Tyrosinase-Mediated Surface Coimmobilization of ... - ACS Publications

Article

Tyrosinase-mediated surface co-immobilization of heparin and silver nanoparticles for antithrombotic and antimicrobial activities Phuong Le Thi, Yunki Lee, Ho Joon Kwon, Kyung Min Park, Mi Hee Lee, Jong-Chul Park, and Ki Dong Park ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 30 May 2017 Downloaded from http://pubs.acs.org on May 30, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Tyrosinase-mediated surface co-immobilization of heparin and silver nanoparticles for antithrombotic and antimicrobial activities

Phuong Le Thi,1† Yunki Lee,1† Ho Joon Kwon,1 Kyung Min Park,2 Mi Hee Lee,3 Jong-Chul Park3 and Ki Dong Park1*

1

Department of Molecular Science and Technology, Ajou University, 5 Woncheon, Yeongtong,

Suwon, 443-749, Republic of Korea 2

Division of Bioengineering, College of Life Sciences and Bioengineering, Incheon National

University, Incheon 22012, Republic of Korea 3

Department of Medical Engineering, Yonsei University College of Medicine, Seoul 120-752,

Republic of Korea

† These authors contributed equally to this work

Submitted to ACS Applied Materials and Interfaces Interfaces

* Corresponding author Department of Molecular Science and Technology, Ajou University, 5 Woncheon, Yeongtong, Suwon 443-749, Republic of Korea. Tel.: +82 31 219 1846; E-mail address: [email protected]

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Thrombus and infections are the most common causes of failure of medical devices, leading to higher hospitalization costs and in some cases, patient morbidity. It is therefore necessary to develop novel strategies to prevent thrombosis and infection caused by medical devices. Herein, we report a simple, highly efficient strategy to impart antithrombotic and antimicrobial properties to substrates, by simultaneously immobilizing heparin and in situ synthesized silver nanoparticles (Ag NPs) via a tyrosinase-catalyzed reaction. This consists of tyrosinase oxidized phenolic groups of a heparin derivative (heparin grafted tyramine, HT) to catechol groups, followed by immobilizing heparin and inducing the in situ Ag NP formation onto poly(urethane) (PU) substrates. The successful immobilization of both heparin and in situ Ag NPs on substrates was confirmed by analyses of water contact angles, XPS, SEM and AFM. The sustained silver release as well as the surface stability was observed for 30 days. Importantly, the antithrombotic potential of the immobilized surfaces was demonstrated by a reduction in fibrinogen absorption, platelet adhesion, and prolonged blood clotting time. Additionally, the modified PU substrates also exhibited remarkable antibacterial properties against both Gram-positive and Gram-negative bacteria. The results from this work suggest a useful, effective, and time-saving method to improve simultaneous antithrombotic and antibacterial performances of a variety of substrate materials for medical devices.

Keywords: Heparin, silver nanoparticles, tyrosinase-mediated reaction, antithrombogenic activities, antibacterial activities.


Page 2 of 25

Page 3 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1. Introduction In the field of biomedical devices (e.g. blood-contacting devices, biosensors, and drug delivery vehicles), there are challenges restricting their applications, such as thrombus formation and microbial contamination.1-3 To overcome these clinical limitations, much attention has been directed towards surface immobilization. In this respect, heparin and silver nanoparticles (Ag NPs) are promising compounds due to their unique anticoagulant and antibacterial properties, respectively.4,5 In past few decades, various methods for surface immobilization have been developed, exploiting either covalent or non-covalent reactions such as physical blending, surface coating and grafting, as well as layer-by-layer assembly.4,6 Among them, mussel-inspired surface modification has recently become a facile method for covalent immobilization of biomolecules onto various inorganic/organic substrates.7 This method is based on the strong adhesion ability of oxidized catechol, a side chain of the amino acid 3,4-dihydroxy-L-phenylalanine (DOPA), to solid substrates under wet conditions.8,9 In addition, the catechol moiety has a reducing ability to induce the formation of noble metal nanoparticles. This finding has led to a green approach in the synthesis of stable nanoparticle cores surrounded by a polymerized shell using catecholconjugated polymers, which acted as both the reducing and stabilizing agents.10,11 Many studies reported the immobilization of biomolecules and nanoparticles onto surfaces via catechol oxidation of DOPA-based materials, but this method has not been used for a one-step immobilization of heparin and Ag NPs on one substrate.12-15 It is well-known that tyrosinase (Tyr) catalyzes the oxidation of phenolic groups to catechols/o-quinones and promotes the formation of new bonds, including reversible coordination bonds to metal ions and covalent bonds to nucleophiles.16 The Tyr-catalyzed oxidative reaction has been exploited in a wide range of fields, such as the in situ formation of hydrogels, drug delivery, biosensors, and protein immobilization.17,18 This reaction takes advantage of its high selectivity, resulting in a reduction of the reaction time compared to the oxidation of DOPA-modified polymers. Using Tyr oxidation, we have developed a facile and fast method for the surface-independent immobilization of phenol-containing bioactive molecules (e.g., mPEG−tyramine and RGD−Y peptide) to precisely control the surface properties for various applications.19,20



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. Schematic representation for molecular conversion from phenol to o-quinone by oxidation reaction of Tyr (a). One-step co-immobilization of heparin and Ag NPs on the PU surfaces via Tyr-mediated reaction is shown in (b). The heparin was conjugated with tyramine (termed “HT”) for the reaction. The phenol moieties of HT polymer are oxidized into o-quinone (or DOPA-quinone) by Tyr, subsequently inducing surface immobilization of Ag NPs (bi) as well as HT molecules (bii) on PU surfaces.

In this study, we report for the first time, the co-immobilization of heparin and Ag NPs on poly(urethane) (PU) surfaces, a biocompatible polymer with good mechanical properties and used extensively in biomedical devices,21 via the Tyr-mediated reaction. We hypothesized that the co-immobilization of heparin and Ag NPs will induce a dual-functional surface that can prevent both thrombosis and infection through the anticoagulant and anti-infective activities of heparin and Ag NPs, respectively. For this approach, we synthesized a phenol rich heparin derivative by grafting the polymer heparin with tyramine (HT polymer) and subsequently adding Tyr into a polymer solution in the presence of silver ions (Ag+). The phenol groups of HT are


Page 4 of 25

Page 5 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


converted to catechol groups via the Tyr-catalyzed oxidative reaction, thus providing the binding moieties for Ag NPs synthesis and immobilization of heparin/Ag NP hybrid biomaterials onto PU surfaces (Figure 1). To confirm the successful co-immobilization of heparin and Ag NPs, the modified surfaces were characterized by water contact angle measurements, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy coupled with energy dispersive X-ray (SEM/EDX) spectroscopy and atomic force microscopy (AFM). The structural stability and silver release from the surfaces were examined for 30 days. The in vitro antithrombogenic and antibacterial properties of the modified surfaces were analyzed in terms of protein absorption, blood clotting time, as well as platelet and bacterial adhesion.

2. Materials and methods 2.1. Materials Heparin sodium salt (molecular weight Mw = 12,000 – 15,000 g/mol), was obtained from Across Organic

(Geel,

Belgium).

1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide

(EDC),

N-hydroxysuccinimide (NHS), tyramine hydrochloride (TA), tris(hydroxymethyl)aminomethane (Tris), silver nitrate (AgNO3, ≥ 99%), tyrosinase from mushroom (Tyr, 3610 units/mg solid), toluidine blue O, N,N-dimethylacetamide (DMAc), tetrahydrofuran (THF), and sodium dodecyl sulfate (SDS) were obtained from Sigma Aldrich (St. Louis, MO, USA). Dulbecco’s modified

eagle

medium

(DMEM),

penicillin−streptomycin

(P/S),

and

trypsin/ethylenediaminetetraacetic acid (EDTA) were purchased from Gibco BRL (Grand Island, NY, USA). Fetal bovine serum (FBS) and Dulbecco’s phosphate buffered saline (DPBS) were purchased from Wisent (SaintBruno, QC, Canada). AlamarBlue® cell viability reagent was purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). EZ-Cytox enhanced cell viability assay kit (WST-1 assay reagent) was purchased from ITSBIO (Seoul, South Korea). Other chemicals and solvents were used without further puriﬁcation.

2.2. Synthesis and characterization of heparin-tyramine (HT) polymer HT polymer was synthesized through amide coupling between the amino groups of tyramine and the carboxylic groups of heparin, using the EDC/NHS-mediated reaction. Briefly, heparin (1 g) was dissolved in 100 mL of deionized water (DIW). EDC (1.52 g, 8 mmol) and NHS (0.575 g, 5 mmol) were subsequently added to the heparin solution to activate the heparin carboxylic acid



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

groups. The pH of the reaction solution was adjusted to 4.5-5 by 5 M HCl. After 1 h, TA (0.55 g, 3 mmol) was added and the resulting mixture was stirred at 25 °C for an additional 24 h. After the reaction, the solution was transferred to a dialysis membrane (MWCO = 3.5 kDa) and dialyzed against DIW for 3 days to remove any unreacted coupling reagents. The dialyzed solution was filtered and the HT polymer was finally obtained by lyophilization. The chemical structure of the HT polymer was characterized by 1H NMR spectroscopy (AS400, OXFORD instruments, UK). The substitution degree of tyramine (termed ‘phenolic content’) was determined by measuring the absorbance of the HT solution at 275 nm, using a UV-Vis spectrophotometer (V-750 UV/vis/NIR, Jasco, Japan).

2.3. Immobilization of HT and Ag NPs onto PU substrates The PU substrates (13 mm in diameter) were immersed in the HT solution (1 wt.%) dissolved in Tris buffer (0.01 M, pH 8.2), followed by the addition of AgNO3 (30 mM) and Tyr (0.4 kU/mL) solutions. After 1 h of incubation at 37 °C, the substrates were removed, cleaned thoroughly with DIW and SDS (1 wt.%) to remove any physical adsorbed matter, and then dried under nitrogen gas. The substrates immobilized with only HT were also prepared under the same conditions, without the AgNO3 addition.

2.4. Characterization of immobilized substrates. The surface wettability of the substrates was characterized by the sessile drop method using a Digidrop contact angle meter (GBX Instrumentation Scientifique, DGD fast 60, France). A droplet of ultrapure water was dropped onto the substrates and visualized using an equipped camera. The surface composition was analyzed by X-ray photoelectron spectroscopy (XPS) (Thermo Electron, K-Alpha, USA). The morphologies of the bare and HT/Ag NPs immobilized surfaces were analyzed by scanning electron microscopy (SEM, S-800, Hitachi) and atomic force microscopy (AFM, XE100, Park system).5 The size of Ag NPs was calculated from SEM images using ImageJ software. The amount of heparin immobilized on the PU surfaces was determined by the toluidine blue (TB) method. Briefly, the samples were immersed in 1 mL phosphate buffer saline (PBS 0.01 M, pH 7.4), and then 1 mL of TB solution (0.0025%) was added. After incubating for 30


Page 6 of 25

Page 7 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


min under gentle shaking at 37 °C, 2 mL of n-hexane was added and the mixture was shaken rigorously for 1 min. The substrates were removed from the solution and the absorbance of the aqueous phase was measured at 620 nm. The amount of immobilized heparin was calculated from a calibration curve constructed by varying the heparin concentration from 0 to 10 µg/mL.

2.5. Surface stability and silver release profile The stability of heparin immobilization was analyzed by TB method. Briefly, the immobilized HT/Ag NPs surfaces were immersed in PBS (pH 7.4) at 37 °C. After 5 and 10 days, the PBS solution was removed and the heparin amount remaining on PU surfaces was measured, using TB method as described above. The amount of silver released from the PU surfaces was determined by Inductively Coupled Plasma – Optical Emission Spectrometry (ICP-OES), using an OPTIMA 5300DV (PerkinElmer, USA). The substrates were immersed in 1 mL of PBS at 37 °C under gentle shaking. After 1, 2, 3, 4, 5, 6, 7, 10, 15, 20, and 30 days, the PBS solutions containing the released silver were collected and analyzed for their silver content.

2.6. In vitro antithrombogenic evaluation. 2.6.1. Protein adsorption Fibrinogen (from human plasma, Sigma) was used as the representative protein to evaluate the protein adsorption on surfaces. The PU substrates were incubated in 1 mL of PBS solution containing fibrinogen (100 µg/mL) at 37 °C for 1 h. After that, the substrates were washed thoroughly with PBS (3 times) to remove any unbound fibrinogen and were then treated with SDS (1 wt.%) for 30 min to collect the adsorbed fibrinogen. The amount of adsorbed fibrinogen was determined using the Micro BCATM protein assay kit (Thermo Scientific Pierce, Rockford, IL, USA).

2.6.2. Platelet adhesion The platelet adhesion on immobilized PU substrates was evaluated using fresh rat whole blood. All animal experiments were performed in accordance with the “Guide for the Care and Use of Laboratory Animals”. The Institutional Animal Care and Use Committee of the Yonsei



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Laboratory Animal Research Center (YLARC) approved all protocols (Permit #: 2015-0052). Animals were maintained in a pathogen-free facility at the YLARC. Fresh rat whole blood was obtained from male Sprague Dawley rats (weights: 350–400 g) from the abdominal vena cava using a syringe containing 0.109 M sodium citrate at a ratio of 4:1. For the anesthetic, 30 mg/kg of Zoletil (Boehringer Ingelheim Agrovet, Hellerup, Denmark) and 10 mg/kg of Rompun (Bayer, Toronto, Canada) were administered by intramuscular injection. The abdominal vena cava of the rats was exposed in order to collect whole blood using a syringe containing 0.109 M sodium citrate at a ratio of 4:1. To isolate high concentration platelet solutions, whole blood was centrifuged at 2,500 rpm for 5 min and the platelet rich plasma (PRP) was separated from the red blood cells and further centrifuged at 2,500 rpm for 5 min to obtain pellets of rich platelets. The PU substrates were sterilized by incubating in ethanol (75 wt.%) for 5 min and rinsing with PBS (0.01 M, pH 7.4) three times. After that, the substrates were immersed in 500 µL of PRP and incubated at 37 °C for 1 h. Subsequently, the samples were rinsed thoroughly three times with PBS and fixed with 2.5% glutaraldehyde for 2 h. The substrates were then lyophilized and coated with an ultra-thin layer of gold/platinum by ion sputtering (E1010, Hitachi, Tokyo, Japan). Finally, the platelet adhesion to the substrates were then observed using SEM. The adhered platelets were counted with images from randomly chosen sections.

2.6.3. Clotting time The thromboresistant potential of the immobilized PU substrates was assessed by the blood clotting analysis, using the fresh rat blood. Blood was collected from the rats using a sterilized syringe and immediately dropped onto the PU surfaces (50 µL/surface). The samples were incubated at 37 °C for predetermined times of 10, 20, 40, and 50 min. After the incubation time, the samples were incubated with 10 mL of DIW for an additional 5 min. The red blood cells not trapped in the thrombus were hemolyzed, releasing free hemoglobin into the water. The blood clotting was analyzed by measuring the absorbance of free hemoglobin in the water at 540 nm. The blood coagulation of HT/Ag NPs immobilized surfaces was evaluated through activated partial thrombin time (APTT), using platelet-poor plasma (PPP). PPP was obtained from rat fresh blood after centrifuging at 4000 rpm for 15 min. Briefly, the modified PU substrates were incubated with 100 µL of fresh PPP at 37 °C for 30 min. Then 50 µL of


Page 8 of 25

Page 9 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


incubated PPP was added into a test cup with 50 µL of APTT agent (Helena Laboratories, Beaumont, Texas, US) and incubated at 37 °C for 5 min. Thereafter, 50 µL of coagulation reagent 0.025 M CaCl2 was added and the timing began. The APTT is recorded as the time for a fibrin clot to form. The APTT values for fresh PPP and bare PU were also measured as control samples.

2.7. Antibacterial activity tests The antibacterial activity of immobilized PU substrates against Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Escherichia coli (E. coli) bacteria was examined. The bacterial strains were grown on the standard methods agar (Becton, Dickinson and Company, Sparks, MD, USA) at 37 °C for 18 h and were maintained at 4 °C prior to their in experiments. These initial cultures were incubated at 37 °C with shaking at 100 rpm for 18 h in a LuriaBertani (LB) broth (Becton, Dickinson and Company) and bacteria were harvested by centrifugation at 4000 rpm and suspended in LB medium. A suspension of bacteria (75 µL, 106 CFU/mL) was added to the samples and incubated for 24 h at 37 °C. Non-adherent bacteria were rinsed off by washing thoroughly with PBS five times. The rinsed samples were then incubated with 500 µL alamarBlue® solution (1 wt.% in LB) for 4 h at 37 °C. The optical density (O.D) was measured at 570 nm, using 600 nm as a reference wavelength to determine the viability of bacteria adhered on the surfaces. To evaluate the bactericidal efficacy, the substrates were incubated with a suspension of bacteria (500 mL, 106 CFU/mL) at 37 °C for 2 h. The substrates were then rinsed with PBS solution and fixed with 4% paraformaldehyde solution for 1 h. The morphologies of the adhered bacterial cells were observed by SEM.

2.8. In vitro cytotoxicity tests The cytotoxicity of modified PU substrates against human dermal fibroblasts (hDFBs) was conducted by means of the WST-1 assay. Extraction medium was obtained by incubating bare/immobilized PU substrates in DMEM medium for 24 h at 37 °C. hDFBs were seeded in 24 well-plates at a concentration of 105 cells/well for 24 h (37 °C and 5% CO2) to allow adhesion, using DMEM supplemented with 10% FBS and 1% PS. After 24 h of culture, the DMEM medium was replaced with extraction medium and cells were incubated for a further 24 h. At the



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 25

prescribed experimental time, 100 µL of WST-1 solution was added to each well and incubated for 1 h at 37 °C. The optical density (O.D) of the live cells was measured at 450 nm. The cell viability was expressed as a percentage according to the equation: Cell viability (%) =

. . . .

x 100%

where the O.D (test), and O.D (control) are the optical density of cells seeded on tissue culture polystyrene (TCPS) with extraction medium and DMEM medium, respectively, and O.D (blank) is the optical density of the DMEM medium without cells.

2.9. Statistical analyses Experimental data from the studies were analyzed using Student’s t-test. Statistical significance is considered as having *P < 0.05. All the experiments were performed in triplicate and data were presented as the mean ± SD.

3. Results and discussion 3.1. Synthesis and characterization of HT HT polymer was synthesized by using the EDC/NHS-mediated reaction as described in Figure 2a. 1H NMR spectroscopy (Figure 2b) clearly indicated the peaks of phenol groups (6.8 – 7.1 ppm), signifying the successful conjugation of TA to the heparin backbone. UV-Vis spectroscopy also confirmed the successful synthesis of HT by the presence of a peak at 275 nm (TA peak), which was absent in the pure heparin polymer (Figure 2c). Using the tyramine standard curve (0.01 – 0.1 mg/mL), the phenolic content of HT polymer was determined to be 355 µmol per 1 g of polymer.


Page 11 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. (a) Reaction scheme to prepare HT polymer; (b) 1H NMR spectrum of HT polymer and (c) UV-Vis spectra of HT (1 mg/mL) and heparin (1 mg/mL) polymer.

3.2. Surface immobilization and characterization HT and Ag NPs were immobilized onto PU surfaces by simply immersing the substrates into the polymer solution (with or without AgNO3) in the presence of Tyr. Tyr oxidases phenol moieties conjugated to the heparin backbone to catechol molecules, subsequently resulting in o-quinone formation. Although the surface bonding mechanism of o-quinone molecules is not fully understood, many studies have proven that surface immobilization occurs on the metal surface via coordinative bonds and surface immobilization occurs on the organic material surfaces by covalent bonding.22 In addition, the π-π interaction between the aromatic groups of PU chains and the catechol groups of the HT polymer can be explained as another molecular mechanism by which the heparin derivative can be immobilized on the PU surface.14 Accordingly, the oquinone conversion of phenol molecules by Tyr-catalyzed oxidative reaction could induce surface immobilization of HT molecules as well as Ag NPs on PU surfaces through chemical



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 25

bonds and physical interactions. Tyr-mediated processes, including the conversion of phenolic moieties to o-quinones, followed by nanoparticle formation and conjugative reaction, were rapidly performed under very mild conditions, within a few minutes (Figure S1). Water contact angle measurements were performed to assess the wettability of PU substrates before and after immobilization (Figure 3a). The bare PU surface is hydrophobic with a contact angle of 78.5o. After the enzymatic treatment, the contact angle decreased dramatically to 28.6o and 32.7o for HT and HT/Ag NPs immobilized PU surfaces, respectively. This result indicated the successful immobilization of hydrophilic heparin molecules onto the substrates. The water contact angle of HT/Ag NPs immobilized surfaces was slightly higher than that of only HT immobilized surfaces and may be due to the consumption of o-quinone molecules for Ag NP formation, consequently reducing the binding moieties for the conjugative reaction. The chemical compositions of bare and immobilized surfaces were further characterized by XPS (Figure 3b). The successful immobilization of HT onto the PU surface was confirmed by the presence of a new peak for sulfur (S 2p, 168 eV) in comparison with bare PU surfaces, which originated from the sulfate groups of heparin molecules. In the case of the HT/Ag NPs immobilized surface, XPS spectra clearly showed peaks for both sulfur (168 eV) and silver (Ag 3d3/2, 374 eV and Ag 3d5/2, 368 eV), indicating that Ag NPs are formed and simultaneously immobilized with HT onto the PU surface via a one-step Tyr-mediated reaction. The EDX results also showed the formation and good distribution of Ag NPs on the PU surfaces (Figure S2). The surface morphology and roughness of the bare and immobilized PU were monitored by using SEM and AFM analysis, respectively. As shown in SEM images (Figure 3c), while the bare and HT immobilized PU surfaces have smooth morphologies, the HT/Ag NPs immobilized PU surface is rougher due to the formation of Ag NPs. The Ag NPs (38.8 ± 6.7 nm in a diameter) are uniformly distributed on the PU substrates. AFM images (Figure 3d) proved that the roughness of PU surfaces was significantly increased after the immobilization of HT and Ag NPs. The root mean square roughness (Rrms) of bare PU was 1.7 nm, compared with that of HT and HT/Ag NPs immobilized PU surfaces of 2.2 and 3.4 nm, respectively. These results further demonstrated that HT and Ag NPs were successfully immobilized on PU surfaces in a homogeneous manner.


Page 13 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


To quantify the amount of HT immobilized on the PU surfaces, the toluidine blue colorimetric method was performed. The amount of HT immobilized on the surface was 0.98 µg/cm2. A further increase in the reaction time and polymer concentration did not significantly increase the amount of immobilized HT (data is not shown). From the obtained results, we showed that the Tyr-catalyzed oxidative reaction is a simple and efficient approach to simultaneously immobilizing heparin and Ag NPs onto substrates for antithrombotic and antimicrobial activities, respectively.

Figure 3. Characterization of integrated HT/Ag NPs immobilized PU surfaces: (a) Water contact angle; (b) XPS analysis for surface composition; (c) SEM images and (d) 3D topographical AFM images of bare (left), HT immobilized (middle) and HT/Ag NPs immobilized (right) PU. The scale bar is 200 nm.

3.3. Surface stability and silver release behavior



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 25

The stability of heparin immobilized on surfaces in physiological conditions was evaluated to confirm the efficiency of immobilization approach.14 As shown in Figure 4b, there was no obvious change in heparin amount after immersion of immobilized surfaces in PBS solution for 5 and 10 days. From this result, it may be concluded that HT was immobilized on PU surface via Tyr-mediated reaction with high stability. The silver release from PU surfaces was evaluated for 30 days using ICP measurement. There is existence of three distinct forms of silver as soon as Ag NPs are released into the solution: Ag0 nanoparticles, free Ag+, and surface-adsorbed Ag+.5,13,23 In general, in aqueous solution containing no other oxidants or reductants, Ag+ releases from Ag NPs by a cooperative oxidation process involving both dissolved oxygen and protons: 1 2Ags + + 2 ! ↔ 2#$! + 2 + Ag release rapidly occurs, gradually decreases and terminates at the equilibrium of reaction, where released Ag+ can be either absorbed or reduced by the original nanoparticles. The Ag+ release from Ag NPs would have an additional contribution to the bactericidal effect of Ag NPs, which was reported by Morones.24 As shown in Figure 4c, the total amount of silver released after 30 days is ~ 1.87 ppm. The silver release profile can be divided into two periods: high, fast release in the initial period (first 10 days) and plateau release from day 15. The SEM images (Figure 4a) also show that the number of spherical Ag NPs reduced, and remained on the PU surfaces after 30 days of being immersed in PBS solution, suggesting the long-term stability of the immobilization method. In general, this release pattern would be beneficial in preventing the initial bacterial infection and minimizing the toxicity of released silver ions.13


Page 15 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. (a) Stability of HT/Ag NPs immobilized PU surfaces performed by SEM: before (left) and after 30 days (right) incubated in physiological environment. The scale bar is 200 nm; (b) Remaining heparin amount after immersing immobilized surfaces in PBS solution; (c) Cumulative silver release profile.

3.4. In vitro evaluation of antithrombogenicity 3.4.1. Fibrinogen adsorption For blood-contacting surfaces, protein adsorption on the surface is one of the most important steps in the coagulation cascade, giving rise to thrombus formation later.6 Among the proteins of blood plasma, fibrinogen is the most abundant protein, besides albumin.25 It is also known as the precursor to fibrin, the key protein of the coagulation cascade. This protein carries negative charges in PBS solution or in the normal blood stream (pH 7.4) and can be easily adsorbed onto many surfaces via hydrophobic, electrostatic or acceptor-donor interactions. As shown in Figure 5a, the immobilized PU surfaces show a significant decrease in fibrinogen adsorption (0.7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 25

µg/cm2) compared with the bare PU surfaces (2.3 µg/cm2). This result confirmed that immobilizing surfaces with highly hydrophilic and negatively charged molecules like heparin, decreased fibrinogen adsorption by reducing the hydrophobic and electrostatic interactions between the surface and fibrinogen, respectively. 3.4.2. Platelet adhesion To evaluate the antithrombotic property of biomaterial surfaces, the platelet adhesion assay is a widely applied method.4 Figure 5b and 5c show the average number of platelets adhering to the bare/modified PU surfaces, which was analyzed from the SEM images. It was observed that the number of platelets adhering to the HT and HT/Ag NPs immobilized PU surfaces, was much lower than those on the bare PU surfaces. The adhered platelet density on bare, HT, HT/Ag NPs immobilized PU was quantitatively determined to be 87.7, 25.0, and 11.3 x 104 cells/cm2, respectively. These results indicated that both immobilized HT and Ag NPs inhibited platelet adhesion, consequently enhancing the blood compatibility of PU surfaces. Although the resistant platelet adhesion of heparin-modified surfaces has been widely reported, there are few studies demonstrating the antiplatelet properties of Ag NPs. Shrivastava et al. first reported that Ag NPs effectively inhibited integrin-mediated platelet functional responses (e.g., aggregation, secretion, and adhesion), resulting in the unique antiplatelet property of Ag NPs, in addition to its wellknown antibacterial properties.26 In a recent study, Xia et al. also reported the effect of Agnanogels reducing platelet adhesion and inhibiting platelet activation.27 From these results, we can conclude that the integrated immobilization of HT and Ag NPs synergistically inhibited platelet adhesion, further improving the antithrombogenic efficiency of PU surfaces compared to only HT immobilized surfaces. 3.4.3. Clotting time The antithrombogenic activity of immobilized surfaces was evaluated by blood clotting potential. The surfaces were incubated with whole fresh blood that allowed clots to form on surfaces and trapped red blood cells (RBCs). The free RBCs that are not trapped in the thrombus were lysed by adding excess DIW and released hemoglobin. As such, the absorbance of hemolyzed hemoglobin from RBCs inversely relates to the clot size, and higher absorbance values indicate better thromboresistance. Figure 5d shows a significantly lower blood clotting rate for HT and


Page 17 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


HT/Ag NPs immobilized PU surfaces after a 50 min test period, compared to that of bare surfaces. It can be explained by HT and Ag NPs on the surface combining or reacting with components in blood (e.g., coagulant factors, fibrinogen, platelet, etc.), therefore inhibit the blood clotting. APTT, which relates mainly to the intrinsic pathway in blood coagulation cascade, is frequently used to evaluate the blood coagulation of heparin modified surfaces.4 We measured APTT values of immobilized PU substrates and the data are shown in Figure 5e. The APTT of HT and HT/Ag NPs immobilized surfaces were 2.6 and 2.7 times longer than those for bare surfaces, respectively, indicating the enhancement in anticoagulant activity. The increased APTT of immobilized surfaces may result from the unique biological properties of heparin, which can inhibit coagulation factors (e.g., Xia, IXa, Xa and IIa (thrombin)) in the coagulation cascade.4 These results prove that our approach can efficiently immobilize heparin derivative (HT) on PU surfaces without influencing the bioactivity of heparin.

Figure 5. In vitro antithrombogenic activity of HT/Ag NPs immobilized PU surfaces: (a) Fibrinogen absorption; (b) SEM images of bare (left), HT immobilized (middle), and HT/Ag NPs immobilized (right) PU surfaces after 1 h platelet adhesion. (The scale bar is 15 µm); (c) Quantitative analysis of platelet adhered on surfaces; (d) The absorbance of hemolyzed



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 25

hemoglobin solution and (e) APTT values for PPP, bare, HT and HT/Ag NPs immobilized PU surfaces. ***P < 0.001 vs. bare PU.

3.5. Antibacterial properties The antibacterial property of immobilized PU surfaces was evaluated by determining the viability of bacteria adhered on surfaces, using the alamarBlue assay. Enterohemorrhagic Escherichia coli (EHEC, gram-negative) and Methicillin-resistant Staphylococcus aureus (MRSA, gram-positive), the most common bacteria causing infection from medical devices and in hospitals, were chosen as model bacteria. As expected, the integrated HT/Ag NP immobilized PU surfaces showed excellent antibacterial activity against both bacterial strains, demonstrated by the reduction of bacteria viability. Compared to bare and HT immobilized surfaces, the bacteria viability was reduced by 94% and 100% for MRSA and EHEC, respectively (Figure 6a and 6c). This result is attributed to the great inhibition capacity of Ag NPs toward both Gramnegative and Gram-positive bacteria, including the antibiotic-resistant strains such as MRSA and EHEC.28


Page 19 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. In vitro antibacterial activity of HT/Ag NP immobilized PU surfaces: Bacteria viability, SEM images of the bacteria morphologies adhered on bare (left) and modified (right) surfaces for S. aureus (a, b) and E. coli (c, d). ***P < 0.001 vs. bare PU. The scale bar is 1 µm.

To test the bactericidal activity of Ag NP immobilized surfaces, SEM analysis was performed to explore the morphological changes of E. coli and S. aureus adhering on the surfaces. As shown in Figure 6b and 6d, most of the bacteria adhering on bare PU surfaces aggregate into clusters and appear as smooth and rounded membrane surfaces, whereas those on the HT/Ag NP immobilized PU exhibited wrinkled, withered, and even broken surfaces. In addition, the bacterial adhesion (also significantly reduced by the HT/Ag NPs immobilization), demonstrated the combination effect of sustained silver release on anti-adhesive and bactericidal activities. Both the results of bacteria viability and morphology suggest that simultaneously immobilizing HT and Ag NPs onto PU surfaces is an excellent strategy to inhibit bacterial infections via anti-adhesion and killing effects.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 25

3.6. Cytotoxicity tests The cytotoxicity test was performed to investigate the influence of bare and modified PU substrates on normal cell viability, according to the ISO/EN 10993 Part 5 guideline.29 In comparison to cells incubated with normal culture media (DMEM), no cytotoxic effects were observed for all groups incubated with extracted media from bare, HT and HT/Ag NP immobilized PU substrates for a culture period of 24 h (Figure 7). The cell morphologies were also observed by microscopy and showed the well spread morphologies for all samples (data is not shown). Similarly, Wang et al. also demonstrated the antibacterial activities of titanium embedded silver nanoparticles without impairing cell compatibility.30 Combining with the results for reduction in protein absorption, platelet adhesion and prolonged clotting time mentioned above, we suggested that the HT/Ag NPs immobilized PU surfaces are potentially used for blood-contacting devices due to their excellent hemocompatibility, antibacterial properties without toxicity to the living fibroblast cells.

Figure 7. In vitro cytocompatibility test for mammalian cells (hDFBs) of modified PU surfaces

4. Conclusion In this study, we have developed a facile method for the fast, in situ formation of Ag NPs and the rapid immobilization of heparin blended Ag NP coatings on PU substrates via a one-step Tyr-triggered reaction. The resulting HT/Ag NPs immobilized surfaces possess improved hydrophilicity and high stability for 30 days. The modified surfaces achieved thromboresistant properties, demonstrated by an inhibition of protein absorption, platelet adhesion, and prolonged


Page 21 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


clotting time. In addition, the integrated HT/Ag NPs immobilized surfaces have excellent antibacterial performances for both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria. This approach is therefore believed to have great potential for the surface modification of various biomedical devices, to achieve antithrombogenic and antibacterial properties. Supporting Information Detailed preparation procedure of PU substrates, UV-Vis spectroscopy for in situ formation of Ag NPs and EDX analysis of HT/Ag NPs immobilized surfaces

5. Acknowledgement This research was supported by the Bio & Medical Technology Development Program of the NRF funded by the Korean government, MSIP (NRF-2015M3A9E2028578)

6. References (1) Hasan, J.; Crawford, R. J.; Ivanova, E. P. Antibacterial Surfaces: The Quest for a New Generation of Biomaterials. Trends Biotechnol. 2013, 31, 295-304. (2) de los Santos Pereira, A.; Sheikh, S.; Blaszykowski, C.; Pop-Georgievski, O.; Fedorov, K.; Thompson, M. Antifouling Polymer Brushes Displaying Antithrombogenic Surface Properties. Biomacromolecules 2016, 17, 1179-1185. (3) Yu, K.; Lo, J. C.; Yan, M.; Yang, X.; Brooks, D. E.; Hancock, R. E. Anti-Adhesive Antimicrobial Peptide Coating Prevents Catheter Associated Infection in a Mouse Urinary Infection Model. Biomaterials 2017, 116, 69-81. (4) Cheng, C.; Sun, S.; Zhao, C. Progress in Heparin and Heparin-Like/Mimicking PolymerFunctionalized Biomedical Membranes. J. Mater. Chem. B 2014, 2, 7649-7672. (5) Agnihotri, S.; Mukherji, S.; Mukherji, S. Immobilized Silver Nanoparticles Enhance Contact Killing and Show Highest Efficacy: Elucidation of the Mechanism of Bactericidal Action of Silver. Nanoscale 2013, 5, 7328-7340.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 25

(6) Ma, L.; Cheng, C.; Nie, C.; He, C.; Deng, J.; Wang, L. Anticoagulant Sodium Alginate Sulfates and Their Mussel-Inspired Heparin-Mimetic Coatings. J. Mater. Chem. B 2016, 4, 32033215. (7) Lee, H.; Rho, J.; Messersmith, P. B. Facile Conjugation of Biomolecules onto Surfaces via Mussel Adhesive Protein Inspired Coatings. Adv. Mater. 2009, 21, 431-434. (8) Li, A.; Mu, Y.; Jiang, W.; Wan, X. A Mussel-Inspired Adhesive with Stronger Bonding Strength under Underwater Conditions than under Dry Conditions. Chem. Commun. 2015, 51, 9117-9120. (9) Fan, C.; Fu, J.; Zhu, W.; Wang, D. A. A Mussel-Inspired Double-Crosslinked Tissue Adhesive Intended for Internal Medical Use. Acta Biomater. 2016, 33, 51-63. (10) GhavamiNejad, A.; Park, C. H.; Kim, C. S. In Situ Synthesis of Antimicrobial Silver Nanoparticles within Antifouling Zwitterionic Hydrogels by Catecholic Redox Chemistry for Wound Healing Application. Biomacromolecules 2016, 17, 1213-1223. (11) Son, H. Y.; Ryu, J. H.; Lee, H.; Nam, Y. S. Silver-Polydopamine Hybrid Coatings of Electrospun Poly(vinyl alcohol) Nanofibers. Macromol. Mater. Eng. 2013, 298, 547-554. (12) Sileika, T. S.; Kim, H. D.; Maniak, P.; Messersmith, P. B. Antibacterial Performance of Polydopamine-Modified Polymer Surfaces Containing Passive and Active Components. ACS Appl. Mater. Interfaces 2011, 3, 4602-4610. (13) Jo, Y. K.; Seo, J. H.; Choi, B. H.; Kim, B. J.; Shin, H. H.; Hwang, B. H. SurfaceIndependent Antibacterial Coating Using Silver Nanoparticle-Generating Engineered Mussel Glue. ACS Appl. Mater. Interfaces 2014, 6, 20242-20253. (14) You, I.; Kang, S. M.; Byun, Y.; Lee, H. Enhancement of Blood Compatibility of Poly(urethane) Substrates by Mussel-Inspired Adhesive Heparin Coating. Bioconjugate Chem. 2011, 22, 1264-1269. (15) Huang, R.; Liu, X.; Ye, H.; Su, R.; Qi, W.; Wang, L. Conjugation of Hyaluronic Acid onto Surfaces via the Interfacial Polymerization of Dopamine to Prevent Protein Adsorption. Langmuir 2015, 31, 12061-12070. (16) Liu, Y.; Zhang, B.; Javvaji, V.; Kim, E.; Lee, M. E.; Raghavan, S. R. Tyrosinase-Mediated Grafting and Crosslinking of Natural Phenols Confers Functional Properties to Chitosan. Biochem. Eng. J. 2014, 89, 21-27.


Page 23 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(17) Faccio, G.; Senkalla, S.; Thöny-Meyer, L.; Richter, M. Enzymatic Multi-Functionalization of Microparticles under Aqueous Neutral Conditions. RSC Adv. 2015, 5, 22319-22325. (18) Jin, R.; Lin, C.; Cao, A. Enzyme-Mediated Fast Injectable Hydrogels Based on Chitosan– Glycolic Acid/Tyrosine: Preparation, Characterization, and Chondrocyte Culture. Polym. Chem. 2014, 5, 391-398. (19) Park, K. M.; Park, K. D. Facile Surface Immobilization of Cell Adhesive Peptide onto TiO2 Substrate via Tyrosinase-Catalyzed Oxidative Reaction. J. Mater. Chem. 2011, 21, 15906. (20) Lee, Y.; Park, K. M.; Bae, J. W.; Park, K. D. Facile Surface PEGylation via TyrosinaseCatalyzed Oxidative Reaction for the Preparation of Non-Fouling Surfaces. Colloids Surf., B 2013, 102, 585-589. (21) Ma, L.; Su, B.; Cheng, C.; Yin, Z.; Qin, H.; Zhao, J. Toward Highly Blood Compatible Hemodialysis Membranes via Blending with Heparin-Mimicking Polyurethane: Study In Vitro and In Vivo. J. Membr. Sci. 2014, 470, 90-101. (22) Lee, H.; Scherer, N. F.; Messersmith, P. B. Single-Molecule Mechanics of Mussel

Adhesion. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 12999-3003 (23) Liu, J.; Hurt, R. H. Ion Release Kinetics and Particle Persistence in Aqueous Nano-Silver Colloids. Environ. Sci. Technol. 2010, 44, 2169−2175. (24) Morones, J. R.; Elechiguerra, J. L.; Camacho, A.; Holt, K.; Kouri, J. B.; Ramirez, J. T. The Bactericidal Effect of Silver Nanoparticles. Nanotechnology 2005, 16, 2346-2353. (25) Xu, F. J.; Li, Y. L.; Kang, E. T.; Neoh, K. G. Heparin-Coupled Poly(poly(ethylene glycol)monomethacrylate)-Si(111)

Hybrids

and

Their

Blood

Compatible

Surfaces.

Biomacromolecules 2005, 6, 1759-1768. (26) Shrivastava, S.; Bera, T.; Singh, S. K.; Singh, G.; Ramachandrarao, P. Characterization of Antiplatelet Properties of Silver Nanoparticels. ACS Nano 2009, 3, 1357-1364. (27) Xia, Y.; Cheng, C.; Wang, R.; Nie, C.; Deng, J.; Zhao, C. Ag-Nanogel Blended Polymeric Membranes with Antifouling, Hemocompatible and Bactericidal Capabilities. J. Mater. Chem. B 2015, 3, 9295-9304. (28) Cavassin, E. D.; de Figueiredo, L. F.; Otoch, J. P.; Seckler, M. M.; de Oliveira, R. A.; Franco, F. F. Comparison of Methods to Detect the In Vitro Activity of Silver Nanoparticles (AgNP) against Multidrug Resistant Bacteria. J. Nanobiotechnol. 2015, 13, 64.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(29) Kim, S. E.; Song, S. H.; Yun, Y. P.; Choi, B. J.; Kwon, I. K.; Bae, M. S. The Effect of

Immobilization of Heparin and Bone Morphogenic Protein-2 (BMP-2) to Titanium Surfaces on Inflammation and Osteoblast Function. Biomaterials 2011, 32, 366-373. (30) Wang, G.; Jin, W.; Qasim, A. M.; Gao, A.; Peng, X.; Li, W. Antibacterial Effects of Titanium Embedded with Silver Nanoparticles Based on Electron-Transfer-Induced Reactive Oxygen Species. Biomaterials 2017, 124, 25-34.

Graphical abstract


Page 24 of 25

Page 25 of 25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

Tyrosinase-Mediated Surface Coimmobilization of ... - ACS Publications

Recommend Documents