Abstract
BackgroundLipoplexes are non-viral vectors based on cationic lipids used to deliver DNA into cells, also known as lipofection. The positively charge of the hydrophilic head-group provides the cationic lipids the ability to condensate the negatively charged DNA into structured complexes. The polar head can carry a large variety of chemical groups including amines as well as guanidino or imidazole groups. In particular, gemini cationic lipids consist of two positive polar heads linked by a spacer with different length. As for the hydrophobic aliphatic chains, they can be unsaturated or saturated and are connected to the polar head-groups. Many other chemical components can be included in the formulation of lipoplexes to improve their transfection efficiency, which often relies on their structural features. Varying these components can drastically change the arrangement of DNA molecules within the lamellar, hexagonal or cubic phases that are provided by the lipid matrix. Lipofection is widely used to deliver genetic material in cell culture experiments but the simpler formulations exhibit major drawbacks related to low transfection, low specificity, low circulation half-life and toxicity when scaled up to in vivo experiments.ResultsSo far, we have explored in cell cultures the transfection ability of lipoplexes based on gemini cationic lipids that consist of two C16 alkyl chains and two imidazolium polar head-groups linked with a polyoxyethylene spacer, (C16Im)2(C4O). Here, PEGylated lipids have been introduced to the lipoplex formulation and the transgene expression of the Opa1 mitochondrial transmembrane protein in mice was assessed. The addition of PEG on the surface of the lipid mixed resulted in the formation of Ia3d bicontinuous cubic phases as determined by small angle X-ray scattering. After a single intramuscular administration, the cubic lipoplexes were accumulated in tissues with tight endothelial barriers such as brain, heart, and lungs for at least 48 h. The transgene expression of Opa1 in those organs was identified by western blotting or RNA expression analysis through quantitative polymerase chain reaction.ConclusionsThe expression reported here is sufficient in magnitude, duration and toxicity to consolidate the bicontinuous cubic structures formed by (C16Im)2(C4O)-based lipoplexes as valuable therapeutic agents in the field of gene delivery.Graphical
Highlights
Lipoplexes are non-viral vectors based on cationic lipids used to deliver Deoxyribonucleic acid (DNA) into cells, known as lipofection
Formation of (C16Im)2(C4O)/DOPE/DSPE‐PEG/Optic atrophy type 1 (OPA1) lipoplexes and structural characterization The mixed lipids used in this work consisted of bis oxyethylene gemini cationic lipid (with two imidazolium polar head linked with a polyoxyethylene spacer of one oxygen and four carbon atoms, (C16Im)2(C4O) [24], the zwitterionic phospholipid DOPE and the PEGylated lipid
Lipoplexes were formed by incubating the mixed lipids (C16Im)2(C4O)/DOPE/DSPE-PEG with increasing amounts of the plasmid DNA coding for the mitochondrial fusion protein Opa1 (MSCV-OPA1)
Summary
Lipoplexes are non-viral vectors based on cationic lipids used to deliver DNA into cells, known as lipofection. The positively charge of the hydrophilic head-group provides the cationic lipids the ability to condensate the negatively charged DNA into structured complexes. Lipidbased phases allow the specific incorporation of multiple drugs along with DNA into its hydrophilic or hydrophobic cores [7] and versatile formulations able to simultaneously address cell targeting, highly concentrated loads and an enhanced endosomal escape [8]. Inverted hexagonal phases result of the presence of non-bilayer lipids such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) [9, 10], which facilitates protein-independent membrane destabilization and fusion and subsequent endosomal escape [9, 10]. PEGylation has been previously used to produce new long-circulating lipoplexes for gene delivery purposes [18,19,20]
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