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Stability of Polyethylene Glycol and Zwitterionic Surface Modifications in PDMS Microfluidic Flow Chambers Thomas James Plegue, Kyle M Kovach, Alex J Thompson, and Joseph A Potkay Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03095 • Publication Date (Web): 12 Dec 2017 Downloaded from http://pubs.acs.org on December 18, 2017
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Stability of Polyethylene Glycol and Zwitterionic Surface Modifications in PDMS Microfluidic Flow Chambers Thomas J. Pleguea, Kyle M. Kovachb, Alex J. Thompsonac, Joseph A. Potkayac* a
b
VA Ann Arbor Healthcare System, Ann Arbor, MI 48105, USA
Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH 44106, USA c
Department of Surgery, University of Michigan, Ann Arbor, MI 48109, USA
Keywords Hemocompatibility, PDMS, Stability, Hydrophilic Coatings, Microfluidic, Zwitterionic, Polyethylene Glycol, Sulfobetaine
Abstract Blood-material interactions are crucial to the lifetime, safety, and overall success of a blood contacting devices. Hydrophilic polymer coatings have been employed to improve device lifetime by shielding blood contacting materials from the natural foreign body response, primarily the 1
intrinsic pathway of the coagulation cascade. These coatings have the ability to repel proteins, cells, bacteria, and other micro-organisms. Coatings are desired to have long-term stability, so that the nonthrombogenic and nonfouling effects gained are long lasting. Unfortunately, there exist limited studies which investigate their stability under dynamic flow conditions as encountered in a physiological setting. In addition, direct comparisons between multiple coatings are lacking in the literature. In this study, we investigate the stability of polyethylene glycol (PEG), zwitterionic sulfobetaine silane (SBSi), and zwitterionic polyethylene glycol sulfobetaine silane (PEG-SBSi) grafted by a room temperature, sequential flow chemistry process on polydimethylsiloxane (PDMS) over time under ambient, static fluid (no flow), and physiologically relevant flow conditions and compare the results to uncoated PDMS controls. PEG, SBSi, and PEG-SBSi coatings maintained contact angles below 20° for up to 35 days under ambient conditions. SBSi and PEG-SBSi showed increased stability and hydrophilicity after 7 days under static conditions. They also retained contact angles ≤ 40° for all shear rates after 7 days under flow, demonstrating their potential for long term stability. The effectiveness of the coatings to resist platelet adhesion was also studied under physiological flow conditions. PEG showed a 69% reduction in adhered platelets, PEG-SBSi a significant 80% reduction, and SBSi a significant 96% reduction compared to uncoated control samples, demonstrating their potential applicability for blood contacting applications. In addition, the presented coatings and their stability under shear may be of interest in other applications including marine coatings, lab on a chip devices, and contact lenses, where it is desirable to reduce surface fouling due to proteins, cells and other organisms.
Introduction
2
Polydimethylsiloxane (PDMS) has a wide range of applications in medical devices because of its biocompatibility, durability, gas permeability, nontoxicity, and ease of fabrication. These silicone based polymers have been used in devices such as catheters, tubing, implants, and extracorporeal devices.1 Research groups have recently used PDMS to create a new class of microfluidic artificial organs, including lungs and kidneys, which promise to more closely mimic their natural counterparts.2–5 Since these are blood contacting devices, controlling the bloodmaterial interactions to minimize activation of the coagulation cascade are crucial to their success. Hydrophobic surfaces are known to increase the level of protein adsorption, which leads to increased platelet activation and conformational changes on the surface.6 This in turn causes activation of the coagulation cascade and clotting.1 Increasing the hydrophilic characteristics of the polymer surface is one of the most widely used approaches to improve device hemocompatibility. Hydrophilic surfaces act by exerting a steric repulsion effect on proteins and cells in the blood.7 It is also entropically favorable for the surface to create a water layer rather than absorbing proteins.8 This can delay activation of the coagulation cascade and extend the lifetime of the device. In other applications, hydrophilic coatings have been shown to reduce bacterial adhesion in lab on a chip and medical devices and fouling on and in marine equipment and surfaces.9–13
Hydrophilic surface properties can be imparted through a variety of methods including plasma treatment, UV/ozone treatment, chemical vapor deposition (CVD), and grafting hydrophilic molecules onto the surface.14 Of these methods, plasma treatment is the quickest method to render PDMS hydrophilic, with contact angles less than 10° in under 30 s.15 Although plasma activation of the surface is quick and simple, PDMS exhibits significant “hydrophobic recovery” within 3
minutes to hours, bringing the surface back to its original hydrophobic state.16 Therefore, hydrophilic coatings are grafted to the surface to help reduce the hydrophobic recovery and create a stable surface. The most common method is to graft the desired molecule to the surface after plasma activation. Plasma activation forms hydroxyl (-OH) groups on the surface, which can then react with the hydrophilic molecule. A variety of surface coatings have been used to increase the hydrophilicity of PDMS including, polyethylene glycol derivatives, zwitterionic molecules, and methacrylates.1,17,18
Polyethylene glycol (PEG) derivatives have been widely used to render PDMS and other materials hydrophilic for improved hemocompatibility.1,7,12,19–22 Siloxy (Si-OR) based PEG molecules have been used to create stable, hydrophilic coatings on PDMS, due to the interactions between the siloxy groups and the PDMS backbone.20,21 These coatings have been shown to exhibit advancing contact angles under 20° after one week in ambient conditions, indicating the potential stability of the coating.20 The siloxy-based PEG also shows much lower contact angles than acrylate-based PEG used in some studies, which indicates its better suitability for hemocompability.23,24
Zwitterionic molecules have great potential to increase surface hydrophilicity due to the highly polar nature of the molecule. Phosphobetaines, sulfobetaines, and carboxybetaines are three widely used types of zwitterionic systems, though sulfobetaines and carboxybetaines are the most commonly used.17 Carboxybetaines in the form of poly(carboxybetaine methacrylate) have shown promising resistance to fibrinogen adsorption for incubation times of up to 74 days.18,25 Wu et al. showed that carboxybetaines observe contact angles of less than 10° when droplets of water are 4
placed on the surface. However, in the same study sulfobetaine contact angles were not able to be measured due to complete wetting of the surface, indicating its superior hydrophilic properties.26 A sulfobetaine compound with a siloxy base group showed incredible stability on PDMS under ambient conditions, keeping a contact angle of less than 20° for over 200 days. This sulfobetaine silane (SBSi) also significantly repelled proteins and had limited bacteria fouling compared to bare PDMS.27 This ability to repel proteins, combined with its stable hydrophilic surface makes SBSi a promising molecule for hemocompatibility.
It has been hypothesized that chain length and grafting density affects the antifouling properties of the grafting molecule. Chang et al. demonstrated that increasing the chain lengths of zwitterionic poly(sulfobetaine methacrylate) resulted in decreasing levels of protein and platelet adsorption.28 Zwitterionic sulfobetaine PEG (PEG-SBSi), a combination of PEG and a zwitterionic molecule in a single molecule with long chain length, demonstrated a significant increase in coagulation time for carbon-coated stents and a decrease in protein adsorption on nitinol alloy stents.29,30 A long chain length zwitterionic sulfoammonium PEG on polycarbonate urethanes achieved similar results.31
While these coating studies have shown promising results in increasing hydrophilicity of their respective materials, most are grafted onto a planar substrate and the stability tested under ambient conditions. These studies are promising, but for use in blood contacting medical devices, marine interfacial equipment, and microfluidic devices, these coatings will need to be stable under dynamic flow conditions. To date, the literature lacks studies demonstrating the stability of these coatings under dynamic flow conditions and lacks a direct comparison between different coatings. 5
Previously, we have shown that PEG-silane can be grafted onto PDMS channel structures using oxygen plasma.20,22 In the current study, we present results for the stability of PEG, SBSi, and PEG-SBSi that are grafted onto the surfaces of enclosed, rectangular PDMS microfluidic flow channels under ambient conditions for 35 days, static fluid conditions for 7 days, and dynamic physiologic flow conditions for 7 days. We also present results of a platelet adhesion test under physiologic flow conditions. For the SBSi and PEG-SBSi coating, we developed a new method to construct complex, long chain length coatings from a single base molecule by sequential reactions (Scheme 1). This process was inspired by the final ring opening reaction performed by Guo et al.31 By using this new method, the time-consuming process of formulating the coating prior to grafting can be avoided, thereby allowing, for the first time, all coatings to be grafted in a single day. For dynamic flow studies, the flow conditions chosen are representative to shear rates of the veins (100 s-1), arteries (650 s-1), and arterioles (1700 s-1).32
Scheme 1. Reaction schemes with approximated chain length a) PEG, b) SBSi, c) PEG-SBSi
6
Materials and Methods Materials Sylgard 184 elastomer and curing agent was purchased from Ellsworth Adhesives (Germantown, WI). [Methoxy(polyethyleneoxy)propyl]trimethoxysilane (PEG-silane), tech-90, 6-9 and (N,Ndimethyl-3-aminopropyl)trimethoxysilane (DMASi) was purchased from Gelest (Morrisville, PA). Acetone and 2-isopropanol was purchased from Fisher Science (Pittsburg, PA). 1,3-Propane sultone (PS), hexamethylene diisocyanate (HDI), dibutyltin dilaurate (DBDTL), heparin, calcium chloride (CaCl2), 3A Molecular Sieves, and 2-dimethylaminoethanol (DMEA) were purchased 7
from Sigma Aldrich (St. Louis, MO). Phosphate-buffered saline (PBS), Calcein AM, and dimethyl sulfoxide (DMSO) was purchased from ThermoFisher Scientific (Hanover Park, IL). SU-8 2035 permanent epoxy negative photoresist and SU-8 developer were purchased from MicroChem (Newton, MA). 4” silicon wafers were purchased from University Wafers (Boston, MA). Citrated bovine platelet rich plasma (PRP) was purchased from Lampire Biological Laboratories (Pipersville, PA). i-STAT Handheld blood analyzer and Celite ACT cartridges were obtained from Abbott Point of Care (Princeton, NJ).
Flow Chamber Assembly Sylgard 184 elastomer was mixed with the curing agent in a 10:1 w/w ratio and poured into a polystyrene dish containing either: 1) an acrylic mold to create a rectangular channel with a height of 840 µm, length of 50 mm, and width of 10 mm (ambient, static, and dynamic flow studies), or 2) a photoresist on silicon mold to create a rectangular channel with a height 100 µm, length of 50 mm, and width of 10 mm (platelet adhesion study). The remaining elastomer was poured into a second dish to create a planar base for the flow channel. Both elastomers were degassed in a vacuum desiccator for 1 h and then cured at 85 °C for 1 h. Holes were punched at each end of the channel and short sections of silicone tubing were bonded to allow for easy access to the channel. The channel and base side were washed with acetone for 15-20 s, dried, then placed in a Nordson MARCH AP-300 Plasma System and exposed to oxygen plasma (900 mtorr, 25 W, 25 s).15 The two sides were then removed, brought into intimate contact to create a permanent bond and form the flow channel, and then a solution was immediately introduced into the channel to initiate one of the desired grafting reactions described below.
8
PEG-Silane Coating Application Prior to the bonding of the two sides of the PDMS flow chamber, PEG-silane solution was made by mixing 1 mL of anhydrous (pre-dried with sieves) acetone and 1 mL of PEG-silane. After bonding, the channel was immediately (within 30 s of bonding) filled with the PEG solution and allowed to react for 1 h at room temperature (RT).20 The chamber was then rinsed with deionized water for 10 minutes at 2 mL/min (0.5 mL/min for the 100 µm chambers) and dried with air.
Zwitterionic Silane (SBSi) Coating Application A molar ratio of 1 PS:1 DMASi was used in calculating the solution formulations.26,27,33 Prior to the bonding the two sides of the PDMS flow chamber, a solution of DMASi was made by mixing 1.51 mL of anhydrous (pre-dried with sieves) acetone and 490 µL of DMASi. After bonding, the channel was immediately (within 30 s of bonding) filled with the DMASi solution and allowed to react for 1 h at RT.27 The channel was washed with anhydrous acetone (10 mL at 2 mL/min or 0.5 mL/min for the 100 µm chambers) and reacted with a solution of 1.80 mL anhydrous acetone and 200 µL of PS for 6 h at RT. After, the channel was rinsed with deionized water for 10 minutes at 2 mL/min (0.5 mL/min for the 100 µm chambers) and dried with air.
Zwitterionic PEG (PEG-SBSi) Coating Application Molar ratios of 2.1 HDI:1 PEG-silane, 0.8 DMEA:1 HDI, and 1.2 PS:1 DMEA were used in calculating solution formulations.29,30 Prior to the bonding the two sides of the PDMS flow chamber, PEG solution was made by mixing 1 mL of anhydrous (pre-dried with molecular sieves) acetone and 1 mL of PEG-silane. After bonding, the channel was immediately (within 30 s of bonding) filled with the PEG solution and allowed to react for 1 h at RT.20,30 The channel was 9
washed with deionized water (10 mL at 2 mL/min or 0.5 mL/min for the 100 µm chambers) and then left to hydrolyze for 1 h at RT. After, the chamber was washed with anhydrous acetone (10 mL at 2 mL/min or 0.5 mL/min for the 100 µm chambers) and reacted with a solution of 1.25 mL anhydrous acetone, 750 µL HDI, and 4 µL DBDTL for 45 min at RT. Next, the chamber was washed with anhydrous acetone and reacted with a solution of 1.621 mL anhydrous acetone, 375 µL DMEA, and 4 µL DBDTL for 30 min at RT. The chamber was then washed with deionized water and reacted with a solution of 1.61 mL anhydrous acetone and 390 µL of PS for 1 h at RT. Finally, the chamber was rinsed with deionized water for 10 minutes at 2 mL/min (0.5 mL/min for the 100 µm chambers) and dried with air.
X-ray photoelectron spectroscopy (XPS) XPS spectra were obtained from a Kratos Axis Ultra XPS with a monochromatic aluminum source. Survey scans were performed over an area of 2 x 1 mm and averaged to create the spectrum. These scans were done with a pass energy of 160 eV and an energy resolution of 0.5 eV. Additional scans were performed centering on the C-1s, O-1s, N-1s, S-2p, and Si-2p binding energies to confirm presence of elemental peaks and chemical shifts. XPS spectra was analyzed using CasaXPS.34
Contact Angle Measurement Advancing contact angles were measured using the sessile drop method (deionized water, 8 µL drop size). Images with the droplets were taken using a custom-built goniometer with an AmScope UCMOS series microscope camera. Images were analyzed using the contact angle plugin for ImageJ.35,36 10
Ambient Study Devices were stored in a covered, but unsealed petri dish until the time of measurement. At specific time points (0, 3, 7, 14, 21, 28, 35 days), three devices were cut open and the contact angles were measured. An additional study was done in ambient where three devices were cut open immediately after coating. The cut open samples were dried with air and stored in a covered, unsealed petri dish. The contact angles of the samples were measured at time points of 0, 3, and 7 days, drying after each measurement.
Static Fluid Study Devices with 840 µm tall flow chambers were made for each coating and allowed 7 days to stabilize under ambient conditions. The purpose of this stabilization period is to allow plasmaactivated PDMS and surfaces with unsuccessful grafting procedures to recover their hydrophobicity, thereby avoiding false positive results. After the stabilization period, each device was filled with PBS and closed off to ensure no leakage of PBS. After 7 days of exposure to static PBS, the devices were dried with air, cut open, and the contact angle was measured. The timing of ambient, static, and dynamic measurements is shown in Scheme 2.
Scheme 2. Schematic of testing, listing where contact angle measurements were taken for the ambient, shear stress, and static studies.
11
Dynamic Flow Study Devices with 840 µm tall flow chambers and each coating were tested under dynamic flow conditions corresponding to venous (100 s-1, 0.9 dynes/cm2), arterial (650 s-1, 5.9 dynes/cm2), and arteriole (1700 s-1, 15.3 dynes/cm2) shear rates.32 Equation 1 was used to calculate the flow rate needed to produce the specified shear rates. Q=
h2 wγw 6
(1)
Where Q is the volumetric flow rate, h is the channel height, w is the channel width, and 𝛾𝑤 is the shear rate. Equation 1 is valid when w>>h. As the channel height in this study was 8% of channel width, we also calculated the shear rate using the general expression for rectangular pipe flow and found the values were within 5%.32,37 The channel dimensions do not change under the wet conditions of the study, as water and therefore PBS does not swell PDMS.38,39
Shear stress is then calculated by multiplying the shear rate by the viscosity of the fluid in poise, assumed for PBS to be 0.9 cP. Three samples per condition and coating were fabricated, stabilized in ambient for 7 days, and then connected to Cole Palmer MasterFlex L/S peristaltic pumps at the appropriate flow and shear rates. Control devices were exposed to the same plasma treatment then
12
allowed to stabilize for 7 days prior to the experiment. All samples were left in the flow circuit for 7 days, then removed and cut open for contact angle measurements (timing in Scheme 2).
Platelet Adhesion Study For this study, devices with 100 µm tall flow chambers were used to limit reagent use. PEG, SBSi, PEG-SBSi, and control devices were stabilized in ambient conditions for 4 days prior to testing. Devices were primed with filtered PBS for 15 min prior to PRP flow to ensure even filling of the channels. CaCl2 was added to PRP at a concentration of 400 µL/10 mL PRP to completely reverse the citrate-based anticoagulation, then heparin was added at 100 µL/10 mL PRP to achieve an active coagulation time (ACT) between 500-600 s. PRP was then flowed through the device for 30 min at 0.65 mL/min to give a shear rate of 650 s-1, shear stress of 22.8 dynes/cm2 (assumed PRP viscosity 3.5 cP32), in a 37 °C water bath. After, devices were washed with cellulose acetate, 0.22 µm filtered PBS at the same flow rate for 5 min, then dried with air. 10 mM Calcein AM in DMSO solution was then added to filtered PBS at 2 µL/mL PBS, and the resulting solution was used to fill each chamber. The filled chambers were incubated at 37 °C for 1 h, then washed with PBS for 5 min at 0.65 mL/min, and dried with air. The resulting stained chambers were imaged using a 10x fluorescent microscope (0.5 s brightfield, 6 s FITC exposure). Four images were taken across the bottom of each chamber; near the inlet and outlet, then left-center and right-center, all centered along the midline. Particles that were >70% in circularity and within range of recorded platelet diameter (1-5 µm)40 were isolated via conversion to gray scale, then thresholding to produce a binary image where platelets are black. The resulting binary images were then quantified via fractional area measurement of the platelet area in ImageJ. The measured FITC images were adjusted by maximizing the brightness and contrast of the images, then decreasing the brightness 13
to yield bright platelets while eliminating background fluorescence. The resulting images were then converted to binary using ImageJ, and the area fraction of the binary images was measured. An example of the image processing is shown in Scheme 3.
Scheme 3. Image processing steps for brightfield and FITC images.
Statistics Three flow chambers for each coating were tested for each study along with a plasma treated, control sample (uncoated). The XPS study compared the uncoated and three coated samples to an unmodified (no plasma treatment) sample. A single area, near the midpoint, on the top and bottom of each chamber was analyzed and the atomic compositions averaged. Three contact angle measurements (one on both ends and one in the middle, centered on the midline) were taken on both the top and bottom of each flow chamber, for a total of six measurements per chamber in the 14
ambient, static, and dynamic flow studies. The measurements across the three chambers were averaged and standard error calculated. For the platelet adhesion study, four images were taken near the midline along the length of the bottom of each chamber. The quantified data was then averaged across all images and outliers were removed using the standard outlier definition (removing points outside 1.5 times the interquartile region ± the highest or lowest value in the sample). Results were analyzed by a student’s t-test in Excel and were significant if p20° compared to 26
PEG-SBSi under ambient exposure on Day 14 and static exposure. At arteriole shear, the contact angle of PEG-SBSi was just above 20° and was a significant increase compared to PEG-SBSi static and ambient Day 14. PEG
SBSi
PEG-SBSi
Uncoated
90 80
Contact Angle (°)
70 60
50 40 30 20 10 0 Ambient Day Ambient Day 7 14
Static (0 s⁻¹)
Venous (100 s⁻¹)
Arterial (650 s⁻¹)
Arterioles (1700 s⁻¹)
Figure 7. Comparison for of contact angles for ambient Day 7 (end of stabilization period for flow samples), ambient Day 14, static PBS, venous shear rate, arterial shear rate, and arterioles shear rate (n=3). While static fluid and ambient tests can demonstrate the stability of a hydrophilic coating in some circumstances, for blood contacting medical devices, the coating needs to be stable under flow. Devices were exposed to shear rates of 100 s-1, 650 s-1, and 1700 s-1 in PBS flow for 7 days, corresponding to venous, arterial, and arteriole conditions.32 Data for PEG coatings demonstrated significant hydrophobic recovery, even at low shear rates. The expected failure point in the molecular structure would be the base silane group because of the precise conditions needed to 27
covalently bond these molecules to the surface of PDMS. However, all three coatings had the same silane group as its reactive base for grafting (Scheme 1) and the other two coatings did not see the same hydrophobic recovery as PEG. In fact, PEG-SBSi had the entire PEG-silane molecule as its base and had contact angles under 20° after venous shear experiments. Therefore, the recovery of the PEG was likely because of interactions of the end methoxy (R-OCH3) group with water. This group can hydrolyze over time which could change the properties of the coating and cause entanglement. Contact angle data for SBSi showed no significant change from ambient Day 7 at low shear. This is hypothesized to be due to the small chain length of SBSi compared to the other coatings, resulting in a compact molecule that is more resilient to shear forces. But, SBSi did experience a transition point where it was effected by shear between the 650 s-1 and 1700 s-1 shear rates. Zwitterionic molecules also have charged elements at the end that can repel each other and help keep the end groups from interacting. This explains the low recovery of PEG-SBSi even through the molecule has a large chain length and has the PEG-silane base molecule. Unexpectedly, the uncoated sample improved under venous conditions likely due to the same reasons it improved under static conditions. This low shear rate provided it similar conditions to static PBS. The higher shear rate having lower hydrophobic recovery than the medium shear rates for PEG-SBSi and PEG was also unexpected. High shear rates have been shown to stretch and tilt polymer chains in the direction of flow, thus decreasing random chain orientation and surface entropy.51 This decrease in entropy allows increased wettability of the surface, during and immediately after flow. This is especially true for the PEG coated samples that do not have charged end groups to keep them upright with decreased entropy.
Platelet Adhesion Study 28
Figure 8 shows representative brightfield images of a chamber for each coating and an uncoated sample before and after PRP flow exposure. Images were taken from the bottom of the chamber near the midline in the direction of flow (inlet, right-center, left-center, outlet). Qualitatively, after comparing the images from before and after PRP flow, PEG-SBSi looks to have the best platelet resistance. After quantifying platelet area using ImageJ, SBSi shows the least platelet area of the brightfield images (Figure 9). Two outlying images were removed from the PEG and PEG-SBSi data, one from the SBSi, and none from the uncoated samples.
29
Figure 8. Brightfield images of surfaces a) before exposure to PRP and b) after to exposure to PRP at a 650 s-1 shear rate for 30 min showing adhered protein and platelets. All coatings showed a significant decrease in adhered platelet area compared to the uncoated samples. PEG, SBSi, and PEG-SBSi decreased adhesion by 82%, 86%, and 82%, respectively compared to the uncoated control.
PEG
SBSi
PEG-SBSi
Uncoated
0.6
Platelet Area Fraction (%)
0.5 0.4 0.3 0.2 0.1 0 1
Figure 9. Comparison of platelet adhesion area of brightfield images after exposure to PRP at a shear rate of 650 s-1 for 30 min (n=3). Representative images of Calcein-labeled platelets for each coating and the uncoated control is shown in Figure 10. Qualitatively, all coatings, showed a reduction in platelet adhesion compared to the uncoated control.
30
Figure 10. Fluorescence platelet adhesion images after exposure to PRP at a shear rate of 650 s-1. Figure 11 shows the average percent area that platelets occupied on the surface of the chamber, after removing one outlier from uncoated, PEG, and PEG-SBSi samples and two from SBSi samples. The uncoated samples displayed the most platelet adhesion, while each coating reduced platelet adhesion by varying amounts. PEG insignificantly reduced adhesion by 69% compared to the uncoated samples, while SBSi and PEG-SBSi significantly reduced platelet adhesion by 96% and 80%, respectively, compared to the uncoated controls.
31
PEG
SBSi
PEG-SBSi
Uncoated
0.035
Platelet Area Fraction (%)
0.03 0.025 0.02 0.015 0.01 0.005 0 0
Figure 11. Comparison of platelet adhesion area of fluorescent images after exposure to PRP at a shear rate of 650 s-1 for 30 min (n=3). An arterial shear rate was used to mimic physiological conditions while providing adequate platelet adhesion, while an ACT of between 500-600 s to provide adequate blood activity in the device, but to avoid clotting in the syringe and tubing.52 Devices with 100 µm tall flow chambers were utilized in this study (in contrast to the 840 µm tall chambers in the ambient, static, and dynamic flow studies) to minimize the amount of required reagents. That is, a smaller flow chamber requires a much smaller flow rate to achieve the same desired shear rate (Eqn. 1) thereby requiring much less PRP during the 30 min testing period and less Calcein AM stain after testing. Another factor which changed in the PRP tests was the stabilization period. In ambient and shear tests, a stabilization period of 7 days was used. During those tests, it became apparent that the 32
uncoated surfaces had exhibited significant recovery after 3 days (Figure 6). Thus, a 4 day stabilization period for the coatings was used in this platelet study. Initial platelet area quantification was done using the brightfield images which yielded the expected results that the coated samples were better than uncoated. This quantification was done by using thresholding to convert the images to binary (platelet areas black, background white), then measuring the fraction of area that is black. However, after images were taken of the coated samples without exposure to PRP, it was observed that the coatings have areas that look like platelets. Thus, an additional analysis was performed on the fluorescence images of the same sample and area of the brightfield images to confirm the quantified results. The dynamic flow study showed that at the arterial shear rate SBSi and PEG-SBSi were the most stable out of the three coatings when characterized by contact angle. This was again demonstrated in the platelet adhesion tests as SBSi and PEG-SBSi had the greatest percent reduction in platelet fluorescence area compared to the uncoated control. SBSi also had the greatest percent reduction in the binary brightfield images. SBSi showed the best resistance to platelet adhesion likely do its high hydrophilicity and stability under arterial shear.27,53 In the dynamic flow study, PEG showed the worst stability at this shear rate, which likely led to the increased platelet adhesion. PEG-SBSi was middle of the three for stability at the shear rate and platelet adhesion. Variance in the uncoated control also occurred in the contact angle measurements, likely due to variances in hydrophobic recovery as low molecular weight species move to the surface at different rates. A longer stabilization period may have thus reduced the variance of the uncoated controls.
Conclusion
33
In this study, we developed a simple, sequential reaction method for the coating of medical and microfluidic devices formed from PDMS. The long-term shelf life of the coatings was demonstrated by a closed channel ambient conditions study, resulting in most coatings with contact angles below 20° for time points up to 35 days. PEG and SBSi showed the longest stability in ambient, resulting in a long shelf life for the coatings. The static fluid study showed the potential additional stability under PBS exposure for the SBSi, PEG-SBSi, and uncoated plasma treated PDMS due to the continuous wetting of the surface. The flow and platelet adhesion study showed the potential use of the coatings in medical and microfluidic devices. PEG was less stable under all shear rates while SBSi and PEG-SBSi showed potential stability under a variety of shear rates. SBSi and PEG-SBSi had the greatest resistance to platelet adhesion, compared to the uncoated control, at the arterial shear rate. PEG had good shelf life, static fluid stability, and platelet resistance but lacked flow stability. SBSi had the most shelf life stability, best platelet resistance, and best stability under the low shear rates. PEG-SBSi had the shortest shelf life but was most stable across all fluidic conditions and showed good platelet resistance, like PEG. Overall, this sequential reaction method and the development of stable coatings under a variety of conditions can be used to coat more complex devices for a variety of applications.
Corresponding Author *Joseph A. Potkay,
[email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
34
Acknowledgments The authors would like to thank the Michigan Center for Materials Characterization and Dr. Kai Sun for their help with XPS analysis. This work was supported by Department of Veterans Affairs Rehabilitation Research and Development (VA RR&D) Award # I01 RX000390 and VA RR&D Award # C3819C, The Advanced Platform Technology Research Center of Excellence. The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.
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