Liposome-A Comprehensive Approach for Researchers

1.1 Composition of liposomes

1. Phospholipids

   1. Derived from natural sources:

      • Phosphatidylcholine

      • Phosphatidylserine

      • Phosphatidylethanolamine

   2. Synthetic phospholipids:

      • Disloyal phosphatidylethanolamine

      • Disloyal phosphatidylcholine

2. Cholesterol

   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].

3.1 Other advantages of using liposomes

• Biodegradability

• Efficient control of the release

• Resemblance to natural membrane structures

• Increased targeting prospects

• Biocompatibility

• Biodegradable

• Liposomes are able to provide both aqueous “milieu internee” and the lipophilic environment in a single system

• It helps in protecting the encapsulated drug.

• 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

• Chelation therapy in case of of heavy metal poisoning

• Diagnostic imaging of tumors

• In enzyme replacement therapy

• Study of membranes

• In gene delivery

• As drug delivery carriers

• In multidrug resistance

• In immunology

• In cosmetology ( Table 4 )

4.1 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.

4.2 Injection methods

4.3 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.

4.4 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.

4.5 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 methanol-chloroform (1:2 v/v) under continuous stirring that is later injected into a PBS or aqueous phase comprising citric-Na₂HPO₄ 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.

4.6 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.

4.7 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.

4.8 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 freeze-thaw of liposomal formulation giving it a final ionic structure.

4.9 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.

4.10 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 ).

6.1 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.

6.2 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.

MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay to evaluate the in-vitro cytotoxicity of the developed formulation.

FACS (fluorescence assisted cell sorting) is used to quantify the cell uptake study.