Ag2[Fe(CN)5NO] Nanoparticles Exhibit Antibacterial Activity and

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Ag2[Fe(CN)5NO] nanoparticles exhibit antibacterial activity and wound healing properties Bonda Rama Rao, Rajesh Kotcherlakota, Susheel Kumar Nethi, Nagaprasad Puvvada, Sreedhar Bojja, Arabinda chaudhuri, and Chitta Ranjan Patra ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00759 • Publication Date (Web): 07 Aug 2018 Downloaded from http://pubs.acs.org on August 7, 2018

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ACS Biomaterials Science & Engineering

Ag2[Fe(CN)5NO] nanoparticles exhibit antibacterial activity and wound healing properties Bonda Rama Rao, Rajesh Kotcherlakota‡, Susheel Kumar Nethi‡, Nagaprasad Puvvada, Bojja Sreedhar ¥, Arabinda Chaudhuri*, Chitta Ranjan Patra* Department of Applied Biology, CSIR-Indian Institute of Chemical Technology, Uppal Road, Tarnaka, Hyderabad - 500007, Telangana State, India. ¥ Inorganic and Physical Chemistry Division, CSIR-Indian Institute of Chemical Technology, Uppal Road, Tarnaka, Hyderabad-500007, Telangana State, India Academy of Scientific and Innovative Research (AcSIR), Training and Development Complex, CSIR Campus, CSIR Road, Taramani, Chennai – 600 113, India ‡These authors contributed equally * E-mail: [email protected], [email protected]. Tel: 91-4027156755, 91-9440040582 (A.C.). * E-mail: [email protected], [email protected]. Phone: 040-27191480. Fax: +91-40-27160387 (C.R.P.).

ABSTRACT: Therapeutic agents harboring both wound healing and antibacterial activities have much demand in biomedical applications. Development of such candidates with clinically approved materials adds more advantages towards these applications. Recently, silver metal complex nano-materials have been playing a major role in medical uses especially for anti-bacterial activity and wound healing. In this report, we designed and synthesized silver nitroprusside complex nanoparticles (abbreviated as AgNNPs) using sodium nitroprusside and silver nitrate (both are FDA approved precursors). The nanoparticles (AgNNPs) were thoroughly characterized by various physicochemical techniques such as XRD, FTIR, TGA, DLS, EDAX, Raman, ICP-OES, HRTEM and FESEM. The cell viability assay in normal cells (EA.hy 926 cells, NIH 3T3) using MTT reagents and CEA assay (CEA: Chick embryo angiogenesis assay) in fertilized eggs demonstrate the biocompatibility of AgNNPs. These nanoparticles show effective antibacterial activity against both Gram positive and Gram negative bacteria through membrane and DNA damage. Additionally, AgNNPs accelerate the wound healing in C57BL6 mice by altering the

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macrophages from M1 to M2. Considering the results together, the current study may offer the development of new silver nano-complex nanomaterials that shows synergistic effect on antibacterial activity and wound healing (2-in-1-system). To the best of our knowledge, this is the first report for the synthesis, characterization and biomedical applications of silver nitroprusside nanoparticles. KEYWORDS: Silver nitroprusside nanoparticles, nanomedicine, biocompatibility, macrophage activation antimicrobial activity, wound healing. 1. INTRODUCTION Microbial infections are global threats often causing diseases like cancers, tuberculosis, leprosy, typhoid fever, diarrhea, etc.1 In 2016, the organisation for economic cooperation and development (OECD) reported around seven hundred thousand deaths worldwide due to antimicrobial resistance. A 2013 report estimated around 58,000 neonatal sepsis deaths attributable to drug resistant infections in India alone.2,3 Various FDA approved new classes of antibiotics (e.g. Teflaro, Veltin, Victoza, Vibativ, Dalvanc) are currently available for treatment of microbial infections. Unfortunately, these drugs exhibit severe side effects such as vomiting, abdominal pain, coughing, wheezing, etc.4 On the other hand, burns and wounds are also the major cause of death due to the microbial infections and other factors. However, the currently available treatment for burns and wounds are highly expensive. In this context, silver based salts/compounds including silver carboxymethyl cellulose, silver alginate, silver sulfadiazine, etc have been used conventionally as anti-microbial agents for long time.5,6,7 Silver impregnated catheters and silver-based wound dressings are also being used for therapeutic applications.8 However, many of these silver composites are less stable and toxic in their native forms. To overcome these limitations, researchers have been developing different silver composites or

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formulations through surface modifications with polyethylene glycols, poly(vinyl alcohols), poly(methyl methacrylate), poly(bisphosphonate-b-2-vinylpyridine), proteins and peptides. 9 More recently, researchers are investigating the therapeutic activities of silver nanoparticles or nano-composites for treating bacterial infections and wounds.10, 11,

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Although there

are numerous silver nanoparticles based products available in the market, they often suffer from certain limitations including toxicity, biosafety, microbial resistance, etc. Hence, there is a need for developing more effective silver nano-composites to overcome these limitations. Herein, we report on design and synthesis of an eco-friendly, simple, template free, stable, and biocompatible silver nitroprusside nanoparticles (AgNNPs) using FDA approved sodium nitroprusside (SNP: anti-hypertensive drug) and silver nitrate. Some of the silver based compounds16 are strictly regulated by FDA for approval because of their toxic effect which is dependent on their chemical composition/formulation/nature of the materials. However, various commercialized silver containing products such as silver sulfadiazine17 are used for the treatment of wound healing and anti-bacterial activity. Furthermore, several research groups including ours are working on development of new silver nanoparticles formulations to minimize these limitations. In the present work, we developed silver nanoparticles using FDA approved precursor, sodium nitroprusside.18 This nanoparticle AgNNPs (Ag2[Fe(CN)5NO]) contains the silver which is complexed with nitroprusside that gives extra stability which allow to release the silver slowly from the complex in biological system (for wound healing and anti-bacterial activity). This complexation of silver may control the reactions with biomolecules that prevents the toxicity. This type of nanomaterials may overcome the limitations of existing silver based products. We show promising anti-bacterial properties of these new classes of AgNNPs against both Gram negative and Gram positive bacterial strains. Importantly, AgNNPs showed effective

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wound healing properties in a mouse model. The AgNNPs described herein may find future therapeutic applications for treatment of bacterial infections and wounds. 2. EXPERIMENTAL PROCEDURE 2.1. Materials 2.1.1 Chemicals: Sodium nitroprusside (Na2[Fe(CN)5(NO)].XH2O: SNP), silver nitrate (AgNO3), penicillin, streptomycin and kanamycin fetal bovine serum (FBS), Dulbecco’s Modified Eagle’s Medium (DMEM), NaOH, NOS, DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride), FITC (Fluorescein isothiocyanate) were purchased from Sigma Aldrich, India. EBM media from Lonza,USA. These chemicals were used directly without further purification. 2.1.2 Antibodies: Ki-67 antibody purchased from (In Vitrogen) Thermo scientific, India. IL-4, IL-13, (interleukins), Arginase-1(Arg-1) were purchase from cell signaling technology (CST), IFN (Interferon)-γ purchase from R&D systems. Initially, stock solutions of AgNO3 (10-3 M) and sodium nitroprusside (SNP)(10-3 M) were prepared in Milli-Q water and used for nanoparticles preparation. The EA.hy 926 cells are kind gift from Dr. Suvro Chatterjee, AU-KBC, Chennai, India after taking permission from Steve Oglesbee, TCF, UNC Lineberger Comprehensive Cancer Center, NC, USA. All bacterial cell lines were purchased from MTCC, Chandigarh, India. Fertile eggs of Vanaraja chicken variety were procured from ICAR-Directorate of Poultry Research, Hyderabad. 2.2. Synthesis of silver nitroprusside nanoparticles (AgNNPs) The AgNNPs were synthesized by reacting two moles of AgNO3 with one mole of sodium nitroprusside (SNP) as shown in equation (1). Initially, the stock solutions of AgNO3 (10-3

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M) and sodium nitroprusside (SNP) (10-3 M) solutions were added at 2:1 ratio (v/v) in a beaker and the reaction mixture was stirred vigorously for 1 h at ambient conditions.

2AgNO3 + Na2[Fe(CN)5NO].XH2O

RT

Ag2[Fe(CN)5NO]

(1)

The resulting pale pinkish suspension was centrifuged (17,700 rpm, 15 °C, 40 min) using Thermo scientific, (Sorvall-WX ultra 100) ultracentrifuge. The materials were purified by washing with Milli-Q water to remove unreacted precursors. Finally, the pellet was dried in hot air oven for 6 h at 75-80 °C and characterized using several physico-chemical techniques such as powder XRD, Raman, HRTEM, FESEM, SEM, FTIR, DLS, EDAX, TGA, BET, ICP-OES etc. This pellet (pale pink color) was used for all in vitro and in vivo experiments. 3. CHARACTERIZATION TECHNIQUES 3.1. X-Ray diffraction analysis The AgNNPs was characterized by X-ray diffraction method (XRD) using Bruker AXS D8 Advance Powder X-ray diffract meter (using CuKα λ=1.5406 °A radiation). 3.2. High resolution transmission electron microscopy (HRTEM) The stock solution (100 µL) of AgNNPs (1 mg/mL: pellet was suspended in water) was further diluted to 1000 µL by adding water. The diluted nanoparticles suspension was sonicated thoroughly and coated on copper grids followed by vacuum drying prior to HRTEM analysis using FEI Tecnai F12 (Philips Electron Optics, Holland) instrument operated at 100 kV 3.3. Field emission scanning electron microscope (FESEM) Morphology and chemical composition of nanoparticles (AgNNPs) were examined by using electron microscope (FESEM, JEOL 7610F). The specimens were mounted in horizontal and

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lateral positions to study the surface topography as well as the fractured cross-sections for evaluating the homogeneity.19 3.4. Dynamic light scattering (DLS) 3.4.1 DLS study for nanoparticles: The hydrodynamic size and dispersity of AgNNPs were measured using DLS instrument (Malvern Instruments Ltd, Zetasizer,Verson 6.20) and zeta potential was measured by Anton Paar (lytesizer). The stock solution of AgNNPs was suspended in 1000 µL Milli Q water and used for the measurement of particle size by DLS. 3.4.2 DLS study of E. coli and B. subtilis after treatment with AgNNPs: The hydrodynamic size and surface charge of the untreated bacterial cells (E. coli and B. subtilis; 1x107 cells/mL) and cells treated with AgNNPs (1µg/mL) were measured after 6 h post treatment using Anton Paar (lytesizer) instrument. The analysis was also carried out for positive control experiment where bacteria cells were incubated with streptomycin (1µg/mL). After 6 h of incubation time, bacterial cells were collected by centrifugation and washed with 1X PBS, fixed in 2% formaldehyde followed by dehydration using subsequent washings with aqueous ethanol (50%, 60%, 70% and 90%) and finally with pure ethanol. The DLS studies of this processed bacteria were performed according to reported literature,20 The processed bacterial samples (E. coli and B. subtilis) were also used for analyzing membrane damage and the bacterial aggregation was determined by staining with dyes (FITC: 1µg/mL and DAPI: 1µg/mL) using confocal microscopy (Nikon, Ti Eclipx). 3.4. Fourier transformed infrared spectroscopy (FTIR) For the characterization of functional groups present in AgNNPs, FTIR technique was employed. The dried sample of AgNNPs powder was employed for FTIR analysis. The spectra

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were recorded using thermo Nicolet Nexus 670 spectrometer in the diffuse reflectance mode at a resolution of 400-4000 cm-1 in KBr pellets. 3.5. Raman spectra of AgNNPs The inelastic changes in sodium nitroprusside (SNP) and silver nitroprusside were determined by using confocal micro-Raman spectroscopy (Horiba Jobin-YvonLabRam HR spectrometer) with a 17 mW internal He–Ne laser source with excitation wavelength of 632.8 nm. 3.6. Thermo gravimetric analysis (TGA) The thermal stability, derivative thermo gravimetric (DTG) analysis andthe presence of water molecules in AgNNPs were examined by TGA using TGA/SDTA851c, from METTLER TOLEDO, Switzerland at 10 ºC/min from 25-800 °C under N2 atmosphere. 3.7. Inductively coupled plasma optical emission spectrometry (ICP-OES) An inductively coupled plasma optical emission spectrometer (ICP-OES, IRIS intrepid II XDL, ThermoJarrel Ash) was used to determine the concentrations of silver ions in the aqueous solutions21. Initially, a series of standard AgNO3 solution with concentration range of 5-50 ppm was prepared for silver standard curve to measure the leaching of Ag+ ion from AgNNPs. The uptake of silver in bacteria cell was also measured by ICPOES. 4. BIOLOGICAL STUDIES 4.1. Biocompatibility study 4.1.1. In vitro experiments:

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The in vitro biocompatibility experiment (cell viability using MTT reagent) was performed according to our previously published,21 and the detailed procedure is described in the supporting information. 4.2.2. Immuno-staining of Ki-67: The immune-staining of Ki-67in EA.hy 926 cells using anti Ki-67 antibodies was performed according to published literature.22 Initially, the EA.hy 926 cells were cultured on sterile glass cover slips and treated with AgNNPs for a period of 24 h. Thereafter, the cells were fixed in 4% paraformaldehyde solution and permeabilized with Triton-X. The cells were blocked with 3% BSA and incubated with anti-Ki-67 antibodies for overnight at 4 °C. Next day, cells were washed with TBST solution and again incubated with Dylight-633 conjugated secondary antibodies for 2 h in room temperature. The cells were washed with TBST buffer and the cover slips were mounted on glass slides using Fluorishield DAPI medium. The processed cells were observed under confocal microscope (Nikon). 4.1.2. Chick embryo angiogenesis (CEA) assay: The chick embryo is a standard in vivo model for studying the effect of any compound/nanomaterials on the growth and development of new blood vessels i.e. angiogenesis.23 The fertile eggs were incubated in vertical egg incubators (Southern) and maintained at a constant conditions of temperature (37 °C) and relative humidity (60–70%) with occasional tilting. On the fourth day of incubation, eggs were taken out and the shell of each egg was peeled on top carefully, to create a window and expose the embryo. The eggs were treated with AgNNPs (1 µg/mL) and images of the embryonic blood vessels of both treated and untreated embryos were captured at 0 h and 4 h post-incubation using a stereo-microscope fitted with a camera (Leica). The images were further processed for evaluating the blood vessel growth

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parameters such as length, size and junctions using the AngioQuant Matlab software (image analysis tool for angiogenesis). 4.2. Biological assays for anti-bacterial activities 4.2.1. Determination of minimum inhibitory concentration (MIC): The minimum concentration at which an antibacterial agent can retain the inhibition of bacterial growth for defined time period is generally considered as MIC. Hence, in order to investigate the MIC of the AgNNPs, we performed the growth curve experiment using E. coli, B. subtilis, S.epidermidis, P. aeruginosa, K. aerogenes bacteria. Briefly, fresh bacterial cultures were inoculated in LB broth medium, cultured under aseptic conditions till OD600 reached the value of 0.6. The bacterial culture was then treated with AgNNPs (0.5-2 µg/mL) and its growth was monitored periodically for 0-24 h by determining the optical density (OD) of the culture at 600 nm using multimode plate reader (Synergy H1). Streptomycin (FDA approved antibiotic) was used as a positive control. Untreated bacteria grown in LB broth and LB broth without bacteria were used as control and blank, respectively. All the experiments were performed in triplicates. The growth curve was constructed by plotting OD600 verses time. 4.2.3. Analysis of zone of inhibition (ZOI): The zone of inhibition assay is one of the basic methods in determining the antibacterial effects of compounds/nanoparticles. The antibacterial effect of AgNNPs was analyzed by zone of inhibition study. Both Gram negative bacteria (E. coli, Klebsiella aerogenes and Pseudomonas aeruginosa) and Gram positive bacteria (Staphylococcus epidermidis, aureus and Bacillus subtilis) were used for this study. LB agar plate with confluence bacteria was prepared by spread plate method and subsequently filter paper disc soaked with AgNNPs (1 µg/mL) was placed over

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the bacterial agar plate. The plates were incubated for 24 h at 37 °C and streptomycin (1 µg/mL) was used as a positive control. 4.2.4. Determination of colony forming units (CFU): The determination of CFU is another method for assessment of antibacterial activity.21 The cultures of E. coli and B. subtilis (O.D. ~0.6) were inoculated on LB agar media using spread plate method. Later, AgNNPs (1 µg/mL) were spread on the bacteria inoculated agar plate and incubated for 24 h. Bacterial culture plates treated with streptomycin (2 µg/mL) was used as a positive control experiment. The number of bacterial colonies and percentage of inhibition by AgNNPs was calculated using the following formula. Antibacterial efficacy (%) = [(Nuntreated– Ntreated)/(Nuntreated– Npositive control)]X100 Where Ntreated, Npositivecontrol and Nuntreated, are members of bacteria colonies grown on agar plates treated with AgNNPs, streptomycin and untreated, respectively. 4.2.5. Analysis of bacterial DNA damage: The effect of AgNNPs on the bacterial DNA was determined by using the agarose gel electrophoresis method as previously reported.24 Freshly cultured E. coli bacteria were incubated with the AgNNPs (0.5-1 µg/mL) for 6 h and the DNA of bacteria was isolated by following the phenol:chloroform method. Briefly, the treated bacterial cell pellet was suspended in lysis buffer and centrifuged at 12,000 rpm for 30 min at 4 °C. Equal volume of phenol:chloroform was added to the supernatant and again centrifuged at 12,000 rpm for 30 min in 4°C. RNAse was added to supernatant of the samples to digest the RNA from the bacterial lysates. Isopropanol was added to the supernatant to precipitate the DNA and recovered by centrifugation at 12,000 rpm for 15 min at 4 °C. Finally, the isolated DNA was dissolved in TE buffer and separated by

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electrophoresis using 0.8% agarose gel in 1XTAE buffer using horizontal electrophoresis system (BioRad) and the bands were visualized in gel doc system (Fusion solo S- vilberlourmat). 4.2.6. Analysis of morphological changes in bacteria by SEM The effect of AgNNPs on the bacterial cell wall damage was studied by SEM analysis in both E. coli and B. subtilis bacteria after treatment with AgNNPs. Initially, E. coli, and B. subtilis were grown through a log phase (OD at 600 nm) in LB growth media and treated with AgNNPs for 30 min. Afterwards, the cells were harvested through centrifugation (3000 rpm, 5 min, 4 °C) and resuspended in 500 µL of PBS. Then bacteria cells were fixed in 2.5% glutaraldehyde (in PBS) for 30 min on ice. Dehydration process was carried out using increasing amounts of ethyl alcohol (50%, 60%, 70%, 80%, 90% and 100%). The samples were processed for SEM (SEM-Hitachi S3000N, Japan) to analyze bacterial morphological changes. 4.2.7. Morphological changes by HRTEM: The HRTEM (TALOS F200X, 200kv) images of AgNNPs treated bacteria were used examining the effect of AgNNPs on the integrity of the bacterial membrane. Briefly, E. coli cells were cultured for overnight (till OD600 reached around 0.6) and incubated with AgNNPs (1 µg/mL) for 30 min. Later, the cells were harvested by centrifugation (3000 rpm, 5 min, 4 °C), followed by fixing with 2.5% glutaraldehyde solution. 4.2.8. Protein degradation study through MALDI spectrum: Bacterial cultures of E. coli and B. subtilis were treated with AgNNPs as mentioned above using both 1 and 2 µg/mL concentrations. The bacterial pellet was subjected to lysis followed by centrifugation at 12000 rpm for 30 min in 4 °C.25 The clear supernatant was collected and submitted for MALDI TOF-MS analysis to study possible protein degradation. Similar experiments were also performed with positive control (streptomycin: 2 µg/mL).

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4.3. Wound healing study and histopathological evaluation 4.3.1. Differentiation of RAW 264.7 cells into M1 and M2: The murine macrophage cell line RAW 264.7 (5X105 cells/well) were seeded in 24 well plate and grown in DMEM supplemented with 10% heat-inactivated FBS containing streptomycin, penicillin, kanamycin and gentamycin (100 mg/L) at 37 ºC. The cells were used for the experiments at 60% confluence in growth. The cells were incubated with lipopolysaccharide (200 ng/mL; Sigma Aldrich) and mouse IFN-γ (10 ng/mL; R & D systems) together for 24 h, which caused differentiation of RAW 264.7 cells to M1 macrophages. Similarly, RAW 264.7 cells were differentiated into M2 macrophage in the presence of IL-4 (20 ng/mL; Cell signaling) and IL-13 (20 ng/mL; Cell signaling). The media was separated from differentiated cells by filtering through a 0.2 µm filter and filtrate used for studying the effects of secreted cytokines on the proliferation of endothelial cell (HUVEC) as reported previously.26 4.3.2. Scratch assay for HUVEC proliferation: HUVEC cells (5X104/well) were seeded in 24-well plate until they attained 80% confluence. In vitro wound model was created using sterile pipette tip by scratching on monolayer of cells.27 HUVECs were washed with 1X PBS thrice and further subjected with 10% (10µL) macrophages differentiated media (collected after 24 h treatment with AgNNPs:1µg/mL to M1 and M2 differentiated macrophages as described above) in starving EBM media for 6 h. The bright field images of the scratched area of cells before and after treatment were taken under an inverted microscope (Nikon, Tokyo, Japan) at 4X magnification. 4.3.3. In vivo wound healing study in C57BL/6J mouse model The wound healing activity of AgNNPs was evaluated in C57BL/6J female mice using punch biopsy model. The mice (3-4 weeks old) were procured from National Institute of

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Nutrition, Hyderabad for wound healing study. The in vivo study (n = 3 per group) was performed with the approval of our Institutional Animal Ethics Committee (IAEC) (IICT/31/2015/19/10/2015), CSIR-IICT, Hyderabad, India. The mice were initially maintained at quarantine area in the animal facility (IICT) for acclimatization for a period of 7 days. The AgNNPs were formulated as a smooth cream at a concentration of 0.1% and 1% (w/w) using commercially available Vaseline cream without any growth factors. The hair over the dorsal surface of the mouse body was removed using a commercially available cream (Veet). A circular wound was created over the skin area using a 6 mm punch biopsy and then skin was peeled out as described earlier.28 Further, AgNNPs formulated cream was applied over the wound area alternately on day 1, 3, 5 and 7 post wound creation. Silverex (0.1%) ointment treated mice were used as a positive control group and Vaseline cream was used as vehicle control group. Images of the wound area of mice were captured using a Canon camera and the diameter of the wound (mm) was monitored using digital Vernier calipers to calculate the % wound healing. 4.3.4. Histological evaluation: After the study period, the mice were euthanized and the skin tissue surrounding the wound was dissected and fixed using 4% formaldehyde solution. Skin tissues were embedded into paraffin wax blocks and thin (3 µm) sections were mounted on clean microscopic glass slides. The slides with the skin sections were washed and stained with Haematoxylin and Eosin (H&E) as per previously reported29 and microscopically examined by an expert pathologist. The microscopic images were captured and the extent of wound healing was determined based on Abramov`s scoring.30 Epithelialization was graded as 0 (None), 1 (Partial) 2 (Complete but immature/thin) and 3 (Complete and mature). Angiogenesis was graded as 0 (None), 1 (≤ 5 vessels/HPF), 2 (6-

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10 vessels/HPF) and 3 (>10 vessels/HPF). Fibrosis was graded as 0 (none to minimal fibroblasts), 1 (few fibroblasts), 2 (more fibroblasts) and 3 (predominantly fibroblasts). Macrophages were graded as 1 (0-25 macrophages), 2 (26-50 macrophages) and 3 (>51 macrophages). 4.3.5. Immuno- histochemical (IHC) analysis At the end of wound healing study, the mice were euthanized and the skin areas around the wound were cut and fixed in 4% formaldehyde solution. Later, these skin tissues were embedded into paraffin wax blocks and 5 µm sections were made and mounted on to the clean microscopic glass slides. For the IHC analysis, the skin sections were de-paraffinized, dehydrated and washed thrice with 1X PBS. The tissues were then blocked with 3% bovine serum albumin for 1 h at room temperature and the slides were washed with 1X TBST. The tissue sections were incubated with primary antibody (anti- rabbit NOS: 1:1000; N217, Sigma Aldrich) for overnight at 4 °C. Then slides were further washed with 1X TBST carefully and incubated with FITC labeled goatanti-rabbit secondary antibody (1:1000; IgG, thermo scientific) for 30 min. The slides were washed with TBST and stained with DAPI (Sigma Aldrich). In a similar manner, type-II macrophages (M2) were examined on AgNNPs treated mice skin tissues by incubating with Arginase-1 goat anti rabbit (1: 1000; Cell Signaling) for overnight at 4 °C. Additional experiments including cell viability assay, BET measurements are described in Supporting Information. 5. RESULTS AND DISCUSSION The AgNNPs were synthesized by interaction of AgNO3 and sodium nitroprusside in aqueous solution and purified by centrifugation. The purified nanomaterials were characterized by various

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physico-chemical techniques used for all in vitro and in vivo assays. Initially, in order to check the crystallinity of the materials, powder XRD was carried out.

The XRD spectrum of AgNNPs (Figure 1a) indicates the crystalline nature with a monoclinic unit cell. The diffraction peaks show the crystalline nature of the material and typical peaks were

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observed at 2θ = 13.2, 13.8, 21.6, 25.3, 30.2, 33.4, 36.2. The results are corroborating with published literature.31 The HRTEM images of AgNNPs (submitted after sonication of the water suspension) show almost spherical nature of nanomaterials (Figure 1b) with an average size of around 20-80 nm in diameter according to the size distribution curve (inset of Figure 1b). The shape and morphology of these nanomaterials with higher resolution was further supported by FESEM (Figure 1c). The actual size of the solid materials observed by FESEM was slightly higher than 100 nm. The hydrodynamic radii of the nanomaterials observed by DLS (260 ± 15 nm with poly dispersity index 0.202, Figure 1d) were significantly higher than the size of nanoparticles measured by HRTEM (20-80 nm, Figure 1b). The results were shown in Figure 1b represents the clear spherical shape of the AgNNPs and its size distribution. The size discrepancy presumably originates from the fact that DLS measures size of the nanoparticles in aqueous solution (which includes large number of water molecules on the surface of the nanoparticles) where as HRTEM measures actual size of the nanoparticles in dried state. The negative zeta potential of AgNNPs observed by DLS studies (-16.6 mV, inserted Figure 1d) prevented aggregation of nanoparticles. Fourier transformed infrared spectroscopy (FTIR) was used to detect functional groups present in the AgNNPs. The two characteristic peaks of nitroprusside at 2145 cm-1 and 1942 cm-1 (attributed to the -C≡N and –N=O functional groups, respectively) were shifted to 2139 cm-1 and 1932 cm-1, respectively, indicating the replacement of sodium ion with silver ion in AgNNPs. Consistent with prior report31, distinguishing peaks for the δ (FeNO) and δ (FeCN) were observed at 658 cm-1 and 512 cm-1, respectively (Figure S1a-b). The two narrow sharp peaks at 3629 cm-1and 3547 cm-1, corresponding to characteristic peaks of water molecules present in the crystalline structure of sodium nitroprusside, were absent in AgNNPs which is further supported

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by TGA of AgNNPs.32 The symmetrical stretching vibrational modes in silver nitroprusside nanoparticles were determined by Raman spectroscopic technique.

Figure 2. Raman spectra of AgNNPs (red) and its precursor sodium nitroprusside (black), respectively. It is based on inelastic scattering of laser photons through molecular vibrations in bond of the molecules. The signals appeared around 1360, 1528 cm-1 in Raman spectra of AgNNPs (Figure 2) may be due to presence of two kinds of M-L bonds (Ag1…N≡C and Ag2…N≡C) as per the published literature.31,33, 34 According to J. Rodríguez-Hernández and co-workers31 there are two kind of silver atoms (Ag1 and Ag2) present in the silver nitroprusside. Both Ag1 and Ag2 are linearly coordinated to –CN ligands (Ag1 is coordinated to N end of the equatorial –CN ligand whereas Ag2 is linked to the axial –CN ligand) of AgNNPs through weak interactions. The peak around 1300-1500 cm-1 is further supported literature.33, 34.

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The shifting of –CN stretching signal from around 2125 cm-1 (AgNNPs) to 2186 cm-1 (SNP) is due to exchange of sodium ion by silver ion in ionic sphere of nitroprusside. The decrease in ν(CN) stretching of AgNNPs compared to that for SNP is due to decline in polarity between AgFe than Na-Fe in nitroprusside complex. As per the literature, the change in wave number (Figure 2) is due to electro negativity of metal atoms in ionic sphere of the complex.35, 36, 37 Due to higher electronegativity of silver (1.93 eV) than sodium (0.93eV) there is shifting at –CN stretching spectrum. TGA and DTG studies were performed for measuring metal complex degradation. At par with prior report31, the TGA and DTG curves of AgNNPs demonstrated that degradation of AgNNPs consisted of three steps involving sequential loss of functional groups as indicated in Figure S2. The absence of degradation peaks at 180-230 °C temperature in TGA of AgNNPs indicated absence of water of crystallization which supported the FTIR data. The elemental composition of the AgNNPs pellet was evaluated by both EDAX and ICP-OES techniques. The values of Ag/Fe ratios were found to be 3.5 and 3.6 by EDAX and ICP-OES, respectively. These values are very close to the theoretically estimated Ag/Fe ratio (3.8) in AgNNPs (Figure S3). The results indicate that sodium ions are completely replaced with silver ions in the ionic sphere of the nitroprusside complex leading to the formation of AgNNPs. The specific surface area of nanoparticles is the summation of areas of the exposed surfaces of the nanoparticles per unit mass. Surface area of particles is inversely proportional to pore size of nanoparticles (Figure S4). Here, nitrogen gas can be used to measure the surface area of powder.38 If the particles are assumed to be spherical, the specific surface area provides an average diameter (in nm) of particles. In nanoparticles

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surface area and pore volumes are determined by BET experiment and these are also important factors for its biomedical applications. The Langmuir surface area of AgNNPs is 18 m2/g and suitable for biomedical applications. All these results suggest that AgNNPs particles are small in size with less pore volume. 5.1. Cell viability assay of AgNNPs

Figure 3. (a- b) Cell viability assay of EA.hy 926 and 3T3 cells using MTT reagent at 24 h. The numerical values on X-axis indicate concentration of silver nitroprusside nanoparticles (µg/mL). (c) Confocal images of nuclei of EA.hy 926 cells stained with Ki-67 (Dylight-633: red color) and DAPI (blue color) Upper panel: untreated control cells, lower Panel: cells treated with AgNNPs (0. 5 µg/mL) for 24 h. Scale Bar: 0-50µm. Cells were observed under 60X objective in confocal microscopy. *Significantly different from control at p

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