Liposomes ethanol injection

Foradada and Estelrich [3.63] studied the encapsulation of thioguanine (TG) in three types of liposomes produced by extrusion, ethanol injection and dehydration-rehydration vesicles. The entrapment has been examined at three different concentrations (1, 0.1 and 0.01 mM) and at three different pH values (4.7,7.4 and 9.2). The dehydration-rehydration vesicles were found to be the optimum method to encapsulate TG, independent of the pH value. At pH 4.7, 12 mmol/mol of lipid were entrapped, while with the other methods a maximum of 3 mmol/mol of lipid has been achieved. The authors related this behavior to the formation of hydrogen bridges between the TG and the liposomes. 

The mixed liposomal solutions were prepared by the ethanol-injection method(13) in order to obtain completely transparent solutions. It is interesting to note that miscibility of the photochromic amphiphiles with DPPC depend on the position of bulky azobenzene. If azobenzene is incorporated close to the end of long alkyl chain, a stable mixed bilayer state cannot be formed. On the other hand, when the azobenzene moiety is located near the head group or at the center of the hydrocarbon tail, the azobenzene amphiphiles are successfully incorporated into the bilayer membrane. No individual micelle formation nor phase separation in the bilayer was observed at 25 °C by absorption spectroscopy. However, the microstructure of the mixed liposomes depends on the type of azobenzene amphiphiles. 

The procedure chosen for the preparation of lipid complexes of AmB was nanoprecipitation. This procedure has been developed in our laboratory for a number of years and can be applied to the formulation of a number of different colloidal systems liposomes, microemulsions, polymeric nanoparticles (nanospheres and nanocapsules), complexes, and pure drug particles (14-16). Briefly, the substances of interest are dissolved in a solvent A and this solution is poured into a nonsolvent B of the substance that is miscible with the solvent A. As the solvent diffuses, the dissolved material is stranded as small particles, typically 100 to 400 nm in diameter. The solvent is usually an alcohol, acetone, or tetrahydrofuran and the nonsolvent A is usually water or aqueous buffer, with or without a hydrophilic surfactant to improve colloid stability after formation. Solvent A can be removed by evaporation under vacuum, which can also be used to concentrate the suspension. The concentration of the substance of interest in the organic solvent and the proportions of the two solvents are the main parameters influencing the final size of the particles. For liposomes, this method is similar to the ethanol injection technique proposed by Batzii and Korn in 1973 (17), which is however limited to 40 mM of lipids in ethanol and 10% of ethanol in final aqueous suspension. 

Gimatecan)-containing liposomes prepared by the ethanol injection method. J. Lipos. Res., 14, 87-109. 

Perhaps the simplest solvent dispersion method is that developed by Batzri and Korn (1973). Phospholipids and other lipids to be a part of the liposomal membrane are first dissolved in ethanol. This ethanolic solution is then rapidly injected into an aqueous solution of 0.16 M KC1 using a Hamilton syringe, resulting in a maximum concentration of no more than 7.5% ethanol. Using this method, single bilayer liposomes of about 25-nm diameter can be created that are indistinguishable from those formed by mechanical sonication techniques. The main disadvantages of ethanolic injection are the limited solubility of some lipids in the solvent (about 40 mM for phosphatidyl choline) and the dilute nature of the resultant liposome suspension. However, for the preparation of small quantities of SUVs, this method may be one of the best available. 

Stano, P, Bufali, S., Pisano, C., Bucci, F., Barbarino, M., Santaniello, M., Carminati, P, and Luisi-Pier, L. (2004). Novel camptothecin analogue (gimatecan)-containing liposomes prepared by the ethanol injection methodJ. Liposome Res., 14, 87-109. 

The manufacturing of nanosized liposomes can be performed using the methods mentioned above. However, the small size of nanoliposomes is difficult to achieved by methods such as film hydration. Molecular self-assembly occurs in the injection method, and then the size and morphology of obtained liposomes can be well controlled. In fact, liposomes that result from the injection method are uniform and small enough, to the nanoscale, and usually SUVs are obtained. Because of the very low toxicity of ethanol, the ethanol injection method is usually used and is described as follows to show the process of manufacturing liposomes [50]. A scale-up manufacturing process of the ethanol injection method has been established [80-82], The obtained liposome size is mostly less than 300 nm ... 

Wagner, A., Vorauer-Uhl, K., Kreismayr, G., and Katinger, H. (2002), The crossflow injection technique—An improvement of the ethanol injection method, J. Liposome... 

Cationic liposomes composed of 3(3- [ N- (N N-dimethylaminoethane (carbamoyl] cholesterol (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) (DC-Chol/DOPE liposome, molar ratio, 1 1 or 3 2) prepared by the dry-film method have been often used as non-vtral gene delivery vectors. We have shown that a more efficient transfection in medium with serum was achieved using DC-Chol/DOPE liposomes (molar ratio, 1 2) than those (3 2), and preparation method by a modified ethanol injection than the dry-film. The most efficient DC-Chol/DOPE liposome for gene transfer was molar ratio (1 2) and prepared by a modified ethanol injection method. The enhanced transfection is related to an increase in the release of DNA in the cytoplasm by the large lipoplex during incubation in opti-MEM 1 reduced-serum medium (optiMEM), not to an increased cellular association with the lipoplex. Cationic liposomes rich in DOPE prepared by a modified ethanol injection method will help to improve the efficacy of liposome vector systems for gene delivery. 

Key words Cationic liposome. Gene transfection, DOPE, DC-Chol, Ethanol injection method. 

We reported that greater transfection efficiency in medium with serum was obtained in human cervical carcinoma HeLa cells, using (I) DC-Chol/DOPE liposomes (molar ratio, 1 2) than liposomes (1 1 or 3 2), (2) a modified ethanol injection (MEI) method to prepare liposomes than the dry-film method (13, 14), and (3) a dilution method to form lipoplex than direct mixing. The physicochemical properties of liposomes and lipoplexes can be examined by measuring particle size. Transfection efficiency was evaluated by using plasmid DNA encoding luciferase gene and the cells. 

Preparation of Liposomes by a Modified Ethanol Injection (MEI) Method (MLs)... 

MEI method of liposome preparation is similar to the reported ethanol injection method (16, 17), proliposome preparation (17, 18), and coacervation methods (19). The differences... 

Cationic liposome (DC-Chol/ DOPE=l 2) and a modified ethanol injection method to prepare liposomes, increased gene expression. Int J Pharm 342 33-39... 

Maitani Y, Soeda H, Wang J, Takayama K (2001) Modified ethanol injection method for liposomes containing (3-sitosterol (3-D-glucoside. J Liposome Res 11 115-125... 

Two important aspects of Uposome formation must be emphasized here. First, in some cases we observe the formation of ordered supramolecular structures starting from a chaotic disordered mixture of surfactants (as in the ethanol injection method . As noticed before, this increase of order is attended by a simultaneous increase of water entropy and a decrease of overall free energy (Upids and solvent). Secondly, every time a liposome forms, there is the anergence of a division, with an inside world that is different from the external environment, even if the two worlds actually interact with each other. The discrimination between inside and outside, appUcable to lipid vesicles, is the first structural pre-requisite for the living cell. It is therefore clear that lipid or fatty acid vesicles may be considered relevant experimental model of simplified cells, and their role on... 

DNA, coding for the GFP was introduced in liposomes composed by POPC, together with the whole T T machinery (T7 RNA polymerase and E. coli cell extracts). GFP was synthesized inside vesicles prepared by the ethanol injection method. 

Practically all available iodinated extracellular X-ray contrast agents have been encapsulated into liposomes using different lipids and methods of preparation. Table 1 gives a short and intentionally incomplete overview of some of the approaches. The first liposomal contrast agent preparation that was tested in humans contained diatrizoate [48]. The injected dose was up to 0.5 ml kg k The preparation was effective even in plain radiography where lesions down to 0.8-1.0 cm could be detected in patients. However, adverse events such as fever and hyperthermia, which occurred in 30% of the patients, limited further use. We have incorporated iopromide into MLVs that were prepared from phosphatidyl choline (PC), cholesterol and stearic acid at a molar ratio of 4 5 1 using the ethanol-evaporation technique [44]. The liposomes can be stored freeze-dried and they are reconstituted before use by... 

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