*6.3.1 Hand shaking or non hand shaking of lipid based film by hydration*

This method was first described by [1] and which is simple for liposomes formation with one limitation of having low drug loading. Phospholipids and cholesterol are to be dispersed in organic solvent and then evaporated by using rotary evaporator at low pressure and vacuum. When solvent is evaporated then a dry film will be formed on the wall of rota flask and that should be hydrated by using aqueous phase buffer. The lipids in film spontaneously gets swell when hydrated to form heterogeneous multilamellar liposomes (MLVs).

#### *6.3.2 Micro-emulsification*

Micro fluidizer helps in preparing small MLVs from liposomal dispersion [23]. Micro fluidizer pumps liposomal fluid at pressure of 10,000 psi through 5 μm orifice and by micro channels that directs two pathways of dispersion to colloid with high velocity. The large MLVs liposomal dispersion or organic medium containing lipids can also be passed through fluidizer. The dispersion collected is replaced through the micro fluidizer until vesicles with spherical dimensions obtained.

## *6.3.3 Sonication method*

When the liposomal vesicles sizes are above 1 μm then sonication is done to reduce size to form SUVs or extrusion done with polycarbonate filters for producing smaller and uniform sized vesicles. Size reduction by ultrasonication for aqueous dispersion can be done mainly by bath or probe sonicators [24].

## *6.3.4 French pressure cell*

The French pressure press breaks cells with appropriate conditions compared with ultrasound techniques [25]. French pressure press is advantage because sonication procedure degrades lipids, proteins and sensitive compounds. This cell can be used for dispersions with low volume of less than 40 ml and not applicable for high volume production batches. Hence, a scale-up-based strategy was established by using micro fluidization technique.

#### *6.3.5 Membrane extrusion*

Another method for liposomes downsizing is extrusion. The vesicles with force are passed through membranes with a lower pressure than french press. Extrusion studies using polycarbonate filters were done and performed on extrusion behavior and membrane properties [26, 27]. Lipex Biomembranes Inc., now called Northern Lipids Inc., invented extrusion vessel from milliliter to several liters. This Lipex extruder allows higher temperatures with jacketed mode. An alternative to this lipex extruder is Maximator device, which is continuous pumping system which was introduced by Schneider et al., 1994. The Maximator has glass vessel which is thermostable and connected directly to pneumatic piston pump. This method consists of preparing liposomes followed by freeze–thaw and finally extrusion. This long process and disadvantage because of high product loss.

## **6.4 Methods on fusion of preformed vesicle**
