LIPOSOMES

• Liposomes are simple microscopic vesicles in

which an aqueous volume is entirely enclos
edby a membrane composed of a lipid molecule
• Structurally, liposomes are concentric
bilayered vesicles in which an aqueous volume
is entirely enclosed by a membraneous lipid
bilayer mainly composed of natural or
synthetic phospholipids
 ADVANTAGES OF LIPOSOMES
• Provides selective passive targeting to tumor
tissues
Eg:liposomal doxorubicin
• Increased efficacy and therapeutic index
• Increased stability of encapsulated drug
• Reduction in toxicity of the encapsulated agent
• Site avoidance effect (avoids non target tissues)
• Improved pharmacokinetic effects (reduced
elimination, increased circulation life times)
• Flexibility to couple with site specific ligands to
achieve active targeting.
 STRUCTURAL COMPONENTS OF
LIPOSOMES

• THE MAIN COMPONENTS OF LIPOSOMES ARE:

• – PHOSPHOLIPIDS
– CHOLESTEROL
PHOSPHOLIPIDS
• Phospholipids are the major structural
components of biological membranes such asthe cell m
embrane.
• TWO TYPES OF PHOSPHOLIPIDS
-- PHOSPHOGLYCERIDES
--SPHINGOLIPIDS
• Molecules of PC are not soluble in water.
• In aqueous media they align themselves closely in
planar bilayer sheets in order to minimize the
unfavourable action between the bulk aqueous
phase and the long hydrocarbon fatty chain. (i.e.
• they orient themselves so that the fatty acid chains
face each other, and the polar heads face the
aqueous phase this reduces the instability which
exists when the molecules exist alone)
• Such unfavourable interactions are completely
eliminated when the sheets fold on themselves to
form closed sealed vesicles.
• SOME OTHER COMMONLY USED PHOSPHOLIPIDS
Naturally occurring phospholipids:
• – PC : Phosphatidylcholine (one chain is oleoyl and th e
other is palmitoyl)
• – PE : Phosphatidylethanolamine
• – PS : Phosphatidylserine
Synthetic phospholipids:
• – DOPC : Dioleoylphosphatidylcholine
• – DSPC : Distearoylphosphatidylcholine
• – DOPE : Dioleoylphosphatidylethanolamine
• – DSPE : Distearoylphosphatidylethanolamine
• CHOLESTEROL
• Incorporation of sterols in liposome bilayer brings
about major changes in the preparation of these
membranes.
• Cholesterol by itself does not form a bilayer
structure.
• cholesterol acts as a fluidity buffer, i.e. Below the
phase transition temperature, it makes the
membrane less ordered and slightly more
permeable
• while above the phase transition temperature it
makes the membrane more ordered and stable.
• It can be incorporated into phospholipid
membranes in very high concentration upto 1:1 or
even 2:1 molar ratios of cholesterol to PC.
• Mechanism of cholesterol acting as a fluidity
buffer.
• Cholesterol incorporation increases the
separation between the choline head groups
and eliminates the normal electrostatic and
hydrogen bonding interactions thus pushing
the phospholipids apart making the layer less
ordered at lower temperatures.
• However, in the higher concentrations the
membrane area occupied by the combination of
acyl chains and cholesterol is greater than (or equal
to) that taken by phosphocholine head group.
• This difference in area retards chain tilt
(the phenomenon responsible for phase transition –
i.e. trans to gauche conformation change). Above
the transition temperature, the reduction in the
freedom of the acyl chains causes the membrane to
remain condensed and rigidized, with a reduction in
area through closer packing and resultant decrease
in fluidity.
Schematic representa ion of liposomc.
• Handling of Liposomes
• The lipids used in the preparation of
liposomes are unsaturated and hence
susceptible to oxidation.
• Also volatile solvents such as chloroform
which are used will tend to evaporate from
the container.
• Thus liposomes must be stored in an inert
atmosphere of nitrogen, and in the dark, in
glass vessels with a securely fastened cap.
 DRYING

• An important step involved in the preparation of

liposomes is the drying of the lipid.
• Large volume of organic solution of lipids is most
easily dried in a rotary evaporator fitted with a
cooling coil and a thermostatically controlled
water bath.
• Rapid evaporation of solvent is carried out by
gentle warming (20 - 40 degrees) under reduced
pressure (400 - 700 mm Hg)
• Rapid rotation of the solvent containing flask
increases the surface area for evaporation
• In cases where sufficient vacuum is not
attainable or if the concentration of lipids is
particularly high, it may be difficult to remove
the last traces of chloroform from the lipid
film.
• Therefore after rotary evaporation, some
further means is employed to bring the
residue to complete dryness.
• Attachment of the flask to the manifold of
lyophilizer, and overnight exposure to high
vacuum is a good method.
4 BASIC METHODS OF PHYSICAL DISPERSION
• Hand shaken multilamellar vesicles
• Non shaking vesicles
• Pro liposomes
• Freeze drying
• AFTER THESE METHODS, OTHER PROCESSING METHODS ARE
USED TO MODIFY THE TYPE OF VESICLES THAT ARE PRODUCED
SUCH AS:
• Micro emulsification liposomes (MEL)
• Sonicated unilamellar vesicles (SUVs)
• French Pressure Cell Liposomes
• Membrane extrusion liposomes
• Dried reconstituted vesicles (DRVs)
• Freeze thaw Sonication (FTS)
• pH induced vesiculation
• Calcium induced fusion
– These methods are known as “the mechanical treatment of
MLVs” or “Processiing of lipids hydrated by physical
Hand shaken multilamellar vesicles
• Simplest and most widely used method
• Disolution of the lipid mixture and charge components
in chloroform:methanol solvent
• Evaporation of the solvent in a rotary
evaporator
or by hand shaking to form a film
• Further drying of the film by attaching the flask to
the manifold of the lyophilizer.
• Casted film is then dispersed in an aqueous medium.
• Upon hydration, lipid swell and peel off the wall of the
flask and vesiculate forming multilamellar vesicles
(MLVs)
Hand shaken method in general
• For preparing pro liposome a special equipment i.e.
Buchi rotary evaporator ‘R’ with water cooled
condensor coil and a stainless steel covered
thermocouple connected to a digital thermometer
is required.
BUCHI Rotary Evaporator R type
 Freeze drying

• Freeze dry the lipid dissolved in a suitable organic

solvent.
• The solvent choice depends on the freeze point
which needs to above the temperature of the
condenser lyophilizers.
• Tertiary butanol is considered to be the most ideal
solvent.
• After obtaining the dry lipid which is an expanded
foam like structure, water or saline can be added
with rapid mixing above the phase transition
temperature to give MLVs.
 I B] Processing of the lipids hydrated
by physical means, or the mechanical
treatment of MLVs

• Micro emulsification liposomes (MEL)

• Sonicated unilamellar vesicles (SUVs)
• French Pressure Cell Liposomes
• Membrane extrusion liposomes
• Dried reconstituted vesicles (DRVs)
• Freeze thaw Sonication (FTS)
• pH induced vesiculation
• Calcium induced fusion
Micro emulsification liposomes (MEL)
• Microfluidiser
• Pumps the fluid at very high pressure through a 5
µmt orifice.
• Forced along defined microchannels – which
direct two streams of fluid to collide together at
right angles at a very high velocity
• Fluid collected recycled through the pump until
vesicles of spherical dimension are obtained
Sonicated unilamellar vesicles (SUVs)
• Exposure of MLV to ultrasonic radiation
• Probe/bath sonicator
• Probe is used for dispersions which require high
energy in a small volume (high conc of
lipids/viscous aq phase)
• Disadvantages : over heating, release titanium
particles into liposome dispersion
• Bath is more suitable for large volumes of diluted
lipids
French Pressure Cell Liposomes
• Extrusion of preformed large liposomes in a french
press under very high pressure(20,000psi) –
uni/oligo lamellar liposomes
• More stable liposomes, leakage of contents is
slower and lower than sonicated liposomes
• High cost of press consists of hydraulic press and
pressure cell
Membrane Extrusion Liposomes
• Size is reduced by gently passing them through
membrane filter of defined pore size – achieved at
much lower pressure (<100psi)
• In this process vesicle contents are exchanged with
the dispersion medium during breaking and
resealing of phospholipid bilayers as they pass
through the polycorbonate membrane
• To achieve high entrapment, the watersoluble
compounds should present in suspending medium
during extrusion process
Dried Reconstituted Vesicles (DRVs)
and Freeze Thaw Sonication (FTS)
• Starts with freeze drying of a dispersion of empty
SUVs and then rehydrating it with the aq fluid
containing the material to be entrapped.
• This leads to dispersion of solid lipids in finely
subdivided form
• Step of freeze drying is used to freeze and lyophilize
a preformed SUVs dispersion rather than to dry the
lipids from an organic solution
• This leads to organized membrane structures as
compared to random matrix structure, which on
addition of water can rehydrate, fuse and reseal to
form vesicles with a high capture efficiency
 pH induced vesiculation

• This method prepares ULVs from MLVs without

sonification or high pressure application.
• They are reassembled by simply changing the
pH.
• It is an electrostatic phenomenon.
• The period of exposure of the phospholipids to
high pH is less than 2 mins and not long enough
to cause detectable degradation of the
phospholipid.
• MLVs or LUVs ( PH 2.5-3)  Add 1 M NaoH
( less than 2 min) PH rises to 11 Now add 0.1
M Hcl PH moves down to 7.5  SUV
• Change in PH brings about an increase in
surface charge density of lipid bilayer, which
induces spontaneous vesiculation
• In this method, dry film of lipids is obtained in
the round bottomflask using a rotary
evaporator and last traces of the solvent are
removed using freeze dryer.
• Then, the film is hydrated with minimum
quantity of water by hand shaking at room
temperature.
• At this stage, material to be entrapped inside
the vesicles may be added in the water before
addition to the lipid.
• The pH of the dispersion will be 2.5 - 3
• Sodium hydroxide solution (1M) is added
rapidly with mixing into the suspension then
the pH is reduced by addition of 0.1M HCl
until a value of pH 7.5 is achieved.
 II] SOLVENT DISPERSION METHODS

• lipids are first dissolved in an organic

solution,which is then brought into contact with
the aqueous phase containing materials to be
entrapped within the liposome.
• At the interface between the organic and aqueous
media, the phospholipids align themselves into a
monolayer which form the bilayer of the liposome.
• Methods employing solvent dispersion fall into
one of three categories
• – The organic solvent is miscible with the aqueous
phase. (e.g. ethanol)
• – The organic solvent is immiscible with the
aqueous phase, the latter being in a large excess.
• – Organic solvent is in large excess, and is again
immiscible with the aqueous phase.
• (1) Ether Infusion Method
• A solution of lipids dissolved in diethyl ether or
ether/methanol mixture is slowly injected to an
aqueous solution of the material to be encapsulated
at 55-65°C or under reduced pressure.
• The subsequent removal of ether under vacuum
leads to the formation of liposomes.
• The main drawbacks of the method
--- heterogeneous(70-190 nm)
--- exposure of compounds to organic
solvents or high temperature
• 2.Ethanol Injection Method
• A lipid solution of ethanol is rapidly injected to a
vast excess of buffer.
• The MLVs are immediately formed.
• The drawbacks of the method
-- population is heterogeneous (30-110 nm),
-- liposomes are very dilute
-- difficult to remove all ethanol because it
forms azeotrope with water
-- possibility of various biologically active
macromolecules to inactivation in the presence of
•3. Water in organic phase OR De emulsification Methods
• The liposome is made up in two steps
•– First the inner leaflet of the bilayer
•– Then the outer half
• It involves the formation of a water in oil emulsion,
produced by introduction of a small quantity of aqueous
medium containing material to be entrapped, into a large
volume of immiscible organic solution of lipid – followed
by mechanical agitation to break up the aqueous phase
into microscopic water droplets.
• These droplets are stabilized by the presence of
phospholipid monolayer at the phase interface.
• The size of the droplet is determined by 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
coalescing with other droplets or with the solvent air
interface.
• The aqueous solution surrounded by the monolayer of
phospholipid forms the central core of the final liposome.
• There are a number of methods which can be used to
prepare the droplets such as
• a) Double emulsion
• b) Reverse phase evaporation
• c) SonicationMethods (Stable Plurilamellar Vesicles –SPVs)
• Double emulsion vesicles:
• Organic solution + Lipid + Aqueous phase
Emulsion (W/O)  Hot aqueous solution of
buffer Multi compartment vesicle W/O/W
(double emulsion) LUVs
• First water in oil emulsion is formed by brief sonication of
a two phase system containing phospholipids in organic
solvent (diethylether or isopropylether or mixture of
isopropyl ether and chloroform) and aqueous buffer.
• The organic solvents are removed under reduced
pressure, resulting in the formation of a viscous gel.
• The liposomes are formed when residual solvent is
removed by continued rotary evaporation under
reduced pressure.
• With this method high encapsulation efficiency up to 65%
can be obtained in a medium of low ionic strength for
example 0.01 M NaCl.
• The method has been used to encapsulate small, large
and macromolecules
• disadvantage of the method is
• the exposure of the materials to be
encapsulated to organic solvents and to brief
periods of sonication.
• These conditions may possibly result in the
denaturation of some proteins or breakage of
DNA strands
• main advantage of the method is that the
liposomes had high encapsulation efficiency
(about 80%).
• c) Sonication Methods (StablePlurilamellar Vesicles – SPVs)
• w/o dispersion is prepared with excess lipid
• drying process continued bath sonication with a
stream of nitrogen.
• The redistribution and equilibration of aqueous solvent
and solute occur during this time in between the various
bilayers in each plurilamellar vesicle.
• The internal structure of SPVs is different from that of MLV
REVs, in that they lack a large aqueous core, the majority of
the entrapped aqueous medium being located in
compartment in between adjacent lamellae.
• The percent entrapment is normally 30%.
 III] DETERGENT SOLUBILIZATION
• The detergents at their critical micelles concentrations
have been used to solubilize lipids.
• As the detergent is removed the micelles become
progressively richer in phospholipid and finally combine to
form LUVs.
• The detergents were removed by dialysis
• . A commercial device called LIPOPREP which is a version of
dialysis system is available for the removal of detergents.
• the dialysis can be performed in dialysis bags engrossed in
large detergent free buffers (equilibrium dialysis)
• advantages of detergent dialysis method
-- excellent reproducibility
-- production of liposome populations which are
homogenous in size.
• The main drawback of the method is the
retention of traces of detergent(s) within the
liposomes.
• Drug loading in liposomes
• Drug loading can be attained either passively (i.e., the drug is
encapsulated during liposome formation) or actively (i.e., after
liposome formation). Hydrophobic drugs, for example
amphotericin B taxol or annamycin, can be directly combined into
liposomes during vesicle formation, and the amount of uptake
and retention is governed by drug-lipid interactions. Trapping
effectiveness of 100% is often achievable, but this is dependent
on the solubility of the drug in the liposome membrane. Passive
encapsulation of water-soluble drugs depends on the ability of
liposomes to trap aqueous buffer containing a dissolved drug
during vesicle formation. Trapping effectiveness (generally <30%)
is limited by the trapped volume delimited in the liposomes and
drug solubility. On the other hand, water-soluble drugs that have
protonizable amine functions can be actively entrapped by
employing pH gradients [39], which can result in trapping
effectiveness approaching 100% [40].
 CHARACTERIZATION OF LIPOSOMES
Evaluation of liposomes

• The behavior of liposomes in both physical and

biological systems is governed by the factors such
as:
• – Physical size
• – Membrane permeability
• – Percent entrapped solutes
• – Chemical composition
• – Quantity and purity of the starting material
• The liposomes are characterized for
physical attributes:
• – Shape, size and its distribution
• --Surface Charge
– Percentage drug capture
• – Entrapped volume
• – Lamellarity
• – Percentage drug release
Chemical Compositions:
• estimation of phospholipids
• phospholipid oxidation
• analysis of cholesterol
 1] PHYSICAL PROPERTIES
• Size and its Distribution
• – Microscopic Methods
• – Laser light scattering
• – Gel permeation
• b) Surface Charge
• Electrophoresis is used to determine the surface
charge of MLVs.

• A technique that separates vesicles on the basis of

their surface charge by electrophoresis on a
cellulose acetate plate in a sodium borate buffer pH
• The lipid samples (5 nmoles) are applied to the plate
and electrophoresis is carried out at 4 degrees celsius
on a flat bed apparatus for 30 mins at 18 V/cm.
• The plate is dried and the phospholipids are
visualized by the molybdenum blue reagent. c)
Percent capture (entrapment)
• Measure the quantity of material entrapped
inside liposomes – because the effects are dose
related.
• Centrifugation method
• Dialysis method
• d) Entrapped Volume
• The percent drug encapsulated in liposomes can
also be determined by entrapped volume per lipid
weight
• The trapped volume is determined by dispersing
lipid in an aqueous medium containing a
radioactive solute.
• Carboxyfluorescein is also used as a marker to
determine the volume of entrapped water per mole
ofphospholipid
• The entrapped volume of a population of liposomes (in uL/mg
phospholipid) can often be deduced from measurements of
the total quantity of solute entrapped inside liposomes
assuring that the concentration of solute in the aqueous
medium inside liposomes is the same as that in the solution
used to start with, and assuming that no solute has leaked out
of the liposomes after separation from unentrapped material.
•
• However, in many cases such assumption is invalid. For e.g., in
two phase methods of preparation, water can be lost from the
internal compartment during the drying down step to remove
organic solvent.
• On other occasions, water may enter or be expelled from the
liposome as a result of unanticipated osmotic differences.
• e) Lamellarity
• The average number of bilayers present in a
liposome can be found by
-- freeze electron microscopy
-- g)31P ‐NMR.
Drug Release
in vitro diffusion cell.
Dialysis method
to predict pharmacokinetics and bioavailability of the drug before
employing costly and time consuming in vivo studies.
• f) Phase Behavior of Liposomes
• An important feature of lipid membrane is the
existence of a temperature dependant,
reversible phase transition
• where the hydrocarbon chains of the phospholipid
undergo a transformation from an ordered (gel)
state to a more disordered fluid (liquid
crystalline) state.
-- freeze fracture electron microscopy
--- differential scanning calorimetery.
a) Quantitative Determination of Phospholipids
• an indirect one in which the phosphate
content of the sample is first measured.
• The phospholipids are measured either using
Bartlett assay or Stewart Assay.
• 2) CHEMICAL PROPERTIES
• Quantitative Determination of Phospholipids
• Phospholipid Hydrolysis
• Phospholipid Oxidation
• Cholesterol Analysis
• Bartlett assay : the phospholipid phosphorous
in the sample is first hydrolyzed to inorganic
phosphate . This is converted to phospho‐
molybdic acid by the addition of ammonium
molybdate and phospho‐molybdic acid is
quantitatively reduced to a blue colored
compound by amino‐naphthyl‐sulfonic acid .
The intensity of the blue color is measured
spectrophotometrically and is compared with
the curve of standards to give phosphorous and
hence phospholipid content.
• Stewart Assay.
• phospholipid forms a complex with ammonium
ferrothiocyanate in organic solution. The advantage
of this method is that the presence of inorganic
phosphate does not interfere with the assay. In this
method, the standard curve is first prepared by
adding ammonium ferrothiocyanate (0.1M) solution
with different known concentrations of
phospholipids in chloroform. Similarly, the samples
are treated and optical density of these solutions is
measured at 485nm and the absorbance of samples
compared with the standard curve of phospholipids
to get the concentration.
• b) Phospholipid Hydrolysis
• The major product of lecithin hydrolysis is lysolecithin
where one fatty acid chain is lost by de esterification.
• estimation of phospholipid hydrolysis by quantitation of
lysolecithin could be carried out by HPLC. techniques
available for determining the oxidation of phospholipids
• UV absorbance method,
• iodometric method (for hydroperoxides)
• GLC method
• c) Phospholipid Oxidation

• Oxidation of the fatty acids of phospholipids in the absence of

specific oxidants occurs via a free radical chain
 Applications of liposomes
• Liposomes as drug delivery vehicles
– Increased therapeutic index.
– Liposomes as a lysosomotropic carriers
– Liposomes in multidrug resistance.
• Liposomes in anti-microbial, anti-fungal (lung therapeutics) and
anti-viral ( anti HIV).
– Amphotericin-B liposomes
• Liposomes in tumor therapy.
– Sterically stabilized stealth liposomes
– Sialic acid conjugated liposomes
– PEG coated liposomes.
– Anthracyclics
• Liposomes in gene therapy.
• Liposomes in anti-sense oligonucleotide therapy.
• Immunological applications of liposomes.
- liposomes as an immunological (vaccines) adjuvant.
- fusogenic liposomes and virosomes.
- Liposomal vaccines
- Liposomes as an carrier of immunomodulators .
• - Liposomes in immunodiagnostic Liposomes
in dermatology and cosmetology.
• Liposomes as radiopharmaceutical and radio
diagnostic carriers.
• Liposomes as red cells substitutes and artificial
RBC’s
• Miscellaneous applications