Lipoplexes formed by the pEGFP-C3 plasmid DNA (pDNA) and lipid mixtures containing cationic gemini surfactant of the 1,2-bis(hexadecyl dimethyl ammonium) alkanes family referred to as C16CnC16, where n=2, 3, 5, or 12, and the zwitterionic helper lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) have been studied from a wide variety of physical, chemical, and biological standpoints. The study has been carried out using several experimental methods, such as zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), cryo-TEM, gene transfection, cell viability/cytotoxicity, and confocal fluorescence microscopy. As reported recently in a communication (J. Am. Chem. Soc. 2011, 133, 18014), the detailed physicochemical and biological studies confirm that, in the presence of the studied series lipid mixtures, plasmid DNA is compacted with a large number of its associated Na+ counterions. This in turn yields a much lower effective negative charge, qpDNA−, a value that has been experimentally obtained for each mixed lipid mixture. Consequently, the cationic lipid (CL) complexes prepared with pDNA and CL/DOPE mixtures to be used in gene transfection require significantly less amount of CL than the one estimated assuming a value of qDNA−=−2. This drives to a considerably lower cytotoxicity of the gene vector. Depending on the CL molar composition, α, of the lipid mixture, and the effective charge ratio of the lipoplex, ρeff, the reported SAXS data indicate the presence of two or three structures in the same lipoplex, one in the DOPE-rich region, other in the CL-rich region, and another one present at any CL composition. Cryo-TEMand SAXS studies with C16CnC16/DOPE-pDNA lipoplexes indicate that pDNA is localized between the mixed lipid bilayers of lamellar structures within a monolayer of ∼2 nm. This is consistent with a highly compacted supercoiled pDNA conformation compared with that of linear DNA. Transfection studies were carried out with HEK293T, HeLa, CHO, U343, and H460 cells. The α and ρeff values for each lipid mixture were optimized on HEK293T cells for transfection, and using these values, the remaining cells were also transfected in absence (-FBS-FBS) and presence (-FBS+FBS) of serum. The transfection efficiency was higher with the CLs of shorter gemini spacers (n=2 or 3). Each formulation expressed GFP on pDNA transfection and confocal fluorescence microscopy corroborated the results. C16C2C16/DOPE mixtures were the most efficient toward transfection among all the lipid mixtures and, in presence of serum, even better than the Lipofectamine2000, a commercial transfecting agent. Each lipid combination was safe and did not show any significant levels of toxicity. Probably, the presence of two coexisting lamellar structures in lipoplexes synergizes the transfection efficiency of the lipid mixtures which are plentiful in the lipoplexes formed by CLs with short spacer (n=2, 3) than those with the long spacer (n=5, 12).

The most important objective of the present study was to explain why cationic lipid (CL)-mediated delivery of plasmid DNA (pDNA) is better than that of linear DNA in gene therapy, a question that, until now, has remained unanswered. Herein for the first time we experimentally show that for different types of CLs, pDNA, in contrast to linear DNA, is compacted with a large amount of its counterions, yielding a lower effective negative charge. This feature has been confirmed through a number of physicochemical and biochemical investigations. This is significant for both in vitro and in vivo transfection studies. For an effective DNA transfection, the lower the amount of the CL, the lower is the cytotoxicity. The study also points out that it is absolutely necessary to consider both effective charge ratios between CL and pDNA and effective pDNA charges, which can be determined from physicochemical experiments.

Lipoplex-type nanoaggregates prepared from pEGFP-C3 plasmid DNA (pDNA) and mixed liposomes, with a gemini cationic lipid (CL) [1,2-bis(hexadecyl imidazolium) alkanes], referred as (C16Im)2Cn (where Cn is the alkane spacer length, n = 2, 3, 5, or 12, between the imidazolium heads) and DOPE zwitterionic lipid, have been analyzed by zeta potential, gel electrophoresis, SAXS, cryo-TEM, fluorescence anisotropy, transfection efficiency, fluorescence confocal microscopy, and cell viability/cytotoxicity experiments to establish a structure-biological activity relationship. The study, carried out at several mixed liposome compositions, α, and effective charge ratios, ρeff, of the lipoplex, demonstrates that the transfection of pDNA using CLs initially requires the determination of the effective charge of both. The electrochemical study confirms that CLs with a delocalizable positive charge in their headgroups yield an effective positive charge that is 90% of their expected nominal one, while pDNA is compacted yielding an effective negative charge which is only 10-25% than that of the linear DNA. SAXS diffractograms show that lipoplexes formed by CLs with shorter spacer (n = 2, 3, or 5) present three lamellar structures, two of them in coexistence, while those formed by CL with longest spacer (n = 12) present two additional inverted hexagonal structures. Cryo-TEM micrographs show nanoaggregates with two multilamellar structures, a cluster-type (at low α value) and a fingerprint-type, that coexist with the cluster-type at moderate α composition. The optimized transfection efficiency (TE) of pDNA, in HEK293T, HeLa, and H1299 cells was higher using lipoplexes containing gemini CLs with shorter spacers at low α value. Each lipid formulation did not show any significant levels of toxicity, the reported lipoplexes being adequate DNA vectors for gene therapy and considerably better than both Lipofectamine 2000 and CLs of the 1,2-bis(hexadecyl ammnoniun) alkane series, recently reported.

The encapsulation processes of sodium dodecyl sulfate (SDS) or sodium perfluorooctanoate (SPFO) monomers into the cavity of /9-Cyclodextrin (9-CD) and its effect in the micellization process of the surfactant itself have been analyzed by measuring the speed of sound, u, at 298.15 K (a) as a function of [surfactant] in the presence of various constant concentrations of /3-CD and (b) as a function of [/9-CD] at different surfactant constant concentrations both in the premicellar and micellar regions. The predominant complex formed (/3-CD:surfactant) in both cases has a stoichiometry of 1:1 and the association constants K have been determined from speed of sound measurements by using a semiempirical model proposed by us previously. The apparent critical micellar concentration, cmc*, is found to increase upon the addition of cyclodextrin, for both systems. However, the concentration of free surfactant available for the micellization process in the postmicellar region when the cyclodextrin is present, [surf]f, remains constant in the case of SDS + 9-CD and presents an overall increase in the case of SPFO + /9-CD.

Lipoplexes of plasmid DNA and mixed liposomes, with a gemini cationic lipid of the 1,2-bis(hexadecyl imidazolium) oxyethylene series, improves their biological activity.

The formation of mixed aggregates has been investigated on a ternary system consisting of two cationic surfactants with similar polar heads and two and/or one 12 carbon atom hydrophobic tail, respectively, didodecyldimethylammonium bromide and dodecylethyldimethylammonium bromide and water. The study has been carried out by means of conductivity, zeta potential, and cryogenic transmission electronic microscopy (cryo-TEM) experiments on the very diluted region. A variety of mixed aggregates, microaggregates, vesicles, and micelles has been found, depending on system composition and total surfactant concentration. Mixed critical microaggregate concentration and mixed critical vesicle concentration have been determined from conductivity data. Furthermore, zeta potential and cryo-TEM experiments allow for the characterization of the aggregates/solution interface and of the shape and size of the aggregates. This experimental evidence has also been analyzed in terms of the theoretical packing parameter, P.

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The compaction of DNA by cationic liposomes constituted by a mixture of a cationic lipid, dioctadecyldimethylammonium bromide (DODAB), and a zwitterionic lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), has been evaluated by means of experimental studies (electrophoretic mobility, conductometry, cryogenic electron transmission microscopy or cryo-TEM, and fluorescence spectroscopy) as well as theoretical calculations. This information reveals that DODAB/DOPE and DODAB/DLPC liposomes are mostly spherical and unilamellar, with a mean diameter of around 70 and 61 nm, respectively, a bilayer thickness of 4.5 nm, and gel-to-fluid transition temperatures, T(m), of around 19 and 28 degrees C, respectively. Their positively charged surfaces efficiently compact the negatively charged DNA by means of a strong entropically driven surface interaction that yields DODAB/DOPE-DNA and DODAB/DLPC-DNA lipoplexes as confirmed by zeta potential and ethidium bromide fluorescence intercalation assays. These experiments have permitted as well the evaluation of the different microenvironments of varying polarity of the DNA helix, liposomes, and/or lipoplexes. DODAB/DOPE-DNA and DODAB/DLPC-DNA lipoplexes have been characterized by isoneutrality ratios (L/D)(phi) of around 4.7 and 4.8, respectively, a more fluid membrane than that of the parent liposomes, and T(m) around 24 and 28 degrees C, respectively, as revealed by fluorescence anisotropy. Cryo-TEM micrographs reveal a rich scenario of nanostructures and morphologies, from unilamellar DNA-coated liposomes to multilamellar lipoplexes passing through cluster-like structures. Phase diagrams (aggregation and re-entrant condensation phenomena), calculated by means of a phenomenological theory, have confirmed the experimental concentration domains and the isoneutrality conditions. The influence of helper lipid in the compaction process, as well as the optimum choice among those herein chosen, has been analyzed.