Using Neutron Reflectometry to Characterize Antimicrobial Protein

May 24, 2017 - when developing active antimicrobial surfaces.20−22 Thus, physical characterization of the polymer−spacer−protein surface in mole...
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Using Neutron Reflectometry to Characterize Antimicrobial Protein Surface Coatings Peter W. Akers,† Andrew J. Dingley,*,‡,§ Simon Swift,¶ Andrew R. J. Nelson,∥ Julie Martin,†,# and Duncan J. McGillivray†,⊥ †

School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand Institute of Complex Systems: Strukturbiochemie (ICS-6), Forschungszentrum Jülich, 52425 Jülich, Germany § Institut für Physikalische Biologie, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany ¶ Department of Molecular Medicine and Pathology, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand ∥ Australian Centre for Neutron Scattering, Australian Nuclear Science and Technology Organisation, Locked Bag 2001, Kirrawee DC, New South Wales 2232, Australia ⊥ MacDiarmid Institute of Advanced Materials and Nanotechnology, Wellington 6140, New Zealand ‡

S Supporting Information *

ABSTRACT: Understanding the interaction of adsorbed or covalently immobilized proteins with solid substrates at the molecular level guides the successful design of functionalized surfaces used in biomedical applications. In this report, neutron reflectometry (NR) was used to characterize the structure of surface-attached antimicrobial protein films, with antimicrobial activity assessed using an adaption of the Japanese Industrial Standard Test JIS Z 2801. NR allowed parameters influencing bioactivity to be measured at nanometer resolution and for conclusions about structural characteristics relating to bioactivity to be drawn. Hydramacin-1 (HM-1) and lysozyme were covalently attached to poly(methyl methacrylate) (PMMA) and 3-aminopropyltriethoxysilane (APTES) films in the presence and absence of a four-unit poly(ethylene glycol) PEG-based spacer and measured using NR, followed by antimicrobial assays. APTES-PEG-protein films were structurally unique, with a layer of 80% water directly beneath the protein layer, and were the only films that displayed antimicrobial activity against Escherichia coli and Bacillus subtilis. The hydration content of these films combined with the subtle difference in the PEG layer thickness of APTES versus PMMA films played a role in defining antimicrobial activity of the prepared surface coatings.



INTRODUCTION Understanding how proteins interact with solid substrates at the molecular level following either adsorption or covalent immobilization guides the design of functionalized surfaces for various applications, including biomaterials used in medical applications. In particular, the development of antimicrobial surface coatings of medical equipment and implant devices is a major global research focus given the emergence of antibioticresistant pathogenic bacteria strains that form biofilms.1−4 Such multicellular aggregates form on adhesive surfaces, such as medical implants, and are difficult to treat because they are adaptively resistant to antibiotics when compared with their planktonic counterparts5,6 and because of weak penetration of antibiotics or host clearance mechanisms.7−9 Therefore, new therapeutic approaches are needed to eradicate biofilm-related infections, with efforts focusing on developing agents with antibiofilm activity.10,11 Covalently immobilized antimicrobial proteins (AMPs) are a promising lead for this challenge because AMPs and peptide derivatives are extremely effective against a broad spectrum of © 2017 American Chemical Society

Gram-negative and Gram-positive bacteria, including multidrug resistant strains,12−19 with picomolar minimum inhibitory concentrations (MICs) observed against planktonic cultures of known human pathogens Escherichia coli20−23 and Staphylococcus aureus.21−23 Interestingly, antimicrobial peptides can display antibiofilm activity at or below their planktonic MIC,17 indicating that antimicrobial activities against planktonic versus biofilm adapted bacteria may differ. There are numerous chemical conjugation methods to covalently attach proteins to surfaces, including AMPs,21,23 and the immobilization chemistry, immobilization surface, longterm stability, and reusability are crucial parameters considered when developing active antimicrobial surfaces.20−22 Thus, physical characterization of the polymer−spacer−protein surface in molecular detail is desirable and should aid in the optimization of AMP-based surface coatings. One particularly Received: March 27, 2017 Revised: May 24, 2017 Published: May 24, 2017 5908

DOI: 10.1021/acs.jpcb.7b02886 J. Phys. Chem. B 2017, 121, 5908−5916

Article

The Journal of Physical Chemistry B

O3 or O2 plasma treatment yielded PMMA films with COOH functionality required for protein deposition. UV/O3 treatment was performed using a Jelight 144AX UVO-CLEANER (Jelight Company Inc., Irvine, CA, USA) equipped with a low pressure mercury vapor grid lamp. According to the manufacturer’s specifications, the lamp output is 28 000 μW cm−2 at a distance of 6 mm. An exposure time of 75 s was optimal for the films prepared. O2 plasma treatment was performed using a March CS-1701 Reactive Ion Etching system at 75 Pa and 50 W with an exposure time of 2−3 s. For APTES deposition the microscope slides or Si wafers were initially cleaned using 10% Micro-90 cleaning solution (Cole-Parmer Instrument Company, Vernon Hills, IL, USA) and sonicated for 2 min in ultrapure water, followed by immersion in a 3:1 mixture of 18.1 M H2SO4 and 30% H2O2 solution (“piranha solution”, 80−100 °C) for >20 min. Substrates were then rinsed with ultrapure water, dried using a stream of N2 and used in experiments within 48 h. Substrates were immersed in a solution of 50 mM APTES in toluene and placed in a sealed vessel under dry N2. The deposition proceeded at room temperature for 8 h. Substrates were then washed with acetone, ethanol, and toluene and sonicated in toluene for 10 min. Curing took place at 120 °C for 1 h. Surface Tethering of PEG, HM-1, and Lysozyme. PEG, HM-1, and lysozyme were covalently attached to both APTES and PMMA films, attaching through −NH2 functional groups on APTES and −COOH functional groups on oxidized PMMA. Oxidized PMMA films were exposed to EDC (50 mg mL−1) and NHS (5 mg mL−1) in 50 mM MES buffer (pH 4.5) for 30 min. PEG was then attached to the surface by adding a 50 mM solution of PEG in 50 mM MES buffer (pH 6) to the reaction vessel and the reaction proceeded for a further 1 h. Surfaces were then removed from solutions, rinsed with ultrapure H2O and dried using a stream of N2. For deposition of the PEG spacer, APTES films were exposed to EDC (50 mg mL−1), NHS (5 mg mL−1) and 20 mM PEG in 50 mM MES buffer (pH 6), and incubated for 1 h. Deposition of protein on either PEG spacer or the PMMA/ APTES films followed the same general procedure. For protein samples prepared in D2O, lyophilized protein material was dissolved in a D2O-based 50 mM MES buffer (pH 6) to a final concentration of 0.1 mg mL−1 and the protein samples were left overnight (15 h) at room temperature to allow hydrogen exchange. APTES or APTES-PEG films were exposed to HM-1 or lysozyme (protein concentration of 0.1 mg mL−1), EDC (50 mg mL−1) and NHS (5 mg mL−1) simultaneously in 50 mM MES buffer (pH 6) and incubated for 1 h. Oxidized PMMA or PMMA−PEG films were exposed to EDC (50 mg mL−1) and NHS (5 mg mL−1) in 50 mM MES (pH 4.5) for 30 min followed by the addition of either HM-1 or lysozyme at 0.1 mg mL−1 in 50 mM MES buffer (pH 6) for 1 h. Substrates were rinsed using ultrapure water, dried using N2, stored under N2, and used within 3 d. For the antimicrobial testing, clean glass slides were used as a control substrate. These slides were presterilized by soaking in 70% ethanol for 1−2 min followed by 100% ethanol for 1 min and air-dried under aseptic conditions. Slides were stored in sterile 50 mL centrifuge tubes before use. Neutron Reflectometry. Time-of-flight neutron reflectometry measurements were performed on the Platypus neutron reflectometer at ANSTO33 using an incident beam spectrum between 2.5 and 19 Å arising from a liquid-D2 cold source on the OPAL Research Reactor. Twenty-four hertz

useful surface characterization technique is neutron reflectivity (NR), which has been used successfully to study protein surface coatings,24,25 because this method provides detailed information at the molecular level about protein layer thickness, hydration, and location in multilayer films. Such information cannot be obtained simultaneously or easily by other techniques. Performing NR measurements under multiple solvent contrasts reduces ambiguities while modeling, and facilitates determination of the fraction of water in layers, offering a powerful means to examine surface coatings. In this report, two antimicrobial proteins were covalently linked to PMMA and APTES films in the presence and absence of a four-unit poly(ethylene glycol) (PEG) spacer and were structurally characterized by neutron reflectometry (NR). Lysozyme, which has been used previously in surface coating studies,26−29 was selected as a benchmark enzyme, whereas the AMP hydramacin-1 (HM-1) was chosen because it interacts with a range of bacteria in a nonenzymatic manner, including clinical isolates of Enterobacter cloacae and multiresistant strains Klebsiella oxytoca and S. aureus.30 Surfaces were tested for antimicrobial activity against E. coli (HM-1-based surfaces) and Bacillus subtilis (lysozyme-based surfaces) using Japanese Industrial Standard JIS Z 2801.31 Activity was observed only for APTES-linked AMPs and examination of the NR data provided evidence for the loss of activity of PMMA coatings, showing that NR combined with biological assays facilitates molecular characterization and development of active AMP coatings.



EXPERIMENTAL SECTION Materials. HM-1 was expressed and purified as described previously.30 N-(3-(Dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide, (NHS), aminopropyltriethyl silane (APTES), poly(methyl methacrylate) (PMMA, ∼ 120 kDa), 2-(N-morpholino)ethanesulfonic acid (MES), phosphate buffered saline (PBS), polyoxyethylene (20) sorbitan monooleate (Tween-20) and lysozyme from chicken egg white were purchased from Sigma-Aldrich (St Louis, MO, USA). PEG (CA(PEG)4) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Analytical grade toluene was obtained from Scharlau S.L (Barcelona, Spain) and was stored over 3 Å molecular sieves before use. Si wafers were obtained from El-Cat Inc. (Ridgefield Park, NJ, USA) and had a diameter of 76.2 mm and thickness of 5 mm. Glass microscope slides were supplied by Waldemar Knittel Glasbearbeitungs GmbH (Braunschweig, Germany). E. coli (ATCC 25922) and B. subtilis (ATCC6633) were purchased from Cryosite (NSW, Australia). Difco Trypticase soy broth (TSB) and Trypticase soy agar (TSA) were supplied by Fort Richard (Auckland, New Zealand). Deposition of PMMA and APTES Films. PMMA (∼120 kDa) films were prepared following a previously used procedure,32 in which PMMA was suspended in toluene at 2.5 g L−1 and heated to 45 °C until no solid particles were visible. The solution was sonicated for 15 min using an Elmasonic S 30 ultrasonic bath (Elma Schmidbauer GmbH, Singen, Germany) and filtered through a 0.2 μM pore size PTFE filter (Raylab Ltd., Auckland, New Zealand). PMMA was then spin coated onto polished and cleaned Si wafers at 2000 rpm for 45 s using a Laurell WS-400B-6NPP Lite spin coater (Laurell Technologies, North Wales, PA, USA) in a clean room facility. Residual toluene was removed by leaving at room temperature overnight followed by curing at 80 °C for 2 h. UV/ 5909

DOI: 10.1021/acs.jpcb.7b02886 J. Phys. Chem. B 2017, 121, 5908−5916

Article

The Journal of Physical Chemistry B

where t is the layer thickness obtained from modeling, nSLD is the neutron SLD of the PEG or protein layer, volf rac is the volume fraction of PEG or protein in the layer, ∑SL is the sum of the neutron scattering lengths of the atoms in the PEG or protein molecules, and multiplication of SC by 1016 converts the value from molecules Å−2 to molecules cm−2. The following equation was used to calculate the mass on the surface (MoS):

neutron pulses were generated from the continuous neutron flux using a disc chopper system operating at 3.3% wavelength resolution. Measurements were made at three angles of incidence, 0.65, 2.5, and 4.5°, allowing a QZ-range of ∼0.01− 0.35 Å−1 with neutron detection using a 3He two-dimensional position sensitive detector inside a vacuum tank. Data was analyzed using the Motofit data analysis program,34 which uses an optical matrix formalism. In this formalism, the surface is modeled as a series of uniform slabs, which are characterized by thickness, neutron scattering length density (nSLD, which has contributions from all materials in each slab), and interfacial roughness. nSLD values for the PMMA and PEG layers were calculated from the bulk densities of the molecules and were fixed during fitting at the calculated values. Recent studies of PMMA32 and PMMA−PEG35 thin films have shown that the nSLD values used are appropriate and characterize the layers well. Since the chemical composition of APTES layers can vary because of variations in the extent of hydrolysis during deposition reactions,36 the nSLD of APTES layers was not fixed during data fitting. nSLDs for the proteins were calculated using the ISIS (Rutherford Appleton Laboratory, Didcot, UK) Biomolecular Scattering Length Density Calculator.37 Variations in nSLD occur when a protein is dissolved in D2O-based buffers due to exchange of labile hydrogens with deuterium present in solution: 93% exchange of labile hydrogens was used for HM-1, and 90% was used for lysozyme. The percent value for lysozyme was determined using available backbone amide hydrogen exchange rates (kex) for lysozyme at pH 7.538 and that labile side chain hydrogens are fully exchanged because these hydrogens have been shown to have kex faster (i.e., > 10−4 s−1) than backbone amide hydrogens in proteins39−41 and are often more solvent accessible than backbone amides. Thus, 236 of a possible 263 labile hydrogens of lysozyme were estimated to have exchanged, i.e., 27 labile amides show negligible exchange during the 15 h incubation because their exchange rates are