Liposomes, Structure of liposome, phospholipids, classification of liposomes, method of preparation of liposomes, mechanism of liposome formation, application of liposomes.

2. INTRODUCTION
 Liposomes are spherical vesicles having an aqueous core enclosed by one or
more phospholipid bilayers.
 Liposome were first produced in England in 1961 by Alec D. Bangham who
was studying phospholipids and blood clotting.
 Liposome is derived from Greek
Lipo- Fatty constitution
Soma- Structure
 The size of a liposome ranges from some 20 nm up to several micrometers.

3. BASIC LIPOSOMESTRUCTURE

4. ADVANTAGES
 Can load hydrophilic as well as hydrophobic drug
 Increased efficacy & therapeutic index.
 Increased stability of encapsulated drug.
 Non-toxic.
 Biodegradable.
 Non-immunogenic.
 Lowers systemic toxicity.

5. CONT.
 Targeted delivery.
 Protection of sensitive drug molecules.
 Low Toxicity Due To Reduced Exposure To Sensitive Tissues.
 Minimum ADR/No Side Effects.
 Possible Formulation- suspension, emulsion, gel, Cream, lotion, Aerosol,
reconstituted Vesicles.

6. DISADVANTAGES
 Long term unstability.
 Some times phospholipids undergoes hydrolysis and oxidation reactions.
 Sensitive to temperature change.
 Leakage of encapsulated drug during storage.
 Production cost is high.

7. STRUCTURALCOMPONENT OF LIPOSOMES
Main component of liposome are:
Phospholipids
Cholesterol

8. PHOSPHOLIPIDS
Phospholipids are the basic molecular building block of the
liposome.
Phospholipids is a lipid which is Ampiphilic molecule and
consist of-
 Hydrophilic polar head
 Hydrophobic tails
 Hence have affinity for both hydrophilic drugs can be
encapsulated in the aqueous phase and hydrophobic drug
molecules can be incorporated in the lipid bilayers.
HYDROPHILIC HEAD
(POLAR)
HYDROPHOBIC TAILS
(NON-POLAR)

9. CONT.
 Examples:
 Dilauryl phosphotidyl choline (DLPC),
 Dimyristoyl phosphotidyl choline (DMPC),
 Dipalmitoy phosphotidyl choline (DPPC),
 Distearoyl phosphotidyl choline (DSPC),
 Dioleolyl phosphotidyl choline (DOPC),
 Dilauryl phosphotidyl glycerol (DLPG),
 Distearoyl phosphotidyl serine (DSPS).

10. STRUCTURE OF LIPOSOME AND
PHOSPHOLIPID

11. CLASSIFICATION

16. Methods of liposome preparation
Solvent dispersion
 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
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

18. PREPARATION OF LIPOSOMES BY LIPID FILM
HYDRATION
Mixture of phospholipid and cholesterol were dispersed in organic solvent.
Then, the organic solvent was removed by means of evaporation (using a Rotary
Evaporator at reduced pressure).
When all the solvent get removed, the solid lipid mixture is hydrated using aqueous
buffer.
The lipids spontaneously swell and hydrate to form liposome.

20. SONICATION
 Disruption of LMV suspensions using sonic energy (sonication) typically
produces small, unilamellar vesicles (SUV) with diameters in the range of 15-
50nm.
 The most common instrumentation for preparation of sonicated particles are-
∞ Bath sonicator
∞ Probe tip sonicator
 Probe tip sonicators deliver high energy input to the lipid suspension but
suffer from overheating of the lipid suspension causing degradation.
Sonication tips also tend to release titanium particles into the lipid suspension
which must be removed by centrifugation prior to use. For these reasons, bath
sonicators are the most widely used instrumentation for preparation of SUV.

21. CONT.
Sonication of an LMV dispersion is accomplished by placing a test tube
containing the suspension in a bath sonicator (or placing the tip of the sonicator in
the test tube)
sonicating for 5-10 minutes above the Tc of the lipid.
The lipid suspension should begin to clarify to yield a slightly hazy transparent
solution.
The haze is due to light scattering induced by residual large particles remaining in
the suspension.
These particles can be removed by centrifugation to yield a clear suspension of
SUV.

23. FRENCH PRESSURE CELL
 French pressure cell involves the extrusion of MLV through a small orifice.
 Dispersions of multilamellar vesicles can be reduced in size by extrusion at
high pressures through a French press.
 Dispersions of lipids are placed in the French press and extruded at 20,000
Ibs/in2 at 4°C.

24. CONT.
 Dispersions extruded only once consist of a heterogeneous collection of
vesicles, including ML V, with approximately 60% of the vesicles occurring in
the 250-500 A size range.
 Multiple extrusion of the preparation resulted in a progressive decrease in the
size heterogeneity, and after four extrusions 94% of the vesicles ranged in size
from 315-525 A in diameter. The resulting vesicles are somewhat larger than
sonicated unilamellar vesicles and the technique should be applicable to a
wide variety of lipid compositions.

25. FREEZE-THAWED LIPOSOMES
 Freeze-thawed liposomes SUVs are rapidly frozen and thawed slowly. The
short-lived sonication disperses aggregated materials to LUV.
 The creation of unilamellar vesicles is as a result of the fusion of SUV
throughout the processes of freezing and thawing.
 This type of synthesis is strongly inhibited by increasing the phospholipid
concentration and by increasing the ionic strength of the medium.
 The encapsulation efficacies from 20% to 30% were obtained.

28. REVERSE-PHASE EVAPORATION (REV) TECHNIQUE
The lipid mixture is added to a round bottom flask and the solvent is removed under
reduced pressure by a rotary evaporator.
The system is purged with nitrogen and lipids are re-dissolved in the organic phase
which is the phase in which the reverse phase vesicle will form.
Diethyl ether and isopropyl ether are the usual solvents of
choice.
Emulsion are obtained

29. CONT.
The solvent is removed from an emulsion by evaporation to a semisolid gel under
reduced pressure.
The resulting liposomes are called reverse phase evaporation vesicles (REV).
 This method is used for the preparation of large uni-lamellar and oligo-lamellar
vesicles formulation and it has the ability to encapsulate large macromolecules
with high efficiency.

30. SOLVENT INJECTION METHOD
 The solvent injection methods involve the dissolution of the lipid into an
organic phase (ethanol or ether), followed by the injection of the lipid solution
into aqueous media, forming liposomes.
 There are two method according to the solvent used:
 Ethanol injection method.
 Ether injection method.
 In ethanol injection method, the lipid is injected rapidly through a fine needle
into an excess of other aqueous medium. In ether injection method the lipid is
injected very slowly through a fine needle into an excess of saline or other
aqueous medium.

32. DETERGENT REMOVAL TECHNIQUE
 In this method, phospholipids and a detergent are mixed together to form
micellar mixtures.
 Then the detergent removed from the preparation while the micelles
progressively become richer in phospholipid content and finally the lipids
come together to form single bilayer vesicles.
 Methods such as dialysis, column chromatography, or adsorption onto bio
beads can be used to remove the detergent from the preparation.
 It was reported that this technique yielded homogeneous population of single
layered vesicles with mean diameters of 50-100 nm.

33. CONT.
 Detergents that are commonly used for this purpose are those that have high
critical micelle concentration (CMC).
 Detergents such as sodium cholate, sodium deoxycholate, and octylglycoside.
 The detergent dialysis method for phospholipid vesicle preparation was initially
introduced by Kagawa & Racker. These authors removed cholate or
deoxycholate from lipid-protein mixtures to form lipid vesicles that incorporated
protein.

34. CONT.
 In Milsmann et al. (1978) technique, detergent was removed by a flow through
dialysis cell from phospholipid detergent mixture. It was reported that this
technique yielded homogeneous population of single layered vesicles with mean
diameters of 50-100 nm.
 Removal of deoxycholate from a mixture of phospholipid-deoxycholate
preparation by column chromatography was reported in the literature (Enoch and
Strittmatter, 1979).
 This procedure yielded 100 nm of single layered phospholipid vesicles. In this
procedure the investigators mixed the deoxycholate with phospholipids in the
ratio of 1:2 and then sonicated them. Subsequently removed the detergent by
passing the preparation through a Sephadex G-25 column.

35. MECHANISM OF LIPOSOME FORMATION
The basic part of liposome is formed by phospholipids, which are amphiphilic
molecules (having a hydrophilic head and hydrophobic tail).
Hydrophilic part
This part mainly phosphoric
acid bound to a water
soluble molecule
Hydrophobic part
This part consists of two
fatty acid chains with 10 –
24 carbon atoms and 0 – 6
double bonds in each chain

36. CONT.
 When these phospholipids are dispersed in aqueous medium, they form
lamellar sheets by organizing in such a way that, the polar head group faces
outwards to the aqueous region while the fatty acid groups face each other
and finally form spherical/ vesicle like structures called as liposomes.

37. THERAPEUTIC APPLICATION OF LIPOSOME
1. Liposome as drug/protein delivery vehicles
 Controlled and sustained drug release.
 Enhanced drug solubilisation.
 Altered pharmacokinetics and biodistribution.
 Enzyme replacement therapy and biodistribution.
 Enzyme replacement therapy and lysosomal storage disorders.
2. Liposome in antimicrobial, antifungal and antiviral therapy
 Liposmal drugs
 Liposomal biological response modifiers

38. CONT.
3. Liposome in tumour therapy
 Carrier of small cytotoxic molecules.
 Vehicle for macromolecules as cytokines orgenes.
4. Liposome in gene delivery
 Gene and antisense therapy.
 Genetic (DNA) vaccination.
5. Liposome in immunology
 Immunoadjuvant.
 Immunomodulator.
 Immunodiagnosis.

39. CONT.
6. Liposome as artificial blood surrogates.
7. Liposome as radiopharmaceutical and radio diagnostic carriers.
8. Liposome in cosmetics and dermatology.
9. Liposome in enzyme immobilization and bioreactor technology.