Large unilamellar vesicle preparation

Wessmann, G. (1978). Comparison of large unilamellar vesicles prepared by a petroleum ether vaporization method with multila-mellar vesicles ESR, diffusion and entrapment analyses, Bio-chim. Biophys. Acta. 542, 137-153. 

Figure 1 Freeze-fracture electron micrographs of egg phosphatidylcholine large unilamellar vesicles prepared by extrusion through polycarbonate filters with pore sizes of (A) 400 nm, (B) 200 run, (Q 100 nm, (D) 50 nm, and (E) 30 nm. The bar in panel (A) represents 150nm. Source From Ref. 7.

Mui BLS, Cullis PR, Evans EA, et al. Osmotic properties of large unilamellar vesicles prepared by extrusion. Biophys J 1993 64 443. 

Fig. 21 Phase-contrast photographs of a fusion of LUVs (large unilamellar vesicles) prepared form a 1 1 mixture of a butadiene lipid with a cationic dimethylammonium bromide headgroup and cholesterol. Vesicle diameter 37 and 45 pm respectively ...

Liposomes are formed due to the amphiphilic character of lipids which assemble into bilayers by the force of hydrophobic interaction. Similar assemblies of lipids form microspheres when neutral lipids, such as triglycerides, are dispersed with phospholipids. Liposomes are conventionally classified into three groups by their morphology, i.e., multilamellar vesicle (MLV), small unilamellar vesicle (SUV), and large unilamellar vesicle (LUV). This classification of liposomes is useful when liposomes are used as models for biomembranes. However, when liposomes are used as capsules for drugs, size and homogeneity of the liposomes are more important than the number of lamellars in a liposome. Therefore, "sized" liposomes are preferred. These are prepared by extrusion through a polycarbonate... 

Preparation of Sphingomyelin Cholesterol (55 45) Large Unilamellar Vesicle by Extrusion... 

DSPC/Chol (55 45) LUVs (diameter = 100 nm) are prepared as described in section Preparation of Sphingomyelin/Cholesterol (55 45) Large Unilamellar Vesicle by Extrusion [(Lipid) = 20 mM, volume = 5mL], using 350 mM citrate pH 4.0 as the hydration buffer, and 20 mM HEPES 1.50 mM NaCl pH 7.5 (HEPES-buffered saline) as the external buffer. In this case, the pH gradient is formed during the final dialysis step. It would also be possible to omit the final dialysis step and form the pH gradient by one of two common column methods. This could be desirable if the LUV... 

MacDonald RC, MacDonald RI, Menco BPM, Takeshita K, Subbarao NK, Hu LR. Small-volume extrusion apparatus for preparation of large, unilamellar vesicles. Biochim Biophys Acta 1991 1061 297-303. 

Fig. 51 a—c. Phase-contrast photographs of a fusion of large unilamellar vesicles (LUVs) prepared from a 1 1 mixture of (26) and cholesterol. Vesicle diameters 37 and 45 pm, respectively, a) orientation in an ac field of about 2 kV/cm b) elongated fused liposome, one second after application of a 30 ps, 140 V/cm field pulse c) spherical new vesicle after turning off ac field new diameter 51 pm83)... 

Liposomes used for transfection are either large unilamellar vesicles (LUVs) of 100 to 200 nm in diameter or small unilamellar vesicles (SUVs) of 20 to 100 nm. Liu et al.124 have reported that for a given liposome composition, multilamellar vesicles (MLVs) of 300 to 700 nm in diameter exhibit higher transfection efficiency than SUVs. However, more recent studies on the nature of the liposome-DNA complex (or lipoplex) revealed that lipoplexes from SUVs or MLVs do not differ significantly in size. On the other hand, the composition of the medium, not the type of the liposome used in the preparation of the lipoplex, plays a key role in determining the final size of the complex. And the transfection efficiency is also shown to depend on the final size of the complexes but not the type of the liposome.125... 

Artificial biomembrane mimetic model systems are used to characterize peptide-membrane interactions using a wide range of methods. Herein, we present the use of selected membrane model systems to investigate peptide-membrane interactions. We describe methods for the preparation of various membrane mimetic media. Our applications will focus on small unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs) as well as on media more suited for nuclear magnetic resonance (NMR) techniques, micelles, and fast-tumbling two-component bilayered micelles (bicelles). 

In order to prepare liposomes, the lipid preparation is dried at low temperature under an inert gas atmosphere (protect the lipid from oxidation). The lipid film is swollen with water or buffered aqueous solution and several freeze-thaw cycles are carried out to get optimal rehydration of the lipid. The rehydrated lipid preparation is filtered using membrane filters with defined pore size. After repeated filtration steps (extrusion) an unilamellar liposome preparation with a defined size distribution is obtained. Large unilamellar vesicles (LUV) are produced in this way. LUV s are about 100 nm in size the thickness of the lipid bilayer is about 4 nm. Even smaller liposomes can be derived from sonication (sonication probe or ultra-sonication bath). Separation of the prepared liposomes... 

Chaimovich and coworkers have prepared large unilamellar vesicles of DODACl by a vaporization technique which gives vesicles of ca 0.5 pm diameter. These vesicles are much larger than those prepared by sonication, where the mean diameter is 30 nm, and their effects on chemical reactivity are very interesting. The reaction of p-nitrophenyl octanoate by thiolate ions is accelerated by a factor of almost 10 by DODACl vesicles (Table 2), but this unusually large effect is due almost completely to increased concentration of the very hydrophobic reactants in the small region of the vesicular surface and an increased extent of deprotonation of the thiol. There is uncertainty as to the volume element of reaction in these vesicles, but it seems that second-order rate constants at the vesicular surface are similar to those in cationic micelles or in water (Cuccovia et al., 1982b Chaimovich et al., 1984). 

Two situations must be distinguished, (i) assays of pure enzyme and (ii) assays of cell extracts. When purified enzyme preparations are available, no labeled substrate is required. Natural or synthetic sphingomyelin is prepared, pure or mixed with other lipids, in the form of extruded large unilamellar vesicles (LUV) ca. 100 nm in diameter. When pure sphingomyelin vesicles are used extrusion must take place at a temperature close to or above the gel-fluid transition temperature of the lipid, i.e. often 45-50 °C. LUV and enzyme are mixed in the appropriate assay buffer and aliquots are removed at fixed time intervals. The aliquots are mixed with chloroform-methanol and, after phase separation, phosphorous (from phosphorylcholine) is assayed in the aqueous phase. The procedure has been described in detail by Ruiz-Arguello et al. [91]. 

Methodology for Liposome Preparation - An informal agreement was reached on the use of a three-letter acronym to designate the type of liposome such as multllamellar vesicles (MLV) or small unilamellar vesicles (SUV) or large unilamellar vesicles (LUV) with the chemical composition in parenthesis after the acronym (Ref. 21, p. 367). The tern liposomes is therefore to be used as a generic name to Include all types of artificial vesicles composed of phospholipids and other amphipathlc lipids. 

The highly complex and variable composition of natural cell membranes makes them a difficult subject for experimental studies. Artificial lipid membranes have consequently been prepared and studied for many years as models of cell membranes [1,3-7], A diverse array of geometries has been developed, including small and large unilamellar vesicles, giant lipid vesicles, lipid membranes supported on solid and polymer-coated substrates, and BLMs. These have been used to study the physical and chemical properties of lipids and lipid mixtures as well as membrane-associated proteins, including reconstituted transmembrane receptors. 

The storage and reactivity of electroactive molecules in polymerized diacetylene vesicles was the subject of studies reported by Stanish, Singh, and coworkers [109, 110], They entrapped ferricyanide in large unilamellar vesicles of photopolymerized PCg PC (1 - palmitoyl - 2 - (tricosa - 10,12-diynoyl)-OT-glycero-3-phosphocholine). Cyclic voltammetry was used to demonstrate that the ferricyanide was electrochem-ically isolated by the poly(lipid) bilayer [110]. At pH7 and 25°C, an anomalously long half-life of 2.4 weeks was calculated for Fe (CN)g- retention in polymerized vesicles. In a subsequent study [109], vesicles with entrapped ferricyanide were prepared from 2-bis(10,12-tricosadiynoyl)-OT-glycero-3-phosphatidylcholine (DCs.gPC) doped with a disulfide-capped lipid (Af-3-(pyridyl-2-dithio)propionyl-2-... 

---