Preparation of Mechanically Stable Self-Assembled Peptides Hydrogels

Through the design of peptide sequences, non-covalent interactions between peptides induce self-assembly, leading to the formation of ordered micro- and nanometer structures, including nanotubes, nanoribbons, nanofibers, and spherical structures¹. When self-assembled into micro- and nanometer fibers/ribbons, these structures macroscopically exhibit hydrogel properties. Peptide self-assembling hydrogels differ from polymer hydrogels in that they self-assemble through non-covalent interactions, their gel form is reversible, and they readily respond to specific conditions to transition between solution and gel phases². For instance, aromatic amino acid peptides can be induced to gelatinize based on solvent switching^3,4,5, RADA16 peptides form gels through cationic and anionic electrostatic interactions⁶, and E1Y9 peptide is induced to form a hydrogel via Ca²⁺ ions⁷. Natural amino acids can be metabolized by the human body and offer excellent biocompatibility, a feature that polymer hydrogels cannot achieve⁸. Proteins are the molecules that execute biological functions, and differences in peptide sequences create their specific biological functions. Therefore, embedding specific biofunctional peptide sequences and endowing them with self-assembling properties can design peptide self-assembling hydrogels with unique biological functions and morphologies^9,10,11. This article introduces three methods for preparing peptide hydrogels, where the gelation process is triggered by environmental responsiveness. It also briefly discusses methods for characterizing the mechanical properties and morphology of peptide hydrogels.

The pH regulates the charge of amino acids, triggering the gelation of some peptides. For instance, positively charged amino acids (e.g., arginine, lysine, histidine) are regulated by pH to attain positive or neutral states. Negatively charged amino acids are regulated by pH to achieve negative or neutral states, moving away from their isoelectric point and thereby altering their hydrophilicity in aqueous solutions. Therefore, controlling electrostatic and hydrophobic interactions between peptides facilitates their ordered self-assembly. Zhang et al. designed an amphiphilic pH-responsive self-assembling peptide, methotrexate-coupled KKFKFEFEF, which responds to slightly acidic environments both in vitro and in vivo, enabling a sol-to-gel phase transition. This leads to efficient cellular uptake and endocytosis, thereby delivering anti-cancer drugs and improving chemotherapy effectiveness¹². Shen et al.¹³ designed the FF8 (KRRFFRRK) peptide, which easily self-assembles into fibers at a pH greater than 9.4. Under neutral conditions, microorganisms neutralize their positive charges due to electrostatic interactions with their negatively charged phospholipid membranes, coordinating with phospholipid molecules to self-assemble, causing membrane rupture and enhancing bactericidal effects¹³.

Triggering peptide supramolecular self-assembly into hydrogels using coordination metals is a relatively rare method¹⁴. When metal ions interact electrostatically with peptides, they form salt bridges that connect peptide molecules, leading to non-covalent interactions and self-assembly, which results in gelation properties. For example, Abul-Haija et al.¹⁵ designed the tripeptide FFD, which transitions from a liquid to a hydrogel upon the addition of copper ions. Tao et al.¹⁶ developed the glutamic acid and phenylalanine-rich peptide E3F3, which self-assembles into fibrous hydrogels in the presence of zinc ions, and is used for prostate drug delivery.

Solvent exchange formation of peptide hydrogels is the most common supramolecular self-assembly triggering condition. After hydrophobic peptides dissolve in organic solvents, their hydrophobic groups are fully exposed. When transferred to an aqueous phase, the hydrophobic groups approach each other, and water molecules facilitate the formation of peptide hydrogen bonds, leading to rapid self-assembly and easy formation of hydrogels. For instance, Zhang et al.¹⁷ designed a peptide that could dissolve stably at high concentrations in polar organic solvents and, upon dilution with water, self-assembled into β-sheet structures to form peptide fiber hydrogels. Shen et al.¹³ designed a reductive peptide ECF-5 (ECAFF), pre-dissolved in dimethyl sulfoxide (DMSO) and then injected into an aqueous phase to form a reductive hydrogel, used for the targeted removal of reactive oxygen species produced by ischemia-reperfusion, which subsequently degraded into a solution after scavenging.

This study selected three simple, rapid, and highly generalizable peptide hydrogel preparation strategies based on previous experiences: (1) pH response method: peptides are dissolved in a solution with a pH far from their isoelectric point, and then the pH is adjusted to near the isoelectric point. This change allows certain self-assembling peptides to form fibers and create peptide hydrogels; (2) Metal ion addition method: coordination cations are added to water-soluble, negatively charged self-assembling peptides. The metal coordination chelation between peptides leads to their self-assembly into hydrogels; (3) Solvent exchange method: high-concentration peptides are dissolved in organic solvents and then diluted into an aqueous phase, inducing gelation behavior.