Molecular organization of the antifungal and anticancer drug 2-(2,4-dihydroxyphenylo)-5,6-dichlorobenzothiazole in solution and in monolayers: an effect of pH

2-(2,4-Dihydroxyphenylo)-5,6-dichlorobenzothiazol (dHBBT) is a new anticancer, antifungal and antibacterial drug characterised also by cytostatic and anticancer activity. The effect of pH on the molecular organization of dHBBT in monomolecular layers and in solution has been studied by electronic absorption and FT-IR spectroscopies. The analysis of the spectroscopic data suggests that at neutral and at high pH values (pH 6-8) dHBBT appears in the anionic form that prevents the formation of dimers due to the electrostatic repulsion between the molecules.     

Effect of tetracaine on model and erythrocyte membranes by DSC and EPR

Summary DSC and EPR experiments were performed on human erythrocyte membranes and DPPC vesicles in order to study the effect of the anaesthetic drug tetracaine on structure and dynamics of the lipid region. Experiments using spin label technique showed that tetracaine induced fluidity changes of the lipid region in the environment of the fatty acid probe molecules incorporated into the membranes in the vicinity of the lipid-water interface. Similarly to EPR observations, DSC measurements reported decrease of the main melting and the pretransition temperature in comparison to control DPPC vesicles, which is the sign of destabilisation of the structure in the head group region of the lipids. Similar effect was observed in the case of erythrocytes where the protein conformation was also controlled in the presence of drug. A separated membrane melting with well distinguished membrane protein phase transition was found that was affected significantly by tetracaine. These results suggest that tetracaine is able to modify not only the internal dynamics of erythrocyte membranes and produce destabilisation of the lipid structure, but the protein system as well. These might lead to further damage of the biological functions.     

Xanthophyll pigments lutein and zeaxanthin in lipid multibilayers formed with dimyristoylphosphatidylcholine

This paper reports the research on the effect of two main carotenoid pigments present in the membranes of macula lutea of the vision apparatus of primates, including humans, lutein and zeaxanthin, on the structure of model membranes formed with dimyristoylphosphatidylcholine (DMPC). The effects observed in DMPC are compared to the effects observed in the membranes formed with other phosphatidylcholines (PC): egg yolk PC (EYPC), and dipalmitoyl-PC (DPPC). The analysis has been focused, in particular, on the following aspects of the organization of lipid-carotenoid membranes: aggregation state of pigments, an effect on a thickness of the bilayer and pigment orientation within the membranes. These problems have been addressed with the application of UV-Vis absorption spectroscopy, linear dichroism measurements and the diffractometric technique. (1) Both lutein and zeaxanthin appear in a partially aggregated form in the oriented DMPC multibilayers, even at molar fractions as low as 2 mol.% with respect to lipid. (2) Orientation of the transition dipole of both xanthophylls with respect to the axis normal to the plane of DMPC membrane is different in the case of a monomeric form (34+/-3 degrees in the case of lutein and 26+/-3 degrees in the case of zeaxanthin) but essentially the same in the case of aggregated forms of both pigments (42+/-3 degrees in the case of lutein and 40+/-5 degrees in the case of zeaxanthin). It was found that only lutein has an effect on the increase in the thickness of the DMPC membranes (by about 3 A at 25 degrees C). A similar effect was observed also in the case of DPPC at the same temperatures despite the differences in the physical state of both membrane systems. The differences between the effects of lutein and zeaxanthin observed are interpreted in terms of differences of stereochemical structure of both xanthophylls leading to the different localization in the lipid phase. The results demonstrate significant differences in the behavior of lutein and zeaxanthin in model membranes, which may contribute to their different physiological functions and different efficacy as membrane antioxidants.     

Comparison of paclitaxel penetration in normal and cancerous cervical model monolayer membranes

The aim of the present study was to evaluate the penetration of paclitaxel in normal as well as cancerous human cervical monolayer membranes and to compare these results with the paclitaxel penetration in a model dipalmitoylphosphatidylcholine (DPPC) monolayer. At physiologically relevant surface pressures of 30 mN/m, equilibrium drug penetration was observed in DPPC model membrane, whereas in cervical lipid model membranes exclusion of the drug and destabilization of the membrane was observed. The maximum surface pressure increment due to penetration (Δπ_max) of 600 nM paclitaxel, for DPPC monolayer was found to be 3.6, 5.4 and 5.0 times higher than those for penetration in the cancerous monolayer at surface pressures 10, 20 and 30 mN/m, respectively. At initial surface pressure 10 mN/m, the maximum surface pressure increment, for 600 nM paclitaxel penetration, of normal cervical lipid membrane was double that of the cancerous cervical lipid membrane. At 30 mN/m initial surface pressure the representative IC₅₀ concentration of the drug produced negligible drug penetration and significant membrane destabilization in cervical lipid model membranes. The difference in penetration profile could be due to differences in composition of the model membranes. The cholesterol level in cancerous cervical membrane was 1.5-folds higher than that in the normal cervical membrane. Apart from PC, another constituent present in 20–32% in cancerous and normal membranes is sphingomyelin (SM). Introduction of 70% SM to the DPPC monolayer decreased the Δπ_max from 4.7 to 1.1 mN/m, revealing the rigidifying effect of SM which was directly proportional to the amount of SM added. Modulation of fluidity of the membranes can alter the penetration of paclitaxel in biological membranes and hence its toxicity profile.     

Dimers of polyene antibiotic amphotericin B detected by means of fluorescence spectroscopy: molecular organization in solution and in lipid membranes

Fluorescence emission from amphotericin B dissolved in 2-propanol-water was recorded in the spectral region 500-650 nm. The fluorescence excitation spectrum corresponds to the absorption spectrum of the monomeric drug. The large energy shift between the excitation and emission bands indicates that emission takes place from an energy level different than that responsible for absorption. These levels were attributed to the 2(1)A(g) and 1(1)B(u) states, respectively. Excitation of the same sample with short wavelength radiation (below 350 nm) yields light emission between 400 and 550 nm. The fluorescence excitation spectrum corresponding to this emission band displays distinct maxima at 350, 334 and 318 nm. This band was analyzed in terms of the exciton splitting theory and assigned to amphotericin B in a dimeric form, in which chromophores are spaced by 4.9 A. The binding energy of the dimers, determined to be 4.9 kJ/mol, indicates that the structures are stabilized by van der Waals interactions. The same type of molecular structures was also detected in the lipid membranes formed with dipalmitoylphosphatidylcholine. Linear dichroism of amphotericin B embedded in lipid multibilayers indicates that molecules are distributed between two fractions: parallel (38%) and perpendicular (62%) with respect to the membrane. The biological importance of such membrane organization is discussed.     

Effects of lipid chain unsaturation and headgroup type on molecular interactions between paclitaxel and phospholipid within model biomembrane

Molecular interactions between paclitaxel, an anticancer drug, and phospholipids of various chain unsaturations and headgroup types were investigated in the present study by Langmuir film balance and differential scanning calorimetry. Both the lipid monolayer at the air-water interface and the lipid bilayer vesicles (liposomes) were employed as model cell membranes. It was found that, regardless of the difference in molecular structure of the lipid chains and headgroup, the drug can form nonideal, miscible systems with the lipids at the air-water interface over a wide range of paclitaxel mole fractions. The interaction between paclitaxel and phospholipid within the monolayer was dependent on the molecular area of the lipids at the interface and can be explained by intermolecular forces or geometric accommodation. Paclitaxel is more likely to form thermodynamically stable systems with 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) and 1,2-dielaidoyl-sn-glycero-3-phosphocholine (DEPC) than with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). Investigation of the drug penetration into the lipid monolayer showed that DPPC and DEPC have higher incorporation abilities for the drug than DPPE and DSPC. A similar trend was also evidenced by DSC investigation with liposomes. While little change of DSC profiles was observed for the DPPE/paclitaxel and DSPC/paclitaxel liposomes, paclitaxel caused noticeable changes in the thermographs of DPPC and DEPC liposomes. Paclitaxel was found to cause broadening of the main phase transition without significant change in the peak melting temperature of the DPPC bilayers, which demonstrates that paclitaxel was localized in the outer hydrophobic cooperative zone of the bilayer, i.e., in the region of the C1-C8 carbon atoms of the acyl chain or binding at the polar headgroup site of the lipids. However, it may penetrate into the deeper hydrophobic zone of the DEPC bilayers. These findings provide useful information for liposomal formulation of anticancer drugs as well as for understanding drug-cell membrane interactions.     

Effects of cholesterol component on molecular interactions between paclitaxel and phospholipid within the lipid monolayer at the air-water interface

Cholesterol is a main component of the cell membrane and could have significant effects on drug-cell membrane interactions and thus the therapeutic efficacy of the drug. It also plays an important role in liposomal formulation of drugs for controlled and targeted delivery. In this research, Langmuir film technique, atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) are employed for a systematic investigation on the effects of cholesterol component on the molecular interactions between a prototype antineoplastic drug (paclitaxel) and 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) within the cell membrane by using the lipid monolayer at the air-water interface as a model of the lipid bilayer membrane and the biological cell membrane. Analysis of the measured surface pressure (pi) versus molecular area (a) isotherms of the mixed DPPC/paclitaxel/cholesterol monolayers at various molar ratios shows that DPPC, paclitaxel and cholesterol can form a non-ideal miscible system at the air-water interface. Cholesterol enhances the intermolecular forces between paclitaxel and DPPC, produces an area-condensing effect and thus makes the mixed monolayer more stable. Investigation of paclitaxel penetration into the mixed DPPC/cholesterol monolayer shows that the existence of cholesterol in the DPPC monolayer can considerably restrict the drug penetration into the monolayer, which may have clinical significance for diseases of high cholesterol. FTIR and AFM investigation on the mixed monolayer deposited on solid surface confirmed the obtained results.     

Structure and function of propranolol: A β-adrenergic blocking drug

In our earlier studies using quantum chemical methods we had proposed that propranolol has an extended structure. These results were confirmed using proton NMR . We have now carried out extensive magnetic resonance and model building studies to examine the interaction of this drug with model membranes. The effect of propranolol on organization of lipid bilayers has been studied using ESR spin labeling technique. Spin label Tempo and spin labeled stearic acid (5 SASL ) have been used to monitor changes in the fluidity of model membranes. Presence of the drug is found to fluidize the lipids. In case of 0.2M dipalmitoyl phosphatidyl choline (DPPC), presence of drug (0.1M) is found to decrease the gel-liquid crystalline phase transition temperature by about 10°C. The order parameter measured from the spectrum of 5 SASL shows a 4% decrease on incorporation of the drug in membranes. ¹³C spin lattice relaxation time (T₁) measurements have been carried out for different nuclear sites of the drug. The aromatic moiety shows a high degree of molecular rigidity when the drug is bound to the lipid bilayers. The oxypropanolamine group is however relatively flexible. It appears from these studies that the aromatic group binds strongly to the hydrophobic regions of the lipid bilayer, while the oxypropanolamine moiety remains relatively free and lies in the hydrophilic region. The ¹³C chemical shifts indicate the involvement of the β-hydroxyl group in hydrogen bonding with the lipids. The NH group may be involved in electrostatic interactions with the negatively charged phosphate group of the lipid bilayers.     

Vibrational spectroscopy of water in hydrated lipid multi-bilayers. II. Two-dimensional infrared and peak shift observables within different theoretical approximations

In a previous report, we calculated the infrared absorption spectrum and both the isotropic and anisotropic pump-probe signals for the OD stretch of isotopically dilute water in dilauroylphosphatidylcholine (DLPC) multi-bilayers as a function of the lipid hydration level. These results were then compared to recent experimental measurements and are in generally good agreement. In this paper, we will further investigate the structure and dynamics of hydration water using molecular dynamics simulations and calculations of the two-dimensional infrared and vibrational echo peak shift observables for hydration water in DLPC membranes. These observables have not yet been measured experimentally, but future comparisons may provide insight into spectral diffusion processes and hydration water heterogeneity. We find that at low hydration levels the motion of water molecules inside the lipid membrane is significantly arrested, resulting in very slow spectral diffusion. At higher hydration levels, spectral diffusion is more rapid, but still slower than in bulk water. We also investigate the effects of several common approximations on the calculation of spectroscopic observables by computing these observables within multiple levels of theory. The impact of these approximations on the resulting spectra affects our interpretation of these measurements and reveals that, for example, the cumulant approximation, which may be valid for certain systems, is not a good approximation for a highly heterogeneous environment such as hydration water in lipid multi-bilayers.     

Antioxidant activity of daidzein, a natural antioxidant, and its spectroscopic properties in organic solvents and phosphatidylcholine liposomes

Daidzein, one of major isoflavones found in soybeans, has a wide spectrum of physiological and pharmacological functions. The observed biological effects involve its interactions with lipid bilayers, usually detected by indirect methods. In this study we use the native fluorescence of daidzein to report changes observed during its interactions with organic solvents and in a phosphatidylcholine membrane.We have investigated interactions of daidzein with lipid bilayers of egg phosphatidylcholine (PC) by absorption and fluorescence methods. The data obtained indicate emission arises from the conjugate anion in excited singlet state. The fluorescence is found to increase with the basicity of the solution and the polarity of the solvent. An increase in fluorescence anisotropy in the presence of membranes suggests partial incorporation of daidzein molecules into the bilayer. Two fluorescence lifetime components, 1.5 ns and 3.5 ns, reflects the partition of daidzein between aqueous and membrane environments, respectively. On the basis of the obtained spectroscopic data we conclude that up to 15% of daidzein is located in hydrophilic region of the membrane whereas the rest is distributed in aqueous bulk and aqueous/membrane interface.For studying the antioxidant activity of daidzein against lipid peroxidation initiated by AAPH the molecule of C11-BODIPY581/591 has been used as a fluorescent oxidation indicator. The results show that the presence of daidzein anions in the membrane interface increases the inhibitory effect on lipid peroxidation compared to the neutral form of daidzein.     

Salicylic acid-induced effects in the mixed-lipid (dipalmitoyl phosphatidylcholine-dipalmitoyl phosphatidylethanolamine) model membrane

The effect of the keratolytic drug salicylic acid (SA) on the thermotropic properties and fluidity of the mixed lipid membrane dipalmitoyl phosphatidylcholine (DPPC)-dipalmitoyl phosphatidylethanolamine (DPPE) had been studied using DSC, (1H and 31P) NMR, SAXS, and dynamic light scattering. The membrane was in multilamellar vesicular (MLV) and unilamellar vesicular (ULV) form with SA/(DPPC+DPPE) molar ratios, R(m), in the range from 0 to 0.5. It was found that the mechanism of interaction of SA with the lipid mixture exhibited similar patterns in both ULV and MLV. Both the NMR and DSC studies indicated that the drug molecules were probably localized in the lipid-water interfacial region neighboring the lipid headgroups or the glycerol moiety. The presence of the drug increased the fluidity of the membrane and the acyl chain order. However, studies on MLV showed that the presence of the drug in high concentration (R(m)0.2), caused destabilization of the DPPC-DPPE mixture, as indicated by the appearance of two endothermic transitions. DSC studies indicated that prolonged equilibration of the membrane led to reduced interaction between the lipid headgroups and the SA molecules. This reduced interaction could be due to the sequestering of the drug molecules into the lipid-water interfacial region, out of proximity to the polar headgroup or glycerol moiety. Effect of inclusion of cholesterol in the membrane systems was also studied.     

Comparison of paclitaxel penetration in normal and cancerous cervical model monolayer membranes

The aim of the present study was to evaluate the penetration of paclitaxel in normal as well as cancerous human cervical monolayer membranes and to compare these results with the paclitaxel penetration in a model dipalmitoylphosphatidylcholine (DPPC) monolayer. At physiologically relevant surface pressures of 30 mN/m, equilibrium drug penetration was observed in DPPC model membrane, whereas in cervical lipid model membranes exclusion of the drug and destabilization of the membrane was observed. The maximum surface pressure increment due to penetration (Δπ_max) of 600 nM paclitaxel, for DPPC monolayer was found to be 3.6, 5.4 and 5.0 times higher than those for penetration in the cancerous monolayer at surface pressures 10, 20 and 30 mN/m, respectively. At initial surface pressure 10 mN/m, the maximum surface pressure increment, for 600 nM paclitaxel penetration, of normal cervical lipid membrane was double that of the cancerous cervical lipid membrane. At 30 mN/m initial surface pressure the representative IC₅₀ concentration of the drug produced negligible drug penetration and significant membrane destabilization in cervical lipid model membranes. The difference in penetration profile could be due to differences in composition of the model membranes. The cholesterol level in cancerous cervical membrane was 1.5-folds higher than that in the normal cervical membrane. Apart from PC, another constituent present in 20–32% in cancerous and normal membranes is sphingomyelin (SM). Introduction of 70% SM to the DPPC monolayer decreased the Δπ_max from 4.7 to 1.1 mN/m, revealing the rigidifying effect of SM which was directly proportional to the amount of SM added. Modulation of fluidity of the membranes can alter the penetration of paclitaxel in biological membranes and hence its toxicity profile.     

Insertion mechanism of a poly(ethylene oxide)-poly(butylene oxide) block copolymer into a DPPC monolayer

Interactions between amphiphilic block copolymers and lipids are of medical interest for applications such as drug delivery and the restoration of damaged cell membranes. A series of monodisperse poly(ethylene oxide)-poly(butylene oxide) (EOBO) block copolymers were obtained with two ratios of hydrophilic/hydrophobic block lengths. We have explored the surface activity of EOBO at a clean interface and under 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayers as a simple cell membrane model. At the same subphase concentration, EOBO achieved higher equilibrium surface pressures under DPPC compared to a bare interface, and the surface activity was improved with longer poly(butylene oxide) blocks. Further investigation of the DPPC/EOBO monolayers showed that combined films exhibited similar surface rheology compared to pure DPPC at the same surface pressures. DPPC/EOBO phase separation was observed in fluorescently doped monolayers, and within the liquid-expanded liquid-condensed coexistence region for DPPC, EOBO did not drastically alter the liquid-condensed domain shapes. Grazing incidence X-ray diffraction (GIXD) and X-ray reflectivity (XRR) quantitatively confirmed that the lattice spacings and tilt of DPPC in lipid-rich regions of the monolayer were nearly equivalent to those of a pure DPPC monolayer at the same surface pressures.     

Advances in Artificial Cell Membrane Systems as a Platform for Reconstituting Ion Channels

An artificial cell membrane that is composed of bilayer lipid membranes (BLMs) with transmembrane proteins incorporated within them represents a well‐defined system for the analysis of membrane proteins, especially ion channel proteins that are major targets for drug design. Because the BLM system has a high compatibility with recently developed cell‐free expression systems, it has attracted attention as a next‐generation drug screening system. However, three issues associated with BLM systems, i. e., their instability, the need for non‐volatile organic solvents and a low efficiency of ion channel incorporation, have limited their use as a drug screening platform. In this personal account, we discuss our recent approaches to address these issues based on microfabrication. We also discuss the potential for using the BLM system combined with cell‐free expression systems as a drug screening system for future personalized medicine.     

SPECTROSCOPIC PROPERTIES OF THE POTENTIOMETRIC PROBE MEROCYANINE-540 IN SOLUTIONS AND IN LIPOSOMES

Absorption, fluorescence and resonance Raman spectra of the membrane dye merocyanine-540 (MC540) were measured. The aggregation of the dye, its binding to lipid membranes and its response to crossmembrane electric potential differences were studied. The dye was found to aggregate even at micromolar concentrations in water, but not in organic solvents. The dimerization constant was evaluated by spectroscopic techniques. The binding constant to liposomes was estimated by a spectroscopic titration method. Resonance Raman spectra of MC540 were measured for the first time. Distinct changes were observed in the vibrational spectrum upon the generation of a valinomycin-induced K⁺ diffusion potential (Nernst potential) on liposomes. The ratio of Raman band intensities, which was found to be related to the membrane potential, can be used to evaluate the absolute value of the electric potential.     

The interaction of a peptide with a scrambled hydrophobic/hydrophilic sequence (Pro-Asp-Ala-Asp-Ala-His-Ala-His-Ala-His-Ala-Ala-Ala-His-Gly) (PADH) with DPPC model membranes: a DSC study

Depending on their hydrophobicity, peptides can interact differently with lipid membranes inducing dramatic modifications into their host systems. In the present paper, the interaction of a synthetic peptide with a scrambled hydrophobic/hydrophilic sequence (Pro-Asp-Ala-Asp-Ala-His-Ala-His-Ala-His-Ala-Ala-Ala-His-Gly) (PADH) with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) model membranes has been investigated by differential scanning calorimetry (DSC), adopting three different experimental approaches. In the first, the peptide is forced to be included into the hydrocarbon region of the lipid bilayer, by codissolving it with the lipid giving rise to mixed multilamellar vesicles–peptide systems; in the second, this system is passed through an extruder, thus producing large unilamellar vesicles–peptide systems; in the third, it is allowed to interact with the external surface of the membrane.

The whole of the DSC results obtained have shown that the incorporation of the peptide into the lipid bilayer by means of the first method induces a decrease in the enthalpy of the gel–liquid crystal transition of the membrane and a shift of the transition to the lower temperatures, thus resembling, in spite of its prevalently hydrophilic nature, the behavior of transbilayer hydrophobic peptides. The extrusion of these systems creates unilamellar vesicles free of peptides but of smaller size as evidenced by the decreased cooperativity of the transition. The peptide, added externally to the DPPC model membrane, has no effect on the phase behavior of the bilayer.

These findings suggest that the effect of the interaction of scrambled hydrophobic/hydrophilic peptides into lipid bilayers strongly affects the thermotropic behavior of the host membrane depending on the preparation method of the lipid/peptide systems. The whole of the results obtained in the present paper can be useful in approaching studies of bioactive peptides/lipids systems.     

Spectroscopic analysis of the interaction of a peptide sequence of Hepatitis G virus with bilayers

Merocyanine 540 (MC540) has been used as external probe to determine the interaction of the peptide sequence 125-139 corresponding to the E2 protein of Hepatitis G virus, with lipid bilayers. The probe was incorporated into large unilamellar vesicles (LUVs) or small unilamellar vesicles (SUVs) of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). When incorporated into bilayers, MC540 shows two absorption maxima corresponding to the monomer (570 nm) and dimer (530 nm) form of the probe. Changes in the probe microenvironment are reflected by a modification in the position and/or intensity of these maxima. Addition of increasing amounts of peptide resulted in a slight decrease of the ratio A570/A530 thus indicating a change in MC540 partition into the membrane, going from a hydrophobic to a more hydrophilic environment. This effect was concomitant with an increase in dimer formation as stated from the values of the apparent dimerization constant (K(app)) obtained. Fluorescence spectra as well as steady state anisotropy measurements were in agreement with the above results indicating that the peptide was able to relocate the probe and displacing MC540 from its initial location into the bilayer. Results with SUVs or LUVs were similar for what curvature does not seem to play any role on peptide activity. These results reflect the ability of peptide to interact with biomimetic membranes in the lipid head group region.     

富勒烯化聚环氧丙基咔唑的合成与表征

通过Ｆｒｉｅｄｅｌ－Ｃｒｒａｆｔｓ反应制备了富勒烯化的聚环氧丙基咔唑，聚合物中Ｃ６０的含量最高可达７．６ｗｔ％。通过凝胶渗透色谱法测定了聚合物分子量，并采用＾上Ｈ和＾１３Ｃ－ＮＭＲ，ＩＲ，热分析及光谱等手段对其结构进行了分析与表征。     

Specific effect of polyunsaturated fatty acids on the cholesterol-poor membrane domain in a model membrane

To understand more fully the effect of polyunsaturated fatty acids (PUFAs) on lipid bilayers, we investigated the effects of treatment with fatty acids on the properties of a model membrane. Three kinds of liposomes comprising dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylcholine (DOPC), and cholesterol (Ch) were used as the model membrane, and the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and detergent insolubility were determined. Characterization of the liposomes clarified that DPPC, DPPC/Ch, and DPPC/DOPC/Ch existed as solid-ordered phase (L beta), liquid-ordered phase (l o), and a mixture of l o and liquid-disordered phase (L alpha) membranes at room temperature. Treatment with unsaturated fatty acids such as oleic acid (OA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) markedly decreased the fluorescence anisotropy value and detergent insolubility. PUFAs and OA had different effects on the model membranes. In DPPC liposomes, the most prominent change was induced by PUFAs, whereas, in DPPC/Ch and DPPC/DOPC/Ch liposomes, OA had a stronger effect than PUFAs. The effect of PUFAs was strongly affected by the amount of Ch in the membrane, which confirmed a specific effect of PUFAs on the Ch-poor membrane domain. We further explored the effect of fatty acids dispersed in a water-in-oil-in-water multiple emulsion and found that unsaturated fatty acids acted on the membranes even when incorporated in emulsion form. These findings suggest that treatment with PUFAs increases the segregation of ordered and disordered phase domains in membranes.     

Porphinato Zinc Complexes Incorporated into the Bilayer Membrane of Lipid Liposomes

3,8,13,17-Tetramethyl-7,12-divinyl-2,18-bis(18-hydroxyoctadecyl propionate) porphinato zinc (BHPZn) and a model compound, dimethyl ester of protoporphinato zinc (DMPZn), were synthesized and incorporated into the hydrophobic region of bilayer membrane of dipalmitoylphosphatidylcholine (DPPC) liposome. The introduction of long alkyl groups onto the porphyrin ring is effective for restriction of porphyrin aggregation in the bilayer membrane of DPPC liposome. When the molar ratio of DPPC lipid to porphyrin is above 100, the spectrum of BHPZn in the liposome suggests that it is in a typically monomeric state. Quenching of BHPZn fluorescence in the hydrophobic bilayer membrane by hydrophilic quenchers is slow and shows smaller Stern-Volmer constants, while the quenching by hydrophobic quenchers shows much larger Stern-Volmer constants than that of the model compound, DMPZn. These results suggest that the location of the porphyrin ring of BHPZn is fixed at a certain depth in the hydrophobic bilayer membrane of DPPC liposome, and that that of DMPZn is widely distributed in the whole hydrophobic region.