Lipoplexes formed by DNA and ferrocenyl lipids: effect of lipid oxidation state on size, internal dynamics, and zeta-potential

The effect of lipid oxidation state on the physical properties of complexes formed by plasmid DNA and the redox-active lipid bis-(11-ferrocenylundecyl)dimethylammonium bromide (BFDMA) is reported. With increasing concentration of BFDMA, the hydrodynamic sizes of complexes formed by BFDMA and DNA (in the presence of 1 mM Li₂SO₄) pass through a maximum and the ζ-potential changes monotonically from −40 mV to +40 mV. In contrast, complexes formed by oxidized BFDMA and DNA exhibit a minimum in size and maintain a negative ζ-potential with increasing concentration of BFDMA. Angle-dependent dynamic light scattering measurements also reveal the presence of relaxation processes within complexes formed by DNA and oxidized BFDMA that are absent for complexes formed by DNA and reduced BFDMA. These results, when combined, reveal that the amphiphilic nature of reduced BFDMA leads to lipoplexes with physical properties resembling those formed by classical cationic lipids, whereas the interaction of oxidized BFDMA with DNA is similar to that of nonamphiphilic cationic molecules bearing multiple charges (e.g., spermidine). In particular, the negative ζ-potential and measurable presence of DNA chain dynamics within complexes formed by oxidized BFDMA and DNA indicate that these complexes are loosely packed with excess charge due to DNA in their outer regions. These results, when combined with additional measurements performed in OptiMEM reduced-serum cell culture medium, lead to the proposition that the strong dependence of transfection efficiency on the oxidation state of BFDMA, as reported previously, is largely a reflection of the substantial change in the ζ-potentials of these complexes with changes in the oxidation state of BFDMA.     

Interaction of cationic liposomes and their DNA complexes with monocytic leukemia cells

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. We examined the relationship between the characteristics of the lipoplexes, their mode of interaction with monocytic THP-1 cells and their ability to transfect these cells. We determined the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and its mixtures with neutral lipids), and lipoplexes at different (+/-) charge ratios. As the (+/-) charge ratio of the lipoplexes decreased to (1/1), a significant reduction in zeta potential and an increase in size was observed. The increase in size resulted from fusion between liposomes promoted by DNA, as demonstrated by a lipid mixing assay, and from aggregation of the complexes. Interaction of liposomes and lipoplexes with THP-1 cells was assessed by monitoring lipid mixing ('fusion') as well as binding and cell association. While no lipid mixing was observed with the 1/2 (+/-) lipid/DNA complexes, lipoplexes with higher (+/-) charge ratios underwent significant fusion in conjunction with extensive cell binding. Liposome binding to cells was dependent on the positive charge of the liposomes, and their fusion could be modulated by the co-lipid. DOTAP/phosphatidylethanolamine (1:1) liposomes fused with THP-1 cells, unlike DOTAP/phosphatidylcholine (1:1) liposomes, although both liposome types bound to the cells to a similar extent. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. The presence of serum increased the size of the cationic liposomes, but not that of the lipoplexes. Low concentrations of serum (3%) completely inhibited the fusion of cationic liposomes with cells, while inhibiting binding by only 20%. Our results suggest that binding of cationic liposomes and lipoplexes to cells is governed primarily by electrostatic interactions, whereas their fusion is regulated by the lipid composition and sterically favorable interactions with cell surface molecules. In addition our results indicate no correlation between fusion of the lipoplexes with the plasma membrane and the levels of transfection.     

Physico-Chemical Characteristics of Lipoplexes Influence Cell Uptake Mechanisms and Transfection Efficacy

Background

Formulation of DNA/cationic lipid complexes (lipoplexes) designed for nucleic acid delivery mostly results in positively charged particles which are thought to enter cells by endocytosis. We recently developed a lipoplex formulation called Neutraplex that allows preparation of both cationic and anionic stable complexes with similar lipid content and ultrastructure.

Methodology/Principal Findings

To assess whether the global net charge could influence cell uptake and activity of the transported oligonucleotides (ON), we prepared lipoplexes with positive and negative charges and compared: (i) their physicochemical properties by zeta potential analysis and dynamic light scattering, (ii) their cell uptake by fluorescence microscopy and flow cytometry, and (iii) the biological activity of the transported ON using a splicing correction assay. We show that positively or negatively charged lipoplexes enter cells cells using both temperature-dependent and -independent uptake mechanisms. Specifically, positively charged lipoplexes predominantly use a temperature-dependent transport when cells are incubated OptiMEM medium. Anionic lipoplexes favour an energy-independent transport and show higher ON activity than cationic lipoplexes in presence of serum. However, lipoplexes with high positive global net charge and OptiMEM medium give the highest uptake and ON activity levels.

Conclusions

These findings suggest that, in addition to endocytosis, lipoplexes may enter cell via a temperature-independent mechanism, which could be mediated by lipid mixing. Such characteristics might arise from the specific lipoplex ultrastructure and should be taken into consideration when developing lipoplexes designed for in vivo or ex vivo nucleic acid transfer.     

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.     

Physico-chemical characterization and transfection efficacy of cationic liposomes containing the pEGFP plasmid

Cationic liposomes-DNA complexes (lipoplexes) are largely used in gene delivery. Deciphering specific chemical and physical properties of lipoplexes is a necessary step to unravel the mechanisms underlying transfection and to improve transfection efficacy in each experimental model. In the present paper we investigated the physico-chemical features of lipoplexes containing a plasmid encoding for the GFP protein, in order to correlate these results with transfection efficacy. Cationic unilamellar vesicles (mean diameter 100 nm) were prepared, from the cationic DC-Chol lipid and the zwitterionic phospholipid DOPE. The two components of the liposome bilayer were used at molar ratio close to unity. ESR spectra were recorded and zeta potential zeta was measured on liposomes complexed with the plasmid. One of the main points of interest in this paper resided in the fact that both kinds of measurements were carried out in the same conditions (i.e. lipid concentration, medium composition, and pH) employed for cell transfection experiments. Transfection was performed on CHO cells; the percentage of fluorescent cells was evaluated and compared with the above physico-chemical features. It emerged that the composition and pH of the medium, the lipoplex/cell ratio, as well as the amount of lipoplex added to the cell culture were critical parameters for transfection efficacy. Finally, lipoplex surface charge played a fundamental role to achieve a high transfection level.     

Gene delivery mediated by cationic liposomes: from biophysical aspects to enhancement of transfection

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. However, the relationship between the features of the lipid-DNA complexes (`lipoplexes') and their mode of interaction with cells, the efficiency of gene transfer and gene expression remain to be clarified. To gain insights into these aspects, the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3- (trimethylammonium) propane (DOTAP) and its mixture with phosphatidylethanolamine (PE)), and their complexes with DNA at different (+/-) charge ratios were determined. A lipid mixing assay was used to assess the interaction of liposomes and lipoplexes with monocytic leukaemia cells. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. However, very limited transfection of these cells was achieved using the above complexes. It is possible that the topology of the cationic liposome-DNA complexes does not allow the entry of DNA into cells through a fusion process at the plasma membrane. In an attempt to enhance transfection mediated by lipoplexes composed of DOTAP and its equimolar mixture with dioleoylphosphatidylethanolamine (DOPE) two different strategies were explored: (i) association of a targeting ligand (transferrin) to the complexes to promote their internalization, presumably by receptor-mediated endocytosis; and (ii) association of synthetic fusogenic peptides (GALA or the influenza haemagglutinin Nterminal peptide HA-2) to the complexes to promote endosomal destabilization and release of the genetic material into the cytoplasm. These strategies were effective in enhancing transfection in a large variety of cells, including epithelial and lymphoid cell lines, as well as human macrophages, especially with the use of optimized lipid/ DNA (+/-) charge ratios. Besides leading to high levels of transfection, the ternary complexes of cationic liposomes, DNA, and protein or peptide, have the advantages of being active in the presence of serum and being non-toxic. Moreover, such ternary complexes present a net negative charge and, thus, are likely to alleviate the problems associated with the use of highly positively charged complexes in vivo, such as avid complexation with serum proteins. Overall, the results indicate that these complexes, and their future derivatives, may constitute viable alternatives to viral vectors for gene delivery in vivo.     

Gene delivery mediated by cationic liposomes: from biophysical aspects to enhancement of transfection.

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. However, the relationship between the features of the lipid-DNA complexes ('lipoplexes') and their mode of interaction with cells, the efficiency of gene transfer and gene expression remain to be clarified. To gain insights into these aspects, the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3- (trimethylammonium) propane (DOTAP) and its mixture with phosphatidylethanolamine (PE)), and their complexes with DNA at different (+/-) charge ratios were determined. A lipid mixing assay was used to assess the interaction of liposomes and lipoplexes with monocytic leukaemia cells. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. However, very limited transfection of these cells was achieved using the above complexes. It is possible that the topology of the cationic liposome-DNA complexes does not allow the entry of DNA into cells through a fusion process at the plasma membrane. In an attempt to enhance transfection mediated by lipoplexes composed of DOTAP and its equimolar mixture with dioleoylphosphatidylethanolamine (DOPE) two different strategies were explored: (i) association of a targeting ligand (transferrin) to the complexes to promote their internalization, presumably by receptor-mediated endocytosis; and (ii) association of synthetic fusogenic peptides (GALA or the influenza haemagglutinin N-terminal peptide HA-2) to the complexes to promote endosomal destabilization and release of the genetic material into the cytoplasm. These strategies were effective in enhancing transfection in a large variety of cells, including epithelial and lymphoid cell lines, as well as human macrophages, especially with the use of optimized lipid/DNA (+/-) charge ratios. Besides leading to high levels of transfection, the ternary complexes of cationic liposomes, DNA, and protein or peptide, have the advantages of being active in the presence of serum and being non-toxic. Moreover, such ternary complexes present a net negative charge and, thus, are likely to alleviate the problems associated with the use of highly positively charged complexes in vivo, such as avid complexation with serum proteins. Overall, the results indicate that these complexes, and their future derivatives, may constitute viable alternatives to viral vectors for gene delivery in vivo.     

Lipid composition of cationic nanoliposomes implicate on transfection efficiency

Abstract

Cationic liposome (CL)-DNA complexes (lipoplexes) have appeared as leading nonviral gene carriers in worldwide gene therapy clinical trials. Arriving at therapeutic dosages requires the full understanding of the mechanism of transfection. However, using CLs to deliver therapeutic nucleic acids and drugs to target organs have some problems, including low transfection efficiency. The aim of this study was developing novel CLs containing four neutral lipids; cholesterol, 1,2-dioleoyl phosphatidylethanolamine, distearoylphosphatidylcholine and dipalmitoylphosphatidylcholine as a helper lipid and dimethyl dioctadecyl ammonium bromide as a cationic lipid to increase transfection efficiency. We have investigated the correlation between number of lipid composition and transfection efficiency. The morphology, size and zeta potential of liposomes and lipoplexes were measured and lipoplexes formation was monitored by gel retardation assay. Transfection efficiency was assessed using firefly luciferase reporter assay. It was found that transfection efficiency markedly depended on liposome to plasmid DNA (pDNA) weight ratio, lipid composition and efficiency of pDNA entrapment. High transfection efficiency of plasmid by four component lipoplexes was achieved. Moreover, lipoplexes showed lower transfection efficiency and less cytotoxicity compared to Lipofectamine?. These results suggest that lipid composition of nanoliposomes is an important factor in control of their physical properties and also yield of transfection.     

Bioreducible liposomes for gene delivery: from the formulation to the mechanism of action

Background

A promising strategy to create stimuli-responsive gene delivery systems is to exploit the redox gradient between the oxidizing extracellular milieu and the reducing cytoplasm in order to disassemble DNA/cationic lipid complexes (lipoplexes). On these premises, we previously described the synthesis of SS14 redox-sensitive gemini surfactant for gene delivery. Although others have attributed the beneficial effects of intracellular reducing environment to reduced glutathione (GSH), these observations cannot rule out the possible implication of the redox milieu in its whole on transfection efficiency of bioreducible transfectants leaving the determinants of DNA release largely undefined.

Methodology/Principal Findings

With the aim of addressing this issue, SS14 was here formulated into binary and ternary 100 nm-extruded liposomes and the effects of the helper lipid composition and of the SS14/helper lipids molar ratio on chemical-physical and structural parameters defining transfection effectiveness were investigated. Among all formulations tested, DOPC/DOPE/SS14 at 25∶50∶25 molar ratio was the most effective in transfection studies owing to the presence of dioleoyl chains and phosphatidylethanolamine head groups in co-lipids. The increase in SS14 content up to 50% along DOPC/DOPE/SS14 liposome series yielded enhanced transfection, up to 2.7-fold higher than that of the benchmark Lipofectamine 2000, without altering cytotoxicity of the corresponding lipoplexes at charge ratio 5. Secondly, we specifically investigated the redox-dependent mechanisms of gene delivery into cells through tailored protocols of transfection in GSH-depleted and repleted vs. increased oxidative stress conditions. Importantly, GSH specifically induced DNA release in batch and in vitro.

Conclusions/Significance

The presence of helper lipids carrying unsaturated dioleoyl chains and phosphatidylethanolamine head groups significantly improved transfection efficiencies of DOPC/DOPE/SS14 lipoplexes. Most importantly, this study shows that intracellular GSH levels linearly correlated with transfection efficiency while oxidative stress levels did not, highlighting for the first time the pivotal role of GSH rather than oxidative stress in its whole in transfection of bioreducible vectors.     

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.     

Transferrin-Associated Lipoplexes as Gene Delivery Systems: Relevance of Mode of Preparation and Biophysical Properties

The successful application of gene therapy depends highly on understanding the properties of gene carriers and their correlation with the ability to mediate transfection. An important parameter that has been described to improve transfection mediated by cationic liposomes involves association of ligands to cationic liposome–DNA complexes (lipoplexes). In this study, ternary complexes composed of 1,2-dioleoyl-3-(trimethylammonium) propane:cholesterol, plasmid DNA and transferrin (Tf, selected as a paradigm of a ligand) were prepared under various conditions, namely, in medium with different ionic strengths (HEPES-buffered saline [HBS] or dextrose), at different lipid/DNA (+/–) charge ratios and using different modes for component addition. We investigated the effect of these formulation parameters on transfection (in the absence and presence of serum), size of the complexes, degree of DNA protection and extent of their association with cells (in terms of both lipid and DNA). Our results show that all the tested parameters influenced to some extent the size of the complexes and their capacity to protect the carried genetic material, as well as the levels of cell association and transfection. The best transfection profile was observed for ternary complexes (Tf-complexes) prepared in high ionic strength solution (HBS), at charge ratios close to neutrality and according to the following order of component addition: cationic liposomes–Tf–DNA. Interestingly, in contrast to what was found for dextrose–Tf-complexes, transfection mediated by HBS-Tf-complexes in the presence of serum was highly enhanced.     

In vitro study of the cytotoxicity of isolated oxidized lipid low-density lipoproteins fractions in human endothelial cells: relationship with the glutathione status and cell morphology

Toxic effects of oxidized lipid compounds contained in oxidized LDL to endothelial cells are involved in the pathogenesis of atherosclerosis. Glutathione (GSH) plays an important role in the redox status of the cell and in the protective effect against oxidant injuries. However, little is known about the respective effect of these different oxidized lipid compounds toward cytotoxicity and GSH status of the cell. In this report, we isolated by high-performance liquid chromatography oxidized lipid compounds from low-density lipoproteins (LDL) oxidized by copper and we examined their effects on cultured endothelial cells. Cytotoxicity and GSH status were determined after incubation of endothelial cells with crude LDL or isolated lipid fractions derived from cholesterol, phospholipids, or cholesteryl esters. Their effects on cell morphology were also assessed. Oxidized lipids coming from cholesteryl esters (hydroperoxides or short-chain polar derivatives) induced a slight but significant GSH depletion without inducing cytotoxicity. The same species coming from phospholipids induced a more pronounced GSH depletion and a cytotoxic effect which is only present for the more polar compounds (short-chain polar derivatives) and corresponding to a total GSH depletion. In contrast, fractions containing oxysterols had a larger cytotoxic effect than their effect on GSH depletion suggesting that their cytotoxic effects are mediated by a GSH-independent pathway. All together, these data suggest that LDL-associated oxidized lipids present in copper-oxidized LDL exert cytotoxicity by an additional or synergistic effect on GSH depletion, but also by another mechanism independent of the redox status of the cell.     

Human serum albumin enhances DNA transfection by lipoplexes and confers resistance to inhibition by serum

Cationic liposome-DNA complexes ('lipoplexes') are used as gene delivery vehicles and may overcome some of the limitations of viral vectors for gene therapy applications. The interaction of highly positively charged lipoplexes with biological macromolecules in blood and tissues is one of the drawbacks of this system. We examined whether coating cationic liposomes with human serum albumin (HSA) could generate complexes that maintained transfection activity. The association of HSA with liposomes composed of 1, 2-dioleoyl-3-(trimethylammonium) propane and dioleoylphosphatidylethanolamine, and subsequent complexation with the plasmid pCMVluc greatly increased luciferase expression in epithelial and lymphocytic cell lines above that obtained with plain lipoplexes. The percentage of cells transfected also increased by an order of magnitude. The zeta potential of the ternary complexes was lower than that of the lipoplexes. Transfection activity by HSA-lipoplexes was not inhibited by up to 30% serum. The combined use of HSA and a pH-sensitive peptide resulted in significant gene expression in human primary macrophages. HSA-lipoplexes mediated significantly higher gene expression than plain lipoplexes or naked DNA in the lungs and spleen of mice. Our results indicate that negatively charged HSA-lipoplexes can facilitate efficient transfection of cultured cells, and that they may overcome some of the problems associated with the use of highly positively charged complexes for gene delivery in vivo.     

In vitro lipofection with novel asymmetric series of 1,2-dialkoylamidopropane-based cytofectins containing single symmetric bis-(2-dimethylaminoethane) polar headgroups

Novel N,N'-diacyl-1,2-diaminopropyl-3-carbamoyl[bis-(2-dimethylaminoethane)] bivalent cationic lipids were synthesized and evaluated for in vitro transfection activity against a murine melanoma cell line. In the absence of the helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), only the dioleoyl derivative 22 (1,2lb5) elicited transfection activity. The transfection activity of this lipid was reduced when formulated with DOPE. Contrary to that, the dimyristoyl derivative 19 (1,2lb2) mediated no activity when used alone but induced the highest levels of marker gene expression in the presence of DOPE. In an effort to correlate the transfection activity with cationic lipid structures, the physicochemical properties of cationic lipids in isolation and of lipoplexes were studied with surface tensiometry, photon correlation spectroscopy, gel electrophoresis mobility shift assay, and fluorescence techniques. In regard to the lipoplex properties, gel electrophoresis mobility shift assay and EtBr exclusion fluorescence assay revealed that the 1,2lb5 was the only lipid to associate and condense plasmid DNA, respectively. Photon correlation spectroscopy analysis found that 1,2lb5/DNA complexes were of relatively small size compared to all other lipoplexes. With respect to the properties of isolated lipids, Langmuir monolayer studies and fluorescence anisotropy on cationic lipid dispersions verified high two-plane elasticity and increased fluidity of the transfection competent dioleoyl derivative 1,2lb5, respectively. The results indicate that high transfection activity is mediated by cationic lipids characterized by an expanded mean molecular area, high molecular elasticity, and increased fluidity.     

Kinetic analysis of the initial steps involved in lipoplex–cell interactions: effect of various factors that influence transfection activity

We investigated the mode of interaction of lipoplexes (DOTAP:DOPE/DNA) with HeLa cells, focusing on the analysis of the initial steps involved in the process of gene delivery. We evaluated the effect of different factors, namely the stoichiometry of cationic lipids and DNA, the presence of serum in the cell culture medium, and the incorporation of the ligand transferrin into the lipoplexes, on the extent of binding, association and fusion (lipid mixing) of the lipoplexes with the cells. Parallel experiments were performed upon cell treatment with inhibitors of endocytosis. Our results indicate that a decrease of the net charge of the complexes (upon addition of DNA) generally leads to a decrease in the extent of binding, cell association and fusion, except for the neutral complexes. Association of transferrin to the lipoplexes resulted in a significant enhancement of the interaction processes referred to above, which correlates well with the promotion of transfection observed under the same conditions. Besides triggering internalization of the complexes, transferrin was also shown to mediate fusion with the endosomal membrane. The extent of fusion of this type of complexes was reduced upon their incubation with cells in the presence of serum, suggesting that serum components limit the transferrin fusogenic properties. Results were analyzed by using a theoretical model which allowed to estimate the kinetic parameters involved in lipoplex–cell interactions. The deduced fusion and endocytosis rate constants are discussed and compared with those obtained for other biological systems. From the kinetic studies we found a twofold enhancement of the fusion rate constant (f) for the ternary lipoplexes. We also concluded that HeLa cells yield a relatively low rate of endocytosis. Overall, our results estimate the relative contribution of fusion of lipoplexes with the plasma membrane, endocytosis and fusion with the endosomal membrane to their interactions with cells, this information being of crucial importance for the development of gene therapy strategies.     

Kinetic analysis of the initial steps involved in lipoplex--cell interactions: effect of various factors that influence transfection activity

We investigated the mode of interaction of lipoplexes (DOTAP:DOPE/DNA) with HeLa cells, focusing on the analysis of the initial steps involved in the process of gene delivery. We evaluated the effect of different factors, namely the stoichiometry of cationic lipids and DNA, the presence of serum in the cell culture medium, and the incorporation of the ligand transferrin into the lipoplexes, on the extent of binding, association and fusion (lipid mixing) of the lipoplexes with the cells. Parallel experiments were performed upon cell treatment with inhibitors of endocytosis. Our results indicate that a decrease of the net charge of the complexes (upon addition of DNA) generally leads to a decrease in the extent of binding, cell association and fusion, except for the neutral complexes. Association of transferrin to the lipoplexes resulted in a significant enhancement of the interaction processes referred to above, which correlates well with the promotion of transfection observed under the same conditions. Besides triggering internalization of the complexes, transferrin was also shown to mediate fusion with the endosomal membrane. The extent of fusion of this type of complexes was reduced upon their incubation with cells in the presence of serum, suggesting that serum components limit the transferrin fusogenic properties. Results were analyzed by using a theoretical model which allowed to estimate the kinetic parameters involved in lipoplex--cell interactions. The deduced fusion and endocytosis rate constants are discussed and compared with those obtained for other biological systems. From the kinetic studies we found a twofold enhancement of the fusion rate constant (f) for the ternary lipoplexes. We also concluded that HeLa cells yield a relatively low rate of endocytosis. Overall, our results estimate the relative contribution of fusion of lipoplexes with the plasma membrane, endocytosis and fusion with the endosomal membrane to their interactions with cells, this information being of crucial importance for the development of gene therapy strategies.     

‘Click’ synthesized sterol-based cationic lipids as gene carriers,and the effect of skeletons and headgroups on gene delivery

In this work, we have successfully prepared a series of new sterol-based cationic lipids (1–4) via an efficient ‘Click’ chemistry approach. The pDNA binding affinity of these lipids was examined by EB displacement and agarose-gel retardant assay. The average particle sizes and surface charges of the sterol-based cationic lipids/pDNA lipoplexes were analyzed by dynamic laser light scattering instrument (DLS), and the morphologies of the lipoplexes were observed by atomic force microscopy (AFM). The cytotoxicity of the lipids were examined by MTT and LDH assay, and the gene transfection efficiencies of these lipid carriers were investigated by luciferase gene transfection assay in various cell lines. In addition, the intracellular uptake and trafficking/localization behavior of the Cy3-DNA loaded lipoplexes were preliminarily studied by fluorescence microscopy. The results demonstrated that the pDNA loading capacity, lipoplex particle size, zeta potential and morphology of the sterol lipids/pDNA lipoplexes depended largely on the molecular structure factors including sterol-skeletons and headgroups. Furthermore, the sterol-based lipids showed quite different cytotoxicity and gene transfection efficacy in A549 and HeLa cells. Interestingly, it was found that the cholesterol-bearing lipids 1 and 2 showed 7–10⁴ times higher transfection capability than their lithocholate-bearing counterparts 3 and 4 in A549 and HeLa cell lines, suggested that the gene transfection capacity strongly relied on the structure of sterol skeletons. Moreover, the study on the structure–activity relationships of these sterol-based cationic lipid gene carriers provided a possible approach for developing low cytotoxic and high efficient lipid gene carriers by selecting suitable sterol hydrophobes and cationic headgroups.     

Polyethylenimine of various molecular weights as adjuvant for transfection mediated by cationic liposomes

Over the last years significant progress has been made in non-viral gene delivery mediated by cationic liposomes. However, the results obtained are still far from being satisfactory regarding transfection efficiency, particularly when compared to that achieved using viral vectors. We have previously demonstrated that association of transferrin with cationic liposomes significantly improves transfection in a large variety of cells, both in vitro and in vivo. In this work, several strategies have been explored in order to further improve transfection mediated by transferrin-associated lipoplexes. To this regard, the effect on transfection of pre-condensation of DNA with polyethylenimine of low MWs (2.7, 2.0 and 0.8 KDa) at various N/P ratios, lipid composition, cationic lipid/DNA (+/-) charge ratio and the presence of a surfactant in the lipoplexes was investigated. Two different modes for preparing the liposomes were tested and the extent of cell association of their complexes with DNA as well as their capacity to protect the carried DNA were evaluated. Our results show that complexes generated from cationic liposomes prepared by the ethanol injection method in which the carried DNA was pre-condensed with low MW polyethylenimine are highly efficient in mediating transfection. The differential modulating effect observed upon association of transferrin to various liposome formulations on transfection mediated by the polyethylenimine-complexes suggests that these complexes enter into the cells through different pathways (involving clathrin versus caveolin), most likely by taking advantage of their intrinsic biophysical properties to escape from the endosome to the cytosol.     

Lamellarity of cationic liposomes and mode of preparation of lipoplexes affect transfection efficiency.

Transfection of NIH-3T3 cells by a human growth hormone expression vector complexed with liposomes composed of N-(1-(2, 3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) with or without helper lipids was studied. The transfection efficiency was dependent on the lamellarity of the liposomes used to prepare the lipoplexes. Multilamellar vesicles (MLV) were more effective than large unilamellar vesicles (LUV) of approximately 100 nm, irrespective of lipid composition. The optimal DNA/DOTAP mole ratio for transfection was 相似文献   

FACTORS INFLUENCING THE EFFICIENCY OF LIPOPLEXES MEDIATED GENE TRANSFER IN LUNG AFTER INTRAVENOUS ADMINISTRATION*

The objectives of this study were to test the influence of different parameters on the in vivo cationic lipid mediated gene transfer in lung after intravenous administration. Luciferase activity was evaluated in lung tissue 24 hours after intravenous administration of different types of lipoplexes. These included lipoplexes prepared using cationic phosphonolipids or DOTAP and various amounts of plasmid DNA. Using two different plasmids we tested the influence of plasmid size on transfection efficiency in vivo. In a last series of experiments, lipoplexes were prepared using different excipients (water, NaCl or 5% glucose solution) and three injection volumes were tested. We demonstrate that chemical structure modifications such as cation substitution and increment of the aliphatic chain length significantly improve transfection efficiency. High luciferase levels are obtained by increasing lipid to DNA charge ratio and plasmid DNA dose and decreasing plasmid size. Lipoplexes prepared in physiological NaCl solution and injected using a volume of 800μl are significantly the most effective.

Cationic lipid mediated gene transfer in lung tissue after intravenous administration is influenced by factors including cationic lipid chemical structure, lipid to DNA ratio and plasmid dose. Nevertheless, plasmid size, injection volume and the excipient, used for the lipoplexes preparation, are also important factors and must be considered for an optimization of in vivo gene delivery using intravenous administration.