Engineered Protein Hydrogels as Biomimetic Cellular Scaffolds

Abstract

The biochemical and biophysical properties of the extracellular matrix (ECM) play a pivotal role in regulating cellular behaviors such as proliferation, migration, and differentiation. Engineered protein-based hydrogels, with highly tunable multifunctional properties, have the potential to replicate key features of the native ECM. Formed by self-assembly or crosslinking, engineered protein-based hydrogels can induce a range of cell behaviors through bioactive and functional domains incorporated into the polymer backbone. Using recombinant techniques, the amino acid sequence of the protein backbone can be designed with precise control over the chain-length, folded structure, and cell-interaction sites. In this review, the modular design of engineered protein-based hydrogels from both a molecular- and network-level perspective are discussed, and summarize recent progress and case studies to highlight the diverse strategies used to construct biomimetic scaffolds. This review focuses on amino acid sequences that form structural blocks, bioactive blocks, and stimuli-responsive blocks designed into the protein backbone for highly precise and tunable control of scaffold properties. Both physical and chemical methods to stabilize dynamic protein networks with defined structure and bioactivity for cell culture applications are discussed. Finally, a discussion of future directions of engineered protein-based hydrogels as biomimetic cellular scaffolds is concluded.

Keywords: bioactive; biomimetic; engineered protein; hydrogel; peptide materials; stimuli‐responsive.

Figure 1.. Molecular- and network-level design considerations for biomimetic, engineered protein hydrogels.

Molecular-level design of recombinant protein backbones can include structural building blocks (e.g. elastin-like, silk-like, or resilin-like peptide sequences) to provide defined mechanical support and/or controlled chain-chain interactions, bioactive building blocks to interact with cell-surface receptors and cell-secreted proteases, and stimuli-responsive building blocks with on-demand, dynamic properties (e.g. in response to ions, light, or enzymes). Network-level design refers to the many different strategies that can be used to crosslink individual protein chains into a stable hydrogel. Physical crosslinking can occur either through self-interactions or interactions between two (or more) engineered components. Chemical crosslinking can be designed to occur by using the intrinsic chemical reactivity of the side chains of certain amino acid residues, by modifying the recombinant protein to present non-canonical chemical functional groups, or by designing peptide sequences into the recombinant protein backbone that are substrates for enzymatic ligation.

Figure 2.

A) Azide side groups used to modify and elastin-like polypeptides (ELP), with increasing hydrophilicity from left to right. B) Unmodified ELP and ELPs modified with their respective azide molecules present distinct lower critical solution temperature (LCST) behaviors as measured by optical density (λ = 300 nm). C) Human neural progenitor cells (hNPCs) exhibit increased spreading when encapsulated in ELP gels with decreased modulus. Reproduced with permission. Copyright 2023, John Wiley and Sons. D) Resilin-like polypeptides (RLP) are recombinant proteins with sequence similarity to native resilin that displays upper critical solution temperature (UCST) behavior. Schematic of photocrosslinkable RLPs and phase separating RLP to form microstructured hydrogels. E) Homogeneous gels were formed above the UCST and then cooled to induce phase separation prior to crosslinking into microstructure gels. Adapted with permission. Copyright 2023, American Chemical Society.

Figure 3.

A) Genetic constructs of recombinant RLP (named RGD-R32) and SRLP (named RGD-RS). B) Representative atomic force microscopy images of amorphous aggregates from RGD-R32 and self-assembled nanofibers from RGD-RS. C) Representative confocal images of the bone marrow mesenchymal stromal cells cultured on amorphous (RGD-R32) and fibrous (RGD-RS) hydrogels with different stiffness for 1 day. The cells were labeled to visualize their actin (Phalloidin-FITC, green) and nuclei (DAPI, blue). Scale bar: 50 μm. Reproduced with permission. Copyright 2022, American Chemical Society.

Figure 4.

A) Four-armed coiled-coil unit bound ELPs (CUBEs) reversibly form temperature-dependent hydrogels that can immobilize growth factors of angiogenic culture of HUVECs. B) Morphologies of HUVECs (red) cultured in CUBEs with soluble or immobilized basic fibroblast growth factor (bFGF) and vascular endothelial growth factor-165 (VEGF₁₆₅). Scale bars represent 100 μm. Reproduced with permission. Copyright 2023, American Chemical Society. C) A library of modular, engineered proteins was constructed with three different ELP domains and for polyalanine helices, resulting in different ratios of helical content from 6.25%-50%. D) Hydrogel microstructure (green) of E1-H5-25% demonstrating thermally-responsive porosity. Scale bars are 50 μm for the main image and 10 μm for the inset. Reproduced with permission. Copyright 2018, Springer Nature.

Figure 5.

A) Design schematic of SHIELD hydrogel with thermos-responsive polymer reinforcement. Component 1 is C7, an engineered protein composed of seven repeats of CC43 WW domains (C, pink) separated by hydrophilic spacers containing cell-adhesive peptides (green). Component 2 is a multi-arm, peptide copolymer, 8-armed polyethylene glycol (PEG) tethered with proline-rich peptides (P, purple) and a thermo-responsive polymer (PNIPAM). Copyright 2020, American Association for the Advancement of Science. B) Representative fluorescent images of Schwann cell morphology in SHIELD gels after 3 days in culture. Green, F-actin; blue, nuclei. Reproduced under the terms of the CC-BY license. Copyright 2020, American Association for the Advancement of Science. C) Design schematic of DnL hydrogels with light-induced photocrosslinking reinforcement. Dock peptides (blue) and Lock peptides (pink) hetero-assemble to form a network with methacrylate functional groups (green) for secondary covalent crosslinking. Copyright 2013, John Wiley and Sons. D) Mesenchymal cells encapsulated and cultured in DnL gels without (left) and with secondary photocrosslinking (right). Reproduced with permission. Copyright 2013, John Wiley and Sons. E) Design schematic of protein-engineered GL5 hydrogels with disulfide bond reinforcement. G_N (red) and G_C (blue) protein fragments hetero-assemble to form GL5 folded structures that can be reversibly stabilized by formation of disulfide bonds (yellow). Reproduced with permission. Copyright 2015, John Wiley and Sons.

Figure 6.

A) A silk protein modified with vinyl sulfone functional groups (SilkVS) has the potential to crosslink via three distinct mechanisms: thiol-ene Michael-type addition (green), enzymatically induced dityrosine formation (orange), and photocrosslinking (blue). The gelation kinetics were tracked using rheometer to monitor shear modulus. Reproduced with permission. Copyright 2023, Elsevier. B) Schematic of an ELP based hydrogel formed by bio-orthogonal cross-linking via a SPAAC reaction. Reproduced with permission. Copyright 2017, American Chemical Society. C) (Top) Schematic of protein hydrogels designed to have static, slow dynamic, or fast dynamic covalent crosslinks. (Bottom) Neural progenitor cells showed enhanced neurite outgrowth in dynamic fast stress-relaxing hydrogels. Reproduced under the terms of the CC-BY license. Copyright 2023, American Association for the Advancement of Science. D) Schematic of the block-copolymer design of two engineered proteins using ELP, SpyTag (A), SpyCatcher (B) and physically assembling P zipper (P) domains. When mixed together, the two proteins form hydrogels with both chemical and physical hydrogels. Reproduced with permission. Copyright 2023, American Chemical Society. E) Schematic of a hydrogel that incorporates de novo-designed protein building blocks. The first recombinant protein construct consists of a dimer core (blue) and complementary crosslinking unit (yellow). Hydrogel formation is achieved either by irreversible, covalent crosslinking with SpyCatcher/SpyTag or by reversible, non-covalent crosslinking through heterodimerization of the LHD101A/B pair. Reproduced under the terms of the CC-BY-NC-ND license. Copyright 2024, National Academy of Sciences.

Figure 7.

A) Schematic of hyaluronan and elastin-like protein (HELP) hydrogel (left). A library of engineered ELPs with different cell-adhesive peptide ligands were incorporated into the HELP hydrogels (middle). A representative digital quantification of neurite out-growth in a HELP hydrogel with RGD peptides (right). Reproduced with permission. Copyright 2023, American Chemical Society. B) Schematic of a hydrogel design that can be decorated with a protein ligand using assembly and disulfide bonding between G_N and G_C protein fragments. Decoration occurs under oxidizing conditions, while ligand release occurs under reducing conditions through the addition of glutathione (GSH). Reproduced with permission. Copyright 2019, Royal Society of Chemistry. C) Design of a coaxial binary ELR tubular scaffold with fast and slow degradation kinetics. The inner layer is fast degrading and conjugated with the VEGF-mimetic peptide (QK), while the outer layer is slow-degrading. D) Morphology of endothelial cells cultured on top of the ELR hydrogels with slow-degrading kinetics (Out) or fast-degrading kinetics with the QK peptide (In) over time. Reproduced with permission. Copyright 2021, Elsevier.

Figure 8.

A) Thermal-responsive hydrogels were created using elastin-like domains, resilin-like domains, and a globular GB1 domain. Upon heating, the elastin-like domains underwent thermal aggregation to reversibly strengthen the hydrogel. Reproduced with permission. Copyright 2023, American Chemical Society. B) Schematic of light responsive SELP sequence design. The proteins are polymerized through SpyTag-SpyCatcher chemistry and assembled into a hydrogel network through light sensitive AdoB₁₂ and CarH_C domain binding. Reproduced with permission. Copyright 2021, Elsevier.

Figure 9.

A) Enzyme-responsive hydrogels are formed by mixing PEG-tetraBCN with azide-flanked peptide crosslinkers that include sortase (SrtA) substrate sequences. Upon exposure to exogenous, substrate-matched SrtA, the gel becomes degraded. B) Spatiotemporally controlled release of HS5 human stromal cells from complex trilayered materials was achieved through multiplexed, sortase-based degradation. Reproduced with permission. Copyright 2023, John Wiley and Sons. C) SrtA-sensitive peptides can be designed into hydrogels to form reversible stiffening and softening biomimetic scaffolds upon application of SrtA or SrtA with a repetitive glycine (Gly) peptide, respectively. D) Cancer spheroids showed higher cell viability after gemcitabine treatment in stiffened gels, while lower cell viability in softened gels. Reproduced with permission. Copyright 2020, Elsevier.