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Antifouling Polymer Brushes Displaying Antithrombogenic Surface Properties Andres de los Santos Pereira, Sonia Sheikh, Christophe Blaszykowski, Ognen PopGeorgievski, Kiril Fedorov, Michael Thompson, and Cesar Rodriguez-Emmenegger Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00019 • Publication Date (Web): 16 Feb 2016 Downloaded from http://pubs.acs.org on February 22, 2016

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Antifouling Polymer Brushes Displaying Antithrombogenic Surface Properties Andres de los Santos Pereira,† Sonia Sheikh,‡ Christophe Blaszykowski,§ Ognen PopGeorgievski, † Kiril Fedorov,∥ Michael Thompson,*,‡ Cesar Rodriguez-Emmenegger*,†,¶ †Department of Chemistry and Physics of Surfaces and Biointerfaces, Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, v.v.i., Heyrovsky sq. 2, 162 06 Prague, Czech Republic. ‡Department of Chemistry − St. George Campus, University of Toronto, 80 St. George Street, Toronto, Ontario, Canada M5S 3H6. §Econous Systems Inc., 80 St. George Street, Toronto, Ontario, Canada M5S 3H6 ∥Institute of Biomaterials & Biomedical Engineering, 164 College Street, University of Toronto, Toronto, Ontario, Canada M5S 3G9 ¶DWI − Leibniz Institute for Interactive Materials and Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, Forckenbeckstraße 50, 52074 Aachen, Germany.

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ABSTRACT

The contact of blood with artificial materials generally leads to immediate protein adsorption (fouling), which mediates subsequent biological processes such as platelet adhesion and activation leading to thrombosis. Recent progress in the preparation of surfaces able to prevent protein fouling offers a potential avenue to mitigate this undesirable effect. In the present contribution, we have prepared several types of state-of-the-art antifouling polymer brushes on polycarbonate plastic substrate, and investigated their ability to prevent platelet adhesion and thrombus formation under dynamic flow conditions using human blood. Moreover, we compared the ability of such brushes —grafted on quartz via an adlayer analogous to that used on polycarbonate— to prevent protein adsorption from human blood plasma, assessed for the first time by means of an ultra-high frequency acoustic wave sensor. Results show that the prevention of such a phenomenon constitutes one promising route towards enhanced resistance to thrombus formation, and suggest that antifouling polymer brushes could be of service in biomedical applications requiring extensive blood-material surface contact.

KEYWORDS Antifouling; Polymer Brush; Platelet Adhesion; Human Blood; Blood Plasma; Acoustic Wave Biosensor


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1. INTRODUCTION Contact of blood with foreign materials that possess different properties from healthy vascular endothelium instigates a set of biological events which include coagulation and the formation of thrombi.1,

2

In the case of implants and indwelling therapeutic or diagnostic

medical devices, these processes dictate the outcome of the intended application and are responsible for its degree of success, notably in terms of performance and durability. Interactions at the surface of medical devices implanted in tissue elicit inflammation, and induce the migration of neutrophils and monocytes to the implant site as well as their activation resulting in the notorious ‘foreign body response’ with formation of an isolating fibrous capsule.3 When employing devices that operate within the vasculature or in contact with circulating blood, the presence of an interface with artificial materials leads to platelet adhesion and activation, blood coagulation and thrombus formation. Concomitantly, these coagulation events themselves can pose a risk to the life of patients as thromboembolic complications may arise with severe consequences such as infarcts and strokes.4 Examples where this may occur include procedure where the extracorporeal circulation (ECC) of blood involves extensive blood-surface contact such as cardiopulmonary bypass, extracorporeal membrane oxygenation, and hemodialysis among others. In order to prevent thrombus formation, the blood of patients is anticoagulated with such agents as heparin during these interventions. However, even under such conditions, these procedures have been associated with adverse outcomes including accelerated neurocognitive deterioration and increased incidence of strokes.5-9 In addition to the obvious hemodynamic changes occurring during such interventions, the extent of the blood–artificial material contact surface has been postulated as a probable factor responsible for the systemic inflammatory response and formation of micro-emboli.8


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Thrombus formation commonly causes implant failure in vascular grafts and cardiovascular stents.10-12 While the introduction of drug-eluting stents has dramatically reduced complications due to in-stent restenosis caused by neointimal hyperplasia, it has actually been shown to increase the incidence of late thrombosis episodes.13,

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The unfortunate

consequence is that the benefits of this life-saving technology are significantly limited due to the lack of control over pathophysiological reactions at the blood-implant interface that lead to thrombosis. In the case of vascular grafts, obstruction due to thrombus formation impairs the use of synthetic vessels of small diameter (less than about 6 mm).15, 16 The first step occurring upon contact of synthetic materials with blood is the spontaneous adsorption of proteins, a phenomenon termed ‘fouling’, which in turn mediates ensuing biological processes/responses.2,

3

The identity, quantity, and conformation of adsorbed

proteins have been shown to influence the activation of the coagulation pathways and platelet adhesion, and the complement system.17,

18

Protein fouling is a complex, dynamic

phenomenon involving the rapid surface adsorption of proteins, their time-dependent exchange with other proteins present in solution that have higher surface affinity, as well as the (gradual) loss of the native protein conformation upon adsorption.19, 20 Thus, the control of the conformational state of proteins on surfaces has been postulated as a potential avenue to achieve surfaces with enhanced hemocompatibility.21 The approaches investigated to date for this purpose have been reviewed elsewhere.4, 22, 23 One method to modify the surface of blood-contacting devices is through passivation with serum albumin, although in vivo studies have not met with a great deal of success.24-26 Other bio-inspired surface modification strategies include surface decoration with saccharide-presenting polymers, in an effort to mimic the highly hydrated structure of the cellular glycocalyx.27 Similarly, zwitterionic phosphorylcholine motifs, whose design take their inspiration from the polar headgroup abundantly found on cellular lipid bilayers, have also been attached onto the surfaces of ECC


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circuits.28 Heparin coatings have also been applied to prevent coagulation, and have shown improved outcomes in terms of inflammation and activation of the blood humoral and cellular systems.29 This effect has been attributed to reduced protein adsorption,30 but in practical terms has resulted in limited clinical benefit.31 Surface modifications that are capable of the prevention of protein fouling offer a promising alternative with respect to the elimination of the triggering of adverse biological reactions.21 Several strategies have been studied and significant advances have been made in the search for surfaces presenting such antifouling properties.32-34 The grafting of poly(ethylene oxide) chains showed a reduction in fouling from single-protein buffered solutions, which can be correlated with increasing layer thickness and grafting densities.35-38 Self-assembled monolayers have been hailed as simple and versatile antifouling surface coatings.39 In many cases, these layers can suppress the adsorption of the main plasma proteins from model buffered solutions.40-42 However, they are not able to efficiently reduce fouling from complex biological media.34,

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A highly successful surface modification

strategy to prevent fouling from complex biological media, including blood plasma and serum, are those based on polymer brushes grown via surface-initiated controlled polymerizations.32,

44

Polymer chains end-grafted to a surface in this fashion achieve a

preferentially stretched conformation, which is considered to be highly beneficial for their remarkable resistance to protein adsorption.36, 45 The recent advances in the short-term prevention of protein fouling open a new set of possibilities to refine the control of interactions at blood-material contacting interfaces. In previous reports, we have demonstrated the noteworthy prevention of surface-induced thrombosis on a polycarbonate substrate by an ultra-thin monoethylene glycol-based adlayer.46 The remarkable antifouling properties of this adlayer have also been confirmed through study of adsorption from serum using the highly sensitive electromagnetic


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piezoelectric acoustic sensor (EMPAS).47 Furthermore, we have studied the ability of polymer brushes to prevent cell adhesion even in serum-containing media, enabling avenues for micrometrically-resolved cell patterns.48 In recent work, we have found that antifouling polymer brushes are capable of sharply reducing the deposition of the cellular components of citrated blood under static conditions.49 In light of these previous results, it is clear that state-of-the-art polymer brushes may hold immense potential as coatings for blood-contacting devices. The goal of the work presented herein is to assess the ability of antifouling polymer brushes to prevent thrombogenesis under dynamic flow conditions at a specified, controlled shear rate. For that purpose, polycarbonate (PC), a common plastic used in biomedical applications, was used as the substrate onto which polymer brushes were grown from a previously immobilized silane-initiator adlayer (Scheme 1). Furthermore, the correlation between the brushes’ antithrombogenicity and antifouling properties was investigated. This was achieved by evaluating, for the first time using the ultra-high frequency EMPAS device, the resistance to blood plasma fouling of brushes grown from the same type of initiator adlayer on quartz resonator discs.

Scheme 1. (a) Chemical structures of the surface modifications performed on polycarbonate. Interaction of (b) bare PC and (c) polymer-brush-modified PC with fluorescently labelled blood platelets. The inserts are typical fluorescence microscopy images recorded on (b) bare PC and (c) polymer brushes.


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2. MATERIALS AND METHODS Materials. CuBr2 (99.999% trace metal basis), CuCl2 (99.999% trace metal basis), CuBr (99.99%),

CuCl

(99.99%),

2,2’-bipyridyl

(99%),

1,4,8,11-tetramethyl-1,4,8,11-

tetraazacyclotetradecane (Me4Cyclam, 98%), 2-hydroxyethyl methacrylate (99%), and oligo(ethylene glycol) methyl ether methacrylate (Mn = 300 g mol−1, MeOEGMA) were purchased from Sigma-Aldrich (Czech Republic). Deionized (DI) water was obtained from a Milli-Q purification system (Merck-Millipore). The ATRP initiator (11-(2-bromo-2methyl)propionyloxy)undecyltrichlorosilane was synthesized according to a procedure reported earlier.50 The monomers N-(2-hydroxypropyl)methacrylamide (HPMA) and (3acryloylaminopropyl)-(2-carboxyethyl)dimethylammonium (carboxybetaine acrylamide or CBAA) were synthetized as previously reported.32 Polycarbonate (PC) plastic sheets, employed as substrates in thrombogenicity assessment experiments, were purchased from McMaster-Carr (USA). AT-cut quartz discs with a fundamental frequency of 20 MHz were acquired from Laptech Precision Inc., Bowmanville, Ontario, Canada. Human blood for thrombogenicity experiments was collected in heparinized Vacutainers from apparently healthy donors. Human blood plasma was obtained by centrifugation from fresh blood of the same donors, collected in citrate Vacutainers. Immobilization of the Initiator Adlayer on the Substrates (Silanization). PC slides were rinsed with DI water, sonicated for 5 min in a 1% solution of sodium dodecylsulfate in DI water, rinsed with DI water, ethanol, and DI water, and dried by a stream of nitrogen. Subsequently, they were activated by exposure to air plasma for 20 min, and then immediately

immersed

in

a

solution

of

ATRP

initiator

(11-(2-bromo-2-

methyl)propionyloxy)undecyltrichlorosilane (1 mg mL-1 in dry hexane). After 3 h, the substrates were removed from the solution, rinsed successively with copious amounts of hexane, ethanol, and then DI water, and finally dried with nitrogen.


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Surface-initiated Polymerizations. The grafting of the polymer brushes from the initiatorfunctionalized substrates was accomplished via atom transfer radical polymerization using our optimized procedures.49,

50

For a detailed description of the polymerization procedure

employed for each monomer, please refer to the accompanying SI. Briefly, the appropriate solvent was degassed either by bubbling Ar or freeze-pump-thaw to remove dissolved oxygen. A solution of degassed monomer, solvent and the Cu(I)- and Cu(II)-salts was prepared under inert atmosphere and was added to sealed Ar-filled reactors containing initiator-coated substrates. After the appropriate polymerization time, the reactors were opened to the atmosphere and filled with water. The substrates were removed and thoroughly rinsed with water and ethanol twice, dried with nitrogen, and stored until further experiments. Surface Characterization. X-ray photoelectron spectroscopy (XPS) measurements were carried out on the modified surfaces of PC substrates after each modification step using a KAlpha+ spectrometer (Thermo Scientific, UK) equipped with a microfocused monochromated Al Kα X-ray source. Charge compensation was used, employing electrons of 8 eV energy and low-energy Ar ions to prevent localized build-up of charge. The spectra were fitted with one or more Voigt profiles (binding energy uncertainty: ±0.2 eV) The analyzer transmission function, Scofield sensitivity factors,51 and effective attenuation lengths (EALs) for photoelectrons were applied for quantification. EALs were calculated using the standard TPP-2M formalism.52 All spectra were referenced to the C 1s peak attributed to C–C, C–H at a binding energy of 285.0 eV, controlled by means of the well-known photoelectron peaks of metallic Cu, Ag, and Au. Advancing and receding contact angle of the surfaces with Milli-Q water were used to assess surface wettability after each modification step and were acquired by the sessile drop method, using a DataPhysics OCA20 instrument. An initial drop of 5 µL was placed on the surface and its volume was increased and subsequently decreased by 10 µL at a constant rate


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of 0.5 µL s–1. The drop profile was extracted from the captured images and fitted to an ellipse, from which the contact angles were obtained. Assessment of the Antifouling Properties of Polymer Brushes with the EMPAS. Fouling from blood plasma was assessed using the electromagnetic piezoelectric acoustic sensor (EMPAS).53 After the quartz resonator discs were cleaned by previously published protocols, the initiator silane layer was then grafted, and the polymer brushes finally grown – the latter two steps following the respective procedures described above for PC substrates. Measurements were conducted at the ultra-high frequency of 0.94 GHz, which represents the 47th harmonic of the employed quartz resonator discs. PBS (pH 7.4) was flowed over through the flow chamber at a rate of 50 µL min–1, which corresponds to a chamber entrance shear rate of 2100 s–1. After stabilization of the baseline frequency, 50 µL of undiluted human blood plasma were injected using an injection loop without interrupting the flow of buffer. PBS flow continued until the frequency re-stabilized, and the fouling behavior of surfaces was then assessed as the shift in frequency before and after contact with blood plasma.47 Assessment of Thrombogenicity in Contact with Whole Blood under Flow. Surfaceinduced platelet adhesion and aggregation and thrombus formation were studied via in situ fluorescence microscopy in a custom-built blood perfusion chamber by flowing freshlyobtained whole human blood with fluorescently labelled cells at a set shear rate of 1000 s–1, according to a protocol previously described.46 A parallel plate single flow cell was employed. Its construction (including a diagram) is described in the Supporting Information. The flow cell was installed in an Axiovert 135 inverted fluorescent microscope (Carl Zeiss) equipped with a DP70 digital camera (Olympus). Slidebook software (Intelligent Imaging Innovations) was utilized to capture image sequences. The sample slides coated with polymer brushes were mounted in the perfusion chamber and bare PC was assessed for comparison (for all surfaces n = 4).


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Blood was collected from healthy donors in heparin-coated Vacutainers and labelled with DiOC6 (3,3’-dihexyloxacarbocyanine iodide, 1 µM) 10 min prior to use. This fluorescent dye binds to mitochondria and vesicle membranes, labelling platelets and leukocytes.54 The perfusion experiments were carried out for at least 10 min at a shear rate of 1000 s−1. Platelet adhesion, aggregation, and thrombus formation were imaged at 32X magnification. The captured image sequences were exported and analyzed with ImageJ software to minimize background fluorescence intensity and only highlight positive signals from adhered platelets and their aggregates. The percentage of the surface covered by platelet aggregation and thrombus formation was assessed using ImageJ software on still images after 5 and 10 min.

3. RESULTS AND DISCUSSION Characterization of Surface Modifications. The modification of polycarbonate (PC) surfaces was assessed via X-ray photoelectron spectroscopy (XPS). The expected chemical structures are presented in Scheme 1. The changes in the spectra of PC samples were recorded following silanization with the ATRP initiator adlayer and subsequent growth of the polymer brushes via ATRP. The C 1s region of the high resolution spectra is shown in Figure 1a. The spectrum of bare plasma-cleaned PC is characterized by components arising from C– C and C–H bonds (285.0 eV), C–O (286.6 eV), and carbonate O–(C=O)–O (290.6 eV).55 The success of the silanization with initiator is shown by the relative increase in the C–C, C–H component at 285.0 eV with respect to the C–O and carbonate peaks owing to the long alkyl backbone of the adlayer’s initiator residues. Further indication of the presence of the initiator on the surface is given by the direct detection of bromine forming C–Br bonds in the Br 3d region of the XPS spectrum (Figure 1b), a signal that shows a typical split of the spin-orbit components appearing at 70.5 eV and 71.6 eV.


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The grafting of the polymer brushes by surface-initiated ATRP is accompanied by pronounced changes in the C 1s region of the XPS spectra (Figure 1a, 3-6). In the case of both methacrylate polymers, poly(HEMA) and poly(MeOEGMA), the ester group linking the polymer side chain to the backbone gives rise to a contribution at 289.0 eV. For poly(CBAA) and poly(HPMA), this occurs via an amide side group whose signal appears at 287.5 eV and 287.9 eV, respectively. The N 1s spectra (Figure 1c) further confirms the presence of the nitrogen forming amide bonds (399.6 eV and 400.0 eV for poly(CBAA) and poly(HPMA), respectively). In the case of poly(CBAA), a further contribution is detected in the N 1s region at 402.5 eV, that can be assigned to the positively charged quaternary ammonium forming the zwitterion side-chain. The side groups of the polymer chains of poly(HEMA) cause an increase in the signal for the C–O component (286.6 eV). This is also observed for poly(MeOEGMA) at the same binding energy, where the increase is more apparent due to the increased in side chain length (that is in EG repeat units). For poly(CBAA), the quaternary ammonium gives rise to a signal for C–N bonds, which overlaps with the C–O component increasing its intensity, while the carboxylate group is observed at 288.2 eV. The spectrum of poly(HPMA) also displays a component at 286.5 eV arising from the C–O and C–N groups in the structure.


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Figure 1. XPS characterization of the brushes obtained on PC substrates. (a) C 1s region of the spectra of bare PC (1), the initiator adlayer on PC (2), and the poly(HEMA) (3), poly(MeOEGMA) (4), poly(CBAA) (5), as well as poly(HPMA) (6) polymer brushes. (b) Br 3d region of the XPS spectrum for the initiator on PC (2). (c) N 1s region of the spectra for poly(CBAA) (5) and poly(HPMA) (6) polymer brushes. Surface water wettability, assessed by means of dynamic contact angle measurements with deionized water by the sessile drop method, was used as a further confirmation of the success of the targeted chemical modifications on the PC substrates (Table 1). Freshly cleaned PC slides show a relatively hydrophilic behavior due to the abundance of polar groups on the surface that were generated/exposed during oxidative plasma treatment. Upon silanization with the initiator adlayer containing grafted molecular residues with a non-polar alkyl backbone, the contact angle increased sharply. Subsequent grafting of the polymer brushes, which contain polar groups in their side chains, led to a decrease in the contact angles. This is especially pronounced in the case of zwitterionic poly(CBAA). In all cases, a large contact


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angle hysteresis —the difference between advancing and receding values— likely is caused by surface roughness, as well as by brush swelling upon contact with water.

Table 1. Dynamic water contact angles Surface

Contact angle (º) Advancing

Receding

Plasma-activated PC

40 ± 2

11 ± 1

Initiator on PC

88 ± 1

58 ± 2

Poly(HEMA)

73 ± 2

31 ± 2

Poly(MeOEGMA)

72 ± 1

46 ± 3

Poly(CBAA)

21 ± 1

0a

Poly(HPMA)

73 ± 2

24 ± 3

a

While decreasing the volume, the contact line was pinned, resulting in continuous decrease in the contact angle until the drop was practically flat and no more volume could be withdrawn.

EMPAS Characterization of Blood Plasma Fouling. Herein, the antifouling behaviour against human blood plasma (full, undiluted) of the various poly(HPMA), poly(HEMA), poly(MeOEGMA), and poly(CBAA) brushes —which were prepared on quartz as the substrate from siloxane adlayer of ATRP initiator— was investigated using, for the first time, the acoustic wave physics of the ‘electromagnetic piezoelectric acoustic sensor’ (EMPAS),56, 57

as opposed to the traditional surface plasmon resonance (SPR) optical technique (for

adlayers prepared, in that case, on a metallic surface of gold).44 The suitability of the EMPAS system for such purpose as the real-time and label-free assessment of surface fouling against full biofluids (i.e. human or animal blood serum/plasma) has recently been validated for a variety of organic surface chemistries.47, 58, 59 In the present study, it is immediately evident from Figure 2 that all polymer brush coatings displayed marked antifouling properties in


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comparison with bare quartz, as assessed through a decrease in the net resonant frequency shift upon plasma injection (∆fbrushes > –3 kHz vs. ∆fquartz ~ –30 kHz). Although there essentially was no statistical difference in this respect among polymer films (Figure 2), the observation was made that only the poly(CBAA) system exposed to full blood plasma repeatedly (i.e. within the series of three replicates) yielded EMPAS profiles featuring no observable net resonant frequency shift. In terms of antifouling performance, these results are overall well in line with those previously reported for polymer brushes grown on SPR gold chips.32, 49, 60

Figure 2. EMPAS net frequency shift upon injection of full, undiluted human blood plasma onto bare or polymer brush-derivatized quartz resonator discs (3 replicates per data set). EMPAS measurements were performed at the ultra-high resonant frequency of 0.94 GHz (47th harmonic), at room temperature. The error bars correspond to the standard deviation. Note: the symbol ‘*’ means that no net frequency shift was observed.


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Prevention of Thrombus Formation on Modified Polycarbonate Plastic. The adhesion and aggregation of platelets and formation of thrombus on bare and polymer-coated surfaces was studied via fluorescence microscopy. Figure 3 shows representative fluorescence micrographs captured while fluorescently labelled blood was being flowed over the various surfaces. Bare PC exhibits significant adhesion/aggregation of platelets at 5 min. The image captured for bare PC at 10 min shows an increase in surface coverage, and that platelet aggregates are still roughly aligned with the direction of the flow (from top to bottom in every image shown). The aggregation is probably induced by the first-adhered, surfaceactivated platelets that recruited more platelets in their vicinity propagating the platelet activation/recruitment process. All antifouling polymer brushes show a substantial reduction in the surface coverage. Interestingly, platelets, as a general trend, appear to attach as isolated entities or form small aggregates at best, no further aggregation being indeed observed during the course of the experiments (10 min). This stands in stark contrast with the growth in size of the aggregates observed on the bare PC surface. Replicate fluorescence images confirming these trends can be found in the accompanying SI.

Figure 3. Typical fluorescence microscopy images obtained for different polymer-brushmodified and bare (control) PC substrates, after 5 and 10 min of contact with DiOC6-labelled blood. The flow direction was approximately from top to bottom.


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The extent of platelet aggregation/thrombus formation was quantified on still images at 5 and 10 min through calculation of surface coverage, as the ratio of pixels brighter than the background (Figure 4). All polymer bushes tested displayed a significant reduction in thrombus formation and platelet aggregation with respect to bare PC substrates. In the case of poly(HEMA), surface coverage was only 7.4% of that observed for the bare substrate at 5 min, this relative value dropping to 2.5% of the surface coverage found on bare PC after 10 min (92.5 and 97.5% reduction, respectively). Poly(CBAA) showed a similar level of reduction in surface coverage —96.2 and 97.3% for 5 and 10 min, respectively. Both poly(MeOEGMA) and poly(HPMA) brushes displayed over 99% reduction in platelet aggregation/thrombus formation, with most of the captured frames actually showing a single spot corresponding to individually-adhered entities (which was taken as a confirmation that the microscope was correctly focused on the surface during perfusion experiments).

Figure 4. Surface coverage by fluorescently labelled aggregated platelets and thrombi, calculated from flow experiments performed at a controlled shear rate of 1000 s–1 after 5 min (blue bars) and 10 min (red bars) of contact with whole human blood. The error bars correspond to the standard deviation.


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In addition to their pronounced resistance to protein adsorption, the marked reduction in thrombogenicity displayed by all antifouling polymer brushes matches well with their previously reported ability to prevent the adhesion and activation of blood cellular components.49,

61

Adsorbed proteins often change their conformation and trigger the

subsequent attachment and activation of platelets and leukocytes.17,

62

Fibrinogen and von

Willebrand clotting factors are among the most studied plasma proteins for their ability to mediate platelet adhesion, but even serum albumin – a protein, which is yet not involved in the coagulation cascade – can undergo surface-induced changes in conformation upon adsorption leading to thrombogenicity.62-64 The ability of antifouling brushes to inhibit the presence of proteins at interfaces appears to play an important role in the suppression of thrombogenicity in vitro. The results presented herein with polymer brushes are well in line with those reported in previous work with ultra-thin, monoethylene glycol adlayers.46 Biochemical analysis of the aggregates deposited on the different surfaces, in terms of protein and cell composition, could provide an interesting avenue to gain insight into the different processes at play and inspire avenues to improve the design of bio-inert coatings. The indepth physicochemical characterization of the surfaces and their interactions with proteins can be accomplished by means of acoustic–wave-based sensors in combination with other methods. These results should be correlated to their resistance to protein fouling and antithrombogenicity to help elucidate the underlying interaction mechanisms. Antifouling polymer brushes are known to reduce the attachment of bacteria in complex biological media and prevent biofilm formation.65, 66 In that case, the prevention of protein fouling is critical, as the fouling layer acts as a conditioning film for bacteria colonization, but it is worth noting that the direct adhesion of bacteria is also strongly prevented by the brushes.50 The combination of antithrombogenicity and prevention of bacterial biofilm formation offers great promise in the biomedical field for implantable devices, such as


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mechanical heart valves, which often suffer from complications arising from both phenomena. Furthermore, polymer brushes feature side- as well as end-chain groups that can be readily functionalized with bioactive motifs such as specific peptides or antibody fragments. These could be used to favor the adhesion of targeted cells, e.g. endothelial progenitor cells, paving the way for potential tissue engineering approaches in order to develop truly biocompatible implants.

4. CONCLUSION Antifouling polymer brushes were grown on polycarbonate substrates and quartz discs from an initiator silane adlayer. The antifouling brushes were found to be capable of drastically reducing fouling from full human blood plasma, as assessed for the first time using the EMPAS acoustic wave device. It was also shown that platelet aggregation and thrombus formation were largely reduced, as visualized by fluorescence microscopy, when the surfaces were exposed to whole human blood in a perfusion chamber under dynamic conditions. Taken together, these results highlight the role of protein adsorption onto artificial materials in mediating subsequent biological reactions, and indicate that the minimization of protein fouling constitutes one promising avenue for the prevention of adverse biological responses in short-term blood contacting applications, such as extracorporeal blood circulation. However, longer-term studies are needed before such surface treatments can be used as coatings for actual implant applications.

ASSOCIATED CONTENT Supporting Information. Grafting of polymer brushes, description and diagram of flow cell construction, additional fluorescence images obtained in the thrombogenicity experiments,


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and time-lapse videos of typical thrombogenicity experiments. This material is available free of charge via the Internet at http://pubs.acs.org.” AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] * E-mail: [email protected]

ACKNOWLEDGMENTS This work was supported by the Grant Agency of the Czech Republic (GACR) under contract no. 15-09368Y and the European Regional Development Funds under the project “BIOCEV − Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University” (CZ.1.05/1.1.00/02.0109) and OPPK CZ.2.16/3.1.00/21545. The authors are also grateful to the Natural Sciences and Engineering Research Council of Canada for partial support of the Canadian component of this work.

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TABLE OF CONTENTS GRAPHIC


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