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Jul 14, 2014 - Adjustable Bioadhesive Control of PEGylated Hyperbranch Brushes on Polystyrene Microplate Interface for the Improved Sensitivity of...
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Adjustable Bioadhesive Control of PEGylated Hyperbranch Brushes on Polystyrene Microplate Interface for the Improved Sensitivity of Human Blood Typing Yan-Wen Chen,†,‡ Yung Chang,*,‡,∥ Rong-Ho Lee,† Wen-Tyng Li,§ Arunachalam Chinnathambi,∥ Sulaiman Ali Alharbi,∥ and Ging-Ho Hsiue*,‡ †

Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan R&D Center for Membrane Technology and Department of Chemical Engineering and §Department of Biomedical Engineering, Chung Yuan Christian University, Chung-Li, Taoyuan 320, Taiwan ∥ Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Kingdom of Saudi Arabia ‡

ABSTRACT: A PEGylated 96-well polystyrene (PS) microplate was first introduced for applications in high-throughput screening for selective blood typing to minimize the risks in blood transfusions. Herein, we present a hemocompatible PS 96well microplate with adjustable PEGylated hyperbranch brush coverage prepared by ozone pretreated activation and thermally induced surface PEGylation. The grafting properties, hydration capacity, and blood compatibility of the PEGylated hyperbrush immobilized PS surfaces in human blood were illustrated by the combined chemical and physical properties of the surface, and the dependence of the specific absorption of human plasma fibrinogen onto the PEGylated surfaces on the grafting density was analyzed by monoclonal antibodies. The surface coverage of PEGylated brushes plays a major role in the bioadhesive properties of modified PS microplates, which in turn control the level of agglutination sensitivity in blood typing. The bioadhesive resistance toward proteins, platelets, and erythrocytes in human whole blood showed a correlation to the controlled hydration properties of the PEGylated hyperbrush-modified surfaces. Therefore, we suggested that the surface coverage of PEGylated hyperbrushes on PS surfaces can increase the sensitivity of cross-matching blood agglutination by up to 16-fold compared to that of the conventional 96-well virgin PS due to the regulated biorecognition of hematocrit and antibodies of the PEGylated hyperbrush-modified surfaces.

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INTRODUCTION Over the past 95 years, blood agglutination has been used as a means for erythrocyte typing, antibody screening, and blood matching. Coming into the modern era, it is essential to enhance the agglutination sensitivity in high-throughput typing before blood transfusions. Antifouling polymer brushes provide blood-contacting interfaces that reduce thrombogenicity via nonspecific plasma protein resistance in nanoscale fouling and may enhance the agglutination sensitivity of blood typing in a microscale spot.1−6 In general, poly(ethylene glycol) (PEG)based polymer brushes have been demonstrated to be ideal antifouling interfaces for their biofouling-resistant characteristics that prevent protein adsorption, cell adhesion, and bacterial attachment.5−9 Several approaches have been shown to immobilize PEGylated brush layers onto the target substrates through surface PEGylation via chemical grafting, such as UV, ozone, or gas plasma pretreatment followed by surface-initiated copolymerization.10,11 Similar to PEGylated grafting, surface PEGylation has also been shown to effectively improve the fouling resistance via physical adsorption such as in selfassembled coatings and spraying phisisorption, but the coating © 2014 American Chemical Society

stability becomes a major concern when applying these methods.10 However, while PEGylated surfaces have effective antifouling properties, earlier studies have also shown that the PEGylated brush coverage needs to be carefully modulated in order to achieve maximum blood compatibility and protein resistance.12−17 Transfusion is an alternative therapy to preventing intraoperative excessive bleeding and severe anemia. The intended use of various blood components (or blood products) including whole blood, packed red blood cell (RBCs), washed RBCs, RBC concentrate, platelet concentrate, and white blood cell (WBC) concentrate is dependent on the individual needs of patients for blood transfusions.18 The error in cross-matching of blood typing leads to hemolytic reactions in recipients, resulting in high mortality,19 and to prevent such errors, most hospital transfusion services use standard tube-type agglutination methods to perform compatibility tests, including ABO/D Received: April 16, 2014 Revised: June 29, 2014 Published: July 14, 2014 9139

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such microplates for blood typing was determined using human whole blood. This research is an original study aimed at revealing the relations between PS-g-PEGMA interfacial bioadhesive characteristics and cross-matching blood agglutination sensitivity.

typing, antibody screening, and cross-matching, due to their simplicity, reliability, and versatility. Previous studies developed the polybrene technique for RBC antibody screening using polystyrene (PS) microplates.18 To date, multiwell microplate screening technology has been adopted for high-throughput testing for selective blood typing to minimize the risks in blood transfusions.20 Importantly, the accuracy and sensitivity of agglutination tests have become important issues in screening blood types. Whether specific interactions between blood cells can be preserved when human blood is stored in hydrophobic PS-well microplates with different interfacial hydrophilicity via the regulated surface coverage of PEGylated polymer brushes is, however, still unclear. In this study, the systematic control of 96-well PS microplates grafted with a varying coverage of PEGylated hyperbranch brushes was developed using ozone-pretreated activation and thermally induced copolymerization, as shown in Figure 1. The surface composition and microstructure of the PS

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MATERIALS AND METHODS

Materials. In this study, nontreated 96-well polystyrene (PS) microplates (Falcon) were used for the base for the PEGMA hyperbrush-modified PS-g-PEGMA surfaces. The poly(ethylene glycol) methacrylate (PEGMA) hyperbrush was prepared using PEGMA with an average molecular weight of 500 Da and 10 ethylene glycol units (Sigma-Aldrich). The bioadhesive properties of the PS-gPEGMA surfaces were analyzed using fibrinogen protein (SigmaAldrich), DiaPanel anti-D antibody (DaiMed Corp), human blood, and a plasma sample provided by the Taiwan Blood Services Foundation. Phosphate-buffered saline (PBS) (Sigma-Aldrich) and deionized water purified to a minimum resistivity of 18.0 MΩ m with a Millipore water purification system were also used for this experiment. Surface PEGylation. In the surface PEGylation process, 96-well PS microplates were treated with a O3/O2 mixture with an O3 concentration of approximately 46 g/L at a flow rate of 6 L/min for 30 min at 25 °C via a custom-built ozone generator (model OG10PWA, Ray-E Creative Co., Ltd., Taiwan). The PEGMA hyperbrushes were then immobilized onto the ozone-treated surfaces via surface-induced thermal graft polymerization, where a 150 μL PEGMA macromonomer solution at concentration of 5 to 30 wt % in deionized water was applied to each well of the 96-well PS microplate and incubated at 80 °C for 24 h, producing different PEGylation coverages and surface hyperbrush structures summarized in Table 1. The surface grafting density of PEGylated hyperbrushes on the PS microplate was determined by the increased weight of the PEGylated PS microplates compare to that of the unmodified PS microplate. Surface Characterization. To evaluate the surface conformational structure of the PS-g-PEGMA surfaces, X-ray photoelectron spectroscopy (XPS) was used. A Thermal Scientific K-α spectrometer was used to measure the energy of photoemitted electrons at a takeoff angle of 45° with respect to the surface produced from the impact of 1487.6 eV photons emitted via a monochromatic Al K X-ray source. The pass energy of the hemispherical energy energy analyzer was set to 150 eV. The high-resolution C 1s spectrum was fitted by Shirley background subtraction and a series of Gaussian peaks, where the peak maximum in the C 1s spectrum was set to 284.6 eV and used as a reference for the binding energy (BE) scale. The amount of PEGMA hyperbrush deposited on the PS surface through the grafting process was determined by the change in dry weight of the respective surfaces in contact with the reaction solutions. The water contact angles were measured by placing a droplet of deionized water onto the sample surface and measuring the angle of

Figure 1. Schematic illustration of the preparation process of PS-gPEGMA via thermally induced surface PEGylation and the grafting chemical structure of PEGMA hyperbrushes grafted on PS well surfaces.

microplates grafted with poly(ethylene glycol) methacrylate (PEGMA) brushes (PS-g-PEGMA) were characterized by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The PEGMA brush layer grafted onto the PS microplate is needed for blood preservation without thrombogenicity. The increase in PEGylated coverage on PSwell microplates may reduce the blood cell attachment and enhance the blood agglutination sensitivity. The agglutination sensitivity of the Rh-D-type antigen from panel cells stored in

Table 1. Characteristic Data of PEGylated Hyperbrushes Grafted on PS-g-PEGMA reaction solution a

sample ID

PEGMA (wt %)

virgin PS PS-g-PEGMA-5 PS-g-PEGMA-10 PS-g-PEGMA-15 PS-g-PEGMA-20 PS-g-PEGMA-25 PS-g-PEGMA-30

0 5 10 15 20 25 30

characterization of PEGylated brushes 2 b

grafting density (mg/cm ) 0.20 0.37 0.61 0.65 0.84 0.87

± ± ± ± ± ±

0.03 0.01 0.04 0.01 0.03 0.02

relative protein adsorption

contact angle (deg)

[C−O]/[C−C, −H]

± ± ± ± ± ± ±

0.17 0.18 0.25 0.27 0.57 0.62

71.3 67.7 67.4 65.2 57.2 49.7 46.7

0.9 1.6 1.5 2.4 2.8 1.3 1.8

c

fibrinogen (%)d 100 44.1 24.3 18.3 15.5 8.0 6.8

± ± ± ± ± ± ±

1.2 9.1 8.4 0.6 9.5 0.4 0.7

human plasma (%)e 100 56.1 51.6 50.6 45.5 42.4 36.1

± ± ± ± ± ± ±

1.6 4.1 3.3 0.8 3.3 1.9 3.5

a

Ninety-six-well PS microplate prepared by ozone pretreatment and thermally induced surface PEGylation. bThe surface grafting weight of PEGylated hyperbrushes on the PS well plate was determined by the extent of weight increase compared to the virgin PS well plate based on the total surface area of the 96-well PS microplate in contact with the reaction solution. cThe ratio of [C−O]/[C−C, C−H] was characterized by XPS. d Relative protein adsorption of fibrinogen on the PS-g-PEGMA surfaces from single fibrinogen in PBS at 37 °C. eRelative protein adsorption of fibrinogen on the PS-g-PEGMA surfaces from 100% human plasma at 37 °C. 9140

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150 μL of PBS at 37 °C for 24 h. Once the PS-g-PEGMA surfaces were equilibrated, 150 μL of RBCs in PBS was added to the PEGylated surfaces. The PEGylated 96-well microplates containing RBCs were incubated in 37 °C water bath for 1 h before the solutions were collected and centrifuged at 2000 rpm for 5 min to remove viable RBCs from the solution. The resulting supernatant containing the released hemoglobin (Hb) from the dead RBCs was measured for absorbance at 541 nm via the PowerWave microplate spectrophotometer as an indicator of the biocompatibility of the tested PS-gPEGMA surfaces. RBCs suspended in deionized water were used as reference for 100% hemolysis, and 108 RBCs suspended in PBS were used as the control. Each hemolysis data point reported was the numerical average of six independent measurements (n = 6). Sensitivity of Blood-Typing Assay. The detailed standard protocol for the polybrene technique assay for blood typing tests is described in the following sections.18

water contact over 10 s at 25 °C via an FTA1000 automatic contact angle meter (First Ten Ångstroms). The hydration capacity (mg/ cm2), defined as the differences in wet weight between PS-g-PEGMA and unmodified PS, was also measured. The dry state PS-g-PEGMA surface conformational structures were observed under a bioatomic force microscope (bio-AFM), and the AFM images were acquired with a NanoWizard scanner on the multimode NanoWizard (JPK Instruments AG). Human Fibrinogen Adsorption. In evaluating the protein absorption of the PS-g-PEGMA surfaces, human fibrinogen protein absorbed onto the PS-g-PEGMA surfaces was measured by enzymelinked immunosorbent assay (ELISA) following standard protocols detailed in previous studies.21 Blood Platelet Adhesion Test. To observed the activated platelet adhesion on the PS-g-PEGMA surface, platelet-rich plasma (PRP) containing roughly 1 × 105 platelet/mL was prepared by centrifuging the human whole blood samples at 120 rpm for 10 min. PRP (150 μL) was then allocated into treated 96-well microplates and incubated for 120 min at 37 °C. After the incubation, the treated well surfaces were washed with 150 μL of PBS two times and cooled to 4 °C in 2.5% glutaraldehyde for 48 h. The fixed platelet on the treated surface was then washed again with 1000 μL of PBS. The washed samples were dried by covering the treated surfaces with 0, 10, 25, 50, 90%, and 100% (v/v) ethanol in PBS for 20 min at each step. The plateletattached samples were air dried before they were cut and sputter coated with gold for analysis with the JSM-5410 scanning electron microscope (SEM) (JEOL) operating at 7 keV. Erythrocyte Adhesion of Human Whole Blood. In order to evaluate the ability of PS-g-PEGMA surfaces to prevent erythrocyte adhesion, a total of 250 mL of fresh human whole blood donated by health volunteers was collected and treated with 25 mL of citrate phosphate dextrose adenine-1 (CPDA-1) to stop blood coagulation. Before the treated blood samples were allocated onto the PEGMA hyperbrush-immobilized surfaces, the PEGylated PS surfaces were equilibrated by 24 h of incubation at 37 °C in 150 μL of PBS. Once the PEGylated PS surfaces were equilibrated, they were placed in direct contact with 150 μL of CPDA-1-treated human whole blood and incubated at 37 °C for 2 h. Following the incubation, the blood solution was removed and the surface was washed two times with 150 μL of PBS. Cells that remained adhered to the treated PS surfaces were fixed with 4.0% formaldehyde in PBS for 15 min at 4 °C and then washed again three times with 150 μL of PBS. The fixed blood cells were stained with 2.5% glutaraldehyde at 4 °C for 24 h and then washed with PBS three times to remove residual glutaraldehyde. The washed samples were imaged under confocal laser scanning microscopy (CLSM) (Nikon, CLSM AIR) at 200× magnification to observe the morphology of the attached cells. For each sample, five independent images were taken. Plasma-Clotting Time Measurement. To study the anticoagulative properties of the prepared membrane, the plasma-clotting time of platelets of human normal plasma containing 20 mM CaCl2 was measured as the start of an absorption transition at 660 nm at 37 °C. The platelet-poor human normal plasma donated by three volunteers was collected by two consecutive 10 min centrifugations at 1200 rpm at 25 °C. The resulting plasma solution was recalcified to contain 20 mM CaCl2 by an added 1 M CaCl2 stock solution. The recalcified human normal plasma was shaken at 37 °C for 30 s before being allocated into 500 μL samples in a nontreated 24-well PS plate, where the absorbance transition was measured via a PowerWave microplate spectrometer under constant temperature control. Each plasma-clotting time reported in this study was the numerical average of six independent measurements (n = 6). Hemolysis of Red Blood Cell. To assess the biocompatibility and hemolysis of red blood cells on PS-g-PEGMA surfaces, red blood cells (RBCs) were isolated from centrifuged blood samples and washed with 0.15 M saline solution three times. The number of RBCs was counted, and they were allocated into 15 mL samples each containing roughly 108 RBCs in PBS. Before the biocompatibility of the PS-gPEGMA surfaces were tested, the surfaces were first equilibrated by

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RESULTS AND DISCUSSION Surface PEGylation and Characterization. In this study, we explore the ability of PEGMA hyperbrush-grafted PS-gPEGMA surfaces to control the bioadhesion of proteins, platelets, and erythrocytes. Varying surface properties, including the grafting density, hydration capacity, and hydrophilicity, were explored for their effects on the bioadhesion of plasma proteins and blood cells on PS-g-PEGMA surfaces. The varying surface properties of the tested PS-g-PEGMA surfaces were prepared under the different polymerization conditions summarized in Table 1. The surface graft polymerization of the PEGMA hyperbrush on PS surfaces is a two-step process as illustrated in Figure 1. To create reactive peroxide sites on 96-well PS microplates, the surfaces were first treated with a gaseous O3/O2 mixture at a controlled flow rate and O3 to O2 ratio. To optimize the amount of peroxide in the treated PS surfaces to around 2.85 nmol/cm2, 2,2-diphenyl-picrylhydrazyl (DPPH) was used to determine the surface peroxide density following a standard protocol detailed in previous publications.7,9 The ozone treatment time was also tested to prevent unfavorable etching and degradation of the PS surfaces as a result of prolonged ozone treatment, and the treatment time was also experimentally optimized to 30 min for the purpose of this study. In the second stage of the preparation of PS-g-PEGMA surfaces, the PEGMA macromonomers were copolymerized onto the surfaces of 96-well PS microplates by thermally induced radical copolymerization. In the thermally induced radical copolymerization, the reaction temperature and time regulate the decomposition of peroxide and the copolymerization of PEGMA macromonomers on the ozone-treated PS surfaces. The reaction condition of 24 h at 80 °C was experimentally shown to be able to completely decompose peroxide on the ozone-treated PS surfaces and copolymerize PEGMA macromonomers. The grafting morphology of the grafted PEGMA hyperbrushes is the primary factor that determines the hydration capacity and hydrophilicity of PS-g-PEGMA, and the surface grafting weight, water contact angle, and surface roughness of the modified surfaces are greatly influenced by the concentration of the PEGMA hyperbrush solution as shown in Figure 2. The water contact angle shown in red indicated that the hydrophilicity of the modified PS microplates surfaces increased with the weight of grafted PEGMA hyperbrushes, and as illustrated in Figure 2, a water contact angle of close to 45° can be achieved. The water contact angles also indicated that 9141

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PEGMA macromonomer reaction solution in thermally induced graft polymerization. To assess the surface conformational structure of the various PS-g-PEGMA surfaces, bio-AFM images were taken in the dry state under atmospheric conditions. The bio-AFM images of samples PS-g-PEGMA-5 through PS-g-PEGMA-30, shown in Figure 4, demonstrated the varying surface microstructual

Figure 2. Changes in the surface grafting weight, water contact angle, and surface roughness for prepared PS-g-PEGMA. (−■−, surface grafting weight; −●−, contact angle; −▲−, surface roughness).

PEGMA polymer brush (PS-g-PEGMA) grafting was effective at increasing the hydrophilicity of PS surfaces whose water contact angle was approximately 70°. To better understand the properties of the PS-g-PEGMA surfaces, we characterized the surface composition and conformational structure by XPS and bio-AFM. The XPS spectroscopy, shown in Figure 3, showed evident C−C and C−

Figure 4. Tapping-mode bio-AFM images of surface morphology and RMS roughness of PS-g-PEGMA: surface images in (a) virgin PS, (b) PS-g-PEGMA-5, (c) PS-g-PEGMA-10, (d) PS-g-PEGMA-15, (e) PS-gPEGMA-20, (f) PS-g-PEGMA-25, and (g) PS-g-PEGMA-30. The dimensions of the scan images are 30.0 μm × 30.0 μm.

properties in PS-g-PEGMA surfaces while unmodified PS surfaces displayed flat surface morphologies across all samples examined. The increased surface roughness and grafting weight, shown in Figure 2, were caused by the increased thickness and coverage of the PEGMA hyperbrush on the hydrophobic PS surfaces. The surface roughness of PS-g-PEGMA, however, peaked for sample PS-g-PEGMA-15, which had a surface coverage of PEGMA hyperbrushes of 0.61 mg/cm2. The increase in the PEGylated surface roughness from ∼13.5 nm (PS-g-PEGMA-5) to ∼203.4 nm (PS-g-PEGMA-15) showed that PEGMA hyperbrushes grafted onto 96-well PS plates were able to keep their flexible conformation in solutions due to the sufficient spacing between polymer brushes. In contrast, the decrease in the PEGylated surface roughness from ∼109.6 nm (PS-g-PEGMA-20) to ∼56.58 nm (PS-g-PEGMA-30) suggested a high surface coverage of the PEGMA hyperbrushes.21 The AFM analysis further supports the statement that the grafting conformation of the PEGylated brushes is strongly associated with their surface packing density, coverage, and flexibility. Correlation of Protein Adsorption and Hydration Capability of the PEGylated PS Microplates. To gain insight into the protein absorption on the hydrated membrane surfaces, we evaluate the hydration capacity and the hydrophilicity of the hydrophilic layer of the prepared sample surfaces.22−25 The hydration capacity of the prepared surfaces exhibited a positive relation to the weight of the PEGMA hyperbrush grafting, i.e., the thickness of the PEGMA grafting affected the hydration capacity of the PEGMA hyperbrushimmobilized surfaces positively as shown in Figure 5. Furthermore, the increased hydration capacity from the increased PEGMA hyperbrush grafting thickness enhanced the resistance of the PS-g-PEGMA surfaces against nonspecific

Figure 3. XPS spectra of the modified PS surfaces with PEGylated hyperbrushes in the C 1s region of the sample: (a) virgin PS, (b) PS-gPEGMA-5, (c) PS-g-PEGMA-10, (d) PS-g-PEGMA-15, (e) PS-gPEGMA-20, (f) PS-g-PEGMA-25, and (g) PS-g-PEGMA-30.

H groups, indicating the presence of a benzene moiety. The grafted PEGMA hyperbrushes on PS-g-PEGMA surfaces, on the other hand, were ascertained by the C 1s core-level peak components for PS-g-PEGMA specific species, [C−C, C−H], [C−O], and [O−CO] at binding energies of 284.3, 286.0, and 288.7 eV, respectively, where the increased surface coverage of PEGMA caused the shifting of characteristic [C− C, C−H] peaks from 283.8 eV in virgin PS to 284.3 eV in PS-gPEGMA. Similarly, the relative intensity of [C−O]/[C−C, C− H] increased from 0.17 to 0.62 when the PEGMA hyperbrush grafting increased to 0.87 mg/cm2 from 0.20 mg/cm2. These results suggested that the surface coverage of the PEGMA hyperbrush grating is dependent on the concentration of the 9142

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protein absorption over those with low PEGylated hyperbrush coverages, PS-g-PEGMA-5. As the human platelet adhesion tests have become an established technique for estimating the hemocompatibility of a prepared substrate surface,28 we examined the SEM images of platelets clinging to the PS-g-PEGMA surfaces by placing the prepared microplates in direct contact with recalcified PRP solution at 37 °C for 120 min in vitro. As shown in Figure 6, the spreading of platelets on an unmodified PS surface was clearly observed, indicating the activation of the platelet. On the other hand, SEM images in Figure 6b−g exhibited suppressed platelet adhesion on PS-g-PEGMA surfaces as compared to an unmodified PS plate. In summary, we observed no platelet adhesion on the PS-gPEGMA surfaces as the PEGMA polymer brush coverage exceeds 0.84 mg/cm2; however, as the PEGMA polymer brush coverage went below 0.37 mg/cm2, a significant number of adhered activated platelets were observed on the PS-g-PEGMA surface. Overall, the PS-g-PEGMA-30 microplates showed outstanding performance in preventing platelet adhesion and blood platelet activation by its ability to prevent nonspecific protein adsorption. The results also suggest that the wellhydrated thermally induced PEGylation of the PEGMA hyperbrush on PS surfaces can effectively lower protein adsorption and platelet adhesion/activation while enhancing hemocompatibility. Erythrocyte Attachment and RBCs Hemolysis. Stable blood compatibility is highly desirable in biomedical devices that may be used for applications that come into contact with human blood.2 In this study, we examined the hemocompatibility of PEGMA hyperbrush-modified PS surfaces by evaluating the extent of erythrocyte attachment from human whole blood in direct blood−membrane contact. The LSCM images of blood cells on PEGMA-grafted membrane after submerging the prepared membrane in 100% human whole blood for 120 min at 37 °C in vitro is shown in Figure 7. Thermally induced surface PEGylation, PS-g-PEGMA-30 shown in Figures 6g and 7, demonstrated a clear blood-inert characteristic where the surface showed high resistance toward platelet adhesion and activation. Statistical quantitative data of relative erythrocyte attachment on the PS-g-PEGMA surfaces, presented in Figure 8, collected from image analysis SimplePCI software demonstrated that the resistance toward erythrocyte attachment of the PS-g-PEGMA surfaces was independent of the grafting density of PEGMA hyperbrushes.

Figure 5. Changes in the relative protein adsorption and hydration capacity for the prepared PS-g-PEGMA as a function of the surface grafting weight of PEGylated hyperbrushes on the PS well surfaces.

fibrinogen absorption in both single protein and 100% plasma solutions. The relative protein absorption on PS-g-PEGMA surfaces was lowered to below 10% of that observed on unmodified PS surfaces when the hydration capacity was increased to 0.8 mg/ cm2. This result indicated that the grafting structure of hyperbranch PEGMA brushes was effective in resisting nonspecific protein adsorption. On the basis of our current understanding of antifouling mechanisms, we hypothesized that the water molecules bound around the antifouling chains of ethylene glycol pendent groups were essential for resisting nonspecific protein adsorption.26,27 As a result, the hydrophilicity, hydration capacity, and PEGMA hyperbrush coverage need to be examined to accurately assess the protein absorption on hydrated PS-g-PEGMA surfaces. Surface Hemocompatibility of the PEGylated PS Microplates. Because the platelet adhesion and activation in blood were controlled by the plasma protein absorption on surfaces, here we evaluate the relative fibrinogen protein absorption onto PS-g-PEGMA microplates in PPP solution by ELISA with monoclonal antibodies. ELISA results, shown in Figure 5, indicated that the PS-g-PEGMA membranes were highly resistant toward fibrinogen absorption compared to that of unmodified PS at 37 °C. In support of this observation, surfaces with a high coverage of PEGylated hyperbrushes, PS-gPEGMA-30, showed improved resistance toward plasma

Figure 6. SEM images of platelets adhered onto the PS-g-PEGMA surfaces of (a) virgin PS, (b) PS-g-PEGMA-5, (c) PS-g-PEGMA-10, (d) PS-gPEGMA-15, (e) PS-g-PEGMA-20, (f) PS-g-PEGMA-25, and (g) PS-g-PEGMA-30. All images were obtained at a magnification of 1000×. 9143

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Figure 7. Camera photographs and LSCM images of erythrocytes adhered onto the PS-g-PEGMA well surfaces of virgin PS, PS-gPEGMA-5, PS-g-PEGMA-15, and PS-g-PEGMA-30. All camera and LSCM images are at magnifications of 8× and 200×, respectively.

Figure 9. Hemolysis of RBC solution in the presence of modified PS well surfaces with grafted PEGMA polymer brushes. (n = 6).

disruption of RBCs to approximately 1.5%. As for PS-gPEGMA surfaces with a grafting density lower that that of 0.37 mg/cm2, strong resistance toward erythrocyte attachment and RBCs homolysis was observed, which suggested that the PS-gPEGMA microplates prepared by simple thermally induced surface PEGylation were able to achieve high hemocompatible if the conformational structure and grafting density of PEGMA hyperbrushes on PS were properly adjusted. Cross-Matching Blood Agglutination. Cross-matching blood agglutination is a test to evaluate the blood compatibility of a donor and a recipient, which decrease the risks associated with blood transfusion. In cross-matching blood agglutination, a sample of the patient’s serum is mixed with a sample of the donor’s red blood cells (RBCs). If agglutination occurs or clumps form upon mixing the samples, then the recipient cannot accept a blood transfusion from this particular donor.30,31 In this study, a 96-well PS microplate was used for highthroughput cross-matching blood screening. PS-g-PEGMA-30 microplates were used to test the Rh-D blood type using manual polybrene cross-matching method,18,32 where the positively charged polybrene was added as an agglutination enhancer. Figure 10 shows the images and quantitative data of blood coagulation caused by reactions between cross-matching solution (3% RBCs solution mixed with a set of D-type antibodies in different dilutions from 1× to 128×) and unmodified PS as well as PS-g-PEGMA-30. As we can see in Figure 10, as the cross-matching solution interacts with the hydrophobic virgin PS microplate, blood coagulation becomes unclear and the agglutination score decreases to +2 as the Dtype antibody dilution factor increases to ×2. The result indicates that the hydrophobic PS well is a highly activating interface which interrupts the specificity between the D-type antibody and RBCs due to erythrocyte attachment and RBC hemolysis on PS microplate wells. As cross-matching solution was added to the PS-g-PEGMA30 microplate, the D-type antibody dilution factor increased to 32× as the agglutination score remained above +2 with clear blood coagulation for easy blood type screening. High resistance toward protein adsorption, platelet adhesion, and blood cell attachment was clearly observed in the anticoagulant activity of PS-g-PEGMA-30, which had a high surface grafting

Figure 8. Number of erythrocytes attached and in the presence of modified PS well surfaces with grafted PEGMA polymer brushes (n = 6).

However, a correlation between surface roughness and the resistance of erythrocyte attachment was also observed, as shown in Figure 3 where enhanced erythrocyte attachment on PS-g-PEGMA surfaces was observed as the surface roughness increased. These results are consistent with previous findings showing favorable erythrocyte attachment on the high-roughness surfaces.29 It is also important to note that an optimal surface roughness for the grafted PEGMA hyperbrush structures on the PS-g-PEGMA-15 surfaces was observed for the attachment of erythrocytes. To assess the correlation of blood compatibility to PEGylation coverage of the PS-g-PEGMA surfaces, we programed an RBCs hemolysis assay where the hemolysis of RBCs in 37 °C deionized water served as the control to which the hemolytic activities of PS-g-PEGMA surfaces of different PEGMA surface coverages were normalized, as shown in Figure 9. In summary, the hydrophobic surfaces were able to interact with blood-cell membranes, causing disruption, which in turn resulted in ∼4% hemolytic activity exhibited by the virgin PS microplate. On the other hand, the PS-g-PEGMA surfaces with a grafting density of 0.37 mg/cm2 or higher displayed minimal hemolytic activity, and the low hemolytic activity was attributed to the antihemolytic activity of the highly hemocompatibility of the PEGMA hyperbrushes capable of limiting the membrane 9144

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RA002-11757), and the Ministry of Science and Technology (100-2628-E-033-001-MY3 and 102-2221-E-033-009-MY3) for their financial support. The Deanship of Scientific Research, College of Science Research Center, King Saud University, Kingdom of Saudi Arabia, is also acknowledged. Yung Chang thanks King Saud University, Riyadh, Kingdom of Saudi Arabia, for a visiting professorship.

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Figure 10. Blood-typing images and blood aggregation areas on the PS-g-PEGMA well surfaces. These measurements were carried out six times for each well (n = 6).

density of the PEGMA hyperbrush. In summary, the highsensitivity, high-throughput screening for cross-matching blood can be performed with ease by the use of thermally induced surface PEGylation, but it is important to keep in mind that the surface grafting density and the PEGylated structures greatly affect the hemocompatible of the surfaces for human blood.

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CONCLUSIONS The effects of surface grafting conditions of PEGMA hyperbrushes on the hemocompatibility of the PEGylated surfaces in human blood were studied. It was found that the resistance to protein absorption, platelet adhesion, and erythrocyte attachment was enhanced by increased surface PEGMA hyperbrush coverage in human blood due to the improved interfacial hydration capacity of the surfaces in aqueous solution. Furthermore, the result indicated that the nonattachment of RBCs can be achieved in human whole blood by modifying the bioadhesive activity of the surfaces via the PEGMA hyperbrushes. The PEGylated hyperbrushes with a grafting coverage of 0.86 mg/cm2 presented cross-matching blood agglutination with a high screening sensitivity that could enhance the 2-fold antibody dilution factor of the virgin PS microplate to the 32-fold antibody dilution factor of the PS-gPEGMA microplate. This study shows that the adjustable surface grafting coverage of PEGylated hyperbrushes on the 96well PS microplate is essential for achieving highly sensitive cross-matching blood in a high-throughput screening.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the Outstanding Professor Research Program of Chung Yuan Christian University, Taiwan (CYCU-00RD9145

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