Prolonged Release of IL ‐10 From Enzyme‐Mediated Poly‐ l ‐(Tyrosine‐ co ‐Phenylalanine) Nanocrystals Enhances Stability and Modulates Inflammatory Responses

Protease‐catalyzed synthesis of a peptide composed of l ‐tyrosine and l ‐phenylalanine (2.2:1 M ratio) was achieved in a natural deep eutectic solvent (NADES) medium and subsequently employed to encapsulate the anti‐inflammatory cytokine interleukin‐10 (IL‐10). IL‐10 was loaded into enzyme‐mediated peptide nanocrystals at doses of 200 and 400 ng, achieving stable nanocomposite formulations. Release studies were conducted in phosphate‐buffered saline (PBS, pH 7.4) at 37°C under gentle agitation, revealing a sustained release profile extending up to 30–31 days, with an initial release of approximately 16% within the first 5 days and near‐complete release between Days 10 and 25, depending on loading. Encapsulation effectively protected IL‐10 from rapid degradation observed for the free cytokine under identical conditions, resulting in markedly enhanced stability. The nanocrystals were further integrated into porcine gelatin–hyaluronic acid (Ge:HA) and microbial poly(hydroxybutyrate‐co‐valerate) (PHBV) matrices, where IL‐10 release was further modulated, reaching up to ~80% release from Ge:HA and ~100% from PHBV‐based systems over 31 days. Cytotoxicity assays using primary human dermal fibroblasts confirmed excellent biocompatibility of all formulations. Moreover, studies in PMA‐activated THP‐1 macrophage‐like cells demonstrated reduced intracellular reactive oxygen species (ROS) and suppression of the pro‐inflammatory cytokine IL‐6, highlighting the combined protective and immunomodulatory effects of IL‐10 encapsulation. The presence of tyrosine residues within the nanocarrier further suggests intrinsic antioxidant contributions. Overall, these results support enzyme‐mediated peptide nanocrystals as an effective platform for the stabilization and prolonged release of IL‐10, with strong potential for treating inflammation‐related skin conditions.