Click chemistry functionalization of self-assembling peptide hydrogels

Abstract

Self-assembling peptide (SAP) hydrogels provide a fibrous microenvironment to cells while also giving users control of biochemical and mechanical cues. Previously, biochemical cues were introduced by physically mixing them with SAPs prior to hydrogel assembly, or by incorporating them into the SAP sequence during peptide synthesis, which limited flexibility and increased costs. To circumvent these limitations, we developed "Click SAPs," a novel formulation that can be easily functionalized via click chemistry thiol-ene reaction. Due to its high cytocompatibility, the thiol-ene click reaction is currently used to crosslink and functionalize other types of polymeric hydrogels. In this study, we developed a click chemistry compatible SAP platform by addition of a modified lysine (lysine-alloc) to the SAP sequence, enabling effective coupling of thiol-containing molecules to the SAP hydrogel network. We demonstrate the flexibility of this approach by incorporating a fluorescent dye, a cellular adhesion peptide, and a matrix metalloproteinase-sensitive biosensor using the thiol-ene reaction in 3D Click SAPs. Using atomic force microscopy, we demonstrate that Click SAPs retain the ability to self-assemble into fibers, similar to previous systems. Additionally, a range of physiologically relevant stiffnesses can be achieved by adjusting SAP concentration. Encapsulated cells maintain high viability in Click SAPs and can interact with adhesion peptides and a matrix metalloproteinase biosensor, demonstrating that incorporated molecules retain their biological activity. The Click SAP platform supports easier functionalization with a wider array of bioactive molecules and enables new investigations with temporal and spatial control of the cellular microenvironment.

Keywords: click chemistry; extracellular matrix; hydrogel; matrix metalloproteinase; self-assembling peptide.

FIGURE 1

Diagram of KFE‐alloc structure and thiol‐ene reaction. (A) Chemical structure of KFE‐Alloc with side‐chain charges indicated. (B) Thiol‐ene click chemistry reaction forms a covalent bond between a thiolated bioactive molecule and KFE‐alloc SAP. SAP, self‐assembling peptide

FIGURE 2

TAMRA dye clicked into SAP hydrogels using thiol‐ene chemistry. Thiol‐ene click chemistry (initiated by UV light) causes an increase in TAMRA retention in alloc‐modified SAP hydrogels compared to gels in which the reaction is not initiated. Mean ± SD, **p < .01 versus no UV control. SAP, self‐assembling peptide; TAMRA, 5(6)‐carboxytetramethylrhodamine

FIGURE 3

SAP mechanical properties are sensitive to peptide concentration but not RGD incorporation by click chemistry. (A) Incorporating RGD into Click SAPs via click chemistry does not significantly change the elastic moduli of the hydrogels, regardless of whether RGD is incorporated into the Click SAP before or after assembly. The densities of KFE‐RGD and KFE‐Alloc used are equivalent at 0.78 mg/ml. Mean ± SD, N = 3–4 experiments, 2 gels per experiment. No significant differences found. (B) Storage moduli of gels containing constant 0.54 mM KFE‐alloc and clicked‐in RGD increases with KFE concentration. SAP, self‐assembling peptide

FIGURE 4

SAP modification does not interfere with fiber assembly. Atomic force microscopy images of 0.1 mg/ml aqueous solutions of (A) pure KFE, (B) pure KFE‐alloc, and (C) a 50/50 mixture of KFE and KFE‐alloc fibers show that modifying the KFE sequence with an alloc group does not inhibit the formation of fibers. Scale bar = 250 nm. Color bar represents the height of each pixel. Images taken after KFE‐alloc was then functionalized with (D) TAMRA, (E) RGD, and (F) QGIW biosensor peptide demonstrate that clicking in these molecules also does not interfere with the formation of a fibrous network. SAP, self‐assembling peptide; TAMRA, 5(6)‐carboxytetramethylrhodamine

FIGURE 5

Incorporation of RGD into SAP gels via click chemistry promotes cell spreading in 2D. (A) Representative images of HT1080 fibrosarcoma cells cultured for 48 h in 2D on top of SAP gels containing non‐adhesive KFE‐RDG or adhesive KFE‐RGD, on TCP, or on gels containing RGD incorporated via thiol‐ene reaction (Click SAP) with and without UV initiation. Actin cytoskeleton labeled with fluorescent phalloidin. Scale bar = 100 μm. (B) Plot indicates the mean and standard deviation of the major axis length on each type of gel. N = 4 replicate experiments. *p < .05 via one‐way ANOVA with Tukey multiple comparisons post‐test. Not all significances shown. (C) Empirical cumulative distribution functions of the major axis lengths of all cells on each substrate across all four replicate experiments. SAP, self‐assembling peptide; TCP, tissue culture plastic; TAMRA, 5(6)‐carboxytetramethylrhodamine

FIGURE 6

Multiple cell types remain highly viable during SAP encapsulation and click reaction. HT1080 cells (A and B) and MSCs (C and D) were encapsulated in SAP gels with RGD clicked in to measure the effects of the SAP, 60 seconds of UV light, and the click reaction on cell viability. Cells were also encapsulated in Matrigel as a control, along with SAPs that were not exposed to UV. 24 h after encapsulation and clicking in RGD, a live/dead assay was performed to quantify the number of live cells (green) and dead cells (red). A custom CellProfiler pipeline was written to quantify the overall percentage of live cells in each experiment (A, C). Points indicate the mean ± SD, N = 3–4 replicate experiments, *p < .05, **p < .01. Representative images demonstrate the effects of gel conditions on cell viability (B, D). Scale bar = 250 μm. MSC, mesenchymal stem cells; SAP, self‐assembling peptide

FIGURE 7

Incorporation of RGD into SAP gels via click chemistry promotes stem cell spreading in 3D. (A) Representative images of MSCs encapsulated for 48 h in 3D SAP gels show that clicking in RGD promotes cell spreading in a non‐MMP dependent manner. Actin cytoskeleton stained with fluorescent phalloidin. GM6001 is a general MMP inhibitor (10 μM). Scale bar = 100 μm. (B) Plots of the mean and standard deviations of the circularity of cells within each type of gel as measured with CellProfiler. N = 3 replicate experiments. *p < .05, ***p < .0005, ****p < .0001. Not all significances shown. (C) Empirical cumulative distribution functions of circularities for all cells across all replicate experiments. MSC, mesenchymal stem cells; SAP, self‐assembling peptide

FIGURE 8

Degradable FRET‐based biosensor incorporated into SAP gels via click chemistry detects MMP activity. (A) Increase in the fluorescence intensity of the FRET‐based biosensor after incubating SAPs in increased concentrations of collagenase in PBS after 24 h. Enzymes degrade the QGIW sequence, which releases the fluorescein dye from the dabcyl quencher. Mean ± SD, N = 3 replicate experiments. *p < .05 versus PBS blank (dotted line). (B) Fluorescence intensity of the biosensor at 0 and 24 h with various seeding densities of MSCs. Mean ± SD, N = 3–4 replicate experiments. *p < .05 versus 0 cells/ml. SAP, self‐assembling peptide