Review on Liposomes: Structure, Composition, Drug Delivery Carrier and Current Status of Approved Drug

Liposomes are tiny, self-made packages made from lipids that form a single layer (uni-lamellar) or several layers (multi-lamellar) around a water-filled centre. They range in size from 30 nanometers to the micrometer level, and the lipid layer itself is about 4 to 5 nanometers thick. The study of liposomes was started by a British scientist named Alec Bangham and his team at Babraham, Cambridge,        in         the       mid-1960s. They first described the structure of liposomes in 1964. Since then, liposomes have been studied a lot as a way to deliver medicines, proteins, genetic material, and imaging tools. Different ways of giving liposomes, like through injection, inhalation, mouth, skin, eyes, and nose, have been developed to make treatments more effective and easier for patients to follow. They are also widely used in food and beauty products. As drug carriers, liposomes possess exceptional properties such as protecting the encapsulated substances from physiological degradation, extending the drug's half-life, controlling the release of drug molecules, and offering excellent biocompatibility and safety. Additionally, liposomes can selectively deliver their payload to the diseased site through passive and/or active targeting, thus reducing systemic side effects, increasing the maximum-tolerated dose, and enhancing therapeutic benefits. A significant advantage of systemic liposomes as drug formulations is their high biocompatibility, low immunogenicity, biodegradability, increased efficiency, prolonged drug half-life, targeted delivery, reduced systemic toxicity, protection of sensitive molecules, and improved pharmacokinetics. The utmost advantage of systemic liposomes is the simultaneous incorporation and release of two different materials with different solubility. Reports from various studies have shown that different types of liposomes are classified based on the number of bilayers, size, and liposomal composition, and are discussed in further sections. Various publications focus on conventional methods, biomedical applications, and recent advances in liposomal methodologies. In this review, we broadly focus on inventive ideas in methods of preparation and commercially available liposomal formulations with different routes of administration, characteristics, and their applications to overcome the limitations of conventional preparations. In addition, this review discusses a broad range from conventional methods to recent advancements in preparation techniques and new innovation technologies in liposomal preparation, along with the mechanism of formation wherever possible, with a mention of specific advantages and limitations of each liposomal methodology. Furthermore, ongoing research on clinical trials and patents approved in recent years is well detailed. Therefore, we anticipate this resource can offer an overall pathway for researchers to choose an optimal method with up-to-date knowledge on various biomedical applications, along with an idea on current research in clinical trials and patents, providing a pathway for liposomes from pre-clinical research to production and clinical use.

Fig 1: Schematic representation of liposomes.

A:1,2-Dipalmitoyl-sn-glycero-3-phosphorylethanolamine (DPPE): This is a type of glycerophospholipid, specifically a phosphatidylethanolamine, where the glycerol backbone is esterified with two palmitic acid chains and a phosphate group linked to ethanolamine.

B:1,2-Dipalmitoyl-sn-glycero-3-phosphatidic acid sodium salt (DPPA): This is also a glycerophospholipid, specifically phosphatidic acid, where the glycerol backbone is esterified with two palmitic acid chains and a phosphate group. The "sodium salt" indicates the counterion for the negatively charged phosphate group.

C: 1,2-Dipalmitoyl-sn-glycero-3-phosphoglycerol sodium salt (DPPG): This structure is a glycerophospholipid, specifically a phosphatidylglycerol, where the phosphate group is linked to another glycerol molecule. Like DPPA, it's shown as a sodium salt.

D: 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC): This is a glycerophospholipid. specifically a phosphatidylcholine, where the phosphate group is linked to a choline molecule. DPPC is a common component of cell membranes.

Fig2:-Palmitic acid-based synthetic phospholipids: A) 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine, B) 1,2-Dipalmitoyl-sn- glycero-3-phosphatidic acid sodium salt, C) 1,2-Dipalmitoyl-sn-glycero-3-phosphorylglycerol sodium salt, andD)1,2-Dipalmitoyl-sn- glycero-3-phosphocholine(DPPC).

Fig3:-Natural phosphatides most commonly used in liposome production include: A) Phosphatidylcholine, B) Phosphatidylethanolamine, C) Phosphatidylserine, D) Phosphatidylinositol, E) Phosphatidylglycerol, and F) Phosphatidic acid.

Liposomes perform their movement through four distinct mechanisms:

• Endocytosis – This occurs via phagocytic cells of the reticuloendothelial system along with neutrophils.
• Adsorption – It occurs on the cellular surface through non-specific electrostatic forces or by the interplay with cell surface components.
• Fusion – It occurs through the insertion of the liposomal bilayer into the plasma membrane, resulting in the continuous release of liposomal content into the cytoplasm.
• Lipid exchange- on this transfer of liposomal lipids to the cellular membrane without association of liposomal contents.

Fig 4: Mechanism of formation of Liposomes.

The main goals of a method for making liposome Nano-formulations are to create particales that are all the same size ( with a narrow size range ), have the desired number of layers, include the drugs efficiently, and stay stable over time. In traditional methods, liposomes are first dissolved in volatile organic solvent and then mixed with an aqueous solution. Using an organic solvent can affect the chemical properties of the active ingredients included or may impact the stability or toxicity of final Nano-formulation. The conventional methods for making liposomes usually include the following meain steps:-

• • • Dissolution of lipids in an organic solvent;
    • Removal of the organic solvent from the lipid solution;
    • Hydration of the lipid in an aqueous medium (followed by agitation or stirring);
    • Reduction in size (and/or alteration in lamellarity);
    • Post-formulation processing (purification, sterilization);
    • Characterization of the final nanoformulation product.

Depending on the specific formation method, the hydration of the lipid (step 3) may occur prior to the removal of the organic solvent from the lipid solution (step 2).

• • Thin-Film Hydration (TFH) Method (Bangham Method):-

The thin-film hydration technique, commonly known as the Bangham method, is the oldest, most widely used, and simplest approach for preparing multilamellar vesicles (MLVs). To achieve a uniform mixture, the main phospholipid components are first dissolved in an organic solvent such as dichloromethane, chloroform, ethanol, or a chloroform-methanol mixture. Subsequently, the solvent is removed under reduced pressure using a vacuum pump at a temperature of 45–60°C. For small volumes (<1 mL), the solvent can be evaporated using a dry nitrogen or argon stream within a fume hood until the residual solvent is entirely removed, while</mark> rotary evaporation is typically used for larger volumes. After the solvent is removed, a homogeneous, dry, thin-lipid film composed of stacked bilayers is formed. The final step involves hydrating the lipid film using an appropriate aqueous solution (buffer).that, for the pharmaceutical formulation, it may consist of a solution of simple distilled water or a normal (phosphate) saline buffer at pH 7.4. The hydration process, which typically lasts 1–2 hours, is generally carried out at a temperature of 60–70 °C, and in any case, above the phase-transition temperature of the component lipids. During this stage, agitation (stirring) may assist in detaching the (swelling) lipids' lamellae from the internal vessel surface. To ensure full lipid hydration, the final liposome suspension is then left overnight at a temperature of T = 4 °C. During the hydration stage, the lipid becomes swollen and hydrated, resulting in the formation of a MLV suspension that is highly heterogeneous in size.

Fig 5: - Thin-Film Hydration (TFH) Technique

• • Mechanical Stirring:
• Process: Phospholipids are directly dissolved in water and undergo vigorous mechanical stirring, often combined with ultrasound to reduce particle size.

Advantages: Avoids toxic organic solvents and is relatively straightforward.

• Solvent Dispersion Methods:
  • Process: Involves dissolving lipids in organic solvents and then dispersing them in an aqueous medium. Techniques include reverse evaporation and microfluidic methods.

Advantages: Can achieve high encapsulation rates and uniform liposome sizes.

• • Supercritical Fluid Methods:
• Process: Utilizes supercritical carbon dioxide to prepare liposomes, which avoids the use of toxic solvents and allows for better control over the process.

Advantages: Non-toxic, recyclable, and can enhance drug encapsulation efficiency.

• Advantages of Liposomes:

• Targeted Drug Delivery: Liposomes can encapsulate drugs and deliver them specifically to target tissues or cells, enabling targeted therapy. This reduces the exposure of healthy tissues to the drug, leading to fewer side effects.
• Cosmetic Applications: Liposomes are used in cosmetics to improve the penetration of active ingredients into the skin, thereby increasing their effectiveness.
• Immunogenicity: Liposomes can enhance the immunogenicity of vaccines, leading to a stronger and more specific immune response.
• Vaccine Delivery: Liposomes play a key role in vaccine development by improving the stability and delivery of antigens, thus enhancing immune responses.
• Reduced Toxicity: Liposomal drug delivery can lower the toxicity of certain drugs by ensuring they target diseased cells while minimizing exposure to healthy tissues.

• Disadvantages of Liposomes:

• Expense: The production of liposomal formulations can be expensive, potentially resulting in higher costs for liposome-based treatments.
• Clinical Translation: Although preclinical studies show promising results, not all liposome-based therapies have successfully moved to clinical application, highlighting challenges in translating research findings into practical use.
• Interaction with Biological Systems: Liposomes may interact with proteins or cells, which can influence their stability, drug release, or overall performance.
• Storage Stability: Liposomes may be unstable during storage, leading to issues such as aggregation, leakage of the encapsulated substances, or changes in size and structure.
• Biodegradability: Based on their composition, liposomes may not be easily biodegradable, raising environmental concerns.
• Complexity: The development of liposomal formulations requires specialized knowledge and can be a complex process, which may limit access for researchers and manufacturers.
• Biological Testing and Evaluation of Liposomes:

Liposomes function as a vital delivery system, especially for targeted drug delivery. They are particularly valuable in the treatment of diseases such as cancer and antiviral conditions. Stealth liposomes are spherical vesicles with a phospholipid bilayer membrane and are employed to transport drugs or genetic materials into cells. A study conducted by Luoetal. investigated the drug release effectiveness of Doxorubicin encapsulated within porphyrin-phospholipid stealth liposomes. Near-infrared light, capable of penetrating tissues, serves as an external stimulus for drug release, enabling precise spatial and temporal regulation. The research indicated that Dox-loaded stealth PoP liposomes exhibited a prolonged circulation half-life in mice, lasting up to 21.9 hours and maintaining stability for months. A single chemophototherapy treatment using these liposomes at a dose of 5-7 mg/kg of Dox effectively eradicated tumors, outperforming conventional chemo- or photodynamic therapies. Thus, stealth liposomes present significant potential in the delivery of drugs for cancer treatment. Recent developments emphasize notable therapeutic improvements through the use of corticosteroid-loaded liposomes in experimental arthritis models. A key element of liposome drug delivery is the encapsulation of drugs into the liposomes, which can occur passively during formation or actively afterward. Hydrophobic drugs such as amphotericin B, taxol, or annamycin can be directly incorporated during liposome creation, and their uptake and retention depend on drug-lipid interactions. Water-soluble drugs with protonizable amine groups can be actively encapsulated using pH gradients. The advantages of drug loading in liposomes include enhanced solubility of lipophilic and amphiphilic drugs, passive and active targeting to immune cells, site-specific targeting, and increased transfer of hydrophilic and charged molecules. Evaluation of liposomes involves pharmacokinetics, in-vitro testing, and efficacy assessment. A study by Wang describes the preparation and in-vitro evaluation of an acidic environment-responsive liposome for paclitaxel tumor targeting. This study employed cholesteryl hemisuccinate (CHEMS) to improve drug accumulation at the tumor site. The in-vitro release characteristics were analyzed using dynamic dialysis, demonstrating acid sensitivity and sustained release properties. In comparison to free paclitaxel, the liposomes showed higher cytotoxicity and improved cellular uptake, making them suitable for targeted cancer therapy with paclitaxel. Microscopic imaging plays a crucial role in analyzing the structural and morphological properties of liposomes. A study by S. Bibi et al. discusses the use of various microscopic imaging techniques to evaluate liposome structure. Larger vesicles such as multilamellar and giant unilamellar vesicles can be observed using light microscopy. Common techniques include light, fluorescence, confocal microscopy, and electron microscopy methods like transmission, cryo, freeze fracture, and environmental scanning electron microscopy. In transmission electron microscopy, a small amount of hydrated specimen is placed on a grid, and a negative stain like Uranyl Acetate or Osmium Tetroxide is used to visualize the vesicles against a stained background. Fluorescent microscopy tracks particulate delivery systems in biological environments and provides information about the structure of bilayer vesicles, allowing the assessment of various parameters since probes can be placed in both the aqueous and bilayer compartments. A review by Klang et al. discusses electron microscopic techniques for pharmaceutical systems. In scanning electron microscopy, images are formed by scanning a focused electron beam across the surface of a solid specimen. One major advantage of SEM is its pronounced depth of focus combined with image formation, where projecting areas cast shadows and recessed areas appear dark, allowing easy interpretation of the information. Another advantage is the absence of sample preparation for solid samples, enabling the investigation of large areas with high depth of focus. However, a major disadvantage is the time required to collect data one pixel at a time, leading to extended exposure to the electron beam.

• Approved drug of Liposomes: