Engineering Injectable Silk Fibroin-Agarose Hydrogels for Localized Sustained Pirfenidone Delivery: Evaluation of Physicochemical Properties and Antifibrotic Efficacy

Dysregulated wound healing, characterized by excessive extracellular matrix (ECM) deposition, leads to fibroproliferative conditions. This presents a significant medical challenge due to its high recurrence rate and the potential to cause organ failure. Fibrosis is driven by overexpression of pro-inflammatory cytokines and ECM regulators, particularly pro-fibrotic matrix metalloproteinases (MMPs), and oxidative stress, leading to abnormal fibrous matrix formation and myofibroblast upregulation. Current antifibrotic drug treatments show limited efficacy due to poor pharmacokinetic and pharmacodynamic properties. To address these limitations, we propose a multifaceted injectable hydrogel that provides sustained and site-activated drug release. This research focuses on developing an in situ injectable hydrogel loaded with an antifibrotic agent, pirfenidone (PFD), and characterizing its physicochemical properties as an antifibrotic drug carrier. The hydrogel was tuned for good cytocompatibility and hemocompatibility without promoting undesired cell proliferation or ECM formation. The PFD-loaded hydrogel exhibits enhanced injectability at 40 °C and transitions to a gel state, as indicated by increased complex viscosity, at 37 °C due to intra- and intermolecular H-bonding between silk fibroin and agarose. It displays excellent shear-thinning and viscoelastic characteristics. The hydrogel provides sustained drug release for 7 days and undergoes pro-fibrotic enzyme-activated degradation, functioning as a site-activated system to effectively prevent fibrosis recurrence. In our lipopolysaccharide (LPS)-induced in vitro fibrosis recurrence model, the PFD-loaded hydrogel suppresses key fibrogenesis markers, such as cell migration; extracellular collagen deposition; and expression of COL1A1, F-actin, and reactive oxygen species. The hydrogel's dual mechanism of action involves utilizing excess MMPs for controlled ECM remodeling and providing prolonged drug release to counteract fibrosis recurrence and promote wound healing.