Poly(2-oxazoline)s One-Pot Polymerization and Surface Coating

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Poly(2-oxazoline)s one-pot polymerization and surface coating: From synthesis to anti-fouling properties out-performing poly(ethylene oxide) Jan Svoboda, Ondrej Sedlacek, Tomáš Riedel, Martin Hruby, and Ognen Pop-Georgievski Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00751 • Publication Date (Web): 22 Jul 2019 Downloaded from pubs.acs.org on July 23, 2019

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Poly(2-oxazoline)s one-pot polymerization and surface coating: From synthesis to anti-fouling properties out-performing poly(ethylene oxide) Jan Svoboda*, Ondřej Sedláček, Tomáš Riedel, Martin Hrubý and Ognen Pop-Georgievski* Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovskeho nam. 2, 162 06 Prague 6, Czech Republic.

KEYWORDS poly(2-oxazoline)s, poly(ethylene oxide), polydopamine, anti-fouling surfaces, polymer brush, surface analytics

ABSTRACT Poly(2-alkyl-2-oxazoline)s (PAOx) represent a class of emerging polymers which can substitute or even outperform poly(ethylene oxide) (PEO) standard in various applications. Despite the great advances in the PAOx research, there is still a gap in the direct experimental comparison of anti-fouling properties between PAOx and the golden standard PEO when exposed to blood. Motivated by this, we developed a straightforward protocol for the one-pot PAOx polymerization and surface coating by “grafting to-” approach. First, we synthesized a library of hydrophilic poly(2-methyl-2-oxazoline)s (PMeOx) and poly(2-ethyl2-oxazoline)s (PEtOx) with molar mass ranging from 1.5 to 10 kg/mol (DP = 16 – 115). The PAOx living chains were directly terminated by amine and hydroxyl groups of polydopamine (PDA) anchor layer providing the highest so far reported grafting densities ranging from 0.2 to 2.1 chains/nm2. In parallel, PEO chains providing the same degree of polymerization 1 ACS Paragon Plus Environment

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(molar mass from 1.2 to 5 kg/mol, DP = 28–116) bearing thiol groups were grafted to PDA. The thickness, surface related parameters, covalent structure and anti-fouling properties of the resulting polymer brushes were determined via various surface sensitive techniques. The comparison of the synthesized PAOx and PEO brushes lead us to the conclusion that at the same surface related parameters PMeOx brushes show significantly better antifouling character when challenged against human blood plasma.

Introduction The recent developments in surface and polymer science have led to the preparation of novel molecular structures with tailored covalent, structural, optical, ferromagnetic, and catalytic properties, to name but a few. Surface-confined architectures have been designed and used for numerous areas and applications.1 The biomaterial research in general –and in particular the areas of biosensing, bioadhesion, drug delivery, and tissue engineering– has devoted great effort for the preparation of structures with precisely controlled interactions with biological systems and in biological media.2-3 This involves the creation of interfaces capable of resisting non-specific protein adsorption (fouling) and cell adhesion,4 as well as preventing bacterial colonization,5 thrombogenicity,6 guiding the formation of new organized tissue,7 capturing specifically recognized biomolecules,8-9 or catalyzing biochemical reactions. The application of such advances in biomaterial science and biosensing is already leading to significantly enhanced outcomes in medical practice. Several approaches have been introduced to achieve the control of interactions at the interfaces of biomaterials and biosensors. Self-assembled monolayers (SAMs) of oligo(ethylene glycol) (OEG) present a well-known strategy to inhibit the protein adsorption.10-11 A series of theoretical and experimental studies have correlated the number of ethylene glycol units per oligomer chain, lateral chain density, interfacial structure,

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conformation, distal-end chemistry, hydrophilicity of the layers, and structure of solvating water to the resistance of these surfaces to fouling from model solutions of single plasma proteins.12 Grafting of preformed poly(ethylene oxide) (PEO) "to-" the material surface often led to the creation of surfaces that outperformed the OEG SAMs in terms of protein resistance and biocompatibility.13-14 The strong steric repulsion forces exerted on the adsorbing proteins by the strongly hydrated PEO chains have been identified as responsible for the improved fouling resistance. Their driving force arises from the entropic penalty from the compression of the polymer chains and energetically unfavorable displacement of polymer-bound water molecules by the approaching proteins.15-16 Thus, for a coating to exhibit antifouling properties, high grafting densities and sufficient polymer layer thickness are simultaneously required. However, there is an increasing debate about the stability of PEO under physiological conditions. Arguably, most of the reports refer to the observed thermal and oxidative instability of PEO surfaces when exposed to extreme conditions.17-18 Additionally, several groups have elaborated that treatment with PEO activates the immune system that produces specific antibodies, leading to “accelerated blood clearance” of the therapeutics based on this polymer.19-20 In animal models, anti-PEO immunity is typically robust but short-lived and consists of a predominantly anti-PEO IgM response.20 Other polymer platforms have been developed to overcome the problems associated with the use of PEO. With the advent of controlled polymerizations and the diversity of employed surface tethering approaches, dense polymer brushes could be achieved based on hydrophilic polypeptoids, N-vinylpyrrolidone, acrylates and methacrylates, acrylamides and methacrylamides, as well as zwitterionic monomers based on phosphorylcholine, carboxy- and sulfo-betaines.6,21-22 Poly(2-alkyl-2-oxazoline)s (PAOx) represent an emerging class of polymers with a wide range of applications.23-33 They are synthesized by living cationic ring-opening



polymerization (CROP) of 2-oxazoline monomers, resulting in polymers with well-defined chain length, narrow molar mass distribution, as well as an excellent end-/sidechain- group functionality and fidelity.34 During the polymerization, the living oxazolinium chain-ends can be terminated by a wide range of nucleophiles, providing a straightforward route to the chainend functionalized PAOx. This strategy was recently used for the synthesis of PAOx-grafted polysaccharides.35-36 The physical properties of PAOx (e.g., hydrophilicity, crystallinity) depend mostly in the structure of the side-chain substituents. PAOx with the shortest side chains, i.e., poly(2-methyl-2-oxazoline) (PMeOx) and poly(2-ethyl-2-oxazoline) (PEtOx) are water-soluble non-fouling polymers with biological properties often surpassing those of PEO.37-38 They form a versatile platform for various biomedical and tissue engineering applications, conjugation of biomolecules, drug delivery and imaging, etc.39-42 Among other applications PAOx have been of great interest for the design on antifouling and biocompatible surfaces.43-44 Most of the reported procedures on PAOx coating rely on two-step protocol consisting in PAOx synthesis by CROP followed by grafting to the surface by an isolated functional polymer.45 Alternatively, PAOx coatings can be prepared by surface-initiated polymerization of the corresponding monomers.46 The obtained PAOx surfaces exhibited high stability and excellent anti-fouling properties when exposed to serum, bacterial and chondrocyte adhesion.24,44,47 Mixed poly(acrylic acid)/PMeOx brushes have been performed by attacking PDA anchoring layer with thiol and amine polymer end-groups. Various codeposition processes utilizing PDA and PMeOx of various architectures have been utilized by Wang et al. to achieve mixed polymer layers that resisted fouling from single protein solutions and prohibited non-specific platelet adhesion.45,48-49 Supposing similar modes of actions as in the case of marine mussel adhesion, a 5-mer peptide of alternating L-3,4dihydroxyphenylalanine (DOPA) and L-lysine has been attached to N-substituted glycine (peptoid) oligomer to suppress the fouling from serum.50


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However, the reports on direct comparison of PAOx-coated surfaces with the PEOylated analogs are still very sparse and are limited to the disseminating works of Textor, Serrano and Benetti.24,38,43 Despite the excellent pioneering work, the method based on adsorption of poly(L-lysine)-graft-PAOx copolymers resulted in polymer brushes not exceeding 0.1–0.2 PAOx chains/nm2 (corresponding to monomer surface densities of 11–15 units/nm2). Even more, the literature lacks reports on the behavior of PAOx when exposed to blood plasma, often considered as the most demanding biological medium when fouling and medical applications are in question. Herein, we report for the first time a simple robust one-pot protocol for the PAOx polymerization followed by direct grafting of the living chains to nucleophilic functional groups of polydopamine (PDA) anchor layer. This protocol is robust and rather universal, enabling the formation of well-defined PAOx brushes of high grafting densities ranging from 0.2–2.1 PAOx chains/nm2 (corresponding to monomer surface densities of 23–45 units/nm2) depending on the molar mass of the used polymer. So far, such dense films have been obtained only when cyclic PAOx polymers have been tethered to poly(glycidyl methacrylate) anchor layer.51 Although very robust, the proposed grafting through modification approach utilizing cyclic PAOx is based on a rather laborious procedure. The physical and anti-fouling properties of prepared PMeOx- and PEtOx-grafted surfaces were compared to the analogic surfaces based on PEO standard. Compared to other protocols24,43,45,51 our simple inexpensive protocol is universal and well suited for various substrates as the lower supporting PDA layer adheres to nearly any substrate, provides chemically fully stable polymer layer as covalent bond in our case is more robust than, e.g., Nb2O5 – poly(L-lysine) electrostatic adhesion24,43. Thus, the method does not suffer from the problems inherent to the usage of PLL backbones and high PAOx chains/L-lysine ratios, leading to: i) reduced the electrostatic adsorption energy since every grafted side chain



eliminates one positively charged amine group of the anchoring PLL backbone (by converting the amine to an amide); ii) reduced adsorption energy of the graft-copolymer due to PAOx chain crowding and the intervening excluded volume effects; and iii) stiffening of the PLL backbone that hampers the surface rearrangement leading to inhomogeneity and incompletely covered surfaces.44,47 Furthermore, the one-pot modification, avoids the creation of highly intermixed films which are formed during the various co-deposition processes.45 As it relies on the direct binding of PAOx chains living end-groups to the nucleophilic groups of PDA it offers minimal disturbance of the initial catechol/quinone ratio which predefines the stability of the anchor layer.45,52 Notably, the robustness of the PDA anchor should not be confused with the short catechol anchors based on DOPA, as the later ones are applicable mainly to metallic surfaces. As the surface adherent PDA film used in our study is not prone to protease degradation and can be further stabilized by thermal crosslinking,53 the developed “one-pot modification” offering the dense attachment of PAOx chains could potentially offer the design of structurally more defined and stable films. The herein reported surface conjunction also allows using our protocol for materials with different surface geometry, morphology and eventually porosity. As it is only limited by the conditions under which the grafting is performed and the actual dimensions of the PAOx chain, the one-pot direct grafting method enables the creation of dense brushes. Last but not least, the developed surface modification method allows detailed characterization of the grafted polymer brushes so defined and reproducible coatings can be synthesized.

Experimental part Chemicals 2-Ethyl-2-oxazoline (EtOx) and 2-methyl-2-oxazoline (MeOx) were purchased from Sigma-Aldrich Ltd. (Prague, Czech Republic) and distilled from calcium hydride prior to use.


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Hetero-bifunctional α-thioethyl-ω-methoxy poly(ethylene oxide) (PEO) with numberaveraged molar mass (Mn) of 1.2, 2.0, and 5.1 kg/mol, were bought from Rapp Polymere. The dispersity (Đ = Mw/Mn) for all PEOs was 1.05 as provided by the producer. The PEO polymers were designated as PEO 1.2k, PEO 2k and PEO 5k according their molar masses. All

other

chemicals,

including

acetonitrile,

dopamine

hydrochloride,

methyl

p-

toluenesulfonate (MeOTs) and triethylamine were purchased from Sigma-Aldrich Ltd. (Prague, Czech Republic) and were used without further purification. Substrate preparation Substrates of size 2 × 1 cm2 were cut from one-side polished silicon wafers (CZ, orientation , p-doped, resistivity 5-20 Ω·cm) bearing 1.6 nm natural oxide over-layer (Siegert Wafer GmbH, Germany). The gold coated substrates for surface plasmon resonance (SPR) analysis and infrared spectroscopy were obtained from Institute of Photonics and Electronics, Academy of Sciences of the Czech Republic, and consisted of glass support, ~2 nm of the titanium adhesion layer and ~50 nm of the gold layer. All substrates were cleaned by sonication in ethanol and water, blow dried with nitrogen, and activated in a UV-ozone cleaner for 20 min. Preparation of PDA anchor layer The anchoring layer was deposited from 2 mg/mL solution of dopamine hydrochloride in an air-saturated 10 mM Tris-HCl (pH 8.5) buffer. The polymerization of PDA on the substrates was performed in open glass dishes under controlled stirring that provided a continuous supply of oxygen from the air. The flat substrates were kept vertical to suppress microparticle sedimentation. After 3 h of polymerization, the PDA coated surfaces were rinsed with water, sonicated in water for 15 min, stabilized at 110°C and blown dry in a stream of purified nitrogen.53



PAOx synthesis and characterization 2-Ethyl-2-oxazoline (EtOx) (10 g) or 2-methyl-2-oxazoline (MeOx) (10 g), respectively, were mixed with dry acetonitrile in the Schlenk flask under argon atmosphere to achieve initial monomer concentration of 4 M (Scheme 1). The calculated amount of MeOTs initiator (Table 1) was added through the septum and the mixture was stirred at 80°C for the period listed in Table 1. The required polymerization time was calculated according to the reference to achieve > 99 % monomer conversion.34 After the polymerization was finished, part of the mixture (200 µL) was taken using a syringe and terminated with methanolic KOH (1 M) for molar mass determination. The main part of the polymerization mixture containing polymers with reactive semitelechelic oxazolinium end-groups was used directly for coating. The molar masses (Mw - weight-averaged molar mass, Mn - number-averaged molar mass) and dispersity (Ɖ = Mw / Mn) of the terminated polymers were determined by size exclusion chromatography (SEC) using an HPLC Ultimate 3000 system (Dionex, USA) equipped with a SEC column (TSKgel SuperAW3000 150 × 6 mm, 4 μm). Three detectors, UV/Vis, refractive index (RI) Optilab®-rEX and multi-angle light scattering (MALS) DAWN EOS (Wyatt Technology Co., USA) were employed; with a methanol and sodium acetate buffer (0.3 M, pH 6.5) mixture (80:20 vol%, flow rate of 0.5 mL/min) as mobile phase using particular refractive index increments (dn/dcPMeOx = 0.181 mL/g; dn/dcPEtOx = 0.179 mL/g).

Scheme 1. Synthesis of PAOx living chains employed for surface grafting


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Table 1. Synthesis and characterization of PAOx grafts.

Polymer

Target Mw [M]0:[I]0a (kg/mol)

Polymerization Mwb (kg/mol) Time (h)

Ɖb

PMeOx 1.5k

1.5

18:1

2

1.62

1.04

PMeOx 3k

3.0

35:1

2

3.19

1.05

PMeOx 6k

6.0

71:1

4

6.22

1.10

PMeOx 10k

10.0

118:1

6

9.80

1.16

PEtOx 1.5k

1.5

15:1

2

1.54

1.05

PEtOx 3k

3.0

30:1

2

2.97

1.03

PEtOx 6k

6.0

61:1

4

6.15

1.07

PEtOx 10k

10.0

101:1

8

10.30

1.11

aInitial

molar ratio of monomer to initiator; bDetermined by SEC-MALS after termination with methanolic KOH, dispersity Ɖ = Mw / Mn.

PAOx grafting PDA-coated samples were immersed under argon atmosphere in the respective polymerization mixtures containing living polymer chains. Dry triethylamine (2 equivalents of the original initiator amount) was added and the system was vortexed at 40°C for 24 h. Then, the grafted substrates were taken out, repeatedly washed with distilled water, methanol and blown dry in a stream of purified nitrogen. PEO grafting The conditions of PEO grafting to PDA-coated samples have been tuned so the surface related parameters of the resulting layers would match the ones observed for their PAOx counterparts. The grafting of PEO 1.2k and PEO 5k was performed under good solvent conditions, i.e., from 1 mg/mL water solutions at 50°C for 2 h. The grafting of PEO 2k was performed from reactive melt at 110°C for 12 h. Afterwards, the grafted substrates were



repeatedly washed with distilled water, methanol and blown dry in a stream of purified nitrogen. Atomic force microscopy (AFM). AFM images were obtained as topographical scans in tapping mode in the air by using a Multimode Atomic Force Microscope NanoScope IIIa (Digital Instruments), using silicon probes OTESPA-R3 (Bruker) with a nominal spring constant of 26 N/m and a tip radius of 7 nm. Areas of 5 × 5 μm2 (512 × 512 pixels) were scanned at a rate of 1 Hz. Gwyddion software was used to analyze the scans. Contact angle goniometry. The static water contact angles were measured with contact angle goniometer OCA 20 (Dataphysics, Germany) equipped with SCA 21 software. 2 μL drops were deposited on tested surfaces and their profiles were fitted with the Young-Laplace equation. Values are averages of at least four measurements recorded at different positions on each sample. Infrared reflection-absorption spectroscopy (IRRAS). The infrared spectra of the dry polymer layers anchored to PDA were recorded using a Thermo Nicolet NEXUS 870 FTIR Spectrometer equipped with a deuterated triglycine sulfate thermoelectric-cooled detector, and a Smart SAGA grazing angle (80°, p-polarization) reflection spectroscopy accessory (Thermo Fisher Scientific). The measurement chamber was continuously purged with dry air. The spectra are averages of 128 scans taken at a resolution of 2 cm−1. The acquisition time was around 15 min. All IRRAS spectra are reported as −log(R/R0), where R is the reflectance of the sample and R0 is the reflectance of bare gold. X-ray photoelectron spectroscopy (XPS). Measurements were carried out with a K-Alpha+ spectrometer (ThermoFisher Scientific, East Grinstead, UK). The samples were analyzed using a micro-focused, monochromated Al Kα X-ray source (400 µm spot size) at an angle of incidence of 30° (measured from the surface) and an emission angle normal to the surface. The kinetic energy of the electrons was measured using a 180° hemispherical energy analyzer


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operated in the constant analyzer energy mode (CAE) at 200 eV and 50 eV pass energy for the survey and high-resolution spectra respectively. All reported spectra are averages of 5 scans taken at a resolution of 0.1 eV and referenced to the C1s peak of hydrocarbons at 285.0 eV. Data acquisition and processing were performed using Thermo Advantage software. The XPS spectra were fitted with Voigt profiles obtained by convolving Lorentzian and Gaussian functions. The analyzer transmission function, Scofield sensitivity factors, and effective attenuation lengths (EALs) for photoelectrons were applied for quantification. EALs were calculated using the standard TPP-2M formalism. The BE scale was controlled by the well-known position of the photoelectron C-C and C-H, C-O and C(=O)-O C1s peaks of polyethylene terephthalate and Cu 2p, Ag 3d, and Au 4f peaks of metallic Cu, Ag and Au, respectively. The BE uncertainty of the reported measurements and analysis is in the range of ±0.1 eV. Surface plasmon resonance spectroscopy (SPR). A custom-built SPR instrument (Institute of Photonics and Electronics, Academy of Sciences of the Czech Republic, Prague) based on the Kretschmann geometry of the attenuated total reflection method and spectral interrogation of the SPR conditions was used. The tested solutions of proteins or body fluids were driven by a peristaltic pump through four independent channels of a flow cell for 15 min., in which the SPR responses were simultaneously measured as shifts in the resonant wavelength, λres. The sensor response (Δλres) was obtained as the difference between the baselines in phosphate buffered saline (PBS) before and after the injection of the tested samples: human serum albumin (HSA, 5 mg/mL), fibrinogen (Fbg, 1 mg/mL) and human blood plasma (diluted to 10% and whole). All dilutions of protein solutions were performed in PBS. The sensor response was calibrated to the mass deposited at the surface of bound molecules. According to a calibration made by Fourier-transform infrared grazing angle specular reflectance, a shift Δλres = 1 nm corresponds to a change in the deposited protein mass of 150 pg/mm2.



Spectroscopic ellipsometry J.A. Woollam M-2000X spectroscopic ellipsometer was used to measure the dry thickness of the polymer brushes to study the kinetics of the polymerization. Ellipsometric data were obtained in the air at room temperature in the wavelength range λ = 245–1000 nm at angles of incidence of 60, 65, and 70°. Focusing optics (producing 120 μm spot) and position-calibrated sample stage enabling repeated measurements over the same sample area have been utilized to increase the measurement precision and exclude errors from the variations of layer thickness throughout the probed area. The data were fitted with CompleteEASE software using a multilayer model. The optical dispersion functions of silicon, natural silicon oxide and PDA were taken from previous studies.52,54 Due to the low penetration depth of used light, the 50 nm thick gold layer was modeled using basis spline function having the complex refractive index of single crystal gold as a seeding value at each measured wavelength. The thickness and refractive index of the PMeOx, PEtOx and PEO layers were obtained from simultaneous fitting of the obtained ellipsometric data using Cauchy dispersion functions. The thicknesses are reported for 3 points on the surfaces of 3 independent samples as mean ± standard deviation. Calculation of grafting density, distance between grafting sites, and structural state of polymer chains bound to PDA. The density 𝜎 = supposing hexagonal packing 𝐷 =

2 3𝜎

ℎ𝜌𝑁𝐴 𝑀n

and the distance between grafting sites

were estimated utilizing the layer thickness in the dry

state as determined by ellipsometry (h),29-31 the bulk densities of PEO and PAOx’s were taken to be 1.09 and 1.14 g·cm-3, respectively, and NA is the Avogadro constant. The radius of gyration Rg for PEO and PAOx’s polymers in water was calculated according to literature reports.32 The overlap parameter

𝐷 2𝑅g ,

can be utilized to describe the state of tethered polymer

𝐷

chains. Values of (i) 2𝑅g > 1.0 indicate that the polymer chains are in a "mushroom" state; (ii)


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𝐷 2𝑅g

= 1.0 are characteristic for a mushroom-to-brush transition; whereas

𝐷 2𝑅g

< 1.0 are

indicative that the chains stretch away from the surface and attain brush conformation.

Results and discussion Generally, the mechanisms by which some polymer brushes display effective antifouling properties and others of apparently similar chemical structure do not prevent fouling are still unclear. The comparison between the different polymer brush systems can be made only in the case of the same surface related parameters. Despite the thorough review comparison of PEO and PAOx when the antifouling properties are in question,24 and the disseminating works of Konradi and Benetti,43,47 there is no direct experimental comparison between these two polymer platforms at the same surface parameters when challenged against the most demanding biological fluid i.e. blood plasma. As such knowledge is crucial for the design of a new generation of biointerfaces with enhanced performance, we focus on the direct experimental comparison of dense polymer brushes based on PEO and its potential replacements based on PMeOx and PEtOx. For this reason, we have grafted PEO and PAOx (PMeOx and PEtOx) brushes of different chain lengths to polydopamine (PDA) anchor layer. This bioinspired anchor multifunctional coating can be prepared on a wide range of inorganic and organic materials, including noble metals, oxides, polymers, semiconductors, and ceramics. Thus our findings can be applied to the surfaces of a wide range of materials. The presence of unsaturated indole rings, catechol and amine groups of the various constituting monomer units of PDA can be utilized for secondary binding reactions for the grafting of add-layers to the various solid surfaces.52,55 PMeOx and PEtOx brushes have been achieved by a novel protocol, which consists in one-pot polymerization and grafting of the polymer living cationic growth centers23 to the available amino groups of PDA under theta conditions. The grafting of PEO to the PDA anchor has



been performed via Michael addition reaction utilizing the thiol groups in the α-position of the chain. By precisely optimizing the PEO grafting conditions we have tuned the surface related parameters to completely match the ones observed for the PAOx counterparts. PDA coating The PDA anchoring layer was deposited on silicon and gold SPR chips from 2 mg/mL solution of dopamine hydrochloride in an air-saturated 10 mM Tris-HCl (pH 8.5) buffer. The 3 h immersion under constant stirring resulted in 15.4 ± 1.5 nm thick layers. PAOx grafting The newly developed one-pot approach was employed for the grafting of PDA-coated surfaces with poly(2-alkyl-2-oxazoline)s (PAOx) (Scheme 2). These were prepared by cationic ring-opening polymerization (CROP) of 2-methyl-2-oxazoline (MeOx), respectively 2-ethyl-2-oxazoline (EtOx), using methyl p-toluenesulfonate as the initiator (Scheme 1). After the polymerization was complete, the PDA-coated wafer was immersed into the reaction mixture and the polymer living chain-ends were terminated with the surface amines and hydroxyl functionalities of the anchor PDA. We successfully synthesized a library of grafted surfaces differing in polymer structure (i.e. PMeOx-, respectively PEtOx-based) and their respective molar mass (Mw varied from 1.54 to 10.3 kg/mol (Table 1); DP varied from 16 to 115 (Table 2)).


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Scheme 2. Preparation of non-fouling poly(2-alkyl-2-oxazoline) (A) and poly(ethylene oxide) (B) brushes. PEO grafting “Standard” PEO layers have been synthesized under various grafting conditions. The careful choice of good solvent and melt grafting conditions led to PEO brushes anchored to PDA-coated samples of practically the same surface related parameters as the ones observed for their PAOx counterparts.52,56 The “grafting to” immobilization reaction proceeds as Michael addition, taking the benefit of the high affinity of thiols to activated double bonds of PDA. PEO’s with an appropriate number of monomeric units were used (Scheme 2B). PEO 1.2k (Mw=1.2 kg/mol, DP=28) was utilized as a standard for the PAOx 3k (DP=16-38), PEO 2k (Mw=2 kg/mol, DP=46) was compared with PAOx 6k (DP=62-73) and PEO 5000 (Mw=5.1 kg/mol, DP=116) was compared with PAOx 10k (DP=104-115). This enabled the experimental comparison of PAOx and PEO polymer brushes of the same DP’s.



Ellipsometry Measurements The obtained thickness and known molar masses of polymers were utilized to calculate the surface related parameters of the PAOx and PEO chains and determine the actual state of the polymer chains when exposed to water environments. The surface related parameters of the obtained polymer layers are summarized in Table 2. The thickness of prepared PAOx was between 3.4 and 5.6 nm and is slightly increasing going from PAOx 1.5k (4.2 – 4.6 nm) to 6k (5.4 – 5.6 nm) and decreasing for PAOx 10k (3.4 – 3.7 nm) to the values even smaller than for PAOx 1.5k. This can be explained by the lower availability of reactive chain-end group in the long polymer coil and the coil’s slower diffusion. The thickness of PEO was lowered for all samples in comparison with PAOx with a similar degree of polymerization. The highest chain density is observed in polymers with the lowest degree of polymerization and decreases from 2.1 chains/nm2 for PEtOx 1.5k to 0.2 chains/nm2 for PEtOx 10k upon prolongation of the polymeric chain. Chain density of PEO is comparable with PAOx within already mentioned groups of polymers with a similar degree of polymerization (1.3 chains/nm2 for PEO 1.2k, 0.8 chains/nm2 for PEO 2k and 0.2 chains/nm2 for PEO 5k). Rather logically, similar surface density (ranging between 20–45 monomer units/nm2) of monomer units (n) has been determined within the individual groups of polymers. However, the grafting of PMeOX 6k resulted in polymer brushes with the highest n i.e. 45 monomer units/nm2. The overlap parameter

𝐷 2𝑅g

was determined to be in the range of 0.4–0.6, indicating that in contact with

water or water-based media the PAOx and PEO polymer chains will adopt the brush conformation.


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Table 2. Surface parameters of polymer brushes: degree of polymerization (DP) of used polymers, ellipsometric thickness h, grafting density σ, surface density of monomer units n, distance between grafting sites D, and structural state D/2Rg and static water contact angles θstatic of polymer chains grafted to PDA anchor layer. σ nb D D/2Rgc,d θstatic [chains/nm2] [units/nm2] [nm] [°]

DP

ha [nm]

PMeOx 1.5k

19

4.2±1.9 1.8±0.8

34±15

0.8

0.5

41.0±9.9

PMeOx 3k

38

4.0±0.7 0.9±0.1

33±4

1.2

0.5

40.1±6.9

PEtOx 1.5k

16

4.6±2.3 2.1±0.5

32±8

0.7

0.5

46.8±6.0

PEtOx 3k

30

4.9±0.7 1.1±0.1

34±3

1.0

0.5

39.6±3.5

PEO 1.2k

28

2.3±0.4 1.3±0.2

35±6

1.0

0.4

27.2±1.3

PMeOx 6k

73

5.6±0.4 0.6±0.1

45±7

1.4

0.4

38.7±9.6

PEtOx 6k

62

5.4±0.7 0.6±0.1

37±6

1.4

0.4

35.9±8.0

PEO 2k

46

2.5±0.5 0.8±0.1

37±5

1.2

0.3

22.0±2.1

PMeOx 10k

115 3.7±1.1 0.3±0.1

30±12

2.1

0.5

38.6±4.0

PEtOx 10k

104 3.4±0.7 0.2±0.1

23±10

2.3

0.5

41.0±5.9

PEO 5k

116 1.4±0.3 0.2±0.1

20±12

2.6

0.4

30.1±2.2

aThe

polymer layers were grafted to 15.4 ± 1.5 nm thick PDA anchor layers showing θstatic=65.0±4.9° bSurface

density of monomer units is reported for better comparison with works of Textor, Konradi and Benetti.24,43 cValues

D/2Rg < 1 indicate that the polymer chains have attained the polymer brush regime.

dThe

radius of gyration Rg for PEO and PAOx polymers in water was calculated according to literature reports.32



Contact angle goniometry The contact angle goniometry further verified the grafting of the PAOx and PEO chains to the PDA anchor layer and the formation of hydrophilic polymer brush layers. The measured water contact angles dropped from the initially observed 65° of neat PDA to about 40° and 30° for the PAOx and PEO brushes, respectively. The increase in the molar mass of the polymer brushes and the changes in grafting densities did not lead to significant changes in the observed hydrophilicity of the PAOx brushes. Interestingly, in the recent study from Benetti et al. a significant difference in the hydrophilicity of PMeOX, PEtOX and PEO brushes was observed.43 The brushes of PMeOX showed remarkable hydrophilicity with water contact angle values of 10°, obviously surpassing their PEO counter parts. In independent studies from our group, highly hydrophilic PEO brushes with water contact angle values well below 10° have been obtained. The hydrophilicity of the PEO brushes increased with increasing molecular weight and grafting density of the polymer brushes.52,56-57 However, despite the fact that the measured values water contact angle values primarily depend on the chemical structure of the surface under investigation, a significant variation stems from the topography, roughness, nanoscopic heterogeneity and the undergoing structural changes of the brush and the anchor layer during the measurement. Therefore, the direct comparison and conclusions on the wettability from various studies is rather troublesome, especially when comparing various surface state and architecture.

X-Ray photoelectron spectroscopy XPS was measured to prove the covalent structure of prepared layers. Representative C 1s, N 1s and O 1s spectra are shown in Figure 1. All other high resolution spectra are presented in Supporting Information (SI Figures S1-S3). The high resolution C 1s spectra of the anchoring PDA layer is characterized by C–C, C–N, C–O, C=O and O–C=O peaks at 285.0,


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286.0, 286.7, 287.9 and 289.0 eV, respectively. The high resolution O 1s spectra showed the C=O quinone and C–O catechol contributions. At the same time the N 1s envelope shows the contributions of C–N=C, C–N–C and C-NH2, and C–NH3+ at 398.3, 400.5 and 402.4 eV respectively. Successful modification with PAOx is confirmed by the increase of relative intensity of peaks of C–N and C=O in C 1s spectra at 286.3 and 288.2 eV, respectively. This is corroborated by the concomitant increases of the intensities of: i) the –C=O–NH– peak in N 1s spectra at 400.4 eV and ii) the peak O=C in O 1s spectra. The successful modification with PEO is proved by the increase of the C–O contributions in the high resolution C 1s and O 1s spectra and decrease of the overall amount of nitrogen (see SI, Table S1). The thickness of anchored PAOx and PEO films is lower than the depth range of XPS measurement and in all spectra the contributions of PDA anchor layer could be seen.



Figure 1. Representative high resolution C 1s, N 1s and O 1s XPS spectra of neat PDA anchor layer (A) and polymer brushes of PMeOx 6k (B), PEtOx 6k (C) and PEO 2k (D) grafted to-PDA. Thicknesses and surface-related parameters of the layers are summarized in Table 2. The composition of the layers is presented in SI, Table S1.

Infrared Reflection Absorption Spectroscopy Further evidence for the immobilization and the structure of prepared layers was provided utilizing IRRAS spectroscopy (Figure 2 and SI, Figures S4 – S6). The spectra of the layers show characteristic regions originating from: (i) the O–H and N–H group vibrations; (ii) the C–H stretching modes of various constituents of PDA, and ethyl, methyl and ether parts of the brushes; (iii) the C=O stretching modes of PDA’s quinones and PAOx’s tertiary amides; and (iv) the different symmetric and antisymmetric stretching, scissoring, rocking, wagging and twisting modes of the polymer brushes. The spectrum of neat PDA layers has a broad character with poorly resolved bands of various overlapping vibrational modes (Figure 2). The spectrum of PDA is characterized by the O–H and N–H group vibrations at about 3320 cm-1. The very weak C–H stretching bands at 2855 and 2925 cm-1 arise from the ethylamine aliphatic chains of dopamine and dopamine 20 ACS Paragon Plus Environment

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quinone monomer units that did not undergo spontaneous oxidation and intramolecular cyclization during the polymerization reaction. The broad shoulder band at about 1710 cm-1 can be assigned to the C=O stretching vibration of quinone groups, respectively. The C=C aromatic ring vibrations of the different PDA subunits give rise to the bands at about 1605, 1560, 1510 and 1450 cm-1. The amine groups contribute to the bending mode bands at 1510 cm-1 and the C–N stretching modes at about 1350 cm-1 which are coupled with the indole ring vibrations. The living cationic groups of the synthesized PAOx library were grafted to the free amine and hydroxyl functionalities of the PDA anchor. The successful immobilization of the PAOx brushes was accompanied by the significant rise in intensity of the CH3 and CH2 bands in the C–H stretching region. At the same time the presence of PAOx chains on the PDA anchor layers gave rise of new dominating C=O band at 1655 cm-1. In addition, the presence of the alkyl side chains led to the appearance of a family of bands at about 1470 and 1380 cm-1 (Figure 2 and SI, Figures S5 and S6). Similarly, the modification of the PDA anchor with PEO brushes was confirmed by the appearance of the strong CH2 bands in the CH stretching region and the dominating two C–O–C stretching bands at about 1137 and 1108 cm-1. Additionally, contributions of CH2 scissoring (1465 cm-1), wagging (1351 cm−1), twisting (1248 cm−1) and rocking (948 cm−1) modes could be resolved. With the exclusion of changes induced due to the binding of the polymer brushes, the IRRAS spectra showed that the initially observed covalent structure of PDA is preserved.



Figure 2. Representative IRRAS spectra of neat PDA anchor layer (A) and polymer brushes of PMeOx 6k (B), PEtOx 6k (C) and PEO 2k (D) grafted to-PDA. Thicknesses and surfacerelated parameters of the layers are summarized in Table 2.

Atom Force Microscopy (AFM) The continuity of the polymer brush layers is a prerequisite for effective protein repellency. Therefore we have investigated the surface topography of polymer modified surfaces utilizing AFM (Figure 3 and SI, Figure S7). The obtained results showed no significant differences between the samples modified with PAOx and PEO brushes. The determined root mean square roughness (Rrms) was about 10 nm, and that irrespectively of the type of brush grafted to the PDA anchor layer. In all cases the chains continuously covered the PDA anchor layer without any surface defects arising from the polymer brush formation. The observed roughness originates mainly from the PDA anchor layer which is known to exhibit grainy 22 ACS Paragon Plus Environment

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structure arising from adsorbed colloidal particles which were formed in the polymerization solution.52,58 This would imply that the differences observed in the ability of these brushes to withstand protein adsorption are mainly connected to the actual covalent structure of the polymer chains and their physical state, and not to the different surface topography defects.24 This is important as our protocol is therefore useable for surfaces with different surface geometry and morphology and of more general use.

Figure 3. Representative AFM surface height topographic map (5 × 5 µm2) of surfaces modified with neat PDA anchor layer (A; Rrms = 8.9 nm) and polymer brushes of PMeOx 6k (B; Rrms = 7.3 nm), PEtOx 6k (C; Rrms = 8.9 nm) and PEO 2k (D; Rrms = 8.5 nm) grafted toPDA.

Direct comparison of PAOx and PEO antifouling properties Single plasma proteins (the most abundant plasma protein human serum albumin (HSA) and often termed “sticky” fibrinogen (Fbg)) and human blood plasma (whole and diluted to



10%) were used to test and compare the antifouling character of the synthesized PAOx library and their representative PEO counterparts at virtually the same surface related parameters (Figure 4). A typical SPR sensogram is presented in the SI, Figure S8. The difference in SPR response was recalculated to the amount of proteins adsorbed on the surface and compared with the amount of proteins adsorbed on unmodified bare gold surface. As expected, all polymer modified surfaces exhibit better antifouling properties in comparison with the bare gold surface. The fouling behavior of the PEO surfaces was different for isolated plasma proteins (HSA and Fbg) and whole plasma, and therefore should be discussed separately. In the case of the isolated single plasma proteins the best antifouling properties were observed for the PEO 2k, i.e. the brushes of intermediate length polymer. This corresponds to the highest thickness and the lowest ratio between the distance between grafting sites and Rg. Upon shortening or prolongation of the polymeric chain the antifouling properties of the PEO brushes were worsened. Both these effects can be related to: i) the secondary adsorption of proteins and which would predominate the fouling on densely packed but short antifouling polymer brushes and ii) the primary adsorption which predominates the fouling when the sparsely organized long polymer chains allow the penetration of the adsorbing protein. As previously observed when comparing PAOx brushes with their PEO analogues, a significantly better fouling resistance was observed for the 2-alkyl-oxazoline counterparts. We have to point to the significant difference between the fouling resistance of the shortest brushes of the highest grafting density, i.e. PMeOx 1.5k and PEtOx 1.5k, when exposed to HSA and Fbg fouling. While the PEtOx 1.5k brushes managed to decrease the fouling from HSA and Fbg to about 5%, their PMeOx 1.5k counterparts led to a decrease in fouling to about 25%. As both types of brushes showed merely the same surface related parameters and hydrophilicity, the difference in the antifouling propertied of the short brushes cannot be easily explained. The observation most probably points to the inability of the PMeOx 1.5k brush with shorter alkyl


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side chains to completely mask the underneath PDA anchoring layer from the adsorbing single proteins. Increasing of the DP to more than 30 monomer units lead to a reduction of the absorbed amounts of HSA and Fbg to values below 10% of the single protein deposits on bare gold. The adsorption of single proteins was within the limits of the measurement error for both PMeOx and PEtOx in the DP range from 30–120 monomer units.

Figure 4. Comparison of the non-fouling properties of PMeOX (■), PEtOX (●) and PEO brushes (▲) of different DP when exposed to HSA (A), Fbg (B), 10% HBP (C) and whole HBP (D). The results are presented as the fraction of the fouling observed on bare gold.



Despite these promising results, the protein adsorption from single plasma protein solutions is not representative and cannot be used for the prediction of the antifouling properties of the polymer brushes when biological media are in question.59 The antifouling properties appear different when the PAOx and PEO brushes are exposed to human blood plasma (HBP, diluted or undiluted). The shortest polymeric chains being anchored to the surface with the highest density, manage to decrease the fouling from HBP to about 70% of the values obtained on bare gold. Upon prolongation of polymeric chains and drop of σ, we observed a decrease of antifouling properties for PEO, as the chains are more sparsely arranged and prone to primary adsorption of the small blood plasma proteins. The prolongation of the PEtOx chain did not lead to significant changes in the antifouling properties when challenged against HBP, although a general decrease in the adsorbed amount could be noticed with increasing molar mass of the brush, especially when challenged against 10% HBP. The prolongation of the PMeOx to 6k and 10k led to decrease of the fouling from whole plasma to about 30% of the values observed on bare gold. The origin of this superior behavior of PMeOx cannot be easily explained, and most probably arises from the concomitant influences of several factors, the number of monomer units per area occupied by single polymer brush, polymer chain flexibility, hydration and resulting steric barrier properties of the layer. However, this is in line with recent findings by Benetti and co-workers43 of increased hydration and flexibility of the PMeOx brushes in comparison to their PEtOx counterparts. Notably, while the PMeOx should have lower flexibility than the PEO brushes at same grafting density, it seems that the imposed steric barrier effects by the chain prolongation, while keeping short side chains, prevail as a contribution which boosts out the antifouling properties of the polymer brush when contacted with biological media. This might explain the surprising progressive increase of antifouling properties of PMeOX with prolongation of the chains despite the observed drop of σ and the induced increase of distance between the chains D.


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Finally, we can conclude that compared to the golden PEO standard and PEtOx at same surface related parameters, PMeOx has been shown to have superior anti-fouling properties when exposed to the most challenging biological media blood plasma. .

Conclusions In this study, we developed a new one-pot easy-to-perform method for polymerization and surface coating to prepare dense poly(2-oxazoline) layers by direct end-tethering of PMeOx and PEtOx chains to PDA modified surfaces. The “grafting to-” reaction utilized the polymer living chain-ends which were terminated with the surface amines and hydroxyls of PDA. The library of grafted PAOx surfaces consisted of polymers with molar mass ranging from about 1.5 to 10 kg/mol (DP varied from 16 to 115). The thickness and surface related parameters of the achieved polymer layers were determined utilizing spectroscopic ellipsometry. The obtained chain densities of the PAOx brushes are the highest so-far reported and varied from 2.1 to 0.2 [chain/nm2] showing pronounced brush character with D/2Rg ≤ 0.5. For the purpose of the comparison of the non-fouling character with the PEO standard, PEO bushes with the same surface related parameters as the PAOx brushes have been synthesized. The grafting of PEO chains with average molar masses of 1.2 – 5.1 kg/mol has been performed under optimized conditions. In all cases we have obtained hydrophilic PAOx or PEO brush layers. The covalent structure of the layers was analyzed by XPS and IRRAS. The AFM topography analysis showed that all brushes lack any visible surface defects, and the main surface topographical features can be related to the PDA anchoring layer. Single plasma proteins, i.e. HSA and Fbg, and HBP (diluted to 10% and whole) were used to compare the antifouling character of the synthesized PAOx brushes and their representative PEO counterparts. The higher thickness and lower ratio between the distance between grafting sites and Rg of intermediate length polymers (PAOx 6k and PEO 2k, average DP 46 – 73) led to their better



antifouling properties compared to their shorter or longer analogues. When exposed to fouling from HBP, the shortest polymeric chains being anchored to the surface with the highest density, managed to decrease the fouling from HBP to about 70 % of the values obtained on bare gold. The prolongation of polymeric chains and the concomitant drop of σ, led to decrease of antifouling properties for PEO, and almost no change for PEtOx. At the same time, the prolongation of the PMeOx to 6k and 10k led to decrease of the fouling from whole HBP to about 30% of the values observed on bare gold. Our results show that the PMeOx brushes have superior anti-fouling properties when compared to the golden PEO standard and PEtOx at same surface related parameters. The observed superiority most probably stems from the imposed steric barrier effects by the chain prolongation, while keeping short side chains, which prevail as a contribution boosting out the antifouling properties of the polymer brush when contacted with biological media. The presented study proves the potential of PAOx and especially PMeOx to take-over the place of PEO as a lead antifouling polymer. With the aid of a combination of PDA and the direct grafting of PAOx polymer chains through their living chains, we can target the surface modification of nearly any solid material. Currently we strive for achieving the maximal densities which have been previously obtained for PEO layers, and thereby completely suppress the fouling from complex biological media such as blood plasma. ASSOCIATED CONTENT Supporting Information. C 1s, N 1s and O 1s XPS high resolution spectra, atomic % of chemical moieties, IRRAS spectra and AFM surface topographic maps for all prepared surfaces and representative SPR sensogram (PDF). ATHOR INFORMATION Corresponding Authors *E-mail: [email protected] (J.S.) 28 ACS Paragon Plus Environment

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*E-mail: [email protected] (O.P.G.) Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT M.H. thanks for financial support to Czech Grant Foundation (grant # 19-01602S). M.H. and O.S. thank for financial support to the BE/FWO-Mobility Projects 2019-2020 (grant # FWO-19-03). O.P.G. and T.R. thank the financial support from the Czech Grant Foundation (grant # 19-02739S). REFERENCES 1.

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Poly(2-oxazoline)s One-Pot Polymerization and Surface Coating

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