Synthesis, characterization and in vitro evaluation of a gelatin-based platform with antioxidant and nitric oxide releasing property

Exogenous supply of signaling molecules such as nitric oxide (NO) has been explored for its therapeutic and multifaceted functions. However, NO released from the material can react with surrounding reactive oxygen species (ROSs), if there is an oxidative stress condition, leading to serious complications. A dually functional polymer with both antioxidant and NO releasing property will not only overcome complications associated with NO/ROSs interplay but also will be beneficial in conditions where both NO and antioxidants are required. A single gelatin-based delivery system including both NO releasing and antioxidant properties is reported here. In the present study N-acetyl cysteine (NAC) was efficiently conjugated with gelatin using two steps EDC/NHS chemistry. Gelatin-NAC (Gel-NAC) conjugates showed characteristic peaks in H-1 NMR and FTIR depicting successful synthesis of Gel-NAC conjugates. The reduced thiol and disulfide groups were quantified using Elmann`s colorimetric assay and with the increase in the ratio of NAC to gelatin, the number of Gel-NAC loaded thiol-groups also increased. Antioxidant potential of the material was validated through 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, with increasing number of reduced thiols in Gel-NAC, it was observed that free radical inhibition property also increased reaching up to 90.3 +/- 0.2%. In vitro studies showed protective role of Gel-NAC in the range of 171 nmol - 1068 nmol while amount higher than 1068 nmol induced toxic effects in both morphological and live-dead assay in H2O2 induced oxidative stress model. Cytotoxicity of Gel-NAC variants were checked through proliferative assay and was found that lower concentrations supported cell proliferation. Gel-NAC was loaded with adequate amount of NO to obtain Gel-NAC-NO conjugates. Gel-NAC-NO was characterized for specific peaks using UV visible spectroscopy and FTIR. Remaining reduced thiols groups in Gel-NAC-NO was quantified using Elmann's assay and retention of at most 74% of thiol-groups after nitration was observed. Gel-NAC-NO variants showed sustained release of NO for 14 days. Functionality assay was performed for NO releasing potential of the system on neuroblastoma cells for neurite extension. Average neurite length of 51 +/- 15 mu m in the presence of Gel-NAC-NO conjugate, was significantly higher as compared to 27 +/- 8 mu m neurite length in control group. Such dually functional polymers will hold great potential in the field of therapeutics and tissue engineering.