Materials Science and Engineering C 53 (2015) 298–309

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Green synthesis, characterization of gold and silver nanoparticles and their potential application for cancer therapeutics Sujata Patra a,1, Sudip Mukherjee a,1,2, Ayan Kumar Barui a,2, Anirban Ganguly a, Bojja Sreedhar b, Chitta Ranjan Patra a,⁎,2 a b

Biomaterials Group, CSIR-Indian Institute of Chemical Technology, Uppal Road, Tarnaka, Hyderabad 500007, Telangana State, India Inorganic and Physical Chemistry Division, CSIR-Indian Institute of Chemical Technology, Uppal Road, Tarnaka, Hyderabad 500007, Telangana State, India

a r t i c l e

i n f o

Available online 1 May 2015 Keywords: Green synthesis Gold and silver nanoparticles Butea monosperma Biocompatible Drug delivery systems Cancer therapy

a b s t r a c t In the present article, we demonstrate the delivery of anti-cancer drug to the cancer cells using biosynthesized gold and silver nanoparticles (b-AuNP & b-AgNP). The nanoparticles synthesized by using Butea monosperma (BM) leaf extract are thoroughly characterized by various analytical techniques. Both b-AuNP and b-AgNP are stable in biological buffers and biocompatible towards normal endothelial cells (HUVEC, ECV-304) as well as cancer cell lines (B16F10, MCF-7, HNGC2 & A549). Administration of nanoparticle based drug delivery systems (DDSs) using doxorubicin (DOX) [b-Au-500-DOX and b-Ag-750-DOX] shows significant inhibition of cancer cell proliferation (B16F10, MCF-7) compared to pristine drug. Therefore, we strongly believe that biosynthesized nanoparticles will be useful for the development of cancer therapy using nanomedicine approach in near future. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Recently, metal nanoparticles especially gold & silver nanoparticles (AuNP & AgNP) are extensively used as medicinal agents for the theranostic applications of several diseases such as cancer, diabetes, Parkinson's, Alzheimer's, HIV/AIDS, arthritis, hepatitis, cirrhosis, spinal cord injury, tuberculosis and cardiovascular diseases (CVD) due to unusual optoelectronic and physicochemical properties [1–5], ease of synthesis, characterization and surface modification in the nanoscale range [2,4,6–13]. Therefore, synthesis of gold and silver nanomaterials through an efficient, economically cheap and environmentally safe method is a very important area of research in biomedical nanotechnology. In this context, green chemistry approach for the synthesis of nanoparticles is always a better selection due to eco-friendliness [13–17].

Abbreviations: b-AuNP, biosynthesized gold nanoparticles using Butea monosperma (BM); b-AgNP, biosynthesized silver nanoparticles using Butea monosperma (BM); BM, Butea monosperma; DOX, Doxorubicin; b-Au-500, biosynthesized gold nanoparticles using 500 μL of Butea monosperma (BM); b-Ag-750, biosynthesized silver nanoparticles using 750 μL of Butea monosperma (BM); DDSs, Drug delivery systems; b-Au-500-DOX, biosynthesized gold nanoparticle based DDS using DOX; b-Ag-750-DOX, biosynthesized silver nanoparticle based DDS using DOX; HUVEC, Human umbilical vein endothelial cell; ECV304, cells [transformed human umbilical vein endothelial cell]; CVD, Cardiovascular diseases; XRD, X-ray diffraction; TEM, Transmission electron microscope; DLS, Dynamic light scattering; FTIR, Fourier transformed infrared spectroscopy; XPS, X-ray photoelectron spectroscopy. ⁎ Corresponding author at: Biomaterials Group, CSIR-Indian Institute of Chemical Technology, Uppal Road, Tarnaka, Hyderabad 500007, Telangana State, India. E-mail addresses: [email protected], [email protected] (C.R. Patra). 1 These authors contributed equally to this work. 2 Academy of Scientific and Innovative Research (AcSIR), 2 Rafi Marg, New Delhi, India.

http://dx.doi.org/10.1016/j.msec.2015.04.048 0928-4931/© 2015 Elsevier B.V. All rights reserved.

Recently, researchers are concentrating in the design and development of most efficient and eco-friendly green chemistry method for the synthesis of metal nanoparticles [2,5,13,16–20]. Among several methods available in the literature, green synthesis approach for the synthesis of metal nanoparticles has several advantages over conventional methods such as it (i) is very simple, clean & efficient, (ii) is eco-friendly & economically cheap as we use bio-resources (plants, fungi, algae, microorganism) that can help as reducing agent as well as stabilizing agent & capping agent, (iii) needs ambient temperature and pressure, (iv) is a non-toxic method due to very less or non- consumption of hazardous materials on the surface of nanomaterials, (v) does not need any external ligand or capping or stabilizing agent for nanoparticles, and (vi) is a low cost method due to minimum or non-requirement of energy [13,17,20–23]. Moreover, biosynthesized nanoparticles are mostly biocompatible and highly applicable for biomedical applications [13,17,20]. In the present report, we demonstrate the green chemistry approach for the synthesis of gold and silver nanoparticles using ‘Butea monosperma (BM)’ leaf extract where BM leaves act as both reducing as well as stabilizing agent/capping agent. This plant is popular as Ayurvedic herb in India as it shows antibacterial, antifungal, hypoglycemic and anti-inflammatory activities. Here, we show that the bio-synthesized nanoparticles are biocompatible towards both different normal endothelial cells as well as cancer cells. Furthermore, we have designed and developed gold and silver nanoparticle based drug delivery systems (DDSs) [b-Au-500-DOX and b-Ag-750-DOX] using doxorubicin (DOX) as an anti-cancer drug and found that the administrations of these nanoparticle based DDSs to cancer cells exhibit significant inhibition of cancer cell proliferation (B16F10, MCF-7) compared to pristine drug (DOX), observed by several in vitro assays.

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Therefore, we strongly believe that biosynthesized nanoparticle based drug delivery systems (DDSs) will be useful towards the development of an alternative treatment strategy for cancer therapy in near future.

2. Experimental procedures

299

2.5. Biosynthesis of gold and silver nanoparticles (b-AuNP and b-AgNP) The synthesis of b-AuNPs & b-AgNPs was carried out with 10−2 M of HAuCl4 and AgNO3 using different volumes of aqueous BM extract. The detailed experimental conditions are presented in Tables 1 & 2.

2.1. Materials

2.6. Chemical synthesis of PEG coated nanoparticles (c-AuNP & c-AgNP)

Tetrachloroauric acid (HAuCl4), silver nitrate (AgNO3), sodium borohydride (NaBH4), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide), poly ethylene glycol (PEG-6000), RNaseA, propidium iodide (PI), Dulbecco's modified eagle medium (DMEM), Dulbecco's phosphate buffered saline (DPBS), Hank's balanced salt solution (HBSS), fetal bovine serum (FBS), penicillin/streptomycin and doxorubicin were purchased from Sigma-Aldrich Chemicals, St. Louis, MO, USA. All chemicals were used without further purification. The human umbilical vein endothelial cells (HUVECs) were obtained from Lonza, USA. The human breast cancer cells (MCF-7), mouse melanoma cells (B16F10), glioblastoma cells (HNGC-2) and human lung carcinoma cells (A549) were purchased from American Type Culture Collection (Manassas, VA). ECV-304 cells were a kind donation from Chair, Dr. V. Shah, Gastroenterology and Hepatology Department, Mayo Clinic, Rochester, MN, USA. ECV-304 cells [transformed human umbilical vein endothelial cell (ECV-304)] were stably transfected with eNOS-GFP (eNOSGFP ECV-304) [24,25].

In order to compare the biocompatibility of biosynthesized gold and silver nanoparticles (b-AuNP and b-AgNP) with chemically synthesized nanoparticles (c-AuNP and c-AgNP), the chemically synthesized nanoparticles were prepared by the interaction of 200 μL (10−2 M) of HAuCl4 or AgNO3 and 2.8 mL of NaBH4 (0.05 mg/mL). The surface of the chemically synthesized nanoparticles (c-AuNP/c-AgNP) was modified by the dropwise addition of PEG-6000 (15 μL of 1% w/v: 10 mg/mL) to the freshly prepared nanoparticle solution.

2.2. B. monosperma (BM) B. monosperma (Fabaceae), known as ‘Flame of forest’ is widely distributed all over the India, Burma and Ceylon. This plant, known as ‘Palas’ has antibacterial, antifungal, hypoglycemic and antiinflammatory activities [26,27]. It is used as tonic, astringent, aphrodisiac and diuretics. According to pharmacological study, the crude extract or isolated product of Butea has anti-stress & anthelmintic activity, antidiarrheal activity, anti-hyperglycemic & anti-hyperlipidemic activity, wound healing and cytotoxic property [28]. Because of numerous medicinal applications, BM was selected for our study. According to published literature, the methanolic/aqueous extract of B. monosperma flower shows cytotoxic capability as well as anticancer activity [29,30]. However, there is no report of cytotoxicity of aqueous leaf extract. Here, the BM leaf extract helps to synthesize stable and biocompatible b-Au-500 and b-Ag-750 (observed in normal cells), that could be useful for delivery of several drugs for cancer therapeutics. Hence, BM leaf extract has been chosen for this work to form stable, biocompatible gold and silver nanoparticles using an efficient, fast, cost effective and ecofriendly route.

2.7. Cell culture studies B16F10, MCF-7, A549 and HNGC-2 cells were maintained in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin, streptomycin) in a humidified 5% CO2 incubator at 37 °C. After 70% confluency, the cells were plated in 96 well plates for MTT assay. HUVEC and ECV 304 cells were cultured according to our published literatures [31–33]. The cells were incubated with BM extract, b-Au-500, c-AuNP, b-Ag-750 and cAgNP in a dose-dependent manner for several in vitro assays. 2.8. Cell viability test using MTT reagent The cell viability assay was carried out using MTT reagent in the presence of different treatments for 24 h in normal cells (HUVEC and ECV-304) and 48 h in cancer cells (B16F10, A549, MCF-7 and HNGC2). The detailed experimental procedure was discussed in the Supporting information. 2.9. Preparation of the standard curve of doxorubicin (DOX) Different concentrations of DOX (1 to 25 μg/mL) were prepared using the supernatant of b-Au-500 and b-Ag-750, obtained after centrifugation. Absorbance was measured for the corresponding solutions and standard curve was plotted against absorbance (at λmax = 490 nm) vs corresponding concentrations of the DOX.

2.10. Conjugation of doxorubicin with b-AuNP and b-AgNP 2.3. Preparation of stock solutions 10−2 (M) of HAuCl4 and 10−2 (M) of AgNO3 in Millipore water were used as stock solutions for the synthesis, characterization and biological activities of b-AuNP and b-AgNP.

2.4. Preparation of aqueous leaf extract of B. monosperma (BM) 100 g of fresh B. monosperma leaves was thoroughly washed with double distilled water followed by sterile Millipore water in a 500 mL beaker. The leaves were kept in 250 mL of Mili-Q water and the mixture was heated for 3 min under microwave irradiation (Samsung, 800 W). The mixture was allowed to keep overnight under stirring conditions at room temperature. A yellowish water extract of B. monosperma was collected after centrifugation at 7000 rpm at 10 °C for 30 min and it was used as stock solution for all in vitro studies.

5 μg/mL DOX was added to b-Au-500 and b-Ag-750 solution and stirred vigorously for 60 min to make b-Au-500-DOX and b-Ag-750DOX, respectively. Both b-Au-500-DOX and b-Ag-750-DOX were purified by ultracentrifugation at 14,000 rpm at 15 °C for 30 min. From the absorbance of supernatants of b-Au-500-DOX and b-Ag-750-DOX, determined by using UV visible spectroscopy, we calculated the % of attachment of DOX in b-Au-500-DOX and b-Ag-750-DOX. 2.11. Cell cycle analysis (using FACS) Cell cycle analysis was carried out in B16F10 cells according to standard staining methods with propidium iodide (PI) [33]. B16F10 cells were incubated with (i) free DOX (0.25 μM), (ii) b-Au-500-DOX (0.25 μM) and (iii) b-Ag-750-DOX (0.25 μM) for 24 h. Untreated B16F10 cells were considered as control experiment. The detailed study was described in the Supporting information.

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Table 1 Reaction conditions for the biosynthesis of gold nanoparticles (b-AuNP) using Butea monosperma leaf extract as reducing agent. Exp. no

BM-Extract (μL)

HAuCl4 (μL)

Water (mL)

Total volume (mL)

Timea (min)

Zeta potential (charge in mV)

1. 2. 3.

250 500 750

200 200 200

4.55 4.30 4.05

5 5 5

~35 ~5 ~10

−12.8 ± 2.5 −16.4 ± 0.7 −13.6 ± 1.3

a

Time was required to appear the red coloration (formation of b-AuNP).

2.12. Fluorescence microscopy: cellular uptake study In order to detect the cellular uptake of doxorubicin present in b-Au500-DOX and b-Ag-750-DOX, B16F10 cells were incubated with doxorubicin (0.25 μM), b-Au-500-DOX (doxorubicin content 0.25 μM) and b-Ag-750-DOX (doxorubicin content 0.25 μM) for 2 h. The detailed study was discussed in the Supporting information. 2.13. Characterization techniques The detailed characterization techniques were described in the Supporting information. 3. Results and discussions The uses of environmentally sustainable solvent systems and ecofriendly reducing as well as stabilizing & capping agents are the most important criteria of green chemistry approach for the synthesis of metal nanoparticles. Scheme 1 demonstrates the green chemistry approach [34] for the synthesis of b-AuNP and b-AgNP, their characterization and applications for drug delivery in medicine and biology. 3.1. Biosynthesis of b-AuNP and b-AgNP and their optimization The green synthesis of gold and silver nanoparticles (b-AuNP and bAgNP) was carried out using HAuCl4 and AgNO3 respectively in the presence of different volumes of B. monosperma (BM) extract (Tables 1 & 2). In order to obtain the optimum reaction conditions, a series of reactions was carried out for the synthesis of b-AuNP (Table 1) & b-AgNP (Table 2). The final volume of reaction mixture was maintained to 5 mL by adjusting the volume of water and leaf extract whereas the volumes of HAuCl4 and AgNO3 were kept constant (200 μL of 10−2 M). Experiment number # 2 in Table 1 and experiment number #2 in Table 2 indicate the optimum reaction conditions for the synthesis of b-Au-500 and b-Ag-750 within 5 min and 2 h, respectively. The formation of gold and silver nanoparticles was observed by the appearance of red and yellowish colors respectively in the reaction mixture and confirmed by several physico-chemical techniques (described later). The optimized nanoparticles (b-Au-500 and b-Ag-750) were used for detailed characterizations and in vitro experiments. It is needless to mention here that we carried out a series of experiments for the synthesis of gold and silver nanoparticles using different amounts of BM extract from 250 μL to 750 μL in order to establish a relationship between the concentrations of BM and the final properties of the gold or silver nanoparticles (surface charge). The reaction was stopped based on the appearance of color of the final solution suggesting the completion of the reaction or formation of the nanoparticles. The experiments do not show the time dependent reaction conditions.

The volume of Butea extract (reactant concentrations) alters the time for the formation of nanoparticles. Higher volume of extract accelerates the reaction for the formation of gold and silver nanoparticles. 3.2. UV visible spectroscopy Absorbance of gold and silver nanoparticles synthesized at 24 h with different concentrations of BM extract (Tables 1 and 2) was monitored by UV visible spectroscopy and presented in Fig. 1.a–b. The surface plasmon resonance (SPR) bands of b-AuNP for the first three sets of reaction with HAuCl4 (Table 1) appeared at around λmax ~534–538 nm clearly suggesting the formation of stable AuNPs (Fig. 1.a) [19,35]. However, the intensity of absorbance of b-AuNP after 24 h is not consistent with the increasing concentration of BM extract (250 μL to 750 μL) (Fig. 1a). The maximum absorbance of b-Au-500 at 24 h was obtained by the reaction of 0.5 mL of BM extract. In the case of b-Au-750, the absorbance is lower than that of b-Au-500 may be due to the aggregation of nanoparticles. Additionally, the red coloration of b-Au-500 appeared within short time (5 min) compared to other two experiments (in Table 1). The inset of Fig. 1a indicates the optical picture of ruby red color of b-AuNP. The UV–visible spectroscopy was utilized to monitor the time dependent formation of b-Au-250 and b-Au-500 using 250 and −500 μL of BM extract, respectively (SI-Fig. 1.a–b). The absorbance of b-Au-250 increases (almost constant λmax) up to a certain time and after that the intensity of absorbance decreases (S.I. Fig. 1.a) may be due to its instability at longer reaction time. However, the absorbance of b-Au-500 increases consistently with time (almost constant λmax) indicating the formation of more AuNPs (S.I. Fig. 1.b) till the completion of the reaction. Based on the faster rate of the formation, stability and color of the nanoparticles, b-Au-500 was selected as optimized gold nanoparticles for further detailed characterization and biological studies. Similarly, the absorbance of b-Ag-500 and b-Ag-750 obtained by the reaction between AgNO3 solution and BM extract (−500 and 750 μL, respectively) at 24 h, was monitored by UV visible spectroscopy (Fig. 1.b). The intense yellow color of the resultant solution and the absorption maxima appeared around 440–475 nm suggest the formation of AgNPs (Fig. 1.b). The inset of Fig. 1.b indicates the optical picture of intense yellow colored b-Ag-500 & b-Ag-750. Similarly, based on the reaction time & stability, b-Ag-750 was selected as optimized silver nanoparticles for further detailed characterization and biological activities. We have observed the maximum absorbance of bio-synthesized AuNPs and AgNPs after 24 h reaction time. The reduction of HAuCl4 and AgNO3 was assumed to complete at 24 h, as there is no further change of absorbance of the nanoparticles, observed by UV visible spectroscopy. Therefore, we have chosen 24 h as optimized time point to determine their UV spectrum (Fig. 1.c–d). It is well established that protein peaks appear at λmax = 350–400 nm [36,37] as can be seen in the inset of Fig. 1d. The AgNPs can't show significant plasmon resonance due to the presence of proteins coated with the surface of silver nanoparticles,

Table 2 Reaction conditions for the biosynthesis of silver nanoparticles (b-AgNP) using Butea monosperma leaf extract as reducing agent. Exp. no

BM-Extract (μL)

AgNO3 (μL)

Water (mL)

Total volume (mL)

Timea (h)

Zeta potential (charge in mV)

1. 2.

500 750

200 200

4.30 4.05

5 5

~4 ~2

−10.2 ± 3.1 −14.7 ± 0.5

a

Time was required to appear the yellow coloration (formation of b-AgNP).

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301

Scheme 1. Overall schematic representation for the green synthesis, characterization of bio-synthesized b-AuNP and b-AgNP and their probable applications in drug delivery.

as the absorptions of silver nanoparticles (λmax = 450 nm) and proteins (λmax = 360 nm) overlap with each other [36–38].

XRD pattern (Fig. 2.a–b) [39]. The particle size distribution, calculated from TEM analysis, suggests the distribution of 30 and 50 nm particle sizes for b-Au-500 & b-Ag-750, respectively (SI-Fig. 2.c–d).

3.3. X-ray diffraction (XRD) spectroscopy The crystal structures of the as synthesized b-Au-500 & b-Ag-750 were analyzed by X-ray diffraction analysis (XRD). According to Bragg's reflections, both b-Au-500 & b-Ag-750 are distinctly indexed to a face centered cubic crystal structures (Fig. 2.a–b). The absence of any extra peak in Fig. 2.a indicates the high purity of the as synthesized b-Au500. In both cases, the diffraction peaks are consistent with the standard data files where JCPDS card no. 04-0784, no. 04-0783 and no. 42-0874 indicate the formation of AuNPs and AgNPs, respectively. The XRD data does not show any peak of Ag2O impurity suggesting that the oxidation state of silver in b-AgNP is zero (Fig. 2.b). 3.4. Transmission electron microscopy (TEM) The size, shape and morphology of as synthesized b-Au-500 & b-Ag750, obtained after 24 h of reactions, were investigated by TEM and presented in Fig. 2.c–f. TEM images of b-Au-250 and b-Au-500 indicate that the nanoparticles are highly monodispersed and consist of mainly spherical gold nanoparticles (10–30 nm), along with few rods (50–75 nm), triangular (30–100 nm) and hexagonal shaped (15–35 nm) b-AuNP (Fig. 2.c–d). Similarly, TEM images of b-Ag-500 and b-Ag-750 clearly show large spherical nanoparticles (20–80 nm) along with few triangular b-AgNP (Fig. 2.e–f). The formation of these large triangular shaped nanoparticles may be due to the presence of relatively weak reducing agents in the B. monosperma leaf extracts, which help in the slow growth process [23]. The SAED patterns of as synthesized b-Au-500 and b-Ag-750 (SI-Fig. 2.a–b) indicate the face centered cubic (FCC) lattice structure of these nanoparticles that correlates with

3.5. Dynamic light scattering (DLS) technique The zeta potential (ξ) of nanoparticles gives an important idea regarding surface charge and stability of nanoparticles [19]. DLS study reveals the negative zeta potential (ξ) values of b-AuNP and b-AgNP (ξ = −10 to −16 mV) (Tables 1 & 2) indicating the stability of these nanoparticles for long time in solution. The negative–negative repulsive force in the solution helps in the stability and high dispersity of these nanoparticles.

3.6. X-ray photoelectron spectroscopy (XPS) The surface chemistry of biosynthesized gold and silver nanoparticles was investigated by XPS and the XPS surveys of b-Au-500 & b-Ag750 are presented in SI-Figs. 3.a and SI-3.c, respectively. These figures show the binding energy (BE) of different atoms (Au, Ag, C, O and N). SI-Fig. 3.b indicates the binding energy peaks for Au4f5/2 and Au4f7/2, which generates due to resolving of Au4f at BE around 88 eV and 83.8 eV, respectively confirming the presence of Au(0) in the metallic state in b-Au-500. Similarly, SI-Fig. 3.d indicates the binding energy peaks for another two spin orbit components of Ag3d3/2 and Ag3d5/2 which generates due to resolving of Ag3d around 374.4 and 368 eV, respectively verifying the occurrence of metallic Ag(0) in the b-Ag-750. The XPS analysis shows the absence of surface oxidation of silver in bAgNPs (SI-Fig. 3.a–d). This was further supported by published literatures [40,41]. The other BE peaks at around 285, 533 and 400 eV generated due to the presence of C(1s), O(1s) and N(1s) elements, respectively (SI-Figs. 4–6).

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Fig. 1. (a–d): UV visible spectra and X ray diffraction pattern of as synthesized gold and silver nanoparticles (b-Au-500 and b-Ag-750). (a) Change of absorbance of gold nanoparticles with different volumes of BM extract after 24 h of reactions. The maximum absorbance for all three reactions is obtained around at λmax = 535 nm. The numerical values in AuNP-250, -500 and -750 denote the corresponding volumes of Butea extract in μL. Inset figure indicates the optical colors of green synthesized b-Au-250, b-Au-500, and b-Au-750, and BM extract only. (b) Change of absorbance of silver nanoparticles with different volumes of BM extract after 24 h of reactions. The maximum absorbance for three reactions is obtained around at λmax = 400–450 nm. The numerical values in b-Ag-500 and -750 denote the corresponding volumes of Butea extract in μL. Inset figure indicates the optical colors of green synthesized b-Ag-500 and b-Ag-750. (c) UV visible spectra of green synthesized b-Au-500 at different time interval. (d) UV visible spectra of green synthesized b-Ag-750 at different time intervals. Inset figure shows the comparative UV spectra of BM, b-Au-500 and b-Ag-750 where arrows indicate the protein peaks. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.7. Fourier transformed infrared (FTIR) spectroscopy FTIR spectroscopy was employed to investigate the role of possible functional groups of phytochemicals present in the Butea leaf that are responsible for the reduction of HAuCl4 and AgNO3 to b-Au-500 & bAg-750 and their stabilization, respectively. SI-Fig. 7.a–c indicates the FTIR spectra of Butea extract (SI-Fig. 7.a), b-Au-500 (SI-Fig. 7.b) and bAg-750 (SI-Fig. 7.c). The major stretching frequency is at ν = 1514.40 cm− 1 due to the presence of amide II in proteins of B. monosperma leaf extract (SI-Fig. 7.a) which has been used during the reduction of Au3+ to b-Au-500 (SI-Fig. 7.b) suggesting the role of proteins in the synthesis of b-Au-500. Again, amide II peak at ν = 1514.40 cm−1 almost disappeared in b-Ag-750 (SI-Fig. 7.c) which confirms the involvement of proteins for the synthesis of b-AgNP (SIFig. 7.c) [21]. The involvement of proteins in the synthesis of gold and silver nanoparticles was further confirmed by SDS gel electrophoresis (discussed later). Additionally, IR peak at ν = 3402.49 cm−1 in BM extract indicates the presence of O–H stretching modes that suggest the presence of polyphenols in BM extract. However, it is shifted towards shorter frequency ν = 3386.31 cm−1 and ν = 3383.65 cm−1 during the formation of b-Au-500 & b-Ag-750 respectively confirming the role of –OH groups as reducing agent for the formation of nanoparticles [42]. The remaining IR peaks in curve-a, -b and -c remain almost unchanged. The above results together indicate the presence of proteins and polyphenols in BM extract and those molecules are responsible for the production of gold and silver nanoparticles and their stabilization

that would be further confirmed by silver nitrate staining (discussed later).

3.8. Silver nitrate staining In order to investigate the role of proteins present in the BM leaf extract for the formation of b-AuNP and b-AgNP (by the reduction of HAuCl 4 and AgNO3 to AuNPs and AgNPs respectively) and their stabilization, SDS-PAGE gel electrophoresis was performed (SIFig. 8.a–b). Butea leaf extract (Lane-1 in SI-Fig. 8.a and SI-b) and concentrated supernatants obtained after centrifugation of b-Au-500 (Lane-2 in SI-Fig. 8.a) and b-Ag-750 (lane-2 in SI-Fig. 8.b) was loaded in the SDS gel for silver nitrate staining. Proteins of around ~ 12 kDa and ~ 22 kDa expressed in lane-1 of SI-Fig. 8.a almost disappeared in lane-2 (SI-Fig. 8.a) indicating that these proteins (~ 12 kDa and ~ 22 kDa from Butea extract) may play an important role for the formation and stabilization of gold nanoparticles. Similar observations are obtained in the case of silver nanoparticles (SI-Fig. 8.b) where proteins around ~ 12 kDa and ~ 22 kDa in lane-1 almost disappeared in lane-2 (SI-Fig. 8.b) indicating the direct involvement of these proteins for the formation and stabilization of silver nanoparticles. The above results altogether suggest that low molecular weight proteins (~ 12 kDa and ~ 22 kDa), present in Butea leaf extract, are responsible for the formation and stabilization of both AuNPs and AgNPs [19,43].

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Fig. 2. (a–f): (a–b) X-ray diffraction patterns of as synthesized gold (b-Au-500) and silver nanoparticles (b-Ag-750). (c–f) TEM images of biosynthesized nanoparticles [b-Au-250 (c), b-Au500 (d), b-Ag-500 (e) and b-Ag-750 (f)] obtained after 24 h of reaction condition.

3.9. In vitro stability studies of AuNPs The stability of nanoconjugates in physiological solutions is one of the most important criteria for drug delivery system in cancer therapeutics. In order to check the in vitro stability of the bio-synthesized b-Au-500, the nanoparticles were incubated in different physiological buffers and solutions like DPBS, buffer saline with pH values = 6, 7 & 8, and fetal bovine serum (FBS) and the stability of b-Au-500 was monitored through UV visible spectroscopy. It is found that b-Au-500 is stable up to 14-days (SI-Fig. 9). Again, the dilution kinetic study was carried out in order to check the stability of b-Au-500 after dilution with water (SI-Fig. 10). In this study, the plasmon wavelength (λmax) and plasmon bandwidth (Δλ) were monitored by recording the absorbance of b-Au-500 on each dilution. On dilution, only a very slight change in the absorbance or plasmon wavelength (λmax) and plasmon bandwidth (Δλ) (b5 nm) [19,44] occurred suggesting high in vitro stability of as synthesized b-Au-500 (SI-Fig. 9). The absorbance of b-Au500 on dilution has linear relationship indicating the high stability even in the concentration range of 10− 5 to 10− 6 M (SI. Fig. 10) [19, 44]. We speculate that several proteins, flavonoids (phenolic compounds), carbohydrates, etc. present in the BM extract support the extra stability of gold nanoconjugates.

experiments [45]. It is well-known that the surface of bio-synthesized nanoparticles are covered with biomolecules (proteins, carbohydrates, polyphenolic compounds etc.) coming from the plant leaf extract.

3.10. Cell viability assay (MTT assay) with biosynthesized and chemically synthesized gold and silver nanoparticles The cytotoxicity assay is one of the basic assays for any materials towards biomedical applications. Therefore, in order to investigate the cytotoxicity level of as synthesized b-Au-500 & b-Ag-750, MTT assay was carried out for both non-cancerous (HUVEC: Fig. 3.a; ECV-304: Fig. 3.b) and cancerous (B16F10: Fig. 5.a–b; MCF-7, A549 & HNGC2) cells (SIFig. 12.a–c). Results reveal that both the biosynthesized gold and silver nanoparticles are biocompatible in nature. In the present article, we compare the cell viability of these biosynthesized nanoparticles with chemically synthesized nanoparticles (c-AuNP and c-AgNP) which were used as gold and silver control

Fig. 3. (a–b): cell viability assay using MTT reagent. Biocompatibility of b-Au-500 and bAg-750 was tested in (a) HUVEC and (b) ECV-304 cells in a dose-dependent manner. Numerical values (0.3, 0.6, 1.2 & 2.5) of BM, b-Au-500, b-Ag-750, indicate the volume in μL.

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Therefore, in order to make similarity, we use chemically synthesized nanoparticles conjugated with PEG-600 (c-Au-PEG and c-Ag-PEG), instead of naked gold nanoparticles. To keep the same gold control in all in vitro experiments we have used PEG-6000 for chemically synthesized nanoparticles. The MTT data with PEG6000 coated nanoparticles are presented in SI-Fig. 11.a–b. According to published literatures, the methanolic/aqueous extract of B. monosperma flower shows cytotoxic activity as well as anticancer activity [29,30]. However, there is no report of cytotoxicity of aqueous BM leaf extract. In our present work, Fig. 5.a–b shows slight cytotoxic effect of BM leaf extract may be due to the presence of some active anti-cancer or cytotoxic bio-molecules present in the extract. On the other hand, the biosynthesized gold and silver nanoparticles (b-Au-500 and b-Ag-750) exhibit their biocompatible nature may be due to the following two reasons: (i) during the formation of nanoparticles, the active anti-cancer agents of the leaf extract are not conjugating with as-synthesized nanoparticles and therefore not showing any cytotoxic effect; (ii) the active anticancer agents of the leaf extract after conjugation with the assynthesized nanoparticles cannot be released in the physiological media and hence do not exhibit their cytotoxic activity. In the present article, the biosynthesized gold and silver nanoparticles do not show cytotoxic effect while the BM leaf extract has slight cytotoxic effect. However, the BM leaf extract helps to synthesize stable and biocompatible b-Au-500 and b-Ag-750, that could be useful for delivery of several drugs for cancer therapeutics. Hence, BM leaf extract has been chosen for this work to form stable, biocompatible gold and silver nanoparticles using an efficient, fast, cost effective and eco-friendly route.

3.11. Attachment of DOX (%) and nature of bonding Considering absorbance of DOX (by measuring the UV absorbance) from the standard curve of DOX in the supernatant of b-Au-500 & bAg-750, the b-Au-500-DOX & b-Ag-750-DOX contain 21% & 13% of DOX, respectively (Fig. 4.a–d). In order to confirm the binding of DOX with b-Au-500 & b-Ag-750, DLS study was performed. The data obtained from DLS clearly indicates that hydrodynamic radius of both b-Au-500DOX & b-Ag-750-DOX is more compared to b-Au-500 & b-Ag-750, respectively. The hydrodynamic radius is increased from 130 nm to 202 nm for b-Au-500 and 82 nm to 107 nm for b-Ag-750 after their conjugation with DOX (Table 3). The probable bonding of DOX with biosynthesized nanoparticles can be explained according to FTIR data of b-Au-500-DOX and b-Ag-750-DOX (SI-Fig. 13.a–d). In the FTIR spectra of b-Au-BM and b-Ag-BM, peak appeared at around 3380 cm−1 is due to the presence of polyphenolic groups over the surface of biosynthesized gold and silver nanoparticles. However, after conjugation with DOX, the peak has been shifted to 3417 cm−1 (SI. Fig. 13.a–d). The increased stretching frequency may happen due to weak dative bond between the surfaces of gold/silver nanoparticles and –OH group present in DOX. This could be explained according to our published literature [46]. Additionally, the bonding of DOX with bio-synthesized gold and silver nanoparticles can further be explained by DLS data. It has been found that the zeta potential values of b-Au-500 (− 14.8 ± 3.5 mV) and b-Ag-750 (− 17.6 ± 2.9 mV) are negative which are slightly increased after conjugation with DOX (b-Au-500-DOX: −12.8 ± 3.1 mV and b-Ag-750-DOX: −15.2 ± 1.4 mV). However, zeta potential value of free DOX is positive (+ 2.6 ± 0.6). Therefore, it can be speculated that weak electrostatic attractive force may be another reason for the

Fig. 4. (a–d): UV visible spectra measurements and standard curve of doxorubicin in water. (a & c) UV visible spectra of b-Au-500 and b-Ag-750 and its conjugation with DOX [b-Au-500DOX and b-Ag-750-DOX]. (b-d) Standard curve of DOX in aqueous solution was determined by measuring the absorbance of DOX at different concentrations. This standard curve of DOX can be used to determine an unknown concentration of pristine DOX or DOX conjugated to b-Au-500 and b-Ag-750.

S. Patra et al. / Materials Science and Engineering C 53 (2015) 298–309 Table 3 Size and charge of the biosynthesized gold & silver nanoparticles (b-AuNP & b-AgNP) and their conjugated form with doxorubicin (b-Au-500-DOX & b-Ag-750-DOX) obtained from DLS study. Sample no

Sample name

Size from DLS (nm)

Zeta potential (charge in mV)

1 2 3 4

b-Au-500 b-Au-500-DOX b-Ag-750 b-Ag-750-DOX

130.5 ± 5.5 202.1 ± 2.7 82.17 ± 1.8 107.32 ± 2.1

−14.8 ± 3.5 −12.8 ± 3.1 −17.6 ± 2.9 −15.2 ± 1.4

DOX attachment towards b-Au-500 & b-Ag-750. So, the attachment of doxorubicin with b-AuNP and b-AgNP occurs mainly due to weak electrostatic attraction and dative bonding. 3.12. Cell viability assay for drug delivery Drug delivery system (DDS) using DOX was developed based on the biocompatible nature of as synthesized b-Au-500 & b-Ag-500. To check the functional activity of DOX in b-Au-500-DOX & b-Ag-750-DOX,

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B16F10 & MCF-7 cancer cells were incubated with these DDSs for a certain time. Administration of gold and silver nanoparticle based drug delivery systems (DDS: b-Au-500-DOX and b-Ag-750-DOX) towards B16F10 cells shows significant inhibition of cell proliferation in a dose dependent manner (0.06–0.25 μM w. r. to DOX) compared to free doxorubicin (DOX) at the same concentration (Fig. 5.a–b). Similar results are observed with these DDS to MCF-7 cells (SI-Fig. 14.a–b). Considering the biocompatible nature of the biosynthesized nanoparticles towards normal cells and therapeutic efficacy of these DDS towards cancer cells, the bio-synthesized b-Au-500 & b-Ag-750 nanoparticles might be applicable as efficient drug delivery vehicle for future cancer therapy and other diseases. It is well established that drug conjugated with nanoparticles (where nanoparticles act as delivery vehicle) or nanoparticle based drug delivery system (DDS) shows better therapeutic efficacy towards cancer cells compared to free or pristine drug at the same concentration. The nanoparticle based DDS enhances anti-cancer efficacy (inhibition of cancer cells proliferation) mainly due to more specificity, retention ability and EPR effects, supported by published literature. This was also further confirmed by more uptake of DOX in cancerous

Fig. 5. (a–c): In vitro anticancer efficacy of DDS and cell cycle analysis in B16F10 cells. Results clearly reveal that b-Au-500-DOX (a) and b-Ag-750-DOX (b) exhibit better anticancer therapeutic efficacy than pristine DOX in a dose dependent manner. (c) Result shows the sub-G1 phase arrest in cells treated with b-Au-500-DOX and b-Ag-750-DOX leading to the apoptotic cell death.

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cells while attached to nanoparticles compared to free DOX (discussed later in Fig. 6). Therefore, both b-Au-500-DOX and b-Ag-750-DOX show more cytotoxicity compared to free DOX. Thus it can be said that biosynthesized AuNPs and AgNPs mainly act as delivery vehicle for the delivery of doxorubicin with better specificity [8,47,48].

[49–51]. These results support the enhanced anticancer effects of DOX in b-Au-500-DOX & b-Ag-750-DOX through enhanced induction of apoptosis than pristine DOX (Figs. 5.c and SI-15.a–d).

3.13. Cell cycle assay (FACS)

Localization of doxorubicin and nanoparticles conjugated with doxorubicin can be traced using fluorescence or confocal microscopy [52]. In order to investigate the cellular uptake of DOX present in bAu-500-DOX and b-Ag-750-DOX, fluorescence microscopy study was carried out in B16F10 cells. First, second and third columns of Fig. 6.a1, a2 and a3: row 1 show the phase image, fluorescent image and merged image of untreated B16F10 cells, respectively. Similarly, Fig. 6b1, b2 and b3: row 2, c1, c2 and c3: row 3 and d1, d2 and d3: row 4 shows the phase image, fluorescent image and merged image of B16F10 cells treated with 0.25 μM free doxorubicin, b-Au-500-DOX (0.25 μM w. r. to DOX) and b-Ag-750-DOX (0.25 μM w. r. to DOX), respectively. There is no red fluorescence in untreated control B16F10 cells indicating the absence of fluorescent DOX and non-specific fluorescent molecule in control cells.

Cell cycle analysis was performed using PI staining method and results are represented through four phases named as sub-G1, G0/G1, S and G2/M (Figs. 5.c and SI-15.a–d). As per published literatures, doxorubicin generally induces cell cycle arrest in G0/G1 or G2/M phase [49–51]. In this present work, DOX induces G2/M arrest in B16F10 melanoma cancer cells treated with b-Au-500-DOX & b-Ag-750-DOX. But, interestingly, B16F10 cells treated with b-Au-500-DOX & b-Ag-750-DOX show increased cell populations in sub-G1 phase (~ 5% & 8% respectively) compared to untreated control cells and consequently decrease in G0/ G1 phase cell population. Several groups showed that accumulation of cells in sub-G1 phase led to the induction of apoptosis or cell death

3.14. Cellular uptake

Fig. 6. Cellular uptake of DOX, b-Au-500-DOX and b-Ag-750-DOX by fluorescence microscopy. (a1–d1) bright field images of untreated B16F10 cells (a1) and cells treated with DOX (b1), b-Au-500-DOX (c1) and b-Ag-750-DOX (d1); (a2–d2) Corresponding fluorescence images of B16F10 cells; (a3–d3) Merged images of bright field and red fluorescence images of the respective cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Again, appearance of red fluorescence in B16F10 cells treated with free doxorubicin (Fig. 6.b2) indicates the incorporation of DOX inside the cells. However, appearance of bright red fluorescence in B16F10 cells (Fig. 6.c2–d2) treated with b-Au-500-DOX and b-Ag-750-DOX, indicate the more internalization/incorporation of DOX in conjugated form with gold and silver nanoparticles, compared to free doxorubicin. These results corroborate with the result of MTT assay in B16F10 cell lines. 3.15. Release of silver ions In order to compare the release of silver ion with existing literatures [38,53–57], we have carried out in vitro silver ion release study by incubating the b-Ag-750 with PBS (pH = 7.4) in a time dependent manner. As all the biological experiments were performed for maximum 48 h, we have quantified the Ag+ ion release up to 48 h. After certain time of incubation the solution was ultra-centrifuged at 22,000 rpm, for 1 h at 10 °C. The supernatant of the corresponding solution was collected and submitted for atomic absorption spectroscopy (AAS). There is a gradual increase of Ag+ ion release up to 24 h starting from 0 h, which is almost saturated at 48 h. The results have been normalized and it has been observed that maximum 10% of Ag+ ion (0.45 ppm) has been generated from the incubated b-Ag-750 (~ 5 ppm) (see Fig. 7.a). Considering the published literatures, it is clear that the release of Ag+ ion is not that much (maximum 10%) which attributes to the fact that the synthesized b-Ag-750 by B. monosperma leaf extract is biocompatible in nature [38,53–57]. The proteins and phytochemicals coated on the silver nanoparticles help to get long term stability and minimum leaching of Ag+ ion from the b-Ag-750. We have presented the pattern of silver ion release in two different units (ppm and nM/mL) (Fig. 7.a– b). 3.16. Plausible mechanism The exact mechanism for the synthesis of nanoparticles using plant extract still remains unclear. However, it is already well established that plant extract helps for the formation and stabilization of nanoparticles. Many investigators have already established the role of plant

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extract as reducing agent as well as stabilizing agent during the formation of metal nanoparticles especially gold and silver nanoparticles [18, 19,23,43,58–62,13,20]. According to published literature, the presence of polyphenolic/alcoholic compounds, aldehydes/ketones and proteins present in the plant extract might be responsible for the reduction of HAuCl4 and AgNO3 to gold and silver nanoparticles and stabilization of these nanoparticles. It is well established by several groups including ours that both low (12–22 kDa) and high molecular weight proteins (~150 kDa) play a major role for the reduction of metal ions to metallic nanoparticles. However, it depends upon the nature of plant as well as the source of leaf. For example, both low (~ 15 kDa) and high (~ 150 kDa) molecular weight proteins of Eclipta alba are responsible for the formation of gold nanoparticles by the reduction of chloroauric acid and their stabilization [17]. Again, low molecular weight proteins (~ 12–15 kDa) of Olax scandens leaf are responsible for the synthesis and stabilization of gold and silver nanoparticles which was confirmed by SDS PAGE analysis [13,20]. Apart from proteins, the presence of polyphenolic/alcoholic compounds, aldehydes/ketones in the plant extract might be responsible for the synthesis of gold and silver nanoparticles [13,17,20]. Recent reports suggest that heat shock proteins (HSP: 10–25 kDa) are present in many leaf extract [65]. Other reports demonstrate the presence of several dehydrin proteins (~12–17 kDa) in plant leaf extract [66]. Therefore, we cannot exclude the possibility of the presence of HSP and dehydrin proteins in the plant extract that might be responsible for the synthesis of gold/silver nanoparticles. However, we do not know whether basic proteins or oxidoreductases are responsible for the fabrication of nanoparticles or not, that needs further investigation which is beyond the scope of this present work. Additionally, we have searched the literatures that demonstrate the presence of polyphenol and acids (Glucoside, Kino-oil containing oleic and linoleic acid, palmitic and lignoceric acid, 3,4,5-trihydroxybenzoic acid, 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one, etc.) in the leaf extract of B. monosperma (BM) (SI. Fig. 16) [26,63]. Rmanjaneyulu et al. demonstrated that ethanolic and aqueous extracts of B. monosperma leaves reveal the presence of alkaloids, tannins, carbohydrates, flavonoids, phenolic compounds and starch [64] (Scheme 2). Formation of gold and silver nanoparticles from HAuCl4 and AgNO3 is an example of redox reactions involving electron transfers. The standard reduction potential values of Au3+/Au0 (E0Au3+/Au0) and Ag+/Ag0 (E0Ag+/Ag0) are 1.50 V and 0.80 V, respectively. On the other hand, the standard reduction potentials for aldehyde/alcohol, acid/aldehyde, quinone/phenol and proteins systems are below (E0) 0.80 V, suggesting that alcohol, aldehyde, and phenols can act as strong reducing agent for the formation of gold and silver nanoparticles [62]. Therefore the phenolic/alcoholic/aldehyde/protein compounds present in Butea extract can help in the reduction of Au3+ (in HAuCl4) to Au0 (gold nanoparticle) and Ag+ (AgNO3) to Ag0 (silver nanoparticles). Additionally, Fourier transformed infrared (FTIR) spectrum of B. monosperma (BM) extract at ν = 3402.49 cm− 1 (O–H stretching modes) suggests the presence of polyphenols in BM extract. However, it is shifted towards shorter frequency at ν = 3386.31 cm−1 and ν = 3383.65 cm−1 during the formation of b-Au-500 & b-Ag-750, respectively confirming the involvement of –OH groups (or polyphenols) as reducing agent for the formation of these nanoparticles. Hence, FTIR analysis supports the presence of polyphenolic compounds that are responsible for the reduction of HAuCl4 and AgNO3. Newman et al. demonstrated the reduction of HAuCl4 to gold nanoparticles using amines through free radical reactions [61]. .

+ − HAuCl4 + 3NR3 = Au0 + 3NR+ 3 + H + 4Cl .

Fig. 7. (a–b): Silver ion release from b-Ag-750 in a time dependent manner — (a) in ppm and (b) in nM/mL.

Published literatures, FTIR data and SDS-PAGE analysis support the presence of amines as well as proteins [low mol. wt. protein: 10–15 kDa and high mol. wt. protein: 100–200 kDa] along with polyphenols in the BM extract, that ultimately help in the reduction of

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Scheme 2. The plausible mechanism for the formation and stabilization of biosynthesized gold and silver nanoparticles from HAuCl4 and AgNO3 solution using Butea monosperma extract.

HAuCl4 and AgNO3 to gold and silver nanoparticles, respectively. The individual isolation of molecules present in Butea leaf extract is already well established by reported literatures [26,63,64]. Again, the isolation of individual active components from BM leaf extract is rigorous and time consuming and that is beyond the scope of our present study. 4. Conclusions We have demonstrated a green chemistry approach for the synthesis of gold and silver nanoparticles at ambient reaction conditions using ‘B. monosperma (BM)’ leaf extract. The leaf extract acts as reducing agent as well as stabilizing/capping agent. The green chemistry approach is clean, efficient, eco-friendly & economically safe. The biosynthesized nanoparticles have been systematically characterized by several physico-chemical techniques. These biosynthesized gold & silver nanoparticles are highly stable for several weeks in different biological buffers. Moreover, they are biocompatible towards various normal and cancer cells. Finally, we have designed and developed biosynthesized gold & silver nanoparticle based drug delivery systems (DDS) using FDA approved anticancer drug doxorubicin and observed that the administrations of these DDS towards cancer cells show better therapeutic efficacy compared to free drug. Considering the biocompatible nature of the nanomaterials towards normal cells and therapeutic efficacy of these DDS towards cancer cells, we believe that our biosynthesized b-Au-500 & b-Ag-750 nanoparticles might be useful as an efficient drug delivery vehicle for future cancer therapy and other diseases. Acknowledgment This research was supported by the ‘Ramanujan Fellowship grant’ (SR/S2/RJN-04/2010; GAP0305), DST-New Delhi and generous financial support by the Council of Scientific and Industrial Research (Project on ‘Advanced Drug Delivery System’: CSC0302), Government of India, New Delhi to CRP. SM and AKB are thankful to CSIR, New Delhi and UGC, New Delhi, respectively for their Senior Research Fellowships. CRP is grateful to Dr. (Ms.) M. Lakshmi Kantam, Director of CSIR-IICT, for her constant help, support and motivation towards nanomedicine research at CSIR-IICT, Hyderabad.

Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2015.04.048.

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