Circular dichroism for the determination of amphotericin B binding to liposomes.

Circular dichroism (CD) of the antifungal antibiotic amphotericin B (AmB) can be used to characterize the liposomal preparations of the drug with regard to the levels of drug bound to the lipids. The very intense dichroic doublet centered around 340 nm of free amphotericin B in water or the dichroism observed above 435 nm can be used to determine the percentages of bound AmB and free AmB in preparations containing high antibiotic/lipid ratios (ranging from 10(-2) to 10(-1] used in these carrier systems. Examples are given for AmB in the presence of small unilamellar vesicles prepared from four saturated fatty acyl chain phosphatidylcholines of different chain lengths, with or without cholesterol. The transfer of AmB from vesicles to two blood components, serum albumin, and lipoproteins can also be monitored by CD under particular conditions.

Two mechanisms of synergism when amphotericin B is used in combination with actinomycin D or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea against the human promyelocytic leukemia cell line HL-60.

The toxic effects of the combinations of amphotericin B (AmB) and actinomycin D or AmB and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea were measured against the human promyelocytic leukemia cells HL-60. The toxicities of both drug combinations were greater than the additive toxicity of each of the drugs used singly, but the exact conditions under which synergism was achieved differed for each combination. The synergism achieved by the AmB-actinomycin combination was accompanied by an AmB-induced increase in uptake of actinomycin D by the HL-60 cells, whereas the synergism of the AmB-1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea combination could be linked to potentiation by 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea of AmB-induced oxidative injury. These results indicate that the synergism of these two drug combinations was caused by different mechanisms.

Transfer of amphotericin B from gel state vesicles to mycoplasma cells: biphasic action on potassium transport and permeability.

The action of amphotericin B on the K+ permeability of Mycoplasma mycoides var. capri cells, containing either cholesterol or ergosterol in their membranes, was studied. When the drug, solubilized in dimethyl sulfoxide, was added directly to the cell suspension, a slightly greater sensitivity to permeabilization was observed for ergosterol-containing cells, confirming the data reported in the literature. When amphotericin B bound to gel state phospholipid vesicles was added to the cell suspension, two effects on cholesterol-containing cells were observed. First, the K+ active transport rates increased; membrane permeabilization and K+ leakage were subsequently detected. For ergosterol-containing cells these sequential events were observed only at amphotericin B concentrations below 10(-6) M. At higher concentrations only K+ leakage was observed. The second permeabilization effect varied with the amphotericin B concentration in different ways in the two types of cells. The permeabilization of ergosterol-containing membranes depended on the amphotericin B/phospholipid molar ratio, whereas the permeabilization of cholesterol-containing membranes did not. In general, the latter remained fairly constant when the total amphotericin B concentration in the medium varied.

Involvement of oxidative damage in erythrocyte lysis induced by amphotericin B.

Lysis of human erythrocytes induced by amphotericin B was retarded when the oxygen tension of the incubation mixture was reduced or when the antioxidant catalase was added; lysis was accelerated when cells were preincubated with the prooxidant ascorbate. In the atmosphere of reduced oxygen tension, the erythrocytes containing carboxyhemoglobin lysed at a slower rate than did the cells containing oxyhemoglobin. Consistent with a role for oxidative damage in lysis, the mixture of erythrocytes and amphotericin B showed an increase in malonyldialdehyde, the product of peroxidation, which paralleled the progression of hemolysis. In contrast, the permeabilizing effect of amphotericin B, measured as a decrease in intracellular K+, was not affected by changes in oxygen tension, catalase, or ascorbate treatment. These results imply that oxidant damage is involved in the lytic, but not in the permeabilizing, action of amphotericin B.

Enhancement of amphotericin B selectivity by antibiotic incorporation into gel state vesicles. A circular dichroism and permeability study.

The permeability induced in cholesterol-or ergosterol-containing phospholipid vesicles by Amphotericin B incorporated into Dipalmitoyl Phosphatidylcholine vesicles has been studied in parallel with Circular Dichroism spectroscopy measurements. In our conditions, Amphotericin B is 5 to 10 times more selective to ergosterol- than to cholesterol-containing vesicles. Such a large difference is not observed when Amphotericin B is directly added to the vesicles suspension as its solution in organic solvent.

Differences in the interaction of the polyene antibiotic amphotericin B with cholesterol- or ergosterol-containing phospholipid vesicles. A circular dichroism and permeability study.

The selective toxicity of the polyene antibiotic amphotericin B between pathogenic eukaryotic organisms and animal cells has often been said to originate in the presence of ergosterol in fungal membranes instead of cholesterol, found in membranes of animal cells. We have tested this hypothesis by measuring the proton efflux induced by amphotericin B in egg yolk phosphatidylcholine small unilamellar vesicles. By measuring circular dichroism under the same conditions, we monitored the interaction of the antibiotic and its conformational changes. Sterol-free vesicles are sensitive to amphotericin B, but the sensitivity of sterol-containing vesicles is always greater and increasingly so with increasing sterol concentration. Ergosterol-containing vesicles are more sensitive than cholesterol-containing vesicles. On the other hand, numerous amphotericin B conformers can be detected in sterol-containing vesicles, depending upon both the concentration of sterol and the amphotericin B sterol ratio. It appears that one conformer, or maybe two at high amphotericin B concentration, is responsible for the induced permeability. From their circular dichroism spectra, these two conformers are the same in the presence of ergosterol or cholesterol. The concentration of amphotericin B necessary to obtain the two conformers is higher with cholesterol than with ergosterol, which agrees with the permeability results.