Table 1.

The most common methods for producing liposomes.

Type of Method	Description of the Method	Ref.
MECHANICAL DISPERSION METHOD
Sonication	Sonication is the best-known and widespread method for the synthesis of liposomes, especially small monolayer vesicles (SUV). Its main disadvantages are very low encapsulation efficiency, elimination of large molecules, metal contamination from the probe tip, possible degradation of phospholipids, and presence of MLV. There are two sonication techniques: (a) probe-tip sonication—the energy needed for lipid dispersion is very high. Accumulation of energy at the tip of the sonicator probe can cause a local rise in temperature. Therefore, the reaction vessel must be immersed in a water or ice bath to control the temperature. During sonication (up to 1 h), more than 5% of the lipids can deesterify. In addition, the use of a titanium-coated probe causes contamination of the solution. (b) sonication using a bath—liposome dispersion is placed in a bath-type sonicator. Controlling the temperature of the dispersion is much easier in this method than in the sonication using a probe. The sonicated liposomes can be secured in a sterile vessel other than the probe or in an inert atmosphere.	[12,22]
French pressure cell: extrusion	The method involves injecting and pushing MLV vesicles through a small hole. It is more advantageous than the sonication method because the resulting liposomes are larger in size and resemble encapsulated substances in form. The production of liposomes requires high temperatures and a limited working volume.	[22,23]
Freeze-thawed liposomes	This method involves freezing the SUV liposomes for a short period of time and then leaving them to thaw for a long time. This technique leads to the dispersion of lipids and the formation of large monolayer vesicles (LUVs). Its greatest limitation is the increase in the phospholipid concentration and ionic strength of the medium.	[22]
High-pressure homogenization	The method is mainly used to obtain small vesicles (SUV) and to reduce the size of multilamellar liposomes.	[24]
Membrane extrusion	A common industrial method for obtaining liposomes and consists of extruding the emulsion through a polycarbonate membrane with a uniform pore size (N × 10,000 Daltons). Lipids are pressed through the membrane, and then they are repeatedly circulated for a specific time, pressure, temperature, and flow rate. This technique requires less energy and fewer mechanical systems (which can cause additional fouling).	[24]
SOLVENT DISPERSION METHOD
Ether injection (solvent vaporization)	Consists of the preparation of solutions of lipids dissolved in diethyl ether or ether-methanol mixture, which are then gradually injected into an aqueous solution of the material, which is encapsulated at 55 to 65 °C or under reduced pressure. Systematic removal of the ether under vacuum leads to liposomes. The main disadvantage of this method is the heterogeneous particle size, ranging from 70 to 200 nm. Compounds that are encapsulated are also exposed to organic solvents at high temperatures.	[12]
Ethanol injection	In this method, a lipid ethanol solution is rapidly injected into an excess of 0.16 M KCl, resulting in the immediate preparation of MLV-type liposomes. A significant disadvantage of this method is the heterogeneous size of the particles obtained (30 to 110 nm), very high dilution of the liposomes, and the need to remove ethanol.	[12]
Reverse-phase evaporation method	The technique allows the production of liposomes with a high water to lipid volume ratio and allows the retention of a significant percentage of aqueous material. It involves the formation of inverted micelles that are shaped by sonication of a mixture of a buffered aqueous phase containing water-soluble molecules (which will be encapsulated in liposomes) and an organic phase (in which amphiphilic molecules are dissolved). In the next step, the organic solvent is removed, and thus the inverted micelles are transformed into a viscous state and gel form. Excess phospholipids in the environment ensure the formation of a complete bilayer around the residual micelles, which results in the formation of liposomes. Liposomes produced by this method can be made from multiple lipid preparations and have a lipid-to-water volume ratio four times higher than that of multilamellar liposomes.	[22,25]
DETERGENT REMOVAL METHOD (REMOVAL OF UNENCAPSULATED MATERIAL)
Dialysis	Detergents are used to solubilize the lipids at their critical micellization concentrations (CMC). These detergents are then removed by dialysis, which can be performed in dialysis bags immersed in buffer solutions that do not contain detergents. While the detergent is removed, the micelles easily bind to the phospholipids and form the LUV structure.	[12]
Detergent removal of mixed micelles	Detergent absorption is possible by shaking the mixed micelle solution with organic polystyrene absorbents. A major advantage of using these types of absorbents is that they can eliminate detergents with very low CMC.	[12]