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Akzeptierter Artikel Titel: Supramolecular Radical Anions Triggered by Bacteria in situ for Selective Photothermal Therapy Autoren: Yuchong Yang, Ping He, Yunxia Wang, Haotian Bai, Shu Wang, Jiang-Fei Xu, and Xi Zhang Dieser Beitrag wurde nach Begutachtung und Überarbeitung sofort als "akzeptierter Artikel" (Accepted Article; AA) publiziert und kann unter Angabe der unten stehenden Digitalobjekt-Identifizierungsnummer (DOI) zitiert werden. Die deutsche Übersetzung wird gemeinsam mit der endgültigen englischen Fassung erscheinen. Die endgültige englische Fassung (Version of Record) wird ehestmöglich nach dem Redigieren und einem Korrekturgang als Early-View-Beitrag erscheinen und kann sich naturgemäß von der AA-Fassung unterscheiden. Leser sollten daher die endgültige Fassung, sobald sie veröffentlicht ist, verwenden. Für die AA-Fassung trägt der Autor die alleinige Verantwortung. Zitierweise: Angew. Chem. Int. Ed. 10.1002/anie.201708971 Angew. Chem. 10.1002/ange.201708971 Link zur VoR: http://dx.doi.org/10.1002/anie.201708971 http://dx.doi.org/10.1002/ange.201708971
10.1002/ange.201708971
Angewandte Chemie
COMMUNICATION Supramolecular Radical Anions Triggered by Bacteria in situ for Selective Photothermal Therapy
Abstract: A supramolecular complex that can be selectively reduced to radical anions in situ by facultative anaerobic bacteria is reported. To this end, a water-soluble bifunctional monomer bearing perylene diimide was synthesized, and its supramolecular complex with cucurbit[7]uril was fabricated on the basis of host-guest complexation, which could be reduced to forming radical anions in the presence of E. coli. It was interestingly found that this supramolecular complex could display different ability of generating radical anions by facultative anaerobic and aerobic bacteria in terms of their various reductive abilities. The selective antibacterial activity of the supramolecular complex could be realized by photothermal performance of radical anions under near-infrared irradiation. It is anticipated that this method may lead to a novel bacteria-responsive photothermal therapy to regulate balance of bacterial flora.
Antibiotics have been widely used in various fields, such as clinical therapy, agricultural utilization, sewage treatment and so forth since penicillin was discovered.[1] However, following the long-period abuse of antibiotics, drug resistant microbes have been becoming a severe threat to human beings.[2] Therefore, developing novel antimicrobial methods is urgently required.[3] In recent years, there is an increasing interest in developing photothermal therapy as a powerful tool to fight against drug resistant microbes. For drug resistant microbes, locally increasing temperature (≥ 50 oC) by photothermal agents can lead to the microbes’ death by denaturation of their proteins.[4] Up to now, various functional materials have been developed as photothermal agents, such as inorganic nanomaterials, carbon based nanomaterials, and conjugated functional polymers and etc.[5] By utilization of these functional materials with good photothermal conversion efficiency, photothermal therapy can overcome drug resistant microbes and become a potential clinical strategy. Although significant progress has been made in photothermal materials, developing novel photothermal agents with excellent selectivity toward various bacteria is still imperative. Bacterial recognition by traditional photothermal materials relies on non-specific interactions, which makes it hard to realize selective inhibition towards bacteria. Herein, a new kind of
[a]
Y. Yang, Dr. J.-F. Xu, Prof. X. Zhang Key Lab of Organic Optoelectronics and Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing100084, China E-mail:
[email protected] [email protected] P. He, Dr. Y. Wang, Dr. H. Bai, Prof. S. Wang Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China Supporting information for this article is given via a link at the end of the document.
bacterial responsive supramolecular complex was designed for photothermal therapy with high selectivity towards facultative anaerobic bacteria. As shown in Scheme 1, the supramolecular complex (CPPDI) was constructed by a perylene diimide derivative (PPDI) and cucurbit[7]uril (CB[7]) through host-guest interactions. It was discovered that facultative anaerobic bacteria like E. coli possessed reductive ability for triggering CPPDI into CPPDI radical anions in situ, which was used for photothermal therapy under the near-infrared (NIR) irradiation, leading to the death of E. coli. On the contrary, the aerobic bacteria like B. subtilis without enough reductive ability could not induce the formation of CPPDI radical anions in situ. Therefore, the selective chemical responses of CPPDI to different bacteria could be employed to selectively and effectively inhibit bacteria by photothermal therapy. The supramolecular complex was fabricated by the host-guest complexation between PPDI and CB[7], which was characterized by 1H NMR, mass spectrum and isothermal titration calorimetry (Figure S1-S3).[6] There are two main advantages for the supramolecular complex comparing with the PPDI itself. Firstly, hydrophobic benzyl groups on PPDI could result in non-specific membranolysis of bacteria. By binding to CB[7], benzyl groups could be prevented from inserting into the membrane of bacteria[7] (Figure S4). Thus, the combination of CB[7] and PPDI could eliminate non-specific antibacterial behavior of PPDI. Secondly, stacking of perylene diimide could be diminished because of the introduction of bulky CB[7], and then the quenching of perylene diimide radical anions could also be decreased.[8] Therefore, the efficiency of photothermal conversion could be enhanced when such perylene diimide radical anions were used as near-infrared materials (Figure S5). Normally, perylene diimide can be reduced to free radicals by chemical reducing agents such as Na2S2O4.[9] We explored that the supramolecular complex CPPDI could also be reduced by E. coli to triggering radical anions. After being incubated with E. coli for 10 hours, the solution containing CPPDI changed into dark blue color. As shown in Figure 1a and Figure S6a, three absorption peaks (735 nm, 782 nm, 820 nm) in the UV-vis spectrum were observed, suggesting that the CPPDI radical anions were generated in the presence of E. coli. To further confirm the formation of CPPDI radical anions, the electron paramagnetic resonance (EPR) spectroscopy was performed to provide direct evidence. As shown in Figure 1a, the EPR signal with g factor of 2.0033 was ascribed to the perylene diimide radical anions.[8b,10] In contrast with E. coli, the aqueous solution of CPPDI did not exhibit color change in the presence of B. subtilis. Moreover, no typical EPR signal was observed, as shown in Figure 1b. The results exhibit that CPPDI radical anions can be triggered by E. coli, while B. subtilis is unable to trigger the formation of radical anions at the same condition.
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Yuchong Yang, Ping He, Yunxia Wang, Haotian Bai, Shu Wang, Jiang-Fei Xu* and Xi Zhang*
10.1002/ange.201708971
Angewandte Chemie
Scheme 1. a) Chemical structures of the designed supramolecular complex (CPPDI) and CPPDI radical anions; b) Diagram of photothermal therapy for CPPDI with high selectivity towards E. coli over B. subtilis.
Based on the fact that CPPDI radical anions present good photothermal conversional property, ascribed to the molecular vibrations under NIR irradiation,[11] and the CPPDI radical anions can be selectively triggered by E. coli, we explored if this supramolecular complex could display selective bacterial inhibition by photothermal therapy. Under NIR irradiation (808 nm), the temperature of the CPPDI aqueous solution increased greatly in the presence of E. coli (Figure 2a). It could be risen up to 65 oC in 30 min, indicating that CPPDI radical anions induced by E. coli can convert optical energy into heat in a short time. As a contrast, there was no clear change of temperature in the presence of B. subtilis without enough reductive ability. The qualitative antibacterial activity was demonstrated by means of plate coating as shown by Figure S7. With the lapse of time, the inhibition to E. coli gradually increased under NIR irradiation. As shown in Figure 3a, upon laser irradiation for 10 min, 20 min and 30 min, the inhibition efficiencies of CPPDI to E. coli were calculated as 33%, 74% and 99%, respectively. On the contrary, no inhibition was observed to B. subtilis under the same conditions (Figure 3b). Therefore, the different capacity of E. coli and B. subtilis for generating CPPDI radical anions could be successfully used to realize selective bacterial inhibition by photothermal therapy.
Figure 1. EPR spectroscopy of CPPDI in the presence of E. coli (a) and B. subtilis (b).
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Figure 2. Temperature changes of the aqueous solution of CPPDI in the presence of E. coli (a) and B. subtilis (b) under NIR irradiation (808 nm).
Figure 3. Colony-forming units (CFU) ratio of E. coli (a), and B. subtilis (b) with CPPDI under NIR irradiation for 30 min; Note: Data shown in (a) are mean ± SD from n = 3. p < 0.05 compared to the 0 min group. Data shown in (b) are mean ± SD from n = 3. p > 0.05 compared to the 0 min group, one way ANOVA.
For designing photothermal agents, their direct contact to the
pathogenic bacteria is very important for photothermal therapy.[12] To get deep insight into the interactions between the CPPDI and bacteria, ζ-potential measurements and confocal laser scanning microscopy (CLSM) were used to investigate the interactions between CPPDI and bacteria. As shown in Table S1, the surface charge of E. coli was changed from -44.5 mV to 2.95 mV with the addition of CPPDI, indicating that positively charged CPPDI was adsorbed onto the surface of E. coli. Such a strong interaction between CPPDI and bacteria was also supported by CLSM (Figure S8). The strong binding between bacteria and CPPDI can assist to trigger the formation of CPPDI radicals in situ, and thus making significant contribution to the effective photothermal therapy. We wondered whether E. coli was the only bacteria for reducing CPPDI to generate radical anions. To answer this question, five kinds of bacteria including E. faecalis, S. aureus, P. aeruginosa, E. coli and B. subtilis were studied in parallel. As shown in Figure 4, EPR signals demonstrated that facultative anaerobes like E. coli and E. faecalis represented the remarkable biochemical response for triggering CPPDI radical anions. S. aureus showed a much weaker reducing capacity than E. coli and E. faecalis. Aerobic bacteria like P. aeruginosa and B. subtilis, however, exhibited no trends for triggering the formation of radical anions under the same condition. It has been reported that, hydrogenases are abundant on the membrane of bacteria for proton transfer, especially in some anaerobic and facultative anaerobic bacteria.[13] Besides, the proton transfer of hydrogenase can reduce methylviologen into free radicals.[14] By comparing the cyclic voltammograms of methylviologen and CPPDI, CPPDI dispalyed a more easily trend to be reduced, as shown in Figure S9. As expected, CPPDI should be reduced by the hydrogenase on the surface of facultative anaerobic bacteria to CPPDI radical anions. Thus, various bacteria with different reductive ability lead to the different biochemical response for the formation of CPPDI radical anions.
Figure 4. EPR spectroscopy of CPPDI in the presence of different bacteria: E. faecalis, S. aureus, P. aeruginosa, E. coli and B. subtilis.
The cytotoxicity of CPPDI on mammalian cells was also
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investigated for further potential applications. In this experiment, a nonmalignant epithelial cell line HaCaT was selected as the representative cell. As shown in Figure S10, CPPDI showed no clear cytotoxicity by MTT assay even at a high concentration (0.5 mM) which was four times higher than the concentration used in antibacterial experiments. It suggests that the use of CPPDI could be a potential safe strategy for photothermal therapy application. In conclusion, we have successfully designed and constructed a supramolecular complex that can be selectively reduced to radical anions in situ by facultative anaerobic bacteria. This novel supramolecular complex displays antibacterial activity with high selectivity towards facultative anaerobic bacteria through photothermal therapy. Such a molecular design could be extended to molecular assemblies, polymers and supramolecular polymers with tunable antibacterial inhibition activity. Moreover, perylene diimide derivative might be replaced by other functional species with extension of NIR absorption. It is anticipated that such a novel bacterial and chemical response could have great potential for regulating microbial balance.
Acknowledgements This work was financially supported by the Foundation for Innovative Research Groups of NSFC (21421064) and the National Basic Research Program (2013CB834502). We are grateful to Prof. Yapei Wang, Zhen Wang and Yonglin He at Renmin University of China for measurements of photothermal conversion.
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COMMUNICATION COMMUNICATION Yuchong Yang, Ping He, Yunxia Wang, Haotian Bai, Shu Wang, Jiang-Fei Xu* and Xi Zhang* Page No. – Page No. Supramolecular Radical Anions Triggered by Bacteria in situ for Selective Photothermal Therapy
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The supramolecular complex of a perylene diimide derivative and cucurbit[7]uril displays different ability of generating radical anions by facultative anaerobic and aerobic bacteria. The selective antibacterial activity of the supramolecular complex can be realized by photothermal performance of radical anions under near-infrared irradiation. This method may lead to a novel bacteriaresponsive photothermal therapy to regulate balance of bacterial flora.
