Therapeutic efficacy of nanostructured lipid carriers co-loaded with simvastatin and adenosine (NLC- SA) in a human epidermal model of diabetic chronic wound

Chronic wounds are characterized by persistent inflammation, elevated oxidative stress, and impaired tissue repair, posing a major clinical challenge, particularly in diabetic patients. In this study, we established a reconstructed human epidermis (RhE) model using primary neonatal human epidermal keratinocytes (NHEKs) to simulate the hyperglycemic and pro-inflammatory milieu typical of diabetic chronic wounds. This model was then used to assess the therapeutic efficacy of nanostructured lipid carriers co-loaded with simvastatin and adenosine (NLC-SA), previously developed and characterized¹. Epidermal tissues were cultured at the air-liquid interface for 14 days to ensure full stratification and differentiation. Subsequently, tissues were exposed to high glucose (40 mM) combined with TNF-α (40 ng/mL) for 5 days via the basolateral compartment, inducing a sustained inflammatory state. This condition was selected based on preserved metabolic activity (WST-1 assay) and the marked increase in IL- 8 and IL-1α secretion, quantified by ELISA. Following inflammatory induction, tissues were treated with NLC-SA (at multiple concentrations), free drug combinations, or unloaded NLCs for 2 or 5 days. Treatment efficacy was evaluated through cell viability, cytokine profiling (IL-8, IL-1α, IL-6), vascular endothelial growth factor (VEGF) secretion (ELISA), and histological analysis. A comparative evaluation of epidermal models generated with either immortalized HaCaT cells or primary NHEKs revealed significant differences in tissue architecture and functional properties. While both models formed multilayered epidermal structures, the NHEK-based epidermis displayed superior stratification, with six viable cell layers and a well-developed stratum corneum, compared to the HaCaT-based model, which exhibited only three viable layers and no evident cornified layer. Additionally, the total epidermal thickness and transepithelial electrical resistance (TEER) values were significantly higher in the NHEK model, indicating enhanced barrier integrity and tissue maturation. These differences were also reflected in the inflammatory response: although both models responded to glucose + TNF-α exposure, the NHEK model exhibited a more consistent and robust cytokine release profile, for IL-1α and IL-8, while HaCaT-based tissues showed greater variability and lower sensitivity to pro-inflammatory stimuli. Treatment with NLC-SA significantly improved cell viability and reduced the secretion of IL-8, IL-1α, and IL-6 in the inflamed NHEK-epidermis model, compared to the untreated group. Moreover, NLC-SA treatment promoted an increase in VEGF production, suggesting a potential pro-angiogenic effect. Histological examination confirmed the protective effects of NLC-SA, with dose-dependent recovery of epidermal architecture. Notably, the highest NLC-SA concentration restored epidermal morphology to a state comparable to that of untreated, non-inflamed controls. In conclusion, the inflammatory NHEK-based epidermis model offers a relevant in-vitro platform for mimicking chronic wound conditions. Compared to HaCaT-based models, the NHEK epidermis better replicates the structural and functional features of native human skin. The NLC-SA system demonstrated therapeutic potential by modulating inflammation and supporting epidermal recovery, providing a promising approach for the topical treatment of chronic wounds.