Enzymatic conjugation of a bioactive peptide into an injectable hyaluronic acid-tyramine hydrogel system to promote the formation of functional vasculature.

Abstract In this study, one-step enzyme-mediated preparation of a multi-functional injectable hyaluronic-acid-based hydrogel system is reported. Hydrogel was formed through the in situ coupling of phenol moieties by horseradish peroxidase (HRP) and hydrogen peroxide (H 2 O 2 ), and bioactive peptides were simultaneously conjugated into the hydrogel during the gel formation process. The preparation of this multi-functional hydrogel was made possible by synthesizing peptides containing phenols which could couple with the phenol moieties of hyaluronic-acid–tyramine (HA–Tyr) during the HRP-mediated crosslinking reaction. Preliminary studies demonstrated that two phenol moieties per molecule resulted in a consistently high degree of conjugation into the HA–Tyr hydrogel network, unlike the one modified with one phenol moiety per molecule. Therefore, an Arg–Gly–Asp (RGD) peptide bearing two phenol moieties (phenol 2 –poly(ethylene glycol)–RGD) was designed for conjugation to endow the HA–Tyr hydrogel with adhesion signals and enhance its bioactivities. Human umbilical vein endothelial cells (HUVECs) cultured on or within the RGD-modified hydrogels showed significantly different adhesion behavior, from non-adherence on the HA–Tyr hydrogel to strong adhesion on hydrogels modified with phenol 2 –poly(ethylene glycol)–RGD. This altered cell adhesion behavior led to improved cell proliferation, migration and formation of capillary-like network in the hydrogel in vitro. More importantly, when HUVECs and human fibroblasts (HFF1) were encapsulated together in the RGD-modified HA–Tyr hydrogel, functional vasculature was observed inside the cell-laden gel after 2 weeks in the subcutaneous tissue. Taken together, the in situ conjugation of phenol 2 –poly(ethylene glycol)–RGD into HA–Tyr hydrogel system, coupled with the ease of incorporating cells, offers a simple and effective means to introduce biological signals for preparation of multi-functional injectable hydrogels for tissue engineering application.