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Structure−Antibacterial Activity Relationships of Imidazolium-Type Ionic Liquid Monomers, Poly(ionic liquids) and Poly(ionic liquid) Membranes: Effect of Alkyl Chain Length and Cations Zhiqiang Zheng,†,∥ Qiming Xu,‡,∥ Jiangna Guo,† Jing Qin,† Hailei Mao,*,‡ Bin Wang,§ and Feng Yan*,† †
Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Department of Polymer Science and Engineering, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, China ‡ Department of Anesthesiology and Critical Care Medicine, Zhongshan Hospital, Fudan University, Shanghai 200438, China § Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China S Supporting Information *
ABSTRACT: The structure−antibacterial activity relationship between the small molecular compounds and polymers are still elusive. Here, imidazoliumtype ionic liquid (IL) monomers and their corresponding poly(ionic liquids) (PILs) and poly(ionic liquid) membranes were synthesized. The effect of chemical structure, including carbon chain length of substitution at the N3 position and charge density of cations (mono- or bis-imidazolium) on the antimicrobial activities against both Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was investigated by determination of minimum inhibitory concentration (MIC). The antibacterial activities of both ILs and PILs were improved with the increase of the alkyl chain length and higher charge density (bis-cations) of imidazolium cations. Moreover, PILs exhibited lower MIC values relative to the IL monomers. However, the antibacterial activities of PIL membranes showed no correlation to those of their analogous small molecule IL monomers and PILs, which increased with the charge density (bis-cations) while decreasing with the increase of alkyl chain length. The results indicated that antibacterial property studies on small molecules and homopolymers may not provide a solid basis for evaluating that in corresponding polymer membranes. KEYWORDS: antibacterial, ionic liquid, poly(ionic liquid) membrane, imidazolium cation, structure−activity relationship, membrane surface
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INTRODUCTION Deaths in hospitalized patients caused by microbial infections are constant challenges to patient safety.1−4 However, an increasing number of infections could not be treated because of growing resistance to antibiotic drugs. The design and synthesis of new antibacterial agents that do not succumb to bacterial resistance are of significant interest.5−8 To enhance the antibacterial activities of materials, antibiotic peptides,9,10 silver,11−13 and cationic compounds (or polymers)14−19 have been intensively studied. The antibacterial mechanisms of these agents generally involve disturbing cell wall (or membrane), interfering with the synthesis of genetic material (DNA or RNA), and releasing metal ions to inhibit certain enzymes.20 Among the antimicrobial agents developed, cationic compounds such as ammonium, imidazolium, pyridinium, and phosphonium salts exhibit biocidal properties against a broad spectrum of bacteria as well as wide usage as disinfectants and cleaning agents in food and pharmaceutical industries and in hospitals.21−24 Therefore, increasing attention has been paid to the antimicrobial polymers substituted with ammonium, imidazolium, pyridinium, or phosphonium cations.25−29 © 2016 American Chemical Society
Due to the unique physicochemical properties, ionic liquids (ILs), which are composed of an organic cation and an inorganic anion, have attracted significant research interest.30,31 ILs are considered as designable solvents due to the possibility to fulfill the technological demands of various applications. Applications of ILs or poly(ionic liquids) (PILs) in the areas of biomaterials and bioengineering have been recently studied.23,32,33 Most of the studies focused on the influence of the cations on the biological activities,18,34 while the effect of the anions has also been recently reported.23,35,36 However, the relationships between the antibacterial activity of IL monomer and corresponding PIL materials have not been systematically investigated as far as we know. In this work, we report the synthesis and characterization of imidazolium cation based antibacterial materials. A series of mono- and bis-imidazolium IL monomers with various substituents were synthesized (see Scheme 1) and characterized Received: March 19, 2016 Accepted: May 4, 2016 Published: May 4, 2016 12684
DOI: 10.1021/acsami.6b03391 ACS Appl. Mater. Interfaces 2016, 8, 12684−12692
Research Article
ACS Applied Materials & Interfaces
Scheme 1. Chemical Structures of Imidazolium-Type Ionic Liquid Monomers and Their Corresponding Poly(ionic liquids)
(DMSO), N,N-dimethylformamide (DMF), ethanol, acrylonitrile, phosphate buffered saline (PBS, pH = 7.4), and polyethylene terephthalate (PET) membranes were purchased from Shanghai Chemical Reagents Co. (Shanghai, China). All reagents were analytical grade and used as received without further purification. All of the vinyl monomer oils were made inhibitor-free by passing through a column filled with neutral alumina. Deionized water was used throughout the experiments. A chain transmit agent (CTA), (S)-2-(ethyl propionate)(O-ethyl xanthate), was kindly provided by Prof. Zhengbiao Zhang (Soochow University, Suzhou, China). S. aureus (ATCC 6538) and E. coli (ATCC 8099) strains were kindly provided by Dr. Shengwen Shao (Huzhou University School of Medicine, China). Synthesis of Ionic Liquid Monomers. 1-Alkyl-3-vinylimidazolium bromide was synthesized via the stirring of bromoalkane (0.025 mol) with 1-vinylimidazole (0.025 mol) at room temperature for 72 h. The product was washed with diethyl ether and ethyl acetate three times and then dried under dynamic vacuum at room temperature for 24 h. 1-Ethyl-3-vinylimidazolium Bromide (IL-C2). 1H NMR (400 MHz, D2O): δ 7.80 (s, 1H), 7.61 (s, 1H), 7.17 (dd, 1H), 5.82 (dd, 1H), 5.45 (d, 1H), 4.1 (dd, 2H), 1.3 (t, 3H). 1-Butyl-3-vinylimidazolium Bromide (IL-C4). 1H NMR (400 MHz, D2O): δ 7.80 (s, 1H), 7.61 (s, 1H), 7.17 (dd, 1H), 5.82 (dd, 1H), 5.45 (d, 1H), 4.27 (t, 2H), 1.99−1.77 (m, 2H), 1.47−1.18 (m, 2H), 0.95 (t, 3H).
with their antibacterial activities. The influence of carbon chain length of substitution at the N3 position and charge density on the antimicrobial activities of IL monomers and analogous PILs against both Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was investigated by determination of minimum inhibitory concentration (MIC). Furthermore, the corresponding PIL membranes (PILMs) were prepared by photo-crosslinking of an imidazolium monomer with acrylonitrile and styrene. The antibacterial properties of resultant PIL membranes were investigated by colony forming units (CFUs) and scanning electron microscopy (SEM), which showed high antibacterial activities against both E. coli and S. aureus. The PIL membranes synthesized in this work also exhibited low cytotoxicity and may have potential applications as ecofriendly and safe antibacterial materials in the area of healthcare. Moreover, the structure−antibacterial activity relationships between IL monomers and their analogous PILs and PIL membranes were investigated.
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EXPERIMENTAL SECTION
Materials. Imidazole, 1-vinylimidazole, 1-bromobutane, 1-bromoctane, 1-bromdodecane, 1-butylimidazole, 1-octylimidazole, 1-dodecylimidazole, 1,6-dibromohexane, styrene, 1,4-divinylbenzene, azobis(isobutyronitrile) (AIBN), benzoin ethyl ether, dimethyl sulfoxide 12685
DOI: 10.1021/acsami.6b03391 ACS Appl. Mater. Interfaces 2016, 8, 12684−12692
Research Article
ACS Applied Materials & Interfaces
molar ratio) was stirred in dried DMF at 80 °C for 24 h. The resultant raw product was purified by precipitating into ether three times to remove unreacted raw materials and other impurities. The prepared polymer solution was dialyzed against alcohol for 48 h using a spectrapor dialysis membrane (molecular weight cutoff, 3500). The yielded polymers were dried in a vacuum oven at 55 °C and characterized by 1H NMR spectroscopy. The degree of the quaternization reaction was determined by the relative peak integration values of protons which belong to methyl groups and imidazolium cations, respectively. All of the quaternization degree was found to be in the range 91−98% (see Figure S3). Poly(1-ethyl-3-vinylimidazolium bromide) (PIL-C2). 1H NMR (400 MHz, DMSO-d6): δ 8.25 (s, 3H), 4.09 (s, 3H), 1.63−1.02 (m, 2H), 1.15 (s, 3H). Poly(1-ethyl-3-(1-vinylimidazolium-3-hexyl)imidazolium bromide) (PIL-C6-Im-C2). 1H NMR (400 MHz, DMSO-d6): δ 9.12 (d, 2H), 7.43 (d, 4H), 4.13 (s, 7H), 1.71−1.35 (m, 10H), 1.29 (s, 3H). Poly(1-butyl-3-vinylimidazolium bromide) (PIL-C4). 1H NMR (400 MHz, DMSO-d6): δ 8.25 (s, 3H), 4.09 (s, 3H), 1.63−1.02 (m, 6H), 1.17 (s, 3H). Poly(1-butyl-3-(1-vinylimidazolium-3-hexyl)imidazolium bromide) (PIL-C6-Im-C4). 1H NMR (400 MHz, DMSO-d6): δ 9.12 (d, 2H), 7.43 (d, 4H), 4.13 (s, 7H), 1.71−1.20 (m, 14H),1.15 (s, 3H). Poly(1-octyl-3-vinylimidazolium bromide) (PIL-C8). 1H NMR (400 MHz, DMSO-d6): δ 8.25 (s, 3H), 4.09 (s, 3H), 1.65 (s, 12H), 1.08 (d, 2H), 1.12 (s, 3H). Poly(1-octyl-3-(1-vinylimidazolium-3-hexyl)imidazolium bromide) (PIL-C6-Im-C8). 1H NMR (400 MHz, DMSO-d6): δ 9.03 (d, 3H), 7.50 (d, 3H), 4.14 (s, 7H), 1.74−1.23 (m, 22H), 1.14 (s, 3H). Poly(1-dodecyl-3-vinylimidazolium bromide) (PIL-C12). 1H NMR (400 MHz, DMSO-d6): δ 8.25 (s, 3H), 4.09 (s, 3H), 1.61−1.18 (m, 22H), 1.16 (s, 3H). Poly(1-dodecyl-3-(1-vinylimidazolium-3-hexyl)imidazolium bromide) (PIL-C6-Im-C12). 1H NMR (400 MHz, DMSO-d6): δ 9.20 (d, 2H), 7.46 (d, 4H), 4.16 (s, 7H), 1.76−1.25 (m, 30H), 1.23 (s, 3H). Preparation of PIL Membranes. A mixture containing IL monomer (20%, molar ratio), acrylonitrile (60%, molar ratio), styrene (20%, molar ratio), divinylbenzene (2 wt % to the formulation based on the weight of monomer), and 1 wt % of benzoin ethyl ether (photoinitiator) was stirred and ultrasonicated until the solution turned transparent and homogeneous. The obtained monomer solution was cast into a glass mold and photo-cross-linked by irradiation with UV light (∼250 nm wavelength) at room temperature. The thickness of the prepared polymer membranes was controlled by standard spacer bars (∼100 μm in diameter). The resultant polymer membranes were further immersed in ethanol at room temperature for 24 h to remove the unreacted monomer. Then the depurated membranes were immersed in deionized water for 24 h and thoroughly washed with deionized water. Characterization. 1H NMR spectra of the synthesized substances were recorded on a Varian 400 MHz spectrometer using DMSO-d6, D2O or CDCl3 as the solvent. The number-average molecular weight (Mn) and molecular weight distribution (Mw/Mn) were estimated by gel permeation chromatography (GPC; Waters 1515). Fourier transform infrared (FT-IR) spectra of the membranes were carried out on a Specode 75 model spectrometer in the range of 400−4000 cm−1. The water contact angle of the polymer membranes was examined by static contact angle measurements (Drop Shape Analysis System DSA10, KRUESS, Germany). A microplate reader was used to record the turbidity of the bacteria suspension. The morphology of the bacteria on the polymer membranes was observed by a field-emission scanning electron microscope (SEM, Hitachi Model S-4700). Bacteria Culture. Bacteria of S. aureus and E. coli were grown overnight in a Luria−Bertani broth medium (LB) at 37 °C up to the exponential growth phase. The bacteria concentration could be assured by measuring the optical density at a wavelength of 600 nm (OD 600). Prior to performing the antibacterial experiments with the membranes, the OD 600 values of bacteria suspending solutions were rectified to 0.1.
1-Octyl-3-vinylimidazolium Bromide (IL-C8). 1H NMR (400 MHz, D2O): δ 7.79 (s, 1H), 7.59 (s, 1H), 7.14 (dt, 1H), 5.96−5.70 (m, 1H), 5.44 (d, 1H), 4.25 (t, 2H), 1.91 (d, 2H), 1.55−1.10 (m, 11H), 0.86 (t, 3H). 1-Dodecyl-3-vinylimidazolium Bromide (IL-C12). 1H NMR (400 MHz, CDCl3): δ 7.93 (d, 1H), 7.71 (d, 1H), 7.23 (dd, 1H), 5.87 (dd, 1H), 5.43 (dd, 1H), 4.32 (t, 2H), 1.90 (s, 2H), 1.23 (d, 19H), 0.76 (t, 3H). 3-(6-Bromohexyl)-1-alkylimidazolium bromide was synthesized via the reaction of 1-akylimidazole (0.025 mol) and 1,6-dibromohexane (0.075 mol) in acetonitrile, and the solution was stirred at 60 °C under a nitrogen atmosphere for 72 h. After the removal of solvent, the product was washed with diethyl ether and ethyl acetate three times, respectively. The obtained compound was then dried under dynamic vacuum at room temperature for 24 h. 3-(6-Bromohexyl)-1-ethylimidazolium Bromide. 1H NMR (400 MHz, D2O): δ 8.78 (s, 1H), 7.48 (s, 2H), 4.19 (dd, 4H), 3.48 (dd, 2H), 1.91−1.82 (dd, 4H), 1.32 (m, 7H). 3-(6-Bromohexyl)-1-butylimidazolium Bromide. 1H NMR (400 MHz, D2O): δ 8.78 (s, 1H), 7.48 (s, 2H), 4.19 (dd, 4H), 3.48 (dd, 2H), 1.87 (dd, 5H), 1.43 (d, 2H), 1.29 (dd, 4H), 0.90 (t, 3H). 3-(6-Bromohexyl)-1-octylimidazolium Bromide. 1H NMR (400 MHz, D2O): δ 8.99 (s, 1H), 7.61 (d, 2H), 4.27 (dt, 4H), 3.46 (t, 1H), 1.87 (dq, 6H), 1.57−1.36 (m, 1H), 1.36−1.09 (m, 4H), 0.80 (t, 3H). 3-(6-Bromohexyl)-1-dodecylimidazolium Bromide. 1H NMR (400 MHz, D2O): δ 9.24 (s, 1H), 7.75 (d, 2H), 4.50−4.31 (m, 4H), 3.47 (t, 1H), 2.05−1.80 (m, 5H), 1.49 (dd, 1H), 1.40−0.95 (m, 7H), 0.77 (d, 3H). Bis-imidazolium ionic liquids, 1-alkyl-3-(1-vinylimidazolium-3hexyl)imidazolium bromide, were synthesized via the reaction of 3(6-bromohexyl)-1-alkylimidazolium bromide (0.016 mol) with 1vinylimidazole (0.024 mol) at room temperature for 96 h. The product was washed with diethyl ether and ethyl acetate three times, respectively, and then dried under dynamic vacuum at room temperature for 24 h. 1-Ethyl-3-(1-vinylimidazolium-3-hexyl)imidazolium Bromide (ILC6-Im-C2). 1H NMR (400 MHz, DMSO-d6): δ 9.57 (s, 1H), 9.26 (s, 1H), 8.16 (s, 1H), 7.91 (s, 1H), 7.77 (s, 2H), 7.26 (dd, 8.7 Hz, 1H), 5.92 (dd, 1H), 5.41−5.33 (m, 1H), 4.13 (dt, 5H), 1.96 (m, 4H), 1.33 (m, 7H). 1-Butyl-3-(1-vinylimidazolium-3-hexyl)imidazolium Bromide (ILC6-Im-C4). 1H NMR (400 MHz, DMSO-d6): δ 9.57 (s, 1H), 9.26 (s, 1H), 8.16 (s, 1H), 7.91 (s, 1H), 7.77 (s, 2H), 7.26 (dd, 8.7 Hz, 1H), 5.92 (dd, 1H), 5.41−5.33 (m, 1H), 4.13 (dt, 5H), 1.85−1.61 (m, 5H), 1.19 (dd, 6H), 0.84 (t, 3H). 1-Octyl-3-(1-vinylimidazolium-3-hexyl)imidazolium Bromide (ILC6-Im-C8). 1H NMR (400 MHz, DMSO-d6): δ 9.65 (s, 1H), 9.33 (s, 1H), 8.25 (s, 1H), 7.99 (s, 1H), 7.85 (s, 2H), 7.34 (dd, 1H), 6.00 (d, 1H), 5.45 (d, 1H), 4.21 (d, 5H), 1.82 (d, 5H), 1.35−1.10 (m, 15H), 0.86 (d, 3H). 1-Dodecyl-3-(1-vinylimidazolium-3-hexyl)imidazolium Bromide (IL-C6-Im-C12). 1H NMR (400 MHz, DMSO-d6): δ 9.60 (s, 1H), 9.28 (s, 1H), 8.21 (s, 1H), 7.95 (s, 1H), 7.81 (s, 2H), 7.30 (dd, 1H), 5.97 (d, 1H), 5.43 (d, 1H), 4.18 (dt, 5H), 1.79 (d, 5H), 1.22 (s, 22H), 0.84 (t, 3H). General Polymerization Procedure of Poly(ionic liquids). A series of homopolymers, poly(vinylimidazole), with desired molecular weight and molecular weight distribution, were synthesized through reversible addition−fragmentation chain transfer (RAFT) polymerization at 60 °C, using AIBN as the initiator, and (S)-2-(ethyl propionate)-(O-ethyl xanthate) as the RAFT agent. The polymerization was stopped after 30 min, and the resultant was poured into the ether to remove unreacted monomers. The obtained products were dried in a vacuum oven at 55 °C for 24 h before the characterization. The molecular weight and molecular weight distribution of synthesized poly(vinylimidazole) are summarized in Supporting Information Table S1. 1H NMR (400 MHz, DMSO-d6): δ 6.82 (s, 3H), 2.12 (d, 2H), 1.16−0.72 (m, 1H). A mixture containing synthesized poly(vinylimidazole) and bromoalkane or 3-(6-bromohexyl)-1-alkylimidazolium bromide (1:3, 12686
DOI: 10.1021/acsami.6b03391 ACS Appl. Mater. Interfaces 2016, 8, 12684−12692
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Figure 1. Minimum inhibitory concentration (MIC) values against both E. coli and S. aureus in the solutions of IL monomers (A) and PILs (B) for 24 h. solution and cultured together at 37 °C for 72 h; then 0.1 mL of MTT solution (5 g/L in PBS) was added into each well. After another 4 h at 37 °C, the MTT medium was removed; 0.75 mL of DMSO was added in each well to dissolve any formazan crystals.. The cell viability was measured by the absorbance of each well at 490 nm (Bio Tek). All of the measurements were repeated for three times. The relative growth rate (RGR) of the human dermal fibroblast cells was calculated according to the following formula with PET as control:39
Colony Forming Unit Counting Method for Antibacterial Activities of the Membranes. Using a typical procedure, the bacteria suspension was dropped onto the surface of the polymeric membrane (1.5 × 1.5 cm2) in the plates and incubated at 37 °C for 4 h. A PET membrane was used as control. Then 20 μL of the bacteria suspension was dropped onto the culture plate containing agar medium. After being incubated at 37 °C for 24 h, the count for viable colonies of bacteria was recorded. Bacterial viabilities after contacting with PIL membranes were compared with the number of colonies from PET control. Morphological Change of the Bacteria. The morphological changes of bacteria onto the polymer membranes was observed by SEM (Hitachi Model S-4700). Then 100 μL of the bacteria suspensions (OD600 = 0.1) was put onto the polymeric membrane surface (1.5 × 1.5 cm2) and incubated at 37 °C for 4 h. Then the membranes were immersed in 2.5 wt % glutaraldehyde solution for 2 h. The membranes were dehydrated by using 10%, 20%, 30%, 50%, 70%, 80%, 90%, and 100% ethanol (about 10 min for each step), respectively. Minimum Inhibitory Concentration Test. The antibacterial activities of IL monomers and PILs were evaluated by determination of MIC values using the microdilution broth format, according to Clinical & Laboratory Standards Institute guidelines. Plates were incubated at 37 °C and read visually at set intervals. The MIC values were determined as the lowest concentrations of the tested agent that inhibited the growth of the detected. E. coli and S. aureus strains by 50%, respectively. Hemolysis Assay. Fresh human blood (3 mL) was collected from healthy donors. Erythrocytes were separated by centrifugation at 1500 rpm for 15 min and washed with PBS until the supernatant was transparent. The precipitate of red blood cells was diluted to 2 vol % in PBS. The sterilized PET and PIL membranes (1.5 × 1.5 cm2) were dipped into the diluted blood (5 mL for each tube) and then incubated at 37 °C for 3 h, respectively. The treated diluted blood samples were centrifuged at 1500 rpm for 15 min, and 100 μL aliquots of the supernatant were then transferred to a 96-well plate. To assess hemoglobin release on the Eon microplate spectrophotometers (Bio Tek Instruments, Inc.), the OD values were recorded at 576 nm. The red blood cells with 2% Triton were used as positive control, while the cells in PBS were used as negative control. The hemolysis rate was calculated according to the following formula: hemolysis rate/% =
ODsample − ODnegative control ODpositive control − ODnegative control
RGR/% =
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ODsample ODcontrol
× 100
RESULTS AND DISCUSSION In order to investigate the antibacterial activities of imidazolium-type IL monomers and PILs, the analogous IL monomers were first studied: 1-ethyl-3-vinylimidazolium bromide (IL-C2), 1-butyl-3-vinylimidazolium bromide (IL-C4), 1octyl-3-vinylimidazolium bromide (IL-C8), 1-dodecyl-3-vinylimidazolium bromide (IL-C12), 1-ethyl-3-(1-vinylimidazolium3-hexyl) imidazolium bromide (IL-C6-Im-C2), 1-butyl-3-(1vinylimidazolium-3-hexyl) imidazolium bromide (IL-C6-ImC4), 1-octyl-3-(1-vinylimidazolium-3-hexyl) imidazolium bromide (IL-C6-Im-C8), and 1-dodecyl-3-(1-vinylimidazolium-3hexyl) imidazolium bromide (IL-C6-Im-C12) (see Scheme 1 and Figure S1). The corresponding PILs with the same molecular weight (Mn = 12000) and distribution (PDI = 1.02) (see Table S1) were further synthesized by RAFT polymerization: poly(1-ethyl-3-vinylimidazolium bromide) (PIL-C2), poly(1-butyl-3-vinylimidazolium bromide) (PIL-C4), poly(1octyl-3-vinylimidazolium bromide) (PIL-C8), poly(1-dodecyl3-vinylimidazolium bromide) (PIL-C12), poly(1-ethyl-3-(1vinylimidazolium-3-hexyl)imidazolium bromide) (PIL-C6-ImC2), poly(1-butyl-3-(1-vinylimidazolium-3-hexyl)imidazolium bromide) (PIL-C6-Im-C4), poly(1-octyl-3-(1-vinylimidazolium-3-hexyl)imidazolium bromide) (PIL-C6-Im-C8), and poly(1-dodecyl-3-(1-vinylimidazolium-3-hexyl)imidazolium bromide) (PIL-C6-Im-C12) (see Figure S2). The chemical structure and purity of these IL monomers and PILs were confirmed by 1H NMR prior to antibacterial activity test. The antibacterial activities of synthesized imidazolium-type IL monomers and PILs were tested using Gram-negative E. coli and Gram-positive S. aureus as model microorganisms. The calculated average minimal inhibitory concentration (MIC) values were listed and summarized in Figure 1 and Table 1. It can be clearly seen that all of the IL monomers and PILs synthesized in this work showed antibacterial activities against both E. coli and S. aureus. The individual MIC values were related to the alkyl chain length of substitution at the N3 position of imidazolium cations. The longer alkyl chain results
× 100
Cytotoxicity Evaluation. Human dermal fibroblasts were provided by Shanghai Ninth People’s Hospital China. The protocols for the isolation and culture of the human dermal fibroblasts follow with the processes reported earlier.37,38 The toxicity of the PILs and PIL membranes against fibroblasts was evaluated via 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, human dermal fibroblasts (1 × 104 CFUs/mL) in 10% fetal calf serum medium were cultured in a 24-well plate for 48 h. The PET and PIL membranes (1.0 × 1.0 cm2) were put into the fibroblast cell 12687
DOI: 10.1021/acsami.6b03391 ACS Appl. Mater. Interfaces 2016, 8, 12684−12692
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membrane poration and cell death.42 In addition, the stronger electrostatic interaction between the cations and the phosphate groups of the cell wall increases the antibacterial efficiency. Here, it can be seen that the antibacterial activities of imidazolium-based PILs against both E. coli and S. aureus are well-consistent with the small molecule IL monomers studied above; that is, PIL-C6-Im-C12 < PIL-C12 < PIL-C6-Im-C8 < PIL -C8 < PIL-C6-Im-C4 < PIL-C4 < PIL-C6-Im-C2 < PILC2. Therefore, PIL-C6-Im-C12 shows the promising activities against both the E. coli and S. aureus. Figure S4 displays the bacteria growth in IL monomers and PILs under the concentration of MIC values for 48 h. The results indicate that the ILs or PILs could kill bacteria and effectively inhibit their growth. Among the antibacterial materials studied, tough and flexible polymer membranes present great advantages. Moreover, from the viewpoint of application, the membranes with high antibacterial properties are requisite for medical settings. On the basis of the antibacterial activities of the IL monomers and PILs studied above, analogous imidazolium-based PIL membranes were prepared by photo-cross-linking of an IL monomer with styrene and acrylonitrile, using divinylbenzene as crosslinking agent (shown in Scheme 2). Acrylonitrile and styrene were chosen as the co-monomers in this work because poly(styrene-co-acrylonitrile) is a type of polymer material with high chemical resistance and expected ability to form robust membranes.43 However, due to the poor solubility of ILC2 and IL-C6-Im-C2 in styrene and acrylonitrile, the synthesis of corresponding PIL membranes by the photopolymerization failed. The synthesized PIL membranes were first characterized by means of FT-IR spectrum. As can be seen from Figure 2, all the membranes show a characteristic peak at 1580−1620 cm−1 due to the stretching vibration of imidazolium cation. An absorption band at about 2230 cm−1 confirms the existence of a cyano (CN) group, while the peaks at 3027−3128 and 1455−1566 cm−1 correspond to the polystyrene units. The
Table 1. Antimicrobial Activities of Imidazolium-Type IL Monomers and PILs Measured as MIC MIC (μmol mL−1) samples
S. aureus
E. coli
IL-C2 IL-C4 IL-C8 IL-C12 IL-C6-Im-C2 IL-C6-Im-C4 IL-C6-Im-C8 IL-C6-Im-C12 PIL-C2 PIL-C4 PIL-C8 PIL-C12 PIL-C6-Im-C2 PIL-C6-Im-C4 PIL-C6-Im-C8 PIL-C6-Im-C12
472.906 54.545 2.983 0.038 110.599 27.273 0.081 0.018 110.345 2.961 1.491 0.061 33.180 0.918 0.081 0.009
945.812 54.545 1.192 0.061 55.300 22.945 0.081 0.018 110.345 5.922 1.192 0.122 33.180 1.853 0.041 0.018
in lower MIC values of IL monomers.36 This result agrees well with the published data.40,41 In addition, the antibacterial activities of IL monomers are also correlated to the charge density of imidazolium cations. The bis-imidazolium ILs exhibit higher antibacterial activities than the mono-imidazolium analogues, indicating that the higher the charge density the lower the MICs of the IL monomers are. As a result, MIC values studied in this work increase in the order: IL-C6-Im-C12 < IL-C12 < IL-C6-Im-C8 < IL-C8 < IL-C6-Im-C4 < IL-C6-ImC2< IL-C4 < IL-C2 (Figure 1A). It has been hypothesized that the antibacterial mechanism of the cationic compounds (or polymers) may involve the electrostatic interaction of the phosphate groups of the cell wall with cation moieties. The hydrophobic segments of the compounds (or polymers) insert into the lipid membrane of bacteria, leading to the cell
Scheme 2. Synthesis of Antibacterial Imidazolium-Type PIL Membranes
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DOI: 10.1021/acsami.6b03391 ACS Appl. Mater. Interfaces 2016, 8, 12684−12692
Research Article
ACS Applied Materials & Interfaces
Panels A′, A″, B′, and B″ of Figure 3 also show the dynamic variation of the antibacterial activities of PIL membranes. The signifigant difference could be observed within the first 1 h. The order of the antibacterial activity for PIL membranes synthesized was determined to be PILM-C4 > PILM-C6-ImC4 > PILM-C6-Im-C8 > PILM-C6-Im-C12 > PILM-C8 > PILM-C12 for S. aureus and PILM-C4 > PILM-C6-Im-C8 > PILM-C6-Im-C4 > PILM-C6-Im-C12 > PILM-C8 > PIL-C12 for E. coli. This result further confirms the relatively poor antibacterial properties of PILM-C8 and PILM-C12 membranes. Therefore, it can be concluded that the antibacterial activities of the bis-imidazolium-based PIL membranes are higher than those of the mono-imidazolium ones. However, the effect of carbon chain length on the antibacterial activities of PIL membranes is quite different with those of IL monomers and PILs. Such an inconsistency may be due to the different orientation of carbon chains in IL monomer (or PIL) suspensions and on the PIL membrane surfaces. It has already been reported that imidazolium-type ILs or PILs substituted with long carbon chains may aggregate in the suspension. The hydrophobic end groups (carbon chains) tend to insert into the hydrophobic cell wall and therefore increase the antimicrobial activities. However, the antimicrobial properties of the prepared PIL membranes can be correlated with not only their chemical composition but also molecular surface structure. It has already been reported that the end groups highly affect the surface of PIL membranes, which causes a completely different composition from the bulk.44,45 In the case of the PIL membranes, the hydrophobic groups (long carbon chains) have the tendency to segregate to the polymer/air interface, and the hydrophilic groups (imidazolium cations) remain in the bulk. However, upon the bacteria suspension being dropped onto the membrane surface, the imidazolium cations are prone to spread to the polymer/water interface, while the long carbon chains partition to the membrane bulk, and thus dramatically reduce the antimicrobial efficiency. Therefore, PIL membranes with
Figure 2. FT-IR spectra of PIL membranes synthesized in this work.
results confirm the successful synthesis of imidazolium-based PIL membranes. In addition, the membranes observed by SEM (Figure S5) show that all the membrane surfaces are smooth and uniform, without any visible pores. Colony forming units counting method was used to characterize the antibacterial properties of the PIL membranes. The antibacterial activities could be determined based on the number of the bacterial colonies on the culture plate. Statistics on the viability of S. aureus and E. coli are shown in Figure 3 and Figure S6. After contacting with PIL membranes for 4 h, the viable colonies of of S. aureus and E. coli decreased sharply onto PILM-C4, PILM-C6-Im-C4, PILM-C6-Im-C8, and PILM-C6Im-C12 membrane surfaces (the relative viability was lower than 0.1%). However, relatively poor antibacterial activities were observed for PILM-C8 and PILM-C12 membranes. These results are inconsistent with the antibacterial properties of corresponding IL monomers and PILs studied above, especially for IL-C4, IL-C8, IL-C12, and PIL-C4, PIL-C8, and PIL-C12 which were investigated in aqueous suspensions.
Figure 3. Bacterial viabilities of (A) S. aureus and (B) E. coli after contacting with PIL membranes for 4h, with PET membranes as controls (left column). Time course of surviving (A′ and A″) S. aureus and (B′ and B″) E. coli bacteria upon contacting with PILs (right two columns, average of five samples). 12689
DOI: 10.1021/acsami.6b03391 ACS Appl. Mater. Interfaces 2016, 8, 12684−12692
Research Article
ACS Applied Materials & Interfaces
Figure 4. Scanning electron microscopy (SEM) images of S. aureus (A−G) and E. coli (a−g), cultured on the PIL membranes for 4 h. PET (A, a), PILM-C4 (B, b), PILM-C8 (C, c), PILM-C12 (D, d), PILM-C6-Im-C4 (E, e), PILM-C6-Im-C8 (F, f), and PILM-C6-Im-C12 (G, g), respectively. Collapses and fusion of bacterial cell membranes on the PIL membranes are observed (indicated by white arrows).
relative longer carbon chains behave with relatively weaker antibacterial properties. The morphological changes of S. aureus and E. coli incubated on the PIL membrane surfaces (for 4 h at 37 °C) were observed by using SEM (Figure 4). In contrast to the smooth and complete surfaces of the bacteria on the PET membranes, distorted and collapsed bacterial walls were clearly observed on the PILM-C4, PILM-C6-Im-C4, PILM-C6-Im-C8, and PILMC6-Im-C12 membrane surfaces, indicating that these bacteria were damaged to such an extent that the cell structure wholly collapsed.42,46−48 The excellent biocompatibility of materials are vital for the practical application of medicine. Herein, the hemolysis and cytotoxicity toward human cells were examined to evaluate the biocompatibility of the PIL-based membranes. The result of the hemolysis assay about PIL membranes toward the fresh human red blood cells (RBCs) is summarized and listed in Table 2.
Figure 5. Relative growth rates (RGR) of PIL membranes to human dermal fibroblast cells detected by MTT assay.
the OD values at 490 nm from the MTT assay, all of the relative growth rates (RGRs) of the dermal fibroblast cells were calculated. RGR values were determined to be 119%, 116%, 99.3%, 72.3%, and 96.1% for PILM-C8, PILM-C12, PILM-C6Im-C4, PILM-C6-Im-C8, and PILM-C6-Im-C12 membranes, respectively, combined with the normal morphology of fibroblast cells under microscopy, indicating low cytotoxicity of these synthesized PIL membranes. However, it should be noted that the RGR value of PILM-C4 membrane is quite low (7.11%), suggesting the high toxicity of the membrane. Although the explanation of these results is still under exploration, it can be concluded here that the higher antibacterial activities of PILM-C4 membrane might also be due to its high toxicity.
Table 2. Hemolysis Rate of Synthesized ILs, PILs, and PIL Membranes (ILs and PILs Were in the Concentration of Their MIC Values) PIL membranes PET PILM-C4 PILM-C8 PILM-C12 PILM-C6-Im-C4 PILM-C6-Im-C8 PILM-C6-Im-C12
hemolysis rate (%) 0.33 2.69 3.14 3.84 3.07 2.42 3.08
± ± ± ± ± ± ±
0.10 0.14 0.64 0.16 0.41 0.14 0.15
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CONCLUSIONS In summary, a series of imidazolium-based IL monomers, PILs, and PIL membranes were designed and synthesized as antibacterial agents. The effect of chemical structures, including the carbon chain length of substituents at the N3 position, and charge density of imidazolium cations on the antibacterial activities against E. coli and S. aureus was investigated. The longer alkyl chain length and higher charge density lead to lower MICs and higher antibacterial properties of both IL monomers and PILs in bacteria suspension. However, the antibacterial activities of the PIL membranes are correlated with not only their chemical composition but also with molecular surface structure, which therefore increased with the charge density while decreasing with the increase of alkyl chain length. Such an inconsistency between IL monomers, PILs, and
After 3 h of contact time, all of the PIL membranes exhibited a low hemolytic activity (
