LIPOSOMES

DEFINITION
The name liposome is derived from two Greek
words:
Lipo: meaning Fat
Soma: meaning body

Liposomes are simple microscopic vesicles in
which an aqueous volume is entirely enclosed
by a membrane composed of a lipid molecule
History
Liposomes were first described by
British haematologist Dr Alec D
Bangham and colleagues in 1961
(published 1964).
Structure
Structurally liposomes are concentric bilayered
vesicles in which an aqueous volume is entirely
enclosed by a membranous lipid bilayer mainly
composed of natural or synthetic phospholipids
COMPONENTS
Phospholipids
major
structural
components of
biological
membranes.

In the membrane
cholesterol
increases
separation
between choline
head groups which
reduces the
normal hydrogen
bonding and
electrostatic
interaction
Drug is encapsulated
in
i. Phospholipid
bilayer
ii. In the entrapped
aqueous
iii. At bilayer
interface.
Activity
Head. Hydrophobic (lipophilic)
Tail. Hydrophillic (lipophobic)
Hydrophobic portion is repelled by water
Hydrophilic portion is attracted by water
Activity of liposomes is enhancesd by modifying
the surface of liposomes with different molecules
such as glycolipids.

ACTIVITY
Hydrophilic drug is
entrapped in hydrophilic portion of liposomes.

Hydrophobic drug is entrapped in lipophillic
portion.

Modes of Liposome/Cell Interaction
Adsorption
Endocytosis
Fusion
Lipid transfer
Mechanism of Drug Release
Liposome attaches to plasma membrane and appears
to fuse with them, releasing their content into cell.

Membrane of phagolysosyme have proton pumps which
decrease pH of phagolysosyme & the enzyme
phospholipase destruct the liposomal membrane
Macrophages engulf liposomes (endocytosis)
Phagosome + lysosyme = phagolysosyme
Liposomes in blood stream
Taken by reticulo-endothelial system
Improved solubility of lipophilic and amphiphilic drugs .
Liposomes have both a lipophilic and aqueous
environment making it useful for delivering
hydrophobic, amphipathic, and hydrophilic medicines
It shows drug effect at its specific targeted site
Shows sustained release action.
Liposomes improved penetration of various drugs into
tissues
Liposomes increases efficacy and therapeutic index.
Liposomes protect the drug from environment and the
sensitive areas from drug.

Need For Liposomes
Disadvantages
High
productio
n cost
Liposome
phospholi
pid may
undergo
oxidation
&
hydrolysis
Shorter
Half life
May have
leakage of
encapsula
ted drug
Liposome
s are
quickly
taken up
by the
reticular
endotheli
al cells
CLASSIFICATION
OF LIPOSOMES
According to Size
Name Diameter No. of lamella
Small unilamellar
vesicles
20-100nm Single
Large unilamellar
vesicles
100 nm - 400 nm Single
Intermediate-sized
unilamellar vesicles
100-200 nm

Single
Giant Unilamellar
Vesicles
1 m & larger Single
Multivesicular
Vesicles
200 nm - ~3 m Multiple
Names Diameter

Number of lamella
Unilamellar vesicles All sizes Single
Multilamellar vesicles 200 nm - ~3 m Multiple(5 & 20)
Multivesicular
liposomes
- -
ACCORDING TO MORPHOLOGY
According To Composition
Immunoliposomes
Liposomes using immunological
molecules particularly,
immunoglobulins for targeting purposes
may be attached to liposomes surface by
covalent linkage through membrane
possessing functional group




s

Proteosomes
A term applied to lipid vesicles
incorporating proteins in or on the outer
membrane






According to Function
Stealth Tranfersomes
Long circulatory liposome
Any liposome that avoids uptake by the
RES as a result of coating the surface of
the liposomes with hydroxylated polymers
Particular composition that are capable
of transferring equeous content across
the skin
There membrane contains a certain
proportion of the bile salt distributed
among the phospholipid, which confers
the increased flexibility on membrane

Methods Of Preparation
METHODS OF PREPARATION
All the methods of preparing the liposomes involve four
basic stages:








The difference between these methods is the steps by which lipids
are drying down from organic solvents and then redispersed in
aqueous media.


1. Drying down lipids
from organic solvent
2. Dispersing the lipid
in aqueous media
3. Purifying the
resultant liposome
4. Analyzing the final
product
SELECTION CRITERIA
Choice of method for liposomes production:

Physicochemical characteristics of drug to be loaded and
ingredients.
Nature of dispersion medium in which liposomes are
dispersed.
Effective concentration and toxicity of entrapped
substance.
Size, dispersity and shelf life of vesicles for intended
application.
Batch to batch reproducibility.
Large-scale production of safe and efficient liposomal
products.

Mechanical Dispersion Method
1. Lipid Hydration Method
2. Lyophillization
3. Proliposomes
To Reduce Liposome
Size
Sonication
French Pressure Cell
Microemulsification
Membrane Extrusion
To Increase Liposome
Size
Freeze thawing
Direct reconstituted
vesicles

1. Mechanical Dispersion Method
Lipid dissolve in organic solvent/co-solvent

Remove organic solvent under vacuum

Film deposition

Solid lipid mixture is hydrated by using aqueous buffer

Lipid spontaneously swell and hydrate

Liposome

Post hydration vortexing, sonication, freeze thawing and high pressure extrusion
i. Lipid Hydration Method
Hand shaken vesicles
most widely used for the preparation of MLV



Drying of lipid solution forms a thin film at bottom
Hydrating this film by adding aqueous buffer
Vortexing it for some time
MLVs prepared
Lipids +Solvent
Non-shaking vesicles
Solution
of Lipids
+ solvent
MLV are
floating on
surface
remaining
fluid
produces
LUV

Nitrogen
gas is
passed
Hydration
Until the opacitiy
of the dried lipid
film disappears.
Flask is slowly
returned to
upright position.
Take care not to
disturb the flask
in any way.
ii. Lyophilization
Freeze-drying involves the removal of water from
products in the frozen state at extremely low
pressures.
The process is generally used for dry products.
The technique has a great potential as a
method to solve long term stability problems
with respect to liposomal stability.
It is exposed that leakage of entrapped
materials may take place during the process of
freeze- drying and on reconstitution.

iii.
PROLIPOSOMES
Lipid is dried over the
finely divided particulate
support i.e.- NaCl,
Sorbitol, or other
polysaccharides.
These dried lipid coated
particulates are called as
proliposomes.
Drying lipid
on finely
divided
support
(NaCl)
Surface area
increases and
continuous
hydration
hydration swelling
MLVs
To Reduce Liposome Size
Sonication
French Pressure Cell
Micro emulsification
Membrane Extrusion

Sonication

most extensively used for the preparation of small
unilamellar vesicle (SUV). Sonication is the act of
applying sound energy to agitate particles in a sample
to reduce the size.


Drawbacks:
Very low encapsulation efficacy
possible degradation of phospholipids
and compounds to be encapsulated
French pressure cell: extrusion
MLV dispersion are placed in the French Pressure
Cell and extruded at about 20,000psi at 45C

95% of MLVs get converted to SUVs which can
be determined by size extrusion chromatoraphy
Dispersion of MLVs can be converted
to SUVs by passage through a small
orifice under high pressure.

MICRO EMULSIFICATION
LIPOSOMES
The lipids can be introduced into fluidizers, either as a
dispersion of large mlvs or as a slurry of unhydrated lipids
in organic medium.
Microfluidizer pumps the fluid at very high pressure
through a 5um orifice.
Forced along defined micro channels,
Two streams of fluid to collide together at right angles
at a very high velocity.
The fluid collected of can be recycled through the pump
and interaction chamber until vesicles of the spherical
dimension are obtained.
Membrane extrusion

Size of liposomes is reduced by gently passing
them through polycarbonate membrane filter of
defined pore size (100nm) at much lower
pressure (<100psi)

To Increase Liposome Size
Freeze thawing
Direct reconstituted vesicles

Freeze-thawing
This method is based
upon freezing of SUV
then thawing by standing
at room temperature for
15mins.
Finally subjecting to a
brief sonication cycle.
It results in a high
proportion of large
unilamellar vesicles
formation.

Freeze
SUVs
Thaw at
room
temp. for
15 min
Sonicate
SUVs
rupture
LUVs
Dried Reconstituted Vesicles

Empty SUVs
Hydrate with
entrapping material
Freeze drying
Rehydrate with material to be
encapsulated
Uni or oligolamellar
vesicles
2. Solvent Dispersion Method:
Lipid dissolve in organic solvent

Excess addition of aqueous phase

Lipids align at interface of aqueous and organic layer

Formation of monolayer and bilayer of phospholipids

Liposome
Solvent
Dispersion
Method
solvent
injection (ether
and ethanol)
Double
emulsification
Reverse phase
evaporation
i. SOLVENT (Ether or Ethanol)
INJECTION TECHNIQUE
Ethanol injection
Lipid +ethanol

Rapid injection

Saline buffer + material to be
entrapped

SUVs
Ether injection
Lipid + ether

Slow injection

In aqueous phase (heat in
water bath 60C)

SUVs

Disadvantage:
Longer time
low efficiency


This method involve the dissolution of the
lipid into an organic phase (ethanol or
ether), followed by the injection of the lipid
solution into aqueous media, forming
liposomes.


ii. Double Emulsification

an active ingredient is first dissolved in an aqueous
phase (w1) which is then emulsified in an organic
solvent of a polymer to make a primary w1/o
emulsion.
This primary emulsion is further mixed in an
emulsifier-containing aqueous solution (w2) to make
a w1/o/w2 double emulsion.
The removal of the solvent leaves microspheres in the
aqueous continuous phase, making it possible to
collect them by filtering or centrifuging.

iv. Reverse phase evaporation
method
Drawback: These conditions may possibly result in the
breakage of DNA strands or the denaturation of some
proteins.
3.Detergent Solubilization

The detergents at their critical micelles concentrations
have been used to solubilize lipids.


Phospholipid brought into intimate contact with aqueous phase
By addition optimized concentration of detergent
Formation of micelles (liposome)
Detergent is removed by following
methods:
i. dialysis
ii. column chromatography
ii. use of biobeads
ACTIVE LOADING
TECHNIQUE
This technique employs the principle that certain type of
compounds with ionizable groups and those with both lipid
and water solubility can be introduced into the liposomes
after the formation of intact vesicle.

Loads drug molecules into preformed liposomes using pH
gradients and potential difference across cell membranes.

The transmembrane pH gradient can be developed using
various methods depending on nature of the drug to be
encapsulated.

2 step process generates the pH imbalance
and remote loading:
The vesicles are prepared in low pH
solution
It is followed by addition of base to the
extraliposomal medium
At the low pH side, molecules are
predominately protonated, which lowers
the concentration of the drug in the
unprotonated form, and thus promote the
diffusion of more drug molecules at the low
pH side of the bilayer, then exchange of
external medium by gel exclusion
chromatography with a neutral solution
ADVANTAGES OVER THE
PASSIVE LOADING METHODS
A high encapsulation efficiency and
capacity.

A reduced leakage of the encapsulated
compounds.