International Journal of Current Pharmaceutical Research

ISSN- 0975-7066

Vol 3, Issue 2, 2011

ReviewArticle

LIPOSOMES:ANOVELDRUGDELIVERYSYSTEM

PRIYANKARKULKARNI*,JAYDEEPDYADAV,KUMARAVAIDYA
DepartmentofPharmaceuticalSciences,N.D.M.V.P.Samajs,CollegeofPharmacy,GangapurRoad,Nasik422002,Maharashtra,India.
Email:pukool32@gmail.com
Received:30Sep2010,RevisedandAccepted:04Nov2010
ABSTRACT
Almostfromthetimeoftheirdiscoverythedemonstrationoftheirentrapmentpotential,liposomalvesicleshavedrawnattentionofresearchersas
potential carriers of various bioactive molecules that could be used for therapeutic applications. Liposomes have been widely investigated since
1970 as drug carriers for improving the delivery of therapeutic agents to specific sites in the body. Liposomes, which are biodegradable and
essentially nontoxic vehicles, can encapsulate both hydrophilic and hydrophobic materials, and are utilized as drug carriers in drug delivery
systems.Asaresult,numerousimprovementshavebeenmade,thusmakingthistechnologypotentiallyusefulforthetreatmentofcertaindiseases
in the clinics. Many liposomebased DNA delivery systems have been described, including molecular components for targeting given cell surface
receptors or for escaping from the lysosomal compartment. The insight gained from clinical use of liposome drug delivery systems can now be
integratedtodesignliposomesthatcanbetargetedontissues,cellsorintracellularcompartmentswithorwithoutexpressionoftargetrecognition
molecules on liposome membranes. The success of liposomes as drug carriers has been reflected in a number of liposomebased formulations,
whicharecommerciallyavailableorarecurrentlyundergoingclinicaltrials.
Keywords:Liposomes,GeneDelivery,Phospholipid.

INTRODUCTION
Themethodbywhichadrugisdeliveredcanhaveasignificanteffect
on its efficacy. Some drugs have an optimum concentration range
withinwhichmaximumbenefitisderived,andconcentrationsabove
orbelowthisrangecanbetoxicorproducenotherapeuticbenefitat
all.Ontheotherhand,the veryslowprogressintheefficacy ofthe
treatment of severe diseases, has suggested a growing need for a
multidisciplinaryapproachtothedeliveryoftherapeuticstotargets
intissues.Fromthis,newideasoncontrollingthepharmacokinetics,
pharmacodynamics, nonspecific toxicity, immunogenicity,
biorecognition, and efficacy of drugs were generated. These new
strategies, often called drug delivery systems (DDS), are based on
interdisciplinary approaches that combine polymer science,
pharmaceutics, bioconjugate chemistry, and molecular biology. To
minimizedrugdegradationandloss,topreventharmfulsideeffects
and to increase drug bioavailability and the fraction of the drug
accumulated in the required zone, various drug delivery and drug
targeting systems are currently under development. Among drug
carriers one can name soluble polymers, microparticles made of
insoluble or biodegradable natural and synthetic polymers,
microcapsules, cells, cell ghosts, lipoproteins, liposomes, and
micelles. The carriers can be made slowly degradable, stimuli
reactive(e.g.,pHortemperaturesensitive),andeventargeted(e.g.,
by conjugating them with specific antibodies against certain
characteristic components of the area of interest). Targeting is the
ability to directthe drugloaded system tothe site of interest.Two
major mechanisms can be distinguished for addressing the desired
sites for drug release: (i) passive and (ii) active targeting. An
example of passive targeting is the preferential accumulation of
chemotherapeuticagentsinsolidtumorsasaresultoftheenhanced
vascular permeability of tumour tissues compared with healthy
tissue. A strategy that could allow active targeting involves the
surface functionalization of drug carriers with ligands that are
selectively recognized by receptors on the surface of the cells of
interest. Since ligandreceptor interactions can be highly selective,
thiscouldallowamoreprecisetargetingofthesiteofinterest.For
over20years,researchershaveappreciatedthepotentialbenefitsof
nanotechnology in providing vast improvements in drug delivery
and drug targeting. Improving delivery techniques that minimize
toxicity and improve efficacy offers great potential benefits to
patients, and opens up new markets for pharmaceutical and drug
delivery companies.Otherapproaches to drug delivery arefocused
on crossing particular physical barriers, such as the blood brain
barrier, in order to better target the drug and improve its
effectiveness;oronfindingalternativeandacceptableroutesforthe

delivery of protein drugs other than via the gastrointestinal tract,

wheredegradationcanoccur.
Liposomesareaformofvesiclesthatconsisteitherofmany,fewor
justonephospholipidbilayers.Thepolarcharacteroftheliposomal
core enables polar drug molecules to be encapsulated. Amphiphilic
and lipophilic molecules are solubilized within the phospholipid
bilayer according to their affinity towards the phospholipids.
Participationofnonionicsurfactantsinsteadofphospholipidsinthe
bilayer formation results in niosomes. Channel proteins can be
incorporated without loss of their activity within the hydrophobic
domain of vesicle membranes, acting as a sizeselective filter, only
allowing passive diffusion of small solutes such as ions, nutrients
and antibiotics. Thus, drugs that are encapsulated in a nanocage
functionalized with channel proteins are effectively protected from
prematuredegradationbyproteolyticenzymes.Thedrugmolecule,
however, is able to diffuse through the channel, driven by the
concentrationdifferencebetweentheinteriorandtheexteriorofthe
nanocage. Liposomes have the distinct advantages of being both
nontoxicandbiodegradablebecausetheyarecomposedofnaturally
occurring substances. Biologically active materials encapsulated
within liposomes are protected to varying extent from immediate
dilution or degradation, suggesting drug carrier systems for the
transport of drugs and other bioactive capsules to diseaseaffected
organs. The unique ability of liposomes to entrap drugs both in an
aqueousandalipidphasemakesuchdeliverysystemsattractivefor
hydrophilicandhydrophobicdrugs.Becauseofadvancementsinthe
methods of preparing and formulating liposomes, highentrapment
efficiencies are possible for incorporating drugs into liposomes,
creating a tremendous pharmaceutical impact. Furthermore, such
encapsulation has been shown to reduce drug toxicity while
retainingorimprovingthetherapeuticefficacy.Severallaboratories
havereportedtheuseofliposomesasdrugcarriersinthetreatment
of cancer, [1,2] leishmaniasis,[3] metabolic disorders, and fungal
diseases.[4] Innovative research in liposomal drugs has led to
commercialization of several anticancer therapeutics such as Doxil,
Myocet, two liposomebased anticancer drugs; doxorubicin; and an
antifungal drug formulation, AmBisome, which is a liposomal
formulationofamphotericinBusedforsystemictherapy.Liposomes
mayhaveauseingenedeliverytocorrectgeneassociateddisorders
or for vaccine therapy.[5] A quantitative entrapment of DNA can be
achieved using the preparation of empty liposomes with cationic
lipidsfollowedbymixingwithDNAoraplasmidofinterest.Because
ofitsconvenienceandefficacy,cationiclipidmediatedgenedelivery
technology is a promising system for in vivo gene therapy. Clinical
trialsoflargesizelipidDNAcomplexeshavemostlyshownalackof

Kulkarnietal.
IntJCurrPharmRes,Vol3,Issue2,1018
adverseeffectsandmoderateexpressioninarelativelylowfraction
ofcells,butnodecisiveclinicaldisadvantages.
ClassificationofLiposomes
Liposomes can be classified either on the basis of their structural
properties or on the basis of the preparation method used. These
twoclassificationsystemareinprinciple,independentofeachother.
Theparametersforthefirsttypeoftheclassificationarementionin
the table 1. Dependent on the selection of lipids, the preparation
technique,andpreparationconditions,liposomescanvarywidelyin
size, number, position of lamellae. These parameters influence the
behaviourofliposomesbothinvivoandinvitro.Classificationbased
onmethodofliposomepreparationismentionedinthetable2.

Table1:Liposomeclassificationbasedonstructuralfeatures
MLV
OLV
UV
SUV
MUV
LUV
GUV
MVV

Multilamellarlargevesicles
Oligolamellarvesicles
Unilamellarvesicles
Smallunilamellarvesicles
Mediumsizedunilamellarvesicles
Largeunilamellarvesicles
Giantunilamellarvesicles
Multivesicularvesicles

Table2:Liposomeclassificationbasedonmethodofliposome
preparation
REV
MLV/REV
SPLV
FATMLV
VET
FUV
FPV
DRV
BSV

Singleoroligolamellarvescilemadeby
reversephaseevaporationmethod.
Multilamellarvesiclesmadebyreversephase
evaporationmethod.
Stableplurilamellarvesicles.
FrozenandthawedMLV
Vesiclespreparedbyextrusionmethod.
Vesiclespreparedbyfusion
Vesiclespreparedbyfrenchpress
Dehydrationrehydrationvesicles
Bubblesomes

MechanismofLiposomeFormation
Liposomes are vesicular structures consisting of hydrated bilayers.
Liposomesstructuresusedforpharmaceuticalpurposesconsistofa
phospholipid baskbone. But other classes of molecules can form
bilayerbasedvesicularstructuresaswell.Ontheotherhandnotall
thehydratedphospholipidsform bilayerstructures.Otherforms of
selfaggregationsuchasinvertedhexagonalphasesormicelleswith
completelydifferentpropertiescanoccur.Thecommonfeaturethat
allbilayerformingcompoundsshareistheiramphilicity.Theyhave
defined polar and nonpolar regions. In water the hydrophobic
regions tend to self aggregate and the polar regions tend to be in
contact with the water phase. Israelachvili and coworkers defined
criticalpackingparameterpby
P=v/a0lc
Where v is hte molecular volume of the hydrophobic part, a0 is the
optimum surface area per molecule at the hydrocarbon water
interface, and lc is the critical half thickness for the hydrocarbon
regionwhichmustbelesstehnthemaximumlengthoftheextended
lipidchains.Forp<1/3,sphericalmicellesareformed.Inthiscategory
fall single chain lipids with large head group areas.eg
lysophosphatidylcholine.For1/3<globularorcylindricalmicelles
areformed.Doublechainfluidstatelipidswithlargeheadarea(1/2
<p<1)formbilayersandvesicles.Thisoccursalsowithdoublechain
gelstatelipidswithsmallheadgroupsandp~1.forp>1inverted
structuressuchastheinvertedhexagonalphasecanbeobserved.An
addtional condition required for bilayer formation is that the
compoundcanbeclassifiedasanonsolubleswellingampiphile.
RawMaterialsForFormationOfLiposomes
Liposomes that are used as carriers for drugs or diagnostic agents
should be prepared from constituents that are safe for use in

humans. Although limited experience is available on the safety of

liposomes. Phosphatidylcholines and phosphatidylglycerols from
natural sources, semisynthetically or fully synthetically produced
and cholestrol and PEGylated phophatidyletanolamine, are
frequently encountered in liposomes designed as drug carriers for
parentral administration or for in vivo diagnostic purposes,
Phosphatidylcholine (PC) is routinely used as a bulk neutral
phospholipid . As a negatively charged lipid,phosphatidylglycerol
(PC) is often selected. Finally , if it is desirable to reduce the
permeabilityoffluidcrystallinestatebilayers,cholesterolisadded
to bilayer structure. Sometimes lipids with a special affinity for
certain target cells in the body are deliberately inserted in bilayer.
This was, for instance, the case when hepatocytic delivery was
aimed for and lactosylceramide, a ligand with a special affinity for
hepatocytes,wasincludedintheliposomalbilayer.[6,7]
Five groups of phospholipids tthat can be used for the liposomal
preparationcanbediscerned[8]
1.

Phospholipidsfromnaturalsoures

2.

Modifiednaturalphospholipids

3.

Semisyntheyicphospholipids

4.

Fullysyntheticphospholipids

5.

Phospholipidswiyhnonnaturalheadgroups

PhospholipidsfromNaturalSoures
The sources for natural phospholipids ,mainly PC, but also
phosphatidylethanolamine(PE),
phsphotidylinositol
(PI),and
sphingomyelin(SPM),are egg yolks and soyabeans. These PCs are
mixed acyl ester phospholipids. Apart from source dependent
diffrences in acyl chain type, considerable interbatch variation has
beenobserevedforeggPC.[9]TheestrifiedaclychainsofeggPCare
diffrentfromthoseofsoybeanPC.
ModifiedNaturalPhospholipids
Naturalphospholipidscanbemodified.Becauseoftheordegreeeof
unsaturation, ehich makes them sensitive to oxidation, PC from
natural sources ca nbe catalytically hydrogenated. Partially or fully
hydrogenatednaturalPCsarereadilyavailable.Theiodinevalueof
these lipids is reduced as teh number of unsaturated C=C bonds
drops. Dependent on the degree of unsaturation left after the
hydrogenation preocess, phase transition temperatures canbe
identified for liposomal dispersions of the patially hydrogenated
PCs. Head group modofications can be performed by using
phospholipase.WiththisenzymeonecanconvertPCintoPG,PE or
phosphatidylyserine(PS).
SemisyntheyicPhospholipids
Theseareacylchainsthatareattachedtophospholipdsfromnatural
sources are often unsaturated. This makes them liable to oxidation
reactions, which may limit liposome shelf life. Moreover as
mentioned above reproducibilty of the quality of the batches in
termsofacylchainsmaybepooraswell,whichmaycausevariation
in stability or liposome properties. Removal of teh original acyl
chainandwithincertasinlimits,replacementbyachosenacylchain
is possible. Phospholipase A2 which cuts the acyl chain at the C2
position of glycerol can be used if only replacement of the C 2 acyl
chainisrequired.
FullySyntheticPhospholipids
Eibyl reviewed different completely chemical pathways for
phospholipidsynthesis[10].
PhospholipidswithNonnatural(Head)Groups
The idea of maintaining the fate of liposomes in the body by
selectingtheappropriatebilayercharacteristicshasledtomodified
phospholipids. The circulation time of liposomes in the blood
compartment
can
be
considerably
prolonged
when
polyethleneglycol chains are attached to bilayer constituents.
Alternatively, for active targetting purposes ligands for cell surface
receptors canbe attached. These ligands can be chemically and
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Kulkarnietal.
IntJCurrPharmRes,Vol3,Issue2,1018
physically widely different structures, such as monoclonal
antibodiesorjustasimplepeptide.PEGhasbeenlinkedtoPEforthe
preparation of long circulating liposomes. Various reactions
schemes have been developed. Molecular weights fractions for
maximum prolongation of circulation times for PEG vary between
1900and5000.allenandcooworkers [11]describedthesynthesisof
a PEG carbonate derivative of PE. Klibanov et al. [12] used a
succinidyl comjugation method, while blume and Cevc [13] adopted
the procedure that Abuchowski and coworkers described for the
preparationofPEGalbuminconjugates(viacyanuricchloride)
TechniquesofLiposomePreparation
Liposome preparation techniques have been described extensively
in a number of review articles. [8,9,14] In different preparation
proceduresageneralpatterncanbedescerned1.thelipidmustbe
hydrated, then 2. liposomes have to be sized, and finally 3.
nonencapsulated drug has to be removed. In some preparation
schemesthehydrationandsizingstepsarecombined.Sometimesall
drugsareliposomesassociatedandnofreedrugcanbefoundafter
stage2thenstage3islacking.
1)Hydrationstage
a)
Mechanical Methods : MLVs were traditionally produced
byhydratingthinlipidsfilmsdepositedfromanorganicsolutionon
aglass wall by shaking at temperaturesabovethe phase transition
temperature of thephospholipidwiththehighest Tc.Thewidesize
distributions of the produced liposome dispersions were usually
narroweddownby(low)pressureextrusionorultrasoncation. [9].
b)
Methods based on replacement of organic solvent by
aqueous media : The lipid constituents are first dissolved in an
organic solvent which is subsequetnly brought in contact with an
aqueous phase. The organic solvent is removed later. During the
removal of the organic phase, liposome are formed. Their
characteristics ( size, organisation of bilayers) depend on the
protocolused.Iftheorganiicsolventwiththedissolvedlipidsisnot
misciblewiththe aqueousphase (ether,chloroform, freons) , then
the intrmediate stage is an emulsion (immiscible solvent). Other
organic solvents containing the dissolved lipid (s) can be mixed
homogenously with the aqueous phase( ethanol) in the first stage.
Then lipoosmes formation occurs when the organic solvent
concentration drops below a certain critical value (miscible
solvents).Thecontentsofresidualorganicsolventthatisexceptable
in thefinished product depends onthe solventin questionand the
routeofadministration.Apartfromevaporation,techniquessimilar
tothoseusedtoremovenonencapsulatedmaterialcanbeselected:
gel permeation, ultracentrifugation, dialysis. Organic solvent may
contain impurities wiht a high affinity for bilayers ; they may be
enriched in the bilayer and cause safety or stability problems.
Diethyl ether for instance, can be contaminated with peroxide that
accumulatesinthe bilayer.Freshly(frombisulphite)distilledether
shouldthereforebeused.
c)
Methods based on detergent removal : (Phospho)lipids,
lipophiliccompoundsandamphiphaticproteinscanbesolubilized
bydetergentsformingmixedmicells.Uponremovalofthedetergent,
vesicle formation can occur. This technique is well established for
preparation of reconstituted virus envelopes (21) or reconstituted
tumor membrane material.[15] Schreier and coworkers described a
two step strategy for insertion of proteins into the outer layer of
liposomes. First liposomes were formed by detergent dialysis
method and subsequently proteins were inserted by partial
resolubalizationofthemembranebythedetergent(deoxycolate)in
thepresenceofprotein.[16]
d)
Method based on size transformation and fusion :
Sonication of phospholipds below their phase transition
temperature (Tc) results in vesicles with defects in the bilayers.
HeatingthedispersiontoTceliminatesthesestructuraldefectsand
causes fusion resulting in large unilamellar liposomes with a wide
sizedistribution.[17]Maindisadvantageofthisprocessisthelimited
number of bilayer composition that reacts and the poor

reproducibility of the particle size distribution of the liposome

dispersionthatisformed.
2)Sizingstage
Therearetwoapproaches,onewithoutaspecialsizingstep[A]and
onewithaspecialsizingstep[B]
[A] In liposome formation process, circumstances are selected and
controlled in such a way that particle size distributions with an
acceptable width are produced. High shear homogenization
producesasizedistributionwhichdependsonoperationalpressure.
[18,19,20]
[B] For small dispersion volumes, the liposome dispersion can be
fractionated by centrifugation as liposome density usually differs
from the density of the medium. Gel permeation chromatography
hasalsobeenusedforsubdividingwideparticlesizedistribution.On
ananalyticalorsemipreparativescale,theselectionoftheporesize
of the chromatographic material provides an opportunity to
manipulatethesizeclassresolutionwithincertainlimits.
3)Removalofnonencapsulatedmaterial
Many lipophilic drugs exhibit a high affinity to the bilayer and are
completelyliposomeassociated.However,forothercompounds,the
encapsulation efficiency is less than 100 percent. The
nonencapsulated fraction of the active compound can cause
unacceptable side effects or physical instability. [21] For removal of
nonencapsulated material, the following techniques are used : a)
dialysisandultracentrifugation,b)Gelpermeationchromatography,
c)Ionexchangereactions.
StabilityOfLiposomes
Liposomes face a number of chemical and physical destabilisation
processes. So liposomes stability is an important consideration
while studying liposomes. This aspect of liposomes stability have
twoaspectsphysicalandchemicalstability.
Physicalstability

Physical processes that effect shelf life include loss of liposome

associated drug and changes in size, aggregation and fusion.
Aggregation is the formation of larger units of liposome material,
these units are still composed of individual liposomes.In principle,
this process is reversible ,e.g., by applying mild shear forces, or by
changing the temperature or by binding metal ions that intiaaly
induced aggregation. Fusion indicates that new colliodal structure
were formed. As fusion is an irreversible process, the original
liposomescanneverberetrieved.
Drug molecules can leakfromliposomes.The leakageratestrongly
dependsonthebilayercompositionandthephysiochemicalnature
ofthedrug.Bilayersinthegelstateorthosecontainigsubstantail(
molar ) fractions of cholesterol tend to lose assocaited drug only
slowly;liquidstatebilayersaremorepronetodruglossandareless
stable during storage. Bilayer permaebility is not necessarily a
constant parameter. Chang in bilayer pernaebility can occur as a
result of chemicaldegredation processes , such as theformation of
lypoPCandFA.
Chemicalstability
a)
Hydrolyiss of the ester bonds : Phosphatidylcholine
possessesforesterbons.Thetwoacylesterbondsaremostliableto
hydrolysis. The glycerophosphate and phosphocholine ester bonds
are more stable. The 1acyllysophosphatidylcholine (LPC) and 2
acylLPCarebothformedatcomparablerates.
b)
Lipid peroxidatin of phospholipids: The polyunsaturated
acyl chains of phospholipids are sensitive to oxidation via free
radical
reactions.
Cyclic
peroxides,
hydroperoxides,
malonilaldehyde, alkanes are the major degradation products. Low
oxygenpressure,absenceofheavymetals,additionofantioxidants,
complexing agents (EDTA,etc), quenchers(betacarotene) of the
photooxidatio reactions improve resistance against lipid
peroxidation.

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IntJCurrPharmRes,Vol3,Issue2,1018
CharacterisationofLiposomes
Both physical and chemical characteristics of liposomes influence
their behaviour in vivo and in vitro. [22] Liposome characterisation
shouldbeperformedimmediatelyafterpreparation.Differenttypes
of chromatography can be used to separate bilayer components
(TLC,GLC,HPTLC).Onemajorproblemisdetectionandquantitation
asUVmolarabsorptivityoflipidsislowanddependsonthedegree
of saturation of the acyl chains. For those phospholipids with only
saturatedacylchains,alternativedetectionsystemsnotbasedonUV
absorption are described i.e. systems based on differential
refractometry, light scattering and flame ionisation. The physical
propertiesofliposomeshaveadirectimpactonthebehaviourofthe
liposomeswithitscontentinvivo.Size,numberoflamellae,internal
mophologycharge,bilayerfluidityarethefactorsthatplaya rolein
theinvivodisposition.Techniquesforcharacterisingsize,numberof
lamellae,charge,andbilayerfluidityarelistedintable3.
[

Table3:Techniquesforphysicalcharacterisationofliposomes
PARAMETER
Size
NamberOfLamellae
BilayerFluidity
Charge

TECHNIQUE
Electronmicrscopylight
scatteringultracentrifugation
Coultercounter
NMRspectroscopy
Smallanglexrayscattering
Electronmicroscopy
Fluorescencepolarization
Microelectrophoresis

Liposomeadministeredviatheparentralroute,ondamagedskin,or
in the eyes must be sterile. Heat sterelization by autoclaving,
sterelization by radiation, aseptic production procedures have
been used to produce sterile products. Large liposome cannot be
sterilized by heat therefore, should be manufactured aseptically.
Assessment of the absence of pyrogens in loposome dispersions is
difficult.ItisnotclearwhetherLAL(limulusamebocyte)test canbe
usedforpyrogendetection.
LiposomesForGeneDelivery
It is important to dissect the overall cell uptake process into
individual steps. In fact different studies have indicated that
successfulgenetransferinvitroinvolves:1)thepackaging ofDNA,
2) the adhesion of packaged DNA to the cell surface, 3)
internalization of DNA, 4) escape of DNA from endosomes if
endocytosisisinvolved,5)DNAexpressionincellnuclei.Toperform
all of the above steps, liposomes have been explored as a delivery
systemforDNAasearlyasin1979. [23]Theencapsulationofplasmid
DNAintoliposomes [24]andtheintroductionofpoliovirusRNAand
SV40 DNA into cells via liposomes [25,26] were reported between
1979and1980.
PhSensitiveLiposomeStrategy
Liposomes of various compositions can extensively bind to cell
surfaces. For gene transfer, it was established that
dioleylphosphatidylethanolamine(DOPE)isbyfarthemostefficient
lipidfor invitro genetransfection forpHsensitive liposomes or as
lipidhelperincationicliposomes. [27,28,29,30]Ithasbeenassumedthat
the function of phosphatidylethanolamine (PE) is that of a
membrane fusion promoter, since in fact this lipid undergoes
changes upon acidification. [31] Cholesterol is often essential to
achieve sufficient stability of these liposomes. The composition of
liposomes may play an important role in their interactions with
cells.Thesizeofliposomesandthetypeofcellsarefundamentalfor
an efficient capture by cells. Generally, liposomes are taken up by
various endocytosic processes. Professional phagocytes such as
macrophagesandneutrophilscantakeupliposomesofvarioussize
and charge through active phagocytosis. The vesicular pathway for
cellular uptake. After binding to the cell surface, liposomes are
internalizedintoendosomeswheretheyencounteramoreacidicpH
than in the external medium. Early endosomes generally have an
internal pH of 6.50.[32,33] Their contents are then transferred to a
moreacidicenvironmentbymaturationorvesicularfusion.Thelast

endosome environment, with an internal pH of 5.56.0, is reached

10 15 min after uptake. The last endocytotic compartment, the
lysosome, is further acidified to pH values of 5.0 or lower and is
reached 20 min or more after uptake. The lysosomes are the main
degrading compartment in the endocytotic pathway. Conventional,
pHinsensitive liposomes and their content are delivered to
lysosomes and degraded. The last requirement for plasmid
liposomes after cell penetration is to avoid accumulation in
particularcellcompartmentssuchaslysosomes.Inordertoprevent
this degradation, pHsensitive liposomes have been proposed. [27,28]
pHsensitive liposomes were designed based on the concept of
viruses that fuse with the endosomal membrane by means of
protein at pH 56, delivering their genetic material to the cytosol
before reaching the lysosomes.[34,35] Generally, the lipid used to
design pH sensitive liposomes is PE. PE represents a lass of lipids
which, when dispersed in pure form, assemble into nonbilayer
structures in n inverted hexagonal phase.[31] To stabilize PE in the
lamellar hase in liposomes a series of stabilizers possessing
titratable acid headgroup such as oleic acid (OA),
palmitoylhomocysteinePHC)andcholesterolhemisuccinateCHEMS)
were used. Liposomes composed of DOPE/OA/chol are capable of
transfecting mouse Ltkcells cells lacking thymidine kinase (TK))
withnexogenousTKgene.Inthisstudy,pHsensitiveiposomeswere
8foldmoreefficientingenedeliverythanpHinsensitiveliposomes.
Interestingly,thesameinvestigatorsalsodemonstratedthatplasmid
DNA adsorbed to preformed empty pHsensitive liposomes can
transfect murine Ltkcells in vitro. In contrast, negligible
transfectionbyfreeplasmidDNAwasobserved.InastudybyZhou
etal.,[36]pUCSV2CATDNAwasusedtoprepareliposomescomposed
of DOPE/dioleylsuccinylglycerol (DOSG) (pHsensitive formulation)
or
of
dioleylphosphatidylcholine/DOSG
(pHinsensitive
formulation).Thedatashowedthattheacidsensitivitywasdirectly
related to the transfection activity. DOPE/DOSG liposome, which
was the most sensitive to pH, transfected cells with the highest
efficiency. Legendre and Szoka [37] compared the transfection
efficiency mediated by pHsensitive, pHinsensitive and cationic
(DOPE/ dioleyloxypropyltrimethylammonium bromide (DOTMA)
liposomesusingtwodifferentgenesandfivedifferentcelllines.For
all cell types investigated, cationic liposomes mediated the highest
transfection level. While pHsensitive liposomes mediated gene
transfer, their efficiency was 130% of that obtained with
DOPE/DOTMA and pHinsensitive liposomes did not induce
transfection.Itisimportanttoemphasizethefactthatseparationof
nonencapsulated (adsorbed or free) DNA was performed by pH
sensitivebutnotbycationicliposomes.Thisfactitselfmayinterfere
withtheperformanceofthetwotypesofliposomes.Akeyquestion
remains concerning the mechanism of pHsensitive liposomes: do
they react as originally intended? Ropert et al. [38,39] encapsulated
antisense oligonucleotides into pHsensitive liposomes, a short
length of DNA directed against the env gene of the murine Friend
retrovirus, to inhibit virus proliferation. They suggested that the
greater activity of oligonucleotides encapsulated into pHsensitive
liposomes was not due to a destabilization of the DOPE liposome
bilayer but to an increased association between pHsensitive
liposomes and cells. They reported that the efficiency of the viral
inhibition obtained with oligonucleotides encapsulated into pH
sensitive liposomes was only twice that of oligonucleotides
encapsulated into nonpHsensitive liposomes. And a twofold
increase in cell association was also observed when pHsensitive
liposomes were compared to pHinsensitive liposomes. In fact, pH
sensitive liposomes are taken up more efficiently by cells than pH
insensitiveliposomes,afactprobablyleadingtoabetteractivity.[34]
CationicLipidStrategy
The encapsulation of DNA into conventional liposomes could be a
technical problem due to the plasmid size, representing a poor
transfection system. On this basis, an alternative technology based
on cationic lipids and PE was developed in the late 1980s. [29] The
idea was to neutralize the negative charge of plasmids with
positivelychargedlipidstocaptureplasmidsmoreefficientlyandto
deliver DNA into the cells. Generally, this is a simple procedure
requiring mixing the cationic lipids with the DNA and adding them
tothecells.Thisresultsintheformationofaggregatescomposedof
DNA and cationic lipids. The cationic lipid DOTMA was first
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Kulkarnietal.
IntJCurrPharmRes,Vol3,Issue2,1018
synthesizedanddescribedbyFelgneretal. [29]Thislipid,eitheralone
or in combination with other neutral lipids, spontaneously forms
multilamellarvesicles(MLV)whichmaybesonicatedtoformsmall
unilamellar vesicles (SUV). DNA interacts spontaneously with
DOTMA to form DNA complexes with 100% of the DNA becoming
associated. It is presumed that complex formation simply results
fromionicinteractionsbetweenthepositivelychargedheadgroupof
DOTMA and the negatively charged phosphate groups of DNA.
DOTMA is commercialized (Lipofectin., GibcoBRL, Gaithersburg,
MD)asaonetoonemixturewithDOPEandhasbeenwidelyusedto
transfect a wide variety of cells.[40,41,42,43] In an effort to reduce the
cytotoxicity of DOTMA, a series of metabolizable quaternary
ammonium salts have been developed whose efficiency is
comparable to that of Lipofectin when dispersed with DOPE. [44] As
stated in the list of requirements, one important step for
transfectionisDNAcompactiontoimprovecellpenetration.Cationic
amphiphilesabletocompactgenomicDNA,namelylipopolyamines,
have been studied. Among them, DOSG (Transfectam.) has been
shown to transfect many animal cells in a highly efficient
manner.[45,46,47] These amphiphiles have been shown to stably
condense DNA into particles. Common detergents of diverse
structures (cetyltrimethylammonium bromide (CTAB), dodecyl
trimethylammonium bromide (DDTAB)) have been compared for
use in combination with DOPE. DDTAB seemed to be the most
promising one and the DTAB/DOPE formulation was patented
(TransfectACE.).AsreportedbyFarhoodetal.,[48]theroleofDOPEin
cationicliposomemediatedgenetransferseemedtobecritical,and
the compound has been extensively used. Since it has been
postulated that the mechanism of DNA/cationic lipids uptake by
cellsisrelatedtoendocytosis,DOPEmayfavortheliberationofDNA
intothecytosolasinpHsensitiveformulations.Electronmicroscopy
observations have shown the endosome destabilizing effect of
DOPEcontaining cationic liposomes, although efforts to synthesize
newcationiclipidsledtothediscoveryofmoreefficienttransfection
agents,theirefficiencydoesnotcorrelatewiththeirabilitytodeliver
DNA after systemic administration to animals. [49] The
physicochemical properties of the DNA/lipid complex may
determine its stability in plasma and its biodistribution or
pharmacokinetics. In an effort to determine the physicochemical
propertiesofthecomplex,cationiclipidsassociatedwithDOPEand
with various amounts of three different cationic surfactants have
beeninvestigatedbycryotransmissionelectronmicroscopy(TEM).
[50] cyoTEM analysis suggests that an excess of lipids in terms of
charge leads to entrapment f the DNA molecules between the
lamellae in clusters of aggregated multilamellar structures. The
choiceofsurfactantdoesnotappeartoaffectthemorphologyofthe
DNAlipidcomplexes. Furthermore, the system containing DOPE
resultsinmorecompactaggregatesthansimilarformulationsusing
egg lecithin. Templeton et al. [48] have proposed a model for the
assembly of DNAlipid (N1(2,3dioleyloxy) propyl, N,N,Ntrimethyl
ammonium methyl sulfate DOTAP)chol complexes in which DNA
adsorbsontotheinvaginatedandtubularliposomesviaelectrostatic
interactions.ThisgeneratesclosedstructuresinwhichDNAmaybe
protected. Farhood et al. [48] proposed the endocytosis s the major
routeforDNAlipidcomplexuptakebycellsduringtransfection.The
surfacebound complex is internalized by endocytosis into
endosomesandlysosomesnwhichalargepartoftheDNAwouldbe
degraded. Accordingto HuiandZhao,[52]the most evident pathway
for DNA entry into CHO cells is also endocytosis and not direct
fusion of the complex with the plasma membrane. Once inside the
cell, how and then DNA and lipids become separate remains in
question.
LiposomeForTargetedDelivery
Use of liposomeencapsulated enzymes for delivery into cells was
first reported in 1971. About the same time, a specific receptor on
hepatocyteswasdemonstratedtomediateclearanceofgalactose
terminated glycoproteins from circulation. A mannosidespecific
receptor was recognized on the cell surface of the RES of rats
(includingtheliversinusiodandmacrophages).Bygraftingdifferent
glycosides on the surface of liposomes, it is possible to direct the
lattertodifferentcelltypesofratliver. [53]Galactosylatedliposomes
are mainly taken up by liver hepatocytes, whereas mannosylated
liposomesaremainlytakenupbynonparenchymalcells. [54]Grafting

specific ligands to the liposome surface facilitates a fusion of the

liposomewithtargetcellsbyendocytosis,thusreleasingmaterialto
be delivered. In cancer chemotherapy, the toxicity of anticancer
drugsisofmajorconcern.Liposomescouldbeusedtodeliversuch
drugs and minimize their toxic effects on healthy cells. Targeted
delivery to cancer cells could be achieved by coating monoclonal
antibodies (MAbs) raised against tumorcell specific antigens. In
vitroandinvivostudiesbyAhmadetal.ofsquamouscellcarcinoma
in mouse models provided evidence that antibodycoated
polyethyleneglycol liposomes containing doxorubicin were more
effective and less toxic than free drugs, drugs incorporated into
antibodyfree liposomes, and antibodycoated conventional
liposomes (1,4).Themajorconcerninantibodygraftedliposomeuse
is the induction of immune response to the grafted antibodies.
Basten et al. suggested a novel approach to overcoming that
difficulty.[55] They used 125Ilabeled antigen to kill the cells
responsibleforimmuneinduction(theantigensuicidetechnique).
Other possible approaches to overcome the immunesystem
problem include immunosuppressive drugs and humanized
antibodiesorestablishingneutralimmunewindowsforsubsequent
injection. Liposomes can be designed to release their entrapped
contents under certain controlled conditions: pHsensitive and
temperaturedependentliposomalsystems.[1,56]Drugtargetingusing
liposomes as carriers holds much promise, especially in reducing
toxicity and targeting delivery to disease sites. The future is bright
forliposomeresearch,withalargenumberofclinicaltrialsongoing
in several countries with liposomal formulations of various
anticancerdrugs,antisense,cytokines,peptidesandproteins.Inthe
nearfuture,severalmoreliposomebaseddrugswillfindtheir way
intothepharmaceuticalmarket.
LiposomesInCosmetics
In the past, the beauty enhancements expected from cosmetic
products were obtained simply by combining the moisturizing,
bleaching, or cell generating agents with the cosmetic base. Now
cosmetic products have reached the stage where liposomes can
encapsulateactive ingredients thought to be necessaryforthe skin
so they may be directly applied to the skin cells. Most useful for
being able to transfer and deliver active ingredients to the
application site of cosmetics. The liposome wall is very similar,
physiologically, to the material of cell membranes. When cosmetic
containing liposomes is applied to the skin, for example, the
liposomes are deposited on the skin and begin to merge with the
cellular membranes. In the process, the liposomes release their
payloadofactivematerialsintothecells.Asaconsequence,notonly
isdeliveryoftheactivesveryspecificdirectlyintotheintendedcells
butthedeliverytakesplaceoveralongerperiodoftime.Liposomes
are typically manufactured from various fatty substances that are
used to encapsulate, or to create a sphere around, cosmetic
materials.Theyactasadeliverysystem.Today,mostoftheexperts
working in the field of liposomal dispersions agree that liposomes
donotpenetrateasintactvesiclesintotheskinorpermeatethrough
the skin. Liposomes are believed to be deformed and transformed
into fragments as a rule. Therefore size, shape, and lamallarity are
notsorelevantfortheapplication,butforthechemicalcomposition
of the total formulation. The multifunctional properties of
phosphatidylcholinesleadtoanumberofdifferentapplications.So,
formulationswithunsaturatedphosphatidylcholinearepreferredto
support skin regeneration, antiaging, acne preventing, and
penetrating other active agents like vitamins and their derivatives
intotheskin.Formulationswithhydrogenatedphosphatidylcholine
may be used for skin and sun protection, but it should be
emphasizedthatinthisrespectnanoparticlesandDMSarestillmore
convenient.[57]
The numerous patents on liposome applications reflect the avid
interest of many companies in this area. A patent involves a skin
whitening lotion in which liposomes, consisting of vitamin E and
complexlipidsaredispersedinalcoholandwater.Thepatentclaims
thatvitaminEremainsstablelongenoughtoexhibitreducingaction.
Asecondpatentrelatedtoskinwhiteningcosmeticsinvolvestheuse
of liposomes encapsulating ascorbic acid and alphatocopherol
(vitamin E). This patent claims that the use of liposomes prevents
theoxidationofascorbicacid.Thestudyshowedthatdecomposition
14

Kulkarnietal.
IntJCurrPharmRes,Vol3,Issue2,1018
of ascorbicacid was inhibited by theuseofliposomes. Onepatent
claims that the stability of a liposome is enhanced by covering its
surfacewithfattyacidestersandpolysaccharides.Thenextpatent
concernsaprocessformanufacturinglargequantitiesofliposomes.
Dissolving an amphiphilic substance in a solvent and spray drying
the resulting solution gives rise to a fine powdery mixture. This
powder, when dispersed in an aqueous medium and mixed, easily
yields large quantities of liposomes. Phosphatidylserine,
sphingomyelin or soybean lecithin may be used as the amphiphilic
components, and yeast, antibiotics, elastin polypeptides, aloe vera
etc., may be used as encapsulated active ingredients. Another
patent claims that a membrane is formed by dissolving lecithin or
phosphatidylcholineinanorganicsolventwhichisthenevaporated.
The homogenization of this membrane with active ingredients can
yield liposomes containing active ingredients which can be used in
the manufacture of cosmetics. The patent also confirms the
percutaneous absorption of the liposome's active ingredients
through the use of a [sup.14] C label. Liposomal dispersions have
provednotonlytobeinnovativeandeffectivecosmeticingredients,
but also to be a very convenient form to work with
phosphatidylcholine.Indermatology,theywillbeusedwithsuccess
for preventing and treating several skin diseases. Complementary
formulations are established where liposomal dispersions come up
againstlimitingfactors.Generally,membersofthemembranefamily
like liposomes, nanoparticles, and DMS are more compatible with
the skin structure than usually applied conventional emulsions.
"Compatible"meansthatformulationsdonotdisturbtheintegrityof
the skin lipid bilayers and are not washed out when the skin is
cleaned. In the sense of modern strategies of cosmetics, these
formulationsgetbywithaminimumofauxiliarycompounds,which
put only a strain on the skin. Moreover, compatibility means
embedding lipids and hydrophilic agents in the horny layer and
being in line with the natural situation. Remarkably,
phosphatidylcholine need not be applied in high concentrations,
because experience shows that formulations are stable at lower
amounts. Also, there is a cumulative effect in the horny layer with
repeated application of phosphatidylcholine. In many cases
liposomes,nanoparticlesandDMSarecompatiblewitheachotherin
a sense that they can be used as a modular system. So these
formulations are believed to still have a great future in cosmetic
science. How far new findings about the importance of the choline
moietyofphosphatidylcholine [58]willimpactskincareresearchand
developmentcannotbeestimated.
TherapeuticApplications
1.OccularApplication
The eye is protected by three highly efficient mechanisms (a) an
epithelial layer that is a formidable barrier to penetration (b) tear
flow(c)theblinkingreflex.Allthreemechanismsareresponsiblefor
poordrugpenetrationintothedeeperlayersofthecoeneaand the
aqueoushumorandfortherapidwashoutofdrugsfromthecorneal
surface.Enhancedefficacyofliposomesencapsulatedidoxuridinein
herpessimplexinfectedcorneallesionsinrabbitswasfirstreported
in1981.[59]Leein1985concludedthatoculardeliveryofdrugscould
be either promoted or impeded by the use of liposome carriers,
depending on the physiochemical properties of the drugs and lipid
mixture employed. Gangliosidecontaining liposomes and wheat
germ agglutinin, a lectin that has a high binding affinity for both
corneaandganglioside,weretestedforcornealadhesion.[60]Corneal
binding as well as accumulation and transcorneal flux of carbachol
wasenhanced2.5to3foldover90minexposuretimes.Daviesetal.
[61]proposedtheuseofmucoadhesivepolymers,carbopol934Pand
carbopol 1342 to retain liposomes at the cornea. While precorneal
retention times were indeed significantly enhanced under
appropriate conditions liposomes even in the presence of the
mucoadhesive had migrated toward the conjuctival sac with very
littleactivityremainingatthecornealsurface.
2.PulmonaryApplication
Pulmonary delivery of liposomes has been explored as a target
selective alternative to systemic administration of antiasthamatic
and antiallergic compounds and for antibiotics used against

pulmonary infections. Liposomes are useful tools for pulmonary

delivery of drugs due to their solubilization capacity for poorly
water soluble substances rendering them more practical for
aerolisation.Theirbiodegradabilityallowsforprolongedpulmonary
residencetimeswithoutdangerofallergicorothersideeffects.The
targetingcapacitytoinfectedorimmunologicallyimpairedalveolar
macrophages is a unique feature of liposomes. The toxicity of
liposomesaerosolshasbeeninvestigatedsystematically.Gonzalez
Rothietal. [62]foundnoinhibitionofphagocyticactivityorviability
upon prolonged exposure of alveolar macrophages to liposomes.
Padmanabhan et al [63] demonstrated high enzyme activity and
prolonged tissue protection following pulmonary instillation of
liposomeincorporatedsuperoxidedismutaseandcatalasealthough
the mechanism respomsible for the observed protection remained
obscure. Another promising application is the pulmonary
application of liposome incorporated antimycobacterial drugs
directedagainstMycobacteriumaviumintracellulare(MAI)infected
alveolar macrophages. Wichert et al [64] demonstrated the superior
killing efficacy of liposomal amikacin against MAI in alveolar
macrophageinvitro.
ClinicalApplications
1.CancerTherapy
Cytotoxicdrugscandistributenonspecificallythroughoutthebody,
leadtodeathofnormalaswellasmalignantcells,therebygivingrise
to a variety of toxic side effects. Entrapment of these drugs into
liposomes resulted in increased circulation lifetime, enhanced
deposition in the infected tissues, protection from the drug
metabolic degradation, altered tissue distribution of the drug, with
itsenhanceduptakeinorgansrichinmononuclearphagocyticcells
(liver,spleenandbonemarrow)anddecreaseduptakeinthekidney,
myocardiumandbrain.Totargettumors,liposomesmustbecapable
of leaving the blood and accessing the tumor. However, because of
their size liposomes cannot normally undergo transcapillary
passage. In spite of this, various studies have demonstrated
accumulation of liposomes in certain tumors in a higher
concentration than found in normal tissues.[65,66] Many research
efforts have been directed towards improving the safety profile of
the anthracycline cytotoxics, doxorubicin (DXR) and daunorubicin
(DNR), along with vincristine (VCR), which are associated with
severe cardiotoxic side effects, although acute gastrointestinal
effectsandothertoxicitiesmayalsooccur.Liposomalentrapmentof
these drugs showed reduced cardiotoxicity, dermal toxicity and
better survival of experimental animals compared to the controls
receiving free drugs.[65] Such beneficial effects of liposomal
anthracyclines have been observed with a variety of liposomal
formulations regardless of their lipid composition provided that
lipids used high cholesterol (Cho) concentration or phospholipids
with high phase transition temperature (Tc) are conducive to drug
retention by the vesicles in the systemic circulation. [67] DXR
entrapped in liposomes shows reduced nonspecific toxicity and
maintains or enhances anticancer effect. DXR hydrochloride
constitutes the first liposomal product (DoxilTM) to be licensed in
the United States. Surface grafted methoxypolyethylene glycol
(MPEG) provides the hydrophilic stealth coating, which allows the
DoxilTM liposomes to circulate in the blood stream for prolonged
periods. The lipid matrix and an internal buffer system combine to
keepvirtuallyalltheDXRencapsulatedduringliposomeresidencein
thecirculation.Thismeansthatthedrugisnotfreetoexertitstoxic
effects.[68] Liposome association alters the drug pharmacokinetics
and thus the liposome has a halflife of approximately 55 hours in
humans, whereas the free drug distributes to the tissues within a
few minutes and is entirely cleared from circulation within 24
hours.[69] Liposomal formulation showed decreased toxic effects of
DXR; a dose higher than the LD50 could be administered without
acutetoxicity,whichsuggeststhattheseliposomesextravasatefrom
the endothelium of tumor tissues and reside around tumor cells
wheretheyreleasethedrugintotheinterstitialfluid.Preclinicaland
clinical investigations have demonstrated significantly increased
efficacy and decreased toxicity of liposomes containing DNR
(DaunoXomeTM)incomparisonwithfreeDNR [70]inthetreatment
of acute leukemia.[71] However, in the treatment of hepatocellular
carcinoma and liver cirrhosis liposomal DNR showed mild
15

Kulkarnietal.
IntJCurrPharmRes,Vol3,Issue2,1018
haematological toxicities and significant hepatic toxicity, which
warns against further assessment of these liposomes in patients
with hepatocellular carcinoma and liver cirrhosis. [72] However,
liposomal DNR showed encouraging results in the treatment of
advanced cutaneous Tcell lymphoma.[73] Furthermore, liposomal
DNR and carboplatin plus etoposide, used to treat children with
recurrent highgrade glioma after surgery and with progressive
teratoid/rhabdoid tumor, showed encouraging results with only
little and transient hematological toxicity.[74] Liposomal
encapsulation of VCR resulted in increased and prolonged plasma
concentration,whichisassociatedwithincreasedantitumoractivity
(murine P388 ascitic tumor) but not increased drug toxicities
compared to the unencapsulated drug.[75] Guthlein et al. [76] found
thatVCRentrappedintoavesicularphospholipidsgelconsisting of
densely packed liposomes was an effective delivery system with
superior antitumor activity compared to conventional VCR against
human small cell lung carcinoma LXFS 650 and the human
mammary carcinoma MX1. Sustained release and passive tumor
targetingcanexplaintheenhancedefficacy.

2.

2.AntimicrobialTherapy

9.

Incorporation of rifabutin in liposomes resulted in a significant

enhancement of activity against Mycobacterium avium infection
comparedto free rifabutin.[77]Moreover,the antitubercularactivity
of rifampin was considerably increased when encapsulated in egg
phosphatidylcholine liposomes. A further increase in the activity
was observed when the macrophage activator tetrapeptide tuftsin
was grafted on the surface of drugloaded liposomes. Rifampin
delivered twice weekly for two weeks in tuftsinbearing liposomes
was at least 2,000 times more effective than the free drug in
lowering the load of lung bacilli in infected animals. [78] Liposome
encapsulated clarithromycin may be more effective than the free
formagainstMycobacteriumaviumintracellular(MAI)infectionsin
vivo, and the use of a combination therapy with ethambutol could
further enhance the efficacy.[79] Furthermore, when the activity of
TLC G65 (liposomal gentamicin preparation), alone and in
combination with rifapentine, clarithromycin, clofazimine and
ethambutol, was evaluated in the beige mouse model of
disseminated Mycobacterium avium infection showed that the
combination of rifapentine and TLC G65 was more active than
either agent alone. The activity of clarithromycin in combination
withTLCG65wassimilartothatofeitheragentalone.Clofazimine
improvedtheactivityofTLCG65withrespecttothespleen,while
ethambutol improved the activity with respect to the liver. [80]
Entrapment of ciprofloxacin in liposomes increases the circulation
halflifeofthedrugwhengivenbyintravenousrouteinmice,which
isassociatedwithenhanceddeliveryofthedrugtotheliver,spleen,
kidneys, and lungs. Furthermore, liposomal entrapment was
associated with increased therapeutic efficacy against the
Salmonella typhimurium infection model in mice. [81] Stevenson and
coworkers [82] showed enhanced activity of streptomycin and
chloramphenicol against Escherichia coli in the cells of the J774
murinemacrophagelinemediatedbyliposomedelivery.

3.
4.
5.
6.
7.
8.

10.
11.

12.
13.
14.
15.

16.
17.
18.
19.

CONCLUSION
Liposomes have developed into a viable pharmaceutical dosage
form. Progress has taken place in quantum leaps, rather than in a
continuum,overthelasttwodecades.Vitalprogesshavebeenmade
in the development of long circulating liposomes that are not
immediately recognized and removed by the cells of mononuclear
phagocytesystem.Despitthislongcirculatingliposomeshaveopned
anewrealmoftherapeuticopportuitiesandwewillseeamultitude
ofnovelapplicationsemergeinfuture.Developmentwillcontinueto
explore the validity of liposomes for the delivery of peptide and
proteins,althoughprogressinthidparticularfieldhasbeenmeager.
These developments will hopefully safeguard against the
overoptimistic and unrealistic ideas and promises of the past and
lead into another highly productive and innovative phase of
liposomeresearch.

20.

21.
22.
23.
24.

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