J. P. Neves Silva

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.

Characterization of Monoolein-Based Lipoplexes Using Fluorescence Spectroscopy

Lipoplexes are commonly used as delivery systems in vitro and in vivo, the role of a neutral lipid as helper being of extreme importance in these systems. Cationic liposomes composed of dioctadecyldimethylammonium bromide (DODAB) with monoolein (MO) as a helper, at different molar ratios (1:2; 1:1 and 1:0.5) were prepared, and subsequently titrated to DNA. The structural and physicochemical properties of the lipid/DNA complexes were assessed by ethidium bromide (EtBr) exclusion, 90° static light scattering (90° SLS) assays and fluorescence resonance energy transfer (FRET). In EtBr exclusion assays, the steady-state fluorescence spectra of EtBr were decomposed into the sum of two lognormal emissions, emanating from two different environments – H2O and DNA, and the effect of charge ratio (+/-) was observed. 90° SLS assays gave an important contribution, detecting size variations in systems with different MO fractions on the lipoplexes. In FRET assays, 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (DPH-HPC) was used as donor and EtBr as acceptor. The DNA component previously calculated by EtBr exclusion, was used to determine the energy transfer efficiency, as an indirect measurement of the lipoplexes structural and physicochemical properties. Our results demonstrate that the inclusion of monoolein in the cationic liposomes formulation significantly modifies the rate of DNA complexation, being DODAB:MO (1:1) the system with higher DNA condensation efficiency.

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 β-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.

Development of dioctadecyldimethylammonium bromide/monoolein liposomes for gene delivery

© 2012 Real Oliveira et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Development of Dioctadecyldimethylammonium Bromide/Monoolein Liposomes for Gene Delivery

How Multi-Step versus One-Step Preparation Method Affects the Physicochemical Properties and Transfection Efficiency of DNA/DODAB:MO Lipoplexes

The consequences for the transfection efficiencies of different lipoplexes preparation methods, largely remain to be explored, but the knowledge of how different experimental approaches can affect the physicochemical properties and transfection efficiency is essential for a proper tailoring of transfection complexes to particular applications. Therefore, the influence of the number of mixing steps (one-step addition versus multi-step addition of liposomes to plasmid DNA (pDNA)) and lipoplex incubation temperature on the final physicochemical properties and transfection efficiency of pDNA/ Dioctadecyldimethylammonium Bromide (DODAB):1-monooleoyl-rac-glycerol (MO) complexes was studied in three distinct DODAB:MO molar ratios: 4:1, 2:1 and 1:1. Dynamic Light Scattering (DLS), Zeta (I¶) Potential, Ethidium Bromide (EtBr) exclusion assays were used to assess the formation, structure and destabilization of the lipoplexes, whereas in vitro transfection assays with pSV-I²-gal plasmid DNA were performed to evaluate their transfection efficiency on the 293T mammalian cell line. Results indicate that the morphology of pDNA/DODAB:MO complexes is dependent on the lipoplex preparation method, resulting in particles of distinct size, surface charge and membrane fluidity. These variations are visible during the complexation dynamics of pDNA and continue throughout the profile of pDNA release from pDNA/DODAB:MO lipoplexes upon incubation with Heparin (HEP), as well as in the in vitro transfection assays. The stepwise addition of DODAB:MO vesicles to pDNA decreases the transfection efficiency of the lipoplexes, while the effect of the lipoplex preparation methods is dependent on the MO content.

Monoolein as helper lipid for non-viral transfection in mammals.

The Portuguese Foundation for Science and Technology (FCT) for the financial support to the Center of Physics and Center of Molecular & Environmental Biology and funding through projects PTDC/QUI/69795/2006 and SFRH/BD/46968/2009 are acknowledged.