Lipid composition of outer leaflet of chloroplast outer envelope determines topology of OEP7.

OEP7, a 6.7-kDa outer envelope protein of spinach chloroplasts inserts into the outer envelope of the organelle independent of a classical cleavable targeting signal. The insertion of OEP7 was studied to describe the determinants for association with, integration into, and orientation of the protein in the outer envelope of chloroplasts. The insertion of OEP7 into the membrane is independent of outer membrane channel proteins and can be reconstituted with the use of protein-free liposomes. In situ, the binding of OEP7 to the membrane surface is not driven by electrostatic interaction because reduction of phosphatidylglycerol or phosphatidylinositol did not reduce the association with the liposomes. The positively charged amino acids flanking the transmembrane domain at the C terminus are essential to retain the native N(in)-C(out) orientation during insertion into chloroplasts. OEP7 inserts with reversed orientation into liposomes containing the average lipid composition of the outer envelopes. The native like N(in)-C(out) orientation is achieved by reduction of the phoshpatidylglycerol concentration mimicking the composition of the outer leaflet of the outer envelope of chloroplasts. We conclude that the unique lipid composition of the outer leaflet due to lipid asymmetry of the outer envelope is essential for the correct topology of OEP7.     

Microfluidic preparation of drug-loaded PEGylated liposomes,and the impact of liposome size on tumour retention and penetration

Understanding the effect of liposome size on tendency for accumulation in tumour tissue requires preparation of defined populations of different sized particles. However, controlling the size distributions without changing the lipid composition is difficult, and differences in compositions itself modify distribution behaviour. Here, a commercial microfluidic format as well as traditional methods was used to prepare doxorubicin-loaded liposomes of different size distributions but with the same lipid composition, and drug retention, biodistribution and localization in tumour tissues were evaluated. The small (～50?nm diameter) liposomes prepared by microfluidics and large (～75?nm diameter) liposomes displayed similar drug retention in in vitro release studies, and similar biodistribution patterns in tumour-bearing mice. However, the extent of extravasation was clearly dependent on size of the liposomes, with the small liposomes showing tissue distribution beyond the vascular area compared to the large liposomes. The use of microfluidics to prepare smaller size distribution liposomes compared to sonication methods is demonstrated, and allowed preparation of different size distribution drug carriers from the same lipid composition to enable new understanding of tissue distribution in compositionally consistent materials is demonstrated.     

Liposome Elimination by Non-Phagocytic Cells of the Liver

Abstract

In this contribution we summarize our observations over a period of nearly two decennia on the role of hepatocytes in the hepatic clearance of intravenously administered liposomes. We demonstrate that, although size is an important parameter, it is not decisive in determining access of liposomes to the hepatocytes. Also lipid composition is an important parameter, including charge, rigidity and headgroup composition. The role of the fenestrated sinusoidal endothelial cells in accessibility is discussed as well as the involvement of opsonizing plasma proteins such as apolipoprotein E. Our observations led us to hypothesize at least four different mechanisms of interaction of liposomes with hepatocytes.     

Fast, efficient reconstitution of the cyclooxygenases into proteoliposomes

To study the physical and catalytic properties of purified membrane proteins, it is often necessary to reconstitute them into lipid bilayers. Here, we describe a fast efficient method for the direct incorporation of cyclooxygenase-1 and -2 (COX-1 and -2) isozymes into liposomes without loss of activity. Purified COX-1 and -2 spontaneously incorporate into large unilamellar vesicles produced from a mixture of DOPC:DOPS (7:3) that has been doped with oleic acid. When incorporation was measured by comparing cyclooxygenase activity to total phospholipid in the proteoliposomes, molar reconstitution ratios of 1000:1 (phospholipid:COX) were obtained. Electron paramagnetic resonance spectroscopic spin counting analysis of proteoliposomes formed with nitroxide spin-labeled COX-2 gave a nearly identical phospholipid:COX ratio, confirming that incorporation had no effect on enzyme activity, and demonstrating that the efficiency of protein incorporation is sufficient for EPR spectroscopic analysis. The spontaneous incorporation of cyclooxygenase into intact liposomes allows only insertion into the outer leaflet for this monotopic enzyme, an orientation confirmed by immunogold staining of the proteoliposomes. This method of reconstitution into liposomes may be generally applicable to the class of monotopic integral membrane proteins typified by the cyclooxygenase isozymes.     

Liposome composition effects on lipid mixing between cells expressing influenza virus hemagglutinin and bound liposomes

The involvement of contacting and distal lipid monolayers in different stages of protein-mediated fusion was studied for fusion mediated by influenza virus hemagglutinin. Inclusion of non-bilayer lipids in the composition of the liposomes bound to hemagglutinin-expressing cells affects fusion triggered by low pH. Lysophosphatidylcholine added to the outer membrane monolayers inhibits fusion. The same lipid added to the inner monolayer of the liposomes promotes both lipid and content mixing. In contrast to the inverted cone-shaped lysophosphatidylcholine, lipids of the opposite effective shape, oleic acid or cardiolipin with calcium, present in the inner monolayers inhibit fusion. These results along with fusion inhibition by a bipolar lipid that does not support peeling of one monolayer of the liposomal membrane from the other substantiate the hypothesis that fusion proceeds through a local hemifusion intermediate. The transition from hemifusion to the opening of an expanding fusion pore allows content mixing and greatly facilitates lipid mixing between liposomes and cells.     

Toward the assembly of a minimal divisome

The construction of an irreducible minimal cell having all essential attributes of a living system is one of the biggest challenges facing synthetic biology. One ubiquitous task accomplished by any living systems is the division of the cell envelope. Hence, the assembly of an elementary, albeit sufficient, molecular machinery that supports compartment division, is a crucial step towards the realization of self-reproducing artificial cells. Looking backward to the molecular nature of possible ancestral, supposedly more rudimentary, cell division systems may help to identify a minimal divisome. In light of a possible evolutionary pathway of division mechanisms from simple lipid vesicles toward modern life, we define two approaches for recapitulating division in primitive cells: the membrane deforming protein route and the lipid biosynthesis route. Having identified possible proteins and working mechanisms participating in membrane shape alteration, we then discuss how they could be integrated into the construction framework of a programmable minimal cell relying on gene expression inside liposomes. The protein synthesis using recombinant elements (PURE) system, a reconstituted minimal gene expression system, is conceivably the most versatile synthesis platform. As a first step towards the de novo synthesis of a divisome, we showed that the N-BAR domain protein produced from its gene could assemble onto the outer surface of liposomes and sculpt the membrane into tubular structures. We finally discuss the remaining challenges for building up a self-reproducing minimal cell, in particular the coupling of the division machinery with volume expansion and genome replication.     

Mimetic Membrane System to Carry Multiple Antigenic Proteins from Leishmania amazonensis

Liposomes have long been used as models for lipid membranes and for the reconstitution of a single or multiple proteins. Also, liposomes have adjuvant activity in vaccines against several protozoan or bacterial organisms. Thus, the main objective of the present study was to obtain a crude extract of detergent-solubilized proteins of Leishmania amazonensis amastigotes and reconstitute them into liposomes. Neutral and zwiterionic detergents were less efficient than an ionic detergent. In order to obtain efficient solubilization using only sodium dodecyl sulfate (SDS), the effects of detergent and protein concentration and incubation time were studied. The maximum of solubilized proteins was obtained instantaneously using a ratio of 0.5 mg/ml of protein to 0.1% (w/v) detergent at 4°C. Dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylserine (DPPS) and cholesterol in a weight ratio of 5:1:4 were used for protein reconstitution into liposomes using the cosolubilization method, yielding 60% of incorporation. The incorporation of multiple parasite proteins results in a vesicular diameter of proteoliposomes of about 140 nm, presenting a final lipid weight ratio for DPPC, DPPS and cholesterol of 1:1:5, with high stability. The detergent-solubilized proteins of L. amazonensis amastigotes present in the proteoliposome, when analyzed by SDS-polyacrylamide gel electrophoresis, include a wide range of parasite-incorporated proteins. BALB/c mice inoculated with these proteoliposomes were able to produce antibodies against the proteins reconstituted in DPPC:DPPS:cholesterol liposomes and were partially resistant to infection with L. amazonensis promastigotes. These results indicate that this system can be used as a possible vaccine against L. amazonensis.     

Effect of lipid compositions on gene transfer into 293 cells using Sendai F/HN-virosomes

Fusogenic liposomes that incorporate Sendai virus envelope proteins, so-called Sendai virosomes, have been developed for in vitro and in vivo genetic modification of animal cells. In this study, several different virosomes of varying lipid compositions were formulated and their in vitro gene-transfer efficiencies compared. The virosomes were prepared by quantitative reconstitution of the Sendai envelope, fusion (F) and hemagglutinin-neuraminidase (HN) proteins into liposomal vesicles. Virosomes that contained luciferase reporter genes were tested in 293 transformed human kidney cells. F/HN-virosomes that were prepared with an artificial Sendai viral envelope (ASVE-virosomes) or phosphatidylserine (PS-virosomes) exhibited an 8- or 6-fold higher gene-transfer efficiency than cationic liposomes that were made with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). F/HNvirosomes that were prepared with phosphatidic acid (PA-virosomes) instead of PS were less efficient in gene transfer than either ASVE- or PS-virosomes. In addition, the gene-transfer capability of ASVE- and PS-virosomes was maximal at a Ca2+ concentration of 510 mM. These results suggest that the incorporated lipid components significantly affect the in vitro gene transfer that is mediated by Sendai F/HN-virosomes.     

Membrane fusion activity of purified SipB, a Salmonella surface protein essential for mammalian cell invasion

An early event in Salmonella infection is the invasion of non-phagocytic intestinal epithelial cells. The pathogen is taken up by macropinocytosis, induced by contact-dependent delivery of bacterial proteins that subvert signalling pathways and promote cytoskeletal rearrangement. SipB, a Salmonella protein required for delivery and invasion, was shown to localize to the cell surface of bacteria invading mammalian target cells and to fractionate with outer membrane proteins. To investigate the properties of SipB, we purified the native full-length protein following expression in recombinant Escherichia coli. Purified SipB assembled into hexamers via an N-terminal protease-resistant domain predicted to form a trimeric coiled coil, reminiscent of viral envelope proteins that direct homotypic membrane fusion. The SipB protein integrated into both mammalian cell membranes and phospholipid vesicles without disturbing bilayer integrity, and it induced liposomal fusion that was optimal at neutral pH and influenced by membrane lipid composition. SipB directed heterotypic fusion, allowing delivery of contents from E. coli-derived liposomes into the cytosol of living mammalian cells.     

Preparation and characterization of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. II. Biochemical characterization

In the previous paper (Block, M. A., Dorne, A.-J., Joyard, J., and Douce, R. (1983) J. Biol. Chem. 258, 13273-13280), we have described a method for the separation of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. The two envelope membranes have a different weight ratio of acyl lipid to protein (2.5-3 for the outer envelope membrane and 0.8-1 for the inner envelope membrane). The two membranes also differ in their polar lipid composition. However, in order to prevent the functioning of the galactolipid:galactolipid galactosyltransferase during the course of envelope membrane separation, we have analyzed the polar lipid composition of each envelope membrane after thermolysin treatment of the intact chloroplasts. The outer envelope membrane is characterized by the presence of high amounts of phosphatidylcholine and digalactosyldiacylglycerol whereas the inner envelope membrane has a polar lipid composition almost identical with that of the thykaloids. No phosphatidylethanolamine or cardiolipin could be detected in either envelope membranes, thus demonstrating that the envelope membranes, and especially the outer membrane, do not resemble extrachloroplastic membranes. No striking differences were found in the fatty acid composition of the polar lipids from either the outer or the inner envelope membrane. The two envelope membranes also differ in their carotenoid composition. Among the different enzymatic activities associated with the chloroplast envelope, we have shown that the Mg2+-dependent ATPase, the UDP-Gal:diacylglycerol galactosyltransferase, the phosphatidic acid phosphatase, and the acyl-CoA thioesterase are associated with the inner envelope from spinach chloroplasts whereas the acyl-CoA synthetase is located on the outer envelope membrane.     

Stabilization of large multilamellar liposomes by human serum in vitro

The leakage of 5,6-carboxyfluorescein from large multilamellar liposomes prepared from dipalmitoylphosphatidylcholine (without or with cholesterol) was investigated in vitro in the presence of human serum. Below the phospholipid phase transition temperature, the rate of dye release is retarted 3–8-fold in the presence of up to 25% human serum in the incubation medium, as compared to the release in isotonic phosphate-buffered saline. This effect is significantly augmented by incorporation of 50 mol% cholesterol into the lipid bilayer. At and above the phase transition temperature, the initial rapid dye leakage in the presence of serum is followed by a slow long-term release. Incubation of the liposomes with serum is assumed to result in the association of serum proteins with the outermost lipid bilayer which in turn will lead to their stabilization, while the inner lamellae are not immediately accessible to the serum proteins. The permeability of the outer protein-rich lipid bilayer appears to be restricted, as concluded from the decreased dye release in the presence of serum. Massive leakage from multilamellar liposomes appears to be primarily due to bilayer defects occurring in the lipid transition region rather than being caused by protein-lipid interactions. The results of our in vitro experiments are discussed in terms of the potential usefulness of multilamellar liposomes as drug carriers in vivo for local and topical applications.     

Incorporation of bacterial membrane proteins into liposomes: factors influencing protein reconstitution

Meningococcal and gonococcal outer membrane proteins were reconstituted into liposomes using detergent-mediated dialysis. The detergents octyl glucopyranoside (OGP), sodium cholate and Empigen BB were compared with respect to efficiency of detergent removal and protein incorporation. The rate of OGP removal was greater than for cholate during dialysis. Isopycnic density gradient centrifugation studies showed that liposomes were not formed and hence no protein incorporation occurred during dialysis from an Empigen BB containing reconstitution mixture. Cholate-mediated reconstitution yielded proteoliposomes with only 75% of the protein associated with the vesicles whereas all of the protein was reconstituted into the lipid bilayer during OGP-mediated reconstitution. Essentially complete protein incorporation was achieved with an initial protein-to-lipid ratio of 0.01:1 (w/w) in the reconstitution mixture; however, at higher initial protein-to-lipid ratios (0.02:1) only 75% protein incorporation was achieved. Reconstituted proteoliposomes were observed as large (>300 nm), multilamellar structures using cryo-electron microscopy. Size reduction of these proteoliposomes by extrusion did not result in significant loss of protein or lipid. Extruded proteoliposomes were unilamellar vesicles with mean diameter of about 100 nm.     

ENHANCED PROTEIN LOADING INTO LIPOSOMES BY THE MULTIPLE CROSSFLOW INJECTION TECHNIQUE

ABSTRACT

Methods for encapsulation of a drug into liposomes should preferably result in a high encapsulation efficiency and a high encapsulation capacity. Our studies were focussed on the establishment of an efficient encapsulation procedure of the radical scavenging protein, rh-Cu/Zn-SOD, into liposomes with the cross flow injection method. Limitations to increase the encapsulation efficiency are caused by the enclosed aqueous volume, by the lipid concentration, the aspired vesicle size and the final ethanol concentration. Our research was performed to maximize the encapsulation following several strategies of injecting higher lipid concentrations into the aqueous phase. The one way triple technique, a sophisticated preparation procedure is presented, which enables three times higher encapsulation rates in comparison to standard procedures. Additionally, scalability studies demonstrate reproducibility independent of the preparation volume. Vesicle size distribution and encapsulation efficiency remain constant. Furthermore, special attention is paid on reproducibility of prepared liposomes, scale-up and on long term stability of the lipid vesicles.     

Liposome-mediated transfer of integral membrane glycoproteins into the plasma membrane of cultured cells

Cultured mouse 3T3 cells treated with phosphatidylserine or phosphatidylserine/phosphatidylcholine (3: 7 mole ratio) liposomes containing ortho- and paramyxovirus envelope glycoproteins become susceptible to killing by virus-specific cytotoxic T lymphocytes indicating that the liposome-derived glycoproteins have been inserted into the cellular plasma membrane. Cells incubated with liposomes of similar lipid composition containing viral antigens plus a dinitrophenylated lipid hapten were killed by both virus- and hapten-specific T lymphocytes indicating that both protein and lipid components are inserted into the plasma membrane. We consider that assimilation of liposome-derived antigens into the plasma membrane results from fusion of liposomes with the plasma membrane. Cells incubated with phosphatidylcholine liposomes containing lipid haptens and viral glycoproteins were not killed by cytotoxic lymphocytes indicating that liposomes of this composition do not fuse with the plasma membrane. Liposome-derived paramyxovirus glycoproteins inserted into the plasma membrane retain their functional activity as shown by their ability to induce cell fusion. These experiments demonstrate the feasibility of using liposomes as carriers for introducing integral membrane (glyco)proteins into the plasma membrane of cultured cells and establish a new approach for studying the role of individual (glyco)proteins in the expression of specific cell surface properties.     

Protein Determinants of SNARE-Mediated Lipid Mixing

Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE)-mediated lipid mixing can be efficiently recapitulated in vitro by the incorporation of purified vesicle membrane (-v) SNARE and target membrane (t-) SNARE proteins into separate liposome populations. Despite the strong correlation between the observed activities in this system and the known SNARE physiology, some recent works have suggested that SNARE-mediated lipid mixing may be limited to circumstances where membrane defects arise from artifactual reconstitution conditions (such as nonphysiological high-protein concentrations or unrealistically small liposome populations). Here, we show that the previously published strategies used to reconstitute SNAREs into liposomes do not significantly affect either the physical parameters of the proteoliposomes or the ability of SNAREs to drive lipid mixing in vitro. The surface density of SNARE proteins turns out to be the most critical parameter, which controls both the rate and the extent of SNARE-mediated liposome fusion. In addition, the specific activity of the t-SNARE complex is significantly influenced by expression and reconstitution protocols, such that we only observe optimal lipid mixing when the t-SNARE proteins are coexpressed before purification.     

Peculiar behaviour of rabbit thymocytes in interaction with liposomes of different compositions shown by fluorescence polarization studies,lipid analysis,and uptake of vesicle-entrapped carboxyfluorescein

In order to obtain more information on membrane phenomena occurring at the cell surface of rabbit thymocytes we have performed experiments aimed at altering the lipid composition of the plasma membrane. Thymocytes were incubated at 37°C with phospholipid vesicles of different compositions. Vesicle-cell interaction was followed by measuring the degree of fluorescence polarization and the uptake of vesicle-entrapped carboxyfluorescein. Neutral and negatively charged liposomes prepared from egg phosphatidylcholine are currently used in investigations of vesicle-cell interaction. In this report we show that these liposomes do not interact with rabbit thymocytes as is evident from unaltered lipid fluidity measured in whole cells and in isolated plasma membranes. This was confirmed by experiments with vesicle-entrapped carboxyfluorescein showing hardly any uptake of the fluorophor from neutral and negatively charged egg phosphatidylcholine liposomes. Using both techniques substantial interaction was found with positively charged egg phosphatidylcholine liposomes and with liposomes prepared from soybean lecithin which is composed of a variety of phospholipids. The results of these experiments were supported by lipid analysis of cells treated with soybean lecithin liposomes. Increase in phosphatidylcholine contents of mixed phospholipid vesicles was further shown to result in decreased vesicle-cell interaction. From measurements of the quantity of carboxyfluorescein inside cells and the total amount of cell-associated carboxyfluorescein it is concluded that adsorption plays a prominent role in interaction between liposomes and rabbit lymphocytes. The grade of maturation of lymphocytes was also found to affect vesicle-cell interaction. The more mature thymocytes took up more vesicle-entrapped carboxyfluorescein from soybean liposomes than immature thymocytes. Mesenteric lymph node cells exhibited a still stronger interaction. The role of vesicle and cell surface charge and membrane fluidity of both vesicles and cells in interaction between liposomes and rabbit thymocytes is discussed.     

Automated formation of multicomponent-encapuslating vesosomes using continuous flow microcentrifugation

Vesosomes – hierarchical assemblies consisting of membrane-bound vesicles of various scales – are potentially powerful models of cellular compartmentalization. Current methods of vesosome fabrication are labor intensive, and offer little control over the size and uniformity of the final product. In this article, we report the development of an automated vesosome formation platform using a microfluidic device and a continuous flow microcentrifuge. In the microfluidic device, water-in-oil droplets containing nanoscale vesicles in the water phase were formed using T-junction geometry, in which a lipid monolayer is formed at the oil/water interface. These water-in-oil droplets were then immediately transferred to the continuous flow microcentrifuge. When a water-in-oil droplet passed through a second lipid monolayer formed in the continuous flow microcentrifuge, a bilayer-encapsulated vesosome was created, which contained all of the contents of the aqueous phase encapsulated within the vesosome. Encapsulation of nanoscale liposomes within the outer vesosome membrane was confirmed by fluorescence microscopy. Laser diffraction analysis showed that the vesosomes we fabricated were uniform (coefficient of variation of 0.029). The yield of the continuous flow microcentrifuge is high, with over 60% of impinging water droplets being converted to vesosomes. Our system provides a fully automatable route for the generation of vesosomes encapsulating arbitrary contents. The method employed in this work is simple and can be readily applied to a variety of systems, providing a facile platform for fabricating multicomponent carriers and model cells.     

Development of a novel fusogenic viral liposome system (HVJ-liposomes) and its applications to the treatment of acquired diseases

Toward human gene therapy and gene analysis in vivo, a novel hybrid vector based on liposome has been developed for more efficient gene delivery and gene expression. The liposome was decorated with HVJ (Sendai virus) envelope fusion proteins to introduce DNA directly into the cytoplasm, and contained DNA and DNA-binding nucelar protein to enhance expression of the gene. Recently, several types of HVJ-liposomes were developed by altering the lipid components of the liposomes. HVJ-cationic liposomes increased gene delivery 100 - 800 times more efficiently in vitro than the conventional HVJ-anionic liposomes. HVJ-cationic liposomes were also more useful for gene expression in restricted portions of organs and for gene therapy of disseminated cancers. It was further discovered that the use of anionic liposomes with a virus-mimicking lipid composition (HVJ-AVE liposomes) increased transfection efficiency by several fold in vivo, especially in liver and muscle. By coupling the Epstein-Barr (EB) virus replicon apparatus to HVJ-liposomes, transgene expression was sustained in vitro and in vivo. Most animal organs were found to be suitable targets for the fusigenicviral liposome system, and numerous gene therapy strategies using this system were successful in animals.     

Development of a novel fusogenic viral liposome system (HVJ-liposomes) and its applications to the treatment of acquired diseases.

Toward human gene therapy and gene analysis in vivo, a novel hybrid vector based on liposome has been developed for more efficient gene delivery and gene expression. The liposome was decorated with HVJ (Sendal virus) envelope fusion proteins to introduce DNA directly into the cytoplasm, and contained DNA and DNA-binding nuclear protein to enhance expression of the gene. Recently, several types of HVJ-liposomes were developed by altering the lipid components of the liposomes. HVJ-cationic liposomes increased gene delivery 100-800 times more efficiently in vitro than the conventional HVJ-anionic liposomes. HVJ-cationic liposomes were also more useful for gene expression in restricted portions of organs and for gene therapy of disseminated cancers. It was further discovered that the use of anionic liposomes with a virus-mimicking lipid composition (HVJ-AVE liposomes) increased transfection efficiency by several fold in vivo, especially in liver and muscle. By coupling the Epstein-Barr (EB) virus replicon apparatus to HVJ-liposomes, transgene expression was sustained in vitro and in vivo. Most animal organs were found to be suitable targets for the fusigenic-viral liposome system, and numerous gene therapy strategies using this system were successful in animals.     

脂质体重组和脂蛋白体在植物生物膜研究中的应用

脂质体是磷脂在一定条件下在水中形成的由脂质双分子层组成的内部为水相的闭合囊泡。在推动生物膜的研究进展中,它作为模式系统起着非常重要的作用,能用于研究膜蛋白的性质和功能;膜脂和膜蛋白的相互关系;膜的电化学性质等。近年来脂质体重组技术开始引入到植物学研究领域,用于对植物膜蛋白的研究。本文简要介绍了脂质体的制备和脂酶体重组的方法及其在植物生物膜研究中的应用。