Design of Stiff, Tough, and Fast-Recovery Protein Hydrogels

Abstract

Proteins are widely used in hydrogel materials due to their excellent biocompatibility, precisely controllable sequence structures, and diverse biochemical and mechanical properties. In recent years, numerous protein hydrogels with tailored mechanical performance have been developed to mimic the mechanical properties of biological tissues such as muscles and cartilages. However, systematic guidelines for the rational design of mechanical properties in protein hydrogels remain scarce. In this review, we comprehensively summarize recent advances in protein hydrogels and explore design strategies for various mechanical properties such as stiff, tough, and fast-recovery protein hydrogels by focusing on crosslinking and hydrogel networks. Subsequently, we briefly summarize the biomedical applications of protein hydrogels. Notably, we discuss the relationship between protein mechanics at the molecular level and bulk hydrogel properties, and highlight the potential of artificial intelligence in guiding protein building block construction and hydrogel design.

In 1998, Tirrell et al. first utilized recombinant proteins in hydrogel preparation by synthesizing reversible protein hydrogels through the physical interactions of leucine zipper domains. This was followed in 1999 by Jindřich Kopeček's development of hybrid hydrogels assembled from synthetic polymers and coiled-coil protein domains, jointly marking the inception of research into recombinant protein-engineered hydrogels. In 2002, David J. Mooney and colleagues covalently conjugated RGD peptide sequences to alginate, a polysaccharide hydrogel matrix, resulting in protein–polysaccharide hydrogels that significantly enhanced chondrocyte and osteoblast adhesion and proliferation, thereby promoting bone tissue regeneration and establishing a benchmark in tissue engineering. In the same year, Timothy J. Deming et al. synthesized diblock copolypeptide amphiphiles comprising charged and hydrophobic segments; the resulting hydrogels exhibited robust mechanical integrity at temperatures up to 90 °C and rapid recovery following stress relaxation. In 2003, David Baker and his team pioneered the field of de novo protein design by creating the protein Top7, later developing numerous de novo proteins for hydrogel fabrication that overcame the limited functionality of natural proteins and enabled precise control from molecular structure to macroscopic mechanics. In 2005, Dixon and Elvin prepared highly elastic hydrogels via photochemical crosslinking of recombinant resilin, catalyzing widespread interest in elastic protein-based materials. In 2006, Hubbell and colleagues incorporated cell-adhesive motifs and protease-sensitive sequences into recombinant protein backbones, which were chemically crosslinked with polyethylene glycol (PEG) to form hydrogels with enhanced biological activity and degradability—representing the first successful integration of functional recombinant protein domains with synthetic polymers. In 2008, Chilkoti et al. designed the first elastin-like polypeptides (ELPs)-based hydrogels by reacting ELPs with β-[tris(hydroxymethyl)phosphino] propionic acid, which were later developed into thermoresponsive, injectable protein hydrogels. In 2009, Heilshorn et al. introduced protein–protein interactions into hydrogel design, creating a mixing-induced two-component hydrogel (MITCH) based on WW domain and proline-rich motif interactions, enabling applications in neural stem cell culture. In 2010, Li et al. linked GB1 domains with resilin to mimic titin, yielding hydrogels with high toughness and fast recovery that reproduced the mechanical behavior of muscle; in 2023, they further advanced this platform to generate protein hydrogels with dense chain entanglement capable of simulating cartilage mechanics. Also in 2010, Khademhosseini demonstrated the strong potential of gelatin methacrylate (GelMA) as a micropatterned cell culture substrate that supported rapid adhesion, proliferation, and migration. In 2011, Kiick et al. developed modular recombinant resilin-like polypeptides (RLPs) and crosslinked them with β-[tris(hydroxymethyl)phosphino] propionic acid to produce hydrogels with excellent and tunable mechanical properties, establishing RLPs as promising bioactive materials. In 2012, Yang et al. engineered a tetrameric protein crosslinker with high affinity for peptide nanofiber termini, significantly enhancing the stiffness of supramolecular hydrogels. In 2015, DeForest et al. employed two orthogonal photochemical bio-click reactions to dynamically pattern proteins within three-dimensional hydrogels, enabling precise spatiotemporal control of stem cell differentiation. In 2017, Stevens et al. developed a β-sheet peptide–poly(γ-glutamic acid) hybrid hydrogel with tunable mechanics and self-healing properties through physical crosslinking by grafted β-sheet peptides; in the same year, Sun et al. reported a B12-dependent photoresponsive protein hydrogel designed for controlled release of stem cells and proteins, where polymeric CarHC proteins self-assembled into elastic hydrogels in the dark and disassembled upon light exposure—offering a general strategy for dynamically tunable protein materials. In 2018, Cao and his team established a framework for rational design and prediction of hydrogel mechanics by tuning the molecular stability of crosslinkers and load-bearing modules, leading to the creation of strong, tough, fast-recovering, and fatigue-resistant hydrogels via mechanisms including metal coordination, tandem crosslinkers, and force-triggered hidden length release; concurrently, Zhang et al. used split intein-mediated protein assembly to biosynthesize high molecular weight proteins like spider silk and mussel foot proteins, yielding materials with greatly enhanced mechanical properties. In 2020, Zhang et al. pioneered slide-ring protein hydrogels using artificially designed lasso proteins, demonstrating the utility of topological protein engineering for hydrogel design. From 2020 to 2024, Liu et al. developed a series of strong and tough protein-based materials, including fibers and bioadhesives, providing multiple strategies for high-performance protein material design. In 2021, Holten-Andersen et al. introduced an in situ mineralization strategy at metal coordination crosslinking sites—such as Ni²⁺/Cu²⁺–histidine and Fe³⁺–catechol—to enhance hydrogel stiffness and durability via nanoparticle nucleation. Most recently, in 2023–2024, He and colleagues strengthened gelatin hydrogels through cyclic mechanical training, creating anisotropic, hierarchical structures with high tensile strength, thus expanding the mechanical versatility of protein hydrogels.

Keywords

Protein hydrogels / Mechanical properties / Crosslinking / Hydrogel network / Molecular design / Protein folding / Noncovalent interactions / Applications

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2025 SIOC, CAS, Shanghai, & WILEY-VCH GmbH