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.
Highlights
Experimental and Personalised Medicines Section, UCL Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK
Biophysical analysis revealed that lipopolyplex formulations containing DOPE and DOPC lipids in variable proportions, were of a consistent size and charge with only marginal affects of alterations to the lipid composition of each formulation
transmission electron microscopy (TEM) images showed that there were no significant differences in lipopolyplex morphology with a mixture of spheres and rods observed in most cases of similar sizes
Summary
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. We have shown previously that the peptide mediates DNA packaging and receptor targeting and so the focus of this study was to investigate the function of the lipids and how they contribute to the transfection efficiency of RTN lipopolyplexes. The neutral lipid DOPE in cationic lipoplex formulations enables higher transfection efficiencies as the conical structure of this lipid promotes the formation of inverted hexagonal structures that rapidly fuse with the endosomal lipid bilayer, independent of charge, enabling cytoplasmic release of the DNA. In vivo transfection efficiencies of RTNs containing DOPE or DOPC were compared to assess the relevance of in vitro studies to in vivo applications
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