Light-triggered in vivo activation of adhesive peptides regulates cell adhesion, inflammation and vascularization of biomaterials

Abstract

Materials engineered to elicit targeted cellular responses in regenerative medicine must display bioligands with precise spatial and temporal control. Although materials with temporally regulated presentation of bioadhesive ligands using external triggers, such as light and electric fields, have recently been realized for cells in culture, the impact of in vivo temporal ligand presentation on cell-material responses is unknown. Here, we present a general strategy to temporally and spatially control the in vivo presentation of bioligands using cell-adhesive peptides with a protecting group that can be easily removed via transdermal light exposure to render the peptide fully active. We demonstrate that non-invasive, transdermal time-regulated activation of cell-adhesive RGD peptide on implanted biomaterials regulates in vivo cell adhesion, inflammation, fibrous encapsulation, and vascularization of the material. This work shows that triggered in vivo presentation of bioligands can be harnessed to direct tissue reparative responses associated with implanted biomaterials.

Figure 1. Light-triggered activation of cell adhesion activity of caged RGD peptide on hydrogels

a, Schematic representation of caged RGD peptide-functionalized PEGDA hydrogels. Light exposure at 350–365 nm cleaves UV-labile caging group to present active cyclic RGD peptide. Magenta/green denotes caged/active RGD peptide. b, Photographs of fluorescently labeled cells cultured on unmodified PEGDA and peptide-modified hydrogels that were either exposed to UV light or not exposed (scale bar, 300 μm). Hydrogels presenting control RGD peptide and UV-exposed caged RGD peptide supported high levels of adherent cells. Unmodified PEGDA gels and hydrogels presenting RDG scrambled peptide and non-exposed caged RGD peptide supported very low numbers of adherent cells with rounded morphology. c, Adherent cell density on hydrogels, box-whisker plot (minimum, 25^th percentile, median, 75^th percentile, and maximum) for 4 samples per group. Kruskal-Wallis p < 0.0026. UV-exposed gels presenting caged RGD supported 4-fold higher adherent cell densities than hydrogels functionalized with caged RGD peptide that were not exposed to UV († p < 0.01), surfaces presenting scrambled RDG peptide, and bare PEGDA hydrogels (p < 0.01). Cell density was higher on control RGD compared to RDG peptide (p < 0.05). No differences in cell adhesion density were observed between UV-exposed caged RGD peptide-presenting gels and hydrogels presenting control RGD peptide (p = 0.08).

Figure 2. Transdermal activation of in vivo inflammatory cell adhesion

a, Schematic representation of timeline for in vivo activation of cell adhesion using transdermal UV exposure. b, Photographs of explanted hydrogels stained for adherent inflammatory cells (green = NIMP-R14 [neutrophil], magenta = CD68 [macrophage], blue = DAPI [DNA], scale bar, 80 μm). Unfunctionalized PEGDA hydrogels exhibited minimal cell adhesion regardless of UV exposure, whereas hydrogels presenting control RGD peptide supported uniformly high numbers of adherent cells. Hydrogels presenting caged RGD peptide that were not UV-exposed displayed background levels of cell adhesion. In contrast, caged RGD-functionalized gels that were exposed to UV transdermal exhibited high numbers of adherent cells that were uniformly distributed over the biomaterial surface. c, Adherent cell density, box-whisker plot (minimum, 25^th percentile, median, 75th percentile, and maximum) for 6–8 mice per group, demonstrating light-based triggering of inflammatory cell adhesion to caged RGD-presenting implants. ANOVA p < 0.0001, * p < 0.05 vs. UV-exposed PEGDA, † p < 0.001 vs. No UV Caged RGD.

Figure 3. Light-triggered spatial patterning of in vivo cell adhesion

a, Schematic representation of patterning experiment using transdermal UV exposure through a mask. b, Photographs of explanted hydrogels stained for adherent cell nuclei for caged RGD and RDG presenting hydrogels at different distances from the center of irradiation (DAPI, color coded cyan, scale bar, 40 μm). c, Left: Composite image of photographs of adherent cell nuclei (cyan). Yellow circle designates exposure spot (scale bar, 200 μm). Right: Adherent cell density vs. distance away from irradiation point. Data represent mean ± standard error for hydrogels explanted for 5 mice per group.

Figure 4. Time-regulated in vivo activation of RGD peptide modulates fibrous encapsulation of implanted biomaterials

Unfunctionalized PEGDA hydrogels and gels presenting either control RGD or caged RGD peptides were implanted subcutaneously and at prescribed time points were exposed to UV transdermally. Biomaterials and associated tissue were explanted at 28 days. a, Photographs of plastic-embedded sections stained by Mason’s trichrome of fibrous capsule formation around implanted hydrogels at 28 days (scale bar, 100 μm). Yellow bars denote fibrous capsule. b, Fibrous capsule thickness around implanted hydrogels, box-whisker plot (minimum, 25^th percentile, median, 75th percentile, and maximum) for 4–6 mice per group. ANOVA p < 0.0001, * p < 0.001 vs. PEGDA, † p < 0.01 vs. No UV Caged RGD, § p < 0.05 vs. Day 0 Caged RGD.

Figure 5. Light-based activation of cell adhesive peptide promotes vascularization of implanted biomaterials

a, Immunostaining images for hydrogels presenting caged RGD or caged RDG peptide for different UV exposure conditions. Top: green = CD31 [endothelial cell], magenta = αSMA [smooth muscle cell], blue = DAPI [DNA], scale bar, 100 μm. Bottom: magenta = CD68 [macrophage], blue = DAPI [DNA], scale bar, 100 μm. b, Fluorescent images of blood vessel ingrowth (green) into PEG-maleimide hydrogels implanted subcutaneously at 14 days (scale bar, 100 μm). PEG-maleimide hydrogels presenting peptides were implanted subcutaneously and exposed to UV transdermally at selected time points. Mice were perfused with DyLight488-conjugated tomato lectin at sacrifice to label functional blood vessels. Hydrogels presenting caged RGD peptides which were exposed to UV transdermally at Day 0 and Day 7 exhibited robust blood vessel growth, similar to gels presenting control RGD. Hydrogels functionalized with scrambled RDG peptide or caged RGD peptide that was not exposed to UV displayed minimal blood vessel infiltration. c, Blood vessel density, box-whisker plot (minimum, 25^th percentile, median, 75th percentile, and maximum) for 4 mice per group for caged RGD conditions, 3 mice per group for control peptides. Kruskal-Wallis p < 0.01, * p < 0.01 vs. RDG, † p < 0.05 vs. No UV Caged RGD.