Efficient Antibacterial Performance and Effect of Structure on Property

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Efficient Antibacterial Performance and Effect of Structure on Property Based on Cationic Conjugated Polymers Liwei Zhai, Ziqi Zhang, Yantao Zhao, and Yanli Tang* Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, P. R. China

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S Supporting Information *

ABSTRACT: Cationic conjugated polymers (CCPs) have attracted more and more attention in antibacteria and tumor treatment based on photodynamic therapy (PDT). However, the main chain structure−antibacterial activities relationship has been rarely reported. Herein, we designed and synthesized four cationic conjugated polymers: one poly(fluorine phenylene) derivative (PFP) and three poly(fluorene-co-phenylene ethynylene) derivatives (PFE-1, PFE-CN-2, and PFE-NP-3). PFP and PFE-1 have the same side chains but bear different conjugated backbones. PFE-CN-2 (π-A) and PFE-NP-3 (A‑π‑A) are modified with electron-donating and/or electron-withdrawing groups. Three PFEs can produce reactive oxygen species (ROS) faster than PFP. The order of antibacterial activities of the four CCPs is as follows: PFE-CN-2 > PFE-1 ≫ PFENP-3 ≈ PFP, which is coincident with the generation rate of ROS. In addition, CCPs with D-π-A structure is more advantageous than A-π-A in ROS production and in antibacterial performance. These results provide an important base for designing more efficient PDT agents for antibacteria and antitumor.

1. INTRODUCTION Bacterial infection can cause major diseases, such as tuberculosis, plague, syphilis, and cholera, which is a severe global health concern. Over the past decades, biotics have been widely used to treat infectious diseases in clinical practice. However, a rise in microbial drug resistance has been becoming a serious threat to human beings due to the longperiod abuse of biotics.1−3 Hence, it is of high importance to develop new antibacterial approaches for killing pathogenic bacteria. It is well-known that photodynamic therapy (PDT) has been considered to be an alternative to the standard antibiotic treatment. PDT involves the use of photosensitizers to generate reactive oxygen species (ROS), especially, singlet oxygen, with light irradiation. ROS induce cell damage or death because ROS can inhibit enzyme activity and disrupt membrane structure.4−11 Compared to traditional therapies, PDT has several advantages, such as a noninvasive nature, broad-spectrum biocidal activity, and the absence of drug resistance.7,12,13 In recent years, cationic conjugated polymers (CCPs) were widely used in biological sensing, cellular imaging, drug release, and cancer diagnosis due to its advantages of good water solubility, high fluorescence quantum yield, and easy modification.10,14−27 Under the light irradiation, CCPs, as good photosensitizers, can sensitize the surrounding oxygen to produce ROS for antibacteria and tumor treatment based on PDT.28 Since Whitten and co-workers explored the antibacterial activities of cationic poly(phenylene ethynylene) for the first time,29 many important investigations about the PDT© XXXX American Chemical Society

based biocidal activities of conjugated polymers have been carried out.11,30−33 For example, it has been reported that CCPs pendant with quaternary ammonium salt groups or modified with different amounts of imidazolium salts on side chain exhibited effective antibacterial capability against different bacteria and spores.8,34−37 Wang and coauthors have shown that the conjugated polythiophenes, combined with porphyrin, can enhance ROS generation by fluorescence resonance energy transfer (FRET) effect.38 In addition, they developed a multifunctional platform for detection and killing of bacteria based on conjugated polyelectrolytes (CPs)−silver nanostructure pair.39 Tang’s group reported a new upconversion nanophosphors/fluorescent conjugated polymers nanohybrid for near-infrared-mediated high-efficiency antibacteria based on FRET.40 Furthermore, Whitten, Schanze, and coauthors have studied the effect of variable chain lengths on the antibacterial activities of symmetrical and asymmetrical cationic oligophenylene ehynylenes and conjugated poly(phenylene ethynylenes).41−43 Because the side group directly modified on the main chain of conjugated polymers also significantly affect the optical properties and ROS generation efficiency, it is important to investigate the structure−activity relationship. In this paper, we designed and synthesized four kinds of cationic conjugated polymers: poly(9,9-bis(6′-N,N,NReceived: July 17, 2018 Revised: August 17, 2018

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DOI: 10.1021/acs.macromol.8b01530 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules

(m, 2 H), 1.38 (m, 4H), 0.93 (t, J = 9.0 Hz, 3 H). 13C NMR (101 MHz, CDCl3) δ: 14.28, 22.78, 25.70, 30.13, 31.80, 74.22, 91.39, 111.45, 115.57, 143.22, 162.52. HRMS (ESI) m/z: calcd for C13H15I2NONa+ 477.9141 [M + Na]+; found 477.9135 [M + Na]+. Monomer 3. Under nitrogen, 3,5-diiodo-4-hydroxybenzonitrile (130 mg, 0.35 mmol) and triethylamine (200 μL) were dissolved in dry CH2Cl2(5 mL), and the mixture was stirred for 15 min at 0 °C. Then the solution of 2,4-dinitrobenzenesulfonyl chloride (224 mg, 0.7 mmol) was added dropwise. The resulting mixture was stirred for another 10 h at room temperature. The solvent was removed under reduced pressure. The residue was subjected to silica gel chromatography with dichloromethane as the eluent, affording monomer 3 (120 mg) as a bright yellow solid (yield: 57.0%). 1H NMR (300 MHz, CDCl3) δ (ppm): 8.79 (s, 1 H), 8.63 (d, J = 9.0 Hz, 1 H), 8.44 (d, J = 9.0 Hz, 1 H), 8.13 (s, 2 H). 13C NMR (101 MHz, DMSO) δ: 93.56, 113.60, 115.05, 121.25, 127.84, 133.64, 134.67, 143.70, 147.80, 151.58, 155.05. HRMS (ESI) m/z: calcd for C13H5I2N3O7SNa+ 623.7835 [M + Na]+; found 623.7830 [M + Na]+. PFE-CN-2. Under nitrogen, monomer 1 (99 mg, 0.15 mmol), monomer 2 (18 mg, 0.03 mmol), and 1,4-diiodobenzene (40 mg, 0.12 mmol) were dissolved completely in degassed DMF (3.2 mL) and triethylamine solution (1.1 mL). Then, Pd(PPh3)2Cl2 (6 mg, 0.0075 mmol) and CuI (1.5 mg. 0.0075 mmol) were added to the above solution. The mixture was stirred at room temperature for 12 h. The reaction mixture was dialyzed using a membrane with molecular cutoff of 3500 for 3 days and lyophilized to afford an orange compound (99 mg, 63%).1H NMR (400 MHz, DMSO-d6) δ: 7.98− 7.38 (m, 9.7 H), 3.19 (br, 4.5 H), 3.00 (s, 18 H), 2.09 (br, 4.4 H), 1.48 (m, 4.4 H). 1.06 (m, 11 H), 0.50 (br, 4.6 H). GPC (The solvent is DMF): Mw = 5074; Mn = 4831; PDI = 1.05. PFE-NP-3. Under nitrogen, monomer 1 (99 mg, 0.15 mmol), monomer 3 (14 mg, 0.03 mmol), and 1,4-diiodobenzene (40 mg, 0.12 mmol) were dissolved completely in degassed DMF (3.2 mL) and triethylamine (1.1 mL), Then, Pd(PPh3)2Cl2 (6 mg, 0.0075 mmol) and CuI (1.5 mg. 0.0075 mmol) were added to the above solution. The mixture was stirred at room temperature for 12 h. The reaction mixture was dialyzed using a membrane with molecular cutoff of 3500 for 3 days and lyophilized to afford an orange compound (87 mg, 57%). 1H NMR (400 MHz, DMSO-d6) δ: 8.86 (s, 0.2 H), 8.60−8.55 (m, 0.2 H), 8.41−8.29 (m, 0.2 H), 8.11−7.36 (m, 11.6 H), 3.17 (br, 4.0 H), 2.98 (s, 18 H), 2.08 (br, 4.4 H), 1.47 (br, 4.5 H), 1.05 (br, 9.2 H), 0.50 (br, 4.2 H). GPC (the solvent is DMF): Mw = 10964; Mn = 10798; PDI = 1.01. 2.3. Bacterial Growth Conditions. A single colony of E. coli on a solid Lysogeny Broth (LB) agar plate was incubated under 25 mL of liquid LB culture medium with shaking at 37 °C for 13 h. Then, bacteria were harvested by centrifuging (4000 rpm for 5 min) and washed with 0.9% NaCl solution twice. The supernatant was discarded, and the remaining bacteria were resuspended in 0.9% NaCl solution and diluted to 1.5 × 107 CFU/mL. 2.4. Antibacterial Experiment. E. coli cells were incubated with the PFP, PFE-1, PFE-CN-2, or PFE-NP-3 for 10 min in the dark. Then the mixture solutions were exposed to white light (90 mW/ cm2) for 30 or 60 min or incubated under the dark for 30 or 60 min. After that, a 1:1 ratio of SYTO 9/PI mixed dyes were added to the samples and kept for 15 min in the dark. The antibacterial activities were measured with a BD Accuri C6 flow cytometer and Olympus IX73 fluorescence microscope. 2.5. Reactive Oxygen Species (ROS) Measurements. 2,7Dichlorofluorescein diacetate (DCFH-DA) was used to probe the production of ROS. Under alkaline conditions, DCFH-DA was transformed into 2,7-dichlorofluorescein (DCFH), which was followed by converting into highly fluorescent 2,7-dichlorofluorescein (DCF, excitation 488 nm, emission at 525 nm,) in the presence of ROS. In the experiments, PFP, PFE-1, PFE-CN-2, PFE-NP-3 (the final concentration is 1.0 μM) was added into 2.0 mL of activated DCFH solution (40 μM). The solutions were irradiated under white light (5 mW/cm2) for 5 min, and the fluorescence intensity of DCF solution in 500−700 nm emission range was recorded every minute with the excitation wavelength of 488 nm.

trimethylammonium)hexyl)fluorine phenylene) (PFP), poly(9,9-bis(6′-N,N,N-trimethylammonium)hexyl)fluorine ethynylene phenylene ethynylene) (PFE-1), and poly(fluorene-cophenylene ethynylene) derivatives (PFE-CN-2 and PFE-NP3). PFP and PFE-1 have the same side chains but bear different conjugated backbones. PFE-CN-2 is modified with an electron-donating alkoxy group and an electron-withdrawing cyano group on the phenylene. PFE-NP-3 contains two strong electron-withdrawing groups, cyano and 2,4-dinitrobenzenesulfonate group, covalently connected in the para of phenylene. The photophysics and ROS production rate of four conjugated polymers were investigated. In addition, their antimicrobial activities against the model bacteria, Escherichia coli, were tested. Finally, the cell cytotoxicity of the four CCPs was studied. These results allows for investigating the effect of different main backbones and side chains on the photophysical properties and antibacterial activity. Interestingly, it was found that poly(fluorene-co-phenyleneethynylene) derivatives can produce ROS faster than poly(fluorene phenylene) derivatives. In addition, CCPs with D-π-A structure are more advantageous than A-π-A in ROS production and in antibacterial performance.

2. EXPERIMENTAL SECTION 2.1. Materials and Measurements. All the chemicals used in the experiments were purchased from J&K Chemical Ltd. (Beijing, China) or Aladdin Industrial Corporation (Shanghai, China) and were used without any further purification. The SYTO 9 was purchased from Thermo Fisher Scientific Inc. The 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and propidium iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO). Medium compositions were purchased from Sangon Biotech (Shanghai) Co., Ltd. All pH values were measured using a Sartorius basic pH meter. UV−vis absorption spectra were taken on a PerkinElmer Lambda 35 spectrophotometer. The fluorescence measurement was conducted with a Hitachi F-7000 spectrophotometer equipped with a xenon lamp excitation source. The Accuri C6 flow cytometer (Becton Dickinson, Franklin Lakes, NJ) was used for testing antibacterial activity. Fluorescence images of E. coli were performed on an Olympus IX73. Molecular weights of conjugated polymers were measured on a Waters Styragel system (columns: styragel HT 4 and 3) with polystyrene as the calibration standard and DMF as the eluent. The absolute quantum yield of the cationic conjugated polymers in water was performed on the quantum efficiency measurement system of C9920-02G (Hamamatsu Photonics). The excitation wavelength was the maximal absorption wavelength of the corresponding conjugated polymers. The white light source (400− 800 nm) was provided by a photoreactor (MVL-210, MejiroGenossen). Time-domain lifetime measurements were measured by a laser flash photolysis spectrometer LP920 (Edinburgh Instruments) with excitation at 355 nm. SEM images were obtained using Hitachi SU-8200 scanning electron microscope. 1H NMR and 13C NMR spectra were recorded on Bruker Avance 300 or 400 MHz spectrometers. 2.2. Synthesis of CCPs. Monomer 1, PFE-1, and PFP were synthesized according to the literature.44,45 Monomer 2. Under nitrogen, 3,5-diiodo-4-hydroxybenzonitrile (371 mg,1 mmol) and K2CO3 (146 mg, 2 mmol) were dissolved in DMF (3.5 mL), and the mixture was stirred at 100 °C for 30 min. Then 1-bromohexane (165 mg, 1 mmol) was added to the above solution. The resulting mixture was stirred for another 30 min. The final mixture was extracted with CH2Cl2, and the collected organic phase was washed with brine and distilled water, dried over MgSO4, and concentrated under vacuum. The residue was subjected to silica gel chromatography, eluted with ethyl acetate, affording monomer 2 (365 mg) as a white solid (yield: 80%). 1H NMR (300 MHz, CDCl3) δ (ppm): 8.05 (s, 2 H), 4.01 (t, J = 6.0 Hz, 2 H), 1.93 (m, 2 H), 1.55 B

DOI: 10.1021/acs.macromol.8b01530 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules Scheme 1. Synthesis of PFE-CN-2 and PFE-NP-3

PBS for three times. Then, the fixed bacteria were dehydrated with a series of ethanol aqueous solutions for 20 min at 4 °C. The dehydrated cells were resuspended with tert-butanol and transferred into a freezing dryer. A small amount of dried samples were added to the conductive adhesive. Finally, morphologies of the specimens were observed by SEM (Hitachi SU-8200).

2.6. Cytotoxicity Test. The cytotoxicity of PFP was operated by MTT assay. MCF-7 cells were cultured in dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C under 5% CO2. MCF-7 cells were seeded into 96-well plate at a density of 5000 cells/well and incubated under 37 °C for 24 h. The solution of PFP was diluted with culture medium to obtain a series of concentrations (0.5, 1.0, 2.0, 3.0, 4.0, 6.0 , 8.0, and 10.0 μM). Then the medium in the well was replaced with the solution of PFP (100 μL), and MCF-7 cells were incubated at 37 °C under 5% CO2 for 24 h. Subsequently, 10 μL of MTT (5 mg/mL in PBS) was added to each well, followed by incubation at 37 °C for 4 h. The supernatant was removed, and 100 μL of DMSO was added to each well to dissolve the produced formazan crystals. After shaking the plates for 20 min on microplate reader, absorbance values of the wells were read by microplate reader (Molecular Devices, Spectra Max M5) at 570 nm. The cytotoxicity of PFE-1, PFE-CN-2, and PFE-NP-3 was operated similar to above. 2.7. SEM Measurements. 1.5 mL of bacterial suspension (∼1.5 × 109 CFU/mL) was mixed with CCPs, followed by the treatment described in antibacterial experiments. Then the mixture was centrifuged at 4000 rpm for 5 min, and the supernatant was removed. E. coli cells were immediately fixed with 2.5% glutaraldehyde in PBS and incubated at 4 °C overnight, followed by washing with 10 mM

3. RESULTS AND DISCUSSION 3.1. Design, Synthesis, and Photophysical Characterization of Cationic Conjugated Polymers. Cationic conjugated polymers have caught wide attention in antibacterial performance because of their high ROS production under light irradiation. Especially, cationic poly(fluorine phenylene) (PFP) and poly(fluorine ethynylene phenylene) (PFE) derivatives were used widely to kill bacteria. Thus, we selected the two kinds of common cationic conjugated polymers (PFP and PFE-1) to study the effect of the main backbone on their antibacterial activity. Furthermore, the side chain modified on the main chain could influence the photophysics and antibacterial ability of conjugated polymers. Roughly, the side chain can be classified into electron-donating C

DOI: 10.1021/acs.macromol.8b01530 Macromolecules XXXX, XXX, XXX−XXX

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Figure 1. (a−d) Absorption spectra (10 μM) and emission spectra (1.0 μM) of the four conjugated polymers ((a) PFE-1, (b) PFE-CN-2, (c) PFENP-3, and (d) PFP) in aqueous solution. (e) Structures of four conjugated polymers.

and electron-withdrawing group. Hence, to investigate the impact of groups on properties, we designed the other PFEs, PFE-CN-2, and PFE-NP-3 that have the same conjugated main chain but bear different side chains. PFE-CN-2 was modified with an electron-donating alkoxy group and an electronwithdrawing cyano group, and PFE-NP-3 was substituted with two strong electron-withdrawing groups: cyano and 2, 4dinitrobenzenesulfonate group on the phenylene. Thus, three PFEs and one PFP were designed and used in our research. The synthesis of conjugated polymers is illustrated in Scheme 1. Monomer 1, PFE-1, and PFP were synthesized according to the literature.44,45 Monomer 2 was obtained by reaction of 3,5-diiodo-4-hydroxybenzonitrile with 1-bromohexane. Similarly, reaction of 3,5-diiodo-4-hydroxybenzonitrile with 2,4-dinitrobenzenesulfonyl chloride affords monomer 3. PFE-CN-2 was prepared by a Sonogashira coupling reaction between monomers 1, 2, and 1,4-diiodobenzene (molar ratio is 1.0, 0.2, and 0.8, respectively) in the presence of Pd(PPh3)2Cl2 and CuI in DMF and triethylamine. PFE-NP-3 was synthesized according to the same procedure except for monomer 3 in place of monomer 2. The 1H NMR spectra of PFE-CN-2 and PFE-NP-3 are shown in Figures S1 and S2. Because of the overlay of hydrogen chemical shift on aromatic rings, it is hard to identify the actual ratio of every monomer. The theoretical ratio was thus used as the proportion of monomer in the PFECN-2 and PFE-NP-3. The UV−vis absorption and fluorescence emission spectra of the four cationic conjugated polymers were measured in water. Figure 1 shows the absorption and emission spectra. The maximal absorption wavelength of PFE-1 is at 393 nm, and the maximal emission wavelength is at 437 nm (Table 1). PFE-CN-2 and PFE-NP-3 exhibit maximal absorption wavelength at 399 and 396 nm and maximal emission wavelength at 443 and 440 nm, respectively. The maximal absorption wavelength of PFP is at 380 nm, and the maximal emission wavelength is at 425 nm. Notably, the fluorescence intensity of PFP is the highest among the four kinds of CCPs. Compared to PFE-1, the fluorescence of PFECN-2 is stronger under the same condition, mainly due to the introduction of cyano and alkoxy groups in the side chain of

Table 1. Photophysical Properties of Four Cationic Conjugated Polymers in Water CPs

λabs (nm)

λem (nm)

absolute ΦF (%)

PFP PFE-1 PFE-CN-2 PFE-NP-3

380 393 399 396

425 437 443 440

14.5 4.5 10.4 0.9

PFE-CN-2. The cyano group is an electron acceptor, and the alkoxy group is an electron donor, making PFE-CN-2 with an electron donor−conjugated system−electron acceptor (D-π-A) structure. This structure makes the fluorescence enhancement of PFE-CN-2 and the maximal absorption/emission redshifting about 6 nm compared to PFE-1, which is consistent with that previously reported in the literature.46,47 Because of 2,4-dinitrobenzenesulfonyl, an electron-withdrawing group, being linked as a side chain of PFE-NP-3 (electron acceptor−conjugated system−electron acceptor, A-π-A), PFE-NP-3 fluorescence is quenched by photoinduced electron transfer (PET) effect, leading to the poorest fluorescence intensity.48−50 To further compare the photophysical properties of the four kinds of CCPs, the absolute fluorescence quantum yield (ΦF) in aqueous solution was measured. The cationic conjugated polymers were excited with the corresponding maximal absorption wavelength. As shown in Table 1, the ΦF of PFP obtained as 14.5% is the highest among them, while that of PFE-NP-3 (0.9%) is the lowest, which is coincident with the results measured by the fluorescence spectrophotometry. 3.2. Antibacterial Activity of CCPs. To explore the role of the main chain structure and substituted side chain group in determining the biocidal activity, the light-induced killing efficiency of four CCPs against the model bacteria, E. coli (Gram-negative bacteria), was studied under white light irradiation by flow cytometry. Previous works have shown that under light irradiation conjugated polymers can sensitize oxygen molecules to generate ROS and therefore efficiently kill nearby bacteria.33,40 As shown in Figure 2, the antibacterial activities of all CCPs against E. coli are dependent on the D

DOI: 10.1021/acs.macromol.8b01530 Macromolecules XXXX, XXX, XXX−XXX

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Figure 2. Antibacterial activities of the four conjugated polymers ((a) PFE-1, (b) PFE-CN-2, (c) PFE-NP-3, and (d) PFP) at various concentrations for 30 or 60 min under white light irradiation (90 mW/cm2) or in the dark.

concentration and irradiation time. When 0.2 μM PFE-CN-2 was incubated with E. coli for 30 and 60 min, 26% and 88% of bacteria are dead, respectively. At the concentration of 0.4 μM, 86% and around 100% of E. coli are dead after incubating for 30 and 60 min, respectively. When PFE-1 was incubated with E. coli for 30 and 60 min, 55% and 92% of bacteria are killed at the concentration of 0.4 μM. When the concentration of PFE-1 was added to 0.8 μM, 88% and 98% of bacteria were dead, respectively. The sterilization effect of PFE-NP-3 is greatly inferior to that of PFE-1 and PFE-CN-2. When the concentration of PFE-NP-3 was 4.0 μM, 73% and 98% of bacteria were dead upon incubating for 30 and 60 min, respectively. Compared to PFE-1, the biocidal activity of PFP is far lower than that of PFE-1. When 3.0 μM PFP was incubated with E. coli for 30 and 60 min, 86% and 100% of bacteria were dead, respectively. When the concentration is added to 4.0 μM, all bacteria were killed after incubation both for 30 min and for 60 min. Thus, the order of antibacterial activities of the four CCPs against E. coli is as follows: PFECN-2 > PFE-1 ≫ PFE-NP-3 ≈ PFP. The half-maximal inhibitory concentration (IC50) values are shown in Table 2. The IC50 of PFE-CN-2 are the smallest, which are 0.26 and 0.11 μM after irradiation for 30 and 60 min, respectively. The IC50 values of PFE-NP-3 (2.0 and 0.98 μM) and PFP (2.1 and 1.3 μM) were very close, but they are much larger than that of PFE-CN-2. Also, the biocidal activities of CCPs against E. coli

in dark were tested. As shown in Figure 2, all CCPs show negligible biocidal activities even when the concentration is added to 1.0 μM. However, when the concentration is increased to 5.0 μM, PFP and PFE-NP-3 shows a certain biocidal activities due to the destruction of the bacterial membrane by the cationic conjugated polymers. Hence, at higher concentration of PFE-NP-3 and PFP, the antibacterial activities result from both PDT and membrane breakage of CCPs. To validate the flow cytometry results, parallel experiments were performed using fluorescence microscopy to visualize the bacteria with the live/dead stains. Figure 3a−d shows the images of E. coli suspension after incubation with PFE-CN-2 at 0.6 μM for 30 or 60 min with irradiation or in dark. All E. coli exhibited green fluorescence (live bacteria) without irradiation. However, all cells emitted red fluorescence that means cells were killed after irradiation for 30 or 60 min. These results showed that PFE-CN-2 exhibited good antibacterial activity under light irradiation. Figures S3−S5 show the fluorescence microscopy images of E. coli suspension with the other three conjugated polymers under light irradiation or in dark. All results are consistent with the flow cytometry data. In addition, to further investigate the interactions between four CCPs and bacteria, zeta (ζ) potentials of bacteria in the absence or presence of CCPs were measured. As shown in Table S1, the model target bacteria, E. coli, became more cationic after incubation with CCPs, which means the strong electrostatic interactions between CCPs and E. coli occur. Also, because CCPs can emit fluorescence upon excitation, the fluorescence images of bacteria after incubation with CCPs were obtained to indicate the interaction between CCPs and bacteria. Figures S6−S9 demonstrate bacteria emit blue fluorescence under excitation, which further proves that

Table 2. IC50 Values of the Four Conjugated Polymers irradiation time (min)

PFE-1 (μM)

PFE-CN-2 (μM)

PFE-NP-3 (μM)

PFP (μM)

30 60

0.38 0.19

0.26 0.11

2.0 0.98

2.1 1.3 E

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Figure 3. Fluorescence microscope images (a−d) and SEM images (e−h) of PFE-CN-2 with E. coli for 30 or 60 min under white light irradiation or in dark. Scale bar: 100 μm (a, b, c, d); 2.0 μm (e, f, g, h).

Figure 4. Transient absorption spectra of (a) PFE-1, (b) PFE-CN-2, (c) PFE-NP-3, and (d) PFP in argon-saturated methanol.

transient absorption experiments were performed in methanol to present the occurrence of triplet states of four conjugated polymers. As shown in Figure 4, four CCPs were excited at 355 nm in argon-saturated methanol to obtain their transient spectra. The triplet−triplet absorption was observed for all CCPs, which demonstrates that the four CCPs can produce ROS under irradiation. It is noted that the triple−triple absorption (TT absorption) of three poly(fluorene-co-phenyleneethynylene) derivatives (PFE) is much stronger than that of PFP. Then, the lifetimes of the triplet states (τtriplet) were calculated from the transient absorption decay curves, which are shown in Table S2. All four cationic conjugated polymers demonstrate good τtriplet. Especially, PFE-CN-2 and PFE-1 have the longer τtriplet among them, which may provide more favorable term for ROS production. To illustrate the difference of bacterial activity among four CCPs, the production of ROS from CCPs was further

bacteria combine with CCPs through electrostatic interaction. Furthermore, scanning electron microscopy (SEM) was employed to visualize the morphological changes of E. coli after incubation with PFE-CN-2 for 30 or 60 min in the dark or under irradiation. As shown in Figure 3e−h, E. coli cells demonstrate the clear edges and the surface integrity when treated with PFE-CN-2 in the dark, while the cells treated with PFE-CN-2 under irradiation appear collapsed and wrinkled. Also, bacterial cells fusion and adhesion are distinctly observed. The SEM images of E. coli suspension with the other three conjugated polymers under light irradiation or in dark are shown in Figures S10−S12. These results further illustrate that under light irradiation the CCPs increase the permeability of the bacterial membrane and significantly change the morphology of the bacteria because of ROS generation. As reported in the literature,39,41,43 the generation of ROS was related closed to the long-lived triplet state. Here, the F

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of PFP is the slowest and least efficient. Its biocidal activity thus is poor. These results demonstrate the antibacterial activity under white light irradiation is significantly dependent on the generation of ROS. Furthermore, poly(fluorene-cophenyleneethynylene) derivatives can produce ROS faster than poly(fluorene phenylene) derivatives. In addition, CCPs with D-π-A structure is more advantageous than A-π-A in ROS production and in antibacterial performance. Interestingly, the ROS production is coincident with the TT absorption and τtriplet of conjugated polymers. These results provide important information for designing new CCPs for more efficient antibacteria and antitumor based on PDT in future. Finally, the cell cytotoxicity of the four CCPs was tested through MTT assay against MCF-7 cells. As shown in Figure 6, four CCPs showed great biocompatibility. The cell viability remains over 94% even if the concentration of the CCPs is increased to 10.0 μM. The results indicate four CCPs exhibit quite low cytotoxicity on MCF-7 cells and can be used as PDT agents for antibacteria.

measured. DCFH-DA was utilized as ROS-sensitive probe that could be converted into highly fluorescent 2,7-dichlorofluorescein (DCF) in the presence of ROS. Therefore, the fluorescence intensity of DCF can be used to monitoring the ROS production. We measured the ROS production rates of the four CCPs upon irradiation with white light. As shown in Figure 5, the control without the CCPs maintains very low

4. CONCLUSIONS In summary, we have successfully designed and synthesized four cationic conjugated polymers and studied their structure− properties relationship. PFE-CN-2 and PFE-1 showed the stronger light-induced antibacterial activity presumably because of their good TT absorption and longer τtriplet, resulting in a high ROS yield. The generation of ROS under light illustration increases the permeability of the bacterial membrane, damages tissue, and causes bacterial killing. Also, the three poly(fluorene-co-phenylene ethynylene) derivatives can produce ROS faster than the poly(fluorene phenylene) derivatives, which brings the corresponding order of antibacterial activities as follows: PFE-CN-2 > PFE-1 ≫ PFE-NP-3 ≈ PFP. Furthermore, CCPs with D-π-A structure is more advantageous than A-π-A in ROS production and in antibacterial performance. In addition, four CCPs show great biocompatibility. This work is significant for designing more efficient PDT agents for antibacteria and antitumor.

Figure 5. Fluorescence intensity of DCF in H2O with four CCPs under white light irradiation (0−5 min) with an excitation wavelength of 488 nm. [DCFH] = 40 μM and [CCPs] = 1.0 μM. The error bars represent the standard deviations of three parallel measurements.

fluorescence intensity at 525 nm with the excitation wavelength at 488 nm. Importantly, in the presence of PFE-1 or PFE-CN-2, the fluorescent intensity of DCF increases dramatically and reaches the plateau after irradiation with white light only for 1 min. Upon irradiation for 2 min, DCF fluorescence intensity in the presence of PFE-CN-2 is a little higher than that in the presence of PFE-1. The high ROS production brings a corresponding high antibacterial activity of PFE-1 and PFE-CN-2. After 2 min, the DCF intensity decreases a little in the presence of PFE-1 or PFE-CN-2, which may result from the photobleaching of DCF. In addition, PFE-NP-3 produced ROS far less and lower than PFE-1 and PFE-CN-2, which may result from 2,4-dinitrobenzenesulfonyl being linked as a side chain of PFE-NP-3. The electron-withdrawing group quenches the fluorescence by PET and make PFE-NP-3 produce ROS inefficiently, resulting in the poorer antibacterial activities of PFE-NP-3. Compared to the other three conjugated polymers, the ROS production rate

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.8b01530. Additional fluorescence images; SEM images; calculated triplet lifetimes; zeta potentials (PDF)

Figure 6. Cell viability of MCF-7 cells in the presence of four CCPs at different concentrations under dark conditions. G

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (Y.T.). ORCID

Yanli Tang: 0000-0002-9979-6808 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 21675106), Natural Science Basic Research Plan in Shaanxi Province of China (No. 2017JM2019), the 111 Project (No. B14041), the Program for Changjiang Scholars and Innovative Research Team in University (No. 14R33), and the Program for Innovative Research Team in Shaanxi Province (No. 2014KCT-28).

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