Research Article www.acsami.org
Zinc Ion Coordinated Poly(Ionic Liquid) Antimicrobial Membranes for Wound Healing Qiming Xu,†,⊥ Zhiqiang Zheng,‡,⊥ Bin Wang,*,§ Hailei Mao,*,† and Feng Yan‡ †
Department of Anesthesiology and Critical Care Medicine, Zhongshan Hospital, Fudan University, Shanghai 200032, China 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 Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200011, China ‡
S Supporting Information *
ABSTRACT: Herein, a series of quaternary ammonium (Qa) or imidazolium (Im) cation-based poly(ionic liquid) (PIL) membranes and their corresponding zinc ion coordinated PIL membranes were synthesized. The effects of chemical structure, including organic cations, alkyl side chain of substitution, and zinc atoms on the antimicrobial activities against Escherichia coli, Staphylococcus aureus, and Candida albicans were investigated. The Zn-containing PIL membranes show higher antibacterial activities compared to those of pristine PIL membranes due to the synergistic attributes of both organic cations (Qa or Im) and zinc atoms. A wound healing test using methicillin-resistant S. aureus infected mouse as the model further demonstrated that zinc ion coordinated PIL membranes were antibacterially active, biologically safe, and may have potential application as an antimicrobial wound dressing in a clinical setting. KEYWORDS: poly(ionic liquid) membrane, antimicrobial, imidazolium cation, zinc ion, mouse model
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INTRODUCTION With the growing emergence of antibiotic-resistant bacteria, microbial infection is attracting more and more concern in hospitals.1−3 Therefore, a variety of materials containing antimicrobial substances such as cationic compounds (or polymers),4−6 graphene,7,8 metal or metal ion containing materials,9−13 and antibiotic peptides14−18 have been extensively studied due to their broad spectrum of activities without inducing resistance. Among the antimicrobial materials studied, cationic polymers substituted with quaternary ammonium (Qa),19−21 imidazolium (Im),22−24 pyridinium,25−27 or phosphonium28,29 groups have attracted much attention due to their unique chemical properties such as the chemical structure similar to that of antibacterial peptides, which leads to high antimicrobial activities.16 In recent years, poly(ionic liquids) (PILs, polymers formed from IL monomers), a novel class of cationic polymers, have aroused considerable attention in the fields of polymer and material science.30−33 Applications of PILs as biomaterials and their antimicrobial activities against a broad spectrum of microorganisms (Gram-positive and Gram-negative bacteria and fungi) have also been investigated.34−39 It is assumed that the cationic groups of PILs could disrupt the bacteria through the electrostatic interaction with the anionic phosphate groups of the cell wall, while the hydrophobic segments of PILs may insert into the hydrophobic regions of the cell membrane, © XXXX American Chemical Society
leading to leakage of the cell membrane and eventually death of the microbes, which results in the selectivity to bacteria over human cells.33,40−43 However, most studies on antimicrobial PIL materials simply focused on the influences of the organic cations, while the effects of anions were rarely investigated.44,45 More recently, imidazolium-type PIL membranes (with Br−) were synthesized and followed by anion exchange with Ltryptophan (Trp−).30−33 The obtained PIL membranes exhibited high antibacterial efficiency due to the synergistic attributes of both the imidazolium cation and Trp− anion. On the other hand, metal-containing PILs may combine the properties of PILs with physical and chemical (or catalytic) properties that emanate from the incorporated metals. However, applications of metal-containing PILs as antimicrobial materials are still rare. In this work, a series of zinc ion coordinated Qa or Im cation-based PIL membranes was synthesized (see Scheme 1A). The effects of chemical structure, including organic cations, alkyl side chain length of substitution, and coordinated metal ions on the antimicrobial activities against representative microorganisms (Gram-positive Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA), GramReceived: February 3, 2017 Accepted: April 12, 2017
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DOI: 10.1021/acsami.7b01677 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Scheme 1. (A) Chemical Structures of Qa or Im Cation-Based PILs and Their Corresponding Zinc Ion Coordinated PILs and (B) Syntheses of Qa or Im Cation-Based Zinc Ion Coordinated PIL Membranes
with vinylbenzyl chloride (3.82 g, 0.025 mol) at room temperature for 72 h (see Figure S1). The product was washed with diethyl ether and ethyl acetate three times and then dried under dynamic vacuum at room temperature for 24 h. 1H NMR (400 MHz, DMSO-d6): δ 9.40 (s, 1H), 7.77 (d, 2H), 7.65−7.30 (m, 4H), 6.71 (dd, 1H), 5.84 (d, 1H), 5.42 (s, 2H), 5.27 (d, 1H), 3.84 (s, 3H) (see Figure S2B). 1-Butyl-3-(4-vinylbenzyl) imidazolium chloride (IL-Im-C4) was synthesized via the stirring of 1-butylimidazole (3.11 g, 0.025 mol) with vinylbenzyl chloride (3.82 g, 0.025 mol) at room temperature for 72 h (see Figure S1). The product was washed with diethyl ether and ethyl acetate three times and then dried under dynamic vacuum at room temperature for 24 h. 1H NMR (400 MHz, DMSO-d6): δ 9.49 (s, 1H), 8.42−7.03 (m, 6H), 7.07−6.49 (m, 1H), 5.87 (d, 1H), 5.70− 4.81 (m, 3H), 4.06 (dt, 6.9 Hz, 2H), 2.18−1.54 (m, 2H), 1.54−1.02 (m, 2H), 0.89 (t, 3H) (see Figure S2C). Preparation of PIL and Corresponding Zinc Ion Coordinated PIL Membranes. A mixture containing IL monomer (10%, molar ratio), acrylonitrile (67.5%, molar ratio), styrene (22.5%, molar ratio), divinylbenzene (0.5 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 UV light (∼250 nm wavelength) at room temperature. The thickness of prepared polymer membranes was controlled by standard spacer bars (∼40 μm in diameter). The resultant polymeric membranes were further immersed in ethanol solution 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 water (see Scheme 1B). Zinc ion coordinated PIL membranes were prepared by immersing the prepared PIL membranes in ZnCl2 saturated ethanol solution at
negative Escherichia coli, and fungal Candida albicans) were investigated. On the basis of the results of in vitro antimicrobial efficacy and biocompatibility, two PIL membranes, PIL-Qa-C1 and PIL-Im-C4-Zn, were evaluated for a skin wound infected with MRSA in a mouse model.
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EXPERIMENTAL SECTION
Materials. 1-Methylimidazole, 1-butylimidazole, N,N-dimethylbutylamine, vinylbenzyl chloride, vinylbenzyl trimethylammonium chloride (IL-Qa-C1), zinc chloride (ZnCl2), acrylonitrile, styrene, 1,4-divinylbenzene, benzoin ethyl ether, and ethanol were purchased from Shanghai Chemical Reagents Co. (Shanghai, China). All reagents were analytic grade and used as received without further purification. The vinyl monomer oils were made inhibitor-free by passing through a column filled with neutral alumina. The water used was deionized throughout the experiments. Strains of S. aureus (ATCC 6538) and MRSA (ATCC 33591), E. coli (ATCC 8099), and C. albicans (ATCC 76615) were kindly provided by Dr. Shengwen Shao (Huzhou University School of Medicine, China). Human dermal fibroblasts were provided by Shanghai Ninth People’s Hospital of China. Synthesis of Ionic Liquid Monomers. Vinylbenzyl dimethylbutylammonium chloride (IL-Qa-C4) was synthesized via the stirring of N,N-dimethylbutylamine (2.53 g, 0.025 mol) with vinylbenzyl chloride (3.82 g, 0.025 mol) at room temperature for 72 h (see Figure S1). 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 H NMR (400 MHz, DMSO-d6): δ 7.57 (dd, 4H), 6.78 (dd, 1H), 5.94 (d, 1H), 5.36 (d, 1H), 4.57 (s, 2H), 3.26 (m, 2H), 2.96 (s, 6H), 1.75 (s, 2H), 1.27 (dd, 2H), 0.93 (t, 3H) (see Figure S2A). 1-Methyl-3-(4-vinylbenzyl) imidazolium chloride (IL-Im-C1) was synthesized via the stirring of 1-methylimidazole (2.10 g, 0.025 mol) B
DOI: 10.1021/acsami.7b01677 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces room temperature for 48 h at 30 °C. Then, the obtained polymer membranes were thoroughly washed with ethanol and water three times (see Scheme 1B). Characterization. 1H NMR spectra of the synthesized substances were recorded on a Varian 400 MHz spectrometer using DMSO-d6 as the solvent. Fourier transform infrared (FT-IR) spectra of the membranes were carried out on a Specode 75 model spectrometer in the range of 4000−400 cm−1. The wettability of the membranes was examined by static contact angle measurements (Drop Shape Analysis System DSA10, KRUESS, Germany). Droplets (1.0 μL) of pure water were placed randomly over the surface. The optical density (OD) values were tested on an Eon microplate spectrophotometer (Bio Tek Instruments, Inc.). The energy-dispersive X-ray spectroscopy (EDX) measurements of the synthesized substances were recorded with the spectrometer attached to a scanning electron microscope (SEM, ZEISS EVO18, Germany) with an accelerating voltage of 10−15 kV. Colony Assay for the Antimicrobial Activities. The S. aureus, MRSA, and E. coli were routinely grown on Luria−Bertani (LB) agar plates at 37 °C, and C. albicans was grown on yeast peptone dextrose (YPD) agar plates at 30 °C with shaking at 150 rpm for 24 h up to the exponential growth phase. After being diluted to ∼1 × 106 CFU/mL, these microbial suspensions (120 μL) in LB or YPD were spread onto sterilized PET and PIL membranes (1.5 × 1.5 cm2). After incubation with the membranes at 37 °C for 4 h at a relative humidity higher than 90%, microbial suspensions (10 μL of each strain) were streaked onto LB or YPD agar plates. The number of the colony-forming units (CFUs) was counted after incubation at 37 °C for 24 h. Each colony assay test was repeated more than three independent times. Bacterial viabilities after contacting with PIL membrane surface for various times were compared with the number of colonies from PET control. Hemolysis Assay. Fresh human blood (3 mL) from healthy human donors was centrifuged at 1500 rpm for 15 min. The precipitate of red blood cells was washed with PBS until the supernatant was transparent and then diluted to 2 vol % in PBS. The sterilized PET and PIL membranes (1.5 × 1.5 cm2) were dipped into the diluted red blood cell solutions (2 mL for each tube) and incubated at 37 °C for 3 h, respectively. The treated blood samples were centrifuged at 1500 rpm for 15 min, and then aliquots of 100 μL supernatant were transferred from each tube into a 96-well plate. The OD values were recorded at 576 nm to assess hemoglobin release on the Eon microplate spectrophotometer. The diluted red blood cell suspensions in 2% Triton and in PBS were applied as the positive and negative controls, respectively. The hemolysis rate was calculated by the percentage of (OD sample−OD negative control)/(OD positive control−OD negative control). The independent experiments were performed in triplicate. Cytotoxicity Evaluation. The toxicity assay of PIL-based membranes was determined against human dermal fibroblast cells via a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). The protocols for the isolation and culture of the human dermal fibroblasts follow the processes reported earlier.32,33 In brief, human dermal fibroblasts (3 × 104) in 1 mL of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum were cultured in a 24-well plate for 72 h. The sterilized PET and PIL membranes (1.0 × 1.0 cm2) were put into the fibroblast cell solution and cultured together at 37 °C for 72 h, and then 0.1 mL of MTT solution (5 g L−1 in PBS) was added into each well. After another 4 h at 37 °C, the MTT medium was removed, and 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 on the Eon microplate spectrophotometer. All the measurements were repeated for three independent times. The relative growth rate (RGR) was calculated using the percentage of ODsample/ODcontrol with PET as control.46,47 Protein Absorption. A Micro BCA protein assay was used to determine the adsorption of a model protein bovine serum albumin (BSA) on the surface of PIL membranes. The sterilized PET and PIL membranes (1.5 × 1.0 cm2) were immersed into 1 mL of BSA (5 wt % in PBS) solution in a 12-well plate at 37 °C for 2 h. The samples were rinsed with PBS three times and transferred into an Eppendorf tube.
The proteins adsorbed on the membrane surface were removed by sonication at 40 kHz for 30 min with 1 mL of PBS solution containing 1 wt % SDS. The amount of BSA adsorbed on the membrane surface was determined by the Micro BCA protein assay reagent kit (Pierece, United States). In Vivo Wound Healing Study. The study using animals was approved by the ethical committee of the Shanghai Jiao Tong University and performed in accordance with the national regulations on animal studies. Adult male BALB/c mice, 6−8 weeks old, were purchased from the Experimental Animal Center at Shanghai Jiao Tong University and housed at constant temperature with 12 h periods of light/dark exposure. After the mice were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg), 1.5 × 1.5 cm2 open excision wound was created to the depth of loose subcutaneous tissue on the dorsal side skin. MRSA suspensions containing 108 CFUs were inoculated over each defined skin area to establish the wound infection model. The wounds with MRSA were covered with to-be-tested PIL-Qa-C1 and PIL-Im-C4-Zn membranes (2.0 × 2.0 cm2) and fixed with sterile gauzes. The wounds treated with PBS or MRSA and covered with sterile gauze directly were set as negative and positive controls, respectively. Six mice were used in PBS negative control group, and 11 mice were used in 3 other groups, including MRSA positive control and the PIL-Qa-C1 and PIL-Im-C4Zn test groups. On postoperative days 0, 4, 7, and 14, the dressings were removed, and the appearance of the wounds was photographed. The unhealed wound rate was calculated by the percentage of At/A0, where At and A0 were the wound areas on the specified day and the day of operation, respectively.48,49 On the 14th day after surgery, the harvested samples, including the entire wound with adjacent normal skin, were excised and fixed in 4% paraformaldehyde for the further histological analysis. The wound area and inflammatory cell counting on the hematoxylin and eosin (H&E) stained sections were determined by the ImageJ 1.41 software provided by National Institute of Health. The body weight and mortality rate of mice were recorded during the process. To further observe the systemic inflammation reaction and organ functions, blood samples were collected by puncturing the retro-orbital plexus and centrifuged at 1500 rpm for 15 min at 4 °C at the 6th, 12th, 24th, and 48th hour post-operation. Four mice were included for each time point in four groups. The serum levels of TNF-α (tumor necrosis factor-α), IL-6 (interleukin-6), IL-1β (interleukin-1β), CRP (Creactive protein), AST (aspartate transaminase), ALT (alanine transaminase), Cr (creatinine), and BUN (blood urea nitrogen) were measured by ELISA (Yousheng, Jiake Biotechnology Co. LTD, China). In addition, 50 mg of liver tissue was put into a sterile homogenizer and ground in 1 mL of PBS, and then an aliquot of 0.1 mL of liver homogenate was plated onto LB agar plate for CFU counting. Statistical Analyses. All numeric data were presented as mean ± SD. Statistical analyses were performed by GraphPad Prism 5.0 (GraphPad, Inc., La Jolla, CA, United States). The unpaired Student’s t test was applied to analyze the difference; P < 0.05 was considered to be statistically significant.
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RESULTS AND DISCUSSION Scheme 1A shows the chemical structures of Qa and Im cationbased PILs uncoordinated and coordinated with zinc chloride (ZnCl2). The analogous IL monomers IL-Qa-C4, IL-Im-C1, and IL-Im-C4 were first synthesized and characterized by 1H NMR prior to the polymerization (see Figure S1 and Figure S2). The PIL membranes were prepared via in situ photo-crosslinking of a mixture containing IL monomer, acrylonitrile, and styrene using divinylbenzene as cross-linking agent (see Scheme 1B). Due to the high chemical resistance and excellent mechanical property of poly(styrene-co-acrylonitrile) membrane, acrylonitrile and styrene were used as the comonomers for the membrane preparation. The prepared PIL membranes C
DOI: 10.1021/acsami.7b01677 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
Figure 1. Microbial viabilities of S. aureus (A), E. coli (B), C. albicans (C), and MRSA (D) after contact with PIL-Qa-C1, PIL-Qa-C4, PIL-Im-C1, PIL-Im-C4, PIL-Qa-C1-Zn, PIL-Qa-C4-Zn, PIL-Im-C1-Zn, and PIL-Im-C4-Zn membranes for 4 h with PET membranes as controls (left column). Time course of surviving (A′, A″) S. aureus, (B′, B″) E. coli, (C′, C″) C. albicans, and (D′, D″) MRSA upon contact with the membranes (right two columns).
ratio) of zinc atoms were incorporated into the PIL membranes. The elemental mapping exhibited a uniform distribution of zinc atoms inside the membranes. All these results indicate the successful preparation of the target PIL membranes. To evaluate the antimicrobial activity of these PIL-based membranes, the membrane surface was dropped with four model microorganisms: Gram-positive S. aureus and MRSA, Gram-negative E. coli, and fungal C. albicans, respectively. The microbial proliferation was assessed by colony assay at various times. The antimicrobial properties of the pristine PIL membranes (uncoordinated with ZnCl2) were first investigated. It can be seen from Figures 1A−D and Figure S7 that the Qatype PIL membranes show antimicrobial efficiencies relatively higher than those of Im-based membranes after 4 h of contact with four microorganisms. In addition, the longer the alkyl side chains of substitution, the lower the antimicrobial activities. Recognition of antimicrobial mechanism for cationic polymers
were then immersed in ZnCl2 solution to form zinc ion containing polymeric membranes. The chemical structures of PIL membranes were first characterized by means of FTIR spectra (see Figure S3). As can be seen, characteristic peaks at 1580−1620 cm−1 are due to the stretching vibration of C−N. Moreover, an absorption band at about 2220 cm−1 confirms the existence of cyano (CN) group, while the peaks at 2950−3000 and 1455−1566 cm−1 correspond to the polystyrene units. However, no obvious changes were observed for the PIL membranes before and after the coordination with ZnCl2. Figure S4 shows the photographs of the obtained PIL membranes. All the free-standing membranes are strong enough to be cut into any desired size. Figure S5 exhibits SEM images of PIL membranes. The surfaces of polymer membranes are uniform and smooth. The prepared PIL membranes were further characterized by energydispersive X-ray spectroscopy (EDX) measurements (see Figure S6). The results indicated that about 0.1% (molar D
DOI: 10.1021/acsami.7b01677 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces is usually based on the interactions between bacteria and substrates; for example, the electrostatic interaction between the phosphate groups of the bacterial (or fungal) cell wall and the cations of PIL membranes as well as the interaction between the hydrophobic regions of the lipid membrane and the hydrophobic segments of the PILs.33,40−43 These interactions boost the cell membrane permeability, leading to the leakage of cytoplasmic material and the lysis of cells. However, the antimicrobial properties of the PIL membranes can also be correlated with their molecular surface structure. Here, the hydrophobic substitutions of carbon chains incline to segregate at the polymer/air interface, while the hydrophilic groups (for example, Qa and Im cations) remain in the membrane bulk. Upon contact with bacterial suspension dropped onto the membrane surface, the long carbon chains are partitioned into the membrane bulk, while the cations spread to the polymer/water interface. Therefore, the antimicrobial efficiency of the PIL membranes with longer carbon chains of substitution is reduced. As a consequence, the antimicrobial activity order of the PIL membranes studied in this work is PIL-Qa-C4 < PIL-Qa-C1 and PIL-Im-C4 < PILIm-C1, respectively (see Figures 1A−D and Figure S7). Furthermore, the concentration of positively charged Qa higher than that of Im cations leads to the higher antimicrobial efficiency of Qa-based PIL membranes due to the stronger electrostatic interaction between the Qa cations and phosphate groups of the cell wall. It should be noted that the viable colonies of S. aureus, E. coli, C. albicans, and MRSA almost completely disappeared after 4 h of contact with zinc-containing PIL membranes (see Figures 1A−D and Figure S7). The results were supported by the dynamic variation of the antibacterial process (see Figures 1A′−D′ and A″−D″), further confirming that the coordination with ZnCl2 highly improves the antibacterial activities of pristine PIL membranes. The supposed antibacterial mechanism of cationic polymers could also be employed to explain the antimicrobial properties of these zinc-containing PIL membranes. In addition to the electrostatic interaction with the teichoic acids in the microorganism membrane,50 it is presumed that Zn2+ can produce reactive oxygen species (ROS) in cells, induce the gene expression related to the oxidative stress, prolong the lag phase of the growth cycle, inhibit the cell wall synthesis, and thus lead to the growth inhibition and death of microbes.13,50−52 Therefore, the high antibacterial efficiency of Zn-containing PIL membranes may be due to the synergistic attributes of both Qa (or Im) cations and Zn2+. Biocompatibility of materials is essential for medical applications. Herein, the in vitro toxicity of all the PIL membranes was evaluated by MTT and hemolysis assays (see Figure 2). It can be seen that Zn-containing PIL membranes showed relatively lower RGR values and higher hemolysis rates than those of corresponding pristine polymers (see Figure 2). The results imply that incorporation of Zn ions slightly increased the in vitro toxicity of PIL membranes to both human dermal fibroblasts and blood red cells. In addition, the relatively higher toxicity of Zn-containing PIL membranes may also be due to the ZnCl3− induced oxidative stress inside the cells.13,50−52 Among four Zn-containing PIL membranes, the longer carbon chain of substitution in Qa or Im cations and the relatively lower toxicity of the Zn-containing PIL membranes was, namely, PIL-Qa-C4-Zn > PIL-Qa-C1-Zn and PIL-Im-C4Zn > PIL-Im-C1-Zn, respectively (see Figure 2). It also can be interpreted that the longer carbon chains of the polymers are
Figure 2. RGR and hemolysis rate of PIL membranes. The toxicities to both human dermal fibroblasts and blood red cells were detected by MTT and hemolysis assays, respectively, with PET membranes as controls.
partitioned into the PIL membrane bulk to weaken the interference to the hydrophobic cell membranes. According to the general acceptance criteria for biocompatibility of medical materials,46,47,53 only the PIL-Qa-C1-Zn membrane shows a relatively lower RGR of 62.13 ± 2.85% and a slightly higher hemolysis rate of 3.88 ± 0.13%, while other PIL membranes exhibit high RGR (>75%) and low hemolysis rate (
