Chapter 6 Chitosan-g-poly(vinyl acetate/vinyl alcohol)shodhganga.inflibnet.ac.in/bitstream/10603/7189/14/14... · 2015-12-04 · focused on the synthesis and characterization of pure

Chapter 6 Chitosan-g-poly(vinyl acetate/vinyl alcohol)

Scope of the Chapter

The focus of this chapter involves the modification of chitosan with a hydrophilic

monomer, vinyl alcohol. For this, vinyl acetate is grafted onto chitosan and the poly(vinyl

acetate) segment is subsequently hydrolysed to poly(vinyl alcohol) with aqueous alkali.

The physico-chemical properties of the chitosan-g-PVAc polymer, the property changes

occurred due to hydrolysis of the PVAc segment to PVOH etc are studied in this chapter.

The mechanically strong chitosan-g-PVAc/PVOH are tested for their preliminary blood

compatibility, cytotoxicity and biodegradability. They have been evaluated for possible

use as haemodialysis membranes by studying their permeability properties to both low

molecular weight and high molecular weight solutes.

The results of the first section of this chapter have been published in Journal of Applied

Polymer Science, Vol. 104, 1852–1859 (2007) ‘Synthesis, Characterisation & Properties of Poly

Vinyl Acetate- and Poly Vinyl Alcohol- Grafted Chitosan’,

The results of the second part of this chapter have been communicated to Journal of

Membranes Science, ‘Modified Chitosan as Artificial Kidney Membrane’.

Chapter Chapter Chapter Chapter 6666

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6.1. Synthesis and Characterisation

6.1.1. Background

In the previous chapters, the attempts on the modification of chitosan using

hydrophilic/ hydrophobic acrylic monomers, HEMA and MMA and also with a PEO-based

macro monomer, PEGm have been described. These systems have moderate hydrophilicity.

The next choice was the modification of chitosan using a highly hydrophilic monomer, vinyl

alcohol. Vinyl alcohol can not be directly grafted onto chitosan. Hence chitosan-g-poly(vinyl

acetate) was prepared first and the poly(vinyl acetate), (PVAc) segment was hydrolysed to

poly(vinyl alcohol), (PVOH) by alkaline hydrolysis. This chapter focuses on the synthesis and

physico-chemical characterization of chitosan-g-PVAc/PVOH and the evaluation of their

preliminary biocompatibility. This chapter also evaluates the permeation properties of the

copolymer films in comparison to the commercial grade cellulose membrane.

Poly(vinyl alcohol) is a hydrophilic biocompatible polymer with several

characteristics desired for biomedical applications [1]. It has good pH stability and

excellent mechanical properties. In view of its semi permeability and biocompatibility it

is of particularly interest for biomedical applications. The graft copolymers of chitosan

and poly(vinyl alcohol) were studied by Huang and Fang [2]. Wang et al explored the

usefulness of chitosan-poly(vinyl alcohol) films for the controlled release of bovine

serum albumin [3]. In a study, Mathews et al showed that the biocompatibility and cell

adhesion characteristics of poly(vinyl alcohol) hydrogel can be improved by blending

chitosan with the hydrogel [4]. In another study, Don et al reported the preparation and

characterization of chitosan-g-poly(vinyl alcohol)/poly (vinyl alcohol) blends and showed

that the cellular compatibility of poly vinyl alcohol was improved due to the

incorporation of chitosan [5]. The studies of Chuang et al also highlighted the potential

of poly(vinyl alcohol)/chitosan blended membranes for biomedical applications [6]. Don

et al reported the mechanical, thermal and swelling properties of chitosan modified with

poly(vinyl acetate) [7]. The copolymers were made by dispersion polymerization of

chitosan and vinyl acetate. In such a case, the polymer will have a mixture of chitosan,

chitosan-g-PVAc and homopolymer of PVAc. No attempt was made to isolate the graft


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copolymer from the homopolymer, poly(vinyl acetate). Therefore, the reported properties

are those of the blends of chitosan-g-PVAc and homopolymer of PVAc, the latter being

present in substantial quantities. The graft-extent is also quite low. This chapter is

focused on the synthesis and characterization of pure chitosan-g-poly(vinyl acetate)

wherein the extent of PVAc grafting is comparatively high.

6.1.2. Preparation of Chitosan-g-poly(vinyl acetate)/poly(vinyl alcohol)

Poly(vinyl acetate) was grafted onto chitosan following the method explained in

chapter 3. A maximum of 92% grafting with a grafting yield of about 32% was achieved on

reacting the system for 5 h. The different compositions were coded as CH-V4B, CH-V6B and

CH-V10B, 4, 6 and 10 being the weight in grams of monomer used for synthesis (Table 6.1).

To prepare chitosan-g-poly(vinyl alcohol), the chitosan-g-poly(vinyl acetate)

copolymers were refluxed with 2% aqueous sodium hydroxide solution for 2 h. The

copolymer was then washed extensively with distilled water and the corresponding

compositions were labeled as CH-V4A, CH-V6A and CH-V10A respectively where ‘A’

denotes after hydrolysis, i.e. chitosan-g-poly(vinyl alcohol) and ‘B’ denotes before

hydrolysis, i.e. chitosan-g-poly(vinyl acetate). The percentage grafting increased

proportionally with the weight of the monomer in the feed. But the percentage yield was

not found to follow the same trend. The extent of grafting was calculated (after the

removal of the homopolymer by soxhlet extraction with methanol), using the equation

given in section 2.4 of chapter 2. The complete removal of the homopolymer was

confirmed by FTIR analysis of the extract.

Table 6.1 Graft copolymerisation of vinyl acetate onto chitosan with Cerium (IV)

No Polymer reference

Weight of Chitosan (g)

Weight of VAc(g)

Monomer conversion (%)

Yield (%)

Grafting %

1 CH-V4B 2 4 12.5 42 25

2 CH-V6B 2 6 19.3 40 58

3 CH-V10B 2 10 18.4 32 92


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The details of the amount of reactants, reaction conditions and grafting yield are

given in Table 6. 1.

6.1.3. FTIR Spectral Analysis

The FTIR spectra were used to prove the grafting between poly(vinyl acetate) and

chitosan and also to confirm the complete hydrolysis of the vinyl acetate segments of the

graft copolymer to vinyl alcohol. The spectra of the chitosan-g-poly(vinyl acetate)

polymers e.g. CH-V10B and CH-V6B showed the presence of the carbonyl absorption

band at around 1733 and 1735 cm-1 which confirmed the presence of PVAc grafts (Fig

6.1a). The C=O peak intensity increases with extent of grafting. Amide I and Amide II

bands of chitosan are also seen at 1642 and 1582 cm-1 respectively and the symmetric

stretching vibrations of the -NH2/-OH groups of chitosan at 3443 cm-1. The characteristic

peaks of chitosan were assigned to be 1643 cm-1 (Amide I), 1582 cm-1 (Amide II) and

3450 cm-1 (primary –NH2) by Gupta and Kumar [8]. In the case of the chitosan-g-

poly(vinyl alcohol), the same peak was absent confirming the complete hydrolysis of the

vinyl acetate segments to vinyl alcohol segments (Fig 6.1b).

Fig 6.1a. FTIR spectra of chitosan, and chitosan-g-PVAc.


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Fig 6.1b. FTIR spectra of chitosan-g-poly(vinyl acetate) (CH-V6B and CH-V10B) and the corresponding chitosan-g-poly(vinyl alcohol)

(CH-V6A and CH-V10A) 6.1.4.X-ray Diffraction Patterns

Wide angle X-ray diffraction patterns of powdered chitosan and the copolymers

are shown in Fig 6.2. Chitosan exhibited the major crystalline peak at 2θ = 20º in

agreement with the literature values [9]. In graft copolymers, additional diffractions are

seen at 2θ = 28º, 48º, and 58º. Though each of these peaks has not been assigned to any

specific planes, the differing pattern of the copolymer vis-à-vis that of the parent

molecule is indicative of a clear structural change in chitosan as a result of grafting with

vinyl acetate/alcohol. On hydrolysis of acetate to alcohol, the intensity of all the peaks

was enhanced. This may be due to the comparatively high crystalline nature of the

chitosan-g-poly(vinyl alcohol).


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Fig 6. 2 XRD pattern of A) chitosan, B) chitosan-g-poly (vinyl alcohol) C) chitosan-g-poly(vinyl acetate)

6.1.5. Thermal Studies

Chitosan shows a glass transition, (Tg) at 62ºC in the second run of DSC scan

conducted at a rate of 20ºCmin-1 in nitrogen atmosphere. For the chitosan-g-poly(vinyl

acetate) when the poly(vinyl acetate) graft is less as in the case of CH-V4B (25%), no

detectable Tg is seen. However, as the poly(vinyl acetate) content gets enhanced, a single

transition is seen in the copolymers CH-V6B and CH-V10B at 28ºC and 29ºC

respectively (Table 6.2). When the vinyl acetate segments are hydrolysed, the glass

transition temperature of the films also increased (36ºC and 41ºC). This increase is due to

hydrogen bonding among the –OH groups. The representative DSC scans of the chitosan-

g-poly(vinyl acetate) and chitosan-g-poly(vinyl alcohol) films are given in Fig (6.3 and

6.4). Don et al reported that the residual ceric ion in the copolymer exhibited an

endothermic peak at 240ºC in the DSC scan of their copolymers [7]. However, in the

present case no such phenomenon was observed implying absence of residual ceric ions

in the copolymers, synthesised.


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Fig 6.3. DSC scans of chitosan-g-poly(vinyl acetate) polymers, heating rate 20ºCmin-1 in nitrogen atmosphere

Fig 6.4. DSC scans of chitosan-g-poly(vinyl alcohol) polymers,

heating rate 20ºCmin-1 in nitrogen atmosphere


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Table 6.2. The Effect of grafting on the glass transition temperature of chitosan

Glass transition temperature ( 0C) Sample codes Before

Hydrolysis (B) After

Hydrolysis (A)

PVAc 28 70

CH-0 62 -

CH-V4 No specific Tg No specific Tg

CH-V6 28 36

CH-V10 29 41

Fig 6.5 Thermograms of chitosan (CH-0) and chitosan-g-poly(vinyl acetate) polymers in nitrogen atmosphere, heating rate = 20 ºCmin-1

The thermograms for chitosan and the chitosan-g-PVAc/PVOH polymers are

shown in Fig 6.5 and 6.6. The two-stage mass-loss pattern of the copolymers confirms

grafting, which is more distinct in DTG thermograms as seen in Fig 6.7 and 6.8.

Data in Table 6.3a and 6.3b compare the decomposition temperatures of virgin

chitosan, chitosan-g-PVAc and chitosan-g-PVOH copolymers. From Table 6.3a, it is


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evident that PVAc grafting did not appreciably affect the initial decomposition temperature (Ti)

and 50% decomposition temperature (T50) of chitosan. On the other hand the initial

decomposition temperature decreased in the case of CH-V6A and CH-V10A compositions of the

chitosan-g-PVOH copolymer. Table 6.3b shows that the 50% decomposition temperature is

substantially increased for the chitosan-g-PVOH copolymers when compared to virgin chitosan.

Fig 6.6 TGA of chitosan (CH-0) and chitosan-g-poly(vinyl alcohol)

polymers in nitrogen atmosphere, heating rate = 10 ºCmin-1

Fig 6.7 DTG of chitosan (CH-0), Poly(vinyl acetate) homopolymer and chitosan-g- poly(vinyl acetate) (CH-10B), in nitrogen atmosphere, heating rate = 20 ºCmin-1


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Fig 6.8 DTG of chitosan (CH-0) and chitosan-g-poly(vinyl alcohol)

(CH-6A), in nitrogen atmosphere, heating rate = 10 ºCmin-1

Table 6.3a Effect of PVAc grafting on the decomposition temperature of chitosan

Copolymer

codes Ti(°C) T50 (°C) Tend (°C) Residue %

CH-0 246 350 683 35

CH-V4B 242 354 684 36

CH-V6B 244 354 677 27

CH-V10B 252 356 677 29

Table 6.3b Effect of PVOH grafting on the decomposition temperature of chitosan

Copolymer

codes Ti (°C) T50 (°C) Tend (°C) Residue %

CH-0 246 322 600 24

CH-V4A 252 431 688 42

CH-V6A 233 343 691 28

CH-V10A 210 398 687 39


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6.1.6. Mechanical Properties

As per the data given in Table 6.4, the grafting results in an enormous increase in

the tensile strength of chitosan film in the dry state with not much change in % elongation. The

tensile strength of the films is marginally decreased on hydrolysis of the vinyl acetate to

vinyl alcohol (Table 6.5). The % elongation increased considerably in the case of the

graft copolymers under wet condition as the vinyl acetate segment retards the rigidity of

the chitosan chain, making the copolymer more soft and extensible.

Table 6.4. Mechanical properties of the chitosan-g-poly(vinyl acetate) films

Tensile Strength (MPa) % Elongation Sample Code

Dry Wet Dry Wet

CH-0 35 ± 11 51 ± 7 15 ± 7 92 ± 7

CH-V4B 123 ± 16 25 ± 4 11 ± 3 153 ± 7

CH-V 6B 96 ± 17 55 ± 7 27 ± 10 151 ± 20

CH-V10B 117 ± 0.9 59 ± 10.2 13 ± 0.4 163 ± 8

Table 6.5. Mechanical properties of the chitosan-g-poly(vinyl alcohol) films

Tensile Strength (MPa) % Elongation Sample Code

Dry Wet Dry Wet

CH-0 35 ± 11 51 ± 7 15 ± 7 92 ± 7

CH-V4A 136 ± 28 63 ± 5 15 ± 3 115 ± 10

CH-V 6A 95 ± 1 38 ± 8 25 ± 2 151 ± 38

CH-V10A 82 ± 23 47 ± 3 23 ± 7 169 ± 12

The increased mechanical property of the chitosan-g-poly(vinyl acetate)

copolymers and their acid resistance are highly beneficial for the fabrication of strong as

well as degradable matrices. Don et al [10] studied the preparation of chitosan-g-

poly(vinyl acetate) copolymers and their adsorption of copper ions. To increase the

mechanical strength and acid resistance, the copolymer membranes were cross linked


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with glutaraldehyde. Wang et al [11] reported the physical and chemical properties of a semi-

interpenetrating polymeric network of chitosan and poly(vinyl alcohol) with glutaraldehyde as

the cross-linker. The authors claimed that the addition of poly(vinyl alcohol) improved the

mechanical properties of the hydrogel. Though the natural polymers can be fabricated into highly

biodegradable matrices, the main disadvantage of them is their poor mechanical properties and

handling inconveniences. Poly(vinyl acetate) grafted chitosan films reported in this work possess

both these qualities even in the uncrosslinked condition and are hence promising candidates for

varied engineering and medical applications.

6.1.7. Swelling Studies

The hydrophilicity of the copolymers is assessed by studying their water

absorption capacity and their under-water octane contact angles. The % swelling of the

samples are studied in phosphate buffer at pH 7.4 and in aqueous acetic acid at pH 1.98.

It is already reported that chitosan is hydrophobic in nature and its swelling index at pH

7.4 is very low. On grafting the hydrophobic monomer (i.e. vinyl acetate), chitosan is

expected to increase hydrophobicity which is confirmed by our results. Fig 6.9 shows that

all the chitosan-g-poly(vinyl acetate) copolymer films except CH-V4B composition have

less percentage swelling when compared to ungrafted chitosan films.

Fig 6.9. % Swelling of chitosan-g-poly(vinyl acetate) at pH 7.4


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In CH-V4B, the percentage grafting is only 25%; it is concluded that moderate PVAc

grafting is conducive to enhance the swelling characteristics. Water absorption occurs by way

of H bonding of NH2 and OH groups on chitosan, with water. When the PVAc content is

enhanced beyond 25%, possibly the OH and NH2 groups are engaged in H bonding with

acetate groups; thereby decreasing their affinity for H bonding with water.

On hydrolysis of the vinyl acetate to the corresponding vinyl alcohol, the

copolymers become more hydrophilic. This is also evident from the data in Fig 6.10

where, all the hydrolysed films show higher percentage swelling at pH 7.4 than chitosan.

Infact the swelling is proportional to the grafting percentage.

Fig 6.10. % Swelling of chitosan-g-poly(vinyl alcohol) at pH 7.4

At pH 1.98, the chitosan and the copolymers of this study show interesting

swelling behaviour (Fig 6.11). Briefly, chitosan has high percentage swelling and tends to

dissolve within 10 minutes of exposure at this pH on account of protonation of the free –NH2

groups. But dissolution time is enhanced to 30-40 minutes in the case of chitosan-g-

poly(vinyl acetate) films for CH-V4B composition, while CH-V6B and CH-V10B

compositions are stable for more than 24 h. The stability of CH-V6B and CH-V10B are


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due to the stabilization effect of vinyl acetate segment, which is grafted to the extent of

58% and 92% respectively.

Fig 6.11 % Swelling of Chitosan-g-poly(vinyl acetate) at pH 1.98

Fig 6.12 % Swelling of Chitosan-g-poly(vinyl alcohol) at pH 1.98

When the chitosan-g-poly(vinyl alcohol) films are kept at pH 1.98, the

compositions CH-V6A and CH-V10A display higher percentage swelling than chitosan.

This is due to the interaction of the pendent vinyl alcohol segments with water. The


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chitosan-g-poly(vinyl alcohol) films also showed increased acid resistance when

compared to virgin chitosan films. The CH-V6A and CH-V10A compositions are stable

for more than two hours at pH 1.98. Fig 6.12 shows the % swelling vs. time of the

hydrolysed copolymer at pH 1.98.

The under-water octane contact angle values also support the fact that the

conversion of the vinyl acetate segment to vinyl alcohol segments increases the

hydrophilicity of the copolymer. Table 6.6 gives the octane contact angle values of the

copolymers before and after hydrolysis.

Table 6. 6 Effect of grafting on octane contact angle of chitosan

Octane contact angle Sample

Before hydrolysis After hydrolysis

CH-0 149 149

CH-V4 145 160

CH-V6 91 117

CH-V10 92 121

Thus, the swelling studies ensure that the hydrophilic/hydrophobic tuning can be

achieved by properly adjusting the extent of hydrolysis of the chitosan-g-poly(vinyl

acetate) copolymers. The synthetic–natural hybrid copolymers having good mechanical

properties and properly tuned hydrophilic/ hydrophobic ratios are promising candidates

which could be exploited for degradable, strong and pH sensitive membrane applications.

6.1.8. In Vitro Screening of Materials

The RBC and WBC counts (initial and after 30 min of contact of the materials

with the blood) are given in the Tables 6.7 and 6.8. 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 test

materials when compared to the reference material. Total number of platelets in the

exposed blood is given in Table 6.9. When compared to the reference material, the test


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samples do not cause a significant change in the platelet counts of the blood samples to

which they are exposed. Table 6.10 tells that all the materials have a percentage

haemolysis value ranging from 0.33% to 0.38%. According to ASTM F 756-00 a material

with a % haemolysis value less than 2% is considered as haemocompatible. The in vitro

screening of the materials shows that the chitosan-g-poly(vinyl acetate) films are suitable

for any type of blood contacting applications. Hence, modification of chitosan through

grafting could result in the preparation of blood compatible membranes. Ching et al also

reported similar observations that chitosan/heparin polyelectrolyte complex

immobilization can endow the poly(acrylonitrile) membrane haemocompatibility and

antibacterial activity while retaining the original permeability [12].

Table 6.7 RBC count in blood upon contact with chitosan-g-PVAc/PVOH polymers

Total cell number (x 10 6/mm3) Sample

Code Sample Details initial After 30 minutes

CH-0 Pure chitosan 6.81+ 0.30 6.12 + 0.30

CH-V4B PVAc grafting 25% 4.31 + 0.03 4.08 + 0.10


CH-V4A PVOH grafting 15% 5.88 + 0.10 5.63 + 0.30

CH-10A PVOH grafting 85% 4.06 + 0.03 3.91+ 0.03

Reference without sample 6.27 + 0.30 5.63 + 0.30

Table 6.8 WBC count in blood upon contact with chitosan-g-PVAc/PVOH polymers

Total cell number (x 10 3/mm3) Sample Code

Sample Details Initial After 30 minutes

CH-0 Pure chitosan 6.55 + 0.30 6.52 + 0.30


CH-V10B PVAc grafting 92% 5.40 +0.60 5.60 + 0.40


CH-10A PVOH grafting 85% 6.60 + 0.10 5.63 + 0.20



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Table 6.9 Platelet count in blood upon contact with chitosan-g-PVAc/PVOH polymers

Platelet count (x 10 3/mm3) Material

Sample

Details Initial After 30 min

CH-0 Pure chitosan 118.60 + 6.30 115 + 7.40


CH-V10B PVAc grafting 92% 119.33 + 15.30 109.33 +12.60


CH-10A PVOH grafting 85% 112.33 + 11.00 133.67 + 7.40


Table 6.10 Percentage haemolysis values for chitosan-g-PVAc/PVOH polymers

Sample code Sample details %Haemolysis

CH-0 Pure chitosan 0.43 + 0.11

CH-V4B PVAc grafting 25% 0.33 + 0.17

CH-V10B PVAc grafting 92% 0.30 + 0.18

CH-V4A PVOH grafting 15% 0.38 + 0.12

CH-10A PVOH grafting 85% 0.53 + 0.21

Reference without sample 0.45 + 0.13

6.1.9. In Vitro Cytotoxicity Test One of the most sensitive toxicity testing protocols is based on the direct contact

of the sample with the cell culture. Fig 6.13 shows the chitosan-g-poly(vinyl acetate) and

chitosan-g-poly(vinyl alcohol) films when exposed to L929 fibroblast cells compared to

the negative control (high density polyethylene). 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.

The membranes also did not induce any deleterious effects such as detachment,

degenerative and lysis with L929 fibroblasts when placed directly on the monolayer of


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the cells. The quantitative determination of cytotoxicity of the materials are done by MTT

assay which reveals that all the copolymer films are highly cytocompatible showing

100% metabolically viable cells when compared to the controls (cells without materials),

the results are given in chapter 7.

Fig 6.13. L929 cells in direct contact with (A) CH-V10B (B) CH-V6B

C) CH-V10A D) negative control for 24 h.

6.1.10. Biodegradation Studies

In vitro biodegradation studies of the films are carried out in enzyme solutions of

trypsin and papain. The biodegradability of the films is assessed by checking the mass-

loss, FTIR spectra and the mechanical property loss of the degraded films. Fig 6.14 and

Fig 6.15 show the % mass-loss of the chitosan-g-PVAc and chitosan-g-PVOH films after

keeping them in enzyme solutions for one month. Chitosan has 25% mass-loss in trypsin

solution. The CH-V4B composition has higher % mass-loss (37%) than chitosan in

trypsin solution. The other compositions CH-V6B and CH-V10B compositions showed

comparatively lower mass-loss (5.3% and 3.4%) than chitosan and the CH-V4B

compositions.


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CH-0 CH-V4B CH-V6B CH-V10B0

5

10

15

20

25

30

35

40

Per

cen

tag

e m

ass-

loss Trypsin control

Papain control Trypsin sample Papain sample

Fig 6.14 Mass-loss behaviour of chitosan and the chitosan-g-PVAc after one month enzyme treatment

CH-0 CH-V4A CH-V6A CH-V10A

0

5

10

15

20

25

Per

cen

tag

e m

ass-

loss

Trypsin control Papain control Trpsin sample Papain sample

Fig 6.15 Mass-loss behaviour of chitosan and the chitosan-g-PVOH after one month enzyme treatment


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CH-0 CH-V4B CH-V6B CH-V10B0

20

40

60

80

100

Trypsin control Papain control Trpsin sample Papain sample

% L

oss

of

stre

ss

Fig 6.16 Stress-loss of chitosan and the chitosan-g-PVAc

after one month enzyme treatment

CH-0 CH-V4A CH-V6A CH-V10A0

20

40

60

80

100

% L

oss

of

stre

ss

Trypsin control Papain control Trypsin sample Papain sample

Fig 6.17 Stress-loss of chitosan and the chitosan-g-PVOH after one month

enzyme treatment

This may be due to the higher % grafting of polyvinyl acetate in these

compositions, nearly 58% and 92% respectively. Another interesting observation was that

only the CH-V4B composition showed a mass-loss in papain solution. All the other


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copolymer films and virgin chitosan film did not show any mass-loss in papain solution.

The chitosan-g-PVOH films also showed a similar degradation behaviour when their

mass-loss in enzyme solutions are studied. The mass-loss is reduced when the CH-V4B is

hydrolysed to CH-V4A (from 37% to 24 %). For CH-V6A and CH-V10A the mass-loss

is 4.2 % and 3.1 % respectively. Fig 6.16 and 6.17 show the percentage stress-loss of

chitosan and the chitosan-g-PVAc/PVOH polymer films in the test solutions including

the buffer solution. It is observed that all the films have undergone loss of tensile strength

in the buffer solution. It is interesting to see that the enzyme solutions trypsin and papain

have no additional effect on the biodegradability of chitosan and chitosan-g-

PVAC/PVOH films. However, as the % grafting increases as we move from CH-V4 to

CH-V10 (both chitosan-PVAc and chitosan-PVOH), the loss of tensile stress decreases

marginally.

Fig 6.18. FTIR Spectra of chitosan-g-PVAc before biodegradation and after keeping in tryspin solution for 1 month

Fig 6.18 and 6.19 are the FTIR-ATR spectra of the chitosan-g-poly(vinyl acetate) and

chitosan-g-poly(vinyl alcohol) films before and after keeping in trypsin solution for

one month. The decrease in intensity of the ester group at around 1730 cm-1, the –OH


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peaks at around 3300 cm-1, -CH2 deformation at 1420, C-C skeleton vibration at 1322 cm-1 and

the deformation of the peaks (C-O-C) at around 1200-1100 cm-1 clearly confirm that

degradation has taken place to both chitosan-g-PVAc and chitosan-g-PVOH polymer

films when they are exposed to enzymatic environment.

Fig 6.19. FTIR Spectra of chitosan-g-PVOH before biodegradation and

after keeping in tryspin solution for 1 month 6.2. Chitosan-g-poly(vinyl acetate) for Dialysis Membrane Applications

6.2.1. Background

Fatal kidney malfunctions, while uncommon, occur mostly in the people of young

age. The kidney removes waste materials from the body, and when this is not achieved

properly, the patient develops a kidney failure. In cases where the conventional methods

can not treat the malfunction, the patient must go through a series of artificial kidney

treatments. The artificial kidney, or dialyzer, is a life support system designed and the

active part of it is the semi permeable membrane itself, for which commercially

regenerated cellulose or cuprophan is still being used due to its good solute permeability


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and mechanical strength. Chitosan membranes have been proposed as artificial kidney

membranes because of their suitable permeability and high tensile strength [13]. All the

properties of chitosan membranes associated with their physico-chemical and biological

properties are particularly interesting for various biomedical applications, such as

pharmaceuticals, wound coverings and tissue engineering [14]. Singh and Ray have

extensively reviewed the application of chitosan and modified chitosans as artificial

kidney membrane [15]. The permeability of modified chitosan membranes has been

reported by many authors [16-18]. One of the most serious problems of using these

artificial membranes is surface-induced thrombosis, where heparinization of blood is

needed to prevent clotting, and people who are liable to internal hemorrhage can be

dialyzed only at great risk. Hence, the most challenging problem still to be solved is the

development of membranes which are inherently blood compatible [15]. Various

modifications are suggested to dramatically improve the blood compatibility of chitosan

membranes without altering its superior permeability [19].

In the previous section we found that the chitosan-g-PVAc films were

mechanically strong, blood compatible, non cytotoxic and biodegradable. It was of

interest to investigate their permeabilities for various solutes. Thus, this section details

the studies conducted to explore the suitability of these films as artificial kidney

membranes.

6.2.2. Creatinine Permeation

As already described in the previous chapters, the permeation characteristics of

chitosan-g-PVAc films were assessed with four different solutes viz; creatinine, urea,

glucose and albumin, in vitro at 37ºC to investigate the suitability of these films as

haemodialysis membranes.

From Fig 6.20, it is clear that the CH-V4B, CH-V6B and CH-V10B copolymer

films show higher permeability to creatinine than the virgin chitosan films and the

commercial cellulose films. In the case of commercial cellulose film, equilibrium is


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attained at 90 minutes itself but for the CH-V4B and CH-V10B films, the amount

permeated goes on increasing even after 4 hours.

0 50 100 150 200 250 1200 14000

10

20

30

40

50

60

70

80

90

Cre

atin

ine

perm

eate

d (%

)

Time (min)

CH-0 CELLULOSE CH-V4B CH-V6B CH-V10B

Fig 6.20. Permeation of creatinine through chitosan-g-PVAc films

In our discussions in the previous chapters a marginal decrease in creatinine

permeation was observed for the graft copolymers of chitosan, modified with the

hydrophilic vinyl monomers, chitosan-g-poly(HEMA) and chitosan-g-PEGm compared

to virgin chitosan films. This was attributed to the solubility of creatinine molecules in

chitosan matrix due to the interaction among several donor groups of creatinine like –NH2,

-C=O etc with the free amino groups in chitosan; thereby increasing its concentration in

the chitosan matrix leading to its high permeability. In the case of chitosan-g-poly(vinyl

acetate), it is interesting to note that all the compositions (CH-V4B, CH-V6B and CH-

V10B) show enhanced permeability to creatinine compared to virgin chitosan films. This

could be interpreted as follows. The chitosan-g-PVAc films behave as rubbery polymer at

the physiological temperature (37ºC) as evident from their glass transition temperatures

(28 to 29ºC). Segmental mobilities are easier in this state. When a penetrant diffuses into

a rubbery polymer above its glass transition temperature, a rapid adjustment of the

polymer chains occurs and, as a result, a fast diffusion results. Fickian behaviour is


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usually exhibited in such cases [20]. The movement of the diffusant is without any

interactions and is simply based on a random walk.

6.2.3. Urea Permeation

In the case of urea permeation (Fig 6.21), the CH-V4B composition permeates

around 35% of the initial amount at 30 minutes itself. The CH-V6B and CH-V10B

compositions show more or less similar permeability as the cellulose membranes. Virgin

chitosan is relatively poor in urea permeability initially upto 200 minutes, but thereafter

the permeability tends to increase.

0 50 100 150 200 250 1200 1600

5

10

15

20

25

30

35

40

Ure

a p

erm

eate

d (%

)

Time (min)

CelluloseCH-0CH-V4BCH-V6BCH-V10B

Fig 6. 21. Permeation of urea through chitosan-g-PVAc films

It is to be noted that the softening of the chitosan-g-poly(vinyl acetate) polymers

above the glass transition temperature has improved the urea permeability of the

chitosan-g-poly(vinyl acetate) films. It may be recalled that in the case of chitosan-g

poly(HEMA) and chitosan-g-PEGm films, initially all the copolymer films showed a

marginal decrease in the amount permeated, vis-à-vis cellulose film.


219

6.2.4. Glucose Permeation

In the case of glucose permeation the CH-V4B copolymer exhibits good permeability.

For the other compositions (CH-V6B and CH-V10B) similar trend of permeation is observed

compared to the parent chitosan (Fig 6.22). But all the copolymer films as well as virgin chitosan

film show higher rate of permeation initially when compared to commercial cellulose film.

0 50 100 150 200 250 300 1200 16000

5

10

15

20

25

30

Glu

cose

per

mea

ted

(%

)

Time (min)

Cellulose CH-0 CH-V4B CH-V6B CH-V10B

Fig 6.22. Permeation of glucose through chitosan-g-PVAc films

Thus, when the low molecular weight permeability is concerned all the chitosan-

g-poly(vinyl acetate) polymer compositions showed higher permeability than the

standard cellulose films.

6.2.5. Albumin Permeation

As reported earlier [21] chitosan is permeable to albumin and the amount

permeated is nearly 2% of the initial concentration (8g/dL). For immunoisolatory

purposes the membrane should be impermeable to albumin and globulins. The chitosan-

g-poly(vinyl acetate) films allowed only trace amount of albumin to pass through them,

e.g. CH-V4B (0.03%), CH-V6B (0.05%) and CH-V10B 0.6%) as shown in Fig 6.23. As

already specified in previous chapters albumin is one of the useful substances, the


220

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 [22]. The albumin permeated by the chitosan-g-PVAc films is

well within the allowed range of albumin leakage.

0 50 100 150 200 250 300 1200 16000.0

0.4

0.8

1.2

1.6

2.0

Alb

umin

per

mea

ted

(%

)

Time (min)

CELLULOSE CH-0 CH-V4B CH-V6B CH-V10B

Fig 6.23. Permeation of albumin through chitosan-g-PVAc films

Several reports are there in the literature relating the polymer properties and semi

permeability. One of the studies stated the usefulness of hydrophilic polymer membranes

as semipermeable membranes as in the case of a film formed from an aqueous solution

containing poly(vinyl alcohol) and chitosan salt [23]. In another study Nasir et al reported

that PEO-chitosan blends have porous structure compared to the pure chitosan

membranes, and this increases the permeability of the blend membranes [24]. Qurashi et al

studied on the modification of chitosan membranes using poly(vinyl pyrrolidone) [16].

According to them, the hydrophilicity of the membranes, molecular weight and chemical

nature of the metabolites are the important parameters in determining transport properties

of the membranes. Amiji, in one of his patents claimed that modified positively charged

polymers such as chitosan provide novel biocompatible and biodegradable

semipermeable membranes [25]. The chitosan-g-poly(vinyl acetate)/poly(vinyl alcohol)


221

films prepared in our lab is non porous as evident from the Environmental Scanning

Electron micrographs (Fig6.24). Hence, the permeation of low molecular weight solutes

through these membranes can not be by pore type mechanism as explained by Singh and

Ray in their study of glucose permeation through Poly(HEMA) modified chitosan

membranes [26]. Amiji studied the permeability properties of chitosan-poly(ethylene

oxide) blends and reported that the increase in permeability through the blend was

dependent more on the hydrophilicity of the membrane than on porosity [25].

Fig 6.24. ESEM micrograph of the virgin chitosan (CH-0), chitosan-g-PVAc (CH-

V6B) and chitosan-g-PVOH (CH-V6A) films

The chitosan-g-poly(vinyl acetate) membranes of our study are hydrophobic than

virgin chitosan membranes. But as reported in section 6.1, the chitosan-g-poly(vinyl

acetate) films are having higher extensibility when compared to chitosan. The % elongation of

the chitosan-g-poly(vinyl acetate) films increased considerably under wet condition as the

vinyl acetate segment retards the rigidity of the chitosan chain, making the copolymer

more soft and extensible. This fact is also confirmed by the decrease in glass transition

temperature of chitosan as an effect of grafting with poly (vinyl acetate). The glass transition

temperature of the CH-V6B and CH-V10B films are 28ºC and 29ºC respectively. This chain

relaxation enables the low molecular weight solutes like urea, creatinine and glucose to

pass through the membranes by a mechanism of ‘random walk’ similar to water

permeability through lipid belayed. These membranes are impermeable to high molecular

weight albumin.


222

From the studies conducted so far it is clear that the membranes prepared from

chitosan-g-poly(vinyl acetate) are superior to the commercial cellulose membranes for

the low molecular weight solute permeability and can be proposed as excellent dialysis

membranes. Moreover these membranes show little permeability to albumin and hence

can be used for immunoisolatory applications. Additionally as reported earlier the films

possess excellent mechanical stability, cytocompatibility and blood compatibility which

again supports its membrane type applications.

6.3. Permeation Properties of Chitosan-g-PVOH

6.3.1. Background In Section 6.2 of this chapter we evaluated the permeation properties of

chitosan-g-poly(vinyl acetate) films. As already mentioned, chitosan-g-poly(vinyl

alcohol) copolymers were derived by the alkaline hydrolysis of the chitosan-g-poly(vinyl

acetate) precursor. It was shown that chitosan-g-PVAc were hydrophobic than pure

chitosan whereas chitosan-g-PVOH exhibited enhanced hydrophilicity as evident from

their swelling characteristics and contact angle measurements. Hence it was of interest to

compare the permeation properties when the hydrophobic polymer is hydrolysed to a

hydrophilic one.

The chitosan-g-PVOH polymers derived from the different compositions of

chitosan-g-PVAc polymers like, CH-V4B, CH-V6B and CH-V10B are coded as CH-V4A,

CH-V6A and CH-V10A respectively. The permeation characteristics of the chitosan-g-

PVOH films were studied using the low molecular weight solutes like creatinine, glucose,

urea and also by the high molecular weight solute albumin.

6.3.2. Creatinine Permeation

As it is observed in the case of chitosan-g-poly(HEMA) and chitosan-g-PEGm,

the creatinine permeability of the chitosan-g-poly(vinyl alcohol) films was also found to

be less than that of the virgin chitosan films initially. However, the permeability

increased and reached upto that of virgin chitosan by 24 h. This increased permeability of


223

creatinine through the copolymer films after 300 min could be due to the swelling of the

hydrophilic chitosan-g-PVOH polymer in the aqueous medium during this period. It is to

be noted that all the copolymer films showed higher permeability than the commercial

cellulose films as can be seen from Fig 6.25.

0 50 100 150 200 250 1200 16000

5

10

15

20

25

30

35

40

Cre

atin

ine

per

mea

ted

(%

)

Time (min)

CH-0 CELLULOSE CH-V4A CH-V6A CH-V10A

Fig 6.25. Permeation of creatinine through chitosan-g-PVOH films

6.3.3. Urea Permeation

The copolymers showed high initial permeability to urea and attained the

equilibrium at 100 min itself. Such stabilization of urea permeation was shown by the

chitosan-g-PVAc films also. The amount permeated is substantially high in the case of

the chitosan-g-PVOH films (48% for CH-V10A) compared to the chitosan-g-PVAc films

(38% for CH-V4B). As noted before, Amiji has reported no significant increase in

creatinine and urea permeability when chitosan films were surface modified with heparin

[24]. In our study chitosan-g-poly(HEMA) and chitosan-g-PEGm films showed only a

marginal increase in urea permeation. However a substantial increase is observed for the

chitosan-g-PVOH films as seen in Fig 6.26.


224

0 50 100 150 200 250 300 1200 16000

10

20

30

40

50

Ure

a p

erm

eate

d (%

)

Time (min)

CELLULOSE CH-0 CH-V4A CH-V6A CH-V10A

Fig 6.26. Permeation of urea through chitosan-g-PVOH films

6.3.4. Glucose Permeation

0 50 100 150 200 250 300 1200 16000

5

10

15

20

25

30

35

Glu

cose

per

mea

ted

(%

)

Time (min)

CELLULOSE CH-0 CH-V4A CH-V6A CH-V10A

Fig 6.27. Permeation of glucose through chitosan-g-PVOH films

The glucose permeability of CH-V10B composition decreased when the PVAc

segment of the copolymer was hydrolysed to PVOH segment (CH-V10A). Moderate


225

grafting is conducive to better transport of glucose through the membranes. As the grafting

extent increases, the possibility for inter molecular “crosslinking” through H bonding

among the PVOH segments increase. This would impede the transport of glucose

molecules through the membranes. Urea is relatively smaller in size. Hence, its

permeability is not affected by the hydrolysis of the chitosan-g-PVAc to chitosan-g-PVOH.

6.3.5. Albumin Permeation

The albumin permeability of the chitosan-g-poly(vinyl acetate) films goes upto

0.5% but it is less than 0.2% for the chitosan-g-poly(vinyl alcohol) films at 24 h. Thus the

restricted permeability of PVOH is advantageous in the sense that the chitosan-g-PVOH

copolymers are better materials than chitosan-g-PVAc for the haemodialysis applications.

0 50 100 150 200 250 300 1200 16000.0

0.4

0.8

1.2

1.6

2.0

Alb

um

in p

erm

eate

d (

%)

Time (min)

Cellulose CH-0 CH-V4A CH-V6A CH-V10A

Fig 6.26 Permeation of albumin through chitosan-g-PVOH films

Thus, modification of chitosan with poly(vinyl alcohol) through poly(vinyl acetate)

grafting and subsequent hydrolysis could be used for the fabrication of strong, semi-

permeable membranes with potential applications in the biomedical field.


226

6.4. Conclusions Chitosan-g-poly(vinyl acetate) copolymers were synthesized by ceric

ammonium nitrate by free radical technique. A maximum of 40% yield with 92% grafting

could be achieved. The hydrolysis of the chitosan-g-poly(vinyl acetate) with aqueous

sodium hydroxide solution led to chitosan-g-poly(vinyl alcohol). Both the copolymers

showed enhanced mechanical properties with tunable hydrophilicity. The hydrolysed

copolymer was more hydrophilic than its precursor. The poor mechanical properties and

the consequent difficulty in processing of chitosan could be obviated by way of graft

copolymerisation. The increased mechanical property of the chitosan-g-poly(vinyl

acetate) is beneficial for the fabrication of strong, degradable matrices. Chitosan-g-

poly(vinyl acetate) films are promising candidates for varied engineering and medical

applications. Biocompatible, biodegradable and mechanically stable membranes with

good mechanical properties and properly tailored hydrophilic/hydrophobic characteristics

could serve as promising candidates for potential applications demanding strong but

degradable and pH sensitive membranes.

The hydrolysis of the chitosan-g-PVAc to chitosan-g-PVOH affected the

permeability of solutes through them. It was observed that creatinine permeability is

higher for all the chitosan-g-PVAc films than that of virgin chitosan films. However, in

chitosan-g-PVOH the trend is reversed and all the films exhibited less permeability than

chitosan in the initial period. Glucose permeation also decreased in the chitosan-g-PVOH

films when the grafting extent is increased. The more ordered arrangement in the

chitosan-g-PVOH copolymers due to internal cross-linking through H bonding restricted

the permeability of the solutes during the initial period. On the otherhand both the

chitosan-g-PVAc and chitosan-g-PVOH films exhibited increased permeability to urea

than virgin chitosan films. This may be due the comparatively small size of the urea

molecules. The albumin permeability through the films also decreased from 0.5% to

0.2% when the chitosan-g-PVAc are hydrolysed to chitosan-g-PVOH. Thus the more

ordered arrangement in the chitosan-g-PVOH polymers are advantageous in controlling

the leakage of albumin like nutrients. Hence the need for the development of


227

biocompatible, mechanically strong dialysis membranes could be addressed with the

fabrication of chitosan-g-poly(vinyl acetate)/poly(vinyl alcohol) membranes. Potential

applications like artificial kidney, artificial pancreas etc are envisaged from these

membranes.

6.5. References

1. Millon, L.E., Wan, W.K., J.of Biomed.Mater Res., Part B, Appl.Biomater.,

2006, 79B, 245.

2. Haung, M., Fang, Y., Biopolymers, 2006, 81, 160.

3. Wang, Q., Du, Y., Fan, L., J.Appl. Polym. Sci., 2005, 96, 808.

4. Mathews, D., Guinness, G.M., Birney, Y., Cahill, P.A., Bioengineering In

Ireland Conference, 2006, Jan 27-28, Clybaun Hotel, Galway.

5. Don, T.M., King, C.F., Chiu, W.Y., Peng, C.A., Carbohydr. Polym., 2006, 63,

3, 331.

6. Chuang, W.Y., Young, T.H., Yao, C.H., Chiu, W.Y., Biomaterials, 1999, 20, 1479.

7. Don, T.M., King, C.F., Chiu, W.Y., J.Appl.Polym.Sci., 2002, 86, 3057

8. Gupta, K.C., Kumar, R., J.Appl. Polym. Sci., 2000, 76, 672.

9. Kim, J.H., Lee, M., Polymer, 1993, 34, 1952.

10. Don, T.M., King, C.H., Chiu, W.Y., Polym. J., 2002, 34, 6, 418.

11. Wang, T., Turhan, M., Gunasekaran, S., Polym. Int., 2004, 53, 911.

12. Lin, W.C., LIU, T.Y., YANG, M.C., Biomaterials, 2004, 25, 10, 1947.

13. Hirano, S., Noshiki, Y., J Biomed. Mater. Res., 1985, 19, 413.

14. Domard, A., and Domard, M., Polymeric Biomaterials, 2nd Edn., (Eds.)

Dumitriu, S., Marcel Dekk., N.Y., 2002.


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15. Singh, D. K., Ray, A.R., J.M.S, Rev. .Macromol.Chem.Phys., 2000, C40, 1, 69.

16. Qurashi, M. T., Blair, H. S., Allen, S. J., J. Appl. Polym. Sci., 1992, 46, 2, 263.

17. Chandy, T., Sharma, C.P., J. Appl. Polym. Sci., 1992, 44, 12, 2145.

18. Amiji, M.M., Biomaterials, 1995, 16, 8, 593.

19. Dutta, Ravikumar and Dutta, JMS-Polym. Rev., 2002, 42, 3, 303.

20. Hajatdoost, S., Yarwood, J. J., Chem. Soc., Faraday Trans, 1997, 93, 8, 1613.

21. Radhakumary, C., Nair, P.D., Mathew, S., Nair, C.P.R., J. Appl. Polym. Sci.,

2006, 101, 2960.

22. Claudio, R., Bernd, B., Sudhir, K. B., Hemodialysis Int., 2006, 10, S48.

23. Mima, Seiichi, Yoshikawa, Susumu, Miya, Masaru, United States Patent

3962158, 1976.

24. Nasir, N.F.M, Zain, N.M., Raha, M. G., Kadri, N.A. Am. J. Appl. Sci., 2005,

2, 12, 1578.

25. Amiji, M.M.,U.S. Patent No. 5,904,927.

26. Singh, D.K., Ray, A.R., J. Appl. Polym. Sci ., 1994, 3, 1115.

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