Cold-Responsive Nanoparticle Enables Intracellular Delivery and Rapid Release of Trehalose for Organic-Solvent-Free Cryopreservation

Conventional cryopreservation of mammalian cells requires the use of toxic organic solvents (e.g., dimethyl sulfoxide) as cryoprotectants. Consequently, the cryopreserved cells must undergo a tedious washing procedure to remove the organic solvents for their further applications in cell-based medicine, and many of the precious cells may be lost or killed during the procedure. Trehalose has been explored as a nontoxic alternative to traditional cryoprotectants. However, mammalian cells do not synthesize trehalose or express trehalose transporters in their membranes, and the lack of an approach for the efficient intracellular delivery of trehalose has been a major hurdle for its use in cell cryopreservation. In this study, a cold-responsive polymer (poly(N-isopropylacrylamide-co-butyl acrylate)) is utilized to synthesize nanoparticles for the encapsulation and intracellular delivery of trehalose. The trehalose-laden nanoparticles can be efficiently taken up by mammalian cells. The nanoparticles quickly and irreversibly disassemble upon cold treatment, enabling the controlled and rapid release of trehalose from the nanoparticles inside cells. The latter is confirmed by an evident increase in cell volume upon cold treatment. This rapid cold-triggered intracellular release of trehalose is crucial to developing a fast protocol to cryopreserve cells using trehalose. Cells with intracellular trehalose delivered using the nanoparticles show comparable postcryopreservation viability compared to that of cells treated with DMSO, eliminating the need for the tedious and cell-damaging washing procedure required for using the DMSO-cryopreserved cells in vivo. This cold-responsive nanoparticle may greatly facilitate the use of trehalose as a nontoxic cryoprotectant for banking cells and tissues to meet their high demand by modern cell-based medicine.