Mechanistic Insight into the Light-Irradiated Carbon Capsules as an

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Biological and Medical Applications of Materials and Interfaces

Mechanistic Insight into the Light-Irradiated Carbon Capsules as an Antibacterial Agent Qiuwen Wu, Gen Wei, Zhuobin Xu, Jing Han, Juqun Xi, Lei Fan, and Lizeng Gao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b04932 • Publication Date (Web): 10 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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ACS Applied Materials & Interfaces

Mechanistic Insight into the Light-Irradiated Carbon Capsules as an Antibacterial Agent

Qiuwen Wu†, Gen Wei†, Zhuobin Xu†, Jing Han†, Juqun Xi *,†,‡, Lei Fan*,§, Lizeng Gao*,†

† Department of Pharmacology, Institute of Translational Medicine, School of Medicine, Yangzhou University, Yangzhou 225001, Jiangsu, China. ‡ Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Senile Diseases, Yangzhou 225001, Jiangsu, China § School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002, Jiangsu, China.

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Abstract Infections caused by bacteria are a growing global challenge for public health as bacteria develop resistance, which will cause the failure of anti-infective treatment eventually. An effective alternative strategy to traditional antibacterial therapy is utilizing reactive oxygen species (ROS) to kill bacteria. Here, we report a simple route to prepare PEGylated nitrogen-doped carbon capsules (PEG-N-CCs) as an antibacterial agent. The PEG-N-CCs can translate near infrared light (NIR) into heat and produce a high concentration of ROS triggered by NIR irradiation. Both heating and ROS are critical to destroy the outer membranes and rupture cell bodies, causing DNA fragmentation and glutathione (GSH) oxidation both in Gram-negative Escherichia coli (E. coli), Gram-positive Staphylococcus aureus (S. aureus) and their multi-drug resistant strains. Moreover, PEG-N-CCs plus NIR irradiation can efficiently scavenge the existing biofilms and prevent the new biofilms formation, killing planktonic bacteria and as well as those within the biofilm. Our studies prove that PEG-N-CCs plus NIR irradiation can provide a simple and effective platform for combating bacteria, employing carbon nanomaterials as an antibacterial alternative for treatment of infectious diseases.

Keywords: nitrogen-doped carbon capsules, antibacterial, ROS, NIR irradiation, biofilm scavenging

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Introduction In recent years, human beings have been attacked more and more violently by harmful microorganisms, with grave consequences. Traditional antibiotics are gradually losing their effect due to the emergence of drug-resistant bacteria.1,2 With the development of nanotechnology, many nanomaterials have been designed for antibacterial applications. For example, nanoparticles of metals (Ag, Cu)3,4 and metal oxides (TiO2, ZnO)5,6 have shown excellent antibacterial activities. However, the antibacterial activities of these nanoparticles are achieved by releasing toxic substances into bacteria, which usually result in heavy side effects to the host cells.7,8 Therefore, developing new nanomaterials possessing good antibacterial effects and low toxicity simultaneously is urgent. Among numerous biomedical materials, carbon materials have attracted more attention owing to their good biocompatibility and excellent chemical stability.9 In particular, carbon nanotubes (CNTs)10,11 and graphene materials (GMs)12-14 are rapidly emerging as new broad-spectrum antibacterial agents. Both GMs and CNTs perform the antibacterial activity through the “chemical” and “physical” effects.15,16 The mechanism of killing effect are attributed to physical and oxidative stress induced by sharp nanoedges, which may cause the mechanical damage to cell membranes, finally leading to the death of bacteria.17-19 However, carbon spheres as one of the most commonly used carbon materials, compared with lamella-like carbon materials, have no sharp edges to rupture the membrane structure of cell and induce the “physical” damage to bacteria. Therefore, both investigating the antibacterial mechanism, including the role of biotic factors (e.g., physiological variations and species/strains tested), and further improving the antibacterial activity of carbon spheres are important for determining the potential application areas of carbon-based materials. The most common method to enhance the antibacterial performances of carbon materials is to prepare hybrid materials, such as ZnO or Ag nanoparticle-decorated graphene.20,21 However, this strategy also introduces new toxicities. In August 2015, Nature Nanotechnology has published a special review (Quick lessons on environmental nanotech), declaring that nanosilvers are harmful to the environment and are recommended to stop using.22 So looking for a novel combined strategy to promote the antimicrobial property of carbon materials is still challenge. Recently, photothermal therapy (PTT), which utilizes light-absorbing agents to absorb light energy and release heat, has been applied in various fields. PTT has many advantages, such as minimal invasiveness, less side effects and the strengths of spatiotemporal selectivity.23 Recently, PTT based on various carbon materials, such as CNTs and GMs, have been widely investigated for cancer therapy, drug delivery and antibacterial treatment.24 Inspired by a preliminary study, we propose to construct a simple and convenient route to synthesize carbon spheres to defeat 3



bacterial strains with the assistance of near infrared light (NIR) irradiation. The potential to construct a photothermal-assisted antibacterial platform may dramatically improve the antibacterial efficiency of carbon-based materials. Here, we designed a simple platform to synthesis PEGylated N-doped carbon nanocapsules (PEG-N-CCs) and evaluated the potential of PEG-N-CCs in antibacterial applications. A mechanism based on thermal-reactive oxygen species (ROS) therapy triggered by NIR irradiation was proposed to fight bacteria. First, exposure of PEG-N-CCs to an 808-nm laser induced hyperthermia, and the heat killed the bacteria directly. Second, brief laser irradiation dramatically increased the intracellular oxidative state, including high levels of ROS and glutathione (GSH) oxidation, leading to cell wall damage, compromise of membrane integrity, intracellular DNA cleavage and finally bacterial death. Although, the carbon capsules have no sharp edges like GMs to insert themselves into cell membranes to cause physical damage to bacteria, they exhibit excellent antibacterial activity with the assistance of NIR exposure. The NIR-irradiated carbon spheres can defect various bacterial strains, including Gram-negative Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli), Gram-positive Streptococcus mutans (S. mutans) and Staphylococcus aureus (S. aureus). Importantly, this antibacterial effect also acts on bacteria with antibiotic-resistance and in biofilms. Thus, our research demonstrates that carbon materials exhibit synergistic effects as a broad-spectrum antibacterial agent through a PTT-ROS mechanism.

Results and Discussion To synthesis N-doped carbon capsules (N-CCs), silica particles were used as template, dopamine were used as carbon and nitrogen precursor.25,26 The obtained carbon capsules were uniform with the size about 170 nm in diameter, characterized by scanning electron microscopy (SEM) (Figure 1a). The transmission electron microscopy (TEM) images (Figure 1b and Figure 1c) indicated that these carbon capsules exhibited a hollow structure with approximately 8 nm thickness of carbon shell. The finally yield of N-CCs was 17.4%. The microstructure of N-CCs was then characterized by X-ray diffraction (XRD) (Figure S1). Peaks appeared at 2θ = 26° and 42° corresponded to the (002) and (100) planes of graphitized carbon, revealing that the N-CCs had graphitic domains similar to reduced graphene.27 The Raman spectroscopy further confirmed the existence of graphitic domains. As displayed in Figure 1d, the peaks centered at 1325 cm-1 and 1585 cm-1 corresponded to the D band (caused by structure defects) and G band (belong to the planar vibration of sp2 carbon in an ideal graphitic layer). The peak intensity ratio of D band to G band (ID/IG=0.9) confirmed that N-CCs had a graphitic character with high graphitization degree.28 The 4


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composition and elemental states of the N-CCs were further investigated by X-ray photoelectron spectroscopy (XPS). The XPS survey spectrum, shown in Figure 1e, identified the existence of C, N and O. The percentage of the N element calculated according to the relevant peak areas was 4.2 at%. The high-resolution N1s peak in Figure 1f was fitted into four peaks at 398.3, 400.1, 401.2 and 403.2 eV, corresponding to pyridinic-N (N-6), pyrrolic-N (N-5), quaternary N (N-Q) and pyridine N-oxides respectively, indicating the successful doping of N atoms into the carbon framework.29

Figure 1. (a) SEM image of N-CCs. (b) TEM image of N-CCs. (c) High magnification TEM image of N-CCs. (d) Raman spectrum of N-CCs. (e) XPS general spectrum of N-CCs. (f) The high-resolution N 1s peaks.

It is generally accepted that PEG modification can prevent the aggregation of nanomaterials in solution and improve their water solubility.30 In our experiments, N-CCs were coated by a kind of PEG derivative (D-α-Tocopherol polyethylene glycol 1000 succinate, TPGS) through hydrophobic interactions in a non-covalent manner.31 The dynamic light scattering (DLS) analysis showed that the average diameter of PEGylated N-CCs (PEG-N-CCs) in water was 254.1 nm, while the average diameter of N-CCs without PEG coating reached 371.9 nm (Figure S2). This result indicated that the modification of PGE decreased the N-CCs aggregation. The zeta potentials of N-CCs with and without PEG modification were also measured. It was found that the zeta potential of naked N-CCs was -17.9 mV in water. After PEG modification, the zeta potential decreased to -24.8 mV (Figure S2). The relatively high negative zeta potential could inhibit the coagulation of nanomaterials. The surface functionalization was further confirmed by the result of Fourier transform-infrared spectroscopy (FTIR). In Figure 5



S3, we observed the C-H stretching and benzene ring skeleton vibration from TPGS of PEG-N-CCs. In addition, the PEG-N-CCs showed excellent compatibility in a variety of solutions (such as saline, serum and cell medium) without showing any obvious macroscopic aggregation over time (Figure S4). With the property to form various covalent bonds (sp2, sp3) between atoms, carbon materials have unique properties with various applications.32 Here, we specifically followed with interest the antibacterial performance of PEG-N-CCs after comprehensive physicochemical characterization. However, PEG-N-CCs did not exhibit an excellent antibacterial effect as expected (Figure S5); the bacteria survival rates were still 70% and 69% for E. coli and S. aureus at 250 µg/mL PEG-N-CCs, respectively. This relative lower antibacterial activity might be attributed to the carbon sphere structure without sharp edges to cause the physical damage to bacteria. In order to improve the antibacterial effect, we sought to utilize the excellent photothermal conversion efficiency of carbon materials to generate heat to kill bacteria. As we expected, the bacteria viabilities could be evidently reduced, with > 99.9% killing, when the bacteria were treated with PEG-N-CCs plus an 808-nm laser irradiation. Compared with the control group, treatment of PEG-N-CCs + Laser towards E. coli and S. aureus caused > 3 –log10 reduction of viable cells in both cases (Figure 2a and 2b). The antibacterial performance of PEG-N-CCs was also examined on LB agar plates shown in Figure 2c and 2d, an obvious decrease in bacterial colonies was achieved after NIR irradiation. Moreover, the antibacterial ability was related to the concentration of PEG-N-CCs (Figure S6a) and the dosage of NIR irradiation (Figure S6b), consistent with other reported antibacterial GMs.33 It should be pointed out that PEG modification also affected the antibacterial activity of carbon materials. As presented in Figure S6c, compared with N-CCs, PEG-N-CCs could cause more growth inhibition of bacteria under the same conditions. The reason was that the hydrophilic shell favored interactions between bacteria and the carbon capsules, endowing the PEG-N-CCs with stronger binding ability to bacteria.34 Thus, the result indicated that PEG modification allowed the carbon capsules not only to have a relatively high stability but also show stronger antibacterial efficiency. In addition, PEG-N-CCs also demonstrated a similar efficiency against Gram-positive S. mutans and Gram-negative P.

aeruginosa (Figure S7), supporting the conclusion that the obtained PEG-N-CCs plus NIR presented a broad-spectrum and efficient antibacterial effect.

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Figure 2. Bacteria viabilities of (a) S. aureus and (b) E. coli after being cultured with PEG-N-CCs with and without 808-nm laser

exposure. Photos of bacterial colonies of (c) S. aureus and (d) E. coli after being treated with (I) Control, (II) Control + Laser, (III) PEG-N-CCs, (IV) PEG-N-CCs + Laser. PEG-N-CCs: 100 µg/mL. Laser irradiation conditions: 2.5 W/cm2, 3 min for E. coli and 6 min for S. aureus.

To further verify the photothermal antibacterial effect of PEG-N-CCs, the 808-nm laser irradiated and non-irradiated PEG-N-CCs treated S. aureus samples were co-stained with SYTOTM 9 and propidium iodide (PI). The fluorescent images were collected by confocal microscopy. SYTOTM 9 nucleic acid stain is a green fluorescent dye, staining live and dead cells to locate bacteria.35 PI is a red fluorescent dye, only staining dead cells.36 As displayed in fluorescent images of S. aureus incubated only with PEG-N-CCs (Figure 3), a very large percentage of S. aureus remained alive. In contrast, after NIR irradiation for 6 min, a significant red fluorescence was observed in the bacterial cells, meaning that almost all bacteria were killed by the photothermal treatment. Similar results for E. coli were presented in Figure S8. Consequently, all the results indicated that the PEG-N-CCs possessed highly effective antibacterial ability with the assistance of laser irradiation.

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Figure 3. Fluorescence images of S. aureus stained with SYTOTM 9-PI. PEG-N-CCs: 100 µg/mL. Laser irradiation conditions: 2.5

W/cm2, 6 min.

We next sought to investigate the antibacterial mechanism of NIR-irradiated PEG-N-CCs. The first possibility was that PEG-N-CCs could convert NIR light to heat and directly kill the bacteria, thus we carefully evaluated the photothermal conversion efficiency of PEG-N-CCs. First, we recorded the temperature rise at different concentrations of PEG-N-CCs under the continuous exposure of NIR (Figure 4a). Ultrapure water was used as a control. When exposed to NIR light for 10 min, the PEG-N-CCs dispersions showed significant temperature increases by 38.6, 36.9, 33.6 and 28.5 °C for 250, 125, 62.5 and 31.3 µg/mL of PEG-N-CCs, respectively. On the contrary, the temperature rise in the water only was not obvious (~ 3.1 °C) under the same conditions. We then drew a curve of a temperature rise in 10 min of NIR exposure against PEG-N-CCs concentration (Figure S9a), and the result suggested a concentration-dependent photothermal profile for the PEG-N-CCs. In addition, PEG-N-CCs dispersion at 100 µg/mL was also irradiated by the NIR at different power densities, and the results indicated that the heat generation was also power intensity-dependent (Figure 4b). To 8


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further assess the photothermal conversion efficacy of carbon materials, PEG-N-CCs dispersion was continuously treated by the NIR laser at 2.5 W/cm2 until the temperature reached to a steady state, and then the laser was turned off for natural cooling (Figure S9b). According to the reported quantification method,37 the photothermal conversion efficacy value (η) of PEG-N-CCs was 15.2% (Figure S9c), which was consistent with a previous result for carbon materials.38 These results verified that PEG-N-CCs could convert the photo-energy into heat efficiently and speedily, showing excellent photothermal conversion ability. This features could be utilized in antibacterial studies. In order to further confirm that the heating can kill bacteria, we carried out a series of comparative tests. First, S. aureus were cultured in LB culture medium at a constant temperature (55 °C, controlled by a water bath) for 6 min. 55 °C was the same temperature as that caused by the 808-nm laser-induced photothermal test for 6 min irradiation. Compared with the control group investigated at room temperature, the external heating from the water bath did cause S. aureus death (Figure 4c), but the antibacterial effect was not as good as that of the laser group. Actually, the heat generation upon NIR irradiation varied continuously (from room temperature to 55°C in 6 min), so we next used a temperature-programmed experiment to more accurately simulate the NIR irradiation process (Figure 4c). It was observed that the external heating produced through the temperature-programmed route did not cause notable death of S. aureus cells. Similar results for E. coli were displayed in Figure 4d. These results made us rethink the effect of heat from the NIR irradiation on the bacterial viability. First, the heating manner using a water bath was external, from outside to inside; the heating upon NIR irradiation was internal, concentrated on the surfaces of PEG-N-CCs. At some point, the surface temperature of PEG-N-CCs might be higher than that in the bulk solution. Considering that PEGylated N-CCs could adhere to the membranes of bacteria, this relatively higher surface temperature was beneficial to the disruption of cellular membranes, finally killing the bacteria. Second, the difference between the two heating routes also reminded us that heating was possibly not the only factor in the photothermal antibacterial therapy.

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Figure 4. (a) Temperature rise at different concentrations of PEG-N-CCs upon 808-nm laser irradiation. (b) Temperature rise of

PEG-N-CCs dispersion (100 µg/mL) upon 808-nm laser irradiation at different power densities. (c) Antibacterial activity of PEG-N-CCs on S. aureus heated in different manners. (d) Antibacterial activity of PEG-N-CCs on E. coli heated in different manners. PEG-N-CCs: 100 µg/mL. Laser irradiation conditions: 2.5 W/cm2, 3 min for E. coli and 6 min for S. aureus.

Considering that sp3 and sp2 carbon structures usually have excellent catalytic ability to produce reactive oxygen species (ROS) through catalyzing small molecule substrates (such as H2O2 and O2) in many systems,39 we conjectured that the toxicity of ROS might be another factor affecting the antibacterial ability of PEG-N-CCs during the photothemal antibacterial therapy. In order to verify our hypothesis, we first used electron spin resonance (ESR) to detect free radicals generated by PEG-N-CCs. As presented in Figure 5a, the ESR spectra in the presence of PEG-N-CCs exhibited four peaks. The peak relative intensity ratio was 1:2:2:1, verifying the generation of hydroxyl radical (•OH).40 Moreover, the PEG-N-CCs could also catalyze O2 to induce the generation of superoxide radicals (O2−•) (Figure 5b), and the signal captured by BMPO exhibited multiplets.41 After NIR irradiation, we observed that the BMPO/•OH and BMPO/ O2−• signals were both strongly increased, meaning the generation of a large number of free radicals enhanced by NIR irradiation. The same conclusion was also verified by spectroscopy experiments. Terephthalic acid (TA) is a fluorescence probe, which can trap •OH to produce 2-hydroxy terephthalic acid (TAOH) and emit fluorescence.42 As show in Figure S10a, compared with the control experiments, the peak intensity increase in the mixed dispersion of PEG-N-CCs and TA with NIR irradiation indicated the •OH generation, and the higher intensity of peak meant the more hydroxyl radicals generation. The level

of

O2 −•

was

tested

by

the

UV-vis

absorption

spectroscopy

of

2,3-bis(2-methoxy-4-nitro-

5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) cultured with PEG-N-CCs.43 Figure S10b 10


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confirmed that the PEG-N-CCs plus NIR irradiation generated a higher level of O2−•. These results illuminated that PEG-N-CCs could produce ROS and this ability could be enhanced by NIR irradiation. Considering the reasons, we attributed this remarkable change of ROS levels upon light exposure to the structure of the carbon materials. PEG-N-CCs have sp3 and sp2 hybridized carbon atoms, the unique chemical and electronic features of carbon structure exhibit the excellent ability to catalyze small molecule substrates to produce ROS,44 such as promoting H2O2 to form ⋅OH. Moreover, NIR irradiation can induce the PEG-N-CCs to produce a more active surface, and in turn affect the electron transport and enhance the ability of PEG-N-CCs to accept and donate electrons, thus generating a higher level of ROS. So the combined hyperthermia-ROS can promote the antibacterial activity of PEG-N-CCs.

Figure 5. (a) ESR spectra of hydroxyl radical (OH•) induced by PEG-N-CCs with and without 808-nm laser at 2.5 W/cm2 for 3 min.

(b) ESR spectra of superoxide radical (O2−•) induced by PEG-N-CCs with and without 808-nm laser at 2.5 W/cm2 for3 min. (c) Fluorescence intensity of DCFH in PEG-N-CCs dispersion with and without GSH when the laser irradiation was off. (d) Fluorescence intensity of DCFH in PEG-N-CCs dispersion with and without GSH with laser irradiation (2.5 W/cm2, 6 min). PEG-N-CCs: 100 µg/mL.

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Figure 6. (a) Fluorescence intensity of DCFH in S. aureus cells in different conditions (PEG-N-CCs: 100 µg/mL. Laser irradiation

conditions: 808-nm, 2.5 W/cm2. (b) The loss of GSH in S. aureus cells in different conditions, as examined with a commercial kit (PEG-N-CCs: 100 µg/mL. Laser irradiation conditions: 808-nm, 2.5 W/cm2, 6 min). The antioxidant, hypotaurine (7%), inhibited antibacterial activity towards S. aureus (c) and E. coli (d) in the presence of PEG-N-CCs (100 µg/mL) plus NIR irradiation (808-nm, 2.5 W/cm2, 6 min for S. aureus and 3 min for E. coli).

Also, it is generally accepted that glutathione (GSH) is crucial in the bacterial antioxidant defense system. Overexpression of GSH diminishes the amount of generated ROS and greatly reduces the efficiency of antibacterial therapy.45 We then evaluated the effect of GSH on ROS generation in the PEG-N-CCs plus NIR system in vivo. DCFH was employed as a probe to evaluate ROS levels. As shown in Figure 5c, when the NIR light was off, the ROS levels in the PEG-N-CCs dispersion were relatively low and the fluorescence intensity of DCFH showed almost no observable change regardless of GSH. When the NIR light was on (Figure 5d), a large number of ROS were produced, which was consistent with the ESR results. However, the ROS levels were decreased in the presence of GSH, confirming that GSH could deplete the ROS generation, as also verified by other research groups.46 It was worth mentioning that we tested the GSH contents before and after NIR irradiation (Figure S11a). It was found that the loss of GSH in the PEG-N-CCs dispersion (100 µg/mL) reached 16% after NIR irradiation for 6 min, which was higher than the corresponding data obtained at a constant temperature of 55 °C in the water bath group. Moreover, the temperature-dependent and time-dependent GSH losses were also measured to evaluate the

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effect of heat (Figure S11b). When the PEG-N-CCs dispersion was kept at 55°C, the loss ratios of GSH reached 9.98 % and 28.78 % in 1 h and 3 h, respectively. However, the loss ratio of GSH obtained at 20°C was much lower than the data measured at 55°C. This result agreed with the fact that organic thiols (R-SH) are catalytic oxidized in a temperature-dependent manner.47 Thus, the loss of GSH might contribute to the stable existence of ROS to kill bacterial strains. Encouraged by the observed production of ROS in vitro, we next investigated ROS levels in bacterial cells. As shown in Figure 6a, PEG-N-CCs induced the generation of intracellular ROS in S. aureus. We noted that PEG-N-CCs plus NIR irradiation could increase the intracellular ROS level obviously. These relatively high ROS levels were crucial for the carbon-based materials to exhibit an effective bactericidal effect. Meanwhile, GSH PEG-N-CCs plus NIR group reached 21%. These results indicated that PEG-N-CCs could oxidize R-SH and NIR irradiation could accelerate the reaction.48 This finding in bacterial cells implied that the GSH oxidation could be enhanced by NIR irradiation upon PEG-N-CCs dispersion, which could explain the rapid death of bacterial strains caused by NIR-induced ROS-hyperthermia. In order to further verify the NIR-triggered ROS generation, we studied the impacts on antibacterial activity of an antioxidant reagent, hypotaurine, which specifically scavenges hydroxyl radicals.49 Figure 6c and 6d showed the antibacterial activity of PEG-N-CCs upon NIR irradiation in the presence of hypotaurine (6%). We observed that the antibacterial activity was lessened after adding hypotaurine, both in S. aureus cells and E. coli cells. These data suggested that the antibacterial activity of PEG-N-CCs was related to the ROS level produced by PEG-N-CCs upon NIR irradiation. Moreover, the GSH oxidation was also accelerated by the laser irradiation, which would potently destruct the redox balance within bacteria and finally result in the bacterial death rapidly.

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Figure 7. (a) Typical SEM images of E. coli and S. aureus cultured with PEG-N-CCs (100 µg/mL) with and without 808-nm laser

irradiation (2.5W/cm2, 3 min for E. coli and 6 min for S. aureus). (b) Cleavage of genomic DNA of E. Coli and S. aureus by PEG-N-CCs (100 µg/mL) plus NIR irradiation. 1-DNA marker; 2-control with buffer only; 3-PEG-N-CCs; 4-PEG-N-CCs under NIR irradiation (2.5 W/cm2, 3 min for E. coli and 6 min for S. aureus). Bacteria viabilities of drug-resistant S. aureus (BW15 (c) and BWMR 26 (d)) after incubation with PEG-N-CCs with and without an 808-nm laser irradiation. Laser irradiation condition: 808-nm at 2.5 W/cm2 for 6 min.

To further investigate the influence of our antibacterial system on bacteria, we used SEM to observe the morphologies of E. coli and S. aureus before and after the PEG-N-CCs plus NIR irradiation. Seen from Figure 7a, the E. coli bacteria in control group showed their typical rod-like shape with intact and smooth cell walls; even after adding PEG-N-CCs, the E. coli bacteria did not show observable membrane damage or cell death. However, most E. coli cells suffered from pronounced membrane damage in the presence of 100 µg/mL PEG-N-CCs plus NIR irradiation, since ROS could oxidize and destruct the membranes of the bacteria, finally causing the death of bacteria. For S. aureus, the SEM results had similarities to those of E. coli. The untreated S. aureus cells were smooth with spherical shapes; in the presence of PEG-N-CCs plus NIR irradiation, S. aureus cells became notably 14


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rough and wrinkled. Although some bacterial cells still kept integrity of their membranes, they were deformed. Thus, these results suggested that in the presence of PEG-N-CCs plus NIR irradiation, an enormous amount of heat and high levels of ROS together caused the damage to bacterial membranes, and the leakage of the intracellular matrix, ultimately leading to the destruction of bacteria. With the deformation and rupture of bacteria, complex mixtures of cellular components (such as proteins and nucleic acids) could release. These released components can accumulate around bacteria, contributing to the formation of biofilms to resist antimicrobial treatments.50 To investigate whether the PEG-N-CCs would degrade this released biomass, we studied the effect of PEG-N-CCs plus NIR irradiation on the collected released nucleic acids. Figure 7b showed that treatment with PEG-N-CCs plus NIR could exactly degrade the released DNA. So, our antibacterial strategy could not only kill bacterial cells efficiently, but also was successful at the degradation of the released-biomass from the cells. Moreover, this combined hyperthermia-ROS therapeutic strategy is also effective for drug-resistant bacteria (Figure 7c, 7d and Table S1). Therefore, the NIR-irradiated PEG-N-CCs were highly efficient for killing bacteria. Since our PEG-N-CCs can degrade the genomic DNA of bacteria, we wondered if the PEG-N-CCs could exhibit the ability to cleave biofilms. So, the effect of PEG-N-CCs plus NIR irradiation on S. aureus biofilm formation and destruction was investigated. It is commonly accepted that the protection provided by the biofilm growth mode makes it difficult to destroy the biofilm completely.51,52 Here, we quantified the biofilm mass by the crystal violet staining assay.53 In the experiments on biofilm destruction, the PEG-N-CCs plus NIR irradiation caused an obvious reduction in the amount of biofilm remaining (Figure 8a). The treatment with only PEG-N-CCs or NIR showed weaker biofilm destruction. In addition (Figure 8b), when using the PEG-N-CCs and NIR irradiation to co-treat the biofilm media, the S. aureus biofilm formation was also inhibited strongly, consistent with the biofilm mass (Figure 8c, 8d) and the bacterial number in biofilms (Figure 8e). Thus, we concluded that PEG-N-CCs could be applied as a ROS generator and an efficient photothemal agent to destroy and inhibit the biofilm formation.

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Figure 8. The effect of the PEG-N-CCs plus NIR irradiation on S. aureus biofilm destruction. (a) The remaining biofilms measured by

crystal violet staining. (b) The generated biofilms measured by crystal violet staining. (c) Schematic of biofilm formation on hydroxyapatite (HA) discs and the NIR irradiation process. (d) Biomass (dry-weight) reduction and (e) antibacterial activity as determined by total viable cell counting. PEG-N-CCs: 100 µg/mL. Laser irradiation conditions: 808-nm at 2.5 W/cm2 for 10 min.

Taken together, the results show that our prepared PEG-N-CCs have efficient photothermal conversion performance and can boost ROS generation upon laser exposure. Although the PEG-N-CCs have no sharp edges, using higher levels of ROS in combination with hyperthermia can also achieve a satisfactory antibacterial effect. A possible antibacterial mechanism was proposed: (1) Hyperthermia of the carbon materials induced by 808-nm laser irradiation causes bacterial death rapidly. (2) High levels of ROS triggered by NIR exposure damage the bacterial membranes to cause leakage of the intracellular matrix, leading to the inactivation of bacterial cells. Meanwhile, the intracellular matrix (DNA) can be broken in response to the NIR irradiation. (3) Hyperthermia triggered by NIR 16


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laser irradiation can accelerate the catalytic oxidation of GSH and potently destruct the redox balance within bacteria, finally leading to the bacterial death rapidly. It is worth noting that the N-doping also plays important role in antibacterial activity of carbon-based material. As reported in our previous studies,54 the N-doped carbon spheres show the stronger ability to produce ROS, compared with non N-doped carbon spheres. In order to further confirm the importance of N doping, we compared the photothermal conversion efficiency, ROS generation and antibacterial activity of these two carbon capsules with or without N doping. The non N-doped carbon capsule was provided by Professor Chen from our college.55 As show in Figure S12, compared with PEG-N-CCs, non N-doped carbon capsule (PEG-CCs) exhibited the similar temperature rising curve under the NIR irradiation. However, the ROS level was much lower than that of PEG-N-CCs. This different efficiency of ROS generation led to the differences in antibacterial performance. Moreover, how to precisely control ROS generation specifically in lesion site but not in normal cells, and how to translate the new ROS generation approaches are the important questions needed to be solved. In our work, NIR irradiation can be used to spatially and temporally control ROS generation by allowing light focusing on the lesion tissue, which may improve the therapeutic efficacy and avoid the potential toxicity. Thus, the investigation of antibacterial mechanism of light-irradiated carbon materials is a fundamental work in the construction of the new efficient antibacterial strategy for biomedical engineering and chemical industries.

Conclusion We designed and tested a simple and effective platform, PEG-N-CCs, for antibacterial applications. Our studies demonstrate that the NIR-irradiated PEG-N-CCs can stimulate the efficient antibacterial performance towards a broad-spectrum of bacteria, including multi-drug resistant strains. Moreover, the PEG-N-CCs antibacterial system also possesses the abilities on scavenging the existing biofilms and inhibiting the formation of new biofilms. Considering these results, the NIR-irradiated PEG-N-CCs can not only generate an enormous amount of heat but also produce high level of ROS through the contact with bacteria, which disrupt the cellular redox balance and damage cellular components. Our studies provided strong evidence that PEG-N-CCs could be applied as an efficient antibacterial agent.

Experimental Methods Materials. Crystal violet and hydrogen peroxide (H2O2) and were bought from Aladdin Chemistry Co., Ltd. 17



(Shanghai, China). Propidium iodide (PI), 2, 7-Dichlorofuorescein diacetate (DCFH-DA) and glutathione (GSH) assay kit were obtained from Beyotime Biotechnology (Shanghai, China). SYTOTM 9 green fluorescent nucleic acid stain was ordered from Thermo Fisher Scientific (Waltham, Massachusetts, USA). 5, 5’-Dithiobis (2-nitrobenzoic acid) (DTNB) and dopamine hydrochloride (DA) were obtained from Adamas Reagent Co., Ltd (Shanghai, China). D-α-Tocopherol polyethylene glycol 1000 succinate and hypotaurine were ordered from Sigma-Aldrich (St. Louis, Missouri, USA). BMPO (3,4-dihydro-2-methyl-1,1-dimethylethyl ester-2H-pyrrole-2-carboxylic acid-1-oxide) was purchased from DOJINDO (Shanghai, China). The bacterial strains were got from Yangzhou University (Yangzhou, Jiangsu, China).

Synthesis of PEG-N-CCs. N-doped carbon capsules (N-CCs) were synthesized through a simple and convenient route. Briefly, 100 mg SiO2 nanoparticles (average diameter 150 nm) and 100 mg DA were dissolved in Tris-HCl solution (30 mL, 50 mM, pH = 8.8), and stirred for 36 h in dark conditions. After centrifugation and washing, the product was dried at 45 °C. Then the obtained powder was carbonized at 800 °C for 2 hours and then immersed in NaOH (1.0 mol/L) at 80 °C to remove the SiO2 template. Finally, the N-CCs powder was obtained after drying at 45 °C overnight. For modification with PEG, 200 µL N-CCs (5.0 mg/mL) and 200 µL D-α-Tocopherol polyethylene glycol 1000 succinate (5.0 mg/mL) were mixed, and then treated with ultrasound for 1 h. After that, the dispersion was harvested for further use.

Antibacterial Effect of PEG-N-Ccs plus NIR Irradiation. The antibacterial activity of PEG-N-CCs was determined through the number of colony-forming units (CFU) using plate counting method. Four groups of S. aureus or E. coli were tested: (I) bacteria; (II) bacteria + Laser; (III) bacteria + PEG-N-CCs; (IV) bacteria + PEG-N-CCs + Laser. Briefly, the bacteria (OD600=0.7) were diluted 10 times and 100 µL of the above diluted bacteria were added into four 1.5 mL centrifuge tubes, respectively. The final concentration of PEG-N-CCs was 100 µg/mL and bacteria were 1.0×107 CFU/mL. After incubation with PEG-N-CCs for 30 min, the bacterial dispersions (100 µL) of group I and III were diluted to 1×103 CFU/mL, and spread on agar culture plates to culture at 37 °C for another 15 h. For the groups II and group IV, S. aureus, after incubating with PEG-N-CCs for 24 min, were exposed to 808-nm laser for another 6 min at 2.5 W/cm2; E. coli, after incubating with PEG-N-CCs for 27 min, were exposed to 808-nm laser for another 3 min at 2.5 W/cm2. The process of the antibacterial experiments exposing PEG-N-CCs to Pseudomonas aeruginosa (P. aeruginosa) and Streptococcus mutans (S. mutans) was similar to the above procedure. 18


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Confocal Fluorescence Assay. After incubating with PEG-N-CCs plus NIR, E. coli and S. aureus cells were stained with PI for 10 min, followed by SYTOTM 9 counterstaining for 5 min. At last, the bacteria were visualized with confocal fluorescence microscope (Leica, TCS SP8 STED, Germany).

GSH Detection. Ellman’s assay: Ellman’s assay was used to examine the Glutathione (GSH) oxidation.56 The disulfide bonds (-S-S-) in Ellman reagent (DTNB) were cleaved through the interaction with the thiol groups in GSH, forming a yellow product. In brief, 100 µg/mL PEG-N-CCs Tris-HCl solution was mixed with 0.8 mM GSH Tris-HCl solution in equal volumes (total volume: 450 µL). The above mixed sample was incubated at 20 °C and 55 °C for 1, 2 and 3 h, respectively. After that, 650 µL Tris-HCl solution and 10 mM DTNB disodium phosphate solution (150 µL, pH = 7.0) was added into the mixture. After a given time, PEG-N-CCs were harvested and record the UV-vis absorbance at 410 nm (Multiskan MK3, Thermo Scientific, USA). The GSH oxidation induced by NIR irradiation was also tested by Ellman’s assay as described above. Before centrifugation, the tube containing GSH and PEG-N-CCs was irradiated by NIR for 30 min intermittently to maintain a constant temperature at 55°C. For the sake of comparison, the GSH oxidation was also carried out at 55 °C and room temperature for 30 min. Determination of intracellular GSH levels: S. aureus suspension was first added into a 1.5 mL tube at a density of 1.0×107 CFU/mL. After being incubated with PEG-N-CCs for 24 min and irradiated with 808-nm laser for another 6 min, S. aureus was collected and mixed with 30 µL protein removal agent solution. After being frozen and thawed twice using liquid nitrogen and water (37 °C) alternately, the samples were harvested for GSH assay according to the manufacturer’s protocol.

ROS Detection. Electron spin resonance (ESR) measurement: The glass capillary tubes containing the irradiated PEG-N-CCs (100 µg/mL, 50 µL) were inserted into the ESR cavity to record ESR signals at selected times. BMPO as the spin trap was utilized to verify the generation of ROS (hydroxyl radical, •OH and superoxide radical, O2−•). PEG-N-CCs without NIR exposure were used as a control group. Intracellular ROS generation analysis: DCFH-DA dye was applied to test the intracellular ROS concentration.57 Briefly, 1.0 mL of stationary growth phase bacterial solution was mixed with 10 µM DCFH-DA. After being cultured at 37 °C for 20 min, the bacteria were centrifuged and re-suspended into four groups: (I) Control, (II) Control + Laser (808-nm, 2.5 W/cm2, 6 min), (III) PEG-N-CCs (100 µg/mL) and (IV) PEG-N-CCs (100 µg/mL) + Laser (808-nm, 2.5 W/cm2, 6 min). Then, the fluorescence intensity of DCFH-DA observed at excitation/emission wavelength of 488/525 nm was recorded to directly reflect the amount of ROS generation. 19



Morphology Observation of Bacteria. Four typical groups of the bacterial dispersions (I) Control, (II) Control + Laser, (III) PEG-N-CCs and (IV) PEG-N-CCs + Laser were harvested and fixed with 2.5 % glutaraldehyde. Then, the cells were further dehydrated by a series of ethanol/water mixtures, and dried by CO2 supercritical drying. Finally, the obtained dried bacteria were observed using scanning electron microscopy (SEM, S-4800II, Hitachi, Japan) observation.

Cleavage of Nucleic Acids. E. coli and S. aureus genome DNA were first extracted with a bacteria genome DNA kit. The degradation of DNA, identified with agarose gel electrophoresis and gel stain assays, were conducted in PEG-N-CCs (100 µg/mL) system plus NIR exposure (808-nm at 2.5 W/cm2, 3 min for E. coli and 6 min for S. aureus). The group of PEG-N-CCs without NIR exposure was also investigated.

Antibiofilm Experiments. The destruction of S. aureus biofilms: The biofilms formed by S. aureus were first incubated with 100 µg/mL PEG-N-CCs for 2 h in LB medium, then the PEG-N-CCs + Laser group was exposed to the 808-nm laser at 2.5 W/cm2 for 10 min/well. The crystal violet stain assay was employed to evaluate the extent of biofilm destruction. Briefly, the amount of remaining biofilm was quantified by adding 33% glacial acetic acid to measure the UV-vis absorbance of crystal violet at 590 nm. S. aureus biofilms without any treatment were used as a control group. The inhibition of formation of S. aureus biofilms: In the process of S. aureus biofilms formation, PEG-N-CCs (100 µg/mL) were added into LB medium containing S. aureus in 96-well microtiter plates, and the group of PEG-N-CCs + laser was further exposed to the 808-nm laser at 2.5 W/cm2 for 10 min. The generation of biofilms was also evaluated by crystal violet staining assay. Wells containing the same concentration of S. aureus without any other treatment were measured as control groups.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Characterizations, bacterial culture, biofilm formation of S. aureus, detection of biofilms mass, calculation of the photothermal conversion efficiency (η) of PEG-N-CCs; Figures S1-S12 and Table S1 (PDF)

Author Information Corresponding Authors *E-mail [email protected] (J.X.). *E-mail [email protected] (L.F). 20


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*E-mail [email protected] (L.G.).

ORCID Juqun Xi: 0000-0003-2622-0151 Lei Fan: 0000-0003-0049-5819

Notes The authors declare no competing financial interest.

Acknowledgments

This project was funded by the National Natural Science Foundation of China (No. 81671810 and 21703198), the Social Development Project of Yangzhou City (YZ2016074), the University Natural Science Foundation of Jiangsu Province (16KJD150004) and the Students’ Technology Innovation Fund of Yangzhou University (X20166779). The authors also gratefully acknowledge financial support from the Top-notch Academic Programs Project of Jiangsu Higher Education Institutions and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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TOC

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Mechanistic Insight into the Light-Irradiated Carbon Capsules as an

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