Active Antibacterial and Antifouling Surface Coating via a Facile One

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Active Antibacterial and Antifouling Surface Coating via a Facile One-step Enzymatic Cross-linking Changzhu Wu, Karin Schwibbert, Katharina Achazi, Petra Landsberger, Anna Gorbushina, and Rainer Haag Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01527 • Publication Date (Web): 05 Dec 2016 Downloaded from http://pubs.acs.org on December 7, 2016

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Active Antibacterial and Antifouling Surface Coating via a Facile One-step Enzymatic Cross-linking

Changzhu Wu,†,⊥ Karin Schwibbert, §,* Katharina Achazi,† Petra Landsberger,§ Anna Gorbushina,§ Rainer Haag †,*

†

Institut für Chemie und Biochemie, Freie Universität Berlin, Takustraße 3, 14195 Berlin,

Germany §

⊥

Federal Institute for Materials Research and Testing (BAM), 12200 Berlin, Germany

Chair of Molecular Biotechnology, Institute of Microbiology, Technische Universität

Dresden, Zellescher Weg 20b, 01217 Dresden, Germany

To whom correspondence should be addressed. Email: [email protected]; [email protected]

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ABSTRACT: Prevention of microbial contamination of surfaces is one of the biggest challenges for biomedical applications. Establishing a stable, easily produced, highly antibacterial surface coating offers an efficient solution but remains a technical difficulty. Here, we report on a new approach to create an in-situ hydrogel film-coating on glass surfaces made by enzymatic cross-linking under physiological conditions. The cross-linking is catalyzed by horseradish peroxidase (HRP)/glucose oxidase (GOD)-coupled cascade reactions in the presence of glucose and results in 3D dendritic polyglycerol (dPG) scaffolds bound to the surface of glass. These scaffolds continuously release H2O2 as long as glucose is present in the system. The resultant polymeric coating is highly stable, bacterial-repellent, and functions under physiological conditions. Challenged with high loads of bacteria (OD540 = 1.0), this novel hydrogel and glucose-amended coating reduced the cell viability of Pseudomonas putida (Gram-negative) by 100% and Staphylococcus aureus (Gram-positive) by ≥ 40%, respectively. Moreover, glucose-stimulated production of H2O2 by the coating system was sufficient to kill both test bacteria (at low titres) with > 99.99% efficiency within 24 h. In the presence of glucose, this platform produces a coating with high effectiveness against bacterial adhesion and survival that can be envisioned for the applications in the glucose-associated medical/oral devices. KEYWORDS: Antifouling, antibacterial, enzymatic cross-linking, dendritic polyglycerol, surface coating


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INTRODUCTION Hospital-acquired bacterial infections are a major cause of morbidity and mortality and consequently a constant threat to patient safety 1. In clinics, even simple oral operations can infect patients and further develop into a number of systemic diseases including cardiovascular disease, bacterial pneumonia, diabetes mellitus, and low birth weight 2. Often these infections are traceable to contaminated medical devices and surgical instruments, which sometimes cause an unacceptably high infection rate. In the past decades, great efforts have been devoted to reduce the infection rate by widespread use of antibiotics on site. However, hospital-associated bacterial infections remain one of the biggest and fastest growing clinical challenges faced in the healthcare industry today. Despite these challenges, coating technologies are providing powerful ways to significantly improve material surfaces to avoid bacterial contamination 3. Typical improvements are achieved either by building an antifouling surface to repel bacterial attachment (the “biopassive” approach) or by killing bacteria via the contact with a bioactive surface (the “bioactive” approach) 4. For examples, grafting polymer brushes onto surfaces can produce hydrophilic, zwitterionic, or super hydrophobic coating that lowers bacterial adhesion

4-5

.

Alternatively, loading surfaces with antibacterial/toxic agents can suppress bacterial survival, in particular at the local surface area. Although these approaches have been successful, they are often limited because antifouling surfaces are sometimes specific to certain strains, for instance, polyethylene glycol (PEG) coating effectively repels hydrophilic strains but not hydrophobic ones 6. Furthermore, the antibacterial efficacy of bioactive coating decreases with time because the active substances are unstable and dilute into the environment 7. Due to these limitations, there have been very few attempts to combine the “bioactive” and “biopassive” strategies to achieve synergistic effects against bacteria. For examples, when Muszanska et al. fabricated surfaces with bifunctional polymers, i.e., pluronic (PEO-PPO3 - Environment ACS Paragon -Plus

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PEO)-lysozyme, by hydrophobic interactions, they observed anti-adhesive and antibacterial effects against Bacillus subtilis 5a. More recently, Yang and co-workers coated surfaces with block copolymers containing cationic polycarbonate and PEG segments by catechol chemistry, which resulted in significant antifouling/antibacterial effects against a number of strains 8. Despite of these successful attempts, there is a general need to further improve such surface coatings. Just to name a few examples, the surface stability needs to be enhanced when coating is made by physical interactions, and chemically bonded coating must avoid use of toxic reagents and should simplify the coating procedure for bio-related applications. Overall, there is a quest to establish a reliable and efficient methodology to simply and quickly coat surfaces under physiological conditions. Besides the pursuit for a simple and efficient approach, there is an increased interest in developing an environmentally friendly, non-toxic, and safe antibacterial coating 9. The immobilization of antibiotics and silver compounds onto surfaces is one of the most common ways that result in bioactive surfaces 10. However, the widespread use of these biocides causes serious bacterial resistance and environmental toxicity. Therefore, there is an urgent demand for safe and drug non-resistant coating. Different from antibiotics and silver ions, hydrogen peroxide (H2O2) is a non-toxic disinfectant that leaves no hazardous residuals, just oxygen and water after degradation 11. Since H2O2 has a broad spectrum of bactericidal activity and does not provoke resistance, it has been widely used in a large number of medical, food, and industrial applications 11. For example, the relatively high level of H2O2 can be commercially applied for antiseptic (882 – 1800 mM) on wound and other surface disinfection, and for dental disinfectant formation (118 – 294 mM), respectively

11

.

Unfortunately, H2O2

molecules are too small and unstable to be efficiently immobilized. But the in-situ generation of H2O2 on surfaces is feasible. For example, surfaces made of specific metal oxides like ZnO can produce H2O2 with an antibacterial effect 12 13, yet the level of H2O2 production is too low


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(≤ 10 µM)

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and leads to low antibacterial efficacy. Moreover, the use of metal oxides

inevitably releases soluble metal ions to the environment. Therefore, a remaining challenge is to build a “surface-bound system” that is bioinert and generates sufficient H2O2 without releasing harmful substances. Here, we describe a simple strategy to generate a stable, in-situ surface coating via an enzyme-coupled cascade cross-linking procedure that also generates H2O2 in the presence of glucose (Scheme 1). The enzymatic cross-linking takes place on dendritic polyglycerol (dPG) derivatives by horseradish peroxidase (HRP) and glucose oxidase (GOD). Since dPG, a multiple hydroxyl bearing polymer, is bioinert

14

, i.e., protein/cell repellent, PG-modified

surfaces can reduce bacterial attachment 15. At the same time, H2O2, generated by GOD from glucose, can actively kill bacteria. It is well known that glucose is the primary energy source in many living systems including bacteria, thus the antibacterial coating can be applied to medical devices and oral filling materials, where certain levels of glucose are constantly present in the applied environment. In general, compared to other traditional coating, this strategy has four distinct advantages: (1) a simple and mild coating approach achieved by one-step enzymatic cross-linking in physiological buffer; (2) in-situ coating functions in the presence of glucose, which makes it particularly interesting for biomedical applications; (3) an efficient H2O2-releasing surface system is produced directly form the starting materials; (4) the combined effect of disinfection and antifouling obtained by a single coating process. To the best of our knowledge, this is the first report of enzymatic cross-linking used to produce stable surface coating with a highly antibacterial and antifouling effect. Moreover, we demonstrate the diverse possibilities of active enzymes for both coating and antibacterial activity.


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contact angle (18.3 ± 3.4o)

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dead NH

HRP

live H2O2

NH

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H2O2

H2O

O2

GOD Glucose

Glucono-δ-lactone

Scheme 1. Design of the antifouling and antibacterial surface coating by enzymatic crosslinking: (A) modification of the surfaces with 3-(4-hydroxyphenyl) propionic acid functionalized dendritic polyglycerol (dPG-HPA) and (B) enzymatic cross-linking for antifouling and antibacterial activity.


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MATERIALS AND METHODS Materials: General chemicals were purchased from Sigma-Aldrich and used without further purification. Anhydrous pyridine and dimethylformamide (DMF) were obtained from Acros Organics. Horseradish peroxidase (HRP, type VI-A, approximately 250 U/mg lyophilized powder using pyrogallol) was bought from Sigma-Aldrich. Glucose oxidase (GOD, 146 U/mg) was kindly donated by Amano Enzyme (Nagoya, Japan). Microscope glass slides were purchased from Thermo Scientific. Microbial strains and growth conditions: Pseudomonas putida (P. putida) BAM 644 and Staphylococcus aureus (S. aureus) BAM 480 were obtained from the strain collection of Bundesanstalt für Materialforschung und –prüfung (BAM), Berlin, Germany. Escherichia coli (E. coli) W3110 was obtained from Freie Universität Berlin, Germany. Bacteria were grown aerobically at 30 °C (37 °C for E. coli); liquid cultures were grown while shaking at 140 rpm. Bacterial medium: P. putida and E. coli were grown in minimal medium MM63 containing 100 mM KH2PO4, 75 mM KOH, 15 mM (NH4)2SO4, 1.7 mM MgSO4x7H2O, 3.6 µM FeSO4x7H2O, 85 mM NaCl, pH 7.2. Unless otherwise stated, glucose concentration was 10 mM. For growth of S. aureus, protein hydrolyzate amicase (acid hydrolyzed casein, 0.5 g/L) and yeast extract (0,5 g/L) were added to the minimal medium MM63 16. CASO Agar (SigmaAldrich) was used to determine colony forming units (CFU) in antibacterial study. All bacterial strains were routinely maintained on Luria-Bertani agar plates (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, 15 g/L agar). All chemicals were purchased from SigmaAldrich, Germany. Growth of bacterial cultures was assessed by reading the optical density at 540 nm (Novaspec Plus, GE Healthcare Life Sciences, Germany). Synthesis and glass modification: Dendritic polyglycerol (dPG, Mw 6 kDa) was synthesized by anionic, ring-opening polymerization of glycidol with 1,1,1-trimethylolpropane as the 7 - Environment ACS Paragon -Plus

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initiator 17. The hydroxyl groups of dPG were modified with 5% amine groups (dPG-5%NH2) via three steps: mesylation, nucleophilic substitution, and reduction (Supporting Information). Some of these amino-functionalized dPG were fully coupled with 3-(4-hydroxyphenyl) propionic acid (HPA) as dPG-5%HPA in dried DMF solution. Others were partially converted as 2.5%HPA-dPG-2.5%NH2 that was performed in phosphate buffer (pH 6) 18. The residual 2.5% amine groups on dPG allowed their further reactions with 3-(triethoxysilyl)propyl isocyanate (TPI) via a stable urea bond formation in order to achieve 2.5%HPA-dPG2.5%TPI which could covalently attach phenolic derivatized dPG onto glass surfaces. The glass surface modification was similar to the literature

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, and the glass slides used for

microscopic study were 20 mm × 28 mm. Typically, glass slides were cleaned and chemically activated by piranha solution (H2SO4/H2O2 = 3:1 (v/v)) for 30 min in order to generate reactive hydroxyl groups on surfaces. The activated slides were immediately used for the silanization process by immersion into the freshly prepared methanolic solution containing 2.5%HPA-dPG-2.5%TPI (0.1 × 10-3 M cm-2 glass surface) and aqueous acetic acid (30% v/v 1 M) for 24 h at reflux condition. Afterwards, the slides were thoroughly rinsed with methanol in order to get rid of residual, unbound silanes and dried in a stream of Ar. Hydrogel film coating: After glass modification, the surfaces were immediately coated with hydrogel films via enzymatic cross-linking in the presence of HPR, GOD, glucose, and dPG5%HPA. Typically, the gel formation was conducted with a total volume of 50 µL precursor solutions containing 100 mg/mL polymers, 0.75 U HRP, 50 or 500 U GOD, and 40 mM glucose. Under such conditions, gelation usually took longer than 20 s. The precursor solutions were quickly transferred to the modified glass surfaces and capped with a thin cover glass until a stable film formed. Subsequently, the coated surfaces were cleaned by 70% ethanol solution twice prior to antibacterial characterization. The non-coated surface was prepared in the same manner but with unmodified bare glass 14b. 8 - Environment ACS Paragon -Plus

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Antibacterial assay: Overnight cultures of bacterial strains were diluted with fresh medium to the required optical density (OD540) 0.03, 0.1, 0.3, or 1.0. Samples were transferred into 100 mL glass flasks and bacterial culture (25 mL) with the indicated optical density was added. All flasks were incubated at 30 °C (37 °C for E. coli) for 24 h. After an initial sedimentation phase of 1 h without shaking, the incubation was done while shaking at 60 rpm. For experiments with “additional glucose”, 40 mM glucose was added to the flask in two portions (2 and 6 h after transfer of the sample to the bacterial culture). Thus, the final glucose concentration throughout these experiments was 50 mM. After 24 h, optical density of the bacterial cultures was measured. To test cell viability, CFU was determined as follows: a serial dilution up to 10-8 in MM63 lacking glucose was made from bacterial cultures; 100 µL of appropriate dilutions were plated onto CASO agar plates, each dilution was plated in triplicate; after incubation at 30 °C (37 °C for E. coli) for 24 h, the colonies were counted. Determination of glucose content: For assessment of the glucose content in the bacterial cultures, the Accu-Chek Aviva test system (Roche) was used. Microscopic characterization: Fixation of bacteria onto glass surfaces was performed in an aqueous formaldehyde (FA) solution (3.7 wt/wt %) overnight at 4 °C. Then the surfaces were thoroughly

washed

with

water

and

stained

with

4,6-diamidino-2-phenylindole-

dihydrochloride (DAPI) solution at a concentration of 10 µg/mL for 10 min in the dark. Afterwards, the surfaces were washed again with water, subsequently treated with mounting media (Mount Fluor from Biocyc GmbH & Co. KG) and covered with a glass cover slide. Confocal laser scanning microscopy (CLSM, Leica DMI6000CSB SP8) was used to quantify bacterial number on the surfaces and characterize their colonization (excitation: 405 nm, detection: 430 – 550 nm).


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RESULTS AND DISCUSSION Synthesis and surface coating. Glass was chosen as the model surfaces for enzymatic coating because of their easy visualization by microscopy and multiple possibilities for further modification. To coat glass with a dendritic polyglycerol (dPG) hydrogel film, both the glass and dPG have to be modified with 3-(4-hydroxyphenyl) propionic acid (HPA) cross-linkers 14a, 14b

. For this, dPG containing 5% HPA moieties (dPG-5%HPA) was synthesized in five

steps for the later film formation (Materials and Methods, Supporting Information). The obtained dPG-5%HPA was further modified with two additional steps to achieve compound 2 (Scheme 1A) that contained 2.5% HPA and 2.5% triethoxysilyl groups, respectively. With 2, the surfaces could be subsequently functionalized to have dPG-2.5%HAP cross-linkers on the top, which eventually resulted in a hydrophilic glass surface with a contact angle at 18.3 ± 3.6o (Scheme 1A). A one-step enzymatic coating of the dPG derivative was directly performed on the top of modified glass surfaces in the presence of HRP, GOD, and glucose (Materials and Methods). The resultant coating was stable in PBS buffer for more than two weeks. In contrast, the same hydrogel film formed on an unmodified (bare) glass surfaces was highly unstable (survived < 24h). This stability difference suggests that enzymatic cross-linking not only takes place on the top of the surfaces to form the gel film but also simultaneously occurs between the film and the modified glass surfaces. Such a high surface stability enables us to perform an extensive antibacterial study. Proof of principle. In order to explore the antibacterial efficacy and antifouling of surfaces, antibacterial assays and cell counts were performed, respectively, as described in Materials and Methods. Briefly, the surface samples were incubated together with bacterial cultures for 24 h. Then, cell density and viability of the bacterial cultures were determined to evaluate the antibacterial effect. At the same time, surface samples were taken from cultures and bacterial 10 - Environment ACS Paragon-Plus

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cells were visualized by confocal laser scanning microscopy (CLSM). The antifouling effect could be directly assessed by microscopic counting of DAPI-stained bacterial cells on surfaces. Pseudomonas putida (P. putida), a Gram-negative rod-shaped bacterium frequently found in the environment, was chosen for the study. Due to its ability to colonize moist and inanimate surfaces, P. putida is known to cause nosocomial infections and therefore is a significant human pathogen 5c. Initially, the antibacterial assay was carried out at a low bacterial optical density (OD540 = 0.03) that mimics slightly contaminated environments. The effect of a GODcoated surface on cell density was tested and compared to the uncoated (bare) and non-GODcoated glass, both of which were used as controls. After 24 h incubation, optical density of controls rose significantly (Figure S2). However, under the same experimental conditions, a coated surface containing 50 U GOD activity (50U-GOD coating) resulted in a 25-fold lower optical density. These findings demonstrate that the GOD coating is the determining factor in suppressing bacterial growth. Cell viability of bacterial cultures after 24 h exposure to differently coated surfaces were determined and the results were expressed as colony forming units (CFU) 20. Figure 1A shows that a high and almost equal number of CFU was observed from cultures after contact with bare glass and non-GOD-coated surfaces, which suggests that non-GOD coating could not suppress bacterial growth. In contrast, no CFU was detected when a GOD-coated surface was used throughout the experiment. These findings demonstrate that the GOD coating has a strong antibacterial effect against P. putida and prove that the remaining bacterial mass in the cultures that had been determined by measurement of the optical density (Figure S2) actually consisted of dead cells. According to our previous work 14b, the antibacterial activity was due to the GOD-catalyzed release of H2O2 from bacterial medium where 10 mM glucose had been supplied. Although hydrogen peroxide (H2O2) is highly toxic to bacteria, it does not 11 - Environment ACS Paragon-Plus

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contribute to immediately antibiotic resistance

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, and thus serves as a perfect disinfection

agent in many biomedical applications.

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0 oat i Dc

ng

s glas

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s g la s

Figure 1. Antibacterial and antifouling effects of GOD-coated surfaces 24 h after exposure to P. putida at low initial OD (OD540 = 0.03). (A) Cell viability of bacterial cultures after contact with different surfaces, determined by colony counting and expressed as percentage of controls (bacterial cultures in contact with bare glass) and (B) bacterial number on different surfaces determined by CLSM and expressed as percentage of controls (bacterial number on bare glass). 4 cm2 surface area was analyzed for each surface. To assess the antifouling effect of the coating, the surface samples were analyzed microscopically. Representative CLSM images are shown in Figure S3 for controls and 50 U GOD-coated surfaces, respectively. Interestingly, although almost identical number of bacteria in control cultures was found (Figure 1A and S2), the number on the surfaces of nonGOD-coated glass was 13 times lower than on bare glass (Figure 1B). Therefore, dPG-coated surfaces themselves possess antifouling properties, which is in line with previous reports 15b

15a,

. Coating with 50U-GOD resulted in an almost clean surface (Figure 1B and S3), a 23-fold

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diminution in bacteria over non-GOD-coated surfaces, highlighting the synergistic effect of antifouling (bio-passive) and disinfection (bioactive). Furthermore, enlarged CLSM images showed that the bacteria on GOD-coated surfaces were much smaller than on the controls, pointing to the stresses inherent to H2O2-producing 50U GOD-coated surfaces (Figure S4). Antibacterial dependency on cell and GOD loads, glucose availability and exposure time. Although 50U-GOD coating is highly antibacterial against low bacterial loads, its application on high bacterial contamination environment is still unclear. Therefore, we further exposed the surfaces to the increased density of P. putida culture ranging from OD540 0.03 to 1.0 over 24 h. Figure 2A shows that 50 U GOD-coated surfaces significantly reduced P. putida levels as compared to non-GOD-coated surfaces, regardless of the initial optical density. Reduction in bacterial number, however, was more pronounced when low initial optical density was used, and only 56 % decrease was observed when bacteria at an OD540 = 1.0 was applied. These findings point out the dependency of antibacterial efficacy on initial bacterial loads. However, even at high initial optical density, the application of GOD coating still led to a severe growth suppression of P. putida. To improve the antibacterial efficiency of 50U-GOD coating, especially at high bacterial loads, we further investigated the conditions that led to a more efficient production of H2O2 that was the bioactive agent in this system. As shown in Scheme 1, the release of H2O2 was directly dependent on the presence of GOD activity and the substrate glucose. Therefore, we increased GOD loads on the surfaces by a factor of 10 from 50 U to 500 U, and meanwhile we monitored the glucose content over time during the antibacterial assay that was performed at high initial bacterial loads of OD540 = 1.0. The glucose profile (Figure 2B) demonstrates that the higher GOD loads of 500 U contributed to the accelerated glucose consumption compared to lower GOD loads and was quickly exhausted after only 4.3 h. With the depletion


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of the glucose supply, H2O2 production came to a standstill and the remaining bacteria survived. Further experiments were carried out to assess the influence of a high GOD load in combination with increased glucose supply on the antibacterial efficacy. The antibacterial assay was performed at high bacterial loads of OD540 = 1.0. Two different GOD loads (50 U or 500 U) with or without extra glucose were also tested. Ten-fold reduction in CFUs was observed 24 h after contact with a 50 U GOD-coated surface (Figure 2C). Increasing the GOD loads to 500 U further increased the antibacterial efficacy by a factor of 3. Glucose addition remarkably enhanced the antibacterial efficacy for both the 50 U and 500 U GOD-coated surfaces. Viable bacteria were not found when 500 U GOD-coated surfaces were used with extra glucose. These results are evidence of a sustained improvement of the antibacterial efficacy due to an increase of the GOD loads and the sufficient continuous glucose supply. Obviously, competition for glucose between GOD and dense bacterial culture was mitigated by extra glucose. The antibacterial assay was also carried out by a shortened incubation time of only 5 h instead of 24 h to assess the usability of the GOD-coated surfaces in a short-term application. Figure 2C reveals that both GOD coating (50 U and 500 U) was very efficient in disinfecting bacteria, even after only 5 h incubation. Moreover, under conditions with 500 U GOD-coated surfaces and additional provision of glucose, the antibacterial efficacy of the surfaces within 5 h was as pronounced as within 24 h, achieving complete loss of bacterial viability.


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g e se ng co s oatin gluco oa oati Dc Dc + glu OD c + -GO 0U-GO oating -G ting n a U o o 0 c n 5 50 Dc OD -GO U- G 50 U 5 00 ting

A

Figure 2. Dependency of antibacterial efficacy on cell loads, glucose availability, GOD surface loads and application time. (A) OD540 of P. putida cultures 24 h after incubation with 50 U GOD-coated surfaces and different initial bacterial loads (as percentages of controls - P. putida cultures incubated with non-GOD coated surfaces). (B) Glucose consumption profile of bacterial cultures (OD540 = 1.0) in contact with different surfaces (as percentages of controls - bacterial cultures in contact with non-GOD coating). (C) Cell viability of bacterial cultures (OD540 = 1.0) after 5 or 24 h contact with different surfaces determined by colony counting and expressed as percentages of control values (bacterial cultures in contact with non-GOD coating). Antibacterial efficacy against other bacteria. To gain insight about the general antibacterial effectiveness, additional bacteria were included into the study. We started with a second Gram-negative bacterium, E. coli that is associated with many infected diseases, such as diarrhea or urinary tract infections

5b

. The antimicrobial assay was performed at low initial

bacterial loads (OD540 = 0.03). Optical density and CFU were determined after 24 h contact with the surfaces. As shown in Figure S5, the results obtained with E. coli are similar to those obtained with P. putida. Incubation for 24 h with a 50U-GOD coating led to a 30 times lower OD compared to the controls. Cell viability was completely lost, and no CFU could be detected from the bacterial culture. CLSM images confirms the high antifouling effects when E. coli was used as the test organism (Figure S6). Bacterial adherence to non-GOD-coated surfaces was less pronounced than bare glass because of the antifouling nature of dPG film on 15 - Environment ACS Paragon-Plus

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the top of the surfaces 15a, 15b. An almost cell-free surface could be again observed when GOD coating was applied, and residual cells displayed a reduced size, which was due to the stressful conditions on this surface (Figure S7). To test the generality of the forgoing observation, we expanded our antibacterial investigation to a Gram-positive bacterium, Staphylococcus aureus (S. aureus). This strain was chosen as the test strain because of its clinical relevance. It is known as a major pathogen of nosocomial infections and often associated with complicated infections such as endocarditis that causes patience prolonged hospital stay and antibiotic therapy. Therefore, it is pivotal to avoid clinical contamination with such pathogenic strain, which is also increasingly resistant to a number of antimicrobial agents 22. Antibacterial assays were carried out with initial bacterial loads ranging from OD540 0.03 to 1.0 and 50 U GOD-coated surfaces for 24 h. Although there was a remarkable reduction of optical density at low initial bacterial loads (OD540 = 0.03 and 0.1), the reduction was less pronounced when loads were increased to OD540 0.3 or 1.0 (Figure S8). This suggests that the growth suppressing effects of GOD coating was significantly weaker as compared to the Gram-negative test organisms, P. putida and E. coli (Figure 2A and S8). Nevertheless, we further evaluated bacterial viability in the assays with the lowest (OD540 = 0.03) and highest (OD540 = 1.0) bacterial loads. As shown in Figure 3A, 50U-GOD coating resulted in a 99.99% reduction of the CFU compared to the control surfaces (non-GOD coating and bare glass). This clearly demonstrates the highly antimicrobial efficacy of the coating against S. aureus. Even at high bacterial loads (OD540 = 1.0), the 50 U GOD-coated surfaces resulted in a 40% reduction of CFU compared to controls (Figure 3B). The findings with P. putida (Figure 2B) suggest this effect might be improved by increasing the GOD loads on the surfaces in combination with the addition of glucose.


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0.0008 ting coa

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e g se ing ing c os a ti n oat oat luco glu -c o Dc Dc + U +g 0 O O g g 50 -G -G ti n ti n non c oa c oa 50U OD OD G G U 50U 500

Figure 3. Antibacterial effects of different surfaces to S. aureus at (A) low loads (OD540 = 0.03) and (B) high bacterial loads (OD540 = 1.0). Cell viability of bacterial cultures after 24 h incubation with different surfaces determined by colony counting and expressed as percentages of controls (bacterial cultures incubated with non-GOD-coated glass).

However, the increase of GOD loads from 50 to 500 U did not further improve the antibacterial efficacy of surfaces (Figure 2C & 3B). A possible explanation might be due to the use of complex substance mixture in the nutrition medium that contains high amounts of phenol groups in the tyrosine residues of proteins. These phenol groups could be oxidized by HRP while constantly consuming the H2O2 level in bacterial culture as scheme 1 14a, 14b, thus offsetting the contribution from the higher GOD loads. This hypothesis was partially proven by the fact that the antibacterial efficacy was significantly improved when extra glucose was provided to ensure the sufficient production of H2O2 in the medium (Figure 3B). Nevertheless, GOD-coated surfaces also exhibited good antibacterial efficacy against the Gram-positive bacterium S. aureus, particularly at low bacterial loads.


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CONCLUSIONS In summary, an enzymatic cascade approach has been established for the in-situ preparation of bioinert as well as bioactive surfaces coatings. This process particularly benefits medicine related biointerfaces, for example, in-situ (re-)coating medical devices or implants. Furthermore, this coating makes full use of two enzymes (HRP and GOD) as catalysts to cross-link the polymer chains onto the surfaces and simultaneously generate H2O2 as a disinfection agent. The success of this design opens up new horizons in coating techniques, as it provides a good example for a multiple use of bioactive compounds for surface applications. In addition, the coating is built up by highly antifouling dPG scaffolds that resulted in significantly reduced bacterial adhesion to surfaces, as demonstrated by P. putida and E. coli. Since dPG bears multiple hydroxyl groups, it could allow surface modification by other active agents like anti-inflammatory drugs to provide the coating with multiple bio-functionality. Moreover, in a glucose-containing environment, this enzymatic bio-coating is highly antibacterial against low cell concentrations of both Gram-negative and positive strains. Remarkably, the disinfection efficacy is still pronounced in highly contaminated bacterial environments, which makes the coating strategy applicable for many medical devices and dental materials that are operated at constant level of glucose environment. ASSOCIATED CONTENT Supporting Information Synthesis of dPG, dPG-5%NH2, 2.5%NH2-dPG-5%NH2 (Scheme S1, S2, S3, S4, and Figure S1); Antifouling and antibacterial study on Pseudomonas putida (Figure S2, S3, and S4); Antifouling and antibacterial study on Escherichia coli (Figure S5, S6, and S7); Antibacterial study on Staphylococcus aureus (Figure S8). This material is available free of charge via the Internet at http://pubs.acs.org. 18 - Environment ACS Paragon-Plus

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AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; [email protected]

Fax: +49-30-838 53357 Notes The authors declare no competing financial interest、

Author Contributions C.W., K.S, and R.H. designed research; C.W., K.S., K.A., and P.L. performed research; C.W. and K.S analyzed data; and C.W. and K.S. wrote the paper.

ACKNOWLEDGEMENTS We thank DFG (SFB 765) and BMBF (Poly4Bio) for financial support. C.W. acknowledges DFG funding program (“Eigene Stelle” WU 814/1-1). We are grateful to Dorothea Thiele for experimental

help,

Dr.

Viola

Boenke

for

project

coordination,

Dr. Pamela

Winchester for proofreading of the manuscript, and Dr. William J. Broughton for his valued help with English editing.

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Active Antibacterial and Antifouling Surface Coating via a Facile One

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