159. RXR-Carbamate – A Novel Molecular Transporter for Skin

Abstract

Cell penetrating peptides are powerful tools for delivery of various therapeutics. One of the critical intrinsic features of these carriers is the presence of minimum 7-9 arginine residues which allows electrostatic interaction with cargos like nucleic acids as well as negatively charged cell surface molecules for facile entry and cargo delivery. However cellular entry might be compromised if the guanidium groups are consecutive and not spaced because of compromised cellular interaction. Another limitation is the amenability of these homoarginine systems to proteolytic cleavage which leads to their low bio-availability. We previously reported a safe and stable homoarginine peptide-based system (R-X-R)-carbamate where amide linkages were replaced by stable carbamate linkages and arginine residues were separated by aminohexanoic acid spacers. This alteration imparted better flexibility and hydrophobicity to these systems in addition to protection against proteolytic cleavage and minimal steric hindrances. These molecules entered cells efficiently and showed high transfection efficiencies in comparison to their amide counterparts. We have now investigated the mechanism behind enhanced transfection efficiency and based on our observations, demonstrate the applicability of this system as an efficient carrier for topical delivery of biomolecules in skin. We demonstrated through flow cytometry studies using chemical inhibitors of different known cellular uptake pathways that (R-X-R)-carbamate- DNA complexes not only show much higher uptake than amide counterpart but the uptake was drastically decreased in presence of inhibitor Methyl-β-Cyclodextrin -a known blocker of lipid raft mediated entry pathway. We have further validated the entry of these carriers using colocalization studies with pathway specific markers. Further, retention, stability and transfection efficiency studies involving depletion of cholesterol (which is a major component of lipid raft structures) using extraction method is being carried out. Comparative transfection efficiency in lipid raft enriched cancerous cell lines is being used for further validation. Lipid rafts act as major mediators of viral infections and are also involved in entry of nanocomplexes in skin. Therefore we explored if the (R-X-R)-carbamate-DNA complexes that are more biased to enter the cells through lipid raft dependant pathways also exhibit efficient entry into skin cells and tissue. The uptake of (R-X-R)-carbamate-DNA complexes and the transfection efficiency were studied in HaCaT cells and human skin tissue. High levels of transfection was achieved in human skin tissue after topical application of these complexes. Further studies on mechanism of interaction of these complexes with skin and the impact on skin integrity is currently ongoing. Ultimately this study will help us in obtaining a carrier system which is safe, more stable and efficient to deliver multiple cargo types in skin in a non-invasive manner for therapeutic applications. Moreover we intend to gain insights into the exact mechanism that it follows during its trajectory inside the skin cells and tissue.