Characterization and biological evaluation of melatonin-loaded silk fibroin-hyaluronic acid films for multifunctional wound dressings.

Chronic wounds pose significant health challenges due to impaired healing and microbial contamination. This study develops multifunctional wound dressings using silk fibroin (SF), hyaluronic acid (HA), and melatonin to enhance healing, manage infections, and restore skin integrity. SF and HA provide mechanical strength and biocompatibility, while melatonin offers anti-inflammatory and antioxidant effects. However, SF and HA exhibit electrostatic repulsion, hindering homogeneous film formation. To overcome this, high-molecular-weight polyethylene glycol was used, enabling the production of blank films (BF). Incorporating melatonin (melatonin-loaded films, MF) improved thermal stability, modulated mechanical and surface properties, and showed no significant cytotoxicity to fibroblasts and keratinocytes. MF reduced surface roughness nearly sixfold and doubled flexibility and strength (Young's modulus) compared to BF. Its surface free energy with water became nearly equal to that with non-polar diiodomethane, indicating improved hydrophilic-lipophilic balance. Melatonin release from MF reached ∼ 37 % in the first 2 h, followed by sustained release for up to 96 h. In vivo, in a rat chronic wound model, BF and MF accelerated healing within the first 7 days, reduced healed skin thickness, and increased vascularization compared to the negative control (NC) treated only with an inert gel. Stress-strain analysis showed that the treatment with the films reduced the Full Width at Half Maximum (FWHM) of the area under the curve by twofold, suggesting a more organized skin structure than NC. BF treatment elevated IFN-γ, IL-2, and IL-6 to levels comparable to the positive control (acute wound) but also increased TNF-α beyond NC levels, suggesting persistent inflammation. In contrast, MF mitigated this response, selectively restoring IL-6 without significantly altering other mediators. MF also modulated the wound microbiota, reducing bacterial diversity while selectively promoting Staphylococcus urealyticus, which may contribute to a favorable healing environment. Overall, the differential impacts of BF and MF treatments on skin healing parameters highlight their potential for addressing distinct aspects of chronic wound management, suggesting tailored applications based on their specific biological effects and therapeutic mechanisms.