The underlying mechanical properties of membranes tune their ability to fuse.

Membrane fusion is a ubiquitous process associated with a multitude of biological events. Although it has long been appreciated that membrane mechanics plays an important role in membrane fusion, the molecular interplay between mechanics and fusion has remained elusive. For example, although different lipids modulate membrane mechanics differently, depending on their composition, molar ratio, and complex interactions, differing lipid compositions may lead to similar mechanical properties. This raises the question of whether (i) the specific lipid composition or (ii) the average mesoscale mechanics of membranes acts as the determining factor for cellular function. Furthermore, little is known about the potential consequences of fusion on membrane disruption. Here, we use a combination of confocal microscopy, time-resolved imaging, and electroporation to shed light onto the underlying mechanical properties of membranes that regulate membrane fusion. Fusion efficiency follows a nearly universal behavior that depends on membrane fluidity parameters, such as membrane viscosity and bending rigidity, rather than on specific lipid composition. This helps explaining why the charged and fluid membranes of the inner leaflet of the plasma membrane are more fusogenic than their outer counterparts. Importantly, we show that physiological levels of cholesterol, a key component of biological membranes, has a mild effect on fusion but significantly enhances membrane mechanical stability against pore formation, suggesting that its high cellular levels buffer the membrane against disruption. The ability of membranes to efficiently fuse while preserving their integrity may have given evolutionary advantages to cells by enabling their function while preserving membrane stability.

Bilayer Charge Asymmetry and Oil Residues Destabilize Membranes upon Poration.

Transmembrane asymmetry is ubiquitous in cells, particularly with respect to lipids, where charged lipids are mainly restricted to one monolayer. We investigate the influence of anionic lipid asymmetry on the stability of giant unilamellar vesicles (GUVs), minimal plasma membrane models. To quantify asymmetry, we apply the fluorescence quenching assay, which is often difficult to reproduce, and caution in handling the quencher is generally underestimated. We first optimize this assay and then apply it to GUVs prepared with the inverted emulsion transfer protocol by using increasing fractions of anionic lipids restricted to one leaflet. This protocol is found to produce highly asymmetric bilayers but with ∼20% interleaflet mixing. To probe the stability of asymmetric versus symmetric membranes, we expose the GUVs to porating electric pulses and monitor the fraction of destabilized vesicles. The pulses open macropores, and the GUVs either completely recover or exhibit leakage or bursting/collapse. Residual oil destabilizes porated membranes, and destabilization is even more pronounced in asymmetrically charged membranes. This is corroborated by the measured pore edge tension, which is also found to decrease with increasing charge asymmetry. Using GUVs with imposed transmembrane pH asymmetry, we confirm that poration-triggered destabilization does not depend on the approach used to generate membrane asymmetry.

Efficient liposome fusion to phase-separated giant vesicles.

Lateral phase heterogeneity in biomembranes can govern cellular functions and may serve as a platform for enrichment or depletion of membrane-anchored molecules. In this work, we address the question of how the process of membrane fusion is affected by the membrane phase state (fluid or gel) and by phase coexistence, as well as the effects of fusion-mediated incorporation of exogeneous lipids on phase separation. Our system is based on the fusion of cationic fluid large unilamellar vesicles (LUVs) composed of dioleoyl trimethylammonium propane (DOTAP) and dioleoyl phosphoethanolamine (DOPE) with neutral and anionic giant unilamellar vesicles (GUVs) composed of phosphatidylcholine and phosphatidylglycerol. By changing the lipid composition of the GUVs, we modulated the phase state and charge of the different phases (charged or neutral, fluid or gel) and identified systems in which we can target fusion to specific domains on phase-separated membranes. Fusion efficiency was quantified using fluorescence microscopy-based lipid and content mixing assays, and flow chamber devices were used to assess the real-time sequence of events of the fusion process. To investigate the bilayer thermal behavior, differential scanning calorimetry (DSC) experiments were performed on LUVs. The results show that fusion is extensive in single-component GUVs only for fluid and negatively charged acceptor membranes. On the other hand, in phase-separated GUVs, high fusion efficiency was observed even when the gel phase was anionic and phase separation somewhat increased the fusion efficiency. Extensive fusion led to dissolution of the gel domains as a result of extensive incorporation of lipids in the fluid state from the fusogenic liposomes. Altogether, these findings have the potential to unravel the important role of membrane phase state, phase separation, charge, and the effects of extensive fusion on membrane organization and may give insights in the regulation of the interactions between cells and liposomes that are used in drug delivery systems.

Membrane Fusion Biophysical Analysis of Fusogenic Liposomes.

Liposomes represent important drug carrier vehicles in biological systems. A fusogenic liposomal system composed of equimolar mixtures of the cationic lipid DOTAP and the phospholipid DOPE showed high fusion and delivery efficiencies with cells and lipid vesicles. However, aspects of the thermodynamics involving the interaction of these fusogenic liposomes and biomimetic systems remain unclear. Here, we investigate the fusion of this system with large unilamellar vesicles (LUVs) composed of the zwitterionic lipid POPC and increasing fractions of the anionic lipid POPG and up to 30 mol % cholesterol. The focus here is to concomitantly follow changes in size, zeta-potential, and enthalpy binding upon membrane interaction and fusion. Isothermal titration calorimetry (ITC) data showed that membrane fusion in our system is an exothermic process in the absence of cholesterol, suggesting that electrostatic attraction is the driving force for fusion. An endothermic component appeared and eventually dominated the titration at 30 mol % cholesterol, which we propose is caused by membrane fluidification when cholesterol is diluted upon fusion. The inflection points of the ITC data occurred around 0.5-0.7 POPG/DOTAP for all systems, the same stoichiometry for which zeta-potential and dynamic light scattering measurements showed an increase in size coupled with charge neutralization of the system, which is consistent with the fact that fusion in our system is charge-mediated. Microscopy observations of the final mixtures revealed the presence of giant vesicles, which is a clear indication of fusion, coexisting with intermediate-sized objects that could be the result of both fusion and/or aggregation. The results show that the fusion efficiency of the DOTAP:DOPE fusogenic system is modulated by the charge and membrane packing of the acceptor membrane and explain why the system fuses very efficiently with cells.

Revealing the Mode of Action of Halictine Antimicrobial Peptides: A Comprehensive Study with Model Membranes.

Antimicrobial peptides are innate host defense molecules with the ability to kill pathogens. They have been widely studied for their membrane lytic activity and their potential to overcome the ever-increasing threat of antimicrobial resistance against conventional antibiotics. Here, we focus on two halictines, antimicrobial peptides first obtained from the venom of the eusocial bee Halictus sexcinctus. The peptides, HAL-1 and HAL-2, are cationic (with +3 and +4 charges, respectively) and amphipathic, have 12 amino acid residues, and exhibit high biological activity. For this study, the mechanism of action of HAL-1 and HAL-2 was studied in detail using large and giant unilamellar vesicles composed of pure palmitoyl oleoyl phosphatidyl choline (POPC) and a mixture of POPC and the anionic lipid palmitoyl oleoyl phosphatidyl glycerol (POPG) as biomimetic models of the membranes of eukaryotes and microorganisms, respectively. A set of complementary techniques was put forward: carboxyfluorescein leakage assay, phase contrast optical microscopy, ζ-potential, static and dynamic light scattering, fluorescence and circular dichroism spectroscopies, and isothermal titration calorimetry. The results show that both halictines are able to interact strongly with anionic membranes: The interaction is exothermic and accompanied by structuring of the peptides as an α-helix and deep insertion into the membrane causing substantial membrane permeabilization at very low peptide/lipid molar ratios. Extensive vesicle aggregation was detected only at a high peptide concentration. On the other hand, the interaction of the halictines with POPC is significantly milder. Yet, the peptides were able to permeabilize the POPC membranes to some extent. Comparing both peptides, HAL-1 showed a somewhat stronger effect on model membranes. Fits to the data revealed apparent binding constants on the order of 103-104 M-1 for anionic membranes and 1 order of magnitude lower for zwitterionic bilayers. When lytic activity results were compared at the same bound peptide/lipid ratio, the halictines exhibited a higher activity toward zwitterionic membranes. As novel peptides, small and with powerful activity, these halictines are potential candidates for becoming antimicrobial agents.

Capsaicin-Cyclodextrin Complex Enhances Mepivacaine Targeting and Improves Local Anesthesia in Inflamed Tissues.

Acidic environments, such as in inflamed tissues, favor the charged form of local anesthetics (LA). Hence, these drugs show less cell permeation and diminished potency. Since the analgesic capsaicin (CAP) triggers opening of the TRPV1 receptor pore, its combination with LAs could result in better uptake and improved anesthesia. We tested the above hypothesis and report here for the first time the analgesia effect of a two-drug combination (LA and CAP) on an inflamed tissue. First, CAP solubility increased up to 20 times with hydroxypropyl-beta-cyclodextrin (HP-ß-CD), as shown by the phase solubility study. The resulting complex (HP-ß-CD-CAP) showed 1:1 stoichiometry and high association constant, according to phase-solubility diagrams and isothermal titration calorimetry data. The inclusion complex formation was also confirmed and characterized by differential scanning calorimetry (DSC), X-ray diffraction, and 1H-NMR. The freeze-dried complex showed physicochemical stability for at least 12 months. To test in vivo performance, we used a pain model based on mouse paw edema. Results showed that 2% mepivacaine injection failed to anesthetize mice inflamed paw, but its combination with complexed CAP resulted in pain control up to 45 min. These promising results encourages deeper research of CAP as an adjuvant for anesthesia in inflamed tissues and cyclodextrin as a solubilizing agent for targeting molecules in drug delivery.

Highly Efficient Protein-free Membrane Fusion: A Giant Vesicle Study.

Membrane fusion is a ubiquitous process in biology and is a prerequisite for many intracellular delivery protocols relying on the use of liposomes as drug carriers. Here, we investigate in detail the process of membrane fusion and the role of opposite charges in a protein-free lipid system based on cationic liposomes (LUVs, large unilamellar vesicles) and anionic giant unilamellar vesicles (GUVs) composed of different palmitoyloleoylphosphatidylcholine (POPC)/palmitoyloleoylphosphatidylglycerol (POPG) molar ratios. By using a set of optical-microscopy- and microfluidics-based methods, we show that liposomes strongly dock to GUVs of pure POPC or low POPG fraction (up to 10 mol%) in a process mainly associated with hemifusion and membrane tension increase, commonly leading to GUV rupture. On the other hand, docked LUVs quickly and very efficiently fuse with negative GUVs of POPG fractions at or above 20 mol%, resulting in dramatic GUV area increase in a charge-dependent manner; the vesicle area increase is deduced from GUV electrodeformation. Importantly, both hemifusion and full fusion are leakage-free. Fusion efficiency is quantified by the lipid transfer from liposomes to GUVs using fluorescence resonance energy transfer (FRET), which leads to consistent results when compared to fluorescence-lifetime-based FRET. We develop an approach to deduce the final composition of single GUVs after fusion based on the FRET efficiency. The results suggest that fusion is driven by membrane charge and appears to proceed up to charge neutralization of the acceptor GUV.

Location of the Positive Charges in Cationic Amphiphiles Modulates Their Mechanism of Action against Model Membranes.

Synthetic cationic amphiphiles (CAms) with physicochemical properties similar to antimicrobial peptides are promising molecules in the search for alternative antibiotics to which pathogens cannot easily develop resistance. Here, we investigate two types of CAms based on tartaric acid and containing two hydrophobic chains (of 7 or 11 carbons) and two positive charges, located either at the end of the acyl chains (bola-like, B7 and B11) or at the tartaric acid backbone (gemini-like, G7 and G11). The interaction of the CAms with biomimetic membrane models (anionic and neutral liposomes) was studied with zeta potential and dynamic light scattering measurements, isothermal titration calorimetry, and a fluorescent-based leakage assay. We show that the type of molecule determines the mechanism of action of the CAms. Gemini-like molecules (G7 and G11) interact mainly via electrostatics (exothermic process) and reside in the external vesicle leaflet, altering substantially the vesicle surface potential but not causing significant membrane lysis. On the other hand, the interaction of bola-like CAms (B7 and B11) is endothermic and thus entropy-driven, and these molecules reach both membrane leaflets and cause substantial membrane permeabilization, likely after clustering of anionic lipids. The lytic ability is clearly higher against anionic membranes as compared with neutral membranes. Within each class of molecule, longer alkyl chains (i.e., B11 and G11) exhibit higher affinity and lytic ability. Overall, the molecule B11 exhibits a high potential as antimicrobial agent, since it has a high membrane affinity and causes substantial membrane permeabilization.

Structure of Rhoptry Neck Protein 2 is essential for the interaction in vitro with Apical Membrane Antigen 1 in Plasmodium vivax.

BACKGROUND: In several Apicomplexa, the formation of moving junctions (MJs) at the interface between the external membranes of the invading parasite and the host cell is essential for the process of parasite invasion. In Plasmodium falciparum and Toxoplasma gondii, the MJ is composed of the Apical Membrane Antigen 1 (AMA1) and Rhoptry Neck Proteins (RONs) complex; specifically, AMA1 interacts with RON2 during host cell invasion. METHODS: Recombinant proteins based on Plasmodium vivax RON2 (A2033-P2100) and its synthetic peptide fragments, one cyclic and one linear, based on PvRON2 (D2035-T2074) were generated and used to evaluate the interaction with P. vivax AMA1 (PvAMA1) by the far western blot, surface plasmon resonance (SPR), and isothermal titration microcalorimetry (ITC) methods. The structural studies of peptides were performed by circular dichroism, and the structural analysis of the complex of PvAMA1 with peptides based on PvRON2 (D2035-T2074) was conducted with small-angle X-ray scattering (SAXS). RESULTS: Surface plasmon resonance (KD = 23.91 ± 2.078 µmol/L) and ITC (K = 3 × 105 mol/L) studies conclusively showed an interaction between the cyclic peptide based on PvRON2 and PvAMA1-His6. In contrast, the linear peptide and recombinant PvRON2 (GST fusion protein) did not show an interaction with PvAMA1. However, the interaction among recombinant proteins PvRON2.2 and PvAMA1-His6 was possible to show by far western blot. CONCLUSIONS: The results show that the PvRON2 structure, particularly the S-S bond between C2051 and C2063, is determinant for the existence of the interaction between PvAMA1 and PvRON2.

Synthesis, biophysical and functional studies of two BP100 analogues modified by a hydrophobic chain and a cyclic peptide.

Antimicrobial peptides (AMPs) work as a primary defense against pathogenic microorganisms. BP100, (KKLFKKILKYL-NH2), a rationally designed short, highly cationic AMP, acts against many bacteria, displaying low toxicity to eukaryotic cells. Previously we found that its mechanism of action depends on membrane surface charge and on peptide-to-lipid ratio. Here we present the synthesis of two BP100 analogs: BP100­alanyl­hexadecyl­1­amine (BP100-Ala-NH-C16H33) and cyclo(1­4)­d­Cys1, Ile2, Leu3, Cys4-BP100 (Cyclo(1­4)­cILC-BP100). We examined their binding to large unilamellar vesicles (LUV), conformational and functional properties, and compared with those of BP100. The analogs bound to membranes with higher affinity and a lesser dependence on electrostatic forces than BP100. In the presence of LUV, BP100 and BP100-Ala-NH-C16H33 acquired α-helical conformation, while Cyclo(1­4)­cILC-BP100) was partly α-helical and partly ß-turn. Taking in conjunction: 1. particle sizes and zeta potential, 2. effects on lipid flip-flop, 3. leakage of LUVs internal contents, and 4. optical microscopy of giant unilamellar vesicles, we concluded that at high concentrations, all three peptides acted by a carpet mechanism, while at low concentrations the peptides acted by disorganizing the lipid bilayer, probably causing membrane thinning. The higher activity and lesser membrane surface charge dependence of the analogs was probably due to their greater hydrophobicity. The MIC values of both analogs towards Gram-positive and Gram-negative bacteria were similar to those of BP100 but both analogues were more hemolytic. Confocal microscopy showed Gram-positive B. subtilis killing with concomitant extensive membrane damage suggestive of lipid clustering, or peptide-lipid aggregation. These results were in agreement with those found in model membranes.

Antimicrobial Peptide K0-W6-Hya1 Induces Stable Structurally Modified Lipid Domains in Anionic Membranes.

Considering the known different mode of action of antimicrobial peptides in zwitterionic and anionic cell membranes, the present work compares the action of the antimicrobial peptide K0-W6-Hya1 (KIFGAIWPLALGALKNLIK-NH2) with zwitterionic and negatively charged model membranes, namely, liposomes composed of phosphatidylcholine (PC) and phosphatidylglycerol (PG) membranes, and a mixture of the two. Differential scanning calorimetry (DSC), steady state fluorescence of the Trp residue, dynamic light scattering (DLS), and measurement of the leakage of an entrapped fluorescent dye (carboxyfluorescein, CF) were performed with large unilamellar vesicles (LUVs). All techniques evidenced the different action of the peptide in zwitterionic and anionic vesicles. Trp fluorescence spectroscopy shows that the differences are related not only to the partition of the cationic peptide in zwitterionic and anionic membranes, but also to the different penetration depth of the peptide into the lipid bilayers: Trp goes deeper into negatively charged membranes, both in the gel and fluid phases, than into zwitterionic ones. DSC shows that the peptide is strongly attached to anionic bilayers, giving rise to the coexistence of two different lipid regions, one depleted of peptide and another one peptide-disturbed, possibly a stable or transient polar pore, considering the leakage of CF. This contrasts with the homogeneous effect produced by the peptide in zwitterionic membranes, probably related to peptide-membrane diffusion. Moreover, in mixed bilayers (PC:PG), the peptide sequesters negatively charged lipids, creating peptide-rich anionic lipid regions, strongly disturbing the membrane. The distinct structural interaction displayed by the peptide in PC and PG membranes could be related to the different mechanisms of action of the peptide in anionic prokaryotic and zwitterionic eukaryotic cell membranes.

Effect of Triton X-100 on Raft-Like Lipid Mixtures: Phase Separation and Selective Solubilization.

Under certain conditions, biological membranes exhibit resistance to solubilization, even at high detergent concentration. These insoluble fragments are enriched in sphingolipids, cholesterol, and certain proteins having a preference for more organized environments. Here we investigated the effect of detergent Triton X-100 (TX-100) on raft-like lipid mixtures composed of POPC (palmitoyl oleoyl phosphatidylcholine, an unsaturated lipid), SM (sphingomyelin, a saturated lipid), and cholesterol, focusing on the detergent-induced phase separation at subsolubilizing concentration and the extent of solubilization at higher concentration. Giant unilamellar vesicles (GUVs) of POPC/SM/chol containing a fluorescent probe known to prefer the liquid-disordered phase were prepared and observed with fluorescence microscopy. A phase diagram constructed in the presence and absence of 0.1 mM TX-100 showed that the detergent induces macroscopic liquid-ordered/liquid-disordered (Lo/Ld) phase separation over a wide range of membrane composition, indicating that TX-100 has the ability to rearrange the lateral heterogeneity of the lipid mixture. The extent of solubilization of the POPC/SM/chol GUVs was quantified by measuring the vesicle size before and after the injection of a high concentration of TX-100. In parallel, the solubilization extent of large unilamellar vesicles (LUVs) was assessed by turbidity measurements. The extent of solubilization decreases significantly as the fractions of SM and cholesterol in the mixture increase. The origin of the detergent resistance is the low partitioning of TX-100 in cholesterol-rich membranes, especially in SM-containing ones, as evidenced by isothermal titration calorimetry experiments on LUVs. Our results provide a guide to future research on the effects of TX-100 on raft-like lipid mixtures.

Gene Transfection Mediated by Catiomers Requires Free Highly Charged Polymer Chains To Overcome Intracellular Barriers.

The prospective use of the block copolymers poly(ethylene oxide)113-b-poly[2-(diethylamino)ethyl methacrylate]50 (PEO113-b-PDEA50) and poly[oligo(ethylene glycol)methyl ether methacrylate]70-b-poly[oligo(ethylene glycol)methyl ether methacrylate10-co-2-(diethylamino)ethyl methacrylate47-co-2-(diisopropylamino)ethyl methacrylate47] (POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47)) as nonviral gene vectors was evaluated. The polymers are able to properly condense DNA into nanosized particles (RH ≈ 75 nm), which are marginally cytotoxic and can be uptaken by cells. However, the green fluorescent protein (GFP) expression assays evidenced that DNA delivery is essentially negligible meaning that intracellular trafficking hampers efficient gene release. Subsequently, we demonstrate that cellular uptake and particularly the quantity of GFP-positive cells are substantially enhanced when the block copolymer polyplexes are produced and further supplemented by BPEI chains (branched polyethylenimine). The dynamic light scattering/electrophoretic light scattering/isothermal titration calorimetry data suggest that such a strategy allows the adsorption of BPEI onto the surface of the polyplexes, and this phenomenon is responsible for increasing the size and surface charge of the assemblies. Nevertheless, most of the BPEI chains remain freely diffusing in the systems. The biological assays confirmed that cellular uptake is enhanced in the presence of BPEI and principally, the free highly charged polymer chains play the central role in intracellular trafficking and gene transfection. These investigations pointed out that the transfection efficiency versus cytotoxicity issue can be balanced by a mixture of BPEI and less cytotoxic agents such as for instance the proposed block copolymers.

Comparative study of the mechanism of action of the antimicrobial peptide gomesin and its linear analogue: The role of the ß-hairpin structure.

Gomesin (Gm) is an antimicrobial peptide first isolated from the hemolymph of a Brazilian spider. Its powerful antimicrobial activity is, however, accompanied by hemolysis. As an alternative to this issue, a linear analogue (named GmL) lacking the disulfide bonds was designed. Here, CD spectroscopy, a fluorescence-based leakage assay, isothermal titration calorimetry (ITC) and light scattering are used to study the interaction of both Gm and GmL with large unilamellar vesicles (LUVs) composed of POPC (palmitoyl oleoyl phosphatidylcholine) with 25 and 50 mol% POPG (palmitoyl oleoyl phosphatidylglycerol). The activities of Gm and GmL in respect to their binding affinity/enthalpy, ability to permeabilize membranes and to induce vesicle aggregation are correlated with peptide secondary structure. Whereas Gm displays a quite stable ß-hairpin motif irrespective of the environment, GmL assumes a random conformation in aqueous solution and in the presence of 25 mol% POPG but adopts a ß-like structure in the presence of 50 mol% POPG. Gm exhibited high lytic activity against both surface charge densities. Instead, the activity of GmL was found to be negligible in the presence of 25 mol% POPG LUVs, but comparable to that of the native peptide against 50 mol% POPG as a consequence of peptide structuring. We conclude that the activity of Gm and its linear analogue is intimately related to the formation of a ß-turn motif, in which the hydrophobic residues form a hydrophobic face able to insert into the membrane and disrupt it.

Efficient Condensation of DNA into Environmentally Responsive Polyplexes Produced from Block Catiomers Carrying Amine or Diamine Groups.

The intracellular delivery of nucleic acids requires a vector system as they cannot diffuse across lipid membranes. Although polymeric transfecting agents have been extensively investigated, none of the proposed gene delivery vehicles fulfill all of the requirements needed for an effective therapy, namely, the ability to bind and compact DNA into polyplexes, stability in the serum environment, endosome-disrupting capacity, efficient intracellular DNA release, and low toxicity. The challenges are mainly attributed to conflicting properties such as stability vs efficient DNA release and toxicity vs efficient endosome-disrupting capacity. Accordingly, investigations aimed at safe and efficient therapies are still essential to achieving gene therapy clinical success. Taking into account the mentioned issues, herein we have evaluated the DNA condensation ability of poly(ethylene oxide)113-b-poly[2-(diisopropylamino)ethyl methacrylate]50 (PEO113-b-PDPA50), poly(ethylene oxide)113-b-poly[2-(diethylamino)ethyl methacrylate]50 (PEO113-b-PDEA50), poly[oligo(ethylene glycol)methyl ether methacrylate]70-b-poly[oligo(ethylene glycol)methyl ether methacrylate10-co-2-(diethylamino)ethyl methacrylate47-co-2-(diisopropylamino)ethyl methacrylate47] (POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47), and poly[oligo(ethylene glycol)methyl ether methacrylate]70-b-poly{oligo(ethylene glycol)methyl ether methacrylate10-co-2-methylacrylic acid 2-[(2-(dimethylamino)ethyl)methylamino]ethyl ester44} (POEGMA70-b-P(OEGMA10-co-DAMA44). Block copolymers PEO113-b-PDEA50 and POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) were evidenced to properly condense DNA into particles with a desirable size for cellular uptake via endocytic pathways (R(H) ≈ 65-85 nm). The structure of the polyplexes was characterized in detail by scattering techniques and atomic force microscopy. The isothermal titration calorimetric data revealed that the polymer/DNA binding is endothermic; therefore, the process in entropically driven. The combination of results supports that POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) condenses DNA more efficiently and with higher thermodynamic outputs than does PEO113-b-PDEA50. Finally, circular dichroism spectroscopy indicated that the conformation of DNA remained the same after complexation and that the polyplexes are very stable in the serum environment.

Peptide:lipid ratio and membrane surface charge determine the mechanism of action of the antimicrobial peptide BP100. Conformational and functional studies.

The cecropin-melittin hybrid antimicrobial peptide BP100 (H-KKLFKKILKYL-NH2) is selective for Gram-negative bacteria, negatively charged membranes, and weakly hemolytic. We studied BP100 conformational and functional properties upon interaction with large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs, containing variable proportions of phosphatidylcholine (PC) and negatively charged phosphatidylglycerol (PG). CD and NMR spectra showed that upon binding to PG-containing LUVs BP100 acquires α-helical conformation, the helix spanning residues 3-11. Theoretical analyses indicated that the helix is amphipathic and surface-seeking. CD and dynamic light scattering data evinced peptide and/or vesicle aggregation, modulated by peptide:lipid ratio and PG content. BP100 decreased the absolute value of the zeta potential (ζ) of LUVs with low PG contents; for higher PG, binding was analyzed as an ion-exchange process. At high salt, BP100-induced LUVS leakage requires higher peptide concentration, indicating that both electrostatic and hydrophobic interactions contribute to peptide binding. While a gradual release took place at low peptide:lipid ratios, instantaneous loss occurred at high ratios, suggesting vesicle disruption. Optical microscopy of GUVs confirmed BP100-promoted disruption of negatively charged membranes. The mechanism of action of BP100 is determined by both peptide:lipid ratio and negatively charged lipid content. While gradual release results from membrane perturbation by a small number of peptide molecules giving rise to changes in acyl chain packing, lipid clustering (leading to membrane defects), and/or membrane thinning, membrane disruption results from a sequence of events - large-scale peptide and lipid clustering, giving rise to peptide-lipid patches that eventually would leave the membrane in a carpet-like mechanism.

Ellipsoidal Relaxation of Deformed Vesicles.

Theoretical analysis and experimental quantification on the ellipsoidal relaxation of vesicles are presented. The current work reveals the simplicity and universal aspects of this process. The Helfrich formula is shown to apply to the dynamic relaxation of moderate-to-high tension membranes, and a closed-form solution is derived which predicts the vesicle aspect ratio as a function of time. Scattered data are unified by a time scale, which leads to a similarity behavior, governed by a distinctive solution for each vesicle type. Two separate regimes in the relaxation are identified, namely, the "entropic" and the "constant-tension" regimes. The bending rigidity and the initial membrane tension can be simultaneously extracted from the data analysis, posing the current approach as an effective means for the mechanical analysis of biomembranes.

Solubilization of binary lipid mixtures by the detergent Triton X-100: the role of cholesterol.

The solubilization of lipid bilayers of different composition and phase by the detergent Triton X-100 (Triton X-100) was investigated using optical and fluorescence microscopy of giant unilamellar vesicles (GUVs) and light scattering of large unilamellar vesicles (LUVs). The compositions explored were 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), in the liquid-disordered (Ld) phase, sphingomyelin (SM), in the gel phase, and binary mixtures of these phospholipids with 30 mol % cholesterol (chol), resulting in bilayers in the Ld and liquid-ordered (Lo) phases, respectively. We show that the phospholipid bilayers are completely soluble in TX-100, but optical microscopy reveals that whereas fluid POPC is gradually solubilized by TX-100, gel SM is first shattered in bilayer fragments. Incorporation of TX-100 in the membrane leads to increase in GUV area, which was quantified and expressed as bound detergent-to-lipid molar ratio. The partition of TX-100 in POPC is very high, decreases in POPC/chol, and is negligible in SM/chol. Fluorescence microscopy shows that TX-100 induces Lo/Ld phase separation in previously homogeneous POPC/chol GUVs, and insoluble bilayer fragments/vesicles are detected with optical microscopy and light scattering. Vesicles of SM/chol, in the Lo phase, are virtually insoluble in TX-100. Taken together, our results show that the presence of cholesterol is the origin of membrane resistance to solubilization, which depending on the specific lipid mixture can result in either partially (POPC/chol) or completely (SM/chol) insoluble mixtures.

Brij detergents reveal new aspects of membrane microdomain in erythrocytes.

Membrane microdomains enriched in cholesterol, sphingolipids (rafts), and specific proteins are involved in important physiological functions. However their structure, size and stability are still controversial. Given that detergent-resistant membranes (DRMs) are in the liquid-ordered state and are rich in raft-like components, they might correspond to rafts at least to some extent. Here we monitor the lateral order of biological membranes by characterizing DRMs from erythrocytes obtained with Brij-98, Brij-58, and TX-100 at 4 °C and 37 °C. All DRMs were enriched in cholesterol and contained the raft markers flotillin-2 and stomatin. However, sphingomyelin (SM) was only found to be enriched in TX-100-DRMs - a detergent that preferentially solubilizes the membrane inner leaflet - while Band 3 was present solely in Brij-DRMs. Electron paramagnetic resonance spectra showed that the acyl chain packing of Brij-DRMs was lower than TX-100-DRMs, providing evidence of their diverse lipid composition. Fatty acid analysis revealed that the SM fraction of the DRMs was enriched in lignoceric acid, which should specifically contribute to the resistance of SM to detergents. These results indicate that lipids from the outer leaflet, particularly SM, are essential for the formation of the liquid-ordered phase of DRMs. At last, the differential solubilization process induced by Brij-98 and TX-100 was monitored using giant unilamellar vesicles. This study suggests that Brij and TX-100-DRMs reflect different degrees of lateral order of the membrane microdomains. Additionally, Brij DRMs are composed by both inner and outer leaflet components, making them more physiologically relevant than TX-100-DRMs to the studies of membrane rafts.