Liposomes preparation methods

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. 

Scaling-up problems Several of the laboratory-scale liposome preparation methods were difficult to scale up to industrial scale. 

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. 

Figure 8.23 Common stages for all liposome preparation methods.

Liposomes belong to the most studied particulate carrier systems. In the past decades, a vast number of liposome preparation methods for the encapsulation of a large variety of molecules have been developed and refined. We refer to the corresponding literamre and to our publications for more information. We recommend the high pressure filter extrusion method for the preparation of peptide or DNA containing liposomes because of its ease, versatility, up-scaling options and high quality of the liposomes produced. 

As a final remark, it is clear that the choice of liposome preparation method depends on the experiment s aim. Since we are dealing with compartmentalized reactions, it is often convenient to reach the maximum entrapment efhciency, in order to have filled vesicles. We will see in next section what are the general strategies to entrap chemicals inside vesicles, and how to feed them with an externally added reactant Another important issue, discussed below, is the chemical compatibility between the hpids, the preparation method, and the (bio)chemicals used in the experiments. 

Lichtenberg, D., and Barenholz, Y. (1988). Liposomes Preparation, characterization and preservation, in Methods of Biological Analysis. Vol. 33 (D Glick, ed.), John Wiley and Sons, New York, pp. 337-461. 

Ohsawa, T., Matsukawa, Y., Takakura, Y., Hashida, M., and Sezaki, H. (1985). Fate of lipid and encapsulated drug after intramuscular administration of liposomes prepared by the freezethawing method in rats, Chem. Pharm. Bull., 5013-5022. 

Regardless of their method of fabrication, most liposome preparations need to be further classified and purified before use. To remove excess aqueous components that were not encapsulated during the vesicle formation process, gel filtration using a column of Sephadex G-50 or dialysis can be employed. To fractionate the liposome population according to size, gel filtration using a column of Sepharose 2B or 4B should be done. 

Jansons, V.K., and Mallet, P.L. (1980) Targeted liposomes A method for preparation and analysis. Anal. Biochem. Ill, 54-59. 

Many types of liposomes of different lipid composition and different sizes having a transmembrane AS gradient were prepared (10). These liposomes varied (i) in their liposome-forming phosphatidylcholine (PC), being with and without cholesterol and/or lipopolymer (ii) in their size and (iii) in their method of preparation. The approaches for preparing these different liposome formulations varies in their lipid hydration and downsizing. Table 1 in Haran et al. (10) gives a partial list of such liposome preparations. In all cases the scheme of liposome preparation can be summarized as described in Table 4. 

Use of the pH-sensitive membrane-impermeable flurophore pyranine based on the ratiometric method, which determines directly level of dissociation of pyranine from the ratio between the charged (nnprotonated) pyranine and total pyranine in the intraliposome aqneons phase Addition of impermeable DPX, which acts as a quencher to pyranine fluorescence, into the liposome external medium ensures lack of contribution of extraliposome medium pyranine fluorescence (18,22). This method is considered invasive as the pyranine has to be added in the hydration medium prior to liposome preparation and cannot be used for pH determination of intraliposome aqueous phase... 

We foimd that the ratiometric method is superior because it is not dependent on pyranine concentration and therefore free of error in pipeting (18,22,54). Calibration curves were constructed by preparing liposomes in which the hydration of the lipids to form MLV was done using solutions of high concentration at the desired pH in the range of 3.0 to 10.0. Gel-exclusion chromatography on a Sephadex column, as mentioned above, yielded a series of liposome preparations with a fixed external pH (pH 7.5), but different internal pH values determined by the buffer used for lipid hydration. Neither KI nor DPX, which quench the fluorescence of aqueous solutions of pyranine, has much effect on the fluorescence intensity of pyranine in the void volume after gel-exclusion chromatography, which indicates the complete removal of the pyranine from the extraliposomal medium. 

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. 

Kirpotin DB. Compound-loaded liposomes and methods for their preparation. United States Patent,6,110,491,2000. 

More recently, a new chelation method based on the technetium chelator, HYNIC, was developed by Laverman et al. (36). HYNIC is well known for its use in labeling peptides and proteins with high efficiency and excellent stability (37). A-hydroxysuccinimidyl hydrazino nicotinate hydrochloride was conjugated to the free amino group of distearoylpho-sphatidyl-ethanolamine (DSPE) and subsequently incorporated in the lipid bilayer during the liposome preparation. 

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). 

D Lichtenberg, Y Barenholz. Liposomes preparation, characterization, and preservation. Methods Biochem Anal 33 337-462, 1988. 

C Pidgeon. Preparation of MLV by the REV method vesicle structure and optimum solute entrapment. In G Gregoriadis, ed. Liposome Technology. 2nd ed. Vol. I. Liposome Preparation and Related Techniques. Boca Raton, PL CRC Press, 1993, pp 99-110. 

Figure 10.10 Transmission electron micrograph of ferritin entrapped in POPC liposomes (palmitoyloleoylphosphatidylcholine). Cryo-TEM micrographs of (a) ferritin-containing POPC liposomes prepared using the reverse-phase evaporation method, followed by a sizing down by extrusion through polycarbonate membranes with 100 nm pore diameters ([POPC] = 6.1 mM) and (b) the vesicle suspension obtained after addition of oleate to pre-formed POPC liposomes ([POPC] = 3 mM, [oleic acid - - oleate] = 3 mM). (Adapted from Berclaz et al, 2001a, b.)...

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

Liposomes are colloidal particles that can be prepared with (phospho)-lipid molecules derived from either natural sources or chemical synthesis (recently reviewed by Lian and Ho [14]). The potential application of liposomes as biodegradable or biocompatible drug carriers to enhance the potency and reduce the toxicity of therapeutic agents was recognized in 1960. In the 1960s and 1970s various methods for liposome preparation were developed as... 

In addition to enzymatic hydrolysis of natural lipids in polymeric membranes as discussed in chapter 4.2.2., other methods have been applied to trigger the release of vesicle-entrapped compounds as depicted in Fig. 37. Based on the investigations of phase-separated and only partially polymerized mixed liposomes 101, methods to uncork polymeric vesicles have been developed. One specific approach makes use of cleavable lipids such as the cystine derivative (63). From this fluorocarbon lipid mixed liposomes with the polymerizable dienoic acid-containing sulfolipid (58) were prepared in a molar ratio of 1 9 101115>. After polymerization of the matrix forming sulfolipids, stable spherically shaped vesicles are obtained as demonstrated in Fig. 54 by scanning electron microscopy 114>. 

The liposomes prepared by this method are usually MLVs, but the structure inside the vesicles can be highly formulation dependent. MLVs composed entirely of neutral lipids tend to be very tightly packed multilayer assemblies with the adjacent bilayers stacked closely on one another, with very little aqueous space between them. The presence of negatively charged lipids in the membram tends to push the bilayers apart from each other and increase the aqueous volume of entrapmer signiLcantly for water-soluble compounds. 

---