Study On Peptide Mediating HEGF Delivery Across Skin And Its Applications

Skin provides a convenient interface for systemic drug delivery. However, it was generally accepted that transdermal delivery of hydrophilic drugs such as peptides and proteins was extremely challenging, as the stratum corneum as a barrier to prevent transdermal penetration of most foreign molecules. The biologically inspired transdermal enhanced peptide TD1and SPACE have been discovered to specifically facilitate transdermal delivery of biological macromolecules via topical co-administration. However, The biological mechanism of transdermal peptide has not been fully defined. In this study, we further develop the applications and meachnism of transdermal peptide. Our works could be of great significance for the future development of promising new transdermal drugs.Firstly, we designed a practical system based on TD1peptide for advanced transdermal delivery. Human epidermal growth factor (hEGF) was selected as the model biological agent and a fusion protein:TD1-hEGF was designed. Study showed that TD1-hEGF not only had the similar bioactivity with native hEGF, but also possessed considerable higher transdermal ability than hEGF and a co-administration of TD1and hEGF. Combination of TD1-hEGF and antibacterial drugs helped reparing the outer mucosaand enhanced the therapeutic of effect for candida albicans vaginitis.Secondly, we also found that energy was required for the TD1-hEGF transdermal delivery through rat and human skins. Results showed that the permeation activity of TD1-hEGF was inhibited by the energy inhibitor and temperature. Adenosine triphosphate (ATP) effectively facilitated the TD1-hEGF into the skin in a dose dependent fashion. We also developed a simple model to describe theoretically the transdermal hEGF delivery mediated by TD1.Thirdly, we discovered that the Na+/K+-ATPase beta-subunit (ATP1B1) on epidermal cells plays a key role in the TD1-medicated transdermal delivery of macromolecular drugs. Using an yeast two-hybrid assay and immunoprecipitation assay, we screened and comfirmed that C-terminus of ATP1B1have specific affinity with TD1in yeast and mammalian cells. Our results showed that the interaction between TD1and ATP1B1affected not only the expression of ATP1B1but also localization. This interaction could be antagonized by the exogenous competitor ATP1B1or be inhibited by ouabain, resulting in the decreased delivery of macromolecular drugs across the skin.At last, we studied the transdermal delivery of the fusion protein which constructed with the skin penetration and cell enter peptide SPACE and hEGF carring siRNA for inducing melanoma cells apoptosis. In vitro test, the SPACE-hEGF not only had the function of skin penetration and cell enter, but also can target to the cancer cells that over-express EGFR. GAPDH and c-Myc siRNA were transfered into melanoma cells by SPACE-hEGF to reduce the transcriptional leverls and induced cell apoptosis.