Research ArticleChitosan-Alginate Nanoparticle System Efficiently DeliversDoxorubicin to MCF-7 Cells

Nuwanthi P Katuwavila12 A D L Chandani Perera13

Sameera R Samarakoon4 Preethi Soysa5 Veranja Karunaratne23

Gehan A J Amaratunga26 and D Nedra Karunaratne13

1Postgraduate Institute of Science University of Peradeniya 20400 Peradeniya Sri Lanka2Sri Lanka Institute of Nanotechnology Mahenwatta Pitipana 10200 Homagama Sri Lanka3Department of Chemistry Faculty of Science University of Peradeniya 20400 Peradeniya Sri Lanka4Institute of Biochemistry Molecular Biology and Biotechnology University of Colombo90 Cumarathunga Munidasa Mawatha 00300 Colombo 3 Sri Lanka5Department of Biochemistry and Molecular Biology Faculty of Medicine University of Colombo 25 Kynsey Road00800 Colombo 8 Sri Lanka6Electrical Engineering Division Department of Engineering University of Cambridge 9 JJ Thomson AvenueCambridge CB3 0FA UK

Correspondence should be addressed to Nuwanthi P Katuwavila nanukanpyahoocom

Received 12 May 2016 Revised 22 July 2016 Accepted 28 July 2016

Academic Editor Yasuhiko Hayashi

Copyright copy 2016 Nuwanthi P Katuwavila et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

A chitosan-alginate nanoparticle system encapsulating doxorubicin (DOX) was prepared by a novel ionic gelation method usingalginate as the crosslinker These nanoparticles were around 100 nm in size and more stable with higher positive zeta potentialand had higher encapsulation efficiency (95) than DOX loaded chitosan nanoparticles (DOX Csn NP) crosslinked withsodium tripolyphosphate (STPP) FTIR spectroscopy and thermogravimetric analysis revealed successful loading of DOX In vitrodrug release showed an initial release phase followed by slow release phase with higher cumulative release obtained with DOXloaded chitosan-alginate nanoparticles (DOX Csn-Alg NP) The in vitro cytotoxicity of DOX released from the two nanoparticlesystems showed a notable difference on comparison with that of free DOX on the MCF-7 cell line The SRB assay AOEB stainingand fluorescence uptake study indicated that free DOX only showed dose dependent cytotoxicity whereas both dose and timedependency were exhibited by the two sets of NPs While both systems show sustained release of DOX from the cell viability plotsDOX Csn-Alg NPs showed their superiority over DOX Csn NPsThe results obtained are useful for developing DOX Csn-Alg NPsas a sustained release carrier system for DOX

1 Introduction

Doxorubicin (DOX) with the trade name Adriamycinalso known as hydroxydaunorubicin and hydroxydauno-mycin is a drug used in cancer chemotherapy and derivedby chemical semisynthesis from a bacterial species [1] DOXan anthracycline antibiotic has been used for treatment ofleukemias Hodgkinrsquos lymphoma bladder breast stomachlung ovarian and thyroid cancers soft tissue sarcoma andmultiplemyeloma [2]The clinical usage ofDOX is associatedwith increased risk of cardiotoxicity which in turn leads to

congestive heart failure [3] In order to increase the anticancerefficacy by prolonging the circulation time in blood streamand reduce its toxicity different carrier systems for DOXhave been developed such as liposomes [4] and polymericnanoparticles [5] The primary goal of using nanotechnologyin medicine is to develop new safety delivery systems havingreduced toxicity and biocompatibility while maintainingtherapeutic effects [6]

At present it has become customary to use naturallyoccurring polymers as source materials for drug delivery[7] Nanoparticles of polymers not only protect the bioactive

Hindawi Publishing CorporationJournal of NanomaterialsVolume 2016 Article ID 3178904 12 pageshttpdxdoiorg10115520163178904

2 Journal of Nanomaterials

material but also facilitate the control release of the material[8] Due to some inherent problems such as low encap-sulation efficiency and rapid leakage of entrapped drugsin liposomes usage of polymeric nanoparticles has gainedmore attention over liposomes [9] Polysaccharides of naturalorigin which offer a wide diversity in their structure andproperties due to their wide range of molecular weight andchemical composition [10] are extensively being researchedin the drug delivery field Further the unique propertiesof polysaccharides such as nontoxicity full biodegradabilitybiocompatibility high stability hydrophilicity and also thehigh abundance in nature and low cost in processing willincrease their usage in synthesizing drug delivery systems[11]

Among these naturally occurring polysaccharides thetwo chosen chitosan and alginate are polyelectrolyte poly-mers of opposite charges [12] They are very promising andhave been extensively exploited in the drug delivery field forcontrolled drug release [13 14] Chitosan a cationic linearnitrogenous polysaccharide composed of glucosamine and N-acetyl-glucosamine linked by (1rarr 4) 120573-glycosidic bonds isthe second most abundant polymer in nature after cellulose[15] It is a hydrophilic polymer obtained from deacetylationof aminoacetyl groups of chitin which is themain componentof the shells of crustaceans the cell walls of the fungi and thecuticle of insects [16] In addition to the main properties ofpolysaccharides such as biocompatibility and biodegradabil-ity [17] the bioadhesiveness of chitosan [18] which facilitatesthe ionic interaction of positively charged amino groupsof chitosan with negatively charged mucous layer [19] isaccountable for its usage as a promising matrix in pharma-ceutical industry Alginate is a hydrophilic anionic copolymerwhich is composed of alternating blocks of (1-4) linked 120573-D-mannuronic acid (M units) and 120572-L-guluronic acid (Gunits) obtained from natural sources such as cell walls andintercellular spaces of marine brown algae and bacteria [19]The wide pharmaceutical applicability of alginate dependson its ability to form hydrogels by chelating with divalentcations [20] The easy gelling property of both chitosan andalginate can be used to prepare polyelectrolyte complexes ofpolycations and polyanions [21] Chitosan-alginate polyioniccomplex is formed via the ionic interactions between theamine group of chitosan and the carboxylic group of alginatethrough ionic gelation [22] Since these interactions reducethe porosity of the complex it protects the encapsulant andslows the release effectively than either chitosan or alginatealone [23] The high solubility of chitosan in low pH isreduced by the poor solubility of alginate network at low pHwhile alginate is stabilized at high pH by chitosan which isless soluble at high pH [11]

In the present study a novel carrier system for DOXwas developedwith chitosan and alginate Several researchershave investigated the delivery properties of chitosan-alginateNPs and nanocomposites for a variety of drugs ThoughDOX in chitosan NPs have been investigated in variousaspects there is no reported literature on chitosan-alginateNP which was directly used as a delivery vehicle for DOXThere are several reports where chitosan and alginate havebeen utilized to prepare carriers to deliver DOX In one

method CMC-predoped porous CaCO3microparticles were

assembled in layered fashion by coating alternatively withchitosan and alginate [24] A second group had synthesizedchitosan-alginate capsules consisting of BSA in a gel capsulefor DOX delivery [25] There is another study in whichmesoporous silica nanoparticles were coated with chitosanand alginate to deliver DOX [26] In the method we reportwe have only used the two biopolymers chitosan and alginateto form nanoparticles of a very small size range using adifferent method following the ionic gelation technique Acomparison with DOX Csn NP revealed an increase inefficiency and potency of Csn-Alg NPs over that of Csn NPsThe cytotoxicity effect of these DOX encapsulated NPs versusfree DOX was investigated with the human breast cancer cellline MCF-7

2 Materials and Methods

21 Materials Chitosan (medium molecular weight dea-cetylation degree sim75) low viscosity sodium alginatesodium tripolyphosphate (STPP) and doxorubicin hydro-chloride (980) were purchased from Sigma-Aldrich Chem-ical Company (St Louis MO USA) All other reagents wereof analytical grade and used directly

MCF-7 cells were purchased fromATCCUSA PowderedDulbeccorsquos Modified Eagle Medium was purchased fromInvitrogen Life Technologies (Carlsbad CA USA) Fetalbovine serum (FBS) streptomycinpenicillin dimethyl sul-foxide (DMSO) agarose and trypsinEDTA were purchasedfrom Sigma Aldrich Chemical Company (St Louis MOUSA)

22 Preparation of DOX Loaded Chitosan-Alginate Nanopar-ticles DOX Csn-Alg NPs were prepared by a novel methodThe chitosan flakes were dissolved in 40mL of acetic acid(03 vv) and the resulting solution (2mgmL) was adjustedto pH 48 The alginate solution (1mgmL) was prepared bydissolving in distilled water at room temperature overnightand adjusting the pH to 52 The drug DOX (30 120583gmL)was stirred with alginate solution for 24 h to form the drug-alginate complex The chitosan solution was stirred withTween 80 (0310 g) at 60∘C for 2 h to obtain a homogeneousmixture and this solution was gradually dropped to thealginate-DOX complex over 1 h while stirring at a high speedThe stirring was continued for another half an hour andthen refrigerated overnightThe nanoparticle suspension wascentrifuged at 9000 rpm for 45min to obtain the nanoparticlepellet Five formulations were prepared by varying chitosancontent to obtain different weight ratios of chitosan toalginate of 05 1 06 1 08 1 1 1 and 2 1

For the comparison DOX Csn NP was prepared accord-ing to a method modified in literature [27] employing atwo-step method that is oil in water emulsion followed bycrosslinking

23 Characterisation of DOX Loaded Nanoparticles Theaverage particle size and size polydispersity of the NPs dis-persed in water were determined by dynamic light scattering

Journal of Nanomaterials 3

technique at 25∘C using a particle size analyzer (ZetasizerNano ZS Malvern Instruments UK) at a fixed scatteringangle of 90∘The zeta potential of nanoparticles wasmeasuredusing the zeta potential analyzer (ZetasizerNanoZSMalvernInstruments UK) All measurements were performed intriplicate Fourier Transform Infrared (FTIR) spectra ofchitosan sodium alginate unloaded nanoparticles andDOXloaded NPs were obtained with a Bruker Vertex 80 IR spec-trometer (Germany) at a resolution of 4 cmminus1 from 4000 to400 cmminus1 Thermal decomposition of chitosan sodium algi-nate unloaded nanoparticles andDOX loaded nanoparticleswere analyzed using a SDTQ600 thermogravimetric analyzer(TA Instruments USA) from 25∘C to 800∘Cusing a ramp rateof 10∘Cmin in air Nanoparticle morphology was examinedby electron microscopy For scanning electron microscopya drop of the particle suspension was placed on a cleanedstub and dried overnight and imaging was done followedby gold sputtering to the specimen by the scanning electronmicroscope (SEM SU6600 Hitachi Japan) operating at 5and 10 kV Further the morphology was recorded by high-resolution transmission electronmicroscopy (HR-TEM JEM2100 JEOL Japan) operated at accelerating voltage 200 kVThe samples prior to their analysis were placed on carboncoated copper grids and dried under ambient conditions

24 Determination of Encapsulation Efficiency Theamount of incorporated DOX in the NPs was determinedby measuring the fluorescent absorbance of DOX remainingin the supernatant (free DOX) after centrifugation ofthe nanoparticle mixture using a fluorometer (FluorologHoriba Japan) with a slit width of 5 nm and excitation andemission wavelengths at 468 nm and 550 nm respectivelyA calibration curve was performed with a series ofconcentrations to obtain the unknown DOX concentrationsThe percent encapsulation efficiency ( EE) was calculatedas follows

EE = Total DOX minus Free DOXTotal DOX

times 100 (1)

25 Evaluation of In Vitro DOX Release The release char-acteristics of DOX from polymeric NPs were studied indistilled water and phosphate buffered saline solution (PBS)(pH sim 74)The centrifuged nanoparticle pellet was dispersedin 5mL of PBS at pH 74 and trapped inside a dialysismembrane (cutoff molecular weight 35 kDa) and immersedin 25mL PBS The temperature of the setup was maintainedat 37∘C in the dark with mild agitation Aliquots (3mL)were withdrawn at predetermined time intervals and thefluorescence emission spectrum was recorded as describedpreviously The release medium was refreshed with 3mLof medium after each withdrawal The release experimentswere repeated three times and average data were reportedUsing the calibration curve the unknown concentrationswere calculated hence the cumulative release of DOX wasdetermined

26 Cell Culture and In Vitro Cytotoxicity Studies Thecytotoxicity activities of DOX Csn and DOX Csn-Alg NPs

against MCF-7 cells were compared to free DOX MCF-7 cells were grown in monolayer cultures in DulbeccorsquosModified Eagle Medium (DMEM) supplemented with 10fetal bovine serum and 50 IUmL penicillin and 50120583gmLstreptomycin Cells were maintained at 37∘C in 95 air5CO2atmosphere with 95 humidity Culture medium was

changed every 2-3 days Next the cells in exponential growthwere removed by trypsinization and seeded at 5 times 103 cellsper well into a 96-well plate and incubated for 24 h forattachment After the incubation period cells were exposedto both DOX loaded NPs (DOX concentration of 001 002005 10 40 and 80 120583gmL) and to free DOX with similarconcentrations for 24 48 and 72 h after incubation

Cell viability was assessed by sulforhodamine B (SRB)assay [28] and the results were expressed as a percentage ofcontrol values All the assays were performed in triplicate inthree different experiments

27 Morphological Observation and Apoptosis Induced byDOX Loaded Nanoparticles Apoptosis was identified mor-phologically by acridine orangeethidium bromide (AOEB)staining MCF-7 cells were seeded separately in 24-well platesat 5 times 104 cellsmLminus1 on cell culture treated coverslips for24 h in a humidified atmosphere at 37∘C in 5 CO

2to allow

cell adherence Cells were then treated with both types ofDOX loaded NPs (DOX concentration of 001 100 and800 120583gmL) and free DOX (001 100 and 800 120583gmL)Treated cells were incubated for 24 48 and 72 h and atthe end of each incubation process cells on the coverslipswere observed under an inverted phase contrast microscope(Olympus CKX41SF Japan) for morphological changes Cellswere then fixed with 4 formaldehyde in PBS at roomtemperature for 15min Cocktail of acridine orange-ethidiumbromide (AOEB) dyes in PBS containing 50mgmLminus1 eachwas added to the fixed cells on coverslips and incubatedfor 10min at room temperature in the dark Cells werethen visualized under a fluorescence microscope (OlympusBX51TRF Japan) Both assays were performed in triplicate inthree different experiments

28 Cellular Uptake of Nanoparticles MCF-7 cells wereseeded separately in 24-well plates at 5 times 104 cellsmLminus1 oncoverslips for 24 h in a humidified atmosphere at 37∘C in 5CO2to allow cell adherence Cells were then treatedwith both

DOX loaded NP systems (DOX concentration of 001 100and 800 120583gmL) and free DOX (001 100 and 800 120583gmL)Treated cells were incubated for 30min and 2 and 24 h andat the end of each incubation process cells were rinsed twicewith PBS and then fixed with 4 formaldehyde in PBS atroom temperature for 15mins The cells on the coverslipswere observed under the fluorescence microscope (OlympusBX51TRF Japan)

3 Results and Discussion

The main effort of this work was to develop a nanodeliverysystem for the drug DOX using natural biodegradable bio-compatible polysaccharides specially chitosan and alginate

4 Journal of Nanomaterials

Table 1 The size zeta potential and EE of DOX Csn and DOX Csn-Alg NPs All analyses were performed using the DOX loaded NPsprepared by using optimized formulations (chitosan STTP 2 1 and chitosan alginate 2 1)

System Average sizenm Zeta potentialmV Encapsulation efficiency Polydispersity indexDOX Csn NPs 100 plusmn 27 26 plusmn 3 65 plusmn 4 0391 plusmn 0048DOX Csn-Alg NPs 100 plusmn 35 35 plusmn 4 95 plusmn 4 0402 plusmn 0071Csn NPs 100 plusmn 25 30 plusmn 5 mdash 0352 plusmn 0046Csn-Alg NPs 100 plusmn 28 36 plusmn 3 mdash 0414 plusmn 0065

The feasibility of using Csn-Alg NPs as a delivery system forDOX was evaluated with comparison to DOX Csn NPs

31 Formation of DOX Loaded Polymeric Nanoparticles Thechitosan NPs are easily formed by the gelation with STPPvia electrostatic interactions At a pH around 48 the pro-tonated amino groups of chitosan interact electrostaticallywith negatively charged phosphate groups of STPP [29]The uncertainty of this encapsulation was the complexationof DOX into the positively charged chitosan since DOXalso exhibits a positive charge However 65 of DOX wasencapsulated into Csn NPs in this experiment overcomingthe charge repulsion Since Csn NPs have shown positiveresults for the DOX delivery an effort was taken to improvethe DOX delivery with the use of alginate polymer as wellThe polyelectrolyte complex Csn-AlgNPswas prepared usinga novel method Initially the chitosan droplet formationoccurred with the stirring with Tween 80 followed by thedroplet solidification by ionically crosslinking of the alginatesolution In aqueous solutions at pH around 4 to 55 theamine groups on chitosan became easily positively chargedAlginate on the other hand dissolved in a neutral pH solu-tionwhere the carboxylate groupswere negatively charged Inaqueous solutions with pH around 52 chitosan amino groupsinteracted with alginate carboxylate groups to form thehydrogel Since alginate is a negatively charged polymer thepositively chargedDOXwas complexed initially with alginateto obtain a higher loading of the drug to the nanoparticleTheformulation (chitosan sodium alginate 2 1) which gavean opalescent suspension with a positive zeta potential wasselected as the optimum ratio for the DOX encapsulationThis formulation resulted in 95 encapsulation efficiency ofDOX

32 Shape Size and Surface Charge of Doxorubicin LoadedNanoparticles The morphology of the polymeric NPs wasobserved by scanning electronmicroscope and TransmissionElectron Microscope and both NPs were below 20 nm andspherical in shape (Figures 1 and 2) Further the TEM imagesclearly showed the well separated well dispersed particlesAfter loading of DOX there was no noticeable change eitherin the shape or in the size in SEM and TEM images ofboth NPs In a study on carvacrol-loaded chitosan NPsKeawchaoon andYoksan have reportedTEM images showing40ndash80 nm sized NPs [27] In a previous study done by Liu etal the particle sizes of Csn-Alg NPs obtained from the trans-mission electron microscopy (TEM) were 20ndash50 nm [11]

A study by Motwani et al had shown the particle sizesof Csn-Alg NPs obtained from TEM to be around 63 nm[22] Thus using the above-mentioned method we were ableto obtain much smaller Csn-Alg NPs The sizes obtainedfrom DLS technique were larger (sim100 nm Table 1) than thesizes obtained from SEM and TEM since DLS measures thehydrodynamic diameter Also the larger particle sizemight bedue to the swellability of polymeric hydrogels in the solution[30] whereas electron microscopy shows the images of driedparticles

The zeta potentials of both DOX Csn and DOX Csn-Alg NPs show large positive values reflecting the stability ofthe colloidal suspensions (Table 1) Having positively chargedsurfaces is an added advantage when using NPs in drugdelivery since they are able to transfer easily through negativechannels in the cell membrane An increased zeta potentialvalue with DOX Csn-Alg NPs when compared to the DOXCsn NPs was caused by the higher amount of positivelycharged DOX encapsulation with Csn-Alg NPs than thatwith Csn NPs Neutralization of the charge on chitosan bythe strong negative charge of STTP versus alginate at theworking pH may have also contributed to the differences inzeta potential

33 Characterisation of DOX Loaded Nanoparticles Thesuccessful loading of DOX into the polymeric NPs wasconfirmed by FTIR and TGA techniques Chemical struc-tures of both reactants and products were analyzed by FTIRspectroscopy In the spectra of pure chitosan (Figure 3(b))characteristic peaks at 3449 cmminus1 for ndashNH

2and ndashOH stretch-

ing 1659 cmminus1 for ndashCO stretching and 1596 cmminus1 for ndashNH2

bending vibrations were observed Crosslinking of the aminegroup of chitosan with phosphate group of STPP depictsa clear shift in the peak at 3565 cmminus1 for ndashNH

2and ndashOH

stretching as shown in the spectrum of CsnNPs (Figure 3(c))On loadingDOX intoCsnNPs the FTIR spectra (Figure 3(e))show peak shifts resulting from hydrogen bonding in theabove modes that is 3578 cmminus1 for ndashNH

2and ndashOH stretch-

ing 1685 cmminus1 for ndashCO stretching and 1578 cmminus1 for ndashNH2

bending vibrations reflecting the successful loading of DOXinto the Csn NPs

In the spectra of pure chitosan the major vibrations ofndashNH2and ndashOH stretching peak appearing at 3449 cmminus1 had

shifted to 3565 cmminus1 in the spectra of Csn-Alg NPs andthe ndashCO stretching vibration at 1659 cmminus1 in pure chitosanhad also shifted to 1651 cmminus1 after interaction of the aminegroup of chitosan with the carboxylic group of alginate

Journal of Nanomaterials 5

(a) (b)

Figure 1 (a) Scanning electron microscopy image of Csn NP (b) Transmission electron microscopy image of Csn NP

(a) (b)

Figure 2 (a) Scanning electron microscopy image of Csn-Alg NP (b) Transmission electron microscopy image of Csn-Alg NP

(Figure 3(d)) Further the ndashNH2bending peak at 1596 cmminus1

disappeared due to binding with alginate to form the NPAfter loading DOX into the Csn-Alg NPs the ndashNH

2and

ndashOH stretching peak at 3565 cmminus1 and the ndashCO stretchingvibration at 1660 cmminus1 shifted to 3356 cmminus1 and to 1637 cmminus1respectively Further the appearance of a small peak at1542 cmminus1 for ndashNH

2bending after loading the DOX may

reflect the physical entrapment of DOX into the Csn-Alg NPs(Figure 3(f))

34 Thermal Behaviour of DOX Loaded Polymeric Nanopar-ticles Nanoparticles with and without DOX were analyzedby thermogravimetry to further evaluate the thermal stabilityand successful loading of DOX In the thermogram ofchitosan NPs a major one-step mass loss about 40 of totalweight was observed at a peaking temperature (inflectionpoint temperature) of 216∘C (Figure 4) Heating up to 800∘Cretained 35 of the weight of the nanoparticles from theresidual sodium ions of the STPP crosslinked Csn NPsSimilarly in DOX loaded chitosan nanoparticles a 50weight loss from 100ndash400∘C was observed in two steps dueto the combined decomposition of chitosan and DOX butthe peaking temperature of 30 weight loss had shiftedto 225∘C proving the loading of the DOX into chitosan

nanoparticles (Figure 4) A new peak with 12 weight lossin the temperature range 600ndash700∘C was observed with theDOXCsnNPs showing entrapment of DOX into the polymermatrix

The decomposition of unloaded Csn-Alg NPs had twomajor and two minor weight losses (Figure 4) The ther-mogram of DOX loaded NPs showed the same pattern ofweight loss as the unloaded particles However when thetemperature reached 350∘C 60 of weight loss was observedwith DOX loaded nanoparticles due to the decompositionof DOX complexed nanoparticles whereas it was only 50with unloaded nanoparticles Further the inflection pointtemperature of the second major step of weight loss ofunloaded composite in the range of 600ndash750∘C peaked at703∘C whereas in the DOX loaded nanocomposite it peakedat 750∘C reflecting the strong physical entrapment of DOXinto the nanoparticles

35 In Vitro Release Kinetics of DOX from PolymericNanoparticles The in vitro release profiles of both optimizedformulations in phosphate buffered saline (PBS) solution(pH-74) are shown in Figure 5 An initial burst release ofDOX from both NPs in PBS solution can be observed upto 24 h This accounts for about 40 of DOX from the totalencapsulated amount Then it followed a more gradual and

6 Journal of Nanomaterials

(c)

(b)

(a)

(d)

(e)

(f)

Wave number (cmminus1)

Tran

smitt

ance

(arb

)

5001000150020002500300035004000

Figure 3 FTIR spectra of (a) pure alginate (b) pure chitosan (c)CsnNPprepared using 2 1weight ratio of chitosan to STPP (d)Csn-Alg NP prepared using 2 1 weight ratio of chitosan to alginate (e)DOX Csn NP and (f) DOX Csn-Alg NP

Temperature (∘C)8007006005004003002001000

0

10

20

30

40

50

60

70

80

90

100

Wei

ght (

)

Csn NPDOX Csn NP

Csn-Alg NPDOX Csn-Alg NP

Figure 4 TGA thermograms of Csn NP DOX Csn NP preparedusing 2 1 weight ratio of chitosan to STPP and Csn-Alg NP andDOX Csn-Alg NP prepared using 2 1 weight ratio of chitosan toalginate

sustained release phase for the next 72 h Both profiles hadfollowed the same pattern of release but the amounts weredifferent The total amount of drug released from Csn-AlgNPs was around 60 In contrast the total amount of drugreleased from Csn NPs was around 45These results clearlyindicated that the release of DOX was retarded significantlydue to the encapsulation in nanoparticles

36 In Vitro Cytotoxicity of DOX Loaded Polymeric Nanopar-ticle Dose response curves were plotted for 3 formulations

Time (hour)

(a)

(b)

100806040200

10

20

30

40

50

60

70

Cum

ulat

ive r

elea

se (

)

Figure 5 Cumulative release of DOX from (a) DOX Csn-Alg and(b) DOXCsnNP at preselected time intervals in pH 74 PBS Resultswere reported as mean plusmn SD 119899 = 3

Free DOXDOX loaded chitosan-alginate NPDOX loaded chitosan NP

2 4 6 80

Concentration (120583gmL)

0

10

20

30

40

50

60

70

80

90

100Pe

rcen

tage

cell

viab

ility

()

Figure 6 Cytotoxicity of free DOX DOX Csn-Alg NP and DOXCsn NP against MCF-7 cells after e24 ◼48 and 99877172 hours oftreatments Cell survival was assessed via SRB assay Results werereported as mean plusmn SD 119899 = 4

free DOX DOX Csn-Alg NPs and DOX Csn NPs (Figure 6)Each experimental curve represents the average of a seriesof 3 experiments With free DOX the inhibition of MCF-7cell proliferation completely depends on the dose of thedrug used After 48 and 72 h 50 cell viability could not beobserved even at the lowest concentration (001120583gmL) offree DOX proving the reduced effect of free DOX after 24 hthat is the free DOX has dose dependent cytotoxicity thus itmight damage the normal cells during therapy whereas thecurrent work demonstrated the dose-dependence as well as

Journal of Nanomaterials 7

Free DOX

Control

Control

Control

DOX Csn NPDOX Csn-Alg NP24 h

48 h

72 h

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

Figure 7 Morphological changes of apoptosis in MCF-7 cells observed under inverted light microscopy exposed to different concentrationsof free DOX DOX Csn-Alg NP and DOX Csn NP after 24 h 48 h and 72 h after incubation

8 Journal of Nanomaterials

Free DOX

Control

Control

Control

DOX Csn NPDOX Csn-Alg NP24 h

48 h

72 h

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

Figure 8 Fluorescence microscopic observations of MCF-7 cells treated with different concentrations of free DOX DOX Csn-Alg NP andDOX Csn NP stained with AOEB after 24 h 48 h and 72 h after incubation

Journal of Nanomaterials 9

Free DOX

Control

Control

Control

DOX Csn NPDOX Csn-Alg NP

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

30min

24 h

2 h

Figure 9 Fluorescence microscopic observations of cellular uptake of MCF-7 cells treated with different concentrations of free DOX DOXCsn-Alg NP and DOX Csn NP after 30min 2 h and 24 h after incubation

10 Journal of Nanomaterials

the time dependence cytotoxicity of DOX loaded NPs withMCF-7 cell line None of the concentrations of DOX in bothNPs that were used caused 50 inhibition of cell growthat 24 h reflecting the slow release from NPs Hence duringthe first 24 h those NPs are capable of circulating in theblood and accumulating at tumour sites before starting theircytotoxic effect Thus DOX encapsulated in NPs achieve itscytotoxicity with time that is there is an effect of both timeand dose on the inhibition of cell proliferation Thereforethis result clearly indicated that the release of DOX wasretarded significantly by the encapsulation in NPs Whencomparing the two delivery systems the cytotoxicity effectof DOX Csn NPs was consistently lower than that of DOXCsn-Alg NPs (Figure 6)This is in accordance with the resultsof in vitro release of DOX being higher from Csn-Alg NPsthan from Csn NPs after 72 h (Figure 5) However bothDOX loaded systems exhibited similar behaviour with theMCF-7 cell line (Figure 6) Same concentration of DOXwhen encapsulated in NPs behaves differently than in thefree state that is encapsulated DOX releases slowly andgradually increases the concentration in the medium Henceit proves that encapsulating DOX in either system hasimproved the efficacy of DOX over free DOX A similarstudy of doxorubicin loaded magnetic NPs grafted to smartcopolymers showed that free DOX has dose dependent butnot time dependent cytotoxicity but DOX loaded magneticNPs have time dependent cytotoxicity [31]

Inverted phase contrast microscopy was used for mor-phological analysis of MCF-7 cells for observation of apop-tosis induced by the DOX loaded NPs and free DOXThese characteristicmorphological changes of apoptosis withtime and dose in MCF-7 cells on treatment after 24 48and 72 h (Figure 7) were clearly visualized Exposure tofree DOX clearly showed a higher cell confluence at lowdoses while higher concentration of free DOX decreased cellconfluence and showed a higher proportion of rounded cellsresulting from detached cells from the culture monolayerWithin the first 24 h most of the cells were damaged bythe induced cytotoxicity of free DOX reflecting its dosedependent cytotoxicity On the other hand bothDOX loadedsystems showed disappearance of the high cell confluencewith the dose of the drug and with time (Figure 7) Thisobservation is further confirmed by the fluorescence micro-scopic results of AOEB stained MCF-7 cells after 24 48and 72 h of incubation following the treatment with differentconcentrations of freeDOXandwith the same concentrationsloaded in Csn and Csn-Alg NPs Nuclei of viable cells werestained uniformly bright green by AO while the apoptoticcells exhibited yellow to orange coloration depending on thedegree of loss of membrane integrity due to costaining withEB (Figure 8) Early apoptotic cells have greenish yellownuclei while late apoptotic cells have orange to red nucleiwith condensed or fragmented chromatin (Figure 8) Thesefigures clearly displayed the difference in behaviour of DOXwhen encapsulated in NPs Free DOX has a dose dependentincrease in induction of apoptosis (Figure 8-24 h) Both thedose and time dependency of DOX encapsulated in NPs are

evident from the alterations in cell staining in all the threeseries (Figure 8) In the case of DOX loadedNPs the drug hasto be released slowly from theNPs before it can exert an effecton the cancer cells but free DOX has an immediate effecton the cells since it has direct contact with the cells initiallyThe mechanism of action of apoptosis of DOX loaded inNPs was not significantly different from that of the free DOXindicating that the encapsulation of DOX in NPs does notmake any changes to its mechanism of action of apoptosis

37 Cellular Uptake of Nanoparticles DOX is a fluorescenceactive compound having a bright red color The red colorindicates the cellular uptake of DOX when compared to theuntreated cells or cells with no internalized DOX Internal-ization of DOX in cancer cells was evaluated on MCF-7 cellsat different time intervals and different concentrations offree and encapsulated DOX Within 30min of incubationall concentrations of free DOX were localized within the cellnucleus (Figure 9 30min) whereas the localization of DOXin NPs could be visualized depending on the concentrationand on time (Figure 9) The efficient uptake of the NPs couldbe facilitated by the positive surface charge of the NPs Asimilar observationwas notedwith the in vitro cellular uptakeof paclitaxel loaded PEGylated PLGA-based NPs [32] Ingeneral NPs are nonspecifically internalized into cells viaendocytosis or phagocytosis while free DOXdiffuses throughthe cell membrane [33]

Morphology analysis suggests that the process of cellgrowth inhibition by DOX loaded in NPs occurs over a muchlonger time than by the free DOX From these observations itcould be inferred that the DOX in NPs sufficiently associateswith the cells with time

4 Conclusion

This study was made to assess the usefulness of the Csn-Alg NP system as a delivery agent for the anticancer drugDOX Comparison with DOX Csn NPs indicated that Csn-Alg NPs were more efficient at entrapment of DOX than theCsnNPsThis successful loadingwas confirmedby both FTIRand TGA data Both nanoparticles were less than 100 nm insize with positive zeta potentials The in vitro release profilesobserved for these NPs were characterized by a burst initialrelease followed by a more gradual and sustained releasephase The in vitro cytotoxicity studies demonstrated thatDOX loaded NPs have potent antigrowth effect on MCF-7cells and inhibited its cell growth in a dose and time depen-dent manner Further the observed cell apoptosis induced bythese DOX loaded NPs by phase contrast microscopy andfluorescence microscopy after AOEB staining proved thatthere is no significant change in the mechanism of action ofDOX after encapsulating in NPs and reflect the slow releaseof DOX from the NPs According to the cytotoxicity resultsDOX Csn-Alg NPs are more cytotoxic than DOX Csn NPs

Competing Interests

The authors declare that they have no competing interests

Journal of Nanomaterials 11

Acknowledgments

This research was financially supported by the HETC QIGWindow 3 Grant University of Peradeniya Peradeniya SriLanka The authors also thank Ms Damayanthi Dahanayakeat Sri Lanka Institute of nanotechnology for her valuablesupport in electron microscopy imaging

References

[1] N Lomovskaya S L Otten Y Doi-Katayama et al ldquoDoxoru-bicin overproduction in Streptomyces peucetius cloning andcharacterization of the dnrU ketoreductase and dnrV genesand the doxA cytochrome P-450 hydroxylase generdquo Journal ofBacteriology vol 181 no 1 pp 305ndash318 1999

[2] K M Laginha S Verwoert G J R Charrois and T MAllen ldquoDetermination of doxorubicin levels in whole tumorand tumor nuclei in murine breast cancer tumorsrdquo ClinicalCancer Research vol 11 no 19 pp 6944ndash6949 2005

[3] G Minotti P Menna E Salvatorelli G Cairo and L GiannildquoAnthracyclines molecular advances and pharmacologic devel-opments in antitumor activity and cardiotoxicityrdquo Pharmaco-logical Reviews vol 56 no 2 pp 185ndash229 2004

[4] M E R OrsquoBrien N Wigler M Inbar et al ldquoReduced car-diotoxicity and comparable efficacy in a phase III trial of pegy-lated liposomal doxorubicin HCl (CAELYXDoxil) versusconventional doxorubicin for first-line treatment of metastaticbreast cancerrdquo Annals of Oncology vol 15 no 3 pp 440ndash4492004

[5] C Zhang W Wang T Liu et al ldquoDoxorubicin-loaded gly-cyrrhetinic acid-modified alginate nanoparticles for liver tumorchemotherapyrdquo Biomaterials vol 33 no 7 pp 2187ndash2196 2012

[6] W H De Jong and P J A Borm ldquoDrug delivery andnanoparticles applications and hazardsrdquo International Journalof Nanomedicine vol 3 no 2 pp 133ndash149 2008

[7] G Vilar J Tulla-Puche and F Albericio ldquoPolymers and drugdelivery systemsrdquo Current Drug Delivery vol 9 no 4 pp 367ndash394 2012

[8] A Kidane and P P Bhatt ldquoRecent advances in small moleculedrug deliveryrdquo Current Opinion in Chemical Biology vol 9 no4 pp 347ndash351 2005

[9] K S Soppimath T M Aminabhavi A R Kulkarni and WE Rudzinski ldquoBiodegradable polymeric nanoparticles as drugdelivery devicesrdquo Journal of Controlled Release vol 70 no 1-2pp 1ndash20 2001

[10] T Coviello P Matricardi C Marianecci and F AlhaiqueldquoPolysaccharide hydrogels for modified release formulationsrdquoJournal of Controlled Release vol 119 no 1 pp 5ndash24 2007

[11] Z Liu Y Jiao Y Wang C Zhou and Z ZhangldquoPolysaccharides-based nanoparticles as drug deliverysystemsrdquo Advanced Drug Delivery Reviews vol 60 no 15 pp1650ndash1662 2008

[12] O Borges G Borchard J C Verhoef A De Sousa andH E Junginger ldquoPreparation of coated nanoparticles for anew mucosal vaccine delivery systemrdquo International Journal ofPharmaceutics vol 299 no 1-2 pp 155ndash166 2005

[13] Y Murata D Jinno D Liu T Isobe K Kofuji and SKawashima ldquoThe drug release profile from calcium-inducedalginate gel beads coated with an alginate hydrolysaterdquoMolecules vol 12 no 11 pp 2559ndash2566 2007

[14] P Li Y-N Dai J-P Zhang A-QWang andQWei ldquoChitosan-alginate nanoparticles as a novel drug delivery system fornifedipinerdquo International Journal of Biomedical Science vol 4no 3 pp 221ndash228 2008

[15] C K S Pillai W Paul and C P Sharma ldquoChitin and chitosanpolymers chemistry solubility and fiber formationrdquo Progress inPolymer Science vol 34 no 7 pp 641ndash678 2009

[16] F Khoushab and M Yamabhai ldquoChitin research revisitedrdquoMarine Drugs vol 8 no 7 pp 1988ndash2012 2010

[17] T Kean and M Thanou ldquoBiodegradation biodistribution andtoxicity of chitosanrdquo Advanced Drug Delivery Reviews vol 62no 1 pp 3ndash11 2010

[18] O Gaseroslashd I G Jolliffe F C Hampson P W Dettmar and GSkjak-Braeligk ldquoThe enhancement of the bioadhesive propertiesof calcium alginate gel beads by coating with chitosanrdquo Inter-national Journal of Pharmaceutics vol 175 no 2 pp 237ndash2461998

[19] M George and T E Abraham ldquoPolyionic hydrocolloids for theintestinal delivery of protein drugs alginate and chitosanmdashareviewrdquo Journal of Controlled Release vol 114 no 1 pp 1ndash142006

[20] A Saha R Yadav and N Rajendran ldquoBiomaterials fromsponges ascidians and other marine organismsrdquo InternationalJournal of Pharmaceutical Sciences Review and Research vol 27no 2 article no 17 pp 100ndash109 2014

[21] S De and D Robinson ldquoPolymer relationships duringpreparation of chitosan-alginate and poly-l-lysine-alginatenanospheresrdquo Journal of Controlled Release vol 89 no 1 pp101ndash112 2003

[22] S K Motwani S Chopra S Talegaonkar K Kohli F J Ahmadand R K Khar ldquoChitosan-sodium alginate nanoparticles assubmicroscopic reservoirs for ocular delivery formulationoptimisation and in vitro characterisationrdquo European Journal ofPharmaceutics and Biopharmaceutics vol 68 no 3 pp 513ndash5252008

[23] F-L Mi H-W Sung and S-S Shyu ldquoDrug release fromchitosan-alginate complex beads reinforced by a naturallyoccurring cross-linking agentrdquo Carbohydrate Polymers vol 48no 1 pp 61ndash72 2002

[24] Q Zhao B Han ZWang C Gao C Peng and J Shen ldquoHollowchitosan-alginate multilayer microcapsules as drug deliveryvehicle doxorubicin loading and in vitro and in vivo studiesrdquoNanomedicine Nanotechnology Biology and Medicine vol 3no 1 pp 63ndash74 2007

[25] H Shen H Shi M Xie et al ldquoBiodegradable chitosanalginateBSA-gel-capsules for pH-controlled loading and release ofdoxorubicin and treatment of pulmonary melanomardquo Journalof Materials Chemistry B vol 1 no 32 pp 3906ndash3917 2013

[26] W Feng W Nie C He et al ldquoEffect of pH-responsive algi-natechitosanmultilayers coating on delivery efficiency cellularuptake and biodistribution of mesoporous silica nanoparticlesbased nanocarriersrdquo ACS Applied Materials amp Interfaces vol 6no 11 pp 8447ndash8460 2014

[27] L Keawchaoon and R Yoksan ldquoPreparation characterizationand in vitro release study of carvacrol-loaded chitosan nanopar-ticlesrdquo Colloids and Surfaces B Biointerfaces vol 84 no 1 pp163ndash171 2011

[28] S R Samarakoon I Thabrew P B Galhena D De Silva andK Tennekoon ldquoA comparison of the cytotoxic potential of stan-dardized aqueous and ethanolic extracts of a polyherbalmixturecomprised ofNigella sativa (seeds)Hemidesmus indicus (roots)

12 Journal of Nanomaterials

and Smilax glabra (rhizome)rdquo Pharmacognosy Research vol 2no 6 pp 335ndash342 2010

[29] K Nagpal S K Singh and D N Mishra ldquoChitosan nanoparti-cles a promising system in novel drug deliveryrdquo Chemical andPharmaceutical Bulletin vol 58 no 11 pp 1423ndash1430 2010

[30] N Bhattarai J Gunn and M Zhang ldquoChitosan-based hydro-gels for controlled localized drug deliveryrdquo Advanced DrugDelivery Reviews vol 62 no 1 pp 83ndash99 2010

[31] A Akbarzadeh M Samiei S W Joo et al ldquoSynthesis charac-terization and in vitro studies of doxorubicin-loaded magneticnanoparticles grafted to smart copolymers on A549 lung cancercell linerdquo Journal of Nanobiotechnology vol 10 article 46 2012

[32] F Danhier N Lecouturier B Vroman et al ldquoPaclitaxel-loadedPEGylated PLGA-based nanoparticles in vitro and in vivoevaluationrdquo Journal of Controlled Release vol 133 no 1 pp 11ndash17 2009

[33] A des Rieux V FievezMGarinot Y-J Schneider andV PreatldquoNanoparticles as potential oral delivery systems of proteins andvaccines amechanistic approachrdquo Journal of Controlled Releasevol 116 no 1 pp 1ndash27 2006

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CeramicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CompositesJournal of

NanoparticlesJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Biomaterials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MetallurgyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

MaterialsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Nano

materials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal ofNanomaterials

2 Journal of Nanomaterials

material but also facilitate the control release of the material[8] Due to some inherent problems such as low encap-sulation efficiency and rapid leakage of entrapped drugsin liposomes usage of polymeric nanoparticles has gainedmore attention over liposomes [9] Polysaccharides of naturalorigin which offer a wide diversity in their structure andproperties due to their wide range of molecular weight andchemical composition [10] are extensively being researchedin the drug delivery field Further the unique propertiesof polysaccharides such as nontoxicity full biodegradabilitybiocompatibility high stability hydrophilicity and also thehigh abundance in nature and low cost in processing willincrease their usage in synthesizing drug delivery systems[11]

Among these naturally occurring polysaccharides thetwo chosen chitosan and alginate are polyelectrolyte poly-mers of opposite charges [12] They are very promising andhave been extensively exploited in the drug delivery field forcontrolled drug release [13 14] Chitosan a cationic linearnitrogenous polysaccharide composed of glucosamine and N-acetyl-glucosamine linked by (1rarr 4) 120573-glycosidic bonds isthe second most abundant polymer in nature after cellulose[15] It is a hydrophilic polymer obtained from deacetylationof aminoacetyl groups of chitin which is themain componentof the shells of crustaceans the cell walls of the fungi and thecuticle of insects [16] In addition to the main properties ofpolysaccharides such as biocompatibility and biodegradabil-ity [17] the bioadhesiveness of chitosan [18] which facilitatesthe ionic interaction of positively charged amino groupsof chitosan with negatively charged mucous layer [19] isaccountable for its usage as a promising matrix in pharma-ceutical industry Alginate is a hydrophilic anionic copolymerwhich is composed of alternating blocks of (1-4) linked 120573-D-mannuronic acid (M units) and 120572-L-guluronic acid (Gunits) obtained from natural sources such as cell walls andintercellular spaces of marine brown algae and bacteria [19]The wide pharmaceutical applicability of alginate dependson its ability to form hydrogels by chelating with divalentcations [20] The easy gelling property of both chitosan andalginate can be used to prepare polyelectrolyte complexes ofpolycations and polyanions [21] Chitosan-alginate polyioniccomplex is formed via the ionic interactions between theamine group of chitosan and the carboxylic group of alginatethrough ionic gelation [22] Since these interactions reducethe porosity of the complex it protects the encapsulant andslows the release effectively than either chitosan or alginatealone [23] The high solubility of chitosan in low pH isreduced by the poor solubility of alginate network at low pHwhile alginate is stabilized at high pH by chitosan which isless soluble at high pH [11]

In the present study a novel carrier system for DOXwas developedwith chitosan and alginate Several researchershave investigated the delivery properties of chitosan-alginateNPs and nanocomposites for a variety of drugs ThoughDOX in chitosan NPs have been investigated in variousaspects there is no reported literature on chitosan-alginateNP which was directly used as a delivery vehicle for DOXThere are several reports where chitosan and alginate havebeen utilized to prepare carriers to deliver DOX In one

method CMC-predoped porous CaCO3microparticles were

assembled in layered fashion by coating alternatively withchitosan and alginate [24] A second group had synthesizedchitosan-alginate capsules consisting of BSA in a gel capsulefor DOX delivery [25] There is another study in whichmesoporous silica nanoparticles were coated with chitosanand alginate to deliver DOX [26] In the method we reportwe have only used the two biopolymers chitosan and alginateto form nanoparticles of a very small size range using adifferent method following the ionic gelation technique Acomparison with DOX Csn NP revealed an increase inefficiency and potency of Csn-Alg NPs over that of Csn NPsThe cytotoxicity effect of these DOX encapsulated NPs versusfree DOX was investigated with the human breast cancer cellline MCF-7

2 Materials and Methods

21 Materials Chitosan (medium molecular weight dea-cetylation degree sim75) low viscosity sodium alginatesodium tripolyphosphate (STPP) and doxorubicin hydro-chloride (980) were purchased from Sigma-Aldrich Chem-ical Company (St Louis MO USA) All other reagents wereof analytical grade and used directly

MCF-7 cells were purchased fromATCCUSA PowderedDulbeccorsquos Modified Eagle Medium was purchased fromInvitrogen Life Technologies (Carlsbad CA USA) Fetalbovine serum (FBS) streptomycinpenicillin dimethyl sul-foxide (DMSO) agarose and trypsinEDTA were purchasedfrom Sigma Aldrich Chemical Company (St Louis MOUSA)

22 Preparation of DOX Loaded Chitosan-Alginate Nanopar-ticles DOX Csn-Alg NPs were prepared by a novel methodThe chitosan flakes were dissolved in 40mL of acetic acid(03 vv) and the resulting solution (2mgmL) was adjustedto pH 48 The alginate solution (1mgmL) was prepared bydissolving in distilled water at room temperature overnightand adjusting the pH to 52 The drug DOX (30 120583gmL)was stirred with alginate solution for 24 h to form the drug-alginate complex The chitosan solution was stirred withTween 80 (0310 g) at 60∘C for 2 h to obtain a homogeneousmixture and this solution was gradually dropped to thealginate-DOX complex over 1 h while stirring at a high speedThe stirring was continued for another half an hour andthen refrigerated overnightThe nanoparticle suspension wascentrifuged at 9000 rpm for 45min to obtain the nanoparticlepellet Five formulations were prepared by varying chitosancontent to obtain different weight ratios of chitosan toalginate of 05 1 06 1 08 1 1 1 and 2 1

For the comparison DOX Csn NP was prepared accord-ing to a method modified in literature [27] employing atwo-step method that is oil in water emulsion followed bycrosslinking

23 Characterisation of DOX Loaded Nanoparticles Theaverage particle size and size polydispersity of the NPs dis-persed in water were determined by dynamic light scattering

Journal of Nanomaterials 3

technique at 25∘C using a particle size analyzer (ZetasizerNano ZS Malvern Instruments UK) at a fixed scatteringangle of 90∘The zeta potential of nanoparticles wasmeasuredusing the zeta potential analyzer (ZetasizerNanoZSMalvernInstruments UK) All measurements were performed intriplicate Fourier Transform Infrared (FTIR) spectra ofchitosan sodium alginate unloaded nanoparticles andDOXloaded NPs were obtained with a Bruker Vertex 80 IR spec-trometer (Germany) at a resolution of 4 cmminus1 from 4000 to400 cmminus1 Thermal decomposition of chitosan sodium algi-nate unloaded nanoparticles andDOX loaded nanoparticleswere analyzed using a SDTQ600 thermogravimetric analyzer(TA Instruments USA) from 25∘C to 800∘Cusing a ramp rateof 10∘Cmin in air Nanoparticle morphology was examinedby electron microscopy For scanning electron microscopya drop of the particle suspension was placed on a cleanedstub and dried overnight and imaging was done followedby gold sputtering to the specimen by the scanning electronmicroscope (SEM SU6600 Hitachi Japan) operating at 5and 10 kV Further the morphology was recorded by high-resolution transmission electronmicroscopy (HR-TEM JEM2100 JEOL Japan) operated at accelerating voltage 200 kVThe samples prior to their analysis were placed on carboncoated copper grids and dried under ambient conditions

24 Determination of Encapsulation Efficiency Theamount of incorporated DOX in the NPs was determinedby measuring the fluorescent absorbance of DOX remainingin the supernatant (free DOX) after centrifugation ofthe nanoparticle mixture using a fluorometer (FluorologHoriba Japan) with a slit width of 5 nm and excitation andemission wavelengths at 468 nm and 550 nm respectivelyA calibration curve was performed with a series ofconcentrations to obtain the unknown DOX concentrationsThe percent encapsulation efficiency ( EE) was calculatedas follows

EE = Total DOX minus Free DOXTotal DOX

times 100 (1)

25 Evaluation of In Vitro DOX Release The release char-acteristics of DOX from polymeric NPs were studied indistilled water and phosphate buffered saline solution (PBS)(pH sim 74)The centrifuged nanoparticle pellet was dispersedin 5mL of PBS at pH 74 and trapped inside a dialysismembrane (cutoff molecular weight 35 kDa) and immersedin 25mL PBS The temperature of the setup was maintainedat 37∘C in the dark with mild agitation Aliquots (3mL)were withdrawn at predetermined time intervals and thefluorescence emission spectrum was recorded as describedpreviously The release medium was refreshed with 3mLof medium after each withdrawal The release experimentswere repeated three times and average data were reportedUsing the calibration curve the unknown concentrationswere calculated hence the cumulative release of DOX wasdetermined

26 Cell Culture and In Vitro Cytotoxicity Studies Thecytotoxicity activities of DOX Csn and DOX Csn-Alg NPs

against MCF-7 cells were compared to free DOX MCF-7 cells were grown in monolayer cultures in DulbeccorsquosModified Eagle Medium (DMEM) supplemented with 10fetal bovine serum and 50 IUmL penicillin and 50120583gmLstreptomycin Cells were maintained at 37∘C in 95 air5CO2atmosphere with 95 humidity Culture medium was

changed every 2-3 days Next the cells in exponential growthwere removed by trypsinization and seeded at 5 times 103 cellsper well into a 96-well plate and incubated for 24 h forattachment After the incubation period cells were exposedto both DOX loaded NPs (DOX concentration of 001 002005 10 40 and 80 120583gmL) and to free DOX with similarconcentrations for 24 48 and 72 h after incubation

Cell viability was assessed by sulforhodamine B (SRB)assay [28] and the results were expressed as a percentage ofcontrol values All the assays were performed in triplicate inthree different experiments

27 Morphological Observation and Apoptosis Induced byDOX Loaded Nanoparticles Apoptosis was identified mor-phologically by acridine orangeethidium bromide (AOEB)staining MCF-7 cells were seeded separately in 24-well platesat 5 times 104 cellsmLminus1 on cell culture treated coverslips for24 h in a humidified atmosphere at 37∘C in 5 CO

2to allow

cell adherence Cells were then treated with both types ofDOX loaded NPs (DOX concentration of 001 100 and800 120583gmL) and free DOX (001 100 and 800 120583gmL)Treated cells were incubated for 24 48 and 72 h and atthe end of each incubation process cells on the coverslipswere observed under an inverted phase contrast microscope(Olympus CKX41SF Japan) for morphological changes Cellswere then fixed with 4 formaldehyde in PBS at roomtemperature for 15min Cocktail of acridine orange-ethidiumbromide (AOEB) dyes in PBS containing 50mgmLminus1 eachwas added to the fixed cells on coverslips and incubatedfor 10min at room temperature in the dark Cells werethen visualized under a fluorescence microscope (OlympusBX51TRF Japan) Both assays were performed in triplicate inthree different experiments

28 Cellular Uptake of Nanoparticles MCF-7 cells wereseeded separately in 24-well plates at 5 times 104 cellsmLminus1 oncoverslips for 24 h in a humidified atmosphere at 37∘C in 5CO2to allow cell adherence Cells were then treatedwith both

DOX loaded NP systems (DOX concentration of 001 100and 800 120583gmL) and free DOX (001 100 and 800 120583gmL)Treated cells were incubated for 30min and 2 and 24 h andat the end of each incubation process cells were rinsed twicewith PBS and then fixed with 4 formaldehyde in PBS atroom temperature for 15mins The cells on the coverslipswere observed under the fluorescence microscope (OlympusBX51TRF Japan)

3 Results and Discussion

The main effort of this work was to develop a nanodeliverysystem for the drug DOX using natural biodegradable bio-compatible polysaccharides specially chitosan and alginate

4 Journal of Nanomaterials

Table 1 The size zeta potential and EE of DOX Csn and DOX Csn-Alg NPs All analyses were performed using the DOX loaded NPsprepared by using optimized formulations (chitosan STTP 2 1 and chitosan alginate 2 1)

System Average sizenm Zeta potentialmV Encapsulation efficiency Polydispersity indexDOX Csn NPs 100 plusmn 27 26 plusmn 3 65 plusmn 4 0391 plusmn 0048DOX Csn-Alg NPs 100 plusmn 35 35 plusmn 4 95 plusmn 4 0402 plusmn 0071Csn NPs 100 plusmn 25 30 plusmn 5 mdash 0352 plusmn 0046Csn-Alg NPs 100 plusmn 28 36 plusmn 3 mdash 0414 plusmn 0065

The feasibility of using Csn-Alg NPs as a delivery system forDOX was evaluated with comparison to DOX Csn NPs

31 Formation of DOX Loaded Polymeric Nanoparticles Thechitosan NPs are easily formed by the gelation with STPPvia electrostatic interactions At a pH around 48 the pro-tonated amino groups of chitosan interact electrostaticallywith negatively charged phosphate groups of STPP [29]The uncertainty of this encapsulation was the complexationof DOX into the positively charged chitosan since DOXalso exhibits a positive charge However 65 of DOX wasencapsulated into Csn NPs in this experiment overcomingthe charge repulsion Since Csn NPs have shown positiveresults for the DOX delivery an effort was taken to improvethe DOX delivery with the use of alginate polymer as wellThe polyelectrolyte complex Csn-AlgNPswas prepared usinga novel method Initially the chitosan droplet formationoccurred with the stirring with Tween 80 followed by thedroplet solidification by ionically crosslinking of the alginatesolution In aqueous solutions at pH around 4 to 55 theamine groups on chitosan became easily positively chargedAlginate on the other hand dissolved in a neutral pH solu-tionwhere the carboxylate groupswere negatively charged Inaqueous solutions with pH around 52 chitosan amino groupsinteracted with alginate carboxylate groups to form thehydrogel Since alginate is a negatively charged polymer thepositively chargedDOXwas complexed initially with alginateto obtain a higher loading of the drug to the nanoparticleTheformulation (chitosan sodium alginate 2 1) which gavean opalescent suspension with a positive zeta potential wasselected as the optimum ratio for the DOX encapsulationThis formulation resulted in 95 encapsulation efficiency ofDOX

32 Shape Size and Surface Charge of Doxorubicin LoadedNanoparticles The morphology of the polymeric NPs wasobserved by scanning electronmicroscope and TransmissionElectron Microscope and both NPs were below 20 nm andspherical in shape (Figures 1 and 2) Further the TEM imagesclearly showed the well separated well dispersed particlesAfter loading of DOX there was no noticeable change eitherin the shape or in the size in SEM and TEM images ofboth NPs In a study on carvacrol-loaded chitosan NPsKeawchaoon andYoksan have reportedTEM images showing40ndash80 nm sized NPs [27] In a previous study done by Liu etal the particle sizes of Csn-Alg NPs obtained from the trans-mission electron microscopy (TEM) were 20ndash50 nm [11]

A study by Motwani et al had shown the particle sizesof Csn-Alg NPs obtained from TEM to be around 63 nm[22] Thus using the above-mentioned method we were ableto obtain much smaller Csn-Alg NPs The sizes obtainedfrom DLS technique were larger (sim100 nm Table 1) than thesizes obtained from SEM and TEM since DLS measures thehydrodynamic diameter Also the larger particle sizemight bedue to the swellability of polymeric hydrogels in the solution[30] whereas electron microscopy shows the images of driedparticles

The zeta potentials of both DOX Csn and DOX Csn-Alg NPs show large positive values reflecting the stability ofthe colloidal suspensions (Table 1) Having positively chargedsurfaces is an added advantage when using NPs in drugdelivery since they are able to transfer easily through negativechannels in the cell membrane An increased zeta potentialvalue with DOX Csn-Alg NPs when compared to the DOXCsn NPs was caused by the higher amount of positivelycharged DOX encapsulation with Csn-Alg NPs than thatwith Csn NPs Neutralization of the charge on chitosan bythe strong negative charge of STTP versus alginate at theworking pH may have also contributed to the differences inzeta potential

33 Characterisation of DOX Loaded Nanoparticles Thesuccessful loading of DOX into the polymeric NPs wasconfirmed by FTIR and TGA techniques Chemical struc-tures of both reactants and products were analyzed by FTIRspectroscopy In the spectra of pure chitosan (Figure 3(b))characteristic peaks at 3449 cmminus1 for ndashNH

2and ndashOH stretch-

ing 1659 cmminus1 for ndashCO stretching and 1596 cmminus1 for ndashNH2

bending vibrations were observed Crosslinking of the aminegroup of chitosan with phosphate group of STPP depictsa clear shift in the peak at 3565 cmminus1 for ndashNH

2and ndashOH

stretching as shown in the spectrum of CsnNPs (Figure 3(c))On loadingDOX intoCsnNPs the FTIR spectra (Figure 3(e))show peak shifts resulting from hydrogen bonding in theabove modes that is 3578 cmminus1 for ndashNH

2and ndashOH stretch-

ing 1685 cmminus1 for ndashCO stretching and 1578 cmminus1 for ndashNH2

bending vibrations reflecting the successful loading of DOXinto the Csn NPs

In the spectra of pure chitosan the major vibrations ofndashNH2and ndashOH stretching peak appearing at 3449 cmminus1 had

shifted to 3565 cmminus1 in the spectra of Csn-Alg NPs andthe ndashCO stretching vibration at 1659 cmminus1 in pure chitosanhad also shifted to 1651 cmminus1 after interaction of the aminegroup of chitosan with the carboxylic group of alginate

Journal of Nanomaterials 5

(a) (b)

Figure 1 (a) Scanning electron microscopy image of Csn NP (b) Transmission electron microscopy image of Csn NP

(a) (b)

Figure 2 (a) Scanning electron microscopy image of Csn-Alg NP (b) Transmission electron microscopy image of Csn-Alg NP

(Figure 3(d)) Further the ndashNH2bending peak at 1596 cmminus1

disappeared due to binding with alginate to form the NPAfter loading DOX into the Csn-Alg NPs the ndashNH

2and

ndashOH stretching peak at 3565 cmminus1 and the ndashCO stretchingvibration at 1660 cmminus1 shifted to 3356 cmminus1 and to 1637 cmminus1respectively Further the appearance of a small peak at1542 cmminus1 for ndashNH

2bending after loading the DOX may

reflect the physical entrapment of DOX into the Csn-Alg NPs(Figure 3(f))

34 Thermal Behaviour of DOX Loaded Polymeric Nanopar-ticles Nanoparticles with and without DOX were analyzedby thermogravimetry to further evaluate the thermal stabilityand successful loading of DOX In the thermogram ofchitosan NPs a major one-step mass loss about 40 of totalweight was observed at a peaking temperature (inflectionpoint temperature) of 216∘C (Figure 4) Heating up to 800∘Cretained 35 of the weight of the nanoparticles from theresidual sodium ions of the STPP crosslinked Csn NPsSimilarly in DOX loaded chitosan nanoparticles a 50weight loss from 100ndash400∘C was observed in two steps dueto the combined decomposition of chitosan and DOX butthe peaking temperature of 30 weight loss had shiftedto 225∘C proving the loading of the DOX into chitosan

nanoparticles (Figure 4) A new peak with 12 weight lossin the temperature range 600ndash700∘C was observed with theDOXCsnNPs showing entrapment of DOX into the polymermatrix

The decomposition of unloaded Csn-Alg NPs had twomajor and two minor weight losses (Figure 4) The ther-mogram of DOX loaded NPs showed the same pattern ofweight loss as the unloaded particles However when thetemperature reached 350∘C 60 of weight loss was observedwith DOX loaded nanoparticles due to the decompositionof DOX complexed nanoparticles whereas it was only 50with unloaded nanoparticles Further the inflection pointtemperature of the second major step of weight loss ofunloaded composite in the range of 600ndash750∘C peaked at703∘C whereas in the DOX loaded nanocomposite it peakedat 750∘C reflecting the strong physical entrapment of DOXinto the nanoparticles

35 In Vitro Release Kinetics of DOX from PolymericNanoparticles The in vitro release profiles of both optimizedformulations in phosphate buffered saline (PBS) solution(pH-74) are shown in Figure 5 An initial burst release ofDOX from both NPs in PBS solution can be observed upto 24 h This accounts for about 40 of DOX from the totalencapsulated amount Then it followed a more gradual and

6 Journal of Nanomaterials

(c)

(b)

(a)

(d)

(e)

(f)

Wave number (cmminus1)

Tran

smitt

ance

(arb

)

5001000150020002500300035004000

Figure 3 FTIR spectra of (a) pure alginate (b) pure chitosan (c)CsnNPprepared using 2 1weight ratio of chitosan to STPP (d)Csn-Alg NP prepared using 2 1 weight ratio of chitosan to alginate (e)DOX Csn NP and (f) DOX Csn-Alg NP

Temperature (∘C)8007006005004003002001000

0

10

20

30

40

50

60

70

80

90

100

Wei

ght (

)

Csn NPDOX Csn NP

Csn-Alg NPDOX Csn-Alg NP

Figure 4 TGA thermograms of Csn NP DOX Csn NP preparedusing 2 1 weight ratio of chitosan to STPP and Csn-Alg NP andDOX Csn-Alg NP prepared using 2 1 weight ratio of chitosan toalginate

sustained release phase for the next 72 h Both profiles hadfollowed the same pattern of release but the amounts weredifferent The total amount of drug released from Csn-AlgNPs was around 60 In contrast the total amount of drugreleased from Csn NPs was around 45These results clearlyindicated that the release of DOX was retarded significantlydue to the encapsulation in nanoparticles

36 In Vitro Cytotoxicity of DOX Loaded Polymeric Nanopar-ticle Dose response curves were plotted for 3 formulations

Time (hour)

(a)

(b)

100806040200

10

20

30

40

50

60

70

Cum

ulat

ive r

elea

se (

)

Figure 5 Cumulative release of DOX from (a) DOX Csn-Alg and(b) DOXCsnNP at preselected time intervals in pH 74 PBS Resultswere reported as mean plusmn SD 119899 = 3

Free DOXDOX loaded chitosan-alginate NPDOX loaded chitosan NP

2 4 6 80

Concentration (120583gmL)

0

10

20

30

40

50

60

70

80

90

100Pe

rcen

tage

cell

viab

ility

()

Figure 6 Cytotoxicity of free DOX DOX Csn-Alg NP and DOXCsn NP against MCF-7 cells after e24 ◼48 and 99877172 hours oftreatments Cell survival was assessed via SRB assay Results werereported as mean plusmn SD 119899 = 4

free DOX DOX Csn-Alg NPs and DOX Csn NPs (Figure 6)Each experimental curve represents the average of a seriesof 3 experiments With free DOX the inhibition of MCF-7cell proliferation completely depends on the dose of thedrug used After 48 and 72 h 50 cell viability could not beobserved even at the lowest concentration (001120583gmL) offree DOX proving the reduced effect of free DOX after 24 hthat is the free DOX has dose dependent cytotoxicity thus itmight damage the normal cells during therapy whereas thecurrent work demonstrated the dose-dependence as well as

Journal of Nanomaterials 7

Free DOX

Control

Control

Control

DOX Csn NPDOX Csn-Alg NP24 h

48 h

72 h

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

Figure 7 Morphological changes of apoptosis in MCF-7 cells observed under inverted light microscopy exposed to different concentrationsof free DOX DOX Csn-Alg NP and DOX Csn NP after 24 h 48 h and 72 h after incubation

8 Journal of Nanomaterials

Free DOX

Control

Control

Control

DOX Csn NPDOX Csn-Alg NP24 h

48 h

72 h

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

Figure 8 Fluorescence microscopic observations of MCF-7 cells treated with different concentrations of free DOX DOX Csn-Alg NP andDOX Csn NP stained with AOEB after 24 h 48 h and 72 h after incubation

Journal of Nanomaterials 9

Free DOX

Control

Control

Control

DOX Csn NPDOX Csn-Alg NP

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

30min

24 h

2 h

Figure 9 Fluorescence microscopic observations of cellular uptake of MCF-7 cells treated with different concentrations of free DOX DOXCsn-Alg NP and DOX Csn NP after 30min 2 h and 24 h after incubation

10 Journal of Nanomaterials

the time dependence cytotoxicity of DOX loaded NPs withMCF-7 cell line None of the concentrations of DOX in bothNPs that were used caused 50 inhibition of cell growthat 24 h reflecting the slow release from NPs Hence duringthe first 24 h those NPs are capable of circulating in theblood and accumulating at tumour sites before starting theircytotoxic effect Thus DOX encapsulated in NPs achieve itscytotoxicity with time that is there is an effect of both timeand dose on the inhibition of cell proliferation Thereforethis result clearly indicated that the release of DOX wasretarded significantly by the encapsulation in NPs Whencomparing the two delivery systems the cytotoxicity effectof DOX Csn NPs was consistently lower than that of DOXCsn-Alg NPs (Figure 6)This is in accordance with the resultsof in vitro release of DOX being higher from Csn-Alg NPsthan from Csn NPs after 72 h (Figure 5) However bothDOX loaded systems exhibited similar behaviour with theMCF-7 cell line (Figure 6) Same concentration of DOXwhen encapsulated in NPs behaves differently than in thefree state that is encapsulated DOX releases slowly andgradually increases the concentration in the medium Henceit proves that encapsulating DOX in either system hasimproved the efficacy of DOX over free DOX A similarstudy of doxorubicin loaded magnetic NPs grafted to smartcopolymers showed that free DOX has dose dependent butnot time dependent cytotoxicity but DOX loaded magneticNPs have time dependent cytotoxicity [31]

Inverted phase contrast microscopy was used for mor-phological analysis of MCF-7 cells for observation of apop-tosis induced by the DOX loaded NPs and free DOXThese characteristicmorphological changes of apoptosis withtime and dose in MCF-7 cells on treatment after 24 48and 72 h (Figure 7) were clearly visualized Exposure tofree DOX clearly showed a higher cell confluence at lowdoses while higher concentration of free DOX decreased cellconfluence and showed a higher proportion of rounded cellsresulting from detached cells from the culture monolayerWithin the first 24 h most of the cells were damaged bythe induced cytotoxicity of free DOX reflecting its dosedependent cytotoxicity On the other hand bothDOX loadedsystems showed disappearance of the high cell confluencewith the dose of the drug and with time (Figure 7) Thisobservation is further confirmed by the fluorescence micro-scopic results of AOEB stained MCF-7 cells after 24 48and 72 h of incubation following the treatment with differentconcentrations of freeDOXandwith the same concentrationsloaded in Csn and Csn-Alg NPs Nuclei of viable cells werestained uniformly bright green by AO while the apoptoticcells exhibited yellow to orange coloration depending on thedegree of loss of membrane integrity due to costaining withEB (Figure 8) Early apoptotic cells have greenish yellownuclei while late apoptotic cells have orange to red nucleiwith condensed or fragmented chromatin (Figure 8) Thesefigures clearly displayed the difference in behaviour of DOXwhen encapsulated in NPs Free DOX has a dose dependentincrease in induction of apoptosis (Figure 8-24 h) Both thedose and time dependency of DOX encapsulated in NPs are

evident from the alterations in cell staining in all the threeseries (Figure 8) In the case of DOX loadedNPs the drug hasto be released slowly from theNPs before it can exert an effecton the cancer cells but free DOX has an immediate effecton the cells since it has direct contact with the cells initiallyThe mechanism of action of apoptosis of DOX loaded inNPs was not significantly different from that of the free DOXindicating that the encapsulation of DOX in NPs does notmake any changes to its mechanism of action of apoptosis

37 Cellular Uptake of Nanoparticles DOX is a fluorescenceactive compound having a bright red color The red colorindicates the cellular uptake of DOX when compared to theuntreated cells or cells with no internalized DOX Internal-ization of DOX in cancer cells was evaluated on MCF-7 cellsat different time intervals and different concentrations offree and encapsulated DOX Within 30min of incubationall concentrations of free DOX were localized within the cellnucleus (Figure 9 30min) whereas the localization of DOXin NPs could be visualized depending on the concentrationand on time (Figure 9) The efficient uptake of the NPs couldbe facilitated by the positive surface charge of the NPs Asimilar observationwas notedwith the in vitro cellular uptakeof paclitaxel loaded PEGylated PLGA-based NPs [32] Ingeneral NPs are nonspecifically internalized into cells viaendocytosis or phagocytosis while free DOXdiffuses throughthe cell membrane [33]

Morphology analysis suggests that the process of cellgrowth inhibition by DOX loaded in NPs occurs over a muchlonger time than by the free DOX From these observations itcould be inferred that the DOX in NPs sufficiently associateswith the cells with time

4 Conclusion

This study was made to assess the usefulness of the Csn-Alg NP system as a delivery agent for the anticancer drugDOX Comparison with DOX Csn NPs indicated that Csn-Alg NPs were more efficient at entrapment of DOX than theCsnNPsThis successful loadingwas confirmedby both FTIRand TGA data Both nanoparticles were less than 100 nm insize with positive zeta potentials The in vitro release profilesobserved for these NPs were characterized by a burst initialrelease followed by a more gradual and sustained releasephase The in vitro cytotoxicity studies demonstrated thatDOX loaded NPs have potent antigrowth effect on MCF-7cells and inhibited its cell growth in a dose and time depen-dent manner Further the observed cell apoptosis induced bythese DOX loaded NPs by phase contrast microscopy andfluorescence microscopy after AOEB staining proved thatthere is no significant change in the mechanism of action ofDOX after encapsulating in NPs and reflect the slow releaseof DOX from the NPs According to the cytotoxicity resultsDOX Csn-Alg NPs are more cytotoxic than DOX Csn NPs

Competing Interests

The authors declare that they have no competing interests

Journal of Nanomaterials 11

Acknowledgments

This research was financially supported by the HETC QIGWindow 3 Grant University of Peradeniya Peradeniya SriLanka The authors also thank Ms Damayanthi Dahanayakeat Sri Lanka Institute of nanotechnology for her valuablesupport in electron microscopy imaging

References

[1] N Lomovskaya S L Otten Y Doi-Katayama et al ldquoDoxoru-bicin overproduction in Streptomyces peucetius cloning andcharacterization of the dnrU ketoreductase and dnrV genesand the doxA cytochrome P-450 hydroxylase generdquo Journal ofBacteriology vol 181 no 1 pp 305ndash318 1999

[2] K M Laginha S Verwoert G J R Charrois and T MAllen ldquoDetermination of doxorubicin levels in whole tumorand tumor nuclei in murine breast cancer tumorsrdquo ClinicalCancer Research vol 11 no 19 pp 6944ndash6949 2005

[3] G Minotti P Menna E Salvatorelli G Cairo and L GiannildquoAnthracyclines molecular advances and pharmacologic devel-opments in antitumor activity and cardiotoxicityrdquo Pharmaco-logical Reviews vol 56 no 2 pp 185ndash229 2004

[4] M E R OrsquoBrien N Wigler M Inbar et al ldquoReduced car-diotoxicity and comparable efficacy in a phase III trial of pegy-lated liposomal doxorubicin HCl (CAELYXDoxil) versusconventional doxorubicin for first-line treatment of metastaticbreast cancerrdquo Annals of Oncology vol 15 no 3 pp 440ndash4492004

[5] C Zhang W Wang T Liu et al ldquoDoxorubicin-loaded gly-cyrrhetinic acid-modified alginate nanoparticles for liver tumorchemotherapyrdquo Biomaterials vol 33 no 7 pp 2187ndash2196 2012

[6] W H De Jong and P J A Borm ldquoDrug delivery andnanoparticles applications and hazardsrdquo International Journalof Nanomedicine vol 3 no 2 pp 133ndash149 2008

[7] G Vilar J Tulla-Puche and F Albericio ldquoPolymers and drugdelivery systemsrdquo Current Drug Delivery vol 9 no 4 pp 367ndash394 2012

[8] A Kidane and P P Bhatt ldquoRecent advances in small moleculedrug deliveryrdquo Current Opinion in Chemical Biology vol 9 no4 pp 347ndash351 2005

[9] K S Soppimath T M Aminabhavi A R Kulkarni and WE Rudzinski ldquoBiodegradable polymeric nanoparticles as drugdelivery devicesrdquo Journal of Controlled Release vol 70 no 1-2pp 1ndash20 2001

[10] T Coviello P Matricardi C Marianecci and F AlhaiqueldquoPolysaccharide hydrogels for modified release formulationsrdquoJournal of Controlled Release vol 119 no 1 pp 5ndash24 2007

[11] Z Liu Y Jiao Y Wang C Zhou and Z ZhangldquoPolysaccharides-based nanoparticles as drug deliverysystemsrdquo Advanced Drug Delivery Reviews vol 60 no 15 pp1650ndash1662 2008

[12] O Borges G Borchard J C Verhoef A De Sousa andH E Junginger ldquoPreparation of coated nanoparticles for anew mucosal vaccine delivery systemrdquo International Journal ofPharmaceutics vol 299 no 1-2 pp 155ndash166 2005

[13] Y Murata D Jinno D Liu T Isobe K Kofuji and SKawashima ldquoThe drug release profile from calcium-inducedalginate gel beads coated with an alginate hydrolysaterdquoMolecules vol 12 no 11 pp 2559ndash2566 2007

[14] P Li Y-N Dai J-P Zhang A-QWang andQWei ldquoChitosan-alginate nanoparticles as a novel drug delivery system fornifedipinerdquo International Journal of Biomedical Science vol 4no 3 pp 221ndash228 2008

[15] C K S Pillai W Paul and C P Sharma ldquoChitin and chitosanpolymers chemistry solubility and fiber formationrdquo Progress inPolymer Science vol 34 no 7 pp 641ndash678 2009

[16] F Khoushab and M Yamabhai ldquoChitin research revisitedrdquoMarine Drugs vol 8 no 7 pp 1988ndash2012 2010

[17] T Kean and M Thanou ldquoBiodegradation biodistribution andtoxicity of chitosanrdquo Advanced Drug Delivery Reviews vol 62no 1 pp 3ndash11 2010

[18] O Gaseroslashd I G Jolliffe F C Hampson P W Dettmar and GSkjak-Braeligk ldquoThe enhancement of the bioadhesive propertiesof calcium alginate gel beads by coating with chitosanrdquo Inter-national Journal of Pharmaceutics vol 175 no 2 pp 237ndash2461998

[19] M George and T E Abraham ldquoPolyionic hydrocolloids for theintestinal delivery of protein drugs alginate and chitosanmdashareviewrdquo Journal of Controlled Release vol 114 no 1 pp 1ndash142006

[20] A Saha R Yadav and N Rajendran ldquoBiomaterials fromsponges ascidians and other marine organismsrdquo InternationalJournal of Pharmaceutical Sciences Review and Research vol 27no 2 article no 17 pp 100ndash109 2014

[21] S De and D Robinson ldquoPolymer relationships duringpreparation of chitosan-alginate and poly-l-lysine-alginatenanospheresrdquo Journal of Controlled Release vol 89 no 1 pp101ndash112 2003

[22] S K Motwani S Chopra S Talegaonkar K Kohli F J Ahmadand R K Khar ldquoChitosan-sodium alginate nanoparticles assubmicroscopic reservoirs for ocular delivery formulationoptimisation and in vitro characterisationrdquo European Journal ofPharmaceutics and Biopharmaceutics vol 68 no 3 pp 513ndash5252008

[23] F-L Mi H-W Sung and S-S Shyu ldquoDrug release fromchitosan-alginate complex beads reinforced by a naturallyoccurring cross-linking agentrdquo Carbohydrate Polymers vol 48no 1 pp 61ndash72 2002

[24] Q Zhao B Han ZWang C Gao C Peng and J Shen ldquoHollowchitosan-alginate multilayer microcapsules as drug deliveryvehicle doxorubicin loading and in vitro and in vivo studiesrdquoNanomedicine Nanotechnology Biology and Medicine vol 3no 1 pp 63ndash74 2007

[25] H Shen H Shi M Xie et al ldquoBiodegradable chitosanalginateBSA-gel-capsules for pH-controlled loading and release ofdoxorubicin and treatment of pulmonary melanomardquo Journalof Materials Chemistry B vol 1 no 32 pp 3906ndash3917 2013

[26] W Feng W Nie C He et al ldquoEffect of pH-responsive algi-natechitosanmultilayers coating on delivery efficiency cellularuptake and biodistribution of mesoporous silica nanoparticlesbased nanocarriersrdquo ACS Applied Materials amp Interfaces vol 6no 11 pp 8447ndash8460 2014

[27] L Keawchaoon and R Yoksan ldquoPreparation characterizationand in vitro release study of carvacrol-loaded chitosan nanopar-ticlesrdquo Colloids and Surfaces B Biointerfaces vol 84 no 1 pp163ndash171 2011

[28] S R Samarakoon I Thabrew P B Galhena D De Silva andK Tennekoon ldquoA comparison of the cytotoxic potential of stan-dardized aqueous and ethanolic extracts of a polyherbalmixturecomprised ofNigella sativa (seeds)Hemidesmus indicus (roots)

12 Journal of Nanomaterials

and Smilax glabra (rhizome)rdquo Pharmacognosy Research vol 2no 6 pp 335ndash342 2010

[29] K Nagpal S K Singh and D N Mishra ldquoChitosan nanoparti-cles a promising system in novel drug deliveryrdquo Chemical andPharmaceutical Bulletin vol 58 no 11 pp 1423ndash1430 2010

[30] N Bhattarai J Gunn and M Zhang ldquoChitosan-based hydro-gels for controlled localized drug deliveryrdquo Advanced DrugDelivery Reviews vol 62 no 1 pp 83ndash99 2010

[31] A Akbarzadeh M Samiei S W Joo et al ldquoSynthesis charac-terization and in vitro studies of doxorubicin-loaded magneticnanoparticles grafted to smart copolymers on A549 lung cancercell linerdquo Journal of Nanobiotechnology vol 10 article 46 2012

[32] F Danhier N Lecouturier B Vroman et al ldquoPaclitaxel-loadedPEGylated PLGA-based nanoparticles in vitro and in vivoevaluationrdquo Journal of Controlled Release vol 133 no 1 pp 11ndash17 2009

[33] A des Rieux V FievezMGarinot Y-J Schneider andV PreatldquoNanoparticles as potential oral delivery systems of proteins andvaccines amechanistic approachrdquo Journal of Controlled Releasevol 116 no 1 pp 1ndash27 2006

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CeramicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CompositesJournal of

NanoparticlesJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Biomaterials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MetallurgyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

MaterialsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Nano

materials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal ofNanomaterials

Journal of Nanomaterials 3

technique at 25∘C using a particle size analyzer (ZetasizerNano ZS Malvern Instruments UK) at a fixed scatteringangle of 90∘The zeta potential of nanoparticles wasmeasuredusing the zeta potential analyzer (ZetasizerNanoZSMalvernInstruments UK) All measurements were performed intriplicate Fourier Transform Infrared (FTIR) spectra ofchitosan sodium alginate unloaded nanoparticles andDOXloaded NPs were obtained with a Bruker Vertex 80 IR spec-trometer (Germany) at a resolution of 4 cmminus1 from 4000 to400 cmminus1 Thermal decomposition of chitosan sodium algi-nate unloaded nanoparticles andDOX loaded nanoparticleswere analyzed using a SDTQ600 thermogravimetric analyzer(TA Instruments USA) from 25∘C to 800∘Cusing a ramp rateof 10∘Cmin in air Nanoparticle morphology was examinedby electron microscopy For scanning electron microscopya drop of the particle suspension was placed on a cleanedstub and dried overnight and imaging was done followedby gold sputtering to the specimen by the scanning electronmicroscope (SEM SU6600 Hitachi Japan) operating at 5and 10 kV Further the morphology was recorded by high-resolution transmission electronmicroscopy (HR-TEM JEM2100 JEOL Japan) operated at accelerating voltage 200 kVThe samples prior to their analysis were placed on carboncoated copper grids and dried under ambient conditions

24 Determination of Encapsulation Efficiency Theamount of incorporated DOX in the NPs was determinedby measuring the fluorescent absorbance of DOX remainingin the supernatant (free DOX) after centrifugation ofthe nanoparticle mixture using a fluorometer (FluorologHoriba Japan) with a slit width of 5 nm and excitation andemission wavelengths at 468 nm and 550 nm respectivelyA calibration curve was performed with a series ofconcentrations to obtain the unknown DOX concentrationsThe percent encapsulation efficiency ( EE) was calculatedas follows

EE = Total DOX minus Free DOXTotal DOX

times 100 (1)

25 Evaluation of In Vitro DOX Release The release char-acteristics of DOX from polymeric NPs were studied indistilled water and phosphate buffered saline solution (PBS)(pH sim 74)The centrifuged nanoparticle pellet was dispersedin 5mL of PBS at pH 74 and trapped inside a dialysismembrane (cutoff molecular weight 35 kDa) and immersedin 25mL PBS The temperature of the setup was maintainedat 37∘C in the dark with mild agitation Aliquots (3mL)were withdrawn at predetermined time intervals and thefluorescence emission spectrum was recorded as describedpreviously The release medium was refreshed with 3mLof medium after each withdrawal The release experimentswere repeated three times and average data were reportedUsing the calibration curve the unknown concentrationswere calculated hence the cumulative release of DOX wasdetermined

26 Cell Culture and In Vitro Cytotoxicity Studies Thecytotoxicity activities of DOX Csn and DOX Csn-Alg NPs

against MCF-7 cells were compared to free DOX MCF-7 cells were grown in monolayer cultures in DulbeccorsquosModified Eagle Medium (DMEM) supplemented with 10fetal bovine serum and 50 IUmL penicillin and 50120583gmLstreptomycin Cells were maintained at 37∘C in 95 air5CO2atmosphere with 95 humidity Culture medium was

changed every 2-3 days Next the cells in exponential growthwere removed by trypsinization and seeded at 5 times 103 cellsper well into a 96-well plate and incubated for 24 h forattachment After the incubation period cells were exposedto both DOX loaded NPs (DOX concentration of 001 002005 10 40 and 80 120583gmL) and to free DOX with similarconcentrations for 24 48 and 72 h after incubation

Cell viability was assessed by sulforhodamine B (SRB)assay [28] and the results were expressed as a percentage ofcontrol values All the assays were performed in triplicate inthree different experiments

27 Morphological Observation and Apoptosis Induced byDOX Loaded Nanoparticles Apoptosis was identified mor-phologically by acridine orangeethidium bromide (AOEB)staining MCF-7 cells were seeded separately in 24-well platesat 5 times 104 cellsmLminus1 on cell culture treated coverslips for24 h in a humidified atmosphere at 37∘C in 5 CO

2to allow

cell adherence Cells were then treated with both types ofDOX loaded NPs (DOX concentration of 001 100 and800 120583gmL) and free DOX (001 100 and 800 120583gmL)Treated cells were incubated for 24 48 and 72 h and atthe end of each incubation process cells on the coverslipswere observed under an inverted phase contrast microscope(Olympus CKX41SF Japan) for morphological changes Cellswere then fixed with 4 formaldehyde in PBS at roomtemperature for 15min Cocktail of acridine orange-ethidiumbromide (AOEB) dyes in PBS containing 50mgmLminus1 eachwas added to the fixed cells on coverslips and incubatedfor 10min at room temperature in the dark Cells werethen visualized under a fluorescence microscope (OlympusBX51TRF Japan) Both assays were performed in triplicate inthree different experiments

28 Cellular Uptake of Nanoparticles MCF-7 cells wereseeded separately in 24-well plates at 5 times 104 cellsmLminus1 oncoverslips for 24 h in a humidified atmosphere at 37∘C in 5CO2to allow cell adherence Cells were then treatedwith both

DOX loaded NP systems (DOX concentration of 001 100and 800 120583gmL) and free DOX (001 100 and 800 120583gmL)Treated cells were incubated for 30min and 2 and 24 h andat the end of each incubation process cells were rinsed twicewith PBS and then fixed with 4 formaldehyde in PBS atroom temperature for 15mins The cells on the coverslipswere observed under the fluorescence microscope (OlympusBX51TRF Japan)

3 Results and Discussion

The main effort of this work was to develop a nanodeliverysystem for the drug DOX using natural biodegradable bio-compatible polysaccharides specially chitosan and alginate

4 Journal of Nanomaterials

Table 1 The size zeta potential and EE of DOX Csn and DOX Csn-Alg NPs All analyses were performed using the DOX loaded NPsprepared by using optimized formulations (chitosan STTP 2 1 and chitosan alginate 2 1)

System Average sizenm Zeta potentialmV Encapsulation efficiency Polydispersity indexDOX Csn NPs 100 plusmn 27 26 plusmn 3 65 plusmn 4 0391 plusmn 0048DOX Csn-Alg NPs 100 plusmn 35 35 plusmn 4 95 plusmn 4 0402 plusmn 0071Csn NPs 100 plusmn 25 30 plusmn 5 mdash 0352 plusmn 0046Csn-Alg NPs 100 plusmn 28 36 plusmn 3 mdash 0414 plusmn 0065

The feasibility of using Csn-Alg NPs as a delivery system forDOX was evaluated with comparison to DOX Csn NPs

31 Formation of DOX Loaded Polymeric Nanoparticles Thechitosan NPs are easily formed by the gelation with STPPvia electrostatic interactions At a pH around 48 the pro-tonated amino groups of chitosan interact electrostaticallywith negatively charged phosphate groups of STPP [29]The uncertainty of this encapsulation was the complexationof DOX into the positively charged chitosan since DOXalso exhibits a positive charge However 65 of DOX wasencapsulated into Csn NPs in this experiment overcomingthe charge repulsion Since Csn NPs have shown positiveresults for the DOX delivery an effort was taken to improvethe DOX delivery with the use of alginate polymer as wellThe polyelectrolyte complex Csn-AlgNPswas prepared usinga novel method Initially the chitosan droplet formationoccurred with the stirring with Tween 80 followed by thedroplet solidification by ionically crosslinking of the alginatesolution In aqueous solutions at pH around 4 to 55 theamine groups on chitosan became easily positively chargedAlginate on the other hand dissolved in a neutral pH solu-tionwhere the carboxylate groupswere negatively charged Inaqueous solutions with pH around 52 chitosan amino groupsinteracted with alginate carboxylate groups to form thehydrogel Since alginate is a negatively charged polymer thepositively chargedDOXwas complexed initially with alginateto obtain a higher loading of the drug to the nanoparticleTheformulation (chitosan sodium alginate 2 1) which gavean opalescent suspension with a positive zeta potential wasselected as the optimum ratio for the DOX encapsulationThis formulation resulted in 95 encapsulation efficiency ofDOX

32 Shape Size and Surface Charge of Doxorubicin LoadedNanoparticles The morphology of the polymeric NPs wasobserved by scanning electronmicroscope and TransmissionElectron Microscope and both NPs were below 20 nm andspherical in shape (Figures 1 and 2) Further the TEM imagesclearly showed the well separated well dispersed particlesAfter loading of DOX there was no noticeable change eitherin the shape or in the size in SEM and TEM images ofboth NPs In a study on carvacrol-loaded chitosan NPsKeawchaoon andYoksan have reportedTEM images showing40ndash80 nm sized NPs [27] In a previous study done by Liu etal the particle sizes of Csn-Alg NPs obtained from the trans-mission electron microscopy (TEM) were 20ndash50 nm [11]

A study by Motwani et al had shown the particle sizesof Csn-Alg NPs obtained from TEM to be around 63 nm[22] Thus using the above-mentioned method we were ableto obtain much smaller Csn-Alg NPs The sizes obtainedfrom DLS technique were larger (sim100 nm Table 1) than thesizes obtained from SEM and TEM since DLS measures thehydrodynamic diameter Also the larger particle sizemight bedue to the swellability of polymeric hydrogels in the solution[30] whereas electron microscopy shows the images of driedparticles

The zeta potentials of both DOX Csn and DOX Csn-Alg NPs show large positive values reflecting the stability ofthe colloidal suspensions (Table 1) Having positively chargedsurfaces is an added advantage when using NPs in drugdelivery since they are able to transfer easily through negativechannels in the cell membrane An increased zeta potentialvalue with DOX Csn-Alg NPs when compared to the DOXCsn NPs was caused by the higher amount of positivelycharged DOX encapsulation with Csn-Alg NPs than thatwith Csn NPs Neutralization of the charge on chitosan bythe strong negative charge of STTP versus alginate at theworking pH may have also contributed to the differences inzeta potential

33 Characterisation of DOX Loaded Nanoparticles Thesuccessful loading of DOX into the polymeric NPs wasconfirmed by FTIR and TGA techniques Chemical struc-tures of both reactants and products were analyzed by FTIRspectroscopy In the spectra of pure chitosan (Figure 3(b))characteristic peaks at 3449 cmminus1 for ndashNH

2and ndashOH stretch-

ing 1659 cmminus1 for ndashCO stretching and 1596 cmminus1 for ndashNH2

bending vibrations were observed Crosslinking of the aminegroup of chitosan with phosphate group of STPP depictsa clear shift in the peak at 3565 cmminus1 for ndashNH

2and ndashOH

stretching as shown in the spectrum of CsnNPs (Figure 3(c))On loadingDOX intoCsnNPs the FTIR spectra (Figure 3(e))show peak shifts resulting from hydrogen bonding in theabove modes that is 3578 cmminus1 for ndashNH

2and ndashOH stretch-

ing 1685 cmminus1 for ndashCO stretching and 1578 cmminus1 for ndashNH2

bending vibrations reflecting the successful loading of DOXinto the Csn NPs

In the spectra of pure chitosan the major vibrations ofndashNH2and ndashOH stretching peak appearing at 3449 cmminus1 had

shifted to 3565 cmminus1 in the spectra of Csn-Alg NPs andthe ndashCO stretching vibration at 1659 cmminus1 in pure chitosanhad also shifted to 1651 cmminus1 after interaction of the aminegroup of chitosan with the carboxylic group of alginate

Journal of Nanomaterials 5

(a) (b)

Figure 1 (a) Scanning electron microscopy image of Csn NP (b) Transmission electron microscopy image of Csn NP

(a) (b)

Figure 2 (a) Scanning electron microscopy image of Csn-Alg NP (b) Transmission electron microscopy image of Csn-Alg NP

(Figure 3(d)) Further the ndashNH2bending peak at 1596 cmminus1

disappeared due to binding with alginate to form the NPAfter loading DOX into the Csn-Alg NPs the ndashNH

2and

ndashOH stretching peak at 3565 cmminus1 and the ndashCO stretchingvibration at 1660 cmminus1 shifted to 3356 cmminus1 and to 1637 cmminus1respectively Further the appearance of a small peak at1542 cmminus1 for ndashNH

2bending after loading the DOX may

reflect the physical entrapment of DOX into the Csn-Alg NPs(Figure 3(f))

34 Thermal Behaviour of DOX Loaded Polymeric Nanopar-ticles Nanoparticles with and without DOX were analyzedby thermogravimetry to further evaluate the thermal stabilityand successful loading of DOX In the thermogram ofchitosan NPs a major one-step mass loss about 40 of totalweight was observed at a peaking temperature (inflectionpoint temperature) of 216∘C (Figure 4) Heating up to 800∘Cretained 35 of the weight of the nanoparticles from theresidual sodium ions of the STPP crosslinked Csn NPsSimilarly in DOX loaded chitosan nanoparticles a 50weight loss from 100ndash400∘C was observed in two steps dueto the combined decomposition of chitosan and DOX butthe peaking temperature of 30 weight loss had shiftedto 225∘C proving the loading of the DOX into chitosan

nanoparticles (Figure 4) A new peak with 12 weight lossin the temperature range 600ndash700∘C was observed with theDOXCsnNPs showing entrapment of DOX into the polymermatrix

The decomposition of unloaded Csn-Alg NPs had twomajor and two minor weight losses (Figure 4) The ther-mogram of DOX loaded NPs showed the same pattern ofweight loss as the unloaded particles However when thetemperature reached 350∘C 60 of weight loss was observedwith DOX loaded nanoparticles due to the decompositionof DOX complexed nanoparticles whereas it was only 50with unloaded nanoparticles Further the inflection pointtemperature of the second major step of weight loss ofunloaded composite in the range of 600ndash750∘C peaked at703∘C whereas in the DOX loaded nanocomposite it peakedat 750∘C reflecting the strong physical entrapment of DOXinto the nanoparticles

35 In Vitro Release Kinetics of DOX from PolymericNanoparticles The in vitro release profiles of both optimizedformulations in phosphate buffered saline (PBS) solution(pH-74) are shown in Figure 5 An initial burst release ofDOX from both NPs in PBS solution can be observed upto 24 h This accounts for about 40 of DOX from the totalencapsulated amount Then it followed a more gradual and

6 Journal of Nanomaterials

(c)

(b)

(a)

(d)

(e)

(f)

Wave number (cmminus1)

Tran

smitt

ance

(arb

)

5001000150020002500300035004000

Figure 3 FTIR spectra of (a) pure alginate (b) pure chitosan (c)CsnNPprepared using 2 1weight ratio of chitosan to STPP (d)Csn-Alg NP prepared using 2 1 weight ratio of chitosan to alginate (e)DOX Csn NP and (f) DOX Csn-Alg NP

Temperature (∘C)8007006005004003002001000

0

10

20

30

40

50

60

70

80

90

100

Wei

ght (

)

Csn NPDOX Csn NP

Csn-Alg NPDOX Csn-Alg NP

Figure 4 TGA thermograms of Csn NP DOX Csn NP preparedusing 2 1 weight ratio of chitosan to STPP and Csn-Alg NP andDOX Csn-Alg NP prepared using 2 1 weight ratio of chitosan toalginate

sustained release phase for the next 72 h Both profiles hadfollowed the same pattern of release but the amounts weredifferent The total amount of drug released from Csn-AlgNPs was around 60 In contrast the total amount of drugreleased from Csn NPs was around 45These results clearlyindicated that the release of DOX was retarded significantlydue to the encapsulation in nanoparticles

36 In Vitro Cytotoxicity of DOX Loaded Polymeric Nanopar-ticle Dose response curves were plotted for 3 formulations

Time (hour)

(a)

(b)

100806040200

10

20

30

40

50

60

70

Cum

ulat

ive r

elea

se (

)

Figure 5 Cumulative release of DOX from (a) DOX Csn-Alg and(b) DOXCsnNP at preselected time intervals in pH 74 PBS Resultswere reported as mean plusmn SD 119899 = 3

Free DOXDOX loaded chitosan-alginate NPDOX loaded chitosan NP

2 4 6 80

Concentration (120583gmL)

0

10

20

30

40

50

60

70

80

90

100Pe

rcen

tage

cell

viab

ility

()

Figure 6 Cytotoxicity of free DOX DOX Csn-Alg NP and DOXCsn NP against MCF-7 cells after e24 ◼48 and 99877172 hours oftreatments Cell survival was assessed via SRB assay Results werereported as mean plusmn SD 119899 = 4

free DOX DOX Csn-Alg NPs and DOX Csn NPs (Figure 6)Each experimental curve represents the average of a seriesof 3 experiments With free DOX the inhibition of MCF-7cell proliferation completely depends on the dose of thedrug used After 48 and 72 h 50 cell viability could not beobserved even at the lowest concentration (001120583gmL) offree DOX proving the reduced effect of free DOX after 24 hthat is the free DOX has dose dependent cytotoxicity thus itmight damage the normal cells during therapy whereas thecurrent work demonstrated the dose-dependence as well as

Journal of Nanomaterials 7

Free DOX

Control

Control

Control

DOX Csn NPDOX Csn-Alg NP24 h

48 h

72 h

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

Figure 7 Morphological changes of apoptosis in MCF-7 cells observed under inverted light microscopy exposed to different concentrationsof free DOX DOX Csn-Alg NP and DOX Csn NP after 24 h 48 h and 72 h after incubation

8 Journal of Nanomaterials

Free DOX

Control

Control

Control

DOX Csn NPDOX Csn-Alg NP24 h

48 h

72 h

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

Figure 8 Fluorescence microscopic observations of MCF-7 cells treated with different concentrations of free DOX DOX Csn-Alg NP andDOX Csn NP stained with AOEB after 24 h 48 h and 72 h after incubation

Journal of Nanomaterials 9

Free DOX

Control

Control

Control

DOX Csn NPDOX Csn-Alg NP

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

30min

24 h

2 h

Figure 9 Fluorescence microscopic observations of cellular uptake of MCF-7 cells treated with different concentrations of free DOX DOXCsn-Alg NP and DOX Csn NP after 30min 2 h and 24 h after incubation

10 Journal of Nanomaterials

the time dependence cytotoxicity of DOX loaded NPs withMCF-7 cell line None of the concentrations of DOX in bothNPs that were used caused 50 inhibition of cell growthat 24 h reflecting the slow release from NPs Hence duringthe first 24 h those NPs are capable of circulating in theblood and accumulating at tumour sites before starting theircytotoxic effect Thus DOX encapsulated in NPs achieve itscytotoxicity with time that is there is an effect of both timeand dose on the inhibition of cell proliferation Thereforethis result clearly indicated that the release of DOX wasretarded significantly by the encapsulation in NPs Whencomparing the two delivery systems the cytotoxicity effectof DOX Csn NPs was consistently lower than that of DOXCsn-Alg NPs (Figure 6)This is in accordance with the resultsof in vitro release of DOX being higher from Csn-Alg NPsthan from Csn NPs after 72 h (Figure 5) However bothDOX loaded systems exhibited similar behaviour with theMCF-7 cell line (Figure 6) Same concentration of DOXwhen encapsulated in NPs behaves differently than in thefree state that is encapsulated DOX releases slowly andgradually increases the concentration in the medium Henceit proves that encapsulating DOX in either system hasimproved the efficacy of DOX over free DOX A similarstudy of doxorubicin loaded magnetic NPs grafted to smartcopolymers showed that free DOX has dose dependent butnot time dependent cytotoxicity but DOX loaded magneticNPs have time dependent cytotoxicity [31]

Inverted phase contrast microscopy was used for mor-phological analysis of MCF-7 cells for observation of apop-tosis induced by the DOX loaded NPs and free DOXThese characteristicmorphological changes of apoptosis withtime and dose in MCF-7 cells on treatment after 24 48and 72 h (Figure 7) were clearly visualized Exposure tofree DOX clearly showed a higher cell confluence at lowdoses while higher concentration of free DOX decreased cellconfluence and showed a higher proportion of rounded cellsresulting from detached cells from the culture monolayerWithin the first 24 h most of the cells were damaged bythe induced cytotoxicity of free DOX reflecting its dosedependent cytotoxicity On the other hand bothDOX loadedsystems showed disappearance of the high cell confluencewith the dose of the drug and with time (Figure 7) Thisobservation is further confirmed by the fluorescence micro-scopic results of AOEB stained MCF-7 cells after 24 48and 72 h of incubation following the treatment with differentconcentrations of freeDOXandwith the same concentrationsloaded in Csn and Csn-Alg NPs Nuclei of viable cells werestained uniformly bright green by AO while the apoptoticcells exhibited yellow to orange coloration depending on thedegree of loss of membrane integrity due to costaining withEB (Figure 8) Early apoptotic cells have greenish yellownuclei while late apoptotic cells have orange to red nucleiwith condensed or fragmented chromatin (Figure 8) Thesefigures clearly displayed the difference in behaviour of DOXwhen encapsulated in NPs Free DOX has a dose dependentincrease in induction of apoptosis (Figure 8-24 h) Both thedose and time dependency of DOX encapsulated in NPs are

evident from the alterations in cell staining in all the threeseries (Figure 8) In the case of DOX loadedNPs the drug hasto be released slowly from theNPs before it can exert an effecton the cancer cells but free DOX has an immediate effecton the cells since it has direct contact with the cells initiallyThe mechanism of action of apoptosis of DOX loaded inNPs was not significantly different from that of the free DOXindicating that the encapsulation of DOX in NPs does notmake any changes to its mechanism of action of apoptosis

37 Cellular Uptake of Nanoparticles DOX is a fluorescenceactive compound having a bright red color The red colorindicates the cellular uptake of DOX when compared to theuntreated cells or cells with no internalized DOX Internal-ization of DOX in cancer cells was evaluated on MCF-7 cellsat different time intervals and different concentrations offree and encapsulated DOX Within 30min of incubationall concentrations of free DOX were localized within the cellnucleus (Figure 9 30min) whereas the localization of DOXin NPs could be visualized depending on the concentrationand on time (Figure 9) The efficient uptake of the NPs couldbe facilitated by the positive surface charge of the NPs Asimilar observationwas notedwith the in vitro cellular uptakeof paclitaxel loaded PEGylated PLGA-based NPs [32] Ingeneral NPs are nonspecifically internalized into cells viaendocytosis or phagocytosis while free DOXdiffuses throughthe cell membrane [33]

Morphology analysis suggests that the process of cellgrowth inhibition by DOX loaded in NPs occurs over a muchlonger time than by the free DOX From these observations itcould be inferred that the DOX in NPs sufficiently associateswith the cells with time

4 Conclusion

This study was made to assess the usefulness of the Csn-Alg NP system as a delivery agent for the anticancer drugDOX Comparison with DOX Csn NPs indicated that Csn-Alg NPs were more efficient at entrapment of DOX than theCsnNPsThis successful loadingwas confirmedby both FTIRand TGA data Both nanoparticles were less than 100 nm insize with positive zeta potentials The in vitro release profilesobserved for these NPs were characterized by a burst initialrelease followed by a more gradual and sustained releasephase The in vitro cytotoxicity studies demonstrated thatDOX loaded NPs have potent antigrowth effect on MCF-7cells and inhibited its cell growth in a dose and time depen-dent manner Further the observed cell apoptosis induced bythese DOX loaded NPs by phase contrast microscopy andfluorescence microscopy after AOEB staining proved thatthere is no significant change in the mechanism of action ofDOX after encapsulating in NPs and reflect the slow releaseof DOX from the NPs According to the cytotoxicity resultsDOX Csn-Alg NPs are more cytotoxic than DOX Csn NPs

Competing Interests

The authors declare that they have no competing interests

Journal of Nanomaterials 11

Acknowledgments

This research was financially supported by the HETC QIGWindow 3 Grant University of Peradeniya Peradeniya SriLanka The authors also thank Ms Damayanthi Dahanayakeat Sri Lanka Institute of nanotechnology for her valuablesupport in electron microscopy imaging

References

[1] N Lomovskaya S L Otten Y Doi-Katayama et al ldquoDoxoru-bicin overproduction in Streptomyces peucetius cloning andcharacterization of the dnrU ketoreductase and dnrV genesand the doxA cytochrome P-450 hydroxylase generdquo Journal ofBacteriology vol 181 no 1 pp 305ndash318 1999

[2] K M Laginha S Verwoert G J R Charrois and T MAllen ldquoDetermination of doxorubicin levels in whole tumorand tumor nuclei in murine breast cancer tumorsrdquo ClinicalCancer Research vol 11 no 19 pp 6944ndash6949 2005

[3] G Minotti P Menna E Salvatorelli G Cairo and L GiannildquoAnthracyclines molecular advances and pharmacologic devel-opments in antitumor activity and cardiotoxicityrdquo Pharmaco-logical Reviews vol 56 no 2 pp 185ndash229 2004

[4] M E R OrsquoBrien N Wigler M Inbar et al ldquoReduced car-diotoxicity and comparable efficacy in a phase III trial of pegy-lated liposomal doxorubicin HCl (CAELYXDoxil) versusconventional doxorubicin for first-line treatment of metastaticbreast cancerrdquo Annals of Oncology vol 15 no 3 pp 440ndash4492004

[5] C Zhang W Wang T Liu et al ldquoDoxorubicin-loaded gly-cyrrhetinic acid-modified alginate nanoparticles for liver tumorchemotherapyrdquo Biomaterials vol 33 no 7 pp 2187ndash2196 2012

[6] W H De Jong and P J A Borm ldquoDrug delivery andnanoparticles applications and hazardsrdquo International Journalof Nanomedicine vol 3 no 2 pp 133ndash149 2008

[7] G Vilar J Tulla-Puche and F Albericio ldquoPolymers and drugdelivery systemsrdquo Current Drug Delivery vol 9 no 4 pp 367ndash394 2012

[8] A Kidane and P P Bhatt ldquoRecent advances in small moleculedrug deliveryrdquo Current Opinion in Chemical Biology vol 9 no4 pp 347ndash351 2005

[9] K S Soppimath T M Aminabhavi A R Kulkarni and WE Rudzinski ldquoBiodegradable polymeric nanoparticles as drugdelivery devicesrdquo Journal of Controlled Release vol 70 no 1-2pp 1ndash20 2001

[10] T Coviello P Matricardi C Marianecci and F AlhaiqueldquoPolysaccharide hydrogels for modified release formulationsrdquoJournal of Controlled Release vol 119 no 1 pp 5ndash24 2007

[11] Z Liu Y Jiao Y Wang C Zhou and Z ZhangldquoPolysaccharides-based nanoparticles as drug deliverysystemsrdquo Advanced Drug Delivery Reviews vol 60 no 15 pp1650ndash1662 2008

[12] O Borges G Borchard J C Verhoef A De Sousa andH E Junginger ldquoPreparation of coated nanoparticles for anew mucosal vaccine delivery systemrdquo International Journal ofPharmaceutics vol 299 no 1-2 pp 155ndash166 2005

[13] Y Murata D Jinno D Liu T Isobe K Kofuji and SKawashima ldquoThe drug release profile from calcium-inducedalginate gel beads coated with an alginate hydrolysaterdquoMolecules vol 12 no 11 pp 2559ndash2566 2007

[14] P Li Y-N Dai J-P Zhang A-QWang andQWei ldquoChitosan-alginate nanoparticles as a novel drug delivery system fornifedipinerdquo International Journal of Biomedical Science vol 4no 3 pp 221ndash228 2008

[15] C K S Pillai W Paul and C P Sharma ldquoChitin and chitosanpolymers chemistry solubility and fiber formationrdquo Progress inPolymer Science vol 34 no 7 pp 641ndash678 2009

[16] F Khoushab and M Yamabhai ldquoChitin research revisitedrdquoMarine Drugs vol 8 no 7 pp 1988ndash2012 2010

[17] T Kean and M Thanou ldquoBiodegradation biodistribution andtoxicity of chitosanrdquo Advanced Drug Delivery Reviews vol 62no 1 pp 3ndash11 2010

[18] O Gaseroslashd I G Jolliffe F C Hampson P W Dettmar and GSkjak-Braeligk ldquoThe enhancement of the bioadhesive propertiesof calcium alginate gel beads by coating with chitosanrdquo Inter-national Journal of Pharmaceutics vol 175 no 2 pp 237ndash2461998

[19] M George and T E Abraham ldquoPolyionic hydrocolloids for theintestinal delivery of protein drugs alginate and chitosanmdashareviewrdquo Journal of Controlled Release vol 114 no 1 pp 1ndash142006

[20] A Saha R Yadav and N Rajendran ldquoBiomaterials fromsponges ascidians and other marine organismsrdquo InternationalJournal of Pharmaceutical Sciences Review and Research vol 27no 2 article no 17 pp 100ndash109 2014

[21] S De and D Robinson ldquoPolymer relationships duringpreparation of chitosan-alginate and poly-l-lysine-alginatenanospheresrdquo Journal of Controlled Release vol 89 no 1 pp101ndash112 2003

[22] S K Motwani S Chopra S Talegaonkar K Kohli F J Ahmadand R K Khar ldquoChitosan-sodium alginate nanoparticles assubmicroscopic reservoirs for ocular delivery formulationoptimisation and in vitro characterisationrdquo European Journal ofPharmaceutics and Biopharmaceutics vol 68 no 3 pp 513ndash5252008

[23] F-L Mi H-W Sung and S-S Shyu ldquoDrug release fromchitosan-alginate complex beads reinforced by a naturallyoccurring cross-linking agentrdquo Carbohydrate Polymers vol 48no 1 pp 61ndash72 2002

[24] Q Zhao B Han ZWang C Gao C Peng and J Shen ldquoHollowchitosan-alginate multilayer microcapsules as drug deliveryvehicle doxorubicin loading and in vitro and in vivo studiesrdquoNanomedicine Nanotechnology Biology and Medicine vol 3no 1 pp 63ndash74 2007

[25] H Shen H Shi M Xie et al ldquoBiodegradable chitosanalginateBSA-gel-capsules for pH-controlled loading and release ofdoxorubicin and treatment of pulmonary melanomardquo Journalof Materials Chemistry B vol 1 no 32 pp 3906ndash3917 2013

[26] W Feng W Nie C He et al ldquoEffect of pH-responsive algi-natechitosanmultilayers coating on delivery efficiency cellularuptake and biodistribution of mesoporous silica nanoparticlesbased nanocarriersrdquo ACS Applied Materials amp Interfaces vol 6no 11 pp 8447ndash8460 2014

[27] L Keawchaoon and R Yoksan ldquoPreparation characterizationand in vitro release study of carvacrol-loaded chitosan nanopar-ticlesrdquo Colloids and Surfaces B Biointerfaces vol 84 no 1 pp163ndash171 2011

[28] S R Samarakoon I Thabrew P B Galhena D De Silva andK Tennekoon ldquoA comparison of the cytotoxic potential of stan-dardized aqueous and ethanolic extracts of a polyherbalmixturecomprised ofNigella sativa (seeds)Hemidesmus indicus (roots)

12 Journal of Nanomaterials

and Smilax glabra (rhizome)rdquo Pharmacognosy Research vol 2no 6 pp 335ndash342 2010

[29] K Nagpal S K Singh and D N Mishra ldquoChitosan nanoparti-cles a promising system in novel drug deliveryrdquo Chemical andPharmaceutical Bulletin vol 58 no 11 pp 1423ndash1430 2010

[30] N Bhattarai J Gunn and M Zhang ldquoChitosan-based hydro-gels for controlled localized drug deliveryrdquo Advanced DrugDelivery Reviews vol 62 no 1 pp 83ndash99 2010

[31] A Akbarzadeh M Samiei S W Joo et al ldquoSynthesis charac-terization and in vitro studies of doxorubicin-loaded magneticnanoparticles grafted to smart copolymers on A549 lung cancercell linerdquo Journal of Nanobiotechnology vol 10 article 46 2012

[32] F Danhier N Lecouturier B Vroman et al ldquoPaclitaxel-loadedPEGylated PLGA-based nanoparticles in vitro and in vivoevaluationrdquo Journal of Controlled Release vol 133 no 1 pp 11ndash17 2009

[33] A des Rieux V FievezMGarinot Y-J Schneider andV PreatldquoNanoparticles as potential oral delivery systems of proteins andvaccines amechanistic approachrdquo Journal of Controlled Releasevol 116 no 1 pp 1ndash27 2006

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CeramicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CompositesJournal of

NanoparticlesJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Biomaterials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MetallurgyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

MaterialsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Nano

materials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal ofNanomaterials

4 Journal of Nanomaterials

Table 1 The size zeta potential and EE of DOX Csn and DOX Csn-Alg NPs All analyses were performed using the DOX loaded NPsprepared by using optimized formulations (chitosan STTP 2 1 and chitosan alginate 2 1)

System Average sizenm Zeta potentialmV Encapsulation efficiency Polydispersity indexDOX Csn NPs 100 plusmn 27 26 plusmn 3 65 plusmn 4 0391 plusmn 0048DOX Csn-Alg NPs 100 plusmn 35 35 plusmn 4 95 plusmn 4 0402 plusmn 0071Csn NPs 100 plusmn 25 30 plusmn 5 mdash 0352 plusmn 0046Csn-Alg NPs 100 plusmn 28 36 plusmn 3 mdash 0414 plusmn 0065

The feasibility of using Csn-Alg NPs as a delivery system forDOX was evaluated with comparison to DOX Csn NPs

31 Formation of DOX Loaded Polymeric Nanoparticles Thechitosan NPs are easily formed by the gelation with STPPvia electrostatic interactions At a pH around 48 the pro-tonated amino groups of chitosan interact electrostaticallywith negatively charged phosphate groups of STPP [29]The uncertainty of this encapsulation was the complexationof DOX into the positively charged chitosan since DOXalso exhibits a positive charge However 65 of DOX wasencapsulated into Csn NPs in this experiment overcomingthe charge repulsion Since Csn NPs have shown positiveresults for the DOX delivery an effort was taken to improvethe DOX delivery with the use of alginate polymer as wellThe polyelectrolyte complex Csn-AlgNPswas prepared usinga novel method Initially the chitosan droplet formationoccurred with the stirring with Tween 80 followed by thedroplet solidification by ionically crosslinking of the alginatesolution In aqueous solutions at pH around 4 to 55 theamine groups on chitosan became easily positively chargedAlginate on the other hand dissolved in a neutral pH solu-tionwhere the carboxylate groupswere negatively charged Inaqueous solutions with pH around 52 chitosan amino groupsinteracted with alginate carboxylate groups to form thehydrogel Since alginate is a negatively charged polymer thepositively chargedDOXwas complexed initially with alginateto obtain a higher loading of the drug to the nanoparticleTheformulation (chitosan sodium alginate 2 1) which gavean opalescent suspension with a positive zeta potential wasselected as the optimum ratio for the DOX encapsulationThis formulation resulted in 95 encapsulation efficiency ofDOX

32 Shape Size and Surface Charge of Doxorubicin LoadedNanoparticles The morphology of the polymeric NPs wasobserved by scanning electronmicroscope and TransmissionElectron Microscope and both NPs were below 20 nm andspherical in shape (Figures 1 and 2) Further the TEM imagesclearly showed the well separated well dispersed particlesAfter loading of DOX there was no noticeable change eitherin the shape or in the size in SEM and TEM images ofboth NPs In a study on carvacrol-loaded chitosan NPsKeawchaoon andYoksan have reportedTEM images showing40ndash80 nm sized NPs [27] In a previous study done by Liu etal the particle sizes of Csn-Alg NPs obtained from the trans-mission electron microscopy (TEM) were 20ndash50 nm [11]

A study by Motwani et al had shown the particle sizesof Csn-Alg NPs obtained from TEM to be around 63 nm[22] Thus using the above-mentioned method we were ableto obtain much smaller Csn-Alg NPs The sizes obtainedfrom DLS technique were larger (sim100 nm Table 1) than thesizes obtained from SEM and TEM since DLS measures thehydrodynamic diameter Also the larger particle sizemight bedue to the swellability of polymeric hydrogels in the solution[30] whereas electron microscopy shows the images of driedparticles

The zeta potentials of both DOX Csn and DOX Csn-Alg NPs show large positive values reflecting the stability ofthe colloidal suspensions (Table 1) Having positively chargedsurfaces is an added advantage when using NPs in drugdelivery since they are able to transfer easily through negativechannels in the cell membrane An increased zeta potentialvalue with DOX Csn-Alg NPs when compared to the DOXCsn NPs was caused by the higher amount of positivelycharged DOX encapsulation with Csn-Alg NPs than thatwith Csn NPs Neutralization of the charge on chitosan bythe strong negative charge of STTP versus alginate at theworking pH may have also contributed to the differences inzeta potential

33 Characterisation of DOX Loaded Nanoparticles Thesuccessful loading of DOX into the polymeric NPs wasconfirmed by FTIR and TGA techniques Chemical struc-tures of both reactants and products were analyzed by FTIRspectroscopy In the spectra of pure chitosan (Figure 3(b))characteristic peaks at 3449 cmminus1 for ndashNH

2and ndashOH stretch-

ing 1659 cmminus1 for ndashCO stretching and 1596 cmminus1 for ndashNH2

bending vibrations were observed Crosslinking of the aminegroup of chitosan with phosphate group of STPP depictsa clear shift in the peak at 3565 cmminus1 for ndashNH

2and ndashOH

stretching as shown in the spectrum of CsnNPs (Figure 3(c))On loadingDOX intoCsnNPs the FTIR spectra (Figure 3(e))show peak shifts resulting from hydrogen bonding in theabove modes that is 3578 cmminus1 for ndashNH

2and ndashOH stretch-

ing 1685 cmminus1 for ndashCO stretching and 1578 cmminus1 for ndashNH2

bending vibrations reflecting the successful loading of DOXinto the Csn NPs

In the spectra of pure chitosan the major vibrations ofndashNH2and ndashOH stretching peak appearing at 3449 cmminus1 had

shifted to 3565 cmminus1 in the spectra of Csn-Alg NPs andthe ndashCO stretching vibration at 1659 cmminus1 in pure chitosanhad also shifted to 1651 cmminus1 after interaction of the aminegroup of chitosan with the carboxylic group of alginate

Journal of Nanomaterials 5

(a) (b)

Figure 1 (a) Scanning electron microscopy image of Csn NP (b) Transmission electron microscopy image of Csn NP

(a) (b)

Figure 2 (a) Scanning electron microscopy image of Csn-Alg NP (b) Transmission electron microscopy image of Csn-Alg NP

(Figure 3(d)) Further the ndashNH2bending peak at 1596 cmminus1

disappeared due to binding with alginate to form the NPAfter loading DOX into the Csn-Alg NPs the ndashNH

2and

ndashOH stretching peak at 3565 cmminus1 and the ndashCO stretchingvibration at 1660 cmminus1 shifted to 3356 cmminus1 and to 1637 cmminus1respectively Further the appearance of a small peak at1542 cmminus1 for ndashNH

2bending after loading the DOX may

reflect the physical entrapment of DOX into the Csn-Alg NPs(Figure 3(f))

34 Thermal Behaviour of DOX Loaded Polymeric Nanopar-ticles Nanoparticles with and without DOX were analyzedby thermogravimetry to further evaluate the thermal stabilityand successful loading of DOX In the thermogram ofchitosan NPs a major one-step mass loss about 40 of totalweight was observed at a peaking temperature (inflectionpoint temperature) of 216∘C (Figure 4) Heating up to 800∘Cretained 35 of the weight of the nanoparticles from theresidual sodium ions of the STPP crosslinked Csn NPsSimilarly in DOX loaded chitosan nanoparticles a 50weight loss from 100ndash400∘C was observed in two steps dueto the combined decomposition of chitosan and DOX butthe peaking temperature of 30 weight loss had shiftedto 225∘C proving the loading of the DOX into chitosan

nanoparticles (Figure 4) A new peak with 12 weight lossin the temperature range 600ndash700∘C was observed with theDOXCsnNPs showing entrapment of DOX into the polymermatrix

The decomposition of unloaded Csn-Alg NPs had twomajor and two minor weight losses (Figure 4) The ther-mogram of DOX loaded NPs showed the same pattern ofweight loss as the unloaded particles However when thetemperature reached 350∘C 60 of weight loss was observedwith DOX loaded nanoparticles due to the decompositionof DOX complexed nanoparticles whereas it was only 50with unloaded nanoparticles Further the inflection pointtemperature of the second major step of weight loss ofunloaded composite in the range of 600ndash750∘C peaked at703∘C whereas in the DOX loaded nanocomposite it peakedat 750∘C reflecting the strong physical entrapment of DOXinto the nanoparticles

35 In Vitro Release Kinetics of DOX from PolymericNanoparticles The in vitro release profiles of both optimizedformulations in phosphate buffered saline (PBS) solution(pH-74) are shown in Figure 5 An initial burst release ofDOX from both NPs in PBS solution can be observed upto 24 h This accounts for about 40 of DOX from the totalencapsulated amount Then it followed a more gradual and

6 Journal of Nanomaterials

(c)

(b)

(a)

(d)

(e)

(f)

Wave number (cmminus1)

Tran

smitt

ance

(arb

)

5001000150020002500300035004000

Figure 3 FTIR spectra of (a) pure alginate (b) pure chitosan (c)CsnNPprepared using 2 1weight ratio of chitosan to STPP (d)Csn-Alg NP prepared using 2 1 weight ratio of chitosan to alginate (e)DOX Csn NP and (f) DOX Csn-Alg NP

Temperature (∘C)8007006005004003002001000

0

10

20

30

40

50

60

70

80

90

100

Wei

ght (

)

Csn NPDOX Csn NP

Csn-Alg NPDOX Csn-Alg NP

Figure 4 TGA thermograms of Csn NP DOX Csn NP preparedusing 2 1 weight ratio of chitosan to STPP and Csn-Alg NP andDOX Csn-Alg NP prepared using 2 1 weight ratio of chitosan toalginate

sustained release phase for the next 72 h Both profiles hadfollowed the same pattern of release but the amounts weredifferent The total amount of drug released from Csn-AlgNPs was around 60 In contrast the total amount of drugreleased from Csn NPs was around 45These results clearlyindicated that the release of DOX was retarded significantlydue to the encapsulation in nanoparticles

36 In Vitro Cytotoxicity of DOX Loaded Polymeric Nanopar-ticle Dose response curves were plotted for 3 formulations

Time (hour)

(a)

(b)

100806040200

10

20

30

40

50

60

70

Cum

ulat

ive r

elea

se (

)

Figure 5 Cumulative release of DOX from (a) DOX Csn-Alg and(b) DOXCsnNP at preselected time intervals in pH 74 PBS Resultswere reported as mean plusmn SD 119899 = 3

Free DOXDOX loaded chitosan-alginate NPDOX loaded chitosan NP

2 4 6 80

Concentration (120583gmL)

0

10

20

30

40

50

60

70

80

90

100Pe

rcen

tage

cell

viab

ility

()

Figure 6 Cytotoxicity of free DOX DOX Csn-Alg NP and DOXCsn NP against MCF-7 cells after e24 ◼48 and 99877172 hours oftreatments Cell survival was assessed via SRB assay Results werereported as mean plusmn SD 119899 = 4

free DOX DOX Csn-Alg NPs and DOX Csn NPs (Figure 6)Each experimental curve represents the average of a seriesof 3 experiments With free DOX the inhibition of MCF-7cell proliferation completely depends on the dose of thedrug used After 48 and 72 h 50 cell viability could not beobserved even at the lowest concentration (001120583gmL) offree DOX proving the reduced effect of free DOX after 24 hthat is the free DOX has dose dependent cytotoxicity thus itmight damage the normal cells during therapy whereas thecurrent work demonstrated the dose-dependence as well as

Journal of Nanomaterials 7

Free DOX

Control

Control

Control

DOX Csn NPDOX Csn-Alg NP24 h

48 h

72 h

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

Figure 7 Morphological changes of apoptosis in MCF-7 cells observed under inverted light microscopy exposed to different concentrationsof free DOX DOX Csn-Alg NP and DOX Csn NP after 24 h 48 h and 72 h after incubation

8 Journal of Nanomaterials

Free DOX

Control

Control

Control

DOX Csn NPDOX Csn-Alg NP24 h

48 h

72 h

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

Figure 8 Fluorescence microscopic observations of MCF-7 cells treated with different concentrations of free DOX DOX Csn-Alg NP andDOX Csn NP stained with AOEB after 24 h 48 h and 72 h after incubation

Journal of Nanomaterials 9

Free DOX

Control

Control

Control

DOX Csn NPDOX Csn-Alg NP

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

30min

24 h

2 h

Figure 9 Fluorescence microscopic observations of cellular uptake of MCF-7 cells treated with different concentrations of free DOX DOXCsn-Alg NP and DOX Csn NP after 30min 2 h and 24 h after incubation

10 Journal of Nanomaterials

the time dependence cytotoxicity of DOX loaded NPs withMCF-7 cell line None of the concentrations of DOX in bothNPs that were used caused 50 inhibition of cell growthat 24 h reflecting the slow release from NPs Hence duringthe first 24 h those NPs are capable of circulating in theblood and accumulating at tumour sites before starting theircytotoxic effect Thus DOX encapsulated in NPs achieve itscytotoxicity with time that is there is an effect of both timeand dose on the inhibition of cell proliferation Thereforethis result clearly indicated that the release of DOX wasretarded significantly by the encapsulation in NPs Whencomparing the two delivery systems the cytotoxicity effectof DOX Csn NPs was consistently lower than that of DOXCsn-Alg NPs (Figure 6)This is in accordance with the resultsof in vitro release of DOX being higher from Csn-Alg NPsthan from Csn NPs after 72 h (Figure 5) However bothDOX loaded systems exhibited similar behaviour with theMCF-7 cell line (Figure 6) Same concentration of DOXwhen encapsulated in NPs behaves differently than in thefree state that is encapsulated DOX releases slowly andgradually increases the concentration in the medium Henceit proves that encapsulating DOX in either system hasimproved the efficacy of DOX over free DOX A similarstudy of doxorubicin loaded magnetic NPs grafted to smartcopolymers showed that free DOX has dose dependent butnot time dependent cytotoxicity but DOX loaded magneticNPs have time dependent cytotoxicity [31]

Inverted phase contrast microscopy was used for mor-phological analysis of MCF-7 cells for observation of apop-tosis induced by the DOX loaded NPs and free DOXThese characteristicmorphological changes of apoptosis withtime and dose in MCF-7 cells on treatment after 24 48and 72 h (Figure 7) were clearly visualized Exposure tofree DOX clearly showed a higher cell confluence at lowdoses while higher concentration of free DOX decreased cellconfluence and showed a higher proportion of rounded cellsresulting from detached cells from the culture monolayerWithin the first 24 h most of the cells were damaged bythe induced cytotoxicity of free DOX reflecting its dosedependent cytotoxicity On the other hand bothDOX loadedsystems showed disappearance of the high cell confluencewith the dose of the drug and with time (Figure 7) Thisobservation is further confirmed by the fluorescence micro-scopic results of AOEB stained MCF-7 cells after 24 48and 72 h of incubation following the treatment with differentconcentrations of freeDOXandwith the same concentrationsloaded in Csn and Csn-Alg NPs Nuclei of viable cells werestained uniformly bright green by AO while the apoptoticcells exhibited yellow to orange coloration depending on thedegree of loss of membrane integrity due to costaining withEB (Figure 8) Early apoptotic cells have greenish yellownuclei while late apoptotic cells have orange to red nucleiwith condensed or fragmented chromatin (Figure 8) Thesefigures clearly displayed the difference in behaviour of DOXwhen encapsulated in NPs Free DOX has a dose dependentincrease in induction of apoptosis (Figure 8-24 h) Both thedose and time dependency of DOX encapsulated in NPs are

evident from the alterations in cell staining in all the threeseries (Figure 8) In the case of DOX loadedNPs the drug hasto be released slowly from theNPs before it can exert an effecton the cancer cells but free DOX has an immediate effecton the cells since it has direct contact with the cells initiallyThe mechanism of action of apoptosis of DOX loaded inNPs was not significantly different from that of the free DOXindicating that the encapsulation of DOX in NPs does notmake any changes to its mechanism of action of apoptosis

37 Cellular Uptake of Nanoparticles DOX is a fluorescenceactive compound having a bright red color The red colorindicates the cellular uptake of DOX when compared to theuntreated cells or cells with no internalized DOX Internal-ization of DOX in cancer cells was evaluated on MCF-7 cellsat different time intervals and different concentrations offree and encapsulated DOX Within 30min of incubationall concentrations of free DOX were localized within the cellnucleus (Figure 9 30min) whereas the localization of DOXin NPs could be visualized depending on the concentrationand on time (Figure 9) The efficient uptake of the NPs couldbe facilitated by the positive surface charge of the NPs Asimilar observationwas notedwith the in vitro cellular uptakeof paclitaxel loaded PEGylated PLGA-based NPs [32] Ingeneral NPs are nonspecifically internalized into cells viaendocytosis or phagocytosis while free DOXdiffuses throughthe cell membrane [33]

Morphology analysis suggests that the process of cellgrowth inhibition by DOX loaded in NPs occurs over a muchlonger time than by the free DOX From these observations itcould be inferred that the DOX in NPs sufficiently associateswith the cells with time

4 Conclusion

This study was made to assess the usefulness of the Csn-Alg NP system as a delivery agent for the anticancer drugDOX Comparison with DOX Csn NPs indicated that Csn-Alg NPs were more efficient at entrapment of DOX than theCsnNPsThis successful loadingwas confirmedby both FTIRand TGA data Both nanoparticles were less than 100 nm insize with positive zeta potentials The in vitro release profilesobserved for these NPs were characterized by a burst initialrelease followed by a more gradual and sustained releasephase The in vitro cytotoxicity studies demonstrated thatDOX loaded NPs have potent antigrowth effect on MCF-7cells and inhibited its cell growth in a dose and time depen-dent manner Further the observed cell apoptosis induced bythese DOX loaded NPs by phase contrast microscopy andfluorescence microscopy after AOEB staining proved thatthere is no significant change in the mechanism of action ofDOX after encapsulating in NPs and reflect the slow releaseof DOX from the NPs According to the cytotoxicity resultsDOX Csn-Alg NPs are more cytotoxic than DOX Csn NPs

Competing Interests

The authors declare that they have no competing interests

Journal of Nanomaterials 11

Acknowledgments

This research was financially supported by the HETC QIGWindow 3 Grant University of Peradeniya Peradeniya SriLanka The authors also thank Ms Damayanthi Dahanayakeat Sri Lanka Institute of nanotechnology for her valuablesupport in electron microscopy imaging

References

[1] N Lomovskaya S L Otten Y Doi-Katayama et al ldquoDoxoru-bicin overproduction in Streptomyces peucetius cloning andcharacterization of the dnrU ketoreductase and dnrV genesand the doxA cytochrome P-450 hydroxylase generdquo Journal ofBacteriology vol 181 no 1 pp 305ndash318 1999

[2] K M Laginha S Verwoert G J R Charrois and T MAllen ldquoDetermination of doxorubicin levels in whole tumorand tumor nuclei in murine breast cancer tumorsrdquo ClinicalCancer Research vol 11 no 19 pp 6944ndash6949 2005

[3] G Minotti P Menna E Salvatorelli G Cairo and L GiannildquoAnthracyclines molecular advances and pharmacologic devel-opments in antitumor activity and cardiotoxicityrdquo Pharmaco-logical Reviews vol 56 no 2 pp 185ndash229 2004

[4] M E R OrsquoBrien N Wigler M Inbar et al ldquoReduced car-diotoxicity and comparable efficacy in a phase III trial of pegy-lated liposomal doxorubicin HCl (CAELYXDoxil) versusconventional doxorubicin for first-line treatment of metastaticbreast cancerrdquo Annals of Oncology vol 15 no 3 pp 440ndash4492004

[5] C Zhang W Wang T Liu et al ldquoDoxorubicin-loaded gly-cyrrhetinic acid-modified alginate nanoparticles for liver tumorchemotherapyrdquo Biomaterials vol 33 no 7 pp 2187ndash2196 2012

[6] W H De Jong and P J A Borm ldquoDrug delivery andnanoparticles applications and hazardsrdquo International Journalof Nanomedicine vol 3 no 2 pp 133ndash149 2008

[7] G Vilar J Tulla-Puche and F Albericio ldquoPolymers and drugdelivery systemsrdquo Current Drug Delivery vol 9 no 4 pp 367ndash394 2012

[8] A Kidane and P P Bhatt ldquoRecent advances in small moleculedrug deliveryrdquo Current Opinion in Chemical Biology vol 9 no4 pp 347ndash351 2005

[9] K S Soppimath T M Aminabhavi A R Kulkarni and WE Rudzinski ldquoBiodegradable polymeric nanoparticles as drugdelivery devicesrdquo Journal of Controlled Release vol 70 no 1-2pp 1ndash20 2001

[10] T Coviello P Matricardi C Marianecci and F AlhaiqueldquoPolysaccharide hydrogels for modified release formulationsrdquoJournal of Controlled Release vol 119 no 1 pp 5ndash24 2007

[11] Z Liu Y Jiao Y Wang C Zhou and Z ZhangldquoPolysaccharides-based nanoparticles as drug deliverysystemsrdquo Advanced Drug Delivery Reviews vol 60 no 15 pp1650ndash1662 2008

[12] O Borges G Borchard J C Verhoef A De Sousa andH E Junginger ldquoPreparation of coated nanoparticles for anew mucosal vaccine delivery systemrdquo International Journal ofPharmaceutics vol 299 no 1-2 pp 155ndash166 2005

[13] Y Murata D Jinno D Liu T Isobe K Kofuji and SKawashima ldquoThe drug release profile from calcium-inducedalginate gel beads coated with an alginate hydrolysaterdquoMolecules vol 12 no 11 pp 2559ndash2566 2007

[14] P Li Y-N Dai J-P Zhang A-QWang andQWei ldquoChitosan-alginate nanoparticles as a novel drug delivery system fornifedipinerdquo International Journal of Biomedical Science vol 4no 3 pp 221ndash228 2008

[15] C K S Pillai W Paul and C P Sharma ldquoChitin and chitosanpolymers chemistry solubility and fiber formationrdquo Progress inPolymer Science vol 34 no 7 pp 641ndash678 2009

[16] F Khoushab and M Yamabhai ldquoChitin research revisitedrdquoMarine Drugs vol 8 no 7 pp 1988ndash2012 2010

[17] T Kean and M Thanou ldquoBiodegradation biodistribution andtoxicity of chitosanrdquo Advanced Drug Delivery Reviews vol 62no 1 pp 3ndash11 2010

[18] O Gaseroslashd I G Jolliffe F C Hampson P W Dettmar and GSkjak-Braeligk ldquoThe enhancement of the bioadhesive propertiesof calcium alginate gel beads by coating with chitosanrdquo Inter-national Journal of Pharmaceutics vol 175 no 2 pp 237ndash2461998

[19] M George and T E Abraham ldquoPolyionic hydrocolloids for theintestinal delivery of protein drugs alginate and chitosanmdashareviewrdquo Journal of Controlled Release vol 114 no 1 pp 1ndash142006

[20] A Saha R Yadav and N Rajendran ldquoBiomaterials fromsponges ascidians and other marine organismsrdquo InternationalJournal of Pharmaceutical Sciences Review and Research vol 27no 2 article no 17 pp 100ndash109 2014

[21] S De and D Robinson ldquoPolymer relationships duringpreparation of chitosan-alginate and poly-l-lysine-alginatenanospheresrdquo Journal of Controlled Release vol 89 no 1 pp101ndash112 2003

[22] S K Motwani S Chopra S Talegaonkar K Kohli F J Ahmadand R K Khar ldquoChitosan-sodium alginate nanoparticles assubmicroscopic reservoirs for ocular delivery formulationoptimisation and in vitro characterisationrdquo European Journal ofPharmaceutics and Biopharmaceutics vol 68 no 3 pp 513ndash5252008

[23] F-L Mi H-W Sung and S-S Shyu ldquoDrug release fromchitosan-alginate complex beads reinforced by a naturallyoccurring cross-linking agentrdquo Carbohydrate Polymers vol 48no 1 pp 61ndash72 2002

[24] Q Zhao B Han ZWang C Gao C Peng and J Shen ldquoHollowchitosan-alginate multilayer microcapsules as drug deliveryvehicle doxorubicin loading and in vitro and in vivo studiesrdquoNanomedicine Nanotechnology Biology and Medicine vol 3no 1 pp 63ndash74 2007

[25] H Shen H Shi M Xie et al ldquoBiodegradable chitosanalginateBSA-gel-capsules for pH-controlled loading and release ofdoxorubicin and treatment of pulmonary melanomardquo Journalof Materials Chemistry B vol 1 no 32 pp 3906ndash3917 2013

[26] W Feng W Nie C He et al ldquoEffect of pH-responsive algi-natechitosanmultilayers coating on delivery efficiency cellularuptake and biodistribution of mesoporous silica nanoparticlesbased nanocarriersrdquo ACS Applied Materials amp Interfaces vol 6no 11 pp 8447ndash8460 2014

[27] L Keawchaoon and R Yoksan ldquoPreparation characterizationand in vitro release study of carvacrol-loaded chitosan nanopar-ticlesrdquo Colloids and Surfaces B Biointerfaces vol 84 no 1 pp163ndash171 2011

[28] S R Samarakoon I Thabrew P B Galhena D De Silva andK Tennekoon ldquoA comparison of the cytotoxic potential of stan-dardized aqueous and ethanolic extracts of a polyherbalmixturecomprised ofNigella sativa (seeds)Hemidesmus indicus (roots)

12 Journal of Nanomaterials

and Smilax glabra (rhizome)rdquo Pharmacognosy Research vol 2no 6 pp 335ndash342 2010

[29] K Nagpal S K Singh and D N Mishra ldquoChitosan nanoparti-cles a promising system in novel drug deliveryrdquo Chemical andPharmaceutical Bulletin vol 58 no 11 pp 1423ndash1430 2010

[30] N Bhattarai J Gunn and M Zhang ldquoChitosan-based hydro-gels for controlled localized drug deliveryrdquo Advanced DrugDelivery Reviews vol 62 no 1 pp 83ndash99 2010

[31] A Akbarzadeh M Samiei S W Joo et al ldquoSynthesis charac-terization and in vitro studies of doxorubicin-loaded magneticnanoparticles grafted to smart copolymers on A549 lung cancercell linerdquo Journal of Nanobiotechnology vol 10 article 46 2012

[32] F Danhier N Lecouturier B Vroman et al ldquoPaclitaxel-loadedPEGylated PLGA-based nanoparticles in vitro and in vivoevaluationrdquo Journal of Controlled Release vol 133 no 1 pp 11ndash17 2009

[33] A des Rieux V FievezMGarinot Y-J Schneider andV PreatldquoNanoparticles as potential oral delivery systems of proteins andvaccines amechanistic approachrdquo Journal of Controlled Releasevol 116 no 1 pp 1ndash27 2006

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CeramicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CompositesJournal of

NanoparticlesJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Biomaterials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MetallurgyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

MaterialsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Nano

materials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal ofNanomaterials

Journal of Nanomaterials 5

(a) (b)

Figure 1 (a) Scanning electron microscopy image of Csn NP (b) Transmission electron microscopy image of Csn NP

(a) (b)

Figure 2 (a) Scanning electron microscopy image of Csn-Alg NP (b) Transmission electron microscopy image of Csn-Alg NP

(Figure 3(d)) Further the ndashNH2bending peak at 1596 cmminus1

disappeared due to binding with alginate to form the NPAfter loading DOX into the Csn-Alg NPs the ndashNH

2and

ndashOH stretching peak at 3565 cmminus1 and the ndashCO stretchingvibration at 1660 cmminus1 shifted to 3356 cmminus1 and to 1637 cmminus1respectively Further the appearance of a small peak at1542 cmminus1 for ndashNH

2bending after loading the DOX may

reflect the physical entrapment of DOX into the Csn-Alg NPs(Figure 3(f))

34 Thermal Behaviour of DOX Loaded Polymeric Nanopar-ticles Nanoparticles with and without DOX were analyzedby thermogravimetry to further evaluate the thermal stabilityand successful loading of DOX In the thermogram ofchitosan NPs a major one-step mass loss about 40 of totalweight was observed at a peaking temperature (inflectionpoint temperature) of 216∘C (Figure 4) Heating up to 800∘Cretained 35 of the weight of the nanoparticles from theresidual sodium ions of the STPP crosslinked Csn NPsSimilarly in DOX loaded chitosan nanoparticles a 50weight loss from 100ndash400∘C was observed in two steps dueto the combined decomposition of chitosan and DOX butthe peaking temperature of 30 weight loss had shiftedto 225∘C proving the loading of the DOX into chitosan

nanoparticles (Figure 4) A new peak with 12 weight lossin the temperature range 600ndash700∘C was observed with theDOXCsnNPs showing entrapment of DOX into the polymermatrix

The decomposition of unloaded Csn-Alg NPs had twomajor and two minor weight losses (Figure 4) The ther-mogram of DOX loaded NPs showed the same pattern ofweight loss as the unloaded particles However when thetemperature reached 350∘C 60 of weight loss was observedwith DOX loaded nanoparticles due to the decompositionof DOX complexed nanoparticles whereas it was only 50with unloaded nanoparticles Further the inflection pointtemperature of the second major step of weight loss ofunloaded composite in the range of 600ndash750∘C peaked at703∘C whereas in the DOX loaded nanocomposite it peakedat 750∘C reflecting the strong physical entrapment of DOXinto the nanoparticles

35 In Vitro Release Kinetics of DOX from PolymericNanoparticles The in vitro release profiles of both optimizedformulations in phosphate buffered saline (PBS) solution(pH-74) are shown in Figure 5 An initial burst release ofDOX from both NPs in PBS solution can be observed upto 24 h This accounts for about 40 of DOX from the totalencapsulated amount Then it followed a more gradual and

6 Journal of Nanomaterials

(c)

(b)

(a)

(d)

(e)

(f)

Wave number (cmminus1)

Tran

smitt

ance

(arb

)

5001000150020002500300035004000

Figure 3 FTIR spectra of (a) pure alginate (b) pure chitosan (c)CsnNPprepared using 2 1weight ratio of chitosan to STPP (d)Csn-Alg NP prepared using 2 1 weight ratio of chitosan to alginate (e)DOX Csn NP and (f) DOX Csn-Alg NP

Temperature (∘C)8007006005004003002001000

0

10

20

30

40

50

60

70

80

90

100

Wei

ght (

)

Csn NPDOX Csn NP

Csn-Alg NPDOX Csn-Alg NP

Figure 4 TGA thermograms of Csn NP DOX Csn NP preparedusing 2 1 weight ratio of chitosan to STPP and Csn-Alg NP andDOX Csn-Alg NP prepared using 2 1 weight ratio of chitosan toalginate

sustained release phase for the next 72 h Both profiles hadfollowed the same pattern of release but the amounts weredifferent The total amount of drug released from Csn-AlgNPs was around 60 In contrast the total amount of drugreleased from Csn NPs was around 45These results clearlyindicated that the release of DOX was retarded significantlydue to the encapsulation in nanoparticles

36 In Vitro Cytotoxicity of DOX Loaded Polymeric Nanopar-ticle Dose response curves were plotted for 3 formulations

Time (hour)

(a)

(b)

100806040200

10

20

30

40

50

60

70

Cum

ulat

ive r

elea

se (

)

Figure 5 Cumulative release of DOX from (a) DOX Csn-Alg and(b) DOXCsnNP at preselected time intervals in pH 74 PBS Resultswere reported as mean plusmn SD 119899 = 3

Free DOXDOX loaded chitosan-alginate NPDOX loaded chitosan NP

2 4 6 80

Concentration (120583gmL)

0

10

20

30

40

50

60

70

80

90

100Pe

rcen

tage

cell

viab

ility

()

Figure 6 Cytotoxicity of free DOX DOX Csn-Alg NP and DOXCsn NP against MCF-7 cells after e24 ◼48 and 99877172 hours oftreatments Cell survival was assessed via SRB assay Results werereported as mean plusmn SD 119899 = 4

free DOX DOX Csn-Alg NPs and DOX Csn NPs (Figure 6)Each experimental curve represents the average of a seriesof 3 experiments With free DOX the inhibition of MCF-7cell proliferation completely depends on the dose of thedrug used After 48 and 72 h 50 cell viability could not beobserved even at the lowest concentration (001120583gmL) offree DOX proving the reduced effect of free DOX after 24 hthat is the free DOX has dose dependent cytotoxicity thus itmight damage the normal cells during therapy whereas thecurrent work demonstrated the dose-dependence as well as

Journal of Nanomaterials 7

Free DOX

Control

Control

Control

DOX Csn NPDOX Csn-Alg NP24 h

48 h

72 h

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

Figure 7 Morphological changes of apoptosis in MCF-7 cells observed under inverted light microscopy exposed to different concentrationsof free DOX DOX Csn-Alg NP and DOX Csn NP after 24 h 48 h and 72 h after incubation

8 Journal of Nanomaterials

Free DOX

Control

Control

Control

DOX Csn NPDOX Csn-Alg NP24 h

48 h

72 h

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

Figure 8 Fluorescence microscopic observations of MCF-7 cells treated with different concentrations of free DOX DOX Csn-Alg NP andDOX Csn NP stained with AOEB after 24 h 48 h and 72 h after incubation

Journal of Nanomaterials 9

Free DOX

Control

Control

Control

DOX Csn NPDOX Csn-Alg NP

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

30min

24 h

2 h

Figure 9 Fluorescence microscopic observations of cellular uptake of MCF-7 cells treated with different concentrations of free DOX DOXCsn-Alg NP and DOX Csn NP after 30min 2 h and 24 h after incubation

10 Journal of Nanomaterials

the time dependence cytotoxicity of DOX loaded NPs withMCF-7 cell line None of the concentrations of DOX in bothNPs that were used caused 50 inhibition of cell growthat 24 h reflecting the slow release from NPs Hence duringthe first 24 h those NPs are capable of circulating in theblood and accumulating at tumour sites before starting theircytotoxic effect Thus DOX encapsulated in NPs achieve itscytotoxicity with time that is there is an effect of both timeand dose on the inhibition of cell proliferation Thereforethis result clearly indicated that the release of DOX wasretarded significantly by the encapsulation in NPs Whencomparing the two delivery systems the cytotoxicity effectof DOX Csn NPs was consistently lower than that of DOXCsn-Alg NPs (Figure 6)This is in accordance with the resultsof in vitro release of DOX being higher from Csn-Alg NPsthan from Csn NPs after 72 h (Figure 5) However bothDOX loaded systems exhibited similar behaviour with theMCF-7 cell line (Figure 6) Same concentration of DOXwhen encapsulated in NPs behaves differently than in thefree state that is encapsulated DOX releases slowly andgradually increases the concentration in the medium Henceit proves that encapsulating DOX in either system hasimproved the efficacy of DOX over free DOX A similarstudy of doxorubicin loaded magnetic NPs grafted to smartcopolymers showed that free DOX has dose dependent butnot time dependent cytotoxicity but DOX loaded magneticNPs have time dependent cytotoxicity [31]

Inverted phase contrast microscopy was used for mor-phological analysis of MCF-7 cells for observation of apop-tosis induced by the DOX loaded NPs and free DOXThese characteristicmorphological changes of apoptosis withtime and dose in MCF-7 cells on treatment after 24 48and 72 h (Figure 7) were clearly visualized Exposure tofree DOX clearly showed a higher cell confluence at lowdoses while higher concentration of free DOX decreased cellconfluence and showed a higher proportion of rounded cellsresulting from detached cells from the culture monolayerWithin the first 24 h most of the cells were damaged bythe induced cytotoxicity of free DOX reflecting its dosedependent cytotoxicity On the other hand bothDOX loadedsystems showed disappearance of the high cell confluencewith the dose of the drug and with time (Figure 7) Thisobservation is further confirmed by the fluorescence micro-scopic results of AOEB stained MCF-7 cells after 24 48and 72 h of incubation following the treatment with differentconcentrations of freeDOXandwith the same concentrationsloaded in Csn and Csn-Alg NPs Nuclei of viable cells werestained uniformly bright green by AO while the apoptoticcells exhibited yellow to orange coloration depending on thedegree of loss of membrane integrity due to costaining withEB (Figure 8) Early apoptotic cells have greenish yellownuclei while late apoptotic cells have orange to red nucleiwith condensed or fragmented chromatin (Figure 8) Thesefigures clearly displayed the difference in behaviour of DOXwhen encapsulated in NPs Free DOX has a dose dependentincrease in induction of apoptosis (Figure 8-24 h) Both thedose and time dependency of DOX encapsulated in NPs are

evident from the alterations in cell staining in all the threeseries (Figure 8) In the case of DOX loadedNPs the drug hasto be released slowly from theNPs before it can exert an effecton the cancer cells but free DOX has an immediate effecton the cells since it has direct contact with the cells initiallyThe mechanism of action of apoptosis of DOX loaded inNPs was not significantly different from that of the free DOXindicating that the encapsulation of DOX in NPs does notmake any changes to its mechanism of action of apoptosis

37 Cellular Uptake of Nanoparticles DOX is a fluorescenceactive compound having a bright red color The red colorindicates the cellular uptake of DOX when compared to theuntreated cells or cells with no internalized DOX Internal-ization of DOX in cancer cells was evaluated on MCF-7 cellsat different time intervals and different concentrations offree and encapsulated DOX Within 30min of incubationall concentrations of free DOX were localized within the cellnucleus (Figure 9 30min) whereas the localization of DOXin NPs could be visualized depending on the concentrationand on time (Figure 9) The efficient uptake of the NPs couldbe facilitated by the positive surface charge of the NPs Asimilar observationwas notedwith the in vitro cellular uptakeof paclitaxel loaded PEGylated PLGA-based NPs [32] Ingeneral NPs are nonspecifically internalized into cells viaendocytosis or phagocytosis while free DOXdiffuses throughthe cell membrane [33]

Morphology analysis suggests that the process of cellgrowth inhibition by DOX loaded in NPs occurs over a muchlonger time than by the free DOX From these observations itcould be inferred that the DOX in NPs sufficiently associateswith the cells with time

4 Conclusion

This study was made to assess the usefulness of the Csn-Alg NP system as a delivery agent for the anticancer drugDOX Comparison with DOX Csn NPs indicated that Csn-Alg NPs were more efficient at entrapment of DOX than theCsnNPsThis successful loadingwas confirmedby both FTIRand TGA data Both nanoparticles were less than 100 nm insize with positive zeta potentials The in vitro release profilesobserved for these NPs were characterized by a burst initialrelease followed by a more gradual and sustained releasephase The in vitro cytotoxicity studies demonstrated thatDOX loaded NPs have potent antigrowth effect on MCF-7cells and inhibited its cell growth in a dose and time depen-dent manner Further the observed cell apoptosis induced bythese DOX loaded NPs by phase contrast microscopy andfluorescence microscopy after AOEB staining proved thatthere is no significant change in the mechanism of action ofDOX after encapsulating in NPs and reflect the slow releaseof DOX from the NPs According to the cytotoxicity resultsDOX Csn-Alg NPs are more cytotoxic than DOX Csn NPs

Competing Interests

The authors declare that they have no competing interests

Journal of Nanomaterials 11

Acknowledgments

This research was financially supported by the HETC QIGWindow 3 Grant University of Peradeniya Peradeniya SriLanka The authors also thank Ms Damayanthi Dahanayakeat Sri Lanka Institute of nanotechnology for her valuablesupport in electron microscopy imaging

References

[1] N Lomovskaya S L Otten Y Doi-Katayama et al ldquoDoxoru-bicin overproduction in Streptomyces peucetius cloning andcharacterization of the dnrU ketoreductase and dnrV genesand the doxA cytochrome P-450 hydroxylase generdquo Journal ofBacteriology vol 181 no 1 pp 305ndash318 1999

[2] K M Laginha S Verwoert G J R Charrois and T MAllen ldquoDetermination of doxorubicin levels in whole tumorand tumor nuclei in murine breast cancer tumorsrdquo ClinicalCancer Research vol 11 no 19 pp 6944ndash6949 2005

[3] G Minotti P Menna E Salvatorelli G Cairo and L GiannildquoAnthracyclines molecular advances and pharmacologic devel-opments in antitumor activity and cardiotoxicityrdquo Pharmaco-logical Reviews vol 56 no 2 pp 185ndash229 2004

[4] M E R OrsquoBrien N Wigler M Inbar et al ldquoReduced car-diotoxicity and comparable efficacy in a phase III trial of pegy-lated liposomal doxorubicin HCl (CAELYXDoxil) versusconventional doxorubicin for first-line treatment of metastaticbreast cancerrdquo Annals of Oncology vol 15 no 3 pp 440ndash4492004

[5] C Zhang W Wang T Liu et al ldquoDoxorubicin-loaded gly-cyrrhetinic acid-modified alginate nanoparticles for liver tumorchemotherapyrdquo Biomaterials vol 33 no 7 pp 2187ndash2196 2012

[6] W H De Jong and P J A Borm ldquoDrug delivery andnanoparticles applications and hazardsrdquo International Journalof Nanomedicine vol 3 no 2 pp 133ndash149 2008

[7] G Vilar J Tulla-Puche and F Albericio ldquoPolymers and drugdelivery systemsrdquo Current Drug Delivery vol 9 no 4 pp 367ndash394 2012

[8] A Kidane and P P Bhatt ldquoRecent advances in small moleculedrug deliveryrdquo Current Opinion in Chemical Biology vol 9 no4 pp 347ndash351 2005

[9] K S Soppimath T M Aminabhavi A R Kulkarni and WE Rudzinski ldquoBiodegradable polymeric nanoparticles as drugdelivery devicesrdquo Journal of Controlled Release vol 70 no 1-2pp 1ndash20 2001

[10] T Coviello P Matricardi C Marianecci and F AlhaiqueldquoPolysaccharide hydrogels for modified release formulationsrdquoJournal of Controlled Release vol 119 no 1 pp 5ndash24 2007

[11] Z Liu Y Jiao Y Wang C Zhou and Z ZhangldquoPolysaccharides-based nanoparticles as drug deliverysystemsrdquo Advanced Drug Delivery Reviews vol 60 no 15 pp1650ndash1662 2008

[12] O Borges G Borchard J C Verhoef A De Sousa andH E Junginger ldquoPreparation of coated nanoparticles for anew mucosal vaccine delivery systemrdquo International Journal ofPharmaceutics vol 299 no 1-2 pp 155ndash166 2005

[13] Y Murata D Jinno D Liu T Isobe K Kofuji and SKawashima ldquoThe drug release profile from calcium-inducedalginate gel beads coated with an alginate hydrolysaterdquoMolecules vol 12 no 11 pp 2559ndash2566 2007

[14] P Li Y-N Dai J-P Zhang A-QWang andQWei ldquoChitosan-alginate nanoparticles as a novel drug delivery system fornifedipinerdquo International Journal of Biomedical Science vol 4no 3 pp 221ndash228 2008

[15] C K S Pillai W Paul and C P Sharma ldquoChitin and chitosanpolymers chemistry solubility and fiber formationrdquo Progress inPolymer Science vol 34 no 7 pp 641ndash678 2009

[16] F Khoushab and M Yamabhai ldquoChitin research revisitedrdquoMarine Drugs vol 8 no 7 pp 1988ndash2012 2010

[17] T Kean and M Thanou ldquoBiodegradation biodistribution andtoxicity of chitosanrdquo Advanced Drug Delivery Reviews vol 62no 1 pp 3ndash11 2010

[18] O Gaseroslashd I G Jolliffe F C Hampson P W Dettmar and GSkjak-Braeligk ldquoThe enhancement of the bioadhesive propertiesof calcium alginate gel beads by coating with chitosanrdquo Inter-national Journal of Pharmaceutics vol 175 no 2 pp 237ndash2461998

[19] M George and T E Abraham ldquoPolyionic hydrocolloids for theintestinal delivery of protein drugs alginate and chitosanmdashareviewrdquo Journal of Controlled Release vol 114 no 1 pp 1ndash142006

[20] A Saha R Yadav and N Rajendran ldquoBiomaterials fromsponges ascidians and other marine organismsrdquo InternationalJournal of Pharmaceutical Sciences Review and Research vol 27no 2 article no 17 pp 100ndash109 2014

[21] S De and D Robinson ldquoPolymer relationships duringpreparation of chitosan-alginate and poly-l-lysine-alginatenanospheresrdquo Journal of Controlled Release vol 89 no 1 pp101ndash112 2003

[22] S K Motwani S Chopra S Talegaonkar K Kohli F J Ahmadand R K Khar ldquoChitosan-sodium alginate nanoparticles assubmicroscopic reservoirs for ocular delivery formulationoptimisation and in vitro characterisationrdquo European Journal ofPharmaceutics and Biopharmaceutics vol 68 no 3 pp 513ndash5252008

[23] F-L Mi H-W Sung and S-S Shyu ldquoDrug release fromchitosan-alginate complex beads reinforced by a naturallyoccurring cross-linking agentrdquo Carbohydrate Polymers vol 48no 1 pp 61ndash72 2002

[24] Q Zhao B Han ZWang C Gao C Peng and J Shen ldquoHollowchitosan-alginate multilayer microcapsules as drug deliveryvehicle doxorubicin loading and in vitro and in vivo studiesrdquoNanomedicine Nanotechnology Biology and Medicine vol 3no 1 pp 63ndash74 2007

[25] H Shen H Shi M Xie et al ldquoBiodegradable chitosanalginateBSA-gel-capsules for pH-controlled loading and release ofdoxorubicin and treatment of pulmonary melanomardquo Journalof Materials Chemistry B vol 1 no 32 pp 3906ndash3917 2013

[26] W Feng W Nie C He et al ldquoEffect of pH-responsive algi-natechitosanmultilayers coating on delivery efficiency cellularuptake and biodistribution of mesoporous silica nanoparticlesbased nanocarriersrdquo ACS Applied Materials amp Interfaces vol 6no 11 pp 8447ndash8460 2014

[27] L Keawchaoon and R Yoksan ldquoPreparation characterizationand in vitro release study of carvacrol-loaded chitosan nanopar-ticlesrdquo Colloids and Surfaces B Biointerfaces vol 84 no 1 pp163ndash171 2011

[28] S R Samarakoon I Thabrew P B Galhena D De Silva andK Tennekoon ldquoA comparison of the cytotoxic potential of stan-dardized aqueous and ethanolic extracts of a polyherbalmixturecomprised ofNigella sativa (seeds)Hemidesmus indicus (roots)

12 Journal of Nanomaterials

and Smilax glabra (rhizome)rdquo Pharmacognosy Research vol 2no 6 pp 335ndash342 2010

[29] K Nagpal S K Singh and D N Mishra ldquoChitosan nanoparti-cles a promising system in novel drug deliveryrdquo Chemical andPharmaceutical Bulletin vol 58 no 11 pp 1423ndash1430 2010

[30] N Bhattarai J Gunn and M Zhang ldquoChitosan-based hydro-gels for controlled localized drug deliveryrdquo Advanced DrugDelivery Reviews vol 62 no 1 pp 83ndash99 2010

[31] A Akbarzadeh M Samiei S W Joo et al ldquoSynthesis charac-terization and in vitro studies of doxorubicin-loaded magneticnanoparticles grafted to smart copolymers on A549 lung cancercell linerdquo Journal of Nanobiotechnology vol 10 article 46 2012

[32] F Danhier N Lecouturier B Vroman et al ldquoPaclitaxel-loadedPEGylated PLGA-based nanoparticles in vitro and in vivoevaluationrdquo Journal of Controlled Release vol 133 no 1 pp 11ndash17 2009

[33] A des Rieux V FievezMGarinot Y-J Schneider andV PreatldquoNanoparticles as potential oral delivery systems of proteins andvaccines amechanistic approachrdquo Journal of Controlled Releasevol 116 no 1 pp 1ndash27 2006

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CeramicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CompositesJournal of

NanoparticlesJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Biomaterials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MetallurgyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

MaterialsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Nano

materials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal ofNanomaterials

6 Journal of Nanomaterials

(c)

(b)

(a)

(d)

(e)

(f)

Wave number (cmminus1)

Tran

smitt

ance

(arb

)

5001000150020002500300035004000

Figure 3 FTIR spectra of (a) pure alginate (b) pure chitosan (c)CsnNPprepared using 2 1weight ratio of chitosan to STPP (d)Csn-Alg NP prepared using 2 1 weight ratio of chitosan to alginate (e)DOX Csn NP and (f) DOX Csn-Alg NP

Temperature (∘C)8007006005004003002001000

0

10

20

30

40

50

60

70

80

90

100

Wei

ght (

)

Csn NPDOX Csn NP

Csn-Alg NPDOX Csn-Alg NP

Figure 4 TGA thermograms of Csn NP DOX Csn NP preparedusing 2 1 weight ratio of chitosan to STPP and Csn-Alg NP andDOX Csn-Alg NP prepared using 2 1 weight ratio of chitosan toalginate

sustained release phase for the next 72 h Both profiles hadfollowed the same pattern of release but the amounts weredifferent The total amount of drug released from Csn-AlgNPs was around 60 In contrast the total amount of drugreleased from Csn NPs was around 45These results clearlyindicated that the release of DOX was retarded significantlydue to the encapsulation in nanoparticles

36 In Vitro Cytotoxicity of DOX Loaded Polymeric Nanopar-ticle Dose response curves were plotted for 3 formulations

Time (hour)

(a)

(b)

100806040200

10

20

30

40

50

60

70

Cum

ulat

ive r

elea

se (

)

Figure 5 Cumulative release of DOX from (a) DOX Csn-Alg and(b) DOXCsnNP at preselected time intervals in pH 74 PBS Resultswere reported as mean plusmn SD 119899 = 3

Free DOXDOX loaded chitosan-alginate NPDOX loaded chitosan NP

2 4 6 80

Concentration (120583gmL)

0

10

20

30

40

50

60

70

80

90

100Pe

rcen

tage

cell

viab

ility

()

Figure 6 Cytotoxicity of free DOX DOX Csn-Alg NP and DOXCsn NP against MCF-7 cells after e24 ◼48 and 99877172 hours oftreatments Cell survival was assessed via SRB assay Results werereported as mean plusmn SD 119899 = 4

free DOX DOX Csn-Alg NPs and DOX Csn NPs (Figure 6)Each experimental curve represents the average of a seriesof 3 experiments With free DOX the inhibition of MCF-7cell proliferation completely depends on the dose of thedrug used After 48 and 72 h 50 cell viability could not beobserved even at the lowest concentration (001120583gmL) offree DOX proving the reduced effect of free DOX after 24 hthat is the free DOX has dose dependent cytotoxicity thus itmight damage the normal cells during therapy whereas thecurrent work demonstrated the dose-dependence as well as

Journal of Nanomaterials 7

Free DOX

Control

Control

Control

DOX Csn NPDOX Csn-Alg NP24 h

48 h

72 h

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

Figure 7 Morphological changes of apoptosis in MCF-7 cells observed under inverted light microscopy exposed to different concentrationsof free DOX DOX Csn-Alg NP and DOX Csn NP after 24 h 48 h and 72 h after incubation

8 Journal of Nanomaterials

Free DOX

Control

Control

Control

DOX Csn NPDOX Csn-Alg NP24 h

48 h

72 h

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

Figure 8 Fluorescence microscopic observations of MCF-7 cells treated with different concentrations of free DOX DOX Csn-Alg NP andDOX Csn NP stained with AOEB after 24 h 48 h and 72 h after incubation

Journal of Nanomaterials 9

Free DOX

Control

Control

Control

DOX Csn NPDOX Csn-Alg NP

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

30min

24 h

2 h

Figure 9 Fluorescence microscopic observations of cellular uptake of MCF-7 cells treated with different concentrations of free DOX DOXCsn-Alg NP and DOX Csn NP after 30min 2 h and 24 h after incubation

10 Journal of Nanomaterials

the time dependence cytotoxicity of DOX loaded NPs withMCF-7 cell line None of the concentrations of DOX in bothNPs that were used caused 50 inhibition of cell growthat 24 h reflecting the slow release from NPs Hence duringthe first 24 h those NPs are capable of circulating in theblood and accumulating at tumour sites before starting theircytotoxic effect Thus DOX encapsulated in NPs achieve itscytotoxicity with time that is there is an effect of both timeand dose on the inhibition of cell proliferation Thereforethis result clearly indicated that the release of DOX wasretarded significantly by the encapsulation in NPs Whencomparing the two delivery systems the cytotoxicity effectof DOX Csn NPs was consistently lower than that of DOXCsn-Alg NPs (Figure 6)This is in accordance with the resultsof in vitro release of DOX being higher from Csn-Alg NPsthan from Csn NPs after 72 h (Figure 5) However bothDOX loaded systems exhibited similar behaviour with theMCF-7 cell line (Figure 6) Same concentration of DOXwhen encapsulated in NPs behaves differently than in thefree state that is encapsulated DOX releases slowly andgradually increases the concentration in the medium Henceit proves that encapsulating DOX in either system hasimproved the efficacy of DOX over free DOX A similarstudy of doxorubicin loaded magnetic NPs grafted to smartcopolymers showed that free DOX has dose dependent butnot time dependent cytotoxicity but DOX loaded magneticNPs have time dependent cytotoxicity [31]

Inverted phase contrast microscopy was used for mor-phological analysis of MCF-7 cells for observation of apop-tosis induced by the DOX loaded NPs and free DOXThese characteristicmorphological changes of apoptosis withtime and dose in MCF-7 cells on treatment after 24 48and 72 h (Figure 7) were clearly visualized Exposure tofree DOX clearly showed a higher cell confluence at lowdoses while higher concentration of free DOX decreased cellconfluence and showed a higher proportion of rounded cellsresulting from detached cells from the culture monolayerWithin the first 24 h most of the cells were damaged bythe induced cytotoxicity of free DOX reflecting its dosedependent cytotoxicity On the other hand bothDOX loadedsystems showed disappearance of the high cell confluencewith the dose of the drug and with time (Figure 7) Thisobservation is further confirmed by the fluorescence micro-scopic results of AOEB stained MCF-7 cells after 24 48and 72 h of incubation following the treatment with differentconcentrations of freeDOXandwith the same concentrationsloaded in Csn and Csn-Alg NPs Nuclei of viable cells werestained uniformly bright green by AO while the apoptoticcells exhibited yellow to orange coloration depending on thedegree of loss of membrane integrity due to costaining withEB (Figure 8) Early apoptotic cells have greenish yellownuclei while late apoptotic cells have orange to red nucleiwith condensed or fragmented chromatin (Figure 8) Thesefigures clearly displayed the difference in behaviour of DOXwhen encapsulated in NPs Free DOX has a dose dependentincrease in induction of apoptosis (Figure 8-24 h) Both thedose and time dependency of DOX encapsulated in NPs are

evident from the alterations in cell staining in all the threeseries (Figure 8) In the case of DOX loadedNPs the drug hasto be released slowly from theNPs before it can exert an effecton the cancer cells but free DOX has an immediate effecton the cells since it has direct contact with the cells initiallyThe mechanism of action of apoptosis of DOX loaded inNPs was not significantly different from that of the free DOXindicating that the encapsulation of DOX in NPs does notmake any changes to its mechanism of action of apoptosis

37 Cellular Uptake of Nanoparticles DOX is a fluorescenceactive compound having a bright red color The red colorindicates the cellular uptake of DOX when compared to theuntreated cells or cells with no internalized DOX Internal-ization of DOX in cancer cells was evaluated on MCF-7 cellsat different time intervals and different concentrations offree and encapsulated DOX Within 30min of incubationall concentrations of free DOX were localized within the cellnucleus (Figure 9 30min) whereas the localization of DOXin NPs could be visualized depending on the concentrationand on time (Figure 9) The efficient uptake of the NPs couldbe facilitated by the positive surface charge of the NPs Asimilar observationwas notedwith the in vitro cellular uptakeof paclitaxel loaded PEGylated PLGA-based NPs [32] Ingeneral NPs are nonspecifically internalized into cells viaendocytosis or phagocytosis while free DOXdiffuses throughthe cell membrane [33]

Morphology analysis suggests that the process of cellgrowth inhibition by DOX loaded in NPs occurs over a muchlonger time than by the free DOX From these observations itcould be inferred that the DOX in NPs sufficiently associateswith the cells with time

4 Conclusion

This study was made to assess the usefulness of the Csn-Alg NP system as a delivery agent for the anticancer drugDOX Comparison with DOX Csn NPs indicated that Csn-Alg NPs were more efficient at entrapment of DOX than theCsnNPsThis successful loadingwas confirmedby both FTIRand TGA data Both nanoparticles were less than 100 nm insize with positive zeta potentials The in vitro release profilesobserved for these NPs were characterized by a burst initialrelease followed by a more gradual and sustained releasephase The in vitro cytotoxicity studies demonstrated thatDOX loaded NPs have potent antigrowth effect on MCF-7cells and inhibited its cell growth in a dose and time depen-dent manner Further the observed cell apoptosis induced bythese DOX loaded NPs by phase contrast microscopy andfluorescence microscopy after AOEB staining proved thatthere is no significant change in the mechanism of action ofDOX after encapsulating in NPs and reflect the slow releaseof DOX from the NPs According to the cytotoxicity resultsDOX Csn-Alg NPs are more cytotoxic than DOX Csn NPs

Competing Interests

The authors declare that they have no competing interests

Journal of Nanomaterials 11

Acknowledgments

This research was financially supported by the HETC QIGWindow 3 Grant University of Peradeniya Peradeniya SriLanka The authors also thank Ms Damayanthi Dahanayakeat Sri Lanka Institute of nanotechnology for her valuablesupport in electron microscopy imaging

References

[1] N Lomovskaya S L Otten Y Doi-Katayama et al ldquoDoxoru-bicin overproduction in Streptomyces peucetius cloning andcharacterization of the dnrU ketoreductase and dnrV genesand the doxA cytochrome P-450 hydroxylase generdquo Journal ofBacteriology vol 181 no 1 pp 305ndash318 1999

[2] K M Laginha S Verwoert G J R Charrois and T MAllen ldquoDetermination of doxorubicin levels in whole tumorand tumor nuclei in murine breast cancer tumorsrdquo ClinicalCancer Research vol 11 no 19 pp 6944ndash6949 2005

[3] G Minotti P Menna E Salvatorelli G Cairo and L GiannildquoAnthracyclines molecular advances and pharmacologic devel-opments in antitumor activity and cardiotoxicityrdquo Pharmaco-logical Reviews vol 56 no 2 pp 185ndash229 2004

[4] M E R OrsquoBrien N Wigler M Inbar et al ldquoReduced car-diotoxicity and comparable efficacy in a phase III trial of pegy-lated liposomal doxorubicin HCl (CAELYXDoxil) versusconventional doxorubicin for first-line treatment of metastaticbreast cancerrdquo Annals of Oncology vol 15 no 3 pp 440ndash4492004

[5] C Zhang W Wang T Liu et al ldquoDoxorubicin-loaded gly-cyrrhetinic acid-modified alginate nanoparticles for liver tumorchemotherapyrdquo Biomaterials vol 33 no 7 pp 2187ndash2196 2012

[6] W H De Jong and P J A Borm ldquoDrug delivery andnanoparticles applications and hazardsrdquo International Journalof Nanomedicine vol 3 no 2 pp 133ndash149 2008

[7] G Vilar J Tulla-Puche and F Albericio ldquoPolymers and drugdelivery systemsrdquo Current Drug Delivery vol 9 no 4 pp 367ndash394 2012

[8] A Kidane and P P Bhatt ldquoRecent advances in small moleculedrug deliveryrdquo Current Opinion in Chemical Biology vol 9 no4 pp 347ndash351 2005

[9] K S Soppimath T M Aminabhavi A R Kulkarni and WE Rudzinski ldquoBiodegradable polymeric nanoparticles as drugdelivery devicesrdquo Journal of Controlled Release vol 70 no 1-2pp 1ndash20 2001

[10] T Coviello P Matricardi C Marianecci and F AlhaiqueldquoPolysaccharide hydrogels for modified release formulationsrdquoJournal of Controlled Release vol 119 no 1 pp 5ndash24 2007

[11] Z Liu Y Jiao Y Wang C Zhou and Z ZhangldquoPolysaccharides-based nanoparticles as drug deliverysystemsrdquo Advanced Drug Delivery Reviews vol 60 no 15 pp1650ndash1662 2008

[12] O Borges G Borchard J C Verhoef A De Sousa andH E Junginger ldquoPreparation of coated nanoparticles for anew mucosal vaccine delivery systemrdquo International Journal ofPharmaceutics vol 299 no 1-2 pp 155ndash166 2005

[13] Y Murata D Jinno D Liu T Isobe K Kofuji and SKawashima ldquoThe drug release profile from calcium-inducedalginate gel beads coated with an alginate hydrolysaterdquoMolecules vol 12 no 11 pp 2559ndash2566 2007

[14] P Li Y-N Dai J-P Zhang A-QWang andQWei ldquoChitosan-alginate nanoparticles as a novel drug delivery system fornifedipinerdquo International Journal of Biomedical Science vol 4no 3 pp 221ndash228 2008

[15] C K S Pillai W Paul and C P Sharma ldquoChitin and chitosanpolymers chemistry solubility and fiber formationrdquo Progress inPolymer Science vol 34 no 7 pp 641ndash678 2009

[16] F Khoushab and M Yamabhai ldquoChitin research revisitedrdquoMarine Drugs vol 8 no 7 pp 1988ndash2012 2010

[17] T Kean and M Thanou ldquoBiodegradation biodistribution andtoxicity of chitosanrdquo Advanced Drug Delivery Reviews vol 62no 1 pp 3ndash11 2010

[18] O Gaseroslashd I G Jolliffe F C Hampson P W Dettmar and GSkjak-Braeligk ldquoThe enhancement of the bioadhesive propertiesof calcium alginate gel beads by coating with chitosanrdquo Inter-national Journal of Pharmaceutics vol 175 no 2 pp 237ndash2461998

[19] M George and T E Abraham ldquoPolyionic hydrocolloids for theintestinal delivery of protein drugs alginate and chitosanmdashareviewrdquo Journal of Controlled Release vol 114 no 1 pp 1ndash142006

[20] A Saha R Yadav and N Rajendran ldquoBiomaterials fromsponges ascidians and other marine organismsrdquo InternationalJournal of Pharmaceutical Sciences Review and Research vol 27no 2 article no 17 pp 100ndash109 2014

[21] S De and D Robinson ldquoPolymer relationships duringpreparation of chitosan-alginate and poly-l-lysine-alginatenanospheresrdquo Journal of Controlled Release vol 89 no 1 pp101ndash112 2003

[22] S K Motwani S Chopra S Talegaonkar K Kohli F J Ahmadand R K Khar ldquoChitosan-sodium alginate nanoparticles assubmicroscopic reservoirs for ocular delivery formulationoptimisation and in vitro characterisationrdquo European Journal ofPharmaceutics and Biopharmaceutics vol 68 no 3 pp 513ndash5252008

[23] F-L Mi H-W Sung and S-S Shyu ldquoDrug release fromchitosan-alginate complex beads reinforced by a naturallyoccurring cross-linking agentrdquo Carbohydrate Polymers vol 48no 1 pp 61ndash72 2002

[24] Q Zhao B Han ZWang C Gao C Peng and J Shen ldquoHollowchitosan-alginate multilayer microcapsules as drug deliveryvehicle doxorubicin loading and in vitro and in vivo studiesrdquoNanomedicine Nanotechnology Biology and Medicine vol 3no 1 pp 63ndash74 2007

[25] H Shen H Shi M Xie et al ldquoBiodegradable chitosanalginateBSA-gel-capsules for pH-controlled loading and release ofdoxorubicin and treatment of pulmonary melanomardquo Journalof Materials Chemistry B vol 1 no 32 pp 3906ndash3917 2013

[26] W Feng W Nie C He et al ldquoEffect of pH-responsive algi-natechitosanmultilayers coating on delivery efficiency cellularuptake and biodistribution of mesoporous silica nanoparticlesbased nanocarriersrdquo ACS Applied Materials amp Interfaces vol 6no 11 pp 8447ndash8460 2014

[27] L Keawchaoon and R Yoksan ldquoPreparation characterizationand in vitro release study of carvacrol-loaded chitosan nanopar-ticlesrdquo Colloids and Surfaces B Biointerfaces vol 84 no 1 pp163ndash171 2011

[28] S R Samarakoon I Thabrew P B Galhena D De Silva andK Tennekoon ldquoA comparison of the cytotoxic potential of stan-dardized aqueous and ethanolic extracts of a polyherbalmixturecomprised ofNigella sativa (seeds)Hemidesmus indicus (roots)

12 Journal of Nanomaterials

and Smilax glabra (rhizome)rdquo Pharmacognosy Research vol 2no 6 pp 335ndash342 2010

[29] K Nagpal S K Singh and D N Mishra ldquoChitosan nanoparti-cles a promising system in novel drug deliveryrdquo Chemical andPharmaceutical Bulletin vol 58 no 11 pp 1423ndash1430 2010

[30] N Bhattarai J Gunn and M Zhang ldquoChitosan-based hydro-gels for controlled localized drug deliveryrdquo Advanced DrugDelivery Reviews vol 62 no 1 pp 83ndash99 2010

[31] A Akbarzadeh M Samiei S W Joo et al ldquoSynthesis charac-terization and in vitro studies of doxorubicin-loaded magneticnanoparticles grafted to smart copolymers on A549 lung cancercell linerdquo Journal of Nanobiotechnology vol 10 article 46 2012

[32] F Danhier N Lecouturier B Vroman et al ldquoPaclitaxel-loadedPEGylated PLGA-based nanoparticles in vitro and in vivoevaluationrdquo Journal of Controlled Release vol 133 no 1 pp 11ndash17 2009

[33] A des Rieux V FievezMGarinot Y-J Schneider andV PreatldquoNanoparticles as potential oral delivery systems of proteins andvaccines amechanistic approachrdquo Journal of Controlled Releasevol 116 no 1 pp 1ndash27 2006

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CeramicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CompositesJournal of

NanoparticlesJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Biomaterials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MetallurgyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

MaterialsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Nano

materials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal ofNanomaterials

Journal of Nanomaterials 7

Free DOX

Control

Control

Control

DOX Csn NPDOX Csn-Alg NP24 h

48 h

72 h

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

Figure 7 Morphological changes of apoptosis in MCF-7 cells observed under inverted light microscopy exposed to different concentrationsof free DOX DOX Csn-Alg NP and DOX Csn NP after 24 h 48 h and 72 h after incubation

8 Journal of Nanomaterials

Free DOX

Control

Control

Control

DOX Csn NPDOX Csn-Alg NP24 h

48 h

72 h

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

Figure 8 Fluorescence microscopic observations of MCF-7 cells treated with different concentrations of free DOX DOX Csn-Alg NP andDOX Csn NP stained with AOEB after 24 h 48 h and 72 h after incubation

Journal of Nanomaterials 9

Free DOX

Control

Control

Control

DOX Csn NPDOX Csn-Alg NP

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

30min

24 h

2 h

Figure 9 Fluorescence microscopic observations of cellular uptake of MCF-7 cells treated with different concentrations of free DOX DOXCsn-Alg NP and DOX Csn NP after 30min 2 h and 24 h after incubation

10 Journal of Nanomaterials

the time dependence cytotoxicity of DOX loaded NPs withMCF-7 cell line None of the concentrations of DOX in bothNPs that were used caused 50 inhibition of cell growthat 24 h reflecting the slow release from NPs Hence duringthe first 24 h those NPs are capable of circulating in theblood and accumulating at tumour sites before starting theircytotoxic effect Thus DOX encapsulated in NPs achieve itscytotoxicity with time that is there is an effect of both timeand dose on the inhibition of cell proliferation Thereforethis result clearly indicated that the release of DOX wasretarded significantly by the encapsulation in NPs Whencomparing the two delivery systems the cytotoxicity effectof DOX Csn NPs was consistently lower than that of DOXCsn-Alg NPs (Figure 6)This is in accordance with the resultsof in vitro release of DOX being higher from Csn-Alg NPsthan from Csn NPs after 72 h (Figure 5) However bothDOX loaded systems exhibited similar behaviour with theMCF-7 cell line (Figure 6) Same concentration of DOXwhen encapsulated in NPs behaves differently than in thefree state that is encapsulated DOX releases slowly andgradually increases the concentration in the medium Henceit proves that encapsulating DOX in either system hasimproved the efficacy of DOX over free DOX A similarstudy of doxorubicin loaded magnetic NPs grafted to smartcopolymers showed that free DOX has dose dependent butnot time dependent cytotoxicity but DOX loaded magneticNPs have time dependent cytotoxicity [31]

Inverted phase contrast microscopy was used for mor-phological analysis of MCF-7 cells for observation of apop-tosis induced by the DOX loaded NPs and free DOXThese characteristicmorphological changes of apoptosis withtime and dose in MCF-7 cells on treatment after 24 48and 72 h (Figure 7) were clearly visualized Exposure tofree DOX clearly showed a higher cell confluence at lowdoses while higher concentration of free DOX decreased cellconfluence and showed a higher proportion of rounded cellsresulting from detached cells from the culture monolayerWithin the first 24 h most of the cells were damaged bythe induced cytotoxicity of free DOX reflecting its dosedependent cytotoxicity On the other hand bothDOX loadedsystems showed disappearance of the high cell confluencewith the dose of the drug and with time (Figure 7) Thisobservation is further confirmed by the fluorescence micro-scopic results of AOEB stained MCF-7 cells after 24 48and 72 h of incubation following the treatment with differentconcentrations of freeDOXandwith the same concentrationsloaded in Csn and Csn-Alg NPs Nuclei of viable cells werestained uniformly bright green by AO while the apoptoticcells exhibited yellow to orange coloration depending on thedegree of loss of membrane integrity due to costaining withEB (Figure 8) Early apoptotic cells have greenish yellownuclei while late apoptotic cells have orange to red nucleiwith condensed or fragmented chromatin (Figure 8) Thesefigures clearly displayed the difference in behaviour of DOXwhen encapsulated in NPs Free DOX has a dose dependentincrease in induction of apoptosis (Figure 8-24 h) Both thedose and time dependency of DOX encapsulated in NPs are

evident from the alterations in cell staining in all the threeseries (Figure 8) In the case of DOX loadedNPs the drug hasto be released slowly from theNPs before it can exert an effecton the cancer cells but free DOX has an immediate effecton the cells since it has direct contact with the cells initiallyThe mechanism of action of apoptosis of DOX loaded inNPs was not significantly different from that of the free DOXindicating that the encapsulation of DOX in NPs does notmake any changes to its mechanism of action of apoptosis

37 Cellular Uptake of Nanoparticles DOX is a fluorescenceactive compound having a bright red color The red colorindicates the cellular uptake of DOX when compared to theuntreated cells or cells with no internalized DOX Internal-ization of DOX in cancer cells was evaluated on MCF-7 cellsat different time intervals and different concentrations offree and encapsulated DOX Within 30min of incubationall concentrations of free DOX were localized within the cellnucleus (Figure 9 30min) whereas the localization of DOXin NPs could be visualized depending on the concentrationand on time (Figure 9) The efficient uptake of the NPs couldbe facilitated by the positive surface charge of the NPs Asimilar observationwas notedwith the in vitro cellular uptakeof paclitaxel loaded PEGylated PLGA-based NPs [32] Ingeneral NPs are nonspecifically internalized into cells viaendocytosis or phagocytosis while free DOXdiffuses throughthe cell membrane [33]

Morphology analysis suggests that the process of cellgrowth inhibition by DOX loaded in NPs occurs over a muchlonger time than by the free DOX From these observations itcould be inferred that the DOX in NPs sufficiently associateswith the cells with time

4 Conclusion

This study was made to assess the usefulness of the Csn-Alg NP system as a delivery agent for the anticancer drugDOX Comparison with DOX Csn NPs indicated that Csn-Alg NPs were more efficient at entrapment of DOX than theCsnNPsThis successful loadingwas confirmedby both FTIRand TGA data Both nanoparticles were less than 100 nm insize with positive zeta potentials The in vitro release profilesobserved for these NPs were characterized by a burst initialrelease followed by a more gradual and sustained releasephase The in vitro cytotoxicity studies demonstrated thatDOX loaded NPs have potent antigrowth effect on MCF-7cells and inhibited its cell growth in a dose and time depen-dent manner Further the observed cell apoptosis induced bythese DOX loaded NPs by phase contrast microscopy andfluorescence microscopy after AOEB staining proved thatthere is no significant change in the mechanism of action ofDOX after encapsulating in NPs and reflect the slow releaseof DOX from the NPs According to the cytotoxicity resultsDOX Csn-Alg NPs are more cytotoxic than DOX Csn NPs

Competing Interests

The authors declare that they have no competing interests

Journal of Nanomaterials 11

Acknowledgments

This research was financially supported by the HETC QIGWindow 3 Grant University of Peradeniya Peradeniya SriLanka The authors also thank Ms Damayanthi Dahanayakeat Sri Lanka Institute of nanotechnology for her valuablesupport in electron microscopy imaging

References

[1] N Lomovskaya S L Otten Y Doi-Katayama et al ldquoDoxoru-bicin overproduction in Streptomyces peucetius cloning andcharacterization of the dnrU ketoreductase and dnrV genesand the doxA cytochrome P-450 hydroxylase generdquo Journal ofBacteriology vol 181 no 1 pp 305ndash318 1999

[2] K M Laginha S Verwoert G J R Charrois and T MAllen ldquoDetermination of doxorubicin levels in whole tumorand tumor nuclei in murine breast cancer tumorsrdquo ClinicalCancer Research vol 11 no 19 pp 6944ndash6949 2005

[3] G Minotti P Menna E Salvatorelli G Cairo and L GiannildquoAnthracyclines molecular advances and pharmacologic devel-opments in antitumor activity and cardiotoxicityrdquo Pharmaco-logical Reviews vol 56 no 2 pp 185ndash229 2004

[4] M E R OrsquoBrien N Wigler M Inbar et al ldquoReduced car-diotoxicity and comparable efficacy in a phase III trial of pegy-lated liposomal doxorubicin HCl (CAELYXDoxil) versusconventional doxorubicin for first-line treatment of metastaticbreast cancerrdquo Annals of Oncology vol 15 no 3 pp 440ndash4492004

[5] C Zhang W Wang T Liu et al ldquoDoxorubicin-loaded gly-cyrrhetinic acid-modified alginate nanoparticles for liver tumorchemotherapyrdquo Biomaterials vol 33 no 7 pp 2187ndash2196 2012

[6] W H De Jong and P J A Borm ldquoDrug delivery andnanoparticles applications and hazardsrdquo International Journalof Nanomedicine vol 3 no 2 pp 133ndash149 2008

[7] G Vilar J Tulla-Puche and F Albericio ldquoPolymers and drugdelivery systemsrdquo Current Drug Delivery vol 9 no 4 pp 367ndash394 2012

[8] A Kidane and P P Bhatt ldquoRecent advances in small moleculedrug deliveryrdquo Current Opinion in Chemical Biology vol 9 no4 pp 347ndash351 2005

[9] K S Soppimath T M Aminabhavi A R Kulkarni and WE Rudzinski ldquoBiodegradable polymeric nanoparticles as drugdelivery devicesrdquo Journal of Controlled Release vol 70 no 1-2pp 1ndash20 2001

[10] T Coviello P Matricardi C Marianecci and F AlhaiqueldquoPolysaccharide hydrogels for modified release formulationsrdquoJournal of Controlled Release vol 119 no 1 pp 5ndash24 2007

[11] Z Liu Y Jiao Y Wang C Zhou and Z ZhangldquoPolysaccharides-based nanoparticles as drug deliverysystemsrdquo Advanced Drug Delivery Reviews vol 60 no 15 pp1650ndash1662 2008

[12] O Borges G Borchard J C Verhoef A De Sousa andH E Junginger ldquoPreparation of coated nanoparticles for anew mucosal vaccine delivery systemrdquo International Journal ofPharmaceutics vol 299 no 1-2 pp 155ndash166 2005

[13] Y Murata D Jinno D Liu T Isobe K Kofuji and SKawashima ldquoThe drug release profile from calcium-inducedalginate gel beads coated with an alginate hydrolysaterdquoMolecules vol 12 no 11 pp 2559ndash2566 2007

[14] P Li Y-N Dai J-P Zhang A-QWang andQWei ldquoChitosan-alginate nanoparticles as a novel drug delivery system fornifedipinerdquo International Journal of Biomedical Science vol 4no 3 pp 221ndash228 2008

[15] C K S Pillai W Paul and C P Sharma ldquoChitin and chitosanpolymers chemistry solubility and fiber formationrdquo Progress inPolymer Science vol 34 no 7 pp 641ndash678 2009

[16] F Khoushab and M Yamabhai ldquoChitin research revisitedrdquoMarine Drugs vol 8 no 7 pp 1988ndash2012 2010

[17] T Kean and M Thanou ldquoBiodegradation biodistribution andtoxicity of chitosanrdquo Advanced Drug Delivery Reviews vol 62no 1 pp 3ndash11 2010

[18] O Gaseroslashd I G Jolliffe F C Hampson P W Dettmar and GSkjak-Braeligk ldquoThe enhancement of the bioadhesive propertiesof calcium alginate gel beads by coating with chitosanrdquo Inter-national Journal of Pharmaceutics vol 175 no 2 pp 237ndash2461998

[19] M George and T E Abraham ldquoPolyionic hydrocolloids for theintestinal delivery of protein drugs alginate and chitosanmdashareviewrdquo Journal of Controlled Release vol 114 no 1 pp 1ndash142006

[20] A Saha R Yadav and N Rajendran ldquoBiomaterials fromsponges ascidians and other marine organismsrdquo InternationalJournal of Pharmaceutical Sciences Review and Research vol 27no 2 article no 17 pp 100ndash109 2014

[21] S De and D Robinson ldquoPolymer relationships duringpreparation of chitosan-alginate and poly-l-lysine-alginatenanospheresrdquo Journal of Controlled Release vol 89 no 1 pp101ndash112 2003

[22] S K Motwani S Chopra S Talegaonkar K Kohli F J Ahmadand R K Khar ldquoChitosan-sodium alginate nanoparticles assubmicroscopic reservoirs for ocular delivery formulationoptimisation and in vitro characterisationrdquo European Journal ofPharmaceutics and Biopharmaceutics vol 68 no 3 pp 513ndash5252008

[23] F-L Mi H-W Sung and S-S Shyu ldquoDrug release fromchitosan-alginate complex beads reinforced by a naturallyoccurring cross-linking agentrdquo Carbohydrate Polymers vol 48no 1 pp 61ndash72 2002

[24] Q Zhao B Han ZWang C Gao C Peng and J Shen ldquoHollowchitosan-alginate multilayer microcapsules as drug deliveryvehicle doxorubicin loading and in vitro and in vivo studiesrdquoNanomedicine Nanotechnology Biology and Medicine vol 3no 1 pp 63ndash74 2007

[25] H Shen H Shi M Xie et al ldquoBiodegradable chitosanalginateBSA-gel-capsules for pH-controlled loading and release ofdoxorubicin and treatment of pulmonary melanomardquo Journalof Materials Chemistry B vol 1 no 32 pp 3906ndash3917 2013

[26] W Feng W Nie C He et al ldquoEffect of pH-responsive algi-natechitosanmultilayers coating on delivery efficiency cellularuptake and biodistribution of mesoporous silica nanoparticlesbased nanocarriersrdquo ACS Applied Materials amp Interfaces vol 6no 11 pp 8447ndash8460 2014

[27] L Keawchaoon and R Yoksan ldquoPreparation characterizationand in vitro release study of carvacrol-loaded chitosan nanopar-ticlesrdquo Colloids and Surfaces B Biointerfaces vol 84 no 1 pp163ndash171 2011

[28] S R Samarakoon I Thabrew P B Galhena D De Silva andK Tennekoon ldquoA comparison of the cytotoxic potential of stan-dardized aqueous and ethanolic extracts of a polyherbalmixturecomprised ofNigella sativa (seeds)Hemidesmus indicus (roots)

12 Journal of Nanomaterials

and Smilax glabra (rhizome)rdquo Pharmacognosy Research vol 2no 6 pp 335ndash342 2010

[29] K Nagpal S K Singh and D N Mishra ldquoChitosan nanoparti-cles a promising system in novel drug deliveryrdquo Chemical andPharmaceutical Bulletin vol 58 no 11 pp 1423ndash1430 2010

[30] N Bhattarai J Gunn and M Zhang ldquoChitosan-based hydro-gels for controlled localized drug deliveryrdquo Advanced DrugDelivery Reviews vol 62 no 1 pp 83ndash99 2010

[31] A Akbarzadeh M Samiei S W Joo et al ldquoSynthesis charac-terization and in vitro studies of doxorubicin-loaded magneticnanoparticles grafted to smart copolymers on A549 lung cancercell linerdquo Journal of Nanobiotechnology vol 10 article 46 2012

[32] F Danhier N Lecouturier B Vroman et al ldquoPaclitaxel-loadedPEGylated PLGA-based nanoparticles in vitro and in vivoevaluationrdquo Journal of Controlled Release vol 133 no 1 pp 11ndash17 2009

[33] A des Rieux V FievezMGarinot Y-J Schneider andV PreatldquoNanoparticles as potential oral delivery systems of proteins andvaccines amechanistic approachrdquo Journal of Controlled Releasevol 116 no 1 pp 1ndash27 2006

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CeramicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CompositesJournal of

NanoparticlesJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Biomaterials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MetallurgyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

MaterialsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Nano

materials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal ofNanomaterials

8 Journal of Nanomaterials

Free DOX

Control

Control

Control

DOX Csn NPDOX Csn-Alg NP24 h

48 h

72 h

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

Figure 8 Fluorescence microscopic observations of MCF-7 cells treated with different concentrations of free DOX DOX Csn-Alg NP andDOX Csn NP stained with AOEB after 24 h 48 h and 72 h after incubation

Journal of Nanomaterials 9

Free DOX

Control

Control

Control

DOX Csn NPDOX Csn-Alg NP

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

30min

24 h

2 h

Figure 9 Fluorescence microscopic observations of cellular uptake of MCF-7 cells treated with different concentrations of free DOX DOXCsn-Alg NP and DOX Csn NP after 30min 2 h and 24 h after incubation

10 Journal of Nanomaterials

the time dependence cytotoxicity of DOX loaded NPs withMCF-7 cell line None of the concentrations of DOX in bothNPs that were used caused 50 inhibition of cell growthat 24 h reflecting the slow release from NPs Hence duringthe first 24 h those NPs are capable of circulating in theblood and accumulating at tumour sites before starting theircytotoxic effect Thus DOX encapsulated in NPs achieve itscytotoxicity with time that is there is an effect of both timeand dose on the inhibition of cell proliferation Thereforethis result clearly indicated that the release of DOX wasretarded significantly by the encapsulation in NPs Whencomparing the two delivery systems the cytotoxicity effectof DOX Csn NPs was consistently lower than that of DOXCsn-Alg NPs (Figure 6)This is in accordance with the resultsof in vitro release of DOX being higher from Csn-Alg NPsthan from Csn NPs after 72 h (Figure 5) However bothDOX loaded systems exhibited similar behaviour with theMCF-7 cell line (Figure 6) Same concentration of DOXwhen encapsulated in NPs behaves differently than in thefree state that is encapsulated DOX releases slowly andgradually increases the concentration in the medium Henceit proves that encapsulating DOX in either system hasimproved the efficacy of DOX over free DOX A similarstudy of doxorubicin loaded magnetic NPs grafted to smartcopolymers showed that free DOX has dose dependent butnot time dependent cytotoxicity but DOX loaded magneticNPs have time dependent cytotoxicity [31]

Inverted phase contrast microscopy was used for mor-phological analysis of MCF-7 cells for observation of apop-tosis induced by the DOX loaded NPs and free DOXThese characteristicmorphological changes of apoptosis withtime and dose in MCF-7 cells on treatment after 24 48and 72 h (Figure 7) were clearly visualized Exposure tofree DOX clearly showed a higher cell confluence at lowdoses while higher concentration of free DOX decreased cellconfluence and showed a higher proportion of rounded cellsresulting from detached cells from the culture monolayerWithin the first 24 h most of the cells were damaged bythe induced cytotoxicity of free DOX reflecting its dosedependent cytotoxicity On the other hand bothDOX loadedsystems showed disappearance of the high cell confluencewith the dose of the drug and with time (Figure 7) Thisobservation is further confirmed by the fluorescence micro-scopic results of AOEB stained MCF-7 cells after 24 48and 72 h of incubation following the treatment with differentconcentrations of freeDOXandwith the same concentrationsloaded in Csn and Csn-Alg NPs Nuclei of viable cells werestained uniformly bright green by AO while the apoptoticcells exhibited yellow to orange coloration depending on thedegree of loss of membrane integrity due to costaining withEB (Figure 8) Early apoptotic cells have greenish yellownuclei while late apoptotic cells have orange to red nucleiwith condensed or fragmented chromatin (Figure 8) Thesefigures clearly displayed the difference in behaviour of DOXwhen encapsulated in NPs Free DOX has a dose dependentincrease in induction of apoptosis (Figure 8-24 h) Both thedose and time dependency of DOX encapsulated in NPs are

evident from the alterations in cell staining in all the threeseries (Figure 8) In the case of DOX loadedNPs the drug hasto be released slowly from theNPs before it can exert an effecton the cancer cells but free DOX has an immediate effecton the cells since it has direct contact with the cells initiallyThe mechanism of action of apoptosis of DOX loaded inNPs was not significantly different from that of the free DOXindicating that the encapsulation of DOX in NPs does notmake any changes to its mechanism of action of apoptosis

37 Cellular Uptake of Nanoparticles DOX is a fluorescenceactive compound having a bright red color The red colorindicates the cellular uptake of DOX when compared to theuntreated cells or cells with no internalized DOX Internal-ization of DOX in cancer cells was evaluated on MCF-7 cellsat different time intervals and different concentrations offree and encapsulated DOX Within 30min of incubationall concentrations of free DOX were localized within the cellnucleus (Figure 9 30min) whereas the localization of DOXin NPs could be visualized depending on the concentrationand on time (Figure 9) The efficient uptake of the NPs couldbe facilitated by the positive surface charge of the NPs Asimilar observationwas notedwith the in vitro cellular uptakeof paclitaxel loaded PEGylated PLGA-based NPs [32] Ingeneral NPs are nonspecifically internalized into cells viaendocytosis or phagocytosis while free DOXdiffuses throughthe cell membrane [33]

Morphology analysis suggests that the process of cellgrowth inhibition by DOX loaded in NPs occurs over a muchlonger time than by the free DOX From these observations itcould be inferred that the DOX in NPs sufficiently associateswith the cells with time

4 Conclusion

This study was made to assess the usefulness of the Csn-Alg NP system as a delivery agent for the anticancer drugDOX Comparison with DOX Csn NPs indicated that Csn-Alg NPs were more efficient at entrapment of DOX than theCsnNPsThis successful loadingwas confirmedby both FTIRand TGA data Both nanoparticles were less than 100 nm insize with positive zeta potentials The in vitro release profilesobserved for these NPs were characterized by a burst initialrelease followed by a more gradual and sustained releasephase The in vitro cytotoxicity studies demonstrated thatDOX loaded NPs have potent antigrowth effect on MCF-7cells and inhibited its cell growth in a dose and time depen-dent manner Further the observed cell apoptosis induced bythese DOX loaded NPs by phase contrast microscopy andfluorescence microscopy after AOEB staining proved thatthere is no significant change in the mechanism of action ofDOX after encapsulating in NPs and reflect the slow releaseof DOX from the NPs According to the cytotoxicity resultsDOX Csn-Alg NPs are more cytotoxic than DOX Csn NPs

Competing Interests

The authors declare that they have no competing interests

Journal of Nanomaterials 11

Acknowledgments

This research was financially supported by the HETC QIGWindow 3 Grant University of Peradeniya Peradeniya SriLanka The authors also thank Ms Damayanthi Dahanayakeat Sri Lanka Institute of nanotechnology for her valuablesupport in electron microscopy imaging

References

[1] N Lomovskaya S L Otten Y Doi-Katayama et al ldquoDoxoru-bicin overproduction in Streptomyces peucetius cloning andcharacterization of the dnrU ketoreductase and dnrV genesand the doxA cytochrome P-450 hydroxylase generdquo Journal ofBacteriology vol 181 no 1 pp 305ndash318 1999

[2] K M Laginha S Verwoert G J R Charrois and T MAllen ldquoDetermination of doxorubicin levels in whole tumorand tumor nuclei in murine breast cancer tumorsrdquo ClinicalCancer Research vol 11 no 19 pp 6944ndash6949 2005

[3] G Minotti P Menna E Salvatorelli G Cairo and L GiannildquoAnthracyclines molecular advances and pharmacologic devel-opments in antitumor activity and cardiotoxicityrdquo Pharmaco-logical Reviews vol 56 no 2 pp 185ndash229 2004

[4] M E R OrsquoBrien N Wigler M Inbar et al ldquoReduced car-diotoxicity and comparable efficacy in a phase III trial of pegy-lated liposomal doxorubicin HCl (CAELYXDoxil) versusconventional doxorubicin for first-line treatment of metastaticbreast cancerrdquo Annals of Oncology vol 15 no 3 pp 440ndash4492004

[5] C Zhang W Wang T Liu et al ldquoDoxorubicin-loaded gly-cyrrhetinic acid-modified alginate nanoparticles for liver tumorchemotherapyrdquo Biomaterials vol 33 no 7 pp 2187ndash2196 2012

[6] W H De Jong and P J A Borm ldquoDrug delivery andnanoparticles applications and hazardsrdquo International Journalof Nanomedicine vol 3 no 2 pp 133ndash149 2008

[7] G Vilar J Tulla-Puche and F Albericio ldquoPolymers and drugdelivery systemsrdquo Current Drug Delivery vol 9 no 4 pp 367ndash394 2012

[8] A Kidane and P P Bhatt ldquoRecent advances in small moleculedrug deliveryrdquo Current Opinion in Chemical Biology vol 9 no4 pp 347ndash351 2005

[9] K S Soppimath T M Aminabhavi A R Kulkarni and WE Rudzinski ldquoBiodegradable polymeric nanoparticles as drugdelivery devicesrdquo Journal of Controlled Release vol 70 no 1-2pp 1ndash20 2001

[10] T Coviello P Matricardi C Marianecci and F AlhaiqueldquoPolysaccharide hydrogels for modified release formulationsrdquoJournal of Controlled Release vol 119 no 1 pp 5ndash24 2007

[11] Z Liu Y Jiao Y Wang C Zhou and Z ZhangldquoPolysaccharides-based nanoparticles as drug deliverysystemsrdquo Advanced Drug Delivery Reviews vol 60 no 15 pp1650ndash1662 2008

[12] O Borges G Borchard J C Verhoef A De Sousa andH E Junginger ldquoPreparation of coated nanoparticles for anew mucosal vaccine delivery systemrdquo International Journal ofPharmaceutics vol 299 no 1-2 pp 155ndash166 2005

[13] Y Murata D Jinno D Liu T Isobe K Kofuji and SKawashima ldquoThe drug release profile from calcium-inducedalginate gel beads coated with an alginate hydrolysaterdquoMolecules vol 12 no 11 pp 2559ndash2566 2007

[14] P Li Y-N Dai J-P Zhang A-QWang andQWei ldquoChitosan-alginate nanoparticles as a novel drug delivery system fornifedipinerdquo International Journal of Biomedical Science vol 4no 3 pp 221ndash228 2008

[15] C K S Pillai W Paul and C P Sharma ldquoChitin and chitosanpolymers chemistry solubility and fiber formationrdquo Progress inPolymer Science vol 34 no 7 pp 641ndash678 2009

[16] F Khoushab and M Yamabhai ldquoChitin research revisitedrdquoMarine Drugs vol 8 no 7 pp 1988ndash2012 2010

[17] T Kean and M Thanou ldquoBiodegradation biodistribution andtoxicity of chitosanrdquo Advanced Drug Delivery Reviews vol 62no 1 pp 3ndash11 2010

[18] O Gaseroslashd I G Jolliffe F C Hampson P W Dettmar and GSkjak-Braeligk ldquoThe enhancement of the bioadhesive propertiesof calcium alginate gel beads by coating with chitosanrdquo Inter-national Journal of Pharmaceutics vol 175 no 2 pp 237ndash2461998

[19] M George and T E Abraham ldquoPolyionic hydrocolloids for theintestinal delivery of protein drugs alginate and chitosanmdashareviewrdquo Journal of Controlled Release vol 114 no 1 pp 1ndash142006

[20] A Saha R Yadav and N Rajendran ldquoBiomaterials fromsponges ascidians and other marine organismsrdquo InternationalJournal of Pharmaceutical Sciences Review and Research vol 27no 2 article no 17 pp 100ndash109 2014

[21] S De and D Robinson ldquoPolymer relationships duringpreparation of chitosan-alginate and poly-l-lysine-alginatenanospheresrdquo Journal of Controlled Release vol 89 no 1 pp101ndash112 2003

[22] S K Motwani S Chopra S Talegaonkar K Kohli F J Ahmadand R K Khar ldquoChitosan-sodium alginate nanoparticles assubmicroscopic reservoirs for ocular delivery formulationoptimisation and in vitro characterisationrdquo European Journal ofPharmaceutics and Biopharmaceutics vol 68 no 3 pp 513ndash5252008

[23] F-L Mi H-W Sung and S-S Shyu ldquoDrug release fromchitosan-alginate complex beads reinforced by a naturallyoccurring cross-linking agentrdquo Carbohydrate Polymers vol 48no 1 pp 61ndash72 2002

[24] Q Zhao B Han ZWang C Gao C Peng and J Shen ldquoHollowchitosan-alginate multilayer microcapsules as drug deliveryvehicle doxorubicin loading and in vitro and in vivo studiesrdquoNanomedicine Nanotechnology Biology and Medicine vol 3no 1 pp 63ndash74 2007

[25] H Shen H Shi M Xie et al ldquoBiodegradable chitosanalginateBSA-gel-capsules for pH-controlled loading and release ofdoxorubicin and treatment of pulmonary melanomardquo Journalof Materials Chemistry B vol 1 no 32 pp 3906ndash3917 2013

[26] W Feng W Nie C He et al ldquoEffect of pH-responsive algi-natechitosanmultilayers coating on delivery efficiency cellularuptake and biodistribution of mesoporous silica nanoparticlesbased nanocarriersrdquo ACS Applied Materials amp Interfaces vol 6no 11 pp 8447ndash8460 2014

[27] L Keawchaoon and R Yoksan ldquoPreparation characterizationand in vitro release study of carvacrol-loaded chitosan nanopar-ticlesrdquo Colloids and Surfaces B Biointerfaces vol 84 no 1 pp163ndash171 2011

[28] S R Samarakoon I Thabrew P B Galhena D De Silva andK Tennekoon ldquoA comparison of the cytotoxic potential of stan-dardized aqueous and ethanolic extracts of a polyherbalmixturecomprised ofNigella sativa (seeds)Hemidesmus indicus (roots)

12 Journal of Nanomaterials

and Smilax glabra (rhizome)rdquo Pharmacognosy Research vol 2no 6 pp 335ndash342 2010

[29] K Nagpal S K Singh and D N Mishra ldquoChitosan nanoparti-cles a promising system in novel drug deliveryrdquo Chemical andPharmaceutical Bulletin vol 58 no 11 pp 1423ndash1430 2010

[30] N Bhattarai J Gunn and M Zhang ldquoChitosan-based hydro-gels for controlled localized drug deliveryrdquo Advanced DrugDelivery Reviews vol 62 no 1 pp 83ndash99 2010

[31] A Akbarzadeh M Samiei S W Joo et al ldquoSynthesis charac-terization and in vitro studies of doxorubicin-loaded magneticnanoparticles grafted to smart copolymers on A549 lung cancercell linerdquo Journal of Nanobiotechnology vol 10 article 46 2012

[32] F Danhier N Lecouturier B Vroman et al ldquoPaclitaxel-loadedPEGylated PLGA-based nanoparticles in vitro and in vivoevaluationrdquo Journal of Controlled Release vol 133 no 1 pp 11ndash17 2009

[33] A des Rieux V FievezMGarinot Y-J Schneider andV PreatldquoNanoparticles as potential oral delivery systems of proteins andvaccines amechanistic approachrdquo Journal of Controlled Releasevol 116 no 1 pp 1ndash27 2006

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CeramicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CompositesJournal of

NanoparticlesJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Biomaterials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MetallurgyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

MaterialsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Nano

materials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal ofNanomaterials

Journal of Nanomaterials 9

Free DOX

Control

Control

Control

DOX Csn NPDOX Csn-Alg NP

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

001 120583gmL

100 120583gmL

800 120583gmL

30min

24 h

2 h

Figure 9 Fluorescence microscopic observations of cellular uptake of MCF-7 cells treated with different concentrations of free DOX DOXCsn-Alg NP and DOX Csn NP after 30min 2 h and 24 h after incubation

10 Journal of Nanomaterials

the time dependence cytotoxicity of DOX loaded NPs withMCF-7 cell line None of the concentrations of DOX in bothNPs that were used caused 50 inhibition of cell growthat 24 h reflecting the slow release from NPs Hence duringthe first 24 h those NPs are capable of circulating in theblood and accumulating at tumour sites before starting theircytotoxic effect Thus DOX encapsulated in NPs achieve itscytotoxicity with time that is there is an effect of both timeand dose on the inhibition of cell proliferation Thereforethis result clearly indicated that the release of DOX wasretarded significantly by the encapsulation in NPs Whencomparing the two delivery systems the cytotoxicity effectof DOX Csn NPs was consistently lower than that of DOXCsn-Alg NPs (Figure 6)This is in accordance with the resultsof in vitro release of DOX being higher from Csn-Alg NPsthan from Csn NPs after 72 h (Figure 5) However bothDOX loaded systems exhibited similar behaviour with theMCF-7 cell line (Figure 6) Same concentration of DOXwhen encapsulated in NPs behaves differently than in thefree state that is encapsulated DOX releases slowly andgradually increases the concentration in the medium Henceit proves that encapsulating DOX in either system hasimproved the efficacy of DOX over free DOX A similarstudy of doxorubicin loaded magnetic NPs grafted to smartcopolymers showed that free DOX has dose dependent butnot time dependent cytotoxicity but DOX loaded magneticNPs have time dependent cytotoxicity [31]

Inverted phase contrast microscopy was used for mor-phological analysis of MCF-7 cells for observation of apop-tosis induced by the DOX loaded NPs and free DOXThese characteristicmorphological changes of apoptosis withtime and dose in MCF-7 cells on treatment after 24 48and 72 h (Figure 7) were clearly visualized Exposure tofree DOX clearly showed a higher cell confluence at lowdoses while higher concentration of free DOX decreased cellconfluence and showed a higher proportion of rounded cellsresulting from detached cells from the culture monolayerWithin the first 24 h most of the cells were damaged bythe induced cytotoxicity of free DOX reflecting its dosedependent cytotoxicity On the other hand bothDOX loadedsystems showed disappearance of the high cell confluencewith the dose of the drug and with time (Figure 7) Thisobservation is further confirmed by the fluorescence micro-scopic results of AOEB stained MCF-7 cells after 24 48and 72 h of incubation following the treatment with differentconcentrations of freeDOXandwith the same concentrationsloaded in Csn and Csn-Alg NPs Nuclei of viable cells werestained uniformly bright green by AO while the apoptoticcells exhibited yellow to orange coloration depending on thedegree of loss of membrane integrity due to costaining withEB (Figure 8) Early apoptotic cells have greenish yellownuclei while late apoptotic cells have orange to red nucleiwith condensed or fragmented chromatin (Figure 8) Thesefigures clearly displayed the difference in behaviour of DOXwhen encapsulated in NPs Free DOX has a dose dependentincrease in induction of apoptosis (Figure 8-24 h) Both thedose and time dependency of DOX encapsulated in NPs are

evident from the alterations in cell staining in all the threeseries (Figure 8) In the case of DOX loadedNPs the drug hasto be released slowly from theNPs before it can exert an effecton the cancer cells but free DOX has an immediate effecton the cells since it has direct contact with the cells initiallyThe mechanism of action of apoptosis of DOX loaded inNPs was not significantly different from that of the free DOXindicating that the encapsulation of DOX in NPs does notmake any changes to its mechanism of action of apoptosis

37 Cellular Uptake of Nanoparticles DOX is a fluorescenceactive compound having a bright red color The red colorindicates the cellular uptake of DOX when compared to theuntreated cells or cells with no internalized DOX Internal-ization of DOX in cancer cells was evaluated on MCF-7 cellsat different time intervals and different concentrations offree and encapsulated DOX Within 30min of incubationall concentrations of free DOX were localized within the cellnucleus (Figure 9 30min) whereas the localization of DOXin NPs could be visualized depending on the concentrationand on time (Figure 9) The efficient uptake of the NPs couldbe facilitated by the positive surface charge of the NPs Asimilar observationwas notedwith the in vitro cellular uptakeof paclitaxel loaded PEGylated PLGA-based NPs [32] Ingeneral NPs are nonspecifically internalized into cells viaendocytosis or phagocytosis while free DOXdiffuses throughthe cell membrane [33]

Morphology analysis suggests that the process of cellgrowth inhibition by DOX loaded in NPs occurs over a muchlonger time than by the free DOX From these observations itcould be inferred that the DOX in NPs sufficiently associateswith the cells with time

4 Conclusion

This study was made to assess the usefulness of the Csn-Alg NP system as a delivery agent for the anticancer drugDOX Comparison with DOX Csn NPs indicated that Csn-Alg NPs were more efficient at entrapment of DOX than theCsnNPsThis successful loadingwas confirmedby both FTIRand TGA data Both nanoparticles were less than 100 nm insize with positive zeta potentials The in vitro release profilesobserved for these NPs were characterized by a burst initialrelease followed by a more gradual and sustained releasephase The in vitro cytotoxicity studies demonstrated thatDOX loaded NPs have potent antigrowth effect on MCF-7cells and inhibited its cell growth in a dose and time depen-dent manner Further the observed cell apoptosis induced bythese DOX loaded NPs by phase contrast microscopy andfluorescence microscopy after AOEB staining proved thatthere is no significant change in the mechanism of action ofDOX after encapsulating in NPs and reflect the slow releaseof DOX from the NPs According to the cytotoxicity resultsDOX Csn-Alg NPs are more cytotoxic than DOX Csn NPs

Competing Interests

The authors declare that they have no competing interests

Journal of Nanomaterials 11

Acknowledgments

This research was financially supported by the HETC QIGWindow 3 Grant University of Peradeniya Peradeniya SriLanka The authors also thank Ms Damayanthi Dahanayakeat Sri Lanka Institute of nanotechnology for her valuablesupport in electron microscopy imaging

References

[1] N Lomovskaya S L Otten Y Doi-Katayama et al ldquoDoxoru-bicin overproduction in Streptomyces peucetius cloning andcharacterization of the dnrU ketoreductase and dnrV genesand the doxA cytochrome P-450 hydroxylase generdquo Journal ofBacteriology vol 181 no 1 pp 305ndash318 1999

[2] K M Laginha S Verwoert G J R Charrois and T MAllen ldquoDetermination of doxorubicin levels in whole tumorand tumor nuclei in murine breast cancer tumorsrdquo ClinicalCancer Research vol 11 no 19 pp 6944ndash6949 2005

[3] G Minotti P Menna E Salvatorelli G Cairo and L GiannildquoAnthracyclines molecular advances and pharmacologic devel-opments in antitumor activity and cardiotoxicityrdquo Pharmaco-logical Reviews vol 56 no 2 pp 185ndash229 2004

[4] M E R OrsquoBrien N Wigler M Inbar et al ldquoReduced car-diotoxicity and comparable efficacy in a phase III trial of pegy-lated liposomal doxorubicin HCl (CAELYXDoxil) versusconventional doxorubicin for first-line treatment of metastaticbreast cancerrdquo Annals of Oncology vol 15 no 3 pp 440ndash4492004

[5] C Zhang W Wang T Liu et al ldquoDoxorubicin-loaded gly-cyrrhetinic acid-modified alginate nanoparticles for liver tumorchemotherapyrdquo Biomaterials vol 33 no 7 pp 2187ndash2196 2012

[6] W H De Jong and P J A Borm ldquoDrug delivery andnanoparticles applications and hazardsrdquo International Journalof Nanomedicine vol 3 no 2 pp 133ndash149 2008

[7] G Vilar J Tulla-Puche and F Albericio ldquoPolymers and drugdelivery systemsrdquo Current Drug Delivery vol 9 no 4 pp 367ndash394 2012

[8] A Kidane and P P Bhatt ldquoRecent advances in small moleculedrug deliveryrdquo Current Opinion in Chemical Biology vol 9 no4 pp 347ndash351 2005

[9] K S Soppimath T M Aminabhavi A R Kulkarni and WE Rudzinski ldquoBiodegradable polymeric nanoparticles as drugdelivery devicesrdquo Journal of Controlled Release vol 70 no 1-2pp 1ndash20 2001

[10] T Coviello P Matricardi C Marianecci and F AlhaiqueldquoPolysaccharide hydrogels for modified release formulationsrdquoJournal of Controlled Release vol 119 no 1 pp 5ndash24 2007

[11] Z Liu Y Jiao Y Wang C Zhou and Z ZhangldquoPolysaccharides-based nanoparticles as drug deliverysystemsrdquo Advanced Drug Delivery Reviews vol 60 no 15 pp1650ndash1662 2008

[12] O Borges G Borchard J C Verhoef A De Sousa andH E Junginger ldquoPreparation of coated nanoparticles for anew mucosal vaccine delivery systemrdquo International Journal ofPharmaceutics vol 299 no 1-2 pp 155ndash166 2005

[13] Y Murata D Jinno D Liu T Isobe K Kofuji and SKawashima ldquoThe drug release profile from calcium-inducedalginate gel beads coated with an alginate hydrolysaterdquoMolecules vol 12 no 11 pp 2559ndash2566 2007

[14] P Li Y-N Dai J-P Zhang A-QWang andQWei ldquoChitosan-alginate nanoparticles as a novel drug delivery system fornifedipinerdquo International Journal of Biomedical Science vol 4no 3 pp 221ndash228 2008

[15] C K S Pillai W Paul and C P Sharma ldquoChitin and chitosanpolymers chemistry solubility and fiber formationrdquo Progress inPolymer Science vol 34 no 7 pp 641ndash678 2009

[16] F Khoushab and M Yamabhai ldquoChitin research revisitedrdquoMarine Drugs vol 8 no 7 pp 1988ndash2012 2010

[17] T Kean and M Thanou ldquoBiodegradation biodistribution andtoxicity of chitosanrdquo Advanced Drug Delivery Reviews vol 62no 1 pp 3ndash11 2010

[18] O Gaseroslashd I G Jolliffe F C Hampson P W Dettmar and GSkjak-Braeligk ldquoThe enhancement of the bioadhesive propertiesof calcium alginate gel beads by coating with chitosanrdquo Inter-national Journal of Pharmaceutics vol 175 no 2 pp 237ndash2461998

[19] M George and T E Abraham ldquoPolyionic hydrocolloids for theintestinal delivery of protein drugs alginate and chitosanmdashareviewrdquo Journal of Controlled Release vol 114 no 1 pp 1ndash142006

[20] A Saha R Yadav and N Rajendran ldquoBiomaterials fromsponges ascidians and other marine organismsrdquo InternationalJournal of Pharmaceutical Sciences Review and Research vol 27no 2 article no 17 pp 100ndash109 2014

[21] S De and D Robinson ldquoPolymer relationships duringpreparation of chitosan-alginate and poly-l-lysine-alginatenanospheresrdquo Journal of Controlled Release vol 89 no 1 pp101ndash112 2003

[22] S K Motwani S Chopra S Talegaonkar K Kohli F J Ahmadand R K Khar ldquoChitosan-sodium alginate nanoparticles assubmicroscopic reservoirs for ocular delivery formulationoptimisation and in vitro characterisationrdquo European Journal ofPharmaceutics and Biopharmaceutics vol 68 no 3 pp 513ndash5252008

[23] F-L Mi H-W Sung and S-S Shyu ldquoDrug release fromchitosan-alginate complex beads reinforced by a naturallyoccurring cross-linking agentrdquo Carbohydrate Polymers vol 48no 1 pp 61ndash72 2002

[24] Q Zhao B Han ZWang C Gao C Peng and J Shen ldquoHollowchitosan-alginate multilayer microcapsules as drug deliveryvehicle doxorubicin loading and in vitro and in vivo studiesrdquoNanomedicine Nanotechnology Biology and Medicine vol 3no 1 pp 63ndash74 2007

[25] H Shen H Shi M Xie et al ldquoBiodegradable chitosanalginateBSA-gel-capsules for pH-controlled loading and release ofdoxorubicin and treatment of pulmonary melanomardquo Journalof Materials Chemistry B vol 1 no 32 pp 3906ndash3917 2013

[26] W Feng W Nie C He et al ldquoEffect of pH-responsive algi-natechitosanmultilayers coating on delivery efficiency cellularuptake and biodistribution of mesoporous silica nanoparticlesbased nanocarriersrdquo ACS Applied Materials amp Interfaces vol 6no 11 pp 8447ndash8460 2014

[27] L Keawchaoon and R Yoksan ldquoPreparation characterizationand in vitro release study of carvacrol-loaded chitosan nanopar-ticlesrdquo Colloids and Surfaces B Biointerfaces vol 84 no 1 pp163ndash171 2011

[28] S R Samarakoon I Thabrew P B Galhena D De Silva andK Tennekoon ldquoA comparison of the cytotoxic potential of stan-dardized aqueous and ethanolic extracts of a polyherbalmixturecomprised ofNigella sativa (seeds)Hemidesmus indicus (roots)

12 Journal of Nanomaterials

and Smilax glabra (rhizome)rdquo Pharmacognosy Research vol 2no 6 pp 335ndash342 2010

[29] K Nagpal S K Singh and D N Mishra ldquoChitosan nanoparti-cles a promising system in novel drug deliveryrdquo Chemical andPharmaceutical Bulletin vol 58 no 11 pp 1423ndash1430 2010

[30] N Bhattarai J Gunn and M Zhang ldquoChitosan-based hydro-gels for controlled localized drug deliveryrdquo Advanced DrugDelivery Reviews vol 62 no 1 pp 83ndash99 2010

[31] A Akbarzadeh M Samiei S W Joo et al ldquoSynthesis charac-terization and in vitro studies of doxorubicin-loaded magneticnanoparticles grafted to smart copolymers on A549 lung cancercell linerdquo Journal of Nanobiotechnology vol 10 article 46 2012

[32] F Danhier N Lecouturier B Vroman et al ldquoPaclitaxel-loadedPEGylated PLGA-based nanoparticles in vitro and in vivoevaluationrdquo Journal of Controlled Release vol 133 no 1 pp 11ndash17 2009

[33] A des Rieux V FievezMGarinot Y-J Schneider andV PreatldquoNanoparticles as potential oral delivery systems of proteins andvaccines amechanistic approachrdquo Journal of Controlled Releasevol 116 no 1 pp 1ndash27 2006

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CeramicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CompositesJournal of

NanoparticlesJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Biomaterials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MetallurgyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

MaterialsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Nano

materials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal ofNanomaterials

10 Journal of Nanomaterials

the time dependence cytotoxicity of DOX loaded NPs withMCF-7 cell line None of the concentrations of DOX in bothNPs that were used caused 50 inhibition of cell growthat 24 h reflecting the slow release from NPs Hence duringthe first 24 h those NPs are capable of circulating in theblood and accumulating at tumour sites before starting theircytotoxic effect Thus DOX encapsulated in NPs achieve itscytotoxicity with time that is there is an effect of both timeand dose on the inhibition of cell proliferation Thereforethis result clearly indicated that the release of DOX wasretarded significantly by the encapsulation in NPs Whencomparing the two delivery systems the cytotoxicity effectof DOX Csn NPs was consistently lower than that of DOXCsn-Alg NPs (Figure 6)This is in accordance with the resultsof in vitro release of DOX being higher from Csn-Alg NPsthan from Csn NPs after 72 h (Figure 5) However bothDOX loaded systems exhibited similar behaviour with theMCF-7 cell line (Figure 6) Same concentration of DOXwhen encapsulated in NPs behaves differently than in thefree state that is encapsulated DOX releases slowly andgradually increases the concentration in the medium Henceit proves that encapsulating DOX in either system hasimproved the efficacy of DOX over free DOX A similarstudy of doxorubicin loaded magnetic NPs grafted to smartcopolymers showed that free DOX has dose dependent butnot time dependent cytotoxicity but DOX loaded magneticNPs have time dependent cytotoxicity [31]

Inverted phase contrast microscopy was used for mor-phological analysis of MCF-7 cells for observation of apop-tosis induced by the DOX loaded NPs and free DOXThese characteristicmorphological changes of apoptosis withtime and dose in MCF-7 cells on treatment after 24 48and 72 h (Figure 7) were clearly visualized Exposure tofree DOX clearly showed a higher cell confluence at lowdoses while higher concentration of free DOX decreased cellconfluence and showed a higher proportion of rounded cellsresulting from detached cells from the culture monolayerWithin the first 24 h most of the cells were damaged bythe induced cytotoxicity of free DOX reflecting its dosedependent cytotoxicity On the other hand bothDOX loadedsystems showed disappearance of the high cell confluencewith the dose of the drug and with time (Figure 7) Thisobservation is further confirmed by the fluorescence micro-scopic results of AOEB stained MCF-7 cells after 24 48and 72 h of incubation following the treatment with differentconcentrations of freeDOXandwith the same concentrationsloaded in Csn and Csn-Alg NPs Nuclei of viable cells werestained uniformly bright green by AO while the apoptoticcells exhibited yellow to orange coloration depending on thedegree of loss of membrane integrity due to costaining withEB (Figure 8) Early apoptotic cells have greenish yellownuclei while late apoptotic cells have orange to red nucleiwith condensed or fragmented chromatin (Figure 8) Thesefigures clearly displayed the difference in behaviour of DOXwhen encapsulated in NPs Free DOX has a dose dependentincrease in induction of apoptosis (Figure 8-24 h) Both thedose and time dependency of DOX encapsulated in NPs are

evident from the alterations in cell staining in all the threeseries (Figure 8) In the case of DOX loadedNPs the drug hasto be released slowly from theNPs before it can exert an effecton the cancer cells but free DOX has an immediate effecton the cells since it has direct contact with the cells initiallyThe mechanism of action of apoptosis of DOX loaded inNPs was not significantly different from that of the free DOXindicating that the encapsulation of DOX in NPs does notmake any changes to its mechanism of action of apoptosis

37 Cellular Uptake of Nanoparticles DOX is a fluorescenceactive compound having a bright red color The red colorindicates the cellular uptake of DOX when compared to theuntreated cells or cells with no internalized DOX Internal-ization of DOX in cancer cells was evaluated on MCF-7 cellsat different time intervals and different concentrations offree and encapsulated DOX Within 30min of incubationall concentrations of free DOX were localized within the cellnucleus (Figure 9 30min) whereas the localization of DOXin NPs could be visualized depending on the concentrationand on time (Figure 9) The efficient uptake of the NPs couldbe facilitated by the positive surface charge of the NPs Asimilar observationwas notedwith the in vitro cellular uptakeof paclitaxel loaded PEGylated PLGA-based NPs [32] Ingeneral NPs are nonspecifically internalized into cells viaendocytosis or phagocytosis while free DOXdiffuses throughthe cell membrane [33]

Morphology analysis suggests that the process of cellgrowth inhibition by DOX loaded in NPs occurs over a muchlonger time than by the free DOX From these observations itcould be inferred that the DOX in NPs sufficiently associateswith the cells with time

4 Conclusion

This study was made to assess the usefulness of the Csn-Alg NP system as a delivery agent for the anticancer drugDOX Comparison with DOX Csn NPs indicated that Csn-Alg NPs were more efficient at entrapment of DOX than theCsnNPsThis successful loadingwas confirmedby both FTIRand TGA data Both nanoparticles were less than 100 nm insize with positive zeta potentials The in vitro release profilesobserved for these NPs were characterized by a burst initialrelease followed by a more gradual and sustained releasephase The in vitro cytotoxicity studies demonstrated thatDOX loaded NPs have potent antigrowth effect on MCF-7cells and inhibited its cell growth in a dose and time depen-dent manner Further the observed cell apoptosis induced bythese DOX loaded NPs by phase contrast microscopy andfluorescence microscopy after AOEB staining proved thatthere is no significant change in the mechanism of action ofDOX after encapsulating in NPs and reflect the slow releaseof DOX from the NPs According to the cytotoxicity resultsDOX Csn-Alg NPs are more cytotoxic than DOX Csn NPs

Competing Interests

The authors declare that they have no competing interests

Journal of Nanomaterials 11

Acknowledgments

This research was financially supported by the HETC QIGWindow 3 Grant University of Peradeniya Peradeniya SriLanka The authors also thank Ms Damayanthi Dahanayakeat Sri Lanka Institute of nanotechnology for her valuablesupport in electron microscopy imaging

References

[1] N Lomovskaya S L Otten Y Doi-Katayama et al ldquoDoxoru-bicin overproduction in Streptomyces peucetius cloning andcharacterization of the dnrU ketoreductase and dnrV genesand the doxA cytochrome P-450 hydroxylase generdquo Journal ofBacteriology vol 181 no 1 pp 305ndash318 1999

[2] K M Laginha S Verwoert G J R Charrois and T MAllen ldquoDetermination of doxorubicin levels in whole tumorand tumor nuclei in murine breast cancer tumorsrdquo ClinicalCancer Research vol 11 no 19 pp 6944ndash6949 2005

[3] G Minotti P Menna E Salvatorelli G Cairo and L GiannildquoAnthracyclines molecular advances and pharmacologic devel-opments in antitumor activity and cardiotoxicityrdquo Pharmaco-logical Reviews vol 56 no 2 pp 185ndash229 2004

[4] M E R OrsquoBrien N Wigler M Inbar et al ldquoReduced car-diotoxicity and comparable efficacy in a phase III trial of pegy-lated liposomal doxorubicin HCl (CAELYXDoxil) versusconventional doxorubicin for first-line treatment of metastaticbreast cancerrdquo Annals of Oncology vol 15 no 3 pp 440ndash4492004

[5] C Zhang W Wang T Liu et al ldquoDoxorubicin-loaded gly-cyrrhetinic acid-modified alginate nanoparticles for liver tumorchemotherapyrdquo Biomaterials vol 33 no 7 pp 2187ndash2196 2012

[6] W H De Jong and P J A Borm ldquoDrug delivery andnanoparticles applications and hazardsrdquo International Journalof Nanomedicine vol 3 no 2 pp 133ndash149 2008

[7] G Vilar J Tulla-Puche and F Albericio ldquoPolymers and drugdelivery systemsrdquo Current Drug Delivery vol 9 no 4 pp 367ndash394 2012

[8] A Kidane and P P Bhatt ldquoRecent advances in small moleculedrug deliveryrdquo Current Opinion in Chemical Biology vol 9 no4 pp 347ndash351 2005

[9] K S Soppimath T M Aminabhavi A R Kulkarni and WE Rudzinski ldquoBiodegradable polymeric nanoparticles as drugdelivery devicesrdquo Journal of Controlled Release vol 70 no 1-2pp 1ndash20 2001

[10] T Coviello P Matricardi C Marianecci and F AlhaiqueldquoPolysaccharide hydrogels for modified release formulationsrdquoJournal of Controlled Release vol 119 no 1 pp 5ndash24 2007

[11] Z Liu Y Jiao Y Wang C Zhou and Z ZhangldquoPolysaccharides-based nanoparticles as drug deliverysystemsrdquo Advanced Drug Delivery Reviews vol 60 no 15 pp1650ndash1662 2008

[12] O Borges G Borchard J C Verhoef A De Sousa andH E Junginger ldquoPreparation of coated nanoparticles for anew mucosal vaccine delivery systemrdquo International Journal ofPharmaceutics vol 299 no 1-2 pp 155ndash166 2005

[13] Y Murata D Jinno D Liu T Isobe K Kofuji and SKawashima ldquoThe drug release profile from calcium-inducedalginate gel beads coated with an alginate hydrolysaterdquoMolecules vol 12 no 11 pp 2559ndash2566 2007

[14] P Li Y-N Dai J-P Zhang A-QWang andQWei ldquoChitosan-alginate nanoparticles as a novel drug delivery system fornifedipinerdquo International Journal of Biomedical Science vol 4no 3 pp 221ndash228 2008

[15] C K S Pillai W Paul and C P Sharma ldquoChitin and chitosanpolymers chemistry solubility and fiber formationrdquo Progress inPolymer Science vol 34 no 7 pp 641ndash678 2009

[16] F Khoushab and M Yamabhai ldquoChitin research revisitedrdquoMarine Drugs vol 8 no 7 pp 1988ndash2012 2010

[17] T Kean and M Thanou ldquoBiodegradation biodistribution andtoxicity of chitosanrdquo Advanced Drug Delivery Reviews vol 62no 1 pp 3ndash11 2010

[18] O Gaseroslashd I G Jolliffe F C Hampson P W Dettmar and GSkjak-Braeligk ldquoThe enhancement of the bioadhesive propertiesof calcium alginate gel beads by coating with chitosanrdquo Inter-national Journal of Pharmaceutics vol 175 no 2 pp 237ndash2461998

[19] M George and T E Abraham ldquoPolyionic hydrocolloids for theintestinal delivery of protein drugs alginate and chitosanmdashareviewrdquo Journal of Controlled Release vol 114 no 1 pp 1ndash142006

[20] A Saha R Yadav and N Rajendran ldquoBiomaterials fromsponges ascidians and other marine organismsrdquo InternationalJournal of Pharmaceutical Sciences Review and Research vol 27no 2 article no 17 pp 100ndash109 2014

[21] S De and D Robinson ldquoPolymer relationships duringpreparation of chitosan-alginate and poly-l-lysine-alginatenanospheresrdquo Journal of Controlled Release vol 89 no 1 pp101ndash112 2003

[22] S K Motwani S Chopra S Talegaonkar K Kohli F J Ahmadand R K Khar ldquoChitosan-sodium alginate nanoparticles assubmicroscopic reservoirs for ocular delivery formulationoptimisation and in vitro characterisationrdquo European Journal ofPharmaceutics and Biopharmaceutics vol 68 no 3 pp 513ndash5252008

[23] F-L Mi H-W Sung and S-S Shyu ldquoDrug release fromchitosan-alginate complex beads reinforced by a naturallyoccurring cross-linking agentrdquo Carbohydrate Polymers vol 48no 1 pp 61ndash72 2002

[24] Q Zhao B Han ZWang C Gao C Peng and J Shen ldquoHollowchitosan-alginate multilayer microcapsules as drug deliveryvehicle doxorubicin loading and in vitro and in vivo studiesrdquoNanomedicine Nanotechnology Biology and Medicine vol 3no 1 pp 63ndash74 2007

[25] H Shen H Shi M Xie et al ldquoBiodegradable chitosanalginateBSA-gel-capsules for pH-controlled loading and release ofdoxorubicin and treatment of pulmonary melanomardquo Journalof Materials Chemistry B vol 1 no 32 pp 3906ndash3917 2013

[26] W Feng W Nie C He et al ldquoEffect of pH-responsive algi-natechitosanmultilayers coating on delivery efficiency cellularuptake and biodistribution of mesoporous silica nanoparticlesbased nanocarriersrdquo ACS Applied Materials amp Interfaces vol 6no 11 pp 8447ndash8460 2014

[27] L Keawchaoon and R Yoksan ldquoPreparation characterizationand in vitro release study of carvacrol-loaded chitosan nanopar-ticlesrdquo Colloids and Surfaces B Biointerfaces vol 84 no 1 pp163ndash171 2011

[28] S R Samarakoon I Thabrew P B Galhena D De Silva andK Tennekoon ldquoA comparison of the cytotoxic potential of stan-dardized aqueous and ethanolic extracts of a polyherbalmixturecomprised ofNigella sativa (seeds)Hemidesmus indicus (roots)

12 Journal of Nanomaterials

and Smilax glabra (rhizome)rdquo Pharmacognosy Research vol 2no 6 pp 335ndash342 2010

[29] K Nagpal S K Singh and D N Mishra ldquoChitosan nanoparti-cles a promising system in novel drug deliveryrdquo Chemical andPharmaceutical Bulletin vol 58 no 11 pp 1423ndash1430 2010

[30] N Bhattarai J Gunn and M Zhang ldquoChitosan-based hydro-gels for controlled localized drug deliveryrdquo Advanced DrugDelivery Reviews vol 62 no 1 pp 83ndash99 2010

[31] A Akbarzadeh M Samiei S W Joo et al ldquoSynthesis charac-terization and in vitro studies of doxorubicin-loaded magneticnanoparticles grafted to smart copolymers on A549 lung cancercell linerdquo Journal of Nanobiotechnology vol 10 article 46 2012

[32] F Danhier N Lecouturier B Vroman et al ldquoPaclitaxel-loadedPEGylated PLGA-based nanoparticles in vitro and in vivoevaluationrdquo Journal of Controlled Release vol 133 no 1 pp 11ndash17 2009

[33] A des Rieux V FievezMGarinot Y-J Schneider andV PreatldquoNanoparticles as potential oral delivery systems of proteins andvaccines amechanistic approachrdquo Journal of Controlled Releasevol 116 no 1 pp 1ndash27 2006

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CeramicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CompositesJournal of

NanoparticlesJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Biomaterials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MetallurgyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

MaterialsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Nano

materials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal ofNanomaterials

Journal of Nanomaterials 11

Acknowledgments

This research was financially supported by the HETC QIGWindow 3 Grant University of Peradeniya Peradeniya SriLanka The authors also thank Ms Damayanthi Dahanayakeat Sri Lanka Institute of nanotechnology for her valuablesupport in electron microscopy imaging

References

[1] N Lomovskaya S L Otten Y Doi-Katayama et al ldquoDoxoru-bicin overproduction in Streptomyces peucetius cloning andcharacterization of the dnrU ketoreductase and dnrV genesand the doxA cytochrome P-450 hydroxylase generdquo Journal ofBacteriology vol 181 no 1 pp 305ndash318 1999

[2] K M Laginha S Verwoert G J R Charrois and T MAllen ldquoDetermination of doxorubicin levels in whole tumorand tumor nuclei in murine breast cancer tumorsrdquo ClinicalCancer Research vol 11 no 19 pp 6944ndash6949 2005

[3] G Minotti P Menna E Salvatorelli G Cairo and L GiannildquoAnthracyclines molecular advances and pharmacologic devel-opments in antitumor activity and cardiotoxicityrdquo Pharmaco-logical Reviews vol 56 no 2 pp 185ndash229 2004

[4] M E R OrsquoBrien N Wigler M Inbar et al ldquoReduced car-diotoxicity and comparable efficacy in a phase III trial of pegy-lated liposomal doxorubicin HCl (CAELYXDoxil) versusconventional doxorubicin for first-line treatment of metastaticbreast cancerrdquo Annals of Oncology vol 15 no 3 pp 440ndash4492004

[5] C Zhang W Wang T Liu et al ldquoDoxorubicin-loaded gly-cyrrhetinic acid-modified alginate nanoparticles for liver tumorchemotherapyrdquo Biomaterials vol 33 no 7 pp 2187ndash2196 2012

[6] W H De Jong and P J A Borm ldquoDrug delivery andnanoparticles applications and hazardsrdquo International Journalof Nanomedicine vol 3 no 2 pp 133ndash149 2008

[7] G Vilar J Tulla-Puche and F Albericio ldquoPolymers and drugdelivery systemsrdquo Current Drug Delivery vol 9 no 4 pp 367ndash394 2012

[8] A Kidane and P P Bhatt ldquoRecent advances in small moleculedrug deliveryrdquo Current Opinion in Chemical Biology vol 9 no4 pp 347ndash351 2005

[9] K S Soppimath T M Aminabhavi A R Kulkarni and WE Rudzinski ldquoBiodegradable polymeric nanoparticles as drugdelivery devicesrdquo Journal of Controlled Release vol 70 no 1-2pp 1ndash20 2001

[10] T Coviello P Matricardi C Marianecci and F AlhaiqueldquoPolysaccharide hydrogels for modified release formulationsrdquoJournal of Controlled Release vol 119 no 1 pp 5ndash24 2007

[11] Z Liu Y Jiao Y Wang C Zhou and Z ZhangldquoPolysaccharides-based nanoparticles as drug deliverysystemsrdquo Advanced Drug Delivery Reviews vol 60 no 15 pp1650ndash1662 2008

[12] O Borges G Borchard J C Verhoef A De Sousa andH E Junginger ldquoPreparation of coated nanoparticles for anew mucosal vaccine delivery systemrdquo International Journal ofPharmaceutics vol 299 no 1-2 pp 155ndash166 2005

[13] Y Murata D Jinno D Liu T Isobe K Kofuji and SKawashima ldquoThe drug release profile from calcium-inducedalginate gel beads coated with an alginate hydrolysaterdquoMolecules vol 12 no 11 pp 2559ndash2566 2007

[14] P Li Y-N Dai J-P Zhang A-QWang andQWei ldquoChitosan-alginate nanoparticles as a novel drug delivery system fornifedipinerdquo International Journal of Biomedical Science vol 4no 3 pp 221ndash228 2008

[15] C K S Pillai W Paul and C P Sharma ldquoChitin and chitosanpolymers chemistry solubility and fiber formationrdquo Progress inPolymer Science vol 34 no 7 pp 641ndash678 2009

[16] F Khoushab and M Yamabhai ldquoChitin research revisitedrdquoMarine Drugs vol 8 no 7 pp 1988ndash2012 2010

[17] T Kean and M Thanou ldquoBiodegradation biodistribution andtoxicity of chitosanrdquo Advanced Drug Delivery Reviews vol 62no 1 pp 3ndash11 2010

[18] O Gaseroslashd I G Jolliffe F C Hampson P W Dettmar and GSkjak-Braeligk ldquoThe enhancement of the bioadhesive propertiesof calcium alginate gel beads by coating with chitosanrdquo Inter-national Journal of Pharmaceutics vol 175 no 2 pp 237ndash2461998

[19] M George and T E Abraham ldquoPolyionic hydrocolloids for theintestinal delivery of protein drugs alginate and chitosanmdashareviewrdquo Journal of Controlled Release vol 114 no 1 pp 1ndash142006

[20] A Saha R Yadav and N Rajendran ldquoBiomaterials fromsponges ascidians and other marine organismsrdquo InternationalJournal of Pharmaceutical Sciences Review and Research vol 27no 2 article no 17 pp 100ndash109 2014

[21] S De and D Robinson ldquoPolymer relationships duringpreparation of chitosan-alginate and poly-l-lysine-alginatenanospheresrdquo Journal of Controlled Release vol 89 no 1 pp101ndash112 2003

[22] S K Motwani S Chopra S Talegaonkar K Kohli F J Ahmadand R K Khar ldquoChitosan-sodium alginate nanoparticles assubmicroscopic reservoirs for ocular delivery formulationoptimisation and in vitro characterisationrdquo European Journal ofPharmaceutics and Biopharmaceutics vol 68 no 3 pp 513ndash5252008

[23] F-L Mi H-W Sung and S-S Shyu ldquoDrug release fromchitosan-alginate complex beads reinforced by a naturallyoccurring cross-linking agentrdquo Carbohydrate Polymers vol 48no 1 pp 61ndash72 2002

[24] Q Zhao B Han ZWang C Gao C Peng and J Shen ldquoHollowchitosan-alginate multilayer microcapsules as drug deliveryvehicle doxorubicin loading and in vitro and in vivo studiesrdquoNanomedicine Nanotechnology Biology and Medicine vol 3no 1 pp 63ndash74 2007

[25] H Shen H Shi M Xie et al ldquoBiodegradable chitosanalginateBSA-gel-capsules for pH-controlled loading and release ofdoxorubicin and treatment of pulmonary melanomardquo Journalof Materials Chemistry B vol 1 no 32 pp 3906ndash3917 2013

[26] W Feng W Nie C He et al ldquoEffect of pH-responsive algi-natechitosanmultilayers coating on delivery efficiency cellularuptake and biodistribution of mesoporous silica nanoparticlesbased nanocarriersrdquo ACS Applied Materials amp Interfaces vol 6no 11 pp 8447ndash8460 2014

[27] L Keawchaoon and R Yoksan ldquoPreparation characterizationand in vitro release study of carvacrol-loaded chitosan nanopar-ticlesrdquo Colloids and Surfaces B Biointerfaces vol 84 no 1 pp163ndash171 2011

[28] S R Samarakoon I Thabrew P B Galhena D De Silva andK Tennekoon ldquoA comparison of the cytotoxic potential of stan-dardized aqueous and ethanolic extracts of a polyherbalmixturecomprised ofNigella sativa (seeds)Hemidesmus indicus (roots)

12 Journal of Nanomaterials

and Smilax glabra (rhizome)rdquo Pharmacognosy Research vol 2no 6 pp 335ndash342 2010

[29] K Nagpal S K Singh and D N Mishra ldquoChitosan nanoparti-cles a promising system in novel drug deliveryrdquo Chemical andPharmaceutical Bulletin vol 58 no 11 pp 1423ndash1430 2010

[30] N Bhattarai J Gunn and M Zhang ldquoChitosan-based hydro-gels for controlled localized drug deliveryrdquo Advanced DrugDelivery Reviews vol 62 no 1 pp 83ndash99 2010

[31] A Akbarzadeh M Samiei S W Joo et al ldquoSynthesis charac-terization and in vitro studies of doxorubicin-loaded magneticnanoparticles grafted to smart copolymers on A549 lung cancercell linerdquo Journal of Nanobiotechnology vol 10 article 46 2012

[32] F Danhier N Lecouturier B Vroman et al ldquoPaclitaxel-loadedPEGylated PLGA-based nanoparticles in vitro and in vivoevaluationrdquo Journal of Controlled Release vol 133 no 1 pp 11ndash17 2009

[33] A des Rieux V FievezMGarinot Y-J Schneider andV PreatldquoNanoparticles as potential oral delivery systems of proteins andvaccines amechanistic approachrdquo Journal of Controlled Releasevol 116 no 1 pp 1ndash27 2006

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CeramicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CompositesJournal of

NanoparticlesJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Biomaterials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MetallurgyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

MaterialsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Nano

materials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal ofNanomaterials

12 Journal of Nanomaterials

and Smilax glabra (rhizome)rdquo Pharmacognosy Research vol 2no 6 pp 335ndash342 2010

[29] K Nagpal S K Singh and D N Mishra ldquoChitosan nanoparti-cles a promising system in novel drug deliveryrdquo Chemical andPharmaceutical Bulletin vol 58 no 11 pp 1423ndash1430 2010

[30] N Bhattarai J Gunn and M Zhang ldquoChitosan-based hydro-gels for controlled localized drug deliveryrdquo Advanced DrugDelivery Reviews vol 62 no 1 pp 83ndash99 2010

[31] A Akbarzadeh M Samiei S W Joo et al ldquoSynthesis charac-terization and in vitro studies of doxorubicin-loaded magneticnanoparticles grafted to smart copolymers on A549 lung cancercell linerdquo Journal of Nanobiotechnology vol 10 article 46 2012

[32] F Danhier N Lecouturier B Vroman et al ldquoPaclitaxel-loadedPEGylated PLGA-based nanoparticles in vitro and in vivoevaluationrdquo Journal of Controlled Release vol 133 no 1 pp 11ndash17 2009

[33] A des Rieux V FievezMGarinot Y-J Schneider andV PreatldquoNanoparticles as potential oral delivery systems of proteins andvaccines amechanistic approachrdquo Journal of Controlled Releasevol 116 no 1 pp 1ndash27 2006

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CeramicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CompositesJournal of

NanoparticlesJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Biomaterials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MetallurgyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

MaterialsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Nano

materials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal ofNanomaterials

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CeramicsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CompositesJournal of

NanoparticlesJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Biomaterials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MetallurgyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

MaterialsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Nano

materials

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal ofNanomaterials