Liposome Preparation Method 1

Phospholipids are generally white or light yellow powders or lumps at room temperature. They are very soluble in chloroform and can also be dissolved in ether, n-hexane or ethanol, and are almost insoluble in water or acetone. Disperse the phospholipid molecules into the aqueous solution by hydration treatment after high dispersion, or changing the solvent. When the concentration reaches the critical micelle concentration (CMC), self-assembly occurs through the hydrophobic interaction to form a bilayer structured liposome bubble. Commonly used preparation methods of liposomes in the laboratory include thin film method, reverse evaporation method, double emulsion method, centrifugal method, injection method, calcium fusion method, ammonium sulfate gradient method, etc. In order to ensure the final smooth transition to mass production, these methods need to verify the key process conditions and process steps to explain the rationality and reproducibility of the process. The process equipment used should be clearly explained and the feasibility of scale-up production should be demonstrated to ensure the safety, effectiveness and quality of the products.

Thin Film Method
The membrane method was first reported by Bangham, the discoverer of liposomes. This is the earliest and still commonly used method. Mix the lipid and the lipid-soluble drug in an appropriate amount of chloromethane or other organic solvents, pass nitrogen gas or decompression rotary evaporation to remove the organic solvent, make the lipid form a thin film on the container wall, add a buffer containing water-soluble drugs. Place the lipids at room temperature for a certain period of time to hydrate the lipids. Shake and disperse the lipids above the phase transition temperature (Tm), the lipid membrane fragments absorb water and swell. And the lipid membrane bending and sealing can form multi-cell liposomes (MLV), its particle size range is 1~5μm. Because MLV prepared by hydrating lipids is too large or very uneven, if you want to change the size and other properties of liposomes, especially to convert MLV into single-chamber liposomes (SUV or LUV), the diameter equalization technique can be used  to disperse the liposomes formed by the thin film method. According to the different dispersion methods, the film method can be divided into the following types: hand-shaken multilamellar vesicles, extrusion vesicles, dried- rehydration vesicles, sonicated vesicles, homogenization vesicles and microflul-dization vesicles.

Reverse Evaporation Method
The reverse evaporation method was originally proposed by Szoka and Papahadjopoulos in 1978, and was a breakthrough in the liposome preparation method. This method is suitable for most phospholipids. It can encapsulate a larger volume of water, and has a higher encapsulation rate for water-soluble drugs. The encapsulation volume is about 30 times larger than that of the single cell liposome of the ultrasonic method, which is larger than the hand-shaken multilamellar vesicles is about 4 times larger; when using a buffer with low ionic strength, it can obtain an encapsulation rate of more than 65% of water-soluble drugs. For some biologically active macromolecular substances such as proteins and alkaline phospholipases can also achieve an encapsulation rate of 30% to 40%. The main disadvantage of this method is that the contact of the drug with an organic solvent may cause denaturation of the drug (such as protein).

The general preparation method is to dissolve membrane materials such as phospholipids in water-insoluble organic solvents such as chloroform, ether, etc.. Add the aqueous solution of the drug to be encapsulated and perform short-term ultrasound until a stable W/O emulsion is formed, and the organic solvent is removed by rotary evaporation under reduced pressure. After reaching the gel state, the buffer solution was added dropwise and rotated to help the gel on the wall of the device to fall off, and then continued to evaporate under reduced pressure to prepare an aqueous suspension. By gel chromatography or ultracentrifugation, the unencapsulated drugs are removed to obtain large unicompartment liposome (LUV). In this method, the main factors affecting the encapsulation rate are the chemical composition of the lipid, the concentration of the lipid, and the ratio of organic phase to water.

Re-emulsion Method
This law was first reported by Matsumoto. First emulsify a small amount of water phase (containing drugs) and a large amount of phospholipid oil phase (soluble in organic solvents) to form a W/O emulsion; remove part of the solvent (or not) under reduced pressure, and then add a larger amount The aqueous phase is emulsified a second time to form a W/O/W double emulsion; the organic solvent is evaporated under reduced pressure to obtain liposomes. The encapsulation rate of this method is 20%~80%. This liposome is very similar to native cells and is an excellent biofilm model.

Centrifugation Method
This method uses centrifugal force to transform W/O emulsion into small unilamellar liposomes. The water droplets in the emulsion are close to the W/O interface under the action of high-speed centrifugal force. When they touch the monomolecular layer at the interface, they coat the second monomolecular lipid and enter the aqueous phase to form the liposome bilayer . The method is simple and fast, the particle size of the prepared liposome is in the range of 50~200nm, and the encapsulation rate of carboxyfluorescein can reach 60%.