Search

Interaction of the pentene antibiotic filipin with dimyristoylphosphatidylcholine (DMPC) membranes has been monitored by 2H-NMR, circular dichroism (CD), electronic absorption and fluorescence in the temperature range 10° to 60°C. Interaction appears to depend on whether filipin is added before or after membrane formation and also upon the temperature of the system.

Sublytic amounts of the pore former Amphotericin B (AmB) induced transient movements of Na and K ions across the hepatocyte plasma membranes without altering the intracellular free Ca ion concentration. The presence of 1-5 microM-AmB induced leakage of up to 80% of the intracellular K+ within 3 min, followed by Na+ entry without loss of cell viability. A repair process occurred after 3-10 min, which restored the initial cationic concentrations. Progressive binding of AmB to the cells could be observed by following the disappearance of the intense excitonic dichroic doublet of free AmB. It was shown that the amount of AmB binding, responsible for the Na+ and K+ movements, was low (approx. 16% of total AmB). The recovery process occurred when higher amounts of AmB bound to the cells, and was mediated by Na+/K+-ATPase. The c.d. spectrum of AmB bound to isolated hepatocyte plasma membranes, indicated that during this step AmB formed a complex with cholesterol, similar to that formed by the binary mixture in water.

Circular dichroism was used to compare the binding of several anthracycline antitumor antibiotics to sonicated phosphatidylcholine vesicles. Daunorubicin analogues, differing from the parent by structural changes in the amino sugar moiety of the molecule, were tested both with vesicles that contained negatively charged phospholipids and with neutral vesicles. The self-association properties of the analogues were also investigated. Binding to negatively charged vesicles was not strictly dependent on electrostatic interactions, since the characteristics of daunorubicin binding were totally different from those of Adriamycin (doxorubicin). Furthermore, the cardiotoxicity of these molecules did not have its origin in their quantitatively preferential electrostatic binding to negatively charged cardiolipin-containing membranes: DR-19, a daunorubicin derivative having lower cardiotoxicity than the parent compound, which bound to negatively charged vesicles in a manner quite similar to that of Adriamycin, whereas DR-10, another daunorubicin derivative with higher cardiotoxicity, bound poorly to negatively charged vesicles.

We compared the anticellular effects of liposomal amphotericin B (AmB) formed from AmB and small unilamellar vesicles. The small unilamellar vesicles with or without cholesterol were prepared from three L-alpha-phosphatidylcholines with saturated acyl chains of different lengths: distearoyl (C18), dipalmitoyl (C16), and dimyristoyl (C14). We found that the anticellular potency of liposomal AmB, compared with that of free AmB, decreased with decreasing length of the acyl chain of the phospholipid and increased with the addition of cholesterol. In a parallel study (S. Jullien, A. Vertut-Croquin, J. Brajtburg, and J. Bolard, Anal. Biochem. 172:197-202, 1988), we found that binding of AmB to vesicles decreased with increasing length of the acyl chain of the phospholipid and decreased with the addition of cholesterol. We conclude that the anticellular effects of liposomal AmB preparations are due to the levels of AmB remaining free (unbound to the lipids) in these preparations.

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.

The aim of this work was to study the interaction of pirarubicin, an anthracycline used in chemotherapy, with planar palmitoyloleoylphosphatidylcholine (POPC) monolayers and bilayers employed as membrane models. Different modes of preparation were used and compared, after a preliminary surface pressure study had been done to determine the characteristics of the pirarubicin−POPC interaction and the level of pirarubicin incorporated in phospholipids. A first method consisted of studying the adsorption of the antibiotic (in aqueous solution) in a POPC monolayer kept at a constant pressure. The final monolayer was composed of almost 11% of pirarubicin in the POPC/pirarubicin mixture at 25 mN/m. The second method consisted of spreading mixtures of POPC/pirarubicin (pirarubicin being now dissolved in an organic solvent) at the air−water interface:  the maximum percentage of pirarubicin in the monolayer was 38% at 25 mN/m. These two kinds of monolayers were then transferred by a Langmuir−Blodgett technique onto prisms previously coated with silver. Surface-enhanced resonance Raman spectroscopy gave information about the orientation of the molecules with respect to the silver coating. These spectra were compared with those of pure POPC bilayers transferred onto the prism and then put in contact with an aqueous solution of pirarubicin. Different results were observed for the orientation of pirarubicin in the bilayers (tilted or parallel to the silver), according to the mode of preparation of the samples, underlining the importance of the choice of the experimental protocol and precautions to take in the examination of the results.