Induction of partial protection against Leishmania donovani by promastigote antigens in negatively charged liposomes

Negatively charged liposomes, proposed as potential vaccine adjuvants, have been extensively studied in association with various antigens. In the present study, we investigated the adjuvanicity of negatively charged liposomes to enhance the protective immunity of membrane antigens of Leishmania donovani promastigotes (LAg). In comparison to the control mice immunized with phosphate-buffered saline and empty liposomes, immunization with free LAg led to significant levels of protection against infection with virulent promastigotes. Encapsulation of LAg in liposomes also induced effective protection. However, the level of protection by LAg-liposome was not significantly different from that induced by free LAg. Investigation of the immune responses showed, in contrast to free LAg, that immunization with LAg-liposome elicited strong antibody responses. IgG isotype analysis revealed the presence of all 4 isotypes. However, the titer of IgG1 was significantly higher than IgG2a, IgG2b, and IgG3. Following infection, stimulation of IgG and IgG isotypes did not differ in the different immunization groups. Delayed-type hypersensitivity (DTH) analysis after immunization showed significant induction by LAg and LAg-liposomes, in comparison to controls. With infection, again, the level of DTH in all the groups became almost comparable. Stimulation of insufficient cellular response, as reflected by DTH and potentiation of IgG1 over IgG2a, IgG2b, and IgG3 suggest a dominance of Th2 response with this liposome-antigen formulation, resulting in weak protection against visceral leishmaniasis.     

Comparison of BCG,MPL and cationic liposome adjuvant systems in leishmanial antigen vaccine formulations against murine visceral leishmaniasis

Background  

The development of an effective vaccine against visceral leishmaniasis (VL) caused by Leishmania donovani is an essential aim for controlling the disease. Use of the right adjuvant is of fundamental importance in vaccine formulations for generation of effective cell-mediated immune response. Earlier we reported the protective efficacy of cationic liposome-associated L. donovani promastigote antigens (LAg) against experimental VL. The aim of the present study was to compare the effectiveness of two very promising adjuvants, Bacille Calmette-Guerin (BCG) and Monophosphoryl lipid A (MPL) plus trehalose dicorynomycolate (TDM) with cationic liposomes, in combination with LAg, to confer protection against murine VL.     

Vaccination against murine cutaneous leishmaniasis by using Leishmania major antigen/liposomes. Optimization and assessment of the requirement for intravenous immunization

Efficacy of vaccination against cutaneous leishmaniasis in highly susceptible BALB/c mice was assessed comparatively by using radiation-attenuated promastigotes and colloidal Ag mixtures generated from a mixed Leishmania major (LV39) isolate (SLV39) and from a virulent cloned line (SVJ2) derived from the Jericho 2 L. major isolate. Dehydration-rehydration vesicle (DRV) liposomes were used as adjuvants. In optimization experiments phospholipid composition of DRV was varied, and the distearoyl derivative (DSPC) (liquid-crystalline phase transition temperature (Tc) 54 degrees C) of egg lecithin L-alpha-phosphatidylcholine was found to be superior to the dipalmitoyl derivative (DPPC, Tc 41.5 degrees C) and underivatized L-alpha-phosphatidylcholine (Tc -10 degrees C). The criteria studied were in vivo priming for a secondary in vitro proliferative response and the prepatency of lesion onset after L. major challenge of mice immunized once i.v. A single s.c. immunization with SLV39 either free or entrapped within DSPC liposomes primed spleen cells to produce significant levels of IL-3 when stimulated with SLV39 in vitro. In contrast, the i.v. route of immunization with the same Ag preparations led to little or no IL-3 production by the spleen cells. Despite development of significant T cell activation, both SLV39 and SVJ2 administered s.c., either free or entrapped within liposomes, were not protective. However, i.v. immunization four times with SVJ2 entrapped within DSPC liposomes induced a level of resistance comparable with that of 2 x 10(7) gamma-irradiated promastigotes in the stringent BALB/c L. major model. Although significant, protection conferred after i.v. immunization with SLV39/DSPC liposomes was less effective. These data therefore show that DSPC/DRV liposomes, although a good adjuvant for inducing protective immunity to cutaneous leishmaniasis, are not able to overcome the requirement for an i.v. route of immunization with the leishmanial Ag preparation. Additionally, they demonstrate a correlation between IL-3 secretion and non-protection. They also suggest that SVJ2 represents a better source of protective Ag than SLV39.     

Inhibition by Liposomal Cholesterol of Streptococcus Pneumoniae Induced Lung Epithelial Cell Damage

Abstract

Streptococcus pneumoniae was shown to be capable of lysing A549 cells in culture. Membrane damage to cells as assessed by trypan blue exclusion increased with increasing concentration of bacteria. After 45 min of incubation with 7.5 × 10⁸ bacteria/ml less than 20% of A549 cells excluded trypan blue. The lytic activity of S. pneumoniae was inhibited by phosphatidylcholine liposomes containing cholesterol. Using an haemolysis assay and S. pneumoniae's culture filtrates, the efficiency of the anti-lytic activity of liposomes was found to be distearoylphosphatidylcholine (DSPC) > dipalmitoylphosphatidylcho-line (DPPC) > dimyristoylphosphatidylcholine (DMPC). Furthermore, the anti-lytic activity also depended on the cholesterol content in a non-trivial manner. There was no protection against haemolytic activity at cholesterol content of less than 20% for DSPC and 35 mole% for DPPC and DMPC liposomes respectively. Above these threshold values inhibition of lytic activity increased sharply. In agreement with the haemolysis results, A549 cells were protected by liposomes against the lytic activity of S. pneumoniae with the efficiency also being DSPC > DPPC > DMPC. Clearly the efficiency of liposomal cholesterol is increased with increasing gel to liquid crystalline phase transition temperature of the lipid matrix. The results suggest that liposomal cholesterol may be used to protect the host against cell damage caused by S. pneumoniae.     

Incorporation of bovine thyroid peroxidase in liposomes

In the microsomal fraction of thyroid glands, the temperature dependence of DPH fluorescence polarization showed a discontinuity in the range of 29-33 degrees C. The transition temperatures of DMPC, DPPC and DSPC are near to the observed for the microsomal fraction. So that, thyroid peroxidase (TPO) was incorporated into liposomes made with these phospholipids. When DPH was incorporated in this peroxidase-liposome complex, a less pronounced phase transition was observed in the profiles of temperature dependence of DPH polarization, and the incorporation of the enzyme decreased the Tc. Arrhenius plots of TPO incorporated into liposomes showed discontinuities at similar temperatures observed by fluorescence polarization. The decrease of transition temperature of liposomes induced by thyroid peroxidase incorporation suggests that this enzyme seems to need a fluid medium for its enzyme activity.     

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.     

Phospholipase C and the physical states of polar head groups of lipids

Summary Activity of phospholipase C fromClostridium perfringens on liposomes made fromsn-3-phosphatidylcholine, dimyristoyl (DMPC), dipalmitoyl (DPPC) or distearoyl (DSPC) was measured at various temperatures and was correlated with their gel/liquid-crystalline phase transitions (T _c : 23, 41.5, 52°C for DMPC, DPPC, DSPC, respectively). In all cases, the activity of phospholipase C was high in the gel phases of the substrates and was almost zero in their liquid-crystalline phases. Fluorescence depolarization measurements of N-dansyl-sn-3-phosphatidylethanolamine (DPE) and 1,6-diphenyl-1,3,5-hexatriene (DPH) incorporated into the liposomes showed that both the head group and the alkyl chains of the lipids were immobilized in the gel phases but were highly mobile in the liquid-crystalline phase. These results indicate that the rotational mobility of lipids (both of the head groups and the alkyl chains) was not a major factor in the phospholipase C reaction. It is inferred that some electrostatic and/or hydrophobic interactions might play important roles in regulation of the phospholipase C activity.     

A region-matched hydrophobic interaction between melittin and dimyristoylphosphatidylcholine in a ternary mixture of phosphatidylcholines

Interaction of melittin with phosphatidylcholine molecules in pure vesicles, binary mixtures and a ternary mixture of dimyristoylphosphatidylcholine IDMPC), dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC) was investigated by differential scanning calorimetry. Melittin binds preferentially with DMPC, and results in segregation of DMPC in binary mixtures of DMPC/DPPC and DMPC/DSPC and in a ternary mixture of DMPC/DPPC/DSPC. The results indicate that the hydrophobic part of peptide interacts preferentially with the phospholipid which has the same size of hydrophobic region or fatty acyl chains.     

Lectin-mediated agglutination of liposomes containing glycophorin: Effects of acyl chain length

Glycophorin from human erythrocytes has been incorporated into liposomes of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC). The thermal properties of unsonicated liposomes with glycophorin/lipid molar ratios up to 4·10^?3 have been studied by differential scanning calorimetry and the numbers of lipids withdrawn from participation in the gel-to-lamellar phase transition were found to be 42±22 (DMPC), 197±28 (DPPC) and 240±64 (DSPC). The initial rates of agglutination of sonicated liposomes with glycophorin/lipid molar ratios up to 4·10^?3 by wheat germ agglutinin in the concentration range 0–7 μM have been measured over a range of temperature. Below the gel-to-lamellar phase transition (T_c) the rates of agglutination increase with acyl chain length in the sequence DMPC < DPPC < DSPC. Agglutination is found to be second order in liposome concentration and is completely reversed on saturation of the wheat germ agglutinin-binding sites by N-acetylglucosamine. Agglutination rates decrease with increasing temperature below T_c and are largely independent of temperature above T_c. The results are discussed in relation to the clustering of glycophorin in the phospholipid bilayers and its effect on binding and subsequent interliposomal bridge formation by wheat germ agglutinin.     

Dye permeability at phase transitions in single and binary component phospholipid bilayers

By encapsulating a pH-sensitive dye, phenol red, in multilamellar liposomes of DMPC, DPPC and DMPC/DPPC mixtures, the permeability of these phospholipid bilayers to dye as a function of temperature has been studied. For both DMPC and DPPC liposomes, dye release begins well below the main gel-to-liquid-crystalline phase transition (24°C and 42°C, respectively) at temperatures corresponding to the onset of the pretransition (about 14°C and 36°C, respectively) with DPPC liposomes exhibiting a permeability anomaly at the main phase transition (42°C). The perturbation occurring in the bilayer structure that allows the release of encapsulated phenol red (approx. 5 Å diameter) is not sufficient to permit the release of encapsulated haemoglobin (approx. 20 Å diameter, negatively charged). In liposomes composed of a range of DMPC/DPPC mixtures, dye release commences at the onset of the pretransition range (determined by optical absorbance measurements) and increases with increasing temperature until the first appearance of liquid crystalline phase after which no further dye release occurs. Interestingly, the dye retaining properties of DMPC and DPPC liposomes well below their respective pretransition temperature regions are very different: DMPC liposomes release much encapsulated dye at incubation temperatures of 5°C whilst DPPC liposomes do not.     

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.     

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.     

Membrane fluidity as affected by the insecticide lindane

Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to study the interaction of lindane with model and native membranes. Lindane disorders the gel phase of liposomes reconstituted with dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC and DSPC), since it broadens and shifts the main phase transition, but no apparent effect is detected in the fluid phase. These effects of lindane are more pronounced in bilayers of short-chain lipids, e.g., DMPC. In equimolar mixtures containing DMPC and DSPC, lindane preferentially interacts with the more fluid lipid species inducing lateral phase separations. However, in mixtures of DMPC and DPPC, the insecticide only broadens and shifts the main phase transition, i.e., an effect similar to that observed in bilayers of pure lipids. Lindane has no apparent effect in DMPC bilayers enriched with high cholesterol content (greater than or equal to 30 mol%), whereas disordering effects can still be detected in bilayers with low cholesterol (less than 30 mol%). Apparently, lindane does not perturb the fluid phase of representative native membranes, namely, mitochondria, sarcoplasmic reticulum, myelin, brain microsomes and erythrocytes in agreement with the results obtained in fluid phospholipid bilayers, despite the reasonable incorporation of the insecticide in these membranes, as previously reported (Antunes-Madeira, M.C. and Madeira, V.M.C. (1985) Biochim. Biophys. Acta 820, 165-172).     

Membrane fluidity as affected by the organochlorine insecticide DDT

Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to study the interaction of DDT with model and native membranes. DDT decreases the phase transition midpoint temperature (Tm) of liposomes reconstituted with dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC and DSPC), and broadens the thermotropic profile of the transition. The effects of DDT are concentration dependent and are more pronounced in bilayers of short-chain lipids, e.g., DMPC. The insecticide fails to alter DPH polarization in the fluid phase of the above lipids. Similar effects were observed in binary mixtures of DMPC plus DPPC. Furthermore, DDT alters the single broad transition of the equimolar mixture of DMPC plus DSPC into a biphasic transition. The lower temperature component has a midpoint at 25 degrees C, i.e., a value close to the Tm of DMPC. DDT inhibits to some extent the cholesterol-induced ordering in DMPC bilayers and high cholesterol concentrations (greater than or equal to 30 mol%) do not prevent insecticide interaction, conversely to the effect observed for lindane (Antunes-Madeira, M.C. and Madeira, V.M.C. (1989) Biochim. Biophys. Acta 982, 161-166). Apparently, the bilayer order is not disturbed by DDT in fluid native membranes of mitochondria and sarcoplasmic reticulum, but moderate disordering effects are noticed in membranes enriched in cholesterol, namely, brain microsomes and erythrocytes.     

Phospholipid miscibility in ternary mixtures

The gel to liquid-crystalline phase transition of aqueous dispersions of phospholipid mixtures was investigated by means of the repartition of the spin label 2,2,6,6-tetramethylpiperidine-I-oxyl between aqueous space and lipid hydrocarbon region. The dimyristoylphosphatidylcholine (DMPC)/dibehenoylphosphatidylcholine (DBPC) and dipalmitoylphosphatidylcholine (DPPC)/DBPC phase diagrams indicate gel phase immiscibility, whereas the distearoylphosphatidylcholine (DSPC)/DBPC phase diagram indicates non-ideal gel phase miscibility at low DBPC molar fractions. Aqueous dispersions of DMPC/DPPC/DBPC ternary mixtures show two distinct phase transitions, the first associated with the melting of a DMPC/DPPC phase and the second with the melting of a DBPC phase. Aqueous dispersions of DMPC/DSPC/DBPC ternary mixtures show to phase transitions at low DSPC molar fractions; the first is probably associated with the melting of a DMPC/DSPC phase, and the second with the melting of a DBPC/DSPC phase. At high DSPC molar fractions, only one phase transition is observed; this suggests that all the lipids are mixed in gel state membranes.     

Partition of lindane in synthetic and native membranes

Partition coefficients of the insecticide gamma-1,2,3,4,5,6-hexachlorocyclohexane (trivially, lindane) were determined in model and native membranes. 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 dramatically decreases partition (2100 falls to 100, at 10 degrees C) and abolishes the temperature dependence. First-order phase transitions of dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC and DSPC) are accompanied by a sharp increase in lindane partition. Apparently, the insecticide is easily accommodated in bilayers of short-aliphatic-chain lipids, since the partitions were 2450, 600 and 50 in DMPC, DPPC and DSPC, respectively, at temperatures 10 Cdeg below the midpoint of their transitions. The lindane partition sequence in native membranes is as follows: mitochondria, sarcoplasmic reticulum, myelin, brain microsomes and erythrocytes. This sequence correlates reasonably well with the relative content of cholesterol and is similar in liposomes of total extracted lipids, although the absolute partitions showed decreased values. Therefore, the presence of proteins in native membranes contributes to the insecticide partition, probably by favouring its interaction with lipids.     

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.     

Leishmania donovani: immunostimulatory cellular responses of membrane and soluble protein fractions of splenic amastigotes in cured patient and hamsters

Visceral leishmaniasis (VL), caused by the intracellular parasite Leishmania donovani, L. chagasi and L. infantum is characterized by defective cell-mediated immunity (CMI) and is usually fatal if not treated properly. An estimated 350 million people worldwide are at risk of acquiring infection with Leishmania parasites with approximately 500,000 cases of VL being reported each year. In the absence of an efficient and cost-effective antileishmanial drug, development of an appropriate long-lasting vaccine against VL is the need of the day. In VL, the development of a CMI, capable of mounting Th1-type of immune responses, play an important role as it correlate with recovery from and resistance to disease. Resolution of infection results in lifelong immunity against the disease which indicates towards the feasibility of a vaccine against the disease. Most of the vaccination studies in Leishmaniasis have been focused on promastigote--an infective stage of parasite with less exploration of pathogenic amastigote form, due to the cumbersome process of its purified isolation. In the present study, we have isolated and purified splenic amastigotes of L. donovani, following the traditional protocol with slight modification. These were fractionated into five membranous and soluble subfractions each i.e MAF1-5 and SAF1-5 and were subjected for evaluation of their ability to induce cellular responses. Out of five sub-fractions from each of membrane and soluble, only four viz. MAF2, MAF3, SAF2 and SAF3 were observed to stimulate remarkable lymphoproliferative, IFN-γ, IL-12 responses and Nitric Oxide production, in Leishmania-infected cured/exposed patients and hamsters. Results suggest the presence of Th-1 type immunostimulatory molecules in these sub-fractions which may further be exploited for developing a successful subunit vaccine from the less explored pathogenic stage against VL.     

A differential scanning calorimetry study of phosphocholines mixed with paclitaxel and its bromoacylated taxanes

High sensitivity differential scanning calorimetry (DSC) was used to investigate the thermotropic phase properties of binary mixtures of disaturated phosphocholines (PCs) and alpha-bromoacyl taxane derivatives. The alpha-bromoacyl taxanes were synthesized as hydrolyzable hydrophobic prodrugs of paclitaxel. The PCs used were 1, 2-dimyristoyl-sn-glycero-3-phosphatidyl-choline (DMPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1, 2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). The bromoacyl chain lengths of the taxane prodrugs were varied from 6 to 12 or 16 carbons. For comparison, paclitaxel and PC mixtures were also examined. DSC data from DPPC and bromoacyl taxane mixtures showed a complete abolition of the pretransition and significant broadening of the main phase transition with increasing amounts of bromoacyl taxane prodrugs. The effects were more pronounced with the long-chain compared to the short-chain prodrugs. Under equivalent DSC conditions, the short-chain DMPC showed greater changes in thermotropic phase behavior than with DPPC on taxane addition, suggesting an enhanced degree of association with the fluid-type bilayers. Under similar conditions, the long-chain DSPC bilayers showed a far less significant change in phase behavior on taxane addition than DPPC. These changes were also chain length-dependent for both the PCs and the taxane prodrugs. In contrast, PC and paclitaxel (lacking the acyl chain) mixtures under similar conditions showed insignificant changes in the endotherms, suggesting only slight insertion of the molecule into the PC bilayers. From the DSC data it is apparent that taxane prodrugs solvated in DMPC bilayers more than in DPPC and DSPC bilayers, and taxane prodrugs with longer acyl chains were able to associate with PCs better than those with shorter chain prodrugs. DSC data also suggest that paclitaxel was poorly associated with any of the PCs. In general, the amount of taxane association with bilayers decreased in order: DMPC > DPPC > DSPC. In contrast, the transition enthalpy (DeltaH) of DMPC, DPPC, and DSPC mixtures with paclitaxel showed significantly lower enthalpies than with taxane prodrugs. Taken together, the DSC data suggest that the acyl chains of paclitaxel prodrugs have some access into the bilayers via alignment with the acyl chain of the PC component.     

Topology of gel-phase domains and lipid mixing properties in phase-separated two-component phosphatidylcholine bilayers.

The influence of the lipid mixing properties on the lateral organization in a two-component, two-phase phosphatidylcholine bilayer was investigated using both an experimental (fluorescence recovery after photobleaching (FRAP)) and a simulated (Monte Carlo) approach. With the FRAP technique, we have examined binary mixtures of 1-stearoyl-2-capryl-phosphatidylcholine/1,2-distearoyl-phosphat idylcholine (C18C10PC/DSPC), and 1-stearoyl-2-capryl-phosphatidylcholine/1,2-dipalmitoyl-phospha tid ylcholine (C18C10PC/DPPC). Comparison with the 1,2-dimyristoyl-phosphatidylcholine/1,2-distearoyl-phosphatidylcholine (DMPC/DSPC) previously investigated by FRAP by Vaz and co-workers (Biophys. J., 1989, 56:869-876) shows that the gel phase domains become more effective in restricting the diffusion coefficient when the ideality of the mixture increases (i.e., in the order C18C10PC/DSPC-->C18C10PC/DPPC-->DMPC/DSPC). However, an increased lipid miscibility is accompanied by an increasing compositional dependence: the higher the proportion of the high-temperature melting component, the less efficient the gel phase is in compartmentalizing the diffusion plane, a trend that is best accounted for by a variation of the gel phase domain shape rather than size. Computer-simulated fluorescence recoveries obtained in a matrix obstructed with obstacle aggregates of various fractal dimension demonstrate that: 1) for a given obstacle size and area fraction, the relative diffusion coefficient increases linearly with the obstacle fractal dimension and 2) aggregates with a lower fractal dimension are more efficient in compartmentalizing the diffusion plane. Comparison of the simulated with the experimental mobile fractions strongly suggests that the fractal dimension of the gel phase domains increases with the proportion of high-temperature melting component in DMPC/DSPC and (slightly) in C18C10PC/DPPC.