INTRODUCTION:
Liposomes were first
described by British haematologist
Dr Alec D Bangham
FRS in 1965 while studying cell membranes. He found that lipsomes
are vesicular structures consisting of hydrated bilalyers
which form spontaneously when phospholipids are dispersed in water.
The name liposome is derived from two Greek words: 'Lipos' meaning fat and 'Soma' meaning body. A liposome can
be formed at a variety of sizes as uni-lamellar or
multi-lamellar construction.
Liposome is a
tiny bubble (vesicle), made out of the same material as a cell membrane. Liposomes can be filled
with drugs, and used to deliver drugs for cancer and other diseases. Liposomes are the smallest
artificial vesicles of spherical shape that can be produced from natural untoxic phospholipids and cholesterol. As shown in the
following schematic drawings of liposomes, the
vesicles can be used as drug carriers and loaded with a great variety of
molecules, such as small drug molecules, proteins, nucleotides.
Fig 1.
Membranes are usually made of phospholipids, which are molecules that have a head group and a tail
group. The head is attracted to water, and the tail, which is made of oil
(hydrocarbon), is repelled by water.
Phospholipids are the main
component of naturally occurring bilayers. These
phospholipids include phosphatidylcholines (PC), phosphatidylethanolamines (PE) and phosphatidylserines
(PS). The key common feature that bilayer-forming
compounds share is their amphiphilicity i.e. they
have defined polar and non-polar regions. This is the reason the non-polar
regions orientate themselves towards the interior away from the aqueous phase,
the polar regions being in contact with it1,3. Fig 13.
MECHANISM
OF LIPOSOME FORMATION:
Liposomes
(lipid vesicles) are formed when thin lipid films or lipid cakes are hydrated
and stacks of liquid crystalline bilayers become
fluid and swell. The hydrated lipid sheets detach during agitation and
self-close to form large, multilamellar vesicles (LMV)
which prevents interaction of water with the hydrocarbon core of the bilayer at the edges. Once these particles have formed,
reducing the size of the particle requires energy input in the form of sonic
energy (sonication) or mechanical energy (extrusion).
1) Mechanical
methods:
A. Film
method:
The original method
of Bangham et al. is still the simplest procedure for
the liposome formation but is limited because of its low encapsulation
efficiency. This technique produces liposomes by
hydrating thin lipid films deposited from an organic solution on a glass wall
by shaking at temperatures above the Tc. The solvent
is removed at reduced pressure in a rotary evaporator. The dry film of lipids
which has been deposited onto the wall of a round-bottom flask is hydrated by
adding a buffer with a water soluble marker. As the lipid becomes hydrated and
starts to form into closed vesicles only a small amount of the solute becomes
entrapped. This method yields a heterogeneous sized population of MLVs over 1mm in diameter2. Fig 22.
Fig 2.
B. Ultrasonication
method:
Ultrasonication of an aqueous
dispersion of phospholipids with a strong bath sonicator
or a probe sonicator will usually yield SUVs with
diameters down to 15-25nm.
2) Methods based on replacement of organic solvent
A. Reverse-phase
evaporation:
In this method,
several phospholipids (pure/mixed with cholesterol) can be used. The lipid
mixture is added to a round bottom flask and the solvent is removed under
reduced pressure by a rotary evaporator. The system is purged with nitrogen and
the lipids are re-dissolved in the organic phase. This is the phase that the
reverse phase vesicles will form. Diethly ether and
isopropyl ether are the usual solvents of choice. After the lipids are
re-dissolved in this phase the aqueous phase (contains compound to be
encapsulated) is added. The system is kept under continuous nitrogen and the
two-phase system is sonicated until the mixture
becomes a clear one-phase dispersion. The mixture is
then placed on the rotary evaporator and the organic solvent removed until a
gel is formed. Non-encapsulated material is removed. The resulting liposomes are called reverse-phase evaporation vesicles
(REV). The large unilamellar and oligolamellar
vesicles formed have the ability to encapsulate large macromolecular vesicles
with high efficiency4.
B: Ethanol
injection method:
In this method a
mixture of lipids in an organic solvent (diethyl ether, ethanol, etc.) is rapid
injected into a aqueous solution. This results in osmotically active, unilamellar
vesicles with a well defined size distribution and high volume trapping
efficiency (about ten times that of sonicated and
hand shaken preparations.)5.
Method shown below in Fig 35.
Fig. 3.
Ethanol injection method:
3) Methods
based on size transformation or fusion of preformed vesicles:
A: Freeze-thaw
extrusion method:
Liposomes formed by the film
method are vortexed with the solute to be entrapped
until the entire film is suspended and the resulting MLVs are frozen in a dry
ice/acetone bath, thawed in lukewarm water and vortexed
again. After two additional cycles of freeze-thaw and vortexing
the sample is extruded three times. This is followed by six freeze-thaw cycles
and an additional eight extrusions. The resulting liposomes
are called large unilamellar vesicles by extrusion
technique (LUVET) and they typically contain internal solute concentrations
which are much higher than external solute concentrations, they have entrapment
ratios greater than one .Proteins can be effectively encapsulated using this
technique6,7.
B: The
dehydration-rehydration method:
This method begins
with empty buffer containing SUVs (hand shaken MLVs can be also be used but are
usually not preferred). These are mixed with the component to be entrapped,
after which they are dried. Freeze-drying is often the method of choice but
other methods such as by vacuum or under a stream of nitrogen can be used. The vesicles are then rehydrated. A mechanism
has been proposed whereby as the vesicles become more concentrated during
dehydration, they flatten and fuse forming multi lamellar planes where the
solute is sandwiched. Therefore on hydration, larger vesicles are formed. This
technique is mild and simple, the main limitation being the heterogeneity of
the size of the size of the liposomes8,9.
APPLICATION:
Liposomes are used for drug delivery due to their unique properties. A
liposome encapsulates a region on aqueous solution inside a hydrophobic membrane; dissolved hydrophilic solutes cannot readily pass through the lipids.
Hydrophobic chemicals can be dissolved into the membrane, and in this way
liposome can carry both hydrophobic molecules and hydrophilic molecules. To
deliver the molecules to sites of action, the lipid bilayer
can fuse with other bilayers such as the cell membrane, thus delivering the liposome
contents. By making liposomes in a solution of DNA
or drugs (which would normally be unable to diffuse through the membrane) they can be
(indiscriminately) delivered past the lipid bilayer.
There are three types of liposomes- MLV (multilamellar vesicles) SUV (Small Unilamellar Vesicles) & LUV (Large Unilamellar Vesicles). These are used to deliver different
types of drugs.
Liposomes can also be designed to deliver drugs in other ways. Liposomes that contain low (or high) pH
can be constructed such that dissolved aqueous drugs will be charged in solution. As the pH naturally
neutralizes within the liposome (protons can pass through
some membranes), the drug will also be neutralized, allowing it to freely pass
through a membrane. These liposomes work to deliver
drug by diffusion rather than by direct cell fusion.
Another strategy for liposome drug delivery is to target endocytosis events.
Liposomes can be made in a particular size range that
makes them viable targets for natural macrophage
phagocytosis. These
liposomes may be digested
while in the macrophage's phagosome,
thus releasing its drug. Liposomes can also be
decorated with opsonins and ligands to activate endocytosis
in other cell types.
The
use of liposomes for transformation or transfection of DNA
into a host cell is known as lipofection.
In addition to gene and drug delivery applications, liposomes can be used as carriers for the delivery of dyes
to textiles, pesticides to plants, enzymes and nutritional supplements to
foods, and cosmetics to the skin10,11.
TARGETING CANCER:
Another interesting property of liposomes
are their natural ability to target cancer.
The endothelial wall of all healthy human blood vessels are encapsulated by
endothelial cells that are bound together by tight junctions. These tight junctions stop any
large particle in the blood from leaking out of the vessel. Tumour vessels do not contain the same
level of seal between cells and are diagnostically leaky. This ability
is known as the Enhanced
Permeability and Retention effect. Liposomes
of certain sizes, typically less than 400nm, can rapidly enter tumour sites from the blood, but are kept in the
bloodstream by the endothelial wall in healthy tissue vasculature. Anti-cancer drugs such as Doxorubicin
(Doxil), Camptothecin and Daunorubicin (Daunoxome) are currently being marketed in liposome
delivery systems.
PROSPECT:
Further advances in liposome research have been able to
allow liposomes to avoid detection by the body's
immune system, specifically, the cells of reticuloendothelial system (RES). These liposomes
are known as "stealth liposomes", and are
constructed with PEG (Polyethylene Glycol) studding the outside of the
membrane. The PEG coating, which is inert in the body, allows
for longer circulatory life for the drug delivery mechanism. However, research
currently seeks to investigate at what amount of PEG coating the PEG actually
hinders binding of the liposome to the delivery site. In addition to a PEG
coating, most stealth liposomes also have some sort
of biological species attached as a ligand to the liposome in order to enable
binding via a specific expression on the targeted drug delivery site. These
targeting ligands could be monoclonal
antibodies (making an immunoliposome), vitamins, or specific antigens.
CONCLUSION:
It
has ability to cross blood brain barrier & can carry both water soluble
drug & lipid soluble drug, for such reason it has been used extensively
throughout world as site specific and targeted drug carriers.
REFERENCES:
1.
Chapman D., Liposomal Technology, 1983: 1: 1-18.
2.
Rongen H.A.H. et.al., J. Immuno.
Methods, 1997: 204: 105-133.
3.
Crommelin D.J.A., Liposomes, 1997: 3: 73-190.
4.
Szoka Jr. F. and Papahadjopoulos D., Proc. Natl.
Acad. Sci., 1978: 60: 4194-4198.
5.
Deamer D. and Bangham A. D., Biochem.
Biophys. Acta, 1976: 443:
629-34.
6.
Chapman C. J., Chem. Physic.
Lipid, 1991: 60: 201-208.
7.
Sou K., Biotechnol. Prog., 2003: 19: 1547-1552.
8.
Kirby C. and Gregoriadis G., Biotechnology, 1984: 35: 979-984.
9.
Olsen F., Biochem. Biophys. Acta,
1979: 557: 9-23.
10.
Crowther J. R., ELISA, theory and practice, 1980: 42: 36-39.
11.
Horton K., Dissertation for degree of Advanced Studies
in Chemical Engineering, Universitat Rovira I Virgili, 2003: 45-49
Received on 16.09.2010
Accepted on 12.10.2010
© A&V Publication all right reserved
Research Journal of Pharmaceutical
Dosage Forms and Technology.
2(6): Nov.-Dec. 2010, 370-373