Single step formation of biocompatible bimetallic alloy nanoparticles of gold and silver using isonicotinylhydrazide

Accepted Manuscript Single step formation of biocompatible bimetallic alloy nanoparticles of gold and silver using isonicotinylhydrazide

P.N. Navya, Harishkumar Madhyastha, Radha Madhyastha, Yuichi Nakajima, Masugi Maruyama, S.P. Srinivas, Devendra Jain, Mohamad Hassan Amin, Suresh K. Bhargava, Hemant Kumar Daima PII: DOI: Reference:

S0928-4931(18)30586-1 https://doi.org/10.1016/j.msec.2018.11.024 MSC 9044

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

24 February 2018 5 November 2018 20 November 2018

Please cite this article as: P.N. Navya, Harishkumar Madhyastha, Radha Madhyastha, Yuichi Nakajima, Masugi Maruyama, S.P. Srinivas, Devendra Jain, Mohamad Hassan Amin, Suresh K. Bhargava, Hemant Kumar Daima , Single step formation of biocompatible bimetallic alloy nanoparticles of gold and silver using isonicotinylhydrazide. Msc (2018), https://doi.org/10.1016/j.msec.2018.11.024

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ACCEPTED MANUSCRIPT

Single step formation of biocompatible bimetallic alloy nanoparticles of gold and silver using isonicotinylhydrazide

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Short Title Designing bimetallic biocompatible nanoparticles with isonicotinylhydrazide

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Navya PN1*, Harishkumar Madhyastha2, Radha Madhyastha2, Yuichi

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Nakajima2, Masugi Maruyama2, S. P. Srinivas3, Devendra Jain4, Mohamad

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Hassan Amin5, Suresh K. Bhargava5, Hemant Kumar Daima1,5,6* 1

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Nano-Bio interfacial research laboratory (NBIRL), Department of Biotechnology, Siddaganga Institute of Technology, Tumkur-572103, Karnataka, India 2 Department of Applied Physiology, Faculty of Medicine, University of Miyazaki, Miyazaki-8891692, Miyazaki, Japan 3 School of Optometry, Indiana University, Bloomington, Indiana-47405, USA 4 Department of Molecular Biology and Biotechnology, Rajasthan College of Agriculture, Maharana Pratap University of Agriculture and Technology, Udaipur-313001, Rajasthan, India 5 Centre for Advanced Materials and Industrial Chemistry, School of Science, RMIT University, Melbourne, VIC 3001, Australia 6 Amity Institute of Biotechnology, Amity University Rajasthan, Kant Kalwar, NH-11C, Jaipur-Delhi highway, Jaipur-303002, Rajasthan, India

*Corresponding authors Email: [email protected], [email protected], [email protected] Phone: +91 8884774863, +91 8764402136; Fax: +91 1426 405679 ORCID: http://orcid.org/0000-0002-9109-430

ACCEPTED MANUSCRIPT ABSTRACT Manufacturing nanoparticles with controlled physicochemical properties using environmentfriendly routes have potential to open new prospects for a variety of applications. Accordingly, several approaches have been established for manufacturing metal nanoparticles. Many of these

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approaches entail the use of hazardous chemicals and could be toxic to the environment, and cannot

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be used readily for biomedical applications. In the present work, we report a single step biofriendly approach to formulate gold (Au), silver (Ag), and Au-Ag alloy nanoparticles with desired

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surface corona and composition isonicotinylhydrazide (INH) as a reducing agent. INH also functioned as a stabilizing agent by enabling a surface corona around the nanoparticles.

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Remarkably, within a single step INH could also provide a handle in regulating the composition

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of Au and Ag in bimetallic systems without any additional chemical modification. The physicochemical and surface properties of the different nanoparticles thus obtained have been

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examined by analytical, spectroscopic and microscopic techniques. Cell cytotoxicity (release of

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lactate dehydrogenase), cell viability and intracellular reactive oxygen species (ROS) assays confirmed that the Au, Ag, and Au-Ag bimetallic nanoparticles prepared with INH are

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biocompatible. Finally, the presence of organic surface corona of INH on the nanoparticles was

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found to impart peroxidase enzyme-like activity and antimycobacterial sensitivity to the nanoparticles.

Keywords: Gold-silver nanoparticles; isonicotinylhydrazide; physicochemical; biocompatible; enzyme-like; antimycobacterial.

ACCEPTED MANUSCRIPT 1 INTRODUCTION Nanoparticles act as a multi-functional platform for a variety of applications due to their unique physicochemical, optoelectrical, and magnetic properties. The unique properties on nanoparticles are imparted as result of nanoscale size, large surface-to-volume ratio, and outer corona (De et al.,

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2008; Kaphle et al., 2018; Navya and Daima, 2016; Umapathi et al., 2018). Interestingly, all the

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physical, chemical, and surface properties of nanoparticles can be fine-tuned to make them suitable for a variety of applications. In biology and medicine, different types of nanoparticles are finding

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their utility and they are at the forefront in diagnosis, sustained drug delivery, targeted gene delivery, molecular imaging, bio-sensing, tissue engineering and management of pathogenic

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microorganisms (Behzadi et al., 2017; Bobo et al., 2016; Campbell et al., 2011; Carnovale et al.,

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2016; Hur and Park, 2016; Navya and Daima, 2016). In this context, efforts to improve the sensitivity, specificity, functionality, and ease of preparation of nanoparticles by tailoring their

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physicochemical and surface properties are imperative. It is anticipated that designer-made

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nanoparticles will have enhanced biomedical capabilities. Therefore, several methods have been established to formulate nanoparticles with predictable characteristics (Euliss et al., 2006;

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Farokhzad and Langer, 2006; Ghosh et al., 2008; Link and El-Sayed, 2000; West and Halas, 2003).

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Previously, synthesis of the metal nanoparticles has often employed harmful chemicals as reducing agents in addition to other compounds for their stability. This had led to undesirable toxic effects on the environment and in the targeted bio-systems. Moreover, surface functionalization is essential for targeted delivery of therapeutic molecules. Thus, post-synthesis functionalization strategies add additional complications, limiting the full potential of metal nanoparticles toward their environmental, medical, and biological applications (Geraci and Castranova, 2010; Holl, 2009; Johnston et al.; Lai, 2012; Sharifi et al., 2012; Siegel et al., 2012). The preparation or surface

ACCEPTED MANUSCRIPT modification

of

metal

nanoparticle

have

thus

necessitated

use

of

biomolecules,

polymer/biopolymer such as amino acids, biocompatible polymer such as poly(l-lactide acid) (PLLA), polyethylene glycol (PEG) or non-woven fabric poly(hydroxybutyrate) (PHB) (Dubey et al., 2015; Siegel et al., 2012; Slepička et al., 2016). Commonly available sugars such as glucose,

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fructose, and sucrose have also been explored as reducing agents to prepare different metal

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nanoparticles (Panigrahi et al., 2004). Other biological resources such as plants, algae, fungi,

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bacteria, and viruses have been employed for the production of low-cost, energy-efficient and nontoxic metallic nanoparticles through green chemistry routes (Bansal et al., 2007; Ramanathan

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et al., 2011; Thakkar et al., 2010; Ugru et al., 2018). It is reported that extracts of a diverse range

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of plant species, plant tissue or fruits can also be used for making nanoparticles, wherein enzymes and various plant metabolites such as alkaloids, phenolic compounds, and terpenoids work as

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reducing agents (Jain et al., 2009; Kharissova et al., 2013; Mittal et al., 2013; Ugru et al., 2018).

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Furthermore, green chemistry routes synthesised nanoparticles are reported to have biomedical effectiveness such as enhanced cell-adhesion, significant proliferation of embryonic fibroblasts,

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tissue engineering, biosensing, drug delivery and bactericidal actions (Dahl et al., 2007; Daima et

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al., 2013; Navya and Daima, 2016; Sharma et al., 2014; Slepička et al., 2015; Slepicka et al., 2015), and enable opportunities for translational research. Therefore, researchers around the globe are

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looking for innovative approaches which will be required to improve the methods for both diagnosis and selective destruction of pathogenic microorganisms for disease management (Mocan et al., 2016; Mocan et al., 2017; Wang et al., 2017; Zhang et al., 2018; Zhu et al., 2014). In the context of treatment of bacterial infections, metal-based nanoparticles, especially of gold (Au) and silver (Ag) nanoparticles have been sought to counter pathogenic microorganisms (Daima and Bansal, 2015; Nasiruddin et al., 2017). In general, Au nanoparticles are considered

ACCEPTED MANUSCRIPT biocompatible; whereas, Ag nanoparticles are known to possess a very high antimicrobial properties. However, fine-tuning the inherent antimicrobial characteristics is possible by careful design and surface modification (Daima et al., 2014; Daima et al., 2013; Daima and Bansal, 2015; Hur and Park, 2016; Ugru et al., 2018). In this context, the role of metal composition within a

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nanoparticle in conjugation with antibiotic surface corona may be of particular interest. Therefore,

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we have engineered Au, Ag and Au-Ag bimetallic nanoparticles using isonicotinylhydrazide

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(INH) as a reducing and stabilizing agent. In and of itself, INH is an antibiotic used in the treatment of tuberculosis and HIV-positive cases. We hypothesize that INH can be not only useful in

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designing metal nanoparticle with suitable composition and antibiotic-corona but also such

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systems would provide be more efficacious in the treatment of tuberculosis through enhanced bioavailability. We believe that antibiotic-corona comprising nanoparticles could be suitable to

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target the mycobacteria, particularly Mycobacterium tuberculosis.

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In the present research, we report on the advantages of INH to formulate antibiotic-corona containing metal nanoparticles. The physicochemical properties, including optical, hydrodynamic

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radius, surface charge, morphology, chemical/elemental nature of nanoparticles are confirmed

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using several techniques. We have assessed the resulting nanoparticles for (a) biocompatibility through cytotoxicity/viability assays and generation of intracellular reactive oxygen species (ROS)

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with fibroblasts, (b) in-vitro peroxidase-like activity, and (c) antimycobacterial sensitivity towards MDR clinical isolate of Mycobacterium tuberculosis.

2 EXPERIMENTAL SECTION 2.1 Materials and reagents

ACCEPTED MANUSCRIPT Tetrachloroauric acid (HAuCl4), silver nitrate (AgNO3), potassium hydroxide (KOH), isonicotinylhydrazide (INH) and 3,3’,5,5’ tetramethylbenzidine (TMB) were obtained from Sigma-Aldrich and used as received. M5S mouse skin fibroblasts were purchased from the RIKEN Cell Bank (Tsukuba, Japan) and grown in alpha-MEM containing 10% heat-inactivated FBS, 2

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mM glutamine, 100 U/ml penicillin G, and 100 µg/ml streptomycin in a humidified chamber (5%

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Intracellular ROS Assay Kit (Cell Biolabs,

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(Promega. Corp., Madison, USA) and OxiSelect

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CO2, 37°C). Various test kits such as CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit

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Inc., San Diego, CA 92126 USA) were acquired and used as per the instruction on the kit. The solutions for the synthesis of nanoparticles, assays evaluation, reagent preparation and

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biological activities were prepared using 'ultrapure' Milli-Q water with a resistivity of 18.2 MΩ·cm

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at 25°C.

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2.2 Isonicotinylhydrazide (INH) mediated synthesis of nanoparticles In a typical 100 mL experiment, aqueous solutions with 1 mM KOH containing 0.5 mM INH were

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boiled for 5 minutes at 90°C. In the boiling solutions, [AuCl4]-, Ag+ or both [AuCl4]- and Ag+ ions

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were added to obtain INH-reduced Au100INH, Au75Ag25INH, Au50Ag50INH, Au25Ag75INH and Ag100INH nanoparticles, respectively. The numers in the subscript designates the relative percentage of Au

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or Ag metal fraction in the nanoparticles. The total metal ion concentrations were kept constant at 0.2 mM in all the reactions (Table S1). 2.3 Characterization of INH-reduced metal nanoparticles The INH-reduced pristine Au100INH, Ag100INH, and bimetallic Au75Ag25INH, Au50Ag50INH and Au25Ag75INH nanoparticles were carefully characterized by spectroscopic and microscopic techniques to establish their optical, size, surface charge, morphological features, and

ACCEPTED MANUSCRIPT chemical/elemental nature. UV–Visible spectrophotometer (Shimadzu UV-1800; resolution of 2 nm) was used to observe optical features and surface plasmon resonance of nanoparticles. Zeta (ζ) potential and dynamic light scattering (DLS) measurements on different nanoparticle solutions were performed at Malvern Instruments Aimil Ltd, Bangalore, India using Malvern Zetasizer Ver.

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7.10 (Serial number MAL1045544) to confirm the surface charge and hydrodynamic radius of all

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the nanoparticles, respectively. Elemental concentration of metals in various nanoparticles was

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estimated by using atomic absorption spectroscopy (Shimadzu AA 7000, Tokyo, Japan). The AAS studies were performed on freshly prepared aqua regia (a corrosive mixture of 1HNO3 + 3HCl)

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digested nanoparticle solutions. For transmission electron microscopy (TEM), 1 µl of

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nanoparticles sample was loaded on to copper grid and allowed to vacuum dry at room temperature. Additionally, the distribution of Au, Ag, N and O species on the surface of Au100INH,

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Au75Ag25INH, Au50Ag50INH Au25Ag75INH, and Ag100INH was estimated using an EDS spectrometer

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(Oxford XMax80T) in a scanning TEM mode using JEM-2100F instrument operated at an accelerating voltage of 200 kV. Fourier-transform infrared spectroscopy (FTIR) spectra were

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recorded on PerkinElmer (Frontier) FT-IR/FIR spectrometer.

2.4 In-vitro peroxidase enzyme-like activity of INH-reduced nanoparticles

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The in-vitro peroxidase enzyme-like activity of Au100INH, Au75Ag25INH, Au50Ag50INH, Au25Ag75INH and Ag100INH nanoparticles was assayed by following oxidation of the a chromogenic substrate 3,3',5,5'-Tetramethylbenzidine (TMB) in the presence of H2O2. Before the biological activity, all nanoparticles were normalized to 100 µg/L concentration for Au in case of bimetallic nanoparticles, whereas Ag fraction varied successively in Au75Ag25INH, Au50Ag50INH and Au25Ag75INH, respectively. In the case of monometallic (Au100INH and Ag100INH) nanoparticles, 100 µg/L metal content concentration was used.

ACCEPTED MANUSCRIPT Briefly, in the dark, an equal volume (200µl) of metal nanoparticles were incubated with TMB and H2O2 for 600 sec to evaluate their in-vitro peroxidase enzyme-like activity. The blue color emerges as the reaction advances over the time, and it was monitored kinetically using an UVvisible spectrophotometer. The conversion of the substrate was measured at 650 nm, and all the

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reactions were performed at room temperature. In the similar dark experimental conditions,

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pristine INH and precursor metal ions ([AuCl4]- and Ag+) were also assessed as a control for their

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2.5 Cell cytotoxicity and viability assays

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potential in-vitro peroxidase enzyme-like activity.

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M5S mouse skin fibroblasts were seeded in 96-well plates at a density of 1×10 cells/well,

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followed by their treatment with nanoparticles at 0, 0.1, 0.5 and 5 µg/ml concentrations for 16 h.

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After incubation, supernatants were collected to determine the released lactate dehydrogenase (LDH), using a Non-Radioactive Cytotoxicity Assay Kit. The amount of enzyme released was

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quantified using UV-Visible spectrophotometer at an absorbance of 490 nm. The assay employs

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the principle of the colorimetric measurement of LDH, a cytosolic enzyme released into the

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medium upon cell lysis.

Likewise, after incubation as specified above, cell viability was determined by 3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. The treated cells were washed with phosphate buffered saline (PBS) and 100 μL 0.1 mM MTT solution was added into each well, followed by incubation at 37°C for 3 h. Dimethyl sulfoxide (DMSO) was used to dissolve formazan crystals. The resulting intracellular purple formazan was quantified with a spectrophotometer at an absorbance of 570 nm.

ACCEPTED MANUSCRIPT 2.6 Quantification of intracellular reactive oxygen species (ROS)

Generation of intracellular ROS was quantified by using OxiSelect

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Intracellular ROS Assay Kit. 3

M5S mouse skin fibroblasts were seeded in 96-well plates at a density of 1×10 cells/well and

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incubated with 0, 0.1, 0.5 and 5 µg/ml nanoparticles followed by washing with Hank’s Balanced

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Salt Solution. 10 μM dichlorofluorescein diacetate (DCFH-DA) was added to the cells at 37°C for

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1 h in the dark. Nonfluorescent DCFH-DA is converted to fluorescent dichlorofluorescein in proportion to the amount of ROS in the cells. The fluorescence signal was quantified using a

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spectrofluorometer (DTX800, Beckman Coulter, Inc., Brea, CA, USA) at excitation and emission

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wavelengths of 485 and 530 nm, respectively.

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2.7 Assessment of antimycobacterial sensitivity

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A susceptible reference strain of Mycobacterium tuberculosis, H37Rv (ATCC 27294) along with one multi-drug resistant (MDR) clinical isolate was used to assess the antimycobacterial

potential

of

INH-synthesised

nanoparticles.

The

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preliminary

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Mycobacteria Growth Indicator Tube (MGIT) with BD BACTEC™ MGIT™ growth supplement in the presence of various nanoparticles were used for susceptibility assessment

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in the MGIT™ instrument (Becton Dickinson). After sterilization in autoclave and sonication, 0.1 ml Au100INH, Ag100INH and bimetallic (Au75Ag25INH, Au50Ag50INH and Au25Ag75INH) nanoparticles were placed in MGIT tubes contaning 7 ml medium. In all the test vials, 0.1 ml of an antibacterial MGIT growth supplement (Becton, Dickinson and Company) was also added to avoid contamination. The INH concentration of 0.1 ml (0.1 µg/ml) in MGIT tube was employed as the positive control, whereas another negative control was inoculated without nanoparticles suspension for the interpretation of the results. It is

ACCEPTED MANUSCRIPT important to state that the culture suspensions for inoculation were well dispersed without any large clumps to avoid false-resistant results. After thorough mixing and homogenization of the culture suspensions, the tubes were allowed to rest for 15 minutes and incubated according to the

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manufacturer’s instructions, and results were interpreted after 25 days.

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3. RESULTS AND DISCUSSION

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In the current research, we reveal the importance of INH to synthesize stable Au, Ag and their

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bimetallic nanoparticles along with a tailor-made surface corona in a single step. As represented in Figure 1 (A), to exert control on the composition of bimetallic nanoparticles, we employed

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different ratios of precursor metal ions of [AuCl4]- and Ag+ at 75:25 (Au rich alloy), 50:50 (equimolar Au=Ag alloy) and 25:75 (Ag rich alloy), respectively, in solutions containing INH.

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INH tends to undergo slow hydrolysis under alkaline medium, resulting in formation of hydrazine,

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which is a strong reducing agent well known to reduce metal ions in an aqueous medium (Maribel

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G Guzmán et al., 2009). Thus, we obtained nanoparticles, functionalized by the oxidized-INH as shown in Figure 1(B). In the past, studies have been carried out to understand the nature of

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interaction between functional groups and nanoparticle’s surface. It has been reported that

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nitrogen/sulphur and carboxylic acid functional groups bind to Au and Ag nanoparticles, respectively (Daima et al., 2014; Jazayeri et al., 2016; Kumar et al., 2003; Selvakannan et al., 2013). Therefore, the oxidised-INH moieties bind on the surface of nanoparticles, wherein carboxylic group binds to Ag and pyridine nitrogen binds to Au to stabilize nanoparticles as shown in Figure 1(C). Previously, UV-Visible spectroscopy has been employed to differentiate alloy and core-shell type of nanomaterials. Accordingly, we employed the method to confirm the alloy nature of Au-Ag

ACCEPTED MANUSCRIPT bimetallic nanoparticles (Figure 2A). Core-shell nanoparticles exhibit two distinct surface plasmon resonance (SPR) bands, wherein the intensity of individual bands depend on the relative percentage of each metal existing in the nanoparticulate system. This is further true for a dispersion containing distinct Au and Ag nanoparticles in a single system. However, only a single SPR feature

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will arise for alloy nanoparticles, that too between those of two independent metal nanoparticles

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(Daima et al., 2011; Hodak et al., 2000; Link et al., 1999). INH-mediated Au100INH and Ag100INH

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nanoparticles displayed their SPR bands at ~525 and ~405 nm, respectively. Similarly, as anticipated a single SPR spectrum was observed for Au75Ag25INH, Au50Ag50INH, and Au25Ag75INH

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due to alloy nature of bimetallic nanoparticles. These alloys indicated their respective SPR features

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at ~499, ~457 and ~430 nm as depicted in Figure 2A. The strong blue shift in SPR character with an increase in Ag fraction provides evidence of an Au-Ag alloy formation. In addition to the

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specific SPR of nanoparticles, it was interesting to observe the presence of supplementary bands

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in the range of ~600-735 nm for Au75Ag25INH, Au50Ag50INH, and Au25Ag75INH attributing due to the non-spherical shapes of nanoparticles, which was further confirmed using TEM imaging (Daima,

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2013; Mulvaney, 1996). Moreover, the spectrum of pure INH exhibits an absorption band at ~260

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nm, which is the characteristic л-л* electronic transition from the pyridine part of INH. This absorption band was observed in all the spectra of nanoparticles (highlighted region), which can

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be observed only when oxidised-INH moieties present on the surface of nanoparticles. It was further confirmed using STEM-EDS elemental mapping and FTIR analysis. As shown in Figure 2B (a-e), Au100INH and Ag100INH nanoparticles are spherical in shape, whereas all the three alloys are present in the quasispherical shape. The TEM micrographs scale is indicative that these nanoparticles are in the nanometer range, which was further confirmed by dynamic light scattering (DLS). The average hydrodynamic radius and zeta (ζ) potential values of

ACCEPTED MANUSCRIPT nanoparticles are shown in Table 1. Synthesis of sub-100 nm nanoparticles and bimetallic alloys was confirmed by DLS as the hydrodynamic radius of all five nanoparticles solutions are falling in the nanoscale range. Au100INH and Au rich alloy display hydrodynamic radius ~30 nm in size, whereas Ag100INH and Ag rich alloy revealed hydrodynamic radius ~50 nm. Further, the ζ potential

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values reflect a surface charge of metal nanoparticles. The negative ζ potential values shown in

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Table 1, were anticipated since these nanoparticles are stabilized with oxidized-INH molecules,

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thus making them negatively charged at physiological pH. The high negative surface charge originates from the carboxylate groups formed after the hydrolysis of INH as shown in Figure 1B.

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All the nanoparticles embrace negative surface charge ca. -38.47, -36.10, -37.93, -28.70 and -36.70

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for Au100INH, Au75Ag25INH, Au50Ag50INH and Au25Ag75INH and Ag100INH, correspondingly. The high negative ζ potential values reflect that these nanoparticles have good stability and do not aggregate.

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It was further confirmed under standard laboratory conditions at room temperature, wherein these

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nanoparticles did not show any sign of aggregation even after twenty months as shown in Figure 2C (a-e) by the digital photographs of metal nanoparticle solutions. Moreover, the snapshots of

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nanoparticle solutions evidently illustrate a transition in visible color from dark wine-red (Au) to

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yellow (Ag), respectively. As shown in Figure 2C, the varying intermediate colors also demonstrate the bimetallic alloy forming capability of INH in a single step. In addition to this, as

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illustrated in Figure 3, the STEM-EDS elemental mapping was also performed on all the nanoparticles solutions to confirm the presence of a homogeneous distribution of Au and Ag elements in bimetallic alloy systems. Furthermore, the existence of INH moieties was also established using corresponding fluorescence signals of N and O, originating from INH corona. Figure 4 shows the infrared spectra of Au100INH, Au75Ag25INH, Au50Ag50INH, Au25Ag75INH, Ag100INH and INH. The FTIR spectrum of pristine INH displays characteristic functional group vibrational

ACCEPTED MANUSCRIPT frequencies (Saifullah et al., 2016), wherein, the main bands are originating from the carbonyl group C=O (1637 cm-1), amino group NH2 wagging (1340 cm-1), N–N single bond (1228 cm-1), pyridine (1416 cm-1), N–H bending (1560 cm-1) and N–H stretching (3247 cm-1). The presence of distinguishing functional groups after nanoparticles formation with shifts in the wave number

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confirmed the presence of oxidised-INH on the surface of nanoparticles. For example, in pure INH

, 3258 cm-1 and 3263 cm-1 for Au100INH, Au75Ag25INH, Au50Ag50INH, Au25Ag75INH and Ag100INH,

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1

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the N–H stretching was observed at 3247 cm-1, which appeared at 3270 cm-1, 3259 cm-1, 3256 cm-

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respectively after nanoparticles formation.

The AAS was used to measure the Au and Ag concentrations in each sample after nanoparticles

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synthesis as illustrated in Table S2. The AAS results specified that Au:Ag ratios in bimetallic alloy nanoparticles showed similarity to the original ratios of metal ions used for the synthesis of

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different compositions containing Au75Ag25INH, Au50Ag50INH, and Au25Ag75INH nanoparticles.

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Further, the AAS analysis illustrated that INH inclines to form Au-rich bimetallic alloy

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nanoparticles during the bimetallic alloy nanoparticles synthesis as shown in Table S2. The metal composition of such bimetallic alloys can be fine-tuned by carefully controlling the initial fraction

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of metal ions. This is interesting to observe that an antibiotic such as INH can provide a handle in

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regulating the composition of bimetallic nanoparticles because, in general, preparation of bimetallic nanoparticles involves a reducing agent along with a capping agent. After detailed physicochemical characterization, to evaluate the patho-cellular changes induced by Au, Ag and their alloy nanoparticles, we assayed various toxicological responsive parameters in M5S skin fibroblast cells following nanoparticles treatment. The fibroblasts were selected as the cell model due to their participation in the processes of wound healing, and their involvement in fibrotic diseases. Since LDH is a highly stable enzyme, it is widely used to evaluate the damage

ACCEPTED MANUSCRIPT and toxicity of tissue and cells. Therefore, as depicted in Figure 5A, LDH assay is used for cell membrane integrity upon stress due to different nanoparticles. All the nanoparticles and alloys were found to be non-toxic as there was very little LDH release into medium up to 1 µg/ml doses. However, Au100INH and Ag100INH nanoparticles exhibited significant (p<0.05) membrane damage

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at higher concentration of 5 µg/ml as there was higher LDH release into the medium. Nevertheless,

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even at the higher concentration of 5 µg/ml, all the three Au75Ag25INH, Au50Ag50INH, and

the biocompatible nature of bimetallic nanoparticles.

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Au25Ag75INH bimetallic alloys did not show significant membrane damage (Figure 5A) confirming

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Since membrane damage by alloy nanoparticles may lead to cytosolic cellular damage by

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fluctuating the mitochondrial activity, we further evaluated the cytotoxic effect by MTT assay (Figure 5B). Exposures to Au100INH and Ag100INH nanoparticles produced enhanced cytotoxic

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responses at a higher dose of 5 µg/ml as there were significant (p < 0.05) decrease in viable cell

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numbers in both cases. However, their lower doses and bimetallic alloy nanoparticles at all the tested doses were not toxic to the cells as compared to control group indicating the dose-dependent

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activity of these nanoparticles and authorizing the biocompatibility of alloy nanoparticles.

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Furthermore, the mitochondrial bleaching due to stress toxicity leads to disturbance in mitochondrial respiratory co-efficient and cellular oxidative stress. Oxidative stress is mediated by

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reactive oxygen species (ROS), and it is caused by an imbalance between the production of free radicals and the activity of the antioxidant defense mechanism. Upon treatment with different nanoparticles only higher doses (5.0 µg/ml) of Au100INH and Ag100INH displayed significant production of ROS in the cell as illustrated in Figure 5C. The free metal ions ([AuCl4]- and Ag+) and pure INH were also assessed for their potential cytotoxicity, viability and ROS production capabilities in similar experimental conditions (Figure S1).

ACCEPTED MANUSCRIPT Additionally, we hypothesize that the presence of INH on the surface may provide biological identity to these nanoparticles, which was verified by in-vitro peroxidase enzyme-like action. Figure 6 (Panel A and B) illustrates metal core reliant kinetics of in-vitro peroxidase enzyme-like activity of pristine Au100INH and Ag100INH nanoparticles at comparable metal concentrations. From

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Panel A, it is apparent that Au100INH have significantly higher catalyzing activity of TMB substrate

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signifying its higher in-vitro peroxidase enzyme-like activity. It is further interesting to notice that

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although both the metal ions ([AuCl4]- and Ag+) did not possess this capacity (Figure S1, Panel D) but in the form of nanoparticulate in conjugation with INH they mimic in-vitro enzyme-like

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activity. INH itself presented intrinsic peroxidase-like activity, which has been imparted on metal

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nanoparticles surface during the synthesis process, and therefore both Au100INH and Ag100INH nanoparticles displayed this activity. Panel B in Figure 6 shows the initial enzyme-like action of

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Au100INH and Ag100INH nanoparticles. This is stimulating to notice that at the beginning of the

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reaction Ag100INH nanoparticles have more activity compare to Au100INH nanoparticles.

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Furthermore, to examine the role of Ag element in the overall in-vitro peroxidase-like activity, the three alloys Au75Ag25INH, Au50Ag50INH and Au25Ag75INH were subjected for study under similar

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experimental conditions (Figure 6, Panel C). Assessment of equivalent stoichiometric bimetallic

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alloys with a different fraction of Ag, concerning constant Au (as discussed in materials and method section), revealed diverse in-vitro peroxidase enzyme-like activities confirming the role of Ag fraction in catalyzing TMB. Here, it was observed that with the increasing fraction of Ag, the in-vitro peroxidase mimicking behavior also enhanced. The control experiments on [AuCl4]- and Ag+ revealed that they do not have any in-vitro peroxidase enzyme-like activity (Figure S1, Panel D) as they could not catalyze oxidation of TMB to produce blue colored reaction product confirming that this in-vitro enzyme-like behaviour was intrinsic in nature for INH-reduced

ACCEPTED MANUSCRIPT nanoparticles, which was introduced during their synthesis from free INH and it was reliant on metalcore along with surface corona. Moreover, in one of the reports, sulfur‐ containing isonicotinoyl hydrazine derivatives were prepared, and it was reported that zinc and antimony 3‐ isonicotinoyl

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dithiocarbazinate were highly active against Mycobacterium tuberculosis, in mice (Strube

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and Stern, 1957). Motivated from the study, preliminary antimycobacterial susceptibility

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assessment was performed with all the INH-synthesised metal nanoparticles, and it was revealed that pristine Au100INH, Ag100INH and their bimetallic nanoparticles are highly

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effective toward reference strain and MDR clinical isolate of M. tuberculosis. It is

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interesting to observe that all the INH-synthesised nanoparticles exhibited very low minimum inhibitory concentration (MIC) for M. tuberculosis. The INH alone was effective;

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however, the required concentration of free INH was significantly high compare to the

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INH-capped nanoparticles. In this context, earlier reports suggest that the clinical isolates of M. tuberculosis can be controlled using Ag nanoparticles at the MIC of 25 μg/ml

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(Agarwal et al., 2013). The results presented here, indicate that the potency of INH and

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nanoparticles can be significantly enhanced by presenting them within a single system. Moreover, the nanoparticles allow the administration of lower dose of medicine that

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ultimately reduces the potential toxic side effects of the drug. Besides, we report that in addition to Ag nanoparticles, the most biocompatible Au nanoparticles can be tuned in a smart manner to be active against clinical isolates of MDR M. tuberculosis by a suitable surface corona of INH. The designing of INH surface corona on nanoparticles not only increases the potency of INH to counter MDR M. tuberculosis but also reduces the toxicity, due to the biocompatibility of Au nanoparticles. Such designer-made metallic nanoparticles

ACCEPTED MANUSCRIPT can serve as innovative antimicrobial agents due to their unique surface corona and physicochemical properties. 4. CONCLUSIONS A single step and environmental friendly synthesis method has been established to prepare Au, Ag

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and bimetallic Au-Ag alloy nanoparticles with a suitable composition. INH has been used as a

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reducing and stabilizing agent, which also develops an organic corona around the nanoparticles

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without any further alteration. After understanding the physicochemical nature of alloys and nanoparticles, their biocompatible nature was confirmed by estimating the cell cytotoxicity, cell

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viability, and intracellular ROS generation assays. Moreover, the inherent in-vitro peroxidase

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enzyme-like activity was discovered due to the existence of organic surface corona of INH on nanoparticles which depends on the Ag fraction of alloys in conjugation to surface corona.

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Furthermore, antimycobacterial susceptibility assessment confirmed that the INH containing metal

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nanoparticles are highly effective towards MDR clinical isolate of M. tuberculosis. Outcomes of this study have potential to open new prospects toward the careful design of biomolecules-

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functionalized metallic nanoparticles with composition control, which will have significant

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potential to be used as the new class of materials for a variety of biological applications.

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ACKNOWLEDGEMENTS

HKD, NPN and DJ acknowledge generous support of Japan Science and Technology (JST) Agency, Japan toward Asia Youth Exchange Program in Science (Sakura Exchange Program). NPN and HKD gratefully acknowledge the support of Malvern Instruments Aimil Ltd, Bangalore for Zeta potential and DLS studies. NPN acknowledge the financial support from Siddaganga Institute of Technology, Tumkur, India (through TEQIP-II). Authors gratefully acknowledge Magnum Diagnostics & Research Centre, Jaipur, India for conducting antimycobacterial assays.

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Figure 1: Illustration of single step synthesis of mono and bimetallic alloy nanoparticles of gold (Au) and silver (Ag) using isonicotinylhydrazide (INH) as reducing and stabilizing agent. The percentage of Au and Ag metal fraction are shown by numerical values in the subscript of the individual nanoparticles. Formation of hydrazine (H2N-NH2) from INH under alkaline conditions as well as reduction of metal ions are shown in Panel B. The molecular orientation of oxidised-

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INH on the surface of Au, and Ag nanoparticles are shown in Panel C.

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Figure 2: Absorbance spectra of INH-mediated Au, bimetallic Au-Ag alloys, Ag nanoparticles and INH (A). TEM micrographs of (a-e) Au100INH, Au75Ag25INH, Au50Ag50INH, Au25Ag75INH and

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Ag100INH nanoparticles. (B). Digital photographs of (a-e) Au100INH, Au75Ag25INH, Au50Ag50INH,

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Au25Ag75INH and Ag100INH nanoparticle solutions synthesised using INH (C).

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Figure 3: Elemental mapping images obtained by STEM-EDS of Au100INH (a, e, m, q), Au75Ag25INH (b, f, j, n, r), Au50Ag50INH (c, g, k, o, s), Au25Ag75INH (d, h, l, p, t) and Ag100INH (i). The scale bar in all micrographs represents 50 nm.

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Figure 4: FTIR spectra of INH-mediated Au, bimetallic Au-Ag alloys, Ag nanoparticles, and INH displaying characteristic functional group vibrational frequencies.

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Figure 5: Cytotoxicity assessments of M5S mouse skin fibroblast cells treated with Au100INH, Au75Ag25INH, Au50Ag50INH, Au25Ag75INH and Ag100INH, respectively. (A) LDH assay to determine the released amount of lactate dehydrogenase as a result of cytotoxicity; (B) fibroblast cells viability determined by 3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay and (C) production of intracellular reactive oxygen species in fibroblast cells determined by ROS kit. Figure 6: In-vitro peroxidase-mimicking activity of Au100INH and Ag100INH nanoparticles are shown in Panel A and B. Panel C shows the in-vitro peroxidase-like activity of Au-Ag bimetallic alloy nanoparticles, confirming the importance of Ag fraction.

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Sample particulars

Hydrodynamic radius (nm)

Zeta (ζ) potential (milliVolts)

Pristine Au

27.83

-38.47

Au75Ag25 INH

Au-rich alloy

29.12

-36.10

Au50Ag50 INH

Equimolar Au and Ag alloy

36.57

-37.93

Au25Ag75 INH

Ag-rich alloy

54.29

Ag100 INH

Pristine Ag

47.35

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Au100INH

-28.70 -36.70

Table 1: Sample particulars, average hydrodynamic radius (size) and zeta (ζ) potential values

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(surface charge) of Au, Ag and Au-Ag bimetallic alloy nanoparticles measured by DLS and zeta

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potential analyzer, respectively.

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Highlights

A single step bio-friendly approach to formulate gold (Au), silver (Ag), and Au-Ag alloy nanoparticles characterized by suitable surface corona and composition is presented.

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Isonicotinylhydrazide (INH) works as reducing, stabilizing and composition regulating agent without any additional modifications.

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Existence of organic surface corona of INH on nanoparticles imparts biological potential on nanoparticles.

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Controlled cytotoxicity reported against M5S mouse skin fibroblast cells along with in-vitro peroxidase enzyme-like activity and antimycobacterial sensitivity.

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