Influence of the Aliphatic Side Chain on the Near Atmospheric

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Influence of the aliphatic side chain on the near atmospheric pressure plasma polymerization of 2-alkyl-2-oxazolines for biomedical applications Joachim Van Guyse, Pieter Cools, Tim Egghe, Mahtab Asadian, Maarten Vergaelen, Petra Rigole, Wenqing Yan, Edmondo Maria Benetti, Valentin Victor Jerca, Heidi A. Declercq, Tom Coenye, Rino Morent, Richard Hoogenboom, and Nathalie De Geyter ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b09999 • Publication Date (Web): 05 Aug 2019 Downloaded from pubs.acs.org on August 8, 2019

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ACS Applied Materials & Interfaces

Influence of the aliphatic side chain on the near atmospheric pressure plasma polymerization of 2-alkyl-2-oxazolines for biomedical applications Joachim Van Guyse1+, Pieter Cools2+, Tim Egghe1, 2+, Mahtab Asadian2, Maarten Vergaelen1, Petra Rigole4, Wenqing Yan3, Edmondo M. Benetti3, Valentin-Victor Jerca1,6, Heidi Declercq5, Tom Coenye4, Rino Morent2, Richard Hoogenboom1* and Nathalie De Geyter2* 1Supramolecular

Chemistry Group, Centre of Macromolecular Chemistry (CMaC), Department of Organic and Macromolecular Chemistry, Faculty of Sciences, Ghent University, Krijgslaan 281 S4, 9000 Ghent, Belgium

2Research

Unit Plasma Technology (RUPT), Department of Applied Physics, Faculty of Engineering and Architecture, Ghent University, Sint-Pietersnieuwstraat 41 B4, 9000 Ghent, Belgium

3 Polymer

Surfaces Group, Laboratory for Surface Science and Technology, Department of Materials, ETH Zürich, VladimirPrelog-Weg 5, CH-8093 Zurich, Switzerland

4Laboratory

of Pharmaceutical Microbiology (LPM), Department of Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium

5Department

6Centre

of Basic Medical Science, Faculty of Medicine and Health Science, Ghent University, De Pintelaan 185 6B3, 9000 Ghent, Belgium

for Organic Chemistry “Costin D. Nenitescu”, Romanian Academy, 202B Spl. Independentei CP 35-108, 060023 Bucharest, Romania +Authors

*Correspondence

contributed equally to the manuscript.

should be addressed to email: [email protected] and [email protected]

KEYWORDS: plasma polymerization, 2-oxazoline, near atmospheric pressure dielectric barrier discharge, polypropylene, tissue engineering

Abstract Plasma polymerization is gaining popularity as a technique for coating surfaces due to the low cost, ease of operation and substrate independent nature. Recently, the plasma polymerization (or deposition) of 2oxazoline monomers was reported resulting in coatings that have potential applications in regenerative medicine. Despite the structural versatility of 2-oxazolines, only a few monomers have been subjected to plasma polymerization. Within this study, however, we explore the near-atmospheric-pressure plasma polymerization of a range of 2-oxazoline monomers, focusing on the influence of the aliphatic side chain length (methyl to butyl) on the plasma polymerization process conditions as well as the properties of the obtained coatings. While side chain length had only minor influences on chemical composition, clear effects on the plasma polymerization conditions were observed, thus gaining valuable insights in the plasma polymerization process as a function of monomer structure. Additionally, cytocompatibility and cell attachment on the coatings obtained by 2-oxazoline plasma polymerization was assessed. The 1 ACS Paragon Plus Environment

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coatings displayed strong cell interactive properties, whereby cytocompatibility increased with increasing aliphatic side chain length of the monomer, reaching up to 93% cell viability after 1 day of cell culture compared to tissue culture plates. As this is in stark contrast to the anti-fouling behavior of the parent polymers, we compared the properties and composition of the plasma polymerized coatings to the parent polymers revealing that a significantly different coating structure was obtained by plasma polymerization.

1. Introduction Plasma polymerization, that is the deposition of monomers on a substrate in plasma, is an attractive technique for the modification of substrates, due to its cost-effective and substrate independent nature, 1–3 while allowing the modulation of the surface properties by variation of the plasma process parameters and by selection of the precursor for the plasma polymerized coating, viz. the monomer. Recently, Bhatt et al. and the group of Vasilev demonstrated the successful deposition of a 2-oxazoline plasma polymerized coating under low pressure conditions. 4–6 When the monomer is introduced in the plasma chamber, reactive species/monomer fragments are formed, which ultimately combine to form a polymeric species that is deposited onto the substrate. Interestingly, this process can be tuned by varying the energy density applied on the monomer, viz. the Yasuda factor, which in turn determines the fragmentation degree of the monomer and therefore the chemical composition of the obtained coating.7 As such, the Yasuda factor can be tuned to obtain the desired chemical functionality in the coatings, leading to 2-oxazoline-based coatings with distinctive properties, either displaying self-sacrificial antifouling, antifouling or biosensing properties.4,5,8–10 Furthermore, plasma polymerized 2-oxazoline coatings have already been applied in medical diagnostics and nanotopography.9,10 Hence, the plasma deposition of 2-oxazoline monomers is an attractive method to obtain various functional coatings for biomedical purposes. Despite the interesting applications, the plasma process and the relationship of monomer structure on the plasma process parameters and eventually the coating properties are still poorly understood. 2-Oxazolines, or in a broader context cyclic imino ethers, are in this respect an ideal monomer class to study the effects of varying monomer structure on the plasma process itself as well as the properties of the obtained coatings, due to the huge variation of reported monomer structures.11–14 The versatile synthetic pathways towards 2-oxazolines have allowed the introduction of multiple chemical functionalities and substitution patterns,11,13,15–18 as well as the successful synthesis of chiral derivatives and larger ring-sizes.12,17,19–21 Despite the great structural versatility of 2-oxazoline monomers, so far only 2-methyl-2-oxazoline (MeOx), 2-ethyl-2-oxazoline (EtOx) and 2-isopropenyl-2-oxazoline (iPRO) have been explored for plasma polymerization.13,14,22 Within this study, we aim to gain a broader understanding of the effect of monomer structure on the plasma process and the obtained coatings thereof. More specifically, we focus on the extension of the aliphatic chain on the 2-position of the monomer and its effect on the plasma polymerization process, therefore effectively extending the scope of plasma polymerization with 2-n-propyl-2-oxazoline (PrOx) and 2-n-butyl-2-oxazoline (BuOx). In addition, we also assessed the obtained coatings in terms of chemical composition, hydrophilicity, stability in aqueous media and finally cell interactivity. The plasma polymerization was performed utilizing a near atmospheric pressure dielectric barrier discharge (DBD) set-up, as depicted in Figure 1. The obtained plasma polymers (pP) were benchmarked versus the polymers obtained via cationic ring-opening polymerization, i.e. poly(2-alkyl/aryl-2-oxazoline)s (PAOx), in 2 ACS Paragon Plus Environment

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terms chemical composition, and hydrophilicity to gain a better understanding of the processes taking place in plasma in relation to the monomer structure.

Figure 1. Schematic representation of the plasma polymerization process and comparison of the plasma deposited films of MeOx, EtOx, PrOx and BuOx monomers to the obtained coatings of the parent polymers via spin coating.

2. Experimental Methods 2.1. Materials All high performance liquid chromatograph (HPLC) grade solvents were purchased from Sigma-Aldrich (diethylether, N,N-dimethylacetamide (DMA), dichloromethane (DCM) and acetonitrile (ACN)). Dry ACN was obtained from a custom made JW Meyer solvent purification system and dried over aluminum oxide columns. The following chemicals were used as received unless otherwise stated: butyronitrile ( ≥ 99%, Sigma-Aldrich), valeronitrile ( ≥ 99%, Sigma-Aldrich), ethanolamine ( ≥ 99%, TCI), zinc acetate dihydrate ( ≥ 98%, Sigma-Aldrich), barium oxide (BaO, 90%, Acros Organics), acetic acid (99.5%, AAc, Sigma-Aldrich), methyl-p-toluenesulfonate (MeOTs, 98%, Sigma-Aldrich). 2-Methyl-2-oxazoline (98%, Chemical point) and 2-ethyl-2-oxazoline ( ≥ 99.8%, Polymer Chemistry Innovations) were both purified by fractional distillation over BaO and ninhydrine under slight argon overpressure. 2-Isopropenyl-2-oxazoline (SigmaAldrich, 98%, iPRO) was distilled over CaH2 under reduce pressure before use. Tetrahydrofuran (SigmaAldrich, THF) was freshly distilled over Na/benzophenone under Ar flow before use. n-Butyl lithium solution 2.5 M in hexane (Sigma-Aldrich, n-BuLi) was used as received. Argon (Ar) (Alphagaz 1) was purchased from Air Liquide and used as supplied. A roll of polypropylene (PP) (thickness: 0.05 mm, biaxial oriented) film and a roll of ultra-high-molecular-weight polyethylene (UHMWPE) (thickness: 0.25 mm) film 3 ACS Paragon Plus Environment


were obtained from Goodfellow Cambridge (United Kingdom). Samples of determined size were cut out for plasma polymerization treatment and used without any pre-treatment.

2.2. Equipment Gas chromatography (GC): samples were measured with GC to determine the monomer conversion from the ratio of the integrals from the monomer and the reaction solvent. GC was performed on an Agilent 7890A system equipped with a VWR Carrier-160 hydrogen generator and an Agilent HP-5 column of 30 m length and 0.320 mm diameter. A flame ionization detector was used and the inlet was set to 250°C with a split injection of ratio 25:1. Hydrogen was used as carrier gas at a flow rate of 2 mL/min. The oven temperature was increased with 20°C/min from 50°C to 120°C, followed by a ramp of 50°C/min to 300°C. 1H nuclear magnetic resonance (1H NMR): NMR spectra were recorded on a Bruker Avance 300 or 400 MHz spectrometer at room temperature. The chemical shifts are given in parts per million (δ) relative to tetramethylsilane. The compounds were dissolved in either CDCl3, D2O or dimethylsulfoxide-d6 (DMSO) from Eurisotop. Spectra of the plasma polymerized coatings were obtained by incubating the films for several days in CDCl3 after which the resulting solution was analyzed. Size-exclusion chromatography (SEC): SEC was performed on a Agilent 1260-series HPLC system equipped with a 1260 online degasser, a 1260 ISO-pump, a 1260 automatic liquid sampler (ALS), a thermostatted column compartment (TCC) set at 50°C equipped with two PLgel 5 µm mixed-D columns and a pre-column in series, a 1260 diode array detector (DAD) and a 1260 refractive index (RI) detector. The used eluent was DMA containing 50 mM of LiCl at a flow rate of 0.500 mL/min. The spectra were analyzed using the Agilent Chemstation software with the gel permeation chromatography add on. Molar mass values and Ð values were calculated against poly(methyl methacrylate) standards from PSS. Liquid chromatography-mass spectrometry (LC-MS): LCMS spectra were acquired on a quadrupole ion trap LC mass spectrometer (Thermo Finnigan MAT LCQ mass spectrometer) equipped with electrospray ionization. X-ray photoelectron spectroscopy (XPS): XPS surface analysis is performed on a PHI 5000 Versaprobe II spectrometer. This equipment uses a monochromatic Al Kα X-ray source (hν = 1486.6 eV) operating at 23.3 W. A vacuum of at least 10-6 Pa was obtained for all measurements. Surveys scans and high-resolution spectra (C1s, N1s and O1s) were recorded with a pass energy of 187.85 eV (eV step = 0.8 eV) and 23.5 eV (eV step = 0.1 eV) respectively at an angle of 45° to the normal of the sample. 4 points per sample were measured. Multipak (v 9.6.1) was used for elemental analysis, using a Shirley background and the relative sensitivity factors supplied by the manufacturer. Prior to analysis, spectra were calibrated at 285.0 eV (C-C bond). Fourier transform-infrared spectroscopy (FT-IR): a Bruker Tensor 27 spectrometer equipped with a single reflection attenuated total reflectance (ATR) accessory (MIRacle™, Pike technology) was used to perform FT-IR analysis on coated PP substrates, using a germanium crystal as internal reflection element. All spectra were recorded using a mercury cadmium telluride (MCT)-detector (liquid N2 cooled) in the spectral region of 4000-700 cm-1 and 64 scans (resolution 4 cm-1) were made for each sample. OPUS 6 software was used to analyse the obtained spectra and to correct for the presence of CO2 peaks within the spectra, originating from the ambient environment. Each sample was supported with 0.25 mm of UHMWPE backing to avoid piercing of the very thin PP substrate. Static water contact angle (WCA): WCA measurements were obtained at room temperature, using a commercial Krüss Easy Drop system (Krüss Gmbh, Germany). A water droplet of 2.0 µL was placed on the sample surface. A minimum of 8 measurements were done per sample within 4 ACS Paragon Plus Environment

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10 minutes of sample manufacturing. Spin coating: samples were spincoated on a SPIN150i spincoater (SPS-Europe) using a 1 w/w% solution of the PAOx in THF. All samples were spun at 3500 rotations per minute (RPM) for 45 seconds at an acceleration speed of 600 RPM/s using a volume of 300 µL.

2.3. Methods 2-Alkyl-2-oxazoline monomer and polymer synthesis and purification While MeOx and EtOx were bought from commercial sources, both PrOx and BuOx were synthesized inhouse following reported procedures.23 Next, all monomers were purified by three fractional distillations over BaO and ninhydrin, sodium, and BaO and ninhydrin, to remove traces of impurities that could interfere with the CROP, such as water and ammonia.24,25 These purified monomers were then used for both the plasma polymerization resulting in pPMeOx, pPEtOx, pPPrOx, pPBuOx and the respective homopolymers by cationic ring-opening polymerization (CROP) following an established procedure yielding poly-2-methyl-2-oxazoline (PMeOx), poly-2-ethyl-2-oxazoline (PEtOx), poly-2-n-propyl-2oxazoline (PPrOx) and poly-2-n-butyl-2-oxazoline (PBuOx).25 In addition to the homopolymers prepared by CROP, iPRO was also polymerized (PiPRO) by living anionic polymerization and subsequently ringopened with acetic acid, to aid in the structural elucidation of the coatings.26 The detailed monomer synthesis, purification and the polymerization of MeOx, EtOx, PrOx, BuOx and iPRO can be found in the supporting information, together with the characterization data.

Plasma polymerization of 2-alkyl-2-oxazoline monomers Plasma reactor set-up and electrical characterization The plasma activation and polymerization are performed in a DBD reactor, similar to what is described in earlier work.27 In short, the cylindrical plasma reactor consists of two electrodes placed 8 mm away from each other. The lower copper electrode is covered with a ceramic layer and the upper electrode is a woven stainless-steel electrode through which the gas is fed. The lower electrode is connected to a 50 kHz AC power source and the upper electrode is connected to ground through a 50 Ω resistor. The composition of the discharge mixture consisting of the different 2-oxazoline monomers and the carrier gas Ar is controlled via a CORI-FLOW mass flow controller and an El-Flow gas mass flow controller (Bronkhorst) respectively. Before the gas enters the plasma reactor through the upper electrode, it first passes through a glass wool filler to distribute the gas flow more evenly before entering the plasma discharge region. The bottom of the plasma reactor is connected to a simple pumping unit, allowing the evacuation of the plasma reactor and subsequent filling with a reproducible atmosphere. After fixation of the PP substrate on the lower electrode, the reactor was pumped down to < 0.5 mbar and subsequently flushed with Ar at 3 standard liter per minute (slm) for 3 min at a pressure of 0.5 bar. This purging step was performed to obtain a reproducible gas composition in the plasma reactor. Then, a plasma pre-activation step was carried out at 0.05 bar (Ar flow of 1 slm) for 30 s at a power of 3.85 ± 0.5 W, to improve the covalent bonding of the first coating layer to the substrate. Plasma polymerization was then performed immediately after plasma pre-activation without exposing the substrates to ambient air. 5 ACS Paragon Plus Environment


Therefore, an Ar flow of 7 slm and a monomer flow of 0.2 g/h was used. The system pressure was 0.5 bar and the gas discharge mixture flowed for 2 minutes before applying the power, in order to obtain a more reproducible equilibrium between in- and out-flow of the gas. The treatment time was constant at 5 min and the applied power was varied between 5 and 50 W. To electrically characterize the DBD, the applied high voltage and the resultant discharge current were measured. The high voltage applied to the lower copper electrode was measured using a 1000:1 high voltage probe (Tektronix P6015A). The discharge current was obtained by measuring the voltage across a 50 Ω resistor connected in series with the reactor to the ground. Via Ohm's law, the discharge current was calculated. The obtained waveforms were recorded using a digital oscilloscope (Picoscope 3204A) and the discharge power was calculated by performing a discrete integration method of the multiplication of voltage and current, which is discussed in more detail in the supporting information (formula S.1.).

Stability of the coating via FT-IR To assess the stability of the coatings, 2 samples (approximately 0.7 x 1 cm2) of each condition were incubated in deionized water for respectively three and seven days, while a control sample was stored under vacuum. An FT-IR measurement was performed on each sample as described in the FT-IR section. By taking the ratio of the area of an absorbance peak that is typical to the coating (1719-1582 cm-1) and an absorbance peak from the PP substrate and the supporting UHMWPE (3000-2842 cm-1), a relative measure of the thickness of the coating was obtained.

Cell and protein adhesion on plasma polymerized coatings To assess the anti-fouling properties of the plasma polymerized coatings, cell tests and bacterial adhesion tests were performed. For the cell tests, all samples were first sterilized by exposure to UV-light for 30 min. Human foreskin fibroblasts (HFF) were seeded onto the samples in a 24-well plate at a density of 10.000 cells/1000 μL of medium per well. Cell culturing was performed using a Dulbecco's modified eagle medium (DMEM) glutamax medium (Gibco Invitrogen) with 15% fetal calf serum (Gibco Invitrogen), 2 mM L-glutamine (Sigma-Aldrich), 10 U/mL penicillin, 10 mg/mL streptomycin and 100 mM sodium-pyruvate (all from Gibco Invitrogen). The cultures were subsequently incubated at 37°C under 5% CO2 for 1 and 3 days. Cell proliferation and viability were examined 1 and 3 days after cell seeding by a 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and by fluorescence imaging (Type URFL-T, Olympus, XCellence Pro software) after live/dead staining. For the MTT assay, all results were normalized to the cell metabolic activity measured on the respective day for tissue culture plates (TCPs) and are the average of 6 independent measurements. For the bacterial adhesion tests, two samples were glued together (Loctite 406+770, which contains ethylcyanoacrylate known for its antibacterial potency)28 to expose only the coating to the bacterial solution. The size of all samples was 0.6 x 0.6 cm³ and 4 samples were tested for each condition. The samples were first sterilized by 30 minutes of UV exposure. The microorganisms (Staphylococcus Aureus and Pseudomonas Aeruginosa) were grown on Tryptic Soy Agar (TSA) (Oxoid, Drongen, Belgium) under aerobic conditions at 37°C. Using sterile forceps, the samples were placed in the wells of a 24-well microtiter plate 6 ACS Paragon Plus Environment

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and subsequently 1 mL of the cell suspension, containing approximately 104 colony forming units (CFU)/mL, was added. The plates were incubated for 4 h at 37°C. Following incubation, the samples were transferred to 10 mL 0.9% (w/v) NaCl and subjected to three cycles of 30 s vortex mixing and 30 s sonication. Tenfold serial dilutions were made in 0.9% (w/v) NaCl and the number of CFU was determined by plate counting. To this end, one mL of each dilution was plated on TSA and the plates were incubated at 37°C for 48 h. To test the protein resistance, each coated polymer film was first immobilised on Si wafers by using a double sticky tape. 1 mg/mL fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (SigmaAldrich) solution and Alexa Fluor 546-labeled fibrinogen from human plasma (Invitrogen) solution were prepared in phosphate buffered saline (PBS) respectively. Subsequently, 20 μL of the protein solution was used to cover each sample. After 1 hour of incubation, the substrates were rinsed with MilliQ water. Afterwards, the fluorescence intensities were measured using a plate reader (with excitation wavelength 495 nm for albumin and 560 nm for fibrinogen) before and after the protein adsorption test. For each sample, six independent positions were taken during each measurement.

3. Results and discussion 3.1. Plasma polymerization of 2-alkyl-2-oxazolines In this study, coatings are deposited by the plasma polymerization of 2-oxazoline precursors with increasing aliphatic side chain length. The precursors were thoroughly purified prior to plasma polymerization to avoid the potential influence of impurities on the 2-alkyl-2-oxazoline plasma polymerization. Polypropylene (PP) is used as a substrate because of its low-interfering character in FT-IR measurements. During the film deposition process, a gas mixture of the evaporated monomer and Ar is flowed into the DBD plasma reactor. A pulsed power is applied to prevent significant electrode heating and monomer fragmentation. The electrical characterization of the DBD indicates that the set-up is operating as pseudo-glow discharge, which is known to lead to a homogeneous plasma treatment of the samples.29 Further details can be found in the supporting information. As described in literature, the conditions of the plasma polymerization process will influence the chemical composition and stability of the resulting coatings.30–32 Therefore, the deposition onto a PP substrate was performed at different powers for all monomers, whereby the chemical composition and stability of the obtained coatings were examined as a function of discharge power. The applied power was varied from 5 to 50 W, while maintaining a deposition pressure of 50 kPa, a monomer flow rate of 0.2 g/h and a deposition time of 5 min.

3.2. Characterization of the plasma polymerized 2-alkyl-2-oxazoline coatings 3.2.1. Hydrophilicity, chemical composition and stability of the coatings The hydrophilicity and stability of the coatings were assessed via WCA and FT-IR measurements, respectively, and the obtained results are shown in Figure 2. Figure 2A shows the WCA evolution as a function of power for each of the used monomers. In this work, similar WCA results were obtained as in 7 ACS Paragon Plus Environment


previous studies dealing with the plasma polymerization of MeOx and EtOx monomers.5,33 In general, lower WCA values are obtained at low discharge powers and the WCA values increase with applied power, varying from 10° to 60°. In addition, each monomer shows a stabilization in WCA value from a certain threshold power on, with stable WCA values of approximately 50° ± 1.5°, 54° ± 0.8°, 56° ± 1.7° and 60° ± 2.2° for MeOx, EtOx, PrOx and BuOx, respectively. This stabilization indicates that the coating composition remains constant in terms of hydrophilicity over a wide range of applied powers (region indicated in green). The power range of this stable region increases with an increasing side chain length, therefore suggesting that a longer aliphatic side chain promotes a more constant plasma deposition process over a wide range of discharge powers. Additionally, the WCA value at which stabilization occurs increases with an increasing aliphatic side chain as could be expected, as an increased aliphatic side chain results in a more hydrophobic coating.

Figure 2. A) WCA for pPMeOx, pPEtOx, pPPrOx and pPBuOx as a function of discharge power. The colors indicate three distinct zones in each graph (green: 'stable', orange: 'metastable' and red: 'unstable'). B) Ratio of the area of the characteristic coating peak between 1719-1582 cm-1 over the area of the substrate (3000-2842 cm-1) after immersion in water for 3 (black) and 7 (red) days on the characteristic coating peak (scaled to substrate peak) for 0 days. Next, the stability of the obtained coatings was assessed after 3 and 7 days of immersion in water as coating stability is desired for most biomedical applications.34 Loss of coating would impair the coating’s intended function, which is undesirable when studying its interaction with cells and bacteria. Figure 2B shows the stability assessment of the coatings obtained via FT-IR by comparing the ratio of the characteristic substrate peaks, i.e. the C-H stretches between 3000-2842 cm-1, and coating peaks between 1719-1582 cm-1 from C=O or C=N stretching and N-H bending, before, and after incubation in water for 3 and 7 days. A decrease in coating thickness, therefore, results in a drop of this ratio. To evaluate the stability of the coatings qualitatively, different boundary values of the FT-IR peak ratio were arbitrarily assigned, which are shown as the horizontal green and red lines in Figure 2B. These boundary values were introduced to account for experimental variations such as inhomogeneity of coating thickness on the sample and fixation of the sample. A first boundary value of 0.55 was identified and all coatings possessing 8 ACS Paragon Plus Environment

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an FT-IR peak ratio above this value are classified as stable coatings. At these high ratios, strong characteristic coating signals are still present in the FT-IR spectra while a minor coating loss could however not be excluded. A second regime is identified between peak ratios of 0.55 and 0.2, indicating a metastable region. Here coating loss occurs, and the strong presence of FT-IR signals related to the coating were not consistently observed after water immersion. At FT-IR peak ratios below 0.2, a considerable amount of coating is lost, resulting in very weak representative FT-IR signals, therefore this third regime is identified as the unstable regime. This comparative FT-IR study clearly shows that for all monomers a stable coating could be obtained, however, their stability is correlated to the aliphatic side chain of the monomer. More specifically, the stability of the coating at lower discharge powers improves with increasing aliphatic side chain length, starting at a discharge power of 7 W for BuOx, 21 W for PrOx, 21 W for EtOx and 30 W for MeOx. This mimics the WCA evolution shown in figure 2A, as similar discharge power values also indicate the boundary of WCA stabilization. Hence, when the coating composition becomes independent of applied power, it can be assumed that a significant amount of the coating is retained on the substrate upon prolonged exposure to aqueous environments. Conversely, when large error bars can be seen in WCA values resulting from coating inhomogeneity, this indicates that the coating has poor stability in aqueous environments, resulting in a significant loss in coating thickness. Monomer Boundary Discharge Power (W) Yasuda factor (105 J mol-1) MeOx 30 7.8 EtOx 21 6.0 PrOx 21 7.2 BuOx 7 2.4 Table 1. Boundary discharge power values and minimal Yasuda factors required to obtain a stable coating for each of the used monomers. Table 1 summarizes the minimal power input and Yasuda factor required to obtain a stable coating for each of the used monomers. MeOx requires the highest discharge power to generate stable coatings followed by EtOx and PrOx. BuOx, on the other hand, requires little power input to obtain stable coatings, therefore suggesting that the increase in the side-chain length of the precursor minimizes required power input. This result, however, is biased by the mass difference between the different monomers used, and therefore the Yasuda factor was calculated to allow a more objective comparison between the monomers.35 The Yasuda factor is expressed by the ratio of the power (W) over the flow rate (F) and molecular weight (M) (Yasuda factor = W/(FM)).35 For MeOx, EtOx and PrOx, the Yasuda factors are significantly higher than for BuOx, which confirms that the size of the side chain still affects the stability of the coating at an equal molar flow rate. This could be attributed to the fact that a longer side chain might be more prone to plasma-induced hydrogen abstraction and subsequent cross-linking of the generated monomer fragments. In addition, as the side-chain length increases, the number of secondary carbons increases, thus increasing the probability of forming secondary radicals, which are more stable than their primary analogs. This phenomenon results in a lower Yasuda factor required to obtain stable coatings. Finally, the increased side chain of BuOx leads to higher hydrophobicity, which also implies that the obtained coatings will less readily dissolve in water, even when not fully cross-linked.

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Figure 3. Chemical composition of the coatings: A) FT-IR spectra of plasma polymerized MeOx at 37.7 W (red), EtOx at 35.9 W (blue), PrOx at 35.9 W (pink), and BuOx at 36.2 W (green). B) XPS spectra showing the carbon, nitrogen and oxygen content in function of applied power for MeOx (red), EtOx (blue), PrOx (pink) and BuOx (green), open symbols denote points in the unstable region for the respective monomers. Next, the chemical composition of the coatings was assessed via FT-IR spectroscopy. Figure 3A shows the FT-IR spectra of the coatings obtained by plasma polymerization of the different monomers at similar discharge powers of approximately 36-38 W. As illustrated, all FT-IR spectra look quite similar, having the same characteristic peaks at 3500-3000 cm-1 (N-H or O-H stretching), between 3000 and 2800 cm-1 (C-H stretching) and between 1700 and 1500 cm-1 (C=O stretch or N-H bending), which coincides with previous reports and indicates that the coatings contain similar chemical functionalities as those obtained in previous reports.1,4,33,36 Increasing the aliphatic side chain length does therefore not entail the generation of additional chemical functionalities in the plasma polymers and suggests a common mechanism for the plasma deposition of all used monomers. Due to the complex mechanisms of fragmentation occurring during the plasma polymerization process, it is difficult to qualitatively assign the different peaks in the FT-IR-spectra to specific chemical functionalities. The FT-IR spectra, however, suggest that the formed coatings contain a variety of chemical functionalities. The small band at 2260-2100 cm-1 points to the minor presence of isocyanides, nitriles and/or other C=N species, which is supported by the strong reduction of this peak upon immersion in water.


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Finally, XPS was used to further characterize the chemical composition of the coatings, especially concentrating on the effects of monomer type and applied discharge power. Figure 3B shows the elemental composition of the different coatings as a function of applied discharge power as obtained from XPS survey scans. From the graphs, it is apparent that the carbon content increases while the nitrogen content decreases when side-chain length increases. This trend is therefore also responsible for the slightly increasing WCA values in the stable regime with increasing aliphatic side chain. For all monomers, a drop in heteroatom content is seen when transitioning from the unstable regime towards the stable regime, which could be expected as functional group retention has been reported to decrease with increasing power.27 In particular, the oxygen content of the coatings tends to decrease with applied discharge power, which seems to lead to increased stability of the coatings in aqueous environment. Additionally, coatings of all applied monomers also show a higher carbon content and lower nitrogen and oxygen content compared to the corresponding monomers. However, the relative difference in elemental composition between the coating and the monomer decreases as side chain length increases. This suggests that the fragmentation of the side chain in plasma occurs more readily with increasing side chain length. While the chemical composition differs slightly for all monomers, the chemical functionalities remain similar, which coincides with the FT-IR data. This was further confirmed by high resolution XPS, showing little to no differences in peak energies for the C1s, N1s and O1s spectra across the different monomers (Figure S11B). Finally, the peak energies show very little variation as a function of power (Figure S11A), suggesting a similar fragmentation mechanism for all monomers. Varying the applied power will therefore only affect the relative abundance of the chemical functionalities, the cross-linking reactions and consequently the coating stability.2,37–39 Next, the chemical inertness of the different coatings in water was assessed by performing XPS survey scans on the coatings before and after 3 and 7 days of incubation in water. Figure 4 shows the O/N ratio of the coatings versus water incubation time. All plasma coatings show similar behavior when exposed to water, the O/N ratio increases up until 3 days of incubation, after which the O/N ratio stabilizes. These results suggest the presence of water labile functionalities in the coatings or hydrolysis of the coatings. From the FT-IR spectra, it is observed that the absorbance peak corresponding to isocyanides (2154 cm-1) disappears upon water exposure, however, the minor presence of these groups in the coating could not explain the dramatic shift in O/N content. Therefore, we hypothesize the presence of other water labile moieties in the plasma polymerized coatings, such as for example imines.



Figure 4. O/N ratios of the plasma polymerized coatings in function of incubation time in water.

3.2.2.Biointeractive properties


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Figure 5. A) Protein adsorption assay of albumin (filled bars) and fibrinogen (empty bars) onto the plasma polymerized 2-oxazoline coatings at different powers for pPMeOx and compared at ±30 W across monomers. B) Results of MTT assays after 1 day (filled bars) and 3 days (empty bars) of HFF cell culture on the plasma polymerized 2-oxazoline coatings. The cell viability is normalized to a tissue culture plate and expressed in percentage. For each monomer, the applied discharge powers are placed under the columns (in W) and arranged in ascending order. C) The numbers of colony forming units (#CFU) on the different 213 ACS Paragon Plus Environment


oxazoline coatings plasma polymerized at different discharge powers. PP is untreated PP that is glued in the same way as the other samples. PP’ is a sample of untreated PP that is glued together on both sides and has the same size as the other samples. Next, we assessed the biointeractive properties of the obtained coatings. First, the adsorption of fluorescently labeled proteins was studied, since the initial adherence of proteins influences the formation of biofilms.40 Initially the protein adhesion as a function of power was investigated on pPMeOx, the most hydrophilic coating, therefore minimizing potential hydrophobic interactions with the proteins. Secondly, the influence of side chain length was investigated by comparing coatings of the different monomers deposited at comparable powers (± 30 W). Figure 5A shows the relative protein adsorption of the coatings versus a pristine PP substrate. Compared to the PP substrate all coatings showed increased protein adsorption. The protein adhesion, however, seems to drop as a function of applied power for pPMeOx, displaying higher protein adsorption for coatings deposited at a lower power, which could be explained by decreased functional group retention as a function of power, thus leading to fewer attachment points for the proteins. When comparing the different monomers, however, no distinguishable trends could be observed as a function of side chain length, which could be explained by the similar chemical composition of the coatings. Overall, the coatings seem to promote protein adhesion, which could be caused by the presence of reactive groups on the surface, as suggested in previous studies.5,41,42 Since the coatings promoted the adhesion of proteins, the proliferation and cellular interaction of the coatings was also investigated with both mammalian cells and bacteria, in order to assess their cytocompatibility. For the mammalian cells, the proliferation and biocompatibility of the coating was quantified via an MTT assay, while a qualitative measure of cell interactivity was obtained via fluorescence microscopy after live/dead staining of the cells seeded on the different plasma polymerized coatings. Figure 5B shows the cell viability of human foreskin fibroblasts after 1 day (full bars) and 3 days (empty bars) of cell culture. The obtained cell viabilities at day 1 can be used as a measure for cellular adhesion to the coatings. Here, the pristine substrate shows very little cell-material interactions, and consequently has a low cell viability. The coatings, however, exhibit strong cell-adhesive properties, where cell adhesion tends to increase with increasing side-chain length, with pPBuOx performing close to a tissue culture plate after 1 day. Following 3 days of cell culture, the obtained cell viabilities relative to day 1 give an indication on the effects of the coatings on cell proliferation. The more hydrophilic coatings, pPMeOx and pPEtOx, display a significant preservation of cell viability compared to the PP substrates, indicating that these coatings have a beneficial effect on cell adaptation during the lag phase. The more hydrophobic coatings, pPPrOx and pPBuOx, display a similar drop in cell viability as the PP substrates, indicating no beneficial effect. While there is no clear correlation between the deposition power and cellular viability from the MTT assays, the fluorescence images taken of the cells seeded on the coatings after live/dead staining (see figure S14) show an improvement in cellular morphology and cell density on coatings deposited at lower discharge powers and with increasing side chain length of the monomer. In summary, the coatings exhibit a remarkable cell-interactive behavior, making them potentially useful in applications for tissue regeneration purposes. The overall substrate independent nature of the plasma process10,38,43 coupled with the cytocompatibility of these plasma polymerized 2-oxazolines may be applied to improve cell viability of different surfaces as well as supports for tissue engineering purposes.1,8,9 14 ACS Paragon Plus Environment

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However, the surfaces do not only promote cellular attachment and proliferation of mammalian cells but also of bacteria such as S. Aureus and P. Aeruginosa (Figure 5C). All plasma polymerized coatings show negligible decreases in bacterial adhesion compared to the bare PP substrate. Therefore, in a tissue engineering context, these coatings could benefit from functionalization with bactericidal functionalities or antibiotics, which could be potentially done by utilizing the reactivity of the coatings as reported by the group of Vasilev.10,42

3.3. Comparison of plasma polymers versus CROP polymers Since the plasma deposition of monomers is a complex mixture of different fragmentation and recombination processes, which are dependent on the plasma conditions, it is difficult to compare and quantify the different chemical functionalities in the chemically complex coating. Since an unambiguous characterization of the different plasma polymerized coatings is challenging, the chemical composition of the obtained plasma polymerized coatings was benchmarked to the well-characterized parent polymers, viz. PAOx. In literature, PMeOx and PEtOx have been reported to possess anti-fouling properties,44–46 which is in contrast to the obtained plasma polymerized coatings. Therefore, a structural comparison between the polymers obtained via CROP and plasma polymerization would be valuable to understand the differences in cell-interactive behavior.



Figure 6. A) WCA measurements on plasma polymerized 2-oxazoline coatings in the stable region on a PP substrate (in red) compared to the spin coated CROP polymers. *For PMeOx a WCA could not be obtained. B) FT-IR measurements of pPEtOx at 34.7 W (black), compared to the CROP polymers of MeOx (red), EtOx (blue), nPrOx (pink) and BuOx (green). C) High resolution C1s, N1s and O1s spectra of pPEtOx (P=35.9 W) (red), PEtOx (black) and ring-opened PIPRO (blue). D) 1H NMR spectra of pPPrOx (black), PPrOx (red) and PrOx (blue). To the authors’ knowledge, no WCA data of the PAOx polymers have been previously reported, despite the extensive literature on the solution behavior of these polymers.47 Only a few WCA values of chemically anchored PAOx brushes have been reported, but since surface and polymer topology could affect the WCA value, spin coating of the high molecular weight PAOx polymers was performed to obtain a PAOx film, allowing a better comparison to the plasma polymer film in terms of topology.48,49 Figure 6A displays the obtained WCA values, revealing a clear difference in wettability between the plasma polymer films and the CROP polymer films. As mentioned earlier, increasing the side chain length of the precursor results in a small increase in WCA value of the plasma polymerized films. This is in strong contrast with the relative difference displayed across the different CROP polymers. For these polymers, the wettability shows a sharp increase with increasing side chain length of the monomer, with WCAs ranging from 0o for PMeOx to 93° for PBuOx. The high contact angle for PBuOx suggests that the sample underwent arrangement of the side-chains towards the polymer-air interface.47 This rearrangement was previously ascribed to the low Tg, which is around room temperature, and may be further facilitated by slower solvent evaporation for the high molecular weight polymers compared to the previously reported rather low molar mass PBuOx, resulting in enhanced chain mobility. In general, the plasma polymers exhibit a relatively constant intermediate WCA as a function of side chain length compared to the CROP polymers, which indicates the high occurrence of side chain fragmentation. This theory is further supported by the XPS measurements of the plasma polymers, where the carbon content of the plasma polymerized films is consistently higher than those of the monomers and the CROP polymers. As such, the WCA does not dramatically increase, which can only be explained by the fragmentation of the monomer side chains. This WCA comparison also confirms the partitioning of heteroatomic fragments, described earlier in the XPS section. The WCA results reveal that the wettability of PEtOx, PPrOx and most probably PMeOx – which could be expected from the reported surface energies47– is higher than the respective plasma polymers, which can only be explained by the partitioning of polar heteroatomic fragments. This partitioning, however, decreases with increasing side chain length of the monomer, possibly due to more side chain fragmentation occurring in favor of ring fragmentation, thus resulting in stable films at lower discharge powers. The FT-IR spectra depicted in Figure 6B also reveal a relatively higher abundance of water, hydroxyl or amine groups in the plasma polymers versus the CROP polymers, which only show a small band from 3600 to 3200 cm-1, which might arise from some absorbed water and partially of the hydroxyl ω-terminus of the polymer. As mentioned before, the plasma polymers also exhibit the presence of isocyanides, nitriles and/or imino-ethers (small band at 2260-2100 cm-1), which are not present in the CROP polymers. Another minor difference is that the amide band of the CROP polymers is much sharper, potentially due to the sole contribution of tertiary amides. This indicates that the plasma polymers may have other chemical 16 ACS Paragon Plus Environment

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functionalities causing C=O stretching or N-H bending in the region 1700-1500 cm-1. Other than these minor differences, FT-IR does not allow the unambiguous distinction of differences in chemical composition. Next, XPS analysis of the plasma polymers, CROP polymers, PiPRO, and its acetic acid ring-opened derivative was performed, allowing the structural comparison of the plasma polymerized films with polymers containing a tertiary amide, an intact oxazoline ring, a secondary amide and ester in their repeating unit structure, respectively. The high resolution C1s, O1s and N1s peaks are shown in Figure 6C for pPEtOx, PEtOx and ring-opened PiPRO, while the high resolution XPS spectra for the other used monomers can be found in the supporting information (Figure S12). From C1s spectra of the known polymer structures, we could conclude that the plasma polymers lack the following functional groups: 1) ester groups, as the strong signal at the peak energies found in acetic acid ring-opened PiPRO is absent in the plasma polymers, and 2) amide groups, as the contribution of the amide binding energy is negligible compared to PEtOx and acetic acid ring-opened PiPRO. From the O1s spectra we can also conclude that there is only a minor presence of tertiary amides, since these would contribute to higher binding energies. Finally, Figure S13 suggests very little oxazoline ring-retention in the plasma polymers, as the peak energies in the O1s and N1s spectra significantly differ from those obtained for the PiPRO polymer. This finding also shows that in the regime where stable coatings can be obtained, little monomer retention can be expected. As a final characterization technique 1H NMR spectroscopy was used to characterize the plasma polymerized coatings and the CROP polymers. In this case, a pPPrOx coating was incubated for several days in CDCl3 and the resulting solution was analyzed. To our surprise, the 1H NMR spectrum of the pPPrOx reveals the presence of discrete peaks, which indicates that the fragmentation and recombination of the reactive species in the plasma follow one or multiple discrete pathways rather than being a random process. The 1H NMR spectrum also shows peaks in the aromatic region, which points to the presence of aromatics or imines, the former could account for the observed autofluorescence of the plasma polymerized coatings. At lower chemical shifts, signals corresponding to aliphatic carbons can also be observed. Most peaks are broad, due to the polymeric nature of the coating, giving rise to longer relaxation times. When pPPrOx is compared to PPrOx and PrOx, it becomes clear that the plasma polymers show very little similarities to the CROP polymer or the monomer, with the characteristic protons of the PPrOx backbone (3.4 ppm) and oxazoline ring (4.2 and 3.8 ppm) being absent in the plasma polymer spectrum. This observation supports the observed differences in coating wettability and peak energies in the high resolution XPS measurements. Together, these results provide an explanation for the large differences observed between the plasma polymerized films and the CROP polymers. WCA analysis reveals a strong partitioning of heteroatomic fragments, resulting in more hydrophobic coatings compared to the CROP polymers (with the exception of BuOx), which is supported by the absence of polar groups such as (tertiary) amide and ester groups as observed via XPS. The absence of tertiary amides, i.e. hydrogen-bond accepting groups, contributes to the major differences in biological properties between PAOx and plasma polymerized polymers. Since the tertiary amide groups and absence of hydrogen-bond donating groups in PAOx contribute to its stealth and antifouling behavior, it is obvious that the absence of tertiary amides and presence of hydroxyl, amine and imines, i.e. hydrogen-bond donating groups, 17 ACS Paragon Plus Environment


would result in cell interactive coating. Also, the presence of reactive groups such as imines, ketones, aldehydes and isocyanates could further promote cellular attachment.

4. Conclusions The near atmospheric pressure plasma polymerization of a set of 2-oxazoline monomers with increasing side chain length was explored in this work. Through this structural investigation, it could be concluded that side chain fragmentation is occurring more frequently during plasma polymerization with increasing side chain length of the monomer relatively to the ring fragmentation. Hence, an increasing side chain length decreases heteroatomic fragment partitioning and has, therefore, minor influences on the overall coating wettability and chemical composition. In addition, an increasing side chain length leads to more stable coatings at lower discharge power, which is overall more favorable, as it leads to a more reproducible coating process that has a higher energy efficiency and allows for lower operating temperatures. Furthermore, increasing the side chain length of the precursor also improved the biointeractive properties of the coatings, showing an improved cell morphology and up to 93% cell viability for pPBuOx relative to a tissue culture plate after 1 day of cell culture. Therefore, the deposition of plasma polymerized 2-oxazoline coatings on a wide range of substrates presents an interesting opportunity for tissue engineering applications. More specifically, these coatings might enhance cellular interactions between substrate and specific cell types50 or be applied for the differentiation of stem cells.51In addition, the developed plasma polymers have been compared to the well-defined CROP polymers obtained from the same precursor in this work. This comparative study revealed clear differences between the plasma and the CROP polymers. Wettability of the plasma polymers remains relatively constant for the different used monomers, while the CROP polymers show large differences for growing aliphatic side chains. Furthermore, by the direct comparison of these polymers, it could also be concluded that the plasma polymers contain very little ester, 2-oxazoline and tertiary amide groups and that surface analysis techniques often fall short to deliver a thorough chemical and structural analysis of plasma deposited films. 1H NMR spectra of the obtained coatings also show the presence of an aliphatic carbon chain, paired with protons in the aromatic region, which could correspond to a semi-aromatic structure or the presence of imines. This finding is also supported by the polymer’s autofluorescence, which was also described in previous reports.33

5. Acknowledgements Pieter Cools would like to thank the Special Research Fund of Ghent University for financing his postdoctoral grant. Tim Egghe acknowledges the support of the Research Foundation Flanders (FWO) for a PhD grant strategic basic research. This work has also received funding from the European Research Council (ERC) under the European Union’s Seventh Framework Program (FP/2007-2013)/ERC Grant Agreement n. 335929 (PLASMATS).

6. Supporting Information


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The Supporting Information is available free of charge on the ACS Publications website at DOI: xxxxxxxxxxxxxx. Synthesis and characterization of monomers and polymers (1H NMR, SEC). Electrical characterization of the DBD. High resolution XPS spectra of plasma polymerized coating and fluorescence images of live/dead staining assays of cell seeding on the plasma polymerized coatings.

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Graphical abstract


Influence of the Aliphatic Side Chain on the Near Atmospheric

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