Synthesis, characterization and investigation of cryoprotective properties of modified polyampholytes

Cryopreservation is a process applied for the long-term preservation of biological and food substances by freezing them at very low temperatures. Consequently, it holds significant importance in regenerative medicine, tissue engineering, and the establishment of gene banks. The primary goal in cryopreservation is to protect the stored materials from damage that may occur during the freezing-thawing stages (e.g., reduced viability, loss of functionality and structure). For this purpose, compounds called cryoprotectants are used during the freezing step. Cryoprotectants are categorized into two classes: cell membrane-permeable and non-permeable. The most commonly used permeable compounds are DMSO and glycerol. As cryopreservation mechanisms become better understood, studies aimed at minimizing the disadvantages caused by these conventional cryoprotectants suggest the development of non-permeable alternatives, particularly polyampholytes. Polyampholytes are generally copolymeric compounds that possess both cationic and anionic groups within their molecules, exhibiting zwitterionic properties, and thus are sensitive to the pH of the medium in which they are used. Research in the literature over the past 15 years, focusing on the synthesis of polyampholytes and evaluating their usability as cryoprotectants, is still largely confined to optimization studies. In the study presented in this thesis, based on the findings and recommendations reported in the literature, p(MAA-DMAEMA) and p(MAA-DEAEMA) copolymer polyampholytes were synthesized in aqueous media using methacrylic acid (MAA) with 2-(dimethylamino)ethyl methacrylate (DMAEMA) and 2-(diethylamino)ethyl methacrylate (DEAEMA) monomers, respectively, via classical free radical addition polymerization. These polymers were characterized by FTIR and GPC-SEC analyses. Their solubility properties and osmotic pressure behaviors were investigated. In vitro cytotoxic properties were determined using the MTT method on human embryonic kidney cell line (HEK293T). Selected poly(MAA-DMAEMA) was further modified with L-cysteine (L-Cys) and L-arginine (L-Arg) amino acids. The structures of the p(MAA-DMAEMA)-Cys and p(MAA-DMAEMA)-Arg products were confirmed by FTIR analysis, and their solubility properties and osmotic pressure behaviors were examined. The in vitro cryoprotective properties and efficacy of all products were investigated using the HEK293T cell line. As a result, post-thaw cell viabilities, when used at the specified concentrations, were obtained at 77,8 % ±9,1 for the control group DMSO (10%), 43 % ±14 for p(MAA-DMAEMA) (75 mg/mL), and very low rates (4-5%) for p(MAA-DMAEMA)-Cys and p(MAA-DMAEMA)-Arg (25, 50, 75 mg/mL). The p(MAA-DMAEMA) product showed significant cryoprotective potential. This study not only demonstrates the potential of polyampholytes in cryopreservation but also elucidates the complex effects of modifications on cell viability through cause-and-effect relationships.