Rational engineering of an antifreeze-based biomaterial with dual-enhanced ice-binding and delivery for high-efficiency cryopreservation

  • J Adv Res. 2025 Dec 8:S2090-1232(25)00998-1. doi: 10.1016/j.jare.2025.12.008.
Yishan Fu  1 ,  Yangyang Li  1 ,  Fei Pan  2 ,  Sahibzada Muhammad Aqeel  1 ,  Song Liu  3 ,  Jian Chen  4
Affiliations
  • 1. Science Center for Future Foods, Jiangnan University, Wuxi 214122, China; School of Biotechnology, Jiangnan University, Wuxi 214122, China.
  • 2. State Key Laboratory of Resource Insects, Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing 100093, China.
  • 3. Science Center for Future Foods, Jiangnan University, Wuxi 214122, China; School of Biotechnology, Jiangnan University, Wuxi 214122, China; JiaXing Institute of Future Food, Jiaxing, Zhejiang 314000, China. Electronic address: [email protected].
  • 4. Science Center for Future Foods, Jiangnan University, Wuxi 214122, China; School of Biotechnology, Jiangnan University, Wuxi 214122, China. Electronic address: [email protected].
PMID: 41371403 DOI: 10.1016/j.jare.2025.12.008
Abstract

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.

Keywords
Cryopreservation; Snow flea antifreeze protein; Structural optimization; Transmembrane delivery; Universality.
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