Enhanced heparan sulfate proteoglycan-mediated uptake of cell-penetrating peptide-modified liposomes

Protein transduction domains (PTDs) are used to enhance cellular uptake of drugs, proteins, polynucleotides or liposomes. In this study, functionalized Antennapedia (Antp, aa 43–-58) and HIV Tat (aa 47–57) peptides were coupled to small unilamellar liposomes via thiol-maleimide linkage. Modified liposomes showed higher uptake into a panel of cell lines including tumor and dendritic cells than unmodified control liposomes. Liposome uptake was time and concentration dependent as analyzed by flow cytometry and live-cell microscopy. At least 100 PTD molecules per small unilamellar liposome (100 ± 30 nm) were necessary for efficient translocation into cells. Cellular uptake of PTD-modified liposomes was 15- to 25-fold increased compared to unmodified liposomes and was inhibited by preincubation of liposomes with heparin. Glycosaminoglycan-deficient CHO cells showed dramatically reduced cell association of PTD-modified liposomes, confirming the important role of heparan sulfate proteoglycans in PTD-mediated uptake. Antp-liposomes used as carriers of the cytotoxic drug N⁴-octadecyl-1--D-arabinofuranosylcytosine-(5- 5)-3-C-ethinylcytidine showed a reduction of the IC₅₀ by 70% on B16F1 melanoma cells compared with unmodified liposomes. PTD-functionalized liposomes, particularly Antp-liposomes, represent an interesting novel carrier system for enhanced cell-specific delivery of a large variety of liposome-entrapped molecules.Received 16 April 2004; received after revision 13 May 2004; accepted 25 May 2004