In vivo safety evaluation of antibacterial silver chloride nanoparticles from Streptomyces exfoliatus ICN25 in zebrafish embryos

Accepted Manuscript In vivo safety evaluation of antibacterial silver chloride nanoparticles from Streptomyces exfoliatus ICN25 in Zebrafish embryos Appadurai Muthamil Iniyan, Rajaretinam Rajesh Kannan, Francis-Joseph Rosemary Sharmila Joseph, Thankaraj Rajam Jabila Mary, Mani Rajasekar, Puthenpurayil Chellappan Sumy, Arul Maximus Rabel, Dasnamoorthy Ramachandran, Samuel Gnana Prakash Vincent PII:

S0882-4010(17)30505-3

DOI:

10.1016/j.micpath.2017.07.054

Reference:

YMPAT 2485

To appear in:

Microbial Pathogenesis

Received Date: 8 May 2017 Revised Date:

8 July 2017

Accepted Date: 26 July 2017

Please cite this article as: Iniyan AM, Kannan RR, Rosemary Sharmila Joseph F-J, Jabila Mary TR, Rajasekar M, Sumy PC, Rabel AM, Ramachandran D, Vincent SGP, In vivo safety evaluation of antibacterial silver chloride nanoparticles from Streptomyces exfoliatus ICN25 in zebrafish embryos, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.07.054. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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In vivo safety evaluation of antibacterial silver chloride nanoparticles from Streptomyces exfoliatus ICN25 in Zebrafish embryos Appadurai Muthamil Iniyan,a Rajaretinam Rajesh Kannan,b Francis-Joseph Rosemary Sharmila Josepha, Thankaraj Rajam Jabila Maryb, Mani Rajasekar,b Puthenpurayil Chellappan Sumy,a Arul Maximus Rabel,b Dasnamoorthy Ramachandran,b Samuel Gnana Prakash Vincenta * a

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International Centre for Nanobiotechnology (ICN), Centre for Marine Science and Technology (CMST), Manonmaniam Sundaranar University, Rajakkamangalam, Kanyakumari Dist-629502, TN, India. b Molecular and Nanomedicine Research Unit, Centre for Nanoscience and Nanotechnology (CNSNT), Sathyabama University, Jeppiaar Nagar, Rajiv Gandhi Road, Chennai-600119, TN, India. *Corresponding author: International Centre for Nanobiotechnology (ICN), Centre for Marine Science and Technology (CMST), Manonmaniam Sundaranar University, Rajakkamangalam, Kanyakumari Dist-629502, TN, India E-mail address: [email protected] (Dr. S. G. Prakash Vincent)

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Abstract Silver chloride nanoparticles were synthesized from the cell-free culture supernatant of Streptomyces strain using green synthesis approach with good yield. The nanoparticles were characterized by UV-Vis, IR, SEM, AFM and XRD techniques. These nanoparticles exhibited broad spectrum of antibacterial activity towards Methicillin-resistant Staphylococcus aureus, Methicillin sensitive S. aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumonia at ≤ 2 µg/ml minimal inhibitory concentrations. In vivo bioassay in nanoparticles treated zebrafish embryos exhibited 16 µg/ml dose as maximal cardiac safety concentration and further increases in concentration revealed adverse effects such as pericardial bulging, mouth protrudation, hemorrhage and yolk sac elongation. The less toxicity of nanoparticles treated embryos in terms of cardiac assessment and lethality analysis was observed. The dose below 5 µg/ml is concluded as an in vitro and in vivo therapeutic dose. The properties of this biosynthesized nanoparticle suggest a path towards developing antibiotic nanoparticles that are likely to avoid development of multidrug resistance. Keywords: Nano-biotechnology, Silver chloride nanoparticle, Streptomyces, Zebrafish, cardiac assessment, MTT assay

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1. Introduction Current drift in infections caused by microbial pathogens resistant to antibiotic drug therapy are major problems worldwide resulting in prolonged illness, greater risk of death and higher costs [1,2]. There is an urgent need of alternative treatment measures to fight against the pathogenic microorganisms. Advances in the manipulation of nanomaterials have permitted the development of nano-biotechnology with enhanced sensitivities and improved response times [3,4]. The synthesis of metal nanoparticles and nanostructure materials especially silver nanoparticles are attractive due to their unusual optical, chemical and biomedical potentials [5,6]. Moreover, the silver nanoparticles (AgNPs) are particularly important which have strong surface plasmon resonance oscillations [7]. It shows very strong bactericidal activity against gram positive as well as gram negative bacteria including multi-resistant strains [8,9]. Several bacterial and fungal species are known to reduce metal ions to the metals. Extracellular metabolites play a major role in the biosynthesis reduction reaction of silver nanoparticles [10,11]. The frequently increasing amount of industrial nanoproducts requires consistent risk management, as contacts with human beings and the environment are becoming more common. A large number of in vitro studies indicate that AgNPs poses toxic effects to the mammalian cells derived from skin, liver, lung, brain, vascular system and reproductive organs. Moreover, AgNPs exerted developmental and structural malformations in non-mammalian model organisms typically used to elucidate human disease and developmental abnormalities. In addition, zebrafish model allowing phenotypic analysis of embryogenesis and organogenesis in vivo have been used for toxicity of silver nanoparticles recently [12,13,14]. These antibacterial nanoparticles possess weak ability of bacteria to develop resistance, the present study is focused on the biosynthesis of silver nanoparticles for effective antimicrobial activity and biocompatibility studies in zebrafish embryos.

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2. Experimental Section The bacterial pathogens such as Methicillin-resistant Staphylococcus aureus ATCC33591, Methicillin sensitive Staphylococcus aureus ATCC29213, Escherichia coli ATCC35218, Pseudomonas aeruginosa ATCC27853 and Klebsiella pneumonia ATCCBAA-1705 were procured from HiMedia Laboratory, India. All the cultures were maintained on nutrient agar slants at 4˚C. HeLa and SiHa cells were purchased from NCCS Pune. UV-Vis spectral analysis was carried out in UV-visible double beam spectrophotometer (Tech Comp 8500 Spectrometer). Field Emission Scanning Electron Microscopy was studied using FESEM-SUPRA 55, Carl Zeiss, Germany with 20 kV and gun vacuum at 2.12 e-009. The XRD patterns were obtained using X-ray diffractometer (Rigaku, Japan) with the CuKα radiation (λ = 1.5418Å) at a scan speed of 3 degree per minute in the 2θ range from 20° - 80°. Atomic force microscopy (Ntegra Prima Modular Mode, Ireland) was performed in the semi-contact mode.

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2.1. Isolation and characterization of the producer strain The bacterial strain was isolated from the rhizome soil region of the mangrove Rhizophora mucronata at Rajakkamangalam estuary, west coast of Kanyakumari using Actinomycetes Isolation Medium (AIM) as described previously [15]. Morphological and physiological characterization studies were carried out by microscopic observation and studies on growth characteristics using International Streptomyces Project (ISP) media [16,17]. The genomic DNA was isolated from the culture broth and the 16S rRNA gene was PCR amplified using the 16S rRNA universal primers (27f and 1429r) and sequenced. The 16S rRNA gene sequence was compared with the GenBank database by using BLAST for the homology sequences [18].

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2.2. Biosynthesis of silver nanoparticles The isolated strain was cultured aerobically in the 100 ml of AIM broth containing pH 7.4 and the fermentation was carried out at 28ºC for 7 days with agitation of 140 rpm. 106 CFU/ml of seed culture was used as the starting inoculums. Cell-free supernatant was prepared from the fermented broth using centrifugation (Eppendorf 5418R) at 8000 rpm for 15 minutes. 1 mM aqueous silver nitrate solution (10 ml) was added to 90 ml of the cell-free culture supernatant and incubated at 37°C in an orbital shaker set at 160 rpm for 24 h in the dark. Bio-reduction of silver ions was observed by the visual appearance of dark brown colour.

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2.3. Antibacterial studies A loop-full of the stock cultures were inoculated to Mueller-Hinton broth and incubated at 37˚C for 24 h. The cultures were diluted with fresh Mueller-Hinton broth to achieve optical densities corresponding to 0.5 (i.e., 105-106 CFU/ml using McFarland’s standard). Mueller-Hinton agar plates were swabbed with a sterile cotton swab that was first dipped in the freshly prepared diluted culture. A 6 mm hole was bored aseptically with a sterile cork borer. The holes were filled with 100 µl of 10 µg/ml synthesized silver chloride nanoparticle solution and the plates were kept for further incubation at 37˚C for 24 h. After incubation, the petri dishes were evaluated for antibacterial activity, which was measured for the inhibition zone diameter. 1 mM silver nitrate and the cell-free supernatant from Streptomyces exfoliatus ICN25 (approximately 20 µg/ml) was also investigated for the antimicrobial assay. MilliQ water was used as a negative control. 2.4. Determination of Minimum Inhibitory Concentration (MIC) The MIC was determined by broth micro-dilution method with the yellow dye 2,3,5-triphenyltetrazolium chloride (TTC) colorimetric assay for detecting the susceptibility [19]. The nanoparticle was serially diluted to obtain different concentrations (0.01, 0.1, 1, 2, 5, 10, 50 and 100 µg/ml) and assayed against each test

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pathogens. The MIC was defined as the lowest concentration of the nanoparticles that inhibited the visual growth of the test cultures on the microtitre plates.

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2.5. Cytotoxic assay The cytotoxicity of the nanoparticles was determined by MTT cell viability assay using HeLa and SiHa cell lines which were maintained in Dulbecco’s modified eagles media supplemented with 10% FBS (Invitrogen) and grown to confluency at 37°C in 5 % CO2 in a humidified atmosphere in a CO2 incubator. Different concentrations of silver chloride nanoparticles at 1 - 25 µg/ml were added to grown cells from a stock solution and incubated for 24 hours. The % difference in viability was determined by standard MTT assay after 24 hours of incubation [20].

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2.6. Safety evaluation of silver chloride nanoparticles in zebrafish embryos Wild type zebrafish were purchased from the aquarium shops and maintained at 26˚C to 28˚C temperature. Six months old zebrafish were used for the breeding to get embryos and were raised in embryo rearing solution [21]. The 48 hours post fertilization (hpf) embryos were treated with various concentrations of silver chloride nanoparticles (10 ng/ml, 100 ng/ml, 1, 2, 5, 10, 20, 30, 40 and 50 µg/ml) in a 24 well plate with 10 embryos in each well containing 1 ml of embryo rearing solution and 1% DMSO as a vehicle. Control embryos were also maintained in the presence of 1% DMSO and the experiments were conducted at a constant temperature (28˚C) in the dark. The following consequent changes in the developing embryos and larvae were noted during the next 96 hours. Within this period any abnormalities in the organ development (brain, eye, heart, ear, somite, notochord, trunk, tail and fin) was also monitored in the Light Microscope (Coslab, India) regularly. The LC50 values were calculated based on the four parameter logistic curve analysis as per the OECD regulations [22].

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2.7. Cardiac assessment Heart Beat Rate assay (HBR) was carried out in 3 days post fertilization (dpf) embryos after 4 hours of treatment with the silver chloride nanoparticles concentrations of 0.001, 0.01, 0.1, 1, 2, 4, 16, 32, 64 and 100 µg/ml [23,24]. Briefly, HBR for 15 sec was recorded in the attached camera at 10X objective lens magnification and processed for heart rate using Adobe premiere 6.5 and ImageJ (NIH). 0.02% tricaine (Sigma) was used to anesthetize the embryos in order to measure the HBR.

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3. Results and Discussion 3.1. Identification of the source bacterium Recently, there has been significant interest in antibacterial nanoparticles to overcome the problem of drug resistance in various pathogenic bacteria [25]. Biodirected syntheses of metal nanoparticles are gaining importance due to their biocompatibility, low toxicity and eco-friendly nature. The aim of this experiment was to produce eco-friendly antibacterial active nanoparticles with less toxicity towards eukaryotic cells. Current approaches with nanoparticles biosynthesis from Streptomyces and its whole genome sequencing studies revealed this genus as a major concern for the biomedical applications [11,26]. Therefore, the Streptomyces species is selected as a suitable candidate for producing nanoparticles. The nanoparticle producing strain was identified as Streptomyces exfoliatus ICN25 (NCBI GenBank accession no. KF017567) using 16S rRNA gene sequence analysis by showing 99.5% similarity to its nearest neighbor Streptomyces exfoliatus strain NBRC13191. The aerial mycelium showed rectus flexibilis type of highly branched long spore chains with smooth surface. It utilized broad range of carbon sources and produced melanin in the ISP6 medium. The strain tolerated upto 5% NaCl in the medium and produced catalase, oxidase, protease, lipase and nitrate reductase enzymes (Table 1). Table 1. Cultural characteristics of Streptomyces exfoliatus ICN25. Characteristics Streptomyces exfoliatus ICN25

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Morphology Aerial mycelium color Substrate mycelium color Pigmentation in the medium Melanin pigment Metabolite Exudation Shape of the aerial hyphae Series Spore chain Spore surface Physiology Growth temperatures Optimum temperature pH tolerance Optimum pH NaCl tolerance Optimum NaCl concentration Carbon source utilization

Tele grey May Green Bracken Green Present Absence Rectus flexibilis (RF) Gray Highly branched, Straight Smooth 20˚C - 37˚C 28˚C 5–9 7 0% - 5% 0.1%

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Positive Positive Positive Negative Positive Positive Negative

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Glucose, Fructose, Maltose, Sucrose, Lactose, Starch, Mannose, Cellulose, Dextrose, Arabinose, Enzyme activity Catalase, Oxidase, Protease, Lipase, Nitrate reductase, Urease

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3.2. Biosynthesis and Characterization of Silver nanoparticles Synthesis of silver nanoparticles was monitored from the addition of cell-free supernatant to silver nitrate solution led to the appearance of dark brown color (Figure 1). The extracts without AgNO3 did not showed any change in color. It is evident that silver nanoparticles exhibit a yellowish-brown color in aqueous solution due to excitation of surface plasmon vibrations in silver nanoparticles [27]. Absorption spectra of nanoparticles formed reaction media has absorbance peak ranging from 400 - 445 nm, broadening of peak indicated that the particles are poly-dispersed. Streptomyces exfoliatus ICN25 showed a maximum absorption at 410 nm and reached the optical density value of 2.726. This is similar to the surface plasmon vibrations with characteristic peaks of the silver nanoparticles prepared by chemical reductions [28].

Figure 1. UV-visible absorption spectra of silver nanoparticles from Streptomyces exfoliatus ICN25. a) Broth culture. b) cell-free supernatant without the addition of silver nitrate. c) Colour change after exposure to silver nitrate for 24 hours.

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The XRD pattern of nanoparticles (Figure 2) suggested that the structure is facing centered cubic shape, which is in good agreement with corresponding to fcc lattice of silver chloride. This indicates that most of Ag+ ions reacted with Cl‒ ions to form AgCl nanoparticles. The silver chloride nanoparticle shows reflections corresponding to (111), (200) and (220) planes. The crystallite size of synthesized silver chloride nanoparticle is estimated from the full width half maximum (FWHM) of high intensity plane (200) by using Scherrer’s formula. The size of the silver chloride nanoparticle ranges around 10 – 40 nm. Most of the nanoparticles aggregated and only a few of them were scattered, as observed under SEM and it exhibit spherical and rod shape (Figure 3).

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Figure 2. X-ray diffraction (XRD) spectrum of silver chloride nanoparticles prepared using culture supernatant of ICN25.

Figure 3. SEM image of silver chloride nanoparticles produced by S. exfoliatus ICN25.

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AFM studies of sprayed silver chloride nanoparticles were carried out and are displayed in Figure 4. A 3D image (5 µm × 5 µm scan) indicates that the average heights of the particles were lie between 5 to 10 nm sizes. The average particle size was observed using histogram analysis and the root mean square (RMS) surface roughness of the particles were calculated and was found to be 1.48 nm. These shows all the particles are in uniform size. Histogram analysis shows that, most of the particles are lie between 6 to 7 nm and the particle size starts from 3 to 12 nm ranges.

Figure 4. AFM images of silver chloride nanoparticles produced by culture supernatant of ICN25.

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It is well known that the results obtained from XRD are always higher than the value obtained from AFM analysis due to particle cluster. Similarly, Gurunathan et al. [29] had biosynthesized the nanoparticles from Allophylus cobbe leaves which is reported to have crystalline structure and face centered cubic crystal structure. The pictures from the FE-SEM experiment showed silver nanoparticles were spherical, quite close distributed with approximately similar diameters. FTIR measurements were carried out to identify the possible biomolecules responsible for the reduction of the Ag+ ions synthesized by actinomycete strain. The major peaks in the FTIR spectrum of silver nanoparticles from Streptomyces exfoliatus ICN25 (Figure 5) were observed at 3464.27, 3431.48, 2063.9, 1637.62 and a minor peak was observed at 586.38 cm-1. The reduction of the silver ions observed may plausibly be due to the protein component resulting from the enzyme nitrate reductase, since nitrate reduction is the phenotypic biochemical characteristic of this culture [30].

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Figure 5. FTIR analysis of the silver nanoparticles produced by culture supernatant of ICN25.

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3.3. Antibacterial activity The use of nanomaterials as antimicrobial agents has received increased attention in recent years. In antibacterial activity testing by well diffusion method, the reduced silver chloride nanoparticles from actinomycete strain showed inhibition zone against all the tested pathogens MRSA, MSSA, E. coli, P. aeruginosa and K. pneumonia (Table 2).

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Table 2. Antimicrobial activity using agar well diffusion method. Silver chloride nanoparticle at 10 µg/ml, silver nitrate at 1 mM and the cell-free culture supernatant 20 µg/ml were used.

MRSA

Culture 12.67±0.29 supernatant AgNO3 4.33±0.29 Silver chloride 15.5±0.5 nanoparticle MilliQ water 0

Zone of Inhibitions (mm) MSSA E. Coli Pseudomonas Klebsiella aeruginosa pneumoniae 3.83±0.58 3.17±0.29 10.5±0.5 4.33±0.58 2.83±0.29 6.33±0.58

3.0±0.5 5.83±0.58

8.17±0.29 12.5±0.5

2.67±0.29 4.33±0.29

0

0

0

0

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The silver chloride nanoparticles exhibited lowest MIC of 1 µg/ml against MRSA and 2 µg/ml against MSSA, E. coli, P. aeruginosa and K. pneumoniae. The antimicrobial activity of silver nanoparticles was reported to be due to the penetration into the bacteria and damage of cell membrane and release of cell contents [31]. The antimicrobial effects are dose-dependent and increased linearly with the increased concentration of the test sample. A lower MIC value exhibited by the silver chloride nanoparticles against MRSA is of great significance in the health care system. Recent studies have shown that the biosynthesized silver nanoparticles to possess antibacterial activity against multiple antibiotic resistant pathogens including Enterococcus faecalis, Vibrio harveyi, Vibrio parahaemolyticus, Bacillus licheniformis and Pseudomonas aeruginosa and for the cytotoxic activity against MCF-7 breast cancer cells and human lung cancer cells (A549) [32-36]. Similarly the biosynthesized silver chloride nanoparticles from our study have shown significant antibacterial property against antibiotic resistant pathogens.

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3.4. Toxicity towards cancer cell lines The cytotoxic viability index was examined using MTT assay of the AgCl NPs. The increasing concentrations favour the antiproliferation of HeLa and SiHa cell lines (Figure 6) which may be due to its nanoscale dimension and the surface functionalization. The AgCl NPs showed potent cytotoxicity over 89.04 ± 0.72 % death of HeLa cells at a treatment concentration of 25 µg/ml and 77.49 ± 0.83 % death of SiHa cells at the concentration of 25 µg/ml. The silver chloride nanoparticle exhibited LC50 value of 4.53 µg/ml against HeLa cells of 4.25 µg/ml against SiHa cells. The green synthesized silver nanorods exhibited anticancer activity in skin cancer cell line with less toxic towards normal cells [37].

Figure 6. MTT based antiproliferative assay of silver chloride nanoparticles on HeLa cell lines (a,b,c,d) and SiHa cell lines (A,B,C,D) after 24 hours incubation under the phase contrast microscope. a,A) Control with 1% DMSO, b,B) 1 µg of AgCl NPs, c,C) 5 µg of AgClNPs d,D) 25 µg of AgClNPs in 1% DMSO.

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3.5. In vivo safety assessment in Zebrafish embryos During the first day of exposure, the fishes did not show any significant changes in behavior as well as internal organ structures and functions. The highest experimental concentration showed mortality in the first day. At third day of treatment 20 µg/ml treated embryos exhibited abnormal tissue formation in the head region and yolk sac elongation (Figure 7a). The same concentration showed cardiac bulging whereas pericardial fluid is accumulated. The embryo showed a hemorrhage near the heart chamber and mouth deformities had observed (Figure 7b).

Figure 7. Effect of AgCl NPs in Zebrafish embryos. a) Abnormal tissue formation in the head region (black line) and yolk sac elongation (arrow). b) White marking shows the embryonic heart with pericardial bulging, mouth perturbation (blue arrow) and hemorrhage below the heart chamber (white arrow). c) Control embryo at 3dpf.

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According to OECD regulations the LC50 value found to be 23.63 µg/ml based on the mortality at 96 hours post treatment. The HBR of the AgCl NPs treated embryos showed decreases in heart rate (Table 3).

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Table 3. Heart Beat Rate assessment of silver chloride nanoparticles in zebrafish embryos after 4 hours of treatment.

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Concentration of Silver chloride Nanoparticle (µg/ml) Control 0.001 0.01 0.1 1 2 4 16 32

Heart Beat Rate (Beats/min) 165.33 ± 4.62 162.67 ± 4.62 165.33 ± 4.62 165.33 ± 4.62 157.33 ± 4.62 157.33 ± 4.62 154.67 ± 4.62 154.67 ± 4.62 126.67 ± 6.11

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64 100

109.33 ± 12.22 0

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The Maximal Cardiac Safety Concentration (MCSC) was found to be 16 µg/ml which show the safety profile. The control embryos showed 168 beats/min and a normal blood flow rate. Slow blood flow rate was also observed at 10 and 50 µg/ml concentrations. Similar study was reported the size dependent mortality and malformations in zebrafish embryos exposed to AgNPs [38]. Lee et al. [39] characterized the transport of a single AgNP into zebrafish embryos and investigated their effects on early embryonic development. CuO nanoparticles reduced the number of transverselyrunning subintestinal vessels in transgenic zebrafish [40]. It is evident from our study that the biosynthesized silver chloride nanoparticles does not showed any notable toxic effect at the MIC dose of microbes but if it exceeds the level above 20 folds, AgCl nanoparticles are found to be toxic to the developing embryos. Notably, the embryos were found to be safe up to 16 µg/ml which is having selectivity index of 23.63 against MRSA and 11.82 against MSSA, E. coli, P. aeruginosa and K. pneumonia towards MIC values. This shows the safety index of the biosynthesized nanoparticles. The dose below 5 µg/ml is considered as an in vitro and in vivo therapeutic dose for killing bacteria and also cancer cell lines. 5. Conclusion In conclusion, the present study highlighted the biosynthesis of less toxic and efficient antibacterial silver chloride nanoparticles from Streptomyces. This shows the actinobacteria mediated synthesis of nanoparticles is an efficient method. The isolate Streptomyces exfoliatus ICN25 could be used as source for metal nanoparticles biosynthesis. Furthermore, the biosynthesized silver chloride nanoparticles exhibited a prominent antibacterial and cytotoxicity activity against test pathogenic bacteria, HeLa and SiHa cancer cell lines. In vivo safety evaluation of synthesized nanoparticles in zebrafish embryos revealed a positive safety index. Taken together, the data collected in this study suggests a path towards developing antibiotic nanoparticles that are likely to avoid development of multidrug resistance. Acknowledgement The authors acknowledge support by Department of Science and Technology, Government of India for AFM facility through DST-FIST, Sathyabama University and University Grants Commission, Government of India through UGC-SAP program to CMST, MSU. References [1] A. Russo, E. Concia, F. Cristini, F.G. De Rosa, S. Esposito, F. Menichetti, N. Petrosillo, M. Tumbarello, M. Venditti, P. Viale, C. Viscoli, M. Bassetti, Current

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[38] O. Bar-Ilan, R.M. Albrecht, V.E. Fako, D.Y. Furgeson, Toxicity assessments of multisized gold and silver nanoparticles in zebrafish embryos, Small. 5 (2009) 1897–1910. [39] K.J. Lee, P.D. Nallathamby, L.M. Browning, C.J. Osgood, X.N. Xu, In vivo imaging of transport and biocompatibility of single silver nanoparticles in early development of zebrafish embryos, ACS Nano. 1 (2007) 133–143. [40] J. Chang, G. Ichihara, Y. Shimada, S. Tada-Oikawa, J. Kuroyanagi, B. Zhang, Y. Suzuki, R. Sehsah, M. Kato, T. Tanaka, S. Ichihara, Copper oxide nanoparticles reduce vasculogenesis in transgenic zebrafish through down-regulation of vascular endothelial growth factor expression and induction of apoptosis, J. Nanosci. Nanotechnol. 15(3) (2015) 2140–2147.

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Antibacterial silver chloride nanoparticles were synthesized from Streptomyces strain using green synthesis approach. In vivo bioassay in zebrafish embryos were studied and cardiac safety concentration was reported. The biosynthesized nanoparticles showed potent cytotoxicity towards HeLa and SiHa Cancer cell lines.

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