Anticancer, antimicrobial and photocatalytic activities of green synthesized magnesium oxide nanoparticles (MgONPs) using aqueous extract of Sargassum wightii

Accepted Manuscript Anticancer, antimicrobial and photocatalytic activities of green synthesized magnesium oxide nanoparticles (MgONPs) using aqueous extract of Sargassum wightii

Arivalagan Pugazhendhi, Raju Prabhu, Kavitha Muruganantham, Rajasree Shanmuganathan, Suganthy Natarajan PII: DOI: Reference:

S1011-1344(18)31209-0 https://doi.org/10.1016/j.jphotobiol.2018.11.014 JPB 11404

To appear in:

Journal of Photochemistry & Photobiology, B: Biology

Received date: Revised date: Accepted date:

25 October 2018 21 November 2018 21 November 2018

Please cite this article as: Arivalagan Pugazhendhi, Raju Prabhu, Kavitha Muruganantham, Rajasree Shanmuganathan, Suganthy Natarajan , Anticancer, antimicrobial and photocatalytic activities of green synthesized magnesium oxide nanoparticles (MgONPs) using aqueous extract of Sargassum wightii. Jpb (2018), https://doi.org/10.1016/ j.jphotobiol.2018.11.014

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ACCEPTED MANUSCRIPT Anticancer, antimicrobial and photocatalytic activities of green synthesized Magnesium oxide nanoparticles (MgONPs) using aqueous extract of Sargassum wightii Arivalagan

Pugazhendhia,*

Murugananthamb,

[email protected], Shanmuganathanc,

Rajasree

Raju

Prabhub,

Suganthy

Kavitha

Natarajanb,*

[email protected] a

Innovative Green Product Synthesis and Renewable Environment Development Research

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Group, Faculty of Environment and Labour Safety, Ton Duc Thang University, Ho Chi Minh

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City, Vietnam. b

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Department of Nanoscience and Technology, Alagappa University, Karaikudi, Tamil Nadu,

India

*Corresponding

authors

at:

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Institute of Research and Development, Duy Tan University, Da Nang 550000, Vietnam Department

University, Karaikudi, Tamil Nadu, India Abstract

of Nanoscience

and

Technology,

Alagappa

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c

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A rapid and ecofriendly fabrication of metal oxide nanoparticles using biogenic sources is the

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current trend being used to replace the toxic chemical method. The present study was carried out to synthesize magnesium oxide nanoparticles (MgONPs) using the marine brown algae Sargassum wighitii as the reducing and capping agent. The as-prepared MgONPs were

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characterized by spectroscopic and microscopic analyses. UV–visible spectrum of the MgONPs showed a sharp absorption peak at 322 nm. X- ray diffraction analysis illustrated that the

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MgONPs were crystalline in nature with a face-centered cubic structure. Presence of magnesium and oxygen were further confirmed by EDX profile. FTIR analysis showed the presence of

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functional groups specific for sulfated polysaccharides, which might be responsible for the synthesis of MgONPs. Zeta potential and dynamic light scattering analysis illustrated that the MgONPs were highly stable at 19.8 mV with an average size of 68.06 nm. MgONPs showed potent antibacterial activity and antifungal activities against human pathogens. Photocatalytic activity of MgONPs was witnessed by the quick degradation of the organic dye methylene blue on exposure to both sunlight and UV irradiation. MgONPs showed significant cytotoxicity against the lung cancer cell lines A549 in a dose dependent manner with the IC50 value of 37.5 ± 0.34 μg/mL. Safety evaluation using peripheral blood mononuclear cells (PBMCs) illustrated the 1

ACCEPTED MANUSCRIPT MgONPs to be non-toxic in nature. Overall, the results concluded that the MgONPs generated using marine algae have exhibited scope for multifaceted biological applications. Keywords Sargassum

wightii

MgONPs Antimicrobial

activity

cell

lines

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A549

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Cytotoxicity

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Photocatalytic activity. 1. Introduction

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Nanotechnology has emerged as a promising multidisciplinary field of 21st century with wide spread applications in the fields of biotechnology, medicine, energy science and material

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science etc. [1-3]. Nanomaterials are widely synthesized using various physical and chemical methods, which require high temperature, vacuum conditions, sophisticated instruments and additives.

Current

advancements

in

chemical methods

for

the

synthesis

of

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chemical

nanomaterials have increased the biological risks to the environment due to the usage of toxic

currently

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chemicals, which remain adhered to the synthesized nanomaterials [4, 5]. Hence, researchers are focusing on the synthesis of nanomaterials using biogenic sources such as

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microorganisms, algae and plants. Recent progress in the green synthesis of nanomaterials due to its rapidity, cost effectiveness and ecofriendly nature has opened a new era for safe

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nanobiotechnology [6, 7]. Metal oxide nanoparticles have gained much interest nowadays due to their exclusive physical and chemical properties in the fields of biosensors, as diagnostic tools,

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catalysts, anticancer and antimicrobial agents [8, 9]. Among the metal oxide nanoparticles, magnesium oxide nanoparticles (MgONPs) have been used for the treatment of various ailments due to their biocompatible nature and remarkable stability under harsh conditions [10, 11]. MgONPs have been used as catalysts, additives in refractories, superconducting products, paints and also as substantial materials in bioremediation [12, 13]. In the field of medicine, MgONPs exhibit remarkable applications such as relief of heart burns, regeneration of bones and as antitumour and antibacterial agents [14, 15].

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ACCEPTED MANUSCRIPT Biogenic synthesis of nanoparticles using seaweeds/marine algae has become prevalent recently due to easy access and efficacy. Rich source of polysaccharides (alginate, laminaran, fucoidan), polyphenols, carotenoids, proteins, amino acids, vitamins and minerals

present in the

algal cell wall act as reducing and capping agents for the fabrication of metal and metal oxide

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nanoparticles under ambient conditions [16, 17]. Sargassum wightii, marine brown algae

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(Phaeophyta) belonging to the family sargassaceae are widely found in the south east coastal

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region of TamilNadu. Presence of secondary metabolites such as polyphenols, isoprenoids (phytol and methyl salicylate), polysaccharides (fucoidans, alginate, laminaran), β carotene and

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 tocopherol makes this seaweed a suitable biofactory for the production of metal and metal

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oxide nanoparticles [16, 18, 19]. Hydroxyl, carboxyl and amino functional groups present in the phytoconstituent serve as both metal-reducing and capping agent providing robust coating on the

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metal NPs in one step synthesis. Literature survey revealed that only few metal oxide

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nanoparticles such as AgNPs and ZnONPs were synthesized using brown seaweed S. wightii [20,

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21] which prompted us to synthesize MgONPs using aqueous extract of Sargasssum wightii as the reducing and capping agent. The as prepared MgONPs were evaluated for their

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antimicrobial, anticancer and dye degradation abilities.

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2. Materials and methods 2.1 Chemicals used

Magnesium nitrate (Mg(NO 3 )2 . 6H2 O), absolute ethanol, sodium hydroxide were purchased from Sisco laboratories, India. Nutrient agar, nutrient broth, Muller Hinton agar, sterile

discs,

Dulbecco's

3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium

Modified

Eagle's

Bromide

medium (DMEM) medium were obtained

laboratories, India. 2.2 Synthesis of MgONPs using aqueous extract of S. wightii 3

(MTT)

and

from Himedia

ACCEPTED MANUSCRIPT Marine brown algae S. wightii was collected from the South Indian coastal area, Tamilnadu during low tide season (June) and the species was identified based on the references of Oza and Zaidi [22]. Seaweeds were collected and subjected to aqueous extraction at 80ºC for 30 min. The extract was filtered with Whatman No.1 filter and the filtrate was stored at 4 ºC. Aqueous extract of seaweed and Mg (NO 3 )2 solution were taken in the ratio 9:1, subjected to continuous stirring for 6 h at 90 ºC and the solution was allowed to stand overnight at room

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temperature. The color transition from yellow to yellowish brown indicated the formation of

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Magnesium oxide nanoparticles (MgONPs). Further, the calcination was done in muffle furnace

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at 500 °C for 3 h.

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2.3 Characterization of MgONPs

Absorption spectrum of the synthesized MgONPs was obtained using a UV-visible

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spectrophotometer (UV 2450, Shimadzu, Kanagawa, Japan) between 200 to 800 nm at the resolution of 1nm. X-ray Diffraction (XRD) analysis was performed with X’ Pert PRO

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Analytical X6 ray diffractometer employing Cu Kα radiation at 40 kV and 30 mA (PANalytical, Netherlands). Bioactive functional groups present in the aqueous extract of S. wightii responsible

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for synthesis of MgONPs were analyzed using a FTIR-Nicolet Thermo spectrophotometer iS5 (USA) instrument in the wave number range of 4000 and 400 cm-1 with a resolution of 4 cm-1 .

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Surface morphology of MgONPs was assessed using Field Emission Scanning Electron Microscopy (FESEM) (Carl Zeiss,

Germany) equipped

with Energy Dispersive X-ray

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spectroscopy (EDX-JEOL, JSM- 5610, Germany). Size and stability of synthesized MgONPs were assessed by dynamic light scattering (DLS) and zeta potential (ZP) analyzer (Malvern

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Zetasizer nano-ZS90, UK).

2.4 Antimicrobial activity of MgONPs Antibacerial activity of MgONPs was assessed against Gram positive ( Staphylococcus aureus (MTCC 96), Escherichia coli (MTCC 40) and Proteus mirabilis (MTCC 7299)) and Gram negative bacterial strains

(Serratia marcescens (MTCC 97), Salmonella typhimurium

(MTCC 3224) and Pseudomonas aeruginosa (MTCC 2642)) based on Kirby–Bauer disk diffusion method [23] with ampicillin (10 μg), kanamycin (10 μg) and gentamycin (10 μg) as 4

ACCEPTED MANUSCRIPT positive control. Sterile discs (6mm diameter) loaded with various concentrations of MgONPs (10-30 µg/mL) were placed on the culture plates and incubated at 37 °C overnight and the zone of inhibition were measured and recorded in mm. Antifungal activity of MgONP (10-30 g/ml) against fungal strains such as Aspergillus niger, Chaetomium sp., Nigrospora oryzae and Fusarium solani were assessed by well-cut diffusion method in comparison with fluconazole as positive control. Minimum inhibitory concentrations (MIC) of MgONPs against Methicillin-

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resistant Staphylococcus aureus (MRSA) and P. aeruginosa were evaluated by microdilution method in Mueller Hinton broth (MHB, HiMedia, India) with gentamycin and vancomycin as

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positive control for P. aeuroginosa and MRSA strains, respectively. MBC is defined as the lowest concentration of the test compound in which no viable bacterial colonies are observed.

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MBC of the MgONPs was determined by plating aliquots of tubes with no visible growth and the first turbid tube in MIC series. Aliquots of treated samples were placed on nutrient agar plates

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and uniformly spread using a sterile L rod and incubated overnight at 37 ºC.

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2.5 Anticancer potential of MgONPs

Human lung cancer cell line A549 was purchased from National Centre for Cell Science

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(NCCS, Pune, India) and the cells were subculture in Dulbecco’s modified Eagle medium (DMEM) (Invitrogen, USA) containing 10 % Fetal Bovine Serum and 1X antibiotic solution

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(10,000 units Penicillin and 10 µg/mL Streptomycin) in a humidified atmosphere with 5 % CO 2 at 37 ºC. Antiproliferative effect of MgONPs on A549 cells was assessed by MTT assay based

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on mitochondrial dehydrogenase activity [24]. Morphological changes in the control and treated cells were visualized using phase contrast microscopy (Nikon ECLIPSE, Ti-E, Japan). In

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addition cell membrane damage and loss of membrane integrity was assessed by lactate dehydrogenase leakage assay [25].

Intracellular reactive oxygen species (ROS) level was

assessed quantitatively and qualitatively using the fluorescent probe DCFH-DA based on the methodology of Laurite et al. [26] using Nikon ECLIPSE fluorescence microscopy (Ti-E, Japan) and microplate reader (Molecular Device Spectramax M3, equipped with Softmax Pro V5 5.4.1 software) at an excitation wavelength of 485 nm and emission wavelength of 535 nm, respectively. Changes in the mitochondrial membrane potential (ΔΨm) upon treatment with MgONPs were analyzed using Rhodamine 123 staining based on methodology of [27]. 5

ACCEPTED MANUSCRIPT Apoptotic effect of MgONPs in A549 cells was assessed based on AO/EtBr dual staining technique. Cells were visualized immediately under Nikon ECLIPSE fluorescence microscope (Ti-E, Japan) with the excitation wavelength of 502 nm and the emission was captured at 525 nm. The cells with condensed or fragmented nuclei were reckoned as apoptotic cells and the experiments were carried out in triplicates, counting about 100 stained cells in 10 randomly chosen fields. Safety evaluation of MgONPs was assessed in peripheral blood mononuclear cells based on

MTT assay [28], one of the simplest and quickest method to evaluate

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(PBMC)

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cytotoxicity based on cell membrane integrity.

2.6. Catalytic activity of MgONPs

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The catalytic activity of MgONPs was assessed based on their ability to degrade the dye methylene blue (MB) in presence of visible and UV radiations. Mercury vapor lamp (120W) was

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used as the UV source. Stock solution (10 mg/L) of methylene blue was prepared. About 10 mg of MgONPs was added to 100 mL of dye solution and subjected to constant stirring in dark for

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half an hour to promote the equilibrium between MB and photocatalyst before exposure to sunlight and UV irradiation. The reaction mixture was exposed to light source and about 2 mL of

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the suspensions were withdrawn at selected time intervals (every 30 min) and suspended particles were removed by ultracentrifugation. The rate of dye degradation was determined using

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the absorbance measured in a UV-visible spectrophotometer (U-2800, Hitachi, Japan) at 664 nm.

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The dye degradation percentage was assessed based on the formula % of degredatio n 

Ci  C f Ci

X 100

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where, Ci and Cf were the initial and final concentrations of dye at time interval “t”respectively. 2.7 Statistical analysis Experiments are carried out in triplicates and the results were expressed as Mean ± SD. One-way ANNOVA followed by Duncan’s multiple range tests using SPSS 17.0 software package were used to assess the statistically significant differences between the treated and control samples. p < 0.05, p < 0.001 were regarded as statistically significant. 3. Results and discussion 3.1 Characterization studies 6

ACCEPTED MANUSCRIPT 3.1.1 UV-visible spectroscopy analysis of MgONPs Surface Plasmon resonance (SPR) band of the synthesized metal nanoparticles depends on the shape, size and distribution of the nanoparticles in colloidal solution [29]. In UV-visible spectroscopy, single SPR band formation at short wavelengths below 300 nm indicates the presence of small sized particles while a band at longer wavelengths indicates the presence of anisotropic nanoparticles [30]. In the present study, the formation of MgONPs was confirmed by

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color transition followed by UV-visible absorption peak. Addition of aqueous extract of S.

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wightii to Mg(NO 3 )2 solution induced color transition from pale yellow to dark brown indicating

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the phycoreduction of Mg(NO 3 )2 to MgONPs. A sharp absorbance peak at 322 nm indicated the formation of small sized MgONPs (Fig. 1A) as reported by 31, 32]. The dynamic absorption

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peak within 200-250 nm indicated the existence of various bioactive compounds such as polyphenols and alginates, which might have been responsible for the reduction of Mg(NO 3 )2 to

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MgONPs.

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3.1.2 Fourier transform infrared spectroscopy (FTIR) analysis of MgONPs FTIR analysis was carried out to identify the bioactive compounds in the aqueous extract

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of S. wightii, which were responsible for the reduction of Mg(NO 3 )2 to MgONPs. Figure 1B shows the MgONPs green synthesized using aqueous extract of S. wightii. The absorption peak

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between 660 and 540 cm-1 indicated Mg-O bond stretching in magnesium hydroxide. The absorption peaks at 660 and 875 cm-1 corresponded to =C–H and N-H bending vibrations. A

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broad absorption peak observed between 1100 and 1000 cm−1 was resultant of the C–H group bending vibrations or C–O or C–C group stretching vibrations of the carbohydrates [33] present in the aqueous extract of S. wightii. Presence of a strong and broad absorption peak between

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3300 to 3500 cm−1 was due to the stretching vibrations of O–H and N–H groups of the amino acids present in the seaweed extract [34]. The absorption peaks at 1481 and 1015 cm-1 corresponded to O-H bending and C-O stretching of the groups in saturated primary alcohol. Peak at 2916 cm-1 could be assigned to C-H stretching of aromatic groups. Moderate absorption peak at 1273 cm−1 implied C–N stretching of chlorophyll molecules. FTIR results suggested that the polysaccharides and pigments present in the seaweed extract might have been involved in the formation of MgONPs from Mg(NO 3 )2 as the reductant and capping agent. 7

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3.1.3 X-ray diffraction (XRD) analysis of MgONPs Cystalline nature of MgONPs was established by X-Ray diffraction (XRD) patterning. The four distinct diffraction peaks at 36.92°, 42.92°, 62.27°, 74.66°, and 78.61° corresponding to the planes (111), (200), (220), (311) and (222) respectively (Fig. 1C) pointed out that the synthesized MgONPs were face centered cubic (FCC) structure and crystalline in nature (JCPDS

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file no. 39-7746). The mean particle size of MgONPs was found to be 43 nm, which was

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calculated based on the width of sharp intense peak corresponding to plane (200) located at

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42.92° using the Debye-Scherrer’s formula. Similar XRD results were reported for MgONPs

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synthesized using other biogenic sources [35].

3.1.4 Scanning electron microscopic (SEM) analysis of MgONPs

magnifications (X4000 and X 10,000).

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Figure. 2A illustrates the SEM images of the phycosynthesised MgONPs at different SEM images showed flower shaped structure and the

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agglomeration observed might be due to the electrostatic attraction of MgONPs. EDX analysis showed strong signals corresponding to the elements Mg and O confirming the formation of

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MgONPs (Fig. 2B). Presence of weak signals for carbon indicated the presence of biomolecules

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bound to the surface of the MgONPs.

3.1.5 Particle size and surface charge analysis of MgONPs

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Size and dispersion ability of the synthesized MgONPs were assessed using dynamic light scattering (DLS) analyzer [36]. The mean size of MgONPs was observed to be 68.06 nm

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(100%) with PDI (Poly dispersity index) of 1.00 and the single peak obtained indicated that the quality of synthesized MgONPs was good (Fig. 2C). Zeta potential (ZP) measurement is based on the movement of nanoparticles under the influence of electric field based on charge and environment. Nanoparticles with high negative or positive ZP never aggregate due to electrostatic force of repulsion while particles with low zeta potential tend to flocculate [37]. In the present study, MgONPs possessed a high negative potential value of -19.8 mv, which indicated that the MgONPs synthesized were highly stable and prevented agglomeration due to strong electrostatic repulsive forces between them. 8

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3.2 Antimicrobial activity of MgONPs 3.2.1 Bactericidal activity The bactericidal activity of MgONPs was assessed by Kirby–Bauer test. Results illustrated that the MgONPs synthesized from brown algae showed potent bactericidal activity against gram positive and negative bacterial strains in a dose dependent manner (Figure 3 and

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Table 1). MgONPs showed potent antibacterial activity against gram positive bacterial strains

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such as Streptococcus pneumonia, MRSA 11, MRSA 56 and gram negative bacterial strains

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including E. coli, Pseudomonas aeruginosa and Aeromonas baumannii when compared to positive control. Among the microbial strains tested, MgONPs exhibited the highest inhibitory

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activity against MRSA 56 and P. aeruginosa with zones of inhibition 9 and 8 mm, respectively at the concentration of 30 µg/mL. The antibacterial efficacy of MgONPs might be due to their

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exhibit potent bactericidal efficiency [38].

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smaller sizes, which was consistent with the report that MgONPs with smaller sizes would

3.2.2 Determination of bacteriostatic and bactericidal concentrations of MgONPs against

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MRSA and P. aeruginosa

Bacteriostatic and bactericidal concentrations of MgONPs were assessed by broth

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dilution method. MIC is the minimum dose of test compound required to inhibit the visible growth of microbes in 24 h [39]. Figure. 4 illustrate the significant (p0.05) reduction in

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bacterial cell viability with an increase in the concentration of MgONPs (4-2048 g/mL). MIC for MRSA 56 and P. aeruoginosa was 256 g/mL (Fig. 4). MBC is the minimum concentration

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of the test compound required to kill the bacterium completely within a fixed period of time under specific conditions [40]. MBC for MRSA and P. aeruoginosa was observed at 256 and 1024 g/mL of MgONPs, respectively (Table 2). Antibacterial activity of MgONPs might have been due to i) spontaneous release of free radicals such as ROS and RNS, inducing oxidative stress mediated cell damage [41], ii) cell membrane damage caused by the electrochemical mode of interaction between the Mg2+ ions with the phosphate group in the LPS, thereby disrupting cell membrane integrity and causing membrane leakage

[35, 42]. In the case of gram positive

bacteria, the thin layer of peptidoglycan with abundant pores might have allowed the penetration 9

ACCEPTED MANUSCRIPT of MgONPs into the cell resulting in membrane damage, cell content release and ultimately leading to cell death [43, 44]. MgONPs were examined for antifungal activity against Aspergillus fumigates, Fusarium solani and Aspergillus niger at three different concentrations (10-30 g/mL). Results in fig. 5 illustrate that MgONPs showed potent antifungal activity when compared to positive control (Fluconozole). Among the three strains, MgONPs inhibited the growth of Fusarium solani and

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Aspergillus niger more effectively when compared to Aspergillus fumigates. The antifungal activity of MgONPs could be due to the electrostatic interaction between the phosphate group in

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the cell membrane and Mg2+, penetration of MgONPs into the cell, followed by binding of Mg2+ with the thiol group of protein leading to denaturation. Moreover, MgONPs might also have

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induced cellular death through ROS mediated oxidative stress [42, 45, 46].

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3.3 Catalytic activity of MgONPs

Catalytic activity of MgONPs was evaluated based on the rate of degradation of organic

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dye pollutant (methylene blue -MB) widely used in dye industry. Rate of MB degradation was calculated as the percentage of decolorization with respect to time based on the absorbance at the

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optimum wavelength of 664 nm. Figure. 5B illustrates the percentage of MB degradation by MgONPs in the presence of both UV and visible lights. Results illustrated that MgONPS

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exhibited potent catalytic activity both under UV and visible lights when compared to control. The as-prepared MgONPs exhibited approximately 8.2 times higher activity than blank in the

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presence of sunlight.

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3.4 Anticancer potential of MgONPs Lung cancer is the most prevalent form of carcinoma incident in both the sexes and it is reported as leading cause for cancer related mortality globally. Epidemiological studies illustrated that among all the cancer types, 12.4% of new cases and 17.6% of total cancer deaths are due to lung cancer [47]. Non-small lung cancer (NSCLC) is the major contributor of total lung cancer, which is further classified into adenocarcinoma (35%), squamous cell carcinoma (20%) and large cell carcinoma (2.9%) [48]. A549 cells (adenocarcinomic human alveolar basal epithelial cells) are used prevalently as in vitro model systems for the study of NSCLC. 10

ACCEPTED MANUSCRIPT Environmental and life style factors such as smoking, lack of physical activity, alcohol, air pollution, diet and occupational exposure were considered as the major risk factors leading to lung cancer [49]. Increase in mortality in lung cancer is due to lack of diagnostics and effective therapeutic strategy. Based on the cancer stages, surgery, chemotherapy and radiation therapy alone or in combination are being used

for lung cancer treatment. FDA approved

chemotherapeutic drugs such as gemcitabine in combination with cisplatin have been used as Currently combinatorial drug therapy i.e cisplatin with

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primary drug for lung cancer therapy.

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drugs such as paclitaxel, docetaxel and gemcitabine have exhibited enhanced therapeutic

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efficiency [50, 51]. Majority of chemotherapeutic drugs are expensive with severe side effects such as neuronal damage, irritation to skin and severe ache; hence, there is an urgent need for

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nontoxic, ecofriendly, cost effective and targeted drugs for the treatment of cancer. Nanoparticles with size less than 100 nm possessing unique physical and chemical properties offer exceptional

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interactions with nucleic acids, proteins and lipids present on the cell surfaces and inside the body cells, which might open up new ways for cancer diagnosis and treatment [52]. Recently,

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various metal oxide nanoparticles such as silver and gold nanoparticles have been reported to be useful in anticancer therapy against several types of cancer [53]. In the present study, the

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anticancer potential of MgONPs synthesized through the biogenic route using seaweeds was

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investigated against lung cancer cell lines (A549).

3.5 Antiprolife rative effect of MgONPs

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In vitro cytotoxic effect of MgONPs on lung cancer cell lines (A549) was assessed by MTT assay and LDH leakage assay. From fig. 6A, it is clear that the MgONPs showed potent

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cytotoxic effects against A549 in a dose dependent manner with an IC50 value of 37.5 ± 0.34 μg/ml in comparison with the positive control cisplatin (IC50 value of 25.4 ± 0.024 μg/mL). Phase contrast microscopic analysis was carried out to assess the changes in cell morphology and membrane damage. Results showed a typical epithelial morphology with high density cell population in control group whereas shrinkage and rounding of cells, chromatin condensation, membrane blabbing and apoptotic body formation with reduced cell population were observed in MgONPs and positive control treated cells (Fig. 6B). Morphological changes might be due to the activation of caspase cascade wherein the substrate poly (ADP-ribose) polymerase (PARP) 11

ACCEPTED MANUSCRIPT required for DNA repair mechanism would be cleaved [54]. Cellular uptake of nanoparticles by endocytosis or macropinocytosis would induce the generation of ROS and activate the apoptotic pathway ultimately leading to cell death [55]. Lactate dehydrogenase (LDH) assay is the most widely used method to assess the tissue or cellular damage based on the LDH level in the extracellular medium. LDH is a soluble cytoplasmic enzyme, which gets released into the extracellular medium on cell membrane

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damage; hence, measurement of LDH is considered as an indicator for cellular toxicity [56]. The

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cytotoxic effect of the MgONPs was further substantiated by the LDH leakage assay. Results

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showed a concentration dependent increase in the percentage of LDH leakage in MgONPs treated cells, exemplifying cell death (Fig. 7 A). The above results were in accordance with the

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report that MgONPs treatment would permeabilize the cell wall allowing the leakage of LDH in

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lung cancer cells, leading to cell death [57].

3.6 MgONPs induced apoptosis via ROS mediated DNA damage

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Chemotherapeutic drugs and radiation therapy induce cancer cell death by activating the ROS mediated apoptotic pathway. Increase in ROS level leads to several pathological events

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such as inflammation, DNA damage, lipid peroxidation and protein oxidation. ROS generation has been proposed to be a key player in various cellular events such as inflammation, senescence

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mutation, DNA damage and apoptosis [58]. In the present study, the intracellular ROS level was assessed in A549 cells to evaluate the mechanism behind the anticancer potential of MgONPs.

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Spectrofluorometric results showed a two-fold increase in fluorescence intensity in the MgONPs treated group when compared to the control group (Fig. 7C). Results were further substantiated

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by fluorescent microscopic analysis, which showed an enhanced green fluorescence intensity in the MgONPs treated groups when compared to the control and the positive control treated cells, representing an increased ROS level (Fig. 7D). These results confirmed that the MgONPs induced apoptosis was associated with the ROS accumulation in A549 cells.

3.7 Assessment of loss of mitochondrial membrane potential induced by MgONPs Loss in mitochondrial membrane potential (MMP m) is one of the key factors in activating the apoptotic pathway [59]. In this study, the disruption of MMP in MgONPs treated 12

ACCEPTED MANUSCRIPT cells was assessed using the Rhodamine 123 dye in A549 cells. Compared to control cells, a significant (p0.05) decrease in fluorescence intensity (three fold) was observed in MgONPs treated cells indicating disruption in MMP (Fig. 7B). Fluorescence microscopic observation showed a decrease in fluorescent intensity in cells treated with MgONPs when compared to the control group; hence, validating spectrofluorometric results and confirming the fact that

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MgONPs would cause significant loss in MMP (Fig. 7A).

3.8 Apoptosis in A549 cells induced by MgONPs

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Triggering the apoptotic pathway to kill the cancer cells is the predominant mechanism of chemotherapeutic drugs used in cancer therapy [60]. In the present study, the rate of apoptosis

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induced by MgONPs was assessed using the AO/EtBr dual staining technique. Figure. 8A shows the presence of uniformly stained green cells in vehicle control, indicating the presence of viable

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cells (Fig. 8A). However, MgONPs treated cells showed the presence of orange colored stained cells, indicating dead cells while in cisplatin treated cells, green and orange dual stained cells

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were present, illustrating necrosis. Results of quantitative analysis exemplified a significant increase of apoptotic cells in the MgONPs treated group (79.5 ± 0.02% apoptotic and 20.5

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±0.03% viable cells) while compared to control (90.88 ± 1.22% viable cells and10.22 ± 0.825% apoptotic cells). Positive control treated cells showed 38.2 ± 0.05% viable cells and 71.8 ±

cell death in A549 cells.

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0.08% apoptotic cells (Fig. 8B). Results illustrated that the MgONPs led to apoptotic mediated

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To gain further insight on the mode of cell death stimulated by MgONPs in A549 cells, 4′,6-diamidino-2-phenylindole (DAPI) staining was carried out. As shown in fig. 8C, the

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MgONPs treatment caused chromatin condensation and morphological alteration in the nucleus of A549 cells, demonstrating the fact that the apoptotic effect of MgONPs completely depended on ROS production, which in turn has led to the oxidative stress mediated cell death in A549 cells. Results concluded that the MgONPs enhanced the intracellular ROS level, disrupting MMP, thereby, activating the intrinsic pathway of apoptosis causing cell death. 3.9 Safety evaluation of MgONPs in PBMC For the assessment of the toxicological profile of the identified drugs, in vitro toxicity studies have been validated as one of the alternate methods to in vivo studies [61] . Human 13

ACCEPTED MANUSCRIPT peripheral mononuclear cells (PBMC) are predominantly used in vitro model systems to predict the immunotoxicity and to determine the dosage limit of new drug compounds [62]. In vitro cytotoxic effects of the MgONPs in PBMC was evaluated using the MTT assay. Figure. 8D illustrated that the MgONPs induced no remarkable changes in cell viability and membrane integrity of PBMC after 24 h incubation and the percentage of cell viabilities were observed to be similar to control cells. However, H2 O2 treated cells, which were used as positive controls

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exhibited 72.5 ± 0.24% cytotoxicity. Results of in vitro toxicological assessment illustrated that

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the MgONPs had no cytotoxic effect similar to control cells; thus, indicating their safe use as

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drug. Further corroboration of safety aspects of drug has to be carried out under in vivo conditions to ensure absolute safer use.

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4. Conclusion

In the present study, MgONPs were synthesized using aqueous extract of the brown

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seaweed Sargassum wightii as a bioreductant and a capping agent. The biosynthesized MgONPs were crystalline in nature with the mean particle size of 68.02 nm. High negative zeta potential

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of the MgONPs indicated the enhanced stability of nanoparticles. The synthesized MgONPs showed potent antimicrobial activities against both human pathogenic bacterial and fungal

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strains in a concentration dependent manner. In addition, the MgONPs showed potent photocatalytic activity in the degradation of the organic dye methylene blue under UV radiation

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and sunlight. MgONPs also showed strong cytotoxic activity against lung cancer cell lines A549, which might be associated with the increase in ROS load, altered mitochondrial membrane

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potential, activated apoptotic pathway causing cell death. Future studies should be focused on the elucidation of molecular mechanism behind the anticancer potential of MgONPs on lung cancer

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cells under in vivo conditions.

Acknowledgement

Dr. N.S thanks University Grants Commission, New Delhi, India, for the financial support through UGC-Startup grant [Ref. No.F.30-381/2017 (BSR), dated 06.07.2017]. R. Prabhu wishes to thank DST, for the financial support through DST-PURSE fellowship [DSTPURSE Phase II/10815/2017].

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ACCEPTED MANUSCRIPT References Figure. 1 (A) UV-visible spectrum (B) FTIR spectrum (C) XRD spectrum of MgONPs synthesized using the aqueous extract of S. wightii Figure. 2 (A) FE-SEM images at 4000x and 16000x magnification (B) EDX spectrum (C) Particle size analysis using DLS (D) Zeta potential measurement of MgONPs Figure. 3 Antibacterial activity of MgONPs against gram positive and gram negative bacteria

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Figure. 4 Minimum bactericidal concentrations (μg/mL) of the MgONPs against (A) MRSA 56

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(B) P. aeruoginosa. Results are expressed as Mean ± S.D of triplicate experiments

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Figure. 5 (A) Antifungal activity of the MgONPs against Aspergillus fumigates, Fusarium solani, A. niger (B) Phtocatalytic activity of the MgONPs expressed as % of methylene blue dye

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degradation. Results are expressed as Mean ± S.D of triplicate experiments Figure. 6 Antiproliferative effect of the MgONPs on A549 cells at 24 h as assessed by (A)

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MTT assay to determine cytotoxicity (B) LDH leakage to determine membrane integrity (C) Phase contrast images illustrating morphological changes. The arrow heads indicate that the cells have undergone apoptosis displaying the characteristic features such as cell shrinkage,

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reduced cell density and formation of apoptotic bodies. The data are presented as Mean ± SD

control and treated groups

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of triplicate experiments. *p<0.05 denotes the statistically significant differences between the

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Figure. 7 (A) Fluorescence microscopic images of A549 cells treated with JC-1 staining to assess the mitochondrial membrane potential (ΔΨm) after treatment with the MgONPs for 24 h

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in comparison with cisplatin (40X) (B) Bar diagram illustrating the percentage of cells with disrupted MMP (C) Fluorescence microscopic images of A549 cells treated with CM-H2

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DCFDA, intracellular ROS indicator after treatment with MgONPs (D) quantification of ROS level using fluorescence spectroscopy. Data are expressed as Mean ± SD of triplicate assays Figure. 8 Apoptotic effect of MgONPs at its IC 50 in A549 cells as depicted by (A) AO/EtBr dual staining (B) Quantification of apoptotic population (C) DAPI staining (D) Safety assessment of MgONPs (25–100 μg/mL) on PBMC in comparison with 100 μM H2 O2 at 24 h. Results are expressed as Mean ± SD of triplicates and the values are considered significant at * # p<0.05 treated vs. control. 15

ACCEPTED MANUSCRIPT Table 1 Antibacterial activity of MgONPs Against Gram positive and Gram negative bacteria Microorganisms

Zone of inhibition (mm) at concentration in (µg/mL) Positive control 20

30

Pseudomonas aeruginosa

0.6Cm

0.6Cm

0.8Cm

1

Aeromonasbaumannii

0.2Cm

0.4Cm

0.7Cm

0.4

MRSA-11

0.3Cm

0.35Cm

0.7Cm

0.5

MRSA-56

0.5Cm

0.9Cm

0.35

Streptococcus pneumoniae

0.1Cm

0.5Cm

0.8Cm

1.2

0.5Cm

0.7Cm

0.4

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0.8Cm

0.3Cm

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E.coli

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10

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MgONPs

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Sample

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Table 2 Minimum bactericidal activity of ZnONPs against MRSA 56 and P. aeruginosa CFU/mL MRSA 56

P. aeruginosa

TNTC

TNTC

4 (g/mL)

TNTC

TNTC

8 (g/mL)

TNTC

TNTC

16 (g/mL)

TNTC

TNTC

Control

16

TNTC

TNTC

64 (g/mL)

TNTC

TNTC

128 (g/mL)

2×102

5×104

256 (g/mL)

158

3×102

51 2(g/mL)

10

152

1024 (g/mL)

5

NIL

2048 (g/mL)

1

CR

IP

32 (g/mL)

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ACCEPTED MANUSCRIPT

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Highlights

Facile green synthesis of MgONPs using aqueous extract of Sargassum wightii

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MgONPs showed significant antibacterial and antifungal activity against human

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

pathogens

MgONPs displayed high photocatalytic activity for dye degradation of methylene blue



MgONPs exhibited potent anticancer effect against lung cancer cell lines A549 cells



MgONPs induced ROS mediated apoptosis in A549 cells

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

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Graphical abstract

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