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Advanced MaterialsVol. 7, pp. 1–9, 2015

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The In Vitro Therapeutic Activity of EllagicAcid-Alginate-Silver Nanoparticles onBreast Cancer Cells (MCF-7) and NormalFibroblast Cell (3T3)Samer Hasan Hussein-Al-Ali1, 2,∗, Sepideh Keshan Balavandy3, Zurina Zainal Abidin3,4,Aminu Umar Kura5, Sharida Fakurazi5, 6, Mohd Zobir Hussein3,∗, and Mohammed Fawaz Silwadi7

1Faculty of Pharmacy, Isra University, P.O. Box 22, Amman 11622, Jordan2Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, Malaysia3Materials Synthesis and Characterization Laboratory, Institute of Advanced Technology (ITMA),Universiti Putra Malaysia, Malaysia4Departments of Chemical and Environmental Engineering, Faculty of Engineering, University Putra Malaysia,43400 Serdang, Selangor, Malaysia5Laboratory of Vaccines and Immunotherapeutics, Institute of Bioscience, Universiti Putra Malaysia, Malaysia6Department of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Malaysia7Applied Chemistry Section, Higher College of Technology, Al-Khwair 33, Muscat, Sultanate of Oman

ABSTRACT

The present work involves the development of EA-Alg-AgNPs nanocomposite based on ellagic acid (EA) asactive compound. Silver nitrate was taken as the metal precursor (AgNPs) and sodium alginate (Alg) as areducing agent. The EA-Alg-AgNPs nanocomposite was characterized using transmission electron microscopyTEM, zeta potential, and in vitro release kinetics. The particles thus obtained were spherical in shape andhaving an average particles size of 10 nm, zeta potentials of −8.2 mV, and the release kinetics of EA fromnanocomposite was following Hixson-Crowell kinetics models with R2 = 0.9956. The cytotoxicity potential offree EA, Alg-AgNPs and the EA-Alg-AgNPs nanocomposite may be determined using a normal mouse fibrob-last cells (3T3) and breast cancer cells (MCF-7). EA-Alg-AgNPs nanocomposite demonstrated a decreasedcytotoxicity effect when compared to free EA on MCF-7 cells with 15.3% cell viability at 128 �g/mL; comparedto 33.5% cell viability in a direct EA exposure. It is worth mentioning the cytotoxicity of Alg-AgNPs againstMCF-7 shows 28% viability at 128 �g/mL.

KEYWORDS: Silver Nanoparticles, Ellagic Acid, Sodium Alginate, Sustained Release, 3T3 Cells, MCF-7 Cells.

1. INTRODUCTIONSynthesis metal nanoparticles has become one of the mostinteresting areas of scientific research in recent years, thisis due to their applications in various fields such as bio-labelling,1 catalysis,2 and drug delivery.3 Silver nanoparti-cles is one of the example on the metal nanoparticles havereceived considerable attention in the photo-catalysts,4

catalysts,5 antibacterial,6 biosensor,7 and bioimaging byenhanced Raman scattering.8 The unique properties ofsilver nanoparticles such as large specific surface area,

∗Authors to whom correspondence should be addressed.Emails: sameralali72@yahoo.com, samer.alali@iu.edu.jo,

mzobir@upm.edu.myReceived: xx Xxxx xxxxAccepted: xx Xxxx xxxx

high fraction of surface atoms and slow-release drug car-riers leads to enhanced antibacterial activity9 and used indrug delivery.10

The common method for the preparation of AgNPswas called chemical method, this method occurs in pres-ence of reducing and stabilizing agents. The reduc-ing agent include sodium borohydride,11�12 hydrazine,13

and N ,N -dimethylformamide.14 In addition, stabiliz-ing agents include triphenylphosphine,15 citrate16 andpolyvinylpyrrolidone.17 Recently, researchers found thatsome of the reducing agents harmful on the environ-ment as well as have biological risks,18 and also thestabilizers are toxic and difficult to dispose.19 With theincreasing of biological risks of reducing and stabilizingagents, different research groups develop green method

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for the preparation of nanoparticles. In this method thenatural polymers was used as reducing and stabilizingagent. Currently, many natural polymers like chitosan andthere derivatives,20 soluble starch,21–23 sodium and calciumalginate,9�23 polyvinyl alcohol,24 and tween 8025 have beenused in the green preparation of nanoparticles.Silver nanoparticles have been extensively investigated

to facilitate the intracellular delivery of therapeutics.10 Cuiwith his group used silver nanoparticles as the antitumordrug (9-aminoacridine-9AA) release materials. The studyshows both silver nanoparticles and 9AA drug can inhibitthe growth of Hela cells. In addition silver nanoparticlescan slow down the antiproliferation effect on Hela cells atlow concentration of antitumor drugs.10

Similar to last study, the antitumor drug ellagic acid ischosen as the drug model in this paper. We have incor-porated alginate on the surface of silver nanoparticles andthen bind the EA with the polymer. The study was alsoinvestigating the cytotoxicity of the nanocomposite andshow if binding the EA with nanocarriers will increase thebiological activity or not.

2. MATERIALS AND METHODS2.1. MaterialsSodium alginate (molecular weight ∼500,000) andhydrated ellagic acid (97% purity) were obtained fromSigma-Aldrich (St. Louis, MO, USA). The AgNO3 wasobtained from Guanghua Sci-Tech Co., Ltd. (Guangzhou,People’s Republic of China). Dimethyl sulfoxide (DMSO)was purchased from Ajax Finechem (Sydney, Australia)with 0.1% water content used as solvent. All reagentswere of analytical grade and used without further purifi-cation. Deionized water was used to prepare all polymersolutions.

2.2. Preparation of the Alginate-Stabilized AgNPs1.5 wt% sodium alginate of was dissolved completelyin 100 ml of deionized water for an hour. Subsequently,50 ml of an aqueous silver nitrate (AgNO3) solution(0.05 M) was dropped into the sodium alginate solutionwith magnetic stirring. These solutions were heated at60 �C. A 1.5 mL solution of NaOH (1 M) was added, thesuspension immediately turned to dark brown color whichindicates the formation of AgNPs. The reaction was con-tinued for 30 Minutes and the obtained suspensions werewashed and centrifuged using a high Centrifuge Machineat 18,000 RPM and AgNPs powder was kept for furtheruse. The product was donated as Alg-AgNPs (alginatecoated silver nanoparticles).

2.3. Preparation of the EA-Alg-AgNPs NanocompositeTypically, the appropriate amount of hydrated EA(0.0013 mol) was dissolved in DMSO followedby 10 minutes stirring and heating for 40 �C.

The EA-Alg-AgNPs nanocomposite was prepared by mix-ing a solution of EA (5 mg/ml) with a known weight ofeach Alg-AgNPs (40 mg/mL). The solution was magneti-cally stirred at room temperature for 18 hours to facilitateEA uptake. The products were separated by the use of cen-trifuge and washed three times using deionized water andwere denoted as EA-Alg-AgNPs (loaded ellagic acid onthe alginate coated silver nanoparticles).

2.4. Ellagic Acid Quantities Loaded and Releasedfrom EA-Alg-AgNPs Nanocomposite

Ellagic acid release profiles from the nanocompositewere determined at room temperature using a phos-phate buffered saline solution (PBS) at a concentration0.01 mol/L at pH 7.4. About 85 mg of the nanocompos-ite was added to 500 mL of the PBS media. The cumu-lative amount of ellagic acid released into the solutionwas measured at preset time intervals at �max = 258.4 nmusing a Shimadzu UV-1601 spectrophotometer at IsraUniversity.To compare the release rate of ellagic acid from the

EA-Alg-AgNPs nanocomposite, with the physical mixturewhich contained ellagic acid with Alg-AgNP, 3.0 mg ofthe physical mixture was obtained by mixing 0.65 mg ofellagic acid with 2.4 mg of Alg-AgNP to compare therelease from EA-Alg-AgNPs nanocomposites. The releaseof the active, ellagic acid was determined as describedabove.

2.5. Cell Culturing and MTT Cytotoxicity AssaysNormal mouse fibroblast cells (3T3) and breast cancercells were obtained from the American Type Culture Col-lection (ATCC, Manassas, VA, USA). The cells weremaintained and cultured in DMEM medium with 10%fetal bovine serum (FBS), 15 mmol L−1 L-glutamine,100 units mL−1 penicillin and 100 �g mL−1 strepto-mycin to maintain cells at 37 �C and 5% CO2 in ahumidified incubator. Media were changed every two daysand at 90%, confluent cells were seeded into a 96-wellplate at 1× 105 cells mL−1 and kept overnight for cellattachment. The old media was discarded and 100 �Lof the new medium containing pure EA, Alg-AgNP, andEA-Alg-AgNPs was used to treat the cells, while wellscontaining media and cells were only used as controls.For each experiment, freshly prepared stock solutions ofEA, Alg-AgNP, and EA-Alg-AgNPs were used for thetreatment. A stock of 10 mg mL−1 (in PBS) was made.Using DMEM, the desired concentrations of the medianeeded for the treatment were made through serial dilu-tion (0–128 �g mL−1). The MTT assay was used 24 hourspost-exposure to determine the toxic effect of theseagents.In the MTT assay, the treatment media were discarded

after 24 hours and MTT-containing media were added at5 mg mL−1 PBS and a volume of 100 �L per well, and the

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plates were incubated at 37 �C in a 5% CO2 humidifiedincubator. Two hours later, a detergent reagent (DMSO)was then added to the cells to stop the conversion and sol-ubilize the formazan. The amount of formazan correlatesdirectly with the number of viable cells after treatment.Absorbance of the formazan that is formed is taken at awavelength of 570 nm using a multiwall microplate reader.The experiment was performed in triplicate, and the resultwas expressed as the mean±SD.

cell viability �%� = Average of treated/average control

×100%

2.6. InstrumentationPowder X-ray diffraction patterns were used to deter-mine the crystal structure of the AgNPs in the range of30–85 degrees on an XRD-6000 diffractometer (Shimadzu,Tokyo, Japan) using CuK� radiation (� = 1.5406 Å) at30 kV and 30 mA. Fourier transforms infrared spec-troscopy (FTIR) spectra of the ellagic acid, Alg-AgNP andEA-Alg-AgNPs materials were recorded over the rangeof 400–4000 cm−1 on a Thermo Nicolet Nexus, SmartOrbit spectrometer using the KBr disc method. Ther-mogravimetric analysis was carried out using a Metter-Toledo 851e instrument (Switzerland) with a heating rateof 10 �C/min, in 150 �L alumina crucibles and in the rangeof 30 �C–900 �C. Scanning Electron Microscopy was usedto observe the surface morphology of the samples usinga NOVA™ Nano SEM 230 (FEI, Hillsboro, OR) scan-ning electron microscope. The zeta potential was measuredat room temperature by dynamic light scattering (DLS),using a Malvern Zetasizer Nano ZS (Malvern Instruments,Malvern, UK). The mean particle size of the samples wereobtained using a transmission electron microscope (HitachiH-7100, Tokyo, Japan) at an accelerating voltage of 80 and200 kV. UV-Vis spectra were measured to determine thecontrolled release study, using an ultraviolet-visible spec-trophotometer Shimadzu UV-1601 at Isra University.

3. RESULTS AND DISCUSSION3.1. X-ray Diffraction (XRD)Figure 1(A and B) shows the XRD patterns of Alg-AgNPsand EA-Alg-AgNPs nanocomposite, respectively. TheXRD peaks for Alg-AgNPs and EA-Alg-AgNPs nanocom-posite in the 30–85 degrees angle range ascertained thatthe peaks in 38.28�, 44.34�, 64.64�, 77.51� and 81.88� canbe attributed to the 111, 200, 220, 311 and 222 plans ofsilver nano-crystalline, respectively. These peaks can bereadily indexed to a face-centered cubic structure of sil-ver (XRD for Ag with Ref. No. 00-087-0719).6 Two peakswas observed at 31.67� and 45.14� in Figure 1, indicatespresence of Ag2O as impurities.26 However, the diffractionpeaks of EA-Alg-AgNPs nanocomposite are broader andlower intensity comparing with Alg-AgNPs.

30 40 50 60 70 80

♦

♦

222

311

220200

A

B

111

Inte

nsity

/a.u

.

2θ/degrees

Fig. 1. Powder X-ray diffraction patterns of the Alg-AgNPs (A) andEA-Alg-AgNPs nanocomposite (B).

The theoretical value of lattice constant “a” for facecubic center of silver can be calculate using Eq. (1)

a= 4/√2× r (1)

Where r for silver was 144 pm. By using the aboveequation, the lattice constant “a” was 4.073 Å. Theexperimental lattice constant ‘a’ was calculated from themost intense peak (111) of the XRD pattern is 4.078 Å.Both theoretical and experimental lattice constant ‘a’ arein very well agreement.The average particle size (L) of silver nanoparticles can

be calculated using Debye-Scherrer equation:

L= 0�94�� cos

(2)

Where 0.94 value is the Scherrer constant, � is the X-raywavelength (1.5418 Å), � is broadening of diffraction linemeasured at half of its maximum intensity (in radian),and is Bragg diffraction angle (in degree). From theScherrer equation, the average particle size of Alg-AgNPsand EA-Alg-AgNPss nanocomposite was 8.4 and 16.2 nm,respectively.

3.2. Infrared Spectroscopy (FTIR)Figure 2(A–C) shows the FTIR spectra of Alg-AgNPs, EAand EA-Alg-AgNPs nanocomposite, respectively. Fromthe literature, sodium alginate given bands at 1609 and1326 cm−1, this is due to asymmetry and symmetrystretching COO−. The absorption band at 1083 cm−1 iscorresponded to C–O–C stretching mode from the gluco-sidic units.27 In the case of Alg-AgNPs (Fig. 2(A)), thebands of asymmetry and symmetry COO− was shifted to

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3600 1500 1000 500

750

1050

1193

1695

758

105811

96

1511

1620

1700

3557

1081

1789

1326

C

B

A

1598

% T

rans

mitt

ance

Wavenumber/cm–1

Fig. 2. FT-IR spectrum of the Alg-AgNPs (A), free EA (B) andEA-Alg-AgNPs nanocomposite (C).

1598 and 1326 cm−1, respectively. The peak of 1083 cm−1

shift to 1081 cm−1 indicating that carboxyl groups areinvolved in the synthesis and stabilization of AgNPs.The appearance of carbonyl functionality after formationof AgNPs from reaction of sodium alginate with silver

Fig. 3. Schematic representation of the interaction between EA, alginate, and AgNPs in the EA-Alg-AgNPs nanocomposite.

(A)

0

50000

100000

150000

200000

250000

–200 –100 0 100 200

Tota

l Cou

nts

Zeta Potential (mV)

Zeta Potential Distribution

(B)

0

100000

200000

300000

400000

–200 –100 0 100 200

Tota

l Cou

nts

Zeta Potential (mV)

Zeta Potential Distribution

(C)

0

100000

200000

300000

400000

–200 –100 0 100 200To

tal C

ount

s

Zeta Potential (mV)

Zeta Potential Distribution

Fig. 4. Zeta potential measurements of Alg-AgNPs (A), EA (B) andEA-Alg-AgNPs nanocomposite (C).

nitrate can be clearly visualized by the appearance of anew band at 1789 cm−1 in the FTIR spectrum of Alg-AgNPs (Fig. 2(A)).28 This indicates the presence of a six-membered cyclic ketone.

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The FTIR spectrum of EA at Figure 2(B) shows anabsorption band at 3557 cm−1, attributed to the stretch-ing mode of OH groups in the phenol.29 A broad absorp-tion bands between 3500–2750 cm−1 due to stretchingof C–H aromatic ring and hydrogen bond between EAmolecules.30 A band at 1700 cm−1 can be attributed tostretching of C O.29�31 Bands at 1620–1511 cm−1 are dueto aromatic rings. A band at 1196 and 1058 cm−1 is dueto ester linkage of C–O stretching29�32 and another band at758 cm−1 is due to C–H aromatic stretching.

FTIR spectrum of EA-Alg-AgNPs nanocomposite(Fig. 2(C)) shows characteristic bands of pure EA togetherwith other band of Alg-AgNPs. This indicates that the EAhas been loaded on the surface of Alg-AgNPs. A bandfor C O slightly shifted in position to 1695 whereas theC–O ester linkage shifted to 1193 and 1050 cm−1 and theC–H aromatic rings bands was shifted to 750 cm−1.

In the producer of AgNPs, sodium alginate moleculesplayed the roles of both reducing and stabilizing agents.When silver nitrate was mixed with sodium alginate solu-tion, silver ions could be bound to alginate moleculesthrow ion–dipole interactions. The dipole was due to theelectron-rich oxygen atoms of polar hydroxyl groups ofalginate. The development of carbonyl functional groupafter reaction of sodium alginate with silver nitrateindicates the presence of a six-membered cyclic ketone.

(A)

5 6 7 8 9 10 11 12 13 14 15 160

3

6

9

12

15

18

21

24

27

30

Fre

quen

cy (

%)

Diameter (nm)

(B)

5 6 7 8 9 10 11 12 13 14 15 16 17 180

3

6

9

12

15

18

Fre

quen

cy (

%)

Diameter (nm)

Fig. 5. TEM images of Alg-AgNPs (A), and EA-Alg-AgNPs (B).

This formation process takes by oxidation of secondaryOH groups present in alginate skeleton. At same time,the silver ions undergo reduction to form AgNPs. There-fore, the electron-rich oxygen atoms in the hydroxyl andether groups of alginate bind tightly with metal clustersand nanoparticles via electrostatic interactions as shown inFigure 3.33 The EA molecules as shown in Figure 3 wasbind with alginate molecules throw hydrogen bonds.

3.3. Determination of the Zeta Potential and SizeDistribution Properties

Figures 4(A–C) shows the zeta potential measurement ofthe Alg-AgNPs, EA and EA-Alg-AgNPs nanocompositedissolved in deionized water, respectively. The zeta poten-tial for Alg-AgNPs, EA and EA-Alg-AgNPs were −24.0,−15.7 and −8.2 mV, respectively. The negative zeta poten-tial of Alg-AgNPs suggest that the surface charge ofthe nanoparticles was dominated by the adsorption layerof alginate, which preventing the Ag nanoparticles fromaggregation through electrostatic repulsion. This result wasvery clear at Figure 5(A). Loaded EA on the surfaceof Alg-AgNPs decreased the negative zeta potential from−24.0 mV to −8.2 mV, therefore the EA-Alg-AgNPsnanocomposite have more aggregation comparing withAlg-AgNPs.

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(A) (B)

Fig. 6. Scanning electron microscopy images of Alg-AgNPs (A), and EA-Alg-AgNP nanocomposite (B).

Figures 5(A and B) shows the TEM morphologies ofAlg-AgNPs, and EA-Alg-AgNPs nanocomposite, respec-tively. The average size of the particles is 10 nm for bothsamples.

3.4. SEM AnalysisFigures 6(A and B) show the SEM images of the Alg-AgNPs and EA-Alg-AgNPs nanocomposite at 10,000×magnification, respectively. Figure 6(A) shows sheets mor-phology, where the morphology of the nanocomposite wasvery different than the Alg-AgNPs. The SEM images forEA-Alg-AgNPs given rod shaped (Fig. 6(B)).

3.5. Thermogravimetric Analysis (TGA)The thermogravimetric analysis thermograms (TGA) offree EA, Alg-AgNPs and EA-Alg-AgNPs nanocompositewere shown at Figure 7. From the literature, the TGAof sodium alginate shows two steps of weight loss. Thefirst weight loss occurred in the temperature range of106–190 �C due to loss of volatile products like dehy-dration. The second-stage of weight loss occurs in the

200 400 600 800

20

40

60

80

100

% W

eigh

t los

s

Temperature (ºC)

EAAlg-AgNPsEA-Alg-AgNPs

Fig. 7. TGA curves are shown free EA, Alg-AgNPs, and EA-Alg-AgNPs nanocomposite.

range of 219–261 �C, which attributed to the decrosslink-ing of polymer networks, formation of a carbonaceousresidue, and finally yields Na2CO3 as char.23 TGA curveof Alg-AgNPs was given in Figure 7. For the peak below100 �C, this is concomitant by 10% weight loss due todesorption of moisture between Ag clusters and alginatebilayer. The dominant weight loss of the sample occurredin temperature region between 190 and 500 �C. It is gen-erally attributed to the evaporation of water and alginateouter layer component. The third step of 16.6% weight-loss in above 500 �C attributed to alginate inner monolayerdecomposition.34–37 The overall, TGA results show a lossof 57.5%.TGA thermogravimetric analysis obtained for EA shows

three weight losses. The first step weight loss can beattributed to the removal of bonded water with hydrogenbond at temperature maxima of 112 �C (8.2%). The sec-ond and third of weight losses of EA occurs at 463 �C(39%) and 596 �C (17.5%).As shown by the TGA curve for the EA-Alg-AgNP

nanocomposite (Fig. 7), the mass loss below 100 �C

0 50 100 150 200 250 3000

20

40

60

80

100

0 2 4 6 8 10 12 140

20

40

60

80

100

% R

elea

se

Time (min)

% R

elea

se

Time (min)

Fig. 8. Release profiles of the ellagic acid from the EA-Alg-AgNPnanocomposite at pH 7.4. The inset provides the release profiles of ellagicacid from the physical mixture of EA, Alg and AgNPs at pH 7.4.

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0 100 200 3000

1

2

3

4

5 Pseudo-firstorder

R2 = 0.9600

ln(q

e-qt

)

Time (min)0 100 200 300

0

1

2

3

Pseudo-secondorder

R2 = 0.9847

t/qt

Time (min)

0 5 10 150

20

40

60

80

100

Higuchi model R2 = 0.9296

% R

elea

se

t^0.50 100 200 300

0

1

2

3

4

5

Hixson modelR2 = 0.9956

Time (min)

(Am

p re

mai

ning

)1/3

(D)(C)

(A) (B)

0.0 0.5 1.0 1.5 2.0 2.5 3.00.0

0.4

0.8

1.2

1.6

2.0

Korsmeyer-peppasmodel

R2 = 0.9767

log

(% R

elea

se)

log (t)

(E)

Fig. 9. Data fitting for ellagic acid release from EA-Alg-AgNP nanocomposite using five different kinetic models at pH 7.4.

was low (2.1%) due to the removal of the absorbedphysical and chemical water. The nanocomposite beganto degrade at approximately 100 �C, and final decom-position occurred at approximately 900 �C, 79% totalmass loss. The mass loss for the Alg-AgNP was lowerthan for the EA-Alg-AgNP nanocomposite; this resultconfirms that the EA was loaded onto the Alg-AgNPssurface.

3.6. In Vitro Study of Ellagic Acid Release from theEA-Alg-AgNP Nanocomposite

The loading percentage of EA-Alg-AgNP nanocompos-ite was analyzed using a UV-Vis spectrophotometer andthe data were calculated using calibration curve. The datashow that the EA-Alg-AgNP nanocomposite is composedof 21%. The sustained release of EA from the nanocom-posite into the release media was performed in 0.01 mol/L

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Table I. The correlation coefficients (R2) obtained by fitting the ellagic acid anion release data from the EA-Alg-AgNP nanocomposite in PBSsolutions at pH 7.4.

R2

Samples Saturation release (%) Pseudo-first order Pseudo-second order Higuchi model Hixson-Crowell model Korsmeyer-Peppas model

EA-Alg-AgNP 100 0.9600 0.9847 0.9296 0.9956 0.9767

concentration pH 7.4 of sodium saline solution. The affin-ity of anions in the aqueous saline solution is higher thanthe EA towards the nanocomposite; therefore, EA anionscan be ion-exchanged with the anions in saline solu-tion. Generally, the release rate is initially rapid, and thenslows until it reaches equilibrium. Maximum release wasachieved at about 300 min. The physical mixture of EAand Alg-AgNPs exposed to pH 7.4 environments showedthe release of EA very quickly, within 14 minutes (inset forFig. 8). The release rate of EA from the nanocompositewas slower than from the physical mixture, indicating thatthe nanocomposite has the potential to be used for thecontrolled release of antimicrobial agents.

3.7. Release Kinetics of Ellagic Acid Anions from theEA-Alg-AgNP Nanocomposite

The data of the cumulative release of the EA fromnanocomposite was fitted to five kinetic models which gen-erally are described as follows:1. The pseudo-first-order kinetic model represented in itslinear form as Eq. (3)38

ln�qe−qt�= ln qe−k1t (3)

in which qe and qt are the quantity released at equilib-rium and the quantity released at any time (t), respectively,and k1 is the rate constant of the pseudo-first-order releasekinetics.2. The pseudo-second-order kinetic equation may be rep-resented in its linear form as Eq. (4)39

t/qt = 1/k2q2e + t/qe (4)

in which k2 is the rate constant of the pseudo-second-orderrelease kinetics.3. The Higuchi model describes the increased release ofthe drug from the nanocomposites with the increasing thesquare root of time40

qt = KH

√t (5)

in which kH is the Higuchi rate constant.4. The Hixson-Crowell model gives the relationshipbetween the cube root of the percentage of drug remainingin the nanocomposites as a function of time40

3√Mo− 3

√qt = Kt (6)

in which Mo is the initial quantity of drug in the nanocom-posite and qt is the quantity released at time t.

5. The Korsmeyer-Peppas model gives the relationshipbetween the log of percentage of drug released and the logof time40

qtq�

= Ktn (7)

in which q� is the release at infinite time.

Among the models used in this work, the correlation coef-ficient (R2) for the Hixson-Crowell kinetic model gave thebest fit with value 0.9956, as shown in Table I.

0

20

40

60

80

100

120

Untra

ted

128643216842

% V

iabi

lity

Concentration (µg/ml)

Alg-AgNPs EA EA-Alg-AgNPs(A)

0

20

40

60

80

100

120

Untra

ted

128643216842

% V

iabi

lity

Concentration (µg/ml)

Alg-AgNPs EA EA-Alg-AgNPs(B)

Fig. 10. The cytotoxicity profiles for the EA, Alg-AgNP and EA-Alg-AgNPs after incubation with 3T3 (A), and MCF-7 (B) cells.

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3.8. Cytotoxicity StudiesThe cytotoxicity of free EA, Alg-AgNP and EA-Alg-AgNPs nanocomposites on 3T3 and MCF-7 cells wasshown at Figure 10. In this study, coated AgNPs withalginate (Alg-AgNPs) did not show enough cytotoxic-ity against 3T3, this result was reported from the lit-erature [41]. The highest concentration of Alg-AgNPs(128 �g mL−1) showed 81.5% viability. Whereas free EAand nanocomposite given 49.5 and 45.3% viability, respec-tively (Fig. 10(A)).42

The MCF-7 cells were highly sensitive to free EA andEA-Alg-AgNPs nanocomposite compared to 3T3. A deeperlook at Figure 10(b) makes it clear that the EA-Alg-AgNPsnanocomposite have a higher tumor suppression effi-ciency compared to EA itself (IC50 values for EA andEA-Alg-AgNPs were 21.4 and 10.5 �g mL−1, respec-tively). The cell viability of the free EA at 128 �g mL−1

shows 33.5%. However, the EA-Alg-AgNPs nanocompos-ite shows a rapid decrease in cell viability as the con-centration increases. The maximum suppression effect ofEA-Alg-AgNPs nanocomposite was observed at the con-centration of 128 �g mL−1 with 15.3% viability.

4. CONCLUSIONThe EA-Alg-AgNPs nanocomposite was prepared by load-ing Ellagic acid on the surface of Alg-AgNPs. Therelease of the drug Ellagic acid from nanocompositewas followed Hixson-Crowell kinetic model. The EAand EA-Alg-AgNPs nanocomposite exhibit MCF-7 cellsin a dose-dependent manner with IC50 value 21.4 and10.5 �g mL−1, respectively.

Declaration of Conflicting InterestsThe authors report no conflicts of interest in this work.

Acknowledgment: The Ministry of Higher Educationof Malaysia (MOHE) provided funding for this researchunder grant No. GP-IPB/2013/9425800. The Isra Univer-sity providing funding for the kinetic studies.

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