TARGETING METHODS
INTRODUCTION, PREPARATION,
EVALUATION OF LIPOSOMES
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INTRODUCTION
Definition
Liposomes are simple microscopic vesicles in which an aqueousvolume is
entirely enclosed by a Phospholipids bilayer molecule.
Liposome were first produced in England in 1961 by Alec D. Bangham
The size of a liposome ranges from some 20 nm up to several micrometers.
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• Structurally liposomes are concentric bilayered vesicles.
• “Liposome” is derived from the Greek word “ lipo” means fatty constitution and
“soma” means structure.
• Membrane composed of phospholipid and cholesterol bilayer.
• In unmodified liposomes, the phospholipid used is phosphatidylcholine.
• The conformation and content of the phosphatidylcholine in the liposome wall is
virtually identical to the walls of the cells in the body and the walls of important
subcellular organelles.
• A liposome can be viewed as an laboratory model for a cell in the body.
• A Phospholipid is an elongated molecule with a water soluble end water seeking end
{polar} and a fat soluble end { non- polar}
• When in water with the appropriate energy force applied, the fat seeking ends of the
phospholipid molecules align against each other to avoid water, forming a bilayer of
molecules that subsequently collapses into a sphere with the water soluble end of the
molecules lining both the inside of the sphere’ cavity as well as covering the outside of
the sphere.
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ADVANTAGES OF LIPOSOMES IN DRUG DELIVERY.
• Liposomes are biocompatible, completely biodegradable, non- toxic, flexible
and nonimmunogenic.
• Liposomes have both a lipophilic and aqueous environment making it useful
for delivering hydrophobic, amphipathic and hydrophilic medicines.
• The encapsulated drug is protected from degradation.
• Liposomes are extremely versatile in the form which they may be
administered. These forms include suspension, aerosol, gel, cream, lotion
and powder which can be administered through most common routes of
medicinal administration.
• Liposomes are also flexible in their size, and as such they can enclose a wide
size range of molecules.
• Liposomes can aide with active targeting as it has flexibility in coupling with
site specific ligands.
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• Provides selective passive targeting to tumor tissues (liposomal doxorubicin) .
• Increased efficacy and therapeutic index.
• Reduction in toxicity of the encapsulated agent.
• Site avoidance effect (avoids non-target tissues).
• Improved pharmacokinetic effects .
• Flexibility to couple with site-specific ligands to achieve active targeting.
• Enhance drug solubilisation [Amphoterecin,Cyclosporins).
• The frequency of drug administration can be decreased with use of liposomes.
• Liposomes can alter tissue distribution of certain drugs by targeting the
elements of reticuloendothelial system.
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DISADVANTAGES OF LIPOSOMES.
• Liposomes may have leakage and fusion of encapsulated drugs.
• The liposome phospholipid may undergo oxidation and hydrolysis.
• Liposomes have a shorter half- life.
• Liposomes have lower solubility.
• Difficult in large scale manufacturing and sterilization.
• Production cost is high.
• Once administered, liposomes can not be removed.
• Liposomes above a certain size range can block the capillaries causing
embolism.
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components of liposomes:
The structural components of liposomes include:
A. Phospholipids
B. cholesterol
A. General representation of phospholipids:
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PHOSPHOLIPIDS :The liposomes are mainly composed of bilayers of phospholipids that are
separated by an aqueous phase within which drug can be incorporated.
• Phospholipids are a special group of lipids containing phosphate.
• The phospholipids are amphiphilic in nature with a hydrophilic head and lipophilic tail.
• The commonly used phospholipids are phosphatidylcholine and phosphatidylglycerol.
• Phospholidids are the major structural components of biological membrane.
• These are amphipathic molecules in which a pair of hydrophobic acyl hydrocarbon chains
and hydrophilic polar head group phosphocholine are linked together with a glycerol
bridge.
• Molecules of PC are not soluble in water.
• In aqueous media they align themselves closely in planar bilayer sheets in order to minimize
the unfavorable action between the bulk aqueous phase and the long hydrocarbon fatty
chain.Such unfavorable interactions are completely eliminated when the sheets fold on
themselves to form closed sealed vesicles
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Phospholipids
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The most common natural phospholipid is the phospatidylcholine (PC ).
Naturally occurring phospholipids used are :
PC: Phosphatidylcholine.
PE: Phosphatidylethanolamine.
PS: Phosphatidylserine
Synthetic phospholipids used are:
DOPC: Dioleoyl phosphatidylcholine
DSPC: Disteroyl phosphatidylcholine
DOPE: Dioleoyl phosphatidylethanolamine
DSPE: Distearoyl phosphatidylethanolamine
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• The interaction between phospholipids and water takes place at a temperature above the
gel to liquid- crystalline phase transition temperature (TC) which represents the melting
point of the acyl chain.
• When fully hydrated, most phospholipids exhibit a phase change from L-β gel crystalline
to the L- α liquid crystalline state at TC.
• All phospholipids of characteristic TC, which depends on the nature of the polar head
group and on length and degree of unsaturation of the acyl chains.
• Above TC phospholipids are in the liquid crystalline phase, characterized by an increased
mobility of the acyl chains.
• Decrease in temperature below TC induces transition to a more rigid state ( Gel state)
resulting in tightly packed acyl chains and the lipid molecules arrange themselves to form
closed planes oh planar head groups.
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B. Cholesterol:
• Cholesterol stabilizes the Membrane Steroid lipid Interdigitates between
phospholipids.i.e. belowTc , it makes membrane less ordered & above Tc
more ordered.
• Being an amphipathic molecule, cholesterol inserts into the membrane
with its hydroxyl group of cholesterol oriented towards the aqueous
surface and aliphatic chain aligned parallel to the acyl chains in the center
of the bilayer .
• Cholesterol act as fluidity buffer .After intercalation with phospholipid
molecules alter the freedom of motion of carbon molecules in the acyl
Chain .Restricts the transformations of trans to gauche
• Conformations. Incorporated into phospholipid membrane upto 1:1 or
2:1 of cholesterol to PC.
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BELOW Tg
• Incorporation of cholesterol at lesser concentration separates the choline head groups
of phospholipid molecule and eliminates electrostatic and H- bonding interactions
hence make membrane less ordered.
• At higher concentration of CH, the area of membrane occupied by acyl side chains and
CH molecules are greater or equal to that of choline head groups. This retards the chain
tilt ( phenomenon responsible for phase transition)
ABOVE Tg
• Above Tg chain tilt happens in acyl chain of PC molecule. This decreases the freedom
of acyl chain hence membrane remains rigid and condensed with decreased fluidity.
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3 Sphingolipids: Sphingolipids are lipids containing long chain amino
alcohol sphingosine and its derivatives. The most abundant sphingolipid is
sphingomylin, which is similar to phospholipids.
4. Charge inducing substances: The charge inducing substances are
incorporated into liposomes to induce a surface charge and prevent
aggregation. Examples are stearylamine and dipalmitoylphosphate glycerol.
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1. STRUCTURAL PARAMETERS
Based on structural
parameters
MLV OLV UV SUV MUV
Multi lamellar Oligolamellar Unilamellar Small Medium sized LUV
large vesicles vesicles vesicles unilamellar unilamellar Large unilamellar
> 0.5 μm 0.1 – 1 μm { all size ranges} vesicle vesicles vesicles
20- 100 nm
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There are four basic methods of physical/mechanical dispersion
• Hand shaken method.
• Non shaking method.
• Pro – liposomes .
• Freeze drying .
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Mechanical dispersion methods of passive loading
Technique begin with a lipid solution in organic solvent & end up with
lipid dispersion in water
Various components are combined by co-dissolving the lipids in organic
solvent which is then removed by film deposition under vacuum.
After solvent removal the solid lipid mixture is hydrated using aqueous
buffer.
The lipids spontaneously swell & hydrate to form liposomes
The post hydration treatments include vortexing, sonication, freeze
thawing & high pressure extrusion.
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Pro-liposomes:
To increase the surface area of dried lipid film & to facilitate instantaneous hydration
• 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.
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Vesicles prepared by extrusion technique :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 ML
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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.
• The prescence of negative lipids tends to decrease their size , while
increasing cholesterol concentrating gives larger liposomes.
• This process is efficient for encapsulation of water-soluble materials.
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Sonication Method
Probe Sonicator: is employed for dispersions, which require high energy in a small
volume (e.g., high concentration of lipids, or a viscous aqueous phase)
Disadvantage- lipid degradation due to high energy and sonication tips release
titanium particles into liposome dispersion
Bath Sonicator: The bath is more suitable for large volumes of diluted lipids.
Method: Placing a test tube containing the dispersion in a bath sonicator and
sonicating for 5- 10min(1,00,000g) which yield a slightly hazy transparent solution.
Using centrifugation to yield a clear SUV dispersion.
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French pressure cell liposomes
The ultrasonic radiation degrades the lipids, other sensitive compounds, macromolecules
for this extrusion of preformed larger liposomes in a French press under very high
pressure is done. This tech. yields unit or oligo lamellar liposomes of size (30-80nm in
dia.) Includes high cost of press that consists of electric hydraulic press & pressure cell
Liposomes prepared by this method are less likely to suffer from structural defects &
instabilities as observed in sonicated vesicles.
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Freeze Thaw Sonication 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
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Extrusion of preformed large liposomes in french press under very high
pressure. uni or oligo lamellar liposomes of intermediate size (30-80nm ) .
Advantages
Less leakage and more stable liposomes are formed compared to sonicated
forms
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Dried-reconstituted Vesicles
Liposomes obtained by this method are usually “uni or oligo lamellar” of the
order of 1.0um or less in diameter.
SUVs in aqueous phase SUVs with solutes to be entrapped Freeze dried
membrane Solutes in uni lamellar vesicles Solutes in uni or oligo lamellar vesicles.
FST method DRV method Rehydration Film stacks dispersion Aqueous phase
Thawing Sonication (15-30 sec)
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FRENCH PRESSURE CELLS(ULV/OLV):
• Method developed by Barenholtz & Hamilton et al.
• Very useful method in which extrusion of preformed large liposomes in
a French Pressure under very high pressure is carried out .
• This technique yields ULV’s/OLV’s of intermediate size(30-
80nm/depending upon applied pressure).
• Liposomes are more stable.
• Free from structural defects.
• Leakage problem is also less.
• However it has high production cost. 16
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Rapid solvent exchange vesicles (RSEVs)
Lipid mixture is transferred between pure solvent & a pure aq.environment.
Organic sol. of lipids through orifice of syringe under vacuum into a tube containing
aqueous buffer. The tube is mounted on vortexed.
It manifest high entrapment volumes
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SOLVENT DISPERSION:
Ethanol and Ether injection:
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DOUBLE EMULSION VESICLES:
In this method, the outer half of the liposome membrane is created at a
second interface between two phases by emulsification of an organic solution in
water. If the organic solution, which already contains water droplet, is
introduced into excess aqueous medium followed by mechanical dispersion,
multicompartment vesicles are obtained. The ordered dispersion so obtained is
described as a WOW system ( that is double emulsion).
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REVERSE PHASE EVAPORATION VESICLES:
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DETERGENT DEPLETION(REMOVAL) METHODS:
Detergents associate with the phospholipid molecules and serve to screen
the hydrophobic portions of of molecule from water.
The structures formed as a result of this association is known as micelles.
A three stage model of interaction for detergents with lipid
bilayers:
Stage1: At low concentration detergents equilibrates between
vesicular lipid and water phase.
stage2: After reaching a critical detergent concentration, membrane
structure tends to unstable and transforms gradually in to micelles.
stage3: All lipid exists in mixed micelle form.
Three methods are applied for removal of detergent and transition
of mixed micelles to concentric bilayered form.
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Active Loading Techniques
Weak amphipathic bases accumulate in the aqueous phase of lipid vesicles in response to a
difference in pH between the inside and outside of the liposomes (pHin & pHout)
Two steps process generates this pH imbalance and active (remote) loading.
Vesicles are prepared in low pH solution, thus generating low pH within the liposomal
interiors, followed by addition of the base to extra liposomal medium.
Basic compounds, carrying amino groups are relatively lipophilic at high pH and hydrophilic
at low pH.
In two chambered aqueous system separated by membrane liposomes, accumulation occurs
at the low pH side, under dynamic equilibrium conditions.
Thus the un protonated form of basic drug can diffuse through the bilayer
The exchange of external medium by gel chromatography with neutral solution
Weak base doxorubicin, Adriamycin and vincristine which co-exist in aqueous solutions in
neutral and charged forms have been successfully loaded into preformed liposomes via the pH
gradient method.
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INCORPORATION OF DRUGS INTO LIPOSOMES
Drug loading can be carried out by three methods depending on the characteristics of
liposomes and the physical and chemical properties of the drug.
1 Encapsulation – The method is useful for water soluble drugs. Encapsulation involves
hydration of a lipid with an aqueous drug solution. A small volume of dissolved drug is
entrapped in the interlamellar spaces, leading to the formation of liposomes.
2. Partitioning- The drug is dissolved along with phospholipids in a suitable organic solvent.
It is either dried first or added directly to the aqueous phase and the residual solvent is removed
under vaccum. The acyl chains of the phospholipids help in solubilizing the drug molecule.
3. Reverse loading- This method is used for certain drugs ( such as weakacids) that may
exist in both ionized and unionized forms depending on the ph of their environment. Such drug
molecules can be added to an aqueous phase in the unionized state to permeate into liposomes
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through the lipid bilayers. Then, the internal ph of the liposomes is adjusted to create a
charge on the drug molecule. Once ionized, the drug substances loses its lipophilicity and
returns to the external medium.
MECHANISM OF DRUG RELEASE FROM LIPOSOMES
When liposomes are injected into the animal or human body, various hydrolytic or lipolytic
enzymes act on the amide or ester bonds formed by the natural lipids. The enzymes cut the
acyl chains to form lysolipids which destabilize the lipid layer and release the entrapped
bioactive components.
The release of the drug from liposome depends on the composition of
liposome, type of drug encapsulated and nature of the cell. Drug is released from liposomes
by the following mechanisms.
1. Stable adsorption to the cell surface.
2. Endocytosis.
3. Fusion with the plasma membrane.
4. Transfer of lipid into the cellular or subcellular membrane.
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Evaluation of liposomes
The liposomes prepared by various techniques are to be evaluated for their physical, chemical
as well as biological properties, has these influence the behavior of liposomes in vivo.
Physical properties
1. Particle size
Both particle size and particle size distribution of liposomes influence their physical stability.
These can be determined by the following method.
a) Laser light scattering
b) Transmission electron microscopy
2. Surface charge
The positive, negative or neutral charge on the surface of the liposomes is due to the
composition of the head groups.
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The surface charge of liposomes governs the kinetic and extent of
distribTuhet isourfnac ei nch avrgiev ofo li,p oasosm wes geolvler nas sth ei nkintetirca ancdt eixotent owf diistthrib utthioen target cells. The
in vivo, as well as interaction with the target cells.
methoTdh ei mnevthodl ivnveovde diin thteh mee amsuremaesntu ofr seumrface nchta rogef i s sbuaserdf oanc freee fclohwarge is based on
electrophoresis of MLVs.
free flow
➢ It eutlileizcest ar coelpluhlosoe raceestaites p loatfe dMippeLd Vin ssod.ium borate buffer of pH 8.8.
It ut➢iliAzbeoust 5an mcoelelsl ouf lliopisd esa mapclees tarae taepp lpielda otne to dthei pplpatee, dwh iicnh iss tohedn ium borate buffer
subjected to electrophoresis at 4 ͦc for 30 mins.
of pH ➢8T.h8e .liposomes get bifurcated depending on their surface charge.
This technique can be used for determining the heterogeneity of charges in the
Abolipuotso 5men s umspeonslioens a so wfel ll aisp toi de tescat amny pimlpeusri tiaesr seuc ha aps pfatltiye acdid son to the plate, which
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is then subjected to electrophoresis at 4 ͦc for 30 mins.
The liposomes get bifurcated depending on their surface charge.
This technique can be used for determining the heterogeneity of
charges in the liposome suspension as well as to detect any impurities
such as fatty acids
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3. Percent drug encapsulated
Quantity of drug entrapped in the liposomes helps to estimate the behavior of
the drug in biological system.
Liposomes are mixture of encapsulated and unencapsulated drug fractions the
% of drug encapsulation is done by first separating the free drug fraction from
encapsulated drug fraction.
The encapsulated fraction is then made to leak off the liposome into aqueous
solution using suitable detergents.
The methods used to separate the free drug from the sample are:
a. Mini column centrifugation method
b. Protamine aggregated method
❑ Determined by ion exchange chromatography, gel exclusion chromatography
and minicolumn centrifugation.
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4. Phase behavior
At transition temperature liposomes undergo reversible phase transition.
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The tc is the in4.d Pihcaaset iboehna voiofr stability permeability and also indicates the
region of drug en➢traApt tmranesintiot.n temperature liposomes undergo reversible
phase transition.
Determined b➢y DThSe Ctc isa tnhed i nfdriceaetizone o-f fstraabiclittyu preer meelaebiclittyr oand amlsoi croscopy.
indicates the region of
drug entrapment.
5. Drug Release ➢RaDteone by DSC.
The rate of drug 5r. eDlreuag sRee lefarsoe mRa ttehe liposomes can be determined by in vivo
The rate of drug release from the liposomes can be determined
assays which helbpy sin t voiv op arsesadyisct the pharmacokinetics and bioavailability of the
drug. However inwh vicihv hoel pss ttuo pdrieedisct athree p hfaormuancodk itnoet icbse a nmd boioraeva iclaobmilityp lete.
of the drug.
However in vivo studies are found to be more complete.
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• Lamellarity – Determined by small angle X- ray scattering freeze
fracture electron microscopy.
• Electrical surface potential and surface ph – Determined by zeta
potential measurements and ph sensitive probes.
Biological Characterization
Sterility – Determined by aerobic or anaerobic cultures.
Pyrogenisity – Determined by LAL test
Animal toxicity- Monitoring survival rates, histology &pathology
Plasma Stability- Cytotoxicity Assay, HPLC Assay
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Chemical properties
1. Determination of phospholipids/ Phospholipid Concentration
The phospholipid content of liposomes can be determined directly by two assays, bartlett
assay and steward assay.
(a) BARTLETT ASSAY
This method of determining the phospholipid is very sensitive and may produce results in
the presence of even trace amounts of inorganic phosphate. Therefore, borosilicate glass
tubes and double-distilled water is used.
(i) Initially the phosphorous present in the lipid bilayer of the sample is hydrolyzed to
inorganic phosphate.
(ii) Then ammonium molybdate is added to convert inorganic phosphate to
phosphomolybdic acid(PMA).
(iii) The sample is then treated with aminonaphthylsulphonic acid to quantitatively reduce
the PMA to a blue-coloured compound.
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(iv) The intensity of the blue colour produced can be measured by spectrophotometric means and
the value is plotted on the standard curve to obtain the content of phospholipids.
(b) STEWARD ASSAY : This assay overcomes the drawbacks of bratlett assay, but cannot
be used to mixture of unknown phospholipids.
(i) A standard curve is prepared by treating known concentration of phospholipids in chloroform
with 0.1 M solution of ammonium ferrothiocyanate reagent).
(ii) The sample are also treated with the same reagent and the optical density is determined at
485 nm.
(iii) The absorbance of the sample can be plotted on the standard curve to obtain the
concentration of phospholipids.
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• Cholesterol analysis
(a) Qualitative analysis : Performed using a capillary column filled with fused silica.
(b) Quantitative analysis : The sample is reacted with a reagent (containing ferric
perchlorate, ethyl acetate and H₂SO₄) and the absorbance of purple coloured complex is
measured at 610 nm.
• Drug concentration- Appropriate methods given in the monograph for the individual
drug.
• Phospholipid peroxidation- Determined by UV absorbance, TBA for endoperoxidase,
iodometric for hydroperoxidase and GLC.
• Phospholipid hydrolysis – Determined by HPLC, TLC and fatty acid concentration.
• Cholesterol auto- oxidation : Determined by Hplc and TLC.
• Osmolarity: Determined using a osmometer.
• Ph : Determined using a ph meter.
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Stability of Liposomes :
❑ Industrially produced liposomes will reach the patient only after a
prolonged time. Thus, during storage or transport, the liposome dispersion
should not change its characterization or lose the associated drug or
antigen. In general, a shelf life of at least one year is a minimum
prerequisite for liposomes.
❑ The stability of any pharmaceutical product is the capability of the delivery
system in the prescribed formulation to remain within defined or pre-
established limits for a predetermined period of time.
The stability in vitro which covers the stability aspects prior to the administration
of the formulation & with regard to the stability of the constitutive lipids.
The stability in vivo which covers the stability aspects once the formulation is
administered via various routes to the biological fluids. It includes stability aspects
in blood if administered by systemic route or in gastrointestinal tract if administered
by oral or per oral routes.
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Chemical stability : Phospholipid form the backbone of the liposome and hence
their chemical stability is important.
The performance of phospholipid bilayer can be affected by hydrolysis of the
ester bonds or peroxidation of unsaturated acyl chain.
Chemical degradation can be prevented by the following.
1. Using freshly prepared and purified solvent.
2. Manufacturing the liposomes in an oxygen- free environment.
3. Avoiding procedures that require high temperatures.
4. Using complexing agents such as EDTA to remove traces of metal that can
potentiate oxidation.
5. Including antioxidants as components of lipid membranes
6. Storing prepared liposomes in an inert atmosphere.
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PHYSICAL STABILITY:
Physical processes that affects shelf life include loss of liposome associated
drug and charge in size and aggregation or fusion of liposome. The liposomes
are considered to be physically stable if the size distribution and the ratio of
lipid to active agent of liposomes remains constant. The stability can be
improved by the use of an aqueous dispersion, by proper selection medium and
bilayer components by freeze drying of liposomes or by the use of proliposomes
approach.
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Stability in vitro:-Stability in vitro mainly covers the chemical stability of
the constitutive lipids under the various accelerated or long term storage
conditions.
Storage temp of these dispersions must be defined & controlled
Liposomal phospholipids can undergo degradation such as oxidation &
hydrolysis. Either as a result of these changes, liposomes maintained in aqueous
dispersion may aggregate fuse and as a result may dump their contents.
Lipid oxidation & Peroxidation :
Lipid peroxidation measurement is based on disappearance of unsaturated fatty
acids or appearance of conjugated dienes.
It can be prevented by minimizing use of unsaturated lipids, use of oxygen,
argon or nitrogen environment, use of antioxidant such as Alpha tocopherols or
BHT or use of light resistant containers for storage of liposomal preparations
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LIPID HYDROLYSIS: The formation of lyso- phospholipid from
phospholipid is a measure of the chemical instability of the lipid leading to the
enhanced permeability of the liposomal contents. This results from the
hydrolysis of the ester bond at the C- position of the glycerol moiety.
The inclusion of a charged molecules in the bilayer shifts the electrophoretic
mobility and makes it positive with the inclusion of the stearylamine or
negative with dicetyl phosphate , thus prevents liposomal fusion or swelling
or aggregation.
LONG TERM ACCELERATED STABILITY:
High temp testing(>25 C) is universally used for heterogeneous products.
Various laboratories store their products at temp ranging from 4 C to 50 C.
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Stability after systemic administration
Two most frequently encountered biological events that the administered
liposomal system undergoes are phagocytosis or antigen presentation via the
macrophages of the RES system.
Opsonins which are proteinaceous components of serum adsorb onto the
surface of liposomes thus making these exogenous materials more palatable &
conductive to phagocytes.
High density lipoprotein removes phospholipid molecules from bilayered
vesicular systems.
The molecular origin of these interactions are mostly long range electrostatic,
Vander waals & short range hydrophobic interactions of particulate surface with
macromolecules in the
Serum.
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Limitations of liposome technology
• Sterilization
• Encapsulation efficiency
• Active targeting
• Gene therapy
• Lysosomal degradation
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Sterilization
• Identification of a suitable method for sterilization of liposome
formulations is a major challenge because phospholipids are thermolabile
and sensitive to sterilization procedures involving the use of heat, radiation
and/or chemical sterilizing agents. The method available for sterilization of
liposome formulations after manufacture is filtration through sterile 0.22
,urn membranes.
• However, filtration is not suitable for large vesicles (>0.2 /zm) and also is
not able to remove viruses.
• Sterilization by other approaches such as irradiation and exposure to
chemical sterilizing agents are not recommended because they can cause
degradation of liposome components and may leave toxic contaminants
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3. Encapsulation efficiency
• Liposome formulation of a drug could only be developed if the encapsulation
efficiency is such that therapeutic doses could be delivered in a reasonable amount
of lipid, since lipids in high doses may be toxic and also cause non-linear (saturable)
pharmacokinetics of liposomal drug formulation.
• Some new approaches that provide high encapsulation efficiencies for
hydrophilic drugs have been developed. For instance, active loading of the
amphipathic weak acidic or basic drugs in empty liposomes can be used to
increase the encapsulation efficiency .
• However, active loading is not suitable for hydrophobic drugs such as
paclitaxel for which encapsulation efficiency is < 3 mole% mainly due to the low
affinity of drugfor the lipid bilayers
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6. Lysosomal degradation
•Once the liposome has reached the target cell the efficacy is determined not only by the
amountof drug associated with the cell, but also by the amount of drug reaching the ‘target
molecule inside the cells.
• Immunoliposomes may deliver the drug to the cells selectively but the pharmacological
activity depends on the ability of intact drug to diffuse into cytoplasm from the endosomes in
sufficient amounts.
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Applications of liposomes in drug delivery
• 1. Formulation aid
• Hydrophobic drugs such as cyclosporin and paclitaxel are usually formulated in surfactants
and organic cosolvents for systemic administration in humans. These solubilizers may cause
toxicity at the doses needed to deliver the drug. In contrast, liposomes are made up of lipids
which are relatively non-toxic, non-immunogenic, biocompatible and biodegradable
molecules, and can encapsulate a broad range of water-insoluble (lipophilic) drugs.
• Currently, liposomes or phospholipid mixtures are being used as excipients for preparing
better tolerated preclinical and clinical formulations of several lipophilic, poorly water
soluble drugs such as amphotericin B. In preclinical studies, liposomes have been evaluated
as a vehicle for the delivery of paclitaxel and its analogs as an alternative to the cremophor/
ethanol vehicle
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• Paclitaxel liposomes were able to deliver the drug systemically and increase the therapeutic
index of paclitaxel in human ovarian tumor models .
• 2. Intracellular drug delivery
• Drugs with intracellular targets/receptors are required to cross the plasma membrane for
pharmacological activity.
• Liposomes can be used to increase cytosolic delivery of certain drugs such as N-
(phosphonacetyl)-L-aspartate (PALA) which are normally poorly taken up into cells.
• 3. Site-specific targeting
• Site-specific delivery, the concept first proposed by Paul Ehrlich which involves the
delivery of a larger fraction of drug to the target site and therefore, reducing exposure to normal
tissues.
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• 3. Sustained release drug delivery
• Sustained release systems are required for drugs such as cytosine
arabinoside (Ara-C) that are rapidly cleared in vivo and require plasma
concentrations at therapeutic levels for a prolonged period for optimum
pharmacological effects.
• 4. Gene therapy
• A number of systemic diseases are caused by lack of enzymes/factors
which are due to missing or defective genes. In recent years, several
attempts have been made to restore gene expression by delivery of the
relevant exogenous DNA or genes to cells
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Site-avoidance delivery
• Drugs used in the treatment of diseases like cancer usually have a narrow
therapeutic index (TI) and can be highly toxic to normal tissues.
• The toxicity of these drugs may be minimized by decreasing delivery to
critical normal organs.
• It has been shown that even a small reduction in distribution of the drug
to critical organs by encapsulation in liposomes can significantly reduce
the drug toxicity .
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Therapeutic uses of liposomes
1. Treatment of cancer- Antitumor drugs such as actinomycin D, vincoblastin,
methotrexate can be enclapsulated into liposomes for drug delivery to the
target tissue.
2. Disease caused by intracellular parasite : Drug loaded liposomes can be used
in the treatment of rickettsial infections, malaria , viral diseases etc.
3. Metal toxicity : Liposomal EDTA can be used in heavy metal poisoning.
Chelating agents cannot pass through the biological membranes, so these are
incorporated in liposomes so that they can easily pass through the biological
membrane.
4. Diabetes: Researchers have studied the potential of liposomes as carriers for
oral administration of insulin. Studies have shown that liposomes have
protective effects against the proteolytic digestive enzymes, pepsin and
pancreatin.
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• Liposomes as radiopharmaceutical carriers: For diagnoistic
purpose liposomes may act as carriers for radio
pharmaceuticals.
• Liposomes in cosmetics and dermatology: the potential of
liposomes as topical drug delivery system has been
explored . These are highly effective in the treatment of
skin disorders.
• Liposomes in immunology: It is used as immunoadjuvant.
Examples are antigen influenza subunit antigens,
immunodiagnostics and immunomodulators.
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Liposomes in Ophthalmic therapy: Liposomes have the
ability to remain in intimate contact with the corneal and
conjuctival surfaces, there by increasing ocular absorption.
Vaccine adjuvant: Vaccine can be prepared by entrapping
microbes, soluble antigen , DNA inside liposomes
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MARKETED PREPERATIONS:
• Liposome ( Doxil™) Doxorubicin = Kaposi’ sarcoma
• Liposome (EVACT ™) = breast cancer
• Liposome(DaunoXome™) Daunosome = Advanced
Kaposi’ sarcoma,small cell lung cancer, leukaemia & solid
tumour .
• Liposome ( VincaXome™) Vincristine = Solid tumour.
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QUESTIONS
• Describe the methods of active and passive targeting using
particulate carriers.Describe use of liposomes for drug
targeting. 20 marks
• Describe the thin film hydration technique of preparation of
liposomes. 5marks
• Discuss the different methods of preparation and
applications of liposomes. 10 marks
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References
• Jain N.K., Controlled & Novel Drug Delivery, CBS Publications, New Delhi
• Jain N.K., Advances in Controlled & Novel Drug Delivery, CBS Publications,
New Delhi.
• Vyas S.P. and Khar R.K., Targeted & Controlled drug delivery- Novel Career
System, CBS Publications, New Delhi.
• Chien Y, Novel Drug Delivery System, Mercel Decker Publications..
• Allen, Theresa M. “Liposomal Drug Formulations: Rationale for Development
and What We Can Expect for the Future.” Drugs 56: 747-756, 1998.
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