Types, Preparation and Evaluation of Liposomes

1. LIPOSOMES
SILAMBARASAN I
M PHARM (PHARMACEUTICS)
MTPG & RIHS

2. CONTENT
➢INTRODUCTION
➢DEFINITION
➢ADVANTAGES AND DISADVANTAGES
➢STRUCTURE OF LIPOSOMES
➢COMPOSITION OF LIPOSOMES
➢MECHANISM OF LIPOSOMES
➢CLASSIFICATION OF LIPOSOMES
➢PREPARATION OF LIPOSOMES
➢MICROENCAPSULATION OFLIOPSOMES
➢REMOVAL OF UNENTRAPPED DRUG
➢CHARACTERIZATION OF LIPOSOMES
➢APPLICATION OF LIPOSOMES
➢LIMITATION OFLIPOSOMES
➢MARKETED PRODUCTS
➢REFERENCES

3. INTRODUCTION
➢Liposomes are nanoparticles, that act as carrier to transfer
the drug to specific site.
➢Liposomes is Greek word means,
LIPO - FAT
SOMES - BODY
➢Liposome were first produced in England in 1961 by
“Alec Douglas Bangham”
➢He is called “FATHER OF LIPOSOMES”.
➢At first they were used to study the diffusion and
permeability study.

4. DEFINITION
➢Liposomes are concentric bilayered vesicles in which an aqueous volume is entirely
enclosed by a membranous lipid bilayers mainly composed of natural or synthetic
phospholipids.
➢The size of a liposomes ranges from some 20 nm upto several micrometres.

5. ADVANTAGES
➢Selective passive targeting to tumour tissue.
Eg: liposomal Doxorubicin
➢Enhanced efficacy and therapeutic index.
➢Decreased toxicity.
➢Deliver both lipophilic and hydrophilic drugs.
➢Flexibility to couple with site specific ligands to achieve active targeting.
➢Increased stability via encapsulation.
➢Naturally occurring lipids are non-toxic & biodegradable.

6. DISADVANTAGES
➢Low solubility in water.
➢ Production cost is high.
➢ Short half-life.
➢ Sometimes phospholipid undergoes oxidation and hydrolysis like reaction.
➢ Leakage and fusion of encapsulated drug / molecules.

9. COMPOSITION OF LIPOSOMES
The major constituents of Liposomes are
1) Phospholipids
2) Cholesterol
HYDROPHILIC HEAD
(POLAR)
HYDROPHOBIC TAILS
(NON-POLAR)

10. 1)PHOSPHOLIPIDS:
➢Phospholipids are the basic molecular “building block” of the liposomes.
➢Phospholipids are amphipathic moieties which consists of
-1 hydrophilic polar head
-2 hydrophobic tails
hence have affinity for both polar and non-polar moiety.
➢Most commonly used phospholipid is
-phosphotidylcholine (Lecithin)
-Phosphotidylethanolamine

11. ➢Hydrophilic polar head
- Phosphoric acid bound to water soluble
molecule
- Glycerol bridge
➢Hydrophobic tail
-2 fatty acid chain containing 10-24 carbon
atoms and 0-6 double bond in each chain.

12. Commonly used phospholipids:
➢Naturally occurring phospholipids used in liposomes are:
◦ Phosphatidylcholine (PC)-LECITIN
◦ Phosphatidylethanolamine (PE)
◦ Phosphatidylserine (PS)
➢Synthetic phospholipids used in the liposomes are:
◦ Dioleoyl phosphatidylcholine(DOPC)
◦ Disteroyl phosphatidylcholine (DSPC)
◦ Dioleoyl phosphatidylethanolamine (DOPE)
◦ Distearoyl phosphatidylethanolamine (DSPE)

13. 2) CHOLESTROL:
➢Steroid lipid
➢Interlock between phospholipids
➢Act as “fluidity buffer”
➢Incorporated into phospholipid membrane
upto 1:1 or 2:1 of cholesterol to PC
➢Enhances the stability of the membrane
➢Enhance the rigidity of the phospholipid
bilayer

14. MECHANISM OF LIPOSOME FORMATION
➢In aqueous media, phospholipids as they are soluble ,align themselves closely in planar
bilayer sheets or lipid cakes which is thermodynamically stable.
➢ In bilayer sheets polar head groups face outwards into the aqueous medium, the lipid
chains turns inwards to avoid the water phase, giving rise to double layer.
➢ For the liposomes to be formed, upon further hydration, the lipid cakes (lamella)
swells eventually they curve to form a closed vesicles in the form of spheres.
➢ These spheres are called as liposomes.

15. LIPOSOMES

16. CLASSIFICATION
Liposomes can be classified based on three different criteria
➢Based on structural parameters
➢Based on method of preparation
➢Based on composition and application

22. METHODOFLIPOSOMEPREPARATION
• Twotypes:
• Passiveloading technique:Loading of theentrapped agentsbefore/ during the
manufactureprocedure.
• Active loading technique:Certain typesof compounds withionizable groups & those
withboth lipid & watersolubility can be introducedinto liposomes after the formation
of intactvesicles.

23. 1)MECHANICAL DISPERSION METHOD
➢ The various components are typically combined by co-dissolving the lipids in an
organic solvent & the organic solvent is then removed by film deposition under
vacuum.
➢ When all the solvent is removed, the solid lipid mixture is hydrated using aqueous
buffer.
➢ The lipids spontaneously swell & hydrate to form liposomes.
➢ These post-hydration treatments include
-Vortexing
-Sonication
-Freeze thawing
-High-pressure extrusion

24. LIPID FILM HYDRATION BY HAND
SHAKING

25. Cont,

26. LIPID FILM HYDRATION BY non- HAND
SHAKING

27. PROLIPOSOMES
➢In order to increase the surface area of dried lipid film & to facilitate instantaneous hydration,
the lipid is dried over a finely divided particulate support, such as powdered sodium chloride, or
sorbitol or other polysaccharides.
➢These dried lipid coated particulates are called pro-liposomes.
➢Pro-liposomes form dispersion of MLVs on adding water into them, where support is rapidly
dissolved & lipid film hydrates to form MLVs.
➢This method also overcomes the stability problems of liposomes encountered during their
storage as dispersion, dry or frozen form.
➢It is ideally suited for preparations where the material to be entrapped incorporates into lipid
membrane.
➢This method is applicable in cases where 100% entrapment of components is not a requirement
rather the stability is preferred.

28. Mechanical treatment of MLVs
➢Multilamellar vesicles formed on hydration of dried lipids could be further engineered
or modified for their size & other characteristics.
➢A large number of methods are devised to reduce their size & to convert liposomes of
large size range into smaller homogeneous vesicles.
These include techniques such as
A)Micro-emulsification
B) Extrusion
C) Ultra sonication
D)French pressure cell

29. ➢A second set of methods is designed to increase the entrapment volume of hydrated
lipids & to reduce the lamellarity of the vesicles formed.
➢These include procedures such as
E)Freeze- drying
F)Freeze- thawing

30. A) SONICATION
The exposure of MLV to ultrasonic irradiation for producing
small vesicles
Probe sonicator
-Used for dispersion of
small volume of high
con lipid.
Bath sonicator
-Large volume of diluted
lipid.
-Mostly used for small
SUVs.

31. MLV
Hazy transparent
solution
SUV
Sonication 5-10 mins
To sediment
Titanium dioxide
and MLV
centrifugation 20 OC,30 mins,100000 g
centrifugation 3-4 hrs,159000 g
DISADVANTAGES OF PROBE
SONICATOR:
➢Probe sonicators deliver high energy & heat
leads to lipid degradation.
➢It may release titanium particles to the lipid
which are to be removed .

32. B)FRENCH PRESSURE CELL
1. French pressure cell is invented by ‟Charles Stacy
French”.
2. In this technique the large vesicles are converted to
small vesicles under very high pressure.
3. This technique yields uni or oligo lamellar
liposomes of intermediate size (25-75nm in diameter
depending on applied pressure).
4. This liposomes are more stable as compared to
sonicated liposomes.
5. Suitable for drugs and compounds which degrade by
ultrasonic radiations.

33. ADVANTAGES:
➢Less leakage
➢More stable liposomes are formed compared to sonicated forms
➢Gentle handling of unstable materials
DISADVANTAGES:
➢High initial cost
➢Working volume is comparatively small(about 50 mL as the maximum)

34. C) MICROEMULSIFICATION METHOD
1. “Micro Fluidizer” is used to prepare small ULV/ MLVs from concentrated lipid dispersion.
2. The lipids are introduced into fluidizers, either as a dispersion of large MLVs or as a slurry of
unhydrated lipids in organic medium.
3. Micro fluidizer pumps the fluid at very high pressure (10,000psi, 600-700 bar) through a 5 µm
orifice.
4. 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.
5. The fluid collected of be recycled through the pump and interaction chamber until vesicles of
the spherical dimension are obtained.
Adv: Samples with high % of lipids can be easily treated.

36. D)MEMBRANE EXTRUSION METHOD
1.Used for preparation of LUVs and MLVs.
2. The size of liposomes is reduced by gently
passing them through polycarbonate membrane
filter of defined pore size at lower pressure
(<100 psi).
3. Before extrusion LMV are disrupted by freeze-
thaw cycles/ pre-filtering through large pore size
(0.2-1 μm).
4. Filter with 100nm pores yield LUV of 120-
140nm.

37. ADVANTAGES:
i) Ease of production
ii) Readily selectable vesicle diameter ( between 50-120 nm for in-vivo experiments)
iii) Batch to batch reproducibility
iv) freedom from solvent / surfactant contamination

38. E)DRIED RECONSTITUTED VESICLES
➢This method involves freeze drying the dispersion of empty SUV followed by
rehydration with aqueous fluid, which have material to be entrapped.
➢Useful for preparation of small uni-lamellar and oligo lamellar vesicles.
ADV: High entrapment of water soluble components and bioactives.

39. F)FREEZE THRAW SONICATION:

41. 2)SOLVENT DISPERSION TECHNIQUE:
➢In this process lipid solution in organic phase is bought in contact with the aqueous phase
including material to be entrapped inside the liposome.
➢Methods employing solvent dispersion can be categorized on the basis of the miscibility of the
organic and aqueous solution.
A)Ethanol injection
B)Ether injection
C)Rapid solvent exchange method
D)De-Emulsification method
E)Double emulsion method
F)Reverse Phase evaporation method

42. A) ETHANOL INJECTION
➢Suitable for incorporating hydrophobic & hydrophilic drugs into liposomes.
➢This is suitable for preparing small & large unilamellar vesicle.
➢Injection size 22 Gauge
Disadvantage:
➢ Limitation of the solubility of lipids in ethanol.
➢Difficult to remove residual ethanol from phospholipid membrane.

43. Lipid+Ethanol
Rapidly inject through fine needle
Saline buffer containing materials to be
entrapped
Dissolution of ethanol
Formation of SUVs

44. B)ETHER INJECTION
➢This was developed by Deamer & Bangham, similar to ethanol injection.
➢Injecting the immiscible organic solution very slowly into aqueous phase through
narrow needle.
➢It is added at the temperature of vaporizing the organic solvent.
➢Less risk of causing oxidative degradation.
DISADVANTAGE:-
➢Low entrapment efficiency
➢Long time consumption

45. Lipid+Ether
Slowly injecting through a narrow needle
In the aqueous phase(On heated water bath
60OC)
Formation of SUVs

47. C)RAPID SOLVENT EXCHANGE METHOD
➢Lipid solution in organic solvent is passed through an orifice of syringe by means of
vacuum in to a tube containing aqueous buffer placed on vortex.
➢ The organic solvent vaporizes due to vacuum before contacting aqueous phase.
➢The lipid mixture precipitates very quickly in aqueous buffer forming liposomes.
ADVANTAGE:
Time consumption is less

48. D)DE-EMULSIFICATION METHOD
Generally the liposomes is made up in 2 steps
➢1st the inner leaflet of the bilayer
➢Then the outer half

49. Aqueous medium +material to be entrapped
High volume of immiscible organic solution of lipid
Agitated mechanically
Break Aq medium to water droplets
W/O emulsion
Stabilized by phospholipid monolayer

50. E)DOUBLE EMULSION METHOD
1.The organic solution, which already contains water droplets (W/O) is introduced into
hot aqueous solution of Tris buffer with the help of 22- gauge hypodermic needle
under vigorous stirring .
2. The (W/O/W) multi component vesicle is formed by evaporation of organic solvent
using strong jet of nitrogen.
3. Removal of organic solvent by centrifugation results in formation of intermediated
sized unilamellar vesicles.

51. Lipid+ Organic solvent + aqueous solution
Sonicate
Formation of W/O emulsion
Evaporate to remove organic solvent
Lipids form phospholipid bilayer
Vigorous Shaking
Water droplets collapse
Formation of SUVs
Rotary Evaporator under reduced pressure
Vortex shaker
F)REVERSE PHASE EVAPORATION METHOD

52. MICROENCAPSULATION OR LOCUS OF
DRUG IN LIPOSOMES

54. REMOVAL OF UNENTRAPPED DRUG
➢It is important to estimate the amount of drug encapsulated within liposome.
➢ This is easier in case of MLVs compared to LUVs and SUVs.
➢ MLVs due to their large size, will be settle down and in pellet form when they will be
centrifuged at high speed, while non-encapsulated drug remains supernatant.

55. LUVs and SUVs are estimated using following methods :-
✓Dialysis.
✓Minimum centrifugation.
✓Protamine aggregation.
✓Gel chromatography.
✓Filtration techniques.

56. CHARACTERIZATION /EVALUATION OF
LIPOSOMES
1)PHYSICAL CHARACTERIZATION:

57. 2)Chemical characterization

58. 3)BULK CHARACTERIZATION

59. 4)STABILITY TESTING OF LIPOSOMES
Liposomal stability can be tested by storing under following six conditions.
1. Highest & lowest temp. for 1 month.
2. Room temp. for 12-24 months
3)12-24 months for various light intensiites
3. 2-3 freeze thaw cycles (-25OC TO 25 OC)
4. 60 cycles/ min in a reciprocating shaker for 24-48 hr
5. 6-8 heat cool cycles (5-45oC, 48 hrs at each temp.)
6. Visual/ microscopic examination.
After storage liposomes are valuated for vesicle size, shape, no. of vesicles/ cubic mm &
residual drug content.

60. ➢Poor quality of raw materials mainly phospholipids
➢Drug loading is too low
➢Shelf life is too short
➢Absence of any data on safety of these carrier
PROBLEMS ENCOUNTERED FOR
SCALEUP

61. APPLICATION OF LIPOSOMES
1. Liposome as drug delivery vehicle
2. Liposome as vaccine carrier
3. Liposome in tumour therapy
4. Liposome in gene delivery
5. Liposome as artificial blood surrogates
6. Liposome as radio-pharmaceutical & radio diagnostic carrier
7. Liposome in cosmetics and dermatology
8. Liposome in enzyme immobilization

62. LIPOSOME AS DRUG DELIVERY VEHICLE
➢Liposomes enhance solublization of drugs
(Amphotericibn-B, paclitaxel, Cyclosporin, Minoxidil).
➢ Provide protection to sensitive drug molecules
(cystone arabinose, DNA, RNA, Ribozymes).

63. LIPOSOMES IN TUMOUR THERAPY
DOXARUBICIN
- For the treatment of refractory tumour and breast cancer.
VINCRISTINE
- for the treatment of solid tumour.

64. LIPOSOME AS ARTIFICIAL BLOOD SURROGATES
➢ Liposome encapsulated hemoglobin products can be used as artificial RBC.
➢Sterically stabilized liposome bearing hemoglobin are better Oxygen carriers.
➢These have low toxicity, less platelet activation& aggregation.

65. LIPOSOME AS RADIO-PHARMACEUTICAL & RADIO-DIAGNOSTIC
CARRIER
➢Liposomal radio-diagnostic applications include imaging of liver, spleen, brain,
lymphatics, tumour, blood pool, cardiovascular pathologies, visualization of
inflammation, infection sites, bone marrow, eye vasculature.
➢Liposome imaging agents are used for ultra sound imaging of tumours.

66. LIPOSOME IN COSMETICS AND DERMATOLOGY
➢ Liposomes with essential oils provide an effective nourishing treatment that
penetrates deeply in to the skin.
➢Liposome based on anti-aging formulations (e.g. creams, lotions, gels and hydrogels)
have been formulated and launched in the cosmetic market by L`oreal in 1986.
➢Liposomal preparation reduce the roughness because of its interaction with
corneocytes, the intracellular lipid resulting in skin softening and smoothing.
➢Various liposome based products for facial and body care, make-up, mascara &
foundation, haircare, sunscreen products & perfumes are in market.

67. LIPOSOMES IN RESPIRATORY DRUG DELIVERY SYSTEM
ISONIAZID & RIFAMPICIN
- improved the effect of drugs for tuberculosis.
CYCLOSPORINS
- preferentially absorbed by lungs & show sustained release
LIPOSOMES AS VACCINE ADJUVANTS
CHOLERA TOXIN
- enhanced antibody level

68. LIPOSOMES FOR BRAIN TARGETING
-Addition of sulphatide group to liposome composition increases their ability to
cross blood-brain barrier.
-Liposomes coated with mannose was found to reach brain tissue easily.

70. REFERENCES
1)Targeted and controlled drug delivery novel carrier system; SP. VYAS & RK.KHAR. 2010, CBS
publishers.
2)www.slideshare.net

71. THANK YOU