Utilization of Freeze Thaw Process for Sodium Alginate and Polyvinyl Alcohol-Based Hydrogel Composite

Sodium alginate and poly vinyl alcohol-based hydrogel was successfully prepared by freeze thaw process. Without crosslinking agent, hydrogel was formed by using 3, 5 and 7 consecutive cycles of freeze thaw. Hydrogel can be formed by hydrogen bonding formation between OH-group of sodium alginate and poly vinyl alcohol throughout network. Scanning electron microscope reported that microstructure of hydrogel was well packed. The interconnect porosity was also observed. Differential scanning calorimetry exhibited that two exothermic peaks at temperature of 110oC and 225oC were observed due to evaporation of water molecule and melting temperature of hydrogel, respectively. The swelling behavior was rapidly increased within initial stage and then it was dimensionally stable. With low sodium alginate content, tensile strength was slightly superior, whereas low in vitro degradation behavior was observed. It was remarkable to note that sodium alginate and poly vinyl alcohol-based hydrogel exhibited outstanding properties for being as a medical material.


Introduction
With the exponential growth of worldwide population, numerous technologies have been extremely designed for various uses. It was typically related to infrastructure, automotive, food packaging, medical material as well as electronic device. Up to the present time, the use of petrochemical based polymeric material was therefore increased for various sectors of industry. Although it provided signi cant advantages in terms of e ciency, it was still limited in particular case due to degradability and biocompatibility. With the utilization of petrochemical based polymer, it can create the disaster for ecological system due to di culty of degradation. Furthermore, the use of petrochemical based polymeric material will be shortage based on the availability of crude oil in the near future. With this regard, numerous bio-based polymeric materials were designed to replace. One effective way to solve this issue was typically related to utilization of eco-friendly material. It provided various advantages such as biodegradability, non-toxicity as well as reduction of environmental pollution. Up to the present time, numerous types of eco-friendly material were developed for various industrial sectors. Among these, medical materials were considered as one of the most important tasks. It can be employed in numerous sectors such as drug delivery system, medical implant, chemical sensor for medical diagnostic and tissue engineering [1]. Recently, the design of eco-friendly materials for medical device was therefore favorite. Page 3/18 One of the most frequent used medical materials was typically related to hydrogel. From the fundamental point of view, hydrogel was de ned as a crosslinked polymer chains swollen in water. It was important to note that hydrogel provided many advantages such as high degree of exibility and moldability. It was biodegradable, biocompatible and injectable. It therefore illustrated the outstanding potential in tissue engineering and drug delivery [2,3]. At the present time, it was notable that hydrogel can be synthesized from both synthetic and natural polymer, which was commonly referred to eco-friendly material. From the fundamental point of view, it was theoretically de ned as non-harmful material, whether in its production, the use and dispose were easily recycled. Furthermore, the use of eco-friendly material was strongly encouraged by "Green policy" [4,5]. It was typically referred to the use of product and process with hazardous chemical reagents should be preferably avoided.
Up to the present time, the occurrence of eco-friendly material was versatile. It can be both synthesized from laboratory and existed from naturally occurring resource. The example of eco-friendly material was typically related to polyvinyl alcohol, polycaprolactone, polyethylene oxide, as well as polyacrylic acid.
One of the most attractive eco-friendly materials was referred to polyvinyl alcohol. It was structurally considered as a linear hydrophilic synthetic polymer, containing pendent hydroxyl group [6]. It illustrated numerous advantages such as non-toxicity, high biocompatibility, high biodegradability as well as high hydrophilicity. It was commonly employed in numerous sectors of industry such as pharmaceutical and cosmetic products, medical technology and wastewater remediation [7][8][9].
To use polyvinyl alcohol with higher e ciency, the design of binary blend-based hydrogel was therefore developed. One of the most important materials for hydrogel development was focused on sodium alginate. From the fundamental point of view, sodium alginate was structurally noted as a polysaccharide found in brown algae. It was hydrophilic and formed a viscous gum when hydrated.
Utilization of sodium alginate was commonly employed in many foods and biomedical application, due to its low toxicity, biocompatibility and low cost. Therefore, to ameliorate performance of polyvinyl alcohol by addition of sodium alginate was favorable. Recently, in 2018, Jiang et al [10] developed polyvinyl alcohol and sodium alginate hydrogel. It can be successfully prepared by freezing/thawing process and socked in NaCl aqueous solution. It was notable that the chain entanglement between polyvinyl alcohol and sodium alginate chain was increased because of the salting-out effect.
Furthermore, hydrogel exhibited tensile strength of 1.32MPa, elongation at break of 400% and electrical conductivity of 3.62S/m. In 2019, Kong et al [11] developed 5-hydroxymethylfurfural-embedded polyvinyl alcohol and sodium alginate-based hydrogels for wound healing application. The presence of 5hydroxymethylfurfural can be effectively provided the excellent antioxidant properties. It can subsequently induce for cell proliferation in wound healing process. The active molecule can be migrated into human skin broblast. Therefore, release mechanism of 5-hydroxymethylfurfural from polyvinyl alcohol and sodium alginate ameliorated the in ammation and enhanced vascularization, extracellular matrix remodeling and re-epithelialization. In 2021, Bialik-Was et al. [12] developed aloe vera loaded into sodium alginate and polyvinyl alcohol-based hydrogel for wound dressing application. It was notable that the presence of aloe vera can signi cantly improve the thermal stability of hydrogel. The release mechanism was gradually complete within a week. The position of aloe vera in hydrogel network can therefore provide the active substance delivery properties of hydrogel lm. Optimal adhesion and cell cytotoxic test were therefore evaluated. This hydrogel can promote cells spreading and proliferation.
Up to the present time, to develop hydrogel for medical material, utilization of freeze thaw technique was employed. This technique was considered as one of the most attractive strategies for hydrogel formation. Without the crosslinking agent, hydrogel was successfully synthesized [13,14]. Florez-Castillo et al [15] also reported that polyvinyl alcohol based hydrogel prepared by freeze thaw technique presented high elasticity and stability. Because the process was typically involved the freezing conditions such as temperature, number of cycle and polymer ratio. It consequently resulted in properties and application of hydrogel. Due to the use of hydrogel related to medical research area, the use of process without crosslinking agent may be appreciated.
Therefore, the objective of this work is to develop polyvinyl alcohol and sodium alginate-based hydrogel composite by freeze thaw process. Characterization by FTIR, SEM and DSC was conducted. Swelling behavior, degradability and mechanical properties were therefore determined.

Chemical reagents
Polyvinyl alcohol (molecular weight 89,000-98,000, 99+% hydrolyzed) was from purchased from Sigma Aldrich Co., Ltd. Sodium alginate was purchased from Sigma Aldrich Co., Ltd. All chemical reagents were used as received, without further puri cation.

Development of sodium alginate and poly vinyl alcohol-based hydrogel composite
Polyvinyl alcohol and sodium alginate-based hydrogel composite by casted onto petri dish. The procedure was conducted based on guideline of previous work reported by Kim et al [16]. Brie y, polyvinyl alcohol (10%w/v) was dissolved in deionized water and gently stirred for 1 h at 80°C. In parallel, sodium alginate (2%w/v) was dissolved in deionized water and gently stirred for 1 h at room temperature. To prepare hydrogel, polyvinyl alcohol solution was poured into sodium alginate solution with a mixed ratio of 5:5, 6:4 and 7:3 v/v. The mixture was casted onto a petri dish, followed by freezing at -20°C for 18 h ang thawing at room temperature for 6 h, for 3, 5 and 7 consecutive cycles. After that the composite hydrogel was freeze dried and stored in desiccator to prevent moisture adsorption.

Swelling behavior
The swelling characteristics of the samples were investigated using a gravimetric technique. Sample was cut into 1cm x 1cm squares with the same weight (W d ) and immersed in deionized water (DI) water for 6 h. Samples were removed from the solution, dried using a lter paper to remove excess water and weighed (W w ). Three samples were investigated following equation, and data were reported as statistical average and standard deviation.
where W w is the weight of the swollen hydrogel at submersion time and W d is the initial weight of the dry hydrogel.

Degradation behavior
The sample was cut into 1 x 1 cm 2 . Then, it was immersed into 10 mL of deionized water at the time intervals of 1, 3, 7, 14, 21 and 28 days. Sample were removed and dried at 80°C at each time point. The percentage degradation was calculated using following equation. The data was reported as a statistical average and standard deviation.
where W 0 and W t are the weights of the sample before and after degradation, respectively.

Fourier transform infrared (FTIR)
The chemical structure was determined using a Fourier transform infrared spectrometer (SPECTRUM ONE, Perkin Elmer, USA). The samples were scanned from 400 cm −1 to 4000 cm −1 at room temperature in attenuated total re ectance mode at a resolution of 4 cm −1 .

Scanning electron microscope (SEM)
The structural features of the hydrogels were monitored by scanning electron microscope (SEM, Quanta 250 microscope, Japan). The specimens were coated with gold using a sputtering device (Jeol, JFC 1200, Japan) prior to the SEM observation. A magni cation of 500 × was used, and micrographs of the samples were recorded.

Differential scanning calorimetry (DSC)
The thermal behavior was investigated using DSC (NETZSCH DSC 204 F1 Phoenix, Germany). The sample were placed in aluminum pans and purged with nitrogen gas at a ow rate of 40 mL/min. The temperature was set to 20℃ − 200℃, with a ow rate of 10℃/min. Characteristic temperature were de ned as the glass transition temperature, melting temperature, and speci c heat capacity.

Tensile testing
The mechanical properties were performed using a universal tensile tester. The hydrogel sheets of 7 cmlength and 1 mm-width were stretched at a tension speed of 30 mm min −1 to calculate the tensile strength and elongation at break. For each sample, 5 specimens were tested. The statistical average and standard deviation were then reported.

Results And Discussion
Polyvinyl alcohol and sodium alginate-based hydrogel composite was successfully prepared from freeze thaw technique. It presented as a whitish-color. It appeared as reformable shape. Figure 1 illustrates the FTIR spectra of polyvinyl alcohol and sodium alginate-based hydrogel prepared by freeze thaw technique.
No signi cant change on functional group of hydrogels was observed when 3, 5 and 7 consecutive cycles were conducted. The functional group of hydrogels was still similar when composition of hydrogel was changed. This was probably due to the fact that FTIR analysis was used to qualitatively analyze the existence of functional group of hydrogels. In Figure 1, the characteristic peak at 3260 cm −1 was observed. This was corresponded to the presence of OH stretching group (hydroxyl group). It referred to the existence of polyvinyl alcohol and sodium alginate. This was in agreement with previous work of Ma et al [17]. It may imply that hydrogel will be well adsorbed moisture. It can create the hydrogen bonding between OH group of hydrogel and water molecule. The use of hydrogel should be therefore stored in desiccator. Furthermore, the presence of wavenumbers at 1410 cm −1 and 2911cm −1 was existed. This was referred to C-H stretching vibration. The occurrence of wavenumber at 1610 cm −1 was also reported due to the presence of CO stretching vibration (carbonyl group), respectively. The presence of carbonyl group was referred to the sodium alginate [18]. These functional groups can create the hydrogen bonding between polyvinyl alcohol and sodium alginate is reported as a schematic diagram in Figure 2. It can create the hydrogen bonding as an intermolecular and intramolecular interaction between sodium alginate and poly vinyl alcohol chain. This is in agreement with previous work of Chhatri et al [19]. Figure 3 exhibits the morphological properties of polyvinyl alcohol and sodium alginate-based hydrogel prepared by freeze thaw technique. The cross-sectional view of hydrogel was reported based on magni cation of 1K and 10K. With high magni cation, it is better to see the porous structure. It was remarkable to note that all of microstructural images presented the porous structure. The pores were interconnected and regularly distributed. The pores can create the due to freeze step. Water part in hydrogel structure was then removed. It can be used to con rm the presence of water in hydrogel structure. This result was similar to previous literature of Zhang et al [20]. Moreover, with 5 and 7 consecutive cycles, the amount of pore was less compared to 3 consecutive cycles. The surface became smoother. It implied that hydrogel was well packed. With high consecutive cycle of freeze thaw, it can be implied that crosslinking reaction between polyvinyl alcohol and sodium alginate was successfully prepared. As a consequence, it therefore provided the di culty when hydrogel will be employed as medical materials. On the other side, it was observed that all hydrogels clearly presented porosity. It was implied that the existence of interconnected porous structure signi cantly provided many bene ts. Water molecule can be adhered and it can be considered as a reservoir of any active molecule. It will be therefore excellent if hydrogel will be employed as medical materials.
Differential scanning calorimetry was considered as an effective instrument to evaluate the existence of water molecule in hydrogel network. As for the hydrogel, water was considered as a large portion. This measurement was allowed to imply the water state change in sodium alginate and poly vinyl alcoholbased hydrogel network, as suggested by Jiang et al [10]. Figure 4 reports the DSC measurement of polyvinyl alcohol and sodium alginate-based hydrogel prepared by freeze thaw technique. Various compositions of sodium alginate and poly vinyl alcohol were evaluated based on 3, 5 and 7 consecutive cycles of freeze thaw. It was notable that all of curves were presented in the similar form. Two exothermic peaks were presented at 110ºC and 225ºC. The peak at 110ºC may involve the evaporation of water molecule in hydrogel network. This was associated with previous work of Wang et al [21]. The strong peak at 225ºC was typically related to melting temperature of sodium alginate and poly vinyl alcohol. Furthermore, it was remarkable to note that the melting temperature was slightly increased respect to increment of sodium alginate content. It was slightly shifted to 230ºC for 3, 5 and 7 consecutive cycles.
The existence of sodium alginate may enhance the compactness of hydrogel by freeze thaw process. This discussion was similar to previous literature of Lut et al [22]. Figure 5 presents the swelling characteristic of polyvinyl alcohol and sodium alginate-based hydrogel composite. The swelling ratio was highly observed within 200 min for all hydrogel structure. After that, the swelling ratio was steady. It can be explained that swelling behavior was occurred due to two reasons. First, it was typically related to hydrophilicity of sodium alginate located inside hydrogel network. It can adsorb water molecule in the system. Second, the swelling behavior was associated with interconnected porous structure in hydrogel network, as suggested by Yu et al [23]. This discussion is in agreement with SEM analysis. Furthermore, it was notable that with high sodium alginate content, the swelling behavior was signi cantly superior. It can be noted that sodium alginate has high water adsorption [24].
To use hydrogel as a medical material, mechanical properties were therefore determined. Figure 6 exhibits the tensile strength of sodium alginate and poly vinyl alcohol-based hydrogel composite. Five measurements for each sample were tested and the data was reported as statistical average and standard deviation. Uniform dispersion of poly vinyl alcohol in sodium alginate was prepared, as suggested in Figure 2. It can create the interfacial adhesion in between, similar to previous work of Hu et al [25]. The level of tensile strength for all hydrogels was in the region of 0.25-1 MPa. It can be indicated that sodium alginate and poly vinyl alcohol-based hydrogel composite can be prepared by freeze thaw technique. No presence of crosslinking agent is necessary. From the result, it was remarkable to note that with high amount of poly vinyl alcohol, tensile strength was slightly high. It can be observed for sodium alginate and poly vinyl alcohol of 50:50 weight ratio for 3, 5 and 7 consecutive cycles. However, with low amount of poly vinyl alcohol content, tensile strength was slightly decreased. It can be implied that poly vinyl alcohol exhibited high dimensional stability. Furthermore, it can be explained that due to the existence of -OH group in both sodium alginate and poly vinyl alcohol, it can effectively create the intermolecular and intramolecular interaction throughout hydrogel network. This is in agreement with previous literature of Mousa et al [26]. No agglomeration was observed. It can be therefore transfer load when external force will be applied [27].
To use hydrogel as a medical material, in vitro degradation test was observed. The data was reported as a statistical average of weight loss of immersed hydrogel into DI water. This was considered as an important key to determine the degradation behavior of hydrogel. Figure 7 reports the percentage of weight loss of sodium alginate and poly vinyl alcohol-based hydrogel versus incubation time. The experiment was conducted at ambient temperature for 28 days. The degradation characteristic of hydrogel was evaluated by mass loss, internal structure transformation and mechanical disintegration, similar to previous work of Zhang et al [28]. Before 3 days, hydrogel can keep its dimensional stability. After that, it initially changes the shape. The results were also typically presented in the similar feature for 3, 5 and 7 consecutive cycles of freeze thaw. No signi cant change of percentage of composition between sodium alginate and poly vinyl alcohol was observed based on variation of consecutive cycles. It was remarkable to note that with high content of sodium alginate, the percentage of degradation behavior is superior. Sodium alginate has high solubility in water, as suggested by previous literature of Wardhani et al [29]. This is also in agreement with swelling behavior reported in Figure 5. The range of degradation precent was estimated to be 70-90%, 30-50% and 10-30% for the composition ratio of sodium alginate and poly vinyl alcohol of 50:50, 60:40 and 70:30, respectively. The degradation mechanism was typically involved high water solubility of sodium alginate. It can dissolve into DI water and thus the hydrogen bonding with poly vinyl alcohol was then disintegrated.

Conclusion
In this study, sodium alginate and poly vinyl alcohol-based hydrogel was successfully prepared by freeze thaw technique. Only 3 consecutive cycles of freeze thaw were optimal for hydrogel formation. Fourier transform infrared con rmed that hydrogen bonding was formed throughout hydrogel network by OH group of sodium alginate and poly vinyl alcohol. The 60:40 composition ratio between sodium alginate and poly vinyl alcohol was optimal. Scanning electron microscope exhibited microstructure of hydrogel. It presented as a porous network. The swelling behavior is relatively high at the initial stage of incubation time. With low content of sodium alginate, tensile strength was slightly enhanced, whereas low in vitro degradation behavior was relatively occurred. Sodium alginate and poly vinyl alcohol-based hydrogel remarkably illustrated extraordinary properties for being as a medical material.