In-Vitro cytotoxicity, antibacterial, and UV protection properties of the biosynthesized Zinc oxide nanoparticles for medical textile applications

Accepted Manuscript In-Vitro cytotoxicity, antibacterial, and UV protection properties of the biosynthesized Zinc oxide nanoparticles for medical textile applications Amr Fouda, Saad EL-Din Hassan, Salem S. Salem, Th I. Shaheen PII:

S0882-4010(17)31498-5

DOI:

10.1016/j.micpath.2018.09.030

Reference:

YMPAT 3180

To appear in:

Microbial Pathogenesis

Received Date: 13 November 2017 Revised Date:

10 June 2018

Accepted Date: 16 September 2018

Please cite this article as: Fouda A, EL-Din Hassan S, Salem SS, Shaheen TI, In-Vitro cytotoxicity, antibacterial, and UV protection properties of the biosynthesized Zinc oxide nanoparticles for medical textile applications, Microbial Pathogenesis (2018), doi: https://doi.org/10.1016/j.micpath.2018.09.030. 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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In-Vitro cytotoxicity, antibacterial, and UV protection properties of the

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biosynthesized Zinc oxide nanoparticles for medical textile applications

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Amr Fouda1, Saad EL-Din Hassan1, Salem S Salem1 and Th. I. Shaheen2*

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Cairo, 11884, Egypt.

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Behouth St. (former El-Tahrir str.), Dokki, P.O. 12622, Giza, Egypt.

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Department of Botany and Microbiology, Faculty of Science, Al-Azhar University, Nasr City,

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National Research Centre (Scopus affiliation ID 60014618), Textile Research Division, El-

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*Corresponding Author: Dr. Tharwat Ibrahim Shaheen (PhD), Email:

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[email protected].

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Abstract

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Nowadays, medical textiles have become the most essential and developing part in

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human healthcare sector. This work was undertaken with a view to harness the bio-active

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macromolecules secreted by fungi e.g. proteins and enzymes in bio-synthesis of ZnO

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nanoparticles for multifunctional textiles such as antibacterial activity and UV protection

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with considering the cytotoxicity limitation. Herein, the isolated fungus, Aspergillus

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terreus, was allowed to produce proteins which has affinity to cape ZnO-NPs. Various

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factors affecting the behavior of the secreted proteins on the formed nanoparticles were

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investigated. Thorough characterizations of the protein capped ZnO-NPs were performed

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by the using of UV-Visible spectroscopy, transmission electron microscope (TEM)

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Fourier Transform-Infra Red (FT-IR) spectroscopy, X-ray diffraction (XRD) analysis and

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Dynamic light scattering analysis (DLS). Prior treatment of cotton fabrics with ZnO-NPs,

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the cytotoxicity of the protein capped ZnO-NPs was examined. After that, the

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antibacterial activity of the ZnO-NPs before and after treating of cotton fabrics, besides,

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the UV-protection (UPF) properties were investigated. Results obviously demonstrated

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the ability of the bio-secreted protein to cape and reduce ZnO to spherical ZnO-NPs with

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particle size lied around 10-45nm, as indicated form UV-vis., spectra TEM, Zeta sizer,

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FTIR and XRD. Regarding to the results of cytotoxicity, the treatment of the cotton

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fabrics with ZnO-NPs were performed at safe dose (20ppm). At this dose, ZnO-NPs

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loaded samples exhibited reasonable antibacterial activity against both Gram positive and

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Gram negative bacteria; besides, good UV-protection with reasonable increase in UVA

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and UVB blocking values. Indeed, nanotechnology based microbiological active

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molecules opens up new opportunities for us to explore novel applications in terms of

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green technology.

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Keywords: Zinc oxide nanoparticles; Biosynthesis; Antibacterial; cytotoxicity; UV

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protection; medical textile.

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1. Introduction

Bio-Nanotechnology is a correlation of both biology and nanotechnology. Most

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recently, bio-nanotechnology is growing up day by day as a quickly developing filed with

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its application in science and technology with the end goal of assembling new materials

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at the nanoscale level [1, 2]. In recent years, different industries as textile, health, food,

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chemicals, and cosmetic products incorporated with nanoparticles (NPs). The 2

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environmental friendly approach for the biosynthesis of nanoparticles is an opportunity to

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be safely applied in medical fields [3]. Combination of textile technology and medical

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science has resulted into a new field called medical textiles. New areas of applications for

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medical textiles have been identified with the development of new either fiber yarns

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technology or the combined functional biomedical materials such as nanoparticles. Green

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synthesis of NPs includes synthesis through plants, bacteria, actinomycetes, fungi [4], and

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algae permit large scale production of NPs free impurities [5].

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Zin oxide nanoparticles (ZnO-NPs) have attracts attention of many researchers for

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it is biological and chemical behavior which can be returned to their morphology. ZnO-

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NPs have been used in various biological application as drug delivery and destroying of

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tumor cell [6], antibacterial and antifungal activity [7], medical textile industry and UV

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filtering prosperities [8].

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There are several methods for ZnO-NPs synthesis as chemical vapor synthesis [9],

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sol-gel method [10] sonochemical [11] and thermal decomposition [12]. Processing steps

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and physical factor as pH, temperature and pressure for these methods are difficult

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controlling beside producing by-product which may be toxic for ecosystem; therefore,

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green synthesis of ZnO-NPs is most preferred. Fungi are simple handling and possess

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different variety of proteins and enzymes in their cell, therefore it is considered excellent

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candidates for ZnO-NPs synthesis.

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Optimization of physic-chemical parameters such as substrate concentration, pH

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and temperature, agitation rate for medium and incubation period play an important roles

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in green synthesis of nanoparticles [13, 14]. The activity of ZnO-NPs against tumor cell

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due to smaller nanoparticle size, generation of reactive oxygen species (ROS) and highly

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specific surface area [15]. In the current study, we aimed to fabricate the multifunctional medical textile

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through using of environmentally benign system. To achieve this goal, this work was

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designed to synthesize and optimize of ZnO-NPs through using of the secreted proteins

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by the isolated fungus Aspergillus terreus AF-1, in concurrent with investigation of their

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MIC (minimum inhibitory concentration) and in-vitro cytotoxicity of the biosynthesized

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ZnO-NPs prior applying of ZnO-NPs onto cotton fabrics. At safe concentration, ZnO-

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NPs were loaded onto cotton fabrics to also investigate both antibacterial activity and

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UV-protection properties.

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2. Materials and Methods

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2.1.Isolation and Identification of fungal isolate

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The soil fungus, Aspergillus terreus AF-1 was isolated from contaminated soil dye,

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Egypt (GPS N: 30o 15 54.51: E: 31o 45 39.95). Isolation of fungi was carried out by

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plating the inoculum on PDA after serial dilutions of soil sample and incubated at 28± 2

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°C for 3-4 days. Bacterial contamination was inhibited by supplementing the medium

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with chloramphenicol at a concentration of 10 µg mL-1 after autoclaving.

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Morphologically differed colonies were individually picked up and reinoculated on PDA

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for purification, and then kept at 4 °C for further study [16].

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Molecular identification was conducted based on amplification and sequencing of

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internal transcribed spacer (ITS) region. Genomic DNA was extracted using the protocol

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of Gene Jet Plant genomic DNA purification Kit (Thermo). The ITS region was amplified

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in polymerase chain reaction (PCR) using the genomic DNA as template and primers of

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ITS1

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TCCGCTTATTGATATGC-3 ̀). The PCR mixture (50µL) contained Maxima Hot Start

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PCR Master Mix (Thermo), 0.5µM of each primer, and 1µL of extracted fungal genomic

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DNA. The PCR was performed in a DNA Engine Thermal Cycler by Sigma Scientific

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Services Company (Cairo, Egypt) with a hot starting performed at 94ºC for 3 min,

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followed by 30 cycles of 94ºC for 0.5 min, 55ºC for 0.5 min, and 72ºC for 1 min,

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followed by a final extension performed at 72ºC for 10 min. The commercial sequencing

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was conducted using ABI 3730x1 DNA sequencer at GATC Company (Germany). The

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ITS sequence was compared against the GenBank database using the NCBI BLAST

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program. Sequences were then compared with ITS sequences in the GenBank database

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using BLASTN. Multiple sequence alignment was done using ClustalX 1.8 software

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package (http://wwwigbmc.u-strasbg.fr/BioInfo/clustalx) and a phylogenetic tree was

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constructed by the neighbor-joining method using MEGA (Version 6.1) software. The

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confidence level of each branch (1,000 repeats) was tested by bootstrap analysis.

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and

ITS4

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TCCGTAGGTGAACCTGCGG

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(5 ̀-

2.2.Biosynthesis of ZnO Nanoparticle using protein secreted fungi Aspergillus terreus AF-1 was grown up in 250 mL Erlenmeyer flask containing 100 mL

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Czapek Dox (CD) fermentative broth medium for 3 days at pH 6.0, 28±2oC, and shaking

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of 150 rpm. After incubation period, the fungal biomass was separated using Whatman

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filter paper No. 1 by filtration method, and washed thrice with sterile distilled H2O to

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remove any medium components. The harvested fungal biomass (15 g) were re-

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suspended in 100 mL sterile distilled H2O in an orbital shaker (150 rpm) for 48 h. at

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28±2oC. The cell-free filtrate was obtained by separating the fungal biomass using

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Whatman filter paper No. 1 and used for ZnO-NPs production as the following. Different ratios of Zinc acetate Zn(CH3CO2)2 were mixed and incubated with cell

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free filtrate at 28±2oC for 24h on orbital shaker (150 rpm). After completion of

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incubation period, a creamy-white precipitate, mainly Zn(OH)2, was indicated. The latter

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was separated and then dried at 150 oC for 48h. The bio-transformed product was

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eventually collected and subjected for further investigation.

2.3.Factors affecting ZnO-NPs production.

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Different variables such as incubation period, Zn (CH3CO2)2 concentrations (0.5mM to

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2.5mM), and pH (5.0-11) which influencing on the production and distribution of the

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particle size of ZnO-NPs were studied using UV-visible spectroscopy (JENWAY 6305

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Spectrophotometer) after resuspension in distilled water.

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2.4.Characterization of the optimized ZnO-NPs

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The particle size and shape of the biosynthesized ZnO-NPs were preliminary

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characterized by using transmission electron microscope (TEM) measurements through

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using drop coating method in which a drop of solution containing nanoparticles was

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placed on the carbon-coated copper grids and kept under vacuum desiccation for

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overnight before loading them onto a specimen holder. For further confirmation, the

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dynamic light scattering analysis (DLS) was employed in order to investigate the particle

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size distribution of synthesized nanoparticles as well as check the stability of the

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nanoparticles by indicating the zeta potential measurement.

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Moreover, the characteristic functional groups present in synthesized nanoparticles

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molecules were analyzed using Fourier Transform-Infra Red (FT-IR) spectroscopy. The

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samples were mixed with KBr (binding agent) and were loaded into discs at high

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pressure. These discs were scanned in the range of 400 to 4000 cm-1 to obtain FT-IR

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spectra. The crystalline structure of ZnO-NPs was characterized by XRD analysis.

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Finally, thermal properties of ZnO NPs were measured using Thermal Gravimetric

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Analysis (TGA/DTA-60H instrument (SHIMADZU) to determine the organic to

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inorganic contents. Decomposition profiles of TGA were recorded at a heating rate of

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10.0 0C/min between room temperature and 800.0 0C in nitrogen gas.

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2.5.Biological activity of the biosynthesized ZnO-NPs Antibacterial activity of ZnO-NPs

The antibacterial activity of biosynthesized ZnO-NPs was evaluated against Gram

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positive (Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6633) and Gram

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negative bacteria (Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 8739)

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using agar well diffusion method [17]. Each bacterial strain was swabbed uniformly onto

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individual Mueller Hinton agar plates. In each plate, wells was cut out using a standard

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cork borer (7 mm diameter). Using a micropipette, 100 µL of Zinc oxide nanoparticles

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(2000 µg/mL) was added into each well. After incubation at 37 °C for 24 h, the diameter

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of inhibition zones was measured in mm and the results were recorded. The experiments

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were performed in three replicates.

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Minimum Inhibitory Concentration (MIC)

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MIC of ZnO-NPs against previous Gram positive and Gram negative bacteria were

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checked by agar well diffusion method. Different concentration of biosynthesized ZnO-

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NPs (1000, 500, 250 and 125 µg/mL) were made. Each bacterial strain was swabbed

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uniformly onto individual Mueller Hinton agar plates, and wells were cut out using a

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standard cork borer (7 mm diameter). A 100 µL of each concentration was poured in to

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wells and the tested plates were incubated at 37 °C for 24 hours. The minimum

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concentration of ZnO-NPs showing inhibition zone was considered to be MIC.

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Experiment was performed in triplicates.

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In vitro cytotoxicity of ZnO-NPs against cancer and normal cells •

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Cell culture

The human colorectal adenocarcinoma cells (Caco-2), normal Vero cells (kidney of

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African green monkey) and normal rat liver epithelial cell (colne-9) were procured from

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ATCC.

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Cell morphology

Morphological changes in epithelial cells in response to ZnO-NPs, after 24 h of

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incubation were assessed by light inverted microscope (Nikon, Japan). Cells were

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cultured in 96-well plates at a density of 1 × 105 cells/well and treated with double fold

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dilution of biosynthesized ZnO-NPs (1000 µg/mL to 3.9 µg/mL).

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MTT assay

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The cytotoxicity of ZnO-NPs was evaluated by cell viability assay MTT [3-(4, 5-

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dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] using Caco-2 cell, Vero cell &

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Clone-9 cell. In brief, the cell (1 X 105 cells/mL) grown in 96-well plates and treated with

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different concentration of ZnO-NPs (double-fold dilution) represented as 1000, 500, 250,

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125, 62.5, 31.25, 15.62, 7.81 and 3.9 µg/mL for 24h at 37oC. The cell were future

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incubated with MTT (5 mg/mL in phosphate buffered saline) at 37oC / 5% CO2 for 1-5 h.

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Adding the formazan (MTT metabolic product dissolved in 200 µL DMSO) to 96-well

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plate. The color intensity was measured at 560 nm using an enzyme linked

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immunosorbent assay (ELISA) reader [18]. The experiments were performed in

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triplicates. The percentage cell viability was then calculated with respect to control (cells

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incubated without zinc oxide nanoparticles) as follows:

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Cell viability % = (sample absorbance / control absorbance) x100.

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Cell toxicity % =100 – [(sample absorbance / control absorbance) x100].

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2.6.Application of ZnO-NPs as a precursor for medical textiles •

Zinc oxide nanoparticles loading onto cotton fabrics based textiles

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Before being used, cotton fabrics were washed and dried. Experiments were performed

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on samples with maximum dimension of 30 cm × 15 cm. Cotton fabrics were padded

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with ZnO-NPs solution at certain safe concentration revealed from both MIC and in-vitro

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cytotoxicity investigation. For the successive treatment of fabrics with colloidal zinc

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oxide, the solution was agitated continuously. All samples were immersed in such colloid

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bath for 5 min then squeezed to 100% wet pick up with laboratory pad at constant

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pressure. Samples were dried at 80°C for 5 min, followed by curing at 150°C for 2 min.

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The following treatments were conducted: (1) Control which A] Trypticasene soya broth

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(TSB) without inoculation by bacterial culture (negative control) B] untreated fabrics

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emerged in TSB inoculated by bacterial culture (positive control) C] untreated fabrics

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emerged in TSB without inoculation (blank) (2) fabrics treated with zinc oxide

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nanoparticles solution [19].

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Scanning electron microscopy (SEM) for cotton fabrics

SEM was studied using a scanning electron –JSM-5400 instrument (Jeol, Japan). The

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specimens in the form of fabrics were mounted on the specimen stabs and coated with

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thin film of gold by the sputtering method.

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Assessment of antimicrobial activity of nanoparticles treated fabric.

The antimicrobial behavior of fabrics was evaluated against Gram positive bacteria

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represented by Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6633 and

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Gram negative represented by Pseudomonas aeruginosa ATCC 9027 and Escherichia

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coli ATCC 8739. Squares of 1 cm of each fabric were prepared in aseptic manner and

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placed in 5 mL TSB inoculated by 100 µL of microbial suspension (adjusted optical

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density for all bacterial culture at O.D. = 1 nm). The efficiency of the antimicrobial

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treatment was determined by comparing the reduction in optical density (O.D.630 nm) of

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the treated samples with that of positive control. Optical density is directly proportional

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to the number of bacteria in the medium. The bacteriostatic activity was evaluated after

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24 h and the percent reduction of bacteria was calculated using the following equation:

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R (%) = [(A - B) / A] × 100

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Where R= the reduction rate, A = the number of optical density for positive control, and

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B = the numbers of optical density for treated fabrics.

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Ultraviolet protection factor of fabrics

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The ability of treated and untreated fabric to block UV light is given by the ultraviolet

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protection factor value (UPF). The UPF value was evaluated in the range of 280 – 400

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nm with difference 10 nm by UV/Visible Spectrophotometer 3101 PC with a software

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version.

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Statistical analysis: The means of three replications and standard error (SEr ±) were calculated for all the results obtained, and the data were subjected to

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analysis of variance means by sigma plot 12.5 program.

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3. Results and discussions

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3.1.

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The fungus isolate Aspergillus terreus AF-1 was identified by molecular methods

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scheme 1. Fungal ITS fragment was identified from PCR and sequencing approaches

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(Amplification and sequencing of ITS region have resulted in approximately 600 bp).

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We constructed a maximum-likelihood phylogenetic tree to correlate our ITS

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sequence with previously described sequences, the results showed that the sequenced

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ITS fragment was related to the topology of Aspergillus terreus with a similarity of

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98%.

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Identification of the isolate Aspergillus terreus AF-1

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Scheme 1: Phylogenetic analysis of ITS sequences of the fungal strain with the

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sequences from NCBI. Symbol ■ refers to ITS fragments retrieved from this

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study. The analysis was conducted with MEGA 6 using neighbor-joining method.

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3.2. Biosynthesis of ZnO NPs using A. terreus AF-1 Over the past few decades, the green bio-synthesis of metallic nanoparticles and their

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oxides has been gained an importance suggested as possible alternatives to chemical and

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physical methods. Proteins and enzymes secreted by fungi have effective role in the

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reduction of ZnO to ZnO NPs, besides stabilizing these formed nanoparticles [20]. In this

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context, A. terreus AF-1 was isolated and identified as aforementioned in scheme 1 and

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then used as a bioreactor for green synthesis of ZnO NPs through harnessing bioactive

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macromolecules such as proteins and enzymes secreted therefrom. Appearance of a

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creamy-white precipitate after contacting of biomass filtrate with zinc acetate at the end

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of reaction indicated the formation of hydrate ZnO (ZnO.H2O). After calcination of the

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latter, ZnO-NPs were obtained as a white powder.

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3.3.Factors affecting on the biosynthesis ZnO-NPs

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Biosynthesis of ZnO-NPs were indicated by UV-visible spectroscopy as represented in

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Figure 1.

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Absorbance

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390 410 Wave length (nm)

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Figure 1: UV-vis spectrophotometer of the biosynthesized ZnO-NPs and their optical

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pictures picked during preparation process of: Zinc acetate solution (A), biomass

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filtrate (B), and white precipitate formed upon addition of zinc acetate to biomass

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filtrate (C).

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It was shown that the maximum absorption peak of the formed ZnO-NPs was occurred at

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wavelength 390 nm, due to its surface plasmon resonance. These observation is in

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agreement with the earlier studies on the biosynthesis of ZnO-NPs, which indicating that

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the characterization peak of ZnO-NPs is lied around wavelength of 320-390nm [21].

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Moreover, the absorption band in Figure 1 is slightly symmetrical with broad in width at

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wavelength 390nm. These could refer to the formation of spherical ZnO with

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polydispersed narrow sizes particles.

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Environmental conditions of the mixture of the fungal biomass filtrate and zinc

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acetate have been the critical component directly affecting the productivity of ZnO-NPs

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and also the process economics. The physical conditions of this mixture directly monitor

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the rate of enzyme activity which reflects on the synthesis of ZnO-NPs. Therefore

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optimization of physical parameters will not only support good growth but also enhance

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the product yield. Hence, in seek of the optimization of the biosynthesized ZnO-NPs, the

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UV-visible spectroscopy was used to determine the proper conditions applied during

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preparation by examining the absorbance of the obtained ZnO-NPs suspensions at

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wavelength 390 nm. Of these variables under investigation Zinc acetate concentration,

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pH and incubation period of biomass filtrate with zinc acetate are shown below in Figure

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2.

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Figure 2: Factors affecting the biosynthesis of ZnO-NPs as a function of absorbance

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at wavelength 390nm. A) Different zinc acetate concentration, B) Different pH

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values and C) Different incubation periods

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Effect of zinc acetate concentration

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Biosynthesis of ZnO-NPs with different concentrations of zinc acetate solution 0.5mM to

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2.5mM was studied. Data represented in figure 2A showed that, by increasing the

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concentration of zinc acetate, the absorbance at wavelength 390nm increases up to 2mM.

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These evidences indicated the high efficiency of the bio-active molecules (proteins and

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enzymes) comprised biomass filtrate on formation of ZnO-NPs at high concentration of

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zinc acetate. Further increasing in the zinc acetate into 2.5mM, the bio-active molecules

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unable to protect the formed ZnO from agglomeration which leads to aggregate to bigger

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sizes and hence, the absorbance decreased significantly [22].

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Effect of different pH values

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The effect of varying different pH values from 5 to 11 onto the biosynthesis of ZnO-NPs

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by A. terreus AF-1 is shown also in Figure 2B, which showed the highest productivity at

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maximum absorbance was occurred at alkaline pH-10. This could be attributed to the

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behavior of the bio-active molecules secreted by A. terreus AF-1 in the solution which

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acting as capping agent of ZnO-NPs is more stable and more reactive at alkaline medium

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than acidic [23].

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Effect of incubation periods

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The incubation period is a critical factor, which not only effect on the secretion of

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bioactive molecules such as proteins and enzymes, but also effect on the reducing affinity

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of ZnO to nanoparticles. Therefore, incubation period of the mixed biomass filtrate with

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zinc acetate of concentration 2.0mM was performed at pH 10. Data illustrated in Figure

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2C revealed that, the best time for extracellular biosynthesis of ZnO-NPs was obtained

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when merely the biomass of A.terreus AF-1 has been mixed with zinc acetate for duration

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of 2 days which the secretion of the bio-active molecules were yielded with highly

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concentration in the biomass filtrate. After 2 days of incubation age, the bio-active

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molecules may be, with the missing of the growth ingredients needed, the affinity of

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fungus biomass to release more of those bio-active molecules was limited and/or most of

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the secreted molecules tend to degrade or deactivate.

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3.4.

Characterizations of ZnO-NPs

TEM measurement was carried out to determine the morphology and the

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approximate size of the biosynthesized ZnO-NPs. Figure 3A revealed that, ZnO-NPs

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with spherical shape were formed successfully using A.terreus AF-1 filtrate with well-

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dispersed narrow sized particles surrounding with capping proteins and enzymes. The

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average size of the formed ZnO-NPs is lied in the range of 10-45nm.

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FT-IR analysis was performed to detect any chemical changes in functional groups.

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Figure 3B is a typical FT-IR for biomass filtrate and ZnO-NPs. Results from biomass

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filtrate spectra showed that, maximum absorption peak seen at near 1650cm-1 which is

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related to (C=O) stretching vibration of peptide bonds and the peak near 3300 cm-1 is

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thought to be N-H bending vibration. On the other side, the maximum spectrum peaks

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picked from ZnO NPs spectra are seen at 3421, 2937, 2359, 1575, 1401, 1025, 940, 676

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and 512 cm-1. Indeed, the peak at 512 cm-1 is the characteristic peak related to the

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absorption of Zn-O bonds, while peak found at 1025cm-1 is corresponding to the C-O

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stretching in amino acids. Maximum absorption peak at 3421 cm-1 may be attributed to

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the characteristic absorption of OH- and NH- groups. Furthermore, the intensity of NH-

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band occurred near 3300 cm-1 were decreased accompanied with shifting of (C=O) amide

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absorption band to lower wavelength 1544cm-1. This obvious refers to the coordinating

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bonds occurred between ZnO and proteins secreted fungi. From above results, it is clearly

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evident that, the proteins secreted biomass filtrate from fungus A. terreus AF-1 have the

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main role in size reduction of ZnO with narrow size distribution in well stabilized

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nanoform.

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On the other hand, the crystalline nature of ZnO nanoparticles were confirmed by XRD

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analysis (Figure 3C). XRD spectra showed well defined peaks at 2θ values 31.6°, 34.5°,

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36.5°, 47.45°, 56.55°, 62.82°, 66.14°, 67.97° and 68.93° corresponding to (100), (002),

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(101), (102), (110), (103) and (112) of ZnO-NPs, which revealing that the sample was

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polycrystalline wurtzite structure (Zincite, JCPDS 5-0664).

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On the other hand, the size and size distribution of ZnO nanoparticles in colloidal

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solution were analyzed by the making use of DLS technique (Figure 3D). The results

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represented in Figure 3D make it evident that, the particles obtained were polydispersed

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mixture with average size of 175.85 nm (95.4%). Considering the presence of the capping

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proteins and enzymes surrounding particles, the hydrodynamic radius obtained from DLS

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does not give more information about the individual ZnO-NPs, however, it is considered

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as a good to measure oval all dimension besides the width of nanoparticles and the

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polymers which covering the particles. Whereas, the stability of colloidal solution which

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depending on the surface charge of the formed nanoparticles was determined by zeta

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potential analysis. In general, the particles keep away each other without any aggregates,

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when the particles in a colloidal solution have large negative or positive zeta potential

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values (greater than +30 mV or smaller than −30 mV) [24]. The biosynthesized ZnO-NPs

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were highly stabled as have a zeta potential found to be -46.6 mV. This high value

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confirms the high stability of colloidal solution.

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Moreover, TGA of ZnO-NPs was shown in Figure 3E. The initial weight loss is assigned

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to be 13.57% which due to water evaporation. The major weight loss occurred between

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temperature 151 0C – 577 0C, which about 14.62% of the original weight. This weight

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loss is attributed to the thermal decomposition of the organic compounds presented in the

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sample under investigation. Whereas, the remaining weight after 800 0C is related to ash

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containing inorganic Zn. The close examination from the TGA results, it would reveal

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that, ZnO-NPs were covered with 14.62% of proteins secreted fungi.

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Figure 3. A) TEM image, B) FTIR spectra, C), XRD pattern, D) Particle size

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distribution and E) TGA of ZnO-NPs synthesized by Aspergillus terreus AF-1.

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•

Antibacterial activity of ZnO-NPs.

The critical importance of ZnO-NPs being in their activity against abroad spectrum of

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human pathogenic bacteria. Therefore, ZnO-NPs synthesized using A. terreus AF-1 were

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investigated for their antibacterial activity against both Gram positive and Gram negative

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bacteria. In general, the inhibitory action for biosynthesized ZnO-NPs which represented

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by diameter of clear zone was more effective against Gram positive than Gram negative

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bacteria. However, the diameter of clear zone formed due to 100 µL of ZnO-NPs (2000

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µg/mL) was 20.2±0.2, 19.1±0.2, 14.2±0.2 and 14.1±0.2 for Bacillus subtilis,

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Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, respectively

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(Figure 4). The inhibitory action for biosynthesized ZnO-NPs may be due to reacting of

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nanoparticles with bacterial protein by combining the thiol (-SH) groups, and hence leads

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to inactivation of proteins and bacterial growth [25]. Gupta, et al., [26] explained the

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mechanism of nanoparticles in bacterial growth reduction through interact with

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phosphorous moieties in DNA, which lead to inactivation of DNA replication and hence

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inhibition of enzymes functions. Whereas, Manke, et al., reported a new mechanism

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which showing that nanoparticles are lead to changes in biological activities including

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reactive oxygen species (ROS) thereby cause membrane damage leading too cell death

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[27]. The exact mechanism of antimicrobial generated by NPs are not completely well

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understood yet.

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3.5.

Minimum Inhibitory Concentration (MIC)

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The diameter of inhibition zone and its relation to the concentration of nanoparticles were

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studied by Shaheen and Fouda [28] to confirm the antibacterial activity was dose 20

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dependent. Therefore, it is compulsory to detect the MIC to ZnO-NPs for each bacterial

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strain. To this end, different concentrations of ZnO-NPs (1000, 500, 250 and 125 µg/mL)

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were used. Regards to the data represented graphically in Figure 4, the obtained MIC for

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Gram Positive Bacillus subtilis, Staphylococcus aureus was 250 µg/mL with clear zone

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11.0±0.1mm and 10.5±0.2mm respectively. Whereas, the obtained MIC for Escherichia

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coli and Pseudomonas aeruginosa was 500 µg/mL with inhibition zone 9.2±0.1mm and

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9.3±0.3mm, respectively.

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Figure 4. Antibacterial activity and MIC for ZnO-NPs at different concentrations (2000, 1000,

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500, 250 and 125 µg/mL) against different human pathogenic bacteria

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3.6.

In vitro cytotoxicity of ZnO-NPs

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Cell morphology

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Either variation in cell shape or cell morphology in culture is the first as well as the most

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notably observation shown after exposure to nanoparticles or any toxic materials.

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Therefore, the light inverted microscope could be used to monitor the cell shape and 21

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morphological changes dependent on ZnO-NPs exposure dose. The normal cell line

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spread continuously on plate and showed epithelial characteristic morphology. The cell

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exposed to different concentrations of ZnO-NPs gradually lost their phenotypic character.

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As shown in Figure 5, at high concentration of ZnO-NPs, the cell become partial or

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complete loss of monolayer, rounding, shrinking or cell granulation when compared to

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untreated control cells. The light inverted microscope image proved, undoubtedly, that

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the damage caused in cell morphology is ZnO-NPs dose dependent.

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Figure 5. Light inverted micrographs proved morphological change for three type of cell

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lines exposure to different concentration of ZnO-NPs. A) Clone-9 cell. B) Vero cell and

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C) Caco-2 cell.

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MTT assay

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Viability assays are basic stage in toxicology that explain the cellular response to a

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toxicant. Also, they give information on the cell death, survival, and metabolic activities

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[29]. MTT assay is sensitive colorimetric assays for the determination of the number of

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viability in cell proliferation and cytotoxicity assays. Data represented in Figure 6

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revealed that, cell mortality for normal and cancer cells were also dose dependent when

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exposure to different concentrations of ZnO-NPs. Data showed that, the IC50 for normal

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cell represented by clone-9 and vero cell were 26.85 µg/mL and 27.05 µg/mL,

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respectively. Whilst, IC50 for cancer cell (Caco-2) was 79.57 µg/mL.

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treatment of cotton fabrics with ZnO-NPs is highly recommended to be performed at

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concentration lower than 26.85 µg/mL, in order to keep the process safe for human.

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Thus, the

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Figure 6. Cell mortality for Clone-9 cell, Vero cell and Caco-2 cell at different

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concentrations of ZnO-NPs synthesized by A. terreus AF-1.

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3.7.Application of the biosynthesized ZnO-NPs into cotton fabric.

Owing the unique properties of ZnO-NPs towards antibacterial activity and UV-

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protection, the biosynthesized ZnO-NPs have been used in multifunctional textile fabrics.

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In the context, ZnO-NPs were applied to cotton fabrics according to method mentioned in

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our previous works [30]. ZnO-NPs were resuspended in distilled water to get final

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concentration of 20 µg/mL (20ppm). Moreover, the deposition of ZnO-NPs on cotton

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fabrics and their chemical composition were demonstrated by SEM-EDX instrument

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(Figure 7A-D). The deposition of ZnO-NPs was clearly shown in the SEM image of the

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treated cotton fabrics (Figure 7B), which indicates the homogenously distribution of

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ZnO-NPs on the cotton fabrics surface whenever compared with control sample (Figure

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7A) which seemed very smooth surface (Figure 7A). Furthermore, the chemical

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compositions of the treated cotton fabrics were determined by Energy Dispersive X-ray

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spectrometer (EDX) as showed in Figure 7C and D. The obtaining results showed that,

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zinc element in zinc oxide nanoparticles occupied 2% of total number of elements found

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in the tested sample as concluded from mapping image (Fig. 7C), whereas the weight

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percent of the Zinc was about 1.03 % as calculated from EDX spectra (Fig. 7D).

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Figure 7. SEM image of: (A) untreated cotton fabrics with ZnO-NPs, (B) treated cotton

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fabrics with ZnO-NPs, and (C) mapping picture of the surface of treated fabrics with

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ZnO-NPs. (D) EDX of treated sample with elemental analysis of the ZnO-NPs contents.

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3.8.Efficiency of the nano-zinc particles as antibacterial agent

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Cotton fabrics treated with nano-sized zinc colloids solution exhibit moderate

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antibacterial efficiency against both Gram positive and negative bacteria. At 20ppm of

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nano-zinc oxide colloid solution used for treatment, the reduction of bacterial growth was

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82.2±0.3 % and 81.9±0.2 % for Staphylococcus aureus and Bacillus subtilis,

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respectively. While bacterial reduction for gram negative bacteria represented by

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Pseudomonas aeruginosa and Escherichia coli were 74.8±0.5 % and 75.4±0.3 %,

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respectively.

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3.9.UV-protection of Cotton fabrics loaded by zinc oxide nanoparticles

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The absorption characteristic of the treated cotton fabrics with ZnO-NPs compared with

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untreated fabrics, which expressed as UPF values and blocking percent for UVA and

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UVB are represented in Table 1. It can be concluded that, the UPF value for treated

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cotton fabrics was 16.1 which is a good protection index for the treated fabrics with ZnO-

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NPs according to Australian/New Zealand Standard AS/NZS 4399:1996 [31], compared

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with UPF value of 4 for untreated fabrics which consider a bad protection index [31]. UV

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is the form of invisible radiation emitted from sun and classified into UVA (315-400nm)

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and UVB (290-315nm) which damages collagen fibres and accelerates skin ageing but

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UVB is more dangerous than UVA because it is directly destroy DNA and causes skin

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cancer [32]. Therefore, data presented in Table 1 revealed that, ZnO-NPs loaded cotton

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fabrics can be blocking UVA and UVB at 76.3% and 85.4%, respectively, when

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compared to untreated fabrics which blocking at 66.9% and 79% respectively.

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Table 1. UPF protection values of untreated and treated cotton fabrics with ZnO-NPs. Fabrics

UPF

protection Transmittance (%)

value

UVA

UVB

(315-400nm)

(290-315nm)

4

29.1

14

Treated

16.1

23.7

10.6

UVA

UVB

66.9

79

76.3

85.4

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Blocking (%)

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4. Conclusion

Although lots of chemical and physical methods were frequently used for the synthesis of

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ZnO-NPs, but these methods possess several disadvantages through either toxic solvents

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used or undesirable by-products yielded in addition to their high energy consumption.

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Therefore, the main objectives of the current study were to produce ZnO-NPs by

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microbial action and to apply these ZnO-NPs to fabricate the multifunctional medical

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textile fabrics with the safe dose during application. To achieve these goals, the work

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was designed to bring into synthesis of ZnO-NPs using biological method prolonging

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with the investigation of their both antibacterial activity and in-vitro cytotoxicity prior

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applying of ZnO-NPs into cotton fabrics for rendering them UV-protection and

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antibacterial properties with safe dose. Therefore, the fungus Aspergillus terreus AF-1

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was successfully used in extracellular biosynthesis of ZnO-NPs using fungus biomass

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filtrate without addition of any hazardous materials, followed by optimization of the

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biosynthesized ZnO-NPs and then characterized by UV-Visible spectroscopy,

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transmission electron microscope (TEM) Fourier Transform-Infra Red (FT-IR)

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spectroscopy, X-ray diffractometry (XRD) analysis and Dynamic light scattering analysis

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(DLS). Results from UV-vis. Spectra revealed that ZnO-NPs were successfully

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synthesized by dint of Aspergillus terreus AF-1 at optimum conditions include incubation

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period of the biomass filtrate with (2.0mM) zinc acetate for 48h at pH 10. At these

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conditions, an absorption peak was observed at maximum wavelength 390nm which

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attributed to the SPR of ZnO-NPs. Further, TEM micrographs confirmed the formation of

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well-dispersed spherical nanoparticles with an average size of 10-45nm. Furthermore, the

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particle size analyzer revealed that ZnO-NPs were formed as a polydispersed mixture

525

with average size of 175.8nm, which could be due to presence of covering layer of

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bioactive secreted molecules surrounding nanoparticles. Thus, the zeta potential was

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raised to be -46.6 mV, indicating the high stability of the nanoparticles formed.

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On the other hand, the antibacterial activity and in-vitro cytotoxicity of biosynthesized

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ZnO-NPs were assessed. Results revealed that, ZnO-NPs have inhibitory action against

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Gram positive ((Staphylococcus aureus and Bacillus subtilis) more than Gram negative

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(Pseudomonas aeruginosa and Escherichia coli) bacteria with MIC 250 and 500 µg/mL,

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respectively. However, ZnO-NPs showed morphological change and cytotoxic effect on

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Clone-9, Vero and Caco-2 cell line at high concentrations. Below this concentration,

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ZnO-NPs were loaded onto cotton fabrics to render them both antibacterial activity and

535

UV blocking properties for application in medical purposes. Our results confirmed that,

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fabrics loaded with biosynthesized

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bacterial growth of Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa

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and Escherichia coli with percent 82.2±0.3, 81.9±0.2, 74.8±0.5

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respectively with UPF value 16.4 (good UV protection) compared to untreated fabrics.

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ZnO-NPs had an ability to inhibit pathogenic

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and 75.4±0.3 %,

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5. References

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[16] Fouda A, Khalil A, El-Sheikh H, Abdel-Rhaman E, Hashem A. Biodegradation and detoxification of bisphenol-A by filamentous fungi screened from nature. J Adv Biol Biotechnol. 2015;2:123-32. [17] Wang Y, Lu Z, Wu H, Lv F. Study on the antibiotic activity of microcapsule curcumin against foodborne pathogens. International journal of food microbiology. 2009;136:71-4. [18] Philip S, Kundu GC. Osteopontin Induces Nuclear Factor κB-mediated Promatrix Metalloproteinase-2 Activation through IκBα/IKK Signaling Pathways, and Curcumin (Diferulolylmethane) Down-regulates These Pathways. Journal of Biological Chemistry. 2003;278:14487-97. [19] Fouda A, Shaheen TI. Silver Nanoparticles: Biosynthesis, Characterization and Application on Cotton Fabrics. Microbiology Research Journal International. 2017;20:14. [20] Bhuyan T, Mishra K, Khanuja M, Prasad R, Varma A. Biosynthesis of zinc oxide nanoparticles from Azadirachta indica for antibacterial and photocatalytic applications. Materials Science in Semiconductor Processing. 2015;32:55-61. [21] Vennila S, Jesurani SS. Eco-friendly green synthesis and characterization of stable ZnO Nanoparticle using small Gooseberry fruits extracts. International Journal of ChemTech Research. 2017;10:5. [22] Rao CR, Trivedi D. Synthesis and characterization of fatty acids passivated silver nanoparticles—their interaction with PPy. Synthetic Metals. 2005;155:324-7. [23] Yedurkar S, Maurya C, Mahanwar P. Biosynthesis of zinc oxide manoparticles using Ixora coccinea leaf extract—A green approach. Open J Synth Theory Appl. 2016;5:1-14. [24] Meléndrez M, Cárdenas G, Arbiol J. Synthesis and characterization of gallium colloidal nanoparticles. Journal of colloid and interface science. 2010;346:279-87. [25] El-Rafie M, Mohamed A, Shaheen TI, Hebeish A. Antimicrobial effect of silver nanoparticles produced by fungal process on cotton fabrics. Carbohydrate polymers. 2010;80:779-82. [26] Gupta P, Bajpai M, Bajpai S. Investigation of antibacterial properties of silver nanoparticleloaded poly (acrylamide-co-itaconic acid)-grafted cotton fabric. Journal of Cotton Science. 2008. [27] Manke A, Wang L, Rojanasakul Y. Mechanisms of nanoparticle-induced oxidative stress and toxicity. BioMed research international. 2013;2013. [28] Shaheen TI, Fouda A. Green Approach for one-pot Synthesis of Silver Nanorod using Cellulose Nanocrystal and their Cytotoxicity and Antibacterial assessment. International Journal of Biological Macromolecules. 2017. [29] Popescu R, Heiss EH, Ferk F, Peschel A, Knasmueller S, Dirsch VM, et al. Ikarugamycin induces DNA damage, intracellular calcium increase, p38 MAP kinase activation and apoptosis in HL-60 human promyelocytic leukemia cells. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis. 2011;709:60-6. [30] El-Rafie MH, Shaheen TI, Mohamed AA, Hebeish A. Bio-synthesis and applications of silver nanoparticles onto cotton fabrics. Carbohydrate polymers. 2012;90:915-20. [31] 4399 AN. Sun protective clothing - evaluation and classification. 1996. [32] Dubrovski PD. Wowen fabric and ultraviolet protection. Woven fabric engineering: InTech; 2010.

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Highlights Spherical narrow-sized ZnO-NPs were prepared using fungus Aspergillus terreus. Optimization of the biosynthesized ZnO-NPs was performed.

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Cytotoxicity dose of ZnO-NPs prior application was studied.

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Antibacterial activity and UV-protection of the treated cotton fabrics were investigated at safe ZnO-NPs dose.