2D Janus peptide-nanostructured materials for enhanced cryopreservation

Abstract

Cryopreservation technology utilizes cryoprotective agents (CPAs) to mitigate cellular and tissue damage caused by ice crystal formation during freeze-thaw cycles, enabling the storage of biological materials at ultra-low temperatures. The increasing demand for cryopreservation in the food industry and biotechnology sectors has stimulated interest in techniques that enhance the organoleptic properties of food and preserve the viability of stem cells, gametes, cell therapeutics, and organs. While synthetic CPAs such as dimethyl sulfoxide, sodium phosphate, glycerol, polyethylene glycol, and polyvinyl alcohol are widely employed due to their scalability and cost-effectiveness, their cytotoxicity at high concentrations and potential to induce genetic modifications limit their application in high-value biomedical fields. Consequently, antifreeze proteins (AFPs) found in extremophilic organisms have emerged as potential alternative CPAs. AFPs exhibit ice-binding properties due to their unique structural characteristics, inhibiting ice recrystallization and growth by forming chemical bonds with specific ice crystal planes through strategically distributed functional groups. Furthermore, AFP-ice interactions modulate the ice-water interfacial energy, increasing chemical potential and inducing thermal hysteresis via the Gibbs-Thomson effect, thereby depressing the phase transition temperature. Despite these advantageous properties, the complex extraction processes and high costs associated with natural AFP production hinder their industrial application. Thus, there is a critical need for the development of synthetic CPAs that are easily synthesized, cost-effective, and highly biocompatible. This study focused on developing biomimetic CPAs inspired by the exceptional ice crystal growth inhibition mechanisms of AFPs. It is crucial to elucidate the relationship between the chemical functionality and structural characteristics of AFPs and their impact on ice crystal growth to design AFP-mimetic CPAs. While previous studies have primarily investigated ice recrystallization inhibition (IRI) activity through localized atomic interactions between ice crystal surfaces and ice-binding moieties (IBMs), this research presents novel nanostructures based on amphiphilic peptides that emulate AFP functionality at both macroscopic and atomic scales. The engineered amphiphilic peptides comprise hexa-phenylalanine, glutamic acid, and an alkyl chain, with IBMs at the hydrophilic terminus. The hexa-phenylalanine segment facilitates β-sheet formation through hydrogen bonding and enhances structural stability via π-π stacking interactions. Electrostatic repulsion between glutamic acid residues and hydrophobic effects from the alkyl tail contribute to the formation of two-dimensional (2D) nanostructures with high specific surface area. The strategic incorporation of IBMs in the hydrophilic region promotes ice-binding, while the hydrophobic segments restrict the access of surrounding water molecules, forming 2D Janus nanosheets. The formation of 2D nanostructures significantly enhanced IRI activity. Evaluation of IRI activity based on the nature of ice-binding residues revealed that amino acid residues capable of hydrogen bonding and hydrophobic interactions at the molecular scale exhibited the most effective IRI activity by synergistic effects. The adsorption process of amphiphilic peptide nanostructures on ice-binding surfaces was confirmed by observing anisotropic ice growth resulting from nanosheet-ice interactions. Adsorption mechanisms and ice-binding planes between peptides and ice were visualized using cryo in-situ imaging techniques, providing direct evidence of antifreeze effects. In conclusion, this study presents a strategy for developing structurally controlled amphiphilic peptide nanostructures as potent ice growth inhibitors by enhancing chemical and physical effects. These easily synthesized, cost-effective, and biocompatible amphiphilic peptides show promise as novel CPAs for preserving the physiological activity of reproductive cells, stem cells, tissues, and organs during cryopreservation, potentially revolutionizing biomedical preservation techniques.