High-Pressure Extrusion Method
This is another method for converting MLV to SUV suspensions.

Work mechanism of High Pressure Extrusion Method is Suspensions of MLVs prepared by the convectional method are repeatedly passed through filters polycarbonate membranes with very small pore diameter (0.8–1.0µm) under high pressure up to 250psi. By choosing filters with appropriate pore sizes, liposomes of desirable diameters can be produced. As the MLVs are forced through the small pores, successive layers are peeled off until only one remains. Besides reducing the liposome size, the extrusion method produces liposomes of homogeneous size distributions. A variety of different lipids can be used to form stable liposomes by this method. Extrusion at low pressures <1Mpa is possible when lipid concentration is low, but the most commonly used pressures are about 5Mpa.


Reverse Phase Evaporation Technique 

It consists of a rapid injection of aqueous solution of the drug into an organic solvent, which contains the lipid dissolved with simultaneous bath sonication of the mixture leading to the formation of water droplets in the organic solvent (i.e., a “water-in-oil” emulsion). The resulting emulsion is dried down to a semi solid gel in a rotary evaporator. The next step is to subject the gel to vigorous mechanical agitation to induce a phase reversal from water-in-oil to oil-in-water dispersion (i.e., an aqueous suspension of the vesicles). During the agitation, some of the water droplets collapse to form the external phase while the remaining portion forms the entrapped aqueous volume. Large unilamellar vesicles (diameter 0.1–1µm) are formed in the process.

This method has been used to encapsulate both small and macromolecules such as RNA and various enzymes without loss of activity. The expected limitation of this method is the exposure of the material to be encapsulated to organic solvents and mechanical agitation, which can lead to the denaturation of some proteins or breakage of DNA strands.

Solubilisation and Detergent Removal Method
Work mechanism of this method is used in the preparation of LUVs and it involves the use of surfactant for the solubilisation of the lipids.

Detergents used in this method :
  1. non-ionic surfactants : n-octyl-bete-D-glucopyranose (octyl gluside)
  2. anionic surfactants : dodecyl sulphate
  3. cationic surfactants : hexadecyltrimethyl ammoniumbromide). 

The procedure involves the solubilisation of the lipids in an aqueous solution of the detergent and the protein to be encapsulated. The detergent should have a high critical micelle concentration (CMC), so that it is easily removed. The detergent is subsequently removed by dialysis or column chromatography. During detergent removal, LUVs of diameter 0.08–0.2µm are produced.

Read more in here : Technology of liposomes production (PART I)

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