Internalization and intracellular trafficking of a PTD-conjugated anti-fibrotic peptide, AZX100, in human dermal keloid fibroblasts.

Abstract

A challenge in advanced drug delivery is selectively traversing the plasma membrane, a barrier that prohibits the intracellular delivery of most peptide and nucleic acid-based therapeutics. A variety of short amino acid sequences termed protein transduction domains (PTDs) first identified in viral proteins have been utilized for over 20 years to deliver proteins nondestructively into cells, however, the mechanisms by which this occurs are varied and cell-specific. Here we describe the results of live cell imaging experiments with AZX100, a cell-permeable anti-fibrotic peptide bearing an "enhanced" PTD (PTD4). We monitored fluorescently labeled AZX100 upon cell surface binding and subsequent intracellular trafficking in the presence of cellular process inhibitors and various well-defined fluorescently labeled cargos. We conclude that AZX100 enters cells via caveolae rapidly, in a manner that is independent of glycoconjugates, actin/microtubule polymerization, dynamins, multiple GTPases, and clathrin, but is associated with lipid rafts as revealed by methyl-beta-cylodextrin. AZX100 treatment increases the expression of phospho-caveolin (Y14), a critical effector of focal adhesion dynamics, suggesting a mechanistic link between caveolin-1 phosphorylation and actin cytoskeleton dynamics. Our results reveal novel and interesting properties of PTD4 and offer new insight into the cellular mechanisms facilitating an advanced drug delivery tool.

• Authors: Charles R Flynn, Joyce Cheung-Flynn, Christopher C Smoke, David Lowry, Robert Roberson, Michael R Sheller, Colleen M Brophy
• Journal: Journal of pharmaceutical sciences (J Pharm Sci) Vol. 99 Issue 7 Pg. 3100-21 (Jul 2010) ISSN: 1520-6017 [Electronic] United States
• PMID: 20140957 (Publication Type: Journal Article)
• Copyright: (c) 2010 Wiley-Liss, Inc. and the American Pharmacists Association

Chemical References

• AZX 100
• Actins
• Heat-Shock Proteins, Small
• Peptides
• Phosphoproteins
• rab GTP-Binding Proteins
• Dynamins

Topics

• Actins (metabolism)
• Amino Acid Sequence
• Caveolae (metabolism)
• Dermis (cytology)
• Dynamins (genetics, metabolism)
• Fibroblasts (cytology, metabolism)
• Heat-Shock Proteins, Small (administration & dosage, chemistry, pharmacokinetics)
• Humans
• Molecular Sequence Data
• Peptides (chemistry)
• Phosphoproteins (administration & dosage, chemistry, pharmacokinetics)
• Protein Transport
• Up-Regulation
• rab GTP-Binding Proteins (genetics, metabolism)