Low‐Temperature Fabrication of Thymosin β4‐Loaded Soluble Microneedles to Promote Wound Healing by Specific Binding to Downregulated Immune Regulators Vsig4 and IL22rɑ2

Skin wound treatment is hindered by the poor penetration of large therapeutics and a lack of treatments that effectively regulate immune environments. While microneedles (MNs) can bypass the skin, they destabilize sensitive peptides, including thymosin β4 (Tβ4), whose immunomodulatory targets are not well understood, limiting their clinical use. This study introduces a novel fabrication method for Tβ4-loaded soluble MNs at low temperatures using chitosan and sucrose, avoiding the denaturing conditions of traditional MN production. These MNs exhibit a uniform shape, high drug capacity (248.15 ± 1.37 µg/patch), quick dissolution within an hour, excellent biocompatibility, and significantly enhanced wound healing in mice. This study examines the mechanism by which Tβ4 accelerates wound healing, identifying the downregulated immune regulators Spp1, Vsig4, and IL22rɑ2 through RNA-seq and DEG analysis. In vitro qPCR, western blot, and surface plasmon resonance (SPR) experiments demonstrate that Tβ4 specifically binds to the downregulated immune regulators Vsig4 (K_D = 3.56 × 10^-6 m) and IL22rɑ2 (K_D = 9.69 × 10^-6 m). This article explores how Tβ4 influences the wound immune microenvironment to aid healing, identifies its specific molecular targets, and moves beyond its general roles to offer new opportunities for drug development.