Liposomes, disks, and spherical micelles: aggregate structure in mixtures of gel phase phosphatidylcholines and poly(ethylene glycol)-phospholipids

Poly(ethylene glycol) (PEG) decorated lipid bilayers are widely used in biomembrane and pharmaceutical research. The success of PEG-lipid stabilized liposomes in drug delivery is one of the key factors for the interest in these polymer/lipid systems. From a more fundamental point of view, it is essential to understand the effect of the surface grafted polymers on the physical-chemical properties of the lipid bilayer. Herein we have used cryo-transmission electron microscopy and dynamic light scattering to characterize the aggregate structure and phase behavior of mixtures of PEG-lipids and distearoylphosphatidylcholine or dipalmitoylphosphatidylcholine. The PEG-lipids contain PEG of molecular weight 2000 or 5000. We show that the transition from a dispersed lamellar phase (liposomes) to a micellar phase consisting of small spherical micelles occurs via the formation of small discoidal micelles. The onset of disk formation already takes place at low PEG-lipid concentrations (<5 mol %) and the size of the disks decreases as more PEG-lipid is added to the lipid mixture. We show that the results from cryo-transmission electron microscopy correlate well with those obtained from dynamic light scattering and that the disks are well described by an ideal disk model. Increasing the temperature, from 25 degrees C to above the gel-to-liquid crystalline phase transition temperature for the respective lipid mixtures, has a relatively small effect on the aggregate structure.     

Polymer-mediated electrostatic interactions between charged lipid assemblies and electrolyte solutions: a tentative model of the polyethylene glycol-induced cell fusion

We developed a theoretical model to investigate the interaction between charged lipid aggregates and a water solution containing ions and uncharged polymers. The local concentration of ions and polymer chains around the lipid aggregate have been treated as variational parameters which can be found by minimizing the total energy of the system. We divided the energy into the following main contributions: (a) Solvation energy of the ions. This depends on the local polymer concentration through the variation of the solvent dielectric properties. (b) Ions-lipid aggregate interactions. These depend on the local concentrations both of the ion cloud and polymer chains. (c) Conformational energy of the polymer. This term is related to the inhomogeneous spatial density of the polymer segments. Any direct interaction between the charged lipid surface and the polymer coils has been intentionally neglected. The minimization procedure leads to a non-linear Poisson-Boltzmann equation coupled with a non-linear algebraic equation describing the polymer distribution. The solution of the above system allows one to calculate the ions and polymer spatial distribution around the lipid aggregate. The knowledge of such parameters is useful to predict the effect of non-ionic polymers on the structure and properties of lipid assemblies such as the mean area per lipid molecule, the aggregation number, the critical micellar concentration and the formation of immiscibility gaps in mixed lipid systems. A possible involvement of these parameters into the fusion process between lipid vesicles is discussed.     

A method to evaluate the effect of liposome lipid composition on its interaction with the erythrocyte plasma membrane

Lipid aggregates are considered promising carriers for macromolecules and toxic drugs. In order to fulfill this function, aggregates should have properties that ensure the efficient delivery of their cargo to the desired location. One of these properties is their stability in blood when accumulating in the targeted tissue. This stability may be affected by a number of factors, including enzymatic activity, protein adsorption, and non-specific lipid exchange between the aggregate and morphological blood components. Since blood cells in the majority consist of erythrocytes, their interaction with aggregates should be carefully analyzed. In this paper, we present a method that allows the exchange of lipid between liposomes and the erythrocyte plasma membrane to be evaluated. The extent of this exchange was measured in terms of the toxicity of a cationic lipid (DOTAP) incorporated into the liposome lipid bilayer, evaluated by plasma membrane mechanical properties. After liposomes were formed from DOTAP/PC or DOTAP/PE mixtures, erythrocyte plasma membranes were destabilized in a manner dependent on DOTAP concentration. A constant quantity of DOTAP mixed with various proportions of SM caused no such effect, indicating very limited lipid exchange with the cell membrane for such liposome formulations.     

Multichain aggregates in dilute solutions of associating polyelectrolyte keeping a constant size at the increase in the chain length of individual macromolecules

Multichain aggregates together with individual macromolecules were detected by light scattering in dilute aqueous solutions of chitosan and of its hydrophobic derivatives bearing 4 mol % of n-dodecyl side groups. It was demonstrated that the size of aggregates and their aggregation numbers increase at the introduction of hydrophobic side groups into polymer chains. The key result concerns the effect of the chain length of individual macromolecules on the aggregation behavior. It was shown that for both unmodified and hydrophobically modified (HM) chitosan, the size of aggregates is independent of the length of single chains, which may result from the electrostatic nature of the stabilization of aggregates. At the same time, the number of macromolecules in one aggregate increases significantly with decreasing length of single chains to provide a sufficient number of associating groups to stabilize the aggregate. The analysis of the light scattering data together with TEM results suggests that the aggregates of chitosan and HM chitosan represent spherical hydrogel particles with denser core and looser shell covered with dangling chains.     

A Transparent Planar Concentrator Using Aggregates of gem‐Pyrene Ethenes

The luminescence properties of pyrene ethenes, both as monomer and aggregate species, are found to depend on the regioisomer structure. Systematic shifts in absorption, emission, and excitation spectra of the gem‐pyrene ethenes, both in solution and in rigid polymer hosts, are consistent with weakly interacting H‐aggregate formation. This aggregation leads to excimer‐like emission with Stokes shifts greater than 1 eV. Planar concentrators fabricated from gem‐pyrene diphenylethenes show comparable performance to previously reported inorganic phosphors. The UV absorption and emission properties of the planar concentrator devices exhibit potential for transparent solar concentrators or visible–blind photodetector applications. This is the first demonstration of exploiting the unusual photophysics of molecular aggregates in planar concentrators.     

Molecular and mesoscopic properties of hydrophilic polymer-grafted phospholipids mixed with phosphatidylcholine in aqueous dispersion: interaction of dipalmitoyl N-poly(ethylene glycol)phosphatidylethanolamine with dipalmitoylphosphatidylcholine studied by spectrophotometry and spin-label electron spin resonance

Spin-label electron spin resonance (ESR) spectroscopy, together with optical density measurements, has been used to investigate, at both the molecular and supramolecular levels, the interactions of N-poly(ethylene glycol)-phosphatidylethanolamines (PEG-PE) with phosphatidylcholine (PC) in aqueous dispersions. PEG-PEs are micelle-forming hydrophilic polymer-grafted lipids that are used extensively for steric stabilization of PC liposomes to increase their lifetimes in the blood circulation. All lipids had dipalmitoyl (C16:0) chains, and the polymer polar group of the PEG-PE lipids had a mean molecular mass of either 350 or 2000 Da. PC/PEG-PE mixtures were investigated over the entire range of relative compositions. Spin-label ESR was used quantitatively to investigate bilayer-micelle conversion with increasing PEG-PE content by measurements at temperatures for which the bilayer membrane component of the mixture was in the gel phase. Both saturation transfer ESR and optical density measurements were used to obtain information on the dependence of lipid aggregate size on PEG-PE content. It is found that the stable state of lipid aggregation is strongly dependent not only on PEG-PE content but also on the size of the hydrophilic polar group. These biophysical properties may be used for optimized design of sterically stabilized liposomes.     

Ultradeformable lipid vesicles can penetrate the skin and other semi-permeable barriers unfragmented. Evidence from double label CLSM experiments and direct size measurements

The stability of various aggregates in the form of lipid bilayer vesicles was tested by three different methods before and after crossing different semi-permeable barriers. First, polymer membranes with pores significantly smaller than the average aggregate diameter were used as the skin barrier model; dynamic light scattering was employed to monitor vesicle size changes after barrier passage for several lipid mixtures with different bilayer elasticities. This revealed that vesicles must adapt their size and/or shape, dependent on bilayer stability and elasto-mechanics, to overcome an otherwise confining pore. For the mixed lipid aggregates with highly flexible bilayers (Transfersomes®), the change is transient and only involves vesicle shape and volume adaptation. The constancy of ultradeformable vesicle size before and after pores penetration proves this. This is remarkable in light of the very strong aggregate deformation during an enforced barrier passage. Simple phosphatidylcholine vesicles, with less flexible bilayers, lack such capability and stability. Conventional liposomes are therefore fractured during transport through a semi-permeable barrier; as reported by other researchers, liposomes are fragmented to the size of a narrow pore if sufficient pressure is applied across the barrier; otherwise, liposomes clog the pores. The precise outcome depends on trans-barrier flux and/or on relative vesicle vs. pore size. Lipid vesicles applied on the skin behave accordingly. Mixed lipid vesicles penetrate the skin if they are sufficiently deformable. If this is the case, they cross inter-cellular constrictions in the organ without significant composition or size modification. To prove this, we labelled vesicles with two different fluorescent markers and applied the suspension on intact murine skin without occlusion. The confocal laser scanning microscopy (CLSM) of the skin then revealed a practically indistinguishable distribution of both labels in the stratum corneum, corroborating the first assumption. To confirm the second postulate, we compared vesicle size in the starting suspension and in the blood after non-invasive transcutaneous aggregate delivery. Size exclusion chromatograms of sera from the mice that received ultradeformable vesicles on the skin were undistinguishable from the results measured with the original vesicle suspension. Taken together, the results support our previous postulate that ultradeformable vesicles penetrate the skin intact, that is, without permanent disintegration.     

Alkyl Chain Regiochemistry of Benzotriazole‐Based Donor Polymers Influencing Morphology and Performances of Non‐Fullerene Organic Solar Cells

The effects of alkyl chain regiochemistry on the properties of donor polymers and performances of non‐fullerene organic solar cells are investigated. Two donor polymers (PfBTAZ and PfBTAZS) are compared that have nearly identical chemical structures except for the regiochemistry of alkyl chains. The optical properties and crystallinity of two polymers are nearly identical yet the PfBTAZ:O‐IDTBR blend exhibits nearly double domain size compared to the blend based on PfBTAZS:O‐IDTBR. To reveal the origins of the very different domain size of two blends, the morphology of neat polymer films is characterized, and it is found that PfBTAZ tends to aggregate into much larger polymer fibers without the presence of O‐IDTBR. This indicates that it is not the polymer:O‐IDTBR interactions but the intrinsic aggregation properties of two polymers that determine the morphology features of neat and blend films. The stronger aggregation tendency of PfBTAZ could be explained by its more co‐planar geometry of the polymer backbone arising from the different alkyl chain regiochemistry. Combined with the similar trend observed in another set of donor polymers (PTFB‐P and PTFB‐PS), the results provide an important understanding of the structure–property relationships that could guide the development of donor polymers for non‐fullerene organic solar cells.     

The effect of chain length on protein solubilization in polymer-based vesicles (polymersomes)

Using a mean-field analysis we derive a consistent model for the perturbation of a symmetric polymeric bilayer due to the incorporation of transmembrane proteins, as a function of the polymer molecular weight and the protein dimensions. We find that the mechanism for the inhibition of protein incorporation in polymeric bilayers differs from that of their inclusion in polymer-carrying lipid vesicles; in polymersomes, the equilibrium concentration of transmembrane proteins decreases as a function of the thickness mismatch between the protein and the bilayer core, whereas in liposomes the presence of polymer chains affects the protein adsorption kinetics. Despite the increased stiffness of polymer bilayers (when compared to lipid ones), their perturbation decay length and range of protein-protein interaction is found to be relatively long. The energetic penalty due to protein adsorption increases relatively slowly as a function of the polymer chain length due to the self-assembled nature of the polymer bilayer. As a result, we predict that transmembrane proteins may be incorporated in significant numbers even in bilayers where the thickness mismatch is large.     

Monitoring the process of HypF fibrillization and liposome permeabilization by protofibrils

Much information has appeared in the last few years on the low resolution structure of amyloid fibrils and on their non-fibrillar precursors formed by a number of proteins and peptides associated with amyloid diseases. The fine structure and the dynamics of the process leading misfolded molecules to aggregate into amyloid assemblies are far from being fully understood. Evidence has been provided in the last five years that protein aggregation and aggregate toxicity are rather generic processes, possibly affecting all polypeptide chains under suitable experimental conditions. This evidence extends the number of model proteins one can investigate to assess the molecular bases and general features of protein aggregation and aggregate toxicity. We have used tapping mode atomic force microscopy to investigate the morphological features of the pre-fibrillar aggregates and of the mature fibrils produced by the aggregation of the hydrogenase maturation factor HypF N-terminal domain (HypF-N), a protein not associated to any amyloid disease. We have also studied the aggregate-induced permeabilization of liposomes by fluorescence techniques. Our results show that HypF-N aggregation follows a hierarchical path whereby initial globules assemble into crescents; these generate large rings, which evolve into ribbons, further organizing into differently supercoiled fibrils. The early pre-fibrillar aggregates were shown to be able to permeabilize synthetic phospholipid membranes, thus showing that this disease-unrelated protein displays the same amyloidogenic behaviour found for the aggregates of most pathological proteins and peptides. These data complement previously reported findings, and support the idea that protein aggregation, aggregate structure and toxicity are generic properties of polypeptide chains.     

Effect of Peg Homopolymer and Grafted Amphiphilic Peg-Palmityl on the Thermotropic Phase Behavior of 1,2-Dipalmitoyl-Sn-Glycero-3-Phosphocholine Bilayer

Abstract

Phospholipids covalently attached to polyethylene glycol (PEG-PE) are routinely used for the preparation of long-circulating liposomes. The common preparation procedure for long-circulating liposomes involves use of organic solvent. Although there is a plethora of studies describing the interaction of PEG-PE with bilayers, little is known about the effects of PEG homopolymers and single chain amphiphilic PEG on liposome structure. In the present investigation the interaction of PEG homopolymer and amphiphilic PEG-palmityl conjugate with large multilamellar liposomes composed of 1,2-dipalmitoyl-sn-glycero-phosphocholine was investigated utilizing differential scanning calorimetry. Vesicle and aggregate sizes were determined by dynamic light scattering. DSC thermograms revealed interaction of PEG homopolymer with DPPC when the two are premixed in organic solvent. The data suggest that PEG interacts with the phospholipid acyl chains deep in the bilayer. Several questions are raised regarding the suitability of the current procedure for preparation of long-circulating liposomes which utilizes organic solvent. Incorporation of only 2 mol% 5 kDa PEG-palmityl conjugate completely solubilized DPPC liposomes. Packing geometry of the lipid anchor, irrespective of the polymer molecular weight, is suggested to be the primary factor for successful grafting of hydrophilic polymers on liposomes. Pure PEG-palmityl formed self-assembled organized structures of potential use in the delivery of poorly soluble drugs.     

Liposomes of dipalmitoylphosphatidylcholine associate with natural surfactant

Unilamellar liposomes of an average diameter of 0.05 micron formed by sonication of dipalmitoylphosphatidylcholine associate in vitro with the large aggregate forms of natural surfactant. The liposomal-surfactant aggregates are stable and previously associated liposomes are not released from the aggregates by the addition of more liposomes. Radiolabeled liposomes, surfactant, and preformed liposomal-surfactant aggregates were injected at a dose of 8-10 mg lipid (about 2-times the endogenous surfactant pool size) into the airways of 3-day-old rabbits. Following airway injection, labeled phosphatidylcholine from the liposomal-surfactant aggregates were recovered in approximately equal amounts by alveolar wash and in the residual lung tissue fractions. This recovery pattern and the clearance kinetics were equivalent for 48 h after airway injection to those measured with radiolabeled surfactant alone. In contrast, following the injection of liposomes alone, labeled phosphatidylcholine from the liposomes was recovered primarily by alveolar wash at 3 and 24 h. The overall clearance of the liposomal-derived phosphatidylcholine from the lung was more rapid than was the clearance of the phosphatidylcholine from the surfactant or liposome-surfactant complexes. Liposomes can interact with surfactant in vitro, and the liposomes associated with the surfactant aggregate have a metabolic fate in vivo similar to surfactant and different from liposomes alone.     

S-layer-supported lipid membranes

Many prokaryotic organisms (archaea and bacteria) are covered by a regularly ordered surface layer (S-layer) as the outermost cell wall component. S-layers are built up of a single protein or glycoprotein species and represent the simplest biological membrane developed during evolution. Pores in S-layers are of regular size and morphology, and functional groups on the protein lattice are aligned in well-defined positions and orientations. Due to the high degree of structural regularity S-layers represent unique systems for studying the structure, morphogenesis, and function of layered supramolecular assemblies. Isolated S-layer subunits of numerous organisms are able to assemble into monomolecular arrays either in suspension, at air/water interfaces, on planar mono- and bilayer lipid films, on liposomes and on solid supports (e.g. silicon wafers). Detailed studies on composite S-layer/lipid structures have been performed with Langmuir films, freestanding bilayer lipid membranes, solid supported lipid membranes, and liposomes. Lipid molecules in planar films and liposomes interact via their head groups with defined domains on the S-layer lattice. Electrostatic interactions are the most prevalent forces. The hydrophobic chains of the lipid monolayers are almost unaffected by the attachment of the S-layer and no impact on the hydrophobic thickness of the membranes has been observed. Upon crystallization of a coherent S-layer lattice on planar and vesicular lipid membranes, an increase in molecular order is observed, which is reflected in a decrease of the membrane tension and an enhanced mobility of probe molecules within an S-layer-supported bilayer. Thus, the terminology 'semifluid membrane' has been introduced for describing S-layer-supported lipid membranes. The most important feature of composite S-layer/lipid membranes is an enhanced stability in comparison to unsupported membranes.     

Phase structure of liposome in lipid mixtures

Gas microbubbles present in ultrasound imaging contrast agents are stabilized by lipid aggregates that typically contain a mixture of lipids. In this study, the phase structure of the lipid mixtures that contained two or three lipids was investigated using three different methods: dynamic light scattering, ¹H NMR, and microfluidity measurements with fluorescence probes. Three lipids that are commonly present in imaging agents (DPPC, DPPE-PEG, and DPPA) were used. Two types of systems, two-lipid model systems and simulated imaging systems were investigated. The results show that liposomes were the dominant aggregates in all the samples studied. The polar PEG side chains from the PEGylated lipid lead to the formation of micelles and micellar aggregates in small sizes. In the ternary lipid systems, almost all the lipids were present in bilayers with micelles absent and free lipids at very low concentration. These results suggest that liposomes, not micelles, contribute to the stabilization of microbubbles in an ultrasound imaging contrast agent.     

A gentle method for the lysis of oral streptococci.

Black lipid planar membranes were prepared by incorporating polymers such as polystyrene in a membrane forming solution. The polymerincorporated planar membranes showed greater stability to applied electric fields and have longer lifertimes. Photopotentials were studied under several conditions; with bacteriorhodopsin in the planar membrane; with bacteriorhodopsin in liposomes; with bacteriorhodopsin fragments in suspension; and with bacteriorhodopsin both in the planar membrane and in liposomes. Skulachev's laboratory has reported that bacteriorhodopsin-liposomes develop potentials across a black lipid planar membrane. In our studies, because the polymer incorporated planar membranes are very stable, it was possible to obtain somewhat larger photopotentials in the presence of bacteriorhodopsin ranging between 30–500 mV. The enhancement of bacteriorhodopsin catalyzed photopotentials, found in the presence of Ca⁺⁺ (or other bivalent cations) and/or applied electric fields, appeared to result from an orientation effect of bacteriorhodopsin at the membrane interface.     

Mimicking a cytoskeleton by coupling poly(N-isopropylacrylamide) to the inner leaflet of liposomal membranes: effects of photopolymerization on vesicle shape and polymer architecture

Networks of N-isopropylacrylamide (NIPAM) copolymers, coupled to spherical phospholipid bilayers, are suitable as a model for the study of the interaction between the cytoskeleton and cellular membranes, as well as for promising new drug delivery systems with triggerable drug release properties and improved stability. In this article, we describe a simple preparation technique for liposomes from egg phosphatidyl choline (EPC) encapsulating a cross-linked NIPAMminus signTEGDM copolymer skeleton (tetraethylene glycol dimethacrylate, TEGDM) which is coupled only to the inner monolayer by a novel membrane anchor monomer. Polymerization in the lipid vesicles was initiated at the inner membrane surface by the radical initiator 2,2-diethoxy-acetophenone (DEAP) permeating through the membrane from the outside. The effects of photopolymerization and polymer formation on vesicle shape and membrane integrity were studied by transmission electron microscopy (TEM), cryo-TEM, and atomic force microscopy (AFM). Upon UV irradiation, approximately 100% of the vesicles contained a polymer gel and only occasional changes in the spherical shape of the liposomes were observed. The architecture of the polymer network inside the liposomal compartment was determined by the conditions of the photopolymerization. Composite structures of polymer hollow spheres or solid spheres, respectively, tethered to spherical membrane vesicles were produced. The increased stability of the polymer-tethered lipid bilayers against solubilization by sodium cholate, compared to pure EPC vesicles, was determined by radiolabeling the lipid membrane.     

Membrane pore formation by pentraxin proteins from Limulus, the American horseshoe crab

The pentraxins are a family of highly conserved plasma proteins of metazoans known to function in immune defence. The canonical members, C-reactive protein and serum amyloid P component, have been identified in arthropods and humans. Mammalian pentraxins are known to bind lipid bilayers, and a pentraxin representative from the American horseshoe crab, Limulus polyphemus, binds and permeabilizes mammalian erythrocytes. Both activities are Ca(2+)-dependent. Utilizing model liposomes and planar lipid bilayers, in the present study we have investigated the membrane-active properties of the three pentraxin representatives from Limulus and show that all of the Limulus pentraxins permeabilize lipid bilayers. Mechanistically, Limulus C-reactive protein forms transmembrane pores in asymmetric planar lipid bilayers that mimic the outer membrane of Gram-negative bacteria and exhibits a Ca(2+)-independent form of membrane binding that may be sufficient for pore formation.     

Molecular dynamics simulation of the evolution of hydrophobic defects in one monolayer of a phosphatidylcholine bilayer: relevance for membrane fusion mechanisms

The spontaneous formation of the phospholipid bilayer underlies the permeability barrier function of the biological membrane. Tears or defects that expose water to the acyl chains are spontaneously healed by lipid lateral diffusion. However, mechanical barriers, e.g., protein aggregates held in place, could sustain hydrophobic defects. Such defects have been postulated to occur in processes such as membrane fusion. This gives rise to a new question in bilayer structure: What do the lipids do in the absence of lipid lateral diffusion to minimize the free energy of a hydrophobic defect? As a first step to understand this rather fundamental question about bilayer structure, we performed molecular dynamic simulations of up to 10 ns of a planar bilayer from which lipids have been deleted randomly from one monolayer. In one set of simulations, approximately one-half of the lipids in the defect monolayer were restrained to form a mechanical barrier. In the second set, lipids were free to diffuse around. The question was simply whether the defects caused by removing a lipid would aggregate together, forming a large hydrophobic cavity, or whether the membrane would adjust in another way. When there are no mechanical barriers, the lipids in the defect monolayer simply spread out and thin with little effect on the other intact monolayer. In the presence of a mechanical barrier, the behavior of the lipids depends on the size of the defect. When 3 of 64 lipids are removed, the remaining lipids adjust the lower one-half of their chains, but the headgroup structure changes little and the intact monolayer is unaffected. When 6 to 12 lipids are removed, the defect monolayer thins, lipid disorder increases, and lipids from the intact monolayer move toward the defect monolayer. Whereas this is a highly simplified model of a fusion site, this engagement of the intact monolayer into the fusion defect is strikingly consistent with recent results for influenza hemagglutinin mediated fusion.     

牛胰多肽(BPP)与脂质体的相互作用——对脂质体通透性的影响

本文报导用测量脂质体内含物羧基荧光素(CF)释放的荧光方法,研究BPP对脂质体通透性的影响,结果表明去氧胆盐(DOC)促进CF的漏出,增加脂质体的通透性,BPP抑制CF的释放,降低通透性,保护膜结构.从而进一步闸明BPP具有细胞保护作用的机理.