Efeito cicatrizante da nanoemulsão de ácido Alfa-lipóico (NanoALA) em cultura de células: determinação do mecanismo de ação

Abstract

Wound healing is a complex biological process that can be impaired by inflammatory imbalances. Alpha-lipoic acid (ALA), due to its antioxidant and anti-inflammatory properties, has been investigated as a therapeutic agent. This study aimed to develop and characterize an ALA-based nanoemulsion (NanoALA) and to evaluate its wound-healing effects in vitro in RAW 264.7, L929, and HaCaT cells. Formulations F1 and F2 exhibited a mean particle size <220 nm, PDI <0.2, zeta potential of approximately −25 mV, and pH ~5.4, remaining stable for up to 90 days. Atomic force microscopy revealed spherical particles with mean sizes of 25.1 nm (F1) and 14.2 nm (F2), and encapsulation efficiencies of 57.19% and 81.20%, respectively. In cytotoxicity assays, both formulations showed concentration-related effects. In RAW 264.7 cells, the IC₅₀ at 24 h was 16.21 µg/mL (F1) and 10.22 µg/mL (F2), with further reduction at 48 h. In L929 cells, no impairment of viability was observed. In HaCaT cells, no cytotoxicity was detected at 24 h, while at 48 h F1 exhibited an IC₅₀ of 46.53 µg/mL, whereas F2 showed no cytotoxic effect. At concentrations of 50 and 100 µg/mL, both NanoALA and blank nanoemulsion increased nitric oxide (NO) levels, whereas free ALA did not alter this parameter and showed no cytotoxicity in any cell line. Based on these findings, NanoALA F1 was selected for subsequent experiments (2.86, 5.72, and 11.44 µg/mL; free ALA: 5 µg/mL). In LPS-stimulated RAW 264.7 cells, NanoALA F1 significantly reduced NO, TNF-α, and IL-1β levels, with a more consistent effect than free ALA. In paracrine models, conditioned medium from NanoALA F1 reduced NO levels, increased TGF-β, and modulated substance P, whereas free ALA exhibited limited effects. Morphological analysis showed that NanoALA F1 attenuated the inflammatory phenotype in RAW 264.7 cells, suggesting a shift from M1 to an M2-like profile, while free ALA showed only partial effects. Morphometric analysis demonstrated that NanoALA F1 reduced inflammatory cell count and restored cell area and circularity, outperforming free ALA. In the scratch assay, NanoALA F1 significantly accelerated monolayer closure, surpassing the effect of free ALA. In conclusion, NanoALA F1 enhances the therapeutic effects of ALA by promoting greater inflammatory modulation, cellular preservation, and re-epithelialization, with efficacy at low concentrations (2.86 µg/mL). Additionally, it exhibits stability at room temperature and superior performance compared to free ALA, representing a promising nanostructured strategy for wound treatment.