Presentation on liposomes

1. A GRADED SEMINAR ON
Presented By- Guided By-
Atish Khilari Dr.(Mrs).Shilpa Chaudhari
M.Ph (2nd sem.)
Pharmaceutics
D.Y
Dr. D. Y. Patil College of Pharmacy,Akurdi,pune-44

2. CONTENTS
Introduction
Composition of liposome
Advantages
Disadvantages
Classification
Methods of preparation
Stability
Characterization / Evaluation
Applications and marketed preparations
References.
2

3. INTRODUCTION
3

4. What is
a
liposome
4

5. INTRODUCTION
 DEFINITION :
Liposomes are concentric bilayered vesicles in which an aqueous volume
is entirely enclosed by a membranous lipid bilayer mainly composed of
natural or synthetic phospholipids.
 Liposome were first produced in England in 1961 by Alec D. Bangham
 The size of a liposome ranges from 20 nm up to several micrometers.
Phospholipid bilayer
Aqueous phase
5
Fig.1-Structure of liposomes

6. Composition
of
LIPOSOME
6

7. COMPOSITION OF LIPOSOMES
• The main components of liposomes are :-
1. Phospholipids
2. Cholesterol
7

8. PHOSPHOLIPIDS
• Phospholipids are the major structural components
of biological membranes such as the cell membrane.
Two types of Phospholipids
(along with their hydrolysis
products)
Phosphoglycerides
Sphingolipids
8

9. PHOSPHOLIPIDS
 Phospholipids are major structural
components of biological membranes in
human body, where 2 types of
phospholipids exist i.e.
phosphoglycerides & sphingolipids.
 Each phospholipid molecule has 3
major parts, 1 head & 2 tails. Head is
made from 3 molecular components:
choline , phosphate & glycerol which is
hydrophilic. Each tail with a long chain
which are hydrophobic.
9
Fig.2- phospholipids

10. COMMONLY USED PHOSPHOLIPIDS
COMMONLY
USED
PHOSPHOLIPIDS
NATURAL
PHOSPHATIDYL
CHOLINE
PHOSPHATIDYL
ETHANOLAMINE
PHOSPHATIDYL
SERINE
SYNTHETIC
DIOLEOYL
PHOSPHATIDYL
CHOLINE
DISTEAROYL
PHOSPHOTIDYL
CHOLINE
DIOLEOYL
PHOSPHATIDYL
ETHANOLAMINE
10

11. CHOLESTEROL
• Cholesterol stabilizes the membrane.
• It plays important role in bilayer formation.
• Cholesterol by itself does not form bilayer structure.
• Cholesterol act as fluidity buffer.
• Enhances the stability of the membrane.
• Enhances the rigidity of the phospholipid bilayer.
• Reduces the permeability of water soluble substance through the
membrane. 11

12. ADVANTAGES
 Non-toxic.
 Biodegradable.
 Increased stability of encapsulated drugs.
 Lowers systemic toxicity.
 Site avoidance effect (avoids non-target tissues).
 Protection of sensitive drug molecules.
 Improved pharmacokinetic effects. 12

13. DISADVANTAGES
• Leakage of encapsulated drug during storage.
• Short half-life.
• Batch to batch variation.
• Difficult in large scale manufacturing and sterilization.
• Production cost is high.
• Once administered, liposomes can not be removed.
• Sometimes phospholipids undergoes hydrolysis and oxidation
reactions.
13

14. CLASSIFICATION:
VESICLE TYPE ABBREVIATION DIAMETER SIZE NO. OF LIPID BI -
LAYER
Unilamellar vesicle UV All size range ONE
Small unilamellar vesicle SUV 20-100 nm ONE
Medium unilamellar vesicle MUV >100µm ONE
Large unilamellar vesicle LUV >1000µm ONE
Giant unilamellar vesicle GUV >1µm ONE
Oligolamellar vesicle OLV 0.1-1µm 5
Multilamellar vesicle MLV >0.5µm 5-25
Multivesicular vesicle MV >1µm Multicompartmental
structure 14

15. PREPARATION OF LIPOSOMES:
Passive loading Active loading
( Remote loading )
1.Mechanical dispersion method
2.Solvent dispersion method
3.Detergent removal method
15

16. MECHANICAL DISPERSION METHODS:
1.Hand-shaking method
2.Sonication
3.French pressure cell
4.Freeze-thaw technique
SOLVENT DISPERSION METHODS:
1.Ethanol injection
2.Ether injection
3.Reverse phase evaporation vesicles
DETERGENT REMOVAL METHODS:
1.Dialysis
2.Gel chromatography dilution
16

17. MECHANICAL DISPERSION METHOD:
Hand-shaking method:
1
• This is one of the simplest method for preparation of liposomes.
• The surfacant /cholesterol mixture is dissolves in diethyl ether in
RBF.
2
• Organic solvent is then removed at room temp. under reduced
pressure.
3
• After releasing vaccum, the flask is flushed with nitrogen then
flask is attached to rotary evaporator rotated at room
temprature at 60 rpm.
4
• The dried surfactant film is hydrated with an aqueous phase at
50°c to 60°c during gentle agitation.
5 • Large multilamellar vesicles are formed.
17

18. Fig.3- Hand shaking method.
18

19. MECHANICAL DISPERSION METHOD:
Sonication: 2 TYPES:
1.BATH SONICATOR 2.PROBE SONICATOR
19

20. BATH SONICATOR PROBE SONICATOR
1.Large volume of diluted lipids
are processed.
1.Small volume of diluted lipids
are processed.
2.Less or no contamination. 2.Chances of contamination.
• At high energy levels, average size of vesicles is further reduced.
• Exposure of MLV’s to ultrasonic irradiations is the most widely
used method for producing small vesicles.
• As chances of contamination are likely to occur in probe sonicator,
bath sonicator is widely used.
20

21. FRENCH PRESSURE CELL
• Construction:
• 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, etc. both the piston and
the bottom seal contain an
O-ring each, which enables
in tight sealing the pressure
cell. 21Fig.4- French pressure cell.

22. 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.
22

23. FREEZE THAW SONICATION(FTS):
Freeze SUV dispersion
Thaw at room temperature for 15 minutes
Sonicate
Rupture of SUV’s occur
Formation of liposomes.
23

24. SOLVENT DISPERSION METHODS
I) ETHANOL INJECTION METHOD:
Lipids ethanol
Rapidly inject through a fine needle
Saline buffer containing materials to be entrapped
dissolution of ethanol
Formation of SUV’s.
24Fig.5- Ethanol injection method.

25. II) ETHER INJECTION METHOD:
Lipid ether
Slowly injecting through a narrow needle
Vapourize temperature at 60˚C
Production of SUV’s.
• Less risk of oxidation as ether is free of peroxides.
• Low efficiency.
• Long time needed for production. 25

26. REVERSE PHASE EVAPORATION VESICLES
Lipid organic solvent aqueous solution
Mix
Sonicate
Formation of w/o emulsion
Evaporate to remove the organic solvent
Lipids form a phospholipid bilayer
Vigorous shaking
Water droplets collapse
Formation of LUV’s.
26

27. STABILITY OF LIPOSOMES:
A. PREVENTION OF CHEMICAL DEGRADATION:
1. Start with freshly purified lipids & freshly distilled solvents.
2. Avoid procedure which involving high temperature.
3. Carry out manufacturing in the absence of oxygen.
4. Deoxygenate aqueous solution with nitrogen.
5. Store liposome suspension in an inert atmosphere.
6. Include an antioxidant as a component.
27

28. STABILITY OF LIPOSOMES:
B. PREVENTION OF PHYSICAL DEGRADATION:
1.‘ANNEALING’ is best method to control physical
degradation i.e incubating the liposomes at a temperature high
enough above the phase transition temperature.
2. The stability of liposomes may also be increased by cross
linking membrane component covalently using Gluteraldehyde
fixation, osmification or polymerization of alkyne containing
phospholipids.
These methods increases mechanical strength of the membrane
& render them less susceptible to disruption.
28

29. CHARACTERIZATION:
Size & its distribution:-
• Electron microscopy is most specific method to determine size
of liposome since it allows us to view individual liposome &
to obtain exact information about profile of liposome
population over the whole range of size. laser light scattering
method is very simple.
Surface charge-
• Free-flow electrophoresis on a cellulose acetate plate in a
sodium borate buffer pH 8.8
• The samples are applied to plate & electrophoresis is carried
out at 4˚C for 30 min.
• The plate is dried and phospholipids are visualised by the
molybdenum blue reagent.
• The liposomes get bifurcated based on the surface charge. 29

30. Percent drug entrapment-
• This can be determined by ‘PROTAMINE AGGREGATE’
& ‘MINICOLUMN CENTRIFUGATION’ method.
Expressed as % entrapment/mg lipid.
Phase behaviour-
• Liposomes at transition temperature undergo reversible phase
transition i.e the polar head groups in gel state become
disordered to form the liquid crystalline state which can be
determined by DSC.
Lamellarity-
• The average no. of bilayers present in liposomes can be find
out by freeze electron microscopy & 31 p-NMR. Now-a-days
freeze fracturing electron microscopy has become a very
popular method to study structural details of aqueous lipid
dispersion. 30

31. APPLICATIONS
• Liposomes as drug or protein delivery vehicles.
• Liposome in antimicrobial, antifungal(lung therapeutics) and antiviral (anti
HIV) therapy.
• In tumor therapy.
• In gene therapy.
• In immunology.
• Liposomes as artificial blood surrogates.
• Liposomes as radio pharmaceutical and radio diagnostic carriers.
• Liposomes in cosmetics and dermatology.
31

32. MARKETED PREPERATIONS:
• Liposome ( Doxil ™) Doxorubicin = Kaposi’s sarcoma
• Liposome (EVACT ™) = breast cancer
• Liposome(DaunoXome™) Daunosome = Advanced
Kaposi’ sarcoma,small cell lung cancer, leukaemia &
solid tumour .
• Liposome ( VincaXome ™) Vincristine = Solid tumour
32

33. Baviskar D.T. and Jain D.K., Novel drug delivery systems,
Nirali Prakashan, pg.no.- 14.15-14.27
Y. Sultana., Liposomal Drug Delivery Systems: An Update
Review., Current Drug Delivery 2007, 4, 297-305.
Sharma Shailesh, Sharma Neelam, Kumar Sandeep, Gupta
GD., Liposomes: A review., Journal of Pharmacy Research
2009, 2(7),1163-1167.
REFERENCES:
33

34. 34