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Fabrication of Semiordered Nanopatterned Diamond-like Carbon and Titania Films for Blood Contacting Applications Deepika Nandakumar, Avi Bendavid, Philip J. Martin, Kenneth D. Harris, Andrew Ruys, and Megan S. Lord ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b11614 • Publication Date (Web): 29 Feb 2016 Downloaded from http://pubs.acs.org on March 4, 2016
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ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Fabrication of Semiordered Nanopatterned Diamond-like Carbon and Titania Films for Blood Contacting Applications Deepika Nandakumar1, Avi Bendavid2, Philip J. Martin2, Kenneth D. Harris3, Andrew J. Ruys1 and Megan S Lord4* 1
Biomedical Engineering, School of AMME, University of Sydney, Sydney, NSW 2007, Australia. 2 Material Science and Engineering, CSIRO, Lindfield, NSW 2070, Australia. 3 National Institute for Nanotechnology, National Research Council, Edmonton, Alberta, Canada. 4 Graduate School of Biomedical Engineering, University of New South Wales, Sydney, NSW 2052, Australia. * Address correspondence to: Megan Lord, phone: +61 2 9385 3910; fax: +61 2 9663 2108; email: [email protected] Abstract Biomaterials with the ability to interface with, but not activate, blood components are essential for a multitude of medical devices. Diamond-like carbon (DLC) and titania (TiO2) have shown promise for these applications, however both support platelet adhesion and activation. This study explored the fabrication of nanostructured DLC and TiO2 thin film coatings using a block co-polymer deposition technique that produced semiordered nanopatterns with low surface roughness (5-8 nm Rrms). These surfaces supported fibrinogen and plasma protein adsorption that predominantly adsorbed between the nanofeatures and reduced the overall surface roughness. The conformation of the adsorbed fibrinogen was altered on the nanopatterned surfaces as compared with the planar surfaces to reveal higher levels of the platelet binding region. Planar DLC and TiO2 coatings supported less platelet adhesion than nanopatterned DLC and TiO2. However, platelets on the nanopatterned DLC coatings were less spread indicating a lower level of platelet activation on the nanostructures DLC coatings compared with the planar DLC coatings. These data indicated that nanostructured DLC coatings may find application in blood contacting medical devices in the future. Keywords: diamond-like carbon, titania, fibrinogen, platelet adhesion, protein adsorption
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1. Introduction The development of next generation biomaterials capable of interfacing with blood without supporting thrombus formation is critical for blood contacting devices such as catheters, blood vessel grafts, vascular stents, artificial heart valves, ventricular assist devices, circulatory support devices and various extracorporeal tubings. These devices typically use metals, such as titanium alloys, pyrolytic carbon, chromium cobalt alloy, nitinol, stainless steel and polymers such as enhanced polytetrafluoroethylene, polyethersulfone, polyethylene oxide, polyethylene glycol, polyethylene, polyvinylchloride, polyurethanes, polysiloxane, polypropylene, DacronTM, and PEBAXTM 1-5. In order to improve the blood compatibility of these surfaces, approaches such as modifying the surface chemistry, or the introduction of nanoscale topographies have been explored6-9. Inorganic coatings including diamond-like carbon (DLC) and titania (TiO2) have gained popularity for their use as effective surface modifiers on implantable devices because of their excellent cytocompatibility, mechanical properties and tribological properties 10. DLC describes a class of materials that are metastable forms of amorphous carbon containing both graphite-like (sp2) and diamond-like (sp3) bonds 11. DLC films have been used to coat materials such as titanium and nitinol, because of their hemocompatible, non-cytotoxic and anti-thrombogenic properties, and have been proven to be wear resistant, chemically inert and low friction, reducing the surface energy of the underlying biomaterial 12. Titanium and its alloys are often used due to the excellent corrosion resistance of the native oxide layer formed on the bulk material13-15. The native oxide layer primarily consists of amorphous TiO2 16-19 that provides a protective and highly stable passive film. In a blood environment, the surface chemistry, wettability, and topography influence complex processes that dictate the adsorption of proteins, their binding affinity, conformation and spatial distribution20. Altering the nanotopography of surfaces changes both the surface energy and wettability, and as such can greatly influence the way in which proteins and cells interact with the surface without compromising on the mechanical properties of the material. Nanofeature spacing, order and height influence cellular adhesion9, 21-24. Techniques such as electron beam lithography (e-beam), chemical etching, glancing angle deposition, and selfassembly nanopatterning by polymer de-mixing or block co-polymer phase segregation have gained considerable attention in recent years for the fabrication of surfaces with nanotopography 25. E-beam lithography is an excellent technique that is able to produce high resolution, ordered arrays of nanofeatures, however, it is a direct-write process, which is time consuming, expensive and limited to the fabrication of nanotopographies on small surface areas. The resolution of nanoparticles is also limited by the wavelength of the light source used for exposure of the coated resist at the wafer 20, 25, and nanofeatures with dimensions in the sub-20 nm range are still being explored 26-27. As such, alternative fabrication processes are being developed as a means to produce low-cost, highly-reproducible nanotopographies 25 . Amongst all known nanopatterning techniques, the directed self-assembly of block copolymers offer several advantages over the nanolithographic techniques including 2 ACS Paragon Plus Environment
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precision of nanoscale patterning, ultrafine line edge roughness, dense packing of nanodomains, low cost of processing, and large area scalable nanopatterning 26. The block copolymer method is able to reproducibly produce large quantities of pseudo-identical substrates with relatively large surface areas. Whilst many studies have analyzed the potential of DLC and TiO2 as blood contacting biomaterials 22, 28-30, few studies have investigated the potential of these materials with surface topography 9, 24, 31 which has the potential to alter both protein adsorption and cell adhesion. This study aimed to develop TiO2 and two forms of DLC-coating, amorphous carbon (a-C:H) and unhydrogenated, tetrahedrally-coordinated amorphous carbon (ta-C), with semiordered surface nanotopographies and investigate their effect on serum protein adsorption and platelet adhesion.
2. Materials and Methods Unless specified otherwise, all chemicals were purchased from Sigma-Aldrich (Castle Hill, Australia). 2.1 Fabrication of test surfaces 2.1.1. Surface preparation Ultra-flat silicon wafers (100) (Silicon Wafers Enterprises LLC, CA, USA) with resistivity 0.008-0.020 Ω.cm and thickness of 0.5-0.55 mm, were cut into 15 mm diameter discs with 0.5 mm tolerance to comfortably fit into 12-well tissue culture plates. The surfaces were pretreated ultrasonically with acetone, followed by ethanol, for 5 min each to clean the surfaces prior to modification. 2.1.2. Surface nanopatterning Silicon wafers were patterned with gold particles using a block copolymer deposition technique based on templating from polystyrene-poly(4-vinylpyridine) (PS-P4VP). Silicon wafers were cleaned using the RCA method 32 before being spin coated with a solution of 1% PS-P4VP (12k-3.2k) diblock copolymers in toluene for 20 s at 3800 rpm. The diblock copolymer films were then soaked in a solution containing gold salts (1.2% HClaq v/v with 200 mM KAuCl4) for 3 h to allow the metal ions to bind to the P4VP phase. The films were then etched with oxygen plasma for 30 s to remove the polymer film and reduce the Au ions to form a pseudo-regular Au nanostructure pattern on the silicon surface before drying with N2 33-34. For the purposes of this study, the Au-nano structured surfaces will be denoted the “nanopatterned surfaces”. 2.1.3. Surface coating Planar and nanopatterned surfaces were coated with either hydrogenated amorphous diamond like carbon (a-C:H), tetrahedral amorphous carbon (ta-C) or titania (TiO2) thin films, and will be termed as “planar a-C:H”, “planar ta-C” and “planar TiO2” and “nanopattened a-C:H”, “nanopattened ta-C and “nanopattened TiO2”. The results from this study will be compared with either planar silicon surfaces, denoted as “non-coated surfaces” or Au-deposited silicon surfaces, denoted as “nanopatterned non-coated surfaces”.
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2.1.3.1 a-C:H deposition Commercially-available a-C:H was coated onto the specimens using RF plasma enhanced chemical vapor deposition to synthesize quality coatings amorphous coatings at low temperatures 35. The deposition system consisted of a plasma reactor equipped with rotary and turbo-molecular pumps with controlled gas supply and pumping system, as previously described 36. A base pressure of 10-3 Pa was attained in the chamber prior to deposition. The DLC films were deposited onto silicon wafers, both planar and nanopatterned. Acetylene, C2H2 and argon, were used as process gases. The gases were introduced into the system through a gas distributor using mass flow controllers. The total pressure was set independently of the gas flow by adjusting a throttle valve. Prior to deposition, the substrates were sputter cleaned in-situ for 15 s in argon plasma operated at 3.3 Pa with the argon flow rate set at 10 sccm at 200 W RF power. The flow rate of C2H2 was kept constant at 60 sccm. The deposition pressure was set at 6.6 Pa at an RF power of 200 W for 10 s. 2.1.3.2 ta-C deposition Hydrogen-free ta-C was deposited using a physical vapor deposition technique called the filtered cathodic vacuum arc, described and characterized in detail elsewhere 37-38. The arc source was operated at a DC arc current of 60 A, producing a positive carbon ion current of 200 mA (measured at the exit of the arc source) with a shutter of 140 mm in diameter. The cathode used for producing the arc was high purity graphite (99.9%). A DC substrate bias voltage of −100 V was applied to the substrate. The deposition was performed at room temperature and the deposition rate was 50 nm/min. 2.1.3.3. TiO2 deposition Crystalline, anatase TiO2 was deposited using a physical vapor deposition technique called the filtered arc cathodic vacuum arc deposition system, described and characterized in detail elsewhere 39. The thin films were deposited from the plasma discharged from the TiO2 electrode (cathode) of the cathodic arc to the substrate. This occurs in vacuum at a low voltage, high current setting. The base pressure of the system was reduced to 2×10– 4 Pa prior to coating. The sample substrates were subjected to the following deposition conditions: 120 A DC arc current; 12 sccm O2 gas flow rate; 0.35 Pa chamber pressure; 200 mA measured titanium ion arc beam current with an area of 8 cm2 biased at -100 V at the substrate position, -50 V substrate bias and a deposition time of 15 s. The deposition process was conducted at room temperature as the intrinsic energy of the depositing metal is sufficiently high to facilitate self-densification of the TiO2 film on the substrate surface. 2.2. Surface Characterization The topographical characteristics of the sterilized nanopatterned and planar surfaces were visualized using scanning electron microscopy (SEM, Zeiss Ultra Plus, Netherlands). The samples were attached to an untilted SEM stage and were imaged under ultra-high vacuum (10-5 Pa) conditions at an accelerating voltage of 10 kV using an InLens detector. Subsections of each sample were visualized using a Zeiss Auriga focused-ion-beam SEM (FIB-SEM) (Zeiss, Netherlands) in ultra-high vacuum with accelerating voltage of 10 kV. High density positively charged gallium ions were bombarded onto the substrate to etch away material and form a cross section of the surface. The etching rate and current were chosen to minimize redeposition of ions. Transmission signals in the form of reflected secondary ions from each 4 ACS Paragon Plus Environment
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point were collated to generate the images representing the depth of deposited thin films on the gold immobilized silicon wafers. The samples were imaged at 53.8° and were corrected for the tilt before calibrating for thickness of the deposited layer. A scan area of 2 µm2 was measured at high beam currents of 50 pA, 30 kV, to mill through the surface. The overall nanoroughness of samples was analyzed by atomic force microscopy (AFM) in air using a Multimode NanoScope IIIa AFM (Dimension 3100, Digital Instruments Inc.) in low voltage tapping mode at 296 K with a tip diameter of 10 nm, scan rate of 1 Hz and scan area of 0.5 µm2 or 1 µm2. Si3N4 cantilevers were used and the force and resonant tip frequency parameters were adjusted continually to obtain line profile and 3-dimensional images. Five spots were scanned for each sample and the surface root mean square roughness (Rrms) was calculated using ∑(Zi−Zaverage)/N; where Zaverage is the average of the Z height values within a given area, Zi is the individual Z-value, and N is the number of pixels within the given area. Image processing analysis was performed using Nanoscope off-line software (Veeco Corp. Santa Barbara, USA). 2.3. Protein adsorption and conformation All protein solutions were diluted in Dulbecco’s phosphate buffered saline (DPBS) at pH 7.4, unless stated otherwise. 2.3.1 AFM imaging of adsorbed fibrinogen and plasma Planar and nanopatterned substrates, in triplicate, were placed in tissue culture well plates precoated with 0.1% (w/v, diluted in DPBS) bovine serum albumin (BSA). The substrates were then incubated at 37 ºC, with either 50 µg/ml fibrinogen (from human plasma, AbCam, Australia) or diluted plasma (diluted 1:9 in DPBS). After an hour of incubation, the samples were washed in DPBS and were imaged in liquid mode using a Catalyst AFM (Bruker Bioscope Catalyst, Australia) in low voltage tapping mode at 296 K with a tip diameter of 2 nm, scan rate of 0.3 Hz and scan area of 1µm2. Si3N4 cantilevers were used and the force and resonant tip frequency parameters were adjusted continually to obtain the 3 dimensional images. Image processing and surface topography analysis was performed using Nanoscope off-line software (Bruker, SA, Australia). 2.3.2. Availability of the platelet binding domain on adsorbed fibrinogen Planar and nanopatterned substrates, in triplicate, were placed in 0.1% (w/v) BSA pre-coated well plates and incubated for 1 h at 37 ºC in a solution containing human fibrinogen (50 µg/ml) diluted in DPBS. After washing with DPBS, the samples were blocked with 0.1% (w/v) BSA for 1 h at 37ºC and washed again with DPBS with 0.01% (v/v) Tween-20 before incubating at 37ºC with mouse monoclonal anti-human fibrinogen antibody (5µg/ml, clone 2C2-G7, Cederlane, USA), diluted in 0.1% (w/v) BSA for 1 h at 37 ºC. This antibody binds to, or sterically hinders access to, the regions of fibrinogen important for platelet adhesion and activation. Wells were then washed twice in DPBS and incubated at room temperature with Alexa Fluor 488 tagged anti-mouse IgG secondary antibody for 1 h at 37 ºC. The samples were then washed twice with DPBS containing 0.5% (v/v) Tween 20, prior to visualizing at 500× magnification using a fluorescence microscope (Axioskop Mot Mat 2, Zeiss, Australia). The availability of the platelet binding domain on surface adsorbed fibrinogen was estimated from the total intensity measured from the number of positive
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pixels in each image using the pixelated computer program, NIH ImageJ (version 1.44p, NIH). 2.4. Platelet Adhesion 2.4.1. Platelet isolation from human blood Human platelets were harvested from healthy adult donors under ethics approval from the University of New South Wales. Blood was collected in 3.2% tri-sodium citrate anticoagulant treated vacutainers. Platelet rich plasma (PRP) was isolated by centrifugation of the blood at 350 g for 20 min at room temperature. The PRP supernatant was then centrifuged for a further 10 min at 350 g to remove any residual red blood cells. The PRP was then transferred to clean centrifuge tubes and centrifuged for 10 min at 1200 g causing the platelets to pellet. The platelet pellet was resuspended in pre-warmed Tyrode’s buffer (1.8 mM CaCl2, 1 mM MgCl2, 2.7 mM KCl, 136.9 mM NaCl, 0.4 mM NaH2PO4, 11.9 mM NaHCO3 and 5.6 mM Dglucose) containing 0.1 U/ml apyrase and the suspension was centrifuged at 1200 g for a further 10 min. The platelets were resuspended in Tyrodes buffer containing 0.1 U/ml apyrase to a concentration of 107 platelets/ml. 2.4.2. Platelet adhesion assay using fluorescence microscopy Surfaces, including tissue culture polystyrene as a control surface, were exposed to platelets (107 platelets/ml in Tyrode’s buffer containing 0.1 U/ml apyrase) for 1 h at 37°C. Unbound platelets were removed by washing with DPBS following the removal of the platelet suspension from wells. The platelets were fixed with 4% (v/v) paraformaldehyde and 1% (w/v) sucrose in DPBS for 15 min at room temperature, followed by permeabilization for 5 min at 4 ºC with 0.3 M sucrose, 0.05 M NaCl, 0.003 M MgCl2, 0.2 M HEPES, 0.5 % (w/v) Triton X-100, at a pH of 7.2. Wells were then blocked with 1% (w/v) BSA/ Tris-buffered saline (TBS) solution for 5 min at 37 ºC. Rhodamine-phallodin (1:100 dilution in 1% w/v BSA/TBS, Invitrogen Corporation, Australia) was used to stain the actin filaments of the permeabilized platelets for 1 h at 37ºC. Following washing with DPBS containing 0.5% (v/v) Tween 20, the samples imaged using a fluorescence microscope (Axioskop Mot Mat 2, Zeiss, Australia). Microscope images were quantitatively analyzed using a pixelated computer program (NIH ImageJ, version 1.44p, NIH) to determine the total number of adhered platelets. 2.5. Statistical Significance Student’s t-test (for two samples, assuming equal variance) was used to compare statistical significance. Results of p < 0.05 were considered significant and the results were expressed as mean ± standard deviation.
3. Results 3.1. Surface characterisation Silicon wafers were deposited with the copolymer, and etching resulted in a pattern of gold nanostructures on the surface replicating the pattern of the phase-separated block copolymer features. This was imaged using SEM to visualize the overall surface nanotopography of the substrate before coating with either a C:H, ta-C or TiO2 layer (Fig. 1A). The pseudo-regular 6 ACS Paragon Plus Environment
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spacing of the gold nanoparticles was determined to be 55.40 ± 14.70 nm over the entire surface. Coatings of either TiO2, a-C:H or ta-C thin films formed a dome-like coverage over the gold particles to produce stochastic nanotopographies, which followed the morphology of the underlying substrate (Fig. 1 B-D).
Figure 1. SEM images of (A and inset) non-coated silicon wafers with surface nanopatterning of gold nanoparticles, followed by coating with (B) TiO2, (C) a-C:H or (D) taC. Scale bars represent 400 nm and 50 nm (panel A inset). The shape and pitch of adjacent gold nanostructures on the non-coated surfaces appeared pseudo-regular with 24.20 ± 2.60 nm in height (z-range). The individual nanostructures varied in their dimensions with an average diameter of 25.40 ± 2.76 nm. Some nanostructures on the TiO2 and a-C:H surfaces coalesced, thus decreasing the pitch between adjacent nanostructures and forming irregular-shaped features. The coating thickness of the deposited films on the non-patterned region of the nanopatterned surfaces was determined using SEMFIB and found to be 32.60 ± 4.45 nm, 37.50 ± 2.95 nm and 34.10 ± 3.50 nm for TiO2, a-C:H, and ta-C deposited layers on nanopatterned surfaces, respectively (Fig. 2). The coating thickness on the semiordered nanofeatures was also determined using SEM-FIB and found to be 18.6 ± 2.4 nm, 25.4 ± 2.5 nm and 24.1 ± 2.8 nm for TiO2, a-C:H, and ta-C deposited layers on nanopatterned surfaces, respectively (Fig. 2), indicating that the TiO2 surface was significantly (p
Article
Fabrication of Semiordered Nanopatterned Diamond-like Carbon and Titania Films for Blood Contacting Applications Deepika Nandakumar, Avi Bendavid, Philip J. Martin, Kenneth D. Harris, Andrew Ruys, and Megan S. Lord ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b11614 • Publication Date (Web): 29 Feb 2016 Downloaded from http://pubs.acs.org on March 4, 2016
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Fabrication of Semiordered Nanopatterned Diamond-like Carbon and Titania Films for Blood Contacting Applications Deepika Nandakumar1, Avi Bendavid2, Philip J. Martin2, Kenneth D. Harris3, Andrew J. Ruys1 and Megan S Lord4* 1
Biomedical Engineering, School of AMME, University of Sydney, Sydney, NSW 2007, Australia. 2 Material Science and Engineering, CSIRO, Lindfield, NSW 2070, Australia. 3 National Institute for Nanotechnology, National Research Council, Edmonton, Alberta, Canada. 4 Graduate School of Biomedical Engineering, University of New South Wales, Sydney, NSW 2052, Australia. * Address correspondence to: Megan Lord, phone: +61 2 9385 3910; fax: +61 2 9663 2108; email: [email protected] Abstract Biomaterials with the ability to interface with, but not activate, blood components are essential for a multitude of medical devices. Diamond-like carbon (DLC) and titania (TiO2) have shown promise for these applications, however both support platelet adhesion and activation. This study explored the fabrication of nanostructured DLC and TiO2 thin film coatings using a block co-polymer deposition technique that produced semiordered nanopatterns with low surface roughness (5-8 nm Rrms). These surfaces supported fibrinogen and plasma protein adsorption that predominantly adsorbed between the nanofeatures and reduced the overall surface roughness. The conformation of the adsorbed fibrinogen was altered on the nanopatterned surfaces as compared with the planar surfaces to reveal higher levels of the platelet binding region. Planar DLC and TiO2 coatings supported less platelet adhesion than nanopatterned DLC and TiO2. However, platelets on the nanopatterned DLC coatings were less spread indicating a lower level of platelet activation on the nanostructures DLC coatings compared with the planar DLC coatings. These data indicated that nanostructured DLC coatings may find application in blood contacting medical devices in the future. Keywords: diamond-like carbon, titania, fibrinogen, platelet adhesion, protein adsorption
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1. Introduction The development of next generation biomaterials capable of interfacing with blood without supporting thrombus formation is critical for blood contacting devices such as catheters, blood vessel grafts, vascular stents, artificial heart valves, ventricular assist devices, circulatory support devices and various extracorporeal tubings. These devices typically use metals, such as titanium alloys, pyrolytic carbon, chromium cobalt alloy, nitinol, stainless steel and polymers such as enhanced polytetrafluoroethylene, polyethersulfone, polyethylene oxide, polyethylene glycol, polyethylene, polyvinylchloride, polyurethanes, polysiloxane, polypropylene, DacronTM, and PEBAXTM 1-5. In order to improve the blood compatibility of these surfaces, approaches such as modifying the surface chemistry, or the introduction of nanoscale topographies have been explored6-9. Inorganic coatings including diamond-like carbon (DLC) and titania (TiO2) have gained popularity for their use as effective surface modifiers on implantable devices because of their excellent cytocompatibility, mechanical properties and tribological properties 10. DLC describes a class of materials that are metastable forms of amorphous carbon containing both graphite-like (sp2) and diamond-like (sp3) bonds 11. DLC films have been used to coat materials such as titanium and nitinol, because of their hemocompatible, non-cytotoxic and anti-thrombogenic properties, and have been proven to be wear resistant, chemically inert and low friction, reducing the surface energy of the underlying biomaterial 12. Titanium and its alloys are often used due to the excellent corrosion resistance of the native oxide layer formed on the bulk material13-15. The native oxide layer primarily consists of amorphous TiO2 16-19 that provides a protective and highly stable passive film. In a blood environment, the surface chemistry, wettability, and topography influence complex processes that dictate the adsorption of proteins, their binding affinity, conformation and spatial distribution20. Altering the nanotopography of surfaces changes both the surface energy and wettability, and as such can greatly influence the way in which proteins and cells interact with the surface without compromising on the mechanical properties of the material. Nanofeature spacing, order and height influence cellular adhesion9, 21-24. Techniques such as electron beam lithography (e-beam), chemical etching, glancing angle deposition, and selfassembly nanopatterning by polymer de-mixing or block co-polymer phase segregation have gained considerable attention in recent years for the fabrication of surfaces with nanotopography 25. E-beam lithography is an excellent technique that is able to produce high resolution, ordered arrays of nanofeatures, however, it is a direct-write process, which is time consuming, expensive and limited to the fabrication of nanotopographies on small surface areas. The resolution of nanoparticles is also limited by the wavelength of the light source used for exposure of the coated resist at the wafer 20, 25, and nanofeatures with dimensions in the sub-20 nm range are still being explored 26-27. As such, alternative fabrication processes are being developed as a means to produce low-cost, highly-reproducible nanotopographies 25 . Amongst all known nanopatterning techniques, the directed self-assembly of block copolymers offer several advantages over the nanolithographic techniques including 2 ACS Paragon Plus Environment
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precision of nanoscale patterning, ultrafine line edge roughness, dense packing of nanodomains, low cost of processing, and large area scalable nanopatterning 26. The block copolymer method is able to reproducibly produce large quantities of pseudo-identical substrates with relatively large surface areas. Whilst many studies have analyzed the potential of DLC and TiO2 as blood contacting biomaterials 22, 28-30, few studies have investigated the potential of these materials with surface topography 9, 24, 31 which has the potential to alter both protein adsorption and cell adhesion. This study aimed to develop TiO2 and two forms of DLC-coating, amorphous carbon (a-C:H) and unhydrogenated, tetrahedrally-coordinated amorphous carbon (ta-C), with semiordered surface nanotopographies and investigate their effect on serum protein adsorption and platelet adhesion.
2. Materials and Methods Unless specified otherwise, all chemicals were purchased from Sigma-Aldrich (Castle Hill, Australia). 2.1 Fabrication of test surfaces 2.1.1. Surface preparation Ultra-flat silicon wafers (100) (Silicon Wafers Enterprises LLC, CA, USA) with resistivity 0.008-0.020 Ω.cm and thickness of 0.5-0.55 mm, were cut into 15 mm diameter discs with 0.5 mm tolerance to comfortably fit into 12-well tissue culture plates. The surfaces were pretreated ultrasonically with acetone, followed by ethanol, for 5 min each to clean the surfaces prior to modification. 2.1.2. Surface nanopatterning Silicon wafers were patterned with gold particles using a block copolymer deposition technique based on templating from polystyrene-poly(4-vinylpyridine) (PS-P4VP). Silicon wafers were cleaned using the RCA method 32 before being spin coated with a solution of 1% PS-P4VP (12k-3.2k) diblock copolymers in toluene for 20 s at 3800 rpm. The diblock copolymer films were then soaked in a solution containing gold salts (1.2% HClaq v/v with 200 mM KAuCl4) for 3 h to allow the metal ions to bind to the P4VP phase. The films were then etched with oxygen plasma for 30 s to remove the polymer film and reduce the Au ions to form a pseudo-regular Au nanostructure pattern on the silicon surface before drying with N2 33-34. For the purposes of this study, the Au-nano structured surfaces will be denoted the “nanopatterned surfaces”. 2.1.3. Surface coating Planar and nanopatterned surfaces were coated with either hydrogenated amorphous diamond like carbon (a-C:H), tetrahedral amorphous carbon (ta-C) or titania (TiO2) thin films, and will be termed as “planar a-C:H”, “planar ta-C” and “planar TiO2” and “nanopattened a-C:H”, “nanopattened ta-C and “nanopattened TiO2”. The results from this study will be compared with either planar silicon surfaces, denoted as “non-coated surfaces” or Au-deposited silicon surfaces, denoted as “nanopatterned non-coated surfaces”.
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2.1.3.1 a-C:H deposition Commercially-available a-C:H was coated onto the specimens using RF plasma enhanced chemical vapor deposition to synthesize quality coatings amorphous coatings at low temperatures 35. The deposition system consisted of a plasma reactor equipped with rotary and turbo-molecular pumps with controlled gas supply and pumping system, as previously described 36. A base pressure of 10-3 Pa was attained in the chamber prior to deposition. The DLC films were deposited onto silicon wafers, both planar and nanopatterned. Acetylene, C2H2 and argon, were used as process gases. The gases were introduced into the system through a gas distributor using mass flow controllers. The total pressure was set independently of the gas flow by adjusting a throttle valve. Prior to deposition, the substrates were sputter cleaned in-situ for 15 s in argon plasma operated at 3.3 Pa with the argon flow rate set at 10 sccm at 200 W RF power. The flow rate of C2H2 was kept constant at 60 sccm. The deposition pressure was set at 6.6 Pa at an RF power of 200 W for 10 s. 2.1.3.2 ta-C deposition Hydrogen-free ta-C was deposited using a physical vapor deposition technique called the filtered cathodic vacuum arc, described and characterized in detail elsewhere 37-38. The arc source was operated at a DC arc current of 60 A, producing a positive carbon ion current of 200 mA (measured at the exit of the arc source) with a shutter of 140 mm in diameter. The cathode used for producing the arc was high purity graphite (99.9%). A DC substrate bias voltage of −100 V was applied to the substrate. The deposition was performed at room temperature and the deposition rate was 50 nm/min. 2.1.3.3. TiO2 deposition Crystalline, anatase TiO2 was deposited using a physical vapor deposition technique called the filtered arc cathodic vacuum arc deposition system, described and characterized in detail elsewhere 39. The thin films were deposited from the plasma discharged from the TiO2 electrode (cathode) of the cathodic arc to the substrate. This occurs in vacuum at a low voltage, high current setting. The base pressure of the system was reduced to 2×10– 4 Pa prior to coating. The sample substrates were subjected to the following deposition conditions: 120 A DC arc current; 12 sccm O2 gas flow rate; 0.35 Pa chamber pressure; 200 mA measured titanium ion arc beam current with an area of 8 cm2 biased at -100 V at the substrate position, -50 V substrate bias and a deposition time of 15 s. The deposition process was conducted at room temperature as the intrinsic energy of the depositing metal is sufficiently high to facilitate self-densification of the TiO2 film on the substrate surface. 2.2. Surface Characterization The topographical characteristics of the sterilized nanopatterned and planar surfaces were visualized using scanning electron microscopy (SEM, Zeiss Ultra Plus, Netherlands). The samples were attached to an untilted SEM stage and were imaged under ultra-high vacuum (10-5 Pa) conditions at an accelerating voltage of 10 kV using an InLens detector. Subsections of each sample were visualized using a Zeiss Auriga focused-ion-beam SEM (FIB-SEM) (Zeiss, Netherlands) in ultra-high vacuum with accelerating voltage of 10 kV. High density positively charged gallium ions were bombarded onto the substrate to etch away material and form a cross section of the surface. The etching rate and current were chosen to minimize redeposition of ions. Transmission signals in the form of reflected secondary ions from each 4 ACS Paragon Plus Environment
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point were collated to generate the images representing the depth of deposited thin films on the gold immobilized silicon wafers. The samples were imaged at 53.8° and were corrected for the tilt before calibrating for thickness of the deposited layer. A scan area of 2 µm2 was measured at high beam currents of 50 pA, 30 kV, to mill through the surface. The overall nanoroughness of samples was analyzed by atomic force microscopy (AFM) in air using a Multimode NanoScope IIIa AFM (Dimension 3100, Digital Instruments Inc.) in low voltage tapping mode at 296 K with a tip diameter of 10 nm, scan rate of 1 Hz and scan area of 0.5 µm2 or 1 µm2. Si3N4 cantilevers were used and the force and resonant tip frequency parameters were adjusted continually to obtain line profile and 3-dimensional images. Five spots were scanned for each sample and the surface root mean square roughness (Rrms) was calculated using ∑(Zi−Zaverage)/N; where Zaverage is the average of the Z height values within a given area, Zi is the individual Z-value, and N is the number of pixels within the given area. Image processing analysis was performed using Nanoscope off-line software (Veeco Corp. Santa Barbara, USA). 2.3. Protein adsorption and conformation All protein solutions were diluted in Dulbecco’s phosphate buffered saline (DPBS) at pH 7.4, unless stated otherwise. 2.3.1 AFM imaging of adsorbed fibrinogen and plasma Planar and nanopatterned substrates, in triplicate, were placed in tissue culture well plates precoated with 0.1% (w/v, diluted in DPBS) bovine serum albumin (BSA). The substrates were then incubated at 37 ºC, with either 50 µg/ml fibrinogen (from human plasma, AbCam, Australia) or diluted plasma (diluted 1:9 in DPBS). After an hour of incubation, the samples were washed in DPBS and were imaged in liquid mode using a Catalyst AFM (Bruker Bioscope Catalyst, Australia) in low voltage tapping mode at 296 K with a tip diameter of 2 nm, scan rate of 0.3 Hz and scan area of 1µm2. Si3N4 cantilevers were used and the force and resonant tip frequency parameters were adjusted continually to obtain the 3 dimensional images. Image processing and surface topography analysis was performed using Nanoscope off-line software (Bruker, SA, Australia). 2.3.2. Availability of the platelet binding domain on adsorbed fibrinogen Planar and nanopatterned substrates, in triplicate, were placed in 0.1% (w/v) BSA pre-coated well plates and incubated for 1 h at 37 ºC in a solution containing human fibrinogen (50 µg/ml) diluted in DPBS. After washing with DPBS, the samples were blocked with 0.1% (w/v) BSA for 1 h at 37ºC and washed again with DPBS with 0.01% (v/v) Tween-20 before incubating at 37ºC with mouse monoclonal anti-human fibrinogen antibody (5µg/ml, clone 2C2-G7, Cederlane, USA), diluted in 0.1% (w/v) BSA for 1 h at 37 ºC. This antibody binds to, or sterically hinders access to, the regions of fibrinogen important for platelet adhesion and activation. Wells were then washed twice in DPBS and incubated at room temperature with Alexa Fluor 488 tagged anti-mouse IgG secondary antibody for 1 h at 37 ºC. The samples were then washed twice with DPBS containing 0.5% (v/v) Tween 20, prior to visualizing at 500× magnification using a fluorescence microscope (Axioskop Mot Mat 2, Zeiss, Australia). The availability of the platelet binding domain on surface adsorbed fibrinogen was estimated from the total intensity measured from the number of positive
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pixels in each image using the pixelated computer program, NIH ImageJ (version 1.44p, NIH). 2.4. Platelet Adhesion 2.4.1. Platelet isolation from human blood Human platelets were harvested from healthy adult donors under ethics approval from the University of New South Wales. Blood was collected in 3.2% tri-sodium citrate anticoagulant treated vacutainers. Platelet rich plasma (PRP) was isolated by centrifugation of the blood at 350 g for 20 min at room temperature. The PRP supernatant was then centrifuged for a further 10 min at 350 g to remove any residual red blood cells. The PRP was then transferred to clean centrifuge tubes and centrifuged for 10 min at 1200 g causing the platelets to pellet. The platelet pellet was resuspended in pre-warmed Tyrode’s buffer (1.8 mM CaCl2, 1 mM MgCl2, 2.7 mM KCl, 136.9 mM NaCl, 0.4 mM NaH2PO4, 11.9 mM NaHCO3 and 5.6 mM Dglucose) containing 0.1 U/ml apyrase and the suspension was centrifuged at 1200 g for a further 10 min. The platelets were resuspended in Tyrodes buffer containing 0.1 U/ml apyrase to a concentration of 107 platelets/ml. 2.4.2. Platelet adhesion assay using fluorescence microscopy Surfaces, including tissue culture polystyrene as a control surface, were exposed to platelets (107 platelets/ml in Tyrode’s buffer containing 0.1 U/ml apyrase) for 1 h at 37°C. Unbound platelets were removed by washing with DPBS following the removal of the platelet suspension from wells. The platelets were fixed with 4% (v/v) paraformaldehyde and 1% (w/v) sucrose in DPBS for 15 min at room temperature, followed by permeabilization for 5 min at 4 ºC with 0.3 M sucrose, 0.05 M NaCl, 0.003 M MgCl2, 0.2 M HEPES, 0.5 % (w/v) Triton X-100, at a pH of 7.2. Wells were then blocked with 1% (w/v) BSA/ Tris-buffered saline (TBS) solution for 5 min at 37 ºC. Rhodamine-phallodin (1:100 dilution in 1% w/v BSA/TBS, Invitrogen Corporation, Australia) was used to stain the actin filaments of the permeabilized platelets for 1 h at 37ºC. Following washing with DPBS containing 0.5% (v/v) Tween 20, the samples imaged using a fluorescence microscope (Axioskop Mot Mat 2, Zeiss, Australia). Microscope images were quantitatively analyzed using a pixelated computer program (NIH ImageJ, version 1.44p, NIH) to determine the total number of adhered platelets. 2.5. Statistical Significance Student’s t-test (for two samples, assuming equal variance) was used to compare statistical significance. Results of p < 0.05 were considered significant and the results were expressed as mean ± standard deviation.
3. Results 3.1. Surface characterisation Silicon wafers were deposited with the copolymer, and etching resulted in a pattern of gold nanostructures on the surface replicating the pattern of the phase-separated block copolymer features. This was imaged using SEM to visualize the overall surface nanotopography of the substrate before coating with either a C:H, ta-C or TiO2 layer (Fig. 1A). The pseudo-regular 6 ACS Paragon Plus Environment
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spacing of the gold nanoparticles was determined to be 55.40 ± 14.70 nm over the entire surface. Coatings of either TiO2, a-C:H or ta-C thin films formed a dome-like coverage over the gold particles to produce stochastic nanotopographies, which followed the morphology of the underlying substrate (Fig. 1 B-D).
Figure 1. SEM images of (A and inset) non-coated silicon wafers with surface nanopatterning of gold nanoparticles, followed by coating with (B) TiO2, (C) a-C:H or (D) taC. Scale bars represent 400 nm and 50 nm (panel A inset). The shape and pitch of adjacent gold nanostructures on the non-coated surfaces appeared pseudo-regular with 24.20 ± 2.60 nm in height (z-range). The individual nanostructures varied in their dimensions with an average diameter of 25.40 ± 2.76 nm. Some nanostructures on the TiO2 and a-C:H surfaces coalesced, thus decreasing the pitch between adjacent nanostructures and forming irregular-shaped features. The coating thickness of the deposited films on the non-patterned region of the nanopatterned surfaces was determined using SEMFIB and found to be 32.60 ± 4.45 nm, 37.50 ± 2.95 nm and 34.10 ± 3.50 nm for TiO2, a-C:H, and ta-C deposited layers on nanopatterned surfaces, respectively (Fig. 2). The coating thickness on the semiordered nanofeatures was also determined using SEM-FIB and found to be 18.6 ± 2.4 nm, 25.4 ± 2.5 nm and 24.1 ± 2.8 nm for TiO2, a-C:H, and ta-C deposited layers on nanopatterned surfaces, respectively (Fig. 2), indicating that the TiO2 surface was significantly (p
