Surface Modification of Silicone for Biomedical Applications Requiring

Nov 2, 2012 - National University Hospital, Kent Ridge, Singapore 117576. •S Supporting Information. ABSTRACT: Silicone has been used for peritoneal...
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Surface Modification of Silicone for Biomedical Applications Requiring Long-Term Antibacterial, Antifouling, and Hemocompatible Properties Min Li,† Koon Gee Neoh,*,† Li Qun Xu,† Rong Wang,† En-Tang Kang,† Titus Lau,‡ Dariusz Piotr Olszyna,‡ and Edmund Chiong‡ †

Department of Chemical and Biomolecular Engineering, National University of Singapore, Kent Ridge, Singapore 117576 National University Hospital, Kent Ridge, Singapore 117576

‡

S Supporting Information *

ABSTRACT: Silicone has been used for peritoneal dialysis (PD) catheters for several decades. However, bacteria, platelets, proteins, and other biomolecules tend to adhere to its hydrophobic surface, which may lead to PD outflow failure, serious infection, or even death. In this work, a crosslinked poly(poly(ethylene glycol) dimethacrylate) (P(PEGDMA)) polymer layer was covalently grafted on medical-grade silicone surface to improve its antibacterial and antifouling properties. The P(PEGDMA)grafted silicone (Silicone-g-P(PEGDMA)) substrate reduced the adhesion of Staphylococcus aureus, Escherichia coli, and Staphylococcus epidermidis, as well as 3T3 fibroblast cells by ≥90%. The antibacterial and antifouling properties were preserved after the modified substrate was aged for 30 days in phosphate buffer saline. Further immobilization of a polysulfobetaine polymer, poly((2-(methacryloyloxy)ethyl)dimethyl-(3-sulfopropyl)ammonium hydroxide) (P(DMAPS)), on the Silicone-g-P(PEGDMA) substrate via thiol−ene click reaction leads to enhanced antifouling efficacy and improved hemocompatibility with the preservation of the antibacterial property. Compared to pristine silicone, the so-obtained Silicone-gP(PEGDMA)-P(DMAPS) substrate reduced the absorption of bovine serum albumin and bovine plasma fibrinogen by ≥80%. It also reduced the number of adherent platelets by ≥90% and significantly prolonged plasma recalcification time. The results indicate that surface grafting with P(PEGDMA) and P(DMAPS) can be potentially useful for the modification of silicone-based PD catheters for long-term applications.

1. INTRODUCTION

our aim is to modify the surface of silicone to improve its antibacterial, antifouling, and hemocompatible properties. Hydrophilization of silicone surface has been reported by many studies to be an effective way to improve its antibacterial and antifouling properties.8,9 The easiest way is to introduce hydroxyl groups directly on the surface by oxygen plasma, ultraviolet light, and corona discharge. However, such modifications are temporary because PDMS chains can rearrange and recover the hydrophobic surface of silicone within a few hours and multiple treatments are necessary to attain stable modifications.10−12 In addition, these modifications are not always very helpful for the inhibition of biofilm formation in vivo.13 A more effective method to reduce the hydrophobicity of silicone is the tethering of functional polymer brushes on its surface via covalent bonding. The highly hydrophilic, nontoxic, and nonimmunogenic poly(ethylene glycol) (PEG) and its derivatives are currently the most commonly used polymers for reducing bacterial adhesion as well as nonspecific protein adsorption on surfaces.14−17

In the past decades, various synthetic materials have been developed for biomedical and biochemical applications, such as microfluidic devices, drug delivery systems, and clinical implant devices.1,2 Among them, silicone, or poly(dimethyl siloxane) (PDMS), is one of the most common choices for biomedical devices (e.g., ureteral stents, catheters, endotracheal tubes, voice prostheses) because it is inert to body fluids and a wide range of medical fluids, and it is also nontoxic and thermally and chemically stable.3,4 However, a major disadvantage of silicone is its highly hydrophobic nature. As a result, bacteria, proteins, and biomolecules tend to readily adhere on the silicone surface. Bacterial adhesion and biofilm formation may lead to severe infection, device failure, or even death.5 In addition, when silicone material comes into contact with blood and living tissues, platelet and cell adhesion may also result in serious problems. For example, omental wrapping of peritoneal dialysis (PD) catheters may lead to extraluminal obstruction and eventual PD outflow failure.6 In PD, the Tenckhoff catheter is considered the “gold standard”, and although new types of catheters have been designed, it has been shown that none of the commonly used catheters are free of complications.7 Thus, © 2012 American Chemical Society

Received: August 26, 2012 Revised: October 24, 2012 Published: November 2, 2012 16408

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to this solution. The solution was stirred for 24 h at room temperature, and then added to excess acetone to precipitate the resulting HSP(DMAPS) and remove the remaining dithioerythritol. The HSP(DMAPS) was further purified by two dissolution−reprecipitation cycles, using double-distilled water (10 mL) to dissolve and acetone (100 mL) to precipitate, prior to being dried under reduced pressure. 2.3. Preparation of P(PEGDMA)-, P(PEGMA)-, and P(DMAPS)Grafted Silicone Films. Medical-grade silicone film (thickness of ∼1 mm) was cut into 2 × 5 cm2 pieces, cleaned ultrasonically in isopropanol once, distilled water twice, for 10 min in each step. The silicone films were exposed to an oxygen−ozone gas mixture generated from an Azcozon ozone generator (model RMU16−04EM) at an oxygen inlet flow rate of 60 L/h, and the ozone production rate was about 2.5 g/h. In this work, the ozone treatment period was set at 20 min since an earlier work reported that, under similar conditions, the peroxide concentration on silicone surfaces increases rapidly in the first 20 min of ozone treatment followed by a very slow increase due to saturation of the surface peroxide concentration.25 For the preparation of the Silicone-g-P(PEGDMA) films, the silicone films after ozone treatment were introduced into an aqueous solution containing 10 wt % of PEGDMA macromonomer and the solution was degassed by bubbling argon through the solution for 30 min. The PEGDMA macromonomer solution containing the ozone-treated silicone film was then irradiated by UV light in a Riko rotary photochemical reactor (model RH400−10W) for 10, 30, and 60 min to obtain the Silicone-gP(PEGDMA)1, Silicone-g-P(PEGDMA)2, and Silicone-g-P(PEGDMA)3 films, respectively. After UV irradiation, the Silicone-gP(PEGDMA) films were washed thoroughly with ethanol and water for an additional 24 h at 60 °C to remove the physically adsorbed macromonomer and homopolymer. The P(PEGMA)-grafted silicone films (Silicone-g-P(PEGMA)1, Silicone-g-P(PEGMA)2, and Siliconeg-P(PEGMA)3) and P(DMAPS)-grafted silicone films (Silicone-gP(DMAPS)) were similarly prepared using PEGMA macromonomer and DMAPS monomer, respectively. 2.4. Coupling of P(DMAPS) to P(PEGDMA)-Grafted Silicone Films (Silicone-g-P(PEGDMA)-P(DMAPS)). For the preparation of Silicone-g-P(PEGDMA)2-P(DMAPS)1 film, the HS-P(DMAPS) polymer was covalently coupled onto the Silicone-g-P(PEGDMA)2 films via thiol−ene click reaction between the thiol groups of HSP(DMAPS) and the residual double bonds of the P(PEGDMA) layer. Briefly, the Silicone-g-P(PEGDMA)2 film was introduced into an aqueous solution containing 0.5 wt % of HS-P(DMAPS) polymer and 5 mg of 2,2-dimethoxy-2-phenylacetophenone. After degassing by bubbling argon through the solution for 30 min, the solution was irradiated by UV light for 10 min. After UV irradiation, the Silicone-gP(PEGDMA)2-P(DMAPS)1 film was washed thoroughly with ethanol and water for an additional 24 h at 60 °C to remove the physically adsorbed HS-P(DMAPS) polymer. Other Silicone-g-P(PEGDMA)P(DMAPS) films (Silicone-g-P(PEGDMA)2-P(DMAPS)2, Silicone-gP(PEGDMA)2-P(DMAPS)3, and Silicone-g-P(PEGDMA)2-P(DMAPS)4) were prepared using similar procedures but with different thiol−ene click reaction times. 2.5. Bacterial Adhesion and Biofilm Formation Assay. For the bacterial adhesion assay, overnight bacterial culture broth (nutrient broth for E. coli, and tryptic soy broth for S. aureus and S. epidermidis) was centrifuged at 2700 rpm for 10 min to remove the supernatant. The bacteria were washed with PBS (pH 7.4) twice and resuspended in PBS at a concentration of 108 cells/mL. The bacterial concentrations were estimated from the optical density of the suspension based on a standard calibration from spread plate counting. An optical density of 0.1 at 540 nm was equivalent to 108 cells/mL. The pristine and modified silicone films of size 1 × 1 cm2 were rinsed with PBS thrice followed by sterilization under UV irradiation for 30 min, and then placed in a 24-well plate and covered with 1 mL bacterial suspension for 4 h at 37 °C. The substrates were then washed three times with PBS to remove any nonadhered or loosely adhered bacteria. For scanning electron microscopy (SEM) visualization, the bacterial cells on the substrates were fixed with 3 vol % glutaraldehyde in PBS overnight at 4 °C, and then subjected to serial dehydration with 25%, 50%, 75%, and 100% ethanol for 10 min each. The substrates

Nevertheless, the PEG polymer brush-modified silicone still suffers from the hydrophobic recovery problem,18 which limits its long-term application (more than 14 days). Recently, polysulfobetaine polymer, which contains the pendant zwitterionic groups of sulfobetaine, has attracted increasing interest due to its excellent biocompatibility and blood compatibility, as well as its ease in synthetic preparation.19−21 Grafting of polysulfobetaine polymer brushes on various substrates for biomedical applications has been reported.22−24 However, reports on surface modification of silicone for long-term improvement of its antibacterial, antifouling, and hemocompatible properties are lacking. In this study, a cross-linked PEG-containing polymer (poly(poly(ethylene glycol) dimethacrylate), P(PEGDMA)) layer was covalently bonded on the medical-grade silicone surface to improve its antibacterial and antifouling properties. The long-term stability of the P(PEGDMA)-grafted silicone (silicone-g-P(PEGDMA)) was tested by aging the substrate in phosphate buffered saline (PBS) for 30 days. In addition, to further improve the silicone’s hemocompatibility, the polysulfobetaine, poly((2-(methacryloyloxy)ethyl)dimethyl-(3sulfopropyl)ammonium hydroxide) (P(DMAPS)), was subsequently anchored onto the silicone-g-P(PEGDMA) surface. Bacterial, cell, and platelet adhesion and protein adsorption on pristine and polymer-modified silicone were evaluated and compared in vitro.

2. EXPERIMENTAL SECTION 2.1. Materials. Medical-grade silicone films were purchased from Bioplexus Inc. (Ventura, CA). Poly(ethylene glycol) methacrylate (PEGMA) macromonomer (Mn ∼526), poly(ethylene glycol) dimethacrylate (PEGDMA) macromonomer (M n ∼550), [2(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (DMAPS, 97%), N,N′-bis(acryloyl)cystamine (BAC, 98%), 4,4′azobis(4-cyanovaleric acid) (98%), folate-free Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, L-glutamine, penicillin, bovine serum albumin (BSA), bovine plasma fibrinogen (FBG), and all solvents used (analytical grade) were purchased from Sigma− Aldrich (St. Louis, MO). PEGMA and PEGDMA macromonomers were passed through a ready-to-use inhibitor-removal column to remove the inhibitor and stored at −20 °C. 3-[4,5-Dimethyl-thiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) was purchased from Alfa Aesar Co. (Ward Hill, MA). Spectra-Por dialysis membranes (molecular weight cutoff: 1000) were obtained from Spectrum Laboratories, Inc. (Rancho Dominguez, CA). Escherichia coli (E. coli, ATCC DH5α), Staphylococcus aureus (S. aureus, ATCC 25923), Staphylococcus epidermidis (S. epidermidis, ATCC 12228), and 3T3 mouse fibroblast cells were purchased from American Type Culture Collection (Manassas, VA). LIVE/DEAD BacLight Bacterial Viability Kit L131152 was purchased from Molecular Probes Inc. (Eugene, OR). 2.2. Synthesis of the Thiolated P(DMAPS) (HS-P(DMAPS)) Polymer. P(DMAPS-co-BAC) copolymer was first synthesized via free radical polymerization of DMAPS and BAC monomers. Briefly, 2 g of DMAPS (7.14 mmol), 0.093 g of BAC (0.36 mmol), and 20 mg of 4,4′-azobis(4-cyanovaleric acid) (7.14 × 10−2 mmol) were added to a flask containing 5 mL of water/ethanol (50:50 vol %) mixed solvent. After purging with argon for 30 min, the flask was sealed and heated to 65 °C. The polymerization was allowed to proceed under continuous stirring at that temperature overnight. After reaction, the reaction mixture was cooled to room temperature. The crude polymer was purified via dialysis in double-distilled water for 72 h followed by freeze−drying. HS-P(DMAPS) was prepared by reduction of the disulfide bond present in BAC moieties of the P(DMAPS-co-BAC) copolymer. The P(DMAPS-co-BAC) copolymer (200 mg) was put in 10 mL doubledistilled water, and an excess of dithioerythritol (100 mg) was added 16409

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2.8. Cell Adhesion. Pristine and modified silicone films of size 1 × 1 cm2 were placed in a 24-well plate and seeded with 1 mL of medium containing 3T3 fibroblasts at a density of 2 × 105 cells/mL. After 24 h of incubation, the substrates were rinsed three times with culture medium to remove cells that did not adhere or adhered loosely, and the adherent cells on the substrates were viewed using a Leica DM IL LED microscope. A standard calibration curve was generated by seeding a known number of 3T3 fibroblasts onto a 24-well plate and incubating for 4 h at 37 °C followed by MTT assay. For quantification of adherent 3T3 fibroblast cells, the silicone films with adherent cells (after rinsing with culture medium) were placed in a 24-well plate followed by MTT assay. The number of 3T3 fibroblasts adhered on the films was calculated from the standard calibration curve. 2.9. Platelet Adhesion. Fresh blood collected from a healthy rabbit was immediately mixed with 3.8 wt % sodium citrate solution at a dilution ratio of 9:1. It was then centrifuged at 700 rpm at 8 °C for 10 min to obtain platelet-rich plasma (PRP). The PRP was diluted with PBS in a 1:1 (v/v) ratio. 0.1 mL of the diluted PRP was then introduced onto the silicone film of size 1 × 1 cm2. The film was incubated at 37 °C for 1 h under static condition. After incubation, the film was gently washed three times with PBS. The adherent platelets were then fixed with 3 vol % glutaraldehyde in PBS solution overnight at 4 °C, and then subjected to serial dehydration with 10%, 25%, 50%, 75%, 90%, and 100% ethanol for 10 min each. The film was dried, coated with platinum, and then observed under SEM. The number of platelets on the films was determined by counting the total number of adherent platelets from representative SEM images at the same magnification (2000×).29 The results obtained from the modified films were normalized by that from pristine silicone. 2.10. Plasma Recalcification Time (PRT). Fresh blood collected from a healthy rabbit was diluted with 3.8 wt % sodium citrate solution as mentioned above. The blood was centrifuged at 3000 rpm, 8 °C for 20 min to obtain the platelet poor plasma (PPP). 0.1 mL of the PPP was then introduced onto the silicone films of size 2 × 2 cm2 (after equilibration in distilled water at 37 °C for 1 h) and incubated at 37 °C under static condition for 10 min. After incubation, 0.1 mL of 0.025 M CaCl2 aqueous solution at 37 °C was then added to the PPP on the silicone films. The plasma solution was monitored for clotting by manually dipping a stainless steel wire hook coated with silicone into the solution to detect fibrin threads. The PRT was recorded at the first appearance of silky fibrin. 2.11. Hemolysis Test. Silicone film of size 1 × 1 cm2 was equilibrated in a BIOLOGIX centrifuge tube containing 10 mL of PBS at 37 °C for 1 h. 0.2 mL of diluted rabbit blood (8 mL of blood diluted with 10 mL of PBS) was then added to the tube, and the tube was incubated at 37 °C for 1 h. Double-distilled water and PBS were used for positive and negative controls, respectively. The tube was centrifuged at 1500 rpm for 10 min and the optical absorbance of the supernatant was measured at 545 nm on a BIO-TEK microplate reader. The hemolysis rate (HR) was calculated as follows:

were dried, coated with platinum, and then observed under SEM. Quantification of adherent bacteria was carried out by the spread plate method as described in the literature.26 The viability of bacterial cells on pristine and modified silicone was assessed using a combination dye (LIVE/DEAD BacLight Bacterial Viability Kit). The substrates were washed with water after the incubation period, stained with 50 μL of the combination dye, and then observed under a Leica DMLM microscope with a 100 W Hg lamp. S. aureus biofilm formation assay was carried out in a similar manner to that described in the literature.26 Briefly, S. aureus bacterial broth after overnight culture was diluted to a concentration of 105 cells/mL with tryptic soy broth. Pristine and modified silicone films were placed in a 24-well plate, and 1 mL of bacterial suspension was added to each well. The plate was incubated at 37 °C for 24 h to allow biofilm growth. After washing with PBS, the films were observed under SEM as described above. 2.6. Protein Adsorption. BSA and FBG were dissolved in CPBS (citrate−phosphate buffer saline, i.e. PBS with 0.01 M sodium citrate, pH 7.4) at a concentration of 1.0 mg/mL, respectively. Pristine and modified silicone films of size 1 × 1 cm2 were first equilibrated for 1 h in CPBS. The CPBS was then replaced by the protein solution, which remained in contact with the test film surface for 4 h at 37 °C. After this period, the films were gently washed twice with the buffer solution and twice with double-distilled water. The amount of protein adsorbed on the films was determined by the modified dye-interaction method using BioRad protein dye reagent (catalog no. 500−0006).27,28 The stock dye solution was diluted five times with double-distilled water before use. Protein solutions (0.2 mL) of known concentration were added to 5 mL of the dye solution. The protein−dye solutions were kept for 10 min and centrifuged at 5000 rpm for 15 min. The absorbance of the supernatant at 465 nm (measured using a Shimadzu UV-1601 PC scanning spectrophotometer) was used to obtain a standard calibration curve. For the quantitative determination of adsorbed protein, the film with adsorbed protein was immersed in 5 mL of the dye solution. The dye would react with the protein which remained either adsorbed on the surface or desorbed into the solution to form a complex. After 3 h of reaction, the film was removed, the remaining solution was centrifuged to remove the protein−dye complex in the solution, and the absorbance of the supernatant was measured at 465 nm. The amount of protein adsorbed on the films was calculated by comparing the absorbance of the supernatant with the standard calibration curve. 2.7. Cytotoxicity Assay. The effect of the substrates on cell viability was investigated using the MTT assay according to the standard protocol stated in ISO 10993−5 for evaluation of in vitro cytotoxicity of medical devices by direct contact. 3T3 fibroblast cells were cultured in DMEM supplemented with 10% fetal bovine serum, 1 mM L-glutamine, and 100 IU/mL penicillin. One milliliter of the medium containing 3T3 fibroblasts at a density of 104 cells/mL was placed in each well in a 24-well plate and incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air for 24 h. The medium was then replaced with a fresh one, and pristine and modified silicone films of size 1 × 1 cm2 were gently placed on top of the cell layer in the well. Since the density of the silicone film is only slightly higher than the medium, the films only touched but did not compress the cell layer. The control experiments were carried out using the complete growth culture medium without substrates (nontoxic control). The cells were then incubated at 37 °C for another 24 h. The culture medium in each well was then removed, and the substrates were very gently taken out without disrupting the cell layer. Observations using a Leica DM IL LED microscope confirmed that there was no obvious cell attachment on the substrates. 900 μL of medium and 100 μL of MTT solution (5 mg/mL in PBS) were then added to each well. After 4 h of incubation at 37 °C, the medium was removed and the formazan crystals were dissolved in 1 mL of dimethyl sulfoxide (DMSO) for 15 min. The optical absorbance was then measured at 570 nm on a BIO-TEK microplate reader (model Powerwave XS). The results were expressed as percentages relative to the optical absorbance obtained in the control experiments.

HR = (AS − AN)/(AP − AN)

(1)

where AS, AN, and AP are the optical absorbance of the supernatant of the solution containing silicone film, the negative control, and the positive control, respectively. 2.12. Stability Test. The modified silicone substrates were aged by immersion in PBS at 37 °C for 30 days. The substrates after the aging period were subjected to the bacterial, cell, and platelet adhesion, and protein adsorption tests described above to assess the stability of the coating. 2.13. Characterization. The surface chemical composition of the silicone films was analyzed by X-ray photoelectron spectroscopy (XPS) on an AXIS UltraDLD spectrometer (Kratos Analytical Ltd., U.K.) with a monochromatic Al Kα X-ray source (1486.71 eV photons). Fourier transform infrared (FT-IR) spectra were obtained in transmission mode on a Shimadzu FT-IR spectrophotometer (model 8400). Gel permeation chromatography (GPC) measurement was performed on a Waters GPC system equipped with Waters Styragel columns, a Waters-2487 dual wavelength UV detector, and a Waters2414 refractive index detector. Water was used as the eluent at a flow 16410

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Figure 1. Schematic illustration of the modification of silicone surface via ozone treatment (Step 1), UV-induced polymerization (Step 2) and thiol− ene click chemistry (Step 3).

Table 1. Surface Composition and Water Contact Angle of the Polymer-Modified Silicone Films sample Pristine silicone Silicone-g-P(PEGMA)1b Silicone-g-P(PEGMA)2b Silicone-g-P(PEGMA)3b Silicone-g-P(PEGDMA)1c Silicone-g-P(PEGDMA)2c Silicone-g-P(PEGDMA)3c Silicone-g-P(PEGDMA)2-P(DMAPS) 1d Silicone-g-P(PEGDMA)2-P(DMAPS) 2d Silicone-g-P(PEGDMA)2-P(DMAPS) 3d Silicone-g-P(PEGDMA)2-P(DMAPS) 4d Silicone-g-P(DMAPS)e

water contact angle (±3°)

UV-induced poymerization time (min)

thiol−ene click reaction time (min)

10 30 60 10 30 60 30

10

1:0.52:-:1:0.41:-:1:0.29:-:1:0.27:-:1:0.24:-:1:0.13:-:1:0.10:-:1:0.13:0.001:0.001

107 52 45 45 47 38 37 38

30

30

1:0.13:0.0019:0.002

38

30

60

1:0.13:0.0024:0.0025

38

30

120

1:0.13:0.0035:0.0038

38

30

-

1:0.32:0.02:0.02

35

[C]:[Si]:[N]:[S]a

a

[C]:[Si]:[N]:[S] molar ratio calculated from the sensitivity factor-corrected XPS C 1s, Si 2p, N 1s, and S 2p core-level spectral area ratio. P(PEGMA)-grafted silicone synthesized by UV-induced graft polymerization of PEGMA macromonomer. cP(PEGDMA)-grafted silicone synthesized by UV-induced graft polymerization of PEGDMA macromonomer. dP(DMAPS)-coupled to P(PEGDMA)-grafted silicone synthesized by thiol−ene click reaction between the HS-P(DMAPS) and silicone-g-P(PEGDMA)2. eP(DMAPS)-grafted silicone synthesized by UV-induced graft polymerization of DMAPS monomer. b

rate of 1.0 mL/min. PEG polymer standards were used to generate the calibration curve. The silicone film surfaces and cross sections were observed by SEM (JEOL, model 5600 LV) and field emission scanning electron microscopy (FESEM, JEOL, model JSM-6700), respectively. To obtain a cross section of the film, a shallow cut was first made on one surface and the film was then immersed in double-distilled water for 1 h. The wetted film was immersed in liquid nitrogen for 5 min and then snapped with tweezers. The film samples were fixed on the SEM metal stubs using carbon tapes and sputter-coated with a thin platinum layer to enhance the contrast and quality of the images prior to SEM and FESEM observation. Static contact angles of the different surfaces were measured at room temperature by the sessile drop method using a 3 μL water droplet in a Rame-Hart telescopic goniometer (model

100-00-(230)). For each sample, three measurements from different regions of a surface were taken, and at least three independent tests were carried out with triplicate samples each time. Tensile tests of the silicone samples were performed using an Instron universal materials testing system (model 5544) with a 10 N load cell in a constant relative humidity (50%) room at 25 °C. Rectangular-shaped samples were cut from the silicone films (5 mm wide with a gauge length of 10 mm). The thickness of the samples was measured with a digital micrometer having a precision of 1 μm. A cross-head speed of 10 mm/ min was used. 2.14. Statistical Analysis. The results were reported as mean ± standard deviation (SD) and were assessed statistically using one-way 16411

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Figure 2. XPS widescan, C 1s and N 1s core-level spectra of the (a−c) Silicone-g-P(PEGMA)2, (d−f) Silicone-g-P(PEGDMA)2, (g−i) Silicone-gP(DMAPS), and (j−l) Silicone-g-P(PEGMA)2-P(DMAPS)3 substrates. The insets show the S 2p core-level spectrum of respective substrate. analysis of variance (ANOVA) with Tukey post hoc test. Statistical significance was accepted at P < 0.05.

monomer. The surface chemical compositions of the pristine and polymer-grafted silicone surfaces as determined by XPS, and the water contact angle are summarized in Table 1. Figure 2a−c shows the widescan, C 1s core-level, and N 1s core-level spectra, respectively, of the Silicone-g-P(PEGMA)2 film. In comparison to the widescan spectrum of the pristine silicone (Figure S1(a), Supporting Information), the intensity of the Si 2p and Si 2s signals in Figure 2a is significantly lower. The [Si]/ [C] ratio of the Silicone-g-P(PEGMA)2 film, as determined from the sensitivity factor-corrected XPS Si 2p and C 1s corelevel spectral area ratio, is about 0.29:1, which is much lower

3. RESULTS AND DISCUSSION 3.1. Surface Characterization. The procedures for the covalent grafting of P(PEGMA), P(PEGDMA), and P(DMAPS) chains on the silicone surfaces are illustrated in Figure 1. The silicone films were first treated with ozone to introduce surface peroxides and hydroxyl peroxides, which served as the initiator30,31 for the subsequent UV-induced polymerization of the PEGMA, PEGDMA, and DMAPS 16412

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Figure 3. FESEM images of the cross section of (a) pristine silicone, (b) Silicone-g-P(PEGMA)2, (c) Silicone-g-P(PEGDMA)2, and (d) Silicone-gP(PEGDMA)2-P(DMAPS)3 films. Scale bar is 10 μm.

than that of the pristine silicone (0.52:1). The Silicone-gP(PEGMA)2 film is hydrophilic with a water contact angle of 45°. In comparison to the hydrophobic pristine silicone (water contact angle of 107°), the significant change in the hydrophilicity after modification indicates the successful grafting of P(PEGMA) polymer on silicone surface. The extent of PEGMA graft copolymerization can be qualitatively assessed from the [Si]/[C] ratio, since Si is present only in silicone and not the grafted polymer. As shown in Table 1, the [Si]/[C] ratios of Silicone-g-P(PEGMA)1, Silicone-g-P(PEGMA)2, and Silicone-g-P(PEGMA)3 films decrease with the increasing UV irradiation time from 10 to 60 min, indicating an increase in the surface density of the grafted P(PEGMA) polymer. However, the density of the grafted P(PEGMA) polymer shows only a small increase with increase in UV irradiation time beyond 30 min. Figure 2d−f shows the wide-scan, C 1s core-level, and N 1s core-level spectra, respectively, of the Silicone-g-P(PEGDMA)2 film. Similar to the P(PEGMA)-grafted silicone, there is a significant reduction in the intensity of the Si 2p and Si 2s signals (Figure 2d) upon PEGDMA graft copolymerization. The [Si]/[C] ratio of the Silicone-g-P(PEGDMA)2 is about 0.13:1, which is lower than that of the Silicone-g-P(PEGMA)2 film (0.29:1) prepared using the same UV irradiation period (30 min). The PEGMA and PEGDMA macromonomers have a similar number of PEG repeat units (n = 10 and 9, respectively). Thus, it can be concluded that there is a higher graft density of PEG chains on Silicone-g-P(PEGDMA)2 than on Silicone-g-P(PEGMA)2. This conclusion is supported by the FESEM images of the cross section of pristine silicone, Silicone-g-P(PEGMA)2, and Silicone-g-P(PEGDMA)2 films in Figure 3a−c, respectively. It can be clearly observed in Figure 3c that the thickness of grafted P(PEGDMA) polymer layer (upper layer) on the Silicone-g-P(PEGDMA)2 film is ∼17 μm, while the grafted P(PEGMA) layer on the Silicone-gP(PEGMA)2 film (Figure 3b) is too thin to be clearly differentiated from the pristine silicone (Figure 3a). The higher

graft density of PEG chains on the Silicone-g-P(PEGDMA)2 also resulted in a more hydrophilic surface (water contact angle of 38°) than the Silicone-g-P(PEGMA)2 film (water contact angle of 45°). The C 1s core-level spectra of the Silicone-gP(PEGMA)2 and Silicone-g-P(PEGDMA)2 in Figure 2b and e, respectively, can be curve-fitted into three peak components with binding energies (BEs) at about 284.6, 286.4, and 288.7 eV, attributable to the C−C (C−Si), C−O, and O−CO species, respectively. The appearance of C−O and O−CO species is associated with the grafted P(PEGMA) and P(PEGDMA) polymer, since these peaks are absent in the C 1s core-level spectrum of the pristine silicone (Figure S1(b)). As expected, there is no discernible nitrogen or sulfur signal in the N 1s and S 2p core-level spectra of the pristine silicone, Silicone-g-P(PEGMA)2, and Silicone-g-P(PEGDMA)2 (SI Figure S1(c), Figure 2c,f, and their insets, respectively) in accordance with their chemical structure. The wide-scan, C 1s core-level, and N 1s core-level spectra of the Silicone-g-P(DMAPS) film are given in Figure 2g−i, respectively. After 30 min of UV-induced graft polymerization of DMAPS, the [Si]/[C] ratio of the Silicone-g-P(DMAPS) film was decreased to 0.32:1, indicating the successful grafting of P(DMAPS) chains. The surface hydrophilicity has significantly improved with a water contact angle of 35°. The C 1s core-level spectrum of the Silicone-g-P(DMAPS) in Figure 2h can be curve-fitted into four peak components with binding energies (BEs) at about 284.6, 285.3, 286.4, and 288.7 eV, attributable to the C−C (C−Si), C−S, C−O (C−N+), and O− CO species, respectively. The C−S, C−O (C−N+), and O− CO species are associated with the grafted P(DMAPS) polymer chains. The appearance of the nitrogen and sulfur signals in the wide-scan, N 1s, and S 2p core-level spectra of the Silicone-g-P(DMAPS) (Figure 2g,i, and the inset of Figure 2i, respectively) also confirm the successful grafting of the P(DMAPS) polymer on silicone surface. The HS-P(DMAPS) polymer was prepared via free radical copolymerization of DMAPS and BAC monomers, followed by 16413

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Figure 4. SEM images of (a) pristine silicone, (b) Silicone-g-P(PEGMA)2, (c) Silicone-g-P(PEGDMA)2, (d) Silicone-g-P(PEGDMA)2P(DMAPS)3, and (e) Silicone-g-P(DMAPS) film surfaces after exposure to a PBS suspension of S. aureus (108 cells/mL) for 4 h. Scale bar is 10 μm. (f) Quantitative count of adherent S. aureus cells per cm2 of the substrate surfaces as determined by the spread plate method after the stated bacterial assay. *Significant differences (P < 0.05) compared with pristine silicone.

Silicone-g-P(PEGDMA)2-P(DMAPS)3 film, respectively. The C 1s core-level spectrum of the Silicone-g-P(PEGDMA)2P(DMAPS)3 in Figure 2k can be curve-fitted into four peak components similar to those in Figure 2h. The nitrogen and sulfur signals in the N 1s and S 2p core-level spectra of the Silicone-g-P(PEGDMA)2-P(DMAPS)3 (Figure 2l and the inset of Figure 2l, respectively) is consistent with the presence of P(DMAPS) chains after the thiol−ene click reaction. The density of the grafted P(DMAPS) polymer chains on the Silicone-g-P(PEGDMA)2 substrate increases with the increasing thiol−ene click reaction time, as indicated by the increase in the [N]/[C] and [S]/[C] ratios of these substrates in Table 1. However, the P(DMAPS) layer is very thin as can be seen from a comparison of Figure 3d with c. This conclusion is supported by the XPS results which show that there is no significant change in the [Si]/[C] ratios of the Silicone-g-P(PEGDMA)2P(DMAPS) substrates as compared to that of Silicone-gP(PEGDMA)2 (Table 1).

reduction of the disulfide bond in the BAC moieties. The resultant HS-P(DMAPS) polymer has a number-average molecular weight (Mn) and weight-average molecular weight (Mw) of about 4470 and 5180, respectively, resulting in a polydispersity index (PDI) of 1.16. The FT-IR spectra of P(DMAPS-co-BAC) copolymer (Figure S2(a)) and HSP(DMAPS) polymer (Figure S2(b)) show the presence of absorption peaks at 1725 cm−1 and 1042 cm−1 associated with the stretching of O−CO and −SO3 group, respectively, of the DMAPS units. The appearance of the −SH stretching band at 2560 cm−1 in Figure S2(b) confirms the successful reduction of disulfide groups, since this peak is absent in Figure S2(a). Monomers with divinyl groups are often used for the preparation of polymer surfaces with residual double bonds.32 Thus, the as-synthesized Silicone-g-P(PEGDMA) films can provide “clickable” sites for the grafting of HS-P(DMAPS) chains via thiol−ene click reaction. Figure 2j−l show the widescan, C 1s core-level, and N 1s core-level spectra of the 16414

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Figure 5. Fluorescence microscopy images of (a, a′) pristine silicone, (b, b′) Silicone-g-P(PEGMA)2, (c, c′) Silicone-g-P(PEGDMA)2, and (d, d′) Silicone-g-P(DMAPS) substrates after exposure to a PBS suspension of S. aureus (108 cells/mL) for 4 h. (a−d) viewed under green filter and (a′−d′) viewed under red filter. Scale bar is 100 μm.

The mechanical properties, Young’s modulus and elongation at break, of the pristine and modified silicone films were derived from the stress−strain curves (Figure S3). The Young’s modulus of Silicone-g-P(PEGDMA)2 and Silicone-g-P(PEGDMA)2-P(DMAPS)3 substrates is 1.02 and 0.97 MPa, respectively, which is similar to that of the pristine substrate (1.04 MPa). The elongation at break values of Silicone-gP(PEGDMA)2 and Silicone-g-P(PEGDMA)2-P(DMAPS)3 substrates are slightly lower than that of the pristine substrate, but still remained more than 1000%. These results indicate that the grafting of P(PEGDMA) and P(DMAPS) chains on the

silicone surface did not result in significant adverse effects on the bulk mechanical properties. 3.2. Bacterial Adhesion and Biofilm Formation Assay. Bacterial adhesion on medical implant devices is a critical problem because it can cause biofilm formation and postoperative infection on the medical implants, which can eventually lead to implant failure or even death.33,34 Studies have shown that the rate of death due to infection in PD patients was about 30 per 1000 dialysis years and S. aureus is a well-documented risk associated with such infections.35,36 Thus, S. aureus was selected as the model bacterium for the detailed 16415

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cell lysis can promote the survival and persistence of remaining bacteria because live cells may derive nutrients from their neighboring dead or lysed ones.42,43 Furthermore, the dead cells can also provide a surface for further bacterial adhesion which protects the bacteria from the P(DMAPS) chains. This may explain the higher number of adherent bacteria on Silicone-g-P(DMAPS) and Silicone-g-P(PEGDMA)2-P(DMAPS) with high P(DMAPS) grafting density as compared to the Silicone-g-P(PEGDMA)2 surface. Stability of the coating layer on PD catheters is of great importance, as these catheters are meant for long-term placement in the abdomen, and the replacement of a PD catheter usually requires surgery. In this work, the stability of the polymer-modified silicone substrates was assessed by conducting the bacterial adhesion assay on the films after they were aged in PBS at 37 °C for 30 days. It can be seen from Figure 4f that the number of adherent bacteria on the aged P(PEGMA) and P(DMAPS) modified substrates increased slightly, but not significantly (P > 0.05), and there was almost no change in the bacterial count on the aged P(PEGDMA) modified substrates (Silicone-g-P(PEGDMA)2 and Silicone-gP(PEGDMA)2-P(DMAPS)3). These results indicate that the P(PEGMA), P(DMAPS), and P(PEGDMA) coatings are able to maintain their antibacterial efficacy after aging in PBS. Furthermore, antibacterial assay using the Silicone-g-P(PEGDMA)2 and Silicone-g-P(PEGDMA)2-P(DMAPS)3 after autoclaving at 121 °C for 20 min also shows that these substrates maintain their antibacterial property after autoclaving (Figure S5). Similar to the results obtained with S. aureus, E. coli and S. epidermidis readily adhere on pristine silicone (Figure S6(a) and S7(a), respectively). After polymer modification, significant reduction in adherent bacteria was achieved (Figures S6(b-e) and S7(b-d)), with the P(PEGDMA) grafted substrates (Silicone-g-P(PEGDMA)2 and Silicone-g-P(PEGDMA)2-P(DMAPS)3) conferring the highest antibacterial efficacy (≥90% reduction in bacteria adherent compared to pristine silicone) even after aging in PBS for 30 days (Figure S6(f) and S7(e), respectively). Bacterial adhesion on surfaces is usually followed by cell multiplication and formation of an extracellular matrix (biofilm) when bacterial cell-growth promoting conditions are present. The biofilm then protects the bacterial cells from the action of antibiotics as well as the host’s natural defense systems.44,45 In this work, the resistance of the polymer-modified silicone substrates to S. aureus biofilm formation was assessed and the results are shown in Figure 6. On pristine silicone, a large number of S. aureus clusters was observed after 24 h in culture medium (Figure 6a). From Figure 6b, it can be seen that there is a very significant reduction in bacterial clusters on Silicone-gP(PEGDMA)2 film. With the immobilization of P(DMAPS) chains on Silicone-g-P(PEGDMA)2 film, the bacterial cell number decreased further on Silicone-g-P(PEGDMA)2-P(DMAPS)3, and most of the cells present as single cells rather than in clusters (Figure 6c). When the incubation period was extended to 72 h, the Silicone-g-P(PEGDMA)2 and Silicone-g -P(PEGDMA)2-P(DMAPS)3 substrates still retained the ability to resist biofilm formation (Figure S8). It has been postulated that nonspecific protein adsorption on surfaces is a crucial step for biofilm formation.46,47 Since the Silicone-gP(PEGDMA)2 and Silicone-g-P(PEGDMA)2-P(DMAPS)3 films are equally effective in inhibiting bacterial adhesion from PBS (Figure 4f), the differences in the antibiofilm

investigation of the antibacterial effects of the polymer-modified silicone substrates in vitro under static conditions. In addition to the Gram-positive S. aureus, the antibacterial effect of the modified silicone was further evaluated with Gram-negative E. coli and Gram-positive S. epidermidis, which are also common species responsible for PD-related infections.37,38 The efficacy of the modified silicone in reducing bacterial adhesion can be seen from the SEM images in Figure 4. Figure 4a shows that S. aureus adhere readily on the pristine film. The hydrophobic silicone surface favors attachment of microbes due to hydrophobic interactions.39 After surface modification with hydrophilic P(PEGMA), P(PEGDMA), and P(DMAPS) polymers, there is a significant reduction in the number of adherent bacteria (Figure 4b−e). The P(PEGDMA)-grafted silicone (Silicone-g-P(PEGDMA)2 and Silicone-g-P(PEGDMA)2-P(DMAPS)3) reduced S. aureus adhesion by ∼93% as compared to that of pristine substrate (Figure 4f), while the Silicone-g-P(PEGMA)2 and Silicone-g-P(DMAPS) substrates are less effective (reduction in adherent bacteria by ∼84% and ∼80%, respectively). The antiadhesive property of the PEG-modified substrates is strongly dependent on the thickness of the hydrophilic layer, which is dependent on the UV-induced polymerization time (Figure S4(a)). For the P(PEGMA)- and P(PEGDMA)-grafted silicone surfaces, the efficacy in inhibiting bacterial adhesion increases with the polymerization time until 30 min, beyond which there is no substantial improvement. This observation is consistent with the results in Table 1 which indicate that the surface graft density of the P(PEGMA) and P(PEGDMA) polymer chains increases rapidly only in the initial 30 min of polymerization. Similarly, the higher efficacy of the P(PEGDMA)-modified surfaces as compared to the P(PEGMA)-modified surfaces prepared using the same polymerization time is attributed to the thicker graft layer on the former (comparing Figure 3c with b). It can be seen from Figure 4 that P(DMAPS) is not as effective in preventing bacterial adhesion as P(PEGDMA). Thus, if P(DMAPS) is conjugated to the Silicone-gP(PEGDMA)2 film in large amounts, an increase in the number of adherent bacteria on the film will result. If the thiol− ene reaction time is restricted to 60 min or less, the bacterial count on Silicone-g-P(PEGDMA)2-P(DMAPS) substrates will not be significantly more than that on the Silicone-gP(PEGDMA)2 substrate (this can be seen by comparing the bacterial count on the substrates obtained using thiol−ene click reaction times of 60 and 0 min in Figure S4(b)). Unlike PEGcontaining polymers which are antiadhesive, P(DMAPS) is bactericidal. Its bactericidal property is attributed to its ammonium groups which can interact with the negatively charged bacterial cell membrane, resulting in bacterial cytoplasmic membrane disruption and cell lysis.40,41 The differences in the antimicrobial action of these two types of polymers can be seen in Figure 5. Large numbers of viable S. aureus (stained green with SYTO 9) and very few dead or membrane-compromised bacteria (stained red with propidium iodide (PI)) can be observed on pristine silicone (Figure 5a,a′, respectively). On the P(PEGMA) and P(PEGDMA) modified surfaces, the number of viable bacteria was greatly reduced (Figure 5b,c, respectively), and few red-stained cells were observed (Figure 5b′,c′, respectively). On the other hand, there is a significant increase in dead or membrane-compromised on Silicone-g-P(DMAPS) surface (Figure 5d′), confirming the bactericidal nature of the grafted P(DMAPS) chains. Bacterial 16416

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Figure 7. BSA and FBG adsorption on pristine silicone, Silicone-gP(PEGMA)2, Silicone-g-P(PEGDMA)2, Silicone-g-P(PEGDMA)2-P(DMAPS)3, and Silicone-g-P(DMAPS) films after the films were treated with 1.0 mg/mL of pure protein solutions for 4 h. *Significant differences (P < 0.05) compared with pristine silicone.

report.50 This limitation of the PEG-modified surfaces may be due to the possibility of interactions between the protein and the PEG chains.27,51 The P(DMAPS) chains are more effective in reducing protein adsorption, with ∼90% and ∼92% reduction in the amount of adsorbed BSA and FBG on the Silicone-g-P(DMAPS) film, respectively, as compared to the pristine silicone. The protein-repelling nature of the P(DMAPS) chains is also evident from the lower amount of BSA and FBG adsorbed on Silicone-g-P(PEGDMA)2-P(DMAPS)3 surface (∼30% and 50% lower, respectively) compared to Silicone-g-P(PEGDMA)2. The protein-repellent behavior of polysulfobetaines can be attributed to the nearsurface hydration layer, induced by the strong water-binding zwitterions.8,52 After aging in PBS for 30 days, the amount of BSA and FBG adsorbed on the modified silicone films was not significantly different from that on the corresponding nonaged ones (P > 0.05), indicating the long-term stability of the polymer coatings. 3T3 mouse fibroblasts, a mammalian cell line, were used as the model cells in this work to study cellular interactions with the silicone films in vitro. Fibroblast cells are widely used in biomedical and biochemical assays, as they play a critical role in wound healing and fibrous encapsulation due to their ability to synthesize extracellular matrix proteins.21,53 Figure 8a−e shows the optical microscopy images of the adherent 3T3 fibroblasts on the pristine and modified silicone surfaces. The number of adherent 3T3 fibroblast cells on the pristine silicone surface is very high (Figure 8a), while few cells can be observed on the modified substrates (Figure 8b−e). Quantification using the MTT assay revealed that all the polymer-modified silicone films inhibited 3T3 fibroblast adhesion by ≥90% even after aging in PBS for 30 days (Figure 8f). The decrease in the fibroblast adhesion may be related to the antiprotein adsorption property, as it has been pointed out by previous studies that reduction of nonspecific protein adsorption is crucial for the inhibition of cellular interaction with biomaterial surfaces.53,54 In view of the potential in vivo application of the modified silicone substrates, it is important to evaluate the potential cytotoxicity of the films. This was carried out by determining the viability of 3T3 fibroblasts after the films were placed in contact with the cells for 24 h, and the results are shown in Figure S9. Cell viabilities were above 95% in all cases and not significantly different from that in the control experiment (P > 0.05). This is not surprising, since silicone, P(PEGMA),

Figure 6. SEM images of S. aureus on (a) pristine, (b) Silicone-gP(PEGDMA)2, and (c) Silicone-g-P(PEGDMA)2-P(DMAPS)3 films after incubation in growth medium containing 105 bacteria cells/mL for 24 h. Scale bar is 100 μm.

formation efficacy of these films as indicated in Figure 6 may be associated with their ability to inhibit protein adsorption, which is discussed in the next section. 3.3. Protein Adsorption and Cell Adhesion. Protein adsorption plays a significant role in bacterial adhesion,8 and our previous work has shown that FBG can enhance S. aureus adhesion while BSA has an opposite effect.27 The ability to inhibit protein adsorption is also a critical property for antifouling surfaces, because when devices contact with blood and living tissue, protein adsorption on the device surface is the first step to induce subsequent platelet and cell adhesion.48,49 The adsorption of two kinds of proteins, BSA and FBG, on the modified silicone substrates was investigated. Figure 7 shows that the pristine silicone surface adsorbs ∼1.9 μg/cm2 of BSA and ∼2.9 μg/cm2 of FBG after 4 h in contact with the protein solution. After grafting with the hydrophilic polymers, the amount of BSA and FBG adsorbed decreased significantly. The Silicone-g-P(PEGMA)2 film reduced BSA and FBG adsorption by ∼75% and ∼70%, respectively. Since proteins favorably adsorb on hydrophobic surfaces, 8 the reduced protein adsorption on the P(PEGMA)-grafted silicone can be attributable to its improved surface hydrophilicity as indicated by its reduced water contact angle. However, it should be noted that, although Silicone-g-P(PEGDMA)2 substrate has a lower water contact angle than Silicone-g-P(PEGMA)2, the reduction in BSA and FBG adsorption is similar (reduction by ∼74% and ∼72%, respectively). These results indicate that increasing the density of PEG chains on silicone may not lead to higher reduction in protein adsorption, consistent with an earlier 16417

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Figure 8. Optical microscopy images of (a) pristine silicone, (b) Silicone-g-P(PEGMA)2, (c) Silicone-g-P(PEGDMA)2, (d) Silicone-gP(PEGDMA)2-P(DMAPS)3, and (e) Silicone-g-P(DMAPS) film surfaces after incubation with 3T3 fibroblasts (2 × 105 cells/mL) for 24 h. Scale bar is 200 μm. (f) Quantitative analysis of adherent 3T3 fibroblast cells per cm2 of substrate surface using MTT assay. *Significant differences (P < 0.05) compared with pristine silicone.

of spreading and pseudopodia. However, after grafting of P(PEGMA), P(PEGDMA) and P(DMAPS) on the silicone surface, the number of adhered platelets is greatly reduced, and these platelets remained round and inactivated (Figure 9b−e). A quantitative comparison of the number of adherent platelets on various substrates is given in Figure 9f. Platelet adhesion on the P(PEGMA) and P(PEGDMA) modified silicone surface was reduced by ∼80% as compared to the pristine surface, while the corresponding reduction for Silicone-g-P(DMAPS) was ∼96%. Consequently, by introducing P(DMAPS) chains on the Silicone-g-P(PEGDMA)2 substrate, the Silicone-gP(PEGDMA)2-P(DMAPS)3 film also shows an improved resistance to platelet adhesion as compared to Silicone-gP(PEGDMA)2. Platelet adhesion on biomaterial surfaces is closely associated with protein adsorption, in particular, the platelet adhesion promoting protein, FBG.60,61 Thus, the resistance to platelet adhesion on the P(PEGMA), P-

P(PEGDMA), and P(DMAPS) polymers are all known to be nontoxic.23,55,56 These results also confirm that the reduction in the number of adherent 3T3 fibroblasts on the polymermodified silicone (Figure 8) arises from the antiadhesive property of the polymer coatings, and not because they are cytotoxic. 3.4. Hemocompatibility Assay. The blood compatibility of the polymer-modified silicone substrates was evaluated from platelet adhesion, plasma recalcification time (PRT), and hemolysis tests. Platelet adhesion and activation have been considered as a major cause of thrombosis,57,58 which leads to intraluminal obstruction of PD catheters and eventual PD catheter malfunction.59 Figure 9a−e shows the SEM images of the adherent platelets on pristine and modified silicone surfaces. It can be seen from Figure 9a that the platelets adhering on the pristine silicone surface have an irregular shape, indicating that they are highly activated with the characteristics 16418

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Figure 9. SEM images of (a) pristine silicone, (b) Silicone-g-P(PEGMA)2, (c) Silicone-g-P(PEGDMA)2, (d) Silicone-g-P(PEGDMA)2P(DMAPS)3, and (e) Silicone-g-P(DMAPS) film surfaces after incubation with platelet-rich plasma for 1 h. Scale bar of (a−e) and the inset of (a) is 10 and 5 μm, respectively. (f) Platelet adhesion on the modified silicone surfaces relative to that on the pristine surface. *Significant differences (P < 0.05) compared with pristine silicone.

modified silicones are shown in Figure 10b. Bare silicone is known to have a low hemolytic effect,12,66 and the degree of hemolysis of the pristine silicone substrate was found to be ∼1%. The modified substrates resulted in even lower degree of hemolysis, ranging from ∼0.7% for the Silicone-g-P(PEGMA)2 to ∼0.4% for the Silicone-g-P(DMAPS). The decrease in the degree of hemolysis of the modified silicone can be attributed to the antiadhesive property of the polymer coatings, which reduces the cellular interactions between RBCs and the modified films. A hemolysis degree of 5% or lower is considered acceptable for biomaterials.67 Although pristine silicone meets this requirement, the polymer-modified substrates in this work have further improved the silicone’s blood compatibility.

(PEGDMA), and P(DMAPS) polymer grafted silicone surfaces is related to the reduction in protein adsorption on the modified substrates as discussed above. Figure 10a shows the results of PRT obtained with the pristine and modified silicone substrates. Grafting of P(PEGMA) and P(PEGDMA) on silicone resulted in only a small increase in PRT from ∼13 min to ∼15 min. Earlier reports have indicated that grafting of PEG chains on surfaces has no or only a slight effect on the inhibition of the bloodclotting factor.62,63 However, the presence of the P(DMAPS) layer increases the PRT significantly by resisting plasma protein adsorption, which results in low fibrin formation.64 Hemolysis is another issue related to the blood compatibility of biomaterials. Hemolysis occurs when red blood cells (RBCs) are exposed to a foreign material surface which causes lysis of the RBCs, leading to the release of hemoglobin and other internal components.65 The released hemoglobin will enhance platelet adhesion and accelerate thrombus formation and clotting. The hemolysis results obtained for the pristine and

4. CONCLUSIONS Medical-grade silicone films were modified with PEGcontaining polymers (P(PEGMA) and P(PEGDMA)) and polysulfobetaine (P(DMAPS) to improve their antibacterial, 16419

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ACKNOWLEDGMENTS

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REFERENCES

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This work was financially supported by the National Kidney Foundation of Singapore Grant NKFRC/2010/01/07.

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Figure 10. (a) PRT and (b) degree of hemolysis on pristine silicone, Silicone-g-P(PEGMA)2, Silicone-g-P(PEGDMA)2, Silicone-g-P(PEGDMA)2-P(DMAPS)3, and Silicone-g-P(DMAPS) surfaces. *Significant differences (P < 0.05) compared with pristine silicone.

antifouling, and hemocompatible properties. The cross-linked P(PEGDMA) graft layer of ∼17 μm thickness can effectively overcome the characteristic hydrophobic recovery of silicone with long-term stability, and the adhesion of fibroblasts and both Gram-negative and Gram-positive bacteria was reduced by >90%. In addition, the P(PEGDMA) layer can serve as an anchor for the attachment of P(DMAPS) chains which effectively reduced platelet and protein adhesion and enhanced the blood compatibility of the substrate. With proper control of the amount of P(DMAPS) immobilized on the surface, the nonspecific antibacterial efficacy can be retained. The favorable antibacterial, antifouling, and hemocompatible properties, as well as long-term stability and noncytotoxicity of the modified silicone, offer promising opportunities for the development of PD catheters that can inhibit infection and omental wrapping. However, since the in vitro assays cannot fully simulate in vivo conditions, animal model studies would be required to verify the efficacy of the modified silicone.

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ASSOCIATED CONTENT

S Supporting Information *

Additional results on materials characterization, bacterial and cytotoxicity assays. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +65-6516-2176; Fax: +65-67791936; E-mail: chenkg@ nus.edu.sg. Notes

The authors declare no competing financial interest. 16420

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