Amniotic Membrane Stem Cell Metabolite Product (AMSCMP) contains a 75.33 kDa protein, making dermal penetration difficult. The Skin Penetrating and Cell Entering (SPACE) peptide facilitates protein molecule penetration reversibly. This study aimed to assess the penetration depth and skin irritation level of AMSCMP combined with SPACE peptide. In vivo experiments were conducted on mice, evaluating penetration via staining depth and irritation through edema, polymorphonuclear (PMN) cell levels, and tissue degeneration. Penetration results showed distinct fluorescence differences, where formulas with the SPACE peptide (F2, F3, F4) achieved penetration depths exceeding 1,200 μm, whereas the control without SPACE (F1) showed no measurable penetration. Statistical analysis confirmed a significant difference in penetration depth among formulas (p < 0.05), proving that SPACE peptide significantly enhanced active ingredient delivery into the skin. Regarding skin safety, F1 and F2 showed an irritation index of zero (no irritation), while F3 and F4 yielded a score of 1.3, falling into the very mild irritation category. However, statistical analysis revealed no significant difference in irritation scores across all formulations (p > 0.05), indicating that the addition of SPACE peptide did not induce meaningful skin irritation. Conclusion: The addition of SPACE peptide significantly enhances the skin penetration of AMSCMP while maintaining an exceptionally mild, non-significant irritation profile suitable for topical application.
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