Liposome

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1. Liposomes
1

2. OUTLINE
•WHAT ARE LIPOSOMES?
•BASIC LIPOSOME STRUCTURE.
•WHY USE LIPOSOMES IN DRUG
DELIVERY?
•ADVANTAGES OF LIPOSOMES.
•STRUCTURAL COMPONENTS OF 
LIPOSOMES.
•CLASSIFICATION OF LIPOSOMES.
•PREPARATION OF LIPOSOMES.
2

3. 3
Liposomes are concentric bilayered vesicles Liposomes are concentric bilayered vesicles
in which an aqueous core is entirely enclosed in which an aqueous core is entirely enclosed
by a membranous lipid bilayer mainly by a membranous lipid bilayer mainly
composed of natural or synthetic composed of natural or synthetic
phospholipids.phospholipids.
Liposomes were first produced in England in Liposomes were first produced in England in
1961 by Alec D. Bangham, who was 1961 by Alec D. Bangham, who was
studying phospholipids and blood clotting.studying phospholipids and blood clotting.
The size of a liposome ranges from some The size of a liposome ranges from some
20 nm up to several micrometers.20 nm up to several micrometers.
Liposomes (= vesicles)Liposomes (= vesicles)

4. The lipid moecules are ususally phospholipids- amphipathic moieties with a
hydrophilic head group and two hydrophobic tails.
On addition of excess water, such lipidic moieties spontaneously originate to give
the most thermodynamically stable conformation.
In which polor head groups face outwords into the aqueous medium, and the lipidic
chains turns inwords to avoid the water phase, giving rise to double layer or bilayer
lamellar stractures.

5. Hydrophobic
Hydrophilic
cavity
5

6. Basic liposome structure
6

7. What is a lamella?
– A Lamella is a flat plate like structure that appears
during the formation of liposomes. The phospholipids
bilayer first exists as a lamella before getting 
converted into spheres.
– Several lamella of phospholipids bilayers 
are stacked 
one on top of the other during formation of liposomes 
 to form a multilamellar structure. 
7

8. Multilamellar vesicles     
 
Unilamellar vesicles 
8

9. Structural Components of
Liposomes
•THE MAIN COMPONENTS OF LIPOSOMES ARE :-
    
1. Phospholipids
2. Cholesterol
9

10. Phospholipids 
•Phospholipids are the major structural compone
nts of biological membranes such as  the 
cell membrane.
Phosphoglyceridesc# msc! dtsMss
mt mgl l"tb$gm s% lys
cdlqs&!"qmg!tltsqm"ufyt M
Two Types Of
Phospholipids(Along With
Their Hydrolysis Products)
Sphingolipids
10

11. Phosphatidylcholine
•Most common phospholipids used is 
    phosphatidylcholine (PC).
•   Phosphatidylcholine is an amphipathic
    molecule in which exists:-
    – a hydrophilic polar head group, 
       phosphocholine.
    – a glycerol bridge.
    – a pair of hydrophobic acyl hydrocarbon 
       chains.
11

12. Generally phospholipids are
represented as follows:-
follows:
12

13. PhospholipidsPhospholipids
Polar Head Groups
Three carbon glycerol
13

14. 14

15. ROLE OF CHOLESTEROL IN BILAYER FORMATIONROLE OF CHOLESTEROL IN BILAYER FORMATION
•Cholesterol by itself does not form bilayer structure.Cholesterol by itself does not form bilayer structure.
•Cholesterol act as fluidity bufferCholesterol act as fluidity buffer
•After intercalation with phospholipid molecules alter the After intercalation with phospholipid molecules alter the
freedom of motion of carbon molecules in the acyl chainfreedom of motion of carbon molecules in the acyl chain
•Restricts the transformations of Restricts the transformations of trans trans to to gauchegauche conformations conformations
•Cholesterol incorporation increases the separation between Cholesterol incorporation increases the separation between
choline head group & eliminates normal electrostatic & choline head group & eliminates normal electrostatic &
hydrogen bonding interactionshydrogen bonding interactions

16. Some other commonly used 
phospholipids
Naturally occurring phospholipids:
      –  PC : Phosphatidylcholine
      –  PE : Phosphatidylethanolamine
      –  PS : Phosphatidylserine
Synthetic phospholipids:
      –  DOPC : Dioleoylphosphatidylcholine
      –  DSPC : Distearoylphosphatidylcholine
16

17. Why Use Liposomes in Drug 
Delivery?
Inactive:  Unmodified liposomes gather in specific tissue 
reticuloendothelial system 
Active:  alter liposome surface with ligand  (antibodies, 
enzymes, protein A, sugars)
Directly to site
Physical: temperature or pH sensitive liposomes 
Drug Targeting
17

18. Protection
Decrease harmful side effects
Pharmacokinetics - efficacy and toxicity
Changes the absorbance and biodistribution
Change where drug accumulates in the body
Protects drug
 Deliver drug in desired form
Multidrug resistance
Why Use Liposomes in 
Drug Delivery?
18

19. Release
Affect the time in which the drug is released 
Prolong time -increase duration of action and 
decrease administration
Dependent on drug and liposome properties
Liposome composition, pH and osmotic gradient, and
environment
Why Use Liposomes in 
Drug Delivery?
19

20. ADVANTAGES OF LIPOSOMESADVANTAGES OF LIPOSOMES
•Provides selective passive targeting to tumor 
      tissues.
•   Increased efficacy and therapeutic index.
•   Increased stability of encapsulated drug.
•   Reduction in toxicity of the encapsulated agent.
•   Site avoidance effect (avoids non-target tissues).
•   Improved pharmacokinetic effects (reduced 
       elimination increased circulation life times).
•  Flexibility to couple with site specific ligands to
      achieve active targetting.  
20

21. DISADVANTAGESDISADVANTAGES
  PHYSICAL/ CHEMICAL STABILITYPHYSICAL/ CHEMICAL STABILITY
  VERY HIGH PRODUTION COSTVERY HIGH PRODUTION COST
  DRUG LEAKEGE/ ENTRAPMENT/ DRUG FUSIONDRUG LEAKEGE/ ENTRAPMENT/ DRUG FUSION
STERILIZATION STERILIZATION 
  SHORT  BIOLOGICAL ACTIVITY / t SHORT  BIOLOGICAL ACTIVITY / t 
½½

  OXIDATION OF BILAYER …LIPIDS AND LOW  SOLUBILITYOXIDATION OF BILAYER …LIPIDS AND LOW  SOLUBILITY
  RATE OF RELEASE  and ALTERED BIODISTRIBUTIONRATE OF RELEASE  and ALTERED BIODISTRIBUTION
    LOW THEARAPEUTIC INDEX and DOSE EFFECTIVENESSLOW THEARAPEUTIC INDEX and DOSE EFFECTIVENESS
  OVERCOMING RESISTANCEOVERCOMING RESISTANCE
  EXTENCIVE CLINICAL  AND LABORATORY  RESEARCH TO  EXTENCIVE CLINICAL  AND LABORATORY  RESEARCH TO  
        ACERTAIN LONG CERCULATING LIPOSOMESACERTAIN LONG CERCULATING LIPOSOMES
REPEATED IV ADMINISTRATION PROBLEMESREPEATED IV ADMINISTRATION PROBLEMES

22. CLASSIFICATION OF LIPOSOMES
•Liposomes are classified on the basis of 
     – Structural parameters
     – Method of preparation
     – Composition and applications
22

23. 1.Based on structural parameters
MLV
Multilamellar 
Large 
vesicles
(>0.5 um)
OLV 
oligolamellar 
vesicles
(>0.1-1.0 um)
UV Unilamellar
Vesicles (all     
size ranges) 
MVV
Multivesicular 
vesicles
(> 1.0 um)
MUV Medium Unilamellar 
Vesicles
GUV Giant Unilamellar Vesicles
>1um
SUV Small Unilamellar 
Vesicles 
20-100nm
LUV Large Unilamellar 
Vesicles
>100nm
Based on structural parameters
23

24. 24

25. 25

26. 3. Based on composition & application
1- Conventional.
2- Stealth.(PEG, increase 
blood circulation time and 
decrease phagocytic 
attack).
3- Cationic.(lipoplex)
4- Targeted.(antibody)
26

27. PREPARATION OF LIPOSOMES
Methods of liposome 
preparation
Passive loading:
Involves loading of  the  
entrapped 
agents before or during the  
manufacturing procedure
Active or remote loading:
certain types of compounds with 
ionisable groups 
and those with both 
manufacturing procedure
lipid and water solubility can be
introduced into the liposomes 
after the 
formation of the intact vesicles 27

28. 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

28

29. SOLVANT SOLVANT
eg.CHCleg.CHCl
33
LIPID e.g.. LIPID e.g..
LICITHIN LICITHIN
DISPERSION DISPERSION
FORMATIONNFORMATIONN
Encapsulated soluteEncapsulated solute
Separate liposome's from Separate liposome's from
supernatatant by supernatatant by
centrifugation, gel centrifugation, gel
filtration/ sonication or filtration/ sonication or
dialysis or by addition of dialysis or by addition of
buffers + drug.buffers + drug.
FILM OF LIPID FILM OF LIPID
OCCURS AT SIDES OCCURS AT SIDES
OF VESSELSOF VESSELS
BASIC METHOD OF FORMULATION OF LIPOSOMESBASIC METHOD OF FORMULATION OF LIPOSOMES

30. MethodMethod VesiclesVesicles
Mechanical methodsMechanical methods
Vortex or hand shaking of phospholipid dispersions MLV
Extrusion through polycarbonate filters at low or medium 
pressure
OLV, LUV
Extrusion through a French press cell “Microfluidizer” 
technique
Mainly SUV
High-pressure homogenization Mainly SUV
Ultrasonic irritation SUV of minimal size
Bubbling of gas BSV
Methods based on replacement of organic solvent(s) by aqueous mediaMethods based on replacement of organic solvent(s) by aqueous media
Removal of organic solvent(s) MLV, OLV, SUV
Use of water-immiscible solvents: ether and petroleumMLV, OLV, LUV
Ethanol injection method LUV
Ether infusion (solvent vaporization) LUV, OLV, MLV
Reverse-phase evaporation
Methods based on detergent removalMethods based on detergent removal
Gel exclusion chromatography SUV
“Slow” dialysis LUV, OLV, MLV
Fast dilution LUV, OLV
Other related techniques MLV, OLV, LUV, SUV

31. PASSIVE LOADING TECHNIQUES PASSIVE LOADING TECHNIQUES
•Mechanical Dispersion method
•Solvent Dispersion method
•Detergent Solubilisation method

32. MECHANICAL DISPERSION MECHANICAL DISPERSION
METHODSMETHODS

33. LIPID HYDRATION METHODLIPID HYDRATION METHOD
The mechanical energy required for swelling of lipids and dispersion
of casted lipid film is imparted by manual agitation (hand shaking
technique)
The % encapsulation efficiency as high as 30% is achieved due to
loss of water soluble component during swelling and entrapped only
10-15%. On other hand lipid soluble drug encapsulated 100%
efficiency.

34. 1.Hand shaken multilamellar vesicles
‐
2.Non shaking vesicles
‐
3.Pro liposomes
‐
4.Freeze drying
 
:–AFTER THESE METHODS, OTHER PROCESSING METHODS ARE
USED TO MODIFY THESE TYPE OF VESICLES THAT ARE
PRODUCED SUCH AS:
•Micro emulsification liposomes (MEL)
•Sonicated unilamellar vesicles (SUVs)
•French Pressure Cell Liposomes
•Membrane extrusion liposomes
•Dried reconstituted vesicles (DRVs)
‐
•Freeze thaw Sonication (FTS)
•pH induced vesiculation
•Calcium induced fusion
    :–These methods are known as “the mechanical treatment of MLVs” or 
“Processing of  lipids by physical means
34

35. Hand shaken multilamellar vesicles
•Simplest and most widely used method of physical dispersion
•Basic method involves
– Dissolution of the lipid mixture and charge components in     
   chloroform:methanol solvent
– Evaporation of the solvent in a rotary evaporator or by hand  
   shaking to form a film shaking to form a
– Further drying of the film by attaching the flask to the  
   manifold of the lyophilizer.
– Casted film is then dispersed in an aqueous medium.
– Upon hydration, lipid swell and peel off the wall of the flask 
   and vesiculate forming multilamellar vesicles (MLVs) 
35

36. PROCESS IN MORE DETAIL –
STEP 1:
•Lipid mixture of different phospholipids and charge components in 
chloroform:methanol solvent mixture (2:1 v/v) is prepared first and 
then introduced into a round bottom flask with a ground glass neck.
•This flask is then attached to a rotary evaporator and rotated at 60 
rpm.
•The organic solvents are evaporated at about 30 degrees Celsius or 
above the transition temperature of the lipids used.
•The rotation is continued for 15 mins after the dry residue first 
appears.
•The evaporator is isolated from the vacuum source by closing the 
tap.
•The nitrogen is introduced into the evaporator and the pressure at 
the cylinder head is gradually raised till there is no difference 
between inside and outside the flask between inside and outside the 
flask.
•The flask is then removed from the evaporator and fixed on to the 
manifold of lyophilizer to remove residual solvents.
36

37. STEP 2 Hydration of lipid layer:
‐
•After releasing the vacuum and removal from the lyophilizer, 
the flask is flushed with nitrogen.
•5ml of saline phosphate buffer (containing solute to be 
entrapped) is added.
•The flask is attached to the evaporator again (flushed with N
2
) 
and rotated at room temperature and pressure at the same 
speed or below 60 rpm.
•The flask is left rotating for 30 minutes or until all lipid has 
been removed from the wall of the flask and has given 
homogenous milky white suspension free of visible particles.
‐
•The suspension is allowed to stand for a further 2 hours at 
room temperature or at a temperature above the transition 
temperature of the lipid in order to complete the swelling 
process to give MLVs.
37

38. Hand shaken method in general
 
38

39. Nonshaking vesicles
‐
•The procedure differs from hand shaken method in that it uses a stream 
of nitrogen to provide agitation rather than the rotationary movements.
•Solution of lipid in chloroform:methanol mixture is spread over the flat 
bottom conical flask.
•The solution is evaporated at room temperature by flow of nitrogen 
through the flask without disturbing the solution.
•After drying, water saturated nitrogen is passed through the flask until 
the opacity of the dried lipid film disappears (15-20mins).
•After hydration, lipid is swelled by addition of bulk fluid. The flask is 
inclined to one side, 10 20 ml of 0.2M sucrose in distilled water 
‐
(degassed) is introduced down the side of the flask, and the flask is 
slowly returned to upright orientation.
•The fluid is allowed to run gently over the lipid layer on the bottom of the 
flask.
•The flask is flushed with nitrogen sealed and allowed to stand for 2 hrs 
at 37 degrees Celsius. Take care not to disturb the flask in any way.
•After swelling, the vesicles are harvested by swirling the contents of the 
flask gently , to yield a milky suspension.
‐
39

40. PROLIPOSOMESPROLIPOSOMES
To increase the surface area of dried lipid film and to
facilitate continuous hydration and lipid is dried over the
finally divided particulate support i.e.- NaCl, Sorbitol, or
other polysaccharides. These dried lipid coated
particulates are called as proliposomes
Proliposomes form dispersion of MLVs on addition of
water, where support is rapidly dissolved and lipid film
hydrate to form MLVs
Methods overcome the stability problem and entrapment
efficiency doesn’t matter when formation of stable
liposome.

41. Freeze drying
 
•Another method of dispersing the lipid in a finely divided 
form, prior to addition of aqueous media, is to freeze dry 
the lipid dissolved in a suitable organic solvent.
•The solvent choice depends on the freeze point which 
needs to above the temperature of the condenser 
lyophilizers. Tertiary butanol is considered to be most 
ideal solvent.
•After obtaining the dry lipid which is an expanded foam 
like structure, water or saline can be added with rapid 
mixing above the phase transition temperature to give 
MLVs.
41

42. SONICATION METHODSONICATION METHOD
PROBE  SONICATORPROBE  SONICATOR :: is employed for is employed for
dispersions, which require high energy in a dispersions, which require high energy in a
small volume (e.g., high concentration of small volume (e.g., high concentration of
lipids, or a viscous aqueous phase) lipids, or a viscous aqueous phase)
DisadvantageDisadvantage- lipid degradation due to high - lipid degradation due to high
energy and sonication tips release titanium energy and sonication tips release titanium
particles into liposome dispersionparticles into liposome dispersion
BATH  SONICATOR: BATH  SONICATOR: The bath is more The bath is more
suitable for large volumes of diluted lipids. suitable for large volumes of diluted lipids.
Method: Method: Placing a test tube containing the Placing a test tube containing the
dispersion in a bath sonicator and sonicating dispersion in a bath sonicator and sonicating
for 5-10min(1,00,000g) which yield a for 5-10min(1,00,000g) which yield a
slightly hazy transparent solution. Using slightly hazy transparent solution. Using
centrifugation to yield a clear SUV centrifugation to yield a clear SUV
dispersiondispersion

43. FRENCH PRESSURE CELL FRENCH PRESSURE CELL
LIPOSOMESLIPOSOMES
This techniques yields rather “uni or
oligo lamellar liposomes” of intermediate
size of 30-80 nm in diameter depending on
the applied pressure.
Dispersion of MLVs can be converted to
SUVs by passage through a small orifice
under high pressure.
MLV dispersion are placed in the French
pressure cell and extruded at about
20,000psi at 45
0
C By multiple extrusion
i.e.., 4.5 passed about 95% of MLVs get
converted into SUVs which can be
determined by size exclusion
chromatography.

44. MICRO EMULSIFICATION LIPOSOMES(MEL)MICRO EMULSIFICATION LIPOSOMES(MEL)
The fluid collected can be recycled through the
pump and interaction chamber until vesicles of
the spherical dimension are obtained.
After a single pass, the size of vesicles is reduced
to a size 0.1 and 0.2um in diameter.
“Micro Fluidizer” is used to prepare small MLVs from Concentrated lipid
dispersion
The lipids can introduced into fluidizers, either as a dispersion of large MLVs
or as a slurry of unhydrated lipids in organic medium.
Microfluidizer pumps the fluid at very high pressure(10,000psi, 600-700 bar)
through a 5um orifice.
Then it is forced along defined micro channels, which direct two streams of
fluid to collide together at right angles at a very high velocity, thereby affecting

an efficient transfer of energy.

45. VESICLES PREPARED BY VESICLES PREPARED BY
EXTRUSION TECHNIQUES EXTRUSION TECHNIQUES (VETs)(VETs)
It is used to process LUVs as well as MLVs.
Liposomes prepared by this tech. are called
as membrane filter extrusion liposomes
The 30% capture volume can be obtained
using high lipid conc. The trapped volume
in this process is 1-2 litre /mole of lipids
 It is due to their ease of production, readily
selectable vesicle diameter, batch to batch
reproducibility & freedom from solvent or
surfactant contamination is possible

46. FREEZE THAW SONICATION FREEZE THAW SONICATION
METHOD (FTS)METHOD (FTS)
The method is based on
freezing of a unilamellar
dispersion & then thawing at
room temp for 15 min.
Thus the process ruptures &
refuses SUVs during which the
solute equilibrates between
inside & outside & liposomes
themselves fuse & increase in
size.
Entrapment volume can be
upto 30% of the total vol. of
dispersion. Sucrose, divalent
metal ions & high ionic
strength salt solutions can not
be entrapped efficiently

47. SOLVENT DISPERSION SOLVENT DISPERSION
METHODSMETHODS
Note:- Organic solvent miscible with aqueous Note:- Organic solvent miscible with aqueous
phasephase

48. SOLVENT DISPERSION METHODS FOR SOLVENT DISPERSION METHODS FOR
PASSIVE LOADINGPASSIVE LOADING

49. •An ethanol solution of lipids is injected rapidly through a fine
needle into an excess of saline or other aq. medium
•This method has low risk of degradation of sensitive lipids
•The vesicles of 100 nm size may be obtained by varying the conc.
Of lipid in ethanol or by changing the rate of injection of ethanol
solution in preheated aqu. solution.
•Limitation-solubility of lipids in ethanol & vol. of ethanol that
can be introduced into medium(7.5%v/v max)
•Difficulty to remove residual ethanol from phospholipid
membrane
•Involves mixing of organic phase into aqu. Phase at the temp. of
vaporizing the organic solvent
•It has low encapsulation efficiency
Ethanol injection:-
Ether injection:-

51. REVERSE PHASE EVAPORATION REVERSE PHASE EVAPORATION
METHODMETHOD

52. DETERGENT SOLUBILISATIOIN DETERGENT SOLUBILISATIOIN
METHODSMETHODS
Note:- Liposome size and shape depend on chemical nature of detergent, Note:- Liposome size and shape depend on chemical nature of detergent,
concentration and other lipid involved concentration and other lipid involved

53. •The phospholipids are brought into intimate contact 
with the aqueous phase via detergents which 
associate with phospholipid molecules
•The structures formed are called as micelles
•The conc. of detergent at which micelles are formed is 
called as CMC
•The detergent methods are not very efficient in % 
entrapment values
•The methods employed for removal of detergent 
include dialysis, column chromatography & use of 
biobeads
Detergent depletion methodDetergent depletion method

54. 54
DialysisDialysis MethodMethod
Detergent commonly use for this purpose exhibit resonably 
high CMC (10 to 20 mM) so that their removal is facilitated 
A commercial version of the dialysis system is available under 
the tradename LIPOREP
Column Chromatography Column Chromatography
Phospholipid inthe form of either sonicated vesicle or as a 
dry film, at a molar ratio of 2:1 with deoxycholate form 
unilamellar vesicles of 100nm on removal of deoxycholate by 
column chromatography 

55. •Weak amphipathic bases accumulate in the aqueous phase of lipid Weak amphipathic bases accumulate in the aqueous phase of lipid
vesicles in response to a difference in pH between the inside and vesicles in response to a difference in pH between the inside and
outside of the liposomes (pHin & pHout)outside of the liposomes (pHin & pHout)
•Two steps process generates this pH imbalance and active Two steps process generates this pH imbalance and active
(remote) loading.(remote) loading.
•Vesicles are prapared in low pH solution, thus generating low pH Vesicles are prapared in low pH solution, thus generating low pH
within the liposomal interiors, followed by addition of the base to within the liposomal interiors, followed by addition of the base to
extraliposomal medium.extraliposomal medium.
•Basic compounds, carrying amino groups are relatively lipophipic Basic compounds, carrying amino groups are relatively lipophipic
at high pH and hydrophilic at low pH.at high pH and hydrophilic at low pH.
•In two chambered aqueous system separated by membrane In two chambered aqueous system separated by membrane
liposomes, accumulation occurs at the low pH side, under liposomes, accumulation occurs at the low pH side, under
dynamic equilibrium conditions.dynamic equilibrium conditions.
•Thus the unprotonated form of basic drug can diffuse through the Thus the unprotonated form of basic drug can diffuse through the
bilayerbilayer
•The exchange of external medium by gel chromatography with The exchange of external medium by gel chromatography with
neutral solutionneutral solution
•Weak base doxorubicine, adriamycin and vincristine which co-Weak base doxorubicine, adriamycin and vincristine which co-
exist in aqueous solutions in neutral and charged forms have been exist in aqueous solutions in neutral and charged forms have been
sucessfully loaded into preformed liposomes via the pH gradient sucessfully loaded into preformed liposomes via the pH gradient
method.method.
ACTIVE LOADING TECHNIQUES ACTIVE LOADING TECHNIQUES
AFTER DRYING IN PROCESS AFTER DRYING IN PROCESS


FILM/CAKE OF LIPID IS FROM FILM/CAKE OF LIPID IS FROM
STACKS OF LIPID STACKS OF LIPID
BILAYER FORM BILAYER FORM

SWELLING IN FLUIDSWELLING IN FLUID


SHEET IS SELF CLOSE SHEET IS SELF CLOSE

LOADING OF DRUG LOADING OF DRUG
ON pH- GRADIENT TECHNIQUEON pH- GRADIENT TECHNIQUE
FORMATION OF BILAYER FORMATION OF BILAYER
(LIPOSOMES) IF DRUG (LIPOSOMES) IF DRUG

56. 56

57. 57

58. 58

59. Reverse phase evaporation vesicles
At this stage-
the monolayers come
close to each other
In rotar y evaporator close to
each other
partial  bilayer 
59

60. Ethanol/Ether injection method
60

61. DRYING
•An important step involved in the preparation of 
liposomes is the drying of the lipid.
•Large volume of organic solution of lipids is most
    easily dried in a rotary evaporator fitted with a drieda 
rotary evaporator fitted with cooling coil and a 
thermostatically controlled water bath.
•Rapid evaporation of solvent is carried out by gentle 
warming (20 40 degrees) under reduced pressure (400 
‐
700 mm Hg)(400 700 mm Hg)
‐
•Rapid rotation of the solvent containing flask increases 
the surface area for evaporation. 
61

62. •In cases where sufficient vacuum is not attainable or if 
the concentration of lipids is particularly high, it may be 
difficult to remove the last traces of chloroform from the 
lipid film.
    Therefore, it is recommended as a matter of routine that 
after  rotary  evaporation,  some  further  means  is 
employed to bring the residue to complete dryness.
Attachment of the flask to the manifold of lyophilizer, and 
overnight exposure to high vacuum is a good method.
62

63. I] Physical Dispersion or
Mechanical Dispersion Methods
•Aqueous volume enclosed using this method is usually 
5 10%, which is very small proportion of the total volume 
‐
used for swelling.
•Therefore large quantity of water soluble compounds are 
‐
wasted during swelling.
•On the other hand, lipid soluble compounds can be 
encapsulated to 100% efficacy, provided they are not 
present in quantities that are greater than the structural 
component of the membrane.
63

64. How LUVs Are Generated From The
Suspension?
•The suspension is centrifuged at 12,000g for 10 
min. in a bench centrifuge at room temperature.
•The layer of multilamellar vesicles floating on the 
surface is removed. To the remaining fluid an 
equal volume of isoosmolar glucose is added 
‐
and centrifuged again at 12,000g. Large 
unilamellar vesicles form a soft pellet which can 
be resuspended in any required medium of 
appropriate osmolarity.
64

65. ProLiposomes
‐
•Method devised to increase the surface of dry lipid while 
keeping the low aqueous volume.
•In this method, the lipids are dried down to a finely divided 
particulate support, such as powdered sodium particulate 
support, such as powdered chloride, or sorbitol or other 
polysaccharide – to give pro liposomes.
‐
•The lipids are swelled upon adding water to dried lipid coated 
powder (pro liposomes), where the support rapidly dissolves 
‐
to give a suspension of MLVs in aqueous solution.
•The size of the carrier influences the size and heterogeneity 
of the liposomes.
65

66. •This method overcomes the problems encountered when 
storing liposomes themselves in either liquid, dry or frozen 
form, and is ideally suited for preparations where the material 
to be entrapped incorporates into lipid membrane.
•In cases where 100% entrapment of aqueous component is 
not essential this method is also of value.
•For preparing pro liposome a special equipment i.e. Buchi 
‐
rotary evaporator ‘R’ with water cooled condenser coil and a 
stainless steel covered thermocouple connected to a digital 
thermometer is required.
•The end of the glass solvent inlet tube is modified to ato fine 
point, so that the solvent is introduced into the flask as a fine 
spray.
66

67. BUCHI  Rotary Evaporator R type
67

68. Method Of Preparation of Proliposomes
•The lipid solution in chloroform (60mg/ml) is prepared and sorbitol powder is 
introduced into 100ml flask.
•The flask is then fitted into the evaporator and rotated slowly so that the 
powder tumbles gently off the walls to ensure good mixing and the solvent is 
evaporated.
•The flask is lowered into a water bath at 50 55 degrees Celsius when a 
‐
good vacuum is developed.
•An aliquot of 5ml of lipid solution is introduced into the flask via the solvent 
inlet tube.
•The solvent is absorbed completely by the powder and the temperature of 
the bed is monitored.
•As evaporation proceeds the temperature will decrease.
•A second aliquot is introduced slowly when the temperature begins to rise 
again.
•The temperature is allowed to rise to 30 degrees Celsius the vacuum is 
released and the drying process is completed by connecting the flask 
containing the powder to lyophilizer, and leaving it evacuated overnight at 
room temperature.
•The powder is transferred into a 10ml glass vial containing 600mg solid 
each (100mg lipid and 500mg sorbitol support) flushed with nitrogen, and 
sealed well and stored.
68

69. Processing of the lipids hydrated
by physical means, or the mechanical
treatment of MLVs
•Micro emulsification liposomes (MEL)
•Sonicated unilamellar vesicles (SUVs)
•French Pressure Cell Liposomes
•Membrane extrusion liposomes
•Dried reconstituted vesicles (DRVs)
‐
•Freeze thaw Sonication (FTS)
•pH induced vesiculation
•Calcium induced fusion
69

70. Micro emulsification liposomes (MEL)
 
70

71. Sonicated unilamellar vesicles (SUVs)
 
 
71

72. French Pressure Cell Liposomes
 
 
72

73. Membrane Extrusion Liposomes
73

74. Liposomes Help ImproveLiposomes Help Improve
Therapeutic index
Rapid metabolism
Unfavorable pharmokinetics
Low solubility
Lack of stability
Irritation
74

75. Current liposomal drug 
preparations
Type of AgentsExamples
Anticancer Drugs
Anti bacterial
Antiviral
DNA material
Enzymes
Radionuclide
Fungicides
Vaccines
*Currently in Clinical Trials or Approved for Clinical Use
Malaria merozoite, Malaria sporozoite
Hepatitis B antigen, Rabies virus glycoprotein
Amphotericin B*
In-111*, Tc-99m
Hexosaminidase A
Glucocerebrosidase, Peroxidase
Duanorubicin,Doxorubicin*, Epirubicin
Methotrexate, Cisplatin*, Cytarabin
Triclosan, Clindamycin hydrochloride,
Ampicillin, peperacillin, rifamicin
AZT
cDNA - CFTR*
75

76. CFTR
Gene Therapy
Deliver cDNA of Cystic Fibrosis Transmembrane Conductance
Regulator (CFTR) to epithelial tissue of respiratory system
Fuse to cell membrane and
incorporate cDNA into cell
Clinical trials - no significant
changein symptoms
Now trying adeno associated
virus
Cationic liposome
76

77. Doxil
Chemotherapy drug doxorubin
Anemia, damage to veins and tissue at injection, decrease
platelet and WBC count, toxic to
Treats Kaposi’s sarcoma lesions or cancer tumors
Modifications of liposome “stealth”
keeps doxorubin in blood for 50 hours instead of
20 minutes
concentrates at KS lesions and tumors
*Just approved by FDA*
77

78. Amphotericin BAmphotericin B
Side effects: nephrotoxicity, chills, and fevers
Systemic fungal
infections in immune compromised patients
Fungizone - AmB with deoxycholate
AmB - kills ergosterol-containing fungal cells, also
kills cholesterol-containing human cells
78

79. No decrease in effectiveness of drug against fungi
Liposomal Formulation of AmB
Decrease in toxicity
Exact Mechanism of liposomes not understood
Cholesterol - only few %moles
Phospholipid:AmB ratio
Diffusion
Lipid transfer
AmB
Lipid
79

80. Problems with Liposomal Preparations of DrugsProblems with Liposomal Preparations of Drugs
 $$$$  
Fungizone $40.58       Amphotec $2334Fungizone $40.58       Amphotec $2334
Doxil $1200 per treatment, twice the cost of normal protocol Doxil $1200 per treatment, twice the cost of normal protocol 
of chemotherapy and drugs of chemotherapy and drugs 
  Lack long term stability (short shelf lifeLack long term stability (short shelf life)
Freeze dry and pH adjustmentFreeze dry and pH adjustment
Low “Pay Load” - poor encapsulaLow “Pay Load” - poor encapsulation
Physical and chemical instability Physical and chemical instability 
Polar drugs and drugs without opposite chargePolar drugs and drugs without opposite charge
ModificationsModifications
Possibility of new side effects
Doxil “hand and foot syndrome”
80

81. Studies with insulin show that liposomes may
be an effective way to package proteins
and peptides for use
Clinical Trials for several liposomal formulations
More studies on the manipulation of liposomes
Future
81

82. THANK YOU
82