liPOsOMes

inTrODUCTiOn
DeFiniTiOn
Liposomes are simple microscopic vesicles in which an aqueous
volume is entirely enclosed by a Phospholipids bilayer molecule.
❖ Liposome were first produced in England in 1961 by
Alec D. Bangham
❖The size of a liposome ranges from some 20 nm up to
several
micrometers.
Phospholipid bilayer

Aqueous phase
  ADVANTAGES
1. Biocompatible & Biodegradable

2. Entrap hydrophilic & hydrophobic pharmaceutical agents.

3. Sustained release depot (Propranolol, cyclosporin )

4. Increased efficacy & therapeutic index of drugs ( Actinomycin D)

5. Can be administered through various routes.

6. Lack of antigenic properties

7. Site avoidance effect (avoids non-target tissues).

8. Flexibility to couple with site-specific ligands to achieve active targeting

5
 DISADVANTAGES

 Less stability

 Low solubility

 Problem of targeting to various tissues due to their large size

 Short half life

 Leakage of encapsulated drug

 High cost of production

 Allergic reactions may occur due to liposomal constituents

6
COMPOsiTiOn
OF
liPOsOMe
 PHOsPHOliPiDs
❖Phospholipids are the basic molecular building block
of the liposome.
❖Phospholipids is a lipid which is Amphiphatic which
consist of
- 1 hydrophilic polar head HYDROPHILIC HEAD
- 2 hydrophobic tails (POLAR)

hence have affinity for both aqueous and

polar moiety i.e. both hydrophilic drugs
can be encapsulated in the aqueous
phase and hydrophobic drug molecules
can be incorporated in the lipid bilayers
❖Most commonly used phospholipid is
-- Phosphotidylcholine or PC
HYDROPHOBIC TAILS
-- phosphatidylethanolamine (NON-POLAR)
sTrUCTUre OF liPOsOMe
PHOSPHOLIPIDS

HYDROPHOBIC REGION
(LIPOPHILIC DRUG
MOLECULES)

AQUEOUS PHASE
(HYDROPHILIC DRUG
MOLECULES)

FATTY ACID CHAINS

NOTE: The temperature at which lipid membrane
undergoes phase transition is called as phase transition
temperature and it is determined by microcalorimetry
Enhances the stability of the membrane
Enhances the rigidity of the phospholipid
b i l ay er
Reduces the permeability of water soluble
sub st ance
Cross-section of liposomes:

Polar Lipids
(Phospholipid)

Lipid Soluble ingredients

(Drugs, Nutrients & vitamins)
H2O Layer Water Soluble
ingredients
(Drugs, Nutrients 5
& vitamins)
A. General representation of
phospholipids:

7
 Phospholipids

Phosphatidylcholine- natural
Amphipathic molecule
Hydrophilic polar head-
Phosphoric acid bound to water
soluble molecule.
Glyceryl bridge
Hydrophobic tail-
2 fatty acid chain containing 10-24 carbon
atoms and 0-6 double bond in each chain.

8
The most common natural phospholipid is the
phospatidylcholine (PC ).
Polar Head Groups
Naturally occurring phospholipids used are :
PC: Phosphatidylcholine.
PE: Phosphatidylethanolamine.
Three carbon glycerol
PS: Phosphatidylserine
Synthetic phospholipids used are:
DOPC: Dioleoyl phosphatidylcholine
DSPC: Disteroyl phosphatidylcholine
DOPE: Dioleoyl phosphatidylethanolamine
DSPE: Distearoyl phosphatidylethanolamine
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MeCHanisM OF FOrMaTiOn
OF liPOsOMe
 MeCHanisM OF FOrMaTiOn OF liPOsOMe
In aqueous media phospholipids as they are not soluble
align themselves closely in planar bilayer sheets or
lipid cakes which is thermodynamically stable.
In which polar head groups face outwards into the
aqueous medium, and the lipidic chains turns inwards
to avoid the water phase, giving rise to double layer or
bilayer

❖ This structure is also called as laMella.

• For the liposomes to be formed, upon further
hydration, the lipid cakes(lamella) swells
eventually they curve to form a closed
vesicles in the form of spheres
• This spheres are called as liposomes.
 BIOLOGICAL FATE OF LIPOSOMES
Liposomes in blood stream taken up by reticulo- endothelial
system.
The macrophages engulf the liposomes which are taken by the
reticulo-endothelial system (endocytosis).
Then phagosome and lysosome combine and form as a
phagolysosome.
The membrane of phagolysosome have proton pumps which
decrease the pH of phagolysosome and the enzyme
phospholipase destruct the liposomal membrane and releases
the drug from destructed liposome.
 CLASSIFICATION
Liposomes can be classified based three different
criteria

Based on structural parameters

Based on method of preparation
Based on composition and applications
 MeThOdS
OF
prepArATION
These methods involve the loading of the entrapped
agents before or during the manufacturing procedure
(Passive loading).
Active:certain type of compounds with ionizable
groups, and those which display both lipid and water
solubility, can be introduced into the liposomes after
the formation of intact vesicles (remote loading).
 MECHANICAL DISPERSION:
Being extremely simple, it is the most widely used method for preparing MLVs by
dispersion method.

Lipid film hydration by hand shaking/non hand shaking:

(i)Initially different phospholipids and charge components are
added to a (2:1 v/v) solvent mixture containing
chloroform:methanol.
(ii)transferred to a round bottom flask RBF, which is attached to a
rotary evaporator and rotated at 60rpm.
(iii)evaporation of org. solvent at 3 ͦc or above the transition temp
of the phospholipid results in the formation of thin film.
(iv)Even after the appearance of the dry film, the rotation of the
flask is continued for another 15 minutes.
(v)The evaporator is then removed from the vaccum sourse and
filled with nitrogen gas. The pressure inside the evaporator is
increased such that the pressure inside and outside the RBF
becomes equal.
(vi)The RBF is then transferred to a lyophilizer to remove any
organic solvent.
(iv)The next step is the dispersion or hydration of the
film. Nitrogen gas is introduced in the RBF and 5 ml
of saline phosphate buffer solution containing the
material to be encapsulated is added.
(v)The RBF is again attached to the evaporator and
rotated (60 rpm) or below.
(vi)The flask is continued to rotate until the lipid adhering
to the walls of the flask get removed.
(vii)A homogenous milky-white, practical-free suspension
obtained is allowed to stand for about 2 hrs at room
temperature or above the Tc of the lipid. Complete
hydration or swelling gives rise to MLVs.
 prOLIpOSOMeS
To increase the surface area of dried lipid film and to
facilitate continuous hydration and lipid is dried over the
finally divided particulate support i.e.- NaCl, Sorbitol, or
other polysaccharides.
These dried lipid coated particulates are called as
proliposomes
Proliposomes form dispersion of MLVs on addition of
water, where support is rapidly dissolved and lipid film
hydrate to form MLVs
Methods overcome the stability problem and
entrapment efficiency
b) Freeze-drying
❖MLVs can also be prepared by freeze drying.
the most commonly employed organic solvent
for this technique is t-butanol as its freezing
point exceeds the operating temperature of
the freeze dryer.
❖Also it does not attack the rubber seals in the
lyophilizer.
❖Phospholipids are dissolved in t-butanol and the
solution is freeze dried to obtain the dried lipid
in a foam like structure. Water or saline is added
to the dry lipid with thorough mixing to obtain
MLVs..
Sonicated SUVs

The small unilamellar vesicles (SUVs) are produced from

MLVs by exposing the MLVs to ultrasonic irradiation.

The production can be achieved by the following two

sonicators.
(i)Bath type sonicator
(ii)Probe sonicator
The production can be achieved by the
following two sonicators.
(i)Bath type sonicator

This sonicator can be used for

processing large volumes of
diluted lipids. An inert
atmosphere in the sonicator
is maintain by means of
nitrogen or oxygen.
(ii) Probe sonicator
It is used for rapid processing of small volumes
lipids with high energy it also utilizes nitrogen
gas and ice bath.
Demerit
there are chances of communication of the
product from the tip of the titanium probe.

Sonication
MLVs hazy transparent
5-10 min solution

centrifugation 30 min

clear SUV
Dispersion.
(b) French pressure cell
it is used for SUVs by passing MLVs through a
narrow orifice under high pressure.
The french pressure cell is constructed from stainless steel and is
capable of withstanding very high pressures, even up to 20,000 -
40,000 psi. the body of the cell contains
• a pressure chamber
• an outlet,
• a piston
• bottom seal
• valve closure
both the piston and the bottom seal contain and O-ring each,
which enables in tight sealing the
pressure cell.
working
(i) Initially the liposome suspension is added to the pressure cell and piston is
pushed into the body.
Then the entire cell is turned upside down i.e., by an angle of 180 ͦ.

(ii)The liquid sample is then filled in the entire cavity till the outlet.

(iii)After filling, the bottom seal is pressed down and the pressure cell is
closed.

(iv)The cell is brought back to upright position and the pressure is developed
in the cell using a hydraulic press.

(v)After sufficient pressure has been developed in the pressure cell, the valve
is opened very slowly and the product is allowed to exit in a drop-wise
manner.
 MICRO EMULSIFICATION LIPOSOMES(MEL)
❖ “Micro Fluidizer” is used to prepare small MLVs from
Concentrated lipid dispersion
❖ The lipids can 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(10,000psi,
600-700 bar) through a 5um orifice.
❖ 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, thereby affecting an efficient transfer of energy.
❖ The fluid collected of be recycled through the pump and
interaction chamber until vesicles of the spherical dimension are
obtained.
❖ After a single pass, the size of vesicles is reduced to a size 0.1
and 0.2um in diameter.
MICRO-EMULSIFICATION:
 VESICLES PREPARED BY EXTRUSION
TECHNIQUES (VETS)
It is used to process LUVs as well as MLVs.

Liposomes prepared by this tech. are called as membrane filter

extrusion liposomes

The 30% capture volume can be obtained using high lipid conc.
The trapped volume in this process is
1-2 litre /mole of lipids

❖It is due to their ease of production, readily selectable vesicle

diameter, batch to batch reproducibility & freedom from solvent or
surfactant contamination is possible
 FREEZE THAW SONICATION METHOD (FTS)

The method is based on freezing of a unilamellar dispersion

& then thawing at room temp for 15 min. Thus the process
ruptures & refuses SUVs during which the solute equilibrates
between inside & outside & liposomes themselves fuse &
increase in size. Entrapment volume can be upto 30% of the
total vol. of dispersion.

❖Sucrose, divalent metal ions & high ionic strength salt

solutions can not be entrapped efficiently
DRIED RECONSTITUTED VESICLES AND
FREEZE THAW SONICATION:
SOLVENT DISPERSION METHODS
SOLVENT DISPERSION:
Ethanol and Ether injection:
De-Emulsification method:

Generally the liposome is made up in 2 steps: Aqueous medium

containing material
1 st the inner leaflet of the bilayer . to be entrapped
Then the outer half.
Add to immiscible
organic solution of
lipid

Mechanical agitation

Microscopic water
droplets
Methods to prepare the droplets:
~Double emulsion vesicles
~Reverse phase evaporation vesicles 38
~Sonication methods
 DOUBLE EMULSION VESICLES:
➢ The organic solution, which already contains water droplets is
introduced in to excess aqueous medium followed by mechanical
dispersion.

➢ The W/O/W double emulsion is formed.

➢ Two aqueous compartments being separated from each other by a

phospholipid monolayer.

➢ Removal of solvent results in an intermediated sized unilamellar

vesicles and entrapment is up to 90%.
REVERSE PHASE EVAPORATION VESICLES:
 DETERGENT SOLUBILISATIOIN METHODS
Phospholipid brought into intimate contact with
aqueous phase

By addition optimized concentration of detergent

Formation of micelles (Liposome)

Below CMC, detergent molecules exist in free solution.

As the concentration is increased, micelles are formed.
Note:- Liposome size and shape
Methods to remove detergents:
depend on chemical nature of
Dialysis
detergent, concentration and other
Column chromatography
lipid involved
Adsorption using bio-beads
DETERGENT DEPLETION(REMOVAL) METHODS:

➢ Detergents associate with the phospholipid molecules and serve to

screen the hydrophobic portions of of molecule from water.
➢ The structures formed as a result of this association is known as
micelles.
➢ A three stage model of interaction for detergents with lipid
bilayers:
Stage1: At low concentration detergents equilibrates between
vesicular lipid and water phase.
stage2: After reaching a critical detergent concentration, membrane
structure tends to unstable and transforms gradually in to micelles.
stage3: All lipid exists in mixed micelle form.
➢ Three methods are applied for removal of detergent and transition
of mixed micelles to concentric bilayered form.
DIALYSIS:
The molecules of detergent are removed from mixed micelle by
dialysis by lowering the concentration of detergent in bulk
aqueous phase.

eg: sodium cholate. octylglucoside.

COLUMN CHROMATOGRAPHY:
Removal of detergent is achieved by by passing the dispersion over
a sephadexg-25 column
pre-saturated with constitutive lipids and pre-equilibrated with
hydrating buffer.
eg: deoxycholate.
 Active/remote loading technique:
The lipid bilayer membrane is impermeable to ions & hydrophilic molecules.
But, Permeation of hydrophobic molecules can be controlled by concentration
gradients.
Some weak acids or bases can be transported due to various transmembrane
gradients
Electrical gradients.
Ionic(pH) gradients.
Chemical potential gradients.
APPLICATION OF LIPOSOMES
Liposomes as drug/protein delivery vehicles
Controlled and sustained drug release in situ.
Enhanced drug solubilization
Altered pharmacokinetics and biodistribution
Enzyme replacement therapy and lysosomal storage disorders

Liposomes in antimicrobial, antifungal and antiviral

therapy
Liposomal drugs
Liposomal biological response modifiers

Liposomes in tumour therapy

Carrier of small cytotoxic molecules
Vehicle for macomolecules as cytokines o genes
Liposomes in gene delivery
Gene and antisense therapy
Genetic vaccination

Liposomes in immunology
Immonoadjuvant
Immunomodulator
Immunodiagnosis

Liposomes as artificial blood surrogates

Liposomes as radio phamaceutical and radio diagnostic
cariers
Liposomes in cosmetics an dermatology
Liposomes in enzyme immobilization and bioreactor
technology
Characterization of liposomes:
PHYSICAL CHARACTERISATION CHEMICA L CHARACTERISATION

→ Vesicles size/shape/morphology → Phospholipids /lipid concentration

→ Surface -charge/electrical potential → Drug concentration

→ Phase behaviour/ lamellarity → PH / Osmomolality

→ Drug release →Antioxidant degradation

→ % capture /free drug → Phospholipids / cholesterols –

peroxidation/oxidation/hydrolysis
BIOLOGICAL CHARACTERISATION
→ Sterility
→ Pyrogenisity
→ Animal toxicity
→Plasma Stability:
Characterization parameters Analytical method/Instrument
1. Vesicle shape and surface morphology Transmission electron microscopy, Freeze-
fracture electron microscopy
2.Mean vesicle size and size distribution Photon correlation spectroscopy, laser light
(submicron and micron range) scattering, gel permeation and gel exclusion

3. Surface charge Free-flow electrophoresis

4. Electrical surface potential and surface pH Zetapotential measurements

5. Lamellarity Small angle X-ray scattering, 31 P-NMR, Freeze-

fracture electron microscopy
6. Phase behavior Freeze-fracture electron microscopy, Differential scanning
calorimetery
7. Percent of free drug/ percent capture Minicolumn centrifugation, ion-exchange
chromatography, radio labelling
8. Drug release Diffusion cell/ dialysis
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Characterization parameters Analytical method/Instrument

1. Phospholipid concentration Barlett assay, stewart assay, HPLC

2. Cholesterol concentration Cholesterol oxidase assay and HPLC

3. Phopholipid peroxidation UV absorbance

4. Phospholipid hydrolysis, HPLC and TLC

Cholesterol auto-oxidation.

5. Osmolarity Osmomete

46
 Characterization parameters Analytical method/Instrument

1. Sterility Aerobic or anaerobic cultures

2. Pyrogenicity Limulus Amebocyte Lysate (LAL) test

3. Animal toxicity Monitoring survival rates, histology and

pathology

STABILITY OF LIPOSOMES:
Stability invitro .
~ Lipid oxidation
~ Lipid peroxidation
~ Long term & accelerated stability
Stability after systemic administration.
47
Encapsulation of drugs in liposomes:
• Encapsulation volume/Trapped volume
Volume of aqueous solution entrapped in liposomes per mole of PL (µL/µmol PL)
• Encapsulation Efficiency
Assessed by mini column centrifugation method & protamine aggregation method.
protamine aggregation method used for neutral and negatively charged liposomes.
Liposome dispersion can be precipitated with protamine solution and subsequent
centrifugation at 2000RPM.
By analyzing the material in supernatant & in liposome pellet ( after disrupting
liposomal pellet with 0.6 ml of 10% triton x-100 ). The encapsulation efficiency of
entrapped material can be estimated.
• % Encapsulation

Drug entrapped in liposomes

x 100
Total drug added
4. Evaluation of liposomes
the liposomes prepared by various techniques are to
be evaluated for their physical, chemical as well as
biological properties, has these influence the behavior of
liposomes in vivo.
Physical properties
1. Particle size
Both particle size and particle size distribution of
liposomes influence their physical stability. These can be
determined by the following method.
a) Laser light scattering
b) Transmission electron microscopy

2. Surface charge
The passive, negative or natural charge on the surface of the
liposomes is due to the composition of the head groups.
The surface charge of liposomes governs the
kinetic and extent of distribution in vivo, as well
as interaction with the target cells.
The method involved in the measurement of
surface charge is based on free-flow
electrophoresis of MLVs.
❖It utilizes a cellulose acetate plate dipped in
sodium borate buffer of pH 8.8.
❖About 5n moles of lipid samples are applied
on to the plate, which is then subjected to
electrophoresis at 4ͦc for 30 mins.
The liposomes get bifurcated depending on their
surface charge.
This technique can be used for determining the heterogeneity of charges in the
liposome suspension as well as to detect any impurities such as fatty acids

3. Percent drug encapsulated.

➢ Quantity of drug entrapped in the liposomes helps to estimate the behavior
of the drug in biological system
➢ Liposomes are misture of encapsulated and unencapsulated drug fractions
➢ the % of drug encapsulation is done by first separating the free drug fraction
from encapsulated drug fraction
➢ The encapsulated fraction is then made to leak off the liposome into
aqueous solution using suitable detergents
➢ The methods used to separate the free drug from the sample are:
a. Mini column centrifugation method
b. Protamine aggregated method

4. Phase behavior
at transition temperature liposomes undergo reversible phase transition
➢ The tc is the indication of stability permeability and also indicates the
region of drug entrapment
➢ Done by DSC
5. Drug Release Rate
The rate of drug release from the liposomes can be
determined by in vivo assays which helps to predict the
pharmacokinetics and bioavailability of the drug. However in
vivo studies are found to be more complete.
Liposome encapsulating the tracer [ᵌH] insulin are employed
for the study. This [ᵌH] insulin is preferred, as it is released
only in the ECF and undergoes rapid renal excretion of the
face tracer coupled to the degradation rate constant o the
tracer released from the liposomes.
Chemical properties
1. Determination of phospholipids
The phospholipid content of liposomes can be
determined directly by two assays, bartlett assay and
steward assay.
(a)Bartlett assay
This method of determining the phospholipid is very
sensitive and may produce erroneous results in the
presence of even trace amounts of inorganic
phosphate. Therefore, borosilicate glass tubes and
double-distilled water is used.
(i)initially the phosphorous present in the lipid
bilayer of the sample is hydrolyzed to inorganic
phosphate.
(ii)Then ammonium molybdate is added to convert
inorganic phosphate to phosphomolybdic
acid(PMA).
(iii)the sample is then treated with
aminonaphthylsulphonic acid to quantitatively
reduce the PMA to a blue-coloured compound.
(iv)the intensity of the blue colour produced can be
measured by spectrophotometric means and the
value is plotted on the standard curve to obtain the
content of phospholipids.
(b) Steward assay
This assay overcomes the drawbacks of bratlett
assay, but cannot be used to mixture of unknown
(i)A standard curve is prepared by treating known
concentration of phospholipids in chloroform with 0.1 M
solution of ammonium ferrothiocyanate9reagent).
(ii)The sample are also treated with the same reagent and the
optical density is determined at 485 nm.
(iii)The absorbance of the sample can be plotted on the
standard curve to obtain the concentration of
phospholipids.
2. Cholesterol analysis
(a)Qualitative analysis
Performed using a capillary column filled with fused silica.
(b)Quantitative analysis
The sample is reacted with a reagent (containing ferric
perchlorate, ethyl acetate and H₂SO₄) and the absorbance of
purple coloured complex is measured at 610 nm.
 MODES OF LIPOSOME AND CELL INTERACTION:

Adsorption Endocytosis

Fusion Lipid transfer

 APPLICATIONS
➢ Liposomes as drug or protein delivery vehicles.
➢ Liposome in antimicrobial, antifungal(lung therapeutics) and
antiviral (anti HIV) therapy.
➢ In tumour therapy.
➢ In gene therapy.
➢ In immunology.
➢ Liposomes as artificial blood surrogates.
➢ Liposomes as radiopharmaceutical and radiodiagnostic
carriers.
➢ Liposomes in cosmetics and dermatology.
 COMMERCIAL PRODUCTS
➢Doxorubicin(DOXIL).
➢Daunorubicin(DAUNOXOME).
➢AmphotericinB(APHOTEC, AMBISOME,
ABELCET).
➢Cytarabine(DEPOCYTE).
 CONCLUSION
Liposome over the years have been
investigated as major drug delivery system.

The use of liposomes in delivery of drugs and

genes to tumour site are promising and may
serve as a handle for focus of future research.