Liposome-A Comprehensive Approach for Researchers

Bangham was first to develop these spherical-shaped nano-vesicles called liposomes in the early 1960s. Today, liposomes have emerged as crucial tools for bettering the delivery of drugs that majorly includes-antifungal drug, peptide hor-mones, enzymes, vaccines antimicrobial agents, drugs against cancer, and genetic materials. Following the different manufacturing practices and versatile properties liposomes can be categorized in various parameters of size, charge, poly-dispersity index, encapsulation efficiency, solubility properties, and lamellarity. Alteration in such parameters elevates the loading and bioavailability of a drug by giving more clear target specification, desired or controlled release. This bibliographic chapter provides a comprehensive overview of methods for the preparation of liposomes with other perspectives that majorly includes—physio-chemical characteristics, dosage regimen, advantages over other delivery systems, approved liposomal based drugs and other ongoing drugs in clinical trials. It will help researchers to break-through more structurally successful delivery vehicles depending upon their various physic-chemical properties.


Introduction
Liposomes can be microscopically examined as the vesicle with spherical structure that comprises one or more bilayer lipid in the aqueous core part of a shell. Liposomes are widely used in the delivery of variety of drugs depending upon its various physic-chemical characteristics. Design and development of liposomes are classified in many ways among which thin film hydration method is the most globally accepted procedure. Liposmes formation occurs when lipids are incorporated into water or buffer solution under continuous stirring, that in return forms the spherically shaped vesicles termed as liposomes. There are many methods to develop liposomes among which thin film hydration method is most common. Recently, lipid film hydration method was used to develop a multilamellar vesicle (MLV) loaded with curcumin (CUR) and Rhodamine B (RhB), [1] as a successful drug delivery approach. Phospholipids and cholesterol are the major components used in the development of liposomes (Figure 1). Where bilayer lipid composes of a hydrophilic head group, i.e., phospholipid and a hydrophilic tail group. Where phospholipids can easily penetrate and localize in the skin thus increases the overall bioavailability in case of many dermal formulations whereas, cholesterol not only increases microviscosity of the bilayer but also defines the stability and rigidity of the formulation [2].
Based on different parameters, liposomes are further classified depending upon method of preparation, structural parameters, biochemistry, cosmetics and medicine composition, and application in biology. Phospholipids can be from natural sources such as soya bean, egg yolk and olive oil. Depending upon various characteristics liposomes can be categorized on the basis of various physical parameters such as-pH, temperature, ionic charges, immunogenicity and stability.
In a recent study performed in 2019, it is revealed that the concentration of phospholipids and cholesterol variates the protein binding of the formulation [4].
Most commonly employed phospholipids in the formulation of liposomes are: phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylethanolamine (PE), dipalmitoyl phosphatidylserine1,2dioleoylsnglyc ero3phosphoserine, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE) [5].   Cholesterol are optimized to be used in the formulation of liposomes up to a wide range with a molar ratios 1:1 or 2:1 against phospholipids. Cholesterol defines a strategic role in liposome composition; although, the adequate quantity to be used in the formulation has not been yet clarified. Thus, we can optimize lipids and cholesterol ratio, to prepare stable and controlled drug release vehicles (Figures 2 and 3) [6].

Applications of liposomes
Role of liposome in drug delivery: • Selective & passive targeting.
• It increases the overall therapeutic index and efficacy of a liposomal formulation.    Table 2.
• Due to the encapsulation of drug, overall stability is increased and reduced the adverse effects of encapsulated drug.
• It helps to improve the pharmacokinetic processes by increasing the circulation lifetime, decreasing elimination and toxic effects thus elevating the overall bioavailability of a drug [7].
• Active targeting can also be achieved by coupling with the site-specific ligands.  • Liposomes are able to provide both aqueous "milieu internee" and the lipophilic environment in a single system • It helps in protecting the encapsulated drug.

Other advantages of using liposomes
• Method of preparation is easy and has no such complicated or expensive procedures involved • Facilitates both active and passive targeting.
• No toxicity in heart as it does not accumulates in the heart.
• Intercepts the oxidation of drug

Methods of preparation
See Figure 4.

Thin film hydration method
This is one of the widely used methods for the preparation of liposomes. As it has no such complicated steps involved in it. Multilamellar vesicles (MLV) are prepared by solubilizing natural or synthesized phospholipid in chloroform, dichloromethane, ethanol or in a mixture of chloroform and methanol in a ratio of 3:1 v/v; 2:1 v/v or 9:1 v/v. A homogeneous thin film forms when this mixture is revolved and dried in a rota-evaporator under vacuum at a temperature around 45-60°C. Layes is kept under nitrogen drying for overnight. Next, comes the hydration process where completely dried thin film is hydrated using aqueous phase-phosphate buffer solution of pH 7.2 for 1-2 h at 60-70°C.
This kind of procedure can be applied to almost any kind of lipid mixtures, but has some drawbacks that majorly includes-low encapsulation space, a bit difficult to scale up and layer formed are not always homogeneous thus shows heterogeneous size distribution during later physio-chemical examination of liposomes through zetasizer.

Ether injection method
Here, the lipid mixture is dissolved in ether or diethyl ether under continuous stirring that is later injected into a PBS or aqueous phase. Which under injection pressure causes the removal of almost all organic solvent that ultimately forms liposomes. This method also suffers with the heterogeneous liposomal formulation defect.

Ethanol injection method
In ethanol injection method the lipid mixture is dissolved in ethanol under continuous stirring that is later injected into a preheated TRIS-HCl buffer or distilled water. Hydrophobicity and hydrophilicity of a drug accounts for drug intake in a liposomal vesicle. It has an advantage of using non-toxic and ethanol and is also easily scalable.

Sonication method
It is the most widely accepted method to develop small unilamellar vesicles (SLV). SLV are prepared by solubilizing natural or synthesized phospholipid in chloroform, dichloromethane, ethanol or in a mixture of chloroform and methanol in a ratio of 3:1 v/v; 2:1 v/v or 9:1 v/v. A homogeneous thin film forms when this mixture is revolved and dried in a rota-evaporator under vaccum at a temperature around 45-60°C. Layes is kept under nitrogen drying for overnight. Next, comes the hydration process where completely dried thin film is hydrated using aquous phase-phosphate buffer solution of pH 7.2 for 1-2 h at 60-70°C. Further the bath sonicator is used to transform the size of vesicles. Lastly, liposomes are centrifuged in order to remove the titanium particles that might got added due to overheating in sonication process. Less encapsulation space is the major drawback of such vesicles.

High-pressure extrusion method
Liposomes are prepared by solubilizing natural or synthesized phospholipid in chloroform, dichloromethane, ethanol or in a mixture of chloroform and methanol in a ratio of 3:1 v/v; 2:1 v/v or 9:1 v/v. A homogeneous thin film forms when this mixture is revolved and dried in a rota-evaporator under vacuum at a temperature around 45-60°C. Layes is kept under nitrogen drying for overnight. Next, comes the hydration process where completely dried thin film is hydrated using aqueous phase-phosphate buffer solution of pH 7.2 for 1-2 h at 60-70°C. In addition, these lipoosmes are passes through high pressure extruder for 10 cycles in order to obtain more uniform and stable liposomes.

Reverse-phase evaporation method
Here, the lipid mixture is dissolved in organic solvents ether or diethyl ether or a mixture of diethyl ether and chloroform (1:1 v/v); a mixture of methanolchloroform (1:2 v/v) under continuous stirring that is later injected into a PBS or aqueous phase comprising citric-Na 2 HPO 4 to improve the overall efficacy of a formulation. Which under injection pressure causes the removal of organic solvent that ultimately leads to the formation of liposomes. This method also suffers with the heterogeneous liposomal formulation defect. Organic solvent is then dried using rota-vapor instrument thus forming homogeneous liposome. The major disadvantage of this procedure is the leftover of remaining organic solvent in the final formulation also faces difficulty in scale up procedures.

Calcium-induced fusion method
Here acidic phospholipids are used to prepare SUV by following the thin film hydration process followed on with the addition of calcium that causes fusion to form MLV. Final addition of ethylenediaminetetraacetic acid (EDTA) to MLV results in the formation of large unilamellar vesicles LUV.

Dehydration-rehydration method
Liposomes are prepared by using the sonication method as explained in Section 4.3. Developed liposomes are freeze dried overnight where the formation of multilamellar vesicles occurs when dry powder gets controlled rehydration.

Freeze-thaw method
Liposomes are prepared by using thin film hydration method as explained in Section 4.1. Developed liposomes are freeze dried overnight and is then thawed  in order to govern the ionic strength and phospholipid concentration of the final liposomal formation. Physical disruption of lamellar structure occurs due to freezethaw of liposomal formulation giving it a final ionic structure.

Microfluidization
Boltic et al. was the first to introduce such method for the preparation of liposomes. Here liposomes are prepared using thin film hydration method as explained in Section 4.1, which is then sonicated and microfluidized in order to obtain partial homogenization. This method has its wide application in industrial formulation of liposomes.

Supercritical fluids (SCF)
Supercritical fluids (SCF) were introduced to replace toxic organic solvents for the preparation of liposomes. Supercritical carbon dioxide is the most widely used supercritical fluid as it has many advantages over conventionally used organic solvents such as-it is not flammable, can be recycled, non-toxic, can be comparatively easily removed from the solvents, requires moderate temperature and also exclude the product degradation in inert surroundings. Karn et al. experimented and explained the comparative study between thin film hydration method and supercritical fluids using method evaluating the non toxicity and better field approaches in term of using super critical fluids for the formulation of liposomes ( Table 5).

Mechanism of liposomal formulation
• Phospholipids shows affinity for polar molecules as well as for aqueous phase due to a hydrophobic tail, that has 2 fatty acids which are made up of 10-24 C atoms comprising of 0-6 double bonds in every chain [8].
• In a phospholipid molecule the polar portion connects with a polar environment of a aqueous medium.
• Phospholipids arrange layers of lipids in close alignment in a planer bilayer sheet. Sufficient amount of energy is required for this planar arrangement (sonication, homogenization, heating, etc.) ( Figure 5).

Morphological and physicochemical characterization of liposomal-formulation
The average size, size distribution, and zeta potential shall be determined by zetasizer.
Transmission electron microscopy is used to study the shape and surface morphology of a liposomal structure.

In vitro performance evaluation and stability studies
Stability studies: stability studies shall be conducted to assess the shelf-life of product as per ICH guidelines.
FACS (fluorescence assisted cell sorting) is used to quantify the cell uptake study.

Marketed liposomal formulations
See Tables 6 and 7.

Conclusions
Liposomes evolved as an extraordinary tool or micro-engineered membranes for the delivery of drugs because of their minimum toxicity and flexibility that can be tailored for various desirable intentions. This unparalleled delivery approach can be used for almost every drug or active pharmaceutical ingredient despite of its varied physicochemical properties and route of administration. Extensive uses of liposome in the delivery of drugs can be starched further by researchers, medical representatives and in scale-up processes in order to develop desired modification and better delivery approaches by holding the promising physio-chemical properties and pharmacokinetics (absorption, distribution, metabolism, and elimination) involved with liposomes, as described in the chapter.  Table 7.
Liposomal cosmetic formulations present in the market [10].

Marketed product Drug used Target diseases Company
Amphotec TM Amphotericin B fungal infections, leishmaniasis SEQUUS, USA Table 6. Liposomal formulations present in the market [9].