Transcript Liposome - PharmaStreet

Liposome
Contents
2  Introduction
 Mechanism of liposome formation
 Classification
 Biological fate of liposome
 Methods of preparation
 Characterization
 Advantages & Disadvantages
 Applications
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Introduction
Lipo – fat or lipid and some-body
Liposomes are simple micro particulate drug
carrier consisting of one or more concentric
bilayered vesicles in which an aqueous volume is
entirely enclosed by a membranous lipid bilayer
mainly composed of natural or synthetic
phospholipids.
When Phospholipid come in contact with water
they formed spherical structure enclosing
aqueous compartment.
Discovered in 1960’s by Bangham and
coworkers.
The structural main components are
phospholipids and cholesterol
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Lets take a look at liposome
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 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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Phospholipids
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 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.
Cholesterol alignment between phospholipid bilayer
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Mechanism of liposome formation
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 Vesicles are formed by hydrophobic effect.
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 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.
 CPP = v/ lc Ap = Ahp / Ap
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Where:
 v = hydrophobic group volume
 lc = hydrophobic group length
 Ap = cross sectional area of hydrophilic head group
 Ahp = cross sectional area of hydrophobic group.
Classification
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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
Different types of liposomes
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Biological fate of liposome
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Liposomes in blood stream
Taken by reticulo-endothelial system
Macrophages engulf liposomes ( endocytosis)
Phagosome + lysosyme = phagolysosyme
Membrane of phagolysosyme have proton pumps
which decrease PH of phagolysosyme & the
enzymes phospholipase destruct the liposomal
membrane
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Method of liposome preparation
18  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
Solvent dispersion method :
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1. Ethanol injection
2. Ether injection
3. Water organic phase:
A) Double emulsion method
B) Reverse phase evaporation
C) Stable plurilamellar vesicles
 Detergent solubilization :
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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Rotary Evaporator
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Non Shaking vesicles
Lipid + solvent
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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
Pro liposome
23 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
Freeze Drying
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Lipid + Solvent ( Tertiary butanol)
Freeze drying
Add Aqueous phase / Saline containing drug
Rapid mixing above phase transition temperature
MLVs
Micro emulsification liposome (MEL)
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 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.
Microfluidizer
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Sonicated unilamellar vesicles
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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
French Pressure Cell
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 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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To increase size of liposome: Freeze thaw sonication
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SUV in aqueous phase + Solute
Freeze drying
FTS method, thawing = melting
Sonication ( 15-30 sec)
Solutes in unilamellar vesicle
Dried reconstituted vesicle
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SUV in aqueous phase + Solute
Freeze drying
DRV method: Rehydration, film stacks dispersed in
aqueous phase
Solute in uni or oligo lamellar vesicles.
PH induced vesiculation
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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
Solvent dispersion method: Ethanol injection
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Lipid + ethanol solution in the syringe
Inject rapidly
In the aqueous phase
Small unilamellar vesicles
Ether injection
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Lipid + ether solution in the syringe
Inject slowly
In the aqueous phase ( On heated water bath, 60ºc)
Large unilamellar vesicles
Water organic phase: Double emulsion
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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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Reverse phase evaporation: (MLV, LUV)
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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”.
Stable plurilamellar vesicle (SPLVs)
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 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.
Detergent dispersion:
 Phospholipids & aqueous phase comes in contact with the
help of detergent
Characterization of Liposome: Physical
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 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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 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
Chemical characterization:
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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
 Phospholipid hydrolysis:
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 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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-PHARMA STREET