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Colloids and Surfaces B: Biointerfaces 104 (2013) 245– 253

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces B: Biointerfaces

jou rn al h om epage: www.elsev ier .com/ locate /co lsur fb

n vitro evaluation of paclitaxel loaded amorphous chitin nanoparticles for colonancer drug delivery

.T. Smithaa, A. Anithaa, T. Furuikeb, H. Tamurab, Shantikumar V. Naira, R. Jayakumara,∗

Amrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham, Kochi 682041, IndiaFaculty of Chemistry, Materials and Bioengineering Research Centre, Kansai University, Osaka 564-8680, Japan

r t i c l e i n f o

rticle history:eceived 26 August 2012eceived in revised form 3 November 2012ccepted 18 November 2012vailable online 20 December 2012

a b s t r a c t

Chitin and its derivatives have been widely used in drug delivery applications due to its biocompatible,biodegradable and non-toxic nature. In this study, we have developed amorphous chitin nanoparticles(150 ± 50 nm) and evaluated its potential as a drug delivery system. Paclitaxel (PTX), a major chemother-apeutic agent was loaded into amorphous chitin nanoparticles (AC NPs) through ionic cross-linkingreaction using TPP. The prepared PTX loaded AC NPs had an average diameter of 200 ± 50 nm. Physico-chemical characterization of the prepared nanoparticles was carried out. These nanoparticles were proven

eywords:anoparticlesolon cancerrug deliverymorphous chitinaclitaxel

to be hemocompatible and in vitro drug release studies showed a sustained release of PTX. Cellular inter-nalization of the NPs was confirmed by fluorescent microscopy as well as by flow cytometry. Anticanceractivity studies proved the toxicity of PTX-AC NPs toward colon cancer cells. These preliminary resultsindicate the potential of PTX-AC NPs in colon cancer drug delivery.

© 2012 Elsevier B.V. All rights reserved.

onic cross-linking

. Introduction

Chitin, poly(�-(1–4)-N-acetyl-d-glucosamine), is one of theidely available natural polymers on earth. Major sources of chitin

re yeast, fungi and the exoskeletons of arthropods like crus-aceans and insects as well as cephalopod molluscs and marineponges [1–4]. This biopolymer has found a plethora of applica-ions in various forms such as gels, membranes, scaffolds, sponges,eads, micro and nanoparticles (NPs) in the biomedical field dueo its biocompatible, biodegradable and non-toxic nature [3,5,6].

ajor applications include its use as carriers for drug deliverynd as scaffolds for tissue engineering and wound healing [3,7,8].he main disadvantage of chitin is its limited solubility in water,hich makes its processing difficult [3,5,9]. It is found to be sol-ble only in a few solvents such as methanol/calcium chloride10,11], sodium hydroxide–urea [12], 35% hydrogen peroxide [13]nd in some fluorinated compounds such as hexafluoroacetonend hexafluoropropanol [6,14]. The hydrophobicity arises from

ts extensive hydrogen bonding between the constituent groupshat result in its rigid crystalline structure [10,15–17]. Studiesave shown that graft copolymerization with hydrophilic polymers18] and chemical substitution [19] can improve its solubility. The

∗ Corresponding author. Tel.: +91 484 2801234; fax: +91 484 2802020.E-mail addresses: rjayakumar@aims.amrita.edu, jayakumar77@yahoo.com

R. Jayakumar).

927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.colsurfb.2012.11.031

solubility of chitin is directly linked with its degree of deacetylation(DD). An increase in DD brings in more number of amino groupsthereby improving its hydrophilicity [6,14,20]. This also leads tothe destruction of the secondary structure of chitin and makes itamorphous [6,14,20,21]. Thus, improving the DD would enhancethe solubility of chitin without the involvement of toxic solventsor any chemical modifications. In this study, we have used amor-phous alpha chitin (source of origin: crab shell) which has a DDof 41.3% and shows solubility in weak acids such as acetic acid.Normal forms of chitin are insoluble in acetic acid and requireextensive processing before their use in biomedical application[11,22–24]. We presume that the usage of amorphous chitin wouldexploit the potential of chitin in biomedical applications to themaximum.

With the advent of nanotechnology, a wide horizon has beenopened up in the field of cancer drug delivery. The enhanced per-meability and retention (EPR) effect of polymeric NPs near thetumor vasculature helps in passive targeting of anticancer drugs[25]. Recent studies have shown the potential of chitin based nano-materials such as chitin nanogels [22–24] and carboxymethyl chitinNPs [26] as carriers for anticancer drugs. The disadvantage asso-ciated with chitin nanogel fabrication is the use of toxic solventsystem and the involvement of repeated sonication and centrifu-

gation, which decreases its encapsulation efficiency and heightensthe chance for drug degradation. Carboxymethylation involvesreactions such as alkalization, etherification [27], which is time con-suming. The preparation of amorphous chitin does not involve any

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f these processing steps which ensure its suitability in biomedicalpplications.

Chitin based polymers can be used as carriers in colon drugelivery. Among the various approaches for colon drug delivery,hese polymers can be used in microflora-activated systems ashe anaerobic microbes present in colon have the ability to breakhe glycosidic linkages of the polymer and thus can degrade them28–33]. As these polymers are not digested in the stomach andmall intestine, the release of maximum amount of drug occurs inhe colon [28,33]. In the current study, we have developed amor-hous chitin nanoparticles (AC NPs) and the ability of the sames a drug delivery vehicle has been assessed using the commonhemotherapeutic drug ‘paclitaxel’ (PTX). PTX exhibits antineo-lastic activity against a wide range of cancers such as breast,varian, head and neck, colon, multiple myeloma, melanoma andaposi’s sarcoma [34–37]. It is an anti mitotic drug that prevents

he depolymerization of microtubules and arrests mitosis at the G2nd M stages of the cell cycle [34,36,37]. PTX is hydrophobic and theommon solvent for it is cremophor EL which causes nephrotoxi-ity, neurotoxicity and hypersensitivity [34–36]. Entrapping PTXnside the NPs would circumvent the toxicity caused by cremophorL thereby reducing its side effects.

The aim of the present study is to evaluate the potentialf PTX loaded amorphous chitin nanoparticles (PTX-AC NPs) inolon cancer. To achieve this, PTX-AC NPs was prepared by ionicross-linking. The bare as well as drug loaded NPs were char-cterized using DLS, SEM, zeta potential, FT-IR and TG/DTA. Inddition, hemocompatibility, cytotoxicity, in vitro drug release,ellular uptake and anticancer studies of the prepared NPs werevaluated.

. Materials and methods

.1. Materials

Amorphous chitin (DD 41.3%, ash content 0.05%, water con-ent 12.6% and viscosity 30 mPa s) was purchased from Koyohemicals, Japan. Pentasodium tripolyphosphate (TPP), PTX, MTT3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium], Mini-

um Essential Medium (MEM), Dulbecco’s Modified Eagle MediumDMEM), Roswell Park Memorial Institute Medium (RPMI) andhodamine 123 was purchased from Sigma Aldrich. Human colo-ectal adenocarcinoma cells (HT-29), human colon carcinoma cellsCOLO-205) and rat intestinal epithelial cells (IEC-6) were pur-hased from NCCS, Pune. All other chemicals used are of analyticalrade.

.2. Methods

.2.1. Preparation of AC NPsAC NPs were prepared by ionic cross-linking reaction using TPP.

.01 wt% amorphous chitin solution was prepared in 1% acetic acid.o 10 mL of this solution, 1 wt% TPP solution was added drop wisender constant stirring. As a result, the clear solution changed inton opalescent suspension. The NPs formed were recovered by cen-rifugation at 20,000 rpm for 45 min. The pellet formed was washedith distilled water and stored at 4 ◦C. The pellets thus obtainedere redispersed in phosphate buffered saline (PBS) prior to the

xperiments and used immediately.

.2.2. Preparation of PTX-AC NPsFor preparing PTX-AC NPs, 0.06 mg/mL of PTX in dimethyl sulf-

xide (DMSO) was added to 0.01 wt% of amorphous chitin solutionnd it was kept for stirring for 4–5 h to allow proper interactionetween the drug and the polymer. Into this solution 1 wt% TPPas added dropwise until the solution became turbid and it was

Biointerfaces 104 (2013) 245– 253

processed in the manner as that of AC NPs mentioned above toobtain PTX-AC NPs.

2.2.3. Preparation of rhodamine 123 labeled PTX-AC NPs(Rh-PTX-AC NPs)

Rhodamine 123 labeling of PTX-AC NPs was carried out basedon a previous report [38]. 10 mL of PTX-AC NPs was prepared, theresulting suspension was centrifuged and the pellet was redis-persed in 5 mL PBS. 50 �L of rhodamine 123 (5 mg/mL) was added tothis and stirred overnight in dark. It was centrifuged and the pelletwas washed thrice to remove the unbound dye. It was redispersedin PBS immediately and used for cell uptake studies.

2.3. Characterization

The size distribution of AC NPs and PTX-AC NPs was determinedby DLS (DLS-ZP/Particle Sizer NicompTM 380 ZLS). The averagesize and surface morphology of the NPs was reconfirmed by SEM(JEOLJSM-6490LA). Surface charge and thereby the stability of NPsystem at a pH of 7.4 was obtained by zeta potential measurements(DLS-ZP/Particle Sizer NicompTM 380 ZLS). The interaction betweenthe constituents of NPs was studied using FT-IR spectra (PerkinElmer Spectrum RXI Fourier Transform Infrared Spectrophotome-ter) using KBr method. X-ray diffraction (XRD) analysis was carriedout using PANanalytical X’Pert PRO X-ray diffractometer. Thermalbehavior of the NPs was studied by thermogravimetric analysis (SIITG/DTA 6200 EXSTAR).

2.4. Validation of the spectrophotometric method

A standard stock solution of PTX (0.01 mg/mL) in DMSO wasprepared. Eight working standard solutions (0.0045, 0.004, 0.0035,0.003, 0.0025, 0.002, 0.0015 and 0.001 mg/mL) were prepared bydiluting the standard stock with PBS. The absorbance values weremeasured at 230 nm (UV-1700 Pharma Spec SHIMADZU) and acalibration curve was plotted to get the linearity and regressionequation (done in triplicates). The Limit of Detection (LOD) andLimit of Quantitation (LOQ) of PTX were determined using cali-bration graphs. LOD and LOQ were calculated as 3.3�/S and 10�/S,respectively, where S is the slope of the calibration curve and � isthe standard deviation of response [39].

2.5. Entrapment efficiency and loading efficiency

To determine the entrapment efficiency (EE) of PTX inside theNPs, PTX-AC NPs were centrifuged (20,000 rpm, 45 min). The pel-let was dissolved in DMSO and sonicated to extract PTX from NPs.This was stirred overnight, centrifuged and the supernatant wasanalyzed spectrophotometrically. EE was calculated with respectto the final amount of drug present in the NPs to the initial amountof drug used for encapsulation studies (n = 3). Loading efficiency(LE) of the system was calculated with respect to the weight of theNPs obtained after centrifugation (n = 3).

EE (%) = Total amount of PTX entrapped inside the pelletInitial amount of PTX taken for drug loading

× 100

LE (%) = Total amount of PTX entrapped inside the pelletTotal amount of NPs obtained

× 100

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.6. In vitro drug release studies

In vitro release profile of PTX from AC NPs was determined underink conditions [40] at a pH of 7.4 at 37 ◦C. The drug-loaded pelletas redispersed in 3 mL of PBS and placed in a dialysis bag (MWCO:

0 kDa) in a beaker containing 30 mL PBS. The whole setup waslaced in a shaker incubator set at 37 ◦C. At predetermined time

ntervals, 3 mL of PBS was removed and replaced with fresh PBS. Theelease was quantified spectrophotometrically. Percentage releaseas quantified as follows:

eleased (%) = Released PTXTotal amount of PTX entrapped inside the NPs

× 100

.7. Blood compatibility studies

.7.1. In vitro hemolysis assayFresh blood was collected from human volunteers into tubes

ontaining acid citrate dextrose (ACD). Different concentrations ofTX-AC NPs (0.01, 0.02, 0.04 and 0.08 mg/mL) were used. The sam-les (100 �L) were treated with 900 �L of blood and incubatedor 3 h at 37 ◦C under shaking. Plasma was collected by centrifu-ation at 4500 rpm for 10 min and the absorbance was measuredsing Beckmann Coulter Elisa plate reader (BioTek Power WaveS). Blood treated with 1% Triton-X was taken as positive controlnd saline treated blood as negative control. This experiment wasepeated thrice to validate the results. Plasma hemoglobin concen-ration (mg/mL) was calculated according to the following equation41]:

lasma Hb = [(2A415) − (A380 + A450)] × 1000 × Dilution factorE × 1.655

n which, A415, A380 and A450 represent the optical density valuesf the hemoglobin at 380, 450 and 415 nm respectively. A415 is thebsorption of hemoglobin based on soret band. A380 and A450 arehe correction factors for uroporphyrin absorption. E is the molarbsorptivity of oxyhemoglobin at 415 nm and 1.655 is the correc-ion factor accounting for plasma turbidity [41]

Hemolysis = Total Hb value of bloodPlasma Hb value of sample

× 100

A graph was plotted with the sample concentration along the-axis and the percentage hemolysis along the Y-axis.

.7.2. Coagulation assay via prothrombin time (PT) and activatedartial thromboplastin time (aPTT) measurements

Fresh blood was collected from human volunteers into ACDontaining tubes. Platelet poor plasma (PPP) was obtained byentrifuging the blood at 4000 rpm at 25 ◦C for 15 min. Differentoncentrations (0.01, 0.02, 0.04 and 0.08 mg/mL) of PTX-AC NPsere prepared in saline. 900 �L of PPP was treated with 100 �L of

ample and kept at 37 ◦C for 20 min. PT and aPTT of the samplesere measured using a coagulation analyzer and reagent kits CK

rest and Fibriprest (Diagnostica Stago). The experiment was donen triplicates using saline treated PPP as negative control.

.8. Cell culture

For cell culture experiments, IEC-6, COLO-205 and HT-29 cellines were used. IEC-6 and HT-29 cells were maintained in DMEMnd COLO-205 in RPMI supplemented with 10% fetal bovineerum (FBS). The cells were incubated in CO2 incubator at 37 ◦C

ith 5% CO2. After attaining the required confluency, the cellsere detached from the flask using Trypsin–EDTA, centrifuged at

000 rpm for 3 min and resuspended in growth medium for furtherxperiments.

Biointerfaces 104 (2013) 245– 253 247

2.8.1. Cytocompatibility studies – MTT assayThe cells were seeded on a 96 well plate at a density of 104 cells

per well. The cells at 90% confluency were incubated with differ-ent concentrations (0.4, 0.8, 1 and 2 mg/mL) of AC NPs (100 �L)for 24 h. The cells in media (untreated with NPs) acted as negativecontrol and cells treated with Triton X-100 acted as positive con-trol. After 24 h, the cells were incubated with MTT solution for 4 hfollowed by 1 h incubation with solubilization buffer. The opticaldensity of the solution was measured at 570 nm using BeckmannCoulter Elisa plate reader (BioTek Power Wave XS). Each samplewas assayed in triplicates; with each experiment repeated threetimes independently. Cell viability was expressed as the percentageof the negative control calculated as

Viability (%) =(

Nt

Nc

)× 100

where, Nt is the optical density of cells treated with sample and Ncis the absorbance of the untreated cells.

2.8.2. Cell uptake studiesCellular internalization of Rh-PTX-AC NPs was evaluated by flu-

orescent microscopy and flow cytometry (in normal and cancercells).

2.8.2.1. Fluorescent imaging. This study was carried out accordingto the previously reported method [41]. Cells were seeded witha density of 5 × 104 cells on to acid etched cover slips placed inindividual wells of a 24-well plate and incubated for 24 h. After24 h, the media was removed and the wells were washed with PBS.Then Rh-PTX-AC NPs were added in triplicates along with mediainto these wells and it was processed accordingly [41]. The coverslips were mounted onto glass slides using DPX and viewed underfluorescence microscope (Olympus DP71).

2.8.2.2. Flow cytometry. The cellular uptake of Rh-PTX-AC NPs bynormal as well as cancer cells was reconfirmed by flow cytome-try. Cells were seeded in a 24 well plate at a density of 5 × 104 cellsper well. After reaching 90% confluency, Rh-PTX-AC NPs (0.04 and0.08 mg/mL) were added and incubated at 37 ◦C for 24 h. The cellswere trypsinized and centrifuged. The pellet was analyzed flowcytometrically after excitation with a 488 nm argon laser usingFACS Aria II (Beckton and Dickinson, Sanjose, CA). Fluorescenceemission above 530 nm from 50,000 cells was collected, amplifiedand scaled to generate single parameter histogram.

2.8.3. In vitro anticancer activity analysis2.8.3.1. MTT assay. MTT assay was carried out in IEC-6, HT-29 andCOLO-205 cell lines. IEC-6 and HT-29 cells were grown in DMEMwhile COLO-205 cells were grown in RPMI. The cells were seededin 96-well plates at the density of 104 cells per well and incubatedfor 24 h followed by addition of AC NPs and PTX-AC NPs (0.01, 0.02,0.04 and 0.08 mg/mL). After 24 h, the cells were incubated with MTTsolution for 4 h followed by 1 h incubation with solubilization buffer[41]. The optical density of the solution was measured at a wave-length of 570 nm using a Beckmann Coulter Elisa plate reader. Eachsample was assayed in triplicates; with each experiment repeatedthree times independently. The cells in media (devoid of NPs) actedas negative control and those treated with Triton X-100 as positivecontrol.

2.8.3.2. Apoptosis assay – annexin V-FITC/PI staining. Anticanceractivity of PTX-AC NPs was evaluated flow cytometrically via apo-

ptosis assay using Annexin V-FITC/PI Vybrant apoptosis assay kit(Molecular Probes, Eugene, OR). The exposure of phosphatidyl ser-ine (PS) residues onto the cell surface is used as an indication todetect and quantify apoptosis. Annexin V has the ability to bind to

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48 K.T. Smitha et al. / Colloids and Surfa

S with high affinity and propidium iodide (PI) can be taken up byecrotic cells. Annexin V is labeled with fluorescein isothiocyanateFITC). Staining the samples with annexin V-FITC as well as PI leadso detection of both apoptotic and necrotic cells. The cells at 90%onfluency were incubated with PTX-AC NPs (0.04 and 0.08 mg/mL)4 h. It was washed with PBS, trypsinized and centrifuged. The pel-

et was resuspended in ice-cold 1× annexin binding buffer (5 × 105

o 5 × 106 cells/mL). 5 �L of annexin V-FITC solution and 1 �L of PI100 �g/mL) were added to 100 �L of the cell suspension, mixednd incubated in dark at room temperature for 15 min. After incu-ation, 400 �L of ice-cold 1× binding buffer was added, gentlyixed and analyzed flow cytometrically. Cells in media (not treatedith NPs) acted as negative control.

.9. Statistical analysis

All the experiments were conducted three times independentlyith triplicates and the results were expressed as mean ± standardeviation. The standard deviation values were indicated as errorars in the corresponding graphs. Student’s t-test was performedo find the statistical significance of these values. A probability of

< 0.05 was considered to be statistically significant.

. Results and discussion

.1. Preparation of AC NPs and PTX-AC NPs

AC NPs were obtained by ionic cross-linking which happensetween the protonated amino groups of amorphous chitin and thehosphate groups of TPP [42–44]. The possible interaction of PTXith amorphous chitin results from the hydrogen bonding between

1) the carbonyl groups of PTX and the protonated amino groups ofhitin, (2) the carbonyl groups of PTX and the hydroxyl groups ofhitin or (3) between the hydroxyl groups of PTX with acetamideroups of chitin. These interactions result in drug loading into thePs.

.2. Nanoparticle size distribution and morphology

The size distribution of bare AC NPs and PTX-AC NPs werebtained using DLS (Fig. 1A and B). It showed that AC NPs lies within

size range of 150 ± 50 nm and PTX-AC NPs lies within a range of50 ± 50 nm. The size and morphology of the prepared NPs wereeconfirmed by SEM. Fig. 1C and D shows the SEM images of ACPs and PTX-AC NPs, which shows spherical particles within a size

ange of 350 nm. The leaky tumor vasculature has a pore size cut offange of 0.2–2 �m [25]. Thus for passive targeting of NPs into theumor cells, the NPs should have an optimum size range such thatt does not escape the leaky tumor vasculature. The prepared NPsave a size below 350 nm, which ensures its suitability in passiveargeting for cancer drug delivery.

.3. Zeta potential measurements

The stability of the prepared NPs systems at physiological pHas analyzed by zeta potential measurements. For AC NPs and PTX-

C NPs, the zeta potential value was found to be +26 ± 3 mV and24 ± 3 mV respectively. This proves the physiological stability andositive surface charge of the NPs. The positive charge could bettributed to the presence of protonated amino groups of chitin. Inhe case of PTX-AC NPs, there was a slight decrease in zeta poten-ial resulting from the interaction of electropositive groups of theolymer with the electronegative groups of PTX.

Biointerfaces 104 (2013) 245– 253

3.4. Nanoparticle characterizations

3.4.1. FT-IRFig. 2A shows the FT-IR spectra of PTX, TPP, PTX-AC NPs, AC NPs

and amorphous chitin. For amorphous chitin, peaks were observedat 3424 ( N H stretching), 2924 ( C H stretching) and 1640 (pri-mary amine and C C stretching). In the spectrum corresponding toTPP, the peak for phosphate group was observed at 1230 cm−1. InAC NPs spectrum, the polymer peaks at 3424, 2924 and 1640 cm−1

have been shifted to 3461, 2940 and 1652 cm−1 respectively. Thesepeak shifts could be due to the cross linking reaction that hap-pened during the NPs formation [44]. Also the phosphate peak at1233 cm−1 has been observed in AC NPs. PTX showed its charac-teristic peaks at 3160 ( N H stretching), 1640 (C C stretching)and 1077 cm−1 ( C O stretching). In PTX-AC NPs, the peaks of PTXwere observed to be shifted from 3160 and 1640 cm−1 to 3494 and1656 cm−1 respectively. The presence of PTX peaks in PTX-AC NPsshows the interaction of PTX with AC NPs. The shift in peaks in PTX-AC NPs could be due to the hydrogen bonding between PTX and thepolymer [45].

3.4.2. Thermal studiesThe thermal behavior of PTX-AC NPs was analyzed in compari-

son with AC NPs and amorphous chitin using TGA and it is explainedin Fig. 2B. In the figure, the initial 20–30% weight reduction canbe attributed to moisture loss. TG curves indicate that amorphouschitin, AC NPs and PTX-AC NPs starts degradation at the same tem-perature, of which bare as well as drug loaded AC NPs degradeat a slower rate than amorphous chitin. An initial 20–30% weightreduction observed could be attributed to moisture loss. At 500 ◦C,amorphous chitin is degraded to 17% whereas bare and drug loadedNPs have degraded to around 30% and 25% respectively.

3.4.3. XRD analysisXRD patterns of amorphous chitin, AC NPs and PTX-AC NPs were

shown in Fig. 2C. XRD patterns of amorphous chitin and AC NPSshowed amorphous nature. PTX has its characteristic peaks of 2�between 5◦ and 21◦ (data not provided) [46,47]. PTX-AC NPs did notshow any prominent crystalline peaks of PTX. This must be due tothe intermolecular interaction between PTX and amorphous chitinwithin the NP matrix. This confirms the amorphous nature of PTXinside the NPs [47].

3.5. Validation, loading efficiency, entrapment efficiency andin vitro drug release

Validation was carried out using the calibration curve (see sup-plementary data: S1A and S1B). It was found that the relationshipbetween drug concentration and UV absorption was linear withinthe measured limits (r2 > 0.995, linear regression analysis). LOD andLOQ values were calculated as 0.228 �g/mL and 0.69 �g/mL respec-tively. The low values of LOD and LOQ obtained indicated goodsensitivity of the proposed method [39].

Supplementary material related to this article can be found,in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2012.11.031.

The drug entrapment efficiency and loading efficiency were cal-culated based on the equations mentioned previously and werefound to be 55% and 6.18% respectively. The in vitro PTX releaseprofile at a pH of 7.4 is shown in Fig. 2D. It shows an initial burstrelease followed by a slow release of PTX. As a result, around 20% ofPTX got released in the first 2 h. This burst release could be due to

the release of PTX which was poorly entrapped inside the polymermatrix or adsorbed onto the surface of NPs. Within the first 24 h,around 40% of the drug got released and this increased to 45% in thenext 12 h. This slow release could be attributed to the diffusion of

K.T. Smitha et al. / Colloids and Surfaces B: Biointerfaces 104 (2013) 245– 253 249

Fig. 1. (A and B) Particle size distribution of AC NPs and PTX-AC NPs by DLS and (C and D) SEM images of AC NPs and PTX-AC NPs.

Fig. 2. (A) FT-IR spectrum showing (a) PTX, (b) TPP, (c) PTX-AC NPs, (d) AC NPs and (e) amorphous chitin. (B) TGA curve showing amorphous chitin, PTX-AC NPs and AC NPs.(C) XRD graph showing amorphous chitin, AC NPs and PTX-AC NPs. (D) Drug release profile of PTX from AC NPs in PBS (pH 7.4) at 37 ◦C. Data shown are the mean values ± SD(n = 3).

250 K.T. Smitha et al. / Colloids and Surfaces B: Biointerfaces 104 (2013) 245– 253

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The cellular internalization of the prepared drug loaded AC NPswas studied using fluorescent microscopy. For this, Rh-PTX-AC NPs

ig. 3. (A) Hemolysis assay graph and (B) photograph of PTX-AC NPs treated with

artial thromboplastin time. Data shown are the mean values ± SD (n = 3).

he drug from the polymer matrix along with the slow degradationf the matrix.

.6. Blood compatibility studies

Blood compatibility of the prepared NPs was analyzed by hemol-sis as well as coagulation studies and the results were shown inig. 3. RBC lysis was measured in terms of hemolysis and coagu-ation studies through PT and aPTT measurements and the results

ere shown in Fig. 3. Fig. 3A represents the hemolysis assay graphsnd Fig. 3B represents the photographs of blood treated with NPamples. From Fig. 3A and B it is clear that the percentage hemol-sis for all the samples was below 5%. This value lies in the criticalafe limit for biomaterials according to ISO/TR 7406. The results ofT and aPTT studies were shown in Fig. 3C. PT and aPTT evaluateshe effect of the samples on the extrinsic and intrinsic coagula-ion pathways. The values obtained confirmed that the PT and aPTTemains within the normal range (normal ranges, PT: 12–15 s andPTT: 25–35 s) for all the samples. Hemolysis as well as coagulationssays proved the blood compatibility of PTX-AC NPs.

.7. Cytocompatibility studies

For evaluating the cytocompatibility of the prepared AC NPs,TT assay was performed. This colorimetric assay measures the

eduction of yellow MTT by mitochondrial succinate dehydroge-ase into purple colored formazan crystals. Since the reduction ofTT can occur only in metabolically active cells, the level of activity

s a measure of the viability of the cells. Formazan crystals have an

bsorbance maximum of 570 nm based on which the percentageell viability was calculated.

IEC-6, COLO-205 and HT-29 cells were used for this assay. Fig. 4hows the graph, where the percentage cell viability was plotted

n blood and (C) coagulation assay graph showing prothrombin time and activated

against NP concentrations. These cells, when exposed to bare ACNPs in the concentration range of 0.4–1 mg/mL showed high cellviability (mean ± SD, n = 3 for each concentration; p > 0.05) whenit is compared with media (devoid of NPs). This indicates that theprepared AC NPs (within the concentration range of 0.4–1 mg/mL)does not impart any toxicity toward these cells. This proves thecytocompatibility of the prepared AC NPs.

3.8. Cell uptake studies using fluorescent microscopy and flow

Fig. 4. Cell viability of IEC-6, COLO-205 and HT-29 cells treated with AC NPs (24 h).Data shown are the mean values ± SD (n = 3).

K.T. Smitha et al. / Colloids and Surfaces B: Biointerfaces 104 (2013) 245– 253 251

Fig. 5. Cellular uptake of rhodamine 123 labeled PTX-AC NPs by fluorescent microscopy (a and c) bright field images of IEC-6 and HT-29 cells exposed to rhodamine labeledPTX-AC NPs and (b and d) IEC-6 and HT-29 cells exposed to rhodamine labeled PTX-AC NPs (6 h). (For interpretation of the references to color in the text, the reader is referredto the web version of the article.)

Fig. 6. Cellular uptake studies of rhodamine 123 labeled PTX-AC NPs by flow cytometry, (A) control HT-29 cells, (B) HT-29 cells exposed to rhodamine 123 labeled of PTX-ACNPs, (C) control IEC-6 cells, and (D) IEC-6 cells exposed to rhodamine 123 labeled PTX-AC NPs (6 h).

252 K.T. Smitha et al. / Colloids and Surfaces B: Biointerfaces 104 (2013) 245– 253

F d PTXt

wflbt

caP

FoHcc

ig. 7. Anticancer activity by MTT assay (24 h). (A) IEC-6 cells treated with PTX anreated with PTX and PTX-AC NPs. Data shown are the mean values ± SD (n = 3).

ere used. The NPs taken up by the cells appear green under theuorescence microscope (Fig. 5). Both IEC-6 and HT-29 cells incu-ated with Rh-PTX-AC NPs showed green fluorescence confirminghe cellular internalization of the NPs (Fig. 5b and d).

The cellular uptake of PTX-AC NPs was reconfirmed using flowytometry. Fig. 6 shows the uptake profile of the NPs by HT-29nd IEC-6 cells. No significant difference is seen in the uptake ofTX-AC NPs when normal cells and cancer cells were compared.

ig. 8. Analysis of anticancer activity by flow cytometry (apoptosis assay) measuring thef PTX-AC NPs by annexin V-FITC/PI staining, (a and d) control HT-29 and IEC-6 cells, (bT-29 and IEC-6 cells exposed to PTX-AC NPs (0.08 mg/mL). X and Y axis shows the FITC aells are located in the right two quadrants [right side lower quadrant: annexin-V positivells (late apoptosis)].

AC NPs, (B) HT-29 cells treated with PTX and PTX-AC NPs, and (C) COLO-205 cells

This suggests that the uptake of these drug loaded NPs by the cellsis non specific.

3.9. Anticancer activity analysis

3.9.1. MTT assayThe cytotoxicity of PTX and PTX-AC NPs were tested on HT-29,

COLO-205 and IEC-6 cells using MTT assay. Fig. 7A–C shows the

apoptotic frequencies of HT-29 and IEC-6 cells exposed to different concentrations and e) HT-29 and IEC-6 cells exposed to PTX-AC NPs (0.04 mg/mL), and (c and f)nd PI emissions respectively. The lower left quadrant shows viable cells; apoptotice cells (early apoptosis), right side upper quadrant: both annexin-V and PI positive

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lot of cell viability against different concentrations of PTX andTX-AC NPs. As the concentration of PTX increases, there is a doseependent toxicity toward all the cell lines tested. There is a signif-

cant difference in toxicity when drug loaded NPs were comparedith the bare drug (bare drug showed enhanced toxicity) in all the

ell lines (mean ± SD, n = 3 for each concentration; p < 0.05). Theecreased toxicity of drug loaded NPs could be due to the slowelease of the drug from PTX-AC NPs.

.9.2. Flow cytometryThe cell death caused by PTX-AC NPs was reconfirmed by apo-

tosis assay in HT-29 and IEC-6 cells using flow cytometry. Fig. 8hows the apoptotic profile of HT-29 cells and IEC-6 cells, wherehe X and Y axis shows the FITC and PI emissions respectively.ig. 8a and b represents the control HT-29 and IEC-6 cells, Fig. 8cnd d, HT-29 and IEC-6 cells treated with PTX-AC NPs contain-ng 0.04 mg/mL of PTX and Fig. 8e and f, IEC-6 and HT-29 cellsreated with PTX-AC NPs containing 0.08 mg/mL of PTX. At both theoncentrations of PTX, significant cell death had occurred. A per-entage apoptosis of 46.1% and 61.6% at a concentration of 0.04 and.08 mg/mL of PTX was observed in HT-29 cells. IEC-6 cells did nothow any significant apoptosis. Previous studies done on HT29-D4ell lines have shown that caspase-8, an important component ofD95 induced apoptotic pathway is activated on exposing the cellso PTX there by causing cell death [48].

. Conclusion

Novel amorphous chitin NPs were prepared via ionicross-linking technique and its efficacy as a drug deliveryehicle for colon cancer was analyzed using paclitaxel. The pre-ared PTX-AC NPs showed a size range below 350 nm with anntrapment efficiency of 55%. This size range is suitable for passiveargeting via EPR effect in cancer drug delivery [25]. AC NPs wereroven to be cytocompatible and the PTX-AC NPs were proven toemocompatible. Cellular internalization of the Rh-PTX-AC NPsas confirmed by fluorescent microscopy. In vitro drug releaseattern showed an initial burst release followed by a sustainedelease of PTX over a period of 48 h at physiological pH. Cytotoxicityssays indicated the anticancer activity of the prepared PTX-ACPs and were proven to cause cell death via apoptosis. Thesereliminary studies prove the potential of AC NPs as a promisingrug delivery vehicle for cancer drug delivery.

cknowledgments

This work was supported by Department of Science andechnology (DST) under the project ‘Nanotheragnostics’SR/NM/NS-99/2009). A. Anitha is thankful to Council of Scientificnd Industrial Research (CSIR), India for providing Senior Researchellowship (SRF award-Ref. No. 9/963 (0005) 2K10378-EMR-I) forarrying out her research work. The authors are also thankful tor. Sajin. P. Ravi, Mrs. P.R. Sreerekha and Mr. P.T. Sudheesh Kumar

or their help in SEM, flow cytometry and TG studies.

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