Biological therapeutics of Pongamia pinnata coated zinc oxide nanoparticles against clinically important pathogenic bacteria, fungi and MCF-7 breast cancer cells

Accepted Manuscript Biological therapeutics of Pongamia pinnata coated zinc oxide nanoparticles against clinically important pathogenic bacteria, fungi and MCF-7 breast cancer cells Balasubramanian Malaikozhundan, Baskaralingam Vaseeharan, Sekar Vijayakumar, Karuppiah Pandiselvi, Rajamohamed Kalanjiam, Kadarkarai Murugan, Giovanni Benelli PII:

S0882-4010(16)30864-6

DOI:

10.1016/j.micpath.2017.01.029

Reference:

YMPAT 2076

To appear in:

Microbial Pathogenesis

Received Date: 12 December 2016 Revised Date:

16 January 2017

Accepted Date: 18 January 2017

Please cite this article as: Malaikozhundan B, Vaseeharan B, Vijayakumar S, Pandiselvi K, Kalanjiam R, Murugan K, Benelli G, Biological therapeutics of Pongamia pinnata coated zinc oxide nanoparticles against clinically important pathogenic bacteria, fungi and MCF-7 breast cancer cells, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.01.029. 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.

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Biological therapeutics of Pongamia pinnata coated zinc oxide nanoparticles against

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clinically important pathogenic bacteria, fungi and MCF-7 breast cancer cells

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Balasubramanian

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Karuppiah Pandiselvia, Rajamohamed Kalanjiamb, Kadarkarai Murugan c, Giovanni Benelli d

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a

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Health Lab, Department of Animal Health and Management, Alagappa University, Karaikudi-

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630004, Tamil Nadu, India.

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b Department of Zoology, Dr.Zakir Husain College, Ilayangudi-630 702, Tamil Nadu, India.

Baskaralingam

Vaseeharana*, Sekar

Vijayakumara,

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Malaikozhundana,

c

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Nadu, India.

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d

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56124 Pisa, Italy

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Nanobiosciences and Nanopharmacology Division, Biomaterials and Biotechnology in Animal

Department of Biotechnology, Thiruvalluvar University, Serkkadu, Vellore 632 115, Tamil

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Department of Agriculture, Food and Environment, University of Pisa, via del Borghetto 80,

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-------------------------------------------------------------------------------------------------------------*Corresponding author:

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Dr.B. Vaseeharan.,

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Tel: + 91 4565 225682.

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Fax: + 91 4565 225202.

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E-mail address: [email protected]

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Abstract

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The overuse of antimicrobics and drugs has led to the development of resistance in a number of

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pathogens and parasites, which leads to great concerns for human health and the environment.

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Furthermore, breast cancer is the second most common cause of cancer death in women. MCF-7

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is a widely used epithelial cancer cell line, derived from breast adenocarcinoma for in vitro

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breast cancer studies because the cell line has retained several ideal characteristics particular to

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the mammary epithelium. In this scenario, the development of novel and eco-friendly drugs are

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of timely importance. Green synthesis of nanoparticles are cost effective, environmental friendly

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and do not involve the use of toxic chemicals or elevate energy inputs. This research focused on

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the antibreast cancer activity of Pongamia pinnata seed extract-fabricated zinc oxide

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nanoparticles (Pp-ZnO NPs) on human MCF-7 breast cancer cells and their antibiofilm activity

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against bacteria and fungi. P. pinnata seed extract-fabricated zinc oxide nanoparticles (Pp-ZnO

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NPs) were characterized by UV–Vis spectroscopy, X-ray diffraction (XRD), Fourier transform

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infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and energy dispersive X-ray

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spectroscopy (EDAX). Pp-ZnO NPs effectively inhibited the growth of Gram positive Bacillus

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licheniformis (zone of inhibition: 17.3 mm) at 25 µg ml-1 than Gram negative Pseudomonas

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aeruginosa (14.2 mm) and Vibrio parahaemolyticus (12.2 mm). Pp-ZnO NPs also effectively

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inhibited the biofilm formation of C. albicans at 50 µg ml-1. Cytotoxicity studies revealed that a

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single treatment with Pp-ZnO NPs significantly reduced the cell viability of breast cancer MCF-

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7 cells at doses higher than 50 µg ml-1. Morphological changes in the Pp-ZnO NPs treated MCF-

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7 breast cancer cells were observed using confocal laser scanning microscopy (CLSM). This

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study concludes that the green synthesized Pp-ZnO NPs may be used as an effective

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antimicrobial and antibreast cancer agents.

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Keywords: Bacillus licheniformis; Candida albicans; ZnO nanoparticle, Pongamia pinnata,

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antibreast cancer.

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1. Introduction

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Recently, nanotechnology has gained increased attention in the field of science and

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technology for the purpose of developing new nanoscale materials [1]. A number of physical,

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chemical, biological, and hybrid methods are employed to synthesize different types of

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nanoparticles [2]. Physical and chemical methods are commonly employed for nanoparticle

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synthesis, however, the risk of toxic compounds limits their applications. To avoid the problem

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of toxicity in nanoparticle synthesis, green methods involving the use of plant materials for

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synthesis are relatively safe and eco-friendly [3]. Moreover, the potentially active molecules in

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the plant mediated synthesis of nanoparticles are more compatible for a wide range of biomedical

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applications [4].

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The advanced features such as biocompatibility and fast electron transfer kinetics

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necessitates the use of zinc oxide to immobilize and modify the biomolecules [5]. Zinc as a

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mineral is essential to human health and ZnO nanoparticles has good biocompatibility to human

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cells [6]. Among metal oxide, ZnO significantly controlled the growth of a broad spectrum of

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bacteria [7]. Currently, ZnO nano powder is used in various products including, ceramics,

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plastics, cement, glass, rubber, paints, lubricants, pigments, food (source of Zn nutrient),

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batteries and fire retardants. Due to the excellent UV absorption and reflective properties, ZnO

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nanoparticles are commonly used in cosmetics and sunscreens.

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Plants as biological materials for the synthesis of zinc oxide nanoparticles has not been

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fully explored. Recently, it has been elucidated that the botanical source of reducing and capping

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agents for nanoparticle synthesis has a strong impact on the bio-physical features of 3

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nanoparticles [8, 9]. In this study, a cheap and non-toxic seed extract of Pongamia pinnata

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(Fabaceae), commonly known as Karanj or Indian beach tree was used for the synthesis of ZnO

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nanoparticles. Different parts of this plant have been used for the treatment of hemorrhoids,

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malignant tumors, skin diseases, ulcers and wounds [10]. The seed of P. pinnata has been

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traditionally used in Asian medicines (ayurveda and unani) as anti-inflammatory, anti-

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plasmodial,

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antioxidants [11]. The antibacterial activity of P. pinnata have been previously reported [12]. P.

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pinnata seed contains 40% oil, 20.5% and 79.4% total saturated and unsaturated fatty acid

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respectively [13]. Among the fatty acids, oleic (46%), linoleic (27%) and linolenic (6%) acids

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were the most abundant. A small amount (0.1%) of low molecular weight fatty acids such as

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lauric and capric acids was present in seeds [14, 15]. Karanjin is a furanoflavonol present in the

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seed and is responsible for the cure of skin diseases [16]. Cancer is the most important cause of

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mortality in the world. Breast cancer is the second most common cause of cancer death in

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women. Currently available chemopreventives and chemotherapeutic agents cause undesirable

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side effects [17, 18]. Therefore, developing a biocompatible and cost-effective method of

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treatment for cancer is indispensable. One of the primary advantages for considering ZnO

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nanoparticles for use in cancer is the inherent preferential cytotoxicity against cancer cells in

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vitro [17, 18]. The anticancer activity of ZnO NPs and Ag/ZnO composite against human lung

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cancer cell lines have been previously reported by Vijayakumar et al. [19] and Thaya et al. [20].

anti-ulcer,

anti-hyperammonic,

and

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anti-hyperglycamic,

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anti-conceptive,

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By considering the above facts, the present study, reports for the first time, the synthesis

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and physico-chemical characterization of zinc oxide nanoparticles using the seed extracts of P.

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pinnata (Pp-ZnO NPs). The antibacterial and antibiofilm activities of Pp-ZnO NPs were

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explored against Gram positive, Gram negative bacteria and fungal pathogens. MCF-7 is a 4

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widely used epithelial cancer cell line, derived from breast adenocarcinoma for in vitro breast

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cancer studies because the cell line has retained several ideal characteristics particular to the

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mammary epithelium (Lee et al., 2015). Taking the above into account, in the present study, the

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antibreast cancer activity of Pp-ZnO NPs was tested against the human MCF-7 breast cancer cell

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lines.

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2. Materials and Methods

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2.1. Collection of plant material

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Pongamia pinnata seeds were collected from in and around the regions of Karaikudi, Tamil

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Nadu, India. The identification of the plant was authenticated by the Botanical Survey of India.

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A voucher specimen of plant has been maintained in the Department (voucher specimen DAHM

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002). Fresh seeds were washed with distilled water, followed by air drying and has been used for

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the present investigation.

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2.2. Preparation of seed extract of P. pinnata

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Aqueous seed extract of P.pinnata was prepared following the method by Vijayakumar et al.

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[21]. The extract was prepared by placing 50g of washed, dried fine seeds in 250 ml glass beaker

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along with 100 ml of distilled water. The mixture was boiled for 60 min until the colour of

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solution changes to light yellow. The extract was cooled, filtered and then stored at 4 ºC for

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further experiments.

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2.3. Green synthesis of nanoparticles

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The synthesis of zinc oxide nanoparticles using the seed extracts of P. pinnata (Pp-ZnO NPs)

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was carried out by the method of Azizi et al. [22]. Zinc acetate dihydrate (99% purity) and

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sodium hydroxide pellet was used as the starting material. Twenty millilitre of 0.02M zinc

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acetate dihydrate was added to 50ml of distilled water with vigorous stirring. After 10 min of

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stirring, 250ml of aqueous seed extract of P. pinnata and aqueous 2.0M NaOH was added. This

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has resulted in a white aqueous solution at pH 12. The contents were then placed in magnetic

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stirrer for 2h to obtain precipitate. The precipitate was then taken out and washed repetitively

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with distilled water, followed by ethanol to remove the impurities of the final product. Finally, a

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white powder of ZnO nanoparticles was obtained after drying at 60 °C in a vacuum oven over

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night.

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2.4. Bio-physical characterization of nanoparticles

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The reduction of Zn+ ions was monitored by measuring the absorption spectrum of the reaction

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medium after 30 min. About 1ml of the sample was collected for UV-Vis spectrum analysis and

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the maximum absorbance spectrum of Pp-ZnO NPs was observed at 300-500 nm [23].

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The particle size of Pp-ZnO NPs were determined using XRD 6000/6100 (Shimadzu

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Corporation, Nakagyo-ku, Kyoto, Japan). X-ray diffraction is a primary analytical technique

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used for phase identification of crystalline material. The analysed material has been finely

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ground, and the average bulk composition was determined. The grain size of the zinc oxide

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nanoparticles was determined using Debye Sherrer’s equation.

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D = 0.94λ / B cos θ

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Where λ is the wavelength (Cu Kα), β is the full width half- maximum (FWHM) of the ZnO

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(101) line and θ is the diffraction angle [24].

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For FTIR spectroscopy,

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and pressed into a pellet. The pellet was placed into the sample holder and FTIR spectra were

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recorded under FTIR spectroscopy (Thermo Scientific Nicolet-iS5, Waltham, USA) at a

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resolution of 4 cm−1 [25].

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Two milligram of Pp-ZnO NPs was mixed with 200 mg potassium bromide (FTIR grade)

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SEM and EDX analysis of Pp-ZnO NPs were done using Hitachi S-4500 (Hitachi,

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Krefeld, Germany). Thin films of synthesized and stabilized Pp-ZnO NPs were prepared on the

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carbon coated copper grid by dropping a small amount of sample on the copper grid. The extra

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solution was removed using a blotting paper and the film on SEM grid was then allowed to dry

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under a mercury lamp for 5min.

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2.5. Antibacterial activity

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The antibacterial activity of Pp-ZnO NPs was tested on Gram positive [Bacillus licheniformis

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(HM235407.1)] and Gram negative [Pseudomonas aeruginosa (HQ693274.1) and Vibrio

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parahaemolyticus (HQ693275.1)] bacteria by Disc Diffusion method [26]. Bacteria was

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inoculated in Luria Bertani broth (pH 7.4) and incubated for 8 h at 37 ºC. The cultures were then

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plated on Luria Bertani agar using sterilized cotton swabs. 25 µg ml-1 of Pp-ZnO NP were used

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to test the antimicrobial activity against bacteria. The antibacterial activity of Pp-ZnO NPs were

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compared with bulk ZnO and seed extract. 100 µl of distilled water was used as negative control.

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The standard antibiotic (Ciprofloxacin) disc was placed on agar surface as a positive control and

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the plates were incubated for 24 h at 37 °C. The experiment was carried out in triplicate plates

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for each organism.

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2.5.1. Minimum Inhibitory Concentration (MIC) of Pp-ZnO NPs

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The Minimal Inhibitory Concentration (MIC) of bulk ZnO, seed extracts and Pp-ZnO NPs were

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determined by the method of Burt [27]. Tubes with 5ml of Luria-Bertani (LB) broth containing

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various concentrations of bulk ZnO, seed extracts and Pp-ZnO NPs ranging from 5 to 9.5µg ml-1

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were inoculated with 200µl of 105 CFU ml-1 of standardized suspensions of bacterial culture. The

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tubes were incubated in orbital shaker (180 rpm) for 24 h at 37 °C. About 100µl from each

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dilution tube was plated in MHA plates and incubated for overnight at 37 °C. The results were

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recorded by comparing plates with positive control (i.e. ciprofloxacin).

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2.6. Antibiofilm activity against bacteria

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The biofilm inhibitory activity of Pp-ZnO NPs on Bacillus licheniformis, Pseudomonas

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aeruginosa and Vibrio parahaemolyticus was analyzed by microtitre plate assay. Bacterial

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colonies (1 x 106 cfu ml-1) were grown on glass pieces (1x1cm) placed in 24-well polystyrene

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plates with 1ml of nutrient broth supplemented with various concentrations of Pp-ZnO NPs (10

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to 25 µg ml-1) and incubated at 37 °C for 24 h. They were then stained with crystal violet and

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examined under Nikon inverted research microscope (ECLIPSE Ti100) at 40x magnification.

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Another set of biofilms grown as above were washed with PBS, stained with acridine orange

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(0.1%) and were examined under a confocal laser scanning microscope (CLSM- Carl Zeiss LSM

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710). The biofilm images were analysed using a Zen 2009 software (Carl Zeiss, Germany).

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2.7. Antibiofilm activity against fungi

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C. albicans MTCC 3017(IMTECH, Chandigarh) was streaked out onto YPD plates containing

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1% yeast extract, 2% peptone, 2% dextrose and 2% Bacto agar. They were grown at 37°C for 24

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to 48 h. For all the experiments performed, YPD medium was used for routine culturing process.

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Glycerol stock (60 %) was prepared and maintained at -20 °C for future use.

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C. albicans biofilms were developed according to Jin et al. [28]. Briefly, washed yeast

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cells (1 × 107 cells/ml) with an optical density of 0.5 Mc Farland Standard was resuspended in

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YPD broth medium containing 100 mM of glucose. The standard cell suspension was used to

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develop biofilms on commercially available presterilized 24 well polystyrene plates (Tarsons,

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India). About 50 µl of cell suspension (1× 107 cells/ml) was added into each well of 24 well

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plates and incubated at 37°C for 90 min in an orbital shaker operated at 75 rpm to allow yeast

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adherence to the well surface (adherence phase). Next, each well was incorporated with various

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concentrations of Pp-ZnO NPs (25 and 50µg mL-1) to investigate the antibiofilm efficacy against

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C. albicans. Similarly, a control was set up in the same manner without the addition of Pp-ZnO

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NPs. Following the adhesion phase, the cell suspensions were aspirated and each well was

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washed with 100 µl of phosphate buffered saline (PBS) to remove the loosely adherent cells.

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About 500 µl of YPD broth containing 100 mM of glucose was then drawn into each of the

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washed wells, and the plates were incubated in an orbital shaker (75 rpm) for 48 h at 37°C. After

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incubation, the biofilm inhibition percentage was calculated.

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Inhibitory percentage = (Absorbance of control – Absorbance of sample)

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Absorbance of control

2.7.1. Confocal laser scanning microscopy (CLSM)

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The morphology and thickness of biofilms formed by C. albicans (MTCC 3017) treated with Pp-

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ZnO NPs were observed under confocal laser scanning microscope (CLSM). The biofilms

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formed on glass pieces were transferred to a 24 well plate containing 2 ml of PBS along with the

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fluorescein isothiocynate-concanavalin A (FITC-ConA; 50 µg ml-1) in 10 mmol-l hydroxyl ethyl

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piperazine ethane sulfonic acid (HEPES) and incubated at 37°C for 30 min. The stained glass

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pieces were visualized under CLSM (Zeiss LSM710 Meta, Germany). The biofilm images were

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analyzed using Zeiss LSM Image Examiner Version 4.2.0.121).

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2.8. Cytotoxicity on human breast cancer cells

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The inhibitory effect of Pp-ZnO NPs on human MCF-7 breast cancer cells were

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evaluated using an MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay

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reported by Manju et al. [29]. Briefly, human MCF-7 breast cancer cell line was procured from

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National Centre for Cell Science (NCCS), Pune, India. The cells were cultured in DMEM

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(Dulbecco’s modified eagle medium) supplemented with 2mM L-glutamine, 100 U/ml penicillin,

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100 µg/ml streptomycin and 10% FBS. The cells were cultured in 75 cm2 cell culture flasks at

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37ºC in CO2 incubator (95% air, 5% CO2 and 100% relative humidity). They were then placed

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into 96 well plates (2×105 cells in each well) and incubated for 24 h. MCF-7 breast cancer cells

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were treated with various concentrations of Pp-ZnO NPs (5, 10, 20, 30, 40 and 50 µg ml-1). A

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respective control (100 µg ml-1) were simultaneously prepared with DMEM medium, saline, seed

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extract and bulk ZnO for comparison of cytotoxic effects. After treatment, the plates were kept

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for 24 h to evaluate the cell viability using 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium

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bromide (MTT) assay. MTT was prepared at a concentration of 5mg ml-1 and 10 µl of MTT was

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added to each well and incubated for 4 h. Purple color formazone crystals formed were dissolved

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in 100 µl of dimethyl sulfoxide (DMSO). The optical density was read at 570 nm in an ELISA

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plate reader. The optical density was subjected to evaluate the percentage of cell viability by

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using the following formula:-

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OD value of experimental samples

Percentage of cell viability =

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____________________________ × 100 OD value of experimental controls

2.8.2. Propidium iodide staining MCF-7 breast cancer cells were placed into a six well chamber plate at 2×105 cells/well.

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At > 90% confluence, the cells were treated with Pp-ZnO NPs (30 µg ml-1) for 24 h. The cells

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were then washed with PBS fixed in methanol: acetic acid (3:1v/v) for 10 min. They were then

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stained with 50 µg ml-1 propidium iodide for 10 min. The morphology of apoptotic cell nuclei

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(condensed/fragmented) was examined under confocal laser scanning microscope (CLSM-710.

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Carl Zeiss. Germany) [29].

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3. Results

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3.1. UV-visible spectroscopy

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UV–vis spectrum of ZnO and Pp-ZnO NPs at different wavelengths ranging from 300 to

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800 nm showed strong absorption peak at 380 nm due to its surface plasmon resonance. ZnO

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exhibited decreased absorbance peak at 380 nm, whereas, after addition of seed extract, an

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increase in the absorbance peak at 380 nm was observed in Pp-ZnO NPs (Fig. 1a).

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3.2. XRD analysis

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The XRD spectrum of Pp-ZnO NPs showed various Bragg’s reflections, which

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corresponds to (102), (103), (110) and (112) set of lattice planes (Fig. 1b). Based on these Bragg

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reflections, it revealed that the synthesized Pp-ZnO nanoparticles are face centered cubic and

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essentially crystalline in nature. The mean crystalline size (D) of the particles were determined

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following Debye Scherrer equation (D = 0.94λ / B cos θ). The calculated crystalline size of the

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Pp-ZnO NPs is about 30.2 nm (JCPDS: 65-3411, system hexagonal and lattice primitive).

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3.3. FTIR spectroscopy

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FTIR spectrum of seed extracts of P. pinnata has shown strong absorption bands at 3465

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and 2388 cm-1 representing C-H and O-H stretching of polyphenols. The peak located at 1652

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cm-1 corresponds to C =C stretching of aromatic rings. The peak at 1070 cm-1 represents C-OH

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group of alkanes (Fig 1c). FTIR spectrum of Pp-ZnO NPs showed the bulk ZnO showing high

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intensity broad band around 3409 cm-1 which corresponds to C-O stretching of amides (Fig. 1c).

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The intense broad band at 2366 and 1736 indicated the presence of –OH stretching of

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intramolecular hydrogen bond, C–C stretching and C=O stretching of alkanes. The bands

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recorded at 1023 and 442cm-1 were assigned to alcohol, phenolic groups and C–N stretching

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vibrations of aliphatic and aromatic amines respectively.

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3.4. Scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analysis The morphological dimensions of Pp-ZnO NPs observed under SEM demonstrated that

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the particle size ranged between 30.4 - 40.8 nm with inter-particle distance. The shape of Pp-

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ZnO NPs were observed to be spherical (Fig. 1d). The EDX spectrum showed that the

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composition of zinc element in Pp-ZnO NPs was 84.24% (Fig. 1e).

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3.5. Antibacterial and biofilm inhibitory activity

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Pp-ZnO NPs exhibited greater activity against bacteria compared to bulk ZnO and seed

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extracts. However, Pp-ZnO NPs showed higher activity against Gram positive Bacillus

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licheniformis than Gram negative Pseudomonas aeruginosa and Vibrio parahaemolyticus (Table

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1). The zone of inhibition against B. licheniformis was 17.3 mm at 25µg ml-1. The zone of

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inhibition against P. aeruginosa was 14.2 mm at 25µg ml-1. The zone of inhibition against V.

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parahaemolyticus was 12.2 mm at 25 µg ml-1. On the otherhand, ciprofloxacin (commerical

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antibiotic) showed 20.3, 18.4 and 14.1 mm of inhibition zones against B. licheniformis, P.

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aeruginosa and V. parahaemolyticus respectively. The minimum inhibitory concentration (MIC)

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of Pp-ZnO NPs were comparatively lesser than that of bulk ZnO and seed extract. The MIC of

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Pp-ZnO NPs against B. licheniformis, P. aeruginosa and V. parahaemolyticus were 1.875, 1.998

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and 3.023 µg ml-1 respectively (Table 2). The light microscopic studies showed that control

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slides exhibited a well developed biofilm growth of tested bacteria. Bacteria treated with 25 µg

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ml-1of Pp-ZnO NPs had developed a poor biofilm growth compared to that of control after 24 h

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(Fig. 2). CLSM studies had also revealed the loose biofilm architecture of bacteria when treated

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with 25 µg ml-1of Pp-ZnO NPs compared to that of control (Fig. 3). These results indicated that

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the biofilm growth was remarakably inhibited at higher concentration (25 µg ml-1) of Pp-ZnO

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NPs (Fig. 4).

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3.6. Inhibition of fungal biofilm formation Pp-ZnO NPs exhibited antibiofilm activity against C. albicans by inhibiting the biofilm

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formation. The biofilm inhibition was greater at 50 µg ml-1 compared to 25 µg ml-1. At 25 µg ml-

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1

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increased to 92% at 50 µg ml-1 compared to control (Fig. 5). The biofilm inhibition potential of

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Pp-ZnO NPs against C. albicans was clearly depicted under CLSM. The control slide showed

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well developed biofilm growth of C. albicans. On the otherhand, the biofilm of C. albicans

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treated with 50 µg ml-1 showed reduced growth compared to that of control (Fig 6).

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3.7. Cytotoxicity on breast cancer cells

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The cytotoxicity of Pp-ZnO NPs were evaluated against human MCF-7 breast cancer cell lines at

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various concentrations (5–50 µg ml-1). The inhibitory concentration (IC50) of Pp-ZnO NPS

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against MCF-7 breast cancer cells were 32.8 µg ml-1. The results showed a significant decrease

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in the viability of MCF-7 breast cancer cells when the concentration of Pp-ZnO NPs was

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increased from 5 to 50 µg ml-1 (Fig.7).. However, the concentration of Pp-ZnO NPs were

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comparatively lesser than that of seed extract and bulk ZnO (100 µg ml-1). This revealed that the

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Pp-ZnO NPs were more effective in controlling the viability of MCF-7 breast cancer cells. To

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authenticate the antibreast cancer effects of Pp-ZnO NPs on the apoptotic cell morphology,

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propidium iodide stained cells were visualized under phase contrast microscope. When compared

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to negative control (DMEM and saline), MCF-7 breast cancer cells treated with seed extract

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(image not shown) and bulk ZnO (positive control) showed nuclear morphological changes like

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clumping of cell and loss of membrane stability. However, treatment with Pp-ZnO NPs, the

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MCF-7 breast cancer cells exhibited cell burst and less viability at 30 µg ml-1 after 24h (Fig.8).

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, the inhibition of fungal biofilm was 48%. However, the biofilm inhibition percentage was

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The cytotoxicity studies revealed that Pp-ZnO NPs were more successful in the control of human

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MCF-7 breast cancer cells compared to the seed extract and bulk ZnO.

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4. Discussion With the growing interest to limit the use of hazardous nanoparticles, the development of

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biological, biomimetic and biochemical approaches for synthesis of nanoparticles is desirable.

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Green method of nanoparticles synthesis are more advantageous than physico-chemical methods

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because of its non-toxic and environmental-friendly nature [30]. The synthesis of Pp-ZnO NPs in

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the present study was established through UV-Vis spectroscopy, XRD, FTIR and SEM analysis.

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It is reported that UV–Vis spectroscopy could be used to determine the size and shape of

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nanoparticles in aqueous solution [31]. In the present study, the UV-Vis absorption spectrum of

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ZnO exhibited decreased absorption spectra at 380 nm, however, after the addition of seed

314

extract, the synthesized Pp-ZnO NPs showed strong absorption peak at 380 nm due to its surface

315

plasmon resonance. The increase in the absorbance peak of Pp-ZnO NPs may be due to the

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functional molecules (keranjin) present in the seed extract. This was in accordance with the

317

findings on the synthesis of zinc oxide nanoparticles using Aloe barbadensis [32] and silver

318

nanoparticles using tea leaf extract [33]. Smitha et al. [34] demonstrated that the size and shape

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of nanoparticles, dielectric constant of the medium and surface adsorbed species determine the

320

spectral position of plasmon band absorption as well as its width. In the present study, the XRD

321

pattern indicates the hexagonal and crystalline nature of Pp-ZnO NPS. The XRD peaks obtained

322

in the present study are confirmed by ZnO hexagonal phase (wurtzite structure) compared with

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JCPDS card No.89-7102. The obtained results are in good agreement with earlier reports of

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Sangeetha et al. [32] and Sivakama Valli & Vaseeharan [35].

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FTIR measurement was performed to identify the biomolecules responsible for capping

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and stabilization of Pp-ZnO NPs. In the present study, the different bands of FTIR have been due

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to primary and secondary alkanes and the stretching of C-H and C = O. The stability of ZnO NPs

328

may be due to the free amino and carboxylic groups that have interacted with the zinc surface.

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The functional groups such as –C–O–, –CO–C–,and –C=C– are derived from heterocyclic

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compounds and the amide bonds derived from the proteins present in the seed extract of P.

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pinnata (karanjin) act as capping ligand which give stability to Pp-ZnO NPs. These results are

332

consistent with the findings of Vijayakumar et al. [21], Sangeetha et al. [32] and Elumalai et al.

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[36] who reported that the stability of ZnO Nps is due to capping agent of plant extract involved

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in synthesis. The particle size and shape of the NPs can be determined by SEM. In this study,

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SEM showed spherical shape of the nanoparticle formed with a surface diameter range of 30.4-

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40.8 nm.

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The element composition of green synthesized nanoparticles were determined by energy

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dispersive X-ray spectra (EDX). EDX spectrum showed that the zinc in the composition of

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nanoparticle was 84%. The size of Pp-ZnO NPs in this study corroborates with findings of Divya

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et al. [37] who reported that the size of ZnO NPs were between 30-56 nm.

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Zhang et al. [38] demonstrated that the primary sizes of zinc nanoparticles are important

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for antibacterial activity. The results of the current study revealed that the green synthesized Pp-

343

ZnO NPs have enhanced antimicrobial activity compared to bulk ZnO and seed extracts. The

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antibacterial activity of Pp-ZnO NPs was very close to that exhibited by ciprofloxacin (i.e.

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commercial antibiotic). This result suggests that Pp-ZnO may be used as an substitute to

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commercial antibiotics. The enhanced antibacterial activity of Pp-ZnO NPs could be due to the

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action of capping agent (karanjin) in the seed extract and relatively the nanosize of the particle.

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Furthermore, Pp-ZnO NPs exhibited stronger activity against Gram positive bacteria (B.

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licheniformis) than that of Gram negative bacteria (P. aeruginosa and V. parahaemolyticus). Our

350

results are supported by the observations of Premanathan et al. [39] who reported that the ZnO

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NP is more toxic to Gram-positive bacteria (S. aureus) than Gram-negative bacteria (E. coli and

352

P. aeruginosa). They further stated that the difference in nanoparticle toxicity may be attributed

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due to the differences in bacteria’s cell membrane structure. The inhibitory activity of Pp-ZnO

354

NPs against bacterial biofilm may be due to the rupture of bacterial cell wall and surface activity

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of Pp-ZnO NPs in contact with bacteria. This observation was well supported by Zhang et al.

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[38] who suggested that the antibacterial activity of ZnO nanoparticle could be due to their

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penetrating ability into the bacterial cell membrane. Stoimenov et al. [40] demonstrated that the

358

contact between nanoparticles and bacterial cell was initiated by the surface charges on the

359

particle and the electrostatic interaction between bacterial surface and nanoparticle. This was

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confirmed by Zhang et al. [41] through electrochemical measurements. After contact with the

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bacterial membrane, ZnO nanoparticle generates high rate of reactive oxygen species that leads

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to the death of bacteria due to chemical interactions between hydrogen peroxide and membrane

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proteins [41, 42].

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The development of resistance to conventional fungicides such as benzimidazoles and

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dicarboximides made the fungi very difficult to control [42]. In the present study, the inhibition

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of C. albicans biofilm was greater at 50 µg ml-1of Pp-ZnO NPs. He et al. [6] repored that ZnO

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NPs at concentration greater than 3 mmol l−1can significantly inhibit the growth of Botrytis

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cinerea and Penicillium

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expansum than B. cinerea. The minimum inhibitory concentration of ZnO NPs against

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Saccharomyces cerevisiae, C. albicans, Aspergillus niger, and Rhizopus stolonifer was above

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100 mg ml−1 [43]. Nanoparticles are promising alternative for treatment of diseases because of their unique

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biological properties. The anticancer activity of ZnO NPs against human myeloblastic leukemia

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and lung carcinoma cells has been reported earlier [19]. In the present study, the inhibition of

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MCF-7 breast cancer cell growth was found to be higher at higher concentrations of Pp-ZnO NPs

376

(50 µg ml-1). This was in close proximity with the findings of Selvakumari et al. [44] who

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reported that 50% reduction of human A549 lung cancer cells and MCF-7 breast cancer cells

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were exhibited at 31.2 µg/ml of ZnO NPs. At a very low concentration, ZnO NPs were found to

379

exhibit activity against liver cancer HepG2 and breast cancer MCF-7 cancer cells in a dose-

380

dependent manner. At 25 µg/mL, the viability of HepG2 cells was less than 10% [45]. In the

381

present study, a significant reduction in the cell viability and also morphological changes in the

382

nucleus of MCF-7 breast cancer cells were observed following treatment with Pp-ZnO NPs. The

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morphological variations such as retardation of cell growth, cell burst, cell clumping and loss of

384

membrane stability were observed in MCF-7 breast cancer cells treated with Pp-ZnO NPs under

385

confocal laser scanning microscopy (CLSM). These suggests that the green synthesized Pp-ZnO

386

NPs has the potential to treat the breast cancer without any harmful effect.

387

5. Conclusions

388

Overall, the present study reported green, environmental friendly and economical approach for

389

the biological synthesis of ZnO nanoparticles using the seed extract of P. pinnata, which

390

function as an effective, reducing and stabilizing capping agent. The green synthesized Pp- ZnO

391

NPs showed a size ranging between 30.4 and 40.8 nm; the nanoparticles were spherical in shape,

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and have a wurtzite structure. The biosynthesized Pp-ZnO NPs have shown good antibacterial

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and antibiofilm activity against pathogenic bacteria and fungi, if compared to the antimicrobics

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currently marketed. Furthermore, Pp-ZnO NPs showed effective toxicity against MCF-7 breast

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cancer cells. Thus, we concluded that the present green synthesis route may be considered further

396

to produce antimicrobial, antibiofilm and anti-breast cancer agent useful in a wide array of

397

biomedical and pharmaceutical applications.

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Conflict of interest statement

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The authors declare no conflict of interest.

400

Acknowledgement

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Dr.B.Malaikozhundan gratefully acknowledges the DST-SERB, New Delhi for support of

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research grant under Young Scientist Scheme (YSS/2015/000645). The corresponding author

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Dr.B.Vaseeharan thank the Professor & Head, Department of Physics and Department of

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Industrial Chemistry, Alagappa University for their help in XRD and SEM analysis.

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Conflict of Interest

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The authors declare no conflict of interest.

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Figure Captions

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Figure 1. (a) UV–Vis absorption spectra of ZnO and Pp-ZnO NPs. (b) X-ray diffraction pattern showing the crystalline nature of Pp-ZnO NPS

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(c) Fourier transform infrared spectra showing the possible functional group of Pp-ZnO

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NPs in comparison with Pongamia pinnata seed extract.

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(d) Scanning electron micrograph showing the shape and size of Pp-ZnO NPs

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(e) Energy dispersive X-ray analysis showing the elemental composition of Pp-ZnO

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NPs

Figure 2. Light microscopy images of bacterial biofilms grown in the presence of Pp-ZnO NPs at 25 µg ml-1. (A) Bacillus licheniformis, (B) Pseudomonas aeruginosa, (C) Vibrio parahaemolyticus.

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Figure 3. Confocal laser scanning microscopy images of bacterial biofilms grown in the presence of Pp-ZnO NPs at 25 µg ml-1. (A) Bacillus licheniformis, (B) Pseudomonas aeruginosa, (C) Vibrio parahaemolyticus.

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Figure 8. Effect of Pp-ZnO NPs (30 µg ml-1) on the morphology of MCF-7 breast cancer cells in comparison with the control (DMEM, saline and bulk ZnO). (a) cells treated with medium showing normal morphology (b) cells treated with saline showing normal morphology (c) cell treated with bulk ZnO showing morphological changes (d) cells treated with Pp-ZnO NPs showing morphological changes. Arrow indicates the morphological changes in the cells.

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Figure 4. Inhibition of biofilm formation of bacterial pathogens by Pp-ZnO NPs. Each bar indicated mean±standard deviations of three replications. (*values are significant at p<0.05 using ANOVA followed by Tukey’s HSD test).

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Figure 5. Inhibition of biofilm formation of C. albicans by Pp-ZnO NPs. Each bar indicated mean±standard deviations of three replications. (*values are significant at p<0.05 using ANOVA followed by Tukey’s HSD test). Figure 6. Confocal laser scanning microscopy images of C. albicans biofilms grown in the presence of Pp-ZnO NPs at 25 and 50 µg ml-1.

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Figure 7. Effect of Pp-ZnO NPs (50 µg ml-1) on the viability of MCF-7 breast cancer cells in comparison with the control (100 µg ml-1 of DMEM, saline, seed extract and bulk ZnO respectively). Each bar indicates mean±standard deviations of three replications. Asterisk indicates statistically significant difference between treatments at P < 0.005 using ANOVA followed by Tukey’s HSD test.

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Table.1. Antibacterial activity of bulk ZnO, seed extracts and Pp-ZnO NPs against different bacteria species, tested at 25 µg ml-1.

Zone of Inhibition (mm)**

Seed extract

Pp-ZnO NPs

SC

ZnO

RI PT

Bacteria

Bacillus licheniformis

10.2±1.1a

17.3±1.2a

Pseudomonous aeruginosa

8.3±1.0ab

10.4±0.8b

14.2±1.4ab

18.4±1.6ab

Vibrio parahaemolyticus

6.3±1.1b

8.5±1.3b

12.2±1.2b

14.1±0.8b

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14.1±1.0a

Ciprofloxacin 5 µg/disc 20.3±0.5a

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Table 2. Minimum inhibitory concentrations of bulk ZnO, seed extracts and Pp-ZnO NPs against different bacteria species.

Bulk ZnO

Seed extract

RI PT

Minimum inhibitory concentration* (µg ml-1)

Bacteria

Pp-ZnO NPs

2.374±0.2ab

2.314±0.4ab

Pseudomonous aeruginosa

2.524±0.5ab

2.420±0.6ab

1.998±0.7ab

Vibrio parahaemolyticus

3.724±0.8a

3.526±0.4a

3.023±0.2ab

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Bacillus licheniformis

1.875±0.3b

* Values are mean ±SE of three replicates. Significant over the control at P<0.05 (ANOVA). Different letters indicate significant differences among values (P<0.05) (ANOVA followed by

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Tukey’s HSD test)

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HIGHLIGHTS • Pongamia pinnataseedextracts based zinc oxide nanoparticle was synthesized. • Pp-ZnO NPswere characterizedby UV spectroscopy, XRD, FTIR, SEMand EDAX.

• Pp-ZnO NPs inhibited the biofilm of C. albicans at 50 µg ml-1.

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• Pp-ZnO NPs controlled the growth of Gram positive bacteria at 25 µg ml-1.

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• Pp-ZnO NPs inhibited the viability of human MCF-7 breast cancer cells at 50 µg ml-1.