Liposomes

1. TO MY
seMinar
PresenTer
MOHaMMeD FaHiM UDDin
M .PHarMaCY
(DeParTMenT OF PHarMaCeUTiCs)
UnDer THe gUiDanCe OF
Dr. YasMin BegUM
Malla reDDY COllege OF PHarMaCY

2. liPOsOMesliPOsOMes

3. COnTenTs
Introduction
Composition of liposome
Mechanism of liposome formation.
Classification.
Methods of preparation.
Characterization./ Evalution
Applications and Commercial products.

4. inTrODUCTiOn

5. WHaT is
a
liPOsOMe

6. 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.
Aqueous phase
Phospholipid bilayer

7. COMPOsiTiOn
OF
liPOsOMe

9. 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
- 2 hydrophobic tails
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
-- phosphatidylethanolamine
HYDROPHILIC HEAD
(POLAR)
HYDROPHOBIC TAILS
(NON-POLAR)

10. HYDROPHOBIC
REGION
(LIPOPHILIC DRUG MOLECULES)
sTrUCTUre OF liPOsOMe
PHOSPHOLIPIDS
AQUEOUS PHASE
(HYDROPHILIC DRUG MOLECULES)
FATTY ACID CHAINS

11. NOTE: The temperature at which lipid membrane
undergoes phase transition is called as phase transition
temperature and it is determined by microcalorimetry

12.  Enhances the stability of the membrane
 Enhances the rigidity of the phospholipid
bilayer
 Reduces the permeability of water soluble
substance
through the membrane

13. MeCHanisM OF FOrMaTiOn MeCHanisM OF FOrMaTiOn
OF liPOsOMeOF liPOsOMe

14. 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.
MeCHanisM OF FOrMaTiOn OF liPOsOMeMeCHanisM OF FOrMaTiOn OF liPOsOMe

15. •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.

17. MeCHanisM OF liPOsOMe FOrMaTiOn

18. ClassiFiCaTiOn

19. CLASSIFICATION
Liposomes can be classified based three different
criteria
Based on structural parameters
Based on method of preparation
Based on composition and applications

25. MeThOdS
OF
prepArATION

27. (a)Hand-shaken MLVs
Being extremely simple, it is the most widely used
method for preparing MLVs by dispersion method.
(i)Initially different phospholipids and charge
components are added to a (2:1 v/v) solvent
mixture containing chloroform:methanol.
(ii) The resulting mixture is then transferred to a round
bottom flask RBF, which is attached to a rotary
evaporator and rotated at 60rpm.
(iii) evaporation of organic solvent at 30 ͦc or above
the transition temperature (Tc) of the phospholipid
results in the formation of a thin film.

28. (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.
(vii) 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.

29. (viii) The RBF is again attached to the evaporator
and rotated (60 rpm) or below.
(ix) The flask is continued to rotate until the lipid
adhering to the walls of the flask get removed.
(x) 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 selling gives rise to
MLVs.

30. MECHANICAL DISPERSION:
Lipid film hydration by hand shaking/non hand
shaking:

31. 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 doesn’t matter when formation of
stable liposome.

32. 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..

34. 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

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

36. The arrangement o sonicator is as shown in the
figure this sonicator can be used for processing
large volumes of diluted lipids. an inert
atmosphere in the sonicator is the maintain by
means of nitrogen or organ.
merit
temperature of the product can be controlled as
the temperature of the ice bath can be
controlled.

37. (ii) Probe sonicator

38. 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.
(b) French pressure cell
it is used for SUVs by passing MLVs through a
narrow orifice under high pressure.

40. 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 etc.
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.

41. 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.

43. 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.

44. MICRO-EMULSIFICATION:

45. 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

47. 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

48. DRIED RECONSTITUTED VESICLES AND
FREEZE THAW SONICATION:

49. SOLVENT DISPERSION METHODS

51. SOLVENT DISPERSION:
Ethanol and Ether injection:

52. 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%.

53. REVERSE PHASE EVAPORATION VESICLES:

54. 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.

55. 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.

57. 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.

58. 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 4 ͦc for 30 mins.
The liposomes get bifurcated depending on their
surface charge.

59. 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

60. 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 [ H] insulin are
ᵌ
employed for the study. This [ H] insulin is preferred, as it is
ᵌ
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.

61. 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.

62. (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
phospholipids.

63. (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.

64. MODES OF LIPOSOME AND CELL INTERACTION:
Adsorption
Endocytosis
Fusion
Lipid transfer

65. 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.

66. COMMERCIAL PRODUCTS
Doxorubicin(DOXIL).
Daunorubicin(DAUNOXOME).
AmphotericinB(APHOTEC, AMBISOME,
ABELCET).
Cytarabine(DEPOCYTE).

67. 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.

68. ANY QUESTIONS??