Dynamic control of hydrogel crosslinking via sortase-mediated reversible transpeptidation

Abstract

Cell-laden hydrogels whose crosslinking density can be dynamically and reversibly tuned are highly sought-after for studying pathophysiological cellular fate processes, including embryogenesis, fibrosis, and tumorigenesis. Special efforts have focused on controlling network crosslinking in poly(ethylene glycol) (PEG) based hydrogels to evaluate the impact of matrix mechanics on cell proliferation, morphogenesis, and differentiation. In this study, we sought to design dynamic PEG-peptide hydrogels that permit cyclic/reversible stiffening and softening. This was achieved by utilizing reversible enzymatic reactions that afford specificity, biorthogonality, and predictable reaction kinetics. To that end, we prepared PEG-peptide conjugates to enable sortase A (SrtA) induced tunable hydrogel crosslinking independent of macromer contents. Uniquely, these hydrogels can be completely degraded by the same enzymatic reactions and the degradation rate can be tuned from hours to days. We further synthesized SrtA-sensitive peptide linker (i.e., KCLPRTGCK) for crosslinking with 8-arm PEG-norbornene (PEG8NB) via thiol-norbornene photocrosslinking. These hydrogels afford diverse softening paradigms through control of network structures during crosslinking or by adjusting enzymatic parameters during on-demand softening. Importantly, user-controlled hydrogel softening promoted spreading of human mesenchymal stem cells (hMSCs) in 3D. Finally, we designed a bis-cysteine-bearing linear peptide flanked with SrtA substrates at the peptide's N- and C-termini (i.e., NH₂-GGGCKGGGKCLPRTG-CONH₂) to enable cyclic/reversible hydrogel stiffening/softening. We show that matrix stiffening and softening play a crucial role in growth and chemoresistance in pancreatic cancer cells. These results represent the first dynamic hydrogel platform that affords cyclic gel stiffening/softening based on reversible enzymatic reactions. More importantly, the chemical motifs that affords such reversible crosslinking were built-in on the linear peptide crosslinker without any post-synthesis modification. STATEMENT OF SIGNIFICANCE: Cell-laden 'dynamic' hydrogels are typically designed to enable externally stimulated stiffening or softening of the hydrogel network. However, no enzymatic reaction has been used to reversibly control matrix crosslinking. The application of SrtA-mediated transpeptidation in crosslinking and post-gelation modification of biomimetic hydrogels is innovative because of the specificity of the reaction and reversible tunability of crosslinking kinetics. While SrtA has been previously used to crosslink and fully degrade hydrogels, matrix softening and reversible stiffening of cell-laden hydrogels has not been reported. By designing simple peptide substrates, this unique enzymatic reaction can be employed to form a primary network, to gradually soften hydrogels, or to reversibly stiffen hydrogels. As a result, this dynamic hydrogel platform can be used to answer important matrix-related biological questions that are otherwise difficult to address.

Keywords: Cancer; Dynamic hydrogels; Extracellular matrix; Sortase A; Tissue stiffening.

Figure 1.

(A) Schematic of reversible SrtA-transpeptidation reaction. SrtA cleaves amide bond between threonine and glycine residues of LPRTG substrate and forms a thioacyl intermediate. Nucleophilic oligoglycine or water resolve intermediate. Note that glycine-containing product, LPRT(G)_n, can undergo multiple cycles of transpeptidation with additional incubation with SrtA and oligoglycine. (B) SrtA-mediated gelation of PEG-peptide hydrogels. SrtA was used to initiate crosslinking between PEG-peptide conjugates. Cysteine containing SrtA substrates (i.e., CLPRTG and GGGGC) were conjugated to PEG-NB in the presence of UV light and photoinitiator LAP. (C) Test tube tilt test to track timing of sol-gel transition using 6 wt% PEG8NB-peptide conjugates, R_GGGG:LPRTG=2). Eosin-Y (1 mM, red dye) was added for image clarity.

Figure 2.

Effect of (A) SrtA and (B) glycinamide concentration on the transpeptidation degradation of PEG-peptide hydrogels (6 wt% PEG-peptide, R_GGGG:LPRTG=2 with 48 mM glycinamide and 50 μM SrtA for (A) and (B), respectively. (C) Effect of SrtA concentration on the hydrolytic degradation of PEG-peptide hydrogels (6 wt%, R_GGGG:LPRTG=2).

Figure 3.

(A) Schematic of SrtA-mediated peptide cleavage. (B) Effect of alanine:glycine composition on softening of PEG8NB-A-G gels (3.0 wt% PEGNB, 25μM SrtA, & 12mM GGGGC). (C) Effect of SrtA concentration on softening of PEG8NB-A-G gels (50%A:50%G, 3.0 wt%, 12mM GGGGC). (D) Effect of initial PEG8NB concentration on softening of PEG8NB-A-G gels (50%A:50%G, 25μM SrtA, & 12mM GGGGC). (E) Effect of treatment time on softening (50%A:50%G, 3.0 wt%, 12mM, 25μM SrtA, and 12mM GGGGC).

Figure 4.

(A) Representative confocal images of encapsulated hMSCs in statically stiff and softened hydrogels. At least three z-stacked images per gel (10 slices, 100 μm thick) were taken. (Scale: 200 μm). (B) Circularity and (C) average cell area measurements of hMSCs in the statically stiff and softened hydrogels on day 14 post-encapsulation.

Figure 5.

(A) Schematic of SrtA-mediated reversible stiffening. (B) Effect of incubation time on stiffening of PEG-peptide hydrogels (2.5 wt% PEGNB, 25 μM SrtA). (C) Effect of SrtA concentration on stiffening of PEG-peptide hydrogels (2.5 wt% PEGNB, 4 hr incubation time). (D) Effect of glycine concentration on softening of PEG-peptide hydrogels (2.5 wt% PEGNB, 25 μM SrtA). (E) SrtA-mediated hydrolytic degradation of PEG-peptide hydrogels (2.5 wt% PEGNB, 16 hour incubation).

Figure 6.

Cyclic stiffening and softening of PEG-peptide hydrogels (2.5 wt% PEGNB). Alternating stiffening and softening correspond to 4 hr. incubations with SrtA (25 μM) and SrtA with glycinamide (15 mM), respectively.

Figure 7.

(A) Timeline of alternate stiffening and softening of COLO-357-laden hydrogels. Hydrogels were stiffened on day 7 and softened on day 14. Time of confocal imaging is indicated by the open arrows. All imaging was completed prior to enzyme treatments (B) Representative confocal images of encapsulated COLO-357 cells in statically soft, stiff, and reversibly stiffened hydrogels. At least three z-stacked images per gel (10 slices, 100 μm thick) were taken. (Scale: 200 μm). Histogram of spheroids diameters for (C) non-dynamic soft, (D) non-dynamic stiff, and (E) reversibly stiffened and softened hydrogels (i.e., soft-stiff-soft).

Figure 8.

(A) Timeline of gel stiffening (at D4) and softening (at D7), as well as three-day gemcitabine treatment (D10-D13). All imaging was completed prior to enzyme or drug treatments. (B-E) Representative live/dead images of cell-laden non-dynamic soft (B), non-dynamic stiff (C), stiffened (D), and softened (E) hydrogels with and without gemcitabine treatment. At least three z-stacked images per gel (10 slices, 100 μm thick) were taken. (Scale: 200 μm). (F) Metabolic activity of encapsulated cells pre- (at D10) and post-gemcitabine treatment (at D13). Data represent Mean ± SEM (n=3, *p<0.05, **p<0.01).