Chapter 4 Chitosan-g-poly(2-Hydroxyethyl...

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

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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.


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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).


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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)


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(%)


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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.


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)


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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


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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


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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.


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.


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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


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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






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






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.


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)


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.


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


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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


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


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


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)


Fig 4.14 Permeation of glucose through chitosan-g-poly(HEMA) films


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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)


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


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)


Fig 4.16. Permeation of albumin through chitosan-g-poly(HEMA) films


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


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.


173

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