SYNTHESIS, CHARACTERIZATION AND BIOCOMPATIBILITY OF PLANT-OIL BASED HYDROGELS

Biocompatible hydrogels are used in a variety of biomedical applications, including tissue scaffolds, drug delivery systems, lab/organ-on-a-chips, biosensors, cell-culture studies and contact lenses. The demand for novel and functional monomers to be used in hydrogel synthesis is increasing as the number of biomedical applications and need for biomaterials increase. The purpose of the study was to develop novel hydrogels from renewable materials. Acrylated methyl ricinoleate, a plant oil-based monomer, was used as the renewable material. The effects of acrylated methyl ricinoleate/N-isopropyl acrylamide molar ratio on hydrogel structural properties, thermal stability and in vitro cytotoxicity were studied. FTIR spectroscopy was used to characterize the structural properties of the hydrogels, while TGA was used to characterize the thermal properties. HEK293 and Cos-7 cell lines were used to test the cytotoxicity of the monomers and hydrogels. IC50 values for acrylated methyl ricinoleate and N-isopropyl acrylamide were found to be greater than 25 mg/mL. Cell viability of hydrogels containing 50% or more acrylated methyl ricinoleate was greater than 60%, while hydrogel biocompatibility decreased with decreasing molar ratio of acrylated methyl ricinoleate. Cells showed a minimum viability of 80% when incubated in hydrogel degradation products. An environmentally friendly synthesis method was developed and novel biocompatible hydrogels from renewable materials were produced for biomedical applications.


Introduction
Hydrogels are crosslinked three-dimensional natural and synthetic polymer networks with hydrophilic properties that can absorb a significant amount of water. Because of their similarity to living tissues, they can be used in a variety of biomedical applications, including drug-delivery systems (Peers et al. 2020), scaffolds (Xu et al. 2019), lab/organ-on-a-chips (Ding et al. 2020), cellculture studies (Bhattacharya et al. 2012) and contact lenses e2 O. Yalcin Capan & P. Cakir Hatir (Peppas & Hoffman 2020). Hydrogels swell in aqueous media because their network structures contain hydrophilic polymers. Depending on the intended application, the swelling profiles of hydrogels may be altered by using more or less hydrophilic monomers. Hydrogels are frequently designed using acrylic-based monomers such as hydroxyethyl methacrylate and methyl methacrylate (Peppas et al. 2000). Furthermore, poly(ethylene oxide) (PEO) and poly(ethylene glycol) (PEG) based hydrogels are gaining popularity due to their biocompatibility and FDA approval. Poly-lactic or glycolic acids are also used in the production of hydrogels, especially for biodegradable systems (Lee & He 2010). Smart hydrogels can also be produced using various monomers such as poly(acrylic acid), poly (methacrylic acid), and poly(vinyl alcohol) (Nisopropyl acrylamide). pH-responsive hydrogel systems are created using acidic and basic monomers (Koetting et al. 2015), while thermoresponsive hydrogels are created using N-isopropyl acrylamide (NIPAM) (Dong & Hoffman 1986). Smart hydrogels are stimuli-responsive hydrogels that can respond to external stimuli by changing their structural conformations and swelling-deswelling behaviors. Several monomers are needed to create novel hydrogels with desired hydrophilicity for a variety of applications. Thus, there will always be a need for novel and functional monomers.
One of the top priorities of researchers is to synthesize functional monomers and novel hydrogels while minimizing environmental impact. Therefore, environmentally friendly green raw materials are favored for the production of monomers and polymers. Plant oilbased materials are often used as renewable resources because they contain a large number of functionalizable hydroxyl groups. Furthermore, fatty acids contain double bonds that are easily converted into epoxy groups, resulting in a variety of reactions with ring-opening reagents to synthesize biocompatible polymers (Miao et al. 2014). Castor oil, for example, is one of the most popular naturally functionalized plant oil triglycerides as it can be used to create a variety of functional monomers (Dupé et al. 2012 In the present study, we aimed to synthesize and characterize novel biocompatible hydrogels derived from renewable resources. We used AMR as the plant-oil based monomer, NIPAM as thermoresponsive monomer and N,N′-Methylenebis (acrylamide) (MBA) as crosslinker. We varied the molar ratio of AMR/NIPAM and evaluated the effects of AMR on structural properties, thermal stability, and in vitro cytotoxicity of hydrogels. We developed a green, environmentally friendly synthesis process in order to create biocompatible hydrogels.

Synthesis of hydrogels
Hydrogels were synthesized by using the photopolymerization method with a radical initiator, DMPA, under UV irradiation at 365 nm. MBA (1.25 mg) and NIPAM (Table 1) were weighed in a vial and dissolved in 400 µl of Phosphate Buffered Saline (PBS). AMR and DMPA (1% with respect to the total number of double bonds in the system) were transferred into the solution. All polymerization reactions were performed under UV irradiation at 365 nm for 60 min at 25°C. After the polymerization, the hydrogels were washed with water and methanol to remove unreacted monomers and kept in vacuum oven at 25°C for 24 hours.

Characterization of hydrogels
The hydrogels were characterized by Fourier Transform Infrared Spectroscopy (FTIR) and Simultaneous Thermogravimetric Analyzer (TGA). Structural characterization was performed by using JASCO 6600 spectrophotometer in the range of 400-4000 cm -1 . All samples were scanned 32 times and FTIR spectra were obtained with 4 cm -1 resolution. Thermal characterization was carried out by HITACHI STA7200 TGA. TGA analyses were carried out under nitrogen atmosphere at a rate of 200 mL/min with a heating rate of 10°C/min from 0°C to 900°C. In vitro cytotoxicity assays of monomers and hydrogels Cytotoxicity assays were performed by using Cos-7 (African green monkey kidney fibroblast-like cell line) and HEK293 cells (Human Embryonic Kidney Cells). The cells were cultured in a complete culture medium consisting of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 2 mM L-Glutamine (Thermo Fisher), 100 IU/mL penicillin-streptomycin (Thermo Fisher) and 10% fetal bovine serum (FBS, Lonza®). Cos-7 and HEK293 cells were seeded in 96-well cell culture plates at 10x10 3 cells/well and then incubated at 37°C in 5% CO2. After 24 h of incubation, 100 μL of different concentrations (0.5-1-2-5-10 mg/mL) of monomers in PBS or only PBS as control were added to the wells. For cytotoxicity assay of hydrogels, 14x10 4 cells/well Cos-7 or HEK293 cells were seeded in 24-well plates which were previously covered with different concentrations of hydrogels. All experiments were carried out in triplicates. After a further incubation for 24 h, cytotoxicity of monomers and hydrogels was measured by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay.
The MTT cytotoxicity assay is a colorimetric assay that measures the number of living cells which have active enzymes converting yellow tetrazolium salt into insoluble purple formazan. The intensity of the purple color is directly proportional to the metabolically active living cells and assessed by colorimetric analysis.
For MTT assay, 10 μL and 100 μL of sterilized MTT (5 mg/mL) reagent was added to each 96-well and 24well, respectively. After incubation of plates at 37°C in 5% CO2 for 4 h, the MTT solution was removed. Purple color formazan products were dissolved in Dimethyl Sulphoxyde (DMSO) by incubating the plates in the dark for 15 min on an orbital shaker. Purple color with an absorbance at 570 nm was measured by using an Eliza Plate Reader. Percentage of cell viability was calculated by taking the ratio of absorbance values for samples and control and multiplied by 100.
In vitro cytotoxicity assays of degradation products 20 mg of hydrogel was weighed and transferred into 1 mL of 0.1 N NaOH solution and incubated at room temperature for 24 h. After the hydrogels were degraded entirely, the pH of the solution was adjusted to 7.4. The solution was filtered to sterilize, afterwards, it was diluted 2, 10 and 100 times with cell culture media. 100 µl of the diluted solutions were transferred to the 96-well plates containing cultured Cos-7 cells and incubated for 24 h at 37℃. Cytotoxicity of the degraded products was evaluated using the MTT assay.

Statistical analysis
For cell viability assays, all experiments were performed as two independent experiments carried out in triplicates. All results are presented as mean ± standard deviation (SD). Statistical significance between different groups were evaluated by using one-way analysis of variance (ANOVA) with Bonferroni correction for post hoc analysis and p<0.05 was considered as significant.
TGA curves are shown in Fig. 2. Experiments were carried out under a nitrogen atmosphere at a rate of 200 mL/min with a heating rate of 10°C/min from 0 to 900°C. Hydrogel H1 exhibits a decomposition between 300°C and 500°C and lost 85% of its weight around 320°C. TGA curves of H2 and H4 show roughly 10% weight loss at 274°C and 55% weight loss at 300°C. TGA curve of H3 exhibits three significant segmental losses. H5 lost 12% of its weight between 30°C and 200°C. Two critical parameters, 5% weight loss temperature and 50% weight loss temperature, were also investigated ( Table 2). The 5% weight loss temperatures were determined as 240°C, 212°C, 216°C, 268°C and 347°C for H1, H2, H3, H4 and H5, respectively, whereas the 50% weight loss temperatures were determined as 344°C, 339°C, 335°C, 348°C and 403°C for H1, H2, H3, H4 and H5, respectively.

In vitro cytotoxicity assays of monomers and hydrogels
The results of the cytotoxicity assay of AMR, NIPAM and MBA monomer solutions with concentrations ranging from 0.5 mg/mL to 10 mg/mL were shown in Fig. 3. Both Cos-7 and HEK293 cells display at least 70% viability in the presence of a wide range of concentrations (0.5-5 mg/mL) of AMR and NIPAM monomer solutions, whereas a significant reduction in cellular viability (less than 70%) was detected with 10 mg/mL concentrations. The toxicity of MBA monomer solution, on the other hand, is significantly high even with 1 mg/mL concentration, less than 50%. IC50 (50% cell growth inhibition) values were also calculated by using the curve constructed by plotting cell viability (%) versus monomer concentration (mg/mL). As shown in Table 3, IC50 values of AMR and NIPAM solutions are higher than 25 mg/mL while MBA IC50 values are very low, ranging between 1.06 mg/mL and 0.05 mg/mL in Cos-7 and HEK293 cells, respectively.   As shown in Fig. 4, cytotoxicity assay for hydrogels on two different cell lines revealed that the viability of the cells was more than 60% in hydrogels with varied molar ratios of AMR:NIPAM (100:0, 80:20 and 50:50). However, as the amount of NIPAM in hydrogels (AMR:NIPAM, 80:20 and 0:100) increased, a reduction in cell viability was detected. The hydrogels made up of solely NIPAM monomers showed less than 45% cell viability in both cell lines.

In vitro cytotoxicity assays of degradation products
Cytotoxicity of degraded hydrogels with different ratios of AMR:NIPAM was analyzed by MTT assay. Hydrogels were kept in 0.1 N NaOH solution at room temperature for 24 h until they are all degraded. Filtered solutions diluted by 2, 10 and 100 times with cell culture media were applied to Cos-7 cells for 24 h. As shown in Fig. 5, cells showed more than 70% viability when exposed to degraded products in different concentrations supporting the biocompatibility of the hydrogels.

Discussion
Hydrogels generated from natural polymers have attracted significant interest in the field of tissue engineering in recent years due to their low cost of production, biocompatible structure, biodegradability and extracellular matrix mimicking capabilities (Geckil et  In the first step of the study, FTIR spectra of the hydrogels were evaluated (Fig. 1). AMR has formerly been shown to exhibit two independent ester carbonyl peaks at 1739 cm -1 and 1722 cm -1 prior to polymerization (Cakir Hatir & Cayli 2019). In the present study, AMR showed two peaks at around 1637 cm -1 and 1620 cm -1 that indicate acrylate double bonds. The absence of these peaks in hydrogels in the former study suggests that acrylate double bonds were consumed during polymerization. Moreover, hydrogels exhibit a strong peak at 1727 cm -1 instead of two separate ester carbonyl peaks, as saturated ester moieties were produced due to polymerization of the acrylate groups. So, the FTIR peaks of the two ester groups shifted to 1727 cm -1 , and one ester carbonyl was detected instead of two peaks. These results proved that AMR was involved in the polymerization processes. To establish that NIPAM was also involved in polymerization, the FTIR spectra of NIPAM were analyzed before and after the reactions. The FTIR spectrum of NIPAM revealed a strong peak at around 3280 cm -1 caused by N-H stretching of secondary amide, which formed a broad band following monomer polymerization (Ilić-Stojanović et al. 2013, Isikci Koca et al. 2020). These broad bands are seen in the FTIR spectra of H4 and H5. The broad bands contain both O-H stretching caused by absorbed water, as proven by TGA data and N-H stretching of secondary amide. Furthermore, there is a peak at 1616 cm -1 caused by the C=C bond, which should not be seen in polymer spectra (Shah et al. 2013). NIPAM hydrogels do not display this peak, instead, they exhibit the bands at 1646 cm -1 and 1539 cm -1 indicating C=O (amide I) and N-H (amide II) stretching, respectively. H3 and H4 have peaks at 1646 cm -1 (H3, H4), H5 has a peak at 1631 cm -1 caused by amide I stretching. The bands at around 2950 cm -1 represent vibrations of -CH3 and -CH2. The FTIR spectra confirms that H1 has AMR, H5 has NIPAM and H2, H3 and H4 have both NIPAM and AMR. Furthermore, increasing intensity of amide I and amide II bands confirm the increasing molar ratio of NIPAM from H2 to H5. The hydrogels synthesized with AMR have bands at 2923 cm -1 and 2854 cm -1 , whereas the hydrogels without AMR have the same bands at around 2969 cm -1 and 2928 cm -1 . Additionally, H4 and H5 have broad bands at around 3292 cm -1 which represent NH stretching of NIPAM. In the spectrum of H3, the broad band can barely be seen at around 3300 cm -1 . On the other hand, H2 does not have a clear band since the molar ratio of NIPAM is relatively low.
In the next step, the thermal stability properties of the hydrogels were investigated by TGA curves (Fig. 2). Hydrogel H1, synthesized from AMR, exhibited a decomposition between 300°C and 500°C. H1 lost 85% of its weight around 320°C, which might be due to the dissociation of the MR moiety from the main chain (Cakir Hatir & Cayli 2019). TGA curves of H2 and H4 show roughly 10% weight loss at 274°C and 55% weight loss at 300°C, respectively. Decomposed sections of H2 and H4 are proportional to the amount of NIPAM used in the polymerization phase. TGA data of H2 and H4 revealed that when monomers are combined in differing molar ratios, the polymer is produced as a building block. TGA curve of H3 shows three major segmental losses, indicating that the crosslinked polymer network was created in the presence of homogeneously dispersed monomers. H5, produced only using NIPAM, lost 12% of its weight between 30°C and 200°C, indicating evaporation of volatile compounds such as water (Ribeiro et al. 2017). H5 is also thermally stable with only a single decomposition step between 340°C and 440°C, as reported in the literature (Ruiz-Rubio et al. 2015). Temperatures for 5% and 50% weight loss were investigated ( Table 2). The 5% weight loss temperatures were determined as 240°C, 212°C, 216°C, 268°C and 347°C for H1, H2, H3, H4 and H5, respectively, whereas the 50% weight loss temperatures were determined as 344°C, 339°C, 335°C, 348°C and 403°C for H1, H2, H3, H4 and H5, respectively. Among all hydrogels, H5 showed the highest temperature for both 5% and 50% weight loss. H1, having only AMR has relatively high temperatures for both 5% and 50% weight loss. On the other hand, the combination of AMR and NIPAM caused a slight decrease at the 5% weight loss temperature. From H2 to H5, it was observed that as the molar ratio of NIPAM increases, the temperature for the 5% weight loss increases as well. However, there is no significant change at the 50% weight loss temperatures.
As the hydrogels, which consist of AMR, NIPAM and MBA, can be used for tissue engineering, biocompatibility is crucial for in vivo application. Therefore, we evaluated the cytotoxicity of these monomers and hydrogels with different ratios of AMR and NIPAM. As cytotoxic effects may vary for different cell types with different origins, two different cell lines (Cos-7, kidney fibroblast and HEK293, embryonic kidney cells) were used to evaluate cell viability (Capella et. al. 2019). We also calculated the IC50 values for each monomer in Cos-7 and HEK293 cell lines (Table 3). IC50 values of AMR and NIPAM are higher than 25 mg/mL while MBA IC50 values are very low, ranging between 1.06 mg/mL and 0.05 mg/mL in Cos-7 and HEK293 cells, respectively. According to the results, MBA has the lowest IC50 value compared to AMR and NIPAM in both cell lines, indicating a high cytotoxic effect for the cells. NIPAM and AMR produced comparable IC50 values in which AMR has higher IC50 with HEK293 cells, whereas NIPAM has higher IC50 with Cos-7 cells.
In addition to the cytotoxicity of monomers, hydrogels were tested as well. The cells grown on hydrogels with increased AMR molar ratio exhibited a higher viability compared to the cells grown on hydrogels with increased NIPAM ratio. Cell viability of hydrogels containing 50% or more AMR was found to be greater than 60%, while biocompatibility of hydrogels reduced with decreasing Biocompatibility of plant-oil based hydrogels e7 Trakya Univ J Nat Sci, Online First for 22 (2): X-X, 2021 molar ratio of AMR. The hydrogels made up of only NIPAM showed less than 45% cell viability in both cell lines.
Although building blocks and hydrogels are biocompatible, we also analyzed the cell viability in the presence of degradation products. Cells showed a minimum 80% of viability when they were incubated in degradation products of hydrogels with high molar ratio of AMR (AMR:NIPAM, 100:0, 80:20, 50:50). Therefore, our results indicate that plant oil-based AMR does not lead to cytotoxicity and can be used in tissue engineering applications. However, a longer exposure of hydrogels and degradation products may confirm reliability of the application.
In conclusion, a green, environmentally friendly synthesis method was successfully developed to design biocompatible hydrogels. A plant oil-based monomer, AMR, was used to synthesize novel hydrogels. Molar ratio of AMR to NIPAM was varied and the effects of AMR on structural characteristics, thermal stability, and in vitro cytotoxicity behaviors of the hydrogels were investigated. The findings demonstrated that hydrogels were biocompatible, although their biocompatibility decreased with decreasing molar ratio of plant oil-based monomer. The developed hydrogels can be used in many biomedical applications such as drug-delivery systems, scaffolds, lab/organ-on-a-chip, cell-culture studies and contact lenses.