Highly efficient and selective antimicrobial isonicotinylhydrazide-coated polyoxometalate-functionalized silver nanoparticles

Colloids and Surfaces B: Biointerfaces 184 (2019) 110522

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Highly efficient and selective antimicrobial isonicotinylhydrazide-coated polyoxometalate-functionalized silver nanoparticles

T

Akhela Umapathia, Navya P. Nagarajub,c, Harishkumar Madhyasthad, Devendra Jaine, ⁎ Sangly P. Srinivasf, Vincent M. Rotellog, Hemant Kumar Daimaa,b, a

Amity Center for Nanobiotechnology and Nanomedicine (ACNN), Amity Institute of Biotechnology, Amity University Rajasthan, Kant Kalwar, NH-11C, Jaipur-Delhi Highway, Jaipur, 303002, Rajasthan, India b Nano-Bio Interfacial Research Laboratory (NBIRL), Department of Biotechnology, Siddaganga Institute of Technology, Tumkur, 572103, Karnataka, India c Department of Biotechnology, Bannari Amman Institute of Technology, Sathyamangalam, Erode – 638401, Tamil Nadu, India d Department of Applied Physiology, Faculty of Medicine, University of Miyazaki, Miyazaki, 8891692, Japan e Department of Molecular Biology and Biotechnology, Rajasthan College of Agriculture, Maharana Pratap University of Agriculture and Technology, Udaipur, 313001, Rajasthan, India f School of Optometry, Indiana University, Bloomington, 47405, IN, USA g Department of Chemistry, University of Massachusetts (UMass) Amherst, 710 North Pleasant Street, Amherst, 01003 MA, USA

A R T I C LE I N FO

A B S T R A C T

Keywords: Isonicotinylhydrazide Polyoxometalates Silver nanoparticles Antimicrobial Nanozyme Cytotoxicity

With the rapidly approaching post-antibiotic era, new and effective combinations of antibiotics are imperative to combat multiple drug resistance (MDR). We have synthesized multimodal antimicrobials that integrate the antibiotic isonicotinylhydrazide (INH), silver nanoparticles (AgNPsINH), and two different polyoxometalates (POMs) namely, phosphotungstic acid (PTA) and phosphomolybdic acid (PMA) to prepare AgNPsINH@PTA and AgNPsINH@PMA, respectively. AgNPsINH have peroxidase-like (nanozyme) activity and very high antibacterial potential toward S. aureus, which was further enhanced upon modification with POMs. The selectivity of these functional nanozymes was evaluated with m5S mouse fibroblasts using WST-8, LDH viability, in vitro reactive oxygen species (ROS) generation assays, and crystal violet morphological studies. These investigations showed very low cytotoxicity for the nanoparticles compared to free metal ions (Ag+), pristine POMs and INH, demonstrating the ability of multifunctional materials to provide efficient and selective antimicrobials.

1. Introduction

therapeutics [12], and other formulations based on nanoparticles have emerged [13–16]. In this context, inorganic nanoparticles (NPs) are particularly promising due to their high surface-to-volume ratio that makes them useful as drug carriers improving pharmacological profiles and providing, targeted administration, sustained therapeutic effect, low immunogenicity, stability, and controlled toxicity [17–19]. Further, the ability to control the morphological characteristics and surface chemistry of inorganic NPs using relatively simple approaches for their synthesis provide versatility for clinical applications [13,20–22]. Beyond serving as a scaffold, inorganic NPs innately possess antimicrobial properties. While bacteria have established multiple mechanisms to develop resistance to metal ions such as reduced uptake, efflux, extracellular/ intracellular sequestration and metabolic bypass [23], development of resistance to certain non-essential metals such as silver (Ag) or tellurium (Te) is challenging for bacteria. In this direction,

The development of multiple drug resistance (MDR) to antibiotics has led to and an emerging healthcare crisis through evolving “superbugs”. The World Health Organization (WHO) has made it clear that multiple species of bacteria have become resistant to standard therapeutic strategies [1,2]. In addition to MDR, microorganisms with no viable treatments options are also emerging, including extensively drug-resistant (XDR) or total drug-resistant (TDR) strains [3]. These wide-ranging forms of drug resistance are being countered continuously by redesigning conventional antibacterial agents such as β-lactam antibiotics, macrolides, aminoglycosides, tetracyclines, and quinolones [4,5]. However, microorganisms are continuing to acquire MDR/XDR/ TDR by numerous mechanisms, in particular horizontal transfer [6–8]. However, as an alternative to antibiotics, antimicrobial peptides [9], phage-based therapies [10], plant-derived compounds [11], RNA-based

⁎ Corresponding author at: Amity Center for Nanobiotechnology and Nanomedicine (ACNN), Amity Institute of Biotechnology, Amity University Rajasthan, Kant Kalwar, NH-11C, Jaipur-Delhi Highway, Jaipur, 303002, Rajasthan, India. E-mail address: [email protected] (H.K. Daima).

https://doi.org/10.1016/j.colsurfb.2019.110522 Received 1 July 2019; Received in revised form 10 September 2019; Accepted 21 September 2019 Available online 25 September 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.

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Fig. 1. Schematic representation of the reduction of metal ions (Ag+) by isonicotinylhydrazide (INH) in the preparation of AgNPsINH. These nanoparticles, which are also stabilized by INH, are treated subsequently with phosphotungstic acid (PTA) and phosphomolybdic acid (PMA) to obtain AgNPsINH@PTA and AgNPsINH@PMA, respectively. The glow (in blue shade) around the nanoparticles indicate the presence of oxidized molecules of INH on their surface. Hydrazine (H2N-NH2) formation from INH under alkaline conditions and reduction of Ag+ is also illustrated (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

2. Materials and methods

Ag NPs, are potent antimicrobials, causing deleterious effects through membrane damage, protein degradation, gene nicking, and oxidative stress [19,24–26]. Many studies have shown that Ag NPs labeled with antibiotics have exhibited synergetic action as antimicrobial agents [27,28]. In most of these cases, the antibiotic is either encapsulating the Ag NPs or associated with the antibiotics through linkers. Amino acids and plant-derived materials provide effective ‘green’ precursors for creating Ag NPs [22,29,30]. Recently, we have developed a strategy for creating Au/Ag alloy NPs using the antibiotic isonicotinylhydrazide (INH), which is a first-line antibiotic for the treatment of tuberculosis [13]. INH is a prodrug that undergoes catalaseperoxidase enzyme (a product of KatG gene) interaction. The prodrug is activated by producing the isonicotinic acyl radical, which is responsible for its efficacy by inhibiting the synthesis of mycolic acid in the bacterium. Resistance to INH is usually developed as a result of mutations in the KatG gene [31], making combination therapeutic strategies essential to evade resistance. We report here the preparation of multifunctional antimicrobials by employing a ‘green’ approach. Here, INH acts as both reducing and capping agent, then the INH-capped Ag NPs (AgNPsINH) are used as a scaffold for polyoxometalates (POMs). These POMs can be readily manipulated in terms of their composition, size, structure, and charge; and have antibacterial, anticancer and antiviral activities [32–36]. We have decorated AgNPsINH with phosphotungstic acid (PTA) and phosphomolybdic acid (PMA) to formulate AgNPsINH@PTA and AgNPsINH@PMA, respectively. The different NPs (i.e., AgNPsINH, AgNPsINH@PTA, and AgNPsINH@PMA) have been evaluated for their antimicrobial properties, antimycobacterial sensitivity and in vitro enzyme-like activity, demonstrating their efficacy. Significantly, in vitro toxicities of AgNPsINH, AgNPsINH@PTA and AgNPsINH@PMA on m5S mouse fibroblast cells indicated minimal toxicity to mammalian cells.

2.1. Chemicals and reagents Silver nitrate (AgNO3), potassium hydroxide (KOH), INH, PTA, PMA, 3,3′,5,5′-Tetramethylbenzidine (TMB), phosphate buffered solution (PBS), fetal bovine serum (FBS), minimal essential Eagle medium (α-MEM), dimethyl sulfoxide, crystal violet blue stain and antibacterial cocktail (A5955) were procured from Sigma Aldrich (St. Louis, MO, USA). Rifamycin and cefixime disks, nutrient agar, and hydrogen peroxide (H2O2) were acquired from HiMedia (Mumbai, Maharashtra, India). The dialysis membrane with MWCO of 12 kDa (Sigma Aldrich) was used to remove the unreacted ions and reducing agents. Hank’s balanced salt solution (HBSS) was procured from Gibco (Carlsbad, CA, USA). Bacterial strains Staphylococcus aureus (ATCC-25923) and Escherichia coli (ATCC-25922), Serotype O6 were procured from ATCC. Mouse skin fibroblast cells (m5S) were purchased from the RIKEN Cell Bank, (Tsukuba, Japan). Cellular ROS detection assay kit (Catalog# 139476) was procured from Abcam (San Francisco, USA). The viability/cytotoxicity multiplex assay kit was obtained from Dojindo Molecular Technologies Inc. Kumamoto, Japan. All the solutions and nanoparticles were prepared using deionized Milli-Q water. All the chemicals used were of analytical grade. 2.2. Preparation of POMs-enriched INH-coated Ag nanoparticles To prepare the INH-coated and POMs functionalized Ag NPs, INH was used to reduce the Ag+ and to later act as a strong capping agent. In a 100 ml typical reaction mixture, 100 mM INH along with 0.1 M KOH was added gently such that the final concentrations were 0.5 mM and 1 mM respectively. The solution was heated gradually under constant stirring followed by controlled addition of 100 mM Ag+. The final concentration of Ag+ in the 100 ml solution was 0.2 mM. This solution 2

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was stirred overnight to produce stable AgNPsINH via Oswald’s ripening. The synthesized AgNPsINH were further subjected to slow heating and constant stirring to increase the metal concentration by ∼10 × . Removal of free ions and residual INH, if any, was carried out by filtration through a 12 kDa cellulose membrane. The resulting AgNPsINH were surface functionalized with PTA or PMA molecules, wherein the final concentration of PTA and PMA was kept at 1 mM to formulate AgNPsINH@PTA and AgNPsINH@PMA, respectively (Fig. 1). AgNPsINH, AgNPsINH@PTA and AgNPsINH@PMA were found to be stable at room temperature for more than nine months.

Company) [13]. NPs (0.1 ml of each) were added to 7 ml medium in the Mycobacteria Growth Indicator Tubes (MGIT), along with 0.1 ml of an antibacterial MGIT growth supplement (Becton Dickinson and Company) to avoid contamination. After thorough mixing of the culture suspensions to avoid false-resistant results, the tubes were allowed to relax for 15 min, and the supernatant was used to inoculate, according to the manufacturer’s instructions and results were interpreted. 2.6. Intracellular ROS detection and cell morphology analysis The generation of ROS by different nanoparticles was assessed by fluorescence microscope (BZ-9000, Keyence, Osaka, Japan) employing 0.5 μg/ml of AgNPsINH, 0.5 μg/ml of AgNPsINH@PTA and 0.2 μg/ml of AgNPsINH@PMA, respectively with m5S skin fibroblasts. The experiment was conducted in a 6-well plate at a cell concentration of 2 × 105 cells/ well. The samples were incubated with 10 μl of DCFH-DA (37 °C, dark conditions) for 16 h. Fluorescence images were captured at excitation and emission wavelengths of 485 and 530 nm, respectively. The crystal violet blue stain in 0.5% methanol was used to stain the m5S cells which were treated with AgNPsINH, AgNPsINH@PTA, and AgNPsINH@PMA. Before capturing the images, the treated cells were fixed with 4% paraformaldehyde and subsequently washed with PBS to remove the unreacted fixative agent. After which distilled water was used to remove the fixative agent.

2.3. Characterization of POMs-enriched AgNPsINH The nanoparticles of AgNPsINH, AgNPsINH@PTA, and AgNPsINH@PMA were characterized by UV–vis spectroscopy (Bio mate 3S, Thermo Scientific) at a resolution of 2 nm. Zeta potential (ζ) and dynamic light scattering (DLS) measurements on different nanoparticles were performed using Malvern Zetasizer Ver. 7.10 (Serial number MAL1045544) after a 10-fold dilution. The shape of the nanoparticles was assessed by transmission electron microscopy (TEM) (Hitachi HT7700, Tokyo, Japan) by placing a 10 μl sonicated sample on a carbon-coated grid. The Ag concentration was determined on aqua regia digested solutions employing inductively coupled plasma mass spectrometry (ICP-MS) (Shimadzu ICPS-8000) with a resolution of 0.0045 nm. The Fourier transform infrared spectroscopy (FTIR) spectra on different nanoparticles were recorded by Shimadzu IR affinity-1 spectrophotometer with a resolution of 4 cm–1.

2.7. Cytotoxicity/survival tests AgNPsINH, AgNPsINH@PTA, and AgNPsINH@PMA were incubated at 37 °C, 5% CO2 with m5S mouse skin fibroblast at 1 × 103 cells/ml. The protocol was followed as specified by the assay kit and the absorbance was recorded after 16 h of incubation. The viability/cytotoxicity multiplex assay kit contains the LDH assay kit and the cell counting kit. The % cytotoxicity was estimated through LDH assay for which absorbance was recorded at 490 nm, whereas the % survival based on WST-8 assay recorded at 450 nm. Data obtained from three independent experiments were analyzed.

2.4. Peroxidase-mimicking enzyme activity of AgNPsINH, AgNPsINH@PTA, and AgNPsINH@PMA AgNPsINH, AgNPsINH@PTA, and AgNPsINH@PMA were evaluated for their peroxidase-mimicking enzyme activity by following the oxidation of TMB as a chromogenic substrate in the presence of H2O2. The reaction mixture contained TMB (0.25 mg/ml), H2O2 (30% w/v), and the nanoparticles (AgNPsINH, AgNPsINH@PTA or AgNPsINH@PMA) in equal amounts. Changes in absorbance were recorded at 650 nm for 600 s using Shimadzu UV-1800.

3. Results and discussion

2.5. Antimicrobial activity and antimycobacterial sensitivity of AgNPsINH, AgNPsINH@PTA, and AgNPsINH@PMA

In the present work, we have prepared INH decorated Ag NPs (AgNPsINH), followed by their surface functionalization with PTA and PMA to formulate AgNPsINH@PTA and AgNPsINH@PMA, respectively (Fig. 1). AgNPsINH were prepared under alkaline conditions in which INH undergoes hydrolysis to form hydrazine (H2N-NH2). The latter reduces Ag+ to form Ag NPs in the aqueous medium. Hydrazine is well known reducing agent [38], and it has potential to reduce metal ions to form metal nanoparticles as illustrated in Fig. 1 [13]. Further, the oxidized-INH moieties also stabilize formed NPs by capping them as shown in Fig. 1. The carboxylate groups in the oxidized form of INH readily form a corona around the Ag clusters giving raise to AgNPsINH as shown by the halo around the NPs (Fig. 1). This self-assembly is governed by electrostatic attractions between the oxidized INH and the metallic clusters [13]. Furthermore, due to reported antibacterial, antiviral and anti-cancerous activities of PTA and PMA, we have chosen them for surface functionalization of AgNPsINH. Nevertheless, it is important to note that PTA/PMA molecules are unstable at physiological pH, leading to their limited applicability in biology and medicine [33,36]. Therefore, improved stability of PTA and PMA by coating them on the surface of AgNPsINH, enhancing their utility in biology and medicine, especially as antimicrobial agents. Fig. 2A (a–c) show the solutions of the different nanoparticles prepared with and without POMs. The AgNPsINH exhibit a pale-yellow color (Fig. 2A-a), whereas functionalization with PTA gives rise to orange colored AgNPsINH@PTA (Fig. 2A-b). A transformation of AgNPsINH@PMA to seaweed green color occurs upon functionalization with PMA (Fig. 2A-c). The visible changes in the solutions suggest surface

Kirby-Bauer disk antibiotic susceptibility test, as described in [37], was performed to determine the antibiotic efficacy of AgNPsINH, AgNPsINH@PTA, and AgNPsINH@PMA. Freshly inoculated strains of E. coli and S. aureus were incubated at 37 °C with constant stirring and allowed to grow until absorbance value reached 0.1. The strains were plated on nutrient agar, and subsequently, disks (approx. 6 mm) prepared from Whatman™ filter paper 2 were placed at equal distance around the circumference of the plate. The disks were treated with 50 μl of 0.1293 μg/ml AgNPsINH, 0.1359 μg/ml AgNPsINH@PTA and 0.106 μg/ml AgNPsINH@PMA and incubated at 37 °C for 24 h. Similar disks of rifamycin (RIF) and cefixime (CFX) were used as positive controls. The inhibition zones were imaged using a Scan®500 camera of 1 megapixel and analyzed with Scan® software. Activity indices (AI) were compared with those for RIF and CFX. Data from three independent experiments were analyzed. Furthermore, to determine the minimum concentration of NPs required for the inhibition of bacterial growth, the broth dilution method was used according to the standard procedure of Clinical and Laboratory Standards Institute [CLSI Guidelines 2012], which facilitates the testing of inhibitory activity at various NPs concentrations. As described previously, antimycobacterial sensitivity assessment of AgNPsINH, AgNPsINH@PTA, and AgNPsINH@PMA were performed on a susceptible strain of M. tuberculosis H37Rv (ATCC 27294) reference strain along with MDR clinical isolate using fluorescence (BACTEC MGIT) method in the MGIT™ instrument (Becton, Dickinson and 3

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Fig. 2. Properties of AgNPsINH and their POMs-enriched nanoparticles: A: Solutions of (a) AgNPsINH, (b) AgNPsINH@PTA and (c) AgNPsINH@PMA nanoparticles. B: UV–vis absorption spectra showing surface plasmon resonance of AgNPsINH, AgNPsINH@PTA and AgNPsINH@PMA. C: TEM micrographs of AgNPsINH (a), AgNPsINH@PTA (b) and AgNPsINH@PMA (c); wherein, the scale bars indicate 100 nm. D: Particle size distribution histogram of (a) AgNPsINH, (b) AgNPsINH@PTA and (c) AgNPsINH@PMA. E: Hydrodynamic radius and surface charge (ζ-potential values) of the nanoparticles in solution.

for AgNPsINH@PTA and AgNPsINH@PMA can be attributed due to the surface modification of AgNPsINH with POMs or may be because of the development of anisotropy. Fig. 2C(a–c) gives the TEM micrographs of NPs, and confirms spherical to pseudo-spherical shaped particles without any noticeable change in the morphology after surface modification. This observation indicates that the additional bands in the range of ca. ∼ 465–598 nm for AgNPsINH@PTA and ca. ∼ 500–720 nm for AgNPsINH@PMA are arising due to the surface modification. Particle size distribution histogram of AgNPsINH, AgNPsINH@PTA and AgNPsINH@PMA are shown in Fig. 2D. The mean particles size of AgNPsINH was found to be ∼18.9 ± 4.4 nm, which increased upon surface functionalization with PTA and PMA as expected. The AgNPsINH@PTA and AgNPsINH@PMA showed their mean particles size ∼21.7 ± 6.3 nm and ∼22.0 ± 7.5 nm, respectively. The functionalization of the NPs was further verified by the surface charge of the NPs, wherein post-surface functionalization the surface charge of NPs must increase in the value

modification due to binding of PTA/PMA on to the surface of the AgNPsINH. We next performed UV–vis spectroscopy to confirm the presence of PTA and PMA moieties on functionalized NPs. As shown in Fig. 2B, the localized surface plasmon resonance (l-SPR) of AgNPsINH was at ∼ 406 nm, which is typical of Ag NPs, and the AgNPsINH did not display any signs of aggregation. However, an additional absorption band at ∼263 nm is observed, possibly corresponding to oxidized-INH on the surface of AgNPsINH. The absorption of pure INH, characteristic л-л* electronic transition of pyridine part of INH (Fig. S1) shows a peak originating at ∼ 270 nm. It was interesting to notice that surface modification with PTA or PMA did not make a significant shift in the SPR of typical Ag nanoparticles. For example, AgNPsINH@PTA and AgNPsINH@PMA show their corresponding spectra at ∼ 408 nm and ∼ 407 nm, respectively. Nevertheless, additional new bands were observed between ca. ∼ 465–598 nm for AgNPsINH@PTA and ca. ∼ 500–720 nm for AgNPsINH@PMA Fig. 2B. The additional bands arising 4

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because the pH of the AgNPsINH is ∼ 8.27. The INH corona of AgNPsINH should theoretically be in the deprotonated state (pKa value of INH is 1.82), which is further validated by the high zeta (ζ) potential values of -27.26 mV (Fig. 2E). Furthermore, the literature indicates that at pH 8 or above, the POMs will be present in the form of their ions. Therefore, at pH ∼ 8.27, PTA must be present as PO43−and WO42− [39] and PMA as PO43− and MoO42- [40]. In the form of WO42−, MoO42- and PO43− the moieties of PTA and PMA will be readily adsorbed on the surface of AgNPsINH to produce AgNPsINH@PTA and AgNPsINH@PMA, correspondingly. This is substantiated by the increase in charge densities from -27.3 mV (AgNPsINH) to values of -35.3 mV and -28.5 mV for AgNPsINH@PTA and AgNPsINH@PMA, respectively; conferring the presence of PTA and PMA on the surface of respective particles. The functionalization of AgNPsINH with POMs leads to a rise in the moiety densities of WO42−, MoO42- and PO43− leading to an increase in surface charge densities for AgNPsINH@PTA and AgNPsINH@PMA. The high ζ potential values of all these NPs also indicate their stability, and validates the observations of UV–vis spectroscopy and TEM, which show no sign of aggregation. It was further established, that these NPs did not show any precipitation or aggregation under standard laboratory storage conditions over nine months. In practice, if the ζ potential values are greater (positive or negative), the repulsive forces are able to overcome the attractive forces, reducing the agglomeration of the colloids [41]. The presence of PTA or PMA moieties is also suggested by the hydrodynamic radii determined using DLS measurements, wherein the size of the AgNPsINH increases as shown in Fig. 2E. The DLS readings display a hydrodynamic radius of ∼54 nm for AgNPsINH with less polydispersity, whereas in the case of AgNPsINH@POMs hydrodynamic radius increased as expected. The data specifies that the AgNPsINH@PMA shows a ∼31% increase in size from AgNPsINH, while AgNPsINH@PTA shows ∼0.4% increase in hydrodynamic radius. The increment variation in the hydrodynamic radius for AgNPsINH@PMA and AgNPsINH@PTA indicates the reorganization of the surface structures, which takes place in order to obtain minimum surface energies. The degree of resurfacing is dependent on the structural organization, charge densities and its interaction in the liquid-solid interface [42,43]. Furthermore, FTIR spectroscopy was employed to confirm the presence of INH on the surface of AgNPsINH, and their modification with PTA and PMA molecules. In the pristine form of INH, it shows distinguishing functional group vibrational frequencies at 3247 cm−1, 1637 cm−1, 1340 cm−1, 1228 cm−1 and 1416 cm−1 originating from NHe stretching, carbonyl group C]O, amino group NH2 wagging, NeN single bond and pyridine, respectively [44]. Presence of characteristic frequencies after AgNPsINH formation with vibrational shifts establishes the existence of oxidised-INH on AgNPsINH [13]. For instance, distinctive functional group vibrational frequencies of NeH stretching, CO] group, NH2 wagging, NeN single bond and pyridine were reported at 3277 cm−1, 1636 cm−1, 1382 cm−1, 1263 cm−1 and 1459 cm−1, correspondingly. Moreover, FTIR spectroscopy provided direct evidence of AgNPsINH surface functionalization with PTA or PMA. The Keggin structures of PTA (H3PW12O40) and PMA (H3PMo12O40) contain oxygen (O) atoms from distinct bonds, which have unique infrared signatures as shown in Table S1 and Fig. 3. Wherein, an asymmetric stretching vibrational mode between P and O atoms at 1077 cm−1 (PTA) and 1070 cm−1 (PMA); bending vibrational modes of O atoms forming bridge between the two tungsten (W) or molybdenum (Mo) atoms at 835 cm-1 (PTA) and 836 cm−1 (PMA); and asymmetric stretching of terminal O atoms at 948 cm−1 (PTA) and 943 cm−1 (PMA) are shown in Fig. 3. The strong binding of PTA or PMA moieties on the surface of AgNPsINH is evident from the shifts in the vibrational modes (PTA or PMA) after formation of AgNPsINH@PTA and AgNPsINH@PMA, as presented in Fig. 3 and Table S1. After confirming the presence and stability of INH and POMs on AgNPs, we next investigated the in vitro peroxidase enzyme-like activity, for all the formulated NPs. Inorganic NPs have emerged as accessible materials for their potential intrinsic enzyme-mimicking

activities, and they are relatively stable compared to the natural enzymatic systems. Our group has established that simple metal NPs with suitable surface coronas can also mimic enzymes [13,22]. As discussed earlier, the INH pro-drug is activated because of the catalase-peroxidase enzyme interaction within the Mycobacteria, and it will be essential to observe if the NPs coated with INH had similar enzyme-like behavior. Therefore, to provide a proof of concept, we have first investigated the in vitro peroxidase enzyme-like activity of NPs and role of POMs after surface modification on AgNPsINH. The peroxidase-like activity of AgNPsINH, AgNPsINH@PTA, and AgNPsINH@PMA were tested using a chromogenic substrate TMB in the presence of H2O2. During the reaction, TMB oxidizes to TMBO by donating an electron to H2O2 which is facilitated by the peroxidases. The oxidized product TMBO can be measured spectrophotometrically at 650 nm. As illustrated in Fig. 4, the AgNPsINH exhibit peroxidase-mimicking nanozyme activity, which further improved upon surface decoration with PTA or PMA. The in vitro peroxidase nanozyme activity of AgNPsINH@PTA and AgNPsINH@PMA is higher than pristine AgNPsINH; with PTA modification has a substantial enhancement in the activity. The difference in the enzyme-like activities between AgNPsINH, AgNPsINH@PTA, and AgNPsINH@PMA can be attributed to the surface corona of the Ag NPs. The results indicate that the surface corona of NPs is vital to control their behavior and decoration of suitable moieties on the corona may improve their potential. To confirm the role of surface moieties, under similar experimental conditions, we have also evaluated the peroxidase-like behavior of pristine INH, free metal (Ag+) ions, PTA and PMA (Fig. S2). It is interesting to observe that the Ag+ and INH have negligible peroxidase-like activity in their native form but during the synthesis of NPs, the AgNPsINH secured inherent peroxidase-like behavior. It has been reported that the PTA is catalytically more active than PMA, and PTA molecules are reported for higher dispersion compare to other POMs including PMA [45]. Therefore, we anticipate that if PTA and PMA have peroxidase-like activity then PTA must display more activity. As anticipated, the free PTA revealed considerably higher peroxidase-like activity as shown in Fig. S2. When we compare the activity of AgNPsINH@PTA and AgNPsINH@PMA, as expected the PTA functionalized particles show superior nanozyme action with respect to PMA modification of Ag NPs (Fig. 4). In the context of higher activity of PTA modified NPs, it is imperative to state that the hydrodynamic radius of AgNPsINH@PTA is ∼54 nm, whereas the hydrodynamic radius of AgNPsINH@PMA is ∼71 nm (Fig. 2E). Therefore, it is apparent that surface area of AgNPsINH@PTA will be higher compare to AgNPsINH@PMA, and it can make PTA functionalized particles more active compare to PMA functionalized particles toward their peroxidase-like activity. After confirming the in vitro peroxidase-mimicking activity, we validated the antibiotic efficiency and antimicrobial potential of POMsenriched AgNPsINH through standard disk diffusion. The effectiveness of AgNPsINH, AgNPsINH@PTA, and AgNPsINH@PMA were compared with the activities of conventional drugs such as RIF and CFX on S. aureus (Gram + ve) and E. coli (Gram -ve) as illustrated in Table 1 and expressed in terms of % activity index (AI). A noticeable change was observed for all the nanoparticles against S. aureus and E. coli. For example, AgNPsINH displays 9.3 ± 0.3 mm and 8.3 ± 0.4 mm; AgNPsINH@PTA shows 10.2 ± 0.1 mm and 9.5 ± 0.1 mm; and AgNPsINH@PMA has 10.0 ± 0.3 mm and 11.1 ± 0.4 mm zone of inhibition for S. aureus and E. coli, respectively (Table 1). From the results, it can be clearly seen that the PTA and PMA functionalization of AgNPsINH has improved antibacterial potential of the NPs for both the studied strains, with these NPs more active against Gram positive bacteria. The difference in activities of PTA and PMA functionalized AgNPsINH can further be attributed to the orientations and inherent behavior of the modifying agents [32]. PTA and PMA have higher action toward Gram +ve bacterium as illustrated in Table S2. Furthermore, the antibiotic efficiency of POMs-enriched AgNPsINH with respect to RIF and CFX was comparable, and an improvement was observed post-modification with POMs as illustrated in Table 1. CFX is active against Enterococci, 5

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Fig. 3. FTIR spectra of POMs and their functionalized AgNPsINH wherein, [A] PTA (curve 1) and AgNPsINH@PTA (curve 2); and [B] PMA (curve 1) and AgNPsINH@PMA (curve 2).

whereas RIF belongs to ansamycin broad-spectrum antibiotic. RIF display pronounced activity against Gram +ve bacterium, and is used in the treatment of several types of bacterial infections, including tuberculosis. RIF inhibits the activity of DNA-dependent RNA-polymerase, but resistance is readily developed for RIF, and therefore, RIF is commonly formulated as a combinatorial medicine with INH [46]. Under similar experimental conditions, the diameter of the zone of inhibition and % AI against RIF and CFX for free Ag+, pristine INH, PTA, and PMA were calculated and are presented in Table S2. Furthermore, the MIC was assessed up to 50 μg/L concentration of different NPs. The MIC of AgNPsINH, AgNPsINH@PTA and AgNPsINH@PMA against Gram bacterial strains are illustrated in Table S3. In the case of Gram + ve S. aureus, a 6.25 μg/L MIC has been observed for all the NPs, which is low compared to Gram -ve E. coli. Nevertheless, the noteworthy improvement in the antibacterial potential of AgNPsINH can be seen post- functionalization with PTA or PMA against E. coli. The required dose of 50 μg/L for AgNPsINH was reduced to 25 μg/L and 12.5 μg/L for AgNPsINH@PTA and AgNPsINH@PMA, respectively. The outcome of MIC studies is validating the results of the zone of inhibition, wherein the higher antibacterial potential can be observed towards Gram + ve bacterium and influence of surface functionalization is evident in Gram -ve bacterium. Antimycobacterial susceptibility assays were performed for AgNPsINH, AgNPsINH@PTA AgNPsINH@PMA against reference strain and MDR clinical isolate of M. tuberculosis. The susceptibility assessment indicated that the AgNPsINH were not effective (at evaluated concentration); however, after post-functionalization with PTA, the same concentration of Ag NPs (AgNPsINH@PTA) became effective. At the same time the functionalization with PMA was not found

Fig. 4. In vitro peroxidase-mimicking activity for AgNPsINH, AgNPsINH@PTA and AgNPsINH@PMA (i.e., nanozyme activity). The coating of AgNPsINH with PTA or PMA enriches the nanozyme action of Ag nanoparticles.

Table 1 Antibiotic efficacy of POMs-enriched AgNPsINH. The diameter of zone of inhibition and % activity index (AI) against RIF and CFX are presented for AgNPsINH, AgNPsINH@PTA, and AgNPsINH@PMA, respectively. Sample

Staphylococcus aureus Zone of inhibition (mm)

AgNPsINH AgNPsINH@PTA AgNPsINH@PMA

9.3 ± 0.3 10.2 ± 0.1 10.0 ± 0.3

Escherichia coli % Activity index

Zone of inhibition (mm)

RIF

CFX

82.0 ± 10.7 89.2 ± 10.3 87.1 ± 7.5

80.2 ± 10.3 87.1 ± 8.5 85.3 ± 7.2

6

8.3 ± 0.4 9.5 ± 0.1 11.1 ± 0.4

% Activity index RIF

CFX

60.2 ± 2.4 69.1 ± 1.7 80.9 ± 2.9

27.1 ± 1.2 31.1 ± 0.8 36.4 ± 1.6

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Fig. 5. ROS generation (A) and the morphology (B) of m5S fibroblasts upon treatment with AgNPsINH, AgNPsINH@PTA and AgNPsINH@PMA (b–d), against wild cells (a). The morphology of fibroblasts treated with AgNPsINH@PTA and AgNPsINH@PMA shows similar appearance. However, Ag+ treated cells display a significant change in morphology indicating severe toxicity; a higher ROS production is also shown in (e). INH treatment did not show significant change in the fibroblasts (f). Scale bars =100 μm.

that increased ROS levels may lead to oxidative damage to cellular biomolecules, including nucleic acids, proteins, and lipids [49]. DNA is likely the most biologically significant target of the oxidative attack, and several studies have shown genotoxicity (DNA damage) induced by Ag NPs in cultured cells leads to death [50]. Nevertheless, these results indicate that the stabilization of INH on the surface of NPs may further provide an opportunity to control the toxicity of free INH, by further reducing its ROS production capacity, whereas its enzyme-like action may be still having upper hand on its therapeutic effects as shown in case of antibiotic efficacy of these particles that can be further controlled using suitable surface corona. Moreover, controlled toxicity may not have a significant influence on the morphological characteristics of cells. We have further evaluated the morphological changes in mammalian cells post-treatment with NPs, free metal ions and pristine INH by crystal violet staining of the mouse fibroblasts as depicted in Fig. 5B. It is interesting to observe that free Ag+ ions displayed morphological changes to the cells, indicating potential toxicity [Fig. 5B(e)], whereas,

to be effective against Mycobacterium, which is due to less activity of PMA moieties. This observation indicates the importance of surface functionalization using suitable molecules to improve the activity of NPs. It has been reported that Ag NPs can damage organs [47], and they can induce apoptosis and restrict DNA synthesis [48] by producing ROS. Inspired by this observation, we further evaluated the role of surface corona in promoting the generation of ROS through DCFH-DA assay. After the diffusion of DCFH-DA into the cells, the fluorogenic dye is deacetylated (product is non-fluorescent) by cellular esterases, and is further oxidized by ROS to DCF, making it highly fluorescent. Fig. 5(A) displays the ROS generation in the m5S mouse skin fibroblasts upon treatment. After incubation with different NPs, compare to untreated cells Fig. 5A(a), AgNPsINH, AgNPsINH@PTA and AgNPsINH@PMA treated fibroblasts [as shown in Fig. 5A(b–d)] revealed moderate ROS generation capabilities. However, the treatment with Ag+ exhibited the highest ROS generations as shown in Fig. 5A(e), whereas the pure INH also leads to moderate ROS production Fig. 5A(f). It is widely accepted

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Fig. 6. Cytotoxicity of AgNPsINH, AgNPsINH@PTA, AgNPsINH@PMA tested with mouse fibroblast m5S cells. Panel A: % Cytotoxicity by LDH assay; Panel B: % Viability by WST-8 assay.

all other treatments of AgNPsINH, AgNPsINH@PTA, and AgNPsINH@PMA did not exhibit any morphological changes as shown in Fig. 5B(b–d) along with free INH [Fig. 5B(f)]. From the morphological changes, it can be attributed that the high-density ROS generation upon treatment with free Ag+ lead to changes in the morphology of the cells, whereas moderate to lower production of ROS does not yield morphological changes in the fibroblast cells. The observed results specify that INH and POMs decorated Ag NPs have capabilities to mimic antibiotics but at the same time they are non-toxic to fibroblast cells. Nevertheless, NPs after entering the cell may change the dynamics and homeostasis of cellular physiology; the internalization of Ag NPs can also induce stress response(s) due to stimulation of free radical production, which in turn, may stimulate the inflammatory signaling pathways [51]. In contrast, our study demonstrated that AgNPsINH, AgNPsINH@PTA and AgNPsINH@PMA did not show toxicity at desirable concentrations confirming the non-lethal property of these particles. In the toxicity context, to further validate the results, we performed LDH assay (cellular death) to assess the membrane damage caused by AgNPsINH, AgNPsINH@PTA, and AgNPsINH@PMA, respectively as shown in Fig. 6(A). The % cytotoxicity exhibited by free Ag+, pristine POMs and pure INH is represented in Fig. S3(A). It was gratifying to observe that AgNPsINH, AgNPsINH@PTA and AgNPsINH@PMA showed negligible membrane damage to fibroblasts until 0.5 μg/ml concentrations, whereas at the concentration of 0.5 μg/ml free Ag+ presented small damage and it was significant for pristine PTA and PMA as shown in Fig. S3. It is notable that the addition of layers of POMs (either PTA or PMA) on the surface of AgNPsINH stabilizes the POMs on NPs and diminishes their toxicity at lower concentrations. However, at higher concentrations of NPs the toxicity to fibroblasts increases, and it was found to be ∼ 41% and ∼ 99% for AgNPsINH, and AgNPsINH@PTA, respectively at 1.0 μg/ml. The sharp rise in toxicity of the NPs at 1.0 μg/ml can be attributed to different effects of NPs on cells [52]. However, significant impact on cell damage in terms of toxicity for AgNPsINH@PMA is reported at 2.0 μg/ ml concentration only. Additionally, the WST-8 assay was conducted to assess the viability of the treated m5S cells. Unlike MTT, the tetrazolium salt WST-8 requires a proton donor to enter the cells. At lower concentrations, as expected the NPs show high cell survival as reflected by high cell viability [Fig. 6(B)], confirming the trends displayed by LDH assay. The observed results of this study show that the INH and POMs decorated Ag NPs have the ability to mimic peroxidase enzymes, and they are comparable to antibiotics in terms of their antibacterial potential. Nevertheless, these INH-coated POMs functionalized Ag NPs have minimal cytotoxicity on fibroblast cells, as measured in terms of fibroblast morphology changes, cell survival and cell death including assessment of ROS production. These studies reveal that stabilization of INH, PTA, and PMA on the surface of Ag NPs assists in diminishing the toxic effects of these systems to mammalian cells, without having an adverse impact of their in vitro enzyme performance and antimicrobial potential.

4. Conclusions We have prepared multifunctional antimicrobials to address the emergence of the drug-resistance strains. The antibiotic INH was employed as a reducing agent to prepare AgNPsINH, following their decoration with POMs to formulate AgNPsINH@PTA, and AgNPsINH@PMA as novel antibiotic NPs. As confirmed by multiple techniques, the particles showed high stability, POM coating at their surfaces, and formation of the suitable antibiotic surface corona. The AgNPsINH, which exhibited peroxidase-like activity, showed significant efficacy against pathogenic bacterial strains. The antimicrobial potential of AgNPsINH enhanced after their surface functionalization with POMs, and they developed antimycobacterial potency. Moreover, tests for ROS generation, morphology analysis and cytotoxicity with mouse skin fibroblasts exhibited that AgNPsINH, AgNPsINH@PTA, and AgNPsINH@PMA are less toxic compared to pristine molecules of INH, POMs, and free metal ions. Taken together, we have generated multifunctional nanoparticles featuring high efficacy and multiple mechanisms of action. These NPs have minimal toxicity to mammalian cells, providing a promising alternative to challenge the nosocomial infections involving MDR strains. Acknowledgments AU acknowledges the scholarship provided by Rao Bahaddur Dharma Pravartha Gubbi Thotadappa Charities (RBDGTC), Bengaluru, India. AU, NPN, DJ, and HKD recognize the generous support of Japan Science and Technology (JST) Agency, Japan toward Asia Youth Exchange Program in Science (Sakura Exchange Program). The technical assistance of Arwa Kaizer Ali of Rajasthan College of Agriculture and Technology, Udaipur, India is duly acknowledged. HKD also acknowledges Centre for Advanced Materials and Industrial Chemistry (CAMIC) at the School of Sciences, RMIT University, Australia for the Honorary Visiting Research Fellowship. VMR acknowledges the National Institutes of Health (NIH, AI13477). Authors gratefully acknowledge Magnum Diagnostics & Research Centre, Jaipur, India for anti-mycobacterium assays. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.110522. References [1] G. Sulis, A. Roggi, A. Matteelli, M.C. Raviglione, Tuberculosis: epidemiology and control, Mediterr. J. Hematol. Infect. Dis. 6 (1) (2014). [2] P.C. Goold, C.J. Bignell, No way back for quinolones in the treatment of gonorrhoea, Sex. Transm. Infect. 82 (3) (2006) 225–226. [3] S. Keshavjee, I.Y. Gelmanova, P.E. Farmer, S.P. Mishustin, A.K. Strelis, Y.G. Andreev, et al., Treatment of extensively drug-resistant tuberculosis in Tomsk, Russia: a retrospective cohort study, Lancet (London, England). 372 (9647) (2008) 1403–1409.

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