Relationship between the action of reactive oxygen and nitrogen species on bilayer membranes and antioxidants

Membrane lipid peroxidation (LPO) induced by hydroxyl (*OH) and ascorbyl (*Asc) radicals and by peroxynitrite (ONOO-) was investigated in asolectin (ASO), egg phosphatidylcholine (PC) and PC/phosphatidic acid mixtures (PC:PA) liposomes and rat liver microsomes (MC). Enthalpy variation (DeltaH) of PC:PA at different molar ratios were obtained by differential scanning calorimetry. It was also evaluated the LPO inhibition by quercetin, melatonin and Vitamin B6. The oxidant effect power follows the order *OH approximately *Asc > ONOO- on PC and MC; whilst on ASO liposomes, it follows *Asc > *OH approximately ONOO-. Increasing amounts of PA in PC liposomes resulted in lower levels of LPO. The DeltaH values indicate a more ordered membrane arrangement as a function of PA amount. The results were discussed in order to provide a complete view involving the influence of membranes, oxidants and antioxidants intrinsic behavior on the LPO dynamics.     

Effect of cisplatin, carboplatin and stobadine on lipid peroxidation of kidney homogenate and phosphatidylcholine liposomes.

The effects of the nephrotoxic, anticancer agents cisplatin (CDDP) and carboplatin (CBDCA), and the free radical scavenger, stobadine, were investigated on lipid peroxidation (LPO) of rat kidney homogenates and phosphatidylcholine (PC) liposomes. Kidney homogenates were incubated in air at 37 degrees C for 6-48 h and lipid peroxidation was detected spectroscopically as absorbance (533 nm) of the thiobarbituric acid-malondialdehyde (TBA-MDA) complex. CDDP (0.3-10 mmol.l-1) increased LPO of the homogenate. CBDCA decreased the TBA-MDA absorbance, yet was found to interfere with MDA, TBA and/or with the TBA-MDA complex. Thus when CBDCA is involved, the TBA-MDA method for detection of LPO is not suitable. Stobadine (0.1 mmol.l-1 and 1 mmol.l-1) inhibited LPO either in the control homogenate and in the homogenate where peroxidation was increased by CDDP. The effect of CDDP and CBDCA on peroxidation of PC liposomes was monitored as oxygen consumption using a Clark-type oxygen electrode. CDDP increased but CBDCA decreased the rate of oxygen consumption during the peroxidation of liposomes induced by FeSO4. The results suggest that the effects of CDDP and CBDCA on LPO may be linked with their nephrotoxicity.     

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.     

The characteristics of the radiation-initiated peroxidation of the phosphatidylcholine making up liposomes containing phospholipids which are susceptible to free-radical fragmentation

The regularities of accumulation of conjugated dienes and thiobarbituric acid (TBA)-reactive substances under gamma-irradiation of liposomes from rat liver phosphatidylcholine (PC) and its mixtures with the resistant to lipid peroxidation saturated phospholipids and bovine brain sphingomyelin (SM) were studied. It was established that the incorporation of negatively charged dipalmitoylphosphatidylglycerol (DPPG) and dipalmitoylphosphatidylethanol (DPPET) into lipid bilayer resulted in the increase of primary and secondary products of LPO, whereas neutral dipalmitoylphosphatidylcholine (DPPC) and SM involving in the phospholipid mixtures inhibited the peroxidation of PC. For anionic phospholipids, DPPG had more profound activating action on LPO, amongst the neutral phospholipids SM was more potent inhibitor of the reaction. Unlike DPPET and DPPC, DPPG and SM were subjected to free radical fragmentation on gamma-radiation. It is suggested that the intermediates and products of free radical fragmentation may modulate the progress of LPO.     

Effect of lysophosphatidylcholine on the radiation-induced lipid peroxidation in liposomes

The effect of lysophosphatidylcholine (LPC) on the lipid peroxidation process (LPO) in liposomes of rat liver phosphatidylcholine initiated by irradiation of 137Cs source was studied. The formation of diene conjugates (DC) is shown to increase dramatically on incorporation of more than 10% LPC into liposomes. The dependence of DC proportion on the irradiation dose is practically linear in the range of 0 to 5 kGy. The DC concentration in the liposomes without LPC grows at least to dose of 3.3 kGy and is unchanged on further irradiation. The malonic dialdehide accumulation follows the similar dependence. The LPC effect is neutralized by the incorporation of cholesterol into liposomes. The product of free radical fragmentation of LPC, palmitoxyacetone, practically has no influence on the DC concentration. The reasons of LPC effect on the irradiation initiated LPO in liposomes is discussed.     

Structural effect of cationic amphiphiles in diacetylenic photopolymerizable membranes

Liposomes have been an excellent option as drug delivery systems, since they are able of incorporating lipophobic and/or lipophilic drugs, reduce drug side effects, increase drug targeting, and control delivery. Also, in the last years, their use reached the field of gene therapy, as non-viral vectors for DNA delivery. As a strategy to increase system stability, the use of polymerizable phospholipids has been proposed in liposomal formulations. In this work, through differential scanning calorimetry (DSC) and electron spin resonance (ESR) of spin labels incorporated into the bilayers, we structurally characterize liposomes formed by a mixture of the polymerizable lipid diacetylenic phosphatidylcholine 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC(8,9)PC) and the zwitterionic lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), in a 1:1 molar ratio. It is shown here that the polymerization efficiency of the mixture (c.a. 60%) is much higher than that of pure DC(8,9)PC bilayers (c.a. 20%). Cationic amphiphiles (CA) were added, in a final molar ratio of 1:1:0.2 (DC(8,9)PC:DMPC:CA), to make the liposomes possible carriers for genetic material, due to their electrostatic interaction with negatively charged DNA. Three amphiphiles were tested, 1,2-dioleoyl-3-trimetylammonium-propane (DOTAP), stearylamine (SA) and trimetyl (2-miristoyloxietyl) ammonium chloride (MCL), and the systems were studied before and after UV irradiation. Interestingly, the presence of the cationic amphiphiles increased liposomes polymerization, MCL displaying the strongest effect. Considering the different structural effects the three cationic amphiphiles cause in DC(8,9)PC bilayers, there seem to be a correlation between the degree of DC(8,9)PC polymerization and the packing of the membrane at the temperature it is irradiated (gel phase). Moreover, at higher temperatures, in the bilayer fluid phase, more polymerized membranes are significantly more rigid. Considering that the structure and stability of liposomes at different temperatures can be crucial for DNA binding and delivery, we expect the study presented here contributes to the production of new carrier systems with potential applications in gene therapy.     

Intermembrane transport and antioxidant action of alpha-tocopherol in liposomes

Studies were made of the ability of alpha-tocopherol, incorporated into unilamellar liposomes from saturated or unsaturated phospholipids (donor liposomes) to inhibit the accumulation of lipid peroxidation (LPO) products in unilamellar liposomes from rat cerebral cortex lipids (acceptor liposomes) in the presence of LPO inducer (Fe + ascorbate). With the molar alpha-tocopherol: phospholipids rations from 1:1000 to 1:100 in donor liposomes, obtained through sonication of lipid dispersions, alpha-tocopherol was incorporated into both monolayers of liposomes and was distributed in monomeric form without forming clusters. Based on the dependencies of LPO inhibition on the alpha-tocopherol concentrations, we chose the ones that completely prevented the accumulation of LPO products in donor liposomes. Under these conditions LPO inhibition in mixtures of donor and acceptors liposomes was fully determined by the antioxidant effect of alpha-tocopherol in acceptor liposomes due to its intermembrane transfer. The efficiency of the "intermembrane" antioxidant action of alpha-tocopherol increased in the course of preincubation of donor and acceptor liposomes (up to 60 min) and this increase was more pronounced when the donor liposomes contained unsaturated phospholipids. Evidence was obtained that the intermembrane transfer of alpha-tocopherol did not result from the fusion of donor and acceptor liposomes during preincubation.     

Mechanical and Electrokinetic Effects of Polyamines/Phospholipid Interactions in Model Membranes

The mechanical and electrical properties of phospholipids layers influenced by interaction with polyamines were determined by measuring surface pressure and compression modulus of monolayers and zeta potential of liposomes. The saturated derivative of phosphatidic acid (DPPA) formed layers of the organization varying with compression degree. Contact of DPPA layers with polyamines present in the subphase resulted in changing their mechanical properties and the conditions in which the layer reorganization appears. The parameters corresponding to the layer reorganization depended on the size and charge of polyamines’ molecules. The values of: area per DPPA molecule, surface pressure at the point of layer structure reorganization, and surface pressure at the point of collapse characterizing of DPPA layers in the studied systems were determined. It was found that polyamines influenced to a much lesser extent the mechanical properties of monolayers formed from unsaturated derivative of phosphatidic acid slightly increasing its mechanical resistance in the range of higher molecular packing. The results of electrokinetic measurements revealed that surface charge of phosphatidic acid liposomes was effectively neutralized in the presence of polyamines. A similar effect was observed for phosphatidyl glycerol and for negatively charged polystyrene latex particles used as a reference. The influence of polyamines on the mechanical properties of DPPA layers was interpreted assuming a possibility of penetration of the lipid layer by polyamines’ molecules. Comparison of action of putrescine and calcium ions and effects of polyamines on phosphatidyl glycerol provided additional justification for the proposed interpretation of the observed effects.     

The biphasic effect of calcium on lipid peroxidation

Mechanisms underlying Ca2+ effects on lipid peroxidation (LPO) induced in liposomes (from egg yolk lecithin) and ufasomes (from linolenic acid and methyl linolenate) with the aid of an O2-(.) -generating system (Fe2+ + ascorbate) were studied. It was shown that stimulation of LPO by low Ca2+ concentrations (10(-6)-10(-5)M) was due to its ability to release Fe2+ ions bound to negatively charged (phosphate or carboxylic) lipid groups (of lecithin or linolenic acid), thus increasing the concentration of catalytically active Fe2+. The inhibitory effect of high Ca2+ concentrations was caused by its interaction with superoxide anion radicals and was not observed in LPO systems independent of O2- generation (e.g., Fe2+ + cumol hydroperoxide).     

Chelating and antiradical effect of rutin during peroxidation of lipids from microsomes and liposomes

The antioxidative effect of rutin (vitamin P) on Fe2+-induced lipid peroxidation (LPO) in bovine heart microsomes and lecithin liposomes was studied. It was shown that the LPO-induced inhibition of microsomes and liposomes in the presence of rutin occurs via two mechanisms, i.e., association of Fe2+ ions to form an inactive complex and a direct interaction between rutin and free radicals. The contribution of these mechanisms depends on the composition of the reaction mixture. In bovine heart microsomes and liposomes, ascorbic acid has a dual activity towards LPO. At high concentrations of Fe2+ necessary for LPO induction (approximately 1 x 10(-3) M), ascorbic acid blocks LPO, whereas at low Fe2+ concentrations (less than 1 x 10(-4) M) it has a prooxidative effect. A combined use of ascorbic acid and rutin results in an additive antioxidative effect at high Fe2+ concentrations (approximately 1.10(-3) M). However, at low Fe2+ concentrations rutin acts as an antagonist of the prooxidative effect of ascorbic acid.     

电子自旋共振波谱技术研究不同pH值对二棕榈酰磷脂酸脂质体相变性质的影响

本文用电子自旋共振(ESR)波谱技术研究了不同pH值对酸性磷脂二棕榈酰磷脂酸(DPPA)脂质体相变性质的影响。结果表明:DPPA脂质体随pH值的增加其相变温度降低。通过对测试条件的选择和波谱参数的测量,TEMPO标记的DPPA脂质体在HEPES溶液中ESR谱不出现疏水峰的原因,是由于仪器分辨率不够以及自旋标记探针TEMPO在DPPA脂质体脂双层疏水相中的超精细偶合常数Ao和各向同性go因子与它们在HEPES溶液中的值相近。     

The Role of Secondary Messengers in the Regulation of Lipid Peroxidation in Rat Liver Microsomes

The effect of phorbol-12-myristate-13-acetate (PMA), an activator of protein kinase C (PK-C) on lipid peroxidation (LPO) in rat liver homogenates and microsomes was studied. PMA (10^?10 to 10^?6M) produced a concentration-dependent inhibition of LPO, which was greatly decreased by polymyxin B (Px B) (an inhibitor of PK-C). The non-active analogue of PMA, 4α-phorbol-12,13-didecanoate (4α-PDD) exerted no inhibitory effect. The adenylate cyclase activator, forskolin (FK) (10^?6 M) abolished the inhibitory effect of PMA on LPO. PMA and FK did not inhibit LPO in liposomes. It is suggested that LPO in biomembranes could be regulated by PK-C, whose inhibitory effect might be prevented by CAMP-dependent protein kinases.     

Headgroup structure and fatty acid chain length of the acidic phospholipids modulate the interaction of membrane mimetic vesicles with the antimicrobial peptide protegrin-1.

The interaction of protegrin-1 (PG-1), a small beta-sheet antimicrobial peptide with acidic phospholipid model membranes was investigated by differential scanning calorimetry. We found that PG-1 can distinguish between liposomes of the anionic phospholipids DPPG, DPPS and DPPA, eventhough the headgroups of these phospholipids all have the same net charge and they carry the same hydrocarbon chains. Specifically, PG-1 had only a minor effect on the thermotropic phase behavior of DPPA liposomes, while it interacted preferentially with the fluid phase of DPPS. Furthermore, PG-1 could induce a phase separation in DPPG liposomes resulting in the formation of peptide-rich domains even at low concentrations of the peptide. However, this peptide-rich domain was not evident when the fatty acyl chains were longer or shorter by two carbon atoms. In addition, PG-1 can also form peptide-rich domains in DPPS vesicles but only at high concentrations of the peptide. These results suggest that in addition to an overall negative charge, the structural features of the phospholipid headgroups, lipid packing and thus membrane fluidity will influence the interaction with PG-1, thereby modulating its biological activity.     

Relevance of lipid polar headgroups on boron-mediated changes in membrane physical properties

Using liposomes composed of either brain phosphatidylcholine (PC), or binary mixtures of PC and phosphatidylserine (PS), galactolipids (GL), phosphatidylinositol (PI), cardiolipin (CL), phosphatidic acid (PA), or phosphatidylethanolamine (PE), we investigated the effects of graded amounts of boric acid (B, 0.5-1000 microM) on the following membrane physical properties: (a) surface potential, (b) lipid rearrangement through lateral phase separation, (c) fluidity, and (d) hydration. Incubation of the different populations of vesicles with B was associated with a small, but statistically significant, increase in membrane surface potential in PC, PC:PS, PC:GL, PC:PI, PC:PA, and PC:PE liposomes. B-induced lipid lateral rearrangement through lateral phase separation in PC, PC:PA, and PC:PE liposomes; but had no effects on PC:PS, PC:GL, and PC:PI liposomes. In PC liposomes B affected membrane fluidity at the water-lipid interface without affecting the hydrophobic core of the bilayer. In all the other binary liposomes studied, B increased membrane fluidity in both, the hydrophobic portion of the membrane and in the anionic domains. The above was associated with a decrease in the fluidity of the cationic domains. B (10-1000 microM) decreased membrane hydration regardless the composition of the liposomes. The obtained results demonstrate the ability of B to interact with membranes, and induce changes in membrane physical properties. Importantly, the extent of B-membrane interactions and the consequent effects were dependent on the nature of the lipid molecule; as such, B had greater affinity with lipids containing polyhydroxylated moieties such as GL and PI. These differential interactions may result in different B-induced modulations of membrane-associated processes in cells.     

Cataract induction by products of lipid peroxidation

Liposome suspension prepared from the unsaturated phospholipids exposed to lipid peroxidation (LPO) induced posterior subcapsular cataracts after injection into the posterior vitreous of rabbit eyes. In the background of this model lies a type of lens opacity formed during retinal degeneration when toxic peroxide substances diffuse anteriorly through the vitreous body resulting in vitreous opacities and complicated cataracts. Saturated liposomes (prepared from beta-oleoyl-gamma-palmitoyl) L-alpha-lecithin) did not induce lens opacities, which is the evidence that a lipid peroxidation mechanism may be responsible for the posterior cataracts. Along with cataract formation accumulation of LPO fluorescent products in vitreous, aqueous humor and lens was observed. It was followed by a decreased level of reduced glutathione in the lens. The obtained results strongly support the hypothesis of LPO initial role in cataracts.     

Comparison of perturbation effect of propranolol, verapamil, chlorpromazine and carbisocaine on lecithin liposomes and brain total lipid liposomes. An EPR spectroscopy study.

Effect of verapamil, propranolol, chlorpromazine and carbisocaine on dynamics and/or order of liposomes (perturbation effect), prepared from different molar ratios of lecithin (PC) and rat brain total lipids (TL) was studied by EPR spectroscopy using spin probes 16-doxyl stearic acid and 14-doxyl phosphatidylcholine. The PC liposomes had higher dynamics and/or lower order than the TL liposomes. The perturbation effect of the drugs depended largely on the lipid composition of the liposomes. The drugs at the drug/lipid molar ratios from 0.1 to 1 increased membrane dynamics and/or decreased membrane order. The drugs had the most pronounced perturbation effect in the liposomes prepared from brain total lipids. The effect of the drugs decreased with decreasing the TL/PC ratio in the liposomes and was lowest, almost diminished, in the PC liposomes. Increasing concentration of the drugs decreased the difference between the dynamics and/or order of the PC and TL liposomes and so eliminated the influence of lipid composition on these membrane parameters. The results emphasize the role of lipid composition in studies concerning drug-lipid interactions in model and biological membranes.     

Hydrogen peroxide production from reactive liposomes encapsulating enzymes.

Reactive cationic and anionic liposomes have been prepared from mixtures of dimyristoylphosphatidylcholine (DMPC) and cholesterol incorporating dimethyldioctadecylammonium bromide and DMPC incorporating phosphatidylinositol, respectively. The liposomes were prepared by the vesicle extrusion technique and had the enzymes glucose oxidase (GO) encapsulated in combination with horseradish peroxidase (HRP) or lactoperoxidase (LPO). The generation of hydrogen peroxide from the liposomes in response to externally added D-glucose substrate was monitored using a Rank electrode system polarised to +650 mV, relative to a standard silver-silver chloride electrode. The effects of encapsulated enzyme concentration, enzyme combinations (GO+HRP, GO+LPO), substrate concentration, electron donor and temperature on the production of hydrogen peroxide have been investigated. The electrode signal (peroxide production) was found to increase linearly with GO incorporation, was reduced on addition of HRP and an electron donor (o-dianisidine) and showed a maximum at the lipid chain-melting temperature from the anionic liposomes containing no cholesterol. To aid interpretation of the results, the permeability of the non-reactive substrate (methyl glucoside) across the bilayer membranes was measured. It was found that the encapsulation of the enzymes effected the permeability coefficients of methyl glucoside, increasing them in the case of anionic liposomes and decreasing them in the case of cationic liposomes. These observations are discussed in terms of enzyme bilayer interactions.     

Effect of oxidized phospholipids on the chemiluminescence of zymosan-activated leukocytes

The effect of liposomes with different degree of oxidation on the zymosan-induced chemiluminescence (CL) of leukocytes was investigated. Non-oxidized liposomes did not influence significantly the CL response of leukocytes. In contrast previously oxidized liposomes increased CL even if liposomes and cells were separated by a dialysis membrane. Based on the observed increase of luminol-activated CL by oxidized liposomes, lipid peroxidation (LPO) products may be suggested to enhance cell activation. Zymosan-activated leukocytes did not affect the amount of malondialdehyde (MDA) in non-oxidized liposomes unless iron salts were added. Fe3+ + ADP added to non-oxidized liposomes triggered LPO. Both catalase and superoxide dismutase (SOD) prevented the effect. In experiments with previously oxidized liposomes the activated oxygen species produced by leukocytes did not increase the amount of MDA; on the contrary, they decreased it both in the presence and in the absence of chelated iron in the liposome suspension. The reaction between lipid hydroperoxide and O2- widely accompanied by CL. SOD decreased CL in this system by a factor of 1.7. On the other hand, peroxidized lipids may "opsonize" initially inactive particles: oxidized liposomes increased CL response of leukocytes similarly as opsonized zymosan routinely used as a phagocyte activator.     

Characterization of diacetylenic liposomes as carriers for oral vaccines

In order to evaluate liposomes as vehicle for oral vaccines the characterization and stability of polymerized and non-polymerized liposomes were examined. Mixtures of 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3 phosphocholine) (DC8,9PC) with saturated 1,2-dimiristoyl-sn-glycero-3-phosphocholine in molar ratio 1:1 were used. Saturated and non-saturated lipids were combined to give a chemically modified membrane by UV polymerization derived from DC8,9PC. Characterization was carried out by electronic microscopy, differential scanning calorimetry (DSC) and by hydrophobicity factor (HF). The stability towards the digestive tract (including saliva): acidic solutions, bile and pancreatin are compared to buffer pH 7.4, measuring the release of Glucose-6-phosphate or bovine plasma albumin entrapment. The polymerized liposomes showed further augmentation of the HF and the size. DSC showed phase separation and lower Tt if compared to data obtained for DC8,9PC. The HF, as main factor is discussed in relation to in vitro stability, suggesting that polymerized and non-polymerized liposomes would serve effectively as an oral delivery vehicle.     

Effect of lipid composition upon fusion of liposomes with Sendai virus membranes

How the lipid composition of liposomes determines their ability to fuse with Sendai virus membranes was tested. Liposomes were made of compositions designed to test postulated mechanisms of membrane fusion that require specific lipids. Fusion does not require the presence of lipids that can form micelles such as gangliosides or lipids that can undergo lamellar to hexagonal phase transitions such as phosphatidylethanolamine (PE), nor is a phosphatidylinositol (PI) to phosphatidic acid (PA) conversion required, since fusion occurs with liposomes containing phosphatidylcholine (PC) and any one of many different negatively charged lipids such as gangliosides, phosphatidylserine (PS), phosphatidylglycerol, dicetyl phosphate, PI, or PA. A negatively charged lipid is required since fusion does not occur with neutral liposomes containing PC and a neutral lipid such as globoside, sphingomyelin, or PE. Fusion of Sendai virus membranes with liposomes that contain PC and PS does not require Ca2+, so an anhydrous complex with Ca2+ or a Ca2+-induced lateral phase separation is not required although the possibility remains that viral binding causes a lateral phase separation. Sendai virus membranes can fuse with liposomes containing only PS, so a packing defect between domains of two different lipids is not required. The concentration of PS required for fusion to occur is approximately 10-fold higher than that required for ganglioside GD1a, which has been shown to act as a Sendai virus receptor. When cholesterol is added as a third lipid to liposomes containing PC and GD1a, the amount of fusion decreases if the GD1a concentration is low.(ABSTRACT TRUNCATED AT 250 WORDS)