Synthesis and Properties of Polyampholytes and their Application to Cell Cryoprotection

Abstract

Polyampholytes are known as polymers comprising both cationic and anionic groups that can have pronounced isoelectric point behavior and strong responses to ionic strength, such as the anti-polyelectrolyte effect. This thesis focusses on exploring the solution properties of fully ionized random polyampholytes, their phase separation behavior, and their application in cell cryoprotection. The reactivity ratios of different monomer pairs were measured by NMR to guide the subsequent formation of a series of polyampholytes with different charge ratios, molecular weights and compositions by free radical copolymerization with little compositional drift. The polyampholytes, comprising different ratios of N-(3-aminopropyl)methacrylamide hydrochloride (APM) and acrylic acid (AA), show phase separation at their isoelectric point, pH(I), anti-polyelectrolyte effects, and temperature responsive solubility. The latter include lower critical solution temperature (LCST) and upper critical solution temperature (UCST) behaviors, depending on composition and conditions. At the pH(I), liquid to solid phase separation is shown to be driven by a combination of electrostatic interactions and hydrogen bonding. The stoichiometric APM/AA polyampholytes with different molecular weights reveal significant molecular weight effects on polyampholyte properties, including their phase separation efficiency, anti-polyelectrolyte effect and LCST, but not their pH(I). High molecular weight polyampholytes were crosslinked into permanent hydrogels using THPC, and used to explore organic dye absorption from water. Stoichiometric polyampholytes based on different monomers showed that the hydrophobicity of the comonomers also contributes to the polyampholyte phase separation. Depending on the balance of electrostatic interaction and polymer hydrophobicity, polyampholytes may show no phase separation, liquid to liquid, and liquid to solid phase separations. Finally, polyampholytes made from N-[3-(dimethylamino)propyl methacrylamide (DMAPMA) and acrylic acid (AA) were used as macromolecular, non-penetrating cryoprotective agents for NIH 3T3 cells. The results show that to work as cryoprotective agents the polyampholytes need to have a moderate excess of negative charge at physiological conditions, with post-thaw cell viability further improved by high MW and with presence of about 10 mol% hydrophobic comonomer. As non-penetrating cryoprotective agents, these polyampholytes show good protection of the cell membrane from interstitial ice damage and 70-80% post-thaw cell viability, by bonding on cell surfaces and phase separating around the cells upon freezing. However, the absence of intracellular protection leads to low cell attachment and growth after thawing, which can be improved by introducing as little as 2% DMSO prior to freezing. As a result, polyampholytes cryoprotective agents can significantly decrease DMSO levels required for cell cryoprotection from 10% to 2%.