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Colloids and Surfaces B: Biointerfaces 95 (2012) 195– 200

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

he anticancer activity of chloroquine-gold nanoparticles against MCF-7 breastancer cells

rachi Joshia,b,1, Soumyananda Chakraborti c,1, Jaime E. Ramirez-Vickd, Z.A. Ansarib, Virendra Shankera,inak Chakrabarti c,∗, Surinder P. Singha,d,∗∗

National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi 110012, IndiaCenter for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, IndiaDepartment of Biochemistry, Bose Institute, P-1/12 CIT Scheme VIIM, Kolkata 700054, IndiaDepartment of Engineering Science and Materials, University of Puerto Rico, Mayaguez, PR 00680, USA

r t i c l e i n f o

rticle history:eceived 9 July 2011eceived in revised form 24 February 2012ccepted 26 February 2012vailable online 6 March 2012

eywords:

a b s t r a c t

In the present study, 11-mercaptoundecanoic acid-modified gold nanoparticles (∼7 nm) were conjugatedwith chloroquine to explore their potential application in cancer therapeutics. The anticancer activity ofchloroquine-gold nanoparticle conjugates (GNP-Chl) was demonstrated in MCF-7 breast cancer cells. TheMCF-7 cells were treated with different concentrations of GNP-Chl conjugates, and the cell viability wasassayed using trypan blue, resulting in an IC50 value of 30 ± 5 �g/mL. Flow cytometry analysis revealedthat the major pathway of cell death was necrosis, which was mediated by autophagy. The drug release

anomedicineold nanoparticleshloroquineCF-7 cells

ell viabilitylow cytometry

kinetics of GNP-Chl conjugates revealed the release of chloroquine at an acidic pH, which was quanti-tatively estimated using optical absorbance spectroscopy. The nature of stimuli-responsive drug releaseand the inhibition of cancer cell growth by GNP-Chl conjugates could pave the way for the design ofcombinatorial therapeutic agents, particularly nanomedicine, for the treatment of cancer.

© 2012 Elsevier B.V. All rights reserved.

ecrosis

. Introduction

Nanotechnology has induced a paradigm shift in biomedical sci-nces, especially in the field of cancer therapy and diagnostics.here have been advances in the development of efficient nano-rug delivery systems with reduced side effects compared withhe use of conventional drugs for cancer treatment [1]. For effec-ive dosing at or near the tumor site, the drug must escape theirculation through the tumor microvasculature, which requireshe delivery system to have dimensions smaller than 100 nm [2].anoparticles offer the ability to engineer the desired particle

ize, and their high surface-to-volume ratio makes them attrac-ive for drug delivery by providing high drug-loading capacity.

he required properties of nanoparticles for therapeutics includenertness, a lack of toxicity, monodispersity, and simplicity in theunctionalization with the desired organic or metallorganic ligands.

∗ Corresponding author.∗∗ Corresponding author at: National Physical Laboratory, Dr. K. S. Krishnan Marg,ew Delhi 110012, India.

E-mail addresses: pinak@boseinst.ernet.in (P. Chakrabarti),urinder.singh@upr.edu, singhsp@mail.nplindia.org (S.P. Singh).

1 These authors contributed equally.

927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2012.02.039

A variety of nanoparticles, such as quantum dots, dendrimers,gold nanoparticles, polymer gels, Fe3O4, and ZnO, have been usedin cancer treatment and imaging [3–10]. Due to their non-toxic andnon-immunogenic properties, gold nanoparticles (GNPs) appear tohave a high potential as drug delivery scaffolds [11]. GNPs exhibitattractive physico-chemical properties, which have been exploitedfor various applications, such as imaging, diagnosing and treat-ing diseases [12–17]. The plasmon properties of gold have beenexploited for radiation therapy to cure cancer, and gold provides anadditional advantage as a tool for contrast imaging [13]. Further-more, the ease of synthesis, the precise control of size and shapeand a wide range of surface chemistry provide multifunctionalityto GNPs, facilitating the attachment of a variety of drugs and targetmolecules to the surface [11]. Several attempts have been made tograft different delivery schemes onto the surface of gold nanopar-ticles to improve drug solubility, regulated release and enhancedlocalized delivery [18,19].

Stimuli-responsive drug carriers based on nanoparticles haveemerged as a new generation of anticancer delivery systems, whichhave proven more effective than the conventional alternatives

[20,21]. A lower pH at or near the tumor site is one of those stimuli,and with nanoparticles as the delivery vehicle, the quick delivery ofthe drug to cancer cells is achieved through lysosomal pH-triggereddrug release [22]. Certain malignancies also cause minor decreases

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n extracellular pH [23]. Chloroquine (C18H26ClN3), a widely usedntimalarial drug, has recently attracted attention due to its anti-ancer effects, as demonstrated in the mouse colon, human lungnd breast cancer cells [24–27]. There is evidence to indicatehat chloroquine (Chl) has excellent potential as a cancer-specifichemosensitizer for combinatorial therapy [28].

The imaging and drug delivery capabilities of GNPs [15,29]ogether with the anticancer effects of Chl [26] have an enormousotential in developing new theranostics platforms for cancer,hich is the main motivation behind the current effort to design aNP-Chl conjugate. In this work, GNP-Chl conjugates were synthe-ized through wet chemistry. The release of Chl from GNP and theH response of GNP-Chl nanoparticles were studied using opticalbsorption spectroscopy at varying pHs at different time intervals,nd the anticancer activity was demonstrated in the MCF-7 breastancer cell line. The viability of MCF-7 cells was assayed using try-an blue, and the cell death mechanism was analyzed with flowytometry.

. Experimental details

.1. Preparation of chloroquine-gold nanoparticles (GNP-Chl)

The gold nanoparticles were prepared through reducing goldIII) ions with sodium borohydride using 11-mercaptoundecanoiccid as the in situ stabilizing agent. Chl was then conjugated to thi-lated gold nanoparticles via EDC/NHS chemistry. The details of thereparation and characterization of GNP-Chl have been describedreviously [30].

.2. Estimation of the loading on and release of chloroquine fromNP-Chl

The amount of Chl bound to GNP and released from GNP-Chlonjugates was estimated using UV–vis absorption spectroscopy.he optical density (OD) spectra were recorded on an Ocean-OpticsR4000 spectrometer equipped with a Toshiba TCD1304AP linearCD-array using a quartz cuvette with a 10-mm path length. TheD of the samples was selectively measured at 343 nm, which is

he characteristic absorption wavelength of Chl. The percentage (%)oading of Chl was estimated using Eq. (1):

loading = A − B

A× 100 (1)

here A is the OD of the total Chl added to the GNP solution, and B ishe OD of unbound Chl in the supernatant of the GNP-Chl solutionfter centrifugation.

To measure the pH response of prepared GNP-Chl conjugates,he conjugates were dispersed in buffers of varying pH, and theirD was measured. At the indicated times, the buffer containingNP-Chl was subjected to ultracentrifugation, and the OD of theupernatant was measured. The percentage (%) release of Chl fromNP-Chl conjugates was estimated with Eq. (2):

release = D

C× 100 (2)

here C is the OD of buffer-dispersed GNP-Chl, and D is the OD ofeleased Chl present in the supernatant after centrifugation.

.3. MCF-7 cell culture

MCF-7 cells were cultured and maintained in DMEM medium

HiMedia, Mumbai, India) supplemented with 10% fetal bovineerum (FBS), insulin (0.1 U/mL), l-glutamine (2 mM), sodium pyru-ate (100 �g/mL), non-essential amino acids (100 �M), antibiotics100 �g/mL streptomycin and 50 U/mL penicillin) and tetracycline

iointerfaces 95 (2012) 195– 200

(1 �g/mL). The medium was adjusted to pH 7.0–7.2, and all of theflasks were incubated at 37 ◦C in a humidified atmosphere contain-ing 5% CO2. Approximately 106 cells per mL were inoculated in 1 mLPetri dishes until 85% cell confluence was achieved.

2.4. Cell viability assay

A total of 1 mL of cells in log phase was seeded in Petri dishes. Dif-ferent concentrations of GNP-Chl were added, and the plates wereincubated for 24 h. Control experiments were performed under thesame conditions but without the addition of GNP-Chl. The cell via-bility was assayed by measuring the penetration of the dye trypanblue. The inhibition concentration IC50, which is the drug concen-tration that inhibits 50% of cell growth (viability), was determinedfrom the graph of the number of visible cells against drug concen-tration. The morphology of MCF-7 cells was studied in the presenceand absence of GNP-Chl conjugates using a normal optical micro-scope at 100×.

2.5. Flow cytometry

The mechanism of cell death was determined with the flowcytometry of cells that were double stained with fluorescein isoth-iocyanate (FITC)-conjugated annexin-V and propidium iodide (PI)using an apoptosis kit (Invitrogen). Flow cytometry was performedusing a FACS Calibur (FACS Calibur; Becton Dickinson) equippedwith Cell Quest Pro software (Becton Dickinson). The data of threeidentical experiments were averaged.

3. Results and discussion

The preparation of GNP-Chl conjugates, as reported previously[30], is illustrated in Fig. 1a. The prepared thiolated gold nanopar-ticles were treated with EDC/NHS for COOH activation and thenconjugated with Chl. The binding of Chl to the gold nanoparticleswas examined with UV–vis absorption measurements (Fig. 1b). Thesimultaneous occurrence of characteristic peaks of Chl at 343 nmand gold nanoparticles at 522 nm for an aqueous GNP-Chl disper-sion indicated the binding of Chl to GNP. The synthesized goldnanoparticles were 7 ± 1.5 nm (Supporting information, Fig. S1).

3.1. Chloroquine binding and pH response of GNP-Chlnanoparticles

The binding and release of Chl (to GNP and from GNP-Chl,respectively) were assayed with optical absorbance measurementsusing Eqs. (1) and (2). The maximum binding (79.8%) occurred atpH 7.4, and a similar level of binding (78.7%) occurred at pH 7 (datanot shown). The pH response of GNP-Chl was measured in termsof the percentage of Chl released at varying pHs and time intervals(Fig. 2a). Chl release was calculated after 2, 10, 24 and 48 h in the pHrange of 5–7. Approximately 18% drug release was observed at pH6 after 2 h, which increased with time and reached 62% after 48 h.At a lower pH, approximately 26% of the drug was released after2 h, which increased to 81% after 48 h, indicating a sustained drugrelease over time. At normal pH 7, the GNP-Chl system was rela-tively stable, with 13% drug release in 2 h and 21% release in 48 h.These levels may be the result of the release of non-specificallybound drug from the surface of the nanoparticles.

The pH response of a thiol-capped GNP solution was alsomeasured for reference, and the representative surface plasmonresonance (SPR) absorbance spectra of GNP-thiol at basic, normal

and acidic pH are shown in Fig. 2b. When GNP-thiol was treatedwith acidic pH, the SPR absorption peak of the gold nanoparticleswas greatly suppressed, and the particles were aggregated (inset).At basic pH (>10), the GNP solution was an intense wine-red color

P. Joshi et al. / Colloids and Surfaces B: Biointerfaces 95 (2012) 195– 200 197

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ig. 1. The formation of chloroquine-conjugated gold nanoparticles (GNP-Chl). (a)

n comparison with unconjugated gold nanoparticles (GNPs) showing the binding o

nd exhibited a sharp SPR peak, suggesting the removal of thioloieties from the surface of gold nanoparticles.

.2. Cell viability using the trypan blue assay

The morphological changes in MCF-7 cells after treatment withNP-Chl for 24 h were compared with the untreated control with

normal optical microscope (Fig. 3a and b). The results werendicative of cell death (rounding and clumping). The trypan bluexclusion assay is a rapid and sensitive method to screen thefficacy of anticancer agents [31–35]. The effect of the GNP-Chloncentration on MCF-7 cell viability is shown in Fig. 3c. Theesults indicate that the unmodified GNP had no effect on celliability compared with GNP-Chl and Chl alone, suggesting annhanced cytotoxicity of GNP-Chl against MCF-7 cells. An IC50 valuef 30 ± 5 �g/mL was determined for GNP-Chl.

Because Chl is released at a lower pH (Fig. 2a), GNP-Chl wouldave to be trafficked to late endosomes or lysosomes, which have aH of 5–6. However, the Chl release from GNP-Chl conjugates in the

ysosome and/or endosome primarily depends on the endocyticathways. Extracellular materials are internalized within cellshrough a variety of endocytic pathways, including phagocytosis,

icropinocytosis, and clathrin- and caveolae-mediated endocyto-is [36]. Wang et al. reported the cellular uptake of doxorubicin-ound GNP in MCF-7 cells via clathrin- and caveolae-mediatedndocytosis and subsequent acid-responsive drug release [37].

atic illustration of GNP-Chl formation. (b) UV–vis absorption spectrum of GNP-Chlroquine to the gold nanoparticles.

Furthermore, pH-responsive doxorubicin-loaded nanoparticleshave been shown to be internalized through caveolae-mediatedendocytosis trafficked to late endosomes/lysosomes [38]. Thus,it is plausible to infer that GNP-Chl conjugates are internalizedthrough clathrin- and/or caveolae-mediated endocytosis, eventu-ally reaching acidic organelles, such as late endosomes/lysosomes,in which Chl can be released to induce cell death. No noticeablecytotoxicity was observed in red blood cells treated with varyingconcentrations of GNP-Chl conjugates (data not shown).

3.3. Flow cytometry

To determine the mechanism of cytotoxicity in MCF-7 cells, theGNP-Chl-treated cells were analyzed using annexin-V flow cytom-etry. Fig. 3d shows that within 24 h of exposure to GNP-Chl, theMCF-7 cell death rate was 31.5%, with 29.3% necrotic and 2.2% lateapoptotic cells, indicating that apoptosis is not the major causeof cell death. It should be noted that the PI signal in this assay isassociated with necrosis, which is an indicator of cell integrity andnonviable cells. Thus, the flow cytometry results of MCF-7 cells inthe presence of GNP-Chl are only indicative of nonviable cells that

have lost plasma membrane integrity. This result is also corrob-orated with the morphological study of GNP-Chl-treated MCF-7cells in Fig. 3b, which shows cell clumping and debris related tononviable cells.

198 P. Joshi et al. / Colloids and Surfaces B: Biointerfaces 95 (2012) 195– 200

Fig. 2. The pH response of GNP-Chl and GNP-thiol. (a) The percentage (%) of drug release from GNP-Chl at varying pHs as a function of increasing time (2–48 h); the valuesare the average of three separate experiments and are expressed as the mean ± standard deviation. (b) The UV–vis absorption of thiol-capped gold nanoparticles at varyingp tures

atCcoda

Hs: basic (pH = 11), normal (pH = 7.4) and acidic (pH = 2.5). The inset shows the pic

Chloroquine is a lysosomotropic agent that neutralizes thecidic pH of lysosomes, preventing autophagic protein degrada-ion and causing autophagosome accumulation [39–41]. Althoughhl has been reported to induce apoptosis in MCF-7 breast

ancer cells through DNA intercalation [42], the results ofur study do not corroborate these findings. Yang et al.emonstrated that Chl killed pancreatic carcinoma cells viautophagy inhibition [43]. Furthermore, it has been shown

of the same samples under ambient light.

that Chl causes autophagic vacuole accumulation and inducesp53-independent death in gliomas, which does not involvecaspase-mediated apoptosis [44]. The necrotic cells observedin the present study could thus be attributed to cells with

extensive autophagosomal accumulation induced by GNP-Chl con-jugates. More detailed study is required to provide any conclusivestatement regarding the GNP-Chl-induced cell death mecha-nism.

P. Joshi et al. / Colloids and Surfaces B: Biointerfaces 95 (2012) 195– 200 199

Fig. 3. The anticancer activity of GNP-Chl against MCF-7 breast cancer cells. (a) MCF-7 controls (without GNP-Chl); (b) MCF-7 cells treated with GNP-Chl (0.1 mg/mL for24 h); (c) concentration-dependent MCF-7 cell killing by GNP-Chl represented as the percentage of dead cells. The values are the average of three separate experiments anda l; 2, tr

4

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re expressed as the mean ± standard deviation. (d) Flow cytometry data (1, contro

. Conclusions

Chloroquine-conjugated gold (GNP-Chl) nanoparticles wereynthesized as a nano-therapeutic drug agent for cancer. Theesults revealed interesting anticancer properties of the GNP-

hl system in vitro. The major results are summarized: (i)he release of chloroquine from GNP-Chl at a lower pH sug-ests lysosomal/endosomal uptake of chloroquine, (ii) GNP-Chlxhibited concentration-dependent cytotoxicity in MCF-7 breast

eated) showing necrosis as the major mechanism of MCF-7 cell death.

cancer cells, and (iii) autophagy is likely the cause of GNP-Chl-induced necrotic cell death. The detailed cell uptake andsignaling pathways are under investigation to elaborate theactual mechanism of cell death. Gold and chloroquine togethercould lead to the design of combinatorial therapy, i.e., drug and

radiation together, for cancer therapeutics. Overall, the under-standing of the nature and selectivity of nanoparticles can haveimportant implications in designing nano-conjugated anticancerdrugs.

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cknowledgments

We thank Dr. T. Das for helpful discussion. PC is supported by JC Bose National Fellowship. PJ and SC are thankful to the Coun-il of Scientific and Industrial Research, India, for fellowships. SPScknowledges the IFN startup grant OIA-0701525.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.colsurfb.2012.02.039.

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