New pH-sensitive liposomes containing phosphatidylethanolamine and a bacterial dirhamnolipid

Phosphatidylethanolamine-based pH-sensitive liposomes of various compositions have been described as efficient systems for cytoplasmic delivery of molecules into cells. Incorporation of an amphiphile of appropriate structure is needed for the stabilization and performance of these vesicles. Among the wide variety of interesting activities displayed by Pseudomonas aeruginosa dirhamnolipids (diRL), is their capacity to stabilize bilayer structures in phosphatidylethanolamine systems. In this work, X-ray scattering, dynamic light scattering, fluorescence spectroscopy and fluorescence microscopy have been used to study the structure and pH-dependent behaviour of phosphatidylethanolamine/diRL liposomes. We show that diRL, in combination with dioleoylphosphatidylethanolamine (DOPE), forms stable multilamellar and unilamellar liposomes. Acidification of DOPE/diRL vesicles leads to membrane destabilization, fusion, and release of entrapped aqueous vesicle contents. Finally, DOPE/diRL pH-sensitive liposomes act as efficient vehicles for the cytoplasmic delivery of fluorescent probes into cultured cells. It is concluded that DOPE/diRL form stable pH-sensitive liposomes, and that these liposomes are incorporated into cultured cells through the endocytic pathway, delivering its contents into the cytoplasm, which means a potential use of these liposomes for the delivery of foreign substances into living cells. Our results establish a new application of diRL as a bilayer stabilizer in phospholipid vesicles, and the use of diRL-containing pH-sensitive liposomes as delivery vehicles.     

Screening the efficiency of intracytoplasmic delivery of materials to HeLa cells by liposomes

Small anionic liposomes containing 6-carboxyfluorescein (6-CF) caused both cytoplasm and endocytotic vacuoles to fluoresce; inhibitors of endocytosis (2-deoxyglucose and sodium azide) prevented vacuoles but not cytoplasm from fluorescing. With small and large cationic liposomes, fluorescence was seen only in vacuoles and this was prevented by the two inhibitors of endocytosis. Both small fluid anionic and small solid cationic liposomes containing diphtheria toxin fragment A (DTA) killed almost all cells in a population of HeLa cells; in contrast, large cationic liposomes containing DTA killed only 25% of treated HeLa cells. DTA is thus superior to 6-CF as a marker molecule for assessing the efficiency with which liposomes deliver their contents to the cytoplasm of cells.     

Reconstitution of purified amphiphilic pig intestinal microvillus aminopeptidase. Mode of membrane insertion and morphology.

Pig intestinal microvillus aminopeptidase (EC 3.4.11.2) was reincorporated into lipid membranes by using either beta-octyl glucoside or sodium deoxycholate. The results showed that for this enzyme the deoxycholate-dialysis method was the preferable one. By using this method, microvillus aminopeptidase was inserted almost quantitatively into either phosphatidylcholine vesicles or microvillus-lipid vesicles. By proteolytic treatment of the vesicles, by probing the aminopeptidase with an inhibitory antibody and by monitoring the positioning of the anchor with the aid of [125I]iodonaphthyl azide, it was demonstrated that the catalytically active part was located outside the liposomes and the anchoring peptide(2) was associated with the membrane. Electron-microscopic observation on this model system demonstrated a dimeric symmetrical structure of aminopeptidase (dimensions about 13.5 nm X 5.5 nm) separated by a 5 nm gap from the membrane. This distance corresponds to a molecular weight of 2000-5000 for this junctional segment of the anchor connecting the intramembrane part of the anchor with the catalytically active main part of the aminopeptidase.     

Fusion of membrane vesicles bearing only the influenza hemagglutinin with erythrocytes, living cultured cells, and liposomes

Membrane vesicles, bearing only the influenza viral hemagglutinin glycoprotein, were reconstituted following solubilization of intact virions with Triton X-100. The viral hemagglutinin glycoprotein was separated from the neuraminidase glycoprotein by agarose sulfanilic acid column. The hemagglutinin glycoprotein obtained was homogenous in gel electrophoresis and devoid of any neuraminidase activity. A quantitative determination revealed that the hemolytic activity of the hemagglutinin vesicles was comparable to that of intact virions. Incubation of fluorescently labeled hemagglutinin vesicles with human erythrocyte ghosts (HEG) or with liposomes composed of phosphatidylcholine/cholesterol or phosphatidylcholine/cholesterol/gangliosides, at pH 5.0 but not at pH 7.4, resulted in fluorescence dequenching. Very little, if any, fluorescence dequenching was observed upon incubation of fluorescently labeled HA vesicles with neuraminidase or glutaraldehyde-treated HEG or with liposomes composed only of phosphatidylcholine. Hemagglutinin vesicles were rendered non-hemolytic by treatment with NH2OH or glutaraldehyde or by incubation at 85 degrees C or low pH. No fluorescence dequenching was observed following incubation of non-hemolytic hemagglutinin vesicles with HEG or liposomes. These results clearly suggest that the fluorescence dequenching observed is due to fusion between the hemagglutinin vesicles and the recipient membranes. Incubation of hemagglutinin vesicles with living cultured cells, i.e. mouse lymphoma S-49 cells, at pH 5.0 as well as at pH 7.4, also resulted in fluorescence dequenching. The fluorescence dequenching observed at pH 7.4 was inhibited by lysosomotropic agents (methylamine and ammonium chloride) as well as by EDTA and NaN3, indicating that it is due to fusion of hemagglutinin vesicles taken into the cells by endocytosis.     

Symbiotic effectiveness, rate of respiration and glutamine synthetase activity of sodium azide-resistant strains of Rhizobium leguminosarum biovar trifolii

Nitrogen fixing efficiency of sodium azide-resistant strains of Rhizobium leguminosarum bv. trifolii was studied in symbiosis with berseem clover plants in chillum jars. Rate of respiration and glutamine synthetase activity were tested in cultured cells and nodules, respectively. It was observed that shoot dry weight and percentage shoot nitrogen were maximum in plants inoculated with strains resistant to 15 μg ml⁻¹ sodium azide. Rate of respiration in cultured cells was lowest in strains resistant to 15 μg ml⁻¹ sodium azide and highest in strains resistant to 5 μg ml⁻¹ sodium azide. A negative correlation was observed between rate of respiration (in cultured cells) and shoot dry weight of host plants. Glutamine synthetase activity was maximum in nodule extracts of host plants inoculated with strains resistant to 5 and 10 μg ml⁻¹ sodium azide, whereas it was minimum for strains resistant to 15 μg ml⁻¹ sodium azide. Hence, resistance to low doses (15 μg ml⁻¹) of sodium azide, together with lower respiratory and glutamine synthetase activities, could be used as a potential method for isolating the symbiotically effective strains of Rh. leguminosarum bv. trifolii.     

Mechanism of formation of malonic dialdehyde during liposome interaction with cells

In the presence of intact Ehrlich ascite carcinoma cells and the supernatant obtained by preincubation and subsequent precipitation of cells, egg phosphatidylcholine is oxidized in liposomes to form malonic dialdehyde (MDA). Catalase and carbon dioxide markedly reduce, whereas sodium azide increases MDA accumulation during liposome incubation with the cells. EDTA, diethylthiocarbonate and alpha-tocopherol effectively inhibit, whereas ascorbate and cysteine strongly activate MDA synthesis in both cases. Superoxide dismutase has no appreciable effect on these processes. It is concluded that metal-containing catalysts and the H2O2 released by intact cells into the incubation medium induce lipid peroxidation in liposomes.     

Transbilayer movement of a fluorescent phosphatidylethanolamine analogue across the plasma membranes of cultured mammalian cells

The internalization of a fluorescent analogue of phosphatidylethanolamine following its insertion into the plasma membrane of cultured Chinese hamster fibroblasts was examined. When liposomes composed of 50 mol % 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidylethanolamine (C6-NBD-PE) and dioleoylphosphatidylcholine were incubated with monolayer cell cultures at 2 degrees C, a spontaneous transfer of the fluorescent lipid from liposomes to cells occurred. As long as the cells were kept at 2 degrees C, the fluorescent lipid remained at the plasma membrane. However, if, after removing the fluorescent liposomes, the cultures were warmed to 37 degrees C, the C6-NBD-PE was internalized and resided in the nuclear envelope, mitochondria, and Golgi apparatus in addition to the plasma membrane. Delivery of the fluorescent lipid to the Golgi apparatus could be blocked by the addition of 2-deoxyglucose plus sodium azide to the incubation medium. Evidence is presented suggesting that while delivery of the fluorescent lipid to the Golgi apparatus was mainly dependent on endocytosis, delivery to the nuclear envelope and mitochondria occurred by rapid transbilayer movement of the lipid across the plasma membrane followed by translocation of lipid monomers. Rapid transbilayer movement of C6-NBD-PE across the plasma membrane was found to be a temperature-dependent process that was blocked below 7 degrees C.     

The improved efficient method for introducing macromolecules into cells using HVJ (Sendai virus) liposomes with gangliosides

Macromolecules such as DNA and RNA can be entrapped within liposomes associated with gangliosides by reverse-phase evaporation. When these liposomes are incubated with HVJ2 (Sendai virus), they deliver their contents into cultured cells efficiently. More than 95% cells of a Ltk- cell line (thymidine kinase-deficient cells) transiently expressed thymidine kinase activity by thymidine kinase gene transfer using HVJ liposomes with gangliosides. Stable transformants could be obtained efficiently from various cell lines by use of HVJ liposomes containing the neoR gene. The neo+ transformants were obtained at frequencies of about 0.2-1.0, 0.06-0.25, and 0.06-0.1% in monolayers of L, CHO-Kl, and HeLa-S3 cells, respectively. Moreover, in Ehrlich ascites tumor cells which grow in suspension, the frequency was more than 0.01%. On introduction of plasmid pTK4 into Ltk- cells, about 0.5-1.0% TK+ transformants were obtained. Cosmid DNA containing the neoR gene (about 45 kbp) was also introduced into L cells by this method and neo+ transformants were obtained at a frequency of 0.1%. When rat liver mRNA was introduced into L cells by HVJ liposomes with gangliosides, immunoprecipitation studies showed that the L cells secreted rat albumin and some other proteins into the cultured medium. Moreover, using erythrocyte membrane vesicles containing IgM that had been incubated with HVJ empty liposomes with gangliosides, the IgM could be introduced into all the L cells.     

Liposome uptake into human colon adenocarcinoma cells in monolayer, spinner, and trypsinized cultures

The purpose of this study was to begin investigating the nature of liposome interactions with colon tumor cells. Thus, experiments were performed to study the uptake and incorporation of multilamellar and of reverse-phase evaporation liposomes of neutral charge into monolayers, suspended spinner cultures, and trypsinized cells of a human colon adenocarcinoma cell line, LS174T. The results showed that the same tumor cells cultured under each condition exhibited a distinct pattern of vesicle uptake as determined at 0, 15, 30, 60, and 120 min. In monolayer cultures of LS174T cells, the uptake of liposomes bearing [3H]actinomycin D in the lipid bilayers was linear throughout the incubation period. In contrast, in trypsinized and spinner suspension cultures, uptake of liposomes was biphasic. There was a proportional uptake of both liposome (labeled with [3H]phosphatidylcholine or [14C]cholesterol) and of actinomycin D (trace labeled with 3H) into the cells under all culture conditions, indicating quantitative delivery of the drug with the intact lipid vesicle. Although the amount of actinomycin D presented to tumor cells by the two liposomes was equivalent, reverse-phase evaporation liposomes were more effective than multilamellar vesicles in inhibiting uridine uptake. In the presence of excess liposomes (10 times the uptake studies), saturation of the tumor cell surface occurred by 120 min. However, the liposomes remained accessible to enzymatic removal for 60 min. Liposome-saturated tumor cells remained refractory to further binding of liposomes for at least 2 hr. The results thus revealed that differences in cell uptake were due to the state of the target cells and not the liposome types, or their differential leakage of labels.     

Transfer of band 3, the erythrocyte anion transporter, between phospholipid vesicles and cells

Band 3, the anion transport protein of human erythrocyte membranes, can be transferred from cells to liposomes and from liposomes back to cell membranes, retaining function and native orientation. After incubation with cells, sonicated phosphatidylcholine vesicles bind a transmembrane protein that comigrates with band 3 on sodium dodecyl sulfate-polyacrylamide gels. Like native red cell band 3, the vesicle-bound protein is cleaved by chymotrypsin into 65- and 30-kdalton fragments and is not cleaved by trypsin. The protein can be cross-linked by copper-phenanthroline oxidation either before or after transfer to vesicles; in either case, the vesicle fractions contain high molecular weight material that is dissociated into 95-kdalton species by mercaptoethanol. Band 3-vesicle complexes contain no detectable cell lipid and are specifically permeable to anions. Greater than 99% of their anion uptake can be blocked by the band 3 inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). Red cells whose band 3 function has been blocked irreversibly by DIDS or eosin maleimide regain part of their anion permeability upon incubation with band 3-vesicle complexes. Under the conditions employed, an average of one copy of functional band 3 is delivered to half of the cells, increasing by 2.3-fold the number of cells containing functional anion transporters. Incubation of pure lipid vesicles or red cell membrane buds with either normal red cells or eosin maleimide inhibited cells has no detectable effect on the cells' anion permeability.     

Introduction of macromolecules into mammalian cells by cell fusion

Proteins with molecular weights of up to 500K can be enclosed in erythrocyte ghosts by exposing the ghosts to hypotonic solution containing these proteins. The proteins can then be introduced into recipient cells by fusing the ghosts with the cells using HVJ, PEG, or influenza virus. Some applications of this method are described. By an improved method, 15 kbp DNA and IgM (900 kDa) can be entrapped in erythrocyte membranes and these are then treated with liposomes containing gangliosides and HVJ. These treated membranes containing large macromolecules fuse with almost 100% of the recipient cells used. Naked liposomes infrequently fuse with cultured cells, so introduction of their contents into cells is very inefficient. However, liposomes constituted from lipid and glycoproteins (HN and F) of HVJ (Sendai virus), by removing a nonionic detergent, fuse with cells about 200 times more efficiently than naked liposomes. Naked liposomes can fuse with specific cells, such as cells infected with subacute sclerosing panencephalitis virus or with human immunodeficiency virus. Plasmid DNA and mRNA of up to about 40 kbp can be entrapped efficiently in liposomes associated with gangliosides formed by reverse-phase evaporation, and then reacted with HVJ. The contents of the resulting liposomes with HVJ can be introduced efficiently into cultured cells in a suspended or plated state, and nearly all the cells then express the gene transiently. This procedure is also effective for obtaining stable transformants of many kinds of cultured cells.     

Chromosomal aberrations induced in human cultured cells by liposome-encapsulated deoxyribonuclease I

Experiments of incorporation of a nucleolytic enzyme into human cells cultured in vitro have been carried out with the aim of inducing structural chromosome variations. Human heteroploid cells, either as asynchronous populations or enriched in mitoses, and PHA-stimulated lymphocytes were used as recipients. We found that all these cells when exposed to pancreatic DNAase I encapsulated in liposomes, either of multilamellar (MLV) or of small unilamellar (SUV) type, show an incidence of chromosome damage higher than that induced by the enzyme free in the incubation buffer. Our results indicate that liposomes are suitable vehicles for the transfer of an exogenous nuclease into human cultured cells. The enzyme remains functionally active and interacts with nuclear DNA, giving rise to chromosome lesions.     

Conformational change and localization of calpactin I complex involved in exocytosis as revealed by quick-freeze, deep-etch electron microscopy and immunocytochemistry

Calpactin I complex, a calcium-dependent phospholipid-binding protein, promotes aggregation of chromaffin vesicles at physiological micromolar calcium ion levels. Calpactin I complex was found to be a globular molecule with a diameter of 10.7 +/- 1.7 (SD) nm on mica. When liposomes were aggregated by calpactin, quick-freeze, deep-etching revealed fine thin strands (6.5 +/- 1.9 [SD] nm long) cross-linking opposing membranes in addition to the globules on the surface of liposomes. Similar fine strands were also observed between aggregated chromaffin vesicles when they were mixed with calpactin in the presence of Ca2+ ion. In cultured chromaffin cells, similar cross-linking short strands (6-10 nm) were found between chromaffin vesicles and the plasma membrane after stimulation with acetylcholine. Plasma membranes also revealed numerous globular structures approximately 10 nm in diameter on their cytoplasmic surface. Immunoelectron microscopy on frozen ultrathin sections showed that calpactin I was closely associated with the inner face of the plasma membranes and was especially conspicuous between plasma membranes and adjacent vesicles in chromaffin cells. These in vivo and in vitro data strongly suggest that calpactin I complex changes its conformation to cross-link vesicles and the plasma membrane after stimulation of cultured chromaffin cells.     

Induction of chromosomal aberrations by restriction endonucleases encapsulated in liposomes

We used liposomes to deliver the restriction endonucleases BamHI and SmaI into human heteroploid HEp-2 cells. With this method very low concentrations of enzymes (2 units/ml) were active in the production of chromosomal aberrations. SmaI, which produces blunt-ended double-strand breaks in the DNA molecule, induces chromosomal aberrations more effectively than BamHI, which produces cohesive ends. Our results indicate that liposomes are suitable vectors for introducing restriction endonucleases into cultured human cells.     

Antibody-mediated targeting of liposomes. Binding to lymphocytes does not ensure incorporation of vesicle contents into the cells

Small sonicated lipid vesicles containing the water-souble fluorescent dye 6-carboxyfluorescein were formed from dioleoyl phosphatidylcholine and the antigenic lipid N-dinitrophenylaminocaproyl phosphatidylethanolamine. When these vesicles were incubated with trinitrophenyl-modified human lymphocytes and divalent anti-trinitrophenyl antibody, the antibody bound 5000 to 15 000 vesicles to each cell. Binding was detected by fluorescence microscopy and quantitated by fluorometry and flow microfluorometry. Binding was three times greater with F(ab')2 fragments than with the whole antibody and, as expected, was almost absent with the monovalent F(ab') fragments. It was also absent or greatly reduced, (i) with control immunoglobulin G, (ii) in the presence of excess soluble trintrophenyl hapten, or (iii) if hapten was omitted from either cells or vesicles. It was unaffected by sodium azide and 2-deoxy-D-glucose but was markedly decreased at 3 degrees C. It was not reversed by incubation at 3 degrees C with excess trinitrophenyl lysine. Self-quenching of the fluorescence of 6-carboxyfluorescein was used to distinguish between release of vesicle contents into the cells and simple binding of intact vesicles (Weinstein, J.N., Yoshikami, S., Henkart, P., Blumenthal, R. and Gagins, W.A. (1977) Science 195, 489--491). Antibody-mediated binding led to little or no increase over spontaneous background levels in the amount of vesicle contents released into the lymphocytes.     

Reconstitution of Neutral Amino Acid Transport System from Ehrlich Ascites Tumor Cells

Amino acid transport systems for alanine and leucine have been reconstituted into artificial lipid vesicles. Purified plasma membrane vesicles from Ehrlich ascites cells were dissolved in 2% sodium cholate, 1 mM dithiothreitol, 0.5 mM EDTA, a mixture which solubilized approximately 50% of the membrane protein. This solubilized protein fraction was further purified by a combination of ammonium sulfate precipitations, gel filtration, and DEAE-cellulose chromatography. A fraction containing approximately 15 Coomassie blue staining bands on sodium dodecyl sulfate gels was obtained. This material was reconstituted into liposomes, and preliminary results demonstrated transport of alanine and leucine dependent on a sodium gradient. In addition, an electrogenic gradient mediated by valinomycin-induced potassium diffusion seemed to stimulate alanine uptake further.     

Reconstitution of Neutral Amino Acid Transport Systems from Ehrlich Ascites Tumor Cells

Amino acid transport systems for alanine and leucine were reconstituted into artificial lipid vesicles. Purified plasma membrane vesicles from Ehrlich ascites cells were dissolved in 2% sodium cholate, 1mM dithiothreitol, and 0.5 mM EDTA a mixture that solubilized approximately 50% of the membrane protein. This solubilized protein fraction was further purified by a combination of ammonium sulfate precipitations, gel filtration, and DEAE-cellulose chromatography. A fraction containing approximately 15 Coomassie blue-staining bands on sodium dodecyl sulfate gels was obtained. This material was reconstituted into liposomes, and preliminary results demonstrated transport of alanine and leucine dependent on a sodium gradient. In addition, an electrogenic gradient mediated by valino-mycin-induced potassium diffusion seemed to stimulate alanine uptake further.     

Reconstitution of neutral amino acid transport system from Ehrlich ascites tumor cells.

Amino acid transport systems for alanine and leucine have been reconstituted into artificial lipid vesicles. Purified plasma membrane vesicles from Ehrlich ascites cells were dissolved in 2% sodium cholate, 1 mM dithiothreitol, 0.5 mM EDTA, a mixture which solubilized approximately 50% of the membrane protein. This solubilized protein fraction was further purified by a combination of ammonium sulfate precipitations, gel filtration, and DEAE-cellulose chromatography. A fraction containing approximately 15 Coomassie blue staining bands on sodium dodecyl sulfate gels was obtained. This material was reconstituted into liposomes, and preliminary results demonstrated transport of alanine and leucine dependent on a sodium gradient. In addition, an electrogenic gradient mediated by valinomycin-induced potassium diffusion seemed to stimulate alanine uptake further.     

Liposomal Hexadecylphosphocholine: Characterization and Effects on Adherent Tumor Cells

Abstract

We describe the preparation of small unilamellar and multilamellar vesicles from hexadecylphosphocholine, cholesterol and 1,2-dipalmitoyl-sn-glycero-phosphoglycerol in the molar ratio 4/5/1. Particle size and chemical stability of two types of liposomes, small unilamellar vesicles and lyophilized, freshly resuspended multilamellar vesicles were proved to be stable for at least 12 months. Compared to hexadecylphosphocholine in free form, liposomal hexadecylphosphocholine showed remarkably reduced hemolysis which did not change during storage. Fluorescence microscopy showed the uptake of propidium iodide containing hexadecylphosphocholine liposomes by KB and MDA-MB 231 tumor cells. Free propidium iodide was not incorporated into these cells. Although cytotoxicity seemed to be reduced in liposomal preparations, hexadecylphosphocholine liposomes still affected cultured tumor cells to a great extent. In relatively low concentrations they induced shape alteration, smoothing of the cell surface and blebbing.     

Efficiency of cytoplasmic delivery by non-cationic liposomes to cells in vitro: A confocal laser scanning microscopy study

It is necessary to understand liposomal uptake mechanisms and intracellular distribution in order to design more efficient gene (drug) carrier systems. Until now, a few studies have been carried out using confocal laser scanning microscopy (CLSM) to investigate the cellular uptake and transfection mediated with liposomes. So, by CLSM, we demonstrated that artificial virus-like envelope (AVE) vesicles labeled with rhodamine-PE (Rh-PE), carbocyanine (DiI) and carboxyfluorescein (CF) were investigated into the cytoplasm of two human cell lines, Mewo (human melanoma cell line) and HepG2 (human hepatoma cell line) cells grown in DMEM medium supplemented with different percentages (0%, 30%, and 100%) fetal calf serum (FCS). The liposome uptake was dependent on the cell line, in view that the whole process of liposomes associated with cells (uptake) is a two-step process involving binding and endocytosis. Based upon the various assays used to measure cellular uptake of liposomes, we conclude the efficacy of cytoplasmic delivery by AVE-liposomes to cells in culture.