Mechanisms of lipoplex formation: dependence of the biological properties of transfection complexes on formulation procedures.

Phospholipid-DNA complexes were made of the cationic triester derivative of phosphatidylcholine, EDOPC (1,2-dioleoyl- sn-glycero-3-ethylphosphocholine), by varying conditions of complex formation, in particular, the rate and direction of mixing, as well as by changing the mode of dispersing the lipid (extrusion or vortexing). The biological effects of variations in the formulation procedure were assessed by measuring transfection activity and cell association in cultures of BHK cells. Formulation procedures generally had little effect on cell association, but had marked effects on transfection efficiency. Transfection varied from effectively nil to extremely efficient with what appeared to be modest changes in formulation procedure. Formulation procedures also had significant effects on average sizes and size distributions of lipoplexes as determined by dynamic light scattering. Among the four possibilities of rapid or slow mixing combined with the two possible directions of mixing, slow addition of DNA to lipid gave results that differed significantly from the other three modes. In the case of vortexed lipid, the latter procedure was much less satisfactory than the other three, whereas in the case of extruded lipid, it was the only mode that produced satisfactory transfection. The factors that determine the difference in lipoplex properties can be identified as both geometric and physical. The geometric factor has to do with the symmetries of the participating units. There are three physical factors that are critical: the difference in vesicle stability upon interaction with DNA, the time dependence of interdiffusion of the components relative to that of vesicle rupture, and difference in input concentrations. These factors determine lipoplex size and, as already also shown by others, lipoplex size influences transfection efficiency.

New strategy for transfection: mixtures of medium-chain and long-chain cationic lipids synergistically enhance transfection.

To date, the primary approach to improve the transfection properties of cationic lipids has been the synthesis of new kinds of cationic amphipaths or the inclusion of noncationic helper lipids. Here, it is reported that an alternative approach can be unusually effective, namely, the combination of two cationic lipid derivatives having the same head group but tails of different chain lengths. Particularly efficient was the combination of dilauroyl (12 carbon chain) and dioleoyl (18 carbon chain) homologues of O-ethylphosphatidylcholine. This mixture transfected DNA into human umbilical artery endothelial cells (HUAEC) more than 30-fold more efficiently than either compound separately. A unique advantage of this kind of combination agent is that transfection can be optimized either in the presence or absence of serum by adjusting the component ratio.

Bilayer mixing, fusion, and lysis following the interaction of populations of cationic and anionic phospholipid bilayer vesicles.

Cationic, O-alkylphosphatidylcholines, recently developed as DNA transfection agents, form bilayers indistinguishable from those of natural phospholipids and undergo fusion with anionic bilayers. Membrane merging (lipid mixing), contents release, and contents mixing between populations of positive vesicles containing O-ethylphosphatidylcholine (EDOPC) and negative vesicles containing dioleolylphosphatidylglycerol (DOPG) have been determined with standard fluorometric vesicle-population assays. Surface-charge densities were varied from zero to full charge. All interactions depended critically on surface-charge density, as expected from the adhesion-condensation mechanism. Membrane mixing ranged from zero to 100%, with significant mixing (>10 <70%) occurring between cationic vesicles that were fully charged and anionic vesicles that had fractional surface charges as low as 0.1. Such mixing with membranes as weakly charged as cell membranes should be relevant to transfection with cationic lipids. Unexpectedly, lipid mixing was higher at high than at low ionic strength when one lipid dispersion was prepared from EDOPC plus DOPG (in different proportions), especially when the other vesicles were of EDOPC; this may somehow be a consequence of the ability of the former mixture to assume non-lamellar phases. Leakage of aqueous contents was also a strong function of charge, with fully charged vesicles releasing essentially all of their contents less than 1 min after mixing. EDOPC was more active in this regard than was DOPG, which probably reflects stronger intermolecular interactions of DOPG. Fusion, as measured by contents mixing, exhibited maximal values of 10% at intermediate surface charge. Reduced fusion at higher charge is attributed to multiple vesicle interactions leading to rupture. The existence of previously published data on individual interactions of vesicles of the same composition made it possible for the first time to compare pairwise with population interactions, confirming the likelihood of population studies to overestimate rupture and hemifusion and underestimate true vesicle fusion.

Further observations on LKB1/STK11 status and cancer risk in Peutz-Jeghers syndrome.

Germline mutations in the LKB1/STK11 tumour suppressor gene cause Peutz-Jeghers syndrome (PJS), a rare dominant disorder. In addition to typical hamartomatous gastrointestinal polyps and pigmented perioral lesions, PJS is associated with an increased risk of tumours at multiple sites. Follow-up information on carriers is limited and genetic heterogeneity makes counselling and management in PJS difficult. Here we report the analysis of the LKB1/STK11 locus in a series of 33 PJS families, and estimation of cancer risks in carriers and noncarriers. Germline mutations of LKB1/STK11 were identified in 52% of cases. This observation reinforces the hypothesis of a second PJS locus. In carriers of LKB1/STK11 mutations, the risk of cancer was markedly elevated. The risk of developing any cancer in carriers by age 65 years was 47% (95% CI: 27-73%) with elevated risks of both gastrointestinal and breast cancer. PJS with germline mutations in LKB1/STK11 are at a very high relative and absolute risk of multiple gastrointestinal and nongastrointestinal cancers. To obtain precise estimates of risk associated with PJS requires further studies of genotype-phenotype especially with respect to LKB1/STK11 negative cases, as this group is likely to be heterogeneous.

Real-time observation of lipoplex formation and interaction with anionic bilayer vesicles.

A novel development has allowed for the direct observation of single, pairwise interactions of linear DNA with cationic vesicles and of DNA-cationic lipid complexes with anionic vesicles. A new cationic phospholipid derivative, l,2-dioleoyl-sn-glycero-3-ethylphosphocholine, was used to prepare giant bilayer vesicles and to form DNA-cationic lipid complexes (lipoplexes). The cationic vesicles were electrophoretically maneuvered into contact with DNA, and similarly, complexes were brought into contact with anionic phospholipid vesicles composed of dioleoylphosphatidylglycerol (DOPG; 100%), DOPG/dioleoylphosphatidylethanolamine (DOPE; 1:1) or DOPG/dioleoylphosphatidylcholine (DOPC; 1:1). Video fluorescence microscopy revealed that upon contact with phospholipid anionic vesicles, lipoplexes exhibited four different types of behavior: adhesion, vesicle rupture, membrane perforation (manifested as vesicle shrinkage and/or content loss), and expansion of DNA (which was always concomitant with membrane perforation.) In one instance, the lipoplex was injected into the target vesicle just prior to DNA expansion. In all other instances, the DNA expanded over the outer surface of the vesicle, and expansion was faster, the larger the area of vesicle over which it expanded. Given the likelihood of incorporation of cellular anionic lipids into lipoplexes, the expansion of the DNA could be important in DNA release during cell transfection. Upon contact with naked DNA, giant cationic vesicles usually ruptured and condensed the DNA into a small particle. Contact of cationic vesicles that were partially coated with DNA usually caused the DNA to wrap around the vesicle, leading to vesicle rupture, vesicle fusion (with other attached vesicles or lipid aggregates), or simply cessation of movement. These behaviors clearly indicated that both DNA and vesicles could be partly or fully covered by the other, thus modifying surface charges, which, among others, allowed adhesion of DNA-coated vesicles with uncoated vesicles and of lipid-coated DNA with uncoated DNA.

Improving ultrasound reflectivity and stability of echogenic liposomal dispersions for use as targeted ultrasound contrast agents.

Targeted echogenic liposome dispersions for ultrasonic enhancement of vasoactive and pathological components of endothelium and atherosclerosis have recently been developed. The component lipids required for acoustic and targeting properties include phosphatidylcholine, phosphatidylethanolamine phosphatidylglycerol (PG), and cholesterol (CH), initially in a 60:8:2:30 mol % ratio. Component lipids, lyophilization, sugars, and freezing conditions were varied to optimize acoustic ultrasound reflectivity and acoustic stability. Echogenic liposome dispersions were made by using the dehydration-rehydration process. The lipid concentrations were varied (CH in the range 1 to 40 mol % and PG from 1 to 16 mol %). Variations in type and concentration of sugars were examined. The effect of freezing conditions and re-lyophilization was examined. Ultrasound reflectivity was assessed by using a 20-MHz intravascular ultrasound catheter and computer-assisted videodensitometry. Ultrasound reflectivity was optimized at a CH concentration of 10 mol %; PG concentration variation had essentially no effect on initial values of echogenicity. Optimal acoustic stability was observed with concentrations of 10-15 mol % CH and with a PG concentration greater than 4 mol %. Preparations made with 0.2 M mannitol were more ultrasound reflective than those made with lactose, trehalose, and sucrose. Re-lyophilization and freezing temperatures below -20 degrees C increased ultrasound reflectivity. We optimized the ultrasound properties of echogenic liposomal dispersions, the conditions of which provide some insight into the underlying lipid structures responsible. The preparations developed are now more stable and acoustically reflective than our previous preparations. This advances the development of echogenic lipid dispersions as targeted ultrasound contrast agents for use in general ultrasound as well as cardiovascular imaging.

Diquaternary ammonium compounds as transfection agents.

Diquaternary ammonium salts constitute a new class of reagent for mediating transfection of DNA in mammalian cell lines. N,N'-dioleyl-N,N,N',N'-tetramethyl-1,2-ethanediamine (TmedEce), N,N'-dioleyl-N,N,N',N'-tetramethyl-1,3-propanediamine (PropEce), N,N'-dioleyl-N,N,N',N'-tetramethyl-1,6-hexanediamine (HexEce), and their corresponding N,N'-dicetyl saturated analogues (TmedAce, PropAce and HexAce) have all been synthesized and characterized. They were prepared via a bis-Menshutkin reaction of the corresponding tetramethyldiamine with 2.2 M equiv of a long-chain alkyl halide (saturated or unsaturated). The reaction was run in anhydrous acetonitrile for ca. 3 days at 60 degrees C, which produced the diquaternary ammonium halides in good to nearly quantitative yields for most derivatives. DNA transfection comparable to commercially available reagents such as Lipofectin, Lipofectace, Lipofectamine, and O-ethyldioleoylphosphatidylcholinium triflate has been achieved in vitro with these new reagents. There was no need to use a colipid for effective transfection, but serum did significantly inhibit transfection. The saturated and the unsaturated derivatives differed with respect to hydration behavior. The saturated derivatives appeared to retain a lamellar-type crystalline array structure upon hydration, whereas the unsaturated versions formed micelles and/or liposomes, depending on the ionic strength: HexEce was micellar in both water and saline; PropEce was micellar in water but lamellar in saline; and TmedEce was lamellar in both. Despite these different hydration patterns, all of these unsaturated derivatives formed productive transfection complexes with DNA. Varying the distance between the quaternary sites affected transfection efficacy in the order HexAce > TmedAce = PropAce for the saturated derivatives and in the order PropEce = HexEce > TmedEce, with a smaller spread, for the unsaturated derivatives.

Cholesterol stabilizes hemifused phospholipid bilayer vesicles.

Cholesterol was found to inhibit full fusion of oppositely charged phospholipid bilayer vesicles by stabilizing the contacting membranes at the stage of the hemifused intermediate. Vesicles of opposite charge containing different amounts of cholesterol were prepared using cationic (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine) and anionic (dioleoylphosphatidylglycerol) phospholipids. Pairwise interactions between such vesicles were observed by fluorescence video microscopy in real time after electrophoretically maneuvering the vesicles into contact. Hemifusion accounted for more than 80% of the observed events when the vesicles contained 33-50 mole% cholesterol. In contrast, vesicles containing only a small proportion of cholesterol (

Atomic force microscopy of the erythrocyte membrane skeleton.

The atomic force microscope was used to examine the cytoplasmic surface of untreated as well as fixed human erythrocyte membranes that had been continuously maintained under aqueous solutions. To assess the effects of drying, some membranes were examined in air. Erythrocytes attached to mica or glass were sheared open with a stream of isotonic buffer, which allowed access to the cytoplasmic membrane face without exposing cells to non-physiological ionic strength solutions. Under these conditions of examination, the unfixed cytoplasmic membrane face revealed an irregular meshwork that appeared to be a mixture largely of triangular and rectilinear openings with mesh sizes that varied from 35 to 100 nm, although few were at the upper limit. Fixed ghosts were similar, but slightly more contracted. These features represent the membrane skeleton, as when the ghosts were treated to extract spectrin and actin, these meshworks were largely removed. Direct measurements of the thickness of the membrane skeleton and of the lateral dimensions of features in the images suggested that, especially when air dried, spectrin can cluster into large, quite regularly distributed aggregates. Aggregation of cytoskeletal components was also favoured when the cells were attached to a polylysine-treated substrate. In contrast, the membrane skeletons of cells attached to substrates rendered positively charged by chemical derivatization with a cationic silane were much more resistant to aggregation. As steps were taken to reduce the possibility of change of the skeleton after opening the cells, the aggregates and voids were eliminated, and the observed structures became shorter and thinner. Ghosts treated with Triton X-100 solutions to remove the bilayer revealed a meshwork having aggregated components resembling those seen in air. These findings support the proposition that the end-to-end distance of spectrin tetramers in the cell in the equilibrium state is much shorter than the contour length of the molecule and that substantial rearrangements of the spectrin-actin network occur when it is expanded by low ionic strength extraction from the cell. This study demonstrates the applicability of AFM for imaging the erythrocyte membrane skeleton at a resolution that appears adequate to identify major components of the membrane skeleton under near-physiological conditions.

Electrostatic control of phospholipid polymorphism.

A regular progression of polymorphic phase behavior was observed for mixtures of the anionic phospholipid, cardiolipin, and the cationic phospholipid derivative, 1, 2-dioleoyl-sn-glycero-3-ethylphosphocholine. As revealed by freeze-fracture electron microscopy and small-angle x-ray diffraction, whereas the two lipids separately assume only lamellar phases, their mixtures exhibit a symmetrical (depending on charge ratio and not polarity) sequence of nonlamellar phases. The inverted hexagonal phase, H(II,) formed from equimolar mixtures of the two lipids, i.e., at net charge neutrality (charge ratio (CR((+/-))) = 1:1). When one type of lipid was in significant excess (CR((+/-)) = 2:1 or CR((+/-)) = 1:2), a bicontinuous cubic structure was observed. These cubic phases were very similar to those sometimes present in cellular organelles that contain cardiolipin. Increasing the excess of cationic or anionic charge to CR((+/-)) = 4:1 or CR((+/-)) = 1:4 led to the appearance of membrane bilayers with numerous interlamellar contacts, i.e., sponge structures. It is evident that interactions between cationic and anionic moieties can influence the packing of polar heads and hence control polymorphic phase transitions. The facile isothermal, polymorphic interconversion of these lipids may have important biological and technical implications.

An assessment of the validity of a computer system for probabilistic record linkage of birth and infant death records in Canada. The Fetal and Infant Health Study Group.

Studies on the validity of probabilistic record linkage are sparse. We performed a probabilistic linkage to link the 1984-1994 birth records (obtained from the Canadian Birth Data Base) with 1984-1995 infant death records (from the Canadian Mortality Data Base) in Canada. We extracted the linked birth-death records for Nova Scotia and Alberta (from January 1990 to December 1991) obtained from Statistics Canada's vital registration data and compared them with corresponding records from provincial data (primarily hospital records). The results showed that over 99% of infant deaths (153/155) in the Nova Scotia provincial data were successfully located in the linked Statistics Canada file; the corresponding figure for Alberta neonatal deaths was also 99% (365/367). The distributions of gestational age and birth weight in matched cases demonstrated high agreement between the two data sources. We conclude that the computer system for probabilistic linkage developed by Statistics Canada using the available personal identifying variables in the Canadian Birth Data Base and the Canadian Mortality Data Base is valid.

O-Alkyl dioleoylphosphatidylcholinium compounds: the effect of varying alkyl chain length on their physical properties and in vitro DNA transfection activity.

1,2-Dioleoyl-sn-3-ethylphosphocholine (EDOPC) has been previously shown be a highly effective DNA transfection reagent in vitro. To assess the effect of alkyl chain length on transfection efficiency, the O-methyl, O-propyl, O-hexyl, O-decyl, and O-octadecyl derivatives have been prepared from dioleoylphosphatidylcholine using the corresponding alkyl trifluoromethylsulfonate. The methyl, ethyl, and propyl derivatives formed liposomes which were very large and unilamellar. The ethyl and propyl derivatives were equally efficient at mediating transfection (even in the presence of serum) of BHK cells, but the chemically labile methyl derivative was a much weaker transfection agent. The O-decyl and O-octadecyl compounds, which assume the inverted hexagonal phase in excess water (as determined by X-ray diffraction), were almost inactive after manual agitation in both water and in saline; however, after sonication, these compounds exhibited good transfection activity. The O-hexyl derivative displayed novel behavior, assuming the lamellar phase at low and a cubic phase at high ionic strength. All compounds, whether lamellar or not, formed lamellar structures when complexed with DNA. In water, where the hexyl compound dispersed well, sonication diminished transfection activity, whereas at physiological ionic strength, which led to poor manual dispersion, sonication was essential for good transfection. These results emphasize the importance of optimal dispersion of a cationic lipid: too little, and interaction with DNA is handicapped, too much, and the resultant particle transfects poorly. Lipid dispersibility is thus an important variable in assessing lipid transfection agents, and caution is advised in attributing too much significance to chemical structure until interaction with DNA has been optimized.

The lymphocytic choriomeningitis virus RING protein Z associates with eukaryotic initiation factor 4E and selectively represses translation in a RING-dependent manner.

Only a few host cell proteins that associate with arenaviruses have been identified. To date, the arenavirus Z protein associates with the promyelocytic leukemia protein PML and the ribosomal P proteins. The majority of PML is present in nuclear bodies which are translocated to the cytoplasm by infection with the arenavirus, lymphocytic choriomeningitis virus (LCMV). The Z protein is a small zinc-binding RING protein with an unknown function which is required for the viral life cycle. Here, we demonstrate an association between Z and the host cell translation factor, eukaryotic initiation factor 4E (eIF-4E) in infected and transfected cells. Z's association with both ribosomal proteins and this translation factor led us to investigate whether Z could modulate host cell translation. In cell culture, Z selectively represses protein production in an eIF-4E-dependent manner. Specifically, we see reduction in cyclin D1 protein production with no effect on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cells transfected with Z. Previous reports indicate that cyclin D1 is sensitive to eIF-4E levels, whereas GAPDH is not. Consistent with this, we observe preferential downregulation of cyclin D1 during infection and no effect on GAPDH. Further, no changes in RNA levels were observed for cyclin D1 or GAPDH transcripts. The interaction between eIF-4E and Z may provide a mechanism for slower growth observed in infected cells and a viral strategy for establishing chronic infection.

Factors governing the assembly of cationic phospholipid-DNA complexes.

The interaction of DNA with a novel cationic phospholipid transfection reagent, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), was investigated by monitoring thermal effects, particle size, vesicle rupture, and lipid mixing. By isothermal titration calorimetry, the heat of interaction between large unilamellar EDOPC vesicles and plasmid DNA was endothermic at both physiological and low ionic strength, although the heat absorbed was slightly larger at the higher ionic strength. The energetic driving force for DNA-EDOPC association is thus an increase in entropy, presumably due to release of counterions and water. The estimated minimum entropy gain per released counterion was 1.4 cal/mole- degrees K (about 0.7 kT), consistent with previous theoretical predictions. All experimental approaches revealed significant differences in the DNA-lipid particle, depending upon whether complexes were formed by the addition of DNA to lipid or vice versa. When EDOPC vesicles were titrated with DNA at physiological ionic strength, particle size increased, vesicles ruptured, and membrane lipids became mixed as the amount of DNA was added up to a 1.6:1 (+:-) charge ratio. This charge ratio also corresponded to the calorimetric end point. In contrast, when lipid was added to DNA, vesicles remained separate and intact until a charge ratio of 1:1 (+:-) was exceeded. Under such conditions, the calorimetric end point was 3:1 (+:-). Thus it is clear that fundamental differences in DNA-cationic lipid complexes exist, depending upon their mode of formation. A model is proposed to explain the major differences between these two situations. Significant effects of ionic strength were observed; these are rationalized in terms of the model. The implications of the analysis are that considerable control can be exerted over the structure of the complex by exploiting vectorial preparation methods and manipulating ionic strength.

Development of echogenic, plasmid-incorporated, tissue-targeted cationic liposomes that can be used for directed gene delivery.

RATIONALE AND OBJECTIVES: Echogenic antibody-conjugated anionic liposomes have been developed that allow directed tissue targeting and acoustic enhancement. These are not efficient for gene delivery. A cationic formulation that allows directed gene delivery while retaining acoustic properties may provide more efficient transfection. METHODS: Cationic liposomes were prepared and acoustic reflectivity was determined. Anti-fibrinogen-conjugated liposomes were laid on fibrin-coated slides and adherence was quantified using fluorescence techniques. Liposomes were combined with a reporter gene and plated on cell cultures. Human umbilical vein endothelial cells were stimulated to upregulate intercellular adhesion molecule-1 (ICAM-1) and were treated with anti-ICAM-1-conjugated liposomes, and gene expression was quantified. RESULTS: Cationic liposomes retained their acoustic reflectivity and demonstrated specific adherence to fibrin under flow conditions. Significant transfection of human umbilical vein endothelial cells was demonstrated, with higher gene expression seen with specific antibody-conjugated liposomes. CONCLUSIONS: Novel acoustic cationic liposomes have been developed that can be antibody conjugated for site-specific adherence and directed cell modification. This presents exciting potential for a vector that allows tissue enhancement and targeted gene delivery.

Physical and biological properties of cationic triesters of phosphatidylcholine.

The properties of a new class of phospholipids, alkyl phosphocholine triesters, are described. These compounds were prepared from phosphatidylcholines through substitution of the phosphate oxygen by reaction with alkyl trifluoromethylsulfonates. Their unusual behavior is ascribed to their net positive charge and absence of intermolecular hydrogen bonding. The O-ethyl, unsaturated derivatives hydrated to generate large, unilamellar liposomes. The phase transition temperature of the saturated derivatives is very similar to that of the precursor phosphatidylcholine and quite insensitive to ionic strength. The dissociation of single molecules from bilayers is unusually facile, as revealed by the surface activity of aqueous liposome dispersions. Vesicles of cationic phospholipids fused with vesicles of anionic lipids. Liquid crystalline cationic phospholipids such as 1, 2-dioleoyl-sn-glycero-3-ethylphosphocholine triflate formed normal lipid bilayers in aqueous phases that interacted with short, linear DNA and supercoiled plasmid DNA to form a sandwich-structured complex in which bilayers were separated by strands of DNA. DNA in a 1:1 (mol) complex with cationic lipid was shielded from the aqueous phase, but was released by neutralizing the cationic charge with anionic lipid. DNA-lipid complexes transfected DNA into cells very effectively. Transfection efficiency depended upon the form of the lipid dispersion used to generate DNA-lipid complexes; in the case of the O-ethyl derivative described here, large vesicle preparations in the liquid crystalline phase were most effective.

O-ethylphosphatidylcholine: A metabolizable cationic phospholipid which is a serum-compatible DNA transfection agent.

1,2-dioleoyl-sn-glycero-3-ethylphosphocholine was prepared in a one-step reaction from phosphatidylcholine by reaction with ethyl trifluoromethanesulfonate. This and related O-alkyl phosphatidylcholines constitute the first chemically stable triesters of biological lipid structures and the first cationic derivatives of phospholipids consisting entirely of biological metabolites linked with ester bonds. The complex of cationic phospholipid and plasmid DNA transfected cells with high efficiency. Maximum efficiency of transfection was obtained with complexes in which the positive charge was a few percent in excess over the negative charge. Modest stimulation of transfection of common cell lines was obtained by continuous culture in the presence of 10% serum. Incubation of the phospholipid complex for at least 2 h at 37 degrees C in nearly pure serum had no deleterious effects on transfection efficiency. The lipid has low toxicity; BHK cells tolerated amounts of 2 mg/2 x 10(6) cells at concentrations of 1 mg/mL. The lipid is biodegradable; it was hydrolyzed by phospholipase A(2) in vitro and was metabolized with a half-life of a few days in cells in culture. The synthetic route to cationic phospholipids is well suited to the preparation of derivatives that are tailor-made to have a wide variety of different properties.

Characterization of vascular gene transfer using a novel cationic lipid.

INTRODUCTION: Cationic liposomes are an alternative non-viral vector for gene therapy, but several factors affect transfection efficiency. A novel cationic lipid, o-ethyldioleoylphosphatidylcholinium (EDOPC), was studied for characterization of the time course and effects of lipid composition, concentration, charge ratio, mixing techniques, passage number, and stimulated state on transfection of human vascular cells, represented by human umbilical vein endothelial cells (HUVEC). METHODS: HUVEC cultures were seeded at a density of 45,000 cells/well in 24-well plates and incubated overnight. Triplicate wells were transfected with samples of EDOPC/reporter plasmid for 2 h, followed by a 24-h expression time, which was the peak expression time point in an initial time-course experiment. Measuring luciferase in cell lysates quantitated gene expression. RESULTS: Transfection of HUVEC with EDOPC was optimal with a concentration of 100 microgram lipid/well, ratio of 3:1 EDOPC:plasmid, fractional mixing of lipid and plasmid, centrifugation, and incubation in serum-free media. Transfections in sequential passages showed striking decreases in gene expression and regression analysis revealed the relationship: RLU = 120,000 - (10, 400 x passage number), r(2) = 0.947. HUVEC activated by cytokine stimulation remain susceptible to gene transfer specifically with EDOPC. SUMMARY: During transfection of HUVEC with cationic lipid species, an increase in passage number is associated with linear reduction in luciferase expression, and hence passage number must be controlled in comparative experiments. Characteristics of EDOPC may permit site-specific efficient transfection of activated human vascular cells that can be isolated from serum by mechanical methods.