Methods for Preparation of Liposomes

Due to its crucial capability in delivering drugs to target sites, liposomes have garnered more extensive attention compared to other delivery systems. The composition and preparation methods of liposomes profoundly influence their fundamental characteristics, including polydispersity index, average size, drug loading efficiency, zeta potential, drug release kinetics, and cellular uptake. Liposome preparation methods can be broadly categorized into conventional methods and large-scale production methods based on the synthesis scale.

Conventional Methods for Liposomes Synthesis

There are various conventional methods for liposome preparation. The differences between these methods mainly lie in how lipids are dried from organic solvents and then dispersed in an aqueous medium. Although conventional methods are easy to implement, their drawback is the substantial use of organic solvents, which pose potential harm to the environment and human health, requiring thorough removal of residual solvents. Furthermore, conventional methods involve multiple homogenization steps that consume significant energy, making them unsuitable for large-scale production.

• Mechanical Mixing Method

In the mechanical stirring method, phospholipids are dissolved directly in water through vigorous mechanical stirring, often with probe ultrasound. Despite its simplicity, the small-sized liposomes produced are not highly stable, and the risk of lipid degradation from probe contact remains unavoidable. Titanium contamination is also a concern during probe ultrasound. While this method avoids toxic organic solvents, limitations like drug leakage and formulation instability hinder its application in drug delivery liposome preparation.

• Film Hydration Method

The film hydration method, also known as the Bangham method, is a widely used technique for liposome preparation. It involves dissolving phospholipids in an organic solvent, forming a lipid film through solvent evaporation. Despite its widespread use and simplicity, the method may yield larger-sized (1-5μm in diameter) multilayered liposomes in the aqueous buffer. Techniques like ultrasonication (SUV formation) or poly-carbonate filtration (LUV formation) are necessary to reduce liposome size, ensuring smaller and more uniform structures. While preferred for achieving a 100% encapsulation rate for lipophilic compounds with sufficient quantity, it should be noted that water-soluble compounds may be washed out during the swelling process, resulting in only 10%-15% encapsulation.

• Reverse Phase Evaporation Method

The reverse evaporation method involves simultaneous introduction of phospholipids and an organic solvent into a round-bottom flask. Evaporation under reduced pressure and controlled temperature follows, using a rotary evaporator. The lipid is then redissolved in an organic phase with nitrogen gas, capable of forming reverse vesicles. Isopropanol and diethyl ether are preferred solvents. After redissolving, an emulsion forms, and under reduced pressure, solvent removal results in a semi-solid gel. The obtained liposomes, after removing free or unencapsulated drugs, are termed reverse evaporation liposomes, known for enhancing drug encapsulation efficiency.

• Solvent Injection Method

In the solvent injection method, phospholipids are first dissolved in an organic solvent, and the solution is thoroughly mixed with an aqueous medium containing the drug to be encapsulated in the liposomes. The arrangement of lipids at the interface between the organic and aqueous phases into a monolayer is a crucial step in the formation of the liposomal bilayer. The organic solvents used in this method may or may not be miscible with the aqueous phase. Solvent injection methods include two different approaches: ethanol injection method and ether injection method. Generally, the ether injection method is preferred over the ethanol injection method.

• Detergent Removal Method

The detergent removal method is a technique used to prepare liposomes of nearly uniform size, based on the formation of detergent-lipid micelles. When the detergent is removed from these micelles, liposomes are formed. The shape and size of the liposomes depend on the rate of detergent removal and the ratio of detergent to lipids. However, this method has some drawbacks, such as a reduction in the final concentration of liposomes in the dispersion, a decrease in drug encapsulation efficiency, and the inability to completely remove the detergent from the final formulation.

• Calcium Fusion Method

In the process of preparing liposomes using the calcium fusion method, calcium is initially added to small unilamellar vesicles (SUVs), leading to the fusion and formation of multilayered liposomes. The addition of ethylenediaminetetraacetic acid (EDTA) to the formulation results in the generation of large unilamellar liposomes.

Large-scale Methods for Liposomes Synthesis

Liposomes have been industrially produced on a large scale and their preparation by various methods, such as supercritical reverse phase evaporation (SCRPE), freeze-drying, heating methods, spray-drying and several other improved ethanol-injection techniques have received increasing attention.

• Heating Method

This method utilizes the hydration of phospholipids with an aqueous solution containing glycerol (3%) to increase the temperature to 60°C or 120°C. Glycerol is isotonic and prevents precipitation and coagulation, increasing the stability of liposome vesicles. This is the preferred method because the lipids do not degrade. This method has been improved and is known as the Mozafari method for encapsulation and targeted delivery of food-grade anti-microbial lactobacillus peptides. This improved method can be used for large-scale single-step preparation of liposomes without prior hydration of the constituent materials and also avoids the use of toxic detergents and organic solvents.

• Freeze-drying Method

Based on the formation of a homogeneous dispersion of lipids from liposomes in a water-soluble carrier material, freeze-drying methods can be used to prepare sterile and non-pyrogenic liposomes of small submicron size. The liposome-forming lipid and a water-soluble carrier material, such as sucrose, are dissolved in an appropriate ratio in a tert-butanol/water cosolvent system to form a clear isotropic single-phase solution, and the single-phase solution is then filter-sterilized and loaded into freeze-drying vials, which are used to form the solid liposome formulation by the freeze-drying process.

• Supercritical Fluid Method

Supercritical fluids are substances in the critical point region that have unique properties, behave like liquids and solvents, and can transport substances like gases, properties that make them an excellent alternative to toxic organic solvents. They are now widely used to enhance preparation techniques, reduce particle size and are also used for purification. Carbon dioxide is one of the most widely used supercritical fluids. It is easily recovered by returning the conditions to atmospheric pressure. Using the supercritical fluid method to reduce the size and sterilize liposomes is an easy method.

• Microjet Method

The method of microjet flow is based on microemulsification and can be used for large-scale production of liposomes. In a microjet device, lipids can be added in the form of dispersions of large multilayered liposomes or dispersed as unhydrated lipids in an organic medium such as a phosphate buffer or a volatile solvent. The collected fluid can be recirculated throughout the processor until spherical liposomes are obtained. The process of the microjet method is reproducible and allows the preparation of liposomes encapsulating water-soluble drugs.

• Membrane Contactor Method

The application of ethanol injection technology in the context of using membrane contactors can be used for large-scale liposome production, and this method is called the membrane contactor method. In this method, the lipid phase (ethanol, phospholipids and cholesterol) is extruded through a membrane with a specific pore size. Nitrogen at a pressure below 5 bar is sufficient to pass the organic phase through the membrane. At the same time, the aqueous phase cuts across the membrane surface and removes the liposomes formed within the membrane device. The advantages of this technology are the simplicity of the design, the ability to adjust the process parameters to control the size of the liposomes, and the enhanced capability for large-scale production.

• High Pressure Homogenization Method

High pressure homogenizers are suitable for the preparation of lipid dispersions and liposomes due to their ability to destroy vesicles. The sample is injected into a specially designed section of the homogenizer at a constant high pressure. Turbulence, shearing or cavitation results in the formation of liposomes. The pressure applied and the number of cycles determine the nature of the liposomes prepared by the high pressure homogenizer. High-pressure homogenization results in the preparation of liposomes of very small size, which makes it suitable for the preparation of liposomes for intravenous injection.

• Electroforming Method

In this method, a phospholipid film is deposited on an electrode, which is subsequently hydrated for several hours under the action of an electric field. Although huge monolayers of liposomes can be formed by applying alternating current (AC) and direct current (DC), the DC field has certain shortcomings and blistering occurs due to water electrolysis. 80% of the liposomes prepared by the electroforming method were more perfect monolayers.