ArticleJournal of advanced research2026
Rational engineering of an antifreeze-based biomaterial with dual-enhanced ice-binding and delivery for high-efficiency cryopreservation.
Article in Journal of advanced research, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
introductionReliable 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.
objectivesThis 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.
methodsrSfAFP-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 (G
resultsThe 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.
conclusionThrough 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.
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