Development and Optimization of Polymeric Self-Emulsifying Nanocapsules for Localized Drug ...

13 Pages·2015·3.19 MB·English

Hindawi Publishing Corporation
e Scientific World Journal
Volume 2014, Article ID 516069, 12 pages
http://dx.doi.org/10.1155/2014/516069
Research Article
Development and Optimization of Polymeric
Self-Emulsifying Nanocapsules for Localized Drug Delivery:
Design of Experiment Approach
JyotiWadhwa,1AbhayAsthana,1SumeetGupta,1
GyatiShilkariAsthana,1andRanjitSingh2
1MMCollegeofPharmacy,MMUniversity,Mullana,Ambala133207,India
2GovernmentCollegeofPharmacy,Rohru,Shimla171207,India
CorrespondenceshouldbeaddressedtoAbhayAsthana;[email protected]
Received23July2014;Accepted22September2014;Published24November2014
AcademicEditor:SanjulaBaboota
Copyright©2014JyotiWadhwaetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Thepurposeofthepresentstudywastoformulatepolymericself-emulsifyingcurcuminnanocapsuleswithhighencapsulation
efficiency,goodemulsificationability,andoptimalglobulesizeforlocalizedtargetinginthecolon.Formulationswereprepared
usingmodifiedquasiemulsionsolventdiffusionmethod.Concentrationofformulationvariables,namely,𝑋1(oil),𝑋2(polymeric
emulsifier),and𝑋3(adsorbent),wasoptimizedbydesignofexperimentsusingBox-Behnkendesign,foritsimpactonmeanglobule
size(𝑌1)andencapsulationefficiency(𝑌2)oftheformulation.Polymericnanocapsuleswithanaveragediameterof100–180nm
andanencapsulationefficiencyof64.85±0.12%wereobtained.Invitrostudiesrevealedthatformulationsreleasedthedrugafter
5hlagtimecorrespondingtothetimetoreachthecolonicregion.Pronouncedlocalizedactionwasinferredfromtheplasma
concentrationprofile(𝐶 200ng/mL)thatdepictslimitedsystemicabsorption.Roentgenographystudyconfirmsthelocalized
max
presenceofcarrier(0–2hinupperGIT;2–4hinsmallintestine;and4–24hinthelowerintestine).Optimizedformulationshowed
significantlyhighercytotoxicity(IC50value20.32𝜇M)inHT29coloniccancercellline.Thepresentstudydemonstratessystematic
developmentofpolymericself-emulsifyingnanocapsuleformulationofcurcuminforlocalizedtargetingincolon.
1.Introduction previously reported studies have aimed at improving
poor aqueous solubility, bioavailability, alkaline stability,
Accumulatedevidencesovertheyearshaveshownthatmany
and/or rapid intestinal metabolism of CUR. These includes
anticancerdrugmolecules,suchassteroidsandnonsteroidal
CUR-impregnated soluble dietary fibres dispersion with
anti-inflammatory drugs, are associated with numerous
enhanced bioavailability (20 times) comparative free CUR
side effects. Perhaps the best instance is the cardiovascular
[6].CURmayalsobecombinedwithpiperine,whichinhibits
impedimentsimposedbytheuseofCOXinhibitors[1,2].As
enzymatic conjugation and allows enhanced absorption of
a result,therehas been an increasingdemandforsaferand
unchangedcurcuminoidsintoportalblood[7].Inaddition,
proficientdrugmolecules.Curcumin,theIndiansolidgold,
variousstrategieshavebeenundertakentodelivercurcumin
has been reported as one of the most promising candidates
to intestine by multiparticulate systems [8], solid lipid
of natural origin, having anticancer properties, with almost
nanoparticles [9, 10], and polymeric micelles [11] in cancer
no reported side effects [3]. Therapeutic applications of
therapy with enhanced systemic bioavailability. However,
curcumin (CUR), however, remain limited due to its poor
absorption and rapid elimination [4]. It has been observed theseaforementionedsystemshavepoorlocalisationefficacy
that more than 60–70% CUR is excreted unchanged in duetorapiddrugabsorptionintothesystemiccirculation.
the faeces and the remainder is metabolized and absorbed A review of literature suggests that the application of
through the intestinal mucosa and liver [5]. Numerous carrier technology is not limited to scientific interest in
2 TheScientificWorldJournal
suchformulationsbutunderlinesthepotentialandversatility Shimadzu HPLC system (LC-2010C HT, Japan) equipped
in addressing the problems associated with poorly aqueous with a reverse phase Phenomenex C18 column (250mm ×
∘
solubledrugsforlocalizeddelivery[12,13].Newapproaches, 4.6mm).Elutionwascarriedoutatroomtemperature(37 C),
such as self-emulsifying drug delivery system, have also withaUV-visibledetectionwavelengthof425nm.Amixture
foundtheirwayinenhancingthesolubilityofCURincolonic ofacetonitrile:HPLCwater(57:43%v/v),pH3.3,wasused
conditionsandhaveseveraladvantagesovertheexistingones asmobilephaseataflowrateof1.0mL/min.
[14,15].Moreover,nanocapsules(polymericwallenveloping
an oil core, confining the drug molecule within the central 2.2.2. Experimental Design. Based on the preliminary stud-
cavity)deliverthedrugtotargetsiteinacontrolledmanner ies, formulation excipients Capryol 90 (oil), HPMCAS-HF
[16]. Novel nanocapsules bearing self-emulsification ability (polymer),andAerosil200(adsorbent)wereselectedasthe
andhighloadingefficiencycaneffectivelyaddressthegastric independent variables 𝑋1, 𝑋2, and 𝑋3, respectively. Box-
resistanceproblemsandsuitablytargetcolonicsites[17]. Behnken design (BBD) was applied to the optimization
Tothebestofourknowledge,polymericself-emulsifying procedureusingDesignExpert(Ver.8.0.7.1)software. Aset
nanocapsules for colon targeting have not been explored of seventeen trial formulations (P1–P17) was prepared by
for curcumin, till date. Nanosized polymeric formulation varyingtheconcentrationofindependentvariablesatthree
transformed to emulsified form when it comes in contact differentlevels(−1,0,and+1)(Table1).Thequadraticmodel
with an alkaline medium (simulating colonic region). As generatedbythedesignhas(1)asfollows:
a result, dissolved drug might be released in a controlled
𝑌=𝑏 +𝑏 𝑋 +𝑏 𝑋 +𝑏 𝑋 +𝑏 𝑋 𝑋 +𝑏 𝑋 𝑋
mannerafterinitialburstrelease,duetodissolutionofpoly- 0 1 1 2 2 3 3 12 1 2 13 1 3
(1)
mericmatrixofthenanoparticulatesystem leadingto local +𝑏 𝑋 𝑋 +𝑏 𝑋 2+𝑏 𝑋 2+𝑏 𝑋 2.
action[18].Presentpaperreportsdevelopmentandoptimiza- 23 2 3 11 1 22 2 33 3
tionofcurcuminpolymericnanocapsuleformulation,using
The above equation comprises the coefficient of the inter-
a pH-sensitive polymer (HPMCAS-HF) possessing self- cept, first-order main effect (𝑋1,𝑋2,𝑋3), interaction terms
emulsifyingabilityandlocalizeddrugdeliveryinthecolon. (𝑋1𝑋2,𝑋1𝑋3,𝑋2𝑋3), and higher order effect (𝑋12,𝑋22,
𝑋32),where𝑌isthemeasuredresponse;responsevariables
2.MaterialsandMethods selectedfortheoptimizationpurposeweremeanglobulesize
(Z-Avg)(𝑌1)andencapsulationefficiency(𝑌2).
2.1.Materials. Curcumin(CUR)waspurchasedfromHiMe-
dia,Mumbai,India.Hydroxypropylmethylcelluloseacetate 2.2.3. Preparation of Curcumin Loaded Polymeric Self-
succinate(HPMCAS-HF)waskindlygiftedbyArihanttrad- Emulsifying Nanocapsules (PSN). CUR loaded PSN formu-
ing Co., Mumbai, India. Capryol 90 (C-90), Lauroglycol lations (P1–P17) were prepared using modified quasiemul-
FCC (LFCC), and Labrafac were obtained as gratis from sion solvent diffusion method (Figure1) [20, 21]. Solu-
Gattefosse Pvt. Ltd (Mumbai, India). Edible oils (isopropyl tion of polymer (HPMCAS-HF) was prepared in ethanol:
myristate,castoroil,oleicacid,ethyloleate,cornoil,Captex dichloromethane (1:1) mixture. CUR was incorporated in
200, apricot oil, olive oil, and soybean oil) were purchased the lipidic phase of Capryol 90, followed by addition of
from HiMedia, Mumbai, India. Aerosil 200 and polyvinyl Aerosil200.CUR-oilmixturewasemulsifiedwithHPMCAS-
alcohol (1,25,000Mol. Wt.) were purchased from CDH, HF solutionusing probe sonicatorto yield a w/o emulsion.
Mumbai, India. 96-well plates (A-U96) were kindly gifted Anaqueouspolyvinylalcoholsolution(0.3%w/v)containing
by the Lipidcure Core NOF Corporation, Japan. Potassium 0.1%sodiumlaurylsulfate(SLS)waspreparedseparatelyand
dihydrogenphosphatewaspurchasedfromMerck,Mumbai, thepreparedw/oemulsionwasemulsifiedinPVAsolution,in
India.Alltheotherchemicalsandreagentsusedinthestudy adropwisemanner.Theresultingw/o/wemulsionwasstirred
wereofanalyticalgrade. magneticallyat500rpmfor4-5hat37∘C.Disperseddroplets
were solidified by diffusion of the solvent into the aqueous
phase. Solidified particles were washed with distilled water
2.2.InitialScreeningofExcipients
thricefollowedbycentrifugation(25,000gfor10minutesat
∘
2.2.1. Solubility Studies. Solubility of CUR was determined 4 C). Solidified PSN was suspended in distilled water and
in oils (C-90, LFCC, Labrafac, isopropyl myristate, castor lyophilized at −18∘C for 24h. Resultant product was stored
∘
oil, oleic acid, ethyl oleate, corn oil, Captex 200, apricot inavacuumdesiccatorat25 C.
oil, olive oil, and soybean oil) using saturated shake flask
methodasreportedbySinghetal.,2010[19].ExcessCURwas 2.3.EvaluationofPolymericSelf-EmulsifyingNanocapsules
suspended in oil in a screw capped glass vial. The mixture
was sonicated for 5min to ensure uniform mixing and 2.3.1. Globule Size. The globule size of the nanocapsule
∘
solubilization of CUR. The mixture was shaken at 37 C for formulations was determined by photon correlation spec-
24hintheshakerwaterbath(AccumaxIndiaPvt.Ltd.,New troscopy (PCS), using Zetasizer Nano S90, Malvern, WR14
Delhi, India), set at 100rpm, and allowed to stand for 48h 1XZ,UK.PSN(10mg)weredispersedin100mLofphosphate
to attain equilibrium. After 72h, mixtures were centrifuged buffer (pH-7.2) using vortex for 1h and filtered through a
at 3000rpm for 10min, followed by filtration through a membranefilter(0.22𝜇m)[15].Thefiltratewasanalyzedfor
0.45𝜇m membrane filter. Analysis was carried out using a Z-Avgandzetapotentialintriplicate.
TheScientificWorldJournal 3
Table1:CompositionofnanocapsuleformulationusingBox-Behnkendesign.
Low Middle High
Levels
Coded Actual(mg) Coded Actual(mg) Coded Actual(mg)
Independentfactors
𝑋:conc.ofCapryol90(oil)(mg) −1 250 0 500 1 750
1
𝑋 :conc.ofHPMCAS-HF(polymericemulsifier)(mg) −1 100 0 150 1 200
2
𝑋 :conc.ofAerosil200(adsorbent)(mg) −1 75 0 100 1 125
3
Dependentvariables
𝑌:meanglobulesize(nm) Minimize
1
𝑌:encapsulationefficiency(%) Maximize
2
Sonication
Polymer CUR-oil- Polyvinyl
w/o n
solution Aemroixsitlu 2re00 emulsion saolcluothioonl catio
ni
o
as
Ultr
Diffusion of organic
Centrifugation solvent out of the
g Quasiemulsion
n droplets
yi droplets
dr
e
z
e
e
Fr
Solidified
nanocapsules
Nanocapsules
Figure1:FormulationstrategyofCUR-PSN.
2.3.2.EncapsulationEfficiency. CURloadedPSNformulation of CUR during the process. A droplet of w/o/w emulsion
theoretically equivalent to 10mg of CUR was weighed and was mounted on a slide and observed by phase contrast
dissolved in 5mL of methanol by vortex [22]. Samples microscope(Metzer-M,Mathura,India).
werecentrifuged(R-4C,Remicentrifuge,Mumbai,India)at
4000rpm for 10min, filtered, diluted, and quantified using
2.3.5. Surface Morphology. The surface morphology of the
HPLC. All studies were conducted in triplicate. Encapsula-
optimized formulation (P5), with and without plasticizer
tionefficiencywascalculatedas
(TEC), was examined using scanning electron microscope,
Encapsulation efficiency (%) SEM(EVO18,Zeiss,Jena,Germany).Thesamplewasfixed
using double-sided adhesive tape to a brass specimen and
=( Actual drug loaded )∗100. (2) madeelectricallyconductivebygoldcoatinginvacuum[24].
Theoretical drug loaded Sampleswereimagedatdifferentresolutions(2-6KX).
2.3.3.EmulsificationEfficiency. AnaccuratelyweighedCUR 2.3.6.DifferentialScanningCalorimetry. Thermalanalysisof
loaded PSN formulation (equivalent to 10mg of CUR) was CUR, C-90, HPMCAS-HF, physical mixture of CUR with
introducedinto100mLofsimulatedcolonicmedium(pH7.2) HPMCAS-HF, and optimized PSN formulation (P5) was
[23]. Medium was agitated at 50rpm for 2h at 37 ± 0.5∘C. carriedoutusingdifferentialscanningcalorimeter(DSC-204
Formationofemulsionwasobservedbyvisualinspection. F1, Phoenix, NETZSCH-Gera¨tebau GmbH, Deutschland,
Germany)undernitrogenpurging(50cc/min).Sampleswere
2.3.4. In Process Particulate Distribution. Trinocular phase placed in crimped aluminium pans and were heated from
∘ ∘
contrastmicroscopywasusedtodeterminethedistribution ambienttemperatureto250 Cat10 C/min[25].
4 TheScientificWorldJournal
2.3.7. X-Ray Powder Diffraction (XRPD). The diffraction controlthathadnotbeentreatedwitheitherformulationor
patternofCUR,HPMCAS-HF,physicalmixtureofCURwith freeCUR,usingthefollowingequation:
HPMCAS-HF,andoptimizedformulation(P5)wasobtained
𝑁
by XRPD (Bruker D8 Advance, Karlsruhe, Germany) to % Cell Viability= 𝑖 ×100, (3)
assess their crystallinity [26]. These were scanned over 2𝜃 𝑁𝑐
rangefrom10to35∘at0.05𝜃/secstepsize.
where 𝑁𝑖 and 𝑁𝑐 are the numbers of surviving cells in
the group treated with CUR loaded formulation and in the
2.3.8. In Vitro Dissolution Studies. In vitro release of CUR
untreatedcellgroup,respectively.
from PSN formulations was determined using USP type II
(Paddle type) dissolution apparatus to study the effect of
2.3.10.InVivoAnimalStudy. InvivostudyoffreeCUR/CUR-
pHondrugrelease.FormulationP1–P17equivalentto10mg
CURwastransferredto325mLofdissolutionmediaat37± loaded PSN formulation was carried out on Duncan Hart-
0.5∘Cfor2hinsimulatedgastricfluid,pH1.2;thepHofthe ley guinea pigs (250–300g), as per the institutional pro-
tocol (MMCP/IAEC/11/23) approved by the animal ethics
dissolution media was then adjusted to 6.8 by the addition
committee of the MM College of Pharmacy. 100mg/kg of
of 125mL of 0.2M trisodium orthophosphate. Dissolution
CUR and equivalent dose of optimized formulation (P5)
was continued in phosphate buffer (pH 7.2) up to 12h.
were administered to guinea pig, in groups of six animals,
Aliquotsof5mLofthedissolutionmediumwerewithdrawn
respectively, in fasting conditions. During the course of the
atpredeterminedtimeintervalsandfilteredthrough0.45𝜇m
studies, waterwasavailable ad libitum.Animalsusedfor in
nylon filter. Concentration of CUR was determined using vivoexperimentsweredividedintothreegroups(𝑛=6).The
HPLCasdescribedpreviously.
PSNsuspension(dose100mg/kg),suspensionofpureCUR
In order to discriminate the formulations showing (dose 100mg/kg), and control were administered by oral
insignificantdifferenceinreleaseprofilecomparativelyopti- routeusingoralfeedingneedlenumber18.Guineapigswere
mizedformulation(P5),adiscriminatingstudywascarried anesthetizedusingchloroform,andbloodsamples(200𝜇L)
outonformulations(P3,P5,andP10)withaforementioned were withdrawn from the femoral vein in EDTA coated
dissolutionconditions,butwithvariedpaddlespeed(50and Eppendorftubesatspecifiedtimeintervals(0,0.5,1,2,4,6,8,
75rpm)andvolumeofdissolutionmedia(phosphatebuffer, 10,12,and24h).Bloodsampleswereimmediatelycentrifuged
pH7.2),500and900mL[27]. for5minandplasmawasseparated.AShimadzuLC-2010C
HT HPLC system equipped with a UV-visible detector
was used to quantitatively determine the concentration of
2.3.9. Cell Viability Assay. The effect of CUR loaded PSN
CURand4-hydroxybenzophenone(internalstandard,IS)in
formulationoncellgrowthwasdeterminedonhumancolon
plasma samples. 100𝜇L of plasma was mixed with 50𝜇L of
carcinoma,HT29cellline.Thecellgrowthinhibitoryactivity
IS working solution (8𝜇g/mL) in an Eppendorf tube. The
ofsampleswasevaluatedby3-(4,5-dimethylthiazol-2-yl)-2,5-
plasmawasthenextractedtwicewith250𝜇Lofacetonitrile
diphenyltetrazoliumbromide(MTT)colorimetricassay[11].
byvigorousmixingfor5minandwascentrifugedat3000g
Cell lines were maintained in folate deficient RPMI 1640
for 15min. 20𝜇L of the supernatant was injected directly
mediumundersuitableconditions(supplementedwith2mM
intothecolumnandwasanalysedquantitatively.Elutionwas
glutamine, 1% Pen-Strep (Sigma Aldrich, St. Louis, USA),
carriedoutat300and425nmfor4-hydroxybenzophenone
and 10% fetal bovine serum (FBS)). Cells were maintained
∘ andCUR,respectively[29].
at37 Cinahumidifiedatmospherecontaining5%CO2/95%
relativehumidity(CO2 incubator;Binder,Germany).When
the cells were confluent, they were trypsinized and seeded 3.Results
into 96-well culture plates at a cell density of 2 × 103 cells
CUR showed a limited aqueous solubility (0.6𝜇g/mL),
per well and the plates were maintained under the condi-
whereasithadsignificantlyhighersolubilityinoils(Figure2).
tionspreviouslymentioned.Twenty-fourhourslater,theold
CURshowedhighestsolubility(11.41±0.23mg/g)inC-90
mediumwascarefullyaspiratedandthecellswereincubated
and thereby selected as the oil phase (independent variable
in a logarithmic growth phase with various concentrations
ranging from 0 to 30𝜇g/mL of free CUR, equivalent CUR 𝑋1)forPSNformulation.Onthebasisofsolubilityofpoly-
merinorganicsolvents(ethanol,acetone,isopropylalcohol,
loaded PSN formulation (P5), equivalent concentration of
and dichloromethane), ethanol and dichloromethane (1:1)
plain PSN formulation, and DMSO (control). After 24h
wereselectedastheorganicphase.
of incubation, the old medium was aspirated and replaced
withfreshmedium.MTTdye(0.5mg/mL,20𝜇L)wasadded
to each well and the plate was incubated for further 4h 3.1.OptimizationofFormulationUsingDesignofExperiments.
∘
at 37 C allowing viable cells to reduce MTT into purple Box-Behnken design (BBD) of experiments was applied to
formazan crystal [28]. After incubation, the medium was the present study to investigate the effect of independent
removed and 200𝜇L of dimethyl sulphoxide (DMSO) was variables 𝑋1, 𝑋2, and 𝑋3 (oil, polymer, and adsorbent)
addedandtheopticaldensitywasmeasuredat450nmusinga concentration, respectively, on dependent variables 𝑌1 and
microplatereader(SunriseTecan,Ma¨nnedorf,Switzerland). 𝑌2 (globule size and encapsulation efficiency, resp.) [30].
Cell viability was expressed as a percentage compared to a Analysis of variance (ANOVA) was applied to determine
TheScientificWorldJournal 5
Table2:CombinationlevelsofindependentvariablesandtheoutcomesofresponsevariablesbyBox-Behnkendesign.
Independentfactors Actualvalue Predictedvalue
Batchnumber
Meanglobule Encapsulation Meanglobule Encapsulation
Capryol90 HPMCAS-HF Aerosil200
size(nm) efficiency(%) size(nm) efficiency(%)
P1 −1 −1 0 173.86 67.42 168.40 68.95
P2 +1 −1 0 194.84 96.18 189.01 94.68
P3 −1 +1 0 140.49 57.81 146.31 59.30
P4 +1 +1 0 172.06 59.15 177.51 57.61
P5 −1 0 −1 127.50 71.82 124.31 64.85
P6 +1 0 −1 180.02 67.05 177.20 63.11
P7 −1 0 +1 164.52 38.41 167.33 42.34
P8 +1 0 +1 163.07 61.16 166.25 68.12
P9 0 −1 −1 156.55 51.86 165.25 57.29
P10 0 +1 −1 164.35 39.90 161.70 45.36
P11 0 −1 +1 191.87 65.44 194.51 59.97
P12 0 +1 +1 173.07 30.61 164.43 25.18
P13 0 0 0 173.07 71.01 176.31 71.31
P14 0 0 0 180.91 70.79 176.31 71.31
P15 0 0 0 169.87 69.76 176.31 71.31
P16 0 0 0 184.81 75.05 176.31 71.31
P17 0 0 0 172.92 69.96 176.31 71.31
∗Standarddeviationoftheobservedresponseswaswithin±5%(𝑛=3).
14 Themodelproposes(4)forglobulesize
12 𝑌 =176.31+12.95𝑋 −8.39𝑋 +8.01𝑋
1 1 2 3
m) 10
g/g −9.343𝑋12+3.34𝑋22−8.20𝑋32+2.64𝑋1𝑋2 (4)
m 8
olubility ( 46 (𝐹 value−=163..54391𝑋, 1𝑅𝑋23=−60.6.859𝑋325𝑋,3a.nd adequate precision =
S
2 11.009.)
Globule size was highest in batch P2 at higher levels of
0
oil,lowlevelofpolymer,andmidlevelofadsorbentandwas
opropyl myristate Ethyl oleate Castor acid Oleic acid Apricot oil 90Capryol Olive oil Corn oil Lauroglycol FCC Labrafac 200Captex lliaoenvvwflaeiuellaseotbnfilcnepeoodblnaybtlmyicnh𝑋eerP1a(5,tF𝑋haigtt2tu,lpor𝑋:we//33d,l(exaav.nd)e)dol.si𝑋R.ooe1frs𝑋gop/i3o1l0n(aS.ns1ue1d5p𝑌5ap1/dl2esw0moa1r4esb/nse5tin1ag6trn0yai6finT9cda)ab.mnlteildy1
Is
Figure2:Solubilityprofileofcurcumininvariousoils(𝑛=3). 3.2.2. Encapsulation Efficiency. Regression analysis for
response 𝑌2 (encapsulation efficiency) suggested quadratic
model and the cubic model was aliased due to insufficient
designpointstoestimatethecoefficients(Table3).ANOVA
datasuggestedregressiontobesignificant(𝑃<0.01).
the significance of regression, correlation coefficient (𝑅-
Themodelproposespolynomialequation(5)forpercent-
Square),adjusted𝑅-squarevalue,andtheadequateprecision
agedrugencapsulationasfollows:
toestimate𝑌1and𝑌2(Table2).
𝑌 =71.31+6.01𝑋 −11.67𝑋 +4.37𝑋 +5.71𝑋
2 1 2 3 12
3.2.EvaluationofCurcuminPSN −6.91𝑋22−17.44𝑋32−6.85𝑋1𝑋2+6.88𝑋1𝑋3 (5)
3.2.1.MeanGlobuleSize. Regressionanalysisforresponse𝑌1 −5.71𝑋2𝑋3.
(meanglobulesize)suggestedlinearandquadraticmodeland
thecubicmodelwasaliasedduetoinsufficientdesignpoints (𝐹value=10.38,𝑅2 =0.9303,andadequateprecision=14.47.)
toestimatethecoefficients(Table3).ANOVAdatasuggested Synergisticeffectsof𝑋1,𝑋1𝑋3,and𝑋12andantagonistic
regressiontobesignificant(𝑃<0.01). effects of 𝑋2, 𝑋3, 𝑋1𝑋2, 𝑋2𝑋3, 𝑋2, and 𝑋32 on 𝑌2 were
6 TheScientificWorldJournal
Table3:Regressionanalysisformeanglobulesizeandencapsulationefficiency.
Response Model Std.dev. 𝑅2 Adjusted𝑅2 Remarks
Linear 12.79 0.532 0.424 Suggested
Secondorder 10.93 0.737 0.579 —
Meanglobulesize(nm)
Quadratic 8.31 0.893 0.756 Suggested
Cube 6.26 0.965 0.862 Aliased
Linear 13.60 0.389 0.248 —
Secondorder 13.77 0.518 0.229 —
Encapsulationefficiency(%)
Quadratic 6.26 0.930 0.840 Suggested
Cube 2.15 0.995 0.981 Aliased
Mean globule size Encapsulation efficiency
152.98 157.195 24.8867 36.9568
B+:200.00 123.267 2 181.452 B+:200.00 58.8342 2 43.3842
nHF 5 nHF 5
Concentratioof HPMCAS- B−:100.00 57.0467 1886.396.021682 1C8−1.:97857.00 CCo+nc:e1nAt2err5aotis.i0oln02 0of0 Concentratioof HPMCAS- B−:100.00 57.0467 45.96699.201682 8C5−.4:57953.00 CC+on:c1enA2ter5raot.is0iol0n2 0of0
A−:250.00 A+:750.00 A−:250.00 A+:750.00
Concentration of Concentration of
Capryol90 Capryol90
(a) (b)
Figure3:CubesurfacegraphsfortheresponsesofCapryol90,HPMCAS-HF,andAerosil200.(a)Meanglobulesize;(b)%encapsulation
efficiency.
observed. Encapsulation efficiency was highest in batch P2 HCl (pH 1.2), whereas less than 20% of drug was released
athigherlevelsofoil,lowlevelofpolymer,andmidlevelof insimulatedintestinalfluid(pH6.8).However,insignificant
adsorbent and was lowest in the batch P12 at mid level of discrimination was observed in selected dissolution test
oil and high level of polymer and adsorbent (Figure3(b)). conditions(simulatedcolonicfluid(pH7.2).
Significance of results has been confirmed by observed 𝑃 Byincreasingthedissolutionmediumvolume(phosphate
value(<0.03)foreachfactor(SupplementaryTable1). buffer,pH7.2)upto900mL,thepercentageofdrugrelease
observed is more than 60% when compared to 500mL of
3.2.3.IdentificationandEvaluationofOptimumFormulation the same medium at both paddle speeds (50 and 75rpm).
UsingtheDesirabilityFunction. PSNformulationwithacom- Dissolution profiles of formulations (P3, P5, and P10) in
positionconsistingof250mgC-90(oil),150mgHPMCAS- 900mLofphosphatebuffer(pH7.2)and50rpmpaddlespeed
HF (polymer), and 75mg A-200 (adsorbent) was observed are shown in Figure6(b). Percentage drug release of three
to be optimal, in terms of desired mean globule size and formulations(P5,P3,andP10)insimulatedphosphatebuffer
encapsulationefficiency(Figure4(a)).Figure4(b)showsthe (pH7.2;900mL)wasobservedat72.19±2.13,65.8±1.58,and
highestdesirabilityfactor1.00andtheoverlayplotsinavaried 60.27±1.72%,respectively,whereas58.15±1.86,56.07±1.12,
rangeofoilandpolymerforoptimizedformulation. and53.99±0.92%ofdrugreleasewereobservedinsimulated
phosphate buffer (pH 7.2; 500mL) from three formulations
(P5,P3,andP10)respectively.
3.2.4.InProcessParticulateDistribution. Figure5showsthat
the drug particles were uniformly distributed within the
nanocapsule.Theshelloftheparticlesappearsasdark,while 3.2.6.DifferentialScanningCalorimetry(DSC). DSCthermo-
theinnercoreappearsasyellowishgreen. gramsofCUR,HPMCAS-HF,C-90,physicalmixtureofCUR
withHPMCAS-HF,andtheoptimizedformulation(P5)are
3.2.5.InVitroDissolutionStudies. Figure6(a)illustratesthe shown in Figure7. A sharp endothermic melting peak of
∘
drug release profile of CUR from PSN formulations (P1– CUR appeared at 180.71 C indicating its crystalline nature.
P17). Arround 6% of drug release was observed in 0.1N HPMCAS-HFandC-90didnotshowanypeakovertheentire
TheScientificWorldJournal 7
Overlay plot
200.00
Desirability
1.000 1.000
F
H
S- 175.00
A
C
B+:300.00 2 M
1.000 1.000 HP
of 150.00 Mean globule size CI: 127.5
ation AS-HF Prediction 1.000 5 ntration
Concentrof HPMC B−:100.00 1.000 1.000 1.0002 C−1:.70500 ConceCnAt+errat:oisoi1ln 72o0f50 Conce 112050..00000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
A−:200 Concentration A+:200 25 30 35 40 45 50 55 60 65 70 75
of Capryol 90 Concentration of Capryol 90
(a) (b)
Figure 4: (a) Cube surface graphs for overall desirability (𝐷); (b)overlay plot for the optimization of concentration of Capryol 90 and
HPMCAS-HF.
3.2.9. Cell Viability Assay. Viability of cells was measured
using the MTT test to evaluate the cytotoxicity of CUR on
Polymer coat HT29 cell lines. Results of cell viability assay are shown in
Figure10.TheIC50 valueoftheoptimizedPSNformulation
Core containing drug (P5) was found to be 20.32 micro molar, while that of free
CUR had 28.56 micro molar. Compared to DMSO treated
cells, no cytotoxicity was observed in the cells exposed to
blank PSN formulation. Insignificant change was observed
afterperformingthetestinsimilarmannerupto72h.
3.2.10.InVivoAnimalStudy. Plasmaconcentrationtimepro-
fileofoptimizedformulationandplainCURarerepresented
Figure 5: Phase contrast microscopy representing in the process
inFigure11.Pharmacokineticprofileofoptimizedformula-
particulatedistributionoftheemulsiondroplets(scale100x).
tion was evaluated, compared to pure CUR. Concentration
of CUR was detectable up to 24h although insignificant
concentrationofdrug(𝐶max200ng/mL)wasobservedwhich
range of temperature. Optimized formulation (P5) did not may be due to the slow clearance rate leading to greater
showanymeltingendothermicpeakowingtoitsamorphous enhancementineliminationhalf-lifeandcorrelateswellwith
nature. invitroreleasedata.
Theresultsaresupportedwithroentgenographicimages
(Figure12) of guinea pig at different time points. Images
3.2.7. X-Ray Powder Diffraction (XRPD). XRPD of CUR
indicated the presence of contrast which represents the
describes its crystalline nature. Majority of peaks for CUR
distributionofformulationinGITatvarioustimepointssuch
occurredat16.97,25.20,16.92,24.19,27.02,25.15,24.24,26.97,
17.02, 27.07, 25.30, 23.03, 24.14, 26.92, 22.98, and 28.59∘ 2𝜃, as from 0 to 2h in upper GIT, from 2 to 4h in the small
intestine,andfrom4to24hinthelowerintestinalregion(up
whereas peak with highest intensity was observed at 25.25
toanus),whereasplainCURshowedmarkedavailabilityin
(Figure8).
lower GIT after 6h which represents the elimination of the
drug.
3.2.8.ScanningElectronMicroscopy(SEM). Thesurfacemor-
phology of the optimized formulation (P5) obtained from 4.Discussion
SEMimagesisshowninFigure9.SEMofoptimizedformu-
lation (P5) with plasticizer appeared with smooth surface, Polymericnanocapsulesofcurcuminwerepreparedbymodi-
whereassignificantporeswithroughsurfacecanbeseenin fiedquasiemulsionsolventdiffusionmethod,usinganenteric
imagescapturedforoptimizedformulation. polymer (HPMCAS-HF), which could obviate the issues
8 TheScientificWorldJournal
100 2 ↑exo D
90 1 A
g release (%) 678000 C (mW/mg) −−201 BBBBBBBBBGGGGGGCCCCC FEE
u 50 S
dr D −3
e 40
ativ 30 −4
ul
m 20 100 200 300 400 500
Cu Temperature (∘C)
10
0 Figure7:DSCscansofA:Aerosil200;B:optimizedformulation,
0 2 4 6 8 10 12
P5;C:physicalmixture(CUR+HPMCAS-HF);D:physicalmixture
Time (h) (CUR+HPMCAS-HF+A-200);E:physicalmixture(CUR+C-90);
P1 P10 F:curcumin;andG:HPMCAS-HF.
P2 P11
P3 P12
P5 P13 14000
P4 P14
12000
P6 P15
P7 P16
P8 P17 nts) 10000
P9 u
o 8000
c
(a) n (
Li 6000 C
D
75
%) 4000 AB
ase ( 60 2000
ele 10 15 20 25 30 35
g r 45 2𝜃
u
dr
ve 30 Figure8:OverlayofXRPDpatternofA.freeCUR;B.HPMCAS-
ati HF;C.physicalmixture;andD.optimizedformulation(P5).
ul
m 15
u
C
0
0 60 120 180 240 300 360 change, of nanocapsule formulation should impact drug’s
release pattern. Release rate is strongly influenced by the
Time (min)
surface morphology of drug-loaded nanocapsules. The for-
P3
mulation was observed to exhibit gastric resistance during
P10
the first 5h and thereafter exhibited a controlled release
P5
pattern up to 12h. The controlled release profile might be
(b) attributed to smooth surface and delivery mechanism of
polymeric nanocapsules probably by surface erosion. It has
Figure6:(a)Plotbetweenmeanpercentcurcuminreleasedforall17
formulationspreparedasper3-factorBox-Behnkendesign(𝑛=3). beenobservedthathigherconcentrationofpolymerextends
(b)DiscriminatorydissolutionstudyonPSNformulations(P3,P5, theduration,duetotheformationofcrystallinegelattheoil-
andP10)inphosphatebufferpH7.2,900mL(𝑛=3). waterinterface.Thisrelationshipagreeswellwiththeresults
ofTrotta,1999,whoproposedtransformationfromoneliquid
crystalline structure to another during the emulsification
relatedtouseofhighconcentrationofsurfactant(30–70%) process [32]. After 5h lag time, enteric polymer dissolved
inconventionalself-emulsifyingformulations. when the pH was changed to 7.2, corresponding to the pH
Formationofafineemulsionisanimportantparameter ofthecolon.Therefore,consideringtheGItransittimefrom
to prevent precipitation and recrystallization of the drug stomachtocolonof4to6h,presentformulationcouldserve
fromformulation.Therateofemulsificationdependsonthe aspotentialcarriersfordeliveryofcurcumintocolon[33].
ratioofpolymerandadsorbentandtheconcentrationofoil Theconcentrationofpolymer(HPMCAS-HF)andadsor-
used in the formulation. The high viscosity of HPMCAS- bent (A-200) was also found to control the rate of drug
HF may contribute to the rate of emulsification. Similarly, release. As shown in Figure6, increasing the amount of
higheroilconcentrationincreasedtheinterfacialfluidityand polymer as well as the adsorbent resulted in a considerable
acceleratestheprogressofemulsification[31]. decreaseindrugrelease.However,insignificantdiscrimina-
Physiochemical properties, namely, surface morphol- tion was observed in selected dissolution test conditions. It
ogy,globulesize,encapsulationefficiency,andpolymorphic may be due to maintenance of nonsink conditions (poor
TheScientificWorldJournal 9
Rough surface
containing pores on the
surface
Smooth surface
without pores on the
surface
(a) (b)
Figure9:Scanningelectronmicroscopy(SEM)imagesofoptimizedformulation(P5)withoutplasticizer(a)andoptimizedformulation(P5)
withplasticizer(b).
120 n 900
o
ati 800
100 entr 700
c
n
co 600
viability (%) 6800 plasma drug 345000000
Cell 40 ative 200
ul 100
m
20 Cu 0
0 2 4 6 8 10 12 14 16 18 20 22 24
0 Time (h)
0 2 4 6 8 10 12 14 16 18 20 22
Concentration (𝜇g/mL) Plain curcumin
Formulation (P5)
Curcumin loaded PSN DMSO
formulation (P5) Plain nanocapsules Figure11:InvivocurcuminreleasefromCURloadedPSNformu-
Curcumin lation(P5)(𝑛=6).
Figure10:CellviabilitystudyofCURloadedPSNformulation(P5)
onHT29celllines(𝑛=3).
discriminatory study showed a significant difference in %
cumulative drug release between selected formulations(P3,
solubilityindissolutionmedia).Toestablishdissolutiontest P5, and P10) with increased volume of dissolution medium
conditions for release characteristics of different formula- (900mL),whereastheslowestpaddlespeed(50rpm)results
tions,discriminatorystudywasincluded.Dataobservedfrom in steeper drug release profile, typically leading to a higher
10 TheScientificWorldJournal
(a) (b)
Figure12:Roentgenographystudyinguineapigs.(a)PlainCUR.(b)CURloadedoptimizedformulation(PSN)(P5)(whereI:blank,II:
after30minadministrationofformulation,III:after1hadministrationofformulation,IV:after2hadministrationofformulation,V:after
4hadministrationofformulation,VI:after6hadministrationofformulation,VII:after12hadministrationofformulation,andVII:after
24hadministrationofformulation).
discriminatingefficiency.Itmaybeduetoincreaseinthesol- Figure 1B). Design suggested formulation (P5) with all
ubilityofdruginenhanceddissolutionmedia(maintenance desirable parameters as optimized formulation. Selection
ofsinkconditions). wasbaseduponthelevelsoffactorsthatyieldedmaximum
DSCthermogramsindicatedachangeinthephysicalstate encapsulationefficiency,optimalglobulesize,andcontrolled
ofthedrugfromthecrystallinetotheamorphousstateinPSN drugrelease.
formulation.AmorphousstateofCURwasfurtherconfirmed Inoptimizedformulation,localizeddeliveryofCURwas
by the presence of a halo pattern in PXRD, a characteristic investigatedbyinvivostudyintheguineapigmodel.Results
of the amorphous form [34]. Appearance of halo pattern obtained from plasma drug concentration time profile rep-
(PXRDspectrum)andabsenceofmeltingendotherm(DSC resent an insignificant amount of drug in plasma (𝐶max
thermogram) show that the drug is dispersed in a polymer = 200ng/mL), suggesting limited systemic uptake of the
matrix at a molecular level and stabilized in its amorphous formulation.
formofpolymer[35]. Cytotoxicity assay on drug loaded nanocapsules was
Literature suggests that systems less than 200𝜇m may conductedonHT29celllinestoestimatethecellviabilitywith
be efficiently engulfed by the macrophages present in the freeCUR,blanknanocapsules,andcontrol(DMSO).Results
colontissue,thusexhibitingeffectivelocalizeddelivery[36]. demonstrated that CUR loaded PSN formulation signifi-
Therefore,apolymerformulationwithglobulesizelessthan cantlyinhibitedthegrowthofthecelllines;however,viability
200𝜇m was chosen for the optimal formulation. Response waslessthanfreeCURandDMSO.Blanknanocapsuleswere
surface analysis as design of experimental approach was treated with cell lines (HT-29) to verify whether a decrease
used to identify the effect of variables on globule size and in % viability was due to polymeric nanocapsule or not. It
percentageencapsulationefficiency.Ithasbeenobservedthat was observed that blank polymeric nanocapsules did not
themeanglobulesizeincreasedwithincreaseinoilconcen- affectthecellularviabilityofHT-29cellsascomparedtoPSN
tration(Capryol90)andtheadsorbent,whereasitdecreased formulations.ThissuggeststhatPSNformulationsreleasethe
withincreasingthepolymerconcentration(HPMCAS-HF). drugincontrolledfashioninthevicinityofproliferatingcell
Standardized effects of the Capryol 90 (oil), HPMCAS-HF linescomparativelyblanknanocapsules.
(polymer),andAerosil200(adsorbent)andtheirinteraction Roentgenographicstudywasperformedonanimalmodel
on mean globule size (𝑌1) are described in Supplementary (guineapig)asthismodelisanalogoustohumanphysiology,
Figure1A. alsohavingsametransittime(GItocolon)4–6hasthatof
Drugencapsulationefficiencywasfoundtobeaffectedby humans.Thestudyhasbeenperformedtoevaluatethekinet-
theconcentrationofemulsifier(HPMCAS-HF)aswellasthe icsofPSNformulationinGI.ImagesindicatethatthePSN
stabilizer (PVA). Formulation with high HPMCAS-HF and formulationsafelyreachedcolon,whichisalsopresentedby
PVA concentration showed poor encapsulation efficiency, formulations, in vitro, with sufficient gastric resistance and
probablyduetoanincreasedformulationviscosity[37].Fur- lag time. The evaluation of the results obtained at different
thermore, encapsulation efficiency, increased with increase stages(timepoints)suggeststhatPSNformulationispresent
in oil concentration, however, decreased with increase in startingfrominitialtimepoint(i.e.,30min)till24thh.Itcan
concentration of polymer and adsorbent (Supplementary be observed that the formulation reaches intestinal region