Membrane damage of liposomes by the mushroom toxin phallolysin

We have investigated the membrane-damaging effect of phallolysin on liposomes varying in phospholipid composition, net charge and physical constitution. Liposomes were prepared from lipids extracted from bovine or human erythrocyte ghosts. The liposomes composed of bovine lipids (the intact cell showing little sensitivity to phallolysin) were found comparably sensitive to those prepared from lipids of human red cells (these cells being of high sensitivity). In addition, artificial mixtures of lipids were used for the preparation of liposomes, consisting of (a) negatively charged phospholipids such as dicetyl phosphate or phosphatidylserine, (b) cholesterol, and (c) either sphingomyelin (as the major component of erythrocytes from ruminants) or phosphatidylcholine (as the major component of erythrocytes from non-ruminants). Again, we found only little difference in the susceptibilities of sphingomyelin- and phosphatidylcholine-containing liposomes. On the other hand, the susceptibility depended on the presence of phospholipids with negative net charges. Omittance of phosphatidylcholine or dicetyl phosphate, or replacement by the positively charged stearylamine, decreased the susceptibility by a factor of more than 20. Finally, we prepared liposomes from dicetyl phosphate, cholesterol and phosphatidylcholine in two physical states: large unilamellar and smaller multilamellar liposomes. The unilamellar liposomes were about 10-times more sensitive to phallolysin. We conclude: (1) Phallolysin damages phospholipid-membranes in the absence of receptor proteins, but high concentrations of the toxin are required. (2) Membrane damage takes place with liposomes containing phosphatidylcholine as well as those containing sphingomyelin. (3) Phallolysin damages only liposomes containing phospholipids with a negative net charge.     

Highly efficient immunoliposomes prepared with a method which is compatible with various lipid compositions

Monoclonal antibody was conjugated to N-glutaryl-phosphatidylethanolamine in the presence of octylglucoside by using N-hydroxysulfosuccinimide as a carboxyl-activation reagent. The conjugated antibody was then incorporated into liposomes by a simple dialysis method. The method is mild and is compatible with various lipid compositions of the liposomes. We have prepared immunoliposomes containing a lung endothelium-specific monoclonal antibody and showed excellent target binding (approximately 75% injected dose) of the immunoliposomes in mouse. Immunoliposomes can be prepared to contain other acidic lipids such as phosphatidylserine and various amounts of cholesterol. The presence of 20% or more cholesterol in liposomes resulted in high level of target binding. We have used in these experiments a new radioactive lipid-phase marker, 111In-DTPA-SA, which was very stable in vivo. The halflife of clearance in mouse exceeded 3 weeks.     

Interactions of the low molecular weight group of surfactant-associated proteins (SP 5-18) with pulmonary surfactant lipids

The interaction of the low molecular weight group of surfactant-associated proteins, SP 5-18, with the major phospholipids of pulmonary surfactant was studied by fluorescence measurements of liposomal permeability and fusion, morphological studies, and surface activity measurements. The ability of SP 5-18 to increase the permeability of large unilamellar lipid vesicles was enhanced by the presence of negatively charged phospholipid. The permeability of these vesicles increased as the protein concentration was raised and the pH was lowered. SP 5-18 also induced leakage from liposomes made both from a synthetic surfactant lipid mixture and from lipids separated from SP 5-18 during its purification from canine sources. When SP 5-18 was added to egg phosphatidylglycerol liposomes, the population of liposomes which became permeable leaked all encapsulated contents, while the remaining liposomes did not leak at all. The extent of leakage was higher in the presence of 3 mM calcium. SP 5-18 also induced lipid mixing between two populations of egg phosphatidylglycerol liposomes in the presence of 3 mM calcium, as monitored by resonance energy transfer between two different fluorescent lipid probes, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine. Negative-staining electron microscopy showed that the addition of SP 5-18 and 3 mM calcium produced vesicles twice the size of control egg phosphatidylglycerol liposomes. In addition, surface balance measurements revealed that the adsorption of liposomal lipids to an air/water interface was enhanced by the presence of SP 5-18, negatively charged phospholipids, and 3 mM calcium. These observations suggest a similar lipid dependence for the interactions observed in the fluorescence and adsorption experiments.     

Optimization of lipase-catalyzed synthesis of novel galactosyl ligands for selective targeting of liposomes to the asialoglycoprotein receptor

The asialoglycoprotein receptor (ASGPR) is a potential target in the search for hepatic cancer drugs. However, application of ASGPR targeting in the clinic is limited by inefficient synthetic methods for the ligand. In this study, we designed and synthesized a novel galactosylated lipid with a mono-galactoside moiety using a lipase. Then we investigated the optimal reaction conditions and analyzed the targeting ability of liposomes modified with the galactosylated lipid. In an organic phase system, different lipases were used as catalysts to synthesize (5-cholesten-3b-yl) [(4-O-β-D-galactopyranosyl)D-glucitol-6] sebacate (CHS-SE-LA). Variables in enzymatic esterification, such as the type of enzyme and solvent, were explored by single-factor experiments. Optimal reaction conditions were determined through response surface methodology. The (CHS-SE-LA)-incorporated galactosylated liposome containing fluorescent dye was then prepared by thin-film hydration and a HepG2 cell transfection test used to confirm the targeting efficiency of galactosylated liposomes to hepatic cancer cells. The structure of CHS-SE-LA was identified by electrospray ionization or ESI and nuclear magnetic resonance or NMR. Under optimal conditions, the predicted yield of CHS-SE-LA was 94.3%, and the actual experimental value was 95.6 ± 1.35%, n = 3. For HepG2 cells, the cellular fluorescence intensities of liposomes modified with CHS-SE-LA (galactosylated liposomes [GAL-FL]) were as much as 2.6-fold (P < 0.01) the control liposomes (FL). Moreover, the presence of excess galactose significantly inhibited the uptake of GAL-FL suggesting ASGPR mediated uptake. The novel galactosylated ligand was synthesized enzymatically with high efficiency under mild conditions. Liposomes containing CHS-SE-LA have great potential as drug delivery carriers for hepatocyte-selective targeting.     

Interactions of antigen-sensitized liposomes with immobilized antibody: a homogeneous solid-phase immunoliposome assay

Dioleoyl phosphatidylethanolamine (DOPE) does not form stable bilayer liposomes at room temperature and neutral pH. However, stable unilamellar liposomes could be prepared by mixing DOPE with a minimum of 12% of a haptenated lipid, N-(dinitrophenylaminocaproyl)-phosphatidylethanolamine (DNP-cap-PE). When the liposomes bound to rabbit anti-DNP IgG that had been adsorbed on a glass surface, lysis of the liposome occurred with the release of the contents into the medium as judged by the fluorescence enhancement of an entrapped self-quenching dye, calcein. On the other hand, incubation of the same liposomes with glass surfaces coated with normal rabbit IgG had little effect. In addition, free anti-DNP IgG induced aggregation of the liposomes but did not cause any dye release. Liposomes composed of dioleoyl phosphatidylcholine (DOPC) and DNP-cap-PE did not lyse when added to the glass surfaces coated with either anti-DNP IgG or normal IgG. A likely mechanism for liposome lysis is that the DNP-cap-PE laterally diffuse to the contact area between the liposome and the glass. Binding of the haptenated lipid with the immobilized and multivalent antibody trap the haptenated lipids in the contact area. As a result of lateral phase separation, lipids may undergo the bilayer to hexagonal phase transition, leading to the leakage of the entrapped dye. Because both the free hapten and the free antibody inhibited the liposome leakage, this process could be used to assay for the free hapten or antibody. We have shown that inhibition assays performed by using this principle can easily detect 10 pmol of free DNP-glycine in 40 microliter. Furthermore, by substituting human glycophorin A, a transmembrane glycoprotein, for the lipid hapten, we have demonstrated that this assay system is also applicable to detect protein antigen with a sensitivity of sub-nanogram level.     

Freeze-thaw immobilization of liposomes in chromatographic gel beads: evaluation by confocal microscopy and effects of freezing rate

Biological membranes immobilized in chromatographic gel beads constitute a multifunctional affinity matrix. Membrane protein-solute interactions and drug partitioning into the lipid bilayers can conveniently be studied. By the use of confocal laser-scanning microscopy (CLSM) the distribution of immobilized model membranes in the beads has been visualized for the first time. Freeze-thaw-immobilized liposomes in Superdex 200 gel beads were situated in a thick shell surrounding a liposome-free core. The amount of phospholipids immobilized by freeze-thawing was dependent on the temperature in the cooling bath and the type of test tube used. A bath temperature of -25 degrees C gave higher immobilization yield than freezing at -75 or -8 degrees C did. Freeze-thawing in the presence of liposomes did not affect the gel bead shape or the refractive index homogeneity of the agarose network of the beads, as shown by confocal microscopy.     

Effect of supplementation of ergosterol on miconazole action in vivo and in vitro in Candida albicans.

Ergosterol was observed to alter the lipid composition of C. albicans 3153 selectively, resulting in steep rise in ergosterol content with marginal changes in other lipids content. Supplementation of ergosterol in presence or absence of cerulenin made cells more protective towards the miconazole drug. However, when the same experiments were carried out with liposomes prepared from the lipid extracts of control and supplemented cells, the pattern obtained show a little deviation from the in vivo experiments thus indicating that in addition to lipids, other cell components like proteins also affect interaction of miconazole with C. albicans.     

Vitamin A in liposomes. Inhibition of complement binding and alteration of membrane structure.

Incorporation of vitamin A aldehyde (retinal) into liposomes had an inhibitory effect on the amount of human complement protein bound in the presence of specific antiserum. The total membrane-bound protein was directly measured on liposomes which were washed after incubation in antiserum and fresh human serum (complement). At every concentration of complement, decreased protein binding was found with liposomes which contained retinal. Binding of the third component of complement (C3) was also measured directly on washed liposomes and was found to be decreased in the presence of retinal. The diminution in protein binding due to retinal was not caused by differences in the amount of antibody bound and this was shown by two experiments. First, specific antibody protein binding to liposomes was directly measured and was essentially unaffected by retinal. Second, liposomes were prepared from lipid extracts of sheep erythrocytes. These liposomes were used as as immunoadsorbants to remove antisheep erythrocyte antibodies. The immunoadsorbant capacity was the same in both the presence and the absence of retinal. A further conclusion from these experiments was that retinal did not change the number of liposomal glycolipid antigen molecules available for antibody binding and thus presumably did not change the total number of lipid molecules present on the outer surface of the liposomes. Retinal did have an effect on the geometric structure of the liposomes. Size distribution measurements were performed in the diameter range of 1-6.35 mum by using an electronic particle size analyzer (Coulter Counter). Liposomes containing retinal were shifted toward smaller sizes and had less total surface area and volume. It was suggested that retinal-containing liposomes may have had a tighter packing of the molecules in the phospholipid bilayer. This effect of retinal on liposomal structure may have been responsible for the observed decreased binding of C3 and total complement protein.     

Estimation of the equilibrium lateral pressure in 1-palmitoyl-2-[6(pyren-1-yl)]hexanoyl-glycerophospholipid liposomes

Compression isotherms for 1-palmitoyl-2-[6(pyren-1-yl)] hexanoyl-sn-glycero-3-phosphocholine (PPHPC), -ethanolamine (PPHPE), -glycerol (PPHPG), -serine (PPHPS) and -phosphatidic acid monomethylester (PPHPM) were recorded at an argon/water interface. Thereafter, the ratios of pyrene excimer to monomer fluorescence emission intensities (Ie/Im) were determined for liposomes of these lipids and were found to be 20.15, 12.30, 11.80, 10.15 and 6.95 for the ethanolamine, choline, monomethylester, glycerol and serine derivatives, respectively. Assuming Ie/Im to depend on the reciprocal of the mean molecular area of the pyrenelipids in liposomes, equilibrium surface pressure to be the same in liposomes of these lipids regardless of the head group structure and neglecting any possible influence due to differences in the orientation of the pyrene moiety, we sought for that surface pressure value in the compression isotherms where the correlation of the reciprocal of mean molecular area in monolayers to Ie/Im values observed in liposomes was maximal. This treatment results in a value of approximately 12 mN m-1 for the equilibrium surface pressure in 1-palmitoyl-2-[6(pyren-1-yl)]hexanoyl-glycerophospholipid liposomes.     

Electrostatically mediated interactions between cationic lipid-DNA particles and an anionic surface.

In an effort to model the interaction of lipid-based DNA delivery systems with anionic surfaces, such as a cell membrane, we have utilized microelectrophoresis to characterize how electrokinetic measurements can provide information on surface charge and binding characteristics. We have established that cationic lipids, specifically N-N-dioleoyl-N,N-dimethylammonium chloride (DODAC), incorporated into liposomes prepared with 1, 2-dioleoyl-i-glycero-3-phosphoethanolamine (DOPE) or 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) at 50 mol%, change the inherent electrophoretic mobility of anionic latex polystyrene beads. Self-assembling lipid-DNA particles (LDPs), prepared at various cationic lipid to negative DNA phosphate charge ratios, effected no changes in bead mobility when the LDP charge ratio (+/-) was equal to or less than 1. Increasing the LDP concentration in a solution of 0.1% (w/v) anionic beads resulted in a charge reversal effect when a net charge of LDP to total bead charge ratio (+/-) of 1:1 was observed. LDP formulations, utilizing either DOPE or DOPC, showed similar titration profiles with a charge reversal observed at a 1:1 net LDP to bead charge ratio (+/-). It was confirmed through centrifugation studies that the DNA in the LDP was associated with the anionic latex beads through electrostatic interactions. LDP binding, rather than the binding of dissociated cationic lipids, resulted in the observed electrophoretic mobility changes of the anionic latex beads.     

Extemporaneous preparation of large unilamellar liposomes

Direct contact between lipids solubilized by octyl glucoside and Amberlite XAD-2 beads yielded large liposomes (240 nm diameter) with no residual detergent molecules, in less than 10 min. This extemporaneous preparation of liposomes was prepared with a detergent/bead ratio no higher than 0.12 (mumol/mg) and a phosphatidylcholine/phosphatidylserine/cholesterol molar ratio of 1:1:1. The liposomes were mainly unilamellar, as deduced from thin section and freeze-fracture electron micrographs and from measurement of calcein incorporation into the vesicles. The relatively large internal volume of these vesicles (8.9 l/mol lipid) accounts for the high percentage of entrapped material observed. The percentage increased with lipid concentration, but could not be increased above 20% corresponding to 20 mM total lipids.     

Pyranine as a sensitive pH probe for liposome interiors and surfaces. pH gradients across phospholipid vesicles.

Pyranine is shown to be a convenient and sensitive probe for reporting pH values, pHi, at the interior of anionic and at the outer surface of cationic liposomes. It is well shielded from the phospholipid headgroups by water molecules in the interior of anionic liposomes, but it is bound to the surface of cationic liposomes. Hydrogen ion concentrations outside the liposomes, 'bulk pH values', pHo, were measured by a combination electrode. While pHi = pHo for neutral, pHi less than pHo for anionic and pHi greater than pHo for cationic liposomes prepared in 5.0 . 10(-3) M phosphate buffers. pKa values for the ionization of pyranine were 7.22 +/- 0.04 and 6.00 +/- 0.05 in water and at the external surface of cationic liposomes. The surface potential for cationic liposomes containing dipalmitoyl-DL-alpha-phosphatidylcholine, cholesterol and octadecylamine in the molar ratio of 1.00 : 0.634 : 1.01, were calcuated to be +72.2 mV. Proton permeabilities were measured for single and multicompartment anionic liposomes. Transfer of anionic liposomes prepared at a given pH to a solution of different pH resulted in a pH gradient if sodium phosphate or borate were used as buffers. In the presence of sodium acetate proton equilibration is promptly established.     

Immunoblotting assays for keratan sulfate

We have bonded glass microbeads (425-600 microm diameter) to the inner walls of polypropylene microcentrifuge tubes. In addition to increasing the surface area of the tubes manyfold, the beads provide surface Si groups which can be reacted with a silane compound such as aminopropyltriethoxysilane, yielding a free amino group. The amino group is reacted with another cross-linking reagent, for example, the homobifunctional compound dimethyl suberimidate, which can form a covalent bond with amine groups of proteins. After binding protein A or G to the dimethyl suberimidate, the beads were used to immunoprecipitate proteins from cell extracts; we show that the protein A/G-coated glass beads yield similar amounts of immunoprecipitated proteins as a standard method using protein A- or G-agarose beads, but with fewer contaminating proteins. In addition, we show that when immunoprecipitating Ras from cell extracts and measuring the amounts of Ras-bound GTP and GDP, the new method yielded higher guanine nucleotide levels than protein G-agarose beads, suggesting that it caused less denaturation of Ras. Because the glass beads are bonded to the walls of the tubes, the immunoprecipitates can be washed rapidly and efficiently, and we show that 20-30 tubes can be washed in 1/10 the time required to wash immunoprecipitates on protein A- or G-agarose beads.     

Spreading of liposomes at the air/water interface

Two types of film structure are formed when liposomes are spread at the air/water interface. At zero surface pressure, there is a slow transformation of the closed bilayered structure into a lipid monolayer. The internal content of the liposomes is released into the aqueous subphase. In contrast, when multilamellar liposomes are spread against a surface pressure, they retain their internal content at the air/water interface by forming multilayered structures. Among the liposomes which dipped through the interface an important fraction loses its internal content. During the spreading process at zero surface pressure, it seems that the outer layer of the liposome spreads with a better yield as compared with the inner layer. It is possible to use this spreading technique to determine the asymmetrical distribution of lipids across bilayers.     

Divalent cation enhancement of the agglutinability by soyabean lectin of liposomes prepared from total lipid of erythrocytes and of erythrocyte membranes

CaCl₂ or MgCl₂ but not NaCl enhances the soyabean lectin-induced agglutination of liposomes prepared from total lipids of erythrocyte membranes. The addition of purified phosphatidylserine to the total lipids of erythrocyte membranes before the formation of liposomes inhibits lectin-induced agglutinability of the preparation in the absence of CaCl₂, but not in its presence. When preformed phosphatidylserine liposomes are added to liposomes of total lipids of erythrocyte ghosts, they do not inhibit agglutination, indicating that phosphatidylserine does not inhibit the lectin directly. CaCl₂ or MgCl₂ but not NaCl also stimulates the soyabean lectin-induced agglutination of human erythrocyte membranes.Electron micrographs indicate that the liposome preparations are multilamellar and separate even in the presence of CaCl₂. When such liposomes are treated with lectin with or without CaCl₂, the electron micrographs show significant agglutination without apparent fusion. The reversal of the agglutination of liposomes by specific sugars followed by turbidimetric and electron microscopic techniques supports the conclusion that CaCl₂ stimulated lectin-induced agglutination is unaccompanied by fusion.The stimulation by divalent cations of lectin-induced agglutination of erythrocyte ghosts or of our liposomes may be due to a decrease in apparent surface charge of these membrane systems.     

The elution profile of immobilized liposome chromatography: determination of association and dissociation rate constants

The interaction of lipophilic cations, tetraphenylphosphonium and triphenylphosphonium homologues with liposomes was investigated using immobilized liposome chromatography (ILC). Large unilamellar liposomes with a mean diameter of 100 nm were stably immobilized in chromatographic gel beads by avidin-biotin. The distribution coefficient calculated from (Ve-V0)/Vs (Ve, retention volume; V0, the void volume; Vs, the stationary phase volume) was found to be independent of flow rate, injection amount and gel bed volume, which is consistent with chromatograph theory. The relationship between the bandwidth and solvent flow rate did not follow band-broadening theories reported thus far. We hypothesized that the solvent might be forced to produce large eddies, spirals or turbulent flow due to the presence of liposomes fixed in the gel. Therefore, we developed a new theory for ILC elution: The column is composed of a number of thin disks containing liposomes and solution, and within each disk the solution is well mixed. This theory accounts for our results, and we were able to use it to estimate the rate constants of association and dissociation of the phosphonium to/from liposomes.     

Studies on osmotic stability of liposomes prepared with bacterial membrane lipids by carboxyfluorescein release.

The authors measured the osmotic stability of liposomes prepared with membrane lipids of bacteria, using the osmotic-shock release of entrapped carboxyfluorescein as an indicator. The sub-second physical changes of liposomes suspended in a solution of low osmotic pressure were examined by stopped flow spectrophotometry. The entrapped carboxyfluorescein was released when the liposomes burst on inflow of excess water. Liposomes prepared with the lipids of a stable Staphylococcus aureus L-form strain were more resistant to low osmotic pressure than those prepared from the wild strain of S. aureus, and liposomes prepared from Mycoplasma orale were even more resistant. Cardiolipin enhanced the lipid membrane stability in S. aureus and cholesterol in M. orale. The stability of lipid membranes to low osmotic pressure could be precisely determined by the present method.     

Effect of sugar-phosphate mixtures on the stability of DPPC membranes in dehydrated systems

The stabilizing role of sugars on dehydrated membranes is well established. The formation of a glassy matrix and the direct interaction between the sugars and the lipids are some of the mechanisms proposed to be involved in this stabilizing effect. Phospholipidic systems have been studied extensively as models for biological membranes and also due to the practical applications of liposomes as vehicles for drug delivery. In this work, we evaluate the effect of sugar-phosphate mixtures on the transition temperature of dehydrated 1,2-dipalmitoylphosphatidylcholine, and also examine some physical characteristics of these mixtures, such as the glass transition temperature and water sorption properties. The addition of phosphate salts to sugar systems has several interesting features that merit its consideration in formulations to protect dehydrated labile biomaterials. In particular, sucrose-phosphate mixtures provide an interesting alternative to pure saccharide formulations due to their high glass transition temperatures and their increased ability to maintain a low melting transition temperature in the presence of small amounts of water.     

Photolabeling of myelin basic protein in lipid vesicles with the hydrophobic reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine

The hydrophobic photolabel 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine([125I]TID) was used to label myelin basic protein or polylysine in aqueous solution and bound to lipid vesicles of different composition. Although myelin basic protein is a water soluble protein which binds electrostatically only to acidic lipids, unlike polylysine it has several short hydrophobic regions. Myelin basic protein was labeled to a significant extent by TID when in aqueous solution indicating that it has a hydrophobic site which can bind the reagent. However, myelin basic protein was labeled 2-4-times more when bound to the acidic lipids phosphatidylglycerol, phosphatidylserine, phosphatidic acid, and cerebroside sulfate than when bound to phosphatidylethanolamine, or when in solution in the presence of phosphatidylcholine vesicles. It was labeled 5-7-times more than polylysine bound to acidic lipids. These results suggest that when myelin basic protein is bound to acidic lipids, it is labeled from the lipid bilayer rather than from the aqueous phase. However, this conclusion is not unequivocal because of the possibility of changes in the protein conformation or degree of aggregation upon binding to lipid. Within this limitation the results are consistent with, but do not prove, the concept that some of its hydrophobic residues penetrate partway into the lipid bilayer. However, it is likely that most of the protein is on the surface of the bilayer with its basic residues bound electrostatically to the lipid head groups.     

Preparation and characterization of F-protein vesicles isolated from Sendai virus by means of octyl glucoside

We have demonstrated that Triton X-100 is always present in F-protein vesicles at concentrations that can provoke cell lysis. In order to avoid any misinterpretation of the fusogenic capacity of this protein, we solubilized the Sendai virus using octyl glucoside, which can be totally removed from the F protein preparation in less than 16 h by dialysis in the presence of absorbent beads. F-glycoprotein preparations preserved their ability to lyse erythrocytes in the presence of lectins and to induce cell-vesicle fusion as demonstrated by ESR studies. These vesicles were characterized by electron microscopy and SDS-polyacrylamide gel electrophoresis. Lipid analysis of these preparations by thin-layer chromatography indicated that they had the same proportion of lipids as virus envelopes, with slight variations in the sphingomyelin content and the cholesterol/phospholipid molar ratio. F-protein vesicles of different sizes can be obtained by adding exogenous lipids before detergent removal. The hemolytic activity of the vesicles was retained over a large range of lipid concentrations. We conclude that F-protein vesicles prepared with octyl glucoside are convenient tools for studying the fusogenic mechanism of this protein and improving the fusion process between liposomes and cells.