Embelin Mediated Green Synthesis of Quasi-spherical and Star

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Embelin Mediated Green Synthesis of Quasi-spherical and Starshaped Plasmonic Nanostructures for Antibacterial Activity, Photothermal Therapy and Computed Tomographic Imaging Sisini Sasidharan, Radhika Poojari, Dhirendra Bahadur, and Rohit Srivastava ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b01894 • Publication Date (Web): 06 Jun 2018 Downloaded from http://pubs.acs.org on June 6, 2018

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ACS Sustainable Chemistry & Engineering

Embelin Mediated Green Synthesis of Quasi-spherical and Star-shaped Plasmonic Nanostructures for Antibacterial Activity, Photothermal Therapy and Computed Tomographic Imaging Sisini Sasidharan,a Radhika Poojari,a Dhirendra Bahadurb* and Rohit Srivastavaa* a. Department of Biosciences and Bioengineering, IIT Bombay, Powai, Mumbai, 400076, India. b. Department of Metallurgical Engineering and Materials Science, IIT Bombay, Powai, Mumbai, 400076, India. AUTHOR INFORMATION Sisini Sasidharan - [email protected], [email protected] Radhika Poojari - [email protected] Corresponding authors* Dhirendra Bahadur- [email protected] Rohit Srivastava - [email protected] ABSTRACT Plasmonic nanostructures of silver and gold synthesized by conventional toxic and cumbersome methodologies raise huge concern for their clinical application, which necessitates the use of a greener approach. Herein, embelin, a benzoquinone derivative extracted from the fruits of Embelia tsjeriam-cottam, with immense medicinal value is used as a reducing and stabilizing agent for the synthesis of quasi-spherical gold and silver nanoparticles as well as gold nanostars. A sunlight assisted synthesis resulted in embelin stabilized silver nanoparticles of bimodal size distribution (~3 and 15 nm) with potent antibacterial activity against Staphylococcus aureus and Escherichia coli. Similarly, embelin was also used for the synthesis of polyhedral gold nanoparticles of 12-15 nm in size and absorbance at 540 nm. These highly faceted and multi-

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twinned gold nanoparticles facilitated the formation of 120 nm sized embelin stabilized gold nanostars absorbing at NIR wavelength (800 nm). The embelin stabilized nanoparticles demonstrated excellent compatibility towards cells and human blood. Additionally, the gold nanostars exhibited superior computed tomographic (CT) contrast characteristics and marked photothermal cytotoxicity towards oral epithelial carcinoma cells. This study thus proposes, for the first time, the synthesis of biocompatible plasmonic nanostructures using embelin and their potential use as antibacterial, CT imaging and photothermal agents. Keywords: Embelin, Gold nanoparticle, Silver nanoparticle, Gold nanostars, Antibacterial, Photothermal, CT imaging, Green synthesis INTRODUCTION Noble metal nanostructures especially silver and gold are highly appealing for medical applications owing to their unique properties such as optical tunability, non-toxicity in comparison to their counterparts, etc. The properties of these nanoparticles and in turn their applications depend on the size of the material, hence, the size-controlled synthesis is considered to be of a key concern. Generally, noble metal nanoparticles are synthesized by chemicals which reduce metal ions in the presence of surfactants to prevent aggregation and control the size. Recently, metal nanoparticles were also synthesized using expensive and sophisticated techniques, such as radiolysis, electrolysis, spray pyrolysis, laser irradiation, etc.; however, their lack of stability necessitate the use of surfactant chemicals.1–3 Considerable research is being carried out in the synthesis of noble metal nanoparticles with biogenic materials in order to reduce toxic substances used during their synthesis. This green chemistry of synthesis is a cheap, efficient and environmentally safe method for producing nanoparticles with specified properties. Plant metabolites such as sugars, proteins, polyphenols, terpenoids and alkaloids play a key role in the reduction of metal ions to yield nanoparticles.4 A number of phytochemicals, such as in tea,5 lemongrass,6 soyabean,7 grape seed extract,8 cocoa9, starch,10 etc. have been widely studied in the synthesis of noble metal nanoparticles. These nanoparticles can also serve as seeds for the development of anisotropic structures such as gold nanostars with unique properties such as surface plasmon resonance (SPR) in the NIR range, high molar extinction coefficient and large absorption cross-section. The aforementioned exceptional properties make gold nanostars the most sought after agent for photothermal therapy. Gold nanostars with its amazing characteristics


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outshine many of its counterparts for other applications as well such as surface enhanced Raman scattering,11,12 photodynamic therapy13 and two-photon luminescence imaging.14 However, gold nanostars are usually synthesized using toxic chemicals such as N,N-dimethylformamide (DMF),11,15 sodium dodecyl sulphate (SDS),16 cetyl trimethylammonium bromide (CTAB),17,18 etc. These hazardous chemicals and lack of functionality hinder its use for biological purposes which warrant the need for a greener approach. Embelin (3-undecyl-2,5-dihydroxy-1,4- benzoquinone), a traditional medicinal compound, is known to form complexes with metal and possess antioxidant properties,19 hence could be a potential natural agent for the reduction of metal ions to form nanoparticles. This quinone derivative is a major constituent in the fruits, seeds and leaves of Embelia tsjeriam-cottam, a Myrsinaceae shrub mainly found in the forests of Western Ghats, Malaysia, China, Srilanka, and India. Embelin has been reported to possess antitumor, antiangiogenic, analgesic, antibacterial, antidiabetic, antiulcer, antihelminthic and anticonvulsant properties.20,21 It is a potent small molecule inhibitor, which permeates the cell, targets the BIR3 (baculoviral inhibitor of apoptosis protein repeat) domain of XIAP (X-linked inhibitor of apoptosis protein),22 scavenges free radicals,23 inhibits lipid peroxidation24 and augments radiation therapy by radiosensitization,25 hence can be classified as a promising anticancer drug. Embelin is a well-established drug in the Ayurvedic system of Medicine and reports no toxic effects on the human body when consumed in normal doses as recommended by Indian Herbal and Ayurvedic Pharmacopoeia.26,27 The safety and toxicity profile of embelin has been studied extensively in rodents and non-rodents. Numerous reports suggest embelin to be safe up to 3 g/kg orally as well as after repeated administration of 10 mg/kg in rats.21,28 Metal complexes of embelin such as potassium embelate, an analgesic drug when administered in rats and mice at a very high dose of 2 g/kg reported no mortality.29 Similarly, no significant toxicity as well as no effect on fertility and reproductive capacities were reported when it was administered for 10 weeks in mice and 24 weeks in monkeys indicating an extremely safe profile of embelin-metal complexes.29 Hence, embelin in addition to being non-toxic, not only reduces metal ions to nanoparticles and stabilize them but also adds medicinal benefits to the structure. A green chemistry of synthesis using phytochemicals for gold nanostars has not been reported till date to the best of our knowledge. So, herein we present, for the first time, the synthesis of quasi-spherical gold, silver nanoparticles



and gold nanostars using embelin as a reducing as well as stabilizing agent and its evaluation for biocompatibility, photothermal therapy, CT imaging and antibacterial properties. EXPERIMENTAL SECTION Note: All the experiments were carried out in triplicates unless mentioned otherwise. Materials Tetrachloroauric acid (HAuCl4), silver nitrate (AgNO3) and ascorbic acid were purchased from Spectrochem, Acros Organics and SD Fine Chemicals, India, respectively. Standard embelin was procured from Sigma Aldrich, India. Fruits of Embelia tsjeriam-cottam were purchased locally and authenticated at National Institute of Science Communication and Information Resources (NISCAIR), New Delhi, India. Extraction of Embelin Embelin was obtained from fruits of Embelia tsjeriam-cottam by ethanolic extraction.30 The fruits were powdered and treated with ethanol under stirring (700 rpm) for 48 h at room temperature. The extract obtained was filtered using Whatman filter paper No. 1 and air-dried to isolate pure embelin which was then stored in an air tight container at 4 °C. Synthesis of Embelin Stabilized Silver Nanoparticles (AgNP) Typically, 10 mg/ml of embelin solution were added to 1mM solution of AgNO3 and the solution was exposed to direct sunlight (~100,000 lux, recorded using a lux meter) on a bright sunny day (12.00 - 1.00 pm) at a high altitude located at 19.13°N and 72.91°E with an elevation of 56 m (1 sun = 100 mW/cm2, at AM 1.5G). The reaction was carried out with different volumes of embelin (10, 20, 30, …to 90 µl), at different pH (3, 5, 7, 9 and 12) and different time duration of sunlight exposure (0, 2, 5, 7, 10, 12 and 15 min). The yellow colored solution obtained, was subjected to differential centrifugation at 4000 and 12000 rpm for 20 min. The pellet was redispersed in deionized water and after repeated washing by centrifugation, was stored at 4 °C. The time and place for sunlight exposure were kept constant during all the experiments to avoid any errors due to changes in intensity. Synthesis of Embelin Stabilized Gold Nanoparticles (AuNP)


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To 1mM solution of HAuCl4, different volumes (10, 20, 30,...to 150 µl) of embelin solution (10 mg/ml) was added and stirred for 24 h in the dark. The red colored solution formed at the end of 24 h was centrifuged (10000 rpm for 10 min), and the pellet after redispersion in deionized water and repeated washing by centrifugation, was stored at 4 °C for subsequent characterization. Synthesis of Embelin Stabilized Gold Nanostars (E-GNS) In a typical experiment, to a solution of 10 ml of 0.25 mM HAuCl4 with 10 µl of 1 M HCl, 100 µl of AuNP was added and stirred at room temperature in the dark. Subsequently, 50 µl of 100 mM ascorbic acid and 100 µl of 3 mM AgNO3 were added simultaneously to the solution and stirred for 30 s. Embelin (10 mg/ml) was added during the formation of greenish blue color, and the solution was further stirred for an hour at pH 12 to yield embelin stabilized gold nanostars (E-GNS). The solution was then centrifuged for 20 min at 12000 rpm, and the pellet redispersed in deionized water was stored at 4 °C for subsequent characterization. Characterization The extracted and standard embelin were dispersed in ethanol and analyzed using U.V–visible spectrophotometry (Lambda 25, Perkin Elmer) and Liquid Chromatograph Mass Spectroscopy (LCMS/MS, Varian Inc.). The formation of AuNP, AgNP and E-GNS was confirmed from its surface plasmon resonance characteristics using spectrophotometric studies (Lambda 25, Perkin Elmer). The conversion yield of Ag+ to Ag and Au3+ to Au during the formation of nanoparticles was evaluated by ICP-AES (inductively coupled plasma atomic emission spectroscopy) analysis after acid-digestion of nanoparticle pellets (obtained by centrifugation) and precursor solutions. Particle size and stability of the nanoparticles were analyzed using dynamic light scattering technique (BI 200SM, Brookhaven Instruments Corporation) and zeta potential analyzer (ZetaPALS, Brookhaven Instruments Corporation), respectively. Nanoparticles (200 µg/ml) in aqueous dispersions as well as in buffers of varying pH were used for analysis. Additionally, nanoparticles were suspended in PBS, saline (0.9%) and complete media for 6 weeks to analyze their long-term stability in the physiological fluids. The nanoparticles were also subjected to hyperspectral imaging using Olympus BX43 microscope integrated with CytoViva 150 Unit. Images were obtained by Dagexcel X16 camera, and HSI system 1.1 with ENVI software was used for hyperspectral imaging (HSI) at 60 X magnification. The size, shape, lattice structure and



elemental composition of nanoparticles were characterized by field-emission-gun transmission electron microscopy (FEG-TEM) (JEM 2100-F, JEOL, 200 kV) along with EDAX analysis for which a diluted drop of sample was placed on a copper grid, dried and viewed under the microscope. Nanoparticles were subjected to X-ray diffraction (XRD) analysis using a Philips X’PERT PRO powder diffractometer (source- Cu-Kα , λ = 1.54056 Å) with spectrum recording over 5°–85° and phase identification with JCPDS database. Functional groups on the samples were analyzed with Fourier Transform infrared Spectrometer (MAGNA 550, Nicolet Instruments Corporation) from a frequency of 4000 to 400 cm-1 using KBr method. The functional groups were also analyzed by NMR spectrometer (NMR mercury plus 300 MHz, Varian) using deuterated chloroform. The amount of embelin attached to nanoparticles was determined by estimating the content of carbon, hydrogen and nitrogen with CHN analyzer (FLASH EA 1112, Thermo Finnigan). To determine the photothermal effect of E-GNS, a NIR laser (808 nm, 1.3 W/cm2, PMC, India) was used for irradiation. In a typical experiment, 200 µl of deionized water (control) and E-GNS (50 µg/ml gold concentration) were placed in distant wells of 96-well plate to avoid heat transfer. The temperature was maintained by keeping the plate in a water bath at 37 °C, and the laser was irradiated on the sample and control wells. A digital thermometer was used to record the temperature at 0, 1, 2.5, 4, 5, 7.5 and 10 min. The photothermal efficiency of E-GNS was also determined from the temperature increment obtained by laser irradiation, as described in supporting information. Similarly, the photothermal conversion efficiency (the ability to absorb energy per unit mass), also known as SAR (specific absorption rate) was determined using the equation described below =

… … … (1)

where, C and V is the specific heat capacity and volume or weight of the individual i component of the sample (C of water and quartz is 4.184 J/ g/°C and 0.839 J/ g/°C, V of water and quartz cuvette is 3 g and 5.9974 g, respectively), m denotes the mass of E-GNS and dT/dt is the temperature increment measured as a function of time during the initial linear slope in the graph.31–34


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A 64-slice cardiac capable PET-CT Scanner (Biograph mCT-Molecular CT, SIEMENS) was employed to analyze the CT contrast efficiency of AuNP and E-GNS. Samples were added into 96-well plates at different concentrations along with water and Omnipaque (clinically approved CT contrast) as the negative control and reference, respectively. The scanner was operated with a tube voltage of 100 kVp, tube current of 200 µA, slice thickness of 1 mm, scan time of 6.4 s, and rotation time of 0.5 s. The contrast was quantified in Hounsfield units by manual selection of regions of interest having an equal diameter. Biological Studies Antibacterial Study of Silver Nanoparticles The bacterial strains, Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) were used as model test strains for Gram-negative and Gram-positive bacteria, respectively. Media, glasswares and filter paper discs (Whatman No. 3 and 6 mm in diameter) were sterilized in an autoclave at 121 °C for 20 min. A single colony of bacteria was grown overnight in nutrient broth, and bacterial suspension was prepared by adjusting the turbidity of the solution to 0.5 McFarland standard. Disc Diffusion Assay The antibacterial activity of silver nanoparticles was tested by the disc diffusion method. Mueller-Hinton agar plates were inoculated with the turbidity adjusted bacterial suspension using spread plate method. The sterile paper discs were dipped in AgNP solution of different concentrations (1, 2.5, 5 and 10 µg/ml) and were air-dried in sterile conditions. They were placed on the top layer of agar plates and a disc dipped in sterile water served as the control. The agar plates were then incubated for 24 h at 37 °C, and the clear zone of inhibition (ZOI) around each paper disc was measured. Morphological Analysis of Bacteria The turbidity adjusted bacterial suspension was incubated with 5 µg/ml of AgNP for 4 h at 37 °C with mild shaking (160 rpm). Untreated bacteria served as the control. Post incubation, the suspension was centrifuged at 3000 rpm for 3 min and the bacterial pellet obtained was further washed twice with PBS. The bacterial cells were then viewed using cryo-field-emission-gun



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scanning electron microscope (Cryo-FEG-SEM) (JSM-7600F with PP3000T cryo preparation unit, JEOL). Cell culture Studies Normal mouse embryonic fibroblast cells (NIH 3T3), mouse fibroblast cells (L929) and oral epithelial carcinoma cells (KB) were purchased from the National Centre for Cell Science (NCCS), Pune, India. Cells were grown in Dulbecco’s modified Eagles Medium (DMEM) (HiMedia, India) supplemented with 10 % fetal bovine serum (FBS), 50 IU/ml penicillin and 50 µg/ml streptomycin (HiMedia, India) at 37 °C and humidified environment of 5% CO2. Biocompatibility Assay A fluorimetric Alamar blue assay was used to analyze the viability of cells on interaction with AuNP, AgNP and E-GNS. The assay is based on the reduction of resazurin (fluorescent blue dye), by viable cells to fluorescent red-colored resorufin. NIH 3T3, L929 and KB cells were grown in 96-well plates at a density of 7 ×103 cells per well. The medium was replaced with different concentrations of nanoparticles in basal media after 24 h of incubation. Cells treated with fresh basal media and Triton X-100 (1%) served as the negative and positive control, respectively. Post incubation for 24 h, the culture medium was removed and PBS was used to wash the wells. Subsequently, 10% Alamar blue solution was added and cells were incubated for 4 h. The fluorescence intensity was measured at an excitation/emission of 560/590 nm with a microplate reader (ThermoScientific, USA) and the percentage of viable cells was determined using the equation: Cell viability (%) =

I sample × 100 … … … (2) I control

where, I control and I sample are the fluorescence intensity values of cells treated with basal medium (negative control) and nanoparticles (sample), respectively. Reactive Oxygen Species (ROS) assay Reactive oxygen species (ROS) assay was performed to evaluate the amount of ROS produced in cells

treated

with

nanoparticles

using

a

cell-permeable

molecule,

2′,7′-

Dichlorodihydrofluorescein diacetate (H2DCFDA), which in the presence of ROS converts to


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highly fluorescent 2′,7′-dichlorofluorescein. In a typical experiment, KB cells were cultured at a density of 105 cells per well into 6-well plates. Post incubation of 24 h, media containing 5 µg/ml of AgNP, 100 µg/ml of AuNP and E-GNS were added into wells after discarding the old media. Cells treated with 30 µM of H2O2 and media alone served as positive and negative controls, respectively. Subsequently, upon incubation for 12 h and followed by a wash with PBS, the cells were subjected to treatment with 10 µM H2DCFDA. Following 30 min of incubation in the dark, cells were washed twice with PBS and trypsinized. The cells resuspended in PBS were then subjected to analysis using flow cytometry (BD FACS Aria, USA). In-vitro Photothermal Therapy The efficiency of E-GNS as a photothermal agent was evaluated by performing in vitro photothermal studies on KB cells treated with E-GNS. Briefly, KB cells were cultured at a density of 7 ×103 cells per well into 96-well plates. Post incubation of 24 h, old culture media was discarded, and the cells were incubated with 100 µl of basal media containing E-GNS (100 µg/ml). Cells treated with Triton X-100 (1%) and fresh media served as positive and negative controls, respectively. Following incubation for 24 h, unbound particles were removed by washing the wells with PBS. Subsequently, following treatment was carried out on control wells; negative control-no treatment, only laser for 2.5, 5 and 7.5 min. Cells treated with E-GNS was irradiated with the laser for 0, 2.5, 5 and 7.5 min. Post incubation for 12 h, cells after washing with PBS, were subjected to Alamar blue assay as described earlier. Qualitative estimation was carried out by staining the treated cells with propidium iodide, and dead cells were visualized using a Nikon Eclipse Ti microscope integrated with filters set of excitation/emission (488/617 nm) wavelength. Blood Compatibility Studies The compatibility studies on blood were performed following IITB-institutional ethical, biosafety clearance (reference number – IITB-IEC/2016/003) and informed consent from healthy volunteers. Five milliliters of whole blood was withdrawn in vials containing sodium citrate from three healthy volunteers of 22-32 yrs of age. Hemolysis Assay



The hemolytic property of nanoparticles on interaction with red blood cell (RBC) was analyzed by estimating free hemoglobin (Hb) released upon lysis into plasma with the help of a spectrophotometer. To 1 ml of whole blood, 100 µl of nanoparticles redispersed in saline (0.9%) at different concentrations (0.5, 1, 2.5 and 5 µg/ml of AgNP; 10, 25, 50, 75 and 100 µg/ml of AuNP and E-GNS) was added and incubated at 37 °C with shaking for 3 h. Blood treated with saline (0.9%) and Triton X-100 (1%) were negative and positive controls, respectively. Post incubation, blood was centrifuged for 10 min at 4500 rpm and spectrophotometer (M200 Pro Tecan, USA) was used to measure the absorbance of diluted plasma (with 0.01% sodium carbonate) at 380, 415, and 450 nm. Amount of plasma hemoglobin was calculated from the equation given below: mg , dl [(2 × A/01 ) − (A345 + A/15 )] × 1000 × dilution factor = … … … (3) E × 1.655

Plasma hemoglobin *

where, A380, A415, and A450 are values of absorbance at 380, 415, and 450 nm, respectively. The correction factors, A380 and A450 are applied due to absorption by uroporphyrin in the same range of wavelength as that of hemoglobin and 1.655 for interference from turbidity of plasma. A415 denotes the sorbent band absorption of oxyhemoglobin and E, the molar absorptivity of oxyhemoglobin at 415 nm ie, 79.46. The percentage hemolysis of AgNP, AuNP and E-GNS was obtained from the equation given below: Hemolysis (%) =

Plasma hemoglobin value of test sample × 100 … … … (4) Total hemoglobin value of blood

RESULTS AND DISCUSSION Embelin, a benzoquinone derivative is a potent antioxidant, hence is able to reduce metal ions to metallic atoms.20,23 This study is focused on the potential of embelin extracted from fruits of Embelia tsjeriam-cottam in the synthesis of quasi-spherical and anisotropic gold nanostructures such as gold nanostars for CT imaging and photothermal therapy as well as silver nanoparticles with antibacterial activity, as depicted in Figure 1.


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Figure 1. Schematic representation of (A) extraction of embelin from the fruits of Embelia tsjeriam-cottam and (B) synthesis of embelin stabilized silver nanoparticles (AgNP), gold nanoparticles (AuNP) and gold nanostars (E-GNS) using embelin as a reducing and stabilizing agent. Embelin crystals of orange in color were extracted from the fruits of Embelia tsjeriam-cottam which readily dispersed in ethanol to form an orange color solution, as displayed in the supporting information (inset of Figure S1). The purity of the extracted compound in comparison to the standard was determined by spectrophotometric studies, wherein the extracted embelin showed a spectrum similar to the standard embelin with λmax at 288 nm, as shown in supporting information (Figure S1). Similarly, the spectra of extracted and standard embelin were also found to be similar in LC and MS-MS as determined in our previous studies.30 The extracted embelin dispersed in ethanol was used for the synthesis of silver and gold nanoparticles at varying reaction conditions. The addition of embelin to silver nitrate solution failed to form silver nanoparticles in the dark as well as ambient conditions of the laboratory. A similar observation was recorded even at high



temperature conditions. However, when the reaction solution was exposed to bright sunlight, the color changed from transparent to yellow and then to black within few minutes, as displayed in the inset of Figure 2A and 2B. The color change indicated the formation of silver nanoparticles which was then confirmed spectrophotometrically with an SPR λmax at 418 nm, as shown in Figure 2A. This signified the crucial role of sunlight in the formation of silver nanoparticles, and embelin, being photostable, did not undergo degradation on irradiation.35 The photoreduction of silver ions to silver nanoparticles is hypothesized to be that of ligand to metal charge transfer (LMCT) as in photoactive complexes of metal.36 Ag ions form complex with embelin due to electrostatic attraction between negatively charged embelin and positively charged Ag ions. Sunlight irradiation of the complex results in the homolytic cleavage of phenolic O-H bond of embelin generating hydrogen radical and transfer of its electron to the bound silver ion (Ag+) forming silver nanoparticle.37 However, the oxygen radical stabilizes itself by extended conjugation in the solution.37,38 A nanoparticle is generally formed by nucleation and their subsequent growth. The nucleation process occurs only when the concentration of reactant species reaches supersaturation. Following the initial process of nucleation, the concentration of the growth species decreases below the specific concentration of supersaturation, resulting in the stoppage of nucleation; however, the growth continues till the growth species attains the equilibrium concentration. The growth process of the nuclei during the formation of nanoparticles is either diffusion controlled or kinetically controlled. The processes of nucleation and growth determine the size and shape of the nanoparticles; hence, the parameters which affect these processes were optimized. The amount of embelin required for the formation of silver nanoparticles for a given concentration of silver nitrate solution were optimized by exposing the reaction solutions to sunlight for a definite time period and then analyzed spectrophotometrically. The absorbance intensity was found to increase with the increasing amount of embelin and was saturated at 60 µl (Figure 2A). The variation in absorption spectra with varying quantities of embelin is attributed to the rate of nucleation and subsequent growth of silver nanoparticles.9 The growth of silver nanoparticles occurs through a kinetically controlled process of Ostwald ripening (growing of larger structures at the expense of smaller ones).39 This growth via Ostwald ripening can be hindered with surface capping agents such as embelin.39 At low concentrations, as less amount of embelin was available for stabilization of formed nanoparticles, the small spherical nanoparticles grew to


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larger anisotropic structures by Ostwald ripening giving rise to spectral peaks in the NIR region, as can be seen in Figure 2A. However, at larger concentrations (above 60 µl), sufficient quantity of embelin was available for appropriate stabilization of formed spherical nanoparticles which accounts for the narrower peak with decreased absorbance in the NIR region (Figure 2A). The reaction parameters such as duration of sunlight exposure and pH were also varied in order to optimize the reaction conditions. As shown in Figure 2B, the intensity of absorbance was found to increase with increasing duration of sunlight exposure up to 15 min. This is attributed to the fact that upon a longer duration of sunlight exposure, a large number of photons interact with embelin resulting in enhanced nucleation and in turn increasing the intensity of absorbance. However, a decrease in intensity was noted after 20 min of exposure which could be due to degradation of embelin by the heat developed from prolonged exposure to sunlight.35 Hence, an exposure to 15 min of sunlight was found to be sufficient for the photoreduction of silver ions. The silver nanoparticles were synthesized at varying pH with 60 µl of embelin and 15 min of exposure to sunlight and analyzed spectrophotometrically, as depicted in Figure 2C. As observed, a pH of 12 was found to be optimum for a high yield synthesis of silver nanoparticles with narrow absorbance spectra. This is attributed to the fact that the protonation of functional groups of embelin at lower pH reduces its magnitude of negative charge which decreases the sites available for association with Ag ions.37,38 The strong binding of Ag ions to embelin at higher pH facilitates enhanced electron shuttling between complexed Ag ions and embelin up on irradiation, resulting in the increased nucleation of silver nanoparticles. This burst of nucleation on sunlight exposure at higher pH (pH 12) results in temporal separation of the nucleation and growth step.39 Hence, a narrow size distribution of nanoparticles with narrow absorbance spectra having no peaks in the NIR region can be observed even with low sunlight exposure (Figure 2D) as well as lower concentrations of embelin (Figure 2E and supporting information, Figure S2). The reaction parameters such as time of sunlight exposure and amount of embelin were again optimized at pH 12. As can be seen from Figure 2D, 2E and supporting information (Figure S2), 50 µl of embelin at pH 12 with 15 min of exposure to sunlight yielded the maximum amount of silver nanoparticles with narrow absorbance spectra. All further studies were carried out with embelin stabilized silver nanoparticles (AgNP) formed by 50 µl of embelin at pH 12 and 15 min of exposure to sunlight. The optical dark field



hyperspectral image of AgNP in Figure 2F revealed a homogeneous dispersion observed as bright spots of different size suggesting the variation in size of the nanostructure. The variation in the size of AgNP was also confirmed by FEG-TEM imaging, wherein the quasi-spherical nanoparticles of two different sizes can be seen. This kind of bimodal distribution of size occurs as all the nuclei are not formed at the same time due to the dependence of nucleation process on sunlight exposure. Moreover, the slow chemical reaction decreases the availability of growth species resulting in growth of nanoparticles via a diffusion controlled process.39 This prevents the variation in size among the two different groups of particles. Larger silver nanoparticles of ~ 15 nm and smaller nanoparticles of ~ 3-4 nm, which are separated by differential centrifugation, can be seen in Figure 3A-C and 3D-E, respectively. The high resolution TEM images observed in Figure 3B-C and 3D clearly depict the highly crystalline nature of the particles. A quasispherical shape of the nanoparticles, especially highly faceted icosahedron can be seen in the high resolution TEM images (Figure 3B and 3C). The formation of this kind of isotropic particles is thermodynamically favored to minimize the interfacial free energy of the system.40 During the growth of particles in materials with low stacking fault energy such as silver and gold, twins or stacking faults, are readily formed, as shown in Figure 3C. Small nanocrystals with multiple twinning are the most favorable crystal structures due to the fact that the most stable (111) faces originating from two subgrains looking like a mirror image and sharing a common crystallographic plane surround such particles.40 The presence of these kinds of planar defects introduces self-propagating ledges which can be active sites for the growth of crystals.40,41 As the multi-twinned seed grows in size, the strain energy caused by twin defects will increase considerably; hence, multi-twinned seeds are only favored by thermodynamics at relatively small sizes.40 This critical dependence on size suggests that the formation of particles is not just controlled by thermodynamics but also experimentally manipulated by reaction kinetics.40 As described earlier, the kinetically controlled synthesis of silver nanoparticles was achieved by Ostwald ripening resulting in the growing of larger structures at the expense of smaller ones.39,40 Hence, a wide variation in size (5-50 nm) was observed with silver nanoparticles synthesized without altering the pH of reaction (pH 5) (Figure 3F). The presence of silver in the nanoparticles was confirmed by EDAX analysis, as shown in supporting information (Figure S3) along with the presence of elements such as carbon, oxygen of embelin; and those associated with the copper grid.


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Figure 2. Absorbance spectra of silver nanoparticles synthesized with (A) variation in volume of embelin (10 mg/ml) at constant precursor concentration (1mM AgNO3) and sunlight exposure (15 min) without alteration of pH (pH 5), (B) variation in duration of sunlight exposure at constant precursor concentration (1mM AgNO3) and volume of embelin (60 µl of 10 mg/ml) without alteration of pH (pH 5), (C) variation in pH at constant precursor concentration (1mM AgNO3), sunlight exposure (15 min) and volume of embelin (60 µl of 10 mg/ml), (D) variation in duration of sunlight exposure at constant precursor concentration



(1mM AgNO3), pH (12) and volume of embelin (60 µl of 10 mg/ml), (E) variation in volume of embelin at constant precursor concentration (1mM AgNO3), pH (12) and sunlight exposure (15 min), (F) Hyperspectral dark field optical image of AgNP. Inset (A-E) shows the photograph of respective silver nanoparticles. Embelin volumes 10, 20, 30,…,90 µl of 10 mg/ml correspond to 0.1, 0.2, 0.3,…,0.9 mg of absolute embelin.

Figure 3. FEG-TEM images at various magnifications of (A-C) larger, (D-E) smaller silver nanoparticles (AgNP) obtained by differential centrifugation after synthesis at pH 12 and (F) silver nanoparticles synthesized without alteration of pH (pH 5). Inset of (B) shows the SAED pattern of AgNP. Multiple twin planes are indicated by white arrows in (C). Similarly, embelin also aids in the formation of gold nanoparticles. However, the reaction did not require any irradiation with sunlight and the gold precursor solution upon the addition of embelin, gradually changed its color from yellow to brownish black and then to purple-red as can be seen in the inset of Figure 4A and supporting information (Figure S4). Initially, reduction of gold ions produced small-sized unstable gold seeds, the nucleation stage, which is marked by the change of color from yellow to brownish black. The color then changed to purple-red when these seeds grew to form stable nanoparticles with characteristic SPR λmax at 540 nm, as shown in Figure 4A. The reaction parameters such as temperature, pH and amount of embelin were varied to optimize the reaction conditions which could yield the maximum amount of gold


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nanoparticles. As deciphered from Figure 4A and supporting information (Figure S4), with the increasing amount of reducing agent, an increase in absorbance intensity is observed which saturates beyond 50 µl. Since the reduction of gold ions occurs at ambient conditions using a weak reducing agent (embelin), nucleation process is slow and embelin is able to stabilize the formed nanoparticles even at lower concentrations resulting in no absorbance in the NIR region. However, when more than 50 µl of embelin was added to the gold precursor solution, reduced gold ions grew in an anisotropic fashion on the gold seed nuclei characterized by longitudinal surface plasmon resonance (LSPR) peak in the NIR region. This was further confirmed by the presence of anisotropic structure in the FEG-TEM image analysis, as shown in supporting information (Figure S5). At higher concentrations of embelin, the nucleation stops due to reduced precursor concentration and only growth continues by the process of secondary reduction on the surface of the preformed nuclei resulting in the formation of larger AuNP.39 The formation of gold nanoparticles was also observed with embelin at higher pH but without any significant increase in the yield; hence, the alteration in pH was not preferred for synthesis. The reaction rate was found to increase with higher temperature resulting in the formation of gold nanoparticles in a shorter period of time. Similarly, gold nanoparticles formed instantaneously within 30 s on microwaving the reaction solution. These gold nanoparticles formed at high temperatures with reduced reaction time exhibited similar absorbance spectra as compared to those formed at room temperature indicating no change in size or shape of nanoparticles. However, as embelin is reported to degrade at higher temperatures,35 gold nanoparticles synthesized at room temperature was preferred for further studies. These embelin stabilized gold nanoparticles (AuNP) formed with 50 µl of embelin at room temperature were then utilized as a seed to synthesize gold nanostars. AuNP was preferred over AgNP as a seed for the synthesis of gold nanostars as these were found to be more biocompatible than silver. AuNP was used as a seed along with silver ions for directional growth and ascorbic acid for instant reduction to synthesize gold nanostars which were further stabilized with embelin yielding embelin stabilized gold nanostars (E-GNS). During the synthesis, ascorbic acid instantly reduced the gold atoms on to AuNP along with silver atoms which adsorbed on the growing gold particle preventing its isotropic growth giving rise to bluish-green colored gold nanostars. Embelin was added during the generation of this particle for stabilization. However, the particles were not found to be stable until the pH was increased to 12. This is attributed to the protonation



of functional groups of embelin at lower pH which reduces its magnitude of negative charge, in turn, decreasing the sites available for its association with the formed nanostars. The increase in pH thus helps in better attachment of embelin to gold nanostars yielding stable nanoparticles. Halide ions (chloride from HCl) along with silver ions aided the shape driven growth of particles.42 Furthermore, the addition of reagents at an appropriate time and ratio played an important role in the generation of E-GNS. Silver ions added early or late to ascorbic acid resulted in precipitation of black colored silver chloride or large gold spheres, respectively with no generation of star-shaped structures.33 The preferred ratio of ascorbic acid to gold precursor solution was above 1.5: 1 for the appropriate reduction and development of E-GNS.43 Similarly, embelin was added only after the appearance of bluish-green color to avoid any hindrance in the proper formation of gold nanostars. E-GNS absorbs over a wide wavelength range from 6001100 nm, however, can be tuned to achieve an SPR λmax of 790-810 nm, the preferred NIR therapeutic window having the least absorbance from biomolecules. This bluish green colored EGNS (inset of Figure 4B) shows an absorbance with SPR λmax at 800 nm, as can be seen in Figure 4B. The hyperspectral imaging of AuNP and E-GNS, as observed in Figure 4C and 4D, shows the homogenous dispersion of white spots with a λmax of scattering spectra at 550 nm and 820 nm, respectively.


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Figure 4. Absorbance spectra of (A) gold nanoparticles synthesized at room temperature with variation in volume of embelin (10 mg/ml) at constant precursor concentration (1mM HAuCl4); and (B) embelin stabilized gold nanostars (E-GNS). Inset shows the photograph of respective nanoparticles. Hyperspectral dark field optical image of (C) embelin stabilized gold nanoparticles (AuNP) and (D) E-GNS. . Embelin volumes 10, 20, 30,…,90 µl of 10 mg/ml correspond to 0.1, 0.2, 0.3,…,0.9 mg of absolute embelin. The TEM images in Figure 5A-B and 5C revealed a quasi-spherical polyhedron morphology with size of ~ 12-15 nm for AuNP and a branched star morphology of ~ 120 nm in size for EGNS, respectively. The high resolution images and SAED pattern (inset) in Figure 5B and 5D depicts the highly crystalline lattice structure of AuNP and E-GNS. The polyhedral morphology of AuNP with multiple twinning can be seen in Figure 5B. These multi-twinned particles as in case of silver are only favored by thermodynamics because of its relatively smaller size.40 These



highly faceted gold nanoparticles with twin planes may furnish proper lattice surfaces as favorable sites for further growth and facilitate the formation of branched morphology.41 The EDAX spectra of AuNP and E-GNS (supporting information, Figure S6A and S6B) revealed the presence of gold along with carbon and oxygen of embelin. The presence of silver can be seen in the EDAX spectra of E-GNS (Figure S6B).

Figure 5. FEG-TEM images of (A-B) AuNP and (C-D) E-GNS at various magnifications. Inset shows the low magnification image (left) and SAED pattern (right) of respective nanoparticles. Synthesis of silver, gold nanoparticles as well as gold nanostars carried out using the standard embelin also yielded similar results (representative absorbance data of gold nanoparticle shown in supporting information, Figure S7) implying no alteration in the mechanism of formation of plasmonic nanostructure synthesized from embelin acquired from other sources or from trace


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quantities of other constituents present in the extracted embelin. The conversion yield of Ag+ to Ag in silver nanoparticles and Au3+ to Au in case of AuNP and E-GNS was found to be 92.2 ± 2%, 94.3 ± 4% and 89 ± 3%, respectively with the help of ICP-AES analysis. The decrease in yield of conversion could be due to loss of nanoparticles in the supernatant upon centrifugation. The aqueous dispersions of AgNP, AuNP and E-GNS were found to be extremely stable with a zeta potential of -30.96 ± 1.13, -46.09 ± 1.08 and -27.03 ± 1.33 mV; and a hydrodynamic diameter of 50 ± 9, 97 ± 7 and 150 ± 5 nm, respectively (data not shown). The nanoparticles exhibited good stability at varying pH conditions (supporting information, Figure S8A) as well as in PBS, saline and culture medium over extended periods as long as 6 weeks (supporting information, Figure S8B and S8C). The high crystallinity of all the nanostructures, namely, AgNP, AuNP and E-GNS was also confirmed by their XRD spectra which depict all characteristic peaks in accordance with the JCPDS database, as can be seen in supporting information (Figure S9 and S10). The broadening of peaks in the spectra further confirms the nanosize of the crystalline structures. The FTIR analysis was performed to analyze the functional groups present on the surface of AuNP, AgNP and E-GNS. As depicted in Figure 6A, the spectra of AuNP, AgNP and E-GNS have absorption bands that are attributed to those of embelin, revealing its presence on the nanoparticles. The band at 3415 cm-1 in the spectra corresponds to O-H stretching vibration and the bands at 2921 and 2850 cm-1 are due to the stretching vibration of C-H group in aromatic and methyl group, respectively.44,45 The bands at 1641 and 1615 cm-1 correspond to the C=O stretching vibration. The decrease in stretching frequency of 1615 cm-1 band in case of AuNP, AgNP and E-GNS indicate the coordination of embelin to metal ion through the carbonyl oxygen atom.44 The bands at 1462, 1375 and 1019 cm-1 attributed to CH bending in –CH2, CH bending in –CH3, C=O stretching vibration, respectively, were retained in all the samples.45 The narrowing of the broad peak at 3415 cm-1 in AuNP, AgNP and E-GNS indicate the loss of phenolic hydrogen on chelation with the metal. This was further confirmed from NMR spectra of embelin, AgNP, AuNP and E-GNS, as shown in supporting information (Figure S11-14). The 1H NMR spectrum of embelin showed triplet peaks at δ0.87 ppm corresponding to three terminal protons suggesting a methyl group.44,46 The chemical shifts observed in the spectra from δ0.9 -1.2 ppm suggest the protons from aliphatic groups and δ2.4 ppm correspond to the aliphatic proton nearest to the ring structure of embelin.44,46 The peak observed at δ5.35 ppm is due to the



phenolic proton (hydroxyl group), and singlet peak at δ7.2 ppm integrates the single proton in the ring.47 The NMR spectra of AuNP, AgNP and E-GNS demonstrated all the chemical shifts similar to embelin except the shift at δ5.35 ppm corresponding to hydrogen of phenolic group suggesting the linkage of embelin to metal at this position with the loss of phenolic hydrogen. These analyses thus confirm the presence of embelin on AuNP, AgNP and E-GNS. The embelin content in AuNP, AgNP and E-GNS were found to be 25 ± 4, 30 ± 3 and 20 ± 5%, as determined by CHN elemental analysis. These plasmonic nanoparticles stabilized with embelin can be used for various biomedical applications. Herein, we analyzed the antibacterial activity of AgNP and the potential of E-GNS in CT imaging as well as photothermal therapy. E-GNS were analyzed for its photothermal efficacy by evaluating the increase in temperature upon laser irradiation, as can be seen in Figure 6B. E-GNS showed a rise in temperature to 43 °C (critical temperature for tumor cell death) within 2.5 min; however, water achieved only a 1 °C rise in temperature (38 °C) in the same time period. This kind of high rise in temperature is due to the thin, elongated as well as sharp branches of gold nanostars which enable the easy penetration of the incoming electric field and results in massive heating up of the entire gold matter.48,49 The photothermal transduction efficiency of E-GNS was evaluated to be 60.2 ± 4% by determining the thermal time constant from graph (time versus natural logarithm of temperature in the cooling period) shown in supporting information (Figure S15), optical density, temperature rise in a given time period, etc. using calculations (supporting information) as described in previous studies.33,50 The specific absorption rate (SAR) of E-GNS was found to be 6.25 ± 0.3 kW/g at 1.3 W/cm2 of laser power. E-GNS did not exhibit any change in SPR λmax and absorbance intensity after the photothermal transduction experiment implying its high photothermal stability (supporting information, Figure S16). For CT imaging applications, E-GNS was evaluated in comparison to the clinically used polyiodinated compound, Omnipaque. Both the materials were imaged at equal concentrations of iodine and gold with a tube current of 200 mA. E-GNS exhibited a visibly brighter contrast in comparison to Omnipaque at concentrations above 0.5 mg ml-1, as can be seen in Figure 6C. EGNS exhibited a 1.5 times higher intensity than Omnipaque of same material concentrations upon quantification in Hounsfield units, as depicted in Figure 6D. The better attenuation of X


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rays by gold due to its high Z-number results in the enhanced contrast of E-GNS, hence, a less amount of material provides the desired contrast thus reducing issues related to toxicity.51

Figure 6. (A) FTIR spectra of embelin, AgNP, AuNP and E-GNS, (B) graphical representation of temperature rise in the photothermal transduction experiment of water (H2O) and E-GNS (50 µg/ml gold concentration) with 808 nm laser (1.3 W/cm2), (C) CT contrast images of E-GNS and Omnipaque with varying concentrations (mg/ml denotes the concentration of gold for E-GNS and iodine for Ominpaque), (D) graphical representation of computed Hounsfield unit values of Omnipaque and E-GNS. Silver nanoparticles were found to show antibacterial activity against Gram-positive bacteria (Staphylococcus aureus) as well as Gram-negative bacteria (Escherichia coli), as shown in



Figure 7(A-F). The antibacterial study was carried out with small-sized AgNP (~3 nm) as smaller silver nanoparticles are reported to possess high antibacterial activity compared to their larger counterparts.52–54 The surface area to volume ratio increases with decreasing size of the nanoparticle and thus in case of smaller silver nanoparticles, large surface area of AgNP is able to come in contact with the bacteria leading to increased permeability and entry of AgNP into the cells in turn enhancing their antibacterial activity. The disc diffusion assay (Figure 7A-B) revealed an increased antibacterial activity with the increase in concentrations of AgNP in both the strains of bacteria. The antibacterial activity of silver nanoparticles has been widely studied; however, its exact mechanism of action is still uncertain. The antibacterial activity of silver nanoparticles is mainly by its attachment to cell membrane, subsequently gaining entry into the cell, leading to damage of membrane, leakage of cell constituents, and finally resulting in the shrinkage of cells.55,56 Another potential mechanism is by the generation of silver ions resulting in protein denaturation by binding to sulfhydryl groups and reduction of the disulfide bonds (S–S → S–H+H–S).55 Moreover, silver ions are also known to complex with electron donor groups such as nitrogen, oxygen or sulfur present in amino acids, phosphates on nucleic acids, thiols, etc.56 Silver nanoparticles are also reported to develop increased levels of ROS, resulting in oxidative stress which causes damage to the cell membrane, DNA, proteins, and intracellular systems such as respiratory chain dehydrogenases, thereby preventing bacterial respiration and reproduction.57–59 However, embelin, being a free radical scavenger, would minimize the levels of ROS in cells (detailed in the ensuing section on ROS) and, silver ions, being complexed with embelin, are not easily released. Hence, the antibacterial activity of AgNP is considered mainly by its damage to bacterial cell membranes, as can be observed in Figure 7(C-F). A typical spherical and rod shaped morphology with smooth surface as well as intact membrane structure was observed in untreated S. aureus (Figure 7C) and E. coli (Figure 7E) bacterial cells, respectively. However, the bacteria treated with AgNP displayed distorted morphologies with loss of membrane integrity. Bacterial cells with multiple perforations were seen in AgNP treated S. aureus (Figure 7D), whereas fragmented membranes and leaky cellular contents of massively ruptured cells were observed in E.coli (Figure 7F). AgNP was observed to exhibit better antibacterial activity against the Gram-negative E. coli than the Gram-positive S. aureus, as shown in Figure 7(A-B), which could be due to the presence of thick peptidoglycan layer of S. aureus preventing the action of silver nanoparticles through the cell wall of bacteria.60 Silver


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nanoparticles unlike conventional antibiotics are active against both Gram-positive and Gramnegative bacteria and do not confront issues such as side effects or multidrug resistance.61 Additionally, embelin, a natural, non-toxic compound with potent antibacterial activity will potentiate the action of silver nanoparticles resulting in a synergistic activity. Hence, embelin stabilized silver nanoparticles (AgNP) could be a better alternative to conventional antibiotics. However, detailed analysis to evaluate the antibacterial efficacy of AgNP needs to be carried out for its potential application.



Figure 7. Representative photographs of agar plates in disc diffusion assay to study the effect of AgNP on (A) S. aureus and (B) E. coli. ZOI - Zone of inhibition. Cryo-FEG-SEM images of (C-D) S. aureus and (E-F) E. coli both untreated (left) and treated (right) with 5 µg/ml of AgNP. Inset (C-F) shows the low magnification images of bacteria. To advocate the use of these nanoparticles for the aforementioned applications, it needs to be non-toxic and biocompatible in nature. The cytocompatible nature of these nanostructures was confirmed by cell viability assay. The cell viability analysis of KB, L929 and NIH3T3 cells using Alamar blue shown in Figure 8, exhibited a cell viability percentage above 80 even at high concentrations for all the nanoparticles implying no major adverse effects on the cellular metabolic activity. The excellent compatibility of AgNP to cells at very high concentrations such as 5-10 µg/ml is quite intriguing, wherein the silver nanoparticles synthesized by conventional methods show toxicity even at a low concentration of 0.5 µg/ml.62 The toxicity of silver nanoparticles is mainly attributed to surface oxidation due to lack of proper coating or its loss or easy displacement due to non-covalent binding.63 The release of free silver ions into cells which were adsorbed on to the nanoparticles and generation of free radicals upon interaction with biological media or macromolecules are also considered to cause toxicity.62,63 Embelin stabilized silver nanoparticles (AgNP) obviate these mechanisms of toxicity by providing a protective surface coating and tight complexation of silver ions with embelin preventing its release as well as by scavenging the free radicals, thus decreasing oxidative stress. Hence, embelin stabilized silver nanoparticles are found to be less toxic in comparison to its counterparts, and thus this kind of green synthetic approach using embelin open up new opportunities for producing nontoxic and potent silver nanoparticles.

Figure 8. Graphical representations of cell viability tests on L929, NIH 3T3 and KB cells treated with (A) AgNP, (B) AuNP and (C) E-GNS at varying concentration for 24 h. NC negative control (untreated cells) and PC - positive control (cells treated with 1% Triton X100).


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The development of reactive oxygen species (ROS) by E-GNS, AuNP and AgNP in KB cells were evaluated by H2DCFDA using flow cytometry to determine and negate the presence of ROS in cells produced by nanostructures. Figure 9 (A-E) shows the high level of ROS with 97.7 % in KB cells treated with H2O2 (positive control), whereas cells treated with high concentrations of E-GNS (100 µg/ml), AuNP (100 µg/ml) and AgNP (5 µg/ml) exhibited only negligible levels (0.1 %) similar to the negative control (cells treated only with media). This may be as a result of radical scavenging and antioxidant property of embelin which is linked to the nanostructures.20,23 The digestion of cells treated with nanostructures by acids and evaluation by ICP-AES analysis confirmed the high uptake (~75%) of nanostructures by cells. The photothermal efficacy of E-GNS on KB cells was evaluated by Alamar blue assay wherein, the cells treated with only laser or only E-GNS did not exhibit any significant cell death. However, laser irradiation of cells treated with E-GNS resulted in marked cell death, and the number of viable cells decreased with increasing duration of laser exposure, as seen in Figure 9F. Similarly, qualitative analysis of photothermal mediated cytotoxicity on KB cells was carried out by microscopic imaging of cells stained with propidium iodide after different modes of treatment. Propidium iodide dye (PI) is taken up specifically by dead cells and stains them red under a fluorescent microscope, however being membrane impermeant, the viable cells remain unaffected. Figure 10 shows the microscopic images of KB cells subjected to different modes of treatment, wherein the left, middle, right columns depict DIC, fluorescent and merged images, respectively. The untreated cells (negative control) and those treated only with the laser for 7.5 min did not exhibit any significant cell death (no red colored cells due to PI in the fluorescent image). However, cells subjected to E-GNS treatment and subsequent laser irradiation for 7.5 min resulted in marked cell death (PI stained dead cells in red color) demonstrating the photothermal efficacy of E-GNS.



Figure 9. Flow cytogram illustrating intracellular ROS generation with dichlorofluorosceindiacetate (DCFH-DA) assay in KB cells treated with (A) media alone (negative control), (B) 30 µM H2O2 (positive control), (C) E-GNS (100 µg/ml), (D) AuNP (100 µg/ml) and (E) AgNP (5 µg/ml). (F) Graphical representation of in vitro photothermal experiment showing cell viability of KB cells treated with E-GNS (P) and laser (L) for different time duration. NC represents negative control (untreated cells), while PC represents positive control (cells treated with 1% Triton X-100).


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Figure 10. Microscopic images showing photothermal mediated cytotoxicity of KB cells treated with (A−C) media alone, (D−F) only laser for 7.5 min, (G−I) E-GNS and (J−L) E-GNS + laser for 7.5 min. Left column shows DIC image, middle column present fluorescent images with PI stain (dead cells in red), and right exhibit the merged images of left and middle showing dead cells due to photothermal treatment. The physiological administration of these nanostructures for its potential biomedical application warrants the need for blood compatibility analysis. The compatibility of these structures to blood



was analyzed by evaluating the hemolysis, i.e., rupturing of red blood cells, as well as microscopic evaluation of blood cell morphology upon contact with the particles. Figure 11A shows the graph depicting no hemolytic response of blood when treated with varying concentrations of AgNP, AuNP and E-GNS, whereas blood treated with 1% Triton X-100 (positive control) showed ~ 100% of hemolysis. This kind of hemolytic response was also observed visually, as can be seen in the representative photograph in the inset of Figure 11A. A clear plasma is seen in the supernatant with undamaged RBC sediment at the bottom in blood samples treated with nanostructures similar to negative control (0.9% saline), whereas a reddish supernatant is observed in positive control due to leakage of hemoglobin from RBC. This was also confirmed by microscopic imaging of blood samples after Leishman staining, as depicted in Figure 11B. Blood samples treated with AgNP, AuNP and E-GNS did not exhibit any change in the morphology of blood cells similar to the negative control; however, positive control revealed rupturing and aggregation of RBC. Hence, these studies clearly demonstrate the excellent compatibility of the silver and gold nanostructures to cells as well as human blood.

Figure 11. (A) Graphical representation of in vitro hemolysis study of AgNP (0.5, 1, 3, and 5 µg/ml), AuNP and E-GNS (10, 25, 50, 75 and 100 µg/ml) with human blood. Inset shows representative photograph of blood samples treated with varying concentrations of E-GNS exhibiting clear plasma indicating no hemolysis similar to the sample treated with 0.9% saline (negative control - NC), whereas blood sample treated with 1% Triton X-100 (positive control)


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shows red colored plasma supernatant indicating leakage of hemoglobin. (B) Microscopic image of Leishman stained RBC treated with 0.9% saline (negative control), 1% Triton X-100 (positive control), AgNP (5 µg/ml) AuNP (100 µg/ml) and E-GNS (100 µg/ml). CONCLUSION Noble metal nanoparticles synthesized conventionally with hazardous chemicals raise huge concern for its use for biomedical applications. Additionally, studies conducted so far using green chemistry mainly focuses on the synthesis and stabilization of isotropic nanoparticles with limited thrust on biological applications. This study hence, reports the synthesis of various biocompatible noble metal nanoparticles such as quasi-spherical gold and silver nanoparticles as well as anisotropic gold nanostars using a medicinal compound - embelin and demonstrated its potential antibacterial, CT imaging and photothermal activity. Embelin stabilized silver nanoparticles of bimodal size distribution (3 and 15 nm) were synthesized by a sunlight assisted method and exhibited marked antibacterial activity against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. Similarly, embelin stabilized gold nanoparticles of 12-15 nm in size and polyhedral morphology with an SPR λmax at 540 nm were synthesized at room temperature. These highly faceted and multi-twinned gold particles then served as a seed for attachment and reduction of gold ions in the presence of embelin to form 120 nm-sized gold nanostars absorbing at NIR wavelength of 800 nm. The nanostructures exhibited excellent compatibility towards cells (L929, NIH3T3 and KB) and human blood. The gold nanostars displayed superior CT imaging characteristics as well as marked photothermal cytotoxicity towards cancerous KB cells. Hence, this study puts forth a green approach for the synthesis of biocompatible noble metal nanoparticles using embelin and its potential use as antibacterial, CT imaging and photothermal agent for cancer. ASSOCIATED CONTENT Supporting Information Absorbance spectra of embelin, silver nanoparticles, gold nanoparticles (synthesized from standard and extracted embelin) and E-GNS (before and after laser irradiation in photothermal transduction experiment); FEG-TEM images of gold nanoparticles; photograph of stability studies, DLS data, zeta potential profile in buffers, EDAX and XRD of AgNP, AuNP and E-



GNS; 1H NMR spectra of embelin, AgNP, AuNP, and E-GNS and calculation of photothermal efficiency of E-GNS. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes Authors declare no competing financial interest. PRESENT ADDRESS * Dhirendra Bahadur - Indian Institute of Technology Goa, Farmagudi, Ponda, Goa, 403401, India.

ACKNOWLEDGMENTS Authors gratefully acknowledge Nanavati Super Speciality Hospital, Mumbai for helping with CT imaging of samples. Authors also thank sophisticated analytical instrument facility (SAIF), IRCC and Department of Physics, IIT Bombay for the characterization facilities. REFERENCES (1)

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ABSTRACT GRAPHIC

SYNOPSIS A green synthesis method using Embelin, a medicinal compound, is developed for synthesis of quasi-spherical and star-shaped plasmonic nanostructures as antibacterial, photothermal and CT imaging agents.


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Embelin Mediated Green Synthesis of Quasi-spherical and Star

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