Lecithin-cholesterol Liposomes Research Articles

Investigation of blood-brain barrier permeability to magnetite-dextran nanoparticles (MD3) after osmotic disruption in rats.

The permeability of experimentally disrupted blood-brain barrier (BBB) to superparamagnetic nanoparticles (MD3) was studied in rats. BBB opening was induced by intracarotid injection of mannitol. One hundred eighty rats were used for the study. Rats were examined at two time points, 30 minutes and 12 hours after intracarotid mannitol injection. Different preparations intravenously injected 30 minutes before rat sacrifice were used for characterization of BBB disruption. BBB integrity was determined with 99mTc-diethylenetriamine pentaacetic acid (DTPA) and 99mTc-albumin. Iron oxide-glucose particles (12-nm mean diameter), 99mTc-labeled lecithin-cholesterol liposomes of three different sizes (50, 100, and 200 nm), and polyethylene glycol (PEG)-coated 99mTc liposomes (50 nm) were used for investigations of the dependence of BBB permeability on particle system size or surface. Magnetite-dextran nanoparticles (MD3) were evaluated as superparamagnetic contrast agent to monitor with magnetic resonance imaging (MRI) the BBB breakdown. In vitro T1 and T2 relaxation times of the brain tissue were measured at 40 MHz and 37 degrees C, and T2-weighted MR images were acquired at 0.5 T. After intracarotid mannitol infusion, as expected, the BBB breakdown was immediate and temporary as judged by soluble molecule diffusion. MD3 nanoparticles crossed the BBB 12 hours after intravenous mannitol injection, at a time when brain permeability for molecules or small particles returns to normal. Magnetite crystals were found in cytoplasmic vesicles of glial cells. On MRI, signal intensity decreased after injection of MD3, even 12 hours after mannitol injection. This particularity could be useful in the study of focal pathological lesions accompanied by BBB permeability modifications. In such conditions, superparamagnetic particle contrast agents could be caught by the BBB, allowing the observation of impaired BBB areas without detectable cellular lesions.

New Synthetic Amphiphilic Polymers for Steric Protection of Liposomes in Vivo

Carboxy group‐terminated synthetic polymers—branched poly‐(ethylene glycol), poly(acryloylmorpholine), and poly(vinylpyrrolidone)—were made amphiphilic by derivatization with phosphatidyl ethanolamine via the terminal carboxy group and then incorporated into lecithincholesterol liposomes prepared by the detergent dialysis method. Following the biodistribution of liposomes in mice, all three polymers were shown to be effective steric protectors for liposomes and were able to sharply increase liposome circulation times in a concentration‐dependent manner. The accumulation of liposomes in the liver decreases. The effects observed are similar to those found for liposomes modified with linear poly(ethylene glycol). At low polymer concentration, amphiphilic branched poly(ethylene glycol) seems to be the most effective liposome protector, most probably, because at the same molar content of anchoring groups, each attachment point carries two polymeric chains and doubles the quantity of liposome‐grafted polymer comparing to linear poly(ethylene glycol).

The Lung Surfactant and Immune System Response to Intratracheal Administration of “Empty” Liposomes

AbstractAfter intratracheal administration of “empty” lecithin-cholesterol liposomes to rats it was found out twofold enhancement of the surfactant content with maximum on the 2nd-3rd day and with normalization to the control level by the 7th day. Phagocytic index of the alveolar macrophages was also increased. It was shown the change of the blast-transformation reaction of bronchoalveolar lavage and blood lymphocytes. Immune complexes content in bronchoalveolar lavage at different period of time after liposomes administration increased 1.5-2-fold. The natural killers (NK) activity of cells obtained from bronhoalveolar lavage and blood was enhanced 10 times and 2 times respectively. It is supposed that enhancement of lung surfactant phospholipid content is caused by substrate stimulation of type II alveolocytes activity. The stimulation of immunocompetent cells might be connected with imitation of bacterial attack by liposomes with proteins adsorbed on their surface.

Modification of lipid composition of neuroblastoma C1300 N18 cells with liposomes alters the cholesterol content

Cell incubation with lecithin-cholesterol liposomes (1:1 mol/mol) caused enhancement of the cholesterol content. The level of cholesterol esters of total and phospholipid unsaturated fatty acids increased in cholesterol enriched cells. Simultaneously the amount of saturated fatty acids decreased and lysophosphatidylcholine appeared in the cells. On the contrary, cell incubation with lecithin liposomes resulted in cholesterol depletion. This effect was accompanied by a decrease of cholesterol esters, of total and phospholipid unsaturated fatty acids. The content of saturated fatty acids was raised in cells with reduced amount of cholesterol. The quantity of n-acylphosphatidylethanolamine and n-acylethanolamine, lipids newly found in neuroblastoma cells, also changed in cells with modified content of cholesterol. The physiological role of cell response to the changes of cholesterol level is discussed.

Liposomes as cyclosporin A carriers: positively charged lecithin-cholesterol liposomes associated with cyclosporin A do not inhibit biosynthesis of mitochondrial polyglycerophosphatides and microsomal phosphatidylinositol.

Phosphatidylcholine (from egg yolk)-cholesterol-stearylamine (7:2:2.25, molar ratio) liposomes when associated with cyclosporin A (phosphatidylcholine:cyclosporin A = 2:0.07, molar ratio) do not inhibit significantly the biosynthesis of [3H]polyglycerophosphatides and [3H]phosphatidylinositol in mitochondrial and microsomal membranes, respectively, isolated from rat liver. These results are explained by possible protection of the liposomes by cyclosporin A against phospholipase A2 hydrolysis.

Purification of human plasma lecithin: cholesterol acyltransferase by covalent chromatography

Human plasma lecithin: cholesterol acyltransferase (LCAT, EC 2.3.1.43) has been purified more than 20 000 fold from plasma in 10% yield. This new procedure is composed for only four steps, including ultracentrifugation of plasma to yield a 1.21–1.25 kg/l density fraction, covalent binding of LCAT in this fraction to thiopropyl-Sepharose followed by adsorption of the enzyme to wheat-germ lectin-Sepharose for elimination of albumin and finally batch-wise treatment of the desorbed LCAT with hydroxyapatite to remove residual impurities. The purified enzyme was free of apolipoprotein A-I, A-II, B, C-I, C-II, C-III and E as checked by double immunodiffusion and SDS-electrophoresis, which latter method also demonstrated the absence of hitherto characterized lipid transfer proteins. Only traces of apolipoprotein D were present in the preparation as detected by immunoblotting. The purified enzyme retained α- and β-LCAT activities. Non-denaturing and denaturing polyacrylamide gel electrophoresis yielded apparent molecular masses of 69 and 66 kDa, respectively, for the enzyme which on isoeletric focusing produced one major and one minor isoform with p I values of 4.20 and 4.25, respectively. Apolipoprotein A-I was required to transform artificial lecithin-cholesterol liposomes into subtrates for the purified LCAT.

Liposomes-mycobacteria incubation systems as a partial model of host-parasite interaction at cell membrane level.

The multilamellar lecithin-cholesterol liposomes entrapping carboxyfluorescence as a specific marker were prepared to be incubated with mycobacteria. The extent of resulting liposomal lysis was measured by marker release. Mycobacteria were highly active in this respect regardless of the species. Staphylococci were much less active and E. coli was completely inactive. Molecular species of lecithin and their ratio to cholesterol were related with the liposome sensitivity to mycobacteria. Ultrastructural and biochemical study showed that liposomes can be in close contact with mycobacteria and lecithins are degraded so that released fatty acids are incorporated into bacterial lipids, especially in virulent species. Liposomes-mycobacteria interaction was discussed as a partial model of the phagocyte-parasite interaction at the membrane level.

Investigations on development of an artificial diet for in vitro rearing of Glossina palpalis palpalis (Diptera: Glossinidae).

Glossina palpalis palpalis (Robineau-Desvoidy) were fed a number of diffcrent diets in a search for the optimal diet for membrane feeding. Flies fed freeze-dried bovine or porcine blood showed intermediate performance with respect to productivity (0.075 fecundity) and offspring weight (26 to 28 mg). Addition of bovine serum albumin overcame some of the deficits in freeze-dried blood. Other additives such as lecithin and cholesterol alone did not improve fly reproductive performance. Lecithin-cholesterol liposomes added to freeze-dried bovine blood increased the fecundity from 0.073 to 0.085 pupa per mature female day. Bovine serum albumin did not completely replace bovine serum, but was an adequate replacement for pig serum when combined with washed pig erythrocytes. Commercial heat-dried blood products were inadequatediets, as were bovine serum albumin and hemoglobin alone.

Preparation and properties of spin-labeled lecithin-cholesterol liposomes.

Lecithin-cholesterol vesicles of various compositions containing membrane-bound spin-labeled cholestane can be prepared by appropriate choice of initial concentrations of components during sonication. Increasing incorporation of spin label increases incorporation of cholesterol and decreases incorporation of lecithin, with the result that liposomes with cholesterol-lecithin molar ratios larger than 2 can be obtained. Besides associating with cholesterol-lecithin complexes in the liposome, the spin label seems to associate with cholesterol. Changes of the paramagnetic resonance spectrum of the liposome-bound spin label due to changes in liposomal cholesterol and spin label mole fractions - assessed by three parameters - can be used in cell-liposome interaction studies.

Action of drugs, detergents, and monomers on liposomes.

Interactions of 14 organic compounds, such as phenols, aromatic carboxylic acids, steroids, and methacrylates with the multi-lamellar lecithin-cholesterol liposomes, were monitored in biological systems. It was found that their actions with our artificial liposomes were quite similar to those of biological membranes described previously.

Characterization of proteoliposomes containing apoprotein A-I: a new substrate for the measurement of lecithin: cholesterol acyltransferase activity.

Proteoliposome vesicles containing apoA-I, lecithin, and cholesterol (including labeled cholesterol) were prepared from various molar ratios of the three components by the cholate dialysis technique. Comparative studies on the sensitivity and efficiency of these proteoliposomes to serve as substrate for lecithin:cholesterol acyltransferase (LCATase) indicated that the proteoliposome with apoA-I:lecithin:cholesterol molar ratio of 0.8:250:12.5 was ideal for assaying LCATase activity of both plasma and purified enzyme. This proteoliposome was shown to be comparable in size by gel filtration (radius, 131.9 +/- 4.8 A, n = 6) and by electron microscopy (radius, 123.4 +/- 5.1 A, n = 100). The proteoliposome preparation was stable as LCATase substrate for at least 3 and 5 weeks, respectively, when stored at 4 degrees C and -20 degrees C, and was a better substrate for the enzyme activity assay than were lecithin-cholesterol liposomes incubated with apoA-I. Under the standardized assay system LCATase activity was a linear function of plasma enzyme added and was independent of the amount of plasma cholesterol added to the proteoliposomes in the range of 3 to 20 microliters of plasma. The mean LCATase activity by this method was 95.1 +/- 14.0 (range 76.5-122.5) nmol/hr per ml of plasma from fifteen normal human subjects. This method of substrate formation using the cholate dialysis technique permits the preparation of large amounts of stable, efficient, homogeneous, and well-defined substrate that is suitable for measuring low levels of enzyme activity, comparative studies, and large scale investigations of plasma LCATase, as well as studies of the mechanism and regulation of LCATase reaction.

Interaction of lecithin:cholesterol acyltransferase and cholesteryl ester transfer protein in the transport of cholesteryl ester into sphingomyelin liposomes.

When isolated lecithin:cholesterol acyltransferase was incubated with cholesterol-lecithin liposomes in the presence of apolipoprotein A-1, cholesteryl ester accumulated until a maximal ester/lecithin weight ratio of 0.03 was reached. This was independent of the amount of enzyme present or the proportion of cholesterol relative to lecithin. The inhibition of transferase associated with accumulation of cholesteryl ester was relieved by additional lecithin-cholesterol liposomes but not by addition of sphingomyelin liposomes containing the same proportion of substrate unesterified cholesterol. These results indicate that it is the accumulation of cholesteryl ester product which directly inhibits transferase activity. When isolated cholesteryl ester transfer protein from human plasma was included in the reaction mixture, cholesteryl ester was transported to sphingomyelin-cholesterol liposomes, with associated release of transferase from product inhibition. Cholesteryl ester incorporated directly into the liposomes or synthesized from free cholesterol via the transferase reaction was equally transferred to sphingomyelin acceptor liposomes, indicating that the cholesteryl ester in these particles formed a single miscible pool for transfer.

In vitro growth of Mycobacterium lepraemurium: Transition from in vivo grown to in vitro adaptation.

For successful in vitro growth of any host-dependent organism the most important thing is the ability of in vivo grown cells to synthesize in vitrotype cell membranes. In case of M. lepraemurium factors responsible for such conversion are α-ketogluterate, hemin reduced oxygen tension, and lecithin-cholesterol liposomes.

Antimycobacterial activity of lecithin-cholesterol liposomes in the presence of phospholipase A2.

Tubercle bacilli were preincubated with lecithin-cholesterol liposomes to be subsequently exposed to phospholipase A2. After further incubation in the environment of acidic buffer, viable units in the final mixture were enumerated by inoculating the serial dilutions of an aliquot onto Kirchner agar medium containing horse serum in 5%. Another aliquot was used for lipid analyses to confirm hydrolysis of lecithin. In addition to this bactericidal type of experiments, bacteriostatic tests were also conducted with Kirchner semi-solid agar medium, into which liposome-treated bacilli were inoculated with the enzyme at a time. Various natural and synthetic lecithins different in constituent fatty acids were employed. The results indicated that toxic fatty acids released from lecithin acted to kill the bacilli or to inhibit their growth.

Interaction of liposomes with polymorphonuclear leukocytes: I. Studies on the mode of interaction

Summary Amphipathic long chain anions (dicetylphosphate) or cations (stearylamine) were inserted into the lipid lamellae of multilayered lecithin-cholesterol liposomes so as to obtain liposomes with varying surface charge. These liposomes were then analysed with respect to their interaction with polymorphonuclear leukocytes (PMN cells) at 37°C. The different liposomes showed different tendencies to associate to the cells: the more negatively charged, the greater the tendency to associate. The association was insensitive to metabolic inhibitors and proceeded without activation of the hexose monophosphate shunt. It was not influenced by omitting divalent cations from the medium. However, lowering the temperature below 30°C and further down inhibited the association. The kinetics of the association process suggests that the presence of dicetylphosphate on the liposome surface increased the maximal rate of association, whereas lowering the temperature decreased the accessibility to appropriate receptor sites in the PMN cell membrane. The results lend support to the hypothesis that controlled manipulations of the liposome composition may be used to engineer the introduction of lipids into PMN cells by different rates and mechanisms. This should have relevance to the possible modification of PMN cell behavior by introduction of components derived from liposomes.

Cholesterol inhibition of the temporary increase of membrane fluidity of lymphocytes induced by mitogenic lectins

The mitogenic response of human peripheral lymphocytes to lectins can be decreased by brief treatment of the cells with lecithin-cholesterol liposomes. This fact indicates that the temporary increase of membrane fluidity, which occurs within 30 min after addition of mitogenic lectins, is an important early event for the subsequent activation of lymphocytes. This temporary increase of membrane fluidity is accompanied by neither a decrease in cellular cholesterol level nor by particular acceleration of the incorporation of polyunsaturated fatty acids into phospholipids. These facts suggest that this change in membrane fluidity is not due to the alteration of membrane lipid composition, but can be regarded as a result of temporary perturbation of membrane lipid bilayers induced by binding of the lectins to their membrane receptors.

An attempt to cultivate mycobacteria in simple synthetic liquid medium containing lecithin-cholesterol liposomes.

An attempt was made to cultivate mycobacteria in a simple synthetic liquid medium containing lecithin-cholesterol liposomes. This lipid complex showed a marked growth-promoting effect on the submerged growth of M. tuberculosis and M. bovis. The role of lecithin as nutrient was suggested. The bacillary growth in such environment retained good viability, strong acid-fastness, and high virulence in mice. An avirulent strain of tubercle bacilli, H37Ra, did not respond to lecithin-cholesterol liposomes unlike the parent virulent strain, H37Rv. However, this was not a general rule for virulence, as a highly virulent strain of M. bovis (Ravenel) and an attenuated strain (BCG) both grew well in the presence of lipsomes. Lipid analysis showed that cholesterol in the liposome medium was esterified to some extent during the bacterial growth. It was discussed that the culture in the liposome-containing medium may present an experimental model for the study of interaction between mycobacteria and the macrophage membrane.

Motional state of spin-labeled stearates in lecithin-cholesterol liposomes and their incorporation capability.

Electron spin resonance spectra of nitroxide-labeled stearates were investigated in the presence of dipalmitoyl or dimyristoyl lecithin-cholesterol liposomes. Beside the broad resonance lines, originated from the incorporated spin probes, the spectra were characterized with the sharp three resonance lines, which were attributed to the spin probes not incorporated in the liposomal membrane. The superposed spectra were analyzed in terms of the peak height ratio of the sharp and broad lines, and also of the order parameter, derived from hyperfine splittings of the broad lines. The two quantities changed depending on the cholesterol content in the liposomes, but appeared to behave entirely different from each other : in the dipalmitoyl lecithin-cholesterol system, the spin probes were more incorporated in the membrane of higher motional state, while, in the dimyristoyl lecithin-cholesterol, they were less incorporated. The relation was discussed and compared with permeability property of the liposomal membranes.

The effect of digitonin on the stimulation by insulin of glucose uptake by isolated fat cells

The effect of digitonin on glucose uptake by isolated fat cells in the presence and absence of insulin has been studied. At low concentrations of digitonin, the stimulation of glucose uptake by insulin was inhibited without severe cell damage as estimated by the leakage of lactate dehydrogenase from the cells. The inhibition of the insulin effect was not reversed by washing the cells or by the addition of cholesterol or lecithin-cholesterol liposomes to the incubation medium of the cells after treatment with digitonin. Cholesterol was shown to be present in the fat cells and it is suggested that the inhibition of the insulin effect is a consequence of the formation of digitonin-cholesterol complexes in the fat cell plasma membrane. Possible ways in which this may result in inhibition of the effect of insulin are discussed.

A nitroxide-sterol derivative potently modifies cholesterol biosynthesis by normal and neoplastic guinea pig lymphocytes

Leukemic guinea pig lymphocytes (L 2 C) synthesise cholesterol in vitro at a forty-fold greater rate than normal cells. Equilibration (18 h) with lecithin or lecithin-cholesterol liposomes, respectively, enhances or suppresses sterol manufacture by normal lymphocytes but does not influence sterol production by L 2 C cells. In contrast, > 5·10 9 molecules/cell of a nitroxide-derivative of androstane, (17 β-hydroxy-4′,4′-dimethylspiro [5 α-androstan-3,2′-oxazolidin]-3′-yloxyl), commonly used as a membrane spin-probe, drastically inhibit sterol production by both normal and leukemic cells (maximum within 2 h). At < 5·10 9 molecules/cell, this sterol stimulates cholesterol synthesis. 25-Hydroxycholesterol at low concentrations also stimulates sterol manufacture, whereas high concentrations are also inhibitory in both cell types.