Journal of Colloid and Interface Science 355 (2011) 402–409

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Journal of Colloid and Interface Science

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Binding of chloroquine–conjugated gold nanoparticles with bovine serum albumin

Prachi Joshi a,b,1, Soumyananda Chakraborty c,1, Sucharita Dey d, Virendra Shanker a, Z.A. Ansari b,Surinder P. Singh a,e,⇑, Pinak Chakrabarti c,⇑a National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi 110 012, Indiab Centre for Interdisciplinary Research in Basic Sciences, JMI, New Delhi 110 025, Indiac Department of Biochemistry, Bose Institute, P-1/12 CIT Scheme VIIM, Kolkata 700 054, Indiad Bioinformatics Center, Bose Institute, P-1/12 CIT Scheme VIIM, Kolkata 700 054, Indiae Department of Engineering Science and Materials, University of Puerto-Rico, Mayaguez Campus, PR 00680, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 29 October 2010Accepted 7 December 2010Available online 15 December 2010

Keywords:Bovine serum albuminGold nanoparticle–chloroquine conjugationProtein–nanoparticle interactionTrp fluorescenceBinding constant measurementDrug docking

0021-9797/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.jcis.2010.12.032

⇑ Corresponding authors. Address: National PhysicaMarg, New Delhi 110 012, India (S.P. Singh).

E-mail addresses: surinder.singh@upr.edu (S.P. Sin(P. Chakrabarti).

1 Contributed equally.

We have conjugated chloroquine, an anti-malarial, antiviral and anti-tumor drug, with thiol-functionalizedgold nanoparticles and studied their binding interaction with bovine serum albumin (BSA) protein. Goldnanoparticles have been synthesized using sodium borohydride as reducing agent and 11-mercaptoun-decanoic acid as thiol functionalizing ligand in aqueous medium. The formation of gold nanoparticleswas confirmed from the characteristic surface plasmon absorption band at 522 nm and transmission elec-tron microscopy revealed the average particle size to be �7 nm. Chloroquine was conjugated to thiolatedgold nanoparticles by using EDC/NHS chemistry and the binding was analyzed using optical density mea-surement and Fourier transform infrared spectroscopy. The chloroquine–conjugated gold nanoparticles(GNP–Chl) were found to interact efficiently with BSA. Thermodynamic parameters suggest that the bindingis driven by both enthalpy and entropy, accompanied with only a minor alteration in protein’s structure.Competitive drug binding assay revealed that the GNP–Chl bind at warfarin binding site I in subdomainIIA of BSA and was further supported by Trp212 fluorescence quenching measurements. Unraveling thenature of interactions of GNP–Chl with BSA would pave the way for the design of nanotherapeutic agentswith improved functionality, enriching the field of nanomedicine.

� 2010 Elsevier Inc. All rights reserved.

1. Introduction

Designing of hybrid nanocarrier systems capable of carryingDNA, proteins, drugs and other biomolecules for various biomedi-cal applications has seen plethora of research efforts in nanotech-nology [1,2]. The high surface to volume ratio of nanoparticlesmakes them attractive for drug delivery as they provide high load-ing capacity for drug. Various nanoparticles have shown potentialin biosensing, bioimaging and targeted drug delivery includingdiagnostics and therapeutics [3–5]. As a promising drug deliverysystem, nanoparticles have been systemically used in oral, pul-monary and other administration routes to study drug targeting,bioavailability, activity and stability [5,6].

Gold nanoparticles (GNP) add onto the group of delivery sys-tems that involves attachment of drugs or ligands to carriers[5,7,8]. The interest for gold lies in its inert and nontoxic nature,

ll rights reserved.

l Laboratory, Dr. K.S. Krishnan

gh), pinak@boseinst.ernet.in

controllable size and ease of functionalization with desired ligands[9]. Due to their size-dependent physico-chemical properties andthe release mechanisms imparted by their functionalized ligands,conjugation of GNP to therapeutic drugs provides attractive vehi-cles for drug delivery applications [10,11]. Another attractive fea-ture of GNP is their affinity for thiols, providing an effective andselective means of controlled surface chemistry and intracellularrelease [7]. As drug delivery vehicles, GNP have been developedto improve bioavailability, efficiency and specificity of pharmaceu-tical drugs, in particular anti-cancer agents [8,12].

Chloroquine (C18H26ClN3) is an inexpensive and well known anti-malarial drug. It has immunomodulatory properties and is used inthe treatment of autoimmune disorders. Chloroquine exhibits theinhibition of glycosylation of viral envelops glycoprotein, and hasbeen proposed as an effective HIV-1 therapeutic agent. It has alsobeen found effective in acute chikungunya infections, severe respira-tory syndrome and several other viral infections including influenza[13–16]. In recent years, metal (Ru, Rh, Au, Zn) complexed chloro-quine has been used against drug-resistant malaria strains and foranti-inflammatory effects [17,18]. Additionally, chloroquine hasbeen shown to display anticancer activity or preventive effect andcan cause enhancement of the potency of other known drugs

P. Joshi et al. / Journal of Colloid and Interface Science 355 (2011) 402–409 403

[19–21]. These novel properties of GNP and chloroquine led us to de-sign chloroquine–conjugated gold nanoparticles (GNP–Chl) for po-tential cancer therapeutic applications.

The pharmacological behavior of any therapeutic drug moleculeplays a critical role in deciding its fate in blood stream. The biodis-tribution, availability and metabolism of therapeutic agentsstrongly depend on their interaction with proteins present in blood[22,23]. Albumins are the most studied serum proteins for theassessment of drug–protein interactions because they transportreversibly-bound drugs to their destination via drug–protein com-plex formation. They are abundant in the circulatory system ofvariety of organisms and have important physiological functionsincluding control of serum osmotic pressure, pH buffering etc.[24]. Serum albumins bind to various exogenous and endogenousligands, such as metabolites, fatty acids, bilirubin, hormones, anes-thetics and commonly used therapeutics agents for their transpor-tation to their appropriate cellular targets. Therefore, the bindinginteraction of GNP–Chl with serum albumins needs to be under-stood for the therapeutic applications of these bioconjugatednanomaterials. The effect on protein structure and stability uponthe binding of nanoparticles would significantly govern their bio-distribution and pharmacokinetics that finally determine their effi-ciency in vivo. With a sequence identity of 76% to that of humanserum albumin, the well characterized bovine serum albumin(BSA) provides a convenient model for serum albumins for under-taking such studies involving protein–nanoparticle interactions[25].

The gold–chloroquine conjugation has been verified usingUV–Vis, Fourier transform infrared spectroscopy (FTIR), and trans-mission electron microscopy techniques. Isothermal titration calo-rimetry, fluorescence and circular dichroism spectropolarimetrymeasurements were performed to understand the interaction ofGNP–Chl with BSA quantitatively and to evaluate contributions ofdifferent forces responsible for the interaction. The specific deliveryof drugs and other ligands by BSA is attributed to the presence of twoprimary drug binding sites, I and II located in its subdomains IIA andIIIA [26]. Since, warfarin and ibuprofen are the known drugs forselectively targeting the binding sites I and II respectively [27], wehave used these drugs to determine the site at which GNP–Chl bindsto BSA by using competitive drug binding assay. The binding site ofGNP–Chl with a tryptophan residue in the vicinity has also beenascertained by docking studies.

2. Materials and methods

2.1. Materials

Gold (III) chloride hydrate (99%), 11-mercaptoundecanoic acid(99%), sodium borohydride (P98%), 1-ethyl-3-[3-dimethylamino-propyl] carbodiimide hydrochloride (98%), N-hydroxysulfosuccini-mide (98%), chloroquine diphosphate salt (P98%) were purchasedfrom Sigma–Aldrich and used as they are. All solvents used were ofanalytical grade and double distilled water has been used as aque-ous medium for all experiments. Bovine serum albumin (BSA),warfarin, and ibuprofen were purchased from Sigma and usedwithout further purification. All other reagents used in theseexperiments were of analytical grade and purchased from Merck,India.

2.2. Preparation of thiol functionalized GNP

The synthesis of mercaptoundecanoic acid (MU)–functionalizedGNP was performed using the previously reported method [28]with minor modifications; methanol had been preferred as reac-tion medium due to the solubility constraints of MU. Sodium boro-

hydride was used as the reductant for gold ions with MU moleculesin their close proximity. Typically, 1 mM gold chloride solution wasprepared in 5 ml of methanol. An amount of 0.044 g of MU was dis-solved into methanol and then added to gold chloride solution. TheMU containing gold chloride solution was stirred for 30 min till thesolution became turbid milky. To this solution, freshly preparedice-cooled solution of 0.1 M sodium borohydride was added dropby drop with continuous stirring. The addition of borohydridewas stopped when a brownish color appeared and the solutionwas kept stirring for another 1–1.5 h. The MU–functionalizedGNP were retrieved by high speed centrifugation and washed withmethanol and copious amount of distilled water. These functional-ized nanoparticles were subsequently re-dispersed in double dis-tilled water and kept for further characterization. All glasswareand magnetic stirring bars were thoroughly cleaned and oven driedto avoid unwanted nucleation and aggregation of GNP duringsynthesis.

2.3. Preparation of GNP–Chl

The synthesized MU–functionalized GNP contacted with 0.01 Mof 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride(EDC) and 0.005 M of N-hydroxysulfosuccinimide (NHS) for theactivation of the ACOOH group. In 5 ml solution of ACOOH acti-vated gold nanoparticles, 2 ml of 100 lM chloroquine diphosphate(with a measured optical density, OD = A) was added and stirredfor 6–7 h for proper binding of chloroquine. The mixer was thencentrifuged and washed several times to remove unbound moie-ties; the supernatant along with the wash was taken for OD mea-surement (providing a value of OD = B). The OD spectra wererecorded on Ocean-Optics HR4000 spectrometer equipped with aToshiba TCD1304AP linear CCD-array detector that enables opticalresolution as precise as 0.02 nm (FWHM) using a quartz cuvette of10 mm path length. The % loading of chloroquine was estimated byputting the values of A and B in Eq. (1). The OD was selectivelymeasured at 343 nm, the characteristic OD of chloroquine:

% loading of chloroquine onto MU-capped GNPs

¼ ½ðA� BÞ � 100�=A ð1Þ

where A is the OD of total chloroquine added into GNP–Chl, B is theOD of chloroquine in supernatant of GNP–Chl, and both A and Bwere measured at 343 nm.

2.4. Binding of GNP–Chl with BSA

BSA protein solution (concentration 5 lM) was exhaustivelydialyzed using dialysis membrane (Spectra biotech membraneMWCO: 3500, Spectrum Lab, CA, USA) against buffer solution at4 �C. A buffer solution, consisting of 0.1 mM of sodium phosphateat pH 7.4, was used in all the experiments. To study the interactionbetween GNP–Chl and BSA, a fixed amount of the GNP–Chl wasadded to the protein solution, mixed by vortexing and incubatedat room temperature for 2 h. We also observed that longer incuba-tion time did not alter the spectroscopic results.

2.5. Electron microscopy

The particle size and dispersity of GNP–Chl were studied usingtransmission electron microscope (TEM). The bright field electronmicrographs of the samples had been recorded on JEM-2010 (de-vice: Orius SC1000 2) at the accelerating voltage of 200 kV. To takemeasurements, a small drop of aqueous sample solution wasplaced on carbon coated copper grid and dried completely in dustfree atmosphere.

404 P. Joshi et al. / Journal of Colloid and Interface Science 355 (2011) 402–409

2.6. FTIR spectroscopy

FTIR technique was used to analyze the conjugation of MU toGNP and chloroquine to MU–GNP. FTIR scanning was performedin transmission mode using Perkin–Elmer spectrometer equippedwith a DTGS KBr detector and a KBr beam splitter with constantnitrogen purging. IR grade KBr was used as scanning matrix. Oneto two milligram of fine sample powder and 90–100 mg of KBrpowder were mixed and dried completely, then transferred to13 mm die to make a nearly transparent and homogeneous pallet.All spectra were taken at 4 cm�1 resolution, averaged over 20 scansin the range 400–4000 cm�1.

2.7. Fluorescence spectroscopy

Fluorescence spectroscopy was used to determine the trypto-phan (Trp212) fluorescence quenching on binding of BSA toGNP–Chl nanoconjugates. All the fluorescence measurements werecarried out using a Hitachi F3000 spectrofluorimeter with 10 lMprotein. For fluorescence quenching measurements, GNP–Chl con-jugate was added to the protein from a 0.72 mM stock solution.The excitation wavelength was set as 295 nm to selectively excitetryptophan residues and the emission was monitored in the rangeof 310–400 nm with the fixed slit width of 5 nm. The fluorescenceintensities were determined at the kmax and after inner filter cor-rection the data analysis was done using the Stern–Volmer equa-tion [29]:

Fo=F ¼ 1þ KSV � ½GNP—Chl� ¼ 1þ Kqs0½GNP—Chl� ð2Þ

where Fo and F denote the steady-state fluorescence intensities inabsence and in presence of the quencher (GNP–Chl), respectively;KSV is the Stern–Volmer quenching constant (also representsStern–Volmer binding constant) and [GNP–Chl] is the concentrationof the quencher. Kq is the bimolecular quenching constant and s0 isthe lifetime of the fluorophore.

For the protein ligand association representing the staticquenching, the following equation was employed for the calcula-tion of the binding constant:

logðFo � FÞ=F ¼ log Kb þ n log½GNP—Chl� ð3Þ

where Kb is the binding constant and n is the number of bindingsites, respectively.

The displacement of the marker drug (warfarin or ibuprofen)was analyzed in a competitive binding study in which the 5 lMof protein was incubated with 5 lM of the respective drug for30 min at room temperature. Samples were selectively excited atthe excitation wavelengths of respective drugs. For example anexcitation wavelength of 310 nm was used to excite warfarin andthe corresponding emission was recorded in the range of 320–440 nm using constant 5 nm slits. Then different concentrationsof GNP–Chl were added to the mixture and the fluorescence spec-tra were measured. The experiments were carried out three timesand the average was taken.

2.8. Circular dichroism (CD) spectropolarimetry

The structural change in BSA induced by the addition of GNP–Chlnanoconjugate was studied using far-UV CD spectroscopy. The CDspectra were obtained using a JASCO-810 spectropolarimeterequipped with a thermostatically controlled cell holder. The temper-ature of the sample was controlled at 25 ± 0.1 �C. A 1 mm path lengthcuvette was used for measurements. For all the measurement a pro-tein concentration of 5 lM was used. The far-UV region was scannedbetween 200 and 260 nm with an average of three scans and a band-width of 5 nm. The final spectra were obtained by subtracting thebuffer contribution from the original protein spectra. This subtrac-

tion (which was also done with the FTIR and fluorescence data)removes the contribution due the scattering from nanoparticles, ifany. The CD results were expressed in terms of mean residual ellip-ticity (MRE) in deg cm2 dmol�1 defined as:

½h� ¼ 100 ½h�obs=lcp ð4Þ

where [h]obs is the observed ellipticity in degrees, cp is the proteinconcentration in mol per liter, and l is the path length of the cell(path of light) in cm. For near-UV CD measurements, 35 lM proteinconcentration was taken, and 5 mm path length cuvette was usedand the scanning was in the range of 260–320 nm.

2.9. Isothermal titration calorimetry (ITC)

Isothermal titration calorimetry measurement was performedon a VP-ITC calorimeter (Microcal Inc., Northampton, MA). BSAwas dialyzed extensively against 0.1 mM sodium phosphate buffer,and GNP–Chl was dissolved in the same dialysate. A typical titra-tion involved 20 injections of the GNP–Chl called titrant (10 llaliquot per injection from a 400 lM stock solution) at 5 min inter-vals into the sample cell (volume 1.4359 ml) containing BSA (con-centration, 35 lM). In order to ensure full occupancy of bindingsites in BSA, sequential titrations were carried out by adding ligandto the sample protein solution. The titration cell was stirred contin-uously at 310 rpm. The heat of the ligand dilution in the bufferalone was subtracted from the titration data for each experiment.The data were analyzed to determine the binding stoichiometry(N), affinity constant (Ka), and other thermodynamic parameterof the reaction using origin software [30]. Titration of protein withGNP–Chl nanoconjugates was carried out at 25 �C. The reportedvalues are the average of two parallel experiments.

2.10. Modeling of chloroquine bound BSA

A homology model of BSA was first constructed to which chol-oroquine was docked. A BLAST search with the BSA sequence[Swissprot ID ALBU_BOVIN (P02769)] revealed 75% identity withhuman serum albumin having the code 2BXI in the ProteinData-Bank (PDB) [31]. The SWISS-MODEL server (http://swissmod-el.expasy.org) was used to obtain the three-dimensional model ofBSA, which was validated using PROCHECK [32]. The atomic coor-dinates of choloroquine was obtained from DrugBank (http://www.drugbank.ca/). PATCHDOCK [33], an automated online serverwas used for docking. The best solution (based on docking score)was retained for further analysis. PyMol (http://www.pymol.org)was used for visualization and for the identification of residuesin the binding pocket (within a distance of 4.5 Å of the drug).

3. Results

3.1. MU–functionalized GNP and GNP–Chl nanoconjugates

3.1.1. Optical properties and particle sizeFig. 1 illustrates the schematic formation of MU–functionalized

GNP (or thiolated-GNP) and their conjugation with chloroquine.The formation of GNP has been confirmed by their representa-

tive surface plasmon resonance (SPR) band measured as OD inUV–Vis absorption spectroscopy. The SPR absorption spectra ofMU–functionalized GNP (GNP–MU) and chloroquine–conjugatedGNP (GNP–Chl) are plotted in comparison to bare GNP in Fig. 2.The SPR absorption band for GNP–MU appears at 522 nm, with asignificant broadening and slight decrease in intensity comparedto the plasmon band for bare GNP at 520 nm. The plasmon reso-nance is surface phenomenon and after attachment of any ligandat surface significantly affects the absorption intensity and full

Au3+ Au3+

Au3+Au3+

Au3+ AuAuNaBH4 EDC/NHS

ChloroquineThiol COOH NH

AuAu

Fig. 1. Schematic illustration representing the formation of 11-mercaptoundecanoic acid functionalized gold nanoparticles and their conjugation to chloroquine.

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Wavelength (nm)

GNP GNP-MU GNP-MU-Chl

Fig. 2. Optical absorption spectra of gold nanoparticles (GNP), 11-mercaptoun-decanoic acid functionalized gold nanoparticles (GNP–MU) and chloroquine–conjugated gold nanoparticles (GNP–MU–Chl). (Note that GNP–Chl in the text hasbeen expanded to GNP–MU–Chl here and in Fig. 4, for clarity.)

10nm

(a)

20nm

(b)

Fig. 3. Transmission electron micrographs of (a) MU–functionalized GNP, and (b)chloroquine–conjugated GNP (GNP–Chl).

P. Joshi et al. / Journal of Colloid and Interface Science 355 (2011) 402–409 405

width at half maximum (FWHM) of absorption band. The observedbroadening in absorption band is attributed to the attachment ofthiol (MU) at the surface of nanoparticles. After attachment ofchloroquine to the MU–functionalized GNP, the intensity of plas-mon absorption band further decreases further with additionalbroadening (Fig. 2).

The absorption spectrum of chloroquine–conjugated GNP revealstwo extra peaks at 329 nm and 343 nm corresponding to aromaticamine transitions in chloroquine, suggesting its binding to GNP–MU. The percentage loading of chloroquine to GNP–MU has been cal-culated using Eq. (1) and found to be 78.9% (or�79%) indicating effi-cient binding of drug to the nanoparticles. The values for A and Bwere obtained as 0.94 and 0.19, respectively, and substituted in Eq.(1).

Fig. 3a shows TEM micrographs of MU–functionalized GNPrevealing that the particles are small in size and uniformly distrib-uted. The particle size of the MU–functionalized GNP is estimatedto be �7 nm. Fig. 3b represents the TEM micrographs of chloro-quine–conjugated GNP (GNP–Chl) revealing the attachment ofchloroquine (and consequent enlargement of the particles) as bio-molecular layer at the surface, resulting in some blurring of themicrograph.

3.1.2. Binding in GNP–Chl nanoconjugatesThe presence of different moieties i.e. thiol MU and chloroquine

at the surface of GNP and their mode of binding have been studied

by FTIR spectroscopy. The FTIR spectra of MU–functionalized GNPand GNP–Chl nanoparticles are plotted in Fig. 4.

The MU–functionalized GNP reveals twin peaks at 1505 and1692 cm�1 that are assigned to the carbonyl CAO stretch of long-chain aliphatic carboxylic acid. The peak at 1335 cm�1 is due toCAOAH in-plane bending and the peaks in 2900–3050 cm�1 regionare corresponding to CAH stretch of methylene (CH2) group. The ab-sence of representative peak of SAH group (in the 2500–2600 cm�1

region) indicates the fact that MU is attached to gold via AuAS bond.In the FTIR spectrum of chloroquine–conjugated GNP, a strong peakat 1711 cm�1 due to the C@O stretch of tertiary amide I (ACONHA) is

500 1000 1500 2000 2500 3000 3500 400035

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3361

2500-2600

30042935

28512926

1234

1335

13961608

1505

1692

1711

% T

Wavenumber (cm-1)

GNP-MU-Chl GNP-MU

Fig. 4. FTIR spectra of MU–functionalized gold nanoparticles (GNP–MU) andchloroquine–conjugated gold nanoparticles (GNP–MU–Chl).

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330

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430

480

530

580

400370340310Wavelength (nm)

Fluo

rese

nce

inte

nsity

(A.U

.)

BSA

BSA + 2 µL GNP-Chl

BSA + 2.25 µL GNP-Chl

BSA + 2.5 µL GNP-Chl

BSA + 2.75 µL GNP-Chl

BSA + 3 µL GNP-Chl

BSA + 3.25 µL GNP-Chl

BSA + 3.5 µL GNP-Chl

BSA + 3.75 µL GNP-Chl

BSA + 4 µL GNP-Chl

Fig. 5. Quenching of tryptophan fluorescence in BSA in presence of varyingconcentrations of GNP–Chl.

406 P. Joshi et al. / Journal of Colloid and Interface Science 355 (2011) 402–409

observed. The peaks observed at 1234, 1396 and 1608 cm�1 areattributed to the overlapped bands of tertiary amide II (ACONHA)and heterocyclic dinuclear aromatic ring stretching vibrations. Theamide II peak generally appears in the region of 1525–1650 cm�1

and the skeleton bands of heterocyclic aromatic ring in the form ofstretching vibrations of C@C, C@N appear as doublet in the regionof 1200–1600 cm�1. The peaks at 2851 and 2926 cm�1 are attributedto CAH stretching of methylene groups. The weak broad peak around3361 cm�1 is assigned to tertiary amine group of chloroquine. Theobserved results clearly suggest that chloroquine is binding toMU–functionalized GNPs through the formation of an amide bondwith the carboxyl group of MU.

y = 1.0253x + 4.78R2 = 0.9867

-1.00

-0.80

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0.00

-5.80 -5.60 -5.40 -5.20 -5.00 -4.80 -4.60log[Q]

log[

(Fo-

F)/F

]

Fig. 6. Plot of log [(Fo � F)/F] against log[Q] derived from the quenching of BSA byGNP–Chl (Q in the plot). The equation of the fitted line is also shown.

3.2. Binding of GNP–Chl with BSA

3.2.1. Intrinsic tryptophan fluorescence quenchingThe intensity and wavelength of tryptophan fluorescence are

sensitive to the environment, and hence indicative of the proteinconformational alterations upon binding. BSA has two Trp residuesat 134 and 212 positions and two binding sites i.e. site I and site II[26]. Trp212 resides inside the hydrophobic patches of subdomainIIA, which also contains the drug binding site I. As such if the bind-ing occurs at this site resulting in a change in the Trp environment,the fluorescence from Trp212 will change. To allocate the proxim-ity of Trp residue in BSA to the binding site of GNP–Chl, we mon-itored the change in intrinsic Trp fluorescence spectra withincreasing GNP–Chl concentration (Fig. 5).

The tryptophan fluorescence of BSA was quenched on additionof GNP–Chl. Moreover, on increasing the concentration of GNP–Chl, the fluorescence intensity decreased gradually. Quenchingcan usually be induced either by a collisional (dynamic) processand/or formation of a complex between the quencher and the fluo-rophore (static quenching). The Stern–Volmer plot showed astraight line with increasing concentration of GNP–Chl nanoconju-gates (Fig. S1, supporting information), which indicates the exis-tence of a single type of quenching. The Stern–Volmer constant(KSV) was found to be 4.15 � 104 M�1. Using this value of KSV,and the standard value of 10�8 s for s0 in Eq. (2), one obtains a va-lue of 4.15 � 1012 M�1 s�1 for Kq (the quenching rate constant). Ingeneral, dynamic and static quenching can be distinguished bytheir different bimolecular quenching rate constants. This valuefor the dynamic process, which depends on the viscosity of themedium, is limited to a maximum of 1010 [34]. An experimentally

determined value of Kq greater than this is indicative of the factthat the quenching was not a dynamic but a static event. Further,Eq. (3) was applied to determine Kb and n by linear regression ofthe log (Fo � F)/F versus log [GNP–Chl] (Fig. 6) and found to be(6.02 ± 0.5) � 104 M�1 and 1.02, respectively.

The binding constant determined by this method is comparableto the value determined from calorimetric experiment.

3.2.2. Secondary and tertiary structures of BSA conjugated GNP–ChlFar-UV CD spectra, containing information on the secondary

structures of protein, are presented in Fig. 7a.The negative peaks appearing at 208 and 222 nm are the char-

acteristics of a-helical structure. The addition of the GNP–Chl intoBSA does not bring about any appreciable perturbation of the pro-tein’s secondary structure, as is the case when chloroquine alone isadded to BSA. Near-UV CD spectra, indicative of tertiary structureof the protein, usually show phenylalanine band at 262–267 nmand tyrosine and tryptophan bands at 282–290 nm. Fig. 7b doesnot indicate any noticeable shift in ellipticity of BSA in the near-UV region upon binding to both chloroquine as well as GNP–Chl.

Thus the binding of GNP–Chl does not lead to much alteration ofthe secondary or the tertiary structure of the protein. However,

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ticity

BSABSA + ChlBSA + GNP-Chl

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250 260 270 280 290 300 310 320Wavelength (nm)

Ellip

ticity

BSA

BSA + ChlBSA + GNP-Chl

a

b

Fig. 7. (a) Far-UV and (b) near-UV CD spectra of BSA in presence of chloroquine(BSA–Chl) and (BSA–GNP–Chl) compared to only BSA.

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nsity

(a.u

.)

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BSA-War BSA-War-0.025mM GNP-Chl BSA-War-0.05mM GNP-Chl

Fig. 8. Displacement of BSA-bound warfarin (5 lM) by chloroquine–conjugatedgold nanoparticles (GNP–Chl) using fluorescence spectroscopy. Fluorescence spec-tra corresponding to warfarin is shown in black.

P. Joshi et al. / Journal of Colloid and Interface Science 355 (2011) 402–409 407

based on the previous fluorescence quenching results on Trp emis-sion behavior it is possible that there is some conformationalchange in the local environment of the fluorophore due to theinteraction with GNP–Chl.

3.2.3. Drug displacement using warfarinTo further investigate the binding site of GNP–Chl and confor-

mation of conjugated protein, we used warfarin and ibuprofen asmodel drugs with known binding sites. Warfarin (3-(a-acetonylb-enzyl)-4-hydroxycoumarin) binds to drug site I in subdomain IIAand ibuprofen binds to drug site II in subdomain IIIA of BSA withhigh affinity [35,36]. Both of these domains are characterized bythe presence of a central cavity formed from six amphipathic heli-ces arranged in a myoglobin-like fold. When another drug is addedto the warfarin/ibuprofen–BSA complex, the drug displaces the al-ready bound drug warfarin/ibuprofen from their respective bind-ing site. The drug displacement is estimated in the form offluorescence quenching characteristic of warfarin or ibuprofen byselectively exciting the drug fluorophores. First, we have addedGNP–Chl to the warfarin–BSA and ibuprofen–BSA complexes indi-vidually. There was no change observed in ibuprofen fluorescencespectra (not shown here), but a quenching was observed in thewarfarin fluorescence implying that GNP–Chl is binding to warfa-rin binding site I. Crystallographic studies had suggested that a sin-gle warfarin molecule binds to BSA [26]. It is established thatwarfarin shares its binding site with a range of other drugs andthus competes with them for binding to BSA [27,36], with theselective excitation wavelength of 310 nm for warfarin, the fluo-rescence from warfarin are found to be quenched upon increasingthe GNP–Chl concentration (Fig. 8).

Similar results were obtained when the experiment was re-peated with only chloroquine suggesting that on its own chloro-quine also binds at warfarin binding site I (data not shown).From the drug displacement experiment, it is concluded that theGNP–Chl nanoconjugate binds at the same site where warfarinbinds (site I, subdomain IIA) and displaces warfarin from the pro-tein complex resulting in the decrease in fluorescence signal com-ing from warfarin.

3.2.4. Thermodynamic properties of GNP–Chl binding to BSAITC has been used to evaluate the binding interaction of GNP–

Chl nanoconjugate with BSA by quantifying the change in enthalpy,entropy, and Gibbs free energy during the binding process. Fig. 9shows the binding isotherm for GNP–Chl nanoconjugate and BSA.At top, the isothermal titration calorimetric thermograms for rawsignals are displayed while at the bottom, the heat flow per molof titrant (GNP–Chl) versus mol ratio (GNP–Chl: BSA) is plotted.

The derived thermodynamic parameters are presented in Table 1.The interaction of GNP–Chl nanoconjugate with BSA is found to beenthalpically, as well as entropically favored (i.e. DH < 0, DS > 0).The negative value of enthalpy is consistent with the characteristicsof weak van der Waals interactions and the positive value of entropyindicates hydrophobic interactions during protein–ligand complexformation [37]. The binding constant is of the order of 104, similarto the value obtained from fluorescence data, Section 3.2.1), showingmoderate binding affinity of GNP–Chl towards BSA.

3.3. Three-dimensional modeling of chloroquine binding with BSA

As the structure of BSA is not known it was modeled based onthe structure of human serum albumin, with which it has a se-quence identity of 75% over 578 residues (Fig. S2, supporting infor-mation). Its complex with chloroquine, as obtained from dockingsimulations, is shown in Fig. 10; the details of the ligand environ-ment are shown in Fig. S3 (supporting information). At this posi-tion of the drug, the point of conjugation to GNP faces theexterior of the molecule, providing enough space in which thenanoparticle can fit. Trp212 is in close proximity to chloroquine,and explains the experimental observation of the quenching oftryptophan fluorescence by GNP–Chl binding. Also this locationcorresponds to the site I binding site of BSA, which again corrobo-rates well with the warfarin displacement experiment.

-1.5

-1.0

-0.5

0.0

-10 0 10 20 30 40 50 60 70 80 90Time (min)

µcal

/sec

0.0 0.5 1.0 1.5 2.0 2.5 3.0

-2

0

Molar Ratio

kcal

/mol

e of

inje

ctan

t

Fig. 9. ITC data from the titration of 35 lM BSA in the presence of 0.35 mM ofchloroquine–conjugated gold nanoparticles. Heat flow versus time during injectionof chloroquine–conjugated gold nanoparticles at 25 �C and heat evolved per mol ofadded nanoparticles (corrected for the heat of chloroquine–conjugated goldnanoparticles dilution) against the molar ratio (GNP–Chl to BSA) for each injection,shown at top and bottom, respectively. The data were fitted to a standard model.

Table 1Thermodynamic parameters for the interaction of GNP–Chl with BSA.

Parameter Value

Ka (binding constant, M�1) 5.6 (±1.3) � 104

DH (binding enthalpy, kcal/mol) �3.48 (±0.4)DS (entropy change, cal/mol K) 10.24�TDS (at 25 �C kcal/mol) �3.05DG (free energy change, kcal/mol) �6.5

Tryptophan

Choloroquine (b)

GNP attachment site

(a)

Fig. 10. Cartoon representation of BSA with bound chloroquine. BSA is shown ingreen and chloroquine is shown in orange: (a) BSA bound chloroquine, where thepoint at which GNP is attached to choloroquine is shown in blue; (b) close-up viewof Trp212 and chloroquine. (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.)

408 P. Joshi et al. / Journal of Colloid and Interface Science 355 (2011) 402–409

The effect of nanoparticle on the structure and stability of a pro-tein depends on the nature and the physiochemical properties ofboth the components [38,39]. There are instances of secondaryand tertiary structural changes in proteins on binding to metalnanoparticles [40,41]. To be useful as a drug delivery vehicle,GNP–Chl should not have any adverse effect on the structure ofBSA, and additionally the binding should be moderate to enablethe smooth release of the nanoconjugate at the appropriate site.There has hardly been any study related to the biodistribution ofnanoconjugates. Our observation on the quenching of Trp fluores-cence due to the interaction with GNP–Chl (Fig. 5) indicates theproximity of the bound nanoconjugate to Trp212, which is locatedat site I of subdomain IIA of BSA. The binding of GNP–Chl at thissite is further confirmed by warfarin displacement assay (Fig. 8).There are no appreciable changes in the secondary and tertiarystructures of BSA brought about by GNP–Chl as revealed by the

CD spectra in the far- and near-UV regions (Fig. 7). The site selec-tive binding studies of variety of drugs and ligands to BSA indicatethat the binding affinity offered by site I is mainly through hydro-phobic interactions [42]. It was found that squaraine dye under-goes competitive binding with dansylamide for the site I in BSAand the complexation involves p stacking and hydrophobic inter-actions [22]. On the basis of results obtained from ITC experiments(Fig. 9 and Table 1) it is concluded that hydrophobic interactionsplay a significant role in the binding of GNP–Chl with BSA. GNP–Chl contains hydrophobic regions in thiol (MU) and chloroquinethat probably interact with protein’s hydrophobic patches (Fig. 10).

4. Conclusions

Experimental results suggest that GNP–Chl binds moderately toBSA, a feature desired for appreciable biodistribution and pharma-cokinetics of GNP–Chl nanoconjugates. The binding occurs withoutany major conformational alteration in the protein. Warfarin dis-placement experiment revealed that the binding of GNP–Chl takesplace at site I of subdomain IIA, which is also supported by fluores-cence quenching of Trp212 that is present in the same subdomain.The demonstration of the optimum binding of GNP–Chl to BSA mayalso lead to the formulation of combinatorial therapy, i.e., drug andradiation together, for example in cancer therapeutics. Overall, theunderstanding of the nature and selectivity of binding interactionsof GNP–Chl with BSA would have important implications in the de-sign of nanoconjugated drugs.

P. Joshi et al. / Journal of Colloid and Interface Science 355 (2011) 402–409 409

Acknowledgments

We thank Dr. Swagata Dasgupta, Prof. B. Bhattacharyya forhelpful discussion. P.C. is supported by JC Bose National Fellow-ship. P.J. is thankful to Council of Scientific and Industrial Research,India for a research Grant. S.P.S. acknowledges NSF Grant No. HRD0833112 (CREST program) and IFN start up Grant OIA-0701525.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jcis.2010.12.032.

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