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Liposomes:
Slow release vehicles
Liposomes
are extensively investigated for selective drug delivery as it can
be utilized on slow release vehicles, say Vrushal S Kelkar, Ramprasad
and Anil Gadage
Liposomes
are microscopic vehicles composed of one or more concentric spheres
of lipid layers separated by water or aqueous buffer compartments.
These spherical structures can be prepared with diameters ranging
from 80-100nm.
Sterilisation
of liposomes
Sterilization
of liposomes is limited to Millipore filters through pore sizes
no greater than 0.22nm since other procedures would destroy the
integrity of the bilayer. The filters are made of different materials,
an asbestos pad in the ‘seitz filter’ diatomaceous earth
in the bulk filled filter, porcelain in the chamber land, pasteur
filter and glass disks in the other filters. Factors which prevent
the passage of organism are: porosity, electric charge of the filter,
the electric charge by the organisms and nature of the fluid being
filtered.
Stability
of liposomes
Physical
stability: Colloidal system can be stabilized electrostatically,
stearically or electrostearically. In addition to normal colloids
self assembling colloids can under go other changes such as fusion
or phase change after aggregation. Therefore, physical stability
means the preservation of liposomes size distribution and amount
of material encapsulated. Obviously this depends on mechanical properties
of liposome membranes, their thermodynamics, and colloidal properties
of the system.
Chemical
stability: Chemically, phospholipids are susceptible to hydrolysis.
Additional phospholipids containing unsaturated fatty acids can
undergo oxidation reaction. Most of the phospholipid liposomal dispersions
used contain unsaturated acyl chain as a part of molecular structure.
These chains are vulnerable to oxidative degradation. The oxidation
reaction can occur during preparation, storage or actual use.
 Oxidative
deterioration of lipids is complex process involving free radical
degeneration and results in the formation of cyclic peroxides and
hydroperoxides. Most of the procedures used to measure lipid perodixation
are non-specific.
Stability
of liposomes in biological fluids: The instability of liposomes
in plasma appears to be result of the transfer of bilayer lipids
to albumin and high density lipoproteins. Additionally some of the
protein is transferred from lipoproteins to the liposome. Both lecithin
and cholesterol also exchange with membranes of RBCs. Liposomes
are most susceptible to HDL attack as their gel to determine by
differential scanning calorimetry where the formulation has a phase
transition temperature close to 370oC. Liposomes prepared with higher
chain length phospholipids are most stable both in buffer and plasma.
Cholesterol is generally very effective in reducing the instability
of liposomes in contact with plasma. Liposomal stability in plasma
increases as the ratio of cholesterol in the liposomes increases.
Quality
control
Internal
volume and encapsulation efficiency: These are the two parameters
used to described entrapment of water-soluble drugs in the aqueous
compartments of liposomes. The internal or trapped or capture volume
is expressed as aqueous entrapped volume per unit quantity of lipid,
it is determined by entrapping a water-soluble marker such as 6-carboxyfluorescein.
C14 OR H3 -glucose or sucrose and then lysing the liposome by the
use of a detergent such as triton X-100. Determination of the amount
of marker that was trapped enables one to back calculate.
The
encapsulation efficiency describes the per cent of the aqueous phase
and hence the per cent of water soluble drug that becomes entrapped
during liposomes preparation. The remaining drug outside of the
liposomes is therefore wasted. The internal or trapped volume and
encapsulation efficiency greatly depend on liposomal content, liquid
concentration, method of preparation, and the drug used.
Lamellarity:
The average number of bilayers present in liposomes can be found
by freeze fracture electron microscopy 31P-NMR. In the latter technique,
signals are recorded before and after the addition of non-permeable
broadening agent such as Mn2. Manganese ions interact with the outer
leaflet of the outer most preparation is unilamellar and at 250oC
reduction in the intensity of the original NMR signal means there
are two bilayers in the liposomes.
Size
and size distribution: The average size and size distribution of
liposomes are important parameters with respect to physical properties
and biological fate of the liposomes and their entrapped substances.
Light
scattering: The popularity of this method depends on its ease of
operation and the speed by which one can obtain data. Such methods
cannot distinguish between a large particle and a flocculated mass
of smaller particles. Most important, it may be necessary to remove
any micron size particle present in the same prior to analysis.
Light
microscopy: This method can be used to examine the gross size distribution
of large vesicle preparations such as multilamellar vesicles. The
inclusion of fluorescent probe in the bilayer permits examination
of liposomes under a fluorescent microscope and is a very convenient
method to obtain an estimate of at least the upper-end of the size
distribution.
Freeze
fracture electron microscopy: This method especially useful for
observation of the morphological structure of liposomes.
DC-chol
assay: The amount of dc-chol is quantified by using Liebermann-Burchard
test. This assay is specific for a 3-hydroxysteriod. Briefly, 100
micltr of liposomes are lyophilized to remove the water and are
later reconstituted with chloroform. To each sample, 900 micltr
of acetic anhydride and 100 micltr of H2S04 are added to the sample
and mixed. This produced a green colour and the absorbance is read
at 625nm.
Endotoxin
test: Batch of liposomes are tested for the presence of endotoxin
by using a QCL-1000 chromogenic limulus amoebocyte lyste (LAL) kit.
Endotoxin levels are quantifiable by this colorimetric assay and
can by expressed as endotoxin units (EU) of internal units (IU).
(1IU=0.75EU)
To
be continued
The
writers are with SGRS Institute of Pharmacy, Saswad
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