Article pubs.acs.org/Langmuir
Prevention of Thrombogenesis from Whole Human Blood on Plastic Polymer by Ultrathin Monoethylene Glycol Silane Adlayer Kiril Fedorov,† Christophe Blaszykowski,‡ Sonia Sheikh,§ Adili Reheman,∥ Alexander Romaschin,⊥ Heyu Ni,∥ and Michael Thompson*,†,‡,§ †
Institute of Biomaterials & Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada Econous Systems Inc., Toronto, Ontario, Canada § Department of Chemistry−St. George Campus, University of Toronto, Toronto, Ontario, Canada ∥ Canadian Blood Services and the Department of Laboratory Medicine and Pathobiology, Keenan Research Centre in the Li Ka Shing Knowledge Institute of St. Michael’s Hospital, University of Toronto, Toronto, Ontario, Canada ⊥ Keenan Research Centre and Clinical Biochemistry, St. Michael’s Hospital, Toronto, Ontario, Canada ‡
S Supporting Information *
ABSTRACT: In contemporary society, a large percentage of medical equipment coming in contact with blood is manufactured from plastic polymers. Unfortunately, exposure may result in undesirable protein−material interactions that can potentially trigger deleterious biological processes such as thrombosis. To address this problem, we have developed an ultrathin antithrombogenic coating based on monoethylene glycol silane surface chemistry. The strategy is exemplified with polycarbonate−a plastic polymer increasingly employed in the biomedical industry. The various straightforward steps of surface modification were characterized with X-ray photoelectron spectroscopy supplemented by contact angle goniometry. Antithrombogenicity was assessed after 5 min exposure to whole human blood dispensed at a shear rate of 1000 s−1. Remarkably, platelet adhesion, aggregation, and thrombus formation on the coated surface was greatly inhibited (>97% decrease in surface coverage) compared to the bare substrate and, most importantly, nearly nonexistent.
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properties.5,7 Chronically raised nevertheless is the question of BPA leaching from the polymer, which constitutes a societal concern since BPA has well-characterized endocrine-disrupting activity with strongly suspected effects on human health, a dearth in epidemiological studies cannot ascertain.7−10 However, considering BPA contamination comes from prolonged/repeated contact with the polymer (mainly from food packaging),8−10 BPA leaching from single-use, disposable medical equipment is not likely to constitute a significant health threat11 since exposure in this case is generally limited in time. Regardless, commercial BPA-PC for a given healthcare application is required to be certified “biocompatible” (i.e., “medical-grade”) by suppliers in compliance with governmental regulations/international standards (ISO 10993).7,12,13 It should not come as a surprise however that, for diverse reasons, there always exists room for improvement/innovation in this domain and that novel, permanent surface modification strategies are always welcome.14,15 In this context, we report herein new monoethylene glycol (MEG-OH) silane adlayer coating for BPA-PC (Figure 1) that displays excellent antithrombogenicity, far exceeding that of the
INTRODUCTION Extracorporeal circulation of blood is a common medical procedureused in hemodialysis and coronary bypass surgerythat requires blood to come in contact with materials foreign to the human body. Interaction of blood components (i.e., proteins) with artificial surfaces may however stimulate biological processes, orchestrated by the immune and coagulation systems,1,2 with deleterious outcomes. Indeed, although the procedure itself may proceed without an immediate serious problem for the patient, postoperative complications such as organ dysfunction are unfortunately a real occurrence.2 Incapacitating brain disorder (e.g., cognitive impairment) would be an example of relative severity.2−4 To avoid having to “trade” a medical condition with another, the search for hemocompatible materials has been and continues to be, understandably, the object of intense efforts. In modern society, synthetic plastic polymers are ubiquitous and have replaced the conventional materials that were wood, metal, and glass in nearly all imaginable applications.5 Polycarbonates (PC)more often than not based on bisphenol A (BPA)are not spared from this trend and actually rank among the fastest prospering plastic polymers.5,6 This also is true in the biomedical industry where thermoplastic BPA-PC resins are highly praised for their many attractive © 2014 American Chemical Society
Received: September 24, 2013 Published: March 13, 2014 3217
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Figure 1. Two-step straightforward surface modification of bisphenol A polycarbonate (BPA-PC) plastic polymer with monoethylene glycol (MEG− OH) silane adlayer. Note: this schematic representation merely depicts the performed surface chemistry, not the actual surface coverage/patchiness of the adlayers nor the anchorage nature and degree of order/packing of the surface-modifying residues within. The acronym “MEG-TFA” stands for monoethylene glycol trifluoroacetyl. The chemical name of the MEG-TFA molecule is 2-(3-trichlorosilylpropyloxy)ethyl trifluoroacetate.
nonderivatized substrate material. This new feature16 for such ultrathin surface chemistrywhich constitutes the novelty and significance of this workadds to the pronounced antifouling character observed in a recent original study, wherein MEGOH coatings imposed on quartz were shown, using acoustic wave physics, to drastically alter the dynamics of full serum adsorption.17 Indeed, antithrombogenicity (prevention of blood clotting) and antifouling (prevention of biological adsorption) are two different surface properties that may not necessarily be related.16 Straightforward BPA-PC surface modifications were characterized using X-ray photoelectron spectroscopy (XPS) supplemented by contact angle goniometry (CAG). Antithrombogenicity was next assessed in real-time using a perfusion chamber and fluorescently labeled whole human blood flown for 5 min at a controlled shear rate of 1000 s−1. Remarkably, platelet adhesion, aggregation, and thrombus formation on the MEG-OH coating was greatly inhibited (>97% decrease in surface coverage) compared to the bare BPA-PC substrate and, most importantly, nearly nonexistent. This work by no means constitutes the first example of chemical surface modification of BPA-PC to improve antithrombogenicity since one strategy, bioinspired, has already been described elsewhere.14,15 In that work,14 the authors mimicked the glycocalyxthe outer surface of biological cell membranes that consists of a highly hydrated carbohydrate-rich mesh18with a polysaccharide coating (dextran) to reduce platelet adhesion on medical-grade Lexan, a BPA-based PC plastic polymer.19 We, on the other hand, employ a novel approach based on nonmimetic, ultrathin MEG silane surface chemistrythe first account of its kind to our knowledge.
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transferred into a humidity chamber (80% RH, room temperature) for overnight surface hydration. MEG-TFA and MEG-OH Adlayer Preparation.17 MEG-TFA surface modifier17,20 (10 μL) was first diluted with hexanes (10 mL) in a glovebox maintained under inert (N2) and anhydrous (P2O5) atmosphere. The resulting solution was portioned in vials (1000 μL) into which preactivated, cleaned BPA-PC slides (vide supra) were then individually soaked [Note: to prevent the undesired reaction of MEGTFA surface modifier with the silanization glassware, the latter was pretreated overnight with a 1/20 (v/v) solution of octadecyltrichlorosilane in anhydrous toluene.] The vials were capped, removed from the glovebox, and then placed on a spinning plate for 1 h at room temperature. Next, the slides were rinsed with hexanes (×3) then sonicated in another portion of solvent for 5 min. This procedure was repeated with 95% ethanol, and the slides finally dried under a gentle stream of N2. Without delay, the slides were individually soaked in 1 mL of a 1/1 (v/v) solution of 95% ethanol and Milli-Q water, overnight at room temperature on a spinning plate. Finally, the slides were rinsed with 95% ethanol (×3) then dried under a gentle N2 stream. For MEG-TFA adlayer formation on BPA-PC sheets, the silanizing solution per sample was 20 μL of MEG-TFA surface modifier diluted in 20 mL of hexanes. The volume of ethanol/water solution for the subsequent conversion to MEG-OH adlayer was 6 mL. Dynamic Flow Assay of Thrombogenicity.21 Human blood was collected in heparinized Vacutainers from apparently healthy donors at St. Michael’s Hospital (Toronto, Ontario, Canada) and used within few hours. Whole blood was freshly labeled with 3,3′dihexyloxacarbocyanine iodide fluorescent dye (DiOC6: 1 μM, 10 min at 37 °C) prior to use. Thrombogenicity experiments were performed for 5 min under high shear conditions (1000 s−1) using a rectangular perfusion chamber (GlycoTech). Platelet adhesion, aggregation, and thrombus formation was visualized and recorded in real-time under an Axiovert 135 inverted fluorescent microscope (Carl Zeiss) equipped with a DP70 digital camera (Olympus) using Slidebook software (Intelligent Imaging Innovations) under 32× magnification. Background fluorescence intensity was defined by this program and subtracted for the entire course of recorded images, only showing positive signals from adhered platelets/aggregates. Surface coverage due to platelet adhesion, aggregation, and thrombus formation was assessed using ImageJ software on still images at 5 min. BPA-PC coated with type I collagen (Nycomed, 100 μg/mL) was used as positive control. X-ray Photoelectron Spectroscopy and Contact Angle Goniometry. XPS was performed with a Theta Probe Instrument (ThermoFisher Scientific). Samples were analyzed with monochromated Al Kα X-rays at a takeoff angle of 90° to the surface. The
MATERIALS AND METHODS
Bisphenol A Polycarbonate (BPA-PC) Substrate Cleaning and Activation. BPA-PC substrates [1.4 cm × 1.4 cm and 1/16 in. thick slides for XPS and CAG analysis (SABIC Polymershapes) or 7.5 cm × 2.5 cm and 0.005 in. thick sheets for blood experiments (McMaster-Carr)] were first copiously rinsed with warm tap water, followed by distilled water, and then soaked under gentle spinning in a 1% sodium dodecyl sulfate (SDS) solution prepared in Milli-Q water (18.1 Mohm). After 5 min, BPA-PC substrates were thoroughly rinsed with 1% SDS (×2) followed by tap water, distilled water, then 95% ethanol. After drying under a gentle N2 stream, BPA-PC substrates were finally air-plasma cleaned/activated for 20 min (Harrick Plasma, 30 W). After this treatment, BPA-PC substrates were immediately 3218
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Figure 2. (a) Overlapped XPS surveys for (top to bottom) plasma-activated bare BPA-PC (red), MEG-TFA (green), and MEG-OH (blue) adlayers. For clarity, the former two profiles have been shifted upward. (b) C1s signal narrow scans showing the various C−C/CC, C−O, CO, and C−F bond contributions and relative compositions for (top to bottom) bare BPA-PC (red), MEG-TFA (green), and MEG-OH (blue) surfaces. binding energy scale was calibrated to the main C1s signal at 285 eV. Peak fitting and data analysis were performed using Avantage software. Static contact angles were measured with a CAM101 goniometer (KSV Instruments) and Milli-Q water as the test liquid.
the latter had effectively cleaved all terminal TFA groups without etching the residual MEG-OH siloxane network from the BPA-PC substrate.17 If not a proof of chemisorption, i.e. of strong covalent anchorage (Figure 1), this certainly is an indicator of great robustness for the MEG-OH adlayer, a definitely desirable attribute for biomaterial coatings. With respect to carbon, the relatively most abundant element (from BPA-PC), the XPS signal decreased upon formation of the MEG-TFA adlayer (Table 1), as expected for a now buried, underlying substrate. Looking closer at its narrow scan (Figure 2b), it can be seen that, depending on the type of surface, the C1s peak is actually composed of up to four different kinds of carbon bonds: C−C/CC, C−O, CO, and C−F. Expectedly (Figure 1), bare BPA-PC only shows XPS peaks for C−C/ CC (at 285 eV), C−O (at 287 eV) and CO (at 291 eV) bonds. For the MEG-TFA adlayer, two new peaks characteristic of the TFA group appeared (CO at 290 eV and C−F at 293 eV; Figure 2b).23 Also, the relative magnitude of the C−O signal at 287 eV significantly increased as a result of the multiple incorporation of MEG backbones and their several ether bonds within the newly formed coating (Figures 1 and 2b). Note as well that, not surprisingly, the CO peaks for the substrate’s carbonate and coating’s TFA ester moieties have slightly different binding energies (291 versus 290 eV, respectively) and actually overlap in the C1s narrow scan of MEG-TFA (Figure 2b). Upon TFA solvolysis to form MEGOH, both C−F and CO peaks (at 290 eV) completely disappeared, whereas the C−O signal from the MEG backbones remained untouched (Figure 2b) confirming that only the TFA terminal groups were cleaved during the aqueous treatment,17 as concluded earlier from the observation of the Si2p signal. Also note here that the substrate’s CO signal at 291 eV is the sole such peak remaining upon conversion of MEG-TFA to MEG-OH adlayer (expectedly attenuated as well compared to bare BPA-PC). These XPS data of BPA-PC
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RESULTS AND DISCUSSION Adlayer Preparation/Characterization. MEG-TFA adlayers were first readily prepared in a simple dip-and-rinse procedure upon immersion of cleaned BPA-PC substrates− preactivated upon exposure to a plasma of highly reactive ionized/radical oxygen species to generate surface hydroxyls22into a 1/1000 (v/v) solution of MEG-TFA surface modifiers in hexanes, for 1 h at room temperature (Figure 1, step I).17 Subsequent solvolysis of the labile trifluoroacetyl (TFA) terminal groupswith a 1/1 (v/v) solution of 95% ethanol and water, overnightsmoothly provided the desired MEG-OH adlayer (Figure 1, step II).17 Both successful transformations were characterized with X-ray photoelectron spectroscopy (XPS) following the appearance/loss of peaks for fluorine (F1s at 688 eV) and silicon (Si2p at 103 eV), the two elements unambiguously attributable to MEG-TFA/MEG-OH adlayers (Figures 1 and 2a, Table 1). Unlike fluorine howeverwhose peak completely disappearedthe signal for silicon was not affected by the subsequent solvolytic treatment (Figure 2a and Table 1), clearly demonstrating that Table 1. XPS Relative Atomic Percentages for the Characteristic Elements of BPA-PC Substrate and MEGTFA/MEG-OH Adlayers relative atomic percentages (%) BPA-PC surface
C1s
O1s
F1s
Si2p
bare (activated) MEG-TFA MEG-OH
76.3 44.7 47.5
22.8 36.8 39.0
0.0 5.9 0.0
0.9 12.6 13.5 3219
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surface modification with MEG-TFA/MEG-OH silane chemistry are well in line with those obtained in the aforementioned study with quartz substrates.17 Both stages of surface modification (MEG-TFA and MEGOH adlayers) as well as bare BPA-PC were further characterized with static contact angle goniometry (CAG) using water as the test liquid. Nonactivated, bare BPA-PC presented a rather hydrophobic surface with a contact angle (CA) of 94 ± 3°, n = 3. Upon plasma activation, the CA significantly decreased to 45 ± 1° (n = 3), a value well consistent with the formation of a more hydrophilic surface presenting hydroxyls and other polar groups.22,24 Pronounced hydrophobicity (CA = 85 ± 2°, n = 5) resumed for the MEGTFA coating owing to the exposure of CF3 groups to the outer environment. [This CA value is consistent with that previously measured for MEG-TFA films on quartz (90°).17] Finally, as expected upon removal of these hydrophobic CF3 moieties during TFA solvolysis to expose polar hydroxyls in MEG-OH coating, some hydrophilicity was recovered as the decreased CA revealed (55 ± 4°, n = 3). Overall, these supplementary CAG data match well with the expected changes in wettability associated with the various modifications of surface functionalities. Hemocompatibility. Antithrombogenicity was next assessed in a real-time manner using a perfusion chamber (Figure 3) and whole human blood−labeled with cell-permeant 3,3′dihexyloxacarbocyanine (DiOC6) fluorescent dye25dispensed for 5 min at a controlled (high) shear rate of 1000 s−1.21 These conditions of hemodynamics provide an environment favorable for platelet accumulation/thrombus growth26 and, hence, appropriate to assess the (short-term) antithrombogenic properties of our MEG-OH exogenous coating. In practice, our experimental setup allows to record videos, from which it is possible to extract frames that can be later computed to access surface coverage due to platelet adhesion, aggregation and thrombus formation.21 As a positive control, in order to test our experimental protocol, we first exposed to blood BPA-PC precoated with type I collagen, a protein exposed on the injured vessel wall that interacts with platelets during hemostasis.27 As can be seen in Figures 4a−b, this control experiment was successful since thrombosis on these collagen surfaces was quite pronounced with a surface coverage calculated to be ∼8.2 ± 2.3% (n = 4). Bare BPA-PC exposed to labeled blood over a course of 5 min also triggered significant thrombus growth (∼5.7 ± 3.6% surface coverage, n = 8 − Figure 4a), as can be seen in Figure 4c. In stark contrast, the MEG-OH coating evidently displayed otherwise more remarkable antithrombogenic properties (Figure 4d), with a barely quantifiable surface coverage of ∼0.1 ± 0.1% (n = 8; Figure 4a), most frames actually showing blank areas (Supporting Information Figure SI-3). Also, in view of the pictures scale (10 μm) and platelets size (∼3 μm), the few observable low-sized microaggregates (marked by arrows) appeared to only encompass a limited number of platelets (Figure 4d and Figure SI-3). This represents for the MEG-OH surface a substantial inhibition of platelet adhesion, aggregation, and thrombus formation of over 97% compared to bare, nonmodified BPA-PC (Figure 4a). This result is quite remarkable considering (1) that blood was not given nor did require any additional anticoagulant treatment to prevent clotting (besides its standard collection and storage in heparinized Vacutainers) despite the conditions of high shear these in vitro blood experiments were performed under and (2)
Figure 3. (top) Experimental setup used to record real-time platelet adhesion, aggregation, and thrombus formation. The perfusion chamber is located at the center of the picture. The microscope camera is situated above. Also shown in the background is the syringepump used to drive blood in. (bottom) Close-up view of the perfusion chamber showing blood in- and outlets.
the structural simplicity of the MEG-TFA surface modifier with which the ultrathin monoethylene glycol MEG-OH coating was prepared.
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CONCLUSION In summary, we have reported herein the excellent antithrombogenic properties of an ultrathin monoethylene glycol (MEG-OH) silane coating deposited on the surface of polycarbonate, an increasingly employed biomedical plastic polymer. The various straightforward steps of surface modification were successfully characterized with X-ray photoelectron spectroscopy supplemented by contact angle goniometry. Antithrombogenicity was assessed in real-time using a perfusion chamber and fluorescently labeled whole human blood dispensed for 5 min at a controlled shear rate of 1000 s−1. A remarkable result was the observation that platelet adhesion, aggregation, and thrombus formation on the MEG-OH coating was greatly inhibited (>97% decrease in surface coverage) compared to the bare, nonderivatized polycarbonate substrate and, most importantly, nearly nonexistent. These results are quite extraordinary considering (1) that blood was submitted to 3220
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ASSOCIATED CONTENT
S Supporting Information *
Replicate antithrombogenicity pictures for all various surfaces. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (M.T.). Author Contributions
K.F. prepared and characterized the various surfaces; C.B. designed and synthesized MEG-TFA surface modifier; S.S. pioneered the original MEG-OH silane chemistry on quartz that led to the work on plastic polymers and also provided technical assistance to K.F., when needed; A.Re. performed the hemocompatibility experiments; A.Ro. provided fresh human blood samples; K.F., C.B., and S.S. analyzed XPS and CAG data; A.Re. and K.F. analyzed the blood experiment data; M.T., H.N., and A.Ro. conceived the study; C.B. wrote the article, which all coauthors edited and approved. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors are grateful to the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canadian Institutes of Health Research (CIHR), and St. Michael’s Hospital (Toronto, Ontario, Canada) for funding this research. S.S. and K.F. also extend their gratitude respectively to the Ontario Graduate Scholarship (OGS) program and the Institute of Biomaterials & Biomedical Engineering (IBBME − University of Toronto) for financial support. The authors also thank Drs. P. M. Brodersen and R. N. S. Sodhi from Surface Interface Ontario (Toronto, Canada) for XPS analysis as well as Prof. C. D. Mazer (Keenan Research Centre − St. Michael’s Hospital & Department of Anesthesia − University of Toronto, Toronto, Ontario, Canada) for stimulating discussions. Special acknowledgments are directed to Lucy Lin for assisting during surface preparation and characterization.
Figure 4. (a) Indicative percentage of surface coverage due to platelet adhesion, aggregation, and thrombus formation on (left to right) collagen-coated, bare, and MEG-OH modified BPA-PC substrates. (b−d) A selection of representative video frames showing platelet adhesion, aggregation, and thrombus formation (or lack of) on collagen-coated, bare, and MEG-OH modified BPA-PC substrates (32× magnification) after 5 min exposure to whole human blood at a shear rate of 1000 s−1. Replicate pictures for all various surfaces can be found in the Supporting Information.
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high shear conditions but did not require (nor was given) any additional anticoagulant treatment to prevent clotting during these in vitro experiments (besides its standard collection and storage in heparinized Vacutainers) and (2) the structural simplicity and ease of preparation of the MEG-OH coating. Several other types of plastic polymerse.g. poly(ethylene terephthalate), poly(vinyl chloride), polyacrylates, and polyurethanes, to name a feware currently being investigated for their ability to be derivatized with MEG-OH surface chemistry. The antithrombogenic properties of the resulting coatings against whole human blood are also being assessed. Most of these plastics will/should not trigger health-related concerns associated with the leaching of toxic chemicals, such as BPA in the case of polycarbonate. Finally, provided it can withstand the conditions used to sterilize biomedical equipment and longterm exposure to blood (under investigation), this unique MEG-OH ultrathin silane surface chemistry could very well revolutionize the world of antithrombogenic coatings.
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