Detection of Nanosized Ordered Domains in DOPC/DPPC and DOPC/Ch Binary Lipid Mixture Systems of Large Unilamellar Vesicles Using a TEMPO Quenching Method

Abstract

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.

Use Liposome as a Designable Platform for Molecular Recognition ~ from “Statistical Separation” to “Recognitive Separation” ~

“…TNS and Laurdan are polar probes, and their emission peaks were blue shifted and their fluorescence intensities increased in proportion to the hydrophobicity of the solvents [27,[29][30]. In the presence of liposomes, the ranges of the TNS emission wavelengths were 438-446 nm, indicating that TNS is inserted at water-membrane interface regions (: [25][26][27][28][29][30][31][32][33][34][35]. Furthermore, the intensity of TNS fluorescence was higher in the 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposome, while it was lower in the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposome at 25 °C, indicating that the membrane surface of DOPC was more hydrophobic than that of DPPC.…”

“…It is concluded that the surface properties of liposome membranes can be monitored using TNS (: 25-35), Laurdan (: 5-30), and DPH (: 4-6), and the characteristics of each probe is summarized in Table 3 and Figure 3. It has been recently reported that the nano-ordered domain can be detected by using the newly developed 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) quenching method [34]. As an example, Figure 4 shows a summary of the physicochemical properties of DOPC/DPPC/Ch binary or ternary lipid mixtures through a schematic illustration of nano-sized ordered domains in LUV.…”

Characterization of the physicochemical properties of phospholipid vesicles prepared in CO2/water systems at high pressure

“…4. The yields for systems (II), (III), and (IV) were as good as that of the conventional vesicle preparation method (i.e., the yield of DPPC vesicles using the thin-film hydration method 22,34 was 92.8 6 3.0%; although a small amount of lipid film existed in the round bottom flask in our experiment). In contrast, the yield for system (I) was lower than the other conditions, and an insoluble lumpy lipid aggregate was observed in the sample.…”

“…Fluorescent probe Laurdan is sensitive to the polarity around itself, which allows the surface polarity of lipid membranes to be determined. 21,22,31 Laurdan emission spectra exhibit a red shift caused by dielectric relaxation. Thus, emission spectra were calculated by measuring the general polarization (GP 340 ) for each emission wavelength as follows:…”

“…20 Based on vesicle characterization protocols, a variety of vesicles prepared by several methods have been investigated. [21][22][23][24] The membrane properties, such as the phase state, membrane fluidity, and membrane polarity, are possible factors to recruit the guest molecules on the membrane surface. 25,26 However, it is assumed that a specific assembled state (i.e., interdigitated membrane) might form in the liquid-liquid-surfactant ternary system.…”