Sonication vesicle preparation using

Mizutani and Whitten, 1985). Vesicles prepared by sonication or vaporization are metastable, and it is necessary to use standard conditions to obtain reproducible kinetic data. 

Submitochondrial particles (membranes from washed sonicated mitochondria) prepared from juice vesicles of Hamlin oranges harvested in September contained KCN-insensitive respiratory activity (46% of total) using a substrate mixture containing 0.05 M raalate, 0.05 M succinate, 0.01 M glutamate and 0.01 M TPP (21). 

Fig. 3 Effect of external pH on internal vesicle pH and MCP hydrolysis. In all experiments, a vesicle sample of 1 ml, was eluted from a Sephadex G-25 column (1 x 50 cm) with the appropriate solution. The column was previously saturated with sonicated vesicles of (DODA)B or lecithin to avoid vesicle disruption due to adsorption on the Sephadex. The vesicles eluted in the void volume were pooled and a 0.05 ml aliquot was added to 1.5 ml buffer. Borate buffer (5 mM) was used at pH 10.2 and NaOH for all other pHs. The samples were maintained at 30 °C and the hydrolysis of MCP was followed at 265 nm. A Effect of pH on the hydrolysis of free (o) and (DODA)B-entrapped MCP ( ). Chloroformic (DODA)B vesicles were prepared in 5 mM MCP chloride and 0.95 M erythritol. The vesicles were eluted from the column with erythritol 0.95 M and NaCl 5 mM. Erythritol 0.95 M was used in all buffers and the ionic strength was maintained at 5 mM with NaCl in order to avoid osmotic and ionic stress. Final concentrations of MCP and (DODA)B were 5 x 10 M and 7 x 10 M, respectively. B Effect of, pH on hydrolysis of free (o) and Lecithin entrapped MCP ( ). Lecithin vesicles were prepared by reverse phase evaporation (0.01 M, with 10% DCP) in 0.16 M KCl and 5 mM MCP, and the sample was eluted from Sephadex with KCl (0.165 M). All buffers contained KCl (0.16 M) and the kinetics were obtained with lecithin (5.6 X 10" M) and MCP (5 x 10" M). (a) Lecithin vesicles eluted from the column were incubated with 5 x 10" M Val for 15 min. After pH change the rate of MCP hydrolysis was the same as in aqueous phase. In a separate experiment, lecithin vesicles containing MCP only in the internal compartment were incubated with Val for 90 min and refiltered in Sephadex. No free MCP was found. An aliquot of the refiltered vesicles was added to NaOH and the rate constant was measured (a). C Kinetics of the effect of pH on the absorbance of intravesicular 2-HTAB. (DODA)B vesicles (5 mM) prepared with 5 mM 2-HTAB, 5 mM NaCl and 0.95 M erythritol were eluted from Sephadex G-25 with NaCl 5 mM and erythritol 0.95 M. Aliquots (0.05 ml) of (DODA)B vesicles were added to 1.5 ml of a solution containing erythritol (0.95 M), NaOH, pH 11.59 and the absorbance was measured at 340 nm. At the end of the reaction, HCl was added. A new addition of NaOH promotes a similar increase of absorbance [42]...

ATP inhibit nonspecific nucleotidases. Another way to prepare [a P] GTP Arfl for the GAP assay is to incubate Arfl with 25 ruM HEPES, pH 7.4, 100 mM NaCl, 3.5 mM MgCl2, 1 mM EDTA, 1 mM ATP, 1 fjM [a PJGTP (specific activity = 50,000-250,000 cpm/pmol), 25 ruM KCl, 1.25 U/ml pyruvate kinase, and 3 mM phosphoenolpyruvate. This buffer contains a GTP regenerating system. If using Arfl that has not been myristoy-lated, include 0.1% (w/v) Triton X-100. For myristoylated Arfl, use either micelles of 3 mM dimyristoylphosphatidylcholine and 0.1% cholate, pH 7.4 or use vesicles prepared by extrusion or sonication (see Chapter 15 of this volume. Assay and Properties of the Arf GAPs AGAPl, ASAPl, and ArfGAPl). 

Figure 1 Representation of the types of sample preparations used for studying membrane lipids and proteins. Micelles and bicelles are usually small (diameters 20-50 nm) structures and bilayers can be sonicated into small (20-50 nm diameter) vesicles, or produced as extended (diameters 100nm) multi-bi-layered or single bilayered closed or open structures, depending upon the method of preparation. Natural membranes are usually as large bilayer fragments or closed structures containing a complex and heterogeneous mixture of lipids and proteins and possibly carbohydrates.

Sonic vesicles from platensis were prepared as described (Owers-Narhi et al. 1979). After washing the membranes with 10 mM sodium pyrophosphate, the Ca-ATPase was extracted either with 2 mM Tricine/1 mM EDTA or with chloroform. The latter was accomplished using the procedure of Piccioni et al. 1981, except that solubilization was done at 4 C. Other procedures essentially followed established methods DEAE sepharose chromatography, sucrose gradient centrifugation, and ATPase assay (Jagendorf 1982), ATP synthesis (Avron 1960), and gel electrophoresis (Laemmli 1970). One unit of ATPase activity is defined as 1 pmole Pi formed per minute. 

Artificial membrane systems can be prepared by appropriate techniques. These systems generally consist of mixtures of one or more phospholipids of natural or synthetic origin that can be treated (eg, by using mild sonication) to form spherical vesicles in which the lipids form a bilayer. Such vesicles, surrounded by a lipid bilayer, are termed liposomes. 

Reverse-phase evaporation in a nitrogen atmosphere was used to prepare lipids. A lipid film previously formed was redissolved in diethyl ether and an aqueous phase containing the dyebath components added to the phospholipid solution. The resulting two-phase system was sonicated at 70 W and 5 °C for 3 minutes to obtain an emulsion. The solvent was removed at 20 °C by rotary evaporation under vacuum, the material forming a viscous gel and then an aqueous solution. The vesicle suspension was extruded through a polycarbonate membrane to obtain a uniform size distribution (400 nm). 

Between 1955 and 1960 various sub-mitochondrial preparations were developed to give vesicles comprising only sealed inner mitochondrial membranes. Cooper and Lehninger used digitonin extraction Lardy and Kielley Bronk prepared sub-mitochondrial particles by sonication. At this time, too, Racker and his colleagues isolated Fq/F1 particles from mitochondria and showed that a separated FI particle behaved as an ATPase. The F0 portion had no enzymic properties but conferred oligomycin sensitivity on the FI ATPase. The orientation of these sub-mitochondrial vesicles (inside-out or vice-versa) was shown by the position in electron micrographs of the dense (FI) particles which in normal intact mitochondria project into the matrix and so define the surface of the inner mitochondrial membrane. 

To prepare DQAsomes or vesicles composed of dequalinium derivatives, the appropriate amount of bola-lipid (10 mM final) was dissolved in methanol, dried using a rotary evaporator, suspended in 2.5 mL 5mM N-2-hydroxyethylpiperazine-N -2-ethane sulfonic acid (HEPES), pH 7.4, bath sonicated for about one hour followed by probe sonication for 45 minutes (10 W). The sample was then centrifuged for 30 minutes at 3000 rpm, the clear, or in some cases, opaque supernatant collected and the remaining non-solubilized residue lyophilized. The concentration of solubilized bola-lipid can be determined spectrophotometrically or can be inferred from the amount of recovered compound after lyophilization. 

The irradiation of a system with sound waves (usually ultrasound). Often used to disrupt cell membranes and in early steps in protein purification, it should also be noted that sonication can increase rates of reaction as well as assist in the preparation of vesicles. 

One of the major drawbacks of liposomes is related to their preparation methods [3,4]. Liposomes for topical delivery are prepared by the same classic methods widely described in the literature for preparation of these vesicles. The majority of the liposome preparation methods are complicated multistep processes. These methods include hydration of a dry lipid film, emulsification, reverse phase evaporation, freeze thaw processes, and solvent injection. Liposome preparation is followed by homogenization and separation of unentrapped drug by centrifugation, gel filtration, or dialysis. These techniques suffer from one or more drawbacks such as the use of solvents (sometimes pharmaceutically unacceptable), an additional sizing process to control the size distribution of final products (sonication, extrusion), multiple-step entrapment procedure for preparing drug-containing liposomes, and the need for special equipment. 

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... 

Liposome Preparation Techniques In most cases, liposomes are named by the preparation method used for their formation, Such as sonicated, dehydrated-rehy-drated vesicle (DRV), reverse-phase evaporation (REV), one step, and extruded. Several reviews have summarized available liposome preparation methods [91,124, 125], Liposome formation happens spontaneously when phospholipids are dispersed in water. However, the preparation of drug-encapsulating liposomes with high drug encapsulation and specific size and lamellarity is not always an easy task. The most important methods are highlighted below. 

For hydration, bidistilled water is used to prepare the liposomes in the concentration of 10 mg/ml. To form the lipid vesicles, a bath sonicator at 45°C is used. After obtaining a dispersion of the lipid in water, sonication is continued with a probe type sonicator to increase the energy input. For the following processes, the sample is transferred into a 50 ml plastic mbe. 

The formation of liposomes [or better arsonoliposomes (ARSL)], composed solely of arsonolipids (Ars with R=lauric acid (C12) myristic acid (C14) palmitic acid (C16) and stearic acid (C18) (Fig. 1) have been used for ARSL construction), mixed or not with cholesterol (Choi) (plain ARSL), or composed of mixtures of Ars and phospholipids (as phosphatidylcholine [PC] or l,2-distearoyl- -glyceroyl-PC [DSPC]) and containing or not Choi (mixed ARSL), was not an easy task. Several liposome preparation techniques (thin-film hydration, sonication, reversed phase evaporation, etc.) were initially tested, but were not successful to form vesicles. Thereby a modification of the so called one step or bubble technique (8), in which the lipids (in powder form) are mixed at high temperature with the aqueous medium, for an extended period of time, was developed. This technique was successfiil for the preparation of arsonoliposomes (plain and mixed) (9). If followed by probe sonication, smaller vesicles (compared to those formed without any sonication [non-sonicated]) could be formed [sonicated ARSL] (9). Additionally, sonicated PEGylated ARSL (ARSL that contain polyethyleneglycol [PEG]-conjugated phospholipids in their lipid bilayers) were prepared by the same modified one-step technique followed by sonication (10). 

Final and definite proof that indeed vesicles are formed after the one step method and that they still exist after sonication can be established by observing the morphology of the arsonoliposome dispersions using different types of electron microscopy (EM), as discussed elsewhere (9-12). Additionally, the ability of the vesicles to encapsulate aqueous soluble markers as carboxyfluorescein or calcein (see below) serves as proof that vesicular structures are present in most of the arsonoliposome dispersions prepared. 

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