Thiol-Reactive Polymers for Titanium Interfaces ... - ACS Publications

Subscriber access provided by University of Rochester | River Campus & Miner Libraries

Article

Thiol-Reactive Polymers for Titanium Interfaces: Fabrication of Antimicrobial Coatings Tugce Nihal Gevrek, Kai Yu, Jayachandran N Kizhakkedathu, and Amitav Sanyal ACS Appl. Polym. Mater., Just Accepted Manuscript • DOI: 10.1021/acsapm.9b00117 • Publication Date (Web): 17 May 2019 Downloaded from http://pubs.acs.org on May 20, 2019

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 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 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.

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 Polymer Materials

Thiol-Reactive Polymers for Titanium Interfaces: Fabrication of Antimicrobial Coatings Tugce Nihal Gevrek,a Kai Yu,c Jayachandran N. Kizhakkedathu,c* Amitav Sanyala,b* a Department b c

of Chemistry, Bogazici University, Bebek, Istanbul, 34342, Turkey

Center for Life Sciences and Technologies, Bogazici University, Istanbul, Turkey

Centre for Blood Research, Life Science Institute, Department of Pathology & Laboratory Medicine and Department of Chemistry, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada Email: [email protected]; [email protected]

ACS Paragon Plus Environment

1

ACS Applied Polymer Materials 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 2 of 25

ABSTRACT Infection associated with surgical implants is a major cause of their failure. Often in such cases, the implant has to be removed and replaced which cause undesirable patient discomfort and complications. Bacterial adhesion and growth on implant surface is the primary reason for such infections. Among the approaches to prevent implant associated infections, the conjugation of antimicrobial peptide (AMP) onto the surface of implant is a very promising approach. In this study, we describe a facile method for the surface modification of titanium (Ti), a widely used material in dental and orthopedic implants, to prevent bacterial adhesion and growth. Thin polymeric films were synthesized on Ti surface using a copolymer containing the maleimide group as a thiol-reactive handle to enable the conjugation of AMPs. Robust attachment of the polymeric coating on Ti surfaces was ensured through installation of catechol moieties on the polymer as surface anchoring groups and the variation of the amount of thiol-reactive maleimide group on titanium surfaces. As a proof-of-concept, to demonstrate a viable application of such thiol reactive surfaces, the antimicrobial peptide E6 (RRWRIVVIRVRRC) was immobilized onto these well characterized thin polymeric layers through Michael addition. Antimicrobial activity of peptide modified surfaces was screened against both gram positive and gram negative bacteria. The hydrophilic polymer coatings decreased the bacterial adhesion, and the immobilized peptide killed more than 80% of the adhered bacteria. The developed surface modification method has broad applicability in terms of the choice of substrates and peptides in the design of bioactive surfaces.

KEYWORDS: reactive polymers, thiol-maleimide click reaction, antimicrobial peptides, catechol anchor, surface modification


2

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


INTRODUCTION Surface modification of materials to render them capable of killing microorganisms is an area of keen interest that finds wide application in everyday materials to sophisticated biomedical implants and devices including that used in dental and orthopedic applications.1,2 Infection associated with such implants and devices is very difficult to treat3,4 and the surface adhesion of bacteria is the primary reason for such infections.5,6 The prevention of surface attachment of bacteria (anti-fouling approaches) and killing of adhered bacteria (bactericidal approaches) on the surfaces provide effective solution to this problem.7 Polymeric materials with anti-biofouling properties are used as surface coatings to enhance in vivo compatibility and provide a layer for inhibition of microbial attachment.8,9 Poly(ethylene glycol) (PEG) is one of the most widely employed polymer used as a surface modification agent to impart them with antifouling characteristics. It resists attachment of proteins,10,11 cells12 and other organisms such as bacteria13 by creating a barrier of structured water associated with PEG and through chain compression which generates entropic barrier.14-16 Another approach for inhibition of microbial attachment is antimicrobial coating. Cationic polymers and polymers conjugated with cationic biocides, antibiotics, or antimicrobial peptides (AMPs) can kill bacteria on contact.17-19 Contrary to other bactericides, antimicrobial peptides are effective to drug resistance strains as well as antibiotics-sensitive bacteria and are biocompatible.20-22 From a mechanistic viewpoint, AMPs target the bacterial cell membranes and disrupt their lipid bilayer structure.23 Their mode of action is more complex than commonly employed cationic groups, since the AMPs can change the function of membrane proteins and kill the bacteria even if complete disruption of their cell wall does not occur.24 Therefore, it is envisaged that such AMP modified surfaces would result in better bactericidal activity than antibiotics containing surfaces.


3


Page 4 of 25

AMPs can be immobilized onto the surfaces either physically or chemically. Physical immobilization of antibacterial peptides involves layer-by-layer approach prepared by loading the peptides between the polyionic polymeric layers.25,26 However, buried peptides in the layers below the upper layer will not be directly in contact with the surrounding bulk and must be released effectively through diffusion. In addition, in this system adhered bacteria may block the release the remaining peptides between the lower layers.27 The main advantage of chemical immobilization of the APMs is that they provide a stable antimicrobial coating whereby AMP leakage is minimal when compared to the commonly utilized physical immobilization methods.28,29 The robust attachment of the polymeric coating on the surface is crucial to warrant reliable performance of surface coated materials under demanding conditions. In recent years, surface chemistry approaches have been inspired by various biological organisms that are able to adhere onto a variety of surfaces in a robust manner by exuding nontoxic and permanent adhesive-like materials.30 In particular, mimicking the surface attachment of mussel adhesive proteins that are rich in dopamine (DOPA) has attracted significant interest. It has been reported that the catechol subunit is primarily responsible for their strong affinity toward metal and metal oxide surfaces. 31-34

To date, the catechol unit has been widely employed as an anchor for strong attachment of

variety of polymeric materials onto inorganic surfaces.35-38 Indeed, a few studies have reported coating copolymers bearing pendant dopamine and reactive groups on solid substrates for biomolecular immobilization.39-41 But most of these reports have focused on conjugation of amine bearing bioactive agents using couplings based on activated esters and epoxides. Development of a robust polymeric coating for the modification of metal and metal oxide based materials where


4

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


conjugation of a bioactive material possessing a naturally occurring thiol group or a synthetically incorporated amino acid would offer an attractive functional interface. Reactive polymeric coatings are designed to afford their facile functionalization with peptides under mild, efficient and reagent-free reaction conditions since harsh conditions may cause degradation of these biologically active motifs. Because of its high reactivity towards thiol containing molecules under mild conditions, maleimide is one of the most widely used functional groups for biomolecular immobilization.42-45 Maleimide containing coatings have been investigated for AMP immobilization. Kizhakkedathu and coworkers reported fabrication of an AMP containing polymer brush on silicon surfaces.46 A side chain amino group containing surface tethered copolymer was appended with maleimide units through post-polymerization modification for attachment of cysteine containing AMPs. These AMP conjugated polymer brushes exhibited broad spectrum antimicrobial activity both in vitro and in vivo. While polymer brushes offer an attractive platform for the fabrication of functional coatings, the process involves several steps such as immobilization of the initiator, and polymerization on the surface. An operationally simpler approach offered by direct modification of the inorganic surface with copolymers containing surface reactive groups is an attractive alternative.47,48 Herein, we report fabrication of thin polymeric coatings anchored onto titanium surface which possess both anti-adhesive and bactericidal properties. A series of copolymers containing pendant PEG chains, furan-protected maleimide groups and dopamine groups were synthesized to modify titanium substrates which is widely used in dental and orthopedic applications (Scheme 1). PEG based copolymers were utilized due to their well-established anti-biofouling property. In order to provide a modular approach for incorporation of different anti-bacterial groups onto the polymer coating, the maleimide functional group was used as a thiol-reactive handle. Furan-


5


Page 6 of 25

protected maleimide group containing polymers were synthesized to incorporate the maleimide groups in their masked form. Upon fabrication of the coating on titanium surface, a thermal treatment enable the unmasking of the maleimide group to their thiol-reactive form via the retro Diels-Alder reaction. Finally, thiol-containing AMPs were conjugated onto these functionalized polymer coating, and adhesion and contact-killing of both gram-negative and gram-positive bacteria were investigated.

Scheme 1. Fabrication of thiol-reactive polymeric layer on titanium surface and antimicrobial peptide modification.

EXPERIMENTAL SECTION Materials. PEGMEMA (Mn: 500 gmol-1) was obtained from Sigma-Aldrich. Dopamine methacrylamide (DMA)49 and furan-protected maleimide monomer (FuMaMA)50 were synthesized

according

to

literature

procedures.

Cysteine-containing

E6

peptide

(RRWRIVVIRVRRC) was synthesized by CanPeptide Corp. (>95% purity by high performance


6

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


liquid chromatography; Montreal, Quebec, Canada) and used as supplied. Solvents are purchased from Merck and used as received. Methods. 1HNMR spectra were recorded on Bruker Avance Ultrashield 400 (400 MHz). Molecular weights of the synthesized polymers were estimated by size exclusion chromatography (SEC) using a PSS-SDV (length/ID 8 × 300 mm, 10 m particle size) gram linear column calibrated with poly(methyl methacrylate) (PMMA) standards using a refractive-index detector with a mobile phase solution of 0.05 M lithium bromide in DMAc as eluent at a flow rate of 1 mL/min at 30 °C. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy was collected on a Thermo Scientific Nicolet 380 FT-IR spectrophotometer. For surface analysis, ATR-FTIR was equipped with Harrick Scientific GATR accessory and a Ge crystal. XPS spectra were recorded on the Kratos Analytical Axis Ultra X-ray photoelectron spectrometer (XPS) with an Al Kα-monochromatized source of 1486.71 eV. Thickness measurement was performed by collecting the variable-angle spectroscopic ellipsometry (VASE) spectra using an M- 2000 V spectroscopic ellipsometer from J. A. Woollam Co. Inc., Lincoln, NE at 50°, 60°, and 70° at wavelengths from 480 to 700 nm with an M-2000 50Wquartz tungsten halogen light source. For water contact angle analysis an image of the 3.5 µL water droplet was captured with Retiga 1300, Q-imaging Co digital camera and its contact angle with surface was analyzed using Northern Eclipse software. For each sample three different regions were tested. Synthesis of Copolymers (P3). Dopamine methacrylamide (1.14 mmol 252.24 mg), PEGMEMA (2.85 mmol, 1425 mg), FuMaMA (1.71 mmol, 497.83mg), AIBN (0.0076 mmol, 1.25 mg) and 4cyano-4-(phenyl carbonothioylthio)pentanoic acid (0.038 mmol, 10.62 mg) were dissolved in 2.8 mL of DMF in a round bottom flask containing a stir bar and the mixture was purged with N2 gas for 20 minutes. The round bottom flask was immersed into an oil bath at 75 oC and stirred for 18


7


Page 8 of 25

hours. The polymer was then precipitated in cold diethyl ether to remove unreacted monomers and reagents. 1H NMR (300 MHz, CDCl3 δ (ppm)): 6.85-6.65 (m, 2H, Ph), 6.51 (s, 2H, CH═CH and m, 1H, Ph) 5.21 (s, 2H, CH bridgehead protons), 4.05 (s, 2H, OCH2 ester protons of PEGMEMA), 3.9 (br s, 2H, OCH2 ester protons of DMA and FuMaMA), 3.81–3.45 (m, (4n+2) H, OCH2 of PEGMEMA), 3.35 (s, 3H, OCH3 of PEGMEMA), 2.85 (s, 2H, CH–CH, bridge protons), (2.932.77 and 2.20-1.55 (m, CH2 and CH3 along polymer backbone). P-0 and P-10 were prepared by altering the ratios of the monomers. (See supporting information). Fabrication of Polymeric Coating on Titanium Surfaces. Polymer solution (25 µL, 400 mg/mL in methanol) was spread over each surface (1x1 cm2 titanium). They were left 1 hour at room temperature to let the solvent evaporate. Surfaces were placed into the vacuum oven at 110 oC for 30 minutes and they were removed after 1.5 hours when the oven’s temperature dropped below 60 oC.

Lastly, surfaces were washed and sonicated in methanol for 30 minutes to remove non-adhered

polymer from surface and then surfaces were dried under stream of nitrogen. Curing the polymer coated substrates at high temperature resulted in strongly attached polymer coating on the surface as well as activation of the maleimide groups via retro Diels-Alder cycloreversion reaction. Bacterial Viability Estimation via Live/Dead Assay. Bacteria were grown in Lysogeny broth (LB; 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl/L) from freezer stocks at 37 °C O/N, and used at approximately 1 × 105 CFU/mL (CFU = colony-forming unit), as determined by OD600 readings using the approximate equation of 0.1 OD600 = 108 in 96-well plates. Live/Dead BacLight bacterial viability kit (L-7012; Molecular Probes, Eugene, OR) was used to determine the bacterial cell viability on polymer coated surfaces. Polymer coated titanium substrates and bare titanium substrates were each placed in a 24-well plate. The samples were then sterilized with 1 mL of 70% ethanol by incubating it for 2 minutes and the process was repeated three times. The


8

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


samples were washed with sterilized water three times. A diluted bacteria suspension (1 mL) was then introduced to each well, and the substrates were placed on a shaker at a speed of 120 rpm to provide a homogeneous liquid environment for the interaction and incubated at 37 °C for 4 h. The substrates were then washed with 1 mL of PBS buffer consecutively. A solution of the SYTO 9 (2.4 μL) and propidium iodide (PI; 12 μL) dyes in PBS buffer (12 mL) was prepared. After incubation of the substrate with a dye solution at room temperature in a dark environment for 15 min, the substrates were washed with sterilized water and dried. The samples were then examined using a fluorescence microscope equipped with a fluorescence illumination system (AttoArc 2 HBO) and appropriate filter sets. Images were taken a 20× objective lens. The pictures were taken using fluorescein isothiocyanate (FITC) and rhodamine filters to visualize the total and dead bacteria respectively. Statistical data analysis was done using GraphPad Prism 5.0. Since surfaces are 2D thin films with 4-5 nm thickness, all bacteria are expected to be in the same plane, so depth profiling was not required.

RESULTS AND DISCUSSION Synthesis and Characterization of Copolymers. The masked maleimide bearing copolymers poly(DOPA-r-PEGMEMA-r-FuMaMA) were synthesized from three different monomers via reversible addition fragmentation chain transfer (RAFT) polymerization (Scheme 2). Utilization of dopamine methacrylamide (DMA), furanprotected maleimide methacrylate (FuMaMA) and poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) as monomers provided these polymers with ability for surface


9


Page 10 of 25

attachment on the metal surface, facile functionalization through thiol-maleimide chemistry and have anti-fouling properties, respectively.

Scheme 2. Synthesis of copolymer poly(DOPA-r-PEGMEMA-r-FuMaMA) via RAFT polymerization.

The synthesized polymers were purified using simple precipitation in cold ether. Monomer composition in copolymers was determined using 1H NMR spectroscopy (Figure 1, Figure S1). The proton resonance at 3.35 ppm (s, 3H, terminal O-CH3 on PEGMEMA), 6.78 ppm and 6.71 ppm (d, 1H, Aryl–Hh and s, 1H, Aryl–Hf in catechol) and 5.22 ppm (s, 2H, CH bridgehead protons in furan-maleimide cycloadduct) were used for calculation of copolymer composition. Actual ratio of monomers incorporated in the copolymers were calculated according to integration values in their 1H NMR spectra (Table 1). The molecular weights of the copolymers P-0, P-10 and P-30 were obtained as 48, 48 and 45 kDa, respectively, with a polydispersity index a 1.3, as measured using size exclusion chromatography (SEC) (Table 1), (Figure S2).


10

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 1. 1H NMR spectrum of copolymer P-30 in CDCl3.

Table 1. Monomer compositions and molecular weight analysis of synthesized copolymers. Copolymera P-0 P-10 P-30

Theoretical ratiob

Obtained ratioc

DMA:PEGMEMA: FuMaMA

DMA:PEGMEMA:FuMaMA

20:80:0 20:70:10 20:50:30

13.9: 86.1:0 15.5: 73.1:11.4 17.2: 57.3: 25.5

Mnd

PDI

48 kDa 48 kDa 45 kDa

1.20 1.36 1.26

a[I]

o/[CTA]o:1/5; [M]o:2M; Initiator: AIBN. CTA: 4-cyano-4-(phenyl carbonothioylthio)pentanoic acid. Reaction time: 18 h; 75°C; solvent: DMF. b Based on feed ratio DMA:PEGMEMA (M = 500 gmol-1): FuMaMA. n c Determined using 1H NMR spectroscopy. d Estimated by SEC eluted with DMAc, using PMMA standards.

Incorporation of the FuMaMA monomer in copolymers was also examined using ATRFTIR (Figure S3). For the copolymer P-0, the FTIR spectrum displayed C=O stretching band belonging to the ester groups at 1726.8 cm-1. For the copolymer P-10, similar stretching band was


11


Page 12 of 25

observed at 1726.34 cm-1, with additional out-of-phase C=O stretching vibration at 1703.68 cm-1 and in-phase C=O stretching vibration at 1770.59 cm-1 corresponding to furan-protected maleimide units of copolymer. Similarly, spectrum belonging to copolymer P-30 displayed a C=O stretching band from polymer backbone at 1724.54 cm-1, and out-of-phase C=O stretching vibration at 1699.17 cm-1 and in-phase C=O stretching vibration at 1771.54 cm-1. As expected, due to higher amount of the masked maleimide monomer, the latter two vibration bands were more intense than those observed for copolymer P-10 (Figure S3). Fabrication and Characterization of Polymer Coatings on Titanium Surfaces Ti-substrates were used for the preparation of the copolymer coating. After the initial spreading of a solution of the copolymer, a curing protocol was used to generate robust thin polymer layers. Curing the polymer coated substrates at high temperature resulted in fabrication of robust polymer film on the surface, as well as activation of the maleimide groups via the retro Diels-Alder cycloreversion reaction (Figure 2-a). Hereafter, copolymer P-0, P-10, and P-30 coated titanium surfaces are referred as S-0, S-10, and S-30, respectively.


12

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


Figure 2. a) Schematic illustration of coating of polymers onto titanium oxide surface and its thermal activation. b) ATR-FTIR spectra of polymers coated surfaces S-0, S-10 and S-30.

The thermal activation of the maleimide functional group during coating process was confirmed via ATR-FTIR analysis of freshly coated and baked surfaces. Analysis was done on surfaces before removing the excess polymers. After the thermal treatment, the carbonyl stretching bands from the ester units of all side chains were observed at 1727.72, 1727.08 and 1725.44 cm-1 for copolymers on titanium surfaces S-0, S-10 and S-30 respectively. Shift of C═O stretching vibration band to 1711.96 from 1703.68 cm-1 and 1707.14 from 1699.17 cm-1 for maleimide containing samples S-10 and S-30 confirms that maleimide groups are unmasked after coating process (Figure 2-b, see also Figure S3). Similar shift in C═O stretching vibration has been observed in other studies upon thermal activation of the maleimide group.51,52


13


Page 14 of 25

Changes in layer thickness and water contact angle data are shown in Table 2. Since reliable thickness data measurement from ellipsometry was challenging due to instability of titanium oxide layer during the coating process, we coated polymers on both Si/SiO2 and titanium substrates at similar conditions and collected thickness data from Si/SiO2 surfaces. Contact angle values of polymer coated titanium and silicon surfaces were similar. Therefore, we assumed that thicknesses of polymeric coatings on titanium surface was around 4-5 nm as those on silicon surfaces. Upon incorporation of maleimide groups and concurrent decrease in PEG content on the surface increased its hydrophobicity; water contact angles of S-0, S-10, S-30 increased from 37° to 43° (Table 1 & Table 2). Surface composition of the polymer coated surfaces was also investigated using X-ray photoelectron spectroscopy (XPS) analysis. Both survey scan and high resolution spectra are shown in Figure 3a. The S-0 surface which bears a coating devoid of any maleimide units showed 0.81% nitrogen content, which originates from the nitrogen in the dopamine units. Incorporation of masked maleimide units on the polymeric surface results in an increase in the nitrogen content. As expected, when the polymeric precursor with higher amount of the FuMaMA monomer was used for surface coating, an increase in the N content on the surface was observed (Figure 3-a and 3-c). Peptide Immobilization on Polymer Coated Titanium Surfaces To investigate the potential utility of maleimide containing polymer coating for surface attachment of biological molecules, we utilized an antibacterial peptide containing a terminal cysteine residue. A cathelicidin-derived peptide E6 (RRWRIVVIRVRRC) was chosen since our earlier studies had established its biocompatibility and efficient antibacterial activity.53,54


14

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


Thermally activated substrates were used for the peptide conjugation. Immobilization of E6 on the surface was confirmed by the change in water contact angle (Table 2) and XPS analyses. Upon peptide conjugation, the water contact angle was increased possibly due to the presence of hydrophobic residues on AMP E6. Table 2. Surface characterization of polymeric surfaces and E6 peptide conjugated surfaces. Surface

Thickness of polymeric coatinga (nm)

CA o

CA o

CA o

(on Si/SiO2)b

(Before peptide conjugation on Ti)c

(After peptide conjugation on Ti)c

S-0

4.2 ± 0.1

36 ± 1

38 ± 1

44 ± 1

S-10

5.2 ± 0.1

39 ± 1

40 ± 1

55 ± 1

S-30

4.5 ± 0.1

42 ± 2

44 ± 1

63 ± 1

a Determined

by ellipsometer using polymeric coating on Si/SiO2 surface. Determined on polymeric coating on Si/SiO2 surface. c Determined on polymeric coating on Ti-surface.

b

XPS analysis was used to further probe the peptide conjugation. As expected, peptide attachment resulted in an increase in nitrogen content (Figure 3) for all surfaces. Interestingly, an increase in the nitrogen content was also observed for surface S-0, where the polymeric coating does not possess any maleimide functional group. The increase in nitrogen content on peptide exposed S-0 surface suggests possible conjugation of thiol-containing peptide with oxidized catechol moiety,55 which may be present to some extent. An increase in maleimide content on the surface (S-10 & S-30) resulted in higher amount of peptide on the surface as evident from the increase in nitrogen content observed in the XPS analysis.


15


Page 16 of 25

Figure 3. (a) XPS survey spectra (b) N1s high resolution spectra and c) relative concentrations of C, O and N polymeric coatings and peptide conjugated polymeric coatings. Antibacterial Activity of Peptide Conjugated Polymer Coatings on Titanium Surfaces The bacterial adhesion and antimicrobial activity of the peptide immobilized surfaces against Gram-positive (S. aureus) and Gram-negative (P. aeruginosa) bacteria were evaluated. The total number of adhered bacteria and dead bacteria on various surface were compared based on an average of three images. All bacteria were counted by zooming into each micrograph that covers 0.18 mm2 area, to obtain the average value. The copolymer coated surfaces (S-0, S-10 and S-30) attracted less bacteria than bare titanium (Figure 4-a-d) due to the presence of hydrophilic PEG in the copolymer. On bare titanium surface, an average 11% of the adhered bacteria were


16

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


dead (Figure 4-d). The bacteria death on bare substrate could be due to the washing procedure after incubation and drying after staining. These procedures possibly bring stress to the bacteria and induce the death. It was observed that E6 conjugated surfaces attracted more bacteria than the control samples, however, importantly about 72 % and 78 % of the adhered bacteria were dead on S-0-E6 and S-10-E6 surfaces, respectively (Figure 4-f). The surface with higher amount of conjugated peptide (S-30-E6) killed about 83 % of adhered bacteria (Figure 4-g). These results show that peptide conjugated surfaces function effectively as an antibacterial coating. Interestingly, even though there were no maleimide groups on S-0 surface, it appears that conjugation of peptides to possibly-oxidized catechol units on the polymer takes place efficiently which resulted in increased adhesion and killing of the bacteria (Figure 4-e). While the micrographs of the live/dead stained bacteria provides a clear idea of the trend, for easier comparison, the numbers of live and dead S. aureus on E6 immobilized surface are presented in Figure 4-h (also see Figure S4). The polymer coated surfaces without AMP displayed antibiofouling characteristics due to the presence of PEG, while the peptide conjugated surfaces attracted more bacteria similar to the published literature.56,57 As expected, the presence of AMP also resulted in higher killing ability of the coating.


17


Page 18 of 25

Figure 4. Fluorescence microscopy images of (a) S-0, (b) S-10, (c) S-30, (d) bare titanium, (e) S0-E6, (f) S-10-E6, and (g) S-30-E6 films on titanium surfaces by live (green)/dead (red) bacteria staining after a 4 h of incubation with S. aureus and number of total and (h) Number of bacteria (S. aureus) on bare titanium and various polymer coated surfaces per 0.18 mm2. Values are average of three images taken with a 20x objective.


18

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


Antibacterial effect of fabricated surfaces was also studied using gram negative bacteria: P. aeruginosa. It was observed that both control and peptide E6 conjugated surfaces attracted more P. aeruginosa than S. aureus. Only 13% of adhered bacteria were dead onto the bare titanium surface (Figure 5-d). The polymer coating was antifouling against P. aeruginosa (Figure 5-a, b and c). Peptide E6 conjugated S-10 and S-30 killed 66% and 81% of adhered bacteria, respectively (Figure 5-f and g); while 61% of bacteria were dead on S-0-E6 surface (Figure 5-e). Killing efficiency of various surface is shown in Figure 5-h (and also Figure S4). Overall, these surface coatings were found be quite effective in killing the adhered bacteria. The antifouling properties of the coating can be further optimized for efficient bacterial killing after AMP attachment by surveying other peptides, as well as preventing the adhesion of bacteria for the highly efficient anti-biofilm coating.58,38 With the next generation of such AMP coating we are aiming to design surfaces with more anti-biofouling characteristics and greater activity. This could be done by increasing the PEG chain length or increasing the graft density of the PEG-based anti-fouling layer. The developed coating method is modular and such modifications are feasible, and could allow for long-term prevention of biofilm formation.


19


Page 20 of 25

Figure 5. Fluorescence microscopy images of (a) S-0, (b) S-10, (c) S-30, (d) bare titanium, (e) S0-E6, (f) S-10-E6, and (g) S-30-E6 on titanium surfaces by live (green)/dead (red) bacteria staining after incubation with P. aeruginosa and number of total and (h) dead P. aeruginosa on bare titanium and polymer coated and E6 immobilized surfaces per 0.18 mm2. Values are average of three images taken with a 20x objective lens.


20

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


CONCLUSIONS In summary, we have described a facile method for preparing a maleimide bearing polymeric coating on titanium surfaces fabricated using copolymers containing a masked maleimide group and dopamine based surface anchoring units. Polymeric precursors were coated on titanium surface and the maleimide groups were activated to their thiol-reactive forms using a simple thermal treatment. The amount of maleimide groups on the surface can be changed by altering the polymer composition. As a proof-of-concept, to demonstrate a potential application of such functional coatings, an antimicrobial peptide was conjugated onto this maleimide containing surfaces. The peptide conjugated surfaces showed both anti-fouling and antimicrobial properties against both gram negative and gram positive bacteria. Importantly both the anti-fouling and antimicrobial properties of the coating can be altered by changing the polymer composition. The modular nature of the polymeric interface is amenable to facile functionalization by any thiolcontaining antibacterial peptide and thus can be adapted to engineer effective anti-bacterial surfaces against various bacteria, among various other possible applications.

ASSOCIATED CONTENT The supporting information is available. Synthesis of copolymers, 1HNMR spectra, FTIR spectra and SEC curves of P-0, P-10 and P-30, and statistical data analysis of bactericidal activity. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected]


21


Page 22 of 25

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS

TNG

acknowledges

fellowship

from

The

Scientific

and

Technological Research Council of Turkey (TUBITAK 2211-D and 2214-A) for financial support. JNK acknowledge the funding from the Canadian Institutes of Health Research (CIHR), Natural Science and Engineering Research Council (NSERC) of Canada. The authors thank the LMB Macromolecular Hub at the UBC Centre for Blood Research for use of the analytical facilities. The infrastructure facility is supported by Canada Foundation for Innovation (CFI) and the British Columbia Knowledge Development Fund (BCKDF). JNK is the recipient of a Career Investigator Scholar award from Michael Smith Foundation of Health Research. REFERENCES 1. Tiller, J. C.; Liao, C. J.; Lewis K.; Klibanov A.M. Designing Surfaces That Kill Bacteria on Contact. Proc. Natl. Acad. Sci. U S A. 2001, 98, 5981-5985. 2. Kaur, R.; Liu S. Antibacterial Surface Design-Contact kill. Prog. Surf. Sci. 2016, 91, 136–153. 3. Klevens, R. M.; Edwards, J. R.; Richards Jr. C. L.; Horan, T. C.; Gaynes, R. P.; Pollock, D. A.; Cardo D. M. Estimating Health Care-Associated Infections and Deaths In U.S. Hospitals, 2002. Public Health Rep., 2007, 122, 160–166. 4. Ribeiro, M., Monteiro, F. J.; Ferraz M. P. Infection of Orthopedic Implants with Emphasis on Bacterial Adhesion Process and Techniques Used in Studying Bacterial-Material Interactions. Biomatter. 2012, 2, 176–194. 5. Stamm, W. E. Infections related to medical devices. Ann. Intern. Med., 1978, 89, 764–769. 6. Ahmed, S.; Darouiche, R. O. Anti‐Biofilm Agents in Control of Device‐Related Infections. Adv. Exp. Med. Biol. 2015, 831, 137–146. 7. Francolini, I.; Vuotto, C.; Piozzi, A.; Donelli, G. Antifouling and Antimicrobial Biomaterials: An Overview. APMIS 2017, 125 (4), 392–417. 8. Buxadera-Palomero, J.; Canal, C.; Torrent-Camarero, S.; Garrido, B.; Javier Gil, F.; Rodríguez, D. Antifouling Coatings for Dental Implants: Polyethylene Glycol-Like Coatings on Titanium by Plasma Polymerization. Biointerphases, 2015, 10, 029505.


22

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


9. Ngo, B. K. D.; Grunlan, M. A. Protein Resistant Polymeric Biomaterials. ACS Macro Lett. 2017, 6, 992–1000. 10. Dalsin, J. L.; Lin, L.; Tosatti, S.; Vörös, J.; Textor, M.; Messersmith, P. B. Protein Resistance of Titanium Oxide Surfaces Modified by Biologically Inspired mPEG-DOPA. Langmuir 2005, 21, 640–646. 11. Gillich, T.; Benetti, E. M.; Rakhmatullina, E.; Konradi, R.; Li, W.; Zhang, A., Schluter, A. D.; Textor, M. SelfAssembly of Focal Point Oligo-catechol Ethylene Glycol Dendrons on Titanium Oxide Surfaces: Adsorption Kinetics, Surface Characterization, and Nonfouling Properties. J. Am. Chem. Soc. 2011, 133, 10940–10950. 12. Fan, X.; Lin, L; Dalsin, J. L.; Messersmith, P. B. Biomimetic Anchor for Surface-Initiated Polymerization from Metal Substrates. J. Am. Chem. Soc. 2005, 127, 15843–15847. 13. Rodriguez-Emmenegger, C., Decker, A.; Surman, F.; Preuss, C. M.; Sedlakova, Z.; Zydziak, N.; Barner-Kowollik, C.; Schwartzd, T.; Barner, L. Suppressing Pseudomonas Aeruginosa Adhesion via Non-Fouling Polymer Brushes. RSC Adv. 2014, 4, 64781–64790. 14. Michelmore, A.; Gross-Kosche, P.; Al-Bataineh, S. A.; Whittle, J. D.; Short, R. D. On the Effect of Monomer Chemistry on Growth Mechanisms of Nonfouling PEG-Like Plasma Polymers. Langmuir, 2013, 29, 2595–2601. 15. Shen, M.; Wagner, M. S.; Castner, D. G.; Ratner; B. D.; Horbett, T. A. Multivariate Surface Analysis of PlasmaDeposited Tetraglyme for Reduction of Protein Adsorption and Monocyte Adhesion. Langmuir, 2003, 19, 1692–1699. 16. Li, Y.; Muir, B. W.; Easton, C. D.; Thomsen, L.; Nisbet, D. R.; Forsythe, J. S. A Study of the Initial Film Growth of PEG-Like Plasma Polymer Films via XPS and NEXAFS. Appl. Surf. Sci. 2014, 288, 288–294. 17. Mukherjee, I.; Ghosh, A.; Bhadury, P.; De, P. Leucine-Based Polymer Architecture-Induced Antimicrobial Properties and Bacterial Cell Morphology Switching ACS Omega 2018, 3, 769-780. 18. Mukherjee, I.; Ghosh, A.; Bhadury, P.; De, P. Matrix-Assisted Regulation of Antimicrobial Properties: Mechanistic Elucidation with Ciprofloxacin-Based Polymeric Hydrogel Against Vibrio Species Bioconjugate Chemistry 2019, 30, 218-230. 19. Huang, K. -S.; Yang, C. -H.; Huang, S. L.; Chen, C. Y.; Lu, Y. Y.; Lin, Y. S. Recent Advances in Antimicrobial Polymers: A Mini-Review. Int. J. Mol. Sci. 2016, 17, 1578. 20.. Okuda, K. I., Zendo, T.; Sugimoto, S.; Iwase, T.; Tajima, A.; Yamada, S.; Sonomoto, K.; Mizunoe, Y. Effects of Bacteriocins on Methicillin-Resistant Staphylococcus Aureus Biofilm. Antimicrob. Agents. Chemother. 2013, 57, 5572–5579. 21. Hirt, H.; Gorr, S. U. Antimicrobial Peptide GL13K is Effective in Reducing Biofilms of Pseudomonas Aeruginosa. Antimicrob. Agents. Chemother. 2013, 57, 4903–4910. 22. Gopal, R.; Lee, J. H.; Kim, Y. G.; Kim, M. S.; Seo, C. H.; Park, Y. Anti-Microbial, Anti-Biofilm Activities and Cell Selectivity of the NRC-16 Peptide Derived from Witch Flounder, Glyptocephalus Cynoglossus. Marine Drugs 2013, 11, 1836–1852. 23. Zhang, L.; Rozek, A.; Hancock, R. E. W. Interaction of Cationic Antimicrobial Peptides with Model Membranes. J. Biol. Chem. 2001, 276, 35714–35722. 24. Bahar, A. A.; Ren, D. Antimicrobial Peptides. Pharmaceuticals 2013, 6, 1543–1575. 25. Etienne, O.; Gasnier, C.; Taddei, C.; Voegel, J. -C.; Aunis, D.; Schaaf, P.; Metz-Boutigue, M. H.; BolcatoBellemin, A. L.; Egles, C. Antifungal Coating by Biofunctionalized Polyelectrolyte Multilayered Films. Biomaterials 2005, 26, 6704–6712. 26. Shukla, A.; Fleming, K. E.; Chuang, H. F.; Chau, T. M.; Loose, C. R.; Stephanopoulos, G. N.; Hammond, P. T. Controlling the Release of Peptide Antimicrobial Agents from Surfaces. Biomaterials 2010, 31, 2348–2357. 27. Onaizi, S. A.; Leong, S. S. Tethering antimicrobial peptides: current status and potential challenges. Biotechnol. Adv. 2011, 29, 67-74. 28. Bagheri, M.; Beyermann, M.; Dathe, M. Immobilization Reduces the Activity of Surface-Bound Cationic Antimicrobial Peptides with No Influence upon the Activity Spectrum. Antimicrob. Agents Chemother. 2009, 53, 1132–1141. 29. Ferreira, L.; Zumbuehl, A. Non-Leaching Surfaces Capable of Killing Microorganisms on Contact. J. Mater. Chem. 2009, 19, 7796–7806. 30. Brubaker, C. E.; Messersmith, P. B. The Present and Future of Biologically Inspired Adhesive Interfaces and Materials. Langmuir 2012, 28, 2200–2205. 31. Lee, H.; Dellatore, S. M.; Miller, W. M.; Messersmith, P. B. Mussel-Inspired Surface Chemistry for Multifunctional Coatings. Science 2007, 318, 426–430. 32. Black, K. C. L.; Liu, Z.; Messersmith, P. B. Catechol Redox Induced Formation of Metal Core-Polymer Shell Nanoparticles. Chem. Mater. 2011, 23, 1130–1135. 33. Lee, Y.; Lee, H.; Messersmith, P. B.; Park, T. G. A Bioinspired Polymeric Template for 1D Assembly of Metallic Nanoparticles, Semiconductor Quantum Dots, and Magnetic Nanoparticles. Macromol. Rapid Commun. 2010, 31, 2109–2114.


23


Page 24 of 25

34. Arslan, M.; Gevrek, T. N.; Lyskawa, J.; Szunerits, S.; Boukherroub, R.; Sanyal, R.; Woisel, P.; Sanyal, A.; Bioinspired Anchorable Thiol-Reactive Polymers: Synthesis and Applications Toward Surface Functionalization of Magnetic Nanoparticles. Macromolecules 2014, 47, 5124–5134. 35. Watson, M. A.; Lyskawa, J.; Zobrist, C.; Fournier, D.; Jimenez, M.; Traisnel, M.; Gengembre, L.; Woisel, P. A ‘Clickable’ Titanium Surface Platform. Langmuir 2010, 26, 15920–15924. 36. Faure, E.; Falentin-Daudréa, C.; Jérôme, C.; Lyskawa, J.; Fournier, D.; Woisel, P.; Detrembleur, C. Catechols as Versatile Platforms in Polymer Chemistry. Prog. Polym. Sci., 2013, 38, 236-270. 37. Ham, H.O.; Liu, Z., Lau, K. H. A., Lee, H., Messersmith, P. B. Facile DNA Immobilization on Surfaces through a Catecholamine Polymer. Angew. Chem. Int. Ed. 2011, 50, 732–736. 38. Mei, Y.; Yu, K.; Lo, J. C. Y.; Takeuchi, L. E.; Hadjesfandiari, N.; Yazdani-Ahmadabadi, H. Brooks, D. E.; Lange, D; Kizhakkedathu, J. N. Polymer-Nanoparticle Interaction as a Design Principle in the Development of a Durable Ultrathin Universal Binary Antibiofilm Coating with Long-Term Activity. ACS Nano 2018, 12, 11881-11891. 39. Xu, L.Q.; Jiang, H.; Neoh, K.-G.; Kang, E.-T.; Fu, G.D. Poly(Dopamine Acrylamide)-co-Poly(Propargyl Acrylamide)-Modified Titanium Surfaces for ‘Click’ Functionalization. Polymer Chemistry, 2012, 3, 920-927. 40. Peng, L.; Li, Z.; Li, X.; Xue, H.; Zhang, W.; Chen, G. Integrating Sugar and Dopamine into One Polymer: Controlled Synthesis and Robust Surface Modification, Macromol. Rapid. Commun. 2016, 38, 1600548. 41. Wen, M.; Liu, M.; Xue, W.; Yang, K.; Chen, G.; Zhang, W. Simple and Green Strategy for the Synthesis of “Pathogen-Mimetic” Glycoadjuvant@AuNPs by Combination of Photoinduced RAFT and Bioinspired Dopamine Chemistry. ACS Macro Lett. 2018, 7, 70–74. 42. Cengiz, N.; Gevrek, T. N.; Sanyal R.; Sanyal, A. Orthogonal Thiol–ene ‘Click’ Reactions: A Powerful Combination for Fabrication and Functionalization of Patterned Hydrogels. Chem. Commun., 2017, 53, 8894-8897. 43. Gevrek, T. N.; Bilgic, T.; Klok, H.-A.; Sanyal, A. Maleimide-Functionalized Thiol Reactive Copolymer Brushes: Fabrication and Post-Polymerization Modification. Macromolecules 2014, 47, 7842−7851. 44. Gok, O.; Kosif, I.; Dispinar, T.; Gevrek, T. N.; Sanyal, R.; Sanyal, A. Design and Synthesis of Water-Soluble Multifunctionalizable Thiol-Reactive Polymeric Supports for Cellular Targeting. Bioconjugate Chem. 2015, 26, 1550−1560. 45. Oz. Y.; Sanyal A. The Taming of the Maleimide: Fabrication of Maleimide-containing 'Clickable' Polymeric Materials. Chem Rec. 2018, 18, 570-586. 46. Gao, G.; Yu, K.; Kindrachuk, J.; Brooks, D. E.; Hancock, R. E. W.; Kizhakkedathu, J. N. Antibacterial Surfaces Based on Polymer Brushes : Investigation on The Influence of Brush Properties on Antimicrobial Peptide Immobilization and Antimicrobial Activity. Biomacromolecules 2011, 12, 3715-3722. 47. Park, S.; Lee, K. -B.; Choi, I. S.; Langer, R.; Jon, S. Dual Functional, Polymeric Self-Assembled Monolayers as a Facile Platform for Construction of Patterns of Biomolecules. Langmuir, 2007, 23, 10902-10905. 48. Gevrek, T. N.; Kosif, I.; Sanyal A. Surface-Anchored Thiol-Reactive Soft Interfaces: Engineering Effective Platforms for Biomolecular Immobilization and Sensing. ACS Appl. Mater. Interfaces 2017, 9, 27946−27954. 49. Lee, H.; Lee, B.P.; Messersmith, B.P. A Reversible Wet/Dry Adhesive Inspired by Mussels and Geckos. Nature 2007, 448, 338-341. 50. Dispinar, T.; Sanyal, R.; Sanyal, A. A Diels-Alder/Retro Diels-Alder Strategy to Synthesize Polymers Bearing Maleimide Side Chains. J. Polym. Sci., Part A: Polym. Chem. 2007, 45, 4545-4551. 51. Dübner, M.; Gevrek, T. N.; Sanyal, A.; Spencer, N. D.; Padeste, C. Fabrication of Thiol–Ene “Clickable” Copolymer-Brush Nanostructures on Polymeric Substrates via Extreme Ultraviolet Interference Lithography. ACS Appl. Mater. Interfaces, 2015, 7, 11337–11345. 52. Mitcova, L.; Buffeteau, T.; Le Bourdon, G.; Babot, O.; Vellutini, L.; Heuze, K. Positive Dendritic Effect on Maleimide Surface Modification of Core-Shell (γ-Fe2O3/Polymer) Nanoparticles for Bio Immobilization. ChemistrySelect 2016, 1, 4350 – 4356. 53. Gao, G. Z.; Lange, D.; Hilpert, K.; Kindrachuk, J.; Zou, Y. Q.; Cheng, J. T. J.; Kazemzadeh-Narbat, M.; Yu, K.; Wang, R. Z.; Straus, S. K.; Brooks, D. E.; Chew, B. H.; Hancock, R. E. W.; Kizhakkedathu, J. N. The Biocompatibility and Biofilm Resistance of Implant Coatings Based on Hydrophilic Polymer Brushes Conjugated with Antimicrobial Peptides. Biomaterials, 2011, 32, 3899−3909. 54. Yu, K.; Lai, B. F. L.; Gani, J.; Mikut, R.; Hilpert, K.; Kizhakkedathu, J. N. Interaction of Blood Components with Cathelicidins and Their Modified Versions. Biomaterials, 2015, 69, 201−211. 55. Xu L. Q.; Yang W. J.; Neoh, K. G.; Kang, E. T.; Fu, G. D. Dopamine-Induced Reduction and Functionalization of Graphene Oxide Nanosheets. Macromolecules 2010, 43, 8336–8339. 56. Glinel, K.; Jonas, A. M.; Jouenne, T.; Leprince, J.; Galas, L.; Huck, W. T. Antibacterial and Antifouling Polymer Brushes Incorporating Antimicrobial Peptide. Bioconjugate Chemistry 2009, 20, 71-77.


24

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


57. Statz, A. R.; Park, J. P.; Chongsiriwatana, N. P. Barron, A. E.; Messersmith, P. B. Surface-Immobilized Antimicrobial Peptoids Biofouling. 2008, 24, 439–448. 58. Yu, K.; Lo, J. C.; Yan, M.; Yang, X.; Brooks, D. E.; Hancock, R. E.; Lange, D.; Kizhakkedathu J. N. AntiAdhesive Antimicrobial Peptide Coating Prevents Catheter Associated Infection in a Mouse Urinary Infection Model. Biomaterials 2017, 116, 69-81.

TABLE OF CONTENT GRAPHIC


25

Thiol-Reactive Polymers for Titanium Interfaces ... - ACS Publications

Recommend Documents