DNA release from lipoplexes by anionic lipids: correlation with lipid mesomorphism, interfacial curvature, and membrane fusion

DNA release from lipoplexes is an essential step during lipofection and is probably a result of charge neutralization by cellular anionic lipids. As a model system to test this possibility, fluorescence resonance energy transfer between DNA and lipid covalently labeled with Cy3 and BODIPY, respectively, was used to monitor the release of DNA from lipid surfaces induced by anionic liposomes. The separation of DNA from lipid measured this way was considerably slower and less complete than that estimated with noncovalently labeled DNA, and depends on the lipid composition of both lipoplexes and anionic liposomes. This result was confirmed by centrifugal separation of released DNA and lipid. X-ray diffraction revealed a clear correlation of the DNA release capacity of the anionic lipids with the interfacial curvature of the mesomorphic structures developed when the anionic and cationic liposomes were mixed. DNA release also correlated with the rate of fusion of anionic liposomes with lipoplexes. It is concluded that the tendency to fuse and the phase preference of the mixed lipid membranes are key factors for the rate and extent of DNA release. The approach presented emphasizes the importance of the lipid composition of both lipoplexes and target membranes and suggests optimal transfection may be obtained by tailoring lipoplex composition to the lipid composition of target cells.     

Interaction of phosphatidylcholine liposomes and plasma lipoproteins with sheep erythrocyte membranes: Preferential transfer of phosphatidylcholine containing unsaturated fatty acids

The interaction of sheep erythrocyte membranes with phosphatidylcholine vesicles (liposomes) or human plasma lipoproteins is described. Isolated sheep red cell membranes were incubated with liposomes containing [¹⁴C]phosphatidylcholine or [^3H]phosphatidylcholine in the presence of EDTA. A time-dependent uptake of phosphatidylcholine into the membranes could be observed. The content of this phospholipid was increased from 2 to 5%. The rate of transfer was dependent on temperature, the amount of phosphatidylcholine present in the incubation mixture and on the fatty acid composition of the liposomal phosphatidylcholine. A possible adsorption of lipid vesicles to the membranes could be monitored by adding cholesteryl [¹⁴C]oleate to the liposomal preparation. As cholesterylesters are not transferred between membranes [1], it was possible to differentiate between transfer of phosphatidylcholine molecules from the liposomes into the membranes and adsorption of liposomes to the membranes. The phosphatidylcholine incorporated in the membranes was isolated, and its fatty acids were analysed by gas chromatography. It could be shown that there was a preferential transfer of phosphatidylcholine molecules containing two unsaturated fatty acids.     

The stimulation by sodium fluoride of plasma-membrane Ca2+ inflow in isolated hepatocytes. Evidence that a GTP-binding regulatory protein is involved in the hormonal stimulation of Ca2+ inflow.

Bilirubin may be transported within intracellular membranes of the hepatocyte and may undergo membrane-membrane transfer to gain access to the conjugating enzyme UDP-glucuronyltransferase in the endoplasmic reticulum. We have demonstrated previously that the lipid composition of liposomal membranes incorporating bilirubin substrate influences the rate of transfer and glucuronidation of bilirubin by hepatic microsomes. To examine the mechanism(s) of substrate transfer, we incorporated radiolabelled bilirubin into small unilamellar model membranes of egg phosphatidylcholine or natural phospholipids in the proportions present in native hepatic microsomes. The rate at which bilirubin was transferred to rat liver microsomes and glucuronidated was then examined in the presence of various endogenous compounds that promote membrane fusion. For bilirubin substrate in membranes of egg phosphatidylcholine, the addition of Ca2+ (2 mM) increased the microsomal glucuronidation rate, whereas retinol enhanced microsomal conjugation rates for bilirubin in membranes of both lipid compositions. When the transfer of [3H]bilirubin from dual-labelled liposomes to microsomes was enhanced by Ca2+ or retinol, there was no associated increase in [14C]phospholipid transfer. Thus it appears likely that bilirubin is transferred to the endoplasmic reticulum by rapid cytosolic diffusion or membrane-membrane collisions, rather than by membrane fusion; this process may be modulated by changes in the lipid microenvironment of the substrate or the effective intracellular concentrations of Ca2+ or retinol. The observation that polymyxin B induced concomitant membrane-membrane transfer of [3H]bilirubin and [14C]phospholipid suggests that under certain circumstances membrane fusion or aggregation may promote the movement of lipophilic substrates in hepatocytes.     

Liposomes as model for taste cells: receptor sites for bitter substances including N-C=S substances and mechanism of membrane potential changes

Various bitter substances were found to depolarize liposomes. The results obtained are as follows: (1) Changes in the membrane potential of azolectin liposomes in response to various bitter substances were monitored by measuring changes in the fluorescence intensity of 3,3'-dipropylthiocarbocyanine iodide [diS-C3(5)]. All the bitter substances examined increased the fluorescence intensity of the liposome-dye suspension, which indicates that the substances depolarize the liposomes. There existed a good correlation between the minimum concentrations of the bitter substances to depolarize the liposomes and the taste thresholds in humans. (2) The effects of changed lipid composition of liposomes on the responses to various bitter substances vary greatly among bitter substances, suggesting that the receptor sites for bitter substances are multiple. The responses to N-C=S substances and sucrose octaacetate especially greatly depended on the lipid composition; these compounds depolarized only liposomes having certain lipid composition, while no or hyperpolarizing responses to these compounds were observed in other liposomes examined. This suggested that the difference in "taster" and "nontaster" for these substances can be explained in terms of difference in the lipid composition of taste receptor membranes. (3) It was confirmed that the membrane potential of the planar lipid bilayer is changed in response to bitter substances. The membrane potential changes in the planar lipid bilayer as well as in liposomes in response to the bitter substances occurred under the condition that there is no ion gradient across the membranes. These results suggested that the membrane potential changes in response to bitter substances stem from the phase boundary potential changes induced by adsorption of the substances on the hydrophobic region of the membranes.     

Cholesterol-dependent insertion of glycosylphosphatidylinositol-anchored enzyme

Evidence is now accumulating that the plasma membrane is organized in different lipid and protein subdomains. Thus, glycosylphosphatidylinositol (GPI)-anchored proteins are proposed to be clustered in membrane microdomains enriched in cholesterol and sphingolipids, called rafts.By a detergent-mediated method, alkaline phosphatase, a GPI-anchored enzyme, was efficiently inserted into the membrane of sphingolipids- and cholesterol-rich liposomes as demonstrated by flotation in sucrose gradients. We have determined the enzyme extraluminal orientation. Using defined lipid components to assess the possible requirements for GPI-anchored protein insertion, we have demonstrated that insertion into membranes was cholesterol-dependent as the cholesterol addition increased the enzyme incorporation in simple phosphatidylcholine liposomes.     

Cholesterol-dependent insertion of glycosylphosphatidylinositol-anchored enzyme

Evidence is now accumulating that the plasma membrane is organized in different lipid and protein subdomains. Thus, glycosylphosphatidylinositol (GPI)-anchored proteins are proposed to be clustered in membrane microdomains enriched in cholesterol and sphingolipids, called rafts.By a detergent-mediated method, alkaline phosphatase, a GPI-anchored enzyme, was efficiently inserted into the membrane of sphingolipids- and cholesterol-rich liposomes as demonstrated by flotation in sucrose gradients. We have determined the enzyme extraluminal orientation. Using defined lipid components to assess the possible requirements for GPI-anchored protein insertion, we have demonstrated that insertion into membranes was cholesterol-dependent as the cholesterol addition increased the enzyme incorporation in simple phosphatidylcholine liposomes.     

Incorporation of adenylate cyclase into membranes of giant liposomes using membrane fusion with recombinant baculovirus-budded virus particles

Recombinant transmembrane adenylate cyclase (AC) was incorporated into membranes of giant liposomes using membrane fusion between liposomes and baculovirus-budded virus (BV). AC genes were constructed into transfer vectors in a form fused with fluorescent protein or polyhistidine at the C-terminus. The recombinant BVs were collected by ultracentrifugation and AC expression was verified using western blotting. The BVs and giant liposomes generated using gentle hydration were fused under acidic conditions; the incorporation of AC into giant liposomes was demonstrated by confocal laser scanning microscopy through the emission of fluorescence from their membranes. The AC-expressing BVs were also fused with liposomes containing the substrate (ATP) with/without a specific inhibitor (SQ 22536). An enzyme immunoassay on extracts of the sample demonstrated that cAMP was produced inside the liposomes. This procedure facilitates direct introduction of large transmembrane proteins into artificial membranes without solubilization.     

Increased affinity of liposomes derived from total spleen and liver lipids to spleen cells

The interaction of liposomes derived from total lipids of mouse spleen and liver with mouse spleen cells was studied. It was shown that the binding of these liposomes is much higher than the binding of liposomes obtained from a model lipid mixture--phosphatidylcholine--phosphatidylethanolamine--cholesterol (2:1:1). Adherent and nonadherent spleen cells were found to have affinity for liposomes derived from total lipids of spleen or liver. Removal of gangliosides and protein contaminants from the liposomes derived from total spleen lipids caused an increased binding of liposomes to spleen cells. Multilamellar liposomes bound more effectively to ultrasonicated vesicles having a homologous lipid composition than the liposomes with a different lipid composition. The increased affinity of liposomes derived from total lipids of spleen or liver for spleen cells may account for the identical fluidity of the lipid bilayer of liposomes and plasma membranes of spleen cells.     

Fluorescent probe studies on binding of glyceraldehyde-3-phosphate dehydrogenase to phosphatidylinositol liposomes. Further evidence for conformational changes

Glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle can be absorbed on charged lipid bilayers by electrostatic forces. Upon binding to phosphatidylinositol liposomes the enzyme modifies its conformational state as it is shown by resonance energy transfer experiments. In the presence of 2-mercaptoethanol o-phthaldialdehyde reacts with amino groups of the protein and the covalently bound fluorophore is an acceptor of excitation energy transferred from tryptophanyl residues of the protein. The observed decrease of energy transfer efficiency upon binding to phosphatidylinositol liposomes is compared with the influence of the urea on the fluorescence spectra of the labelled protein. Significance of conformational changes of the enzyme upon adsorption on liposomes in the regulating function of cell membranes is discussed.     

Endocytosis of liposomes by Entamoeba histolytica and transport of encapsulated molecules toward the cytoplasm

We have examined the interaction of a highly phagocytosing cell: Entamoeba histolytica with liposomes of different lipid compositions, and followed, by a semi-quantitative method, the intracellular fate of the entrapped molecules. Liposomes containing a small molecule, 6-carboxyfluorescein, are first phagocytosed. Then the encapsulated compound migrates from the vacuoles to the cytoplasm. Liposomes containing macromolecular substances, such as fluorescent albumin or ferritin, are also phagocytosed, but the encapsulated molecules remain within the vacuoles. We conclude that the transfer of carboxyfluorescein does not involve a fusion between liposomes and vacuoles, but more likely occurs via diffusion through membranes. The lipid composition of the liposomes does not affect phagocytosis of liposomes. In contrast, oleic acid greatly increases the transfer of carboxyfluorescein from vacuole to cytoplasm.     

Effect of lipid composition on HVJ-mediated fusion of glycophorin liposomes to erythrocytes

We previously reported that liposomes containing glycophorin or gangliosides, both of which were isolated from human erythrocytes, are efficiently fused to erythrocyte membranes in the presence of HVJ (Umeda, M. et al., J. Biochem. 94, 1955-1966 (1983), and Virology 133, 172-182 (1984]. In the present work, the effect of lipid composition in glycophorin liposomes on their sensitivity to fusion with erythrocytes was studied. Very little fusion occurred when glycophorin liposomes composed of dipalmitoylphosphatidylcholine-dicetylphosphate (9:1), dimyristoylphosphatidylcholine-dicetylphosphate (9:1), or egg yolk phosphatidylcholine-dicetylphosphate (9:1) were incubated with human erythrocytes in the presence of HVJ at 37 degrees C. Addition of cholesterol into these liposomal membranes greatly enhanced the sensitivity of the liposomes to fusion. The presence of phosphatidic acid and phosphatidylethanolamine in liposomes also enhanced the sensitivity, whereas the presence of lysophosphatidylcholine had no significant effect on the ability of the liposomes to fuse. The fusion efficiency of liposomes was also enhanced by the presence of glucosylceramide. Change of lipid composition in liposomes had, however, no appreciable influence on the HVJ-mediated binding of liposomes to erythrocytes, suggesting that the interaction between HANA protein of HVJ and glycophorin in liposomes was not affected by the lipid composition of the liposomes.     

Stability of protein-encapsulating DRV liposomes after freeze-drying: A study with BSA and t-PA

Stability of protein-encapsulating DRV (dried-rehydrated vesicle) liposomes is evaluated after freeze-drying vesicles in presence (or not) of trehalose. Two proteins, bovine serum albumin (BSA) and tissue-type plasminogen activator (t-PA), are used, and protein-encapsulating liposomes with different lipid compositions are prepared by DRV technique. Encapsulation efficiencies are calculated, after measuring BSA with a fluorescence technique and t-PA's amidolytic activity toward a chromogenic substrate.Experimental results show that encapsulation of BSA in vesicles ranges between 35 and 53% of the protein and is only slightly affected by lipid composition. For t-PA, entrapment efficiencies are lower, ranging between 2 and 16%, while lipid composition has substantial effect on entrapment (cholesterol inclusion is very important). After freeze-drying, some lipid compositions remain stable, retaining most of initially entrapped proteins, while others do not, but they may be stabilized by trehalose. In the case of BSA, liposome behavior cannot be explained based on lipid membrane rigidity (more rigid = more stable). This may be connected with previously demonstrated interactions of BSA with membranes. Oppositely, t-PA behavior is more predictable, meaning that the lipid composition selected for the specific therapeutic application determines the need for cryoprotectant addition before freeze-drying t-PA containing DRV liposomes, perhaps due to the fact that under conditions applying minimum or no interactions between t-PA and lipid membranes occur.Thereby, interactions between proteins and membranes determine not only the encapsulation efficiency but also the need for cryopreservation of liposomal protein formulations.     

Membrane fusion by proline-rich Rz1 lipoprotein, the bacteriophage lambda Rz1 gene product.

The fusogenic properties of Rz1, the proline-rich lipoprotein that is the bacteriophage lambda Rz1 gene product, were studied. Light scattering was used to monitor Rz1-induced aggregation of artificial neutral (dipalmitoylphosphatidylcholine/cholesterol) and negatively charged (dipalmitoylphosphatidylcholine/cholesterol/dioleoylphosphatidylserin e) liposomes. Fluorescence assays [the resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)dihexadecanol-sn-glycero-3-phosphoethanolamine lipid fluorescent probes, as well as fluorescent complex formation between terbium ions and dipicolinic acid encapsulated in two liposome populations and calcein fluorescence] were used to monitor Rz1-induced lipid mixing, contents mixing and leakage of neutral and negatively charged liposomes. The results demonstrated that Rz1 caused adhesion of neutral and negatively charged liposomes with concomitant lipid mixing; membrane distortion, leading to the fusion of liposomes and hence their internal content mixing; and local destruction of the membrane accompanied by leakage of the liposome contents. The use of artificial membranes showed that Rz1 induced the fusion of membranes devoid of any proteins. This might mean that the proline stretch of Rz1 allowed interaction with membrane lipids. It is suggested that Rz1-induced liposome fusion was mediated primarily by the generation of local perturbation in the bilayer lipid membrane and to a lesser extent by electrostatic forces.     

Stability of Protein-Encapsulating DRV Liposomes After Freeze-Drying: A Study with BSA and t-PA

Stability of protein-encapsulating DRV (dried-rehydrated vesicle) liposomes is evaluated after freeze-drying vesicles in presence (or not) of trehalose. Two proteins, bovine serum albumin (BSA) and tissue-type plasminogen activator (t-PA), are used, and protein-encapsulating liposomes with different lipid compositions are prepared by DRV technique. Encapsulation efficiencies are calculated, after measuring BSA with a fluorescence technique and t-PA's amidolytic activity toward a chromogenic substrate.

Experimental results show that encapsulation of BSA in vesicles ranges between 35 and 53% of the protein and is only slightly affected by lipid composition. For t-PA, entrapment efficiencies are lower, ranging between 2 and 16%, while lipid composition has substantial effect on entrapment (cholesterol inclusion is very important). After freeze-drying, some lipid compositions remain stable, retaining most of initially entrapped proteins, while others do not, but they may be stabilized by trehalose. In the case of BSA, liposome behavior cannot be explained based on lipid membrane rigidity (more rigid?=?more stable). This may be connected with previously demonstrated interactions of BSA with membranes. Oppositely, t-PA behavior is more predictable, meaning that the lipid composition selected for the specific therapeutic application determines the need for cryoprotectant addition before freeze-drying t-PA containing DRV liposomes, perhaps due to the fact that under conditions applying minimum or no interactions between t-PA and lipid membranes occur.

Thereby, interactions between proteins and membranes determine not only the encapsulation efficiency but also the need for cryopreservation of liposomal protein formulations.     

Investigation of mechanisms of synaptic vesicles fusion with acceptor membranes in the model of exocytosis

Fusion of synaptic vesicles with various target membranes was investigated on the cell-free model system that reflects the final step of exocytosis. Plasma membranes, synaptic vesicles and liposomes were used as acceptor membranes. The process of membrane fusion was triggered by Ca2+. We have demonstrated that synaptic vesicles are prone to fuse with liposomes in buffer solution. This process was strongly dependent on ionic force of medium and phospholipid composition of liposomes. Cytosolic proteins of synaptosomes inhibited the fusion of synaptic vesicles with liposomes, while these were required for the fusion of synaptic vesicles with native membrane structures. Trypsinolysis of acceptor membranes markedly inhibited the fusion response. It means protein components of target membrane are necessary for realization of the final step of exocytosis.     

Fusion of artificial lipid membranes induced by synthetic "fusion peptide" of arenaviruses

The fusing capacity of lipid membranes of a synthetic 23-member peptide was studied. This hydrophobic peptide represents an analog of a predicted functional site ("fusion peptide") of the GP2 envelope protein of the Lassa virus (family Arenaviridae). Fusion of small monolayer liposomes was detected by the method of resonance energy transfer between the fluorescent derivatives of the lipid, NBD-PE (donor) and Rd-PE (acceptor). Using this peptide, the pH-dependent fusing activity was found in liposomes having different phospholipid composition. The rate and efficiency of liposome fusion increased with a decrease in pH and the lipid/peptide ratio as well as with a temperature increase. The increase in the ionic strength and Ca2+ concentration in the reaction mixture led to the inhibition of the peptide-induced fusion of liposomes. Neither the phospholipid charge, nor the transmembrane proton gradient of liposomes had any appreciable effect on the kinetics of the peptide-induced fusion. Neutralization of the medium in the course of the fusion reaction sharply decelerated, whereas repeated acidification activated this process. This finding suggests that peptide protonation plays a role in fusion reactions. It was suggested that acidification causes conformational changes in the peptide structure, thus activating the peptide-induced fusion of liposomes. The fusing capacity of the predicted Lassa virus fusion peptide is similar to that of viruses characterized by a pH-dependent step at the initial stages of the viral infection.     

Optimizing and quantifying fusion of liposomes to mammalian sperm using resonance energy transfer and flow cytometric methods

BACKGROUND: Liposomes are used to carry pharmaceutical agents and to alter the lipid composition of cell membranes. This study compared resonance energy transfer (RET), fluorescence dequenching, and flow cytometry as monitors and quantifiers of fusion between liposomes and mammalian spermatozoa. METHODS: Preliminary experiments used RET to determine the optimum sperm concentration for fusion of DL-alpha-phosphatidylcholine dipalmitoyl (PC)/DL-alpha-phosphatidylethanolamine dipalmitoyl (PE) liposomes at 35 degrees C +/- 5 mM Ca2+. Microscopy confirmed the fusion of liposomes, not just adhesion (n = 3). Dequenching tested the time-dependent fusion of liposomes of two different lipid compositions to sperm, both, (n = 3) +/- 1 mM Ca2+ and (n = 3) without Ca2+ at two sperm concentrations. Finally, flow cytometry absolutely quantified the percentage of sperm fusing to liposomes at different liposome-to-sperm ratios (n = 4) and with sperm from different donors (n = 3). RESULTS: RET detected fusion of liposomes with sperm and microscopy confirmed the interaction to be true fusion. Dequenching detected more fusion of liposomes with sperm at 100 x 10(6) sperm per milliliter than at lower concentrations (P < 0.05). Fusion dynamics differed with lipid composition but Ca2+ had no effect. Flow cytometry reliably quantified the percentage of sperm fusing with liposomes, which varied from bull to bull (P < 0.05). CONCLUSION: Liposome fusion with mammalian sperm membranes can be quantified cytometrically and varies with lipid composition, sperm-to-liposome ratio, and individual animals.     

Competition of solid and fluid liposomes for binding and metabolism with lipids from the cell surface

The competitive behavior of solid vs. fluid liposomes in liposome-cell adsorption and cell-to-liposome lipid transfer processes was investigated with L cells and FBT epithelial sheets. Binding and transfer experiments have demonstrated that: solid liposomes adhere to the cell surface as integral vesicles retaining the entrapped substance; fluid liposomes are partly disintegrated at the cell surface with concomitant entry of entrapped substances into the cytoplasm, while their lipids remain on the cell surface; fluid liposomes that escape lysis dissociate from the cell taking away cell lipid molecules. No lipid transfer occurs between the plasma membrane and solid liposomes. Cell-bound solid liposomes interfere with the transfer of cell lipids to fluid liposomes, while these in turn inhibit the binding of solid liposomes to the cell surface.     

Effect of lipid composition of liposomes on their sensitivity to peroxidation

The effect of lipid composition of liposomes on peroxidation induced by ferrous ion and ascorbate was examined. Temperature affects the sensitivity of liposomes; the peroxidation rate was increased with increase of the incubation temperature. With liposomes consisting of 1-palmitoyl-2-arachidonyl phosphatidylcholine (substrate) and a peroxidation-insensitive lipid, 1-palmitoyl-2-oleoyl phosphatidylcholine, peroxidation was dependent on the density of the substrate. No appreciable peroxidation was observed with liposomes containing less than 10 mol% of the substrate at 37 degrees C. When 1 mol substrate was mixed with 9 mol dimyristoyl phosphatidylcholine, peroxidation occurred below 10 degrees C, but not above 20 degrees C. Above 20 degrees C, the substrates should be located homogeneously on the membranes, whereas they should be clustered below 10 degrees C, since the gel-liquid crystalline phase transition temperature of matrix membrane of dimyristoylphosphatidylcholine was 17-21 degrees C. Peroxidation of liposomes consisting of 1-palmitoyl-2-arachidonyl phosphatidylcholine was also suppressed by cholesterol. These findings indicate that the lateral distribution as well as the density of the substrate on membranes affects the sensitivity of the substrate to peroxidation. It was also found that alpha-tocopherol is preferentially located in the 1-palmitoyl-2-arachidonyl phosphatidylcholine-rich regions of membranes consisting of mixed phospholipids, and efficiently suppresses peroxidation of liposomal lipids.