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   

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.     

Effects of hemagglutinin fusion peptide on poly(ethylene glycol)-mediated fusion of phosphatidylcholine vesicles.

The effects of hemagglutinin (HA) fusion peptide (X-31) on poly(ethylene glycol)- (PEG-) mediated vesicle fusion in three different vesicle systems have been compared: dioleoylphosphatidylcholine (DOPC) small unilamellar vesicles (SUV) and large unilamellar vesicles (LUV) and palmitoyloleoylphosphatidylcholine (POPC) large unilamellar perturbed vesicles (pert. LUV). POPC LUVs were asymmetrically perturbed by hydrolyzing 2.5% of the outer leaflet lipid with phospholipase A(2) and removing hydrolysis products with BSA. The mixing of vesicle contents showed that these perturbed vesicles fused in the presence of PEG as did DOPC SUV, but unperturbed LUV did not. Fusion peptide had different effects on the fusion of these different types of vesicles: fusion was not induced in the absence of PEG or in unperturbed DOPC LUV even in the presence of PEG. Fusion was enhanced in DOPC SUV at low peptide surface occupancy but hindered at high surface occupancy. Finally, fusion was hindered in proportion to peptide concentration in perturbed POPC LUV. Contents leakage assays demonstrated that the peptide enhanced leakage in all vesicles. The peptide enhanced lipid transfer between both fusogenic and nonfusogenic vesicles. Peptide binding was detected in terms of enhanced tryptophan fluorescence or through transfer of tryptophan excited-state energy to membrane-bound diphenylhexatriene (DPH). The peptide had a higher affinity for vesicles with packing defects (SUV and perturbed LUV). Quasi-elastic light scattering (QELS) indicated that the peptide caused vesicles to aggregate. We conclude that binding of the fusion peptide to vesicle membranes has a significant effect on membrane properties but does not induce fusion. Indeed, the fusion peptide inhibited fusion of perturbed LUV. It can, however, enhance fusion between highly curved membranes that normally fuse when brought into close contact by PEG.     

Protein-induced fusion of phospholipid vesicles of heterogeneous sizes.

We have investigated the fusion of phospholipid vesicles induced by lysozyme and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Vesicles were composed of dimyristoylphosphatidylcholine/dioleoylphosphatidylethanolamine/ cholesterol (DMPC:DOPE:Chol, 2:1:1). Small unilamellar vesicles (SUV, diameter ca. 30 nm) obtained by extensive sonication or large unilamellar vesicles (LUV, diameters ranged from 100 to 400 nm) obtained by extrusion methods were used. Fusion of LUV induced by lysozyme and GAPDH was drastically decreased when the diameter of the vesicles increased over a value of 100 nm. Lysozyme effect was stopped at the aggregation step while GAPDH effect was stopped at the fusion (lipid mixing) step. Fusion of heterogeneous vesicle populations (SUV with LUV) was observed only with GAPDH and this happened only when the lipids were in the liquid-crystalline state.     

Gangliosides reduce leakage of aqueous-space markers from liposomes in the presence of human plasma

We have studied the role of glycolipids in reducing leakage of aqueous-space markers from liposomes, composed primarily of egg phosphatidylcholine, in the presence of human plasma. Liposomes were either small unilamellar (SUV) or large unilamellar (LUV). Leakage of liposome contents as affected by the incorporation into the liposomal bilayer of mono-, di-, or trisialogangliosides (GM, GD, GT) at different molar ratios in the presence or absence of cholesterol was examined. Leakage from liposomes decreased with increasing ganglioside sialic acid. Asialogangliosides had no effect on calcein leakage in the presence of plasma. The stabilizing effect of gangliosides and cholesterol was synergistic, and SUV containing 10 mol% GT and 33 mol% cholesterol had a half-life for leakage of calcein in plasma at 37 degrees C approaching 24 hours. LUV in the presence of plasma retained their contents longer than SUV, and gangliosides had an additional stabilizing effect. Phosphatidylserine and sulfatides were also capable of substituting for gangliosides in stabilizing liposomes to plasma-induced leakage. It appears that gangliosides stabilize liposomes in plasma at least in part through their ability to impart surface negative charge.     

Interaction of the polyene antibiotic amphotericin B with model membranes: differences between small and large unilamellar vesicles

The interaction of the polyene antibiotic amphotericin B (AmB) (Fig. 1) with large unilamellar vesicles (LUV) was monitored by circular dichroism (CD) and carboxyfluorescein (CF) release. LUV afford a far better model for biological membranes than small unilamellar vesicles (SUV) which have been used until now. With dimyristoyl phosphatidyl choline (DMPC) LUV (i.e., containing saturated acyl chains), a strong and not saturable binding for AmB/lipid ratios up to 0.5 was observed both above and below the phase transition temperature. Incorporation of cholesterol into the vesicles did not significantly change the interaction. With egg PC (EPC) LUV (i.e., containing unsaturated acyl chains), quite a different picture emerged: the binding reached saturation for AmB/lipid ratios of about 5 x 10(-3), a result not observed with EPC SUV. When sterols were introduced into membranes, the CD spectral features obtained in the presence of ergosterol were different from those obtained in the presence of cholesterol. Such a different behavior was not observed with SUV. We suggest that species whose CD spectrum was observed after 15 min in the presence of ergosterol-containing EPC LUV is the particular one which forms wide channels and induces a Ca2+ release. (H. Ramos, A. Attias, B.E. Cohen and J. Bolard, submitted for publication). The CF release from EPC LUV induced by AmB was very low, even at very high concentrations of the antibiotic (3 x 10(-4)M). In contrast, an important release of the fluorescent dye was observed with DMPC LUV at concentrations of approximately 10(-5)M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exchange and flip-flop of dimyristoylphosphatidylcholine in liquid-crystalline, gel, and two-component, two-phase large unilamellar vesicles

The rate and extent of spontaneous exchange of dimyristoylphosphatidylcholine (DMPC) from large unilamellar vesicles (LUV) composed of either DMPC or mixtures of DMPC/distearoylphosphatidylcholine (DSPC) have been examined under equilibrium conditions. The phase state of the vesicles ranged from all-liquid-crystalline through mixed gel/liquid-crystalline to all-gel. The exchange rate of DMPC between liquid-crystalline DMPC LUV, measured between 25 and 55 degrees C, was found to have an Arrhenius activation energy of 24.9 +/- 1.4 kcal/mol. This activation energy and the exchange rates are very similar to those obtained for the exchange of DMPC between DMPC small unilamellar vesicles (SUV). The extent of exchange of DMPC in LUV was found to be approximately 90%. This is in direct contrast to the situation in DMPC SUV where only the lipid in the outer monolayer is available for exchange. Thus, transbilayer movement (flip-flop) is substantially faster in liquid-crystalline DMPC LUV than in SUV. Desorption from gel-phase LUV has a much lower rate than gel-phase SUV with an activation energy of 31.7 +/- 3.7 kcal/mol compared to 11.5 +/- 2 kcal/mol reported for SUV. A defect-mediated exchange in gel-phase SUV, which is not the major pathway for exchange in LUV, is proposed on the basis of the thermodynamic parameters of the activation process. Surprisingly, the rates of DMPC exchange between DMPC/DSPC two-component LUV, measured over a wide range of compositions and temperatures, were found to exhibit very little dependence on the composition or phase configuration of the vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Free fatty acids modulate intermembrane trafficking of cholesterol by increasing lipid mobilities: novel 13C NMR analyses of free cholesterol partitioning

Cholesterol and free fatty acids in membranes modulate major biological processes, and their cellular metabolism and actions are often coordinately regulated. However, effects of free fatty acid on cholesterol-membrane interactions have proven difficult to monitor in real time in intact systems. We developed a novel (13)C NMR method to assess effects of free fatty acids on molecular interactions of cholesterol within--and transfer between--model membranes. An important advantage of this method is the ability to acquire kinetic data without separation of donor and acceptor membranes. Large unilamellar phospholipid vesicles (LUV) with phosphatidylcholine/cholesterol ratios of 4:1 served as cholesterol donors. Small unilamellar vesicles (SUV) made with phosphatidylcholine were acceptors. The (13)C(4)-cholesterol peak is narrow in SUV, but very broad in LUV, spectra; the increase in intensity of this peak over time monitored transfer. Oleic acid and other long chain free fatty acids [saturated (C12-18) and unsaturated (C18)] dose-dependently increased mobilities of lipids in LUV (phospholipid and cholesterol) and cholesterol transfer rates, whereas short (C8-10) and very long (C24) chain free fatty acids did not. Decreasing pH from 7.4 to 6.5 (+/-oleic acid) had no effect on cholesterol transfer, and 5 mol % fatty acyl-CoAs increased transfer rates, demonstrating greater importance of the fatty-acyl tail over the headgroup. In LUV containing sphingomyelin, transfer rates decreased, but the presence of oleic acid increased transfer 1.3-fold. These results demonstrate free fatty acid-facilitated cholesterol movement within and between membranes, which may contribute to their multiple biological effects.     

Physicochemical characterization of large unilamellar phospholipid vesicles prepared by reverse-phase evaporation

Properties of large unilamellar vesicles (LUV), composed of phosphatidylcholine and prepared by reverse-phase evaporation and subsequent extrusion through Unipore polycarbonate membranes, have been investigated and compared with those of small unilamellar vesicles (SUV) and of multilamellar vesicles (MLV). The unilamellar nature of the LUV is shown by 1H-NMR using Pr3+ as a shift reagent. The gel to liquid-crystalline phase transition of LUV composed of dipalmitoylphosphatidylcholine (DPPC) monitored by differential scanning calorimetry, fluorescence polarization of diphenylhexatriene and 90 degrees light scattering, occurs at a slight lower temperature (40.8 degrees C) than that of MLV (42 degrees C) and is broadened by about 50%. The phase transition of SUV is shifted to considerably lower temperatures (mid-point, 38 degrees C) and extends over a wide temperature range. In LUV a well-defined pretransition is not observed. The permeability of LUV (DPPC) monitored by leakage of carboxyfluorescein, increases sharply at the phase transition temperature, and the extent of release is greater than that from MLV. Leakage from SUV occurs in a wide temperature range. Freeze-fracture electron microscopy of LUV (DPPC) reveals vesicles of 0.1-0.2 micron diameter with mostly smooth fracture faces. At temperatures below the phase transition, the larger vesicles in the population have angled faces, as do extruded MLV. A banded pattern, seen in MLV at temperatures between the pretransition and the main transition, is not observed in the smaller LUV, although the larger vesicles reveal a dimpled appearance.     

Implications of surface charge and curvature for the binding orientation of Thermomyces lanuginosus lipase on negatively charged or zwitterionic phospholipid vesicles as studied by ESR spectroscopy

The triglyceride lipase (EC 3.1.1.3) Thermomyces lanuginosus lipase (TLL) binds with high affinity to unilamellar phospholipid vesicles that serve as a diluent interface for both lipase and substrate, but it displays interfacial activation on only small and negatively charged such vesicles [Cajal, Y., et al. (2000) Biochemistry 39, 413-423]. The productive-mode binding orientation of TLL at the lipid-water interface of small unilamellar vesicles (SUV) consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) was previously determined using electron spin resonance (ESR) spectroscopy in combination with site-directed spin-labeling [Hedin, E. M. K., et al. (2002) Biochemistry 41, 14185-14196]. In our investigation, we have studied the interfacial orientation of TLL when bound to large unilamellar vesicles (LUV) consisting of POPG, and bound to SUV consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC). Eleven single-cysteine TLL mutants were spin-labeled as previously described, and studied upon membrane binding using the water soluble spin-relaxation agent chromium(III) oxalate (Crox). Furthermore, dansyl-labeled vesicles revealed the intermolecular fluorescence quenching efficiency between each spin-label positioned on TLL, and the lipid membrane. ESR exposure and fluorescence quenching data show that TLL associates closer to the negatively charged PG surface than the zwitterionic PC surface, and binds to both POPG LUV and POPC SUV predominantly through the concave backside of TLL opposite the active site, as revealed by the contact residues K74C-SL, R209C-SL, and T192C-SL. This orientation is significantly different compared to that on the POPG SUV, and might explain the differences in activation of the lipase. Evidently, both the charge and accessibility (curvature) of the vesicle surface determine the TLL orientation at the phospholipid interface.     

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

Abstract

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

Factors contributing to the distribution of free fatty acids among phospholipid vesicles

The distribution of free fatty acids at equilibrium after incubation of small sonicated unilamellar vesicles (SUV) with large unilamellar vesicles (LUV) of different lipid composition has been determined. Stearic acid (SA) and oleic acid (OA) showed similar preferences for SUV and LUV of egg yolk phosphatidylcholine (EYPC). Both ionized and protonated forms of the free fatty acids (FFAs) behaved similarly with respect to the equilibrium distribution between EYPC of different size. The charge of the vesicles was found, however, to be important, since both FFAs in their ionized form preferentially associated to vesicles of phosphatidylcholine (PC) as compared with vesicles of phosphatidylglycerol (PC). While SA preferred membranes in the gel state, OA showed preference for the membrane in fluid state. The insertion of both OA and SA in phosphatidylethanolamine (PE)/phosphatidylcholine vesicles is less favourable than in vesicles of pure PC. All these data suggest that membrane lipid content may play a role in determining the distribution of free fatty acids among the membranes of a cell.     

Liposome-entrapped plasmid DNA: characterisation studies

Plasmid DNA pRc/CMV HBS (5.6 kb) (100 microg) encoding the S (small) region of hepatitis B surface antigen was incorporated by the dehydration-rehydration method into liposomes composed of 16 micromol egg phosphatidylcholine (PC), 8 micromol dioleoylphosphatidylcholine (DOPE) and 1, 2-diodeoyl-3-(trimethylammonium)propane (DOTAP) (cationic liposomes) or phosphatidylglycerol (anionic liposomes) in a variety of molar ratios. The method, entailing mixing of small unilamellar vesicles (SUV) with the DNA, followed by dehydration and rehydration, yielded incorporation values of 95-97 and 48-54% of the DNA used, respectively. Mixing of preformed cationic liposomes with 100 microg plasmid DNA also led to high complexation values of 73-97%. As expected, the association of DNA with preformed anionic liposomes was low (9%). Further work with cationic PC/DOPE/DOTAP liposomes attempted to establish differences in the nature of DNA association with the vesicles after complexation and the constructs generated by the process of dehydration/rehydration. Several lines of evidence obtained from studies on vesicle size and zeta-potential, fluorescent microscopy and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, interaction of DNA with preformed cationic SUV as above, or with cationic SUV made of DOPE and DOTAP (1:1 molar ratio; ESCORT Transfection Reagent), leads to the formation of large complexes with externally bound DNA. For instance, such DNA is accessible to and can be dissociated by competing anionic SDS molecules. However, dehydration of the DNA-SUV complexes and subsequent rehydration, generates submicron size liposomes incorporating most of the DNA in a fashion that prevents DNA displacement through anion competition. It is suggested that, in this case, DNA is entrapped within the aqueous compartments, in between bilayers, presumably bound to the cationic charges.     

Ultrasonic absorption and permeability for liposomes near phase transition

The specific ultrasonic absorption coefficient per wavelength as a function of temperature in the vicinity of the phase transition of liposomes, composed of a 4:1 mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG), of different sizes was determined using an acoustic interferometer. Small unilamellar vesicles (SUV) and multilamellar vesicles (MLV) yielded results similar to those in the literature, viz., an absorption maximum at the transition temperature. Seven intermediate sizes including several size distributions of large unilamellar vesicles (LUV) were studied, yielding information on size dependencies of the temperatures at which the peaks occur, the widths at half peak amplitude, and the peak amplitudes. All liposome sizes except the SUV exhibited approximately the same transition temperature as did the largest MLV. The widths of the peaks were inversely related to liposome size, with a strong dependence for the smallest vesicles and an approach to independence for the largest vesicles. The amplitudes of the peaks exhibited a general increase with size with two exceptions, viz., the SUV and the vesicles with average diameters of 90-100 nm. It was also found that the membrane permeability increased near the transition temperature. The temperature dependencies of ultrasonic absorption and membrane permeability are compared.     

How to measure and analyze tryptophan fluorescence in membranes properly, and why bother?

Tryptophan fluorescence is a powerful tool for studying protein structure and function, especially membrane-active proteins and peptides. It is arguably the most frequently used tool for examining the interactions of proteins and peptides with vesicular unilamellar model membranes. However, high light scattering associated with vesicular membrane systems presents special challenges. Because of their reduced light scattering compared to large unilamellar vesicles (LUV), small unilamellar vesicles (SUV) produced by sonication are widely used membrane models. Unfortunately, SUV, unlike LUV, are metastable and consequently unsuitable for equilibrium thermodynamic measurements. We present simple and easily implemented experimental procedures for the accurate determination of tryptophan (Trp) fluorescence in either LUV or SUV. Specifically, we show that Trp spectra can be obtained in the presence of up to 6 mM LUV that are virtually identical to spectra obtained in buffer alone, which obviates the use of SUV. We show how the widths and peak positions of such spectra can be used to evaluate the heterogeneity of the membrane conformation and penetration of peptides. Finally, we show how to use a reference fluorophore for the correction of intensity measurements so that the energetics of peptide partitioning into membranes can be accurately determined.     

Membrane charge and curvature determine interaction with acyl-CoA binding protein (ACBP) and fatty acyl-CoA targeting

Although acyl-CoA binding protein (ACBP) stimulates utilization of long-chain fatty acyl-CoA by a variety of membrane-bound enzymes, it is not known whether ACBP directly interacts with membranes. To test this hypothesis, mouse recombinant (mr) ACBP was engineered to contain the native mouse ACBP amino acid sequence expressed as a fusion protein at high levels (>150 mg/L) in Escherichia coli. Purification and cleavage of the fusion tag resulted in mrACBP identical to native ACBP as shown by mass (10000.5 Da) and amino acid sequence (peptide mapping after proteolysis) determined by matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectroscopy. The mrACBP was functionally active as shown by binding of cis-parinaroyl-CoA with high affinity, K(d) = 12 +/- 2 nM, at a single binding site, stimulating oleoyl-CoA utilization by microsomal glycerol-3-phosphate acyltransferase 3.2-fold and protecting oleoyl-CoA from microsomal acyl-CoA hydrolase. Direct interaction of mrACBP with membranes was demonstrated by two independent methods: (i) Circular dichroism showed an 8% increase in alpha-helix content of mrACBP in the presence of anionic phospholipid-rich, but not neutral, small unilamellar vesicles (SUV). (ii) Membrane filtration confirmed that mrACBP bound to anionic phospholipid-rich SUV but only weakly interacted with neutral SUV or large unilamellar vesicles (LUV), regardless of charge. (iii) The mrACBP-oleoyl-CoA complex transferred 2-3-fold more oleoyl-CoA to anionic phospholipid-rich SUV than to anionic phospholipid-rich LUV and neutral SUV or LUV. Conversely, mrACBP extracted less oleoyl-CoA from anionic phospholipid-rich SUV. Taken together, these data indicated for the first time that mrACBP interacted preferentially with anionic phospholipid-rich, highly curved membranes to facilitate transfer of ACBP-bound ligands.     

Lipid mixing during membrane aggregation and fusion: why fusion assays disagree

The kinetics of lipid mixing during membrane aggregation and fusion was monitored by two assays employing resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) and N-(lissamine Rhodamine B sulfonyl)phosphatidylethanolamine (Rh-PE). For the "probe mixing" assay, NBD-PE and Rh-PE were incorporated into separate populations of phospholipid vesicles. For the "probe dilution" assay, both probes were incorporated into one population of vesicles, and the assay monitored the dilution of the molecules into the membrane of unlabeled vesicles. The former assay was found to be very sensitive to aggregation, even when the internal aqueous contents of the vesicles did not intermix. Examples of this case were large unilamellar vesicles (LUV) composed of phosphatidylserine (PS) in the presence of Mg2+ and small unilamellar vesicles (SUV) composed of phosphatidylserine in the presence of high concentrations of Na+. No lipid mixing was detected in these cases by the probe dilution assay. Under conditions where membrane fusion (defined as the intermixing of aqueous contents with concomitant membrane mixing) was observed, such as LUV (PS) in the presence of Ca2+, the rate of probe mixing was faster than that of probe dilution, which in turn was faster than the rate of contents mixing. Two assays monitoring the intermixing of aqueous contents were also compared. The Tb/dipicolinic acid assay reported slower fusion rates than the 1-aminonaphthalene-3,6,8-trisulfonic acid/N,N'-p-xylylene-bis(pyridinium bromide) assay for PS LUV undergoing fusion in the presence of Ca2+. These observations point to the importance of utilizing contents mixing assays in conjunction with lipid mixing assays to obtain the rates of membrane destabilization and fusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on lung surfactant replacement in respiratory distress syndrome. Rapid film formation from binary mixed liposomes

Binary mixed liposomes were prepared from dipalmitoylphosphatidylcholine (DPPC) and a minor compound, e.g., egg phosphatidylglycerol (PG) at a ratio of 9:1. Using different preparative techniques, large unilamellar vesicles (LUV), small unilamellar vesicles (SUV) or multilamellar vesicles (MLV) were obtained and were studied with an electron microscope for morphology, with a Wilhelmy balance for spreading and surface tension lowering potential, and in the surfactant-depleted isolated rat lung for their ability to restore expiratory lung capacity. Only the simultaneous investigation of phospholipids by negative staining and thin sectioning allows unequivocal classification of liposomes. The surface-active structures prepared with the technique of Bangham et al. (Bangham, A.D., Hill, M.W. and Miller, N.G.A. (1974) in Methods in Membrane Biology (Korn, E., ed.), Vol. 1, pp. 1-68, Plenum Press, New York) at room temperature are LUV. LUV containing DPPC:PG at a ratio of 9:1 rapidly spread to a film with high surface tension lowering potential. Within 5 min after injection into the subphase they rise to the surface and form a film at the air/liquid interface able to lower the surface tension to less than 1 mN/m at compression. SUV of the same chemical composition, however, are immediately surface-active only when spread directly onto the surface. MLV exhibit poor surface activity. LUV or pure DPPC, applied onto the surface, are weakly surface active within 5 min. DPPC vesicles injected into the subphase at 37 degrees C do not adsorb to any film with surface tension lowering potential in this time. The minor compounds PE, PI, PS, PA, lysoPC enable DPPC to form surface-active films after application on saline at 37 degrees C. Removal of surfactant decreases the expiratory lung capacity of the isolated rat lung from 49.7 to 12.4% at 4 cmH2O. After substitution with natural surfactant, the expiratory lung capacity is twice that of the washed lung (25.9%), but the original distensibility of the native lung is not restituted. The effect of LUV containing DPPC:PG at a ratio of 9:1 is also remarkable (21.2%).     

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.