Synthesis and characterisation of morin reduced gold nanoparticles and its cytotoxicity in MCF-7 cells

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Contents lists available at ScienceDirect

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Synthesis and characterisation of morin reduced gold nanoparticles and its cytotoxicity in MCF-7 cells

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Biochemistry Laboratory, Central Leather Research Institute, Adyar, Chennai, India Bio-Physics Laboratory, Central Leather Research Institute, Adyar, Chennai, India

a r t i c l e

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K. Sindhu a, S.R. Bhuvanasree a, A. Rajaram b, Rama Rajaram a,⇑

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Article history: Received 20 June 2014 Received in revised form 9 September 2014 Accepted 21 September 2014 Available online xxxx

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Keywords: Morin Gold nanoparticles Biocompatibility Nanoparticle internalization Breast cancer Apoptosis Secondary necrosis

a b s t r a c t There is significant interest in investigating the therapeutic potential of phytochemical reduced and bound gold nanoparticles (AuNPs) as it bridges the gap between nanotechnology and therapy. In the present study, AuNPs prepared using the flavonoid morin (mAuNPs) are characterised and have been studied for their anti-cancer effects. The –OH groups of morin reduce Au3+ and stabilize Au0 to form spherical and crystalline mAuNPs. These mAuNPs are biocompatible towards normal human blood cells and breast epithelial cells. Through TEM analysis, we report that they are readily taken up by breast cancer cells (MCF-7) to induce cell death. Apoptosis has also been assessed by other morphological observations and cell viability studies. Flow cytometric studies reveal that the cells undergo a transient phase of apoptosis progressing towards secondary necrosis as the dose and time of mAuNPs treatment increases. The ability of mAuNPs to induce cell death in MCF-7 cells indicates its potential as an anti-cancer agent. Ó 2014 Elsevier Ireland Ltd. All rights reserved.

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1. Introduction

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AuNPs are gaining much attention due to their ease in preparation and wide-ranging applications. They are being used as catalysts, diagnostic tools, in materials design, in therapeutics and more recently in nanomedicine [1–3]. Even though AuNPs synthesized using chemical methods cause cell death in many cancer cell lines [4–6], their biocompatibility towards normal human cells is of concern. Toxicity is induced by citrate reduced AuNPs

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Abbreviations: AO, acridine orange; AuNPs, gold nanoparticles; ctAuNPs, citrate reduced AuNPs; DDSA, dodecenyl succinic acid; DMEM, Dulbecco’s modified Eagles medium; DLS, dynamic light scattering; DMSO, dimethylsulphoxide; EtBr, ethidium bromide; FBS, fetal bovine serum; FFT, fast Fourier transform; FITC, fluorescein isothiocyanate; FTIR, Fourier transform infrared spectroscopy; HR-TEM, high resolution transmission electron microscopy; HBL-100, human breast lactating donor 100; HP, human plasma; HSA, human serum albumin; ICP-OES, inductively coupled plasmon-optical emission spectroscopy; IRB, Institutional Review Board; mAuNPs, morin conjugated AuNPs; MCF-7, Michigan Cancer Foundation-7 (human breast cancer cells); MR, molar ratio; NMA, nadic methyl anhydride; PBMCs, peripheral blood mononuclear cells; PBS, phosphate buffered saline; PCM, phase contrast microscopy; PI, propidium iodide; RBC, red blood corpuscles; RNase A, ribonuclease A; RPMI, Rosewell Park Memorial Institute medium; RT, room temperature; SAED, selected area electron diffraction; TGA, Thermo gravimetric analysis; XRD, X-ray powder diffraction. ⇑ Corresponding author at: Biochemistry Laboratory, Central Leather Research Institute, Adyar, Chennai 600 020, India. Tel.: +91 (44) 24437177; fax: +91 (44) 24911589. E-mail address: [email protected] (R. Rajaram).

in lymphocytes and in epithelial and endothelial cell lines [7,8]. Hence development of biocompatible AuNPs synthesised using phytochemicals with appropriate reactive groups which are toxic to cancer cells hold great potential in nanomedicine [9,10]. A possible application of these AuNPs is in the treatment of breast cancer. The incidence of breast cancer has increased in Asian countries in the past few decades with over 90,000 cases diagnosed annually in India [11,12], a large portion of which being hormone dependent [13]. Hormonal or chemotherapy involves the administration of both steroidal and non-steroidal drugs which act as anti-estrogens suppressing proliferation [14–16]. Flavonoids, such as quercetin, kaempferol, genistein, apigenin, epigallocatechin gallate (EGCG), etc. show anti-mutagenic, anti-estrogenic and anti-carcinogenic properties by acting on cell signalling pathways which inhibit cell proliferation and induce apoptosis [17–19]. Studies show a 13% decrease in breast cancer risk when 0.5 mg of flavones per day are included in the diet [20]. Their relative nontoxic nature towards human cells makes them effective in cancer therapy [21]. Morin (3, 20 , 40 , 5, 7-pentahydroxyflavone) (Fig. 1c) is a common flavonol present in plants of the family Moraceae. The structure is characterized by two aromatic rings (A and B) linked by an oxygen containing heterocycle (ring C) with the five –OH groups (3, 20 , 40 , 5 and 7) positions. It is a light yellow coloured natural dye with a broad spectrum of biological activities such as being anti-oxidant, free radical scavenging, anti-inflammatory and anti-carcinogenic

http://dx.doi.org/10.1016/j.cbi.2014.09.025 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.

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[22,23]. The anti-inflammatory activity is due to inhibition of 20 S proteasome and blocking of NF-jB activation which leads to decreased cytokine production. It also causes apoptosis by caspase-activated mitochondrial dependent or independent pathway resulting in cell cycle arrest and inhibition of proliferation [24–26]. Drugs such as tamoxifen, paclitaxel, doxorubicin etc., though approved by FDA and currently in use, exhibit disadvantages like intrinsic or acquired resistance by the cells [27,28]. Since, morin not only inhibits the multi drug resistant proteins but also increases the accumulation of drugs into cells [29–31], its potential as an anti-carcinogenic agent is being investigated. Utilization of morin, however, is difficult due to its low bioavailability. Conjugation with cyclodextrin, phospholipids and nanoemulsions has been tried for overcoming the problem [32–34]. Another possible method to increase bioavailability may be by conjugating it to AuNPs. Very recently, conjugation of morin to citrate reduced AuNPs has been shown to increase its interaction efficiency with bio-macromolecules [35]. A green approach for synthesising AuNPs using morin alone without employing toxic chemicals will be advantageous in biomedicine and has been attempted in this study. The mAuNPs formed are found to be spherical and stable. Its biocompatibility towards normal human blood cells and HBL-100 cells is also established. We show further that cell death can be brought about in MCF-7 cancer cells by the use of these mAuNPs.

heparin, sodium bicarbonate, penicillin, streptomycin, gentamycin, amphotericin B, dextran, DMSO, DMEM, trypan blue, resazurin (Alamar blue), EtBr, AO, PI and RNAse A were purchased from Sigma–Aldrich, USA. Annexin V apoptosis detection kit was bought from BD Pharmingen, San Diego; CA. FBS was purchased from GIBCO, USA, potassium carbonate (K2CO3) from Merck, India and tri-sodium citrate from Qualigens, India. All the materials for TEM were bought from EM Sciences, USA. MCF-7 cells were a gift from Dr. D. Karuranagaran, Indian Institute of Technology (IIT), Chennai. HBL-100 cells were purchased from King Institute for Preventive Medicine and Research, Chennai.

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2.2. Synthesis of morin conjugated AuNPs (mAuNPs)

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2. Materials and methods

Morin was solubilized in milli Q water by increasing its pH to 8 and above using K2CO3. To this solution, HAuCl4 was added drop wise with simultaneous shaking. The concentration of morin ranged from 62.5 lmol/L to 500 lmol/L and that of HAuCl4 from 62.5 lmol/L to 2 mmol/L. In order to optimize the formation, synthesis was carried out at varying pHs of morin (8–11) and at different MRs of morin to HAuCl4 (1:1 to 1:8). The formed mAuNPs were isolated by centrifuging the solution at 12,000 rpm for 17 min (Sigma 3–30 K). The pellet obtained was washed twice in milli Q water at the same speed to remove any unreacted substances. For comparison, in cell culture experiments, ctAuNPs were also prepared following the inverse method of Ojea-Jimenez et al. using sodium citrate and HAuCl4 in the ratio of 1:6.8 [36].

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2.1. Materials

2.3. Physico-chemical characterisation

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Gold (III) chloride hydrate (HAuCl4) 99.99% pure, Morin hydrate, PBS, RPMI 1640 medium, HBSS, Ficoll-Hypaque (Histopaque 1077),

UV–Visible and fluorescence measurements for all experiments were made in a multimode plate reader (Tecan Infinite M200).

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Fig. 1. (a) UV–Visible spectra of morin and mAuNPs. The solid line shows the spectra of morin (62.5 lmol/L), at pH 4.8, dashed line shows morin at alkaline pH and dotted line shows mAuNP spectra with SPR peak at 522 nm. (Concentration of HAuCl4 is 0.375 mmol/L). (b) FTIR spectra of morin and mAuNPs. (c) Schematic representation of the reduction and capping of Au3+ by morin.

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Infrared spectra of the mAuNPs were measured in Nicolet Impact spectrometer. Thermal analysis of the mAuNPs was done in TGA Q50 V20.13 instrument in the temperature range of 200–800 °C. The concentration of Au was determined by ICP-OES analysis in a Perkin-Elmer Optima 5300 DV instrument. The hydrodynamic size and zeta potential of mAuNPs were measured by Zetasizer Nano ZS, Malvern. For HR-TEM measurements, mAuNPs solution dropped on a carbon coated copper grid was allowed to dry and observed through Tecnai electron microscope at 300 kV. XRD measurements of the powdered mAuNPs were carried out in a Seifert ISO Bebyeflex 2002.

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2.4. Cell culture

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Blood was drawn from healthy volunteers after obtaining informed consent following the procedure approved by IRB. Heparinized tubes (50 lg/mL) were used to collect the blood which was then carefully layered on to Ficoll-Hypaque solution and centrifuged at 1800 rpm for 30 min. This gave an upper layer of plasma followed by a thin layer of buffy coat and a pellet consisting of RBCs and granulocytes. To the pellet obtained, equal volume of 6% dextran in PBS was added and centrifuged at 1800 rpm for 20 min [37]. The pellet containing RBCs was washed in PBS, diluted 10 times in PBS and used for hemocompatibility studies. The buffy coat containing PBMCs was centrifuged and washed twice with HBSS. To the PBMCs, RPMI 1640 medium with 10% HP (autologous), sodium bicarbonate (2 mg/mL) and antibiotics [penicillin (100 IU/mL), streptomycin (100 lg/mL), gentamicin (30 lg/mL), amphotericin B (2.5 lg/mL)] were added. PBMCs were treated with 19.7, 39.4 and 78.8 lg/mL of mAuNPs for different time intervals. A parallel set of experiments was also conducted using PBMCs cultured in RPMI 1640 medium without HP. The cells were maintained at 37 °C with 5% CO2 in a CO2 incubator (Binder, Germany). For culturing MCF-7 and HBL-100 cells, DMEM medium containing 10% FBS and antibiotics was used. Upon reaching 80% confluence, the cells were trypsinized and seeded for the experiments. The cells were allowed to attach overnight and then treated with mAuNPs while untreated cells served as control. The cells were maintained at 37 °C with 5% CO2 in a CO2 incubator (Binder, Germany).

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2.5. Hemocompatibility assay

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Hemocompatibility test was conducted according to the protocol of Zhao et al. [38]. Briefly, to 100 lL of diluted RBCs, 400 lL of mAuNPs (19.7, 38.4 and 78.8 lg/mL) in PBS were added. 400 lL of PBS and milli Q water served as the negative and positive controls respectively. The solutions were vortexed and incubated for 4 h at RT. Then, the solutions were vortexed and centrifuged at 1800 rpm for 10 min. The supernatant was collected and the absorbance of hemoglobin measured at 577 nm. The percentage hemolysis was calculated by the formula [(sample absorbance  negative control absorbance)/(positive control absorbance  negative control absorbance)]  100.

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2.6. Microscopic analysis

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For observing morphological changes in MCF-7 cells, about 50  103 cells were seeded on to 12 well plates and treated. After the treatment period, the cells were washed twice with PBS and then observed through Phase Contrast Microscope (Nikon Eclipse TS100) for any morphological changes at 200 magnification and images were acquired using NIS Elements software. PBMCs and MCF-7 cells were also double stained with 0.25 lg/10 lL each of EtBr and AO. Cells that internalize AO were considered to be viable while EtBr enters cells with compromised membrane integrity. The

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stained cells were observed using confocal laser scanning microscope (Nikon Eclipse E600, Japan) (Ex 450–490 nm) and images acquired using EZ-C1 software.

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2.7. Uptake studies

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For TEM analysis, 1  106 cells were plated into a T25 flask. After treatment with19.7 lg/mL of mAuNPs for 24 h, the cells were washed thrice with PBS. The cells were then trypsinised and fixed with 2.5% of glutaraldehyde prepared in 0.1 M sodium cacodylate buffer at a pH of 7.4 for 4 h. The pellet was then centrifuged and washed with the same buffer thrice for 10 min each. They were then post fixed in 0.1% osmium tetroxide prepared in the same buffer for 2 h at 8 °C and further washed as above. The cells were pelleted through centrifugation after each step and further dehydrated through a graded series of acetone 30%, 50%, 70%, 80%, and 90% for 10 min. It was then treated with 100% acetone twice for 10 min, each time followed by propylene oxide treatment for 10 min. The pellet was then infiltrated with resin mixture consisting of Epon 812 resin, DDSA and NMA starting with 25%, 50% and 75% for 2 h and 100% overnight. It was then embedded in the same resin mixture with catalyst (DMP 30) in ‘‘easymoulds’’ at 60 °C for 48 h. Ultra thin sections from the resin block were cut and stained with saturated solutions of uranyl acetate and lead citrate. After air drying, the sections were visualized in Jeol JEM 1400 transmission electron microscope at 80 kV and micrographs acquired using Olympus Keenview CCD camera. For ICP analysis, 2  105 cells were seeded onto a 12 well plate and grown for 24 h in DMEM medium containing 10% FBS. The cells were treated with 19.7 lg/mL of mAuNPs for 24 h. They were then washed twice with PBS to remove the excess mAuNPs, trypsinised and digested with aqua-regia. The solution was filtered through a 0.22 lm filter and Au content was measured in Perkin Elmer Optima 5300 DV instrument.

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2.8. Viability assay

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Alamar blue assay was conducted to measure the viability of PBMCs, HBL-100 and MCF-7 cells. For PBMCs, 2  105 cells and for HBL-100 and MCF-7 cells about 20  103 cells were seeded and treated with mAuNPs at different concentrations for 24, 48 and 72 h. Before 6 h of the culmination of treatment period, 10 lL of Alamar blue dye was added and fluorescence measured (Ex 530 nm and Em 590 nm). The experiment was also performed after treating MCF-7 cells with equivalent concentrations of ctAuNPs and 1.5, 3 and 6 lg/mL of morin. The concentration of morin was calculated based on TGA results and employed for cell culture experiments.

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2.9. Flow cytometric analysis

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2  105 cells were grown in 6 well plates and treated for 24 and 48 h. For annexin V FITC/PI analysis, treated cells were incubated with 2 lL each of annexin V FITC and PI stain for 20 min in the dark. For cell cycle analysis, cells were fixed in 70% ice cold ethanol and left overnight at 4 °C. After washing off the ethanol with PBS, 50 lg each of PI and RNAse were added and incubated for 30 min in the dark. The fluorescence of FITC (FL-1 channel (530 nm)) and PI (FL-2 channel (585 nm)) of the stained cells was measured using flow cytometer (FACS Calibur, Beckton Dickinson, Inc., USA). 10,000 events for annexin V FITC/PI analysis and 20,000 events for cell cycle analysis were collected. The data acquired was analysed by CellQuest Pro software.

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2.10. Statistical analysis

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GraphPad Prism 5 software was used for statistical analysis. The results are given as mean ± SD of three independent experiments. For viability studies, means of data were compared with respect to control using analysis of variance (ANOVA) and Bonferroni post tests conducted. ⁄p-values 6 0.05 were considered as statistically significant.

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3. Results

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3.1. Synthesis of mAuNPs

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In this study, morin is used as the reductant to prepare mAuNPs in a single step process. The formation of mAuNPs can be visually monitored as the pale yellow coloured solution turns burgundy red with an SPR centred at 522 nm when HAuCl4 is added drop wise to morin solution. The mAuNPs formed at pH of morin within a range of 9.5–11 and MR for morin to HAuCl4 at 1:6 is stable in solution for a period of 3 months and represents optimum condition for the formation of mAuNPs (Fig. S1a). Hence, for further characterisation of mAuNPs, these parameters were employed. The spectral kinetics demonstrates a broad SPR spectrum initially, which narrows gradually with increase in intensity reaching a maximum within 2 h. (Fig. S1b). The narrowing of SPR spectra with time has been correlated to the temporal evolution of smaller sized and uniform AuNPs from larger sized particles as observed during the formation of curcumin conjugated AuNPs [39,40].

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3.2. Characterisation

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The mAuNPs have been characterized by UV–Visible and FTIR spectroscopic methods to analyse the chemical bonds involved in the formation. The UV–Visible absorption spectrum for morin in DMSO shows two absorption maxima (Fig. 1a, solid line). The peak at 344 nm is due to the absorption of the cinnamoyl part (B + C), while that at 248 nm is assigned to the benzoyl part (A + C). On adding K2CO3 and increasing the pH of morin above 9, a red shift in both the peaks with increase in intensity is observed along with the appearance of a new peak at 320 nm which might be due to deprotonation at higher pH (Fig 1a, dashed line). H atom abstraction at 3 –OH, 40 –OH and 20 –OH of the cinnamoyl ring and 70 –OH of the benzoyl ring are effected [22,41]. Similar red shift and decrease in intensity of the absorption spectra of morin is also observed when HSA binds and interacts with –OH groups of morin [22]. When reacted with HAuCl4, both the peaks disappear and the appearance of a new peak at 522 nm due to the plasmon resonance of electrons indicates the formation of mAuNPs (Fig. 1a, dotted line). The interaction of morin with Au is further confirmed by FTIR analysis (Fig. 1b). The bands at 1379 cm1, 1258 cm1, 1174 cm1 and 1104 cm1 in the spectra of morin are due to the vibrations of C-OH bonds. The bathochromic shift of these bands in mAuNPs indicates the participation of the OH groups in forming Au0. The characteristic stretching mode vibrations of the C@O group at 1662 cm1 also show a bathochromic shift to 1639 cm1 reflecting the involvement of the carbonyl group in the reaction. Such a shift is observed during complexation of morin with metals, indicating the interaction of these bonds with Au3+ [42,43]. The bands at 1508 cm1 and 1459 cm1 attributed to the stretching C@C vibrations of the aromatic ring and the ring vibrations suggest the presence of morin in mAuNPs. A schematic representation of the chemical groups of morin involved in the reduction of HAuCl4 to form mAuNPs, as deduced from the above results, is given in Fig. 1c.

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In order to further confirm and quantitatively estimate the presence of morin, TGA has been performed. The weight loss at a range of 100 °C to 200 °C in both morin and mAuNPs thermograms are due to desorption of water molecules (Fig. S2). The weight loss between 297–374 °C and 431–523 °C is characteristic for the decomposition of pure chemical morin [44]. The thermogram of mAuNPs shows the onset of weight loss from 200 °C with an inflexion point at 674 °C. The increase in temperature of the inflexion point indicates the strong interaction of morin on AuNPs, indicating its coating on the surface. The weight loss in mAuNPs after decomposition is 7%, indicating the amount of morin present in mAuNPs. DLS results obtained show the hydrodynamic diameter of mAuNPs to be 48 nm (Fig. S3). HRTEM results indicate that all the particles formed are spherical in shape (Fig. 2). A majority of the particles are around 20 ± 6 nm in size as indicated by the size distribution histogram (Fig. 2a inset). The micrograph of a single nanoparticle shows parallel lattice fringes. The corresponding FFT suggests the distance between these lattice fringes to be 0.22 nm and 0.20 nm which are the (1 1 1) and (2 0 0) lattice planes of Au respectively (Fig. 2b). These results along with the SAED pattern (Fig. 2c) indicate the crystalline nature of mAuNPs. The results obtained from XRD pattern show three peaks characteristic to AuNPs positioned at 2h values of 38.13°, 44.28° and 64.35°, which are due to the (1 1 1), (2 0 0) and (2 2 0) lattice planes of gold respectively (Fig. 2d) confirming the crystalline nature of Au core in the mAuNPs. A zeta potential value of 20.8 mV obtained for mAuNPs shows that they are stable. The absence of any change in the spectral properties of the mAuNPs when suspended in different buffers, nutrient media and over a wide range of pHs, indicates its stability in cell culture conditions (Fig. S4).

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3.3. Biocompatibility in normal human blood cells

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The blood compatibility of mAuNPs has been evaluated using human RBCs (Table 1). The results obtained show that the percentage of hemolysis by mAuNPs at a concentration of 78.8 lg/mL is 4.65% which is lesser than the acceptable level of 5%. The nontoxicity towards PBMCs, as assessed by Alamar blue assay shows a viability of 90% on treatment with 78.8 lg/mL at 72 h. Cells grown in medium without HP shows a much lesser viability for both control and treated cells (Fig. S5). To further confirm the non-toxicity of mAuNPs to PBMCs, the percentage of viable cells after treatment with mAuNPs has been assessed by EtBr/AO staining. The percentage of live cells in control is 96 ± 1.4 and treated is 95.5 ± 6.3 indicating the non-toxic nature of mAuNPs (Fig. 3).

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3.4. Uptake studies

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Upon observation using TEM, mAuNPs are found to get readily internalised into the cells (Fig. 4). The mode of internalization is found to be through endocytosis as indicated by the formation of pseudopodia around the mAuNPs (Fig. 4b). The internalised mAuNPs reside in endo-lysosomal structures scattered throughout the cytoplasm (Fig. 4c). The presence of mAuNPs could not be detected in other cellular organelles or nucleus. Significantly, no aggregation of the mAuNPs was noticed. Quantitation of mAuNPs through ICP-OES analysis indicates that approximately 0.317 lg of mAuNPs are taken up by the cells and this corresponds to a 16% uptake by the cells.

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3.5. Viability studies

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After treatment of MCF-7 cells with varied concentrations of mAuNPs for 24 and 48 h, they have been observed in PCM to find out whether any distinctive morphological changes occur. The

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Fig. 2. (a) Spherical mAuNPs with average size distribution histogram in the inset. (b) A single mAuNP depicting lattice fringes and corresponding FFT in the inset. (c) SAED pattern depicting the lattice planes of Au in mAuNPs. (d) XRD spectrum illustrating crystalline nature of the mAuNPs.

Table 1 Hemocompatibility of mAuNPs.

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Concentration of mAuNPs (lg/mL)

% Hemolysis (mean ± sd)

19.7 39.4 78.8

1.6 ± 0.28 3.8 ± 0.14 4.65 ± 0.07

MCF-7 control cells are elongated in shape with a round nucleus and many spherical nucleoli. On treatment with 19.7 lg/mL of mAuNPs, shrinkage of cells and detachment from the neighbouring cells are observed. On doubling the concentration, the cells get rounded off, rupture and release their contents (inset, Fig. 5, middle panel). Other features like membrane dissolution, condensation of chromatin leading to pyknotic nuclei formation (inset, Fig. 5, last panel) are visualized when the concentration is increased to 78.8 lg/mL. The effect of mAuNPs on viability of MCF-7 and HBL-100 cells has also been investigated through Alamar blue assay after treatment for 24, 48 and 72 h. It is noticed that in MCF-7 cells, an increase in concentration of mAuNPs decreases the percentage of viable cells (Fig. 6a). The viability decreases to 61 ± 2.1% and 48 ± 5.6% at 24 h on treatment with 19.7 and 39.4 lg/mL respectively. The highest concentration of mAuNPs induces significant decrease in cell viability with only 28 ± 0.7% of the cells being viable after 72 h. On the other hand, with a concentration of 19.7 lg/mL, the viability of HBL-100 cells is 89 ± 19% after 72 h

(Fig. 6b). Even though the viability of HBL-100 cells decreases to 67 ± 15% after treatment with the highest concentration for 72 h, the decreases are much less compared to that for MCF-7 cells. In the presence of free morin (6 lg/mL) and ctAuNPs (80 lg/mL), the viability values are 90 ± 1.6% and 75 ± 1.4% respectively (Fig. 6c and d) indicating mAuNPs are more effective in inducing toxicity in MCF-7 cells when compared to either free morin or ctAuNPs.

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3.6. Mode of cell death

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For this experiment, the cells are double stained with EtBr/AO and observed through fluorescence microscopy (Fig. 7). Cells in the control group show green fluorescence with intact nuclei and no membrane damage, indicating they are viable. Cells treated for 24 h have diffused or orange coloured nuclei and are present as clusters. However, after 48 h, higher percentage of red or orange red coloured cells are seen, depicting membrane disruption leading to significant uptake of EtBr and binding with fragmented DNA. The percentage of EtBr stained cells increases in a concentration dependent manner and at 48 h, maximum red fluorescence is observed when cells are treated with 78.8 lg/mL of mAuNPs. Cells treated with different concentrations of mAuNPs for 24 and 48 h, stained with annexin-V FITC/PI and analysed by flow cytometry are shown in Fig. 8. Dots in the lower left quadrant represent live cells, while dots in the lower and upper right are stained cells denoting early and late apoptotic cell population

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Fig. 3. EtBr/AO pictures show both (a) control and (b) 78.8 lg/mL of mAuNPs treated PBMCs fluorescing green. (c) depicts the quantitated graph of the images with a mean ± SD of 96 ± 1.4 for control and 95.5 ± 6.3 for treated cells. (Three independent experiments were conducted and a total of 100 cells were counted in each experiment).

Fig. 4. Uptake of mAuNPs. (a) TEM images showing internalization of mAuNPs through endocytosis ( ) and within lysosomes ( ). Magnified image shows (b) the endocytosis and (c) the presence of dispersed mAuNPs in lysosomes.

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Fig. 5. Morphological assessment. Control MCF-7 cells are illustrated in the first panel and the changes in morphology on treatment with different concentrations of mAuNPs at 24 and 48 h are as indicated Nucleoli, rounded off cells, dissolution of membrane, pyknotic nucleus.

Fig. 6. Viability assay. Alamar blue assay indicates the differential toxicity of mAuNPs towards (a) MCF-7 and (b) HBL-100 cells. The decrease in percentage viability caused by (c) morin alone (mor) and (d) ctAuNPs are also illustrated. The experiments are performed three times in triplicates. The differences are considered to be statistically significant when compared to control cells, denoted by ⁄⁄⁄ for p 6 0.001, ⁄⁄ for p 6 0.01 and ⁄ for p 6 0.05.

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Fig. 7. Fluorescence microscopy analysis. EtBr/AO staining indicates a concentration and time dependent increase in intake of EtBr by cells on treatment with mAuNPs, while control cells fluoresce green.

Fig. 8. AnnexinV FITC/PI staining of MCF-7 cells treated with mAuNPs (a) 24 h and (b) 48 h, the percentage of cells stained with annexin V/FITC increases on treatment with 39.4 lg/mL and nearly 50% of the population is stained with PI at 78.8 lg/mL. (c) Representative dot plots showing mAuNP induced cell death at 48 h treatment.

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respectively. Cells in the upper left quadrant are stained by PI alone, representing cells in necrotic stage. The percentage of live cells is found to be 78 ± 13% and 75 ± 5% on treatment with 34.4 lg/mL and 78.8 lg/mL respectively for 24 h. This decreases

to 67.5 ± 12% and 46.5 ± 23% for the same concentrations after 48 h. Corresponding increase in early and late apoptotic fraction of cells by 12% is also noticed indicating that the cells undergo apoptosis at 24 h. After 48 h of treatment, even though the early

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Fig. 9. Cell cycle analysis. (a) MCF-7 cells exposed to different concentrations of mAuNPs show an increase in sub G1 population at 24 h. (b) Exposure for up to 48 h increases the population of cells in G2/M phase with a concomitant decrease in G0/G1 population. (c) Representative histogram showing G2/M arrest at 48 h.

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apoptotic population remained unchanged the percentage of late apoptotic and necrotic population increases to 35.9 ± 18% and 13.9 ± 7% respectively. Through cell cycle analysis, a serial decrease in G0/G1 phase and a corresponding increase in cells in sub G1 fraction are noticed after 24 h treatment with mAuNPs (Fig. 9). An increase of sub G1 population from 2.99 ± 0.5% in control to 15.04 ± 1.4% (39.4 lg/mL) and 30.95 ± 14% (78.8 lg/mL) indicates a concentration dependent effect of mAuNPs. The percentage of cells in S and G2/M phase are similar to that of control. However, after 48 h of treatment with 78.8 lg/mL of mAuNPs, about 63.1 ± 8.4% of cells are in G2/M phase with a concomitant decrease in G0/G1 (23 ± 9.6%) and sub G1 (12 ± 1.7%) phase.

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4. Discussion

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In this study, stable AuNPs prepared with morin as the reducing and stabilizing agent are found to be biocompatible towards normal cells but show anti-carcinogenic activity in breast cancer cells. The ability of morin to form anions at a pH of 9.8 and above has been exploited to reduce Au3+ to Au0 and cap the AuNPs [22]. This method of synthesis is different from that reported by Yue et al. where morin is conjugated to citrate of the citrate reduced AuNPs [35]. The compatibility of mAuNPs with human blood cells and breast epithelial cells has been investigated. No significant lysis of RBCs or decrease in viability of PBMCs are observed at mAuNP concentrations (19.7–78.8 lg/mL) indicating their high compatibility towards blood cells. AuNPs coated with phytochemicals such as curcumin and EGCG also show good hemocompatibility [39,45]. mAuNPs exhibit low toxicity towards PBMCs and HBL-100 cells while being highly toxic to MCF-7 cells. This is in accordance with other reports suggesting biomolecules reduced AuNPs to be

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non-toxic towards normal cells as a consequence of reductant presence on the surface [46,47]. This is in contrast to the citrate reduced AuNPs, which have been reported to cause cyto- and genotoxicity in PBMCs and other normal human cells [7,48]. The ability of morin to protect RBCs from lysis by anti-tumour drugs like doxorubicin or mitomycin C when co-administered indicates the protective role of morin [49]. The biocompatibility of mAuNPs towards normal human blood cells and breast epithelial cells support the use of these nanoparticles as possible therapeutics against breast cancer. The uptake of AuNPs and its effect on cells are dependent on size, shape and uniformity of the particles. Through TEM and ICP-OES analyses, it is observed that a considerable amount of mAuNPs are taken up by MCF-7 cells. The internalization is observed as endocytosis and mAuNPs are found to be localized inside the lysosomes. Such endocytotic uptake and lysosomal localization have been observed when MCF-7 cells are treated with glucose capped AuNPs [50]. The entrapped mAuNPs are assumed to activate lysosomal enzymes affecting the viability of cells as evaluated through cell viability assays. The absence of any aggregation of mAuNPs inside the cells reflects a high degree of stability. Citrate reduced AuNPs are also reported to undergo such an endocytotic fate in macrophage cells [51]. Cho et al. have investigated the different parameters affecting the uptake of nanoparticles into cells [52]. Uniformly dispersed, spherical citrate reduced gold nanoparticles of size < 40 nm are found to be easily taken up by cells due to high diffusion rates [53,54]. The mAuNPs made in this study are spherical of 20 nm size and satisfy the entire criteria for uptake into cells. Some studies note that AuNPs of size around 20 nm might get widely distributed in vital organs and are found to cross the brain epithelial cells in vitro [55,56] but other in vivo studies report that 21 nm sized AuNPs are safer to vital organs [57]. The results by Zhang et al. states that PEG coated AuNPs of size 10 and 60 nm are toxic; while 5 and 30 nm sized

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particles show lesser toxicity in vivo indicating that toxicity cannot be even attributed to a particular size range of the particles [58]. Based on the fact that morin is non-toxic to normal cells, it can be that the side effects caused, may be limited [21,23,49]. By a quantitative analysis, we find that mAuNPs at a concentration of 78.8 lg/mL decreases the viability percentage to 33 at 24 h. Selim et al. have reported that 70% of MCF-7 cells are viable in the presence of similar concentration and treatment periods (75 lg/mL of AuNPs 24 h) [6]. Thus, the results are suggestive of the greater potential of the mAuNPs to kill cancer cells. MCF-7 cells acted upon by mAuNPs, show apoptotic features at 24 h (sub G1 population and annexin V/FITC positive cells) through flow cytometry. This transits to necrosis (G2/M arrest and PI uptake) at 48 h. Pan et al. have also detected a transient population of apoptotic cells followed by a steady increase in secondary necrotic cells on treatment of HeLa cells with AuNPs synthesised using triphenylphosphinemonosulfonate [59]. Treatment with steroids such as exmestane also shows G2/M arrest attributed to enhanced apoptosis and cytotoxicity [13]. Studies show that cells cultured in vitro reach an advanced apoptotic stage, followed by a transition towards necrotic stage called secondary necrosis in the nonavailability of scavengers [60,61]. Growth inhibition involves extensive damage to the DNA, which interrupts the cell cycle progression. The increase in population of cells in G2/M region also signifies the arrest of cells, indicative of massive damage to the DNA. Similar changes have also been observed on treatment of A549 cells with chitosan reduced AuNPs and radio sensitisation of ovarian cancer cells treated with thio-glucose capped AuNPs [5,62]. The values from our viability assays of free morin with MCF-7 cells are also supported by other reports where 15 lg/mL of morin produces nearly 30% decrease in cell survival [63]. Morin is also reported to induce G2/M arrest of HL- 60 cells and squamous cell carcinoma at concentrations around 30–120 lg/mL [26,64]. Considering all results, we believe that the morin moiety present in the mAuNPs, in synergistic action with the AuNP, is responsible for its anti-carcinogenic activity towards MCF-7 cells.

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5. Conclusion

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Homogenous, spherical and stable mAuNPs have been prepared using the flavonoid morin as the reducing and stabilizing agent. The mAuNPs are found to be biocompatible towards normal human blood cells and HBL-100 cells, while showing potential to cause cell death in MCF-7 cells. The mAuNPs are endocytosed by the cells and get sequestered in lysosomes. The mechanism of cell death in MCF-7 cells is found to begin by events leading to apoptosis which then progresses and culminates in secondary necrosis. The ability of mAuNPs to cause G2/M arrest at higher concentrations and longer time points suggests the extent of toxicity caused by mAuNPs. This is particularly important as the arrest in G2/M is Q3 found to be an important target to check proliferation of cancer cells. The synergistic action of morin and AuNP in causing MCF-7 cell death is significant. Conflict of Interest

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The authors declare that there are no conflicts of interest.

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Acknowledgement

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The authors thank CSIR, New Delhi, India for financial support through the XII five year plan project (ADD-CSC0302). One of the author K. Sindhu thanks CSIR, India for the Senior Research Q5 Fellowship. The authors also acknowledge National Institute of Q4

Interdisciplinary Science and Technology, Trivandrum for HRTEM analysis, IIT Chennai for ICP analysis and Anna University for XRD analysis. We are very thankful to Dr. Pushpa Vishwanathan, Cancer Institute, Chennai, for her support in TEM analysis of the biological samples.

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Appendix A. Supplementary data

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cbi.2014.09.025.

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