Novel polymeric hydrogel composites: Synthesis, physicochemical, mechanical and biocompatible properties

Poly (vinyl alcohol) (molecular weight of 1,70,000) was gained from Spectrum India, and KC was purchased from TCI Chemicals Pvt Ltd., Japan. Glutaraldehyde was purchased from Spectrochem, Mumbai, India, and Lysozyme enzyme (ex. white egg) and phosphate buffer saline (PBS) from SRL Pvt Ltd. India.

Preparation of PVA solution was obtained by dissolving the PVA in the weight percent ratio of (100, 90, 80, 70, and 60% w/w) in DI water and stirred on a magnetic stirrer at 75 ± 2 °C and attempted to cool in room temperature. Similarly, the solution of KC prepared by dissolving increasing weight percent (10, 20, 30, 40, and 100% w/w) was prepared in distilled water and stirred for 4 h until obtaining a homogenous viscous solution. After PVA solution and KC were combined and mixed well then added crosslink agent glutaraldehyde (0.025%) and kept overnight stirring at room temperature and the total weight of the mixture was maintained to 2 gm. After a complete homogeneous solution of PVA/KC, then subjected to sonication to remove the bubbles, later solution was poured on the petri plates and permitted to evaporate the hydrogel and formed gel films were dried under an oven at 40 °C for 12 h to complete the removal of solvent from petri plates. Films were removed from the petri plates washed with ethanol and PBS (Phosphate Buffer Saline) later kept in a polythene bag and stored in a desiccator. Samples were named PVA90/KC10, PVA80/KC20, PVA70/KC30, PVA60/KC40, and also pure PVA and KC films were prepared for comparative study. Plausible schematic interaction between PVA and KC crosslinked with glutaraldehyde is presented in figure 1.

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The mechanical properties of the film samples were determined by using a universal testing machine (UTM, LLOYD Instrument). Tensile strength (T_S), elongation at break (E_b), and Youngs Modulus (YM) were determined according to ASTM D882-91 (ASTM, 2009). The hydrogel films were stretched to a rate of crosshead speed of 5 mm min⁻¹ at room temperature.

The surface morphology of the hydrogel films was examined by field emission scanning electron microscopy (FESEM) JSM- 6360, JEOL, Germany. Images of hydrogel were collected by using a voltage of 10 kV. Samples were sputter-coated with a gold layer and by using double-sided sticky carbon tapes samples were mounted on metal stubs.

The molecular interaction between components was successfully analyzed with an attenuated total reflection (ATR) method of IR spectrometer FT-IR spectroscopy-attenuated total reflection, Prestige 21, Shimadzu, Japan.

Atomic force microscopy (AFM) images were investigated by software Nanosurf Easyscan 2, Switzerland, and instruments were used to obtain the morphological structure of the hydrogel film. Topographic figures were viewed by contact angle mode.

A thermal investigation of the material was performed under equipment SDT Q600 V20.9 TA instruments. The samples weight about 5–10 mg and warmed up to 700 °C at a heating rate of 10 °C min⁻¹ under N₂ atmosphere (flow rate 100 ml min⁻¹).

WCA measurements were examined to collect information on the nature of hydrophilicity of the polymeric material (samples). The instrument used for water contact is named SEO Phoenix instrument.

The swelling behaviour of the samples was analyzed in phosphate buffer saline (PBS) media at different intervals of time. The weight of wet (W_s, swollen weight) and dry weight (W_d, dried weight) film samples were measured. Then, calculate the index of swelling by using the following equation.

$$\begin{eqnarray*}&&{\rm{Swelling}}\left( \% \right)=\displaystyle \frac{{\rm{Ws}}-{\rm{Wd}}}{\mathrm{Wd}\,}\times 100\end{eqnarray*}$$

A study of enzyme degradation was performed in the PBS/lysozyme enzyme media. A known quantity (W_i) of prepared hydrogel films were immersed in phosphate buffer solution at 37 °C ± 2 (pH 7.4) containing enzyme lysozyme (Muramidase, white hen egg) enzyme (g/ml) in a water bath. Films were removed after a week, later dried, and noted the weight of dried films (W_f). The loss of weight percent calculated using the following formula.

$$\begin{eqnarray*}&&{\rm{Degradation}}\left( \% \right)=\displaystyle \frac{{\rm{Wi}}-{\rm{Wf}}}{\mathrm{Wi}\,}\times 100\end{eqnarray*}$$

Cell viability study was performed using a protocol of MTT (MTT Cell Proliferation Assay Instruction Guide –ATCC, VA, USA) 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) as a substrate. Briefly, human embryonic kidney-293 cells (HEK-293) were cultured in the flask T-25, and then trypsinized and aspirated in a centrifuge tube (5 ml). The cell pellets were obtained by centrifugation at 300 × g, after a number of cells were counted and regulated with Dulbecco's Modified Eagle Medium (DMEM) high glucose (HG) medium, such that using a suspension of 200 μl, which contained 1 × 10⁴ cells approximately. The hydrogel samples were cut and placed into the wells. Each well (96 well microtitre plate) added 200 μl of cell suspension and later incubated at temperature 37 °C and atmosphere 5% CO₂ for 24 h. Then spent medium was aspirated and were added 200 μl of a medium that contained MTT reagent (10%) to each well to produce a final concentration of 0.5 mg ml⁻¹ and incubated at the temperature 37 °C and atmosphere 5% CO₂ for 3 h. After the cultured medium was removed without altered the crystals formed. Then 100 μl of solubilization solution dimethyl sulfoxide (DMSO) was added and gently plate was shaken in a gyratory shaker to solubilize the formazan, then polymeric material (hydrogel film) were removed from the microtitre plate wells and recorded absorbance by using a microplate reader at 570 nm wavelength, finally calculated the percent of cell viability.

The Ts, Eb%, and YM of the hydrogel films were investigated and stress-strain curves are depicted in figure 2, and the results are reported in table 1. The highest tensile strength showed in the film PVA60/KC40 and lowest Ts for PVA80/KC20 up to 21.71 MPa and 9.99 MPa respectively, as compared to pure PVA hydrogel film yielded 6 MPa, but the Eb% for the pure PVA showed highest 124.58% among all tested films. For instance, the modulus of the hydrogel film sample increased as an increase in the content of kappa-carrageenan from 26.18 MPa (pure PVA100) to 551 MPa (PVA60/KC40). In addition, stress-strain curves indicated that an increase in the content of KC increased Ts up to 21 MPa for PVA60/KC40, at the same time Eb% showed descending order (11.93%), this might be attributed to intermolecular interactions between the PVA and KC. The sample PVA80KC20 exhibited lower Ts among PVA/KC blend films may due to variation in the temperature during the evaporation and drying process.

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Pure PVA is soft and flexible and whereas PVA/KC hydrogel film becomes harder with decreased elasticity due to higher crystallinity associated with the KC. The phenomenon revealed that the addition of k-carrageenan made the sample more hard and rigid in the dry state because KC interrelates with PVA to enable stress transfer and hydrogen bonding of KC is the key role associated with self-aggregation that supports the emergence of the rigid network structure. The Eb%, significantly decreased as the KC increased, it indicated the film is less elastic and showed good miscibility, the results of the study have close conformity with FT-IR and SEM. Relatively resemble results were occurred by another group for polysaccharide/PVA hydrogel film in the drug release study [23].

The surface morphological structure of pure and hybrid hydrogel films was depicted in figure 3. The surface morphologies of pure PVA, KC, and PVA/KC blend hydrogel films are significantly different from each other. The pure PVA, KC hydrogel film presented a more or less smooth continuous with uniform and some particles were aggregated on the surface structure of the PVA film, which may be attributed to the phase separation and crystallization [27]. However, PVA/KC hydrogel film showed a rough surface with minute pores present on the surface of film, this might be due to the place of water permeation and the connection points of external stimuli with the hydrophilic group's relation with the KC (SO₃ ⁻ ). SEM micrographs revealed that the surface structure of the hydrogel film became heterogeneous.

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Micrometre sized agglomerations were found most probably due to the dispersion of KC particles in the PVA/KC film. Hydrogel film surface exhibited heterogeneous morphology with a rough surface after mixing with PVA and KC, which indicates the appreciable interaction between PVA and KC. The results have good conformity with other reports showed in the literature for KC mixed with agar [28]. At higher magnification, some scattered bulges and ridges with highly network form were confirmed which are likely to bring about by a few phase separations that may have obtained due to different crosslinking kinetics of KC compared to PVA (Figure PVA90/KC10 and PVA60/KC40). Few researchers noted similar results in the literature [29]. Overall SEM results suggested that the PVA/KC samples were successfully crosslinked by glutaraldehyde which showed the absence of surface holes but observed ridge formation in PVA90/KC10. This elaborated the formation of rough surface and phase segregation with network formation as compared to other proportions of PVA/KC (80:20 and 70:30). We predicted that significant changes were recorded in PVA60/KC40. Results show the formation of some droplet structures due to miscibility and high compatibility between crosslinking agent glutaraldehyde and PVA/KC film. In summary KC in lower concentration was mostly physically entangled but formed a network with glutaraldehyde in a higher concentration of KC [30].

Figure 4 summarizes the FT-IR spectra of hydrogel film PVA, PVA/KC, and crosslinked PVA/KC films. Certainly, the spectrum of the PVA hydrogel film displayed typical hydroxyl and acetate group peaks [31]. Briefly, the peak observed between 3,450 to 3,200 cm⁻¹ is related to the stretching O–H group and stretching band of C–H noticed at 2920 cm⁻¹ and 2880 cm⁻¹. The peak occurred at 1083 cm⁻¹ associated with O–H bending. The peak occurred at 1428 cm⁻¹ and 1378 cm⁻¹ corresponds to bending and wagging of CH₂ vibrations respectively [32, 33] and the peaks 1731 for pure PVA and crosslinked PVA 1735 to 1720 cm⁻¹ corresponded to C=O and C–O by acetate group of PVA [34, 35]. The incorporation of KC induced many changes to the vibrational peaks of the PVA film. The hydroxyl groups present in KC chains may cause a shift in the peak value and possible crosslink (GA) has also influenced PVA/KC film. The FTIR spectrum of the film PVA/KC crosslinked GA is shown in figure 4. It is observed that tested film samples of PVA/KC exhibited an intense absorption peak at 1254 cm⁻¹, related to S–O of sulphate esters from KC. Similarly, the occurrence of peaks at 844 cm⁻¹ is corresponding to galactose-4-sulphate. However, the presence of a band at 1070 cm⁻¹ belongs to the presence of glycoside linkage (C–O–C (3, 6anhydro-D-Galactose) [36, 37]. The sharp peak observed at 2921 cm⁻¹ is associated with C–H alkyl stretching and −OH stretching appears at between 3319–3397 cm⁻¹. However, −OH stretching band (3600–3674 cm⁻¹) disappeared in the spectra of PVA/KC film. We believed that non-bonded −OH from PVA might have involved in the crosslinking reaction. Further, it is also noted that there is no increase in the C=O band in the vicinity of 1715 cm⁻¹, it is assumed that the aldehyde groups of GA are may consumed in crosslinking reaction with −OH groups from PVA and KC.

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The AFM hydrogel film images of different ratios were shown in figure 5. The PVA/KC films with composition 60/40 showed a decrease in roughness than pure PVA. The presence of a higher concentration of PVA showed an increase in the surface roughness (PVA90/KC10). It suggests that KC is miscible well at a molecular range when present in the high concentration with lower PVA concentration (PVA60/KC40) exhibited small micrometric ridges with a uniform surface. At a higher concentration of PVA (PVA90/KC10), dispersion and aggregation of PVA were observed in the film matrix which related to the rough surface and phase separation, the obtained results are consistent with the SEM.

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TGA curves of PVA, KC pure, and PVA/KC blend are depicted in figure 6. It is clear from the thermal analysis that the weight of hydrogel films was decreased as the temperature increased. The significant differences were recorded in the thermal degradation of pure polyvinyl alcohol (A) and kappa-carrageenan (F) when compared to PVA/KC (B, C, D, and E). The thermogram of PVA exhibited a three-step degradation process. The peak occurred around 270 °C in PVA this might be associated with the breakage of cross-links during the first phase of decomposition. Similar results were also reported by other researchers [38]. The major degradation process in the second phase occurred from 250 °C to 450 °C, but phase degradation gradually decreased in blend films compared to PVA. In all blend films the second degradation process phase occurred at 240 °C–430 °C, 230 °C−410 °C, 250 °C–400 °C and 220 °C–240 °C for PVA/KC (B, C, D, and E respectively). In PVA a third degradation step occurred at 470 °C−550 °C, which is attributed to the by-products generated by poly (vinyl alcohol) during the thermal degradation process [39]. In contrast, third thermal degradation occurred at 530 °C, 510 °C, 505 °C and 500 °C for PVA/KC (B, C, D, and E respectively) due to a residue of the film and also ash content in all blend film. It is slightly higher when compared to PVA and this could be possibly due to KC which consists of SO₃ ⁻ group which was left as ash. The overall result suggests that increased concentration of KC with respect to a decreased ratio of PVA in blend leads to decreased stability of the hydrogel film (PVA/KC). We believed that the PVA concentration has a significant influence on the stability of matrix.

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The water contact angle of the PVA pure and hydrogel film (PVA/KC) are illustrated in figure 7. The hydrogel film (PVA90/KC10) showed the highest contact angle of 83° which indicated that affinity towards water increased to a small extent with the increased concentration of KC (PVA60/KC40, 71°). This suggests that hydrophilicity increased with an increase in the concentration of KC and decrease in the concentration PVA as compare to pure PVA (showed 85°). Similar results were reported by Fanrong et al [24]. The lowest contact angle was observed for the PVA60/KC40 film which implies that the KC significantly improved the wettability of the sample surface.

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Fluid absorption behaviour studies are of significance for the preliminary analysis of biodegradable materials. For fluid uptake quantification, all the specimens of the PVA/KC hydrogel films with different weight ratios (PVA100/KC00, PVA90KC10, PVA80KC20, PVA70/KC30, PVA60/KC40, and PVA00/KC100) were prepared as described in the previous section. Swelling behaviour experiment conducted with pure PVA, KC, and cross-linked PVA/KC with GA. A typical swelling behaviour is shown in figures 8(a) and (b). The swelling phenomenon happened to each cured film after dipping in PBS at different intervals of time (1, 2, 4, & 8 h) at room temperature. Briefly, the observed results indicated that initial rapid mass uptake was observed approximately at 1 h, followed by mass stabilization over a 3 h period. Visual examination of the samples also showed an appreciable increase in volume. The pure PVA and KC exhibited higher water uptake with 307 and 750% respectively, as illustrated in figure 8(a) (a) and (f), and in the cross-linked hydrogel film the mixture of PVA and KC reduced the swelling rate. Perhaps, the highest swelling rate exhibited for the film PVA60KC40 (286%) and lowest for PVA80KC20 (196%). Overall results indicated that hydrogel film (B, C, D, and E) maintained their water uptake behaviour up to 2 h as compared to PVA and KC pure are initially increased the water uptake up to 1 h. Later on, decreased due to higher mobility of interconnected molecules and finally, the swelling ratio percent of the polymeric material was measured using the following formula [40].

$$\begin{eqnarray*}&&{\rm{Swelling}}\,{\rm{percentage}}=\displaystyle \frac{Ws-Wd}{Wd\,}\times 100\end{eqnarray*}$$

Where W_d and W_s represent the weight of hydrogel film i.e. dried and water uptake before and after immersed in PBS media respectively.

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The bio-degradation study was conducted in PBS media with lysozyme enzyme. The dried hydrogel films were cut and the initial weight was recorded. Then films were placed in lysozyme/PBS (g/ml) and incubated at 37 ± 1 °C in a water bath. The wet films were dried and weighed after two weeks (14 days). The resulted data obtained from the degradation study were reported in figure 9. The lowest degradation rate of the PVA/KC film was 6% for PVA90KC20 and the highest 22% for PVA60KC40 after 14 days, this degradation was less than pure components PVA and KC. The overall result of the study indicated that KC concentration in the film was more affected on the mobility of molecules leads to easy degradation of film. The degradation percentage was measured using the following equation [40].

$$\begin{eqnarray*}&&Degradation\,percentage=\displaystyle \frac{Wi-Wf}{Wi\,}\times 100\end{eqnarray*}$$

Where W_i and W_f represent the initial and final weight of film, respectively.

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Biocompatibility or viability study was analyzed with MTT after 24 h cells seed by incorporating HEK-293 and NIH3T3 fibroblast cell line. Figures 10 and 11 show the cell viability (%) and cell attachment of optical images, these samples showed comparable acceptable biocompatibility. Finally, cells seeded on a culture plate contain without sample as a control. Results from this study showed that cell viability percent varying from PVA90KC10 (90.1%) to PVA60KC40 (70%) (Compared to control as 100%) for HEK-293 and NIH3T3 about 86% (PVA90KC10) to 98% (PVA60KC40), in this comparison NIH3T3 showed more compatible than HEK-293. For further study extended to the proliferation of cells on hydrogel film by using NIH3T3 from 24 to 72 h, the study revealed that cell growth was increased about 92 and 99% for PVA90KC10 and PVA60KC40 respectively, this indicated that cells are having viable tendency over the matrices. It is believed that a large number of cell attachments over the matrices due to micrometric ridges and rough surface structure of the film and also because of more number of chemical cues to the cell for attachment and growth over the hydrogel film. Results were related to the growth of cells on a polymeric material that starts with the attachment of cells on a template, layout, proliferates, and differentiate [41]. An overall study result suggested NIH3T3 fibroblast cells are more biocompatible than HEK-293 cells.

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