Reduced Blood Cell Adhesion on Polypropylene Substrates through a

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Reduced blood cell adhesion on polypropylene substrates by a simple surface zwitterionization Sheng-Han Chen, Yung Chang, and Kazuhiko Ishihara Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b03295 • Publication Date (Web): 01 Nov 2016 Downloaded from http://pubs.acs.org on November 2, 2016

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Reduced blood cell adhesion on polypropylene substrates by a simple surface zwitterionization

Sheng-Han Chen,1, 2 Yung Chang,1, 2* and Kazuhiko Ishihara3, 4*

1

R&D Center for Membrane Technology, 2Department of Chemical Engineering, Chung Yuan University, Chung-Li, Taoyuan 320, Taiwan 3

Department of Materials Engineering, 4Department of Bioengineering,

School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

*To whom all correspondence should be addressed

ACS Paragon Plus Environment

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ABSTRACT To conquer thrombogenic reactions of hydrocarbon-based biomaterials in clinical blood treatment, we introduce a model study of a surface zwitterionization of polypropylene (PP) substrate using a set of well-defined copolymers for controlling the adhesion of blood cells in vitro. Random and block copolymers containing zwitterionic units

of

2-methacryloyloxyethyl

phosphorylcholine

(MPC),

[3-(methacryloylamino)propyl] dimethyl(3-sulfopropyl)ammonium hydroxide inner salt (SBAA), or non-ionic units of 2-hydroxyethyl methacrylate (HEMA) with a controlled hydrophobic segment of 70% n-butyl methacrylate (BMA) units in these polymers were synthesized by reversible addition-fragmentation chain transfer polymerization. A systematic study of how zwitterionic and non-ionic copolymer architectures associated with chain orientation control via hydration processes affect blood compatibility is reported. The surface wettability of PP substrates coated with the block copolymer with poly(MPC) (PMPC) segments was higher than that of the random copolymer, poly(MPC-random-BMA). However, only the random copolymers with SBAA units demonstrate the higher surface wettability. The PP substrate coated with non-ionic copolymers containing HEMA units showed relative lower hydration capability associated with higher protein adsorption, platelet adhesion, and leukocyte attachment than

those

with

zwitterionic

copolymers.

The

random

copolymer

of

poly(SBAA-random-BMA) coated on PP substrates exhibited resistance to cell adhesion in human whole blood at a level comparable to that of the MPC copolymers. An ideal zwitterionic PP substrate could be obtained by coating it with block copolymer composed of PMPC and poly(BMA) (PBMA) segments, PMPC-block-PBMA. The water contact angle decreased dramatically from ca. 100° on the original PP to 11° within 30 s. The number of blood cells attached on the PMPC-block-PBMA decreased significantly to less than 2.5% that on original PP. These results supported that the rational design of zwitterionic polymers incorporated with a hydrophobic anchoring portion provides a promising approach to reduce blood cell adhesion and protein adsorption of hydrocarbon-based biomaterials applied in directly contacting with human whole blood.


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KEYWORDS Zwitterionic polymers; blood compatibility; hydration; protein adsorption; cell adhesion.


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INTRODUCTION

Blood cells-inert interface from antifouling materials to resist the adsorption, adhesion, and activation of plasma proteins, platelets, erythrocytes, and leukocytes are crucial in the design of blood-contacting biomaterials for antithrombogenic implants or devices.1-3 General molecular characteristics of blood-inert functional groups are hydrophilic, charge neutral, hydrogen bond acceptors, but not hydrogen bond donors.4 In this category, hydroxyl group and poly(oxyethylene) chain have been examined.5-9 These functional groups tightly bind water molecules via hydrogen bonding. This water hydration barrier believed to make reduction of protein adsorption and blood cell adhesion. Previously, as these polymers, poly(2-hydroxyethyl methacrylate (HEMA) (PHEMA) and poly(ethylene glycol) (PEG) have been applied for preventing the thrombus formation. However, the blood-contacting durability of these polymers is always a major concern. Bioinspired zwitterionic polymers with a pair of opposite charges in pendant groups, which can be classified as phosphorylcholine (PC: PO4− and N+(CH3)3), sulfobetaine (SB: N+(CH3)3 and SO3−), and carboxybetaine (CB: N+(CH3)3 and COO−), have become the new generation of robust blood-inert biomaterial systems beyond HEMA copolymers and PEG containing polymers.10-12 2-Methacryloyloxyethyl phosphorylcholine (MPC) bearing a PC group in the side chain is the most widely used zwitterionic compound in the preparation of biocompatible phospholipid polymers.13-15 Sulfobetaine methacrylate (SBMA) is also a potential zwitterionic material, which can form an ultra-hydrated interface to resist non-specific protein adsorption, blood cell attachment, human tissue adhesion, and bacterial attachment.16-18 Previous studies of poly(MPC) (PMPC)- and poly(SBMA) (PSBMA)-grafted substrates showed that surface packing density, grafting coverage, polymer chain conformation, and chemistry are critically important in the design of a zwitterionic interface that exhibits excellent blood compatibility at a molecular level.19-21 Thus, efficient surface zwitterionizaiton, such as photo-, thermal-, and plasma-induced grafting from polymerization have been developed to improve the blood compatibility of general hydrophobic biomaterials. The common methods of surface zwitterionization are generally developed from the typical approaches of “grafting from” and “grafting onto” for the design and creation of protein-resistant and blood-inert interfaces. Surface zwitterionization via the grafting


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from approach of surface-initiated controlled living radical polymerization techniques provides the high surface coverage and low packing defects of zwitterionic polymer brushes, such as PMPC and PSBMA brushes, on various biomaterial surfaces.22-25 Although the bio-inert performance of protein adsorption, blood cell adhesion, bacterial attachment, and biofilm formation are almost perfect on those surfaces, tubular-shape, patterned-spot, and large-scale surface zwitterionization are still difficult because surface pretreatment, long reaction time, and multiple preparation steps are usually required during the modification processes. A simple, versatile, and effective method of surface zwitterionization via the grafting onto approach through physical coating takes several attractive advantages in an easy operation and suitable preparation for large-scale surface modification.26-27 Poly(MPC-co-n-butyl methacrylate (BMA)) is the most well-studied case to modify various materials including polymers, metals, and ceramics. The good blood compatibility of those modified surfaces was performed when the copolymer composition, segment sequence, and polymer density of poly(MPC-co-BMA) are precisely controlled and optimized. Importantly, the adhesion and activation of blood cells on the interface of the MPC polymer-modified substrate were suppressed when the MPC unit composition was above 25 mol%.28 Although zwitterionic MPC polymer coating on general hydrophobic substrates has been well studied, it is still unclear that the blood cells-inert control of zwitterionic copolymers coated on polypropylene (PP) substrates in human blood. It is also important to figure out that the effects of zwitterionic and non-ionic copolymer architectures with controlled chain orientation via hydration process on protein adsorption and blood cell attachment. In this study, random and block copolymers synthesized by zwitterionic monomers (MPC and SBAA) and non-ionic monomers (HEMA) with a theoretical composition control of 70 unit mol% BMA were coated onto PP substrates, as shown in Figure 1. The hydrophobic BMA unit is required for zwitterionic or non-ionic copolymer anchoring onto the hydrophobic PP substrates, and the MPC, SBAA, or HEMA units are compared in terms of their role in controlling blood compatibility in human blood. Although an increase in the BMA unit composition in the prepared copolymers might enhance polymer anchoring to the target substrates, blood cell resistance may be decreased because of hydrophobicity. The proper control in chain orientation of MPC, SBAA, or HEMA units associated with wetting properties


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needs to be determined to optimize overall blood compatibility. The random sequence and block-type monomer unit architectures of copolymer-coated PP substrates were also considered and compared. Human fibrinogen was used to test the protein-level antifouling property of the substrates. Blood cell-contact tests were performed using platelets, erythrocytes, and leukocytes adhesion from human blood to evaluate blood cell resistance.

EXPERIMENTAL METHODS

Materials. MPC was purchased from NOF Co., Ltd. (Tokyo, Japan), which was synthesized

as

previously

reported

method.13

[3-(Methacryloylamino)propyl]dimethyl(3-sulfopropyl) ammonium hydroxide inner salt (SBAA) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). BMA, HEMA, and α, α’-azobisisobutyronitrile (AIBN) were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). 4-Cyano-4-(thiobenzoylthio)pentanoic acid (CPD) was obtained from Stream Chemicals Inc. (Newburyport, Massachusetts, USA). Other organic reagents and solvents were commercially available in extra-pure grade and used without further purification. Phosphate buffered saline (PBS) was purchased from Sigma-Aldrich. Deionized water used in the experiments was purified using a Millipore water purification system with a minimum resistivity of 18.0 MΩ·m. PP substrates with a thickness of 530 µm were provided by LCY Chemical Corp. (Kaohsiung, Taiwan).

Polymer synthesis. Random copolymers of poly(MPC-random-BMA) (PMB), poly(SBAA-random-BMA) (PSB), and poly(HEMA-random-BMA) (PHB) were synthesized via conventional free radical polymerization. In general, the monomers and AIBN as an initiator were dissolved in a solvent system of ethanol, methanol, and tetrahydrofuran (THF) at a total monomer concentration of 1.0 mol/L, respectively. The feed solution was put into the glass tube and bubbled with argon gas for 15 min to remove the residual oxygen. After sealing the glass tube, the polymerization was carried out at 60°C for 12 h. As the glass tube cooled to 25°C, the copolymer was purified and precipitated with poor solvents which summarized in Table 1. The collected copolymer was dried overnight in a glass vacuum container to remove the residual solvent and


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stored as a powder. Diblock copolymers were synthesized via reversible addition-fragmentation chain transfer polymerization (RAFT). For example, MPC, AIBN, and CPD as RAFT reagent (mole fraction: [total monomer]/[CPD]/[AIBN] = 30/1.0/0.20) were dissolved in the solvent (ethanol) at a total monomer concentration of 0.50 mol/L. After the polymerization was run at 60°C for 24 h, the collected PMPC was precipitated and used in the synthesis of diblock copolymers as a polymeric initiator. Next, the diblock copolymer was prepared using PMPC as a macroinitiator for RAFT polymerization. The PMPC macroinitiator and AIBN (mole fraction: [total monomer]/[polymeric initiator]/[AIBN] = 70/1.0/0.20) were dissolved in a cosolvent (ethanol/ THF = 3/ 1)at a total monomer concentration of 0.70 mol/L. After sealing the glass tube, the monomer solution was polymerized at 60°C for 30 h. As the glass tube cooled to room temperature, the copolymer was purified by reprecipitation with a poor solvent. Finally, the collected polymer PMPC-block-poly(BMA) (PMPC-block-PBMA) was dried in a glass vacuum container. Instead of MPC, SBMA and HEMA were dissolved in methanol and THF to synthesize the macroinitiator for the synthesis of the diblock copolymers

poly(SBAA)

(PSBAA)-block-PBMA

and

PHEMA-block-PBMA,

respectively. In addition, the solvent used to synthesize PSBAA-block-PBMA and PHEMA-block-PBMA were 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and ethanol. The chemical structure and chain ratio of the synthesized random and diblock copolymers were analyzed by 1H-NMR spectroscopy (NMR 400 MHz, JNM-GX400, JEOL Tokyo, Japan). The weight-averaged (Mw) and number-averaged (Mn) molecular weight of the synthesized random and diblock copolymers were estimated via gel permeation chromatography (GPC, JASCO Co., Ltd, Tokyo, Japan). The synthetic results are summarized in the Table 1.

Surface modification and characterization. Synthesized copolymers were coated on the PP substrates via a simple dip-coating process. The substrates were immersed into 0.50 wt% copolymer solutions for 1.0 min and dried at room temperature. This process was carried out twice for each polymer coating. The solvent used in the preparation of the polymer solution was methanol or ethanol. All samples were dried in a vacuum drying chamber. The water contact angle was measured (Automatic Contact Angle


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Meter, model FTA1000, First Ten Ångstroms Co, Ltd., Portsmouth, Virginia, USA) by dropping 4.0 µL of deionized (DI) water in air to evaluate the surface hydrophilicity. Contact angles were measured continuously for 10 min. X-ray photoelectron spectroscopy

(XPS,

Thermal

Scientific

K-Alpha,

Waltham,

USA)

with

a

monochromated Al K X-ray source (1486.6 eV photons) was used to evaluate the chemical composition on the prepared surfaces. The nano-scan modes of C1s, O1s, N1s, P2p, and S2p spectra were acquired at a photoelectron take-off angle of 45°. All binding energy (BE) were referred to the C1s peak at 284.6 eV. The surface morphology and roughness of copolymer anchoring-coating surfaces were examined by atomic force microscopy (AFM), and the images were acquired with a JPK Instruments AG multimode NanoWizard (Berlin, Germany). This work describes the difference in surface hydration using a wet-state scanning mode. Commercial Si cantilevers (SEIKO SI-DF20, Tokyo, Japan and MikroMasch, CSC37/AIBS, Watsonville, California) were used at a resonant frequency of 320 kHz in tapping mode to observe the dry and hydrated surfaces in a scan area of 10 µm × 10 µm.

Plasma protein adsorption test. The Micro BCA protein assay (Thermo Scientific Inc., Waltham, Massachusetts, USA) was used to evaluate fibrinogen adsorption on the prepared substrates. Flat PP substrates with a surface area of 1.32 cm2 were placed in individual wells of a 24-well tissue culture plate and then pre-wetted with DI water overnight at room temperature. After removing the DI water, 2.0 mL of fibrinogen solution at a concentration of 0.30 mg/mL was added into each well and incubated at 37°C. After incubation for 2 h, samples were rinsed in PBS several times to remove the non-adsorbed protein and detach the adsorbed protein in sodium n-dodecyl sulfate (SDS) solution. Then, 150 µL of SDS solution was gently mixed with BCA reagents in a 96-well plate. After the reaction was run at 37°C for 2.0 h, the absorbance was determined at 562 nm by a microplate reader (BioTek Instruments Inc., Winooski, Vermont). The amount of protein adsorbed on the substrates was calculated using a calibration curve. Every measurement was carried out six times for each sample (n = 6).

Blood cell attachment test. Human blood for research was donated by a healthy volunteer who legally signed the agreement of the institutional review board (IRB) of


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the Taiwan Blood Services Foundation. Platelet-rich plasma (PRP) was obtained from whole blood, which was prepared in a centrifuge (Model: 5804R, 15 amp version, Eppendorf, Hauppauge, USA) at 1200 rpm for 10 min. For the preparation of leukocytes concentration (also called white blood cells, WBCs), the Ficoll-Paque™ PLUS reagent was used and the standard protocol was followed. Density gradient centrifugation was carried out at a speed of 1940 rpm for 30 min. In order to enhance the purity of the leukocytes, 5.0 mL of D-PBS (D-PBS reduces platelet activation to extend the storage time of leukocytes by removing the calcium and magnesium ions) was added into the leukocytes and centrifuged twice at 300 rpm for 10 min. In this work, the pre-wetted substrates were equilibrated in individual wells of a 24-well tissue culture plate with 1000 µL of DI water overnight at 25°C. After removing the DI water, the prepared samples in each well were put in direct contact with 1000 µL of PRP, leukocytes, or whole blood and incubated for 2.0 h at 37°C. After rinsing with PBS three times, the blood cells that have adhered on the surfaces were stained and fixed with 3.0 µL of CD3-FITC, CD14-FTIC, CD45-FITC, and DAPI in 270 µL of PBS with 2.5% glutaraldehyde at 4°C for 15 min. The blood cells attached on the PP substrates were sealed with coverslips and their morphology was observed by confocal laser scanning microscopy (CLSM, Nikon Co., Model A1R, Tokyo, Japan) at magnifications of 200× and 400×. Lasers with wavelengths of 403 and 488 nm equipped in the CLSM instrument were used to observe the blood cell attachment in blue (DAPI, a fluorescent dye used to stain the cell nuclei of human leukocytes) and green (FITC, a fluorescent dye used to stain the cell membrane of human whole blood) fluorescence. Finally, the numbers of adhered whole blood cells, platelets, and leukocytes were determined by the ImagePro 7.0 software (Media Cybernetics, Inc., MD, USA). The quantitative results of the adhered blood cells are calculated as the average value of repeated measurements of four images (n = 4).

RESULTS AND DISCUSSION

Characterization of zwitterionic random and block copolymers. Regarding the surface zwitterionization via self-assembly anchoring and hydrated chain orientation,


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the molecular design of copolymers becomes a critical factor to enable biomaterial interfaces with good anti-fouling performance as contacting with biomolecules. In this work, conventional free radical polymerization and RAFT were used to synthesize copolymers with the random- and diblock- architectures, respectively, as agents for the coating process. Previous studies revealed that PMB with 30 mol% MPC unit composition exhibited outstanding blood compatibility and surface coating stability on polymeric and metallic substrates.13-14 PMB was built as an ideal case of blood compatible reference coating on PP substrates in comparison with PSB and PHB coatings.

Furthermore,

PP

substrates

coated

with

diblock

copolymers

of

PMPC-block-PBMA, PSBAA-block-PBMA, and PHEMA-block-PBMA containing the ideal fixed monomer unit composition of 30 mol% were introduced to discuss the effect of chain architecture on their blood compatibility. In general, the increase in hydrophobic BMA unit composition in the copolymers might enhance polymer anchoring to the target substrates but reduce blood cell resistance. Importantly, this study investigated the proper control of polymer chain orientation in hydrophilic units associated with the defined pre-wetting process. The effects of hydrophilic unit orientation and hydration which exposure facing out from hydrophobic unit anchoring interfaces was discussed on the overall blood compatibility through contact with plasma proteins and human blood. The hydrophilic monomer unit compositions were controlled at an ideal amount of 30 mol% in the feed monomer solution. Table 1 shows the physical properties of random and diblock copolymers. The results indicate the homogeneous reaction between comonomers in random sequence with an ideal composition ratio approaching approximately 30/70 by conventional free radical polymerization. Good living characteristics of block polymer chains were also shown with a narrow polydispersity of less than 1.1 by RAFT polymerization.

Characterization of surface zwitterionization via copolymer anchoring. For the analysis of specific chemical compositions of copolymers anchored on the PP substrates, the core-level electronic spectra of C1s, O1s, N1s, P2p, and S2p were determined by XPS measurements. All XPS charts are shown in Figure S-1 (Supplementary Information). In the pre-wetting condition after copolymer coating with subsequent


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hydration by DI water immersion for 24 h, obvious reductions of the N1s and S2p peak signals were observed in the case of PSBAA-block-PBMA-coated PP substrates, indicating that copolymer detachment might have occurred. The coating stability of zwitterionic copolymers on the hydrophobic PP substrates needs to be considered to sustain and improve the blood-inert performance of the copolymer-coated PP substrates. Thus, the stability of copolymer-coated PP substrates was investigated by the surface elemental ratios of P/C, S/C, and O/C estimated from the XPS spectra. Freeze-drying treatment was applied to fix the polymer segments in the hydrated state before XPS characterization. All glass transition temperatures (Tg) of the polymers are higher than room temperature. As shown in Figure 2a, the O/C ratio may show the anchoring stability of PBMA on the PP substrates. The change in the O/C ratio between dry and pre-wetting conditions was observed to be less than 15%, indicating that a partial amount of coated polymer might be washed away due to weak anchoring in the multilayer portion. The result also provides evidence that PBMA may stably anchor on the surface of PP substrates via hydrophobic-driven interaction. For the cases of random copolymers in Figure 2b−d, the change in the P/C, S/C, and O/C ratios of PMB-, PSB-, and PHB-coated PP substrates through the pre-wetting process resulted in similar levels of reduction as well as amounts of detached copolymer compared to PBMA-coated PP substrates. This reveals that the prepared random copolymers containing a range from 61 to 76 mol% BMA units provided the anchoring stability to keep the MPC, SBAA, and HEMA units on the PP substrate. In the cases of well-defined diblock copolymers in Figures 2b and 2d, the changes in the P/C and O/C ratios of PMPC-block-PBMA- and PHEMA-block-PBMA-coated PP substrates were less than 5.0%. It was pointed out that the block copolymer architecture had stronger anchoring stability on PP substrates than the copolymers with random sequence, even when the composition of hydrophobic BMA units was in a lower range from 45 to 64 mol%. However, in the case of PSBAA-block-PBMA with 14 mol% BMA units, the obvious reduction in the S/C ratio of over 90% after pre-wetting treatment proved the unstable anchoring of PSBAA-block-PBMA on PP substrates, which indicates that the less hydrophobic PBMA segments resulted in weak coating stability. Moreover, the initial amount of the immobilized copolymers depends on the composition and architecture of BMA segments in the prepared polymer. For PMPC-based copolymers, the immobilization


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amount of PMPC-block-PBMA is less than random copolymer form, indicating the good self-assembling structures of block copolymer architecture. However, for the case of PSBAA-based copolymer, the less coating amount of PSBAA-block-PBMA is due to the low BMA composition of hydrophobic anchoring block, providing low stability and antifouling coverage. It is accepted that surface hydrophilicity plays an important role to affect the blood compatibility of biomaterial interfaces. According to previous studies, zwitterionic materials provided super hydrophilic interfaces covered with a strong hydration layer to resist general biofouling adhesion such as protein, cells, and bacteria.7, 29-30 Dynamic water contact angle is a good index used in this study to estimate the surface hydrophilicity associated with the anchored chain orientation. Figure 3 shows the dynamic changes in contact angle on the various copolymer-coated PP substrates during 10 min of successive measurements in the dry sample state. The water contact angles of the virgin PP and PBMA-coated substrates were over 90°, indicating their hydrophobic, water-repellent property. In the cases of random copolymer-coated surfaces, there is a significant difference in the contact angles between PMB-, PSB-, and PHB-coated PP substrates. PMB-coated surfaces underwent a gradual decrease in contact angle from 102° to 34° in 10 min, showing the chain orientation of MPC units from coating the inside layer to the water-contacting interface. In general, it is quite crucial to maintain low surface energy on biomaterial interfaces in both air and the aqueous phase.31 Therefore, to minimize the surface energy of PMB-coated surfaces, the nature thermodynamic tendency derives that the polar groups of MPC units orient away from the surface in air but toward the more polar phase at aqueous interfaces. PSB-coated surfaces showed a relatively lower variation of contact angles from 42° to 18° but a higher surface hydrophilic response in random copolymer sequence than the PMB-coated surfaces. However, the PHB-coated surface kept the low chain orientation of HEMA units toward the aqueous interfaces to limit its surface hydrophilic state. In the cases of block copolymer-coated surfaces with well-defined brush architectures, the PMPC-block-PBMA-coated surface perform high rapid wettability with an ultra-low contact angle of 11° during a 10-min period, indicating the easier chain orientation of PMPC units in block form than in random sequence. A contact angle of 71° was also observed on the PHEMA-block-PBMA-coated surfaces, which was lower than that of


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81° on PHB-coated surfaces. To measure the equilibrium water contact angles, the copolymer-coated PP substrates were treated in the pre-wetting condition at 25°C for 24 h, as summarized in Table 1. In the case of the PMPC-block-PBMA-coated surface, the measured contact angle after pre-wetting almost approached the same super-hydrophilic level as that of 11° in 10 min from the initial dry state. This supported the highly rapid wettability associated with the strong hydration in the block architecture of PMPC brushes anchored on PP substrate surfaces. It was observed that the PMB-coated surfaces had an equilibrium water contact angle of 20°, explaining that the polar groups of MPC units in random sequence with hydrophobic BMA units needed enough chain relaxation time to orient the MPC units toward the aqueous phase and improved the surface hydrophilicity. However, the PHB-coated surface still exhibited poor chain orientation of HEMA units incubated in the aqueous phase, but the advantage of the block architecture of PHEMA brushes drove the PHEMA-block-PBMA-coated surface to exhibit a reduced contact angle of 60° after pre-wetting for 24 h. The stable anchoring of PSB on PP substrates showed a steady contact angle of 17° in pre-wetting condition. A very slight change in the water contact angle was observed from 80° (in the initial dry state) to 85° (in the pre-wetting state) on the PSBAA-block-PBMA-coated surface. This corresponded to the detachment of copolymers containing 14 mol% PBMA units, which was supported by XPS analysis in Figure 2. The surface morphology and topography of the copolymer-coated PP substrates were determined by AFM in both dry and hydrated states as shown in Figure 4. The hydrophobic PP substrate coated with PBMA revealed no significant change in the surface roughness between the dry state (49.5 nm) and hydrated state (54.9 nm). It is interesting to note that the PMB- and PMPC-block-PBMA-coated surfaces in the dry state showed uniform coverage with very low root-mean-square (RMS) roughness values of 11.1 and 16.3 nm, respectively. After the prepared surfaces were treated in pre-wetting condition with pure water immersion at 25°C for 24 h, obvious increases in the RMS roughness were observed in the range of 32.8 and 38.4 nm, indicating that the surface-anchored PMPC chains took on an excellent swelling conformation in the aqueous phase as a result of well hydration by water molecules. The results also delivery important information that the random sequence of MPC units in the coating


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layer successfully retained the chain flexibility to orient the zwitterionic units toward the aqueous phase and improved surface hydrophilicity. For PSB-coated surfaces in the dry state, the image in Figure 4h shows the spherical structures formed by submicron particles of ~500 nm. After the pre-wetting treatment, a decrease was observed in the RMS roughness from 147.5 nm to 44.7 nm, illustrating the rearrangement of copolymer chains into a uniform and well-extended conformation, enhancing the hydrophilic property as indicated in Figure 3. Figure 4i shows the nano-scale spherical structures on coated PP surfaces in the dry state by micelles of PSBAA-block-PBMA. The detachment of these copolymers was demonstrated by the decrease in RMS roughness from 44.8 nm to 31.8 nm. The results were also supported by XPS analysis and contact angle measurements. On the surfaces modified with PHB (Figures 4j and 4n) and PHEMA-block-PBMA (Figures 4k and 4o) copolymers, there was no obvious change in the surface topography, and the roughness slightly decreased by less than 15 nm from the dry state to the hydrated state.

Protein adsorption resistance observed on the surface. In general, the adsorption of a monolayer of protein on hydrocarbon-based surfaces is driven by hydrophobic interactions.32-33 Thus, hydrocarbon-based biomaterials easily induce plasma protein adsorption when the interfaces are in contact with human blood. Protein resistance of human fibrinogen is one of the key issues required in the development of blood compatible biomaterials. In this work, the amounts of fibrinogen adsorbed on the prepared samples were quantitatively determined by the MicroBCA assay at 37°C for 2 h. As expected, in Figure 5, the hydrophobic surfaces of the original PP substrate and PBMA-coated substrate led to high protein adsorption levels of 3.9 and 3.4 µg/cm2, respectively. The zwitterionic surfaces anchored with PMB and PSB under a pre-wetting state showed lower protein adsorption levels of 0.39 and 0.23 µg/cm2, respectively. In the case of block copolymer-coated surfaces with well-defined brush architectures, the PMPC-block-PBMA-coated surface perform the highest protein resistance with fibrinogen adsorption at 0.17 µg/cm2 as well as rapid wettability with an ultra-low contact angle of 11°. It is attributed to the easier chain orientation of MPC units in block form than that in random sequence to effectively resist more protein adsorption. However, the PSBAA-block-PBMA-coated surface showed high protein


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adsorption at 3.0 µg/cm2 due to the detachment of the copolymers in the pre-wetting state, as supported by the XPS, AFM, and contact angle analyses. Similar to the case of PMPC-block-BMA

with

well-defined

brush

architecture,

the

PHEMA-block-PBMA-coated surface with protein adsorption of 0.70 µg/ cm2 was also observed in lower value than that of 2.8 µg/ cm2 on PHB coated surface. The results of protein adsorption are summarized as the following important viewpoints: (1) zwitterionic groups as hydrophilic units are effective to reduce protein adsorption compared with HEMA units; (2) the surface properties of SBAA copolymers depend on the polymer architecture; (3) MPC copolymers exhibit stronger and more satisfying protein resistance than non-ionic HEMA copolymers.

Reduction of blood cell attachment on the surface. The resistance of blood cell adhesion and activation is an important factor in the design of a stable biomaterial as it avoids thrombotic responses in human whole blood. These responses constitute complicated activation processes involving leukocyte adhesion, platelet adhesion and activation, and fibrin network formation. The correlations between plasma protein adsorption of human fibrinogen and blood cell attachment from human whole blood are further

investigated

to

illustrate

the

blood

compatibility

of

the

prepared

copolymer-modified PP substrates in this study. As shown in Figures 6 and 7, the blood cells attached on the copolymer-coated surfaces from undiluted human whole blood were qualitatively observed by CLSM and quantitatively determined using ImageJ software. Platelets (diameter: 2-3 µm), erythrocytes (diameter: 6-8 µm) and leukocytes (diameter: 9-20 µm) attachment in color green were observed in Figure 6. On the original PP substrates and PBMA-coated substrates, highly number of blood cells as 3.2 × 103 and 2.2 × 103 cells/mm2 were attached, respectively. On the zwitterionic random copolymer-coated surfaces, only less than 4.0 % of blood cells adhered on the surface compared with the PP substrate. This corresponded to the amount of fibrinogen adsorption as shown in Figure 5. The PMPC-block-PBMA-coated surface showed extremely strong bio-inert property with minimal attachment at 80 cells/mm2 (about 2.5% of the number of cells observed on the PP substrate) as well as the highest protein resistance with the lowest adsorption at 0.17 µg/cm2 and an ultra-low contact angle of 11°. On the other hand, PSBAA-block-PBMA-coated surfaces demonstrated significant


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blood cell attachment at over 1.3 × 103 cells/mm2. The possible reason for this performance was the detachment of the copolymer during pre-wetting state, which was demonstrated by XPS, AFM, and water contact angle measurements. However, it was observed that the PP surfaces coated with PHB and PHEMA-block-PBMA lost their bio-inert property while in contact with whole blood. Figure 7 indicates that the blood compatibility of the hydrophobic PP substrates in human whole blood can be well controlled via the rational chain architecture design of the zwitterionic copolymers. It is worth noting that only PHB-coated PP substrate surfaces induced significant attachment of leukocytes from human whole blood, as shown in Figure 6g. In general, blood transfusion is an important way of saving human lives. However, transfusion-related immunomodulation might increase the risk of infections and cancer recurrence because of immune depression. The mechanism of the transient depression of the immune system in the human body is still unclear. For safety reasons, the Food and Drug Administration in the USA recommends that all transfused blood products undergo leukocyte reduction in order to offset the contribution of donor white blood cells to immune suppression.34-35 In this study, the evaluation of leukocyte attachment from 100% human whole blood onto modified PP substrates coated with HEMA-based random copolymer inspired our research. A further detail analysis of platelet adhesion and thus leukocyte attachment was carried out to assess the effects of the physical and chemical architecture of these amphiphilic copolymers on preferential blood cell attachment. In the case of hydrophobic surfaces, a large number of platelets at approximately 33 × 103 and 22 × 103 cells/mm2 (Figures 8a and 8b) adhered on original PP and PBMA-coated PP substrates, respectively, when incubated in 100% PRP solution at 37°C. This observation is consistent with previous studies that have shown that fibrinogen adsorption subsequently induced platelet adhesion, which is also demonstrated in Figures 5 and 9. Interestingly, the higher number of platelets resulted in a lower level of leukocyte attachment at ~0.07 × 103 cells/mm2 on the original hydrophobic PP substrates than that at ~0.11 × 103 cells/mm2 on PBMA-coated PP substrates. This might be due to the denaturation and activated level of attached leukocytes, which were attributed to the dependence of surface hydrophobicity. When the zwitterionic copolymer-modified surfaces were in contact with 100% PRP, platelets hardly adhered on surfaces with PMB, PMPC-block-PBMA, and PSB, as shown in


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Figures 8 and 9. Consistent with our previous work, hydrophilic surfaces prepared with proper control of the surface coverage of zwitterionic layers resulted in modified PP substrates with an ideal blood cell-inert interface.30 For the PHEMA-modified surfaces, it was shown that the PHEMA-block-PBMA-coated PP substrates with well-defined polymer chains bearing –OH groups provided effective resistance to platelet adhesion and leukocyte attachment in a relatively low biofouling level compared to the hydrophobic PBMA-coated surfaces. However, it should be noted that the PP substrate modified with PHB, HEMA, and BMA units induced preferential leukocyte attachment at approximately 0.38 × 103 cells/mm2 but lower platelet adhesion at ~11 × 103 cells/mm2. It can be relatively compared with the hydrophobic PBMA-coated surfaces with higher numbers of platelets at over ~22 × 103 cells/mm2 and lower numbers of leukocytes at below ~0.11 × 103 cells/mm2. The results indicate an important finding that the control of preferential leukocyte attached interfaces should be considered from the balance of hydrophilicity between hydroxyl groups and hydrocarbon groups. This could bring the potential impact in the rational design of next generation biomaterials used in transfusion filters for blood products.

CONCLUSIONS

In this study, we demonstrated a set of well-defined zwitterionic and non-ionic copolymers for the blood-inert control of PP by surface treatment of self-assembled anchoring

processes.

Six

different

random

and

block

copolymers,

PMB,

PMPC-block-PBMA, PSB, PSBAA-block-PBMA, PHB, and PHEMA-block-PBMA, were successfully prepared using conventional free radical polymerization and RAFT polymerization. The chain orientation of these zwitterionic copolymers anchored on hydrophobic PP substrates can be controlled via pre-wetting treatment to obtain optimized protein-resistant interfaces.

The zwitterionization on the PP substrate by

simple coating with the diblock copolymer PMPC-block-PBMA performs excellent bio-inert properties with satisfying resistance to protein adsorption, platelet adhesion, and leukocyte attachment. The random copolymers of PSB also indicated good blood compatibility in human whole blood at a level comparable to that of PMB. It was noted


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that preferential blood cell-attached interfaces could be formed from PHB-coated PP substrates for enhanced leukocyte attachment, which was dependent on platelet adhesion. Surface anchoring of zwitterionic copolymers offer a promising opportunity for the development of blood compatible PP-based biomaterial interfaces in the design of blood-contacting devices.


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AUTHOR INFORMATION Corresponding Author *Yung Chang: E-mail: +886-3-265-4199

[email protected].

Phone:

+886-3-265-4122.

Fax:

* Kazuhiko Ishihara: E-mail: [email protected]. Phone: +81-3-5841-7124. Fax: +81-3-5841-8647

ACKNOWLEDGMENTS The authors would like to acknowledge the project of Outstanding Professor Research Program in the Chung Yuan Christian University, Taiwan, and the Ministry of Science and Technology (MOST 104-2622-B-033-002 and 105-2622-B-033-001) for their financial support. Sheng-Han Chen would like to express the sincere appreciation to Interchange Association, Japan (IAJ) and Ministry of Science and Technology (MOST), Taiwan to provide the funding and opportunity in the jointed Summer Program in 2014.


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25, 11911–11916. (12) Shao, Q.; Jiang, S., Molecular Understanding and Design of Zwitterionic Materials. Adv. Mater. 2015, 27, 15–26. (13) Ishihara, K.; Ueda, T.; Nakabayashi, N., Preparation of Phospholipid Polylners and Their Properties as Polymer Hydrogel Membranes. Polym. J. 1990, 22, 355–360. (14) Iwasaki, Y.; Ishihara, K., Cell Membrane-Inspired Phospholipid Polymers for Developing Medical Devices with Excellent Biointerfaces. Sci. Technol. Adv. Mater. 2012, 13, 064101. (15) Ishihara, K.; Fukazawa, K., 2-Methacryloyloxyethyl Phosphorylcholine Polymers. In Phosphorus-Based Polymers: From Synthesis to Applications. Ed S. Monge, and G. RSC Publishing, Cambrige UK, 2014, 68–96. (16) Chang, Y.; Chen, S. F.; Zhang, Z.; Jiang, S., Highly Protein-Resistant Coatings from Well-Defined Diblock Copolymers Containing Sulfobetaines. Langmuir 2006, 22, 2222–2226. (17) Brzozowska, A. M.; Parra-Velandia, F. J.; Quintana, R.; Xiaoying, Z.; Lee, S. S. C.; Chin-Sing, L.; Jańczewski, D.; Teo, S. L. M.; Vancso, J. G., Biomimicking Micropatterned Surfaces and Their Effect on Marine Biofouling. Langmuir 2014, 30, 9165–9175. (18) Shen, C. H.; Cho, Y. J.; Lin, Y. C.; Chien, L. C.; Lee, T. M.; Chuang, W. H.; Lin, J. C., Surface Modification of Titanium Substrate with a Novel Covalently-Bound Copolymer Thin Film for Improving its Platelet Compatibility. J. Mater. Sci.: Mater. Med. 2015, 26, 1–13. (19) Chang, Y.; Shih, Y. J.; Lai, C. J.; Kung, H. H.; Jiang, S., Blood-Inert Surfaces via Ion-Pair Anchoring of Zwitterionic Copolymer Brushes in Human Whole Blood. Adv. Funct. Mater. 2013, 23, 1100–1110. (20) Inoue, Y.; Onodera, Y.; Ishihara, K., Preparation of a Thick Polymer Brush Layer Composed

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Poly((3-(methacryloylamino)propyl)-dimethyl(3-sulfopropyl)ammonium hydroxide). Langmuir 2007, 23, 5678–5682. (24) Liu, P. S.; Chen, Q.; Liu, X.; Yuan, B.; Wu, S. S.; Shen, J.; Lin, S. C., Grafting of Zwitterion from Cellulose Membranes via ATRP for Improving Blood Compatibility. Biomacromolecules 2009, 10, 2809–2816. (25) Yang, J.; Chen, H.; Xiao, S.; Shen, M.; Chen, F.; Fan, P.; Zhong, M.; Zheng, J., Salt-Responsive Zwitterionic Polymer Brushes with Tunable Friction and Antifouling Properties. Langmuir 2015, 31, 9125–9133. (26) Chen, S. H.; Chang, Y.; Lee, K. R.; Wei, T. C.; Higuchi, A.; Ho, F. M.; Tsou, C. C.; Ho, H. T.; Lai, J. Y., Hemocompatible Control of Sulfobetaine-Grafted Polypropylene Fibrous Membranes in Human Whole Blood via Plasma-Induced Surface Zwitterionization. Langmuir 2012, 28, 17733–17742. (27) Sundaram, H. S.; Han, X.; Nowinski, A. K.; Ella-Menye, J. R.; Wimbish, C.; Marek, P.; Senecal, K.; Jiang, S., One-Step Dip Coating of Zwitterionic Sulfobetaine Polymers on Hydrophobic and Hydrophilic Surfaces. ACS Appl. Mater. Interfaces 2014, 6, 6664–6671. (28) Ishihara, K.; Oshida, H.; Endo, Y.; Ueda, T.; Watanabe, A.; Nakabayashi, N., Hemocompatibility of Human Whole Blood on Polymers with a Phospholipid Polar Group and Its Mechanism. J. Biomed. Mater. Res. 1992, 26, 1543–1552. (29) Hower, J. C.; Bernards, M. T.; Chen, S.; Tsao, H. K.; Sheng, Y. J.; Jiang, S., Hydration of “Nonfouling” Functional Groups. J. Phys. Chem. B 2009, 113, 197– 201. (30) Jiang, S.; Cao, Z., Ultralow-Fouling, Functionalizable, and Hydrolyzable Zwitterionic Materials and Their Derivatives for Biological Applications. Adv. Mater. 2010, 22, 920–932. (31) Callow, M. E.; Fletcher, R. L., Special Issue Marine Biofouling and Corrosion the Influence of Low Surface Energy Materials on Bioadhesion — A Review. Int. Biodeterior. Biodegrad. 1994, 34, 333–348.


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(32) Prime, K.; Whitesides, G., Self-Assembled Organic Monolayers: Model Systems for Studying Adsorption of Proteins at Surfaces. Science 1991, 252, 1164–1167. (33) Van Oss, C. J.; Good, R. J.; Chaudhury, M. K., The Role of Van Der Waals Forces and Hydrogen Bonds in “Hydrophobic Interactions” between Biopolymers and Low Energy Surfaces. J. Colloid Interface Sci. 1986, 111, 378–390. (34) Bordin, J.; Heddle, N.; Blajchman, M., Biologic Effects of Leukocytes Present in Transfused Cellular Blood Products. Blood 1994, 84, 1703–1721. (35) Singh, S.; Kumar, A., Leukocyte Depletion for Safe Blood Transfusion. Biotechnol. J. 2009, 4, 1140–1151.


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Figure 1. Schematic illustration of chain orientation of random and diblock copolymer-coated PP substrates in dry and pre-wetting states. 127x51mm (300 x 300 DPI)


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Figure 2. Elemental ratios determined by XPS measurement. (a) the original PP and PP substrates coated with PBMA, (b) PMB and PMPC-block-PBMA, (c) PSB and PSBAA-block-PBMAA, and (d) PHB and PHEMAblock-PBMA in the dry (closed column) and pre-wetting (open column) conditions. 138x96mm (300 x 300 DPI)


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Figure 3. Time dependence of water contact angles of PP substrates coated with various polymers. Open circle ○: original PP, closed circle ●: PBMA, open inverted triangle▽: PHB, closed inverted triangle ▼: PHEMA-block-PBMA, open square □: PSB, closed square ■: PSBAA-block-PBMA, open triangle △: PMB, closed triangle ▲: PMPC-block-BMA. 151x105mm (300 x 300 DPI)


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Figure 4. Tapping-mode AFM images of surface RMS roughness of (a) the original PP and PP substrates coated with (b and e) PBMA, (c and f) PMB, (d and g) PMPC-block-PBMA, (h and l) PSB, (i and m) PSBAAblock-PBMA, (j and n) PHB, and (k and o) PHEMA-block-PBMA in dry and pre-wetting states, respectively. Dimensions of the scan area are 10 µm × 10 µm 179x143mm (300 x 300 DPI)


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Figure 5. Amount of human fibrinogen adsorbed on the PP substrates coated with various polymers. 123x86mm (300 x 300 DPI)


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Figure 6. CLSM images of blood cells attached on (a) the original PP substrate and PP substrates coated with (b) PBMA, (c) PMB, (d) PMPC-block-PBMA, (e) PSB, (f) PSBAA-block-PBMA, (g) PHB, and (h) PHEMA-blockPBMA in the pre-wetting condition after contact with whole blood for 120 min. All images captured by the CLSM are shown at magnifications of 200× and 400×. 151x57mm (300 x 300 DPI)


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Figure 7. Number of blood cells attached on the PP substrates coated with various polymers after contact with whole blood for 120 min as determined from the CLSM images. 123x86mm (300 x 300 DPI)


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Figure 8. CLSM images of platelets and leukocytes attached on (a and e) the original PP substrate and PP substrates coated with (b and f) PBMA, (c and g) PMB, (d and h) PMPC-block-PBMA, (i and m) PSB, (j and n) PSBAA-block-PBMA, (k and o) PHB, and (l and p) PHEMA-block-PBMA in the pre-wetting condition. All images captured by CLSM are shown at magnifications of 400× and 800×. 145x137mm (300 x 300 DPI)


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Figure 9. Number of platelets adhered and leukocytes attached on the PP substrates coated with various polymers as determined from the CLSM images. Numbers of platelet and leukocyte adhered on the surface are indicated as open column and closed column, respectively. 123x86mm (300 x 300 DPI)


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Table 1. Synthetic results and characteristics of random and diblock copolymers Composition (mol%) In polymer b

In feed

Copolymers a

Static water Yield c

Mw d

M w/

(%)

(×104)

Mn d

Initial

Pre-

state

wetting e

2

102 ± 2

20.0 ± 1.0

6.9

1.4

41.7 ± 2.1

16.8 ± 1.5

80

5.2

1.7

91.2 ± 2.5

80.6 ± 2.1

64

65

9.4

1.1

52.8 ± 0.7

10.6 ± 1.1

0

14

20

3.1

1.1

80.6 ± 3.3

85.3 ± 4.2

55

45

42

4.2

1.1

83.4 ± 2.6

60.2 ± 3.4

MPC

SBAA

HEMA

BMA

MPC

SBAA

HEMA

BMA

30

0

0

70

32

0

0

68

95

20

0

30

0

70

0

24

0

76

68

0

0

30

70

0

0

39

61

PMPC-block-PBMA i

30

0

0

70

36

0

0

PSBAA-block-PBMA j

0

30

0

70

0

86

PHEMA-block-PBMA k

0

0

30

70

0

0

poly(MPC-random-BMA) (PMB) f poly(SBAA-random-BMA) (PSB) g poly(HEMA-random-BMA) (PHB) h

a

contact angle (o)

Random copolymers and diblock copolymers were synthesized at 60 oC for 24 h via conventional free radical polymerization and RAFT polymerization,

respectively. b Compositions of each monomer units in the copolymers were determined by 1H-NMR. c The yield of copolymers were estimated by the real obtained mass. d Mw and Mn were determined by GPC measurement. e Pre-wetting treatment was carried out in water for 24 h. The prepared copolymers were precipitated with f diethyl ether/ chloroform (80/ 20, v/v), g acetone/ methanol (50/ 50, v/v), h water/ acetone/ methanol (30/ 30/ 30, v/v), i diethyl ether (1st units) and n-hexane (2nd units), j membrane dialysis (Spectra/Por® 7 Dialysis Membranes, MWCO: 1kD) in water (1st units) and acetone/methanol (50/ 50, v/v) (2nd units), k diethyl ether (1st units) and n-hexane (2nd units) for purification.


Reduced Blood Cell Adhesion on Polypropylene Substrates through a

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