Liposome preparation procedure

Liposomes made of pure phospholipids will not form at temperatures below T of the phospholipid. This temperature requirement is reduced to some extent, but not eliminated, by the addition of cholesterol (17). In some cases, it is recommended that liposome preparation be carried out at temperatures well above T of the vesicles. For instance, in the case of vesicles con-taining dipalmitoyl phosphatidylcholine (DPPC, T = 41°C), it has been suggested that the liposome preparation procedure be carried out at 10°C higher than the T at 51°C (18, 19). This is in order to make sure that all the phospholipids are dissolved in the suspension medium homogenously and have sufficient flexibility to align themselves in the structure of lipid vesicles. Following termination of the preparation procedure, usually nanoliposomes are allowed to anneal and stabilize for certain periods of time (e.g. 30-60 min), at a temperature above T, before storage. 

The liposome preparation procedure is based on film formation and hydration. After transferring the mixture of Phospholipon and GDNT solutions into 10 ml round bottom flasks, the chloroform-methanol solution is removed by evaporation at 300 mbar and 45°C to obtain a lipid film. 

Liposomes are members of a family of vesicular structures which can vary widely in their physicochemical properties. Basically, a liposome is built of one or more lipid bilayers surrounding an aqueous core. The backbone of the bilayer consists of phospholipids the major phospholipid is usually phosphatidylcholine (PC), a neutral lipid. Size, number of bilayers, bilayer charge, and bilayer rigidity are critical parameters controlling the fate of liposomes in vitro and in vivo. Dependent on the preparation procedure unilamellar or multilamellar vesicles can be produced. The diameter of these vesicles can range from 25 nm up to 50 ym—a 2000-fold size difference. 

As illustrated above there exist a large variety of techniques for preparing liposomes. From a pharmaceutical point of view, optimum liposome preparation techniques would avoid the use of organic solvent and detergents (which are difficult to remove), would exhibit a high trapping efficiency, would yield well-defined vesicles which can be produced in a reproducible way, and would be rapid and amenable to scale-up procedures (see Sec. VIII). 

The biotinylated liposomes prepared by this procedure may be stored under an inert-gas atmosphere at 4°C for long periods without degradation. 

Liposome Formation. The pioneering investigations of Bang-ham (5) have shown that thin films of natural phospholipids form bilayer assemblies if they are lyophilized in excess water by simple handshaking above the phase transition temperature. While this procedure results in the formation of large, multibilayered spherical structures, by ultrasonication of such lipid dispersions small unilamellar liposomes are formed (16), which are schematically shown in Figure 10. Additional metTiods for liposome preparation are described in a number of reviews (17,44,45,46). 

Phospholipid concentration was determined using our modification of Bartlett s procedure (49,53). Cholesterol concentration and purity were determined by HPLC or enzymatically by cholesterol oxidase (49,53). Purity of phospholipids as raw materials, and the extent of their hydrolysis during various steps of liposome preparation and liposome storage, were assessed by TLC and enzymatic determination of the increase in level of nonesterified fatty acids (10,38,49-51,53). 

For the design of mitochondriotropic liposomes, we have used a method, that has been a standard procedure in liposome technology for over 30 years the lipid-mediated anchoring of artificially hydrophobized water-soluble molecules into liposomal membranes (25-28). We have hydrophobized mitochondriotropic TPP cations by conjugating them to long alkyl residues specifically, we have synthesized stearyl TPP (STPP) salts (29). Following liposome preparation in the presence of STPP, the liposomal surface became covalently modified with TPP cations, thereby rendering these liposomes mitochondriotropic as verified in vitro by fluorescence microscopy (30). 

The dialysis method is well suited for the encapsulation of lipophilic drugs. The liposomes prepared by this method are usually homogeneous in size with good reproducibility, and the encapsulation condition is mild compared with other methods. The drawbacks include (i) low entrapment efLciency for hydrophilic molecules, (ii) the complete removal of residual detergent is impossible, (iii) the procedure is lengthy, and (iv) scale-up is difLcult. 

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 general, liposomes due to their physicochemical characteristics, may cause adverse effects i.e., blockage of capillaries, pulmonary embolism etc. The new technological knowledge and industrial improvements on liposomal production as well as the high quality control of the preparation procedure of the liposomal formulations can eliminate most of their undesired side effects. 

The following section thoroughly describes polymer synthesis and purification, as well as liposome preparation, purification, and basic characterization. To provide detailed protocols, the number of experiments was narrowed down to the most general procedures. However, creative investigators will find plenty of ways to modify and adjust the protocols to achieve their drug delivery needs and objectives. 

Mehnert implicates micelles, mixed micelles, liposomes, and drug-nanoparticles, depending on composition, as possible structures resulting from SLN preparation methods, apart from the main particulate carrier. He calls for control samples such as a liposome formulation prepared under identical conditions [40], Often, liposphere preparation procedures include a washing step with phosphate-buffered saline (PBS) to remove unencapsulated drug which possibly partly removes by-products as well. 

During the preparation procedure of the samples for SEM, certain deviations from the values of the mean vesicle diameters are possible. Referring to a liposome dispersion standard sample (Natipide II), it was concluded that the values of the vesicle diameters are in agreement with the standard sample diameter values within the standard deviation. This could be acceptable, for the aim of this paper was to investigate this influence of various factors (PRO concentration, homogenization terms) on changes in the mean diameter of liposomes. 

While the precision in the detennination of phase transition temperatures is generally excellent, so that 7 m-values obtained with different instruments by different groups agree fairly well, this is not so for the transition enthalpies A//, the other thermodynamic quantity obtainable by calorimetry. The agreement between data reported by different groups and with different instruments is usually not better than 5% for the A7/-values. There are several reasons for these discrepancies, namely different purity of samples, different preparation procedures of the liposomal suspensions, inaccuracies in sample concentrations (the concentration is usually calculated from dry weight or from phosphorous analysis), different scanning speeds and incubation times at low temperature, and finally, differences in base line subtraction procedures. 

Liposome size can range from around 20 nm to around 50 pm. To a certain extent, the mean diameter and distribution of the diameters can be controlled by sizing procedures after the formation of the initial liposome dispersion or by a careful selection of the preparation conditions (cf. Sec. II). Several techniques can be used to determine mean particle size and particle size distribution (Groves, 1984). 

Kirby, C. J., and Gregoriadis, G. (1984). A simple procedure for preparing liposomes capable of high encapsulation efficiency under mild conditions, in Liposome Technology, Vol. 1 (G. Gregoriadis, ed.), CRC Press, Boca Raton, pp. 19-27. 

Szoka, F., and Papahadjopoulos, D. (1978). Procedure for preparation of liposomes with large aqueous space and high capture by reverse-phase evaporation, Proc. Natl. Acad. Sci. USA. 75, 4194-4198. 

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