Mechanism of improved gene transfer by the N-terminal stearylation of octaarginine: enhanced cellular association by hydrophobic core formation

Abstract

The internalization mechanisms associated with octaarginine and stearyl-octaarginine were investigated using confocal laser microscopy and flow cytometric analysis. Octaarginine is able to translocate through cell membranes in a manner that does not exactly involve the classical endocytic pathways of internalization. However, when a stearyl moiety is attached to the N-terminus of octaarginine, the internalization shifts mainly to an endocytosis-dependent pathway. The transfection efficiency of stearyl-octaarginine was significantly higher than that of octaarginin. To understand the mechanism of the improved gene transfer by the N-terminal stearylation of octaarginine, the gene transfer processes mediated by octaarginine or stearyl-octaarginine were compared. Both octaarginine and stearyl-octaarginine are able to carry plasmid DNA into cells. The amount of plasmid DNA internalized as well as that delivered to the nucleus was higher in the case of stearyl-octaarginine. Even though the internalization mechanisms of octaarginine and stearyl-octaarginine were different, their complexes with plasmid DNA were internalized via the same pathway, presumably, the clathrin-mediated pathway of endocytosis. The results of the atomic force microscopy revealed that stearyl-octaarginine, but not octaarginine, can completely condense the DNA into stable complexes that can be highly adsorbed to the cell surface and subsequently highly internalized. Therefore, using stearylated-octaarginine provided higher internalization of plasmid DNA into cells, due to enhanced cellular association, as well as higher nuclear delivery. The results presented in this study provide a better understanding of the mechanisms of improved transfection using stearylated-octaarginine. The concept of using stearylated peptides may aid in the development of more efficient nonviral gene vectors.

Topology of octaarginines (R8) or IRQ ligand on liposomes affects the contribution of macropinocytosis- and caveolae-mediated cellular uptake

“…Based on the high arginine content in the Tat sequence, Futaki et al synthesized a polypeptide composed solely of arginine residues [8], which can deliver macromolecules as efficiently as a Tat peptide [9,10]. Stearylated octaarginine (STR-R8) is a multifunctional device.…”

A new peptide motif present in the protective antigen of anthrax toxin exerts its efficiency on the cellular uptake of liposomes and applications for a dual-ligand system

“…Egg phosphatidylcholine (EPC), cholesterol (Chol), N-(lissamine rhodamine B sulfonyl)-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (rhodamine-DOPE), the cells were washed 3 times with 1 ml of ice-cold phosphate buffer saline (PBS) supplemented with heparin (20 units/ml) to completely remove the surface-bound KYND-PEG-LP, as reported previously (Khalil et al, 2004) and then treated with Reporter Lysis Buffer (Promega Corp., Madison, WI, USA) followed by centrifugation at 12,000 rpm for 5 min at 4 °C to remove debris. The cellular 140 uptake efficiency of the prepared rhodamine labeled LPs were determined by measuring the fluorescence intensity of rhodamine (excitation at 550 nm and emission at 590nm) using FP-750 Spectrofluorometer (JAS Co, Tokyo, Japan).…”

“…Before transfection, the cells were washed with 1 ml of PBS and were pre-incubated with Kreb's buffer in the absence or presence of the following 175 inhibitors for various times: Amiloride (5 mM) for 30 min; Sucrose (1 M) for 30 min or Filipin III (400 g/ml) for 1 h at 37 °C (Mudhakir et al, 2007, Khalil et al, 2004, Hansen et al, 1993. KYND modified PEG-LP was added and the cells were incubated for 1 hr at 37 °C in the presence or absence of inhibitors.…”

“Programmed packaging” for gene delivery

“…While the corresponding particle exhibited a high level of gene expression in dividing cells [66,67], nearly background level of transfection activity was observed by the R8-modified MEND (R8-MEND) in JAWS II cells derived from murine dendritic cells. Furthermore, some efforts directed to the use of the NLS failed to enhance the transfection efficacy in JAWS II cells or primary mouse bone marrow-derived dendritic cells (BMDCs).…”