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Biological and Medical Applications of Materials and Interfaces

Surface characterization, antimicrobial effectiveness, and human cell response of a biomedical grade polyurethane blended with a mixed soft block PTMO-Quat/PEG copolyoxetane polyurethane Chenyu Wang, Olga Yu Zolotarskaya, Kayesh M. Ashraf, Xuejun Wen, Dennis E. Ohman, and Kenneth J. Wynne ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 22 May 2019 Downloaded from http://pubs.acs.org on May 22, 2019

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ACS Applied Materials & Interfaces

Surface characterization, antimicrobial effectiveness, and human cell response for a biomedical grade polyurethane blended with a mixed soft block PTMO-Quat/PEG copolyoxetane polyurethane Chenyu Wang,a,# Olga Zolotarskaya,a,# Kayesh M. Ashraf,a,# Xuejun Wen,a Dennis E. Ohmanb and Kenneth J. Wynnea* a Department

of Chemical and Life Science Engineering, Virginia Commonwealth University, Biotech8, 737 N 5th St, Richmond, VA 23219, E-mail: [email protected] b

Institute for Engineering and Medicine, Department of Chemical and Life Science Engineering

Virginia Commonwealth University, 601 West Main Street, Room # 403, Richmond, Virginia 23284-3028 c Department

of Microbiology and Immunology, VCU School of Medicine, 1101 East Marshall Street, Richmond, VA 23298 and McGuire Veterans Affairs Medical Center, Richmond, VA 23249 # Each

of these authors contributed equally.

Abstract Infection is a serious medical complication associated with health care environments. Despite advances, the 5-10% incidence of infections for hospital patients is well documented. Sources of pathogenic organisms include medical devices such as catheters and endotracheal tubes. Offering guidance for curbing the spread of such infections, a model antimicrobial coating is described herein that kills bacteria on contact but is compatible in with human cells. To achieve these characteristics, a novel blend of a conventional biomedical grade polyurethane (Tecoflex) with mixed soft block polyurethane is described. The functional polyurethane (UPC12-50-T) has a copolyoxetane soft block P-C12-50 with quaternary ammonium (C12) and PEG-like side chains and a conventional poly(tetramethylene oxide) (PTMO, T) soft block. DSC and DMA data point to limited miscibility of UP-C12-50-T with Tecoflex. The blend of Tecoflex with 10 wt% UP-C12-50-T designated UP-C12-50-T-10 radically changed surface properties. Evidence for surface concentration of the P-C12-50 soft block was obtained by 1 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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

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atomic force microscopy (AFM), dynamic contact angles (DCA), zeta potentials () and X-ray photoelectron spectroscopy (XPS). Antimicrobial effectiveness of the blend coatings was established by the ASTM E2149 “shake flask” test for challenges of E. coli and a methicillin resistant strain of S. epidermidis. Cytocompatibility was demonstrated with an in vitro test designed for direct contact (ISO 10993-5). Growth of human mesenchymal stem cells (MSCs) beside and under UP-C12-50-T-10 indicated remarkable biocompatibility for a composition that is also strongly antimicrobial. Overall, the results point to a model coating with a level of PC12-50 that combines high antimicrobial effectiveness and low toxicity to human cells.

Introduction Excellent mechanical properties and low cost contribute to the use of polyurethanes for medical devices including catheters,1-3 endotracheal tubes,4 PICC lines5 and ureteral stents.6-7 However, like other biomedical materials used in applications that traverse the extracorporealintracorporeal interface in placement and/or use, bacterial adhesion and biofilm formation are problematic. In view of the continuing prevalence of infections acquired in nosocomial environments8-12 and the attendant problem of increased bacterial resistance to antibiotics,13-14 we report an advanced blend approach for polyurethanes for antimicrobial surface modification and biocompatibility. A net positive charge of alkylammonium moieties results in contact bacterial kill on glass, paper and other substrates.15-18 The mechanism by which alkylammonium functionalized surfaces interact with pathogenic organisms for contact kill has been discussed extensively.18-20 For surfaces, analogies have been drawn to cell wall disruption in liquid media by antimicrobial peptide (AMP) model polycations.21-26 In this regard, E. coli cell wall disruption by AMP-like

2 ACS Paragon Plus Environment

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co-polyoxetane P-C12-50 1 is supported by real time AFM observations. Nanoscale pores and bulges were associated with cytoplasm leakage and cell death. AFM-based hydrophobicity mapping further supported the model of P-C12-50 binding in a carpet-like fashion with quaternary side chains conjugated to the cell wall and PEG-like side chains interfacing with the PBS solution.27

Me

-

Requirements for contact kill antimicrobial coatings

Br +N

OMe

presumably include bacterial cell wall disruption but

4

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O

have the challenge of remaining free of biofouling.

O

Gottenbos showed that strains of Gram-negative E. coli

O

O

O

0.53

0.47

P[(C12)0.47(ME2Ox)0.53] = P-C12-50

and P. aeruginosa did not grow after four hours contact

1

with a quaternary functionalized acrylate but were adherent.28 Bacterial adhesion and the development of a biofilm is a serious problem for contact kill as a protective biofilm prevents access to surface charge. Antimicrobial effectiveness and resistance to biofouling were addressed by Zhang with the synthesis of a polyurethane having a pendant quaternary ammonium groups on the chain extender and polyethylene glycol (PEG) soft blocks.29-30 The proposed surface model for the multicomponent, crosslinked polyurethane suggested an outermost antibacterial quaternary ammonium layer and an antifouling PEG-like sub-layer. Importantly, in vivo biocompatibility was shown. Rather than a single polymer system, a blend approach for coatings is appealing in combining a robust commodity polymer with a specialty polymer containing functionality that surface-concentrates during processing. Co-processing holds the promise of durability, adhesion to substrate and biocompatibility.31 Interesting progress for a blend approach was described by



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Ishihara combining an acrylate surface modifier with a thermoplastic polyurethane. A minor amount of poly(methacryloyloxyethyl phosphorylcholine-co-cyclohexyl methacrylate (poly(MPC-CHMA) was combined with Tecoflex 60 polyurethane in a common solvent followed by evaporative coating. Surface concentration of poly(MPC-CHMA) was verified by XPS and blood compatibility was improved by surface concentration of the phospholipid polar groups.32-33 The water solubility of UP-C12-50, which contains only soft block 1 led to the synthesis of a polyurethane with P-C12-50 and PTMO blocks designated UP-C12-50-T (Table 1, Scheme 2). Herein we describe blends containing 2 and 10 wt% UP-C12-50-T and the biomedical grade polyurethane Tecoflex (Schemes 1, 2). The blends are designated UP-C12-50-T-2 and UP-C1250-T-10 based on the weight percent UP-C12-50-T. DSC and DMA were used for characterization of bulk properties while surface functionality was assessed with XPS, zeta potentials and contact angles. Antimicrobial effectiveness via contact kill was established with challenges of Gram-negative Escherichia coli and Grampositive Staphylococcus epidermidis. Growth of human mesenchymal stem cells (MSCs) adjacent to and under UP-C12-50-T-10 indicated remarkable biocompatibility for a composition that is also strongly antimicrobial.

Experimental Section Materials. Tecoflex 80A, an aliphatic polyurethane, was a generous gift from Lubrizol. 3Methyl-3-oxetanemethanol (Ox) was purchased from Combi-Blocks Inc. 3-(Bromomethyl)-3methyloxetane (BrOx) was purchased from Chemada Fine Chemicals. Sodium hydride NaH (60% dispersion in oil), diethylene glycol monomethyl ether, 1,4-dibromobutane, boron trifluoride


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diethyl ether (BF3), sodium hydroxide, 1,4-butanediol (BD), tetra-n-butylammonium bromide (TBAB), N,N-dimethyldodecylamine, 4, 4-methylenebis(cyclohexyl isocyanate) (HMDI), poly(tetramethylene oxide (PTMO, MW = 2 kDa), and anhydrous magnesium sulfate (MgSO4) were purchased from Aldrich. Dibutyltin diacetate was purchased from TCI America. Prior to use, 3-(bromomethyl)-3-methyloxetane and 1,4-dibromobutane were distilled under reduced pressure. Diethylene glycol monomethyl ether was dried over anhydrous MgSO4 and distilled under reduced pressure. Tetrahydrofuran (THF) was distilled over sodium in the presence of benzophenone. 1,4-Butanediol was dried with activated molecular sieves. Boron trifluoride diethyl ether was used as received. A standard purification procedure for dichloromethane (DCM) included shaking 100 mL with 5 mL of concentrated H2SO4, separating the organic phase and washing with a sequence of water, aqueous 5% NaHCO3, drying over MgSO4, and distillation at atmospheric pressure over CaH2. The purified solvent was stored under nitrogen. M9 media components were purchased from Aldrich (constituents are shown in Table S1). Luria Broth (LB) was purchased from Fisher Scientific. Synthesis of 3-((2-(2methoxyethoxy)ethoxy)methyl)-3methyloxetane (ME2Ox). Monomer ME2Ox was synthesized following a

Scheme 1. Synthesis of BBOx, ME2Ox and P[(BBOx)0.47(ME2Ox)]0.53 designated P-Br-50.

modification of the method described by Kurt (Scheme 1, Eq 1).34 Briefly, a solution of diethylene glycol monomethyl ether (40 g, 0.33 mol) in 25 mL of THF was added dropwise to a



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suspension of NaH (14.5 g, 0.36 mol) in 50 mL of THF over 2 h under nitrogen. During the addition the temperature increased to 32 oC along with hydrogen generation. The reaction mixture was stirred for 1 h at ambient temperature followed by dropwise addition of 3(bromomethyl)-3-methyloxetane (55 g, 0.33 mol) in 50 mL THF. The resulting white suspension was stirred overnight at room temperature followed by quenching with 60 mL water. Upon removal THF under reduced pressure, the product was extracted with ether, washed with brine, and dried over MgSO4. Ether was removed under reduced pressure and the remaining crude ME2Ox, was distilled (60 oC, 0.1 mm Hg). 1H-NMR (CDCl3, 600 MHz): δ (ppm) 1.31 (s, 3H), 3.38 (s, 3H), 3.54 (m, 4H), 3.65 (m, 6H),), 4.34 (d, 2H, J = 5.7 Hz,), 4.51 (d, 2H, J = 5.7 Hz). Synthesis of 3-((4-bromobutoxy)methyl)-3-methyloxetane (BBOx). Monomer BBOx was synthesized following a modification of the method described by Kawakami (Scheme 1, Eq 2).35 To a solution of 3-methyl-3-oxetanemethanol (31 g, 0.3 mol) and 1,4-dibromobutane (200 g, 0.92 mol) in 80 mL of hexane was added a solution of TBAB (12 g, 37.3 mmol) in 80 mL of water. To the two-phase system, NaOH (192 g, 4.8 mol) was added gradually during 4 h at 65-70 oC.

The reaction mixture was stirred at room temperature for 36 h and heated to reflux for 1 h.

The organic layer was separated and the aqueous portion was extracted with hexane. Combined organic fractions were dried over MgSO4. Upon removal of hexane, the remaining crude product was distilled at 67 oC (0.2 mm Hg). 1H-NMR (CDCl3, 600 MHz): δ (ppm) 1.3 (s, 3H), 1.74 (m, 2H), 1.96 (m, 2H), 3.45 (t, 2H, J= 6.72 Hz), 3.47 (s, 2H), 3.50 (t, 2H, J = 6.20 Hz), 4.35 (d, 2H, J = 5.76 Hz), 4.50 (d, 2H, J = 5.76Hz).


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Synthesis of polyol P-Br-50, Scheme 1, Eq 3. BF3 etherate (920 µL; 7.5 mmol) was added to a solution of 1, 4-butanediol (332 µg, 3.72 mmol) in 20 mL dichloromethane followed by stirring for 40 min at room temperature and cooling to 0 oC. BBOx (6 g, 25.3 mmol) and ME2Ox (6.3 g, 30.84 mmol) in 20 mL DCM were added dropwise under nitrogen to this solution over 1 h. After stirring overnight, the reaction was quenched with 10 mL 3% HCl. The separated organic layer was washed with brine and

Scheme 2. Synthesis of UP-C12-50-T.

dried over MgSO4. DCM was removed under reduced pressure and the remaining crude product was dried for 24 h under vacuum at ambient temperature. Hexane (50 mL) was added to the crude P-Br-50 followed by stirring under reflux for 30 min. After cooling the polymer/hexane mixture to -40 oC for 1 h, hexane was decanted. The hexane addition, heating, cooling and decantation process was repeated six times. After heating with hexane (50 mL) for a seventh time, the whole was poured while hot into a separatory funnel. After cooling overnight at ambient temperature, the bottom P-Br-50 layer was collected and



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dried overnight under vacuum. Purification was monitored by GPC, which gave Mn = 3.0 kDa, PDI = 1.3, while end group analysis by 1H-NMR spectroscopy36 gave Mn = 5.5 kDa. NMR (CDCl3, 600 MHz): δ (ppm) 0.91 (s, 6H), 1.68 (m, 2H), 1.94 (m, 2H), 3.19 (br. s, 8H), 3.24 (br. s, 2 H), 3.30 (br. s, 2 H), 3.38 (m, 5H), 3.44 (t, 2H), 3.53-3.58 (m, 4H), 3.60-3.65 (m, 4H). By 1HNMR analysis, P[(BBOx)(ME2Ox)] contained 47 mol% BBOx, which was close to the targeted 1:1 mole ratio. The latter is the basis for the P-Br-50 designation used herein. Synthesis of polyurethane HMDI-BD(35)[(PTMO)96(P-Br-50)4], UP-Br-50-T, Scheme 2, Eq 4. Reactions were monitored by FTIR spectroscopy, which showed disappearance of the HMDI isocyanate absorption at 2267 cm-1 and appearance of carbonyl (C=O) and N-H bands at 1715 cm-1 3316 cm-1, respectively. To a solution of PTMO (4.5 g; 2.25 mmol; Mn = 2 kDa) in 20 ml of THF, HMDI (2.16 g; 8.2 mmol) was added followed by 200 µL catalyst (dibutyltin diacetate in THF, 1:10 V/V)). The mixture was refluxed for 2 h under nitrogen. In a second step, 1, 4butanediol (0.42 g, 4.7 mmol) was added and refluxing was continued for 3 h. P-Br-50 (0.5 g, 0.09 mmol) in 10 ml of THF was added followed by refluxing for 45 min. Finally, 1, 4budanediol (0.11 g, 1.22 mmol) in 20 ml of THF was added dropwise and the mixture was heated under reflux for 12 h. The total 1,4-BD (0.42 g and 0.11 g) was 0.53 g, 5.72 mM. To the viscous mixture, 70 ml THF was added followed by stirring for 4 h. The solution was poured into methanol/water (200 ml / 600 ml) giving a white solid that was filtered, dissolved in THF and reprecipitated in methanol/water (1 /3, v /v). The product is represented as HMDIBD(35)[(PTMO)96(P-Br-50)4] based on feed and 1H-NMR and designated UP-Br-50-T. GPC showed Mn = 39 kDa and PDI = 2.3 with a THF mobile phase.


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Synthesis of UP-C12-50-T, Scheme 2, Eq 5. 3 g UP-Br-50-T was dissolved in 48 mL THF / acetonitrile (1/1, v/v). After addition of 1.5 mL N, N-dimethyl dodecylamine the whole was stirred at 60 oC overnight under nitrogen. After removing solvents under reduced pressure, crude UP-C12-50-T was dissolved in 25 mL THF and reprecipitated in 650 mL hexane. Pale yellow UP-C12-50-T was dissolved in 20 mL of THF and reprecipitated in water twice. Coatings.

Table 1. Polymer coating compositions Composition Tecoflex UP-C12-50-T-2 UP-C12-50-T-10 UP-C12-50-T

Mixing ratio: UP-C12-50-T / Tecoflex 0 / 100 2 / 98 10 / 90 100 / 0

wt% of HDMI-BD hard block

wt% of PTMO soft block

N/Aa

N/Aa

34%

56%

wt% of UPC12-50-T soft block 0% 0.2% 1% 10%

a. Tecoflex is an HMDI/BD-PTMO thermoplastic elastomer; the composition is unknown.

Tecoflex was dissolved in THF (5 wt%) and poured into methanol/water (1/3, v/v) for precipitation / purification (two cycles). Solvents were removed in a vacuum oven at 60 °C for 48 hr. Purified Tecoflex was dissolved in THF (5 wt%). Separately, a solution of UP-C12-50-T in THF was prepared (5 wt%). UP-C12-50-T-2 and UP-C12-50-T-10 solutions were prepared by mixing a 5 wt% UP-C12-50-T solutions and 5 wt% Tecoflex polyurethane (Table 1). The mass ratios of UP-C12-50-T solution to Tecoflex solution were 2/98 and 10/90 for UP-C12-50-T-2 and UPC12-50-T-10 solutions, respectively. THF solutions of Tecoflex, UP-C12-50-T-2, UP-C12-50-T10 and neat UP-C12-50-T were poured in aluminum weighing dishes (64 mm diameter). Solvent was removed at ambient temperature overnight and subsequently in vacuum at 60 °C for 72 h to obtain bubble-free thin films. Tecoflex films were transparent and colorless while UP-C12-50-T



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was transparent and pale yellow. UP-C12-50-T-2 and UP-C12-50-T-10 were also transparent, with color increasing with increasing UP-C12-50-T content. Instrumental Methods. Gel permeation chromatography (GPC). A Viscotek TDAmax GPC system was used for molecular weight determinations. A set of columns suitable for polar polymers (GRAM columns from Polymer Standards Service-USA Inc.) calibrated with polyethylene oxide (PEO) standards were used. PDI’s and Mn’s for all measurements have only relative accuracy. Wilhelmy plate method for dynamic contact angle (DCA) analysis. The Wilhelmy plate method37 employed dip coated cover slips and a Cahn Model 312 Analyzer. Glass containers used for DCA analysis were cleaned by rinsing with Nanopure water and treatment with a gas/oxygen flame. Water surface tension (72.6 ± 0.4 dyne cm-1) was checked before each experiment using a flamed glass cover slip. By analyzing force versus distance curves (fdc’s), advancing (θA) and receding (θR) contact angles were obtained via the relationship F = m/g = Pγ(cos θ), where F is the force derived from respective mass (mg) changes on immersion and emersion, g is the gravitational constant, γ is the liquid surface tension, and θ is the contact angle.38 Extrapolating the fdc to the point of immersion eliminates the need for a buoyancy correction to F. Cycles were conducted with stage speeds of 100 μm/s. Water was tested for purity after each sample analysis to examine the extent of water contamination due to leached species.38-39 Atomic Force Microscopy (AFM). Morphological investigations were carried out using a Bioscope Catalyst atomic force microscope (Veeco Instruments Inc., model #BIOII). Tapping mode imaging was performed in air using microfabricated silicon cantilevers (40 N/m, Veeco, Santa Barbara, CA). Unless otherwise noted, tapping force corresponded to a setpoint ratio rsp of


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0.8, where rsp = Aexp/Ao, Ao is free oscillation amplitude and Aexp is the experimental oscillation amplitude. Images were analyzed by using NanoScope v710r1 software. For comparisons AFM images were normalized to the same phase scale (z, deg) as indicated in figure legends. The phase scale was chosen to optimize image quality and consistency with topographical images. X-ray photoelectron spectroscopy (XPS). Data were obtained with a PHI 5000 versa probe 11 X-ray photoelectron spectrometer, Al Kα source (1486.68 eV), 200 μm spot size, 55 eV pass energy, 0.05 eV step size and 45° take off angle. Deconvolution of the N1s peak area utilized Multipak Version 9.8.0.19 using a Gaussian−Lorentzian (70:30) function after “smart” background subtraction. C, O, Br and N atom percent was obtained with the same software. The low at% N and Br was addressed by averaging 20 scans. Zeta potential (ζ) measurements. An Anton-Paar SurPASS instrument with a clamping cell was used to obtain zeta potentials as previously described.40 Samples used for ζ measurements were coatings on glass microscope slides (7.5 cm × 2.5 cm). Polypropylene (PP) sheets (0.0125 in., 5 × 2 cm2, McMaster-Carr) were used as a reference. Measurements were obtained at pH 5.6, as monitored by two Ag/AgCl electrodes at 20 ± 2 °C, and at a maximum pressure difference of 500 mbar. An average of four measurements was recorded. To be compatible with the alkylammonium bromide surface modifier, the electrolyte was 0.001 M KBr (99%, SigmaAldrich). Zeta potentials at the solid/liquid interface are based on measurements of streaming potentials and streaming currents. The KBr electrolyte flows through the measuring cell that contains the coated microscope slide. The gap created between sample and polypropylene reference is adjusted to generate a pressure difference between the inlet and outlet of the measuring cell. During a measurement, pressure increases continuously and the pressure



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difference across the measuring cell (p) and streaming potential (U) are used to calculate ζ according to the method by Fairbrother-Mastin (Eq 6).41

𝜁=

∆𝑈 ∆𝑝

𝜂

× 𝜀 × 𝜀0 × 𝐾 𝐵

Eq 6

with ζ = zeta potential U/p = slope of streaming potential vs differential pressure  = electrolyte viscosity  = dielectric coefficient of solvent 0 = vacuum permittivity KB = electrolyte conductivity. Differential Scanning Calorimetry (DSC). Thermograms were obtained with a TA-Q1000 SeriesTM Instrument (TA Instruments). Samples (5-10 mg) were equilibrated at -90 °C followed by a heating ramp of 10 °C/min to 100 °C. A cooling ramp of 10 °C/min was applied back to -90 °C and a second heating cycle of 10 °C/min to 150 °C was employed. The consecutive heating cycles were followed to identify any changes with heating. In vitro Antimicrobial Assay, ASTM-2149, “shake flask” test. Medium. As reported previously, M9 growth medium was chosen to evaluate the antimicrobial activity of the coatings.42 M9 growth medium was prepared containing 20 wt % glucose as the carbon source. Table S1 shows components used in preparing the respective media. Bacterial challenges. A Gram-negative HfrH Escherichia coli (E. coli) strain43 and a Gram positive Staphylococcus epidermidis (S. epidermidis) RP62A methicillin resistant strain (MRSE)44 were used for antimicrobial tests. Cultures were streaked on Luria Agar plates and incubated overnight at 37 °C. A single colony from each strain was used to inoculate 10 mL of M9 medium followed by overnight incubation at 37 °C with shaking (225 rpm). Bacterial


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concentrations usually reached 1-3 × 108 colony forming units per milliliter (CFU/mL). These bacterial sources were then diluted with fresh M9 medium to obtain challenge concentrations of 1-3 × 105 CFU/mL. Shake flask test. Antimicrobial activity was determined by the ASTM E2149 “shake flask test”.45 Tecoflex and UP-C12-50-T-based films (0.2 g, 56 cm2) were sterilized with UV light for 30 min and placed in 20 mL glass test tubes. The respective bacterial stock suspensions (6 ml, ~1 x 10-5 cfu/mL) in M9 medium were added to each test tube. A test tube with only medium served as blank control. Tubes were capped and shaken at 37 °C for 24 hr. A visual observation of solution transparency (kill) or turbidity (growth) provided a qualitative test. A quantitative test for bacterial growth involved measuring solution optical density at λ = 600 nm with a Beckman Coulter DU 640 UV-visible spectrophotometer. To determine the log reductions for E. coli or S. epidermidis, the medium from respective flasks were serially diluted, plated on LB agar Petri dishes in triplicate and incubated at 37 °C for 24 h. Colonies were counted with reported results being the average of three Petri dish counts. Log reduction by UPC12-50-T-based polyurethanes compared to Tecoflex is calculated via Eq 7.

𝐿𝑜𝑔 𝑟𝑒𝑑𝑢𝑐𝑡𝑖𝑜𝑛 = 𝐿𝑜𝑔

𝐴 𝐵

Eq. 7

Where: A = CFU/mL for the medium containing UP-C12-50-T-based coatings and B = CFU/mL for the medium containing Tecoflex medical grade polyurethane.



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Viability of adherent bacteria. UP-C12-50-T-based coatings and Tecoflex films (0.2 g, 56 cm2) were shaken with E. coli stock suspensions (6 ml, ~1 x 10-5 cfu/mL) in M9 medium at 37 °C for 24 h. A log reduction > 2 was found for UP-C12-50-T-based coatings compared to Tecoflex. To test the viability of adherent bacteria, the samples were removed and rinsed briefly with PBS buffer and transferred into 50 mL Erlenmeyer flasks with 10 mL of PBS. A 5 min sonication was carried out to detach bacteria from the surface. 100 µL medium from respective flasks were plated on LB agar Petri dishes and incubated at 37 °C for 24 h. Triplicate plates were prepared for each sample. The results are shown in Figure S9. Test for Leaching. The presence or absence of antimicrobial leaching was determined by the test method specified in ASTM E2149-01 (Figure 1) using E. coli. (1) Tecoflex, UP-C12-50-T-2, UP-C12-50-T-10 and UP-C12-50-T films were extracted with PBS in individual flasks at 37 °C for 24 h. The surface to volume ratio (1 cm-1) was the same as that used in shake flask tests. (2) 100 µL E. coli stock solution (1.1×105 CFU/mL) was uniformly spread on M9 glucose medium agar plates to form a “lawn” of bacteria, (3) A 3/8 inch diameter hole was bored in center of each inoculated plate and the plug was removed, (4) 100 µL of extract solution for polyurethane films (step 1) was dripped into the agar holes, (5) 100 µL of trimethoprim in PBS solution (10 µg/mL) was used as positive control and (6) samples were incubated for 24 h at 37 °C. Figure 1 depicts the process and formation of a zone of inhibition (ZOI) characteristic of leached antimicrobial (or none). Figure 1. Test procedure for antimicrobial leaching (ASTM E2149). 14 ACS Paragon Plus Environment

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Cytotoxicity Assay: ISO 10993-5. Sample preparation. Tecoflex and UP-C12-50-T-based films from 5% THF solutions were obtained as described above. A circular microscope cover glass (Fisher brand, Dia: 12 mm,) was encapsulated in each polyurethane to prevent floating in the cell culture medium. As illustrated in Figure 2, 5 wt% Tecoflex in THF (20 g) was poured into a Teflon nonstick pan (Dia: 20 cm) with a flat inner surface. The pan was then covered with aluminum foil to slow solvent evaporation and avoid bubble formation. The solvent was allowed to evaporate for 5 h at ambient temperature until films were solid but tacky. Then ~60 circular microscope cover glasses were placed on top of the Tecoflex layer taking care that cover glasses did not touch. The Tecoflex solution (25 g) was poured into the pan so as to cover all the cover glasses. The pan was again covered with aluminum foil and solvent removed at ambient temperature overnight, followed by 60 °C in a vacuum oven. The polyurethane film was peeled off the pan and cut along the edge of the microscope cover glasses to obtain Tecoflex encapsulation. Lastly, Tecoflex and UP-C12-50T-based polyurethanes in THF solutions (5 wt%) were dripped on the Tecoflex polyurethane substrates to cover the entire surface. Solvent was removed at 60 °C in a vacuum oven for 72 h.

Figure 2. Procedure used to prepare samples for ISO 10993-5 direct contact cytotoxicity assay.

Specimens were sterilized with UV light for 2 h prior to testing. Pieces of latex rubber gloves (Kimberly-Clark™ Professional Comfort Latex Powder-Free Exam Gloves) were used as positive controls for the ISO 10993-5 direct contact test. Circular



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latex films (12 mm diameter) were cut from the gloves and attached to microscope cover glasses with medical grade glue (LOCTITE 3311TM) to prevent floating in the medium. ISO 10993-5, direct contact test. The test for cytocompatibility was carried out in vitro by an ISO 10993-5 (direct contact) test, which specifies incubation of cultured cells in contact with a “device”. Mesenchymal stem cells (MSCs) were obtained from American Type Culture Collection ATCC. The growth medium was 89% DMEM/F12+Gluta MAX-1(1x) (GibcoTM), 10% fetal bovine serum (ScienceCellTM) and 1% antibiotic

Figure 3. Modified ISO 10993-5 (direct contact) test for in vitro cytotoxicity.

antimycotic solution (100x), stabilized (SigmaTM). MSCs were evenly seeded in 6-well cell culture plates and grown to 80-90% confluency (Figure 3). The culture medium in each well was removed and Tecoflex, UP-C12-50-T-2, UP-C12-50-T-10 and UP-C12-50-T overcoated disks were gently placed at the center of the wells with the coated side or the latex side facing downward for cell contact. Three replicates were prepared for each sample. Fresh medium (1.5 mL) was slowly added into each well. Culture plates were carefully placed in 37 °C incubator avoiding any unnecessary movement. Optical microscope images (Olympus CKX41) were obtained 24 and 48 h after introducing specimens. Confocal microscope (Olympus IX81) images were obtained after 48 h.


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Results and Discussion The water solubility of UP-C12-50 containing only soft block 1 led to the synthesis and characterization of a polyurethane with P-C12-50 and PTMO blocks designated UP-C12-50-T (Experimental, Table 1, Scheme 2). Bulk properties of Tecoflex, UP-C12-50-T blends and UPC12-50-T modifier are described below in the context of qualitative assessments of miscibility for the minor component and the impact on surface modification. Bulk characterization. Thermal transitions and mechanical properties. DSC (-90 to 100 °C) at a heating rate of 10 °C /min was used to assess thermal transitions (Figure S4, Table 2). For the first run, R1, a change in slope at -77 °C for Tecoflex and UP-C12-50-T blends is assigned to the PTMO Tg (Table 2, Figure S4-R1).46-47 A second transition at 15 °C for Tecoflex is assigned to Tm for PTMO. This transition is weak in run R2 that was carried out after re-cooling. The attenuated endotherm is attributed to slow PTMO recrystallization. The PTMO melting point is depressed to

Table 2. DSC and DMA data for Tecoflex, UP-C12-50-T-2, UP-C12-50T-10 and UP-C12-50-T. Composition

PTMO Tg (°C)

DSC PTMO Tm (°C)

tan δ (°C)a

Tecoflex -78 15 -54 UP-C12-50-T-2 -76 3 -53 UP-C12-50-T-10 -76 -1 -67 UP-C12-50-T -78 -2 -55 a. Temperature corresponding to the tan  peak.

DMA E' at 25 °C E' at 37 °C (Pa) (Pa) 6.5 × 106

6.8 × 106

5.3 × 106

4.9 × 106

2.8 × 106

2.1 × 106

3.3 × 106

3.0 × 106

~3 °C for UPC12-50-T-2 and to -1 °C for UP-C12-50-T-10. The PTMO melting point depression of 12-16 °C



Page 18 of 43

for modified Tecoflex is significant and suggests limited miscibility for the respective UP-C1250-T modifiers. The similar Tg’s for Tecoflex and the blends also suggests some miscibility for the PTMO and P-C12-50 soft blocks. The balance is interesting as AFM imaging (vida infra) shows surface phase separation of a modifier domain that drives contact angles, zeta potentials and antimicrobial effectiveness.

+

CF3

N

H 2C

Phase separation for Tecoflex and P-C12-50-T compositions may be compared to that for Tecoflex 60 blends with acrylate surface modifying

copolymers.32

H 3C C C H2

O C H2

Br-

H 3C

O

C C 0.11 H2

CH3 CH211

O C H2

0.89

2

Phase separation played a key role in the success of (poly(MPC-CHMA) as a surface modifier that reduced thrombogenicity of Tecoflex 60 polyurethane. In contrast, poly-(MPC-co-EHMA) (poly(methacryloyloxyethyl phosphorylcholine-co-ethylhexyl methacrylate)) was miscible with the bulk and failed as a surface modifier.32 Previously, we described the synthesis of a copolyoxetane soft blocks with semifluorinated side chains as “chaperones” for concentrating antimicrobial quaternary function on a conventional polyurethane.34 Copolyoxetane soft block 2 appeared to be promising in this regard, but after a few days at ambient temperature, microscale phase separation of the fluorous modifier took place and antimicrobial effectiveness was lost with sequestration of quaternary moieties.48 Morphological instability of a blend with a minor component is therefore another mechanism for foiling surface functionality. Dynamic mechanical analysis data are summarized in Table 2. The scans from -100 to 100 °C at 1 Hz are shown in Figure S5. Blends soften with increasing concentration of UP-C1250-T. E’ at 25 °C decreases from 6.5 × 106 Pa for Tecoflex to 3.3 x 106 Pa for UP-C12-50-T. E’ at 37 °C decreases from 6.8 × 106 Pa for Tecoflex to 3.0 x 106 Pa for UP-C12-50-T. The


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mechanical instability of the soft films results in scatter in E’’ and tan δ curves above ~ 0 °C. As a result, Tm for PTMO is not detected. Water absorption. The optical transparency of Tecoflex, UP-C12-50-T-2 and UP-C12-50T-10 coatings was not changed after overnight immersion in water or PBS buffer. To obtain weight percent water uptake, films were immersed overnight at 37°C. The uptake of water for Tecoflex was 1% while that for UP-C12-50-T-2 and UP-C12-50-T-10 was 2%. The slight increase in water uptake due to the hydrophilic surface modifier is similar to that previously

Phase

observed.49 In keeping with its hydrophilic

1 nm

character, mass uptake for the neat modifier UP-C12-50-T was 8% and the coating became

Height

A

opaque.

2 nm

The same mass uptake was found for Tecoflex and UP-C12-50-T-based coatings after

B

immersion in PBS overnight. Mass uptake for the neat modifier UP-C12-50-T was 8% in water and 12% in PBS. The difference may be

6 nm

C

due problems in removing water due to film wrinkling.

6 nm

D Surface Characterization Atomic Force Microscopy (AFM). Tapping Mode AFM reveals an evolution of surface morphologies for Tecoflex and UP-C12-50-T

Figure 4. 5 µm × 5µm AFM phase (left) and height (right) images for A, Tecoflex; B, UPC12-50-T-2; C, UP-C12-50-T-10; D, UP-C1250-T; rsp, 0.8. Rq is shown on height images.

Z= 200 nm, phase angle, 60º. 19 ACS Paragon Plus Environment


Page 20 of 43

compositions at the microscale and nanoscale. Tecoflex shows phase separated 20-50 nm features that are characteristic of well-known hard block / soft block nanoscale phase separation. Lighter features in false color imaging are assigned to hard block-rich domains with higher modulus, while soft segment domains are assigned to the darker nano-features (Figure 4A, phase).50-54 Phase imaging for UP-C12-50-T-2 (Figure 4B) reveals rounded 0.1-0.2 μm features superposed on a background characteristic of Tecoflex (Figure 4A). Figure 4C shows much larger features that have sizes ranging from nanoscale to ~1.5 µm and occupy about half of the image. The increased prominence of these lighter features for UP-C12-50-T-10 compared to UPC12-50-T-2 is consistent with a UP-C12-50-T mixed soft block domain “blooming” to the surface. While there is some modest degree of phase mixing based on DSC analysis (vida supra), the micron-scale features are attributed to UP-C12-50-T or a domain rich in this component. The lighter color for the features associated with the UP-C12-50-T domains is opposite that expected based on mechanical property measurements that show Tecoflex has a bulk modulus double that for UP-C12-50-T (Table 2). The usual correlation is associating lighter areas of phase images due to more elastic tip-sample interactions.50 However, the lighter features emerging in phase images are clearly associated with domains rich in UP-C12-50-T. Previously, we observed false color images “flipping” even during a single scans and discussed uncertainties associated with phase images.55 It seems unlikely, but in the present case, it is possible that the modulus of surface constrained UP-C12-50-T domains is lower than the bulk. Whether or not this is the case will be the subject of future studies by Peakforce Quantitative Nanomechanical Mapping (PF-QNM), an AFM mode.56


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AFM images for UP-C12-50-T at the same scan settings show phase separated features (Figure 4D). Zeta potentials are consistent with a high charge density assigned to surface concentration of P-C12-50 but how to interpret the AFM images is unclear. The root-mean-square (rms) height deviation Rq is commonly used to evaluate surface roughness. By this measure, surfaces of Tecoflex (Figure 4A, height) are topologically flat on the nanoscale (Rq ~ 1 nm). A slightly higher Rq (2 nm) is observed for UP-C12-50-T-2 (Figure 4B, height), while higher 6 nm Rq’s are found for UP-C12-50-T-10 (Figure 4C, height) and UPC12-50-T (Figure 4D, height). The increasing Rq with increasing P-C12-50 concentration is consistent with phase separation resulting from increased UP-C12-50-T content. Dynamic Contact Angles (DCA). To elucidate further changes accompanying UP-C12-50-T content, contact angles were investigated by Wilhelmy plate DCA measurements.39 Considering that the soft block P-C12-50 is soluble in water and growth media,57-58 and that increased water absorption occurred (vida supra), contact angles were expected to decrease with increasing UP-C1250-T content. Figure 5 shows force distance curves (fdc’s) and advancing θA and receding θR contact angles for Tecoflex and UP-C12-50-T based coatings. Contact angles for UP-C12-50-T-2 are similar to those for Tecoflex, with a contact angle hysteresis of 50° (θΔ = θA - θR), which is usual for aliphatic polyurethanes.39 Contact angles for UP-C12-50-T-10 (θA 80°, θR 22°) reflect an amphiphilic character

21 Figure 5. DCA force distance curves for ACS Paragon Plus Environment A, Tecoflex; B, UP-C12-50-T-2; C, UPC12-50- T-10; D, UP-C12-50-T.


Page 22 of 43

compared to Tecoflex (θA 95°, θR 45°). The receding contact angle of 22° is particularly low for UP-C12-50-T-10 compared to the other compositions and suggests that this blend has relatively high near-surface P-C12-50 concentration driven by contact with water (amphiphilicity).59. This hydrophilic character is consistent with the phase separated morphology revealed by AFM (Figure 4C). The receding contact angle for UP-C12-50-T is ~20° higher than UP-C12-50-T-10 and close to that for Tecoflex. This result seems at odds with greater water uptake (8%) but the zeta potential for UP-C1250-T is in line with a high surface concentration of hydrophilic P-C12-50 soft blocks as is antimicrobial effectiveness. X-ray photoelectron spectroscopy (XPS). The atomic percentage of quaternary nitrogen (N+) provides a quantitative assessment of P-C12-50 in the outermost 3-4 nm. Figure 6 shows spectra for UP-C12-50-T-based coatings with a peak for quaternary nitrogen at 402 eV60-61 and urethane

Figure 6. N1s XPS for A, UP-C1250-T-2; B, UP-C12-50-T-10 and C, UP-C12-50-T: quaternary nitrogen, 402 eV; urethane nitrogen, 399.5 eV.

nitrogen at 399.5 eV.62-63 The increased intensity of the N1s peak area with increasing P-C12-50 Table 3. Calculated and observed atom percent for Tecoflex and UP-C12-50-T-coatings. C Tecoflexa UP-C12-50-T-2a UP-C12-50-T-10a UP-C12-50-T P-C12-50

Calc Found Calc 78.3 79.34 75.9 17.61 79.31 76.82 17.60 79.01 75.7 17.53 80.49 14.63

N N N+ Br (total) (urethane) Found Calc Found Calc Found Calc Found Calc Found 20.5 1.07 1.07 0 0 22.4 3.05 1.62 3.05 0.60 0.002 0.06 0.002 0.06 0.02 0.3 21.12 3.06 1.77 3.04 1.36 0.02 0.41 0.21 0.6 21.4 3.24 2.32 3.03 1.26 0.21 1.06 2.44 2.44 22 0 2.44 -

O

ACS is Paragon Plus Environment a. Estimated because the Tecoflex composition not known.

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content correlates with the change in surface morphology observed by AFM (Figure 4B, C) and supports the notion of surface modifier “blooming”. Table 3 lists calculated and observed atom percent for Tecoflex and UP-C12-50-T-coatings. For UP-C12-50-T-2, N+ and Br- atom percentages are near the limits of detection (Figure 6A) but the presence of near surface P-C12-50 is evident from zeta potentials that are stable over the course of ~1 h (Figure 7). Representative high-resolution C, N, O, and Br XPS spectra for UP-C12-50-T-10 are shown in Figure S6. The 0.41 atom percent N+ for UP-C12-50-T-10 (0.41) exceeds the estimated atom% by a factor of 20 (0.02) and is double that calculated for UP-C12-50-T (0.71). The atom percent N+ for UP-C12-T-10 is a factor of 5 lower than the P-C12-50 soft block alone (2.44). The atom% N+ is matched by a similar atom% for Br- (0.3). Overall, the XPS data for UP-C12-50-T-10 is consistent with AFM imaging that shows a phase separated UP-C12-50-T domain occupying about 50% of the image (Figure 4C). The impact of the high surface concentration of the P-C12-50 soft block is clear from the high zeta potential and robust antimicrobial effectiveness. The nitrogen XPS spectrum for UP-C12-50-T is shown in Figure 6C. The N+ atom% is highest among the UP-50-T-based coatings. The N+ atom% found (1.06) is about 5 times that calculated for the UP-C12-50-T (0.21) giving strong evidence for surface concentration of the PC12-50 soft block illustrated in Figure 8. Zeta potentials. Surface charge density has been estimated by dye adsorption/desorption.45 Because of inconsistent results from dye adsorption, zeta potentials were used previously to analyze surface charge density for a polyurethane blend with a polyurethane modifier containing a soft block having quaternary and fluorous side chains.64 Focusing on 1% modification, a



Page 24 of 43

decrease in streaming potential with immersion time in KBr electrolyte or after annealing signaled instability of surface charge. A custom-built system resulted in instrument-specific differential values, but these results along with AFM imaging and loss of antimicrobial effectiveness were correlated with phase separation and sequestration of quaternary groups driven by the fluorous moiety that was meant to enhance quaternary function. In this prior investigation, the biocidal performance of a polymer surface modifier (PSM) having a copolyoxetane soft block with C12-quat and short PEG-like side chains was about the same as the fluorous analog.34 This finding prompted the present examination of zeta potentials to determine effects of microscale phase separation shown by AFM studies (Figure 4) and increase in N+ observed by XPS analysis (Figure 6). In contrast to high vacuum XPS measurements, zeta potentials provide insight into surface charge in an aqueous electrolyte. The surface concentration of functional groups, adsorption of ions, effects of pressure and pH are among variables that influence zeta potentials. To compare zeta potentials for Tecoflex and UP-C12-50-T-based polyurethanes, the sample geometry, pressure, temperature, pH and electrolyte concentration were held constant. An Anton Paar SurPAAS instrument was used with a clamping cell that provides an average zeta potential for a coating area exposed to the electrolyte.40 Potassium bromide (10-3 M) was used to provide a common ion with the quatbromide functionalized UP-P-C12-50-T coatings. Zeta potentials are specific to measurement geometries and parameters noted above. However, for univalent electrolytes, Kirby proposed the empirical relationship ξ ~ -log[C} = pC, where C is the electrolyte concentration. Using this relationship, zeta potentials obtained from published sources were compared for a number of polymers. For pC = 3, the zeta potential for Tecoflex (-86 mV) gives ξ/pC = 25 mV. This compares favorably with ξ/pC of 26 mV for a


Page 25 of 43 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


PTMO based polyurethane studied by Ikada.65 This finding gives some confidence in analytical results for a method not often used for polymer surface analysis. The net charge density on a polymer coating in contact with an aqueous solution gives rise to an electrical double layer (EDL) and electroosmosis.66 In the Guoy-Chapman-Stern (GCS) model67 of the double layer,68-69 the electrical double layer (EDL) is comprised of an innermost Stern layer and an outer diffuse layer. The zeta potential is reflective of polarization in an electric field associated with the outer diffuse or slippage layer. The negative zeta potential for nominally neutral Tecoflex or other polymers such as nylons is not intuitively obvious, but is attributed to aqueous hydroxide or electrolyte anion

Figure 7. Zeta potentials () as a function of time for A, Tecoflex; B, UP-C12-50-T-2; C, UP-C12-50-T-10; D, UPC12-50-T.

adsorption.70-71 Aqueous hydroxide adsorption by hydrogen bonding to polar moieties such as near surface amide or oxygen in PTMO could well form a negatively charged innermost layer. For charge balance, the next layer is a non-mobile net-positively charged layer. Together, these layers constitute the Stern-layer.67 Under an electric field, the next diffuse or slippage/mobile layer has a negative charge under applied voltage, that is, a zeta potential. The increasingly negative zeta potential for polyurethanes and polyamides with increasing pH supports the notion of increased adsorption of hydroxide ion for the innermost charged layer.72-73 For this discussion, is convenient to designate the layered sequence Ns1-Ps2 for negative surface-1, positive surface-



Page 26 of 43

2 that makes up the Stern layer and Nm for the diffuse mobile layer that is polarized by an electric field to give a Negative . Zeta potentials for Tecoflex and the UP-C12-50-T-based coatings were determined over ~1 h to assess stability (Figure 7). Little change was observed showing stability of charge density in 0.001 M KBr. This result stands in contrast to those noted above for a polyurethane modified by a PSM with a copolyoxetane soft block having fluorous and quat side chains designated “1% PU-1” where a drop of streaming potential from 112 to 34 mV took place in 2 min.64 With increasing UP-C12-50-T content the zeta potential becomes increasingly positive. (Table 4). This trend is marked by a striking increase of 100 mV from -44 mV for UP-C12-50T-2 to +39 mV for UP-C12-50-T-10. The highest  in this series was found for neat UP-C12-50T,  +130 mV. From these findings, we propose that a crossover occurs whereby the innermost layer for UP-C12-50-T-10 can be designated Ps1-Ns2, which corresponds to a quaternary charge layer (Ps1) and the next Ns2 bromide layer. This is followed by a Pm designation for the diffuse mobile layer that is polarized by an electric field giving a positive . UP-C12-50-T became translucent upon immersion in water. This is attributed to surface concentration of UP-C12-50-T, which has a PTMO content that is just enough to insolubilize the mixed soft block polyurethane. Thus, added to experimental variables noted above, the outermost surface composition and water content are changing with composition. For soft material interfaces such as biomembranes, estimating charge densities from zeta potentials is complex.74 For UP-C12-50-T modified Tecoflex, electroosmotic flow within the swollen Ps1Ns2 Stern layer may lead to significant differences between the observed electrokinetic response and that analyzed on the basis of hard surface Guoy-Chapman model.75 Despite these


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considerations, the Gouy equation (Eq 8, Table S3) was used to estimate the surface charge density.76 𝑧𝑒𝜁

Eq. 8

𝜎 = √(8𝜀𝜀𝜀0𝑘𝑇𝑁𝑐)sinh (2𝑘𝑇)

where σ = charge density (C/m2), k = Boltzmann constant (1.38 × 10−23 J/K), T = 293 K, N = Avogadro’s number (6.02 × 1023 mol−1), z = counterion charge (+1), e is the charge on the electron, and c = bulk electrolyte concentration (mol/m3). Constants used to calculate the charge density were ε = 80 and ε0 = 8.85 × 10−12 F/m. A spreadsheet for charge density calculations is provided in Supporting Information (Table S3). Table 4 summaries  and charge densities for Tecoflex and the UP-C12-50-T-based coatings. The negative charge density for Tecoflex follows from the negative zeta potential (85.78 mV) which confirms previous

Table 4. Zeta potentials and charge densities at pH 5.5.

findings and the calculation of ξ/pC described above.64-65 Charge densities follow  that

 (mV)

Calculated charge density (charge/cm2)

Differential charge density (charge/cm2)a

Tecoflex

-85.78

-6.1E+12

0

UP-C12-50-T-2

-44.06

-2.3E+12

3.8E+12

UP-C12-50-T-10

38.65

2.0E+12

8.1E+12

UP-C12-50-T

129.02

1.5E+13

2.1E+13

a. Differential charge density = charge density of sample – charge density of Tecoflex

increase with increasing UP-C12-50-T content. A crossover from negative to positive charge density is found for UP-C12-50-T-2 (-2.3 x 1012 charge/cm2) compared to UP-C12-50-T-10 (2.0 × 1012 charge/cm2). Charge density increases further to 1.5 × 1013 charge/cm2 for neat UP-C1250-T. The value for UP-C12-50-T-10 and UP-C12-50-T are in the range associated with the threshold value for contact antimicrobial kill (1013 - 1014 charge/cm2) reported by Kugler.77 However, they are significantly lower than the threshold value reported by Matyjaszewski (≥5 × 27 ACS Paragon Plus Environment


Page 28 of 43

1015 charge/cm2).45 The substrates in these studies were polycation nanofilms where charge density was determined by dye adsorption/desorption. The charge density calculation employed herein is based on different physical principles compared to dye adsorption, but interestingly, antimicrobial effectiveness described in the next section indicated UP-C12-50-T-10 was highly effective and durable compared to UP-C12-50-T-2. Thus, empirically, it seems that a positive ξ that corresponds to a charge density of ~1012 charge/cm2 is required for biocidal effectiveness against E. coli by a contact kill mechanism. Table 4 also provides differential charge density calculations based on subtraction of the negative charge density for Tecoflex from the values for UP-C12-50-T coatings. These values are based on the notion that the surfaces are heterogeneous at the nanoscale (AFM Figure 4). While only loosely grounded in theory, the results in Table 4, column 3 are satisfying in that charge densities are positive for all UP-C12-50-T based coatings and thus account for the relative order of effectiveness for antimicrobial kill discussed in the next section. Model for Surface Modification. Based on physical characterization, a model for PC12-50 surface concentration is shown in Figure 8 where the bulk is comprised of and UPC12-50Figure 8. Model for a polyurethane blend with P-C12-50 soft block surface concentration.


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Antimicrobial effectiveness. The ASTM E2149 test (shake flask test) described in the Experimental section is a direct way of quantifying surface antimicrobial activity. E. coli34 and S. epidermidis RP62A, a methicillin resistant strain (MRSE), were used for antimicrobial tests.44 In order to evaluate durability of surface accessible charge, the shake flask test was repeated sequentially three times for E. coli challenges (Figure 9, Trials 1, 2 and 3). A typical observation (from Trial 2) for E. coli growth after 24 h is shown in Figure S7, where a turbid medium was observed for a blank

10-4

A

10-2

B

10-2

C

10-2

D

Trial 1

control (without sample), Tecoflex and

10-5

10-5

10-5

10-5

≥4

≥4

≥4

10-2

10-2

Trial 2

UP-C12-50T-2. In

0

10-2

≥4

10-2

≥4

contrast, the medium for UP-C12-50T-10 and UPC12-50-T remained

Trial 3

0

≥4

≥4

Figure 9. Three sequential “shake flask” trials on the same coating for establishing stability. Log reduction is shown beneath the plates: A, Tecoflex; B, UP-C12-50-T-2; C, UP-C12-50-T-10; D, UP-C12-50-T. The medium dilution ratio is in the upper left corner of images.

clear after the test signaling negligible growth of E. coli.



Page 30 of 43

Log reductions are listed in Table S2 and shown beneath plate images in Figure 9. High antimicrobial effectiveness (log reduction > 4) was achieved for UP-C12-50-T-2, UP-C12-50-T10 and UP-C12-50-T coatings for Trial 1, showing that the accessible surface charge density for the blend coatings with low UP-C12-50 content (~0.2%) was adequate to inhibit E. coli growth. However, by three tests in sequence on the same specimens stability of antimicrobial effectiveness was demonstrated only for UPC12-50-T-10 and UP-

Figure 10. Plated media aliquots (dilution x100) from representative shake flask tests for S. epidermidis. A, Tecoflex; B, UP-C12-T-2; C, UP-C12-50-T-10; D, UP-C12-50-T. Log reductions are shown beneath the plates.

C12-50-T coatings (Figure 9). That is, UP-C12-50-T-2 became ineffective on the second test. This finding correlates with lower quaternary nitrogen analysis by XPS (Table 3), a surface morphology with a small area fraction of UP-C12-50-T (Figure 4B), and a low zeta potential or charge density (Figure 7, Table 4). To investigate antimicrobial effectiveness against Gram-positive bacteria, the shake flask test was used for a challenge of S. epidermidis RP62A, a methicillin resistant strain (MRSE).44 Figure 10 shows a representative result for a challenge of 1 x 105 CFU/mL in M9 growth medium. A trend for increased biocidal effectiveness for UP-C12-50-T-based coatings with increased surface concentration of UP-C12-50-T is reflected in increasing log reductions. For triplicate determinations, an average 3.7 log reduction of was found against S. epidermidis (Table S4) while a reduction of > 4 log was achieved against E. coli (Table S2). The result may be compared with Zhang’s finding for quaternary ammonium modified


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polyurethanes described above (5- 6 log reductions for E. coli).30 Yagci prepared network polyurethanes functionalized with quaternary amine compounds and reported similar log reductions for Gram-positive (S. aureus) and Gram-negative (E. coli) challenges.78 Although there has been considerable discussion concerning the susceptibility of Gram-positive and Gramnegative bacteria to contact kill,79 within experimental error there is little difference in log reductions for the bacterial strains used herein or by others.30 Viability of adherent bacteria. For the shake flask test, the concentration of bacteria in the medium is assessed. However, after the test, there can be bacteria adhering to the samples. A viability test for adherent bacteria establishes whether contact kill is complete. This test for viability of adherent bacteria was carried out by Zhang for a series of cross-linked polyurethanes described above.30 No live bacteria (E. coli or S. aureus) were found on sample surfaces while a great number of bacteria (4.4 × 106 CFU/cm2 for S. aureus and 3.4 × 106 CFU/cm2 for E. coli) were observed on controls. A similar test for viability of adherent bacteria was carried out after a shake flask test for E. coli. Bacteria were detached with sonication and analyzed by plating on LB agar. As shown in Figure S9, live E. coli cells were not found after sonicating UP-C12-50-T-based shake flask films while substantial E. coli growth was observed for Tecoflex. This result indicates that bacteria are killed by direct contact with the UP-C12-50-T-based coatings. However, release of dead bacteria is important for coatings on biomedical devices as dead bacteria may accumulate on the surface and compromise the antimicrobial surface with a biofilm forming on dead cells. The degree to which strongly hydrophilic UP-C12-50-T-based coatings resist fouling will be the subject of future studies.



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Leaching studies. Leaching of antimicrobial species was investigated by following a test method specified in ASTM E2149-01. To mimic the diffusion of leachable biocidal species in M9 glucose medium used in shake flask tests, 0.5% agar was added to M9 glucose medium to prepare the agar

Figure 11. Leaching antimicrobial test (specified in ASTM E2149) results for A, medium control; B, Tecoflex (negative control); C, Trimethoprim (10 µg/mL, positive control, ZOI circled); D, UP-C12-50-T-2; E, UP-C12-50T-10; F, UP-C12-50-T.

plates. Figure 11 shows representative results for E. coli. Bacterial growth occurred on the agar surfaces near the hole filled with medium control and medium extraction of Tecoflex and UP-C12-50-T-based coatings (Figure 11A, B, D, E and F). A zone of inhibition was observed only for the trimethoprim positive control due to leaching of this biocidal antibiotic (10 µg/mL, Figure 11C). The MIC of trimethoprim for E. coli is ~ 1 µg/mL80 but the lowest concentration which gave a zone of inhibition was 10 µg/mL (Figure S8). This limitation means that slight leaching of antimicrobial species cannot be rigorously excluded for UP-C12-50-T-based coatings despite the absence of a ZOI.


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Cell compatibility. Cell compatibility of UP-C12-50-T-based coatings toward human mesenchymal stem cells (MSCs) was investigated as a metric for biocompatibility. This assay was done by direct cell contact with the coatings. Evidence for cytotoxicity is cell viability being compromised near coating edges. Results after sample introduction are shown for 0 h (Figure 12, left column), 24 h (Figure 12, middle column) and 48 h (Figure 12, right column). Noticeable cell death or morphological changes were not observed for the medium alone, the

Figure 12. ISO 10993-5 test, MSC cells for direct contact with A, no specimen (medium control); B, Tecoflex (negative control); C, latex (positive control); D, UP-C12-50-T-2; E, UP-C12-50-T-10; F, UP-C1250-T for 0 h (left column), 24 h (middle column) and 48 h (right column). 33 ACS Paragon Plus Environment


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Tecoflex negative control or for UP-C12-50-T-based coatings (Figure 12D-F). According to the ISO 10993-5 test procedure, passing the test is denoted by five grades. Grade 0 was observed, which corresponds to “no detectable zone around or under specimen”. In contrast, most cells were killed by the latex positive control resulting in detachment from the Petri dish within 24 h. These results demonstrate promising biocompatibility for the UP-C12-50-T-based coatings. MSC cells were stained with DAPI (4',6-diamidino-2-phenylindole) for cell nucleus and Alexa Fluor 546 for cytoskeleton at the end of cell compatibility tests. Images shown in Figure 13 were obtained using confocal microscopy. The overall blue color of sample slides is due to polyurethane autofluorescence. No differences were observed for Tecoflex (negative control, Figure 13A) and UP-C12-50-based coatings (Figure 13B-D). Remarkably, a large amount of viable cells were observed not only near the edge of sample slides, but also underneath the samples. Interestingly, the appearance of cells under the sample slides was changed from red to magenta due to polyurethane autofluorescence. In summary, these results demonstrate superior in vitro cell compatibility of the mixed soft Figure 13. Confocal microscopy of MSC cells in 48 hr contact with A, Tecoflex; B, UP-C12-50-T-2; C, UPC12-50-T-10 and D, UP-C12-50-T. The slides are blue due to Tecoflex polyurethane autofluorescence. Cells were stained with DAPI (4',6-diamidino-2phenylindole) for cell nucleus (blue), and Alexa Fluor 546 for cytoskeleton (red).

block UP-C12-50-T-based polyurethanes.

Conclusion. To explore the surface modifier

concept (Figure 8), polyurethane UP-C12-50-T with PTMO and P-C12-50 soft blocks was 34 ACS Paragon Plus Environment

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prepared where P-C12-50 1 is a copolyoxetane soft block with antimicrobial C12-quaternary and cytocompatible PEG-like side chains (Schemes 1 and 2). Two blends were prepared, UP-C1250-T-2 and UP-C12-50-T-10, containing 0.2 and 1 wt%, P-C12-50 respectively (Table 1). DSC for UP-C12-50-T-based coatings suggest limited bulk miscibility with Tecoflex, which was signaled by a modest reduction in the Tecoflex PTMO Tm. UP-C12-50-T-based modifiers reduce the modulus of Tecoflex into the 3 MPa range typical of silicones (Table 2). This softening is likely beneficial for biomedical applications such as wound care. AFM showed that limited miscibility of UP-C12-50-T in Tecoflex results in favorable surface phase separation in the blends. The phase image of UP-C12-50-T-10 (Figure 4C) is noteworthy with ~50 area% of the surface assigned to domains rich in UP-C12-50-T-10. X-ray photoelectron N1s spectra showed surface enrichment of quaternary N+ that increased with UPC12-50-T content. At 10 wt% UP-C12-50-T, the N+ atom% (0.41) is about half that for the neat modifier (0.71). Thus, the XPS data supports conclusions from AFM imaging. The trend for increasing zeta potentials  with increased UP-C12-50-T content was marked by a striking increase of 100 mV from -44 mV for UP-C12-50-T-2 to +39 mV for UP-C12-50-T10 (Figure 7). Taken together, zeta potentials, estimates of charge densities, AFM, XPS, and DCA analyses for UP-C12-50-T based polyurethane coatings account for the relative order of effectiveness for antimicrobial kill summarized below. In a shake flask test (ASTM E2149) against a Gram-negative E. coli challenge, a >4 log reduction (>99.99% kill) was observed for UP-C12-50-T-10, which is noteworthy as this blend contains only 1 wt% P-C12-50. Similarly, a 3.7 log reduction was found against a Gram-positive S. epidermidis strain that is methicillin resistant. Effectiveness for surface concentration of quaternary species against both Gram-positive and Gram-negative bacteria agree with prior



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studies of polyurethane surface modification30, 78 In solution, effectiveness against Gramnegative bacteria with thicker cell walls is attributed to cations with hydrophobic alkyl chains.81 It is assumed that a similar influence is operative in contact kill and is responsible for equal Gram-negative/Gram-positive antimicrobial effectiveness. Growth of human mesenchymal stem cells (MSCs) beside and under the polyurethane coatings indicates remarkable biocompatibility (Figures 12, 13). Of particular note is MSC cell growth under and beside the UP-C12-50-T-10 coating (Figure 13C) that is strongly antimicrobial (Figure 9C). Bulk and surface characterization, antimicrobial effectiveness and cytocompatibility with only 1% P-C12-50 make UP-C12-50-T-10 a candidate for future studies. Surface modification of polyurethanes with a minor constituent that surface-concentrates shows promise for stopping bacterial contamination of without release of antibiotics or other agents. Limitations include multistep polymerization processes for the surface modifier described herein and for those of others.30 Introducing a more easily prepared reactive species that copolymerizes and surface-concentrates is attractive and lends itself to network formation that can stabilize surface functionalization.78 Limiting bacterial adhesion is the greatest challenge to long term use of polycation-based contact-kill coatings for medical devices that cross the extracorporeal-intracorporeal interface. Surfaces functionalized with zwitterions have remarkable resistance to biofouling82 but do not provide antimicrobial effectiveness. The challenge of resistance to biofouling together with antimicrobial effectiveness has been discussed,79 but to our knowledge has not yet been achieved. Our future studies are aimed at addressing this challenge.


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Acknowledgement. KJW thanks the National Science Foundation, Division of Materials Research, Polymers Program (DMR-1206259) and Biomaterials Program (DMR-1608022) and the School of Engineering Foundation for partial support of this research.

The authors declare no competing financial interest.

Supporting Information. M9 glucose media formulation, process flow diagram for ASTM E2149 shake flask test, NMR, DSC, DMA, a typical observation of shake flask test, colony count, and leaching test.

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