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Exosomes are small extracellular vesicles (sEVs) involved in distal cell-cell communication and cancer migration by transferring functional cargo molecules. Membrane domains similar to lipid rafts are assumed to occur in exosome membranes and are involved in interactions with target cells. However, the bilayer membrane properties of these small vesicles have not been fully investigated. Therefore, we examined the fluidity, lateral domain separation, and transbilayer asymmetry of exosome membranes using fluorescence spectroscopy. Although there were some differences between the exosomes, TMA-DPH anisotropy showing moderate lipid chain order indicated that ordered phases comprised a significant proportion of exosome membranes. Selective TEMPO quenching of the TMA-DPH fluorescence in the liquid-disordered phase indicated that 40-50% of the exosome membrane area belonged to the ordered phase based on a phase-separated model. Furthermore, NBD-PC in the outer leaflet showed longer fluorescence lifetimes than those in the inner leaflets. Therefore, the exosome membranes maintained transbilayer asymmetry with a topology similar to that of the plasma membranes. In addition, the lateral and transbilayer orders of exosome membranes obtained from different cell lines varied, probably depending on the different membrane lipid components and compositions partially derived from donor cells. As these higher membrane orders and asymmetric topologies are similar to those of cell membranes with lipid rafts, raft-like functional domains are possibly enriched on exosome membranes. These domains likely play key roles in the biological functions and cellular uptake of exosomes by facilitating selective membrane interactions with target organs.

The Belousov-Zhabotinsky (BZ) reaction is an oscillating reaction due to periodic oscillations that happen in the concentration of some intermediates. Such systems can be applied together with hydrophobic membranes to create an autonomous behavior in artificial systems. However, because of a complex set of reactions happening in such systems, the interferences caused by hydrophobic membranes are not easily understood. In this study, we tested lipid membranes composed of trimethylammonium-propane (TAP) and phosphate (PA) lipids in an attempt to break down how the polar region of phosphatidylcholine (PC) lipid membranes affect the BZ reaction. According to our findings, the trimethylammonium group and membrane fluidity are crucial to change the frequency of oscillations in the reaction. In addition, the results also indicate a possible complexation of cerium ions with membranes with a phosphate head group.

Aqueous dispersions of oleic acid (OA) and those modified with 1-oleoylglycerol (monoolein, MO) form various kinds of self-assembled structures: micelles, vesicles, oil-in-water (O/W) emulsions, hexagonal phases, and dispersed cubic phases. Conventionally, these self-assembled structures have been characterized using cryogenic transmission electron microscopy or X-ray diffraction spectroscopy. However, these methodologies require specialized treatment before they can be used, which may lead to the self-assemblies not adopting their true equilibrium state. Herein, we systematically characterized the self-assemblies composed of OA and MO in aqueous solution using Raman spectroscopy and fluorescent probe 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan). The OA/MO dispersions at pH 5.0 showed increased chain packing in comparison to the OA micelle at pH 11 or OA vesicle at pH 9.0, which were characterized by the intensity ratio of the Raman peaks at 2850 and 2890 cm-1, R = I2890/I2850. In the Laurdan fluorescence measurements, the obtained spectra were deconvoluted to two peak fractions (A1: λem= 490 nm; A2: λem = 440 nm), and the peak area ratio, A1/(A1 + A2), was defined as the membrane hydrophilicity Om. The Om value of the OA/MO dispersion at pH 5.0 was similar to that of the OA O/W emulsion, indicating that the membrane surfaces of these self-assemblies were relatively dehydrated compared to the OA micelle or OA vesicle. To categorize the type of self-assembly dispersion, a Cartesian diagram plot was systematically drawn: R on the x axis and Om on the y axis, with the cross point at x = 1, y = 0.5. By comparing the membrane properties of the OA-based micelles, O/W emulsions, and dispersed cubic phases, we determined that the OA/MO dispersion at pH 5.0 possessed higher chain packing (R > 1) and a dehydrated membrane surface (Om < 0.5), which is similar to that of the ordered membranes in gel phases. This characterization method can be useful in evaluating the ordered membrane properties in dispersed self-assemblies in aqueous media.

Artificial vesicles formed from sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in aqueous solution are used successfully as additives for enzymatic oligomerizations or polymerizations of aniline or the aniline dimer p-aminodiphenylamine (PADPA) under slightly acidic conditions (e.g., pH 4.3 with horseradish peroxidase and hydrogen peroxide as oxidants). In these systems, the reactions occur membrane surface-confined. Therefore, (i) the physicochemical properties of the vesicle membrane and (ii) the interaction of aniline or PADPA with the AOT membrane play crucial roles in the progress and final outcome of the reactions. For this reason, the properties of AOT vesicles with and without added aniline or PADPA were investigated by using two fluorescent membrane probes: 1,6-diphenyl-1,3,5-hexatriene (DPH) and 6-lauroyl-2-dimethylaminonaphthalene (Laurdan). DPH and Laurdan were used as "sensors" of the membrane fluidity, surface polarity, and membrane phase state. Moreover, the effect of hexanol, alone or in combination with aniline or PADPA, as a possible modifier of the AOT membrane, was also studied with the aim of evaluating whether the membrane fluidity and surface polarity is altered significantly by hexanol, which, in turn, may have an influence on the mentioned types of reactions. The data obtained indicate that the AOT vesicle membrane at room temperature and pH 4.3 (0.1 M NaH

In order to investigate the interaction of hydrophilic molecules with liposomal membranes, we employed 1-(4-(trimethylamino)phenyl)-6-phenyl-1,3,5-hexatriene and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(5-dimethylamino-1-naphthalenesulfonyl) as fluorescent probes to monitor the surface regions of the membrane, and the results for various liposomes were plotted in correlation diagrams. According to the formation of a variety of phase states, different tendencies of decreasing surface hydrophobicity were observed in the liposomes that were modified with high concentrations of cholesterol or in the liposomes that were composed of ternary components. These liposomes, with hydrophobic surfaces, also showed preferential adsorption of l-histidine (l-His), and the hydrophobicity of the liposomal membrane at the surface changed during l-His adsorption regardless of the initial liposomal properties. Furthermore, we revealed that accelerated adsorption of l-His and preferential binding was induced in ternary liposomes forming boundaries between two separate phases.

Liposomes are considered an ideal biomimetic environment and are potential functional carriers for important molecules such as steroids and sterols. With respect to the regulation of self-assembly via sterol insertion, several pathways such as the sterol biosynthesis pathway are affected by the physicochemical properties of the membranes. However, the behavior of steroid or sterol molecules (except cholesterol (Chl)) in the self-assembled membranes has not been thoroughly investigated. In this study, to analyze the fundamental behavior of steroid molecules in fluid membranes, Chl, lanosterol, and ergosterol were used as representative sterols in order to clarify how they regulate the physicochemical properties of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes. Membrane properties such as surface membrane fluidity, hydrophobicity, surface membrane polarity, inner membrane polarity, and inner membrane fluidity were investigated using fluorescent probes, including 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, 8-anilino-1-naphthalenesulfonic acid, 6-propionyl-2-(dimethylamino) naphthalene, 6-dodecanoyl-2-dimethylaminonaphthalene, and 1,6-diphenyl-1,3,5-hexatriene. The results indicated that each sterol derivative could regulate the membrane properties in different ways. Specifically, Chl successfully increased the packing of the DOPC/Chl membrane proportional to its concentration, and lanosterol and ergosterol showed lower efficiencies in ordering the membrane in hydrophobic regions. Given the different binding positions of the probes in the membranes, the differences in membrane properties reflected the relationship between sterol derivatives and their locations in the membrane.

Fatty acids (FAs) are known to form vesicle structures, depending on the surrounding pH conditions. In this study, we prepared vesicles by mixing FAs and a cationic surfactant, and then investigated their physicochemical properties using fluorescence spectroscopy and dielectric dispersion analysis (DDA). The assemblies formed from oleic acid (OA) and linoleic acid (LA) were modified by adding didecyldimethylammonium bromide (DDAB). The phase state of FA/DDAB mixtures was investigated with pH titration curves and turbidity measurements. The trigonal diagram of FA/ionized FA/DDAB was successfully drawn to understand the phase behaviors of FA/DDAB systems. The analysis of fluidities in the interior of the membrane with use of 1,6-diphenyl-1,3,5-hexatriene (DPH) indicated that the membrane fluidities of OA/DDAB and LA/DDAB at pH 8.5 slightly decreased in proportion to the molar ratio of DDAB in FA/DDAB systems. The fluorescent probe 6-lauroyl-2-dimethylamino naphthalene (Laurdan) indicated that the LA vesicle possessed a dehydrated surface, while the OA vesicle surface was hydrated. Modification of LA vesicles with DDAB induced the hydration of membrane surfaces, whereas modification of OA vesicles by DDAB had the opposite effect. DDA analysis indicated that the membrane surfaces were hydrated in the presence of DDAB, suggesting that the surface properties of FA vesicles are tunable by DDAB modification.

Nanosized ordered domains formed in 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DOPC/DPPC) and DOPC/cholesterol (Ch) liposomes were characterized using a newly developed (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) quenching method. The membrane fluidity of the DOPC/DPPC liposomes, evaluated by the use of 1,6-diphenyl-1,3,5-hexatriene (DPH), increased significantly above their phase-transition temperature. The fluorescence spectra of 6-lauroyl-2-dimethylamino naphthalene (Laurdan) indicated the formation of an immiscible ordered phase in the DOPC/DPPC (50/50) liposomal membrane at 30 °C. The analysis of the membrane polarity indicated that the surface of the liquid-disordered phase was hydrated whereas that of the ordered phase was dehydrated. DOPC/DPPC and DOPC/Ch (70/30) liposomes exhibited heterogeneous membranes, indicating that nanosized ordered domains formed on the surface of the DOPC/DPPC liposomes. The size of these nanosized ordered domains was estimated using the TEMPO quenching method. Because TEMPO can quench DPH distributed in the disordered phases, the remaining fluorescence from DPH is proportional to the size of the ordered domain. The domain sizes calculated for DOPC/DPPC (50/50), DOPC/DPPC (25/75), DOPC/Ch (70/30), and DOPC/DPPC/Ch (40/40/20) were 13.9, 36.2, 13.2, and 35.5 Å, respectively.

The effect of cationic liposomes (CLs) on messenger RNA(mRNA) conformation and translation was studied, focusing on membrane heterogeneity. CLs, composed of 1,2-dioleoyl-sn-glycerol-3-phosphocholine/1,2-dioleoyl-3-timethylammonium propane (DOPC/DOTAP) and DOPC/3β-{N-[(N',N'-dimethylamino)ethyl]carbamoyl}cholesterol (DOPC/DC-Ch), inhibited mRNA translation in an Escherichia coli cell-free translation system. Analysis of the membrane fluidity and polarity indicated a heterogeneous DOPC/DC-Ch (70/30) membrane, while other CLs exhibited homogeneous disordered membranes. mRNA adsorbed onto DOPC/DC-Ch liposomes showed translational activity, while DOPC/DOTAP liposomes inhibited mRNA translation in proportion to its adsorption onto membranes. Dehydration of DOPC/DOTAP (70/30) and DOPC/DC-Ch (70/30) was observed in the presence of mRNA but not in the case of zwitterionic DOPC liposomes, indicating that mRNA binds in regions between the phosphate [-PO(2)(-)-] and carbonyl [-C=O-] moieties of lipids. UV resonance Raman spectroscopy suggests that adenine, cytosine, and guanine interact with DOPC/DOTAP (70/30) and DOPC/DC-Ch (70/30) but not with DOPC. Circular dichroism indicates that DOPC/DOTAP (70/30) extensively denatured the mRNA. In contrast, heterogeneous DOPC/DC-Ch (70/30) induced partial conformational changes but maintained the translational activity of mRNA.