Adding Dynamic Biomolecule Signaling to Hydrogel Systems via Tethered Photolabile Cell-Adhesive Proteins

Abstract

Sequential biochemical signaling events direct key native tissue processes including disease progression, wound healing and angiogenesis, and tissue regeneration. While in vitro modeling of these processes is critical to understanding endogenous tissue behavior and improving therapeutic outcomes, current models inadequately recapitulate the dynamism of these signaling events. Even the most advanced current synthetic tissue culture constructs are restricted in their capability to sequentially add and remove the same molecule to model transient signaling.Here, we developed a genetically encoded method for reversible biochemical signaling within poly(ethylene glycol) (PEG)-based hydrogels for greater accuracy of modeling tissue regeneration within a reductionist environment. We designed and implemented a recombinant protein with a SpyCatcher domain connected to a cell-adhesive RGDS peptide domain by a light-cleavable domain known as PhoCl. This protein was shown to bind to SpyTagfunctionalized PEG-matrices via SpyTag-SpyCatcher isopeptide bonding to present RGDS adhesive ligands to cells. Upon 405 nm light exposure, the PhoCl domain was cleaved to subsequently release the RGDS peptide, which diffused out of the matrix. This system was implemented to confer reversible adhesion of 3T3 fibroblasts to the PEG-based hydrogel surface in 2D culture (73.36 ± 21.47% cell removal upon cell-compatible light exposure) and temporal control over cell spreading over time in 3D culture within cell-degradable PEG-based hydrogels, demonstrating the capability of this system to present dynamic signaling events to cells toward modeling native tissue processes within in a controlled, ECM-mimetic matrix.

Bioactive Polyurethane–Poly(ethylene Glycol) Diacrylate Hydrogels for Applications in Tissue Engineering

“…HDF adhesion on photocured 5% w/v PUDA (Figure 5a, panel (i)) and PUDA-PQ films (Figure 5a, panel (ii)) is lower than that observed on 5% w/v PEG-PUDA (Figure 5a, panel (iii)) and PEG-PUDA-PQ (Figure 5a, panel (iv)) hydrogels, likely due to hindered presentation of the PEG-RGDS within the thin films. Within the water-swollen 3D matrix of the hydrogels, the PEGylated RGDS is grafted into the hydrogel backbone, leaving the adhesion peptide free to interact with cells [3,4,66], which has been shown to increase cell spreading, migration, and survival [45,[67][68][69][70][71][72]. HDFs under all conditions show an elongated morphology, indicating cell spreading and significant levels of adhesion through interactions of cell-surface integrins with RGDS.…”

4D Biochemical Photocustomization of Hydrogel Scaffolds for Biomimetic Tissue Engineering

“…Tethered PhoCl-epidermal growth factor (EGF) chimera proteins were selectively released from hydrogels to spatially confine spheroid formation in HeLa cells (Figure e). A follow-up study from the West lab replaced the chemoenzymatically introduced azide cross-linking handle with an N-terminal SpyCatcher, offering a fully canonical protein-based approach through immobilization in a SpyTag-decorated gel …”

“…A follow-up study from the West lab replaced the chemoenzymatically introduced azide cross-linking handle with an N-terminal SpyCatcher, offering a fully canonical protein-based approach through immobilization in a SpyTag-decorated gel. 51…”

PhoCoil: An Injectable and Photodegradable Single-component Recombinant Protein Hydrogel for Localized Therapeutic Cell Delivery

“…PhoCl is a photocleavable protein whose peptide backbone undergoes irreversible cleavage at the chromophore in response to 405 nm light, providing both a cytocompatible and highly bioorthogonal trigger for 4D control of protein cleavage (10)(11)(12). Previous work from our group and others has shown PhoCl's utility in introducing photoresponsivity to hydrogels (13)(14)(15). The resulting material, referred to as "PhoCoil", achieves the intended combination of chemical, physical, and stimuli-responsive properties (biocompatible, injectable, photodegradable) which are all directly specified in its molecular design.…”