A Photon-Responsive Antibacterial Nanoplatform for Synergistic

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Biological and Medical Applications of Materials and Interfaces

A Photon-Responsive Antibacterial Nanoplatform for Synergistic Photothermal-/Pharmaco- Therapy of Skin Infection Lingling Zhang, Yingqian Wang, Jie Wang, Yulan Wang, Aoying Chen, Can Wang, Wenting Mo, Yingxue Li, Quan Yuan, and Yufeng Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b18146 • Publication Date (Web): 06 Dec 2018 Downloaded from http://pubs.acs.org on December 6, 2018

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ACS Applied Materials & Interfaces

A Photon-Responsive Antibacterial Nanoplatform for Synergistic Photothermal-/Pharmaco- Therapy of Skin Infection Lingling Zhang,†,Yingqian Wang,‡, Jie Wang,‡, Yulan Wang,† Aoying Chen,† Can Wang,† Wenting Mo,† Yingxue Li,‡ Quan Yuan‡,* and Yufeng Zhang†,* †

State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key

Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan 430079, China Medical Research Institute, School of Medicine, Wuhan University, Wuhan 430071, China ‡

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education),

College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China *Yufeng Zhang: [email protected] *Quan Yuan: [email protected]

KEYWORDS: black phosphorus, photothermal therapy, liposome, drug-resistant bacteria, skin abscess

ACS Paragon Plus Environment

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ABSTRACT Abuse of antibiotics and their residues in the environment result in the emergence and prevalence of drug-resistant bacteria and lead to serious health problems. Herein, a photon-controlled antibacterial platform that can efficiently kill drug-resistant bacteria and avoid the generation of new bacterial resistance was designed by encapsulating black phosphorus quantum dots (BPQDs) and pharmaceutical inside a thermal-sensitive liposome. The antibacterial platform can release pharmaceutical in a spatial-, temporal- and dosage-controlled fashion since the BPQDs can delicately generate heat under near-infrared (NIR) light stimulation to disrupt the liposome. This user-defined delivery of drug can greatly reduce the antibiotic dosage, thus avoiding the indiscriminate use of antibiotics and preventing the generation of superbugs. Moreover, by coupling the photothermal effect with antibiotics, this antibacterial platform achieved a synergistic photothermal-/pharmacotherapy with significantly improved antibacterial efficiency towards drug-resistant bacteria. The antibacterial platform was further employed to treat antibiotic-resistant bacteria-caused skin abscess and it displayed excellent antibacterial activity in vivo, promising its potential clinical applications. Additionally, the antibacterial mechanism was further investigated. The developed photon-controlled antibacterial platform can open new possibilities for avoiding bacterial resistance and efficiently killing antibiotic-resistant bacteria, making it valuable in fields ranging from anti-infective therapy to precision medicine.


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Bacterial infection seriously threatens public health, with an estimated death toll of 10 million people per year by 2050.1-3 The usage of antibiotics is a main alternative for treating patients suffering from bacterial infections.4 However, the indiscriminate utilization of antibiotics have led to the emergence and spread of the drug-resistant bacteria, especially superbugs, thus making antibiotic treatments ineffective.5-7 Although large efforts have been made to combat bacterial resistance, there is still a wide gap between the sustainable appearance of drug-resistant bacteria and the development of new antibiotics.8 Therefore, it is urgent to develop new antibacterial strategies to effectively treat diseases caused by drug-resistant bacteria. Precise release of pharmaceutical, which means the accurate control of the time, area, and dosage, is a promising way to avoid the abuse of antibiotics and further inhibit bacterial resistance.9-11 Furthermore, photothermal therapy is also reported to be a promising method for broad-spectrum antimicrobial therapy, especially drug-resistant bacteria.12-14 The combination of phototherapy with pharmacotherapy can not only significantly improve the antibacterial efficiency towards drug-resistant bacteria, but also readily reduce the antibiotic dosage, thus providing a potential solution to the problems of drug-resistant bacteria.15-17 Consequently, developing a new antibacterial platform that can realize controlled release of pharmaceuticals and efficient synergistic therapy can function as a promising strategy to combat drug-resistant bacteria. Photothermal therapy, which applies photo-induced heat to medical treatment, has been widely utilized in the fields of biomedicine owing to its spatiotemporally controlled photothermal effect.18-23 Particularly, photothermal therapy mediated by near-infrared (NIR) light has many other special advantages including deep tissue penetration and little photodamage, making it a good supplement to conventional pharmacotherapy.24-29 Black phosphorus quantum dots (BPQDs), which refer to black phosphorus (BP) with several nanometers, is a conceptually new


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photothermal reagent with two-dimensional metal-free layered structure that have high photothermal conversion efficiency under NIR light irradiation.30-35 Moreover, it is worth noting that BPQDs can gradually degrade into nontoxic phosphate in physiological environment, suggesting its excellent biocompability.36-42 Therefore, BPQDs can be used as an excellent photothermal reagent with NIR controllability for precise synergistic phototherapy and pharmacotherapy.

Scheme 1. Schematic illustration of the photon-controlled antibacterial platform for synergistic treatment of bacteria-infected subcutaneous abscess.


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Here, by using a thermal-sensitive liposome as the carrier for BPQDs and antibiotics vancomycin, we designed a photon-responsive antibacterial platform (denoted as BPQDsvanco@liposome) that displays precise delivery of drugs and efficient photothermal/pharmaco synergistic therapy for anti-bacteria. As shown in Scheme 1, under NIR irradiation, the thermalsensitive liposome is disrupted owing to the photothermal effect of BPQDs, which leads to the release of the encapsulated antibiotics vancomycin to kill bacteria. At the same time, the local temperature rise caused by photothermal effect of BPQDs can also result in the efficient ablation of bacteria, including drug-resistant bacteria. The photon-responsive antibacterial platform was further applied in treating the drug-resistant bacteria-infected skin abscess. In vivo results showed that the skin abscess caused by drug-resistant bacteria can be efficiently cured by local injection of the NIR-responsive liposomes. The proposed photon-responsive antibacterial platform can achieve NIR-mediated remote control of drug delivery as well as synergistic antibacteria, which can open new ways for areas including nanomedicine, anti-infective therapy and precise cancer therapy. RESULTS AND DISCUSSION

Characterization of the BPQDs-vanco@liposome Antibacterial Platform. As illustrated in Figure 1a, the photon-controlled antibacterial platform is prepared by encapsulating BPQDs and vancomycin inside the liposome. Transmission electron microscopy (TEM) image (Figure 1b) shows the obtained BPQDs with uniform morphology. Based on the statistical TEM analysis of 100 counts of BPQDs, the average lateral size is determined to be  3.0 nm (Figure S1). The atomic force microscopy (AFM) image in Figure 1c exhibits the topographic morphology of the obtained BPQDs. The thickness of the BPQDs is measured by cross-sectional analysis and the


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Figure 1. Characterization of BPQDs and BPQDs-vanco@liposome. (a) Schematic illustration of the preparation of BPQDs-vanco@liposome. TEM (b) and AFM (c) image of BPQDs. (d) Height profiles along the white lines in (c). (e) CLSM bright field image of BPQDsvanco@liposome. (f) Elemental mapping images of the BPQDs-vanco@liposome obtained on SEM. (g) Raman spectra of the BPQDs and BPQDs-vanco@liposome. height profiles are shown in Figure 1d. The BPQDs have a thickness of around 1.93.4 nm, corresponding to 36 layers. Confocal laser scanning microscopy (CLSM) image in Figure 1e shows the uniform and well-dispersed BPQDs-vanco@liposome. Elemental mapping was


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conducted on scanning electron microscope (SEM) to verify the formation of the designed BPQDs-vanco@liposome nanostructure. As shown in Figure 1f, C and O elements from the liposome co-distribute around the limbic region, whereas the abundant P element locates in the central area, indicating that the BPQDs are successfully loaded inside the liposome. In addition, three well resolved Raman peaks at 359.7, 436.2 and 463.5 cm1 can be identified in the spectra of both BPQDs and BPQDs-vanco@liposome (Figure 1g), which correspond to one out-of-plane phonon mode (A1g) and two in-plane modes (B2g and A2g) of BP, respectively.43,44 These results confirm the successful formation of the designed BPQD-vanco@liposome nanostructure. Photothermal Performance and NIR-triggered Release of BPQDs-vanco@liposome. As show in Figure 2a, the BPQDs-vanco@liposome shows a very broad absorption band, from ultraviolet region to near-infrared region.45,46 The absorption intensities increase with the elevated concentration of BPQDs-vanco@liposome. The extinction coefficient of the ontained BPQDs-vanco@liposome at 808 nm is measured to be 9.6 L g1 cm1 (Figure S2). The photothermal performance of the BPQDs-vanco@liposome at different concentrations was further examined. As show in Figure 2b, the temperature of the BPQDs-vanco@liposome colloid dispersions increases instantly upon exposure to NIR irradiation. Importantly, the solution temperature increases by 35 ºC at a very low concentration (50 ppm) of the BPQDsvanco@liposome, demonstrating that BPQDs-vanco@liposome can convert NIR into heat rapidly and efficiently.47-50 The photothermal stability of the BPQDs-vanco@liposome was also investigated. As shown in Figure 2c, the temperature elevation during several cycles of irradiation almost remains unchanged, indicating that the BPQDs-vanco@liposome is stable enough to serve as an excellent photothermal agent. The long-term stability of BPQDsvanco@liposome was also studied. Figure 2d shows that the photothermal capacity of bare


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BPQDs deteriorates significantly over time and the solution temperature only increases by  3 ºC under NIR irradiation after 8 days’ storage, indicating the rapid degradation of the bare BPQDs in humid environment.51 In contrast, the temperature variation of BPQDs-vanco@liposome colloid dispersion stored for 8 days is close to that of the freshely prepared BPQDsvanco@liposome colloid dispersion (Figure 2e). These results manifest that liposome encapsulation can effectively prevent BPQDs degradation and retain their photothermal performance, which could be ascribed to the protection of BPQDs from oxygen and water by the liposome encapsulation.52 Since precisely controlled release is desired in drug delivery, the NIRcontrolled release of encapsulated guest molecules from the liposome is monitored. Fluorescein and BPQDs co-loaded liposome (denoted as FITC-BPQDs@liposome) was prepared to investigate the release behavior of FITC under NIR irradiation (Figure 2f). As shown in Figure 2g, when exposed to pulse NIR irradiation, a rapid release of FITC is observed upon irradiation. Whereas the follow-up release of FITC is negligible once the NIR irradiation was switched off. In addition, Figure 2g shows that no obvious FITC release from FITC-BPQDs@liposome is observed during the whole course of the tests without NIR irradiation. Also, FITC-doped liposome (labeled as FITC@liposome) displays insignificant release of FITC under NIR irradiation (Figure 2g), verifying the integrity and stability of lisoposome in the absence of photothermal reagents. These results indicate that the NIR-controlled release of guest molecules from the liposome can be realized, which is attributed to the heat produced by BPQDs under NIR irradiation that can change the film layer fluidity of the thermal-sensitive liposome to release the inner guest molecules.53-56 Furthermore, CLSM characterization was carried out to investigate the photon-controlled release performance of FITC-BPQDs@liposome. Without NIR irradiation, the encapsulated FITC is concentrated inside the liposome (Figure 2h). However, upon NIR


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irradiation, diffused FITC is seen the entire field of vision (Figure 2i), indicating the NIR triggered release of FITC. All of the above results demonstrated that the designed

Figure 2. Photothermal performance and photon controlled release behavior of the BPQDsvanco@liposome. Absorption spectra (a) and photothermal performance (b) of BPQDsvanco@liposome

at

different

concentrations.

(c)

Photothermal

cycle

of

BPQDs-

vanco@liposome (20 ppm). Photothermal curves of bare BPQDs (d) and BPQDsvanco@liposome (e) dispersed in PBS for different periods of time under 808 nm laser irradiation (1 W cm-2) for 15 min. (f) Schematic representation of FITC-BPQDs@liposome. (g) NIR-responsive release curve. (h) CLSM images of FITC-BPQDs@liposome before and (i) after NIR irradiation.


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BPQDs-vanco@liposome displays excellent photothermal effect and NIR light controlled release of drug, making it possible to achieve synergistic photothermal therapy and pharmacotherapy of bacterial infections. In Vitro Antibacterial Activity towards Drug-resistant Bacteria. Methicillin-resistant Staphylococcus aureus (MRSA), one of the leading dangerous pathogens associated with the emergence and prevalence of antibiotic-resistance, has spread worldwide since it emerged in the 1960s and has become a major cause of bacterial infections in health-care and community settings.57,58 Here MRSA cells were utilized as the model bacteria to estimate the antibacterial performance of the proposed platform. Figure 3a illustrates the antibacterial application of the photon-controlled antibacterial platform. Due to the photothermal effect of BPQDs loaded in BPQDs-vanco@liposome, the stability of the thermal-sensitive liposomes is changed under NIR irradiation, causing the release of the loaded vancomycin. Moreover, the local temperature rise caused by the photothermal effect of BPQDs can act simultaneously with the antibiotics to kill bacteria. The growth kinetics of MRSA were teasted to quantitatively evaluate the antibacterial activity of the BPQDs-vanco@liposome. The MRSA bacterial proliferation was monitored by detecting the optical density at 600 nm (OD600 nm). As shown in Figure 3b and 3c, MRSA treated with PBS experiences an exponential growth after 12 h of incubation and the number of MRSA keeps stationary till 15 h. When MRSA is exposed to PBS and NIR light, similar growth curve is observed, indicating that NIR irradiation itself has no effect on bacterial activity. Vancomycin loaded liposome (denoted as Vanco@liposome) also causes no obvious delay to the growth of MRSA either with or without NIR irradiation, which suggests that NIR itself cannot trigger vancomycin release in the absence of BPQDs. The bacterial proliferation of MRSA treated with BPQDs loaded liposome (denoted as BPQDs@liposome) is also not delayed compared to that of


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MRSA treated with PBS. However, when the BPQDs@liposome group is irradiated with the NIR laser, the bacteria growth is seriously delayed, showing that the NIR-triggered photothermal effect has a pronounced effect on inactivation of bacteria. When MRSA is treated with BPQDsvanco@liposome without NIR irradiation, no obvious growth delay is observed. By contrast, in the presence of both BPQDs-vanco@liposome and NIR irradiation, the growth of MRSA is completely inhibited. The above results thus demonstrate that the designed BPQDsvanco@liposome antibacterial platform exhibits outstanding antibacterial activity by coupling the photothermal effect with pharmacological action.

Figure 3. In vitro antibacterial activity. (a) Schematic illustration of the interaction of BPQDsvanco@liposome with MRSA. (b) MRSA growth curves after incubation with BPQDsvanco@liposome, BPQDs@liposome, Vanco@liposome and PBS with NIR irradiation and (c) without NIR irradiation.


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To further confirm the synergistic antibacterial efficacy of the photon-controlled antibacterial platform, the standard spread plate method was performed to calculate the viable colonies. As shown in Figure 4a and 4b, a large number of viable colonies are visible on the Luria-Bertani (LB) agar plate in the blank group after culturing overnight in a 37 C incubator. In accordance with the previous growth curve data, BPQDs@liposome can inhibit MRSA growth to a certain degree under NIR irridiation. Notably, when the BPQDs-vanco@liposome group is irradiated with NIR light, no viable colony is observed, demonstrating that the photon-controlled antibacterial platform possesses the best antibacterial ability among all of the tested groups. Fluorescent-based tests of live and dead bacterial cells were further performed to visually demonstrate the bactericidal effect of the photon-controlled antibacterial platform, as shown in Figure 4c. Live MRSA is stained by Calcein acetoxymethylester (Calcein-AM) and displays green fluorescence, whereas dead MRSA is labelled by propidium iodide (PI) and shows red fluorescence.59 In the group of PBS treated MRSA either with or without NIR irradiation, bacteria are all stained green. Both red and green fluorescence were seen in the BPQDs@liposome group with NIR irradiation, indicating the photothermal effect of BPQDs has a certain germicidal efficacy. Significantly, in the BPQDs-vanco@liposome group with NIR irradiation, all bacteria are stained red, which suggests that the photon-controlled antibacterial platform has excellent bactericidal effect. The fluorescence intensity of PI and Calcein AM were further measured to quantify the bacterial livability (Figure 4d and Figure 4e). Almost all of the bacteria are killed in the BPQDs-vanco@liposome group exposed to NIR irradiation, further confirming that the proposed antibacterial platform with photothermal effect and pharmacological action has outstanding antibacterial efficiency. Consequently, one can draw the conclusion that the NIR-


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triggered on demand release of antibiotics can synergistically act with the photothermal therapy to achieve more potent antibacterial capability.

Figure 4. In vitro antibacterial activity. (a) Photographic images of the colonies of MRSA treated at different conditions (scale bar = 1 cm). (b) CFU counting of photographic images shown in (a). (c) Live (green fluorescence, Calcein-AM) and dead (red fluorescence, PI) MRSA under various treatments (scale bar = 100 μm). Fluorescence intensity of PI (d) and Calcein-AM (e) in images shown in (c). *Statistical significance at P < 0.05. In Vivo Antibacterial Activity. Infectious model was further built to estimate the in vivo antibacterial potential of the photon-controlled antibacterial platform. Subcutaneous abscesses


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are created in the mice model by local injection of MRSA.60 These mice with focal infections were divided into 8 groups randomly and accept different treatments for 5 days, including 1) injected with BPQDs-vanco@liposome, 2) injected with BPQDs-vanco@liposome and exposed to NIR irradiation, 3) injected with BPQDs@liposome, 4) injected with BPQDs@liposome and exposed to NIR irradiation, 5) injected with Vanco@liposome, 6) injected with Vanco@liposome and exposed to NIR irradiation, 7) injected with PBS, and 8) injected with PBS and exposed to NIR irradiation. As illustrated in Figure 5a, temperature changes in the subcutaneous abscesses are examined. Figure 5b and Figure 5c exhibit the thermographic images

Figure 5. In vivo photothermal performance. (a) Schematic illustration of the photothermal effect of BPQDs-vanco@liposome in vivo upon NIR irradiation. (b) Infrared images of the local site with various treatment conditions. (c) Local temperature rise during treatment. *Statistical significance at P < 0.05.


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The treatment of subcutaneous abscesses with the BPQDs-vanco@liposome is illustrated in Figure 6a. Under NIR laser irradiation, the encapsulated drug can be released in a controllable manner and the site of subcutaneous abscess yields an elevated temperature, making it possible to realize the synergistic bactericidal effects of hyperthermia and antibiotics. Figure 6b shows the established subcutaneous abscess that is locally injected with MRSA. The health conditions of the mice skin were systematically recorded using image analysis. As shown in Figure 6c, when the mice are subjected to both BPQD-Vanco@liposome and NIR irradiation, no scars or ulcerations are observed in the skin of the abscess site. As for mice treated with BPQDs@liposome and NIR irradiation, the wounds become apparently smaller compared with that on mice without treatment. These results demonstrate that the established antibacterial platform with photothermal effect and pharmacological action has excellent antibacterial capability in vivo. Subsequent to treatment after 5 days, the abscess tissues on the above mice were excised and mechanically homogenized under sterile conditions. The bactericidal effect of the antibacterial platform was evaluated by counting the number of CFUs in the obtained homogenate with the standard plate counting method.61 As demonstrated in Figure 6d, almost no colonies are seen in the group of mice treated with BPQDs-vanco@liposome and NIR irradiation. Compared to the group of mice treated with PBS, the CFU count is decreased sharply to 9.4% in the group of mice treated with BPQDs-vanco@liposome and NIR irradiation (Figure 6e), demonstrating that the photon-controlled antibacterial platform possesses an outstanding antibacterial effect. In addition, the group of mice treated with BPQDs@liposome and NIR irradiation also present a relatively strong antibacterial effect, evidenced by the normalized CFU count of 42.1%, which suggests that photothermal therapy exhibits good antibacterial capability towards drug-resistant bacteria. The above results demonstrate that the photon-controlled antibacterial platform is an


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efficient antibacterial tool and can effectively inhibit bacterial survival upon NIR irradiation both in vitro and in vivo.

Figure 6. In vivo antibacterial activity. (a) Schematic illustration of the synergistic bactericidal effect of BPQDs-vanco@liposome in vivo under NIR irradiation. (b) Bacterial infectious site of mouse before therapy. (c) Photographs of the infectious site after various treatments for 5 days. (d) Photographs of bacterial colonies obtained from infected tissues of mice (scale bar = 1 cm) and (e) related quantitative results (n = 3). *Statistical significance at P < 0.05. Antibacterial Mechanisms. The possible antibacterial mechanisms of the photon-controlled antibacterial platform were further investigated. SEM measurements were first conducted to explore the antibacterial behavior of the antibacterial platform. As demonstrated in Figure 7a, the MRSA subjected to PBS alone has intact morphologies and smooth surfaces. Also, MRSA treated with PBS and exposed to NIR irradiation is sphere-shaped with smooth surface. When the MRSA cells are incubated with Vanco@liposome, no obvious disruption of the cell membrane occurs


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either with or without NIR irradiation, indicating that vancomycin is efficiently encapsulated in liposome in the absence of photothermal effect. After treatment with BPQDs@liposome, the cell membrane of MRSA is also integrated. After treatment with BPQDs@liposome and NIR irradiation, the cell membrane becomes wrinkled but still integrate, indicating that BPQDs@liposome upon NIR irradiation can affect the integrity of cell membrane to a certain degree. When the MRSA cells are treated with BPQDs-vanco@liposome alone, the bacterial morphologies are similar to those of the control cells with smooth surface. However, as for MRSA treated with BPQDs-vanco@liposome and NIR irradiation, the MRSA cells become amorphous and fragmentized. It is generally believed that the destruction of bacterial membrane is an important process for disinfection.62 Thus the destroy of MRSA membrane may be one of the reasons for bacteria death induced by the photon-controlled antibacterial platform. SEM results show that MRSA cells lost their structural integrity due to the membrane damage caused by the photon-controlled antibacterial platform under NIR irradiation. It is widely accepted that the components in bacterial cytoplasm such as nucleic acid will be released once the membrane integrity has been destroyed.63 The nucleic acids leakage from MRSA was further tested to investigate the antibacterial mechanism of the photon-controlled antibacterial platform. When the MRSA cells are treated with BPQDs-vanco@liposome and exposed to NIR irradiation, the DNA concentration in the collected supernatant significantly increases in comparison with the control MRSA treated with PBS (Figure 7b). The results of DNA leakage tests are in good coincidence with SEM results, implying that nucleic acid leakage induced by the damage of the bacterial membrane may be another cause for the antibacterial effect. Reactive oxygen species (ROS) are usually generated as natural products of respiration but ROS at elevated levels is harmful to cell.64 Enhanced generation of ROS is thought be one of the major antibacterial


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Figure 7. Antibacterial mechanisms. (a) SEM images of MRSA after receiving various treatments (scale bar = 200 nm). (b) DNA concentration released from bacteria after various treatments. (c) DFCH-DA test of MRSA after treatment. Inset: Fluorescence intensity of ROS. (d) Schematic illustration of the proposed mechanisms of synergistic bactericidal effect of BPQDsvanco@liposome. mechanisms of many nanocomposites, such as graphene and ZnO.65,66 An oxidation-sensitive fluorescent probe 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA) was applied to test the level of ROS generated in MRSA.67 As exhibited in Figure 7c, the fluorescence intensity of MRSA cells treated with BPQDs@liposome and NIR irradiation is almost double that of MRSA cells treated with PBS alone. What’s more, after treatment with BPQDs-vanco@liposome and NIR irradiation, the fluorescence intensity is almost 4 times that of MRSA cells treated with PBS. Thus, it appears that the photon-controlled antibacterial platform can effectively elevate the ROS level in bacteria and this may also play a vital part in the superior bactericidal performance of


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BPQDs-vanco@liposome. Previous study has demonstrated that the relatively high level of ROS may cause DNA damage.68 Thus the integrity of the DNA from MRSA was further assessed by agarose gel electrophoresis. After treatment with BPQDs-vanco@liposome and NIR irradiation, the isolated DNA exhibits no lane (Figure S10), indicating that no macromolecule DNA exists. Whereas extracted DNA from MRSA treated with BPQDs-vanco@liposome alone or PBS show apparent lane. These results indicate that the BPQDs-vanco@liposome coupled with NIR irradiation can destruct DNA molecules into small fragments or even oligonucleotide. Thus, it can be speculated that the destruction of DNA integrity induced by elevated ROS may be one of reason for killing bacteria. Thus the proposed mechanism for the antibacterial behaviors of the photon-controlled antibacterial platform is illustrated in Figure 7d. That is, the membrane destruction, nucleic acid leakage, elevated ROS level and DNA damage are all responsive for the ablation of MRSA treated with BPQDs-vanco@liposome coupled with NIR irradiation. CONCLUSION In summary, a photon-controlled antibacterial platform was constructed for treatment of the drug-resistant bacteria-infected skin abscess. Since BPQDs have high photothermal conversion efficiency upon NIR irradiation, the thermal-sensitive liposomes loaded with BPQDs can serve as efficient stimuli-responsive nanocarriers for precise drug release. By combining photothermal effect of BPQDs with pharmaco-therapy, this proposed antibacterial system achieved a synergistic antibacterial effect. Importantly, subcutaneous abscess treatment results indicated that the stimuli-responsive liposome have an outstanding performance in killing drug-resistant bacteria in vivo. Additionally, the possible antibacterial mechanism was also investigated, which can provide valuable instructions for the design of antibacterial methods to combat drug-resistant bacteria. This work highlights the tremendous potential of the photon-controlled antibacterial


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platform for killing drug-resistant bacteria. The proposed photon-manipulated platform with precise delivery of loaded molecules and efficient synergistic therapy may further find wide applications in cancer therapy and precision medicine. EXPERIMENTAL SECTION Materials and Reagents. The BP crystals were obtained from Nanjing XFNANO Materials Tech Co., Ltd (Nanjing, China). Vancomycin was obtained from Sigma-Aldrich. The cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine

(DPPC)

and

1,2-dipalmitoyl-sn-glycero-3-

phosphoethanolamine-N-[methoxy-(polyethyleneglycol)-amine] (DPPE-PEG-NH2, MW 2000) were provided by Avanti Polar Lipids, Inc. The N-methyl-2-pyrrolidone (NMP) and chloroform were purchased from Sinopharm Chemical Reagent Co., Ltd., China. Calcein-AM was obtained from Dojindo Molecular Technologies. The propidium iodide (PI) and Cell Counting Kit-8 (CCK-8) were obtained from Beyotime Institute of biotechnology. The fetal bovine serum (FBS) and dulbecco's modified eagle medium (DMEM) were provided by Thermo Fisher Scientific Inc. Sample Characterization. A transmission electron microscope with working voltage of 200 kV (JEOL, JEM-2100, Japan) and an atomic force microscopy in tapping mode (Bruker Multimode 8) were used to observe the morphologies of BPQDs. An OLYMPUS biological confocal laser scanning microscope (model: FV1200) was utilized to obtain the light microscopy (LM) images and confocal fluorescence images. A RM 2000 Microscopic Confocal Raman Spectrometer (Renishaw PLC, England) was used to record the Raman spectra. An UV-vis absorbance spectrometer (UV-2550, Shimadzu, Japan) was used to record the UV-vis spectra. The FLIR A35 infrared thermal imaging camera (USA) was employed to monitor the NIR laserinduced heat conversion curves. A plate reader (PowerWave XS2, BioTek, USA) was used to test the optical density. The fluorescence results were obtained by using a fluorescence


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spectrometer (Hitachi. Ltd., F-4600, Japan). The bacterial morphology was observed using a scanning electron microscope (SEM, Zeiss, Germany). Synthesis of BPQDs. A previously reported liquid exfoliation technique was used to synthesize the BPQDs.31 A mixture containing BP powders (200 mg) and NMP (300 mL) was sonicated (ultrasonic frequency: 1925 kHz) in an ice bath under argon protection for total 14 h. In the first 8 h, the mixture was sonicated in the mode of period of 2 s with the interval of 0.1 s. For another 6 h, the ultraonic mode was changed to a period of 2 s with the interval of 4 s. The supernatant containing BPQDs was obtained after centrifugation for 10 min at 6000rpm. Preparation of BPQDs-vanco@liposome. The previously reported thin lipid film hydrated methods was applied to synthesize the liposomes.69 Chloroform solution containing DPPC, cholesterol, and DPPE-PEG-NH2 (100:50:5, molar ratio, 26 mg in total) was mixed and dried in a vacuum for 2 h in a round bottom flask. Then, 3 mL of PBS (0.01 M, pH 7.4) was added to hydrate the formed film and then the solution was vortexed at 50 C. Then 0.05 mL of ethanol was added to the phospholipids suspension, and the suspension was stirred at 125 rpm at 4 C overnight. The suspension centrifuged and washed three times with PBS (0.01 M, pH 7.4) to remove ethanol. After that, a certain amount of BPQDs and vancomycin were added into the obtained phospholipids suspension and heated at 50 C for 2 h under vortex mixing. The final concentration of BPQDs and vancomycin were 20 ppm and 0.01 mg mL1, respectively. The BPQDs-vanco@liposome was obtained after centrifugation and re-dispersing in PBS (0.01 M, pH 7.4). NIR Laser-Induced Heat Conversion. Sample solutions of different concentrations (10, 20, 50 ppm) were prepared to estimate the photothermal conversion performance. PBS solution was used as control. These solutions were irradiated with an 808 nm laser (1 W cm2, BWT Beijing


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Ltd, Beijing, China) for 15 min. During irradiation, the FLIR A35 infrared thermal imaging camera was employed to monitor the temperature variation. NIR-triggered Vancomycin Release Using Dialysis Devices. NIR-triggered release of vancomycin was monitored using the FITC-BPQDs@liposome. The dialysis devices containing 1 mL of sample was irradiated for different time. The released FITC was monitored by the fluorescence spectrometer. Bacteria Preparation. The model organism used in all antibacterial experiments was Methicillin Resistant Staphylococcus Aureus (MRSA, ATCC 43300). MRSA cells were cultured in an aerophilic environment at 37 °C in LB broth (10 g Bacto peptone, 15 g agar, 5 g yeast extract, and 10 g NaCl per litre of ultrapure water) to grow into the mid-exponential growth phase. The bacterial suspension was then diluted to the concentration of around 1 × 106 colony forming units (CFU) per mL. Evaluation of Growth Curve of Bacteria. MRSA cells in the mid-exponential phase were diluted to about 106 CFU per mL of bacteria with culture medium. Then the MRSA cells were treated with various samples (20 ppm) with or without NIR irradiation. After incubation for 2 h, the MRSA suspensions were diluted 105 times and then cultured in an aerophilic environment at 37 °C. The OD

600 nm

of 100 μL of bacterial suspension for each group was tested and recorded

by Power Wave XS2 every hour for 24 hours. Observation of the Colony Forming Units after Treatment. First, the bacterial suspensions were diluted to 1.0 × 106 CFU per mL and were further treated with various conditions, including 1)

BPQDs-vanco@liposome,

2)

BPQDs-vanco@liposome

and

NIR

irradiation,

3)

BPQDs@liposome, 4) BPQDs@liposome and NIR irradiation, 5) Vanco@liposome, 6) Vanco@liposome and NIR irradiation, 7) PBS, 8) PBS and NIR irradiation. The NIR irradiaion


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groups were irradiated with an 808 nm laser (1 W cm2) to reach 45 °C and maintained for 3 min. After incubation at 37 C and shaking for 2 h, 100 μL bacterial suspension was taken from each group and diluted 105 times. Thereafter, 50 μL of the diluted solutions were spread evenly onto LB plates. After culturing overnight at 37 °C in an aerophilic environment, the number of the colonies was counted and recorded. Bacterial Staining. Differently treated MRSA cells were double-stained by incubation for 15 min in PBS containing 2 μM Calcein acetoxymethylester (Calcein-AM) and 1 μM propidium iodide (PI). The Olympus D71 fluorescence microscope (Olympus Co, Japan) was used to characterize the live and dead cells. Live MRSA is stained by Calcein-AM and displays green fluorescence, whereas dead MRSA is labelled by PI and shows red fluorescence.59 The fluorescence intensity was measured by the Image J software. In Vitro Cytotoxicity Experiments. Bone marrow mesenchymal stem cells (BMSCs) and gingival fibroblasts (GFs) of Kuming mice were used to study the cytotoxicity of the BPQDsvanco@liposome. A standard CCK-8 assay was performed to measure the in vitro cytotoxicity. BMSCs and GF cells were cultured in a humidified incubator (37 °C, 5% CO2) in 96-well plate (about 5000 cells/well, 6 wells for each concentration) for 24 h. Then the cells in each well were washed with PBS (0.01 M, pH = 7.4), followed by adding different concentrations of BPQDsvanco@liposome solutions (0, 2, 5, 10, 20, 50, and 100 ppm). After co-incubated for 72 h, CCK8 solution was added and co-incubated with cells for another 1.5 h. Finally, absorbance at 450 nm was examined by a micro-plate reader (SpectraMax M2, MDC, USA) to evaluate cell viability. The cells without BPQDs-vanco@liposome were applied as control. Culture medium containing CCK-8 solution but with no cells was used as background.


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In Vivo Antibacterial Experiment Using Mice Abscess Model. Four kinds of antibacterial solutions including 100 ppm of BPQDs-vanco@liposome, BPQDs@liposome, Vanco@liposome and PBS were prepared using sterile water. All the solutions were prepared under sterilized environment. Kunming mice (8 weeks, 18−23 g) were divided randomly into eight groups with six mice in each group: 1) injected with BPQDs-vanco@liposome, 2) injected with BPQDsvanco@liposome and exposed to NIR irradiation, 3) injected with BPQDs@liposome, 4) injected with BPQDs@liposome and exposed to NIR irradiation, 5) injected with Vanco@liposome, 6) injected with Vanco@liposome and exposed to NIR irradiation, 7) injected with PBS, 8) injected with PBS and exposed to NIR irradiation. Subcutaneous abscesses were obtained by locally injecting MRSA on the back of the mice under anesthesia following previously reported protocol.60 After injection for 24 h, 50 μL of antibacterial solutions were injected separately into the abscess at the corresponding groups. 5 min after injection, the abscess areas were exposed to an 808 nm laser (1.5 W cm−2) to reach 45 °C and maintained for 3 min in the four irradiation treated groups. The other four groups were handled under the same condition, but with no NIR irradiation. Photographs of the abscess were taken after treatment of 24 h and 5 days, respectively. After that, the abscess tissues were excised from the euthanasia-treated mice for further analysis. The obtained abscess tissues were homogenated to quantify the amount of bacteria in these infected tissues.61 Tipically, 50 μL of bacterial suspension of the grinded tissue was dropped and spread evenly on the LB agar plate and cultured for 24 h at 37 °C. The relative CFU were obtained based on these colony countings. The CFU of PBS-treated group was regarded as 100 %. All mice were treated on account of the guidelines of the Institutional Animal Care and Use Committee. After finishing the experiment, the mice were discarded on the basis of the standard approved protocol.


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Observation of Morphological Destruction of Bacteria. SEM was used to observe the morphological changes of the differently treated bacteria. The bacteria were treated with various condition, including 1) BPQDs-vanco@liposome, 2) BPQDs-vanco@liposome and NIR irradiation,

3)

BPQDs@liposome,

4)

BPQDs@liposome

and

NIR

irradiation,

5)

Vanco@liposome, 6) Vanco@liposome and NIR irradiation, 7) PBS, 8) PBS and NIR irradiation. The bacteria were incubated at 37 °C for 2 hours and further condensed by centrifugation at 6000rpm. After re-suspending in 50 μL of PBS, the bacterial suspensions were gently dropped onto clean glass-slides and air drying. Then 2.5% glutaraldehyde were used to quickly fix the bacteria. After that, 30, 50, 70, 80, 90 and 100% ethanol as well as HMDS were used sequentially for 15min respectively to dehydrate the glass-sliedes. After room temperature drying, the obtained samples were finally sputter-coated with gold and observed using SEM. Evaluation of the Leakage of Nucleic Acid. Cytoplasmic constituents would flow out into the ambient environment once bacterial membranes were broken or destroyed. The amount of the released nucleic acids was determinated by the absorbance at 260 nm to indicate the integrality of bacterial cell membrane. After treatment with various conditions for 2 hours, the bacterial suspensions were centrifuged at 12,000 rpm for 5 min and the OD 260 nm of the supernatants was examined by Nano Drop 2000 UV-Vis spectrophotometer. Measurement of ROS Production in Bacteria. The 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was utilized to detcet the produced ROS according to the previous study.67 In short, DFCH-DA solution was added into diluted MRSA suspensions (1 × 106 CFU per mL) in a proportion of 1:1000 and the final working concentration of DFCH-DA is 10 μM. The suspensions were then kept in dark and incubated at 37 °C for 20 min with the bacterial suspensions inverted every 3 min. Different materials were added to the bacterial solution and


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the fluorescence intensity at 488/525 nm was tested by a fluorospectrophotometer (FL4500, Hitachi, Japan). Investigation of DNA Destruction of Bacteria. MRSA cells were subjected to various conditions and then incubated for 2 h. Then 1 mL bacterial cells (108 CFU per mL) of each group were centrifuged (8000 rpm, 10 min, 4 °C) to obtain the supernatant. After that, apoptosis, DNA Ladder Extraction Kit (TIANGEN, China) was used to extract DNA. The acquired DNA was finally electrophoresed in 0.5% agarose gel and visualized by GelRed staining. The photograph of the gel was taken under ultraviolet light. Statistical Analysis. The data in this experiment were presented as mean ± SD. All the experiments were repeated three times, with at least three replicates in each group. One-way ANOVA and a post-hoc t-test were applied to analyze the statistical significance among each group. Graphpad Software v.4 (Graphpad Software, USA) was employed in statistical analysis. A difference of P < 0.05 was deemed to have statistically significance, while P < 0.01 was thought of high statistically significance.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Additional figures, including average lateral size of 100 counts of BPQDs, extinction coefficient of BPQDs at 808 nm, enhanced stability of the BPQDs-vanco@liposome under ambient conditions compared with bare BPQDs, FITC release under continuous NIR irradiation, in vivo temperature evolution of BPQDs-vanco@liposome, surgical photos of subcutaneous


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abscess, pictures of in vivo anctibacterial procedure, the photothermal image of the mouse during antibacterial therapy, in vitro cytotoxicity experiments, and agarose gel for DNA measurement. AUTHOR INFORMATION Corresponding Author *(Y.Z.) E-mail: [email protected] *(Q.Y.) E-mail: [email protected] ORCID Jie Wang: 0000-0003-4170-8470 Quan Yuan: 0000-0002-3085-431X Yufeng Zhang: 0000-0001-8702-5291 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. These authors contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the funds of the National Natural Science Foundation of China (81771050, 81271108, 81600906 and 21675120), the Technical Innovation of Hubei Province (2017CFA025 and 2017AHB046), National Key R&D Program of China (2017YFA0208000), the National Postdoctoral Program for Innovative Talents (BX20180223), Fundamental Research


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A Photon-Responsive Antibacterial Nanoplatform for Synergistic

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