Uptake and intracellular trafficking of novel ternary lipoplexes for gene delivery

Laila Kudsiova, Amit Vassa, Helen C. Hailes, Alethea B. Tabor

Research output: Contribution to conferenceAbstract

Abstract

Objectives To date a large number of vesicle-forming cationic lipids and peptides have been synthesized and used for the delivery of DNA with varying degrees of success. To meet t herapeutic requirements, however, the DNA-delivering efficiency of those non-viral vectors needs major improvement. One possible strategy is to gain better understanding of their mechanisms of entry in relation to their eventual transfection. Endocytosis is thought to be the major internalization pathway for most non-viral gene-delivery vectors; however, the relative contribution of each distinctive endocytic pathway, including clathrin- and caveolae-mediated endocytosis and/or macropinocytosis, is not yet fully under- stood. In this study the transfection efficiency and intracellular uptake and trafficking of novel lipid ternary vectors composed of a series of C 14 glycerol- based analogues of N-(1-(2,3-dioleyloxy)prop yl)-trimethyl-ammonium chloride (DOTMA) were studied using various endocytotic pathway inhibitors. Methods The novel C 14 DOTMA analogues (Writer et al 2006), incorporating cis, trans and alkyne moieties at C-9 and C-11 positions of the alkyl chain, were synthesized, formulated into vesicles with the neutral lipid dioleoylphosphatidyl- ethanolamine (DOPE) and mixed with pGL-3 plasmid in the presence or absence of an integrin-targeting peptide, Pep6. The novel peptide contains a Lys16 domain at the N-terminus designed to bind and condense the DNA, and a cyclic integrin- targeting recognition site known to bind specifically to the cell-surface protein 51 i ntegrin. Lipid–DNA complexation efficiency was assessed using gel electrophoresis, light scattering and zeta potential. Transfection studies of the lipoplexes in the presence or absence of Pep6 were performed in MDA-MB-231 breast cancer cells in the presence of endocytic inhibitors to determine the route of complex internalization and the relative contributions of each endocytic pathway (Khalil et al 2006). Endocytic inhibitor toxicity studies were also carried out using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Results Zeta potentials, particle-size measurements and gel electrophoresis indicated that complete complexation of DNA occurred between lipid/DNA charge ratios of 2:1 and 4:1, offering partial protection of DNA from DNAseI enzymatic degradation. The presence of Pep6 showed highly improved transfection efficiency compared to the lipoplexes alone, showing up to 3-fold higher transfection efficiencies in some of the lipids when compared with Lipofectamine ® .The transfection efficiency of lipoplexes with and without Pep6 was totally inhibited in the presence of chlorpromazine, a clathrin-mediated endocytic inhibitor. No inhibition of transfection was observed in the presence of filipin III or nystatin, both used as inhibitors for caveolae-mediated endocytosis. Some concentration and time-dependent cytotoxicity was observed with chlorpromazine (with T 50 of 25.2 and 9.2 mg/mL for 30 and 60 minute incubation times respectively); however, any reduction in cell viability was accounted for during the transfection experiments. Conclusions The proposed ternary lipoplexes are promising candidates for gene delivery due to their high transfection efficiency, although slight structural variations in the lipids also play a major role in their eventual transfection. Clathrin-mediated endocytosis appears to be the major pathway for complex internalization in both the presence and absence of Pep6.
Original languageEnglish
Pages0-0
Number of pages1
Publication statusPublished - 1 Sep 2008
EventBritish Pharmaceutical Conference - Manchester, UK, 2008
Duration: 1 Sep 2008 → …

Conference

ConferenceBritish Pharmaceutical Conference
Period1/09/08 → …

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