Fibroblast-derived small extracellular vesicles loaded with tacrolimus enhances dermal delivery and alleviates cytokine-overdriven skin inflammation

Long COVID has been increasingly linked to chronic inflammatory skin conditions driven by cytokine overproduction. Topical tacrolimus, a calcineurin inhibitor, is commonly used to manage such conditions due to its immunosuppressive properties. However, due to poor dermal penetration, tacrolimus oftens produce to suboptimal efficacy and adverse effects such as local irritation and burning sensation. Effective management of chronic inflammatory skin conditions linked to long COVID necessitates targeted, controlled drug delivery into deeper skin layers to modulate excessive cytokine production and attenuate localized inflammation. This study explores fibroblast-derived small extracellular vesicles (sEVs) as a new controlled delivery vehicle for tacrolimus. The sEVs were isolated using sucrose-cushioned density ultracentrifugation and characterized by TEM, NTA, Dot blot, and MicroBCA assay, confirming their successful isolation and purity. Tacrolimus was encapsulated into sEVs via sonication, with successful drug loading confirmed by morphological and physicochemical characterization. The resulting Tac-sEVs exhibited an encapsulation efficiency of 79.19% ± 0.01. Franz diffusion studies revealed a rapid initial release within the first 10 h, followed by sustained higher release over time. Tape-stripping demonstrated significantly deeper dermal penetration of tacrolimus loaded sEVs (Tac-sEVs) compared with commercial tacrolimus ointment and free drug. Both tacrolimus and Tac-sEVs downregulated IFN-γ, GCS-F, IL-2, and IL-4 expression, indicating potent suppression of SARS-CoV-2 spike glycoprotein-induced cytokine overproduction. PKH-26 fluorescence labelling confirmed efficient cellular uptake, while cytotoxicity assays (Alamar Blue, CCK-8) showed high cell viability for both formulations. In summary, these results position Tac-sEVs as a safe and promising therapeutic platform for cytokine-driven inflammatory skin diseases associated with long COVID, meriting further clinical investigation.