Charge‐Tuning Ice Inhibition in Antifreeze Peptides

ABSTRACT Antifreeze peptides (AFPTs) have garnered significant interest as promising ice growth inhibitors. However, the rational design of AFPTs is hindered by the unclear functional roles of non‐ice‐binding sites (NIBSs), especially whether the charge‐specific effect on ice inhibition exists. Here, we design a series of charged AFPTs and clarify the hidden contributions of the “dark matter” NIBSs, revealing their essential charge impact on the cryoprotective performance. Moderate net negative charges of NIBSs create a hydration‐mediated interfacial environment that facilitates AFPT adsorption onto ice crystals and enhances ice growth inhibition. However, excessive negative charges induce overly strong hydration layers that hinder effective interfacial contact and reduce solution pH, thereby leading to cytotoxicity. Positive charges promote ordered water arrangements conducive to ice growth. The optimal charged AFPT (a net charge of ‐2) exhibits a 3.5‐fold ice growth inhibition rate compared with other AFPTs, as well as possessing biocompatibility, thus enhancing the post‐thaw red blood cells recovery rate to above 93%. This work offers new insights on elucidating the structure‐activity relationship of AFPTs and provides a promising approach for developing a next‐generation cryoprotectant in clinical applications.