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Degradable Carbon Dots from Cigarette Smoking with BroadSpectrum Antimicrobial Activities against Drug-Resistant Bacteria Yuxiang Song,† Fang Lu,‡ Hao Li,† Huibo Wang,† Mengling Zhang,† Yang Liu,*,† and Zhenhui Kang*,† †
Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Institute of Functional Nano & Soft Materials (FUNSOM), Soochow University, 199 Ren’ai Road, Suzhou 215123, Jiangsu P. R. China ‡ School of Basic Medical Sciences, Beijing University of Chinese Medicine, Beijing 100029, China
ACS Appl. Bio Mater. Downloaded from pubs.acs.org by UNIV OF WINNIPEG on 11/29/18. For personal use only.
S Supporting Information *
ABSTRACT: Infection of pathogenic bacteria and the abuse of antibiotics are the main factors in worldwide health issues. A number of nanomaterials have been fabricated to directly battle drug-resistant bacteria. However, few studies have referred to the relation between common daily carbon nanoparticles (e.g., automobile exhaust and cigarette smoke) and antimicrobial activities. Herein, carbon dots (CDs) extracted from cigarette smoke are introduced, showing low in vivo and in vitro toxicity. These CDs show broadspectrum antimicrobial activities that originate from the destruction of the double helix structure of DNA. CDs can degrade to smaller particles and organic fragments with the existence of horseradish peroxidase (HRP) and H2O2 in 7 days, during which CDs can function as an effective antibiotic within the early days and then gradually degrade and lose the antimicrobial effects until finally being eliminated through metabolism. Our findings relate common daily carbon nanoparticles from cigarette smoke with antimicrobial activities, suggesting that the CDs can function as an effective broadspectrum antibiotic, even against drug-resistant bacteria. KEYWORDS: carbon dots, cigarette smoking, antimicrobial activities, degradable, low toxicity
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INTRODUCTION Pathogenic bacteria, especially some drug-resistant strains, are responsible for the severe threat to food safety and human health.1−4 Because of the widespread abuse of antibiotics, the emergence of drug-resistance generation in bacteria is increasingly highlighted.5,6 Every year in the U.S., more than 2 million infections and 23 000 deaths are caused by drugresistant bacteria, based on the data from the U.S. Centers for Disease Control and Prevention.7 Thus, the urgent demand for developing novel and effective antibiotics has become a huge challenge. Recently, a number of materials have been designed to directly battle drug-resistant bacteria or enhance the antibiotic efficacy of existed antibiotics, such as gold nanoparticles, ruthenium complexes, cationic conjugated polymers, and antimicrobial peptides.8−11 Carbon dots (CDs) have been an emerging nanomaterial in recent years that can be widely applied in bioimaging, biosensing, nanomedicine, drug delivery, cancer therapy, etc.12−20 Moreover, some studies have revealed that CDs can function as an effective antibiotic, showing inhibitory effects on a broad spectrum.21−24 These materials truly gain antimicrobial activities, but all of them are artificially synthesized through chemicals. On the other hand, we can always meet carbon nanoparticles in our everyday life, for example, automobile exhaust, cigarette smoke, chimney smoke, plant ash, and carbon black ink.25−30 However, as far as we know, none of these studies referred to the relation © XXXX American Chemical Society
between common daily carbon nanoparticles and antimicrobial activities, which is more important as such a worldwide issue influences every single human being. In this work, we extracted a novel kind of CDs via cigarette smoking method for the first time. The smoke was pumped through water to form a brownish yellow solution of CDs, which exhibit nanoscale size and bright fluorescence, and most importantly, low in vitro and in vivo toxicity. More than 85% HeLa cells incubated with up to 1200 μg/mL of CDs remain alive, and the biochemical parameters of blood and tissue sections with CDs show no histological abnormity in lung, spleen, liver, kidney, and heart. In addition, CDs show antimicrobial activities to Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), ampicillin-resistant E. coli (AREC), and kanamycin-resistant E. coli (KREC) within the maximum concentration of 1000 μg/mL. The antimicrobial property originates from the destruction of the double helix structure of DNA, whereas the morphology of the bacteria can remain unbroken. Moreover, CDs can degrade to smaller particles and organic fragments with the existence of horseradish peroxidase (HRP) and H2O2 in 7 days, proved by a drop of ultraviolet− visible (UV−vis) absorbance, reduce of transmission electron Received: August 13, 2018 Accepted: November 16, 2018
A
DOI: 10.1021/acsabm.8b00421 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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ACS Applied Bio Materials
Figure 1. (a) TEM image of CDs and HR-TEM image as inset. (b) Size distribution histogram of CDs. (c) FT-IR (red curve) and UV−vis (black curve) spectra of CDs. (d) PL spectra of CDs with different excitation wavelengths from 350 to 410 nm with 20 nm as an interval, with the photograph of CDs solution under sunlight and UV light (365 nm) as inset. (e) Viability evaluation of HeLa cells incubated with different concentrations of CDs for 24 and 48 h. Synthesis of CDs. CDs were synthesized via a one-step cigarette smoking method. 200 cigarettes were ignited and connected to a gas washing bottle one by one. The smoke was pumped through the filter tips using a circulating water vacuum pump connected to the end of the gas washing bottle, and dissolve into 1000 mL deionized water to form a brownish yellow solution. (Experimental setups can be seen in Figure S1) Then a 0.22 μm filter membrane was used to remove undissolved substances and large particles in the raw solution. To remove the impurities, the solution was then dialyzed using a semipermeable membrane (MWCO 1000). The purified CDs solution was stored at 4 °C for further characterization and experiments. Characterization Methods. The TEM and HR-TEM analysis was conducted on a FEI/Philips Tecnai G2 F20 TWIN TEM. The FT-IR spectrum was characterized by a Bruker Fourier Transform Infrared Spectrometer (Hyperion). The UV−vis measurement was conducted with a PerkinElmer UV−vis spectrophotometer (Lambda 750). The PL spectra were collected using a Horiba Jobin Yvon (Fluoro Max-4) luminescence spectrometer. The XPS images were obtained on a KRATOS Axis ultra-DLD X-ray photoelectron spectrometer. The dynamic light scattering measurement was conducted to obtain the size distribution of CDs by using a
microscopy (TEM) size distribution and decrease of m/z value of matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS). During the degradation process, CDs can still function as an effective antibiotic within the early 2−4 days. Then CDs gradually degrade and lose the antimicrobial effects until finally may being eliminated through metabolism. Our findings relate common daily carbon nanoparticles from cigarette smoke with antimicrobial activities for the first time, suggesting that the CDs extracted from cigarette smoke can function as an effective broad-spectrum antibiotic, including drug-resistant bacteria, and have the potential to be applied as a neoteric therapy in clinical medicine.
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EXPERIMENTAL SECTION
Materials. All the chemical reagents were purchased from Adamas-beta and Sigma-Aldrich, and used as-is. The HeLa cell line was purchased from the Cell Bank of Chinese Academy of Science. Phage λ DNA was purchased from Toyobo Co., Ltd. B
DOI: 10.1021/acsabm.8b00421 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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Figure 2. (a) Curve of XPS survey scan of CDs. High-resolution XPS spectra of (b) C 1s, (c) N 1s, and (d) O 1s of CDs. h. After that, an initial 8 mL of 800 μM H2O2 was added into the mixed liquor to start the biodegradation reaction, followed by daily additions of 250 μL of 800 μM H2O2 for 7 days. The whole procedure was conducted under dark conditions to avoid enzyme denaturation and photolysis of H2O2. The final solution was filtered through a 0.22 μm filter membrane to remove HRP and stored at 4 °C for further test. In Vivo Cytotoxicity Analysis. To test for acute toxicity, we divided 32 Kunming mice (30 ± 3g) for tail injections into two groups of 16 each, and each group had 8 female and 8 male mice. One group of mice was injected intravenously through a single tail injection of CDs at a dose of 300 mg/kg body weight. The other group of mice was injected with 0.9% NaCl aqueous solution as a control group. After CDs treatment for 3 days, blood samples for blood chemistry tests were collected from each mouse to determine the biochemical parameters of the mice. Data were presented as mean ± standard deviation (SD). Statistical analyses were performed by one-way ANOVA. Differences with a P < 0.05 were considered significant. Meanwhile, the lung, spleen, liver, kidney, and heart were harvested and 4% of paraformaldehyde was prepared to fix the tissues for another 24 h before they were dehydrated in elevated concentrations of ethanol and embedded in paraffin. Five micrometer sections were applied for H&E staining to evaluate potential edema or inflammation caused by CDs.
ZEN3690 zetasizer (Malvern, U.K.). The CLSM images were acquired with a laser-scanning confocal fluorescence microscope (Leica, TCS-SP5). The SEM images were analyzed to examine the bacterial morphology with and without the treatment of CDs by using a Zeiss Supra 55 SEM. The circular dichroism spectra were collected on a JASCO J-815 spectropolarimeter. MALDI-TOF MS experiments were performed on a Bruker Ultraflextreme MALDI TOF/TOF mass spectrometer (Bruker Daltonics, Inc., Billarica, MA) with an Nd:YAG laser (355 nm, the laser spot size of 50−100 μm, 2000 kHz), using a software package of Bruker DataAnalysis 3.3. Bacteria Culture. E. coli, S. aureus, AREC, and KREC were all shaking cultured at 180 rpm in aqueous lysogeny broth (LB) medium at 37 °C. The obtained CDs solution was diluted to a series of known concentrations and added into the raw bacteria solution, while the control groups were free of CDs. Then the CDs-bacteria solution was cocultured and all the bacteria were obtained simultaneously with optical density at 600 nm (OD600) of control groups being 0.6−0.8. The bacteria coated plates were acquired by painting the bacteria solution obtained as above on concretionary LB medium and cultured at 37 °C. In Vitro Cytotoxicity Analysis. The HeLa cell line was cultured in the standard medium in 5% CO2 at 37 °C. Two separate groups of cells were incubated in a 96-well plate, and then a series of diluted solutions of CDs with known concentrations were added to them. After 24 and 48 h, the relative viabilities of the cells were determined using colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays to assess the metabolic activity of the cells. The cells were lysed with acidulated sodium dodecyl sulfate (SDS), and a microplate reader (Bio-Rad 680, USA) was used to measure the absorbance at 570 nm. At least three independent experiments were carried out to guarantee the accuracy of the data. Phage λ DNA Treatment. Two equal parts of DNA were diluted with 0.01 M PBS and 1000 μg/mL CDs solution to the same concentration. The two parts were then heated in 95 °C water bath for 10 min, followed by shaking at 25 °C for 1−2 h. Phage λ DNA was stored at 4 °C before circular dichroism spectrum analysis. Biodegradation Procedure. Four milliliters of 0.385 mg/mL HRP and 4 mL of 1000 μg/mL CDs were incubated together for 24
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RESULTS AND DISCUSSION Characterization of CDs. CDs were first characterized with a series of methods. Figure 1a shows the TEM image as well as the inset high resolution TEM (HR-TEM) image of CDs, revealing that CDs disperse uniformly at nanoscale. The crystal lattice spacing is 0.21 nm according to the inset, demonstrating the (100) lattice plane of graphitic carbon.31 Figure 1b exhibits the size distribution histogram of CDs, from which we can observe that the diameter of CDs distributes from 4.5 to 7 nm, with the weighted average of diameter as 5.4 nm. The Fourier transform infrared (FT-IR) and the UV−vis C
DOI: 10.1021/acsabm.8b00421 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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multiple drug-resistant bacteria were researched in the following experiments. E. coli (Gram-negative bacteria) and S. aureus (Gram-positive bacteria) were first examined to evaluate the antimicrobial activities of CDs. Serial dilutions of CDs solution (200, 400, 600, 800, and 1000 μg/mL corresponding to in vitro cytotoxicity test) were added into the bacterial solution and cultured following the procedure shown in the Methods section. At 1000 μg/mL, CDs were able to completely inhibit the proliferation of E. coli, whereas a dramatic decrease in the number of bacterial colonies of S. aureus can also be observed, demonstrating that CDs own antimicrobial activities against both Gram-negative and Grampositive bacteria without selectivity. Next, the antimicrobial activities of CDs were further tested by using multiple drugresistant E. coli (AREC and KREC). CDs were diluted to the same concentrations as above, and at 1000 μg/mL excellent antimicrobial effects of CDs could be observed on both strains of the drug-resistant bacteria. The photographs of the coated plates of E. coli, S. aureus, AREC, and KREC in different concentrations of CDs solution are shown in Figure 3a. As the
spectra of CDs are presented in Figure 1c, where the red curve represents the FT-IR spectrum and the black curve represents the UV−vis spectrum. From the FT-IR spectrum, the broad adsorption peak from 3700 to 3030 cm−1 can be assigned to the stretching vibrations of − OH and − NH group, while the peak at 2978 cm−1 can be attributed to the stretching vibrations of −CH2− group. The adsorption peaks of the stretching vibrations of C = O, C−N, and C−OH groups can be observed at 1630, 1397, and 1234 cm−1, respectively. The peak located at 1049 cm−1 corresponds to the asymmetric and symmetric stretching vibrations of C−O−C group.32,33 From the UV−vis spectrum, two adsorption peaks located at ca. 220 nm and ca. 260 nm can be observed, which represent the π−π* transition and n−π* transition of carbonyl/amine functional groups.34 Moreover, strong photoluminescence (PL) can be visually observed under the excitation from 320 to 420 nm (Figure S2). As shown in Figure 1d, the maximum fluorescence intensity is obtained at 370 nm excitation, and a red shift of the fluorescence intensity can be seen as the excitation wavelength elevates, which can be assigned to defect luminescence.13 The inset of Figure 1d shows the photographs of the same solution of CDs under sunlight (left) and 365 nm UV light (right). The PL spectra of CDs in 0.01 M phosphate buffered saline (PBS) were also collected and displayed in Figure S3. Because CDs are extracted directly from cigarette smoke, the prior property to make clear is the cytotoxicity of CDs. Varied concentrations of CDs were added into the HeLa cells and cocultured in 96-well plate for 24 and 48 h. At the same time, a standard assay was set to evaluate the influence of CDs on the metabolic activities of HeLa cells, and the results are shown in Figure 1e. With the concentration set to be 200, 400, 600, 800, 1000, and 1200 μg/mL, more than 85% of cells can survive at all the concentrations in range, revealing the low in vitro toxicity of CDs at the concentration as high as 1200 μg/mL. As concentrations below 1000 μg/mL are considered nontoxic (more than 90% cells alive), CDs of 200, 400, 600, 800, and 1000 μg/mL will be set at the following antimicrobial experiments. Confocal laser scanning microscopy (CLSM) images of HeLa cells incubated with CDs (1000 μg/mL) at 37 °C for 3 h are displayed in Figure S4, from which it can be clearly observed that CDs can enter HeLa cells without changing the morphology of them, indicating that CDs have excellent biocompatibility. The structure and chemical components of CDs are further studied with X-ray photoelectron spectroscopy (XPS) analysis. Figure 2a depicts the XPS survey scan of CDs, from which three main elements can be found as C, N and O. The highresolution C 1s, N 1s, and O 1s XPS spectra are shown in Figure 2b−d, respectively. The partial XPS spectrum of C 1s spectrum in Figure 2b can be divided into three peaks, where the peak located at 284.5 eV corresponds well to sp2 C and the peaks at 285.9 and 287.3 eV can be ascribed to C−O/C−N and C=O/C=N bonding. The high-resolution N 1s in Figure 2c is divided into two peaks at 399.3 and 400.1 eV, revealing the existence of C−N and C=N bonding. In the highresolution O 1s spectrum in Figure 2d, two peaks located at 530.5 and 532.1 eV can be attributed to C−O and C=O bonding.35,36 To sum up, the obtained CDs have graphite structure cores with multiple N and O containing functional groups on the surfaces, such as amino groups, carbonyl groups, and hydroxyl groups. Antimicrobial Activities of CDs. The antimicrobial activities of CDs on typical pathogenic bacteria along with
Figure 3. (a) Photographs of bacterial colonies of E. coli, S. aureus, AREC, and KREC at elevating concentrations of CDs. (b) Calculated alive ratio of E. coli, S. aureus, AREC, and KREC at corresponding concentrations of CDs.
concentration of CDs increases, the number of bacterial colonies of all strains/species visually decreases. CDs are capable to reach completely inhibitory effects on E. coli and KREC at 1000 μg/mL, whereas the number of bacterial colonies of S. aureus and AREC is the minimum at the same concentration (Completely inhibitory effects can be reached at 1200 μg/mL of CDs, Figure S5). We next tested the alive ratio of all four strains/species by measuring the OD600 value at different concentrations of CDs and then divided by the value of control group. The histogram graph in Figure 3b depicts the same phenomenon as the above D
DOI: 10.1021/acsabm.8b00421 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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Figure 4. (a) Circular dichroism spectrum of DNA with and without CDs treatment. (b) Curves of UV−vis spectra of the CDs before and after biodegradation along with the photographs at corresponding periods as inset. (c) TEM image and size distribution of CDs after biodegradation. (d) MALDI-TOF MS spectra of CDs before and after biodegradation. (e) Antimicrobial activities on E. coli, S. aureus, AREC, and KREC with CDs at different degradation periods.
photographs show. The alive ratio at 0 μg/mL of CDs (control group) was normalized to 100%, and the alive ratio at each concentration was determined by dividing the corresponding OD600 value by that of control group. As the concentration of CDs increases, the alive ratio of all strains/species drops continuously, and then reaches the minimum at 1000 μg/mL. The lowest alive ratio is 1.69% for E. coli, 13.5% for S. aureus, 7.55% for AREC, and 0.48% for KREC. We also tested the antimicrobial activities of CDs at completely inhibitory concentrations for E. coli, S. aureus, AREC and KREC in various time. The results are shown in Figure S6, from which we can observe that the alive ratio of all four kinds of bacteria remain under 5% for up to 48 h, indicating that the antimicrobial effects of CDs are not temporary. To fully
demonstrate the antimicrobial activities of CDs, more species of bacteria are used to test their alive ratio with CDs. Proteusbacillus vulgaris (P. vulgaris, Gram-negative), Bacillus subtilis (B. subtilis, Gram-positive) and Pseudomonas aeruginosa (P. aeruginosa, Gram-negative) are treated the same way as above and the test results are displayed in Figure S7, in which we can come to the conclusion that CDs are still effective. Moreover, a comparison between CDs and clinically useful ampicillin was made by measuring the alive ratio of E. coli and AREC in ampicillin shown in Figure S8. Although ampicillin is more effective at low concentrations on E. coli, the bacteria gain drug resistance to clinically useful antibiotics still in existence, proven by the high alive ratio of AREC in ampicillin. However, according to the above results, CDs can completely E
DOI: 10.1021/acsabm.8b00421 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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electrostatic attraction. Thanks to the nanoscale size, CDs are capable of diffusing into the bacteria after adsorption. Then, because the bacterial DNA is naked, CDs can make the double helix structure of DNA loose, and finally inhibit the proliferation of the bacteria. The ζ potential of the other four kinds of CDs derived from camphor tree leaves, mulberry leaves, lalang grass rhizome, and schizonepeta tenuifolia was also measured, and the values are −11.4, −7.64, −9.05, and −1.40 mV, respectively (Figure S17), which demonstrates that the ζ potential of CDs is a factor contributing to the antimicrobial activities. Biodegradation Study of CDs. Whether the CDs can degrade is a key factor to evaluate their biological properties.39 Because hydrogen peroxide can be generated by cell activities as well as peroxidase can be found in a wide variety of organisms,40−42 biodegradation of CDs under these circumstances will reduce their toxicity and side effects after they function as antibiotics. For this reason, we tested the biodegradation ability of CDs according to the reported method.43,44 Four milliliters of 1000 μg/mL CDs were incubated with 4 mL of 0.385 mg/mL HRP for 1 day, followed by an initial 8 mL of 800 μM H2O2 and daily additions of 250 μL of 800 μM H2O2 for a week. UV−vis, TEM, size distribution and MALDI-TOF MS analysis were carried out to characterize the biodegradation of CDs. Figure 4b depicts the UV−vis spectra of CDs before and after biodegradation. A dramatic decrease can be observed, indicating that the concentration of CDs decreases during the biodegradation process, which is further proved by a visual decrease in the color of CDs solution from the inset. TEM image of CDs after biodegradation is exhibited in Figure 4c, as well as corresponding size distribution histogram. Compared with original CDs, the biodegradation process makes CDs visually smaller in size, and the weighted average of diameter reduces from 5.4 to 1.8 nm. From the MALDI-TOF MS spectra in Figure 4d, multiple additional peaks can be found at m/z value from 0 to 150, indicating a variety of organic fragments. These lower m/z peaks along with the lack of signals over m/z 300 prove that CDs can reach complete degradation after some time. In addition, the stability of CDs was tested under 37 °C and 2.84 mW/cm2 visible light radiation. The results in Figure S18 indicate that CDs are stable at 37 °C and change within 15% under 2.84 mW/cm2 visible light radiation, for which the stability is not a main factor for the degradation of CDs. In summary, CDs can degrade to smaller particles and organic fragments within 1 week, because of the homolytic cleavage of H2O2 with HRP to produce free radicals that oxidize the CDs.44 Further, the antimicrobial activities of CDs at different degradation periods are tested on the same strains/species of bacteria as experiments above. Figure 4e presents the alive ratio of E. coli, S. aureus, AREC, and KREC incubated with 2day, 4-day, and 6-day degradation of original 1000 μg/mL CDs. Despite that the concentration of CDs reduces during degradation process, CDs can still function as an effective antibiotic at preliminary days. After that, CDs gradually degrade to smaller particles and organic fragments and lose the antibiotic effects until finally may being eliminated through metabolism.45 To sum up, the process of CDs inhibiting bacterial growth can be explained as three steps: (i) CDs adsorb on the surface of the bacteria due to electrostatic attraction; (ii) CDs diffuse into the bacteria as they are in nanoscale size; (iii) CDs make
inhibit the growth of both kinds of bacteria, which is an advantage compared with clinically useful antibiotics. CDs are capable of suppressing the growth of both Grampositive and Gram-negative bacteria, as well as some strains of drug-resistant bacteria at 1000 μg/mL. However, with the same concentration CDs barely show any cytotoxicity to HeLa cells, which can be explained as the following two factors: (i) the DNA of cells is covered by karyotheca while the DNA of bacteria is naked, so it is more difficult for CDs to affect the DNA of cells; (ii) bacteria are much smaller than cells and lack organelles, so bacteria are more susceptible than cells with the same concentration of CDs. In comparison, some other common plants (camphor tree leaves, mulberry leaves, lalang grass rhizome, and schizonepeta tenuifolia) were also dried and treated by a similar method as cigarette smoking. The obtained four smoke-water solutions were filtered and dialyzed as the cigarette smoking CDs and their basic characterization (TEM, UV−vis and FT-IR) were displayed in Figure S9−S11. Then, the antimicrobial activities of the four kinds of CDs were also tested using E. coli, S. aureus, AREC, and KREC. The alive ratios of these bacteria were determined by UV−vis spectrophotometer, and the results are displayed in Figure S12. All the four kinds of CDs do not show any inhibitory effects on all species of bacteria, which could explain the uniqueness of cigarette smoking to obtain a broadspectrum antibiotic. The reasons may be explained as CDs extracted from other plants are lack of the peak at 1234 cm−1 according to FT-IR results in Figure S13, which represents C− OH stretching vibrations. CDs with rich oxygen containing groups can be easily adsorbed on the bacterial cell walls and then able to enter them through diffusion, which may lead to a better antimicrobial performance.37 All the above results suggest that CDs can suppress the growth of drug-resistant bacteria, and the mechanism of the antimicrobial activities of CDs will be studied next. Mechanism of the Antimicrobial Activities. Next, we investigated the interaction between CDs and the bacterial organelles to seek for the generation of the as-shown antimicrobial activities. First, we studied the morphology of the bacteria to determine if CDs would destroy the bacterial cell walls or membranes. Living E. coli and S. aureus were cultured with 1000 μg/mL of CDs for 6 h, and the samples were then treated for scanning electron microscopy (SEM) analysis (see methods in the Supporting Information). The images in Figure S14 demonstrate that CDs can make E. coli shorter in length, which is a sign of bacterial inhibition. Except that, CDs barely have influence on the bacterial cell walls or membranes of both E. coli and S. aureus. From the insets, it can be seen that the bacteria only shrink a little with CDs. This result is further proved by propidium iodide (PI) staining experiments, from which we can observe that PI cannot stain E. coli treated by CDs, indicating that CDs are able to pass through the bacterial cell walls and membranes without destroying them (Figure S15 and methods in Supporting Information). Next, we checked the influence of CDs on DNA, and the circular dichroism spectrum is depicted in Figure 4a, where the peak of CDs group is much lower than the one of control group as the arrow points, which reveals the fact that CDs can make the double helix structure of DNA loose. The ζ potential of CDs was further measured, and the statistic value is 4.24 mV (Figure S16). Considering the cell walls of both Gram-positive and Gram-negative bacteria possess negative charges,38 CDs can adsorb on the surface of the bacteria due to F
DOI: 10.1021/acsabm.8b00421 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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Figure 5. H&E stained tissue sections (lung, spleen, liver, kidney, and heart) of mice injected with 300 mg/kg of CDs for histological evaluation of toxicity in vivo.
degrade and lose the antimicrobial effects until finally may being eliminated through metabolism. This work for the first time relate common daily carbon nanoparticles from cigarette smoke with antimicrobial activities, revealing that a complex synthesis route using chemicals is not the only way to obtain antimicrobial CDs. Our findings suggest that the CDs extracted from cigarette smoke can function as an effective broad-spectrum antibiotic, including drug-resistant bacteria, and have the potential to be applied as a neoteric therapy in clinical medicine. Last but not the least, although the results based on the experiments provide the perspective that the CDs extracted from cigarette smoke have antimicrobial activities, this work does not mean smoking behavior is beneficial to human health.
the double helix structure of DNA loose, while at the same time keep the morphology of the bacteria unbroken. What is more, CDs can degrade to smaller particles and organic fragments with the existence of horseradish peroxidase (HRP) and H2O2 in 7 days. During the degradation process, CDs can still function as an effective antibiotic within the early 2−4 days. CDs then gradually degrade and lose the antimicrobial effects until finally being eliminated through metabolism. In Vivo Toxicity of CDs. In vivo toxicology of CDs was further studied to see if CDs can cause potential edema or inflammation, which is more important for antibiotics to be applied in living bodies.46 Kunming mice labeled as control group and CDs group were used for biochemical parameters of blood and acute toxicity tests. The biochemical parameters of blood are listed in Table S1, and the tissue sections can be found in Figure 5. CDs group shows no significant difference compared with control group, according to Table S1, with p values for all indices greater than 0.05. Thus, CDs barely influence the functions of liver and kidney after 3 days from injection. As depicted in Figure 5, compared with control group, no histological abnormity can be found in all the tissue sections from samples of the CDs group. On the basis of all the above data, the conclusion is drawn that CDs are biocompatible and have the potential for in vivo applications.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.8b00421.
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CONCLUSION In conclusion, we extracted a novel kind of CDs via the cigarette smoke method. The obtained CDs exhibit nanoscale size, bright fluorescence, and most importantly, low toxicity in vitro and in vivo. Moreover, CDs show inhibitory effects on E. coli, S. aureus, AREC, and KREC within the maximum concentration of 1000 μg/mL. The antimicrobial property originates from the destruction of double helix structure of DNA, whereas the morphology of the bacteria can remain unbroken. In comparison, four other kinds of CDs were prepared from camphor tree leaves, mulberry leaves, lalang grass rhizome and schizonepeta tenuifolia, and they were characterized to be different from the CDs from cigarette smoke. Also, they failed to inhibit bacterial growth, which could explain the uniqueness of cigarette smoke to obtain a broad-spectrum antibiotic. Moreover, CDs can degrade to smaller particles and organic fragments with the existence of horseradish peroxidase (HRP) and H2O2 in 7 days. During the degradation process, CDs can still function as an effective antibiotic within the early 2−4 days. Then CDs gradually
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Additional methods including bacterial morphology study and propidium iodide (PI) staining procedure; additional figures including experimental setups, PL spectra in water and PBS, cell imaging, completely inhibitory effects on S. aureus and AREC, antimicrobial effects on 3 more species of bacteria, characterization of CDs extracted from other plants and their antimicrobial effects, effects of CDs on bacterial cell walls, ζ potential distribution and stability test of CDs; a table of biochemical parameters of blood of mice after 3-day treatment of CDs (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (Y.L.). *E-mail:
[email protected] (Z.H.K.). ORCID
Zhenhui Kang: 0000-0001-6989-5840 Author Contributions
Y.X.S. performed experiments and wrote the manuscript. Y.X.S. and H.L. revised the manuscript. F.L. performed biochemical parameters of blood and acute toxicity tests. H.B.W. performed TEM test. M.L.Z. performed circular dichroism test. Y.L. and Z.H.K. designed, conceived, and supervised the project. G
DOI: 10.1021/acsabm.8b00421 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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ACS Applied Bio Materials Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is supported by the Collaborative Innovation Center of Suzhou Nano Science and Technology, the National Natural Science Foundation of China (51725204, 51572179, 21471106, 21771132, 21501126), the Natural Science Foundation of Jiangsu Province (BK20161216), the 111 Project, and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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DOI: 10.1021/acsabm.8b00421 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
