In 1965, British researchers dispersed phospholipids in water and observed spherical structures under an electron microscope, naming them “liposomes” (from “lip,” meaning lipid, and “som,” meaning body). Since 1971, liposomes have been widely accepted as “biological missiles” and have become a novel drug delivery system. Various technologies have emerged, driving the rapid development of liposome technology. Over the years, more than ten liposome-based products have been commercialized, including doxorubicin, vincristine, daunorubicin, paclitaxel, irinotecan, mifamurtide, cytarabine/daunorubicin, amphotericin B, bupivacaine, and amikacin.
Discovery and Structure of Liposomes
Liposomes are closed spherical structures formed by the self-assembly of lipids into single or multiple concentric bilayers, enclosing an aqueous core. Their size ranges from 30 nm to micrometers, with a phospholipid bilayer thickness of approximately 4–5 nm. Liposomes were first discovered in 1961 by British scientist Alec Bangham and his colleagues, who observed that phospholipids dispersed in aqueous media spontaneously form closed vesicles. In 1964, they published the structure of liposomes, and by 1968, the term “liposomes” was officially adopted and has been used ever since. The basic components of liposomes are typically amphiphilic phospholipids and cholesterol. Amphiphilic phospholipids form the bilayer structure, while cholesterol supports and stabilizes it. Commonly used phospholipids include sphingomyelin and glycerophospholipids, both of which have hydrophilic heads and hydrophobic tails. In aqueous environments, phospholipid molecules spontaneously arrange into liposomes driven by hydrophobic interactions and other intermolecular forces. Cholesterol enhances lipid chain packing, stabilizes the bilayer, reduces membrane fluidity, and minimizes the transmembrane transport of water-soluble drugs. Additionally, cholesterol reduces interactions between liposomes and proteins in the body, preventing phospholipid loss and improving liposome stability.
Advantages of Liposomes
Biocompatibility and Biodegradability: Liposomes closely resemble cell membranes, making them highly biocompatible and biodegradable. They protect drugs from enzymatic degradation before reaching the target site, enhance drug stability, reduce toxicity, and allow for higher dosing, improving therapeutic efficacy.
Targeted Delivery: The amphiphilic phospholipid bilayer can be modified physically or chemically with ligands or functional groups to confer tissue-targeting properties. This prolongs the retention time of liposomes at the target site and enables efficient targeted drug delivery.
Versatility in Drug Delivery: By altering the surface charge of the bilayer, liposomes can encapsulate and deliver DNA and RNA drugs, such as cationic liposomes.
Liposome Preparation Techniques
Solvent Injection Method
This is the most widely used method due to its simplicity, cost-effectiveness, and lack of requirement for full aseptic production. Lipophilic excipients are dissolved in organic solvents (e.g., ethanol, ether, methanol, dichloromethane) and rapidly injected into an aqueous medium to form crude liposomes. Residual organic solvents are removed using membrane filtration or tangential flow ultrafiltration. For lyophilized liposome products, liposomes containing organic solvents can be directly freeze-dried. Many commercial liposome products, such as doxorubicin and irinotecan liposomes, are prepared using this method.
Thin-Film Hydration Method (Bangham Method)
Named after Alec Bangham, this is the most classic liposome preparation method. Phospholipids are dissolved in an organic solvent, which is then evaporated under reduced pressure to form a thin lipid film. The film is hydrated with an aqueous medium at the phospholipid’s phase transition temperature, leading to the formation of liposomes. This method is simple and achieves nearly 100% encapsulation for most lipophilic drugs. However, the resulting liposomes have uneven size distributions, requiring size control techniques such as polycarbonate membrane extrusion or homogenization. Full aseptic conditions are necessary to ensure product sterility.
Reverse-Phase Evaporation Method
Similar to the thin-film hydration method, this technique involves dissolving lipophilic excipients in an organic solvent and emulsifying them with an aqueous drug solution. The organic solvent is then evaporated under reduced pressure to form liposomes. If the organic solvent volume significantly exceeds the aqueous phase, additional aqueous solution is added post-evaporation to disperse the liposomes. This method is limited to water-soluble drugs and macromolecular active substances and is unsuitable for highly toxic drugs due to low encapsulation efficiency and safety risks from free drugs.
Double Emulsion Method
Lipophilic excipients are dissolved in an organic solvent and mixed with a small amount of aqueous solution to form a stable water-in-oil (W/O) emulsion using mechanical forces (e.g., stirring, shearing, sonication). A large volume of aqueous solution is then added for secondary emulsification, forming a water-in-oil-in-water (W/O/W) emulsion. Residual organic solvents are removed to obtain a liposome suspension. This method typically produces multivesicular liposomes for sustained drug release. Although complex and with narrow process parameter windows, it achieves high encapsulation efficiency and is used to prepare commercial multivesicular liposomes.
Microfluidic Method
Microfluidic technology, based on fluid dynamics, enables continuous mixing, emulsification, and purification within microchannels. In liposome preparation, lipid and aqueous phases are pumped at controlled rates into a microfluidic chip for emulsification. Different channel designs create turbulent, laminar, or atomized flows, and high-pressure pumps reduce particle size through impact and shear forces. This method offers excellent reproducibility and process control.
Additional Techniques for Liposome Production
Additional techniques include freeze-drying hydration, calcium-induced fusion, detergent removal, and supercritical fluid methods. While numerous methods exist, the challenge lies in selecting the most suitable technique based on the drug’s properties, encapsulation efficiency, and desired release profile. Researchers can leverage these methods to optimize liposome formulations for specific experimental or therapeutic goals, ensuring high reproducibility and scalability in production.
Conclusion
Liposomes represent a versatile and transformative drug delivery system with a rich history of development. From solvent injection to microfluidic techniques, the preparation methods have evolved to meet diverse research and pharmaceutical needs. The choice of technique depends on the drug’s characteristics and the desired experimental outcomes, underscoring the importance of method optimization for specific applications. As research continues, liposomes are poised to play an even greater role in advancing drug delivery systems and therapeutic efficacy.