liposomes

1. FORMULATION AND
EVALUATION OF LIPOSOMES
Presented by:
Madhu k s
1st YEAR M PHARM
Dept,of Ind pharmacy
Srinivas college of pharmacy
Mangalore4/1/2019 1

2.  Definition
 Mechanism of action
 Classification
 Method of preparation
 Purification
 Evaluation
 Applications
 References
Content
2

3. LIPOSOMES
 The term liposome’s (meaning lipid body) was
derived on the basis of names of sub cellular
particles like lysosome and ribosome. It is
defined as a spherule/vesicle of lipid bilayers
enclosing an aqueous compartment. The lipid
most commonly used is phospholipids.
Sphingolipids, glycolipids and sterols have also
been used to prepare liposomes. Their size
ranges from 25 to 5000 nm
4/1/2019 3

4. 4/1/2019 4

5. 4/1/2019 5

6.  Adsorption to cell surface
 Endocytosis
 Fusion with plasma cell membrane
 Transfer of liposomal content
6
MECHANISM OF ACTION

7. 4/1/2019 7

8. 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 DRV4/1/2019 8

9. Preparation of liposomes
4/1/2019 9

10. Methods of liposome preparation
Solvent dispersion
methods
Ethanol injection
Ether injection
Double emulsion
vesicles
Stable plurilamellar
Vesicles
Reverse phase
evaporation vesicles
Detergent removal
methods
Passive loading techniques
Detergent(Cholate,
Alkyl glycoside,
Triton X-100) removal
from mixed micelles by
Dialysis
Column
chromatography
Dilution
Reconstituted sendai
virus enveloped
vesicles
Active loading techniques
Lipid film hydration by
hand shaking non-hand
shaking and freeze drying
Micro emulsification
Sonication
French pressure cell
Membrane extrusion
Dried reconstituted
vesicles
Freeze thawed liposomes
Mechanical dispersion
methods
4/1/2019 10

11. Mechanical Dispersion Methods
Rotary Evaporator
4/1/2019 11

12. Solid lipid mixture is hydrated by using aqueous
buffer
Film deposition
Remove organic solvent under vacuum
Lipid dissolve in organic solvent/co-solvent
Post Hydration vortexing, sonication, freeze thawing &
high pressure extrusion
Liposome
Lipid spontaneously swell & Hydrate
4/1/2019 12

13. 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
4/1/2019 13

14. 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
4/1/2019 14

15. Freeze Drying
Lipid + Solvent ( Tertiary butanol)
Freeze drying
Add Aqueous phase / Saline containing drug
Rapid mixing above phase transition temperature
MLVs
4/1/2019 15

16. 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.
4/1/2019 16

17. 4/1/2019 17

18. Microfluidizer
4/1/2019 18

19. Sonicated unilamellar vesicles
MLV in test tube
Sonicate for 5-10 min above phase transition temp
Filter & centrifuge at 100000g for 30 min at 20º c
Decant top layer to get Sonicated unilamellar vesicles
4/1/2019 19

20. BATH SONICATOR
4/1/2019 20

21. French Pressure Cell
 French pressure cell is invented by ‘Charles Stacey
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.
4/1/2019 21

22. 4/1/2019 22

23. 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).
4/1/2019 23

24. 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
4/1/2019 24

25. 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
4/1/2019 25

26. Ether injection
Lipid + ether solution in the syringe
Inject slowly
In the aqueous phase ( On heated water bath, 60ºc)
Large unilamellar vesicles
4/1/2019 26

27. Ethenol and ether injection
4/1/2019 27

28. 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
4/1/2019 28

29. 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”.
4/1/2019 29

30. • Commonly purified by gel filtration column
chromatography or dialysis or centrifugation.
• In column chromatographic separation, Sephadex G-
50 is most widely used material.
• In dialysis method, hollow fiber dialysis cartridge may
be used.
• The separation of liposomes by centrifugation
method depends on the size as well as the
composition of the bilayers.
30
PURIFICATION OF LIPOSOMES

31. Evaluation of liposome: physical
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)
4/1/2019 31

32. Vesicle shape & lamellarity ( No. of bilayers):
 Sample + 31 p 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
4/1/2019 32

33. Chemical
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
4/1/2019 33

34. 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.
4/1/2019 34

35.  Enzyme replacement therapy
 Delivery of antibodies, antigens and vaccines
 Hormones and blood factors
 Blood substitutes
 Interferon
35
APPLICATIONS

36. References
 Kant S, Kumar S, Prasdar V. A complete review on
liposomes. Int Res J Pharm 2013; 3(7): 10-16.
 S.P.Vyas, V.K.Disit Advance in Liposomal Therapeutics
2009; 4(6): 51-73.
 Mansoori MA, Agrawal S, Jawade S, Khan MI. a review
on liposome. Int J pharm Pharm 2012; 2(4): 453-461.
 priyanka RK, Jaydeep DY, kumar a review arrival on
liposomes: a novel drug delivery system 2011; 3 (2): 31-
45.
4/1/2019 36

37. 4/1/2019 37