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Crosslinking Polymer Brushes With Ethylene Glycol-Containing Segments: Influence on Physico-Chemical and Antifouling Properties Ella S. Dehghani, Nicholas D. Spencer, Shivaprakash N. Ramakrishna, and Edmondo Maria Benetti Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b02958 • Publication Date (Web): 19 Sep 2016 Downloaded from http://pubs.acs.org on September 23, 2016

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Crosslinking Polymer Brushes With Ethylene Glycol-Containing Segments: Influence on PhysicoChemical and Antifouling Properties Ella S. Dehghani,† Nicholas. D. Spencer,† Shivaprakash N. Ramakrishna,† Edmondo. M. Benetti† † Laboratory for Surface Science and Technology, Department of Materials, ETH Zurich, Vladimir-Prelog-Weg 5, CH-8093 Zurich, Switzerland. KEYWORDS protein adsorption, polymer brushes, surface-initiated polymerization, atomic force microscopy

The introduction of different types and concentrations of crosslinks within poly(hydroxyethyl methacrylate) (PHEMA) brushes influences their interfacial, physico-chemical properties, ultimately governing their adsorption of proteins. PHEMA brushes and brush-hydrogels were synthesized by surface-initiated, atom-transfer radical polymerization (SI-ATRP) from HEMA, with and without the addition of di(ethylene glycol)dimethacrylate (DEGDMA) or tetra(ethylene glycol)dimethacrylate (TEGDMA) as crosslinkers. Linear (pure PHEMA) brushes show high hydration, low modulus and additionally provide an efficient barrier against non-specific protein adsorption. In contrast, brush-hydrogels are stiffer and less hydrated, and the presence of

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crosslinks affects the entropy-driven, conformational barrier that hinders the surface interaction of biomolecules with brushes. This leads to the physisorption of proteins at low concentrations of short crosslinks. At higher contents of DEGDMA or in the presence of longer TEGDMA-based crosslinks, brush-hydrogels recover their antifouling properties due to the increase in interfacial water association by the higher concentration of ethylene glycol (EG) units.

INTRODUCTION Surface contamination by nonspecific protein adsorption is a major concern in the design, fabrication and application of biomaterials, including implants, catheters, contact lenses and, generally, bioassay devices and biosensors.1 In all these cases, the adsorption of proteins can mediate the attachment of bacteria, eventually leading to infection and compromising of the biomaterial’s performance. In contrast, in other applications, enhanced adsorption of specific biomolecules is required, with the aim of stimulating both adhesion and proliferation of cells on the modified supports. Examples include scaffolds for regenerative medicine, where opportunely protein-decorated surfaces can not only trigger cell adhesion but can also direct cell differentiation towards a particular tissue type.2 In both these scenarios, precise control of the interaction between proteins in the biological medium and the exposed surface is highly desirable and can determine the performance of the entire biomaterial. Surface grafting of a number of hydrophilic polymers to form polymer “brush” films3 has been exploited to inhibit and/or control non-specific surface interactions of biomolecules. Notably, surface-initiated polymerizations (SIP) from previously initiator-functionalized surfaces have enabled the synthesis of densely grafted polymer brush films on a variety of materials, including inorganic2,4 and polymeric supports5,6. When SIP is applied in conjunction with controlled


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radical polymerization, precise tuning of brush grafting density and grafted-chain length (brush thickness) can be achieved.7 Among the different controlled radical SIPs, surface-initiated atom transfer radical polymerization (SI-ATRP) has proven to be an effective technique to synthesize hydrophilic polymer brushes when applied as protein-repellent coatings. These have included poly(ethylene

glycol)

(PEG)-,8,9

polyoxazoline

(POX)-,10,11

polyzwitterionic-,12,13

and

poly(hydroxyethyl methacrylate) (PHEMA)-based brushes14–17. The biopassivity of these and other hydrophilic polymer brushes, in the form of densely grafted, relatively thick films, has been ascribed to two main contributions: an entropy-regulated steric repulsion by the brush conformational barrier18–21 and an energetic obstacle, due to the formation of a hydration layer by hydrogen-bonded or ionically coupled water22,23. Despite the efficiency of the above mentioned polymer brushes as antifouling layers, the interfacial, physico-chemical properties of the subsequently formed coatings are often difficult to control, most of all when the polymers are grafted on diverse biomaterial surfaces. It is of particular interest to be able to adjust the mechanical and swelling properties of the films to obtain the desired robustness, or, alternatively, to tune the response to the surrounding physiological medium when the coatings are applied on supports for tissue engineering.24 This can be achieved by the simple introduction of crosslinks within antifouling polymer brushes, to yield brush-hydrogels with the desired composition and properties. In this work we show how a modulation of brush structure by introducing different types and numbers of crosslinks can significantly influence the biopassivity and interfacial properties of brush films. We have especially focused on PHEMA brushes and brush-hydrogels, synthesized by SI-ATRP from previously initiator-functionalized silicon oxide surfaces.25 PHEMA brushes were shown to efficiently repel the adsorption of proteins and attachment of bacteria,15,26,27 and


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display good blood compatibility28. Interestingly, PHEMA is just marginally soluble in aqueous media, hence PHEMA brushes adopt a compact configuration on the surface, whereas their hydroxyl-group-rich interface can form a diffuse hydration layer via hydrogen bonding with water molecules, hindering protein-surface interactions. Due to these distinctive features, PHEMA-brush short-time protein repellency (until 72 hours of incubation) has been found to directly correlate with the total number of polymer chains covering the functionalized substrate, i.e. the brush thickness.27,29 This implies that the antifouling character of PHEMA brushes in a theta or poor solvent such as water, is governed by the capability of the brush for substrate shielding, coupled with the formation of a water-bound layer at the brush interface. The influence of brush conformational freedom and its modulation by precise tuning of crosslinking architecture has only been very marginally addressed—both in the case of PHEMA and for other grafted-from brushes. Hence, in order to systematically investigate the relationship between crosslinking and protein adsorption, different monomer feeds were applied during the SI-ATRP process, to yield linear PHEMA brushes, as well PHEMA brush-hydrogels with two different (1 mol% and 2 mol%) concentrations of either a short, di(ethylene glycol)dimethacrylate (DEGDMA) crosslinker or a longer, tetra(ethylene glycol)dimethacrylate (TEGDMA) crosslinker. The interfacial properties of both linear brushes and covalently crosslinked brush-hydrogels were studied by a combination of surface-sensitive and scanning-probe techniques. SI-ATRP kinetics and the swelling properties of PHEMA brush and brush-hydrogels were monitored by means of the quartz crystal microbalance with dissipation (QCM-D), in conjunction with variable-angle spectroscopic ellipsometry (VASE). Colloidal-probe atomic force microscopy (CPM) was applied to study the conformational, steric barrier exerted by PHEMA films and their


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adhesion properties as a function of the content and type of crosslinks introduced between the grafted chains.30,31 Finally, these physico-chemical properties of PHEMA brushes and brushhydrogels were correlated with their antifouling characteristics. Full human serum (FS) was employed as the test protein source, the most abundant component being albumin—a small, globular protein. FS was additionally complemented with fibrinogen (Fgn), which is a large, coil-like protein particularly sensitive to surface defects.32–34 The mass of protein adsorbed on the different PHEMA films was evaluated by a combination of VASE and QCM-D. Additionally, the interaction between FS proteins and the polymer films was quantitatively evaluated by CPM, utilizing a colloidal probe that had formerly been coated with proteins, and measuring the repulsive/attractive forces between the functionalized colloid and the different brush layers.35–38 The results obtained from these studies demonstrate how the structure of brush films in aqueous medium can be precisely varied to tune both the interfacial, physico-chemical properties and the antifouling character of the coatings, almost independently. Brush crosslinking reduces the solvent content and stiffens the obtained films. Simultaneously, the presence of covalent crosslinks, as well as their chemical nature and water-associating capability, cooperatively regulate the resistance of the films towards protein adsorption. If these characteristics are altered, together or separately, the brush biopassivity can either be compromised or enhanced. On the one hand, crosslinking of PHEMA brushes reduces the effectiveness of the conformational steric barrier towards protein contamination, while, on the other hand, the introduction of ethylene glycol (EG)-containing crosslinkers enhances water association at the brush interface. Protein interactions with the PHEMA films are directed by the balance of these two contributions. EXPERIMENTAL Materials


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10-undecen-1-ol (Aldrich-Fine Chemicals, Switzerland), THF (99.5% extra dry, Acros, Germany),

bromoisobutyryl

bromide

(Aldrich-Fine

Chemicals,

Switzerland),

dimethylchlorosilane (Aldrich-Fine Chemicals, Switzerland), hydrochloric acid (Sigma-Aldrich, Germany), Phosphate-buffered saline (PBS) (pH 7.4, Sigma-Aldrich, Germany), copper(II) bromide, copper(I) chloride (Aldrich-Fine Chemicals, Switzerland), 2,2’-bipyridyl (Aldrich-Fine Chemicals, Switzerland), fibrinogen from human serum (Aldrich-fine chemicals, Switzerland), Alexa Fluor® 546-labeled fibrinogen from human plasma (Thermo Fischer scientific, Germany) and Alexa Fluor® 546-labeled albumin from human plasma (Thermo Fischer scientific, Germany) were used as received. Hydroxyethyl methacrylate was purified to remove any diacrylate present, according to the procedure described by Baker et al.39 Diethylene glycol dimethacrylate (DEGDMA) and tetraethylene glycol dimethacrylate (TEGDMA) (Aldrich-Fine Chemicals, Switzerland) were passed through a column of basic alumina prior to use, in order to remove the hydroquinone inhibitors. The water used for the swelling experiments and the AFM tests was ultra-pure grade, produced with a GenPure filtration system (18.2 MU cm, TKA, Switzerland). Each vial of human serum (cobas® integra 400 plus, Roche diagnostic, Germany) was mixed with 5 ml of milliQ water. Silicon wafers (P/B , Si-Mat Silicon Wafers, Germany) were used as received. Surface-initiated, atom-transfer radical polymerization (SI-ATRP) The synthesis of the silane-based initiator for SI-ATRP was carried out in accordance with the already reported protocol by Sanjuan et al.40 Briefly, a 25 mmol solution of 10-undecen-1-ol in 25 ml dry THF was added drop-wise to a 0.35 mM THF solution of bromoisobutyryl bromide, containing 4.5 mmol of triethylamine. After stirring for 24 hours, the solution was washed with 2M HCl and subsequently passed through a silica column to remove unreacted 10-undecen-1-ol.


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The colorless oily product was later added to dimethylchlorosilane in the presence of chloroplatinic acid and it was stirred overnight. The final product (11-(2-bromo-2-methylpropionyl)-dimethylchlorosilane (BPCS) was filtered through a silica plug and kept under high vacuum overnight (H-NMR spectrum of the initiator is provided in the Supporting Information). Silicon wafers were cleaned with piranha solution (warning: piranha solution is very reactive and corrosive; use extreme caution!), and placed overnight under a 10 mM solution of BPCS in dry toluene under nitrogen, to form initiator SAMs. SiO2 coated QCM-D sensors were cleaned by sonication in ethanol (10 minutes) and toluene (10 minutes), followed by ozone cleaning (UV/Ozone ProCleanerTM and ProCleanerTM Plus, BioForce, IA, USA) for 40 minutes, prior to the immobilization of the BPCS initiator from dry toluene solutions. PHEMA brushes and brushhydrogels

were

grown

from

initiator

SAMs

by

SI-ATRP

of

different

HEMA/DEGDMA/TEGDMA mixtures, applying a catalytic system comprising CuCl (55 mg, 0.55 mmol), CuBr2 (36 mg, 0.16 mmol), and 2,2′-bipyridine (bipyr) (244 mg, 1.56 mmol) in a mixture of monomer:water (4:4 mL).41 The monomer mixture, solvent and ligand were deoxygenated by nitrogen bubbling for 30 minutes, after which the solution was transferred to a flask kept under nitrogen and containing the copper species (CuCl/CuBr2). This solution was allowed to stir for 30 minutes following complete formation of the catalyst complex (characterized by a dark-brown solution and complete dissolution of the copper salts). The polymerization solution was finally transferred to the flask containing the BPCS-functionalized silicon dioxide substrates and the QCM liquid cell, and SI-ATRP was performed for the desired time at room temperature. PHEMA brush-hydrogels were synthesized following the same experimental procedure, alternatively adding 1 and 2 mol% of DEGDMA and TEGDMA to the monomer mixtures.


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Characterization FT-IR transmission spectra were recorded with an infrared spectrometer (Bruker, IFS 66 V) equipped with a liquid-nitrogen-cooled MCT detector. A background spectrum was collected from a freshly cleaned, bare silicon wafer. The chemical composition of the interfacial layer of PHEMA brushes and brush-hydrogels was studied by X–ray photoelectron spectroscopy (XPS). XPS measurements were performed with a Theta-Probe X–ray photoelectron spectrometer (ARXPS, Thermo Fisher Scientific, Waltham MA, USA), equipped with a monochromatic Al Kα source and a beam diameter of 400 µm. The pass energy was 100 eV for the high-resolution spectra of carbon and oxygen elemental analysis, while the pass energy of the survey spectrum was 200 eV. An electron-argon-ion flood gun was used to compensate for the charging occurring at the surface. Three measurements were performed for each sample in the standard lens mode with an emission angle of 53° to the surface and an acceptance angle of ± 30°. The elemental composition determined from the survey scan (69.9% carbon and 30.1% oxygen) reflected, within the experimental error, the composition expected for a PHEMA film (66.7 % carbon and 33.3 % oxygen, 5% error). The C1s peak for PHEMA was resolved into five component peaks: a hydrocarbon (C-C) peak at 285 eV, an ether (C-O) peak at 287 eV, a quaternary carbon (q-C) peak at 285.6 eV, a hydroxyl (C-OH) peak at 286.4 eV and a carbonyl (O-C=O) peak at 289.1 eV (see Supporting Information). The assignments, binding energies, and relative areas of all these peaks were consistent with the structure of PHEMA, as previously reported.42 The C1s peak recorded for each brush-hydrogel was also resolved with the same component peaks and their relative ratios were used to estimate the crosslinker concentration. Namely, we applied Eq.1, where A is the calculated area of C-OH peak from XPS C1s (summarized for each film in Table S1).


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Mol% of Crosslinker = (APHEMA – Ahydrogel)/APHEMA

Eq.1

Variable-angle spectroscopic ellipsometry (VASE) (M-2000F, LOT Oriel GmbH, Darmstadt, Germany) was used to measure the thickness of the polymer films under both dry and wet conditions, and to estimate the amount of protein adsorbed on the films. For the measurements of dry thickness, the recorded amplitude (Ψ) and phase (∆) components as a function of wavelength (275-827 nm) were fitted by means of a four-layer model (Si/SiO2/Cauchy (BPCS initiator)/Cauchy (PHEMA film)) with known thicknesses and refractive indices of the Si, SiO2 and initiator layers (software WVASE32, LOT Oriel GmbH, Darmstadt, Germany).43 A Cauchy model, n = A + B/ λ2, was used to describe the refractive index of the PHEMA films by means of two fitting parameters: offset (A = 1.45) and wavelength dispersion (B = 0.01). The swelling characteristics of the different PHEMA films were studied by VASE in ultra-pure water, using a custom-made liquid cell, applying a six-layer model to fit Ψ and ∆ (Si/SiO2/Cauchy (BPCS initiator)/Cauchy (PHEMA)/swollen PHEMA/ambient (water)), with the refractive index of water set as 1.33. The swollen PHEMA was modeled with an effectivemedium approximation (EMA) model, provided by the instrument software, constituting of both Cauchy and water components. Static and dynamic contact angles were studied by the sessile-drop method, employing a RameHart goniometer (RameHart Instrument Co., Model-100, Netcong, NJ). A 5 µL drop was placed on the surface and the advancing (ΘA°) and receding (ΘR°) contact angles were measured by addition to and withdrawal from the drop at a rate of 4 and 7 µL.min-1, respectively. QCM-D (E4 instrument, Q-Sense, Västra Frölunda, Sweden) was used to monitor the grafting of PHEMA films in situ, and subsequently the protein adsorption on the already-formed films. The instrument was equipped with Q-Soft 301 software (Q-Sense AB, Göteborg, Sweden). SiO2-


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coated sensors were cleaned and functionalized ex situ with initiating SAMs by following the same procedure reported in the previous section. Later on, they were mounted in an instrument equipped with a liquid-flow system. After starting the measurements, ultra-pure water was injected into the chambers and while collecting the data the instrument was allowed to equilibrate. Once a drift-free signal was recorded for 20 to 40 minutes, ethanol was injected and a new baseline was acquired. For monitoring SI-ATRP, the cell was filled with a 1 mL mixture of the polymerization solution (monomer/crosslinker/solvent/ligand/catalysts) via an external syringe. After 10 minutes of SI-ATRP, the reaction was terminated by injection of ethanol, followed by ultra-pure water. An extended viscoelastic model44 was used to fit the frequency and dissipation shifts (∆f and ∆D respectively) using three overtones, to obtain the change in hydrated polymer mass during SIATRP.45 The polymer-coated sensors were subsequently incubated with the protein solutions and the corresponding ∆f and ∆D variations were recorded. After 90 minutes of incubation, the sensors were rinsed with ultra-pure water to wash off any loosely bound protein. Also in this case, the overall decrease of ∆f and increase of ∆D after rinsing was translated into adsorbed protein hydrated mass by applying an extended viscoelastic model to fit three overtones. In order to investigate the surface roughness and topography of polymer brushes and brushhydrogels, a Bruker Dimension Icon® atomic force microscope (AFM) was used in tapping mode. The cantilever resonance and the spring constant were 300 kHz and 40 N·m-1, respectively. The nanomechanical properties of the PHEMA brushes and brush-hydrogels were evaluated in ultra-pure water by colloidal probe microscopy (CPM), using an Asylum MFP-3D (Santa


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Barbara, CA, US). The normal spring constants of Au-coated, tipless cantilevers (NSC-36, Bruker, US) were measured by the thermal-noise method before attaching the colloidal microspheres. Silica microparticles with radius of 8 µm (EKA chemicals AB, Kromasil, Sweden) were glued with UV-curable glue (Norland optical adhesive 63, New Jersey, USA) to the end of the cantilever by means of a home-built micromanipulator. The elasticity and adhesive properties of brush and brush-hydrogel films were measured from the approaching and the retracting profiles of the recorded force-vs-distance (FD) curves (30 forces curves over 20 µm × 20 µm area, in a minimum of three areas). The apparent Young’s modulus of the different films was calculated with a Hertzian model, fitting the approaching profiles of the FD curves between the contact point and a penetration depth corresponding to less than 5% of the film swollen thickness, by applying the following equation: F = (4 E R0.5 δ1.5 ) / (3 – 3ν2)

Eq.2

where F is the applied load, R is the radius of the colloid used as a probe and ν is the Poisson’s ratio (considered as 0.5). Protein-adsorption experiments Full human serum (FS) and full human serum complemented with fibrinogen (FS + Fgn) were used as protein sources to study the biopassivity of the different PHEMA films. Four different sets of samples were studied to assure the reproducibility of the protein adsorption results. Silicon dioxide surfaces were used as references. For both VASE and QCM-D, the adsorption tests were carried out for 90 minutes. For the quantitative evaluation of the surface-protein interaction forces, a silica colloidal bead (16 µm in diameter) glued onto a tipless cantilever (1 Nm-1 spring constant) was UV-cleaned for 20 minutes and subsequently immersed in FS for 2 hours, prior to use. The formation of a uniform film of proteins on the colloid was confirmed by


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incubating the probe in fluorescently labeled albumin, and imaging the surface of the colloid by fluorescence microscopy (see Supporting Information). The interaction forces between the different brush films and the protein-coated probe were evaluated from the approaching and the retracting profiles of the recorded FD curves. All the measurements were performed in PBS, applying a normal load within the range 5-10 nN. RESULTS AND DISCUSSION Physico-Chemical Characterization of PHEMA Brushes and Brush-Hydrogels PHEMA brushes and brush-hydrogels were synthesized by SI-ATRP under aqueous conditions and applying a relatively high concentration of deactivator Cu(II)-based species (Experimental Section). This allowed a fast and controlled grafting of thick films within a relatively short reaction time.59

Scheme 1. Schematic structure of the polymer brushes and brush-hydrogels fabricated in this study. PHEMA brushes (depicted in black) (a), PHEMA brushes crosslinked with 1 mol% of DEGDMA (b) and 2 mol% of DEGDMA (c) (depicted in red), PHEMA brushes crosslinked with 1 mol% of TEGDMA (d) and 2 mol% of TEGDMA (e) (depicted in blue).


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The polymerization time was adjusted in order to obtain films presenting similar dry thicknesses for the different brush and brush-hydrogel types. In particular, PHEMA brushes and brush-hydrogels with average dry thicknesses between 20 and 25 nm were synthesized during 7 to 12 minutes of SI-ATRP (Table 1 and Experimental Section for details). The preparation of the brush-hydrogels involved the use of two types of crosslinker, DEGDMA and TEGDMA, which contain two or four EG units between the methacrylate functions, respectively (Scheme 1). PHEMA brush-hydrogels synthesized with 1 and 2 mol% of DEGDMA were named PHEMADEGDMA-1 and PHEMA-DEGDMA-2, respectively. Brush-hydrogels prepared by adding 1 and 2 mol% of TEGDMA were named PHEMA-TEGDMA-1 and PHEMA-TEGDMA-2, respectively. Table 1. Dry thickness Dt, swollen thickness St and swelling ratio Sr (%) = 100.(St – Dt)·Dt-1 of PHEMA brushes and brush-hydrogels measured by VASE. Dt (nm)

St (nm)

Sr (%)

PHEMA

19 ± 1

30 ± 3

58 ± 7

PHEMA-DEGDMA-1

25 ± 1

35 ± 4

40 ± 6

PHEMA-DEGDMA-2

23 ± 1

30 ± 2

30 ± 3

PHEMA-TEGDMA-1

19 ± 1

27 ± 3

40 ± 7

PHEMA-TEGDMA-2

21 ± 1

28 ± 2

33 ± 4

` The chemical composition of the synthesized films was verified by Fourier-transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). The analysis of the obtained FTIR spectra reflected the typical patterns of PHEMA, with basically no differences between brushes and brush-hydrogels (Supporting Information).


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In order to evaluate the effective incorporation of crosslinks with respect to the relative concentration of crosslinker molecules introduced in the polymerization media, XPS was performed on each film. The resulting C 1s peaks were resolved into five component peaks, originating from the functional groups within the monomer units and the crosslinker (see Experimental Section and Supporting Information for a detailed XPS analysis). According to Equation 1, the effective concentration of DEGDMA and TEGDMA within each brush-hydrogel could be obtained. As summarized in Table 2, the presence of 1 and 2 mol% of DEGDMA during SI-ATRP translated into a crosslinker concentration within the corresponding brush-hydrogels of 6 ± 1 and 11 ± 3 mol%, respectively. In a similar way, polymerizations performed in the presence of 1 and 2 mol% of TEGDMA resulted in 4 ± 2 and 9 ± 2 mol% of crosslinker incorporated in the PHEMA-TEGDMA-1 and PHEMA-TEGDMA-2, respectively. The higher concentrations of crosslinkers in the films, compared to those present in the SI-ATRP reaction mixtures, were presumably due to the higher reactivity of both DEGDMA and TEGDMA compared to HEMA.30 The corresponding concentration (mol%) of EG units within the films is summarized in Table 2. Due to the sampling depth of XPS under the experimental conditions followed in this study (~ 6 nm), the derived EG concentrations refer to the interfacial composition of the different brushhydrogels. Table 2. Relative concentrations of the different crosslinker types introduced in the polymerization media and present in the films as derived from the XPS analysis. The corresponding content of EG units in the different brush and brush-hydrogel films is also provided. mol

%

in mol % crosslinker in mol % EG units


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feed

the films (XPS)

in the films

PHEMA-DEGDMA-1

1

6±1

11 ± 2

PHEMA-DEGDMA-2

2

11 ± 3

21 ± 5

PHEMA-TEGDMA-1

1

4±2

14 ± 6

PHEMA-TEGDMA-2

2

9±2

28 ± 5

The influence of the specific polymer architecture on the wettability of the films was evaluated by measuring the static and the dynamic water contact angles (CA). Static (ΘS°), advancing (ΘA°) and receding CA (ΘR°), together with the corresponding contact-angle hysteresis (δ = ΘA° - ΘR°) are reported in Table 3. For all the brush films studied, ΘS° values were all measurable and varied between 35° and 42°, confirming the expected amphiphilic character of PHEMA, which generally swells but does not dissolve in aqueous media.6,20 Interestingly, both PHEMA brushes and brush-hydrogels showed high values of δ, δ = 48° being recorded for PHEMA brushes and the various brush-hydrogels displaying markedly higher δ values, which lay between 60° and 68° (Table 3). A high value of δ for PHEMA films has been ascribed to the asymmetrical environment at the polymer-air interface, which compels the grafted polymer chains to orient and expose their hydrophobic moieties towards the gas phase.46 When the films are exposed to air, it is energetically favorable to bury the polar hydroxyl groups of HEMA within the brush layer, causing high values of ΘA°. However, when PHEMA films are brought into contact with water, the polymer chains reorient, exposing their hydroxyl functions, which are capable of strongly interacting with water molecules via hydrogen bonding. This phenomenon minimizes the interfacial tension and very low values of ΘR°, varying from 11 to 14°, are usually measured for all the different PHEMA films.


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The larger hysteresis values recorded for all the brush-hydrogels originated from the relatively higher values of ΘA°, which indicated a decrease of interfacial concentration of hydrogenbonding donors (hydroxyl groups of HEMA) when DEGDMA and TEGDMA are copolymerized together with HEMA. On the other hand, incorporation of the crosslinkers does not affect ΘR°, since EG has the unusual property of hydrogen bonding strongly to water (for steric reasons), while being intrinsically hydrophobic.47 Like HEMA, the EG can rearrange, minimizing the interfacial tension under either air or water. The fact that the crosslinking does not appear to inhibit the rearrangement-induced hysteresis in CA suggests that the phenomenon occurs on a local, monomer-scale level, rather than involving chain rearrangement. A reduction of tethered-chain mobility upon brush crosslinking was already demonstrated in the case of polyacrylamide (PAAm) brushes, which were synthesized in the presence of increasing concentrations of bis-acrylamide (bisAAm).30 PAAm brushes, which are highly hydrophilic, show a non-zero contact angle, due to bridging effects of the grafted chains at the vapor-liquid interface. ΘS° showed a progressive decrease with increasing crosslinker content, as covalently crosslinked grafts cannot efficiently “bridge” the vapor-liquid interface of a spreading drop.48 In contrast to these previous findings, no difference in the static wettability was found between the PHEMA brushes and brush-hydrogels, presumably due to the limited hydrophilicity of PHEMA (which swells in water, but is insoluble). Hence, any effect on the measured ΘS° produced by the introduction of lateral constraints between grafts was masked, the static wettability of the films being uniquely determined by the surface chemistry. Table 3. CA analysis on PHEMA brush and brush-hydrogels. ΘS° indicates static water contact angle; ΘA° advancing water contact angle; ΘR° receding water contact angle; the water contact angle hysteresis is expressed as δ = ΘA° - ΘR°.


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ΘS°

ΘA°

ΘR °

δ

PHEMA

35 ± 2

59 ± 1

11 ± 5

48 ± 6

PHEMA-DEGDMA-1

39 ± 2

73 ± 3

13 ± 1

60 ± 4

PHEMA-DEGDMA-2

41 ± 1

82 ± 3

14 ± 1

68 ± 4

PHEMA-TEGDMA-1

42 ± 2

74 ± 2

13 ± 2

61 ± 4

PHEMA-TEGDMA-2

37 ± 2

76 ± 4

12 ± 1

64 ± 5

In order to monitor the grafting process of PHEMA brushes and brush-hydrogels in situ, and to study the polymer-architecture-dependent swelling and viscoelastic properties of the films, the SI-ATRP was monitored in situ by QCM-D (see Experimental Section for details).49 As shown in Figure 1, for all the brush structures studied, a steady decrease of ∆f, accompanied by a concomitant increase of ∆D was observed during polymerization. This general result indicated that the hydrated mass (polymer mass and coupled water) of all PHEMA films increased with the polymerization time, while the thickening of the films was accompanied by a progressive increase in their viscoelastic character. However, the application of different contents and types of crosslinker led to diverse decays in frequency and increments in dissipation.


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Figure 1. Frequency (a, c) and dissipation (b, d) variations recorded in situ by QCM-D during SI-ATRP of different monomer/crosslinker mixtures. ∆f and ∆D recorded during the grafting of PHEMA brushes are reported in black; PHEMA-DEGDMA-1 and PHEMA-DEGDMA-2 are reported in red and green, respectively (in graph a, b); PHEMA-TEGDMA-1 and PHEMATEGDMA-2 are reported in violet and blue, respectively (in graph c, d). As shown in Figure 1, the grafting of PHEMA brushes produced the more marked shift of ∆f among all the different films, and this was also coupled to the highest increase in ∆D. This was due to the high amount of coupled water and the marked viscoelastic character of linear PHEMA brushes. The introduction of crosslinks during SI-ATRP of HEMA induced a markedly smaller variation of ∆f and ∆D compared to linear PHEMA brushes. In addition, an increase in concentration, from 1 to 2% of both DEGDMA and TEGDMA, caused further reductions in the variation of ∆f and, simultaneously, smaller increases of ∆D. These phenomena were presumably due to a combination of reduced swelling and enhanced rigidity by more crosslinked grafts. As a confirmation, the swelling ratios of the different brush films (defined as Sr and reported in Table 1), measured by VASE, showed a progressive decrease with increasing crosslinker concentration


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for each crosslinker type. Linear PHEMA brushes showed a swelling ratio (Sr) = 58%, while PHEMA-DEGDMA-1 and PHEMA-DEGDMA-2 displayed Sr values of 45 and 33%, respectively. A similar decrease of Sr was also found by increasing the concentration of TEGDMA from 1 to 2 mol%, which caused a shift of Sr by 12%, from 50 to 38% (Table 1). It has to be emphasized that the different profiles of ∆f and ∆D recorded in situ for SI-ATRP of PHEMA films with different polymer architectures are primarily not due to differences in grafted polymer masses (dry). In fact, the PHEMA brushes and brush-hydrogels showed very similar dry thickness values, as measured by VASE, after the same polymerization times (and conditions) applied during the QCM-D experiments (Table 1). Hence, differences in frequency and dissipation shifts can be predominantly attributed to variations in the amount of coupled water and viscoelasticity for the different film structures.50 In order to evaluate how the viscoelasticity of the layers varied during the SI-ATRP process for each different PHEMA film, the variation of dissipation was plotted against the corresponding frequency change (Figure 2).

Figure 2. ∆D-vs-∆f plots during SI-ATRP for PHEMA brushes (black trace in a and b) PHEMA-DEGDMA-1 (red trace in a), PHEMA-DEGDMA-2 (green trace in a), PHEMATEGDMA-1 (violet trace in b) and PHEMA-TEGDMA-2 (blue trace in b).


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As reported in Figure 2, following a progressive increment in polymer/coupled water mass (increase of ∆f), ∆D showed a more pronounced increase in the case of linear PHEMA brushes. In contrast, a lower ∆D growth was observed when DEGDMA was present in the polymerization medium. Similarly, brush crosslinking by increasing concentrations of TEGDMA caused an analogous decrease of the slope of ∆D-vs-∆f curves (Figure 2). However, the difference between ∆D-vs-∆f curves recorded for PHEMA, PHEMA-TEGDMA-1 and PHEMA-TEGDMA-2 was less marked, presumably due to the higher hydration and viscoelasticity provided by longer, TEG-based crosslinks. In order to investigate how the introduction of different crosslinker types and contents affected the nanomechanical properties of PHEMA grafts, CPM was subsequently performed in aqueous media. The resulting force-vs-indentation profiles (FI) highlighted how the introduction of crosslinks generally enhanced film stiffness, as reported in Figures 3a and 3b. The presence of either DEGDMA or TEGDMA was shown to cause a steepening of the FI profiles. These can be fitted by Hertz model (see Experimental Section for details) in order to estimate the Young’s modulus (E) of the different films. The values of E increased from 1.1 ± 0.4 MPa of PHEMA brushes to 2.1 ± 0.6 MPa of PHEMA-DEGDMA-1 and 2.9 ± 0.9 MPa of PHEMA-DEGDMA-2. A similar increase was also found for PHEMA brushes crosslinked with TEGDMA, which showed E values of 1.7 ± 0.4 and 2.4 ± 0.6 MPa in the case of PHEMA-TEGDMA-1 and PHEMA-TEGDMA-2, respectively.


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Figure 3. (a,b) FI profiles recorded by CPM (spring constant of the cantilever 1 N·m-1, radius of the silica probe 8 µm) on PHEMA brushes (black), PHEMA-DEGDMA-1 (red), PHEMADEGDMA-2 (green), PHEMA-TEGDMA-1 (violet) and PHEMA-TEGDMA-2 (blue). (c,d) distributions of the Young’s moduli estimated by Hertz-model fitting of the indentation profiles. The retracting portions of the FI curves recorded in ultra-pure water were analyzed in order to evaluate the adhesion force between the colloidal silica probe and the different brush films. As displayed in Figure 4, adhesion was also markedly affected by the concentration and the length of the crosslinker functions within the PHEMA films. In particular, PHEMA brushes showed the highest pull-off force, while the presence of an increasing concentration of DEGDMA caused a marked decrease of adhesion (Figure 4a and 4c). This variation is presumably due to the introduction of lateral constraints between the grafted chains, which hamper tethered-chain freedom, hindering probe penetration and reducing the contact area between the probe and the film.51,52 In the case of PHEMA-TEGDMA brush-hydrogels, the reduction in the measured pulloff force was found less pronounced, as evidenced in Figure 4d. We believe that this was due to


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the presence of the longer, TEG-based crosslinker, leading to the formation of more compliant and penetrable brush-hydrogel films compared to the PHEMA-DEGDMA ones.

Figure 4. Retraction profiles from FI curves recorded on PHEMA brush and brush-hydrogels by CPM (a, b). The measured pull-off force distributions for each film type are reported in (c) and (d). In summary, the swelling, viscoelastic and nanomechanical characteristics of PHEMA grafts were shown to be markedly altered by the introduction of crosslinks. In addition, these physicochemical properties could be effectively tuned by varying the length and the relative concentration of the added crosslinker. Intrigued about how the modulation of the polymer-brush structure would regulate the antifouling character of the generated surfaces, we subsequently systematically studied the interaction of serum proteins and Fgn with PHEMA brushes and brush-hydrogels. Protein Adsorption on PHEMA Films Protein adsorption on PHEMA brushes and brush-hydrogels was studied by a combination of VASE, QCM-D and CPM. VASE and QCM-D provided information on protein dry thickness


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(correlated to the dry protein mass) and the hydrated protein mass adsorbed on the films, respectively. In addition, FS-modified colloidal probes (in CPM) were used to evaluate the attractive/repulsive forces between proteins and the exposed surface of the different PHEMA films. The adsorption of proteins on PHEMA brushes and brush-hydrogels was tested ex situ by VASE.15 Specifically, all the fabricated films were incubated for 90 min in FS and FS + Fgn, and the dry thickness of the adsorbed protein layer was measured. As shown in Figure 5, all the PHEMA films displayed a substantial reduction in protein adsorption compared to the bare silicon oxide surface, used as a control. This result was consistent with the picture of an extended hydration layer being present on all the PHEMA-based interfaces, and which efficiently reduced surface contamination by biomolecules.14–17 Comparing protein adsorption among the different PHEMA films (Figure 5a and 5b), the PHEMA brushes showed the lowest FS adsorption, while the introduction of crosslinks generally caused an increase in adsorbed protein thickness. QCMD confirmed the results of VASE, displaying an analogous trend for the adsorbed hydrated protein masses among the different PHEMA films (Figure 5b). Interestingly, the results from both these techniques indicated that the highest amount of adsorbed serum was recorded for PHEMA-DEGDMA-1, which showed a twofold increase in adsorbed protein compared to PHEMA brushes.


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Figure 5. Adsorbed protein thickness measured by VASE (a) and adsorbed hydrated protein mass measured by QCM-D (b) on the different PHEMA films following 90 minutes of incubation in FS (violet bars) and FS + Fgn (grey bars). The error bars indicate the standard deviation of four independent experiments. * indicates a statistically significant difference analyzed by ANOVA test P < 0.0001, § refers to P < 0.001, # indicates P < 0.05. In the presence of Fgn, the amount of adsorbed proteins followed a very similar trend compared to FS alone (Figure 5a and 5b, grey bars). Yet, the total thickness (directly correlated to protein mass) of adsorbed FS + Fgn was generally lower than that of FS alone. Considering that the most abundant protein in FS is albumin, previous studies34,53 highlighted how in the competitive adsorption of albumin and Fgn, albumin adheres first on the surface due to its faster diffusion, while Fgn, a bulkier protein characterized by a slower movement in solution, replaces albumin over time. This phenomenon, known as the “Vroman effect” describes the competitive exchange between proteins with a large difference in molar mass when adsorbed on a surface.53,54 However, in the present study, the exchange between Fgn and albumin appeared to take place within a very short time of incubation and involved a limited amount of physisorbed


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proteins, as confirmed by the nearly constant ∆f recorded by QCM-D55 (Figure 6). Once Fgn stably attached on the PHEMA surface, the exposed protein interface became more hydrophilic, presumably hindering the re-adsorption of albumin due to enhanced surface hydration.56 As a result, the total amount of physisorbed FS + Fgn showed a substantial reduction on all the PHEMA films when compared to FS alone. As a further evidence of the protein exchange process in the presence of Fgn, the variation of ∆f recorded on a bare silicon dioxide sensor showed a progressive decay during protein incubation with respect to the adsorption of just FS (Supporting Information). This could be more clearly highlighted on an unfunctionalized surface due to the high protein affinity of the bare silicon dioxide.

Figure 6. ∆f shifts recorded during protein adsorption on PHEMA (red trace), PHEMADEGDMA-1 (green trace) and silicon dioxide (black trace). In all the three sensograms, position “1” corresponds to the baseline taken in ultra-pure water, “2” the injection of FS into the QCMD fluid cells, and “3” the washing step by injection of ultra-pure water. Since protein adsorption appears to be dependent on the concentration and type of crosslinks within PHEMA grafts, we applied FS-modified CPM57,58 (FS-CPM) to investigate the interfacial forces between FS proteins and the different tested films. Recording FD curves by FS-CPM and concentrating on the retraction profiles, strong adhesive forces between brush-hydrogels and FS


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proteins on the colloids were generally recorded, as shown in Figures 7a and 7b. FS proteins on the colloidal probe could interact with the polymer surface via multiple contact points due to the flat appearance of the probe on the biomolecular scale, thus long-range rupture/protein unfolding events were observed from the retracting profiles.35,59 In all cases, the recorded rupture distance was higher than the polymer swollen thickness and the protein size in solution, suggesting that the proteins on the probe presumably unfolded and finally detached from the surface.59–61 Among the studied films, PHEMA brushes showed the lowest adhesion force (2.1 ± 1 nN), confirming the repulsion against protein contamination by a hydrated, linear PHEMA brush. In contrast, PHEMA-DEGDMA-1 showed the highest adhesion force recorded (9.3 ± 4 nN), indicating that FS proteins more strongly interacted with the exposed surface of this brushhydrogel, and confirming the results of VASE and QCM-D. Adhesion decreased with increasing crosslinker concentration, with PHEMA-DEGDMA-2 showing a value of 5.2 ± 3 nN and also displaying several rupture events at long distances. In addition, PHEMA-TEGDMA-1 and PHEMA-TEGDMA-2 showed lower adhesion forces compared to PHEMA-DEGDMA-1 and PHEMA-DEGDMA-2, with average values of 4.4 ± 2, and 4.5 ± 1 nN, respectively. Generally, these results are consistent with the hypothesis that PHEMA brushes are the most protein-resistant surfaces among the different brush architectures studied, due to both the brush steric repulsion and the high interfacial hydration of the polymer. Nevertheless, the relatively small but relevant adhesion force recorded for the uncrosslinked brushes indicated how linear PHEMA chains interact with proteins via hydrogen bonding and van der Waals interactions when the functionalized probe is compressing and perturbing the brush. As a result, PHEMA grafts can be pulled away from their equilibrium configuration by the retracting probe. A similar


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observation was previously mentioned by Leckband and coworkers in the case of PEG brushes, when their conformation is perturbed by an approaching, protein-functionalized countersurface.35 In the case of covalently crosslinked brush-hydrogels, brush conformational freedom is “frozen” due to the presence of lateral constraints. In these cases, surface chemistry determined the extent of interaction between the polymer films and the proteins. The results of FS-CPM suggest that the films presenting a relatively high interfacial concentration of hydroxyl groups, i.e. containing shorter crosslink type or lower concentration of crosslinks, generated higher adhesion forces with proteins. This was presumably due to the formation of hydrogen bonds between biomolecules and the hydroxyl moieties of HEMA.35,37 The strength and the range of interactions between the FS-modified probe and the surfaces were thus extremely sensitive to the composition and the graft structure of the polymer films. Thus, the combination of these two parameters ultimately determined the protein-repellent character of PHEMA brush-hydrogels. The steric repulsion exerted by the films upon contact with proteins was especially highlighted in the approaching profiles from the FD curves obtained by FS-modified CPM. The linear PHEMA brush showed the most pronounced repulsion force, originating at a relative vertical separation of ~ 80 nm, (Figure 7c and 7d). PHEMA-DEGDMA-2 showed much less pronounced repulsion forces (starting at a separation of ~ 60 nm) while PHEMA-DEGDMA-1 showed a typical “jump to contact” behavior, indicating the presence of an attractive force between the approaching colloid and the polymer surface (Figure 7c).62–64 PHEMA-TEGDMA-1 and PHEMA-TEGDMA-2 showed similar protein-repulsion character to the PHEMA brush, with repulsive forces starting at separations of ~ 80 nm for both these films. The observed long-range repulsion towards proteins observed for the PHEMA brush was due to a combination of disruption of the water-bound layer, which induced an enthalpic penalty, and


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steric resistance against compression by a densely grafted, linear brush, which generated an entropic barrier against protein adsorption.37,38,61 This last, steric component was presumably markedly reduced by introducing 1 mol% DEGDMA crosslinker within the film. Accordingly, PHEMA-DEGDMA-1 showed the highest amount of adsorbed proteins, which also showed stronger interactions with this particular polymer surface if compared to other brush and brushhydrogel films. The brush-hydrogels containing 2 mol% of TEGDMA and DEGDMA, generally showed better protein resistance with respect to PHEMA-DEGDMA-1. The reasons for this phenomenon are likely twofold. Firstly, the number of ethylene glycol units exposed at the interface in the cases of PHEMA-DEGDMA-2 and PHEMA-TEGDMA-2 were substantially increased (from 11 mol% of PHEMA-DEGDMA-1 and PHEMA-TEGDMA-1, to 21 and 28 mol% of PHEMA-DEGDMA-2 and PHEMA-TEGDMA-2, as reported in Table 2), therefore enhancing water association (and consequently hindering protein binding through an enthalpy loss). Secondly, especially in the cases of PHEMA-TEGDMA-1 and PHEMA-TEGDMA-2 brush-hydrogels, looser and more hydrated networks of grafted chains generated by longer crosslinks might provide a steric repulsion towards protein adsorption more similar to the one exerted by a linear, uncrosslinked brush. In agreement with this hypothesis, the approaching profiles from FD curves by FS-modified CPM recorded on PHEMA-TEGDMA brush-hydrogels almost overlapped with those on PHEMA brushes (Figure 7d).


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Figure 7. (a, b) Retracting profiles from FD curves recorded in PBS solution on the different PHEMA brush and brush-hydrogels by a FS-modified colloidal AFM probe; (c, d) approaching profiles extracted from FD curves recorded under the same conditions on the synthesized polymer films. In summary, the combination of VASE, QCM-D and FS-modified CPM have confirmed that linear PHEMA brushes efficiently repel proteins and display a biopassive character towards both FS and FS + Fgn. The protein repellency of the fabricated brush-hydrogels was determined by the relative content and the type of lateral constraints, i.e. the interfacial concentration of EGbased crosslinkers and their length. Conclusions In this work, the influence of polymer-brush architecture, i.e. content and type of crosslinks, on the physico-chemical and antifouling properties of the PHEMA-based films were systematically addressed by a variety of surface-sensitive and scanning probe techniques. PHEMA brushes and brush-hydrogels featuring different concentrations of DEGDMA and TEGDMA crosslinkers were fabricated by SI-ATRP. The grafting process and the swelling properties of the films were


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comprehensively studied by a combination of VASE and QCM-D, revealing how homogeneously crosslinked brush-hydrogels films could be obtained. The nanomechanical properties of the different films were investigated by CPM. Linear PHEMA brushes were more compliant when immersed in water, while the Young’s modulus of the brush-hydrogels progressively increased with the crosslinker concentration. The protein resistance of PHEMA brushes and brush-hydrogels was found to be directly related to the specific film architecture. Linear PHEMA brushes showed good biopassive properties towards both FS and FS + Fgn, while protein adsorption on brush-hydrogels was enhanced at low concentration of the shorter, DEGDMA crosslinker. In this specific case, the entropy-related steric barrier provided by the brush configuration was hindered by the presence of crosslinks, and proteins physisorbed on the films. At higher EG-containing crosslinker concentrations, the biopassive character of PHEMA-based brush-hydrogels was progressively recovered. This was presumably an effect of the higher content of water-associating EG units from the crosslinker functions at the film interface,65,66 which compensated the loss of conformational freedom (due to crosslinking) with an extended hydration layer preventing protein adsorption. In summary, we demonstrated that the precise tuning of brush architecture by introduction of different concentrations and lengths of crosslinkers allowed the modulation of both the physicochemical and biopassive properties of the obtained films. These findings demonstrate that brush crosslinking by segments with specific compositions can be applied to fabricate biopassive coatings with the desired mechanical properties, keeping thickness and brush grafting density constant. Brush films featuring different structures but interacting differently with adsorbing biomolecules could thus be potentially applied to regulate the settlement of larger biological


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objects (e.g. stem cells or bacteria) and to develop coatings with variable mechanical and swelling properties.

ASSOCIATED CONTENT Supporting Information Detailed XPS analysis and raw data, detailed QCM-D data and analysis, in addition to AFM topography images and FTIR spectra of synthesized coatings, further results from fluorescence microscopy

and

VASE,

NMR

of

ATRP-initiator.

AUTHOR INFORMATION Corresponding Author *Edmondo M. Benetti, Laboratory for Surface Science and Technology, Department of Materials, ETH Zurich, Vladimir-Prelog-Weg 5, CH-8093 Zurich, Switzerland Funding Sources Funding from the ETH Research Commission is gratefully acknowledged. EMB acknowledges financial support from the Swiss National Science Foundation (SNSF “Ambizione” PZ00P2148156). ACKNOWLEDGMENT The authors thank Prof. Antonella Rossi and Giovanni Cossu (ETH Zürich) for their support on the XPS measurements.


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For Table of Contents Only antifouling BRUSH

fouling BRUSH-HYDROGEL antifouling BRUSH-HYDROGEL


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Crosslinking Polymer Brushes with Ethylene Glycol ... - ACS Publications

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