Binding of a pleurotolysin ortholog from Pleurotus eryngii to sphingomyelin and cholesterol-rich membrane domains

A mixture of sphingomyelin (SM) and cholesterol (Chol) exhibits a characteristic lipid raft domain of the cell membranes that provides a platform to which various signal molecules as well as virus and bacterial proteins are recruited. Several proteins capable of specifically binding either SM or Chol have been reported. However, proteins that selectively bind to SM/Chol mixtures are less well characterized. In our screening for proteins specifically binding to SM/Chol liposomes, we identified a novel ortholog of Pleurotus ostreatus, pleurotolysin (Ply)A, from the extract of edible mushroom Pleurotus eryngii, named PlyA2. Enhanced green fluorescent protein (EGFP)-conjugated PlyA2 bound to SM/Chol but not to phosphatidylcholine/Chol liposomes. Cell surface labeling of PlyA2-EGFP was abolished after sphingomyelinase as well as methyl-β-cyclodextrin treatment, removing SM and Chol, respectively, indicating that PlyA2-EGFP specifically binds cell surface SM/Chol rafts. Tryptophan to alanine point mutation of PlyA2 revealed the importance of C-terminal tryptophan residues for SM/Chol binding. Our results indicate that PlyA2-EGFP is a novel protein probe to label SM/Chol lipid domains both in cell and model membranes.     

Effect of reconstituted discoidal high-density lipoproteins on lipid mobilization in RAW 264.7 and CHOK1 cells

Reconstituted discoidal high‐density lipoproteins (rHDL) resemble nascent HDL, which are formed at the early reverse cholesterol transport steps, and constitute the initial cholesterol (Chol) acceptors from cell membranes. We have used different sized rHDL containing or not Chol, to test their abilities to promote cholesterol and phospholipid efflux from two different cell lines: Raw 264.7 macrophages and CHOK1 cells. All rHDL and lipid‐free apolipoprotein A‐I (apoA‐I) were found to be bound to CHO and RAW cells. In RAW cells, a positive correlation between cellular binding and Chol removal was found for 78 and 96 Å rHDL. Chol‐free rHDL were more effective than Chol‐containing ones in binding to RAW cells and promoting Chol removal. These results were more evident in the 96 Å rHDL. On the other hand, rHDL binding to CHO cells was relatively independent of disc size and Chol content. In spite of the fact that apoA‐I and rHDL promoted Chol efflux from both cellular lines, only in CHOK1 cells this result was also associated to decrease Chol esterification. Among choline‐containing phospholipids, only phosphatidylcholine (PC) (but not sphingomyelin) was detected to be effuxed from both cellular lines. With the only exception of Chol‐free 96 Å discs, the other rHDL as well as apoA‐I promoted PC efflux from RAW cells. Chol‐containing rHDL were more active than Chol‐free ones of comparable size to promote PC efflux from RAW macrophages. Regarding CHO cells, only apoA‐I and Chol‐free 78 Å rHDL were active enough to remove PC. J. Cell. Biochem. 113: 1208–1216, 2012. © 2011 Wiley Periodicals, Inc.     

Ethanol-based proliposome delivery systems of paclitaxel for in vitro application against brain cancer cells

In this study the anticancer activity of paclitaxel-loaded nano-liposomes on glioma cell lines was investigated. Soya phosphatidylcholine:cholesterol (SPC:Chol), hydrogenated soya phosphatidylcholine:cholesterol (HSPC:Chol) or dipalmitoylphosphatidylcholine:cholesterol (DPPC:Chol) in 1:1?mole ratio were used to prepare ethanol-based proliposomes. Following hydration of proliposomes, the size of resulting vesicles was subsequently reduced to nanometer scale via probe-sonication. The resulting formulations were characterized in terms of size, zeta potential and morphology of the vesicles, and entrapment efficiency of paclitaxel (PX) as well as the final pH of the preparations. DPPC-liposomes entrapped 35–92% of PX compared to 27–74% and 25–60% entrapped by liposomes made from SPC and HSPC formulations respectively, depending on drug concentration. The entrapment efficiency of liposomes was dependent on the lipid bilayer properties and ability of PX to modify surface charge of the vesicles. In vitro cytotoxicity studies revealed that PX-liposome formulations were more selective at inhibiting the malignant cells. The cytotoxicity of PX-liposomes was dependent on their drug-entrapment efficiency. This study has shown PX-liposomes generated from proliposomes have selective activity against glioma cell lines, and the synthetic DPPC phospholipid was most suitable for maximized drug entrapment and highest activity against the malignant cells in vitro.     

Preparation of long-circulating immunoliposomes using PEG-cholesterol conjugates: effect of the spacer arm between PEG and cholesterol on liposomal characteristics.

Poly(ethylene glycol)-coated liposomes were prepared with two new synthesised pegylated cholesterol (Chol) derivatives linked via carbamate bond. Poly(ethylene glycol) (PEG) was directly linked to Chol (PEG-Chol) or through a space arm of diaminebutane (PEG-L-Chol). In buffer, the physicochemical properties of PC/Chol liposomes (2/1, molar ratio) containing up to 10 mol% of pegylated Chol derivatives did not change significantly and the PEG layer at liposome surface inhibited the agglutination of biotin-liposomes induced by streptavidin. On the other hand, in serum, PEG-L-Chol seemed to reduce the interactions of liposomes with serum proteins, much more than PEG-Chol. The low steric hindrance of PEG-Chol derivative may be due to the slow conformational transition rate of the polymer, since PEG may be deeper located in the membrane. The coupling efficiency of the ligand to the functionalised amino group at the polymer end was also affected, but, its antigen-binding activity was preserved. The basic physical-chemical characteristics studied in this work are relevant to assess the application of pegylated Chol liposomes as drug delivery systems.     

Cholesterol efflux from bovine sperm. I. Induction of the acrosome reaction with lysophosphatidylcholine after reducing sperm cholesterol

Methods were developed to quantitatively reduce the cholesterol (Chol)/phospholipid (PL) ratio of bovine sperm and to determine the effectiveness of this treatment in capacitating sperm. Washed sperm (2 × 10⁸) were incubated in 1.0 ml of modified Tyrode's solution (TS) containing unilamellar liposomes of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and [¹⁴C]-Chol (35:35:30 molar ratio, 300 nmol total PL). [³H]-triolein was included as a nonexchangable marker. After 90 min at 39°C, a 13% net exchange of [¹⁴C]-Chol from liposomes to sperm was observed (n = 4), and sperm motility was 80%. Sperm were then washed and 50 × 10⁶ sperm were incubated as before with PC/PE liposomes containing no Chol. After 90 min, sperm were separated from liposomes by centrifugation. Measurement of [¹⁴C]-Chol in the liposomes (supernatant) and parallel gas chromatographic analysis of extracted, saponified liposomes (n = 4) indicated that 30% of sperm Chol was removed by this procedure. Chol efflux decreased percent motile sperm by less than 10% but reduced sperm velocity by more than 50%. Sperm incubated with no liposomes (control), with liposomes containing Chol ( + Chol), and with Chol-free liposomes (—Chol) were washed and resuspended in TS with 0.2% BSA and 30 μg lysophosphatidylcholine (LPC)/mg bovine serum albumin (BSA). Percent sperm undergoing the acrosome reaction (AR) upon incubation with LPC-BSA was used as a measure of sperm capacitation. After 60 min of exposure to LPC-BSA at 39°C, the mean (± SE) percent motile sperm for control, +Chol, and —Chol treatments was 57.0 ± 4.9, 60.0 ± 4.7, and 57.0 ± 6.8, respectively. Corresponding values for percent AR were 14.0 ± 3.4, 20.3 ± 4.4, and 39.7 ± 1.2. These results suggest that loss of Chol from bovine sperm may be an early step in sperm capacitation in this species.     

Potential effect of cationic liposomes on interactions with oral bacterial cells and biofilms

Context: Although oral infectious diseases have been attributed to bacteria, drug treatments remain ineffective because bacteria and their products exist as biofilms. Cationic liposomes have been suggested to electrostatically interact with the negative charge on the bacterial surface, thereby improving the effects of conventional drug therapies. However, the electrostatic interaction between oral bacteria and cationic liposomes has not yet been examined in detail.

Objective: The aim of the present study was to examine the behavior of cationic liposomes and Streptococcus mutans in planktonic cells and biofilms.

Materials and methods: Liposomes with or without cationic lipid were prepared using a reverse-phase evaporation method. The zeta potentials of conventional liposomes (without cationic lipid) and cationic liposomes were ?13 and 8?mV, respectively, and both had a mean particle size of approximately 180?nm. We first assessed the interaction between liposomes and planktonic bacterial cells with a flow cytometer. We then used a surface plasmon resonance method to examine the binding of liposomes to biofilms. We confirmed the binding behavior of liposomes with biofilms using confocal laser scanning microscopy.

Results: The interactions between cationic liposomes and S. mutans cells and biofilms were stronger than those of conventional liposomes. Microscopic observations revealed that many cationic liposomes interacted with the bacterial mass and penetrated the deep layers of biofilms.

Discussion and conclusion: In this study, we demonstrated that cationic liposomes had higher affinity not only to oral bacterial cells, but also biofilms than conventional liposomes. This electrostatic interaction may be useful as a potential drug delivery system to biofilms.     

Cholesterol efflux from bovine sperm: II. Effect of reducing sperm cholesterol on penetration of zona-free hamster and in vitro matured bovine ova

Several reports have indicated that sperm capacitation includes loss of membrane cholesterol (Chol) with a concomitant decrease in the Chol-to-phospholipid (PL) ratio. Methods were developed for quantifiable removal of bovine sperm Chol, which predisposed sperm to induction of the acrosome reaction upon addition of lysophosphatidylcholine (LPC). The objective of this study was to evaluate the effect of Chol removal from bovine sperm on penetration of zona-free hamster and intact bovine ova in vitro. Washed ejaculated bovine sperm were incubated (2 h, 39°C) in a modified Tyrode's solution (TALP) containing (1) Chol-free liposomes (—Chol, 50 × 10⁶ sperm and 600 nmol phospholipid/ml); (2) liposomes containing 30 mol% Chol (+ Chol, 2 × 10⁸ sperm and 300 nmol total lipid/ml); or (3) no liposomes (Control). We have previously shown that net Chol efflux from sperm is 31% of the total sperm Chol with —Chol liposomes and less than 1% with control media. Sperm were then washed twice and challenged with LPC bound to bovine serum albumin (BSA) using celite as a carrier. Treated sperm (25 × 10⁶) were incubated immediately with either zona-free hamster ova (HO) or in vitro matured bovine ova (BO) in 50-μl droplets of TALP under medical fluid in an atmosphere of 5% CO₂ in air (3 h, 39°C). Ova were fixed in ethanol:acetic acid, stained with 1% orcein, and examined. Percent penetration (%P) of HO (X ± SEM) for 30 and 40 μg of LPC/mg BSA was 59.4 ± 5.3 and 82.9 ± 5.4; 38.5 ± 5.6 and 52.3 ± 4.7; and 16.0 ± 4.6; and 23.2 ± 5.6 for —Chol, Control, and + Chol treatments, respectively (n = 3). %P of BO (X ± SEM) for 30, 35, and 40 μg of LPC/mg BSA was 43.3 ± 5.4, 70.7 ± 7.5, and 81.5 ± 5.1 for —Chol and 16.4 ± 6.9, 36.2 ± 6.9, and 44.2 ± 8.6 for Control treatments, respectively (n = 3). In a second set of experiments %P of BO (X ± SEM) was 63.6 ± 6.8, 31.8 ± 4.9, and 10.5 ± 3.4 for —Chol, Control, and +Chol treatments, respectively, when 40 μg LPC/mg BSA was added (n = 2). %P and the number of sperm per fertilized ovum were consistently higher for the —Chol treatment for both HO and BO (P < .01). These results demonstrate that Chol removal from bovine sperm facilitates penetration of ova in vitro suggesting a potential role in bovine sperm capacitation.     

Topical delivery and in vivo antileishmanial activity of paromomycin-loaded liposomes for treatment of cutaneous leishmaniasis

The present study aimed to evaluate the potential of liposomes loaded with paromomycin (PA), an aminoglycoside antibiotic associated with poor skin penetration, for the topical treatment of cutaneous leishmaniasis (CL). Fluid liposomes were prepared and characterized for particle size, zeta potential, and drug entrapment. Permeation studies were performed with two in vitro models: intact and stripped skin. The antileishmanial activity of free and liposomal PA was evaluated in BALB/c mice infected by Leishmania (L.) major. Drug entrapment ranged from 10 to 14%, and the type of vesicle had little influence on this parameter. Particle size and polydispersity index of the vesicles composed by phosphatidylcholine (PC) and PC/cholesterol (Chol) ranged from of 516 to 362?nm and 0.7 to 0.4, respectively. PA permeation across intact skin was low, regardless of the formulation tested, while drug penetration into skin (percent of the applied dose) from PC (7.2?±?0.2%) and PC/Chol (4.8?±?0.2%) liposomes was higher than solution (1.9?±?0.1%). PA-loaded liposomes enhanced in vitro drug permeation across stripped skin and improved the in vivo antileishmanial activity in experimentally infected mice. Our findings suggest that the liposomes represent a promising alternative for the topical treatment of CL using PA.     

Biodistribution and immunotargetability of ganglioside-stabilized dioleoylphosphatidylethanolamine liposomes.

The biodistribution and immunotargetability of liposomes composed primarily of dioleoylphosphatidylethanolamine (DOPE) or dioleoylphosphatidylcholine (DOPC) in mice injected via the tail vein were examined and compared. The ganglioside GM1 (7 mol%) prolonged the circulation of DOPC but not DOPE liposomes. Gangliosides GD1a and GT1b (7 mol%) also increased the amount of DOPC liposomes remaining in circulation, and to a similar extent as GM1, at 15 min post injection. However, these liposomes were cleared from the circulation by 2.5 h. Monoclonal antibody 34A, which specifically binds to a surface glycoprotein (gp 112) of the pulmonary endothelial cell surface, was coupled with N-glutarylphosphatidylethanolamine and incorporated into liposomes by a dialysis procedure. These 34A-immunoliposomes, composed of DOPE and GM1 (7 mol%), but not the antibody-free liposomes, accumulated efficiently (approximately 24% of the injected dose) in the lungs. Inclusion of cholesterol (31 mol%) enhanced the lung accumulation of both DOPE/GM1 immunoliposomes and DOPC/GM1 immunoliposomes to 33% and 51% of the injected dose, respectively. The transient increase in DOPC liposome circulation provided by GD1a and GT1b was sufficient to enhance DOPC immunoliposome binding, where 44% and 43% of the injected dose of DOPC/Chol/GD1a and DOPC/Chol/GT1b immunoliposomes accumulated in lung at 15 min after injection, respectively. In general, cholesterol-containing DOPC liposomes were more targetable than DOPE liposomes, and the degree to which these liposomes avoid RES uptake influences their targetability. The results presented here are relevant to the design of targetable drug delivery vehicles.     

Effect of Cholesterol on the Properties of Spray-Dried Lysozyme-Loaded Liposomal Powders

The influence of cholesterol (Chol) in the liposomal bilayer on the properties of inhalable protein-loaded liposomal powders prepared by spray-drying technique was investigated. Lysozyme (LSZ) was used as a model protein. Feed solution for spray drying was prepared by direct mixing of aqueous solution of LSZ with mannitol solution and empty liposome dispersions composed of hydrogenated phosphatidylcholine and Chol at various molar ratios. The spray-dried powders were characterized with respect to morphology, thermal property, and crystallinity using scanning electron microscopy, differential scanning calorimetry, and X-ray diffraction, respectively. Most formulations gave slightly aggregated, spherical particles, and percentage yields of the spray-dried powders decreased with increasing Chol content. Degree of particle aggregation depended on the powder composition. The powders spontaneously formed liposomes which efficiently entrapped LSZ after reconstitution with HEPES buffered saline (HBS) at 37°C. Lysozyme entrapment efficiency and size distribution of the reconstituted liposomes were evaluated after the powders were reconstituted with HBS. Increasing Chol content resulted in a decrease in size of the reconstituted liposomes and an increase in entrapment efficiency of LSZ. These results correlated with thermal behaviors of the reconstituted liposomes. Biological activity of LSZ was not affected by the spray-drying process. It was also demonstrated that LSZ-loaded liposomal powders could be produced without the need to preload the LSZ into liposomes prior to spray-drying process.     

Receptor-Specific Delivery of Liposomes Via Folate-Peg-Chol

Abstract

A novel lipophilic conjugate of folate, folate-PEG-Chol, was synthesized and evaluated for receptor-mediated targeting of liposomes to tumor cells. Liposomes composed of DSPC/Chol/PEG-DSPE/folate-PEG-Chol (60/ 34/5/1, m/m) were taken up by cultured folate receptor-bearing KB cells via a saturable mechanism. Cellular binding of these liposomes could be competitively inhibited by free folic acid with an IC₅₀ of 0.39 mM, indicating an extraordinarily high binding affinity. Fluorescence micrographs of KB cells treated with targeted liposomes encapsulating calcein showed that they were distributed both on the cell surface and in intracellular vesicular compartments. Targeted liposomes carrying doxorubicin were shown to be 38 times more toxic to KB cells than non-targeted control liposomes. A biodistribution study in receptor-positive tumor-bearing C57BL/6 mice showed no significant differences between the tumor uptake of folate-PEG-liposomes and non-targeted control liposomes. This study has demonstrated that cholesterol could be used as an alternative to phospholipids as an effective anchor for incorporation of a targeting ligand into liposomes.     

The Delivery of Benzyl Penicillin to Staphylococcus aureus Biofilms by Use of Liposomes

The delivery of benzyl penicillin [penicillin G (pen‐G)] encapsulated in cationic liposomes to a pen‐G‐sensitive strain of Staphylococcus aureus immobilized in biofilms has been investigated. The cationic liposomes prepared by extrusion (VETs, diameter ～ 140 nm) were composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol, and dimethylammonium ethane carbamoyl cholesterol (DC‐chol) at a molar ratio of 1.0 :0.49 :0.43. This composition containing 22 mole% of the cationic lipid DC‐chol has been found previously (Kim et al. Colloids Surfaces A 1999, 149, 561–570) to be optimum for adsorption of the liposomes on S. aureus biofilms. The effectiveness of the liposomes to deliver pen‐G to the biofilms immobilized on microtitre plates was assessed from the rate of growth of the cells after exposure to the liposomal drug carrier relative to free pen‐G at the same concentration. The time to reach maximum growth rate from biofilms was investigated as a function of overall drug concentration in a range 2.9 × 10^{? 3} mM to 1.09 mM and as a function of time of exposure to liposomal drug in a range 1.5 s to 2 h. Liposomal drug delivery was most effective relative to free drug at low overall drug concentrations and short times of exposure. The time to reach maximum growth rate from S. aureus biofilms could be extended by a factor of approximately 4 relative to free drug by the use of liposomally encapsulated pen‐G. The results were supported by direct measurements of the distribution of pen‐G between biofilm and supernatant which showed enhanced values relative to free drug and a transient preferential uptake of drug induced by the liposomes. The study demonstrates that for low drug concentrations and short exposure times liposomal drug delivery greatly enhances the effectiveness of pen‐G for inhibiting the growth of bacterial biofilms of the potentially pathogenic bacterium Staphylococcus aureus.     

Detection of liposomal cholesterol and monophosphoryl lipid A by QS-21 saponin and Limulus polyphemus amebocyte lysate

Liposomes containing cholesterol (Chol) have long been used as an important membrane system for modeling the complex interactions of Chol with adjacent phospholipids or other lipids in a membrane environment. In this study we utilize a probe composed of QS-21, a saponin molecule that recognizes liposomal Chol and causes hemolysis of erythrocytes. The interaction of QS-21 with liposomal Chol results in a stable formulation which, after injection into the tissues of an animal, lacks toxic effects of QS-21 on neighboring cells that contain Chol, such as erythrocytes. Here we have used liposomes containing different saturated phospholipid fatty acyl groups and Chol, with or without monophosphoryl lipid A (MPLA), as model membranes. QS-21 is then employed as a probe to study the interactions of liposomal lipids on the visibility of membrane Chol. We demonstrate that changes either in the mole fraction of Chol in liposomes, or with different chain lengths of phospholipid fatty acyl groups, can have a substantial impact on the detection of Chol by the QS-21. We further show that liposomal MPLA can partially inhibit detection of the liposomal Chol by QS-21. The Limulus amebocyte lysate assay is used for binding to and detection of MPLA. Previous work has demonstrated that sequestration of MPLA into the liposomal lipid bilayer can block detection by the Limulus assay, but the binding site on the MPLA to which the Limulus protein binds is unknown. Changes in liposomal Chol concentration and phospholipid fatty acyl chain length influenced the detection of the liposome-embedded MPLA.     

Inhibitory effect of liposomes containing sulfatide or cholesterol sulfate on syncytium formation induced by bovine immunodeficiency virus-infected cells

The effect of galactocerebroside 3'-sulfate (sulfatide) or cholesterol sulfate on syncytium formation induced by bovine immunodeficiency virus (BIV)-infected cells was investigated in vitro. Sulfatide was purified from bovine brain and incorporated in liposomes which were composed of egg phosphatidylcholine (PC), cholesterol (Chol), and dipalmitoylphosphatidic acid (DPPA). Either sulfatide- or cholesterol sulfate-containing liposomes effectively prevented syncytium formation induced by BIV-infected cells, but the inhibitory effect of sulfatide alone on syncytium formation was low. On the other hand, neither liposomes containing galactocerebroside nor liposomes composed of egg PC, Chol, and DPPA had any effect on syncytium formation induced by BIV-infected cells. These results suggest that liposomes containing sulfatide or cholesterol sulfate are an efficient agent to inhibit syncytium formation induced by BIV-infected cells, and that sulfate residue might play an important role in the inhibition of syncytium formation.     

Arsenic Trioxide Liposomes: Encapsulation Efficiency and In Vitro Stability

The use of arsenic‐containing compounds in cancer therapy is currently being re‐considered, after the recent approval of arsenic trioxide (Trisenox®) for the treatment of relapsed promyelocytic leukemia (PML). In an attempt to prepare a carrier system to minimize the toxicity of this drug, the aim of this study is to prepare and characterize liposomes encapsulating arsenic trioxide (ATO). For this, we prepared different types of liposomes entrapping ATO: large multilamellar (MLV), sonicated (SUV) and dried reconstituted vesicles (DRV). The techniques used were: thin film hydration, sonication and the DRV method, respectively. Two lipid compositions were studied for each liposome type, EggPC/Chol (1:1) and DSPC/Chol (1:1). After liposome preparation, drug encapsulation was evaluated by measuring arsenic in liposomes. For this, energy‐dispersive X‐ray fluorescence spectroscopy or atomic absorption was used. In addition, the retention of the drug in the liposomes was evaluated after incubating the liposomes in buffer at 37°C. The experimental results reveal that encapsulation of ATO in liposomes ranges between 0.003 and 0.506 mol/ mol of lipid, and is highest in the DRV vesicles and lowest in the small unilamellar vesicles, as anticipated. Considering the in vitro stability of ATO‐encapsulating liposomes: 1) For the PC/Chol liposomes (DRV and MLV), after 24 hours of incubation, more than 70% (or 90% in some cases) of the initially encapsulated amount of ATO was released. 2) The liposomes composed of DSPC/Chol could retain substantially higher amounts of ATO, especially the DRV liposomes (54% retained after 24 h). 3) In the case of PC/Chol, temperature of incubation has no effect on the ATO release after 24 hours, but affects the rate of ATO release in the MLV liposomes, while for the DSPC/Chol liposomes there is a slight increase (statistically insignificant) of ATO release at higher temperature.     

Gadolinium Effects on Gigaseal Formation and the Adhesive Properties of a Fungal Amoeboid Cell,the <Emphasis Type="BoldItalic">Slime</Emphasis> Mutant of <Emphasis Type="Italic">Neurospora crassa</Emphasis>

Low gadolinium concentrations induce rapid gigaseal formation and cell adhesion to glass and plastic (polystyrene) substrates in the slime mutant of Neurospora crassa. Cellular adhesion is independent of an integrin-mediated mechanism, because pretreatment with the oligopeptide ARG-GLY-ASP-SER (RGDS) did not inhibit it, and there was no spatial correlation between integrin and adhesions. In contrast, concanavalin A and beta-galactosidase both inhibit adhesion, suggesting that adhesion is mediated by sugar moeities at the cell surface. The adhesion sites are motile in the plasma membrane, as shown by the movement of polystyrene microspheres on the cell surface. In addition to an integrin-based adhesive system, which has already been characterized in walled hyphal cells, hyphae have evolved at least two different plasma membrane-based adhesion mechanisms. The relatively non-specific sugar-mediated adhesion caused by gadolinium may be part of the mechanism of gigaseal formation in other cells. In the absence of sugar-mediated adhesion, gadolinium increases the magnitude of the gigaseal in giant unilamellar liposomes composed of phosphatidylcholine, phosphatidylethanolamine, and cholesterol, with or without the negatively charged phosphatidylserine. Thus, gigaseal formation involves at least two different mechanisms. Abbreviations: BTP, bis-tris-propane; MES, morpholinoethanesulfonic acid; RGDS and RGDE, oligopeptides ARG-GLY-ASP-SER and ARG-GLY-GLU-SER, respectively; BS, bath solution; BSA, bovine serum albumin; PBS, phosphate-buffered saline; GULs, giant unilamellar liposomes; PC, Palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine; PE, 1-Palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine; PS, 1-Palmitoyl-2-linoleoyl-sn-glycero-3-[phospho-L-serine] (sodium salt); Ch, Cholesterol.     

Effect of the cholesterol content of small unilamellar liposomes on their stability in vivo and in vitro

Small unilamellar neutral, negatively and positively charged liposomes composed of egg phosphatidylcholine, various amounts of cholesterol and, when appropriate, phosphatidic acid or stearylamine and containing 6-carboxyfluorescein were injected into mice, incubated with mouse whole blood, plasma or serum or stored at 4°C. Liposomal stability, i.e. the extent to which 6-carboxyfluorescein is retained by liposomes, was dependent on their cholesterol content. (1) Cholesterol-rich (egg phosphatidylcholine/cholesterol, 7:7 molar ratio) liposomes, regardless of surface charge, remained stable in the blood of intravenously injected animals for up to at least 400min. In addition, stability of cholesterol-rich liposomes was largely maintained in vitro in the presence of whole blood, plasma or serum for at least 90min. (2) Cholesterol-poor (egg phosphatidylcholine/cholesterol, 7:2 molar ratio) or cholesterol-free (egg phosphatidylcholine) liposomes lost very rapidly (at most within 2min) much of their stability after intravenous injection or upon contact with whole blood, plasma or serum. Whole blood and to some extent plasma were less detrimental to stability than was serum. (3) After intraperitoneal injection, neutral cholesterol-rich liposomes survived in the peritoneal cavity to enter the blood circulation in their intact form. Liposomes injected intramuscularly also entered the circulation, although with somewhat diminished stability. (4) Stability of neutral and negatively charged cholesterol-rich liposomes stored at 4°C was maintained for several days, and by 53 days it had declined only moderately. Stored liposomes retained their unilamellar structure and their ability to remain stable in the blood after intravenous injection. (5) Control of liposomal stability by adjusting their cholesterol content may help in the design of liposomes for effective use in biological systems in vivo and in vitro.     

Kinetics of the interaction of hemin liposomes with heme binding proteins

As a model for the transport of hemin across biological membranes, sonicated phosphatidylcholine liposomes with incorporated hemin were characterized. The interaction of the hemin liposomes with the heme binding proteins albumin, apomyoglobin, and hemopexin was examined as a function of liposome charge and cholesterol content. In all cases, there was an almost complete transfer of hemin from liposome to protein; a rapid phase and a slow phase were observed for the transfer. For negatively charged liposomes (with 11% dicetyl phosphate), the rapid and slow phases showed observed rates of transfer of ca. 2 and 0.01 s-1, respectively, for all three proteins. The presence of cholesterol in the liposomes decreased the observed rates by a factor of 2, and positively charged liposomes (with 11% stearylamine) showed about one-fifth the observed rates of negatively charged liposomes. The observed rates were independent of protein concentration, indicating that the rate-determining step is hemin efflux from the lipid bilayer. The hemin interaction with the phospholipid bilayer is suggested to be primarily hydrophobic with some electrostatic character. The two phases are suggested to arise from two different populations of hemin within the liposomes and are interpreted as arising from two different orientations of hemin within the bilayer.     

Membrane fusion by proline-rich Rz1 lipoprotein, the bacteriophage lambda Rz1 gene product.

The fusogenic properties of Rz1, the proline-rich lipoprotein that is the bacteriophage lambda Rz1 gene product, were studied. Light scattering was used to monitor Rz1-induced aggregation of artificial neutral (dipalmitoylphosphatidylcholine/cholesterol) and negatively charged (dipalmitoylphosphatidylcholine/cholesterol/dioleoylphosphatidylserin e) liposomes. Fluorescence assays [the resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)dihexadecanol-sn-glycero-3-phosphoethanolamine lipid fluorescent probes, as well as fluorescent complex formation between terbium ions and dipicolinic acid encapsulated in two liposome populations and calcein fluorescence] were used to monitor Rz1-induced lipid mixing, contents mixing and leakage of neutral and negatively charged liposomes. The results demonstrated that Rz1 caused adhesion of neutral and negatively charged liposomes with concomitant lipid mixing; membrane distortion, leading to the fusion of liposomes and hence their internal content mixing; and local destruction of the membrane accompanied by leakage of the liposome contents. The use of artificial membranes showed that Rz1 induced the fusion of membranes devoid of any proteins. This might mean that the proline stretch of Rz1 allowed interaction with membrane lipids. It is suggested that Rz1-induced liposome fusion was mediated primarily by the generation of local perturbation in the bilayer lipid membrane and to a lesser extent by electrostatic forces.     

Adhesion of Positively Charged Liposomes to Mucosal Tissues

Abstract

A series of positively charged phospholipid and cholesterol derivatives was synthesized and evaluated as membrane components for liposomes. Small unilamellar liposomes containing up to 40 mole% of the synthetic lipids were prepared by sonication. Selected liposome preparations containing these synthetic lipid materials were found to be noncytotoxic in vitro by using a cell growth inhibition assay, whereas liposomes containing more classic positively charged components (stearylamine and cetyltrimethylammonium bromide) showed considerable cytotoxicity. Using an unanesthetized rabbit eye model, we have found that inclusion of the positively charged lipid derivatives into the liposomes significantly enhanced the ocular retention compared to neutral or negatively charged liposomes, presumably by molecular association with poly anionic corneal and conjunctival surface mucoglycoproteins. the increased retention was dependent on charge density and rigidity of the lipid bilayer. An assay for primary amino groups in these liposomes suggested that the distribution of the charged molecules between the inner and outer leaflets of the bilayer could be manipulated by lipid composition. Studies of liposomes containing cholesteryl esters of amino acids of various carbon chain lengths indicated that the charged amino groups need to extend from the surface of the lipid bilayers for better adhesion and retention. the ocular surface was saturable with respect to applied liposomes, which were cleared slowly from the eye with a half-time of clearance of about 2 hr. these data suggest a specific adhesion of the cationic liposomes to the surface of mucosal tissues.