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Development and Evaluation of a Nanometer-Scale Hemocompatible and Antithrombotic Coating Technology Platform for Commercial Intracranial Stents and Flow Diverters Anna Schumacher, Chad Gilmer, Keerthi Atluri, Joun Lee, Aju S Jugessur, Aliasger K. Salem, Ned B Bowden, Madhavan L. Raghavan, and David Hasan ACS Appl. Nano Mater., Just Accepted Manuscript • Publication Date (Web): 15 Nov 2017 Downloaded from http://pubs.acs.org on November 26, 2017

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ACS Applied Nano Materials

Development and Evaluation of a Nanometer-Scale Hemocompatible and Antithrombotic Coating Technology Platform for Commercial Intracranial Stents and Flow Diverters Anna L. Schumacher1‡, Chad M. Gilmer2‡, Keerthi Atluri3, Joun Lee4, Aju S. Jugessur5, Aliasger K. Salem3, Ned B. Bowden2, Madhavan L. Raghavan1, David M. Hasan6* 1

The University of Iowa Department of Biomedical Engineering, 2The University of Iowa

Department of Chemistry, 3The University of Iowa Department of Pharmaceutical Sciences and Experimental Therapeutics, 4The University of Iowa Department of Chemical and Biochemical Engineering, 5The University of Iowa Optical Science and Technology Center, 6The University of Iowa Department of Neurosurgery KEYWORDS: flow diverter, aneurysm, thrombomodulin, thrombosis, plasma-enhanced atomic layer deposition, anticoagulation, nanometer-scale coating

ACS Paragon Plus Environment

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ABSTRACT: An intracranial aneurysm is a local dilation of an artery in the cerebral circulation and can be endovascularly treated with two types of medical devices known as intracranial stents or flow diverters – both are metallic devices that help redirect blood from the diseased arterial segment; yet the placement of intracranial devices in the cerebral circulation mandates the adjunctive administration of dual anti-platelet pharmaceuticals to the patient to minimize thromboembolic events, despite being associated with increased patient risk. We present a new multilayer, nanometer-scale coating technology platform suitable for commercial intracranial flow diverters to minimize the use of dual anti-platelet therapy in the elective setting and expand the use of intracranial devices in the acute setting of ruptured intracranial aneurysms. A combination of qualitative and quantitative characterization techniques including scanning electron microscopy, ellipsometry, confocal microscopy, x-ray photoelectron spectroscopy, and focused ion beam milling coupled with scanning electron microscopy were used to assess the composition, uniformity, and thickness of each coating layer on commercially available flow diverting devices. Overall the coating was found to be relatively uniform, less than 50 nm thick, and conformal to device microwires. X-ray photoelectron spectroscopy data further indicates the developed nanoscale coating technology can be modified for use as a platform for the attachment of human recombinant thrombomodulin, a naturally occurring glycoprotein with antithrombotic functionality. The in-vitro thrombin generation capacity of commercial intracranial flow diverters coated with the technology was assessed using the Calibrated Automated Thrombogram Assay; further, platelet and fibrin deposition on coated commercial flow diverters was assessed ex-vivo via a primate arteriovenous shunt model. The in-vitro and ex-vivo test results suggest potential hemocompatible and antithrombotic properties.


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INTRODUCTION Thrombus, or blood clot, formation on commercial medical devices used to treat intracranial aneurysms is a significant clinical problem.1, 2 An intracranial aneurysm is a local dilation of an artery in the brain; while intracranial aneurysm formation, growth, and rupture are not well understood, a complex set of factors including inflammation, abnormal vascular wall remodeling, and hemodynamic-associated stress likely contribute to the disease.3, 4 Despite the uncertainty in its progression, intracranial aneurysm rupture is a catastrophic event and can lead to subarachnoid hemorrhage (SAH), brain damage, or death (mortality rate of 50%).5 The annual incidence of SAH in the United States is approximately 30,000 cases, a prevalence which has pushed practitioners to aggressively treat the disease using both surgical and endovascular techniques.3 In 2002 the international subarachnoid hemorrhage aneurysm trial (ISAT) found an endovascular treatment technique known as embolization coiling to be superior to microsurgical aneurysm clipping; this finding spurred the increased use of endovascular devices like intracranial flow diverters and stents.6, 7 However introducing intracranial stents or flow diverters into the cerebral circulation mandates the administration of dual anti-platelet therapy (DAPT) to the patient to prevent intra-device thrombosis or embolic complications that can cause stroke or death. Furthermore DAPT must be administered both pre- and post-device placement and is associated with increased patient risk because it lowers the ability of the body to respond to cuts or other injuries.8-10 Despite the strict administration of DAPT to patients treated with intracranial devices, thromboembolic complications that lead to brain injury or death are still significant; two large multicenter retrospective studies indicate that thromboembolic complications occur in 4.7% of intracranial aneurysm patients treated with the Pipeline™ flow diverting device and DAPT1, and in 7.1% of patients treated with DAPT and stent-supported coil embolization.2

Likewise,


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practitioners are increasingly using intracranial flow diverters in patients suffering from acute SAH, despite the fact that they are not FDA approved for use in this patient population.11-14 Therefore we sought to develop a hemocompatible and antithrombotic nanometer-scale coating platform suitable for both commercially available intracranial flow diverting devices and stents with the long term goal of decreasing the use of DAPT pharmaceuticals, or completely eliminating their use. Several research groups have recognized the revolutionizing impact surface-modified intracranial devices could have on aneurysm patients and have published promising results. Specifically, researchers have published using the Cordis Bx Velocity™ coronary stent with Hepacoat™ in the human cerebral circulation15, or else on the improved in-vitro thrombogenicity of heparin and human serum albumin surface coatings for the Acandis Acclino® stent-assisted coiling device.16 Likewise, medical device companies have recognized the significant patient-care and market impact a hemocompatible and antithrombotic technology holds. In 2015 Girdhar et al. published on a nanometer-scale coating developed explicitly for the intracranial Pipeline™ Flex flow diverting device called Shield Technology™ that may be an effective coating technology to mitigate thrombotic complications.17

A United Kingdom (UK) clinical trial assessing the

incidences of stroke and neurological adversities or death associated with the Shield Technology™ has been conducted and the results will be released soon.18 Despite this work, DAPT is still the standard of care for intracranial device placement and thromboembolic events will likely occur even with the next generation of intracranial devices. Intracranial devices are complex and present unique challenges to coating deposition in terms of device geometry, composition, and deployment mechanics – for instance, intracranial stents possess between 5-15% metal to blood vessel surface area coverage and act to promote


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longitudinal aneurysmal occlusion while serving as a rigid scaffold for embolization coils (Figure 1A).19 Flow diverting devices are a second type of intracranial device that possess approximately 30% metal to blood vessel surface area coverage, are stand-alone devices, and are used to facilitate longitudinal aneurysm occlusion in selected cases when intracranial stents are not appropriate (Figure 1B).19 A challenge of surface-modifying either intracranial device is that the composition varies among manufacturers. Commercial intracranial stents are generally composed of one type of continuous metal, but commercial flow diverters possess individual microwires of varying compositions that are woven to form a crossed, cylindrical pattern (Figure 1B). Because the woven wires must move independently to facilitate deployment in the brain, microwire surface functionalization is particularly challenging. Furthermore, because both types of commercial intracranial devices are often deployed in tortuous vasculature, it is important that any surface modifications not significantly alter the underlying device mechanics. Therefore a targeted effort was made to develop a nanometer-scale coating, conformal to commercial flow diverter microwires.


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Figure 1. Brightfield microscopy images at 4x of (A) a commercial intracranial stent-assisted coiling device and (B) a commercial intracranial flow diverting device. In (B) the flow diverter is composed of two different wires that are woven together. While the inner mesh geometries and size scales of (A) and (B) are different, it is important to note each device comes in several diameters and lengths to accommodate different sizes of cerebral arteries and aneurysms. We developed a nanometer-scale, multilayer coating platform that was attached in a conformal manner to commercial intracranial flow diverting devices. In particular, we developed this technology to serve as a platform for the attachment of recombinant glycoprotein human thrombomodulin (hTM). We chose hTM to conjugate to our coating platform since it is an integral membrane protein expressed in-vivo on the human endothelial cell surface and actively disrupts


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coagulation as a protein cofactor in the thrombin-catalyzed activation of protein C, though it lacks FDA approval.20 The primary goal of this work was to design and develop a nanometer-scale coating technology with hemocompatible and antithrombotic properties suitable for deposition on commercial intracranial flow diverters. Such a coating is needed because flow diverters have a documented propensity for thromboembolic complications in-vivo,1, 2 in addition to the increased patient risk associated with the DAPT standard of care.10 In particular, we fabricated a nanoscale coating technology platform suitable for deposition on commercial intracranial flow diverting devices. The hemocompatibility of this coating on commercial flow diverters was investigated by the calibrated automated thrombogram (CAT) assay, an in-vitro thrombin generation test, as well as platelet and fibrin deposition in an ex-vivo primate shunt model.

EXPERIMENTAL SECTION Materials. Trimethylaluminum, 3-Aminopropyl-triethoxysilane, 2,4,6-trichloro-1,3,5-triazine, human recombinant thrombomodulin, Dulbecco’s 1X PBS buffer, Alexa Fluor 488 5-TFP fluorophore, and all solvents were purchased from Sigma-Aldrich. All chemicals were used as received.

Plasma Enhanced-Atomic Layer Deposition (PE-ALD) of Al2O3. Commercial intracranial flow diverting devices were sonicated in acetone, isopropyl alcohol, methanol, and deionized water (4 min each). The devices were rinsed with acetone and dried with nitrogen gas (high purity semiconductor grade 5) at room temperature (10 min). In a class 100 (ISO 5) cleanroom environment, the aluminum oxide layer was deposited on the devices by plasma-enhanced atomic


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layer deposition (PE-ALD) using an OpAL instrument (Oxford Instruments). The precursors used in the deposition were trimethylaluminum (TMA) and oxygen plasma, with a chamber temperature of 200C and an exposure cycle number of 300 at a deposition rate of approximately 0.11 nm/cycle.

Silanization of the Al2O3 Layer. The silanization reaction conditions were adapted from Ploetz et al.21 Each intracranial device was sonicated in methanol, ethanol, DI H2O, and acetone, and dried with a flow of N2. Toluene (60 mL) was heated to 65°C in an Erlenmeyer flask and the intracranial devices were added to the flask. 3-Aminopropyl-triethoxysilane (1.8 mL, 7.7 mmol) was added to the flask and the reaction was stirred for 20 min. The reaction was then cooled to room temperature and the reaction mixture was decanted. The devices were washed with toluene and methanol (three times each) and dried with a flow of N2.

Attachment of 2,4,6-Trichloro-1,3,5-Triazine (TCT) to the Silane Layer. Reaction conditions were adapted from Yeh and Lin.22 A Schlenk flask containing a stirbar was evacuated and backfilled with N2 three times to create an inert atmosphere. TCT (1.25 g, 6.8 mmol) was dissolved in toluene (25 mL) to create a 0.27 M solution. A silanized device and 25 mL of TCT solution was added to the Schlenk flask and placed in a 70°C oil bath under flowing N2. The reaction was allowed to stir for 4 h and 15 min and then cooled to room temperature. The reaction mixture was decanted, devices were washed with toluene and methanol (three times each) and dried with a stream of N2.

Attachment of Thrombomodulin to the TCT Layer. Conjugation of thrombomodulin was based on reaction parameters adapted from Yeh and Lin.22 A 0.02 mg/mL solution of human


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recombinant thrombomodulin (hTM) in 1X Dulbecco’s PBS buffer (DPBS) was prepared under a sterile cell culture hood. A TCT functionalized device was added and allowed to react at 4°C for 24 h. The hTM solution was decanted and the device was rinsed with DPBS and allowed to air dry under a sterile cell culture hood.

Scanning Electron Microscopy (SEM). Micrographs of uncoated and PE-ALD coated flow diverting devices were taken using a Hitachi S-4800 SEM (with 2.0 nm resolution). An electron beam intensity of 3 kV (or approximately 11,000 nA emission current) and magnification of 300x were applied. To maintain the integrity of the respective platform coating layers no additional surface conducting layers were applied to the devices.

Ellipsometry. Ellipsometry measurements were taken using a Woollam M-2000 spectroscopic instrument with a 75 W xenon arc lamp and focused beam size of 150 m. Specifically, measurements were taken at 10 discrete locations on p-doped silicon wafers (1x1 cm2) coated with each layer in the developed coating technology platform (n=10). This was done using sequential Cauchy models and associated constants determined in consultation with the instrument manufacturer and work published by Gunda et al.23

Confocal Microscopy. Confocal microscopy imaging was performed using a Leica SP8 STED Super Resolution Microscope with a continuum fiber laser and the Alexa Fluor 488 5-TFP fluorophore. For the labeling reaction, Alexa Fluor 488 5-TFP (1 mg, 1 mol) was first dissolved in dried dimethylformamide (DMF, 500 L) protected from light. Both TCT and hTM coated commercial flow diverting device pieces were placed into individual wells of a flat-bottom 96 well


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microplate and incubated in 250 L of fluorophore solution in the dark for 1 h at room temperature. A microplate shaker, set at moderate speed, was used to stir the fluorophore solutions during the incubation period. Afterward the device pieces were transferred to sterile plastic test tubes, washed with dry DMF three times, and dried with a stream of N2. Confocal microscopy was performed while devices were horizontally oriented using a 10x dry objective lens. The autofluorescentcapability of an uncoated commercial flow diverting device piece was also tested and imaged in the same orientation and magnification.

X-Ray Photoelectron Spectroscopy (XPS). XPS measurements were performed using a Kratos Axis Ultra DLD spectrometer with a monochromatic aluminum x-ray source operating at 15 kV accelerating voltage and 10mA of emission current. Photoelectrons were analyzed from the outermost layer and at takeoff angles of  = 90. Low energy electrons were used for charge compensation to neutralize the samples. Survey spectra were acquired with pass energies of 160 eV; high resolution spectra were collected with pass energies of 20 eV. These scans were further resolved into individual Gaussian peaks using the CasaXPS software package version 2.3.17; calibration was done using the adventitious carbon C 1s peak at 284.8 eV, after which the relative atomic concentrations of the elements present in each survey scan were calculated from the respective scan peak areas and the Kratos library relative sensitivity factors (Table S1). A Shirley type background was routinely used to account for inelastically scattered electrons that may contribute to the broad background. For each platform coating layer, XPS elemental intensity maps were generated by directing the irradiating x-rays through a slot aperture (~300x700 m2) and limiting the detection electron spectrometer to output only the signal from electrons detected within an energy range characteristic of an element of interest.24, 25


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Focused Ion Beam (FIB)-SEM Imaging. FIB-SEM images were acquired using a FEI Helios NanoLab DualBeam system with a high resolution SEM electron column, field emission gun electron source, and gallium ion source for FIB nano-machining. Micrographs of a nano-etched aluminum oxide layer on a commercial flow diverting device were taken with an electron beam intensity of 5 kV (accelerating voltage) at magnifications of 16,000x and 500,000x at the University of Notre Dame. To preserve the integrity of the aluminum oxide platform coating layer, platinum metal was initially deposited on top – through both electron beam and ion beam processes – followed by subsequent nano-etching.

Functional Ex-Vivo Primate Shunt Testing: An established primate model26-31 was used to assess the extent of platelet and fibrin accumulation on two different commercially available flow diverters coated with hTM, as well as control devices, in an ex-vivo arteriovenous (AV) shunt. All primate experimentation was performed at the Oregon National Primate Research Center (ONPRC) in Beaverton, Oregon under the umbrella of an IACUC-approved protocol (#0681). The first experiment consisted of comparing both the platelet and fibrin deposition of the following devices deployed in the AV shunt of the same primate to limit variability (male, approximately four years old): an uncoated flow diverting device (n=2), a hTM coated device (n=2), and an uncoated device deployed in combination with DAPT (n=2). In contrast, the second experiment consisted of comparing both the platelet and fibrin deposition of the following devices, produced by a different manufacturer (approximately 2.6 times as long as the devices in the first experiment) and deployed in the same AV shunt of a different primate (male, approximately four years old): an uncoated flow diverting device (n=2), a hTM coated device (n=2), a hTM coated device


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deployed in combination with aspirin only, and an uncoated device deployed in combination with DAPT (n=2). Prior to shunt deployment in both experiments, all devices were re-inserted into their deployment catheters and sterilized by the respective device manufacturer. Once each device was deployed in the AV shunt, the shunt blood flow was held constant at 100 ml/min by an external screw clamp; a Doppler ultrasonic flow meter was also used to continuously measure the mean blood flow rate through the shunt during each experiment. To measure platelet deposition in each deployed device, autologous platelets were radiolabeled with Indium-111 (111In) and re-injected into the primate. Platelet deposition was then measured over a one-hour perfusion period using a high sensitivity 99Tc collimator and scintillation camera (GE 400T, General Electric); imaging of the 172 keV

111

In photon peaks was done at approximately three-minute intervals and recorded

over the perfusion period. Fibrin deposition was also measured during the same hour long perfusion period; to do this, homologous fibrinogen labeled with Iodine-125 was first injected 24 hours prior to experimentation. Following the perfusion period, each device was removed from the shunt and the amount of fibrin deposition was measured after the Iodine-125 decayed using a Wizard gamma counter and standard curves.32 For comparison, each device was subsequently dehydrated in increasing concentrations of ethanol, critical point dried, and weighed; the device weight was then compared to the weight prior to deployment in the shunt to verify the amount of fibrin accumulation over time.

Functional In-Vitro Thrombin Generation Testing: To assess in-vitro thrombin generation the Calibrated Automated Thrombogram (CAT) assay was used, originally developed by Hemker et al.33, utilizing a fluorogenic substrate specific to thrombin. A third-party vendor performed


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preliminary CAT assay testing using the Thrombinoscope (Maastricht, Netherlands) protocol, reagents, and software package on commercial flow diverting devices coated with the developed platform and conjugated hTM compared to similar flow diverting devices coated with a commercial antithrombotic surface modification. In this study, all devices or controls were added to assay microwells containing re-calcified human platelet-rich plasma (PRP), the fluorogenic substrate, and a tissue factor coagulation trigger. Specifically, uncoated commercial flow diverter pieces (n=4) and hTM coated device pieces (n=9) were added to individual microwells and compared to flow diverter pieces coated with the commercial surface modification (n=4). Glass pieces (n=6) and tissue factor triggered re-calcified human PRP (blank, n=5) were used as controls. During testing the temperature was kept constant at 37˚C. The resulting fluorescent signals were measured by a 390 nm excitation/460 nm emission filter set on a microplate reader and converted to thrombin generation time courses by the Thrombinoscope software. Results were reported as the peak thrombin concentration associated with each individual thrombin generation curve (nM).

RESULTS AND DISCUSSION Overview of the Platform Coating Technology: Our process to coat commercially available intracranial flow diverters with the platform technology is shown in Figure 2. The first step is the deposition of aluminum oxide (Al2O3) by plasma-enhanced atomic layer deposition (PE-ALD).34 PE-ALD deposition is batch-based, in which gaseous precursors are dosed to a substrate in a time sequence of pulses and purges.35 The next layer in the coating technology platform is the assembly of a layer of 3-aminopropyl-triethoxysilane (APTES), which binds to the aluminum oxide via its ethoxy groups, forms a multilayer, and leaves an amino-terminated surface.


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The final layer in the developed coating platform is 2,4,6-trichloro-1,3,5-triazine (TCT), which attaches to the APTES free amines. The hTM is attached to the TCT-terminated surface.

Figure 2. Schematic illustration of the developed coating technology platform for intracranial flow diverters. Fabrication of the Al2O3, Aminosilane, and TCT Layers: The aluminum oxide layer was deposited on each intracranial flow diverter via a PE-ALD process. The PE-ALD technique was chosen since it allows for highly uniform and defect-free film growth on substrates with complex geometries. In PE-ALD a substrate is pulsed with Al(CH3)3 followed by an oxygen plasma to yield an Al2O3 film built up layer by layer.34 Further, deposition of Al2O3 provides a uniform oxide surface that can be functionalized in a consistent manner regardless of the material composition of the underlying device; this is of particular importance since commercial intracranial devices may be composed of multiple metallic alloys which can vary among manufacturers. SEM micrographs of uncoated (Figure 3A) and aluminum oxide coated (Figure 3B) commercially available flow diverting devices indicate that the PE-ALD deposited aluminum oxide layer of this technology does not significantly alter the flow diverter device mesh


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morphology or wire diameter. The thickness of this aluminum oxide layer was determined using ellipsometry and FIB etching. Since ellipsometry requires a planar substrate, silicon wafers were initially coated with the aluminum oxide layer concurrent with the intracranial flow diverters; the thickness of this layer on the wafers was found to be 31.860.26 nm. To verify the aluminum oxide layer thickness on an intracranial device, a commercial flow diverter was coated with platinum and a rectangular area was etched by FIB. A SEM micrograph of the FIB etch (Figure 3C) and resulting thickness measurements via an image processing software (Figure 3D) reveal an aluminum oxide thickness of 31.2 nm and 33.7 nm at two points along the flow diverter surface.

Figure 3. (A) SEM micrograph of an uncoated commercially available flow diverting device. (B) SEM micrograph of a similar device coated with 300 cycles of Al2O3 deposited by PE-ALD. (C) FIB rectangular etch on a commercially available flow diverting device coated with 300 cycles of PE-ALD deposited Al2O3 and platinum, the white box denotes the approximate location of the SEM cross-sectional image. (D) SEM cross-sectional image of the FIB etch. The aluminum oxide layer was functionalized with APTES followed by reaction with TCT as described in the experimental section and shown in Equation 1.


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(1) Reaction of APTES with Al2O3 is known to result in the formation of a multilayer; ellipsometric thickness measurements from silicon wafers first coated with Al2O3 and then APTES were 1.590.14 nm. The reaction of TCT on the APTES-terminated surface, shown in Equation 2, also placed a thin layer of TCT on the surface. This was verified by XPS (Figure 4D), but did not add to the APTES layer thickness as measured by ellipsometry, likely due to the presence of the APTES multilayer. Further, we estimated the triazine layer surface coverage on a commercial flow diverter using the XPS data and the Gries formula for inelastic mean free path36 (see supporting information). In keeping with this estimation, we found that approximately 0.57 triazine molecules are dispersed per nm2 on a flow diverter device.

(2) Coated commercial flow diverting devices were further investigated by XPS to determine the composition of each layer (Figure 4); specifically, the XPS spectra reported the elements expected on each sample (the full elemental compositions of the XPS spectra are found in the supporting information Figure S1). The XPS spectrum of an uncoated commercial flow diverter (Figure 4A) had peaks for cobalt, chromium, nickel, and platinum which demonstrated that the specific device microwires are composed of multiple metals.

The XPS background signal for metals is

significantly higher than that of non-metals due to the nature of resonant inelastic electrons.37 Therefore the presence of multiple elements will cumulatively contribute to the background of a


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XPS spectrum37, as indicated by the significant background signal in Figure 4A. The XPS spectrum of the aluminum oxide coated commercial flow diverter (Figure 4B) indicated that the surface was composed of aluminum (30.4%), oxygen (52.2%), and carbon (17.1%) without any peaks for the microwire metals (Table S1). The lack of peaks associated with the underlying metals was consistent with the measured thickness of the aluminum oxide layer, approximately 30 nm. The XPS spectrum for the APTES coated commercial flow diverter (Figure 4C) had peaks for aluminum (15.9%), oxygen (34.6%), carbon (42.2%), nitrogen (3.6%), and silicon (3.2%) (Table S1). This spectrum is consistent with assembly of an APTES multilayer as shown by the decrease in intensity of the aluminum peak and a nearly identical intensity of the nitrogen and silicon peaks, which are unique to APTES. The XPS spectrum of the TCT coated commercial flow diverter (Figure 4D) had peaks for chlorine (0.7%) and an elevated ratio of nitrogen (6.5%) to silicon (4.1%), which is consistent with the attachment of TCT to the surface (Table S1).


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Figure 4. (A) XPS survey spectrum of an uncoated commercially available flow diverting device. (B) XPS survey spectrum associated with a commercial flow diverter coated with 300 cycles of Al2O3 deposited by PE-ALD. (C) XPS survey spectrum associated a commercial flow diverter coated with Al2O3 and APTES. (D) XPS survey spectrum associated with a commercial flow diverter coated with Al2O3, APTES, and TCT. Coated commercial flow diverting devices were further characterized by XPS via elemental intensity maps (Figure 5). The intensity maps of the uncoated diverter (Figure 5A) indicate that at least two different microwire types comprise the device, since platinum was most intense for


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only 25% of the microwires. The use of multiple wires to fabricate a flow diverter further demonstrates the need to first assemble a uniform oxide layer on each microwire to ensure equivalent functionalization. Figures 5B and 5C indicate a relatively uniform aluminum oxide deposition on the flow diverter surface and a relatively uniform attachment of the APTES layer respectively. The intensity map associated with the TCT-terminate layer (Figure 5D) indicates that chlorine, an element unique to TCT, was attached to the device mircowires; the weak signal intensity of chlorine is as expected due to its low concentration within the monolayer.

Figure 5. (A) XPS elemental intensity maps for an uncoated commercially available flow diverter. (B) XPS elemental intensity maps for a similar flow diverter coated with 300 cycles of Al2O3 deposited by PE-ALD. (C) XPS elemental intensity maps for a similar flow diverter coated with Al2O3 and APTES. (D) XPS elemental intensity maps for a similar flow diverter coated with Al2O3, APTES, and TCT. Attachment of Thrombomodulin to the TCT Layer. The glycoprotein hTM was chosen to conjugate to the coating platform since it is an integral membrane protein expressed in-vivo on


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the human endothelial cell surface and actively disrupts coagulation as a protein cofactor in the thrombin-catalyzed activation of protein C.20 It was expected that attachment of hTM on a flow diverter would prevent the formation of clots by actively preventing thrombosis. In this work the extracellular domain of hTM was conjugated to the TCT layer via a free amine coupling as described in the experimental section and shown in Equation 3. Use of hTM containing only the extracellular domain was motivated by the fact that hTM likely binds thrombin, its key antithrombotic function, in the extracellular domain. 20

(3) The composition of an hTM coated commercial flow diverting device surface was probed by XPS (Figure 6). The decreased signal intensity of the aluminum peak (11.0%) in the XPS spectrum for the hTM coated device (Figure 6A, Table S1) compared to the aluminum peak (18.6%) in the XPS spectrum for the TCT coated device (Figure 4D, Table S1) suggests the attachment of hTM. This was further investigated by XPS core level scans of the nitrogen (N 1s) peak for both the TCT and hTM coated flow diverter surfaces (Figure 6B). The number of –N= bonds, characteristic of the TCT molecule, decreases by more than half from the TCT coated device (42.8%, Table S2) compared to the hTM coated device (14.2%, Table S2). Furthermore, the intensity of the peak associated with amide nitrogens increases for the hTM coated device (Table S2). Together this information indicates that some hTM protein is conjugated to the flow diverter surface. Due to the cost of hTM, it was not possible to coat the silicon wafer and measure thickness by ellipsometry.


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Figure 6. (A) XPS survey spectrum associated with a commercial flow diverting device coated with 300 cycles of Al2O3 deposited by PE-ALD, APTES, TCT, and hTM. (B) XPS nitrogen core level spectra associated with a similar flow diverter coated with Al2O3, APTES, and TCT with nitrogen binding chemistry indicated, as well as a similar flow diverter coated with Al2O3, APTES, TCT, and hTM with the nitrogen binding chemistry indicated. To further assess the conjugation of hTM onto the commercial flow diverting devices they were labeled with a fluorophore reactive to primary amines, emitting a green fluorescence as described in the experimental section; these fluorophore-labeled flow diverters were imaged by confocal microscopy (Figure 7). Figures 7C and 7D indicate substantive hTM conjugation on both


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the interior and exterior surfaces of the hTM-labeled flow diverter. The TCT terminal layer on a flow diverter was also exposed to the same fluorophore and imaged (Figures 7A and 7B); a relatively diminished and inconsistent fluorescent signal was observed due to a small number of unreacted primary amines from the underlying APTES layer. The difference in fluorescence intensity between the hTM and TCT-labeled device surfaces is consistent with the conjugation of hTM. An additional control experiment was conducted where the same fluorophore was exposed to an uncoated flow diverter and no fluorescence was observed (Figure 7E).

Figure 7. Confocal microscopy images taken with a 10x dry objective lens of commercial flow diverters exposed to a fluorescent dye reactive with primary amines. The (A) exterior and (B) interior of a TCT coated commercial flow diverter and the (C) exterior and (D) interior of a hTM coated commercial flow diverter are shown after exposure to the fluoroscent dye. (E) A commercial flow diverter that was not coated but exposed to the fluoroscent dye did not show any fluoroscence.


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Evaluation of Thrombomodulin Coated Devices in an Ex-Vivo Primate Shunt. An established primate model26-31 was used to assess the extent of platelet and fibrin accumulation in two different commercially available flow diverters coated with hTM, as well as control devices. This primate shunt model has been used extensively to quantify the hemocompatibility of biomaterials, including stents,26-31 as well as the antithrombotic efficacy of both established and novel antithrombotic drugs.38-41 Specifically a baboon is a good thrombosis model because of its hemostatic similarity to humans, its large size, the logistical ease of acquiring frequent blood samples, as well as the animals’ general acceptance of chronically patent arteriovenous (AV) cannulas.32 The extent of fibrin accumulation and platelet deposition on two different types of commercial flow diverters were measured post deployment in the primate AV shunt. Platelet deposition can be used as a biomarker of coagulation since changes in the platelet architecture during surface adhesion act to promote thrombin formation, accelerating thrombosis.42 Increased platelet deposition indicates increased thrombosis. Likewise, fibrin is an important biomarker of coagulation since it helps stabilize a blood clot, thus increased fibrin accumulation indicates increased clotting activity.43 One type of commercial flow diverter was tested in the AV shunt with three different strategies including uncoated flow diverters deployed alone (labeled “Bare” in Figure 8), hTM coated flow diverters deployed alone (labeled “hTM coated” in Figure 8), and uncoated flow diverters deployed in combination with DAPT (labeled “Bare+DAPT” in Figure 8). A second set of commercial flow diverters were also investigated, these flow diverters were approximately 2.6 times as long as the first set. The second set of flow diverters were uncoated flow diverters, hTM coated flow diverters, hTM coated flow diverters deployed in combination with aspirin (labeled as “hTM Coated+ASA”), and uncoated flow diverters in combination with


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DAPT. For both sets of flow diverters no aspirin or DAPT were given to the primates unless explicitly stated.

Figure 8. (A) Fibrin accumulation for one type of commercial flow diverting device deployed in an established primate AV shunt model. The black circles indicate the fibrin accumulation for each individual device tested, while the bars indicate the average. (B) Fibrin accumulation for a second type of commercial flow diverting device deployed in the primate model. The black circles indicate the fibrin accumulation for each individual device tested, while the bars indicate the average. (C) The corresponding average platelet accumulation for each device deployed in the primate model and shown in panel A. (D) The corresponding average platelet accumulation for each device deployed in the primate model and shown in panel B.


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Several trends were observed from these two ex-vivo primate shunt studies. Short hTM coated flow diverters deployed alone accumulated fewer platelets (Figure 8C) and had decreased fibrin deposition (Figure 8A) when compared to uncoated flow diverters. This result indicates that the hTM coating had an effect. However, short hTM coated flow diverters deployed with DAPT still accumulated more platelets and fibrin over time than uncoated devices deployed in combination with DAPT. For long flow diverters, the hTM coating led to a smaller amount of fibrin adsorption compared to uncoated flow diverters (Figure 8B). There was less difference between the longer uncoated and hTM coated flow diverters when the platelet accumulation was investigated, but when aspirin was used with the hTM coated flow diverters fewer platelets were accumulated (Figure 8D). These results are promising and are considered preliminary because of the small number of primates investigated (due to the cost and complexity of the experiments). Evaluation of Coated Devices by In-Vitro Thrombin Generation. To assess the invitro thrombin generation capacity of the developed coating technology, the CAT Assay was performed by an independent vendor. This assay utilizes a fluorogenic substrate specific to the serine protease thrombin, which is produced as part of the coagulation cascade and catalyzes the formation of fibrin within the blood clot, as well as catalyzing many other coagulation-related reactions.43 In particular, the fluorogenic substrate is added to a mixture of re-calcified human plasma and the flow diverter (or drug) of interest; when cleaved by thrombin, the substrate fluoresces.

In these experiments the time-varying fluorescence signal was measured by a

microplate reader and converted to individual thrombin generation curves by commercial Thrombinoscope software.44 Others have measured complement activation, namely C3a and C5a, and used the measurements to characterize biomaterial-associated thrombosis since this pathway works in tandem.45 We have chosen to investigate thrombin generation herein as a first step in


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understanding the developed platform coating’s thrombotic response since it is a direct thrombosis metric. Commercial flow diverters with the platform coating and conjugated hTM were investigated and compared to controls. Figure 9 shows the peak in-vitro thrombin concentrations generated in the CAT assay performed by an independent vendor with hTM coated commercial flow diverters as compared to similar devices coated with a commercial antithrombotic surface modification and controls. In these studies, all devices or controls were added to microwells containing re-calcified human plasma, the fluorogenic substrate, and a tissue factor coagulation trigger. Specifically, glass was added to some microwells and used as a positive control while blank microwells containing no glass or devices were used as a negative control. Likewise, other microwells contained either a piece of hTM coated flow diverter, a piece of flow diverter coated with a commercial antithrombotic coating, or a piece of uncoated flow diverter. These results (Figure 9), suggest that the thrombin generating capacity of hTM coated commercial flow diverters in a static, in-vitro environment is comparable to similar flow diverting devices coated with a commercial surface modification. In an effort to quantify the amount of hTM attached to a flow diverter a linear relationship was assumed to model peak thrombin generation. Using this linear relationship, we estimate that there are 353 functional molecules of hTM attached per µm2. Additionally, the hTM coated flow diverters had similar responses as the blank microwells, indicating that these flow diverters do not cause significant thrombosis.


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Figure 9. The in-vitro peak thrombin concentrations generated in the CAT assay by hTM coated commercial flow diverters as compared to similar devices coated with a commercial antithrombotic surface modification and controls. This assay was performed by a third-party vendor.

CONCLUSION We developed a nanometer-scale coating for commercial intracranial flow diverters that may be used as a platform for assembly of hTM, a naturally occurring small molecule chosen specifically to address a key device challenge of enhancing hemocompatibility. Furthermore, the entire coating technology is less than 50 nm thick, suggesting it will have a minimal impact on the mechanics of the 30 m diameter microwires when coated commercial flow diverters are deployed in the brain. Coated commercial flow diverters were then investigated by in-vivo and ex-vitro methods with promising preliminary results indicating the hTM platform modification enhanced device hemocompatibility relative to uncoated devices. With further optimization and testing this technology has the potential to minimize the adjunctive use of DAPT in the endovascular treatment


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of intracranial aneurysms. In other words, the technology has the potential to make a significant patient-care impact. In future work, we intend to investigate the flexibility of the APTESterminated and the amine reactive TCT platform layers; the triazine group will allow for facile attachment of other synthetic or natural polymers and small molecules that may further enhance the coating hemocompatibility and inhibit complement activation.


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ASSOCIATED CONTENT Supporting Information. XPS survey spectra associated with the developed coating technology on commercial flow diverters; relative atomic concentrations of each XPS survey spectra; nitrogen binding chemistries of the XPS core level spectra associated with hTM and TCT coated commercial flow diverters; and the estimation of TCT coverage on the flow diverter surface. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *David Hasan, MD Email: [email protected] Tel: 319-400-9455 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Funding Sources This study was supported by grants from: The Lyle and Sharon Bighley Professorship (A.K.S); the USDA NIFA-2014-03660 (N.B.B); NIH K08NS082363-01A1(D.M.H). ACKNOWLEDGMENT The authors would like to acknowledge that this work utilized the Hitachi S-4800 SEM instrument in the University of Iowa Central Microscopy Research Facilities (UI CMRF) that was purchased


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with funding from the NIH SIG grant 1 S10 RR022498-01. Additionally, the authors would like to thank Dr. Alexander Mukasyan for his help in the acquisition of the FIB-SEM images of the aluminum oxide coated flow diverters and the UI CMRF staff for training resources. The authors would also like to thank the University of Iowa Microfabrication Facility Optical Science and Technology Center for the training and use of their PE-ALD and ellipsometer and Dr. Monica Hinds and the ONPRC for performing the ex-vivo primate shunt experiments. CONFLICT OF INTEREST All authors, except J. Lee, own equity in Advanced Endovascular Therapeutics, LLC.

ABBREVIATIONS subarachnoid hemorrhage (SAH); international subarachnoid hemorrhage aneurysm trial (ISAT); dual anti-platelet therapy (DAPT); United Kingdom (UK); Recombinant human thrombomodulin (hTM); methoxy-poly(ethylene glycol) amine (mPEG); Plasma-enhanced atomic layer deposition (PE-ALD); trimethylaluminum (TMA); 3-aminopropyl-triethoxysilane (APTES); 2,4,6-trichloro-1,3,5-triazine (TCT); Dulbecco’s PBS buffer (DPBS); calibrated automated thrombogram (CAT) assay; scanning electron microscopy (SEM); x-ray photoelectron spectroscopy (XPS); focused ion beam (FIB); Oregon National Primate Research Center (ONPRC); arteriovenous (AV); Indium-111 (111In); University of Iowa Central Microscopy Research Facilities (UI CMRF).

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TABLE OF CONTENTS GRAPHIC


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Development and Evaluation of a Nanometer ... - ACS Publications

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