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Cationic Oligo(thiophene ethynylene) with Broad-Spectrum and High Antibacterial Efficiency under White Light and Specific Biocidal Activity against S. aureus in Dark Qi Zhao, Junting Li, Xiaoqian Zhang, Zhengping Li, 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 S Supporting Information *
ABSTRACT: We designed and synthesized a novel oligo(thiophene ethynylene) (OTE) to investigate the antibacterial activities against Grampositive (Staphylococcus aureus and Staphylococcus epidermidis) and Gramnegative (Ralstonia solanacearum and Escherichia coli) bacteria in vitro by photodynamic therapy (PDT). Notably, OTE presents broad-spectrum and greatly high antibacterial activities after white light irradiation at nanogram per milliliter concentrations. The half inhibitory concentrations (IC50) values obtained for S. aureus, S. epidermidis, E. coli, and R. solanacearum are 8, 13, 24, and 52 ng/mL after illumination for 30 min, respectively, which are lower than that of other PDT agents. Interestingly, OTE shows the specific and very strong dark killing capability against S. aureus at the concentration of 180 ng/mL for 30 min, which is the highest efficiency biocide against S. aureus without the need of irradiation to date. The antibacterial mechanism investigated demonstrated that reactive oxygen species or singlet-oxygen generated by OTE kills bacteria irreversibly upon white light irradiation, and OTE as a v-type oligomer exerts its toxicity directly on destroying bacterial cytoplasmic membrane in the dark. Importantly, the OTE shows no cell cytotoxicity and excellent biocompatibility. The results indicate that it is potential to provide versatile applications in the efficient control of pathogenic organisms and specific application for killing S. aureus. KEYWORDS: antibiotics, conjugated oligomer, singlet oxygen, photodynamic therapy, photosensitizer
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INTRODUCTION In the past few years, the introduction of antibiotics had made a striking impact on the treatment of infectious diseases and had dramatically decreased the mortality. However, the emergence of multiple antibiotics-resistant bacteria poses a new threat to human health.1−4 The photodynamic therapy (PDT) had been suggested to be an alternative to the standard antibiotic treatment of some infections.5−9 PDT involves the use of photosensitizers to generate reactive oxygen species (ROS), especially singlet oxygen (1O2), upon exposure to light at a suitable wavelength.10−13 Recently, conjugated polyelectrolytes as admirable photosensitizers have demonstrated striking lightactivated antimicrobial and antitumor due to their unique optical properties, such as efficient light-harvesting ability, high fluorescence quantum yields, and amplified fluorescence quenching effect.11,14−20 However, the uncentainty of structure and content of materials makes it difficult to study the structure−activity relationship. Whitten and co-workers have reported a series of oligo-(p-phenylene ethynylene)s (OPEs) with controlled chain lengths and functional side groups that exhibit UV light-activated biocidal activity.21−25 Nevertheless, the weakness of the OPEs photosensitizers is that they require UV-light illumination, which limits their practical application because UV light itself is harmful to living organisms.26 The © 2015 American Chemical Society
donor−acceptor oligomers prepared by Schanze and coworkers can absorb visible light, whereas their antibacterial activity is still not satisfying.26 Therefore, the design and synthesis of novel photosensitizers with definite molecular weight, strong absorbance at visible light region, high singlet oxygen quantum yields, and low cytotoxicity is highly significant. As reported, thiophene derivatives have shown antimicrobial activity as a new photodynamic agent.15 To develop lightactivated biocidal oligomers that have strong absorption in the visible region, we introduced the thiophene units to replace all the benzenes in the OPE backbones.22,27 The introduction of thiophene unit should possess two advantages, such as inducing the absorption of oligomers red-shift to visible light region and taking on fascinating killing efficiency. In this work, we reported the design, synthesis, and photophysical properties of the cationic water-soluble oligo(thiopheneethynylene) (OTE). In addition, the Gram-positive bacteria (Staphylococcus aureus and Staphylococcus epidermidis and Gram-negative bacteria (Escherichia coli and a soilborne Received: November 22, 2015 Accepted: December 16, 2015 Published: December 16, 2015 1019
DOI: 10.1021/acsami.5b11264 ACS Appl. Mater. Interfaces 2016, 8, 1019−1024
Research Article
ACS Applied Materials & Interfaces Scheme 1. Mechanism for OTE Cytotoxicity under White Light and in Dark
The absorption and fluorescence spectra of OTE in water are shown in Figure S1. The maximal absorption wavelength is at 395 nm, and the maximal emission wavelength is at 488 nm. Obviously, the absorption band extends well into the visible region. To compare the physical properties with the OPEs reported previously, we synthesized the EO-OPE-1(C3) and EO-OPE-1(Th) according to the literature.22 They exhibit maximal absorption wavelength at 330 and 353 nm, respectively. Compared with OPEs, OTE has a wider absorption wavelength in the visible region owing to the introduction of the thiophene unit, which can avoid the use of UV light. The property of OTE indicates that OTE are promising for PDT under white light, which enhances greatly the practical application. The antibacterial mechanism of OTE under white light and in dark is shown in Scheme 1. As we reported previously, the generation of ROS by poly(phenylene ethynylene)s or OPEs after irradiation with visible light or UV, respectively, was the cause of bacterial killing.24,30 Both peroxyl radicals and subsequently produced singlet oxygen can lead to hydroperoxides by reacting with phospholipids, which may contribute to tissue damage and result in bacterial killing. OTE is watersoluble due to its cationic side groups and exhibits high affinity toward bacteria through electrostatic interactions. Moreover, OTE also can absorb visible light as a photosensitizer, probably taking on the same antibacterial process.24,30 In addition, dark killing efficiency of OTE was detected to investigate the application prospect without light. Ikeda and co-workers have investigated the dark mechanism of cationic biocides, who suggested several steps in bacteria killing: cationic biocide adsorption onto a bacterial surface, diffusion through the cell wall, cytoplasmic membrane disruption after the biocide binds to it, and the bacteria death upon release of the cytoplasmic constituents of the cell.31 OTE is a cationic oligomer with quaternary ammonium salt side chains. It is reasonable to speculate that OTE may kill bacteria following a similar darkkilling mechanism. The smaller and unique v-type structure of OTE may enable it to easily penetrate cell walls and
plant-pathogenic bacterium Ralstonia solanacearum28) were used to investigate the antimicrobial activity of OTE under white light illumination. Furthermore, the dark killing ability of OTE against organisms and the cell cytotoxicity of OTE were studied.
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MATERIALS AND METHODS
Glutaric dialdehyde (50%), 2-iodothiophene, 2,5-diiodothiophene, trimethylsilylacetylene, N,N-dimethyl ethanolamine, N-iodosuccinimide, trans-dichlorobis (triphenyl-phosphine) palladium(II), copper(I) iodide, and 4-dimethylaminopyridine were purchased from J&K Chemical Ltd. and Aladdin Industrial Corporation and used without further purification. The SYTO9 and SYTO24 were purchased from Thermo Fisher Scientific Inc. The 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was purchased from Sigma. The propidium iodide (PI) and medium components were purchased from Sangon Biotech (Shanghai) Co., Ltd. All solutions were prepared with ultrapure water purified using a Millipore filtration system. UV−vis absorption spectra were taken on a PerkinElmer Lambda 35 spectrophotometer. The fluorescence spectra were recorded on a Hitachi F-7000 spectrophotometer equipped with a xenon lamp excitation source. The Accuri C6 flow cytometer (Becton Dickinson, Franklin Lakes, NJ) used was equipped with a blue laser that excites at 488 nm, as well as two filters: a green fluorescence filter (FL-1; 520 nm) and a red fluorescence filter (FL-2; 610 nm). Fluorescence images were obtained from fluorescence microscope (Olympus, IX73). Bacterial killing assay processed under visible light via a Mejiro Genossen MVL-210 photoreactor.
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RESULTS AND DISCUSSION The synthesis of OTE was illustrated in Scheme S1. The compounds 1 and 2 were synthesized according to the literature.22 Reaction of 2-iodothiophene with N,N-dimethylethanolamine affords compound 3.29 Compound 4 is obtained by iodination reaction of 3 with N-iodosuccinimide in the presence of CH2Cl2/AcOH at room temperature. Compound 5 is prepared by a Sonogashira coupling reaction between 2 and 4 in the presence of Pd(PPh3)2Cl2/CuI in diethylamine/CHCl3. OTE is obtained by quaternarization reaction of 5 with CH3I in CHCl3. 1020
DOI: 10.1021/acsami.5b11264 ACS Appl. Mater. Interfaces 2016, 8, 1019−1024
Research Article
ACS Applied Materials & Interfaces
Figure 1. (a−d) S. aureus, S. epidermidis, R. solanacearum, and E. coli viability against OTE upon exposure to visible light at various concentrations for 30 and 60 min. (e) Antibacterial activity of OTE, EO-OPE-1(C3), and EO-OPE-1(Th) against E. coli at the concentration of 90 ng mL−1 when incubated under irradiation for 30 and 60 min. (f) OTE, EO-OPE-1(C3), and EO-OPE-1(Th) sensitized ROS upon white light irradiation (0−5 min) with an excitation of 488 nm. The error bars represent the standard deviations of three parallel measurements.
bacterium E. coli shows the similar results to that observed against R. solanacearum (Figure 1d). After irradiation for 30 min, 98.8% of cells were dead at the concentration of 90 ng/ mL. Upon irradiation for 60 min, 99.6% cells were killed. The IC50 are 24 and 17 ng/mL after irradiation for 30 and 60 min, respectively. All IC50 obtained against Gram-negative are higher than that gained against Gram-positive bacteria. These results demonstrated that Gram-negative bacteria are more difficult to inactivate by OTE because the Gram-negative outer membrane better protects the bacteria from antibiotics, dye, and detergents that would otherwise damage the inner membrane or cell wall. 30 To obtain the same biocidal effect, a higher concentration of OTE must be used. It is worthy to note that OTE shows broad-spectrum and highly efficient lightactivated antibacterial activity against organisms at low concentration (≤120 ng/mL), which is promising for developing a new PDT agent. To compare the antibacterial activity of OTE with that of OPEs after white light irradiation, we studied the biocidal activity of OTE, EO-OPE-1(C3), and EO-OPE-1(Th) against E. coli by the similar procedure. The concentration of all oligomers was fixed at 90 ng/mL, because OTE showed the maximum antibacterial activity against E. coli at this concentration, which is above-mentioned. As shown in Figure 1e, upon irradiation with white light either for 30 min or for 60 min, almost no antibacterial activity is observed for both EOOPE-1(C3) and EO-OPE-1(Th). The main cause is the maximal absorption wavelength of OPEs appears in the UV region, which leads to OPEs absorbing white light difficultly. Apparently, the OTE demonstrates the superior antibacterial activity under white light, which is attributed to its particular thiophene ethynylene structure making its maximal absorption wavelength red-shifting to 395 nm. It was speculated that efficient antibacterial activity of OTE against organisms is likely due to rapid and efficient generation of ROS by OTE after irradiation with white light. Therefore, to prove the production of ROS of OTE upon irradiation with white light, we utilized DCFH-DA as a cell-permeable fluorogenic ROS-sensitive probe in our experiment.9 As
membranes and subsequently disrupt these structures by electrostatic and hydrophobic interactions. Finally bacteria showed antibacterial activity after cytoplasmic constituents releasing from cell.32 We found that OTE showed greater activity against S. aureus than other either Gram-negative or Gram-positive organisms in dark, which may be attributed to unique structure of OTE and the extraordinary interactions between OTE and S. aureus membrane, which is promising for developing highly efficient and specific biocide of S. aureus. First, the biocidal activity of OTE against Gram-positive bacteria, S. aureus, was studied under white light illumination by flow cytometry. As shown in Figure 1a, the activities of OTE against S. aureus show concentration-dependent. After irradiation with white light for 30 min, 62% of S. aureus cells were dead at the OTE concentration of 10 ng/mL. When the concentration of OTE was added to 60 ng/mL, all bacteria were killed after white light irradiation for 30 min. We also studied the bacterial activity of OTE against S. aureus after exposure under white light for 60 min. The similar result was obtained to that for 30 min. The half inhibitory concentrations (IC50; shown in Table S1) are 8 and 7 ng/mL after irradiation for 30 and 60 min, respectively, which is the lowest reported inhibitory concentration of a PDT antibacterial agent.33 The biocidal activity of the OTE against another Gram-positive bacterium S. epidermidis shows the similar results to that observed against S. aureus (Figure 1b). After irradiation for 30 and 60 min, 95.5% and 97% of cells were dead at the concentration of 60 ng/mL. The IC50 are 13 and 10 ng/mL after irradiation for 30 and 60 min, respectively. Then we investigated the antibacterial activity of OTE against Gram-negative R. solanacearum (a soilborne plantpathogenic bacterium28) by the same procedure. As shown in Figure 1c, OTE displays a trend similar to that of observed against S. aureus, which were time- and concentrationdependent. All bacterial cells are killed at the concentration of 120 ng/mL, and the IC50 are 52 and 28 ng/mL after irradiation for 30 and 60 min, respectively, which shows OTE has potential application as a highly efficient agriculture biocide. The biocidal activity of the OTE against another Gram-negative 1021
DOI: 10.1021/acsami.5b11264 ACS Appl. Mater. Interfaces 2016, 8, 1019−1024
Research Article
ACS Applied Materials & Interfaces shown in Figure 1f, upon irradiating DCFH-DA in the presence of OTE under white light (400−800 nm), an apparent emission at 525 nm was detected (excitation: 488 nm), while the control without OTE is maintained at the basic level, which confirms the efficient generation of ROS by OTE. As a comparison, the fluorescence intensity after DCFH-DA incubating with either EO-OPE-1(Th) or EO-OPE-1(C3) at the same condition was detected. The intensity of DCFH induced by OPEs increases a little, which shows that both of them hardly produce ROS with exposure under white light. These results demonstrate the death of organism under white light irradiation is significantly related to the generation of ROS. The antibacterial efficiency of OTE was also proved by fluorescence microscopy. Figure 2 shows overlapping images of
Figure 3. (a) S. aureus viability (1 × 107 CFU/mL) against OTE at various concentrations for 30 and 60 min in the dark. The error bars represent the standard deviations of three parallel measurements. (b) SEM image of S. aureus incubated with OTE ((3) and (4)) for 60 min in the dark. A sample without OTE was used as the control ((1) and (2)). Scale bars: 5 μm (1, 3) and 2 μm (2, 4).
Our previous study showed that the morphology was changed upon the bacteria being killed at high concentration of OPEs in dark.32 Here, scanning electron microscope (SEM) was used to image the structural changes on S. aureus cells upon incubation with the OTE at very low concentration (180 ng/ mL). As shown in Figure 3b, the strain of S. aureus cells alone in phosphate-buffered saline maintain their integrity with a smooth cell surface, while S. aureus cells treated with OTE appear wrinkled and cracked compared to the untreated sample. The remarkable characteristic of the OTE-treated cells is the appearance of abundant amorphous material, presumably cell content released from damaged cells. These results indicate that the cells were disrupted and killed after incubation with OTE in dark at such low concentration, probably because the unique v-type structure of OTE contributes to breaking the membrane of S. aureus efficiently. Our research indicates that the OTE is promising for becoming a specific and highly efficient biocide for S. aureus without the need of irradiation. An ideal PDT antibacterial agent requires low cell cytotoxicity at appropriate concentration. Herein, the cell cytotoxicity of the OTE was tested using MTT assay against HeLa cells. The absorbance of MTT assay at 595 nm is dependent upon the degree of activation of the cells. The cell viability is expressed by the ratio of the absorbance of the cells incubated with OTE to the cells incubated with culture medium only. As shown in Figure 4, OTE showed excellent
Figure 2. Fluorescence microscope images of OTE with S. aureus after 30 and 60 min of irradiation with visible light. Green staining indicates live bacteria, and red staining indicates dead bacteria. Scale bar, 50 μm.
S. aureus suspensions under the green fluorescence field and red fluorescence field, where the cells that appeared green/red were indicative of live/dead bacteria, respectively. After incubation with OTE at 60 ng/mL for 30 or 60 min, all cells emit red fluorescence that means cells were killed. The results showed that the OTE exhibited efficient biocidal activity for living S. aureus under white light illumination, which is highly consistent with that obtained by flow cytometry. The same results shown in Figure S2 were obtained for other organisms. To understand the light-induced antibacterial activity of OTE better, the OTE dark killing effects is detected. OTE was mixed with bacteria and kept in the dark for tens of minutes, and then flow cytometry data were obtained. As shown in Figure S3, at low concentrations (
