Role of lipid polymorphism in pulmonary surfactant.

The development of artificial surfactants for the treatment of respiratory distress syndrome (RDS) requires lipid systems that can spread rapidly from solution to the air-water interface. Because hydration-repulsion forces stabilize liposomal bilayers and oppose spreading, liposome systems that undergo geometric rearrangement from the bilayer (lamellar) phase to the hexagonal II (HII) phase could hasten lipid transfer to the air-water interface through unstable transition intermediates. A liposome system containing dipalmitoylphosphatidylcholine was designed; the system is stable at 23 degrees C but undergoes transformation to the HII phase as the temperature increases to 37 degrees C. The spreading of lipid from this system to the air-water interface was rapid at 37 degrees C but slow at 23 degrees C. When tested in vivo in a neonatal rabbit model, such systems elicited an onset of action equal to that of native human surfactant. These findings suggest that lipid polymorphic phase behavior may have a crucial role in the effective functioning of pulmonary surfactant.

Biofilm penetration, triggered release and in vivo activity of inhaled liposomal amikacin in chronic Pseudomonas aeruginosa lung infections.

OBJECTIVES: Chronic infections of Pseudomonas aeruginosa in the lungs of cystic fibrosis patients are intractable antibiotic targets because of their biofilm mode of growth. We have investigated the biofilm penetration, mechanism of drug release and in vivo antimicrobial activity of a unique nanoscale liposomal formulation of amikacin designed specifically for nebulization and inhaled delivery. METHODS: Penetration of fluorescently labelled liposomes into sputum or P. aeruginosa (PA3064) biofilms was monitored by a filter assay and by epifluorescence or confocal scanning laser microscopy. Amikacin release in vitro and rat lung levels after inhalation of nebulized material were measured by fluorescence polarization immunoassay. A 14 day agar bead model of chronic Pseudomonas lung infection in rats was used to assess the efficacy of liposomal amikacin versus free aminoglycosides in the reduction of bacterial count. RESULTS: Fluorescent liposomes penetrated readily into biofilms and infected mucus, whereas larger (1 microm) fluorescent beads did not. Amikacin release from liposomes was mediated by sputum or Pseudomonas biofilm supernatants. Rhamnolipids were implicated as the major releasing factors in these supernatants, active at one rhamnolipid per several hundred lipids within the liposomes. Inhaled liposomal amikacin was released in a slow, sustained manner in normal rat lungs and was orders of magnitude more efficacious than inhaled free amikacin in infected lungs. CONCLUSIONS: Penetration of biofilm and targeted, sustained release from liposomes can explain the superior in vivo efficacy of inhaled liposomal amikacin versus free drug observed in a 14 day infection model. Inhaled liposomal amikacin may represent an important therapy for chronic lung infections.

The captured volume of multilamellar vesicles.

Characterization of classical 'hand-shaken' multilamellar lipid vesicles (MLVs) confirmed that these systems exclude solute during formation thus confounding previous captured volume measurements which typically have utilized solute as a merker of the occluded aqueous space. We used solvent rather than solute to determine the captured volume of these systems and obtained values at least twice those previously reported. We present here a captured volume and lamellarity profile of 'hand-shaken' MLVs and suggest that these parameters are dependent on the lipid concentration present during hydration.

Liposomal entrapment of the neutrophil-derived peptide indolicidin endows it with in vivo antifungal activity.

Indolicidin, a cationic tridecapeptide amide isolated from the granules of bovine neutrophils, has been found to possess potent antimicrobial activity in vitro but its nonselective toxicity could restrict its therapeutic utility. We found that the concentration at which indolicidin disrupts washed human red blood cell membranes coincided with the concentration at which indolicidin self associates. Because of a preponderance of hydrophobic residues, we believed that indolicidin would partition into liposomes which would restrict its exchange with biological tissues and consequently reduce its toxicity. Fluorescence spectroscopy of indolicidin added to 100 nm liposomes comprised of POPC, POPC/cholesterol (60:40 mol%), DPPC, or DPPC/cholesterol (60:40) revealed a large blue-shift and an increase in intensity of the emission profile indicating insertion into the bilayer. Of the lipids tested, POPC exhibited the highest degree of indolicidin binding as determined by fluorescence and encapsulation efficiency. By sequestering indolicidin within the lipid bilayer of 100 nm POPC liposomes we significantly reduced its toxicity to CHO/K1 cells. Likewise, the systemic toxicity of liposomal indolicidin in Balb/c mice was decreased dramatically relative to aqueous solutions; the maximum dose at which no deaths occurred was 0.4 mg/kg for free indolicidin versus 40 mg/kg for indolicidin-POPC. Because of this decrease in toxicity, we were able to administer liposomally encapsulated material at significantly higher concentrations than unencapsulated aqueous material and achieve efficacy in treating animals systemically infected with Aspergillus fumigatus. Liposomal but not free indolicidin was found to be effective in obtaining cures. This report is the first description of the in vivo therapeutic activity of a neutrophil-derived antimicrobial peptide and suggests that liposomal treatment modalities will provide effective strategies for endowing this class of compounds with pharmacological utility.

Interdigitation-fusion: a new method for producing lipid vesicles of high internal volume.

Previously we demonstrated that fused phospholipid sheets can be formed from small unilamellar vesicles (SUVs) comprised of saturated symmetric chain lipids by exposing them to concentrations of ethanol sufficient to cause bilayer interdigitation (Boni et al. (1993) Biochim. Biophys. Acta 1146, 247-257). Here we report that these sheets spontaneously form large, predominately unilamellar vesicles, when exposed to temperatures above their main phase transition temperature (Tm). These vesicles, termed interdigitation-fusion vesicles (IFVs), have mean diameters between 1 and 6 microns, and, once produced, are stable both above and below the Tm of the lipid. The average captured volume of IFVs is dependent upon lipid chain length, the concentration of ethanol used to induce interdigitation-fusion, and size of the precursor liposomes. IFVs comprised of DPPC and DSPC had averaged captured volumes of 20-25 microliters/mumol lipid. IFVs produced from SUVs containing only DPPG or DPPC/DPPG mixtures had captured volumes equivalent to those made from pure DPPC SUVs indicating that charge can be introduced without consequence to the IFV process. Inclusion of cholesterol in precursor vesicles reduced IFV captured volume in a concentration dependent fashion by interfering with interdigitation. Cholesterol could be incorporated, however, into IFVs through admixture with the already formed phospholipid sheets producing far less comprise to captured volume. IFVs are useful as model systems or drug carriers, since their large internal volume allows for efficient encapsulation particularly with regard to compounds such as iodinated radiocontrast agents which otherwise interfere with vesicularization.

Amphotericin B-phospholipid interactions responsible for reduced mammalian cell toxicity.

When interacting with phospholipid in an aqueous environment, amphotericin B forms unusual structures of markedly reduced toxicity (Janoff et al. (1988) Proc. Natl. Acad. Sci. USA 85, 6122-6126). These structures, which appear ribbon-like by freeze-fracture electron microscopy (EM), are found exclusively at amphotericin B to lipid mole ratios of 1:3 to 1:1. At lower mole ratios they occur in combination with liposomes. Circular dichroism (CD) spectra revealed two distinct modes of lipid-amphotericin B interaction, one for liposomes and one for the ribbon-like structures. In isolated liposomes, amphotericin B which comprised 3-4 mole percent of the bulk lipid was monomeric and exhibited a hemolytic activity comparable to amphotericin B suspended in deoxycholate. Above 3-4 mole percent amphotericin B, ribbon-like structures emerged and CD spectra indicated drug-lipid complexation. Minimal inhibitory concentrations for Candida albicans of liposomal and complexed amphotericin B were comparable and could be attributed to amphotericin a release as a result of lipid breakdown within the ribbon-like material by a heat labile extracellular yeast product (lipase). Negative stain EM of the ribbon-like structures indicated that the ribbon-like appearance seen by freeze-fracture EM arises as a consequence of the cross-fracturing of what are aggregated, collapsed single lamellar, presumably interdigitated, membranes. Studies examining complexation of amphotericin B with either DMPC or DMPG demonstrated that headgroup interactions played little role in the formation of the ribbon-like structures. With these results we propose that ribbon-like structures result from phase separation of amphotericin B-phospholipid complexes within the phospholipid matrix such that amphotericin B release, and thus acute toxicity, is curtailed. Formation of amphotericin B-lipid structures such as those described here indicates a possible new role for lipid as a stabilizing matrix for drug delivery of lipophilic substances, specifically where a highly ordered packing arrangement between lipid and compound can be achieved.

Solute-induced shift of phase transition temperature in Di-saturated PC liposomes: adoption of ripple phase creates osmotic stress.

We have examined the calorimetric behavior of large liposomes consisting of symmetric saturated chain phosphatidylcholines. Most notably, for systems made in solutions containing solute (e.g., NaCl, glucose, etc.) there was an additional major endotherm just below the main phase transition temperature. The new endotherm was found to represent a population of lipid whose main phase transition was shifted to lower temperature due to an induced osmotic stress across the membrane. Absent for isoosmotic systems, the osmotic stress was created when the liposome internal volume decreased, a consequence of the Lbeta' (gel) to Pbeta' (rippled) phase transition. That is, rippling of the membrane caused vesicle volume to decrease (> or = 28%) and because the free flow of water outward was restricted by solute, an osmotic gradient was created where none had existed before. The distribution of enthalpy between the new shifted Tm and the expected Tm correlated with the percent of lipid in the outer bilayer and it was concluded that only the outer bilayer sensed the induced stress. Internalized liposome structures were shielded, thus explaining the persistence of the expected Tm in preparations made in solute. The shift in Tm (deltaTm) was discrete and linearly dependent upon lipid chain length for the PC series di-17:0 (deltaTm approximately 1.4 degrees C) through di-20:0 (deltaTm approximately 0.6 degrees C), suggesting a structural change (i.e., lipid packing/orientation) was involved. Although freeze-fracture electron microscopy of stressed and unstressed bilayers revealed no differences in ripple periodicity there were differences in surface features and in vesicle shape. The fact that this phenomenon has gone unnoticed for MLVs is probably due to the fact that these systems are known to exclude solute and thus exist under osmotic compression.

Combination of antitumor ether lipid with lipids of complementary molecular shape reduces its hemolytic activity.

Because the therapeutic use of the antitumor ether lipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH3) is restricted by its hemolytic activity we explored the use of lipid packing parameters to reduce this toxicity by creating structurally optimized ET-18-OCH3 liposomes. We postulated that combination of ET-18-OCH3, which is similar in structure to lysophosphatidylcholine, with lipid molecules of complementary molecular shape (opposite headgroup/chain volume) would likely yield a stable lamellar phase from which ET-18-OCH3 exchange to red blood cell membranes would be curtailed. To quantitate the degree of shape complementarity, we used a Langmuir trough and measured the mean molecular area per molecule (MMAM) for monolayers comprised of ET-18-OCH3, the host lipids, and binary mixtures of varying mole percentage ET-18-OCH3. The degree of complementarity was taken as the reduction in MMAM from the value expected based on simple additivity of the individual components. The greatest degree of shape complementarity was observed with cholesterol: the order of complementarity for the ET-18-OCH3-lipid mixtures examined was cholesterol >> DOPE > POPC approximately DOPC. Phosphorus NMR and TLC analysis of aqueous suspensions of ET-18-OCH3 (40 mol%) with the host lipids revealed them to all be lamellar phase. For ET-18-OCH3 at 40 mol% in liposomes, the hemolytic activity followed the trend of the reduction in MMAM and was least for the ET-18-OCH3/cholesterol system (H50 = 661 microM ET-18-OCH3) followed by ET-18-OCH3/DOPE (H50 = 91 microM) and mixtures with POPC and DOPC which were comparable at H50 = 26 microM and 38 microM, respectively: the H50 concentration for free ET-18-OCH3 was 16 microM. This experimental strategy for designing optimized liposomes with a reduction in exchange, and hence toxicity, may be useful for other amphipathic/lipophilic drugs that are dimensionally compatible with lipid bilayers.

Enhancement of the in vivo circulation lifetime of L-alpha-distearoylphosphatidylcholine liposomes: importance of liposomal aggregation versus complement opsonization.

Incorporation of N-(omega-carboxy)acylamido-phosphatidylethanolamines (-PEs) into large unilamellar vesicles (LUVs) of L-alpha-distearoylphosphatidylcholine (DSPC) was found to dramatically increase the in vivo liposomal circulation lifetime in rats, reaching a maximal effect at 10 mol.% of the total phospholipid. Neither pure DSPC liposomes nor those with the longest circulating derivative, N-glutaryl-dipalmitoylphosphatidylethanolamine (-DPPE), were found to significantly bind complement from serum. Therefore, the relatively short circulation time of pure DSPC liposomes did not appear to be related to greater complement opsonization leading to uptake by the reticuloendothelial system. However, N-(omega-carboxy)acylamido-PEs were particularly efficient inhibitors of a limited aggregation detected for pure DSPC liposomes. The aggregation tendency of DSPC liposomes incorporating various structural analogs of N-glutaryl-DPPE correlated inversely with the circulation lifetimes. Therefore, it is concluded that such PE derivatives enhance the circulation time by preventing liposomal aggregation and avoiding a poorly understood mechanism of clearance that is dependent on size but is independent of complement opsonization. At high concentrations of N-glutaryl-DPPE (above 10 mol.%), the liposomes exhibited strong complement opsonization and were cleared from circulation rapidly, as were other highly negatively charged liposomes. These data demonstrate that both the lack of opsonization and the lack of a tendency to aggregate are required for long circulation. Liposomal disaggregation via N-(omega-carboxy)acylamido-PEs yields a new class of large unilamellar DSPC liposomes with circulation lifetimes that are comparable to those of sterically stabilized liposomes.

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 microM for free ET-18-OCH3, it was approximately 250 microM for DOPC (dioleoylphosphatidylcholine):Chol (cholesterol):DOPE-GA (glutaric acid derivatized DOPE):ET-18-OCH3, (4:3:1:2) and 640 microM 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.

Curvature dependent induction of the interdigitated gel phase in DPPC vesicles.

Ethanol causes biphasic melting behavior in saturated lecithins (Rowe (1983) Biochemistry 22, 3299-3305), a consequence of the formation of the stable interdigitated phase (Simon, S.A. and McIntosh, T.J. (1984) Biochim. Biophys. Acta 773, 169-172). The membrane systems studied to date have been large vesicle systems in which the membrane surface can be assumed to be locally planar. An immediate question arises as to whether surfaces of higher curvature interdigitate. To address this question we have prepared DPPC vesicles of varying diameters which we employed to determine the limiting size at which interdigitation occurs using ethanol as the inducer. We find that with decreasing vesicle size the concentration of ethanol necessary for the onset of interdigitation increases. Small isolated vesicles, at inducing concentrations of ethanol, do not stably interdigitate but rupture and coalesce into a viscous gel comprised of interdigitated lipid sheets. As discussed elsewhere (Ahl et al. (1992) Biophys. J. 243a) these sheets can be used as precursors for producing liposomes of large size and high internal volumes useful in drug delivery or modeling applications.

Doxorubicin physical state in solution and inside liposomes loaded via a pH gradient.

We have examined doxorubicin's (DOX) physical state in solution and inside EPC/cholesterol liposomes that were loaded via a transmembrane pH gradient. Using cryogenic electron microscopy (cryo-EM) we noted that DOX loaded to 200-300 mM internal concentrations in citrate containing liposomes formed linear, curved, and circular bundles of fibers with no significant interaction/perturbation of the vesicle membrane. The individual DOX fibers are putatively comprised of stacked DOX molecules. From end-on views of bundles of fibers it appeared that they are aligned longitudinally in a hexagonal array with a separation between fibers of approx. 3-3.5 nm. Two distinct small angle X-ray diffraction patterns (oblique and simple hexagonal) were observed for DOX-citrate fiber aggregates that had been concentrated from solution at either pH 4 or 5. The doxorubicin fibers were also present in citrate liposomes loaded with only one-tenth the amount of doxorubicin used above (approx. 20 mM internal DOX concentration) indicating that the threshold concentration at which these structures form is relatively low. In fact, from cryo-EM and circular dichroism spectra, we estimate that the DOX-citrate fiber bundles can account for the vast majority (>99%) of DOX loaded via a pH gradient into citrate buffered liposomes. DOX loaded into liposomes containing lactobionic acid (LBA), a monoanionic buffer to control the internal pH, remained disaggregated at internal DOX concentrations of approx. 20 mM but formed uncondensed fibers (no bundles) when the internal DOX concentration was approx. 200 mM. This finding suggests that in the citrate containing liposomes the citrate multianion electrostatically bridged adjacent fibers to form the observed bundles. 13C-NMR measurements of [1,5-13C]citrate inside liposomes suggested that citrate 'bound' to the DOX complex and 'free' citrate rapidly exchange indicating that the citrate-DOX interaction is quite dynamic. DOX release into buffer was relatively slow (<4% at 1 h) from liposomes containing DOX fibers (in citrate loaded to a low or high DOX concentration or in LBA liposomes loaded to a high internal DOX concentration). LBA containing liposomes loaded with disaggregated DOX, where the internal DOX concentration was only approx. 20 mM, experienced an osmotic stress induced vesicle rupture with as much as 18% DOX leakage in less than 10 min. The possible implications for this in vivo are discussed.

Streptokinase entrapment in interdigitation-fusion liposomes improves thrombolysis in an experimental rabbit model.

The successful design of new thrombolytic agents depends on providing these agents with increased clot selectivity. As recently demonstrated (10), entrapment of tissue plasminogen activator into liposomes apparently provided the selective targeting needed to improve the efficacy of this fibrinolytic agent. To test whether liposomal entrapment would benefit streptokinase, a fibrinolytic agent with a different mode of action and inactivation, we compared liposomal streptokinase with free streptokinase in an experimental rabbit model of thrombolysis. First we adapted a new method to produce liposomes of high entrapment efficiency, termed interdigitation-fusion (IF) liposomes, for the encapsulation of streptokinase. This system was then tested in an in vivo rabbit model of thrombolysis where animals with established clots were infused with either free streptokinase (40,000 U/kg), liposomally entrapped streptokinase, free streptokinase+empty liposomes, or the corresponding amount of empty liposomes or saline. Significant differences (p < 0.05) in the percent clot lysis were observed between saline control (22.4 +/- 3.3%; mean +/- S.E.), free streptokinase (36.3 +/- 3.4%), and liposomal streptokinase (47.4 +/- 1.4%). Importantly, animals treated with empty liposomes experienced a level of thrombolysis (32.4 +/- 2.8%) not different to that produced by free streptokinase or empty liposomes plus free streptokinase (38.0 +/- 2.0%). We believe the effect of liposomes alone is due to a transient redistribution or margination of circulating platelets. When tested in rabbits immunized against streptokinase, liposomal (33.8 +/- 1.5%) but not free streptokinase (29.3 +/- 2.1%) showed significant thrombolytic activity compared to saline (22.4 +/- 3.3%) (p < 0.05). The thrombolytic activity was comparable to free streptokinase in non-immunized rabbits. This suggests liposomal streptokinase would have better thrombolytic activity than streptokinase alone and still provide to those patients possessing high levels of anti-streptokinase antibodies (5% of the population) the equivalent degree of therapy expected from free streptokinase.

The determination of liposome captured volume.

Manipulating the process by which lipids assemble to form bilayer membranes has produced a myriad of protocol-dependent liposome types. For each of these systems the arrangement of bilayers is characteristic and can be described by parameters such as aqueous entrapment per mole lipid or captured volume, vesicle size distribution, the average number of lamellae per vesicle and shape. For specific applications as model systems or drug delivery systems specific characteristics are desired. Consequently over the years many techniques have evolved to better quantitate these parameters. Here we focus on and detail several methods to quantitate liposome captured volume. We also briefly describe the available methods to measure the other aforementioned physical properties and discuss their interdependency with captured volume.

Novel therapeutic nano-particles (lipocores): trapping poorly water soluble compounds.

The development of stable spherical lipid-coated drug particles that are termed 'lipocores' is reported here. Unlike conventional lipid-based particles (i.e. liposomes, emulsions, micelles), these particles are comprised solely of a core of a poorly water soluble drug surrounded by polyethyleneglycol conjugated lipid (PEG-lipid) and are formed via a 'kinetic' trapping process. These lipocore particles were made with the acyl chain of 16 carbon length (C16) acyl-chain derivatives of paclitaxel or vinblastine and with the polyene antifungal hamycin. Formation of the particles occurred regardless of the type of PEG-phospholipid used (i.e. acyl chain length, chain saturation, and polymer length) and could also be formed with the negatively charged lipid N-glutaryl-dioleoyl-phosphatidylethanolamine (DOPE-GA). Images from both freeze-fracture electron microscopy and electron cryo-microscopy revealed solid spherical structures with no internal lamellae for the PEG-lipid particles made with the C16 derivatives of paclitaxel (BrC16-T) or vinblastine (C16-Vin). From a solute distribution study of lipocores made with BrC16-T and distearoyl-phosphatidylethanolamine-PEG2000 (DSPE-PEG2000), the particles were found to have no measurable aqueous captured volume. Fluorescence anisotropy and order parameter measurements revealed the core material of these particles to be highly immobilized. The mole ratio of BrC16-T:lipid in the lipocores was typically > 90: < 10 and as high as 98:2, and the refrigerated lipocores were stable for several months. BrC16-T/DSPE-PEG2000 lipocores of 50-100 nm particle size were far less toxic than paclitaxel (Taxol) after intraperitoneally (i.p.) or intravenously (i.v.) administration in mice and were active against i.p. and subcutaneously (s.c.) planted human (OvCar3) ovarian carcinoma grown in SCID mice. It is believed the high drug:lipid ratio, the stability, and therapeutic efficacy of these novel particles make them a paradigm for delivery of poorly water soluble drugs and/or their hydrophobic derivatives.

An electrical and structural characterization of H+/OH- currents in phospholipid vesicles.

Paramagnetic amphiphiles have been utilized to measure and characterize electrogenic H+/OH- ion transport in a series of model membrane systems. Membrane conductivity to H+/OH- ions varies with the method of vesicle preparation and with the level of saturation of the membrane phospholipid. Small sonicated vesicles have the lowest conductivities by approximately an order of magnitude compared to reverse-phase or ether-injection vesicle systems. This conductivity is particularly sensitive to the presence of polyunsaturated lipids in the vesicle membrane. The current-delta pH dependence of the H+/OH- conductivity shows a nonideal behavior and renders the phenomenological membrane permeability dependent upon the experimental value of delta pH that is chosen. These factors can account for much, if not all, of the variability in the published values for the H+/OH- permeability of model membranes. A procedure has been developed to establish and estimate changes in the dipole potential of vesicle bilayers. Using this method, we demonstrate that H+/OH- currents are insensitive to alterations in the membrane dipole field, a result that suggests that these currents are not rate limited by diffusion over simple electrostatic barriers in the membrane interior. In addition, conduction in D2O has been examined, and we find that there is little difference in the magnitudes of D+/OD- currents compared to H+/OH- currents in vesicle systems.

Characterization of H+/OH- currents in phospholipid vesicles.

In pure phospholipid vesicles, the conductivity of H+/OH- ions exceeds that for other simple inorganic ions. Protons achieve electrochemical equilibrium across egg phosphatidylcholine vesicles within tens of minutes. When pH gradients are established across vesicles, transmembrane potentials develop. Conversely, the establishment of transmembrane potentials leads to the formation of pH gradients. When the phenomenological permeability of H+/OH- ions in vesicles is estimated, values are obtained that are much greater (six orders of magnitude larger) than those for Na+ or K+. A wide range in the values for this permeability has been reported; however, much of the discrepancy can be attributed to differences in the vesicle systems and experimental conditions. The H+/OH- current appears to be modulated by changes in membrane dielectric constant. However, the dependence of this current on the pH gradient and on the membrane voltage argues against simple diffusion mechanisms as the source of the H+/OH- current. In addition, in vesicle systems the H+/OH- current shows a surprising invariance to changes in the membrane dipole potential, an observation that argues against the role of simple carriers for H+ and OH- ions.

A selective cholesterol-dependent induction of H+/OH- currents in phospholipid vesicles by amphotericin B.

The effect of amphotericin B on the proton/hydroxide permeability of small unilamellar vesicles has been investigated by using potential-dependent paramagnetic probes. Amphotericin B at 1-10 molecules/vesicle causes a modest 4-8-fold increase in the background H+/OH- permeability of egg phosphatidylcholine (egg PC) vesicles. However, in the presence of cholesterol, amphotericin B promotes a dramatic increase in the H+/OH- permeability of more than 2 orders of magnitude. Surprisingly, this is not observed in vesicle membranes containing ergosterol. In membranes composed of 5-15 mol% ergosterol, amphotericin B is even less effective at promoting H+/OH- currents than in pure egg PC vesicles. The K+ current promoted by amphotericin B in vesicles formed from egg PC and from egg PC plus cholesterol or ergosterol was measured. No significant sterol dependence was found for the K+ current. These results strongly suggest that different mechanisms, or amphotericin B/sterol complexes, are responsible for the induction of H+/OH- and K+ currents. These results have important implications for understanding the therapeutic and toxic effects of amphotericin B.

In vitro and in vivo antifungal activity of amphotericin B lipid complex: are phospholipases important?

Amphotericin B lipid complex for injection (ABLC) is a suspension of amphotericin B complexed with the lipids L-alpha-dimyristoylphosphatidylcholine (DMPC) and L-alpha-dimyristoylphosphatidylglycerol. ABLC is less toxic than amphotericin B deoxycholate (AmB-d), while it maintains the antifungal activity of AmB-d. Active amphotericin B can be released from ABLC by exogenously added (snake venom, bacteria, or Candida-derived) phospholipases or by phospholipases derived from activated mammalian vascular tissue (rat arteries). Such extracellular phospholipases are capable of hydrolyzing the major lipid in ABLC. Mutants of C. albicans that were resistant to ABLC but not AmB-d in vitro were deficient in extracellular phospholipase activity, as measured on egg yolk agar or as measured by their ability to hydrolyze DMPC in ABLC. ABLC was nevertheless effective in the treatment of experimental murine infections produced by these mutants. Isolates of Aspergillus species, apparently resistant to ABLC in vitro (but susceptible to AmB-d), were also susceptible to ABLC in vivo. We suggest that routine in vitro susceptibility tests with ABLC itself as the test material may not accurately predict the in vivo activity of ABLC and that the enhanced therapeutic index of ABLC relative to that of AmB-d in vivo may be due, in part, to the selective release of active amphotericin B from the complex at sites of fungal infection through the action of fungal or host cell-derived phospholipases.