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Oligonucleotide delivery by a cationic derivative of the polyene antibiotic amphotericin B
Abstract
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.
Modern conceptions of the physicochemical properties of dimethylsulfoxide and polyene antibiotics are reviewed. The results of investigations of independent and mutual effects of polyene antibiotics and dimethylsulfoxide on membrane permeability were analysed. The own experimental data of radioprotective and antitumour action of complex dimethylsulfoxide -polyene antibiotics are presented, and the perspectives of their use in medicine are described.
Using a pore- and channel-forming peptide, TV-XIIa, which is an 11-residual peptaibol isolated from the fungus Trichoderma viride, we developed a vehicle for the cellular delivery of such polar biologically active agents as antisense oligodeoxynucleotides (ODNs). To function as an ODN carrier, basic amino acids, 10-mer of lysine, were conjugated to the C-terminus of TV-XIIa and the designed carrier peptide, Ac-U-N-I-I-U-P-L-L-U-P-I-K-K-K-K-K-K-K-K-K-K-OH (U: α-aminoisobutyric acid), was synthesized by the Fmoc-based solid-phase method. The complex between the carrier peptide and ODNs, which was electrostatically formed, was capable of crossing the membranes of NIH3T3 cells and the ODNs were accumulated in the cytoplasm and the nucleus. However, the complex was not taken up by A549 cells. The translocation of the complex occurred at both 4 and 37 °C in NIH3T3 cells and did not seem to involve an energy-dependent endocytic process.
Cationic lipids are promising non-viral vectors for the cellular delivery of nucleic acids. Important considerations for the development of new delivery vectors are enhanced uptake efficiency, low toxicity and traceability. Traceable gene transfer systems however typically require the inclusion of a labeled excipient, and highly sensitive imaging instrumentation to detect the presence of the label. Recently, we reported the synthesis and characterization of colored, polyene cationic phospholipidoids composed of a rigid, polyeneoic acid of predetermined dimension (C20:5 and C30:9) paired with flexible saturated alkyl chains of varying lengths (12:0, 14:0, 16:0, 18:0, 20:0 carbons). Herein, the potential of these cationic phospholipids as siRNA carriers was evaluated through standard liposomal formulations in combination with a neutral helper lipid, DOPE. The polyene-based lipids were compared with a standard cationic lipid for siRNA-delivery into luciferase expressing HR5-CL11 cells. Within the series of lipids screened, knockdown results indicated that polyene cationic phospholipids paired with longer saturated alkyl chains are more effective as gene transfer agents, and perform comparably with the commercial lipid EPC. Furthermore, the chromophore associated with the polyene chain allowed tracking of the siRNA delivery using direct observation. The polyene lipoplexes were tracked both on a macroscopic and microscopic level as either a single-component or as a multi-component lipoplex formulation. When combined with a reference EPC, effective knockdown and tracking abilities were combined in a single preparation.
Copyright © 2014. Published by Elsevier B.V.
Nucleic acids have relevance in the therapy of various disorders like cancer, stroke, trauma, myocardial infarction, autoimmune disorders, and pregnancy-associated complications. However, nucleic acid delivery is still a challenge, since the nucleic acid has to traverse both the endothelial and cellular barrier, combat the endolysosomal degradative pathway, as well as cross the nuclear barrier. To overcome these problems, advanced delivery strategies have been developed using nanovectors which could be viral, non-viral and hybrid, providing several advantages, not only traversing the cellular barrier but also modulating intracellular trafficking. The chapter would address the challenges in the delivery of nucleic acids by nanomedicine delivery strategies and therapeutic applications of nucleic acids, with a focus on relevant intellectual property rights on this subject.
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.
Stock solutions of the polyene antibiotic amphotericin B (AmB) were prepared at pHs from 4.0 to 5.4 in acetic acid/acetate buffer, at 7.4 in phosphate-buffered saline (PBS), and from 10.0 to 10.8 in carbonate/bicarbonate buffer. Their antifungal activity was tested on Candida albicans cells and their toxicity on human erythrocytes. Antifungal activity was observed for all preparations. Surprisingly, the toxicity (K+ release and hemolysis) drastically decreased for the two preparations at pH 5.4 and 10.8. This effect was attributed, in the first case, to a screening of the positively charged aggregated antibiotic by the negatively charged acetate ions of the buffer, and, in the second case, to a neat modification of the mode of AmB self-association.
Nucleic acids show immense potential to treat cancer, acquired immune deficiency syndrome, neurological diseases and other incurable human diseases. Upon systemic administration, they encounter a series of barriers and hence barely reach the site of action, the cell. Intracellular delivery of nucleic acids is facilitated by nanovectors, both viral and non-viral. A major advantage of non-viral vectors over viral vectors is safety. Nanovectors evaluated specifically for nucleic acid delivery include polyplexes, lipoplexes and other cationic carrier-based vectors. However, more recently there is an increased interest in inorganic nanovectors for nucleic acid delivery. Nevertheless, there is no comprehensive review on the subject. The present review would cover in detail specific properties and types of inorganic nanovectors, their preparation techniques and various biomedical applications as therapeutics, diagnostics and theranostics. Future prospects are also suggested.
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.
Amphotericin B (AmB) is a life-saving antibiotic, used to treat deep-seated mycotic infections. Both the therapeutic and toxic side effects of AmB are directly dependent on its molecular organization. Organization of AmB was studied in monocomponent monomolecular layers formed at the argon-water interface, by means of polarized and non-polarized electronic absorption spectroscopy and analyzed in terms of the exciton splitting theory. The results provide direct indication that AmB forms spontaneously dimers that can be assembled into molecular structures characterized by homogeneous orientational distribution in the monolayer, interpreted as cylindrical pores. The structures are not stable at surface pressures higher than 20 mN/m and therefore dimers are concluded as abundant molecular organization forms of AmB in biomembranes. Possibility of stabilization of the cylindrical structures, at higher surface pressures, by other molecules, e.g. sterols, is also discussed.
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.
In vitro, the efficacy of the antisense approach is strongly increased by systems delivering oligodeoxyribonucleotides (ODNs) to cells. Up to now, most of the developed vectors favor ODN entrance by a mechanism based on endocytosis. Such is the case for particulate systems, including liposomes (cationic or non-cationic), cationic polyelectrolytes, and delivery systems targeted to specific receptors. Under these conditions, endosomal compartments may represent a dead end for ODNs. Current research attempts to develop conditions for escaping from these compartments. A new class of vectors acts by passive permeabilization of the plasma membrane. It includes peptides, streptolysin O, and cationic derivatives of polyene antibiotics. In vivo, the interest of a delivery system, up to now, has appeared limited. Development of vectors insensitive to the presence of serum seems to be a prerequisite for future improvements.
Despite extensive investigations into oligonucleotide lipoplexes, virtually no work has addressed whether the physicochemical properties of these assemblies vary as a function of the constituent oligonucleotide (ODN) sequence and/or composition. The present study was aimed at answering this question. To this end, we complexed N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) liposomes, in dispersion, with either 18-mer phosphorothiote homo-oligonucleotides composed of either adenine, thymidine or cytosine; or one of three structurally related 18-mer phosphorothioate oligonucleotides (S-ODNs) (G3139, its reverse sequence and its two-base mismatch). After ODN addition to vesicles at different mole ratios, changes in pH and electrical surface potential at the lipid-water interface were analyzed by using the fluorophore heptadecyl-7-hydroxycoumarin while particle size distributions were analyzed by static-light scattering. The results indicate that each homo-oligonucleotide does indeed exhibit different complexation behavior. In particular, the maximal level of DOTAP neutralization by the polyadenine S-ODN is much lower than that for the two other homo-oligonucleotides and hence its lipoplex is much more positively charged. Much smaller electrostatic differences are also apparent between lipoplexes formed from each of the G3139-related ODNs. This paper identifies nucleotide base selection and sequence as a variable that can affect the physicochemical properties of oligonucleotide lipoplexes and hence probably their transfection competency.
Fluorescence emission from amphotericin B dissolved in 2-propanol-water was recorded in the spectral region 500-650 nm. The fluorescence excitation spectrum corresponds to the absorption spectrum of the monomeric drug. The large energy shift between the excitation and emission bands indicates that emission takes place from an energy level different than that responsible for absorption. These levels were attributed to the 2(1)A(g) and 1(1)B(u) states, respectively. Excitation of the same sample with short wavelength radiation (below 350 nm) yields light emission between 400 and 550 nm. The fluorescence excitation spectrum corresponding to this emission band displays distinct maxima at 350, 334 and 318 nm. This band was analyzed in terms of the exciton splitting theory and assigned to amphotericin B in a dimeric form, in which chromophores are spaced by 4.9 A. The binding energy of the dimers, determined to be 4.9 kJ/mol, indicates that the structures are stabilized by van der Waals interactions. The same type of molecular structures was also detected in the lipid membranes formed with dipalmitoylphosphatidylcholine. Linear dichroism of amphotericin B embedded in lipid multibilayers indicates that molecules are distributed between two fractions: parallel (38%) and perpendicular (62%) with respect to the membrane. The biological importance of such membrane organization is discussed.
Using a pore- and channel-forming peptide, TV-XIIa, which is an 11-residual peptaibol isolated from the fungus Trichoderma viride, we developed a vehicle for the cellular delivery of such polar biologically active agents as antisense oligodeoxynucleotides (ODNs). To function as an ODN carrier, basic amino acids, 10-mer of lysine, were conjugated to the C-terminus of TV-XIIa and the designed carrier peptide, Ac-U-N-I-I-U-P-L-L-U-P-I-K-K-K-K-K-K-K-K-K-K-OH (U: alpha-aminoisobutyric acid), was synthesized by the Fmoc-based solid-phase method. The complex between the carrier peptide and ODNs, which was electrostatically formed, was capable of crossing the membranes of NIH3T3 cells and the ODNs were accumulated in the cytoplasm and the nucleus. However, the complex was not taken up by A549 cells. The translocation of the complex occurred at both 4 and 37 degrees C in NIH3T3 cells and did not seem to involve an energy-dependent endocytic process.
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.
The toxicity of the antifungal polyene antibiotic amphotericin B (AMB) has been related to its low solubility, more specifically to a self-associated form termed toxic aggregate. In addition, AMB in aqueous medium gives rise to concentration, ionic strength, and time-dependent polydisperse systems. For this reason different approaches, including the use of several lipid aggregates, have been used in attempts to improve the drug's solubility and increase its therapeutic index. In this context, understanding AMB's self-association properties should help in the preparation of less toxic formulations. Ions from the Hofmeister series alter water properties: while kosmotropes (water structure makers-sulfate, citrate, phosphate) decrease solute solubility, chaotropes (water structure breakers-perchlorate, thiocyanate, trichloroacetate, and the neutral molecule urea) have opposite effects. This work reports a study of the effect of Hofmeister ions and urea on the self-aggregation of AMB and some of its derivatives. Optical absorption and circular dichroism spectra were used to monitor monomeric and aggregated antibiotic. While kosmotropes increased aggregation in a concentration-dependent manner, the opposite was observed for chaotropes. It is shown, for the first time, that thiocyanate and trichloroacetate can induce complete AMB monomerization. The understanding of these processes at the physicochemical and molecular levels and the possibility of modulating the aggregation state of AMB and its derivatives should contribute to elucidate the mechanisms of action and toxicity of this widely used antibiotic and to develop more efficient and less toxic preparations.
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.
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.
In aqueous suspensions of amphotericin B (AmB), a polyene antibiotic and antifungal agent, three forms of AmB coexist: monomers, water-soluble oligomers, and non-water-soluble aggregates. The toxicity of the water-soluble self-associated form of AmB compared with that of the non-water-soluble self-associated form was tested by measuring induction of K+ leakage from human erythrocytes, using different suspensions containing the antibiotic and phosphate-buffered saline. These suspensions were obtained from various stock solutions of the antibiotic in dimethyl formamide or dimethyl sulfoxide. Their circular dichroism spectra around 340 nm, indicative of the degree of AmB self-association, were strongly dependent on the concentration of organic solvent in the suspensions. The nonsoluble self-associated form was separated from the water-soluble form by centrifugation. The nonsoluble form was favored by a high concentration of AmB of the stock solution. The kinetics of AmB-induced K+ leakage from human erythrocytes also appeared to be strongly dependent on the AmB concentration of the stock solution being much weaker with concentrated stock solutions. It was concluded that the only form of AmB toxic to human erythrocytes is the water-soluble self-associated form (in contrast with fungal cells on which the monomeric form is also active). This result may be important in the design of new less toxic AmB derivatives and in the understanding of the mechanism of action of liposomal AmB.
Gene therapy is based on the vectorization of genes to target cells and their subsequent expression. Cationic amphiphile-mediated delivery of plasmid DNA is the nonviral gene transfer method most often used. We examined the supramolecular structure of lipopolyamine/plasmid DNA complexes under various condensing conditions. Plasmid DNA complexation with lipopolyamine micelles whose mean diameter was 5 nm revealed three domains, depending on the lipopolyamine/plasmid DNA ratio. These domains respectively corresponded to negatively, neutrally, and positively charged complexes. Transmission electron microscopy and x-ray scattering experiments on complexes originating from these three domains showed that although their morphology depends on the lipopolyamine/plasmid DNA ratio, their particle structure consists of ordered domains characterized by even spacing of 80 A, irrespective of the lipid/DNA ratio. The most active lipopolyamine/DNA complexes for gene transfer were positively charged. They were characterized by fully condensed DNA inside spherical particles (diameter: 50 nm) sandwiched between lipid bilayers. These results show that supercoiled plasmid DNA is able to transform lipopolyamine micelles into a supramolecular organization characterized by ordered lamellar domains.
We have developed a practical analytical treatment of the non-linear Poisson-Boltzmann (P-B) equation to characterize the strong but non-specific binding of charged ligands to DNA and other highly charged macromolecules. These reactions are notable for their strong salt dependence and anti-cooperativity, features which the theory fully explains. We summarize analytical results for concentration profiles and ion binding in various regimes of surface curvature and ionic strength, and show how counterion size and charge distribution may influence competitive binding. We present several practical applications of the formalism, showing how to estimate the ligand concentration needed to effectively compete with a given buffer salt, and how to calculate the amounts of counterion species bound at various distances from the DNA surface under given bulk solution conditions. We cast our results into the form of a Scatchard binding isotherm, showing how the apparent binding constant K(obs) and S = -dlog K (obs )d log[M (+)] can be predicted from the basic theory. Anti-cooperativity arises naturally without steric repulsion, and binding curves can be fitted with K(obs) and effective charge as the only free parameters. We extend the analytical P-B analysis to an arbitrary number of counterion species, and apply the results to fit and predict three-ion competition data.
This chapter describes cationic liposomes, DNA, and gene delivery. Gene therapy—the concept, which was initially proposed 30 years ago—theoretically offers healing of human diseases at their cause rather than treating their symptoms. For a successful gene therapy, an effective and safe delivery of plasmids, antisense oligonucleotides, ribozymes, or other nucleic acid sequences into appropriate cells, preferentially in vivo is required. Cationic lipids and DNA both exhibit rich polymorphic behavior at higher concentrations or upon interaction with specific agents. In concentrated solutions, DNA forms a variety of liquid crystalline phases, similar to lipids. While lipid phases are mostly lamellar and inverse hexagonal, DNA is packed into hexatic phases, whereby the DNA helices are arranged into normal hexagonal phase. A conclusive observation of in vitro transfection is that an increased amount of cationic lipids increases transfection as well as cytotoxicity. Therefore, the balance between activity and toxicity determines transfection activity. A majority of researchers are trying to improve transfection by synthesizing novel lipids, the colloidal properties of DNA–lipid complexes are also as important. This claim can be strengthened by the fact that, despite a decade of work, no clear molecular structure–transfection activity correlations have been found.
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.
Electron microscopy is used to show the morphology of liposome/DNA complexes as related to their cationic component, the molar ratio of the helper lipid (usually DOPE1), the nature of the DNA-component, as well as the composition of the media. Liposomes made of monovalent cationic amphiphiles adhere and fuse during interaction with negatively charged DNA thereby complexing the DNA. The size of the resulting complexes is depending upon charge neutralization and is smallest at a slightly positive net charge. At molar ratios of DOPE, to the cationic component of ≥ 1.5, hexagonal lipid tubules are formed, especially in media containing high salt concentrations, and even in the control lipid mixture, not interacting with any DNA or oligonucleotide. Complexes, made of plasmid-DNA, monovalent cationic amphiphiles, and DOPE at a lower molar ratio, show additionally to the semifused or fused liposomes a new structure, called spaghetti-like structure, representing a bilayer-coated, supercoiled DNA. Single-strand and short oligonucleotides seem not to form such structures during interaction with monovalent cationic liposomes. Neither fusion nor spaghetti formation is observed during interaction of DNA with liposomes made of polyvalent cationic amphiphiles. In general, small complexes consisting of some few semifused liposomes bearing the self-encapsulated nucleic acid and additionally the spaghetti-like structure, free or connected with these complexes, seem to be candidates for the transfectionactive structure rather than large extended HII1-lipid arrangements.
Binding of dodecyltrimethylammonium cation to calf thymus DNA was studied by using the surfactant-selective electrode as a function of added electrolyte concentration at various temperatures. Binding is highly cooperative and biphasic. An abruptly occurring binding attains a slight shoulder within a very narrow equilibrium concentration range followed by a further augmentation in binding, as the equilibrium concentration is increased. Temperature caused relatively small shift in binding isotherm, but a distinct inversion point was found around 27 °C. Above this temperature the isotherm shifts to the left and then to the right below it. Analogous binding studies as above were repeated with heat-denatured DNA, which binds the surfactant with higher intrinsic affinity but less cooperatively than the intact DNA. A fluorescent probe, pyrene, was employed to assess the microenvironmental polarity of pyrene in DNA before and after surfactant binding. The surfactant–DNA complex has a hydrophobic region akin to ordinary micelle.
The relationship between polyene macrolide-induced early membrane damage and cytotoxicity in B1 (hamster), B82 (mouse), and RAG (mouse) cells has been investigated. Filipin (FIL) induced the greatest immediate damage, as monitored by 51Cr release, followed by mediocidin (MED), amphotericin B-deoxycholate (Fungizone) (FZ) and pimaricin (PIM). For long term effect, PIM was the least toxic followed by MED, FZ, and FIL as indicated by 24-hour survival, 72-hour viability, and growth rate of cells. In evaluating polyene macrolide-induced permeability alterations and cytotoxicity two types of interactions with mammalian cells were found: (1) cell toxicity at polyene macrolide levels not eliciting immediate membrane permeability changes; and (2) immediate membrane damage without long range toxicity.
The binding of cationic lipids to DNA induces the condensation of complexes of the lipid and polyelectrolyte. This paper presents data on the binding of the simple cationic lipid cetyltrimethylammonium bromide (CTAB) to DNA prior to the condensation process. The complete thermodynamics of the binding of CTAB to both helical and single strand DNA are evaluated with use of isothermal titration calorimetery data and analysis of UV melting transitions. The binding to the helical form involves a two-step process: first, binding to an isolated phosphate site on the DNA strand with a binding constant of 1.5 × 103 M-1, and second, a highly cooperative binding event that seems to involve hydrophobic intereactions between hydrocarbon chains of the bound CTAB. The cooperativity parameter is 56, leading to a cooperative binding constant of 8.7 × 104 M-1. The enthalpies of the two binding events on the helix sites are resolved: −20 kJ/mol for the isolated site and +3.3 kJ/mol for contiguous sites. Binding to the single strand DNA is also quite strong with an estimated equilibrium constant of about 1.3 × 104 M-1. Because CTAB binds both to helical and single strand DNA, biphasic melting transitions are observed. An analysis of the behavior of the melting curves and the thermodynamic data allows reasonable models of the binding processes to be constructed.
Interaction between cetyltrimethylammonium bromide (CTAB) and large T4DNA was studied by potentiometric titration using an ion-selective membrane electrode. The data obtained were assessed in terms of recently reported results regarding the higher order structure of T4DNA with the addition of CTAB: individual DNA chains exhibit discrete phase-transition between random coil and compact globule states. The apparent cooperativity represented as a sigmoidal function is attributed to bimodality in the distribution between the elongated coil and compact globule states of T4DNA chains. In addition, the globule-coil transition of single T4DNA is reversible, demonstrated by single-molecule observation with fluorescence microscopy. At a fixed concentration of CTAB, transition from the globule to the coil state is brought about by the addition of polyacrylic acid, suggesting that this reversible transition may be used for the purification of giant DNA molecules.
It becomes clear that large DNA molecules exhibit discrete conformational change between the coil and globule states with the addition of a very small amount (with the order of 10(-5) M) of cationic surfactant, cetyltrimethylammonium bromide (CTAB). We use fluorescence microscopy as a tool of single molecular observation of double-stranded T4DNA in an aqueous environment. When the concentration of CTAB is less than 9.4 x 10(-6) M, all DNA molecules exhibit the extended coil state. Whereas, when the CTAB concentration is higher than 2.0 x 10(-5) M, only compacted DNA molecules in the globular state are observed. In the region between these two critical concentrations, the coil and globule states coexist in the solution. A small but apparent increase of the size of the DNA globule is noticed at the CTAB concentration higher than 10(-3) M, due to the penetration of CTAB molecules into the DNA globule. To study the dynamical aspect of coil-globule transition, the process of the structural change from the coil into the globule state is observed under the spatial gradient of the CTAB concentration. The formation of aggregates from two or more globules is noticed at high concentrations of surfactant above 1.6 x 10(-4) M. Below this concentration, the globules do not coalesce into an aggregate even if they collide with each other. The translational diffusion constant D of DNA molecules is measured from the time series of video frames of the fluorescence image. The hydrodynamic gyration radius xi(H) is evaluated from the D and the viscosity of the bulk aqueous solutions. The increase of the globule size at the higher CTAB concentrations above 10(-3) M is confirmed by the increase of xi(H) values.
Cationic liposomes are a potential delivery system for antisense oligonucleotides, but physical chemistry of this system is still poorly understood. We studied physicochemical properties (size distribution, lipid mixing reactions) and the morphology of oligonucleotide–cationic lipid complexes in buffer and in cell culture medium using quasi-elastic light scattering (QELS), resonance energy transfer (RET) and freeze-fracture electron microscopy. In addition, interaction of the complexes with model membranes was studied at extracellular (pH 7.4), intracellular (pH 7.1) and endosomal (pH 5 and 6) conditions. Phosphorothioate oligonucleotides (ODN, 15 mer) were complexed with cationic liposomes composed of DOTAP, or DOTAP with DOPE at 1/1 and 1/2 molar ratios and investigated at different charge ratios (−/+, ODN–cationic lipid). The size of the complexes formed in water increased prominently in buffer and DMEM and it was maximal at charge ratio (−/+) of 1.2–1.5. In the case of sole DOTAP liposomes the complex sizes were smaller at −/+ ratios <0.5 and >1.7 in all media. In the presence of DMEM the DOPE-containing complexes gave average diameter in μm range irrespect of the charge ratio. Lipid mixing, in general, increased with increasing −/+ ratio and with DOPE in the presence of buffer or DMEM. Freeze-fracture electron micrographs showed cationic liposomes undergoing aggregation and fusion during interaction with ODNs. DOPE in cationic liposomes induced hexagonal lipid tubule formation that was most pronounced in cell culture medium. Upon incubation with endosomal model liposomes ODN was partly released from the complexes. The release was more pronounced when the liposomes contained DOPE. It appears that DOPE as a helper lipid affects the behaviour of ODN/lipid complexes at several stages.
The aggregation state of amphotericin B (AmB) was previously reported to modulate its therapeutic efficiency. As a preliminary study to test the biological effects of ‘superaggregates’ generated by heat treatment, we present spectroscopic data related to their formation in aqueous solutions. Drastic changes in the AmB aggregation state in water were shown to occur on heating at 50–60°C. The concentration of the aggregates formed at high (At) or room (A) temperature, and the concentration of the monomeric form (M) of AmB were calculated by processing absorption data. The thermally induced conversion from A to At depends on the AmB concentration. Rayleigh scattering measurements suggest that the At aggregates are larger than the A aggregates. At room temperature, the condensation rate of A with M—leading to the ‘superaggregated’ form At—was slower and depended on the concentration of M. The superaggregated species At was shown to be the most chemically stable species. Physico-chemical properties of these superaggregates are discussed as a potential new solution to improve the therapeutic efficacy of AmB.
It is proved that the absorption spectrum of a suspension of particles is flattened, as compared with that of a solution of the pigments contained in the particles. This flattening is due to the enhanced mutual shading of the pigment molecules in the suspension. A quantitative theory of this flattening is given. If the flattening is measured experimentally, the optical density of the individual particles can be computed, at least in principle (for spherical particles a nomograph is given to this purpose). This optical density may be used for the calculation of the “true” absorption spectrum of a pigment in suspension (which can be used, for example, for an improved analysis of a composite spectrum, in terms of overlapping absorption spectra of the several pigments present in the particle). Such an analysis is generally required when relative photochemical efficiencies of these pigments must be determined.
The synthesis of new N-alkyl amphotericin B derivatives obtained in the Michael addition reaction of the antibiotic with N-substituted maleimides is described and in vitro biological data are presented.
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.
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.
In the 1970's great strides were made in understanding the mechanism of action of amphotericin B and nystatin: the formation of transmembrane pores was clearly demonstrated in planar lipid monolayers, in multilamellar phospholipid vesicles and in Acholeplasma laidlawii cells and the importance of the presence and of the nature of the membrane sterol was analyzed. For polyene antibiotics with shorter chains, a mechanism of membrane disruption was proposed. However, recently obtained data on unilamellar vesicles have complicated the situation. It has been shown that: membranes in the gel state (which is not common in cells), even if they do not contain sterols may be made permeable by polyene antibiotics, several mechanisms may operate, simultaneously or sequentially, depending on the antibiotic/lipid ratio, the time elapsed after mixing and the mode of addition of the antibiotic, there is a rapid exchange of the antibiotic molecules between the vesicles. Although pore formation is apparently involved in the toxicity of amphotericin B and nystatin, it is not the sole factor which contributes to cell death, since K+ leakage induced by these antibiotics is separate from their lethal action. The peroxidation of membrane lipids, which has been demonstrated for erythrocytes and Candida albicans cells in the presence of amphotericin B, may play a determining role in toxicity concurrently with colloid osmotic effect. On the other hand, it has been shown that the action of polyene antibiotics on cells is not always detrimental: at sub-lethal concentrations these drugs stimulate either the activity of some membrane enzymes or cellular metabolism. In particular, some cells of the immune system are stimulated. Furthermore, polyene antibiotics may act synergistically with other drugs, such as antitumor or antifungal compounds. This may occur either by an increased incorporation of the drug, under the influence of a polyene antibiotic-induced change of membrane potential, for example, or by a direct interaction of both drugs. That fungal membranes contain ergosterol while mammalian cell membranes contain cholesterol, has generally been considered the basis for the selective toxicity of amphotericin B and nystatin for fungi. Actually, in vitro studies have not always borne out this assumption, thereby casting doubt on the use of polyene antibiotics as antifungal agents in mammalian cell culture media.(ABSTRACT TRUNCATED AT 400 WORDS)
Natural polyene macrolide antibiotics and their N-acyl and methyl ester derivatives, which differ mainly in their electric net charge, were compared for their ability to increase the ionic permeability of large unilamella vesicles, using the proton-cation exchange method and 31P-NMR spectroscopy. The zwitterionic (amphotericin B, vacidin A) and negatively charged (N-N'-diacetyl vacidin) compounds induced permeability according to an all-or-none process on both cholesterol- and ergosterol-containing membranes. The same mechanism of permeability induction is obtained only on ergosterol-containing vesicles for positively charged antibiotics (perimycin A, vacidin A methyl ester, amphotericin B methyl ester). A different type of action is observed for the latter group of ionophores in cholesterol-containing vesicles. In this case, a progressive proton efflux occurs in which all of the vesicle population is involved. This qualitative difference in the kinetics of ionic fluxes induced by antibiotics without a free carboxyl group in cholesterol-containing as compared to ergosterol-containing membranes was ascribed to differences in polyene-sterol interactions as well as in the life time of the ionic path formed. This difference may provide a basis for the improvement of selective toxicity of this group of antifungal agents by rational modifications.
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.
Isotherms for the binding of dodecyltrimethylammonium (DTA+) and tetradecyltrimethylammonium (TTA+) ions by DNA in aqueous solution at 30 degrees C are reported. The binding isotherms were determined using a potentiometric technique with cationic surfactant-selective electrodes. The DNA concentrations used are 5 X 10(-4) and 10(-3) equiv./kg, surfactant concentrations varying from 3 X 10(-6) M to the critical micelle concentration. The influence of added NaCl (0.01 M) on the binding process is studied. The binding process is shown to be highly cooperative. Applying the binding theory of Schwarz and of Satake and Yang, binding constants and cooperativity parameters can be calculated. The binding constant K is found to be 1.2kT larger for TTA+ than for DTA+ in salt-free solution, and 1.4kT larger for TTA+ than for DTA+ in 0.01 M NaCl. The cooperativity parameter mu is about 1.1kT larger for TTA+ in salt-free solution, and 1.2kT larger in 0.01 M NaCl. It is concluded that the hydrophobic part of the bound surfactant is not completely immersed in the hydrophobic DNA core, but also interacts with other surfactant molecules. This situation is compared to the case of micelle formation.
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.
The paper contains data on the induction of K+ efflux and viability of baby hamster kidney (BHK-21) cells after their treatment with macrolide antibiotics inducing specific pores in membrane. New water-soluble semisynthetic derivatives of amphotericin B and aureofacin (N-glycosyl and trimethylammonium methyl ester derivatives) as well as the parent compounds were used to compare the concentration of antibiotics inducing permeabilizing and cytostatic effects. We found that a two- to eight-times-higher concentration of polyene antibiotic was required to observe a cytostatic effect than for release of 50% of the cellular potassium (K50 concentration) from BHK-21 cells. These differences were larger for water-soluble derivatives than for the parent compounds. The amount of intracellular potassium in treated cells incubated under optimal growth conditions was higher than in cells which had been further washed with K+-free maintenance medium. The membrane permeability changes induced by low concentrations of specific polyenes were observed to be reversible. BHK-21 cells were able to repair polyene-induced membrane permeability within 3 to 12 h under optimal growth conditions, after cell treatment with K50 concentration of specific macrolide antibiotics. The repair phenomenon is postulated as an explanation for the dissociation observed between permeabilizing and cytostatic effect of specific polyenes in BHK-21 cells.
We develop and test a Poisson-Boltzmann model of the electrostatics of the B-Z transition of DNA. Starting from the detailed geometries of the two forms, we compute at each radius the fractions of DNA matter, of volume forbidden (for nonpoint-like ions), and of volume accessible to the center of ions. These radial distributions are incorporated in a composite cylinder model; availability to ions (porosity) and the dielectric constant at each radial distance are then obtained. The phosphate charge is distributed with cylindrical symmetry on two layers at the appropriate radial distances. The porous sheath, between the axis and the charge distribution, provides much more room for ions in B-DNA than in Z-DNA. By using previously developed methods, the Poisson-Boltzmann problem of such cylinders is easily solved. The computational load is small, so that results can be obtained for a large set of salt concentrations and for a number of ionic radii. The variation of the electrostatic free energy difference with salt concentration compares favorably with the experimental value (it is half as large). There is also qualitative agreement with experiments on supercoiled DNA, including a maximum of the free energy difference at submolar salt concentrations. The results for this cylinder with porous sheath are in line with those of the earlier simple planar model and of a plain cylinder with sheath, which is also presented here. They are thus insensitive to details of the model. They support the proposition that the main electrostatic feature of the B-Z transition is the better immersion of the B-DNA phosphates into the solution. They also give confidence in the validity of the Poisson-Boltzmann approach, despite the large salt concentrations involved. Prior studies using an approach based on the potential of mean force are discussed.
We recently demonstrated that cationic lipids, added in monomer or micellar form, bind to DNA, resulting in the formation of a hydrophobic complex. This complex can serve as a well-defined intermediate in the preparation of DNA-lipid particles (DLPs) with many potential applications for delivery of polynucleotides in vitro and in vivo. To develop a better understanding of the factors governing complex formation, we have characterized the cationic lipid/DNA binding reaction. This was evaluated by measuring DNA and cationic lipid (DODAC) complex formation using the Bligh and Dyer extraction procedure. Efficient recovery of DNA (> 95%) in the organic phase was achieved when sufficient monocationic lipids interact with DNA phosphate groups. The rate of binding depends on the amount of DNA or cationic lipid present in the system. The time required to generate the hydrophobic complex was increased when < 10 micrograms of DNA or < 40 nmol of DODAC was present. Surprisingly, the rate of complex formation was contingent on the incubation period after partitioning the DNA/lipid mixture into organic and aqueous phases. These results suggest that the cationic lipid/DNA complex forms at the aqueous/organic interface and that DNA/lipid binding is dependent on multivalent interactions at this interface. A Scatchard analysis of DNA/DODAC binding demonstrated that the binding reaction exhibits a high degree of positive cooperativity. The apparent dissociation constant (Kn), using data obtained under conditions where DODAC binding to DNA approached saturation, indicated a high-affinity reaction (Kn > 10(-11) mol L-1). At this point, approximately 8400 mol of DODAC was bound per mole of DNA, which is equivalent to a charge ratio (+/-) of 0.585 for the 7.2 kb plasmid used and suggests that formation of the hydrophobic complex occurs at a stage prior to charge neutralization. The influence of other lipids on DNA/cationic lipid binding at the aqueous/organic interface was also studied. Cholesterol and DOPC had little effect on DNA/DODAC binding while the anionic lipids LPI, DOPS, and DMPG inhibited complex formation. The zwitterionic lipid DOPE, however, had a concentration-dependent effect on cationic lipid binding that was also dependent on the mixing order. We believe that this approach for evaluating lipid/DNA binding provides an effective procedure for assessing factors which control the dissociation of lipids from DNA and may be beneficial in the selection of lipids for effective use in gene transfection studies.
The effect of synthetic cationic lipids on the structure of DNA was studied. The fluorescence enhancement of ethidium bromide on intercalation into DNA was suppressed by the addition of bilayer-forming lipids, but not by micellar ones. Results on the fluorescence depolarization index suggest that ethidium bromide is not released from DNA by lipids intercalated into DNA. CD spectra of the DNA-lipid complexes revealed that the structure of DNA was changed only by bilayer-forming lipids at temperatures lower than their Tc values. Thus, the conformation of DNA is forced to change by cationic lipids forming the rigid bilayer membrane so that ethidium bromide fluorescence might be reduced, and the conformation can be controlled by selection of the appropriate lipid and temperature.
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.
Conditions of double-stranded DNA precipitation by the polyamines spermidine and spermine have been determined experimentally and compared to theoretical predictions. The influence of the concentrations of DNA and added monovalent salt, and of the DNA length has been investigated in a systematic manner. Three regimes of DNA concentrations are observed. We clarify the dependence of these regimes on the monovalent salt concentration and on the DNA length. Our observations make possible a rationalization of the experimental results reported in the literature. A comparison of the precipitation conditions of different kinds of polyelectrolytes suggests a general process. Our experimental data are compared to the "ion-bridging" model based on short-range electrostatic attractions. By starting from the spinodal equation, predicted by this model, and using the limiting form of Manning's fractions of condensed counterions, analytical expressions of the precipitation conditions have been found in the three regimes. Experimental and theoretical results are in good agreement.
It is proved that the absorption spectrum of a suspension of particles is flattened, as compared with that of a solution of the pigments contained in the particles. This flattening is due to the enhanced mutual shading of the pigment molecules in the suspension. A quantitative theory of this flattening is given. If the flattening is measured experimentally, the optical density of the individual particles can be computed, at least in principle (for spherical particles a nomograph is given to this purpose). This optical density may be used for the calculation of the “true” absorption spectrum of a pigment in suspension (which can be used, for example, for an improved analysis of a composite spectrum, in terms of overlapping absorption spectra of the several pigments present in the particle). Such an analysis is generally required when relative photochemical efficiencies of these pigments must be determined.