| Summary: | <i>Achyrocline satureioides</i> (Lam.) DC Asteraceae extracts (ASEs) have been investigated for the treatment of various skin disorders. This study reports the effects of ASE-loaded nanoemulsions (NE<sub>ASE</sub>) on the cellular viability, death by necrosis, and migration of immortalized human keratinocytes (HaCaT cell line), as well as the irritant potential through the hen’s egg chorioallantoic membrane test (HET-CAM). NE<sub>ASE</sub> exhibited a polydispersity index above 0.12, with a droplet size of 300 nm, ζ-potential of −40 mV, and content of flavonoids close to 1 mg/mL. No cytotoxicity of the ASE was observed on HaCaT by MTT assay (up to 10 µg/mL). A significant increase of HaCaT viability was observed to NE<sub>ASE</sub> (up to 5 μg/mL of flavonoids), compared to treatment with the ASE. The necrosis death evaluation demonstrated that only NE<sub>ASE</sub> did not lead to cell death at all the tested concentrations. The scratch assay demonstrated that NE<sub>ASE</sub> was able to increase the cell migration at low flavonoid concentrations. Finally, the HET-CAM test proved the non-irritative potential of NE<sub>ASE</sub>. Overall, the results indicate the potential of the proposed formulations for topical use in wound healing, in view of their promising effects on proliferation and migration in keratinocytes, combined with an indication of the absence of cytotoxicity and non-irritating potential.
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