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.