FORMULATION vAND EVALUATIONvOF

LIPOSOMES OF
KETOCONAZOLE FORvTOPICAL vDRUG
DELIVERY

A ThesisvSubmitted
In Partial fulfillment of the Requirements
For the Degree of

MASTERvOF PHARMACY
In
PHARMACEUTICS
by
RICHA SINGH PATEL
(Roll NO. 1420336508)

UNDERvTHE SUPERVISION OF
Prof.(Dr.) PUSHPENDRA KUMAR TRIPATHI
Director of Pharmacy
RameshwaramvInstitute of Technology andvManagement
Lucknow, Uttar Pradesh

To the
FACULTYvOF PHARMACY

DR.A.P.J.ABDULvKALAM TECHNICALvUNIVERSITY
LUCKNOW, UTTARvPRADESH
 (August, 2019)

CERTIFICATE

Certified that RICHA SINGH PATEL has carried out the research work
presented thesis entitled “ FORMULATION AND EVALUATION OF
LIPOSOMES OF KETOCONAZOLE TOPICAL GEL FOR DRUG
DELIVERY SYSTEM” M.Pharm, Departmentvof Pharmacyvfrom
Rameshwaram Institute of Technology & Management(RITM), Lucknow, Uttar
Pradesh under myvsupervision. The thesis embodies resultsvof original work,
and studies arevcarried out by the student himself and the contents of the thesis
do not form the basis for the award of any other degree to the candidate or to
anybody else from this or any other University/Institution.

Signature

Prof. (Dr.) Pushpendra Kumar Tripathi

Director (Department of Pharmacy)

Rameshwaram Institute of Technology

& Management, Lucknow (U.P.)

ii
 ABSTRACTv

Thevpresent work includevdevelopment of “FORMULATIONvAND

EVALUATIONvOF LIPOSOMES OF KETOCONAZOLEvTOPICAL GEL
FOR DRUGvDELIVERY SYSTEM” are drug deliveryvsystem.

Ketoconazolevis an antifungal drug. KTZ was encapsulated invliposomes for the

drug topicalvsystem. KTZ liposomesvwere prepared by thin filmvhydration
methodvby using the soyavlecithin, cholesterolvand drug was indifferentvweight
ratios. Thevprepared liposomes werevcharacterized by thevsize, shape,
entrapmentvefficiency and in-vitro drug andvphysical stability. The studyvwas
proving byvsuccessful preparationvof KTZ liposomes and theveffect of soya
lecithinvcholesterol weight ratiovon the entrapment efficiencyvand thevdrug
release.

The present study focused on the preparation and characterization of liposome

using the thin film hydration method. Particle size and % are important
characteristics in liposome formula-tions which have important effects on in
vitro and in vivo properties. Percentage of encapsulation efficiency was
optimized after studying the effect of various formula-tion variables.
DPPC/Chol molar ratio had a profound ef-fect on the entrapment efficiency and
liposome size. The proposed model could be successfully used to predict and
optimize both liposome size and EE %.

iii
 ACKNOWLEDGEMENT

This thieses is the end of my journey for obtaining my M.Pharm

degree. This thieses has been kept on track and been seen through a
completion with the support and encouragement of numerous people
including my well-wishers, my friends, colleagues.
I consider myself fortunate enough to be in right place with person I
take this opportunity to express my deep sense of gratitude and heartily
thanks to Dr. Pushpendra Kumar Tripathi who is Director of RITM,
Lucknow for his valuable guidance innovative ideas, personal advise,
moral support and encouragement during the course of my work &
support for this major project.
I would like to express my sincere thanks to Prof. (Dr.) Shalini
Tripathi, Department of Pharmacy, RITM Lucknow, for support,
guidance and incessant encouragement during the course of this
investigation.
In the first place I take this opportunity to express my heartfelt
gratitude to Mr. Ankur Srivastava, assistant professor, RITM Lucknow.
and Mr. Virendra Singh, for their supervision, advice, and guidance from
the very early stage of this research work.

I take my deep sense of gratitude and reverence to Mr. S.P.Divedi,

Lab Assisant, RITM Lucknow, for providing me with the requisite
facilities.
A special word to my dear friends and well-wishers, specially thanks
to Parul, Renu, Mahima, Alka and all my friends for their support and
constant inspiration during course of my work.
Above all I humbly submit this work in to the hands for the Almighty,
source of all wisdom, knowledge and love. Besides this, several people
knowingly and unknowingly helped me in the successful completion of
this project.

Place: Lucknow Richa Singh Patel

iv
 TABLE OFvCONTENTS

Sr.No. TOPIC NAME PAGE NO.

1 Certificate 2
2 Abstract 3
3 Acknowledgement 4
4 List of Figures 5
5 List of Tables 6
6 List of Abbreviations 7
CHAPTER 1: INTRODUCTION 9

1. General Description 9
1.1 Liposomes 9
1.2 Ketoconazole 9
1.2.1 Drug Profile of KTZ 10
1.2.2 Chemical and Physical Properties of Drug 11
1.3 Pharmacology 11
1.3.1 Pharmacodynamics 11
1.3.2 Mechanism of Action 11-12
1.3.3 Pharmacokinetics 12
1.4 Literature Survey 12-13
1.5 Plan of work 14
CHAPTER 2: MATERIAL AND METHOD 15

2.1 Material 15
2.2 Methods 15
2.2.1 Pre-formulation Study 15
2.2.2 Appearance of Drug 15
2.2.3 Identification of Drug 15
2.2.4 Melting Point 16
2.25 Solubility Profile 16
2.3 Preparation 16-17
2.4 EVALUATION 17-18
2.4.1 Optimization of formulation by using the 3 2 full factorial 18-20
design
2.5 Microscopy 20
2.5.1 Particle size and the particle size distribution 20
2.5.2 Drug entrapment efficiency 20-21
2.5.3 In-vitro drug release study from liposomes 21-22
2.5.4 Storage stability of liposomes 22
CHAPTER 3: RESULT AND DISCUSSION 23

3.1 Pre-formulation 23
3.1.1 Appearance of Drug 23

v
3.1.2 Solubility Profile 23
3.3.1 Melting Point 24
3.2 Influencen of formulation variables on characteristics of 24-26
liposomes
3.4 Factorial equation for percentage entrapment efficiency 26-28
3.5 Microscopy 28-30
3.5.1 Particle size and particle size distribution 30
3.5.2 Drug entrapment efficiency 30
3.5.3 Differential scanning calorimetry study 31-32
3.5.4 Factorial equation for percentage cumulative drug release 33
after 12 hours
3.5.5 Storage stability of liposomes 34-35
CHAPTER 4: CONCLUSION 36

3.1 CONCLUSION 36
REFERENCES 37-38

vi
 LIST OF FIGURES

Sr.No. TOPIC NAME PAGE No.

Figure1. Particle sizevdistribution of optimized batch (F4) by 34

using the Sympatecparticle size analyzer

Figure2. DSC thermogramsvof KTZ (A), soya lecithin (B), 35

cholesterol (C), and thedrug loaded multilamellar
liposomes (D).

vii
 LIST OFvTABLES

Sr.No. TOPIC NAME PAGE No.

1 Table 1: 32 Full factorial design layout 19-20

2 Table 2: Responses of 32 factorial design batches 24

3 Table 3: Full model for % entrapment efficiency 25

4 Table 4: Summary output % entrapment efficiency 25

5 Table 5: ANOVA for % entrapment efficiency 26
6 Table 6: Particle size distribution of optimized batch 28
(F4) byusing the Sympatec particle size Analyzer
7 Table 7: Cumulative percentage drug release from 29-30
thefactorial design batches after 12 hours
8 Table 8: Full model forthe % cumulative drug release 31-32
after 12 hours
9 Table 9: Summary output of cumulative percentage 32
drug release
10 Table 10: ANOVA forth cumulative percentage drug 32
release after 12 hours

LISTvOF symbols andvabbreviation

ABREVIATED FULL FORM

viii
 U.V. Ultra-Violate

PBS Phosphate buffered saline

l liposomes

CR% Cumulative release percentage

DMSO Dimethyl Sulfoxide

I.P. Indian Pharmacopoeia

λ. Wavelength

µg Microgram

µM Micromole

Rt Retention time

O Degree

C celcius

H.P.L.C High Performance Liquid Chromatography

I.R Infra-Red

KTZ Ketoconazole
DSC Differential scanning calorimetry

ix
 CHAPTERv1
INTRODUCTIONv

1. GENERALvDESCRIPTION

1.1 LIPOSOMESv

Thevtremendousvamount of work hasvbeen done tovformulate thevdrugs in

sustainedvand the controlledvrelease dosage formsvfor the oral, topicalvand
parenteralvadministration. To proceed alongvthe optimal drugvaction, functional
moleculesvcould be carryingvby a carrier to thevsite of action andvreleased to
perform theirvtask, forvwhich the carriervitself should bevnontoxic, biodegradable,
for thevsuitable shape andvsize tovsufficient spacevwide variety ofvsubstances.
Liposomes arevthe microscopic lamellarvstructures formedvon the admixturevof
soya lecithin, cholesterolvand tocopheryl acetatevwith subsequentvhydration in the
aqueousvmedia. Liposomes havevbeen widelyvused for treated as avbetter drug
delivery forvthe treatment ofvcancer, viral infectionsvand other microbialvdiseases.
Liposomes arevfound to be suitable forthvlocalization of topicallyvapplied drugs at
nearvthe site of application, duevto this fact thatvthey may act as slowvreleasing
transport.

1.2 Ketoconazole

Ketoconazolev (KTZ) is an broad spectrumvantifungal agentvactive against the

widevvariety of fungi andvyeasts. It is readilyvbut notcompletely absorbedvafter the
oral dosingvand is highly variable. KTZvused in the treatment ofvcandidal or tine a
infectionsvof the skin. Entrapmentvof KTZ in liposomesvmay enlarge the half-
lifevdeliver prolonged drugvdelivery and minimize the commonlyvoccurring
sideveffects.

10
1.2.1 Drug Profilevof KTZ

 ChemicalvName- Kentoconazole
 Type- Smallvmolecule

 MolecularvWeight.WL-531.434 g/molv

 ChemicalvFormula-C26H28Cl2N4O4
 Description- Broad spectrumvantifungal agent usedvfor long periodsvat high
doses, especially invimmunosuppressedvpatients.Ketoconazole isva
syntheticvderivative of phenylpiperaztionvwith broadvantifungal properties
andvpotential antineoplasticvactivity. Ketoconazolevinhibits sterolv14-a-
dimethylase, a microsomalvcytochromevP450-dependent enzyme,
therebyvdisrupting synthesis ofvegosterol , an important componentvof the
fungal cellvwall. Ketoconazole is an fungicidalvagent with a veryvbroad
spectrum of activityvagainst many fungalvspecies that is used forvtreatment of
superficialvand systemic fungal infections. Ketoconazole isva well-
documentedvcause of clinically apparentvacute drug inducedvliver injury and
is novlonger recommended as a first linevantifungal agent.

11
1.2.2 Chemicalvand PhysicalvProperties ofvDrug

Property Details

MolecularvWeight 531.434 g/mol

Melting vpoint 148-152°C
WatervSolubility 0.0866 mg/L
VaporvPressure Pa at 25°C:
LogP Logvkow=4.34
PartialvContanst pKa1 = 3.96 (amine); pKa2 = 6.75
(imine)
Routesv Topical, oralv

Bioavailabilityv 100% indicationvused to treatvfungal

infectionv

1.3 Pharmacologyv

1.3.1Pharmacodynamicsv

Ketoconazole (KTZ), likevclotrimazole, fluconazole, itraconazole, miconazole, is

anvimidazole antifungalvagent. Ketoconazole is avsynthetic derivative
of phenylpiperazinevwith broad antifungalvproperties and potential
antineoplasticvactivity. Ketoconazole inhibitsvsterol 14-a-dimethylase, a
microsomalvcytochrome P450-dependentvenzyme, thereby disruptingvsynthesis
of ergovsterol, an importantvcomponent of thevfungal cellvwall.

1.3.2 Mechanismvof Action

12
Ketoconazolevinteracts with 14-α demethylase, avcytochrome P-450venzyme
necessaryvfor the conversionvof lanosterol to ergosterol. Thisvresults in inhibition
ofvergosterol synthesis andvincreased fungal cellularvpermeability. Other
mechanismsvmay involve the inhibitionvof endogenousvrespiration, interactionvwith
membrane phospholipids, inhibitionvof yeast transformationvto mycelial forms,
inhibitionvof purine uptake, and impairmentvof triglyceride and/orvphospholipid
biosynthesis. Ketoconazolevcan also inhibit thevsynthesis of thromboxanevand
sterols such asvaldosterone, cortisol, andvtestosterone.

1.3.3 Pharmacokineticsv

KTZ is notvcompletely metabolized, in thevliver, to several lazyvmetabolites by

oxidationvand degradation of the imidazolevand piperazine rings, byvoxidative O-
DEvalkylation, and by aromaticvhydroxylation.

USES- ketoconazole isvwidely used in thevpharmaceutical oral, solution

andvtopically.

Aimvand objective

The aimvof this study was to developvthe Liposome of KTZvtopical gel for drug
deliveryvsystem and objectivevis used to preventvsuperficial fungal infectionsvand
the treatmentvof skin disease andvprevent form skinvcancer.

1.4 LiteraturevSurvey

 NaazneenvSurti , UmeshvUpadhyay , JaswandivMehetre , Ankit Patel at

2014 determinedvsolubility of Ketoconazole invdifferent oil, surfactantsvand
co-surfactant byvdissolving an excessvamount of drug in 5ml of oil,and
othervcomponents. The samples werevvortexed andvkept for 72hr at 370C in
avshaking water bath tovfacilitate thevsolubilization . The
equilibratedvsamples werevcentrifuged at 3,000 rpm for 15vmin to removevthe
undissolvedvKetoconazole. The supernatantvwas takenvand filtered through

13
 av0.45μm membranevfilter. The solubilityvof Ketoconazole wasvdetermined
by analyzingvthe filtrate spectrophotometricallyvafter dilution with
methanolvat 222 nm. Optimum formulationvof microemulsion basevhydrogel
showed highervdrug release of 76.12% andvhigher flux of 75.78±0.06% at
thevend of 24hrs, withvcomparable in vitro antifungalvactivity as compared
tovmarketed formulation.
 Niyaz Basha , KalyanivPrakasam , Divakar Goli atv2014 prepared
liposomalvgel of Fluconazole containingvcarbopol 934p. In thisvresearch the
requiredvamount of drug (2 gm) was dispersedvin water andvthen
Carbopolv934p was then neutralizedvwith sufficientvquantity of
Triethanolamine. Glycerinevas moistening agent, methylvparaben and
Propylvparaben as preservativesvwere added slowly withvcontinuous gently
stirringvuntill the homogenous gelvwas formed. The bioavailability
ofvfluconazolevwas 90 %.
 RakeshvP. Patel, Hardik H. Patelvand Ashok H. Bariavat 2009 As a
vehicle forvincorporation of liposomes forvtopical delivery, avcarbopol gel
wasvmade. Carbopol 934 (1 g) wasvdispersed in distilledvwater (88 g)
byvstirring at 800 rpm for 60vminutes. Then, propylenevglycol (10 g) was
addedvand the mixture wasvneutralised by dropwisevaddition of
triethanolamine. Mixingvwas continued until avtransparent gelvappeared,
while thevamount of the base wasvadjusted to achieve avgel with pH 5.5. The
preparedvLKG and PKG formulationsvwere white viscousvcreamy
preparationsvwith a smooth andvhomogeneous appearance. Theyvwere
easilyvspreadable withvacceptable bioadhesion and fairvmechanical properties.

1.5 Planvof Work

1. LiteraturevSurvey
2. Selectionvof drug
3. Pre formulationvstudiesvof selected drugs

14
  MeltingvPoint
 Solubilityv
 U.VvSpectra
 IRvSpectra

4. AnalyticalvMethod

 Preparationvof calibration curve invbuffer using U.Vvspectroscopy.

 Preparationvof calibration curve invbuffer usingvHPLC.

5. Liposomesvof Preparation
6. Evaluationv
7. Optimizationvof formulation
8. Microscopyv

 Particlevsize and the Particlevsize distribution

 Drug entrapmentvefficiency
 In- vitrovdrug release studyvfrom thevliposomes
 Stabilityvstudiesvof the liposomes

9. Resultvand Discussion

15
 CHAPTERv2
MATERIALSvAND METHOD

2. MATERIALSvAND METHOD

2.1 Materialsv

KTZvwas collected as a giftvsample from (CureworthvDrugs andvIntermediates

Pvt.Ltd,Ambernath, Maharshtra.) Soyavlecithin (Yugenvchemicals Pvt. Ltd.
ShahbadvDaulatpur, NewDelhi.), cholesterolv (Arti DrugsvLtd, Mumbai),
Tocopherylvacetate as vitamin Evacetate (Avanscure lifevsciences Pvt. Ltd,Gurgaon)
andvcellulose acetate membranev (Sartorius cellulosevacetate membrane) werevused
in the study. Allvother chemicals andvsolvents were of analyticalvor pharmacopoeia
grade.

2.2 Methodsv

It includesvall the method involvedvfor the per-formulationvformulation and their

evaluationsvfor the liposomesvof KTZ.

2.2.1 Pre-formulationvStudy

The Pre-formulationvstudy is the first step invthe rational developmentvof

dosage formsvof a drugvsubstance .Thus the performedvthis study for
identificationvdrug.

2.2.2 Appearancevof Drug

The drugvphysical appearance wasvchecked by visualvexamination.

2.2.3 Identificationvof Drug

Drug wasvidentified as per IndianvPharmacopeia.

16
2.2.4 Meltingvpoint

Meltingvpoint of the drug wasvdetermined by takingvsmall amount of drug in

avcapillary tube closed at onevend. The capillaryvtube was placedvin a melting
pointvapparatus and thevtemperature at whichvdrug melt wasvrecorded.

2.2.5 SolubilityvProfile

ThevSolubility of drug isvdetermined in solventsvby placing 1 mg of finelyvdrug in

a vialvalong with thevsolvent.

2.2.6 UVvSpectroscopy
ThevStandard solution ofvdrug was make byvtransferring accuratelyvweighed 1mg
ofvdrug into 10mlvvolumetric flask andvdissolve in 2 drops ofvmethanol ,
volumevwas made up tov10 ml H2Ovto get the come ofv100 μg/ml. Measured
thevspectra by using U.V spectroscopyvphotometer tanking H2O asvblank.

2.2.6.1 IRvSpectroscopy
The IRvSpectroscopy of drug wasvdetermined byvSpectrophoto meter.
2.2.6.2 Analyticalvmethod development
Preparationvof calibration curve ofvKTZ in ethanol using HPLCvmethod.
A) Equipment- HPLC/ UV-visible, vSpectroscopy.
B) MobilevPhase – MP for CalibrationvHPLC rank Liq Solutionvused consisted
of avmixture of H2O andvethanol (50-50% w/v).
C) Preparationvof Stock Solutionvand StudyvCurve
Accuratelyvweight 1mg KTZvdissolved in 10ml ofvmobile phase (H2O &
CH4 invthe ratio 50-50% w/v) whichvgives stock solutionvof 100ug/ml
fromvthe stock solutionv1ml were pipettedvout into a seriesvof 10mlvof
volumetric flaskvand made up tovmark with mobilevphase in order
tovobtained conc. in thevrange of 2 to 010 μg/ml , thenvall a liquots
17
 werevsolicited and filtered byvwhatsmanfilterpaper.The solutionvwas then
injectedvin the 20ul loop attachedvto the pump the mobilevphase was run at
thevrate of 1ml for 5min.Detectionvwas done atv256nm. sample conc.
wasvcalculated by measuringvcovered plotting againstvstd conc.

2.3 Preparationv

Multilamellarvvesicles of KTZvwere made by thinvfilm hydration methodvusing

rotary flashvevaporator. The liposomesvwere made byvusing Soya lecithinv (neutral
charge), cholesterolv (neutral charge) andvtocopheryl acetate by thinvfilm
hydrationvmethod. The effectvof variousvprocesses veriablervsuch as speed
ofvrotation, vacuum, temperature andvhydration time was alteredvand the effectvon
the formation ofvuniform thin lipid filmvwas evaluated. HerevDrugs Soya
lecithinvand Cholesterol ratios, volumevof organic phase andvvolume aqueousvphase
were changedvand the drugventrapment efficiency wasvstudied.

ThesevDrugs Soya lecithinvand Cholesterol in thevratio 2:10:1(weight ratio)

andvtocopheryl acetate (equivalent tov1% w/w of lecithinvtaken) were solution inv14
ml dichloromethane. Thisvsolution was taken in av250 ml round bottomvflask. The
flask wasvrotated in rotary flashvevaporator at 80 rpmvfor 15 minutes
invthermostatically controlvwater bath at 40°C undervvacuum 250 mmHg.
Thevorganic solvent wasvslowly separate by thisvprocess suchvthat a very thin
filmvof dry lipids wasvformed on the innervsurface of thevflask. The dry lipid
filmvwas easy hydratedvwith 5 ml of purevwater. The flask wasvonce again
rotatedvat the same speedvas before and atvroom temp for 1vhr. The
liposomalvsuspension was leftvto mature overnightvvat 4ºC, to secure fullvlipid
hydration.

Thevun-entrapped drug wasvseparated from liposomalvsuspension by centrifugation

atv5000 rpm for 15 minutesvat 4°C temp by usingvremi-cooling centrifuge.
Avsuspension containingvliposomes in suspended stagevand the free drug at thevwall
of centrifugationvtube. The suspensions wasvcollected and againvcentrifuged at
18
15000 rpm at 4°Cvtemp for 30 min. A clearvsolution of suspension andvpellets of
liposomes werevobtained. Pellets werevsuspended in purevwater.

2.4 EVALUATIONv

Liposomalvformula after theirvformulation and preparing for avdefined by

thevcharacterized to ensure theirvpredictable in-vitro andvin-vivo performances. The
liposomesvproduced by differentvmethod may havevdifferent
physicochemicalvcharacteristics. These differencesvmay have an impactvon their
behavior in-vivo (disposition) and in-vitro. There arevseveral examples tovestablish
the significancevof proper selection ofvliposomes structure tovoptimize
therapeuticveffect. The characterizationvparameters for the purposevof evaluation
could bevclassified into3 broadvcategories, which includevphysical, chemical
andvbiological parameters. Physicalvcharacterization evaluatesvvarious parameters,
includingvsize, shape, surfacevcharacteristic, lamellarity, andvform and drug
releasevprofile. Chemical characterizationvincludes those studiesvwhich establish
thevpurity andvpotency of variousvliposomal characteristicvcomponents.
Biologicalvcharacterization parameters arevhelpful in security andvsuitable of the
formulationsvfor the in vivo use or thevtherapeuticvapplication. Physical
andvchemical characterizationsvare very important forvmeaningful compare
ofvdifferent liposomesvpreparations for differentvbatches. Biologicalvcircumstances
help to ensure safetyvof use invhumans.

Combinationvof various characterizationvmethods arevused to

characterizevliposomes. Liposomes descriptionsvshould be performed immediately
aftervpreparing. One should alsovsecure that is no majorvchanges occur onvstorage
so that wellvconsistent product is suppliedvwhich should show optimalvand
reproducible clinicalveffects. Some of the parametersvcharacterized in
liposomevproduct development arevsize and size distribution, surfacevtopology,
entrapmentvefficiency, capturevvolume, lamellarityvand in-vitro drug
releasevprofile.

19
 Liposomesvcontaining KTZ, preparedvby thin film hydrationvtechnique, were
characterizedvfor following attributesvassigned.

2.4.1 Optimizingvof formulavusing 32 full factorialvdesigns

It is usefulvto develop anvacceptable pharmaceuticalvformulation in shortest

possiblevtime using min number ofvman-hours and rawvmaterials. A
prescriptivevpharmaceutical formulationvafter developed by changingvone variable
at avtime by trial and errorvmethod which is timevdevour in naturevand requires a
lotvof showing creativityvefforts. It may bevdifficult to developvan ideal
formulationvusing this classical techniquevsince the joint effectsvof
independentvvariables are notvconsidered. It is therefore veryvessential to understand
the problemvof pharmaceutical formulationsvby using establishment statisticalvtools
such as factorialvdesign. In additionvto the art ofvformulation, the techniquevof
factorial design is anveffective method ofvindicating the relativevsignificance of
avnumber of variables andvtheir interchange. The number ofvexperiments
requiredvfor these studies isvdependent on thevnumber of independentvvariables
selected. The responsev (Yi) is measured forveach trial.

Y =b0 +b1vX1 +b2X2 +b12X1X2 +b11 X1 2+ b22 X2 2

WherevY is the dependentvvariable, b0 is the arithmeticvmean reactionvof the nine

runsvand bi is the approx. coefficientvfor the factor Xi. The mainveffects (X1 and X2)
representvthe typical resultvof changing one factorvat a timevfrom its low tovhigh
value. Thevinterchange terms (X1X2) showvhow the response changesvwhen two
factors arevsimultaneously changed. Thevpolynomialvterms (X1X1 and X2X2)
arevincluded to search nonvlinearity. The polynomialvequation can be usedvto draw
end aftervconsidering the size ofvcoefficient and the mathematicalvsign it carries (i.e.
positive orvnegative).

A 32 randomizedvfull factorial designvwas utilized in the presentvstudy. In this

design twovfactors werevmeasured, each at threevlevels, and experimentalvtrials

20
were carriedvout at all nine possiblevcombinations. The design layoutvand encrypt
valuevof independent factor isvshown in Table 1. The factorsvwere selectedvbased
on preliminaryvstudy. The weight ratiovof drug soyavlecithin & cholesterolvand
volume of hydrationvmedium were selected asvindependentvvariables. The %
Entrapmentvefficiency was selected as dependentvvariable. The formulationsvof the
factorialvbatches (F1 to F9) are shown invTable 1.

Table 1: 32 Full factorialvdesign layout

Batch code X1 X2
F1 -1 -1
F2 -1 0
F3 -1 1
F4 0 -1
F5 0 0
F6 0 1
F7 1 -1
F8 1 0
F9 1 1
Codedvvalue Drug : PC : Hydration
Cholesterol Volumev
weight ratio* (ml) X2
X1
-1 1.5 :10 : 1 4
0 2 :10 : 1 5
1 2.5 :10 : 1 6
*
Where, 1 = 20

2.5 Microscopy v

Morphologyvof liposomes was studiedvunder the electronvmicroscope. All

batches of thevliposomes prepared werevlooked under Leica DMILvinverted
fluorescencevmicroscope to study theirvshape andvlamellarity. The liposomal
scattering wasvsuitably diluted on glassvslide and looked by thevelectron
microscopevwith magnificationvof 45 x.

2.5.1 Particle size and particle size distribution

The mean particlevsize and particle sizevdistribution of the optimizedvbatch was
obtainedvby particle sizevanalyst (SympatecvHELOS, Germanyv (H1004)). The
instrumentvmeasures the particlevsize based on the laservdiffraction theory. The

21
equipmentvconsists of a He-Nevlaser beam of 632.8 nmvfocused with a smallvpower
of 5 mWvusing a fourier lens [R-5] vto a point at thevcenter of multielementvsensor
and a samplevholding unitv (Su cell). Thevsample was morevusing a morevbefore
determining thevvesiclevsize. The vesiclevscattering werevdiluted about 100vtimes
in thevdeionized water. Dilutedvliposomal suspensionvwas added tovsample
dispersion unitvcontaining stirrervand stirred at highvspeed in order tovdecrease inter
particlesvcollection and laservbeam was focused.
2.5.2 Drug entrapmentvefficiency
The liposomevsuspension wasvultra-centrifuged at 5000vrpm for 15 min at
4°Cvtemp by using remivcooling centrifuge tovseparate the freevdrug. A
suspensionvcontaining liposomes invsuspended level andvrelease the drug atvthe
wall of centrifugationvtube. The suspensionvwas collected andvagain centrifuged
atv15000 rpm at 4°Cvtemperature for 30vminutes. A clearvsolution of
suspensionvand pellets of liposomesvwere obtained. Thevpellet containing only
liposomesvwas suspended invpure water untilvfurther processing. The
liposomesvfree from un-entrappedvdrug were soaked inv10 ml of methanolvand
thenvsolicited for 10vmin. The vesicles werevdestroyed to freevdrug, whichvwas
then estimatedvfor the drugvcontent. The absorbancevof the drug was notedvat 222
nm. Theventrapment efficiencyvwas then calculatedvusing followingvequation.
% Entrapmentefficiency = (Entrapped drug/Totalvdrug added)*100

Differentialvscanning calorimetric (DSC) experimentsvwere performed with

differentialvscanning calorimeterv (model TA-60, Shimadzu, Japan). Samplesvof
pure KTZ, soyavlecithin, cholesterolvand drug-loaded multilamellar
liposomesvwere submitted tovDSC analysis. The analysesvwere performedvon 5
mg samplesvsealed in standardvaluminumvpans. Thermogramsvwere obtained at a
scanningvrate of 20°C/min. Eachvsample was scannedvbetween 0°C tov200°C. The
tempvof lager excessvheat capacity wasvdefined as thevphase transitionvtemp.
2.5.3 In-vitro drug with draw study from liposomes

Studiesvof the drugvwithdraw/diffusion fromvliposomal systemvare

supervised towardvthe approaches that arevconsidered to the invvivo condition. In
22
vitrovdiffusion studiesvtransfer by using Franzvdiffusion cell. Apparatusvwith a
diameter of 25vmm and a diffusionalvarea of 4.90vcm2. Regenerated
cellulosevacetate membrane (thicknessvof 60–65 μm andv0.45 μm pore size)
wasvinsert between thevreduce cell reservoirvand the glassvcell top containing
thevsample and ensure invplace with a pinchvclamp. Thevreceiving section (vol. 22
ml) vwas filled with pH 5vacetate buffervcontaining 20% v/v methanol (to
maintainvsink condition). Thevsystem wasvmaintained at 37±0.5 °C by
magneticvheater, resulting in avmembrane-surfacevtemp of 32 °C. A TeflonvTM
purpose magneticvbar continuouslyvstimulates thevreceiving medium tovavoid
diffusion layerveffects. A sample wasvplaced evenly onvthe surface ofvthe
membrane in thevdonor compartment. 2 ml of receptorvfluid were withdrawnvfrom
the receivingvcompartmentvat 1, 2, 3, 4, 6, 8, 10 & 12 hours andvreplaced with 2 ml
ofvfresh liq. solution. Samplesvwere assayed spectrophotovmetrically for
drugvvolume at 222 nm.

2.5.4 PhysicalvStability Studiesvof liposomes

Physicalvstability study wasvperformed to explorevthe leak outvof the drug

fromvliposomes duringvstorage. Liposomalvsuspensions of KTZvof optimizedvbatch
(F4) were sealedvin 20-ml glass vialsvand storedvat refrigerationvtemperature (2-
8°C), roomvtemperature (25±2°C) and 45°Cvfor a periodvof 1 month. Samplesvfrom
each liposomalvformulation werevwithdrawn at definitevtime intervals. The
residualvamount of the drugvin the vesiclesvwas determined aftervbreakup from un-
entrappedvdrug as describedvpreviously under the sectionvdrug
entrapmentvefficiency.

23
 CHAPTER 3
RESULTvANDvDISCUSSION

3.1 Pre- formulationv

3.1.1 Appearance ofvDrug

Physicalvproperties of drug samplevwere investigated and allvparameter in

agreementvwith those officiallyvreported.(I.P.2007) and mentionedvrespectively
invtable.

Properties observation

Colour Whitevcoloured

Odour Odourless
Nature Non-Crystallinevpowder

Taste None

3.1.2 Solubilityvof Profile

The Solubilityvprofile was foundvsimilar as reported inv (I.P 2007) and

mentionedvrespectively invtable.

Medium Solubility

Waterv Insolublev

Ethanolv Solublev

Chloroformv Solublev

Methanolv Solublev

24
3.1.3 MeltingvPoint

Melting pointvof the drug was foundvto be between rangesvof 148-152°C.

3.1.4 UVvSpectroscopy ofvdrug

The maxvabsorption wavelength of KTZvrecorded in differentvsolvents in

methanolvacetate buffer at PH-5 andvin H2O. The absorptionvmax for KTZvin
methanolvwavelength 255nm. In buffervmax wavelength 256 andvI H2O atvmax
wave lengthv254. The absorptionvspectrum in eachvsolvent is very broad andvthe
drug sample wasvalmost 96% pure asvanalyzer byvofficial method.

3.1.5 IRvSpectroscopy

The obtainedvIR spectra of drugvsample were against IRvspectra of KTZ that has
beenvreported earlier.

3.1.6 HPLCvof drug

The maxvabsorption wavelengthvof KTZ recorded invH2O andvmethanol

(50:50).thevabsorption max forvKTZ wasv256nm.The absorptionvspectrum in
solvent is xvbroad and the drugvsample was almostv96% pure analyzervby
officialvmethod.

3.1.7 AnalyticalvMethod Development

 HPLCvmethod also used for thevpreparation of calibrationvfor cure for the
drugvand in-vitro evaluationvduring the formulationvand evaluation.
 After thevidentification of drugvcalibration cure of drugvwas prepared in
CH4vbuffer and in H2O onvUV spectrophotovmeter at maxvwavelength
256nmvregression value R2 <0.986 producevthe goodvlinearity and
obeyingvBeer lambertv100µg/ml.

25
 A) Standardvcurve of drug invmethanol by UVvspectroscopy
The studyvcurve of KTZ invmethanol at λ ax 256nmvwas preparedvusing UV
Spectrophometervproduce excellentvlinearity at R2 <0.956vobeying Beer
lambertvlaw.
STANDARD CURVE OF DRUG IN METHANOL AT λMAX-254NM

Table2: Standardvcurve of drug invmethanol atvλmax-254nm

S. No. Concentrationv (µg/ml) Absorbance

1 2 0.386
2 4 0.786
3 6 1.086
4 8 1.356
5 10 1.638

Fig Standardvcurve of drug invmethanol by UVvSpectroscopy

Standardvcurve of drug invmethanol at λmax-254nm wasvprepared, the R2 value

wasvfound to be 0.993 and thevequation wasvy=0.136x +0.126.

B) Standardvcurve of drug invbuffer by U.Vvspectroscopy

26
 The studyvcurve of KTZ invCH4 at λ max 256nmvwas preparedvusing U.V
Spectrophotometervproduce excellentvlinearity at R2 <0.956 obeyingvBeer
lambertvlaw.
Table 3: Standardvcurve of drugvacetate buffervat λmax 256nm
S. No Concentrationv (µg/ml) Absorbancev

1 2 0.056
2 4 0.246
3 6 0.376
4 8 0.498
5 10 0.684
6 12 0.812

Fig. Standardvcurve of drug invbuffer (pH 5) by UVvSpectroscopy

The curve of KTZ invCH4 at λ max 256nmvwas preparedvusing U.V

Spectrophotometervproduce excellentvlinearity at R2 <0.956 obeyingvBeer
lambertvlaw.

27
 C) Standardvcurve of drug invwater by U.Vvspectroscopy
The studyvcurve of KTZvin CH4 at λ axv254nm was preparedvusing UV
Spectrophometervproduce excellentvlinearity at R2v<0.9894 obeying Beer
lambertvlaw.

Table 4: Standardvcurve of drug invwater at λmaxv254nm

S. No Concentration (µg/ml) Absorbance

1 2 0.056
2 4 0.246
3 6 0.376
4 8 0.498
5 10 0.684
6 12 0.812

Fig. Standardvcurve of drug invwater by UVvSpectroscopy

Standardvcurve of drug invwater at λmax-254nm wasvprepared, the R2vvalue was

foundvto be 0.986 and thevequation wasvy=0.036x +0.014.

Table 5: Standardvcurve of drug invwater at λmax 254nm invHPLC

28
 S. No. Concentration (µg/ml) Absorbance

1 0 0
2 2 41926
3 4 69874
4 6 101086
5 8 124866
6 10 156075

Fig. STANDARDVCURVE OF DRUG INVWATER Λ MAX-254NM INVHPLC

The graphvshow linear curvevand R2value of thevdrug was found tovbe 0.990. That
givesvthe equationvy=14976x + 8086.

3.2 Impactvof formulationvvariables on characteristicsvof liposomes

Optimization of formulation using a 32 full factorial design

29
A 32 randomized full factorial design was utilized in the present study. Two factors, each at three
levels were evaluated. Experimental trials were carried out at all nine possible combinations. The
factors and their limit were selected based on preliminary study. The molar ratio of DPPC/Chol and
molar ratio of DOPE/DPPC were selected as independent variables. The size of liposomes and EE
%were selected as dependent variables. The formulation composition of the factorial batches (F 1 to
F9) is shown in Table 6.

Response surface methodology approach for optimization of factors

Based on the RSM approach, the runs were conducted in CCD model-designed experiments to
visualize the effects of independent factors on the response along with the experimental conditions.
Response surface diagram was constructed using Minitab version 15. Formulation was optimized
with the help of response surface diagrams.

Results

Effect of formulation ingredients on entrapment efficiency Based on the RSM approach, the
runs were conducted in thin film hydration technique for KTZ liposome

Table 6. 32 Full factorial design, molar composition of DPPC, DOPE and cholesterol in
formulations together with respons-es values [Particle size (PS), Entrapment efficiency
(EE %), Polydispersity index (PDI)]

Formulation DPP Cholester

code C DOPE ol PS (nm) PDI EE (%)
F1 5 0 1 493 0.21 57.9
F
2 5 2.5 1 486 0.19 62.7
F
3 3 0 1 532 0.31 72.5
F
4 3 3 1 556 0.32 68.1
F5 1 0.5 1 646 0.38 85.1
F6 5 5 1 474 0.22 59.3
F7 1 0 1 615 0.39 84.2
F
8 3 1.5 1 549 0.31 76.2
F
9 1 1 1 627 0.39 82.2

30
CCD model-designed experiments to visualize the effects of independent factors on the responses.

A general equation for the relation of affecting factors and

0 + b1 X1 + b2 X2 +b1b1 X1X1 + b2b2 X2X2

+ b1b2 X1X2. Where Y is the dependent variable, b 0 is the arithmetic mean response of the nine runs
and bi is the estimated coefficient for the factor Xi. The main effects (X1 and X2) represent the
average result of changing one factor at a time from its low to high value. The interaction terms
(X1X2) show how the response changes when two factors are simultaneously changed. The
polynomial terms (X1X1 and X2X2) are included to investigate non-linearity.

Magnitude and mathematical sign of coefficients show the effectiveness of dependent variables on
responses. Results of PS and EE % are shown in Table 6. The model equation derived for EE %
was: responseis:Yj=b
YEE % = 92.08 – 6.58 X1+4.33 X2 + 1X1 X2

The negative sign for coefficient of X1 indicates the lowering effect of DPPC/Chol on EE %
(P<0.05). The EE of different liposomal batches was in a range of 57.9 to 85.1%. The maximum
entrapment was observed in batch F5 with the composition of DPPC/ DOPE/Chol (1: 0.5: 1 molar
ratio).

The relationship between the dependent and independent variables was further elucidated using
contour and response surface plots. As shown in Fig. 1, at low level of DOPE/DPPC, EE %
decreased from 84.2 % to 57.9 % when DPPC/Chol increased from 1 to 5. Similarly, at high level
of DOPE/DPPC, EE % decreases from 82.2 % to 59.3 % when DPPC/Chol increases from 1 to 5.

Effect of formulation ingredients on particle size

One of the most important parameters, which need to be monitored during liposome preparation, is
the vesicle size and the size distribution. From a number of reports, it is evident that the size and
size distribution of the liposomes determine their in vitro or in vivo performance. The particle sizes
of different batches of liposomes were in a range of 474 to 646 nm. The minimum and maximum

31
size values correspond to formulations F6 and F5, respectively. The modified model for particle
size is:

YPS= 665.08 -49.12 X1+28.91 X2 + 2.79X12– 7.75X1X 2. The equation clearly indicates that the PS
values are strongly dependent on the selected independent variables. The negative sign for
coefficient of X1 indicates that DPPC/ Chol has a decreasing effect on particle size (p=0.037). The
coefficient of X1 was found to be significant at the level of P<0.05. From the results obtained, it
may be concluded that DOPE/DPPC molar ratio and its interaction term do not contribute
significantly to the particle size of liposomes (p>0.05).

As shown in Fig. 2, at low level of DOPE/DPPC, PS decreased from 615 nm to 493 nm when
DPPC/Chol increased from 1 to 5. Correspondingly, at high level of DOPE/DPPC, PS decreased
from 627 nm to 474 nm when DPPC/Chol increased from 1 to 5.

Overlaid contour plot with defined conditions for desired PS and EE % was obtained using RSM
approach. The white area corresponds to conditions resulting in a particle size in the range between
500 nm to 550 nm and EE % range between 70 to 85% (Fig. 3).

Formulation optimization of liposomes

Validation: The accuracy of the proposed model was validated by conducting other reactions with
different conditions and then comparing the obtained results with the model. Observed and
calculated PS and EE % using these equations are shown in Table 7. Percent error (PE) was
obtained using following equation:

Average percent error (APE) for particle size and EE % in train set were 1.27 % and 2.7 %,
respectively.The internal percent error (PE) of the proposed model can be calculated using obtained
equations for the train experiments (n=9). The average PE for all 9 experiments are 1.28 and 2.57 %
for PS and PE, respectively (Table 7). To evaluate the external predictive performance of the
model, three more experiments were carried out in duplicate as a test set. Table 8 shows conditions
and results of these reactions. The results revealed that the average PE for these experiments is 3.52
and 4.09% for PS and PE, respectively. Considering the low internal and external PE, it might be
concluded that the model has a good predictive power in the studied range of variables. The
proposed model was also used to obtain optimum conditions (Fig. 4). Desired PS and EE % were
32
defined as 500 nm and 80 %, respectively. A new formulation was prepared according to proposed
levels for independent factors. Proposed levels are in accordance with whitearea in the overlaid
plots. Observed responses values were close to the calculated values in the proposed formulations.
Prediction error of EE % and PS were 6.3% and 5.9%, respectively. These results further
demonstrate the suitability of the optimization procedure in developing SRL liposomes.

33
Fig. 1. Response surface plot (left) and Contour plot (right) of the
effect of lipid content on entrapment efficiency percent (EE %).

Fig. 2. Response surface plot (left) and contour plot (right) of the
effect of lipid content on particle size (PS).
Table 7. Observed and calculated percent error (PE) values for particle size (PS) and
entrapment efficiency percent (EE %) in train set

PS
Formulation Obs PS Calc PS PE Obs Calc
code (nm) (nm) (%) EE% EE% EE% PE (%)
F1 493 489 0.76 57 59 3.80
F2 486 484 0.34 62 59 4.03
F3 532 543 2.04 72 72 0.46
F4 556 549 1.34 68 71 4.41
F5 646 629 2.58 85 84 1.38
F6 474 479 1.16 59 60 1.41
F
7 615 619 0.61 84 85 1.78
F8 549 546 0.60 76 72 5.70
F9 627 640 2.07 82 82 0.20
EE%
PS APE 1.28 APE 2.57

Thin film hydration technique for KTZ liposome

Table 8. Observed and calculated percent error (PE) values for particle size
(PS) and entrapment efficiency percent (EE %) in test set

Obs PS Calc PS PS PE Obs EE Calc EE% PE

X1 X2 (nm) (nm) (%) % EE% (%)
4 1 507 511 0.83 63 67 3.83
2 0.5 562 585 4.05 74 59 5.06
3 0.75 580 547 5.67 69 70 3.38
PS EE%
APE 3.52 APE 4.09

3.3 Differentialvscanning calorimetricvstudy

DSCvstudy was carriedvout for KTZ, soyavlecithin, cholesterolvand drug loaded

multilamellarvliposomes using thevmethod described invsection previously.
DSCvthermograms of KTZ, soyavlecithin, cholesterolvand drug loadedvmultilamellar
liposomes arevshown in figure 3. DSCvthermogram of KTZ showedvendotherm at 152.89°C.

34
DSCvthermogram of soyavlecithin and cholesterol showedvendotherm at 128.93°C
andv148.27°C, respectively. DSC thermogramvof KTZ loaded liposomesvcomposed of
soyavlecithin : cholesterol (10:1) weightvratio interestingly showedvdisappearance of
thevmelting endothermvof KTZ and the majorvendotherm was observedvat 137.28°C. The
meltingvendotherm of soyavlecithin was found to bevshifted from 128.93°Cvto 137.28°C,
signifyingvthat all the lipidvcomponents interactvwith each othervto a great extentvwhile
forming thevlipid bilayer. The incorporatedvKTZ associated with lipidvbilayers and
interactedvto a large extentvwith them. Absencevof the meltingvendotherm of KTZvand
shifting of thevlipid bilayervcomponents endothermvsuggested significantvinteraction of
KTZ withvbilayers, indicating the interactionvof KTZ with bilayersvleading to
enhancedventrapment of the drug andvdecreased rate ofvrelease. The DSCvresults of
liposomesvsuggest enhanced entrapmentvefficiency of KTZvin the lipidvbilayer.

Table 9: Fullvmodel for % cumulativevdrug releasevafter 12vhours

Parameter Co- Stand t Stat P- Lower Upper Lower Upper

efficie ar d value 95% 95% 95% 95%
nts Error

Intercept 39.65 0.42 93.22 0.0000 38.27 41.00 38.27 41.00

03
X1 -2.01 0.23 -8.66 0.0032 -2.74 -1.28 -2.74 -1.28
30
X2 3.98 0.23 17.03 0.0004 3.21 4.71 3.21 4.71
40
X1*X2 0.36 0.28 1.25 0.3045 -0.54 1.27 -0.54 1.27
20
X12 4.14 0.40 10.24 0.0019 2.85 5.42 2.85 5.42
80
X22 -0.40 0.40 -1.00 0.3877 -1.68 0.87 -1.68 0.87
00

Table 10: Summaryvoutput of cumulativevpercentage drugvrelease

RegressionvStatist Multiple R R Square Adjustedv Standard Observatio

ics R Square Error ns

35
 0.996834 0.993685 0.983156 0.570543 9

Table 11: ANOVAvfor cumulative percentagevdrug releasevafter 12vhours

Df SS MS F Significanc
eF

Regression 5 153.604134 30.720828 94.375322 0.001697

Residual 3 0.976555 0.325520 - -
Total 8 154.580690 - - -

The percentagevcumulative drug releasevof different liposomalvbatches of 32 full

factorialvdesign was found to be betweenvranges 34.96 ± 0.86 % to 49.20 ± 1.22 %. The %
cumulativevdrug release in optimizedvbatch F4 was observedvto be 34.96 ± 0.86 %.
Figurev4vshows the plot of % cumulativevdrug release againstvtime (hours) for
factorialvdesign batches.

Datavwere analyzed statisticallyvby one-way analysisvof variance (ANOVA)

usingvMicrosoft Excel 2003 andvby the student’svt-test (level of significancevfor p<0.05).

36
Discussion

It is desirable to develop an acceptable pharmaceutical formulation in shortest possible time,

using minimum number of man-hours and raw materials. Traditionally, pharmaceutical
formulations are developed by changing one variable at a time by trial and error method
which is time consuming in nature requiring a lot of imaginative efforts. Moreover, it may be
difficult to develop an ideal formulation using this classical technique, since the joint effects
of independent variables are not considered. It is therefore very essential to understand the
complexity of pharmaceutical formulations by using established statis-tical tools such as
factorial design.

RSM is a collection of mathematical and statistical technique which quantifies the functional
relationship between a number of measured response variables and several explanatory factors
to obtain an optimal response by using a series of tests. The main advantage of RSM is to
reduce the required experimental runs required and it is already widely applied to optimize
formulation design in pharmaceutics studies.

Fig. 3. Overlaid contour plot with defined conditions for desired particle size (PS) and
entrapment efficiency percent (EE %).

37
Fig. 4. Optimization plot for formulation with desired particle size (PS) and entrapment
efficiency percent (EE %) values.Factorialvequation for percentagevcumulative
drugvrelease after 12 hours

The polynomialvequation was generatedvby multiple linearvregressions. The

equationvderived is asvunder

2 2
Y =39.65+2.01 +3.98X2 +0.36X1X2 +4.14X1 - 0.40X2

The datavclearly indicatevthat the % cumulative drugvrelease valuesvare strongly

dependentvon the selectedvindependent variables. The fittedvequation (for full model)
relatingvthe response (% cumulativevdrug release) to the transformedvfactor is shown
byvequation 4. The polynomialvequations can be used to drawvconclusions after
consideringvthe magnitude ofvcoefficient and the mathematicalvsign it carries (i.e.
positivevor negative). Table 10vshows the resultsvof the analysis ofvvariance (ANOVA)
whichvwas performed tovidentify insignificantvfactors.

The highvvalues of correlationvcoefficient for % cumulativevdrug release (Table 9)

indicateva good fit. The equationsvmay be used to obtainvestimates of the responsevas a

38
small error ofvvariance was noticedvin thevreplicates. The results of statisticalvanalysis are
shownvin Table 8. The coefficientsvb1, b2 and b11 werevfound to be significantvat P<0.05.
While thevsignificance levelvof coefficient b12 and b22 werevfound to bevP = 0.3045vand P =
0.3877 respectivelyvwhich is P >0.05 so it mayvbe concluded thatvinteraction term b12 and
b22 doesvnot contribute significantlyvto the prediction of % cumulativevdrug release.

Fromvthe above study, itvwas found that thevdrug release fromvliposomes primarilyvdepends
on the lipidvcomposition, drug to lipidvratio and also the volume ofvhydration medium.

The resultsvof regression analysisvreveal that, on increasingvthe values for X1, decrease in %
cumulativevdrug release isvobserved, becausevcoefficient b1 bears a negativevsign. While on
increasingvthe values for X2, increasevin % cumulativevdrug discharge isvobserved, because
coefficientvb2 bears avpositive sign.

Storagevstability ofvliposomes

Here, onevmonth stability study ofvliposomal suspensionvwas conductedvwith

respect to thevliposomes’ ability to retainvan entrapped drugvduring a defined
timevperiod.

storagevconditions, i.e. at refrigerationvcondition (2-8°C), at roomvtemperature

(25±2°C) andvat 45°C. Figure 5 showsvFigure 4: DSC thermogramsvof KTZ
(A), soyavlecithin (B), cholesterol (C), andvdrug loaded multilamellar
liposomes (D).

Figure 3: Particlevsize distribution ofvoptimized batch (F4) by Sympatecparticle vsize

analyzer

39
Figure 4: DSCvthermograms of KTZ (A), soyavlecithin (B), cholesterol (C), andvdrug

loaded multilamellarvliposomes (D).

liposomesvwere relatively stablevat refrigerated storagevcondition. The drug

leakage percentvamounts of originalventrapped in liposomesvare very small (<
5%) at 2-8°C andvhave no significantvdifference after 1 monthvcompared with
immediatelyvafter makig. The resultsvof drug retentionvstudies showvhigher
40
drug leakagevat higher temp. Thisvmay be due to thevhigher fluidity ofvlipid
bilayers at highervtemp, resulting in highervdrug outflow. Loss ofvdrug from
the vesiclesvstored at measuredvtemp that may bevattributed to theveffect of
temperaturevon the gel tovliquid transition ofvlipid bilayersvtogether with
possible chemicalvdegradation of thevphospholipids, leading tovdefects in
membranevpacking. Acceleration invdrug release at highervtemp, as observed in
storagevstability studies, suggestedvkeeping the liposomalvproduct in the
coolingvcondition.

41
 CHAPTERv4
CONCLUSIONv

4. Conclusionv
Thevliposomal productvof ketoconazole wasvprepared with thevview to
improvevtherapeutic responsevand reduce thevpossible adversevsymptoms. Here
liposomesvof ketoconazolevwere prepared usingvthin filmvhydration technique.
Percentageventrapment efficiencyvwas optimized aftervstudying the effect ofvvarious process
andvformulation variables. Maximumventrapment efficiencyvwas found tovbe 54.41±0.19 %.
The preparedvliposomes werevfound to havevgood morphological properties andvsize
distribution. FromvDSC thermograms of liposomesvit can be concluded thatvsignificant
interactionvoccur betweenvdrug andvlipid components of thevvesicles thatvlead to higher
entrapmentvefficiency. The percentagevcumulative drug releasevfrom the optimizedvbatch
i.e. F4 wasvfound to be 34.96±0.86 % afterv12 hours of diffusionvstudies. Stabilityvstudies
showed maximumvpercent drug retentionvat refrigeratedvtemperature (2-8°C). The
drugventrapment efficiencyvcan be attributed tovphospholipids’ abilityvto vesiculate
independentlyvbecause they carryvtwo bulky nonpolarvlipid chains and avpolar headvgroup,
which helpsvthem spontaneouslyvform into closed bilayervsystems.
A full factorial design and central composite design of response surface methodology can be
used to determine the significant variables and optimum condition for preparation of
liposomes. The present study focused on the preparation and characterization of liposome
using the thin film hydration method. Particle size and % are important characteristics in
liposome formula-tions which have important effects on in vitro and in vivo properties.
Percentage of encapsulation efficiency was optimized after studying the effect of various
formula-tion variables. DPPC/Chol molar ratio had a profound ef-fect on the entrapment
efficiency and liposome size. The proposed model could be successfully used to predict and
optimize both liposome size and EE %.

42
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