1. Dr. B. Sree Giri Prasad
Associate Professor
Nalla Narasimha Reddy Education Society’s Group of Institutions
2.
3. DE-EMULSIFICATION METHODS:
First the inner leaflet of the bilayer, then the outer
half.
The common feature of this method is the
formation of “W/O” emulsion by introducing small
quantity of aqueous medium containing material to
be entrapped into large volume of immiscible
organic solution of lipid.
This method requires two steps for the liposomes.
The was followed by mechanical agitation to
break up the aqueous phase into microscopic water
droplets.
4. These droplets are stabilized by the presence of
phospholipid monolayer at the interface.
The size of droplets is determined the intensity of
mechanical energy used to form the emulsion and
amount of lipid relative to the volume of aqueous
phase, since each droplet requires a complete
monolayer of phospholipid covering its surface in
order to prevent the possible coalescene with other
droplets.
The aqueous solution surrounded by the
monolayer of phospholipid forms the central core of
the final liposome.
5.
6. There are number of methods, which could be
used for preparing droplets including double
emulsion, reverse phase evaporation and sonication
methods.
DOUBLE EMULSON VESICLES (DEV’s):
In this method, the outer half of the liposome
membrane is created at a second interface between
two phases by emulsification of organic solution in
water.
if the organic solution, which is already contains
water droplet, is introduced in to excess aqueous
medium followed by mechanical dispersion, results
into Multi compartment vesicles are obtained.
7. The ordered dispersion so obtained is described as
a W/O/W system (i.e double emulsion).
These vesicles with aqueous core are suspended in
aqueous medium, the two aqueous compartments
being separated from each other by a pair of
phospholipid monolayers whose hydrophobic
surfaces face each other across a thin film of organic
solvent.
Removal of this, solvent clearly results in an
intermediate sized unilamellar vesicles.
The theoretical entrapment may reach upto 90%.
8. Preparation of DEV’s:
Prepared by rapidly injecting the dispersion of
micro-droplets in to hot aqueous solution of TRIS-
Buffer with the help of 22 guage hypodermic needle
under vigorous stirring.
The organic solvent is evaporated using strong jet
of nitrogen thus forming double emulsion.
The last traces of organic solvent are removed by
evaporation and finally the volume is adjusted by
adding extra-dsitilled water and then the product is
centrifuged at 200C for 30 min at 37,000g to
remove lipid aggregates.
9. REVERSE PHASE EVAPORATION VESICLES
(RPEV’s):
The essential feature of this method is the removal
of solvent from an emulsion by evaporation.
Here the droplets are formed by bath sonication of
mixture of two phases, then the emulsion is dried
down to a semisolid gel in a rotary evaporator under
reduced pressure.
At this stage, the monolayers of phospholipids
surrounding each water compartment are closely
opposed by each other and in some cases probably
already form part of a bilayer membrane separating
adjacent compartment.
10.
11. Next step, to bring about the collapse of a certain
proportion of water droplets by vigorous mechanical
shaking using a vortex mixer.
In these circumstances, the lipid monolayer, which
enclosed the collapsed vesicle, is contributed to
adjacent intact vesicle to form the outer leaflet of the
bilayer of a large unilamelar liposome.
The aqueous content of the collapsed droplet
provides the medium required for dispersion of these
newely formed liposomes.
After conversion of the gel into a homogeneous
free flowing fluid, the dispersion is dialyzed to
remove the last traces of solvent.
12. The vesicles formed are unilamellar and have an
average diameter of 0.5µm.
The encapsulation percentage is found to be nearly
50%.
