Multilamellar Phosphatidylcholine Liposomes Research Articles

Liposomes Integrated with Amyloid Hydrogels: a Novel Composite Drug Delivery Platform

The present study was undertaken to evaluate the possibility of designing the composite drug delivery platform by integration of the liposomes into the network of amyloid hydrogels. The efficiency of drug entrapment into the combined lipid-protein systems was assessed using the two anticancer agents, viz., the hydrophilic positively charged drug doxorubicin (DOX) and the hydrophobic europium coordination complex, referred to here as V7. The lipid vesicles were composed of the neutral lipid phosphatidylcholine (PC) and its mixture with anionic lipid cardiolipin (CL, 10 mol%), while the amyloid fibrils were obtained from the egg yolk lysozyme (LzF) and the bovine serum albumin (BSAF). The cumulative results from the fluorescence and equilibrium dialysis studies revealed that the negatively charged albumin fibrils favor the DOX encapsulation into PC multilamellar liposomes but exert opposite effect in the case of CL-containing liposomes. However, no sensitivity to the presence of fibrillar protein was observed in the systems PC/DOX/LzF and CL10/DOX/LzF. Likewise, neither LzF nor BSAF affected the extent of V7 encapsulation into the lipid vesicles. These findings highlight the possibility of increasing the payload efficiency of hydrophilic drugs through combining the liposomes with amyloid hydrogels.

Ground and excited state proton transfer and antioxidant activity of 7-hydroxyflavone in model membranes: Absorption and fluorescence spectroscopic studies

Steady state and time resolved fluorescence spectroscopy have been used to probe microenvironments of the therapeutically active intrinsically fluorescent flavonoid, 7-hydroxyflavone (7-HF), in model membranes consisting of multilamellar phosphatidylcholine liposomes. Additionally, the antioxidant effects of 7-HF against lipid peroxidation have been evaluated using spectrophotometric assay. Large Stokes shifted emissions with distinct spectroscopic signatures, are observed from the excited state proton transfer (ESPT) tautomer (which is generated by a solvent mediated mechanism) and the ground state anion of 7-HF. The neutral (7-HFN) and anionic (7-HFA) species' appear to be located in the non-polar acyl chain and the polar head group regions of the lipid vesicles respectively. The partition coefficients of 7-HFN and 7-HFA in these vesicles have also been estimated using their intrinsic fluorescence. Anisotropy ( r) versus temperature (T) measurements reveal the utility of the tautomer fluorescence anisotropy as a sensitive parameter for exploring structural changes in the membranes. Fluorescence decay kinetics studies indicate heterogeneity in the microenvironments of both 7-HFN and 7-HFA. Furthermore, we demonstrate that lipid peroxidation of the model membranes is partially arrested upon 7-HF binding, suggesting its potential usefulness as an inhibitor of peroxidative damage of cell membranes.

Phosphatidylcholine-rich acceptors, but not native HDL or its apolipoproteins, mobilize cholesterol from cholesterol-rich insoluble components of human atherosclerotic plaques

To examine the potential of high density lipoproteins (HDL) to ameliorate atherosclerotic plaques in vivo, we examined the ability of native HDL, lipid-free HDL apolipoproteins (apo HDL), cholesterol-free discoidal reconstituted HDL (R-HDL) comprised of apo HDL and phosphatidylcholine (PC) and PC liposomes to release cholesterol from cholesterol-rich insoluble components of plaques (ICP) isolated from atherosclerotic human aorta. Isolated ICP had a free cholesterol (FC) to phospholipid (PL) mass ratio (0.8–3.1) and a sphingomyelin (SPM) to PC mass ratio (1.2–4.2) that exceeded those of plasma membranes of cultured cells. Suprisingly, native HDL and its apolipoproteins were not able to release cholesterol from ICP. However, R-HDL and PC liposomes were effectively released cholesterol from ICP. The release of ICP cholesterol by R-HDL was dose-dependent and accompanied by the transfer of >8× more PC in the reverse direction (i.e., from R-HDL to ICP), resulting in a marked enrichment of ICP with PC. Compared to R-HDL, PC liposomes were significantly less effective in releasing cholesterol from ICP but were somewhat more effective in enriching ICP with PC. Native HDL was minimally effective in enriching ICP with PC, but became effective after prior in vitro enrichment of HDL with PC from multilamellar PC liposomes. The enrichment of ICP with PC resulted in the dissolution of cholesterol crystals on ICP and allowed the removal of ICP cholesterol by apo HDL and plasma. Our study revealed that the removal of cholesterol from ICP in vivo will be possible through a change in the level, composition, and physical state of ICP lipids mediated by PC-enriched HDL.

In Vitro Antioxidant and Antibacterial Activities of Shallot and Scallion

ABSTRACT: In vitro antioxidant and antibacterial protection of shallot and scallion were examined. Water extract and oil of shallot and scallion significantly delayed lipid oxidation in multilamellar phosphatidylcholine liposomes and human RBC membranes (p < 0.05). Shallot oil at 5 and 10 mM showed marked antioxidant activity at 75 °C (p < 0.05). Shallot and scallion oils significantly inhibited the growth of 4 food‐borne bacteria, Salmonella typhimurium DT104, E.coli O157:H7, Listeria monocytogenes and Staphylococcus aureus, and 4 nosocomial bacteria, methicillin‐resistant Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii (p < 0.05). All observed antioxidant and antibacterial activities were dose‐dependent (p < 0.05). These results suggested the use of shallot and scallion oils in food systems could enhance lipid and microbial stability.

Different effects of propofol and nitrosopropofol on DMPC multilamellar liposomes

The mechanisms of reaction of propofol with nitrosoglutathione lead to the formation of an active species which was identified, and then synthesised, as 2,6-diisopropyl-4-nitrosophenol. In the present work, we demonstrate the in vitro formation of 2,6-diisopropyl-4-nitrosophenol, then we discuss the interaction of propofol and 2,6-diisopropyl-4-nitrosophenol with dimyristoylphosphatidylcholine and egg yolk phosphatidylcholine multilamellar liposomes using differential scanning calorimetry and spin labelling techniques. It was demonstrated that both molecules are highly lipophylic and absorb almost entirely in the lipid phase. The thermotropic profiles showed that these molecules affect the temperature and the co-operativity of the gel-to-fluid state transition of the liposomes differently: the effects of 2,6-diisopropylphenol on the lipid organisation are quite similar to phenol and coherently interpretable in terms of the disorder produced in the membrane by a bulky group; 2,6-diisopropyl-4-nitrosophenol is a stronger perturbing agent, and ESR spectra suggest that this is due to a relative accumulation of the molecule into the interfacial region of the bilayer.

The role of drug-lipid interactions on the disposition of liposome-formulated opioid analgesics in vitro and in vivo.

Although liposome encapsulation prolongs the duration of action of epidurally administered drugs, little is known about how liposome encapsulation affects opioids differently, or about how lipid content of liposomes alters the bioavailability of epidurally-administered opioids. To address these issues, morphine, alfentanil, fentanyl, and sufentanil were loaded into D-alpha-dipalmitoyl phosphatidylcholine multilamellar liposomes, and incorporation efficiency and in vitro release rates were determined. We then determined epidural morphine and sufentanil liposomes, at two different lipid/opioid ratios, in vivo in a pig model in which epidural and intrathecal spaces were continuously sampled via microdialysis. Liposome encapsulation efficiency was significantly more for sufentanil (100%) than for the other opioids (25%-30%). The in vitro release rate was slowest for morphine, intermediate for fentanyl and alfentanil, and fastest for sufentanil. In vivo, morphine was released more slowly than sufentanil. It is most important to note that increasing the lipid content of morphine liposomes increased the proportion of drug reaching the intrathecal space. In contrast, increasing the lipid content of sufentanil liposomes did not alter intrathecal movement but did decrease movement into plasma. Therefore, increasing drug hydrophobicity and lipid content of the liposomes modulates drug distribution in vivo. The degree of interaction between opioids and lipid bilayers in liposome-formulated opioids dictates the rates at which epidurally-administered drugs distribute into the intrathecal compartment and blood in potentiating analgesic effects.

Astaxanthin and Peridinin Inhibit Oxidative Damage in Fe2+-Loaded Liposomes: Scavenging Oxyradicals or Changing Membrane Permeability?

Astaxanthin and peridinin, two typical carotenoids of marine microalgae, and lycopene were incorporated in phosphatidylcholine multilamellar liposomes and tested as inhibitors of lipid oxidation. Contrarily to peridinin results, astaxanthin strongly reduced lipid damage when the lipoperoxidation promoters—H2O2, tert-butyl hydroperoxide (t-ButOOH) or ascorbate—and Fe2+:EDTA were added simultaneously to the liposomes. In order to check if the antioxidant activity of carotenoids was also related to their effect on membrane permeability, the peroxidation processes were initiated by adding the promoters to Fe2+-loaded liposomes (encapsulated in the inner aqueous solution). Despite that the rigidifying effect of carotenoids in membranes was not directly measured here, peridinin probably has decreased membrane permeability to initiators (t-ButOOH > ascorbate > H2O2) since its incorporation limited oxidative damage on iron-liposomes. On the other hand, the antioxidant activity of astaxanthin in iron-containing vesicles might be derived from its known rigidifying effect and the inherent scavenging ability.

EPR study of spermine interaction with multilamellar phosphatidylcholine liposomes

The interaction of spermine with egg-yolk phosphatidylcholine liposomes was investigated. The EPR spin labeling technique evidenced that spermine induces modifications of some membrane functions of biological interest like water permeability and is a possible modulator of diffusion processes for charged and polar molecules. The association constant for a hypothesized complex between spermine and the phosphate group of phosphatidylcholine was evaluated by enzymatic methods.

In vitro reverse cholesterol transport from THP-1-derived macrophage-like cells with synthetic HDL particles consisting of proapolipoprotein A1 or apolipoprotein A1 and phosphatidylcholine.

The human monocytic leukaemia cell line THP-1 was induced to differentiate to macrophage-like cells by the addition of phorbol myristoyl acetate (PMA). Subsequently, the cells were enriched in cholesterol and these cholesterol laden cells were used to study the capability of reconstituted discoidal complexes (RDCs), consisting of either human apolipoprotein A1 (apo A1) or recombinant human proapolipoprotein A1 (proapo A1) and phosphatidylcholine (PC), to promote cholesterol efflux. RDCs containing apo A1 and proapo A1 were both effective in the mobilization of intracellular cholesterol, whether this was measured by intracellular cholesterol mass or by the appearance of radiolabelled cholesterol in the supernatant. Using the radiolabelling technique, the activity was saturable and followed Michaelis-Menten kinetics. For both types of complexes and for native HDL the maximum rate of cholesterol removed was approximately 0.5 nmol h-1 per 10(6) cells. For RDCs of proapo A1 and apo A1 and for native HDL the Km values were 3.7, 2.9 and 64.8 micrograms ml-1 respectively. A significant in vitro cholesterol efflux could only be achieved with protein-lipid complexes; no significant export was observed with either free proapo A1 or multilamellar PC liposomes without apolipoprotein. Both RDCs were found to be more active in the mobilization of intracellular cholesterol than HDL isolated from human plasma. The combined results demonstrate that synthetic complexes consisting either of apo A1 or proapo A1 and PC are both active in the in vitro reverse transport of cholesterol.

Immunogenicity of ribonucleic acid-protein fraction of Mycobacterium tuberculosis encapsulated in liposomes

Immunologically potent RNA-protein extracted from Mycobacterium tuberculosis strain H37Ra, when entrapped in phosphatidylcholine multilamellar liposomes and injected into mice, induced both cellular and humoral immune responses. Significant protection against infection with M. tuberculosis H37Rv was also induced in the immunised mice, as monitored by (i) higher survival rates, (ii) decreased viable counts of M. tuberculosis H37Rv in lungs, livers and spleens, (iii) lower lung density, and (iv) lower root specific lung weight, in comparison with a control group of unimmunized mice.

Stoichiometry of the binding of Ca 2+ on the inner and the outer monolayers of sonicated vesicle bilayers

The binding constant and the stoichiometry for Ca 2+ interacting with the inner and outer monolayers of egg phosphatidylcholine sonicated vesicle bilayers have been evaluated from 31P nuclear magnetic resonance and fluorescence anisotropy measurements. The values obtained for the binding on each monolayer can be compared to those reported in the literature for multilamellar phosphatidylcholine liposomes using a variety of experimental procedures. It is concluded that the Ca 2+ binding on the inside is different from that on the outside both in strength and in stoichiometry. The 2:1 phospholipid:Ca 2+ ratio obtained for the inner monolayer promotes conformational changes at the hydrocarbon chain region and the external head-groups. This enhances the binding of Ca 2+ on the outer monolayer. No conformational change in the hydrocarbon core was found for Ca 2+ adsorbing on the outer surface. In good agreement with this observation, the 31P NMR data for the outward facing phosphate groups indicate that Ca 2+ binds with a 1:1 stoichiometry. Moreover, the binding constant values depend on the region chosen for detecting the Ca 2+ action.

Stoichiometry of the binding of Ca 2+ on the inner and the outer monolayers of sonicated vesicle bilayers

The binding constant and the stoichiometry for Ca 2+ interacting with the inner and outer monolayers of egg phosphatidylcholine sonicated vesicle bilayers have been evaluated from 31P nuclear magnetic resonance and fluorescence anisotropy measurements. The values obtained for the binding on each monolayer can be compared to those reported in the literature for multilamellar phosphatidylcholine liposomes using a variety of experimental procedures. It is concluded that the Ca 2+ binding on the inside is different from that on the outside both in strength and in stoichiometry. The 2:1 phospholipid:Ca 2+ ratio obtained for the inner monolayer promotes conformational changes at the hydrocarbon chain region and the external head-groups. This enhances the binding of Ca 2+ on the outer monolayer. No conformational change in the hydrocarbon core was found for Ca 2+ adsorbing on the outer surface. In good agreement with this observation, the 31P NMR data for the outward facing phosphate groups indicate that Ca 2+ binds with a 1:1 stoichiometry. Moreover, the binding constant values depend on the region chosen for detecting the Ca 2+ action.

Sublytic and lytic effects of the zwitterionic bile derivative 3-((3-Deoxycholamidopropyl)dimethylammonio)-1-propanesulfonate on phosphatidylcholine liposomes

The effects of the zwitterionic bile derivative 3-((3-deoxycholamidopropyl)dimethyl-ammonio)-1-propanesulfonate (Chaps) on multilamellar phosphatidylcholine liposomes have been characterized. When the surfactant is added to preformed liposome suspensions, equilibrium is attained in less than 6 h. Fifty percent solubilization, as measured by analysis of lipid P in supernatants after solubilization, occurs at a 0.32 lipid/detergent mole ratio for a 1 m m phospholipid concentration. Fifty percent release of entrapped glucose occurs at the same detergent concentration, suggesting that, in this system, no increase in permeability occurs prior to solubilization. A linear relationship is found between phospholipid concentration and amount of surfactant producing 50% solubilization. No lytic effect of Chaps is seen below 2 m m surfactant, this being probably near the critical micellar concentration of the amphiphile under our conditions. In the sublytic range of detergent concentrations, Chaps binds the lipid bilayers with high affinity, so that, at least at 1 m m phospholipid, the amount of free Chaps is negligible; solubilization starts when about two surfactant molecules are incorporated per phospholipid molecule. Differential scanning calorimetry shows that incorporation of Chaps into saturated phosphatidylcholine bilayers, even at concentrations below those producing solubilization, causes a decrease in the T c gel-to-liquid crystalline main transition temperature of the phospholipid, and a decrease in the transition enthalpy; at the same time, a “shoulder” appears on the low-temperature side of the main endotherm. The ensemble of our data suggests that the behavior of Chaps toward phospholipid bilayers is intermediate between that of the natural bile derivatives and that of some well-known nonionic synthetic surfactants.

Immunoprotective behaviour of plasma-membrane-associated antigens of axenic Entamoeba histolytica

Immunisation of golden hamsters with plasma-membrane-associated antigens of a virulent subline of axenic Entamoeba histolytica strain NIH 200 V, entrapped in multilamellar phosphatidylcholine liposomes (MPL) or Freund's complete adjuvant (FCA), afforded protection against intrahepatic challenge with axenic amoebic trophozoites of the same strain. Amoebic liver abscess developed in 86% and 80% of the animals that received empty liposomes or buffer emulsified in FCA but in none of the animals that received plasma-membrane-antigen vaccines. All the immunised animals had significantly higher levels (p less than 0.001) of antibodies to plasma-membrane components and significantly higher levels (p less than 0.001) of cellular sensitisation. Antibody-dependent macrophage-mediated cytotoxicity against trophozoites was also found to be significantly greater (p less than 0.001) in immunised animals. Liposome-entrapped antigens stimulated the immune system of the host as well as, or better than, antigens administered with FCA.

Elicitation of protective immunity to Entamoeba histolytica--an experimental study.

Immunization of hamsters with plasma membrane (PM) antigens of virulent subline of axenic Entamoeba histolytica (NIH: 200 V) entrapped in multilamellar phosphatidyl choline liposomes conferred 100% protection to a subsequent intrahepatic amoebic challenge. In contrast, vaccination of hamsters with live amoebic trophozoites injected intradermally failed to protect any of the animals. The protected animals had significantly high levels of anti-PM anti-amoebic antibodies, cellular sensitization and macrophage-mediated antibody-dependent cytotoxicity against amoebic trophozoites. However, none of the intradermally immunized animals had anti-PM anti-amoebic antibodies at the time of challenge. Such animals also had significantly low macrophage-mediated antibody-dependent cellular cytotoxicity. The data indicate the potential prophylactic use of PM antigens against hepatic amoebic infection.

X-ray diffractometry and calorimetry studies of structural modifications induced by gamma-irradiation in phosphatidylcholine multilamellar liposomes.

Experimental results are reported on structural and thermodynamic modifications induced by gamma-irradiation in model membranes. Differential scanning calorimetry (DSC) and X-ray diffraction were used to study the different phases and associated transitions of dipalmitoylphosphatidylcholine after 60Co gamma-irradiation. Changes were observed in the shape of calorimetric peaks and in the corresponding enthalpy. The repetition distance of the layers increases while the distances related to the aliphatic chains decrease as a function of gamma-irradiation time. Moreover, an increase in the hexagonal symmetry with increasing dose was detected. No disappearance of a pre-transition was detected even at high doses.

Nonelectrolyte substitution for water in phosphatidylcholine bilayers

Glycerol substitutes for water in multilamellar phosphatidylcholine liposomes in that the fluid spaces between bilayers, as well as their main transition temperatures, heat capacities, and ethalpies are very similar in water and in pure glycerol. One major difference is that the gel state phase of dipalmitoylphosphatidylcholine (DPPC) in glycerol consists of bilayers with fully interdigitated hydrocarbon chains. Interdigitated DPPC phases are also formed in ethylene glycol or in methanol (at low methanol content). In solutions of glycerol and water, the fluid spacing between bilayers is a function of mole fraction of glycerol X g, reaching maximum values at X g ≌ 0.1 for lipid in the liquid crystalline phase and at X g ≌ 0.3 for the gel phase. These changes are explained in terms of a modification of the long-range Van der Waals attractive forces by glycerol.

Organization of the glycosphingolipid asialo-G M1 in phosphatidylcholine bilayers

An affinity purified monovalent ferritin conjugate of Ricinus communis agglutinin (RCA 60) is used with freeze-etch electron microscopy to study the ultrastructural localization of the glycosphingolipid asialo-G M1 in multilamellar phosphatidylcholine liposomes. Dimyristoylphosphatidylcholine (DMPC) liposomes containing up to 20 mol% asialo-G M1 and quenched below the main transition temperature show a striking linear localization of ferritin-RCA 60 between phospholipid ridges. The glycosphingolipid localization is similar to that postulated for up to 20 mol% cholesterol in pure phosphatidylcholine bilayers by Copeland, B.R. and McConnell, H.M. (Biochim. Biophys. Acta, 599, 95–109 (1980)). Above the main phase transition temperature, asialo-G M1 appears to be organized into clusters, especially in palmitoyloleoylphosphatidylcholine (POPC) liposomes. This clustered distribution of glycosphingolipids seen above the phase transition temperature suggests that this type of lipid may exhibit compositional domain structure in biological membranes.

Liposomes in immunology: multilamellar phosphatidylcholine liposomes as a simple, biodegradable and harmless adjuvant without any immunogenic activity of its own.

Multilamellar phosphatidylcholine liposomes were coated with the antigens human serum albumin (HSA) or bovine gamma globulin (BGG) simply by suspending the liposomes in a solution of the antigens. Antigen coated phosphatidylcholine liposomes showed nearly the same adjuvant activity after intravenous injection as liposomes of a more complex phospholipid composition. Since phosphatidylcholine liposomes are biodegradable, harmless, easily obtainable, have no immunogenic activity of their own and may be administered intravenously, they seem to be a promising immunoadjuvant.

Carbon-13 nuclear magnetic resonance studies of the cholesteryl ester – phosphatidylcholine system

Using difference spectroscopy, 13C spin–lattice relaxation measurements on 40% egg phosphatidylcholine multilamellar liposomes (containing 25 mol% cholesteryl palmitate)–60% D2O indicate the ester contributes negligibly to the microviscosity of the acyl chains in the bilayers.At 37 °C, spin–spin and spin–lattice relaxation measurements have been obtained for 25 mol% cholesteryl linoleate in both egg phosphatidylcholine and dipalmitoyl phosphatidylcholine multilayers and indicate: (a) T1 values of the linoleate ester chain are 0.5 times those of the lecithin chain carbons, and (b) T2* = 16 ± 6 ms for all discernable linoleate carbons. At 52 °C, cholesteryl ring carbons C5 and C6 yield T2* values of 23 and 20 ms, respectively. These studies suggest the esters reside in an environment whose "fluidity" approaches that found in very low density lipoprotein (VLDL).