Rational engineering of an antifreeze-based biomaterial with dual-enhanced ice-binding and delivery for
Yishan Fu1, Yangyang Li1, Fei Pan2
1Science Center for Future Foods, Jiangnan University, Wuxi 214122, China; School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Introduction:
Reliable cell cryopreservation is essential for biomedical research and cell-based therapies. Although dimethyl sulfoxide (DMSO) is widely used as a cryoprotectant, its cytotoxicity and clinical incompatibility limit its application, creating an urgent need for safer alternatives. Antifreeze proteins (AFPs) offer favorable biocompatibility and reduced cytotoxicity, making them promising candidates for next-generation cryoprotectants.
Objectives:
This study aims to develop a high-performance cryoprotective biomaterial via rational engineering of snow flea AFP (SfAFP), establish an effective DMSO-free cryopreservation strategy, and verify its applicability across multiple cell lines.
Methods:
rSfAFP-MU was generated by site-directed mutagenesis (T16G, A19G, S22G) of SfAFP. Comprehensive physicochemical (thermal hysteresis, particle size, hydrophobicity, morphology) and functional (EA.hy926 post-thaw recovery) characterizations were performed. Molecular dynamics simulations identified key ice-binding interfaces, guiding alanine substitutions at D75, P78, P81 to test geometry. To enhance intracellular delivery, the TAT peptide was fused to rSfAFP-MU's N-terminus via a (G4S)3 linker (yielding rTAT-GS-MU), whose localization was confirmed by confocal microscopy. Cryopreservation efficacy was tested in EA.hy926, A549, and HEK293 cells.
Results:
The rSfAFP-MU mutant demonstrated significantly enhanced thermal hysteresis (5 °C vs. 2.19 °C for wild-type SfAFP) and increased EA.hy926 post-thaw recovery by 10.20 % compared to SfAFP. Molecular dynamics simulations revealed that enhanced ice-binding affinity stems from optimized hydrophobic surface intersection angles between monomers, a finding validated by activity reductions in targeted mutants. Confocal imaging confirmed efficient intracellular uptake of rTAT-GS-MU, which correlated with improved cryopreservation efficacy. rTAT-GS-MU achieved 74.77 % EA.hy926 post-thaw recovery (comparable to DMSO) with improved mitochondrial and nuclear DNA integrity and demonstrated efficacy in A549 and HEK293 cells.
Conclusion:
Through rational design, we converted a natural AFP into a high-performance bioactive material that enables efficient cryopreservation of multiple cell lines. This DMSO-free strategy addresses key limitations of traditional methods, offering a valuable alternative for biomedical use.
