Overview of Liposome Preparation Process

In 1965, Bangham et al. first discovered that when phospholipid molecules were dispersed in aqueous solution, phospholipid molecules spontaneously formed closed double-layer vesicles. Subsequently, the closed spherical vesicles composed of phospholipid bilayers are called liposomes. The discovery of liposomes has aroused widespread interest among scientists. The hydrophilic environment in liposomes can encapsulate hydrophilic drugs, while lipophilic drugs can be loaded between the phospholipid molecular layers that constitute liposomes. Liposomes as carriers have a biofilm structure and low immunogenicity, which can prolong the half-life of drugs, reduce drug toxicity, and improve drug delivery efficiency.

Schematic diagram of DMPC unilamellar liposomesFig. 1. Schematic diagram of DMPC unilamellar liposomes (Pharmaceutics. 2022, 14(3): 543).

Since liposomes have attracted the attention of researchers, further innovations have been made in liposome formulation design, structural modification, and preparation processes. Problems such as poor stability and low encapsulation efficiency of traditional liposomes are gradually being solved. The application of liposomes in drug delivery has been continuously expanded, covering multiple therapeutic areas such as anti-tumor, anti-fungal, analgesia, vaccines and photodynamic therapy. In recent years, many pharmaceutical companies have also successfully developed and launched several liposome preparations, such as paclitaxel liposome injection, doxorubicin liposome injection, amphotericin B liposome injection, etc. Since different formulations and preparation processes will have different impacts on the safety and effectiveness of liposome drugs, it is necessary to understand the critical quality attributes of liposome drugs and establish an effective quality control strategy.

Lipid Products for Liposome Preparation

CatalogProduct NameCAS NumberCategory
BPG-3286Cholesterol57-88-5Glycosylated Sterols
BPG-302427-alkyne cholesterol1527467-07-7Click Reagents
BPG-3028alkyne-cholesterol1631985-09-5Click Reagents
BPG-32827α-Hydroxy Cholesterol17954-98-2Cholesteryl Esters
BPG-310514:0 PE-DTPA384832-89-7Lipids For Bicelle Formation
BPG-310912:0 Diether PC72593-72-7Loadable Liposomes
BPG-310706:0 Diether PC79645-39-9Liposomal Doxorubicin Nano-Drug
BPG-310813:0 Diether PC328250-31-3Liposomal Doxorubicin Nano-Drug

Preparation Process of Liposomes

In order to achieve good clinical effects of liposome preparations, it is inseparable from the research on the liposome preparation process. Small changes in the drug preparation process will have an important impact on the safety and effectiveness of liposome preparations. Currently, thin film hydration method, solvent injection method, multiple emulsion method, active drug loading method, microfluidic technology, etc. are all liposome preparation methods used to produce liposome drugs with different particle sizes, structures and functions.

Thin Film Hydration Method

Thin film hydration method is one of the traditional methods for preparing liposomes. This method is to mix lipids and organic solvents such as chloroform, and remove the organic solvent by rotary evaporation or other methods. When a hydrophilic medium such as phosphate buffer is added to the formed lipid film, the lipids will spontaneously hydrate and aggregate to form non-homogenized liposomes with larger particle sizes. It is usually a multi-layered structure. By combining ultrasound and membrane extrusion, the liposome particle size can be reduced and liposomes with a relatively uniform particle size can be obtained. This method is relatively mature and has simple equipment, but because the method uses harmful organic solvents such as chloroform and methanol, residues of these chemicals may remain in the final liposome preparation, causing potential toxicity.

Thin film hydration method for liposome preparationFig. 2. Thin film hydration method for liposome preparation (Pharmaceutics. 2022, 14(3): 543).

In order to avoid the use of toxic reagents, Mortazavi et al. proposed using an improved heating method to prepare liposomes. Each component of the liposome, dipalmitate phosphatidylcholine (DPPC), dishexadecylphosphate (DPC), and cholesterol, was hydrated in phosphate buffer at room temperature for 2h. Then add glycerol (3%, v/v) to the cholesterol suspension, heat to 120°C and stir for 20 min. Then lower the temperature to 60°C, add other components of the liposomes to the cholesterol suspension, stir and leave at room temperature for 30 minutes to form liposomes. This method does not involve the use of toxic reagents, does not require high shear homogenization and ultrasonic treatment, and the encapsulation rate of the prepared anionic liposome-Ca2+-DNA ternary complex is as high as 81%. Nowadays, in addition to the traditional thin film hydration method, a variety of fast, organic solvent-free, and homogenization-free hydration methods have been developed.

Solvent Injection Method

The solvent injection method is to dissolve phospholipids and cholesterol in ethanol or ether to form an organic phase. Inject the organic phase into a certain volume of aqueous phase using a syringe pump under magnetic stirring. Liposomes are spontaneously formed due to their hydrophobic interaction when organic solvents come into contact with water, and the organic solvent in the liposomes is finally removed by rotary evaporation. Parameters such as lipid concentration, stirring speed, injection flow rate, solvent injection volume, and temperature will all affect liposome particle size.

Solvent injection method for liposome preparationFig. 3. Solvent injection method for liposome preparation (Pharmaceutics. 2022, 14(3): 543).

For example, Jaafar-Maalej et al. successfully prepared liposome formulations encapsulating lipophilic and hydrophilic drugs using the ethanol injection method, and studied the effects of key process parameters and formulation parameters on liposomes. The results show that the stirring rate of the aqueous phase and the concentration of phospholipids in the organic phase are key parameters affecting liposome size. Gentine et al. used an optimized ethanol injection method to heat the ethanol solution containing lipids to 60°C (higher than the phase transition temperature of the lipids); then inject it into the aqueous phase at 70°C. After stirring, the ethanol is removed by rotary evaporation. This optimized ethanol injection method can effectively control the liposome particle size. The results showed that when the volume ratio of ethanol was 33% (v/v), lipid concentration, lipid charge and water phase type had little effect on the vesicle diameter.

Multiple Emulsion Method

The multiple emulsion method, also called the secondary emulsification method, refers to dissolving lipids in an appropriate amount of organic solvent and adding a small amount of water phase in proportion, and obtaining a W/O emulsion after sonication or shaking. Then add a large amount of aqueous phase solution and perform a second emulsification to obtain a W/O/W emulsion. Finally, remove excess organic solvent and water phase by rotary evaporation. The multiple emulsion method is a common method for preparing multivesicular liposomes.

Luo et al. prepared oleanolic acid polyvesicular liposomes through the multiple emulsion method, which showed sustained release effects both in vivo and in vitro. In vivo studies of naltrexone hydrochloride multivesicular liposomes prepared by Sun et al. through the Depofoam double-emulsion method have shown that the plasma concentration can be maintained at a relatively constant level above 10 ng·mL-1 for about 120 h. The Depofoam technology, a patent for multivesicular liposome preparation technology launched by an American pharmaceutical company, uses the multiple emulsion method. This technology has been developed for more than 20 years and has been successfully used in multiple marketed liposome preparations, such as cytarabine liposome injection, morphine sulfate sustained-release liposome injection, bupivacaine liposome injection, etc. Due to the particularity of the structure, the particle size of multivesicular liposomes prepared by the multiple emulsion method is larger than that of ordinary liposomes, usually in the range of 5-50 μm.

Active Drug Loading Method

The above liposome preparation methods are all passive drug loading methods, while the active drug loading method is to use the pH gradient or ion gradient of the internal and external aqueous phases of the liposome to make the water-soluble or amphiphilic drugs actively cross the lipid molecular layer into the internal aqueous phase of the liposome. The active drug loading method has a high encapsulation rate for water-soluble drugs and amphipathic drugs. The pH gradient method, the ammonium sulfate gradient method, the calcium acetate gradient method, etc. are all liposome preparation methods used to encapsulate weakly alkaline and weakly acidic drugs. In the active drug loading method, transmembrane gradient (pH gradient/ion gradient), incubation temperature and time, drug-lipid ratio, logarithm of drug oil-water partition coefficient (logP) and acidity coefficient (pKa) are all influencing factors of active drug loading. For example, doxorubicin liposome preparations are prepared using the ammonium sulfate gradient method, and the encapsulation rate reaches about 97%. The irinotecan liposomes, cytarabine and daunorubicin complex liposomes approved by the FDA are also prepared using active drug loading methods.


Since traditional liposome production methods such as thin film hydration and solvent injection mainly rely on the spontaneous aggregation of lipids in solvents, the process is uncontrollable. Therefore, the synthesized liposomes are polydisperse and multilayered, and usually need to be further processed by extrusion, ultrasound or high-pressure homogenization. Microfluidic technology is a multifunctional liposome preparation technology. The lipid phase and the water phase pass through a channel with a diameter of tens to hundreds of microns at different inlets. The lipid phase flow is focused into a narrow sheet under hydrodynamic action. By adjusting the volume flow rate ratio of the water phase and the lipid phase, the length of the fluid channel, temperature, mixing conditions, and lipid concentration, a variable liposome particle size (tens of nanometers to hundreds of microns) and a narrow particle size distribution are achieved.

Microfluidic (channel) method for liposome preparationFig. 4. Microfluidic (channel) method for liposome preparation (Pharmaceutics. 2022, 14(3): 543).

Elsana et al. prepared new cationic liposomes by thin film hydration and microfluidic method, respectively. The results showed that compared with the liposomes prepared by thin film hydration, the liposomes prepared by microfluidic method had smaller and more uniform particle size and Zeta potential, and the encapsulation efficiency was higher. Deng et al. used surfactant-assisted microfluidic technology to prepare monodisperse single-chamber and multi-chamber liposomes, achieving high-yield, high-throughput production of single-layer and multi-layer liposomes.

Supercritical Fluid Technology

Supercritical fluid (usually economical and non-toxic CO2) has the characteristics of gas and liquid phases at the critical point, that is, low viscosity, high density, good fluidity and solubility, and has special advantages in liposome preparation. Supercritical fluids can be used as solvents or dispersants for lipids. When used as a solvent, it can replace the use of organic solvents to combine the lipid phase with water, and remove CO2 into gas by changing the pressure and temperature, thereby avoiding the residue of organic solvents in the final product. When used as a dispersant, the lipids dispersed in pure water are pressurized by supercritical fluid, so that the lipids are better dispersed in the medium due to collision and shear forces. Not only that, supercritical fluid technology has a fast and simple one-step preparation process. By controlling important parameters such as lipid concentration, dispersion volume, pressure and temperature, it can prepare and industrially produce liposomes with specific physical and chemical properties. A variety of supercritical fluid technologies have been applied to the preparation of liposomes, such as injection decompression method, supercritical antisolvent method, supercritical solution rapid expansion method, supercritical fluid reverse phase evaporation method, etc.

Supercritical reversed-phase evaporation method for liposome preparationFig. 5. Supercritical reversed-phase evaporation method for liposome preparation (Pharmaceutics. 2022, 14(3): 543).

In order to simplify the preparation method of amphotericin B liposomes and reduce production costs, Lim et al. used a supercritical antisolvent method to prepare amphotericin B liposomes with a particle size of 100~150nm and an encapsulation rate of about 90%. There were no significant changes in the particle size, encapsulation efficiency, and particle size distribution of the liposomes before and after freeze-drying. This shows that the liposomes prepared by this method have uniform particle size and strong stability, and can be used as a potential method for preparing amphotericin liposomes.

Thin Film Hydration MethodSuitable for preparing multilamellar liposomes with a particle size of 1~5m, the most basic and widely used.Organic solvent remains and needs to be homogenized.
Solvent Injection MethodIt is suitable for preparing 30~200 mm unilamellar liposomes with simple conditions and high operational feasibility.Organic solvent remains and needs to be homogenized.
Multiple Emulsion MethodSuitable for preparing polysaccharide liposomes with a diameter of 5~50 μm.Encapsulation efficiency is low.
Active Drug Loading MethodSuitable for encapsulation of water-soluble and amphiphilic drugs with high encapsulation rate.The process is cumbersome and requires steps such as dialysis and desalination.
Microfluidie TechnologyWide application, high encapsulation rate, uniform particle size, and simple steps.Organic solvents remain and the technical conditions are demanding.
Supercritical Fluid TechnologyWide application, uniform particle size, no organic solvent residue, and simple steps.Technical requirements are demanding.

Table 1. Comparison of advantages and disadvantages of liposome preparation methods.

What Can We Offer for Liposome Preparations?

With extensive manufacturing capabilities in lipids, BOC Sciences can synthesize custom liposomes based on specific requirements such as size, composition, and surface functionalization to facilitate drug delivery, vaccine development, cosmetics, and other research advancements. We can also provide comprehensive characterization of liposomes, including size, zeta potential, morphology and encapsulation efficiency.

If you are interested in our liposome preparation support services, please contact us for more information.


  1. Lombardo, D. et al. Methods of Liposomes Preparation: Formation and Control Factors of Versatile Nanocarriers for Biomedical and Nanomedicine Application. Pharmaceutics. 2022, 14(3): 543.

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