Airborne Nanoparticle Release and Toxicological Risk from Metal

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Airborne nanoparticle release and toxicological risk from metal oxide-coated textiles: toward a multi-scale safe-by-design approach Paride Mantecca, Kaja Kasemets, Archana Deokar, Ilana Perelshtein, Aharon Gedanken, Yeon Kyoung Bahk, Baharh Kianfar, and Jing Wang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b02390 • Publication Date (Web): 17 Jul 2017 Downloaded from http://pubs.acs.org on July 18, 2017

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Environmental Science & Technology

Airborne nanoparticle release and toxicological risk from metal oxide-coated textiles: toward a multi-scale safe-by-design approach P. Mantecca1*, K. Kasemets1,2, A. Deokar3, I. Perelshtein3, A. Gedanken3, Y.K. Bahk4,5, B. Kianfar5, J. Wang4,5 1

Department of Earth and Environmental Sciences, Research Center POLARIS, University of Milano-Bicocca, Milan, Italy 2

Laboratory of Environmental Toxicology, National Institute of Chemical Physics and Biophysics, Tallinn, Estonia 3

Department of Chemistry and Nanomaterials, Bar-Ilan University Center for Advanced Materials and Nanotechnology, Ramat-Gan, Israel

4

Laboratory for Advanced Analytical Technologies, Empa - Swiss Federal Laboratories for Materials Science and Technology, Dübendorf, Switzerland 5

Institute of Environmental Engineering, ETH Zurich, Zurich, Switzerland

*Corresponding author: Paride Mantecca, Department of Earth and Environmental Sciences, Research Centre POLARIS, University of Milano Bicocca, 20126- I, Milan. Tel. +39 02 64482916; email: [email protected] 1 ACS Paragon Plus Environment


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Abstract

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Nano metal oxides have been proposed as alternatives to Silver (Ag) nanoparticles (NPs) for

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antibacterial coatings. Here cotton and polyester-cotton fabrics were sonochemically coated with

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Zinc oxide (ZnO) and Copper oxide (CuO) NPs. By varying the reaction solvent (water or

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ethanol), NPs with different sizes and shapes were synthesized. The cytotoxic and pro-inflammatory

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effects of studied NPs were investigated in vitro in human alveolar epithelial A549 and

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macrophage-like THP1 cells. To understand the potential respiratory impact of the NPs, the coated

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textiles were subjected to the abrasion tests and the released airborne particles were measured.

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Very little amount of the studied metal oxides NPs were released from abrasion of the textiles

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coated by the ethanol-based sonochemical process. The release from the water-based coating was

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comparably higher. Lung and immune cells viability decreased after 24 h exposure only at the

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highest studied NPs concentration (100 µg/mL). Differently from the ZnO NPs, both formulations

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of CuO NPs induced IL-8 release in the lung epithelial cells already at sub-toxic concentrations (1-

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10 µg/mL) but not in immune cells. All the studied NPs did not induce IL-6 release by the lung and

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immune cells. Calculations revealed that the exposures of the NPs to human lung due to the

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abrasion of the textiles were lower or comparable to the minimum doses in the cell viability tests

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(0.1 µg/mL), at which acute cytotoxicity was not observed.

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The results alleviate the concerns regarding the potential risk of these metal oxide NPs in their

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applications for the textile coating and provide insight for the safe-by-design approach.

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Keywords: metal oxide nanoparticles, antibacterial coated textiles, airborne particles, toxicity, safe-by-design

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1. Introduction

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The increase of infectious diseases is at the forefront among the concerns to human health at a

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global scale with extremely high social and economic costs, especially the nosocomial infections

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typically derived from pathogen bacterial exposure in hospitals or healthcare service units. It has

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been estimated that over 4 million people are under risk to contract a healthcare associated

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infection, with 37,000 deaths estimated annually.1 The development and use of antibacterial

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coatings has been identified as an important mitigation strategy against infectious diseases and

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today represents an intensively explored research field, with very promising possibility for

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industrial upscaling.2 The goal of the material science in this field is primarily the achievement of

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the most effective antimicrobial coatings (AMCs) and up to now the devices impregnated with

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antibiotics or silver represent the most widely-used ones.2 Even after the nanotechnology boom,

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silver (Ag) has been identified as the most promising element in coating technologies to fight

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against bacteria, with the toxicity properties even enhanced by the manipulation of the materials at

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the nanoscale.3 Effective nanoAg-coated textiles and other materials have been indeed marketed but

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recently many doubts have been expressed by the scientific community toward the environmental

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and health safety of nanoAg containing materials, based on the abundant literature reporting the

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high toxicity on environmental organisms and humans.4 A COST Action CA15114 network has

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been established to address the sustainable development of antibacterial coatings especially in

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healthcare settings and a recently published opinion paper points out the strategies to design and use

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safe antimicrobial coatings.5 The safe-by-design approach has been especially considered in that

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paper as a pivotal tool to achieve the goal of producing and using new generation safer AMCs.

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Textiles are among the materials with the highest demand for antimicrobial functional coating, due

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to their ubiquitous use in both healthcare settings (e.g. pajamas, bedsheets) and in house furniture

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(e.g. upholstery). The growing need for antibacterial textiles has resulted in revolutionary progress

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in the textile industry6, leading to new technologies and products able to improve the antibacterial

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efficacy and to concomitantly reduce the environmental and health hazard. 3 ACS Paragon Plus Environment


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In the last decade, the design of new methods of fabric finishing has included the use of metal and

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metal oxide nanoparticles (NPs) that have high surface areas, biocidal properties and can be finely

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spread on the surface of the substrates. The metal oxides can be deposited as a separate phase or in a

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combination of composite nanostructured materials. Most of the methods for antibacterial finishing

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of textiles are based on multistage procedures and require toxic templating and binding agents for

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the anchoring of the nanoparticles on the substrate.7 In a previous review8 , the development of the

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sonochemical technique for doping/deposition of nanoscale metal and metal oxides on ceramic and

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polymer substrates was described and explained. The unique properties that make ultrasound

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radiation an excellent technique for the adherence of nanoparticles to a large variety of substrates

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were reported, as well as the advantages of sonochemistry as a one-step, environmentally-friendly

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method for antimicrobial finishing of textiles.9

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To impart antibacterial properties to textiles, also in view of nanoAg replacement, we have

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employed metal oxide NPs of CuO, ZnO, and lately Cu0.89Zn0.11O10-12, all formed by the basic

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hydrolysis of the corresponding metal acetates.

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In this work, the sonochemical process for the synthesis and simultaneous deposition of metal oxide

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NPs on fabrics has been improved by making the synthetic route safer and more cost efficient using

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water as alternative to ethanol:water solution. Previously, the metal oxide NPs were prepared by the

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basic hydrolysis of the corresponding metal acetates in 9:1 ethanol:water (v:v). In addition to

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ethanol-based traditional coating, in the current study water-based metal oxides NPs coated cotton

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and polyester-cotton (65/35) were produced. Coatings with NPs differing in size and shape have

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been obtained, making the toxicological and the adhesion to the surface studies of great importance

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to address the safety of such new materials.

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The consumer products with NPs – metal oxides included - as coatings or additives are indeed

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subject to mechanical impact such as wear and tear, washing, scratching, etc., during their whole

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life cycle and this may lead to release of NPs and human exposure. Release of AgNPs from fabrics 4 ACS Paragon Plus Environment

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and textiles have been widely reported13-18 and release to water by washing has been the most

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studied process. The sonochemical technique is a relatively new method to coat NPs on textiles and

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the release from such textiles into air has not been investigated to our knowledge.

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The released NPs in the airborne form are particularly worrisome due to their high mobility in the

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air and the possibility to enter human body via inhalation. Airborne NP exposure with inhalable

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uptake is viewed as the most critical exposure route among various pathways including ingestion

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and dermal penetration.19 For example, more realistic in vivo inhalation studies were recommended

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with aerosolized carbon nanotubes.20, 21 Airborne released NPs may directly target lung epithelial

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and immune cells. This is why in the present work the effects of CuO and ZnO NPs have been

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investigated in human lung epithelial A549 and THP-1– macrophage-like cells.

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The Taber Abraser is a widely used device to study abrasion resistances of coated fabrics or flexible

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materials with its own international standards (ISO 5470-1:2016; ASTM G195-08). Several studies

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have used the Taber Abraser to generate particles from nanocomposites, to measure the particle size

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distribution, to search for released NPs and to quantify them.22-26 In the present study, the

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sonochemically coated textiles were subjected to abrasion tests with the Taber Abraser and the

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generated airborne particles were measured. The results gave us the opportunity to investigate how

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to reduce the NP release and toxicity at the synthesis step, in line with the safe-by-design approach.

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Both the eco-toxicity and hazards to human by metal oxide NPs have been critically investigated.4,

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27-31

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and size and shape have been indicated as relevant NP properties able to modulate the adverse

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biological effects.30, 31 Inhalation is likely one of the most significant routes of exposure not only

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from an occupational standpoint, but also by consideration of the use and disposal of nano-enabled

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materials. Nano-sized particles indeed have a potentially high efficiency for deposition in the

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respiratory system, and can target both the upper and the lower parts of it. They are also retained in

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the deep lung for long periods and are able to induce stronger oxidative stress and inflammatory

CuO and ZnO NPs have been reported to induce significant toxicity in in vitro and in vivo30,



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burden compared to their fine-sized equivalents.32 These aspects make the inhalation toxicity

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studies mandatory for those nanomaterials (NMs) that are potentially exposed to human in the

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airborne form.

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Oxidative stress, lipid peroxidation, cell membrane damage and oxidative DNA damage were

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reported to affect human lung epithelial cells when exposed in vitro to CuO and ZnO NPs at

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increasing doses and time, with a relevant contribution to the toxicity coming from the nano-

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structure and the solubilized metal ions.33-38 The adverse effects on the respiratory system may

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change according to the NP physical and chemical properties and many evidences exist on the

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possibility to weaken the adverse effects of these two NPs by controlling the influential factors, e.g.

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the NP size and surface properties. 39

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By comparing the results obtained from the physical and chemical characterization of the various

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NPs and coated textiles, the toxicity responses to the synthesized NPs with different sizes and

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shapes and the abrasion experiments pointing to processes leading to less airborne particle release.

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This study aims to address one of the main safety aspects related to the use of nano-antimicrobial

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coated textiles. The results are helpful for design and development of safer NP coated textiles.

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2. Materials and Methods

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2.1 Coating of fabrics with ZnO and CuO NPs

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The coating of NPs on fabric was done using the roll to roll sonochemical installation.40 Two types

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of textiles were used for coating: 100% cotton and hybrid fabric polyester-cotton (65/35).

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Copper/zinc acetates (0.01M) were dissolved in 400 ml double distilled water (ddH2O). A 9/1

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ethanol:water (E) volume ratio solution was obtained after adding 3.6 L of ethanol. In case of water

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synthesis (W), ethanol was replaced by ddH2O. The solutions were heated by the ultrasonic

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transducers and after a temperature of 600C was reached, an aqueous solution of ammonium

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hydroxide (28-30%) was injected into the reaction cell to adjust the pH to ~8.0. At the end of the 6 ACS Paragon Plus Environment

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reaction, the color of the fabric changed from white to brown in the case of CuO NPs, whereas in

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the case of ZnO NPs it remained white due to curdy white precipitate of ZnO NPs. The coated

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fabrics were washed once with ddH2O and once with ethanol, and then dried under vacuum.

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2.2 ZnO and CuO NP powders: collection and characterization

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In parallel to coating process, powder of corresponding metal oxides NPs was formed during the

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reaction. At the end of the reaction, the NPs were collected, centrifuged and dried. The following

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NPs samples were obtained: water-based ZnO and CuO (wZnO, wCuO) and ethanol-based ZnO and

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CuO (eZnO, eCuO). The same acronyms are used to indicate the differently coated fabrics.

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The X-ray diffraction (XRD) patterns of the powders were determined using a Bruker D8

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diffractometer with Cu Kα radiation. The particle morphology and size distribution were studied

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with a high-resolution scanning electron microscope (HRSEM), FEI. The Cu and Zn concentrations

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on the fabric were determined by ICP analysis, ULTIMA 2 model, by dissolving the coating from

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the fabric using 0.5 M HNO3.

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2.3 Abrasion test

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The experimental set-up for the abrasion experiments is shown in Figure 1. In the left most part, the

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samples were abraded with a Taber Abraser (Model 5135, Taber, NY, USA). We used one abrasive

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wheel on the Taber Abraser to abrade the sample continuously. The sample rotated 60 times per

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minute, and the applied weight on the wheel was 0.75 kg. The samples using cotton as the base

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fabric were abraded using the abrasive wheel CS-10 composed of resilient binder, aluminum oxide

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and silicon carbide abrasive particles; the polyester-cotton based samples were abraded using a

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rubber wheel CS-0 with S-42 sandpaper strip consisting of very fine aluminum oxide grit. The slow

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dry abrasion process was conducted for 400 cycles.

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For the particle measurement and collection, a small enclosure chamber was constructed to reduce

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the background particle concentration. A tube for particle collection was inserted in the enclosure



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chamber from the top and placed close to the abrasion area. The details of the enclosure chamber,

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tube inlet and particle loss in the tubing can be found elsewhere.25

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2.4 Airborne particle measurements

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The released particles were characterized by aerosol measurement devices as shown in Figure 1. In

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order to determine the full size range of the abraded particles, two different aerosol instruments

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were employed. For particles with sizes in the nanometer range (∼13-520 nm) a scanning mobility

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particle sizer (SMPS, Model 3080, TSI, MN, USA) was used. For particles in the micrometer range

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(∼0.5-20 µm) an aerodynamic particle sizer (APS, Model 3321, TSI, MN, USA) was applied. The

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scanning time was set for APS and SMPS 30 sec and 60 sec, respectively. To obtain reliable data,

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each sample was measured more than three times, each time using a new abrasive of the same

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wheel type. The background of each experiment was measured in the enclosure chamber without

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the abrasion operation for three times and averaged.

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A filter was used for collection of particles either for toxicity tests or for characterization by SEM.

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SEM samples were collected on a Nuclepore track-etch membrane filter (pore size 0.2 or 0.4 µm,

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Whatman, UK) and coated with Pt. The imaging was carried out using Nova NanoSEM 230 (FEI,

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Hillsboro, OR, USA). The flow in the experimental set-up was provided by a vacuum pump with

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the fixed flow rate 15 L/min.

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2.5 Data analysis and modeling using the measurement results of the released particles

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The number-size data of the released particles measured by the APS and SMPS were statistically

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analyzed by the analysis of variance (ANOVA) method, which is a common method for more than

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three groups of data to determine whether they are significantly different from each other.

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The multiple-path particle dosimetry model (MPPD2)41, 42 was employed to simulate inhalation and

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deposition of released particles in a human lung. Bi-modal particle distributions combining the

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SMPS and APS measurement results were used in the model. The SMPS and APS distributions

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were simply combined without any adjustment. The parameters used in the calculation, such as the 8 ACS Paragon Plus Environment

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lung physiological parameters and particle statistical data, are presented in Table S4. Potential

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particle depositions on the lung alveolar cells were estimated based on the total surface area of

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human lung and mean surface area of alveolar cell 57.22 m2 and 5100 µm2, respectively.43, 44 The

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calculated particle depositions on the lung cells were used as reference values for the doses in the

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cell toxicity tests.

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2.6 Cell cultures and NPs treatment

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Human pulmonary adenocarcinomic alveolar basal epithelial cell line A549 (ECACC 86012804)

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and human monocytic leukemia cell line THP-1 (ATCC TIB-202) were used for NPs toxicity

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testingA549 cells grow as an adherent monolayer and THP-1 monocytes as a suspension culture.

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Phorbol-12-myristate-13-acetate (PMA) and 1,25-dihydroxyvitamin D3 (VD3) are commonly used

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to differentiate THP-1 monocytes to macrophage-like cells.45 During the differentiation THP-1 cells

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become adherent.

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Both cell lines were maintained and cultured in OptiMEM medium (Invitrogen, Italy) supplemented

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with 10% fetal bovine serum (FBS) and penicillin-streptomycin solution (100 U or µg/mL,

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respectively) (Life Technology)at 37°C in a humidified atmosphere with 5% CO2. For the cell

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viability, and interleukins IL-6 and IL-8 release determination, A549 cells were seeded on the 6-

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well plates (Corning) (2 mL per well) at initial cell density of 0.75×105 cells/mL A549 cells were

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cultivated before the NPs exposure experiments for ~24 h, reaching a cell density of ~1.5×105/ml

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(~3.0×105 cells/well) at approximately 75-90 % confluency.

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THP-1 monocytes were differentiated into macrophage-like cells by incubation with 200 nM PMA

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(Sigma Aldrich) for 72 h. Initial cell density was 2.0×105/mL (2 ml per well of the 6-well plates).

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PMA-supplemented medium was then removed and macrophage-like cells, attached onto the

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surface of the culture plates were washed once with phosphate-buffer saline (PBS) and incubated

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for 24 h in PMA-free Opti-MEM medium with 10% FBS. Cell density of the PMA-differentiated

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THP-1 cells before the NPs exposure experiments was ~1.8×105/mL (~3.6×105 cells/well). Cell 9 ACS Paragon Plus Environment


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morphology and the expression of the cell surface receptor CD14, specific marker for the

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macrophage

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immunofluorescence (see Supporting Information file). Results showed the presence of

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heterogeneous population of cells showing the macrophage-like morphology and expressing CD14

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at different extent (Fig. S1), which is characteristic for the PMA-differentiated THP-1 cells.45 To

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prepare the stock suspensions (2 mg/mL), NPs were weighed using a micro-balance, suspended in

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ultrapure (MQ) water (18 MΩ, Milli-Q, Millipore) and sonicated for 30 min in the ultrasonic bath

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(Sonica® Ultrasonic Extractor, Soltec, Italy). Aliquots from the stock solution were pre-diluted in

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MQ water (20-fold intermediate stock solution of NPs) and then added to the microplate wells (100

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µl per 2 mL) to achieve the following NPs test concentrations: 0.1, 1.0, 10 and 100 µg/mL in

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serum-free OptiMEM medium. The NP treatments were set to cover a broad range of

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concentrations from very low to very high ones, considering the lack of previous data on these

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particles and to match the uncertainty of the NP release and lung deposition calculations for these

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new coated materials. A549 and macrophage-like THP-1 were treated with wZnO, eZnO, wCuO

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and eCuO NPs for 24 h at 37°C, 5% CO2. Control cells were exposed to NPs-free medium.

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2.7 Cell viability assay

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The cell viability was screened by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

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bromide] assay. At the end of NPs treatments, the exposure media were removed and cells were

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rinsed once with PBS and 1 ml of MTT at a final concentration of 0.3 mg/mL in OptiMEM with

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10% FBS added into the wells and incubated for 1 h at 37°C, 5% CO2. Then the MTT-medium was

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removed and the purple MTT reduction product (formazan crystals) was dissolved in 1 mL DMSO

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(Sigma Aldrich). The absorbance (OD) of each sample, proportional to the cell viability, was

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measured with a microplate spectrophotometer (Thermo Multiskan Ascent) at 570 nm using 690 nm

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as a reference wavelength. Cell viability was expressed as OD percent in comparison to the control

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± standard error (SE) of the mean. Toxicity experiments were repeated 3 times.

differentiation,

were

examined

by

conventional

light

microscopy

and


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2.8 Interleukins release detection

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As a marker of the pro-inflammatory potential, the release of the interleukins IL-6 and IL-8 by the

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A549 and THP-1 macrophage-like cells was assayed. At the end of the NPs exposure, the cell

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media were collected and centrifuged at 1200 rpm, 4°C for 6 min (Thermo Scientific TM, SL 8R).

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The supernatants were collected and stored frozen at -20°C until use. Human IL-6 and IL-8

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CytoSetTM ELISA kits (Invitrogen Corporation, USA) were used according to the manufacturer's

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protocol. Briefly, 96-well plates were coated with capture antibody and incubated overnight at room

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temperature. Supernatants were added on the plate and incubated for 2 h. Then the detection

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antibody was loaded to each well for a further incubation. Finally, horseradish conjugated

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streptavidin and the peroxidase substrate were added. The reaction was stopped by 1.8 N H2SO4 and

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OD was measured with a microplate spectrophotometer (Thermo Multiskan Ascent) at 450 nm,

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using 650 nm as a reference wavelength. Since IL-6 measurements resulted in the range of the

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lowest detection limit of the ELISA kit and never showed significant increase in respect of the

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control, only IL-8 results are reported. The results represent the mean of 3 independent experiments

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± SE. For the cell viability and IL-8 assay, statistical comparisons were made by the t-test.

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3. Results and Discussion

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3.1 Antibacterial nano-metal oxides and coated textiles

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One of the motivations for the current study is the evaluation of potential toxicity of metal oxide

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NPs of various sizes and shapes. From our observation, solvent is one of the factors that influences

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the shape and size of the sonochemically produced metal oxide NPs. Therefore, the synthesis of

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NPs and the coating of textiles were carried out from a mixture of ethanol-water (E) solution as well

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as from water (W). CuO/ZnO NPs coated fabrics in water (W) and ethanol (E) were analyzed by

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HRSEM (Figure 2). All the fabrics coated with NPs (Figure 2b-e) demonstrated homogenous and

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dense coating of metal oxides on the surface as compared to smooth texture of the pristine fabric 11 ACS Paragon Plus Environment


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(Figure 2a). The inset images were taken at higher magnification, which indicate that the particle

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size of the deposited CuO was ~80 nm both in water- and ethanol-based synthesis. The size of

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coated ZnO was ~150 nm and there was no visible influence of the solvent on the particle size. The

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amount of coating was determined by ICP and estimated to be less than 1% wt in all 4 samples.

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Antibacterial tests revealed that in case of CuO NPs coated bandages both in water or ethanol

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complete eradication of both gram-positive S. aureus and gram-negative E. coli bacteria was

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observed in 30 min, whereas in 60 min, ZnO NPs coated bandages in water or ethanol revealed 5

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log reductions for both bacterial strains.

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In order to evaluate more precisely the size and shape of NPs, the characterization of the powders

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collected after the coating process was performed. Figure 3 represents the XRD patterns of CuO and

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ZnO NPs synthesized in water and ethanol. The crystalline structure of neither ZnO nor CuO was

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influenced by the solvent used during the hydrolysis process. Identical widths for the reflection

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peaks were obtained in both solvents and the peaks match well the patterns of the PDF files 89-

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7102 and 80-1916. A detailed analysis of the shape and size of metal oxide NPs was carried out by

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HR-TEM and images are shown in Figure 3. The inset in Figure 3 presents the SAED (selected

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area electron diffraction) of the NPs. All the synthesized NPs are crystalline and typical ring

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diffraction pattern was observed. The diffractions of each metal oxide match very well with the

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XRD result presented in Figure 3. Water based ZnO NPs (Fig. 3a) demonstrate a rugby ball shape

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with an average length ~109 nm and a width ~70 nm (aspect ratio ~1.5). Whereas, ethanol-based

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ZnO NPs (Fig. 3c) are rectangular with an average length and width of ~270 and ~166 nm,

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respectively, i.e. an aspect ratio ~1.6.

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Leaf-like structure was observed for CuO NPs synthesized in water with an average length and

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width of ~113 and ~25 nm, respectively, with an aspect ratio ~4.52 (Fig. 3b). In addition,

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aggregates of ~130 nm were observed in case of ethanol-based CuO NPs with an individual particle

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size of ~7 nm (Fig. 3d). CuO NPs synthesized in ethanol demonstrate much smaller particle size 12 ACS Paragon Plus Environment

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compared with water synthesized particles. It is clearly seen that the nature of the hydrolyzing

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solvent of the metal acetates has an impact on the shape and the size of the obtained NPs.

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3.2 Release of airborne NPs from textile abrasion and exposure estimation

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Five different types of cotton fabric samples were tested. The uncoated one was referred to as the

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reference sample. Four samples were coated with either ZnO or CuO using either water or ethanol

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as the reaction solvent. Five types of polyester-cotton samples were also used, with the same

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coatings as described above for the cotton fabric samples.

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The size distributions of the released particles from the cotton samples in the ranges of 13–520 nm

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measured by the SMPS are shown in Figure 4a. For the reference material and the cotton coated in

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ethanol solution, no obvious difference between the background and abrasion experiments was

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observed. However, clear difference was observed between the background and abrasion tests on

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the cotton coated in water solution. The peak size was about 100–200 nm for both ZnO and CuO

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coated samples. The peak concentration for the ZnO samples was higher than for the CuO samples.

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Figure 4b shows the size distributions of the released particles from the cotton fabric samples in the

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range of 0.5–20 µm measured by the APS. Similar to the SMPS results, concentrations of the

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released particles from the samples coated with NPs in water are significantly higher than in ethanol

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solution. For the ZnO and CuO samples coated in water, two modes around 1 and 3 µm can be

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clearly identified from the size distributions.

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The mechanism of sonochemical coating46,

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chemical reaction between the coating and the surface. One of the factors that might have an impact

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on the adhesion of particles is the intensity of cavitation collapse during the sonochemical process.

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The type of the solvent, particularly its surface tension, has a direct influence on the amount of the

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collapsing bubbles. Stronger and more intensive cavitation occurs in a solvent with smaller surface

295

tension. In our case, the surface tension of ethanol is smaller than of water and as a result the

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adhesion of the particles in ethanol-based synthesis is expected to be stronger. This is why in case

47

is based on physical phenomenon rather than on



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of ethanol based coating, very little release of nanoparticles was observed while in water based

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coating, the abrasion test indicated a release of aggregates in the micrometer size range.

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Example SEM images of the particles released from the ZnO coated cotton fabrics are shown in

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Figure 5. Particles with various sizes and shapes can be seen. The particles from 1 to several µm

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seem to be mainly fabric flakes loaded with ZnO particles. They correspond to the modes at 1 and 3

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µm in the size distributions measured by the APS. The particles below 1 µm seem to be ZnO

303

particles, their agglomerates and possibly their agglomerates with pieces of the fabrics. These

304

particles correspond to the 100–200 nm peak in the SMPS measurement. Released particles from

305

the uncoated polyester-cotton fabric, coated polyester-cottons with water-based CuO and ZnO were

306

collected on Nuclepore filters and analyzed by SEM-EDX; the resultant spectrums are shown in

307

Figure 6. As expected, Cu and Zn elements were observed in the corresponding samples and no

308

metal peak was visible in the uncoated sample.

309

The results for the polyester-cotton samples are shown in Figure 7. Panels (a) and (b) show the

310

SMPS and APS results, respectively. No obvious difference between the abrasion results for the

311

reference material and coated polyester-cotton samples was observed with SMPS scan. As shown in

312

panel (b), APS scan showed similar results with those of the cotton fabric samples. The coated

313

samples with NPs in water showed higher particle release than in ethanol. ZnO samples showed

314

significantly higher peak concentrations than CuO samples and the peak sizes were around 1 µm for

315

all polyester-cotton samples including the reference material.

316

The total number and mass concentrations of released particles are shown in Table 2. The mass

317

concentrations corresponding to the SMPS and APS data were calculated with the polyester density

318

(1.39 g/cm3) and unit density (1 g/cm3), respectively; spherical particle assumption was used in both

319

cases. Although the airborne mass concentration included the abraded fabric flakes and NPs, only

320

the polyester density was considered for calculation using the SMPS data. This is because the

321

amount of NP coating was determined by ICP and estimated to be less than 1 wt % (Table 1), thus 14 ACS Paragon Plus Environment

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322

the contribution of the NPs to the airborne mass concentration was considered low. The unit density

323

was used for the APS data due to the definition of aerodynamic diameter. The spherical assumption

324

may lead to certain errors, but it is consistent with the assumptions made for the SMPS and APS

325

measurement. Therefore, we consider the spherical assumption as a reasonable and practical way to

326

compute the mass concentrations.

327

The results of ANOVA analysis were summarized in Table 3. Two factors, the textile material and

328

particle size, were considered in order to see how the textile material influence the particle release

329

at different particle sizes. For instance, in the APS data for cotton based samples, all measurement

330

results for 5 different samples at 0.542 µm size were compared and the comparison showed that the

331

factor is significant. It means that the textile material affected significantly the results at size 0.542

332

µm. As shown in Table 3, for the cotton based samples, the material has a significant influence on

333

the particle release in the entire analyzed range of APS data (0.542-5.048 µm) and 51.4-310.6 nm

334

size range for SMPS data. In contrast, the polyester-cotton results showed weaker material effect on

335

the particle release for both APS and SMPS measurements than the cotton samples. In the

336

polyester-cotton case, the material showed a significant effect on the result in the smaller particle

337

size range (20.2-101.8 nm) of SMPS measurement.

338

The calculated results for lung deposition using the MPPD2 model are presented in Table 4. In

339

agreement with the particle release data, the samples coated with water based NPs showed higher

340

deposited mass fluxes than the reference and ethanol based samples. More details regarding the

341

calculated results can be found in Tables S5-S6 in the supplementary information.

342

Particle depositions on the alveolar cells were calculated to provide references for the doses in the

343

toxicity tests. The calculation used exposure time of 24 hours as in the toxicity tests and the results

344

are presented in Table 5.

345

NP concentrations of 0.1, 1, 10 and 100 µg/ml were used in the toxicity tests and we estimated the

346

amounts of the NPs exposed to the cells. Rischitor et al.48 investigated the deposition of gold NPs in 15 ACS Paragon Plus Environment


Page 16 of 37

347

the size range 10-80 nm on the cells in order to characterize NP transport during the toxicity test.

348

Depending on their sizes, 12-75% of gold NPs were deposited on the cells in 72 hours of exposure.

349

They also showed that NP depositions increased with increasing exposure time, but stabilized after

350

certain time. Although that study showed 12-75% of NPs were finally deposited on the cells, we set

351

the deposition fraction of metal oxide particles on the cells as 1% and 100% for the minimum and

352

maximum dose scenarios, respectively. With this broad range, the effects of the different particle

353

densities, sizes, and the time dependence could be accounted for and we assume that the actual

354

deposition fractions in our toxicity tests were covered in this range. The densities of A549 and

355

THP1 cells in the wells in Table 5 were estimated based on the cell densities in the NPs exposure

356

experiments. The calculated lung depositions for all cotton and polyester-cotton samples showed

357

lower or comparable values than the minimum studied doses in cytotoxicity tests as shown in Table

358

5. The maximum doses were even 5 to 6 orders of magnitude higher than the calculated lung

359

deposition values. Therefore the calculation demonstrated that the doses applied in the toxicity tests

360

represented worse cases than the potential lung exposure to the NPs from abrasion of the textiles.

361 362

3.3 Comparative toxicity of water- and ethanol-based ZnO and CuO NPs

363

No cell viability decrease was observed in either A549 or THP-1 macrophage-like cells exposed for

364

24 h to all the studied CuO and ZnO NPs until the concentration of 10 µg/mL. Only exposure to

365

100 µg/mL resulted in significant decrease of cell viability (Fig. 8a). Under the experimental

366

conditions used in this study, no specific cytotoxicity/cell viability response can be related to the

367

studied NPs physical-chemical properties (i.e. metal form, size, shape), since no significant

368

differences were observed between water- and ethanol-based ZnO and CuO NPs from the MTT

369

assay.

370

Different from ZnO NPs, both water- and ethanol-based CuO NPs induced IL-8 release already at

371

the sub-toxic concentrations (1 µg/mL) in A549 cells (Fig. 8b). Compared to A549 cells, the PMA16 ACS Paragon Plus Environment

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372

induced macrophage-like THP-1 cells exhibited many times higher initial concentration of cytokine

373

excretion (397±57 pg/ml in control cells) and the exposure to either CuO or ZnO NPs did not

374

induce increase in IL-8 release (Fig. 8c). Increased level of IL-8 release by PMA-differentiated

375

THP-1 cells has been shown in response to treatment with silica NPs,49 carbon nanotubes50 and

376

ultrafine particles51, 52 and also by the ZnO NPs.53 A significant decrease in IL-8 secretion by the

377

lung and immune cells was evident after exposure to the highest studied concentrations of both

378

formulations of ZnO and CuO NPs (Fig. 8b, c). Also the cell viability decreased sharply at this

379

concentration (Fig. 8a). Such negative effect on the IL-8 release could be explained by the

380

magnitude and specificity of cell damages, which in turn may negatively affect cell catabolic and

381

anabolic activity, e.g. synthesis and release of cytokines. Toxicity results demonstrated that the

382

sonochemically synthesized ZnO and CuO NPs did not induce acute toxicity on lung epithelial and

383

macrophage cells at low exposure concentrations (up to 10 µg/ml). Only at the highest studied NPs

384

concentration (100 µg/mL) the cytotoxic effects become relevant. As abundantly reported in

385

literature, dissolved cations significantly contribute to the CuO and ZnO NP toxicity. Anyway our

386

previous data suggest that only 50% of the total cell viability decrease at 24h exposure in A549

387

cells can be attributed to the extracellular dissolution of CuO NPs, with a great secondary

388

contribution deriving from the intracellular reacting and dissolving NPs.4, 36 Preliminary studies

389

indeed revealed the capacity of A549 and differentiated THP-1 cells to internalize the

390

sonochemically synthesized CuO and ZnO NPs, similarly to what occurred for commercially

391

available nano metal oxides. In Fig. S2 is reported the light microscopy evaluation of the eZnO NPs

392

interactions with A549 cells, clearly showing the role of NP internalization in cell toxicity. Similar

393

results are expected for the other metal oxides. Although the comprehension of the mode-of-action

394

is beyond the scope of the present paper, these observations support the hypothesis that also for the

395

NPs here studied a combined effect of internalized NPs and dissolved ions may be responsible for

396

the cytotoxic action.



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397

Considering the lung exposures to NPs from abrasion of textiles were lower or comparable to the

398

minimum doses in the toxicity tests (see Table 5), we may regard the nano metal oxide

399

sonochemical coating technology as safe at least for short term – acute – inhalation exposure. Of

400

course more studies are needed to ensure the safety over long term exposure at low NPs loading.

401

Although the studied CuO and ZnO NPs were not toxic at the low concentrations to the A549 and

402

THP-1 cells, both water- and ethanol-based CuO NPs induced the release of IL-8 in the A549 cells

403

already at quite low exposure concentration as 1 µg/ml. This suggests CuO NPs may be considered

404

less safe than ZnO NPs in antibacterial-coating applications. With respect to the capacity to kill

405

bacteria, the CuO and ZnO sonochemically coated textiles provided comparable results.54

406 407

Implications

408

The sonochemical technology provides a new way to efficiently coat textiles with NPs and the

409

product safety need to be assessed by holistic approaches. The coating process, rather than the NP

410

physical and chemical properties, is shown to be the main factor in modulating the NP release. The

411

ethanol-based process led to less release than the water-based process. However, water-based

412

process is safer for the manufacturing phase. This study provides data for the safe-by-design

413

approach and balance of the risks during the manufacturing and product usage phases, with the

414

eventual goal of applying the nano-enabled antibacterial textiles in the local and global mitigation

415

strategies against the spread of infectious diseases.

416 417

Acknowledgements

418

This work was supported by the grant 2013-0987 of Fondazione Cariplo to P.M. for the project

419

“OverNanotox”, by the Estonian Research Council projects IUT23-5, and by the EU grant 720851

420

for the project PROTECT, call H2020-NMBP-PILOTS-2016.

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Supporting Information Available

423 424

References

425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469

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52. Corsini, E.; Ozgen, S.; Papale, A.; Galbiati, V.; Lonati, G.; Fermo, P.; Corbella, L.; Valli, G.; Bernardoni, V.; Dell'Acqua, M.; Becagli, S.; Caruso, D.; Vecchi, R.; Galli, C. L.; Marinovich, M., Insights on wood combustion generated proinflammatory ultrafine particles (UFP). Toxicology Letters 2017, 266, 74-84. 53. O'Keefe, S. J.; Feltis, B. N.; Piva, T. J.; Turney, T. W.; Wright, P. F. A., ZnO nanoparticles and organic chemical UV-filters are equally well tolerated by human immune cells. Nanotoxicology 2016, 10, (9), 12871296. 54. Perelshtein, I.; Lipovsky, A.; Perkas, N.; Gedanken, A.; Moschini, E.; Mantecca, P., The influence of the crystalline nature of nano-metal oxides on their antibacterial and toxicity properties. Nano Research 2015, 8, (2), 695-707.

579


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580

Figure captions

581

Fig. 1. Schematic of the experimental set-up, consisting of the abrasion area, the particle analyzing devices, a

582

filter for particle collection, and a pump. The air flow directions are indicated by arrows.

583

Fig. 2. SEM images of (a) pristine cotton fabric, fabric coated after water-based reaction with ZnO (b) and

584

CuO (c) and after ethanol-based reaction with ZnO (d) and CuO (e)

585

Fig. 3. Characterization of the metal oxide nanoparticles obtained after the sonochemical reaction. HR-TEM

586

images show water-based NPs, ZnO (a) and CuO (b) and ethanol-based NPs, ZnO (c) and CuO (d); (e) XRD

587

spectra of the different NPs.

588

Fig. 4. The size distributions of the released particles from the coated cotton fabrics during the abrasion

589

experiments in the range of 13–520 nm measured by the SMPS (a) and in the range of 0.5–20 µm measured

590

by the APS (b).

591

Fig. 5. Example SEM images of the particles released from the ZnO coated cotton fabrics. The particles were

592

collected on Nuclepore filters with flat surfaces and relatively regular capillary holes of about 0.2 µm.

593

Fig. 6. SEM-EDX spectrum for released particle from (a) the polyester-cotton sample without NP coating,

594

(b) polyester-cotton sample with water synthesized CuO NP coating and (c) polyester-cotton sample with

595

water synthesized ZnO NP coating.

596

Fig. 7. The size distributions of the released particles from the polyester-cotton samples during the abrasion

597

experiments in the range of 13–520 nm measured by the SMPS (a) and in the range of 0.5–20 µm measured

598

by the APS (b).

599

Fig. 8. Cytotoxic and pro-inflammatory response of lung epithelial A549 and PMA-induced macrophage-like

600

THP-1 cells after 24-h exposure to water- and ethanol-based ZnO and CuO, (a) MTT-based viability and (b,

601

c) release of interleukin-8 (IL-8) by the A549 and THP-1 cells. *statistically significant difference from

602

control (not-treated) according to t-test (α=0.05).



Abstract art 85x47mm (300 x 300 DPI)

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Fig. 1. Schematic of the experimental set-up, consisting of the abrasion area, the particle analyzing devices, a filter for particle collection, and a pump. The air flow directions are indicated by arrows. 180x56mm (300 x 300 DPI)



Fig. 2. SEM images of (a) pristine cotton fabric, fabric coated after water-based reaction with ZnO (b) and CuO (c) and after ethanol-based reaction with ZnO (d) and CuO (e) 148x169mm (300 x 300 DPI)


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Fig. 3. Characterization of the metal oxide nanoparticles obtained after the sonochemical reaction. HR-TEM images show water-based NPs, ZnO (a) and CuO (b) and ethanol-based NPs, ZnO (c) and CuO (d); (e) XRD spectra of the different NPs. 150x289mm (300 x 300 DPI)



Fig. 4. The size distributions of the released particles from the coated cotton fabrics during the abrasion experiments in the range of 13–520 nm measured by the SMPS (a) and in the range of 0.5–20 µm measured by the APS (b). 180x60mm (300 x 300 DPI)


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Fig. 5. Example SEM images of the particles released from the ZnO coated cotton fabrics. The particles were collected on Nuclepore filters with flat surfaces and relatively regular capillary holes of about 0.2 µm 151x139mm (300 x 300 DPI)



Fig. 6. SEM-EDX spectrum for released particle from (a) the polyester-cotton sample without NP coating, (b) polyester-cotton sample with water synthesized CuO NP coating and (c) polyester-cotton sample with water synthesized ZnO NP coating. 150x55mm (300 x 300 DPI)


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Fig. 7. The size distributions of the released particles from the polyester-cotton samples during the abrasion experiments in the range of 13–520 nm measured by the SMPS (a) and in the range of 0.5–20 µm measured by the APS (b). 189x60mm (300 x 300 DPI)



Fig. 8. Cytotoxic and pro-inflammatory response of lung epithelial A549 and PMA-induced macrophage-like THP-1 cells after 24-h exposure to water- and ethanol-based ZnO and CuO, (a) MTT-based viability and (b, c) release of interleukin-8 (IL-8) by the A549 and THP-1 cells. *statistically significant difference from control (not-treated) according to t-test (a=0.05). 127x185mm (300 x 300 DPI)


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Table 1 103x71mm (300 x 300 DPI)



Table 2 150x159mm (300 x 300 DPI)


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Table 3 150x141mm (300 x 300 DPI)



Table 4 150x74mm (300 x 300 DPI)


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Table 5 175x166mm (300 x 300 DPI)


Airborne Nanoparticle Release and Toxicological Risk from Metal

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