Amino Acid-Based Zwitterionic Polymer Surfaces Highly Resist Long

---

Article pubs.acs.org/Langmuir

Amino Acid-Based Zwitterionic Polymer Surfaces Highly Resist Long-Term Bacterial Adhesion Qingsheng Liu,§,† Wenchen Li,§,† Hua Wang,† Bi-min Zhang Newby,† Fang Cheng,‡ and Lingyun Liu*,† †

Department of Chemical and Biomolecular Engineering, The University of Akron, Akron, Ohio 44325, United States School of Pharmaceutical Science and Technology, Dalian University of Technology, Dalian, Liaoning, China 116024

‡

S Supporting Information *

ABSTRACT: The surfaces or coatings that can effectively suppress bacterial adhesion in the long term are of critical importance for biomedical applications. Herein, a group of amino acid-based zwitterionic polymers (pAAZ) were investigated for their long-term resistance to bacterial adhesion. The polymers were derived from natural amino acids including serine, ornithine, lysine, aspartic acid, and glutamic acid. The pAAZ brushes were grafted on gold via the surface-initiated photoiniferter-mediated polymerization (SI-PIMP). Results show that the pAAZ coatings highly suppressed adsorption from the undiluted human serum and plasma. Long-term bacterial adhesion on these surfaces was investigated, using two kinds of representative bacteria [Gram-positive Staphylococcus epidermidis and Gram-negative Pseudomonas aeruginosa] as the model species. Results demonstrate that the pAAZ surfaces were highly resistant to bacterial adhesion after culturing for 1, 5, 9, or even 14 days, representing at least 95% reduction at all time points compared to the control unmodified surfaces. The bacterial accumulation on the pAAZ surfaces after 9 or 14 days was even lower than on the surfaces grafted with poly[poly(ethyl glycol) methyl ether methacrylate] (pPEGMA), one of the most common antifouling materials known to date. The pAAZ brushes also exhibited excellent structural stability in phosphate-buffered saline after incubation for 4 weeks. The bacterial resistance and stability of pAAZ polymers suggest they have good potential to be used for those applications where longterm suppression to bacterial attachment is desired.

1. INTRODUCTION Unintended biofilm formation on surfaces will cause negative effects for biomedical,1 industrial,2 and marine applications.3 To inhibit biofilm formation, much effort has been devoted to developing microbicidal coatings via release-based or nonreleasebased approaches.4 In the release-based method, silver ion or particle embedded polymer coatings have been widely used to form antibacterial films.5Antibiotic or antiseptic can also be incorporated into polymer coatings to kill bacteria or prevent bacterial attachment.6 However, toxicity of silver particles7 and fast exhaustion of the antibacterial agents8 are the drawbacks existing in the release-based approach. To overcome these issues, surfaces can be functionalized with cationic quaternary amine moieties, which kill bacteria in a nonrelease-based manner.9 The destructive interaction of polycations with the cell wall and/or cytoplasmic membrane is the generally accepted mechanism to explain their antibacterial activity.10 However, polycations may fail to destruct certain cells, resulting in further bacterial accumulation on surfaces when encountering the Gramnegative bacterial cell, which has an additional membrane to protect its inner cytoplasmic membrane.11 Therefore, new strategies need to be come up with to inhibit biofilm formation. In addition to the methods mentioned above, another commonly used strategy to suppress bacterial adhesion and © 2016 American Chemical Society

accumulation is to graft antifouling polymers on surfaces with hydrophilic or zwitterionic polymers. Poly(ethylene glycol) (PEG)-based coatings are widely used as hydrophilic antifouling surfaces and capable of resisting bacterial adhesion.12 However, in the presence of transition metal ions or oxygen, PEG is unstable and liable to degrade. Zwitterionic polymers are promising candidates to replace PEG in resisting bacterial adhesion. Methacryloyloxyethyl phosphorylcholine (MPC) copolymers can prevent biofilm formation for 24 h.13 Other zwitterionic polymers, such as poly(sulfobetaine methacrylate) (pSBMA)14 and poly(carboxybetaine methacrylate) (pCBMA),15 are also able to resist bacterial adhesion for both short and long-term. Amino acids are well-known natural zwitterions, having a carboxyl group (−COOH) and an amine group (−NH2) directly linked to the central α-carbon. Previously we have developed zwitterionic vinyl monomers based on serine,16 ornithine, lysine,17 glutamic acid, and aspartic acid18 (structures shown in Table 1). Each monomer was then grafted from surfaces to form antifouling polymer coatings, which have been proven to Received: April 6, 2016 Revised: June 30, 2016 Published: July 11, 2016 7866

DOI: 10.1021/acs.langmuir.6b01329 Langmuir 2016, 32, 7866−7874

Article

Langmuir Table 1. Chemical Structures of SerMA, LysAA, OrnAA, AspAA, and GluAA

Scheme 1. Experimental Setup of the Parallel Flow Chamber System for Biofilm Formation Assay

N,N′-carbonyldiimidazole (CDI), trifluoroacetic acid (99%), tetrahydrofuran (THF), and phosphate-buffered saline (PBS, pH 7.4) were purchased from Sigma-Aldrich. Boc-L-glutamic acid α-tert-butyl ester and boc-L-aspartic acid α-tert-butyl ester were purchased from Chem-Impex (Wood Dale, IL). O-methacryloyl L-serine (SerMA), N-ε-methacryloyl lysine (LysAA), N-δ-methacryloyl ornithine (OrnAA), N4-(2-methacrylamidoethyl) asparagine (AspAA), and N5-(2-methacrylamidoethyl) glutamine (GluAA) were synthesized using methods published before.17−19 Detailed information about how to synthesize these five amino acid-based zwitterionic (AAZ) monomers can be found in the Supporting Information. Pooled human blood serum and plasma were purchased from BioChemed Services (Winchester, VA). Sodium chloride, tryptone, and yeast extract were purchased from EMD Millipore to prepare the Luria−Bertani (LB) medium. Water in the experiments was purified by a Millipore system to reach a resistivity above 18MΩ·cm. Grafting Polymers on Surfaces. The pAAZ brushes were grafted from gold surfaces via surface-initiated photoiniferter-mediated polymerization (SI-PIMP). The photoiniferter 11-mercaptorundecane1-[4({[(diethylamino)-carbonothioyl] thioethyl}phenyl)carbamate] (DTCA) was synthesized according to a previously reported method.23 Self-assembled monolayer (SAM) of DTCA was first formed on the gold surface. The gold-coated glass chip was prepared by coating a 2 nm chromium adhesion layer and a 48 nm gold layer on a glass substrate by e-beam evaporation. The chip was cleaned by acetone, ethanol, and water successively, treated by UV/ozone for 20 min, washed by water and ethanol, and air-dried. The cleaned chip was then

be effective in combating biofouling such as nonspecific protein adsorption and cell attachment.17−20 Nevertheless, whether these amino acid-based zwitterionic polymers (pAAZ) can resist long-term bacterial adhesion remains unknown. Considering that the biofilm formation on medical devices (e.g., artificial kidney21 and intraocular lenses22) can cause infections and failed performance of devices in clinical use, the surfaces that can resist bacterial adhesion for long-term are highly desirable. The objective of this work is to explore the ability of pAAZ polymers to suppress bacterial adhesion for long-term, using both Gram-positive and Gram-negative bacteria as model. To the best of our knowledge, it is the first work to study long-term bacterial resistance of the zwitterionic amino acid-based polymers. The stability of polymer brushes under physiological conditions will also be investigated and correlated with their long-term antibacterial performance. The five pAAZ polymers, as well as the respective monomers, exhibited very low cytotoxicity,20 although they have not been approved to be used in vivo.

2. EXPERIMENTAL SECTION Materials. L-Serine (99+%), L-lysine hydrochloride (99+%), hydrochloride (99%), methacryloyl chloride (97%), 8-hydroxyquinoline, ethylenediamine (99%), and triethylamine (99%) were obtained from Alfa Aesar. Poly(ethylene glycol) methyl ether methacrylate (PEGMA, Mn = 300), copper carbonate basic,

L-ornithine

7867

DOI: 10.1021/acs.langmuir.6b01329 Langmuir 2016, 32, 7866−7874

Article

Langmuir soaked in 1 mM photoiniferter solution in THF overnight at room temperature, washed with THF, and dried with filtered air. The SAM-modified chip was placed into a quartz tube for polymerization. The tube was sealed with a rubber septum stopper and filled with N2 for protection. Ten milliliters of 0.2 M AAZ monomer solution in PBS was purged with N2 for 30 min and transferred to the quartz tube. The sample was then irradiated under a 302 nm UV lamp (UVP, model UVM-57), coupled with a 280 nm cutoff filter, for the desired polymerization time. After reaction, the chip was rinsed with water and dried before use. X-ray Photoelectron Spectroscopy (XPS). The chemical composition of the polymer brush grafted surfaces was determined by XPS (Versa Probe II Scanning XPS Microprobe, Physical Electronics). All samples were dried overnight in vacuo before measurement. C 1s spectrum at 285.0 eV was set as reference to correct the binding energy (BE) scale. Protein Adsorption by Surface Plasmon Resonance (SPR). A four-channel SPR sensor (PLASMON-IV, Institute of Photonics and Electronics, Academy of Sciences, Czech Republic) was used to measure protein adsorption on the pAAZ-grafted gold chips. The protein adsorption amount was determined by the change in the resonant wavelength at a fixed light incident angle. A preadsorptive baseline was established by flowing PBS buffer at 50 μL/min over the chip for 10 min. Then the undiluted human blood serum or plasma was flowed through different channels for another 10 min. The postadsorptive baseline was established by flowing PBS buffer again to remove unbound molecules. The wavelength shift between postadsorptive and preadsorptive baselines was finally used to quantify the protein adsorption amount. A 1 nm SPR wavelength shift at 750 nm can be converted to a protein adsorption of 15 ng/cm2.24 Ellipsometry. Thicknesses of polymer brushes grafted on gold surfaces were measured in air by an α-SE ellipsometer (J. A. Woollam Co., Lincoln, NE) equipped with a 632.8 nm He−Ne laser at incident angles of 65−75°. The refractive index of 1.45 was assigned to the polymer layer. To evaluate the stability of pAAZ brushes in PBS, the five kinds of pAAZ-grafted gold surfaces with respective optimal polymer film thickness were incubated in PBS for 1, 3, 5, 9, 14, or 28 days. The chips were then rinsed by DI water and dried, and film thicknesses were measured again using ellipsometry in air. Herein, the optimal film thickness corresponds to the minimal protein adsorption on each pAAZ surface. Biofilm Formation Assay. Staphylococcus epidermidis (S. epidermidis) or Pseudomonas aeruginosa (P. aeruginosa) was cultured on Luria− Bertani agar plates (BD, USA) at 37 °C overnight. Several colonies of each species were then used to inoculate 5 mL LB medium separately, followed by 8-h incubation under 37 °C at 280 rpm. Next the 5 mL grown culture was transferred to 45 mL LB medium and incubated for another 18 h under the same culture conditions. The culture solution was finally diluted to the concentration of 106 cells/mL and used as bacterial suspension for biofilm formation assay. A parallel flow four-chamber system (schematic diagram shown in Scheme 1) was used to evaluate biofilm formation on polymer-grafted chips. All glassware was sterilized by autoclave before use. Each glass chamber (15 cm (L) × 12 mm (W) × 2 mm (H)) contained seven chips placed next to each other: bare gold, pSerMA-, pOrnAA-, pLysAA-, pAspAA-, pGluAA-, and pPEGMA- modified substrates. Bacterial suspension (100 mL) was first continuously circulated at 2 mL/min between the bacterial suspension flask and the chamber system for 3 h for the initial bacterial attachment. The circulation loop is shown by the dashed line in Scheme 1. After 3 h, the sterile LB medium was delivered to four parallel chambers by pumps at 1 mL/min. The medium washed away the unattached bacteria and provided nutrients for the attached bacteria. After 1, 5, 9, or 14 days, at each time point, one set of chips were taken out from one chamber and rinsed with sterile PBS. Since 2 weeks are enough for biofilms to reach mature thickness and stable community structure in transcutaneous devices such as catheters,25,26 we chose to incubate the chips in flow chamber for up to 14 days. The chips were then stained with LIVE/DEAD BacLight bacterial viability kit (Invitrogen) for

observation. Five images from each chip were randomly captured by fluorescence microscopy (Olympus IX70). The number of accumulated bacteria was counted from images and averaged. The experiment was repeated three times. Statistical Analysis. The experiments were carried out at least three times for each sample, and the mean value (±standard deviation) is reported. Statistical analysis was performed using analysis of variance and Student’s t test. P values of