Caffeic Acid Phenethyl Ester Loaded PLGA Nanoparticles: Effect of Various Process Parameters ...

13 Pages·2015·2.17 MB·English

Hindawi Publishing Corporation
Journal of Nanomaterials
Volume 2015, Article ID 341848, 12 pages
http://dx.doi.org/10.1155/2015/341848
Research Article
Caffeic Acid Phenethyl Ester Loaded PLGA Nanoparticles:
Effect of Various Process Parameters on Reaction Yield,
Encapsulation Efficiency, and Particle Size
SerapDerman
BioengineeringDepartment,ChemicalandMetallurgicalEngineeringFaculty,YildizTechnicalUniversity,
Esenler,34220Istanbul,Turkey
CorrespondenceshouldbeaddressedtoSerapDerman;[email protected]
Received22July2015;Accepted13September2015
AcademicEditor:IlariaArmentano
Copyright©2015SerapDerman.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
CAPEloadedPLGAnanoparticleswerepreparedusingtheoilinwater(o/w)singleemulsionsolventevaporationmethods.Five
differentprocessingparametersincludinginitialCAPEamount,initialPLGAamount,PVAconcentrationinaqueousphase,PVA
volume,andsolventtypewerescreenedsystematicallytoimproveencapsulationofhydrophobicCAPEmolecule,simultaneously
minimizeparticlesize,andraisethereactionyield.Obtainedresultsshowedthattheencapsulationefficiencyofthenanoparticles
significantly increased with the increase of the initial CAPE amount (𝑝 < 0.05) and particle size (𝑝 < 0.05). Furthermore,
theparticlesizeissignificantlyinfluencedbyinitialpolymeramount(𝑝 < 0.05)andsurfactantconcentration(𝑝 < 0.05).By
the optimization of process parameters, the nanoparticles produced 70±6% reaction yield, 89±3% encapsulation efficiency,
−34.4±2.5mVzetapotential,and163±2nmparticlesizewithlowpolydispersityindex0.119±0.002.Theparticlesizeandsurface
morphologyofoptimizednanoparticleswerestudiedandanalysesshowedthatthenanoparticleshaveuniformsizedistribution,
smoothsurface,andsphericalshape.LyophilizednanoparticleswithdifferentCAPEandPLGAconcentrationinformulationwere
examinedforinvitroreleaseatphysiologicalpH.Interestingly,theoptimizednanoparticlesshowedahigh(83.08%)andsustained
CAPErelease(lastingfor16days)comparedtononoptimizednanoparticle.
1.Introduction humanB-lymphoma[15],prostatecancerPC-3[16],myeloid
leukemia U-937 [17], and colon cancer HT-29, 26-L5 [18]
Caffeic acid phenethyl ester (CAPE), a flavonoid-like com- cells.
pound (Figure1), is one of the most active components of On the other hand, CAPE can be hydrolyzed in plasma
honeybeepropolis[1].Numerousbiologicalandpharmaco- enzymesbyanesterase[19]andthishydrolysisleadstorapid
logicaleffectsofCAPEhavebeenreported,suchasantiviral clearanceandshorthalf-lifeandresultsinpoorbioavailability
[2], antioxidant [3], antiallergic [4], anticarcinogenic [5], and poor biological performances [20, 21]. Wang et al.
anti-inflammatory [6], antimicrobial [7], immunomodula- reportedthatthehalf-lifeof5𝜇g/mLCAPEat37∘Cwas0.35
tory[8],andanticancer[9,10]activities.CAPEhasnopoten- hours [22]. Additionally, poorly water soluble character of
tially harmful effects on normal cells [11] but also has been CAPElimitsitsinvivoandinvitroefficacy[1].
showntoinhibitthegrowthofdifferenttypesoftransformed Intherecentyears,manystrategieshavebeendeveloped
cells[12].Duetotheinhibitingpotentialofthetranscription toimprovebioavailabilityandwatersolubilityofhydrophobic
factor nuclear factor-kappa B (NF-𝜅B), CAPE has strong drugs including drug carrier systems such as antibodies,
antitumoractivityincancercells[9,10].Miscellaneousstudy liposomes, or nanoparticles [23]. Several disease related
hasshowedtheNF-𝜅Binhibitingpotentialindifferentcancer drugs/bioactive molecules are successfully encapsulated to
cell lines, including breast cancer MCF-7 [13], malignant improve solubility, bioavailability, and bioactivity [23, 24].
peripheralnervesheathtumors,astrocytomaGRT-MG[14], Among the biocompatible and biodegradable polymeric
2 JournalofNanomaterials
further purification. Ultra-pure water was obtained from
O MilliporeMilliQGradientsystem.
The in vitro release measurements were carried out at
HO ∘
O 37 CinphosphatebuffersolutionatpH=7.4andpH5.2.
2.2. Nanoparticle Preparation Method. The CAPE loaded
HO
PLGAnanoparticleswerepreparedbymodifiedoilinwater
Figure1:Chemicalstructureofcaffeicacidphenethylester(CAPE). (o/w) single emulsion solvent evaporation according to
method described by Song et al. [28] with minor modifi-
cations. The organic phases consisted of PLGA and CAPE
which were dissolved into DCM and ethanol, respectively.
nanoparticlesPLGA(poly-d,l-lactide-co-glycolide)isoneof
Briefly, known amount of CAPE and PLGA (Table1) was
the most commonly and successfully used biodegradable
mixedandstirredtoensurethatallmaterialsweredissolved.
nanosystemsforencapsulationofvarioustherapeuticagents
Thisorganicsolutionwasemulsifiedwiththe4mLaqueous
becauseofitshighbiodegradability,biocompatibility,andlow
solutionofPVA(3%w/v)bysonication(outputpower70W,
toxicity[23],andalsofinaldegradationproducts(lacticacid
power of 80%, and 2 minutes) using a microtip probe son-
andglycolicacid)arecompletelysafebecausetheyarefinally
icator (Bandelin Sonopuls, Germany) over an ice bath. The
eliminatedascarbondioxideandwater[25].
o/w single emulsions were stirred overnight on a magnetic
PLGA based polymeric nanoparticles are studied to
stirreratroomtemperatureforevaporationoforganicphase.
enhancethebiologicalactivity,watersolubility,andbioavail-
The resulting particles were collected by centrifugation at
abilityofvariousdrugandnaturalproductsbecauseoftheir
9.000xrpm for 40min (Hettich-Universal 32 R), washed
small particle size and large surface area [10]. For instance,
three times with ultra-pure water to remove excess PVA,
Singh et al. showed that tea polyphenol loaded PLGA
and then lyophilized. The free nanoparticles were prepared
nanoparticlesprovide30-foldhigherpreventionontheDNA
withsimilarmethodwithoutusingCAPEandalllyophilized
damage than free tee polyphenols [26]. Chaowanachan et nanoparticleswerestoredat−80∘Cuntilused.
al. synthesized efavirenz loaded PLGA nanoparticles and
In this study, the effect of various process parameters
researchershowedthattheHIVinhibitoryeffectofnanopar-
on reaction yield (RY), encapsulation efficiency (EE), the
ticleshoweduptoa50-foldreductioninthe50%inhibitory meanparticlesize(𝑍-Ave),polydispersityindex(PDI),and
concentration(IC50)comparedtofreedrug[27]. zeta potential were investigated, including the initial CAPE
Despite the above positive features of nanoparticle sys-
amount,initialPLGAamount,PVAconcentrationinaqueous
tem, there is only one study in the literature concern-
phase,PVAvolume(aqueous-to-organicphasevolumeratio)
ing encapsulation of CAPE into polymeric nanoparticle.
andsolventtype(acetone/DCM+ethanol)volumeratio.
Hyo-YoungandcolleaguesencapsulatedCAPEintopoly(𝜀-
caprolactone)/poly(ethylene glycol) block copolymer and
2.3.CharacterizationofPolymericNanoparticles
they showed that CAPE incorporated nanoparticles have
superiorantimetastaticefficiencyagainstpulmonarymetas-
2.3.1.ReactionYield(RY). Thenanoparticlepreparationtech-
tasismodel.However,tothebestofourknowledge,thereare
nique with a high reaction yield would reduce chemicals
nostudiesintheliteratureaboutencapsulationofCAPEto
loss and product cost. The reaction yield was calculated
PLGAbasednanoparticularsystem.
gravimetricallyusingtheformulagivenbelow[29]:
Thusthefirstpurposeofthepresentstudywastoexamine
the initial CAPE amount, initial PLGA amount, stabilizer RY (%)= Amount of nanoparticle produced ×100. (1)
concentrationinaqueousphase,oil/aqueousphaseratio,and Amount of initial polymer+CAPE
solventtypeforoptimizingthenanoparticleformulationin
respect to reaction yield, encapsulation efficiency, particle 2.3.2.EncapsulationEfficiency(EE). TheencapsulatedCAPE
size,polydispersityindex(PDI),andzetapotential.Tosyn- intheNPswasdetectedintriplicatedindirectquantification
thesizethePLGAnanoparticlesinoptimumformulationfor methods by using UV-Vis Spectroscopy at 323nm. EE was
the first time by oil in water (o/w) single emulsion solvent determined by analyzing the supernatant obtained from
evaporation method and detailed characterize improved the ultracentrifugation of each nanoparticle formulation.
drugencapsulatednanoparticlesweretheotheraimsofthe CAPE concentration in the supernatant was determined by
existingstudy. comparing the concentration to a previously constructed
standard calibration curve. The concentration of loading
CAPEinnanoparticleswascalculatedfromthetotalamount
2.MaterialsandMethods
ofCAPEandtheamountofCAPEthatwasnotencapsulated.
The CAPE encapsulation efficiency (EE) was calculated
2.1. Materials. PLGA (lactide:glycolide = 50:50; inherent
viscosity 0.45–0.60dL/g, Mw ∼ 38–54kDa P50/50), poly- usingtheformulasgivenbelow:
vinyl alcohol, CAPE, acetone, and ethanol were purchased Amount of CAPE encapsulated in NPs
EE (%)=
from Sigma Aldrich (St. Louis, USA); dichloromethane Initial CAPE added (2)
(DCM)wasobtainedfromRideldeHaen.Allthechemicals
×100.
and solvents were of analytical grade and used without
JournalofNanomaterials 3
Table1:VariousprocessparametersofCAPEloadedNPs.
CAPEandPLGAamount PVA Solventtype
Nanoparticle
Aseton DCM Et-OH
number CAPE(mg) PLGA(mg) (%) mL
(mL) (mL) (mL)
NP1 10 100 3 4 — 1.5 0.5
NP2 20 100 3 4 — 1.5 0.5
NP3 30 100 3 4 — 1.5 0.5
NP4 40 100 3 4 — 1.5 0.5
NP5 50 100 3 4 — 1.5 0.5
NP6 20 100 0.1 4 — 1.5 0.5
NP7 20 100 0.5 4 — 1.5 0.5
NP8 20 100 1 4 — 1.5 0.5
NP9 20 100 2 4 — 1.5 0.5
NP10 20 100 3 6 — 1.5 0.5
NP11 20 100 3 8 — 1.5 0.5
NP12 20 100 3 10 — 1.5 0.5
NP13 20 100 3 20 — 1.5 0.5
NP14 20 100 3 4 0.5 1 0.5
NP15 20 100 3 4 0.75 0.75 0.5
NP16 20 100 3 4 1 0.5 0.5
NP17 20 100 3 4 1.5 — 0.5
NP18 20 200 3 4 — 1.5 0.5
NP19 20 300 3 4 — 1.5 0.5
NP20 20 400 3 4 — 1.5 0.5
OptimizedNP 50 100 2 4 — 1.5 0.5
A standard calibration curve of the absorbance as a previouslydescribedbyHalayqaandDoman´ska[31].Adrop
functionofCAPEconcentrationwasstudiedat323nm.All of nanoparticles suspensions was placed on a black carbon
experimentswereperformedintriplicate. tapewithadoubleside.Afterdryingthesampleswerecoated
withgoldlayerundervacuumandanalyzedwithSEM(Zeiss
2.3.3. Mean Particle Size (Z-Ave), Zeta Potential, and Poly- EVOLS10,Germany)at5kV.
dispersity Index (PDI). Dynamic light scattering technique
wasusedfordeterminingofthe𝑍-average(𝑍-Ave)andPDI 2.3.5.FourierTransformsInfrared(FT-IR)Spectrometry. IR-
valuesofnanoparticlesusingaZetasizer(ZetasizerNanoZS, Prestige21FTIRspectrophotometer(Shimadzu,Japan)was
Malvern,UK)instrumentequippedwith4.0mVHe-Nelaser usedforchemicalanalysesofthefunctionalgroupspresentin
(633nm) [30]. Measurements were carried out in triplicate, nanoparticles[32].MeasurementswerecarriedoutforPLGA,
at 25 ± 0.1∘C with using 0.8872cP of viscosity and 1.330 of CAPE, and NPs in universal attenuation total reflectance
refractive index for the solutions. The number of runs and (ATR)mode.TheFT-IRspectrawereobtainedwith16scans
−1
rundurationswerechoseautomatically. persamplerangingfrom4000to750cm andaresolution
Zetapotential(𝜁)valueofnanoparticleswasdetermined of4cm−1.
by electrophoreticlight scattering (ELS) technique and car-
ried out in the folded capillary cell at 25 ± 0.1∘C [30]. 2.3.6. In Vitro CAPE Release. The in vitro CAPE release
The measurements were performed in triplicate, with the fromnanoparticleswasstudiedusingamodifieddissolution
followingparameters:viscosity,0.8872cP;dielectricconstant, method[33]inphosphatebuffersolutionsatpH7.4.ThepH
79;𝑓(𝑘𝑎),1.50(Smoluchowski).Themeasurementdurations valuewasselectedtosimulatethephysiologicalpH(7.4)[34].
andvoltageselectionsweresettoautomaticmode. In a typical release experiment, 5mg of the CAPE loaded
All samples were prepared by diluting with phosphate PLGA nanoparticles was suspended in 10mL of PBS with
buffer saline (PBS), filtered with a 0.20𝜇m RC-membrane 0.01%sodiumazideandthesuspensionwasincubatedat37∘C
filter(Sartorius)beforemeasurement. in a shaking incubator (60rpm) at pH 7.4. At selected time
intervals(1hour,2hours,3hoursand1,2,3,4,7,11,13,and
2.3.4.ScanningElectronMicroscopy(SEM). Thesurfacemor- 16 days), the release medium was centrifuged at 9000rpm,
phology and shape of the CAPE loaded nanoparticles were 20min, the supernatant was collected, and the pellet was
observed using scanning electron microscopy (SEM) as resuspendedwith10mLfreshPBS.TheCAPEconcentration
4 JournalofNanomaterials
Table2:Effectofprocessparametersonthereactionyield,encapsulationefficiency,meanparticlesize,PDI,andzetapotential.
Encapsulation
Particlenumber Reactionyield(%) Size(nm) PDI Zetapotential(mV)
efficiency(%)
NP1 39±7 60±3 218±9 0.077±0.005 −22.4±0.7
NP2 40±6 76±4 208±8 0.065±0.006 −18.7±0.3
NP3 48±7 86±5 214±8 0.101±0.008 −18.1±1.0
NP4 49±6 89±2 207±10 0.055±0.005 −19.2±1.3
NP5 53±5 92±5 214±9 0.079±0.003 −17.7±1.6
NP6 74±7 91±5 469±15 0.461±0.038 −16.3±1.8
NP7 80±8 91±3 324±10 0.113±0.007 −20.2±3.1
NP8 64±6 86±3 246±12 0.119±0.005 −19.6±0.7
NP9 63±9 83±5 243±13 0.138±0.010 −15.8±1.9
NP10 68±8 84±7 260±7 0.139±0.006 −14.0±2.9
NP11 70±7 85±6 284±9 0.177±0.005 −15.6±1.8
NP12 68±6 84±3 271±7 0.150±0.009 −14.2±1.8
NP13 64±8 80±5 248±8 0.164±0.008 −12.7±1.3
NP14 15±4 65±4 180±7 0.087±0.005 −18.7±2.0
NP15 7±4 66±2 158±9 0.057±0.002 −16.6±1.8
NP16 12±6 64±4 158±10 0.103±0.008 −16.2±2.0
NP17 15±7 83±4 187±6 0.143±0.014 −16.3±2.6
NP18 83±6 84±6 215±5 0.128±0.018 −17.5±1.6
NP19 86±7 81±4 404±9 0.403±0.042 −21.4±2.5
NP20 92±5 85±5 437±9 0.499±0.066 −19.4±2.0
OptimizedNP 70±6 89±3 163±2 0.119±0.002 −34.4±2.5
inthesupernatantwasdeterminedwithUV-VisSpectroscopy Particularly, initial PLGA and CAPE amount had strongly
at 323nm by comparing the concentration to a previously and significantly positive effects on reaction yield while
constructedstandardcalibrationcurve. acetone/DCM+ethanolconcentrationreducedthereaction
yield.
2.4. Statistical Analysis. All experiments were repeated at Figure2(a)showsthattheyieldrosefrom39±7%to53±
least three times. Data were expressed as mean ± standard 5%withtheincreaseintheinitialCAPEamountfrom10mg
deviation.SPSS15.0softwarewasusedforstatisticalanalyses to50mg.
[35].NonparametricanalysiswithMann-Whitney𝑈testwas Additionally, it is clearly seen in Figure2(b) that initial
carriedoutforcomparisonoftheresults.𝑝valueslessthan PLGAamounthadagreatereffectonthereactionyieldwhen
0.05(𝑝<0.05)wereconsideredsignificant. theothersystemparameterssetataconstantvalue.Asshown
inTable2thereactionyieldincreasedfrom40±6%to92±5
with the increase in initial PLGA amount from 100mg to
3.Results
400mg, indicating that the higher the PLGA amount used
In this study, the o/w single emulsion solvent evaporation in the formulation, the greater the yield. Our results are
method was used for fabrication of CAPE loaded PLGA in agreement with the recent observations of Hussein et al.
nanoparticles.Theeffectoffiveprocessparametersonreac- [29] who found that the reaction yield increased twofold
tionyield(RY),encapsulationefficiency(EE),meanparticle (from40%to80%)withariseinPLGAconcentrationsfrom
size (𝑍-Ave), polydispersity index (PDI), and zeta potential 2.5mg/mLto10mg/mL.TheincreaseinthePLGAamount
were investigated. The basic characteristics of the CAPE givesrisetotheviscosityofsolutions,causingtheformation
loadedPLGAnanoparticleswerepresentedinTable2. oflargeranddenserparticles,whichwillafterwardincrease
thereactionyield[29,36,37].
3.1. Effect of Process Parameters on Reaction Yield. High TheeffectofPVAvolumeonthereactionyieldwasshown
efficiency nanoparticle preparation methods considerably in Figure2(c). The reaction yield first increased up to 68 ±
prevent material loss, improve particle production, and 8 (for 6mL of PVA), then reached a plateau, and slowly
decrease manufacturing cost [29]. In this study, all the decreasedwithanincreaseinPVAvolume.
formulatedbatchesgavewideyieldsthatrangedbetween7± Figure2(d) shows the effect of PVA concentration in
4%and92±5%.Obtainedresultsindicatedthatthereaction the aqueous phase on the reaction yield. As depicted from
yield responses strongly depend on process parameters. the figure, at low PVA concentration (<1w/v) the increase
JournalofNanomaterials 5
100 100 100 100
80 80 %) 80 80 %)
%) ncy ( %) ncy (
Reaction yield ( 4600 4600 psulation efficie Reaction yield ( 4600 4600 psulation efficie
20 20 nca 20 20 nca
E E
0 0 0 0
10 20 30 40 50 0 100 200 300 400
Initial CAPE amount (mg) Initial PLGA amount (mg)
(a) (b)
100 100 100 100
80 80 %) 80 80 %)
Reaction yield (%) 462000 246000 ncapsulation efficiency ( Reaction yield (%) 462000 246000 ncapsulation efficiency (
E E
0 0 0 0
4 6 8 10 20 0.1 0.5 1.0 2.0 3.0
PVA volume (mL) PVA concentration w/v (%)
(c) (d)
100 100
80 80 %)
%) ncy (
eld ( 60 60 fficie
yi e
n n
Reactio 40 40 psulatio
20 20 ca
n
E
0 0
0.0 0.333 0.6 1.0 3.0
Acetone/DCM + Et-OH
(e)
Figure2:Effectofvariousprocessparametersonreactionyield(line)andencapsulationefficiency(column),includingtheinitialCAPE
amount(a),initialPLGAamount(b),PVAvolume(c),PVAconcentrationinaqueousphase(d),andsolventtype(e)(𝑛=3).
in the yield was confined to 80 ± 8% and then system- yieldof7±4%atanacetone:DCM:ethanolratioof3:3:2
atically decreased with increase in the PVA concentration (for2mL);thenaweakincrease(15±7)wasobservedinthe
(𝑝<0.05). absenceofDCMandtheratioofacetone:ethanolof3:1.
Finally,theeffectofsolventtypeonthereactionyieldwas Hence, it can be thought that the reaction yield had a
showninFigure2(e).Itcanbeclearlyseenthatthereaction positive relationship with the initial CAPE amount, initial
yieldissignificantlyaffectedfromthesolventtype(𝑝<0.05). PLGAamount,andPVAvolumeandanegativerelationship
Theincreaseintheacetoneratioinsolventmixtureledtoa with PVA concentration and the presence of acetone in
decreasethereactionyield.Thereactionachievedaminimum solventmixture.
6 JournalofNanomaterials
3.2. Effect of Process Parameters on Encapsulation Efficiency. PVAvolume.Actually,encapsulationefficiencyisindirectly
TheresultsoftheeffectofinitialCAPEandPLGAamount, affectedbyaqueousphasevolumeduetotheinfluenceofPVA
PVAvolumeandconcentration,andsolventtypeonencapsu- volumeonparticlesize,whichdirectlyaffectsencapsulation
lationefficiencyarelistedinTable2andillustratedinFigures efficiency.
2(a)∼2(e)respectively. Figure2(d) shows the effect of PVA concentration on
WhenaconstantinitialamountofPLGA(100mg),PVA encapsulationefficiencyofCAPE.PVAformulatednanopar-
volume (4mL), PVA concentration (3%w/v), and solvent ticlesreachedmaximumencapsulationatlow(0,1%w/v)sta-
type (only DCM and ethanol) was maintained, the amount bilizerconcentration.Encapsulationefficiencylevelreached
ofCAPEvariedfrom10to50mg(Figure2(a)).Theincrease as high as 91 ± 5% and was seen at lowest PVA con-
in the initial CAPE amount in organic phase resulted in a centration. When the concentration of stabilizer increased,
significant increase in entrapment efficiency of CAPE (𝑝 < a linear and small reduction in overall CAPE encapsula-
0.05), which was in accordance with the results reported tion was seen (Table2). Obtained results are in agreement
[28,31].InthefirstplacetheEEremarkablyincreased(from with literature [28, 34, 43, 44]. Cooper and Harirforoosh
60 ± 3% to 86 ± 5%) and then reached a plateau when the synthesized diclofenac encapsulated PLGA nanoparticles
amountofCAPEwasbetween30and50mg. with polyvinylalcohol and didodecyldimethylammonium
Itwasreportedthatthedrugpartitioncoefficientininter- bromide(DMAB)asastabilizer.Thesegroupobservedthat
nalandexternalphasessignificantlyaffectstheencapsulation encapsulation of diclofenac decreases with increase in the
efficiency of particles using an o/w method [38, 39]. Boury concentrationofeachofthetwostabilizers[44].
et al. showed that the drug polymer interaction contributes Theeffectofthesolventtype(acetonetoDCM+ethanol
to increasing encapsulation efficiency [40]. Panyam et al. volume ratio is in the range of 0 to 3.0) on encapsulation
discussed the importance of drug miscibility in polymer efficiency of CAPE was shown in Figure2(e). It was seen
for hydrophobic flutamide and reported that higher drug in Figure2(e) and Table2 that encapsulation efficiency is
polymermiscibilityleadstoahigherdrugencapsulation[41]. significantly decreased by addition of acetone to organic
Similar results established by Budhian et al. indicated that solventmixtureandreachedaplateauwiththeriseofacetone
the increase in the polymer drug interaction also increases volumeratio(from0.333to1.0)andthenincreaseforacetone
thedrugcontentofparticlesasaresultofhighencapsulation to DCM + ethanol volume ratio was equal to 3.0. This
efficiency[24]. occurredpossiblybecauseofthechangeofAcetone/DCM+
It was observed in our results that with the increase ethanol volume ratio affecting the dispersion of CAPE in
in initial CAPE amount, the concentration of CAPE in the organic phase. Encapsulation efficiency reached maximum
organic phase increased and more drug molecules could foracetone/DCM+ethanolvolumeratiobeing3.0;however
interact with the polymer molecules. This results in the in this condition the significant reduction in reaction yield
increaseinencapsulationefficiencyofCAPE. (15±7%)wasobserved.
Figure2(b) shows the effect of initial PLGA amount on
the encapsulation efficiency of CAPE. It is clearly seen that 3.3.EffectofProcessParametersonParticleSizeandPolydis-
theencapsulationefficiencyofCAPEincreasedsignificantly persity Index. In this study the effects of initial CAPE and
(𝑝 < 0.05) with increase in the PLGA concentration in PLGA amount, PVA concentration, PVA volume (aqueous-
organicphase.TheriseintheinitialPLGAamountleadsto to-organic phase volume ratio), and solvent type on mean
an increase of organic phase viscosity, which causes more particlesizeofnanoparticleswereinvestigated.Theparticle
diffusionalresistancetodrugmoleculesfromorganicphase sizevaluesforallbatchesshowawidevariationinresponse
totheaqueousphase[42].Additionally,increaseintheinitial thatrangedfromaminimumof158±9nmtoamaximum
PLGA amount resulted in a rise in the particle size. As of 469 ± 15nm. Obtained results openly indicate that the
shownintheliterature,largerparticlesprovidehigherdrug mean particle size is strongly related on selected process
encapsulationefficiency[24,28,31,43].Moreover,duetothe parameters.
increaseofparticlesizethelengthofdiffusionalpathwayof TheeffectofinitialCAPEamountonnanoparticlessize
drugsfromorganicphasetoaqueousphaseincreasesandas was shown in Figure3(a). Initial CAPE amount in organic
aresultofthisincreasethedruglossalsodecreases.Thereby, phase had no significant effect on the mean particle size
reducingthedruglossthroughdiffusionprovidesincreased whichwassimilartomanyearlierstudieswithhydrophobic
encapsulationefficiency[42].Obtainedresultsinaccordance molecules[24,28].
withtheresultsreportedbyHalayqaandDoman´skaandthe Figure3(b) shows that the mean particle size of CAPE
authorsshowthatincreaseinthepolymeramountfrom0.8% loaded PLGA nanoparticlesincreasedsignificantlywith the
to1.6%increasestheencapsulationefficiency46.6%to71.6% increase of initial PLGA concentration. It can be observed
for perphenazine and 44.8% to 61.6% for chlorpromazine that increase of the PLGA amount from 100mg to 400mg
hydrochloride[31]. increases the mean particle size from 208 ± 8nm to 437 ±
Theeffectoftheaqueousphasevolumeontheencapsu- 9nm.Thesameresultswerealsopreviouslyreportedbyother
lationefficiencyofCAPEisshowninFigure2(c).Theencap- researcher[28,31,45–48].Thiscouldexplainthatincreaseof
sulation efficiency of CAPE firstly increased with increase thePLGAamountinorganicphaseleadstoriseofsolution
of PVA volume and then reached a plateau that ranged viscosity and decrease of net shear stress. As a result of
from 76 ± 4% to 85 ± 6%. It was clearly seen from results reducing shear stress larger particles are formed. Addition-
that encapsulation efficiency is not significantly affected by ally,increaseintheviscositycouldpreventquickdispersion
JournalofNanomaterials 7
250 500
0.5
0.5
200 400
0.4
m) m) 0.4
n150 n 300
article size (100 00..23 PDI article size ( 200 00..23 PDI
P P
50 0.1 100 0.1
0 0.0 0 0.0
10 20 30 40 50 0 100 200 300 400
Initial CAPE amount (mg) Initial PLGA amount (mg)
(a) (b)
350 0.5 500 0.5
300
0.4 400 0.4
250
m) m)
n 0.3 n 300 0.3
e ( 200 e (
cle siz 150 0.2 PDI cle siz 200 0.2 PDI
arti 100 arti
P P
0.1 100 0.1
50
0 0.0 0 0.0
4 6 8 10 20 0.1 0.5 1.0 2.0 3.0
PVA volume (mL) PVA concentration w/v (%)
(c) (d)
250 0.5
200 0.4
m)
n 150 0.3
e (
cle siz 100 0.2 PDI
arti
P
50 0.1
0 0.0
0.0 0.333 0.6 1.0 3.0
Acetone/DCM + Et-OH
(e)
Figure3:Effectofvariousprocessparametersonparticlesize(column)andPDI(line),includingtheinitialCAPEamount(a),initialPLGA
amount(b),PVAvolume(c),PVAconcentrationinaqueousphase(d),andsolventtype(e)(𝑛=3).
of polymer solution into the aqueous PVA phase, resulting the mean particle size [28]. Furthermore, the increased the
in bigger droplets which formed larger nanoparticles after totalsystemvolumewouldreducethenetshearstressbecause
evaporationofoilphase. of a constant energy source causing to formation of larger
Figure3(c)showsthatthemeanparticlesizedependence particles[28,47].
toPVAvolume(aqueous-to-organicphasevolumeratio)in In this study PVA was applied as emulsifier in the
aqueousphaseforCAPEloadednanoparticles.Itcanbeseen aqueousphase(w)tosimplifythepreparationofo/wsingle
in the figure that the mean particle size was first increased emulsion.Figure3(d)showstheeffectofPVAconcentration
andthendecreasedwithincreaseinthePVAvolume.Increase inaqueousphaseonthemeanparticlessize.DifferentPVA
in PVA volume causes an increase in the PVA amount, concentrations are selected while keeping the other system
resulting in interfacial tension reduction thence decreasing parameters constant and PVA was studied between 0.1%
8 JournalofNanomaterials
and 3.0%(w/v) concentrations. It can be observed that the Also, PVA concentration generally exhibited a negative
mean particle size first decreased significantly (𝑝 < 0.05) influence on PDI (Figure3(d)). It was observed in our
withincreaseofPVAconcentrationupto1%(w/v)andthen results that the polydispersity index decreased when PVA
graduallydecreasedfrom246±12to208±8nm(3%w/v). concentrationincreasesfrom0.1%to3%.
ThiscanexplainthatPVAplayremarkableroleinreducing Figure3(e)showstheeffectofsolventtypeonPDIvalue
theinterfacialtensionandinthiswaydispersetheemulsion ofCAPEloadednanoparticles.Itwasseeninthefigurethat
nanodropletsandpreventthemfromagglomeration[31,49]. PDI is affected from additionof acetone in solvent mixture
ItwasreportedthatincreaseinthePVAconcentrationmay especially in the lack of DCM. In the absence of acetone
lead to the decrease of particle size due to tight surface (DCMtoethanolratioof3:1)thePDIvalueofnanoparticles
that was formed from PVA macromolecular chains in high was obtained 0.065 ± 0.006, addition of acetone increased
surfactant concentrations [31, 43, 45, 50, 51]. Otherwise the PDI up to 0.103 ± 0.008, and this value reached maximum
viscosityofthesystemincreaseswiththeincreaseofthePVA (0.143±0.014)foracetonetoDCM+ethanolratioequalto
concentration;thisleadstodecreaseofnetsharestresswhich 3.0.
resultsinformationoflargermolecules[24].Conversely,in
our results the decreases in the mean particles size reached 3.4. Effect of Process Parameters on Zeta Potential. Zeta
a plateau after the concentration of PVA is between 1 and potential is another important physicochemical parameter
3%w/v. The same results observed Budhian and colleagues in nanoparticles that influences stability of nanoparticle
studywhichshowthatthesizeofHaloperidolloadedPLGA suspension. Extremely negative or positive zeta potential
nanoparticles first decreases (up to 1%w/v), then reached a causes high repulsive forces and prevents agglomeration of
plateau(upto5%w/v),andgraduallyincreases(highercon- nanoparticles[55].Undertheseconditionslong-termstabil-
centrationthan5%w/v)byincreasingofPVAconcentration ityofthenanoparticlescanbeanticipated[55].Itcanbeseen
forsonicationmethods[24].Accordingtoourresultsitcanbe in Table2 that the zeta potential values of all nanoparticles
concludethatincreaseinthePVAconcentrationmorethan werenegativeandrangedbetween−12.7±1.3mVand−22.4±
3%(w/v)mayresultinanincreaseofthemeanparticlesize 0.7mV. This expected negativity was based on presence of
ofnanoparticles. ionized carboxyl groups [51] and PVA [55] on the surface
The solvent type also has a remarkable effect on the ofthenanoparticles.Also,highernegativezetapotentialcan
mean particle size. Figure3(e) shows the significant (𝑝 < enhancethedispersionofthenanoparticlesinphysiological
0.05)reductionofmeanparticlesizewithacetone-to-DCM+ systemsandwater[54].
ethanol ratio. Addition of water-miscible organic solvent
leadstosignificantdecreaseofinterfacialtensionbecauseof 3.5. Optimization of CAPE Loaded PLGA Nanoparticles.
rapiddispersionofacetoneintotheexternalaqueousphase, Obtained results show that CAPE was successfully encap-
therebydecreasingtheparticlesizewhichwasconsistentwith sulated into the PLGA nanoparticles. On the other hand,
literatures[28,43,52]. according to our criteria for lower particle size and higher
ThePDIisanimportantpropertyofparticlesandrefersto entrapment efficiency, we prepared CAPE loaded PLGA
broadnessofameanparticlesizedistributionfordispersion nanoparticles as follows: 50mg of CAPE and 100mg of
ofnanoparticle[53].Therefore,effectofprocessparameters PLGA were dissolved into 0.5mL of ethanol and 1.5mL of
on PDI values of nanoparticles was examined in this study. DCM,respectively.Themixedorganicphasewasemulsified
ThepositiveandnegativeeffectofprocessparametersonPDI with 6mL of aqueous PVA solution (2%w/v) by sonication
ofnanoparticlescanbeseeninTable2andFigures3(a)∼3(e) (output power 70W, power of 80%, and 2 minutes) using a
respectively. microtipprobesonicatorinicebath.Theemulsionwasstirred
Generally, PDI < 0.3 is considered a requested value overnight for evaporation of organic phase. The resulting
foranacceptablenarrowrangeofnanoparticlesize[54].In particleswerecollectedandlyophilized.
the first place particles prepared by increasing amounts of
CAPE (10mg to 50mg) were narrowly distributed and had 3.6. Detailed Characterization of Optimized Nanoparticles.
the lowest PDI values (varied between 0.055 ± 0.005 and Forthedetailedcharacterizationofoptimizednanoparticles
0.101±0.008)(Figure3(a)). firstly, we determined reaction yield, encapsulation effi-
ObtainedresultsshowedthatthePLGAconcentrationis ciency,andloadingcapacity.Lyophilizednanoparticleswere
themostimportantfactoraffectingthePDIofnanoparticles. weighed and yield of the process was calculated to be 70 ±
ThePDIvaluesvariedbetween0.065±0.006and0.499±0.066 6%.Thecorrespondingencapsulationefficiencyandloading
for100mgto400mgPLGAamount,respectively.Fromthe capacityofoptimizednanoparticleswerecalculated89±3and
resultsitcanbeseenthatalmostmonodispersenanoparticles 42±4,respectively.
were produced for 100mg polymer amount and the mean
particlesizedistributionwasbroadenedwithincreaseofthe 3.6.1. DLS and ELS Analysis. Dynamic and electrophoretic
initialPLGAamount(Figure3(b)). lightscatteringtechniqueswereusedforparticlesizeandzeta
FromFigure3(c)andTable2,itcanbeclearlyseenthat potentialmeasurementofnanoparticles.Figure4depictsthe
all of PVA volume was sufficient for reducing PDI below formation of nearly monodisperse CAPE nanoparticle with
0.3.InthisgroupPDIvalueofparticlesfirstincreasedfrom themeanparticlesizebeing163±2nm(PDI=0.119±0.002).
0.065 ± 0.006 to 0.177 ± 0.005 (for 8mL of PVA) and then ThezetapotentialofCAPEloadednanoparticleswasequalto
slowlydecreasedbyincreaseintheaqueousphasevolume. −34.4±2.5(Figure4).
JournalofNanomaterials 9
×104
30
18 Size distribution by intensity
16
14
20
12
%) nts
Intensity ( 1680 Total cou 10
4
2
0 0
1 10 100 1000 10000 −60 −50 −40 −30 −20 −10 0
Particle size (nm) Apperent zeta potential (mV)
(a) (b)
Figure4:Particlesize(a)andzetapotential(b)distributionofoptimizednanoparticle.
(a)
2993 2989
(b)
1751
%) 1165 1087
e ( 3320
nc 3471 2993 2989
a
mitt (c) 175116001273
ns 1165 1087
a
Tr 3050-2840
1𝜇m 3471 3320
1681
Figure 5: Scanning electron microscopy image of optimized
nanoparticles. 1600 1273 1172
4000 3500 3000 2500 2000 1500 1000 500
−1
Wavenumbers (cm )
3.6.2. SEM Analysis. The surface morphology of the opti-
Figure6:FTIRspectrumofPLGA(a),optimizednanoparticle(b),
mized nanoparticle was determined by SEM. As shown in
andfreeCAPE(c).
Figure5, smooth and spherical shape nanoparticles with
uniform distribution were obtained. SEM results are also
in agreement with DLS results that the particles optimized
nanoparticles have a uniform size distribution and low C–O stretching, respectively. However, in the FTIR spectra
polydispersityindex. of CAPE loaded nanoparticles, the major peaks of CAPE
−1 −1 −1
at 3471cm , 3320cm , and 1600cm were significantly
3.6.3.FTIRAnalysis. Figure6showsFTIRspectraofPLGA decreasedandthepresenceofthesecharacteristicpeaksisa
(a), optimized nanoparticle (b), and pure CAPE (c). In the confirmationofCAPEencapsulationonPLGAnanoparticles
FTIR spectrum of PLGA and nanoparticles the bands at successfully.
−1 −1
2993cm and2989cm wereC–HstretchofCH2andC–H
stretchof–C–H–,respectively.
3.6.4.ReleaseStudy. Figure7illustratesinvitroreleasepro-
−1
A band at 1751cm was assigned to the stretching
filesofCAPEfromnanoparticlespreparedatdifferentprocess
vibrationofC=Oofesterbond(strongandnarrow)and1165–
parameters.Thenanoparticlesshowedatypicalthree-phase
−1
1087cm was attributed to C–O stretching, which belongs releasepatterninpH7.4.
to the characteristic peaks of PLGA molecule [56]. It can Forallthenanoparticlespreparedwithdifferentprocess
be seen from the FT-IR spectrum of free CAPE that the parameters, an initial burst release, caused by diffusion of
−1 −1
bandsobtainedat3471cm and3320cm wereassignedto drug, continuing for 24h of incubation was observed. On
−1
–OHstretching.Thestrongandnarrowpeaksat1681cm , theotherhand,asseeninFigure7dependingontheprocess
−1 −1
1600cm ,and1172cm werealsoattributedC=O,C=C,and parameters, the percentage of burst release varies between
10 JournalofNanomaterials
100 forlow-watersolublecompounds.Thesmallparticlesizeand
controlled release properties of nanoparticles can improve
the water solubility, bioavailability, biocompatibility, and
80
absorptionofthedeliveredbioactivemolecules.
%)
ase ( 60 esteThrlouasd,eindtPhLisGsAtundya,nfoopratrhteicfilersstwtiemreescuacffceeiscsfauclildypphroendeutcheydl
e
el by a single emulsion solvent evaporation method. Addi-
e r
v tionally, the influences of different process parameters on
ati 40
ul 60 reaction yield, particle size, encapsulation efficiency, poly-
Cum 20 mulative release (%)5432100000 dswyiessrpteeemrdsaeitttyiecraminllyidneiexnd,v.eaPsntadirgtaizcteeutdalaarplnyod,tetonhpteitaiimlnioatiflalpnrCaonAcoePpsEsarptaiancrdlaemsPLwetGeerrAes
Cu 0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 amount, PVA concentration, and solvent type were found
0 Time (h)
tohaveplayedoverpoweringroleinencapsulationefficiency
0 50 100 150 200 250 300 350 400
andparticlesize.
Time (h)
The final optimal value for the initial CAPE and PLGA
Optm. NP amount,PVAvolume,andconcentrationweredeterminedat
NP 2 50mg,100mg,6mL,and2%w/v,respectively.Aparticlesize
NP 20 equal to 163 ± 2nm with low polydispersity index 0.119 ±
Figure7:InvitroreleaseprofileofCAPEinpH7.4phosphatebuffer 0.002,−34.4±2.5mVzetapotential,andpercentageencap-
fromCAPEloadedPLGAnanoparticlescorrespondingtodifferent sulationof89±3%wasachievedundertheoptimalcondition
polymer/drugratios. employed.Interestingly,theoptimizednanoparticlesshowed
a high (83.08%) and sustained CAPE release (lasting for 16
days)comparedtononoptimizednanoparticle.
In conclusion, high encapsulation efficiency with small
32.59% and 66.72%. In the second phase of release, caused
size and sustained release make CAPE loaded nanoparti-
bydrugdiffusionandpolymerchaincleavage,approximately
cles a suitable candidate for the further development of
constantandlowlevelofCAPEreleaseratewasobserved.In
nanomedicine.Additionallystudiesarecurrentlyinprogress
thelaststepofCAPErelease,continuousreleasealmostclose
to test the antimicrobial activity of CAPE loaded PLGA
to linear was observed over 7–16 days and reached 42.65%,
nanoparticleagainstdifferentGrampositiveandGramneg-
46.26%, and 83.08% for NP 2, NP 20, and optimized NP,
ativebacteria.
respectively,attheendof16thday.
InFigure7itwasclearlyseenthatwhenthepolymer/drug
ConflictofInterests
ratiodecreasedtheburstreleaseofCAPEincreases.Usually
initialburstreleaseoccurredduetothefastdiffusionofdrug
The author declares that there is no conflict of interests
absorbedonthesurfaceorlocalizednearsurfaceofparticles
regardingthepublicationofthispaper.
[57].Inthelowpolymer/drugratio,PLGAwasnotenoughto
encapsulatethedrug;thusmostofthedrugwasadsorbedon
orlocalizednearthesurfaceofnanoparticles,whichresulted Acknowledgment
inhigherburstrelease[57].Theminimumburstreleasewas
observed for NP 20 (polymer/drug ratio equal to 0.05) and ThisresearchhasbeensupportedbyYildizTechnicalUniver-
thehighestwasoptimizedNP(polymer/drugratioequalto sity Scientific Research Projects Coordination Department,
0.5).Inaccordancewiththeseresultstheinitialburstrelease Projectno.2014-07-04-GEP02.
ofNP2(drug/polymerratioequalto0.2)higherthanNP20
andlowerthanoptimizedNPwasobtained. References
[1] H.-Y.Lee,Y.-I.Jeong,E.J.Kimetal.,“Preparationofcaffeicacid
4.Conclusion
phenethylester-incorporatednanoparticlesandtheirbiological
activity,”JournalofPharmaceuticalSciences,vol.104,no.1,pp.
CAPEhasnumerousbiologicalandpharmacologicaleffects,
144–154,2015.
such as antiviral, antioxidant, and anticancer activities.
[2] M. R. Fesen, Y. Pommier, F. Leteurtre, S. Hiroguchi, J. Yung,
Although CAPE has many biological properties, its usage
and K. W. Kohn, “Inhibition of HIV-1 integrase by flavones,
in pharmaceutical area is limited due to its low aqueous caffeic acid phenethyl ester (CAPE) and related compounds,”
solubility and instability in biological system. One of the BiochemicalPharmacology,vol.48,no.3,pp.595–608,1994.
methodstoovercometheseproblemsistoencapsulationof
[3] G. F. Sud’Ina, O. K. Mirzoeva, M. A. Pushkareva, G. A. Kor-
theCAPEintobiodegradablenanoparticularsystem. shunova,N.V.Sumbatyan,andS.D.Varfolomeev,“Caffeicacid
Poly(DL, lactic-co-glycolic acid) is the most frequently phenethyl ester as a lipoxygenase inhibitor with antioxidant
usedbiodegradable,biocompatiblecopolymerfordeveloping properties,”FEBSLetters,vol.329,no.1-2,pp.21–24,1993.
nanoparticlesincontrolledrelease(CR)applications.PLGA [4] S.-G.Park,D.-Y.Lee,S.-K.Seoetal.,“Evaluationofanti-allergic
nanoparticlesareespeciallybeneficialindrugdeliverysystem properties of caffeic acid phenethyl ester in a murine model