biomedical applications of the chitosan–g
poly(HEMA).
Poly(2-Hydroxyethyl methacrylate) or poly(HEMA), is one of the most important
hydrogels in the biomaterials world since it has many advantages over other hydrogels. These
include a water-content similar to living tissue, inertness to biological processes, resistance to
degradation, permeability to metabolites, resistance to absorption by the body. Hydrogels are
routinely used for biomedical and pharmaceutical applications, such as drug release, artificial
tendons, wound-healing bioadhesives, artificial kidney membranes, artificial skin and contact
lenses. The most common example of poly(HEMA) is its use as contact lenses [1].
4.1.2. Synthesis of Chitosan-g-poly(2-Hydroxyethyl methacrylate)
HEMA was grafted onto chitosan following the same method used for MMA as
explained in chapter 3. Gurruchaga et al used a similar method to prepare HEMA-
g-starch [2]. Reports on grafting HEMA onto chitosan are rare. Singh and Ray grafted
HEMA onto chitosan using 60Co gamma radiation and studied the blood compatibility of
the products [3]. This method could lead to uncontrolled grafting and possible
crosslinking.
Chapter Chapter Chapter Chapter 4444
151
0 100 200 300 400 500 600 7000
100
200
300
400
500
Gra
ft p
erce
nta
ge
Concentration of HEMA (wt% of chitosan)
Fig 4 .1. Dependency of grafting efficiency on monomer concentration
Table 4.1 Graft copolymerisation of HEMA onto chitosan \
No Polymer reference
Wt of chitosan (g)
Wt of HEMA (g)
Monomer conversion %
Yield %
Graft-ing %
1 CH-HE5 2 5 48 63.3 121
2 CH-HE7.5 2 7.5 60 68.7 225
3 CH-HE10 2 10 77 81.0 385
4 CH-HE12.5 2 12.5 68 72.4 425
5 CH-HE15 2 15 91 92.4 685
6 CH-HE 25 2 25 86 87 1080
The preparation of chitosan-g-poly(HEMA) was done according to the procedure
detailed in section 2.3 of chapter 2. In all the experiments, 2 g chitosan dissolved in 100
ml of 2% aqueous acetic acid and 0.1M ceric ammonium nitrate in 10 ml of 1 N HNO3
were added. The reaction time was varied. Maximum yield (92.4%) was obtained at 5 h
with 15 g of HEMA. The extent of grafting was calculated after the removal of the
homopolymer by soxhlet extraction with methanol. The complete removal of the
homopolymer was confirmed by FTIR analysis of the extract as explained in the
experimental part. Enhancing the weight of HEMA upto 15g led to enhanced yield (Table 4.1).
Chapter Chapter Chapter Chapter 4444
152
But when the weight of HEMA was increased further, say to 25g, the % yield decreased
to 87%. Grafting increased with reaction time. Under the conditions employed here, the
yield did not increase significantly beyond 5 h of reaction. The conversion was in the
range of 48 to 91% for the different compositions. But the percentage conversion
decreased as the monomer weight increased further upto 25g. The graft polymers were
obtained as off-white powdery materials. The different compositions were coded as CH-
HE5, CH-HE7.5, CH-HE10 & CH-HE12.5 respectively. A plot of apparent graft ratio as
a function of the relative concentration of HEMA in the medium is shown in Fig 4.1.
4.1.3. FTIR Spectral Analysis
The FTIR study was conducted to investigate the graft copolymerisation between
chitosan and HEMA. The grafting of HEMA onto chitosan was ascertained by the presence of
the characteristic absorption peak of the carbonyl group of poly(HEMA) segment in the
copolymer. Typical spectra are given in Fig 4.2. Amide I and Amide II bands of pure chitosan are
located at 1649 cm-1 and 1593 cm-1 respectively. The carbonyl absorption band appeared at 1718 cm-1 for
the homopolymer, poly(HEMA). On the other hand, the carbonyl absorption band of the
chitosan-g-poly(HEMA) shifted to lower frequency. Ahn etal noted a similar shifting of the
carbonyl peak to lower frequency for poly acrylic acid-chitosan complex [4]. This is ascribed to
the hydrogen bonding of the carbonyl with the amino and hydroxyl groups of chitosan.
Fig 4.2. FTIR Spectra of poly(HEMA), chitosan and chitosan-g-poly(HEMA)
Chapter Chapter Chapter Chapter 4444
153
4.1.4. Thermal Studies
The glass transition temperature (Tg) of the graft copolymer was measured by
differential scanning calorimetry as explained in the previous chapter. The values were
taken from the second run after heating and cooling the samples at a rate of 10ºCmin-1 in
nitrogen atmosphere. The copolymer Tg is invariant with the extent of grafting beyond a
grafting of 225%. This is shown in Fig 4.3. Grafting led to enhanced Tg (108-112ºC).
This implies a stronger intermolecular interaction in the copolymer between the graft and
the core by way possibly of H-bonding. Zhang et al studied the variation of glass
transition temperature of chitosan blended with PEG [5] and observed little difference
between chitosan and its blend. Don etal could not observe any Tg in the DSC
thermogram of chitosan because of its highly rigid chains [6]. In the present study we got
a very distinct Tg for chitosan in the second heat cycle (at a rate of 10ºCmin-1) at 82.5ºC
and this value increased with extent of grafting (Fig 4.4). The graft copolymers showed a
single Tg indicating the formation of a homogeneous matrix containing the chitosan core
and poly(HEMA) grafts. The Tg values, in between those of chitosan and poly(HEMA)
confirmed the miscibility of the segments.
Fig4.3. Effect of grafting on glass transition temperature
0 100 200 300 400 50080
85
90
95
100
105
110
115
Tg
(°C
)
Graft(%)
Chapter Chapter Chapter Chapter 4444
154
Fig 4.4. DSC scan of A) chitosan, B)CH-HE5, C)CH-HE7.5, D)CH-HE10, E) CH-HE 12.5 F) poly(HEMA) (at 10ºCmin-1, in nitrogen atmosphere)
The grafting with HEMA enhanced the thermal stability of chitosan as seen from
the TGA of the polymers performed at 20ºCmin-1 in nitrogen atmosphere. In pure
chitosan, the initial decomposition temperature was observed at 248ºC with 83% weight
remaining at this stage. At 582ºC, 36% of the chitosan residue remained. The TGA of
chitosan, poly(HEMA) and the copolymers of varying compositions are shown in Fig 4.5.
It is seen that the copolymer undergoes a two-stage decomposition corresponding to the
core and the graft. However, the overall decomposition temperature of the graft
copolymers are elevated vis-à-vis pure chitosan. This is attributed to the thermal
stabilization of the core-ring by way of H-bonding with the pendant grafts.
Chapter Chapter Chapter Chapter 4444
155
Fig 4.5. TGA of chitosan, chitosan-g-poly(HEMA) and Poly(HEMA) (at 20ºCmin-1, in nitrogen atmosphere)
The second stage of decomposition occurring at~420ºC is attributed to the HEMA
segments as this is comparable to the thermogram of pure poly(HEMA). The thermal
decomposition data are compiled in Table 4.2. Grafting resulted in an increase in the initial
decomposition temperature (Ti) and the 50% decomposition temperature (T50). As the HEMA
content increases, the char residue of the graft copolymer decreases at the end of decomposition
(Tend). This implied a decreased thermal stability of the graft copolymer at elevated temperature.
The corresponding DTG of all the grafted films confirmed the two-stage decompositions. The
peak at around 415ºC is due to the decomposition of poly(HEMA) segment which is proved from
the DTG of pure poly (HEMA) shown in Fig 4.6.
Fig 4.6. DTG of chitosan, chitosan-g-poly(HEMA) and poly(HEMA) (at 20ºCmin -1, in nitrogen atmosphere)
Chapter Chapter Chapter Chapter 4444
156
Table 4.2. The effect of grafting on the decomposition temperature of chitosan
Films Ti (°C) T50 (°C) Tend (°C) Residue %
CH-0 248 335 582 36
CH-HE 5 261 413 584 25
CH-HE 7.5 268 408 583 26
CH-HE 10 292 422 583 15
CH-HE 12.5 268 420 582 9.8
4.1.5. Mechanical Properties
It is seen from Table 4.3 that the grafting results in a decrease in the tensile
strength of chitosan, particularly beyond about 425% grafting. The elongation also
decreases. HEMA leads to rigidification of the graft copolymer by way of strong
intermolecular H-bonding. Under wet conditions, graft copolymers showed a further
decrease in property. Hydrogen bonding with water breaks down intermolecular
interaction through hydrogen bonding among the polymer molecules. This plasticization
effect is reflected in a higher elongation in the wet condition for the graft copolymer. In
the case of virgin chitosan the plasticization effect of water increases the tensile strength
of the films. The % elongation also increases proportionally.
Table 4.3. Mechanical Properties of the Grafted Films
Dry Wet
Sample Code
% Grafting
(%) Tensile Strength (MPa)
%
Elongation
Tensile Strength (MPa)
%
Elongation
CH-0 0 34.5 ± 11.2 14.9 ± 7 51.1 ± 6.8 92.3 ± 6.8
CH-HE5 121 33.1 ± 2.7 2.9 ± 0.4 7.9 ± 3.3 89.7 ± 25.6
CH-HE7.5 225 29.0 ± 6.9 6.4 ± 3.4 12.3 ± 1.9 55.1 ± 4.5
CH-HE10 385 33.6 ± 8.3 2.0 ± 0.2 17.6 ± 2.8 89.9 ± 12.6
CH-HE12.5 425 22.1 ± 0.2 3.2 ± 1 12.8 ± 0.6 65.0 ± 2.7
Chapter Chapter Chapter Chapter 4444
157
Some studies report that blending or grafting of chitosan with hydrophilic
monomer decrease the mechanical property of the chitosan film. Zhang et al found that
when the PEG concentration is increased, the mechanical properties of the chitosan-PEG
blends deteriorated [5]. On the other hand Chandy etal reported improved mechanical
properties for chitosan-PVA blends under wet conditions [7]. Blair etal reported that
when chitosan is blended with PVA, none of the blend membranes was as strong as the
chitosan or the poly(vinyl alcohol) membranes [8]. A small amount of PVA in the blend
produced a large reduction in strength and elasticity of chitosan. Our observation is
similar to that of Blair et al who also used a very hydrophilic monomer (vinyl alcohol)
like HEMA in the present study. It may be concluded that grafts of hydrophilic polymers
leads to deterioration of mechanical strength particularly in wet conditions.
4.1.6. Hydrophilicity of Graft Copolymers
The swelling characteristics of the graft copolymers were studied at pH 7.4 and pH
1.98. At pH 7.4, the swelling increased to a maximum of 93% (with respect to chitosan)
0 10 20 30 40 500
20
40
60
80
100
120
140
160
180
200
Sw
ellin
g (
%)
Time (min)
CH-0 CH-HE5 CH-HE7.5 CH-HE10 CH-HE12.5
Fig 4.7a. Swelling properties of the chitosan-g-poly(HEMA) films at pH 7.4
Chapter Chapter Chapter Chapter 4444
158
0 2 4 6 8 100
400
800
1200
1600
2000
2400
2800
Sw
ellin
g (%
)
Time (min)
CH-0 CH-HE5 CH-HE7.5 CH-HE10 CH-HE12.5
Fig 4.7b. Swelling properties of the chitosan-g-poly (HEMA) films at pH 1.98
during 1h in the case of CH-12.5. At pH 1.98, it increased considerably with respect to
chitosan for the same composition when studied for 10 minutes. The swelling response of
chitosan and the copolymer films at pH 7.4 and 1.98 are shown in Fig 4.7a and Fig 4.7b
respectively. The swelling reaches a stable equilibrium more rapidly at pH 7.4 than at pH
1.98. The swelling % of the films at pH 1.98 could not be determined accurately beyond
10 minutes, as the films started degrading at this stage. This indicated that the dissolution
tendency of the films exceeded the swelling. A similar behaviour was observed by Gupta
et al while studying the pH dependency of the hydrolysis of the chitosan/PEG polymer
network microspheres [9]. The observed swelling of the copolymer films increases with
the extent of grafting. The increase in swelling could be due to the high diffusivity of
water into poly(HEMA) matrix. The % swelling of the pure chitosan film is significantly
less at pH 7.4, which shows its inherent hydrophobicity at high pH value. Swelling
studies imply that the hydrophilicity is significantly improved by graft co-polymerisation
of HEMA onto chitosan.
Chapter Chapter Chapter Chapter 4444
159
Fig 4.8. Octane contact angle variation of the films with % grafting
This is further confirmed from the octane contact angle studies [10]. As the
percentage grafting increases, the contact angle increases implying a proportionally
improved hydrophilicity Fig (4.8).
4.1.7. X-ray Diffraction Patterns
Wide angle X-ray diffraction patterns of powdered poly-HEMA, chitosan and the
graft co-polymers are shown in Fig 4.9. Chitosan exhibited a typical peak that appeared at
2θ =20º. These peaks were assigned to a mixture of the planes (0 0 1) and (1 0 0) of (1 0 1)
and (0 0 2) respectively [11]. The intensity of this peak decreases with the increase in
concentration of HEMA in the medium. When chitosan-g-poly(HEMA) polymers are
formed in presence of Ce (IV), the amino and hydroxyl groups form a complex to initiate
polymerization (ref scheme 2.1 in chapter2). This may break the hydrogen bonding
between amino and hydroxyl groups in chitosan and this results in the amorphous
structure of the graft copolymer. The decrease in the intensity of the peaks indirectly
implied grafting of HEMA on to chitosan.
Chapter Chapter Chapter Chapter 4444
160
Fig 4.9. XRD Pattern of a) poly (HEMA) b) chitosan c) CH-HE 7.5 and d) CH-HE 12.5
4.1.8. In Vitro Screening of Materials
The RBC (red blood cell) and WBC (white blood cell) counts (initial and after 30
min of contact of the materials with the blood) are given in the Tables 4.4 and 4.5. From
the data, it is clear that none of the materials caused a reduction in the cell counts as an
effect of contact with the blood. Total number of platelets in the exposed blood is given
in Table 4.6. When compared to the reference material, the test samples do not cause a
significant change in the platelet counts of the blood samples to which they are exposed.
Table 4.4. RBC count in blood upon contact with chitosan-g-Poly(HEMA) polymers
RBC Count (x10 9/ml) Materials Sample details
initial 30 min
CH-0 Pure chitosan 6.81 6.12
CH-HE5 Poly(HEMA) grafting 121% 6.01 6.16
CH-HE 7.5 Poly(HEMA) grafting 225% 6.25 6.16
CH-HE 12.5 Poly(HEMA) grafting 425 % 6.21 6.30
Reference Without sample 6.21 6.60
Chapter Chapter Chapter Chapter 4444
161
Table 4.5 WBC count in blood upon contact with chitosan-g-Poly(HEMA) polymers
WBC Count (x 106/ml) Materials Sample details
initial 30 min
CH-0 Pure chitosan 11.10 13.05
CH-HE5 Poly(HEMA) grafting 121% 11.85 14.10
CH-HE 7.5 Poly(HEMA) grafting 225% 11.70 12.75
CH-HE 12.5 Poly(HEMA) grafting 425 % 12.15 12.0
Reference Without sample 11.70 12.9
Table 4.6 Platelet count in blood upon contact with chitosan-g-Poly(HEMA) polymers
Platelet Count (x 108/ml) Materials Sample details
initial 30 min
CH-0 Pure chitosan 3.54 3.04
CH-HE5 Poly(HEMA) grafting 121% 4.18 3.15
CH-HE 7.5 Poly(HEMA) grafting 225% 4.14 3.09
CH-HE 12.5 Poly(HEMA) grafting 425 % 3.93 3.15
Reference Without sample 4.02 2.70
The percentage haemolysis induced by the chitosan-g-poly(HEMA) films to the
blood samples are given in Table 4.7. Compared to the reference material neither
chitosan nor the copolymer films have an effect on the haemolysis of the blood sample to
which they are exposed. As per ASTM F 756-00 the percentage haemolysis value less
than 2% is considered as nonhaemolytic [12]. Hence, the in vitro screening of the materials
shows that the chitosan-g-poly(HEMA) films are suitable for any type of blood contacting
applications.
Chapter Chapter Chapter Chapter 4444
162
Table 4.7 Percentage haemolysis upon contact with chitosan-g-Poly(HEMA) polymers
Sample code Sample details % Haemolysis
CH-0 Pure chitosan 0.28 + 0.09
CH-HE5 Poly(HEMA) grafting 121% 0.26 ± 0.27
CH-HE 7.5 Poly(HEMA) grafting 225% 0.27 ± 0.08
CH-HE 12.5 Poly(HEMA) grafting 425 % 0.26 ± 0.10
Reference Without sample 0.37 ± 0.13
4.1.9. In Vitro Cytotoxicity Test Assessment of the cytotoxicity of the membranes was carried out according to
ISO 10993-5, 1999 as mentioned in the previous chapter. The fibroblast cells are spindle
shaped and were evaluated for general morphology, vacuolization, detachment, cell lysis
and degeneration when in contact with the materials. The L929 cells in contact with test
samples retained their spindle-shaped morphology when compared to negative control
(high density polyethylene). The membranes also did not induce deleterious effects such
as detachment, degenerative and lysis with L929 fibroblasts when placed directly on the
monolayer of the cells. Fig 4.10 shows the chitosan and the chitosan-g-poly(HEMA)
films when exposed to L929 fibroblast cells. The figure also shows the morphology of
the L929, normal fibroblast cells.
Fig 4.10. Cytotoxicity evaluation of virgin chitosan and chitosan-g-Poly(HEMA)
Chapter Chapter Chapter Chapter 4444
163
4.1.10. Biodegradation Studies
0
5
10
15
20
25
% M
ass-
loss
CH-0 CH-HE5 CH-HE7.5 CH-HE10 CH-HE12.5Sample code
Trypsin sampleTrypsin controlPapain samplePapain control
Fig 4.11 a. Mass-loss of chitosan and the chitosan-g-Poly(HEMA)
after one month enzyme treatment
In vitro biodegradation studies of the films are carried out in enzyme solutions,
trypsin in tris buffer at pH 8.1 and papain in citrate buffer at pH 6 as detailed in section 2.14.
The biodegradability of the films is assessed by checking the mass-loss, the mechanical
property-loss and FTIR spectra of the films after enzyme treatment. Fig 4.11a gives the
histogram showing the percentage mass-loss of the different films after biodegradation. The
films kept in trypsin solution only have suffered mass-loss. The enzyme, papain did not cause
any significant reduction in mass as evident from the histogram. We can see that in trypsin
solution the chitosan films have undergone the highest % mass-loss (25%) than the
grafted ones. For the CH-HE5 polymer film, the mass-loss decreases to 9.4%. When the
percentage grafting increases further, the mass-loss decreases to 5.2%, 5.5% and 4.6% in
CH-HE7.5, CH-HE10, and CH-HE12.5 respectively. Hence, grafting can have a control
over the biodegradability of chitosan. In other words, the grafted films can remain in the
enzymatic environment for a prolonged time than the virgin chitosan films.
Chapter Chapter Chapter Chapter 4444
164
Fig 4.11b shows the percentage loss of tensile strength of chitosan and the
chitosan-g-poly(HEMA) polymer films after keeping in the test solutions. The films kept
in trypsin solution have undergone loss of tensile strength. For chitosan films, all the test
solutions, (buffers and enzyme solutions) caused loss of tensile strength. But the effect
decreases as the extent of grafting increases. The CH-HE12.5 films in which the grafting
% is the maximum, has the minimum loss of mechanical property. Fig 4.12 is the FTIR-
ATR spectrum of the chitosan-g-poly(HEMA) films before and after keeping in enzyme
solution (trypsin) for one month. The decrease in intensity of the -C=O stretching (1710
cm-1), -OH stretching (3300 cm-1) etc clearly confirm that degradation has been taken
place to the copolymer films when they are exposed to enzymatic environment.
0
20
40
60
80
100
% S
tres
s-lo
ss
CH-0 CH-HE5 CH-HE7.5 CH-HE10 CH-HE12.5Sample code
Trypsin controlTrypsin samplePapain controlPapain sample
Fig 4.11 b. Stress-loss of chitosan and chitosan-g-poly(HEMA)
after one month enzyme treatment
Chapter Chapter Chapter Chapter 4444
165
Fig 4.12 FTIR Spectra CH-HE12.5 (untreated), after keeping in
tris buffer and trypsin solution for 1 month
4.2. Chitosan-g- poly(HEMA) for Dialysis Membrane Applications
4.2.1. Background
The design of artificial kidney systems has made possible repetitive haemodialysis
and sustaining the life of chronic kidney failure patients. Chitosan membranes have been
proposed as an artificial kidney membrane because of their favourable permeability and
high tensile strength [13-16]. The most important part of the artificial kidney is the semi
permeable membrane. Regenerated cellulose and cuprophane are the commercially
available dialysis membranes. But, there is little selectivity in the separation of two
closely related molecules [17]. Hence, novel membranes need to be developed for better
control of transport, ease of formability and inherent blood compatibility. Chitosan is said
to be haemostatic, i.e., aids in blood clotting- that can compromise the permeability
characteristics [18]. So, attempts were made to improve the blood compatibility of
chitosan membranes via surface modification with least interference to their permeability
properties [19-22]. A non-thrombogenic albumin blended chitosan membrane was
derived by immobilizing this bioactive complex via carbodiimide [23]. Reports are
Chapter Chapter Chapter Chapter 4444
166
available regarding the permeability of various molecules through chitosan membranes
[23-25] which contain different immobilized and modified biomolecules.
This section covers the detailed studies carried out on the permeation characteristics
of chitosan and chitosan-g-poly(HEMA) membranes using the low molecular weight solutes
creatinine, urea, glucose and the high molecular weight solute albumin.
4.2.2. Permeation Studies
The experimental conditions and the description of the apparatus used for the
permeation study are detailed in section 2.15 of chapter 2. The chitosan-g-poly(HEMA)
polymer films of different compositions, e.g. CH-HE5, CH-HE7.5, and CH-HE12.5 were
used for the study. The percentage of initial amount permeated, Ct/C0 x 100, where Ct is
the concentration of the solute in the receptor cell diffused at time t (minute), C0 is the
initial concentration of the solute in the donor cell, was calculated for each membrane
and the results were compared with that of the virgin chitosan and cellulose films.
4.2.2.1. Creatinine Permeation
Scheme 1. Creatinine
Figure 4.13 shows the % permeation of creatinine vs. time through chitosan and the
chitosan-g-poly(HEMA) films. In the case of chitosan-g-poly(HEMA) films, grafting of
HEMA onto chitosan did not affect the creatinine permeability considerably. There is only a
marginal difference in the amount permeated between chitosan and the copolymer films during
the initial period. Virgin chitosan film permeated 32% of the initial amount at 150 min where
the other copolymer films permeated 27 to 31 % at the same time period. CH-HE12.5
composition attained the equilibrium at the 45th min itself. For the other copolymer films the
Chapter Chapter Chapter Chapter 4444
167
amount permeated reached upto that of chitosan by 300 min, and attained the equilibrium
condition. The chitosan-g-poly(HEMA) are hydrophilic, which eventually swells in the
aqueous medium leading to the increased permeability of creatinine after a period of 300 min.
The permeability of molecules across a membrane can be described as a linear
function of partition coefficient P = KD/∆x, where K is the partition coefficient (which is
a measure of the solubility of the substance), D is the diffusion coefficient and ∆x is the
thickness of the cell membrane. In the case of diffusion through the cell membrane, the
substrates that dissolve in lipids (lipid bilayer-cell membrane) pass more easily into the
cell. In a similar way the creatinine molecules are soluble in chitosan matrix due to the
interaction among the donor groups in creatinine like–NH2, and -C=O (Scheme 1) with
the free amino groups in chitosan; thereby increasing its concentration in the chitosan
matrix leading to its permeability. Similar observations have been reported where the
electrostatic attraction between uric acid and chitosan enhanced the permeation of uric
acid through the chitosan/PVA blended hydrogel membranes [26]. It is evident from Fig
4.13 that all the chitosan-g-poly(HEMA) films showed better permeability to creatinine
when compared to the cellulose dialysis membranes.
0 100 200 300 400 5000
5
10
15
20
25
30
35
% C
reat
inin
e p
erm
eate
d
Time (min)
CH-0 CH-HE5 CH-HE7.5 CH-HE12.5 Cellulose
Fig 4.13. Permeation of creatinine through chitosan-g-poly(HEMA) films
Chapter Chapter Chapter Chapter 4444
168
4.2.2.2. Glucose Permeation
Scheme 2. Glucose
Fig 4.14 shows the percentage permeation of glucose vs. time for chitosan and the
chitosan-g-poly(HEMA) films. The hydrophilic CH-HE12.5 composition showed high
permeation (38%) at 15th minutes itself establishing the equilibrium condition afterwards.
The permeability of CH-HE7.5 composition increased linearly with time and attained the
equilibrium at 150 min. The enhanced glucose permeability of these chitosan-g-
poly(HEMA) polymers may be attributed to the improved hydrophilicity due to
poly(HEMA) pendant graft. The strong interaction between the OH groups of HEMA
with that of glucose increases its concentration in the copolymer matrix leading to its
high permeability. The structure of glucose is depicted in Scheme 2.
0 100 200 300 400 5000
5
10
15
20
25
30
35
40
% G
luco
se p
erm
eate
d
Time (min)
CH-0 CH-HE5 CH-HE7.5 CH-HE12.5 Cellulose
Fig 4.14 Permeation of glucose through chitosan-g-poly(HEMA) films
Chapter Chapter Chapter Chapter 4444
169
4.2.2.3. Urea Permeation
Scheme 3. Urea
Fig 4.15 shows the permeation of urea vs. time for chitosan and the chitosan-g-
poly(HEMA) films. The initial amount permeated by all the chitosan-g-poly(HEMA)
films at the 5th, 15th, 30th and 45th minutes is higher than that of the virgin chitosan
membrane, showing an improved permeability for the copolymer films. However, the
amount permeated by all the films is less than the cellulose film upto 100 min, thereafter
it increases. The maximum amount permeated is the highest (i.e.43%) for CH-HE12.5
composition. The structure of urea is given in Scheme 3.
0 20 40 60 80 100 120 140 160 180 2000
10
20
30
40
50
% U
rea
perm
eate
d
Time (min)
CH-0 CH-HE5 CH-HE7.5 CH-HE12.5 Cellulose
Fig 4.15. Permeation of urea through chitosan-g-poly(HEMA) films
It is established that modification of chitosan through grafting with poly(HEMA)
has improved the permeability of low molecular weight solutes in compositions like, CH-
HE12.5 and CH-HE7.5, where the extent of grafting is high (425% and 225%). In the
Chapter Chapter Chapter Chapter 4444
170
case of CH-HE5 where the extent of grafting is the lowest (121%), poly(HEMA) grafting did
not significantly aid the permeation properties of chitosan. Amiji noted no significant increase
in creatinine and urea permeability when chitosan films are surface modified with heparin [24].
4.2.2.4. Albumin Permeation
Chitosan permeated a maximum of 1.8 % of the initial amount of albumin (Fig
4.16). The chitosan-g-poly(HEMA) films CH-HE7.5 and CH-HE12.5 showed 20% and
7.6% permeability to albumin. Commercial cellulose film showed 0.45% permeability.
The CH-HE5 composition showed the same trend as that of virgin chitosan.
For haemodialysis purposes, the uremic toxins like urea, creatinine etc should be
cleared out and the loss of substances essential to the patient should be prevented. Albumin is
one of these useful substances, the leakage of which if left unchecked, may compromise the
nutritional status of the patient; as a generalization, loss of over about 2–3 g of albumin per
treatment session is considered undesirable [27]. The albumin permeability of the graft
copolymers are found to be well within the desired range. The maximum permeability was
shown by the CH-HE7.5 (20%) and CH-HE12.5 (7.6%) compositions, which amount to be
1.6g and 0.6g albumin respectively (the concentration of albumin in the donor cell is
8g/dl) which is less than the allowed range, 2-3 g of albumin.
0 100 200 300 400 5000
5
10
15
20
% A
lbu
min
per
mea
ted
Time (min)
CH-0 CH-HE5 CH-HE7.5 CH-HE12.5 Cellulose
Fig 4.16. Permeation of albumin through chitosan-g-poly(HEMA) films
Chapter Chapter Chapter Chapter 4444
171
It is evident from Fig 4.17, the ESEM micrograph at 4000x magnification, that both the
virgin chitosan and the chitosan-g-poly(HEMA) films are non porous. Hence, the
permeation of solutes through these films is not by a pore-type mechanism. Here, the
permeation of solutes takes place due to swelling of the matrix in the aqueous medium.
Miller and Bruening correlate swelling and permeability of polyelectrolyte multilayer
Fig 4.17. ESEM micrograph of the virgin chitosan (CH-0) and chitosan-g-
poly(HEMA) (CH-HE7.5) films
films in one of their studies. The hayaluronic acid/chitosan polyelectrolyte films having
high degree of swelling index permit a 250 fold greater fractional passage of glucose
[28]. In general, films prepared from polyelectrolytes with a high charge density show
low swelling and slow solute transport, presumably because of a high degree of ionic
cross-linking. The chitosan-g-poly(HEMA) films are glassy at room temperature as
evident from their glass transition temperatures which ranges from 108-112ºC. The
polymer rigidity packing is promoted by the strong intermolecular hydrogen bonding
between the core and the graft. The glassy polymer responds relatively slowly initially.
Eventually it swells in the diffusion medium and the polymer structure changes as
diffusion proceeds [29]. Diffusion in glassy polymers is known as non-fickian “case II
diffusion” which is usually observed in such polymers subjected to penetration by a low
molecular weight solvent. The most characteristic feature of this phenomenon is the
development of a sharp front in the concentration profile, which advances linearly in
time, and ahead of which the penetrant concentration is very low. As soon as the slow
Chapter Chapter Chapter Chapter 4444
172
molecules are plasticized at the front, they quickly make room for the penetrant, which in
turn is rapidly transported into the thus opened free volume [30].
Thus grafting with poly(HEMA) could improve the hydrophilicity, blood
compatibility and cyto compatibility of chitosan without sacrificing its low molecular
weight solute permeability. Moreover grafting can have a control on the biodegradability
of chitosan. In certain compositions, CH-HE7.5 and CH-HE 12.5 the albumin
permeability was found to be increased, but the amount permeated is well within the
desired range.
4.3. Conclusions
HEMA was successfully grafted onto chitosan with ceric ammonium nitrate as
initiator under controlled conditions. Grafting was enhanced by HEMA concentration in
the medium. The graft copolymerisation was confirmed by FTIR, thermal and XRD
studies. The Tg of the graft copolymer was found to be in between those of chitosan and
poly(HEMA). The grafted products showed good film forming property. Grafting
resulted in rigid polymer whose mechanical properties in wet conditions were
significantly impaired due to the plasticizing effect of water. The copolymer films
showed enhanced hydrophilicity and overall thermal stability compared to chitosan. The
copolymers showed very high swelling in acid pH and limited swelling in physiological
pH which is an essential requirement for developing gastrointestinal drug delivery
systems using these polymers. Moreover grafting with the synthetic polymer improved
the stability of chitosan films in the enzymatic environment without sacrificing its blood
compatibility and cyto compatibility.
The HEMA-modified chitosan films were studied for their suitability to be
applied for dialysis membranes. It is proved that the permeability of all the copolymer
films is unaffected due to grafting, and in certain compositions grafting resulted in better
permeability in comparison to virgin chitosan. All the compositions showed higher %
permeation for the solutes than that for standard cellulose films.
Chapter Chapter Chapter Chapter 4444
173
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