Bioreducible Liposomes for Gene Delivery: From the Formulation to the Mechanism of Action

Abstract

BackgroundA promising strategy to create stimuli-responsive gene delivery systems is to exploit the redox gradient between the oxidizing extracellular milieu and the reducing cytoplasm in order to disassemble DNA/cationic lipid complexes (lipoplexes). On these premises, we previously described the synthesis of SS14 redox-sensitive gemini surfactant for gene delivery. Although others have attributed the beneficial effects of intracellular reducing environment to reduced glutathione (GSH), these observations cannot rule out the possible implication of the redox milieu in its whole on transfection efficiency of bioreducible transfectants leaving the determinants of DNA release largely undefined.Methodology/Principal FindingsWith the aim of addressing this issue, SS14 was here formulated into binary and ternary 100 nm-extruded liposomes and the effects of the helper lipid composition and of the SS14/helper lipids molar ratio on chemical-physical and structural parameters defining transfection effectiveness were investigated. Among all formulations tested, DOPC/DOPE/SS14 at 25∶50∶25 molar ratio was the most effective in transfection studies owing to the presence of dioleoyl chains and phosphatidylethanolamine head groups in co-lipids. The increase in SS14 content up to 50% along DOPC/DOPE/SS14 liposome series yielded enhanced transfection, up to 2.7-fold higher than that of the benchmark Lipofectamine 2000, without altering cytotoxicity of the corresponding lipoplexes at charge ratio 5. Secondly, we specifically investigated the redox-dependent mechanisms of gene delivery into cells through tailored protocols of transfection in GSH-depleted and repleted vs. increased oxidative stress conditions. Importantly, GSH specifically induced DNA release in batch and in vitro.Conclusions/SignificanceThe presence of helper lipids carrying unsaturated dioleoyl chains and phosphatidylethanolamine head groups significantly improved transfection efficiencies of DOPC/DOPE/SS14 lipoplexes. Most importantly, this study shows that intracellular GSH levels linearly correlated with transfection efficiency while oxidative stress levels did not, highlighting for the first time the pivotal role of GSH rather than oxidative stress in its whole in transfection of bioreducible vectors.

Splenic Delivery System of pDNA through Complexes Electrostatically Constructed with Protamine and Chondroitin Sulfate

“…Complexes with positive charges on the particle surface interacted electrostatically with the cell membrane and led to high gene expression. 22) Gene expression was increased with an increase in the protamine charge ratio, and the highest level was observed at charge ratios of 12.8 and 16 (Fig. 2).…”

A peptide-mediated targeting gene delivery system for malignant glioma cells

“…6,7,11,14 It has been reported that many carriers can augment the efficiency of introducing drugs or nucleotides into glioma cells. 20,[26][27][28][29] One peptide, called VTW, which can selectively bind to GBM cells, indicates another promising approach. 22 One report showed that the VTW peptide specifically increased the uptake of nitric oxide by GBM cells.…”

Recent progress in gene therapy to deliver nucleic acids with multivalent cationic vectors

“…The cationic groups commonly used are ammonium [53,54], imidazolium [55][56][57], pyridinium [58,59], lysine or arginine [60][61][62], and/or amine/polyamines [63][64][65][66][67].…”

“…The electrostatic interaction in lipoplexes formed between GCLs and DNA or RNA, in the presence of a helper lipid as DOPE, DOPC, DMPC and others, have been fully investigated recently. Thus, several groups have studied lipoplexes containing the GCL series formed by two alkyl hydrophobic chains, two ammonium headgroups and an alkyl spacer between them and either DNA [54,66,71,91,[109][110][111][112][113] or siRNA [114]. Other groups have reported the electrostatic interaction when the GCL contains an oligo-oxyethylene spacer [93,115], and/or other headgroups as imidazolium or pyridinium [45, 57-59, 75, 99, 116].…”