Orthogonal enzymatic reactions for rapid crosslinking and dynamic tuning of PEG–peptide hydrogels

Abstract

Stiffening of the extracellular matrix is a hallmark in cancer progression, embryonic development, and wound healing. To mimic this dynamic process, our work explored orthogonal enzymatic reactions capable of modulating the properties of poly(ethylene glycol) (PEG)-peptide hydrogels. A hepta-mutant bacterial transpeptidase sortase A (SrtA) was used to ligate two PEG-peptide macromers (i.e., PEG-YLPRTG and NH-GGGG-PEG) into a primary hydrogel network. The hydrogels were dynamically stiffened using mushroom tyrosinase (MT), which oxidized tyrosine residues into di-tyrosine and led to increased matrix stiffness. After confirming the expression and enhanced catalytic activity of SrtA, we investigated the cytocompatibility of the enzymatic reaction with a mouse insulinoma cell line, MIN6. In addition, we altered peptide substrate concentrations and evaluated their influence on primary hydrogel network properties and MT-triggered stiffening. Using a pancreatic cancer cell line, COLO-357, the effect of MT-triggered stiffening on spheroid formation was investigated. We found that cell spheroids formed in hydrogels that were exposed to MT were significantly smaller than spheroids formed without MT incubation, suggesting that matrix stiffening played a crucial role in the sizes of cancer cell spheroids. Through utilizing highly specific and orthogonal enzymatic reactions, this hydrogel platform permits rapid and mild in situ cell encapsulation, as well as dynamic control of matrix stiffness for investigating the role of matrix stiffening on cell fate processes.

Linkage Groups within Thiol–Ene Photoclickable PEG Hydrogels Control In Vivo Stability

“…In addition, thiol–ene PEG hydrogels exhibit high cytocompatibility, precise spatiotemporal control of gelation, tunable degradation, and versatile modulation of biophysical and biochemical properties. The versatility of these hydrogels is demonstrated through extensive applications, ranging from protein and drug delivery, cartilage development, osteogenic differentiation, endothelial tubulogenesis, brain tumor models, synthetic matrix mimics, 2D culture substrates, and islet and cell encapsulation . However, these applications have been exclusively conducted in vitro, yielding little insight into the in vivo behavior of these hydrogels.…”

Enzyme‐catalysed polymer cross‐linking: Biocatalytic tools for chemical biology, materials science and beyond

“…[45,46] SrtA catalysed polymer cross-linking has been used for the generation of hydrogels for human tissue culture, as first demonstrated by Arkenberg and Lin in 2017. [38] In this study, a non-calcium-dependent mutant of S.…”

“…These proteins are stable, and do not exhibit a loss in enzyme activity following incubation at room temperature for >24 hours. [38,39] The ability of sortases to proficiently fuse proteins or peptides to poly(glycine) containing substrates has encouraged researchers to explore the use of these enzymes across a range of application areas. [40][41][42] It should be noted that the enzyme in most of these applications is linking linear segments to create a longer chain rather than catalysing cross-linking between chains through non-terminal locations.…”

“…also been shown to be comparable to those produced by Matrigel, or chemically cross-linked PEG-based hydrogels. [38,39] One additional intriguing observation is that SrtA may also be employed as an agent of hydrogel dissolution ( Figure 4). [47] Valdez et al…”

“…[77] Of the enzyme classes mentioned in this review, trans- the ability to mimic ECM stiffening. [38] Initial cross-linking of the PEG polymers was performed using SrtA to establish a hydrogel network, which was subsequently stiffened by the introduction of additional cross-links using tyrosinase. The secondary rigidification of the hydrogel scaffold closely mimics effects observed in the ECM during cancer progression and wound healing.…”