INTRODUCTION

      Bacterial infectious diseases are severe challenges to human health, leading to significantly social and medical concerns for a long time [1,2]. As a primary category of clinically significant pathogens, Gram-positive bacteria induce serious infections with high morbidity and mortality [3,4], such as sepsis that annually accounts for nearly 1.7 million cases and 270,000 deaths in the United States, as well as 30 million cases and 6 million deaths all around the world [5,6]. Generally, bacterial infections are cured by broad-spectrum antibiotics, while their global overuse or abuse has accelerated the evolution and spread of drug resistance [7–9]. In addition, this situation is deteriorated as the production of novel antimicrobial agents is increasingly difficult and costly [10]. As reported by the World Health Organization (WHO), the drug-resistant bacterial infections claim more than 700,000 lives every year, and if current trends continue, the number could rise to 10 million by 2050 with an estimated economic cost of 100 trillion dollars [11,12]. Therefore, it is highly desirable to develop effective antimicrobial agents incapable of inducing drug resistance in the post-antibiotic era, especially those specific to Gram-positive bacteria [13].

      Recently, photothermal therapy (PTT) has attracted extensive attention for bactericidal applications, on the basis of generating localized hyperthermia to inactivate enzymes or proteins, disrupt membrane permeability, and disorder metabolic signals upon near-infrared (NIR) light irradiation with photothermal agents [14–17]. PTT holds advantageous characteristics of spatiotemporal control, noninvasiveness, and little possibility to induce drug resistance. Up to now, various photothermal agents have been developed to ablate bacteria, including inorganic gold-silver nanocages, copper sulfide, graphene, and black phosphorus, as well as organic IR-780 iodide (IR780), indocyanine green, polydopamine, and polypyrrole [18–22]. However, PTT suffers from the inability to specifically inactivate bacteria and toxic side effects against adjacent mammalian cells, which seriously limit its practical usage.

      Metabolic labelling with unnatural precursors is an emerging technique to engineer cells with chemical tags for subsequent modification of complementary molecules via efficient chemistries [23–25], which has been extensively applied in various fields, including cancer labelling and targeting [24], microbiota identification [26–28], and bacterial inactivation [29–31]. A variety of artificial precursors such as chemically modified galactosamine, mannosamine, sialic acid, and fucose are used for cell membrane labelling. Benefitting from the specific components of bacterial cell wall, D-alanine and 3-deoxy-D-mannooctulosonic acid derivatives are metabolized into the peptidoglycan and lipopolysaccharide, respectively [32,33]. There-after, functional fluorescent probes and therapeutic molecules are introduced by bioorthogonal reactions, such as click chemistry between azide and alkyne [31,34]. Thus, metabolic labelling could be utilized to precisely conjugate photothermal agents on bacteria for selective pathogenic ablation by PPT.

      In this contribution, IR780, a lipophilic cationic heptamethine dye with excellent degradability, low cytotoxicity, and superior photothermal conversion efficiency [35–37], was functionalized with dibenzocyclooctyne (DBCO) to prepare a bioorthogonal photothermal agent (IR780-DBCO) for specific killing of Grampositive bacteria in vitro and in vivo (Fig. 1). Upon metabolizing 3-azido-D-alanine (D-Ala-N3) into bacterial peptidoglycan, IR780-DBCO was selectively labelled on the surface of Grampositive bacteria rather than Gram-negative ones via copper-free click chemistry between azide and DBCO. The specific labelling resulted from the poor permeability of Gram-negative bacterial outer membrane against IR780-DBCO with a molecular weight larger than 650 Da [31,38]. Therefore, typical Gram-positive pathogenic bacteria, including Staphylococcus aureus (S. aureus) and vancomycin-resistant Enterococcus faecalis (VRE), were selectively ablated under NIR light irradiation in vitro and in vivo.

EXPERIMENTAL SECTION

Materials and characterizations

      The experimental details of materials, characterizations, synthesis of IR780-(3-MPA) and IR780-DBCO, photothermal heating curves measurement, bacteria culture and metabolic modification, morphology observation, cytotoxicity and hemolysis assays are described in the Supplementary information.

Bacterial labelling

      One milliliter of D-Ala-N3-pretreated or unpretreated bacterial suspension (S. aureus, VRE, Escherichia coli (E. coli), or Pseudomonas aeruginosa (P. aeruginosa)) in a 1.5-mL Eppendorf tube was centrifuged at 2300 ×g for 5 min. After the supernatant was discarded, bacteria were resuspended in 200 µL of DBCOCy3 solution (20 µmol L−1 , phosphate buffer saline (PBS)/dimethyl sulfoxide (DMSO) (v/v) = 99/1) and incubated at room temperature in the dark for 30 min. The labelled bacteria were harvested and washed with PBS for four times. Upon fixing in 1 mL of 4% paraformaldehyde at 4°C overnight, the bacteria were collected and washed with PBS for four times. Then 1 mL PBS was added to resuspend the labelled bacteria. The above bacterial suspension (10 µL) was transferred into a confocal dish and covered by a clean cover glass for imaging. The images were recorded by an inverted fluorescence microscope with excitation wavelength of 530 nm and emission wavelength of 593 nm, and a structured illumination microscope (SIM) with excitation wavelength of 561 nm and emission wavelength of 600–660 nm.

In vitro selective antibacterial assays

       The antibacterial activity of IR780-DBCO towards S. aureus, VRE, E. coli, or P. aeruginosa was evaluated by a standard agar plating method. First, 1 mL of the pretreated bacteria in a 1.5-mL Eppendorf tube was centrifuged at 2300 ×g for 5 min to discard the supernatant. Second, the bacteria were suspended in 200 µL of IR780-DBCO solutions with various concentrations (0, 2.5, 5, or 10 µmol L−1 , PBS/DMSO (v/v) = 99/1), and incubated at room temperature in the dark for 30 min. Then the labelled bacteria were harvested and washed with PBS for three times, and subsequently resuspended in 400 µL PBS. For the antibacterial activity of IR780-DBCO under NIR light irradiation, 200 µL of the above-prepared bacterial suspension was transferred to a sterile disposable petri dish and irradiated by 808-nm NIR laser (1.0 W cm−2 ) for 15 min. For the antibacterial activity of IR780- DBCO in the dark, 200 µL of the above-prepared bacterial suspension was placed in the dark for 15 min under the same conditions. Afterwards, 1.8 mL PBS was added to the petri dish, followed by sonication for 3 min to disperse the bacteria. One hundred microliter of the suspension was taken for 10-fold gradient dilution. Ten microliter of the diluted bacterial solution was inoculated in an agar plate, and the colony forming units (CFUs) were counted after incubation at 37°C overnight. In addition, 50 µL of the suspension diluted by 100 folds was spread on the agar plate. The bacterial viability (%) = CFUexperiment/ CFUcontrol × 100%, where CFUexperiment and CFUcontrol were the average CFU of the experimental group with IR780-DBCO treatment and the control group with PBS treatment, respectively. All experiments were repeated for three times. Using the similar method, the antibacterial activity of IR780-DBCO (0 and 5 µmol L−1 , PBS/DMSO (v/v) = 99/1) towards S. aureus, VRE, E. coli, or P. aeruginosa without D-Ala-N3 pretreatment was evaluated in the dark or under NIR laser irradiation. In addition, the killing effect of IR780, IR780-(3-MPA), or IR780-DBCO (0 and 5 µmol L−1 , PBS/DMSO (v/v) = 99/1) against D-Ala-N3-pretreated or unpretreated VRE was measured in the dark or under NIR light illumination.

In vivo anti-infective assay

      The in vivo anti-infective ability of IR780-DBCO was assessed in a mouse model with VRE-infected wound. All procedures were approved by the Animal Ethical Committee of Northwestern Polytechnical University. Female BALB/c mice (6–8 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

      Twelve BALB/c mice were randomly divided into four groups: (1) control group only with bacterial infection, (2) bacteriainfected group under 808-nm NIR laser irradiation, (3) bacteriainfected group treated with IR780-DBCO, and (4) bacteriainfected group treated with IR780-DBCO and 808-nm NIR laser irradiation. The mice were anesthetized using isoflurane and then the open excision wounds were created on the dorsal skin using an 8-mm diameter biopsy punch. The bacteria-infected skin wound models were established by inoculating 50 µL of the pretreated VRE suspension (104 CFU) on the wound area. For the groups with IR780-DBCO treatment, a total of 50 µL of the pretreated VRE suspension and IR780-DBCO (the final concentration was 20 µmol L−1 ) were added onto each wound and kept for 30 min. Afterwards, the wounds were irradiated by 808-nm NIR laser (1.0 W cm−2 ) for 15 min or kept in the dark. The treatments by IR780-DBCO and with or without 808-nm NIR laser irradiation were performed for total three times. The wound areas were photographed using an 8-mm rubber ring as reference every day. After four days, the mice were sacrificed and the entire wounds with the adjacent skin tissues were harvested and placed in 1 mL PBS followed by sonication for 15 min. The CFUs of bacteria in infectious tissues were evaluated by the standard agar plating method. The collected wound tissues were fixed in 4% paraformaldehyde at 4°C, embedded in paraffin, and cut into slices for histopathological hematoxylin-eosin (H&E) staining. 

RESULTS AND DISCUSSION

Synthesis and characterization of IR780-DBCO

      To enable photothermal property and the reactivity with azide group, IR780 was firstly conjugated with 3-mercaptopropionic acid (3-MPA) through nucleophilic substitution reaction between sulfhydryl and chlorine groups to produce IR780-(3- MPA) in a 64% yield, and subsequently reacted with DBCOamine (DBCO-NH2) through amide condensation reaction to prepare IR780-DBCO in a 43% yield (Fig. S1 and Fig. 2a). As confirmed by nuclear magnetic resonance and mass spectra, IR780-DBCO was successfully synthesized. The optical characteristic of IR780-DBCO was displayed in Fig. 2b, with the absorption lying in the broad ultra violet-visible-NIR (UV-VisNIR) range from 200 to 900 nm and the maximal peak located at 785 nm. Moreover, the temperature evolution curves of IR780-DBCO were measured under 808-nm NIR laser irradiation with a power density of 1.0 W cm−2 (Fig. 2c). At a concentration of 100 µg mL−1 , the temperature increased by 26.5°C upon irradiation for 60 s, while it had negligible change at 0 µg mL−1 under the same condition. Upon IR780-DBCO reacting with D-Ala-N3 through click chemistry, there were no significant differences for the UV-Vis-NIR spectrum and temperature evolution curve when compared with those for IR780-DBCO alone (Fig. S2). These results revealed that IR780-DBCO possessed an outstanding photothermal effect, which was not influenced by the click reaction, and held great potential for eliminating bacteria.

Selective bacterial labelling

      In order to evaluate the specific labelling of Gram-positive bacteria by metabolic engineering, two Gram-positive bacteria (S. aureus and VRE) and two Gram-negative bacteria (E. coli and P. aeruginosa) were pretreated with D-Ala-N3 to integrate azide groups into the peptidoglycan of bacteria, followed by bioorthogonal clicking with fluorescent DBCO-Cy3, a commercial dye containing alkyne groups. As shown in Fig. 3, Gram-positive S. aureus and VRE were stained with strong red fluorescence, while Gram-negative E. coli and P. aeruginosa presented almost no fluorescence. Without pretreatment with D-Ala-N3, all types of bacteria showed negligible fluorescence, indicating the efficient click reaction between DBCO-Cy3 and metabolic precursor (Fig. S3). Most importantly, to demonstrate the exact region of labelled DBCO-Cy3, super resolution imaging of bacteria was performed by SIM. The obvious fluorescence was only observed around the periphery of Gram-positive bacteria (S. aureus and VRE), indicating the preferential labelling of their cell walls (Fig. 4). The underlying mechanism for selective imaging of Gram-positive bacteria could be explained as follows: D-Ala-N3 was able to metabolize into peptidoglycan of bacterial cell wall to endow azide groups owing to the presence of Dalanine motif [32]. The outer membrane of Gram-negative bacteria is less permeable to molecules with molecular weight larger than 650 Da [31], and thus DBCO-Cy3 with molecular weight of 1186 Da was specifically labelled on Gram-positive bacteria through copper-free click chemistry between azide and DBCO groups. Moreover, while traditional culture-based methods for bacterial identification usually require several days, this metabolic labelling approach is much faster and effective.

Photothermal antibacterial study

      Because of the similar chemical structure to DBCO-Cy3, IR780-DBCO with a molecular weight of 994 Da could be specifically loaded on the cell envelope of D-Ala-N3-pretreated Grampositive bacteria, which is advantageous for photothermal inactivation of pathogens. We then assessed the selective antibacterial capability of IR780-DBCO using the standard plate counting method [20]. Gram-positive bacteria (S. aureus and VRE) and Gram-negative ones (E. coli and P. aeruginosa) were metabolized with D-Ala-N3 and incubated with IR780-DBCO at different concentrations, followed by treatment in dark or under 808-nm NIR laser irradiation at a power density of 1.0 W cm−2 for 15 min. For Gram-positive bacteria, IR780-DBCO showed concentration-dependent inhibition under NIR light irradiation, with the killing efficiency of 99.7% for S. aureus or 100% for VRE, and few bacterial colonies formed on the agar plates at a concentration of 10 µmol L−1 (Fig. 5a–d). Nevertheless, NIR light irradiation or IR780-DBCO alone presented negligible killing effect on Gram-positive bacteria, which grew smoothly on the agar plates. For Gram-negative bacteria, the killing effect was insignificant upon treatment with NIR light irradiation, IR780- DBCO, or IR780-DBCO plus NIR light irradiation, resulting in obvious colony formation similar to the control group (Fig. 5e– h). Without D-Ala-N3 pretreatment, the viability of all types of bacteria was much higher upon incubation with equivalent IR780-DBCO or plus NIR light irradiation (Fig. S4). Moreover, for Gram-positive VRE, the killing effect of IR780 and IR780-(3- MPA) without or with D-Ala-N3 pretreatment was much lower than that incubated with D-Ala-N3 and equivalent IR780-DBCO under NIR light illumination (Fig. S5). These results suggested that Gram-positive bacteria rather than Gram-negative ones could be selectively and effectively eliminated by PTT through metabolic labelling of IR780-DBCO on the cell wall.

      Moreover, in order to elucidate the antibacterial mechanism, scanning electron microscopy (SEM) was utilized to observe the morphological changes of bacteria after different treatments. Upon metabolic labelling with IR780-DBCO and irradiation with NIR light, the cell envelopes of Gram-positive bacteria were seriously destroyed, with obvious deformation and collapse for S. aureus and obvious wrinkle for VRE, as indicated by the white arrows in Fig. 6 and Fig. S6. However, the morphology of Grampositive bacteria treated with NIR light irradiation or IR780- DBCO or Gram-negative bacteria with any treatment had insignificant change, displaying intact cell wall and relatively smooth surface. These results clearly proved that IR780-DBCO conjugated on the bacterial surface could damage the integrity of bacterial cell walls through PTT [36,39], inducing the selective elimination of Gram-positive bacteria.

In vitro biocompatibility study

      To evaluate the potential of IR780-DBCO as a practical antimicrobial agent, the cytotoxicity of IR780-DBCO against mouse embryonic fibroblast cells (NIH3T3) was determined [40]. As shown by alamarBlue assay, IR780-DBCO exhibited negligible cytotoxicity even at a concentration of 10 µmol L−1 (Fig. 7a). Moreover, on the basis of live/dead staining results, NIH3T3 cells incubated with IR780-DBCO were almost alive with analogous morphology to the control cells (Fig. 7b). In addition, blood compatibility of IR780-DBCO was assessed [41]. After incubating rabbit red blood cells (RBCs) with D-Ala-N3 and different concentrations of IR780-DBCO, colorless supernatant similar to normal saline group was observed with the hemolysis ratios lower than 1% (Fig. 7c and Fig. S7). Nevertheless, the supernatant of RBCs treated with hemolytic 0.1% Triton X-100 showed a red color due to the rupture of cell membrane of RBCs and the release of a large amount of hemoglobin. These results indicated that IR780-DBCO had outstanding biocompatibility towards mammalian cells.

In vivo anti-infective assay

      To further demonstrate the bactericidal efficacy of IR780-DBCO in vivo, we established an animal model with azide-labelled VRE-infected wounds on the back skin of BALB/c mice. Upon different treatments with NIR light irradiation, IR780-DBCO, or IR780-DBCO plus NIR light irradiation, the photographs of wounds were recorded at varied time points. As depicted in Fig. 8a, all the VRE-infected wounds had a certain degree of suppuration on day 2. Moreover, compared with the groups with no treatment or treated with only NIR light irradiation or IR780- DBCO, the suppuration in the group treated with IR780-DBCO plus NIR light irradiation was obviously diminished on day 4, proving that IR780-DBCO was capable of inhibiting the bacterial infection at the wound site through PTT. Meanwhile, VRE in the homogenized wound tissues on day 4 was quantified to evaluate the anti-infective effect of IR780-DBCO. The number of bacteria in wound tissues for the control group, NIR light irradiation group, or IR780-DBCO group was 10.87 × 106 , 8.97 × 106 , or 6.30 × 106 CFU g−1 , respectively, which was consistent with the result of a large amount of bacterial colonies presented on the agar plate (Fig. 8b, c). However, the bacterial number detected in IR780-DBCO plus NIR light irradiation treated group reduced by three orders of magnitude, with only 2.76 × 103CFU g−1 , which indicated the excellent photothermal bactericidal performance of IR780-DBCO. In addition, the wound tissues of mice were collected on day 4 and stained with H&E to investigate the anti-infective ability of IR780-DBCO towards VRE. Generally, infected tissues can generate large numbers of neutrophils, which are stained blue by H&E staining [42]. As revealed in Fig. 8d, VRE-infected wounds treated with IR780-DBCO plus NIR light irradiation showed much fewer neutrophils than the other groups, implying that IR780-DBCO under NIR laser illumination could significantly eliminate Gram-positive VRE and effectively suppress bacterial infection at the wounds.

CONCLUSIONS

      In summary, Gram-positive bacteria were selectively inactivated by a bioorthogonal photothermal agent (IR780-DBCO) via metabolic engineering. Followed by pretreating bacteria with metabolic precursor D-Ala-N3, the bioorthogonal IR780-DBCO could be specifically conjugated on the surface of Gram-positive bacteria instead of Gram-negative ones through click reaction. Benefitting from the outstanding photothermal conversion performance and excellent biocompatibility of IR780-DBCO, precise inactivation of Gram-positive bacteria, such as S. aureus and superbug VRE, was demonstrated under NIR light irradiation. Most importantly, in vivo anti-infective capability was successfully verified in a VRE-infected mouse skin wound model. As compared with the reported nanoplatforms [43–45] or electrostatic and hydrophobic interaction-based strategies [46–48] for selective bacterial imaging and elimination, our method is advantageous in the stable covalent conjugation of photothermal agent on the cell wall with short distance for enhanced and spatiotemporally controlled PTT, as well as the defined or tunable chemical structures and the easy diffusion to infectious sites of small-molecule reagents. This study not only provides valuable practice for designing bioorthogonal photothermal molecules and one-step metabolic labelling phototheranostic agents to specifically sterilize Gram-positive bacteria, but also promotes the development of next-generation antimicrobial agents in the post-antibiotic era.

Received 3 April 2021; accepted 10 June 2021;

published online 18 August 2021

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (52003222 and 21875189), Ningbo Natural Science Foundation (202003N4064), the Natural Science Foundation of Chongqing (cstc2020jcyj-msxmX0752), the Joint Research Funds of Department of Science & Technology of Shaanxi Province and Northwestern Polytechnical University (2020GXLH-Z-013), and the Fundamental Research Funds for the Central Universities.

Author contributions

Feng T, Li P, and Huang W conceived the idea and supervised the study; Feng T, Ye X, and Lu H performed the experiments and wrote the manuscript; Nie C, Zhang J, Yu L, and Jin H participated in the in vivo anti-infective assay. All authors contributed to the general discussion.

Conflict of interest

The authors declare that they have no conflict of

Supplementary information Experimental details and supporting data are

Tao Feng received her BSc and MSc degrees from Jilin University in 2010 and 2013, and PhD degree from Nanyang Technological University in 2018. She is currently an associate professor at Northwestern Polytechnical University. Her research focuses on the design of fluorescent probes for selective discrimination and accurate killing of bacteria.

Hui Lu received his BSc degree from Anhui University of Technology in 2019. Currently, he is a graduate student at the Frontiers Science Center for Flexible Electronics, Northwestern Polytechnical University. His research interest is developing new phototheranostic agents to selectively inactivate bacteria.

Xiaoting Ye received her BSc degree from Nanjing Tech University in 2018 and MSc degree from the Frontiers Science Center for Flexible Electronics, Northwestern Polytechnical University in 2021. Her research focuses on preparing novel photothermal materials for specific killing of bacteria.

Peng Li received his BSc from Tianjin University in 2006 and PhD degree from Nanyang Technological University in 2013. He has been a professor of the Frontiers Science Center for Flexible Electronics at Northwestern Polytechnical University since 2018. His research interest is developing innovative antibacterial materials and strategies for infectious diseases.

Wei Huang received his BSc degree from Peking University in 1983, followed by MSc and PhD degrees from the same university. Then he did his postdoctoral research at the National University of Singapore, where he participated in the foundation of the Institute of Materials Research and Engineering in 1995. In 2011, he was elected as academician of the Chinese Academy of Sciences. Now, he is the chief scientist of the Frontiers Science Center for Flexible Electronics at Northwestern Polytechnical University. His research interests include polymer sciences, bioelectronics, nanoelectronics, and organic/plastic/flexible electronics. available in the online version of the paper.

This article is excerpted from the Sci China Mater by Wound World.