Graphene Based Composite Hydrogel for Biomedical Applications

In this work, composite hydrogel consisting of poly(vinyl alcohol), graphene and silver nanoparticles, (Ag/PVA/Gr), was prepared by the immobilization of silver nanoparticles (AgNPs) in PVA/Gr hydrogel matrix in two steps. The first step was cross linking of the PVA/Gr colloid solution by the freezing/thawing method, while in the second step, in situ electrochemical method was used to incorporate AgNPs inside the PVA/Gr hydrogel matrix. We used UV–vis spectroscopy, cyclic voltammetry, Raman spectroscopy, DSC analysis, as well as test of cytotoxicity and antibacterial activity, to characterize obtained Ag/PVA/Gr hydrogel. It was shown that graphene-based composite hydrogel with incorporated AgNPs is non toxic biomaterial with antibacterial activity, with a potential for use in biomedical purposes.


INTRODUCTION
[3][4] Polymers, synthetic or natural, are prevalent foundation of such materials, because of their similarities to real tissue, capability to take on many shapes and forms, and almost immeasurable possibilities to tailor their properties to the unique requirements of target use.Hydrogels based on synthetic and natural polymers or their blends, interpenetrating networks and copolymers, are widely used and researched for biomedical purposes, because of their biocompatibility, hydrophilicity and malleability, which allow relatively easy processing and wide range of applications.Among others, poly(vinyl alcohol) (PVA) is a biocompatible synthetic polymer that has been widely used for preparation of biomedical composite materials, such as wound dressings [5,6] and scaffolds for tissue engineering. [7,8]PVA hydrogels are hydrophilic, containing many hydroxyl groups, which allow them to readily swell in aqueous solutions, thus enabling the regulation of the moisture of the wound surroundings.Cross linking of PVA can be done by simple freezingthawing method, [9,10] eliminating the need to use toxic cross linkers which could be difficult to remove from the hydrogel.Additionally, PVA is a known stabilizer often used to synthesize and load metal nanoparticles, which bind to -OH groups on the PVA chains and their surface energy is lowered via interactions with active sites on polymer chains. [11]Silver nanoparticles (AgNPs) are particularly interesting for use in medical applications, because of their unique and efficient antimicrobial activity against many microorganisms, including bacteria, viruses and eukaryota. [12,13]Graphene (Gr) is a relatively new material that has seen applications in many branches of materials science, from electronic and thermoregulatory devices, [14][15][16] to biomedical applications. [17,18]As a 2D carbon-atom monolayer, Gr has high mechanical strength and excellent thermal and electrical conductivity, [19,20] which makes it strong candidate for improvement of many properties of compo-I site materials.Indeed, graphene has been reported to improve many physico-mechanical properties of polymer composites, e.g.tensile strength, thermal stability and conductivity. [21,22]By incorporating graphene into the PVA polymer matrix, we attempted to improve specific properties of Ag/PVA/Gr nanocomposite hydrogels with respect to their topical application.The wound dressings for severe wounds are aimed for prolonged use, over the course of several weeks, during which the dressing is supposed to retain most of its mechanical strength, elasticity, exudate absorption ability and antibacterial activity.Graphene, as a nanofiller in PVA hydrogels, has been shown to improve the overall mechanical strength and elasticity of the polymer matrix. [23]This was also confirmed for PVA/Gr and Ag/PVA/Gr nanocomposites in our previous work. [21,24,25]dditionally, several studies indicated that graphene possesses a certain degree of antibacterial activity, with the ability to induce membrane rupture and oxidative stress of bacterial cells. [26,27]This could be an important property, especially in the initial period of Ag/PVA/Gr wound dressing application, when Gr presence could enhance the antibacterial properties of AgNPs and help prevent wound infection and biofilm formation.
The aim of this work was to produce nanocomposite graphene-based biomaterial with incorporated silver nanoparticles using in situ electrochemical method, aimed for soft tissue implants, wound dressings and drug delivery.The electrochemical route of nanoparticles synthesis is especially attractive for biomedical applications due to high purity and precise size control of metal particles and the absence of chemical cross linking agents and undesired products.

Electrochemical Synthesis of Composite Hydrogel
Electrochemical synthesis of composite Ag/PVA/Gr hydrogel was performed according to the procedure we published elsewhere. [24]In brief, an aqueous solution of the PVA (10 wt%) was prepared by dissolving the PVA powder in hot dH2O (90 °C).PVA/Gr dispersion was prepared by adding graphene under vigorous stirring, to obtain a final concentration of 10 wt% PVA and 0.01 wt% Gr.After being cooled, the PVA/Gr colloid dispersion was subjected to successive freezing and thawing for five cycles, each of which consisted of freezing at -18 °C for 16 h, and thawing at 4°C for 8 h.The obtained hydrogel was cut into small discs (diameter d  10 mm; thickness δ  5 mm) and subjected to swelling for 48 h in a precursor solution containing different concentrations of AgNO3 (0.25, 0.5 or 3.9 mM) and 0.1 M KNO3 at 25°C.In situ electrochemical reduction of Ag + at a constant voltage (90 V) was carried out in swollen PVA/Gr hydrogel, to obtain Ag/PVA/Gr nanocomposite with incorporated AgNPs.The hydrogel discs were placed between the two Pt electrodes in the special glass holder.The synthesis was carried out for 4 min, using DC power source MA 8903 Electrophoresis Power Supply (Iskra d.d., Ljubljana, Slovenia).were prepared according to the protocol we published elsewhere. [24]In brief, the experimental procedure involved seeding PBMC suspension in a 24-well plate in nutrient medium.Control was cell suspension without biomaterial samples.PVA/Gr and Ag/PVA/Gr hydrogel samples (2 g) were placed into the 24-well plates, and either PBMC suspension or blank nutrient was added.Tetrazolium dye MTT test was employed for determination of target cell survival.The number of viable cells was assessed based on the change of optical absorbance of MTT treated cells, due to dehydrogenase activity of their mitochondria. [28]The percent of cell survival (S) was DOI: 10.5562/cca3133 Croat.Chem.Acta 2017, 90 (2)   calculated using the following equation:

Methods of characterization
where Au is the absorbance of PBMCs in the presence of Ag/PVA/Gr hydrogel and Ac is the absorbance of control PBMCs.Cytotoxicity of the samples was described according to the following scale, based on the cell viability relative to control: > 90 % survival -non-cytotoxic, 60-90 % survivalslightly cytotoxic, 30-59 % survival -moderately cytotoxic, ≤ 30 % survival -severely cytotoxic. [28,29]ntibacterial activity.Ag/PVA/Gr hydrogel, obtained from 0.25 mM AgNO3 swelling solution, was tested against Gram-positive Staphylococcus aureus TL (culture collection-FTM, University of Belgrade, Serbia) and Gramnegative Escherichia coli (ATTCC 25922) bacteria strains.The evaluation of antibacterial activity was carried out using test in suspension, via the spread-plate protocol we published elsewhere. [24]The number of viable colonies (CFU mL −1 ) of S. aureus and E. coli was determined using a colony counter at the start of the experiment and after incubation at 37 °C for up to 24 h.

Preparation of PVA/Gr hydrogels
Prior to electrochemical synthesis of AgNPs, the PVA/Gr hydrogels were prepared from the colloid dispersions using a simple cyclic freezing-thawing method.It has been long known that PVA colloids, frozen and subjected to subsequent cycles of freezing and thawing, result in the formation of physical, thermo-reversible hydrogels. [30,31]The properties of the obtained hydrogels, such as swelling behavior, elasticity and mechanical strength, depend strongly on the initial colloid concentration, as well as the freezing and thawing temperature, duration and number of cycles. [30]In our work, the freezing parts of the cycles were performed for 16 h at −18 °C, and the thawing temperature was 4 °C in order to allow for a slower thawing process, resulting in a more ordered structure, as opposed to shock-melting at room temperature.[33][34] LS phase separation is believed to occur via diffusion of water and formation of ice crystals during the freezing parts of the cycle, leaving out PVA macromolecules as impurities from the formed ice.After each cycle, the water ice phase and PVA phase become purer and are separated, whereas during thawing process the pores are formed at the sites of ice crystals. [31,34]Finally, LL phase separation is explained by the spinoidal decomposition process during the gelation, leaving polymer-poor and polymer-rich regions, where afterwards gel formation takes place via hydrogen bonding. [32]Most probably the formation of PVA/Gr hydrogels is achieved through the combination of aforementioned processes, and well-dispersed graphene remains entrapped in the formed PVA cross linked network.The advantages of the described technique for preparation of PVA hydrogels, especially for biomedical applications, include ability to avoid toxic aldehyde cross linkers, higher mechanical strength than the gels obtained by irradiation cross linking, good elasticity and high swelling degree. [35]

Electrochemical synthesis
Electrochemical synthesis of AgNPs inside PVA/Gr hydrogel matrix is founded on in situ electrochemical reduction of Ag + precursor ions in hydrogel disc swollen in 3.9 mM AgNO3 solution prior to synthesis.The reduction was carried at 90 V, during 4 min.The appearance of a dark brown color of Ag/PVA/Gr disc (Figure 1a) is evidence of incorporation of AgNPs within PVA/Gr hydrogel.The effect of polymer matrix on stabilization of nanoparticles and hindering the undesired deposition of a silver layer at the cathode surface has been explained elsewhere. [24,36]Namely, -OH groups of PVA chain interact with Ag + , allowing for the formation of Agm m+ -PVA complex, which undergoes electrochemical reduction at the cathode surface.Resulting Agm 0 -PVA complex is then stabilized by PVA chains.Figure 1b depicts FE-SEM microphotograph of Ag/ PVA/Gr hydrogel.The AgNPs are well dispersed inside the polymer matrix of the hydrogel, adopting spherical morphology with dimensions in the nanometer range (10−30 nm).Therefore, the FE-SEM analysis confirmed incorporation of silver nanoparticles in Ag/PVA/Gr nanocomposite hydrogels.

Cyclic Voltammetry
Cyclic voltammetry was employed in order to investigate the redox processes of the Ag/PVA/Gr hydrogels on the surface of Pt electrodes.Figure 2 represents stationary voltammograms (CVs which are acquired after several cycles and remain unchanged in subsequent cycles) of Ag/PVA/Gr hydrogels obtained from AgNO3 swelling solutions with two concentrations of Ag + (0.25 mM and 0.5 mM).In the anodic sweep, both CVs contain one prominent peak, at 345 mV for Ag/PVA/Gr from 0.25 mM AgNO3, and at 405 mV for Ag/PVA/Gr from 0.5 mM AgNO3.These peaks can be ascribed to the oxidation of silver nanoparticles from the hydrogel on the surface of Pt electrode. [25,41]Since the anodic current density of a peak in cyclic voltammograms is proportional to the concentration of the reactive species, it is evident from Figure 2 that Ag/PVA/Gr obtained from 0.5 mM AgNO3 swelling solution contains nearly six times higher amount of AgNPs than Ag/PVA/Gr from 0.25 mM AgNO3, indicating more successful synthesis of Ag/PVA/Gr from 0.5 mM AgNO3.Another anodic process occurs at potentials more positive than 800 mV, seen as a shoulder with high current densities on the CVs.This potential region covers oxygen evolution reaction and formation of surface oxides on the Pt electrode, [42,43] but here is also possible formation of higher oxides of silver.The cathodic sweep contains a broad peak at 430 mV for Ag/PVA/Gr from 0.25 mM AgNO3, and at 395 mV for Ag/PVA/Gr from 0.5 mM AgNO3, which is ascribed to the reduction reaction of Pt and Ag oxides, formed in the anodic sweep.Figure 3 depicts five cyclic voltammetric sweeps of Ag/PVA/Gr hydrogels obtained from 0.25 mM (Figure 3a) and 0.5 mM (Figure 3b) AgNO3 swelling solutions.Voltammograms of both hydrogels quickly reach steady state after several cycles.During cycles 1-5, a continuous decrease of the anodic current density can be observed for the peak of oxidation of AgNPs at 320-340 mV for Ag/PVA/Gr from 0.25 mM AgNO3, and at 370-390 mV for Ag/PVA/Gr from 0.25 mM AgNO3, followed by an increase in current density of oxygen evolution peak at potentials higher than 700 mV for both hydrogels.The major differences are between the first and subsequent cycles, indicating that polymer/Pt interface is quickly depleted of AgNPs and there is a much smaller amount available for oxidation in the subsequent scans.In the cathodic region for both hydrogels, cycle 1 contains peaks at ~ 50 mV, which probably involve reduction of residual Ag + ions, remaining unreduced in the hydrogel after the synthesis.The disappearance of this peak in cycles 2−5 hints that almost all Ag + ions are reduced in the first cycle of the CV sweep.

DSC Measurements
The DSC thermograms of both PVA/Gr (as reference) and Ag/PVA/Gr hydrogels exhibit endothermic changes in the range of 20-1000 °C (Figure 4).The first endothermic peak, at 119 °C for PVA/Gr and at 120 °C for Ag/PVA/Gr, is related to the evaporation and loss of water from the hydrogel network upon heating the samples.The high temperature of this event (~ 120 °C) indicates that water molecules are strongly hydrogen bonded to the hydroxyl groups on the PVA chain, as well as to oxidized groups in graphene structure.This is bound water with highly oriented and ordered dipoles that usually evaporates at higher temperatures. [44]The area of this peak does not change significantly upon addition of Ag to PVA/Gr hydrogel, which implies that AgNPs have small influence on the binding and orientation of H2O molecules in the polymer matrix.The loss of free water at temperatures lower than 100 °C did not result in any significant peaks on both DSC curves, so we conclude that the main state of water in hydrogels is bound H2O, due to the presence of a large number of oxygen-containing groups in PVA macromolecules.
The second endothermic peak on DSC curves of PVA/Gr and Ag/PVA/Gr is located at 287 °C and 311 °C, respectively, and is related to the melting of the polymer.[47] Tm of PVA/Gr hydrogel is shifted to significantly higher temperatures (287 °C) with respect to PVA, indicating increased thermal stability due to incorporated graphene.Graphene and graphene-oxide nanofillers are known to improve thermal properties of polymer materials, often owing to the incorporation inside the hydrogel structure, as well as interactions with polymer chains that lead to lowered mobility and higher stability of the polymer matrix.It is well known that graphene always possesses a certain amount of oxygen-containing groups, present as impurities or residues from the manufacturing process, which allow it to be dispersed in aqueous media and form bonds with various materials.The interactions with PVA are achieved via hydrogen bonding between these oxygenated groups of graphene, and -OH groups of PVA.Several studies have shown the influence of graphene fillers on thermal stability and thermal degradation of polymeric networks, such as poly(methyl methacrylate) (PMMA), [48,49] PVA films [23] and PVA/starch blends. [50,51]urthermore, melting of Ag/PVA/Gr hydrogel occurs at even higher Tm = 311 °C, exhibiting much better thermal stability due to presence of AgNPs together with graphene.This corroborates the assumption that AgNPs form complexes mainly with -OH groups of PVA macromolecule, which has a strong influence on both thermal stability and crystallinity of PVA polymer.
Finally, at temperatures higher than 400 °C, the DSC curves exhibit stable endothermic regions with small change over the analyzed temperature range.This region of the curve probably covers mostly slow thermal oxidation of graphene sheets incorporated in the hydrogels, as also seen by small-area endothermic peak on the DSC curve of PVA/Gr at around 825 °C.

Biological Properties
Although more silver nanoparticles (AgNPs) are synthesized in the hydrogel swollen in 0.5 mM AgNO3, we chose the lowest concentration of silver (0.25 mM) for biological evaluation, in order to prove that our nanocomposite Ag/PVA/Gr hydrogels possess antibacterial properties even with lowest AgNPs content.Moreover, in our previous research, [24,25] we found that hydrogels synthesized from