Daptomycin and A54145 are homologous lipopeptide antibiotics that permeabilize the cell membranes of Gram-positive bacteria. Membrane permeabilization depends on the presence of both phosphatidylglycerol (PG) and calcium, and it involves the formation of oligomeric transmembrane pores that consist of approximately 6-8 subunits. We here show that each lipopeptide molecule binds two calcium ions in separable, successive steps. The first calcium ion causes the lipopeptide molecule to bind to the target membrane, and likely to form a loosely associated oligomer. Higher calcium concentrations induce binding of a second ion, which produces the more tightly associated and more deeply membrane-inserted final, functional form of the oligomer. Both calcium-dependent steps are accompanied by fluorescence signals that indicate transition of specific amino acid residues into less polar environments, suggestive of insertion into the target membrane. Our findings agree with the earlier observation that two of the four acidic amino acid residues in the daptomycin molecule are essential for antibacterial activity.

Long-circulating liposomes are typically prepared with poly(ethylene glycol)- (PEG-) modified lipids, where the lipid portion is inserted in the lipid bilayers as an anchor and the hydrophilic PEG coats the surface to prevent liposome aggregation and rapid clearance in vivo. However, these steric protection effects are compromised upon systemic administration due to low retention of PEGylated lipids within liposome membranes upon dilution. Hence, a series of PEGylated bolaamphiphiles (PEG-bolas) were for the first time developed to increase retention in the lipid bilayer, presumably leading to enhanced integrity of the PEG protective layer upon dilution. We hypothesized that PEG-bolas with a sufficiently long hydrophobic domain and rigid central group could predominantly adopt a membrane-spanning configuration, taking full advantage of steric protection offered by PEG and enhanced retention in liposomes enabled by the bola geometry. In this paper, liposomes stabilized by PEG-bolas comprised of a biphenyl core and twelve-carbon alkyl chain not only exhibited similar storage and biological stability compared to conventional PEGylated lipid stabilized liposomes, but also significantly improved retention upon dilution. Our findings facilitate new designs of liposome-stabilizing agents and can be applied to improve the delivery efficiency of liposomal delivery vehicles in vivo.

Cationic amphiphiles have received increasing attention as antimicrobials given their unique ability to disrupt bacteria cell membranes. While extensive research has demonstrated that amphiphiles' hydrophobic-to-charge ratio significantly modulates antibacterial activity, less work has focused on elucidating the specific impact of charge location on amphiphile bioactivity. In this study, two series of cationic amphiphiles, termed bola-like and gemini-like, were synthesized with analogous hydrophobic-to-charge ratios yet differing charge location, and their resulting antibacterial activity was assessed. Bola-like amphiphiles exhibited preferential activity against two Gram-positive bacteria, with activity increasing with increasing hydrophobicity, whereas gemini-like amphiphiles were active against both Gram-positive and Gram-negative bacteria, with activity decreasing with increasing hydrophobicity. After identifying lead compounds from each amphiphile series (bola- and gemini-like), biophysical experiments indicated that both amphiphiles were membrane-active; notably, the lead gemini-like amphiphile exhibited a strong dependence on electrostatic interactions for membrane interaction. In contrast, the lead bola-like amphiphile exhibited a reliance on both hydrophobic and electrostatic contributions. These results demonstrate that charge location significantly impacts cationic amphiphiles' antibacterial and membrane activity.

Background: Daptomycin forms oligomeric pores in bacterial cell membranes. Cardiolipin is a membrane lipid associated with bacterial resistance to the antibiotic. Results: Cardiolipin makes liposomes impervious to daptomycin permeabilization, and it confines daptomycin to the outer membrane leaflet. Conclusion: Preventing daptomycin from reaching the inner membrane leaflet inhibits pore formation. Significance: Bacteria may become resistant to daptomycin by changing their membrane lipid composition.Daptomycin is an acidic lipopeptide antibiotic that, in the presence of calcium, forms oligomeric pores on membranes containing phosphatidylglycerol. It is clinically used against various Gram-positive bacteria such as Staphylococcus aureus and Enterococcus species. Genetic studies have indicated that an increased content of cardiolipin in the bacterial membrane may contribute to bacterial resistance against the drug. Here, we used a liposome model to demonstrate that cardiolipin directly inhibits membrane permeabilization by daptomycin. When cardiolipin is added at molar fractions of 10 or 20% to membranes containing phosphatidylglycerol, daptomycin no longer forms pores or translocates to the inner membrane leaflet. Under the same conditions, daptomycin continues to form oligomers; however, these oligomers contain only close to four subunits, which is approximately half as many as observed on membranes without cardiolipin. The collective findings lead us to propose that a daptomycin pore consists of two aligned tetramers in opposite leaflets and that cardiolipin prevents the translocation of tetramers to the inner leaflet, thereby forestalling the formation of complete, octameric pores. Our findings suggest a possible mechanism by which cardiolipin may mediate resistance to daptomycin, and they provide new insights into the action mode of this important antibiotic.

The accumulated evidence has shown that lipids and polymers each have distinct advantages as carriers for siRNA delivery. Composite materials comprising both lipids and polymers may present improved properties that combine the advantage of each. Cationic amphiphilic macromolecules (CAMs) containing a hydrophobic alkylated mucic acid segment and a hydrophilic poly(ethylene glycol) (PEG) tail were non-covalently complexed with two lipids, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), to serve as a siRNA delivery vehicle. By varying the weight ratio of CAM to lipid, cationic complexes with varying compositions were obtained in aqueous media and their properties evaluated. CAM–lipid complex sizes were relatively independent of composition, ranging from 100 to 200nm, and zeta potentials varied from 10 to 30mV. Transmission electron microscopy confirmed the spherical morphology of the complexes. The optimal N/P ratio was 50 as determined by electrophoretic mobility shift assay. The ability to achieve gene silencing was evaluated by anti-luciferase siRNA delivery to a U87-luciferase cell line. Several weight ratios of CAM–lipid complexes were found to have similar delivery efficiency compared to the gold standard, Lipofectamine. Isothermal titration calorimetry revealed that siRNA binds more tightly at pH=7.4 than pH=5 to CAM–lipid (1:10 w/w). Further intracellular trafficking studies monitored the siRNA escape from the endosomes at 24h following transfection of cells. The findings in the paper indicate that CAM–lipid complexes can serve as a novel and efficient siRNA delivery vehicle.

Daptomycin is an anionic membrane active antimicrobial lipopeptide used to treat serious infections caused by gram-positive bacteria. It causes target membrane depolarization by forming oligomeric pores that allow leakage of potassium ions, but the complete mechanism of action is unresolved. Antibiotic function is calcium dependant and requires the presence of anionic phospholipids like phosphatidylglycerol (PG) in the target membrane. Because of its efficacy in treating infections resistant to many front-line antibiotics, the recent emergence of strains displaying reduced daptomycin susceptibility is troubling. These quasi-resistant strains have modified membrane lipid content, including an increased presence of cardiolipin (CL) and lysyl-PG, the latter possibly due to the observed up-regulation of mprF, a gene that codes for a protein with lysyl-PG synthase and flippase activity. Using monolayer and bilayer model systems, we sought here to study the effects of the presence of CL on daptomycin binding to PG-containing membranes. Surprisingly, isothermal titration calorimetry (ITC) revealed that daptomycin-membrane affinity increased when small amounts of CL was present and continued to increase until 10 mol%, above which the trend reversed drastically. Results from Langmuir monolayer insertion experiments also show that CL affects the degree of drug insertion into PG-containing lipid films as determined by greater increases in surface pressure after daptomycin was injected into the aqueous subphase when CL was present. Preliminary results from monolayer insertion experiments in which lysyl-PG was included in the films indicate that this lipid also significantly affects drug-monolayer interactions. We hypothesize from these results that alteration of the lipid content of bacterial membranes represents an important component of the potential resistance mechanism and should be considered in the development and formulation of the next generation of membrane targeting antibiotics.

Daptomycin, an antimicrobial lipopeptide used to treat infections caused by Gram-positive bacteria that are resistant to many conventional therapies, acts through calcium-mediated binding to and rapid depolarization of the target bacterial membrane. Convincing evidence has recently been reported suggesting that small daptomycin oligomers form at the membrane surface and that these complexes represent the active state of the drug. Daptomycin's activity is closely correlated with the presence of phosphatidylglycerol (PG) in the target membrane. Although there have no cases of clinical resistance to daptomycin reported, troubling signs are emerging indicating that changes in lipid composition of bacterial membranes cause decreased susceptibility to the drug. It is therefore of interest to gain a more profound understanding of the details of daptomycin's mechanism of activity at the membrane level and the possible causes of potential resistance and their relationship to lipid composition. In the current study, we report on our investigation into the surface and membrane binding properties of daptomycin. From the Gibb's adsorption isotherm, we estimate the molecular area of daptomycin at the air-aqueous interface. Using Langmuir monolayers as membrane models, we also report limiting surface pressures and kinetics for daptomycin insertion to lipid films comprised of pure PG or PG-phosphatidylcholine mixtures. Finally, we attempt to correlate daptomycin's binding behavior in monolayers to that in bilayers, in the form of unilamellar vesicles, by presenting results from isothermal titration experiments. The results represent, for the first time, thermodynamic binding parameters for daptomycin-membrane interactions.

Daptomycin is a clinically important lipopeptide antibiotic that kills Gram-positive bacteria through membrane depolarization. Its activity requires calcium and the presence of phosphatidylglycerol in the target membrane. Calcium and phosphatidylglycerol also promote the formation of daptomycin oligomers, which have been assumed but not proven to be required for the bactericidal effect. Daptomycin shares substantial structural similarity with another lipopeptide antibiotic, A54145; the two have identical amino acid residues in 5 out of 13 positions and similar ones in 4 more positions. We here examined whether these conserved residues are sufficient for oligomer formation. To this end, we used fluorescence energy transfer and excimer fluorescence to detect hybrid oligomers of daptomycin and CB-182,462, a semisynthetic derivative of A54145. Mixtures of the two compounds indeed produced hybrid oligomers, but at the same time displayed a significantly less than additive antibacterial activity against Bacillus subtilis. The existence of functionally impaired oligomers indicates that oligomer formation is indeed important for antibacterial function. However, it also shows that oligomerization is not sufficient; once formed, the oligomers must take another step in order to acquire antibacterial activity. Thus, the amino acid residues shared between daptomycin and CB-182,462 suffice for formation of the oligomer, but not for its subsequent activation.

Conjugated linoleic acids (CLA) are found naturally in dairy products. Two isomers of CLA, that differ only in the location of cis and trans double bonds, are found to have distinct and different biological effects. The cis 9 trans 11 (C9T11) isomer is believed to have anti-carcinogenic effects, while the trans 10 cis 12 (T10C12) isomer is believed to be associated with anti-obesity effects. In this paper we extend earlier molecular dynamics (MD) simulations of pure CLA-phosphatidylcholine bilayers to investigate the comparative effects of cholesterol on bilayers composed of the two respective isomers. Simulations of phosphatidylcholine lipid bilayers in which the sn-2 chains contained one of the two isomers of CLA were performed in which, for each isomer, the simulated bilayers contained 10% and 30% cholesterol (Chol). From MD trajectories we calculate and compare structural properties of the bilayers, including areas per molecule, thickness of bilayers, tilt angle of cholesterols, order parameter profiles, and one and two-dimensional radial distribution function (RDF), as functions of Chol concentration. While the structural effect of cholesterol is approximately the same for both isomers, we find differences at an atomistic level in order parameter profiles and in two-dimensional radial distribution functions.

Surfactant amphiphilic macromolecules (AMs) were complexed with a 1:1 ratio of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), either by a coevaporation (CE) or postaddition (PA) method, to form AM-lipid complexes with enhanced drug delivery applications. By characterizing the surfactant-lipid interactions, these heterogeneous drug delivery systems can be better controlled and engineered for optimal therapeutic outcomes. In this study, the physical interactions between DOPE:DOTAP liposomes and AM surfactants were investigated. Langmuir film balance and isothermal calorimetry studies showed cooperative intermolecular interactions between pure lipids and AM in monolayers and high thermostability of structure formed by the addition of AM micelles to DOTAP:DOPE vesicles in buffer solution respectively. Increasing the AM weight ratio in the complexes via the CE method led to complete vesicle solubilization--from lamellar aggregates, to a mixture of coexisting vesicles and micelles, to mixed micelles. Isothermal calorimetry evaluation of AM-lipid complexes shows that, at higher AM weight ratios, PA-produced complexes exhibit greater stability than complexes at lower AM weight ratios. Similar studies show that AM-lipid complexes produced by the CE methods display stronger interactions between AM-lipid components than complexes produced by the PA method. The results suggest that the PA method produces vesicles with AM molecules associated with its outer leaflet only (i.e., an AM-coated vesicle), while the CE method produces complexes ranging from mixed vesicles to mixed micelle in which the AM-lipid components are more intimately associated. These results will be helpful in the design of AM-lipid complexes as structurally defined, stable, and effective drug delivery systems.

Oxidized analogs of cholesterol (oxysterols) are produced through both enzymatic and non-enzymatic pathways and have been shown to perturb membrane properties in vitro and in vivo. In the present study, the membrane behavior of two naturally occurring oxysterols, 25-hydroxycholesterol and 7-ketocholesterol, was examined in two model systems. The presence of an additional oxygen moiety was found to alter membrane properties compared to native cholesterol and to each other in lipid monolayers, composed of either pure sterol or sterol-glycerophospholipid and sterol-sphingomyelin binary films, as well as in mixed multilamellar vesicles. The ability of oxysterols to condense phosphatidylcholine and sphingomyelin films, their capacity to cause changes in in-plane elasticity moduli, and their propensity to form detergent-resistant membrane domains were all found to be dependant on the location of the oxygen functionality in the oxysterol, the chemical nature of the phospholipid in the model systems, and the oxysterol/phospholipid ratio in the membrane. The findings described in this study with respect to their biophysical/biophysiological implications provide additional insight into the activity of cytotoxic oxysterols in model membranes.

Lysophosphatidic acid (LPA), the structurally simplest of the glycerophospholipids, is a potent second messenger whose functional diversity makes it a compelling target in lipid research. LPA, the effects of which include cell motility and proliferation, platelet activation, fertility and development, and neuropathic pain, is believed to act through a family of G protein-coupled receptors (GPCR). Since some members of this family of proteins are localized in ordered lipid domains (membrane rafts), a role for LPA in altering and re-ordering membranes as part of regulation of the signaling pathway cannot be discounted. As part of a series of efforts to obtain biophysical information about the effects of LPA on membranes, we have employed the Langmuir monolayer technique and isothermal titration calorimetry (ITC) to measure the kinetics and thermodynamics of LPA intercalation into lipid films and bilayers of various compositions representing different physical phases known to exist in biomembranes. Our results indicate that LPA-membrane interactions depend on initial surface pressure, phospholipid headgroup and degree of acyl chain saturation, presence and amount of cholesterol, aqueous media conditions, and aggregation state of LPA. These data suggest that, in addition to its function as a ligand for specific GPCR, LPA interacts directly with the target membrane, constituting a role for this phospholipid as a physical regulatory molecule for LPA cellular signaling pathways.

Conjugated linoleic acids (CLA) are known to exert several isomer-specific biological effects, but their mechanisms of action are unclear. In order to determine whether the physicochemical effects of CLA on membranes play a role in their isomer-specific effects, we synthesized phosphatidylcholines (PCs) with 16:0 at sn-1 position and one of four CLA isomers (trans 10 cis 12 (A), trans 9 trans 11 (B), cis 9 trans 11 (C), and cis 9 cis 11 (D)) at sn-2, and determined their biophysical properties in monolayers and bilayers. The surface areas of the PCs with the two natural CLA (A and C) were similar at all pressures, but they differed significantly in the presence of cholesterol, with PC-A condensing more than PC-C. Liposomes of PC-A similarly showed increased binding of cholesterol compared to PC-C liposomes. PC-A liposomes were less permeable to carboxyfluorescein compared to PC-C liposomes. The PC with two trans double bonds (B) showed the highest affinity to cholesterol and lowest permeability. The two natural CLA-PCs (A and C) stimulated lecithin-cholesterol acyltransferase activity by 2-fold, whereas the unnatural CLA-PCs (B and D) were inhibitory. These results suggest that the differences in the biophysical properties of CLA isomers A and C may partly contribute to the known differences in their biological effects.

Cholesterol's role in ordering lipid membrane domains is well known. Even subtle changes in the structure of this sterol greatly affect the biophysical dynamics of membranes, usually because of perturbations in the interactions between the sterol and other membrane lipids that chemical modifications cause. Cholesterol oxidation products (oxysterols), which result from enzymatic and non-enzymatic mechanisms, are cytotoxic and found in atherosclerotic plaques. Previous studies have shown that the membrane properties of oxysterols vary, depending on the specific site of the oxygen-containing moiety. In this study, we examined the interactions of two oxysterols, one formed through non-specific oxidation (7-ketocholesterol), and one produced enzymatically (25-hydroxycholesterol) with two common membrane lipids, 1-palmitoyl-2-oleoyl-sn-phosphocholine (POPC) and brain-derived sphingomyelin. Analysis of force-area isotherms obtained by compression of pure sterol monolayers and of binary monomolecular films at the air-water interface, comprised of varying mole fractions of POPC or sphingomyelin and either oxysterol, reveals significant differences in surface behavior with respect to each other and to native cholesterol. Both oxysterols condensed POPC and sphingomyelin films to a lesser degree than cholesterol, and an expansion of sphingomyelin films was observed with low mole fraction 7-ketocholesterol. Additionally, surface compression moduli data obtained from the force-area isotherms reveal a decreased ability of both oxysterols to mitigate the phase transition of sphingomyelin compared to cholesterol. The changes of membrane behavior in the presence of oxysterols reported here suggest a relation of their toxicity to the propensity of lipids membranes to form liquid-ordered domains (rafts).

The binding of the gelsolin P2 peptide (residues 150–169) with lysophosphatidic acid (LPA) and lipopolysaccharide (LPS) was investigated by isothermal titration calorimetry. P2 binds to LPS with higher affinity than to LPA. For the interaction of 1-oleoyl-LPA with P2 in the absence of salt, Kd and ΔH° were 920 nM and −2.07 kcal/mol, respectively, at pH 7.4 and 25 °C. For the interaction of lipopolysaccharide (LPS) from P. aeruginosa with P2 under the same conditions, Kd was 177 nM and ΔH° was −7.6 kcal/mol.

Analogues of cholesterol (compounds 1 and 2) and coprostanol (compound 3) containing the BODIPY fluorophore in the aliphatic tail of the free sterol have been synthesized starting with bisnorcholenic acid, cholenic acid 3beta-acetate, and lithocholic acid, respectively. An ester linkage joining the fluorophore to the sterol nucleus interfered with the ability of the fluorescent sterol to pack with phospholipids in monolayers. However, an analogue in which the linker was devoid of polar atoms exhibited a substantially similar physical behavior to cholesterol in model membranes with respect to localization in raft domains.

The critical micelle concentrations (CMC) of lysophosphatidic acid (LPA) and sphingosylphosphorylcholine (SPC) were measured by isothermal titration calorimetry. The CMC of LPA decreases with salt concentration and acyl chain length. In water at 25°C, the CMC values of 1-acyl-2-lyso-sn-glycero-3-phosphatidic acid are 1.850, 0.540, 0.082, and 0.346mM, respectively, when the acyl group is myristoyl, palmitoyl, stearoyl, and oleoyl. The CMC of SPC in 10mM sodium phosphate buffer, pH 7.4, at 25°C was 0.158mM, and did not change with an increase in salt concentration.

6‐Photocholesterol, a new photoactivatable analog of cholesterol in which a diazirine functionality replaces the 5,6‐double bond in the steroid nucleus, was used recently to identify cholesterol‐binding proteins in neuroendocrine cells [Thiele, C., Hannah, M.J., Farenholz, F. and Huttner, W.B. (2000) Nat. Cell Biol. 2, 42–49], to track the distribution and transport of cholesterol in Caenorhabditis elegans [Matyash, V., Geier, C., Henske, A., Mukherjee, S., Hirsh, D., Thiele, C., Grant, B., Maxfield, F.R. and Kurzchalia, T.V. (2001) Mol. Biol. Cell 12, 1725–1736], and to probe lipid–protein interactions in oligodendrocytes [Simons, M., Kramer, E.M., Thiele, C., Stoffel, W. and Trotter, J. (2000) J. Cell Biol. 151, 143–154]. To determine whether 6‐photocholesterol is a faithful mimetic of cholesterol we analyzed the ability of this probe, under conditions in which it is not photoactivated to a carbene, to substitute for cholesterol in two unrelated assays: (1) to condense 1‐palmitoyl‐2‐oleoyl‐ sn ‐glycero‐3‐phosphocholine monomolecular films and (2) to mediate the fusion of two alphaviruses (Semliki Forest and Sindbis) with liposomes. The results suggest that this analog is a suitable photoprobe of cholesterol.