Materials Science and Engineering C 80 (2017) 659–669

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Materials Science and Engineering C

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Multifunctional biosynthesized silver nanoparticles exhibiting excellentantimicrobial potential against multi-drug resistant microbes along withremarkable anticancerous properties

Diksha Jha a, Prasanna Kumar Thiruveedula a, Rajiv Pathak a, Bipul Kumar a, Hemant K. Gautam a,⁎⁎,Shrish Agnihotri b, Ashwani Kumar Sharma b, Pradeep Kumar b,⁎a Microbial Biotechnology Laboratory, CSIR- Institute of Genomics and Integrative Biology, Sukhdev Vihar, Mathura Road, Delhi 110025, Indiab Nucleic Acids Research Laboratory, CSIR-Institute of Genomics and Integrative Biology, Mall Road, Delhi 110007, India

⁎ Correspondence to: P Kumar, CSIR-Institute of GenDelhi University Campus, Mall Road, Delhi 110007, India.⁎⁎ Correspondence to: H.K. Gautam, CSIR-Institute of GeSukhdev Vihar, New Delhi 110020, India.

E-mail addresses: hemant@igib.res.in (H.K. Gautam), p

http://dx.doi.org/10.1016/j.msec.2017.07.0110928-4931/© 2017 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 27 April 2017Received in revised form 2 June 2017Accepted 9 July 2017Available online 10 July 2017

This study demonstrates the therapeutic potential of silver nanoparticles (AgNPs), which were biosynthesizedusing the extracts of Citrus maxima plant. Characterization through UV–Vis spectrophotometry, Dynamic LightScattering (DLS), Fourier Transform Infrared spectroscopy (FTIR), X-rayDiffraction (XRD) and Transmission Elec-tronMicroscopy (TEM) confirmed the formation of AgNps in nano-size range. These nanoparticles exhibited en-hanced antioxidative activity and showed commendable antimicrobial activity against wide range of microbesincludingmulti-drug resistant bacteria that were later confirmed by TEM. These particles exhibitedminimal tox-icity when cytotoxicity study was performed on normal human lung fibroblast cell line as well as human redblood cells. It was quite noteworthy that these particles showed remarkable cytotoxicity on human fibrosarcomaandmousemelanoma cell line (B16-F10). Additionally, the apoptotic topographies of B16-F10 cells treated withAgNps were confirmed by using acridine orange and ethidium bromide dual dye staining, caspase-3 assay, DNAfragmentation assay followed by cell cycle analysis using fluorescence-activated cell sorting. Taken together,these results advocate promising potential of the biosynthesized AgNps for their use in therapeutic applications.

© 2017 Elsevier B.V. All rights reserved.

Keywords:Citrus maximaSilver nanoparticlesAntioxidantAntimicrobialMulti-drug resistant microorganismsAnti-cancerous

1. Introduction

Nanotechnology is amultidisciplinary area that dealswith synthesis,designing and manipulation of particles at nano-size range. Thesenanoparticles exhibit unique physicochemical, mechanical, electronic,catalytic, optical and thermal properties due to increased surface area-to-volume ratio, which are not present in the bulk form of materials[1,2]. Thus, nanoparticles have triumphed substantial deliberations invarious fields such as drug and nucleic acids delivery, catalysis, singleelectron transistor, food, cosmetics, health, chemical industries, and bio-medical sciences [3]. Amid numerous nanoparticles, silver nanoparticles(AgNps) have extensively been used in the various fields includingmedicine and biology [4,5]. Physical methods produce low yields ofthe particles and chemical methodologies are toxic, expensive andeco-unfriendly [6,7]. Green strategies utilizingnatural capping, reducingand stabilizing agents to synthesize nanoparticles with craved

omics and Integrative Biology,

nomics and Integrative Biology,

kumar@igib.res.in (P. Kumar).

morphology and size is a noteworthy angle for scientists. Thesemethods include the utilization of bacteria, algae, fungi and plants [8–10]. However, synthesis, using plants, affords a better platform as theyoffer natural and non-toxic capping and stabilizing agents along withminimizing the expenditure ofmicroorganism's isolation. The synthesisof nanoparticles using plants is less time consuming, biocompatible,fetches high yield and the size of nanoparticles can bemanaged by con-trolling parameters such as temperature, concentration of the reactants,speed of stirring and pH [11].

Emergence of multi-drug resistant pathogenic microorganisms de-mands implementation of novel antimicrobials [12]. Methicillin-resis-tant Staphylococcus aureus, multi-drug resistant (MDR) Pseudomonasaeruginosa and Salmonella enteritidis are known for their drug dodgingmechanisms [13,14]. Not only these, but some bacteria residing on thehuman skin such as Propionibacterium acnes, a skin commensal, turnspathogenic in the case of acne vulgaris, developing resistance againstantibiotics like tetracycline, minocycline, clindamycin, etc., hence chal-lenging scientists to develop novel and effective drugs [15,16]. Thus, itbecomesmandatory to come upwith strategies that can facilitate pivot-al bacterial killing. Traditional antimicrobial approaches suggest silveras an antimicrobial agent, thus its potential need to be explored [17,18]. Along with these, there are several reports to support the efficient

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660 D. Jha et al. / Materials Science and Engineering C 80 (2017) 659–669

antimicrobial potential of silver nanoparticles having rare chance for thedevelopment of drug-resistance [19–25]. Along with antimicrobial po-tential, these nanoparticles have also shown to impart their role as po-tential anticancerous agents [26–29].

In the present study, extracts from various parts of Citrus maximaplant were used as novel and green source of precursors in order tobiosynthesize the silver nanoparticles (AgNps). These particles showedenhanced antioxidant activity, excellent antimicrobial as well asanticancerous activity without much affecting the normal human celllines and RBCs. Taken together, these biosynthesized silver nanoparti-cles might serve as drugs next door for various outrageous diseases in-cluding cancer.

2. Materials and methods

2.1. Biosynthesis of silver nanoparticles using C. maxima extracts

The plant extract preparationwas performed, as described previous-ly [30,31]. Briefly, healthy fruit, leaves and peel were collected andcleaned with autoclaved Milli Q water. Fruit pulp was crushed andjuice was strained through Whatman filter paper No. 1 (HiMedia)followed by centrifugation at 12000 rpm for 45min at 4 °C and then fil-tered using 0.2 μm membrane (HiMedia). Whereas, dried leaves andpeel were crushed in a mixer. Fine leaf and peel powder (~10 g) weresuspended in separate vessels in 100 ml of Milli Q water and heated inan oil bath at 90 °C for a period of 3–5 h. Then, these were cooled, fil-tered, lyophilized and stored at 4 °C. In order to biosynthesize silvernanoparticles, fruit juice (10ml) was added dropwise in a flask contain-ing 200 ml of 1 mM AgNO3 solution with constant stirring at 1000 rpmat room temperature. For leaf and peel extracts too, 10ml of the extractwas added to other flasks containing 200 ml of 1 mM AgNO3 solutions,respectively. Color of the solution changed from colorless to brownishblackwithin 4–6 h. Itwas centrifuged at 12000 rpm for 45min to obtainthe AgNps pellets, followed by its extensive washings with water andlyophilization [32]. In addition, chemically synthesized silver nanoparti-cles were also prepared by using sodium borohydride (NaBH4) (Sigma),which were used as a control.

2.2. Characterization of synthesized nanoparticles

Synthesis of AgNps was confirmed by recording their spectra (200–850 nm) using UV–Vis spectrophotometer. Aqueous solutions of syn-thesized AgNps (1 mg/ml) were used for Dynamic Light Scattering(DLS) measurement using Zetasizer Nano-ZS (Malvern Inc., UK). Typi-cally, AgNps (1 mg) were dispersed in 1 ml of Milli Q water by sonica-tion (3 × 5 min). After formation of homogeneous solution, size andzeta potential measurements were performed. Average values of theseparameters were obtained in automatic mode for 20 and 30 runs, re-spectively. Fourier Transform Infrared (FTIR) spectra of these nanopar-ticles were recorded using a single-beam Spectrum RXI-MID-IR(PerkinElmer, USA), with the following scan parameters: scan range,4400–400 cm−1; number of scans, 16; resolution, 4 cm−1; interval,1 cm−1; unit %T. Lyophilized samples of synthesized AgNps were sub-jected to X-ray diffraction (XRD) measurements with an X'pert PROXRD (Analytical BV, Almelo, Netherlands), operating in transmissionmode at 30 kV, 20 mA with Cu-Ka radiation [32]. The size and shapeof AgNps were analyzed using Transmission Electron Microscopy(TEM), as described previously [33]. The TEM micrographs were takenat an accelerating voltage of 200 kV (Tecnai G2 30 U-twin, 300 kVUltra-twin microscope).

2.3. Antioxidant activity

The antioxidant activity of synthesized AgNps was performed fol-lowing a previously reported method [34]. Briefly, the dilutions of C.maxima extract as well as biosynthesized silver nanoparticles were

made in the range of 50 to 1000 μg/ml. 100 μl of each diluted sampleswas mixed with 100 μl of 2,2-diphenyl-1-picrylhydrazyl solution (80μg/ml, DPPH, HiMedia) in a 96-well plate (Corning) and the final vol-ume was made up to 200 μl. The plate was incubated in dark for30min at room temperature. Quercetin andmethanolwere used as pos-itive and negative controls, respectively. Finally, the DPPH free-radicalscavenging activity (%) was determined at 517 nm using Tecan micro-plate reader.

2.4. Antimicrobial activity

The antimicrobial activity of these synthesized AgNps was per-formed against various pathogenic microorganisms such as Escherichiacoli MG1655 (MTCC 1586), Bacillus cereus (MTCC 430), Bacillus subtilis(MTCC 121), Klebsiella pneumoniae (MTCC 3384), Pseudomonasaeruginosa (MTCC 741), Staphylococcus aureus (MTCC 740), multi-drugresistant Pseudomonas aeruginosa (ATCC 27853), Salmonella enteritidis(ATCC 13076), methicillin-resistant Staphylococcus aureus (MRSA)(ATCC 43300). The antimicrobial activity was also performed againstseveral clinically isolated pathogenicmicroorganisms from acne lesions,viz., Klebsiella pneumoniae (MCC 2748), Acinetobacter radioresistens (KF954714), Enterobacter xiangfanfensis (MCC 2770), Propionibacteriumacnes (KF 268368) and Staphylococcus haemolyticus (KF 268371). Allthe multi-drug resistant (MDR) strains and acne pathogens were cul-tured in Brain Heart Infusionmedia (HiMedia) at 37 °C.Whereas, othersmicroorganisms were maintained in Luria Bertani media (HiMedia) at37 °C. Gentamicin (0.01 mg/ml) (HiMedia) and autoclaved Milli Qwater were used as positive and negative controls, respectively. Inocu-lum of optical density 0.5 (105 colony forming units/ml) was preparedby taking pure colony with a sterile inoculating loop from agar plate insterile broth, incubated overnight at 37 °C.

The antimicrobial activity was performed by using Kirby-Bauer diskdiffusion assay as described previously [35,36] Sterile disks of 6mm, im-pregnated with the synthesized AgNps (100 μg/disk), C. maxima ex-tracts (1 mg/ml), AgNO3 (1 M), Gentamicin (positive control) andsterile Milli Q water (negative control), were placed on Mueller Hintonagar plates seeded with test microorganism followed by an overnightincubation at 37 °C. The appeared zone of inhibition (mm) aroundeach discwasmeasured usingHiAntibiotic zone scale (HiMedia). To an-alyze theminimum inhibitory concentration of AgNps against thesemi-crobes, microbroth dilution assay was performed in 96-well plates [33].Serial dilutions of AgNps (1 to 0.0005 mg/ml) with 10 μl of mid-logphase bacterial culture andMueller Hinton Broth (HiMedia) were final-ly made to 200 μl. Wells with only media and bacterial culture served asgrowth controls. Absorbance was measured by using TECAN spectro-photometer at 600 nm, before and after overnight incubation at 37 °C.Experiment was done in triplicates. After overnight incubation, 50 μlof 200 μg/ml of p-iodonitrotetrazolium chloride (HiMedia) was addedto the each well and further incubated at 37 °C for 30 min to monitorthe bacterial growth.

2.5. Transmission Electron Microscopy (TEM)

Transmission electron microscopic analysis of AgNps treated patho-genicmicroorganismswas performed as carried out previously [33–36].Briefly, fresh culture of B. cereus and S. enteritidis were treated withAgFeNps, AgLeNps and AgPeNps (1 mg/ml) for 30 min at 37 °C andthen centrifuged at 4000 rpm for 10min. The untreated bacteria servedas control. The pellet, so obtained,waswashedwith1XPBS (pH7.4) andthen fixed with 4% paraformaldehyde (Sigma) at 4 °C overnight. Later,the dehydration of samples was done by washing with gradually in-creasing concentration of ethanol (Merck) (10–100%). Bacterial pelletswere resuspended in 200 μl of autoclaved Milli Q water followed bystainingwith 1% uranyl acetate (HiMedia) for 5min. 10 μl of the stainedbacterial cells were loaded on carbon-coated TEM grids (200 mesh Cu,TED Pella Inc., USA), air dried and TEM micrographs were taken.

Fig. 1. Synthesis and characterization of biosynthesized silver nanoparticles (AgNps): a) Showing the formation of AgNps as represented by change in color formation before and after thereaction; b) UV–Vis absorbance peaks in the range of 400–500 nm showing the synthesis of AgNps; c) Transmission electron microscopy (TEM) images showing the AgFeNps atmagnification of 6500×, 25,000× and 50,000×, respectively.

661D. Jha et al. / Materials Science and Engineering C 80 (2017) 659–669

2.6. Hemolytic activity

Hemolytic assay was performed following the standard protocol[36]. Briefly, human red blood cells (RBCs) were washed thricewith 1X PBS, centrifuged at 1500 rpm for 10 min at 4 °C. 100 μl of4% RBCs was added in each well of a 96-well plate, containing 160,800 and 1600 μg/ml of AgFeNps, AgLeNps, and AgPeNps. TritonX-100 (1%) (Sigma) and PBS were used as positive and negative con-trols, respectively. Plate was incubated at 37 °C for 1 h, centrifuged at2000 rpm for 10 min at 4 °C and then 100 μl of supernatant wastransferred to a fresh plate. Absorbance was measured at 540 nmusing TECAN multimode plate reader. Experiment was performedin triplicates.

2.7. Cell cytotoxicity assay

Normal human lung fibroblast (MRC-5), human fibrosarcoma (HT-1080) and mouse melanoma cell lines (B16-F10) were purchasedfrom American Type Culture Collection (ATCC). Out of these three celllines, MRC-5 and HT-1080 were cultured and maintained in Dulbecco'sModified Eagle's Medium (DMEM), whereas B16-F10 cell line wasmaintained in DMEM-F12 medium (Gibco). These cell lines were

Table 1DLS analysis representing the size, PdI and zeta potential of biosynthesized silver nanopar-ticles (AgNps).

aAgNps bSize (d·nm) ± SD (nm) cPdI ± SD Zeta Potential (mV) ± SD

AgFeNp 121.0 ± 5.4 0.42 ± 0.02 −17.8 ± 0.87AgLeNp 396.0 ± 22.0 0.32 ± 0.05 −15.4 ± 0.40AgPeNp 243.1 ± 30.0 0.22 ± 0.01 −14.4 ± 0.36

a Biosynthesized silver nanoparticles (AgNps) from C. maxima fruit (AgFeNp), leaf(AgLeNp) and peel extracts (AgPeNp).

b Size of AgNps in nanometers.c PdI: Polydispersity Index.

supplemented with 10% fetal bovine serum (FBS, Gibco) and weremaintained in a humidified 5% CO2 incubator at 37 °C. In order to deter-mine the cell viability of the treated cell lines, CellTiter-Glo assay kit(Promega) was used following the standard protocol [36]. Briefly, 1× 104 cells/well (MRC-5, HT-1080 and B16-F10 cells) were seeded inNunc 96-microwell plates. These cells were treatedwith biosynthesizednanoparticles (10–250 μg/ml of AgFeNps, AgLeNps, AgPeNps) and ex-tracts of C. maxima plants followed by 48 h of incubation. Untreatedcells in media were used as control for all the assays. Wells containingmedia alone were used to set baseline during measurements. Substratewas used for generating luminescence and it was recorded in TECANplate reader with zero integration time. All the experiments were per-formed in triplicates.

2.8. Characterization of cytotoxicity of AgNps on B16-F10 cell line

The morphological evidence for the detection of apoptosis in B16-F10 cell line, treated with biosynthesized AgFeNps, was carried outusing Acridine orange and Ethidium bromide (HiMedia) dual dye stain-ing [37]. Briefly, B16-F10 cell line was treated with biosynthesizedAgFeNps (10 μg/ml), fruit extracts (50 μg/ml) and AgNO3 followed byincubation for 48 h. Cells were harvested, centrifuged at 1500 rpm for10 min at 4 °C, washed thrice and resuspended in 1X PBS (pH 7.4). 10μg/ml of acridine orange and 100 μg/ml of ethidium bromide in 1XPBS were mixed to prepare AO/EtBr mix. Cell suspension was stainedwith AO/EtBr mix and examined immediately under fluorescence mi-croscopy (Zeiss) using fluorescein filter. On the other hand, the cell via-bility of B16-F10 cell line- under similar conditions was assessed usingtrypan blue assay [37]. Since, DNA fragmentation is known to occurduring apoptosis due to the activation of caspase-3, we have carriedout DNA fragmentation analysis. Whole genomic DNA from treatedB16-F10 cell line was isolated using DNeasy blood and tissue kit(QIAGEN) and analyzed on 1% agarose gel in order to scrutinize theinternucleosomal DNA laddering in the treated cells [38].

Fig. 2. Functional characterization of biosynthesized silver nanoparticles (AgNps): a) Fourier transform infrared spectroscopy (FTIR) data showing the presence of various functionalgroups in the fruit extract, biosynthesized silver nanoparticles from fruit (AgFeNps) and leaf (AgLeNps), respectively; X-ray powder diffraction (XRD) data showing the crystallinenature of biosynthesized silver nanoparticles from fruit (b), leaf (c) and peel extract (d), respectively.

662 D. Jha et al. / Materials Science and Engineering C 80 (2017) 659–669

2.9. Caspase-3 activity assay

Caspase-3 activity was measured in biosynthesized AgNps treatedB16-F10 cell line as described previously [39,40]. Briefly, 1.5 × 105

cells were seeded in a 6-well plate and treated with 10 μg/ml AgNps,50 μg/ml extract, 1 mM AgNO3 and untreated cells were taken as con-trol. After 48 h of incubation, Caspase-3 activity was performed usingCaspase-Glo® 3/7 Assay (Promega). Cleavage of the fluorogenic substratewas measured in TECAN fluorometer (excitation: 380 nm; emission:

Fig. 3. Enhanced antioxidant activity of biosynthesized silver nanoparticles: DPPH free-radical scalong with their respective C. maxima extracts (fruit, leaf and peel extracts).

460 nm) and the activity was represented as Relative FluorescenceUnits (RFU).

2.10. Cell cycle analysis using fluorescence-activated cell sorting (FACS)

Cell cycle analysis using flow cytometry (BD Biosciences) wasperformed for the biosynthesized nanoparticles treated B16-F10 cells.Treatment and harvesting of B16-F10 cells were done as describedearlier. Pellet was resuspended and fixed in 200 μl of 70% ethanol at

avenging activity (%) of biosynthesized silver nanoparticles (AgFeNps, AgLeNps, AgPeNps)

Fig. 4. Antimicrobial activity of biosynthesized silver nanoparticles: Relative survival (%) of several pathogenic microorganisms in response to AgFeNp, AgPeNp and AgLeNp at theirminimum inhibitory concentrations.

663D. Jha et al. / Materials Science and Engineering C 80 (2017) 659–669

4 °C for 24 h followed by washing with 1× PBS and centrifugation at2000 rpm for 5 min. 0.1% Triton X-100 (Sigma) and RNase (40 μg/ml)(HiMedia) were added to the pellet and incubated at 37 °C for 45 min.Cells were stained with 50 μg/ml propidium iodide, incubated for15 min in dark at room temperature and then cell cycle was analyzedby flow cytometry (BD FACSAria™ III) as mentioned earlier [41]. Thenumber of cells present in each growth phase was analyzed the usingModFit software.

3. Results and discussion

3.1. Green approach for synthesis of silver nanoparticles (AgNPs)

C. maxima fruit, leaf and peel extracts were used to biosynthesizeAgFeNps, AgLeNps and AgPeNps, respectively. Series of reactions withvarying amounts of C. maxima fruit, leaf and peel extracts were carriedout in order to get well-stabilized nanoparticles. Initially, the reactionmixtures were colorless (AgNO3), but as the reaction proceeds, themix-ture turned to dark brown color, indicating the synthesis of AgNps (Fig.

Table 2Zone of Inhibition (mm) for biosynthesized silver nanoparticles against various pathogen-ic microorganisms in (i) tabular and (ii) histogram format. Fe: fruit; Le: leaf; Pe: peel.

0

5

10

15

20

25

AgFeNp

AgLeNp

AgPeNp

Microbial pathogensZone of Inhibition ± SD (mm)

AgFeNp AgLeNp AgPeNp

Escherichia coli 19±1.4 14±1.3 16±1.4

Bacillus cereus 18±1.5 11±1.4 13±1.4

Bacillus subtilis 20±1.4 12±1.5 15±1.5

Klebsiella pneumoniae 17±0.9 11±1.1 14±1.3

Pseudomonas aeruginosa 21±1.3 12±1.2 14±0.8

Staphylococcus aureus 19±0.9 12±1.1 15±0.9

(i)

(ii)

1a). Change in the color is due to the excitation of surface plasmon res-onance exhibited by the synthesized nanoparticles [42–44]. Synthesis ofAgNps (i.e. color change) was observed within 30 min of addition ofplant extracts. Therefore, this approach can be used as a perfect modelfor rapid synthesis of nanoparticles.

3.2. Characterization of biosynthesized silver nanoparticles (AgNps)

Varying amounts of fruit juicewere used to optimize the synthesis ofnanoparticles. In order to achieve the same, fruit juice (Fe2, 2 ml; Fe4,4 ml; Fe6, 6 ml) was used to obtain silver nanoparticles. UV–Vis spec-troscopywas carried out in order to establish the synthesis and stabilityof silver nanoparticles. Due to their surface plasmon resonance, AgNpsshowed distinct peak around 400–460 nm, confirming the synthesis ofsilver nanoparticles (Fig. 1b). AgNps (1 mg/ml) were sonicated so asto disperse the particles homogeneously in the aqueous solution, andused for size (d, nm), polydispersity index (PdI) and zeta potential(mV) measurements using DLS. The size of these nanoparticles wasfound in the range of ~120 to 400nm. The negative values of zeta poten-tial represent the negatively charged AgNps (Table 1). The size andshape of nanoparticles was further confirmed through TEM. The elec-tron micrographs showed that the nanoparticles were spherical inshape and size was found to be in between 2 and 50 nm (Fig. 1c). Thesize of these TEM analyzed nanoparticles was found to be smaller ascompared to that analyzed by DLS. This might be attributed to the hy-dration of particles during DLS measurements. Further, Fourier Trans-form Infrared (FTIR) spectroscopy confirmed the presence of severalfunctional groupswithin the fruit juice aswell as AgFeNps andAgLeNps.The sharp absorption peaks observed in the fruit juice indicated thepresence of large number of functional groups. A broad absorptionpeak at 3300 cm−1 showed the presence of hydroxyl (−OH) groupsand a relatively sharp peak in native fruit juice revealed the presenceof both amino (−NH2) and hydroxyl groups. Peak at stretching fre-quency 2950 cm−1 showed the presence of –CH, CH2 and CH3 groups,which caused C\\H bond to stretch. Another peak at 1738 cm−1 con-firmed the presence of –C_O group of ester linkage in the fruit juice.An intense peak at 1160 cm−1 was due to the C\\O bond stretching(Fig. 2a). Thus, it is evident that the presence of –C_O, −NH2 and –OH plays an important role in the reduction of silver ions to silver nano-particles. X-ray diffraction (XRD) of the nanoparticles gave rise toBragg's reflections. More than one Bragg's reflections were observedconfirming the crystalline nature of the synthesized AgNPs (Fig. 2b-d).On comparing the observed pattern with the standard XRD pattern ofAgNps, the crystalline nature was confirmed by the peaks at 2θ valuesof 38, 44 and 65 corresponding to Bragg's reflections (111), (200) and(220) of the standard XRD pattern of silver nanoparticles.

Table 3Minimum Inhibitory Concentrations (μg/ml) of biosynthesized silver nanoparticles against various clinical as well asmulti-drug resistant microorganisms in (i) tabular and (ii) histogramformats.

Silver nanoparticles

Minimum Inhibitory Concentrations (µµg/ml)

Clinical pathogens Multi-drug resistantBacteria Acne associated microbial pathogens

EC BS BC KP SA PA MDR -SE MRSAMDR -

PA KP AR EX SE SH PAc

AgFeNp 50 25 25 35 25 20 65 90 75 80 70 80 90 95 85AgLeNp 75 65 70 60 55 55 160 145 135 125 115 125 150 160 155AgPeNp 75 40 50 45 45 35 130 135 125 95 80 115 125 145 150

AgNp* >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200

MIC against variousclinical as well as multi-drug resistant microorganisms; EC: Escherichia coli; BS: Bacillus subtilis; BC: Bacillus cereus; KP: Klebsiella pneumoniae; SA: Staphylococcus aureus; PA: Pseudomonasaeruginosa; MDR-SE: multi-drug resistant-Salmonella enteritidis; AR: Acinetobacterradioresistens; EX: Enterobacter xiangfanfensis; SE: Staphylococcus epidermidis; SH: Staphylococcus haemolyticus; PAc: Propionibacterium acnes; *Chemically synthesized AgNp was used as a control.

0

20

40

60

80

100

120

140

160

180

MIC

(µg/

ml)

AgFeNp

AgLeNp

AgPeNp

Clinical pathogensMDR bacteria

Acne -isolated microbes

(i)

(ii)

MIC against various clinical as well as multi-drug resistant microorganisms; EC: Escherichia coli; BS: Bacillus subtilis; BC: Bacillus cereus; KP: Klebsiella pneumoniae; SA: Staphylococcus au-reus; PA: Pseudomonas aeruginosa;MDR-SE:multi-drug resistant-Salmonella enteritidis; AR: Acinetobacter radioresistens; EX: Enterobacter xiangfanfensis; SE: Staphylococcus epidermidis; SH:Staphylococcus haemolyticus; PAc: Propionibacterium acnes; *Chemically synthesized AgNp was used as a control.

Fig. 5.Transmission electronmicroscopy (TEM) images of Bacillus cereus (a–e) andMDR- Salmonella enteritidis (f–j): (a, f) untreatedbacteria; (b, g)AgFeNps entering into the bacterial cell;(c, h) breakdown of bacterial cell wall; (d, i) release of bacterial cell contents; (e, j) complete breakdown of bacterial cell.

664 D. Jha et al. / Materials Science and Engineering C 80 (2017) 659–669

Fig. 6. Hemolytic activity: Hemolysis (%) of human red blood cells (RBCs) treated withbiosynthesized silver nanoparticles prepared from fruit (AgFeNps), leaf (AgLeNps) andpeel extracts (AgPeNps), respectively.

665D. Jha et al. / Materials Science and Engineering C 80 (2017) 659–669

3.3. Biosynthesized silver nanoparticles exhibited enhanced antioxidativepotential

Being a member of Rutaceae family, it was anticipated that the ex-tracts of C. maxima plant would be rich in antioxidants and hence theAgNps synthesized from C. maxima extracts should have high antioxi-dant potential. To validate this, DPPH quantitative estimation was

Fig. 7. In vitro cell cytotoxicity assays: Cell cytotoxicity observed after 24 h of treatmentwith bioextracts and AgNO3 on a) HT-1080; b) B16-F10; c) MRC-5 cell lines; Microscopic images of d) H

performed. Interestingly, all the biosynthesized silver nanoparticles ex-hibited higher antioxidant potential as compared to their respective ex-tracts. Out of these, AgFeNps (1000 μg/ml) showed the strongestantioxidant activity (~96%) followed by AgLeNps (~82%) and AgPeNps(~80%), ensuring the fruit as the rich source of antioxidants (Fig. 3).

3.4. Biosynthesized silver nanoparticles exhibited remarkable antimicrobialactivity against multi-drug resistant microorganisms

In order to comprehend the action of nanoparticles as an antimicro-bial agent, both Gram-positive and Gram-negative bacteria were used.We observed the appeared zone of inhibition in the range of 11 to21 mm against these pathogenic microorganisms (Fig. 4(i)). Out ofthese three silver nanoparticles, AgFeNps showed the highest antimi-crobial activity followed by AgPeNp, AgLeNp (Table 2). The quantitativeestimation of exhibited antimicrobial potential was done by usingmicrobroth dilution assay. Multi-drug resistant bacteria as well asacne associated pathogens were included in this study so as to identifyif these nanoparticles can serve as alternative drug against these deadlypathogens (Table 3). The observation reflected that at MIC, nanoparti-cles were successful in combating the growth of these pathogens,along with Methicillin-resistant S. aureus, MDR\\P. aeruginosa and S.enteritidis (Table 3), stating one more effective property of thebiosynthesized nanoparticles. The relative survival (%) of these mi-crobes was endorsed in Fig. 4(ii). The reason of this activity could bethe interaction of nano-sized particles on the comparatively larger sur-face area ofmicroorganisms.Most interestingly, chemically synthesizedsilver nanoparticle (AgNp) which was used as a control, showed negli-gible zone of inhibition and MIC observed for each pathogen was

synthesized silver nanoparticles (AgFeNp, AgLeNp, AgPeNp), C. maxima fruit, leaf and peelT-1080; e) B16-F10; f) MRC-5 cells, before and after treatment with AgNps, respectively.

Fig. 8. Detection of apoptosis within B16-F10 cells treated with biosynthesized silver nanoparticle (AgNp): a) Relative survival (%) of B16-F10 cells after 48 h of treatment with AgFeNp,fruit extract and AgNO3 as was shown by the uptake of trypan blue dye; b) AO/EtBr dual dye staining assay to assess apoptosis throughmorphological variations and fluorescence emittedfrom the B16-F10 cells nuclei after 48 h treatment with AgFeNps, i) untreated cells, ii) cells treated with C. maxima extract (50 μg/ml), iii) cells treated with AgNO3 (1 mM), iv–vi) cellstreated with AgFeNps (10 μg/ml) showing higher number of early, mid/late apoptotic bodies with highly condensed fragmented nuclei, respectively; c) Gel electrophoresis of genomicDNA of B16-F10 cells treated for 48 h: L) DNA ladder, i–ii) treated with AgFeNps, iii) untreated sample, iv) treated with fruit extract, v–vi) treated with AgNO3; d) Highest RFU shownby AgFeNps indicating maximum activation of Caspase-3 as compared to fruit extract and AgNO3, respectively; e) Cell cycle analysis by using Flow cytometry showing the blockage ofcells at G0–G1 phase in response to AgFeNps treatment leading to apoptosis.

666 D. Jha et al. / Materials Science and Engineering C 80 (2017) 659–669

N200 μg/ml. These biosynthesized nanoparticles can be tried directly orin conjugationwith the available antibiotics so as to reduce the antibiot-ic load on the systemandhence inhibit the bacteria to acquire resistanceagainst these drugs. To perceive themorphological changes in the nano-particle-treated B. cereus andMDR- S. enteritidis, TEM images were cap-tured and analyzed. TEM results showed that these microbes werecompletely ruptured in the presence of biosynthesized nanoparticlesand thus exhibited excellent antimicrobial potential (Fig. 5a-j). Bunchof particles dumped on the surface and inside the bacterial cells wereseen in the images. The main reason behind this could be the reactionof silver particles with –SH group of the bacterial proteins, inhibiting re-spiratory chain enzymes or negatively regulating the proton and phos-phate permeability in the cell membrane. Accumulation of silver andsulphur in such dumps indicated hampering of DNA replication andhence, killing the bacteria [45–47]. Studies also suggest that nanoparti-cles can distort cell membrane, rendering them permeable to these par-ticles. Accumulation of these particles on the membrane created spacebetween the bilayer, thus inducing the entry of particles inside the bac-terial cell and hence, causing death [48]. Our study also concludes thatthese particles disrupted themembrane and caused bacterial cell death.

3.5. Biosynthesized silver nanoparticles showedminimal toxicity on normalhuman red blood cells

In order to ascertain the safety of biosynthesized nanoparticles onhumans, these were tested on human red blood cells by performing he-molysis assay. Study was conducted with 1X, 5X and 10X concentra-tions of biosynthesized nanoparticles. Here, the highest MIC,considering all the tested pathogens (160 μg/ml), was taken as 1X. AtMIC concentration, the hemolysis was found to be less than ~25%. Con-sidering the lowest MIC as was observed in the case of P. aeruginosa (20μg/ml), this toxicity seems to be negligible. However, at maximum

concentration (1600 μg/ml), which was 10 times of the highest ob-served MICs and 80 times of lowest observed MICs, the hemolysis wasless than ~55%. These observations clearly state that these nanoparticlescan be used directly (at lower doses) or in combination with a vehicle,which can reduce its toxicity on human cells and thus increase its actionon the target cells (Fig. 6).

3.6. Biosynthesized silver nanoparticles exhibited remarkable cytotoxicityon cancerous cell lines

Nanoparticles showed N60% of cytotoxicity in cancer cell line, HT-1080, when these cells were exposed to nanoparticles (100 μg/ml) for48 h (Fig. 7a). Similarly, N75% of cytotoxicity was observed in anothercancer cell line, B16-F10, using 100 μg/ml dose of nanoparticles (Fig.7b).Most convincingly, the cytotoxicity assay showed very lowcytotox-icity (N80% viable cells) towards normal cell lines (MRC-5) under simi-lar conditions (Fig. 7c). Although, we observed cytotoxicity of thesenanoparticles on normal human cell lines (MRC-5) at higher concentra-tion, thiswas very low as compared to that observed on cancer cell lines.Microscopic images of treated cancerous cells showed retorted growth(Fig. 7d–e), whereas in case of normal cells, there was no significant dif-ference observed as compared to their respective untreated cells (Fig.7f).

3.7. Detection of apoptosis in B16-F10 cells treated with biosynthesized sil-ver nanoparticles

In order to assess the apoptosis potential of these nanoparticles, B16-F10 cell line was chosen, based on the cytotoxicity profiling. Moving inthis direction, induction of cytotoxicity by these nanoparticles wasfirst quantified using trypan blue assay. N60% of cytotoxicity was ob-served in case of AgFeNps treatment, whereas cells treated with

Fig. 9. Pictorial representation showing the antimicrobial as well as anticancerous effect of biosynthesized silver nanoparticles with minimal toxicity towards the normal human cells.

667D. Jha et al. / Materials Science and Engineering C 80 (2017) 659–669

controls showed no significant toxicity (Fig. 8a). The apoptosis potentialwas assessed throughAO/EtBr staining of treated B16-F10 cells followedby their microscopic analysis (Fig. 8b). Highly condensed chromatinstructure along with membrane blabbing were clearly observed incase of B16-F10 cells treated with biosynthesized silver nanoparticles,indicating the cellular apoptosis leading to cell death [Fig. 8b(iv–vi)].No morphological changes in the nuclei of B16-F10 cells, treated withC. maxima extracts or AgNO3, were observed [Fig. 8b(i–iii)]. Cell deathmight have caused due to the massive DNA breakage and chromatincondensation, because of ROS generation by these nanoparticles on itspenetration inside the cell. Characteristic feature of apoptosis is consid-ered as the activation of endonucleases resulting in the fragmentation ofgenomic DNA at internucleosomal linker regions [49,50]. To observe theactivation of endonucleases, DNA fragmentation analysis was per-formed for all the samples. AgFeNps (10 μg/ml) treated B16-F10 cellsshowed clear fragmentation of genomic DNA, on ethidium bromidepre-stained agarose gel [Fig. 8c(i–ii)] but not with either fruit extract(50 μg/ml) or AgNO3 (1 mM) [Fig. 8c(iii–vi)], indicating that AgNpscaused apoptosis by prompting internucleosomal DNA fragmentation.Further, Caspase-3 activity assay was performed in order to investigate,whether the internucleosomal DNA fragmentation was due to the acti-vation of Caspase-3, a hallmark feature of apoptosis. AgFeNps treatedB16-F10 cells (after 48 h) showed higher activation of Caspase-3 withrespect to untreated, fruit extract and AgNO3 treated cells (Fig. 8d).

These results indicated the ability of AgFeNps to cause cell deaththrough apoptosis which might be due to oxidative stress caused bythe release of metal ions from AgNps. Further, cell cycle analysis forthe biosynthesized silver nanoparticle-treated B16-F10 cells was per-formed by using FACS. Based on the FACS data, the inhibitory effect ofAgFeNps (10 μg/ml) was clearly observed in case of treated B16-F10cells (Fig. 8e). An increased cell population was observed in Go–G1phase than S-phase after treatment, as compared to untreated B16-F10 cells. The cells treated with fruit extract showed no significantchange in the cell population. However, the cell cycle progression wasblocked in the Go–G1 phase, when cells were treated with AgNps (Fig.8e). Thus, it can be interpreted that Go–G1 growth phase arrested cellsmight enter sub-Go phase that may further lead to cell death throughapoptosis. Hence, these nanoparticles can be used as an anticancer ortargeted delivery agent.

4. Conclusions

Ordinance of nanoparticles as an alternative drug due to their non-resistant properties in organisms becomes enchanting in this era ofevolving multi-drug resistant microbes and not so successful cancerdrugs. Herein, we demonstrated a novel, eco-friendly and green ap-proach for the biosynthesis of silver nanoparticles using C. maximafruit, leaf and peel extracts as bioreducing agents. These biosynthesized

668 D. Jha et al. / Materials Science and Engineering C 80 (2017) 659–669

AgNps exhibited excellent antimicrobial activity against several patho-genic microorganisms which have developed resistant for most of thedrugs available during prolonged drug therapy. The exact mechanismsbehind the mode of action of silver nanoparticles on these pathogenicmicroorganisms are not yet effusively explicated. However, based onTEM analysis, we can conclude that these biosynthesized nanoparticlesmay trigger structural alterations in the bacterial cell membrane and af-fect the permeability which leads to demise of the bacterial cell. Alongwith such antimicrobial efficiency, these biosynthesized nanoparticlesshowed remarkable anti-cancerous activity against mammalian cancer-ous cell lines. The synthesizednanoparticleswere found to beminimallytoxic towards both human RBCs aswell as normal human cell line, indi-cating the biocompatibility with normal mammalian cells. The pictorialrepresentation depicting the overall findings of this study has beenshown in Fig. 9. This study beholds anticipation that biosynthesizednanoparticles may impart their role in medical field as novel antimicro-bial as well as anticancerous agents in the near future. Studies, cloudingthese properties of nanoparticles,would definitely help the scientific so-ciety to mediate effective drugs for the mankind.

Author contributions

DJ, PKT and SA designed and carried out the experiments and ana-lyzed the data. DJ and RPwrote themanuscript and prepared all figures.PK, AKS andHKG proposed the basic idea and supervised the project. BKand SA participated in the interpretation of the experiments anddiscussed the results. All authors reviewed and approved the finalmanuscript.

Notes

The authors declare that they have nofinancial or non-financial con-flicts of interest related to this article.

Acknowledgement

Authors gratefully acknowledge thefinancial support fromCSIRNet-work Project, BSC0302. Authors also thank University Science Instru-mentation Centre, University of Delhi, Delhi, for spectroscopic analysisof the samples.

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Multifunctional biosynthesized silver nanoparticles exhibiting excellent antimicrobial potential against multi-drug resist...1. Introduction2. Materials and methods2.1. Biosynthesis of silver nanoparticles using C. maxima extracts2.2. Characterization of synthesized nanoparticles2.3. Antioxidant activity2.4. Antimicrobial activity2.5. Transmission Electron Microscopy (TEM)2.6. Hemolytic activity2.7. Cell cytotoxicity assay2.8. Characterization of cytotoxicity of AgNps on B16-F10 cell line2.9. Caspase-3 activity assay2.10. Cell cycle analysis using fluorescence-activated cell sorting (FACS)

3. Results and discussion3.1. Green approach for synthesis of silver nanoparticles (AgNPs)3.2. Characterization of biosynthesized silver nanoparticles (AgNps)3.3. Biosynthesized silver nanoparticles exhibited enhanced antioxidative potential3.4. Biosynthesized silver nanoparticles exhibited remarkable antimicrobial activity against multi-drug resistant microorganisms3.5. Biosynthesized silver nanoparticles showed minimal toxicity on normal human red blood cells3.6. Biosynthesized silver nanoparticles exhibited remarkable cytotoxicity on cancerous cell lines3.7. Detection of apoptosis in B16-F10 cells treated with biosynthesized silver nanoparticles

4. ConclusionsAuthor contributionsNotesAcknowledgementReferences

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