This document provides an overview of liposomes. It begins with an introduction describing liposomes as concentric bilayer vesicles composed mainly of phospholipids and cholesterol. It then covers the mechanism of liposome formation, classifications, biological fate, preparation methods, characterization techniques, advantages and disadvantages, and applications. Preparation methods discussed include physical dispersion, solvent dispersion, detergent solubilization, and various size reduction/increase techniques. Characterization includes assessing size, shape, lamellarity, surface charge, drug release, and encapsulation efficiency using tools like microscopy, NMR, and chromatography.

1. Liposome
Presented by : Riteksha Patel

2. Contents
 Introduction
 Mechanism of liposome formation
 Classification
 Biological fate of liposome
 Methods of preparation
 Characterization
 Advantages & Disadvantages
 Applications
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3. Introduction
 Liposomes are simple
microscopic, concentric bilayered vesicles in
which an aqueous volume is entirely enclosed
by a membranous lipid bilayer mainly
composed of natural or synthetic
phospholipids.
 Discovered in 1960‟s by Bangham and
coworkers.
 The structural main components are
phospholipids and cholesterol
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4. Lets take a look at liposome
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5.  Phospholipids are amphipathic molecule i.e.
having affinity for both aqueous & polar
moieties, as they have a hydrophobic tail &
hydrophilic head.
 The tail portion consist of 2 fatty acid
chains having 10-24 carbon atoms & 0-6
double bonds in each chain.
 The head or polar portion consist of
phosphoric acid bound to a water soluble
molecule.
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6. Phospholipids
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7.  Cholesterol by itself do not form a bilayer
structure, it acts as fluidity buffer.
 That means below phase transition
temperature it makes the membrane less
ordered & slightly more permeable while
above phase transition temperature it
makes the membrane more ordered &
stable.
 It inserts into membrane with hydroxyl
group oriented towards aqueous surface &
aliphatic chain aligned parallel to acyl
chains in the centre of bilayer.
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8. Cholesterol alignment between phospholipid bilayer
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9. Mechanism of liposome formation
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10.  Vesicles are formed by hydrophobic effect.
 Ratio of hydrophilic & hydrophobic
moieties.
 CPP ( Critical packing parameter)
 If CPP value is less than 0.5 than liposomes
are formed by hydrophobic effect.
 If CPP value is more than 0.5 than
liposomes are formed by hydrophilic effect.
 If CPP value is between 0.5-1.0 than the
liposomes are formed by surfactant effect.
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11.  CPP = v/ lc Ap = Ahp / Ap
Where:
 v = hydrophobic group volume
 lc = hydrophobic group length
 Ap = cross sectional area of hydrophilic
head group
 Ahp = cross sectional area of hydrophobic
group.
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12. Classification
On the basis of structural parameters:
 Multilamellar vesicles (> 0.5 um) MLV
 Oligolamellar vesicles (0.1-1 um) OLV
 Unilamellar vesicles (all size range) UV
 Small unilamellar vesicles (20-100 nm) SUV
 Medium sized unilamellar vesicles MUV
 Large unilamellar vesicles (> 100 um) LUV
 Giant unilamellar vesicles (>1 um) GUV
 Multi vesicular vesicles (>1 um) MVV
On the basis of liposome preparation:
 Vesicles prepared by reverse phase evaporation method REV
 Multi lamellar vesicle by REV MLV-REV
 Stable plurilamellar vesicle SPLV
 Frozen & thawed MLV FATMLV
 Vesicles prepared by extrusion techniques VET
 Dried reconstituted vesicles DRV
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13. Different types of liposomes
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14. Biological fate of liposome
Membrane of phagolysosyme have proton pumps
which decrease PH of phagolysosyme & the
enzymes phospholipase destruct the liposomal
membrane
Macrophages engulf liposomes ( endocytosis)
Phagosome + lysosyme = phagolysosyme
Liposomes in blood stream
Taken by reticulo-endothelial system
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17. Method of liposome preparation
 Physical dispersion method:-
1. Hand shaking MLVs
2. Non-shaking LUVs
3. Freeze drying
4. Pro-liposomes
 To reduce liposome size:
1. Micro emulsification
2. Membrane extrusion
3. Ultrasonication
4. French pressure cell
 To increase liposome size:
1. Dried reconstituted vesicle
2. Freeze thawing
3. Induction of vesiculation by PH change
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18.  Solvent dispersion method :
1. Ethanol injection
2. Ether injection
3. Water organic phase:
A) Double emulsion method
B) Reverse phase evaporation
C) Stable plurilamellar vesicles
 Detergent solubilization :
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19. Hand shaken MLV‟s
Lipids + solvent ( chloroform: Methanol)
( In 250 ml RBF)
Evaporate for 15 min above phase transition temperature
(Flush with nitrogen)
Till residues dry
Add 5 ml buffer containing material to be entrapped
Rotate flask at room temp, at 60 RPM for 30 min until lipid
removes from wall of RBF
Milky white dispersion (stand for 2 hours to get MLV)
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20. Rotary Evaporator
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21. Non Shaking vesicles
Lipid + solvent
Evaporate at room temperature by flow of nitrogen for drying
Add water saturated nitrogen until opacity disappears
Add bulk fluid (drug) & 10-20 ml 0.2M sucrose solution to swell
(Flush again with nitrogen)
Stand for 2 hrs at 37º c, do not disturb for 2 hrs
(Swirl to yield milky dispersion )
Centrifuge at 12000 rpm for 10 min at room temp
(MLV on surface is removed)
To remaining fluid add iso-osmolar glucose solution
( centrifuge at 12000 rpm)
LUV is formed
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22. Pro liposome
Sorbitol / Nacl ( increase surface area of lipid film)
+ 5ml lipid solution ( fitted to evaporator )
(Evaporation)
Again add lipid solution
Dry the content using Lyophilizer ( freeze dryer)
(Stand over night at room temp)
Flushed with nitrogen for drying properly
MLVs
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23. Freeze Drying
Lipid + Solvent ( Tertiary butanol)
Freeze drying
Add Aqueous phase / Saline containing drug
Rapid mixing above phase transition temperature
MLVs
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24. Micro emulsification liposome (MEL)
 MEL is prepared by the “Micro fluidizer”, which pumps
fluid at very high pressure (10,000 psi) through a 5 um
orifice.
 Then, it is forced along defined micro channels, which
direct two streams of fluid to colloid together at right
angle at very high velocity.
 After a single pass, size reduced to a size 0.1& 0.2 um in
diameter.
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25. Microfluidizer
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26. Sonicated unilamellar vesicles
MLV in test tube
Sonicate for 5-10 min above phase transition temp
Filter & centrifuge at 100000 rpm for 30 min at 20º c
Decant top layer to get Sonicated unilamellar vesicles
BATH SONICATOR PROBE SONICATOR
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28. French Pressure Cell
 French pressure cell is invented by „Charles stacy French‟.
 In this technique the large vesicles are converted to small
vesicles under very high pressure.
 This technique yields uni or oligo lamellar liposomes of
intermediate size (30-80 nm in diameter depending on
applied pressure).
 This liposomes are more stable as compared to sonicated
liposomes.
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30. Membrane extrusion liposomes
 In this technique vesicle contents are exchanged
with dispersion medium during breaking &
resealing of phosphate lipid bilayer as they pass
through polycarbonate membrane.
 Less pressure is required here (> 100 psi), as
compare to French pressure cell.
 Use to process MLVs and LUVs.
 Two types of membrane one is Tortuous ( zigzag)
and another is Nucleation trach ( vertically
parallel).
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31. To increase size of liposome: Freeze thaw sonication
SUV in aqueous phase + Solute
Freeze drying
FTS method, thawing = melting
Sonication ( 15-30 sec)
Solutes in unilamellar vesicle
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32. Dried reconstituted vesicle
SUV in aqueous phase + Solute
Freeze drying
DRV method: Rehydration, film stacks dispersed in
aqueous phase
Solute in uni or oligo lamellar vesicles.
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33. PH induced vesiculation
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
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34. Solvent dispersion method: Ethanol injection
Lipid + ethanol solution in the syringe
Inject rapidly
In the aqueous phase
Small unilamellar vesicles
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35. Ether injection
Lipid + ether solution in the syringe
Inject slowly
In the aqueous phase ( On heated water bath, 60ºc)
Large unilamellar vesicles
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36. Water organic phase: Double emulsion
Organic solution + Lipid + Aqueous phase
Emulsion (W/O)
Hot aqueous solution of buffer
Multi compartment vesicle W/O/W (double emulsion)
LUVs
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38. Reverse phase evaporation: (MLV, LUV)
Emulsion
Evaporation under reduced pressure, rotary evaporator
Semi solid gel
Shake to get LUVs
“Lipid monolayer which enclosed the collapsed
vesicle, is contributed to adjacent intact vesicle to
form the outer leaflet of bilayer of LUV”.
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39. Stable plurilamellar vesicle (SPLVs)
 It involves preparation of water in organic phase
dispersion with an excess of lipid followed by drying
under continued bath sonication with stream of nitrogen.
 The internal SPLV is different from that of MLV-REVs, in
that they lack a large aqueous core.
 The internal environment of both the vesicle is different
from each other.
Detergent dispersion:
 Phospholipids & aqueous phase comes in contact with the
help of detergent
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40. Characterization of Liposome: Physical
 Vesicle shape & lamellarity ( No. of bilayers):
 Sample + 31p NMR + Mangnese (affect signal intensity)
 If intensity is decrease by 50% = unilamellar vesicle are
formed
 If intensity is decrease by more intensity = MLVs are formed
 Freeze fracture electron microscopy.
 Vesicle Size: Determined by:-
 Light microscopy
 Fluorescent microscopy
 Electron microscopy: SEM, TEM
 Laser light scattering
 Gel permeation
 Ultracentrifugation
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41. Surface charge: Determined by Electrophoresis
Drug release: Dissolution
Entrapped volume: (water content is determined)
 Water is replaced with deuterium oxide & is
analyzed by NMR
Encapsulation efficiency:
 Protamine aggregation method:
 Liposome + Protamine = Precipitation
 Centrifuge (2000 rpm), remove supernatant
 Liposome pellet + Trixon x-100 (surface breaker)
 The encapsulation efficiency can be determined
(Analytically)
 Mini column centrifugation
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42.  Chemical characterization:
1. Quantitative determination of phospholipids
2. Phospholipid hydrolysis
3. Phospholipid oxidation
4. Cholesterol analysis
 Phospholipid determination: (Bartlett assay)
 Phospholipid phosphorous + Hydrolysis=
Inorganic phosphate.
 Inorganic phosphate +ammonium molybdate=
phospho molybdic acid
 phospho molybdic acid + Amino naphthyl
sulfonic acid= reduced to blue color whose
intensity is measured & compared with standard
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43.  Phospholipid hydrolysis:
 Phospholipids + Hydrolysis= Lysolecithin
 One chain is lost by desterification
 Determined by HPLC
 Phospholipid oxidation:
 Free radical determination by UV, iodometric
method, GLC etc.
 Cholesterol analysis:
 Cholesterol + Iron + Reagent (Ferric per
chlorate, ethyl acetate & Sulfuric acid= Purple
complex, which is determined at 610 nm.
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