No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Oligonucleotide delivery by a cationic derivative of the polyene antibiotic amphotericin B
Abstract
We report a study of the behavior of oligodeoxyribonucleotide (ODN)/amphotericin B3-(N′-dimethylamino)propylamide (AMA) complexes, in the presence of lipid monolayers and large unilamellar vesicles. This study follows the recent discovery of the capacity of AMA, as a new cationic vector, to enhance ODN cellular uptake and efficacy. It aims at investigating the internalization mode of a nucleic acid by AMA. A first study at the air–water interface of AMA and AMA/ODN by surface pressure measurement shows that only free AMA would adsorb at the air–water interface. Second, in the presence of zwitterionic phospholipid- and sterol-containing mixture, ODN–AMA interactions in solution would be higher than lipid–AMA interactions at the interface. In monolayer or with large unilamellar vesicles, AMA monomers adsorb mainly at the phospholipid interface. These results favor a crossing mechanism through AMA channel formation, despite the size of ODN.
... At the presence of these cations, so-called triple complexes are formed [1]. Also, the transfer of DNA into the cell is more effective when nucleic acid is in complexes with cationic lipids [3], cationic antibiotic [4] or cationic surfactants [5]. ...
The method of electrostriction has been applied to study the physical properties of supported lipid membranes (sBLM) during membrane formation at application of negative potential. Application of negative potential -350 mV to the sBLM during its formation resulted in more compact membrane structure as revealed by higher elastic modulus in comparison with sBLM formed without application of this potential. We also studied interaction with sBLM cationic surfactant hexadecylamine (HDA), HDA-DNA and DNA-Mg(2+) complexes. Interaction of HDA with sBLM resulted in decrease of membrane capacitance and two-directional effect on elasticity modulus (increase or decrease), which can be caused by different aggregation state of surfactant at the surface of sBLM. In contrast with effect of HDA, the complexes of HDA-DNA resulted, in most cases, increase of elasticity modulus and increase of membrane capacitance, which can be caused by incorporation of these complexes into the hydrophobic interior of the membrane. Certain part of these complexes can, however, be adsorbed on the sBLM surface. DNA itself does not cause substantial changes of physical properties of sBLM; however, addition of bivalent cations Mg(2+) to the electrolyte-contained DNA caused substantial increase of elasticity modulus and surface potential. These changes are, however, much slower than that observed for HDA-DNA complexes, which can be caused by slow competitive exchange between Na(+) and Mg(2+) ions.
This review reports the significance of bilayer lipid membranes on a solid support (sBLM) for the construction of biosensors. The methods of formation of lipid membranes on different solid supports, including different metals (silver, gold, stainless steel, mercury), agar, and conducting polymers, are presented. Several examples of the application of electrostriction and dielectric relaxation methods for the study of the mechanical properties and dynamics of solid-supported bilayers are shown. We demonstrate that these methods are useful for studying the physical properties of chemically modified supported membranes, the interaction of surfactants and nucleic acids with lipid membranes, the binding of enzymes and antibodies to sBLM, and hybridization of nucleic acids on the membrane surface. A comparison of the mechanical properties of sBLM of various compositions and method of formation is presented.
In this paper we present the characteristics of the Langmuir monolayers formed by amphotericin B 3-(N',N'-dimethylamino) propyl amide (AMA) that is a derivative of amphotericin B (AmB), a polyene antifungal antibiotic. Also, the interactions of AMA with sterols (cholesterol, ergosterol) and a phospholipid (l-α Phosphatidylcholine dipalmitoyl — DPPC) were studied in the mixed films. The experiments were based on measurements of the surface pressure during film compression and the results were analyzed with the isothermal compressibility, the excess area per molecule, the excess free energy of mixing and the total free energy of mixing values. It was found that the ionic state of AMA molecule affects its biological properties. Moreover, the size of the antibiotic molecule influences its interactions with sterols. Thus, AMA that is larger molecule than AmB reflects the higher selectivity as compared to the parent antibiotic. Interestingly, contrary to AmB derivatives investigated previously, the interactions between DPPC and AMA are not important from the point of view of biological properties of the antibiotic studied.
Biomembranes belong to the most important structures of the cell and the cell organels. They play not only structural role of the barrier separating the external and internal part of the membrane but contain also various functional molecules, like receptors, ionic channels, car-riers and enzymes. The cell membrane also preserves non-equillibrium state in a cell which is crucial for maintaining its excitability and other signaling functions. The growing interest to the biomembranes is also due to their unique physical properties. From physical point of view the biomembranes, that are composed of lipid bilayer into which are incorporated inte-gral proteins and on their surface are anchored peripheral proteins and polysaccharides, rep-resent liquid scrystal of smectic type. The biomembranes are characterized by anisotropy of structural and physical properties. The complex structure of biomembranes makes the study of their physical properties rather difficult. Therefore several model systems that mimic the structure of biomembranes were developed. Among them the lipid monolayers at an air-water interphase, bilayer lipid membranes (BLM), supported bilayer lipid membranes (sBLM) and liposomes are most known. This work is focused on the introduction into the ,,physical word" of the biomembranes and their models. After introduction to the membrane structure and the history of its establishment, the physical properties of the biomembranes and their models areare stepwise presented. The most focus is on the properties of lipid monolayers, BLM, sBLM and liposomes that were most detailed studied. This contribution has tutorial char-acter that may be usefull for undergraduate and graduate students in the area of biophysics, biochemistry, molecular biology and bioengineering, however it contains also original work of the author and his co-worker and PhD students, that may be usefull also for specialists working in the field of biomembranes and model membranes.
Structural analogues of the natural polyamines which contain a Si(CH3)2 group in the central carbon chain have previously been found to be cytostatic to various tumor cell lines and to inhibit tumor cell growth in experimentally-grafted animals. We show that membrane damages induced by hexamine in chinese ovary cells are the earliest cytotoxic events. We report the interaction of natural and dimethylsilane polyamines with membrane models. These polyamines protect the red blood cell membrane from hypotonic-induced cell lysis. In the case of dimethylsilane derivatives and to a lesser extent for putrescine, this stabilizing effect is followed, for longer times, by the progressive lysis of cell membranes, suggesting a biphasic interaction of polyamines with membrane components. The Langmuir's monolayer method shows that the first step of the electrostatic interaction of spermine with negatively charged phospholipids, egg phosphatidic acid (EPA), is characterised by a decrease of the mean molecular area of phospholipid. Over longer times, an increase of the mean molecular area is observed for all polyamines suggesting the insertion of their hydrocarbon chains into the monolayers. Probably as a consequence of their higher hydrophobicity, the maximal increase is obtained with dimethylsilane derivatives.
This study focuses on the importance of sterols in the action of miltefosine (hexadecylphosphocholine, HePC) against Leishmania donovani.
Plasma membranes of L. donovani promastigotes were depleted of sterol using methyl-beta-cyclodextrin (MCD) and cholesterol oxidase (CH-OX). Sterols were quantified and HePC susceptibility was assessed using the MTT test. A biomimetic model of the outer leaflet of a Leishmania plasma membrane was used to decipher the HePC-lipid interactions.
CH-OX, which is known to act more specifically on condensed membranes, therefore at the level of lipid rafts, gave a better extraction yield in HePC-resistant parasites, confirming the more rigid structure of their membranes than those of wild-type parasites. Sterol depletion was responsible for a 40% decrease in HePC susceptibility in both wild-type and HePC-resistant parasites. Sterol repletion of the sterol-depleted parasites restored HePC susceptibility. The biomimetic model of the outer leaflet of a Leishmania plasma membrane confirmed that condensed microdomains were able to incorporate higher quantities of HePC than fluid ones and this result was amplified when the sterol concentration was increased.
Sterol and lipid rafts probably play a significant role as an HePC reservoir providing a constant supply to the previously described transporter. In addition, (1)H NMR experiments suggested that HePC stimulated lipid trafficking in parasites.
A novel approach based on a plasma membrane permeability-disturbing agent was proposed as an antisense oligonucleotide delivery system. AMA, a derivative of the polyene antibiotic amphotericin B, formed a stable complex when mixed with phosphodiester oligodeoxynucleotides and enhanced the intracellular uptake of a 5' fluoresceinated anti-mdr1 20-mer into NIH-MDR-G185 cells. The nonlabeled phosphorothioate form of the oligodeoxynucleotide, complexed to AMA, inhibited P-glycoprotein expression with better efficiency and less nonspecific effects than when vectorized by Lipofectin. AMA may thus be a good agent for antisense strategy.
We have recently described how oligonucleotide (ON) stability and release from O/W cationic emulsions are governed by the lipid composition. The aim of the present paper was to investigate the properties of the ON/lipid complexes through fluorescence resonance energy transfer (FRET), size, surface tension measurements and cryomicroscopy. Starting from a typical emulsion containing stearylamine as a cationic lipid, the influence of the lipid structure (monocationic molecules bearing mono or diacyl chains, or polycations) as well as of the presence of PEGylated lipids, were studied. The presence of a positive charge on the droplet surface clearly contributed to enhance the ON interaction with lipid monolayers and to bring the ON molecules closer to the interface. Hydrophobic interactions through the acyl chains were shown to further enhance the anchorage of the ON/lipid complexes. In contrast, the incorporation of PEGylated lipids acted as a barrier against the establishment of electrostatic bindings, the polyethyleneglycol chains acting themselves as interaction sites for the ON leading to hydrophilic complexes. Similar features were observed for the polycationic lipid, and cryomicroscopy revealed the existence of bridges of various intensities between the droplets of the emulsion containing either PEG or the polycation, probably because of the configuration of the ON at the interface.
This work presents an analysis of the physicochemical interactions of natural and dimethylsilyl polyamines with an anionic deuterated phospholipid monolayer, d(62)DPPG (dipalmitoyl phosphatidyl glycerol), at the air-water interface. It was motivated by previous studies, which suggested an antitumour strategy based on the accumulation of derivatives such as bis(7-amino-4-azaheptyl) dimethylsilyl (azhepSi), in order to diminish the concentration of natural polyamines (spermine and putrescine) whose metabolism is strongly activated in tumour cells. Our results, obtained by the surface-pressure technique, Brewster angle microscopy (BAM) and polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS), support the idea of an interaction between the polar head groups of d(62)DPPG and amino groups followed by an adsorption of polyamines up to the carbonyl group. Moreover, an insertion of the dimethylsilyl group up to the alkyl chains occurs with azhepSi, in agreement with the observation that the cohesion of the alkyl chain is lower in this case, as compared with the effect of natural polyamines.
The phospholipid analogue miltefosine or hexadecylphosphocholine (HePC) is a drug of high interest in the treatment for fatal visceral leishmaniasis (VL) due to Leishmania donovani particularly because of its activity by oral route. In this study, the interaction of HePC with a monolayer of beta-palmitoyl-gamma-oleyl-phosphatidylcholine (POPC) as membrane model or sterol (ergosterol or cholesterol) was investigated. At a constant pressure of 25 mN/m, the adsorption kinetics of HePC into the monolayers showed that HePC molecules are inserted into the monolayer of lipids as monomers until the critical micellar concentration (CMC). At HePC concentrations superior to the CMC, the micelles of HePC are deployed at the interface as groups of monomers into the POPC or sterol monolayer. The study of mixture of HePC/(POPC or sterol), spread at the air-water interface, shows that a simple miscibility between HePC and POPC is observed, whereas a high condensation appears between HePC and sterols showing a high affinity between HePC and sterols. In addition, HePC does not act as detergent disturbing membrane integrity.
The novel group of amphotericin B (AmB) cationic derivatives has been examined in terms of relationship between self-association and selective toxicity. In all determinations AmB has been used as reference compound. In vitro toxic effects of the compounds on human erythrocytes were determined by measuring leakage of intracellular potassium ions and hemolysis. Antifungal effects were determined as MIC and intracellular potassium loss. The compounds self-association was followed by UV-Vis spectroscopy. The results suggested that: i) unlike AmB the monomer/self-associated species ratio is not an essential in governing the selective toxicity of the derivatives studied; ii) the presence of a bulky substituent in the AmB molecule, preferably located at the amino group of mycosamine moiety is the structural factor essential for the selective toxicity improvement.
Miltefosine (hexadecylphosphocholine [HePC]) is the first orally active drug approved for the treatment of visceral leishmaniasis.
In order to investigate the biochemical modifications occurring in HePC-resistant (HePC-R) Leishmania donovani promastigotes, taking into account the lipid nature of HePC, we investigated their fatty acid and sterol metabolisms. We
found that the content of unsaturated phospholipid alkyl chains was lower in HePC-R parasite plasma membranes than in those
of the wild type, suggesting a lower fluidity of HePC-R parasite membranes. We also demonstrated that HePC insertion within
an external monolayer was more difficult when the proportion of unsaturated phospholipids decreased, rendering the HePC interaction
with the external monolayer of HePC-R parasites more difficult. Furthermore, HePC-R parasite membranes displayed a higher
content of short alkyl chain fatty acids, suggesting a partial inactivation of the fatty acid elongation enzyme system in
HePC-R parasites. Sterol biosynthesis was found to be modified in HePC-R parasites, since the 24-alkylated sterol content
was halved in HePC-R parasites; however, this modification was not related to HePC sensitivity. In conclusion, HePC resistance
affects three lipid biochemical pathways: fatty acid elongation, the desaturase system responsible for fatty acid alkyl chain
unsaturation, and the C-24-alkylation of sterols.
The positively charged polyene molecule amphotericin B 3-dimethylaminopropylamide (AMA) is an efficient agent for the delivery of antisense oligodeoxyribonucleotides (ODN) into target cells. In the present study, bilayer lipid membrane (BLM) conductance, elasticity modulus perpendicular to the membrane plane, surface potential and electrical capacitance were measured by conductance and electrostriction methods in the presence of AMA, pure or complexed to 20-mer single stranded ODN at different ratios. Pure AMA did not induce changes in conductance of cholesterol-containing BLM, but did induce an increase in elasticity modulus and surface potential. ODN/AMA complexes changed BLM properties depending on the charge ratio. The most pronounced effect on membrane conductance was observed for positively charged ODN/AMA complexes (charge ratio rho-/+=0.1), while for negatively charged complexes these changes were less marked/apparent, correlating to substantially lower binding constants. The effect of ODN/AMA complexes on elasticity modulus and charge potential was biphasic. After an increase in both values, a decrease was observed for higher incubation times and ODN/AMA concentrations. These results are interpreted as indicating that the membrane property changes result from the large AMA aggregates induced by the presence of the negatively charged ODN, which condensate on these aggregates. It is suggested that the decrease of elasticity modulus and surface potential in the presence of increasing incubation time and AMA concentration result from desorption of the complexes in the complex-free compartment of the BLM cell, or appearance of a non-linear conductance of the lipid bilayer. The first alternative would explain the AMA-induced transmembrane transfer of ODN.
Novel cationic amphotericin B derivatives as highly potent antifungal agents are reported. These semi-synthetic derivatives of amphotericin B were elaborated through a series of modifications both on the nitrogen atom of the mycosamine and on the C-16 carboxylic acid moiety. The antifungal activity of the new conjugates was tested against Saccharomyces cerevisiae and also against nine different strains of Candida albicans and Candida glabrata, including an amphotericin resistant strain. High potency was observed in the case of polyamine derivatives bearing two 3-aminopropyl chains on the mycosamine. The evaluation of the biological properties also included the determination of the hemolytic activity of the compounds by measuring the EH50 values.
Amphotericin B (AmB) is a polyene antibiotic used to treat deep-seated mycoses. Both the therapeutic action and the toxic side effects of this drug are dependent on its molecular organization. AmB appears as a zwitterion at neutral pH owing to NH(3)(+) and COO(-) groups. The results obtained with electronic absorption, fluorescence, resonance light scattering and infrared absorption spectroscopic analyses show that in the aqueous medium at pH above 10 AmB appears in the monomeric form owing to the negative net electric charge of the molecule. On the contrary, anomalously high aggregation level has been observed at pH below 2, despite the positive net electric charge. The effect is interpreted in terms of the permanent polarization of the polyene chain at low pH, associated with relative rotational freedom of the charged mycosamine fragment of the molecule. The pH-dependent aggregation of AmB is discussed in aspect of pharmacological action of the drug.
Large unilamellar and oligolamellar vesicles are formed when an aqueous buffer is introduced into a mixture of phospholipid and organic solvent and the organic solvent is subsequently removed by evaporation under reduced pressure. These vesicles can be made from various lipids or mixtures of lipids and have aqueous volume to lipid ratios that are 30 times higher than sonicated preparations and 4 times higher than multilamellar vesicles. Most importantly, a substantial fraction of the aqueous phase (up to 62% at low salt concentrations) is entrapped within the vesicles, encapsulating even large macromolecular assemblies with high efficiency. Thus, this relatively simple technique has unique advantages for encapsulating valuable water-soluble materials such as drugs, proteins, nucleic acids, and other biochemical reagents. The preparation and properties of the vesicles are described in detail.
Nystatin and amphotericin B induce a cation-selective conductance when added to one side of a lipid bilayer membrane and an anion-selective conductance when added to both sides. The concentrations of antibiotic required for the one-sided action are comparable to those employed on plasma membranes and are considerably larger than those required for the two-sided action. We propose that the two-sided effect results from the formation of aqueous pores formed by the hydrogen bonding in the middle of the bilayer of two "half pores," whereas the one-sided effect results from the half pores alone. We discuss, in terms of the flexibility of bilayer structure and its thickness, how it is possible to have conducting half pores and "complete pores" in the same membrane. The role of sterol (cholesterol and ergosterol) in pore formation is also examined.
We have identified a 45-kDa protein purified from rat renal brush border membrane that binds short single-stranded nucleic
acid sequences. This activity was purified, reconstituted in proteoliposomes, and then fused with model planar lipid bilayers.
In voltage-clamp experiments, the reconstituted 45-kDa protein functioned as a gated channel that allows the passage of nucleic
acids. Channel activity was observed immediately after addition of oligonucleotide. Channel activity was not observed in the
absence of purified protein or of oligonucleotide or when protein was heat-inactivated prior to forming proteoliposomes. In
the presence of symmetrical buffered solution and oligonucleotide, current passed linearly over the range of holding potentials
tested. Conductance was 10.4 ± 0.4 picosiemens (pS) and reversal potential was 0.2 ± 1.7 mV. There was no difference in channel
conductance or reversal potential between phosphodiester and phosphorothioate oligonucleotides. Ion-substitution experiments
documented a shift in reversal potential only when a concentration gradient for oligonucleotide was established, indicating
that movement of oligonucleotide alone was responsible for current. Movement of oligonucleotide across the bilayer was confirmed
by using 32P-labeled oligonucleotides. Channel open probability decreased significantly in the presence of heparan sulfate. These studies
provide evidence for a cell surface channel that conducts nucleic acids.
This present work is devoted to the study of the behavior in water of important antifungal drug, the amphiphilic Amphotericin-B (AmB) molecule. Experimental spectroscopy data, namely, the modification of the AmB UV absorption spectrum in water (hypsochromic frequency shift) with the increasing AmB concentration, indicate the self-association of the molecule in water. In this work, a possible three-dimensional structure of the AmB self-associated species present in water has been determined by using in concert both spectroscopy and energy information and not by fitting the calculated UV absorption spectrum to the experimental one. The calculations have been performed upon AmB dimers and helicoidal oligomers involving 2−8 unit cells. The total energy of any helicoidal oligomer has been evaluated taking into account, besides the intermolecular interactions (computed using an accurate intermolecular interaction potential), (i) the “thermal” effects evaluated utilizing the well-known formulas of statistical thermodynamics and (ii) “solvent” effects calculated in the framework of an adequate continuum model. It has been shown that all energy results obtained for the different helicoidal oligomers may be extended to infinite helicoidal aggregates. In conclusion, (i) The study of the evolution with Nuc (the number of unit cells) of the total energy (involving all here-above cited terms) of both single and double helices has shown that the highest energy gain ( per AmB molecule) is obtained with the double helix. (ii) Such a stabilization mainly proceeds from van der Waals forces together with entropy factors. (iii) This optimized double helix, which is characterized by an helicoidal angle, θ = 30°, and an intercell distance R as 5.7 Å, (thus a pitch of 62.7 Å), leads to an hypsochromic frequency shift (Δν+) calculated as 3950 cm-1, a value which is quite close to the experimental one, 4368 cm-1.
The inducement of K+ permeability through membranes by the polyene antibiotic amphotericin B (AmB) has been analyzed as a measure of the antibiotic activity. Dose-response curves have been obtained with cholesterol- and ergosterol-containing egg yolk phosphatidylcholine large unilamellar vesicles (LUVs), human erythrocytes, and Saccharomyces cerevisiae cells. Conductance changes induced by AmB in sterol-containing planar bilayer membranes have also been studied. AmB self-association in aqueous buffer was determined by circular dichroism (CD) as a function of the antibiotic concentration. Electronic absorption and CD spectra of AmB were recorded in the presence of LUVs. For given AmB concentrations, the extent of permeability inducement is dependent on the lipid concentration. On the other hand, for cholesterol-containing LUVs or erythrocytes, a critical AmB concentration had to be reached before any permeability is observed. Independent of lipid concentration, this concentration was directly related to antibiotic self-association in the aqueous buffer. The same observation was made for erythrocytes and nystatin. The AmB absorption and CD spectra were totally different for ergosterol- and cholesterol-containing LUVs. Formation of single channels by one-sided addition of AmB could be observed only in ergosterol-containing membranes. These data lead us to propose that the permeability pathways induced by amphotericin B or nystatin, in ergosterol- and in cholesterol-containing membranes, are of different natures. In the latter case the antibiotics are only active, by single-sided addition, in the self-associated form. These findings offer important clues for the design of less toxic derivatives of AmB: they should have a low degree of self-association in water.
The interaction of the polyene antibiotic amphotericin B (AmB) (Fig. 1) with large unilamellar vesicles (LUV) was monitored by circular dichroism (CD) and carboxyfluorescein (CF) release. LUV afford a far better model for biological membranes than small unilamellar vesicles (SUV) which have been used until now. With dimyristoyl phosphatidyl choline (DMPC) LUV (i.e., containing saturated acyl chains), a strong and not saturable binding for AmB/lipid ratios up to 0.5 was observed both above and below the phase transition temperature. Incorporation of cholesterol into the vesicles did not significantly change the interaction. With egg PC (EPC) LUV (i.e., containing unsaturated acyl chains), quite a different picture emerged: the binding reached saturation for AmB/lipid ratios of about 5 x 10(-3), a result not observed with EPC SUV. When sterols were introduced into membranes, the CD spectral features obtained in the presence of ergosterol were different from those obtained in the presence of cholesterol. Such a different behavior was not observed with SUV. We suggest that species whose CD spectrum was observed after 15 min in the presence of ergosterol-containing EPC LUV is the particular one which forms wide channels and induces a Ca2+ release. (H. Ramos, A. Attias, B.E. Cohen and J. Bolard, submitted for publication). The CF release from EPC LUV induced by AmB was very low, even at very high concentrations of the antibiotic (3 x 10(-4)M). In contrast, an important release of the fluorescent dye was observed with DMPC LUV at concentrations of approximately 10(-5)M.(ABSTRACT TRUNCATED AT 250 WORDS)
The conditions of formation of amphotericin B-cholesterol or -ergosterol complexes in monolayers are investigated by the penetration into a monolayer of egg phosphatidylcholine/sterol of 14C-labelled N-fructosyl-amphotericin B dissolved in the aqueous subphase. An increase of both surface pressure and radioactivity as a function of concentration are observed simultaneously while a 'saturation' effect occurs only for the surface pressure. The experiments are not accurate enough to make conclusions about the number of actually penetrated amphotericin B molecules. Therefore, the existence of an amphotericin B-sterol complex was evidenced from a study of surface pressure area per molecule isotherm. The results indicate that a complex with a 2:1 stoichiometry is formed and that the amphotericin B-ergosterol interaction is larger than the amphotericin B-cholesterol interaction. The complex is dissociated by addition of egg phosphatidylcholine due to a competition between egg phosphatidylcholine and amphotericin B for sterol.
The quantitative structure-activity relationships studies of amphotericin B and its 16 semisynthetic derivatives obtained by modification at carboxyl and amino groups have been done. The results of five biological tests were subjected to principal component analysis, a numerical method useful in the investigation of large sets of data. For some compounds, also, interaction with lipidic vesicles was investigated by spectroscopic methods. The results obtained indicate that: (i) The presence of positively charged nitrogen atom (protonable or bearing fixed charge) is indispensable for biological activity and antibiotic-sterol interaction; (ii) The lack of free carboxyl group in the molecule favours the differentiation between cholesterol and ergosterol containing cells.
This paper summarizes effects of amphotericin B on the permeability properties of lipid bilayer membranes. The polyene antibiotic reduced the electrical resistance of bilayer membranes and produced anion permselectivity. Osmotic volume flow was nondiffusional, and there were graded increments in nonelectrolyte permeability; sucrose (radius = 5.2 Å) was excluded from the membranes. The amphotericin B effects on membrane permeability were dependent on interactions with cholesterol or related sterols that had an equatorial 3 OH group and a rigid phenanthrene nucleus. The data were rationalized by assuming that amphotericin B cholesterol interactions resulted in the formation of pores with 5 Å radii. A tentative hypothesis for the structure of amphotericin B cholesterol pores is presented, in which the rod components of the respective molecules are oriented in parallel in the hydrocarbon phase; the carboxyl, mycosamine, and some hydroxyl groups of amphotericin B and the 3 OH group of cholesterol are anchored at the membrane and water interface, and there is hydrogen bonding at the membrane and water interface between the hydroxyl group of cholesterol and carbonyl and/or hydroxyl groups of amphotericin B.
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.
A series of aliphatic amides of amphotericin B have been synthesized. The structure of the derivatives which were obtained has been established by mass spectrometry. Their biological properties: inhibition of growth of Saccharomyces cerevisiae and hemolytic activity have been determined. The quantitative relationships between the activity of amides of amphotericin B against S. cerevisiae and their lipophilicity can be expressed by a parabolic function, whereas that between hemolytic activity and lipophilicity--by polynominal expression of fourth degree.
Interest in amphotericin B has undergone a renaissance of sorts over the past few years despite the advent of the newer less-toxic azole antifungal drugs. This is, in part, owing to the unfortunate increase in fungal diseases worldwide. It is also, however, owing to the reduction of toxicity via innovative liposomal delivery systems, better understanding of drug mechanism and distribution and a surprising expansion of the antibiotic spectrum of amphotericin B to include select virus, parasite and possibly prion infections. In this article, Scott Hartsel and Jacques Bolard summarize the recent leaps in pharmaceutics, spectrum and molecular mechanistic knowledge of this surprising molecule.
The ability of amphotericin B (AmB) to form ion-permeable channels in cholesterol containing lipid bilayers was studied by UV/visible absorbance, circular dichroism, and fluorescence spectroscopy. Stable liposomes composed of distearoylphosphatidylcholine, cholesterol, distearoylphosphatidylglycerol, and AmB were prepared so that a wide range of AmB concentrations in the bilayer could be studied. Singular value decomposition analysis (Henry & Hofrichter, 1992) of the circular dichroism spectra of AmB at different AmB/lipid ratios suggests that AmB exists primarily in only two states in the bilayer, a "monomeric" state and an "aggregated" state. The transition from the "monomeric" to the "aggregated" state begins to occur at a critical concentration of 1 AmB per 1000 lipids in the membrane and coincides with the appearance of channel activity. The data support the recent theoretical conclusions of Weakliem et al. (1995) which predict that pore formation in the lipid bilayer will occur when the drug molecule concentration exceeds a critical value. At this critical concentration, it is calculated that a minimum number of 16 AmB molecules per liposome are required to observe channel activity. The results are consistent with the sterol-dependent AmB channel models proposed by de Kruijff and Demel (1974), Andreoli (1974), and Khutorsky (1992). To further elucidate the effects of sterol on AmB-mediated channel formation, liposomes were prepared with varying ratios of cholesterol and AmB. At cholesterol mole percentages greater than 1, channel activity was observed to occur at AmB concentrations just above the critical value. Previous reports show that cholesterol forms "tail-to-tail" dimers at mole percentages greater than 2 (Harris et al., 1995). This suggests that formation of the bilayer-spanning channels by AmB is initiated most efficiently when the tail-to-tail dimer of cholesterol is present. Although the structural nature of the AmB channel could not be unambiguously determined, these experiments provide further evidence in support of the widely held view that AmB's primary mechanism of killing fungal cells occurs by forming ion-permeable channels.
Spectroscopic studies of four amphotericin B (AmB) lipid preparations--small negatively charged unilamellar vesicles "AmBisome", positively charged oligolamellar liposomes "Ampholiposomes", AmB Lipid Complex "L-AmpB33"--and AmB association with gamma cyclodextrin demonstrated that the composition of drug delivery system directly influences AmB organization. The aggregation state and release in external medium of AmB was monitored by circular dichroism and UV-visible absorption. AmB short-term activity against Candida albicans (K+ leakage) was found to be correlated with the amount of free AmB released from lipid preparations. These data seem to indicate that lipid composition influences anti-Candida albicans activity, by modulation of AmB binding to lipids.
I consider a vesicle with an open pore of small radius rp, exposed to a DNA solution. The crucial moment is the entry, when a chain end faces the pore and enters it. I discuss qualitatively the following three characteristic times: (i) the duration of the entry of one chain end (defining the minimum lifetime of the pore) taue approximately 10(-4) sec, (ii) the transfection time taut (the time required to be sure that one chain has gone in) taut approximately hours, and (iii) the sliding time tauS (the time between entry of one end and entry of the other end) approximately 1 sec. A fortunate feature is that sliding may proceed even if the pore tends to close itself after entry.
Antisense strategy requires efficient systems for the delivery of oligodeoxyribonucleotides (ODN) into target cells. Cationic amphiphiles have shown good efficiency in vitro and a lot of attention is currently paid to their interaction with nucleic acids. In the present study, this interaction was, for the first time, analysed at the molecular level, taking advantage of the spectroscopic properties of the positively charged chiral polyene molecule amphotericin B 3-dimethylaminopropyl amide (AMA), the efficiency of which, as delivery system, has been demonstrated [Garcia et al., Pharmacol. Ther. (2000), in press]. By UV-visible absorption and circular dichroism (CD) we studied its self-association properties in pure water, saline and RPMI medium. Drastic changes were observed upon ODN addition, stronger in pure water than in media of high ionic strength. At low AMA concentration (<10(-6) M), the strong increase of the CD signal, characteristic of self-association, indicated condensation of AMA on the ODN molecules. At a higher concentration (10(-4) M), and for a nucleic acid negative charge/AMA positive charge ratio higher than 1, spectra were interpreted as a reorganisation of free self-associated AMA species into smaller ones 'decorating' the nucleic acid molecule. Electron microscopy data were interpreted according to this scheme.
- J Bolard
- P Legrand
- F Heitz
- B Cybulska
J. Bolard, P. Legrand, F. Heitz, B. Cybulska, J. Am. Chem.
Soc. 30 (1991) 5707^5715.
[12] F.Jr. Szoka, D. Papahadjopoulos, Proc. Natl. Acad. Sci.
USA 75 (1978) 4194^4198.
- A Jarzebski
- L Falkowski
- E Borowski
A. Jarzebski, L. Falkowski, E. Borowski, J. Antibiot. 35
(1982) 220^229.
- A Vertut-Croquin
- J Bolard
- M Chabbert
- C Gary-Bobo
A. Vertut-Croquin, J. Bolard, M. Chabbert, C. Gary-Bobo,
Biochemistry 22 (1983) 2939^2944.
- I Blanc
- M.-H. Buena Da Costa
- J Bolard
I. Blanc, M.-H. Buena Da Costa, J. Bolard, M. Saint-Pierre
Chazalet, Biochim. Biophys. Acta 1464 (2000) 299^308.
- A Marty
- A Finkelstein
- J Gen
A. Marty, A. Finkelstein, J. Gen. Physiol. 65 (1975) 515^
526.
- B Cybulska
- M Hervë
- E Borowski
- C M Gary-Bobo
B. Cybulska, M. Hervë, E. Borowski, C.M. Gary-Bobo,
Mol. Pharmacol. 29 (1985) 293^298.
- M Chëron
- B Cybulska
- J Mazerski
- J Grzybowska
- A Czerwinski
- E Borowski
M. Chëron, B. Cybulska, J. Mazerski, J. Grzybowska, A.
Czerwinski, E. Borowski, Biochem. Pharmacol. 37 (1988)
827^836.
- S Hartsel
- J Bolard
S. Hartsel, J. Bolard, Trends Pharmacol. Sci. 17 (1996) 4454
49.
- C Garcia
- O Seksek
- J Grzybowska
- E Borowski
- J Bolard
C. Garcia, O. Seksek, J. Grzybowska, E. Borowski, J. Bolard, Pharmacol. Ther. (2000), in press.
- I Blanc
- M.-H. Buena Da Costa
- J Bolard
- M Saint-Pierre
- Chazalet
I. Blanc, M.-H. Buena Da Costa, J. Bolard, M. Saint-Pierre
Chazalet, Biochim. Biophys. Acta 1464 (2000) 299^308.
- G Fujii
- J.-E Chang
- T Coley
- B Steere
G. Fujii, J.-E. Chang, T. Coley, B. Steere, Biochemistry 36
(1997) 4959^4968.
- P Legrand
- M Cheron
- L Leroy
- J Bolard
P. Legrand, M. Cheron, L. Leroy, J. Bolard, J. Drug Target
4 (1997) 311^319.
- J Bolard
- P Legrand
- F Heitz
- B Cybulska
J. Bolard, P. Legrand, F. Heitz, B. Cybulska, J. Am. Chem.
Soc. 30 (1991) 5707^5715.
- F Szoka
- D Papahadjopoulos
F.Jr. Szoka, D. Papahadjopoulos, Proc. Natl. Acad. Sci.
USA 75 (1978) 4194^4198.
- T E Andreoli
T.E. Andreoli, Ann. N. Y. Acad. Sci. 235 (1974) 448^468.
- R W Holz
R.W. Holz, Ann. N. Y. Acad. Sci. 235 (1974) 469^479.