Biofilm Impeding AgNPs Target Skin Carcinoma by Inducing

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Biofilm impeding AgNPs target skin carcinoma by inducing mitochondrial membrane depolarization mediated through ROS production Debasis Nayak, Manisha Kumari, Sripathi Rajachandar, Sarbani Ashe, Neethi Chandra Thathapudi, and Bismita Nayak ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b11391 • Publication Date (Web): 07 Oct 2016 Downloaded from http://pubs.acs.org on October 9, 2016

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ACS Applied Materials & Interfaces

Biofilm impeding AgNPs target skin carcinoma by inducing mitochondrial membrane depolarization mediated through ROS production Debasis Nayak, Manisha Kumari, Sripathi Rajachandar, Sarbani Ashe, Neethi Chandra Thathapudi, Bismita Nayak* Immunology and Molecular Medicine Laboratory, Department of Life Science, National Institute of Technology, Rourkela, Odisha-769008, India

Abstract Reactive oxygen species (ROS) are double edged sword that possesses both beneficial and harmful effects. Although basic research on skin cancer prevention has undergone a huge transformation, cases of recurrence with higher rates of drug resistance are some of its drawbacks. Therefore, targeting mitochondria by ROS overproduction provides an alternate approach for anticancer therapy. In the present study green synthesized silver nanoparticles (AgNPs) were explored for triggering the ROS production in A431 skin carcinoma cells. The synthesized AgNPs were characterized for size, charge, morphology and phase through high throughput DLS, Fe-SEM, XRD and ATR-FTIR techniques. Their physiochemical properties with hemoglobin and blood plasma were screened through hemolysis, hemagglutination assay, and Circular dichroism spectroscopy confirmed their nontoxic nature. The AgNPs also exhibited additional efficacy in inhibiting biofilm produced by V. cholerae and B. subtilis thereby facilitating better applicability in wound healing biomaterials. The depolarization of mitochondrial membrane potential ∆Ψm through excess ROS production was deduced to be the triggering force behind the apoptotic cell death mechanism of the skin carcinoma. Subsequent experimentation through DNA fragmentation, comet tail formation, cell membrane blebbing and reduced invasiveness potentials through scratch assay confirmed the physiological hallmarks of apoptosis. Thus, depolarizing mitochondrial membrane potential through green synthesized AgNPs provides an economic, non-toxic, specific approach for targeting skin carcinoma with additional benefits of antibacterial activities. Keywords: Green synthesis, Silver nanoparticles, Reactive oxygen species, Apoptosis, Mitochondria, membrane potential

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1. Introduction Reactive oxygen species (ROS) are a small group of highly reactive oxygen-derived species (hydroxyl radical, singlet oxygen, hydrogen peroxide, and superoxide) that are produced normally in cell membranes, mitochondria, peroxisomes, and endoplasmic reticulum 1. They can clearly be designated as double edged sword having both beneficial and harmful effects. Targeting cancer cells by modulating the ROS production and mitochondrial membrane potential (∆Ψm) provides a promising approach for anticancer therapy. The ∆Ψm is maintained through the pumped protons in the intermembrane space by the complex I, III and IV of the electron transport chain (ETC) 2. During ATP generation, free oxygen radicals (O2.) are the main by-products produced that are highly reactive in nature and multiply in exponential rate owing to its unstable reactive form. Under normal physiological conditions the ∆Ψm is generally maintained in higher proportion whereas the membrane potential of permeability transition pore complex (PTPC) is found to be lower. The lower membrane potential of PTPC maintains the influx and efflux of solutes, but during stress conditions (ROS overproduction), the conductance of PTPC is tremendously increased leading to the unregulated influx of solutes inside the mitochondrial membrane. Due to the amplified inner osmotic pressure the permeability of the outer membrane changes which releases the proapoptotic proteins into the cytoplasm leading to the programmed cell death (apoptosis) 3. The mitochondrial ROS (mROS) possess many beneficial effects such as secondary messenger in cell-cell signaling, catalyzing the synthesis of thyroxine and NADPH oxidase, phagocytosis, folding of proteins in the endoplasmic reticulum and activating the tumor suppressor genes 4. But contrary to that, at elevated levels the same free radicals can cause cell and mitochondrial membrane damage leading to the progression of inflammation, tumor/cancer development and neurodegenerative diseases 5. In a normal cell, the level of mROS is balanced due to the activities of internal antioxidants: superoxide dismutase (SOD1, SOD2 and SOD3), glutathione peroxidase and catalase. During mutation in mitochondrial DNA (mtDNA), the level of ATP synthesis gets hampered. As a consequence these sequences triggers a cascade that leads to the over production of ROS which ultimately causes oxidative damage to the mitochondrial and cell membrane. Successive ROS production eventually leads to progression of cancer through the destruction of cellular machinery such as DNA, lipids and proteins

6

. An extra load of ROS in the

microenvironment helps in maintaining the initial tumor/cancer cell development. The


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quantity of ROS is always retained below its threshold level through the aid of in-house antioxidants. However, at exaggerated conditions, overproduction of ROS stimulates the destruction of the same cancer cells following apoptotic or non-apoptotic pathways. The perspective of ROS overproduction for cancer therapy using metallic components of various anticancer drugs like cisplatin (mainly composed of platinum) has been appreciated for its therapeutic value in chemotherapy 7. But cisplatin and other such metal containing complexes show higher rate of drug resistance with additional side effects on kidney (nephrotoxicity), neurons (neurotoxicity), ear or specifically cochlea and auditory nerve (ototoxicity), and inducing frequent vomiting (emetogenesis) 8. Among various types of cancer, the rate of epidermal cancer is increasing in a very fast pace worldwide 9. Skin cancer is mainly classified into non-melanoma skin cancer (NMSC) and cutaneous malignant melanoma (CMM). The exact incidence of cutaneous cancer in India is not known but NMSC is more common among Asians. Various reports convey that the factors responsible for skin cancer are UV rays but other socioeconomic factors may also contribute for their rising distribution among Indian diaspora. Conventional chemo and radiotherapy are the most commonly used therapy for the treatment of skin cancer. But the chemotherapeutic agents are usually associated with high toxicity and side effects. Currently, there are very few reports on mitochondrial membrane potential (∆Ψm) playing a significant role in targeting skin cancer using biologically synthesized AgNPs. The main objective of this investigation is to explore the possibilities of synthesized As-AgNPs and EpAgNPs in channeling overproduction of ROS and modulating the physiological cascade that can trigger the process of apoptosis by depolarization of the mitochondrial membrane in A431 skin carcinoma cells. Detailed step wise experiments were designed to elucidate the effects of these biologically synthesized AgNPs on blood plasma, hemoglobin, DNA machinery and cell membrane. Additional experiments were conducted to explore the efficacy of the synthesized AgNPs in impeding the formation of biofilms in nosocomial strains of Bacillus subtilis and Vibrio cholerae. 2. Materials and methods 3-(4,5-dimethylthiazol-2-yl)-2,5 di-phenyl-tetrazolium bromide (MTT), Dulbecco's Modified Eagle's medium (DMEM), Fetal bovine serum (FBS), Antibiotic solution (penicillinstreptomycin), Dichloro-dihydro-fluorescein diacetate (DCHF-DA), Rhodamine 123, Propidium iodide, Acridine orange were purchased from Sigma-Aldrich (Mumbai, India). 3 ACS Paragon Plus Environment


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Silver nitrate, Mueller-Hinton agar and Mueller-Hinton broth, Agarose, 100bp DNA ladder and all other reagents of analytical grade were purchased from Hi-Media (Mumbai, India). Distilled water was used throughout the experiments.

2.1 Plant material collection Whole plant parts of Eclipta prostrata and Alternanthera sessilis were collected in the month of August 2015 from the campus of National Institute of Technology, Rourkela. The whole plants were first washed with tap water followed by successive washing with distilled water to remove any traces of soil and dust. The plant samples were then shade dried and using a mechanical grinder a fine powder was obtained. The powdered samples were then kept in air tight containers for further use.

2.2 Preparation of silver nanoparticles The nanoparticles were synthesized as reported earlier by our group 10. Briefly, 90ml of silver nitrate solution (1 M) was mixed with 10 ml of plant extract and the reaction mixture was kept in a water bath at different temperature until the appearance of reddish brown colour. The same procedure was applied to observe the time and pH required for the synthesis of AgNPs with periodic monitoring of the reaction mixture in a UV–visible spectrophotometer (Lambda 35® PerkinElmer, Waltham, MS, USA) operated at a resolution of 1 nm at room temperature scanned in the wavelength range of 400–600nm. The resultant coloured reaction mixture was then centrifuged at 10,000 rpm for 45 min (C24-BL centrifuge, REMI, India). The obtained pellet was washed thrice with deionized water to remove the unwanted phytoconstituents of the plant extract. Then the resultant pellets were lyophilized and stored for further characterizations. All the conditions were optimized for its reproducibility.

2.3 Characterization of silver nanoparticles The hydrodynamic (Z-Average) size, polydispersity index (PDI) and surface zeta potential (charge) of the synthesized As-AgNPs and Ep-AgNPs were analyzed by Zetasizer (ZS 90, Malvern Instruments Ltd., Malvern, UK) and the results were obtained by the Malvern ZS nano software. The surface morphology of the synthesized nanoparticles was investigated by field emission scanning electron microscopy (Nova NanoSEM 450/ FEI, USA). The nanoparticles were fixed on adequate support and coated with gold using gold sputter module in a vacuum evaporator. Observations under different magnifications were performed at 10 kV. The X-ray powder diffraction (XRD) patterns of AgNPs were obtained using X-ray 4 ACS Paragon Plus Environment

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diffractometer (Rigaku Ultima IV, Japan) equipped with Ni filter and Cu Kα (l = 1.54056 Å) radiation source. The diffraction angle was varied in the range of 10–80° while the scanning rate was 5°/s. The Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy analysis was conducted to corroborate the possible role of various phytochemicals present in the plant extract for surface modification during the nanoparticle synthesis. The ATR-FTIR was performed on a Bruker ALPHA spectrophotometer (Ettlinger, Germany) with a resolution of 4cm-1 the spectral region between 4000 and 500cm

-1

average

of 25 scans per sample. 1 drop of sample was kept on the sample holder and scanned; the result obtained was analyzed through OPUS software. 2.4. Hemocompatibility of the silver nanoparticles 2.4.1 Hemolysis activity Compatibility analysis with blood was done following standard protocol

11

. Briefly, blood

was drawn from a healthy donor from CWS, Hospital, Rourkela, Odisha, in a 15 mL falcon tube containing 3.8% sodium citrate. The whole blood was diluted with 0.9% normal saline (NS) solution. For checking hemolysis, 1 mg of each lyophilized plant extract and their synthesized AgNPs were added to 10 mL of NS and 0.2 mL of the diluted blood was mixed gently and incubated for 60 min at 37°C in an incubator. After 60 min of incubation, all the samples were centrifuged for 5 min at 3000 rpm and the supernatant was carefully removed. The optical density was measured at 545 nm in a UV-vis spectrophotometer. Similarly, for positive control, 0.2 mL of diluted blood was taken in 10 mL of 0.1% sodium carbonate solution and for negative control; 0.2 mL of diluted blood was taken in 10 mL of normal saline solution and incubated for 60 min at 37°C. The % hemolysis rate was calculated using the formula: % =

− 100 −

Where ODs is OD of the sample; ODnc is the OD of negative control; ODpc is OD of the positive control. 2.4.2 Hemagglutination activity The citrated blood was centrifuged at 1000 rpm for 10 min at room temperature. 100 µL of the pellet was mixed with 10 mL of PBS (pH 7.4) and again centrifuged for 10 minutes at 1000 rpm. Finally, 100 µL of the pellet was mixed with 10 mL PBS which was used in the



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assay. In a 96 ‘U’ shaped plate 100 µL of PBS was added to each well except the 1st column which served as the negative control containing only RBCs and nanoparticles whereas the 12th column served as a positive control containing only PBS and RBCs. In all wells, the AsAgNPs and Ep-AgNPs (1mg/mL) were serially diluted from 1st well to the 11th well and lastly diluted RBCs (100 µL) were added to each well. After incubation for 2 hours the button like formation were observed visually.

2.5. Plasma interaction studies To observe the interaction of blood plasma with As-AgNPs and Ep-AgNPs 1mg/mL concentration of nanoparticles were incubated with the plasma for 1 h at 37°C. The incubated reaction mixture was scanned in ATR-FTIR to observe any transformation in their functional groups before and after interaction with blood plasma. The Circular dichroism (CD) spectra of blood plasma before and after incubation with As-AgNPs and Ep-AgNPs and their respective PE (plant extract) in aqueous medium were recorded at room temperature on a JASCO J-815 CD spectrometer (Model No J-815-150 S) with 1 mm path length. All spectra were collected from 190 to 260 nm and average of three scans was adopted to increase the signal-to-noise ratio of the CD spectra.

2.6. Effect of nanoparticles on biofilm activity of bacteria To check the quantitative assessment of biofilm formation against the synthesized As-AgNPs and Ep-AgNPs the tissue culture plate (TCP) assay

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was followed where one ideal gram-

positive and one gram-negative bacterial strain were selected for the study. Bacillus subtilis was collected from SCB Medical College, Cuttack, Odisha, India, and Vibrio cholerae (classical 0139) Strain no. 3906 obtained from MTCC, Chandigarh, India. Briefly, isolates from fresh MHA plates were inoculated in MHB containing test tubes and incubated overnight at 37°C in a static condition. This overnight culture was diluted 100 times and 200µL aliquots of the diluted culture were added to individual wells of sterile, polystyrene, 96 well flat bottom plates. Control was maintained by adding only MHB (without bacterial culture). The plates were incubated for 48 hours at 37°C. After incubation, the contents of each well were gently removed by slightly tapping the plates. The wells were then washed with PBS to remove any free-floating bacteria and stained with 0.1 % (w/v) crystal violet solution. Excess stain was removed by 95 % ethanol and the plates were dried. Optical density (OD595) of the wells was determined with a microplate reader (2030 Multilabel Processor VictorTMX3 Perkin Elmer, USA). OD values ≥ 0.01 were considered as an index 6 ACS Paragon Plus Environment

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of attachment to surface and biofilm formation. The experiment was performed in triplicate and the mean ± S.E of OD value was calculated. The isolates with OD595 ≥ 0.01 were considered biofilm positive and isolates with OD595 ≥ 0.04 was considered good biofilm formers.

2.7 Assessment of Anticancer Activity 2.7.1 Determination of cell viability by MTT Assay To test the cytotoxic effect of the green synthesized AgNPs, cell viability study was done with the conventional MTT reduction assay

10

. Briefly, A431 skin carcinoma and HaCaT

cells were purchased from NCCS, Pune, India. The cells were seeded in 96 well plates at the density of 3000 cells/well based on the doubling time in the presence of 200µL DMEM supplemented with 10% FBS and 1% penicillin-Streptomycin solution and incubated for 24 h in an incubator containing 5% CO2 at 37 °C. After 24 h of seeding, the existing media was removed and replaced by fresh media along with various concentrations of As-AgNPs and Ep-AgNPs viz., 10, 30, 40, 50, 60, 70, 80 and 100µg/mL and incubated for 24 h at 37 °C, 5% CO2. To detect the cell viability, MTT working solution was prepared from a stock solution of 5 mg/mL in growth medium without FBS to the final concentration of 0.8 mg/mL. 100 µL of MTT solution was added and incubated for 4 h. After 4 h of incubation, the MTT solution was discarded and 100 µL of DMSO solvent was added to each well under dark followed by an incubation of 15 min and the optical density of the formazan product was read at 595 nm in a microplate reader (PerkinElmer, Waltham, MS, USA). In all the cell culture experiments cisplatin was used as positive control. AgNO3 and the respective plant extracts of A. sessilis and E. prostrata were also used to compare the obtained results against the skin cancer cell line.

2.7.2 Cell cycle analysis by flow cytometry The cell cycle analysis was performed by flow cytometry using Propidium Iodide (PI) dye. Briefly, after the treatment of cells with As-AgNPs and Ep-AgNPs for 24 hours in CO2 incubator, they were trypsinized and collected by centrifugation (1500 rpm, 7 min at 4°C), washed twice with PBS and then fixed in 90% ice-cold ethanol. After incubation at -20°C for 1 h, cells were centrifuged and resuspended in PBS followed by treatment with RNase A (500 U/mL) to digest the residual RNAs and stained with PI (10 µg/mL). Samples were incubated for 30 min at 4°C and cell cycle analysis was performed with a BD accuri6 flow cytometer (BD bioscience) 13. 7 ACS Paragon Plus Environment


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2.7.3 Determination of internal ROS activity In vitro ROS activity was measured by using standard Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay

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. The intracellular ROS production was monitored after incubating the

treated cells for 4 h with DCFH-DA. DCFH-DA is a non-polar dye which is converted into the polar derivative DCFH by cellular esterases. The intracellular ROS was observed through flow cytometry using the dye DCHF-DA and their fluorescent intensity was observed in the FL-1 filter. Similarly, the cells after treatment were incubated with the dye and their images were captured in Epifluorescent Microscope (Olympus IX71, Olympus, Tokyo, Japan).

2.7.4 Determination of mitochondrial membrane potential The mitochondrial membrane potential (MMP) of the AgNPs treated cells were analyzed using flow cytometry and fluorescence microscopy. Briefly, for the flow cytometric analysis approx. 5×105 cells were seeded in 6 well plates and after 80% confluency, the cells were treated with the green synthesized As-AgNPs and Ep-AgNPs. After 4 h of incubation, the cells were harvested and washed with PBS. 15µL of 10 mM Rhodamine 123 dye (Rh 123) was added to each tubes containing 1 ml of culture media with the harvested cells. After 30 min of incubation, the cells were washed with PBS and their fluorescence intensity was measured using Flow cytometer (BD bioscience). For fluorescence microscopy, 15µL of Rh 123 dye was added to each treated wells and were incubated in dark for 30 min. After incubation, the cells were washed with PBS and were later fixed with 4% paraformaldehyde. The cells were observed in Epifluorescent Microscope (Olympus IX71, Olympus, Tokyo, Japan). The fluorescence intensity of the cells was analyzed through ImageJ software.

2.7.5 Fluorescence microscopy The effect of As-AgNPs and Ep-AgNPs on A431 skin carcinoma cells was studied by fluorescence microscopy using Propidium iodide (PI) and Acridine orange (AO) staining. After treatment, the cells were stained with 15µl of PI (1mg/mL) and 15µl of AO (1mg/mL) and incubated in dark for 30 min. Subsequently, the cells were washed with PBS thrice and then were fixed with 4% paraformaldehyde for 30 min. After fixing the cells were further washed with PBS and later they were visualized under Epifluorescent Microscope (Olympus IX71) 15. The relative fluorescence intensity of the cells was analyzed using ImageJ software.


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2.7.6 Morphology study of the A431 cells after treatment using SEM The morphological changes of the A431 skin carcinoma cells after their treatment with the synthesized AgNPs were observed through scanning electron microscopy (SEM) (Jeol 6480LV JSM Microscope). Briefly, the treated cells after harvesting were fixed onto small glass slides and were fixed with 4% paraformaldehyde followed by successive washing in increasing concentration of alcohol up to absolute 100% alcohol. The cells were later visualized under different magnifications at 20 kV.

2.7.7 DNA damage studies: DNA fragmentation and comet assay To determine cell death qualitatively, DNA fragmentation assay was performed as reported earlier

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. Briefly, 5×105 cells treated with As-AgNPs and Ep-AgNPs were harvested and

centrifuged at 2000 rpm for 5 min at 4˚C. After removing the supernatant, 20µL of TES lysis buffer were added to the pellet and mixed well by stirring with a wide-bore pipette tip. 10µL of RNase cocktail were added and mixed well by flipping the tip of the tube and incubated for 2 h at 37˚C. Then 10µL of proteinase K were added and incubated at 50˚C for 90 min. After incubation, the samples were run on a 1.5% agarose gel. The DNA bands were observed though the gel documentation system (Bio-Rad). To further validate the DNA damage in single cell level the comet assay was performed following the protocol of Rath et al,17 . Briefly, the harvested cells were mixed with low melting agarose and a layer was made on a previously agarose coated glass slide. The glass slides were pre-incubated in an alkaline lysis solution followed by electrophoresis and PI staining for 15 min. Finally, the images of the comet were visualized using an epifluorescent microscope (Olympus IX71, Olympus, Tokyo, Japan) and its parameters were analyzed using the ImageJ openComet utility tool. 2.7.8 Scratch assay In the scratch assay, cells were grown up to 90% confluency and a scratch was made on a uniform layer of cells using a sterile 200µL micropipette tip. The cells were then washed with PBS to remove the cellular debris. After treatment with the synthesized AgNPs photographs were taken after 0th, 12th and 24 hours of incubation.

3. Results The first inference for silver nanoparticle formation was visualized from the colour change of the reaction mixture to dark reddish. This is one of the characteristic phenomena of nanoparticles where with the reduction in size there is an overall change in the optical 9 ACS Paragon Plus Environment


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inference of the reaction mixture. To further validate the inference from the colour change phenomenon

the

reaction

mixtures

were

monitored

periodically

in

a

UV-Vis

spectrophotometer.

3.1 UV-Vis spectroscopy The progress of the reaction leading to the conversion of Ag+ from AgNO3 to reduced nano silver was monitored by observing the colour change inference and absorbance maxima peak in the range of 420-450 nm. For maximum and rapid yield of nanoparticles, optimization of various parameters such as temperature, pH and incubation time are some of the essential requisites. To optimize the above-mentioned parameters the reaction mixture was incubated at different temperature conditions with different range of pH. The samples were monitored periodically in UV-Vis spectrophotometer at different time intervals along with their colour change inference. In biological systems all primary biological reactions such as activation of enzymes, homeostatic balance of the cytoplasm and blood are governed by the alteration in pH. Thus, during the synthesis of AgNPs, the reaction mixture was monitored at different pH values (pH 2, 4, 6, 8 and 10) and temperature conditions (20°C, 40°C, 60°C and 80°C) until the colour change inference. Simultaneously the samples were scanned in UV-Vis spectroscopy and the appearance of the broad peak at around 430 nm supported the results of visual observation. In A. sessilis extracts, the nanoparticles were synthesized after 30 min of incubation in pH 8 at 80°C which was visible from the colour change inference as well as the appearance of an absorbance peak at around 430 nm. At all other pH range i.e. pH 2, 4 and 6 the alteration in temperature conditions did not showed any effect on the colour change of the reaction mixture and flat absorbance lines were detected.


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Figure 1: Optimization of pH, temperature and incubation time for the synthesis of: (i) A. sessilis AgNPs (a) 20°C; (b) 40°C; (c) 60°C; (d) 80°C ; (ii) E. prostrata AgNPs (a) 20°C; (b) 40°C; (c) 60°C; (d) 80°C

But contrary to this, when E. prostrata reaction mixture was incubated at different temperature conditions broad absorbance peak was witnessed at around 435 nm within 15 minutes of incubation at 80°C. Interestingly at both pH 8 and 10 sharp absorbance peaks were observed, but at 80°C the colour change of reaction mixture was significantly very rapid which was quantified in the UV-vis spectroscopy. Qualitative screenings of the synthesized As-AgNPs and Ep-AgNPs exhibited the presence of phenols and flavonoid groups which demonstrated their significance during the nanoparticle synthesis. Upon qualitative assessment, the phenolic content was found to be 30µg of GAE/g for As-AgNPs and 42.39µg GAE/g for Es-AgNPs whereas their flavonoids content was around 104µg RUE/g and 100µg RUE/g for As-AgNPs and Ep-AgNPs respectively.

3.2 Dynamic light scattering (DLS) studies Dynamic light scattering (DLS) studies were conducted to investigate the hydrodynamic size, polydispersity index and surface zeta potential of the synthesized AgNPs in a colloidal aqueous environment. The principle of DLS study relies on the collective assessments of particles undergoing Brownian motion in the suspended liquid solution that measures the fluctuations in the intensity of scattering light. By applying Stokes-Einstein equation, the Zaverage size (hydrodynamic diameter) is obtained. Fig 2 i-(a) shows the hydrodynamic size of the As-AgNPs and Ep-AgNPs at different temperature conditions. As seen in fig 1, with a gradual increase in temperature the size of the synthesized AgNPs gradually decreased. Therefore, with gradual change in temperature (20°C→80°C), the variation in nanoparticle size was prominently observed. Similarly, Ep-AgNPs also showed their inversely proportional relationship with temperature; with gradual increase in temperature, a pattern decrease in size in a linear fashion was observed. Figure 2 i-(b) shows the relation of temperature with the surface zeta potential during the nanoparticle synthesis. Surface zeta potential indirectly correlates with the stability of the nanoparticles in a colloidal aqueous solution. From the graph, it can be clearly deduced that with a gradual increase in temperature, the size of the nanoparticles decreases and the surface zeta potential increases. This indicates that temperature plays a pivotal role in nanoparticle synthesis. The 11 ACS Paragon Plus Environment


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polydispersity index (PDI) is the measure of the width of the particle size distribution calculated from a cumulant analysis of the DLS measured intensity autocorrelation function where a single particle size is assumed and a single exponential fit is applied to the autocorrelation function 18.

3.3 Morphology study The morphology of the synthesized AgNPs was observed through Fe-SEM. Fig 2(ii) shows the roughly spherical morphology of the green synthesized AgNPs having a rough surface on its outer layer.

Figure 2: i- (a) Effect on the size of the synthesized As-AgNPs and Ep-AgNPs at different temperature conditions. (b) Effect on surface zeta potentials of the prepared As-AgNPs and Ep-AgNPs at different temperature conditions; ii- Fe-SEM images of (a) As-AgNPs; (b) EpAgNPs; iii- (a) FTIR analysis of the synthesized AgNPs with their respective plant extracts (b) XRD analysis of the synthesized AgNPs.

3.4 ATR-FTIR analysis The ATR-FTIR analysis was conducted to corroborate the role of phytochemicals present in the plant extracts during the synthesis of As-AgNPs and Ep-AgNPs. Hence, it provides a unique molecular fingerprint that correlates with the presence of specific spectral bands 12 ACS Paragon Plus Environment

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unique for that sample. In As-PE, spectral bands were detected at 3731 cm-1, 2928 cm-1, 1533 cm-1, 1309 cm-1 and 1019 cm-1 whereas in the synthesized As-AgNPs most of the spectral bands were shifted to left yielding spectral bands at 3740 cm-1, 3189 cm-1, 2341 cm-1 and 1566 cm-1with a common band occurring at 1019 cm-1. These bands corresponded to the presence of -N-H stretching, ≡C-H stretching, C-C stretching, ≡C-N stretching, -N=O stretching and –C-H wag respectively. Similarly in Ep-PE, bands were detected at 3740 cm-1, 2332 cm-1, 1031 cm-1 and 1533 cm-1 whereas in Ep-AgNPs bands were observed at 3740 cm1

, 2341 cm-1, 1676 cm-1, 1529 cm-1, 1035 cm-1 that corresponded to the presence of N-H

stretching, ≡C-N stretching, -N stretching, C=O stretching, N-O asymmetrical stretching respectively. From the fig 2 iii-(a) it can be perceived that the PE acts on the silver salt reducing them to Ag0 by the presence of various phytochemicals that confirmed by their ATR- FTIR spectral emission bands. The presence of amides (C=O stretch), aromatic ring compounds (C-C stretch), aliphatic amines (C-N stretch) and nitro compounds (N-O asymmetrical stretch) provided a confirmatory result for the presence of flavonoids and phenolic compounds which were also confirmed by their qualitative and quantitative assessment results.

3.5 XRD analysis X-ray powder diffraction (XRD) analysis was employed to identify the phase, orientation and grain size of the synthesized nanoparticles. Fig 2-iii(b) clearly showed the characteristic diffraction peaks associated with crystalline silver. The Braggs peaks at 2θ degrees were observed at 27°, 32°, 38°, 44°, 46°, 64° and 77° which corresponds to the miller indices (111), (200), (220), (311), (420) and (422) for both the synthesized Ep-AgNPs and AsAgNPs confirming the face-centered cubic (FCC) crystalline elemental silver. The obtained results were matched with JCPDS (Joint commission of powder diffraction standards) database bearing file no (04-0783). Further the crystalline grain size of the synthesized AgNPs were calculated using Scherer’s equation: D = (Kλ/βCosθ) where ‘D’ is the mean crystalline size of the particle, ‘K’ is the shape factor whose value is 0.9, ‘λ’ is the wavelength of the X-ray radiation source i.e. 0.154 nm, ‘β’ is (π/180)* FWHM and ‘θ’ is the Bragg angle

19

. The average size obtained using the Scherer’s equation was 22.12 nm and

24.6 nm for synthesized Ep-AgNPs and As-AgNPs respectively.



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3.6 Hemocompatibility activity Hemocompatibility with the components of blood, especially with haemoglobin, is a standard procedure for screening any drugs/pharmaceutical product to study their level of toxicity with blood. Fig 3(i) shows the hemocompatibility of the synthesized As-AgNPs and Ep-AgNPs. In the crude form the As-PE and Ep-PE were supposed to contain a consortium of phytochemicals favoring higher hemolysis activity therefore, they were used as control in the hemocompatibility experiments. Higher hemotoxicity levels might produce adverse effects on different cells or other cellular components. As expected the synthesized AgNPs showed negligible toxicity towards hemoglobin when compared with their crude plant extracts (PE) (Fig 3 i-a). Further, the nanoparticles were serially diluted with blood to examine the toxicity of AgNPs in dose dependent fashion. The formation of uniform ‘button like’ structure showed their hemocompatibility with the blood components (fig 3 i-b). Both As-PE and EpPE showed hemolysis in their initial concentration during hemagglutination assay but, EsAgNPs was more hemocompatilbe than As-AgNPs as the ‘button-like’ structure formation was visible from 2nd and 3rd dilution. As the synthesized AgNPs had % hemolysis value less than 5% which is considered as the critical safe hemolytic ratio for biomaterials according to ISO/TR 7406 20, the synthesized As-AgNPs and Es-AgNPs can be considered to be relatively safe.

3.7 Nanoparticle interaction studies with blood plasma Circular dichroism (CD) spectroscopy is a powerful technique used for predicting the secondary structure/conformational changes that might occur during protein nanoparticle interactions and can predict any abnormality or presence of any disease in the body 21. It can monitor the protein-nanoparticle interaction and the resulting unfolding of the protein in the specific biosystem. In the present study, the characteristic CD properties of normal blood plasma and nanoparticle-treated plasma were evaluated to investigate the changes in secondary structure of native plasma and the subsequent changes after their incubation with As-AgNPs and Ep-AgNPs. CD spectra of incubated plasma showed negligible change in the percentage of alpha and beta helix of the incubated plasma when compared with CD spectra of untreated plasma (Fig 3ii-a). After incubating the blood plasma with As-AgNPs and Ep-AgNPs their % ellipicity of alpha and beta sheets were calculated using the K2D3 algorithm where the data is compared with that of the protein data bank (PDB) using //http//www.ogic.ca/projects/k2d3/orainaldia.html and the values were found out to be α2.27%, β-29.58% (normal blood plasma); α-1.72%, β-30.88% (A. sessilis PE); α-1.71%, β14 ACS Paragon Plus Environment

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30.31% (E. prostrata PE); α-2.3%, β-30.22% (As-AgNPs) and α-1.71%, β-30.21% (EpAgNPs).

Since proteins have confirmation sensitive spectral fingerprint region in the infrared region of the electromagnetic spectra, the ATR-FTIR spectroscopy was employed to identify the changes occurring in the nanoparticle-treated plasma proteins with that of untreated normal blood plasma proteins. Fig 3ii-b shows the ATR-FTIR spectra of the treated and untreated blood plasma proteins. In all the samples only two spectral bands were observed at 2952 cm-1 and 1553 cm-1 which correspond to the presence of C-H asymmetrical stretching and N-H stretching were the only bands that were present in untreated control plasma as well as plasma treated with As-AgNPs and Ep-AgNPs. In case of plasma treated with As-PE and EpPE some additional bands were observed at 1394 cm-1, 1052 cm-1 and 730 cm-1 which might be due to the presence of different phytochemicals in the crude extracts of the plants used. From fig 3ii it can be clearly illustrated that neither the synthesized AgNPs nor their crude plant extracts had any drastic effect on the configurative structure of plasma after their treatment.



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Figure 3: i- Hemocompatibility assay of the synthesized As-AgNPs and Ep-AgNPs (a) hemolysis assay; (b) hemagglutination assay; ii- Interaction study of blood plasma with AsAgNPs and Ep-AgNPs through (a) CD spectroscopy; (b) FTIR spectroscopy; iii- Anti-biofilm activity of As-AgNPs and Ep-AgNPs against strains of (a) B. subtilis and (b) V. cholera


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3.8 Anti-biofilm activity In present study, the anti-biofilm activity of the synthesized As-AgNPs and Ep-AgNPs was examined against the pathogenic strains of B. subtilis and V. cholerae. The respective plant extracts were kept as controls to observe their potency with respect to the synthesized AgNPs. Reports associating the exclusive ability of AgNPs in inhibition of biofilm formation by several pathogenic bacteria like E. coli, P. aeruginosa, and S. aureus, have been reported by various groups

22

. The test organisms were grown in microtiter plate wells with and

without AgNPs to form biofilm for 24 h. The wells with no AgNPs were considered negative control while the wells with only bacterial cultures were considered positive control. With the increase in the concentration of AgNPs, the absorbance decreased indicating reduced biofilm formation. The As-AgNPs (100µg/mL) reduced biofilm formation up to 55.9% in V. cholerae and 52.5% in B. subtilis. Similarly, Ep-AgNPs (100 µg/mL) reduced biofilm formation up to 45.3% in V. cholerae and 54.4% in B. subtilis. In comparison biofilm formation in both gramnegative and gram-positive bacteria was less in the PE of the same concentration. As-PE inhibited biofilm formation by 37% approximately in both the bacteria while Ep-PE reduced biofilm formation by 31% in V. cholerae and 45.7% in B. subtilis (Fig. 3iii). The above data clearly indicates that nanoparticles are better anti-biofilm agents as compared to the plant extracts. The presence of nanosilver may increase the antibiofilm activity due to physical properties like NP size, which facilitates better penetration into a bacterial cell and chemical properties like affinity between the NPs and the biofilms that increase biosorption

23

as

compared to only plant extracts. Thus the AgNPs can be further explored for their antibiofilm activities. 3.9 Elucidation the anticancer potentials of AgNPs in skin carcinoma 3.9.1. MTT colorimetric assay In this era of advanced technology rapid preclinical screening of novel therapeutics is very essential for targeting the ever growing tumours/cancer cells. For prior development and screening of new compounds MTT assay provides an economic, convenient and rapid inference for the viable and dead cells. MTT assay employs a colorimetric method for determining the no of viable cells based on mitochondrial dehydrogenase activity measurement at 595 nm. Thus, MTT assay was employed for the preliminary screening of the green synthesized As-AgNPs and Ep-AgNPs. Based on their toxicity Fig 4 (i-a) shows the % cell viability of A431 skin carcinoma cells upon their treatment with As-AgNPs and EpAgNPs. 17 ACS Paragon Plus Environment


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Figure 4: i MTT assay (a) A431 skin carcinoma cells treated with the biologically synthesized As-AgNPs and Ep-AgNPs and their respective plant extracts; (b) normal HaCaT cells treated with the biologically synthesized As-AgNPs and Ep-AgNPs and their respective plant extracts; ii Cell cycle analysis of A431 skin carcinoma cells treated with (a) control; (b) As-PE; (c) Ep-PE; (d) As-AgNPs; (e) Ep-AgNPs; (f) AgNO3; (g) Cisplatin

The IC50 values were calculated to be 68.94±0.72µg/mL (As-PE), 69.82±1.29µg/mL (EpPE), 70.52±0.24µg/mL (As-AgNPs), 70.59±1.02µg/mL (Ep-AgNPs), 58.69±0.91 µg/mL (AgNO3) and 54.01±0.29 µg/mL (cisplatin) where both the PE exhibited approximately same IC50 value and their synthesized AgNPs also exhibited similar IC50 values against the A431 skin carcinoma cell lines. In cancer therapy, apart from identifying the effect of the drugs on cancerous cells testing their effects on normal cells is equally important. Therefore, in the 18 ACS Paragon Plus Environment

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current study the effects of the biologically synthesized nanoparticles in the normal keratinocyte HaCaT cells were investigated. From fig 4 (i-b) it was clearly observed that the biologically synthesized nanoparticles (As-AgNPs and Ep-AgNPs) exhibited negligible toxicity when compared with their activities in the skin carcinoma cells. From the results on HaCaT cells we can draw an inference that as the cancerous cells are very metabolically active they readily uptake the nanoparticles present in their vicinity whereas the normal cells take time to engulf the foreign substance using its cellular machinery24. For all other experiments the respective IC50 values of the synthesized AgNPs were used. After confirming the IC50 values, the cell cycle analysis of AgNPs treated A431 cells were carried to detect the cell cycle arresting potentials of the synthesized As-AgNPs and Ep-AgNPs in the A431 skin carcinoma cells.

3.9.2 Cell cycle analysis Many cytotoxic and DNA-damaging agents arrest cell cycle at G1, S or G2/M phase depending on their property. G2 checkpoints control the entrance to mitosis and progression through the cell cycle or G2/M arrest in response to DNA damage. To observe the changes in cell cycle flow cytometry based cell cycle analysis was performed after treatment of A431 skin carcinoma cells with AgNPs, where cisplatin and the respective plant extracts (PE) were used as controls (Fig 4 ii). From the figure it can be postulated that upon treatment with AgNPs cancer cells exhibit chromosome instability and mitotic arrest under in vitro conditions which were evident from the expression profile of G2/M phase. According to Dziedzic’s group, the Ag+ ions released from the nanoparticle surface may be involved in cell-signaling cascades where they help in the activation of Ca2+ release that activates catabolic enzymes which latter leads to the mitochondrial membrane damage making the cells deficient of ATP and thus causing cell death

25

. To further confirm that green

synthesized AgNPs induced cell cycle arrest in the A431 skin carcinoma cells, the intensity of ROS production was measured to draw a connection between the results obtained through MTT assay and cell cycle analysis.

3.9.3 Detection of intercellular ROS generation ROS plays a prominent role in regulating various biochemical functions in our body where any deviation in their activity leads to development of pathological conditions including cancer. The role of nanoparticles in instigating cellular ROS production for anticancer potential is well reported

26,27

. In the present study, the internal ROS production after 19

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treatment with green synthesized As-AgNPs and Ep-AgNPs was monitored by flow cytometry and fluorescence microscopy using DCFH-DA which is an oxidation-sensitive fluorescent dye. From fig 5 it can be clearly observed that in both flow cytometry and fluorescence microscopy analysis the mean fluorescence intensity of As-AgNPs and EpAgNPs treated cells showed more intracellular ROS production than their respective PE which might be due to the ROS quenching ability of the PE owing to the presence of phytochemicals. Upon comparison with controlled untreated cells AgNPs treated cells exhibited higher production of intracellular ROS which signifies greater damage to the cellular machinery specifically DNA, lipids, mitochondria and cellular proteins. Mitochondrion being the primary source of ROS production, over accumulation of ROS in its internal membrane possesses major damaging effect to its mtDNA and outer membrane potential. Therefore to confirm the effects of ROS production on mitochondrial membrane potentials Rhodamine 123 dye was employed to quantify the charge depolarization in the outer membrane of mitochondria.


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Figure 5: In vitro ROS detection through (i) (a) Flow cytometry for ROS detection using FL1 filter; (b) mean fluorescence intensity of the flow cytometer results; (ii) Fluorescence microscopy (a) control; (b) As-PE; (c) Ep-PE; (d) As-AgNPs; (e) Ep-AgNPs; (f) AgNO3; (g) Cisplatin

3.9.4 Detection of mitochondrial membrane potential From fig 6 it was evidently observed that upon incubation with the synthesized AgNPs the mean fluorescence intensity of the cells decrease. As Rh123 dye is known for its ability to be actively taken up by the intact mitochondria, the untreated control cells showed higher fluorescence intensity in flow cytometry and fluorescence microscopy when compared with the AgNPs treated groups. Subsequently, the AgNPs treated cells exhibited a lower rate of fluorescence which occurred due to the depolarization of the permeability transition pore complex (PTPC) which regulates the permeability of the mitochondrial membrane. Upon heavy ROS production the depolarized PTPC facilitates the influx of solutes and ROS molecules that damages the mtDNA and thereby leading to the various significant changes in transcriptional level as well as in physiology of the cell. The damage to the mitochondria directly affects the ATP generation thus perturbing energy depletion in the cells. From both flow cytometric and fluorescence microscopy images it was prominently revealed that the synthesized AgNPs triggers generation of intracellular ROS which causes the change in ∆Ψm. Thus, from ROS production and ∆Ψm studies we can draw an inference that ROS plays a very significant role in affecting the redox potentials of ATP/ADP translocators in mitochondria which thereby causes the opening of PTP facilitating the induction of apoptosis through the intrinsic pathway.



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Figure 6: Determination of mitochondrial membrane potential using Rh123 dye through (i) (a) Flow cytometry for ROS detection using FL-1 filter; (b) mean fluorescence intensity of the flow cytometer results; (ii) fluorescence microscopy- (a) control; (b) As-PE; (c) Ep-PE; (d) As-AgNPs; (e) Ep-AgNPs; (f) AgNO3; (g) Cisplatin After observing the effect of ROS induced by the synthesized AgNPs in mitochondrial membrane potential and cell cycle analysis the morphological effects related to apoptosis and their induction of cell death process was observed through fluorescent microscopy employing two nucleic acid stains Propidium iodide (PI) and Acridine orange (AO).

3.9.5 PI and AO staining PI is a membrane impermeant dye which is generally excluded from the cell membrane of viable cells and thus it stains necrotic and late apoptosis cells. In dead cells, it enters the cell and intercalates inside ds-DNA to emit fluorescence 28. From fig 7(i) it can be observed that As-AgNPs and Ep-AgNPs have the ability of inducing nuclear material condensation and subsequently swollen nucleolar membrane as well as small nuclear bodies in the cytoplasm was prominently visible. Further the mean fluorescence intensity (fig 7i-g) of the AgNPs


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treated cells exhibited the higher mean fluorescence intensity when compared to the control untreated cells. AO is also an intercalating nucleic acid specific fluorochrome that emits green fluorescence. AO can penetrate the plasma membrane of viable cells and early apoptotic cells. The early apoptotic cells exhibit a very bright green nucleus showing condensed form of chromatin (Fig 7 ii) 29. Thus when both the results of PI and AO staining were compared it showed some preliminary insights on the onset of apoptosis in the AgNPs treated A431 skin carcinoma cells.

Figure 7: Fluorescence microscopy of A431 skin carcinoma cells after being treated with AsAgNPs and Ep-AgNPs with respect to the control untreated cells and cells treated with the respective plant extracts and cisplatin using (i) Propidium iodide dye (a) control; (b) As-PE; 23 ACS Paragon Plus Environment


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(c) Ep-PE; (d) As-AgNPs; (e) Ep-AgNPs; (f) Cisplatin; (g); mean fluorescence intensity of the fluorescence images; (ii) Acridine orange dye (a) control; (b) As-PE; (c) Ep-PE; (d) AsAgNPs; (e) Ep-AgNPs; (f) Cisplatin; (g); mean fluorescence intensity of the fluorescence images.

3.9.6 Morphological changes by scanning electron microscopy After the fluorometric assessment through PI and AO staining, there were strong indications for apoptosis being the probable mechanism for the anticancer activity of the synthesized AsAgNPs and Ep-AgNPs. Thus, another important hallmark of apoptosis i.e. morphological changes in the plasma membrane through blebbing was taken into account. The membrane blebbing activity is one of the most prominent features that occurs during apoptosis where cytoskeleton of the cells are broken down thereby causing the plasma membrane to bulge outside

30

. Fig 8 shows the characteristic membrane blebbing of the A431 skin carcinoma

cells after treatment with the synthesized AgNPs whereas intact plasma membrane was observed in the untreated control cells.


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Figure 8: SEM images of the A431 skin carcinoma cells after treatment with (a) PBS; (b) As-PE; (c) Ep-PE; (d) As-AgNPs; (e) Ep-AgNPs; (f) AgNO3; (g) Cisplatin

3.9.7 Detection of DNA damage DNA ladder assay, a form of DNA degradation is acknowledged as a key biomolecular alteration of the cellular DNA observed during the late stages of apoptosis in which caspase-3 plays a vital role. As observed in fig 9 (a) upon treatment with As-AgNPs and Ep-AgNPs the fragmentation of the A431 skin carcinoma DNA was observed that showed the same more or less fragmented patterns in both the synthesized AgNPs as well as the positive control cisplatin. When compared with the respective plant extracts (PE) the results indicated that the phytochemicals present in the plant extracts were not modified or lost during the nanoparticle synthesis. Thus, confirming the capping and reducing ability of the phytochemicals during the nanoparticle synthesis.

To further confirm the results observed in the above experiment the DNA damage at the single cell level was quantified using the single cell electrophoresis ‘Comet’ assay. Comet assay is considered as a very rapid, sensitive and reliable assay that provides a detailed report on the damage the nanoparticles has conferred to the DNA of the cancer cell after their treatment

31

. Fig 9 (b) shows the fluorescence images of the comets observed through

fluorescence microscopy. To confirm the visual observations the fluorescence images were analyzed using the openComet plugin in the ImageJ software which provided an array of measures on the DNA migration. The three most commonly used parameters of tail length, tail intensity and olive tail moment were taken into consideration to plot the graph (Fig 9 c). The tail length determines the distance the DNA has migrated out of the cell which is directly related to the fragmented size of the DNA inside the cells. The tail intensity of the % of DNA in the tail provides the amount of DNA that has migrated out of the nucleus and thus it is directly proportional to the amount of DNA damage inside the cell. Among the three parameters the olive tail moment data is considered to be the most statistically significant measurement as it provides an insight into the DNA distribution profile within the tail thus confirming the most DNA concentrated region inside the formed tail region 32.



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Figure 9:(a) Formation of DNA fragmentation assay L1: A. sessilis PE; L1: E. prostrata PE; L3: As-AgNPs; L4: Ep-AgNPs; L5: AgNO3; L6: Cisplatin; (b) Fluorescence microscopy images of the comets after alkaline gel electrophoresis; (c) Analysis of Tail length, tail moment and olive moment of the comets obtained through openComet plugin in ImageJ software. After getting a clear understanding of ROS being the primary source for charge depolarization in the outer membrane of mitochondria succeeding experiments confirmed the apoptotic cell death in AgNPs treated cells. Uncontrolled growth and invasive potentials are some of the abnormal characteristics of the cancerous cells, therefore to validate the inhibitory effects of our synthesized AgNPs upon incubation in the A431 skin cancer cells the in vitro starch assay was performed.

3.9.8 Scratch assay The in vitro wound healing assay also known as scratch assay is one the most simple and cost effective assay employed to perceive the invasive behaviour of the cells after being treated with drugs/anticancer agents. Fig 10 shows the wound healing activity of the A431 skin carcinoma cells after being treated with As-AgNPs and Ep-AgNPs at different time intervals of 0th, 12th and 24th hours of treatment with respect to the untreated cells and the positive control cisplatin. When compared with the control it can be clearly visualized that the synthesized AgNPs were able to arrest their further migration with slow invasive rate. When aligned in the ascending order of arresting the growth profile of A431 skin carcinoma the EpAgNPs were more competent to inhibit the migration of the cancer cells with respect to AsAgNPs which exhibited significantly slower migratory rates.


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Figure 10: Scratch wound healing assay of A431 skin carcinoma cells after treatment with the synthesized As-AgNPs, Ep-AgNPs, and their respective plant extracts and cisplatin as a positive control. 4. Discussion In cancer therapy, multidrug resistance is a severe problem arising due to the repeated reoccurrence of tumor/cancer at the primary site of incidence. To tackle this problem, metalbased nanoparticles can be utilized as a novel alternative to the drug-resistant cancer cells. Nanotechnology is one of the most rapidly emerging interdisciplinary sciences that have opened new frontiers in various fundamental and applied aspects

33–35

. Engineered

nanomaterials (1-100 nm) exhibit novel properties that are distinctively different from their conventional forms and are responsible for their unique physical, chemical, and biological behavior. Engineered nanoparticles can be classified into 4 groups i.e. carbon based (fullerene); metal based (nano-zinc, nano-gold); polymer based (PLGA, PLA nanoparticles) and composites (zinc/iron oxide)

36

. Among these four types, metallic nanoparticles have

gained immense advancement owing to their application in biomedical sciences as potential therapeutic and contrasting agents. Silver nanoparticles (AgNPs) have generated much interest in routinely used materials (textiles, water filters, air purifiers) to medicinal products (ointments, gels, antimicrobial bandages, sutures)

37,38

. Recently various studies reported the

anticancer potentials of herbal extracts and their mediated synthesized AgNPs with minimal toxicity to normal cells 39–41. Taking into consideration these facts, two plants were selected based on their therapeutic potentials against skin diseases described in Ayurveda folk fare medicinal system 42. Eclipta 27 ACS Paragon Plus Environment


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prostrata (family: Asteraceae) commonly known as bhringaraja which is used for the treatment of hair loss, body pain, diminision of vision, indigestion, jaundice, liver diseases, spleen, skin diseases, memory impairment, atherosclerosis, hepatic disorders and anti-snake venoms 43. Alternanthera sessilis (family: Amaranthaceae) commonly known as gudrisag is a perennial herb found near ponds and reservoirs. It has antipyretic, hepatoprotective, antiulcer, antibacterial, hematinic, and diuretic activities 44. Using the dye DCHF-DA in flow cytometry and fluorescence imaging the accumulation of ROS in the As-AgNPs and Ep-AgNPs treated cells were observed. Mitochondria are known as the powerhouse of the cell and play a very vital role in regulating the cell cycle. During ATP generation some free radicals are produced which are taken care by the in-house antioxidants such as catalase, superoxide dismutase, and glutathione reductase. The concept of surplus generation of ROS through metallic nanoparticles as anticancer therapeutics is quite well reported

45–47

. Thus mitochondria obviously become

venerable to its own produced ROS which not only damages the mtDNA but also attacks lipids, proteins, and cellular DNA. When the cell undergoes severe conditions of oxidative stress, mitochondria seems to adapt in its new surroundings resulting in mutations in mtDNA. Therefore, triggering overproduction of ROS in cancer cells can provide exciting venture for initiating a crusade against cancer. In pathological conditions generally, the ROS production is at its peak thereby damaging surrounding tissues and creating a hypoxia-like situation inside the tumor/cancer cells and causes oncogenic stimulation, increased metabolic activity and mitochondrial malfunction. Rhodamine 123, a lipophilic cationic fluorochrome was employed to investigate the changes occurring in the mitochondrial membrane potential (∆Ψm) after treatment with AgNPs. As ∆Ψm is very critical for maintaining the physiological function of the respiratory chain to generate ATP any type of charge polarization may indirectly lead to energy depletion in the cells. As seen in fig 6, after the treatment with AgNPs the mean fluorescence intensity of the cells was decreased thus, confirming the loss of integrity of the mitochondrial membrane. The change in mitochondrial membrane permeability also leads to the formation of mitochondrial permeability transition pores (MPTP) which facilitates unchecked inflow of solutes and free radicals inside the cell. This uncontrolled inflow causes increase of osmotic pressure inside the inner mitochondrial complex and thus finally leads to the overall collapse of the mitochondrial membrane which is considered as the initial step for the activation of the intrinsic cell death pathways

48

.

Hence, disruption of the barrier function in the outer mitochondrial membrane causes loss of 28 ACS Paragon Plus Environment

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function in the bioenergetics level by charge depolarization. Thereby, the redox homeostatic balance between inner and outer mitochondrial membrane is disturbed and the adjustment in ion channels leads to the overall destruction of the cancer cell. Consequent analysis of cell cycle arrest, nuclear condensation staining though PI and early apoptotic vacuoles observation though AO staining, DNA damage analysis through fragmentation and comet assay confirmed the apoptotic cell death process. The formation of DNA fragmentation clearly indicated the role of AgNPs in causing structural damage to the DNA double helical structure by increased oxidative stress in the AgNPs treated cells with respect to the control untreated group. Further, the formation of membrane blebbing after exposure of AgNPs confirms the fingerprint of apoptosis which gets triggered by the ROS production

49

. Our green synthesized As-AgNPs and Ep-AgNPs exhibited all the typical

hallmarks of apoptosis as shown in fig 8 and fig 9 which we can interlink with the flow cytometry data of the intercellular ROS production (fig 5) whereas control group did not display any additional stress levels when compared with the AgNPs treated cells. ROS also plays a very notorious role in the production of additional by-products of lipid peroxidation, which may also attack the DNA structure thereby forming a joint venture of ROS products in the overall cancer cell death. Satapathy et al.,

50

and Chairuangkitti et al.,

51

have reported

over production of ROS being the major cause of cell death when cancer cells were subjected to treatment with AgNPs. The results observed in the present study clearly justify our rationale that overproduction of ROS being the prominent cause for cell death in A431 skin carcinoma cells after treatment with the biologically synthesized As-AgNPs and Ep-AgNPs. In the healthcare industry, multidrug-resistant microbes lead the formation of biofilms. These biofilms are consortia of microbes that functions as a reservoir for pathogenic organisms which forms the primary source of disease outbreaks. These biofilms cause a wide variety of nosocomial infections through the surfaces of catheters, medical implants, wound dressing materials and other routinely used medical devices used for diagnostics

52,53

time the utilization of silver as an antibacterial agent is well documented

. From ancient

54,55

. There are

numerous reports on the efficacy of silver nanoparticles as excellent antimicrobial agents and many burn and wound healing agents are available in the market (Acticoat™, Silverline®, Silvasorb®, Bioglass, ON-Q-SilverSoaker) containing nano silver formulations. Our group has previously reported the bacteriostatic and bactericidal activity of biologically synthesized AgNPs against pathogenic hospital isolates

18,39

. In skin cancer, the patient becomes



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immunocompromised and vulnerable during the course of therapy for attack by any facultative opportunistic microbes to invade the wounded site. Thus, anti-biofilm activity of the synthesized As-AgNPs and Ep-AgNPs were explored as an additional characterization for testing their overall utility in multiple targeting against skin cancer and nosocomial strains. Nanoparticles are known to form various complexes with plasma proteins. Hence, to quantify the toxicity profile of our synthesized As-AgNPs and Ep-AgNPs, they were incubated with blood plasma proteins and were analyzed for their interaction by CD spectroscopy and ATRFTIR studies. The protein interaction studies showed the non-reactivity of our synthesized nanoparticles when compared with the native untreated blood plasma which mainly contains albumin, immunoglobulin, fibrinogen, and apolipoproteins. Similar kind of results were also reported while using TiO2 and ZnO nanoparticles

56,57

. The biological reactivity of the

nanoparticles is usually dictated by their rate of mutual attraction and adsorption of the plasma proteins on the surface of the nanoparticle. Thus, surface charge, size and surface morphology play a very significant role in directing the compatibility of the nanoparticles with the bodily fluids specifically blood plasma. 5. Conclusion Discovery and development of novel therapeutic agents for enhancing the survival rates with minimal chances of recurrence rate in skin cancer was the main goal in the present investigation. The applicability of biologically synthesized AgNPs was explored against epidermoid A431 skin carcinoma cells. The As-AgNPs and Ep-AgNPs exhibited dose dependent toxicity with controlled growth phase in the cell cycle process. Upon treatment with AgNPs overall quantity of ROS production was increased that caused the depolarization of mitochondrial membrane. The overproduction of ROS triggers the change in ∆Ψm which might have instigated the intrinsic pathway of apoptosis. The overall nuclear condensation and formation of apoptotic bodies with membrane blebbing showed the clear signs of apoptosis in the AgNPs treated A431 skin carcinoma cells. The As-AgNPs and Ep-AgNPs upon incubation with blood plasma did not showed any additional conformational changes in α-helices and β sheets of the blood plasma proteins confirming their inertness with various arrays of proteins. Further, tests with hemoglobin exhibited negligible toxicity of the synthesized AgNPs. Our study also presented the efficacy of the prepared AgNPs in inhibiting the formation of biofilm in standard gram-negative V. cholerae and gram positive B. subtilis pathogenic strains facilitating their applicability to control bacterial load and their use in skin healing/treatment purposes. Hence, biologically synthesized Ag nanoparticles 30 ACS Paragon Plus Environment

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could be used in newer directions of treating skin cancer by modulating overproduction of ROS. Author Information *Corresponding author: Dr. Bismita Nayak, Assistant Professor, Department of life Science, National Institute of Technology, Rourkela Email ID: [email protected]

Acknowledgement The authors would like to acknowledge National Institute of Technology, Rourkela for providing all the necessary equipment and financial support for carrying out the work on anticancer activity of biologically synthesized silver nanoparticles.

Conflict of Interest The authors declare no conflict of interest

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