Epoxylated Zwitterionic Triblock Copolymers Grafted onto Metallic

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Epoxylated Zwitterionic Triblock Copolymers Grafted Onto Metallic Surfaces for General Biofouling Mitigation Ying-Nien Chou, Antoine Venault, Chia-Ho Cho, Mei-Chan Sin, Lu-Chen Yeh, Jheng-Fong Jhong, Arunachalam Chinnathambi, Yu Chang, and Yung Chang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02164 • Publication Date (Web): 22 Aug 2017 Downloaded from http://pubs.acs.org on August 26, 2017

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Epoxylated Zwitterionic Triblock Copolymers Grafted Onto Metallic Surfaces for General Biofouling Mitigation Ying-Nien Chou1, Antoine Venault1, Chia-Ho Cho1, Mei-Chan Sin1, Lu-Chen Yeh1, JhengFong Jhong1, Arunachalam Chinnathambi3, Yu Chang*,2, Yung Chang*,1,3 1

R&D Center for Membrane Technology and Department of Chemical Engineering, Chung Yuan Christian University, Chung-Li, Taoyuan 320, Taiwan, R.O.C.

2

Department of Obstetrics and Gynecology, E-Da Hospital, I-Shou University, Kaohsiung City 82445, Taiwan, R.O.C.

3

Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia

ABSTRACT Titanium and stainless steel materials are widely used in numerous devices or in custom parts for their excellent mechanical properties. However, their lack of biocompatibility seriously limits their usage in the biomedical field. This study focuses on the grafting of triblock copolymers on titanium and stainless steel metal susbtrates for improving their general biofouling resistance. The series of copolymers that we designed is composed of two blocks of zwitterionic sulfobetaine (SBMA) monomers and one block of glycidyl methacrylate (GMA). The number of repeat units forming each block, n, was finely tuned and controlled to 25, 50, 75 or 100, permitting to regulate the grafting thickness, the morphology, and the dependent properties such as the surface hydrophilicity and biofouling resistance. It was shown that the copolymer possessing n = 50 repeat units in each block, corresponding to a molecular weight of about 15.2 kDa, led to the best nonfouling properties, assessed using plasma proteins, blood cells, fibroblasts cells and various bacteria. This was explained by an optimized grafting degree and chain organization of the copolymer. Lower value (n = 25) and higher values (n = 75, 100) led to low surface coverage and the formation of aggregates, respectively. The best copolymer was grafted onto scalpels (steel) and dental roots (titanium), and antifouling properties demonstrated using Escherichia coli and HT1080 cells. Results of this work show that this unique triblock copolymer holds promise as a potential material for 1 ACS Paragon Plus Environment

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surface modification of biomedical metallic devices, provided a fine tuning of the blocks organization and length.

KEYWORDS: zwitterionic triblock copolymer • poly(GMA-tb-PSBMA) • grafting onto • biocompatible metal • antibiofouling surfaces

INTRODUCTION Blood-contacting implants, hemodialysis membranes, drug (or gene) delivery carriers, diagnostic biosensors, vascular stents heart valves, and ventricular assist devices must have one essential property in common: their antifouling nature which in turns provides biocompatibility to the medical device they are used in.1-5 Generally, to achieve minimum interactions with the surrounding environment, resistance to non-specific protein is required. As earlier defined and demonstrated,6 protein-resistance materials must meet three prerequisites: (i) they must not be hydrophobic, (ii) they should be electrically neutral, and (iii) they should contain hydrogen bond acceptors instead of hydrogen bond donors. These rules have been successfully used for the design of antifouling polymers.7,8 Some important materials that fit in these important criteria and which are extremly attractive for antibiofouling studies are zwitterionic materials such as phosphobetaine, carboxylbetaine, and sulfobetaine with conjunction of positive and negative charges.9-17 Among them, poly(sulfobetaine methacrylate) (PSBMA) is probably the zwitterionic material that has been receiving the most attention because of its highly efficient anti-fouling character combined to its ease of production. Moreover, numerous techniques have been applied to improve its range of application in harsher conditions, as well as its anchoring techniques: some recent examples of our group are the development of thermo-responsive antifouling materials,18,19 the design of copolymers for hydrophobicity-driven surface assembling20 and the synthesis of ionic-anchoring copolymers.21 Metals have always been frequently utilized in biomedical materials designs, for their chemical and physical properties. For example, stainless steel, titanium and titanium alloys are largely utilized as dental or orthopaedics biomaterials.22,23 Stainless steel is easy to process at a relatively low cost, and is commonly utilized as surgical and orthopaedic implant materials, as well as in pulmonary stents, screws or artery stents.24,25 Titanium and titanium alloys are utilized in dental applications for their corrosion resistance, biocompatibility, relatively low modulus of elasticity and notably light weight.26,27 Besides, they have also 2 ACS Paragon Plus Environment

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been used in various artificial implant body parts such as joints and fingers, as well as in pacemakers or heart valves. In summary, stainless steel and titanium are widely utilized for as biomataerials in vivo.23,28 However, for chronic usage (e.g. cardiovascular devices), titanium and stainless steel are sensitive to protein adsorption and cells attachment, which can induce foreign body responses such as thrombosis orthromboembolism. Thus, it is critical to decrease cells or interfacial tissue adhesion onto the implant surface.29-32 A way to mitigate or even prevent the cells-metal interactions is to graft zwitterionic polymers at the surface of the metals, before using them in vivo. Grafting from and grafting to are two methods used to introduce the polymers on the substrate to form an effective antifouling surface. Grafting from methods consist in growing a polymer from a momoner mixture at the surface of the material to modify, and bond it covalently to the surface. Surface-induced atom transfer radical polymerization (SI-ATRP) and surface-induced reversible addition-fragmentation chain transfer (SI-RAFT) polymerization are common grafting from methods.33,34 But although surface zwitterionization using grafting from approaches provides good fouling resistance in regard to protein-level adsorption, it is still difficult to modify versatile substrates, complicated geometric shapes, and large-scale biomaterial surfaces. The grafting to (or onto) methods consist in bonding a polymer at the surface of the material at play,35,36 and an efficient method to graft zwitterionic heads by this technique is to use glycidyl methacrylate, a biomimetic anchoring group.37-39 Unlike in grafting from methods, the surface is contacted directly with the final polymer, rather than with the monomer solution. But like in the grafting from techniques, the polymer is covalently bonded to the surface even though some authors also list the methods involving physical sorption in this class, while others refer to them as the coating methods. In any case, whether chemisorption or physisorption is considered, the polymer has been pre-prepared before surface modification, which permits to better control its composition, configuration or chain length. Other than the technique used to attach a polymer at a surface to make it antifouling, the configuration of the polymer plays a critical role in the final antifouling performances. Indeed, we recently investigated the effect of the polymer configuration, random, di-block or tri-block, on the antifouling properties of model tissue culture polystyrene interfaces, and showed that if much higher coating densities were achievable using a random configuration, it did not reflect on the antifouling properties of the interfaces.40 Instead, the block configurations performed better regardless of the nature of the biofoulants (protein, bacteria, human cells). Because of the randomness organization of the copolymer chains, some 3 ACS Paragon Plus Environment

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antifouling groups of the random copolymer were unfavorably oriented, and thus, were unable to participate in the creation of a hydrophilic protective layer. From there, we could hypothesize that choosing block copolymers over random copolymers for modifying any type of material surfaces to be used in biomedical devices, including metallic surfaces, would be a reasonable choice. However, current state of the art lacks of information on stable and efficient triblock copolymer grafting/coating for metal surfaces. A recent study made use of PEO–PPO–PEO triblock copolymer for the surface modification of titanium and stainless steel metals.41 However, this triblock copolymer is also known to be water-soluble. In addition, the surface modification at play was a coating process in which low-energy interactions bonded the copolymer to the metal. As a result, the stability of the antifouling properties can be questioned because many antifouling materials are used in aqueous environments (water, plasma, serum, etc…). Therefore, it is essential to control not only the configuration of the copolymer, but also to make sure that strong interactions are established between the metal and the copolymer, in order to optimize the antifouling properties, and particularly if a water-soluble copolymer is used. Clearly, literature lacks of reports on stable surface modification of metals for fouling mitigation. We recently presented a random copolymer of glycidyl methacrylate and sulfobetaine methacrylate that could efficiently modify versatile surfaces including polymers, ceramics and metals, despite its randomness nature.39 From the knowledge acquired from these previous studies, we decided to move onto the design of tri-block copolymers, to further improve the antifouling properties of metal surfaces. Nonetheless, another important design factor to take into consideration concerns the optimized length of each block forming the copolymer to provide the best antifouling proeprties as possible. Even though the longer the better seems to apply to PEG-based designs,40 the conclusions cannot be extrapolated to zwitterionic systems, given the intramolecular and intermolecular electrostatic interactions that can be formed, especially for long brushes that could potentially lead to the crash of the structure and their failure to provide antifouling property. So, it is also essential to investigate the important role of the blocks length on the surface modification. Taken this into account, this work reports on the design of a set of symmetric triblock copolymers made of glycidyl methacrylate (GMA) and sulfobetaine methacrylate (SBMA) monomers. The final triblock copolymers contain two hydrophilic PSBMA blocks surrounding one hydrophobic anchoring PGMA group and will be referred to as poly(GMAtb-SBMA). In addition, each distinct block contains a similar number of repeat units. The epoxide group can undergo a nucleophilic attack base-catalyzed, leading to the opening of the 4 ACS Paragon Plus Environment

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ring making possible covalent bonding with a variety of functional groups on a surface such as amine or hydroxyl groups. From this knowledge, we first pre-treated stainless steel (SUS) and titanium surfaces with UV/Ozone, to generate hydroxyl groups. We then exposed the hydroxylated

surfaces

with

poly(GMA-tb-SBMA)

in

alkaline

conditions

(using

triethylamine). As a result, the deprotonated hydroxyl groups could react with GMA segments leading to the final chemisorption of the triblock copolymers onto the surfaces. Different copolymers containing n = 25, 50, 75 and 100 repeat units in each block were grafted onto SUS and titanium surfaces. At first, we aimed at determining which of these copolymers would lead to an optimized combination of hydration and protein resistance. Then, the general antifouling properties of the surfaces were carefully investigated using leukocytes, platelets and whole blood, as well as several bacteria and HT1080 fibroblasts. Eventually, we moved onto the surface modification of a dental root (titanium-based) and a scalpel (stainless steel-based) to demonstrate the efficiency of the optimized polymer coating in practical application. The proposed combination of this unique sulfobetaine formulation and grafting to method provide a potential strategy to effectively generate an antifouling zwitterionic layer on various activated metallic surfaces to control non-specific biofouling to very low levels.

EXPERIMENTAL SECTION

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Materials. Glycidyl methacrylate (GMA), triethylamine (TEA), phosphate buffered saline (PBS) (P3813), 4,4′-Azobis(4-cyanovaleric acid) (ACVA), ethanol, chloroform, acetone, ttrabutylammoium hydrogen sulfate, mineral spirits, sodium hydroxide, hydrochloric acid, carbon disulfide, human plasma fibrinogen (fraction I), primary monoclonal antibody, and secondary monoclonal antibody (anti-mouse IgG) were purchased from Sigma Chemical Co. [2-(Methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide and sulfobetaine methacrylate (SBMA) were purchased from Monomer-Polymer & Dajac Laboratories, Inc., U.S. Glutaraldehyde (25wt%, solution in water) was purchased from ACROS Co. Beef extract was purchased from Scharlau Co. Stainless steel (SUS 316L) and titanium (Class IVpTi) disks were purchased from Walsin Lihwa Corporation (Taiwan). Whole blood was collected in Taipei medical blood center (Beitou, Taipei, Taiwan). It was obtained from a pool of at least five healthy human volunteers. 35 mL of anticoagulant (citrate phosphate dextrose adenine-1) were added to the blood. Deionized water (DI water) was obtained from a Millipore purification system (minimum resistivity: 18.0 MΩ.cm). Synthesis of RAFT Reagent. A mixture containing 0.9 mole of chloroform, 0.36 mole of carbon disulfide, 7.1 millimoles of tetrabutylammonium hydrogen sulfate and 0.9 moles of acetone was prepared in 0.12 L of mineral spirits, in a double walled glass reactor cooled with tap water and under inert atmosphere of nitrogen. Then, 2.52 moles of sodium hydroxide was added dropwise to the mixture, over 1.5h, so as to keep the temperature of the solution below 25oC. The reaction mixture was then stirred overnight, before adding 0.9 L of DI-water (aiming at dissolving the solid formed), followed by 0.12 L of concentrated hydrochloric acid. Then the mixture was stirred for 30 min and purged with nitrogen. Afterwards, the solution was filtered and the obtained solid rinsed thoroughly with water. It was dried under vaccum to constant weigth (41.3 g) to yield a earth colored product. It can be further purified in toluene/acetone (4/1) or by recrystallization from 60% 2-propanol or acetone to yield a yellow crystalline solid. Its 1H, 13C NMR and MS (chemical ionization) characterizations has been documented elsewhere.42 Synthesis of Poly(GMA-tb-SBMA) Copolymers. A schematic of the synthesis process is presented in Scheme 1. A total 15 wt% solid content of SBMA monomer, RAFT-reagent and ACVA initiator was dissolved in 20 mL of DI-water. The molar

[SBMA]:[RAFT-

reagent]:[ACVA] ratios were as follows: 200:1:0.2, 150:1:0.2, 100:1:0.2 and 50:1:0.2). The mixture was purged by nitrogen to remove residual oxygen. The reaction was performed at 60oC for 16 hr under an inert atmosphere of nitrogen. Afterwards, the reaction solution was cooled down by immersing the flask in an ice bath for 1 hr. Methanol was added to 6 ACS Paragon Plus Environment

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precipitate the product formed, which was then dried with a vacuum pumping system for 1 day, and finally freeze-dried at -45 oC for 8 hr. It yielded a yellow powder, which composition was confirmed by 1H NMR (500 MHz spectrometer, Bruker) using D2O as solvent. Once confirmed that the compound formed was PSBMA-RAFT-reagent, we proceeded with the copolymerization of GMA. A total 15 wt% solid content of GMA monomer, PSBMA-RAFTreagent and ACVA initiator was dissolved in water. The molar [GMA]:[PSBMA-RAFTreagent]:[ACVA] ratios were 200:100:0.2, 150:75:0.2, 100:50:0.2 or 50:25:0.2. The reaction was performed at 60 oC for 16 hr under an inert atmosphere of nitrogen, then cooled down by immersing the flask in an ice bath for 1 hr. The product of the reaction was precipitated using acetone, and then redisolved in ethanol to remove residual initiator. Precipitation and redissolution steps were repeated once. Then, the product was dried with a vacuum pumping system for 1 day and freeze-dried at -50 oC for 8 hr to yield a white powder. 1H NMR was also used, as detailed in following sections, to confirm that the compound obtained was the triblock copolymer PSBMAn-b-PGMAn-b-PSBMAn (poly(GMA-tb-PEGMA)) with n = 25, 50, 75, 100, and as defined in Table 1.


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S

nS

O

n

S

O

O

ACVA, 60°C, H2O, 16h

N+

CH3

O

CH3

CH3

N+

CH3

O

O S -

SBMA monomer

ACVA, 60°C, H2O, 16h

S O

O

O

O-

PSBMA-RAFT reagent

PSBMA-b-PGMA-b-PSBMA

n = 25, 50, 75, 100 m = n/2

Scheme 1. Copolymerization of the poly(GMA-tb-SBMA) triblock copolymer by RAFT reaction.

Table 1. Characterization of poly(GMA-tb-SBMA) copolymers. Polymer ID G25 G50 G75 G100

Initial molar ratio of monomers [GMA] : [SBMA] 1:2 1:2 1:2 1:2

Actual composition of copolymers a

Average molecular weight Mw (kDa) 14.7 15.2 18.3 21.8

Mw/Mn 2.4 2.5 2.7 2.3

PGMA (mol%) 30.4 30.7 27.1 26.6

PSBMA (mol%) 69.6 69.3 72.9 73.4

[PGMA] : [PSBMA] 1 ： 2.29 1 ： 2.25 1 ： 2.69 1 ： 2.75

a

Estimated by 1H NMR in D2O from the relative peak area of proton resonance of the epoxide group (PGMA) in the δ range 3.6-3.8 ppm and that of proton resonance of (CH3)2N+ groups (PSBMA) in the δ range 3-3.4 ppm.

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Characterization of Poly(GMA-tb-SBMA) Copolymers. The molecular weight (Mw) and polydisperisty index (Mw/Mn) of the different triblock copolymers were determined using aqueous gel-permeation chromatography (GPC; Viscotek GPCmax Module, USA), equipped with a Viscogel column, type G6000 PW XL, that permits to analyze molecular weights ranging between 2 kDa and 8000 kDa. The system was calibrated using poly(ethylene oxide) standards. The column was maintained at 23 oC and the elution flow rate fixed to 1.0 mL/min. The detector used was a Viscotec refractive-index detector. The eluent was a 0.1 M NaNO3 aqueous solution. Prior to the analysis, all samples were pre-filtered through PTFE membranes (with a 0.2 µm-average pore size). The chemical structure of the triblock copolymer was characterized by 1H NMR, performed on a Bruker 500 MHz instrument. D2O was used as solvent. Grafting of

Poly(GMA-tb-SBMA) onto Titanium and Stainless Steel Surfaces. The

titanium and stainless steel (SUS) metallic surfaces were disposed in glass tubes filled with ethanol or DI water, and the tubes immersed in ultrasonic baths. Then the samples were exposed for 20 min to an UV light ozone cleaner, operated at 110 W, to generate hydroxyl groups at the surface of the metals. Afterwards, the samples were immersed in DI water for 24h and at a temperature of 80°C. The hydroxylated metals were then stored in DI water until surface modification. Before grafting of the copolymers, the metals were first dried with nitrogen gas. Then, the dry substrates were immersed into the aqueous solutions of triblock copolymers at a fixed concentration of 3 mg/mL. A volume V = 100 µL of triethylamine was added to each copolymer solution to catalyze the ring-opening reaction leading to the surface grafting of the copolymer onto the metals. The surface modification was performed at 60 oC for 24 h. Finally, the metals were thoroughly cleaned with DI water to remove the unreacted or loosely adhering polymers, and stored in PBS until use.


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-

O

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O

OO

S

S

O

O

N+

H3C

CH3

N+

H3C

O

O S

O

O

S

n

O

S

m

m O

O

Metallic surface

OH

OH

OH

OH

OH

Hydroxylated metallic surface UV Ozone 20 min 110 W

OH

n

O

O

OH

CH3

O

O-

O-

OH

O-

OH

Activated metallic surface

OH

OH

OH

Grafted metallic surface

Poly(GMA-tb-SBMA) Triethylamine 24h 60°C

Scheme 2. Schematic illustration of the zwitterionic surface grafting on titanium and stainless steel metal substrates.

10


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Characterization of the Poly(GMA-tb-SBMA)-grafted Metallic Surfaces. The effect of the copolymer grafting on the surface hydrophilicity of the metals was evaluated by water contact angle (WCA) measurements performed at 25°C with an automatic contact angle meter instrument (Kyowa Interface Science Co., Model: CA-VP, 209). For these measurements, a 4-µL DI-water droplet was dropped at the surface of each virgin and grafted metallic surface, at 5 different sites. In addition, three independent samples were considered for each metallic surface, and the average obtained considered as the WCA of the material. X-ray photoelectron spectroscopy measurements were conducted with PHI Quantera SXM/Auger spectrometer according to a method previously described.5 A spectroscopic ellipsometer (HORIBA scientific, France), operated at an incident angle of 70°, was used to measure the thickness of the grafted layers onto the modified SUS and titanium metallic surfaces. The surface of the samples was observed by field emission scanning electronic microscope (FE-SEM) using a Hitachi S-4800 instrument. After fixing the metallic samples on a SEM holder with double-sided adhesive tape, the chamber was closed and an accelerating voltage of 1 keV was applied. All images presented were taken at x50K magnification. The virgin and grafted metallic surfaces were examined by atomic force microscopy on a JPK Instruments AG NanoWizard operated in liquid state (DI water), with a commercial Si cantilever purchased from TESP tip. The temperature of the instrument was maintained constant with an AFM Zeiss Loop. In addition, the amplitude at which the cantilever probe oscillates and the scan rate were some of the improtant adjusted parameters. All images presented have a 30 µm x 30 µm surface area. General Biofouling Resistance of the Poly(GMA-tb-SBMA)-grafted Metallic Surfaces Tests Protein adsorption tests were carried out with fibrinogen using a standard enzymelinked immunosorbent assay (ELISA) according to a similar protocol to that described in literature.43 The resitance of grafted stainless steel and titanium surfaces to human blood cells was studied according to the following procedure. Blood fractions (platelet-rich-plasma solution and leukocyte concentrates) were prepared by centrifugation of 250 mL of whole blood at 1200 revolutions per minute, for a total duration of 10 min. 3 replicates of each metal and grafted metal sample were placed in individual wells of a 24 mutliwell plate, and incubated at 37°C with 1 mL of PBS for 3h. Thereafter, PBS was replaced by either 200 µL of recalcified (with 1M CaCl2, 5 µL/well) platelet-rich-plama solution, 1 mL of leukocytes concentrate or 1 mL of whole blood. Incubation of the metallic surfaces with the blood fraction or whole blood was performed at 37°C for 2h. Thereafter, the blood fraction/whole blood was removed 11 ACS Paragon Plus Environment

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and the samples were thouroughly washed with PBS. The adhering blood cells were fixed to the subtrates using a diluted solution of glutaraldehyde (2.5% v/v in PBS, 0.8 mL). This operation was performed at 4°C and lasted 48h. The cells adhering to the metallic surfaces could then be obesrved by confocal microscope using a CLSM A1R instrument (Nikon, Japan), at 5 different positions. For the observation, the excitation and emission wavelengths were fixed to 488 nm and 520 nm, respectively. Finally, the images were analyzed using the open source image analysis software ImageJ®, in order to quantify cell adhesion. The adhesion of tissue cells (HT-1080 fibroblasts) onto the different grafted metallic surfaces was studied as follows. First the cells were grown at 37°C in a Dulbecco’s Modified Eagle medium to which was added fetal calf serum 10%, nonessential amino acids 1%, penicillin streptomycin 1% and sodium pyruvate 1%. Then, 3 replicates of each surface to test were placed in a 24 multiwell plate. The metals were rinsed 3 times with PBS. Then, the samples were incubated with 1 mL of HT1080 cells suspension at 37°C for 1 day in a humidified atmosphere containing 5% carbon dioxide. The observation of adhering cells onto the metals was performed using an Olympus IX-71 microscope. Prior to observation, the cells were fixed with a 4% paraformaldehyde solution, then rinsed with PBS and finally permeabilized with 0.5% t-octylphenoxypolyethoxyethanol. As in blood cell tests, the quantitative analysis was done using the open source ImageJ® software. Bacterial adhesion tests were carried out using Escherichia coli species. Bacteria were first grown similarly to an experimental protocol earlier presented.20 The stationary phase corresponded to a bacterial concentration of 108 cells/mL reached after 12 h of culture. The metallic samples were washed with ethanol, DI-water and PBS, before being placed in a 24 multiwell plate. 1 mL of Escherichia coli solution was poured in each well, and the surfaces incubated with the bacteria for 24h at 37°C. During the incubation period, the bacterial solution was changed 3 times at regular time steps (every 6 hrs). At the end of the incubation period, the bacteria solution was removed and the metallic surfaces washed with PBS. Bacteria adhering to the substrates were stained with 200µL of Live BacLight (10 minincubation). Then, the metals were washed with PBS. The observation of adhering bacteria onto the metals was performed with an Olympus BX51, using an excitation fluorescence filter (450-490 nm). Surface Modification of Medical Devices and Assessment of their Biofouling Resistance. Six artificial tooth roots (titanium-based) and six surgical scalpel (stainless steel-based) were all purchased at a local general medical store (Med First medical supply store, Taiwan). They were washed and surface-modified with poly(GMA-tb-SBMA) according to the procedure 12 ACS Paragon Plus Environment

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above-detailed for model surfaces. The modified roots were incubated with HT1080 cells while the modified scalpels were incubated with Escherichia coli suspensions. The procedures for HT1080 cells adhesion and bacterial attachment, as well as those related to the observation of the samples, are the same as above-mentionned.

RESULTS AND DISCUSSION

Polymerization of Poly(GMA-tb-SBMA) Copolymers via RAFT reaction. We designed a triblock epoxylated

sulfobetaine

copolymer,

poly(GMA-tb-SBMA), in

which the

hydrophobic and hydrophilic blocks are similar to those reported in a previous work in which a random polymer was prepared.33 However, the block structure is expected to outperform the random copolymer for several reasons: (1) all hydrophobic moieties should interact with the hydrophobic surface only, hence minimizing the intermolecular interactions and the chances for heterogeneous multilayer coating; (2) all hydrophilic moieties are expected to be favorably oriented toward the surrounding environment to perform nonfouling, unlike random copolymers in which some hydrophilic segments are entangled within the bulk of the architecture, which prevents them to contribute to water trapping. Moreover, it has to be noted that the triblock architecture was chosen over the diblock configuration for solubility reasons: the large polarity difference between GMA and SBMA prevents solubility of the diblock copolymer in polar solvents. In the triblock configuration, the presence of the two outward hydrophilic blocks surrounding the central hydrophobic block enhances the copolymer solubility. Moreover, RAFT polymerization was chosen because it is a metal-free process that can be carried out with numerous functional groups,44 including zwitterionic functional groups.45 To achieve our target triblock structure of pSBMA-pGMA-pSBMA, the first RAFT-block was synthesized using SBMA repeat units with controlled molar ratio of monomer, initiator and RAFT reagent earlier synthesized according to the work of Lai et al.42 This SBMA-RAFT-block was then used to react with GMA monomer (Scheme 1). and four poly(GMA-tb-SBMA) copolymers, G25, G50, G75 and G100 (named after the theoretical number of repeat units forming each PGMA anchoring block), were formed and characterized (Table 1). The actual compositions of the different copolymers, assessed from 1H NMR analysis (Figure 1) and reported in Table 1 reveal a fair control of the copolymer synthesis with a 13 ACS Paragon Plus Environment

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slightly higher reactivity of GMA. Indeed the PGMA:PSBMA composition ranged between 1:2.25 and 1:2.75, while the theoretical composition corresponding to the initial molar composition in the reaction solution was 1:2. More precisely, the composition of the different triblock copolymers was estimated from the relative peak areas of the proton signal in the epoxide side groups (GMA) (δ ranging between 3.6 ppm and 3.8 ppm) and that of the (CH3)2N+ proton resonance (SBMA), located between δ = 3 ppm and δ = 3.4 ppm. In addition, the molecular weigths of G25, G50, G75 and G100 were found to be 14.7, 15.2, 18.3 and 21.8 kDa, consistently with an increasing of initial molar content of monomers in the initial reaction solution.

#

4, 4‘, 4’’ 7

5+6

3

2

1

S100-b-G100-b-S100

SBMAn-b-GMAn-b-SBMAn

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S

S75-b-G75-b-S75 O

O

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m

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m

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CH3

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4’

CH3

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CH3

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O S

S25-b-G25-b-S25

-

O S

O

O

O

O-

n = 25, 50, 75, 100 m = n/2

δ (ppm)

Figure 1. 1H NMR spectra of the poly(GMA-b-SBMA) copolymers. #: solvent peak.

Preparation and Characterization of Poly(GMA-tb-SBMA) Grafted Metallic Surfaces. The anchoring of the copolymers onto metallic surface relies on the reactivity between the epoxide groups of GMA and the pre-generated hydroxyl groups of the surface, through a base catalysed ring opening reaction. UV-ozone treatment was used to generate hydroxyl groups during the activation step of the surface. Triethylamine (TEA) catalyzed the deprotonation of


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the hydropxyl groups and the nucleophilic attack on the epoxide groups, resulting in a ringopening reaction and in the covalent bonding of the triblock copolymer with the metal surface (Scheme 2). It has to be noted that the grafting conditions (including the process conditions related to the surface activation, the TEA content, etc…) were chosen accordingly to our previous study starring a random copolymer.39 Following the surface-modification process, an XPS analysis was conducted to confirm the grafting of the triblock poly(GMA-tb-SBMA) copolymer onto the surfaces. Figure 2 shows the C1s, O1s, N1s, and S2p core-level spectra of the virgin and grafted metallic surfaces. The nitrogen (N) and sulfur (S) peaks are normally used for characterizing the existence of zwitterionic sulfobetaine on the surface.46, 47 The spectra corresponding to SUS and grafted SUS are displayed in Figure 2a, while those related to titanium and grafted titanium surfaces are presented in Figure 2b. The C1s signal was deconvoluted into three peaks at maximum binding energy of 284.8, 286.3 and 289.1 eV, corresponding to [C-C], [C-O] and [C=O], respectively. The virgin surfaces present one major peak, consistently with the dominating presence of C-C metal bonds and C elements in the alloy forming these materials. The grafting of the triblock copolymer arises in the presence of carboxylate groups attributed to pSBMA blocks. These bonds contribute to the [C-O] and [C=O] signal observed at 286.3 eV and 289.1 eV, respectively. We also noticed that this signal was more intense for G50 polymer, while it seemed weeker for G25, G75 and G100, regardless of the nature of the metal. In addition, it is seen from the N1s that a very week signal is detected for the virgin surfaces, which suggests the presence of nitrogen in the composition of the alloys. Grafting of poly(GMA-tb-SBMA) led to an intense signal located at higher binding energie, 403 eV, corresponding to the the quaternary ammonium group forming SBMA (CH2SO3 -). It is also seen that a strong N1s signal was obtained using G50 polymer only, while it was much weaker for G25, G75 and G100-grafted surfaces. A similar observation was made from the analysis of the S2p spectra: a stong signal was observed at 168.6 eV, attributed to the presence of sulfonate groups, but this signal was more intense on G50-grafted metals than on G25-, G75- and G100-grafted metals. Although we lack of evidences at this stage, these combined observations suggest that the grafting of G50 is more efficient or that it leads to a grafted layer in which acrylate and ziwtterionic groups are more readily detectable and so available to provide the materials with a protective hydration layer. Low intensity signal obtained using G25 might be due to poor grafting or attributed to too short PBSMA segments, while low intensity signals observed on the spectra of G75 and G100 may be ascribed to irregular grafting or polymer aggregation. These hypotheses and observations will 15 ACS Paragon Plus Environment

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have to be further confirmed in subsequent sections. Finally, it is worth noting that the present surface-modification of SUS and Ti metals using this triblock copolymer seems to lead to relatively stable interfaces, as no important increase of surface water contact angle could be measured after 10 days immersion of G50-g-SUS and G50-g-Ti in PBS, as seen in Figure S1 (Supporting Information). The slight increase of WCA observed may correspond to the loss of a loosely adhering superficial layer. Once removed, the coating appears to be stable with a steady and low WCA.

S2p

(a)

C1s

N1s

virgin SUS

N alloy

G25-g-SUS [SO3-]

G50-g-SUS

[C-C], C alloy

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G100-g-SUS 162

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G75-g-Ti

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166

168

170

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282

284

286

288

290

396

398

400

402

404

406

408

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Figure 2. C1s, S2p and N1s core-level spectra of the (a) stainless steel and (b) titanium surfaces modified with the different triblock copolymers.


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Antifouling Capability of the Grafted Surfaces – Effect of the Chain Length of Poly(GMA-tb-SBMA) Copolymers. Fibrinogen is a very sticky protein mediating blood clotting once adsorbed to a surface in contact with blood.48,49 In order to be considered as bioinert, fibrinogen adsorption onto the devices must be minimized. Here, we studied the results of the adsorption of human fibrinogen on the various virgin and grafted surfaces, using polystyrene (100% adsorption) and SBMA hydrogel as positive and negative controls. Figure 3 shows that maximal resistance to protein adsorption was observed on both SUS and titanium surfaces modified with G50 polymer, for which the highest surface hydrophilicity was also obtained. It corresponded to a reduction of 54% (Ti) and 64% (SUS), compared to the unmodified metal surfaces, or 63% (Ti) and 77% (SUS) compared to polystyrene. However, shorter (G25) and longer (G75, G100) were almost inefficient to protect the surfaces. It is also seen that the grafting with G25 is associated with the lowest film thickness (18.2 ± 1.4 nm for G25-g-SUS and 16.7 ± 2.4 nm for G25-g-SUS). These results confirm those of Figure 2, in which the signal of the zwitterionic moieites could be barely detected, which implies that the hydrophobic block is too short to efficiently interact with the metal surfaces. The higher film thickness measured on G50 surface (60.5 ± 5.34 nm for G50-g-SUS and 53.2 ± 2.8 nm for G50-g-SUS) is a physical evidence of the efficient chemical surface modification earlier observed from the C1s, N1s and S2p core-level spectra (Figure 2), leading to an improved surface hydrophilicity, which in turn explains the lowest fibrinogen adsorption given the strong correlation between surface hydrophilicity and nonfouling ability.50 On the other hand, large chain length of poly(GMA-tb-SBMA) did not permit to prepare surfaces efficiently resisting biofouling, regardless of the nature of the metallic substrate. This suggested that the grafting was either inefficient (no chemical interaction of the copolymer with the metal after the grafting process) or that grafted surfaces were heterogeneous with the formation of large polymer aggregates which could not effectively protect the surfaces from biofouling. However, the presence of a quite strong signal on the C1s core-level spectrum at 286.3 eV revealed the presence of the copolymer. So we decided to further analyze the surface structure of the different grafted materials.


Langmuir

(a)

(b) 100

40

40

20

20

15

0

0

0 p PS

e lat

in Vrig

S S S S el US SU rog -SU -SU -SU g-S 5-g 0- g 5-g hyd 00G7 G5 G2 A G1 SBM

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l Ti Ti te -Ti -Ti -Ti oge -gpla gin 5-g 0-g 5-g 00 ydr PS Vri G2 G5 G7 G1 Ah M SB

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Realtive fibrinogen adsorption (%)

80 80

100 Fibrinogen adsorption Water contact angle Thickness

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Fibrinogen adsorption Water contact angle Thickness

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Water contact angle (°)

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Figure 3. The effect of poly(GMA-tb-SBMA) grafting on (1) the resistance of metallic surfaces to fribinogen, (2) the surface hydrophilicity of the metallic surfaces and (3) the thickness of the the metallic surfaces. (a) Results obtained with the stainless steel surfaces; (b) Results obtained with the titanium surfaces. Tissue culture polystyrene (PS) and SBMA hydrogel were used as controls.

Figures 4 and 5 present the different surface morphologies before and after surface modification. Unlike virgin surfaces which are rather smooth, the grafted surfaces all exhibit the presence of grain-like clusters but the characteristic dimensions of the clusters vary importantly from one surface to another. Both the cluster dimension and density are crucial for optimizing the antifouling properties of a metallic surface.39 Virgin surfaces look quite smooth, in particular the stainless steel surface, while the polymer-grafted surfaces exhibit a rougher topography with numerous grains of various sizes distributed all over the metallic surfaces. This increase of surface roughness explains why the WCA did not decrease in an important extent, as it went from 82 ± 2° for virgin SUS down to 38 ± 5° for G100-g-SUS, and from 82 ± 2° for virgin Ti down to 46 ± 9° for G100-g-Ti. Globally, surfaces of G25 and G50 were uniformly covered with very small grains of characteristic length ranging between 50 and 100 nm, which suggests uniform organization of the copolymer layer at the surface of the metals. It is seen, however, that the stainless steel surface modified with G25 exhibits fewer grains, compared with other surfaces, probably explaining the low thickness (18.2 ± 1.4 nm for G25-g-SUS and 16.7 ± 2.4 nm for G25-g-SUS), which can reasonably be associated to a low surface coverage of the triblock copolymer,51,52 as well as the weak XPS signal detection. On the other hand, the titanium surface covered with G50 polymer reveals the presence of larger elliptical-shape grains, probably formed at the locations where the virgin surface exhibits rugosities, which eventually contributed to the higher thickness of 18 ACS Paragon Plus Environment

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these samples and higher surface coverage. Interestingly, surface grafting with larger molecular weight-triblock copolymers (G75 and G100) led to surfaces presenting much larger grains, particularly well seen on the 30 µm x 30 µm AFM images, organized in clusters which characteristic size was at least 1 µm. The titanium surface covered with G100 even shows a larger cluster which almost entirely covered the surface area observed on the SEM image at the magnification chosen (x 50k), with numerous surface cracks on each side. These clusters indeed contributed to the quite thick grafted layer evidenced in Figure 3, but these surfaces were either too physically irregular or too chemically heterogeneous to protect the surfaces from biofouling. virgin SUS

G25-g-SUS

1 µm virgin Ti

G50-g-SUS

1 µm G25-g-Ti

1 µm

G75-g-SUS

1 µm G50-g-Ti

1 µm

G100-g-SUS

1 µm

1 µm

G75-g-Ti

1 µm

G100-g-Ti

1 µm

1 µm

Figure 4. FE-SEM images of the different virgin and grafted metallic surface.

Virgin SUS

G25-g-SUS

G50-g-SUS

G75-g-SUS

G100-g-SUS 750 nm

30 µm

Virgin Ti

30 µm

G25-g-Ti

30 µm

30 µm

G25-g-Ti

G75-g-Ti

30 µm

0 nm

G100-g-Ti 950 nm

30 µm

30 µm

30 µm

30 µm

30 µm

0 nm

Figure 5. 30µm x 30µm AFM images of the different virgin and grafted metallic surface performed in wet state.

Based on the results of Figures 2-5, we propose a possible mechanism to explain the structure/properties relationship based on conformational changes of the different polymers grafted on the metal surfaces, themselves directly depending on the molecular weight of the grafted material, as shown in Scheme 3. The G25 copolymer is too short to (1) efficiently 19 ACS Paragon Plus Environment

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interact with the surfaces and (2) establish intra-molecular interaction through the hydrophilic side blocks. Even if some polymeric molecules are efficiently grafted, the lack of intramolecular interactions prevents surface packing. As a result of this low surface packing (or surface density), little resistance to fouling is obtained. Oppositely, larger molecular weigth polymers (G75 and G100) lead to numerous interactions between the metallic surface and the hydrophobic blocks of the polymer, but also strong electrostatic forces between the SBMA moieties distributed along the long hydrophilic side chains. It results in the crash of the structure followed by the formation of

aggregates that assembled into larger clusters.

Consequently, numerous zwitterionic moieties entrapped within the aggregates could not participate in the protection of the surfaces from biofouling. The G50 copolymer stands in between, and the related grafted surfaces were regarded as presenting the ideal combination of (i) hydrophobic-hydrophobic interactions between the surface and the copolymer, and (ii) intra-molecular interactions between the side chains of the copolymers ensuring a surface packing dense enough to protect the surfaces from biofouling, wihout leading to polymer aggregation. As a conclusion and even though more tests should be performed with other zwitterionic copolymers, the results of this investigation tend to prove that each block copolymer containing zwitterionic hydrophilic moieties present an optimized chain length, on which depend the extent of interactions between the surface to modify and the polymer, as well as the polymer surface arrangement and packing. Both factors are critical to finely tune the fouling resistance of the surfaces at play.


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O O O O

O O O O O O O O O O O O

O O O O

G25-g-metal

G50-g-metal

Low surface coverage Short hydrophilic chains

High surface coverage Expansion of the hydrophilic segments

O O O O O O O O O O O O O O O O O O O O

O O O O O O O O OO O O O O O O

G75-g-metal

G100-g-metal

High surface coverage Long hydrophilic segments start to interact Hydrophilic chains begin to collapse Onset of aggregates formation

High surface coverage Numerous inter-chain interactions Complete collapsing of hydrophilic chains Large aggregates are formed

Scheme 3. A possible mechanism for explaining the chain length dependence of the poly(GMA-tb-SBMA) configuration at the surface of the grafted metals.

General Anti-biofouling Property Resistance of Grafted Metallic Surfaces. Surfaces foreign body reactions are common when a material is not entirely biocompatible. The formation of blood clots arising from blood cells adhesion on the material, so called interface-induced coagulation,21, 47 but also tissue inflammation after the early tissue cellmetal interactions, or various tissue infections due to biofilm formation, are some evidences of the lack of biocompatibility of the metallic devices. Clearly, biocompatibility and biofouling resistance are closely related to each other. Taking that into account, we incubated our materials with blood cells (from individual concentrates or from whole blood), fibroblasts and various species of bacteria. Starting with 21 ACS Paragon Plus Environment

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the evaluation of the resistance to blood cells of the different materials, results reported in Figure 6 shows that a high amount of adhered platelets and leukocytes can be observed on both the virgin SUS and titanium samples. With the grafting of poly(GMA-tb-SBMA) triblock copolymers, platelets and leukocytes attachment significantly decreased to reach a minimum using G50 polymer, hence supporting previous results on fibrinogen adsorption. However, using G75 and G100 polymers had very little effect on the hemocompatibility of the different metals. In addition, even though less cells from whole blood were detected on the different virgin and grafted metals, because of lower cell concentration than in the platelets or leukocytes concentrates used in individual cell attachment tests, grafting the surface with G50 still led to a noticeably important decrease of cell adhesion from whole blood. From an image analysis of the confocal results, the numbers of adhered platelets, leukocytes, and cells from whole blood on the virgin SUS surfaces were found to be 2979 ± 564 cells/mm2, 1910 ± 205 cells/mm2 and 374 ± 158 cells/mm2, respectively. A significant decrease of the number of platelets, leukocytes and cells from whole blood cells were observed on the G50 grafted surface, with only 558 ± 177 cells per mm2 adhering platelets, 51 ± 25 cells/mm2 attached leukocytes, and 7 ± 4 cells/mm2 after incubation with whole blood. For titanium samples, the virgin surface also exhibited high cell adhesion, with densities found to be approximately 4774 ± 601 cells/mm2, 1537 ± 307 cells/mm2 and 710 ± 122 cells/mm2, using platelets, leukocytes and cells from whole blood, respectively. Similarly to the results obtained grafting G50 on SUS, the G50-modified titanium surfaces showed minimized cell adhesion, measured to be 706 ± 83 cells/mm2 using platelets concentrate, 62 ± 28 cells/mm2 with leukocytes concentrates and 50 ± 23 cells/mm2 after incubation of the surfaces with whole blood. These results correspond to an overall cell adhesion reduction ranging between 80 and 97% using G50 surfaces, and compared with unmodified metals.


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(a)

virgin SUS

G25-g-SUS

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Platelets Leukocytes Cells from whole blood

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500 0 Virgin Ti

G25-g-Ti

G50-g-Ti

G75-g-Ti

G100-g-Ti

Scale bar: 100 µm

Figure 6. The effect of surface grafting with the poly(GMA-tb-SBMA) polymers on the resistnce to blood cell of (a) stainless steel and (b) titanium surfaces. 23


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Metals including SUS and titanium alloy are broadly used in surgical materials. These surgical materials should efficiently resist the adhesion of bacteria, to avoid bacterial-induced infections. Escherichia coli (EC) and Staphylococcus epidermidis (SE) are common bacterial species able to cause these infections; 53,54 thus we incubated the different virgin metals and grafted metals with EC and SE solutions. Figure 7 shows the fluorescence microscope images and related quantitative analysis of the attached EC and SE on SUS, titanium and poly(GMAtb-SBMA) grafted SUS and titanium surfaces. Large EC and SE densities can be osberved at the surface of the virgin SUS and titanium surfaces. All grafted surfaces evidence a significant decreasing of bacterial attachment, but the best results were again obtained using G50 polymer. From the quantitative analysis, the reduction of bacterial attachment on poly(GMA-tb-SBMA)-grafted SUS and titanium surfaces ranged between 93 and 96%, compared with the unmodified metallic surfaces. EC and SE density onto SUS surface covered with G50 were found to be about 1911 cells/mm3 and 2491 cells/mm3, respectively while for a similar grafting, densities on the grafted titanium surfaces were also low and measured to be 1174 cells/mm3 and 799 cells/mm3 for EC and SE, respectively. These tests correlate with the results of protein adsorption and blood cells adhesion, and thus, tend to further show the general antifouling property of G50 polymer.


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virgin SUS

G25-g-SUS

G50-g-SUS

G75-g-SUS

G100-g-SUS

25 Escherichia coli Staphylococcus epidermidis

Cell density (x10-3 cells/mm2)

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25 Escherichia coli Staphylococcus epidermidis

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G25-g-Ti

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Scale bar: 50 µm

Figure 7. The effect of surface grafting with the poly(GMA-tb-SBMA) polymers on the resistance to bacteria of (a) stainless steel and (b) titanium surfaces.

25


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To further prove the general antifouling property of the G50 surfaces, we performed fibroblast tissue cells adhesion tests. Fibroblasts represent the most common cell population likely to be in contact with biomaterials, in particular in the fields of wound healing,55 tissue engineering,56 or regenerative medicine.57 As seen in Figure 8, numerous cells can be observed on the virgin SUS and titanium surfaces, evidencing their lack of biocompability. Grafting the different metals with the triblock copolymers enabled to significantly reduce the extent of fibrobalsts adhesion, and minimum attachment was observed using G50 polymer. More precisely, HT1080 densities on virgin SUS and titanium were found to be 1387 ± 34 cells/mm2 and 1418 ± 32 cells/mm2, respectively. These densities felt to 54 ± 8 cells/mm2 and 25 ± 13 cells/mm2 on the related grafted metals modified with G50.

virgin SUS

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Scale bar: 50 µm

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Ti

1200 1000 800 600 400 200 0

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G25-g-metal

G50-g-metal

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Figure 8. The effect of surface grafting with the poly(GMA-tb-SBMA) polymers on the resistance to HT1080 fibroblast attachment on the various metallic surfaces.


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In conclusion, the series of biofouling tests have proven the general antifouling property of the triblock copolymer provided a fine tuning of the molecular design, later influencing the quality of the grafting. It is clear that we manage to take advantage of the well-established antifouling properties of SBMA,3,11,12 providing the optimization of the surface grafting conditions through the control of the triblock copolymer chain length. The chain length plays a dominant role on the physical organization of the brushes at the surface, and was proven to control the extent of biofouling. This also suggests that the formation of an effective protective hydration layer preventing biofouling,58 is also controlled by this parameter.

Surface Grafting of Poly(GMA-tb-SBMA) Copolymers Onto a Dental Root and a Surgical Scalpel. To demonstrate the efficiency of the triblock epoxylated sulfobetaine copolymers in practical usage, we surface-modified a dental root (titanium metal) and a surgery scalpel (stainless steel metal). Titanium root is used as a dental implant for its excellent mechanical properties and overall fair hemocompatibility.26,27 It is directly in contact with the mandible bone and the surrounding muscle cells. To prevent potential infection arising from uncontrolled cell-metal interactions or biofilm formation, the implanted root should ideally resist biofouling. Here, we coated the root with G50 polymer, and carried HT1080 fibrobalsts adhesion tests, to evaluate the cell compatibility of the root. As shown in Figure 9a, an important decrease of HT1080 attachment was observed on the G50-grafted titanium root surface, compared with the unmodified titatnium root, hence evidencing an improvement of the biocompatibility of the implant. Less than 28 cells/mm2 were counted on the implanted root surface grafted with G50, corresponding to a reduction of 95%, compared with the virgin surface. This first example proved the good resistance to tissue cells of the zwitterionic copolymer in a more practical case. Secondly, we grafted a surgical scalpel using G50 polymer. Surgical knifes and tools must resist totally the adhesion of bacteria, in particular, as infections arising from biofilm formation may have dramatic consequences. After incubating the unmodified stainless steel scalpel with EC, numerous cells could still be observed on the realted confocal image in Figure 9b. On the other hand, after grafting the tool with G50, a 96% reduction of EC attachment was obtained, correpsonind to a cell density as low as 2 cells/mm2. The results indicate that the grafting onto approach using poly(GMA-tb-SBMA) triblock copolymer can be practically and succesffully applied in the surface modification of stainless steel and titatnium based materials commonly used in the biomedical field, in order to achieve lowbifouling. 27 ACS Paragon Plus Environment

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(a)

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(b)

Virgin root

Virgin scalpel

Grafted root

Grafted scalpel

Figure 9. Evaluation of the poly(GMA-tb-SBMA) grafting efficiency (G50-grafting) to provide biofouling resistance to (a) titanium dental roots and (b) stainless steel scalpel. The antifouling properties were tested using (a) HT1080 fibroblasts and (b) Escherichia coli bacteria.

CONCLUSION In this study, we designed a series of unique triblock epoxylated sulfobetaine copolymers, poly(GMA-tb-SBMA), containing one epoxylated group and two zwitterionic blocks. These copolymers with varying moelcular weight and constant GMA/SBMA mole ratio were grafted onto metallic surfaces of titanium and stainless steel, in order to improve and optimize their antifouling properties. It was first shown that the block copolymer synthesis was failry well controlled, from the initial molar ratio in the polymerization reaction bath. A preliminary investigation of antifouling properties using fibrinogen protein revealed that the copolymer with a 15.2 kDa molecular weight, corresponding to 50 repeat units in each block, provided the best fouling resistance. From a physicochemical analysis of the surfaces, it was ascribed to the formation of homogeneous and well-dispersed copolymer domains at the surfaces of the metals. On the contrary, a polymer with a lower molecular weight led to a lower surface coverage while larger molecular weights led to the crash of the polymeric sturctures and the formation of aggregates resulting from numerous intra- and intermolecular electrostatic interactions. The metallic surfaces grafted with the optimized copolymer were then shown to efficiently resist biofouling by a number of biofoulants including leukocytes 28 ACS Paragon Plus Environment

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concentrates, platelets concentrates, cells from whole blood, Escherichia coli, Staphylococcus epidermidis and HT1080 fibrobalsts cells. Finally, dental roots and surgical scalpels were modified with poly(GMA-tb-SBMA) and cell adhesion tests confirmed the conclusions drawn from the analysis of biofouling on model surfaces. As a conclusion, poly(GMA-tbSBMA) is a viable solution to efficiently modify metallic surfaces used in biomedical devices.

ASSOCIATED CONTENT

Supporting information The following Supporting Information is available free of charge on the ACS Publications website: stability tests.

AUTHOR INFORMATION

Corresponding Author *Yung Chang: E-mail: [email protected]. Phone: 886-3-265-4122. Fax: 886-3-265-4199 Author Contributions This manuscript was written through contributions of all authors. Notes The authors declare no competing financial interest.

ACKNLOWDGEMENT The authors would like to acknowledge the project of Outstanding Professor Research Program at Chung Yuan Christian University, Taiwan, and the Ministry of Science and Technology (MOST 102-2221-E-006-219-MY3, 102-2221-E-033-009-MY3 and 103-222129 ACS Paragon Plus Environment

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E-033-078-MY3) for their financial support. The authors extend their appreciation to the International Scientific Partnership Program ISPP at King Saud University for funding this research work through ISPP# 0051.

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Figure Captions Scheme 1. Copolymerization of the poly(GMA-tb-SBMA) triblock copolymer by RAFT reaction. Scheme 2. Schematic illustration of the zwitterionic surface grafting on titanium and stainless steel metal substrates. Figure 1. 1H NMR spectra of the poly(GMA-b-SBMA) copolymers. #: solvent peak. Figure 2. C1s, S2p and N1s core-level spectra of the (a) stainless steel and (b) titanium surfaces modified with the different triblock copolymers. Figure 3. The effect of poly(GMA-tb-SBMA) grafting on (1) the resistance of metallic surfaces to fribinogen, (2) the surface hydrophilicity of the metallic surfaces and (3) the thickness of the the metallic surfaces. (a) Results obtained with the stainless steel surfaces; (b) Results obtained with the titanium surfaces. Tissue culture polystyrene (PS) and SBMA hydrogel were used as controls. Figure 4. FE-SEM images of the different virgin and grafted metallic surface. Scheme 3. A possible mechanism for explaining the chain length-dependence of the poly(GMA-tb-SBMA) configuration at the surface of the grafted metals. 33 ACS Paragon Plus Environment

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Figure 5. 30µm x 30µm AFM images of the different virgin and grafted metallic surface performed in wet state. Figure 6. The effect of surface grafting with the poly(GMA-tb-SBMA) polymers on the resistnce to blood cell of (a) stainless steel and (b) titanium surfaces. Figure 7. The effect of surface grafting with the poly(GMA-tb-SBMA) polymers on the resistance to bacteria of (a) stainless steel and (b) titanium surfaces. Figure 8. The effect of surface grafting with the poly(GMA-tb-SBMA) polymers on the resistance to HT1080 fibroblast attachment on the various metallic surfaces. Figure 9. Evaluation of the poly(GMA-tb-SBMA) grafting efficiency (G50-grafting) to provide biofouling resistance to (a) titanium dental roots and (b) stainless steel scalpel. The antifouling properties were tested using (a) HT1080 fibroblasts and (b) Escherichia coli bacteria.


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TOC Figure

virgin Bacteria

Blood

Fibroblasts

pSBMA-b-pGMA-b-pSBMA + - + - + - + - + - + + - + - + OH O

O

O

O

O

grafted Bacteria

Blood

Fibroblasts

Titanium/stainless steel


Epoxylated Zwitterionic Triblock Copolymers Grafted onto Metallic

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