Mechanistic Analysis of Cellular Internalization of a Cell- and Skin-Penetrating Peptide

Abstract

Background: Keratinocytes are a targeted cell-type for treating skin disease. However, delivering drugs into the skin and keratinocytes remains challenging due to transport barriers posed by both the stratum corneum and cell membrane. Nonetheless, SPACE™ peptide has been shown effective in delivering a number of macromolecules into the skin and cells. Accordingly, we sought to explore the mechanism of SPACE™ peptide internalization under conditions relevant for skin delivery. Methods: Mechanistic analysis was performed using live cell confocal microscopy. Potential binding targets were assessed by Western blot and mass spectrometry. Results: Experimental data suggest an energy-dependent mechanism for the cellular entry of SPACE™ peptide into human epidermal keratinocytes. SPACE™ peptide associates with the cell membrane within seconds and then is internalized rapidly. Co-incubation with heparin sulfate or serum suggests that association with the cell membrane may be specific in nature. Western blot and mass spectrometry studies are consistent with earlier findings that keratin may play an important role in SPACE™ peptide internalization. Conclusions: This work highlights the potential for delivering drug into the cytoplasm of keratinocytes for the treatment of skin disease through topical application. Further, it highlights an ongoing need for studies involving cell-penetrating peptides under clinically relevant conditions. Lay Summary: SPACE™ peptide has been shown to enhance delivery of a wide variety of macromolecules into the skin and cells. However, the mechanism of cell entry of SPACE™ peptide under conditions relevant for skin delivery is not understood. Accordingly, we sought to explore the mechanism of SPACE™ peptide internalization in keratinocytes under conditions relevant for skin delivery. A better understanding of the dominating pathways and major molecular players involved in SPACE™ peptide internalization under clinically relevant conditions will aid the development and translation of SPACE™ peptide-based technologies for treating skin disease.