Intracellular accumulation of secreted proteoglycans inhibits cationic lipid-mediated gene transfer. Co-transfer of glycosaminoglycans to the nucleus.

Molecules secreted by potential target cells may interfere with cationic lipid-mediated gene transfer. This has been studied using human lung fibroblasts and human epidermoid lung cancer cells. Secreted cell medium components caused a substantial decrease both in the uptake of cationic lipid-DNA complexes (2-4-fold) and in reporter gene expression (100-1000-fold). Metabolic labeling of the cell medium showed that especially [35S]sulfate-labeled macromolecules competed with DNA for binding to the cationic lipid. Release of DNA from the cationic lipid by cell medium components was demonstrated by an ethidium bromide intercalation assay. In the presence of the cationic lipid, the secreted macromolecules were internalized by the cells. By enzymatic digestions, it was shown that the competing macromolecules consist of chondroitin/dermatan sulfate and heparan sulfate proteoglycans and that the effects on transfection were mediated by the polyanionic glycosaminoglycan portion of the proteoglycan. Accordingly, pretreatment of cell medium with the polycationic peptide protamine sulfate abrogated the inhibitory effects on gene transfer. Fluorescence microscopy studies revealed that heparan sulfate, internalized as a complex with cationic lipids, accumulated in the cell nuclei. These results support the view that the lack of specificity of this type of gene transfer vehicle is a major hindrance to efficient and safe in vivo administration.     

Effect of spacer attachment sites and pH-sensitive headgroup expansion on cationic lipid-mediated gene delivery of three novel myristoyl derivatives

The transfection activity and physicochemical properties of the dimyristoyl derivatives from three novel series of double-chained tertiary cationic lipids were compared. Two of the derivatives were constructed as isomers with different linkages of the same bis-(2-dimethylaminoethane) polar headgroup and hydrophobic chains to the diaminopropanol backbone, while the third was designed with a hydrophilic region containing only a single ionizable amine group. Such systematic molecular changes offer a great opportunity to delineate factors critical for transfection activity, which in this work include the intramolecular distance between the hydrophobic chains and pH-expandability of the polar headgroup. The physical studies comprised a variety of techniques, including pKa determination, Langmuir monolayer studies, fluorescence anisotropy, gel electrophoresis mobility shift assay, ethidium bromide displacement assay, particle size distribution, and zeta potential. These studies are crucial in the development of lipid-based gene delivery systems with improved efficacy. Physicochemical characterization revealed that a symmetric bivalent pH-expandable polar headgroup in combination with greater intramolecular space between the hydrophobic chains provide for high transfection activity through efficient binding and compaction of pDNA, increased acyl chain fluidity, and high molecular elasticity.     

In vitro cationic lipid-mediated gene delivery with fluorinated glycerophosphoethanolamine helper lipids.

There is a need for the development of nonviral gene transfer systems with improved and original properties. "Fluorinated" lipoplexes are such candidates, as supported by the remarkably higher in vitro and in vivo transfection potency found for such fluorinated lipoplexes as compared with conventional ones or even with PEI-based polyplexes (Boussif, O., Gaucheron, J., Boulanger, C., Santaella, C., Kolbe, H. V. J., Vierling, P. (2001) Enhanced in vitro and in vivo cationic lipid-mediated gene delivery with a fluorinated glycerophosphoethanolamine helper lipid. J. Gene Med. 3, 109-114). Here, we describe the synthesis of fluorinated glycerophosphoethanolamines (F-PEs), close analogues of dioleoylphosphatidylethanolamine (DOPE), and report on their lipid helper properties vs that of DOPE, as in vitro gene transfer components of fluorinated lipoplexes based on pcTG90, DOGS (Transfectam), or DOTAP. To evaluate the contribution of the F-PEs to in vitro lipoplex-mediated gene transfer, we examined the effect of including the F-PEs in lipoplexes formulated with these cationic lipids (CL) for various CL:DOPE:F-PE molar ratios [1:(1 - x):x with x = 0, 0.5 and 1; 1:(2 - y):y with y = 0, 1, 1.5, and 2], and various N/P ratios (from 10 to 0.8, N = number of CL amines, P = number of DNA phosphates). Irrespective of the F-PE chemical structure, of the colipid F-PE:DOPE composition, and of the N/P ratio, comparable transfection levels to those of their respective control DOPE lipoplexes were most frequently obtained when using one of the F-PEs as colipid of DOGS, pcTG90, or DOTAP in place of part of or of all DOPE. However, a large proportion of DOGS-based lipoplexes were found to display a higher transfection efficiency when formulated with the F-PEs rather than with DOPE alone while the opposite tendency was evidenced for the DOTAP-based lipoplexes. The present work indicates that "fluorinated" lipoplexes formulated with fluorinated helper lipids and conventional cationic lipids are very attractive candidates for gene delivery. It confirms further that lipophobicity and restricted miscibility of the lipoplex lipids with the endogenous lipids does not preclude efficient gene transfer and expression. Their transfection potency is rather attributable to their unique lipophobic and hydrophobic character (resulting from the formulation of DNA with fluorinated lipids), thus preventing to some extent DNA from interactions with lipophilic and hydrophilic biocompounds, and from degradation.     

Poly(propylacrylic acid) enhances cationic lipid-mediated delivery of antisense oligonucleotides

The use of antisense oligodeoxynucleotides (ODNs) to inhibit the expression of specific mRNA targets represents a powerful technology for control of gene expression. Cationic lipids and polymers are frequently used to improve the delivery of ODNs to cells, but the resulting complexes often aggregate, bind to serum components, and are trafficked poorly within cells. We show that the addition of a synthetic, pH-sensitive, membrane-disrupting polyanion, poly(propylacrylic acid) (PPAA), improves the in vitro efficiency of the cationic lipid, DOTAP, with regard to oligonucleotide delivery and antisense activity. In characterization studies, ODN complexation with DOTAP/ODN was maintained even when substantial amounts of PPAA were added. The formulation also exhibited partial protection of phosphodiester oligonucleotides against enzymatic digestion. In Chinese hamster ovary (CHO) cells, incorporation of PPAA in DOTAP/ODN complexes improved 2- to 3-fold the cellular uptake of fluorescently tagged oligonucleotides. DOTAP/ODN complexes containing PPAA also maintained high levels of uptake into cells upon exposure to serum. Addition of PPAA to DOTAP/ODN complexes enhanced the antisense activity (using GFP as the target) over a range of PPAA concentrations in both serum-free, and to a lesser extent, serum-containing media. Thus, PPAA is a useful adjunct that improves the lipid-mediated delivery of oligonucleotides.     

pH-sensitive cationic polymer gene delivery vehicle: N-Ac-poly(L-histidine)-graft-poly(L-lysine) comb shaped polymer

Advancing biotechnology spurs the development of new pharmaceutically engineered gene delivery vehicles. Poly(L-histidine) ?PLH? has been shown to induce membrane fusion at endosomal pH values, whereas PLL has a well documented efficacy in polyplex formation. Therefore, N-Ac-poly(L-histidine)-graft-poly(L-lysine) ?PLH-g-PLL? was synthesized by grafting poly(L-histidine) to poly(L-lysine) ?PLL?. PLH-g-PLL formed polyplex particles by electrostatic interactions with plasmid DNA ?pDNA?. The mean particle size of the polyplexes was in the range of 117 +/- 6 nm to 306 +/- 77 nm. PLH-g-PLL gene carrier demonstrated higher transfection efficacy in 293T cells than PLL at all equivalent weight ratios with pDNA. The inclusion of chloroquine as an endosomolytic agent enhanced transfection for both PLL and PLH-g-PLL gene carriers. PLH-g-PLL enhanced beta-galactosidase expression compared to PLL, but still increased in efficacy when chloroquine was included.     

Zinc improves gene transfer mediated by DNA/cationic polymer complexes

Background

The weak efficiency of plasmid transfer into the cytosol remains one of the major limiting factors to achieve an efficient transfection with DNA/cationic polymer complexes. We found that divalent metal Zn²⁺ can improve the polyfection efficiency, especially with DNA/histidylated polylysine (His‐pLK) complexes.

Methods and results

The supplementation of the transfection medium with 250 µM ZnCl₂ increased the polyfection of human hepatocarcinoma (HepG2) cells with a plasmid encoding EGFP complexed with pLK, polyethyleneimine and His‐pLK. Zn²⁺ is more efficient on DNA/His‐pLK complexes: the number of EGFP‐positive cells increased from 1% to more than 40%. This phenomenon is selective to Zn²⁺ because no effect was obtained with other divalent cations. The effect of zinc varies from cell to cell. The binding of Zn²⁺ to histidyl residues might increase zinc endosomal concentration favoring membrane fusion. Flow cytometry and confocal microscopy studies clearly indicate that with His‐pLK, the plasmid is better delivered in the cytosol as well as in the cell nucleus in zinc‐treated cells. An investigation conducted with the histidine‐rich peptide H5WYG showed that zinc inhibits membrane permeabilization but promotes membrane fusion as evidenced by resonance energy transfer.

Conclusions

Data reported here imply that the addition of zinc ions in the transfection medium can trigger an increase of the fusion of endosomes containing polyplexes which is more effective in the presence of histidine‐rich molecules. Consequently, the amount of plasmid in the cytosol available to reach the nucleus is increased leading to an improvement of polyfection. Copyright © 2002 John Wiley & Sons, Ltd.

     

Poly[Lys-(AEDTP)]: a cationic polymer that allows dissociation of pDNA/cationic polymer complexes in a reductive medium and enhances polyfection.

Polyplexes of high stability resulting from the condensation of a plasmid DNA by a cationic polymer are widely used to develop polymer-based gene delivery systems. However, the plasmid must be released from its vector once inside the cells for an efficient expression of the exogenous gene in the cell nucleus. We have designed a disulfide-containing cationic polymer termed poly[Lys-(AEDTP)] which allowed for the formation of polyplexes and the release of the plasmid in a reductive medium. The amino groups of polylysine were substituted with 3-(2-aminoethyldithio)propionyl residues in order to have each amino group of poly[Lys-(AEDTP)] interacting with a phosphate DNA linked to the polymer backbone via a disulfide bond. As evidenced by agarose gel electrophoresis and ethidium bromide/pDNA fluorescence restoration, poly[Lys-(AEDTP)] polyplexes were decondensed and the plasmid released upon treatment with either dithiothreitol, glutathione in the presence of glutathione reductase, or the thioredoxin reductase. Electron microscopy showed that polyplexes exhibiting spherical particles of a mean size at about 100 nm were decondensed in the presence of glutathione and exhibited filamentous aggregates. Finally, we found that the transfection of 293T7 and HepG2 cells was 10- and 50-fold more efficient with poly[Lys-(AEDTP)] polyplexes, respectively, than with poly[Lys] polyplexes. These results indicate that disulfide-containing cationic polymers must be borne in mind for developing polymer-base gene delivery systems.     

The potential role of proteoglycans in cationic lipid-mediated gene delivery. Studies of the interaction of cationic lipid-DNA complexes with model glycosaminoglycans.

Recent evidence supports a role for proteoglycans in polycation-mediated gene delivery. Therefore, the interaction of glycosaminoglycans with cationic lipid-DNA complexes (CLDCs) has been characterized using a combination of biophysical approaches. At low ionic strength, CLDCs bind to heparin-derivatized Sepharose particles, with the ratio of cationic lipid to DNA controlling the binding. Incorporation of the helper lipids cholesterol or 1,2-dioleoyl-phosphatidylethanolamine increases the amount of bound CLDC. Heparin also induces the aggregation of CLDCs, with cholesterol reducing this effect. Isothermal titration calorimetry demonstrates an endothermic heat for the binding of heparin to CLDCs at low ionic strength, whereas circular dichroism studies suggest a heparin-stimulated release of DNA from CLDCs at a greater than 20-fold charge excess. Increasing the ionic strength to 0.11 reduces CLDC binding to heparin beads, and greatly enhances the release of DNA from CLDCs by heparin. The ability of the cell surface glycosaminoglycan heparan sulfate to release DNA from CLDCs is more sensitive than heparin to the incorporation of the cholesterol or 1,2-dioleoyl-phosphatidylethanolamine. Titration calorimetry reveals an exothermic heat for the interaction glycosaminoglycans with CLDCs at higher ionic strength. These results are consistent with the direct involvement of proteoglycans in transfection.     

Ultrasound enhances retrovirus-mediated gene transfer

Viral vector systems are efficient for transfection of foreign genes into many tissues. Especially, retrovirus based vectors integrate the transgene into the genome of the target cells, which can sustain long term expression. However, it has been demonstrated that the transduction efficiency using retrovirus is relatively lower than those of other viruses. Ultrasound was recently reported to increase gene expression using plasmid DNA, with or without, a delivery vehicle. However, there are no reports, which show an ultrasound effect to retrovirus-mediated gene transfer efficiency. Retrovirus-mediated gene transfer systems were used for transfection of 293T cells, bovine aortic endothelial cells (BAECs), rat aortic smooth muscle cells (RASMCs), and rat skeletal muscle myoblasts (L6 cells) with beta-galactosidase (beta-Gal) genes. Transduction efficiency and cell viability assay were performed on 293T cells that were exposed to varying durations (5 to 30 seconds) and power levels (1.0 watts/cm(2) to 4.0 watts/cm(2)) of ultrasound after being transduced by a retrovirus. Effects of ultrasound to the retrovirus itself was evaluated by transduction efficiency of 293T cells. After exposure to varying power levels of ultrasound to a retrovirus for 5 seconds, 293T cells were transduced by a retrovirus, and transduction efficiency was evaluated. Below 1.0 watts/cm(2) and 5 seconds exposure, ultrasound showed increased transduction efficiency and no cytotoxicity to 293T cells transduced by a retrovirus. Also, ultrasound showed no toxicity to the virus itself at the same condition. Exposure of 5 seconds at the power of 1.0 watts/cm(2) of an ultrasound resulted in significant increases in retrovirus-mediated gene expression in all four cell types tested in this experiment. Transduction efficiencies by ultrasound were enhanced 6.6-fold, 4.8-fold, 2.3-fold, and 3.2-fold in 293T cells, BAECs, RASMCs, and L6 cells, respectively. Furthermore, beta-Gal activities were also increased by the retrovirus with ultrasound exposure in these cells. Adjunctive ultrasound exposure was associated with enhanced retrovirus-mediated transgene expression in vitro. Ultrasound associated local gene therapy has potential for not only plasmid-DNA-, but also retrovirus-mediated gene transfer.     

Inhibitory effects of tumor necrosis factor-alpha on cationic lipid-mediated gene delivery to airway cells in vitro

Cationic liposomes have been used successfully for DNA delivery to airway cells in vitro and are being tested in human clinical trials for their efficacy in cystic fibrosis transmembrane conductance regulator (CFTR) gene delivery in cystic fibrosis patients. While cationic liposomes are effective for transfection of airway cells in culture, they have not been effectively used for gene delivery to human airway cells in vivo. Several barriers in cystic fibrosis lungs, including increased amounts of mucus, phagocytic cell activity and cytokine-rich milieu caused by inflammation, may cause inhibition of gene transfection. As presented in this paper, we examined the effects of inflammatory cytokines on cationic lipid-mediated transfection of model airway cells. The results of these experiments indicate that tumor necrosis factor (TNF)-alpha dramatically inhibits Lipofectin-mediated transfection efficiency of H441 cells. Addition of anti-TNF-alpha neutralizing antibody results in recovery of efficiency. Results of temporal studies are consistent with the concept that TNF-alpha reduces transfection efficiency by a mechanism(s) other than or in addition to gene expression. These results are corroborated by fluorescence microscopic experiments which demonstrate that endocytosis of lipoplex is altered in the presence of TNF-alpha.     

The role of helper lipids in cationic liposome-mediated gene transfer.

In the procedure for cationic liposome-mediated transfection, the cationic lipid is usually mixed with a "helper lipid" to increase its transfection potency. The importance of helper lipids, including dioleoylphosphatidylcholine (DOPC) and phosphatidylethanolamine (dioleoyl PE), DO was examined. Freeze-fracture electron microscopy of DNA:cationic complexes containing the pSV-beta-GAL plasmid DNA, the cationic lipid dioleoyl trimethylammonium propane, and these helper lipids showed that the most efficient mixtures were aggregates of ensheathed DNA and fused liposomes. PE-containing complexes aggregated rapidly when added to culture media containing polyanions, whereas PC-containing complexes did not. However, more granules of PC-containing complexes were formed on cell surfaces after the complexes were added to Chinese hamster ovary (CHO) cells in transfection media. Pronase treatment inhibited transfection, whereas dilute poly-L-lysine enhanced transfection, indicating that the attachment of DNA:liposome complexes to cell surfaces was mediated by electrostatic interaction. Fluorescence spectroscopy studies confirmed that more PC-containing complexes than PE-containing complexes were associated with CHO cells, and that more PC-containing complexes were located in a low pH environment (likely to be within endosomes) with time. Cytochalasin-B had a stronger inhibitory effect on PC-containing liposome-mediated than on PE-containing liposome-mediated transfection. Confocal microscopic recording of the fluorescently label lipid and DNA uptake process indicated that many granules of DNA:cationic liposome complexes were internalized as a whole, whereas some DNA aggregates were left out on the cell surfaces after liposomes of the complexes fused with the plasma membranes. For CHO cells, endocytosis seems to be the main uptake pathway of DNA:cationic liposome complexes. More PC-containing granules than PE-containing granules were formed on cell surfaces by cytoskeleton-directed membrane motion, after their respective DNA:liposome complexes attached to cell surfaces by electrostatic means. Formation of granules on the cell surface facilitated and/or triggered endocytosis. Fusion between cationic liposomes and the cell membrane played a secondary role in determining transfection efficiency.     

Unique features of a pH-sensitive fusogenic peptide that improves the transfection efficiency of cationic liposomes

BACKGROUND: One of the critical steps in intracellular gene delivery using cationic liposomes is the endosomal escape of the plasmid/liposome complexes to the cytosol. The addition of GALA, a pH-sensitive fusogenic peptide, is a promising method to accelerate this step in order to enhance the expression of the desired proteins. Detailed studies on the methods of enhancement would broaden the horizon of its application. METHODS: Using representative commercially available cationic liposomes (Lipofectin, Lipofectamine, and Lipofectamine 2000), the effects of GALA on transfection efficiency were studied by luciferase assay and confocal microscopic observations. RESULTS: A concentration-dependent increase in the transfection efficiency was observed for GALA. Addition of 0.1 microM GALA to the plasmid/liposome complex significantly increased the transfection efficiency, especially in the case of Lipofectin, but higher concentration of GALA decreased transfection efficiency. Successful reduction in the liposomal dosage was attained by employing GALA while maintaining a high transfection efficiency. Interestingly, although the transfection efficiency was higher in the presence of GALA, a lower amount of the plasmid DNA was taken up by the cells. Confocal microscopic observations of the rhodamine-labeled plasmid did not show a significant difference in the cellular localization among cells incubated in the presence or absence of GALA, suggesting that a slight increase in GALA-induced release of the plasmid to the cytosol may cause a significant change in the transfection efficiency. CONCLUSION: The unique features of GALA to mediate improved transfection efficiencies were identified.     

Evaluation of cationic liposome suitable for gene transfer into pregnant animals.

Cationic liposome-mediated in vivo gene transfer represents a promising approach for somatic gene therapy. To assess the most suitable liposome for gene delivery into a wide range of organs and fetuses in mice, we have explored several types of cationic liposomes conjugated with plasmid DNA carrying the beta-galactosidase gene through intravenous injection into pregnant animals. Transduction efficiency was assessed by Southern blot analysis and expression of the transferred gene was evaluated by enzymatic demonstration of beta-galactosidase activity. Through the analysis of several types of recently synthesized cationic liposome/lipid formulations, DMRIE-C reagent, a liposome formulation of the cationic lipid DMRIE (1, 2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide) and cholesterol in membrane-filtered water met our requirements. When the plasmid DNA/DMRIE-C complexes were administered intravenously into pregnant mice at day 11.5 post coitus (p.c.), transferred genes were observed in several organs in dams and were expressed. Furthermore, although the copy numbers transferred into embryos were low, we observed reporter gene expression in the progeny.     

A series of cationic sterol lipids with gene transfer and bactericidal activity

A family of cationic lipids was synthesized via direct amide coupling of spermine to the C-24 position of cholic acid analogs. Four monosubstituted spermines and a bis-substituted spermine were evaluated as plasmid transfection reagents, as bacteriostatic agents, and as bactericidal agents. The incorporation of a double bond in the sterol moiety enhanced transfection efficiency significantly and produced two compounds with little cytotoxicity and transfection potency comparable to Lipofectamine2000^?. Inclusion of the double bond had no effect on the general trend of increasing bactericidal activity with increasing sterol hydrophobicity. Co-formulation of the most hydrophilic of the compounds with its bis-substituted analogue led to enhancement in transfection activity. The bis-substituted compound, when tested alone, emerged as the most bacteriostatic compound in the family with minimum inhibitory concentrations (MIC) of 4 μM against Bacillus subtilis and 16 μM against Escherichia coli and therapeutic indexes (minimum hemolytic concentration/minimum inhibitory concentration) of 61 and 15, respectively. Cationic lipids can be optimized for both gene delivery and antibacterial applications by similar modifications.     

Airway gene transfer using cationic emulsion as a mucosal gene carrier

BACKGROUND: Delivery of genes to airway mucosa would be a very valuable method for gene therapy and vaccination. However, there have been few reports on suitable gene delivery systems for administration. In this study, we use a cationic emulsion system, which is physically stable and facilitates the transfer of genes in the presence of up to 90% serum, as a mucosal gene carrier. METHODS AND RESULTS: Cationic lipid emulsion was formulated with squalene and 1,2-dioleoyl-sn-glycero-3-trimethylammoniumpropane (DOTAP) as major components. Emulsions formed stable complexes with DNA and protected and transferred DNA to target cells against DNase I digestion in the presence of mucosal destabilizers such as heparin sulfate (a polysaccharide of the glycosaminoglycan family in mucosa) and Newfectan (a natural lung extract of bovine) in an in vitro system. In contrast, commercial liposomes and counter liposomes, made with an identical lipid composition of emulsions, failed. After in vivo intranasal instillation, the cationic emulsion showed at least 200 times better transfection activity than the liposomal carriers in both nasal tissue and lung. CONCLUSIONS: These findings show that cationic emulsions can mediate gene transfection into airway epithelium, making it a good choice for transferring therapeutic genes and for genetic vaccination against an pathogenic infection via an airway route.     

Novel ortho ester-based,pH-sensitive cationic lipid for gene delivery in vitro and in vivo

Context: Cationic lipoplexes are less toxic than viral gene vectors and more convenient to prepare but their efficiencies of gene delivery are generally lower.

Objective: To develop ortho ester-based, pH-sensitive lipoplexes for efficient gene delivery both in cultured cells and in vivo.

Materials and methods: A novel cationic and acid-labile lipid (DOC) containing a cationic headgroup and a cholesterol-derived lipid tail joined together by an acid-labile ortho ester linker was designed and synthesized. DOC was formulated into liposomes with the conical helper lipid DOPE, and then into lipoplexes with plasmid DNA encoding a luciferase reporter gene. The physicochemical properties of the lipoplexes (size, surface charge and pH-sensitivity) were characterized. Gene delivery by DOC/DOPE/DNA lipoplexes was also evaluated in CV-1 cells and in CD-1 mice following intratracheal injection. Lipoplexes consisting of the acid-stable cationic lipid DC-Chol were characterized as a control.

Results: DOC formed cationic lipoplexes with DOPE and DNA. After incubation at acidic pH 4.6, DOC/DOPE/DNA lipoplexes lost their positive charges and aggregated with one another as a result of DOC hydrolysis. Both in CV-1 cell culture and in CD-1 mice, DOC/DOPE/DNA lipoplexes increased the luciferase gene expression by 5- to 10-fold compared with the analogous but acid-stable DC-Chol/DOPE/DNA lipoplexes.

Discussion and conclusion: Incorporation of an acid-labile ortho ester linker into a cationic lipid is a viable approach to enhance gene delivery by the corresponding lipoplexes both in cultured cells and in vivo.     

Cationic lipid-mediated gene transfer: Analysis of cellular uptake and nuclear import of plasmid DNA

Cationic lipids are widely used for gene transfer in vitro and show promise as vectors for in vivo gene therapy applications. However, there is limited understanding of the cellular mechanisms involved in nonviral gene transfer. We investigated two major steps that could be limiting barriers to cationic lipid-mediated gene transfer in vitro. We used a fluorescent plasmid to study the cellular uptake and the intracellular fate of lipoplexes during in vitro transfection of fibroblast cells and found that 100% of the cells take up lipoplexes. The intracellular staining observed with lipoplexes was clearly different from that obtained with endocytosed fluorescent dextran. This suggests that cells readily take up lipoplexes by a mechanism that could be different from endocytosis in our conditions. However, the escape of DNA from intracellular vesicles could be a major limiting barrier to gene transfer. Direct injection of plasmid DNA into the nucleus and cytoplasm of cells indicated that DNA traffic from the cytoplasm to the nucleus might be also an important limiting step.