Characterization of qCQDs.
The size distribution of carbon nanomaterials is not only the simple physical change, but also is able to affect their antibacterial activity31. Transmission electron microscope (TEM) image showed that the average diameter of qCQDs was about 3 nm with homogeneous size distribution (Fig. 1a). High-resolution TEM (HRTEM) showed that the well-resolved lattice fringe with intercrystalline spacings of qCQDs was 0.22 nm (Fig. 1b), corresponding to the (100) facets of graphitic carbon18, 32. The particle size of qCQDs measured by dynamic light scattering (DLS) was centered at 4.5 nm with relatively concentrated distribution, suggesting the consistent and uniform morphology of qCQDs confirmed from TEM.
The optical property of qCQDs was evaluated. UV-vis absorption spectrum (Fig. 1d) of qCQDs showed π→π* transition of C=C (aromatic sp2 domains) at 225 nm and n→π* transition of C=O and C-O at 283 nm33. The maximum emission wavelength of qCQDs fluorescence increased with the red shift of excitation wavelength (Fig. 1e), suggesting the excitation-dependent luminescence behavior32. And qCQDs generated a fluorescence spectrum with the maximum emission wavelength at 455 nm under the excitation wavelength of 355 nm (red arrows in Fig. 1e). Furthermore, Fig. 1f showed the typical mirror symmetry of maximum emission spectrum (black) and the maximum excitation spectrum (red). Using quinine sulfate in 0.5 mol/L H2SO4 as standard reference34, the fluorescent quantum yield of qCQDs was calculated to be 4.61% when the excitation wavelength was at 355 nm, suggesting the acceptable luminescence property.
The molecular functional groups of qCQDs was investigated by fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR), as shown in Fig. 2. The FTIR absorption peaks of DDA at 3027 cm-1 and 2983 cm-1 and glucose at 2944 cm-1 and 2913 cm-1, were attributed to asymmetric telescopic vibration and symmetric telescopic vibration of C-H bond, respectively29. The qCQDs formed by DDA reacting with glucose still exhibited the absorption peaks of asymmetric telescopic vibration and symmetric telescopic vibration of C-H bond at 3029 cm-1 and 2933 cm-1, while the characteristic absorption peaks attributing to the aldehyde group in glucose at 2796 cm-1 and 2694 cm-1 disappeared in qCQDs, suggesting that the aldehyde group was destroyed in the synthetic process of qCQDs. The absorption peaks of qCQDs at 1646 cm-1 and DDA at 1643 cm-1 were attributed to the contraction vibration generated by the C=C double bond, indicating the new formation of C=C double bond in qCQDs or the retaining of C=C double bond of DDA in qCQDs. The absorption peaks of qCQDs at 1473 cm-1 was attributed to the shear plane bending vibration of C-H in -N+(CH3)2-, which was consistent with the absorption peak of -N+(CH3)2- in DDA at 1479 cm-1, confirming that qCQDs was functionalized with quaternary ammonium groups27. The absorption peaks at 1380 cm-1 and 1340 cm-1 caused by in-plane bending vibration of O-H single bond in glucose, and a series of absorption peak at 1295 cm-1, 1224 cm-1, 1203 cm-1 1149 cm-1 and 1110 cm-1 (yellow area in Fig. 2a) generated by the C-O-H bond in glucose, disappeared in qCQDs. The absorption peaks of symmetric telescopic vibration at 1078 cm -1 and 1035 cm-1 in qCQDs were ascribed to alkyl aromatic ether, indicating that aromatic ether structure existed in qCQDs. The absorption peaks of qCQDs at 962 cm-1 and 879 cm-1 might be generated by the telescopic vibration of C-C and C-O, respectively. NMR was further applied to investigate glucose, DDA and qCQDs using D2O as solvent27. The 1H NMR spectra in Fig. 2b revealed that the peaks of the proton signals of glucose at green area almost disappeared in qCQDs. Nevertheless, the proton peaks of C=C (6.21 and 5.89 ppm), -CH2- (4.07 ppm) and -N+(CH3)2- (3.18 ppm) in DDA remained in qCQDs with a chemical shift of about 0.13 ppm. The results of 1H NMR spectra signified that glucose dehydroxylated and deprotonated to form C=C double bonds and polymerized with DDA to obtain carbon quantum dots modified by quaternary ammonium groups. Compared with DDA, the chemical shift of the proton peaks of C=C (6.08 and 5.75 ppm), -CH2- (3.93 ppm) and -N+(CH3)2- (3.05 ppm) in qCQDs to high field may be due to the effect that the qCQDs contained more quaternary ammonium groups and formed more conjugated systems, which weakened the electronegativity in their structures and generated shielding effect, causing the proton peaks to shift toward high field35.
The percentage content, chemical state and chemical bond of elements in qCQDs were further analyzed by X-ray photoelectron spectroscopy (XPS), as shown in Fig. 2. The XPS spectrum of qCQDs (Fig. 2c) showed the three main elements, i.e. C, N and O, with the corresponding percentages of 72.45%, 6.21% and 21.23%, respectively. Fig. 2d showed that the high resolution peaks of C1s at the positions of 284.2 and 284.8 eV corresponded to C-C single bond/C=C double bond of graphene carbon32. And the peaks at the positions of 285.6, 286.1 and 286.8 eV corresponded to C-N single bond (sp3 hybrid carbon), C-O single bond (sp3 hybrid carbon) and C=O double bond, respectively36, 37. Fig. 2e showed that the peaks of N1s at 399.6 eV and 401.3 eV were attributed to C-N-C structure like pyridine type, and the peaks at 401.9 eV and 402.6 eV were assigned to the positively charged quaternary ammonium group (-N+(CH3)2-), indicating the doping effect of N element and quaternary ammonium group in qCQDs26, 38, 39. The high-resolution results of O1s (Fig. 2f) showed the peak of C=O double bond (carbonyl oxygen) at the binding energy of 531.3 eV and the peaks C-O single bond (sp2 hybrid oxygen) at the position of 531.9 eV and 532.5 eV. According to the results of TEM, FTIR, NMR and XPS, it was deduced that qCQDs contained carbon core and functional groups including quaternary ammonium group (-N+(CH3)2-), carbonyl group (C=O) and typical graphene carbon (C-C/C=C).
In vitro antibacterial activity of qCQDs.
The antibacterial activity of qCQDs on different species of bacteria was investigated (Fig. 3). Disk a, b and c on MHA plates contained 0.2 mg of qCQDs, 0.2 mg of glucose and 0.2 mg of DDA, respectively. Four species of gram-positive bacteria (S. aureus, MRSA, S. epidermidis and E. faecalis) and two species of gram-negative bacteria (E. coli and P. aeruginosa) were used as model bacteria to evaluate the antibacterial activity of qCQDs. Obvious inhibition zones appeared around disk a (qCQDs) on all the MHA plates with incubated bacteria, and the diameters of inhibition zones were more than 10 mm. In contrast, there was no inhibition zone around disk b (glucose) or c (DDA), indicating no antibacterial activity of two reaction substrates. Moreover, the minimum inhibitory concentration (MIC) of qCQDs on the above six species of bacteria was determined by broth dilution method, as shown in Supplementary Table 1. The MIC of qCQDs was 12.5 µg/mL for S. epidermidis, 25 µg/mL for S. aureus, MRSA and E. faecalis, and 50 µg/mL for both E. coli and P. aeruginosa. The above results confirmed that qCQDs exhibited antibacterial action on both gram-positive and gram-negative bacteria, implying the broad-spectrum antibacterial properties.
In order to better understand bactericidal and bacteriostatic effect of qCQDs, we investigated the number of colonies on nutrient agar plates after the six species of bacteria were exposed to different concentrations of qCQDs in different periods, as shown in Supplementary Fig. 1. When the concentration was up to 200 μg/mL, the changing tendency of the six species of bacteria was similar to that of 100 μg/mL, indicating that qCQDs can effectively inhibit and kill the gram-positive and gram-negative bacteria at 100 μg/mL. The detailed description for Supplementary Fig. 1 was provided in Supplementary Information.
Antimicrobial mechanism of qCQDs against gram-positive bacteria and gram-negative bacteria.
The morphological changes of the six species of bacteria before and after the treatment of qCQDs were characterized by TEM, as shown in Fig. 4. Without the treatment of qCQDs, all of the bacteria exhibited normal cell structure, complete cell walls, and uniform density of substance inside the cells (Fig. 4a, 4c, 4e, 4g, 4i and 4k). In comparison, after the treatment of 100 μg/mL qCQDs for 12 h, bacterial cells were significantly changed with obviously broken cell wall and cell membrane (Fig. 4b, 4d, 4f, 4h, 4j and 4l). More specifically, the bacterial cells treated with qCQDs were disintegrated to different degrees, the cytoplasmic density of bacterial cells obviously decreased, and even the phenomenon of cavities and substance agglomeration occurred in the bacterial cells. And bacterial cells lost their integrity, causing irreversible damage to bacteria cells and resulting in death of bacteria40, 41, which was completely different from the untreated bacteria. The morphology of the treated bacteria indicated that qCQDs had the significant destructive effects on the above six species of bacterial cells, confirming the killing effect of qCQDs on both gram-positive and gram-negative bacteria. Furthermore, using S. aureus and E. coli as the representative bacteria, flow nanoanalyzer was applied to measure the changes of particle size of bacteria before and after the treatment of qCQDs, as shown in Supplementary Fig. 2. For untreated S. aureus and E. coli, particles larger than 1000 nm accounted for 78.3% and 92.2% of the total particles, respectively. After S. aureus and E. coli were treated with qCQDs, particles larger than 1000 nm were significantly reduced in both bacteria. In addition, with the increase of qCQDs concentration from 50 to 100 μg/mL and then to 200 μg/mL, particles larger than 1000 nm in the two species of bacteria decreased from 52%/64.2% to 16.8%/21.1% and then to 10.3%/1.0%, respectively, showing gradual decreasing trend. It is further confirmed that qCQDs could make bacterial cells lyse or disintegrate, resulting in death of the bacteria, which is consistent with the results observed by TEM.
The 2’,7’-dichlorofluorescin-diacetate (DCFH-DA) as ROS probe was used to investigate whether or not ROS was generated in the process of qCQDs interacting with bacteria42. As shown in Supplementary Fig. 3, DCFH-DA had little fluorescence at 520 nm with the interaction with S. aureus or E. coli. After H2O2 (ROS representative substance) was added, obvious fluorescence at 520 nm appeared in the both bacterial suspension. However, for the direct interacting system of qCQDs and bacteria, there was hardly any fluorescence at 520 nm in the both bacterial suspension containing DCFH-DA. Then, the above both bacterial suspension containing DCFH-DA and qCQDs showed obvious fluorescence at 520 nm with the addition of H2O2. Therefore, it is preliminarily inferred that almost no ROS generated in the process of qCQDs combating bacteria.
In order to elucidate the antibacterial mechanism of qCQDs against bacteria, S. aureus and E. coli were used as the representative gram-positive and gram-negative bacteria, respectively. And then, tandem mass tag (TMT)-based quantitative proteomics was performed to analyze the protein changes of bacteria before and after the treatment of qCQDs43, 44. Control group (C), low concentration group (L) and high concentration group (H) were set up in the test of quantitative proteomics. After S. aureus and E. coli were treated with qCQDs, the analysis of differential expressed proteins was conducted by comparing L with C and H with C. With the increase of qCQDs concentration, the amount of the differential proteins with both up-regulated and down-regulated expression increased in S. aureus and E. coli, respectively, and the differential expressed proteins in S. aureus were significantly more than those in E. coli (Supplementary Fig. 4). After S. aureus and E. coli were treated with qCQDs, the identified proteins of the two bacteria were annotated in Cluster of Orthologous Groups of proteins (COG), showing that the functions of the proteins were mainly annotated in energy production and conversion, amino acid transport and metabolism, carbohydrate transport and metabolism, and translation, ribosomal structure and biogenesis (Supplementary Fig. 5). The enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway revealed the proteins pathways with significant statistical differences. In the KEGG pathway for S. aureus, the differentially expressed proteins were mainly enriched in ribosome, RNA degradation, aminoacyl-tRNA biosynthesis and carbon fixation in photosynthetic organisms by comparing L with C and H with C (Supplementary Fig. 6). Ribosome, RNA degradation and aminoacyl-tRNA biosynthesis were mainly related to protein synthesis, among which the enrichment of ribosomal proteins showed the most significant difference (p=3.34×10-5). The 46 differentially expressed proteins were enriched in ribosomes by further analysis of KEGG chord diagram (Fig. 5a and Supplementary Table 2). The corresponding clustering heat map was drawn, and 33 ribosomal proteins were up-regulated and 13 were down-regulated by comparing H with C (Fig. 5c). Correspondingly, for E. coli, the differentially expressed proteins in the KEGG pathway were mainly enriched in biosynthesis of antibiotics, microbial metabolism in diverse environments, citrate cycle, sulfur metabolism, metabolic pathways, biosynthesis of secondary metabolites, tryptophan metabolism, propanoate metabolism, carbon metabolism, lysine degradation, butanoate metabolism, benzoate degradation and nitrogen metabolism by comparing L with C and H with C (Supplementary Fig. S8). Among them, further analysis of KEGG chord diagram showed that 10 differentially expressed proteins were enriched in the following six pathways (Fig. 5b and Table 3), including biosynthesis of antibiotics, microbial metabolism in diverse environments, citrate cycle, metabolic pathways, biosynthesis of secondary metabolites and carbon metabolism. And these 10 proteins are essential enzymes for the citrate cycle (p=3.01×10-5) (Supplementary Fig. 8). The corresponding clustering heat map showed that the 10 differentially expressed proteins in citrate cycle were down-regulated by comparing H with C (Fig. 5d). After the differentially expressed proteins associated with ribosome in S. aureus and citrate cycle in E. coli were annotated in Gene Ontology (GO), the two types of proteins were classified according to cellular component, molecular function and biological processes, respectively. All the 46 ribosomal proteins belonged to intracellular cellular component which were mainly related to ribosomes, including large and small subunit, and involved in the biological process of protein translation, and their molecular functions were mainly focused on structural constituent of ribosome and RNA/rRNA binding (Supplementary Fig. 9). In contrast, the molecular function of the 8 proteins associated with citrate cycle were mainly concentrated in the activity of various enzymes, including aconitate hydratase, isocitrate dehydrogenase (NADP+), oxoglutarate dehydrogenase (succinyl-transferring), oxidoreductase and electron carrier, and the binding of thiamine pyrophosphate, metalion, cofactor and iron-sulfur cluster, and involved in the biological process including tricarboxylic acid cycle, metabolic process and oxidation-reduction process (Supplementary Fig. 10). Meanwhile, interaction analysis of differentially expressed proteins showed that 22 of the 46 ribosomal proteins were in the three protein functional interaction networks (Fig. 5e) and all the 10 proteins associated with citrate cycle were at the hub positions of the one protein functional interaction networks (Fig. 5f).
In order to gain insight into the contributions of ribosome and citrate cycle to the antibacterial action of qCQDs against S. aureus and E. coli, gene set enrichment analysis (GSEA) was conducted to further analyze the genes of differentially expressed proteins in KEGG pathways, and real-time quantitative PCR (RT-qPCR) was used to verify the expression of related protein genes. GSEA showed that there were positive and negative correlations in the function set of ribosomal proteins (Fig. 6a), but all the proteins in the function set of citrate cycle were negatively correlated (Fig. 6b), which were consistent with the differentially expressed proteins in Fig. 5c and 5d, respectively. At the same time, GSEA gave the sequence of the corresponding genes of the proteins in the function set. According to the rank in gene list, six proteins in S. aureus and E. coli were selected from the corresponding differentially expressed proteins, respectively, and the information of selected proteins was shown in Supplementary Table 5. The proteomics analysis showed that three ribosomal proteins were down-regulated and the other three were up-regulated after S. aureus was treated with qCQDs (Supplementary Fig. 11A). Fig. 6c showed the RT-qPCR results of the corresponding genes of six ribosomal proteins in S. aureus. The gene expression of the six ribosomal proteins had no significant difference at the low-concentration of qCQDs. When S. aureus was treated with high-concentration of qCQDs, the gene expression of down-regulated and up-regulated ribosomal proteins changed significantly, which was consistent with the variation trend in the proteomics. The proteomics analysis showed that all six proteins associated with citrate cycle in E. coli were down-regulated (Supplementary Fig. 11B). The RT-qPCR results in Fig. 6d showed that the gene expression of these six proteins had no significant difference at the low-concentration of qCQDs, while the gene expression of these six proteins significantly down-regulated at the high-concentration of qCQDs, corresponding to the variation trend of the six proteins in the proteomics.
In vivo antibacterial activity of qCQDs.
When the skin is compromised, bacteria from environment and skin surface are able to infiltrate into the subcutaneous tissue that is physiologically optimum for colonization and growth, eventually leading to wound infection. The impact of wound infections on health care is enormous, including burns, surgical-site infections, and nonhealing diabetic foot ulcers. S. aureus and P. aeruginosa among the most common organisms isolated from both acute and chronic wounds of various etiologies have been attributed to causing infections of penetrating trauma and burn wounds in surgical site infections as well as in the military setting45. Double infections with S. aureus and P. aeruginosa is more virulent and/or results in worse patient outcomes than single infections, and both species are notorious for their resistance to antimicrobials46. Therefore, we established the rat trauma model with mixed infection of S. aureus and P. aeruginosa to evaluate the in vivo antibacterial activity of qCQDs. The experimental process was shown in Fig. 7a.
Fig. 7b and Supplementary Fig. 12 showed the healing process of infected wounds and the bacterial culture of wound exudate at different times, respectively. A full-layer skin with a diameter of about 18 mm was resected with a scalpel on the back of the clean grade Sprague-Dawley (SD) rats to form the round wounds. After that, the application containing 2 mL of mixed bacterial suspension was adhered to the wound surface and fixed with mesh elastic bandage. The concentrations of both S. aureus and P. aeruginosa were about 3.0×108 CFU/mL in the mixed bacterial suspension. After 48 h, the massive abscess appeared on the wounds. The qCQDs in the experimental group, levofloxacin in the positive control group, and normal saline in the negative control group were employed to treat the infected skin wounds, respectively, investigating the in vivo antibacterial activity of qCQDs. After treatment for 3 days, compared with the negative control group, the obvious scab was observed on the infected skin wounds with decreased exudate in the experimental group and the positive control group. After treatment for 5 days, although exudate appeared in all three groups, purulent lesions and the number of bacteria in the experimental group and the positive control group were significantly lower than that in the negative control group. After treatment for 7 days, exudate, purulent lesions and the number of bacteria significantly reduced in both the experimental group and the positive control group. After treatment for 10 days, the wound area in the experimental group and the positive control group significantly decreased, but the wound size in the negative control group was similar to that of the other two groups on the seventh day and there was still a large amount of exudate and numerous bacteria. After treatment for 14 days, the wounds in the two treated groups completely had no exudate and basically healed without detectable bacteria, but the wound size of the negative control group was the same as that of the two treated groups on the tenth days with the still present bacteria colonies. The above results indicated that qCQDs could promote the recovery of the wound infected with mixed bacteria, which had the same therapeutic effect as levofloxacin at a certain concentration.
Fig. 8a showed that three groups of SD rats lost weight after the wounds were infected with mixed bacteria for 48 hours, and the weight of the rats in the experimental group and the positive control group increased gradually after intervention. The increasing trend continued within 14 days. Meanwhile, the weight of the negative control group increased slowly in the first 6 days intervention, and always were lower than that of the experimental group and the positive control group. Within the whole course of intervention, the dead rats appeared in the negative control group on 2nd, 3rd, 5th, 6th and 9th day, as shown in Fig. 8b, with a final survival rate of 56.4%. All the rats of experimental group and positive control group survived in the treating process. The above results indicated that qCQDs could effectively combat the infection with mixed bacteria of S. aureus and P. aeruginosa, greatly contribute to the weight recovery of experimental rats, and significantly reduce the death rate of the rats.
H&E staining was carried out on the pathological sections of the wound tissues after 48 hours infection and 14 days treatment to evaluate the recovery of the infected wounds in different groups, as shown in Fig. 8c. For the infected wounds, a large number of neutrophils appeared in the wound tissues, with enlarged blood vessels and increased blood cells (black arrow in Fig. 8c), which confirmed the successfully infected wounds with S. aureus and P. aeruginosa. After intervention for 14 days, capillaries in the wound tissues of the experimental group and the positive control group were significantly reduced, neutrophils basically disappeared, and obvious connective tissue formed on the surface of the wound (red arrow in Fig. 8c). In the negative control group, there were still a large number of neutrophils in wound tissues and many blood cells in the enlarged capillaries accompanying with the uncontrolled inflammation in the wounds after intervention for 14 days. The above results indicated that qCQDs were beneficial for the abatement of inflammation in the infected wound tissues and had similar therapeutic effect to levofloxacin in the wounds infected with mixed bacteria.
Biosafety evaluation of qCQDs.
At first, the possible induced drug resistance of qCQDs to bacteria was evaluated to assess the biosafety ascribed to the serious and common problem of bacterial antibiotic resistance. Supplementary Fig. 13 showed that S. aureus, E. coli and MRSA did not develop resistance to qCQDs after induced resistance for 30 days at sub-MIC level, meaning that bacteria could not easily develop new or secondary resistance to qCQDs. It may be due to the fact that the qCQDs can play an antimicrobial role via new signaling pathways or effect targets that is different from the acting targets of traditional antibiotics. In vitro cytotoxicity of qCQDs was further investigated by MTT test based on the effects of qCQDs at different concentrations on macrophages and HepG2 cells at different times. As shown in Supplementary Fig. 14, when the concentration of qCQDs was 150 μg/mL, the survival rates of macrophages (Supplementary Fig. 14a) and HepG2 (Supplementary Fig. 14b) were above 80% within 6 to 12 hours, and 67.3% and 70.7% after exposed for 24 h, respectively. When the concentration reached 200 μg/mL, the survival rates of the two kinds of cells were somewhat reduced with 63.2% and 42.9% after exposed for 24 h. The above results showed that certain cytotoxic effects of qCQDs on eukaryotic cells would also occur in vitro when the concentration reached a high level. The reason might be the poor selectivity of qCQDs for target ascribed to the broad-spectrum antibacterial activity. In vitro hemolysis test was also conducted to verify whether qCQDs could cause erythrolysis. As can be seen from Supplementary Fig. 14c, when red blood cells were added to the sterilized water, the solution changed from colorless to red after centrifugation, meaning that red blood cells were ruptured due to different osmotic pressures inside and outside the cells. Red blood cells were added to normal saline containing different concentrations of qCQDs, and subjected to centrifugation. Red blood cells were deposited at the bottom of the centrifuge tube, and the solutions were still transparent and colorless. Even if the concentration of qCQDs reached 400 μg/mL, compared with the negative control, the hemolysis rate is only 1.9% (<5%), as shown in Supplementary Fig. 14d, indicating that qCQDs would not cause red blood cells rupture and hemolysis in physiological conditions and has good blood compatibility.
The in vivo toxicity of qCQDs to the main organs (including heart, liver, spleen, lung and kidney) of rats was evaluated by pathomorphology, as shown in Supplementary Fig. 15. Rats in the experimental group were given with 4 mg/mL of qCQDs solution by continuous gavage at the dose of 5 mL/day for 7 days, and the control group was given with the same dose of sterilized normal saline by gavage. Within 7 days, the rats in the two groups showed normal activity, diet and mental state. H&E-stained pathological sections of heart, liver, spleen, lung and kidney showed no significant difference in the tissue structure between the two groups, meaning that qCQDs would not cause pathological damage to main organs of rats by intragastric administration for 7 days. It may be the reason that qCQDs does not generate toxicity and side effects on the main organs of the body, or qCQDs can be converted into non-toxic substances for the main organs in the body. Thus, it is preliminarily concluded that qCQDs may be a kind of carbon nanomaterial without detectable in vivo toxicity for in vivo antibacterial applications.