Efficient Cellular Knockdown Mediated by siRNA Nanovectors of Gemini Cationic Lipids Having Delocalizable Headgroups and Oligo-Oxyethylene Spacers

The use of small interfering RNAs (siRNAs) to silence specific genes is one of the most promising approaches in gene therapy, but it requires efficient nanovectors for successful cellular delivery. Recently, we reported liposomal gene carriers derived from a gemini cationic lipid (GCL) of the 1,2-bis­(hexadecyl dimethyl imidazolium) oligo-oxyethylene series ((C₁₆Im)₂(C₂H₄O)_<i>n</i>C₂H₄ with <i>n</i> = 1, 2, or 3) and 1,2-dioleyol phosphatidylethanolamine as highly efficient cytofectins for pDNA. On the basis of the satisfactory outcomes of the previous study, the present work focuses on the utility of coliposomes of these gemini lipids with the biocompatible neutral lipid mono oleoyl glycerol (MOG) as highly potent vectors for siRNA cellular transport in the presence of serum. The (C₁₆Im)₂(C₂H₄O)_<i>n</i>C₂H₄/MOG-siRNA lipoplexes were characterized through (i) a physicochemical study (zeta potential, cryo-transmission electron microscopy, small-angle X-ray scattering, and fluorescence anisotropy) to establish the relationship between size, structure, fluidity, and the interaction between siRNA and the GCL/MOG gene vectors and (ii) a biological analysis (flow cytometry, fluorescence microscopy, and cell viability) to report the anti-GFP siRNA transfections in HEK 293T, HeLa, and H1299 cancer cell lines. The in vitro biological analysis confirms the cellular uptake and indicates that a short spacer, a very low molar fraction of GCL in the mixed lipid, and a moderate effective charge ratio of the lipoplex yielded maximum silencing efficacy. At these experimental conditions, the siRNA used in this work is compacted by the GCL/MOG nanovectors by forming two cubic structures (<i>Ia</i>3<i>d</i> and <i>Pm</i>3<i>n</i>) that are correlated with excellent silencing activity. These liposomal nanocarriers possess high silencing activity with a negligible cytotoxicity, which strongly supports their practical use for in vivo knockdown studies

How Does the Spacer Length of Cationic Gemini Lipids Influence the Lipoplex Formation with Plasmid DNA? Physicochemical and Biochemical Characterizations and their Relevance in Gene Therapy

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 C₁₆C_<i>n</i>C₁₆, where <i>n</i> = 2, 3, 5, or 12, and the zwitterionic helper lipid, 1,2-dioleoyl-<i>sn</i>-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 (<i>J. Am. Chem. Soc.</i> <b>2011</b>, <i>133</i>, 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, <i>q</i>_pDNA^–, 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 <i>q</i>_DNA^– = −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, ρ_eff, 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-TEM and SAXS studies with C₁₆C_<i>n</i>C₁₆/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 ρ_eff 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 (<i>n</i> = 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 (<i>n</i> = 2, 3) than those with the long spacer (<i>n</i> = 5, 12)

Effects of a Delocalizable Cation on the Headgroup of Gemini Lipids on the Lipoplex-Type Nanoaggregates Directly Formed from Plasmid DNA

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)₂C_<i>n</i> (where C_<i>n</i> is the alkane spacer length, <i>n</i> = 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, ρ_eff, 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 (<i>n</i> = 2, 3, or 5) present three lamellar structures, two of them in coexistence, while those formed by CL with longest spacer (<i>n</i> = 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

Magnetic Silica Nanoparticle Cellular Uptake and Cytotoxicity Regulated by Electrostatic Polyelectrolytes–DNA Loading at Their Surface

Magnetic silica nanoparticles show great promise for drug delivery. The major advantages correspond to their magnetic nature and ease of biofunctionalization, which favors their ability to interact with cells and tissues. We have prepared magnetic silica nanoparticles with DNA fragments attached on their previously polyelectrolyte-primed surface. The remarkable feature of these materials is the compromise between the positive charges of the polyelectrolytes and the negative charges of the DNA. This dual-agent formulation dramatically changes the overall cytotoxicity and chemical degradation of the nanoparticles, revealing the key role that surface functionalization plays in regulating the mechanisms involved