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1.
We describe the synthesis of liposomes with an artificial membrane skeleton as a model of the native cellular cytoskeleton. Similar to natural conditions, a flat polymer network is coupled to the inner membrane leaflet like a suspended ceiling via membrane-inserted anchor monomers with a spacer. The polymer is composed of DMAPMA (N-(3-N,N-dimethylaminopropyl) methacrylamide) and TEGDM (tetraethylene glycol dimethacrylate) as a linker and is coupled to the membrane anchor DOGM (1,2-distearyl-3-octaethylene glycol glycerol ether methacrylate). In the first step of the synthesis, DMAPMA and TEGDM are encapsulated into liposomes composed of egg phosphatidylcholine (EPC), and free monomers are removed by gel chromatography. At pH 10, DMAPMA adsorbs to the inner membrane surface, as demonstrated in parallel studies with lipid monolayers using a Langmuir film balance. The polymerization by UV irradiation was initiated with DEAP (2,2-diethoxyacetophenone) as the initiator and was shown to be complete after 15 min. At pH 6, polymer was desorbed from the inner membrane surface to form a lamellar structure similar to that of the cellular cytoskeleton, as shown by electron microscopy. In comparison to NIPAM (N-isopropylacrylamide), which was used as a monomer in a recent study (Stauch, O.; Uhlmann, T.; Frohlich, M.; Thomann, R.; El-Badry, M.; Kim, Y.-K.; Schubert, R. Biomacromolecules 2002, 3, 324-32), DMAPMA shows much slower membrane permeation leading to an essential restriction of the formed polymer to the liposomal interior. The DMAPMA-based composite structure stabilizes the lipid membrane against sodium cholate by a factor of 2.5 as compared to plain EPC liposomes. This is discussed in the context of the situation in the liver, where the cytoskeleton probably plays a crucial role in the stabilization of the membrane against high bile salt concentration.  相似文献   

2.
The structure of three types of liposomes (egg yolk phosphatidylcholine (EPC) without modification and EPC vesicles containing cross-linked N-isopropylacrylamide (NIPAM) networks of low and a high concentration inside the vesicles) were analyzed by static and dynamic light scattering. Upon polymerization the network was assumed to become attached to the membrane by reactive anchoring monomers. For the sample of high poly(NIPAM) content the polymer network was assumed to fill the whole space in the vesicles. The issue of the present study was to examine hard and hollow sphere behavior of the liposomes with networks of high and low poly(NIPAM) content. The theoretical scattering curves differ markedly for uniform hard and uniform hollow spheres by the presence of specific peaks. However, polydispersity washed out the peaks and led to smoothed asymptotes with fractal dimensions of df = 2 for hollow and df = 4 for hard spheres. The experimental data could efficiently be fitted with weakly polydisperse hollow spheres. No clear conclusion could be drawn from the angular dependence alone for the liposome of high poly(NIPAM) content. The two wavelengths from the HeNe and Ar lasers proved to be too long for the studied liposomes of about 100 nm in radius. However, evidence for hollow sphere behavior was found for fractionated liposomes from the ratio rho = Rg/Rh = 1.04 +/- 0.02 (theory rho = 1.00 for hollow spheres). Finally, from the molar mass and the sphere radius, an apparent density was determined. The analysis gave the expected density for the pure EPC lecithin vesicles and a poly(NIPAM) network density of 0.244 g/mL. For the liposome of low poly(NIPAM) content the network appeared to be attached to the inner surface of the lecithin shell to form a layer of about 18 nm thickness.  相似文献   

3.
The morphology and curvature of biological bilayers are determined by the packing shapes and interactions of their participant molecules. Bacteria, except photosynthetic groups, usually lack intracellular membrane organelles. Strong overexpression in Escherichia coli of a foreign monotopic glycosyltransferase (named monoglycosyldiacylglycerol synthase), synthesizing a nonbilayer-prone glucolipid, induced massive formation of membrane vesicles in the cytoplasm. Vesicle assemblies were visualized in cytoplasmic zones by fluorescence microscopy. These have a very low buoyant density, substantially different from inner membranes, with a lipid content of ≥60% (w/w). Cryo-transmission electron microscopy revealed cells to be filled with membrane vesicles of various sizes and shapes, which when released were mostly spherical (diameter ≈100 nm). The protein repertoire was similar in vesicle and inner membranes and dominated by the glycosyltransferase. Membrane polar lipid composition was similar too, including the foreign glucolipid. A related glycosyltransferase and an inactive monoglycosyldiacylglycerol synthase mutant also yielded membrane vesicles, but without glucolipid synthesis, strongly indicating that vesiculation is induced by the protein itself. The high capacity for membrane vesicle formation seems inherent in the glycosyltransferase structure, and it depends on the following: (i) lateral expansion of the inner monolayer by interface binding of many molecules; (ii) membrane expansion through stimulation of phospholipid synthesis, by electrostatic binding and sequestration of anionic lipids; (iii) bilayer bending by the packing shape of excess nonbilayer-prone phospholipid or glucolipid; and (iv) potentially also the shape or penetration profile of the glycosyltransferase binding surface. These features seem to apply to several other proteins able to achieve an analogous membrane expansion.  相似文献   

4.
Recently, we have reported the discovery of block liposomes (BLs), a new class of liquid (chain-melted) vesicles, formed in mixtures of the curvature-stabilizing hexadecavalent cationic lipid MVLBG2, the neutral lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), and water with no added salt. BLs consist of connected spheres, pears, tubes, or rods. Unlike in typical liposome systems, where spherical vesicles, tubular vesicles, and cylindrical micelles are separated on the macroscopic scale, shapes remain connected and are separated only on the nanometer scale within a single BL. Here, we report structural studies of the effect of salt and pH on the BL phase, carried out using differential interference contrast microscopy (DIC) and cryogenic transmission electron microscopy (cryo-TEM). Addition of salt screens the electrostatic interactions; in low-salt conditions, partial screening of electrostatic interactions leads to a shape transition from BLs to bilamellar vesicles, while in the high-salt regime, a shape transition from BLs to liposomes with spherical morphologies occurs. This demonstrates that strong electrostatic interactions are essential for BL formation. Understanding the control of liposome shape evolution is of high interest because such shape changes play an important role in many intracellular processes such as endocytosis, endoplasmatic reticulum-associated vesiculation, vesicle recycling and signaling.  相似文献   

5.
The work presented here demonstrates that the phenomenon of spontaneous vesiculation is not restricted to charged lipids and lipid mixtures, but occurs also in isoelectric phospholipid mixtures consisting of egg phosphatidylcholine (EPC) and egg lysophosphatidylcholine (lyso-EPC). 1H high-resolution NMR and freeze-fracture electron microscopy have been used to characterize the mixed EPC/lyso EPC dispersions in excess H2O. The predominant phase in these mixed phospholipid dispersions is smectic (lamellar) at least up to approximately 70% lysophosphatidylcholine. The type of phospholipid aggregate formed in excess H2O depends on the mole ratio diacyl to monoacyl phosphatidylcholine. The dispersive (lytic) action of lysophosphatidylcholine on phosphatidylcholine bilayers becomes effective at lysophospholipid contents in excess of approximately 10%. Large multilamellar liposomes are disrupted and replaced by smaller particles, mainly unilamellar vesicles. Between 30 and 70% lysophosphatidylcholine a significant proportion of the total phospholipid is present as small unilamellar vesicles (SUV) of a diameter of 23 nm (range: 20-70 nm). At even higher lysophosphatidylcholine contents the fraction of phospholipid present as small mixed micelles with a diameter smaller than about 14 nm grows at the expense of the vesicular structures. There is a second effect of increasing the quantity of lysophosphatidylcholine in phosphatidylcholine bilayers: the presence of lysophosphatidylcholine in excess of 10% renders the phospholipid bilayer more permeable to ions as compared to pure phosphatidylcholine bilayers. The key factor in inducing spontaneous vesiculation is probably not the charge but the wedge-like shape of the lysophospholipid molecule. The molecular shape may give rise to an asymmetric distribution of lysophosphatidylcholine between the two halves of the bilayer, thus stabilizing highly curved bilayers as present in SUV.  相似文献   

6.
The method of atomic force microscopy has been used to investigate the morphology of mica-supported bilayer lipid membranes and stability of their complexes with a cationic polymer, poly-(N-ethyl-4-vinylpyridinium bromide). Lipid bilayers with a minimum of defects were obtained by the fusion of monolamellar neutral or mixed anionic bilayer vesicles (liposomes) on the mica surface, followed by excessive solvent removal by means of rapid rotation of a plate in horizontal plane (spin-coating). It has been shown that the cationic polymer does not interact with the bilayers, where the outer leaflet (i.e., the monolayer exposed to the surrounding aqueous solution) is made of an electroneutral phosphatidylcholine (PC). At the same time, the polymer irreversibly binds to the bilayer containing an anionic lipid.  相似文献   

7.
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.  相似文献   

8.
The pharmacological activity of several amphiphilic drugs is often related to their ability to interact with biological membranes. Propranolol is an efficient multidrug resistance (MDR) modulator; it is a nonselective β‐blocker and is thought to reduce hypertension by decreasing the cardiac frequency and thus blood pressure. It is used in drug delivery studies in order to treat systemic hypertension. We are interested in the interaction of propranolol with artificial membranes, as liposomes of controllable size are used as biocompatible and protective structures to encapsulate labile molecules, such as proteins, nucleic acids or drugs, for pharmaceutical, cosmetic or chemical applications. We present here a study of the interaction of propranolol, a cationic surfactant, with pure egg phosphatidylcholine (EPC) vesicles. The gradual transition from liposome to micelle of EPC vesicles in the presence of propranolol was monitored by time‐resolved electron cryo‐microscopy (cryo‐EM) under different experimental conditions. The liposome–drug interaction was studied with varying drug/lipid (D/L) ratios and different stages were captured by direct thin‐film vitrification. The time‐series cryo‐EM data clearly illustrate the mechanism of action of propranolol on the liposome structure: the drug disrupts the lipid bilayer by perturbing the local organization of the phospholipids. This is followed by the formation of thread‐like micelles, also called worm‐like micelles (WLM), and ends with the formation of spherical (globular) micelles. The overall reaction is slow, with the process taking almost two hours to be completed. The effect of a monovalent salt was also investigated by repeating the lipid–surfactant interaction experiments in the presence of KCl as an additive to the lipid/drug suspension. When KCl was added in the presence of propranolol the overall reaction was the same but with slower kinetics, suggesting that this monovalent salt affects the general lipid‐to‐micelle transition by stabilizing the membrane, presumably by binding to the carbonyl chains of the phosphatidylcholine.  相似文献   

9.
Lipid bilayer vesicles (liposomes) with and without glycoprotein incorporated into the membranes were tested for stability during freezing and thawing, in presence and absence of the cryoprotective agents (CPA) glycerol and dimethyl sulfoxide. Changes in turbidity and loss of energy transfer between fluorescent probes present in the bilayers were used to estimate membrane integrity.Freezing caused a 30 to 40% destruction of protein-free liposomes, in absence of CPA. CPA at 10 to 20% concentration prevented such losses, but at higher concentrations destabilized liposomes even without freezing. Protein-containing liposomes suffered no loss on freezing in absence or presence of CPA at moderate concentrations.Lowering of the storage temperature of frozen samples within the range of ?5 to ?27 °C increased the freeze damage. Slower rates of cooling and warming caused a slightly greater loss.The results are interpreted in terms of the liquid mosaic model for lipid bilayers. CPA at higher concentrations destabilize bilayers by dissolving phospholipids. At moderate concentrations, however, they prevent the damaging effect of dehydration of the lipid on freezing. Proteins appear to stabilize bilayers by providing increased hydration at the membrane surface, and by additional hydrophobic binding in the membrane interior.  相似文献   

10.
The structural effects of Hg(II) ions on the erythrocyte membrane were studied through the interactions of HgCl2 with human erythrocytes and their isolated resealed membranes. Studies were carried out by scanning electron microscopy and fluorescence spectroscopy, respectively. Hg(II) induced shape changes in erythrocytes, which took the form of echinocytes and stomatocytes. This finding means that Hg(II) locates in both the outer and inner monolayers of the erythrocyte membrane. Fluorescence spectroscopy results indicate strong interactions of Hg(II) ions with phospholipid amino groups, which also affected the packing of the lipid acyl chains at the deep hydrophobic core of the membrane. HgCl2 also interacted with bilayers of dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine, representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively. X-ray diffraction indicated that Hg(II) ions induced molecular disorder to both phospholipid bilayers, while fluorescence spectroscopy of dimyristoylphosphatidylcholine large unilamellar vesicles confirmed the interaction of Hg(II) ions with the lipid polar head groups. All these findings point to the important role of the phospholipid bilayers in the interaction of Hg(II) on cell membranes.  相似文献   

11.
The effect of synthetic polycations, polyallylamine, and polyethylenimine, on liposomes containing phosphatidylserine was investigated along with that of polylysine and divalent cations. The addition of polycations caused aggregation of sonicated vesicles composed of phosphatidylserine and phosphatidylcholine (molar ratio 1:4) as determined by measuring the turbidity changes. Liposomal turbidity increased 10 times compared with that of control liposomes at charge ratios of polymer/vesicle from 0.23 (polylysine) to 2.5 (linear polyethylenimine), while the turbidity was unchanged by the addition of Ca2+ or Mg2+ at charge ratios up to 500. These polycations also induced intermixing of liposomal membranes as indicated by resonance energy transfer between fluorescent lipids incorporated in lipid bilayers, without inducing drastic permeability changes as determined from the calcein release. Fifty percent intermixing of liposomes (0.05 mM as lipid concentration) was induced by these polycations at charge ratios of around 1.0. However, the highest resonance energy transfer was produced by the addition of polyallylamine, which caused multicycles of membrane intermixing between vesicles. Polycation-induced membrane intermixing and permeability changes of phosphatidylserine liposomes were also investigated. At charge ratios of around 1.0, these polymers caused resonance energy transfer of fluorescent lipids incorporated in separate vesicles; however, polyallylamine and branched polyethylenimine also caused permeability increases of liposomal membranes. Membrane intermixing and permeability changes of phosphatidylserine vesicles induced by polyallylamine were dependent on the polymer/vesicle charge ratio, and were different from those induced by Ca2+ since the latter caused half-maximal membrane intermixing or permeability change of phosphatidylserine vesicles at about 1 mM at the liposomal concentrations investigated.  相似文献   

12.
N Oku  S Shibamoto  F Ito  H Gondo  M Nango 《Biochemistry》1987,26(25):8145-8150
For the purpose of cytoplasmic delivery of aqueous content in liposomes through endosomes, we synthesized a pH-sensitive polymer, cetylacetyl(imidazol-4-ylmethyl)polyethylenimine (CAIPEI), which generates polycations at acidic pH. CAIPEI in its aqueous phase caused aggregation of sonicated vesicles composed of phosphatidylserine (PS) and phosphatidylcholine (PC) (molar ratio 1:4) when the pH of the solution was lowered. The polymer also induced membrane intermixing as measured by resonance energy transfer between vesicles containing N-(7-nitro-2,1,3-benz[d]oxadiazol-4-yl)phosphatidylethanolamine and those containing N-Rhodamine phosphatidylethanolamine at pH 4-5, while the addition of CAIPEI caused neither aggregation of PC vesicles nor the intermixing of liposomal membranes between PC and PC/PS vesicles at any pH. The CAIPEI-induced membrane intermixing was dependent on the polymer/vesicle ratio rather than on the polymer concentration. Then the polymer was incorporated into the bilayers of PC vesicles. These CAIPEI vesicles also caused membrane intermixing with liposomes containing PS under acidic conditions. The reconstituted CAIPEI did not reduce the trapping efficiency of vesicles or increase their permeability to glucose even at low pH. The vesicles caused the low pH induced aggregation and membrane intermixing with other negatively charged liposomes containing phosphatidic acid or phosphatidylglycerol. These results suggest that the protonation of the polymer at acidic pH endows the CAIPEI vesicles with the activity to fuse with negatively charged liposomes.  相似文献   

13.
We show that the three core histones H2A, H3 and H4 can transverse lipid bilayers of large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). In contrast, the histone H2B, although able to bind to the liposomes, fails to penetrate the unilamellar and the multilamellar vesicles. Translocation across the lipid bilayer was determined using biotin-labeled histones and an ELISA-based system. Following incubation with the liposomes, external membrane-bound biotin molecules were neutralized by the addition of avidin. Penetrating biotin-histone conjugates were exposed by Triton treatment of the neutralized liposomes. The intraliposomal biotin-histone conjugates, in contrast to those attached only to the external surface, were attached to the detergent lysed lipid molecules. Thus, biotinylated histone molecules that were exposed only following detergent treatment of the liposomes were considered to be located at the inner leaflet of the lipid bilayers. The penetrating histone molecules failed to mediate translocation of BSA molecules covalently attached to them. Translocation of the core histones, including H2B, was also observed across mycoplasma cell membranes. The extent of this translocation was inversely related to the degree of membrane cholesterol. The addition of cholesterol also reduced the extent of histone penetration into the MLVs. Although able to bind biotinylated histones, human erythrocytes, erythrocyte ghosts and Escherichia coli cells were impermeable to them. Based on the present and previous data histones appear to be characterized by the same features that characterize cell penetrating peptides and proteins (CPPs).  相似文献   

14.
Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters <100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter <100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes <100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.  相似文献   

15.
Reaction of the melanotropin hormone analogs [Nle(4),D-Phe(7)]-alpha-MSH and [Nle(4),D-Phe(7)]-alpha-MSH(4-10), which were extended at their N-terminus by a thiol-functionalized spacer arm, with preformed liposomes containing thiol-reactive (phospho)lipid derivatives resulted in the aggregation of the vesicles and in a partial leakage of their inner contents. This aggregation/leakage effect, which was only observed when the peptides were covalently conjugated to the surface of the liposomes, was correlated with the fusion of the vesicles as demonstrated by the observed decrease in resonance energy transfer between probes in a membrane lipid mixing assay. A limited fusion was confirmed by monitoring the mixing of the liposome inner contents (formation of 1-aminonaphthalene-3,6,8-trisulfonic acid/p-xylene bis(pyridinium bromide) complex). The membrane-active properties of the peptides could be correlated with changes in the fluorescence emission spectra of their tryptophan residue, which suggested that after their covalent binding to the outer surface of the liposomes they can partition within the core of the bilayers. A blue shift of 10 nm was observed for [Nle(4),D-Phe(7)]-alpha-MSH which was correlated with an increase in fluorescence anisotropy and with changes in the accessibility of the coupled peptide as assessed by the quenching of fluorescence of its tryptophan residue by iodide (Stern-Volmer plots). These results should be related to the previously described capacity of alpha-MSH, and analogs, to interact with membranes and with the favored conformation of these peptides which, via a beta-turn, segregate their central hydrophobic residues into a domain that could insert into membranes and, as shown here, trigger their destabilization.  相似文献   

16.
The dependence of geometric structure and thermal stability of liposomes on their component phospholipid molecules and distribution of molecules in the inner and the outer layers of the liposome is investigated by conducting molecular simulations in explicit water for the eight types of liposomes constructed from different phospholipids. Using molecular mechanics structure-relaxation based on the coarse grained (CG) model, stable structures of the solvated liposomes are obtained. In addition, the molecular dynamics (MD) simulations based on the CG model are carried out at 310 and 360 K for elucidating the change in structure of the solvated liposomes. The MD simulations reveal that liposomes having the same number of lipids (SNL) in both the inner and the outer layers keep their spherical structures even at 360 K. In particular, the SNLs composed of palmitoyloleoyl-phosphatidyl-ethanolamine1 or dimyristoylglycero-phosphatidyl-choline lipid exhibit a compact spherical shape. In contrast, liposomes having the same density of lipids in the inner and the outer layers cannot keep their spherical shapes at 360 K. The obtained results contribute toward developing novel liposomes with enhanced thermal stability.  相似文献   

17.
The effect of increasing concentrations of lipid X (2,3-bis(3-hydroxymyristoyl)-alpha-D-glucosamine 1-phosphate) on the phase behaviour of EPC (egg phosphatidylcholine) and EPE (egg phosphatidylethanolamine) is studied at a pH greater than or equal to 7 where lipid X carries one to two negative charges. Small amounts of lipid X (molar ratio approximately 0.01) induce continuous swelling of EPC and EPE bilayers and consequently the formation of large unilamellar vesicles in excess water. In many respects, the effect of lipid X on EPC and EPE bilayers is similar to that of phosphatidic acid. However, lipid X/EPC mixtures form micelles in excess lipid X whereas mixtures of phosphatidic acid/EPC vesiculate at all ratios. The same is true for lipid X/EPE mixtures. Small unilamellar vesicles of an average diameter of 40 nm form spontaneously upon dispersion of a dry lipid X/EPE film (molar ratio = 10). Unsonicated dispersions of lipid X/EPC (molar ratio = 1) are subjected to pH-jump treatment which involves raising of the pH to 11-12 and subsequent lowering of the pH to between 7.5 and 8.5. Such a treatment has little effect on the vesicle size and size distribution as compared to a control dispersion at pH 8.2. The mean size is determined to be 92 +/- 60 nm. Electron micrographs of freeze-fractured samples of lipid X/EPC (molar ratio = 1) reveal the presence of mainly micelles at pH 12. Upon lowering the pH to neutrality these micelles become unstable and aggregate/fuse rapidly to unilamellar vesicles (average diameter 95 +/- 40 nm). Sonication of equimolar mixtures of lipid X and EPC at pH 7 yields small unilamellar vesicles of a diameter of 20-25 nm as well as mixed micelles of a size between 15 and 17 nm. This behaviour is again different from that of mixed EPC/phosphatidic acid dispersions which form small unilamellar vesicles. The presence of lipid X in such mixtures does not prevent the aggregation/fusion to larger vesicles during freezing of the dispersion. As with pure EPC bilayers, stabilization is, however, achieved in the presence of 10% sucrose. This indicates that the covalently bonded glucosamine group of lipid X cannot substitute water of hydration in neighbouring EPC molecules.  相似文献   

18.
Peptide AS-48 induces ion permeation, which is accompanied by the collapse of the cytoplasmic membrane potential, in sensitive bacteria. Active transport by cytoplasmic membrane vesicles is also impaired by AS-48. At low concentrations, this peptide also causes permeability of liposomes to low-molecular-weight compounds without a requirement for a membrane potential. Higher antibiotic concentrations induce severe disorganization, which is visualized under electron microscopy as aggregation and formation of multilamellar structures. Electrical measurements suggest that AS-48 can form channels in lipid bilayers.  相似文献   

19.
20.
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.  相似文献   

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