INTRODUCTION:

Hydrogels are three dimensional networks of hydrophilic polymers that are known to swell in aqueous solution without dissolving or losing their structural integrity^[1]. Due to unique properties, hydrogels have a wide range of biomedical applications in drug delivery^[2-5], wound dressing^[6-8], contact lens^[9-11], tissue engineering^[12-15], artificial implants^[16], biosensors^[17]and surgical prostheses^[18]. Many synthetic and naturally derived materials have been reported to form well characterized hydrogels. In the present study, polyvinyl alcohol (PVA) has been chosen as one of the components of the hydrogel due to its popularity in the biomedical community^[19-21], non toxicity, non carcinogenicity and biocompatibility. PVA also has been found to demonstrate some desirable physical properties such as high degree of swelling in aqueous media, good film-forming property and elastic nature.

Further hydrogels of PVA are known for their reduced protein adsorption and cell adhesion^[22-24]. Another monomer chosen for the preparation of the hydrogels is acryl amide (AM) and 2-acrylamide-2-methylpropane sulfonic acid (AMPS). Both AM and AMPS have large applications in medical

field as well as other applications^[25]. AMPS have attractive application as wound dressing material^[26,27] since it adheres to healthy skin but not to the wound surface and is easily replaceable without any damage to the healing wound.

The blood compatibility of materials is an important factor in the development of various medical devices. It is well known that the major problem of artificial implants is that blood clot occurs after a certain period of implantation^[28]. During the past decades, many studies have been performed to understand the interaction of platelets with synthetic polymers, hoping to find materials that are blood compatible^[29-36]. It has been shown that polymer surfaces modified by grafted polymers or anti-thrombogenic mediators reduce platelet adhesion^[37-42]. However, blood compatibility of the modified polymers decrease with blood contacting time due to desquamation of the modified materials.

The uniqueness of nanomaterials is that as the size decreases their surface area per unit mass increases. This may partly explain as to why unique properties can be found at the nanoscale that is not observed in the same bulk material. In the field of biomedical applications, as the nanoparticles enter the body and directly come in contact with tissues and cells, thus it is significant to explore their level of biocompatibility. The response of blood in contact with the material depends on various physicochemical features such as surface area, surface charge, hydrophobicity/hydrophilicity, etc^[43,44].

EXPERIMENTAL:

MATERIALS AND METHODS:

Chloroauric acid (HAuCl₄) was purchased from Himedia, India Acrylamido-2-methyl-1-propane sulfonic acid (AMPS) was purchased from Merck, India and its sodium salt (AMPS-Na) was prepared by neutralizing with 1 mole of NaOH solution. Acryl amide (AM) (Merck, India) was purified twice by recrystalling with ethanol. Polyvinyl alcohol (PVA), potassium peroxodisulfate (KPS) were purchased from Merck, India. N,N^I-methylenebisacrylamide (MBA) was obtained from Central drug House (P) Ltd. India.

Preparation of the Pure Composite Hydrogel:

The hydrogels are prepared by free radical polymerization method. The monomers are mixed by dissolving in 5 ml of double distilled water and a homogeneous solution is prepared. To this solution 0.073 mM KPS as a reaction initiator and 0.129 mM MBA as a reaction cross-linker are added and are mixed with continuous stirring. The solution is then transferred into PVC and polymerization is carried out at 60-80⁰C for 15-30 minutes. After complete polymerization the hydrogels are dried at 50⁰C for 7-8 hours.

Preparation of Gold Nanoparticles by Citrate Reduction of HAuCl₄:

100 ml of 1mM HAuCl₄ was boiled with continuous stirring. Then 10 ml of 38.8 mM sodium citrate was added to the vortex of the solution rapidly. The pale yellow color of the solution was found to gradually change into wine red. Boiling was continued for another 10 minutes and stirred for 30 minutes. The colloidal gold Nanoparticle solution is stored at room temperature in a dark bottle and characterized.

Preparation of Gold nanoparticle Composite Hydrogel:

0.25 g of polyvinyl alcohol (PVA) is initially made to dissolve in 5 ml of gold Nanoparticle solution by continuous stirring in a beaker. To this solution 4.68 mM AMPS-Na and 7.03 mM AM are added and a homogeneous solution is prepared. To this solution 0.073 mM KPS as a reaction initiator and 0.129 mM MBA as a cross-linker are added and mixed with continuous stirring. The solution is then transferred into PVC straw and the polymerization is carried out at 60⁰C for 20 minutes. After complete polymerization, the hydrogel is dried at 50⁰C for 8-10 hours and finally purified by allowing it to equilibrate in double distilled water for 10 days.

Characterisation of hydrogel:

IR-spectra of the pure and nanoparticle composite hydrogels were recorded in Shimadzu-8400s Ft-IR spectrophotometer. The morphology of both the hydrogels was investigated by using Scanning electron microscope (SEM) (Carlzeissieo Leo 1430VP). The thermal property of the gel was investigated by thermo gravimetric analysis (TGA) (Perkin Elmer 4000). The optical behavior of Au nanoparticle is studied in UV-visible spectrophotometer Shimadzu 1700.

Swelling study:

The progress of swelling was monitored gravimetrically as described by other workers^[45]. 40 mg of dry hydrogel was immersed in 100 ml of double distilled water. After every 30 minutes interval of time the hydrogel was taken out and excess surface adhered water was removed by blotting and the weights of swollen gels were recorded. The swelling ratio was calculated by following equation.

Weight of swollen gel

Swelling Ratio=------------------------------ ……..(1)

Weight of dry gel

To understand the effect of monomer composition and external environment on the swelling, the swelling experiment is carried out by varying monomer composition, pH and temperature of the swelling medium.

Blood Compatibility Study:

The assessment of biocompatibility has been made on the basis of 3 in vitro tests viz BSA adsorption test, blood clot formation and hemolysis assay.

a)Clot formation test:

The antithrombogenic potential of the blend surface was judged by the blood clot formation test as described elsewhere^[46]. In brief, the PVA blends under investigation were hydrated in 0.9% saline water at 30⁰C in a constant temperature bath. To these swollen gels, 0.2ml of acid citrate dextrose (ACD) blood was added, followed by an addition of 0.02ml of a 4M CaCl₂ solution to start the thrombus formation. The thrombus formed was dried at 35⁰Cfor 48 hours and weighed.

b)Haemolysis assay:

Haemolysis experiments were performed on the surfaces of the prepared blends as reported elsewhere^[47]. The hydrogels were well equilibrated in normal saline water (0.9% w/v) for 48 hrs at 30⁰C and human ACD blood (0.20ml) was added to the surface of the hydrogels. After 20mins, 2ml of a 0.9% NaCl solution was added to each sample to stop the hemolysis and the samples were incubated for 60min at 37⁰C. Positive and negative controls were obtained by the addition of a 0.2ml of human ACD blood and 0.9% NaCl solution, respectively, to 2ml of triple-distilled water. Incubated samples were centrifuged for 45min, the supernatant was taken, and its absorbance was recorded at 545nm. The hemolysis % was calculated as

Hemolysis % = A test sample – A(-)-control/ A(+)-control– A(-)-control (2)

c) Protein adsorption:

To judge the blood compatibility of the prepared hydrogels, blood protein-hydrogel interactions were investigated by the adsorption of BSA onto the hydrogel surfaces with the batch-contact method ^[48]. In a typical experiment, a 20ml BSA solution (0.2% w/v), prepared in phosphate buffered saline (pH 7.4) containing pre-/weighted and pre-swollen hydrogel pieces, was mildly shaken for 30 min to prevent foam formation at the solution-air interface. After being shaken, the supernatant solution was analyzed for the residual BSA concentration by recording its absorbance at 277nm (UV-Vis double beam spectrophotometer). The amount of BSA remaining in the solution was calculated by the construction of a calibration plot, and the amount of adsorbed BSA (mg/g) was calculated with the following equation

Adsorbed BSA (mg/g) = (C_o - C_a) / w × V (3)

Where C_o and C_a are the BSA concentrations (mg/ml) before and after adsorption, w is the weight of the swollen gel (g) and V is the volume of the protein solution.

RESULTS AND DISCUSSION:

FT-IR analysis:

FT-IR spectra of hydrogel and Au nanoparticle composite hydrogels are compared in fig.1. FT-IR spectra of hydrogel (fig.1 a) shows combined spectral properties of different functional groups. The peak at 3631 cm^-1 is due to N-H stretching of AM, Na-AMPS and MBA. The peaks from 2788 to 3271 cm^-1 for C-H (symmetric and asymmetric) stretching confirms the present of AM, Na-AMPS in the network of polymer. The N-H bending and N-C stretching is observed at 1533 cm^-1 and 1218 cm^-1 respectively. The C=O stretching for AM, Na-AMPS and MBA is observed at 1697 cm^-1. The characteristic peak of Na-AMPS units can be seen at 1041 cm^-1 due to SO group. The evidence of cross linker in the hydrogel is confirmed by the peak at 682 cm^-1(secondary amide) for MBA. In case of FT-IR spectra of Au composite hydrogel, all the peaks present in the pure hydrogel are also present in Au composite hydrogel. No characteristic changes in functional group peaks are observed. This indicates that the Au deposition is only physical in nature.

Figure 1: IR spectra of (a) pure hydrogel (b) Au Nanoparticle composite hydrogel

Scanning Electronic Microscope (SEM) of Hydrogel:

The scanning electron micrograph (SEM) of prepared hydrogel is shown in fig.2 (a & b) from the micrograph it is observed that the morphological surface of the hydrogel is heterogeneous in nature having some pores. The porosity nature of hydrogel may be due to grafting of PVA on AM-AMPS chain. Au nanoparticle composite hydrogel(fig.2 b) contains small spherical particles are well dispersed into polymer network.

Thermo Gravimetric Analysis of Hydrogel:

The thermo gravimetric analysis (TGA) thermogram is depicted in fig.3. The Au nanocomposite hydrogels were characterized by thermogravimetrical analysis to determine percentage of weight loss of hydrogel as well as Au nanocomposite hydrogel matrix. Fig.3 shows that both the composite and blank composite hydrogel followed three steps decomposition. 33.8 % degradation of the pure hydrogel chains occurred below 500⁰C. But only 24% weight loss were occurred even above 500⁰C in the case of Au nanocomposite hydrogel. The difference in decomposition between the hydrogel and Au nanocomposite hydrogel confirms the presence of Au nanoparticles in the hydrogel.

Figure 3: TGA of the GNP hydrogel(a) and Pure Hydrogel (b)

UV-visible study:

The existence of Au nanoparticles in the hydrogel networks are investigated by using UV-visible spectra. UV-visible spectra shown in fig4 illustrates that the colloidal Au nanoparticle solution have absorption peak at 529 nm (fig.b) and Au nanocomposite gel (GNP) have distinct peak at 536 nm(Fig.a), which arises from the surface plasmon resonance effect of gold nanoparticles. The absorption band is slightly shifted to higher wave number after encapsulation to the polymer matrix. This indicates the divergence of nano crystals in the polymer matrix. In case of pure hydrogel, no such peak is observed at 536 nm (fig c). This spectral analysis confirmed the formation of Au nanoparticles in the hydrogels.

X-ray Diffraction Study:

The formation of Au nanoporticles is verified by using X-ray diffraction. The X-ray diffraction pattern of hydrogel and Au nanocomposite hydrogel are demonstrated in fig.5. The diffractogram of Au nanocomposites shows a broad diffraction peak (2θ) at 38.362^o with FWHM value 0.768. The broadening of the diffraction peaks indicated the nanocrystalline nature. No such peaks are observed at pure hydrogel due to amorphous nature of hydrogel.

Figure 4: UV-visible spectra of Au nanocomposite hydrogel(a), colloidal Au nanoparticle solution(b) and pure hydrogel(c)

Fig5 XRD of Pure and gold nanoparticle composite hydrogel (GNP)

Swelling study:

Effect of Water:

On comparison of the swelling behavior of the gold nanoparticle-composite hydrogel(GNP) with pure hydrogel, it is found that the equilibrium swelling ratio of GNP hydrogel is lower (equilibrium SR 27.35) than pure hydrogel (equilibrium SR 37.1). This decrease in swelling ratio in the GNP hydrogel is due to the formation of physical interaction between nano-particles and polymer chain. As such, overall cross-link density of the hydrogel increases and forms a compact network structure. This factor prevents the swelling of the GNP hydrogel. However, most importantly its mechanical strength is found to increase. The results are depicted in fig.6.

Effect of pH:

Environmental pH value has large effect on the Swelling ratio especially for the hydrogel composed of ionic networks and containing large pendant groups ^[49] like these hydrogels. The swelling experiment is carried out a pH range of 2-9 by adjusting pH with NaOH and HCl solution. The results in fig 7 indicate that both the hydrogels are sensitive to pH and optimum swelling is observed at around pH 7. The swelling pattern of GNP hydrogel is similar with its pure hydrogel counterpart in terms of different pH. Here also optimum swelling is observed at pH 7 This observation implies that Au NP do not play any role in pH sensitiveness to the prepared hydrogels.

Fig 6: Comparison of the Swelling study

Fig 7: Comparison of the Swelling study in terms of pH

Effect of Temperature:

The effect of temperature of the swelling medium on the water uptake potential of the hydrogel is investigated by allowing the hydrogel to equilibrate at different temperatures ranging from 10^oC to 60^oC. The results are shown in fig.8. The swelling ratio increased with rise in temperature. This observation may be explained by the fact that with increasing temperature, the segmental mobility of the hydrogel chains increase effectively, and consequently, the water sorption capacity of the hydrogel also increase. Optimum swelling is observed at 60^oC. Au NP does not play any role in thermal sensitiveness to the prepared hydrogels.

Fig 8: Comparison of the Swelling study in terms of Temperature

Evaluation of Blood Compatibility:

The selection of a material to be employed as a biomaterial for a specific end use must meet several criteria such as physicochemical properties, function desired, nature of the physiological environment, adverse effects in the case of failure, expected durability and consideration relating to cost and case of production. Whatever the type of materials, the biocompatibility is the foremost requirement for all biomaterials. In the present study, the assessment of biocompatibility has been made on the basis of 3 in vitro tests viz BSA adsorption test, blood clot formation and hemolysis assay as discussed below. In the present study PVA based hydrogel and its Gold nanoparticle composite have been studied for their blood-compatible behavior.

a)Protein (BSA) adsorption test:

The importance of protein adsorption on the hydrogel surface lies in the fact that it determines the mechanism and extent of intrinsic coagulation and adhesion of platelets. The adsorption of proteins onto a polymer surface is a complex process and the extent of adsorption is determined by many factors such as hydrophilicity, hydrophobicity, polar, non polar, charged or uncharged parts of the polymer and protein content.

In the present work, in vitro biocompatibility has been judged by monitoring the amount of BSA protein adsorbed by the blend. The effect of PVA content in the blend has been investigated on the blood compatibility parameters in the concentration range of 0.1-0.25g. Results are shown in table1 which clearly shows that with increasing concentration of PVA in the blends, the amount of adsorbed BSA decreases implying an enhanced blood compatibility of the material. The observed decrease is consistent with the blood clot formation results.

The observed findings in the decrease in the amount of adsorbed protein maybe explained by the fact that as the amount of hydrophilic components in the blend increases, it imparts greater hydrophilicity to the blend which thus makes the blend surface more protein resistant, ensuring blood compatible nature of the blend. Moreover due to the inert and flexible nature of the PVA chains, enhanced blood compatibility has been observed.

b)Blood Clot formation:

One of the main problems of blood contacting materials is thrombus formation on the artificial surface. The response of blood to contact with a material depends on physicochemical features such as surface area, crystallinity and hydrophilicity/hydrophobicity of the surface.

Blood clot data summarized in table1 indicates that on increasing concentrations of AMPS in the blend, the amount of blood clots formed decreases. This obviously implies an antithrombogenic nature of the hydrogel surfaces. The observed decrease in the amount of blood clots formed maybe attributed to the fact that as the components in the blend are hydrophilic in nature, their increased amount in the blend imparts greater hydrophilicity to the blend, which makes the blend surface more blood compatible.

c) Haemolysis test:

The prepared hydrogels were also tested for their haemolytic activity and the results obtained are shown in table1. Results clearly indicate that on increasing AMPS content, the extent of haemolysis decreases. The observed results are attributed to the reason that with increasing concentration of AMPS in the blend, surface composition favourably changes which improves the blood compatible quality of the material. However, it is to be noted from the summarized data that the haemolysis results are not very consistent with those of the blood clot and protein adsorption experiments.

The blood compatibility behavior of the GNP hydrogel is studied similarly to that of the pure hydrogel. The weight of the blood clots formed, protein adsorbed and percentage of hemolysis were found to have comparatively much lower values than that of the pure hydrogel. Results are displayed in table 2. The increase in blood compatibility nature after Au NP encapsulation into the polymer matrix may be due to the physical interaction between Au NP and the polymer network. It is also to be noted that this favorable decrease in percentage of hemolysis, adsorbed protein and low blood clot weights were observed at this particular composition of the gel (GNP) only.

Comparison of the in-vitro Blood compatibility study between the pure and nanocomposite gel:

On comparing the in-vitro blood compatibility property, it has been found that the biological parameters required for judging the biocompatibility of any polymer system has been satisfied by both the pure and the nanocomposite gels. However, the gold nanocomposite gel (GNP) displays much better result in contrast to the pure gel. This may be attributed to interaction between Au NP and the network structure of the gel. The Comparative study is displayed in fig.9.

Table 1: Data showing the weights of Blood Clots formed, Percentages of Hemolysis and Amounts of BSA adsorbed by PVA based Gels of different compositions.

Sample	PVA(g)	AAc(mM)	AMPS(mM)	MBA(mM)	Weight of blood clots formed(g)	Hemolysis (%)	BSA adsorbed(mg/g)
1	0.25	7.03	4.68	0.12	0.036	89.16	17.42
2	0.25	14.06	4.68	0.12	0.031	92.13	16.13
3	0.25	17.6	4.68	0.12	0.030	91.27	11.23
4	0.5	7.03	4.68	0.12	0.032	88.5	22.38
5	0.75	7.03	4.68	0.12	0.025	89.7	16.48
6	1	7.03	4.68	0.12	0.019	81.6	15.05
7	0.25	7.03	3.51	0.12	0.047	93.17	21.22
8	0.25	7.03	7.02	0.12	0.033	88.7	15.29
9	0.25	7.03	9.36	0.12	0.029	85.42	15.11
10	Glass surface				0.039	88.62	32.77
11	Glass surface				0.041	--	27.21

Table 2: Data showing the weights of Blood Clots formed, Percentages of Hemolysis and Amounts of BSA adsorbed by PVA based Gold nanoparticle composite Gels of different compositions.

Sample No	AMPS-Na	AM	PVA	KPS	MBA	Weight of blood clot(gm)	Protein adsorbed	Hemolysis (%)
PG1	4.68mM	3.51mM	0.25gm	0.12mM	0.12mM	0.036	17.32	89.36
*GNP*	*4.68mM*	*7.03mM*	*0.25gm*	*0.12mM*	*0.12mM*	*0.031*	*16.13*	*90.27*
PG2	4.68mM	14.06mM	0.25gm	0.12mM	0.12mM	0.027	13.43	91.53
PG3	4.68mM	21.1mM	0.25gm	0.12mM	0.12mM	0.022	11.23	90.14
PG4	7.02mM	7.03mM	0.25gm	0.12mM	0.12mM	0.026	12.11	86.62
PG5	9.36mM	7.03mM	0.25gm	0.12mM	0.12mM	0.021	10.05	85.14
PG6	11.7mM	7.03mM	0.25gm	0.12mM	0.12mM	0.017	9.43	84.13
PG7	4.68mM	7.03mM	0.1gm	0.12mM	0.12mM	0.023	22.21	86.14

Fig 9: Comparison of the Blood Compatibility study

CONCLUSIONS:

In the present work we have synthesized PVA containing AMPS based hydrogel and its Gold nanoparticle composite hydrogel (GNP) by free radical polymerization and Citrate Reduction method using MBA as a cross linking agent, characterized and studied the swelling and blood compatibility behavior. Both the pure and gold nanocomposite hydrogel are well characterized by XRD, UV-visible spectra, FT-IR, and SEM techniques. The XRD data and UV-visible spectra confirm the formation of gold nanocomposite in the hydrogel. The SEM and XRD data show small spherical shaped Au NP well dispersed within the polymer matrix. The swelling of the prepared hydrogel is sensitive to pH and temperature. In vitro blood compatibility experiments shows that the hydrogel has sustained level of blood compatibility. Comparing GNP hydrogel and pure hydrogel we can conclude that though the swelling of GNP hydrogel decreased but its mechanical strength, percentage of blood compatibility is increased on Au NP encapsulation. The hydrogel might possess further scope of research in the field of drug release and tissue engineering.

ACKNOWLEDGEMENT:

The authors are grateful to the Departments of Chemistry and Bio-Technology, Cotton University for the facilities.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 24.08.2017 Modified on 11.09.2017

Asian J. Research Chem. 2017; 10(6):750-756.

DOI: 10.5958/0974-4150.2017.00127.4