This review discusses various aspects of protein aggregation and inhibition strategies, emphasizing the use of polymers, which is one of the most promising approaches to combat various aggregation-induced complications in neurodegenerative diseases and protein therapeutics.

Polyampholytes are emerging macromolecular membrane non-penetrating cryoprotectants; however, the mechanism behind their cryopreservation remains unclear. Here, we investigated the mechanism using solid-state NMR spectroscopy. The polymer-chain dynamics and the water and ion mobilities in the presence of various membrane penetrating and non-penetrating cryoprotectants were monitored at low temperatures to mimic cryopreservation conditions. NMR experiments revealed that the water, Sodium-ion, and polymer-chain signals in a carboxylated poly-ʟ-lysine (COOH-PLL) solution broadened upon cooling, indicating increasingly restricted mobility and increased solution viscosity. Moreover, strong intermolecular interactions facilitated the COOH-PLL glass transition, trapping water and salt in the gaps of the reversible matrix, preventing intracellular ice formation and osmotic shock during freezing; this reduced cell stress is responsible for cryoprotection. This simple NMR technique enabled the correlation of the cryoprotective properties of polymers that operate through mechanisms different from those of current cryoprotectants, and will facilitate the future molecular design of cryoprotectants.

A completely synthetic polyampholyte cryoprotectant was developed with cationic and anionic monomers by reversible addition-fragmentation chain transfer polymerization. The neutralized random polyampholyte, which had an equal composition ratio of monomers, showed high cryoprotective properties in mammalian cells. Introduction of a small amount of hydrophobic monomer enhanced cell viability after cryopreservation, indicating the importance of hydrophobicity. Leakage experiments confirmed that these polyampholytes protected the cell membrane during cryopreservation. Due to low cytotoxicity, this polyampholyte has the potential to replace the convention cryoprotective agent dimethyl sulfoxide. The present study is the first to show that we can design a polymeric cryoprotectant that will protect the cell membrane during freezing using appropriate polymerization techniques.

Hydrogels are promising substrates for tissue engineering applications because of their unique biocompatibility, flexible methods of synthesis, range of constituents, and desirable physical characteristics. Cryopreservation of cell-containing constructs using such hydrogel scaffolds is in high demand in tissue-engineering applications for the production of “off-the-shelf” tissue-engineered products. However, cryopreservation of regenerated tissues including cell sheets and cell constructs is not easy compared to the preservation of cell suspensions, even when cryoprotectants are used. Here, we report a dextran-based polyampholyte hydrogel that itself shows cryoprotective properties, which could be useful for cell encapsulation and tissue engineering applications involving hydrogel formation. Amination was performed by introducing poly-L-lysine onto azide groups conjugated with dextran, and a portion of the amino groups was converted into carboxyl groups. These dextran-based polyampholytes showed good cryoprotective properties for mammalian cells, and the addition of dextran substituted with dibenzylcyclooctyne acid induced in situ hydrogel formation via Cu-free click chemistry with high biocompatibility. Cells encapsulated with such in situ hydrogels can be cryopreserved well without the addition of any cryoprotectants. Thus, these hydrogels can serve as scaffolds with cryoprotective properties that also provide structural integrity to tissue constructs.

We report the novel one-step synthesis of a zwitterionic polymer, polysulfobetaine, via living reversible addition fragmentation chain transfer (RAFT) polymerization. Lysozyme did not aggregate when heated in the presence of this polymer. Amyloid formation, the cause of many diseases, was also suppressed. The zwitterionic polymer was significantly more efficient than previously described inhibitors of protein aggregation. Lysozyme heated in the presence of polysulfobetaine retained its solubility and very high enzymatic efficiency, even after prolonged heating. The secondary structures of lysozyme change with increasing temperature, accompanied by an increase in the β-structure. This change was prevented by mixing the polymer with lysozyme. ¹H-NMR before and after aggregation revealed the conformational changes taking place in the lysozyme: during aggregation, lysozyme is transformed into a random coil conformation, thus losing its secondary structure. Presence of the polymer facilitates retention of partial higher order structures and lysozyme solubility at higher temperatures. The high efficiency of the polyampholyte was ascribed to its ability to prevent collisions between aggregating species by acting as a molecular shield.