Specific binding of different vesicle populations by the hybridization of membrane-anchored DNA

We demonstrate a method of heterogeneous vesicle binding using membrane-anchored, single-stranded DNA that can be used over several orders of magnitude in vesicle size, as demonstrated for large 100 nm vesicles and giant vesicles several microns in diameter. The aggregation behavior is studied for a range of DNA surface concentrations and solution ionic strengths. Three analogous states of aggregation are observed on both vesicle size scales. We explain the existence of these three regimes by a combination of DNA binding favorability, vesicle collision kinetics, and lateral diffusion of the DNA within the fluid membrane. The reversibility of the DNA hybridization allows dissociation of the structures formed and can be achieved either thermally or by a reduction in the ionic strength of the external aqueous environment. Difficulty is found in fully unbinding giant vesicles by thermal dehybridization, possibly frustrated by the attractive van der Waals minimum in the intermembrane potential when brought into close contact by DNA binding. This obstacle can be overcome by the isothermal reduction of the ionic strength of the solution: this reduces the Debye screening length, coupling the effects of DNA dehybridization and intermembrane repulsion due to the increased electrostatic repulsion between the highly charged DNA backbones.     

Interaction and aggregation of lipid vesicles (DLVO theory versus modified DLVO theory)

Interaction and aggregation of acidic phospholipid (phosphatidylserine) vesicles were studied with variation of cation species and their concentrations in vesicle suspensions, and of vesicle sizes. Aggregation was determined by measuring turbidity of vesicle suspension. The experimental results of aggregation of vesicles induced by monovalent cations (Na⁺, K⁺, Cs⁺ and TMA⁺) were explained well in terms of the interaction energy of two interacting vesicles using the ordinary Derjaguin–Landau–Verwey–Overbeek (DLVO) theory for both small and large lipid vesicles. However, the experimental results of aggregation of vesicles induced by divalent cations (Ca²⁺, Mg²⁺ and Ba²⁺) were not explained by the ordinary DLVO theory. In order to explain the experimental results of these vesicle aggregation phenomena, it was necessary to modify the theory by including hydration interaction energies which are due to hydrated water at membrane surfaces, and their magnitude and sign depend upon the nature (hydrophobicity) of the membrane surface.     

Engineering bio-mimicking functional vesicles with multiple compartments for quantifying molecular transport

Controlled design of giant unilamellar vesicles under defined conditions has vast applications in the field of membrane and synthetic biology. Here, we bio-engineer bacterial-membrane mimicking models of controlled size under defined salt conditions over a range of pH. A complex bacterial lipid extract is used for construction of physiologically relevant Gram-negative membrane mimicking vesicles whereas a ternary mixture of charged lipids (DOPG, cardiolipin and lysyl-PG) is used for building Gram-positive bacterial-membrane vesicles. Furthermore, we construct stable multi-compartment biomimicking vesicles using the gel-assisted swelling method. Importantly, we validate the bio-application of the bacterial vesicle models by quantifying diffusion of chemically synthetic amphoteric antibiotics. The transport rate is pH-responsive and depends on the lipid composition, based on which a permeation model is proposed. The permeability properties of antimicrobial peptides reveal pH dependent pore-forming activity in the model vesicles. Finally, we demonstrate the functionality of the vesicles by quantifying the uptake of membrane-impermeable molecules facilitated by embedded pore-forming proteins. We suggest that the bacterial vesicle models developed here can be used to understand fundamental biological processes like the peptide assembly mechanism or bacterial cell division and will have a multitude of applications in the bottom-up assembly of a protocell.

Giant vesicle functional models mimicking a bacterial membrane under physiological conditions are constructed.     

Modulation of interaction of polycations with negative unilamellar lipid vesicles

Interactions of small unilamellar negative vesicles composed of diphosphatidylglycerol (cardiolipin, CL²⁻), 20 mol%, and phosphatidylcholine (egg yolk lecithin, EL), 80 mol%, with various cationic polymers (CP) derived from poly(4-vinylpyridine) (PVP) were studied in water and water–salt solutions by means of photon correlation spectroscopy, microelectrophoresis, conductometry, and fluorescence techniques. The linear charge density and hydrophilic lipophilic balance of CPs were varied by quaternization of PVP with various amounts of different alkyl bromides (ethyl-(2), heptyl-(7), dodecyl-(12), cetyl-(16)). Substantial differences were observed in the behavior of exhaustingly N-ethylated PVP (CP2) and PVP N-ethylated to 50 mol% (CP2(50)) or 30 mol% (CP2(30)). All of them adsorb to the CL²⁻/EL vesicle membrane, neutralizing the surface negative charge and causing aggregation of the vesicles. However, CP2, a polycation with a maximum linear charge density, strongly enhances transfer of the negative lipid ions from the inner to outer bilayer leaflet, while CP2(50) and CP2(30) do not. Adsorbed CP2 does not disturb integrity of the vesicle membrane and can be completely removed from the surface of aggregated vesicles by adding a simple salt (NaCl) or a negative linear polyelectrolyte (polyacrylic acid (PAA) sodium salt). Such removal is followed by release of the original vesicles. In contrast to that, adsorbed CP2(50) or CP2(30) produce some leak through the lipid bilayer and cannot be completely desorbed either by increasing ionic strength or adding an excess of PAA. The probable reason of these differences is discussed. PVP partially N-alkylated with dodecyl or cetyl bromides (3 mol%) and then completely N-ethylated (CP2,12 and CP2,16), also having a maximum linear charge density, adsorbs to the negative vesicle surface as a result of both electrostatic binding and hydrophobic interaction. Bulky hydrocarbon pendant groups incorporate into the inner bilayer compartment. Similarly to CP2(50) and CP2(30), CP2,12 and CP2,16 cannot be removed from the surface either by adding the simple salt, or an excess of PAA. However, in contrast to CP2(50) and CP2(30), the polycations with the bulky hydrocarbon pendant groups do not cause any leak through the vesicle membrane. Finally, we have succeeded to prepare the ternary vesicles also composed of 20 mol% of CL²⁻, but partially replacing EL for polyoxyethylene 20 cetyl ether (Brij 58) (up to 30 mol%). The CL²⁻/EL/Brij vesicle carries a hydrophilic corona formed by polyoxyethylene chains exposed into water, while hydrophobic cetyl radicals are incorporated in the lipid bilayer. The CL²⁻/EL/Brij vesicles adsorb all studied CPs similar to the binary CL²⁻/EL vesicles. This means that polyoxyethylene corona is permeable for polycationic species restricting neither electrostatic binding nor incorporation of bulky hydrocarbon groups of CP2,16 into the membrane. However, the corona effectively stabilizes the CP-vesicle complexes against aggregation when the membrane surface is neutralized.     

Lipid nanotube formation from streptavidin-membrane binding

A novel transformation of giant lipid vesicles to produce nanotubular structures was observed upon the binding of streptavidin to biotinylated membranes. Unlike membrane budding and tubulation processes caused by proteins involved with endocytosis and vesicle fusion, streptavidin is known to crystallize at near the isoelectric point (pI 5 to 6) into planar sheets against biotinylated films. We have found, however, that at neutral pH membranes of low bending rigidity (<10kT), such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), spontaneously produce tubular structures with widths ranging from micrometers to below the diffraction limit (<250 nm) and lengths spanning up to hundreds of micrometers. The nanotubes were typically held taut between surface-bound vesicles suggesting high membrane tension, yet the lipid nanotubes exhibited a fluidic nature that enabled the transport of entrained vesicles. Confocal microscopy confirmed the uniform coating of streptavidin over the vesicles and nanotubes. Giant vesicles composed of lipid membranes of higher bending energy exhibited only aggregation in the presence of streptavidin. Routes toward the development of these highly curved membrane structures are discussed in terms of general protein-membrane interactions.     

具有双活性序列的新型抗菌肽的设计及性质

设计合成了具有2个活性序列的线性和环状多肽及具有单个活性序列的短链多肽, 研究了它们的杀菌活性、 细胞毒性及溶血性. 结果表明, 线性肽和环状肽的杀菌活性高于短链肽. 利用计算模拟的方法计算了多肽与细菌细胞膜中一种重要的成分磷脂酰甘油(DMPG)的结合能. 结果表明, 多肽-DMPG的结合能与多肽的杀菌活性具有较高的相关性, 线性和环状多肽与DMPG的结合能大于短链肽. 线性和环状多肽均含有2个活性序列, 可提供多个荷正电氨基酸与荷负电的磷脂结合, 结合能较大, 杀菌活性较强. 采用模拟生物膜对其中几条多肽的作用机理进行了初步研究. 结果表明, 该类多肽有可能使正常哺乳动物细胞的细胞膜产生孔洞; 而对于细菌细胞膜, 多肽并未在膜上产生明显孔洞, 而是引起了细菌细胞膜的聚集.     

Influence of lipid composition on membrane activity of antimicrobial phenylene ethynylene oligomers

Host defense peptides (HDPs), part of the innate immune system, selectively target the membranes of bacterial cells over that of host cells. As a result, their antimicrobial properties have been under intense study. Their selectivity strongly depends on the chemical and mostly structural properties of the lipids that make up different cell membranes. The ability to synthesize HDP mimics has recently been demonstrated. To better understand how these HDP mimics interact with bilayer membranes, three homologous antimicrobial oligomers (AMOs) 1-3 with an m-phenylene ethynylene backbone and alkyl amine side chains were studied. Among them, AMO 1 is nonactive, AMO 2 is specifically active, and AMO 3 is nonspecifically active against bacteria over human red blood cells, a standard model for mammalian cells. The interactions of these three AMOs with liposomes having different lipid compositions are characterized in detail using a fluorescent dye leakage assay. AMO 2 and AMO 3 caused more leakage than AMO 1 from bacteria membrane mimic liposomes composed of PE/PG lipids. The use of E. coli lipid vesicles gave the same results. Further changes of the lipid compositions revealed that AMO 2 has selectively higher affinity toward PE/PG and E. coli lipids than PC, PC/PG or PC/PS lipids, the major components of mammalian cell membranes. In contrast, AMO 3 is devoid of this lipid selectivity and interacts with all liposomes with equal ease; AMO 1 remains inactive. These observations suggest that lipid type and structure are more important in determining membrane selectivity than lipid headgroup charges for this series of HDP mimics.     

Binding of chitosan to phospholipid vesicles studied with isothermal titration calorimetry

We thermodynamically characterize the interaction of chitosan with small liposomes and the binding and organization of the polysaccharide on the membrane of the vesicles. By means of isothermal titration calorimetry (ITC), we obtain the enthalpy variations arising from binding of the positively ionized chitosan to neutral and negatively charged liposomes. The strong electrostatic interaction of the polysaccharide with the negative charges at the membrane gives rise to highly exothermic signal until charge compensation is reached. The equilibrium constant, the interaction stoichiometry, and the molar enthalpy of binding chitosan monomers to phospholipids from the external leaflet of the vesicle membrane are obtained from the isotherm curve fitting assuming independent binding sites. The strong exothermic signal indicates that the electrostatically driven binding of chitosan to the membrane is energetically favored, leading to further stabilization of the vesicle suspension. The higher the net negative charge of the vesicles, the more pronounced the adsorption of chitosan is, leading to weaker chain organization of the adsorbed chitosan at the membrane. At the point of charge saturation, vesicle aggregation takes place and we show that this behavior does not always lead to charge reversal at the membrane. Models for the binding behavior and structural organization of chitosan are proposed based on the experimental results from ITC, ζ-potential, and dynamic light scattering.     

Use of poly(ethylene glycol) to control cell aggregation and fusion

Although poly(ethylene glycol) (PEG) has been widely used as an agent to induce cell aggregation and fusion, the physicochemical principles of its function are only becoming understood recently. PEG has an extremely high affinity for water. The PEG commonly used for these applications is in the molecular weight range of 8000 to 10 000. At low concentrations (0–15 wt.%), PEG in this molecular weight range tends to deplete from cell or lipid surfaces, creating an osmotic gradient which brings cells or lipid vesicles together. The depletion force is measured using a surface force apparatus. The corresponding reduction of surface viscosity is verified by shear viscosity measurements and by vesicle tumbling experiments. At higher concentrations (15–45 wt.%), the extremely high osmotic pressure generated by PEG compresses apposing surfaces of aggregated cells or vesicles to within limits where the membrane is no longer stable, and fusion occurs at point defects. A fusion lumen is formed with the help of cell swelling. If PEG is adsorbed or covalently link to the cell or vesicle surface, the surface force profile becomes entirely repulsive, and aggregation and fusion is inhibited. The repulsion is accountable by steric and electrostatic forces. Therefore, the fusogenic function of PEG can be explained quantitatively by colloidal stability theories.     

Coarse-grained transmembrane proteins: hydrophobic matching, aggregation, and their effect on fusion

Molecular transport between organelles is predominantly governed by vesicle fission and fusion. Unlike experimental vesicles, the fused vesicles in molecular dynamics simulations do not become spherical readily, because the lipid and water distribution is inappropriate for the fused state and spontaneous amendment is slow. Here, we study the hypothesis that enhanced transport across the membrane of water, lipids, or both is required to produce spherical vesicles. This is done by adding several kinds of model proteins to fusing vesicles. The results show that equilibration of both water and lipid content is a requirement for spherical vesicles. In addition, the effect of these transmembrane proteins is studied in bilayers and vesicles, including investigations into hydrophobic matching and aggregation. Our simulations show that the level of aggregation does not only depend on hydrophobic mismatch, but also on protein shape. Additionally, one of the proteins promotes fusion by inducing pore formation. Incorporation of these proteins allows even flat membranes to fuse spontaneously. Moreover, we encountered a novel spontaneous vesicle enlargement mechanism we call the engulfing lobe, which may explain how lipids added to a vesicle solution are quickly incorporated into the inner monolayer.     

Construction of macroscopic cytomimetic vesicle aggregates based on click chemistry: controllable vesicle fusion and phase separation

Vesicle-vesicle aggregation to mimic cell-cell aggregation has attracted much attention. Here, hyperbranched polymer vesicles (branched-polymersomes, BPs) with a cell-like size were selected as model membranes, and the vesicle aggregation process, triggered by click chemistry of the copper-catalysed azide-alkyne cycloaddition reaction, was systematically studied. For this purpose, azide and alkynyl groups were loaded on the membranes of BPs through the co-assembly method to obtain N(3)-BPs and Alk-BPs, respectively. Subsequently, macroscopic vesicle aggregates were obtained when these two kinds of functional BPs were mixed together with the ratio of azide to alkynyl groups of about 1:1. Both the vesicle fusion events and lateral phase separation on the vesicle membrane occurred during such a vesicle aggregation process, and the fusion rate and phase-separation degree could be controlled by adjusting the clickable group content. The vesicle aggregation process with N(3) -micelles as desmosome mimics to connect with Alk-BPs through click-chemistry reaction was also studied, and large-scale vesicle aggregates without vesicle fusion were obtained in this process. The present work has extended the controllable cytomimetic vesicle aggregation process with the use of covalent bonds, instead of noncovalent bonds, as the driving force.     

The Role of Hydrophobicity in the Antimicrobial and Hemolytic Activities of Polymethacrylate Derivatives

We synthesized cationic random amphiphilic copolymers by radical copolymerization of methacrylate monomers with cationic or hydrophobic groups and evaluated their antimicrobial and hemolytic activities. The nature of the hydrophobic groups, and polymer composition and length were systematically varied to investigate how structural parameters affect polymer activity. This allowed us to obtain the optimal composition of polymers suitable to act as non-toxic antimicrobials as well as non-selective polymeric biocides. The antimicrobial activity depends sigmoidally on the mole fraction of hydrophobic groups (f_HB). The hemolytic activity increases as f_HB increases and levels off at high values of f_HB, especially for the high-molecular-weight polymers. Plots of HC₅₀ values versus the number of hydrophobic side chains in a polymer chain for each polymer series showed a good correlation and linear relationship in the log–log plots. We also developed a theoretical model to analyze the hemolytic activity of polymers and demonstrated that the hemolytic activity can be described as a balance of membrane binding of polymers through partitioning of hydrophobic side chains into lipid layers and the hydrophobic collapsing of polymer chains. The study on the membrane binding of dye-labeled polymers to large, unilamellar vesicles showed that the hydrophobicity of polymers enhances their binding to lipid bilayers and induces collapse of the polymer chain in solution, reducing the apparent affinity of polymers for the membranes.     

Formation and activity of template-assembled receptor signaling complexes

Problems in membrane biology require methods to recreate the interactions between receptors and cytoplasmic signaling proteins at the membrane surface. Here, unilamellar vesicles composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine and a nickel-chelating lipid were used as templates to direct the assembly of proteins from the Escherichia coli chemotaxis signaling pathway. The bacterial chemoreceptors are known to form clusters, which promote the binding of the adaptor protein (CheW) and the kinase (CheA). When CheA was incubated with vesicles, CheW, and a histidine-tagged cytoplasmic domain fragment of the aspartate chemoreceptor (CF), the kinase activity was stimulated approximately 300-fold. Activity and pull-down assays were used with dynamic light scattering and electron microscopy to characterize the protein-vesicle compositions that were correlated with the high levels of activity, which demonstrated that CF-CheW-CheA complexes on the vesicle surface were the active entities. Assembly and stimulation occurred with vesicles of different sizes and CFs in different extents of glutamine substitution (in place of glutamate) at physiologically relevant sites. An exception was the combination of sonicated vesicles with the unsubstituted CF, which displayed lower CheA activity. The lower activity was attributed to the high curvature of the sonicated vesicles and a weaker tendency of the unsubstituted CF to self-assemble. Electron micrographs of the vesicle-protein assemblies revealed that protein binding induced pronounced changes in vesicle shape, which was consistent with the introduction of positive curvature in the outer leaflet of the bilayer. Overall, vesicle-mediated template-directed assembly is shown to be an effective way to form functional complexes of membrane-associated proteins and suggests that significant changes in membrane shape can be involved in the process of transmembrane signaling.     

pH-induced destabilization of lipid bilayers by a peptide from the VP3 protein of the capsid of hepatitis A virus.

The membrane destabilizing and fusogenic properties of the synthetic peptide VP3(110-121), corresponding to an immunogenic sequence of the hepatitis A virus (HAV) VP3 capsid protein, were studied. By tryptophan fluorescence and acryalmide quenching it was demonstrated that the peptide binds liposomes of POPC-SM-DPPE (47 + 39 + 14) and POPC-SM-DPPE-DOTAP (40 + 33 + 12 + 15) and penetrates the membrane, at both neutral and acidic pH (POPC = 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine; SM = sphingomyelin; DPPE = 1,2-dipalmitoylphosphatidylethanolamine; DOTAP = 1,2-dioleoyl-3-trimethylammoniumpropane). VP3(110-121) did not have membrane-destabilizing properties at neutral pH. Acid-induced destabilization of the vesicles was demonstrated by fluorescence techniques and dynamic light scattering. VP3(110-121) induced aggregation of POPC-SM-DPPE-DOTAP (40 + 33 + 12 + 15) vesicles, lipid mixing and leakage of vesicle contents, all consistent with fusion of vesicles. In POPC-SM-DPPE (47 + 39 + 14) vesicles, at acidic pH, VP3(110-121) induced membrane destabilization with leakage of contents but without aggregation of vesicles or lipid mixing. The peptide only showed fusogenic properties when bound to the vesicles at neutral pH before acidification to pH below 6.0, and no effect was seen if the peptide was added to vesicles already set at acidic pH. These results may have physiological significance in the mechanism of infection of host hepatic cells by HAV.     

Aggregation Behavior of Nonionic Clycolipid Vesicles in Acidic Region

Large unilamellar vesicles composed of a nonionic synthetic glycotipid, 1,3 - di- 0 - phylanyl -2-0-(β- maltotriosyl ) glycerol show pH-dependent aggregation - dissocialion process, that is, vesicle aggregation occurs in the lower pH region and vesicle dissociation occurs in the higher pH region. This process is almost reversible and the aggregation threshold pH is dependent on NaCl concentration. Fluorescence technique has been applied to study whether the vesicle fusion occurs or not during the aggregation-dissociation process. It is concluded pH can only induce the aggregation of this nonionic glycoltpid vesicle.     

Preparation of primary amine-based block copolymer vesicles by direct dissolution in water and subsequent stabilization by sol-gel chemistry

A new amphiphilic biocompatible diblock copolymer, poly(epsilon-caprolactone)-block-poly(2-aminoethyl methacrylate), PCL-b-PAMA, was synthesized in three steps by (i) ring-opening polymerization of epsilon-caprolactone, (ii) end-group modification by esterification, and (iii) atom transfer radical polymerization (ATRP) of 2-aminoethyl methacrylate hydrochloride (AMA) in its hydrochloride salt form. This copolymer forms block copolymer vesicles with the hydrophobic PCL block forming the vesicle membrane. Unusually, these vesicles are easily prepared by direct dissolution in water without using organic co-solvents, pH adjustment, or even stirring. These vesicles can be stabilized by aqueous sol-gel chemistry using tetramethyl orthosilicate (TMOS) as the silica precursor. It is well-known that cationic polymers can catalyze silica formation, but in this particular case, it seems that the TMOS precursor is solubilized within the hydrophobic PCL membrane. Thus, the neutral membrane actually directs silica formation, rather than the cationic PAMA chains. The final vesicle morphology and the silica content depend on the silicification conditions. Provided that the TMOS/AMA molar ratio does not exceed 10:1, silicification is solely confined within the PCL membrane. At higher ratios, silica nanoparticles (5-12 nm) are also observed on the outer surface of the silicified vesicles. However, these nanoparticles appear to be only weakly adsorbed, since they can be easily removed by dialysis. The mean hydrodynamic diameter of the silicified vesicles varies from 175 to 205 nm with solution pH due to (de)protonation of the externally expressed PAMA chains. Calcination of the silicified vesicles at 800 degrees C leads to the formation of hollow silica particles. 1H NMR, transmission electron microscopy (TEM), dynamic light scattering (DLS), aqueous electrophoresis, and thermogravimetric analysis (TGA) were employed to characterize the vesicles, both before and after silicification.     

Membrane electroporation and electromechanical deformation of vesicles and cells

Analysis of the reduced turbidity (delta T-/T0) and absorbance (delta A-/A0) relaxations of unilamellar lipid vesicles, doped with the diphenylhexatrienyl-phosphatidylcholine (beta-DPH pPC) lipids in high-voltage rectangular electrical field pulses, demonstrates that the major part of the turbidity and absorbance dichroism is caused by vesicle elongation under electric Maxwell stress. The kinetics of this electrochemomechanical shape deformation (time constants 0.1 < or = tau/microsecond < or = 3) is determined both by the entrance of water and ions into the bulk membrane phase to form local electropores, and by the faster processes of membrane stretching and smoothing of thermal undulations. Moreover, the absorbance dichroism indicates local displacements of the chromophore relative to the membrane normal in the field. The slightly slower relaxations of the chemical turbidity (delta T+/T0) and absorbance (delta A+/A0) modes are both associated with the entrance of solvent into the interface membrane/medium, caused by the alignment of the bipolar lipid head groups in one of the leaflets at the pole caps of the vesicle bilayer. In addition, (delta T+/T0) indicates changes in vesicle shape and volume. The results for lipid vesicles provide guidelines for the analysis of electroporative deformations of biological cells.     

Electrochemical characterization of pore formation by bacterial protein toxins on hybrid supported membranes

The interaction of pore-forming streptolysin O (SLO) with biomimetic lipid membranes has been studied by electrochemical methods. Phosphatidylcholine lipid vesicles were deposited onto gold electrodes modified with supporting layers of hexyl thioctate (HT) or thioctic acid tri(ethylene glycol) ester (TA-TEGE), and integrity and permeability of the resulting membranes were characterized by cyclic voltammetry and impedance spectroscopy. Both positively and negatively charged electrochemical probes, potassium ferrocyanide, hexaammineruthenium(III) chloride, and ferrocene carboxylic acid (FCA), were employed to evaluate their suitability to probe the membrane permeability properties, with FCA exhibiting ideal behavior and thus employed throughout the work. Fusion of vesicles incubated with SLO on the electrodes yielded membranes that showed a distinctive response pattern for FCA as a function of SLO concentration. A direct dependence of both the currents and peak separation of FCA in the cyclic voltammograms was observed over a concentration range of 0-10 hemolytic units (HU)/microL of the toxin. The interaction of SLO with preformed supported lipid membranes was also investigated, and much lower response was observed, suggesting a different extent of membrane-toxin interactions on such an interface. Nonionic surfactant Triton was found to disrupt the vesicle structure but could not completely remove a preformed membrane to fully restore the electrode response. The information reported here offers some unique insight into toxin-surface interactions on a hybrid membrane, facilitating the development of electrochemically based sensing platforms for detecting trace amounts of bacterial toxins via the perforation process.     

Comparison of Facially Amphiphilic versus Segregated Monomers in the Design of Antibacterial Copolymers

A direct comparison of two strategies for designing antimicrobial polymers is presented. Previously, we published several reports on the use of facially amphiphilic (FA) monomers which led to polynorbornenes with excellent antimicrobial activities and selectivities. Our polymers obtained by copolymerization of structurally similar segregated monomers, in which cationic and non‐polar moieties reside on separate repeat units, led to polymers with less pronounced activities. A wide range of polymer amphiphilicities was surveyed by pairing a cationic oxanorbornene with eleven different non‐polar monomers and varying the comonomer feed ratios. Their properties were tested using antimicrobial assays and copolymers possessing intermediate hydrophobicities were the most active. Polymer‐induced leakage of dye‐filled liposomes and microscopy of polymer‐treated bacteria support a membrane‐based mode of action. From these results there appears to be profound differences in how a polymer made from FA monomers interacts with the phospholipid bilayer compared with copolymers from segregated monomers. We conclude that a well‐defined spatial relationship of the whole polymer is crucial to obtain synthetic mimics of antimicrobial peptides (SMAMPs): charged and non‐polar moieties need to be balanced locally, for example, at the monomer level, and not just globally. We advocate the use of FA monomers for better control of biological properties. It is expected that this principle will be usefully applied to other backbones such as the polyacrylates, polystyrenes, and non‐natural polyamides.     

Phospholipase D-mediated aggregation, fusion, and precipitation of phospholipid vesicles

Large unilamellar vesicles with a diameter of 100 nm were prepared from the zwitterionic phospholipid POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) at pH 8.0. After addition to these vesicles of the enzyme phospholipase D (PLD) from Streptomyces sp. AA586 at 40 degrees C, the terminal phosphate ester bond of POPC was hydrolyzed, yielding the negatively charged POPA (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid) and the positively charged choline. While the reaction yield in the presence of 1 mM Ca2+ reached 100%, the yield was only approximately 68% in the absence of Ca2+. Furthermore, in the absence of Ca2+, the size of the vesicles did not change significantly with time upon PLD addition, as judged from turbidity, dynamic light scattering, and electron microscopy measurements. In the presence of 1 mM Ca2+, however, PLD addition resulted in vesicle aggregation, fusion, and precipitation, originating from the interaction of Ca2+ ions with the negatively charged phospholipids formed in the membranes. Vesicle fusion was monitored by using a novel fusion assay system involving vesicles containing entrapped trypsin and vesicles containing entrapped chymotrypsinogen A. After vesicle fusion, chymotrypsinogen A transformed into a-chymotrypsin, catalyzed by trypsin inside the fused vesicles. The alpha-chymotrypsin formed could be detected with benzoyl-L-Tyr-p-nitroanilide as a membrane permeable chymotrypsin substrate. The observed vesicle precipitation occurring after vesicle fusion in the presence of 1 mM Ca2+ was correlated with an increase of the main phase transition temperature, Tm, of POPA to values above 40 degrees C.