M. E. C. D. Real Oliveira

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.

Effect of temperature and surfactant on the control release of microencapsulated dye in lecithin liposomes. I.

Abstract The objective of our work has been the microencapsulation of dyes with lecithin from soybean, with the formation of liposomes, as a substitute for synthetic auxiliaries so as to improve the quality of the effluent. Current scenarios promote the disintegration and leakage of the liposomes, such as, changes in temperature, pH and the use of surfactants. Since dyeing process is a mix of all these parameters, we pretended to study each one separately. Rhodamine 6G fluorescence is known to be concentration quenched through the formation of non-fluorescent dimmers and, additionally, through the energy transfer from rhodamine monomer to these dimmers (Baptista ALF, Coutinho PJG, Real Oliveira MECD, Gomes JINR. Proceedings of 13th International Symposium of Surfactants, SIS 2000, Gainesville, USA, 2000). The temperature, the surfactant and pH induce a release of the encapsulated dye resulting in rhodamine dilution and consequently alterations in the dimerization/binding equilibrium. The experimental spectra indicate that rhodamine binds almost completely to liposomes. The decomposition of the rhodamine fluorescence spectra allowed us to determine the percentage of released dye during a simulated dyeing process, and allowed us to conclude that the dimerization process occurs mainly at the inner interfaces. The amount of dye released induced by temperature changes was greater in the presence of surfactant.

Effect of Surfactants in Soybean Lecithin Liposomes Studied by Energy Transfer Between NBD-PE and N-Rh-PE

Abstract The effect of C12E8 (polyoxyethylene 8 lauryl ether) and sodium laurate on the structural properties of soybean lecithin vesicles was studied in different concentrations of surfactants, using the fluorescent probes NBD-PE (N-7-nitro-2,1,3-benzodiazoyl phosphatidylethanolamine) and N-Rh-PE (N-lissamine rhodamine B sulfonyl phosphatidylethanolamine). Direct energy transfer studies were carried out in labelled vesicles with addition of surfactants. Rhodamine emission with maximum at β585 nm was detected by excitation of NBD at λ = 473 nm. This fact is caused by direct energy transfer process from NBD to rhodamine. The yield of this process decreases with increasing amounts of surfactants, indicating that the average spatial separation of the lipid probes increases, as a result of the enlargement of the vesicle size and due to alteration of its structure to mixed micelles.

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

Monitoring the Phase Transition of C12E5/Water/Alkane Microemulsions Through Excimer Formation

The phase transition of microemulsions involving the nonionic surfactant C12E5 [C12H25(OCH2-CH2)5OH], water, and alkanes (heptane, decane and tetradecane) has been investigated through the excimer formation of pyrene. On going to the microemulsion bicontinuous phase, by changing either composition or temperature, pronounced changes in the pyrene excimer-to-monomer fluorescence intensity ratio, IE/IM, are observed. Several differences in the steady-state emission spectra and in fluorescence decay curves show that as a probe pyrene is well suited to follow the transition from the water continuous to the oil continuous phase, through an intermediate bicontinuous (continuous in both water and oil) region. The results provide information about the different characteristics and structure of these three regions (water continuous, bicontinuous, and oil continuous) of the phase diagram for C12E5/water/alkane systems.

Role of counter-ion and helper lipid content in the design and properties of nanocarrier systems: a biophysical study in 2D and 3D lipid assemblies

There is a direct correlation between the physicochemical properties of nanocarrier systems and their biological performance, including stability under physiological conditions, cellular internalization and transfection efficiency. Therefore, understanding the biophysical aspects that affect self-assembled nanocarriers is determinant for a rational design of efficient formulations. In this study, a comprehensive evaluation of the effects of each component on the molecular organization of aggregates formed by the cationic lipids dioctadecyldimethylammonium bromide and chloride (DODAB and DODAC) and the neutral lipid monoolein (MO) was made. Specifically, the effects of the helper lipid content (MO) and the role of the counter-ion of the cationic lipids were evaluated in 2D and 3D assemblies by Langmuir surface pressure–molecular area (π–A) isotherms, Brewster Angle Microscopy (BAM), infrared reflection absorption spectroscopy (IRRAS), confocal Raman microscopy, and Small Angle X-ray Scattering (SAXS). The results show that MO has a different distribution on the DODAC and DODAB bilayers, and a fluidizing effect dependent on the MO content. For low MO molar ratios, the fluidizing effect was more pronounced in DODAC : MO mixtures, indicating a more homogeneous distribution of MO in DODAC than in DODAB bilayers. For high MO molar ratios, packing of membranes was similar for both cationic lipids, and the effect of the counter-ion is attenuated. The distribution of MO in the two cationic systems is closely related with the efficiency of the counter-ions in the screening of the charged group.

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.