Structural dynamics and physicochemical properties of pDNA/DODAB:MO lipoplexes: effect of pH and anionic lipids in inverted non-lamellar phases versus lamellar phases.

Dioctadecyldimethylammonium bromide (DODAB):Monoolein (MO) lipoplexes have mainly been studied within the range of high molar ratios of DODAB, with noticeable transfection efficiencies in the Human Embryonic Kidney (HEK, a.k.a. 293T) cell line. In this work, we intend to study the effect of high MO content on the structure and physicochemical properties of pDNA/DODAB:MO lipoplexes to achieve some correlation with their transfection efficiency. Static/Dynamic Light Scattering and Cryo-TEM imaging were used to characterize the size/morphology of DNA/DODAB:MO lipoplexes at different DODAB:MO contents (2:1, 1:1, 1:2) and charge ratios (CRs) (+/-). Nile Red fluorescence emission was performed to detect changes in microviscosity, hydration and polarity of DNA/DODAB:MO systems. Lipoplexes stability at physiological pH values and in the presence of anionic lipids was evaluated by Förster Resonance Energy Transfer (FRET). Physicochemical/structural data were complemented with transfection studies in HEK cells using the ß-galactosidase reporter gene activity assay. This work reports the coexistence of multilamellar and non-lamellar inverted phases in MO-richer lipoplexes (DODAB:MO 1:2 and 1:4), leading to transfection efficiencies comparable to those of multilamellar (DODAB-richer) lipoplexes, but at higher charge ratios [CR (+/-)=6.0] and without dose-effect response. These results may be related to the structural changes of lipoplexes promoted by high MO content.

DODAB:monoolein-based lipoplexes as non-viral vectors for transfection of mammalian cells.

DNA/Cationic liposome complexes (lipoplexes) have been widely used as non-viral vectors for transfection. Neutral lipids in liposomal formulation are determinant for transfection efficiency using these vectors. In this work, we studied the potential of monoolein (MO) as helper lipid for cellular transfection. Lipoplexes composed of pDNA and dioctadecyldimethylammonium bromide (DODAB)/1-monooleoyl-rac-glycerol (MO) at different molar ratios (4:1, 2:1 and 1:1) and at different cationic lipid/DNA ratios were investigated. The physicochemical properties of the lipoplexes (size, charge and structure), were studied by Dynamic Light Scattering (DLS), Zeta Potential (ζ) and cryo-transmission electron microscopy (cryo-TEM). The effect of MO on pDNA condensation and the effect of heparin and heparan sulphate on the percentage of pDNA release from the lipoplexes were also studied by Ethidium Bromide (EtBr) exclusion assays and electrophoresis. Cytotoxicity and transfection efficiency of these lipoplexes were evaluated using 293T cells and compared with the golden standard helper lipids 1,2-dioleoyl-sn-glycero-3-hosphoethanolamine (DOPE) and cholesterol (Chol) as well as with a commercial transfection agent (Lipofectamine™ LTX). The internalization of transfected fluorescently-labeled pDNA was also visualized using the same cell line. The results demonstrate that the presence of MO not only increases pDNA compactation efficiency, but also affects the physicochemical properties of the lipoplexes, which can interfere with lipoplex-cell interactions. The DODAB:MO formulations tested showed little toxicity and successfully mediated in vitro cell transfection. These results were supported by fluorescence microscopy studies, which illustrated that lipoplexes were able to access the cytosol and deliver pDNA to the nucleus. DODAB:MO-based lipoplexes were thus validated as non-toxic, efficient lipofection vectors for genetic modification of mammalian cells. Understanding the relation between structure and activity of MO-based lipoplexes will further strengthen the development of these novel delivery systems.