Molecular shape of the cationic lipid controls the structure of cationic lipid/dioleylphosphatidylethanolamine-DNA complexes and the efficiency of gene delivery

Pyridinium amphiphiles, abbreviated as SAINT, are highly efficient vectors for delivery of DNA into cells. Within a group of structurally related compounds that differ in transfection capacity, we have investigated the role of the shape and structure of the pyridinium molecule on the stability of bilayers formed from a given SAINT and dioleoylphosphatidylethanolamine (DOPE) and on the polymorphism of SAINT/DOPE-DNA complexes. Using electron microscopy and small angle x-ray scattering, a relationship was established between the structure, stability, and morphology of the lipoplexes and their transfection efficiency. The structure with the lowest ratio of the cross-sectional area occupied by polar over hydrophobic domains (SAINT-2) formed the most unstable bilayers when mixed with DOPE and tended to convert into the hexagonal structure. In SAINT-2-containing lipoplexes, a hexagonal topology was apparent, provided that DOPE was present and complex assembly occurred in 150 mm NaCl. If not, a lamellar phase was obtained, as for lipoplexes prepared from geometrically more balanced SAINT structures. The hexagonal topology strongly promotes transfection efficiency, whereas a strongly reduced activity is seen for complexes displaying the lamellar topology. We conclude that in the DOPE-containing complexes the molecular shape and the nonbilayer preferences of the cationic lipid control the topology of the lipoplex and thereby the transfection efficiency.     

The role of lipid charge density in the serum stability of cationic lipid/DNA complexes

To evaluate the role of lipid charge density in the serum stability of DOTAP-Chol/DNA complexes (lipoplexes), lipid-DNA interactions, extent of aggregation, supercoil content, and in vitro transfection efficiency of lipoplexes were investigated. In general, higher serum concentration destabilized, and increasing molar charge ratio of DOTAP to negatively charged phosphates in the DNA (DOTAP(+)/DNA(-)) stabilized lipoplexes in serum as assessed by the criteria used in this study. The increase of cholesterol content led to increased serum stability, and DOTAP:Chol (mol/mol 1:4)/DNA lipoplex with DOTAP(+)/DNA(-) ratio 4 was the most serum stable formulation of all the formulations examined, and maintained lipid-DNA interactions, did not aggregate and exhibited high in vitro transfection efficiency in 50% (v/v) serum. The increased stability of this formulation could not be explained by the decreased charge density of the lipid component. Furthermore, no single parameter examined in the study could be used to consistently predict the in vitro transfection efficiency of lipoplexes in serum. Surprisingly, no correlation between the maintenance of supercoiled DNA content and in vitro transfection efficiency was found in the study.     

Phase behavior of cationic amphiphiles and their mixtures with helper lipid influences lipoplex shape,DNA translocation,and transfection efficiency

Cationic lipids are widely used for gene transfection, but their mechanism of action is still poorly understood. To improve this knowledge, a structure-function study was carried out with two pyridinium-based lipid analogs with identical headgroups but differing in alkyl chain (un)saturation, i.e., SAINT-2 (diC18:1) and SAINT-5 (diC18:0). Although both amphiphiles display transfection activity per se, DOPE strongly promotes SAINT-2-mediated transfection, but not that of SAINT-5, despite the fact that DOPE effectively facilitates plasmid dissociation from either lipoplex. This difference appears to correlate with membrane stiffness, dictated by the cationic lipid packing in the donor liposomes, which governs the kinetics of lipid recruitment by the plasmid upon lipoplex assembly. Because of its interaction with the relatively rigid SAINT-5 membranes, the plasmid becomes inappropriately condensed, which results in formation of structurally deformed lipoplexes. This structural deformation does not affect its cellular uptake but, rather, hampers plasmid translocation across endosomal and/or nuclear membranes. This is inferred from the observation that both lipoplexes effectively translocate much smaller oligonucleotides into cells. In fact, SAINT-5/DOPE-mediated transfection is greatly improved when, before lipoplex assembly, the plasmid is stabilized by condensation with polylysine. The results emphasize a role of the structural shape of the plasmid in gaining cytosolic/nuclear access. Moreover, it has been proposed that such a translocation is promoted when the lipoplex adopts the hexagonal phase, and data are presented that demonstrate that the lamellar SAINT-5/DOPE lipoplex adopts such a phase after its interaction with acidic phospholipid-containing membranes.     

Biophysical characterization of anionic lipoplexes

Transfection efficiency of liposomal gene delivery vectors depends on an optimal balance in the electro-chemical and structural properties of the transfection-capable complexes. We have recently reported a novel anionic lipoplex DNA delivery system composed of a ternary complex of endogenous occurring non-toxic anionic lipids, physiological Ca²⁺ cations, and plasmid DNA encoding a gene of interest with high transfection efficiency and low toxicity. In this work, we investigate the electro-chemical and structural properties anionic lipoplexes and compare them with those of Ca²⁺-DNA complexes. Biophysical characterization is used to explain the transfection efficiency of anionic lipoplexes in mammalian CHO-K1 cells. Circular dichroism and fluorescence spectroscopy showed that the plasmid DNA underwent conformational transition from native B-DNA to Z-DNA due to compaction and condensation upon Ca²⁺-mediated complexation with anionic liposomes. Zeta potential measurements and gel electrophoresis studies demonstrated that Ca²⁺ interaction with plasmid DNA during the formation of lipoplexes also led to increased association of supercoiled plasmid DNA with the lipoplexes, leading to charge neutralization which is expected to facilitate transfection. However, even 10-fold higher concentrations of Ca²⁺ alone (in the absence of the anionic liposomes) were unable to induce these changes in plasmid DNA molecules. A model explaining the possible mechanism of anionic lipoplex formation and the correlation of high transfection efficiency to biophysical properties was proposed. These studies confirm the utility of biophysical studies to identify optimal formulation conditions to design efficient liposomal gene delivery vectors.     

Lipoplex size determines lipofection efficiency with or without serum

In order to identify factors affecting cationic liposome-mediated gene transfer, the relationships were examined among cationic liposome/DNA complex (lipoplex)-cell interactions, lipoplex size and lipoplex-mediated transfection (lipofection) efficiency. It was found that lipofection efficiency was determined mainly by lipoplex size, but not by the extent of lipoplex-cell interactions including binding, uptake or fusion. In addition, it was found that serum affected mainly lipoplex size, but not lipoplex-cell interactions, which effect was the major reason behind the inhibitory effect of serum on lipofection efficiency. It was concluded that, in the presence or absence of serum, lipoplex size is a major factor determining lipofection efficiency. Moreover, in the presence or absence of serum, lipoplex size was found to affect lipofection efficiency by controlling the size of the intracellular vesicles containing lipoplexes after internalization, but not by affecting lipoplex-cell interactions. In addition, large lipoplex particles showed, in general, higher lipofection efficiency than small particles. These results imply that, by controlling lipoplex size, an efficient lipid delivery system may be achieved for in vitro and in vivo gene therapy.     

Lipoplex-mediated transfection of mammalian cells occurs through the cholesterol-dependent clathrin-mediated pathway of endocytosis

Synthetic amphiphiles are widely used as a carrier system. However, to match transfection efficiencies as obtained for viral vectors, further insight is required into the properties of lipoplexes that dictate transfection efficiency, including the mechanism of delivery. Although endocytosis is often referred to as the pathway of lipoplex entry and transfection, its precise nature has been poorly defined. Here, we demonstrate that lipoplex-mediated transfection is inhibited by more than 80%, when plasma membrane cholesterol is depleted with methyl-beta-cyclodextrin. Cholesterol replenishment restores the transfection capacity. Investigation of the cellular distribution of lipoplexes after cholesterol depletion revealed an exclusive inhibition of internalization, whereas cell-association remained unaffected. These data strongly support the notion that complex internalization, rather than the direct translocation of plasmid across the plasma membrane, is a prerequisite for accomplishing effective lipoplex-mediated transfection. We demonstrate that internalized lipoplexes colocalize with transferrin in early endocytic compartments and that lipoplex internalization is inhibited in potassium-depleted cells and in cells overexpressing dominant negative Eps15 mutants. In conjunction with the notion that caveolae-mediated internalization can be excluded, we conclude that efficient lipoplex-mediated transfection requires complex internalization via the cholesterol-dependent clathrin-mediated pathway of endocytosis.     

Surface adsorption of protein corona controls the cell internalization mechanism of DC-Chol-DOPE/DNA lipoplexes in serum

Serum has often been reported as a barrier to efficient lipid-mediated transfection. Here we found that the transfection efficiency of DC-Chol-DOPE/DNA lipoplexes increases in serum. To provide insight into the mechanism of lipoplex-serum interaction, several state-of-the-art methodologies have been applied. The nanostructure of DC-Chol-DOPE/DNA lipoplexes was found to be serum-resistant as revealed by high resolution synchrotron small angle X-ray scattering, while dynamic light scattering measurements showed a marked size increase of complexes. The structural stability of DC-Chol-DOPE/DNA lipoplexes was confirmed by electrophoresis on agarose gel demonstrating that plasmid DNA remained well protected by lipids. Proteomics experiments showed that serum proteins competed for the cationic surface of lipid membranes leading to the formation of a rich a ‘protein corona’. Combining structural results with proteomics findings, we suggest that such a protein corona can promote large aggregation of intact lipoplexes. According to a recently proposed size-dependent mechanism of lipoplex entry within cells, protein corona-induced formation of large aggregates most likely results in a switch from a clathrin-dependent to caveolae-mediated entry pathway into the cells which is likely to be responsible for the observed transfection efficiency boost. As a consequence, we suggest that surface adsorption of protein corona can have a high biological impact on serum-resistant cationic formulations for in vitro and in vivo lipid-mediated gene delivery applications.     

Multilamellar Cationic Liposomes are Efficient Vectors for in Vitro Gene Transfer in Serum

Abstract

Multilamellar vesicles (MLVs) containing the cationic lipid DOTAP were used as vectors to lipofect a number of culture cell lines in the presence of serum. The lipofection efficiency of lipoplexes made of MLVs and the plasmid pSV-β galactosidase are much less sensitive to the lipofection-inhibitory effect of serum than the conventionally used lipoplexes made of sonicated small unilamellar vesicles (SUVs). In order to determine the factors favoring the lipofection efficiency of MLVs, we measured the size, as well as the cellular association and uptake of MLV and SUV lipoplexes containing DOTAP alone or DOTAP:DOPE (1:1). Electron microscope images of these complexes were taken to confirm their structure and size. The single most important factor that correlates with transfection efficiency in serum is the size of the lipoplex. SUV lipoplexes remain smaller than 300 nm in the presence of serum, and the lipofection efficiencies are low. MLV lipoplexes are larger (>300 nm) and the lipofection efficiency, as well as cellular association and uptake, are much higher than those of SUV lipoplexes. Exceptions are those lipoplexes made of MLVs of DOTAP and DOPE (1:1) combined with DNA at higher charge ratios, which form hexagonal structures and show poor lipofection as well as cellular association and uptake, even if their lipoplex size exceeds 300 nm. This finding lends credence to our theory of the serum inhibition effect upon lipofection, and suggests ways to improve the transfection efficiency in the presence of serum, by fabricating lipoplexes of defined sizes.     

Lipid mixing between lipoplexes and plasma lipoproteins is a major barrier for intravenous transfection mediated by cationic lipids

It has been previously shown that transfection activity of cationic liposome/DNA lipoplexes delivered systemically is drastically inhibited by lipoproteins (Tandia, B. M., Vandenbranden, M., Wattiez, R., Lakhdar, Z., Ruysschaert, J. M., and Elouahabi, A. (2003) Mol Ther. 8, 264-273). In this work, we have compared the binding/uptake and transfection activities of DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride) and diC14-amidine (3-tetradecylamino-N-tert-butyl-N'-tetra-decylpropionamidine)-containing lipoplexes in the presence or absence of purified low density lipoproteins and high density lipoprotein. Binding/uptake of both lipoplexes by the mouse lung endothelial cell line was inhibited to a similar extent in the presence of lipoproteins. In contrast, transfection activity of diC14-amidine-containing lipoplexes was almost completely inhibited (approximately by 95%), whereas approximately 40% transfection activity of DOTAP-containing lipoplexes was preserved in the presence of lipoproteins. Interestingly, the ability of lipoproteins to inhibit the transfection efficiency of lipoplexes was well correlated with their ability to undergo lipid mixing with the cationic lipid bilayer as revealed by fluorescence resonance energy transfer assay. Incubation of lipoplexes with increased doses of lipoproteins resulted in enhanced lipid mixing and reduced transfection activity of the lipoplexes in mouse lung endothelial cells. The role of lipid mixing in transfection was further demonstrated using lipid-mixing inhibitor, lyso-phosphatidylcholine, or activator (dioleoylphosphatidylethanolamine). Incorporation of Lyso-PC into diC14-amidine-containing lipoplexes completely abolished their capacity to undergo lipid mixing with lipoproteins and allowed them to reach a high transfection efficiency in the presence of lipoproteins. On the other hand, the incorporation of dioleoylphosphatidylethanolamine into DOTAP/DNA lipoplex activated lipid mixing with the lipoproteins and was shown to be detrimental toward the transfection activity of these lipoplexes. Taken together, these results indicate that fusion of lipoplexes with lipoproteins is a limiting factor for in vivo transfection.     

Lipoplex size determines lipofection efficiency with or without serum

In order to identify factors affecting cationic Iiposome-mediated gene transfer, the relationships were examined among cationic liposome/DNA complex (lipoplex)-cell interactions, lipoplex size and lipoplex-mediated transfection (lipofection) efficiency. It was found that lipofection efficiency was determined mainly by lipoplex size, but not by the extent of lipoplex-cell interactions including binding, uptake or fusion. In addition, it was found that serum affected mainly lipoplex size, but not lipoplex-cell interactions, which effect was the major reason behind the inhibitory effect of serum on lipofection efficiency. It was concluded that, in the presence or absence of serum, lipoplex size is a major factor determining Iipofection efficiency. Moreover, in the presence or absence of serum, lipoplex size was found to affect lipofection efficiency by controlling the size of the intracellular vesicles containing lipoplexes after internalization, but not by affecting lipoplex-cell interactions. In addition, large lipoplex particles showed, in general, higher lipofection efficiency than small particles. These results imply that, by controlling lipoplex size, an efficient lipid delivery system may be achieved for in vitro and in vivo gene therapy.     

Phase behavior, DNA ordering, and size instability of cationic lipoplexes. Relevance to optimal transfection activity

Mechanisms of cationic lipid-based nucleic acid delivery are receiving increasing attention, but despite this the factors that determine high or low activity of lipoplexes are poorly understood. This study is focused on the fine structure of cationic lipid-DNA complexes (lipoplexes) and its relevance to transfection efficiency. Monocationic (N-(1-(2,3-dioleoyloxy)propyl),N,N,N-trimethylammonium chloride, N-(1-(2,3-dimyristyloxypropyl)-N,N-dimethyl-(2-hydroxyethyl)ammonium bromide) and polycationic (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanammonium trifluoroacetate) lipid-based assemblies, with or without neutral lipid (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, cholesterol) were used to prepare lipoplexes of different L(+)/DNA(-) charge ratios. Circular dichroism, cryogenic-transmission electron microscopy, and static light scattering were used for lipoplex characterization, whereas expression of human growth hormone or green fluorescent protein was used to quantify transfection efficiency. All monocationic lipids in the presence of inverted hexagonal phase-promoting helper lipids (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, cholesterol) induced appearance of Psi(-) DNA, a chiral tertiary DNA structure. The formation of Psi(-) DNA was also dependent on cationic lipid-DNA charge ratio. On the other hand, monocationic lipids either alone or with 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine as helper lipid, or polycationic 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanammonium trifluoroacetate-based assemblies, neither of which promotes a lipid-DNA hexagonal phase, did not induce the formation of Psi(-) DNA. Parallel transfection studies reveal that the size and phase instability of the lipoplexes, and not the formation of Psi(-) DNA structure, correlate with optimal transfection.     

Characterization and transfection properties of lipoplexes stabilized with novel exchangeable polyethylene glycol-lipid conjugates

The positive charge of cationic-lipid/DNA complexes (lipoplexes) renders them highly susceptible to interactions with the biological milieu, leading to aggregation and destabilization, and rapid clearance from the blood circulation. In this study we synthesized and characterized a set of novel amphiphiles, based on N-methyl-4-alkylpyridinium chlorides (SAINTs), to which a PEG moiety is coupled. Plasmids were fully protected in lipoplexes prepared from cationic SAINT-2 lipid and stabilized with SAINT-PEGs. Our results demonstrate that SAINT-PEG stabilization is transient, and permits DNA to be released from these lipoplexes. The rate of SAINT-PEG transfer from lipoplexes to acceptor liposomes was determined by the nature of the lipid anchor. Increased hydrophobicity, by lengthening the alkyl chain, resulted in a decrease of the rate of DNA release from the lipoplexes. Chain unsaturation had the opposite effect. Similarly, the in vitro transfection potency of lipoplexes containing PEG-SAINT derivatives was sensitive to the length and (un)saturation of the alkyl chain. However, the internalization of SAINT-PEG stabilized lipoplexes is determined by their charge, rather than by the concentration of the polymer conjugate. Lipoplexes targeted to cell-surface epithelial glycoprotein 2, by means of a covalently coupled monoclonal antibody, were specifically internalized by cells expressing this antigen.     

Efficacy of immobilized polyplexes and lipoplexes for substrate‐mediated gene delivery

Non‐viral gene delivery by immobilization of complexes to cell‐adhesive biomaterials, a process termed substrate‐mediated delivery, has many in vitro research applications such as transfected cell arrays or models of tissue growth. In this report, we quantitatively investigate the efficiency of gene delivery by surface immobilization, and compare this efficiency to the more typical bolus delivery. The ability to immobilize vectors while allowing cellular internalization is impacted by the biomaterial and vector properties. Thus, to compare this efficiency between vector types and delivery methods, transfection conditions were initially identified that maximized transgene expression. For surface delivery from tissue culture polystyrene, DNA complexes were immobilized to pre‐adsorbed serum proteins prior to cell seeding, while for bolus delivery, complexes were added to the media above adherent cells. Mathematical modeling of vector binding, release, and cell association using a two‐site model indicated that the kinetics of polyplex binding to cells was faster than for lipoplexes, yet both vectors have a half‐life on the surface of approximately 17 min. For bolus and surface delivery, the majority of the DNA in the system remained in solution or on the surface, respectively. For polyplexes, the efficiency of trafficking of cell‐associated polyplexes to the nucleus for surface delivery is similar or less than bolus delivery, suggesting that surface immobilization may decrease the activity of the complex. The efficiency of nuclear association for cell‐associated lipoplexes is similar or greater for surface delivery relative to bolus. These studies suggest that strategies to enhance surface delivery for polyplexes should target the vector design to enhance its potency, whereas enhancing lipoplex delivery should target the material design to increase internalization. Biotechnol. Bioeng. 2009;102: 1679–1691. © 2008 Wiley Periodicals, Inc.     

Physicochemical characterization and purification of cationic lipoplexes.

Cationic lipid-nucleic acid complexes (lipoplexes) consisting of dioleoyltrimethylammoniumpropane (DOTAP) liposomes and plasmid DNA were prepared at various charge ratios (cationic group to nucleotide phosphate), and the excess component was separated from the lipoplex. We measured the stoichiometry of the lipoplex, noted its colloidal properties, and observed its morphology and structure by electron microscopy. The colloidal properties of the lipoplexes were principally determined by the cationic lipid/DNA charge ratio and were independent of the lipid composition. In lipoplexes, the lipid membranes as observed in freeze-fracture electron microscopy were deformed into high-radius-of-curvature features whose characteristics depended on the lipid composition. Lipoplexes prepared at a threefold or greater excess of either DOTAP or DNA could be resolved into complexes with a defined stoichiometry and the excess component by sedimentation to equilibrium on sucrose gradients. The separated, positively charged complex retained high transfection activity and had reduced toxicity. The negatively charged lipoplex showed increased transfection activity compared to the starting mixture. In cryoelectron micrographs the positively charged complex was spherical and contained a condensed but indistinct interior structure. In contrast, the separated negatively charged lipoplexes had a prominent internal 5.9 +/- 0.1-nm periodic feature with material projecting as spikes from the spherical structure into the solution. It is likely that these two lipoplexes represent structures with different lipid and DNA packing.     

The role of organ vascularization and lipoplex-serum initial contact in intravenous murine lipofection

Following intravenous administration of cationic lipid-DNA complexes (lipoplexes) into mice, transfection (lipofection) occurs predominantly in the lungs. This was attributed to high entrapment of lipoplexes in the extended lung vascular tree. To determine whether lipofection in other organs could be enhanced by increasing the degree of vascularization, we used a transgenic mouse model with tissue-specific angiogenesis in liver. Tail vein injection of N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP)/cholesterol lipoplexes resulted in increased lipoplex entrapment in hypervascularized liver but did not boost luciferase expression, suggesting that lipoplex delivery is not a sufficient condition for efficient organ lipofection. Because the intravenously injected lipoplexes migrated within seconds to lungs, we checked whether the effects of immediate contact with serum correlate with lung lipofection efficiency of different DOTAP-based formulations. Under conditions mimicking the injection environment, the lipoplex-serum interaction was strongly dependent on helper lipid and ionic strength: lipoplexes prepared in 150 mM NaCl or lipoplexes with high (>33 mol%) cholesterol were found to aggregate immediately. This aggregation process was irreversible and was inversely correlated with the percentage of lung cells that took up lipoplexes and with the efficiency of lipofection. No other structural changes in serum were observed for cholesterol-based lipoplexes. Dioleoyl phosphatidylethanolamine-based lipoplexes were found to give low expression, apparently because of an immediate loss of integrity in serum, without lipid-DNA dissociation. Our study suggests that efficient in vivo lipofection is the result of cross-talk between lipoplex composition, interaction with serum, hemodynamics, and target tissue "susceptibility" to transfection.     

Chemical activation of lipoplexes formed from DNA and a redox-active, ferrocene-containing cationic lipid

We recently reported that the ferrocene-containing cationic lipid BFDMA [bis(11-ferrocenylundecyl)dimethylammonium bromide] can be used to mediate cell transfection, and that levels of transfection depend critically upon the oxidation state of the ferrocenyl groups of the lipid. Here, we report that the redox activity of BFDMA can be exploited to transform lipoplexes formed from oxidized BFDMA (which do not transfect cells) to lipoplexes that are "active" (and thus mediate high levels of transgene expression) by treatment with the chemical reducing agent glutathione (GSH). We demonstrate that GSH can be used to reduce the ferrocenium groups of oxidized BFDMA rapidly both (i) in solution and (ii) in lipoplexes formed by mixing oxidized BFDMA and DNA. Lipoplexes transformed in this manner mediate levels of cell transfection in vitro that are comparable to levels of transfection mediated by lipoplexes prepared by mixing DNA and reduced BFDMA. We demonstrate further that the chemical reduction of oxidized BFDMA leads to changes in the zeta potentials of these lipoplexes (e.g., from negative to positive). Characterization of lipoplex internalization using confocal microscopy demonstrated that these changes in zeta potential correlate to differences in the extents to which these lipoplexes are internalized by cells. These results provide a framework from which to interpret differences in cell transfection mediated by reduced and oxidized BFDMA. When combined, the results of this study suggest the basis of an approach that could be used to transform lipoplexes actively or "on-demand" and provide spatial and/or temporal control over the transfection of cells in a range of different fundamental and applied contexts.     

Enhanced gene expression in lung by a stabilized lipoplex using sodium chloride for complex formation

BACKGROUND: In this study, we investigated the in vivo gene transfection efficacy of a 'surface charge regulated' (SCR) lipoplex, dispersed in the presence of an essential amount of NaCl during lipoplex formation. METHODS: SCR lipoplexes were prepared and their physicochemical properties were analyzed. After intravenous (i.v.) administration, transfection efficacy, distribution characteristics, and liver toxicity were evaluated in mice. RESULTS: At NaCl concentrations of 10 mM, the particle sizes of the SCR lipoplexes were about 120 nm and were compatible with a conventional lipoplex. However, fluorescent resonance energy transfer analysis revealed that cationic liposomes in the SCR lipoplexes increased fusion. After i.v. administration, the transfection activity in the lung of the SCR lipoplex (10 mM NaCl solution in the lipoplex) was approximately 10-fold higher than that of the conventional lipoplex. Pharmacokinetic studies demonstrated a higher distribution in lung by the SCR lipoplex. When the gene expression levels of the SCR lipoplex and conventional lipoplex were compared, the SCR lipoplex at a dose of 30 microg was compatible with that of the conventional lipoplex at a dose of 50 microg. A significantly higher serum alanine aminotransferase (ALT) activity and TNFalpha concentration was observed by the conventional lipoplex (pDNA dose; 50 microg), but this was not the case for the SCR lipoplex (pDNA dose; 30 microg). CONCLUSIONS: We demonstrated that the SCR lipoplex could enhance the transfection efficacy in the lung without increasing the liver toxicity. Hence, the information will be valuable for the future use, design, and development of lipoplexes for in vivo applications.     

Dependence of the cellular internalization and transfection efficiency on the structure and physicochemical properties of cationic detergent/DNA/liposomes

BACKGROUND: Control of the structure and physicochemical properties of DNA complexed with nonviral vectors is essential for efficient biodistribution and gene delivery to cells. Cationic liposomes interact with DNA giving transfection competent but large and heterogeneous aggregates. On the other hand, cationic detergents condense DNA into small homogeneous but reversible complexes inefficient for transfection. METHODS: In order to combine the favorable features of both vectors, ternary complexes were prepared by adding cationic liposomes to plasmid DNA condensed by cationic detergents. The structure and physicochemical properties of these complexes were investigated by electron microscopy, quasi-elastic light scattering, gel electrophoresis and fluorescence techniques. These data were then correlated with the transfection efficiency and intracellular trafficking of the ternary complexes determined by luciferase gene expression and confocal microscopy, respectively. RESULTS: The ternary complexes were found to form small, homogeneous, globular, stable and positively charged particles with a highly dense and packed lamellar internal structure differing from the multilamellar structure (L(alpha)(C)) of the corresponding lipoplexes. In the presence of serum, the ternary complexes were more efficiently internalized into cells, less toxic and showed 20-fold higher transfection efficiency than lipoplexes. CONCLUSIONS: This study showed that small, monodisperse and highly stable complexes could be obtained by precompaction of DNA with cetyltrimethylammonium bromide, followed by addition of cationic lipids. The higher efficiency of the ternary complexes with respect to their corresponding lipoplexes was related to their internal structure which prevents their dissociation by serum proteins and allows efficient internalization in the target cells.     

Biophysical and lipofection studies of DOTAP analogs

In order to investigate the relationship between lipid structure and liposome-mediated gene transfer, we have studied biophysical parameters and transfection properties of monocationic DOTAP analogs, systematically modified in their non-polar hydrocarbon chains. Stability, size and (by means of anisotropy profiles) membrane fluidity of liposomes and lipoplexes were determined, and lipofection efficiency was tested in a luciferase reporter gene assay. DOTAP analogs were used as single components or combined with a helper lipid, either DOPE or cholesterol. Stability of liposomes was a precondition for formation of temporarily stable lipoplexes. Addition of DOPE or cholesterol improved liposome and lipoplex stability. Transfection efficiencies of lipoplexes based on pure DOTAP analogs could be correlated with stability data and membrane fluidity at transfection temperature. Inclusion of DOPE led to rather uniform transfection and anisotropy profiles, corresponding to lipoplex stability. Cholesterol-containing lipoplexes were generally stable, showing high transfection efficiency at low relative fluidity. Our results demonstrate that the efficiency of gene transfer mediated by monocationic lipids is greatly influenced by lipoplex biophysics due to lipid composition. The measurement of fluorescence anisotropy is an appropriate method to characterize membrane fluidity within a defined system of liposomes or lipoplexes and may be helpful to elucidate structure-activity relationships.     

Transfection efficiency boost by designer multicomponent lipoplexes

Cationic liposome-DNA complexes (lipoplexes) have emerged as leading nonviral gene carriers in worldwide gene therapy clinical trials. Arriving at therapeutic dosages requires the full understanding of the mechanism of transfection. We investigated the correlation between structural evolution of multicomponent lipoplexes when interacting with cellular lipids, the extent of DNA release and the efficiency in transfecting mouse fibroblast (NIH 3T3), ovarian (CHO) and tumoral myofibroblast-like (A17) cell lines. We show, for the first time, that the transfection pattern increases monotonically with the number of lipid components and further demonstrate by means of synchrotron small angle X- ray scattering (SAXS) that structural changes of lipoplexes induced by cellular lipids correlate with the transfection efficiency. Specifically, inefficient lipoplexes either fused too rapidly upon interaction with anionic lipids or, alternatively, are found to be extremely resistant to solubilization. The most efficient lipoplex formulations exhibited an intermediate behaviour. The extent of DNA unbinding (measured by electrophoresis on agarose gel) correlates with structural evolution of the lipoplexes but DNA-release does not scale with the extent of transfection. The general meaning of our results is of broad interest in the field of non-viral gene delivery: rational adjusting of lipoplex composition to generate the proper interaction between lipoplexes and cellular lipids may be the most appropriate strategy in optimizing synthetic lipid transfection agents.