Design and Evaluation of Peptide-Functionalized Gelatin Hydrogels for Therapeutic Vascularization

Abstract

The development of bioactive hydrogels that promote vascular integration has great potential for advancing tissue regeneration and therapeutic delivery in cardiovascular diseases. Here, we have created a hydrogels that utilize gelatin, a naturally occurring polymer with known capacity for growing blood vessels (pro-angiogenic) and enhanced it by chemically conjugating additional pro-angiogenic peptides to the backbone. The first generation of this hydrogel, GelCad, was applied in a mouse model of peripheral artery disease. PAD is the leading cause of amputations in the United States and is a CVD that occurs when plaque buildup in the extremities leads to reduced blood flow to the limbs, killing the native tissue due to lack of oxygen and nutrients. In our initial study, we induced PAD by reducing blood flow to the hindlimb with a surgical procedure. Mice treated with gelatin or saline, lost their limb within two weeks, while mice treated with GelCad had their blood flow fully restored in two weeks, preventing limb loss. Further, we found that mice remined healthy five months post treatment with GelCad. Building on the first hydrogel, we synthesized a library of additional gelatin-based hydrogels functionalized with pro-angiogenic peptides and evaluated their vascularization potential in vitro and in vivo. Each hydrogel was prepared at three peptide concentrations and validated using NMR and quantitative fluorescent tagging, resulting in nine unique formulations. In a murine fat pad injection model, QK (high concentration) and HepPep (low concentration) hydrogels significantly enhanced vessel length and volume compared to unmodified gelatin. A combination hydrogel (QKCad), integrating QK and a cadherin-mimetic peptide, further improved vascular metrics, suggesting a synergistic effect between endothelial and smooth muscle cell signaling cues. In vitro assays using primary human smooth muscle cells revealed that peptide-functionalized hydrogels supported greater cell proliferation, reduced apoptosis under stress, and enhanced wound closure compared to gelatin alone. A pilot study in pigs confirmed hydrogel retention, perfusion, and biocompatibility over 14 days, with localized vascular responses observed particularly in the QKCad formulation.