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

Abstract

Abstract. 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 unmod-CMLS, Cell. Mol. Life Sci. 61 (2004) 1785 -1794 1420-682X/04/141785-10 DOI 10.1007/s00018-004-4166-0 © Birkhäuser Verlag, Basel, 2004 CMLS Cellular and Molecular Life Sciencesified 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 N 4 -octadecyl-1-b-D-arabinofuranosylcytosine-(5¢-5¢)-3¢-C-ethinylcytidine showed a reduction of the IC 50 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.

Clodronate-liposome-mediated depletion of tumour-associated macrophages: a new and highly effective antiangiogenic therapy approach

“…The in vitro cytotoxicity of clodronate was assessed as described before (Marty et al, 2004). Briefly, cells were incubated in 96-well plates with liposomes, clodronate and clodrolip (6 h, 371C, 1 mg clodronate ml À1 ) and cell viability was determined by addition of WST-1 reagent (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's recommendations.…”

Cell-penetrating and cell-targeting peptides in drug delivery

“…In several studies, initial ionic interactions at the cell membrane 7 surface have been however shown to be key determinants for the uptake of all the cationic CPPs since the peptide entry could be strongly reduced by competition with polyanionic compounds [51][52][53][54] or by stringent cell washes with solutions at acidic pH [55].…”

“…Park et al described the equivalent cell translocation potency of a short protamine derivative rich in arginine residues which was equivalent to that of the Tat peptide combined with the absence of toxicity [85]. Cardozo et al showed that concentrations of up to 100 M of Tat (48)(49)(50)(51)(52)(53)(54)(55)(56)(57) were essentially harmless in all cells tested, whereas Antp(43-58) was significantly more toxic [86]. In addition, it is noteworthy that peptide concentrations used in toxicity tests are in their very large majority far above the concentrations used for delivering various drugs into cells (which range from 1 to 10 M).…”

The taming of the cell penetrating domain of the HIV Tat: Myths and realities

“…In a previous study, however, no PTD mediated transduction in brain was observed, [46] possibly because beta-gal was chemically conjugated to PTD instead of recombinant fusion protein. Similarly, another study also failed to deliver PTD- 99 Tc to the brain [76]. Since PTD is conjugated to Tc or beta-gal, this might have resulted in structural modifications in the PTD and hence no transduction was observed.…”