Toward a Molecular Understanding of the Mechanism of Cryopreservation by Polyampholytes: Cell Membrane Interactions and Hydrophobicity

Cryopreservation enables long-term preservation of cells at ultra-low temperatures. Current cryoprotective agents (CPAs) have several limitations, making it imperative to develop CPAs with advanced properties. Previously, we developed a novel synthetic polyampholyte based CPA, copolymer of 2-(dimethylamino)ethyl methacrylate (DMAEMA) and methacrylic acid(MAA), (poly-(MAA-DMAEMA)) which showed excellent efficiency and biocompatibility. Introduction of hydrophobicity increased its efficiency significantly. Herein, we investigated the activity of other polyampholytes. We prepared two zwitterionic polymers, poly-sulfobetaine (SPB) and poly-carboxymethyl betaine (CMB) and compared its efficiency with poly-(MAA-DMAEMA). Poly-SPB showed only intermediate property and poly-CMB showed no cryoprotective property. These data suggested that the polymer structure strongly influences cryoprotection, providing an impetus to elucidate the molecular mechanism of cryopreservation. We investigated the mechanism by studying the interaction of polymers with cell membrane, which allowed us to identify the interactions responsible for imparting different properties. Results unambiguously demonstrated that polyampholytes cryopreserve cells by strongly interacting with cell membrane, with hydrophobicity increasing the affinity for membrane interaction, which enable it to protect the membrane from various freezing induced damages. Additionally cryoprotective polymers, especially their hydrophobic derivatives, inhibit the recrystallization of ice, thus averting cell death. Hence, our results provide an important insight into the complex mechanism of cryopreservation, which might facilitate the rational design of polymeric CPAs with improved efficiency