Synthesis of a Novel Quinoline Skeleton Introduced Cationic

Beijing National Laboratory for Molecular Science, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100...
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Synthesis of a Novel Quinoline Skeleton Introduced Cationic Polyfluorene Derivative for Multimodal Antimicrobial Application Han Sun,† Bohan Yin,† Hongli Ma,† Huanxiang Yuan,‡ Bin Fu,*,† and Libing Liu*,‡ †

Department of Applied Chemistry, China Agricultural University, Beijing, 100193, P. R. China Beijing National Laboratory for Molecular Science, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, P. R. China

‡

S Supporting Information *

ABSTRACT: A new functional polyfluorene derivative containing quinoline skeleton and quarternary ammonium group (QAG) modified side chains (PFPQ) was synthesized and characterized. The multimodal antimicrobial effect toward Gram-negative E. coli was achieved by the dark toxicity resulting from the quinoline skeleton, QAG, and light toxicity resulting from reactive oxygen species (ROS) produced by the main backbone of PFPQ under white light. The mechanism of interaction between PFPQ and bacteria was also demonstrated. PFPQ bound to E. coli mainly through electrostatic interactions causing nearly 50% bacterial death in the absence of light irradiation, and the huge capability of PFPQ to generate ROS under white light opened another bactericidal mode. The killing efficiency was more than 99% upon relatively mild irradiation under white light (400−800 nm) with a light dose of 18 J·cm−2. PFPQ with the incorporation of quinoline into the backbones will provide a new versatile strategy to achieve the multimodal antimicrobial effect to fight against resistant bacteria. KEYWORDS: polyfluorene, quinoline, QAG, ROS, multimodal, antimicrobial activity reported to have strong light-harvesting properties.18 As numerous quinoline derivatives have been widely used as microbiocides commercially,19 we incorporated quinoline skeleton into polyfluorene backbone, and the polymer side chains were modified by quaternary ammonium groups that were conducive to binding negatively charged bacteria. Thus, in this work, we developed a novel antimicrobial polymer PFPQ containing quinoline skeleton and quaternary ammonium group which contributed to the dark toxicity. The main backbone of PFPQ produced ROS under white light contributing to the light toxicity. As expected, our newly established system shows excellent antibacterial activity to Gram-negative bacteria which may cause severe infections and even death.20−22 The killing efficiency was more than 99% upon relatively mild irradiation under white light (400−800 nm). It should be noted that both the light intensity (20 mW· cm−2) and the light dose (18 J·cm−2) in our experiment were less than those of reported PACT systems.9,23−25 Moreover, we also demonstrated the mechanism of interaction between PFPQ and bacteria through CLSM, ζ potential, and scanning electron microscopy (SEM) experiments. To the best of our knowledge, this is the first reported antimicrobial system based on polyfluorene derivative containing a quinoline skeleton.

1. INTRODUCTION As the outbreak of infectious diseases by pathogenic bacteria has become a significant global public health threat,1,2 there is growing interest in the design and development of novel antimicrobial materials. Nanoparticles,3−5 antimicrobial peptides,6,7 bacteriophages,8 and synthetic polymers9 have been widely studied. Photodynamic antimicrobial chemotherapy (PACT) as a new therapy modality has been already proposed for localized pathogen infections.10,11 The light, photosensitizer, and molecular oxygen are the three primary components in PACT to generate reactive oxygen species (ROS) that are mainly responsible for the excellent broad spectrum antimicrobial activities and prevent the bacteria from readily developing resistance.12,13 Although much work in the application of PACT has been reported, the development of more efficient photosensitizers, using more gentle light conditions and achieving higher sterilization rate, is still a research hotspot.14,15 Compared to the toxicity of low-molecular-weight antimicrobial agents to the environment and short-term antimicrobial ability,16 the introduction of antimicrobial functional groups to polymers offers promise for enhancing the efficacy of antimicrobial agents and minimizing the environmental problems by reducing the residual toxicity of the agents.17 Because of the facile substitution at the fluorene C9 position and excellent optical properties, polyfluorene derivatives are among the most common species in biological applications among water-soluble conjugated polymers (CPs) that are © 2015 American Chemical Society

Received: August 26, 2015 Accepted: October 23, 2015 Published: October 23, 2015 25390

DOI: 10.1021/acsami.5b07890 ACS Appl. Mater. Interfaces 2015, 7, 25390−25395

Research Article

ACS Applied Materials & Interfaces Scheme 1. Synthesis of PFPQ

Figure 1. (A) Normalized UV−vis absorption and emission spectra of PFPQ in PBS (1 mM, pH 7.4). The excitation wavelength for emission spectra is 374 nm. (B) Fluorescence intensity of DCFH (40 μM) in PBS with and without PFPQ (10 μM) under white light irradiation (0−4 min). The excitation wavelength is 488 nm.

PFPQ provides a new strategy to achieve the multimodal antimicrobial effect to fight against resistant bacteria.

phenylenebisboronic acid through Suzuki coupling reaction in a 57% yield. The ratio of the two copolymer units was 0.48/0.52 calculated from 1H NMR. Gel permeation chromatography (GPC) analysis showed that the weight-average molecular weight (Mw) of polymer 5 was 15 280 with a polydispersity index (PDI = Mw/Mn) of 2.60. The targeted polymer PFPQ was achieved by quaternization of polymer 5 with trimethylamine in a 89% yield. With QAG modified side chains, the cationic PFPQ was readily soluble in water containing 1% DMSO (v/v) at room temperature. The optical characterization of PFPQ was measured in phosphate buffered saline (PBS). As shown in

2. RESULTS AND DISCUSSION 2.1. Preparation and Characterization of PFPQ. The synthetic procedures of PFPQ were shown in Scheme 1. Reaction of quinolinone 1 with methylamine produced a white solid amide compound; without further purification, direct alkylation of the crude product with 1,6-dibromohexane provided compound 2. Subsequently, polymer 5 was prepared by copolymerization of compound 2, compound 3, and 1,425391

DOI: 10.1021/acsami.5b07890 ACS Appl. Mater. Interfaces 2015, 7, 25390−25395

Research Article

ACS Applied Materials & Interfaces

Figure 2. (A) Biocidal activity of PFPQ toward Ampr E. coli in the dark and under white light irradiation (20 mW·cm−2, 15 min). Values are expressed as means ± SD (n = 3, P < 0.01). (B) Biocidal activity of PFPQ (10 μM) for different irradiation time. Values are expressed as means ± SD (n = 3, P < 0.01). (C) Plate photographs for Ampr E. coli on LB agar plate treated with PFPQ with and without white light irradiation (20 mW· cm−2, 15 min).

of PFPQ ([PFPQ] = 10 μM) toward E. coli was nearly 50% in the dark and more than 99% upon irradiation, respectively. The results displayed the synergistic effects of dark toxicity from the structural characteristics of PFPQ and light toxicity from ROS. It should be noted that the light intensity (20 mW·cm−2) and the light dose (18 J·cm−2) in our experiment were less than those in reported PACT systems.9,23−25 2.3. Mechanism of Bacterial Killing of PFPQ. In order to demonstrate the mechanism of interaction between PFPQ and bacteria, confocal laser scanning microscopy (CLSM) was utilized to directly visualize the binding of PFPQ to E. coli. As shown in Figure 3, after incubating E. coli with PFPQ for 10 min, PFPQ bound to the membrane of E. coli cells, and caused the E. coli cells to aggregate. Zeta potentials (ζ) were employed to obtain deeper insights into the interactions between PFPQ and E. coli. As shown in Figure 4, after incubation with PFPQ and washing by water, the ζ potential of E. coli became more positive. When the mixture of E. coli and PFPQ was washed by 500 mM NaCl, the ζ potential almost turned back to the same situation as the control group in the absence of PFPQ, and the fluorescence intensity of PFPQ also decreased after the mixture was washed by 500 mM NaCl. Therefore, the interaction of PFPQ with Ampr E. coli was influenced obviously by the ionic strength, which indicated that the binding of PFPQ to E. coli was mainly dominated by electrostatic interactions. In combination with CLSM experiments, a reasonable explanation could be proposed. The cell wall of E. coli is protected by an outer membrane which contains high levels of negatively charged lipopolysaccharide.27 The cationic PFPQ could bind to E. coli membrane surface through electrostatic interactions, attracting

Figure 1A, PFPQ showed a maximum absorption peak at 374 nm and a maximum emission peak at 460 nm in PBS with a fluorescence quantum yield of 9.2% using quinine sulfate as the standard. The molar absorption coefficient of PFPQ is 2.9 × 104 L·mol−1·cm−1. The generation of ROS can be probed by 2,7-dichlorofluorescein diacetate (DCFH-DA).26 DCFH-DA was hydrolyzed by sodium hydroxide to afford DCFH, which was very sensitive to ROS and could be quickly converted into highly fluorescent 2,7-dichlorofluorescein in the presence of ROS, leading to the obvious increase of fluorescence intensity around 525 nm. As shown in Figure 1B, the generation ability of ROS by PFPQ was evaluated upon exposure to white light with a light intensity of 1 mW·cm−2. After DCFH was irradiated without PFPQ, there was no obvious change for the fluorescence spectra. With the addition of PFPQ, emission at 525 nm was detected and the fluorescence intensity increased gradually. These results indicated the powerful capability of PFPQ for the generation of ROS in a relatively low light intensity. 2.2. Bacterial Killing. On the basis of the antibacterial groups of PFPQ and its good ability to produce ROS, we subsequently studied the bacterial killing efficiency of PFPQ. Bacterial survival experiments were performed separately in the dark and under white light irradiation by a traditional plate count method and the results were displayed in Figure 2. With the increase of PFPQ concentration, the killing efficiency gradually improved for both the light and dark cases. In comparison to dark conditions, the killing efficiency under white light increased about one time (Figure 2A,C). With the extension of irradiation time, the killing efficiency also increased (Figure 2B). Colony counting showed that the killing efficiency 25392

DOI: 10.1021/acsami.5b07890 ACS Appl. Mater. Interfaces 2015, 7, 25390−25395

Research Article

ACS Applied Materials & Interfaces

interaction between PFPQ and bacteria was also demonstrated. In the dark situation, PFPQ bound to E. coli mainly through electrostatic interactions, causing nearly 50% bacterial death. The huge capability of PFPQ to produce ROS under white light provides another bactericidal mode. The killing efficiency was more than 99% upon relatively mild irradiation under white light (400−800 nm) with a light dose of 18 J·cm−2. PFPQ with the incorporation of quinoline into the backbones will provide a new versatile strategy to achieve the multimodal antimicrobial effect to fight against resistant bacteria. PFPQ has potential applications in surgical equipment, protective apparel, and medical implants to avoid pathogen infections.

4. EXPERIMENTAL SECTION Materials. Solvents were obtained from Beijing Lan Yi Co., Ltd. and were used without further purification. Methylamine, 1, 6dibromohexane, 1, 4-phenylenebisboronic acid, and Pd(dppf)Cl2 were purchased from Ou He Chem. Compound 1 and Compound 3 were synthesized according to the literature.28,29 DCFH-DA was purchased from Sigma-Aldrich. The bacteria E. coli (TOP10) was obtained from Beijing Bio-Med Technology Development Co., Ltd. and transfected by ampicillin-resistant plasmids (pcDNA3, Invitrogen). Measurements. NMR spectra were recorded with a Bruker Avance DPX300 spectrometer with tetramethylsilane as the internal standard. GPC analysis was performed on a Waters-410 system against polystyrene standard with THF as eluent. Mass spectra (MALDI) were measured on a Bruker Apex IV FTMS for high resolution mass spectra (HRMS). UV−vis spectra were measured using a JASCO V550 spectrophotometer. Fluorescence spectra were recorded on a Hitachi F-4500 fluorometer. Plate counting photographs were taken by a Bio-Rad Molecular Imager ChemiDoc XRS system. CLSM experiments were performed with a confocal laser scanning biological microscope (FV1000-IX81, Olympus, Japan). The white light source (400−800 nm) for photosensitized damage of bacteria was provided by a metal halogen lamp (MVL-210, Mejiro Genossen, Japan). The intensities of incident beams were measured by a radiometer (Photoelectric Instrument Factory of Beijing Normal University). Zeta potentials were taken on a Nano ZS (ZEN3600) system. SEM data were generated in the Hitachi S-4800 scanning electron microscope. Synthesis and Characterization of Compounds. Compound 2. A mixture of compound 1 (1.03 g, 2.9 mmol) and 20 mL of 40% methylamine methanol solution was stirred for 6 h at room temperature. 20 mL of ethyl acetate was added in the reaction medium. The crude product was filtered under reduced pressure and washed with ethyl acetate there times and then dried to give a white

Figure 3. CLSM images of Ampr E. coli incubated with PFPQ. Left: phase contrast bright-field image. Right: fluorescence image. [PFPQ] = 10 μM.

the negatively charged E. coli, thus causing the E. coli cells to aggregate together. The direct visualization of the morphological changes of E. coli was observed via field-emission SEM. As shown in Figure 5, the control group exhibited a smooth cell surface and clear bacterial edges. In the dark condition, obvious aggregation of E. coli with the addition of PFPQ could be observed, and about half of the E. coli displayed collapsed, split, and merged membranes. Upon treatment with light irradiation, catastrophic structural damages to the bacteria were visualized, and almost all the bacteria were collapsed and fused. SEM images further verified the striking damage caused by PFPQ, and were consistent with the CLSM and antibacterial experiments.

3. CONCLUSION In conclusion, a functional cationic polyfluorene derivative with quinoline skeleton introduced to the main backbone and quaternary ammonium groups modified side chains (PFPQ) was synthesized and characterized. The excellent collaborative antibacterial activity toward Gram-negative E. coli was achieved by the dark toxicity resulting from quinoline skeleton and QAG and light toxicity resulting from ROS produced by the main backbone of PFPQ under white light. The mechanism of

Figure 4. (A) ζ potentials of Ampr E. coli in the absence and presence of PFPQ. Final measurements were performed in 1 mM PBS solution at 25 °C. (B) Fluorescence spectra of the mixture washed by water and 500 mM NaCl. 25393

DOI: 10.1021/acsami.5b07890 ACS Appl. Mater. Interfaces 2015, 7, 25390−25395

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ACS Applied Materials & Interfaces

Figure 5. Morphology of bacteria incubation with PFPQ: Ampr E. coli without PFPQ and without light (A, D), Ampr E. coli with PFPQ but without light (B, E), and Ampr E. coli with PFPQ and with light (C, F). D, E, F are enlarged pictures of A, B, C. solid compound (0.88 g). A mixture of this compound (0.88 g, 2.4 mmol), 1,6-dibromohexane (1.2 g, 4.9 mmol), and K2CO3 (0.68 g, 4.9 mmol) in N,N-dimethylformamide (DMF; 20 mL) was heated at 70 °C for 2 h. After cooling to room temperature, 20 mL of distilled water was added, and the mixture was extracted with ethyl acetate (3 × 30 mL), dried over anhydrous Na2SO4, and then concentrated in vacuo. The residue was purified by column chromatography with ethyl acetate and petroleum ether (1:3, v/v) as eluent to give compound 2 (0.76 g). White solid, yield: 50%. 1H NMR (300 MHz, CDCl3, δ): 8.27 (d, J = 4.2, 1H), 7.81 (d, J = 8.1, 1H), 7.74 (s, 1H), 7.66 (d, J = 8.1, 1H), 4.29 (t, J = 6.2, 2H), 3.44 (t, J = 6.7, 2H), 3.11 (d, J = 5.1, 3H), 1.97 (m, 4H), 1.60 (m, 4H). 13C NMR (300 MHz, CDCl3, δ): 164.01, 163.58, 150.98, 145.61, 133.47, 133.18, 124.79, 121.38, 115.53, 98.88, 69.85, 33.32, 32.27, 28.26, 27.51, 26.13, 25.13. HRMALDI-MS m/z (%): [M + H] + calcd: for C17H20Br3N2O2, 520.9075; found: 520.9069. Polymer 5. A solution of monomer 2 (0.2000 g, 0.38 mmol), monomer 3 (0.2486 g, 0.38 mmol), and 1,4-phenylenebisboronic acid (0.1268 g, 0.76 mmol), Pd(dppf)Cl2 (30 mg) in 15 mL THF was degassed with nitrogen. Then, 2 mL of aqueous potassium carbonate (2.0 M) was added to the mixture. The resulting mixture was stirred at 80 °C for 48 h. After cooling to room temperature, 15 mL of distilled water was added and the mixture was extracted three times with chloroform and dried over anhydrous Na2SO4. After the organic solvent was removed, the residue was precipitated into methanol to give desired polymer 5 (0.3312 g). Yellowish brown solid, yield:57%. 1 H NMR (300 MHz, CDCl3, δ): 7.74 (m, 18H), 4.07 (s, 2H), 3.33 (m, 6H), 3.06 (m, 3H), 2.10 (m, 4H), 1.71 (m, 6H), 1.18 (m, 14H), 0.79 (m, 4H). PFPQ. A solution of polymer 5 (0.1206 g, 0.09 mmol) and trimethylamine (3 mL, 3.2 M Me3N solution in methanol, 9.6 mmol) in CH2Cl2 was stirred for 24 h at room temperature. After the organic solvent was removed by vacuum distillation, the residue was washed with acetone to give PFPQ (0.1078g). Yellowish brown solid, yield: 89%. 1H NMR (300 MHz, DMSO-d6, δ): 7.77 (m, 18H), 4.08 (s, 2H), 3.28 (m, 6H), 3.08 (m, 30H), 2.08 (m, 4H), 1.50 (m, 6H), 1.11 (m, 14H), 0.70 (m, 4H). Reactive Oxygen Species (ROS) Measurements. PFPQ was added to activated DCFH solution (40 μM) to a final concentration of 10 μM. The fluorescence spectra were recorded at 1.0 min intervals after the specimens were irradiated under white light (1 mW·cm−2). The excitation wavelength is 488 nm and the fluorescence spectra was measured in the 500−700 nm emission range. The fluorescence intensity of DCFH at 525 nm was plotted as a function of the irradiation time. Preparation of Bacterial Solutions. A single colony of Ampr E. coli from a solid Luria Broth (LB) agar plate was added to 10 mL of liquid LB culture medium containing 100 μg·mL−1 ampicillin and grown at 37 °C for 6−8 h. Bacteria were harvested through centrifuging (7100 g

for 1 min) and washed by PBS (1 mM, pH = 7.4) three times. After discarding the supernatant, the remaining Amp r E. coli was resuspended in PBS and diluted to an optical density of 1.0 at 600 nm (OD600 = 1.0). Antibacterial Experiments. To measure the light toxicity of PFPQ, PFPQ was incubated with E. coli for 10 min at 37 °C in the dark, and then exposed to 20 mW·cm−2 white light for 15 min. The mixture was serially diluted 10 000-fold with PBS. A 100 μL portion of diluted bacterial suspension was evenly extended onto solid LB agar plate which contained 100 μg·mL−1 ampicillin. After incubating for 16 h at 37 °C, the bacterial colonies were formed. The number of colonyforming units (cfu) of Ampr E. coli incubated with PFPQ divided by the number of cfu of control group conducted in the absence of PFPQ was the survival fraction. For the dark toxicity of PFPQ, the illumination step was replaced by holding in the dark for 15 min. Other conditions were the same as the measurement of light toxicity. Confocal Laser Scanning Microscopy (CLSM) Characterization. The Ampr E. coli in PBS was incubated with 10 mM PFPQ at 37 °C for 10 min. After discarding the unbound PFPQ in media by centrifugation (7100 g for 1 min), the residue was resuspended in PBS and kept on ice. 10 μL aliquots of suspensions were added to clean glass slides, and then the coverslips were slightly covered. The specimens were observed under confocal laser scanning microscopy using a 405 nm laser. The control group was Ampr E. coli incubated without PFPQ. The fluorescence of PFPQ was highlighted in cyan. Zeta Potential Measurements. The Ampr E. coli in PBS was incubated with 10 mM PFPQ at 37 °C for 10 min. After that, the unbound PFPQ was removed with centrifugation (7100 g for 1 min). The obtained pellets were washed with water and 500 mM NaCl of PBS solution, respectively. After centrifugation (7100 g for 1 min), the pellets were resuspended in 1 mL PBS and kept on ice. The specimens were prepared for zeta potential measurements. The Ampr E. coli incubated without PFPQ as the control group was conducted under the same condition. SEM Measurements. To directly visualize the morphological changes of Ampr E. coli incubated with PFPQ in the absence and presence of white light, SEM characterization was employed to this study. After the operation according to antibacterial experiments, the mixture of E. coli and PFPQ was centrifuged (3500 g for 10 min). The supernatants were removed, and the bacterial pellets were fixed with 0.5% glutaraldehyde in PBS at room temperature for 30 min. Then, 2 μL aliquots of bacterial suspensions were transferred onto clean silicon slices. As soon as the specimens became dried, 0.1% glutaraldehyde was added for further fixation for 3 h. Next, the specimens were washed with sterile water twice and then dehydrated with increasing concentrations of ethanol (70% for 6 min, 90% for 6 min, and 100% for 6 min). At last, the dried specimens were coated with platinum for SEM measurements. 25394

DOI: 10.1021/acsami.5b07890 ACS Appl. Mater. Interfaces 2015, 7, 25390−25395

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b07890. ROS measurements, 1H NMR and 13C NMR spectra, and GPC data (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We greatly acknowledge the financial support by The National Natural Science Foundation of China (No. 21172255) and National Science Foundation for Fostering Talents in Basic Research of China (No. J1210064), and the National S&T Pillar Program of China (2012BAK25B03).

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DOI: 10.1021/acsami.5b07890 ACS Appl. Mater. Interfaces 2015, 7, 25390−25395