Colloids and Surfaces B: Biointerfaces 117 (2014) 128134

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Colloids and Surfaces B: Biointerfaces

jo ur nal ho me p ag e: www.elsev ier .com/ locate /co lsur fb

Curcum natreatm

Malathi SBalasubra Department ob Biomaterials

a r t i c l

Article history:Received 19 AReceived in reAccepted 8 FeAvailable onlin

Keywords:Poly (dl-lacticMelt polycondElectrospinningNanoberCurcuminCarcinoma

A] co 10,0oadeon obing b

resuetics s Ko

drug dissolution and non-Fickian diffusion as a major drug release mechanism. The effect of CPNF on cellviability was assessed by the MTT (3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyltetrazolium bromide) assayto examine the cytotoxic effect of released curcumin on A431 cells in vitro.

2014 Elsevier B.V. All rights reserved.

1. Introdu

Melanomwith skin cmetastatic mortality ra12% of all cgrowth of iand treatme1, 7-bis (4-ha polyphenolonga (turmsuch as Indidiseases, wand is also eand pharm[4], antioxidhave been the treatmepancreatic

CorresponE-mail add

http://dx.doi.o0927-7765/ ction

a and carcinoma are the main cause of death of patientsancer. Melanoma is a cutaneous tumor and is fatal instage [1]. The malignant melanoma has the highestte (1015%) among all skin disorders and accounts forancer-related deaths among Caucasians. The aggressivenvasive malignant melanoma calls for early diagnosisnt. The natural curcumin [diferuloylmethane; (1E, 6E)-ydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione]lic pigment isolated from the dry rhizomes of Curcumaeric), has found use in traditional medicine in countriesa and China [2] for the treatment of common cold, skinound healing, abdominal spasms and inammation [3]mployed as a dietary herbal supplement. The biologicalacological properties of curcumin such as antitumorant [58], anti-inammatory [911] and anti-HIV [12]reported. There are several ongoing clinical trials fornt of different types of cancers such as skin cancer,cancer, multiple myeloma and colorectal cancer. The

ding author. Tel.: +91 4422202794.ress: bala2010@yahoo.com (B. Sengottuvelan).

remarkable pharmacological activity of curcumin is attributed toits ability to act on multiple intracellular targets and patients withcolorectal cancer could achieve efcient chemopreventive effect[1315]. The large dose of curcumin and frequent administrationsshowed an increase in side effects and lower compliance from theusers [16]. The factors that limit the use of free curcumin for cancertherapy are its poor aqueous solubility, poor absorption and rapidmetabolism [17,18]. In an effort to improve the bioavailabilityof the hydrophobic drug curcumin, it was encapsulated in PLGApolymeric nanoparticles or nanobers which are known for theirbiodegradability [1921], biocompatibility, solubility and goodmechanical strength [22,23]. Several delivery systems have beenemployed to enhance the effectiveness of drug to reduce thedosage and to overcome the problem of poor solubility of the drugin water as well as its reduced bioavailability [24,25].

The fabrication of nanobers can be achieved by a numberof techniques such as template [26], self-assembly [27] phaseseparation [28], melt-blown [29] and electrospinning [30]. Electro-spinning is an important technique for generating bers directlyfrom polymers, composites, ceramic and metal nanobers. It isa simple and versatile method to produce large scale continuousbers and the ber diameter can be adjusted from nanometer tomicron. Dalton et al. have reported that electrospun PEO-block-PCLnanobers can eliminate the entry of cytotoxic solvents into the

rg/10.1016/j.colsurfb.2014.02.0202014 Elsevier B.V. All rights reserved.in loaded poly (lactic-co-glycolic) acident of carcinoma

ampatha, Rachita Lakrab, PurnaSai Korrapatib,amanian Sengottuvelana,

f Inorganic Chemistry, University of Madras, Guindy Campus, Chennai 600 025, Indiadivision, CSIR-CLRI, TICEL Biopark, Chennai 600 113, India

e i n f o

pril 2013vised form 8 February 2014bruary 2014e 19 February 2014

-co-glycolic) acidensation

a b s t r a c t

Poly (dl-lactic-co-glycolic) acid [PLGmolecular weight (15,400, 11,000 andGPC and TGA-DTA studies. Curcumin lspinning in which no visible aggregatiobtained from the topographical imagindicate that an increase in GA contentin vitro release prole and release kinfrom CPNF. The release prole follownober for the

polymers with different ratios (78/22, 68/32 and 61/39) and00 Da) were synthesized and characterized by 1H NMR, FTIR,d PLGA with the size of 100300 nm were obtained by electro-served on the surface. The diameter of CPNF (61/39) nanobery AFM is 160 10 nm. The water contact angle measurementslts in increase in the hydrophilicity of the PLGA copolymer. Thefrom the CPNF demonstrated a sustained release of curcuminrsmeyerPeppas model suggesting a combination of surface

M. Sampath et al. / Colloids and Surfaces B: Biointerfaces 117 (2014) 128134 129

human broblasts cells when the bers are deposited directly ontobroblasts [31]. The polymeric nanobers are employed as carriersfor controlled drug delivery, wound healing, biocatalysts and alsoas active components for biosensing [32] due to their large sur-face area to volume ratio and also they exhibit good capability ofsupporting cell adhesion and differentiation.

Fibers can interact with skin at cellular level as an adjuvant.The nanoberskin interaction can be used to enhance immuneresponse for topical applications. The nanobers can facilitate slowrelease of curcumin for its wound healing and antimicrobial activ-ities [33]. The curcumin released from nanober not only inhibitsmicrobial proliferation, but also accelerates wound healing. Thiscontrolled release of curcumin while the nanobers remain on theskin surfaceery systems

Langer arelease of pof antiangioPLGA degratrolled by tmolecular wtallinity. In by followinsion througenvironmendation.

Curcumiexhibit gooreports on cof curcuminis to synthemolar ratiosby melt polynanobers

2. Experim

2.1. Materi

dl-Lacticdichloromemethanol (Stannous oreported prout the expout at pH 7.microbiologand were u

2.2. Method

2.2.1. Melt PLGA w

(80.025kPand glycoli70/30 and

connected to a liquid nitrogen trap. Stannous octoate was addedin catalytic amount. The mixture of lactic acid and glycolic acidwas dehydrated at 120 C for 6 h at 8.0 kPa. In the second stage, thecondensation of LA and GA occurs with the elimination of waterresulting in the formation of the ester when the mixture of lacticand glycolic acid was heated at 150 C for 12 h under high vacuum(2.5 kPa). In the third stage viscous and brous polymer wasproduced under very low pressure (0.025 kPa). The crude brousPLGA was soluble in chloroform and precipitated with diethylether as a non-solvent. The precipitate was dried under vacuum toyield transparent yellow ber. The PLGA copolymers with differentcompositions were prepared using the same procedure.

Prepa encrriedlutioer in issolvmoguouse nan

to he n

solvber wThe fm [E

electforceed ong prry trie 2)

Chara drutomer Tratic Red o

wehlororpho

FielHITAHI). Ted bof ther donio

cm ed aremeer suh m

copoly highlights one of the most successful topical drug deliv-.nd Folkman were the rst to demonstrate the controlledroteins using polymers which enabled the developmentgenic drug delivery systems for cancer therapy [34].dation and drug release kinetics can be precisely con-he physicochemical properties of the polymer, such aseight, polydispersity index, hydrophobicity and crys-general, drugs can be released in a controlled mannerg Fickian or non-Fickian kinetics due to drug diffu-h the polymeric matrix, or be triggered in response total stimuli or released in the course of chemical degra-

n loaded PLGA nanoparticles have been reported tod anticancer activity [35,36]. However, there are nourcumin loaded PLGA nanobers for controlled delivery

in squamous carcinoma. The aim of the present studysize a series of lactic/glycolic acid polymers with various

of lactic to glycolic acid and various molecular weightscondensation method and study their curcumin loaded

for the treatment of squamous carcinoma.

ental

als

acid and glycolic acid (Merck), curcumin (Sigma),thane, acetone, diethyl ether (Fischer Scientic),SRL) were used as such without further purication.ctoate was prepared with slight modication of theocedure in Ref. [37]. Deionized water was used through-eriment. The in vitro release measurement was carried4 in PBS medium. Nutrient agar (Himedia) was used forical tests. All other reagents were of analytical gradesed as received.

s

poly condensation of PLGA copolymersas synthesized in three stages under vacuum

a) (Scheme 1). In the rst stage, dl-lactic acid (0.08 M)c acid (0.02 M) with different molar ratio (80/20,60/40) were placed in a ask with a distillation head

2.2.2. The

was camer sopolymwas dthe hocontinpreparappliedfrom tnied bynanoment. of 10 cstrongtional collectresultilimina(Schem

2.2.3. The

trophoFourieMagnerecordspectraated cThe mousing a6600, HITACexaminbicity of watangle gfoil (1preparmeasuthe bfor eac

Scheme 1. Synthesis of the PLGA ration of curcumin loaded PLGA nanobersapsulation of PLGA copolymer with curcumin (10:1)

out by electrospinning technique. The PLGA copoly-n was prepared at room temperature by dissolving the40% chloroform under gentle stirring for 2 h. Curcumined in 60% methanol and was gradually poured intoeneous polymer solution. The solution was sonicatedly for 1 h and it was lled in a 5 mL plastic syringe. Toobers by electrospinning, a high voltage (24 kV) wasthe polymer solution and a charged jet is driven outeedle which undergoes stretching/thinning accompa-ent evaporation (Supporting information, Fig. SF1). Theas collected on a drum, which is attached to the instru-eed-rate was 0.4 mL/h, with a tip-to-collector distancespin Nano]. The electrospinning process is governed byrical forces, viscoelastic forces, surface tension, gravita-s and frictional force of the air drag [38]. The ber wasn an aluminum foil and was dried under vacuum. Theoduct was stored in a freezer for further studies. Pre-als were carried out to adjust the processing parameters.

cterizationg concentration was determined by UVvisible spec-try at 425 nm. The polymer was characterized bynsform Infrared Spectroscopy (FTIR) and Nuclearesonance spectroscopy (1H-NMR). FTIR spectra weren a Bruker Tensor 27 IR spectrometer and 1H-NMRre obtained on a Bruker NMR spectrometer in deuter-form with tetramethylsilane as an internal standard.logy of curcumin loaded PLGA nanober was analyzedd Emission Scanning Electron Microscope (FESEM, SU-CHI) and Scanning Electron Microscope (SEM, S3400,he bers were coated with a thin layer of gold and theny FESEM and SEM. The hydrophilicity and hydropho-e CPNF was measured by contact angle relaxationroplet by Kruss easy drop method using a contactmeter (KRUSS, DSA II GmbH, Germany). The aluminum

1 cm) containing electrospun nanober was freshlynd then attached to a glass slide for contact anglents. A droplet of deionized water was pipetted ontorface and the contact angle was measured at 25 C

easurement. The images of the solution droplet were

mers.

130 M. Sampath et al. / Colloids and Surfaces B: Biointerfaces 117 (2014) 128134

ded PL

obtained usvalues werwas ascertaBruker X-ration sourcegravimetriccarried out lyzer, underrate of 10 Croughness dSystem Atonanobers by non-con

2.2.4. DeterCurcumi

rately weigmethanol iwere then tor (Remi ofor 2 h afte1 h, for comthreefold wspectrophospectrophosulation ef

Encapsulati

2.2.5. In vitThe CPN

at pH 7.4. TThe sampleincubator. intervals atcumin fromof methanoThe concena calibratio

2.2.6. ReleaThe drug

the followin

1) Higuchi 2) Power la

where Q is ing the desreleased at nent. The nIf the n valu

on, aFicki

Cytot celyl) armin. Thech 8

wit and t samn 5% owthas dxprestand

ults

direus oc

efcc acid

undeiffereigh

andmersrom2). Wease ed. Wolect al. tio o/50 a

inhsperser r

encytocodicald. ThScheme 2. Schematic illustration of curcumin loa

ing a high speed digital camera and the contact anglee determined. The crystalline nature of the polymerined by powder X-ray diffraction measurements usingy diffractometer with a Cu K-monochromatic radia-

( = 1.5406 A) operating at 30 kV and 15 mA. Thermo (TG) and differential thermal analysis (DTA) wereon a Pyris 1 TGA (Perkin Elmer) thermo gravimetric ana-

nitrogen atmosphere, with 4 mg of sample, at a heating/min in the temperature range from 40 to 600 C. Theata were obtained from the images recorded by Parkmic Force Microscope (AFM) XE 100. The drug loadedwere coated on to a mica substrate and scanned in airtact mode for the AFM studies.

mination of drug encapsulation efciencyn was extracted from CPNF by suspending an accu-hed amount (10 mg) of curcumin loaded CPNF in 10 mln well-closed screw-capped 15 ml vials. The samplesmaintained at 37 C in a shaker (100 rpm) incuba-rbital shaker). The samples were stirred continuouslyr the incubation and were sonicated continuously forplete drug dissolution. A 1-ml sample was dilutedith methanol, and curcumin content was determinedtometrically in methanol at 425 nm using UVvisibletometer (Perkin Elmer -35). The percentage of encap-ciency was calculated using the following equation:

on efciency (%) = Weight of drug in CPNFTheoretical drug loading

100

ro drug releaseF (100 mg) was dispersed in 100 ml phosphate bufferhe solution was distributed in 1.5 ml eppendorf tubes.s were then maintained at 37 C in a shaker (100 rpm)The solution was centrifuged at predetermined time

4000 rpm for 10 min to separate the released cur- CPNF. The released curcumin was dissolved in 1 mll, and then analyzed spectrophotometrically at 425 nm.tration of the released curcumin was determined fromn curve of curcumin in methanol.

se kinetics release kinetics and mechanism were investigated byg mathematical models.

diffusia non-

2.2.7. The

diphento detegrowthin whitreatedcuminThe tes37 C itive grcells) wwere eples

3. Res

Thestannoits highglycolifor 4 hwith dtheir w11,000copolytive chFig. SFa decrobservwith mZhou efeed raand 50

Thepolydimonomused toof its cbiomemethoModel Q = Kt1/2.w (or) KorsmeyerPeppas model Mt/M = Ktn.

the cumulative drug release, K is the constant reect-ign variables of the system. Mt/M is fraction of drugtime t, K is the rate constant and n is the release expo-

value is used to identify different release mechanisms.e is 0.45 or less, the release mechanism follows Fickian

such as confrom the suoptimized chloroforma medium tis also highformation oout with dithe conditiGA nanober (CPNF).

nd higher values 0.45 < n < 0.89 for mass transfer followan model (anomalous transport).

oxicity studiesl viability MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-ssay was carried out with skin cancer (A431) cell linese the effect of curcumin loaded PLGA nanobers on cell

inhibition in cell growth was determined by MTT assay000 cells/well were taken in a 48-well plate and wereh 10, 25, 50, 100 and 250 m concentration of free cur-equivalent doses of curcumin-loaded PLGA nanobers.ples were kept in triplicate. The plate was incubated at

CO2. The assay was terminated after 48 h and the rela- inhibition compared to that of control cells (untreatedetermined spectrophotometrically at 570 nm. The datassed as mean absorbance value (OD) of triplicate sam-ard deviation of the mean.

and discussion

ct melt polycondensation of LA and GA was initiated bytoate as a catalyst. This catalyst was chosen because ofiency in promoting the polymerization of lactic acid and. The polymerization reaction was carried out at 150 Cr reduced pressure (0.025 kPa). PLGA was synthesizedent molar feed ratios (78/22, 68/32 and 61/39) andt average molecular weights were found to be 15,400,

10,000 Da respectively. The molecular weights of the were determined by GPC method and the representa-atogram is reproduced (vide: Supporting information,hen the ratio of glycolic acid monomer was increased,in the molecular weight of the PLGA copolymer wasang et al. have reported the direct synthesis of PLGA

ular weight ranging from 900 to 1400 Da [39]. Recently,have reported the direct synthesis of PLGA with molarf dl-lactic acid to glycolic acid 100/0, 85/15, 75/25, 65/35nd its application in protein delivery systems[40].erent viscosity, melting point, molecular weight andity index of a series of PLGA copolymer with differentatios are tabulated (Table 1). The PLGA nanober wasapsulate curcumin in the present investigation becausempatibility, biodegradability and suitability for various

applications. The CPNF was obtained by electrospinninge control over different electrospinning parameters

centration, applied voltage, distance of source electrodebstrate, temperature, and solvent volatility have beenfor good spinnability to get nanobers. A mixture of

and methanol in the ratio 40/60 (v/v) was chosen aso dissolve PLGA and curcumin and this solvent mixturely volatile. When curcumin content was increased, thef nanobers was difcult. The experiment was carriedfferent concentration of curcumin in order to optimizeon for forming smooth nanobers and nally the ratio

M. Sampath et al. / Colloids and Surfaces B: Biointerfaces 117 (2014) 128134 131

Table 1Feed monomer ratio, calculated monomer ratio, inherent viscosity, molecular weight and polydispersity index of the PLGA copolymer.

Sl. no Feed monomerratio (LA/GA)

Calculated monomerratio (LA/GA)a

Inherentviscosity (dL/g)

Melting point(C)

Molecularweight (Da)

Poly dispersityindex (Mw/Mn)

1 80/20 78/22 0.23 96 2 70/30 68/32 0.18 52 3 60/40 61/39 0.16 nd

nd: not detected. Inherent viscosity was determined using tetrahydrofuran as solvent with a solution ca Calculated by NMR.

of curcumin and PLGA was maintained at 1:10. Said et al. havereported fusidic acid loaded PLGA ultrane bers for antimicrobialand wound bacterial studies [41].

Fig. 1 shows the characteristic FTIR spectrum of PLGA copoly-mer. A strong band at 1759 cm1 is due to the C O stretchingfrequency of ester group and the bands at 3002 and 2987 cm1

are attributed to CH stretching frequency. The strong band at3558 cm1 is assigned to the OH stretching frequency. The bandsat 1463 and 862 cm1 are assigned to OH in-plane and out-of-plane bending vibrations respectively. The bands at 1202 and1097 cm1 are assigned to CO stretching while the band at750 cm1 is assigned to out-of-plane bending vibration of CH. Thisspectral study conrms the formation of PLGA copolymer.

The FTIR spectrum of curcumin (Fig. 1) shows the characteristicpeak at 3500 cm1 which can be attributed to phenolic OH stretch-ing vibration [42] and this band was observed at 3661 cm1inCPNF. The bands at 1510 and 1426 cm1 are due to the stretchingvibrations of CC of benzene ring and olenic bending vibrationof CH group bound to the benzene ring of curcumin respectively[43]. These peaks were shifted to1524 and 1454 cm1 in CPNF andthus, conrming the curcumin was encapsulated by PLGA poly-mer. The peak at 865 cm1 is due to the stretching vibration ofCO in CCPNF. A strbonyl stretcAll major b

Fig. 1. F

frequenciestogether tomation, Tab

NMR spPLGA copolcidated bydifferent ra(vide: Suppof PLGA coassigned tois assigned observed a61/39) is caton signals.GA from theCH2 peak nCH2, CH proare tabulate

The LA/Gthe integral(CH, LA), ac[45]. The ar

.79 pere o

wer encrting

therin thhe co

of th00

Suppted iopolre shopolted t

lacti therserie

XRD as se) and). Th9.7, mainses wOCH3. The phenyl ring was observed at 871 cm1 inong and sharp peak at 1750 cm1 is attributed to car-hing (C O) which was shifted to1752 cm1 in CPNF.ands of both curcumin and PLGA are shifted to higher

7.117tons wprotoning theSuppo

Theascertaity of tcurvesup to 6(vide: presenPLGA cperatuPLGA cattribution ofhigherof the

Theas well(68/32mation19.1, 2the redecreaTIR spectra of (A) Curcumin (B) PLGA copolymer and (C) CPNF.

informationPLGA (68/3

Several gthe surfacetact anglescontact angfaces with 15400 2.011000 2.310000 2.1

oncentration of 0.1 g/dL.

in CPNF indicating that curcumin and PLGA are bound form more stable nanobers (vide: Supporting infor-le ST1).ectroscopy was used to analyze the composition of theymers. The structure of the PLGA copolymers was elu-1H NMR spectrum. The 1H NMR spectra of PLGA withtios viz. (61/39), (68/22) and (78/22) are reproducedorting information, Fig. SF3). The 1H NMR spectrumpolymer (78/22) shows a signal at 5.16 ppm which is

CH protons of GA unit while the signal at 1.50 ppmto CH3 protons of LA unit. The CH2 protons of GA ist 4.76 ppm. The ratio of LA/GA of PLGA (78/22, 68/32,lculated from the intensities of CH(LA) and CH2(GA) pro-

Wang et al. have determined the molar ratio of LA and integral data of the CH peak near 5.19 ppm and that ofear 4.85 ppm [44]. The chemical shift values of the CH3,tons observed in the 1H NMR spectra of the copolymersd (vide: Supporting information, Table ST1).A monomer ratio of the copolymer was calculated using

values of the peak at 4.29 ppm (CH2, GA) and 4.51 ppmcording to the method described by Gilding and Reedomatic protons of curcumin exhibit a signal in the regionpm. However, in the case of CPNF, the aromatic CH pro-bserved at 7.35 ppm while the CH2 (GA) and CH (LA)e observed at 4.35 and 4.7 ppm respectively indicat-apsulation of curcumin in the PLGA copolymer (vide:

information, Fig. SF4).mal behavior of PLGA copolymers was investigated toe inuence of the monomer ratio on the thermal stabil-polymers. The weight loss and differential weight losse copolymers were obtained by heating the samples

C at the rate of 10 C/min under nitrogen atmosphereorting information, Fig. SF5) and the relevant data aren Table ST2 (Supporting information). The TGA plots forymers are similar, with the initial decomposition tem-ifting to higher values as the GA component increases inymer (Supporting information, Fig. SF5). This process iso the decomposition of PLGA copolymer with the forma-de and glycolide monomers. The PLGA (68/32) exhibitsmal stability when compared to that of other monomerss.

pattern of the copolymers indicates their amorphousmi crystalline nature. The XRD results of PLGA (78/22),

(61/39) are reproduced in Fig. SF6 (Supporting infor-e PLGA (78/22) exhibits ve peaks (2 = 17.5, 18.2,and 30.8), whereas the peak intensity decreases ining copolymers indicating that polymer crystallinityith increase in the glycolic acid ratio (vide: Supporting

, Fig. SF6). The PLGA78/22 is semi crystalline whereas2) and (61/39) are amorphous in nature.roups have employed experimental results to compare

roughness of nanomaterial with the observed con- [46,47] and it has been suggested that surfaces withle < 90 exhibit hydrophilic properties whereas sur-contact angle > 90 exhibit hydrophobic properties.

132 M. Sampath et al. / Colloids and Surfaces B: Biointerfaces 117 (2014) 128134

Fig. 2. Water cresults are giv

The hydropwith the incCPNF nanothe GA contact angle oindicating i60.1 0.89 (78/22) resin these twhydrophobiof lactic achydrophobiF-108 can cpared to tha

The SEM images of CPNF (61/39) (Fig. 3) and other ratios arereproduced in supporting information (Fig. SF7A and B). The effectof curcumin content of PLGA (1:2, 1:10) on the morphology of

s observed in these photographs. As decreasing curcumint resulted in progressively reduced beading, increased berter, and reduced diameter distribution were observed andorphology of the ber was smooth without the presence

beads. The images observed in the SEM indicate that the non-uniform because of its lower thermal stability (vide:rting information, Fig. SF8A and B), which is reected byelting of nanobers on the SEM grid itself. The polymerbers employed in the present investigation essentially hasolecuLGA.o 10/curc

showses wCPNF icontendiamethe mof anyber isSuppothe mnanolow mmer P1000 tof PLAwhichdecreaontact angle measurements of (A) 78/22, (B) 68/32 and (C) 61/39. Theen in degrees as a mean value standard deviation (n = 5).

hilic nature of PLGA nanobers increases substantiallyrease in GA content. The contact angle measurements ofbers indicate the change in surface hydrophilicity whentent in the copolymer was increased (Fig. 2). The con-f water droplets containing CPNF (61/39) is 51.5 0.18ts hydrophilic nature. The contact angle increases toand 88.1 0.97 in the case of CPNF (68/32) and CPNFpectively suggesting that the hydrophilicity decreaseso systems. PLGA copolymer exhibits a wide range ofc/hydrophilic property depending on the molar ratioid and glycolic acid. Vasita et al. have reported thatc PLGA ber meshes blended with hydrophilic pluronicause a decrease in the surface contact angle when com-t of pure PLGA meshes [48].

mainly attrexplained bmore solubthe curcumshaped, non

The AFMof CPNF (61The scan raAFM [50,51ent molecupresence ofof electrospticles encapdescribed bimage of AFSurface rousurface comsurface rousurface rouRp-v (peaksured from variation ingated usingdue to the fimage showsurface. Thsurface of tdistributionwith a mea

The rele37 C in PBSdrug was dmeasured adays with nincreases tstrength anelectrospinrmed the study. The

Table 2Surface roughThe results are

Sample code

CPNF 78/22 CPNF 68/32 CPNF 61/39 lar weight and also has lower ratio of LA in the copoly- The average diameter of nanober decreased from0 nm. Lin et al. [49] have reported average diameterumin composite nanobers between 756 and 971 nm,s that the average diameter of composite nanoberith increase in curcumin content. The results were

ibuted to the solubility of the curcumin, and can bey the fact that curcumin is hydrophobic drug and isle in PLGA/dichloromethane/methanol solution. Whenin content is higher, the nanobers formed are beaduniform and unstable.

non-contact mode two and three dimensional images/39) are shown in Fig. SF9 (Supporting information).nges are 5 m 5 m and 5 m 5 m respectively.] lets one to qualitatively visualize the surface depend-lar event in 3D on a nanometer spatial resoltuion in the

oxygen. Xu et al. have studied the structural evolutionun nanobers using AFM [52]. Chitosan/siRNA nanopar-sulated in PLGA nanobers for siRNA delivery has beeny Chen et al. [53]. It is evident from the topographicalM that the curcumin is impregnated in the PLGA matrix.ghness (Ra) analysis provides a method of assessing theposition and the drug distribution of the ber mats. Theghness parameters of the CPNF (61/39) viz., Ra (averageghness), Rq (root means square surface roughness) and

to valley surface roughness) of the system (SF9) mea-the 2D images are 115, 140 and 497 nm respectively. The

wettability due to the surface roughness was investi- water contact angle measurement (Table 2). Lower Raaster internalization and the corresponding amplitudeed the presence of only a few particles on the ber

e Ra value shows that most of the drug is beneath thehe ber and to a lesser extent on the surface. The size

curve from the AFM image indicates the size of bern diameter of 160 10 nm.ase kinetics of curcumin from CPNF was observed at

(pH 7.4). At predetermined time intervals, the releasedissolved in 1 ml of methanol and its absorbance wast 425 nm. Curcumin release was sustained at least for 8o burst effect. The absence of beads in the nanobershe surface area to volume ratio, better mechanicald also the drug compatibility with the polymer/solventning solution while the sustained release pattern con-structural reliability of the nanobers throughout therelease kinetics of curcumin from the PLGA nanobers

ness parameters using AFM and water contact angle measurements. given in degrees, as a mean value and standard deviation (n = 5).

Ra (nm) Rq (nm) Rp-v (nm) Contact angle ()

320 371 1392 88.1 0.97235 282 1121 60.1 0.89115 140 397 51.5 0.18

M. Sampath et al. / Colloids and Surfaces B: Biointerfaces 117 (2014) 128134 133

Fig. 3. SEM images of (A) CPNF bead (1:2) and (B) CPNF (61/39) nanober (1:10).

Table 3Drug loading and release prole of curcumin loaded PLGA nanobers (CPNF). Theloading efciency and drug release data presented as percentage. Data are presentedin mean value and showing standard deviation (n = 3).

Sl. no Sample code Drug releasetime (h)

Drug loadingefciency (%)

Drug release(%)

1 CPN2 CPN3 CPN

was followenanober wCurcumin isin hydrophloading ef78/22 (81%)in GA conteity of the m(Table 3) orelease rateof rifampiciage of drugin the amouin glycolic aPLGA with ysis becaussterically hemployed cemulsicatidrug loadinnanoparticlrelease for 7

The kinebetween drvalues of retting the ralong with

vitrond sho

ationST3)., was evaluated from the regression coefcients obtained byi and KorsmeyerPeppas (power law) kinetic models. Thee indicates that the KorsmeyerPeppas model is best suited

release kinetics of curcumin. The release exponent value sustained release of curcumin is 0.45 < n > 0.89, which lies

the limits of KorsmeyerPeppas model (vide: Supportingation, Table ST3). The release exponent is a clear indicationomalous diffusion (non-Fickian model) could be the princi-ase mechanism for curcumin, which is due to the combinedf diffusion and erosion mechanism for drug release [59].F 78/22 264 81 0.57 25 0.11F 68/32 264 70 1.50 45 0.50F 61/39 264 66 1.00 96 1.02

d for more than 10 days. The drug content (%) of theas calculated and the values are tabulated (Table 3).

a hydrophobic drug and hence it is expected to resideobic LA domain of the PLGA copolymer. The curcuminciency of the polymer is in the following order: CPNF:

> CPNF: 69/31 (70%) > CPNF: 61/39 (66%). The increasent of the polymer results in increase in the hydrophilic-atrix and it is reected in lesser loading percentage

f the hydrophobic drug curcumin and also its faster (Fig. 4). A similar observation has been made in the casen which is also a hydrophobic drug [54]. The percent-

release in the CPNF nanober increases with increasent of GA content in the PLGA copolymers. The increasecid percentage results in faster degradation, whereas

higher lactic acid content is less susceptible to hydrol-e the pendant methyl group on the lactic acid moietyinders the attack of water molecules. Tsai et al. haveurcumin loaded PLGA nanoparticles by high-pressureon-solvent evaporation method and found that theg efciency was 46.9%. The release prole of curcumines was found to be an initial burst followed by sustaineddays [55].

Fig. 4. Invalues a

informTable (CPNF)HiguchR2 valufor thefor thewithininformthat anpal releeffect otic modeling on drug release shows the relationshipug dissolution and drug release pattern [5658]. Thelease constant and release exponent (n) determined byelease data into their respective mathematical models,regression coefcient (R2) are provided in supporting

A431 cemeric nanoand Vishwagested thatpotential a

Fig. 5. Cytotoxicity prole of CPNF (61/39). Data are presented as mean valu drug release prole of PLGA copolymers. Data are presented as meanwing standard deviation (n = 3).

(vide: Supporting information, Figs. SF10SF15 and The curcumin release prole from nanober matrixlls were used to assess the potential of PLGA poly-bers as anticancer drug delivery vehicle. Mukerjeenatha formulated curcumin-loaded PLGA NPs and sug-

a nanoparticle-based formulation of curcumin has highdjuvant therapy in prostate cancer [60]. Cell viability

es and showing standard deviation (n = 3).

134 M. Sampath et al. / Colloids and Surfaces B: Biointerfaces 117 (2014) 128134

(MTT) assays were performed using equivalent dosages of free cur-cumin and curcumin-loaded PLGA nanober (CPNF: 61/39). ThePLGA (61/39) nanober was taken as blank and untreated cellsserved as controls. The cell death was maximum when the unboundcurcumin content was increased from 10 to 250 M while CPNFexhibits only marginal decrease in cell death when curcumin con-tent was increased indicating the controlled release of curcuminfrom the polymer matrix (Fig. 5). However, the PLGA did not showany effect on the cell viability. The assay was terminated after 48 hand the colthat curcumgrowth of ccancer cell PLGA nanop

4. Conclus

The biodwith low mdensation m1HNMR, GPfree curcumelectrospincurcumin cacterized bmeasuremeconcentraticumin loadhydrophilicrelease rateage diamethigh drug estudies fromsustained mThe cell viananobers CPNF is capcontrolled m

Acknowled

The nafully acknoNanosciencMadras for

Appendix A

Supplemfound, in th2014.02.02

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Curcumin loaded poly (lactic-co-glycolic) acid nanofiber for the treatment of carcinoma1 Introduction2 Experimental2.1 Materials2.2 Methods2.2.1 Melt poly condensation of PLGA copolymers2.2.2 Preparation of curcumin loaded PLGA nanofibers2.2.3 Characterization2.2.4 Determination of drug encapsulation efficiency2.2.5 In vitro drug release2.2.6 Release kinetics2.2.7 Cytotoxicity studies

3 Results and discussion4 ConclusionsAcknowledgementsAppendix A Supplementary dataAppendix A Supplementary data