Thionine Conjugated Gold Nanoparticles Trigger Apoptotic Activity

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Thionine Conjugated Gold Nanoparticles Triggers Apoptotic Activity Towards HepG2 Cancer Cell Line Puja Paul, Sabyasachi Chatterjee, Arindam Pramanik, Parimal Karmakar, Subhash Chandra Bhattacharya, and Gopinatha Suresh Kumar ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 11 Dec 2017 Downloaded from http://pubs.acs.org on December 16, 2017

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ACS Biomaterials Science & Engineering

Thionine Conjugated Gold Nanoparticles Triggers Apoptotic Activity Towards HepG2 Cancer Cell Line

Puja Paul,

ab,†£

a,†

Sabyasachi Chatterjee, Arindam Pramanik,c§ Parimal Karmakar,c Subhash b

a,*

Chandra Bhattacharya and Gopinatha Suresh Kumar a

Organic and Medicinal Chemistry Division, CSIR-Indian Institute of Chemical Biology, Kolkata 700 032, India b

Department of Chemistry, Jadavpur University, Kolkata 700 032, India c

Department of Life Science and Bio-technology, Jadavpur University, Kolkata 700 032, India

*Dr. Gopinatha Suresh Kumar, Ph.D, FAScT, FRSC, FAMS Chief Scientist CSIR-Indian Institute of Chemical-Biology Kolkata 700032, INDIA Phone: +91 33 2499 5723 Electronic mail: [email protected] *author to whom all correspondence should be addressed. † First two authors contributed equally. £ Current address: Department of Chemistry, Dinabandhu Mahavidyalaya, Bongaon, West Bengal 743235, INDIA. § Current address: School of Bio-medical Sciences, University of Leeds, LS2 9JT, UNITED KINGDOM.

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ABSTRACT Cancer cells could be locally damaged using targeted gold nanoparticles (GNP) conjugated with therapeutic dye thionine (TN). GNP was prepared by citrate reduction method, and the two complexes, namely GTN1 and GTN2 were synthesized by mixing GNP and TN at different ratios - at room temperature and at 80 oC, respectively. It is expected that GTN1 is formed when stabilizer TN participates in the reduction of Au3+ ions to Au0 nanocrystallites, while GTN2 is synthesized when the cationic dye TN adsorb onto the GNP surfaces due to the electrostatic attraction. The compounds were characterized by strong plasmon resonance absorption, FTIR spectroscopy, dynamic light scattering technique, zeta potential measurement, transmission electron microscopy and atomic force microscopy. Crystallinity of the NPs was ascertained by X-ray diffraction. Strong binding of GTN1 to DNA and the structural perturbation prompted us to study the cytotoxic activity of the compounds on hepatocellular carcinoma cell lines (HepG2) by MTT assay. The mode of cytotoxicity was found due to reactive oxygen species (ROS) generation inside the cells. Fluorescence microscopy analysis revealed nuclear fragmentation which was caused due to the ROS. The GTN1 induced fragmentation led to the apoptosis mediated cell death as found from cell cycle study. Conclusions drawn from these studies emphasised GTN1 to be capable of inhibiting proliferation in cancer cells in greater amount than other compounds. The importance of the work lies in the exploration of effectiveness of nanoparticles to cancer cells in vitro which can take a progressive step towards novel biomedical applications. Keywords: Gold nanoparticles, Surface plasmon, Anticancer, Apoptosis, HepG2


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INTRODUCTION One of the major causes of mortality in humans is cancer necessitating the development of cancer therapy as effectual and fruitful way to treat tumour cells. But lack of specificity showed by conventional chemotherapeutic agents bar them from differentiating normal and cancer cells. Owing to this serious impediment to progress in the field of cancer therapy, a diversion towards active targeting strategies utilising nanomedicine has instigated many researchers for the past two decades or so. Research in the biomedical field has been amplified related to use of nano vaccines and nano drugs following the seminal works of Maeda and Matsumura on enhanced permeability and retention (EPR) effect.1 Nonspecific biodistribution, low therapeutic indices and high toxicity are successfully overcome by nanoparticles owing to their increased cellular uptake and minimal side-effects to normal cells. Due to their tunable attributes such as size, shape, nanoparticles provide fascinating scaffolds for enveloped chemotherapeutic agents and deliver them to tumour cells effectively. Gold, endowed with properties like biocompatibility, non-toxicity and inertness can surpass many membrane barriers and hence prove to be a promising therapeutic particle towards successful cell specific targeting. Gold nanoparticles (GNPs) and functionalized GNPs have multitude of application in chemical sensing, cellular probe, bio-imaging and preferential administration of electromagnetic radiation to disease sites. Gold nanostructures have widespread popularity for hypothermic treatment of cancer, being efficient in raising specifically the tumor temperature sparing healthy cells.2 Much scientific interest has been spurred when the potential of GNP loaded with drugs, as a delivery vehicle, overcoming many biological barriers has been understood. Recently, attention has been paid to GNPs for providing bioconjugation platform to molecular probes and help in targeting cell surface receptors by endocytic mechanism.3,4 GNP synthesis has been carried out by amine-



containing polyelectrolytes exhibiting twofold functions of reducing as well as stabilizing agent.5 Inhibition of cell growth and death of human cancer cells induced by a chemotherapeutic drug paclitaxel is aggravated in presence of GNPs.6 GNPs have been coupled with a variety of therapeutics like ciprofloxacin, doxorubicin, curcumin and chloroquine enhancing therapeutic efficacy of the drugs and limiting target toxicity.7-10 Based on earlier reports, it can be said that toluidine blue O when coupled with GNP shows enhanced antibacterial properties as well as photodynamic therapy of cultured tumour cells.11,12 GNP-MB conjugation manifested destruction of Candida biofilm while cell wall integrity was lost by fragmentation of nucleus.13 The high affinity of GNP towards sulphur or nitrogen is well known and this knowledge is harnessed by conjugating an already studied DNA intercalator and singlet oxygen generator thionine (TN) on its surface. The phenothiazinium moiety of TN bearing both sulphur and nitrogen along with two amino substituents would serve as a good probe to be trapped on GNP surfaces in a facile way. Photodynamic destruction and inhibition of growth of Escherichia coli, Saccharomyces cerevisiae, Mycoplasma and Acholeplasma species are exhibited by TN on absorption of light.14,15 TN, as polymerization photoinitiator has rendered inactive bladder cancer cells by light dependent therapy and is also used as a fluorescent probe for the detection of Ag+ ion.16 TN can undergo potential genotoxic and cytotoxic activities in prokaryotic cells, show photoinduced DNA damage, delayed cell senescence and effective in combatting rickettsial infections.17-19 Herein, for the first time, we tried to establish if TN which is an established toxic dye, after being conjugated to GNP can aggravate cancer cell killing because successful DNA targeting in vivo is still a daunting task; even due to the hydrophobic nature of the photosensitiser TN intravenous administration through the blood stream in a targeted fashion towards cancer cells is severely hampered.


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To get a coherent picture of the efficacy of GNP, TN and nanoconjugates as chemotherapeutic agent, application to HepG2 in vitro has been done. This can detect the effectiveness of the synthesized nanoconjugates in antiproliferation and initiation of apoptosis in vitro in comparison to GNP and TN. This can be applied in vivo as future diagnostic tool which may hold the potential for a “magic bullet” against cancer. EXPERIMENTAL SECTION Materials DNA from Herring Testes (HT) (Type IV, CAS No. 438545-06-3, 50% Guanine+Cytosine base pair content), thionine (TN, CAS No. 78338-22-4, Color Index No. 52,000, purity > 85%), chloroauric acid (HAuCl4), tri-sodium citrate were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). TN was purified by recrystallization from water followed by chromatography on alumina using chloroform as eluting solvent. The sample was found to be pure from chromatographic analysis.20 Cell culture medium DMEM and Fetal Bovine serum were procured from Invitrogen-Life technologies, India. Thiazolyl blue formazan (MTT), and 2′,7′-Dichlorofluorescin diacetate (DCF-DA) were also from Sigma-Aldrich. Annexin V FITC with 7AAD was product of Biolegend, USA. DNA absorption spectrum revealed an A260/A280 ratio in the reange1.89 -1.93 and an A260/A230 ratio between 2.13 - 2.23. DNA was solubilized in the experimental buffer and dialyzed against the buffer several times. DNA and TN concentrations were determined from UV-Vis optical density using molar absorption coefficient (ε) value of 13,200 M−1 cm−1 (base pairs) at 260 nm and 54,200 M−1 cm−1 at 598 nm, respectively. All other chemicals and materials used were of high purity. Experiments were conducted at twenty degree Celsius in Na-phosphate buffer (20 mM), pH 7.2 prepared from deionized and triple distilled H2O. All glass wares used in this study were cleaned with aqua-regia



and thereafter extensively rinsed with ultrapure H2O. pH measurements were made on a high precision pH-meter fitted with a semi-micro electrode (Sartorius AG, Germany) which rendered an accuracy of > ±0.01 units. Membrane filters (0.22 µm pore size) were employed to clarify the buffer solution (Millipore India Private. Limited, Bengaluru, INDIA). Synthesis of NPs GNP was synthesized as: 1000 µM of chloroauric acid was heated to boiling followed by addition of 1% tri-sodium citrate. The heating was continued for 30 min. with constant stirring. The solution gradually turned light blue and then wine red color [Supporting Information Fig. S1a] of GNP appeared owing to the excitation of surface plasmon vibrations. Collection of the GNPs was done by centrifuging the solution (12,000 rpm, 10 min., 15°C) after washing thoroughly with water to confirm removal of unbound citrate ions in the NP dispersion. To synthesize GTN1, 50 µM of TN aqueous solution and 0.02M of HAuCl4 were mixed in a ratio of 1:2 and stirred vigorously overnight at room temperature. The networked structure [Supporting Information Fig. S1b] got converted to blackish yellow solution [Supporting Information Fig. S1c]. To synthesize GTN2, 50 µM of TN aqueous solution and the prepared GNP were mixed and heated at 80oC for 3 hours. Violet colored TN-covered GNPs [Supporting Information Fig. S1d] were obtained on centrifugation at 12000 rpm. This was washed with cold water to ensure removal of un-adsorbed TN. Proposed mechanism Visual inspection of colour change is the preliminary analysis for the onset of the reaction. There have been reports of possessing redox properties by several hydroquinones and amines in


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reduction of metals.21,22 For the synthesis of GTN1, TN acts as the reducing as well as capping agent for chloroauric acid in situ. Thus TN was responsible for both the synthesis and

Scheme 1. Proposed mechanism for preparation of GTN1 (upper panel) and GTN2 (lower panel).

stabilization of the GNPs. Back bonding is usually observed from the electron rich centers of a compound, possessing hydroxyls or amino group to electron deficient metal centers. This phenomenon, which is commonly called chelation, depends on the symmetrical placement of neighboring electron rich groups so as to grab the electron acceptor metal centre. This electron donation acceptance property has been manifested in case of GTN1 where two symmetrically placed amino groups of ligand TN coordinate Au3+ ions by forming a stable cyclic structure. An electron transfer reaction is observed as a consequence of this encapsulation such that a simultaneous oxidation-reduction reaction is followed- in situ reduction of Au3+ ions to Au0 atoms, while TN's amine groups are oxidized to the tertiary amine groups [Scheme 1]. The initiation of formation of gold nanoparticles are presumed to be due to nucleation and growth of metallic Au but agglomeration is prevented as TN acts as a stabiliser.23 This is demonstrated by the simultaneous discharge of dark blue networked structure to blackish-yellow color. A coordination complex of TN with Au3+ ions is formed which is ascribed to transfer of electron from amino groups into the vacant orbital of electron deficient Au center. After being reduced by TN, the Au nuclei associated with GNPs self-assembled onto the surface of TN which is the 7 ACS Paragon Plus Environment


outcome of both van der Waals force and the high affinity between the amino group and the Au particles. Similar type of mechanism was also reported for polyphenol compounds, curcumin and genistein.24-26 The change of color obviously points to the conjugation of GNP-TN which was confirmed from UV-Vis spectroscopy as the 520 nm peak in case of GNP only (Supporting Information Fig. S2 b) shifted to 570 nm (Supporting Information Fig. S2 c). This 50 nm bathochromic shift in the peak of plasmon absorption is associated to the change in the local dielectric constant around the GNPs due to TN conjugation.26 The essential issue for the progress of the reaction is that the reduction potential of HAuCl4 must be lower than the oxidation potential of the studied amine. The reduction potential of HAuCl4 to Au0 has been reported as 0.853 V in aqueous solution.27 When the reduction potential of HAuCl4 is higher than oxidation potential of amine reducing agent, the reduction reaction of HAuCl4 to Au0 would be followed by simultaneous oxidation reaction of Au0 to Au1+, prohibiting GNP formation.27 Because the oxidation potential of TN is .253V, we propose that the redox reaction occurring between TN and HAuCl4 generates Au0 in tandem to oxidation of TN to a radical cation. The possibility of equilibrium between the Au0 in the form of GNPs and Au1+ in solution has been reported along with postulated equilibrium between oxidised and reduced form of TN.27 In case of GTN2, the negative charge on GNP surface owing to citrate coating welcomes the oppositely charged (positive) dye TN to adsorb on its’ surface by ion-ion attraction. Ding et al predicted that this might lead to the formation of closely packed TN on a charged particle surface promoting dipolar or hydrophobic interactions between dye molecules converting the monomeric form of TN to H-type dimeric state.28 Methods Absorbance spectral studies


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A Jasco V 660 spectrometer (Jasco Company, Japan) was used for absorbance studies at (20±0.5) °C. This unit has a thermoelectrically controlled absorbance monitoring set-up for doing measurements at any pre-fixed temperature. XRD, DLS and Zeta potential measurements The average particle size of the NPs was determined by a dynamic light scattering instrument (Model DLS-nano ZS90, Zetasizer Nanoseries, Malvern Instruments). Dilution of NP's was done with distilled water. The incident light was projected at an angle of 173°. The mean size and zeta potential were evaluated from the five measurements allotted to each specimen. X-ray diffraction patterns (XRD; D-8 Advanced, Bruker) collected at 0.02° intervals (2θ) assessed the crystalline nature of the prepared NP samples. Transmission electron microscopy Electron microscopy images were obtained with a Tecnai G2 Spirit Biotwin (type: FP5018/40) at an accelerating voltage of 80 kV and under bright field conditions. A drop of the solution was placed on a 300 mesh copper grid (Allied Scientific Product, USA) coated with a thin amorphous carbon film after sonication for 10 min. After five minutes of deposition, a strip of filter paper was used to blot away the excess of solution while the rest was allowed to dry by placing in a vacuum desiccator for 24 h. The specimen was eventually examined under the electron microscope to study the structural features of the NPs. Atomic force microscopy An aliquot of 10 µL, from a final concentration 10 nM, was used for AFM imaging of the NPs. This was adsorbed onto muscovite Ruby mica sheet (freshly cleaved, ASTM V1 grade Ruby Mica from MICAFAB, Chennai, India) and dried for 20 min. under vacuum in nitrogen gas atmosphere. AFM (AAC mode) was performed using a Pico plus 5500 ILM AFM (Agilent



Technologies, USA) with a piezo scanner having a maximum range of 9 µm. Micro fabricated silicon cantilevers from Nano-Sensors (USA), 225 µm in length with a nominal spring force constant of 21-98 N/m and nominal radius of curvature less than 10 nm. were used. The cantilever oscillation frequency was a nominal resonance of 146-236 kHz and scan rate of 1 line/s. All AFM images were captured at 512 × 512 pixels with a scan size 0.5 to 5 µm at the scan speed rate of 0.5 lines/S in tapping mode at ambient condition. Processing and manipulation of the images were done through software, "Picoview Version 1.1" and "Pico Image Advanced" (Agilent Technologies), respectively. Fourier transform infrared (FTIR) spectroscopy FTIR spectroscopy studies were performed on a Bruker Tensor 27 FTIR spectrometer (Bruker Inc. United States of America) in the range 4000-500 cm-1. Forty eight scans were performed for each sample and the result is presented as their average. Bruker software was used for data processing. Spectropolarimetric studies Circular dichroism (CD) spectral recording was performed on a J-815 model unit of Jasco Company equipped with their PFD 425L/15a model temperature controller at (20±0.5) oC as per previous descriptions.29 The solutions were housed in a rectangular quartz cuvette of one centimeter path-length. Recording parameters for data acquisition were, scan speed of fifty nm/min., bandwidth of one nm, and sensitivity of one hundred milli degree. The ellipticity (molar) values are expressed in terms of DNA nucleotides (210-400 nm). Isothermal titration calorimetry For performing calorimetry experiments we used a MicroCal unit (Model VP-ITC, MicroCal, Inc., Northampton, MA, United States of America, now Malvern Instruments, United Kingdom)


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following elaborate protocols developed in our laboratory for dye-DNA titrations.29,30 Degassed titrant solution (the DNA) from the rotating syringe (292 rpm) was injected into the dye solutions (1.424 mL). This protocol was adopted to avoid any dye aggregation possibility. Concomitantly a experiment (control) to determine the heat of dilution of DNA (titrant) was performed. Dyedilution was experimentally found to generate no significant heat. Determination of the area under each heat burst spike was performed by integration using the software of the unit to provide the measure of the heat associated with the injection. The heat of DNA-dye binding was obtained after subtracting the heat of DNA dilution from the heat of dye-DNA binding. Corrected injection heats thus obtained were plotted against molar ratio and fit with a binding model of "one set of binding sites" yielding the binding affinity (K) and the binding enthalpy change (∆Ho). The binding Gibbs energy (∆Go) and the entropic contribution (T∆So) of the association were then deduced from thermodynamic equations described previously.30 Cell cultures Hepatocellular carcinoma (HepG2) cells were purchased from National Institute of Cell Science, Pune, India. Cells were grown in Dulbecco's Modified Eagle's medium with 10% fetal bovine serum, streptomycin/penicillin (100 units/mL) at 5% CO2, 37° C. Cell viability study HepG2 cells were tested for cytotoxicity with the NPs at various concentrations. Cells were grown in 48-well plates overnight with average cell number of 1×104 cells per well prior to treatment with the compounds. After treatment, cells were left for 24 h and then gently washed with 1X PBS. Working concentration of MTT dye at (0.5mg/ml) was added into the wells and left for incubation for 3 hrs. Finally, the MTT dye was discarded and MTT extraction buffer was added and the cells put on a shaker for 10mins. The cell viability absorbance was determined at



595 nm in a spectrophotometer (Epoch Micro-plate, United States of America). LD50 dose was considered to be that concentration at which 50% of cell population is either viable or dead. Reactive oxygen species estimation ROS was estimated by DFC-DA. This dye once inside the cells, reacts with the reactive oxygen to give a green-fluorescent complex. To describe briefly, a working concentration of DCF-DA (100 µM) was prepared in 1X PBS and cells were incubated with it for 30mins prior to treatment. Further cells were treated with LD50 dose of NPs for 8 hrs. On treatment, cells were washed with 1X PBS and fluorescence intensity was measured (excitation : 485 nm, emission: 520 nm (Hitachi Co,, Japan). Cells were also imaged using fluorescent microscope (Leica, Germany). DNA damage analysis HepG2 Cells were treated for 24 h with LD50 dose of the NPs. After 24 h of incubation with NPs Cells were washed with cold 1× PBS and were fixed with paraformaldehyde (4% in PBS) for 20 min. at room temperature. After fixation, the cells were extensively cleaned with ice-cold 1× PBS and then incubated with permeabilising buffer containing 0.2% Triton X. Cells were further blocked with 5% BSA and incubated overnight in moist chamber at 4°C with the anti-γH2AX antibody diluted in wash buffer (1:100) containing 0.1% BSA and 0.05% Tween-20 in PBS. Next day, the cells were washed with wash buffer and labelled with appropriate secondary antibodies conjugated to Alexa Fluor 568. The cells were imaged under fluorescence microscope (Leica DM2500, Germany). Cell death analysis and apoptosis Upon treatment of HepG2 cells with the NPs for 24 h at LD50 dose, the cells were washed with 1X PBS and fixed with 70% ethyl alcohol for 12 h at 4°C. Prior to staining with 50 µg/ml


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concentration of propidium iodide (Sigma), the cellular RNA was degraded by treating with 100 µg/ml concentration of RNAse A (Thermo-Fisher Scientific) at 37°C for 1 h. To study apoptosis, cells were treated with the NPs for the 24 hr at LD50 dose and then stained with DAPI which selectively stains the nucleus. One step further, treated HepG2 cells were washed with ice-cold 1X PBS and re-suspending in 100 µL of annexin binding buffer to which Annexin V-FITC (2 µg/ml) was added and incubated for 30 min at room temperature. After incubation, 400 µl of annexin binding buffer was added to which 5 µl (0.5 µg/mL) of 7-AAD was further supplemented prior to analysis. All FACS analysis were done with BD Biosciences, FACSCalibur, United States of America. RESULTS AND DISCUSSION Characterization by absorbance spectroscopy Strong plasmon resonance absorption characteristics of GNPs is a major determinant of the size and shape of the plasmonic nanoparticles. Plasmon excitation occurs with the whole particle jumping through transition bands. The literature value for plasmon bands is usually a between 520 and 530 nm for spherical GNPs ranging between 15-20 nm.31 The characteristic spectra of the synthesized GNP and GTN1 showed maximum at 520 nm [Supporting Information Fig. S2 b] and 570 nm [Supporting Information Fig. S2 c], respectively. The maximum absorbance of TN is centred at 598 nm [Supporting Information Fig. S2a] which depicts the existence of the monomeric form; the shoulder at 557 nm indicates presence H-type dimer aggregate.32 GTN2, formed as a result of adsorption of TN on GNP, has two absorption bands at 585 nm along with a prominent shoulder at 560 nm [Supporting Information Fig. S2 d]. This dictates presence of both transverse and longitudinal plasmon modes, in turn, specifying the probable formation of non-



spherical nanoparticles.33 TEM measurements can suitably confirm this anisotropy. The conversion to H- type dimeric form of TN from monomeric form is facilitated by small particles. XRD, DLS and Zeta potential measurements The synthesized GNPs exhibited face centered cubic nanocrystals form. The symmetry as predicted from XRD pattern [Fig. 1a] was an Fm3m with 2θ values of 38.270, 44.330, 64.920,

Figure 1. (a) XRD patterns of GNP (black), GTN1 (green) and GTN2 (red), DLS data of (a) GNP, (b) GTN1 and (c) GTN2 revealing particle size distribution. and 77.59o, which can be assigned to (111), (200), (220), and (311) planes, respectively.34 Crystallinity of GNP in GTN2 increased which suggested that GNPs must have agglomerated in the presence of TN compared to that in GTN1; all the peak positions of GNPs are though intact. Dynamic light scattering (DLS) technique is an adopted technique used for characterization of particle size of the synthesized nanoparticles. Figure 1b,c,d below is representation of typical DLS plot of the prepared samples. The average size of GNP [Fig. 1b] and GTN1 [Fig. 1c], as displayed by the outcome of the experiment varied in the range 10-100 nm (with maximum distribution at 40 nm for GNP and 60 nm for GTN1), while that for GTN2 [Fig. 1d] showed maximum distribution at 90 nm. The variation in size cannot be pinpointed exactly by DLS measurement since it measures hydrodynamic diameter and gives indication about the average dimension in the form of an equivalent sphere regardless of the original shape of the particles. However, DLS analysis needs to be compared with other reliable qualitative techniques for 14 ACS Paragon Plus Environment

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characterization purpose due to dearth of robustness consequent to other cumulants analysis.35 Absorbance and TEM measurements were used in corroboration to the results obtained from DLS studies. Electrophoretic Light Scattering (ELS) is a widely used method for measuring the electrophoretic mobility of dispersed particles in solution which can be utilized in the conversion to Zeta potential. Surface charge can be measured by Zeta potential and is widely used to monitor adsorption of TN on the surface of synthesized GNP.36 GNPs showed a zeta potential of about -18.9 mV, owing to weakly bound citrate coating which is in congruous with previously reported data.37 The negative zeta potential value suggested repulsive interaction between nanoparticles, preventing agglomeration, exhibiting excellent stability and suggesting less toxicity to normal cells.38 The ζ value measured for GTN1 and GTN2 were -16.9 mV and +5.1 mV, respectively. The slight decrement in the zeta potential from -18.9 mV to -16.9 mV for GTN1 is credited to the partial neutralization of charge, as cationic TN dye molecules weakly interact with negatively charged AuCl4- anions. Similar observation of acridine orange and cyanine dye adsorption on GNP surface was reported previously.39 The change in the sign of the potential for GTN2 is an indication of adsorption of TN on GNP surfaces, as was supported by FTIR analysis. Reversal of the charge from -18.9 mV to +5.1 mV ensures successful adsorption of a layer on the surface of the nanoparticle.36 Additionally, GNPs rendered a conductance value of 1.41 mS cm−1 from conductivity measurement. However, GTN1 and GTN2 showed a increase from 1.40 mS cm−1 to 2.61 mS cm−1 and 1.87 mS cm−1, respectively. Earlier data reporting similar increment in conductivity values as a consequence of adsorption of dye on the surface of GNPs have been found.39,40 4.3. Transmission electron microscopy



It is well known that the shape and size of any nanoparticle is a key parameter to gain better understanding of cellular uptake in vitro. Following the study of Chan et al., it can be said that nanoparticles with spherical shape showed more efficient uptake than elongated rod shaped

Figure 2. TEM images of (a) GTN1 and (b) GTN2 at a scale of 100 nm illustrating particle distribution of nanoparticles. particles.41 TEM images can conclusively confirm the shape and size of the prepared samples. GNPs were monodispersed, uniformly distributed and spherical in shape with size ranging from 25±4 nm. GNPs were scattered with only few of them present as aggregates, as observed under this microscope (Figure not shown). The shape of GTN1 and GTN2 were different from that of GNP. GTN1 was somewhat spherical [Fig. 2a] while GTN2 was dice shaped as seen in Fig. 2b. The average size of GTN1 and GTN2 are about 40±4 nm and 60±4 nm, respectively. Presence of some clustered nanoparticles, glued together, due to TN dye being acting as stabilizer (in case of GTN1) or being adsorbed on the surfaces of GNP (in case of GTN2) with an effective inter particle linkage is evident from Fig. 2 a,b. Adsorption of 5,5-disulfopropyl-3,3dichlorothiacyanine dye and acridine orange on the surface of GNPs yielded very close resemblance as was reported here.39,40 It is required to mention that particle size domain as predicted by TEM and DLS does not exactly match as they are different size predicting methods. 16 ACS Paragon Plus Environment

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TEM gives the number-average diameter of the dried state particle, whereas DLS give a zaverage diameter of the solvated particle. Atomic force microscopy Additional morphological information of the prepared samples was gathered by analyzing AFM images. Figure 3a,c,e represents the AFM image of GNP, GTN1 and GTN2 within a scan area of 1.4 µm × 1.4 µm, 0.7 µm × 0.7 µm and 0.7 µm × 0.7 µm, respectively. Figure 3b,d,f are the three-dimensional representation of Figure 3a,c,e.. GNP [Fig. 3 a,b] and GTN 1 [Fig. 3 c,d] were spherical in shape. On careful inspection, faceted monocrystals of GTN2 of ellipsoidal shape were seen in Fig. 3 e,f. The average size of GNP, GTN1 and GTN2 were about 25±4 nm, 42±4 nm and 61±4 nm, respectively. The reason for the two plasmon bands of GTN2 as was observed from UV-vis spectra is clear after the shape analysis of ellipsoidal GTN2. This stems from coherent oscillation of free metallic electrons along the short and long axes of the Figure 3. AFM images: (a) image of GNP within a scan area of 1.4 µm × 1.4 µm, (b) 3D image of GNP, (c) image of GTN1 within a scan area of 0.7 µm × 0.7 µm, (d) 3D image of GTN1, (e) image of GTN2 within a scan area of 0.7 µm × 0.7 µm, (f) 3D image of GTN2.



GTN2.42 Furthermore, there is a slight variation observed in the shapes as analysed by TEM and AFM techniques as the modes of measurement are different and effective contrast present in the TEM case cannot be ruled out. Shape, sizes, and distribution of GNP and GTN2 from TEM show congruency with the data obtained from spectral analysis. FTIR analysis FTIR is an effectual technique to demonstrate changes in vibrational frequencies of the adsorbed molecule bound to a metal surface.43The inequality of intensities in the spectra of the bound molecules in comparison to the unbound molecules furnish necessary details concerning interactive pattern and stability of a ligand on association. The adsorption characterisation of TN on GNP surfaces was demonstrated by FTIR spectroscopy. The IR spectra of free TN and synthesized complexes GTN1 and GTN2 are shown in Fig. 4 a,b,c. The skeletal vibration of the heterocyclic ring of TN manifested absorption bands at 1600 and 1500 cm-1.44 The existence of TN on the GNP surfaces in GTN1 and GTN2 has been confirmed by the presence of abovementioned bands. It is assessed that nitrogen atoms of both the amino moieties of TN was somehow bound, being observed from N-H stretching vibrational frequency of 3310 cm-1 in Fig. 4a. This absorption

Figure 4. FT-IR spectra of (a) GNP, (b) GTN1 and (c) GTN2.


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band was observed with a reduced intensity in GTN1 (Fig. 4b) and negligible intensity in IR spectrum of GTN2 (Fig. 4c). Moreover, there is absence of absorption band at 850 cm-1 in the IR spectra of GTN1 [Fig. 4b] and GTN2 [Fig. 4c]. This band stems from N-H bending vibration of the amino moieties of TN [Fig. 4a] and hence confirms that the nitrogen atoms of the exocyclic amino groups are not free. However, comparison of the weak and broad absorption band of C-S bond in the range of 710-570 cm-1 is of no use as this cannot bring out any meaningful difference between the IR spectrum of free TN and that of GTN1 and GTN2.44 The ability of nitrogen and sulfur atoms present in the central heterocyclic ring of TN to act as binding centres on nanoparticle surfaces has not been considered due to their electron deficient property.7 Ding et al performed optimized a model for TN-GNP complex based on quantum chemical calculations.28 Their calculation showed that the electron rich nitrogen centre of the amino group of planar TN favourably donate their lone pair of electrons. This is accepted by the electron deficient GNPs which facilitates the association of the -NH2 on the surfaces of nanoparticle. Thus, the bond distance of N-H and C-N bonds at both the -NH2 ends of the complex is increased. Rarely any change is observed in the bond angle of C-S-C and C-N-C at the central heterocyclic ring, indicating that the central nitrogen and sulfur atoms do not have any role in binding to the gold particle surfaces.28 Spectropolarimetric studies Targeting the DNA of cancer cells with NPs is the stepping stone for many budding anticancer therapies. The potential of GNPs to alter genetic codes linked to continuation of cell cycle is the causative factor of DNA damage. This occurs as gold atoms are very prone to physicochemical interaction with functional groups of intracellular proteins, on one hand, nitrogen bases and



phosphate groups in DNA, on the other.45 Thus an initial assessment of binding strength of the NPs with DNA is required before their application to cell line. Circular dichroism (CD) spectroscopy is a strong tool for probing subtle variations of DNA chiral confirmation during NP-DNA interactions. The spectrum 1 in Figure 5 shows a positive band around 275 nm region and a negative band around 248 nm which is the characteristic feature of B-form DNA. Figure 5 a,b,c portrays the impact of the NPs on the conformation of

Figure 5. Intrinsic CD spectral changed of HT DNA in the presence of (a) GNP, (b) GTN1 and (c) GTN2. DNA molecule in the region 210-400 nm. The observed signal changes that surfaced in the DNA molecule can obviously be pointed to the interaction phenomenon since achiral NPs lack any CD signal in the above region. This lead to another observation of increment in the ellipticity of the positive band and negligible amount of decrement of the negative band. Thus, a simple conclusion that can be deduced based on the binding event is that the right-handed helicity of DNA remained intact. However, compactness of DNA enhanced due to substantial increase in the base stacking degree. Change in the conformation of the nucleotide resulting in B-A transition of DNA is the immediate consequence of the interaction.46 From the figure it is evident that highest interaction of DNA is with GTN1 [Fig. 5b] followed by GTN2 [Fig. 5c] and GNP [Fig. 5a]. 20 ACS Paragon Plus Environment

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Isothermal titration calorimetry Top panel of Fig. 6 a,b,c presents the representative primary data from the isothermal

Figure 6. ITC patterns of DNA interaction with a) GNP, (b) GTN1 and (c) GTN2. The top segment (a-c) represent the raw data obtained after sequential injection of DNA into the nanoparticles. The bottom section (d-f) depicts the fitted curve with the experimental points to a one-site model against the molar ratio of DNA /NPs.

calorimetric titration of each of the DNA samples (500 µM) into GNP, GTN1 and GTN2 solutions (10 µM). Each heat burst curves here corresponds to a single injection het generation. These injection heats were then corrected by subtracting the control heats derived from DNA titration into the buffer alone (shown in the upper segment of figure, spikes off set for clarity). The bottom panels in Fig. 6 presents the resulting corrected heats plotted against the molar ratio. The data points here are the actual experimental input data and the smooth line is the calculated best fit to the data. The titration profile in all the three cases showed single binding event and was characterized by exothermic heats. The ITC profiles were fit to a single site model. The results of the analysis are presented in Table 1. The binding affinity value obtained



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for TN-DNA was of the order of 105 M−1.47 The GTN1-DNA [Fig. 6b] afforded the highest binding affinity followed by GTN2-DNA Table 1 Thermodynamic data for the binding of GNP, GTN1 and GTN2 to DNA. Compounds studied

K×10-5 (M-1)

∆Go (kcal /mole)

∆Ho (kcal / mole)

GNP 1.51±0.04 -6.61±0.04 -1.06±0.04 GTN1 14.3±0.02 -7.73±0.02 -1.21±0.02 GTN2 6.22±0.06 -7.38±0.06 -1.57±0.06 a The values in this table are average of four experiments under identical conditions.

T∆So (kcal/mole) 5.55±0.04 6.52±0.02 5.81±0.06

[Fig. 6c] and GNP-DNA [Fig. 6a]. Thus GTN1 has a strong affinity towards DNA as well as it is capable of disrupting the native conformation of DNA. These findings stemming from circular dichroism and isothermal titration calorimetry prompted us to investigate the propensity of the NPs as therapeutic entities. Cell viability study

The cell viability was measured using MTT assay.48 It is a NADPH dependent cellular oxidoreductase enzyme which indicates the number of viable cells present as this enzyme cannot penetrate non-viable cells. Cell viability of HepG2 cells was analyzed for GNP, TN, GTN1 and GTN2 [Fig. 7]. From the result it was observed that these compounds had a dose dependent cytotoxicity on the HepG2 cells. But we found that HepG2 cells were more resistant to TN as compared to MCF-7 cells as reported by Manivel et al.49 Interestingly, the synthesized derivatives of TN (GTN1 & GTN2) were more effective than TN in inhibiting HepG2 cell growth. From the MTT assay, GTN1 was the most efficient for showing anticancer activity among all the compounds having LD50 dose of 42 µM followed by GTN2 and TN which had the LD50 dose of 60 µM and 160 µM respectively. GNP as already reported by various research groups did not have any toxicity at such significant dose.50 22 ACS Paragon Plus Environment

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Figure 7. Cell cytotoxicity effect of GNP, TN, GTN1 and GTN2 on HepG2 cells after 24hrs (MTT assay).

Reactive oxygen species assay Many compounds having anticancer activity are observed to induce ROS generation.51 ROS is majorly responsible to cause various cellular damages in the DNA and mitochondria which finally leads to the cell death. After studying the cytotoxic role of the compounds on HepG2 cancer cells, we wanted to explore, if the compounds generated reactive oxygen inside the cells by using DCF-DA. Once diffused into the cells, deacetylation of the dye DCF-DA occurs by cellular esterases to a non-fluorescent compound, which on oxidation by ROS is converted into highly fluorescent 2’, 7’-dichlorofluorescein (DCF) and hence can be easily detected using fluorimeter or fluorescent microscope.52 Upon treating HepG2 cells with the compounds at their respective LD50 dose it was found that TN [Fig. 8c], GTN1 [Fig. 8e] and GTN2 [Fig. 8d] did show indications of ROS generations as observed using a fluorescence spectrophotometer. The control cells of HepG2 whereas did not show any fluorescence indicating the induction of ROS was due to the treatment alone [Fig. 8a]. Among the compounds GTN1 had the maximum fluorescence intensity indicating its reason to show the maximum cytotoxic effect inside the



cancer cells [Fig. 8e]. The green fluorescence of the DCF was also observed under the fluorescent microscope for all the compounds. The comparative study has been depicted graphically [Fig. 8f].

Figure 8. Detection of reactive oxygen species (ROS) for (a) untreated (b) GNP (c) TN (d) GTN2 (e) GTN1 on HepG2 cells by DCF-DA under fluorescence microscope and (f) the quantitative analysis profile.

DNA damage One of the consequences of ROS production is the formation of double strand breaks in DNA. This event triggers activation of many proteins, one of which is histone variant H2AX which is phosphorylated and known as γH2AX.53 By measuring the amount of cells positive for γH2AX foci, the extent of DNA damage can be measured. From our study it was observed that GTN1 had the maximum effect for DNA damage as evident by foci inside its nucleus compared to GTN2 or TN [Fig 9].


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Figure 9. Fluorescence microscope image of γH2AX foci inside the nucleus of HepG2 cells upon treatment.

Cell cycle analysis Cell cycle is an important study to know the effect of a compound on the phase of cell growth.54 The cell cycle analysis generally divides the cells into 3 different phases namely G1 phase (growth phase), S phase (DNA synthesis phase) and G2/M phase. Cell cycle analysis can reveal a lot of information especially if any cell is proceeding towards apoptosis which then causes the cell cycle to arrest just before the G1 phase (sub G1).55 One of the major targets of reactive oxygen is DNA and our compound was also found to disrupt the native DNA structure which combined effect may leads to apoptosis mediated cell death.56 After 24hr of treatment of the compounds on HepG2 cells at LD50 dose of GTN1, GTN2 and TN did show a higher percentage of cells arrested in the sub G1 phase compared to GNP [Fig. 10A]; whereas the control cells had almost no cell cycle arrest (3%) at sub G1. The quantitative value of the cells in subG1 phase was 71.6, 36 and 25.6 for GTN1, GTN2 and TN, respectively [Fig. 10D]. Thus GTN1 was found 25 ACS Paragon Plus Environment


to have an enhanced anticancer activity compared to GTN2 and TN. Further to confirm apoptosis we studied the nucleus of the cells using DAPI. Apoptosis study An underlying mechanistic principle of a chemotherapeutic drug is based on its propensity to suppress cell proliferation or directly kill cancer affected cells. The prime target of such anticancer agents is still a matter of debate, but based on the rationale that DNA is the hub of genetic information storage, it can be speculated that their cytotoxic property is the outcome of their interactive ability with DNA (vide infra). Apoptosis is thought to be the foremost cause of MCF-7 inhibition.57 Rely on cancer specific targets like DNA by small molecules has earned impetus as they may be responsible for triggering cell cycle arrest whose immediate consequence is cell death.58 During apoptosis the DNA inside the nucleus starts fragmenting and produces apoptotic bodies.59 DAPI staining can throw light on the same. We compared GTN1 treated HepG2 cancer cells with the other compound and studied the morphology of the nuclear DNA at the respective LD50 dose [Fig. 10b]. At 24hr of treatment TN, GTN1 and GTN2 showed occurrence of apoptosis inside the nucleus as evident by apoptotic bodies and fragmentation of DNA. The control cells and the GNP treated cells on the other hand had almost no effect on the nuclear DNA. Thus the compounds confirmed the induction of apoptosis which was jointly responsible due to the generation of ROS and the DNA disruption prohibiting it to replicate further. The fluorescence microscopy analysis reveals the nuclear changes in the form of condensed chromatins and shrinking morphology in the drug treated HepG2 cancer cells, which is another important feature of apoptosis. Baicalin-conjugated GNP and thionine-serum albumin complex inhibited the proliferation of cancerous


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Figure 10. (a) Cell cycle analysis detecting DNA fragmentation in HepG2 cells. (b) DAPI staining of HepG2 cells to detect presence of apoptotic bodies . (c) Annexin V FITC analysis to detect apoptsis. Quantitative plots to show percentage of cells in (d) sub G1 phase and (e) apoptotic phase.

MCF-7 cells by inducing apoptosis in a similar manner.49,60 The percentage of cells in sub G1 phase is given graphically.



Another important event which occurs during apoptosis is the translocation of phosphatidylserine (PTS) from the inner cytoplasmic side of the phospholipid bilayer to the outer membrane.51 In the in-vivo condition this translocation of PTS signals the macrophages to engulf the cell and cause apoptosis mediated cell death. For the purpose of pathological detection of apoptosis, Annexin V molecule is used. Annexin V selectively binds to the PTS expressing cells and thus can be used to determine occurrence of apoptosis when tagged with a marker. From our study we found that cells treated with the GTN1 induced maximum percentage of apoptosis in HepG2 cells [Fig 10c]. The quantitative value of percent of cells in apoptotic phase were 58, 35 and 31 for GTN1, GTN2 and TN, respectively [Fig 10e]. A schematic representation of the possible mechanism for the cytotoxic activity of GTN1 on HepG2 cancer cells is shown in Scheme 2.

Scheme 2. Schematic pictorial depiction of the cytotoxic activity of GTN1 on HepG2 cancer cells. CONCLUSIONS Although many big strides in the field of nanomedicine have been made yet many hurdles like selective targeting, lack of specificity etc remain to be accomplished. The GNPs were prepared by reduction procedure while utilizing the stabilizing and adsorption property of thionine, two


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other complexes of different sizes were also synthesized. The synthesized GNP and complexes were characterized in terms of particle size, surface charge, morphology and identified by several techniques including UV-Vis absorption spectroscopy, zeta potential, dynamic light scattering transmission electron microscopy, atomic force microscopy. The geometry of the NPs established from XRD analysis were face centered cubic patterned. Results from TEM, DLS and AFM gave idea regarding the shape and size of the NPs. Zeta potential measurements confirmed partial surface charge neutralization of negatively charged GNPs by cationic TN, also suggesting a high stability of the nanoparticles in case of GTN1. The mechanism proposed for the formation of GTN1 suggests TN to be both reducing and capping agent, stabilizing the gold sol. Thus, the surface functionalization of GNPs with TN indicates possibility of an advanced procedure of using the phenothiazinium dye as drug delivery vehicles. FTIR confirmed the adsorption of TN on GNP surface. Circular dichroism augments possibility of conformational distortion in native conformation of DNA while isothermal titration calorimetry confirmed strong binding of the complexes with DNA. Cytotoxic activity of the complexes clearly showed a decrease in the percentage of cell viability in a concentration-dependent manner. Upon studying the mechanism it was found that ROS was one of the major factors responsible for the cytotoxicity. Reactive oxygen has various targets in the cell organelles, one of which is DNA damage inside the nucleus as evident from the γH2AX foci inside the nucleus. From nuclear staining it was evident that the GTN1 and GTN2 did show nuclear fragmentation which is a hallmark of apoptosis. Further, from flow cytometry the cell cycle arrest in sub G1 indicates the occurrence of apoptosis in the HepG2 cells which was significantly enhanced in GTN1 and GTN2 compared to TN and GNP. Further the annexin V FITC data confirms it. This in situ knowledge would serve as a



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precedent in showing the progressive route for the design of engineered nanoparticles with improved functionality, to be implemented in the arena of nanomedicine. Supporting Information Available The following files are available free of charge: Fig. S1 and Fig. S2 depicting characteristic colour and spectra of the nanoparticles, respectively.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Phone: +91 33 2499 5723. Fax: +91 33 2473 5197. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the Council of Scientific and Industrial Research (CSIR) FYP, BSC0123. Dr. Puja Paul has been a Woman post-doctoral fellow under UGC scheme at Jadavpur University. All the colleagues at the Biophysical Chemistry Laboratory, CSIR-IICB, Physical Chemistry Laboratory, Jadavpur University and Department of Life Science and Bio-technology, Jadavpur University, rendered immense help in performing the work described here. REFERENCES 1) Matsumura, Y.; Maeda, H. A new concept for macromolecular therapeutics in cancer chemotherapy:mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res.1986, 46 (12), 6387-6392. 2) Habash, R.W.Y.; Bansal, R.; Krewski, D.; Alhafid, H.T. Thermal therapy, Part 2: hyperthermia

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TABLE OF CONTENTS (TOC) GRAPHIC

Thionine Conjugated Gold Nanoparticles Triggers Apoptotic Activity Towards HepG2 Cancer Cell Line ab,

a

Puja Paul, Sabyasachi Chatterjee, Arindam Pramanik,c Parimal Karmakar,c Subhash Chandra b a, Bhattacharya and Gopinatha Suresh Kumar * a

Organic and Medicinal Chemistry Division, CSIR-Indian Institute of Chemical Biology, Kolkata 700 032, India b

Department of Chemistry, Jadavpur University, Kolkata 700 032, India c

Department of Life Science and Bio-technology, Jadavpur University, Kolkata 700 032, India



84x31mm (300 x 300 DPI)

ACS Paragon Plus Environment

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Thionine Conjugated Gold Nanoparticles Trigger Apoptotic Activity

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