Arsonoliposomes: effect of lipid composition on their stability and morphology

The influence of the lipid composition of arsonoliposomes on their membrane integrity was investigated to evaluate whether it is possible to combine their action with drugs that can be encapsulated in their aqueous interior. This was investigated by measuring the retention of vesicle-encapsulated calcein (100 mM) during incubation, in the absence and presence of serum proteins. Liposomes containing various concentrations of arsonolipid (with the palmitoyl side chain) as well as egg-lecithin (phosphatidylcholine, PC) and cholesterol (lipid/chol 2:1 mol:mol) were prepared. In some experiments, PC was replaced by the synthetic phospholipid DSPC. All PC/arsonoliposomes tested are stable after 24 h of incubation in buffer at 37 degrees C. After incubation in the presence of serum proteins, arsonoliposomes that contain low amounts of arsonolipid (up to 5 mol% of the lipid content without cholesterol) are stable, whereas increased release of calcein is observed when vesicle arsonolipid concentration is raised (from 5 to 15 mol%). Further increase of arsonolipid content results in immediate decrease of calcein latency while the remaining calcein is rapidly released during incubation. DSPC/arsonoliposomes are comparably more stable, and membrane integrity is independent of the vesicle arsonolipid content, in the range investigated (15-40 mol% of the lipid content without cholesterol). Thereby, we conclude that more stable arsonoliposomes that incorporate high arsonolipid concentrations may be produced when PC is replaced by DSPC. The latter arsonoliposomes provide a system that may be used for combining arsonolipid activity with the activity of other drugs.     

Incorporation of PEG-lipids in arsonoliposomes results in formation of highly stable arsenic-containing vesicles

We investigated the effect of pegylation on the physical stability, morphology and membrane integrity of arsonoliposomes. Arsonoliposomes composed of distearoylglycerophosphocholine (DSPC), cholesterol (Chol) and the palmitoyl side chain arsonolipid (with concentrations ranging from 0 mol% [DSPC/Chol vesicles] to 53 mol% of total lipid) containing either 4 or 8 mol% DPPE-PEG2000 or DSPE-PEG2000, were prepared by sonication. Arsonoliposome membrane integrity was evaluated by measuring the retention of encapsulated calcein in vesicles (during incubation in buffer or fetal calf serum [FCS]) while physical stability was evaluated by measuring vesicle dispersion turbidity (during incubation in water or CaCl(2)). Vesicle morphology was studied by cryo-electron microscopy. Experimental results show that: (i) PEG-lipids are incorporated in arsonoliposomes (as confirmed by the vesicle zeta potential modulation), (ii) pegylation of arsonoliposomes prevents their aggregation and fusion in the presence of calcium ions and (iii) when 8 mol% of PEG-DSPE is incorporated in arsonoliposomes based on their arsonolipid content, two groups of pegylated vesicles are formed: low content arsonoliposomes (<20 mol% arsonolipid) which are highly leaky and high content arsonoliposomes (>27 mol% arsonolipid) which are highly stable (70% calcein retention after 24h incubation in fetal calf serum [FCS]). In addition to high membrane integrity, the high content pegylated arsonoliposomes are morphologically perfect round-shaped vesicles without the sharp edges typically observed with non-pegylated DSPC-containing arsonoliposomes.     

Stability and aggregation studies of non-sonicated arsonolipid-containing vesicles

In this study, we investigated non-sonicated arsonolipid-containing liposomes (arsonoliposomes) in terms of the influence of lipid composition on their stability, assessed as membrane integrity and physical stability [size]. Vesicles consisting of plain arsonolipids or mixtures of arsonolipids with cholesterol [Chol] or with distearoyl-phospatidylcholine [DSPC] were studied. Membrane integrity was evaluated by measuring the retention of incorporated 5-(6)carboxyfluorescein [CF] during incubation of the vesicles in Tris buffer, pH 7.4. Photon correlation spectroscopy was used to investigate the time-dependent aggregation of arsonoliposomes in the absence and presence of Ca(2+)ions. Vesicles composed of plain C18 (acyl fatty chain) arsonolipids were found to be unstable, with only 15% of the initially incorporated CF remaining in the vesicles after 24 hours. The addition of Chol to the membrane (1:1 mol/mol) significantly increased the stability of arsonoliposomes, while the addition of DSPC to the lipid bilayer (1:1 mol/mol) increased vesicle stability to a lower extent. The results of particle size analysis showed that non-sonicated arsonoliposomes consisting of plain arsonolipid Ars/Stearic are highly and rapidly aggregated, while calcium-induced aggregation is also significant, but slower. Aggregation could not be always explained on the basis of zeta potential changes, indicating that the process is complex.     

Temperature dependence of Na+/Ca2+ exchange activity in beef-heart sarcolemmal vesicles and proteoliposomes

Temperature dependence of Na+/Ca2+ exchange activity was studied in beef cardiac sarcolemmal vesicles in the absence and presence of the inhibitor amiloride and in proteoliposomes reconstituted with different lipid mixtures. Arrhenius plots for Na+/Ca2+ exchange activity in both control and amiloride-treated vesicles revealed an apparent energy of activation of 9665 +/- 585 (SE, n = 4) cal/mol, corresponding to a temperature coefficient (Q10) value of 1.70 +/- 0.05 (SE, n = 4) over the range 25-37 degrees C. When Na+/Ca2+ exchange was reconstituted into phosphatidylcholine (PC):phosphatidylserine (PS) (52:48, mol/mol), PC:PS:cholesterol (25:39:36, mol/mol), and PC:PS:distearoylphosphatidylcholine (DSPC) (31:48:21, mol/mol) proteoliposomes, the highest activity was found in PC:PS:cholesterol proteoliposomes. Arrhenius plots of Na+/Ca2+ exchange activity exhibited breakpoints at 23 degrees C (PC:PS), 33 degrees C (PC:PS:cholesterol), and 23 degrees C (PC:PS:DSPC). The increase in the thermotropic transition temperature with cholesterol could result from the condensing effect of this sterol, whereas the breaks observed with PC:PS and PC:PS:DSPC could be caused by a non-lipid-mediated membrane protein conformational change. These results indicate that the lipid microenvironment around the Na+/Ca2+ exchanger and the nature of the specific lipid-protein interactions influence the activity of this antiporter. Further evidence supporting the hypothesis that cholesterol behaves as a specific positive effector for the exchanger is also given.     

Interaction between ether glycerophospholipid vesicles and serum proteins in vitro

The effect of blood serum on the stability of small unilamellar vesicles consisting of 1-O-(1'-alkenyl)-2-acyl-sn-glycerophosphocholine (choline plasmalogen) or of the alkylacyl-, dialkyl- and diacyl analogs was evaluated by measuring either release of entrapped calcein or transfer of phospholipids from vesicles to serum high-density lipoproteins. The following order of stability was found: alkenyloleoylGPC greater than dioleoylGPC greater than di-O-octadecenylGPC greater than acyloleoylGPC = egg phosphatidylcholine = alkyloleoylGPC. AlkyloleoylGPC and acyloleoylGPC had aliphatic chain compositions similar to that of alkenyloleoylGPC. From the results obtained it is concluded that stability of vesicles in the presence of serum depends on vesicle size (larger vesicles are more stable) and on the type of bond (ether or ester) in position 2 of glycerol. Dioctadecenyl vesicles are about the same size as alkylacylGPC vesicles, but are significantly more stable in the presence of serum. Thus, it appears that an ester bond in position 2 of glycerol (which is replaced by an ether bond in dioctadecenylglycerol) favors the interaction of phospholipids with serum high-density lipoproteins or lipid-exchange proteins. The addition of cholesterol greatly enhances vesicle stability; among the vesicles used in this study those composed of alkenylacylGPC plus 30 mol% cholesterol were most resistant to disruption by serum. Experiments with sn-1 and sn-3 enantiomers of alkylacylGPC and diacylGPC have shown that interaction of vesicle membranes with serum components is independent of the steric configuration of vesicle phospholipids.     

Effects of ethanol on lipid bilayers with and without cholesterol: the distearoylphosphatidylcholine system

Differential scanning calorimetry (DSC) and fluorescence spectroscopy are useful techniques for investigating the phase transitions of phospholipid bilayers. In this study, these methods have been extended to determine the effects of ethanol on DSPC and DSPC/2 mol.% cholesterol bilayers. The biphasic effect of the main transition was observed on the DSC heating scans above 0.60 M ethanol. In addition, the concentration at which the biphasic effect occurs is not significantly changed in the presence of 2 mol.% cholesterol. For the fluorescence studies, 1,6-diphenyl-1,3,5-hexatriene (DPH) has been incorporated into the bilayer to monitor the phase transitions through the displacement of DPH. This fluorescent probe is used to directly determine the onset of interdigitation in the bilayer systems as indicated by a large decrease in the DPH fluorescence intensity. The addition of cholesterol lowered and broadened the transition temperatures of the phosphatidylcholine (PC) system. However, 2 mol.% cholesterol did not have a significant effect on the induction of the interdigitated phase in DSPC as observed from the small difference in ethanol threshold concentration for the two systems. This suggests that DSPC forms a more stable interdigitated gel phase than other PCs with shorter acyl chains.     

Preparation and properties of arsonolipid containing liposomes

Arsonolipids are analogs of phosphonolipids which have a chemically versatile head group. In preliminary cell culture studies, liposomes composed solely of arsonolipids or of phosholipid-arsonolipid mixtures, demonstrate a specific toxicity against cancer cells (Gortzi et al., unpublished results). The possibility of using such formulations as an alternative of arsenic trioxide with or without combination of other cytostatic agents (encapsulated in their aqueous interior) prompted the investigation of their physicochemical characteristics. Herein we compared the characteristics of arsonolipid containing vesicles with different lipid compositions. Experimental results and morphological observations reveal that non-sonicated formulations have different structures and stability (when both membrane integrity and aggregation are taken into account) depending on the acyl chain length of the arsonolipid. When phospholipids and especially cholesterol are included in their membranes almost all arsonolipids studied produce more stable vesicles. An interesting aspect of these arsonolipid containing vesicles is also their negative surface charge, which may be modulated by mixing phospholipids with arsonolipids. Sonicated vesicles have smaller sizes and profoundly higher stability, especially when containing cholesterol and phosphatidylcholine mixed with arsonolipids. The only exception is that of the arsonolipid with the C(12) acyl chain which was observed to produce long tubes which break down to cubes by sonication. In conclusion, these initial studies demonstrate that sonicated vesicles composed of arsonolipid and phospholipid mixtures mixed with cholesterol posses the stability required to be used as an arsonolipid delivery system. In addition, although cryo-electron microscopy demonstrated that the sonicated vesicles are elliptical in shape, their encapsulation efficiency is not significantly lower than sonicated phospholipid liposomes. Thereby, these vesicles may be also used for the delivery of other drug molecules which can be sufficiently retained in their aqueous interior.     

Thermal behavior of novel non-sonicated arsonolipid-containing liposomes

The thermal properties of novel arsonolipid-containing liposomes in PBS pH 7.4 and in water in absence and presence of Ca(2+) ions are reported. Liposomes composed of arsonolipids with different acyl chains (C(12), C(16) and C(18)) were prepared by the one step method. Microcalorimetry results showed that (i) the thermotropic transitions of arsonoliposomes (in PBS, pH 7.4, and in water) increase as a function of arsonolipid fatty acyl chain length, (ii) arsonoliposomes of long fatty acyl chain arsonolipids (C(16) and C(18)) showed higher enthalpy and transition temperature in the buffer compared to those observed in water (for arsonoliposomes of C(12)-fatty acyl chain arsonolipid, the order was reversed which might be attributed to their different structure), and (iii) the presence of 2 mM CaCl(2) has more pronounced effects on the thermal properties of arsonoliposomes in distilled water than in buffer, which suggests that the ionic strength of the dispersion medium plays an important role in determining the thermal properties of arsonoliposomes.     

Liposome (Lipodine)-mediated DNA vaccination by the oral route

Plasmid DNA pRc/CMV HBS encoding the S (small) region of hepatitis B surface antigen (HBsAg) was incorporated by the dehydration-rehydration method into Lipodine liposomes composed of 16 micro moles phosphatidylcholine (PC) or distearoyl phosphatidylcholine (DSPC), 8 micro moles of (dioleoyl phosphatidylethanolamine (DOPE) or cholesterol and 4 micro moles of the cationic lipid 1,2-dioleoyl-3-(trimethylammonium propane (DOTAP) (molar ratios 1 : 0.5 : 0.25). Incorporation efficiency was high (89-93% of the amount of DNA used) in all four formulations tested and incorporated DNA was shown to be resistant to displacement in the presence of the competing anionic sodium dodecyl sulphate molecules. This is consistent with the notion that most of the DNA is incorporated within the multilamellar vesicles structure rather than being vesicle surface-complexed. Stability studies performed in simulated intestinal media also demonstrated that dehydration-rehydration vesicles (DRV) incorporating DNA (DRV(DNA)) were able to retain significantly more of their DNA content compared to DNA complexed with preformed small unilamellar vesicles (SUV-DNA) of the same composition. Moreover, after 4h incubation in the media, DNA loss for DSPC DRV(DNA) was only minimal, suggesting this to be the most stable formulation. Oral (intragastric) liposome-mediated DNA immunisation studies employing a variety of DRV(DNA) formulations as well as naked DNA revealed that secreted IgA responses against the encoded HBsAg were (as early as three weeks after the first dose) substantially higher after dosing with 100 micro g liposome-entrapped DNA compared to naked DNA. Throughout the fourteen week investigation, IgA responses in mice were consistently higher with the DSPC DRV(DNA) liposomes compared to naked DNA and correlated well with their improved DNA retention when exposed to model intestinal fluids. To investigate gene expression after oral (intragastric) administration, mice were given 100 micro g of naked or DSPC DRV liposome-entrapped plasmid DNA expressing the enhanced green fluorescent protein (pCMV.EGFP). Expression of the gene, in terms of fluorescence intensity in the draining mesenteric lymph nodes, was much greater in mice dosed with liposomal DNA than in animals dosed with the naked DNA. These results suggest that DSPC DRV liposomes containing DNA (Lipodine) may be a useful system for the oral delivery of DNA vaccines.     

Partition of malathion in synthetic and native membranes

Partition coefficients of [14C]malathion in model and native membranes are affected by temperature, cholesterol content, and lipid chain length. Partition in egg phosphatidylcholine bilayers decreases linearly with temperature, over a range (10-40 degrees C) at which the lipid is in the liquid-crystalline state. Addition of 50 mol% cholesterol severely decreases partition and practically abolishes the temperature dependence. First-order phase transitions of dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC and DSPC) are accompanied by a sharp increase in malathion partition. Apparently, the insecticide is easily accommodated in bilayers of short-aliphatic-chain lipids, since the partitions were 225, 135 and 48 in DMPC, DPPC and DSPC, respectively, at temperatures 10 Cdeg below the midpoint of their transitions. Partition values in native membranes decrease sequentially as follows: sarcoplasmic reticulum, mitochondria, brain microsomes, myelin and erythrocytes. This dependence parallels the relative content of cholesterol and is similar in liposomes of total extracted lipids, although the absolute partitions showed decreased values.     

Processing of Doxorubicin-Containing Liposomes by Liver Macrophages in Vitro

Abstract

Previous results suggested that drug formation in macrophages is an important aspect of the mode of action of doxorubicin (DXR)-containing liposomes. Intracellular degradation of DXR-liposomes may result in the liberation of DXR molecules that subsequently are released from the macrophages. We investigated whether the rate of intracellular degradation of DXR-liposomes phagocytosed by rat liver macrophages (Kupffer's cells) in monolayer culture is dependent on the type of DXR-liposomes internalized and whether differences in degradation rate of DXR-liposomes are reflected in different DXR release profiles. Two DXR-liposome types that were previously shown to differ markedly both in antitumor activity and degradation rate in vivo were selected for this investigation: a liposome composed of egg-phosphatidylcholine (PC), phosphatidylserine (PS), and cholesterol (chol), and a liposome composed of distearoylphosphatidylcholine (DSPC), dipalmitoyl-phosphatidylglycerol (DPPG), and chol. To monitor the rate of intracellular degradation of DXR-liposomes, cholesterol-1-[¹⁴Cjoleate was used as marker of the liposomal lipid phase. DXR was monitored with the use of a high-performance liquid chromatography (HPLC) method capable of detecting not only intact DXR but also major metabolites.

Comparable amounts of both types of DXR-liposomes were taken up by in vitro cultured Kupffer's cells. Liposome-associated cholesteryloleate was metabolized by the cells in a liposome-type-dependent pattern. During the first 30 min after start of the incubation, degradation of cholesteryloleate occurred at a similar rate for both types of DXR-liposomes. During continued incubation, however, PC/PS/chol DXR-liposomes were degraded at a considerably higher rate than DSPC/DPPG/ chol DXR-liposomes. the difference in susceptibility to lysosomal degradation of the two liposome preparations was also demonstrated by incubating the DXR-liposomes with lysosomal fractions isolated from rat liver homogenates: PC/PS/chol DXR-liposomes were much more sensitive to lysosomal esterase than DSPC/DPPG/chol DXR-liposomes. DXR either free or in liposomal form was chemically stable for up to 26 hr during incubation with the lysosomal fractions. Following uptake of DXR-liposomes by the cells, DXR was released from the cells into the medium. the release of DXR from cells that internalized DSPC/DPPG/chol DXR-liposomes was significantly delayed compared to the release of DXR from cells that internalized PC/PS/chol DXR-liposomes. Correlation of the relatively slow intracellular degradation of the DSPC/DPPG/chol DXR-liposomes with the delayed release of DXR from the cells suggests that by varying the type of DXR-liposomes, the rate of intracellular degradation can be manipulated, which, in turn, determines the rate of extracellular DXR release and thereby the therapeutic availability of the drug.     

Protection of liposomes during dehydration or freezing.

When liposomes are subjected to dehydration or freeze-thawing, vesicle fusion and/or leakage of vesicle contents can occur. The disaccharide, trehalose and the cryoprotectant, glycerol, are known to protect vesicle integrity during dehydration and freezing respectively. Here we examine their protective abilities as a function of vesicle size and lipid composition. It is shown that fatty acyl composition, cholesterol content and, with the exception of phosphatidylglycerol, acidic lipid content do not significantly alter the retention of aqueous contents by vesicles dehydrated and rehydrated in the presence of trehalose. The susceptibility to leakage induced by both dehydration and freezing is, however, critically dependent upon vesicle size with the smallest systems (70-100 nm diameter) being most stable. The mechanism whereby trehalose protects against vesicle fusion and leakage is also discussed.     

Lipid chain length effect on the phase behaviour of PCs/PEG:2000-PEs mixtures. A spin label electron spin resonance and spectrophotometric study

Spin-label electron spin resonance (ESR) spectroscopy and spectrophotometry at fixed wavelength are used to study fully hydrated aqueous dispersions of phosphatidylcholines (PCs) with poly(ethylene glycol:2000)-phosphatidylethanolamines (PEG:2000-PEs). PEG:2000-PE is a micelle-forming polymer-lipid that is extensively used for increasing the lifetime of PC liposomes in the blood circulation through a steric stabilisation effect. The PC lipids and the PEG:2000-PE polymer-lipids have the same acyl chain length of either dimiristoyl (DM) or distearoyl (DS) chains. DMPC/PEG:2000-DMPE and DSPC/PEG:2000-DSPE mixtures were investigated over the entire range of relative compositions (0-100 mol%). In both dispersions, the low-temperature conventional spin label ESR spectra and the temperature dependence of the absorbance at 400 nm give an indication of the conversion from lamellae to micelles with increasing PEG:2000-PEs content. The physical state of the lipid assemblies, lamellar or micellar, is dependent not only on PEG:2000-PEs content, but also on the length of hydrocarbon chain of the lipid matrix. Micellisation is attained more readily in dispersions with longer hydrocarbon chains (i.e. in DSPC/PEG:2000-DSPE mixtures) than in those with shorter acyl chains (i.e. in DMPC/PEG:2000-DMPE mixtures). Saturation transfer ESR (ST-ESR) and absorbance measurements reflect the disaggregation of the bilayers and a reduction in the size of the lipid aggregates by PEG:2000-PEs at low content.     

LIPOSOME (LIPODINE™)-MEDIATED DNA VACCINATION BY THE ORAL ROUTE

ABSTRACT

Plasmid DNA pRc/CMV HBS encoding the S (small) region of hepatitis B surface antigen (HBsAg) was incorporated by the dehydration–rehydration method into Lipodine? liposomes composed of 16 µmoles phosphatidylcholine (PC) or distearoyl phosphatidylcholine (DSPC), 8 µmoles of (dioleoyl phosphatidylethanolamine (DOPE) or cholesterol and 4 µmoles of the cationic lipid 1,2-dioleoyl-3-(trimethylammonium propane (DOTAP) (molar ratios 1 : 0.5 : 0.25). Incorporation efficiency was high (89–93% of the amount of DNA used) in all four formulations tested and incorporated DNA was shown to be resistant to displacement in the presence of the competing anionic sodium dodecyl sulphate molecules. This is consistent with the notion that most of the DNA is incorporated within the multilamellar vesicles structure rather than being vesicle surface-complexed. Stability studies performed in simulated intestinal media also demonstrated that dehydration–rehydration vesicles (DRV) incorporating DNA (DRV(DNA)) were able to retain significantly more of their DNA content compared to DNA complexed with preformed small unilamellar vesicles (SUV–DNA) of the same composition. Moreover, after 4h incubation in the media, DNA loss for DSPC DRV(DNA) was only minimal, suggesting this to be the most stable formulation. Oral (intragastric) liposome-mediated DNA immunisation studies employing a variety of DRV(DNA) formulations as well as naked DNA revealed that secreted IgA responses against the encoded HBsAg were (as early as three weeks after the first dose) substantially higher after dosing with 100 µg liposome-entrapped DNA compared to naked DNA. Throughout the fourteen week investigation, IgA responses in mice were consistently higher with the DSPC DRV(DNA) liposomes compared to naked DNA and correlated well with their improved DNA retention when exposed to model intestinal fluids. To investigate gene expression after oral (intragastric) administration, mice were given 100 µg of naked or DSPC DRV liposome-entrapped plasmid DNA expressing the enhanced green fluorescent protein (pCMV.EGFP). Expression of the gene, in terms of fluorescence intensity in the draining mesenteric lymph nodes, was much greater in mice dosed with liposomal DNA than in animals dosed with the naked DNA. These results suggest that DSPC DRV liposomes containing DNA (Lipodine?) may be a useful system for the oral delivery of DNA vaccines.     

The effects of bilirubin on the thermal properties of phosphatidylcholine bilayers.

The thermotropic properties of multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC), as a function of the concentration of bilirubin in the range of 0.1 to 1 mol%, were measured. The exact effects of bilirubin depended on the chain length of the polymethylene chains. But the general effects of bilirubin were the same in all systems. At the lowest concentrations tested (0.1 mol bilirubin/100 mol phospholipid (0.1 mol%)), bilirubin broadened and shifted to higher temperatures the main phase transitions of all bilayers. For DPPC and DSPC, but not DMPC, this concentration of bilirubin was associated with a new transition at 25 degrees C (DPPC) or 34 degrees C (DSPC). Bilirubin at 0.2 mol% was required for the detection of a similar transition (at 13.7 degrees C) in DMPC. Higher concentrations of bilirubin (> 0.2 mol%) suppressed completely the main phase transitions in all bilayers but increased the enthalpy of the new transition. Maximal values of delta H for these transitions were reached at 0.5, 0.25, and 0.2 mol% bilirubin in DMPC, DPPC, and DSPC, respectively. Values of delta H and delta S for these transitions were far larger than for the corresponding gel-to-liquid crystal transitions in pure lipid bilayers but were equal to those expected for a transition between crystalline and liquid crystalline phases.     

Triggered release of doxorubicin following mixing of cationic and anionic liposomes

In many applications, an ability of liposomes to retain drug and then rapidly release it at some later time would be of benefit. In this work, we investigate the ability of cationic large unilamellar vesicles (LUV) to promote rapid release of doxorubicin from anionic LUV. It is shown that the addition of cationic liposomes containing cholesterol, dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC) and the cationic lipid N,N-dioleyl-N,N-dimethylammonium chloride (DODAC) to doxorubicin-containing LUV composed of cholesterol, DOPE, DSPC and the anionic lipid dioleoyphosphatidylglycerol (DOPG) can result in release of more than 90% of the drug in times of 30 s or less. Further, it is shown that these release characteristics are exquisitely dependent on the presence of DOPE and cholesterol. In the absence of DOPE, much slower release rates are observed, with maximum release levels of 50% after a 2-h incubation at 20 degrees C. Remarkably, threshold levels of more than 10 mol% cholesterol are required before any appreciable release is observed. [31P]NMR spectroscopy and freeze-fracture electron microscopy studies reveal that systems giving rise to rapid release of doxorubicin exhibit limited formation of inverted hexagonal (H(II)) phase, suggesting that these lipids facilitate drug release by formation of local regions of non-bilayer structure. It is concluded that drug release triggered by mixing anionic and cationic liposomes could be of utility in drug delivery applications.     

Pardaxin Permeabilizes Vesicles More Efficiently by Pore Formation than by Disruption

Pardaxin is a 33-amino-acid neurotoxin from the Red Sea Moses sole Pardachirus marmoratus, whose mode of action shows remarkable sensitivity to lipid chain length and charge, although the effect of pH is unclear. Here we combine optical spectroscopy and dye release experiments with laser scanning confocal microscopy and natural abundance ¹³C solid-state nuclear magnetic resonance to provide a more complete picture of how pardaxin interacts with lipids. The kinetics and efficiency of release of entrapped calcein is highly sensitive to pH. In vesicles containing zwitterionic lipids (PC), release occurs most rapidly at low pH, whereas in vesicles containing 20% anionic lipid (PG), release occurs most rapidly at high pH. Pardaxin forms stable or transient pores in PC vesicles that allow release of contents without loss of vesicle integrity, whereas the inclusion of PG promotes total vesicle collapse. In agreement with this, solid-state nuclear magnetic resonance reveals that pardaxin takes up a trans-membrane orientation in 14-O-PC/6-O-PC bicelles, whereas the inclusion of 14-0-PG restricts it to contacts with lipid headgroups, promoting membrane lysis. Pore formation in zwitterionic vesicles is more efficient than lysis of anionic vesicles, suggesting that electrostatic interactions may trap pardaxin in several suboptimal interconverting conformations on the membrane surface.     

Ether phosphatidylcholines: comparison of miscibility with ester phosphatidylcholines and sphingomyelin, vesicle fusion, and association with apolipoprotein A-I

Nonhydrolyzable matrices of ether-linked phosphatidylcholines (PCs) and sphingomyelin have been used to study the mechanism of action of lipolytic enzymes. Since ether PCs, sphingomyelin, and ester PCs vary in the number of hydrogen bond donors and acceptors in the carbonyl region of the bilayer, we have examined several physical properties of ether PCs and sphingomyelin in model systems to validate their suitability as nonhydrolyzable lipid matrices. The intermolecular interactions of ether PCs with ester PCs, sphingomyelin, and cholesterol were investigated by differential scanning calorimetry. Phase diagrams constructed from the temperature dependence of the gel to liquid-crystalline phase transition of 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine (DPPC-ether) and 1,2-O-ditetradecyl-sn-glycero-3-phosphocholine (DMPC-ether) with both 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) demonstrated complete lipid miscibility in the gel and liquid-crystalline phases. Additionally, phase diagrams of egg yolk sphingomyelin (EYSM) with DMPC or DMPC-ether and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-O-dioctadecyl-sn-glycero-3-phosphocholine (DSPC-ether) demonstrated no major differences in miscibility of EYSM in ester and ether PCs. The effect of 10 mol % cholesterol on the thermal transitions of mixtures of ester and ether PCs also indicates little preference of cholesterol for either lipid. The fusion of small single bilayer vesicles of DMPC, DMPC-ether, DPPC, and DPPC-ether to larger aggregates as determined by gel filtration indicated that the ester PC vesicles were somewhat more stable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Triggered release of doxorubicin following mixing of cationic and anionic liposomes

In many applications, an ability of liposomes to retain drug and then rapidly release it at some later time would be of benefit. In this work, we investigate the ability of cationic large unilamellar vesicles (LUV) to promote rapid release of doxorubicin from anionic LUV. It is shown that the addition of cationic liposomes containing cholesterol, dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC) and the cationic lipid N,N-dioleyl-N,N-dimethylammonium chloride (DODAC) to doxorubicin-containing LUV composed of cholesterol, DOPE, DSPC and the anionic lipid dioleoyphosphatidylglycerol (DOPG) can result in release of more than 90% of the drug in times of 30 s or less. Further, it is shown that these release characteristics are exquisitely dependent on the presence of DOPE and cholesterol. In the absence of DOPE, much slower release rates are observed, with maximum release levels of 50% after a 2-h incubation at 20 °C. Remarkably, threshold levels of more than 10 mol% cholesterol are required before any appreciable release is observed. [³¹P]NMR spectroscopy and freeze-fracture electron microscopy studies reveal that systems giving rise to rapid release of doxorubicin exhibit limited formation of inverted hexagonal (H_II) phase, suggesting that these lipids facilitate drug release by formation of local regions of non-bilayer structure. It is concluded that drug release triggered by mixing anionic and cationic liposomes could be of utility in drug delivery applications.     

Preparation and characterization of gas-filled liposomes: can they improve oil recovery?

Although well known for delivering various pharmaceutical agents, liposomes can be prepared to entrap gas rather than aqueous media and have the potential to be used as pressure probes in magnetic resonance imaging (MRI). Using these gas-filled liposomes (GFL) as tracers, MRI imaging of pressure regions of a fluid flowing through a porous medium could be established. This knowledge can be exploited to enhance recovery of oil from the porous rock regions within oil fields. In the preliminary studies, we have optimized the lipid composition of GFL prepared using a simple homogenization technique and investigated key physico-chemical characteristics (size and the physical stability) and their efficacy as pressure probes. In contrast to the liposomes possessing an aqueous core which are prepared at temperatures above their phase transition temperature (T(c)), homogenization of the phospholipids such as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocoline (DSPC) in aqueous medium below their T(c) was found to be crucial in formation of stable GFL. DSPC based preparations yielded a GFL volume of more than five times compared to their DPPC counter part. Although the initial vesicle sizes of both DSPC and DPPC based GFL were about 10 microm, after 7 days storage at 25 degrees C, the vesicle sizes of both formulations significantly (p < 0.05) increased to 28.3 +/- 0.3 mum and 12.3 +/- 1.0 microm, respectively. When the DPPC preparation was supplemented with cholesterol at a 1:0.5 or 1:1 molar ratio, significantly (p < 0.05) larger vesicles were formed (12-13 microm), however, compared to DPPC only vesicles, both cholesterol supplemented formulations displayed enhanced stability on storage indicating a stabilizing effect of cholesterol on these gas-filled vesicles. In order to induce surface charge on the GFL, DPPC and cholesterol (1: 0.5 molar ratio) liposomes were supplemented with a cationic surfactant, stearylamine, at a molar ratio of 0.25 or 0.125. Interestingly, the zeta potential values remained around neutrality at both stearylamine ratios suggesting the cationic surfactant was not incorporated within the bilayers of the GFL. Microscopic analysis of GFL confirmed the presence of spherical structures with a size distribution between 1-8 microm. This study has identified that DSPC based GFL in aqueous medium dispersed in 2% w/v methyl cellulose although yielded higher vesicle sizes over time were most stable under high pressures exerted in MRI.