Transgene expression in mice of the Opa1 mitochondrial transmembrane protein through bicontinuous cubic lipoplexes containing gemini imidazolium surfactants.

BACKGROUND: Lipoplexes are non-viral vectors based on cationic lipids used to deliver DNA into cells, also known as lipofection. The positively charge of the hydrophilic head-group provides the cationic lipids the ability to condensate the negatively charged DNA into structured complexes. The polar head can carry a large variety of chemical groups including amines as well as guanidino or imidazole groups. In particular, gemini cationic lipids consist of two positive polar heads linked by a spacer with different length. As for the hydrophobic aliphatic chains, they can be unsaturated or saturated and are connected to the polar head-groups. Many other chemical components can be included in the formulation of lipoplexes to improve their transfection efficiency, which often relies on their structural features. Varying these components can drastically change the arrangement of DNA molecules within the lamellar, hexagonal or cubic phases that are provided by the lipid matrix. Lipofection is widely used to deliver genetic material in cell culture experiments but the simpler formulations exhibit major drawbacks related to low transfection, low specificity, low circulation half-life and toxicity when scaled up to in vivo experiments. RESULTS: So far, we have explored in cell cultures the transfection ability of lipoplexes based on gemini cationic lipids that consist of two C16 alkyl chains and two imidazolium polar head-groups linked with a polyoxyethylene spacer, (C16Im)2(C4O). Here, PEGylated lipids have been introduced to the lipoplex formulation and the transgene expression of the Opa1 mitochondrial transmembrane protein in mice was assessed. The addition of PEG on the surface of the lipid mixed resulted in the formation of Ia3d bicontinuous cubic phases as determined by small angle X-ray scattering. After a single intramuscular administration, the cubic lipoplexes were accumulated in tissues with tight endothelial barriers such as brain, heart, and lungs for at least 48 h. The transgene expression of Opa1 in those organs was identified by western blotting or RNA expression analysis through quantitative polymerase chain reaction. CONCLUSIONS: The expression reported here is sufficient in magnitude, duration and toxicity to consolidate the bicontinuous cubic structures formed by (C16Im)2(C4O)-based lipoplexes as valuable therapeutic agents in the field of gene delivery.

Gemini-Based Lipoplexes Complement the Mitochondrial Phenotype in MFN1-Knockout Mouse Embryonic Fibroblasts.

Mitochondria form a dynamic network of constantly dividing and fusing organelles. The balance between these antagonistic processes is crucial for normal cellular function and requires the action of specialized proteins. The mitochondrial membrane proteins mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2) are responsible for the fusion of the outer membrane of adjacent mitochondria. Mutations within Mfn1 or Mfn2 impair mitochondrial fusion and lead to some severe mitochondrial dysfunctions and mitochondrial diseases (MDs). A characteristic phenotype of cells carrying defective Mfn1 or Mfn2 is the presence of a highly fragmented mitochondrial network. Here, we use a biocompatible mixture of lipids, consisting on synthetic gemini cationic lipids (GCLs) and the zwitterionic phospholipid (DOPE), to complex, transport, and deliver intact copies of MFN1 gene into MFN1-Knockout mouse embryonic fibroblasts (MFN1-KO MEFs). We demonstrate that the GCL/DOPE-DNA lipoplexes are able to introduce the intact MFN1 gene into the cells and ectopically produce functional Mfn1. A four-fold increase of the Mfn1 levels is necessary to revert the MFN1-KO phenotype and to partially restore a mitochondrial network. This phenotype complementation was correlated with the transfection of GCL/DOPE-MFN1 lipoplexes that exhibited a high proportion of highly packaged hexagonal phase. GCL/DOPE-DNA lipoplexes are formulated as efficient therapeutic agents against MDs.

Plasmid-Templated Control of DNA-Cyclodextrin Nanoparticle Morphology through Molecular Vector Design for Effective Gene Delivery.

Engineering self-assembled superstructures through complexation of plasmid DNA (pDNA) and single-isomer nanometric size macromolecules (molecular nanoparticles) is a promising strategy for gene delivery. Notably, the functionality and overall architecture of the vector can be precisely molded at the atomic level by chemical tailoring, thereby enabling unprecedented opportunities for structure/self-assembling/pDNA delivery relationship studies. Beyond this notion, by judiciously preorganizing the functional elements in cyclodextrin (CD)-based molecular nanoparticles through covalent dimerization, here we demonstrate that the morphology of the resulting nanocomplexes (CDplexes) can be tuned, from spherical to ellipsoidal, rod-type, or worm-like nanoparticles, which makes it possible to gain understanding of their shape-dependent transfection properties. The experimental findings are in agreement with a shift from chelate to cross-linking interactions on going from primary-face- to secondary-face-linked CD dimers, the pDNA partner acting as an active payload and as a template. Most interestingly, the transfection efficiency in different cells was shown to be differently impacted by modifications of the CDplex morphology, which has led to the identification of an optimal prototype for tissue-selective DNA delivery to the spleen in vivo.

Ca(2+)-mediated anionic lipid-plasmid DNA lipoplexes. Electrochemical, structural, and biochemical studies.

Several experimental methods, such as zeta potential, gel electrophoresis, small-angle X-ray scattering, gene transfection, fluorescence microscopy, flow cytometry, and cell viability/cytotoxicity assays, have been used to analyze the potential of anionic lipids (AL) as effective nontoxic and nonviral DNA vectors, assisted by divalent cations. The lipoplexes studied are those comprised of the green fluorescent protein-encoding plasmid DNA pEGFP-C3, an anionic lipid as 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) or 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and a zwitterionic lipid, the 1,2-dioleoyl-sn -glycero-3-phosphatidylethanolamine (DOPE, not charged at physiological pH). The studies have been carried on at different liposome and lipoplex compositions and in the presence of a variety of [Ca2+]. Electrochemical experiments reveal that DOPG/DOPE and DOPS/DOPE anionic liposomes may compact more effectively pDNA at low molar fractions (with an excess of DOPE) and at AL/pDNA ratios ≈20. Calcium concentrations around 15-20 mM are needed to yield lipoplexes neutral or slightly positive. From a structural standpoint, DOPG/DOPE-Ca2+-pDNA lipoplexes are self-assembled into a HIIc phase (inverted cylindrical micelles in hexagonal ordering with plasmid supercoils inside the cylinders), while DOPS/DOPE-Ca2+-pDNA lipoplexes show two phases in coexistence: one classical HIIc phase which contains pDNA supercoils and one Lα phase without pDNA among the lamellae, i.e., a lamellar stack of lipidic bilayers held together by Ca2+ bridges. Transfection and cell viability studies were done with HEK293T and HeLa cells in the presence of serum. Lipoplexes herein studied show moderate-to-low transfection levels combined with moderate-to-high cell viability, comparable to those yield by Lipofectamine2000*, which is a cationic lipid (CL) standard formulation, but none of them improve the output of typical CL gen vectors, mostly if they are gemini or dendritic. This fact would be indicating that, nowadays, lipofection via anionic lipids and divalent cations as mediators still needs to enhance transfection levels in order to be considered as a real and plausible alternative to lipofection through improved CLs-based lipoplexes.

Lipid nanoparticles as nano-Trojan-horses for siRNA delivery and gene-knockdown.

The therapeutic messenger RNA strategies, such as those using small interfering RNAs, take several advantages (versatility, efficiency and selectivity) over plasmid DNA-based strategies. However, the challenge remains to find nanovectors capable of properly loading the genetic material, transporting it through troublesome environments, like a tumoral site, and delivering it into the cytoplasm of target cells. Here, lipid nanoparticles, consisting of a gemini cationic/neutral helper lipid mixture, are proposed as siRNA nanovector. Cells from cervical and brain cancer overexpressing the green fluorescent protein (GFP) were chosen to analyse the biological response as well as the efficiency and safety of the siRNA-loaded nanovector according to the cell phenotype. Flow cytometry and epifluorescence or confocal microscopy were used to follow the gene knockdown in these overexpressed cells. The effect of the nanovector on cellular proliferation was evaluated with cytotoxicity assays while their potential oxidative stress generation was determined by quantifying the generation of reactive oxygen species. To explore the mechanism of cellular uptake, different inhibitors of endocytic pathways were used during incubation with cells. Finally, nanovectors were incubated in 3D-grown cells (spheroids) to see whether they can penetrate the complex tumoral microenvironments, their efficiency to knockdown GFP expression being monitored by confocal microscopy.

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)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.

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 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).

Why is less cationic lipid required to prepare lipoplexes from plasmid DNA than linear DNA in gene therapy?

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.

Insights into colloidal nanoparticle-protein corona interactions for nanomedicine applications.

Colloidal nanoparticles (NPs) have attracted significant attention due to their unique physicochemical properties suitable for diagnosing and treating different human diseases. Nevertheless, the successful implementation of NPs in medicine demands a proper understanding of their interactions with the different proteins found in biological fluids. Once introduced into the body, NPs are covered by a protein corona (PC) that determines the biological behavior of the NPs. The formation of the PC can eventually favor the rapid clearance of the NPs from the body before fulfilling the desired objective or lead to increased cytotoxicity. The PC nature varies as a function of the different repulsive and attractive forces that govern the NP-protein interaction and their colloidal stability. This review focuses on the phenomenon of PC formation on NPs from a physicochemical perspective, aiming to provide a general overview of this critical process. Main issues related to NP toxicity and clearance from the body as a result of protein adsorption are covered, including the most promising strategies to control PC formation and, thereby, ensure the successful application of NPs in nanomedicine.

Controlled pDNA Release in Gemini Cationic Lipoplexes by Femtosecond Laser Irradiation of Gold Nanostars.

The design of nanovectors able to overcome biological barriers is one of the main challenges in biomedicine. Gemini cationic lipids are considered potential candidates for gene therapy due to their high biocompatibility and capacity to condense nucleic acids safely in the form of lipoplexes. However, this approach presents difficulties regarding genetic unpacking and, therefore, control over this process becomes crucial to ensure successful transfection. In this work, gemini cationic lipoplexes were prepared in the presence of plasmonic gold nanostars (AuNSs) to afford a nanovector that efficiently releases plasmid DNA (pDNA) upon irradiation with near-infrared femtosecond laser pulses. A critical AuNSs concentration of 50 pM and optimized laser power density of 400 mW led to successful pDNA release, whose efficiency could be further improved by increasing the irradiation time. Agarose gel electrophoresis was used to confirm pDNA release. UV-Vis-NIR spectroscopy and transmission electron microscopy studies were performed to monitor changes in the morphology of the AuNSs and lipoplexes after irradiation. From a physicochemical point of view, this study demonstrates that the use of AuNSs combined with gemini cationic lipoplexes allows control over pDNA release under ultrafast laser irradiation.

Gemini Cationic Lipid-Type Nanovectors Suitable for the Transfection of Therapeutic Plasmid DNA Encoding for Pro-Inflammatory Cytokine Interleukin-12.

Ample evidence exists on the role of interleukin-12 (IL-12) in the response against many pathogens, as well as on its remarkable antitumor properties. However, the unexpected toxicity and disappointing results in some clinical trials are prompting the design of new strategies and/or vectors for IL-12 delivery. This study was conceived to further endorse the use of gemini cationic lipids (GCLs) in combination with zwitterionic helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanol amine) as nanovectors for the insertion of plasmid DNA encoding for IL-12 (pCMV-IL12) into cells. Optimal GCL formulations previously reported by us were selected for IL-12-based biophysical experiments. In vitro studies demonstrated efficient pCMV-IL12 transfection by GCLs with comparable or superior cytokine levels than those obtained with commercial control Lipofectamine2000*. Furthermore, the nanovectors did not present significant toxicity, showing high cell viability values. The proteins adsorbed on the nanovector surface were found to be mostly lipoproteins and serum albumin, which are both beneficial to increase the blood circulation time. These outstanding results are accompanied by an initial physicochemical characterization to confirm DNA compaction and protection by the lipid mixture. Although further studies would be necessary, the present GCLs exhibit promising characteristics as candidates for pCMV-IL12 transfection in future in vivo applications.

Experimental and theoretical approach to the sodium decanoate-dodecanoate mixed surfactant system in aqueous solution.

The mixed system consisting of two anionic surfactants of identical headgroups but with 10 and 12 carbon atoms on the hydrophobic tail, sodium decanoate (C(10)Na) and sodium dodecanoate (C(12)Na), has been studied in aqueous solution at 298.15 K by means of conductivity and fluorescence spectroscopy experiments and from a theoretical point of view. The monomeric and micellar phases of the mixed aggregates were analyzed through the experimental determination of the total critical micelle concentration, cmc*, the degree of ionization of the mixed micelle, beta, and the total aggregation number, N*. Results indicate that, compared to the ideal behavior, the mixed system with two anionic surfactants differing only in two methylenes in the hydrophobic tail shows a negative deviation in the cmc* and a positive one in N*. Pure surfactants (C(10)Na and C(12)Na) form spherical micelles, but mixed micelles must aggregate with a rodlike shape to allow more surfactant molecules than expected. In addition, rodlike micelles result in more compacted aggregation (i.e., less area per polar head). From the experimental data in this work, several theoretical models for mixed surfactant systems have been checked: Rubingh's model predicts lower deviations from ideality than Motomura's model. The stability of the micelles has been analyzed by computing the standard Gibbs energy of micelle formation, Delta G(mic,0), of pure and mixed micelles. Results of this work reinforce the feature that mixed systems formed by alkylsurfactants with the same polar head that differ in the hydrocarbon length, usually admitted as roughly ideal systems, may show nonideal behavior. This deviation, being mostly related to the difference in the chain length, Delta n(c), between surfactants can be analyzed only when very accurate experimental techniques as well as adequate theoretical models are used.

Effect of lipid composition on the structure and theoretical phase diagrams of DC-Chol/DOPE-DNA lipoplexes.

Lipoplexes constituted by calf-thymus DNA (CT-DNA) and mixed cationic liposomes consisting of varying proportions of the cationic lipid 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol) and the zwitterionic lipid, 1,2-dioleoyl-sn-glycero-3-phosphoetanolamine (DOPE) have been analyzed by means of electrophoretic mobility, SAXS, and fluorescence anisotropy experiments, as well as by theoretically calculated phase diagrams. Both experimental and theoretical studies have been run at several liposome and lipoplex compositions, defined in terms of cationic lipid molar fraction, α, and either the mass or charge ratios of the lipoplex, respectively. The experimental electrochemical results indicate that DC-Chol/DOPE liposomes, with a mean hydrodynamic diameter of around (120 ± 10) nm, compact and condense DNA fragments at their cationic surfaces by means of a strong entropically driven electrostatic interaction. Furthermore, the positive charges of cationic liposomes are compensated by the negative charges of DNA phosphate groups at the isoneutrality L/D ratio, (L/D)(ϕ), which decreases with the cationic lipid content of the mixed liposome, for a given DNA concentration. This inversion of sign process has been also studied by means of the phase diagrams calculated with the theoretical model, which confirms all the experimental results. SAXS diffractograms, run at several lipoplex compositions, reveal that, irrespectively of the lipoplex charge ratio, DC-Chol/DOPE-DNA lipoplexes show a lamellar structure, L(α), when the cationic lipid content on the mixed liposomes α ≥ 0.4, while for a lower content (α = 0.2) the lipoplexes show an inverted hexagonal structure, H(II), usually related with improved cell transfection efficiency. A similar conclusion is reached from fluorescence anisotropy results, which indicate that the fluidity on liposome and lipoplexes membrane, also related with better transfection results, increases as long as the cationic lipid content decreases.

Biocompatible Nanovector of siRNA Consisting of Arginine-Based Cationic Lipid for Gene Knockdown in Cancer Cells.

Despite the use of small interfering RNAs (siRNAs) as therapeutic agents through the knockdown expression of pathogenic proteins, transportation and delivery of such siRNAs into cells continue to be under investigation. Within nonviral vectors, cationic lipids that include amino acid residues in their structures, and that have already demonstrated their suitability as plasmid DNA nanocarriers, may be also considered as potential siRNA vehicles. A double-chain cationic lipid based on the amino acid arginine mixed with a helper lipid has been the object of this biophysical study. First, ζ-potential measurements and agarose gel electrophoresis experiments confirmed the siRNA compaction, while small-angle X-ray scattering analysis (SAXS) revealed the structural pattern of the lipoplexes. Two bicontinuous cubic phases were found to coexist: the double-gyroid phase (QIIG) and the double-diamond phase (QIID), with Pn3m and Ia3d as crystallographic space groups, respectively; the siRNA is known to be located inside their bicontinuous aqueous channels. Second, in vitro studies in HeLa-green fluorescent protein (GFP) and T731-GFP cell lines (modified for GFP overexpression) showed moderate to high gene knockdown levels (determined by flow cytometry and epifluorescence microscopy) with remarkable cell viabilities (CCK-8 assay). Finally, nano-liquid chromatography/mass spectrometry (nanoLC-MS/MS) was used to identify the nature of the proteins adhered to the surface of the lipoplexes after incubation with human serum, simulating their behavior in biological fluids. The abundant presence of lipoproteins and serum albumin in such protein corona, together with the coexistence of the bicontinuous cubic phases, may be behind the remarkable silencing activity of these lipoplexes. The results reported herein show that the use of amino-acid-based cationic lipids mixed with a suitable helper lipid, which have already provided good results as DNA plasmid nanocarriers in cellular transfection processes, may also be a biocompatible option, and so far little investigated, in gene silencing in vitro strategies.

Protein Expression Knockdown in Cancer Cells Induced by a Gemini Cationic Lipid Nanovector with Histidine-Based Polar Heads.

A histidine-based gemini cationic lipid, which had already demonstrated its efficiency as a plasmid DNA (pDNA) nanocarrier, has been used in this work to transfect a small interfering RNA (siRNA) into cancer cells. In combination with the helper lipid monoolein glycerol (MOG), the cationic lipid was used as an antiGFP-siRNA nanovector in a multidisciplinary study. Initially, a biophysical characterization by zeta potential (ζ) and agarose gel electrophoresis experiments was performed to determine the lipid effective charge and confirm siRNA compaction. The lipoplexes formed were arranged in Lα lamellar lyotropic liquid crystal phases with a cluster-type morphology, as cryo-transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering (SAXS) studies revealed. Additionally, in vitro experiments confirmed the high gene knockdown efficiency of the lipid-based nanovehicle as detected by flow cytometry (FC) and epifluorescence microscopy, even better than that of Lipofectamine2000*, the transfecting reagent commonly used as a positive control. Cytotoxicity assays indicated that the nanovector is non-toxic to cells. Finally, using nano-liquid chromatography tandem mass spectrometry (nanoLC-MS/MS), apolipoprotein A-I and A-II followed by serum albumin were identified as the proteins with higher affinity for the surface of the lipoplexes. This fact could be beyond the remarkable silencing activity of the histidine-based lipid nanocarrier herein presented.

A Non-Viral Plasmid DNA Delivery System Consisting on a Lysine-Derived Cationic Lipid Mixed with a Fusogenic Lipid.

The insertion of biocompatible amino acid moieties in non-viral gene nanocarriers is an attractive approach that has been recently gaining interest. In this work, a cationic lipid, consisting of a lysine-derived moiety linked to a C12 chain (LYCl) was combined with a common fusogenic helper lipid (DOPE) and evaluated as a potential vehicle to transfect two plasmid DNAs (encoding green fluorescent protein GFP and luciferase) into COS-7 cells. A multidisciplinary approach has been followed: (i) biophysical characterization based on zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), and cryo-transmission electronic microscopy (cryo-TEM); (ii) biological studies by fluorescence assisted cell sorting (FACS), luminometry, and cytotoxicity experiments; and (iii) a computational study of the formation of lipid bilayers and their subsequent stabilization with DNA. The results indicate that LYCl/DOPE nanocarriers are capable of compacting the pDNAs and protecting them efficiently against DNase I degradation, by forming Lα lyotropic liquid crystal phases, with an average size of ~200 nm and low polydispersity that facilitate the cellular uptake process. The computational results confirmed that the LYCl/DOPE lipid bilayers are stable and also capable of stabilizing DNA fragments via lipoplex formation, with dimensions consistent with experimental values. The optimum formulations (found at 20% of LYCl content) were able to complete the transfection process efficiently and with high cell viabilities, even improving the outcomes of the positive control Lipo2000*.

Compaction process of calf thymus DNA by mixed cationic-zwitterionic liposomes: a physicochemical study.

The compaction of calf thymus DNA (CT-DNA) by cationic liposomes constituted by a 1:1 mixture of a cationic lipid, 1,2-distearoyl-3-(trimethylammonio)propane chloride (DSTAP), and a zwitterionic lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE, null net charge at pH = 7.4), has been evaluated in aqueous buffered solution at 298.15 K by means of conductometry, electrophoretic mobility, cryo-TEM, and fluorescence spectroscopy techniques. The results reveal that DSTAP/DOPE liposomes are mostly spherical and unilamelar, with a mean diameter of around 77 +/- 20 nm and a positively charged surface with a charge density of sigmazeta = (21 +/- 1) x 10(-3) C m(-2). When CT-DNA is present, the genosomes DSTAP/DOPE/CT-DNA, formed by means of a surface electrostatic interaction, are generally smaller than the liposomes. Furthermore, they show a tendency to fuse forming cluster-type structures when approaching isoneutrality, which has been determined by the electrochemical methods at around (L/D)phi = 5.6. The analysis of the decrease on the fluorescence emission of the fluorophore ethidium bromide, EtBr, initially intercalated between DNA base pairs, as long as the genosomes are formed has permitted us to confirm the electrostatic character of the DNA-liposome interaction.

A physicochemical characterization of the interaction between DC-Chol/DOPE cationic liposomes and DNA.

A 1:1 mixture of the cationic lipid 3beta-[ N-( N', N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol) and the zwitterionic lipid, 1,2-dioleoyl- sn-glycero-3-phosphoetanolamine (DOPE), has been used to compact calf-thymus DNA (CT-DNA) in aqueous buffered solution at 298.15 K. The formation process of this lipoplex has been analyzed by means of electrophoretic mobility, cryo-TEM, dynamic light scattering, and fluorescence spectroscopy techniques. The experimental results indicate that DC-Chol/DOPE liposomes are mostly spherical and unilamellar, with a mean diameter of around 99 +/- 10 nm and a bilayer with a thickness of 4.5 +/- 0.5 nm. In the presence of CT-DNA, DC-Chol/DOPE/CT-DNA lipoplexes are formed by means of a strong entropically driven surface electrostatic interaction, as confirmed by zeta potential and fluorescence results, as a consequence of which DNA is compacted and condensed at the surface of the cationic liposomes. The negative charges of DNA phosphate groups are neutralized by the positive charges of cationic liposomes at the isoneutrality L/ D ratio, ( L/ D) varphi around 4, obtained from electrophoretic, fluorescence, and DLS measurements. The decrease in the fluorescence emission intensity of ethidium bromide, EtBr, initially intercalated between DNA base pairs, as long as the association between the biopolymer and the cationic liposomes takes place has permitted one to confirm its electrostatic character as well as to evaluate the different microenvironments of varying polarity of DNA-double helix, liposomes, and/or lipoplexes. Electronic microscopy reveals a rich scenario of possible nanostructures and morphologies for the lipoplexes, from unilamellar DNA-coated liposomes to multilamellar lipoplexes passing through cluster-like structures and several intermediate morphologies.

Multidisciplinary Approach to the Transfection of Plasmid DNA by a Nonviral Nanocarrier Based on a Gemini-Bolaamphiphilic Hybrid Lipid.

A multidisciplinary strategy, including both biochemical and biophysical studies, was proposed here to evaluate the potential of lipid nanoaggregates consisting of a mixture of a gemini-bolaamphiphilic lipid (C6C22C6) and the well-known helper lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) to transfect plasmid DNA into living cells in an efficient and safe way. For that purpose, several experimental techniques were employed, such as zeta potential (phase analysis light scattering methodology), agarose gel electrophoresis (pDNA compaction and pDNA protection assays), small-angle X-ray scattering, cryo-transmission electron microscopy, atomic force microscopy, fluorescence-assisted cell sorting, luminometry, and cytotoxicity assays. The results revealed that the cationic lipid and plasmid offer only 70 and 30% of their nominal positive () and negative charges (), respectively. Upon mixing with DOPE, they form lipoplexes that self-aggregate in typical multilamellar Lα lyotropic liquid-crystal nanostructures with sizes in the range of 100-200 nm and low polydispersities, very suitably fitted to remain in the bloodstream and cross the cell membrane. Interestingly, these nanoaggregates were able to compact, protect (from the degrading effect of DNase I), and transfect two DNA plasmids (pEGFP-C3, encoding the green fluorescent protein, and pCMV-Luc, encoding luciferase) into COS-7 cells, with an efficiency equal or even superior to that of the universal control Lipo2000*, as long as the effective +/- charge ratio was maintained higher than 1 but reasonably close to electroneutrality. Moreover, this transfection process was not cytotoxic because the viability of COS-7 cells remained at high levels, greater than 80%. All of these features make the C6C22C6/DOPE nanosystem an optimal nonviral gene nanocarrier in vitro and a potentially interesting candidate for future in vivo experiments.

A Gemini Cationic Lipid with Histidine Residues as a Novel Lipid-Based Gene Nanocarrier: A Biophysical and Biochemical Study.

This work reports the synthesis of a novel gemini cationic lipid that incorporates two histidine-type head groups (C3(C16His)2). Mixed with a helper lipid 1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanol amine (DOPE), it was used to transfect three different types of plasmid DNA: one encoding the green fluorescence protein (pEGFP-C3), one encoding a luciferase (pCMV-Luc), and a therapeutic anti-tumoral agent encoding interleukin-12 (pCMV-IL12). Complementary biophysical experiments (zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), and fluorescence anisotropy) and biological studies (FACS, luminometry, and cytotoxicity) of these C3(C16His)2/DOPE-pDNA lipoplexes provided vast insight into their outcomes as gene carriers. They were found to efficiently compact and protect pDNA against DNase I degradation by forming nanoaggregates of 120⁻290 nm in size, which were further characterized as very fluidic lamellar structures based in a sandwich-type phase, with alternating layers of mixed lipids and an aqueous monolayer where the pDNA and counterions are located. The optimum formulations of these nanoaggregates were able to transfect the pDNAs into COS-7 and HeLa cells with high cell viability, comparable or superior to that of the standard Lipo2000*. The vast amount of information collected from the in vitro studies points to this histidine-based lipid nanocarrier as a potentially interesting candidate for future in vivo studies investigating specific gene therapies.