chapter 7 comparative study of the properties and...

Chapter 7

Comparative study of the properties and applications of chitosan-g-polymers

Scope of the Chapter

In this chapter, a comparative study of all the chitosan-g-vinyl polymers is given.

The DSC and XRD studies are compared to reveal the crystalline/amorphous nature and

the thermograms to foretell the thermal stability as an index of extent of grafting. The

swelling index and under-water octane contact angles are measured to determine the

hydrophilicity and the tensile stress and elongation at break, to evaluate the mechanical

stability of the copolymers. The haemolysis induced by the copolymers to the blood

samples, the cytotoxicity test on L929 cells by MTT assay and live-dead assay, the mass-

loss and tensile stress-loss of the copolymer films due to enzymatic degradation etc are

compared. The low molecular weight solute permeability of the films are compared to

choose the best film synthesized, to be proposed as a haemodialysis membrane which has

a performance equal to or better than commercial cellulose films.

Chapter Chapter Chapter Chapter 7777

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7.1. Background

This chapter deals with the comparative study of the polymers in terms of their

physico-chemical properties, biocompatibilities and their possible biomedical

applications. As chitosan modified with HEMA, PEGm and PVAC/PVOH were

evaluated for possible haemodialysis applications, a comparative study of their

permeation properties has also been made. Though chitosan-g-PMMA could not be

processed into films for evaluation as membranes for haemodialysis applications, it could

be processed into porous microspheres and evaluated for drug release applications.

7.2. Comparison of Physico-Chemical Properties

All the copolymers were synthesized by the same technique by a redox initiated

free radical route using ceric ammonium nitrate as the redox initiator. The products were

purified by precipitation using aqueous sodium hydroxide solution, washing extensively

with water and drying. The copolymers were soxhelet-extracted with a suitable solvent to

remove the homopolymer formed during the reaction process. The purified copolymers

were dissolved in aqueous acetic acid solution and films were cast by evaporating the

solvent. The copolymers were characterized by various physico-chemical

characterization techniques and a comparative study of the properties of the different

copolymers was carried out.

FTIR spectroscopy was found to be very useful for the confirmation of the

grafting reaction between chitosan and the vinyl monomers. The presence of the -C=O

stretching vibrations of the carbonyl group of the vinyl polymers at around 1730-1740 cm-1 in

the copolymer spectra, was used for the identification of the pendant vinyl graft. In all

the copolymer spectra the intensity of this peak increased as the amount of monomer in

the graft segment increased. The characteristic peaks of chitosan, the Amide I and Amide II

bands at around 1642 and 1580 cm-1 respectively and the symmetric stretching vibrations

of the -NH2/-OH groups at 3443 cm-1 are distinctly visible in the copolymer spectra.


231

The thermal properties of the copolymers were studied using the techniques like

DSC, TGA, and DTG. The DSC was used to get the glass transition temperature of the

copolymers. Poly(HEMA) and PMMA when grafted onto chitosan the copolymers gave

a Tg at around 111ºC, and 120ºC respectively, while chitosan-g-PVAc copolymers

exhibited a glass transition temperature at 28-29ºC and chitosan-g-PVOH at 36-40ºC

which is well below the glass transition temperature of virgin chitosan. Chitosan-g-

PEGm copolymers did not show any distinct glass transition temperature, but they

manifested a clear melting endotherm of the PEO segment. As the PEO concentration

increased, these endotherms became more prominent. Hence, the glass transition

temperatures of the copolymers foretell that the chitosan-g-poly(vinyl acetate)

copolymers will be soft while the chitosan-g PMMA/PHEMA brittle at room

temperature. The chitosan-g-PEGm copolymers were predominantly of soft nature

contributed by PEG graft. The DSC studies revealed that the chitosan-g-PEGm

copolymers have a crystalline nature as the copolymers manifested a melting endotherm

without showing any significant glass transition temperature. The SEM micrographs and

XRD studies also supported this.

Though thermal stability is not of concern for the projected applications of the

polymers, these are finger prints for the molecular configuration and composition, and

thus serve as effective characteristics. The thermo gravimetric analysis (TGA) showed

that grafting with vinyl monomers did not cause any adverse effect on the thermal

stability of chitosan, on the other hand in certain cases the thermal stability is enhanced

due to grafting. Grafting with poly(HEMA) and PMMA enhanced the thermal stability

of chitosan, while grafting with PVAc and PEGm did not affect its thermal stability.

The two-stage decomposition temperature of the copolymers compared to the

single stage decomposition temperature of virgin chitosan confirms the grafting reaction

between chitosan and the vinyl monomer. This is further confirmed from the DTG of the

copolymers which gave an additional peak due to the decomposition of the pendant vinyl graft.


232

The crystalline structure of chitosan is lost due to grafting with vinyl monomers

as evident from the X-ray diffraction pattern of chitosan and the copolymers. The

amorphous PHEMA, PMMA and PVAc when grafted on to chitosan, the crystalline peak

intensity of chitosan (2 θ= 20º) decreased. In the case of chitosan grafted with PEGm, the

graft polymer manifested additional diffraction pattern at 2θ = 40º and 70º due to the

pendant comblike graft of PEO, where PEO forms crystallites.

The mechanical property evaluation revealed that grafting with poly(HEMA) led

to rigidification of the graft copolymer by way of strong intermolecular H-bonding.

Under wet conditions, the copolymers showed a further decrease in property. PMMA

grafting also caused an enormous decrease in the tensile strength of chitosan under wet

condition. The elongation also decreased. The mechanical property of the films in the dry

condition was not studied as the films were weak and fragile. The grafting of PVAc

resulted in an enormous increase in the tensile strength of chitosan film both in the dry

and wet states. Though there was not much change in elongation under dry state, it

increased considerably under wet condition as the vinyl acetate segments are more soft

and extensible. On the otherhand, the grafting with PEGm resulted only in a marginal

increase in the tensile strength of chitosan film in the dry state. But a moderate grafting

helped improve the tensile strength of the copolymer films. However, under wet

conditions, grafted polymers showed a decrease in tensile strength.

It can be noted that certain compositions of chitosan-g-poly(vinyl acetate) and

chitosan-g-poly(vinyl alcohol) films achieved the highest tensile strength in the dry

condition, (123 MPa for the former and 136 MPa for the latter, the value for virgin

chitosan in the same conditions being 35 MPa). Chitosan-g-PEGm films gained 46 MPa.

In the case of chitosan-g-PMMA films the tensile property of the dry films could not be

evaluated at all due to its brittle nature. The maximum wet strength of the chitosan-g-

PHEMA films is 18 MPa and chitosan-g-PEGm films is 11 MPa. The chitosan-g-PVAc

films exhibited the maximum wet strength of 59 MPa and chitosan-g-PVOH films,

63MPa. Thus, the copolymers of chitosan with poly(HEMA), PVAc, PVOH and PEGm possess


233

the required strength to be proposed for any type of membrane applications. The mechanical

property data of the different copolymers compared to chitosan films are given in Table 7.1.

Table7.1. The maximum achieved tensile properties of the copolymer films in

comparison to chitosan films

Tensile strength (MPa) % Elongation Films

Grafting (wt %) Dry Wet Dry Wet

CH-0 0 35+ 11 51+7 15 ± 7 92 ± 7

CH-PMMA 177 - 6.7 ± 2.3 - 43 ± 4

CH-PHEMA 385 34 +8 18 + 3 2 ± 0.2 90 ± 13

CH-PVAc 92 117 + 1 59 + 10 13 ± 0.4 163 ± 8

CH-PVOH 85 82+ 23 47+ 3 23 ± 7 169 ± 12

CH-PEGm 164 45+73 6.5+ 1 9.3 ± 3 45.4 ± 1

The hydrophilicity of the copolymers was checked by the water swelling and

under water contact angle studies. Swelling studies imply that the hydrophilicity is

significantly improved by graft co-polymerisation of HEMA onto chitosan. Though

PMMA is a hydrophobic polymer, the chitosan-g-PMMA with a medium extent of

grafting was found to be more hydrophilic than virgin chitosan. Chitosan is highly

crystalline due to the strong intermolecular hydrogen bonding among the –NH2 and –OH

groups of the adjacent linear chains. Grafting with PMMA, to a certain extent, will

disrupt this strong intermolecular hydrogen bonding and facilitates hydrogen bonding

with water and leads to increased hydrophilicity of the medium-grafted copolymers. As

the PMMA content increases the organic nature of PMMA predominates conferring

hydrophobicity to the copolymer (CH-M10). On grafting the hydrophobic monomer,

vinyl acetate, chitosan is expected to increase hydrophobicity which is confirmed by the

results. In this case also, the moderate PVAc grafting is conducive to enhancing the

swelling characteristics. A dramatic increase in the swellability of chitosan-g-PEGm was

observed at pH 7.4 for the graft copolymer with 320 % grafting. The swelling % of the

PHEMA and PMMA grafted chitosan films at pH 1.98 could not be determined

accurately beyond 10 minutes, as the films started degrading at this stage. This indicated the


234

dissolution tendency of the films, as the swelling exceeded a certain level. But in the case of

chitosan-g-PVAc, the films showed higher stability in the acidic pH. The chitosan-g-PVOH

films also showed increased acid resistance when compared to virgin chitosan films.

0

100

200

300

400

500

600

CH-0 CH-HE12.5 CH-M10 CH-V10B CH-V10A CH-P7.5

Copolymer codes

% S

wel

ling

Fig 7.1. % Swelling of the copolymer films at pH 7.4 (24 h study)

The under-water octane contact angle values also supported the above findings in

all the cases (Fig 7.2).

60

80

100

120

140

160

180

200

CH-0 CH-HE12.5 CH-M10 CH-V10B CH-V10A CH-P7.5

Fig 7.2. Octane contact angle values of the copolymer films


235

Thus, the physico-chemical characterization studies concluded that the

copolymers were of variable mechanical strengths and hydrophobic/hydrophilic

characteristics. A proper choice of the appropriate vinyl monomers and adjusting the

extent of grafting and transformation of the pendant grafts (as in the case of chitosan-g-

PVAc and chitosan-g-PVOH) would be effective in the synthesis of tailor-made, natural-

synthetic hybrids for varied engineering and bio-medical applications.

7.3. Preliminary Biocompatibility Evaluation

The preliminary biocompatibility evaluation was done by studying the blood

material interaction and cytotoxic response of the chitosan copolymers. The blood

material interaction studies included, evaluating the % haemolysis, reduction in cell

counts like, WBC, RBC and platelets as an effect of the material contact with the blood.

The studies were conducted following the standard protocol, ISO 10993-4. The simplest

and the most commonly conducted blood compatibility test is the haemolysis test. Fig 7.3

illustrates the % haemolysis of the blood samples which are exposed to the copolymer

films compared with the reference (empty polystyrene dishes) material. None of the

samples induced haemolysis to the blood samples when compared to the reference.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

CH-0

CH-HE5

CH-HE7.5

CH-HE10

CH-HE12

.5

CH-M4

CH-M10

CH-V4B

CH-V10

B

CH-V4A

CH-V10

A

CH-P5

Refer

ence

% H

aem

oly

sis

Fig 7.3. Percentage haemolysis of the copolymers


236

According to ASTM standards, ASTM F 756-00, a material with a haemolysis

value less than 2% is considered as haemocompatible. Fig 7.3 shows that all the

copolymers have a haemolysis value less than 2% (ranging from 0.25% to 0.55%) and

are suitable for blood contacting applications. As far as the cell count changes of WBC,

RBC and platelets are concerned, none of the parameters showed any significant

reduction as an effect of material contact with the blood.

The cytotoxicity evaluation was done using L929 mouse fibroblast cells. L929 is

an established and well characterized mammalian cell line that has demonstrated

reproducible results. One of the most sensitive toxicity testing protocols is based on the

direct contact of the sample with the cell culture. The test on extract study was also used

for the cytotoxicity evaluation of the microspheres. The test protocols were as per ISO

10993-5. All the copolymers were found to be highly cytocompatible proving that these

materials could be used for any of the biomedical applications.

The viability of the fibroblast cells after incubating with the materials for 48h was

assessed by a dual staining technique using a 1:1 mixture of acridine orange and

ethidium bromide as detailed in chapter 2. The live cells stain green and the dead cells

deep red when observed under the fluorescent microscope.


237

Fig 7.4a. Live-dead assay with L929 mouse fibroblast cells on contact with chitosan (1-3), chitosan-g-poly(HEMA) (4-6), chitosan-g-PEGm (7-9), chitosan-g-PVAc (9-12)

and chitosan-g-PVOH (13-15) with different magnifications (of the objective)

Fig 7.4a is the fluorescent microscopic image of the stained cells after culturing on

chitosan and the copolymer films for 48 h. All the cells on contact with the materials are

green confirming that the materials are cytocompatible to L929 mouse fibroblast cells.

The quantitative determination of the cytotoxicity of the materials was done by

MTT assay. The Fig 7.4b depicts that all the copolymer films are highly cytocompatible

showing 100% metabolically active cells when compared to the controls (cells without

materials). The percentage viability (living cells) values greater than 100% indicate the

cell proliferation. As the chitosan-g-PMMA is used for the preparation of microspheres,


238

the MTT assay was done on the microspheres to assess the cytocompatibility of them

quantitatively.

0

40

80

120

160

200

CH-0 CH-HE7.5 CH-V6B CH-V6A CH-P5 Reference

%V

iab

ility

Fig 7.4b. Cytotoxicity evaluation of chitosan copolymer films by MTT

assay using L929 mouse fibroblast cells

The biodegradability of the films was assessed by analyzing the FTIR spectra of

the films, mass-loss of the films and also from the reduction in the mechanical properties

of the films after exposure to test solutions. All the graft copolymer films were more

stable in the enzymatic environment than the virgin chitosan films. The FTIR spectra of

the films showed signs of degradation as evident by the absence or reduction in the

intensity of certain characteristic peaks. The percentage mass-loss and percentage tensile

strength-loss observed for the different copolymer films are given in Table 7.2. The

chitosan-g-PVAc and chitosan-g-PVOH films underwent the maximum mass-loss, (37

and 25%) compared to other copolymer films. Chitosan grafted with poly(HEMA),

PMMA, and PEGm displayed almost similar range of percentage mass loss, viz; 5%, 3-

5% and 5-13% respectively. The tensile properties of the copolymer films when

examined after keeping in the enzyme solution for one month showed a decrease in the

range of 35 to 93% whereas the tensile strength loss of virgin chitosan film is 91%.


239

Table 7.2. The mass-loss and tensile stress-loss of the films after keeping in enzyme solution

S.No Films Mass- loss (%) Tensile Stress-loss

(%)

1 Chitosan 25 91

2 Chitosan-g-PHEMA 5 85 to 93

3 Chitosan-g-PMMA 5 to 9 54 to 84

4 Chitosan-g-PVAc 3 to 37 56 to 81

5 Chitosan-g PVOH 4 to 25 35 to70

6 Chitosan-g-PEGm 5 to 13 Not done

Hence, it is concluded that the copolymer films, except CH-V4B and CH-V4A

retained their shape and mass in the enzyme solution more than the virgin chitosan films

but the strength of the films underwent deterioration to the same extent or less than

chitosan films.

7.4. Assessment of the Copolymers for Biomedical Applications

7.4.1. Copolymers as Haemodialysis Membranes

7.4.1.1. Background of the Study

Permeability is a measure of the clearance rate of molecules of medium molecular

weight. 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 and

mechanical strength. Since the primary action of the cellulose membrane is that of a

sieve, there is little selectivity in the separation of two closely related molecules. As

explained earlier, the membranes with desirable mechanical strength, e.g. chitosan-g-

PHEMA, chitosan-g-PVAc, chitosan-g-PVOH and chitosan-g-PEGm were explored for

their permeability to small molecular weight solutes like creatinine, urea, glucose and the

high molecular weight solute, albumin. Fig 7.5 to 7.8 show a comparative evaluation of

the maximum amount of the solutes permeated through each copolymer films (of


240

comparable thickness) for 24 h. Within 24 h, all the copolymer films and virgin chitosan

film permeated > 30% of the initial amount of creatinine present in the donor

compartment. The chitosan-g-PVAc films showed 41-82%, chitosan-g-PEGm 37-41%

and the chitosan-g-PVOH films 35-39% permeability. In the case of chitosan-g-PHEMA

films, all the copolymer films exhibited almost same permeability of 31%. The

permeability of commercial cellulose films studied under similar experimental conditions

was found to be 16% while virgin chitosan films had a value of 37% (Fig 7.5). Hence, it

is established that certain chemical modification through grafting enhanced the creatinine

permeability of chitosan. The PVAc grafted system exhibited comparatively better

performance among the series.

0

10

20

30

40

50

60

70

80

90

% C

reat

inin

e P

erm

eate

d

Cellulo

se

Chitosa

n

CH-HE5

CH-HE7.5

CH-HE12

.5

CH-V4B

CH-V6B

CH-V10

B

CH-V4A

CH-V6A

CH-V10

A

CH-P3

CH-P5

Creatinine

Fig 7.5. The percentage of creatinine permeated at a 24 h study

Fig 7.6 high lights the glucose permeability comparison among the copolymers.

All the copolymer films except the CH-HE5 composition showed better permeability to

glucose than virgin chitosan and cellulose films.


241

0

5

10

15

20

25

30

35

40

% G

luco

se P

erm

eate

d

Cellulo

se

Chitosa

n

CH-HE5

CH-HE7.5

CH-HE12

.5

CH-V4B

CH-V6B

CH-V10

B

CH-V4A

CH-V6A

CH-V10

A

CH-P3

CH-P5

Glucose

Fig 7.6. The percentage of glucose permeated at a 24 h study

Fig 7.7 depicts the maximum amount of urea permeated through the copolymer

films for 24 h. The urea permeability through the copolymer films when compared with

chitosan and cellulose films revealed the fact that all except the CH-V6A film are equally

good or better than chitosan and cellulose.

0

5

10

15

20

25

30

35

40

45

50

% U

rea

Per

mea

ted

Cellulo

se

Chitosa

n

CH-HE5

CH-HE7.5

CH-HE12

.5

CH-V4B

CH-V6B

CH-V10

B

CH-V4A

CH-V6A

CH-V10

A

CH-P3

CH-P5

Urea

Fig 7.7. The percentage of urea permeated at a 24 h study


242

Fig 7.8 illustrates the albumin permeability of the chitosan-g-vinyl polymer films.

The chitosan-g-PHEMA films CH-HE7.5 and CH-HE12.5 showed 20% and 7.6%

permeability to albumin. Virgin chitosan and cellulose films had 1.8% and 0.45%

permeability respectively while all other chitosan copolymers exhibited albumin

permeability in the range of 0.01 to 1.6%.

0

2

4

6

8

10

12

14

16

18

20

% A

lbu

min

Per

mea

ted

Cellulo

se

Chitosa

n

CH-HE5

CH-HE7.5

CH-HE12

.5

CH-V4B

CH-V6B

CH-V10

B

CH-V4A

CH-V6A

CH-V10

A

CH-P3

CH-P5

Albumin

Fig 7.8. The percentage of albumin permeated at a 24 h study

It is realised that modification of chitosan through graft copolymerisation is an

excellent technique for the designing of blood compatible, cell friendly, mechanically

strong, degradable membranes with superior permeability to low molecular weight

solutes and little leakage of albumin like nutrients. Albumin is one of the 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 [1]. The albumin permeability of the graft

copolymers are found to be well within the desired range. The slight augmented

permeability was shown by the CH-HE7.5 (20%) and CH-HE12.5 (7.6%) compositions

which amount to be 1.6 g and 0.6 g albumin respectively (the concentration of albumin

in the donor cell is 8g/dl) and the permeability of other compositions are highly


243

acceptable as they range from 0.8 mg to 130 mg which is significantly very less. On

verification of the permeability of the chitosan-g-vinyl polymers it is obvious that the

chitosan-g-PMMA, chitosan-g-PVAc, and chitosan-g-PVOH copolymer films are equal

to or better than the commercial cellulose films for application as haemodialysis

membranes. For the chitosan-g-PEGm films, the permeability for low molecular weight

solutes was comparatively less initially due to the crystalline phases of PEO grafts that

impede their transport. However, this was a blessing in disguise for preventing absolutely

the transport of high molecular weight solute like albumin.

It can be concluded that modification of chitosan through grafting is an excellent

technique for the designing of blood compatible, cell friendly, mechanically strong,

degradable membranes with superior permeability to low molecular weight solutes and

little leakage of albumin like nutrients.

7.4.2. Chitosan-g-PMMA Microspheres for Controlled Release of Ampicillin

Numerous controlled or sustained delivery systems with chitosan and its

derivatives have been described in the literature. Chandy and Sharma reported that the

release of drugs from chitosan beads at pH 7.4 is very slow and attributed it to an effect

of crystal structure formation of ampicillin within chitosan beads [2]. In oral

administrations, the use of chitosan is restricted by its fast dissolution in the stomach and

limited capacity for controlling the release of drugs [3]. When chitosan was used as a

drug carrier, the reactive functional groups on the polymer matrix were bonded through

the functional groups on the drug. The rate of cleavage of the bond linking the drug to

the matrix controlled the drug release and in some cases, the activity of the drug also [4].

We hypothesize that the availability of functional groups of chitosan to which the drugs

bind can be controlled by chemical modification through grafting. Chitosan molecule

contains a primary amino group at C2 and hydroxyl group at C6 positions, and hence is

amenable for a host of chemical reactions under mild conditions. PMMA is one of the

synthetic materials that is currently being used for controlled drug delivery [5-9]. It was

expected that the modification of chitosan by PMMA could control its fast degradation


244

and also to modulate the in vitro release of the encapsulated drug, without sacrificing its

bioactivity. The section 3.2 of chapter 3 details the preparation of chitosan and

chitosan-g-PMMA microspheres, the blood compatibility, cytotoxicity, biodegradability

and the drug absorption and drug release properties and the antibacterial properties of the

drug carrying microspheres.

Microspheres were prepared from aqueous solution of the copolymer using

hexane-paraffin mixture. Particle size was controlled by stirring at 600 rpm at room

temperature. The copolymer microspheres were prepared form a homogeneous blend of

virgin chitosan and chitosan-g-PMMA. The drug (ampicillin) encapsulation, controlled

release of the drug, blood compatibility, cytotoxicity, and biodegradability of the

microspheres were studied in detail. The results promised that the chitosan–g-PMMA are

superior materials for the processing of porous microspheres which serve as excellent

carriers for drug showing controlled release of the incorporated drug for several days

with strong inhibition for both the gram positive and gram negative bacteria in contrast to

virgin chitosan microspheres which showed lesser release and lower anti microbial

properties. Potential applications like oral drug delivery, wound dressings and tissue

engineering are envisaged from these microspheres.

7.5. Conclusions

The objective of the study was to synthesise natural–synthetic hybrid materials

from chitosan and vinyl polymers with tailor made properties depending upon the nature

and concentration of the grafts. The method chosen for the modification was a chemical

route, i.e. graft polymerization of chitosan with the vinyl monomers through a redox

initiation with ceric ammonium nitrate. The objective was achieved through the

copolymerisation of chitosan with the selected vinyl monomers, MMA, HEMA, VAc,

VOH and the methacrylate terminated PEG. The copolymers were found to be strong,

biocompatible and qualified for the proposed end use like haemodialysis membranes and

controlled delivery systems as evident from the data described in preceeding chapters.


245

7.6. References

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

2. Chandy, T., Sharma, C. P., Biomaterials, 1993, 14, 12, 939.

3. Remunan-Lopez, Lorenzo-Lamosa, M.L, Vila-Jato, J.L., Alonso, M.J. Eur. J.

Pharm. Biopharm., 1988 Jan, 45, 1, 49.

4. Jayakrishnan, A., Latha, M.S., Biodegradable polymeric microspheres as drug

carriers, in Controlled and novel drug delivery (Ed.), N.K.Jain, CBS

publishers, India, 1997, 236.

5. Calhoun, J. H., Mader, J.T., Am. J. Surg., 1989, 157, 443.

6. Gerhart, T.N., Roux, R. D., Hanff, P. A, Horowitz, G. L., Renshaw, A. A.,

Hayes, W. C. J., Orthop. Res., 1993, Mar; 11, 2, 250.

7. Sivakumar, M., and Rao, K. P., Biomater. Sci. Polym. Edn., 2002, 13, 2, 111.

8. Del Real, R.P., Padilla, S.,Vallet-Regi, M., J. Biomed. Mater. Res., 2000, 52, 1.

9. Yang, J.M., Su, W.Y., Leu, T.L., Yang, M.C., J. Membr. Sci., 2004, 236, 1-2, 39.

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