- Email: [email protected]
Integration of antifouling and bactericidal moieties for optimizing the efficacy of antibacterial coatings
Journal of Colloid and Interface Science 438 (2015) 138–148
Contents lists available at ScienceDirect
Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Integration of antifouling and bactericidal moieties for optimizing the efficacy of antibacterial coatings Rong Wang, Koon Gee Neoh ⇑, En-Tang Kang Department of Chemical and Biomolecular Engineering, National University of Singapore, Kent Ridge, Singapore 117576, Singapore
a r t i c l e
i n f o
Article history: Received 23 August 2014 Accepted 25 September 2014 Available online 5 October 2014 Keywords: Bifunctional coating Bacterial colonization Antifouling Bactericidal Hemocompatibility
a b s t r a c t Hypothesis: Surface coatings that are either antifouling or bactericidal can reduce bacterial colonization, but either type has certain drawbacks. We hypothesize that by integrating an antifouling polymer, poly(sulfobetaine methacrylate) (pSBMA), and a bactericidal polymer, N-[(2-hydroxy-3-trimethylammonium)propyl] chitosan chloride (HTCC), in one coating, these drawbacks can be overcome and the antibacterial efficacy can be greatly improved compared to an antifouling or bactericidal coating. Experiments: A copolymer comprising chitosan and pSBMA in almost equal molar ratio was grafted onto an aminolyzed silicone surface via genipin-induced crosslinking reaction, and treated with glycidyltrimethylammonium chloride to endow the surface with quaternary ammonium groups. The antibacterial property and cytotoxicity of the resultant coating (HTCC-b-pSBMA) were tested. The possibility of incorporating heparin in the coating to improve blood compatibility was also investigated. Findings: The HTCC-b-pSBMA coating reduced colonization by both planktonic bacteria from aqueous medium and aerosolized bacteria by approximately two orders of magnitude compared with the pristine surface. In comparison, surfaces coated with either the pSBMA or HTCC component are unable to achieve such efficacy. The heparin-loaded HTCC-b-pSBMA coating improved hemocompatibility without adversely affecting the antibacterial efficacy. No significant cytotoxicity of the coatings was observed with mammalian cells. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Medical devices are essential components in modern healthcare, but their surfaces are prone to bacterial colonization. For example, devices before implantation may be contaminated by bacteria from droplets, air or hands of healthcare workers and patients. The deposited bacteria could survive on the surface and multiply to form colonies after implantation. Furthermore, the surface of indwelling devices will contact body fluid (e.g. blood, urine), where pathogens may be present, and the planktonic bacteria will approach and attach to the surface via various bacteria–surface interactions (e.g. van der Waals forces, hydrogen bonding, ionic interactions, hydrophobic interactions) [1]. The adherent bacteria will subsequently multiply and colonize the surface, and eventually this may lead to infection of the device [2]. Therefore, inhibiting bacterial colonization is of critical importance in reducing the probability of nosocomial infections. ⇑ Corresponding author at: Blk E5, 4 Engineering Drive 4, #02-34, Singapore 117576, Singapore. Fax: +65 67791936. E-mail addresses: [email protected] (R. Wang), [email protected] (K.G. Neoh), [email protected] (E.-T. Kang). http://dx.doi.org/10.1016/j.jcis.2014.09.070 0021-9797/Ó 2014 Elsevier Inc. All rights reserved.
Two common strategies have been used to inhibit bacterial colonization on surfaces: (1) by coating the surface with an antifouling layer to repel bacteria, or (2) by coating with a non-leaching bactericidal layer to kill the bacterial cells on contact [3]. Antifouling coatings rely mainly on the formation of a hydrated barrier which prevents bacterial attachment [4–7]. Such antifouling coating delays biofilm formation but will not kill bacterial cells that are already deposited on the surface [8]. On the other hand, a surface with covalently immobilized bactericidal agents, such as quaternary ammonium compounds and antimicrobial peptides, kills bacteria on contact and exhibits a self-sterilizing effect [9–12]. However, the accumulation of dead bacteria on the bactericidal coating over the course of exposure would result in blanketing of the active groups [13], and the coating efficacy would eventually be compromised. Since both antifouling and bactericidal coatings have certain advantages and drawbacks, we hypothesize that by integrating both types of moieties in one coating, the advantages of the two complementary mechanisms can be combined to optimize the efficacy of the surface in preventing bacterial colonization. Most such bifunctional coatings reported in the literature rely on the conjugation of antifouling polymers with antibiotics [14], antimicrobial
R. Wang et al. / Journal of Colloid and Interface Science 438 (2015) 138–148
enzymes [15], or antimicrobial peptides [16]. However, the use of antibiotics is controversial due to the potential for development of antibiotic-resistant bacteria, and antimicrobial enzymes and peptides are usually costly. It has been reported that poly(sulfobetaine methacrylate) (pSBMA) coating inhibits protein adsorption and bacterial adhesion in aqueous medium [4,17,18]. On the other hand, chitosan (CS) and its derivatives with quaternary ammonium groups have bactericidal property attributable to the positively charged groups that alter the permeability of the bacterial membrane [19,20]. Hence, to obtain a copolymer which has antifouling as well as bactericidal properties, sulfobetaine methacrylate (SBMA) was block-copolymerized onto CS in this study. The resultant CS-block-pSBMA (CS-b-pSBMA) copolymer was grafted and crosslinked onto an aminolyzed silicone surface using genipin. Since it has been reported that CS derivatives with quaternary ammonium groups have higher bactericidal activity than CS [20], glycidyltrimethylammonium chloride (GTMAC) was grafted onto the resultant coating to endow the surface with quaternary ammonium groups. The corresponding antifouling pSBMA coating and bactericidal CS treated with GTMAC (N-[(2-hydroxy-3-trimethylammonium)propyl] chitosan chloride (HTCC)) coating were also prepared. The antifouling and antibacterial properties of the bifunctional coating were compared with the pSBMA coating and HTCC coating. Heparin was further loaded in the coating to evaluate the potential application of the bifunctional coating in blood-contacting devices. 2. Materials and methods 2.1. Materials Medical grade silicone sheets (1 mm thickness) were obtained from BioPlexus Inc., US. Chitosan (degree of deacetylation = 75–85%), (3-aminopropyl)triethoxysilane (APTES), ammonium persulfate, glycidyltrimethylammonium chloride (GTMAC), [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl) ammonium hydroxide (sulfobetaine methacrylate, SBMA), heparin sodium salt from porcine intestinal mucosa (Grade I-A, P180 USP units/mg), toluidine blue O, bovine plasma fibrinogen (FBG) and 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich, US. Genipin is a product from Challenge Bioproducts, Taiwan. Escherichia coli (E. coli ATCC DH5a), Proteus mirabilis (P. mirabilis ATCC 51286, a strain isolated from a patient with urinary catheter infection), Staphylococcus aureus (S. aureus ATCC 25923) and 3T3 fibroblasts were obtained from American Type Culture Collection (ATCC, US). Pseudomonas aeruginosa (P. aeruginosa PAO1) was purchased from National Collection of Industrial Food and Marine Bacteria (NCIMB, UK). 2.2. Synthesis and characterization of CS-b-pSBMA, pSBMA and HTCC polymers CS-b-pSBMA copolymer was synthesized in a similar fashion as the procedure outlined in the literature for the preparation of CS-poly(ethylene glycol) block copolymer (Fig. 1a) [21]. CS powder (0.5 g) was dissolved in 1% (v/v) aqueous acetic acid solution (30 mL) under stirring at 500 rpm. This solution was heated to 60 °C and maintained at this temperature throughout the reaction period. An oxygen-free environment was then established in the system by degassing with argon prior to the introduction of ammonium persulfate (100 mg). Argon was bubbled through the mixture for another 30 min. SBMA aqueous solution (at a [SBMA]/[CS] molar ratio of 1:5, 1:1 or 5:1 in 20 mL distilled water) was then gradually added to the mixture using a syringe over a 30-min period. The reaction was allowed to proceed under argon atmosphere
139
with stirring for 6 h. pSBMA homopolymer was synthesized in a similar manner using 0.87 g of SBMA without CS. HTCC polymer was prepared according to the method reported earlier via the reaction of CS with GTMAC (Fig. S1) [22]. Briefly, CS (3 g) was dispersed in distilled water (50 mL), and GTMAC (5.65 mL) was added dropwise to the suspension over 30 min. The reaction was allowed to proceed under continuous mechanical stirring at 500 rpm at 85 °C for 10 h. After reaction, small oligomers, salts, and unreacted reagents were removed by dialysis using a cellulose membrane (molecular weight cut-off of 12,000, Sigma–Aldrich) over three days, and the products were collected after lyophilization. The polymers were characterized using Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS), and the experimental details are given in the Supporting Information (SI). 2.3. Surface modification and characterization of HTCC-b-pSBMAcoated silicone Silicone sheets (cut into 1 10 cm2) were first cleaned by ultrasonication in isopropanol for 10 min and then in distilled water for 20 min to remove surface impurities. An oxygen-ozone gas mixture (generated from an Azcozon ozone generator (Model VMUS-4PSE, AZCO Inc., Canada)) was passed over the surface of the silicone sheet for 20 min to generate the necessary surface reactive groups. To obtain an ozone production rate of 6 g/h with an ozone concentration of 25 g/m3, the oxygen inlet flow rate was fixed at 4 L/min. After ozonization, the silicone sheet was immediately immersed in 15 mL freshly prepared solution containing APTES (5%, v/v), distilled water (5%, v/v) and ethanol (90%, v/v). The solution with the silicone sheet was placed in a shaker at 500 rpm for 1 h at room temperature. The silanized sheet was rinsed with ethanol and distilled water, dried under a flow of nitrogen, and heated in a 60 °C oven for 2 h to promote the formation of a crosslinked silane layer [23]. The silanized silicone sheet was then immersed in a CS-b-pSBMA aqueous solution (13.5 mL distilled water containing 150 mg copolymer), and a genipin ethanol solution (1.5 mL ethanol containing 150 mg genipin) was immediately added. The mixture was placed in a 37 °C water bath shaker at 100 rpm for 24 h. The silicone sheet was then removed from the reaction mixture and washed in ethanol overnight in a 37 °C shaker at 100 rpm, dried under a nitrogen flow, and denoted as CS-bpSBMA-coated sheet. The CS-b-pSBMA-coated sheet was immersed in 15 mL GTMAC aqueous solution (5%, v/v) at 70 °C for 24 h. The sheet was then washed with distilled water, dried under a nitrogen flow, and denoted as HTCC-b-pSBMA-coated sheet (Fig. 1b). The preparation of heparin-loaded coatings was carried out in a similar manner using a heparin-loaded CS-b-pSBMA polymer solution, and treatment with GTMAC to obtain a HTCC-b-pSBMA-Hep coating. To prepare a heparin-loaded CS-b-pSBMA polymer solution, a heparin aqueous solution (300 lL, 10% w/v) was added dropwise to a CS-b-pSBMA polymer solution (13.2 mL containing 150 mg CS-b-pSBMA) over 10 min under continuous mechanical stirring. To simulate in vivo application of the HTCC-b-pSBMA-Hep-coated substrate where it would be immersed in body fluid, a piece of the HTCC-b-pSBMA-Hep-coated substrate (1 1 cm2) was incubated in 50 mL phosphate buffered saline (PBS, 10 mM, pH 7.4) at 37 °C in a shaker at 100 rpm for 7 days. The amount of heparin in the coating before and after incubation was quantified using the toluidine blue method as described in an earlier publication [24]. HTCC-coated surface was prepared in a similar manner as the HTCC-b-pSBMA-coated surface using a mixed solution of CS and genipin, and pSBMA-coated surface was prepared by UV-induced graft-polymerization of SBMA on an ozonized silicone surface. The details of this procedure are given in the SI. The surfaces of
140
R. Wang et al. / Journal of Colloid and Interface Science 438 (2015) 138–148
Fig. 1. Schematic diagram illustrating (a) synthesis of CS-b-pSBMA copolymer, and (b) steps for modifying silicone sheet surface.
R. Wang et al. / Journal of Colloid and Interface Science 438 (2015) 138–148
the pristine and modified silicone sheets were characterized using XPS and contact angle measurement. The cross-sections of the silicone sheets were prepared and the coating thickness was determined using field emission scanning electron microscopy (FESEM). The details of these experimental procedures are given in the SI. 2.4. Biofilm formation on substrates immersed in bacterial suspension Bacteria were cultured in an appropriate broth (nutrient broth for E. coli, tryptic soy broth for P. mirabilis and S. aureus, lysogeny broth for P. aeruginosa) overnight. The bacterial cells were collected by centrifugation (2700 rpm, 10 min) and then washed twice using PBS (10 mM, pH 7.4). The cells were resuspended in PBS (10 mM, pH 7.4), and the cell density was determined by measuring the absorbance at 540 nm (A540nm of 1.0 unit corresponds to 1 109 cells/mL based on spread plate counting) [25]. The bacterial suspension was further diluted using the appropriate culture medium to a concentration of 105 cells/mL. One mL of the prepared bacterial medium suspension was added to each pristine or modified silicone sheet (cut into 1 1 cm2 size), which were pre-sterilized by UV irradiation and placed in a 24-well plate (Greiner Bio-one, Germany). The plate with the silicone sheets were incubated at 37 °C for 24 h or 72 h. For experiments carried out with an incubation period of 72 h, the medium was replaced with fresh culture medium every 24 h. After the incubation period, the bacterial suspension in each well was discarded and the substrate was washed using PBS for three times to remove any non-adherent or loosely adhered bacteria on the surface. Scanning electron microscopy (SEM, JEOL, Model 5600LV, Japan) was used to observe biofilms on the surfaces, and the viable bacterial cells on each surface were enumerated using the spread plate method as described in an earlier publication [25]. 2.5. Bacterial colonization on substrates exposed to bacterial aerosol Harvested bacterial cells were suspended in PBS (10 mM, pH 7.4) at a concentration of 108 cells/mL as described above. The prepared bacterial PBS suspension (100 lL) was sprayed onto silicone sheet surfaces (cut into 1 1 cm2 size) using a commercial sprayer. The diameter of the droplets from the sprayer was estimated to be in the range of 10–200 lm from observations using an optical microscope. The substrates were then dried in air for 5 min, and placed in a 24-well plate. One mL of melted growth agar (0.7% agar in yeast-dextrose broth, autoclaved and cooled to 37 °C) was poured over the substrates, and the plate was incubated at 37 °C for 24 h [26]. The silicone sheets after incubation were gently removed from the agar, placed in a new 24-well plate, and washed thrice with PBS. The silicone surfaces were observed using SEM and the number of viable adherent cells was quantified using the spread plate method. 2.6. Protein adsorption Protein adsorption assay was carried out by immersing the pristine and modified silicone substrates in a FBG protein solution (1 mg/mL in citrate–phosphate buffered saline (CPBS, 10 mM sodium citrate in 10 mM PBS, pH 7.4)) for 4 h. The amount of adsorbed protein on the substrate surface was quantified using the modified dye-interaction method with Bio-Rad protein dye reagent (Catalog No. 500-0006, Bio-Rad, US) in a similar manner as described in an earlier publication (details in SI) [27]. To investigate the effect of pre-adsorbed protein on bacterial adhesion and biofilm formation, pristine and modified substrates after treatment of FBG protein for 4 h at 37 °C were incubated in bacterial PBS suspension (108 P. mirabilis cells/mL, for bacterial adhesion assay) for 4 h, or bacterial culture medium (105 P. mirabilis
141
cells/mL, for biofilm assay) for 24 h, and the bacterial cells on the surface was observed using SEM as described above.
2.7. Platelet adhesion and plasma recalcification time (PRT) assays Platelet adhesion and PRT assays were carried out to assess the hemocompatibility of the surfaces in the same manner as described in a previous publication [27]. The platelet adhesion test was conducted by introducing diluted platelet-rich plasma (PRP) on the substrates followed by incubation at 37 °C for 1 h. The platelets adhering on the surfaces were observed using SEM. For the PRT assay, platelet poor plasma (PPP) was introduced on substrates followed by addition of CaCl2 solution. Clotting of the PPP solution was monitored by detecting the formation of fibrin threads in the solution, and the PRT (defined as the period from the introduction of CaCl2 to the first observation of silky fibrin) on each surface was recorded. The details of the assays are given in the SI.
2.8. Cytotoxicity assay To investigate the effect of the pristine and modified surfaces on cell viability, MTT assay which measures mitochondrial activity was carried out according to the standard protocol stated in ISO 10993-5 for evaluation of in vitro cytotoxicity of medical devices by direct contact [28]. Briefly, 3T3 fibroblast cells were cultured in a 75 cm2 tissue culture flask (Corning, US) using Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 1 mM L-glutamine, and 100 IU/mL penicillin. The medium was changed every 2 days over a period of 7–10 days until the cells reached 80% confluence. After the incubation period, the cells were detached from the flask using a trypsin–EDTA solution (5.3 mM, 12 mL), harvested by centrifugation (200g, 10 min), and suspended in fresh culture medium at a concentration of 104 cells/mL. One mL of the cell suspension was then added to each well of a 24-well plate. After incubation at 37 °C in a humidified atmosphere of 5% CO2 and 95% air for 24 h, the medium in the plate was completely refreshed, and pristine and modified silicone sheets (1 1 cm2, pre-sterilized by UV irradiation for 30 min) were gently placed on top of the cell layer in the well. Since the density of the silicone sheet is slightly higher than the medium, the silicone sheet would only contact the cell layer but not compress it. In the non-toxic control group, the cells were incubated with the complete growth culture medium without the silicone sheet. The plate was then incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air for 24 h or 72 h. After the incubation period, the silicone sheet was very gently taken out to avoid disrupting the cell layer. No cell attachment on the silicone surface was discernible under an optical microscope. The culture medium in each well was then replaced with equal volume of freshly prepared MTT solution (0.5 mg/mL in medium). After incubation at 37 °C for 4 h, the medium was discarded. The formazan crystals in each well were dissolved in 1 mL dimethyl sulfoxide for 15 min in the dark. The absorbance of the solutions at 570 nm was then measured using a microplate reader (Tecan GENios, Switzerland). All samples were tested in triplicate, and the results were reported as percentage of absorbance of the experimental group relative to that of the non-toxic control.
2.9. Statistical analysis The results were reported as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) with Tukey post hoc test was employed in the statistical assessment of the experimental data. A P value of <0.05 was considered as statistically significant.
142
R. Wang et al. / Journal of Colloid and Interface Science 438 (2015) 138–148
3. Results and discussion 3.1. Polymer synthesis and characterization CS-b-pSBMA copolymer was synthesized in a similar manner as a previously reported method of preparing CS-poly(ethylene glycol) block copolymer [21]. When ammonium persulfate was added to an aqueous solution of CS at 60 °C, the free radicals generated from the decomposition of the persulfate anions cleaved the CS chain into two shorter chains with one chain having a free radical at the end and the other chain having a carbonyl group (Fig. 1a) [29]. The free radicals on the CS chains serve as initiator for subsequent block copolymerization with SBMA to produce CS-b-pSBMA (Fig. 1a). The chemical composition of the resultant copolymers prepared with different [SBMA]/[CS] feed ratios is shown in Table S1. With a [SBMA]/[CS] feed ratio of 1:1, a CS-b-pSBMA copolymer with a [SBMA]/[CS] ratio of 0.92 was obtained, and further increase in the [SBMA]/[CS] feed ratio to 5:1 resulted in only a small increase in the [SBMA]/[CS] ratio of the copolymer. Hence, the CS-b-pSBMA copolymer with a [SBMA]/[CS] ratio of 0.92 was used for the preparation of the coated surface. The FTIR spectra of CS, pSBMA and CS-b-pSBMA are shown in Fig. S2a. The main characteristic adsorption bands of CS are located at 1034 cm 1 (–C–O–), 1420 cm 1 (–C–N–), 1651 cm 1 (–N–H), and 3464 cm 1 (–O–H) [30]. In the spectrum of the CS-b-pSBMA copolymer ([SBMA]/[CS] ratio: 0.92), new adsorption bands appear at 1041/1188 cm 1, 1481 cm 1, and 1728 cm 1, which are attributed to the –SO3 , N+–CH3 and –COO– species, respectively [31]. The curve-fitted XPS N 1s core-level spectra of CS, pSBMA and CS-b-pSBMA are given in Fig. S2b. The peak at 399.8 eV is attributed to the NH species in the CS units [32,33], and the peaks at higher binding energy (above 400 eV) can be assigned to positively charged nitrogen, i.e. protonated N+H species in the CS units (401.7 eV) and quaternary N+ species in the SBMA units (402.5 eV) [34]. These three peak components are also observed in the N 1s core-level spectrum of HTCC (Fig. S3).
3.2. Surface coating and characterization When the silicone sheet was exposed to an oxygen–ozone gas mixture, hydroxyl groups, peroxides and hydroxyl peroxides were generated on its surface [23,35]. These functional groups on the ozonized surface were used to react with alkoxy groups in APTES to form an aminolyzed surface [23]. CS-b-pSBMA polymer was then grafted on the surface with crosslinking between the polymer chains via reaction between genipin and the primary amino groups on the aminolyzed silicone surface and in the polymer, as illustrated by Fig. 1b. The amino groups in the polymer may be crosslinked by either a single molecule of genipin, or a short chain of condensed genipin units [36,37]. Since it has been reported that CS derivative with quaternary ammonium groups has higher bactericidal activity than CS [20,30], GTMAC was grafted to the CS moiety via reaction of the amino groups in CS and the epoxy groups in GTMAC. GTMAC may also react with the amino groups on the aminolyzed silicone surface. XPS N 1s core-level signals of the pristine and modified silicone surfaces are shown in Fig. 2. As expected, no discernible nitrogen signal was observed on the pristine silicone surface (Fig. 2a). After the APTES treatment, the presence of nitrogen peaks at 399.8 eV (NH) and 401.7 eV (N+H) confirmed that the surface was aminolyzed (Fig. 2b). The N 1s spectrum of the aminolyzed silicone surface after treatment with CS-b-pSBMA (Fig. 2c) shows the component peaks of the copolymer (Fig. S2b), indicating that the grafting was successful. The higher intensity of the NH peak at 399.8 eV in Fig. 2c compared to that in Fig. S2b may be due to contribution from
the APTES layer. The intensity of the N+ peak at 402.5 eV further increased on the HTCC-b-pSBMA-coated surface due to the introduction of GTMAC moieties (Fig. 2d). On the HTCC-coated surface, the dominant peaks are the NH (at 399.8 eV) and N+ (at 402.5 eV) peaks (Fig. 2e). For the pSBMA-coated surface, the dominant peak at 402.5 eV (Fig. 2f) is consistent with the quaternary N+ peak of pSBMA (Fig. S2b). The ratio of positively charged nitrogen species to total nitrogen species ({[N+H]+[N+]}/[N]) and the ratio of sulfur to nitrogen ([S]/[N]) on the surfaces were estimated from the XPS spectra and given in Table S2. With the introduction of GTMAC moieties, the {[N+H]+[N+]}/[N] ratio increased from 0.47 on the CSb-pSBMA coating to 0.61 on the HTCC-b-pSBMA coating, whereas the [S]/[N] ratio decreased correspondingly from 0.30 to 0.26. Water contact angles on the pristine and modified surfaces were measured using the sessile drop method and the results are given in Table S2. The pristine silicone surface is hydrophobic with a contact angle of 109°, and modification of silicone with APTES resulted in a decrease in the contact angle to 85°. The CS-b-pSBMA and HTCC-b-pSBMA coatings improved the surface hydrophilicity significantly and the contact angle decreased to 34° and 36°, respectively. The HTCC-coated surface showed a relatively high contact angle of 51°. It has been reported that genipin crosslinked CS hydrogel showed a relatively higher contact angle compared to uncrosslinked CS hydrogel, since genipininduced crosslinked CS hydrogel is hydrated in a smaller extent compared to the uncrosslinked CS hydrogel [38]. The pSBMA coating is very hydrophilic with a contact angle of 10°, due to the zwitterionic polymer being fully hydrated in water. The lower contact angles of the CS-b-pSBMA and HTCC-b-pSBMA coatings compared to the HTCC coating is thus attributed to the SBMA moiety in the CS-b-pSBMA copolymer. The thickness of the HTCC-b-pSBMA, HTCC and pSBMA coatings is <1 lm (0.5–0.7 lm) (Fig. S4b–d). The HTCC coating (Fig. S4c) is thicker than the HTCC-b-pSBMA and pSBMA coatings (Fig. S4b and d). For the same weight of CS and CS-b-pSBMA polymers used in the preparation of the coating, there are more –NH2 groups in the former for reaction with genipin than the latter, leading to a higher degree of crosslinking and grafting of the CS chains onto the surface compared to the CS-b-pSBMA copolymer. Since the three coatings, HTCC-b-pSBMA, HTCC and pSBMA, have comparable thicknesses, the differences in their antibacterial property (as discussed below) are not likely to be due to coating thickness. 3.3. Bacterial colonization on silicone surface via different routes Surfaces of medical devices are subjected to possible colonization by bacteria which can be transmitted by contact with contaminated fluid, droplet and air. To investigate the effectiveness of the coatings against bacterial colonization, two representative assays were carried out. Biofilm formation on substrate immersed in bacterial suspension: Pristine and modified silicone sheet surfaces were immersed in culture medium inoculated with either E. coli, P. mirabilis, or P. aeruginosa at a concentration of 105 cells/mL for 24 h to study biofilm formation on the surfaces by these bacterial cells. Under this condition, the planktonic bacterial cells in the aqueous medium have to approach and attach on the surface to form colonies [1]. Fig. 3 shows the SEM images of pristine and modified silicone surfaces after the incubation period. The pristine silicone surfaces were readily colonized by these three microorganisms, although the biofilm coverage was different due to different biofilm formation capability of the bacterial species. Bacterial colonization was reduced on the HTCC-coated surface, but numerous bacterial clusters can still be observed. Quantitative counts of viable bacterial cells on the HTCC-coated surface shows that the HTCC coating reduced bacterial colonization by 89% for E. coli, 94% for
R. Wang et al. / Journal of Colloid and Interface Science 438 (2015) 138–148
143
Fig. 2. XPS N 1s core-level spectra of (a) pristine, (b) ATPES-, (c) CS-b-pSBMA-, (d) HTCC-b-pSBMA-, (e) HTCC-, and (f) pSBMA-modified silicone surfaces. CS-b-pSBMA- and HTCC-b-pSBMA-coated surfaces were prepared from CS-b-pSBMA copolymer with a [SBMA]/[CS] ratio of 0.92.
P. mirabilis, and 85% for P. aeruginosa compared to that on the pristine surface (Fig. 4). The positively charged N+H and N+ groups on the HTCC coating interact with the negatively charged bacterial cells, disrupt the cell membranes, which lead to cell death [19]. However, the positively charged groups also attract bacterial debris. In addition, the HTCC-coated surface is relatively hydrophobic (contact angle of 51°, Table S2), and hydrophobic–hydrophobic interaction between the substrate surface and surface of bacterial cells may increase debris deposition and accumulation of dead bacteria. As a result, the active groups in the coating will be blanketed, and planktonic cells from the medium can then attach and multiply on the surface [13]. On the other hand, the pSBMA coating effectively inhibited bacterial colonization over 24 h, as few cell clusters were found on the surface and the remaining bacteria exist mostly as single cells (Fig. 3). Bacterial colonization by the three microorganisms was reduced by >99% on the pSBMA-coated surface (Fig. 4). Two factors may contribute to the observed reduction in bacterial colonization: the bactericidal property of the quaternary N+ groups in the coating (each SBMA unit has a quaternary N+ group), and/or the hydrophilic property of the surface. The bactericidal property of pSBMA was evaluated from the minimum inhibitory concentrations (MICs) of pSBMA against E. coli, P. mirabilis, P. aeruginosa and S. aureus. For all these bacteria, the MIC values were determined to be >2048 mg/L. This value is much higher than the corresponding values for CS and HTCC (degree of substitution: 67%) (details in SI, Table S3). This is consistent with an earlier finding that the bactericidal activity of polymers containing sulfobetaine groups is much lower than antimicrobial polycations based on poly(trialkylvinylbenzylammonium chlorides) [39]. The presence of the –SO3 group in close proximity to the quaternary
N+ group may diminish the degree of interaction between the N+ group and the bacterial cell wall. On the other hand, the pSBMAcoated surface is highly hydrophilic (contact angle of 10°, Table S2), and this surface hydration layer serves as a physical and energy barrier to discourage bacterial adhesion on the surface [40]. Therefore, the antibacterial property of the pSBMA coating as shown in Figs. 3 and 4 is attributed primarily to its anti-adhesive nature rather than the bactericidal activity of its quaternary N+ groups. Fig. 3 shows that the CS-b-pSBMA coating is as effective as the pSBMA coating in reducing bacterial colonization, and the HTCC-b-pSBMA coating is the most effective in reducing bacterial colonization (P99.7% reduction in bacterial colonization by the three tested microorganisms compared to pristine silicone, Fig. 4). Thus, the high antibacterial efficacy of the HTCC-b-pSBMA coating resulted from the combination of an abundance of bactericidal quaternary ammonium groups in the HTCC polymer, and the anti-adhesive property of pSBMA, which prevented the deposition of bacterial cells and debris. From Figs. 3 and 4, it can be seen that the efficacy of the coatings against P. mirabilis biofilm formation after 24 h is the lowest. Thus, the efficacy of the coatings was further tested with P. mirabilis over a 72 h period. A thick P. mirabilis biofilm was observed on the pristine surface (Fig. S5a). The bacterial cells on the HTCCcoated surface also developed into a dense biofilm after 3 days of incubation (Fig. S5b). On the pSBMA-coated surface, many more bacterial cells were present after 3 days compared to the corresponding surface after 1 day (comparing Fig. S5c and Fig. 3). As discussed above, the pSBMA coating mainly relies on the hydration barrier to reduce bacterial attachment, and the bactericidal activity of SBMA is low. As a result, any bacterial cell that succeeds in
144
R. Wang et al. / Journal of Colloid and Interface Science 438 (2015) 138–148
Fig. 3. SEM images of in vitro bacterial colonization on pristine and modified silicone sheet surfaces after incubation in bacterial culture medium for 24 h (initial inoculum was 105 cells/mL). Scale bars represent 10 lm.
Fig. 4. Number of viable adherent bacterial cells per cm2 of substrate surface after incubation in bacterial culture medium for 24 h (initial inoculum was 105 cells/mL). ⁄ denotes significant difference with P < 0.01 compared with pristine silicone surface. #, + denote significant difference with P < 0.01 and P < 0.05, respectively, compared with HTCC-b-pSBMA-coated silicone surface.
breaching the hydration barrier and attaches on the pSBMA-coated surface will likely proliferate and form a colony over a longer duration. For the HTCC-b-pSBMA-coated surface, although there is an
increase in the number of adherent bacteria after 3 days compared with the corresponding surface after 1 day (comparing Fig. S5d and Fig. 3), the reduction in bacterial colonization is still substantial compared to the pristine surface (Fig. S5a) and also the pSBMAcoated surface (Fig. S5c) over the same period of incubation. These results confirmed that the bifunctional coating integrating antifouling pSBMA and bactericidal HTCC moieties showed better performance in resisting colonization by planktonic bacteria in aqueous medium than the corresponding antifouling pSBMA coating and bactericidal HTCC coating. Bacterial colonization on substrates exposed to bacterial aerosol: To simulate bacterial contamination from air and droplets, a bacterial PBS suspension (containing 108 cells/mL of E. coli, P. mirabilis, or P. aeruginosa) was sprayed onto the surface of pristine and modified silicone sheets. The contaminated surface was then incubated under growth agar for 24 h. SEM images of the surfaces after the incubation period are shown in Fig. 5. Numerous distinct clusters of bacteria can be observed on the pristine surfaces. The HTCC coating very effectively inhibited bacterial colonization as few bacterial clusters were observed on the surface. The number of viable bacterial cells on the HTCC-coated surface was reduced by 99% (Fig. 6). This demonstrates the efficacy of the positively charged groups of HTCC in killing the deposited bacterial cells upon contact.
R. Wang et al. / Journal of Colloid and Interface Science 438 (2015) 138–148
145
Fig. 5. SEM images of in vitro bacterial colonization on pristine and modified silicone sheet surfaces after spraying with bacterial PBS suspension (108 cells/mL) and subsequent incubation under solid growth agar for 24 h. Scale bars represent 10 lm.
Fig. 6. Number of viable adherent bacterial cells per cm2 of substrate surface after spraying with bacterial PBS suspension (108 cells/mL) and subsequent incubation under solid growth agar for 24 h. ⁄ and # denote significant difference with P < 0.01 compared with pristine silicone surface and HTCC-b-pSBMA-coated silicone surface, respectively.
Fig. 7. FBG adsorption on pristine, and HTCC-, pSBMA-, HTCC-b-pSBMA-coated silicone surfaces after the substrates were treated with 1 mg/mL of FBG solutions for 4 h. ⁄, @ denote significant difference with P < 0.01 and P < 0.05, respectively, compared with pristine silicone surface. # denotes significant difference with P < 0.01 compared with HTCC-b-pSBMA-coated silicone surface.
146
R. Wang et al. / Journal of Colloid and Interface Science 438 (2015) 138–148
On the pSBMA-coated surface, the number of bacterial clusters was substantial, indicating the deposited bacterial cells were viable. These results are consistent with the MIC results which show that the bactericidal activity of quaternary N+ groups of pSBMA is much lower than those of HTCC. The CS-b-pSBMA coating reduced bacterial colonization, and further reduction in bacterial colonization was observed on the surface with HTCC-b-pSBMA coating (Fig. 5). Fig. 6 shows that incorporating the antifouling pSBMA moieties in almost equal proportion as HTCC in the bifunctional coating did not diminish the HTCC’s bactericidal activity against aerosolized bacteria.
From the above observations of bacterial colonization via two very different routes, it can be seen that coatings with pSBMA alone are effective in preventing planktonic cells in aqueous medium from approaching the surface and colonizing it but are not effective against aerosolized bacteria. On the other hand, coatings with HTCC alone can effectively kill aerosolized bacteria on contact but are not effective against biofilm formation by bacteria from aqueous medium over extended periods of time. The bifunctional HTCC-b-pSBMA coating combines the two complementary antibacterial mechanisms, and shows high efficacy in inhibiting bacterial colonization under both conditions.
Fig. 8. SEM images of adherent platelets on (a) pristine, (b) HTCC-, (c) pSBMA-, (d) HTCC-b-pSBMA-, (e) HTCC-b-pSBMA-Hep-coated surfaces, and (f) HTCC-b-pSBMA-Hepcoated surface after aging in PBS for 7 days. Insets show a higher magnification of platelet on the corresponding surfaces. Scale bars represent 10 lm in main images, and 5 lm in insets. (g) Platelet adhesion on the modified silicone surfaces relative to that on the pristine silicone surface, and PRT on pristine and modified silicone surfaces. ⁄, @ denote significant difference with P < 0.01 and P < 0.05, respectively, compared with pristine silicone surface. #, + denote significant difference with P < 0.01 and P < 0.05, respectively, compared with HTCC-b-pSBMA-coated silicone surface.
R. Wang et al. / Journal of Colloid and Interface Science 438 (2015) 138–148
3.4. Protein adsorption Implanted medical devices are exposed to body fluids or tissues containing proteins. FBG is a major protein in blood plasma and when a surface first comes to contact with blood, adsorption of FBG rapidly occurs [41]. Subsequent consequences such as bacterial adhesion and platelet and cell adhesion induced by the adsorbed FBG may adversely affect the functionality of the device [41,42]. Fig. 7 compares the amount of FBG adsorbed on the pristine and modified surfaces from 1 mg/mL protein solution after 4 h. The pristine silicone surface adsorbed 3.1 lg/cm2 of FBG. After HTCC coating on the surface, the adsorption of FBG increased by 29%, and protein aggregates were observed on the HTCC surface (Fig. S6). The isoelectric point of FBG is 5.1 [43], and thus FBG is negatively charged under the experimental condition in this study (10 mM CPBS, pH 7.4). Electrostatic interactions between the FBG molecules and positively charged HTCC coating may enhance protein adsorption, and also change the conformation of the adsorbed FBG [44]. As expected, the pSBMA coating with its surface hydration layer [40] is effective in decreasing FBG adsorption (86% reduction compared to pristine surface). The HTCC-b-pSBMAcoated surface decreased FBG adsorption by 60%, indicating that integration of SBMA moiety in the copolymer surface coating at a [SBMA]/[CS] ratio of 0.92 counteracted the effect of HTCC, and improved the surface antifouling property. It has been reported that adsorbed FBG promotes bacterial adhesion on the surface [45–47]. Hence, bacterial adhesion and biofilm formation by P. mirabilis on pristine silicone surface and HTCC-b-pSBMA-coated surface which were pretreated with FBG solution were further investigated. An increase in the number of adherent P. mirabilis cells was observed on the pristine surface after it was pretreated with FBG (comparing Fig. S7a and c). It has been reported that pre-adsorbed FBG on surface promotes S. aureus adhesion due to the expression of FBG binding protein (clumping factor) by the S. aureus cells [47,48]. However, for P. mirabilis, fimbriae play an important role in the bacterial adhesion process [49]. The presence of blood components in the medium is reported to enhance expression of fimbriae by P. mirabilis cells [50]. Therefore, it is likely that the adsorbed FBG increases fimbriae expression by the cells and subsequently promotes P. mirabilis adhesion on the surface. The number of adherent P. mirabilis cells also increased on the HTCC-b-pSBMA-coated surface pretreated with FBG, compared to the corresponding surface without FBG pretreatment (comparing Fig. S7b and d). With the increase in number of adherent bacteria on both the pristine and HTCC-b-pSBMAcoated surfaces after FBG pretreatment, it is expected that biofilm formation would also be enhanced (comparing Fig. 3 and Fig. S8b). Nevertheless, reduction in biofilm formation by P. mirabilis on the FBG-treated HTCC-b-pSBMA-coated surface was substantial compared to the pristine surface after similar treatment (Fig. S8a).
147
was prepared. Since CVCs are expected to stay in the body for a median duration of 7 days [54], the HTCC-b-pSBMA-Hep coating was incubated in PBS for 7 days. The hemocompatibility of the surfaces before and after incubation in PBS was evaluated using platelet adhesion and PRT assays. Fig. 8a to f show the SEM images of the adherent platelets on pristine and modified silicone surfaces after incubation for 1 h. As can be seen, the pristine silicone surface and HTCC-coated surface are prone to platelet adhesion, and the adherent platelets showed pseudopodia and spreading, indicating that they were activated by these surfaces (Fig. 8a and b). The small white dots on the HTCC-coated surface in Fig. 8b are probably adsorbed proteins and biomolecules from PRP, since the HTCC coating promotes protein adsorption and formation of aggregates on the surface (Fig. S6d). Platelet adhesion on the pSBMA-, HTCCb-pSBMA-, and HTCC-b-pSBMA-Hep-coated surfaces was greatly inhibited, and the adherent platelets were round and not activated (Fig. 8c–f). A quantitative comparison of the relative number of adherent platelets on the different surfaces is given in Fig. 8g. An increase in platelet adhesion by 12% was observed on the HTCC-coated surface compared to the pristine surface. In contrast, the pSBMA-coated surface reduced platelet adhesion by 95%. This is consistent with an earlier finding that pSBMA coating can effectively reduce platelet adhesion [18]. As expected, with the presence of HTCC in the HTCC-b-pSBMA coating, the number of adherent platelets was more than on the pSBMA coating. However, platelet adhesion on the HTCC-b-pSBMA-coating is 95% lower than the HTCC coating. The extent of platelet adhesion correlates with FBG adsorption since FBG plays an important role in mediating the process of platelet adhesion [41]. The heparin-loaded coating, HTCC-b-pSBMA-Hep, decreased platelet adhesion on the surface to the same level as the pSBMA coating. After incubation in PBS for 7 days, 0.4 ± 0.1 lg/cm2 heparin remained in the HTCC-bpSBMA-Hep coating, and with this amount of heparin, the coating still achieved 91% reduction in platelet adhesion. The PRT results obtained with the pristine and modified surfaces are shown in Fig. 8g. The PRT was 12 min and 10 min on the pristine silicone surface and HTCC-coated surface, respectively. With the pSBMA coating, the PRT increased significantly to 21 min. The HTCC-bpSBMA coating resulted in a PRT of 15 min. On the surface coated with HTCC-b-pSBMA-Hep, the PRT further increased to 23 min. Even after 7 days of incubation of the HTCC-b-pSBMA-Hep coating in PBS, a PRT of 17 min was achieved on this surface. Bacterial colonization assays carried out with HTCC-b-pSBMA-Hep-coated surface showed that this coating inhibited bacterial colonization
3.5. Heparin-loaded coating for blood-contacting surface Since the bifunctional coating demonstrates favorable antifouling and antibacterial properties, it may potentially be useful for preventing bacterial colonization of medical devices. A promising application of such coating may be for central venous catheters (CVCs), where infections associated with these devices result in significant patient morbidity, mortality and healthcare cost [51]. Due to the blood-contacting nature of CVCs, the surface coating also has to be blood compatible. Heparin has been reported to have strong anticoagulant activity [52], and the high density of negative charges in the heparin molecule allows it to be loaded in the HTCCb-pSBMA coating via ionic interactions with the positive charges in the polymer [53]. Using this method, a heparin-loaded coating (HTCC-b-pSBMA-Hep) with a heparin loading of 2.2 ± 0.3 lg/cm2
Fig. 9. Viability of 3T3 fibroblast cells incubated with pristine and modified silicone sheets for 24 h and 72 h. MTT assay was employed to evaluate cell viability through the measurement of mitochondrial activity. Viability is expressed as a percentage relative to the result obtained with the non-toxic control (3T3 fibroblast cells incubated in the absence of silicone sheet).
148
R. Wang et al. / Journal of Colloid and Interface Science 438 (2015) 138–148
by S. aureus, a common pathogen related to bloodstream infections, as effectively as the HTCC-b-pSBMA coating under both conditions (in aqueous medium and from bacterial aerosol) (Fig. S9). Therefore, it can be concluded that the heparin loaded into the HTCCb-pSBMA coating did not compromise its antibacterial property, and such coating is promising for blood-contacting catheter surfaces which have to be both antibacterial and hemocompatible. 3.6. Cytotoxicity assay Since implanted medical devices are in contact with body tissue, it is crucial that the surface coatings are non-cytotoxic. MTT assay, which measures mitochondrial activity, was used to evaluate the cytotoxic properties of the surfaces. The direct contact method was used, whereby the silicone surfaces were contactincubated on top of a pre-seeded fibroblast cell layer [28]. Fig. 9 shows that the pristine silicone sheet and the modified silicone sheets (CS-b-pSBMA-, HTCC-b-pSBMA-, and HTCC-b-pSBMA-Hepcoated) posed no significant cytotoxicity to the mammalian cells over 24 h and 72 h of contact-incubation. Therefore, it can be concluded that the coatings are non-cytotoxic and can be potentially used for in vivo applications. 4. Conclusion A bifunctional coating with both antifouling and bactericidal moieties was prepared by grafting a CS-b-pSBMA copolymer with a [SBMA]/[CS] ratio of 0.92 onto an aminolyzed silicone surface using genipin-induced crosslinking. Subsequent treatment of the coating with GTMAC resulted in a surface {[N+H]+[N+]}/[N] ratio of 0.61. The efficacy of the so-obtained coating against bacterial colonization on surfaces immersed in bacterial suspension or exposed to bacterial aerosol were evaluated and compared with the antifouling pSBMA coating and the bactericidal HTCC coating. The pSBMA coating was not effective against aerosolized bacterial cells, whereas the HTCC coating has limited capability in preventing bacterial colonization in aqueous medium. The bifunctional coating, which combines the advantages of the two complementary mechanisms, inhibited bacterial colonization by approximately two orders of magnitude under both conditions compared with the pristine silicone surface, and it also reduced protein adsorption significantly. The loading of heparin in the bifunctional coating improved the surface hemocompatibility and did not compromise the antibacterial properties. Cytotoxicity assay confirmed that the HTCC-b-pSBMA coating does not pose significant cytotoxicity to mammalian cells. Therefore, the HTCC-b-pSBMA coating offers a promising strategy for inhibiting bacterial adhesion and colonization as well as improving hemocompatibility of medical device surfaces. Acknowledgment This work was financially supported by the National Medical Research Council of Singapore Grant NMRC/BnB/12sep040. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcis.2014.09.070. References [1] M. Katsikogianni, Y.F. Missirlis, Eur. Cells Mater. 8 (2004) 37. [2] R.M. Donlan, Emerg. Infect. Dis. 7 (2001) 277. [3] J. Hasan, R.J. Crawford, E.P. Ivanova, Trands Biotechnol. 31 (2013) 295.
[4] G. Cheng, Z. Zhang, S.F. Chen, J.D. Bryers, S.Y. Jiang, Biomaterials 28 (2007) 4192. [5] M.L. Hawkins, F. Faÿ, K. Rénel, I. Linossier, M.A. Grunlan, Biofouling 30 (2014) 247. [6] J. Wu, W. Lin, Z. Wang, S. Chen, Y. Chang, Langmuir 28 (2012) 7436. [7] R.L. Meyer, A. Arpanaei, S. Pillai, N. Bernbom, J.J. Enghild, Y.Y. Ng, L. Gram, F. Besenbacher, P. Kingshott, Colloids Surf. B 102 (2013) 504. [8] M.R. Nejadnik, H.C. van der Mei, W. Norde, H.J. Busscher, Biomaterials 29 (2008) 4117. [9] L. Ferreira, A. Zumbuehl, J. Mater. Chem. 19 (2009) 7796. [10] J.C. Tiller, C.J. Liao, K. Lewis, A.M. Klibanov, Proc. Natl. Acad. Sci. USA 98 (2001) 5981. [11] M.D.P. Willcox, E.B.H. Hume, Y. Aliwarga, N. Kumar, N. Cole, J. Appl. Microbiol. 105 (2008) 1817. [12] R.X. Chen, N. Cole, M.D.P. Willcox, J. Park, R. Rasul, E. Carter, N. Kumar, Biofouling 25 (2009) 517. [13] A.M. Klibanov, J. Mater. Chem. 17 (2007) 2479. [14] N. Aumsuwan, R.C. Danyus, S. Heinhorst, M.W. Urban, Biomacromolecules 9 (2008) 1712. [15] S. Yuan, D. Wan, B. Liang, S.O. Pehkonen, Y.P. Ting, K.G. Neoh, E.T. Kang, Langmuir 27 (2011) 2761. [16] A.K. Muszanska, E.T.J. Rochford, A. Gruszka, A.A. Bastian, H.J. Busscher, W. Norde, H.C. van der Mei, A. Herrmann, Biomacromolecules 15 (2014) 2019. [17] R. Lalani, L. Liu, Biomacromolecules 13 (2012) 1853. [18] R.S. Smith, Z. Zhang, M. Bouchard, J. Li, H.S. Lapp, G.R. Brotske, D.L. Lucchino, D. Weaver, L.A. Roth, A. Coury, J. Biggerstaff, S. Sukavaneshvar, R. Langer, C. Loose, Sci. Transl. Med. 4 (2012) 153ra132. [19] E.I. Rabea, M.E.T. Badawy, C.V. Stevens, G. Smagghe, W. Steurbaut, Biomacromolecules 4 (2003) 1457. [20] W. Sajomsang, Carbohydr. Polym. 80 (2010) 631. [21] F. Ganji, M.J. Abdekhodaie, Carbohydr. Polym. 74 (2008) 435. [22] S.M. Alipour, M. Nouri, J. Mokhtari, S.H. Bahrami, Carbohydr. Res. 344 (2009) 2496. [23] G.A. Diaz-Quijada, D.D.M. Wayner, Langmuir 20 (2004) 9607. [24] F.J. Xu, Y.L. Li, E.T. Kang, K.G. Neoh, Biomacromolecules 6 (2005) 1759. [25] R. Wang, K.G. Neoh, Z.L. Shi, E.T. Kang, P.A. Tambyah, E. Chiong, Biotechnol. Bioeng. 109 (2012) 336. [26] L. Cen, K.G. Neoh, E.T. Kang, Langmuir 19 (2003) 10295. [27] M. Li, K.G. Neoh, E.T. Kang, T. Lau, E. Chiong, Adv. Funct. Mater. 24 (2014) 1631. [28] International Organization for Standardization (ISO), Biological evaluation of medical devices – part 5: Tests for in vitro cytotoxicity. ISO 10993–5:2009(e), 2009, 1. [29] S.C. Hsu, T.M. Don, W.Y. Chiu, Polym. Degrad. Stab. 75 (2002) 73. [30] Z.L. Shi, K.G. Neoh, E.T. Kang, W. Wang, Biomaterials 27 (2006) 2440. [31] S.G. Chen, S.J. Chen, S. Jiang, Y.M. Mo, J.X. Luo, J.N. Tang, Z.C. Ge, Colloids Surf. B 85 (2011) 323. [32] I.F. Amaral, P.L. Granja, M.A. Barbosa, J. Biomater. Sci. Polym. Ed. 16 (2005) 1575. [33] S.H. Hsu, C.H. Lin, C.S. Tseng, Biofabrication 4 (2012) 015002. [34] R. Wang, K.G. Neoh, E.T. Kang, P.A. Tambyah, E. Chiong, J. Biomed. Mater. Res. B (2014), http://dx.doi.org/10.1002/jbm.b.33230. [35] M. Li, K.G. Neoh, L.Q. Xu, R. Wang, E.T. Kang, T. Lau, D.P. Olszyna, E. Chiong, Langmuir 28 (2012) 16408. [36] R.A.A. Muzzarelli, Carbohydr. Polym. 77 (2009) 1. [37] A.H. Wang, Y. Cui, J.B. Li, J.C.M. van Hest, Adv. Funct. Mater. 22 (2012) 2673. [38] B.S. Liu, C.H. Yao, S.S. Fang, Macromol. Biosci. 8 (2008) 432. [39] M. Ward, M. Sanchez, M.O. Elasri, A.B. Lowe, J. Appl. Polym. Sci. 101 (2006) 1036. [40] S.F. Chen, L.Y. Li, C. Zhao, J. Zheng, Polymer 51 (2010) 5283. [41] W.B. Tsai, J.M. Grunkemeier, T.A. Horbett, J. Biomed. Mater. Res. 44 (1999) 130. [42] H. Chen, L. Yuan, W. Song, Z. Wu, D. Li, Prog. Polym. Sci. 33 (2008) 1059. [43] V.P. Hoven, V. Tangpasuthadol, Y. Angkitpaiboon, N. Vallapa, S. Kiatkamjornwong, Carbohydr. Polym. 68 (2007) 44. [44] I. Van De Keere, R. Willaert, A. Hubin, J. Vereecken, Langmuir 24 (2008) 1844. [45] Y.H. An, R.J. Friedman, J. Biomed. Mater. Res. B 43 (1998) 338. [46] C. Tedjo, K.G. Neoh, E.T. Kang, N. Fang, V. Chan, J. Biomed. Mater. Res. A 82 (2007) 479. [47] Y. Wang, G. Subbiahdoss, J. de Vries, M. Libera, H.C. van der Mei, H.J. Busscher, Biofouling 28 (2012) 1011. [48] C. Wolz, D. McDevitt, T.J. Foster, A.L. Cheung, Infect. Immun. 64 (1996) 3142. [49] S.M. Jacobsen, D.J. Stickler, H.L.T. Mobley, M.E. Shirtliff, Clin. Microbiol. Rev. 21 (2008) 26. [50] R.K. Latta, M.J. Schur, D.L. Tolson, E. Altman, Can. J. Microbiol. 44 (1998) 896. [51] N.P. O’Grady, M. Alexander, L.A. Burns, E.P. Dellinger, J. Garland, S.O. Heard, P.A. Lipsett, H. Masur, L.A. Mermel, M.L. Pearson, I.I. Raad, A. Randolph, M.E. Rupp, S. Saint, the Healthcare Infection Control Practices Advisory Committee, Guidelines for the prevention of intravascular catheter-related infections, 2011. [52] J. Hirsh, R. Raschke, T.E. Warkentin, J.E. Dalen, D. Deykin, L. Poller, Chest 108 (1995) 258S. [53] K. Kamin´ski, K. Zazakowny, K. Szczubiałka, M. Nowakowska, Biomacromolecules 9 (2008) 3127. [54] O. Traoré, J. Liotier, B. Souweine, Crit. Care. Med. 33 (2005) 1276.
Contents lists available at ScienceDirect
Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Integration of antifouling and bactericidal moieties for optimizing the efficacy of antibacterial coatings Rong Wang, Koon Gee Neoh ⇑, En-Tang Kang Department of Chemical and Biomolecular Engineering, National University of Singapore, Kent Ridge, Singapore 117576, Singapore
a r t i c l e
i n f o
Article history: Received 23 August 2014 Accepted 25 September 2014 Available online 5 October 2014 Keywords: Bifunctional coating Bacterial colonization Antifouling Bactericidal Hemocompatibility
a b s t r a c t Hypothesis: Surface coatings that are either antifouling or bactericidal can reduce bacterial colonization, but either type has certain drawbacks. We hypothesize that by integrating an antifouling polymer, poly(sulfobetaine methacrylate) (pSBMA), and a bactericidal polymer, N-[(2-hydroxy-3-trimethylammonium)propyl] chitosan chloride (HTCC), in one coating, these drawbacks can be overcome and the antibacterial efficacy can be greatly improved compared to an antifouling or bactericidal coating. Experiments: A copolymer comprising chitosan and pSBMA in almost equal molar ratio was grafted onto an aminolyzed silicone surface via genipin-induced crosslinking reaction, and treated with glycidyltrimethylammonium chloride to endow the surface with quaternary ammonium groups. The antibacterial property and cytotoxicity of the resultant coating (HTCC-b-pSBMA) were tested. The possibility of incorporating heparin in the coating to improve blood compatibility was also investigated. Findings: The HTCC-b-pSBMA coating reduced colonization by both planktonic bacteria from aqueous medium and aerosolized bacteria by approximately two orders of magnitude compared with the pristine surface. In comparison, surfaces coated with either the pSBMA or HTCC component are unable to achieve such efficacy. The heparin-loaded HTCC-b-pSBMA coating improved hemocompatibility without adversely affecting the antibacterial efficacy. No significant cytotoxicity of the coatings was observed with mammalian cells. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Medical devices are essential components in modern healthcare, but their surfaces are prone to bacterial colonization. For example, devices before implantation may be contaminated by bacteria from droplets, air or hands of healthcare workers and patients. The deposited bacteria could survive on the surface and multiply to form colonies after implantation. Furthermore, the surface of indwelling devices will contact body fluid (e.g. blood, urine), where pathogens may be present, and the planktonic bacteria will approach and attach to the surface via various bacteria–surface interactions (e.g. van der Waals forces, hydrogen bonding, ionic interactions, hydrophobic interactions) [1]. The adherent bacteria will subsequently multiply and colonize the surface, and eventually this may lead to infection of the device [2]. Therefore, inhibiting bacterial colonization is of critical importance in reducing the probability of nosocomial infections. ⇑ Corresponding author at: Blk E5, 4 Engineering Drive 4, #02-34, Singapore 117576, Singapore. Fax: +65 67791936. E-mail addresses: [email protected] (R. Wang), [email protected] (K.G. Neoh), [email protected] (E.-T. Kang). http://dx.doi.org/10.1016/j.jcis.2014.09.070 0021-9797/Ó 2014 Elsevier Inc. All rights reserved.
Two common strategies have been used to inhibit bacterial colonization on surfaces: (1) by coating the surface with an antifouling layer to repel bacteria, or (2) by coating with a non-leaching bactericidal layer to kill the bacterial cells on contact [3]. Antifouling coatings rely mainly on the formation of a hydrated barrier which prevents bacterial attachment [4–7]. Such antifouling coating delays biofilm formation but will not kill bacterial cells that are already deposited on the surface [8]. On the other hand, a surface with covalently immobilized bactericidal agents, such as quaternary ammonium compounds and antimicrobial peptides, kills bacteria on contact and exhibits a self-sterilizing effect [9–12]. However, the accumulation of dead bacteria on the bactericidal coating over the course of exposure would result in blanketing of the active groups [13], and the coating efficacy would eventually be compromised. Since both antifouling and bactericidal coatings have certain advantages and drawbacks, we hypothesize that by integrating both types of moieties in one coating, the advantages of the two complementary mechanisms can be combined to optimize the efficacy of the surface in preventing bacterial colonization. Most such bifunctional coatings reported in the literature rely on the conjugation of antifouling polymers with antibiotics [14], antimicrobial
R. Wang et al. / Journal of Colloid and Interface Science 438 (2015) 138–148
enzymes [15], or antimicrobial peptides [16]. However, the use of antibiotics is controversial due to the potential for development of antibiotic-resistant bacteria, and antimicrobial enzymes and peptides are usually costly. It has been reported that poly(sulfobetaine methacrylate) (pSBMA) coating inhibits protein adsorption and bacterial adhesion in aqueous medium [4,17,18]. On the other hand, chitosan (CS) and its derivatives with quaternary ammonium groups have bactericidal property attributable to the positively charged groups that alter the permeability of the bacterial membrane [19,20]. Hence, to obtain a copolymer which has antifouling as well as bactericidal properties, sulfobetaine methacrylate (SBMA) was block-copolymerized onto CS in this study. The resultant CS-block-pSBMA (CS-b-pSBMA) copolymer was grafted and crosslinked onto an aminolyzed silicone surface using genipin. Since it has been reported that CS derivatives with quaternary ammonium groups have higher bactericidal activity than CS [20], glycidyltrimethylammonium chloride (GTMAC) was grafted onto the resultant coating to endow the surface with quaternary ammonium groups. The corresponding antifouling pSBMA coating and bactericidal CS treated with GTMAC (N-[(2-hydroxy-3-trimethylammonium)propyl] chitosan chloride (HTCC)) coating were also prepared. The antifouling and antibacterial properties of the bifunctional coating were compared with the pSBMA coating and HTCC coating. Heparin was further loaded in the coating to evaluate the potential application of the bifunctional coating in blood-contacting devices. 2. Materials and methods 2.1. Materials Medical grade silicone sheets (1 mm thickness) were obtained from BioPlexus Inc., US. Chitosan (degree of deacetylation = 75–85%), (3-aminopropyl)triethoxysilane (APTES), ammonium persulfate, glycidyltrimethylammonium chloride (GTMAC), [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl) ammonium hydroxide (sulfobetaine methacrylate, SBMA), heparin sodium salt from porcine intestinal mucosa (Grade I-A, P180 USP units/mg), toluidine blue O, bovine plasma fibrinogen (FBG) and 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich, US. Genipin is a product from Challenge Bioproducts, Taiwan. Escherichia coli (E. coli ATCC DH5a), Proteus mirabilis (P. mirabilis ATCC 51286, a strain isolated from a patient with urinary catheter infection), Staphylococcus aureus (S. aureus ATCC 25923) and 3T3 fibroblasts were obtained from American Type Culture Collection (ATCC, US). Pseudomonas aeruginosa (P. aeruginosa PAO1) was purchased from National Collection of Industrial Food and Marine Bacteria (NCIMB, UK). 2.2. Synthesis and characterization of CS-b-pSBMA, pSBMA and HTCC polymers CS-b-pSBMA copolymer was synthesized in a similar fashion as the procedure outlined in the literature for the preparation of CS-poly(ethylene glycol) block copolymer (Fig. 1a) [21]. CS powder (0.5 g) was dissolved in 1% (v/v) aqueous acetic acid solution (30 mL) under stirring at 500 rpm. This solution was heated to 60 °C and maintained at this temperature throughout the reaction period. An oxygen-free environment was then established in the system by degassing with argon prior to the introduction of ammonium persulfate (100 mg). Argon was bubbled through the mixture for another 30 min. SBMA aqueous solution (at a [SBMA]/[CS] molar ratio of 1:5, 1:1 or 5:1 in 20 mL distilled water) was then gradually added to the mixture using a syringe over a 30-min period. The reaction was allowed to proceed under argon atmosphere
139
with stirring for 6 h. pSBMA homopolymer was synthesized in a similar manner using 0.87 g of SBMA without CS. HTCC polymer was prepared according to the method reported earlier via the reaction of CS with GTMAC (Fig. S1) [22]. Briefly, CS (3 g) was dispersed in distilled water (50 mL), and GTMAC (5.65 mL) was added dropwise to the suspension over 30 min. The reaction was allowed to proceed under continuous mechanical stirring at 500 rpm at 85 °C for 10 h. After reaction, small oligomers, salts, and unreacted reagents were removed by dialysis using a cellulose membrane (molecular weight cut-off of 12,000, Sigma–Aldrich) over three days, and the products were collected after lyophilization. The polymers were characterized using Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS), and the experimental details are given in the Supporting Information (SI). 2.3. Surface modification and characterization of HTCC-b-pSBMAcoated silicone Silicone sheets (cut into 1 10 cm2) were first cleaned by ultrasonication in isopropanol for 10 min and then in distilled water for 20 min to remove surface impurities. An oxygen-ozone gas mixture (generated from an Azcozon ozone generator (Model VMUS-4PSE, AZCO Inc., Canada)) was passed over the surface of the silicone sheet for 20 min to generate the necessary surface reactive groups. To obtain an ozone production rate of 6 g/h with an ozone concentration of 25 g/m3, the oxygen inlet flow rate was fixed at 4 L/min. After ozonization, the silicone sheet was immediately immersed in 15 mL freshly prepared solution containing APTES (5%, v/v), distilled water (5%, v/v) and ethanol (90%, v/v). The solution with the silicone sheet was placed in a shaker at 500 rpm for 1 h at room temperature. The silanized sheet was rinsed with ethanol and distilled water, dried under a flow of nitrogen, and heated in a 60 °C oven for 2 h to promote the formation of a crosslinked silane layer [23]. The silanized silicone sheet was then immersed in a CS-b-pSBMA aqueous solution (13.5 mL distilled water containing 150 mg copolymer), and a genipin ethanol solution (1.5 mL ethanol containing 150 mg genipin) was immediately added. The mixture was placed in a 37 °C water bath shaker at 100 rpm for 24 h. The silicone sheet was then removed from the reaction mixture and washed in ethanol overnight in a 37 °C shaker at 100 rpm, dried under a nitrogen flow, and denoted as CS-bpSBMA-coated sheet. The CS-b-pSBMA-coated sheet was immersed in 15 mL GTMAC aqueous solution (5%, v/v) at 70 °C for 24 h. The sheet was then washed with distilled water, dried under a nitrogen flow, and denoted as HTCC-b-pSBMA-coated sheet (Fig. 1b). The preparation of heparin-loaded coatings was carried out in a similar manner using a heparin-loaded CS-b-pSBMA polymer solution, and treatment with GTMAC to obtain a HTCC-b-pSBMA-Hep coating. To prepare a heparin-loaded CS-b-pSBMA polymer solution, a heparin aqueous solution (300 lL, 10% w/v) was added dropwise to a CS-b-pSBMA polymer solution (13.2 mL containing 150 mg CS-b-pSBMA) over 10 min under continuous mechanical stirring. To simulate in vivo application of the HTCC-b-pSBMA-Hep-coated substrate where it would be immersed in body fluid, a piece of the HTCC-b-pSBMA-Hep-coated substrate (1 1 cm2) was incubated in 50 mL phosphate buffered saline (PBS, 10 mM, pH 7.4) at 37 °C in a shaker at 100 rpm for 7 days. The amount of heparin in the coating before and after incubation was quantified using the toluidine blue method as described in an earlier publication [24]. HTCC-coated surface was prepared in a similar manner as the HTCC-b-pSBMA-coated surface using a mixed solution of CS and genipin, and pSBMA-coated surface was prepared by UV-induced graft-polymerization of SBMA on an ozonized silicone surface. The details of this procedure are given in the SI. The surfaces of
140
R. Wang et al. / Journal of Colloid and Interface Science 438 (2015) 138–148
Fig. 1. Schematic diagram illustrating (a) synthesis of CS-b-pSBMA copolymer, and (b) steps for modifying silicone sheet surface.
R. Wang et al. / Journal of Colloid and Interface Science 438 (2015) 138–148
the pristine and modified silicone sheets were characterized using XPS and contact angle measurement. The cross-sections of the silicone sheets were prepared and the coating thickness was determined using field emission scanning electron microscopy (FESEM). The details of these experimental procedures are given in the SI. 2.4. Biofilm formation on substrates immersed in bacterial suspension Bacteria were cultured in an appropriate broth (nutrient broth for E. coli, tryptic soy broth for P. mirabilis and S. aureus, lysogeny broth for P. aeruginosa) overnight. The bacterial cells were collected by centrifugation (2700 rpm, 10 min) and then washed twice using PBS (10 mM, pH 7.4). The cells were resuspended in PBS (10 mM, pH 7.4), and the cell density was determined by measuring the absorbance at 540 nm (A540nm of 1.0 unit corresponds to 1 109 cells/mL based on spread plate counting) [25]. The bacterial suspension was further diluted using the appropriate culture medium to a concentration of 105 cells/mL. One mL of the prepared bacterial medium suspension was added to each pristine or modified silicone sheet (cut into 1 1 cm2 size), which were pre-sterilized by UV irradiation and placed in a 24-well plate (Greiner Bio-one, Germany). The plate with the silicone sheets were incubated at 37 °C for 24 h or 72 h. For experiments carried out with an incubation period of 72 h, the medium was replaced with fresh culture medium every 24 h. After the incubation period, the bacterial suspension in each well was discarded and the substrate was washed using PBS for three times to remove any non-adherent or loosely adhered bacteria on the surface. Scanning electron microscopy (SEM, JEOL, Model 5600LV, Japan) was used to observe biofilms on the surfaces, and the viable bacterial cells on each surface were enumerated using the spread plate method as described in an earlier publication [25]. 2.5. Bacterial colonization on substrates exposed to bacterial aerosol Harvested bacterial cells were suspended in PBS (10 mM, pH 7.4) at a concentration of 108 cells/mL as described above. The prepared bacterial PBS suspension (100 lL) was sprayed onto silicone sheet surfaces (cut into 1 1 cm2 size) using a commercial sprayer. The diameter of the droplets from the sprayer was estimated to be in the range of 10–200 lm from observations using an optical microscope. The substrates were then dried in air for 5 min, and placed in a 24-well plate. One mL of melted growth agar (0.7% agar in yeast-dextrose broth, autoclaved and cooled to 37 °C) was poured over the substrates, and the plate was incubated at 37 °C for 24 h [26]. The silicone sheets after incubation were gently removed from the agar, placed in a new 24-well plate, and washed thrice with PBS. The silicone surfaces were observed using SEM and the number of viable adherent cells was quantified using the spread plate method. 2.6. Protein adsorption Protein adsorption assay was carried out by immersing the pristine and modified silicone substrates in a FBG protein solution (1 mg/mL in citrate–phosphate buffered saline (CPBS, 10 mM sodium citrate in 10 mM PBS, pH 7.4)) for 4 h. The amount of adsorbed protein on the substrate surface was quantified using the modified dye-interaction method with Bio-Rad protein dye reagent (Catalog No. 500-0006, Bio-Rad, US) in a similar manner as described in an earlier publication (details in SI) [27]. To investigate the effect of pre-adsorbed protein on bacterial adhesion and biofilm formation, pristine and modified substrates after treatment of FBG protein for 4 h at 37 °C were incubated in bacterial PBS suspension (108 P. mirabilis cells/mL, for bacterial adhesion assay) for 4 h, or bacterial culture medium (105 P. mirabilis
141
cells/mL, for biofilm assay) for 24 h, and the bacterial cells on the surface was observed using SEM as described above.
2.7. Platelet adhesion and plasma recalcification time (PRT) assays Platelet adhesion and PRT assays were carried out to assess the hemocompatibility of the surfaces in the same manner as described in a previous publication [27]. The platelet adhesion test was conducted by introducing diluted platelet-rich plasma (PRP) on the substrates followed by incubation at 37 °C for 1 h. The platelets adhering on the surfaces were observed using SEM. For the PRT assay, platelet poor plasma (PPP) was introduced on substrates followed by addition of CaCl2 solution. Clotting of the PPP solution was monitored by detecting the formation of fibrin threads in the solution, and the PRT (defined as the period from the introduction of CaCl2 to the first observation of silky fibrin) on each surface was recorded. The details of the assays are given in the SI.
2.8. Cytotoxicity assay To investigate the effect of the pristine and modified surfaces on cell viability, MTT assay which measures mitochondrial activity was carried out according to the standard protocol stated in ISO 10993-5 for evaluation of in vitro cytotoxicity of medical devices by direct contact [28]. Briefly, 3T3 fibroblast cells were cultured in a 75 cm2 tissue culture flask (Corning, US) using Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 1 mM L-glutamine, and 100 IU/mL penicillin. The medium was changed every 2 days over a period of 7–10 days until the cells reached 80% confluence. After the incubation period, the cells were detached from the flask using a trypsin–EDTA solution (5.3 mM, 12 mL), harvested by centrifugation (200g, 10 min), and suspended in fresh culture medium at a concentration of 104 cells/mL. One mL of the cell suspension was then added to each well of a 24-well plate. After incubation at 37 °C in a humidified atmosphere of 5% CO2 and 95% air for 24 h, the medium in the plate was completely refreshed, and pristine and modified silicone sheets (1 1 cm2, pre-sterilized by UV irradiation for 30 min) were gently placed on top of the cell layer in the well. Since the density of the silicone sheet is slightly higher than the medium, the silicone sheet would only contact the cell layer but not compress it. In the non-toxic control group, the cells were incubated with the complete growth culture medium without the silicone sheet. The plate was then incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air for 24 h or 72 h. After the incubation period, the silicone sheet was very gently taken out to avoid disrupting the cell layer. No cell attachment on the silicone surface was discernible under an optical microscope. The culture medium in each well was then replaced with equal volume of freshly prepared MTT solution (0.5 mg/mL in medium). After incubation at 37 °C for 4 h, the medium was discarded. The formazan crystals in each well were dissolved in 1 mL dimethyl sulfoxide for 15 min in the dark. The absorbance of the solutions at 570 nm was then measured using a microplate reader (Tecan GENios, Switzerland). All samples were tested in triplicate, and the results were reported as percentage of absorbance of the experimental group relative to that of the non-toxic control.
2.9. Statistical analysis The results were reported as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) with Tukey post hoc test was employed in the statistical assessment of the experimental data. A P value of <0.05 was considered as statistically significant.
142
R. Wang et al. / Journal of Colloid and Interface Science 438 (2015) 138–148
3. Results and discussion 3.1. Polymer synthesis and characterization CS-b-pSBMA copolymer was synthesized in a similar manner as a previously reported method of preparing CS-poly(ethylene glycol) block copolymer [21]. When ammonium persulfate was added to an aqueous solution of CS at 60 °C, the free radicals generated from the decomposition of the persulfate anions cleaved the CS chain into two shorter chains with one chain having a free radical at the end and the other chain having a carbonyl group (Fig. 1a) [29]. The free radicals on the CS chains serve as initiator for subsequent block copolymerization with SBMA to produce CS-b-pSBMA (Fig. 1a). The chemical composition of the resultant copolymers prepared with different [SBMA]/[CS] feed ratios is shown in Table S1. With a [SBMA]/[CS] feed ratio of 1:1, a CS-b-pSBMA copolymer with a [SBMA]/[CS] ratio of 0.92 was obtained, and further increase in the [SBMA]/[CS] feed ratio to 5:1 resulted in only a small increase in the [SBMA]/[CS] ratio of the copolymer. Hence, the CS-b-pSBMA copolymer with a [SBMA]/[CS] ratio of 0.92 was used for the preparation of the coated surface. The FTIR spectra of CS, pSBMA and CS-b-pSBMA are shown in Fig. S2a. The main characteristic adsorption bands of CS are located at 1034 cm 1 (–C–O–), 1420 cm 1 (–C–N–), 1651 cm 1 (–N–H), and 3464 cm 1 (–O–H) [30]. In the spectrum of the CS-b-pSBMA copolymer ([SBMA]/[CS] ratio: 0.92), new adsorption bands appear at 1041/1188 cm 1, 1481 cm 1, and 1728 cm 1, which are attributed to the –SO3 , N+–CH3 and –COO– species, respectively [31]. The curve-fitted XPS N 1s core-level spectra of CS, pSBMA and CS-b-pSBMA are given in Fig. S2b. The peak at 399.8 eV is attributed to the NH species in the CS units [32,33], and the peaks at higher binding energy (above 400 eV) can be assigned to positively charged nitrogen, i.e. protonated N+H species in the CS units (401.7 eV) and quaternary N+ species in the SBMA units (402.5 eV) [34]. These three peak components are also observed in the N 1s core-level spectrum of HTCC (Fig. S3).
3.2. Surface coating and characterization When the silicone sheet was exposed to an oxygen–ozone gas mixture, hydroxyl groups, peroxides and hydroxyl peroxides were generated on its surface [23,35]. These functional groups on the ozonized surface were used to react with alkoxy groups in APTES to form an aminolyzed surface [23]. CS-b-pSBMA polymer was then grafted on the surface with crosslinking between the polymer chains via reaction between genipin and the primary amino groups on the aminolyzed silicone surface and in the polymer, as illustrated by Fig. 1b. The amino groups in the polymer may be crosslinked by either a single molecule of genipin, or a short chain of condensed genipin units [36,37]. Since it has been reported that CS derivative with quaternary ammonium groups has higher bactericidal activity than CS [20,30], GTMAC was grafted to the CS moiety via reaction of the amino groups in CS and the epoxy groups in GTMAC. GTMAC may also react with the amino groups on the aminolyzed silicone surface. XPS N 1s core-level signals of the pristine and modified silicone surfaces are shown in Fig. 2. As expected, no discernible nitrogen signal was observed on the pristine silicone surface (Fig. 2a). After the APTES treatment, the presence of nitrogen peaks at 399.8 eV (NH) and 401.7 eV (N+H) confirmed that the surface was aminolyzed (Fig. 2b). The N 1s spectrum of the aminolyzed silicone surface after treatment with CS-b-pSBMA (Fig. 2c) shows the component peaks of the copolymer (Fig. S2b), indicating that the grafting was successful. The higher intensity of the NH peak at 399.8 eV in Fig. 2c compared to that in Fig. S2b may be due to contribution from
the APTES layer. The intensity of the N+ peak at 402.5 eV further increased on the HTCC-b-pSBMA-coated surface due to the introduction of GTMAC moieties (Fig. 2d). On the HTCC-coated surface, the dominant peaks are the NH (at 399.8 eV) and N+ (at 402.5 eV) peaks (Fig. 2e). For the pSBMA-coated surface, the dominant peak at 402.5 eV (Fig. 2f) is consistent with the quaternary N+ peak of pSBMA (Fig. S2b). The ratio of positively charged nitrogen species to total nitrogen species ({[N+H]+[N+]}/[N]) and the ratio of sulfur to nitrogen ([S]/[N]) on the surfaces were estimated from the XPS spectra and given in Table S2. With the introduction of GTMAC moieties, the {[N+H]+[N+]}/[N] ratio increased from 0.47 on the CSb-pSBMA coating to 0.61 on the HTCC-b-pSBMA coating, whereas the [S]/[N] ratio decreased correspondingly from 0.30 to 0.26. Water contact angles on the pristine and modified surfaces were measured using the sessile drop method and the results are given in Table S2. The pristine silicone surface is hydrophobic with a contact angle of 109°, and modification of silicone with APTES resulted in a decrease in the contact angle to 85°. The CS-b-pSBMA and HTCC-b-pSBMA coatings improved the surface hydrophilicity significantly and the contact angle decreased to 34° and 36°, respectively. The HTCC-coated surface showed a relatively high contact angle of 51°. It has been reported that genipin crosslinked CS hydrogel showed a relatively higher contact angle compared to uncrosslinked CS hydrogel, since genipininduced crosslinked CS hydrogel is hydrated in a smaller extent compared to the uncrosslinked CS hydrogel [38]. The pSBMA coating is very hydrophilic with a contact angle of 10°, due to the zwitterionic polymer being fully hydrated in water. The lower contact angles of the CS-b-pSBMA and HTCC-b-pSBMA coatings compared to the HTCC coating is thus attributed to the SBMA moiety in the CS-b-pSBMA copolymer. The thickness of the HTCC-b-pSBMA, HTCC and pSBMA coatings is <1 lm (0.5–0.7 lm) (Fig. S4b–d). The HTCC coating (Fig. S4c) is thicker than the HTCC-b-pSBMA and pSBMA coatings (Fig. S4b and d). For the same weight of CS and CS-b-pSBMA polymers used in the preparation of the coating, there are more –NH2 groups in the former for reaction with genipin than the latter, leading to a higher degree of crosslinking and grafting of the CS chains onto the surface compared to the CS-b-pSBMA copolymer. Since the three coatings, HTCC-b-pSBMA, HTCC and pSBMA, have comparable thicknesses, the differences in their antibacterial property (as discussed below) are not likely to be due to coating thickness. 3.3. Bacterial colonization on silicone surface via different routes Surfaces of medical devices are subjected to possible colonization by bacteria which can be transmitted by contact with contaminated fluid, droplet and air. To investigate the effectiveness of the coatings against bacterial colonization, two representative assays were carried out. Biofilm formation on substrate immersed in bacterial suspension: Pristine and modified silicone sheet surfaces were immersed in culture medium inoculated with either E. coli, P. mirabilis, or P. aeruginosa at a concentration of 105 cells/mL for 24 h to study biofilm formation on the surfaces by these bacterial cells. Under this condition, the planktonic bacterial cells in the aqueous medium have to approach and attach on the surface to form colonies [1]. Fig. 3 shows the SEM images of pristine and modified silicone surfaces after the incubation period. The pristine silicone surfaces were readily colonized by these three microorganisms, although the biofilm coverage was different due to different biofilm formation capability of the bacterial species. Bacterial colonization was reduced on the HTCC-coated surface, but numerous bacterial clusters can still be observed. Quantitative counts of viable bacterial cells on the HTCC-coated surface shows that the HTCC coating reduced bacterial colonization by 89% for E. coli, 94% for
R. Wang et al. / Journal of Colloid and Interface Science 438 (2015) 138–148
143
Fig. 2. XPS N 1s core-level spectra of (a) pristine, (b) ATPES-, (c) CS-b-pSBMA-, (d) HTCC-b-pSBMA-, (e) HTCC-, and (f) pSBMA-modified silicone surfaces. CS-b-pSBMA- and HTCC-b-pSBMA-coated surfaces were prepared from CS-b-pSBMA copolymer with a [SBMA]/[CS] ratio of 0.92.
P. mirabilis, and 85% for P. aeruginosa compared to that on the pristine surface (Fig. 4). The positively charged N+H and N+ groups on the HTCC coating interact with the negatively charged bacterial cells, disrupt the cell membranes, which lead to cell death [19]. However, the positively charged groups also attract bacterial debris. In addition, the HTCC-coated surface is relatively hydrophobic (contact angle of 51°, Table S2), and hydrophobic–hydrophobic interaction between the substrate surface and surface of bacterial cells may increase debris deposition and accumulation of dead bacteria. As a result, the active groups in the coating will be blanketed, and planktonic cells from the medium can then attach and multiply on the surface [13]. On the other hand, the pSBMA coating effectively inhibited bacterial colonization over 24 h, as few cell clusters were found on the surface and the remaining bacteria exist mostly as single cells (Fig. 3). Bacterial colonization by the three microorganisms was reduced by >99% on the pSBMA-coated surface (Fig. 4). Two factors may contribute to the observed reduction in bacterial colonization: the bactericidal property of the quaternary N+ groups in the coating (each SBMA unit has a quaternary N+ group), and/or the hydrophilic property of the surface. The bactericidal property of pSBMA was evaluated from the minimum inhibitory concentrations (MICs) of pSBMA against E. coli, P. mirabilis, P. aeruginosa and S. aureus. For all these bacteria, the MIC values were determined to be >2048 mg/L. This value is much higher than the corresponding values for CS and HTCC (degree of substitution: 67%) (details in SI, Table S3). This is consistent with an earlier finding that the bactericidal activity of polymers containing sulfobetaine groups is much lower than antimicrobial polycations based on poly(trialkylvinylbenzylammonium chlorides) [39]. The presence of the –SO3 group in close proximity to the quaternary
N+ group may diminish the degree of interaction between the N+ group and the bacterial cell wall. On the other hand, the pSBMAcoated surface is highly hydrophilic (contact angle of 10°, Table S2), and this surface hydration layer serves as a physical and energy barrier to discourage bacterial adhesion on the surface [40]. Therefore, the antibacterial property of the pSBMA coating as shown in Figs. 3 and 4 is attributed primarily to its anti-adhesive nature rather than the bactericidal activity of its quaternary N+ groups. Fig. 3 shows that the CS-b-pSBMA coating is as effective as the pSBMA coating in reducing bacterial colonization, and the HTCC-b-pSBMA coating is the most effective in reducing bacterial colonization (P99.7% reduction in bacterial colonization by the three tested microorganisms compared to pristine silicone, Fig. 4). Thus, the high antibacterial efficacy of the HTCC-b-pSBMA coating resulted from the combination of an abundance of bactericidal quaternary ammonium groups in the HTCC polymer, and the anti-adhesive property of pSBMA, which prevented the deposition of bacterial cells and debris. From Figs. 3 and 4, it can be seen that the efficacy of the coatings against P. mirabilis biofilm formation after 24 h is the lowest. Thus, the efficacy of the coatings was further tested with P. mirabilis over a 72 h period. A thick P. mirabilis biofilm was observed on the pristine surface (Fig. S5a). The bacterial cells on the HTCCcoated surface also developed into a dense biofilm after 3 days of incubation (Fig. S5b). On the pSBMA-coated surface, many more bacterial cells were present after 3 days compared to the corresponding surface after 1 day (comparing Fig. S5c and Fig. 3). As discussed above, the pSBMA coating mainly relies on the hydration barrier to reduce bacterial attachment, and the bactericidal activity of SBMA is low. As a result, any bacterial cell that succeeds in
144
R. Wang et al. / Journal of Colloid and Interface Science 438 (2015) 138–148
Fig. 3. SEM images of in vitro bacterial colonization on pristine and modified silicone sheet surfaces after incubation in bacterial culture medium for 24 h (initial inoculum was 105 cells/mL). Scale bars represent 10 lm.
Fig. 4. Number of viable adherent bacterial cells per cm2 of substrate surface after incubation in bacterial culture medium for 24 h (initial inoculum was 105 cells/mL). ⁄ denotes significant difference with P < 0.01 compared with pristine silicone surface. #, + denote significant difference with P < 0.01 and P < 0.05, respectively, compared with HTCC-b-pSBMA-coated silicone surface.
breaching the hydration barrier and attaches on the pSBMA-coated surface will likely proliferate and form a colony over a longer duration. For the HTCC-b-pSBMA-coated surface, although there is an
increase in the number of adherent bacteria after 3 days compared with the corresponding surface after 1 day (comparing Fig. S5d and Fig. 3), the reduction in bacterial colonization is still substantial compared to the pristine surface (Fig. S5a) and also the pSBMAcoated surface (Fig. S5c) over the same period of incubation. These results confirmed that the bifunctional coating integrating antifouling pSBMA and bactericidal HTCC moieties showed better performance in resisting colonization by planktonic bacteria in aqueous medium than the corresponding antifouling pSBMA coating and bactericidal HTCC coating. Bacterial colonization on substrates exposed to bacterial aerosol: To simulate bacterial contamination from air and droplets, a bacterial PBS suspension (containing 108 cells/mL of E. coli, P. mirabilis, or P. aeruginosa) was sprayed onto the surface of pristine and modified silicone sheets. The contaminated surface was then incubated under growth agar for 24 h. SEM images of the surfaces after the incubation period are shown in Fig. 5. Numerous distinct clusters of bacteria can be observed on the pristine surfaces. The HTCC coating very effectively inhibited bacterial colonization as few bacterial clusters were observed on the surface. The number of viable bacterial cells on the HTCC-coated surface was reduced by 99% (Fig. 6). This demonstrates the efficacy of the positively charged groups of HTCC in killing the deposited bacterial cells upon contact.
R. Wang et al. / Journal of Colloid and Interface Science 438 (2015) 138–148
145
Fig. 5. SEM images of in vitro bacterial colonization on pristine and modified silicone sheet surfaces after spraying with bacterial PBS suspension (108 cells/mL) and subsequent incubation under solid growth agar for 24 h. Scale bars represent 10 lm.
Fig. 6. Number of viable adherent bacterial cells per cm2 of substrate surface after spraying with bacterial PBS suspension (108 cells/mL) and subsequent incubation under solid growth agar for 24 h. ⁄ and # denote significant difference with P < 0.01 compared with pristine silicone surface and HTCC-b-pSBMA-coated silicone surface, respectively.
Fig. 7. FBG adsorption on pristine, and HTCC-, pSBMA-, HTCC-b-pSBMA-coated silicone surfaces after the substrates were treated with 1 mg/mL of FBG solutions for 4 h. ⁄, @ denote significant difference with P < 0.01 and P < 0.05, respectively, compared with pristine silicone surface. # denotes significant difference with P < 0.01 compared with HTCC-b-pSBMA-coated silicone surface.
146
R. Wang et al. / Journal of Colloid and Interface Science 438 (2015) 138–148
On the pSBMA-coated surface, the number of bacterial clusters was substantial, indicating the deposited bacterial cells were viable. These results are consistent with the MIC results which show that the bactericidal activity of quaternary N+ groups of pSBMA is much lower than those of HTCC. The CS-b-pSBMA coating reduced bacterial colonization, and further reduction in bacterial colonization was observed on the surface with HTCC-b-pSBMA coating (Fig. 5). Fig. 6 shows that incorporating the antifouling pSBMA moieties in almost equal proportion as HTCC in the bifunctional coating did not diminish the HTCC’s bactericidal activity against aerosolized bacteria.
From the above observations of bacterial colonization via two very different routes, it can be seen that coatings with pSBMA alone are effective in preventing planktonic cells in aqueous medium from approaching the surface and colonizing it but are not effective against aerosolized bacteria. On the other hand, coatings with HTCC alone can effectively kill aerosolized bacteria on contact but are not effective against biofilm formation by bacteria from aqueous medium over extended periods of time. The bifunctional HTCC-b-pSBMA coating combines the two complementary antibacterial mechanisms, and shows high efficacy in inhibiting bacterial colonization under both conditions.
Fig. 8. SEM images of adherent platelets on (a) pristine, (b) HTCC-, (c) pSBMA-, (d) HTCC-b-pSBMA-, (e) HTCC-b-pSBMA-Hep-coated surfaces, and (f) HTCC-b-pSBMA-Hepcoated surface after aging in PBS for 7 days. Insets show a higher magnification of platelet on the corresponding surfaces. Scale bars represent 10 lm in main images, and 5 lm in insets. (g) Platelet adhesion on the modified silicone surfaces relative to that on the pristine silicone surface, and PRT on pristine and modified silicone surfaces. ⁄, @ denote significant difference with P < 0.01 and P < 0.05, respectively, compared with pristine silicone surface. #, + denote significant difference with P < 0.01 and P < 0.05, respectively, compared with HTCC-b-pSBMA-coated silicone surface.
R. Wang et al. / Journal of Colloid and Interface Science 438 (2015) 138–148
3.4. Protein adsorption Implanted medical devices are exposed to body fluids or tissues containing proteins. FBG is a major protein in blood plasma and when a surface first comes to contact with blood, adsorption of FBG rapidly occurs [41]. Subsequent consequences such as bacterial adhesion and platelet and cell adhesion induced by the adsorbed FBG may adversely affect the functionality of the device [41,42]. Fig. 7 compares the amount of FBG adsorbed on the pristine and modified surfaces from 1 mg/mL protein solution after 4 h. The pristine silicone surface adsorbed 3.1 lg/cm2 of FBG. After HTCC coating on the surface, the adsorption of FBG increased by 29%, and protein aggregates were observed on the HTCC surface (Fig. S6). The isoelectric point of FBG is 5.1 [43], and thus FBG is negatively charged under the experimental condition in this study (10 mM CPBS, pH 7.4). Electrostatic interactions between the FBG molecules and positively charged HTCC coating may enhance protein adsorption, and also change the conformation of the adsorbed FBG [44]. As expected, the pSBMA coating with its surface hydration layer [40] is effective in decreasing FBG adsorption (86% reduction compared to pristine surface). The HTCC-b-pSBMAcoated surface decreased FBG adsorption by 60%, indicating that integration of SBMA moiety in the copolymer surface coating at a [SBMA]/[CS] ratio of 0.92 counteracted the effect of HTCC, and improved the surface antifouling property. It has been reported that adsorbed FBG promotes bacterial adhesion on the surface [45–47]. Hence, bacterial adhesion and biofilm formation by P. mirabilis on pristine silicone surface and HTCC-b-pSBMA-coated surface which were pretreated with FBG solution were further investigated. An increase in the number of adherent P. mirabilis cells was observed on the pristine surface after it was pretreated with FBG (comparing Fig. S7a and c). It has been reported that pre-adsorbed FBG on surface promotes S. aureus adhesion due to the expression of FBG binding protein (clumping factor) by the S. aureus cells [47,48]. However, for P. mirabilis, fimbriae play an important role in the bacterial adhesion process [49]. The presence of blood components in the medium is reported to enhance expression of fimbriae by P. mirabilis cells [50]. Therefore, it is likely that the adsorbed FBG increases fimbriae expression by the cells and subsequently promotes P. mirabilis adhesion on the surface. The number of adherent P. mirabilis cells also increased on the HTCC-b-pSBMA-coated surface pretreated with FBG, compared to the corresponding surface without FBG pretreatment (comparing Fig. S7b and d). With the increase in number of adherent bacteria on both the pristine and HTCC-b-pSBMAcoated surfaces after FBG pretreatment, it is expected that biofilm formation would also be enhanced (comparing Fig. 3 and Fig. S8b). Nevertheless, reduction in biofilm formation by P. mirabilis on the FBG-treated HTCC-b-pSBMA-coated surface was substantial compared to the pristine surface after similar treatment (Fig. S8a).
147
was prepared. Since CVCs are expected to stay in the body for a median duration of 7 days [54], the HTCC-b-pSBMA-Hep coating was incubated in PBS for 7 days. The hemocompatibility of the surfaces before and after incubation in PBS was evaluated using platelet adhesion and PRT assays. Fig. 8a to f show the SEM images of the adherent platelets on pristine and modified silicone surfaces after incubation for 1 h. As can be seen, the pristine silicone surface and HTCC-coated surface are prone to platelet adhesion, and the adherent platelets showed pseudopodia and spreading, indicating that they were activated by these surfaces (Fig. 8a and b). The small white dots on the HTCC-coated surface in Fig. 8b are probably adsorbed proteins and biomolecules from PRP, since the HTCC coating promotes protein adsorption and formation of aggregates on the surface (Fig. S6d). Platelet adhesion on the pSBMA-, HTCCb-pSBMA-, and HTCC-b-pSBMA-Hep-coated surfaces was greatly inhibited, and the adherent platelets were round and not activated (Fig. 8c–f). A quantitative comparison of the relative number of adherent platelets on the different surfaces is given in Fig. 8g. An increase in platelet adhesion by 12% was observed on the HTCC-coated surface compared to the pristine surface. In contrast, the pSBMA-coated surface reduced platelet adhesion by 95%. This is consistent with an earlier finding that pSBMA coating can effectively reduce platelet adhesion [18]. As expected, with the presence of HTCC in the HTCC-b-pSBMA coating, the number of adherent platelets was more than on the pSBMA coating. However, platelet adhesion on the HTCC-b-pSBMA-coating is 95% lower than the HTCC coating. The extent of platelet adhesion correlates with FBG adsorption since FBG plays an important role in mediating the process of platelet adhesion [41]. The heparin-loaded coating, HTCC-b-pSBMA-Hep, decreased platelet adhesion on the surface to the same level as the pSBMA coating. After incubation in PBS for 7 days, 0.4 ± 0.1 lg/cm2 heparin remained in the HTCC-bpSBMA-Hep coating, and with this amount of heparin, the coating still achieved 91% reduction in platelet adhesion. The PRT results obtained with the pristine and modified surfaces are shown in Fig. 8g. The PRT was 12 min and 10 min on the pristine silicone surface and HTCC-coated surface, respectively. With the pSBMA coating, the PRT increased significantly to 21 min. The HTCC-bpSBMA coating resulted in a PRT of 15 min. On the surface coated with HTCC-b-pSBMA-Hep, the PRT further increased to 23 min. Even after 7 days of incubation of the HTCC-b-pSBMA-Hep coating in PBS, a PRT of 17 min was achieved on this surface. Bacterial colonization assays carried out with HTCC-b-pSBMA-Hep-coated surface showed that this coating inhibited bacterial colonization
3.5. Heparin-loaded coating for blood-contacting surface Since the bifunctional coating demonstrates favorable antifouling and antibacterial properties, it may potentially be useful for preventing bacterial colonization of medical devices. A promising application of such coating may be for central venous catheters (CVCs), where infections associated with these devices result in significant patient morbidity, mortality and healthcare cost [51]. Due to the blood-contacting nature of CVCs, the surface coating also has to be blood compatible. Heparin has been reported to have strong anticoagulant activity [52], and the high density of negative charges in the heparin molecule allows it to be loaded in the HTCCb-pSBMA coating via ionic interactions with the positive charges in the polymer [53]. Using this method, a heparin-loaded coating (HTCC-b-pSBMA-Hep) with a heparin loading of 2.2 ± 0.3 lg/cm2
Fig. 9. Viability of 3T3 fibroblast cells incubated with pristine and modified silicone sheets for 24 h and 72 h. MTT assay was employed to evaluate cell viability through the measurement of mitochondrial activity. Viability is expressed as a percentage relative to the result obtained with the non-toxic control (3T3 fibroblast cells incubated in the absence of silicone sheet).
148
R. Wang et al. / Journal of Colloid and Interface Science 438 (2015) 138–148
by S. aureus, a common pathogen related to bloodstream infections, as effectively as the HTCC-b-pSBMA coating under both conditions (in aqueous medium and from bacterial aerosol) (Fig. S9). Therefore, it can be concluded that the heparin loaded into the HTCCb-pSBMA coating did not compromise its antibacterial property, and such coating is promising for blood-contacting catheter surfaces which have to be both antibacterial and hemocompatible. 3.6. Cytotoxicity assay Since implanted medical devices are in contact with body tissue, it is crucial that the surface coatings are non-cytotoxic. MTT assay, which measures mitochondrial activity, was used to evaluate the cytotoxic properties of the surfaces. The direct contact method was used, whereby the silicone surfaces were contactincubated on top of a pre-seeded fibroblast cell layer [28]. Fig. 9 shows that the pristine silicone sheet and the modified silicone sheets (CS-b-pSBMA-, HTCC-b-pSBMA-, and HTCC-b-pSBMA-Hepcoated) posed no significant cytotoxicity to the mammalian cells over 24 h and 72 h of contact-incubation. Therefore, it can be concluded that the coatings are non-cytotoxic and can be potentially used for in vivo applications. 4. Conclusion A bifunctional coating with both antifouling and bactericidal moieties was prepared by grafting a CS-b-pSBMA copolymer with a [SBMA]/[CS] ratio of 0.92 onto an aminolyzed silicone surface using genipin-induced crosslinking. Subsequent treatment of the coating with GTMAC resulted in a surface {[N+H]+[N+]}/[N] ratio of 0.61. The efficacy of the so-obtained coating against bacterial colonization on surfaces immersed in bacterial suspension or exposed to bacterial aerosol were evaluated and compared with the antifouling pSBMA coating and the bactericidal HTCC coating. The pSBMA coating was not effective against aerosolized bacterial cells, whereas the HTCC coating has limited capability in preventing bacterial colonization in aqueous medium. The bifunctional coating, which combines the advantages of the two complementary mechanisms, inhibited bacterial colonization by approximately two orders of magnitude under both conditions compared with the pristine silicone surface, and it also reduced protein adsorption significantly. The loading of heparin in the bifunctional coating improved the surface hemocompatibility and did not compromise the antibacterial properties. Cytotoxicity assay confirmed that the HTCC-b-pSBMA coating does not pose significant cytotoxicity to mammalian cells. Therefore, the HTCC-b-pSBMA coating offers a promising strategy for inhibiting bacterial adhesion and colonization as well as improving hemocompatibility of medical device surfaces. Acknowledgment This work was financially supported by the National Medical Research Council of Singapore Grant NMRC/BnB/12sep040. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcis.2014.09.070. References [1] M. Katsikogianni, Y.F. Missirlis, Eur. Cells Mater. 8 (2004) 37. [2] R.M. Donlan, Emerg. Infect. Dis. 7 (2001) 277. [3] J. Hasan, R.J. Crawford, E.P. Ivanova, Trands Biotechnol. 31 (2013) 295.
[4] G. Cheng, Z. Zhang, S.F. Chen, J.D. Bryers, S.Y. Jiang, Biomaterials 28 (2007) 4192. [5] M.L. Hawkins, F. Faÿ, K. Rénel, I. Linossier, M.A. Grunlan, Biofouling 30 (2014) 247. [6] J. Wu, W. Lin, Z. Wang, S. Chen, Y. Chang, Langmuir 28 (2012) 7436. [7] R.L. Meyer, A. Arpanaei, S. Pillai, N. Bernbom, J.J. Enghild, Y.Y. Ng, L. Gram, F. Besenbacher, P. Kingshott, Colloids Surf. B 102 (2013) 504. [8] M.R. Nejadnik, H.C. van der Mei, W. Norde, H.J. Busscher, Biomaterials 29 (2008) 4117. [9] L. Ferreira, A. Zumbuehl, J. Mater. Chem. 19 (2009) 7796. [10] J.C. Tiller, C.J. Liao, K. Lewis, A.M. Klibanov, Proc. Natl. Acad. Sci. USA 98 (2001) 5981. [11] M.D.P. Willcox, E.B.H. Hume, Y. Aliwarga, N. Kumar, N. Cole, J. Appl. Microbiol. 105 (2008) 1817. [12] R.X. Chen, N. Cole, M.D.P. Willcox, J. Park, R. Rasul, E. Carter, N. Kumar, Biofouling 25 (2009) 517. [13] A.M. Klibanov, J. Mater. Chem. 17 (2007) 2479. [14] N. Aumsuwan, R.C. Danyus, S. Heinhorst, M.W. Urban, Biomacromolecules 9 (2008) 1712. [15] S. Yuan, D. Wan, B. Liang, S.O. Pehkonen, Y.P. Ting, K.G. Neoh, E.T. Kang, Langmuir 27 (2011) 2761. [16] A.K. Muszanska, E.T.J. Rochford, A. Gruszka, A.A. Bastian, H.J. Busscher, W. Norde, H.C. van der Mei, A. Herrmann, Biomacromolecules 15 (2014) 2019. [17] R. Lalani, L. Liu, Biomacromolecules 13 (2012) 1853. [18] R.S. Smith, Z. Zhang, M. Bouchard, J. Li, H.S. Lapp, G.R. Brotske, D.L. Lucchino, D. Weaver, L.A. Roth, A. Coury, J. Biggerstaff, S. Sukavaneshvar, R. Langer, C. Loose, Sci. Transl. Med. 4 (2012) 153ra132. [19] E.I. Rabea, M.E.T. Badawy, C.V. Stevens, G. Smagghe, W. Steurbaut, Biomacromolecules 4 (2003) 1457. [20] W. Sajomsang, Carbohydr. Polym. 80 (2010) 631. [21] F. Ganji, M.J. Abdekhodaie, Carbohydr. Polym. 74 (2008) 435. [22] S.M. Alipour, M. Nouri, J. Mokhtari, S.H. Bahrami, Carbohydr. Res. 344 (2009) 2496. [23] G.A. Diaz-Quijada, D.D.M. Wayner, Langmuir 20 (2004) 9607. [24] F.J. Xu, Y.L. Li, E.T. Kang, K.G. Neoh, Biomacromolecules 6 (2005) 1759. [25] R. Wang, K.G. Neoh, Z.L. Shi, E.T. Kang, P.A. Tambyah, E. Chiong, Biotechnol. Bioeng. 109 (2012) 336. [26] L. Cen, K.G. Neoh, E.T. Kang, Langmuir 19 (2003) 10295. [27] M. Li, K.G. Neoh, E.T. Kang, T. Lau, E. Chiong, Adv. Funct. Mater. 24 (2014) 1631. [28] International Organization for Standardization (ISO), Biological evaluation of medical devices – part 5: Tests for in vitro cytotoxicity. ISO 10993–5:2009(e), 2009, 1. [29] S.C. Hsu, T.M. Don, W.Y. Chiu, Polym. Degrad. Stab. 75 (2002) 73. [30] Z.L. Shi, K.G. Neoh, E.T. Kang, W. Wang, Biomaterials 27 (2006) 2440. [31] S.G. Chen, S.J. Chen, S. Jiang, Y.M. Mo, J.X. Luo, J.N. Tang, Z.C. Ge, Colloids Surf. B 85 (2011) 323. [32] I.F. Amaral, P.L. Granja, M.A. Barbosa, J. Biomater. Sci. Polym. Ed. 16 (2005) 1575. [33] S.H. Hsu, C.H. Lin, C.S. Tseng, Biofabrication 4 (2012) 015002. [34] R. Wang, K.G. Neoh, E.T. Kang, P.A. Tambyah, E. Chiong, J. Biomed. Mater. Res. B (2014), http://dx.doi.org/10.1002/jbm.b.33230. [35] M. Li, K.G. Neoh, L.Q. Xu, R. Wang, E.T. Kang, T. Lau, D.P. Olszyna, E. Chiong, Langmuir 28 (2012) 16408. [36] R.A.A. Muzzarelli, Carbohydr. Polym. 77 (2009) 1. [37] A.H. Wang, Y. Cui, J.B. Li, J.C.M. van Hest, Adv. Funct. Mater. 22 (2012) 2673. [38] B.S. Liu, C.H. Yao, S.S. Fang, Macromol. Biosci. 8 (2008) 432. [39] M. Ward, M. Sanchez, M.O. Elasri, A.B. Lowe, J. Appl. Polym. Sci. 101 (2006) 1036. [40] S.F. Chen, L.Y. Li, C. Zhao, J. Zheng, Polymer 51 (2010) 5283. [41] W.B. Tsai, J.M. Grunkemeier, T.A. Horbett, J. Biomed. Mater. Res. 44 (1999) 130. [42] H. Chen, L. Yuan, W. Song, Z. Wu, D. Li, Prog. Polym. Sci. 33 (2008) 1059. [43] V.P. Hoven, V. Tangpasuthadol, Y. Angkitpaiboon, N. Vallapa, S. Kiatkamjornwong, Carbohydr. Polym. 68 (2007) 44. [44] I. Van De Keere, R. Willaert, A. Hubin, J. Vereecken, Langmuir 24 (2008) 1844. [45] Y.H. An, R.J. Friedman, J. Biomed. Mater. Res. B 43 (1998) 338. [46] C. Tedjo, K.G. Neoh, E.T. Kang, N. Fang, V. Chan, J. Biomed. Mater. Res. A 82 (2007) 479. [47] Y. Wang, G. Subbiahdoss, J. de Vries, M. Libera, H.C. van der Mei, H.J. Busscher, Biofouling 28 (2012) 1011. [48] C. Wolz, D. McDevitt, T.J. Foster, A.L. Cheung, Infect. Immun. 64 (1996) 3142. [49] S.M. Jacobsen, D.J. Stickler, H.L.T. Mobley, M.E. Shirtliff, Clin. Microbiol. Rev. 21 (2008) 26. [50] R.K. Latta, M.J. Schur, D.L. Tolson, E. Altman, Can. J. Microbiol. 44 (1998) 896. [51] N.P. O’Grady, M. Alexander, L.A. Burns, E.P. Dellinger, J. Garland, S.O. Heard, P.A. Lipsett, H. Masur, L.A. Mermel, M.L. Pearson, I.I. Raad, A. Randolph, M.E. Rupp, S. Saint, the Healthcare Infection Control Practices Advisory Committee, Guidelines for the prevention of intravascular catheter-related infections, 2011. [52] J. Hirsh, R. Raschke, T.E. Warkentin, J.E. Dalen, D. Deykin, L. Poller, Chest 108 (1995) 258S. [53] K. Kamin´ski, K. Zazakowny, K. Szczubiałka, M. Nowakowska, Biomacromolecules 9 (2008) 3127. [54] O. Traoré, J. Liotier, B. Souweine, Crit. Care. Med. 33 (2005) 1276.