Modulation of amphotericin B membrane interaction by cholesterol and ergosterol--a molecular dynamics study

Amphotericin B (AmB) is a well-known polyene macrolide antibiotic used to treat systemic fungal infections. According to a well-documented hypothesis, molecules of AmB form ionic membrane channels that are responsible for chemotherapeutic action. These channels disturb the barrier function of the cell membrane which, in consequence, leads to cell death. The presence of sterols in the cell membrane is necessary for full manifestation of the antibiotic's ionophoric activity, at least in vivo. Ergosterol-containing fungal membranes are targeted more efficiently by AmB than mammalian membranes containing cholesterol. However, a similar level of disturbance of fungal and mammalian membranes is responsible for serious toxicity of the antibiotic. Due to the importance of AmB and lack of better antifungal alternatives, the search for new less toxic derivatives of this antibiotic still continues. Therefore, studies of the AmB-membrane interaction are very important. The present work constitutes a continuation of a broad program of study on AmB mode of action in our group. In particular, molecular dynamics simulations of AmB monomers inside the bilayers of three different compositions (pure dimiristoylphosphatidylcholine (DMPC) and DMPC bilayer containing approximately 25 mol % of cholesterol or ergosterol) were carried out. In general, analysis of generated trajectories resulted in identifying many significant differences in the behavior of AmB monomers depending on the membrane environment. In particular, it was established that the antibiotic increases the internal order of DMPC bilayer containing 25 mol % of cholesterol, while it has no effect on the order of the bilayer with the same amount of ergosterol. Performed calculations also revealed that relatively rigid and elongated AmB molecules exhibit higher affinity toward the sterol-containing lo phases and, therefore, may be cumulated in ordered membrane domains (e.g., lipid rafts). Since the partition coefficient between the ld and lo phase appears to be greater in the case of the ergosterol- compared to cholesterol-containing membrane, this effect can be also discussed as the possible origin of AmB-selective toxicity and indirect sterol involvement in expression of AmB activity.     

Conformational properties of amphotericin B amide derivatives – impact on selective toxicity

Even though it is highly toxic, Amphotericin B (AmB), an amphipathic polyene macrolide antibiotic, is used in the treatment of severe systemic fungal infections as a life-saving drug. To examine the influence of conformational factors on selective toxicity of these compounds, we have investigated the conformational properties of five AmB amide derivatives. It was found that the extended conformation with torsional angles (,)=(290°,180° ) is a common minimum of the potential energy surfaces (PES) of unsubstituted AmB and its amide derivatives. The extended conformation of the studied compounds allows for the formation of an intermolecular hydrogen bond network between adjacent antibiotic molecules in the open channel configuration. Therefore, the extended conformation is expected to be the dominant conformer in an open AmB (or its amide derivatives) membrane channel. The derivative compounds for calculations were chosen according to their selective toxicity compared to AmB and they had a wide range of selective toxicity. Except for two AmB derivatives, the PES maps of the derivatives reveal that the molecules can coexist in more than one conformer. Taking into account the cumulative conclusions drawn from the earlier MD simulation studies of AmB membrane channel, the results of the potential energy surface maps, and the physical considerations of the molecular structures, we hypothesize a new model of structure-selective toxicity of AmB derivatives. In this proposed model the presence of the extended conformation as the only well defined global conformer for AmB derivatives is taken as the indicator of their higher selective toxicity. This model successfully explains our results. To further test our model, we also investigated an AmB derivative whose selective toxicity has not been experimentally measured before. Our prediction for the selective toxicity of this compound can be tested in experiments to validate or invalidate the proposed model.     

How do sterols determine the antifungal activity of amphotericin B? Free energy of binding between the drug and its membrane targets

Amphotericin B (AmB) is a well-known polyene antibiotic used to treat systemic fungal infections. It is commonly accepted that the presence of sterols in the membrane is essential for the AmB biological activity, that is, for the formation of transmembrane ion channels. The selective toxicity of AmB for fungal cells is attributed to the fact that it is more potent against fungal cell membranes containing ergosterol than against the mammalian membranes with cholesterol. According to the "primary complex" hypothesis, AmB associates with sterols in a membrane to form binary complexes, which may subsequently assemble into a barrel-stave channel. To elucidate the molecular nature of the AmB selectivity for ergosterol-containing membranes, in the present work, we used computational methods to study the formation of the putative AmB/sterol complexes in a lipid bilayer. The free energy profiles for the AmB-sterol association in phospholipid bilayers containing 30 mol % of sterols were calculated and thoroughly analyzed. The results obtained confirm the formation of specific AmB/ergosterol complexes and are used to determine the energetic and structural origin of the enhanced affinity of AmB for ergosterol than for cholesterol. The significance of this affinity difference for the mechanism of action of AmB is discussed. The data obtained allowed us also to suggest a possible origin of the increased selectivity of a novel class of less toxic AmB derivatives.     

How does the N-acylation and esterification of amphotericin B molecule affect its interactions with cellular membrane components-the Langmuir monolayer study

This work presents the results of Langmuir monolayers study of two amphotericin B derivatives obtained by N-acylation (N-acetylamphotericin B, Ac-AmB) and esterification (amphotericin B methyl ester, AME) of the parent AmB molecule. The main objective of present investigations was to examine the strength and nature of interactions of Ac-AmB and AME with natural membrane components as compared to AmB, and verify the monolayer results with biological studies in vitro. Our experiments were based on surface pressure-area measurements of mixed monolayers formed by the investigated antibiotics and sterols/DPPC. The interactions were analyzed with the following dependencies: compression modulus-surface pressure, mean molecular area-composition, excess molecular area-composition and excess free energy-composition plots. It has been found that both Ac-AmB and AME form monolayers of a liquid expanded state and their stability is highest as compared to AmB films. The investigated compounds mix in monolayers with natural membrane components within the whole range of the antibiotic mole fraction. The quantitative analysis of the interactions of the investigated antibiotics with sterols and DPPC as well as sterols/DPPC interactions allow us to verify the monolayer results with biological results. A good correlation between both kinds of studies has been found.     

Mycosamine orientation of amphotericin B controlling interaction with ergosterol: sterol-dependent activity of conformation-restricted derivatives with an amino-carbonyl bridge

Amphotericin B (AmB 1) is known to assemble together and form an ion channel across biomembranes. The antibiotic consists of mycosamine and macrolactone moieties, whose relative geometry is speculated to be determinant for the drug's channel activity and sterol selectivity. To better understand the relationship between the amino-sugar orientation and drug's activity, we prepared conformation-restricted derivatives 2-4, in which the amino and carboxyl groups were bridged together with various lengths of alkyl chains. K+ influx assays across the lipid-bilayer membrane revealed that ergosterol selectivity was markedly different among derivatives; short-bridged derivative 2 almost lost the selectivity, while 3 showed higher ergosterol preference than AmB itself. Monte Carlo conformational analysis of 2-4 based on NOE-derived distances indicated that the amino-sugar moiety of 2 comes close to C41 because of the short bridge, whereas those of 3 and 4 are pointing outward. The mutual orientation of the amino-sugar moiety and macrolide ring is so rigid in derivatives 2 and 3 that these conformations should be unchanged upon complex formation in lipid membranes. These results strongly suggest that the large difference in sterol preference between derivatives 2 and 3 is ascribed to the different orientation of amino-sugar moieties. These findings allowed us to propose a simple model accounting for AmB-sterol interactions, in which hydrogen bonding between 2'-OH of AmB and 3beta-OH of ergosterol plays an important role.     

Ion selectivity, transport properties and dynamics of amphotericin B channels studied over a wide range of acidity changes

Amphotericin B (AmB) is an antifungal antibiotic which, despite the severe side effects, is still used for the treatment of systemic fungal infections. Herein we studied the influence of pH upon the selectivity and the transport properties of AmB channels inserted in reconstituted, ergosterol-containing zwitterionic lipid membranes. Our electrophysiology experiments carried out on single and multiple AmB channels prove that at pH 2.8 these channels are anion selective, whereas at neutral and alkaline pH's (pH 7 and pH 11) they become cation selective. We attribute this to the pH-dependent ionization state of the carboxyl and amino groups present at the mouth of AmB molecules. Surprisingly, our data reveal that the single-molecule ionic conductance of AmB channels varies in a non-monotonic fashion with pH changes, which we attribute to the pH-dependent variation of the surface and dipole membrane potential. We demonstrate that when added only from one side of the membrane, in symmetrical salt solutions across the membrane and low pH values, AmB channels display a strong rectifying behavior, and their insertion is strongly favored when positive potentials are present on the side of their addition.     

Modulating the structure and properties of cell membranes: the molecular mechanism of action of dimethyl sulfoxide

Dimethyl sulfoxide (DMSO) is a small amphiphilic molecule which is widely employed in cell biology as an effective penetration enhancer, cell fusogen, and cryoprotectant. Despite the vast number of experimental studies, the molecular basis of its action on lipid membranes is still obscure. A recent simulation study employing coarse-grained models has suggested that DMSO induces pores in the membrane (Notman, R.; Noro, M.; O'Malley, B.; Anwar, J. J. Am. Chem. Soc. 2006, 128, 13982-13983). We report here the molecular mechanism for DMSO's interaction with phospholipid membranes ascertained from atomic-scale molecular dynamics simulations. DMSO is observed to exhibit three distinct modes of action, each over a different concentration range. At low concentrations, DMSO induces membrane thinning and increases fluidity of the membrane's hydrophobic core. At higher concentrations, DMSO induces transient water pores into the membrane. At still higher concentrations, individual lipid molecules are desorbed from the membrane followed by disintegration of the bilayer structure. The study provides further evidence that a key aspect of DMSO's mechanism of action is pore formation, which explains the significant enhancement in permeability of membranes to hydrophilic molecules by DMSO as well as DMSO's cryoprotectant activity. The reduction in the rigidity and the general disruption of the membrane induced by DMSO are considered to be prerequisites for membrane fusion processes. The findings also indicate that the choice of DMSO concentration for a given application is critical, as the concentration defines the specific mode of the solvent's action. Knowledge of the distinct modes of action of DMSO and associated concentration dependency should enable optimization of current application protocols on a rational basis and also promote new applications for DMSO.     

Influence of polymyxins on the structural dynamics of Escherichia coli lipid membranes

Polymyxins are a family of nonribosomic cationic peptide antibiotics highly effective against Gram-negative bacteria. Two members of this family, Polymyxins B and E (PxB, PxE), form molecular vesicle-vesicle contacts and promote a selective exchange of phospholipids at very low concentrations in the membrane, a biophysical phenomenon that can be the basis of their antibiotic mode of action. To get more insight into the interaction of these antibiotics with the lipid membrane, their effect on the structural dynamics of bilayers prepared with lipids extracted from the membrane of Escherichia coli was determined using fluorescently labeled phopholipids. Steady-state anisotropy measurements with probes that localize at different positions in the membrane give information on the effects of polymyxins on the mobility of the phospholipids. Results with PxB, PxE, colymycin M and polymyxin B nonapeptide (PxB-NP), a deacylated derivative with no antibiotic properties, are compared. At low peptide concentrations (<2 mol%) PxB and PxE bind to the membranes superficially, affecting very slightly the ordering of the lipids at the outermost part of the bilayer. Above this concentration, PxB and PxE insert more deeply in the bilayer, increasing lipid order both in the gel and liquid-crystal states and modifying phase transitions. Fluorescence experiments with pyrene labeled phospholipids indicate that the increase in lipid packing is accompanied by an enrichment of phospholipids in the bilayers. In contrast, colymycin M and PxB-NP did not modify lipid packing or phase transition, nor did they induce microdomain formation. The possible significance of these results in the antibiotic mode of action of PxB and PxE is discussed. The combination of spectroscopic techniques described here can be useful as part of a general method of screening for new antibiotics that act on the membrane by the same mechanism as polymyxins.     

Facilitated permeation of antibiotics across membrane channels--interaction of the quinolone moxifloxacin with the OmpF channel

The facilitated influx of moxifloxacin through the most abundant channel in the outer cell wall of gram-negative bacteria was investigated. Molecular modeling provided atomic details of the interaction with the channel surface, revealed the preferred orientation of the antibiotic along its pathway, and gave an estimated time necessary for translocation. High-resolution conductance measurements on single OmpF trimers allowed the passages of individual moxifloxacin molecules to be counted. The average mean residence time of 50 micros is in agreement with the predicted strong interaction from the modeling. In contrast, control measurements with nalidixic acid, a hydrophobic antibiotic that rather permeates across the lipid membrane, revealed a negligible interaction. The spectral overlap of tryptophan with moxifloxacin was suitable for a FRET study of the protein-antibiotic interaction. Combining molecular dynamics simulations with selective quenching identified an interaction of moxifloxacin with Trp61 inside the OmpF channel, whereas nalidixic acid showed preferential interaction with Trp214 on the channel exterior. An understanding of the detailed molecular interactions between the antibiotic and its preferred channel may be used to develop new antibiotics with improved uptake kinetics.     

Conformation and position of membrane-bound amphotericin B deduced from NMR in SDS micelles

Amphotericin B (AmB) is known to self-assemble to form an ion channel across lipid bilayer membranes. To gain insight into the conformation of AmB in lipidic environments, AmB in SDS micelles was subjected to high-resolution NMR and CD measurements, and the NMR-derived conformation thus obtained was refined by molecular mechanics calculations. These results indicate that AmB in SDS micelles is conformationally fixed particularly for the macrolide moiety. Paramagnetic relaxation experiments with the use of Mn2+ reveal that AmB is shallowly embedded in the micelle with the polyhydroxyl chain being close to the water interface and the side of polyene portion facing to the micelle interior. CD measurements demonstrate that AmB is in a monomeric form in SDS micelles. The structure of AmB in the micelles obtained in the present study may reproduce the initial stage of membrane interaction of AmB prior to the assembly formation in biomembranes.     

Large molecular assembly of amphotericin B formed in ergosterol-containing membrane evidenced by solid-state NMR of intramolecular bridged derivative

Amphotericin B (AmB 1) is known to assemble and form an ion channel across biomembranes. We have recently reported that conformation-restricted derivatives of AmB 2-4 show different ergosterol preferences in ion-channel assays, which suggested that the orientation of the mycosamine strongly affects the sterol selectivity of AmB. The data allowed us to assume that compound 3 showing the highest selectivity would reflect the active conformation of AmB in the channel assembly. In this study, to gain further insight into the active conformation of AmB, we prepared a new intramolecular-bridged derivative 5, where the linker encompassed a hydrophilic glycine moiety. The derivative has almost equivalent ion-channel activity to those of AmB and 3. The antifungal activity of 5 compared with 3 improves significantly, possibly because the increasing hydrophilicity in the linker enhances the penetrability through the fungal cell wall. Conformation of 5 was well converged and very similar to that of 3, thus further supporting the notion that the conformations of these derivatives reproduce the active structure of AmB in the channel complex. Then we used the derivative to probe the mobility of AmB in the membrane by solid-state NMR. To measure dipolar couplings and chemical shift anisotropies, we incorporated [1-(13)C,(15)N]glycine into the linker. The results indicate that 5 is mostly immobilized in ergosterol-containing DMPC bilayers, implying formation of large aggregates of 5. Meanwhile some fraction of 5 remains mobile in sterol-free DMPC bilayers, suggesting promotion of AmB aggregation by ergosterol.     

Amphotericin B covalent dimers forming sterol-dependent ion-permeable membrane channels

Polyenemacrolides such as amphotericin B (AmB) were thought to assemble together and form an ion channel across plasma membranes. Their antimicrobial activity has been accounted for by this assemblage, whose stability and activity are dependent on sterol constituents of lipid bilayer membranes. The structure of this channel-like assemblage formed in biomembranes has been a target of extensive investigations for a long time. For the first step to this goal, we prepared several AmB dimers with various linkers and tested for their channel-forming activity. Among these, AmB dimers that bore an aminoalkyl-dicarboxylate tether covalently linked between amino groups of AmB showed potent hemolytic activity. Furthermore, K+ influx actions monitored by measuring the pH of the liposome lumen by 31P NMR revealed that the dimers formed the molecular assemblage similar to that of AmB in phospholipid membrane. Judging from changes in 31P NMR spectra, the dimers appeared to induce "all-or-none"-type ion flux across the liposome membrane in the presence of ergosterol, which suggested that the ion channel formed by ergosterol/dimer is similar to that of AmB. With these data in hand, we are now trying to elucidate the structure of the ion-channel complex by making the labeled conjugates of AmB for NMR measurements.     

Design and photochemical characterization of a biomimetic light-driven Z/E switcher

Protonated Schiff bases (PSBs) of polyenals constitute a class of light-driven switchers selected by biological evolution that provide model compounds for the development of artificial light-driven molecular devices or motors. In the present paper, our primary target is to show, through combined computational and experimental studies, that it is possible to approach the design of artificial PSBs suitable for such applications. Below, we use the methods of computational photochemistry to design and characterize the prototype biomimetic molecular switchers 4-cyclopenten-2'-enylidene-3,4-dihydro-2H-pyrrolinium and its 5,5'-dimethyl derivative both containing the penta-2,4-dieniminium chromophore. To find support for the predicted behavior, we also report the photochemical reaction path of the synthetically accessible compound 4-benzylidene-3,4-dihydro-2H-pyrrolinium. We show that the preparation and photochemical characterization of this compound (together with three different N-methyl derivatives) provide both support for the predicted photoisomerization mechanism and information on its sensitivity to the molecular environment.     

Rationalizing the generation of broad spectrum antibiotics with the addition of a positive charge

Antibiotic resistance of Gram-negative bacteria is largely attributed to the low permeability of their outer membrane (OM). Recently, we disclosed the eNTRy rules, a key lesson of which is that the introduction of a primary amine enhances OM permeation in certain contexts. To understand the molecular basis for this finding, we perform an extensive set of molecular dynamics (MD) simulations and free energy calculations comparing the permeation of aminated and amine-free antibiotic derivatives through the most abundant OM porin of E. coli, OmpF. To improve sampling of conformationally flexible drugs in MD simulations, we developed a novel, Monte Carlo and graph theory based algorithm to probe more efficiently the rotational and translational degrees of freedom visited during the permeation of the antibiotic molecule through OmpF. The resulting pathways were then used for free-energy calculations, revealing a lower barrier against the permeation of the aminated compound, substantiating its greater OM permeability. Further analysis revealed that the amine facilitates permeation by enabling the antibiotic to align its dipole to the luminal electric field of the porin and form favorable electrostatic interactions with specific, highly-conserved charged residues. The importance of these interactions in permeation was further validated with experimental mutagenesis and whole cell accumulation assays. Overall, this study provides insights on the importance of the primary amine for antibiotic permeation into Gram-negative pathogens that could help the design of future antibiotics. We also offer a new computational approach for calculating free-energy of processes where relevant molecular conformations cannot be efficiently captured.

A rapid pathway sampling method combining Monte Carlo and graph theory, developed to describe permeation pathways through outer membrane porins, can distinguish between structurally similar analogs with different permeabilities.     

Phospholipid Bilayers as Molecular Models for Drug-Cell Membrana Interactions. The Case of the Antiinflamatory Drug Diclofenac

Phospholipids are amphipatic molecules with long hydrophobic acyl chains and zwitterionic polar heads which assemble into different types of molecular aggregates. The most relevant is the bilayer because of its relation with cell membranes, which are very complex entities. For this reason, simpler molecular models based on phospholipids bilayers are widely used. We have determined the bilayer structure of phospholipids located in the outer and inner monolayers of most cell membranes, and use them as molecular models to study the way different chemicals of biological interest interact with cell membranes. We present the results of our studies on the nonsteroidal anti-inflammatory drug diclofenac, from which little is known about its effects on human erythrocytes. This report presents the following evidence that diclofenac interacts with the human red cell membrane: a) X-ray diffraction and fluorescence spectroscopy of phospholipids bilayers show that diclofenac interacts with a class of lipids found in the outer moiety of the erythrocyte membrane; b) in isolated unsealed human erythrocyte membranes the drug induced a disordering effect on the acyl chains of the membrane lipid bilayer; c) in scanning electron microscopy studies on human erythrocytes it was observed that the drug induced morphological changes different from their normal biconcave shape.     

Molecular mechanisms of membrane perturbation by antimicrobial peptides and the use of biophysical studies in the design of novel peptide antibiotics

Antibiotic resistant bacterial strains represent a global health problem with a strong social and economic impact. Thus, there is an urgent need for the development of antibiotics with novel mechanisms of action. There is currently an extensive effort to understand the mode of action of antimicrobial peptides which are considered as one alternative to classical antibiotics. The main advantage of this class of substances, when considering bacterial resistance, is that they rapidly, within minutes, kill bacteria. Antimicrobial peptides can be found in every organism and display a wide spectrum of activity. Hence, the goal is to engineer peptides with an improved therapeutic index, i.e. high efficacy and target specificity. For the rational design of such novel antibiotics it is essential to elucidate the molecular mechanism of action. Biophysical studies have been performed using to a large extent membrane model systems demonstrating that there are distinctive different mechanisms of bacterial killing by antimicrobial peptides. One can distinguish between peptides that permeabilize and/or disrupt the bacterial cell membrane and peptides that translocate through the cell membrane and interact with a cytosolic target. Lantibiotics exhibit specific mechanisms, e.g. binding to lipid II, a precursor of the peptidoglycan layer, either resulting in membrane rupture by pore formation or preventing cell wall biosynthesis. The classical models of membrane perturbation, pore formation and carpet mechanism, are discussed and related to other mechanisms that may lead to membrane dysfunction such as formation of lipid-peptide domains or membrane disruption by formation of non-lamellar phases. Emphasis is on the role of membrane lipid composition in these processes and in the translocation of antimicrobial peptides.     

Influence of K+, Na+ or Ca2+ Ions on the Interaction Between AmB and Saturated Phospholipids by Langmuir Technique

Interaction between amphotericin B(AmB) and cell membrane is influenced by different metal cations. In the presence of K⁺, Na⁺ or Ca²⁺ ions, the surface pressure-area isotherms and the elastic modulus of an amphotericindipalmitoylphosphatidylcholine(AmB-DPPC) mixed monolayer were discussed. And the excess free energy and entropies of mixing were calculated according to the surface pressure-area isotherms. The phase transition of the mixed monolayer needed a higher concentration of AmB in the sequence Na⁺ > pure buffer > K⁺ > Ca²⁺. When the molar fraction of AmB(x_AmB) was 0.5, the molecular interaction changed from attraction to repulsion and the mixed monolayer turned to ordered state from disorder state under the induction of K⁺ or Ca²⁺ ions at all surface pressure in our experiment. At high surface pressure, the disorder of monolayer enhanced in the presence of Na⁺ ions at x_AmB > 0.1. At different molar ratios of AmB, the influences of these metal cations were discrepant. These cations may influence AmB molecules to form pores on the monolayer. It is helpful to understand the reduction of AmB's toxicity as theoretical reference.     

Stimulus Dependent Redistribution of Membrane Raft Cholesterol in Human Platelets

Cell membranes provide a requisite dynamic interface to facilitate communication between the extracellular environment and the intracellular milieu. These membranes contain proteins that span and/or are loosely associated with the lipid bilayer. The organization of lipids and proteins components into membrane micro-domains provides a temporal and spatial signaling platform for communication. Recently, cholesterol and sphingomyelin enriched membrane micro-domains known as lipid rafts have been implicated in cell signaling events. In these studies we have advanced our hypothesis that stimulus dependent rearrangement of cholesterol into and out of membrane rafts provides a unique lipid–mediated regulatory mechanism. Using fluorescent derivatives of cholesterol, we have shown that membrane raft associated cholesterol was altered in response to collagen-induced platelet aggregatory stimulation. Collagen stimulation resulted in a rapid redistribution of cholesterol from the outer to the inner membrane monolayer. The reorganization of the outer membrane monolayer resulted in a concomitant increase in outer monolayer fluidity. These studies are the first to show that membrane cholesterol was released from the exchangeable membrane raft pool in response to physiological stimuli.     

Computer simulation studies on the interactions between nanoparticles and cell membrane

In recent times,nanoparticles(NPs)have received intense attention not only due to their potential applications as a candidate for drug delivery,but also because of their undesirable effects on human health.Although extensive experimental studies have been carried out in literature in order to understand the interaction between NPs and a plasma membrane,much less is known about the molecular details of the interaction mechanisms and pathways.As complimentary tools,coarse grained molecular dynamics(CGMD)and dissipative particle dynamics(DPD)simulations have been extensively used on the interaction mechanism and evolution pathway.In the present review we summarize computer simulation studies on the NP-membrane interaction,which developed over the last few years,and particularly evaluate the results from the DPD technique.Those studies undoubtedly deepen our understanding of the NP-membrane interaction mechanisms and provide a design guideline for new NPs.     

Lipid Nanoparticles Containing siRNA Synthesized by Microfluidic Mixing Exhibit an Electron-Dense Nanostructured Core

Lipid nanoparticles (LNP) containing ionizable cationic lipids are the leading systems for enabling therapeutic applications of siRNA; however, the structure of these systems has not been defined. Here we examine the structure of LNP siRNA systems containing DLinKC2-DMA(an ionizable cationic lipid), phospholipid, cholesterol and a polyethylene glycol (PEG) lipid formed using a rapid microfluidic mixing process. Techniques employed include cryo-transmission electron microscopy, (31)P NMR, membrane fusion assays, density measurements, and molecular modeling. The experimental results indicate that these LNP siRNA systems have an interior lipid core containing siRNA duplexes complexed to cationic lipid and that the interior core also contains phospholipid and cholesterol. Consistent with experimental observations, molecular modeling calculations indicate that the interior of LNP siRNA systems exhibits a periodic structure of aqueous compartments, where some compartments contain siRNA. It is concluded that LNP siRNA systems formulated by rapid mixing of an ethanol solution of lipid with an aqueous medium containing siRNA exhibit a nanostructured core. The results give insight into the mechanism whereby LNP siRNA systems are formed, providing an understanding of the high encapsulation efficiencies that can be achieved and information on methods of constructing more sophisticated LNP systems.