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May 29, 2018 - ... Jamia Millia Islamia, New Delhi, India; gCancer Research Division, Rajiv Gandhi. Centre for Biotechnology, Thiruvananthapuram, Kerala, ...

Drug and Chemical Toxicology

ISSN: 0148-0545 (Print) 1525-6014 (Online) Journal homepage: http://www.tandfonline.com/loi/idct20

Biosynthesized composites of Au-Ag nanoparticles using Trapa peel extract induced ROS-mediated p53 independent apoptosis in cancer cells Naheed Ahmad, Abhay K. Sharma, Seema Sharma, Imran Khan, Dhananjay K. Sharma, Ayesha Shamsi, T. R. Santhosh Kumar & Mahendra Seervi To cite this article: Naheed Ahmad, Abhay K. Sharma, Seema Sharma, Imran Khan, Dhananjay K. Sharma, Ayesha Shamsi, T. R. Santhosh Kumar & Mahendra Seervi (2018): Biosynthesized composites of Au-Ag nanoparticles using Trapa peel extract induced ROSmediated p53 independent apoptosis in cancer cells, Drug and Chemical Toxicology, DOI: 10.1080/01480545.2018.1463241 To link to this article: https://doi.org/10.1080/01480545.2018.1463241

Published online: 29 May 2018.

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DRUG AND CHEMICAL TOXICOLOGY, 2018 https://doi.org/10.1080/01480545.2018.1463241

RESEARCH ARTICLE

Biosynthesized composites of Au-Ag nanoparticles using Trapa peel extract induced ROS-mediated p53 independent apoptosis in cancer cells Naheed Ahmada, Abhay K. Sharmaa, Seema Sharmab, Imran Khanc,d, Dhananjay K. Sharmad,e, Ayesha Shamsif, T. R. Santhosh Kumarg and Mahendra Seervia a Department of Botany/Biotechnology, Patna University, Patna, Bihar, India; bDepartment of Physics, A. N. College, Magadh University, Patna, Bihar, India; cDepartment of Chemistry, College of Science, Sultan Qaboos University, Muscat, Oman; dCICECO – Aveiro Institute of Materials, Chemistry Department, University of Aveiro, Aveiro, Portugal; eTEMA – Department of Mechanical Engineering, University of Aveiro, Aveiro, Portugal; fDepartment of Electrical and Electronic Engineering, Jamia Millia Islamia, New Delhi, India; gCancer Research Division, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India

ABSTRACT

ARTICLE HISTORY

The current study highlights rapid, sustainable, and cost-effective biosynthesis of silver (Ag), gold (Au) nanoparticles (NPs), and bimetallic Au-AgNPs composites using bio-waste extract of Trapa natans. Growth of the NPs was monitored spectrophotometrically and peak was observed at 525 nm, 450 nm, and 495 nm corresponding to Plasmon absorbance of AuNPs, AgNPs, and Au-AgNPs, respectively. Transmission electron microscopy (TEM) revealed the size of AgNPs (15 nm), AuNPs (25 nm), and Au-AgNPs (26–90 nm). Synthesized NPs follow the Gaussian bell curve and its crystalline nature was identified by X-ray diffraction (XRD). Furthermore, Au-AgNPs induced cytotoxicity in various cancer cells (HCT116, MDA-MB-231, and HeLa) effectively at 200 lg/mL. Au-AgNPs-exposed cancer cells exhibited apoptotic features such as nuclear condensation, mitochondrial membrane potential loss, and cleavage of casp-3 and poly (ADP-ribose) polymerase-1 (PARP). Au-AgNPs exposure enhanced reactive oxygen species (ROS) and upon inhibition of ROS, apoptosis was reduced effectively. NPs treatment killed HCT116 WT and p53 knockout cells without any significant difference. Mechanistically, AuAgNPs derived with Trapa peel extract significantly enhance ROS which trigger p53-independent apoptosis in various cancer cells effectively. Our study explores the use of bio-waste for the green synthesis of NPs, which can be attractive candidates for cancer therapy.

Received 13 February 2018 Revised 5 April 2018 Accepted 6 April 2018

Introduction Metal nanoparticles (NPs) especially gold and silver nanoparticles (AuNPs & AgNPs) are utilized in cancer diagnosis and treatment owing to their unusual optoelectronic and physiochemical attributes such as size dependent properties, tunable surface chemistry, and solvent stability stability (Gil and Parak 2008). From past one decade, bimetallic functional composite of NPs have emerged as a better tool for bioimaging and drug delivery in cancer treatment (Song et al. 2016). Such composite has an assembly of various properties of both the metals, expanding its potential use. Green biosynthesis of NPs involving plant sources is nontoxic, cost-effective, and biocompatible method and is an attractive alternative to existing chemical, physical, and microbial methods. Plants accumulate variety of phenolic compounds as a defense mechanism to counter stress. Due to their redox properties, these phenolic compounds have antioxidant activity and play an important role in absorbing and neutralizing free radicals, quenching singlet and triplet oxygen, and decomposition of peroxides (Kumar 2012). This property of phenolic compounds is useful for the synthesis of NPs from metal ions (Ahmad and Sharma 2012a). The CONTACT Mahendra Seervi [email protected]; Naheed Ahmad Ashok Rajpath Road, Patna, Bihar 800005 India ß 2018 Informa UK Limited, trading as Taylor & Francis Group

KEYWORDS

Trapa; Au-Ag nanoparticles; green-synthesis; bio-waste; apoptosis; p53

Biosynthesized NPs can be capped or functionalized with various biomolecules which transforms them as important tool for the development of next generation diagnosis and therapies (Dykman and Bogatyrev 2007). Surface functionalization is possible due to the presence of various active groups (\\COOH, \\NH2, \\OH, etc.) of the capped phytochemicals. Plant-derived NPs is conjugated with different bio-moieties such as antibodies, proteins, antibiotics, etc. to achieve the targeted drug delivery especially in cancer therapy. Many reports have hitherto suggested the green synthesis of silver or gold NPs and their effect on cancer cells (He et al. 2016, Safwat et al. 2016). Even though plant-derived bimetallic NPs have been synthesized earlier, its effect and mode of action on cancer cells is not yet clearly explored (Shankar et al. 2004, Song et al. 2016). Sustainable, eco-friendly green technology has become the mantra of new world. There is a burgeoning field in nanotechnology where bio-wastes are utilized by adding value to its components for production of NPs (Makarov et al. 2014). Bio-agro-wastes often have no further use except for fodder or as the starting point of the food chain in the detritus cycle and need to be utilized or managed. Previously, synthesis of NPs has been reported from the peels of

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Department of Botany/Biotechnology, Patna University,

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N. AHMAD ET AL.

pomegranate (Ahmad et al. 2012b), Annanas squamosa (Kumar et al. 2012), egg shells (Aal et al. 2011), etc. In this study, we demonstrate the green chemistry approach for the synthesis of AuNPs, AgNPs, and composite of Au-AgNPs synthesized using Trapa natans peel extract. In this synthesis, peel extract acts as both reducing as well as stabilizing agent/capping agent. Trapa natans var. bispinosa Roxb. (Singhara) belongs to the family Trapaceae commonly known as water chestnut (Adkar et al. 2014). It is an aquatic floating herb which has a large edible seed enclosed within the peel. Trapa is a famous ayurvedic herb in India as it shows anti-fungal, anti-bacterial, and anti-inflammatory activities and cancer protective ability (Gani et al. 2015). Considering the excellent medicinal values of Trapa, we utilized its bio-waste (peel) for the synthesis of functional NPs. The physiochemical properties of AuNPs, AgNPs, and composites of Au-AgNPs were studied. This composite of Au-AgNPs induced apoptosis effectively in various cancer cells suggesting its significant potential in cancer therapy. Detailed mechanistic studies indicate that these composite NPs induced reactive oxygen species (ROS)-mediated p53 independent apoptosis in cancer cells. This study successfully biosynthesized functional composites of Au-AgNPs, which can be very useful in cancer therapy.

spectrophotometer (Double Beam UV-VIS Systronics 2202) in the wavelength of 200–800 nm. Bioreduction was done by mixing the extracted broth with the prepared solutions of Au, Ag, and Au-Ag. The effects of reaction conditions such as the broth amount, metal ion concentration, and contact time were also studied. The solution containing bio-reduced AgNPs (dark brown) and AuNP (mauve/pink) and Au-AgNPs (overlay of pink and brown-finally a purplish tinge) composites was then poured out into petri-dishes and left in the oven for drying at 250  C for 24 h.

Analysis of NPs by XRD and TEM The formation and quality of compounds were checked by the X-ray diffraction (XRD) pattern measurements of dropcoated film of AgNPs, AuNPs, and Au-AgNPs on glass substrate. The XRD pattern were recorded in a wide range of Bragg angles H at a scanning rate of 2 min1, carried out at a voltage of 40 kV and a current of 30 mA with Cu Ka radiation (1.5405 Å) in a Philips PW 1830. High resolution transmission electron microscopy (HRTEM) was performed by TECHNAIG20-STWIN (200 kV) machine with a line resolution 2.32 Å. These images were taken by drop coating AgNPs, AuNPs, and Au-AgNPs on a carbon-coated copper grid.

Materials and methods Chemicals and reagents Tetrachloroauric acid (HAuCl4), silver nitrate (AgNO3), DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) of purity 99.99%, 99.0%, and 95.0%, respectively, Hoechst 33342, 3–(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), and N-acetyl-L-cysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), Opti-MEM serum-free medium, and 100 antibiotic-anti-mycotic cocktail were purchased from Invitrogen (Carlsbad, CA).

Preparation of plant extract and green synthesis of nanoparticles Peel of fresh fruit (edible corm) of Trapa was separated, weighed (5 gm), and air dried. Dried plant material was pulverized into fine powder and stored in airtight bottles. Dried peel powder was boiled with 200 mL double-distilled water in steam bath for 15–20 min. The extract obtained was cooled to room temperature and filtered. This solution was treated as source extract and was further used for the synthesis of NPs. Aqueous solutions (0.025 M) of silver nitrate and auric acid were prepared. For preparation of the Au-Ag composite, solution of Agþ and AuCl4 was mixed in 1:1 proportion.

Spectral analysis of NPs by UV-Vis spectroscopy Spectral analyses of synthesized NPs and composites at different reaction concentrations were observed. The optical absorbance was recorded on ultraviolet–visible (UV-vis)

Antioxidant assay About 1 mL of broth extract and 0.5 mL of 0.15 mM DPPH solution (in methanol) were mixed vigorously, incubated at room temperature for 30 min before recording the absorbance at 517 nm. The measurements were run in triplicate and average was read out. Ascorbic acid was used as control (Malviya et al. 2010). The scavenging activity was calculated using the equation. % scavenging Absorbance of control  Absorbance of test sample  100 ¼ Absorbance of control

Cell lines HeLa, MDA-MB-231, HCT116, and its p53 knockout (KO) were maintained in DMEM supplemented with 10% FBS and 1 antibiotic–anti-mycotic cocktail in a humidified CO2 incubator at 37  C with 5% CO2.

Cell viability (MTT) assay MTT assay was carried out to measure cell viability under different treatments as described previously (He et al. 2016). Cell viability was calculated as follows: Cell viability ¼ Mean OD (treated samples)/ Mean OD (untreated sample)  100

DRUG AND CHEMICAL TOXICOLOGY

Assessment of nuclear condensation and mitochondrial membrane potential (Dwm) Cells were grown on 96-well culture plates and after treatment with NPs (200 lg/mL), stained with 0.5 mg/mL of nuclear stain Hoechst 33342 for 10 min and images were captured using UV-filter, Nikon Ti-U Inverted fluorescent microscope (Tokyo, Japan) with DS-Qi2 camera, and analyzed with NIS element software. Cells with apoptotic-condensed nuclei were scored in percentage per sample. After capturing images for Hoechst stain, the medium was gently removed and 100 lL of Opti-MEM containing Dwm specific dye Tetramethylrhodamine, methyl ester (TMRM) (50 nM) was added and cells were incubated for 10 min at 37  C. The images were captured with Tetramethylrhodamine-isothiocyanate (TRITC) filter of microscope.

Measurement of ROS Untreated control and treated cells were stained with 5 lM Cell ROX deep-red reagent (Molecular Probes, Oregon, USA) as per manufacturer’s instructions and analyzed on flow cytometer (BD FACS Aria, CA, USA).

Analysis of ROS induced cell death by PI staining Cells grown in 24-wells plate were treated with Au-AgNPs at a concentration of 200 lg/mL in the absence and presence of ROS inhibitor NAC (5 mM) for 36 h. Cells were trypsinized and centrifuged for 3000 rpm for 5 min and incubated with PI (1 lg/mL) for 5 min in dark and washed with PBS and subjected to flow cytometer analysis.

Western blot Cells with or without treatment were harvested, protein extracted, and Western blot was performed as described earlier (He et al. 2016). Antibodies used were b-actin (M1000120), p53 (K000465P) (ImmunoTag, MO, USA), cleaved casp-3, and cleaved PARP (apoptosis sampler kit-9915, Cell Signaling Technology (Boston, MA). The signal was detected by using Pierce ECL Plus reagents (Thermo Fisher Scientific Co., Waltham, MA) or diaminobenzidine (DAB; Sigma-Aldrich, St. Louis, MO) substrate in Tris buffer/H2O2.

Statistical analysis All values were expressed as means ± SEM of three independent in vitro experiments which had three technical replicates. Two-tailed unpaired Student’s t-tests were performed and a minimum p values of

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