Liposomes, as drug carriers, offer several advantages such as high targeting capability, effective protection of encapsulated drugs, controlled and sustained drug release, significant improvement in therapeutic index, and reduction of adverse drug reactions. In the previous article, we introduced the structure and types of liposomes. Today, we will share the methods for preparing liposomes!
Overview of Liposome Preparation Methods
To achieve good clinical outcomes with liposome formulations, research on liposome preparation techniques is essential. Minor changes in the preparation process can significantly impact the safety and efficacy of liposome formulations. Methods such as thin-film hydration, reverse-phase evaporation, solvent injection, surfactant removal, and microfluidic technology are used to produce liposome drugs with different sizes, structures, and functions.
Liposome Preparation Methods
2.1 Thin-Film Hydration Method
The thin-film hydration method, also known as the Bangham method, was proposed by Bangham et al. in 1967. It is the simplest and earliest method for preparing liposomes. In this method, lipid materials are first dissolved in an organic solvent, and then the organic solvent is removed using rotary evaporation to produce a lipid film. The lipid film is then hydrated to produce dispersed liposomes. The hydration conditions directly affect the structure of the formed vesicles. Mild hydration conditions form large unilamellar vesicles, while vigorous shaking produces multilamellar vesicles with less uniform sizes. Sonication methods, such as probe sonication and bath sonication, can be used to produce unilamellar vesicles with small, uniform sizes. However, probe sonication can cause localized high temperatures, potentially damaging the structure and function of liposome active parts and causing metal contamination from the probe. Therefore, bath sonication is a better choice for obtaining uniform small unilamellar vesicles at appropriate temperatures and sonication parameters.
2.2 Reverse-Phase Evaporation Method
The reverse-phase evaporation method was first proposed by Szoka et al. In this method, lipid components are dissolved in an organic solvent immiscible with water, a small amount of aqueous solution is added, and then the solvent is removed by rotary evaporation to obtain a liposome suspension. This process forms reverse micelles or water-in-oil emulsions, with the aqueous phase carrying the target drug and the organic phase consisting of lipids. The addition of the aqueous phase allows rapid sonication to form uniformly dispersed liposomes. Compared to the thin-film hydration method, this method provides higher internal aqueous phase loading, resulting in more stable liposomes that are less prone to aggregation and precipitation. Residual solvents can be removed by dialysis or centrifugation. However, the drawback is that the drug to be encapsulated in the vesicles comes into contact with the organic solvent.
2.3 Solvent Injection Method
The principle of the solvent injection method is to dissolve lipid components in an organic solvent and then inject the desired drug at a certain rate into the organic phase to induce liposome formation. Liposomes prepared by this method maintain relatively high polydispersity. The removal of the organic solvent is the main challenge for large-scale production, especially when ethanol is used as the organic solvent, as some lipids have poor solubility in ethanol and may form heterogeneous liposomes if not mixed thoroughly. This affects the stability and safety of liposome products. Additionally, factors such as drug type, lipid concentration, ratio of organic solvent to lipids, mixing rate, injection port diameter, and injection rate determine the size of the produced liposomes.
2.4 Surfactant Removal Method
The surfactant removal method is a gentle technique for producing various types of vesicles and highly homogeneous liposomes. In this method, lipids are dissolved in an aqueous solution containing surfactants to form surfactant-lipid micelles. When the surfactant is at its critical micelle concentration, it is removed by dialysis or other methods to form liposomes. The size and uniformity of the produced liposomes depend on the rate of surfactant removal and the initial ratio of surfactant to lipids. This method does not involve organic solvents but has the drawback of low liposome concentration in the final solution, potential surfactant residues, and possible removal of other small hydrophilic compounds during the surfactant removal process.
2.5 Microfluidic Technology
Traditional liposome production methods, such as thin-film hydration and solvent injection, rely primarily on the spontaneous aggregation of lipids in solvents, which is an uncontrollable process. Therefore, the synthesized liposomes are polydisperse and multilamellar, often requiring further processing by extrusion, sonication, or high-pressure homogenization. Microfluidic technology, however, is a versatile technique for liposome preparation. The lipid phase and aqueous phase are introduced through different inlets into a channel with a diameter of tens to hundreds of micrometers. The lipid phase is focused into a narrow sheet by hydrodynamic forces, and by adjusting the volume flow rate ratio of the aqueous phase to the lipid phase, channel length, temperature, mixing conditions, and lipid concentration, variable liposome sizes (ranging from tens of nanometers to several hundred micrometers) and narrow size distributions can be achieved.
Conclusion
In addition to the methods mentioned above, there are many other techniques for preparing liposomes, such as active drug loading, double emulsification, and supercritical fluid methods. The ever-maturing and diverse preparation techniques make liposome preparation more complete. However, the key issue is which type of liposome is suitable for the encapsulated drug, which method can better encapsulate the drug, and whether the ideal in vivo release profile can be achieved. Therefore, the choice of liposome preparation technology should be tailored to the specific drug.
