1.
LIPOSOMES
SIVASWAROOP YARASI

2.
DEFINITION:
• Liposomes are concentric bilayer vesicles in which an aqueous volume is entirely
enclosed by a membranous lipid bilayer mainly composed of natural or
synthetic phospholipids.
• Liposomes were first produced in England in 1961 by Alec D. Bangham.
• The size of a liposome ranges from some 20 nm up to several micrometres.

3.
ADVANTAGES:
• Provides selective passive targeting to tumor tissues (Liposomal doxorubicin).
• Increased efficacy and therapeutic index.
• Increased stability via encapsulation.
• Reduction in toxicity of the encapsulated agents.
• Site avoidance effect.
• Improved pharmacokinetic effects (reduced elimination, increased circulation life
times).
• Flexibility to couple with site specific ligands to achieve active targeting.
• Biocompatible and completely biodegradable

4.
DISADVANTAGES:
• Production cost higher.
• Leakage and fusion of encapsulated drug.
• Short half life.
• Sometimes phospholipid undergoes oxidation and hydrolysis like reaction.
• Low solubility.

5.
STRUCTURE AND COMPONENTS OF LIPOSOME:
• The lipid molecules are usually phospholipids-amphipathic moieties with a
hydrophilic head group and two hydrophobic tails.
• Composition of liposomes:
• The basic components of liposomes are phospholipids which are stabilised by
cholesterol, with other stabilisers sometimes added to the mixture depending on the
specific use of the liposome.
• Phospholipids
• Cholesterol
• Polymers.

6.
• Phospholipids:
• Major component
• Two types
• Synthetic: Examples DOPC: Dioleoyl phosphatidylcholine, DSPC: Disteroyl phosphatidylcholine, DOPE:
phosphatidylethanolamine, DSPE: Distearoyl phosphatidylethanolamine.
• Natural: most common is the phosphatidylcholine (PC) also known as lecithin. Originated from animal (hen
egg) and vegetable (soya bean). Other examples are phosphatidyl serine, phosphatidyl inositol,
ethanolamine.
• Cholesterol:
• Cholesterol and its derivatives are often included in liposomes for
• Decreasing the fluidity or micro viscosity of the bilayer.
• Reducing the permeability of the membrane to water soluble molecules.
• Stabilizing the membrane in the presence of biological fluids such as plasma.

7.
• Polymers:
• Synthetic phospholipids with diacetylene group in the hydrocarbon chain polymerize
when exposed to U.V, leading to formation of polymerized liposomes having
significantly higher permeability barriers to entrapped aqueous drugs. E.g. for other
Polymerizable lipids are – lipids containing conjugated diene, methacrylate etc.

8.
MECHANISM OF LIPOSOME FORMATION:
• The basic part of liposome is formed by phospholipids, which are amphiphilic molecule
(having a hydrophilic head and hydrophobic tail).
• The hydrophilic part is mainly phosphoric acid bound to a water soluble molecule, whereas,
the hydrophobic part consists of two fatty acid chains with 10 – 24 carbon atoms and 0 – 6
double bonds in each chain.
• 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.
• The polar portion remains in contact with aqueous region along with shielding of the non-
polar part.

9.
• When phospholipids are hydrated in water, along with the input of energy like sonication,
shaking, heating, homogenization, etc.
• It is the hydrophilic/ hydrophobic interactions between lipid – lipid, lipid – water molecules
that lead to the formation of bilayered vesicles in order to achieve a thermodynamic
equilibrium in the aqueous phase,
• The reasons for bilayered formation include:
• The unfavorable interactions created between hydrophilic and hydrophobic phase can be minimized
by folding into closed concentric vesicles.
• Large bilayered vesicle formation promotes the reduction of large free energy difference present
between the hydrophilic and hydrophobic environment.
• Maximum stability to supramolecular self assembled structure can be attained by forming into
vesicles.

10.
CLASSIFICATION:
• Based on the structural parameters.
• Unilamellar vesicles:
• Small unilamellar vesicles (SUV): size ranges from 20-40 nm.
• Medium unilamellar vesicles (MUV): size ranges from 40-80 nm.
• Large unilamellar vesicles (LUV): size ranges from 100-1000 nm.
• Oligolamellar vesicles (OLV): these are made up of 2-10 bilayers of lipids surrounding
a large internal volume.
• Multilamellar vesicles (MLV): they have several bilayers. They can compartmentalize
the aqueous volume in an infinite number of ways. They differ according to way by
which they are prepared.

11.
• Based on method of liposome preparation:
Type Size and made of
REV Single or oligolamellar vesicles made
by reverse-phase evaporation
method.
MLV-REV Multilamellar vesicles made by reverse
phase evaporation method.
SPLV Stable plurilamellar vesicles.
FATMLV Frozen and thawed MLV.
VET Vesicles prepared by extrusion
technique.
DRV Dehydration-rehydration method.

12.
• Based on composition and application:
Type Property
Conventional liposomes Neutral or negatively charged phospholipids and
cholesterol.
Fusogenic liposomes (RSVE) Reconstituted sendai virus envelops.
pH sensitive liposomes Phospholipid such as PE and DOPE with either
CHEMS or OA.
Cationic liposomes Cationic lipids with DOPE.
Long circulatory (stealth) liposomes
(LCL)
Neutral high Tc˚, cholesterol and 5-10% of PEG-
DSPE.
Immuno-liposomes CL or LCL with attached monoclonal antibody or
recognition sequence.

13.
METHODS OF LIPOSOMES PREPARATION:
• Two types:
• Passive loading technique: Loading of the entrapped agents before/ during the
manufacture procedure.
• Active loading technique: Certain types of compounds with ionizable groups & those
with both lipid & water solubility can be Introduced into liposomes after the
formation of intact vesicles.

14.
PASSIVE LOADING TECHNIQUE:
• These are classified into three types based on the modes of dispersion. They are:
• Mechanical dispersion methods
• Solvent dispersion methods
• Detergent solubilizing methods.

15.
MECHANICAL DISPERSION METHODS:
• Lipid is solubilised in organic solvent, drug to be entrapped is solubilise in
aqueous solvent, the lipid phase is hydrated at high speed stirring due to affinity
of aqueous phase to polar head it is trapped in lipid vesicles.
• Types:
• Lipid film hydration by hand shaking, freeze drying or non-hand shaking
• Micro emulsification
• Sonication
• French pressure cell
• Membrane extrusion
• Dried reconstituted vesicles.

16.
THIN FILM HYDRATION USING HAND SHAKING AND NON HAND SHAKING
METHODS:
• Lipids are casted as stacks of film from their organic solution using flash rotatory
evaporator under reduced pressure (or by hand shaking) and then casted film is
dispersed in an aqueous medium.
• Upon hydration the lipids swell and peel off from the wall of the RBF and
vesiculate forming MLVs.
• Mechanical energy required for the swelling of lipids and dispersion of casted
lipid film is imparted by hand shaking or by exposing the film to a steam of water
saturated nitrogen for 15 min followed by swelling in aqueous medium without
shaking.

17.
SONICATION:
• Most extensively used method for the preparation of SUV.
• MLVs are sonicated either with a bath type sonicator or a probe sonicator under a
passive atmosphere.
• Disadvantages:
• Very low internal volume/encapsulation efficacy.
• Degradation of phospholipids and compounds to be encapsulated.
• Elimination of large molecules.
• Metal pollution from probe tip.
• Presence of MLV along with SUV.

18.
Probe sonicator:
• Tip of a sonicator is directly engrossed into the liposome dispersion.
• Energy input into lipid dispersion is very high results in local hotness.
• With the probe sonicator, titanium will slough off and pollute the solution.
Bath sonication:
• The liposome dispersion in a cylinder is placed into a bath sonicator.
• Controlling temperature usually easier in this method.
• The material being sonicated can be protected in a sterile vessel, dissimilar the
probe units, or under an inert atmosphere

19.
FRENCH PRESSURE CELL: EXTRUSION:
• Involves the extrusion of MLV through a small orifice.
• The size of liposomes is reduced by gently passing them through polycarbonate
membrane filter of defined pore size at lower pressure.
• Used for preparation of LUVs and MLVs
• French press vesicle appears to recall entrapped solutes significantly longer than
SUVs do, produced by sonication or detergent removal.

20.
Advantages:
• Less leakage.
• More stable liposomes are formed compared to sonicated forms.
• Gentle handling of unstable materials.
Disadvantages:
• High temperature is difficult to attain.
• Working volumes are comparatively small (about 50 mL as the maximum).

21.
SOLVENT DISPERSION METHODS
• In this method, lipids are first dissolved in organic solvent, which is then brought
into contact with aqueous phase containing material to be entrapped in
liposome under rapid dilution and rapid evaporation of organic solvent.
• Types:
• ethanol injection
• ether injection
• double emulsion
• reverse phase evaporation vesicles
• stable pluri lamellar vesicles

22.
ETHER INJECTION:
• A solution of lipids dissolved in diethyl ether or ether/methanol mixture is slowly
injected to an aqueous solution of the material to be encapsulated at 55-65°C or
under reduced pressure.
• The subsequent removal of ether under vacuum leads to the formation of
liposomes.
• The main drawbacks of the method are population is heterogeneous (70-190 nm)
and the exposure of compounds to be encapsulated to organic solvents or high
temperature.

23.
ETHANOL INJECTION:
• A lipid solution of ethanol is rapidly injected to a vast excess of buffer.
• The MLVs are immediately formed.
• The drawbacks of the method are that the population is heterogeneous (30-110
nm), liposomes are very dilute, it is difficult to remove all ethanol because it forms
azeotrope with water and the possibility of various biologically active
macromolecules to inactivation in the presence of even low amounts of ethanol.

24.
REVERSE PHASE EVAPORATION:
• First water in oil emulsion is formed by brief sonication of a two phase system
containing phospholipids in organic solvent (diethylether or sopropylether or
mixture of isopropyl ether and chloroform) and aqueous buffer.
• The organic solvents are removed under reduced pressure, resulting in the
formation of a viscous gel.
• The liposomes are formed when residual solvent is removed by continued rotary
evaporation under reduced pressure. The method has been used to encapsulate
small and large macromolecules.

25.
DETERGENT SOLUBILIZING METHODS.
• In this method the phospholipids are brought into intimate contact with
aqueous phase via detergent which associate with phospholipids molecules and
serve to screen the hydrophobic portions of the molecules from water.
Detergent (cholate, alkyl glycoside, Triton x-100) removed from mixed micelles
by
• Dialysis
• Column chromatography
• Dilution.

26.
BY DIALYSIS:
• The detergents at their critical micelles concentrations have been used to solubilize
lipids.
• As the detergent is removed the micelles become progressively richer in phospholipid
and finally combine to form LUVs.
• The detergents can be removed by dialysis.
• The advantages of detergent dialysis method are excellent reproducibility and
production of liposome populations which are homogenous in size.
• The main drawback of the method is the retention of traces of detergent(s) within the
liposomes.

27.
GEL-PERMEATION CHROMATOGRAPHY:
• In this method, the detergent is depleted by size special chromatography.
Sephadex G-50, Sephadex G-l 00, Sepharose 2B-6B, and Sephacryl S200-S1000
can be used for gel filtration.
• The liposomes do not penetrate into the pores of the beads packed in a
column.
• At slow flow rates, the separation of liposomes from detergent monomers is
very good.

28.
CHARACTERIZATION OF LIPOSOMES:
• Liposomes produced by different methods have varying physicochemical
characteristics, which leads to differences in their
• in vitro (sterilization and shelf life) and
• in vivo (disposition) performances.

29.
SIZE AND SIZE DISTRIBUTION:
• When liposomes are intended for inhalation or parenteral administration, the size
distribution is of primary consideration.
• Various techniques of determing the size of the vesicles include:
• microscopy (optical microscopy, negative stain transmission electron microscopy ,
cryo-transmission electron microscopy, freeze fracture electron microscopy and
scanning electron microscopy ),
• Diffraction and scattering techniques (laser light scattering, and photon correlation
spectroscopy)and
• hydrodynamic techniques (field flow fractionation, gel permeation and
ultracentrifugation).

30.
PERCENT DRUG ENCAPSULATION
• The amount of drug encapsulated/ entrapped in liposome vesicle is given by percent drug
encapsulation.
• The formulation consists of both free (unencapsulated) and encapsulated drug.
• Column chromatography can be used to estimate the percent drug encapsulation of liposomes.
• So as to know the exact amount of drug encapsulated, the free drug is separated from the
encapsulated one.
• Then the fraction of liposomes containing the encapsulated drug is treated with a detergent, so
as to attain lysis, which leads to the discharge of the drug from the vesicles into the surrounding
medium.
• This exposed drug is assayed by a suitable technique which gives the percent drug encapsulated
from which encapsulation efficiency can be calculated

31.
• Trapped volume per lipid weight can also give the percent drug encapsulated in a
liposome vesicle.
• It is generally expressed as aqueous volume entrapped per unit quantity of lipid,
μl/μmol or μg/mg of total lipid.
• In order to determine the trapped volume, various materials like radioactive
markers, fluorescent markers and spectroscopically inert fluid can be used.
• % Encapsulation
• Drug entrapped in liposomes/ Total drug added x 100

32.
SURFACE CHARGE:
• Charge on the liposome surface plays a key role in the in vivo disposition, it is
essential to know the surface charge on the vesicle surface.
• Two methods namely:
• free-flow electrophoresis
• zeta potential measurement.
• The surface charge can be calculated by estimating the mobility of the liposomal
dispersion in a suitable buffer (determined using Helmholtz– Smolochowski
equation)

33.
VESICLE SHAPE AND LAMELLARITY
• Various electron microscopic techniques can be used to assess the shape of the
vesicles.
• The number of bilayers present in the liposome, i.e., lamellarity can be
determined using:
• Freeze fracture electron microscopy and
• 31P-Nuclear magnetic resonance analysis.
• The surface morphology of liposomes can be assessed using
• freeze-fracture and
• freeze-etch electron microscopy .

34.
PHOSPHOLIPID IDENTIFICATION AND ASSAY:
• The chemical components of liposomes must be analyzed prior to and after the
preparation.
• To estimate the phospholipid concentration:
• Barlett assay , Stewart assay and thin layer chromatography can be used
• A spectrophotometric method is used to quantify total phosphorous, which
measure the intensity of blue color developed at 825 nm against water.
• Techniques can be used to determine the cholesterol concentration:
• Cholesterol oxidase assay or ferric perchlorate method and Gas liquid chromatography

35.
STABILITY OF LIPOSOMES:
• The therapeutic activity of the drug is governed by the stability of the liposomes
right from the manufacturing steps to storage to delivery.
• A well designed stability study includes the evaluation of its physical, chemical
and microbial parameters along with the assurance of product’s integrity
throughout its storage period.
• Physical:
• morphology, size and size distribution of the vesicles are important parameters to
assess the physical stability.
• In order to monitor this, a variety of techniques like light scattering and electron
microscopy can be used to estimate the visual appearance (morphology) and size of
the vesicles.

36.
• Chemical:
• Phospholipids are chemically unsaturated fatty acids that are prone to oxidation and
hydrolysis, which may alter the stability of the drug product.
• Indeed chemical reaction can be induced even by light, oxygen, temperature and
heavy metal ion
• Liposomes can be prevented from oxidative degradation by protecting them from
light, by adding anti-oxidants such as alpha – tocopherol or butylated hydroxyl
toluene (BHT), producing the product in an inert environment (presence of nitrogen or
Argon) or by adding EDTA to remove trace heavy metals

37.
IN-VITRO DRUG RELEASE:
• In vitro drug release can be performed using the dialysis tube diffusion
technique.
• The dialysis bag membrane should be selected following screening of various
membrane, no drug adsorption may occur and the membrane should be freely
permeable to the active ingredient.
• The entire system is kept at 37 degree C under continuous magnetic stirring
and the receptor medium is closed to avoid evaporation of the dissolution
medium.
• Samples of the dialysate are taken at various time intervals and assayed for the
drug by HPLC, spectrophotometer or any other convenient method.

38.
APPLICATIONS:
• As drug delivery carriers.
• Site-avoidance delivery
• Site specific targeting:
• Intracellular drug delivery
• Sustained release drug delivery
• Immunological adjuvants in vaccine
• In gene delivery.
• Enzyme replacement therapy.
• Chelation therapy for treatment of heavy metal poisoning.
• Liposomes in antiviral/anti microbial therapy.
• In multi drug resistance.
• In cosmetology