The Role of the Helper Lipid on the DNA Transfection Efficiency of Lipopolyplex Formulations

Abstract

Multifunctional, lipopolyplex formulations comprising a mixture of cationic liposomes and cationic, receptor-targeting peptides have potential use in gene therapy applications. Lipopolyplex formulations described here are typically far more efficient transfection agents than binary lipoplex or polyplex formulations. It has been shown previously that the peptide component mediates both DNA packaging and targeting of the nanoparticle while in this report we investigate the contribution of the lipid component. We hypothesised that the lipid components synergise with the peptides in the transfection process by promoting endosomal escape after lipid bilayer fusion. Lipopolyplexes were prepared with cationic liposomes comprising DOTAP with either neutral lipid DOPE or DOPC. DOPE promotes fusogenic, inverted hexagonal lipid structures while DOPC promotes more stable laminar structures. Lipopolyplexes containing DOPE showed substantially higher transfection efficiency than those formulated with DOPC, both in vitro and in vivo. DOPE-containing lipopolyplexes showed rapid endosomal trafficking and nuclear accumulation of DNA while DOPC-containing formulations remained within the late endo-lysosomal compartments. These findings are consistent with previous finding for the role of DOPE in lipoplexes and support the hypothesis regarding the function of the lipid components in lipopolyplexes. These findings will help to inform future lipopolyplex design, strategies and clinical development processes.

Lipid rafts-mediated endocytosis and physiology-based cell membrane traffic models of doxorubicin liposomes

“…However, in both cells the elevated scopes of TC, B and I time courses from C40-1-A to C40-1-B were shortened when compared with those from C40-1-0 to C40-1-A, which suggest that ≥ 20 mol% of DOPE in liposomes is effective for improving the internalization of both cells. The inverted hexagonal structure of the DOPE lipid enabled cell/endosome membrane fusion, compared with the lamellar DPPC structure [18].…”

“…DDAB and DOTAP contribute positive surface charge of liposomes. DOPE promotes the fusion of liposomes and cell/endosome membranes, while DPPC promotes more stable laminar structures [18,21,22]. PEG-DSPE was used in some formulations to achieve surface pegylation.…”

“…Due to siRNA-mediated gene silencing been completely abolished in cationic liposomes containing 5 mol%, but not 1-2 mol%, of PEG-DSPE [17], the cationic liposomes containing 1 mol% of PEG-DSPE were adopted in current study to achieve surface pegylation [2]. The fusogenic lipid DOPE helps liposomes to efficiently enter into cytosol, but not endosomes, since it adopts a hexagonal inverted phase structure allowing liposome fusion with the endosomal membrane [18]. Due to the restrict effects of lipid structures, i.e., cationic lipid, fusogenic lipid, and pegylation, on the ability of liposomes to target intracellular sites, the understanding of the quantitative relationships between these parameters and the determination of the kinetic processes of liposomes in intracellular compartments are critical for optimizing the formulation.…”

Effect of inserted spacer in hepatic cell-penetrating multifunctional peptide component on the DNA intracellular delivery of quaternary complexes based on modular design

“…19 Briefly, the required amounts of DOTAP and DOPE were first dissolved in chloroform at the weight ratio of 1:1, and a thin lipid film was produced via slowly evaporating the chloroform overnight with a rotary evaporator under vacuum conditions. The lipid film was subsequently hydrated with double-distilled water at room temperature to produce a crude suspension of 1 mg/mL.…”

A method for concentrating lipid peptide DNA and siRNA nanocomplexes that retains their structure and transfection efficiency

“…Empirically optimized ratios of these components are required to produce monodisperse nanoparticles with peak transfection efficiency. The formulation used in this study comprises a mixture of liposomes and targeting peptides that self-assemble on mixing with DNA [11][12][13][14][15][16][17][18] or siRNA [19][20][21] to form LPD or LPR receptor-targeted nanocomplexes (RTNs). Based on this platform, we have described both cationic and anionic RTN formulations and shown their in vivo potential by delivery The formulation of RTNs is more complicated than simple lipoplex or polyplexes and becomes increasingly problematic in our experience at higher nucleic acid concentrations of more than 0.5 mg/mL with large aggregates and precipitates forming, which leads to poor biodistribution, systemic toxicity, and low transfection efficiency in vivo.…”