Application of silver oxide nanoparticles for the treatment of cancer

Accepted Manuscript Application of Silver oxide Nanoparticles for the Treatment of Cancer

Seemab Iqbal, Muhammad Fakhar-e-Alam, Fozia Akbar, M. Shafiq, M. Atif, N. Amin, Muhammad Ismail, Atif Hanif, W. Aslam Farooq PII:

S0022-2860(19)30436-3

DOI:

10.1016/j.molstruc.2019.04.041

Reference:

MOLSTR 26413

To appear in:

Journal of Molecular Structure

Received Date:

04 February 2019

Accepted Date:

09 April 2019

Please cite this article as: Seemab Iqbal, Muhammad Fakhar-e-Alam, Fozia Akbar, M. Shafiq, M. Atif, N. Amin, Muhammad Ismail, Atif Hanif, W. Aslam Farooq, Application of Silver oxide Nanoparticles for the Treatment of Cancer, Journal of Molecular Structure (2019), doi: 10.1016/j. molstruc.2019.04.041

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Graphical Abstract

ACCEPTED MANUSCRIPT

Application of Silver oxide Nanoparticles for the Treatment of Cancer Seemab Iqbala, Muhammad Fakhar-e-Alam a, Fozia Akbara, M. Shafiqb, M. Atif c, N. Amina, Muhammad Ismaild, Atif Hanif e, W. Aslam Farooqc aDepartment

of Physics, GC University, Allama Iqbal Road, 38000, Faisalabad, Pakistan, of Physics, Allama Iqbal Open University,H-8 Islamabad, Pakistan cDepartment of Physics and Astronomy, College of Science, King Saud University, Riyadh 11451, Saudi Arabia dInstitute of Biomedical and Genetic, Engineering (IBGE), 24 Mauve Area, G-9/1, Islamabad, Pakistan eBotany and Microbiology Department, College of Science, King Saud University, Riyadh11451, KSA bDepartment

*Correspondence: [email protected], [email protected] Abstract Silver oxide nanoparticles are wonderful material and having great potential towards biomedical applications. Silver oxide nanoparticle were synthesized via Chemical Aqueous method and characterized by applying manifold available techniques. X-ray diffraction (XRD) was used for analyzing structural property of nanoparticle crystals, the morphology of synthesized nanoparticles was studied by scanning electron microscope (SEM), elemental analysis of the composition was observed by energy dispersive X-ray spectra (EDXS) and the optical properties was analyzed by the Uv-Vis spectrometer. Spectroscopic analysis confirmed the spherical morphology of the nanoparticles with the effect of calcined temperature. Phototoxic and cytotoxic effects of grown particles were examined by conducting various relevant experimental techniques on hepatocellular (HepG2 Cell line) model. The obtained results were verified by applying polynomial fit which confirmed the goodness of fit. Silver oxide NPs has unique bio interaction characteristics and physicochemical properties as anticancer agent. This research will be beneficial particularly for cancer therapeutics.

Keywords Hepatocellular Model (HepG2 Cell line); Silver oxide nanoparticles; Cytotoxicity; Statistical analysis

ACCEPTED MANUSCRIPT

1. Introduction Nanoparticles gain much popularity due to biological, therapeutic and medical applications in the present era [1]. Among the nanomaterials, silver oxide nanoparticles are much more popular in the fields of textile, food industry, electronics, construction materials, pharmaceutical products, antimicrobial agent, cosmetic and paints [2, 3]. The cost effective and environmental friendly synthesis of silver oxide nanoparticles is the hot topic of current research studies. Silver oxide nanoparticles have strong antibacterial, antiviral and antifungal effects [4]. Nanoparticles are being rapidly used as nano medicine, nano therapeutic, nano theranostic and cancer treatment [5]. Nanomaterials/nanoparticles cause cell death when taken up by the human tissues and cellular system and hence cell died causes via oxidative stress led by toxicity of nanoparticles. Size of nanoparticles depends on kinetics of uptake process, localization in cells and exocytose, physicochemical, surficial properties and on the ability to bond. Kinetics of uptake of nanoparticles also depends upon morphology of cell and its nature [[6], [7], [8], [9]]. Silver oxide nanoparticles are used as antiviral therapeutics against such type of viruses that causes disease. Silver nanoparticles are mainly focused to study for possible destruction against bacteria. It is also realized that silver oxide nanoparticles are beneficial against several types of viruses. Synthesized nanomaterials can be applied as Nano medicinal and Nano therapeutic drugs due to their anti-cancer, anti-viral properties and other surface charge property which might be beneficial for treating cancer and other viral/bacterial diseases. Due to strong antibacterial property it may be used for living organisms, including the human body, in the food, and also through skin or the respiratory system [10]. Silver nanoparticles go into individuals by oral absorption, inhalation, through damage skin still during hurdle of retina in mature and transmission or endocytosis through the skin of embryos. Ag nanoparticles penetrate into female genital tract and commonly female use different hygienic products consisting of silver (Ag) NPs [[11], [12], [13], [14]].Thus, nanomaterials provide an opportunity to overcome this challenge of drug delivery in the target site [15]. It is expected that nanotechnology will bring a central change in biological applications such as drug delivery and diagnostics in the coming few decades [16].

ACCEPTED MANUSCRIPT

In this experimental study, after successful synthesis and characterization steps, authors are interested to explore the real cytotoxicity and photo toxicity mechanism via proper pathway/chain reaction for cell killing effect.

2. Experimental 2.1. Materials and Methods In current work co-precipitation method was applied for synthesis of silver oxide nanoparticles. This method is simple, direct and straight forward method. The detail for the preparation of silver oxide nanoparticles is discussed in detail in the sample preparation. 2.2. Sample preparation Silver oxide nanoparticles were prepared via well-developed co-precipitation method using silver nitrate (1.70g), de-ionized water (10 mL). AgNO3 was mixed with de-ionized water and stirred to have homogeneous solution by magnetic stirrer. Triethylamine (10 mL) included in the solution and stirred for 200 minutes. Centrifugation process of obtained precipitates proceeded at 6500 rpm for 7 to10 minutes. These precipitates are washed with distilled water or ethanol (10 mL). Washed precipitates were dried at 30 C° then placed for 24h in an incubator. Prepared amount of precipitates were placed in an electric oven at 200 C° for 4h than again in furnace at 800 C° for 24h. Furnace can be used to dry particles and also to minimize the size of particles. By controlling the morality ratio of solution, pH, time, temperature and addition of possible seed solution the desired shape and size of silver oxide nanoparticles can be achieved. For cancer cell toxicity the size of developed nanoparticles must be within the range of 100 nm which is less than the pore size of Biological model. 2.3.

Cell Culturing and labelling Conditions

In cell culturing process, HepG2 cell line was cultivated in tissue cultured plastic flasks (Nunc Wiesbaden Germany) in Minimum Essential Medium (MEM) with Hanks salts, supplemented with 10% fetal bovine serum (FBS), 2 mL glutamine and with some nonessential amino acids. Furthermore, for attachment to the substratum properly, the cells were incubated at 37°C for 24h. The cells were also sub cultured for two or three times in a week. After that, the cells were harvested via trypsin 0.25% once reached to the confluence of 75-85 % [6]. HepG2 cells having concentration of 1×105 cells/well were incubated with different concentrations ranging from 0-90 μg/mL of silver oxide nanoparticles.

ACCEPTED MANUSCRIPT

2.4.

Cell viability assay

Cell life is determined by using dye (neutral red assay) [5, 6]. Culture media was replaced after 24h with fresh 200µL MEM as well as 50µL (50 mg/mL) of neutral red assay. In first four columns of 96 well plates poured concentrated neutral red assay and in eleventh and twelfth column remains empty. The 96-well-plates were placed in an incubator for 3h and then media was removed and washed 2-3 times each well with 250μL PBS, and evaluated the neutral red by using a mixture 1:1 of 50vol % ethanol and 1vol % acetic acid. The plate was shaken for 60sec and then left for 15 minutes at room temperature. With the help of micro plate reader absorbance of the dye determined. Independent experiments were repeated at least three times to confirm the data. Quantification of the extracted dye was correlated with the live cell number. The percentage of viable cells in the cell population at each concentration was calculated by using the following formula: 𝑝𝑒𝑟𝑐𝑒𝑛𝑡 𝑐𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 =

𝑃𝑡𝑟𝑒𝑎𝑡𝑒𝑑 ― 𝑃𝑒𝑚𝑝𝑡𝑦 𝑃𝑐𝑜𝑛𝑡𝑟𝑜𝑙 ― 𝑃𝑒𝑚𝑝𝑡𝑦

× 100

(1)

Where Ptreated is the mean absorbance of silver oxide nanoparticles, Pempty is the mean absorbance of empty wells, and Pcontrol is the absorbance of control cells.

3. Results and Discussions The crystal structural properties of the synthesized silver oxide nanoparticles were characterized by X-Ray diffractometer (P’Analytical, XPERT-Pro system) operated at λ= 1.54Å having voltage of 40 kV using CuKα as a radiation source. The topography of the prepared sample was studied by (Jeol JSM-840) scanning electron microscope (SEM) operated at voltage 25 kV. The Energy Dispersive X-ray spectroscopy (EDS) system attached to the SEM (Oxford –x-act Analytical Silicon Drift Detector) was used to study the composition of different elements present in the prepared sample. Lambda 35 spectrometer was used to study the optical absorption spectrum. 3.1. Structural Analysis Fig. 1 showed the crystal structure patterns of the silver oxide nanoparticles synthesized by aqueous (co precipitation) method at 800oC for 24 h. The silver oxide synthesized material showed the cubic crystal structure. It is very clearly seen that x-ray diffraction pattern have a

ACCEPTED MANUSCRIPT

single phase. All the peaks are indexed with computer software GSAS, the Miller indices of the highest intensity peaks labeled with plane [111] at the about diffraction angle 38.51o while the rest of plans revealed in this manner [200], [220], [311] and [222] respectively. It is observed that the highest intensity peaks have a broader width, which means that the formation of the material growth has completed and has a small crystallite size. Moreover, the structural parameters such as lattice constant (a), unit cell volume (Vcell), crystallite size (D) and X-ray density (dx-ray) were calculated from XRD data using the following formulae, respectively.

d=

a

(2)

h2 + k2 + l2

Vcell = a3

(3) 𝐾𝜆

(4)

𝑛.𝑀

(5)

𝐷ℎ𝑘𝑙 = 𝛽ℎ𝑘𝑙cos 𝜃ℎ𝑘𝑙 𝜌 = 𝑎3𝑁

𝐴

Where λ is the wavelength of X-ray XRD (λ=1.54 Å), θ is the Bragg’s diffraction angle, k is the shape factor. Z represents molecules per unit cell of the spinel structure, M is the molecular weight of the sample and NA is the Avogadro’s number (6.02x1023 g/mol) Table 1. The structural Parameters, Miller indices, Diffraction angle, Inter plain distance, Lattice constant a (Ǻ) , volume of the unit cell ( Ǻ3), crystallite size (nm), X-ray density, dx (g/cm3) of synthesized silver oxide nanoparticles at 800 oC / 24 h via aqueous method. The lattice parameter of the synthesized sample are calculated by Equation 2 and listed in the (Table 1) which are in excellent agreement with the earlier reported values for the similar structure. It is observed that the lattice parameter have variation in each planes, overall average value of the samples is found to be 4.0632 Ǻ. In addition, unit cell volume is also calculated using the Equation 3 for all the peaks and the values are listed in Table 1. The crystallite size of all the x-ray diffraction peaks are calculated using Scherrer’s formula and is found to be 64.3 nm as listed in Table 1. In the present study, the observed crystallite size lies in the desired nanometer scale so that the NPs are suitable for biomedical applications.

ACCEPTED MANUSCRIPT

Miller Indices (h,k,l)

[111]

[200]

[220]

[311]

[222]

Diffraction Angle 2θ (Theta)

38.51

44.69

64.78

77.70

81.84

Inter-plain distance (d)

2.337

2.027

1.437

1.228

1.176

Lattice constant, a (Ǻ)

4.048

4.055

4.067

4.072

4.074

66.3493

66.6695

67.2587

67.5443

67.6038

Crystallite size (nm)

41.1

55.9

75.4

81.7

67.4

X-ray Density, dx (g/cm3)

6.00

6.03

6.03

6.03

6.03

Volume of the unit cell ( Ǻ3)

Table1. The structural Parameters, Miller indices, Diffraction angle, Inter plain distance, Lattice constant a (Ǻ) , volume of the unit cell ( Ǻ3), crystallite size (nm), X-ray density, dx (g/cm3) of synthesized silver oxide nanoparticles at 800 oC / 24 h via wet chemical method.

4500

Silver oxide nanoparticles [111]

4000

3500

Intensity(a.u)

3000

2500

[200]

2000

1500

[220]

[311]

1000

[222]

500 [200]

[220]

0 35

40

45

50

55

60

65

70

75

80

Angle2 (Degree)

Fig.1. XRD Analysis of silver oxide nanoparticles

85

ACCEPTED MANUSCRIPT

3.2.

Scanning Electron Microscopy (SEM) Analysis

Fig. 2 shows the scanning electron microscope which is used to examine the morphology and grain size of the investigated silver oxide powders. It is clear from the SEM micrograph that the material has fine grain uniformly distributed. The average grain size lies in the nanoscale range. It is also evident from the micrograph that silver oxide nanoparticles have almost spherical shapes of different sizes.

Fig.2. Morphological analysis for silver oxide nanoparticles prepared at 800 OC / 24 h 3.3.

Elemental Analysis

Fig. 3 showed the EDX (energy dispersive X-ray spectra) of the silver oxide of the prepared sample. The study of this spectrum shows that the formation of the required oxide material after mixed oxides have completed their chemical reaction successively. So, the analysis of obtained results have shown that our desired product is clean from extra bi products in the form of nitrates or other metal oxides , ions. The spectrum shows the obtained ratio of the silver oxide atoms is close to the reported data values [17]. Silver (Ag) is indicated in black color and O in red color with notation in yellow.

ACCEPTED MANUSCRIPT

Fig.3. EDX Spectrum for silver oxide nanoparticles at 800OC / 24 h (Ag is indicated in black color and O in red color) 3.4.

Uv-Vis spectroscopy analysis

Lambda 35 spectrometer was used to study the optical absorption spectrum. Silver oxide nanoparticles sample was dispersed in distilled water to measure the absorption spectrum in the range of 300nm to 1000 nm at room temperature. Calcination effect has studied by optical spectrum of silver oxide nanoparticles as shown in Fig.4. As the colloidal particles reduce in size, blue shift in absorption band is observed. It is clearly seen from the spectrum that the maximum wavelength of absorption (blue shift) seen at 500 nm to 390nm due to the calcination temperature at 800 C° [18]. Graphs has shown the characteristic peak of silver at 400 nm corresponding to resonating oscillations by surface plasmon and inter band transitions correspond to broad band region indicated by the tail of UV region. The spherical shape of particles is implied by the single surface plasmon peak in the graph [19].

ACCEPTED MANUSCRIPT

Fig.4. Uv-Vis spectrum of silver oxide nanoparticles 3.5.

Cellular absorption

In a 96 well flat bottomed microtiter plate, 1 × 105 HepG2 cells were incubated with 0, 20, 30, 40, 50, 60, 70, 80, 90, and 100µg/mL for 0 to 47h. Cellular assimilation of silver oxide nanoparticles were determined by measuring the optical density of 450nm light using microwell-plate reader (AMP PLATOS R-496) after 24h, 26h, 28 h, 45 h and 47h explored in hepatocellular model. Fig.5. (a) shows the snapshot of HepG2 cell line cultured in MEM. The trends in graph rise in absorbance plot were shown in fig. 5.(b), the data recorded between 2426h of time shown increasing trend in cellular uptake collected from 10 µg/mL-30 µg/mL of concentration of nanoparticles and homogeneity in the uptake line were investigated at 40 µg/mL. The plot shows the absorbance vs silver oxide nanoparticles concentration (0-80 microgram/mL). However, significant peak quantity in absorbance was recorded against 50 microgram/mL Silver oxide nanoparticles dispersed solution. Our data match with previous reported work [5]. Then steeply increasing trend in hepatocellular uptake was found above 50 µg/mL concentration. Maximum cellular absorbance is at 50 µg/mL concentration and abruptly decline in absorbance. It is novelty of our work not such kind of trend was reported in previous published data [6]. After 45h- 47h, the optical density of silver oxide nanoparticles in HepG2 cell

ACCEPTED MANUSCRIPT

model shown sudden homogeneous/saturation trend in the absorbance line indicating the maximum absorbance into cells at concentration of 60 µg/mL.

Fig. 5 (a) shows the snapshot of HepG2 cell line cultured in MEM. (b) Shows the cellular uptake of nanoparticles into HepG2 cell line at different time of incubation. 3.6.

Photo toxicity analysis

Fig.6 showed the significant loss in cell viability when cells were labeled with 60 microgram/mL concentration of silver oxide nanoparticles. After this concentration the resistance in cell viability loss was investigated. At optimal concentration (60 µg/mL) of silver oxide nanoparticles about 70% cell viability losses were recorded even in the absence of laser light. In the initial (0-40 µg/mL) cell viability loss was about 50 %. This loss reaches to 70% by using 6070 µg/mL of said solution. Many researcher reported that nonsignificant cell killing pattern of RD labeled with different concentrations of TF-59 were recorded as mentioned before (in case of 5-ALA), this loss in cell viability reaches to almost 30%. Some of the researchers agreed with this opinion that approximately 25% cell death might occur due to mechanical stress/trauma of the shape and configuration of different sizes of nanomaterials e.g. ZnO NRs, TF-200 NPs, TF59 NPs can have ability of cell membrane rupture/trauma [[11], [12], [13],[14],[15],[16],[20]].

ACCEPTED MANUSCRIPT

Loss in Cell Viability

Percent Loss in Cell Viability

100

80

60

40

20

0 0

20

40

60

80

Ag2O NPs Concentrations

Fig.6. Percent cell viability loss in HepG2 Cell viability 3.7.

Reactive oxygen species Analysis

After optimized dose of silver oxide nanoparticles concentration (from 0 µg/mL to 80 µg/mL) concentration, HepG2 Cells were incubated with CMH2DCFDA for tracing the actual cells killing mechanism via oxidative stress or reactive oxygen species. Significant ROS fluorescence was depicted in Fig.8. Similar kind of study was already reported by F.shaheen et al. and S.Iqbal et al [[21],[22]] for detection ROS liberation in term of nanomaterial’s exposed and their absence. The marvelous trend of rising fluorescence was found from 1000 a.u to 5200 a.u when the silver oxide nanoparticles concentration increases from 0 to 80 µg/mL. Results depicted the excellent from of pattern with previous published data [[23], [24], [25], [26]. The results implement that after certain concentration of silver oxide nanoparticles depicts very toxic might be helpful for biomedical and clinical applications. In addition ROS fluorescence beard treated HepG2 cells revealed significant reactive oxygen fluorescence and necrosis form of treated HepG2 cells as when in ROS micrograph Fig. 7, where (a) shows ROS Fluorescence and (b)

ACCEPTED MANUSCRIPT

shows Necrosed cells treated with silver oxide nanoparticle having concentration of 60 microgram/mL[[27],[28]].

(b)

Fig.7. (ROS micrograph (a) ROS Fluorescence (b) Necrosed cells treated with silver oxide nanoparticle (60 microgram/mL)

6000

ROS Fluorescence vs silver oxide nanoparticles Conc.

ROS Fluorescence (a.u)

5000

4000

3000

2000

1000

0 0

10

20

30

40

50

60

70

80

90

silver oxide nanoparticles Concentrations (mg/mL)

Fig.8. ROS Fluorescence (a.u) for silver oxide nanoparticles exposed in vitro HepG2 Model.

ACCEPTED MANUSCRIPT

3.8.

Statistical Analysis

Fig.9 was the verification of our experimental results and data by applying statistical regression analysis using polynomial fit, coefficient of determination and p value. With respect to concentration of silver oxide nanoparticles into HepG2 cell line, the significant percentage of cell viability loss has been estimated by this curve fit. R2 is the coefficient of determination that measures the proportion of variability in y (response) due to change in x (input variable). The proposed model of cell viability is close to the exponential decay with some unknown variables as described in equation (6). y = Intercept + B1*x^1 + B2*x^2

(6)

The values of unknown are extracted from the method of polynomial fit using origin Pro 9.0 fitting tools and given as follows. Intercept = 99.2473, B1 = -2.07922, B2 = 0.0155

(7) (8) (9)

Loss in Cell Viability

Percent Loss in Cell Viability

100

Polynomial Fit of B

80

Model

Polynomial

Equation

y = Intercept + B1*x^1 + B2*x^2

Weight

Instrumental 30.36764

Residual Sum of Squares

0.98003

Adj. R-Square

Value

60

Intercept B

Standard Erro

99.2473

1.24377

B1

-2.07922

0.07749

B2

0.0155

9.55981E-4

40

20

0 0

20

40

60

80

Silver oxide nanoparticles Concentrations (g/mL)

Fig.9. Statistical Analysis of the experimental verification

ACCEPTED MANUSCRIPT

Where, y is percent cell viability loss and is dependent variable, x is concentration of nanoparticles and is independent variable, B1 and B2 are the statistical constants found in model fit. The adjusted R2 is found 0.98003 which is goodness of fit for our estimated model and experimental data and the significance of applied model is found via ANOVA which is found to have p value 0.001. The graph and the equation show that the loss in % cell viability increases with the increase in nanoparticles concentration and the decay rate is measured. It means that the live cell quantity is decreasing with the increase in amount of nanoparticle in cell line in the current study [[22],[23],[24]]. 4. Conclusion Silver oxide nanoparticles were successfully synthesized by applying Chemical Aqueous method. The synthesized nanoparticles were characterized by applying XRD, SEM, EDS, and UV-Vis analysis. Some optimized parameters helped to analyze the correct physiochemical response of silver oxide nanoparticles, like 47h of time of incubation were selected as suitable uptake time and 60µg/mL were supposed to be threshold value of silver oxide nanoparticles concentration because cell viability loss about 70 % were assessed with given optimized parameters. A polynomial fit was applied to confirm the goodness of fit of the experimental results. It is concluded that silver oxide nanoparticles shows promising anticancer agent (nano medicinal/nano therapeutic effect) due to its localized drug characteristics at required site.

Acknowledgments The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the Research Group Project No. RG-1439-293.

ACCEPTED MANUSCRIPT

References [1]

R Jain, M. Seder-Colomina, N. Jordan, P. Dessi, P, I. Cosmidis, E. D. van Hullebusch, S.Weiss, F. Farges, P.N. Lens Journal of hazardous materials., 295(2015),pp.193-200.

[2]

A.Diallo, B.D. Ngom, E. Park, M. Maaza Journal of Alloys and Compounds., 646 (2015), pp.425-430.

[3]

H. Bouwmeester, S. Dekkers, M.Y. Noordam S, W.I. Hagens , A.S. Bulder ,C. de Heer C, S.E.C.G. ten Voorde ,S.W.P. Wijnhoven , H.J.P. Marvin, J.A. Sips M., Regul. Toxico. and Pharma., 53 (2009), pp. 52-62.

[4]

M. Murphy,K. Ting , X. Zhang, C. Soo, Z. Zheng Journal of Nanomaterials.,2015 (2015), p.12

[5]

M. Atif ,M. F. Alam , S. Firdous ,S. S. Z. Zaidi , R. Suleman R, M. Ikram Laser Phys. Lett., 7 (2010), pp.757-764.

[6]

M. fakhar e Alam, M.Atif, M.S Alsalhi , M.Siddique, K. Sultana.,M.I. Qadir ,M. Willander Laser Phys.,21 (2011), pp. 972-982.

[7]

S. Zhang , H.Gao, G. Bao ACS Nano., 9(2015), pp. 8655-8671

[8]

D. H.Jo, J. H. Kim ,T.G. Lee ,J.G. Kim Nanotech., Biolog. and Med., 11 (2015), pp. 1603-11

[9]

K. B. Narayanan, H. H. Park , S.S.Han Chemosphere., 141 ( 2015), pp. 169-75

[10]

A.M.

Schrand,

L.

K.Braydich-Stolle.

J.J.Schlager,

L.Dai,

S.M.Husaain

Nanotechnology.,19 (2008), 235104 , pp. 13 [11]

A.M.Atta , G.A. El-Mahdy ,H.A. Al-Lohedan ,A.R.O. Ezzat Molecules., 20 (2015), pp. 11131-11153

[12]

J.Kang ,Y. Li , Y. Chen ,A. Wang, B.Yue.,Y. Qu ,Y. Zhao, H.Chu Mater. Res. Bull., 71 ( 2015), pp.116-121

[13]

J.L.West,N.J. Halas Current Opinion in Biotechnology., 11 (2000),pp.215-217

[14]

E. M. Renehan , F.K. Enneking , M. Varshney, R. Partch, D. M. Dennis ,T.E. Morey Reg. Anesth. and Pain Med., 30 (2005),pp. 380-384

[15]

A. S. Singh,S. S. Shendage, J. Nagarkar M.,.Tetrahed. Lett., 55(2014), pp.6319-6323

[16]

M. Fatima,M. Fakhar e Alam, M. Atif,M. N. Shakoor , M. Afzal, M. W. Waseem, M. H.Aziz, Laser Phys., 24 (2014) 125602,pp.5

ACCEPTED MANUSCRIPT

[17]

J.Baharara , F.Namvar ,T. Ramezani, N.Hosseini , R.Mohamad Molecule., 19 (2014), pp. 4624-4634

[18]

L. Gharibshahi, E.Saion , E.Gharibshahi , A.H.Shaari , M.K. Amin. Materials., 10(2017) pp.402

[19]

N. Haram, N. Ahmad J Clust Sci., 2015(26), pp. 713–725

[20]

M. N. Shakoor ,M.Fakhar e Alam ,N. Abbas, U.Tariq , A.U.Ahmad ,N. Amin ,M.H. Aziz, M. Atif M.,W.A. Farooq J. of Nanoelect. and Optoelect., 11 (2016), pp. 339-341

[21]

F. Shaheen, M. H. Aziz, M. Fakhar-e-Alam, M. Atif, M. Fatima, R. Ahmad, A. Hanif, S. Anwar, F. Zafar, G. Abbas, S.M. Ali, M. A Ahmed Nanomaterials., 7 (2017), pp. 1-18

[22] S. Iqbal, M. Fakhar-e-Alam, M .Atif, N. Ahmed,N. Amin,A. Hanif, W.A.Farooq Micromachines., 2019(10), pp.60 [23]

M. W. Akram, M. Fakhar-e-Alam, M. Atif ,A.R. Butt ,A. Asghar,Y. Jamil ,K.S. Alimgeer, Z.M. Wang Scientific Reports., 8 (2018), pp. 4576

[24]

M. Atif, A. R. Malik, M. Fakhar-e-Alam, S. S. Hayat, S. S. Z. Zaidi, R. Suleman, and M. Ikram Laser Physics 22 (2012), pp. 286-293.

[25]

M. Atif, S. Firdous, R. Mahmood, M. Fakhar-e-Alam, S. S. Z. Zaidi, R. Suleman, M. Ikram, and M. Nawaz, Laser Physics 21(2011), pp. 1235-42.

[26]

M. Atif, S. Firdous, A. Khurshid, L. Noreen, S.S.Z. Zaidi and M. Ikram Laser Physics Letters 6(2009), pp. 886-891

[27]

S. Iqbal, M. Fakhar-e-Alam, N. Amin, M.Ismail , G. Mustafa, M. Raza Ahmad, M. Atif Int. J. Mater. Res. IJMR_MK111759 – 26.2.19/reemers., pp. 1-3

[28]

M. Atif, M. Fakhar-e-Alam, N.Abbas, M.A. Siddiqui, A.A. Ansari, A.A. Al-Khedhairy, Z.M. Wang Journal Of Nanomaterials., (2016) Article ID 7691861, pp. 6

ACCEPTED MANUSCRIPT

Highlights 

Silver oxide nanoparticles were synthesized by chemical aqueous method



Invitro Cytotoxicity and ROS Activity under dark and light exposure



70 % cell viability loss at 60µg/mL



Polynomial fit confirmed experimental results.