Ice Recrystallization Inhibition via 2D Janus Peptide Nanosheets—A Molecular to Macroscales Approach

Abstract

Antifreeze proteins (AFPs) are crucial for the survival of polar organisms under subzero temperatures, as they selectively bind to specific planes of ice crystals and regulate formation and growth of the ice. To develop cryoprotective agents that effectively replicate the function of AFPs, it is essential to understand how their chemical functionalities and structural features influence ice crystal formation and growth. Previous studies have focused on the atomic-level interactions between ice-binding moieties (IBMs) and ice crystal surfaces to explain ice recrystallization inhibition (IRI) activity. Self-assembled nanostructures composed of amphiphilic peptides were designed to mimic the functional characteristics of AFPs both locally and structurally on a macro-scale and atomic-scale. The amphiphilic peptides are composed of hexa-phenylalanine, glutamic acid, and an alkyl tail, with the IBMs incorporated at the hydrophilic terminus. The hexa-phenylalanine segment facilitates β-sheet formation via hydrogen bonding and enhances structural stability through π-π stacking of aromatic rings. Simultaneously, the electrostatic repulsion between glutamic acid residues and the hydrophobic effect from the alkyl tail promotes the self-assembly of peptides into two-dimensional (2D) nanostructure with a high specific surface area. The presence of IBMs at the hydrophilic segment enhances interaction with ice, whereas the hydrophobic face acts as a barrier that limits water penetration. These features give rise to well-defined 2D Janus nanosheets. The formation of 2D nanostructures markedly enhanced ice recrystallization inhibition (IRI) activity. Assessment of IRI activity across different ice-binding residues showed that those enabling both hydrogen bonding and hydrophobic interactions led to the highest inhibition efficiency via synergistic molecular effect. Anisotropic ice crystal growth was observed in the presence of amphiphilic peptide nanosheets, indicating interaction between the nanosheet and the ice. Ice nucleation occurred at a lower temperature in the presence of amphiphilic peptide nanosheets than in pure water, implying their modulatory effect on ice formation. Cryo in-situ imaging techniques enabled direct visualization of the adsorption behavior and ice-binding interfaces between the peptides and ice, offering clear structural evidence of their antifreeze activity. Amphiphilic peptide nanoagents can induce chemical interactions at the atomic and molecular scales, mimicking the structural function of natural AFPs by 2D structural formation. These results suggest that self-assembled 2D amphiphilic peptide nanostructures serve as potent ice growth inhibitors and represent a cryoprotective agents for the preservation of sperm, oocytes, stem cells, tissues, and organs.