Designing biocompatible spin-coated multiwall carbon nanotubes-polymer composite coatings

Journal Pre-proof Designing biocompatible spin-coated nanotubes-polymer composite coatings

multiwall

carbon

Damian Maziukiewicz, Barbara M. Maciejewska, Jagoda Litowczenko, Mikołaj Kościński, Alicja Warowicka, Jacek K. Wychowaniec, Stefan Jurga PII:

S0257-8972(19)31189-2

DOI:

https://doi.org/10.1016/j.surfcoat.2019.125199

Reference:

SCT 125199

To appear in:

Surface & Coatings Technology

Received date:

20 September 2019

Revised date:

5 November 2019

Accepted date:

26 November 2019

Please cite this article as: D. Maziukiewicz, B.M. Maciejewska, J. Litowczenko, et al., Designing biocompatible spin-coated multiwall carbon nanotubes-polymer composite coatings, Surface & Coatings Technology (2018), https://doi.org/10.1016/ j.surfcoat.2019.125199

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© 2018 Published by Elsevier.

Journal Pre-proof Designing biocompatible spin-coated multiwall carbon nanotubes-polymer composite coatings

Damian Maziukiewicza◊, Barbara M. Maciejewskaa*◊, Jagoda Litowczenkoa,b, Mikołaj Kościńskia,c, Alicja Warowickaa, Jacek K. Wychowanieca,§, Stefan Jurgaa

a

NanoBioMedical Centre, Adam Mickiewicz University, Wszechnicy Piastowskiej 3,

Department of Molecular Virology, Faculty of Biology, Adam Mickiewicz University,

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b

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PL61614 Poznań, Poland

Department of Physics and Biophysics, Faculty of Food Science and Nutrition, Poznań

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c

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Uniwersytetu Poznańskiego 6, PL61614 Poznań, Poland

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University of Life Sciences, Wojska Polskiego 38/42, PL-60637 Poznań, Poland

These authors contributed equally to this work.

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*corresponding author: [email protected]

Present addresses §

Jacek K. Wychowaniec School of Chemistry, University College Dublin, Belfield, Dublin

4, Ireland, email: [email protected]

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Table of Content Graphics:

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Journal Pre-proof Abstract Composite coatings provide a promising way of in vitro cell behavior screening. Previous efforts included fabrication of single-walled carbon nanotubes (SWCNTs)-polymer materials, however in this work, for the first time we combined two polymers of different hydrophobic character: poly(maleic-alt-1-octadecene) and polyvinylpyrrolidone with multiwalled carbon nanotubes (MWCNTs) to produce composite coatings with varied hydrophobicity. Prior to their incorporation, MWCNTs were characterized using Raman

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spectroscopy during ball-milling procedure at different times to establish an ideal fabrication

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conditions for their best quality. Electrostatic force microscopy was then used to look at the

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distribution and MWCNTs networks formation in the composite coatings. Atomic force and

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scanning electron microscopies were used to establish the topographical features and thicknesses of produced coatings, which varied with the content of MWCNTs. All

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composites as well as control pure polymer coatings proved to be biocompatible and

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exhibited a viability of >80% on two human cell lines: cancerous osteosarcoma (U2OS) and fibroblast (MSU-1.1) that varied by their tumorigenicity, irrespectively of the hydrophobicity

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of the coating. Both cell lines were further shown by scanning electron microscopy to remain in the typical morphological state with high proliferation and attachment to all formed composites. These results show the potential of formation of MWCNTs-polymer composites by facile preparation way (spin-coating) and their potential as coatings for 2D in vitro cell culture platforms.

Keywords: Biocompatible coating, poly(maleic-alt-1-octadecene), polyvinylpyrrolidone, multi-walled carbon nanotubes, spin-coating

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Journal Pre-proof 1. Introduction Composite biocompatible coatings, especially patterned in a particular way [1]–[3], can serve as promising 2D cell contact in vitro models for cell culture [4], [5] or serve as antibacterial surfaces [6]. They can also be used to coat surface of 3D objects, such as commonly used medical implants to increase their duration, modulate degradation rate, prevent corrosion and or increase biocompatibility aspect, especially in vivo [7], [8]. There are many techniques of thin films fabrication, such as spray-coating[8], plasma

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enhanced chemical vapor deposition [9], electrospinning [6], dip-coating and spin-

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coating[10]. Out of all techniques, spin-coating is simplest, costless and quick method of

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covering a flat substrate with a coating solution, where the applied centrifugal force spreads

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the solution evenly over a surface. In particular, physicochemical properties (chemistry and topography) of the fabricated coatings’ surfaces is known to influence the adhesion,

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proliferation and viability of the cultured cells [11], [12].

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One of the recent strategies to tune the physicochemical surface properties of the coatings is to incorporate functional (nano)fillers that will modulate properties such as

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stiffness, hydrophobicity, exposed chemical groups or roughness [13], [14]. In particular, in recent years carbon nanomaterials attracted significant interested and use as nanofillers due to their superior properties including electrical conductivity, stiffness and size [15]–[18]. Out of all families of carbon nanomaterials, carbon nanotubes (CNTs) possess high mechanical strength [19] (Young’s modulus reaching up to 1TPa) and electrical conductivity [20], [21], high aspect ratio and hydrophobic surface prone for interaction with other species, that together constitute them as promising fillers for formation of biocompatible composite coatings [22], [23]. The unique combination of CNTs properties (i.e. high mechano-electrical performance and unique shape) in combination with various matrices (such as hydrogels,

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Journal Pre-proof polymers and elastomers) was found to be particularly attractive for restoration and augmentation of conductive and highly stretchable tissues as flexible bioelectronics [24]– [27]. The elongated shape of carbon nanotubes and propensity to align them along a given direction are typically found to be superior properties over graphene-based materials for fabrication of two-dimensional muscle and nerve tissues engineering constructs [25], [28]. However, many fundamental issues still oscillate with respect to the use of CNTs for fabrication of biocompatible and functional tissue engineering constructs, including: i) their

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aggregation propensity in aqueous environments due to intrinsic hydrophobicity; ii)

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reproducibility of materials due to large variety of CNT available products (e.g. single-walled

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versus multi-walled CNTs); and iii) toxicity due to impurities (e.g. leftover metal catalysts

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from chemical vapour deposition) and intrinsic structural shape with high aspect ratio and sharp edges. For effective reinforcement of CNTs-polymer composite materials, proper

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dispersion and appropriate interfacial adhesion between the CNTs and polymer matrix have

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to be guaranteed. To this day methods for dispersing CNTs oscillate between mechanical treatments (e.g. ball-milling, ultrasonication) and chemical dispersants and functionalization,

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however the most effective is typically found to be combination of both, e.g. mechanical treatment in the presence of surfactant or mechanical treatment of functionalized CNTs, as summarized in the recent review by Ma et al.[29]. Over the last decade, CNTs carried out a ‘disgrace’ of carrying similar toxicity to asbestos [30], however only very recent year studies elucidated exact routes and toxicity mechanisms of these materials, both in vitro and in vivo [31], [32]. In fact, some composite versions of CNTs were found to have high blood circulation times that suggested greatly delayed clearance by the reticuloendothelial system (RES) of mice, a highly desired property for in vivo applications of nanomaterials [33]. As summarized in the recent report[34], the exact toxicity of nanomaterials including CNTs in various forms and composites needs to be

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Journal Pre-proof assessed and still remains to be fully elucidated. In case of spin-coated films, the addition of hydrophobic/hydrophilic component into the precursor solution may change the general character of the film [35]. The nanofillers present in the coatings can increase the roughness as they become coated with the polymeric solution creating focal point for cells to adhere to the surface [36]. Moreover, various nanomaterials trapped in coatings can be continuously and steadily released and delivered to the cells through the appropriate choice of soluble polymer [37], such as PVP, which is

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commonly used in cosmetics, pharmaceuticals (binder in drugs), ophthalmology [38]. Since

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PVP is soluble in water and other polar solvents and has good wetting properties it is

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generally found to be biocompatible synthetic polymer, which can serve as a coating or

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additive to coatings. PMHC18, on the other hand, is a rarely used amphiphilic, water-insoluble polymer which was previously used as a solubilizing agent [39], [40]. Both polymers possess

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unique properties and were already used in combination with carbon nanotubes (CNTs) for

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biomedical applications such as increase of circulation time in blood [33]. Therefore, in this work, we fabricated spin-coated composite coatings composed of

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MWCNTs embedded in two polymers of different physicochemical character: PVP and PMHC18. Thus far, only SWCNTs were used in combination with those polymers and for fabrication of functional materials [32], [33], [41]–[44] and no attempts were made to fabricate MWCNTs-polymer coatings that remain biocompatible. Due to known problems of CNTs aggregation, we used ball-milling in the presence of surfactant as one of the methods for detangling, de-agglomerating and cutting carpeted CNTs into shorter tubules [29], [45]. The influence of milling time on CNTs structure quality was then determined by Raman spectroscopy measurements. To establish the influence of the MWCNTs content on the overall physicochemical properties and interactions between MWCNTs and polymers we investigated the composite coatings stability, surface roughness, thickness and wettability.

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Journal Pre-proof The agglomeration tendency and distribution of MWCNTs in the coatings were visualized by Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). To evaluate biocompatibility, we have then cultured two cell types (cancerous U2OS and non-cancerous MSU-1.1) on MWCNTs-polymer composite coatings and evaluated their viability and morphology over the course of 2 days. We finally visualized cells attachment on the coatings

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using SEM.

MWCNTs synthesis

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2.1.

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2. Materials and methods

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MWCNTs were synthesized by means of floating catalyst Chemical Vapor Deposition route. Ferrocene was used as a catalyst and toluene as a carbon source. The synthesis was carried

Ball-milling

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2.2.

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out in argon atmosphere at the temperature of 760°C similarly to our previous work [46].

MWCNTs underwent a milling process from 1 to 30 hours in a Retsch ball-mill with the

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commercially available Nanosperse AQ (Nano-Lab) surfactant solution. Afterwards samples were dried for 24 hours in order to remove the liquid phase. 2.3.

Coatings fabrication

Three different amounts (1, 2 and 4 mg) of MWCNTs after 10 hrs of ball-billing (see main text) were added to 5%wt. polyvinylpyrrolidone (PVP; Mw = 360 000), 20%wt. and 5%wt. poly(maleic-alt-1-octadecene) (PMHC18; Mw = 30 000 – 50 000) in chloroform solutions in order to functionalize the MWCNTs. All samples were sonicated and thoroughly mixed using the magnetic stirrer. The exact relative concentrations of MWCNTs versus polymers are presented in the ESI Table 2).

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Journal Pre-proof The polymer/MWCNTs solutions were deposited on coverslips in a SPIN200i-INT Single Substrate Spin Processor. Initially for pure polymers the spinning speed ranged from 1000 to 8000 RPM. 6000 RPM was then fixed for fabrication of all MWCNTs-polymer composites (see main text). All samples were spin coated for 30 seconds at room temperature (RT=21°C). 2.4.

Raman spectroscopy

Raman spectroscopy measurements were performed on an in Via Renishaw Raman

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Microscopy with a 514 nm argon laser and 1800 g/mm grating. The laser beam was focused

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on the sample with a 50x/0.75 Leica microscope objective. All Raman spectra were obtained

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from 250 cm-1 to 2000 cm-1 with 10 s acquisition time and 5 accumulations. All spectra

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corrections and peaks position measurements were done by fitting Lorentzian profile in an Origin Pro 9.0 software. All intensities were normalized to the G peak (1347 cm-1). Atomic/Electrostatic force microscopy (AFM/EFM)

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2.5.

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AFM/EFM measurements were performed using the Icon Scanning Probe Microscope (Bruker, USA). Samples were scanned in air, at room temperature (RT) using tapping and lift

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modes with antimony N-type doped Si (MESP) tips (Bruker, USA). The images were acquired at 512 × 512 pixels resolution over 50 × 50 µm areas at a scan rate of 0.5 Hz. Each set of the samples was initially scanned in tapping mode to obtain the samples topography, then the Electrostatic Force data were collected by measuring the variations in the phase signal. Electrostatic Force Microscopy worked at Lift mode with lift height range set to 80 nm. The Si substrates were coated with thin gold layer and then the composite films were deposited on the top of gold layer. Substrates with the samples were fixed and grounded prior to EFM measurements. During all experiments voltage was applied to the sample and set to 5V. The surface roughness (Ra, Rq) of the composite films were calculated based on obtained

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Journal Pre-proof tapping mode height images using NanoScope Analysis software. All images were 2nd order flatten using Nanoscope software. 2.6.

Scanning electron microscopy

Composite films were firstly frozen under liquid nitrogen (-80◦C) and then carefully broken to small pieces, which were subsequently spattered with a thin layer of Au (10 nm). Scanning electron microscopy images were then gathered using Jeol 7001TTLS microscope with the accelerating voltage between 2 and 3 kV. Sample was placed with a tilt of 45° to allow

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determination of thickness of small pieces of the composite films. All composite films were

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sterilized before cell seeding using UV irradiation (λ≈254nm). Then U2OS and MSU-1.1

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cells were seeded on the top of all films in an amount of 1.5 x 105 cells and incubated for

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48 h. After cultivation samples were fixed with 1% glutaraldehyde in PBS and sodium cacodylate buffer (0.1 M) and rinsed with PBS. Finally, samples were dehydrated by series of

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washes with graded ethanol (25% - 90%). Prior to SEM images, samples were sputtered with

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a thin gold layer (10 nm). Contact angle

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Wettability of composite films was investigated on Phoenix 300 Contact Angle tensiometer using the sessile drop method for deionized water and culture medium (Dulbecco's Modified Eagle's medium - DMEM). Fluid droplets were added until a plateau in the contact angle was reached. Analysis and calculations of contact angle were done using SEO Surface 7 version 1.0 software. 2.8.

Cell culture

Human fibroblasts (MSU 1.1) and human osteosarcoma (U2OS) cell lines were maintained in Dulbecco’s Modified Eagle’s culture medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich) along with 1% antibiotic solution (including 100 U ml-1 penicillin, 100 µg ml-1 streptomycin, Sigma-Aldrich) at 37°C in a humidified

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Journal Pre-proof atmosphere of 5% CO2. After reaching 80% of confluence, cells were washed with Hank’s Balanced Salt Solution (HBSS, Sigma-Aldrich) and subcultured by trypsinization (1% Trypsin-EDTA solution in PBS). Cells were observed under the light microscope (Leica DM IL LED) and counted by Automated Cell Counter (BioRad). Human osteosarcoma (U2OS) cell line was purchased from the American Type Culture Collection (ATCC, USA). MSU-1.1 human fibroblast cells were obtained through the courtesy of Prof C. Kieda (CBM, CNRS, Orleans, France). Viability tests

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2.9.

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The cell viability was evaluated using Muse™ Count & Viability Kit with the Muse Cell

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Analyzer (Millipore, Merck), which differentially stains viable and non-viable cells based on

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their permeability to two DNA binding dyes. Prior to cell viability analysis on the Muse™ Cell Analyzer, glass cover slips (22 x 22 mm) were covered by MWCNTs-polymer

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composite films and MWCNTs-polymer films were firstly sterilized upon UV light

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irradiation (λ=254 nm). Films were placed in 6-well plates and next MSU-1.1 and U2OS cells were seeded in each well at a density of 5 x 105 cells/well and incubated for 24 and 48 hours

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under required conditions. Cells cultured on sterile glass cover slips were taken as negative control. For positive control, DMSO solution (10 % - dimethyl sulfoxide in DMEM) was added to cells cultured on glass cover slips. After 24 h of incubation in humidified atmosphere (37 °C, 5% CO2) the medium was discarded, and cells plated on cover slips were collected by trypsinization (1% Trypsin-EDTA solution in PBS). Finally, at each time point, obtained cells (50 µl) were harvested and mixed with Count & Mouse Viability Reagent (450 µl). The experiment was run in triplicate on Muse™ Cell Analyzer. All data were gathered and analyzed according to the manufacturer's protocol (Millipore). 3. Results and discussion

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Journal Pre-proof We have firstly prepared the selection of pure polymeric coatings (from PMHC18 and PVP) via their spin-coating on glass cover slips covered by a thin gold layer at a consecutively increasing rotational speed (from 1000 rpm to 8000 rpm with 1000 rpm steps). The surface of the polymer coatings produced at 6000 rpm were most uniform and presented least number of defects (Figure 1) in comparison to films produced at all other rotational speeds (ESI Figure 1). Thus, we selected 6000 RPM speed as the constant parameter for composites formation with MWCNTs. The average thicknesses (calculated

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from SEM images) of obtained films were 0.31 ± 0.04 µm and 2.01 ± 0.38 µm for PMHC18

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and PVP, respectively (ESI Figure 2). The significant differences between the average

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thicknesses of PMHC18 and PVP coatings at any rotational speed were a result of different

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wetting properties of these two polymers. The contact angle measurements revealed that PVP

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0.17) (Table 1).

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coatings are more hydrophilic (Φ=51.33 ± 1.59) than films made from PMHC18 (Φ=78.17 ±

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Figure 1: SEM images of: A) PMHC18, B) PVP films. AFM images of C) PMHC18, D) PVP

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films, produced via their spin-coating at 6000 rpm.

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Typically, one can obtain a uniform dispersion of MWCNTs from their CVD synthesized carpet form using ball-milling.[50] To form composite films, we firstly assessed

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the ball-milling conditions to obtain MWCNTs of highest quality. Their quality was quantified using Raman spectroscopy before and after the ball-milling process with varied ball-milling time. The ratio of intensities (ID/IG) of D and G bands explicitly shows the quality of the carbon materials subjected to the ball-milling process among other processes[47] We therefore evaluated the quality of MWCNTs using ID/IG since it well describes the number of defects in carbon nanomaterials, as a function of ball-milling time (ESI Figure 2). The mean ID/IG ratio initially decreases in time up to 10 hours, which can be attributed to detangling, mechanical purification and separation of MWCNTs. The minimum (lowest ID/IG=0.33 ratio) indicating highest purity was observed at 10 hours and therefore we used this time to produce all MWCNTs to be embedded in the spin-coated polymer coatings.

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Journal Pre-proof It is worth to note that further increase of the milling time beyond 10 hours resulted in deterioration of MWCNTs quality and the increase of their ID/IG ratio. This was unexpected since, according to previous reports, ball-milling of MWCNTs has no influence on their quality even after 120 h of milling.[48] We hypothesize that this difference arises from the fact that in our case surfactant (Nanosperse AQ from Nano-Lab) was present in the MWCNTs dispersion, however further detailed investigation of this phenomena is out of the

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scope of the current article.

Figure 2: AFM/EFM images showing topography and distribution of MWCTNs of: A) MWCNT0.1%-PMHC18, B) MWCNT0.3%-PMHC18, C) MWCNT0.6%-PMHC18, D) MWCNT0.1%PVP, E) MWCNT0.3%-PVP, F) MWCNT0.6%-PVP films. (Colour version of this figure may only be visible online). Next, we have prepared selection of composite spin-coated MWCNTs-polymer coatings at an increasing MWCNTs concentration with respect to the polymer (0.1, 0.3, 0.6, w/w%). Again, we spin-coated all CHCl3 solutions on glass cover slips in order to assess the

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Journal Pre-proof influence of MWCNTs addition on the integrity, hydrophobicity, thickness, and roughness of the obtained films. Overall, all fabricated composite MWCNT-polymer coatings were homogeneous (ESI Figure 3). The roughness and nano-topography remain one of the essential parameters influencing the cell adhesion [49]–[51]. The topography of all formed MWCNTs-PMHC18/PVP composite and control polymer coatings is shown on the AFM images in the Figure 2 and on the corresponding SEM images (ESI Figure 3). It can be clearly observed that the roughness of formed composite MWCNTs-polymer films increases

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with the increased content of MWCNTs, even though the average roughness of all the

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samples was lower than 20 nm (Ra<10 nm, Rq<20 nm) (ESI Table 1).

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We further imaged all the obtained samples using electrostatic force microscopy mode

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(EFM) to look at the distribution of MWCNTs within polymeric samples (Figure 2). Due to their unique electrical properties [52] MWCNTs were easily identified on the phase images

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by distinct color contrasting the non-conductive polymer background. For all used

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concentrations in both polymer composite samples, MWCNTs were homogenously distributed, which is typically most important factor in composite reinforcement[16], with

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very little agglomeration of MWCNTs observed for both MWCNTs-polymer samples at the highest 0.6 w/w% MWCNTs concentration (Figure 2C and 2F). It appears that in most cases MWCNTs were randomly aligned in the polymer matrix, however we noticed that in some cases and in some regions of the sample, MWCNTs tend to stay aligned due to rotational forces used in spin-coater during fabrication (ESI Figure 4A). Similar conductivity was obtained for all MWCNT-PMHC18 composites due to the maximum phase angle (º) observed on the EFM images (Figure 2) indicating good percolation network and diffused electron transfer from MWCNTs to the surrounding PMHC18 matrix (Figure 2C). On the other hand, it seems that only highest concentration of MWCNT in their PVP composites formed highly agglomerated MWCNT network with similar phase angle (Figure 2F) to that of MWCNT-

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Journal Pre-proof PMHC18 composites. Lengths of MWCNTs embedded in the polymers were estimated based on the obtained SEM and EFM images and their average lognormal size distribution was

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0.81 ± 0.07 µm (ESI Figure 4B).

Figure 3: Raman spectra of A) 10h milled MWCNTs, MWCNT0.1%-PMHC18, MWCNT0.3%PMHC18, MWCNT0.6%-PMHC18, and PMHC18 powder; and B) 10h milled MWCNTs, MWCNT0.1%-PVP0, MWCNT0.3%-PVP, MWCNT0.6%-PVP and PVP powder. We further measured Raman spectra of all samples in order to examine the molecular interactions between the MWCNTs and polymers (Figure 3). Typical D (1347 cm-1) and G (1572 cm-1) bands were present for MWCNTs, and subsequently observed in each MWCNTpolymer coating. Both bands were observed to shift, broaden and the overall ID/IG ratio was observed to increase. For MWCNTs-PMHC18 it was evident that the addition of 0.1 w/w%

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Journal Pre-proof MWCNTs (lowest concentration) already yielded strong interactions and changes (shifts of ∆ID and ∆IG=11 cm-1) (Figure 3A). Further increase of the MWCNTs content did not further affect the shift of the G band, which remained at the same peak position (∆IG=10-11 cm-1, with resolution of 0.7 cm-1) but caused smaller shifts in D band of ∆ID=5-6 cm-1. The shift of the G band peak can be assigned to the apparent doping effect coming from close hydrophobic interactions with the polymers [53]. These close interactions affect the local surface charge density, which can impede vibrational modes of sp2 carbon atoms in outermost

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MWCNTs wall, overall increasing the energy required for vibrations to occur [54], [55]. We

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further observed similar D and G band shifts in all MWCNTs-PVP composite coatings

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(Figure 3B). Again, the addition of low content of MWCNTs (0.1 w/w%) yielded blue shifts

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of ∆ID= ∆IG=11 cm-1, and further addition of MWCNTs (0.3 and 0.6w/w%) shifts of ∆IG=1011 cm-1, and ∆ID=5-6 cm-1. The ID/IG ratio for 0.1 and 0.3 w/w% of MWCNTs however did

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not significantly increase suggesting lower structural coverage of PVP polymer than in the

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case of PMHC18. In fact, unlike in the MWCNT-PMHC18 composites, the signal for pure PVP was not observed for the 0.3 and 0.6% of MWCNTs in composites (Figure 4B). Since

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PVP is more hydrophilic than PMHC18, during the spin-coating coatings preparation, higher quantities of it would be able to ‘fall off’, which overall could be evident by thinner films made from PVP in comparison to PMHC18. Nevertheless, the fact that we observed strong shifts of MWCTNs G band suggests that the PVP polymer is still present in the composite and interacts with MWCNTs, but most likely the w/w% has changed in favor of MWCNTs in comparison to our initial preparation ratios.

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Figure 4: Thickness of PMHC18 and PVP coatings with different amounts of MWCNTs.

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deviations (SD).

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Samples were spin coated at 6000 RPM. All values are presented as average ± standard

Previously we observed high discrepancy between the thicknesses of pure polymeric

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coatings (ESI Figure 1). Interestingly, the addition of low content of MWCNTs 0.1 w/w% equalized that thickness (dPMHC18= 2.35 ± 0.34, dPVP=2.55 ± 0.23, Figure 4). Further increase of the MWCTNs content in the coatings led to an increase of thicknesses of both films, with higher rate of increase for PMHC18 (mPMHC18=1.84±0.41 > mPVP=0.58±0.12, P<0.05, Figure 4). This behavior suggested stronger hydrophobic attraction between PMHC18 monomers and MWCNTs than for PVP, stemming from their intrinsic hydrophobicity (HPMHC18>HPVP) and evidenced by the calculated gradients of the thicknesses increase in the produced coatings (Figure 4). Other researches have already studied hydrophobic interactions between polymers and carbon nanomaterials including polydopamine with MWCNTs [56], phenylalanine containing peptides with graphene oxide-based derivatives [15], or PVP

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Journal Pre-proof interactions with carbon nanomaterials [57], [58]. In pure PVP films, the non-ionic PVP polymer weakly interacted with glass cover slips leading to small surface coverage and deposition during spin-coating process. The addition of MWCNT-PVP led to formation of polymer-wrapped MWCTNs, which resulted in the formation of thicker composite coatings that stuck glass cover slips. Here, due to their affinity for hydrophobic interactions MWCNTs acted as nanofiller that held both polymers and allowed their thicker deposition during spincoating process.

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Table 1: Contact angle (Φ) of all spin-coated films.

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Contact Angle ± SD [degree] Sample

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H2O

78.17 ± 0.17

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PMHC18

MWCNT0.3%-PMHC18

78.42 ± 0.27

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MWCNT0.1%-PMHC18

DMEM 78.64 ± 1.05 79.17 ± 0.17

76.42 ± 0.18

79.29 ± 0.29

74.64 ± 0.17

75.29 ± 0.16

51.33 ± 1.59

59.88 ± 1.07

MWCNT0.1%-PVP

52.90 ± 0.99

62.80 ± 1.71

MWCNT0.3%-PVP

53.74 ± 1.12

64.69 ± 2.20

MWCNT0.6%-PVP

53.82 ± 0.71

65.30 ± 2.54

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PVP

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MWCNT0.6%-PMHC18

Indeed, the MWCTNs-polymer interactions will affect the formed composite coatings bulk surface wetting properties and reflect the hydrophobicity (H) of the structure. Wettability of the surface also plays an important role in cell proliferation and protein adsorption [50], [51], [59]. Therefore, we evaluated the contact angle of all produced coatings with respect to deionized water and DMEM solution (+10% FBS +1% antibiotic solution), that should well-reflect the environmental conditions in which cells are typically cultured.

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Journal Pre-proof PMHC18 as a water-insoluble polymer exhibits much more hydrophobic behavior than watersoluble PVP. Contact angles for PMHC18 and its subsequent MWCTNs- PMHC18 composites for both water and DMEM have only decreased for the highest MWCNTs concentration of 0.03 w/w%, (Table 1). On the other hand, the interactions between DMEM medium components (including proteins) and PVP coating and MWCNTs-PVP composite coatings were more apparent with the significantly increased contact angle value for DMEM, in comparison to H2O, in each case by around 10º (P<0.05, Table 2). The significant increase

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(P<0.05) of the contact angle of MWCNTs-PVP with the increasing content of MWCNTs

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suggests stronger interactions between DMEM components and PVP polymer than in the

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PMHC18 case. The overall surface bulk hydrophobicity reflected by a contact angle increases

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from Φ=53.82 ± 0.71 for H2O on MWCNT0.03%-PVP to Φ=65.30° ± 2.54° for DMEM on MWCNT0.03%-PVP indicating that protein-rich medium has different surface tension than

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40 0 100 80 60 40

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Viability (%)

60

U2OS

80

20

24h

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100

48h

MSU

pure water.

20

C +

M

M

W

C

N

C P T0 W M .1 C % HC N T0 -P 18 .3 M M % H W C -P C M 18 N T0 H C .6 1 % -P 8 M H M C W 18 C N T0 PV M P W . C 1% N T0 -PV M P .3 W % C N T0 PV P .6 % -P V P

0

Figure 5: Viability of osteosarcoma (U2OS) and fibroblast (MSU-1.1) cell lines after incubation at 24 and 48 hours on all films (controls, pure polymeric films and MWCNT19

Journal Pre-proof polymer composites with different amounts of MWCNTs). Positive controls (C+) was cells cultured with 10% DMSO and negative controls (C-) used here were cells cultured on glass cover slips in DMEM. Error bars are SD. Next, to evaluate the biocompatibility of all produced coatings, we cultured two human cell lines (U2OS and MSU-1.1), and subsequently evaluated their viability using Muse™ cell analyzer (Figure 5). Generally, the viability of both cell lines over the course of 48 hours was found to remain over 80% suggesting acceptable biocompatibility of all formed

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coatings, as per current ISO legislations (10993-5: above 80% considered as non-cytotoxic;

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80%–60% weak; 60%–40% moderate; below 40% strong cytotoxicity)[60]. After 24 hours of

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incubation fibroblasts seeded on the PMHC18-based coatings exhibited a good survival rate of

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~88% for PMHC18, ~86% for MWCNT0.1%-PMHC18, ~88% for MWCNT0.3%-PMHC18, and ~90% for MWCNT0.6%-PMHC18 (Figure 5). Similar results were obtained for cancer cell line

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(U2OS) where cells seeded on PMHC18 coatings exhibited 80% viability, while ~84% for

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MWCNT0.1%-PMHC18, 85% for MWCNT0.3%-PMHC18, and 81% for MWCNT0.6%- PMHC18. Both cell lines incubated with PVP-based coatings show a constant survival of approximately

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92% for MSU-1.1 and 89% for U2OS cells and a loss in viability down to ~86% for fibroblasts and 81% for osteosarcoma cultured on MWCNT0.6%-PVP after a period of 24h. After 48 h treatment PMHC18 films exhibited ~94% viability for U2OS (and ~88% for MSU1.1 respectively), while ~83% for MWCNT0.1%-PMHC18 (~75% for MSU-1.1), ~86% for MWCNT0.3%-PMHC18 (~89% for MSU-1.1), and ~85% for MWCNT0.6%-PMHC18 (~88% MSU-1.1) indicating similar level of non-cytotoxicity for both cell lines. PVP-based coatings exhibited an overall better survival rate for osteosarcoma than cultured on PMHC18 with ~90% viability for PVP (~90% for MSU-1.1), ~91% for MWCNT0.1%-PVP (~90% for MSU1.1), ~91% for MWCNT0.3%-PVP (~90% for MSU-1.1), and ~88% for MWCNT0.6%-PVP (~90% for MSU-1.1), again indicating strikingly similar level of cytotoxicity for both cell

20

Journal Pre-proof lines. It is known that cancer cells exhibit higher proliferation rates and multiply in an uncontrollable manner in comparison to normal cell lines. This typically leads to different cytotoxic responses when one compares biocompatibility of composites on cancer versus non-cancer cell lines [61]. However, in our case, there was no apparent difference between the two cell lines and the two formed polymer-composite coatings with different amounts of MWCNTs. This showed that fabricated coatings had cell-type independent minimal

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cytotoxicity.

Figure 6: SEM images of U2OS cell line cultured on PMHC18 (A),MWCNT-PMHC18 composites (B-C) and PVP (D) and MWCNT-PVP(E-F) coatings. Scale bar represents 30 µm. 21

Journal Pre-proof The cell-surface interactions are crucial for proper cells behavior such as adhesion, proliferation, spreading, maturation and differentiation [50] Interaction of cells with substrates begins by cells attachment to the surface of material through contact sites. Cells then strongly interact with the surface, which is essential for their communication and further tissue formation[62]. We have therefore used SEM imaging on fixed osteosarcoma (Figure 6) and fibroblast (ESI Figures 5 and 6) cells at 48h to compare their morphology across all formed polymer-composite coatings (Figure 6). PMHC18-based coatings were stable in the

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culture medium and provided a good surface for the cellular attachment, growth and

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spreading (Figure 6A-C). Osteosarcoma cultured on PMHC18 as well as MWCNT- PMHC18

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composite coatings proliferated rapidly and covered entire samples (Figure 6A-C). U2OS

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cultured on pure PVP and PMHC18 have similar elongated morphology (Figure 6A, D). We did not observe any morphological changes of the cells cultured on composites with an

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addition of MWCNT compared to cells cultivated only on pure polymeric coatings indicating

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that addition of MWCNTs does not influence cell morphology. At low MWNCTs concentrations (0% and 0.1%), PVP in PVP-based coatings may have been dissolved in cell

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medium during fixation procedure leaving patches of undissolved polymer and densely packed cells around them (Figure 6D-F). At higher MWNCTs concentrations of 0.6%, MWCNTs were strongly attracting PVP monomers holding it still in place overall contributing to higher integrity of the coating in the cell culture media and similar osteosarcoma proliferation and coverage to that on MWCNTs-PMHC18 composites. Composite coatings with 0.6% content of MWCNTs could therefore be used as in vitro screening platforms for further biomedical applications offering MWCNTs as protein or biomolecules bindings sites, similarly to recently published reports [63].

4. Conclusions

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Journal Pre-proof To sum up, in this work, we have used two polymers of different hydrophobicity to form composite MWCNTs-polymer coatings for the first time using facile spin-coating technique, as opposed to typically used SWCNTs. Raman spectroscopy not only allowed us to choose MWCNTs of highest quality from the ball-milling process but also suggested that non-covalent molecular interactions occur between the polymers and MWCNTs and are due to hydrophobic attraction between the surface of MWCNTs and polymer monomers, evident by the shifts in the G band (1600 cm-1). Due to varying strength of these hydrophobic

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interactions between PMHC18, PVP and MWCNTs, the spin-coated coatings varied

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significantly in their thickness using the same, fixed spin-coating set of parameters.

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Electrostatic force microscopy was used to visualize and establish MWCNTs networks

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presents in the formed coatings with homogenous dispersions of fillers achieved in polymer matrices. Contact-angle measurements of water and cell culture medium revealed differences

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in hydrophobicity between coatings fabricated from PMHC18 and PVP. The cell culture

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viability tests then showed that survival rate of both non-cancerous and cancerous cells was over 80% on all MWCNTs-polymer over 2 days culture cells cultured suggesting promising

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biocompatibility of the coatings. Finally, SEM micrographs showed that both cell lines attached more facilely to the surface of all coatings and remained in the typical for this type of cells morphology. The PMHC18-based coatings remained most stable in the cell culture providing a good support for cellular growth and spreading and thus were found promising cell contact coatings for future in vitro screening studies for biomedical applications, including in vitro coatings for conductive tissue studies and nerve regeneration. In particular, we envisage continuing studies on such composite coatings with additional well-defined nano- and micro- topographies using aligned MWCNTs and better understanding activation of specific proteins and biochemical cascades over longer cell culture periods.

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Journal Pre-proof Supplementary information The Supporting Information is available free of charge at XYZ. Graphs and tables: Thickness of PMHC18 and PVP thin films vs rotatory speed (ESI Figure 1); Influence of ball-milling time on MWCNTs quality (ESI Figure 2); SEM images showing MWCNT-polymer composite films (ESI Figure 3); Roughness of MWCNTs-polymer films (ESI Table 1); SEM micrograph of aligned MWCNTs with corresponding MWCNTs size histogram (ESI Figure 4). SEM micrographs of cells cultured on PMHC18 (ESI Figure 5) and

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PVP (ESI Figure 6).

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Author Contributions: D.M., B.M.M. and J.L. prepared original version of this manuscript.

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M.K. performed AFM and contact angle measurements. J.L and A.W. performed cell-culturebased experiments. B.M.M. performed SEM imaging and synthesized MWCNTs. D.M. and

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B.M.M prepared all samples. J.K.W., B.M.M., J.L. and D.M. prepared graphics. J.K.W.

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prepared final version of the manuscript. All authors commented on the final version of this

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manuscript and approved its submission.

Funding sources: D.M and J.L. acknowledge the financial support from the National Science

Centre

(NSC)

grants:

OPUS

(2016/21/B/ST8/00477)

(2016/23/N/ST5/00955).

ORCID numbers of authors: Damian Maziukiewicz: https://orcid.org/0000-0003-1540-094X Barbara M. Maciejewska: https://orcid.org/0000-0002-3101-366X Jagoda Litowczenko: https://orcid.org/0000-0002-8515-2171 Alicja Warowicka: https://orcid.org/0000-0003-4301-9855

24

and

PRELUDIUM

Journal Pre-proof Jacek K. Wychowaniec: https://orcid.org/0000-0002-6597-5242 Stefan Jurga: https://orcid.org/0000-0002-1665-6077

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Journal Pre-proof Author Contributions: D.M., B.M.M. and J.L. prepared original version of this manuscript. M.K. performed AFM and contact angle measurements. J.L and A.W. performed cell-culturebased experiments. B.M.M. performed SEM imaging and synthesized MWCNTs. D.M. and B.M.M prepared all samples. J.K.W., B.M.M., J.L. and D.M. prepared graphics. J.K.W. prepared final version of the manuscript. All authors commented on the final version of this

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manuscript and approved its submission.

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Journal Pre-proof Highlights  Multiwall carbon nanotubes (MWCNTs)-polymer composite coatings are fabricated using facile spin-coating approach.  Ball-milling process strongly affects quality of MWCNTs.

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 The varied hydrophobicity of the MWCNTs-polymer does not affect cell viability.

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