Improved DNA: liposome complexes for increased systemic delivery and gene expression

To increase cationic liposome-mediated intravenous DNA delivery extruded DOTAP:cholesterol liposomes were used to form complexes with DNA, resulting in enhanced expression of the chloramphenicol acetyltransferase gene in most tissues examined. The DNA:liposome ratio, and mild sonication, heating, and extrusion steps used for liposome preparation were crucial for improved systemic delivery. Size fractionation studies showed that maximal gene expression was produced by a homogeneous population of DNA:liposome complexes between 200 to 450 nm in size. Cryo-electron microscopy examination demonstrates that the DNA:liposome complexes have a novel morphology, and that the DNA is condensed on the interior of invaginated liposomes between two lipid bilayers. This structure could account for the high efficiency of gene delivery in vivo and for the broad tissue distribution of the DNA:liposome complexes. Ligands can be placed on the outside of this structure to provide for targeted gene delivery.     

Liposome-cell interactions in vitro: effect of liposome surface charge on the binding and endocytosis of conventional and sterically stabilized liposomes

The cellular uptake of liposomes is generally believed to be mediated by adsorption of liposomes onto the cell surface and subsequent endocytosis. This report examines the effect of liposome surface charge on liposomal binding and endocytosis in two different cell lines: a human ovarian carcinoma cell line (HeLa) and a murine derived mononuclear macrophage cell line (J774). The large unilamellar liposomes were composed of 1, 2-dioleolyl-sn-glycero-3-phosphatidylcholine with and without the addition of either a positively charged lipid, 1, 2-dioleoyl-3-dimethylammonium propanediol (DODAP), or a negatively charged lipid, 1,2-dioleolyl-sn-glycero-3-phosphatidylserine. In some experiments 5 mol % of the anionic PEG2000-PE or a neutral PEG lipid of the same molecular weight was added. HeLa cells were found to endocytose positively charged liposomes to a greater extent than either neutral or negatively charged liposomes. This preference was not lipid-specific since inclusion of a cationic cyanine dye, DiIC18(3), to impart positive charge in place of DODAP resulted in a similar extent of endocytosis. In contrast the extent of liposome interaction with J774 cells was greater for both cationic and anionic liposomes than for neutral liposomes. The greater uptake of positively charged liposomes by HeLa cells was also observed with sterically stabilized liposomes (PEG liposomes). Although the overall amount of endocytosis for all the PEG liposomes examined was attenuated relative to conventional liposomes, the extent of endocytosis was greatest for positively charged PEG liposomes, whereas negatively charged PEG2000-PE liposomes were hardly endocytosed by the HeLa cells. Incorporation of a neutral PEG lipid into liposomes permits the independent variation of liposome steric and electrostatic effects in a manner that may allow interactions with cells of the reticuloendothelial system to be minimized, yet permit strong interactions between liposomes and proliferating cells.     

A novel DNA-peptide complex for efficient gene transfer and expression in mammalian cells

To develop a nonviral gene delivery system for treatment of diseases, our strategy is to construct DNA complexes with short synthetic peptides that mimic the functions of viral proteins. We have designed and synthesized two peptides which emulate viral functions - a DNA condensing agent, YKAK(8)WK, and an amphipathic, pH-dependent endosomal releasing agent, GLFEALLELLESLWELLLEA. The active gene delivery complex was constructed step-wise through a spontaneous self-assembly process involving oppositely charged, electrostatic interactions. To assemble DNA-peptide complexes with different overall net charges, only the negative charges of DNA phosphate, the positive charges of the 10 epsilon-amino groups of YKAK(8)WK and the negative charges of the 5 gamma-carboxyl groups of GLFEALLELLESLWELLLEA were considered. In the first step, negatively charged DNA was rapidly-mixed with an excess of YKAK(8)WK to form positively charged DNA-YKAK(8)WK complexes, which gave little gene transfer. In the second step and to form the active complex,the cationic DNA complex was rapidly mixed with spontaneously incorporated through electrostatic interactions. Transfection using these complexes of CMV-luc, YKAK(8)WK and GLFEALLELLESLWELLLEA gave high-levels of gene expression in a variety of cell lines. These simple DNA complexes, which contain only three molecularly defined components, have general utility for gene delivery and can replace viral vectors and cationic lipids for some applications in gene therapy.     

Biodistribution and gene expression of lipid/plasmid complexes after systemic administration

The objectives of this study were to investigate the influence of physicochemical properties of lipid/plasmid complexes on in vivo gene transfer and biodistribution characteristics. Formulations based on 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and novel biodegradable cationic lipids, such as ethyl dioleoyl phosphatidylcholine (EDOPC), ethyl palmitoyl myristyl phosphatidylcholine (EPMPC), myristyl myristoyl carnitine ester (MMCE), and oleyl oleoyl L-carnitine ester (DOLCE), were assessed for gene expression after tail vein injection of lipid/plasmid complexes in mice. Gene expression was influenced by cationic lipid structure, cationic lipid-to-colipid molar ratios, plasmid-to-lipid charge ratios, and precondensation liposome size. Detectable levels of human growth hormone (hGH) in serum, human factor IX (hFIX) in plasma, and chloramphenicol acetyltransferase (CAT) in the lung and liver were observed with positively charged lipid/plasmid complexes prepared from 400-nm extruded liposomes with a cationic lipid-to-colipid ratio of 4:1 (mol/mol). Intravenous administration of lipid/CAT plasmid complexes resulted in distribution of plasmid DNA mainly to the lung at 15 min after injection. Plasmid DNA accumulation in the liver increased with time up to 24 hr postinjection. There was a 10-fold decrease in the amount of plasmid DNA in the lung at 15 min after injection, when the lipid/plasmid complex charge ratio was decreased from 3:1 to 0.5:1 (+/-). Bright fluorescent aggregates were evident in in vivo-transfected lung with the positively charged pCMV-CAT/DOLCE:dioleyl phosphatidylethanolamine (DOPE) (1:1, mol/mol) complexes, while more discrete punctate fluorescence was observed with a 4:1 molar ratio of cationic lipid:colipid formulations. Preinjection of polyanions such as plasmid, dextran sulfate, polycytidic acid, and polyinosinic acid decreased hGH expression, whereas the preinjection of both positively charged and neutral liposomes had no effect on hGH serum levels. Of the cationic lipids tested, DOLCE was found to be the most effective potentially biodegradable cationic lipid. A correlation between gene expression and cationic lipid:colipid ratios and lipid-to-plasmid charge ratio was also observed for DOTMA- and DOLCE-based formulations.     

Cationic liposomes in gene delivery

Cationic liposomes have been extensively explored as gene delivery vector for several reasons. It is because disadvantages of viral vectors include risk of replication, possible immunogenicity, and the difficulty of obtaining a large quantity of viral vectors. Currently, a variety of cationic components for liposome formulations have been developed. The components are broadly divided into two classes based on the chemical structure of hydrophobic moieties: long aliphatic (saturated or unsaturated) hydrocarbons and cholesterol ring. A variety of hydrophilic moieties also include tertiary amines, ammonium salt and spermine. The role of liposomes is to condense DNA to form complexes with high affinity to cell surfaces where possible fusion or destabilization of the membrane and/or endocytosis are involved. However, at present, little structure-activity relationships are known. Some vectors are on clinical trials approved by NIH.     

Intratumoral pharmacokinetics and in vivo gene expression of naked plasmid DNA and its cationic liposome complexes after direct gene transfer

The pharmacokinetic properties and gene expression of naked plasmid DNA and its cationic liposome complexes were studied after direct intratumoral injection. Using a Walker 256 tissue-isolated tumor perfusion system, we quantified the recovery of naked plasmid DNA and cationic liposome complexes in the tumor, leakage from the tumor surface, and the venous outflow after intratumoral injection. Approximately 50% of naked plasmid DNA had been eliminated from the tumor 2 h after injection, whereas more than 90% of plasmid DNA was retained in the tumor when it was complexed with cationic liposomes. However, the distribution of these complexes in the tumor was restricted to the tissue surrounding the injection site. Pharmacokinetic analysis of the venous outflow profiles suggested that the rate-limiting process that determines the retention of plasmid DNA in the tumor is transferred from the injection site in the tumor tissue and that complexation with cationic liposomes may retard this process. Furthermore, we examined the gene expression of chloramphenicol acetyltransferase DNA constructs (naked pCMV-CAT) and the corresponding cationic liposome [3-beta-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol] complexes. A similar level of gene expression was observed in vivo after direct intratumoral injection of naked DNA and its cationic liposome complexes. In both cases, a great variation was observed between tumors, and localization of gene-transduced cells in the tumor tissue was limited to the area in the vicinity of the injection site. Thus, these pharmacokinetic and gene expression studies have demonstrated that cationic liposomes can enhance the retention of injected DNA in the tumor model, whereas cationic liposome complex does not necessarily improve gene expression because of its poor dissemination in this tumor. The present study also suggested that there is a need to control the behavior of the injected naked plasmid DNA and its cationic liposome complexes to ensure better distribution throughout the tumor.     

Stable and monodisperse lipoplex formulations for gene delivery

A stable single vial lipoplex formulation has been developed that can be stored frozen without losing either biological activity or physical stability. This formulation was identified by systematically controlling several formulation variables and without introducing either stabilizers or surfactants. Analytical assays were used to unambiguously characterize the formulations. The critical formulation parameters were: (1) the size of the cationic liposomes; (2) the rate and method of DNA and cationic liposome mixing; and (3) the ionic strength of the suspending vehicle. The mixing conditions were precisely controlled by using a novel, specially designed continuous flow pumping system in which the DNA and liposome solutions were mixed at the junction of a T-connector. Homogenous cationic liposome preparations were prepared by extrusion in two different size ranges of either 400 or 100 nm. Extruded liposomes produced more monodisperse and physically stable lipoplex formulations than unextruded liposomes, but the formulations prepared with 100 nm liposomes were less active in in vitro transfection assays than either the 400 nm or unextruded liposomes. Low ionic strength and 5% sorbitol were required for the lipoplex formulations to survive freezing and thawing. A frozen lipoplex formulation stored for more than a year maintained its biological activity. These results have broad implications for the pharmaceutical development of lipoplex formulations for gene delivery.     

Dental procedure fees 1975 through 1995: how much have they changed?

Generally, cationic vector-based intravenous delivery of DNA is hindered by interactions of positively charged complexes with serum proteins. However, if optimally formulated, cationic vectors can provide reasonable levels of transfection in the lung either by intravenous or intrapulmonary routes. We investigated the in vivo transfection capacity of a cationic polymer: linear, 22 kDa polyethylenimine. PEI/DNA complexes were formulated in 5% glucose and delivered into adult mice through the tail vein. Two marker genes were used, beta-galactosidase and luciferase. High levels of luciferase expression (10(7) RLU/mg protein) were found in the lung when DNA was complexed with PEI at a ratio of 4 nitrogen equivalents per DNA phosphate. Lower levels of transfection were found in the heart, spleen, liver and kidney. Expression was dose- and time-dependent in all tissues examined. In the lung, beta-galactosidase staining showed transgene expression in clusters of 10 or more pulmonary cells including the alveolar endothelium, squamous and great alveolar epithelial cells (type I and II pneumocytes) and septal cells. These findings indicate that the complexes pass the capillary barrier in the lung. Although the delivery mechanism requires elucidation, linear PEI has promise as a vector for intravenous transfer of therapeutic genes.     

Liposomes enhance delivery and expression of an RGD-oligolysine gene transfer vector in human tracheal cells

Nonviral gene delivery systems consist predominantly of lipoplexes or receptor-targeting and nontargeting polyplexes. We examined integrin-mediated gene delivery using an Arg-Gly-Asp/oligo-L-lysine ([K]16RGD) cyclic peptide and investigated its gene transfer efficiency when associated with a cationic liposome. We demonstrated that human cystic fibrosis and noncystic fibrosis tracheal epithelial cells in culture express integrins that recognise the RGD integrin-binding motif. We found a 10-fold (P < 0.01) increased expression of a luciferase encoding plasmid in these cells when complexing the plasmid to the [K]16RGD peptide as compared with plasmid alone. This increase was specific to the [K]16RGD peptide since neither a [K]16RGE nor a [K]16 peptide gave a comparable increase. Expression was further enhanced 30-fold (P < 0.01) with lipofectamine and the ratio of DNA/peptide/lipofectamine was critical for specificity and expression. Fluorescence and radioactive labelling of the complex showed that the [K]16RGD peptide increased the endocytic uptake of DNA into cells. The cell association of both DNA and peptide increased even further with lipofectamine. Confocal microscopy showed that the [K]16RGD peptide and the DNA internalised together within 30 min and localised to vesicles in the perinuclear region. These results show that an integrin-binding ligand can deliver genetic material to airway cells and that a cationic liposome can enhance the efficacy of this nonviral vector system.     

An RGD-oligolysine peptide: a prototype construct for integrin-mediated gene delivery

We have synthesized a linear, bifunctional peptide that comprises an integrin-targeting domain containing an arginine-glycine-aspartic acid tripeptide motif and a DNA-binding moiety consisting of a short stretch of 16 lysine residues. This peptide can form distinctive, condensed complexes with DNA and is capable of mediating its delivery and expression in a variety of mammalian cells in culture. Internalization is mediated by cell surface integrin receptors via a mechanism that is known to be phagocytic. We have analyzed the relationship between DNA and peptide and have investigated the conditions suitable for optimal gene delivery. The formation of condensed peptide DNA complexes leads to resistance to nuclease degradation. The level of reporter gene expression obtained is dependent on the peptide-to-DNA ratio and is enhanced in the presence of the endosomal buffer chloroquine, polyethyleneimine, and deactivated adenovirus during gene delivery. Under optimal conditions the levels of reporter gene expression obtained approach or even exceed those obtained with DNA delivered with the commercial liposome Lipofectamine. The ability to produce an efficient gene delivery system using small, easily modified, and well-defined constructs that have no constraint of particle size demonstrates the advantages of integrin-targeting peptides for gene transfer.     

Inhibitor and temperature effect on catalase in the liver of adult diploid and haploid Rana rugosa

Cellular uptake and gene expression of plasmid DNA and its cationic liposome complexes were studied using primary cultures of bovine brain microvessel endothelial cells (BMEC) developed as an in vitro model of the blood-brain barrier. An avid association of naked plasmid DNA with the BMEC monolayer was observed at 37 degreesC, which is comparable to that of the DNA/liposome complex. The cellular association significantly decreased at low temperature (4 degreesC). The binding at 4 degreesC was saturable and significantly inhibited by polyanions involving polyinosinic acid and dextran sulfate, typical ligands for the macrophage scavenger receptors, but not by polycytidylic acid or in the presence of EDTA. Unexpectedly, a significant gene expression in the BMEC was obtained by transfection with naked plasmid DNA although the expression level was lower than that obtained by plasmid DNA/cationic liposome complex. Taken together, cultured capillary endothelial cells derived from the brain are able to take up naked plasmid DNA via a scavenger receptor like-mediated mechanism for polyanions and gene expression in the cells takes place.     

Transfection of cultured myoblasts in high serum concentration with DODAC:DOPE liposomes

The inhibitory effect of serum is one of the main obstacles to the in vivo use of cationic liposomes as a DNA delivery system. We have found that a novel liposome formulation, DODAC:DOPE (1:1) is totally resistant to the inhibitory effects of serum for transfection of cultured myoblasts and myotubes. Transfection with a lacZ reporter gene in the presence of 95% fetal bovine serum gave up to 25% beta-gal-positive cells in C2C12 myoblasts and about six-fold less in primary human myoblasts. The lower transgene expression in primary cells does not appear to be a result of less DNA uptake but might result from differences in intracellular trafficking of the complexes. DODAC-based liposomes are unique in their resistance to serum inhibition and may therefore be valuable for the systemic delivery of genetic information to muscle and other tissues.     

Gene transfer by guanidinium-cholesterol cationic lipids into airway epithelial cells in vitro and in vivo

Synthetic vectors represent an attractive alternative approach to viral vectors for gene transfer, in particular into airway epithelial cells for lung-directed gene therapy for cystic fibrosis. Having recently found that guanidinium-cholesterol cationic lipids are efficient reagents for gene transfer into mammalian cell lines in vitro, we have investigated their use for gene delivery into primary airway epithelial cells in vitro and in vivo. The results obtained indicate that the lipid bis(guanidinium)-tren-cholesterol (BGTC) can be used to transfer a reporter gene into primary human airway epithelial cells in culture. Furthermore, liposomes composed of BGTC and dioleoyl phosphatidylethanolamine (DOPE) are efficient for gene delivery to the mouse airway epithelium in vivo. Transfected cells were detected both in the surface epithelium and in submucosal glands. In addition, the transfection efficiency of BGTC/DOPE liposomes in vivo was quantitatively assessed by using the luciferase reporter gene system.     

Cationic liposomes coated with polyethylene glycol as carriers for oligonucleotides

Modification of liposome surface with polyethylene glycol was used to improve oligodeoxyribonucleotide (ODN) loading, stability of the resulting complexes, and specificity of cellular delivery of ODN by cationic liposomes. Liposomes composed of a cationic lipid (DOTAP, DOGS, DDAB), a neutral lipid (DOPE), and a phospholipid derivative of polyethylene glycol (PEG-PE) formed a complex with 18-mer phosphorothioate up to ODN/lipid molar ratio of 0.25. The complexes showed intact vesicular structures similar to original liposomes and their size (100-130 nm) was unchanged after several weeks of storage, whereas complexes lacking PEG-PE showed progressive aggregation and/or precipitation. After exposure to human plasma, PEG-modified cationic liposomes retained over 60% of the originally bound ODN. PEG-coated complexes resulted in 4-13-fold enhancement of the ODN uptake by human breast cancer cells in serum-supplemented growth medium, relative to free ODN. Complexes containing conjugated anti-HER2 F(ab') fragments at the distal termini of PEG chains efficiently delivered ODN primarily into the cytoplasm and nuclei of HER2 overexpressing cancer cells and greatly enhanced the biological activity of antisense ODN. The development of PEG-modified cationic liposomes may lead to improved ODN potency in vivo.     

NMR characterization of a single-cysteine mutant of Escherichia coli thioredoxin and a covalent thioredoxin-peptide complex

Cationic liposomes are used as the carriers of polyanionic genes for combating against hereditary diseases in gene therapy. Studies directed to careful biophysical characterizations of the cationic liposomes commonly used in gene delivery have just begun. Herein, we report on a novel liposomal exo-surface bound indazolization reaction of an amphiphilic arenediazonium salt as evidence for the existence of remarkably alkaline exo-surface of cationic liposomes commonly used in gene transfection. Our results demonstrate that formation of 5-hexadecyl-7-methylindazole in thermal indazolization of 2,6-dimethyl-4-hexadecylbenzenediazonium tetrafluoroborate bound to liposome surface is a strong indication for the existence of significantly high exo-surface pH for cationic liposomes commonly used in gene delivery. The present method can be used in determining the relative exo-surface basicities of various cationic liposomes used in gene transfection and subsequently to find any possible correlation between the transfection efficiencies of these liposomes and their exo-surface basicities.     

Gene expression of interleukin-2 in purified human peripheral blood eosinophils

Liposomes have been used as carriers of various materials and as tools for gene transfer: for the latter purpose, positively charged liposomes are usually used. To evaluate the stability in the presence of serum and the in vivo behavior of such liposomes as well as those aspects of neutral and negatively charged liposomes, we investigated liposomal agglutinability in the presence of serum, serum protein binding to these liposomes, and real-time liposomal trafficking by a non-invasive method using positron emission tomography (PET). Liposomes composed of dipalmitoylphosphatidylcholine, cholesterol without or with charged lipid were prepared in the presence of mannitol, and the turbidity change in the presence of serum was determined. Turbidity increase was not observed for so-called long-circulating liposomes, i.e., liposomes modified with glucuronic acid or with poly(ethylene glycol), or for negatively charged liposomes containing dicetyl phosphate (DCP), phosphatidylglycerol, or phosphatidylserine. On the contrary, a significant turbidity increase was observed when positively charged liposomes modified with stearylamine, stearyltrimethylammonium chloride or 1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl bromide (DMRIE), which is known as a component of liposomes for gene transfer, were used. These liposomes were found to have bound a high amount of serum proteins after separation of unbound serum proteins by use of a spin column. The liposomal trafficking in vivo was determined for three kinds of liposomes, i.e., liposomes with DMRIE, those with DCP, and those without charged lipids. These liposomes were prepared in the presence of 2-[18F]fluoro-2-deoxy-D-glucose ([2-18F]FDG), and the [2-18F]FDG-labeled liposomes were administered to mice to perform PET scans. Positively charged liposomes containing DMRIE showed high accumulation in the liver compared with neutral and negatively charged liposomes. Since DMRIE-liposomes tended to aggregate in the presence of serum, and to be associated with serum protein, these characteristics may lead to the high uptake of DMRIE-liposomes by the liver.     

High-efficiency entrapment of superoxide dismutase into cationic liposomes containing synthetic aminoglycolipid

A monofatty acid ester of glucosamine (PGlcN) was synthesized to provide liposomal membranes with a positive charge, and the trapping efficiency of negatively charged substances (superoxide dismutases, SODs) into cationic liposomes containing PGlcN or stearylamine (SA) prepared by various methods was compared to find the most efficient trapping methods. We demonstrated that cationic liposomes, which were prepared in a buffer of low ionic strength containing sorbitol by a simple hydration method, could entrap a large amount of negatively charged SODs which retained their activity, as compared with cationic liposomes prepared in a buffer of high ionic strength. We also showed a reverse-phase evaporation method entrapped a large amount of SODs. However, SODs were inactivated during the preparation; therefore, this method was not suitable to entrap the enzyme. Freeze-thaw method induced the formation of cationic liposomes which were smaller than extruded liposomes and could entrap the SODs in a buffer of low ionic strength. Dehydration-rehydration method with a buffer of low ionic strength also entrapped a large amount of SODs, indicating that the integrity of liposomes was lost in the lipid bilayer after freeze-drying and the SODs were entrapped in the reconstruction of liposomes during rehydration. These findings showed that the hydration method based on electrostatic attraction with a buffer of low ionic strength was simple and the most effective for entrapping SODs without loss of their activity.     

Mucosal immunoadjuvant activity of liposomes: role of alveolar macrophages

Previously, we have reported on a liposomal adjuvant system for stimulation of both systemic IgG and mucosal s-IgA responses against viral antigens (influenza virus subunit antigen or whole inactivated measles virus) administered intranasally to mice. Immune stimulation is observed with negatively charged, but not with zwitterionic, liposomes and is independent of a physical association of the antigen with the liposomes. Furthermore, liposome-mediated immune stimulation requires deposition of the liposomes and the antigen in the lower respiratory tract. In the present study, it is shown that alveolar macrophages (AM) are the main target cells for negatively charged liposomes administered to the lungs of mice. AM isolated from animals, to which negatively charged liposomes were administered beforehand, showed large intracellular vacuoles, suggestive of massive liposome uptake. Under ex vivo conditions, both AM and RAW 264 cells exhibited a high capacity to take up negatively charged liposomes. The deposition of negatively charged liposomes, but not zwitterionic, liposomes in the lung reduced the phagocytic and migratory behaviour of AM, as assessed on the basis of transport of carbon particles to the draining lymph nodes of the lungs. Depletion of AM in vivo with dichloromethylene diphosphonate, facilitated an enhanced systemic and local antibody response against influenza subunit antigen deposited subsequently to the lower respiratory tract. In conclusion, these data provide support for the hypothesis that uptake of negatively charged liposomes blocks the immunosuppressive activity of AM, thereby facilitating local and systemic antibody responses.     

Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells

The beta-galactosidase reporter gene, either free or complexed with various cationic vectors, was microinjected into mammalian cells. Cationic lipids but not polyethylenimine or polylysine prevent transgene expression when complexes are injected in the nucleus. Polyethylenimine and to a lesser extent polylysine, but not cationic lipids, enhance transgene expression when complexes are injected into the cytoplasm. This latter effect was independent of the polymer vector/cDNA ionic charge ratio, suggesting that nucleic acid compaction rather than surface charge was critical for efficient nuclear trafficking. Cell division was not required for nuclear entry. Finally, comparative transfection and microinjection experiments with various cell lines confirm that barriers to gene transfer vary with cell type. We conclude that polymers but not cationic lipids promote gene delivery from the cytoplasm to the nucleus and that transgene expression in the nucleus is prevented by complexation with cationic lipids but not with cationic polymers.     

Surfactant inhibits cationic liposome-mediated gene transfer

We have demonstrated that tracheal insufflation of recombinant plasmid DNA results in transfection of rat lungs to the same extent as insufflation of plasmid-cationic liposome complex. To understand this observation better, we investigated the in vitro gene transfer of plasmid DNA in the presence and absence of cationic liposome and the effect of surfactant on gene transfer. The chloramphenicol acetyltransferase (CAT) expression plasmids pBL-CAT and pSV-CAT were studied in three cell types: rat fetal lung fibroblast (RFL-6), calf pulmonary artery endothelial cell (CPAE), and rat type II alveolar epithelial cell (type II AE). Three cationic liposomes were tested: DDAB (dimethyl-dioctadecyl ammonium bromide)-liposome, DOTAP (dioleoyltrimethyl ammonium propane)-liposome, and lipofectin. The results revealed that (i) plasmid DNA alone caused a dose-dependent, low-level transfection, most efficiently in RFL-6 followed by CPAE and type II AE, (ii) DDAB-liposome markedly enhanced gene transfer, most efficiently in RFL-6 followed by CPAE and type II AE, (iii) Survanta, a naturally derived surfactant preparation, and Exosurf, a synthetic surfactant, while having no effect on in vitro gene transfer by plasmid DNA alone, markedly inhibited cationic liposome-mediated gene transfer, (iv) dipalmitoyl phosphatidylcholine was responsible for the inhibitory effect of Exosurf, and (v) inhibition of cationic liposome-mediated gene transfer by Exosurf was not due to inhibition of plasmid DNA-cationic liposome complex uptake or interference with the promoter and enhancer. The observed inhibition of cationic liposome-mediated gene transfer by surfactant may in part explain our previous observation that tracheal insufflation of plasmid DNA and plasmid-cationic liposome complex results in equal lung gene transfer.