3. LIPOSOMES:
Liposomes are cocentric bilayered
vesicles in which an aqueous
volume is entirely enclosed by
a membranous lipid bilayer mainly
composed of natural or
synthetic phospholipids.
3
4. • Liposomes were discovered about 40 years ago by Alec
Bingham.
• Liposomes can be produced from cholesterols, non toxic
surfactants,sphingolipids,glycolipids,long chain fatty acids &
even membrane proteins.
• Liposomes are the drug carrier loaded with great variety of
molecules such as small drug molecules, proteins,
nucleotides & even plasmids.
• Considerable progress was made during 1970s and 1980s in
the field of liposome stability leading to long circulation
times of liposomes
4
7. • Phospholipids are major structural components of
biological membranes in human body, where 2 types of
phospholipids exist i.e. phosphodiglycerides &
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 EFA which are hydrophobic.
• Most commonly used phospholipids – PC an amphipathic
molecule with a hydrophilic polar head group,
phosphocholine . PC, also known as “lecithin”, can be
derived from natural and synthetic sources.
7
8. The lipid bi-layer used in the liposomes are usually made of
phospholipids and cholesterol.
Following are the
A) Naturally occurring phospholipids used in liposomes are:
• Phosphatidylcholine (PC),
• Phosphatidylethanolamine (PE),
• Phosphatidylserine (PS).
B) Synthetic phospholipids used in the liposomes are:
• Dioleoyl phosphatidylcholine (DOPC),
• Distearoyl phosphatidylcholine (DSPC),
• Dioleoyl phosphatidylethanolamine (DOPE),
• Distearoyl phosphatidylethanolamine (DSPE).
8
9. CLASSIFICATION:
VESICLE TYPE ABBREVIATI
ON
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
9
10. FORMATION OF LIPOSOME:
When phospholipids are
dispersed in water, they
spontaneously form closed
structure with internal aqueous
environment bounded by
phospholipid bilayer membranes,
this vesicular system is called as
liposome.
10
15. 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.
15
16. II) FRENCH PRESSURE CELLS(ULV/OLV):
• Method developed by Barenholtz & Hamilton et al.
• Very useful method in which extrusion of preformed large
liposomes in a French Pressure under very high pressure is
carried out .
• This technique yields ULV’s/OLV’s of intermediate size(30-
80nm/depending upon applied pressure).
• Liposomes are more stable.
• Free from structural defects.
• Leakage problem is also less.
• However it has high production cost. 16
17. III) FREEZE THAW SONICATION(FTS):
Freeze SUV dispersion
thaw at room temperature for 15 minutes
sonicate
rupture of SUV’s occur
Formation of liposomes
17
18. 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.
18
19. 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. 19
20. 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.
20
21. CHARACTERIZATION:
PARTICLE SIZE ANALYSIS-
• Laser light scattering, transmission electron microscopy
determines the particle size & its distribution.
SURFACE CHARGE-
• Free-flow electrophoresis on a cellulose acetate plate in a sodium
borate buffer pH 8.8 & a zeta potential measurement.
• 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.
21
22. 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-Freeze-
fracture electron microscopy & freeze-etch electron
microscopy & P-NMR method.
22
23. 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.
7.Iron chelater is used to prevent initiation of free radical
chain reaction.
23
24. 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 to allow differences in
packing density between opposite sides of the bilayers to
equalize by trans membrane flip-flop .
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.
24
25. APPLICATIONS:
1.The therapeutic value of liposomes as drug carriers,
particularly for anticancer, antifungal, and antibacterial
agents.
2.As anticancer , cytotoxic drugs like Cytarabine, alkylating
agents .
3.As vaccine adjuvants i.e. when administered by IM route,
they slowly release the antigens and accumulate in lymph
nodes.
4.In ophthalmic drug delivery systems,Idoxuridine used in
acute & chronic keratitis . 25
26. 5. Sustained release system of systemically or locally
administered liposomes. Ex biological proteins or peptides
such as vasopressin.
6. Site specific targeting: in certain cases liposomes with
surface attached ligands can bind to target cells (‘key and
lock’ mechanism). Ex antineos, anti infectors &
antiinflammatory drugs.
7. Improved transfer of hydrophilic, charged molecules like
chelators , antibiotics, plasmids & genes into the cells.
26
27. RECENT ADVANCES & ON GOING
CLINICAL TRIALS:
Antigens as Liposomal Preparation Applications:
• Diphtheria toxoid = Superior immunoadjuvant
• Herpes simplex virus = Enhanced Ab level
• Hepatitis B virus = Higher Ab response
• Bacterial polysaccharides = Superior immunoadjuvants
Tetanus toxoids = Increased Ab titre
• Influenza subunit antigen = Intranasal, protects animal
from virus
• Carbohydrate antigen = Increased Ab titre in salivary
gland
27
