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Biomaterials 32 (2011) 3794e3806

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Biomaterials

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The potential of celecoxib-loaded hydroxyapatite-chitosan nanocomposite for thetreatment of colon cancer

P. Venkatesan a, Nagaprasad Puvvada b, Rupesh Dash c, B.N. Prashanth Kumar a, Devanand Sarkar c,Belal Azab c, Amita Pathak b, Subhas C. Kundu d, Paul B. Fisher c, Mahitosh Mandal a,*a School of Medical Science and Technology, Indian Institute of Technology, Kharagpur 721302, IndiabDepartment of Chemistry, Indian Institute of Technology, Kharagpur 721302, IndiacDepartment of Human and Molecular Genetics, VCU Institute of Molecular Medicine, VCU Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USAdDepartment of Biotechnology, Indian Institute of Technology, Kharagpur 721302, India

a r t i c l e i n f o

Article history:Received 3 November 2010Accepted 12 January 2011Available online 10 March 2011

Keywords:NanocompositeCelecoxibColon cancerInhibitionXenograft

* Corresponding author. Tel.: þ91 3222 283578; faxE-mail address: mahitosh@smst.iitkgp.ernet.in (M

0142-9612/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.biomaterials.2011.01.027

a b s t r a c t

Celecoxib has shown potential anticancer activity against most carcinomas, especially in patients withfamilial adenomatous polyposis and precancerous disease of the colon. However, serious side effects ofcelecoxib restrict its generalized use for cancer therapy. In order to resolve these issues and develop analternative strategy/preliminary approach, chitosan modified hydroxyapatite nanocarriers-mediatedcelecoxib delivery represents a viable strategy. We characterized the nanoparticle for morphology,particle size, zeta potential, crystalinity, functional group analysis, entrapment efficiency, drug releaseand hemocompatibility. The effects of celecoxib-loaded nanoparticles on colon cancer cell proliferation,morphology, cytoskeleton, cellular uptake and apoptosis were analysed in vitro. Further, we evaluatedthe antiproliferative, apoptotic and tumor inhibitory efficacy of celecoxib-loaded nanocarriers in a nudemouse human xenograft model. Nanoparticles exhibited small, narrow hydrodynamic size distributions,hemocompatibility, high entrapment efficiencies and sustained release profiles. In vitro studies showedsignificant antiproliferation, apoptosis and time-dependent cytoplasmic uptake of celecoxib-loaded Hap-Cht nanoparticles in HCT 15 and HT 29 colon cancer cells. Additional in vivo studies demonstratedsignificantly greater inhibition of tumor growth following treatment with this modified nanoparticlesystem. The present study indicates a promising, effective and safe means of using celecoxib, andpotentially other therapeutic agents for colon cancer therapy.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Colon cancer is the second leading cause of cancer-relateddeaths worldwide and accounts for 677,000 deaths per year [1,2].Major risk factors for colon cancer are diet and smoking whichcontain aromatic and heterocyclic amines [3]. Standard first-linechemotherapeutic regimens for colon cancer involve a combinationof infusional 5-fluorouracil, leucovorin and oxaliplatin with bev-acizumab or a combination of infusional 5-fluorouracil, leucovorinand irinotecan with bevacizumab [2]. However, severe side effectsassociated with these chemotherapeutic strategies decrease thepatient’s quality of life and can be fatal at times. Hence, treatmentstrategies displaying minimal or no toxicity are vital to effectivelytreat this deadly disease. Efficacy of an anticancer agent in patient

: þ91 3222 282221.. Mandal).

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depends on both its potency and application of an effective drugdelivery system. Hence, a current focus of cancer chemotherapy istargeted delivery of anticancer agents to the tumor site bymanipulating pharmacokinetic and biodistribution properties ofnanocarriers [4,5]. Various drug delivery carrier systems such asnanoparticles, liposomes, polymeric micelles and parentral emul-sions have been evaluated for cancer chemotherapy [6e8]. Amongthese systems, nanoparticle-mediated delivery provides a numberof advantages including small particle size, increased drug efficacy,lowered toxicity, enhanced drug solubility and stability, and anability to achieve steady-state therapeutic levels over an extendedtime frame [9]. In cancer tissues, the phenomenon of “Enhancedpermeability and retention” (EPR) uniquely modifies the tumormicroenvironment promoting angiogenesis, hypervascularization,defective vascular architecture, impaired lymphatic drainage/recovery system and increased production of permeability media-tors [10]. This passive mechanism can be exploited for selectivetargeting of cancer tissue by nanoparticles. These nanoparticles can

P. Venkatesan et al. / Biomaterials 32 (2011) 3794e3806 3795

be prepared from organic polymers, inorganic compounds orbiomolecules such as gelatin and albumin, which are non-toxic [11].

Hydroxyapatite (Hap) is a calcium phosphate salt that possessesvarious useful properties such as biocompatibility, bioactivity, andosteoconductivity, and is non-toxic, noninflammatory, and non-immunogenicity with ultrafine structure similar to physiologicalhydroxyapatite, which can be exploited in various drug deliveryapplications [12]. Some calcium (Hap)-based drug delivery systemshave already been reported in the literature for antibiotics (cipro-floxin) and anticancer agents [13,14]. Hap has reported to producecytotoxicity against sarcomas and carcinomas, particularly humanhepatomas, colon carcinomas and gastric carcinomas [14,15]. Thehydroxyapatite prepared through a chemical precipitation methodusing ‘capping agent’ offers advantages of forming nanocrystalswith different morphologies and sizes [16]. Furthermore, thesurface functionality and mechanical properties of Hap nano-particles can be improved by forming a composite of polymer layeron its surface [16]. Chitosan (Cht) is one such polymer used tomodify the Hap to form different sized nanocomposite materialswith improved mechanical properties [17,18]. Chitosan is a linearpolysaccharide molecule, which is extensively used as a pharma-ceutical carrier in controlled release due to chemical modifications,biocompatibility, biodegradability and low cytotoxicity [19]. Chi-tosan has also been reported to improve sustained release and slowelimination of paclitaxel, which was attributed to the amphiphilicnature of the polymer [6]. Particle size, surface charge and materialcomposition of nanoparticles play vital roles in incorporation ofdrugs and ligands, and in diminishing cytotoxicity. In this regard,the chitosan nanocarriers (that are positively charged) exhibitincreased uptake efficiency by negatively charged cancer cellmembranes due to electrostatic attraction [19,20]. Previous studieshave reported the application of hydroxyapatite/chitosancomposites for antibiotic delivery [14,21].

Anticancer agents with hydrophobicity and narrow therapeuticindexes are difficult to formulate as drug products in conventionalmethods for treating cancer [22]. Hence, these drugs representpotential candidates for targeted drug delivery [23]. Among thisgroup NSAID’s (non steroidal antinflammatory drugs), Celecoxibhas been extensively evaluated for its anticancer activity againstseveral cancer models [24,25]. Preclinical studies of celecoxibreported prominent anticancer activity in head and neck squamouscell carcinoma, colon cancer, breast cancer and lung cancer [24,25].Hence, celecoxib was approved by the FDA of the USA for adjuvanttreatment of patients with colon cancer [1]. Regardless of thesuccess of celecoxib in colorectal cancer therapy, associations ofside effects including thromboembolism and cardiovascular risk,and poor water solubility have restricted its usage in cancerchemotherapy [26,27]. Celecoxib is also rapidly eliminated fromplasma, which might limit the therapeutic concentration at thetumor site [28]. In addition, in vitro studies mainly employ DMSO(dimethyl sulfoxide) as a solvent for solubility of celecoxib, which isnot suitable for in vivo use [3,24e26,29]. Consequently, it is crucialto define differentmethods of administering celecoxib, i.e., targeteddrug delivery systems (DDS) specific for cancer cells. To the best ofour knowledge, no study has been reported on the anticanceractivity of celecoxib-loaded Hap-Cht nanocomposites in humancolon cancer. Based on these considerations, we hypothesized thatthe celecoxib-loaded nanocomposites might provide a novel andeffective approach to circumvent toxicity and solubility bymeans oftargeted delivery to the tumor site through the EPR phenomenon.

In the present study, Hap, Hap-Cht and celecoxib-loaded Hap-Cht nanoparticles were prepared using a coprecipitation method.These nanoparticles were characterized by XRD (X-ray powderdiffraction), FTIR (Fourier transformed infrared), DLS (Dynamiclight scatter) and HRTEM (high resolution transmission electron

microscopy). Entrapment efficiency and hemocompatibility of thenanoparticles was estimated by reverse-phase HPLC (High Perfor-mance Liquid Chromatography) and hemolytic assays, respectively.Anticancer activity of the nanoparticle on colon cancer cell HCT 15was analysed in vitro by cell proliferation assays and by morpho-logical, nuclear and cell cycle analyses. Intracellular uptake of thenanoparticle was investigated using flow cytometry and epi-fluo-rescence microscopy. In vivo tumor inhibition efficacy of thenanoparticle formulation was evaluated in a human colon cancerxenograft mouse model.

2. Materials and methods

Calcium chloride (98%), tartaric acid (99%), orthophosphoric acid (85%) andammonia (25%) were purchased from Merck Pvt Ltd Bombay. Chitosan (85%deacetylated form), propidium iodide (PI), Hochest 33258, RNase (Ribonuclease),rhodamine-phalloidin, FITC (fluorescein isothiocyanate), MTT ((3-(4,5-Dimethylth-iazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent, DAPI (40 ,6-diamidino-2-phenylindole) and DMSO were obtained from Sigma Aldrich, USA.Celecoxib wasgenerously provided by Aarthy Pharmaceutical Ltd Mumbai. RPMI 1640 medium,glutamine, penicillin, streptomycin and FBS were purchased from Himedia andGibco� Invitrogen India, respectively. All other chemicals used in this study were ofanalytical grade.

2.1. Cell culture conditions

Human colon cancer cell lines HCT 15 and HT 29 (National Centre for Cell Science(NCCS) Pune, India) were grown as adherent cultures in RPMI 1640 medium sup-plemented with 10% FBS, 2 mM l-glutamine, 100 units/ml penicillin and 0.1 mg/mlstreptomycin at 37 �C and 5% CO2 in air. After cells became 80% confluent (usuallyafter 3 days), they were trypsinized (0.25% Trypsin þ 0.1% EDTA), centrifuged(Heraeus Table top Centrifuge E003, Germany) and suspended in RPMI medium.Cells were for experiments displayed >95% viability. For subsequent experiments,the cells were seeded in 96-well plates, cover slips and 60 mm petridishes.

2.2. Preparation of Hap-Cht nanocomposites

Hap was prepared using calcium chloride (CaCl2) and orthophosphoric acid asa source of calcium and phosphate ions, respectively. Equal volume aliquots offreshly prepared aqueous solutions of CaCl2 (0.1 M) and tartaric acid (0.2 M) weretaken (stoichiometric ratio) and mixed together to obtain a solution of water-esoluble complex of calcium tartrate. Appropriate volume of orthophosphoric acid(0.06 M), requisite for maintaining the Ca/P mole ratio at 1.67 in the mixture, wasthen mixed into the solution under vigorous stirring. Finally, a white gelatinousprecipitate was obtained when the pH of the resultant solution mixture wasadjusted to 11 by drop-wise addition of ammonia. The obtained precipitate wasseparated by filtration and repeatedly washed with de-ionized water to remove anytraces of dilute ammonia (0.2 M) from the residue. The precipitate was eventuallyvacuum-dried at 80 �C and ground to a fine powder for further characterization andanalysis.

For the preparation of the (Hap-Cht) nanocomposites, a clear and homogeneoussolution of chitosan was made in dilute acetic acid [21]. At first, a clear solution ofchitosan was obtained by dissolving 200 mg of chitosan in 10 ml of 1% (v/v) aceticacid. Next, 1.0 gm of the dried hydroxyapatite powders was accurately weighed andmixed with 25 ml of de-ionized water to form slurry. The slurry was then added intothe chitosan solution under vigorous stirring to obtain a uniform dispersion ofhydroxyapatite (0.2 w/w%) in the solution mixture. Drop-wise addition of diluteammonia (0.2 M) into the dispersed mixture under vigorous stirring eventuallyturned it opalescent at pH 9, suggesting the formation of (Hap-Cht) nanocompositeparticles. The excess acetic acid and ammonia was removed from the resultantmixture through dialysis and the separated (Hap-Cht) nanocomposite particles wasused for characterization.

2.3. Preparation of celecoxib-loaded Hap-Cht nanocomposites

The procedure for preparing the celecoxib-loaded Hap-Cht nanoparticles issimilar to the method described above. In this method, celecoxib was first dissolvedin ethanol and it was then added to the dispersion of hydroxyapatite (0.2 w/w) inchitosan solution (dilute acetic acid). The mixture was centrifuged and redispersedin ethanol to remove traces of free celecoxib. For confocal laser fluorescencemicroscopic (CLSM, Olympus FV 1000 attached with inverted microscope IX 81, Japan)analysis, green fluorescent FITC was conjugated with free amine group present onthe surface of the Hap-Cht nanoparticles [20].

For in vitro studies, a stock solution of Hap-Cht and celecoxib-loaded Hap-Chtnanoparticles was dispersed in culture medium (RPMI 1640 incomplete medium)using a sonicator (Cole Parmer ultra sonic probe CP-18, USA) and then sterilized bypassage through a 0.2 mm membrane filter. During filtration, some amount of

P. Venkatesan et al. / Biomaterials 32 (2011) 3794e38063796

nanoparticles was retained due to its large particle size. According to the weight lossmeasurement, a higher initial concentration was used to equilibrate the lost nano-particles to guarantee a final stock concentration. Then the stock suspension wasserially diluted to produce desired concentrations of the complex.

2.4. Characterization of nanoparticles

Phase analysis of the nanoparticles was carried out by XRD (XRD-PhilipsPW1710 diffractometer) using Ni-filtered CuKa radiation within the scanning rangeof 2q ¼ 20� to 60� , scan rate of 1.1� min�1, sampling interval of 0.02 at 30 mA and40 kV current. The functional group analysis of nanoparticle was carried out usingFTIR Spectroscopy (Perkin Elmer RX-II, Model No 73713, USA) within the wavenumber range of 4000e450 cm�1. Thermogravimetric analysis of all the sampleswas done by Pyris Diamond TG/DTA machine (NETZSCH STA 409 PC, Germany)heating the sample at a heating rate 10�/min in an alumina crucible in presence ofair. Morphology and particle size of the nanoparticles were analyzed using HRTEM(JEM-2100, JEOL, Japan) with an acceleration voltage of 200 kV. Particle size distri-bution, mean particle size and zeta potential of the nanoparticles were determinedusing Zetasizer Nanoseries (Malvere instruments, USA) equipped for the measure-ment of both particle size and zeta potential. Prior to the measurement of particlesize and zeta potential, the nanoparticles (w1 mg/ml) were freshly dispersed in PBSat pH 7.4.

2.5. Entrapment efficiency of celecoxib and its release profile

Entrapment efficiency of celecoxib in Hap-Cht nanoparticles was determined byextracting celecoxib from a defined mass of celecoxib-loaded Hap-Cht nanoparticleswith 1 ml of methanol containing 0.1 N HCl (pH 1.0). Then the extract was filteredthrough a syringe filter and analyzed by reverse-phase HPLC [30]. HPLC analysis wascarried out using a Perkin Elmer HPLC system (USA) equipped with a Perkin Elmerseries 200 pump, C18 column (4.6 mm � 250 mm, 5 mm) preceded by a C18 guardcolumn and UV detectors at a wavelength of 250 nm. The columnwas maintained atroom temperature. Amixture of methanol: water (75:25 v/v) was used as themobilephase. The flow rate of 20 ml sample injection was maintained at 1.25 ml min�1. Acalibration curve was constructed using a celecoxib concentration range from 0 to50 mg/ml. Dilution factor was taken into consideration for calculating the entrap-ment efficiency (EE) of celecoxib in the nanoparticles. Celecoxib encapsulationefficiency was determined in triplicate, and the values were reported asmeans� SD.The percentage of the drug entrapped in nanoparticles was calculated by thefollowing expression:

EEðEntrapment efficiencyÞ ¼ ½Amount of celecoxib in the nanoparticle=Total amount of celecoxib� � 100%

To obtain the celecoxib release profile from Hap-Cht nanoparticles, 10 mg ofcelecoxib-loaded Hap-Cht nanoparticles (n¼ 3) were suspended in 3ml PBS (pH 7.4)and stirred [100 strokes/min] continuously at 37 �C. The amount of released cele-coxib was determined by studying aliquot amounts of the sample solutions with-drawn at selected time intervals (1e15 days). At each time interval the sample waswithdrawn, centrifuged at 8000 rpm for 10 min and absorbance of the supernatantwas detected using a UV spectrophotometer (Japan) at 250 nm [13,19].

2.6. Hemocompatibility study

In this study, free celecoxib, Cht, Hap, Hap-Cht and celecoxib-loaded Hap-Chtnanoparticles, each were individually suspended in 10 mM HEPES buffer saline.Hemocompatibility of these samples were analyzed using a modified protocol [31].In brief, blood was obtained from 6 week old BALB/c male mice and red blood cells(RBC) were collected by centrifugation (1500 g for 5 min at 4 �C) and a ficoll densitygradient. The collected RBC pellet was diluted in 20 mM HEPES buffered saline (pH7.4) to 5% v/v solution. The RBC suspension was added to HEPES saline, 1% Triton X-100 and other samples, and incubated at 37 �C for 30 and 60 min. After incubation,all the samples were centrifuged (Heraeus table top centrifuge 5805R, Germany) at12,000 rpm at 4 �C and supernatants were transferred to a 96-well plate. Thehemolytic activity was determined by measuring the absorption at 570 nm (Bioradmicroplate reader model 550, Japan). Control samples of 0% lysis (in HEPES buffer)and 100% lysis (in 1% Triton X-100) were employed in the experiment [18]. Hemo-lytic effect of each sample was expressed as percentage of cell lysis relative to theuntreated control cells, which can be given by the following expression: percentageof hemolysis ¼ [[OD 570 nm samples]/[OD 570 nm control cells]] � 100, whereoptical density is abbreviated as OD. Data is reported as mean � SD (n ¼ 3).

2.7. Cell proliferation assay

Cytotoxicity of free celecoxib, Hap-Cht and celecoxib-loaded Hap-Cht nano-particles on HCT 15 cells was determined by conventional MTT assay, as describedpreviously [32,33]. Briefly, HCT 15 cells in the exponential growth phase wereseeded in 96-well flat-bottom culture plates at a density of 5 � 103 cells per well in0.1 ml RPMI 1640 complete medium. The cells were allowed to adhere and grow for

24 h at 37 �C in an incubator (Heraeus Hera Cell, Germany), after which the mediumwas aspirated and replaced with 0.1 ml fresh medium containing various concen-trations of free celecoxib, Hap-Cht and celecoxib-loaded Hap-Cht nanoparticlesdispersion. Control wells were treated with equivalent volumes of celecoxib freemedia. After 72 h of incubation, the culture medium was removed, and 100 ml of1 mg/ml MTT reagent in PBS was added to each well. After 4e5 h incubation, theunreduced MTT solution was discarded. Then, DMSO (100 ml) was added into eachwell to dissolve the purple formazan precipitate which was reduced from MTT byactive mitochondria of viable cells. Plates were shaken and formazan dye wasmeasured spectrophotometrically using a benchmark microplate reader. The assaywas performed in triplicate. The cytotoxic effect of each treatment was expressed aspercentage of cell viability relative to the untreated control cells (% control) definedas: [[OD 550 nm treated cells]/[OD 550 nm control cells]] � 100.

2.8. Morphological analysis

HCT 15 cells at a density of 6 � 103 were grown on sterile poly-L-lysine-coatedcover glass and treated without (control) or in the presence of an IC50 concentrationof free celecoxib, celecoxib-loaded Hap-Cht nanoparticles or Hap-Cht (equivalent toweight of celecoxib-loaded Hap-Cht nanoparticles) for 48 h. After incubation, thecells were observed under a phase contrast microscope (Leica 20X, Germany) [11,14].

The morphological analysis of cancer cells using high resolution SEM wouldprovide clear evidence of an effect of these nanoparticles on filopodia and lamelli-podia [34]. After treatment, the cells were washed three times in 0.1 M cacodylatebuffer (pH 7.4) and then fixed in ice-cold 1% OsO4 for 1 h. The cells were thendehydrated with ethanol (50%, 70%, 95% and 100%). The samples were placed inHMDS (1,1,1,3,3, 3-Hexamethyl disilazane) for 5 min to overcome drying effects.Samples were then air dried at room temperature andmounted on a stub. Next, theywere placed in a vacuum chamber of SEM gold coating apparatus and gold wascoated at 2.5 kV, 20e25mA for 120 s. Themorphogram of the HCT 15 cells were thenobserved using a scanning electron microscope (JEOL JSM-5800, Japan) using 20 kVacceleration voltage.

2.9. Cytoskeleton and nuclear analysis

Cytoskeleton analysis of nanoparticle-treated HCT 15 cells was performed usingCLSM [34]. Briefly, HCT 15 cells were grown on poly-L-lysine-coated cover glassslides and treated with free celecoxib, Hap-Cht or celecoxib-loaded Hap-Cht nano-particles for 48 h. The medium was removed, slides were washed three times withPBS (pH 7.4), fixed with ice-cold 4% paraformaldehyde in PBS (pH 7.4) and per-meabilized with 0.1% Triton X-100. Then non-specific binding sites were blockedusing PBS containing 10% FBS for 1 h. The cells were stained with blue fluorescentDNA stain (Hoechst 33258) and the red fluorescent dye (rhodamine-phalloidin) tovisualize nuclei and cytoskeletal actin, respectively. After staining, the adhering cellswere washed with PBS, air dried and mounted on slides. Fluorescent images fromthe stained constructs were obtained using a CLSM at 40X (CLSM, Olympus FV 1000,Japan) equipped with Argon (488 nm) and He-Ne (534 nm) lasers.

For nuclear analysis, the above protocol was followed with slight modification.Briefly, cells were treated with nanoparticles for 48 h, fixed, permeabilized, stainedwith 0.2 mg/ml of DAPI (4, 6-diamidino-2-phenylindole) in PBS at room temperaturefor 15 min and mounted onto glass slides. The nuclear morphology of the cells wasanalyzed using epi-fluorescence microscope at 20X (Leica DMR, Germany).Apoptotic cells were evaluated based on nuclear morphology, chromatin conden-sation and fragmentation [35].

2.10. Cell cycle analysis

HCT 15 cells were cultured in 60 mm petridishes for 24 h at a density of 2 � 105

and then treated with IC50 concentration of free celecoxib, Hap-Cht or celecoxib-loaded Hap-Cht nanoparticles for 48 h. In this study, untreated and Hap-Chtnanoparticle-treated cells were used as reference control and blank formulation,respectively. After incubation, cells were trypsinized and centrifuged at 1200 rpmfor 5 min at 4 �C. The pellet was suspended in 5 ml of PBS and then centrifuged at1200 rpm for 10 min at 4 �C. The supernatant was discarded and the pellet was fixedwith 2 ml of ice-cold ethanol solution (70% v/v in PBS) at 4 �C overnight. Fixed cellswere centrifuged at 1200 rpm for 10min at 4 �C and the pellet was incubatedwith PImixture (10 mg/ml RNase, 20 mg/ml propidium iodide dissolved in cold PBS) for30 min at 37 �C. DNA content analysis was carried out on a FACS Calibur (BDBioscience, USA) flow cytometer (10,000 events were acquired for each sample). Thedata obtained were processed for cell cycle analysis with the cell quest pro softwarepackage [20]. The amount of propidium iodide intercalating to DNAwas used as theparameter to determine the cell cycle distribution phases. Apoptosis fraction wasconsidered as DNA loss resulting in a sub-G1 peak.

2.11. Cellular localization/uptake of nanoparticles by fluorescence microscopy andflow cytometry

To determine cellular localization of celecoxib-loaded nanoparticles, HCT 15 andHT 29 cells were seeded at a density of 3�104 and 5�104 cells on sterile glass cover

P. Venkatesan et al. / Biomaterials 32 (2011) 3794e3806 3797

slips, respectively. The cover slips were pretreated with 0.01% poly-L-lysine beforeseeding. The cells were then treatedwith 250 mg/ml of FITC labeled celecoxib-loadedHap-Cht nanoparticles for different time periods. After incubation, the cover slipswere removed and washed twice with PBS solution. Then cells were fixed using 4%paraformaldehyde for 20 min. The cells were washed with an additional 2X PBS,counterstained with propidium iodide for 30 min and mounted in fluorescentmounting medium. The HCT 15 and HT 29 cells were analyzed using epi-fluores-cence and CLSM, respectively [20,36].

To further verify the uptake of nanoparticles by HCT 15 and HT 29 cells, flowcytometeric analysis was performed [20,23]. HCT 15 and HT 29 cells were cultured in24-well culture plates at a density of 1 �104 and 3 � 104 cells per each well in RPMI1640 medium at 37 �C for 24 h, respectively. The cells were next treated with250 mg/ml of FITC labeled celecoxib-loaded Hap-Cht nanoparticles for different timeperiods. The culturemediumwas then removed and each well was washed with PBSat pH 7.4. The cells were fixed by adding 300 ml of paraformaldehyde (4% v/v) for15 min. The samples were determined using FACS (FACS Calibur BD Biosciences,USA). Then the cell associated FITC labeled nanoparticles were analyzed in greenchannel [excitation 490 nm, emission 520 nm]. Data were collected from10,000gated events and analyzed using the CELL Quest pro software program (BD Biosci-ences, USA).

2.12. Changes in cell surface potential

Surface charge of nanoparticle-treated HCT 15 cells was determined to validatethe cellular localization of the nanoparticles [20]. HCT 15 cells grown on petridisheswere incubated with 250 mg/ml celecoxib-loaded Hap-Cht nanoparticles at twodifferent time periods of 1 and 4 h. Then the cells were trypsinized to prepare a cellsuspension in PBS at pH 7.4. The surface charge of the nanoparticles localized in cellswas determined using DLS and analysis was performed at 25 �C.

2.13. Tumor inhibition in a human colon cancer xenograft mouse model

Nude mice (6e8 week old and 18e22 g of weight) provided by the animal carecenter, at the Virginia Commonwealth University were maintained under pathogenfree condition. All procedures performed in animals were approved by the institu-tional animal use and investigation committee at Virginia Commonwealth Univer-sity, School of Medicine, Richmond, VA, USA. HCT 15 cells were collected at theirlogarithmic growth phase and resuspended in serum-free medium at a density ofapproximately 106 cells in 0.2 ml suspension and were injected subcutaneously intothe right flank of mice [9]. When tumor volume reachedw100 mm3 (after 14 days),the animals were randomly assigned to four groups (6 animals per group). Prior totreatment, body weight and tumor volume of all mice were measured. Then, thetumor bearing mice were injected by tail vein as follows: (i) sterile normal saline(control group); (ii) celecoxib (100 mg/kg celecoxib); (iii) celecoxib-loaded Hap-Cht

Fig. 1. Schematic representation of celecoxib lo

nanoparticles (100 mg/kg celecoxib eq.); and (iv) Hap-Cht nanoparticles (weight ofthe celecoxib-loaded nanoparticles). Injections were performed 3 times in first 5days, 1 day spaced between two administrations.

Following treatment (14th day after tumor cell inoculation), animals weremonitored regularly for tumor growth, survival, visible toxicity and any change atthe injection sites for the remainder of the study. At the end of the 4-week treatmentperiod (30 days), the mice were asphyxiated using carbon dioxide. The tumors wereexcised and measured with vernier calipers in two dimensions. The tumor volume(V in mm3) was calculated with following formula [35,37]: V¼ (LxW2)/2, where L(mm) is the longest diameter and W (mm) is perpendicular to L. For immunohis-tochemical analysis, one part of the tumor was fixed in formalin and embedded inparaffin. Another part was embedded in optimal cutting temperature (OCT)compound, rapidly frozen in liquid nitrogen and stored at �80 �C.

2.13.1. Immunohistochemical analysis of cellular proliferation (Ki-67 antigen) andapoptosis (TUNEL and DAPI)

Analysis of celecoxib, Hap-Cht and celecoxib-loaded Hap-Cht nanoparticles onproliferation of human colon cancer xenografted mice tumors was performed usingKi-67 antibody. Tissue specimens were processed for immunohistochemical anal-yses as described previously [38e40]. Neutral buffered formalin-fixed tissue wasembedded in paraffin. Tissue sections (5 mm) were prepared using a microtome andmounted on slides. Immunohistochemical analysis was done within 24 h of thesections being cut. Sections were deparaffinized in xylene, rehydrated in gradedalcohols (100%, 95% and 80% v/v) and washed in distilled water. Endogenousperoxidase activity was quenched with 0.01% H2O2. Further, sections for Ki-67analysis were treated with 0.05% trypsin, 0.05% CaCl2 in TriseHCl (pH 7.6) for 5 minat 37 �C. Antigen retrieval was done by microwaving the sections in 10 mM/l citricacid (pH 6.0) for 30 min. The slides were washed thrice in PBS and blocked with 10%normal horse serum for 30min. Tissue sections were then incubatedwith antiserumto Ki-67 (1:50) for 3 h at room temperature. After being washed thrice with PBS, thesections were incubated with biotinylated anti-mouse immunoglobulins (1:500) for30 min at room temperature. The slides were then washed thrice in PBS, labeledusing avidin-biotin peroxidase complexes (1:25) for 30 min at room temperatureand then washed with 2X PBS. Immunoreactivity was determined using dia-minobenzidine as the final chromogen. Finally, sections were counterstained withMeyer’s hematoxylin, dehydrated through a sequence of increasing concentrationsof alcohol, cleared in xylene and mounted with epoxidic medium.

Effect of celecoxib, Hap-Cht and celecoxib-loaded Hap-Cht nanoparticles onapoptosis of xenografted mouse tumors was performed using commercially avail-able TUNEL kit (Promega Corporation, USA) and DAPI (4, 6-diamidino-2-phenyl-indole) staining. TUNEL assay was described above with the following modifications[39]. Sections from frozen tissues were fixed with 4% paraformaldehyde for 10 min,washed twice with PBS for 5 min and then incubated with 0.2% Triton X-100 for15 min. After two 5-min washes with PBS, the samples were incubated with

ading and FITC tagging in nanocomposites.

P. Venkatesan et al. / Biomaterials 32 (2011) 3794e38063798

equilibration buffer for 10 min. The equilibration buffer was drained, reaction buffer(44 ml equilibration buffer, 5 ml nucleotide mix and 1 ml terminal deoxynucleotidyltransferase) was added to the sections and incubated at 37 �C for 1 h in the dark. Thereaction was terminated by immersing the samples in 2X SSC for 15 min. Sampleswere then washed with PBS to remove unincorporated fluorescein-dUTP. TUNEL-positive apoptotic cells were detected by localized green fluorescence within the cellnuclei. Further some tissue sections were stained with 0.2 mg/ml DAPI in PBS for15 min at room temperature, washed twice each with PBS and water, and mounted.In this study, epi-fluorescence microscope equipped with narrow band pass exci-tation filters mounted in a filter wheel was used. We captured images by usinga chilled, cooled, charge-coupled device camera and SmartCapture software. Fromthe DAPI stained images, the apoptotic cells were evaluated based on the nuclearmorphological changes, chromatin condensation and fragmentation [41].

2.14. Statistical analysis

All the statistical analysis was performed by graphpad prism 5 software. Datawere presented using mean � SD. The statistical significance was determined byusing one-way analysis of variance (ANOVA). ***P < 0.001 and **P < 0.05 wereconsidered significant.

3. Results and discussion

3.1. Formation of nanoparticles

The formation of Hap nanoparticles involves following chemicalreaction:

10Ca2þþ 6PO3�4 þ 2OH� / Ca10ðPO4Þ6ðOHÞ2 (1)

In this reaction, calcium chloride was suitably complexedwith tartaric acid in the presence of orthophosphoric acid and

Fig. 2. (A) XRD pattern of Hap (a) and Hap-Cht (b) nanoparticles. (B) FTIR spectra of Hap (acurves of (a) Hap, (b) Hap-Cht and (c) celecoxib-loaded Hap-Cht nanoparticles.

ammonium hydroxide to form Hap nanoparticles. The formednanoparticles were further modified by addition of chitosan poly-mer, which introduced reactive functional group (NH2) on thesurface of the nanoparticles [21]. Further, FITC molecules wereadded to bind with free amine groups on surface of the nano-particles for cellular uptake studies [42]. Fig. 1 shows a schematic ofcelecoxib-loaded Hap-Cht nanoparticles preparation. A previousstudy has reported the potential of loading both hydrophilic andhydrophobic drugs in a chitosan nanoparticle system [19]. Based onthis consideration, hydrophobic celecoxib might be incorporatedinto Hap-Cht nanoparticles either by embedding physically into theexternal surface or adsorbing onto the surface of the nanoparticle.Furthermore, charge density is an important factor in determiningthe electrostatic interaction with cells. Approaching of positivelycharged celecoxib and chitosan towards the negatively chargedHap (�13.9 mV) core might be driven by electrostatic attraction.This electrostatic interaction was further corroborated by anincrease in zeta potential from �1.8 mV (Hap-Cht nanoparticles) to6.36 mV (celecoxib-loaded Hap-Cht nanoparticles). The zetapotential is a good index of degree of repulsive interaction betweennanoparticles. It depends on the surface charge of the nano-composite, especially on concentration of polymer and the incor-porated drug [31].

3.2. Characterization of nanoparticles

Phase analysis of nanoparticles was performed by X-raydiffraction (XRD). The diffraction data are consistent with JCPDS file

), Hap-Cht (b) and celecoxib-loaded Hap-Cht (c) nanoparticles. (C) Thermogravimetry

Fig. 3. (a) TEM micrographs of Hap, Hap-Cht and celecoxib-loaded Hap-Cht nanoparticles. (b) Drug release profile of celecoxib-loaded Hap-Cht nanoparticles. Values shown hererepresent � SD (n ¼ 3).

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no: 09e432. Fig. 2 (Aa and Ab) show the identical XRD patterns forboth Hap and Hap-Cht nanoparticles, respectively. The XRD peakswere broad, which revealed the nanocrystalline nature of Hap andHap-Cht nanoparticles. In Hap nanoparticles, broadening of thepeaks might be attributed to lattice strain and low crystalline size.Both Hap and Hap-Cht nanoparticles produced broader XRD lines,which indicate nanocrystalline composition. The crystallite sizewas calculated using Scherrer’s equation i.e.,

D ¼ 0:9 l

b cos q

Where, D is crystallite size, l is wavelength of target material(1.5406 Ǻ), b is full width at half height (FWHH) in radians and q isdiffraction angle. The mean crystallite size of Hap and Hap-Chtnanoparticles was calculated as 15 nm and 17 nm respectively.Generally, crystalline nanocomposites are required due to theirhigh in vivo resorbability.

Functional group analysis was carried out using FTIR spectros-copy. From the Hap analysis (Fig. 2Ba), stretching vibration ofhydroxyl group band is assigned to 3571 cm�1 and 634 cm�1, whilebroad band at 3427 cm�1 is attributed to absorbedwatermolecules.Additionally, bands at 1096, 1032, 962, 603 and 564 cm�1 can beattributed to PO4

3- group which is in agreement with valuesreported in the literature [12]. Hap-Cht analysis revealed weakbands at 2925 cm�1 and 1653 cm�1 which can be ascribed tostretching vibration of CeH and amide bond in the chitosanmolecules, respectively (Fig. 2Bb). In addition, the band at3568 cm�1 was nearly identical with those of hydroxyapatite-gelatin nanocomposites [21]. Interestingly, celecoxib-loaded Hap-Cht nanoparticles exhibited bands at 1333 and 1147 cm�1 that canbe attributed to S]O stretch (asymmetry) and S]O stretch(symmetry), respectively. While 1284 and 1234 cm�1 correspond toCeF stretch (asymmetry) and CeF stretch (symmetry). Hence,these results represent the incorporation of celecoxib into Hap-Cht

nanocomposites (Fig. 2Bc). Further, thermogravimetric analysiswas performed to confirm the Cht coating formation on the surfaceof Hap nanoparticles. Fig. 2c (a, b and c) show the comparativeweight loss for Hap, Hap-Cht and celecoxib-loaded Hap-Chtnanoparticles, respectively. Hap nanoparticles have demonstratednegligible amount of weight loss. But, Hap-cht nanoparticles dis-played weight loss between 50 and 170 �C which is due to loss ofphysically adsorbed water molecule on the surface. Further theweight loss in the second regime corresponds to decomposition ofCht coating over the Hap nanoparticles. In celecoxib-loaded Hap-Cht nanoparticles, the weight loss can be ascribed to removal ofphysically adsorbed moisture (50e170 �C) and decomposition ofthe celecoxib and chitosan (200e550 �C) that contribute to majorportion of total weight the nanoparticles.

TEM analysis of nanoparticles is a semi quantitative technique,which provides sufficient information on morphology and the sizeof nanoparticles [43]. Fig. 3a represents HRTEM images of Hap,Hap-Cht and celecoxib-loaded Hap-Cht nanoparticles. The resu-lts confirm the nanoscale size with acicular needle like shapeand spherical shape of Hap nanoparticles (4e7 nm in diameter and25e39 nm length) and both Hap-Cht (45e65 nm in diameter) andcelecoxib-loaded Hap-Cht (60e75 nm in diameter) nanoparticles,respectively. As these particles are so small, they tend to aggregatedue to their high specific surface energy and form unclear images[44].

Particle size distribution and mean particle sizes of the nano-particles were measured by DLS in PBS at pH 7.4. The results indi-cate a well dispersed colloidal system of nanoparticles. The meanparticle size determined for Hap, Hap-Cht and celecoxib-loadedHap-Cht nanoparticles as 120.8 nm, 144.6 nm and 164.1 nm,respectively. In addition, Hap, Hap-Cht and celecoxib-loaded Hap-Cht nanoparticles exhibited particle size distribution in the range of97e135 nm, 125e158 nm and 146e180 nm, respectively. Thehydrodynamic particle size measured using nano zetasizer wasgreater than TEM analysis, which might be due to aggregation of

Fig. 4. (a) Hemolytic assay of Hap, Cht, celecoxib, Hap-Cht and celecoxib-loaded Hap-Cht nanoparticles. eve control of 0% lysis (in HEPES buffer) and þve control of 100% lysis (in 1%Triton X-100) were employed in this experiment. All the samples (excluding celecoxib) showing insignificant amount of hemolysis with respect to the þ ve control. The bars indicatethe means � SD (n ¼ 3). Significant difference is shown as ***P < 0.001 versus þ ve control. (b) Antiproliferative effect of celecoxib and celecoxib-loaded Hap-Cht nanoparticles onHCT 15 cells. (c) Phase contrast microscopic (20X) and (d) Scanning electron microscopic images of HCT 15 cells treated with control, free celecoxib (IC50), Hap-Cht (equivalent toweight of the celecoxib-loaded Hap-Cht nanoparticles) and celecoxib-loaded Hap-Cht nanoparticles (IC50) for 48 h. In the SEM images, healthy filopodia and lamellipodia of controlHCT 15 cells are marked with short arrows. In treated HCT 15 cells, truncated cytoplasmic extensions (lamellipodia and filopodia) are marked with long arrows.

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particles. The size of the anoparticles plays a vital role in interac-tions with cells and toxicity. It is also reported that the smaller sizedparticles permit increased cellular interaction and therefore maypossess enhanced intrinsic toxicity [45]. Keeping nanoparticles insolution for longer periods results in aggregation. Therefore, toguarantee nanosized particles for subsequent studies, the formu-lations had to be prepared fresh or prevented from settling byconstant gentle mixing prior to use [32].

3.3. Entrapment efficiency and in vitro release of celecoxib

Efficient encapsulation of celecoxib in Hap-Cht nanoparticleswas observed. Approximately 59% of entrapment efficiency wasdetermined from HPLC analysis. The in vitro release profile of cel-ecoxib from Hap-Cht nanoparticles is shown in Fig. 3b. About13.78 � 5.6% of the celecoxib was released in the first 24 h. It issuggested that the surface embedded celecoxib would be releasedearlier than the celecoxib-loaded inside the nanoparticles. Therelease rate gradually decreased to a sustained release from day 6(w54.07 � 5.4% of celecoxib) through day 15 (w83.56 � 15.23% ofcelecoxib) which can be attributed to low solubility and highviscosity of the chitosan gel layer formed around the nanoparticleupon contact with dissolution medium [19]. It was also reportedthat chitosan immersed in dissolution medium led to gradualdegradation and formation of interconnected pores on the surfaceand inside the Hap-Cht nanocomposites [21]. This porous structuremight facilitate slow release of celecoxib. Furthermore, the attrac-tive forces between celecoxib and the nanoparticles could sustain

release. These results suggest that release of celecoxib from thenanoparticles follows diffusion mechanism.

3.4. Hemocompatibility study

Intravenous administration of a drug is limited by its solubility,hemolytic activity and side effects. Nanoparticle formulation ofhydrophobic drugs has been reported to overcome the solubilityand hemolytic issues [18,31]. Hemolysis is an important factor toassess the biocompatibility of a material [9]. Hence, hemolyticassays were performed to examine interaction of nanoparticleswith red blood cell membranes by measuring the released hemo-globin. The membrane of red blood cells (RBC) consists of two-sublayers, the external sublayer being negatively charged and theinternal one being positively charged. It has been reported thatproperties such as structure, size, surface chemistry, softness,surface charge and agglomeration state of nanoparticles play a vitalrole in interaction with RBC [9,45]. In this study, free celecoxibexhibited significant hemolysis, which may be due to SO2NH2groups present in celecoxib (Fig. 4a) [18]. The interaction betweencell and free celecoxib might induce membrane twists, whichresults in rupture and release of hemoglobin. However, Hap-Chtand celecoxib-loaded Hap-Cht nanoparticles exhibited significantlylow hemolytic activities (less than 2%), which can be attributed torigid nature of the nanoparticles [36]. A rigid molecule is less proneto attach to the RBC membrane than a flexible molecule, whichwould explain low hemolytic activity of Hap-Cht and celecoxib-loaded Hap-Cht nanoparticles. Generally, a percentage of hemolysisless than 5% was regarded as non-toxic material [9].

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3.5. Cell proliferation assay

In recent years, MTT assays have been routinely used asa preliminary screen for evaluation of in vitro cytotoxicity ofnanocomposite materials. It is a quick and effective method fortesting mitochondrial impairment and correlates quite well withcell proliferation. Nanoparticles can cause cytotoxicity throughadhering to cell membranes and subsequent release of cytotoxicdrugs [9]. To observe cytotoxicity of free celecoxib, celecoxib fromcelecoxib-loaded Hap-Cht and drug-free Hap-Cht nanoparticles,the cell proliferation assay against the colon cancer cell HCT 15 wasperformed. The range of concentrations of celecoxib used in theexperiment was 0e200 mM. Cell viability of 25% was achieved withfree celecoxib at the concentration of 200 mM upon 72 h of treat-ment. At the same concentration and incubation period, the cele-coxib-loaded Hap-Cht nanoparticles greatly reduced cell viability to8%. Results of MTT assays revealed a concentration-dependentdecrease of mitochondrial activities after treatment with free cel-ecoxib and celecoxib-loaded Hap-Cht nanoparticles (Fig. 4b)Interestingly, IC50 values of HCT 15 cells decreased from 102.7 mMfor celecoxib to 66.7 mM for the celecoxib-loaded Hap-Cht nano-particles after 72 h of incubation.

This higher toxicity suggests the possibility of increased cellularassociation and/or uptake of positively charged Hap-Cht nano-particles due to non-specific interaction with the negativelycharged cell surface [45]. In addition, a recent study also reportedthat the chitosan nanoparticles induced apoptosis in A2780 cancercells [20]. Hence, it is worth mentioning that the cytotoxicity ofcelecoxib-loaded Hap-Cht nanoparticles on HCT 15 cells can beattributed to the combination of celecoxib, chitosan and hydroxy-apatite. Minimal reduction in cell viability of Hap-Cht nanoparticle-treated HCT 15 cells [data not shown] is most likely due to highconcentration of free amines at the surfaces of the nanoparticlepresent in the cytosol [46].

3.6. Morphological analysis

Light microscopy and SEM analysis were used to view the effectsof nanoparticles on the morphology of HCT 15 cells after 48 h ofincubation. Morphology of HCT 15 cells treated with free celecoxib,Hap-Cht and celecoxib-loaded Hap-Cht nanoparticles wereobserved under a phase contrast microscope. Untreated controlHCT 15 cells displayed a well spread and flattened morphology(Fig. 4c). Conversely, free celecoxib and celecoxib-loaded Hap-Chtnanoparticles treated HCT 15 cells had prominent morphologicalchanges, which included rounding, reduced spreading, shrunkencells, and retraction of cellular processes. In case of Hap-Chtnanoparticles, treatment resulted in minimal morphologicalchanges, which can be attributed to cytotoxic effects of both chi-tosan and hydroxyapatite on HCT 15 cells. Phase contrast micros-copy has technical drawbacks limiting its application for viewingchanges in cellular architecture [34]. Consequently, it is difficult toexamine fine morphological changes in cancer cells mediated byanticancer agents using phase contrast microscopy [47].

High resolution SEM is a vital tool for analysis of surface andmorphological features of cancer cell. Though morphologicalcharacteristics of normal and cancer cells have been well studiedusing SEM, researchers have infrequently used SEM to examinemorphological changes in cancer cells mediated by anticanceragents [48]. We have used SEM to analyze the morphology treatedHCT 15 cells treated with free celecoxib, Hap-Cht and celecoxib-loaded Hap-Cht nanoparticles (Fig. 4d). Control cells are flat andhave a large number of smooth, slender and filamentous lateral cellmembrane extensions. This observation suggests that HCT 15 cellsare highly motile. The free celecoxib and celecoxib-loaded Hap-Cht

nanoparticle-treated HCT 15 cells had a thickened morphology,small ruffles, irregular retraction of cytoplasm from substratum andfewer cell membrane extensions. In contrast, Hap-Cht nano-particles treatment resulted in minimal morphological changes.These results suggest that the biologically active hydroxyapatiteand chitosan not only interact with HCT 15 cells, but they alsostimulate interaction between the celecoxib and HCT 15 cells.

3.7. Effect on the cytoskeleton and nucleus

In general, morphological features and submicroscopic struc-tures of cells obtained from CLSM images provide advantages overtraditional phase contrast microscopic images. Such images withCLSM include, but are not restricted to cell morphology, cytoskel-etal elements, organelles and cell volume. Therefore, many earlierstudies have utilized CLSM to examine structureefunction rela-tionships in biological systems [34]. In this study, we have usedCLSM to evaluate effects on morphology and actin organization oftreated HCT 15 cells. The untreated HCT 15 cells displayed anelongated shape and a dense network of actin, forming organizedparallel filamentous structures in the cytoplasm (Fig. 5a). After 48 hof treatment with free celecoxib (IC50) and celecoxib-loaded Hap-Cht nanoaparticles (IC50), the cells appeared round in shape andwere sparse and irregular with no striations in actin filamentsorganization [34]. Conversely, Hap-Cht nanoparticles (equivalent tothe weight of the celecoxib-loaded Hap-Cht Nanoparticle) treat-ment for 48 h produced minimal effects on cell shape and actinfilament organization (Fig. 5a). These results confirm that cele-coxib-loaded Hap-Cht produces more apoptotic activity than freecelecoxib, which might be attributed to both the celecoxib andHap-Cht nanocomposite.

DAPI, is a nuclear stain, that complexes with double-strandedDNA and produces fluorescence that is specific to adenine-thymine(AT), adenine-uracil (AU) and inosine-cytosine (IC) molecules [49].To examine the ability of celecoxib to induce apoptosis afterencapsulation in Hap-Cht nanoparticles, DAPI staining of DNA wasperformed [35]. The nuclei of control HCT 15 cells showedhomogenous fluorescence with no evidence of segmentation orfragmentation (Fig. 5b). Exposure of the cells to celecoxib-loadedHap-Cht nanoparticles (IC50) for 48 h led to characteristic changesin nuclear shape which included separation of cell nuclei intosegments and DNA condensation.

3.8. Cell cycle analysis

Celecoxib-induced apoptosis in HCT 15 cells was demonstratedby cell cycle analysis. The percentage of apoptotic cells was countedusing a FACs Calibur flow cytometer. As shown in (Fig. 5c), HCT 15cells untreated (Control), or treated with free celecoxib, Hap-Cht orcelecoxib-loaded Hap-Cht nanoparticles had 0.71 � 0.34%,39.14 � 0.47%, 6.21 � 0.75% and 62.33 � 5.22% of apoptosis,respectively. Empty nanoparticles have shown minimal apoptoticactivity when compared to medium-treated controls. It is evidentthat free celecoxib and celecoxib-loaded Hap-Cht nanoparticlestreatment of HCT 15 cells for 48 h induced more apoptosis thanHap-Cht nanoparticles. The higher apoptotic activity of celecoxib-loaded Hap-Cht nanoparticles than free celecoxib can be attributedto slow release of celecoxib from nanoparticles and the cytotoxiceffects of Hap-Cht on cancer cells [20]. The results suggest thatcelecoxib-loaded Hap-Cht nanoparticles might be internalized intocells and release celecoxib into the cytoplasm for a sustained periodof time resulting in apoptosis. Hence, celecoxib-loaded Hap-Chtnanoparticles can significantly enhance the efficiency of the intra-cellular delivery and the apoptosis-inducing effects of celecoxib.

Fig. 5. Effect of free celecoxib (IC50), celecoxib-loaded Hap-Cht (IC50) and Hap-Cht (equivalent to weight of the celecoxib-loaded Hap-Cht nanoparticles) on HCT 15 cell’s (a)cytoskeleton organization (CLSM image at 40X and scale bars: 10 mm) (b) nuclear morphology (epi-fluorescence microscopic image at 20X and scale bars: 10 mm) and (c) apoptosis(cell cycle analysis by FACS caliber) after 48 h of treatment. Percentage of apoptotic cells are indicated as the proportion of cells that contained sub-G1 phase. Values shown hererepresent � SD (n ¼ 3).

P. Venkatesan et al. / Biomaterials 32 (2011) 3794e38063802

3.9. Cellular uptake of nanoparticles

Cellular uptake of polymeric nanoparticles is mainly dependenton properties such as particle size, surface charge, shape, molecularweight and composition [50]. Nanoparticles of small size, uniformsize distribution and cationic surface charge have significantlyincreased cellular uptake and tissue localization [45]. In addition,the cellular uptake and in vitro cytotoxicity of cationic (þve)charged silica and paclitaxel loaded chitosan nanoparticles wereattributed to the electrostatic interaction of these nanoparticleswith anionic (-ve) cells [20,36,45]. In our study, cellular uptakeefficiency of fluorescently labeled celecoxib-loaded Hap-Chtnanoparticles by HCT 15 cells at different time periods was visu-alized using epi-fluorescence microscopy (Fig. 6a). After 3 h ofincubationwith nanoparticles, the HCT 15 cells showedweak greenfluorescence in their cytoplasm. Interestingly, a slight increase influorescence was observed as incubation times were increased. Inparticular, 24 h of incubation resulted in a more intensive dottedpattern of green fluorescent particles inside the cell.

Cellular uptake of FITC tagged celecoxib-loaded Hap-Cht nano-particles was also studied in HT 29 cell using CLSM. Incubation ofHT 29 cells with nanoparticles for 30 min resulted in intense andweak fluorescent signal along the cell membrane and cytoplasm,respectively, which signifies strong binding of Hap-Cht nano-particles at the cellular surface (Fig. 6b). Interestingly, more intensefluorescence with homogeneous dispersion in the cytoplasm andaround the nucleus was observed after 24 h of incubation. This

observation substantiates localization of nanoparticles in HT 29cells. This could be a consequence of non-specific interactions ofcationic Hap-Cht nanoparticles with the negatively charged cellsurface. Additionally, the uptake might also be mediated by non-specific absorptive endocytosis mechanisms, which results inrelease of celecoxib from the nanoparticles. However, entry ofcelecoxib-loaded Hap-Cht nanoparticles into the nucleus was notreadily apparent after extended incubation times, which may bea consequence of the small nuclear membrane pores preventingentry of the nanoparticles.

To further validate intercellular uptake of nanoparticles by HCT15 and HT 29 cells, flow cytometry analysis was performed [23,51].Mean fluorescence intensity (MFI) of HCT 15 cells was increasedwith incubation time. Maximal MFI was achieved after 24 h ofincubation, which indicates more localization of nanoparticlesinside the cell that is similar to the observation made by fluores-cence microscopy (Fig. 6c). MFI of HT 29 cells is also in agreementwith observation made using CLSM (Fig. 6d). Thus, the surfacecharge of nanoparticles is a vital part of nanoparticle-mediateddrug delivery [45].

3.10. Changes in surface potential of cancer cells

Previous studies have reported that cytotoxicity and intracel-lular uptake of nanoparticles are mainly dependent on surfacepotential of the nanoparticles [20,45]. Zeta potential is defined asthe difference of electrical potential between surface of the cell and

Fig. 6. (a) Localization of FITC-tagged celecoxib-loaded Hap-Cht nanoparticles by HCT 15 cells at different times of incubation using epi-fluorescence microscopy (scale bars: 20 mmat 20X). The left image was obtained from propidium iodide (PI) channel (red), which shows the nuclei; the right one was from FITC channel (green) and PI channel (red), whichshows localization of nanoparticles (white arrows) in the cytoplasm and nucleus. (b) Localization of nanoparticles in HT 29 cells using CLSM (scale bars: 10 mm at 40X). The leftimage was obtained from FITC channel (green), which shows nanoparticles localization in cytoplasm and nucleus; the middle one was from propidium iodide (PI) channel (red),which shows the nuclei; and the right one from combination of both PI channel (red) and FITC channel (green). The white arrow indicates the localization of nanoparticles. Meanfluorescence intensity (MFI) of FITC tagged celecoxib-loaded Hap-Cht nanoparticles localized in HCT 15 (c) and HT 29 (d) cells at varying time period as measured by flow cytometry.Mean and � SD are shown (n ¼ 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

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bulk-surrounding medium. Changes in zeta potential of celecoxib-loaded Hap-Cht nanoparticle-treated HCT 15 cells were determinedusing Zetasizer. The results indicated that zeta potential of HCT 15cells increased from �9.86 mV (Control HCT 15 cell) to 1.4 mV(nanoparticles treated cell). This increase in zeta potential of HCT 15cells correlates with uptake of the nanoparticles, which neutralizesthe negative surface charge of the cells.

3.11. Inhibition of cell proliferation (Ki-67) and tumor growth, andinduction of apoptosis (TUNEL and DAPI) in a human colon mousexenograft model

On the basis of promising in vitro cytotoxicity and enhancedcellular uptake of celecoxib-loaded Hap-Cht nanoparticles on HCT15 cells, we evaluated tumor inhibition by celecoxib-loaded Hap-Cht nanoparticles in a nude mouse human colon tumor xenograftmodel Fig. 7a. The maximum tolerated dose of celecoxib in rodentshas been reported to be 1 mg/kg of body weight [28]. The dosage ofthe celecoxib-loaded formulation used in this study was 100 mg/kgFig. 7b shows decreases in the tumor volume with Hap-Cht < freecelecoxib < celecoxib-loaded Hap-Cht nanoparticles when

compared with untreated controls. An anti-tumor effect of thechitosan carrier has been observed in A2780 cells [20]. Celecoxib-loaded Hap-Cht nanoparticles displayed greater tumor growthinhibition and less toxicity than free celecoxib following injectionthrough the tail vein at an equivalent dose of 100 mg/kg of celecoxib.These observations further corroborate the results of our in vitrocytotoxicity studies. It is also speculated that celecoxib-loaded Hap-Cht nanoparticles might circulate in blood vessels for prolongedperiods, increase the availability at tumor target sites through leakyblood vessels (EPR effect) and maintain effective therapeuticconcentrations for a longer period of time. In addition, the death ofcancer cells might lower transport resistance around blood vesselsthereby facilitating the movement of nanoparticles deeper into thetumor tissue and its microenvironment.

The cellular uptake study showed stronger affinity of nano-particles towards HCT 15 cells. Photographs of representativeexcised tumors from control and treated groups are shown inFig. 7c. In all of the treatment groups, including control mice, nosignificant change in body weight was observed during the courseof the experiment. During the observation period, all the groups(control and treated) displayed no signs of visible toxicity, death, or

Fig. 7. Mice human colon tumor xenografts were established by injecting HCT 15 cells subcutaneously into the right flank of a nude mouse. Tumor inhibitory effect of tail veininjections of free celecoxib, Hap-cht and celecoxib-loaded Hap-Cht nanoparticles on nude mice (a) bearing xenografted HCT 15 carcinoma cells, tumor volume (b) and the image ofexcised tumors (c) at the time of sacrifice (30 days post treatment). The results are provided as means � SD (n ¼ 3). ***P < 0.001; **P < 0.05, when compared with the cancercontrol group. Tumors from different treatment groups underwent immunohistochemical analysis for expression of Ki-67 (cell proliferation) and apoptosis (TUNEL and DAPI).Compared with tumors from the control mice, treatment with celecoxib decreased the number of Ki-67 positive cells (c) and, increased the number of TUNEL-positive cells (d) andnuclear fragmentation (d). These changes were even more prominent when animals were treated with celecoxib-loaded Hap-Cht nanoparticles. All images were taken under theidentical instrumental conditions and presented at the same intensity scale.

P. Venkatesan et al. / Biomaterials 32 (2011) 3794e38063804

obvious necrosis at the injection sites. Hence, this injectablenanocomposite system provides a promising delivery vehicle forcelecoxib to increase its efficacy and therapeutic effect in coloncancer treatment.

We also evaluated the effect of celecoxib-loaded nanoparticleson cell proliferation and apoptosis by Ki-67 (proliferation) and,TUNEL and DAPI staining (apoptosis), respectively. Tumors wereharvested, formalin-fixed, paraffin-embedded, sectioned and sub-jected to immunohistochemistry. Hap-cht, free celecoxib and cel-ecoxib-loaded Hap-Cht nanoparticle-treated mice xenografttumors showed decreased expression of Ki-67 and increasedTUNEL staining compared to the untreated controls (Fig. 7d and e).These results are in agreement with earlier reports of inhibition oftumor growth and Ki-67 expression, and apoptosis of prostatecancer cells by celecoxib [38]. Control tumors contain hypercellularareas with a high expression of Ki-67. Hap-Cht nanoparticlestreatment showed minimal reduction in cell proliferation, butcelecoxib-loaded Hap-Cht nanoparticles treatment significantlyreduced cellular proliferation. A progressive increase in greenfluorescent apoptotic cells (TUNEL þ ve tumor cells) were detectedin the celecoxib-loaded Hap-Cht nanoparticle-treated group when

compared to controls. This can be attributed to a combination effectof both Hap-Cht carrier and celecoxib on inhibition of tumor cellproliferation with concomitant induction of tumor cell apoptosis.Further, nuclei with homogenous fluorescence and evidence ofsegmentation or fragmentation was observed in both celecoxiband celecoxib-loaded Hap-Cht nanoparticle-treated group whencompared to controls.

4. Conclusions

In this study, a celecoxib-loaded Hap-Cht nanocomposite wasprepared by a coprecipitation method. The nanoparticles werenanosized and monodispersed with less agglomerative particles.Moreover, the nanoparticles showed high encapsulation efficie-ncy, sustained release patterns, desirable hemocompatibility andenhanced cytotoxicity on HCT 15 colon cancer cells. A time-dependent cytoplasmic uptake of Hap-Cht nanoparticles by HCT 15and HT 29 cells was observed. In vivo human colon tumor xenograftnude mouse tumor studies proved that the celecoxib-loaded Hap-Cht nanoparticles were more potent in inhibiting tumor growththan free celecoxib and this approach did not elicit any serious side

P. Venkatesan et al. / Biomaterials 32 (2011) 3794e3806 3805

effects. Based on these results, it is concluded that the Hap-Chtnanocomposite can be an effective and safe vehicle for celecoxibdelivery in colon cancer chemotherapy. This preliminary approachof nanoparticle-mediated targeted delivery might overcome sideeffects caused by administering unmodified celecoxib. The presentwork is encouraging and supports performance of detailed pre-clinical studies with celecoxib-loaded Hap-Cht nanoparticles asa prerequisite for potentially advancing into human (clinical)applications.

Acknowledgements

We are grateful to Aarthi Drug Ltd., for generously providingcelecoxib. We wish to thank Mr. Debashis Gayen for skilled tech-nical assistance in confocal laser fluorescence microscopy. Thiswork was supported by funds from the School of Medical Scienceand Technology, Indian Institute of Technology, Kharagpur, India.Further, the Department of Science and Technology and Depart-ment of Biotechnology, New Delhi supported our work financially(to SCK and MM). SCK wishes to acknowledge to Indo-US science &Technology Forum, New Delhi for his visit to USA. Dr. DevanandSarkar is a Harrison Scholar and Dr. Paul B. Fisher holds the ThelmaNewmeyer Corman Chair in Cancer Research in the VCU MasseyCancer Center, Virginia Commonwealth University, School ofMedicine. SCK and MM are also grateful to professor Paul Fisher,VCU Institute of Molecular Genetics for his kind hospitality duringtheir short stay.

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