The mechanics of the extracellular matrix (ECM) dynamically influences cancer cell behaviors and stem cell differentiation. Therefore, cell encapsulation systems capable of presenting dynamic mechanics are suitable for studying matrix-guided cell fate. Traditionally, temporally tunable crosslinking is achieved by incorporating photoresponsive, thermoreversible, or host-guest interactive linkers into the primary hydrogel network. In this study, an enzymatic reaction was used to crosslink and reversibly stiffen (Fig. 1A) poly(ethylene glycol) (PEG)-peptide hydrogels. Specifically, sortase A (SrtA), a cysteine transpeptidase that ligates an LPXTG and Gn peptide (where X is any amino acid except proline) was used due to its highly specific substrates and mild/predictable reaction kinetics. The gelation time was tuned over a broad scale (minutes to hours) and the gels could be fully degraded using the same enzymatic reaction for crosslinking. We further synthesized a bis-cysteine-bearing linear peptide with flanking SrtA substrates to enable multiple cycles of high-magnitude (ΔG’ ~2 kPa) reversible hydrogel stiffening/softening (Fig. 1B). The dynamically and reversibly stiffened/softened matrices were successfully used to investigate the effect of matrix stiffening/softening on pancreatic cancer cell growth and chemoresistance. Specifically, matrix stiffening restricted growth of cancer cell spheroids and reduced their susceptibility to gemcitabine treatment. Upon dynamic matrix softening, the majority of the cells remained alive following gemcitabine treatment. This suggests that local matrix degradation may not be an effective strategy as the cells would have been ‘de-sensitized’ to drug treatment after experiencing a period of stiffened matrix.

Dynamic control of hydrogel crosslinking through reversible sortase-mediated transpeptidation

2019 Spring National Meeting

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