Changes in the morphology of cell-size liposomes in the presence of cholesterol: Formation of neuron-like tubes and liposome networks

Spontaneous changes in the morphology of cell-size liposomes (dioleoylphosphatidylcholine, DOPC and egg PC) as model cells were investigated in the presence of cholesterol. Tube structures and liposome networks connected by the tubes were observed in the presence of 5-30% cholesterol by dark-field and laser-scanning microscopy. Furthermore, in the presence of more than 40 mol% of cholesterol, the tubes disappeared and changed to small liposomes. Thus, cholesterol induced a morphological change in giant liposomes from tubes to small liposomes. These phenomena may be related to the role of cholesterol in the morphological changes in living cells such as neurons.     

Changes in the morphology of cell-size liposomes in the presence of cholesterol: formation of neuron-like tubes and liposome networks

Spontaneous changes in the morphology of cell-size liposomes (dioleoylphosphatidylcholine, DOPC and egg PC) as model cells were investigated in the presence of cholesterol. Tube structures and liposome networks connected by the tubes were observed in the presence of 5-30% cholesterol by dark-field and laser-scanning microscopy. Furthermore, in the presence of more than 40 mol% of cholesterol, the tubes disappeared and changed to small liposomes. Thus, cholesterol induced a morphological change in giant liposomes from tubes to small liposomes. These phenomena may be related to the role of cholesterol in the morphological changes in living cells such as neurons.     

Stability of small unilamellar liposomes in serum and clearance from the circulation: The effect of the phospholipid and cholesterol components

Small unilamellar liposomes containing carboxyfluorescein (CF) and composed of various unsaturated and saturated phospholipids with or without cholesterol were incubated in the presence of mouse serum at 37°C. Liposomes composed of egg L-α-phosphatidylcholine (PC), L-α-dioleoylphosphatidylcholine (DOPC) or sphingomyelin (SM) became rapidly permeable to entrapped CF but incorporation of cholesterol into such liposomes reduced CF leakage. Under similar conditions, CF leakage from cholesterol-free liposomes composed of saturated phospholipids of increasing fatty acid chain length was dependant on the liquid-crystalline phase transition temperature (Tc) of the phospholipid component. Thus, L-α-dilaureoylphos-phatidylcholine (DLPC), L-α-dimyristoyl phosphatidylcholine (DMPC) and L-α-dipalmitoylphosphatidylcholine (DPPC) with Tc's below or near the temperature of the incubation (37°C) released CF rapidly whereas L-α-diheptedecanoyl phosphatidylcholine (DHPC), L-α-distearoylphosphatidylcholine (DSPC) and hydrogenated egg PC (HPC) liposomes with Tc's above 37°C retained the dye quantitatively. After incorporation of cholesterol into liposomes composed of saturated phospholipids, CF release was reduced for DLPC and DMPC and increased for DPPC, DSPC, DHPC and HPC vesicles. Liposomes with or without cholesterol exhibiting greatest stability (in terms of CF retention) in the presence of serum were injected intravenously into mice and rates of clearance of quenched CF from the circulation measured. Observed clearance rates were linear and, when liposomes contained tritiated phospholipid, identical to those of the radiolabel suggesting retention of liposomal integrity in the intravascular space. However, half-lifes of liposomes ranging from 0.1 to 16 h did not correlate with the physical characteristics of their phospholipid component. After intraperitoneal injection, there was quantitative entry of quenched CF (stable liposomes) into the blood from which it was eliminated at rates corresponding to those observed after intravenous injection. These results suggest that solute retention by liposomes and their half-life in the circulation can be controlled by the appropriate manipulation of liposomal membrane fluidity and composition.     

Liposomes for cryopreservation of bovine sperm

In this study, the effect of various unilamellar liposomes on cryopreservation of bovine spermatozoa has been investigated. Liposomes were composed of saturated lipids with various acyl chain lengths: DSPC (18:0), DPPC (16:0), DMPC (14:0), or DLPC (12:0). Alternatively, liposomes were prepared using unsaturated egg phosphatidylcholine (EPC) or DOPC (18:1, neutral), alone or in combination with lipids with various head groups: DOPS (negatively charged), DOPG (negatively charged), and DOPE (neutral). Fourier transform infrared spectroscopy studies showed that bovine sperm membranes display a gradual phase transition from 10 to 24 ^oC. Phase transition temperatures of the liposomes varied from −20 to +53 ^oC. Sperm was incubated in the presence of liposomes for either 6 or 24 h at 4 °C prior to freezing. Postfreeze survival rates were determined based on the percentage of progressively motile cells as well as the percentage of acrosome- and plasma membrane-intact cells. With DOPC liposomes a postthaw progressive motility of 43% was obtained compared with 59% using standard egg yolk freezing extender. Postthaw progressive motility increased up to 52% using DOPC:DOPG (9:1) liposomes, whereas DOPC:DOPS or DOPC:DOPE liposomes did not increase survival compared with DOPC liposomes. Among the saturated lipids, only DMPC was found to increase cryosurvival, up to 44% based on progressive motility. DLPC liposomes caused a complete loss in cell viability, already prior to freezing, whereas DPPC and DSPC liposomes neither positively nor negatively affected cryosurvival. Taken together, the higher postthaw survival obtained with DOPC:DOPG liposomes as compared with DOPC liposomes can likely be attributed to increased liposome-sperm interactions between the charged phosphatidylglycerol groups and charged regions in the sperm membranes. Interestingly, the lipid phase state of the liposomes during preincubation is not the decisive factor for their cryoprotective action.     

Biodistribution and immunotargetability of ganglioside-stabilized dioleoylphosphatidylethanolamine liposomes.

The biodistribution and immunotargetability of liposomes composed primarily of dioleoylphosphatidylethanolamine (DOPE) or dioleoylphosphatidylcholine (DOPC) in mice injected via the tail vein were examined and compared. The ganglioside GM1 (7 mol%) prolonged the circulation of DOPC but not DOPE liposomes. Gangliosides GD1a and GT1b (7 mol%) also increased the amount of DOPC liposomes remaining in circulation, and to a similar extent as GM1, at 15 min post injection. However, these liposomes were cleared from the circulation by 2.5 h. Monoclonal antibody 34A, which specifically binds to a surface glycoprotein (gp 112) of the pulmonary endothelial cell surface, was coupled with N-glutarylphosphatidylethanolamine and incorporated into liposomes by a dialysis procedure. These 34A-immunoliposomes, composed of DOPE and GM1 (7 mol%), but not the antibody-free liposomes, accumulated efficiently (approximately 24% of the injected dose) in the lungs. Inclusion of cholesterol (31 mol%) enhanced the lung accumulation of both DOPE/GM1 immunoliposomes and DOPC/GM1 immunoliposomes to 33% and 51% of the injected dose, respectively. The transient increase in DOPC liposome circulation provided by GD1a and GT1b was sufficient to enhance DOPC immunoliposome binding, where 44% and 43% of the injected dose of DOPC/Chol/GD1a and DOPC/Chol/GT1b immunoliposomes accumulated in lung at 15 min after injection, respectively. In general, cholesterol-containing DOPC liposomes were more targetable than DOPE liposomes, and the degree to which these liposomes avoid RES uptake influences their targetability. The results presented here are relevant to the design of targetable drug delivery vehicles.     

A multi-step lipid mixing assay to model structural changes in cationic lipoplexes used for in vitro transfection.

Formation of liposome/polynucleotide complexes (lipoplexes) involves electrostatic interactions, which induce changes in liposome structure. The ability of these complexes to transfer DNA into cells is dependent on the physicochemical attributes of the complexes, therefore characterization of binding-induced changes in liposomes is critical for the development of lipid-based DNA delivery systems. To clarify the apparent lack of correlation between membrane fusion and in vitro transfection previously observed, we performed a multi-step lipid mixing assay to model the sequential steps involved in transfection. The roles of anion charge density, charge ratio and presence of salt on lipid mixing and liposome aggregation were investigated. The resonance-energy transfer method was used to monitor lipid mixing as cationic liposomes (DODAC/DOPE and DODAC/DOPC; 1:1 mole ratio) were combined with plasmid, oligonucleotides or Na(2)HPO(4). Cryo-transmission electron microscopy was performed to assess morphology. As plasmid or oligonucleotide concentration increased, lipid mixing and aggregation increased, but with Na(2)HPO(4) only aggregation occurred. NaCl (150 mM) reduced the extent of lipid mixing. Transfection studies suggest that the presence of salt during complexation had minimal effects on in vitro transfection. These data give new information about the effects of polynucleotide binding to cationic liposomes, illustrating the complicated nature of anion induced changes in liposome morphology and membrane behavior.     

Improvement of in vivo stability of phosphodiester oligonucleotide using anionic liposomes in mice

Antisense phosphodiester oligonucleotides (ODN) are unstable in biological fluids due to nuclease-mediated degradation and therefore cannot be used in most antisense therapeutic applications. We describe here an in vitro and in vivo stabilization of a 15 mer phosphodiester sequence using anionic liposomes. Two formulations have been studied: DOPC/OA/CHOL and DOPE/OA/CHOL (pH-sensitive liposomes). Our in vitro findings reveal the same stabilization effect in mouse plasma for both anionic liposomes. In vivo investigation showed a great protective effect for both formulations after intravenous administration to mice. By contrast with in vitro results, a higher protection of ODN was observed with DOPC/OA/CHOL liposomes compared to the DOPE/OA/CHOL formulation. The latter was degraded in blood (75% of the injected dose at 5 min) probably due to interactions with blood components, and the remaining (25% at 5 min) was distributed mostly to the liver and spleen. DOPC liposomes were remarkably stable in blood and were distributed more slowly to all studied organs (liver, spleen, kidneys and lungs). Intact ODN was still observed in some organs (liver, spleen, lungs), but not in blood, 24 hours after DOPC liposome administration. These results suggest that this antisense strategy using carrier systems may be applicable to the treatment of diseases involving the reticuloendothelial system.     

Purification, characterization and substrate specificity of rabbit lung phospholipid transfer proteins.

Three phospholipid transfer proteins, namely proteins I, II and III, were purified from the rabbit lung cytosolic fraction. The molecular masses of phospholipid transfer proteins I, II and III are 32 kilodaltons (kDa), 22 kDa and 32 kDa, respectively; their isoelectric point values are 6.5, 7.0 and 6.8, respectively. Phospholipid transfer proteins I and III transferred phosphatidylcholine (PC) and phosphatidylinositol (PI) from donor unilamellar liposomes to acceptor multilamellar liposomes; protein II transferred PC but not PI. All the three phospholipid transfer proteins transferred phosphatidylethanolamine poorly and showed no tendency to transfer triolein. The transfer of [14C]PC from unilamellar liposomes to multilamellar liposomes facilitated by each protein was affected differently by the presence of acidic phospholipids in the PC unilamellar liposomes. In an equal molar ratio of acidic phospholipid and PC, phosphatidylglycerol (PG) reduced the activities of proteins I and III by 70% (P = 0.0004 and 0.0032, respectively) whereas PI and phosphatidylserine (PS) had an insignificant effect. In contrast, the protein II activity was stimulated 2-3-times more by either PG (P = 0.0024), PI (P = 0.0006) or PS (P = 0.0038). In addition, protein II transferred dioleoylPC (DOPC) about 2-times more effectively than dipalmitoylPC (DPPC) (P = 0.0002), whereas proteins I and III transferred DPPC 20-40% more effectively than DOPC but this was statistically insignificant. The markedly different substrate specificities of the three lung phospholipid transfer proteins suggest that these proteins may play an important role in sorting intracellular membrane phospholipids, possibly including lung surfactant phospholipids.     

Lipolysis in model membranes in the presence of positively charged soluble proteins]

Phospholipase A2 hydrolysis of neutral and negatively charged lipid membranes modified by positively charged proteins has been studied using liposomes composed of either dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylglycerol (DOPG) alone or their equimolar mixture in the presence of cytochrome c, histone H1, cytochrome b5, and polylysine. Twenty minutes after the reaction had been initiated, DOPC hydrolysis was 58%, while that in the equimolar mixture with DOPG was 35%. DOPG hydrolysis was more complete in binary mixtures of liposomes. The same was observed for liposomes in the presence of cytochrome c. Hydrolysis of phospholipids in binary liposomes in the presence of histone H1 was 3 times faster than that in protein-free liposomes. In the presence of polylysine the rate of DOPG hydrolysis was decreased. The results obtained are suggestive of electrostatic interactions between hydrophilic proteins and negatively charged phospholipids, the phospholipase A2 catalytic activity being affected by these interactions.     

Stability of Desmopressin Loaded in Liposomes

Abstract

Desmopressin-containing liposome formulations have been developed for intranasal administration previously. Positively charged liposomes were found to be an efficient delivery system for desmopressin. In this study, stability of the loaded desmopressin in positively charged liposomes was further investigated. Comparison of the stability of desmopressin in solution and liposomes was made. Degradation of desmopressin was shown to follow a pseudo-first-order reaction. Degradation of desmopressin in both solution and liposomes demonstrated the same kinetic behavior and exhibited no significant difference in half-lives. Similar v-shape pH-rate profile was found for desmopressin degradation in solution and liposomes. At pH 4.0, the inflection point of the v-shape pH-rate curve, the reaction rate of desmopressin was lowest and the stability was greatest. The stability of lipid ingredients of dioleoylphosphatidylcholine (DOPC), cholesterol (C), and stearylamine (S) in the liposome dispersion at pH 4.0 was studied. Results demonstrated that DOPC, C, and S were relatively stable in the liposome structure when formulated with desmopressin. The degradation of desmopressin in solution and liposomes in the presence of α-chymotrypsin was investigated. A longer half-life for desmopressin in liposomes than in solution was observed. It was suggested that desmopressin was protected by the liposomes against α-chymotrypsin digestion.     

Stability of desmopressin loaded in liposomes

Desmopressin-containing liposome formulations have been developed for intranasal administration previously. Positively charged liposomes were found to be an efficient delivery system for desmopressin. In this study, stability of the loaded desmopressin in positively charged liposomes was further investigated. Comparison of the stability of desmopressin in solution and liposomes was made. Degradation of desmopressin was shown to follow a pseudo-first-order reaction. Degradation of desmopressin in both solution and liposomes demonstrated the same kinetic behavior and exhibited no significant difference in half-lives. Similar v-shape pH-rate profile was found for desmopressin degradation in solution and liposomes. At pH 4.0, the inflection point of the v-shape pH-rate curve, the reaction rate of desmopressin was lowest and the stability was greatest. The stability of lipid ingredients of dioleoylphosphatidylcholine (DOPC), cholesterol (C), and stearylamine (S) in the liposome dispersion at pH 4.0 was studied. Results demonstrated that DOPC, C, and S were relatively stable in the liposome structure when formulated with desmopressin. The degradation of desmopressin in solution and liposomes in the presence of alpha-chymotrypsin was investigated. A longer half-life for desmopressin in liposomes than in solution was observed. It was suggested that desmopressin was protected by the liposomes against alpha-chymotrypsin digestion.     

Small-angle neutron scattering study of the n-decane effect on the bilayer thickness in extruded unilamellar dioleoylphosphatidylcholine liposomes

Dioleoylphosphatidylcholine (DOPC) and n-decane were mixed and hydrated afterwards in an excess of heavy water at 1 wt.% of DOPC. From this dispersion, unilamellar liposomes were prepared by extrusion through polycarbonate filter with 500-A pores. Small-angle neutron scattering (SANS) was conducted on these liposomes. From the Kratky-Porod plot ln[I(Q)Q2] vs. Q2 of SANS intensity I(Q) in the range of scattering vectors Q corresponding to the interval 0.001 A(-2) < or = Q2 < or = 0.006 A(-2), the liposome bilayer radius of gyration Rg and the bilayer thickness parameter d(g) = 12(0.5)Rg were obtained. The values of d(g) indicated that the bilayer thickness is within the experimental error constant up to n-decane/DOPC approximately 0.5 molar ratio, and then increases by 2.4 +/- 1.3 A up to n-decane/DOPC = 1.2 molar ratio.     

Interaction study between maltose-modified PPI dendrimers and lipidic model membranes

The influence of maltose-modified poly(propylene imine) (PPI) dendrimers on dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) (3%) liposomes was studied. Fourth generation (G4) PPI dendrimers with primary amino surface groups were partially (open shell glycodendrimers — OS) or completely (dense shell glycodendrimers — DS) modified with maltose residues. As a model membrane, two types of 100 nm diameter liposomes were used to observe differences in the interactions between neutral DMPC and negatively charged DMPC/DMPG bilayers. Interactions were studied using fluorescence spectroscopy to evaluate the membrane fluidity of both the hydrophobic and hydrophilic parts of the lipid bilayer and using differential scanning calorimetry to investigate thermodynamic parameter changes. Pulsed-filed gradient NMR experiments were carried out to evaluate common diffusion coefficient of DMPG and DS PPI in D₂O when using below critical micelle concentration of DMPG. Both OS and DS PPI G4 dendrimers show interactions with liposomes. Neutral DS dendrimers exhibit stronger changes in membrane fluidity compared to OS dendrimers. The bilayer structure seems more rigid in the case of anionic DMPC/DMPG liposomes in comparison to pure and neutral DMPC liposomes. Generally, interactions of dendrimers with anionic DMPC/DMPG and neutral DMPC liposomes were at the same level. Higher concentrations of positively charged OS dendrimers induced the aggregation process with negatively charged liposomes. For all types of experiments, the presence of NaCl decreased the strength of the interactions between glycodendrimers and liposomes. Based on NMR diffusion experiments we suggest that apart from electrostatic interactions for OS PPI hydrogen bonds play a major role in maltose-modified PPI dendrimer interactions with anionic and neutral model membranes where a contact surface is needed for undergoing multiple H-bond interactions between maltose shell of glycodendrimers and surface membrane of liposome.     

Transport of actin-decorated liposomes along myosin molecules in vitro

We examined whether actin filaments bound to positively charged liposomes could interact with myosin molecules and induce liposome motility. When liposomes were constructed from the mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cationic N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP), actin filaments bound to the liposomes. The actin-bound liposomes exhibited movement on myosin molecules in the presence of adenosine-5'-triphosphate (ATP). The displacement was almost linearly increased with time and the behavior differed from that of Brownian motion. Furthermore, the presence of 30% DOTAP in liposomes was most effective for transport. These data show that the actomyosin system was successfully integrated into the liposomes and possesses the ability to actively transport useful agents enclosed within the liposomes.     

Detection of DNA hybridization on a liposome surface using ultrasound velocimetry and turbidimetry methods

19-mer oligonucleotides with oleylamine tethered at 3' and 5' terminal, respectively, were incorporated into unilamellar liposomes of dioleoylphosphatidylcholine (DOPC). Addition of complementary nucleotide resulted in hybridization with oligonucleotides located on different liposomes and caused liposome aggregation. Significant changes of sound velocimetry and turbidity were readily observed at 10 nM concentration of the complementary chain.     

Stabilization of liposomal membranes by thermozeaxanthins: carotenoid-glucoside esters.

Thermozeaxanthins (TZS) are novel carotenoid-glucoside esters existing in the cell membranes of the thermophilic bacterium, Thermus thermophilus. The effect of TZS on membrane permeability was studied by measuring the leakage of the fluorescent dye from calcein-entrapped large unilamellar liposomes (LUVs). The LUVs were composed of a small portion (0.2-1.0 mol%) of TZS and phosphatidylcholine (PC) of various length and saturation degree of hydrocarbon chains. Incorporation of TZS in egg PC LUVs stabilized the liposomes in the temperature range from 30 to 80 degrees C, as only 2.6% of the entrapped calcein leaked out in contrast to 10% release from the egg PC liposomes without TZS. LUVs composed of dipalmitoylphosphatidylcholine (DPPC) or dioleoylphosphatidylcholine (DOPC) were stabilized by the incorporation of TZS at a temperature below 30 degrees C. Inclusion of TZS in LUVs composed of dimyristoylphosphatidylcholine, whose hydrocarbon chains are shorter than both DPPC and DOPC, did not stabilize the liposomes. About 90% of the entrapped dye was lost indicating defects of the liposomal membranes. Matching of the lipid bilayer thickness with the molecular length of TZS in the bilayers is discussed.     

Phospholipid-cationic lipid interactions: influences on membrane and vesicle properties

Liposomes composed of synthetic dialkyl cationic lipids and zwitterionic phospholipids such as dioleoylphosphatidylethanolamine have been studied extensively as vehicles for gene delivery, but the broader potentials of these cationic liposomes for drug delivery have not. An understanding of phospholipid-cationic lipid interactions is essential for rational development of this potential. We evaluated the effect of the cationic lipid DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium) on liposome physical properties such as size and membrane domain structure. DSC (differential scanning calorimetry) showed progressive decrease and broadening of the phase transition temperature of dipalmitoylphosphatidylcholine (DPPC) with increasing fraction of DOTAP, in the range of 0.4-20 mol%. Laurdan (6-dodecanolyldimethylamino-naphthalene), a fluorescent probe of membrane domain structure, showed that DOTAP and DPPC remained miscible at all ratios tested. DOTAP reduced the size of spontaneously-forming PC-containing liposomes, regardless of the acyl chain length and degree of saturation. The anionic lipid DOPG (dioleoylphosphatidylglycerol) had similar effects on DPPC membrane fluidity and size. However, DOTAP/DOPC (50/50) vesicles were taken up avidly by OVCAR-3 human ovarian tumor cells, in contrast to DOPG/DOPC (50/50) liposomes. Overall, DOTAP exerts potent effects on bilayer physical properties, and may provide advantages for drug delivery.     

Interactions of ciprofloxacin with DPPC and DPPG: fluorescence anisotropy, ATR-FTIR and 31P NMR spectroscopies and conformational analysis

The interactions between a drug and lipids may be critical for the pharmacological activity. We previously showed that the ability of a fluoroquinolone antibiotic, ciprofloxacin, to induce disorder and modify the orientation of the acyl chains is related to its propensity to be expelled from a monolayer upon compression [1]. Here, we compared the binding of ciprofloxacin on DPPC and DPPG liposomes (or mixtures of phospholipids [DOPC:DPPC], and [DOPC:DPPG]) using quasi-elastic light scattering and steady-state fluorescence anisotropy. We also investigated ciprofloxacin effects on the transition temperature (T(m)) of lipids and on the mobility of phosphate head groups using Attenuated Total Reflection Fourier Transform Infrared-Red Spectroscopy (ATR-FTIR) and (31)P Nuclear Magnetic Resonance (NMR) respectively. In the presence of ciprofloxacin we observed a dose-dependent increase of the size of the DPPG liposomes whereas no effect was evidenced for DPPC liposomes. The binding constants K(app) were in the order of 10(5) M(-1) and the affinity appeared dependent on the negative charge of liposomes: DPPG>DOPC:DPPG (1:1; M:M)>DPPC>DOPC:DPPC (1:1; M:M). As compared to the control samples, the chemical shift anisotropy (Deltasigma) values determined by (31)P NMR showed an increase of 5 and 9 ppm for DPPC:CIP (1:1; M:M) and DPPG:CIP (1:1; M:M) respectively. ATR-FTIR experiments showed that ciprofloxacin had no effect on the T(m) of DPPC but increased the order of the acyl chains both below and above this temperature. In contrast, with DPPG, ciprofloxacin induced a marked broadening effect on the transition with a decrease of the acyl chain order below its T(m) and an increase above this temperature. Altogether with the results from the conformational analysis, these data demonstrated that the interactions of ciprofloxacin with lipids depend markedly on the nature of their phosphate head groups and that ciprofloxacin interacts preferentially with anionic lipid compounds, like phosphatidylglycerol, present at a high content in these membranes.     

Arsenic trioxide liposomes: encapsulation efficiency and in vitro stability

The use of arsenic-containing compounds in cancer therapy is currently being re-considered, after the recent approval of arsenic trioxide (Trisenox) for the treatment of relapsed promyelocytic leukemia (PML). In an attempt to prepare a carrier system to minimize the toxicity of this drug, the aim of this study is to prepare and characterize liposomes encapsulating arsenic trioxide (ATO). For this, we prepared different types of liposomes entrapping ATO: large multilamellar (MLV), sonicated (SUV) and dried reconstituted vesicles (DRV). The techniques used were: thin film hydration, sonication and the DRV method, respectively. Two lipid compositions were studied for each liposome type, EggPC/Chol (1:1) and DSPC/Chol (1:1). After liposome preparation, drug encapsulation was evaluated by measuring arsenic in liposomes. For this, energy-dispersive X-ray fluorescence spectroscopy or atomic absorption was used. In addition, the retention of the drug in the liposomes was evaluated after incubating the liposomes in buffer at 37 degrees C. The experimental results reveal that encapsulation of ATO in liposomes ranges between 0.003 and 0.506 mol/ mol of lipid, and is highest in the DRV vesicles and lowest in the small unilamellar vesicles, as anticipated. Considering the in vitro stability of ATO-encapsulating liposomes: 1) For the PC/Chol liposomes (DRV and MLV), after 24 hours of incubation, more than 70% (or 90% in some cases) of the initially encapsulated amount of ATO was released. 2) The liposomes composed of DSPC/Chol could retain substantially higher amounts of ATO, especially the DRV liposomes (54% retained after 24 h). 3) In the case of PC/Chol, temperature of incubation has no effect on the ATO release after 24 hours, but affects the rate of ATO release in the MLV liposomes, while for the DSPC/Chol liposomes there is a slight increase (statistically insignificant) of ATO release at higher temperature.     

Stability of association of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine with liposomes is composition dependent

The ether lipid, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH₃), has anticancer activity, but it has serious side-effects, including hemolysis, which prevent its optimal use. We surmised if ET-18-OCH₃ could be stably associated with liposomes, less free ET-18-OCH₃ would be available for lytic interaction with red cells. Liposome composition variables investigated included acyl chain saturation, phospholipid head group and mole ratio of Chol and ET-18-OCH₃. It was found that attenuation of hemolysis was strongly liposome composition dependent. Some ET-18-OCH₃ liposome compositions were minimally hemolytic. For example, whereas the HI₅ (drug concentration required to cause 5% human red cell lysis) was 5–6 μM for free ET-18-OCH₃, it was approximately 250 μM for DOPC (dioleoylphosphatidylcholine):Chol (cholesterol):DOPE-GA (glutaric acid derivatized DOPE):ET-18-OCH₃, (4:3:1:2) and 640 μM for DOPE (dioleyolphosphatidylethanolamine):Chol:DOPE-GA:ET-18-OCH₃ (4:3:1:2) liposomes. Efflux of carboxyfluorescein (CF) from liposomes and Langmuir trough determinations of mean molecular area of lipids in monolayers (MMAM) were used as indicators of membrane packing and stability. Incorporation of ET-18-OCH₃ in liposomes reduced the MMAM. Reduction in CF permeation was correlated with reduction in hemolysis. The most stable liposomes included components, such as cholesterol, DOPC and DOPE, which have complementary shapes to ET-18-OCH₃.