Role of Crosslinker Chemistry and Comonomer Type on Cryopreservation Efficacies of Nanogels

Nonpermeating cryoprotective agents (CPAs) represent an alternative to reduce the dependence on small molecules such as dimethyl sulfoxide and glycerol that may cause toxic effects in sensitive cell lines. In this work, a library of lactone-modified poly(vitamin B5 analogous monomer (B5AMA))-based nanogels with systematically tuned amphipathicity and crosslinker chemistry were synthesized and evaluated as extracellular CPAs for the cryopreservation of human red blood cells and murine breast cancer cells. Three γ-hydroxy(amino)amide methacrylamide comonomers possessing variable alkyl chain lengths were prepared and incorporated into poly(B5AMA) nanogel networks utilizing three structurally distinct bifunctional crosslinkers. Nanogels were characterized by dynamic light scattering, zeta potential, and transmission electron microscopy, demonstrating sub-100 nm dimensions and a porous structure. The nanogel formulations prepared with different crosslinkers and comonomers possessed different volume phase transition temperatures and hence overall amphipathicity and demonstrated enhanced IRI activities relative to hydrophilic analogues. The nanogel synthesized with different crosslinkers clearly demonstrated the role of crosslinker amphipathicity in the cryopreservation efficacies of mammalian cells. Meanwhile, amphipathic formulations prepared by copolymerizing B5AMA with γ-hydroxy(amino)amide methacrylamide comonomers in the presence of a specific crosslinker demonstrated crosslinker- and comonomer-type-dependent cryopreservation efficacies in different cell types. Collectively, these findings demonstrate that amphipathicity, crosslinker chemistry, polymer network architecture, and macromolecular crowding jointly govern cryoprotective outcomes, positioning lactone-modified poly(B5AMA) nanogels as promising macromolecular CPAs for use in the preservation of cell-based therapies.