Lipid Biomembranes Research Articles

Antitumor lipids in biomembranes modeled with the Langmuir monolayer technique

Studies on interactions between physiologically active compounds and cell membrane components in Langmuir monolayers, which provide suitable models of biomembranes, can be considered as one of the crucial factors leading to the understanding of the mode of action of many drugs that act on membrane level. A good example of membrane-targeted drugs are synthetic analogues of lysophosphatidylcholine, generally named as antitumor lipids (ATLs), due to their anticancer properties. They have advantages over traditional cytostatics as they do not target DNA but cellular membrane. Apart from many studies on cell lines, little is known about the transport of ATLs to the cells, and it is still unclear which membrane components target drug molecules to the tumor cell membrane, sparing the normal cells. This review provides examples of the successful application of the Langmuir monolayer technique in getting insight into biological action and selectivity of representative ATL – edelfosine.

Effect of curcumin on lateral diffusion of phosphatidylcholines in saturated and unsaturated bilayers.

Curcumin, a dietary polyphenol, is a natural spice with preventive and therapeutic potential for neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Curcumin possesses a spectrum of antioxidant, anti-inflammatory, anticarcinogenic, and antimutagenic properties. Because of this broad spectrum of pharmacological activity, it has been suggested that, like cholesterol, curcumin exerts its effect on a rather basic biological level, such as on lipid bilayers of biomembranes. The effect of curcumin on translational mobility of lipids in biomembranes has not yet been studied. In this work, we used (1)H NMR diffusometry to explore lateral diffusion in planar-oriented bilayers of dimyristoylphosphatidylcholine (DMPC) and dioleoylphosphatidylcholine (DOPC) at curcumin concentrations of up to 40 mol % and in the temperature range of 298-333 K. The presence of curcumin at much lower concentrations (∼7 mol %) leads to a decrease in the lateral diffusion coefficient of DOPC by a factor of 1.3 at lower temperatures and by a factor of 1.14 at higher temperatures. For DMPC, the diffusion coefficient decreases by a factor of 1.5 at lower temperatures and by a factor of 1.2 at higher temperatures. Further increasing the curcumin concentration has no effect. Comparison with cholesterol showed that curcumin and cholesterol influence lateral diffusion of lipids differently. The effect of curcumin is determined by its solubility in lipid bilayers, which is as low as 10 mol % that is much less than that of cholesteroĺs 66 mol %.

Watching individual molecules flex within lipid membranes using SERS

Interrogating individual molecules within bio-membranes is key to deepening our understanding of biological processes essential for life. Using Raman spectroscopy to map molecular vibrations is ideal to non-destructively ‘fingerprint’ biomolecules for dynamic information on their molecular structure, composition and conformation. Such tag-free tracking of molecules within lipid bio-membranes can directly connect structure and function. In this paper, stable co-assembly with gold nano-components in a ‘nanoparticle-on-mirror’ geometry strongly enhances the local optical field and reduces the volume probed to a few nm3, enabling repeated measurements for many tens of minutes on the same molecules. The intense gap plasmons are assembled around model bio-membranes providing molecular identification of the diffusing lipids. Our experiments clearly evidence measurement of individual lipids flexing through telltale rapid correlated vibrational shifts and intensity fluctuations in the Raman spectrum. These track molecules that undergo bending and conformational changes within the probe volume, through their interactions with the environment. This technique allows for in situ high-speed single-molecule investigations of the molecules embedded within lipid bio-membranes. It thus offers a new way to investigate the hidden dynamics of cell membranes important to a myriad of life processes.

Phenolic content and biological activity of extracts of blackcurrant fruit and leaves

The studies were designed to determine the polyphenolic composition and biological activity of extracts from fruit and leaves of blackcurrant in relation to biological and lipid membranes. A detailed quantitative and qualitative analysis of extracts was conducted, using the UPLC-DAD and UPLC-PDA–Q/TOF-MS methods. Furthermore, stability of the phenolic content in the extracts was determined using the Folin–Ciocalteu method. The antioxidant activity of the extracts in relation to the membrane of erythrocytes and lipids extracted from red blood cell membranes (RBCL) exposed to chemical oxidizing agents (AAPH) was determined, and the effects of blackcurrant extracts on the properties of the membrane of erythrocytes and liposomes (DPPC and RBCL) were examined. Antioxidant activity of the extracts was studied fluorometrically, while effects of the extracts on the properties of membranes were examined using calorimetric, IR spectroscopy and fluorimetric methods. In particular, the effects of the extracts on packing order, membrane fluidity and the main phase transition temperature were determined. Additionally, the affinity of extracts to organic or aqueous media was determined on the basis of their partition coefficient between octanol and phosphate buffer. The results showed that the tested extracts are rich sources of polyphenols, primarily from the group of flavonoids; in leaves flavonols dominate, while in fruit anthocyanins dominate. Their polyphenolic content was quite stable and only slightly changed within 12months. The substances used markedly protect the membranes against oxidation and to varying degrees modify properties of the hydrophilic regions of membranes. They localize mainly in the area of lipid polar heads of membrane, changing their arrangement, which is consistent with their disclosed hydrophilic character. Due to the rich polyphenolic composition and high antioxidant activity, extracts from the leaves and fruits of blackcurrant protect the organism or food products from the harmful effects of free radicals. In addition, they slightly change the properties of biomembrane lipids and lipid vesicles once they incorporate these extracts. This kind of knowledge is not only of pro-health importance, but can also be used in the food, pharmaceuticals or cosmetics industry, for designing liposomal structures as carriers of drugs, dietary supplements and extracts as biologically active substances.

Effect of penehyclidine hydrochloride on β-arrestin-1 expression in lipopolysaccharide-induced human pulmonary microvascular endothelial cells

β-arrestins are expressed proteins that were first described, and are well-known, as negative regulators of G protein-coupled receptor signaling. Penehyclidine hydrochloride (PHC) is a new anti-cholinergic drug that can inhibit biomembrane lipid peroxidation, and decrease cytokines and oxyradicals. However, to date, no reports on the effects of PHC on β-arrestin-1 in cells have been published. The aim of this study was to investigate the effect of PHC on β-arrestin-1 expression in lipopolysaccharide (LPS)-induced human pulmonary microvascular endothelial cells (HPMEC). Cultured HPMEC were pretreated with PHC, followed by LPS treatment. Muscarinic receptor mRNAs were assayed by real-time quantitative PCR. Cell viability was assayed by the methyl thiazolyl tetrazolium (MTT) conversion test. The dose and time effects of PHC on β-arrestin-1 expression in LPS-induced HPMEC were determined by Western blot analysis. Cell malondialdehyde (MDA) level and superoxide dismutase (SOD) activity were measured. It was found that the M3 receptor was the one most highly expressed, and was activated 5 min after LPS challenge. Furthermore, 2 μg/mL PHC significantly upregulated expression of β-arrestin-1 within 10 to 15 min. Compared with the control group, MDA levels in cells were remarkably increased and SOD activities were significantly decreased in LPS pretreated cells, while PHC markedly decreased MDA levels and increased SOD activities. We conclude that PHC attenuated ROS injury by upregulating β-arrestin-1 expression, thereby implicating a mechanism by which PHC may exert its protective effects against LPS-induced pulmonary microvascular endothelial cell injury.

Concluding remarks Back to the future: mechanics and thermodynamics of lipid biomembranes

To some extent, Faraday Discussion 161 on Lipids andMembrane Biophysics is an off spring of the earlier Faraday Discussion 81 on Lipid Vesicles and Membranes in 1986, as brought to our attention by John Nagle in his Introductory Lecture. Yet, in my view, Discussion 161 has been far more polished in its content—revealing novel materials and challenging experiments, new instrumentation and computational methods—with very well organized presentations (plus posters). Personally, I was wowed by every 5 min abstract of a paper—all were succinct and each set the stage for a well balanced 25 min period of discussion. On the other hand, with the notable exception of an excellent Introductory Lecture and within the caveat of my failing memory, it seemed to me that there was less focus on the underlying physics of membranes than in Discussion 81. At the same time, there was more attention, in my opinion, than needed to the parochial issue of ‘‘raft–lipid membranes’’. Also, I was left with an uncertain feeling about whether coarse-graining in molecular-scale simulations will manage (in my lifetime) to completely fulfil its agonizing quest for ‘‘material reality’’. Beyond this hubris, I feel unqualified to pick and choose amongst the 24 excellent Discussion papers to praise or criticize. Instead, the objective of my Concluding remarks will be to expand on/add to the membrane biophysics emphasized in John Nagle’s Introductory Lecture which, although well appreciated by the ‘‘old guard’’, was perhaps too esoteric for many of the bright ‘‘new comers’’ to the subject. In particular, my aim is to provide some additional background, a kind of classroom ‘‘tutorial’’, regarding important mechanical and thermodynamic features of vesicle bilayers, lore established over the past two decades since Discussion 81. At the same time, I want to highlight some new directions in the biophysics of membranes suggested by these insights. My intent is not to give a balanced review of the literature. Rather, I have chosen references that have played seminal roles in the developments I will describe. I dedicate this paper to our dear friend Myer Bloom, soon to be 84 years of age (07/12/2012)—ever bright in mind, even though encumbered by Parkinson’s disease. Also, I want to express my sincere thanks to the Organizing Committee for the opportunity to participate in Faraday Discussion 161 and for the hospitality shown by their wonderful RSC associates. Evan Evans

Phospholipase-Catalyzed Hydrolysis in an Artificial Cell Membrane in the Presence of Melittin

Biomimicry involves the use of the structure and function of biological systems as models for the design and engineering of materials and machines. An artificial cell membrane was developed using biomembrane components, and the membrane, formed by a lipid bilayer, was analyzed using surface plasmon resonance (SPR) to monitor hydrolysis by phospholipase (PL). The simultaneous atomic force microscope (AFM) images show that PL catalyzed the nanometer-scale hydrolysis of the artificial lipid biomembranes through enzymatic hydrolysis. In addition, it was confirmed that the combination of PL and melittin allowed the control of enzyme hydrolysis for the degradation of the lipid bilayer. Regarding the expected activating effect of melittin on hydrolysis, no difference with respect to the non-treated lipid membrane was observed in the AFM images. It is assumed that the partitioning of melittin into the membrane might prevent the binding or hydrolysis of Phospholipase A2 (PLA2). This study provides basic knowledge on a new approach for patterning biomimicking lipid membranes on a nano-scale.

A Comprehensive View on the Brownian Motion of Quantum Dots in Electrolytic Solution, Lipid Bilayer and Their Aggregated State in the Lipid Biomembrane

A Comprehensive View on the Brownian Motion of Quantum Dots in Electrolytic Solution, Lipid Bilayer and Their Aggregated State in the Lipid Biomembrane

Cubic phases in membrane lipids

On the basis of data obtained by time-resolved X-ray diffraction, we consider in the present article the occurrence and formation pathways of inverted bicontinuous cubic phases, or bilayer cubic phases, Q (II)(B) , in diluted dispersions of lipids representing major biomembrane lipid classes [phosphatidylethanolamines (PEs), mixtures of PEs and phosphatidylcholines (PCs) with other lipids, glycolipids]. We show that Q (II)(B) formation proceeds much more easily upon cooling from the H(II) phase than upon heating or isothermal conversion from the L(α) phase, thus identifying an indirect but faster route for Q (II)(B) phase induction in lipids. The data collected consistently show that the ability to convert into cubic phase upon temperature cycling appears to be a general property of all lipids exhibiting an L(α) ↔ H(II) phase transition. Admixtures of charged phospholipids, both anionic and cationic, strongly facilitate Q (II)(B) formation in PEs. Their effect may be attributed to increased electrostatic repulsion between the lipid bilayers that reduces the unbinding energy and facilitates the dissipation of the L(α) phase required for its conversion into bilayer cubic phase.

The membrane interaction of drugs as one of mechanisms for their enantioselective effects

The discrimination between different enantiomers of chiral compounds by the biological system is medically important as the pharmacological and toxicological effects of enantiomeric drugs significantly differ depending on their stereostructures. One enantiomer is preferred over its enantiomeric counterpart and a racemic mixture for higher activity or lower toxicity. Such enantioselectivity has been exclusively explained by the stereostructure-specific interactions with receptors, channels and enzymes of drugs including general and local anesthetics, sedatives, hypnotics, anti-inflammatory drugs, analgesics and β-adrenergic antagonists. These drugs can act on not only protein targets but also lipid biomembranes. Almost all of the relevant proteins are embedded in or associated with membrane lipid bilayers. Therefore, we propose one of possible mechanisms that drugs might enantioselectively interact with membrane lipids and induce changes in membrane property like fluidity which are discriminable between enantiomers. If the induced changes are different between enantiomers, enantiomeric drugs would differently influence the membrane lipid environments for receptors, channels and enzymes, resulting in the enantioselectivity of drug effects. The enantioselective membrane interactions of drugs could be mediated by membrane component cholesterol and phospholipids, both of which have chiral centers in structure as well as drug enantiomers. Chiral membrane lipids possibly exhibit the preference for the interactions with drug molecules of either the same chirality or the different chirality, producing the selectivity to one drug enantiomer. The proposed hypothesis may be available to investigate more useful medicines based on the novel concept of drug enantioselectivity.

Abstract 597: Response of reactive oxygen species to eicosapentaenoic and calorie restriction in relation to mammary tumor prevention

Abstract Exposure of lipid biomembranes to reactive oxygen species (ROS) results in the generation of a wide variety of lipid peroxidation products. Some of these products, including 4-hydroxy-2-nonenal (4HNE) can form protein adducts which may impact pathways associated with tumorigenesis. We have reported that calorie restriction protects against spontaneous mammary tumor development in mice and have recently shown that this is accompanied by a decrease in serum levels of lipid peroxidation products. Eicosapentaenoic acid (EPA) is known to inhibit mammary tumorigenesis; however, this type of highly unsaturated omega-3 fatty acid has also been shown to increase in vivo levels of lipid peroxidation products. In the present study, the interaction of calorie restriction and dietary EPA was investigated with respect to mammary tumor formation. Lipid peroxidation products of ROS were also measured. From 10 weeks of age MMTV-Her2/neu mice were fed a modified AIN-93M diet (10.2% kcal from fat) which had either 100% soybean oil (SO) as the fat source or a blend of EPA (72%) and SO (18%). Mice were further divided into groups which were ad libitum-fed, (AL); chronic calorie restricted (CCR, received 75% of AL) or intermittently calorie restricted (ICR, received 50% of AL for 3 weeks followed by 3 weeks at 100% of AL) until 60 weeks of age or until mammary tumor size required euthanasia. Mice were weighed and examined for mammary tumors weekly. Serum thiobarbituric acid reactive substances (TBARS) and 4-hydroxy-2-nonenal (4HNE)-protein adducts in mammary tissues were measured to assess the extent of lipid peroxidation. In both diet groups, mammary tumor incidence was highest in the AL group (SO 87%, EPA 63%). These groups also had the highest levels of serum TBARS and the AL-SO group had the highest level of mammary tissue 4HNE-protein adducts. The calorie restricted mice had lower levels of mammary tumor incidence (CCR-SO 47%, CCR-EPA 40%, ICR-SO 59%) with the lowest level in the ICR-EPA group (15%). The ICR-SO group had serum TBARS and mammary tissue 4HNE-protein adduct levels that were significantly lower than the AL-SO group (p<0.05 for both). In the EPA groups, there was no difference between the AL, CCR, and ICR groups in either serum TBARS or mammary tissue 4HNE-protein adducts. These results suggest that higher levels of lipid peroxidation products are associated with a higher incidence of mammary tumors. However, although the dietary combination of ICR and EPA resulted in the greatest amount of mammary tumor inhibition, this combination was not associated with the lowest level of lipid peroxidation products. The relationship between EPA, calorie restriction, and reactive oxygen species appears to be quite complex. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 597. doi:1538-7445.AM2012-597

From simple to complex: investigating the effects of lipid composition and phase on the membrane interactions of biomolecules using in situ atomic force microscopy

Biomembrane lipid composition and lateral heterogeneity vary significantly both spatially and temporally and have been shown to be associated with cell functions. Lipid raft-based membrane heterogeneity might be involved with cell physiological and pathological processes. Therefore, there is a great need to investigate the roles of lipid composition and lateral heterogeneity in membrane-related physiological and pathological processes. Supported lipid bilayers (SLBs) of increasing complexity are excellent membrane model systems to investigate the effects of lipid composition and phase on the membrane interactions of biomolecules. In situ AFM is a powerful tool to examine the dynamic interactions between SLBs and biomolecules on a nanoscale. Therefore, the in situ AFM measurements between SLBs of increasing complexity and biomolecules are excellent ways to investigate the effects of lipid composition and phase on the membrane-related processes. In this review, the following basic knowledge is first discussed: biomembrane lipid composition, lipid raft, lipid phase separation, SLBs, and AFM. Then the biological applications of in situ AFM to visualize the interactions between SLBs of increasing complexity and biomolecules are discussed.

Fourth-Order Curvature Energy Model for the Stability of Bicontinuous Inverted Cubic Phases in Amphiphile−Water Systems

The bicontinuous inverted cubic (Q(II)) phases of amphiphiles in water have many practical applications. It is necessary to understand the stability of these phases as a function of composition and ambient conditions in order to make the best use of them. Moreover, many biomembrane lipids and some biomembrane lipid extracts form Q(II) phases. The stability of Q(II) phases in a given lipid composition is closely related to the susceptibility of that composition to membrane fusion: changes in composition that stabilize Q(II) phases usually increase the rate of membrane fusion. However, the factors determining Q(II) phase stability are not fully understood. Previously, an expression was derived for the curvature free energy of Q(II) phases with respect to that of the lamellar (L(alpha)) phase using a model for the curvature energy with terms up to fourth order in curvature as formulated by Mitov. Here this model is extended to account for the effects of water content on Q(II) phase stability. It is shown that the observed L(alpha)/Q(II) phase-transition temperature, transition enthalpy, and transition kinetics are all sensitive to water content. The same observables also become sensitive to small noncurvature energy contributions to the total free-energy difference between the Q(II) and L(alpha) phases, especially the unbinding energy in the L(alpha) phase. These predictions rationalize earlier observations of Q(II) phase formation in N-monomethylated dioleoylphosphatidylethanolamine that otherwise appear to be inconsistent. The model also provides a fundamental explanation of the hysteresis typically observed in transitions between the L(alpha) and Q(II) phases. It is an accurate model of Q(II) phase stability when the ratio of the volume fraction of the lipid in the Q(II) phase unit cell is < or = 0.5.

バイオ材料としての部分含フッ素脂質液晶膜の設計と特性化

This article reviews our approaches for designing fluorinated lipid liquid–crystal membranes suitable for biomaterials such as membrane protein reconstitution matrices. We proposed several partially fluorinated fatty acids as well as phospholipids bearing long (C18) hydrophobic chains comparable to biomembrane lipids. The fatty acids and/or phospholipids containing perfluoroalkyl group was easily synthesized in good yields. Judging from the collapse (equilibrium spreading) pressures of the proposed fatty aids and/or phospholipids at the air–water interface, the partial fluorination of hydrophobic chains was enough to improve their monolayer stability. The fluorinated phospholipid with C–C triple bonds formed flexible structures such as stable vesicles in water at a room temperature. The partially fluorinated phospholipids are applicable to protein reconstitution matrices, e.g., reconstitution of photosynthetic membrane proteins.

Regulation of catalytic activity of acid phosphatase by lipids in a reverse micellar system

The influence of biomembrane lipids on the catalytic activity of a peripheral membrane enzyme, acid phosphatase (AP), was studied in a reverse micellar system. It was found that the interaction of AP with lipids led to a number of kinetic effects depending on lipid nature on enzyme function. The observed effects might be caused by the formation of lipoprotein complexes as well as by the influence of lipids on structure and properties of the micellar matrix. The results are important for clear understanding of molecular mechanisms of regulation of the catalytic activity of the membrane-associated enzyme in vivo. These data can also be used as a physicochemical basis for application of AP in medical fields as a diagnostic tool for diseases caused by changes in lipid metabolism, e.g. urinary, orthopedic, and allergic diseases.

Trans-monolayer coupling of fluid domains in lipid bilayers

Lipid membranes that exhibit fluid–fluid phase coexistence are widely studied as models for the physical state of lipids in raft-forming biomembranes. Yet, whereas model membranes are typically symmetric, biomembranes maintain different lipid compositions in their two leaflets. In the plasma membrane, only the extracellular but not the cytoplasmic leaf contains a raft-competent lipid mixture. This raises the question if a raft-like state in the extracellular layer is able to induce a similar state also on the cytoplasmic side. Recent studies have thus begun to investigate the problem of trans-monolayer domain coupling in model membranes. Experimental work on asymmetric membranes has revealed that a tendency to phase-separate in one leaf can induce phase separation on the other leaf. Conversely, the lack of such a tendency can prevent phase separation in the apposed leaf. The strength of the coupling between domains can be described by a composition-dependent surface tension or, for small compositional changes, by a single coupling constant. Theoretical work has demonstrated how the coupling constant affects the phase behavior of a binary membrane. We summarize previous experimental findings and modeling efforts. In addition, three possible physical mechanisms of trans-monolayer domain coupling are discussed: electrostatic coupling, cholesterol flip-flop, and dynamic chain interdigitation. We argue that dynamic chain interdigitation likely provides the main contribution to the coupling constant. The charges of acidic lipids tend to reduce the coupling constant – but the reduction is insignificant even at low salt content.

Biological detection and analysis of mercury toxicity to alfalfa ( Medicago sativa) plants

Mercury has become one of the major causes of toxic metal pollution in agricultural lands. Accumulation of mercury by plants may disrupt many cellular functions and block growth and development. To assess mercury toxicity, we performed an experiment focusing on the responses of alfalfa ( Medicago sativa) to Hg 2+-induced oxidative stress. Alfalfa plants were treated with 0–40 μM HgCl 2 for 7 d. The concentrations of Hg 2+ were positively correlated with the generation of O 2 − and H 2O 2 in leaves. Treatment with Hg 2+ increased the activities of NADH oxidase and lipoxygenase (LOX) and damaged the biomembrane lipids. To understand biochemical responses under Hg stress, activities of several antioxidant enzymes, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) were assayed. Analysis of SOD activity by non-denaturing polyacrylamide gel electrophoresis revealed five isoforms in leaves, but they showed different patterns. Also, eight isoenzymes of APX and seven of POD in leaves were detected. However, only one isoform of CAT was visualized. The total activities of APX, POD and CAT were generally enhanced. We also measured several antioxidative metabolites such as ascorbate and glutathione (GSH), and found that both differentially accumulated in leaves. These results indicate that the increased levels of O 2 − and H 2O 2 under Hg stress were closely linked to the improved capacity of antioxidant enzymes. The data not only provide the important information for better understanding of the toxic and tolerance mechanisms, but as well can be used as a bio-indicator for soil contamination by Hg.

Employing an Amphipathic Viral Peptide to Create a Lipid Bilayer on Au and TiO2

Supported lipid bilayers formed by the fusion of small unilamellar vesicles onto SiO2 or organic film-modified surfaces serve as model membranes in both scientific research and practical applications. Here, we describe the use of an amphipathic α-helical viral peptide derived from the hepatitis C virus NS5A protein (AH peptide) to destabilize the vesicles leading to lipid bilayer formation on gold and TiO2 solid substrates. Whereas previous researchers have been limited in their selection of surface materials for lipid biomembranes, the use of such peptides as destabilizing agents will allow the freedom to choose a broader variety of solid substrates to support planar bilayers. In particular, the favorable electrical properties of gold and the beneficial biocompatibility of TiO2 make these substrates attractive. The formation of model lipid bilayers supported on gold and TiO2 substrates can be utilized in many membrane-associated biological, physiological, or electrochemical applications with the advantag...

Natural lipid extracts and biomembrane-mimicking lipid compositions are disposed to form nonlamellar phases, and they release DNA from lipoplexes most efficiently

A viewpoint now emerging is that a critical factor in lipid-mediated transfection (lipofection) is the structural evolution of lipoplexes upon interacting and mixing with cellular lipids. Here we report our finding that lipid mixtures mimicking biomembrane lipid compositions are superior to pure anionic liposomes in their ability to release DNA from lipoplexes (cationic lipid/DNA complexes), even though they have a much lower negative charge density (and thus lower capacity to neutralize the positive charge of the lipoplex lipids). Flow fluorometry revealed that the portion of DNA released after a 30-min incubation of the cationic O-ethylphosphatidylcholine lipoplexes with the anionic phosphatidylserine or phosphatidylglycerol was 19% and 37%, respectively, whereas a mixture mimicking biomembranes (MM: phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine /cholesterol 45:20:20:15 w/w) and polar lipid extract from bovine liver released 62% and 74%, respectively, of the DNA content. A possible reason for this superior power in releasing DNA by the natural lipid mixtures was suggested by structural experiments: while pure anionic lipids typically form lamellae, the natural lipid mixtures exhibited a surprising predilection to form nonlamellar phases. Thus, the MM mixture arranged into lamellar arrays at physiological temperature, but began to convert to the hexagonal phase at a slightly higher temperature, ∼ 40–45 °C. A propensity to form nonlamellar phases (hexagonal, cubic, micellar) at close to physiological temperatures was also found with the lipid extracts from natural tissues (from bovine liver, brain, and heart). This result reveals that electrostatic interactions are only one of the factors involved in lipid-mediated DNA delivery. The tendency of lipid bilayers to form nonlamellar phases has been described in terms of bilayer “frustration” which imposes a nonzero intrinsic curvature of the two opposing monolayers. Because the stored curvature elastic energy in a “frustrated” bilayer seems to be comparable to the binding energy between cationic lipid and DNA, the balance between these two energies could play a significant role in the lipoplex–membrane interactions and DNA release energetics.

Oxidation of liposomal cholesterol and its effect on phospholipid peroxidation

Lipid peroxidation is believed to play an important role in the pathogenesis of many diseases. Much research has therefore been devoted to peroxidation of different lipids in biomembranes and in model systems (liposomes) of different compositions. Yet, in spite of the relative simplicity of the liposomes, the existing literature is insufficient to reach definite conclusions regarding basic questions including the susceptibility of cholesterol to oxidation, its effect on the peroxidation of polyunsaturated phospholipids such as palmitoyllinoleoylphosphatidylcholine (PLPC) and how cholesterol influences the effect of water-soluble antioxidants such as urate on the peroxidation. The aim of the present study was to clarify these issues. Its major findings are that: (i) AAPH-induced peroxidation of cholesterol is slow and independent of the peroxidation of PLPC. In turn, AAPH-induced peroxidation of PLPC is not affected by cholesterol, independent of the presence of urate in the system. (ii) Cholesterol is not susceptible to copper-induced oxidation, but its inclusion in PLPC liposomes affects the peroxidation of PLPC, slowing down the initial stage of oxidation but promoting later stages. (iii) Addition of urate accelerates copper-induced peroxidation of PLPC in the absence of cholesterol, whereas in cholesterol-containing liposomes it inhibits PLPC oxidation. We attribute the complexity of the observed kinetics to the known cholesterol-induced rigidization of liquid crystalline bilayers.