Active Release of an Antimicrobial and Antiplatelet Agent from a

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Active release of an antimicrobial and antiplatelet agent from a non-fouling surface modification Marcus James Goudie, Priyadarshini Singha, Sean P. Hopkins, Elizabeth J. Brisbois, and Hitesh Handa ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b16819 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 6, 2019

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ACS Applied Materials & Interfaces

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Active release of an antimicrobial and antiplatelet agent from a nonfouling surface modification

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Marcus J. Goudie†,a, Priyadarshini Singha†,a, Sean P. Hopkinsa, Elizabeth J. Brisboisb, Hitesh Handa*a

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a School

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b Department

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of Chemical, Materials and Biomedical Engineering, College of Engineering, University of Georgia, Athens, GA, USA of Materials Science and Engineering, University of Central Florida, Orlando, FL,

USA † Authors

contributed equally to this work

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*Corresponding Author:

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Dr. Hitesh Handa

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Assistant Professor

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University of Georgia

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220 Riverbend Road

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Athens, GA 30602

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Telephone: (706) 542-8109

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E-mail: [email protected]

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Keywords: nitric oxide, antimicrobial, antithrombotic, antifouling, surface chemistry

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Abstract

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Two major challenges faced by medical devices are thrombus formation and infection. In this

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work, surface tethered nitric oxide (NO) releasing molecules are presented as a solution to combat

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infection and thrombosis. These materials possess a robust NO release capacity lasting ca. 1 month

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while simultaneously improving the non-fouling nature of the material by preventing platelet,

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protein, and/or bacteria adhesion. Nitric oxide’s potent bactericidal function has been implemented

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by a facile surface covalent attachment method to fabricate a triple action surface - surface

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immobilized S-nitroso-N-acetylpenicillamine (SIM-S). Comparison of NO loading amongst the

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various branching configurations is shown through the NO release kinetics over time and the

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cumulative NO release. Biological characterization is performed using in vitro fibrinogen and

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Staphylococcus aureus assays. The material with the highest NO release, SIM-S2, is also able to

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reduce protein adhesion by 65.8 ± 8.9% when compared to unmodified silicone. SIM-S2

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demonstrates a 99.99% (i.e. ~4 log) reduction for Staphylococcus aureus over 24 h. The various

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functionalized surfaces significantly reduce platelet adhesion in vitro, for both NO releasing and

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non-NO releasing surfaces (up to 89.1 ± 0.9%), demonstrating the non-fouling nature of the surface

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immobilized functionalities. The ability of the SIM-S surfaces to retain antifouling properties

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despite gradual depletion of the bactericidal source, NO, demonstrates its potential use in long

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term medical implants.


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Introduction

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Fouling of materials used in medical devices leads to increased risk of infection and device

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failure, and is the result of the adherence of proteins, bacteria, or thrombus formation. These

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complications can lead to large increases in healthcare costs and mortality.1-2 While systemic

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heparin is often administered to aid in the prevention of thrombus formation, its prolonged use can

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result in morbidity and mortality while providing no activity to prevent infection.3 In addition to

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this, the increasing use of antibiotics has led to the development of resistant strains of bacteria,

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which can be attributed to the high dosages required to be effective against established biofilms.4

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The degree of bacteria or platelet adhesion is highly influenced by the ability to prevent protein

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adhesion to the materials surface.5-7 A number of strategies have been used to develop adhesion

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resistant materials,8 such as increasing the hydrophilicity,5,

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surfaces,10-13 liquid-infused materials,14-16 or grafting of polymer brushes.17-24

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materials are suitable for decreasing the adhesion of protein and bacteria through “passive”

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mechanisms, they provide no “active” mechanism to prevent platelet activation and adhesion or

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any bactericidal activity towards bacteria that have adhered, which ultimately leads to proliferation

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and biofilm formation.

9

super hydrophobic or patterned While these

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Nitric oxide (NO) is an endogenous, gaseous, free radical that is produced naturally by

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macrophages and by endothelial cells lining the vascular walls, and is involved in various

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biological processes, such as preventing platelet activation and adhesion, while also being a potent,

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broad spectrum bactericidal agent.25 To take advantage of these properties, NO donors (e.g. S-

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nitrosothiols or diazeniumdiolates) have been developed to allow for the storage and localized

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delivery of NO, and are particularly advantageous for polymeric materials typically used for

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medical devices, such as polyurethanes, silicones, or polyvinyl chloride.26-27 The addition of these



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donors at various levels also provides a simple method for controlling the level of NO that is

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delivered from the materials.28 Materials releasing NO have been shown to significantly reduce

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thrombus formation in both extracorporeal circuits and vascular catheter models, and have been

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shown to provide significant reductions in viable bacteria during long term catheterization.2, 29-30

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In this work, a novel method to prepare a material surface with the ability to not only offer

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an improved steric ability to prevent platelet and protein adhesion through a passive mechanism,

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but also utilize the active biocidal mechanism of NO. In addition to combining these mechanisms,

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the antifouling capability of the material is retained after the entire NO payload has been released

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from the surface, making it attractive for long-term applications. Specifically, this is achieved by

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the immobilization of the NO donor precursor N-acetyl-D-penicillamine (NAP) to various amine

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functionalized silicone surfaces (Figure 1A).

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Materials and Methods

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Materials: N-Acetyl-D-penicillamine (NAP), sodium chloride, potassium chloride, sodium

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phosphate dibasic, potassium phosphate monobasic, phosphate buffered saline (PBS, pH 7.4 at

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25°C), ethylenediaminetetraacetic acid (EDTA), tetrahydrofuran (THF), tert-butyl nitrite, and

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sulfuric acid were purchased from Sigma-Aldrich (St. Louis, MO). Aminopropyl trimethoxy silane

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(APTMES) was purchased from Gelest.

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polydimethylsiloxane Sylgard 184 (Dow Corning). Methanol, hydrochloric acid, and sulfuric acid

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were obtained from Fisher Scientific (Pittsburgh, PA).

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modification of Eagle’s medium (DMEM) were obtained from Corning (Manassas, VA 20109).

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The bacterial Staphylococcus aureus (ATCC 5538) strain was obtained from American Type

All silicone substrates were fabricated with


Trypsin-EDTA and Dulbecco’s

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86

Culture Collection (ATCC). Luria Agar (LA), Miller and Luria broth (LB), Lennox were

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purchased from Fischer BioReagents (Fair Lawn, NJ). All aqueous solutions were prepared with

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18.2 MΩ deionized water using a Milli-Q filter (Millipore Corp., Billerica, MA).

89 90

Synthesis of SIM material: Silicone films were first fabricated by mixing Sylgard 184 base to

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curing agent (ratio of 10:1). The solution was cast into Teflon molds and placed under vacuum for

92

degassing. The casted solution was then placed in an oven (80°C, 90 min) for curing. To create a

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hydroxyl group functionalized surface, the silicone films were submerged in a mixture of 13 N

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HCl : 30 wt.% H2O2 (50:50) in H2O under mild agitation (15 min). The surfaces were then rinsed

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with DI H2O and dried under vacuum. The amine functionalization was then achieved by

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submerging the hydroxyl-functionalized surfaces in 5 wt.% APTMES in extra dry acetone for 2 h.

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Films were then rinsed with extra dry acetone to remove any non-covalently attached silane from

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the surface, and vacuum dried for 24 h. Branching of the immobilized moieties was achieved

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through incubation of the amine functionalized surface in 2:1 (v/v) methanol:methyl acrylate (24

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h) followed by 2:1 (v/v) methanol: ethylenediamine (24h) as shown in Figure 1A. Samples were

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rinsed twice with methanol (20 mL) between incubating solutions. Amine-functionalized surfaces

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were then submerged in 10 mg mL-1 NAP-thiolactone in toluene for 24 h, allowing for the ring

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opening reaction of thiolactone to bind to free amines.31-32 The samples were then air-dried for 5

104

h to completely remove any residual solvent. Nitrosation of the immobilized NAP was achieved

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by incubation in neat tert-Butyl nitrite for 2 h. The resultant SIMS samples were stored at -20°C

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for further experiments.

107



108

Contact angle and FTIR analysis: Surface properties and proof of attachment of nitric oxide

109

donors to silicone surfaces was analyzed using contact angle measurements and FTIR. Static

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contact angle was measured using a DSA 100 drop shape analysis system (KRÜSS) with a

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computer-controlled liquid dispensing system (Krüss). A 3 µL droplet of water was placed on

112

various silicone films, and the average of left and right contact angles were measured via the Krüss

113

software. Infrared spectroscopy studies of the samples were done using a Thermo-Nicolet model

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6700 spectrometer with a grazing angle attenuated total reflectance accessory at 64 scans with a 6

115

cm−1 resolution.

116 117

Nitric Oxide Release Characteristics: Nitric oxide release from the films containing SNAP was

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measured using a Sievers Chemiluminescence Nitric Oxide Analyzer (NOA) 280i (Boulder, CO).

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The Sievers chemiluminescence Nitric Oxide analyzer is considered as the gold standard for

120

detecting nitric oxide and is widely used due to its ability to limit interfering species, such as

121

nitrates and nitrites, as they are not transferred from the sample vessel to the reaction cell. Films

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were then placed in the sample vessel immersed in PBS (pH 7.4, 37°C) containing 100 µM EDTA.

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Nitric oxide was continuously purged from the buffer and swept from the headspace using nitrogen

124

sweep gas and bubbler into the chemiluminescence detection chamber.

125

Samples were analyzed for NO release and stored in the same conditions as found physiologically

126

for medical implants/devices (shielded from light and at 37 °C). For each measurement, NO release

127

was allowed to plateau so that burst effect of NO release was not included in the average flux for

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each time point measurement (approximately 0.5 h for each measurement). The detection limit

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of the NOA for measurement of gas-phase NO is ~0.5 part per billion by volume (~1 picomole).


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Thiol quantification by Ellman's assay: The covalent attachment of NAP-thiolactone can be

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directly related to the amount of free sulfhydryl groups on the surface of the PDMS. Measurement

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of these functional groups was done using Ellman's assay, which reacts 5,5'-dithio-bis-(2-

134

nitrobenzoic acid) (DTNB) with free sulfhydryl groups to form conjugated disulfide and 2-nitro-

135

5-thiobenzoic acid (TNB). While in solution, TNB's extinction coefficient has been recorded to be

136

14,150M-1 at 412 nm. Surface functionalized films were first cut to a recorded surface area before

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being placed in a solution containing 50 µL of DNTB stock solution (50 mM sodium acetate and

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2 mM DTNB in DI water), 100 µL of PBS, and 850 µL of DI water. The samples were then

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thoroughly mixed and allowed to incubate at room temperature for 5 minutes. Optical absorbance

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was then recorded at 412 nm using a UV-Vis spectrophotometer. A standard calibration curve

141

using acetyl cysteine was made to correlate thiol concentration with absorbance.

142 143

Protein Repulsion Quantification: Levels of protein adhesion were quantified for the various

144

materials using a modified version of a previously reported method.7 FITC-human fibrinogen (13

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mg/mL, Molecular Innovations) was diluted to achieve 2 mg mL-1 in PBS (pH 7.4). Silicone disks

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were incubated at 37°C for 30 min in a 96-well plate, followed by the addition of the stock protein

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solution to achieve a concentration of 2 mg mL-1.7 Following 2h of incubation, infinite dilution of

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the well contents was carried out to wash away the bulk and any loosely bound protein from the

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materials. The fluorescence of each well (n=8) was then measured using a 96-well plate reader

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(Biotek Cytation 5), and the amount of protein adsorbed was determined via a calibration curve.

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The excitation and emission wavelength for FITC are 495 and 519 nm.

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Bacterial Adhesion Assay: The ability of the samples to inhibit growth and promote killing of the

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adhered bacteria on the polymer surface was tested following guidelines based on American

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Society for Testing and Materials E2180 protocol with the commonly found nosocomial pathogen,

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Gram-positive S. aureus (ATCC 6538). A single colony of bacteria was isolated from a previously

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cultured LB-agar plate and incubated in LB Broth (37°C,150 rpm, 14-16h). The optical density of

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the culture was measured at a wavelength of 600 nm using a UV-vis spectrophotometer

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(Thermoscientific Genesys 10S UV-Vis) to ensure the presence of ~108 CFU mL-1. The overnight

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culture was then centrifuged at 2500 rpm for 7 min to obtain the bacterial pellet. The bacterial

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pellet obtained was resuspended in sterile PBS. The polymer samples (SR control, SIM-N1, SIM-

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S1, SIM-N2 and SIM-S2) were then incubated in the bacterial suspension (37°C, 24h, 140 rpm).

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After incubation, samples were removed from the bacterial suspension and rinsed with sterile PBS

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to remove any unbound bacteria. They were then sonicated for 1 min each using an Omni Tip

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homogenizer for 1 min to collect adhered bacteria in sterile PBS. To ensure proper homogenization

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of the collected bacteria, the samples were vortexed for 45s each. The solutions were serially

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diluted, plated on LB agar medium and incubated at 37°C. After 24h, the total CFUs for serially

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diluted and plated bacterial solutions were counted.

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A second antimicrobial test was done, in addition to the one mentioned above, to evaluate the

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robustness of the NO-releasing materials when compared to antifouling materials. In this method,

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all steps were followed according to the process mentioned above except for the addition of an

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initial 24 h incubation in fibrinogen (2 mg/mL) from human serum. This incubation step was added

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before the bacterial incubation step due to the sequence of exposure seen in physiological

174

conditions.33 The results are shown on Figure S3.


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The efficiency of the sample to inhibit bacterial attachment was calculated according to the

176

following formula shown in Equation 1.

177

𝐵𝑎𝑐𝑡𝑒𝑟𝑖𝑎 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 (%) =

(𝐶𝐹𝑈 𝑐𝑚 ―2 𝑜𝑛 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑠𝑎𝑚𝑝𝑙𝑒 ― 𝐶𝐹𝑈 𝑐𝑚 ―2 𝑜𝑛 𝑡𝑒𝑠𝑡 𝑠𝑎𝑚𝑝𝑙𝑒 ) (𝐶𝐹𝑈 𝑐𝑚 ―2 𝑜𝑛 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑠𝑎𝑚𝑝𝑙𝑒)

× 100

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(Equation 1)

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Platelet Adhesion Assay: Freshly drawn citrated (3.8%, 9:1 citrate: whole blood) porcine blood

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was purchased from Lampire Biologicals. The anticoagulated blood was centrifuged (1100 rpm,

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12 min) using the Eppendorf Centrifuge 5702. The platelet rich plasma (PRP) portion was

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collected carefully with a pipet as to not disturb the buffy coat. The remaining samples were then

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centrifuged (4000 rpm, 20 min) to retrieve platelet poor plasma (PPP). Total platelet counts in both

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PRP and PPP fractions were determined using a hemocytometer (Fisher). The PRP and PPP were

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combined in a ratio to give a final platelet concentration ca. 2 x 108 mL-1. Calcium chloride (CaCl2)

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was added to the final platelet solution to achieve a final concentration of 2.5 mM.7 Disks of each

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respective surface were placed in a 5 mL blood tube. Approximately 4 mL of the calcified PRP

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was added to each tube and incubated (37°C, 90 min) with mild rocking (25 rpm). Following the

189

incubation, the tubes were infinitely diluted with normal saline. The degree of platelet adhesion

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was determined using the lactate dehydrogenase (LDH) released when the adherent platelets were

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lysed with a Triton-PBS buffer using a Roche Cytotoxicity Detection Kit (LDH). The silicone

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disks were then incubated in 1 mL of Triton-PBS buffer. After 25 min, 100 μL of the buffer was

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transferred to a 96-well plate and combined with 100μL of the LDH reagent buffer per the supplier

194

specifications. The absorbance of each well (duplicates of n=6,492 nm and 690 nm) was further

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duplicated) was then measured using a 96-well plate reader (Biotek Cytation 5), and the number

196

of platelets adhered was determined using the calibration curve.



197 198

Results and Discussion

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Functionalization and characterization of silicone surfaces for non-fouling coatings with active

200

release of NO

201

A detailed schematic of the reactions used for branching of the initial alkyl-amine spacer

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is included in Supplement Figure S1. Briefly, the increasing grafting density of free amines was

203

done through branching free amines using sequential reactions of 1:2 methyl acrylate: methanol

204

and 1:2 ethylene diamine:methanol for 24 h. The surfaces were then incubated for 24 h in 10

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mg/mL NAP-thiolactone (dissolved in toluene). Following immobilization, the free thiols of the

206

grafted donor are nitrosated to its NO-rich form S-nitroso-N-acetyl-D-penicillamine (SNAP) using

207

tert-butyl nitrite. To ensure covalent bonding of the surface modifications, FTIR measurements

208

were carried out (Figure 1B). Since nitrogen atoms overlap in terms of FTIR peaks, appearance

209

and disappearance of amide and primary amines were observed as the reaction steps were

210

completed. This was followed by measurement of water contact angle (Table 1) to evaluate any

211

significant differences in hydrophilicity of the functionalized surface. It is interesting to note here

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that hydrophilicity of the surfaces increased with increased NO-release (will be discussed in the

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next section). This could be attributed to lower availability of amine functionalized surfaces as the

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reaction is more complete.

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As seen in the design strategy, the NO-load and release capacity of the materials was varied

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by branching of the initial alkyl spacer to increase the number of free amines. This variation in

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NO-load and release capacity was measured by using a chemiluminescence nitric oxide analyzer

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(NOA). The NOA is the gold standard for measurement of NO flux from materials and is a very


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efficient and sensitive instrument which can analyze NO release down to 1/10th of ppb.34 Samples

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are analyzed for NO release by shielding them from light and incubated in a phosphate buffered

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saline at 37 °C. These parameters mimic the physiological conditions of indwelling medical

222

devices and have been used as a guideline in previous research.5,

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expectations was to see increasing NO-load and release measurements with an increase in

224

branching. However, as seen in Figure 2, NO release measurements were significantly higher for

225

SIM-S2 (cumulative release: 4. 34 ± 0.04 μmol cm-2) when compared to SIM-S4 (cumulative

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release: 2.33 ± 0.03 μmol cm-2) over the 25-d period. Tabulated values for instantaneous and

227

cumulative NO release are in Supplementary Tables S1 and Table S2, respectively, and were

228

comparable to total free thiol content as measured via Ellman’s assay (Figure S2).37 There could

229

be two possible explanations for this: steric hindrance in the case of higher branching and hence

230

NAP thiolactone was not able to completely bind to the amine groups, and/or more branching

231

increases the probability of chain interactions within the polymer during reactions with ethylene

232

diamine, decreasing the total free amines available for binding with NAP thiolactone. Therefore,

233

the conclusion from this study was that increased branching does not necessarily increase the total

234

NO-load or release. Further, this enables the designed surface to release NO up to 25 d at effective

235

flux levels (ability to reduce platelet activation and bacterial adhesion significantly).5, 35-36 This

236

increasing branching method is a novel technique to increase NO release characteristics much like

237

the function of metal ions when added to NO releasing polymers.38 However, this material proves

238

to be more advantageous as it also imparts antifouling characteristics to the material as seen in the

239

following studies conducted. To ensure NO release was only from the surface functionalization

240

and not from the bulk material due to possible swelling of the diamine group during the reaction

241

period, control measurements were done on samples using the same reaction scheme without


35-36

One of the theoretical


242

immobilization of the aminosilane. The results are not shown in figures or tables since no NO

243

release was observed without the immobilization of the aminosilane. This proved that the

244

aminosilanes immobilization was a necessary step for NO release and that NO release was not due

245

to any swelling of the material from the solvents/reactions used for immobilization of the NO

246

donor.

247

Assessment of the Non-fouling nature of various SIM surfaces against protein, bacteria, and

248

platelet rich plasma

249

One common method for assessing the fouling of materials in vitro is to examine the ability

250

of the material to resist non-specific protein adhesion, or if intended for blood contacting

251

applications more specifically, fibrinogen (Fg). The adsorption of Fg to the material surface greatly

252

aids in the ability for activated platelets or bacteria to bind to the surface, leading to higher risks

253

of thrombus formation or infection.6 While the orientation of Fg adsorption has been shown to

254

determine the degree of platelet adhesion, limiting protein adhesion regardless of orientation is

255

generally considered to be an improvement in the hemocompatibility of a material.7 Developing

256

NO-releasing materials that can reduce protein adsorption could provide drastic improvements in

257

the overall hemocompatibility and antibacterial nature of these materials. To examine if the surface

258

immobilized NO donors (both nitrosated and non-nitrosated) can provide a decrease in protein

259

adhesion observed on NO-releasing materials, 2 h exposure to FITC-labeled fibrinogen (2 mg mL-

260

1)

261

observed, increasing the branched nature of the surface grafted NAP groups decreased the degree

262

of Fg adsorption, and is hypothesized to result from increases in steric hindrance.34 However,

263

altering the chemistry of the linkages to the amine functionalized surface could greatly increase

264

the non-fouling ability of these materials. Overall, reductions in protein adsorption were observed

was conducted at 37°C (Figure 3A, Table 2). While minimal changes in contact angle were


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to reach 65.8 ± 8.9% for SIM-S2 when compared to the unmodified SR. It is also interesting to

266

note that the release of NO from the surface had no significant effect on the amount of adsorbed

267

Fg. Previous reports have suggested that increasing NO release levels from other NO donors lead

268

to increased fibrinogen adsorption, where additions of 1 and 9.2 wt.% N-diazeniumdiolated

269

dibutyl-hexanediamine were added to polyvinylchloride films.39 The NO release levels of 10 x10-10

270

mol cm-2 and 15 x10-10 mol cm-2 were shown to increase protein adsorption compared to PVC

271

(226 ± 99% and 2334 ± 496%, respectively). However, extensive studies regarding the interaction

272

of proteins and various NO donors have yet to be studied, specifically the differences between S-

273

nitrosothiols and diazeniumdiolates, as well as a broader examination of the levels of NO release.

274

However, the immobilized SIM-S surfaces demonstrate the effectiveness of the surface

275

immobilized NO donors as providing active NO release while decreasing protein adsorption.

276

Bacterial adhesion, which ultimately results in biofilm formation, is a predominant issue

277

for implanted devices aided by the moist and microbiome sustaining milieu. Coupled with fouling

278

proteins, implants can become hosts to several pathogens that ultimately leads to medical device

279

failure, infection (including bloodstream infection), and sometimes death.8 Antimicrobial efficacy

280

of the designed non-fouling antimicrobial coating SIM-S materials was compared to the SR control

281

samples to confirm their superior bacterial repulsion properties. The samples were incubated in

282

bacterial solutions containing ~108 CFU mL-1 of S. aureus, which is one of the most commonly

283

found nosocomial infection bacteria.35, 40 These infections are most commonly associated with

284

catheters, stents, and prosthetic devices among other implants. As mentioned in the Introduction

285

Section, our hypothesis was that the immobilized structure was expected to repel proteins and

286

bacteria while simultaneously releasing NO to actively kill bacteria, thus enhancing the

287

biocompatibility of the material even after all the NO load was depleted. The antimicrobial efficacy



288

of the SIM-S surfaces was clearly observed after 24 h of incubation, the crucial time for initiation

289

of bacterial adhesion on the surface that leads to infection. The CFU cm-2 of viable S. aureus

290

adhered to each sample was determined by plate counting (Figure 3B). SIM-S2 showed the highest

291

bactericidal efficiency with a reduction of 99.99 ±0.002 % (Table 3) when compared to the SR

292

samples, where a growth of ~108 CFU cm-2 was observed. This reduction is higher as compared

293

to samples with only NAP thiolactone functionalization (SIM-N1= 82.14 ±22.20 % and SIM-N2=

294

96.86 ±0.50 %) and SIM-S1 (85.71 ±24.74 %). It can also be concluded from the results that NAP

295

thiolactone functionalized surfaces alone only reduces bacteria adhesion because it cannot kill

296

bacteria as it does not have any bactericidal property. However, the presence of protein is also

297

known to drastically increase the bacterial adhesion, particularly those associated with medical

298

devices. To further demonstrate the robustness of the antimicrobial activity of NO-releasing SIM-S

299

surfaces and compare to only non-fouling SIM-N surfaces, in the presence of protein attachment

300

similar to the methods described by Smith et. al (details in Supplementary section, Figure S3).41

301

These surfaces functionalized with NO-releasing moieties make effective bactericidal-releasing

302

coatings which significantly enhance the antimicrobial efficacy. From the protein and bacterial

303

adhesion assays, the varying effects of the modifiable NO-release kinetics from SR surface were

304

observed which in turn can help significantly reduce dire clinical consequences of a medical

305

implantation.

306

Platelet activation and adhesion are important considerations when determining the

307

hemocompatibility of materials. Upon activation, platelets release several coagulation agonists,

308

such as phospholipase A2 (which is then converted into thromboxane A2), which further increase

309

platelet activation, the coagulation cascade, and thrombin generation.42 One key predecessor of

310

platelet activation is the adsorption of fibrinogen to the materials surface, where changes in the


Page 14 of 32

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311

protein conformation allows for binding to the Gp IIb/IIIa receptors on platelets. Therefore,

312

reducing protein adhesion alone can act as a mechanism to reduce platelet activation. The aim of

313

this study was to confirm that while NO-releasing SIM-S materials have been shown to

314

significantly reduce platelet adhesion, the functionality of the modified surface is not lost after all

315

the NO payload has been released. In fact, small molecules with free thiol groups (similar to NAP)

316

have been shown to provide potent thrombolytic effects when administered systemically by

317

binding to von Willebrand factor crosslinks of adhered platelets in arterial thrombi.43 Both

318

nitrosated and non-nitrosated surfaces were incubated in porcine platelet rich plasma for 90 min,

319

where the degree platelet adhesion was then determined using a lactate dehydrogenase (LDH)

320

assay (Figure 3C). Each variation of the surface modifications was able to provide significant

321

reductions in platelet adhesion when compared to unmodified SR controls (Table 4). The SIM-N1

322

and SIM-S2 modifications provided the highest reductions, but were not statistically significant

323

when compared to each other (p value > 0.5). However, as the brush size increased from the SIM-

324

N1 to the SIM-N2 configuration, the ability to prevent platelet adhesion begins to decrease (p =

325

0.001). This may stem from the surface wetting characteristics of the methacrylate and diamine

326

linkages, as a decrease in contact angle from 94.36 to 64.95 ± 11.87 was observed. The hypothesis

327

is that the increase in platelet adhesion stems from the confirmation of fibrinogen adsorption, even

328

though the overall protein adsorption was decreased.7, 44 While the SIM-S2 configuration did not

329

provide significant reductions in platelet adhesion when compared to SIM-N1 or SIM-S1, the

330

significant increase in NO release can provide increased bactericidal activity for extended

331

durations.

332

Conclusion



333

In summary, this study presents a facile method to attach various amounts of the NO-

334

releasing donor, SNAP, to any polymer material in order to provide both bactericidal/antiplatelet

335

activity while adding a non-fouling nature to the material surface. Through the covalent

336

attachments, the NO release characteristics from the modified surface were tunable by varying the

337

branching linkers for NO donating structures. This enabled detailed analysis of the protein

338

repelling, antithrombotic, and antibacterial properties of each of the modified surfaces that had

339

varying degrees of NO release for different spans of time. It is a significant progress towards

340

quantitatively controlling the release of NO and hence use this technique to further study NO-

341

releasing properties for applications including microfluidic devices for high-throughput assays.

342

This method will also be highly applicable for biomedical device materials that are prone to

343

infection- and thrombosis-related failures, and can easily be coupled with existing NO-releasing

344

polymers.

345 346

Supporting Information

347

Figure S1: Detailed schematic of various SIM surfaces

348 349

Figure S2: A) Quantification of free thiols on various SIM surfaces. B) Calibration curve of free thiols

350 351

Figure S3: CFU of S. aureus per cm2 of sample after 24 h exposure to fibrinogen from human serum followed by 24 h of S. aureus incubation under physiological conditions (n=5)

352

Table S1: Day by day NO release measurements for 600 h/25 d. (n=3)

353

Table S2: Cumulative NO release over 600 h/25 d. (n=3)

354 355 356

Acknowledgements


Page 16 of 32

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357

M.G. and P.S. contributed equally to this work. The authors acknowledge the financial support of

358

the National Institutes of Health (K25HL111213 and R01HL134899), and the University of

359

Georgia start-up funds. MG would like to thank the ARCS Foundation of Atlanta for their support.

360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383

References



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1. Ratner, B. D., The Catastrophe Revisited: Blood Compatibility in the 21st Century. Biomaterials 2007, 28 (34), 5144-5147. 2. Brisbois, E. J.; Davis, R. P.; Jones, A. M.; Major, T. C.; Bartlett, R. H.; Meyerhoff, M. E.; Handa, H., Reduction in Thrombosis and Bacterial Adhesion with 7 day Implantation of S-nitroso-Nacetylpenicillamine (SNAP)-Doped Elast-eon E2As Catheters in Sheep. Journal of Materials Chemistry B 2015, 3, 1639-1645. 3. Cronin, R. E.; Reilly, R. F. In Unfractionated Heparin for Hemodialysis: Still the Best Option, Seminars in Dialysis, Wiley Online Library: 2010; 510-515. 4. Høiby, N.; Ciofu, O.; Johansen, H. K.; Song, Z.-j.; Moser, C.; Jensen, P. Ø.; Molin, S.; Givskov, M.; Tolker-Nielsen, T.; Bjarnsholt, T., The Clinical Impact of Bacterial Biofilms. International journal of oral science 2011, 3 (2), 55. 5. Singha, P.; Pant, J.; Goudie, M. J.; Workman, C. D.; Handa, H., Enhanced Antibacterial Efficacy of Nitric Oxide Releasing Thermoplastic Polyurethanes with Antifouling Hydrophilic Topcoats. Biomaterials Science 2017. 6. Charville, G. W.; Hetrick, E. M.; Geer, C. B.; Schoenfisch, M. H., Reduced Bacterial Adhesion to Fibrinogen-Coated Substrates via Nitric Oxide Release. Biomaterials 2008, 29 (30), 4039-4044. 7. Sivaraman, B.; Latour, R. A., The Relationship Between Platelet Adhesion on Surfaces and the Structure Versus the Amount of Adsorbed Fibrinogen. Biomaterials 2010, 31 (5), 832-839. 8. Singha, P.; Locklin, J.; Handa, H., A Review of the Recent Advances in Antimicrobial Coatings for Urinary Catheters. Acta Biomaterialia 2017, 50, 20-40. 9. Xu, L.-C.; Siedlecki, C. A., Effects of Surface Wettability and Contact Time on Protein Adhesion to Biomaterial Surfaces. Biomaterials 2007, 28 (22), 3273-3283. 10. Hou, X.; Wang, X.; Zhu, Q.; Bao, J.; Mao, C.; Jiang, L.; Shen, J., Preparation of Polypropylene Superhydrophobic Surface and its Blood Compatibility. Colloids and Surfaces B: Biointerfaces 2010, 80 (2), 247-250. 11. Ueda, E.; Levkin, P. A., Micropatterning Hydrophobic Liquid on a Porous Polymer Surface for Long-Term Selective Cell-Repellency. Advanced healthcare materials 2013, 2 (11), 1425-1429. 12. Xu, L. C.; Siedlecki, C. A., Protein Adsorption, Platelet Adhesion, and Bacterial Adhesion to Polyethylene-Glycol-Textured Polyurethane Biomaterial Surfaces. Journal of Biomedical Materials Research Part B: Applied Biomaterials 2017, 105 (3), 668-678. 13. Falde, E. J.; Yohe, S. T.; Colson, Y. L.; Grinstaff, M. W., Superhydrophobic Materials for Biomedical Applications. Biomaterials 2016, 104, 87-103. 14. MacCallum, N.; Howell, C.; Kim, P.; Sun, D.; Friedlander, R.; Ranisau, J.; Ahanotu, O.; Lin, J. J.; Vena, A.; Hatton, B., Liquid-Infused Silicone as a Biofouling-free Medical Material. ACS Biomaterials Science & Engineering 2014, 1 (1), 43-51. 15. Leslie, D. C.; Waterhouse, A.; Berthet, J. B.; Valentin, T. M.; Watters, A. L.; Jain, A.; Kim, P.; Hatton, B. D.; Nedder, A.; Donovan, K., A Bioinspired Omniphobic Surface Coating on Medical Devices Prevents Thrombosis and Biofouling. Nature biotechnology 2014, 32 (11), 1134-1140. 16. Goudie, M. J.; Pant, J.; Handa, H., Liquid-Infused Nitric Oxide-Releasing (LINORel) Silicone for Decreased Fouling, Thrombosis, and Infection of Medical Devices. Scientific Reports 2017, 7, 13623. 17. Alcantar, N. A.; Aydil, E. S.; Israelachvili, J. N., Polyethylene Glycol-Coated Biocompatible Surfaces. Journal of biomedical materials research 2000, 51 (3), 343-351. 18. Holmberg, K.; Bergström, K.; Stark, M.-B., Immobilization of Proteins via PEG Chains. In Poly (Ethylene Glycol) Chemistry, Springer: 1992, pp 303-324. 19. Gölander, C.-G.; Herron, J. N.; Lim, K.; Claesson, P.; Stenius, P.; Andrade, J., Properties of Immobilized PEG Films and the Interaction with Proteins. In Poly (ethylene glycol) Chemistry, Springer: 1992, pp 221-245.


Page 18 of 32

Page 19 of 32 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

431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477


20. Lee, J. H.; Lee, H. B.; Andrade, J. D., Blood Compatibility of Polyethylene Oxide Surfaces. Progress in Polymer Science 1995, 20 (6), 1043-1079. 21. Andrade, J.; Hlady, V.; Jeon, S.-I., Polyethylene Oxide and Protein Resistance: Principles, Problems, and Possibilities. Polymeric Materials: Science and Engineering 1993, 60-61. 22. Harris, J. M., Poly (ethylene glycol) Chemistry: Biotechnical and Biomedical Applications. Springer Science & Business Media: 1992. 23. Brisbois, E. J.; Handa, H.; Meyerhoff, M. E., Recent Advances in Hemocompatible Polymers for Biomedical Applications. In Advanced Polymers in Medicine, Springer: 2015, pp 481-511. 24. Kolobow, T.; Stool, E.; Weathersby, P.; Pierce, J.; Hayano, F.; Suaudeau, J., Superior blood Compatibility of Silicone Rubber Free of Silica Filler in the Membrane Lung. Transactions-American Society for Artificial Internal Organs 1974, 20, 269-276. 25. De Groote, M. A.; Fang, F. C., NO inhibitions: Antimicrobial Properties of Nitric Oxide. Clinical Infectious Diseases 1995, 21 (Supplement 2), S162-S165. 26. Goudie, M. J.; Brisbois, E. J.; Pant, J.; Thompson, A.; Potkay, J. A.; Handa, H., Characterization of an S-nitroso-N-acetylpenicillamine–Based Nitric Oxide Releasing Polymer from a Translational Perspective. International Journal of Polymeric Materials and Polymeric Biomaterials 2016, 65 (15), 769778. 27. Brisbois, E. J.; Major, T. C.; Goudie, M. J.; Meyerhoff, M. E.; Bartlett, R. H.; Handa, H., Attenuation of Thrombosis and Bacterial Infection Using Dual Function Nitric Oxide Releasing Central Venous Catheters in a 9day Rabbit Model. Acta Biomaterialia 2016, 44, 304-312. 28. Vaughn, M. W.; Kuo, L.; Liao, J. C., Estimation of Nitric Oxide Production and Reaction Rates in Tissue by Use of a Mathematical Model. American Journal of Physiology-Heart and Circulatory Physiology 1998, 274 (6), H2163-H2176. 29. Brisbois, E. J.; Major, T. C.; Goudie, M. J.; Bartlett, R. H.; Meyerhoff, M. E.; Handa, H., Improved Hemocompatibility of Silicone Rubber Extracorporeal Tubing via Solvent Swelling-Impregnation of Snitroso-N-acetylpenicillamine (SNAP) and Evaluation in Rabbit Thrombogenicity Model. Acta biomaterialia 2016, 37, 111-119. 30. Brisbois, E. J.; Major, T. C.; Goudie, M. J.; Meyerhoff, M. E.; Bartlett, R. H.; Handa, H., Attenuation of Thrombosis and Bacterial Infection Using Dual Function Nitric Oxide Releasing Central Venous Catheters in a 9day Rabbit Model. Acta Biomaterialia 2016, 44, 304-312. 31. Moynihan, H. A.; Roberts, S. M., Preparation of Some Novel S-nitroso Compounds as Potential Slow-Release Agents of Nitric Oxide In Vivo. Journal of the Chemical Society, Perkin Transactions 1 1994, (7), 797-805. 32. Paryzek, Z.; Skiera, I., Synthesis and Cleavage of Lactones and Thiolactones. Applications in Organic Synthesis. A review. Organic preparations and procedures international 2007, 39 (3), 203-296. 33. Smith, R. S.; Zhang, Z.; Bouchard, M.; Li, J.; Lapp, H. S.; Brotske, G. R.; Lucchino, D. L.; Weaver, D.; Roth, L. A.; Coury, A.; Biggerstaff, J.; Sukavaneshvar, S.; Langer, R.; Loose, C., Vascular Catheters with a Nonleaching Poly-Sulfobetaine Surface Modification Reduce Thrombus Formation and Microbial Attachment. Sci Transl Med 2012, 4 (153), 153ra132. 34. Brisbois, E. J.; Handa, H.; Meyerhoff, M. E., Recent Advances in Hemocompatible Polymers for Biomedical Applications. In Advanced Polymers in Medicine, Puoci, F., Ed. Springer International Publishing Switzerland: Switzerland, 2015, 481-511. 35. Liu, Q.; Singha, P.; Handa, H.; Locklin, J., Covalent Grafting of Antifouling Phosphorylcholine-Based Copolymers with Antimicrobial Nitric Oxide Releasing Polymers to Enhance Infection-Resistant Properties of Medical Device Coatings. Langmuir 2017, 33(45), 13105-13113. 36. Goudie, M. J.; Pant, J.; Handa, H., Liquid-Infused Nitric Oxide-Releasing (LINORel) Silicone for Decreased Fouling, Thrombosis, and Infection of Medical Devices. Scientific Reports 2017, 7 (1), 13623.



478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499

37. Ellman, G. L., Tissue Sulfhydryl Groups. Archives of Biochemistry and Biophysics 1959, 82 (1), 7077. 38. Pant, J.; Goudie, M. J.; Hopkins, S. P.; Brisbois, E. J.; Handa, H., Tunable Nitric Oxide Release from S-Nitroso-N-acetylpenicillamine via Catalytic Copper Nanoparticles for Biomedical Applications. ACS Applied Materials & Interfaces 2017, 9 (18), 15254-15264. 39. Lantvit, S. M.; Barrett, B. J.; Reynolds, M. M., Nitric Oxide Releasing Material Adsorbs More Fibrinogen. Journal of Biomedical Materials Research Part A 2013, 101 (11), 3201-3210. 40. Tong, S. Y.; Davis, J. S.; Eichenberger, E.; Holland, T. L.; Fowler, V. G., Staphylococcus Aureus Infections: Epidemiology, Pathophysiology, Clinical Manifestations, and Management. Clinical microbiology reviews 2015, 28 (3), 603-661. 41. Smith, R. S.; Zhang, Z.; Bouchard, M.; Li, J.; Lapp, H. S.; Brotske, G. R.; Lucchino, D. L.; Weaver, D.; Roth, L. A.; Coury, A., Vascular Catheters with a Nonleaching Poly-Sulfobetaine Surface Modification Reduce Thrombus Formation and Microbial Attachment. Science translational medicine 2012, 4 (153), 153ra132-153ra132. 42. Ferguson, J. J.; Harrington, R. A.; Chronos, N. A., Antiplatelet Therapy in Clinical Practice. Taylor & Francis: 1999. 43. de Lizarrondo, S. M.; Gakuba, C.; Herbig, B. A.; Repessé, Y.; Ali, C.; Denis, C. V.; Lenting, P.; Touzé, E.; Diamond, S. L.; Vivien, D., Potent Thrombolytic Effect of N-Acetylcysteine on Arterial Thrombi. Circulation 2017, CIRCULATIONAHA. 117.027290. 44. Zhang, L.; Casey, B.; Galanakis, D. K.; Marmorat, C.; Skoog, S.; Vorvolakos, K.; Simon, M.; Rafailovich, M. H., The Influence of Surface Chemistry on Adsorbed Fibrinogen Conformation, Orientation, Fiber Formation and Platelet Adhesion. Acta biomaterialia 2017, 54, 164-174.

500 501 502 503 504 505 506 507 508 509 510 511 512 513 514

Tables and Figures


Page 20 of 32

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515 516 517 518 519 520 521 522 523 524 525 526


Figure 1: A) Preparation of surface immobilized S-nitroso-N-acetyl-d-penicillamine (II. SIM-S1, III. SIM-S2, IV. SIM-S4) Structure I is a product of functionalization of PDMS surface with hydroxyl groups by submerging it in 50:50 ratio of 13 N HCl:30 wt.% H2O2 in H2O and treatment with APTMES for amine functionalization. Structure II is a product of nitrosation of thiol groups with tert-butyl nitrite. Structure III and IV are synthesized after branching of primary amine via reaction with methyl acrylate and amine functionalization of branched site using ethylene diamine. B) FTIR spectra for different samples. Amine-1, Amine-2 and Amine-4 correspond to amine functionalized surfaces with unbranched and branched surfaces. 3500-2500 represents unreacted –COOH groups present after amine-functionalization for SIMN4 and Amine-2. 2950 represents alkyl groups present in abundance in SR and aminated surfaces of SR. Double peaks of 1650,1550 represent primary amine groups in Amine-1, Amine-2 and Amine-4. 1650 represents saturated amide groups in SIM-N1, SIM-N2, SIM-N4, SIM-S1, SIM-S2 and SIM-S4. 1550 in SIMS2 represents nitroso group of the NO-donor attached.

527 528

Figure 1A

529

530 531

Figure 1B



532 533 534


Page 22 of 32

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535 536


Figure 2: A) Comparison of day by day NO release measurements between SIM-S1, SIM-S2, and SIM-S4. (n=3). B) Comparison of cumulative NO release from SIM-S1, SIM-S2, and SIM-S4. (n=3)

537 538

Figure 2A

539

540 541 542 543 544



545

Figure 2B

546

547 548


Page 24 of 32

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549 550 551 552

Figure 3: A) Adsorption of fibrinogen to modified SR surfaces over a 2 h period. Values are expressed as mean ± standard error. Measurements were conducted using n=8 per group. B) SIM-S2 was able to reduce bacteria adhesion by ~4 log when compared to control samples. C) Comparison of adsorbed platelets per surface area between SR, SIM-N1, SIM-S1, SIM-N2 and SIM-S2.

553

Figure 3A

554

555



556

Figure 3B


Page 26 of 32

Page 27 of 32 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

557


Figure 3C

558 559 560 561 562 563 564



565 566

Table 1: Contact angle measurements compared between all NAP-thiolactone and nitroso group functionalized surfaces.

Material

Static Water Contact Angle (°)

SR

106.77 ± 3.36

SIM-N1

94.36 ± 3.36

SIM-S1

101.56 ± 4.48

SIM-N2

64.95 ± 11.87

SIM-S2

53.78 ± 5.23

SIM-N4

93.09 ± 2.91

SIM-S4

90.90 ± 7.97

567 568


Page 28 of 32

Page 29 of 32 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

569 570


Table 2: Ability of various surface modified SR substrates to reduce nonspecific protein adsorption over 2h.

SR

SIM-N1

SIM-S1

SIM-N2

SIM-S2

72.4 ± 16.4

74.5 ± 13.7

51.0 ± 15.5

33.0 ± 11.0

24.7 ± 3.2

Reduction (%)

-

-

29.6 ± 26.7

54.4 ± 18.3

65.8 ± 8.9

p value vs control

-

NS

0.024

1.94 ×10

6.59 ×10

0.027

NS

-

Fg Adsorption -2

(µg cm )

p value vs SIM-S2

-5

6.59 ×10

-5

1.43 ×10

571 572


-4

-5


573

Page 30 of 32

Table 3: Ability of various surfaces to decrease bacterial adhesion over 24 h.

SR Average CFU of S. aureus cm

-2

SIM-N1 6

6.81 x10

SIM-S1 6

1.22 x10

SIM-N2 5

9.73 x10

SIM-S2 5

2.14 x10

2

3.89 x10

Reduction (%)

-

82.14 ± 22.20 85.71 ± 24.74 96.86 ± 0.49 99.99 ± .002

p value vs control

-

0.01

0.01

0.02

0.02

p value vs SIM-S2

0.02

NS

NS

0.01

-

574 575


Page 31 of 32 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

576


Table 4: Platelets adsorbed per surface area over a period of 90 mins.

Platelets cm

Control SR

SIM-N1

SIM-S1

SIM-N2

SIM-S2

13.5 ± 0.4

1.5 ± 0.1

2.8 ± 0.1

3.6 ± 0.2

1.6 ± 0.1

-

89.1 ± 0.9

79.1 ± 1.0

73.4 ± 1.3

87.7 ± 0.7

-

0.1

1.7

0.5

0.2

-2

6

(x10 ) Reduction (%) 6

p value (x10 ) 577



578

Graphical Abstract

579 580


Page 32 of 32

Active Release of an Antimicrobial and Antiplatelet Agent from a

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