Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Nanoscale Characteristics and Antimicrobial Properties of (SI-ATRP)Seeded Polymer Brush Surfaces Yoo Jin Oh,*,† Essak S. Khan,‡,§ Arań zazu del Campo,‡,§ Peter Hinterdorfer,† and Bin Li*,‡ †
Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, A-4020 Linz, Austria INM−Leibniz Institute for New Materials, Campus D2.2, 66123 Saarbrücken, Germany § Chemistry Department, Saarland University, 66123 Saarbrücken, Germany ‡
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ABSTRACT: Microbial resistant coatings have raised considerable interest in the biotechnological industry and clinical scenarios to combat the spreading of infections, in particular in implanted medical devices. Polymer brushes covalently attached to surfaces represent a useful platform to identify ideal compositions for preventing bacterial settlement by quantifying bacteria−surface interactions. In this work, a series of polymer brushes with different charges, positively charged poly[2(methacryloyloxy)ethyl trimethylammonium chloride] (PMETAC), negatively charged poly(3-sulfopropyl methacrylate potassium salt) (PSPMA), and neutral poly(2-hydroxyethyl methacrylate) (PHEMA) were grafted onto glass surfaces by surface-initiated atom transfer radical polymerization in aqueous conditions. The antimicrobial activity of the polymer brushes against Gram-negative Escherichia coli was tested at the nano- and microscopic level on different time scales, that is, from nm to 100 μm, and ms to 24 h, respectively. The interaction between the polymer brushes and E. coli was studied by single-cell force spectroscopy (SCFS) and by quantification of the bacterial density on surfaces incubated with bacterial suspensions. E. coli firmly attached to positive PMETAC brushes with high work required for de-adhesion of 28 ± 9 nN·nm, but did not significantly bind to negatively charged PSPMA and neutral PHEMA brushes. Our studies of bacterial adhesion using polymer brushes with controllable chemistry provide essential insights into bacterial surface interactions and the origins of bacterial adhesion. KEYWORDS: polymer brush, single-cell force spectroscopy, bacterial adhesion, E. coli, electrostatic interaction
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INTRODUCTION The settlement of bacteria on synthetic material surfaces and the subsequent biofilm formation are crucial in many fields, such as the fouling of naval ship hulls, devices for medical diagnostics, and food packaging.1−3 Material-related fouling starts with the initial adhesion of organic molecules, such as polysaccharides, proteins, and proteoglycans, as well as with inorganic compounds that rapidly accumulate on the surface to form a conditioning film for the subsequent colonization.4 Specific ligand−receptor interactions of membrane receptors and the physicochemical properties of the materials surface together determine the type and strength of interactions with bacteria.5 Therefore, the understanding and the control of these interactions are central questions to prevent bacterial adhesion. However, when it comes to understanding the underlying mechanisms of this dynamic phenomenon in detail, many questions remain unanswered to date because of the complexity of the dynamic fouling process and the different factors involved. A lot of effort has been undertaken to develop and identify antifouling coatings and antimicrobial materials, including self-cleaning coatings and incorporation of biocidal agents (silver, antibiotics, and nanoparticles).5−8 However, the main problems of these strategies always lead to increased © XXXX American Chemical Society
bacteria resistance or pollution to the environment. Hydrophilic polymer coatings, with a highly hydrated polymeric layer, show a strong capability to bind water around the polymer chains, leading to a repulsive osmotic force to suppress the uptake of biological entities (e.g., macromolecules, cells, larvae) and prevent biofilm formation from pathogenic bacteria.9−11 Polymer brushes, a “forest” of densely end-grafted polymer chains onto a substrate,12,13 are of great interest with respect to medical and bioengineering applications because of their ability of preventing bacterial adhesion. Poly(ethylene glycol) is widely used as a nonfouling coating and is extensively discussed in literature.6,8,14 Other examples include polyethylene oxide,15 peptoids,16−19 glycerol, and carbohydrate derivatives.20−24 The hydrophobicity and the charge of the polymer brushes are the most two important parameters for bacteria adhesion.25−28 Cationic polymer chains can penetrate cells and thereby disrupt membrane integrity. Negatively charged polymer chains inhibit bacterial adhesion due to Received: June 6, 2019 Accepted: July 1, 2019 Published: July 1, 2019 A
DOI: 10.1021/acsami.9b09885 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces Table 1. Thickness and Grafting Density of Polymer Brush Films Prepared by SI-ATRP PMETAC PSPMA PHEMA
hdry (nm)
rms roughness (nm)
hswell (nm)
swelling ratio (hswell/hdry)
N
d (chains/nm2)
D (nm)
50.1 ± 0.9 56.7 ± 1.6 42.6 ± 0.7
0.24 ± 0.02 1.08 ± 0.03 0.86 ± 0.03
128.4 ± 4.9 132.9 ± 2.6 144.1 ± 1.2
2.56 2.34 3.38
2207 2185 2846
0.08 0.07 0.08
4.1 4.3 4.0
with nitrogen for half an hour. CuBr (36 mg, 0.25 mmol) and 2,2′bipyridine (95 mg, 0.6 mmol) were added. In a three-necked flask containing the ATRP initiator-coated glass coverslip and purged with argon, the polymerization precursor solution was syringed into the flask. The coverslip was completely covered by the monomer solution, and the polymerization was allowed to proceed for 1 h under an argon atmosphere. After polymerization, the substrates were washed with water and ethanol and dried with a stream of N2. The procedures for poly(3-sulfopropyl methacrylate potassium salt (PSPMA) and poly(2hydroxyethyl methacrylate) (PHEMA) brushes were similar. Reaction conditions were as follows: SPMA (1.5 g), CuBr (36 mg, 0.25 mmol), 2,2′-bipyridine (95 mg, 0.6 mmol), 2 mL H2O, 1 mL MeOH, 45 min polymerization time. HEMA (2 mL), CuBr (36 mg, 0.25 mmol), 2,2′bipyridine (95 mg, 0.6 mmol), 2 mL H2O, 1 mL MeOH, 15 min polymerization time. Characterization. Thickness Measurement. A variable angle (65°/70°/75°) spectroscopic ellipsometer (M-2000 DI; J.A. Woollam) was used to measure the thickness of the films at the ambient condition (RH 20−25%, RT 25 °C). The data were fit to a Cauchy model using the commercial modeling software (WVASE32, JA Woollam). Ellipsometric optical quantities, the phase (Δ) and amplitude (ψ), were obtained by acquiring spectra for 65°, 70°, and 75° incidence angles using wavelengths from 300 to 900 nm. In the Cauchy model, the top layer’s thickness and refractive index were determined by fitting experimental data with the model. The swelling thickness of the polymer brushes was measured on a null-ellipsometer (Multiscope, Optrel Berlin, Germany) in a polarizer-compensatorsample-analyzer configuration. The swelling ratio was measured at an incident angle of 68° using a cuvette filled with water. The reported thickness values are the average values from at least three samples. Measurements were taken at different positions within each sample. Surface Characterization of Polymer Brushes by AFM. The morphology of the brush surfaces was imaged with a commercial AFM (JPK Instruments, Nanowizard III, Berlin, Germany). All the images were acquired using tapping mode under ambient conditions (ca. 20% relative humidity, 25 °C) using silicon cantilevers (Olympus OMCL-AC240-TN) with a nominal spring constant of 2 N m−1 and a resonance frequency of 70 kHz. Images were plane-leveled by mean plane subtraction before root mean square (rms) roughness was calculated. Polymerization Degree and Grafting Density. The polymerization degree (N) was estimated following a published method.37,38 N = [1.074(hswell)3/2]/[hdry (Å2)1/2]; from the dry thickness of the polymer brush (hdry), the density of the polymer (ρ), approximately 1.15 g/cm3 for poly(methyl methacrylate)-type polymers, and the polymerization degree of the grafted polymer chains (N), the grafting density d can be roughly calculated according to d = (ρhdryNA)/M0N; M0: molecular weight of monomer, NA: Avogadro constant. The distance between grafting sites (D) can be obtained by D = [4/ (πd)]1/2 (Table 1). Bacteria Adhesion Assay. E. coli NEB 5-alpha were grown in Luria−Bertani (LB) medium overnight at 37 °C under shaking at 250 rpm. Bacteria culture was diluted with LB medium to final OD value of 0.1 and incubated on polymer brush functionalized glass coverslips surfaces for different time intervals. After 1, 5, 12, and 24 h, the substrates were washed once with 0.87% NaCl and three times with phosphate buffer saline (PBS) for removing loosely attached bacteria. The nucleus of the bacterial cells was stained with 0.1% Hoechst solution (Thermo Fisher Scientific) for 15 min for imaging and quantification of bacterial attachment. The substrates were imaged using a Zeiss Axio Observer epi-fluorescence microscope, and the number of bacteria was determined by ImageJ software. Ten samples
electrostatic repulsion between the negatively charged bacterial surface and the negatively charged polymer surface. Existing strategies to study this phenomenon rely on biochemical and biochemistry assays and optical microscopy imaging techniques such as atomic force microscopy (AFM) and scanning electron microscope (SEM).29−33 However, these strategies do not provide extensive insights into the parameters governing the interactions of bacteria and surfaces at the microscale level, or even down to the nanoscale that a single cell senses and responses at the physiological condition. Exploring bacterial binding at the single-cell level provides useful information for the understanding of the fundamentals of the initial bacterial adhesion to surfaces. Recently, AFM-based single-cell force spectroscopy (SCFS) was applied to quantify bacterial adhesion on polymer brush coatings, which provides information on the interactions between bacteria and surfaces down to the molecular level.34,35 To study the influence of surface charge of hydrophilic polymers in the regulation of bacterial adhesion, and to provide a research repertoire for conducting such studies, we present a polymer brush-based platform in which surface properties can be tailored and effects on bacterial adhesion can be studied at different scales. We show quantitatively, at the single cell level, how adhesion force and binding capabilities of Escherichia coli adapt to differently charged polymer brush surfaces. We directly assessed single bacterium−surface interactions to gain insights into the initial stage of adhesion as a perspective for the nonfouling performances of synthetic materials. Long-term macroscopic adhesion was also performed by a bacteria adhesion assay, which revealed that longer incubation time leads to pronounced adhesion of the bacteria onto surfaces. Our results highlight the importance of electrostatic interactions governing bacterial adhesion dynamics. Tuning surface coatings properties considering electrostatic interaction will inspire the effective design of antimicrobial materials. Moreover, the technique of using SCFS can aid in constructing antibacterial materials with variable and defined properties and in exploring quantitative information contributing to individual factors orchestrating the bacterial adhesion kinetics.
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EXPERIMENTAL SECTION
Synthesis of Polymer Brush Surfaces. ATRP Initiator Immobilization to Glass Coverslips. ATRP initiator chlorosilane 3(trichlorosilyl)propyl 2-bromo-2-methylpropanoate was synthesized following a previously published procedure.36 The initiator was immobilized on glass coverslips (15 mm in diameter, VWR International, LLC.) via the vapor deposition. Piranha solution (H2SO4/H2O2, 7:3, v/v) reacts violently with many organic materials and should be used with extreme care. Cleaned glass coverslips were placed into a vacuum desiccator with a vial containing 10 μL of the chlorosilane ATRP initiator. The chamber was evacuated to
