Liposomes : Types, Preparation & Evaluation

1. Seminar on ,
Presented By: :
Mr. Badhe Shubham Pradip
M. Pharm 1st year
(Dept. of P’ceutics))
Guided By ::
Dr. P.K.Deshmukh
(Dept. of P’ceutics)
Department Of Pharmaceutics
H.R.Patel Institute Of Pharmaceutical
Education& Research, Shirpur

2. LIPOSOMES

3. Introduction Of Liposomes
Composition Of Liposomes
Advantages / Dis-advantages Of Liposomes
Types/Classification Of Liposomes
Method Of Preparation Of Liposomes
Evaluation Of Liposomes
Conclusion
References

4.  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
Fig.1-Structure of liposomes
INTRODUCTION

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

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

7. Phospholipids Are Major Structural
Component Of Biological Membranes In
Human Body, Where Two Types Of
Phospholipids Exist i.e.
Phosphoglycerides & Sphingolipids.
Each Phospholipid Molecule Has Two
Major Parts, 1 Head & 2 Tails. Head Is
Made From Three Molecular
Components: Choline, Phosphate &
Glycerol Which Is Hydrophilic. Each
Tail Which A Long Tail Which Are
Hydrophobic.
Fig.2- Phospholipids
PHOSPHOLIPIDS

8. 1. Cholesterol stabilizes the membrane.
2. It plays important role in bilayer formation.
3. Cholesterol by itself does not form bilayer structure.
4. Cholesterol act as fluidity buffer.
5. Enhances the stability of the membrane.
6. Enhances the rigidity of the phospholipid bilayer.
7. Reduces the permeability of water soluble substance through the
membrane.
CHOLESTEROL

9. Provides selective passive targeting
to tumour tissues.
Increased efficacy & therapeutic
index.
Increased stability via
encapsulation.
Reduction in toxicity of the
encapsulated agent.
Site avoidance effect (avoid non-
target tissues).
Improved pharmacokinetic effects.
Production cost is high
Leakage and fusion of
encapsulated drug/molecules.
Sometimes phospholipids
undergoes oxidation and hydrolysis
like reaction.
Short half-life.
Low solubility.
Advantages Of
Liposomes
Dis-advantages
Of Liposomes

10. 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 (>100um) LUV
Giant Unilamellar Vesicles (>1um) GUV
Multivesicular Vesicles (>1um) MUV
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 Reconstitued Vesicles DRV
Classification

11. Different types of liposomes

12. Physical Dispersion Method :-
1. Hand Shaking MLV’S
2. Non-shaking LUV’S
3. Freeze -Drying
4. Pro-liposomes
 To Reduce Liposome Size :
1. Microemulsification
2. Membrane extrusion
3. Ultrasonification
4. French pressure cell
 To Increase Liposome Size :
1. Dried Reconstitued vesicle
2. Freeze thawing
3. Induction Of Vesiculation by Ph Change
Method Of liposome preparation

13. Solvent Dispersion Method :
1. Ethanol Injection
2. Ether Injection
3. Water Organic Phase :
Double emulsion method
Reverse phase evaporation
Stable plurilamellar vesicles
 Detergent dispersion :

14. Lipids + Solvent (Chloroform : Methanol)
(In 250 ml RBF)
Evaporate For 15 min Above Phase Transition Temperature
(Flush With Nitrogen)
Till Residues By
Add 5 ml Buffer Containing Material To Be Entrapped
Rotate Flask At Room Temperature At 60 RPM For 30 min Until Lipid Removes From
Wall Of RBF
Milky White Dispersion (Stand For 2 Hours To Get MLV)
Hand shaken MLV’S

16. 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 0.2 m Sucrose Solution To Swell
(Flush Again With Nitrogen)
Stand For 2 Hrs At 30⁰ 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
Non shaking vesicles

17. Sorbitol / Nacl (Increase Surface Area Of Lipid Film)
+ 5ml Lipid Solution (Fitted To Evaporater)
(Evaporation)
Again Add Lipid Solution
Dry The Content Using Lyophilizer (Freeze Dryer)
(Stand Over Night At Room Temp)
Flushed With Nitrogen For Drying Properly)
MLV’s
PRO LIPOSOME

18. Lipid + Solvent (Tertiary Butanol)
Freeze Drying
Add Aqueous Phase / Saline Containing Drug
Rapid Mixing Above Phase Transition Temperature
MLV’s
FREEZE DRYING

19. • MEL Is Prepared By The “Micro Fluidizer”, Which Pumps Fluid At
Very High Pressure (10,000 Psi) Through At 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.
MICRO EMULSIFICATION LIPOSOME (MEL)

20. MICROFLUIDIZER

21. MLV In Test Tube
Sonicate For 5-10 min Above Phase Transition Temp
Filter & Centrifuge At 100000 Rpm For 30 min At 20⁰ C
Decant Top Layer To Get Sonicated Unilamellar Vesicles
SONICATED UNILAMELLAR VESICLES

23. 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 Liposome Of Intermediate
Size (30-80 nm In Diameter Depending On Applied Pressure).
This Liposome Are More Stable As Compared To Sonicated Liposomes.
FRENCH PRESSURE CELL

25. • In This Technique Vesicle Contents Are Exchanges 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 & LUVs.
• Two Types Of Membrane One Is Tortuous (Zigzag) And Another Is Nucleation
Track (Vertically Parallel).
MEMBRANE EXTRUSION LIPOSOMES

26. SUV In Aqueous Phase + Solute
Freeze Drying
FTS Method, Thawing = Melting
Sonication (15-30 Sec)
Solutes In Unilamellar Vesicle
To Increase Size Of Liposome : Freeze Thaw
Sonication

27. SUV In Aqueous Phase + Solute
Freeze Drying
DRV Method : Rehydration, Film Stacks Dispersed In Aqueous Phase
Solute In Unit Or Oligo Lamellar Vesicles.
Dried Reconstituted Vesicle

28. 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.
PH Induced Vesiculation

29. Lipid + Ethanol Solution In The Syringe
Inject Rapidly
In The Aqueous Phase
Small Unilamellar Vesicles
Solvent dispersion method :
ethanol injection

30. Lipid + Ether Solution In The Syringe
Inject Slowly
In The Aqueous Phase (On Heated Water Bath, 60⁰ C)
Large Unilamellar Vesicles
Ether Injection

31. Organic Solution + Lipid + Aqueous Phase
Emulsion (W/O)
Hot Aqueous Solution Of Buffer
Multi Compartment Vesicle W/O/W (Double Emulsion)
LUVs
Water Organic Phase: Double Emulsion

33. 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”.
Reverse Phase Evaporation: (MLV, LUV)

34. 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-REV, 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.

35. Physical Characterization :
 Vesicle shape & lamellarity (no. of bilayers) :
• If intensity is decrease by 50 % = unilamellar vesicle are formed.
• If intensity is decrease by more intensity = MLV are formed.
• Freeze fracture electron microscopy.
 Vesicle Size: Determind By :-
• Light microscopy.
• Fluorescent microscopy.
• Electron microscopy: SEM,TEM.
• Laser light scattering.
• Gel permeation.
• Ultracentrifugation.
EVALUATION OF LIPOSOMES

36. Surface Charge : Determined By Electrophoresis.
Drug Release : Dissolution.
Entrapped Volume : (Water Content Is Determined).
• Water Is Replaced With Deuterium Oxide & Is Analysed 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.

37. 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 phophate + ammonium molybdate = phosphomolybdic acid.
 Phospho molybdic acid + amino naphthyl sulfonic acid = Reduced to blue
color whose intensity is measured & compared with standard.
Phospholipid Hydrolysis :
 Phospholipids + hydrolysis = lysolecithin.
 One chain is lost by desterification.
 Determined by HPLC.

38. 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.

39. Liposome Are One Of The Unique Drug Delivery System, Which Can Be
Of Potential Use In Controlling And Targeting Drug Delivery.
Liposomes Are Administered Orally, Parenterally And Topically As Well
As Used In Cosmetic And Hair Technologies, Sustained Release
Formulations, Diagnostic Purpose And As Good Carriers In Gene
Delivery.
Nowadays Liposomes Are Used As Versatile Carriers For Targeted
Delivery Of Drug.
CONCLUSION

40. Targeted And Controlled Drug Delivery, S.P.Vyas And R.K.Khar, CBS
Publication, 2008, Page No.173 To 248.
Text Book Of Industrial Pharmacy, Shobha Rani Hiremath, Orient
Longman Private Ltd, Page No. 97 To 110.
Liposome : A Versatile Platform For targeted Delivery Of Drug By Sanjay
S. Patel.
Kant shashi, Kumar satinder, Prashar bharat “International research
journal of pharmacy” A complete review on liposome, ISSN 2230-8407,
IRJP 2013, 3 (7).
REFERENCES