liposomes

LIPOSOMES
Controlled and novel drug delivery system
INTRODUCTION:

HISTORY

Ideal drug delivery system delivers drug at a specific rate dedicated by need of body over
the period of treatment to the site of action. The targeted drug delivery system achieves the site
specific delivery but is unable to control the release kinetic of drug in predictable manner.
1) PAUL EHRLICH in 1906 initiated development for targeted delivery.
2) Number of carriers are utilized afterwards which are serum proteins by GREGORIADIS
in 1977, Immunoglobulin by GOLD BERG in 1983, Synthetic polymers by POST el.al in
1983, lipid vesicles(liposomes) by POZNANSKY & JULIANO in 1984.
3) Other carriers are microspheres, erythrocytes, niosomes, pharmacosomes, reverse
micelles. Few drugs have reached the stage of clinical development.
What are Liposomes?
These carriers are biologically inert in nature, devoid of any antigenic, pyrogenic or allergic
reactions and their components are used as the components of biological membrane. Drugs
incorporated do not cause unfavorable side effects as well.
Liposomes are simple microscopic vesicles in which an aqueous volume is entirely enclosed
by a membrane composed of lipid molecule.
So the drug molecule can either be encapsulated in aqueous space or intercalated (coated) into
lipid bilayer.

Advantages of liposomes:

Provides selective passive targeting to tumor tissues (liposomal doxorubicin).

Increased efficacy and therapeutic index.
Increased stability via encapsulation.
Reduction in toxicity of the encapsulated agent.
Site avoidance effect
Improved pharmacokinetic effects ( reduced elimination, increased circulation life times)
Flexibility to couple with site specific ligands to achieve active targeting.

Characteristics of Liposomes
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Lipid bilayer nature (amphiphilic also called amphipathic nature): polar

head covalently attached to one or two hydrophobic hydrocarbon tails.

When these lipids exposed to an aqueous environment, interactions

between themselves (hydrophilic interactions between polar head groups
and vander-waals interactions between hydrocarbon chains and hydrogen
bonding with water molecules) lead to formation of closed bilayer.

LIPOSOME CLASSIFICATION BASED ON PHARMACEUTICAL AND

THERAPEUTIC ASPECTS:

Classification:
Lamella: A Lamella is a flat plate like structure that appears during the formation of
liposomes. The Phospholipid bilayer first exists as a lamella before getting converted
into spheres. Based on number of lamellaes there are two types

Unilamellar Vesicle
Multilamellar Vesicles

Based upon composition and application:

Conventional liposomes(CL)
Fusogenic liposomes(RSVE)
Cationic liposomes
Immuno-liposomes

Types:
Based on structural parameters:
MLV
OLV
UV
SUV
MUV
LUV
GUV
MV

Specifications:
Multilamellar large vesicles>0.5m
Oligolamellar vesicles>0.1-1m
Unilamellar vesicles(all size range)
Small unilamellar vesicles 20-100nm
Medium sized unilamellar vesicles
Large unilamellar vesicles> 100nm
Giant unilamellar vesicles> 1m
Multivesicular vesicles>1m

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Based on method of liposome

preparations:
REV
MLV-REV
SPLV
FATMLV
VET
DRV

Single or oligolemellar vesicles made

by reverse phase evaporation
method
Multilamellar vesicles made by
reverse phase evaporation method
Stable plurilamellar vesicles
Frozen and thawed MLV
Vesicles prepared by extrusion
technique
Dehydration-rehydration method

STRUCTURAL COMPONENTS OF LIPOSOMES:

Cholesterol
Phospholipids( phosphodiglycerides, sphingolipids)

Phospholipids: Most common phospholipid is the phosphotidylcholine molecule. It is a

molecule in which a glycerol bridge links a pair of hydrophobic acyl hydrocarbon chains
with a hydrophilic polar head group phospho choline).
Head group
Common name
Abbreviations
O-CH2-CH2-N-3Methyl
Choline
PC
O-CH2-CH2-NH3
Ethanolamine
PE
O-H
Acid
PA
Most common of them is PC also known as lecithin, can be derived from natural and
synthetic sources. At various temperature lecithin can exist in different phases. At low
temperature it will be in solid form, at high temperature lipid membrane passes from tightly
ordered gel to liquid crystal phase.
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Liposome membranes are semipermeable in that diffusion of molecules and ions acrozz
membranes varies considerably for molecules with high solubility in both organic and
aqueous media. A phospholipid membrane constitutes a very denser barrier, on other hand
polar solutes such as glucose and higher molecular weight compounds pass across membrane
very slowly. Smaller molecules with natural charge (e.g H2O and urea) can diffuse across
quite rapidly while charged ions differ greatly in this behavior. Na+ and K+ ions diffuse very
slowly. Proteins and metal ions diffuse at phase transition temperature.
Cholestrol: To make phospholipid hard and thick cholesterol is added (solidification).
Cholesterol is added into phospholipid at ratio of 1:1 , 2:1 cholesterol to phosphotidyl
choline.
Role of cholesterol in bilayer formation:

Acts as fluidity buffer

After intercalation with phospholipid molecules alters the freedom of motion of carbon
molecules in the acyl chain
Restricts the transformation of trans- to gauche- confirmations

Handling of liposomes: Standard composition of liposome is

Egg lecithin: Cholesterol: Phophotidyl glycerol
These lipids can be stored either as solids or in organic solution at -20 to -70C in order to
reduce chances of oxidation. The solvent most widely used is a mixture of chloroform and
methanol in a volume ratio of 2:1. Compounds which are sparingly soluble in either
chloroform or methanol alone often dissolve readily in this 2:1 solvent mixture. Chloroform
give rise to phosgene on standing (degraded product). This can be prevented by addition of
1% ethanol to stabilize the chloroform. All lipid solutions should be stored in dark in glass
vessels with securely fastened ground glass stopper. Polypropylene containers may also be
used. To reduce the possibility of oxidation of lipids nitrogen is commonly used.

Drying:
Large volume of organic solution of lipid is mostly dried in rotary evaporator fitted with a
cooling coil and a thermostatically controlled water bath. Rapid evaporation of solvent is
carried out by gentle warming (20-40C) at reduced pressure (400-700mmHg).Rapid rotation
of solvent containing flask increases the surface area for evaporation. If the concentration of
lipid is particularly high it may be difficult to remove last traces of chloroform from lipid
film. So it is recommended that after rotary evaporation, some further mean is employed to
bring the residue to complete dryness. Attachment of flask to lyophilizer and overnight
exposure to high vacuum is a good method.

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METHOD OF LIPOSOME PREPARATION:

All methods for preparing liposomes involve three or four basic stages:
1)
2)
3)
4)

Drying down lipids from the organic solvent.

Dispersion of lipids in the aqueous media.
Purification of resultant liposomes.
Analysis of final product.

Steps:
1)
2)
3)
4)
5)
6)
7)
8)

Cholesterol, lecithin( charged)

Dissolved in organic solvent( chloroform and methanol)
Solution in organic solvent
Drying( solvent removed)
Thin film of liposomes( lipid bilayer is formed)
Dispersion in aqueous media
Hydration
Liposome suspension is formed

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Loading of Drug in Liposomes

Two methods are followed:

Passive loading
Remote loading

Passive loading:
It involves the loading of entrapped agents before or during manufacture
procedure.
Remote loading:
Certain types of compounds with ionisable groups, and those which display both
lipid and water solubility, can be introduced into the liposomes after the formation of intact
vesicles.

PHYSICAL DISPERSIONS OR MECHANICAL DISPERSION

There are four basic methods of physical dispersion
1. Hand shaking or rotary evaporator method.
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2. Non shaking
3. Freeze drying
4. Proliposomes
1. HAND SHAKING OR ROTARY EVAPORATOR METHOD:
Lipid film hydration by hand shaking is most widely used physical dispersion method to
prepare multi lamellar vesicle (MLVS).

Method of preparation:
1. Mixing
2. Drying
3. Hydration of lipid
1. Mixing
Lipid mixtures of different phospholipids and charge components in chloroform:
methanol (2:1 v/v) solvent mixture is prepared and introduced in round bottom flask.

2. Drying
It is then attached to rotary evaporator and rotated at 60 rpm.Organic solvent is removed
at 30 C or above the transition temperature of lipids within 15 minutes. Nitrogen is
introduced in evaporator. Flask is removed form evaporator and Residual of organic
solvent is removed with the help of lyophilizer.
3. Hydration of lipids:
After drying the flask is flushed with nitrogen and 5 ml of saline phosphate buffer
(containing solute to be entrapped) is added. The flask is again attached with the
evaporator and rotated at room temperature and pressure at 60 rmp. The flask is left
rotating for 30 minutes until lipids has been removed from the wall of the flask and has
given homogenous milky white suspension free of visible particles.
The suspension is allowed to stand for further 2 hours at room temperature or at the
temperature above the transition temperature of the lipids in order to give the swelling
process to give MLVS.

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2. NON SHAKING METHOD: (FOR LUVS)

Same method as in hand shaking but care is taken over swelling process.
Hydration and swelling are carried out in two separate steps. Hydration is
initiated by exposing the film with stream of water saturated nitrogen for 15
minutes which is followed by swelling in aqueous medium without shaking.
Drying:
Solution of lipid in chloroform: methanol (2:1v/v) spread over flat bottom conical
flask. Solution is evaporated at room temperature by the flow of nitrogen through
the flask without any disturbance. After drying water saturated nitrogen is passed
through the flask until opacity of lipid layer disappeared within 15 to 20 minute.
3. FREEZE DRYING: (MLVS)
Lipoids dissolved in suitable organic solvent are freeze dried. Solvent added should be
near the freeze point, which should be above the temperature of condense of freeze dried.
Tertiary butyl alcohol is widely used solvent. Water or saline is added to lyophilized lipid
with rapid mixing above the phase transition temperature to give MLVS.
4. PROLIPOSOMES:(MLVS)
What are proliposomes?
In order the increase the surface area of dried lipid film and to facilitate instantaneous
hydration the lipid is dried over finely divided particulate support such as powdered
sodium chloride or sorbitol or other polysaccharides. These dried lipid coated particulates
are called proliposomes.
Pro liposomes form dispersions of MLV on adding water in to them where the support is
rapidly dissolved and lipid film hydrates to form MLV.

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ADVANTAGES:
1. This method over comes the stability problem of liposomes encounter during their
storage as dispersion, dry or frozen form.
2. It is ideally suited for the preparations where the material to be entrapped
incorporates in to lipid membrane.
3. It is suitable in the cases where 100 % entrapment of component is not required
rather the stability is preferred.
PREPARATION OF PROLIPOSOMES:
Procedure: Buchi rotary evaporator:
1. Lipid solution in chloroform (60mg/ml) is prepared and sorbitol is introduced in flask.
2. Fit the flask to the evaporator and rotate slowly so that powder tumbles gently off the
walls and ensure good mixing and solvent is evaporated.
3. Flask is lowered in water bath at 50 to 55 C when a good vacuum has developed. An
aliquot of 5 ml of lipid solution is introduced in the flask via the solvent inlet tube.
4. Solvent is absorbed completely by the powder.
5. Vacuum is released. Connect the flask to lyophilizer for drying.
6. Powder is transferred to 10ml glass vial containing 600mg solid (100 mg lipid and 500mg
sorbitol) flushed with nitrogen sealed well and stored.
How to use?
For use introduce 10 ml of distilled water in vial mix on a whirl mixer for 30 sec or in a shaking
water bath above Tc (phase transition temperature.
PROCESSING OF LIPIDS HYDRATED BY PHYSICAL MEANS
OR
OR MECHANICAL TREATMENT OF MLVS
A large number of methods have been developed to reduce their sizes and to convert liposomes
of large size to smaller homogenous vesicle. Such techniques are

1.

Microemulsification
Sonication
Extrusion
French pressure cell
Dried reconstituted vesicles (DRVS)
Freeze thaw sonication method (FTS)
MICRO ENCAPSULATION OR MICRO EMULSIFICATION:

MICROFLUDIZER
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Microfludizer used for microemusification of liposomes. In this method micro fluidizer pump are
used. It consists of following parts

Pump
Interaction chamber
Reservoir
Filter

Procedure:
1. The lipids are introduced in the fludizer either as a suspension of lager MLVS or as a
slurry of unhydrated lipid in organic medium.
2. Microfludizer pumps the fluid at a very high pressure (10,000 psi, 600 -700bar) through a
5micrometer orifice.
3. Then it is forced along defined micro channels which direct two streams of fluid to
collide together at right angles at a very high velocity, therefore affecting an efficient
transfer of energy.
4. The fluid collected can be recycled through the pump and interaction chamber until
vesicles of spherical dimensions are obtained.
After a single cycle size of vesicles is reduced to a size of 0.1 and 0.2 micrometer in diameter.
Advantages:

High rate of production

Process the samples having a high proportion of lipids (20% or more by weight)
This process is sufficient for encapsulation of water soluble drugs.

2. SONICATED UNILAMELLAR VESICLES (SUVS)

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At a high energy level the size of vesicles is further reduced. There are two methods of
sonication base on used of either probe or bath ultrasonic disintegrators.

1. Probe ultrasonic disintegrator:

Method:

Sonication of MLVS is achieved by placing the tip of probe sonicator in test tube
containing the lipid dispersions and sonicationg for 5 to 10 minutes above the phase
transition temperature. The lipid dispersions begin to clarify to yield slightly hazy
transparent solution. These particles can be removed by centrifugation to yield clear SUV
dispersion.
Centrifugation:
Liposomal dispersion after sonication is placed in clear plastic walled ultracentrifuge
tube. It is centrifuged at 100,000g (30 min at 20 C) to sediment titanium particles and
large MLVS followed by high speed centrifugation (1, 59,000g for 3-4 hour)
After spinning tube is removed liquid at the top clear layer is decanted which contains
pure dispersions of SUVS.

Use:
It is employed for the dispersions which require high energy in small volume i.e high
concentration of lipids or viscous aqueous phase.
Disadvantage:
1. Probe tip sonicator supply a high energy in to liquid dispersions but suffer from
overheating of liposomal dispersions resulting in lipid degradation.
2. Sonication tip also tend to release titanium particles in liposomal dispersions which must
be removed by centrifugation.
1. Bath ultrasonic disintegrator:
Method:
Sonication of MLVS dispersion is accomplished by placing the test tube containing lipid
dispersions in bath sonicatorfor 5 to 10 minutes above the phase transition temperature.
Use:

Bath sonicators are used for suitable large volumes of diluted lipids.
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Widely used to prepare SUVS.

It overcomes all the disadvantages associated with probe sonicator.

2. FRENCH PRESSURE CELL LIPOSOMES:

One of the first and very useful methods is the extrusion of preformed large liposomes in French
pressure under very high pressure (Barenholt et al 1979, Hamilton et al 1980)
Advantages:
1. This technique yield rather uni or oligo lamellar liposomes of intermediate size (30 to 80
nm in diameter depending on applied pressure)
2. Liposomes prepared by this method are less likely to suffer from structural defects and
instabilities observed in sonaicated vesicles
3. Leakage of contents form vesicles is slower and lower than sonicated vesicles.
4. It reduces the heterogeneity of population of liposomes obtained by detergent dialysis.

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Draw backs: Initial high cost of press that consists of an electric hydraulic press and pressure
cell.

Apparatus:
The heart of French pressure cell is pressure cell, manufactured in stainless steel and designed to
resist up to 20,000 or even 40,000psi.
It consists of following parts:

Precision bored cylindrical cavity with small outlet orifice (valve)

A bottom seal
A piston
Valve closure and outlet rubbing.

Method:

Liposomal suspension is filled in pressure chamber.

The liquid sample is introduced in cavity.
Height of piston is adjusted so that chamber is completely filled with the liquid.
Complete filling is important to prevent compression of trapped air which will result in
uncontrollable splashing during extrusion.
Upon filling the bottom seal is inserted.
Pressure is adjusted with hydraulic valves.
At high force smaller liposomes are formed.

VESICLES PREPARED BY EXTRUSION TECHNIQUES: (VETS)

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The membrane extrusion method the size of liposome is reduced by passing them through
membrane filter of defined pore size. It can be achieved at lower pressure <100psi. Than
required in French pressure cell.
Types of membrane filters:
1. Tortuous path type membrane
2. Nucleation path type of membrane
1. Tortuous path type membrane:
In this type random paths arise between cris crossed fibers. Liposomes which are larger
than channels diameter get stuck when one tries to pass them. Fibers blocked easily
because of convoluted nature of channels.
2. Nucleation path type membrane:
In this type liposomes can pass easily through membrane even they are larger than pore
diameter. The membrane consists of continuous sheet of polymer usually polycarbonate
in which straight side pore holes of exact diameter are present so they offer much less
resistance to the material passing through.

Uses:
This technique is used to process LUVand MLV as well as to produce SUV and LUV due to their
ease of production readily selectable diameter batch to batch reproducibility and freedom from
solvent and surfactant contamination.

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5. DRIED RECONTITUTED VESICLES (DRVS):

This method starts with freeze drying of a dispersion of empty SUV and then rehydrating it with
aqueous fluid containing the material to be entrapped. This leads to a dispersion of lipid in finely
divided form. liposomes obtained by this method are uni or oligo lamellar of the order of 1.0m
or less in diameter.
Reason of using freeze drying?

The step of freeze drying is used to freeze and lyophilized preformed SUVS dispersion
rather than to dry a lipid from organic solvent this leads to organized membrane structure
as compared to random matrix which on addition of water can rehydrate fuse and reseal
to form vesicles with high capture efficiency.

Advantages:
1. High entrapment of water soluble drug.
2. Use of mild conditions for loading and preparation of bioactive.

6. FREEZE THAW SONICATION (FTS):

Its an extension of DRVS method. This method is based upon freezing of a uni lamellar(mainly
SUVs) dispersion and then thawing at room temperature for about 15 minutes and finely
subjecting to sonication cycle.
Method:
1. Empty liposomes are prepared by sonication for about 15 to 30 sec.
2. It is then followed by thawing.
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3. Liposomes are again subjected to sonication. This step reduces the permeability of
liposome membrane by accelerating the rate at which packing defects are eliminated.
Advantages:

It is simple rapid and mill for entrapped solute and result in high proportion of large
unilamellar formation which is use full for study of membrane transport phenomenon.
Inclusion of some negatively charged lipid give higher entrapped volume (20l/mg) as
compared to (10l/mg) for neutral lipid.

Disadvantages:

Neutral lipid cannot be subjected to freeze thaw sonaication.

Sucrose divalent metal ions and high ionic strength salt solution cannot entrap.

2. SOLVENT DISPERSION METHOD:

PRINCIPLE:

Lipid first dissolve in organic solution

Aqueous phase should mix with material to be entrapped within
liposomes
Lipid are then brought into contact with aqueous phase

METHOD INVOLVING SOLVENT DISPERSION:

1. Organic solvent miscible with aqueous phase
2. Organic solvent immiscible with aqueous phase but aqueous phase is
in excess.
3. Organic solvent immiscible with aqueous phase but organic solvent is
in excess.
TECHNIQUES:
1. ETHER INJECTION:
It involves injecting the immiscible organic solution
very slowly into the aqueous phase. Method also treats sensitive lipids very
gently and a little risk of causing oxidative degradation because ether is free
from peroxides. For substances that degrade at high temperature fluorinated
hydrocarbons are used.
2. ETHANOL INJECTION:

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It involves injecting ethanol rapidly in aqueous

phase. As a result of this ethanol dissolve and phospholipids molecules are
dispersed. There is no need to increase the temperature as in the case with
ether injection method.

3. WATER IN ORGANIC PHASE:

Liposomes are made in two steps
1. First the inner leaflet of lipd bilayer is form
2. Then the outer half is form
The common feature of this method Is formation of water in oil emulsion
produced by introduction of small quantity of aqueous medium containing
material to be entrapped intolarge volume of immiscible organic solution of
lipid followed by mechanical agitation to break up the aqueous phase into
microscopic water droplet
4. DOUBLE EMULSION VESICLES:
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The organic solution which already

contains water droplet is introduced into excess of aqueous medium followed
by mechanical agitation a multicompartment vesicles are obtained
It may be described as w/o/w system
The organic solvent is evaporated by strong jet of nitrogen in double
emulsion. and final volume is adjusted by adding distilled water.
5. REVERSE PHASE EVAPORATION METHOD:

6. STABLE PLURILAMELLAR VESICLES:

In this method water in oil dispersion
is prepared but drying process is accompanied by continuos bath sonication
with stream of nitrogen.

3. DETERGENT SOLUBILIZATION:
In this method phospholipids are brought
into contact with aqueous phase with the intermediary of detergent. The
structure formed as a result of this is known as micelle and can be composed
of several hundred components of molecules

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Micelle containing components in addition to detergent is known as mixed

micelles.

PURIFICATION OF LIPOSOMES:

Gel Filtration
Coloumn Chromatography
Dialysis
Centrifugation

CHARACTERIZATION OF LIPOSOMES:
Behaviour of liposomes in both physical and biological systems is goverened by the
factors:

physical size
membrane permeability
percent entrapped solute
chemical composition
quantity of starting material
percentage drug release

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CHEMICAL PROPERTIES

Quantitative determination of phspholipids;

Phspholipids hydrolysis:
Phspholipids oxidation:
Cholestrol analysis

1-QUANTITATIVE DETERMINATION OF PHSPHOLIPIDS;

It is difficult to measure directly the phospholipids concentration since dried
lipids often contain considerable quantities of residual solvents. So the most
widely used methods are indirect, which includes
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Bartlett assay
Stewart assay
TCL (thin layer chromatography)
A) Bartlett Assay:
phospholipids phosphorous in the sample is first hydrolyzed to
inorganic phosphate, which is then treated with ammonium molybdate
to form phosphomolybdis acid, which is then reduced by aminonaphtyl-sulphonic acid to a blue color compound. Its intensity is
measured spectrophotometrically.
B) Stewart Assay;
In this method first prepare the stdandard dilutions. Add lipids in
chloroform and make different dilutions and then in each dilution add
0.1 M Ammonium ferrothiocyanate which result in formation of
complex. Measure density at 485 nm and compare it with sample.
C) TLC:
It is also employed for determining the purity and concentration of
phospholipids.
2-PHSPHOLIPIDS HYDROLYSIS:
Major product of lecithin hydrolysis is lysolecithin. Phospholipids hydrolysis is
measured by approximation of lysolecithin by HPLC.
3- PHSPHOLIPIDS OXIDATION:
In the absence of specific oxidant, oxidation takes place through free radical
chain mechanism. Initiation step for this is removal of H-atom from fatty acid
chain and it is brought about by
1. Electromagnatic radiation
2. Transition metals
Techniques available for determining oxidation are

UV absorbance method
TBA method
Iodometric method
GLC method

4) CHOLESTROL ANALYSIS:
It is qualitatively analyzed by capillary column of fused silica.

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Quantitatively estimated by measuring absorbance of purple colour

complex form by treating the sample with a combined reagent consisting
of ethyl acetate, ferric perchlorate and sulfuric acid. Absorbance is
measured at 610 nm.

PHARMACOKINETICS OF LIPOSOMAL ENCAPSULATED

DRUGS:
Most of the drug delivery system are designed to control one of the
following parameters
1. Rate of input of drug into particular compartment
2. Distribution and localization of drug in the body
3. Rate of metabolism of drug
Liposomes used one possible approach to control the drug delivery.
Comparison of liposomes and polymeric devices?
1-Similarity: both are drug reservoir with an output controlled and limited
by the permeability of membrane
2-Differences:

Liposomes give sustained effect from hrs-days while polymeric devices

give effect from days to months
Drug disposition can be changed by liposomes but not by polymeric
devices

CLEARANCE AND DISTRIBUTION OF LIPOSOMES IN VIVO

Liposomes injected into circulation are gradually sequestered into different
organs and tissues. Distribution depend on two factors

Size of liposomes: smaller the size longer will be persistence in

circulation and larger the size rapid will be clearance. That is why
SUVs persist in circulation for longer time and MLVs are rapidly
cleared
Surface charge of liposomes: SUVs with positive and negative
charge persist in circulation for longer time while SUVs with negative
charge are rapidly cleared.

After clearance from circulation liposomes are sequestered into Different

tissues and organs. In case of MLVs primary Site of uptake is Liver and
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Spleen. Out of MLVs which are of larger size retained in capillaries of lungs
Because of physical entrapment.
SUVs have broader tissue distribution as compared to MLVs however at the
end they also accumulate in liver and spleen.

PHARMACODYNAMICS OF LIPOSOME ENCAPSULATED DRUG

Behaviour of drug is largely determined by the behavior of Carrier.Inshort
encapsulated drugs will have following pharmacodynamic effects
1. Retardation of drug clearance from circulation
2. High drug accumulation in tissues rich in reticulo endothelial cells
especially liver and spleen
3. Retention of drug in tissues for longer time
4. Protection of drug against metabolic degradation

STABILITY OF LIPOSOMES:
Liposomes stability is much and more severe. Liposomes are liable to fusion
aggregation, leakage, hydrolysis and oxidation.
A) PREVENTION OF CHEMICAL DEGRADATION:
Following precautions should be followed
Use freshly purified lipids
Use freshly distilled solvents
Avoid the procedure which involve high temperature
Store Liposomes in inert atmosphere
Deoxygenate all aqueous solution with nitrogen
Add an antioxidant as a component of lipid membrane e.g Toccopherol
Add iron chelator.
Slow down the hydrolysis by carrying the procedure at neutral pH
Use the lipids which have ether linkage instead of ester linkage
Enzymatic hydrolysis can be prevented by using sphingomyelins or
phospholipid derivatives.
B) PREVENTION OF PHYSICAL DEGRADATION:

1. Leakage and fusion:

Leakage and fusion of vesicles occur as a result of packing defects in the
membrane introduced during their manufacture. These irregularities can
be dispensed by a process called annealing in which simply incubate the
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vesicles at a temp. High enough above the transition temp to allow

differences in packing density b/w the opposite sides of the bilayer to
equalize by trans-membrane flip-flop of lipid molecules.
SUVs are more prone to fusion. They do so for relieving stress which
arises from high curvature of membrane.
2-Aggregation and sedimentation
Aggregation of neutral liposomes is brought about by van der waals
interaction and is more pronounced in larger vesicles because of inc.
planarity of the membrane which allows the greater area of the
membrane to come in contact with each other.
TECNIQUES TO OVERCOME THE INSTABILITY:
1-Store liposome suspension at a temp. Away from transition temp.
2-Use more polar and hydrophilic solutes
3-Lyophilization: In this liposomes are converted into anhydrous or solid
form, where chemical degradation is less likely to occur.
4-Cryo preservation: cryo protectants(liquid Nitrogen) are used. it is most
successful method for liposomes.

APPLICATION OF LIPOSOMES:
Mode of action
Intracellular uptake

Application
Microbial disease Metal storage diseases Gene
manipulation
Uptake by tumor cell
Slow release of drug near Tumor near fixed macrophage, Macrophage
the target area
activation to
tumoricidal/microbicidal state
Circulating Reservoirs
Blood surrogates
Facilitation
of
drug Drug delivery to skin ,lungs.eyes,mucosal tissue
uptake by certain routes
Avoidance
of
tissue Cardiotoxicity of doxorubicin
sensitivity
The following are the few more properties which make liposomes
applicable in various fields
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Cell liposome interaction: SUVs can interact with the cell in 4 ways;
1. Stable adsorption: Association of intact vesicles cell
surface.This association is mediated by non-specific
electrostatic,hydrophobic or other forces
2. Endocytosis: It is the uptake of intact vesicles into endocytic
vesicles and their delivery to lysosomal apparatus.
3. Fusion: It is the merging of the vesicle bilayer with the plasma
membrane bilayer,with the resultant release of vesicle contents
into cytoplasm.
4. Lipid exchange: It is the transfer of individual lipid molecules
b/w vesicles and cell surface,without the cell association with the
aqueous vesicle content.
Localized drug effects: Liposomes help in depositing the drug within
selected sites or selected cell types. This is because of their larger size
and low degree of penetration in epithelial and connective tissue
barrier.
Enhanced drug uptake: It occurs by vesicle cell fusion or via
endocytosis.
Molecules with different range of solubilities can be accommodated.
Flexibility in structural characteristics.

1-CANCER CHEMOTHERAPY AND NEOPLASIA:

Anticancer drugs are usually non selective and result into toxicity to normal
cells.Liposomes have successfully been used for such drugs and it has
following advantages;
1. Inc. circulation life time
2. Protects the metabolic degradation of drug
3. Altered the Tissue distribution of the drug with enhanced uptake in
liver and spleen and dec. uptake in brain myocardium and kidney.
2-LIPOSOMES AS CARRIER FOR VACCINES:
a) Liposomes as immunological adjuvants: Liposomes have adjuvant
effect on protein antigen.Liposomes cab be effectively used as carrier
for antigen. It has the following advantages;
1. A non-immunogenic substance can be converted into immunogenic
2. Hydrophobic antigens can be reconstituted
3. Small amounts may be suitable as immunogens.
4. Multiple antigens may be incorporated into the single liposome.
5. Adjuvants may be incorporated with antigen
6. Longer duration of action may be achieved
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M.Phil Pharmaceutics semester 01

liposomes

b) Liposomes as carrier for antigens: Liposomes recognized as an

efficient carrier for delivering biologically active material to specific
cells. However on administration major fraction is uptaken by liver and
spleen. This uptake can be retarded by Following approaches;
1. Rate of uptake of liposomes by RES is minimized by using small,
neutral unilammelar vesicles having higher Tc and cholesterol.
2. By coating the surface of liposomes which make them less
recognizable by RES.
3. Coupling appropriate ligand on the liposome surface which can bind to
the receptors on the surface of target cells.
c) Liposomes as carrier of drug in oral treatment: Liposomes can be
effectively used for delivering the drugs by oral route
1. Arthritis: Steroids are used in the treatment of arthritis but their effect
is transient bcoz of rapid destruction in periphery. When encapsulated
in liopsomes produces
Effect for longer duration
Localized effect
2. Diabetes: Insulin encapsulated in liposome when administered to
hyperglycemic animals via oral route, of glucose level was observed.
Liposomes provided protection to insulin against the pepsin and
pancretin.
d) Liposomes for topical applications: Liposomes have great potential
as topical drug delivery. Liopsomes based formulation of NSAIDS, local
anesthetics, antimicrobials, and anti tumor drugs are effective in the
treatment of respective disorders.
e) Leishmaniasis: It is a parasitic disease affecting millions of people. It
become lethal if parasite invades into liver and if it is left untreated.
Even many people die with the treatment, bcoz commonly prescribed
drugs antimonials are related to arsenic are very toxic to Heart, liver
and kidney. Their liposome encapsulation produces following
advantages
1-Toxicity is reduced
2-Localized effect in liver is achieved.
3-Amount of the drug needed is reduced
f) Lysosomal storage disease: These diseases mostly results from the
genetically determined enzymatic deficits of particular lysosomal
enzymes. So liposomes are suitable candidate for lysomotropis enzyme
replacement. This includes Gaucher disease (-Glucosidase deficiency)
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M.Phil Pharmaceutics semester 01

liposomes

where site of pathological catabolite accumulation is RES and Pomps

disease (-glucosidase deficiency) where the primary affected tissues
are liver and muscle.

References:

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M.Phil Pharmaceutics semester 01