Hydrazide derivative-based carbon dots as well as preparation method and application thereof

Abstract

The invention discloses a hydrazide derivative-based carbon dot and a preparation method and application thereof, and relates to the technical field of antibacterial materials.The preparation method comprises the following steps that a hydrazide derivative is used as a raw material, and the hydrazide derivative-based carbon dot is prepared through a one-step hydrothermal method; the hydrazide derivative is at least one of phenylhydrazine, methylhydrazine, isophthaloyl hydrazine, thiodihydrazide, salicylhydrazine, acethydrazide, 3-pyridine formylhydrazine, benzoyl hydrazine, p-carboxyphenylhydrazine and carbohydrazide; the invention also protects the hydrazide derivative-based carbon dots prepared by the method, and application of the hydrazide derivative-based carbon dots in preparation of antibacterial materials and antibacterial drug-resistant materials. The preparation method has the beneficial effects that 10 hydrazide derivatives are taken as precursors, 10 hydrazide derivative-based carbon dots are prepared through a simple one-step hydrothermal method, all the 10 hydrazide derivative-based carbon dots have antibacterial activity, and the carbon hydrazide-based carbon dots have excellent antibacterial activity and can effectively eradicate biological membranes.

Description

Technical Field

[0001] The invention relates to the technical field of antibacterial materials, and in particular to a hydrazide derivative-based carbon dot and a preparation method and application thereof. Background Art

[0002] Antibiotics have been widely used to treat bacterial infections, which may lead to the emergence and rapid spread of bacterial resistance. Typical antibiotics can effectively eradicate common bacteria, but their efficacy against resistant bacteria is significantly reduced. However, the development of alternative treatments cannot keep up with the evolution of bacteria. In particular, with the emergence of biofilms, bacteria aggregate in their own secreted extracellular polymers, further increasing their resistance to various antimicrobial drugs. It has been reported that a variety of bacteria and chronic infections are associated with bacterial biofilms. In the face of the current serious problems of bacterial infection and drug resistance, effective plans should be formulated to regulate the use of antibiotics to slow down the development of bacterial resistance and maintain the effectiveness of key medically available antibiotics. Many countries have enacted laws and regulations on the rational use of antibiotics. The discovery of new antimicrobial agents is of great significance for treating bacterial infections and alleviating or changing the emergency situation caused by antimicrobial resistance.

[0003] With the development of nanotechnology, nanomaterials with antibacterial activity have been widely developed. Nanomaterials exhibit some unique functions due to their extremely small particle size and large specific surface area, which have been widely explored in the field of antibacterial. To date, a variety of antibacterial nanoformulations have been reported, including metal-based nanoparticles, graphene oxide, dendrimers, organic nanoparticles, etc. Among them, carbon dots (CDs) are a multifunctional carbon-based nanomaterial that shows great potential in bioimaging, drug delivery, biosensing, tumor therapy, antibacterial, etc. The properties of CDs are closely related to different synthesis methods and precursors. For example, doping elements can endow CDs with unique properties. Previous studies have shown that N-doped CDs exhibit a variety of structures, adjustable structural defects, and enhanced optoelectronic properties. However, few reports in the prior art focus on the antibacterial and anti-biofilm properties of CDs derived from such precursors. Summary of the invention

[0004] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to provide a hydrazide derivative-based carbon dot and a preparation method and application thereof.

[0005] The present invention uses a variety of hydrazide derivatives as precursors. Hydrazide derivatives are common raw materials for organic synthesis containing carbon and nitrogen elements, and are ideal precursors for preparing N-doped CDs derived from single precursors. The present invention adopts a simple hydrothermal synthesis method to prepare new N-doped CDs. The 10 hydrazide derivative-based carbon dots synthesized by the present invention all have certain antibacterial properties. Among them, CDs derived from cheap, safe and environmentally friendly carbon hydrazides (CDH-CDs) stand out for their excellent antibacterial properties. CDH-CDs show the best antibacterial activity compared with other CDs based on hydrazide derivatives, and have efficient anti-biofilm performance, low ability to induce bacterial resistance and good biocompatibility. In addition, CDH-CDs can induce oxidative stress to produce endogenous ROS, thereby destroying the bacterial membrane structure and affecting gene expression, thereby hindering the normal reproduction and growth of bacteria.

[0006] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for preparing hydrazide derivative-based carbon dots, comprising the following steps: Hydrazide derivative-based carbon dots were prepared using hydrazide derivatives as raw materials through a one-step hydrothermal method.

[0007] The present invention uses 10 hydrazide derivatives as precursors and prepares 10 hydrazide derivative-based carbon dots through a simple one-step hydrothermal method. All of the 10 hydrazide derivative-based carbon dots have antibacterial activity.

[0008] In a preferred embodiment of the present invention, the hydrazide derivative is at least one of phenylhydrazine, methylhydrazine, isophthalic acid hydrazide, thiodihydrazide, salicylic acid hydrazide, acetic acid hydrazide, 3-pyridine carboxyhydrazide, benzoic acid hydrazide, p-carboxyphenylhydrazine and carbohydrazide.

[0009] In a preferred embodiment of the present invention, the preparation method comprises the following specific steps: dissolving the hydrazide derivative in water and subjecting it to ultrasonic treatment to obtain a mixture; The mixture is heated to react to obtain hydrazide derivative-based carbon dots.

[0010] In a preferred embodiment of the present invention, the addition ratio of the hydrazide derivative to water is 100 mg to 140 mg: 10 mL to 14 mL. Preferably, it is 110 mg to 130 mg: 11 mL to 13 mL. More preferably, it is 115 mg to 125 mg: 11.5 mL to 12.5 mL. In a preferred embodiment of the present invention, the ultrasonic treatment time is 20 min to 40 min, preferably 25 min to 35 min, and more preferably 28 min to 32 min.

[0011] In a preferred embodiment of the present invention, the temperature of the heating reaction is 160° C. to 200° C. Preferably, the temperature of the heating reaction is 170° C. to 190° C. More preferably, the temperature of the heating reaction is 175° C. to 185° C.

[0012] In a preferred embodiment of the present invention, the heating reaction time is 10 h to 14 h. Preferably, the heating reaction time is 11 h to 13 h. More preferably, the heating reaction time is 11.5 h to 12.5 h.

[0013] In a second aspect, the present invention provides hydrazide derivative-based carbon dots obtained by the above-mentioned preparation method.

[0014] In a third aspect, the present invention provides the hydrazide derivative-based carbon dots obtained by the above-mentioned preparation method or the use of the above-mentioned hydrazide derivative-based carbon dots in the preparation of antibacterial materials.

[0015] In a fourth aspect, the present invention provides the hydrazide derivative-based carbon dots obtained by the above-mentioned preparation method or the use of the above-mentioned hydrazide derivative-based carbon dots in the preparation of antibacterial and drug-resistant materials.

[0016] The present invention has at least one of the following beneficial effects: The present invention uses 10 hydrazide derivatives as precursors and prepares 10 hydrazide derivative-based carbon dots by a simple one-step hydrothermal method. All 10 hydrazide derivative-based carbon dots have antibacterial activity. The best antibacterial activity is carbon hydrazide carbon dots, followed by methyl hydrazide carbon dots, acetyl hydrazide carbon dots and benzoyl hydrazide carbon dots, then thiodihydrazide carbon dots, 3-pyridinecarboxyhydrazide carbon dots, p-carboxyphenylhydrazide carbon dots, then phenylhydrazide carbon dots, and finally isophthaloyldihydrazide carbon dots and salicylic hydrazide carbon dots. Among them, compared with other CDs based on hydrazide derivatives, carbon hydrazide carbon dots (CDH-CDs) show excellent antibacterial activity and can effectively eradicate biofilms. Compared with two typical antibiotics, rifampicin and kanamycin, the two resistant bacteria have no obvious resistance to CDH-CDs. Mechanism studies have shown that CDH-CDs kill bacteria by producing endogenous ROS and affecting gene expression. In vivo experiments further demonstrated the good antibacterial properties and biocompatibility of CDH-CDs. The present invention proposes a simple method to overcome bacterial infection and reveals a new perspective on the potential application of CDs. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG1 shows the structural formulas of 10 hydrazide derivatives.

[0018] Figure 2 shows the synthesis and characterization of CDH-CDs. (A) One-step hydrothermal synthesis of CDH-CDs. (B) TEM image of CDH-CDs (inset: HRTEM image). (C) Histogram of particle size distribution in B, analyzed by ImageJ. (D) XRD pattern of CDH-CDs. (E) FT-IR spectra of CDH-CDs and precursor CDH. XPS total curve (F), C1s (G), N1s (H) and O1s (I) high-resolution XPS curves of CDH-CDs.

[0019] Figure 3 shows the zeta potential of CDH-CD in PBS (pH 7.4 or 5.6). The results are expressed as mean ± SD, n = 3.

[0020] FIG4 shows the UV-visible absorption spectra of the precursors CDH and CDH-CD.

[0021] Figure 5 In vitro antimicrobial properties of CDH-CDs. Inhibition rates of different concentrations of CDH-CDs or CDH against (A) MRSA, (B) ESBL-E, (C) Staphylococcus aureus, and (D) E. coli. Results are expressed as mean ± SD, n = 5. Photographic images of ZOIs developed during the paper disk diffusion test of CDH and CDH-CDs against MRSA (E and G) and ESBL-E (F and H), respectively, and the corresponding bar graphs (indicated by black circles). Results are expressed as mean ± SD, n = 3.

[0022] Figure 6 In vitro antimicrobial efficacy and bacterial resistance of CDH-CDs. Growth inhibition curves of MRSA (A) and ESBL-E (B) at different concentrations of CDH-CDs. Results are presented as mean ± SD, n = 5. MRSA (C) and ESBL-E (D) developed resistance to CDH-CDs, rifampicin, and kanamycin, respectively. (E) Live / dead staining of MRSA or ESBL-E incubated with or without CDH-CDs. Green channel, DMAO, live and dead bacteria, λex = 488 nm, λem = 500-550 nm. Red channel, PI, dead bacteria, λex = 552 nm, λem = 560-620 nm. Scale bar, 50 μm. (F) SEM images of MRSA or ESBL-E incubated with or without CDH-CDs. Surface contraction and fusion with the pore are indicated by blue arrows. Scale bar, 1 μm.

[0023] Figure 7 In vitro antibiofilm performance of CDH-CDs. Crystal violet staining photos (A) and inhibition rates (B) of MRSA or ESBL-E biofilms formed in the presence of different concentrations of CDH-CDs. Results are expressed as mean ± SD, n = 5. Crystal violet staining photos (C) and removal rates (D) of MRSA or ESBL-E biofilms after incubation with different concentrations of CDH-CDs. Results are expressed as mean ± SD, n = 5. (E) Live / dead staining of MRSA or ESBL-E biofilms after incubation with different concentrations of CDH-CDs. Green channel, DMAO, live and dead bacteria, λex = 488 nm, λem = 500-550 nm. Red channel, PI, dead bacteria, λex = 552 nm, λem = 560-620 nm. Scale bar, 50 μm.

[0024] Figure 8 The antibacterial mechanism of CDH-CDs. (A) ROS produced by MRSA after incubation with sterile water (control), CDH-CDs, H2O2 or Rosup using DCFH-DA as a probe. Excitation: 488 nm. (B) Fluorescence spectra of MRSA after incubation with TA, CDH-CDs or CDH-CDs and TA. The increase in fluorescence intensity of TA at 410 nm indicates the generation of -OH. Excitation: 305 nm. (C) 1O2 produced by incubation of MRSA with different concentrations of CDH-CDs using DPBF as a probe. (D) O2 produced by incubation of MRSA with different concentrations of CDH-CDs using cytochrome c as a probe. The results are expressed as mean ± SD, n = 3. ***P < 0.001, using Student's t test. (E) Relative expression levels of mRNA in MRSA incubated with CDH-CDs. The results are expressed as mean ± SD, n = 3. (F) Proposed antibacterial mechanism of CDH-CDs.

[0025] Fig. 9 ROS production by ESBL-E cultured with sterile water (control), CDH-CDs, H2O2, or Rosup using DCFH-DA as a probe. Excitation: 488 nm.

[0026] Fig.10 Agarose gel electrophoresis images of bacterial DNA incubated with different concentrations of CDH-CD.

[0027] Fig.11 The weight changes of MRSA-infected mice after different treatments. The results are expressed as mean ± SD, n = 5.

[0028] Fig.12 Figure 2 Hemolysis test of different concentrations of CDH-CD. The results are expressed as mean ± SD, n = 3.

[0029] Fig.13 In vivo antimicrobial activity of CDH-CDs. (A) Schematic diagram of the treatment scheme for MRSA-infected mice. (B) Photos of wounds in MRSA-infected mice after different treatments. (C) Relative wound areas in MRSA-infected mice after different treatments. Results are presented as mean ± SD, n = 5. (D) Schematic diagram of wounds stained with Masson's trichrome staining of B. (E) H&E and infected wounds at the end of treatment. Scale bar, 200 μm.

[0030] Fig.14 H&E staining of major organs (heart, liver, spleen, lung, and kidney) of healthy mice or MRSA-infected mice after different treatments. Scale bar, 200 μm. DETAILED DESCRIPTION

[0031] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0032] Example 1 1. CDs synthesis This embodiment uses the structural formula Figure 1 The 10 hydrazide derivatives (phenylhydrazine, methylhydrazine, isophthalic acid hydrazide, thiodihydrazide, salicylic acid hydrazide, acetic acid hydrazide, 3-pyridine carboxyhydrazide, benzoylhydrazide, p-carboxyphenylhydrazide, carbohydrazide) shown in the figure were used as precursors to synthesize CDs based on hydrazide derivatives through a one-pot hydrothermal method. Specifically, they include: 120 mg of 10 hydrazide derivatives were dissolved in 12 mL of double distilled water (ddwater). After ultrasonic treatment for 30 min, the mixture was maintained in an oven at 180 °C in a poly(tetrafluoroethylene)-lined autoclave for 12 h and then cooled to room temperature. The resulting mixture was lyophilized and CDs were dispersed in ddwater at a concentration of 10 mg / mL for further use.

[0033] 2. Determination of the Minimum Inhibitory Concentration (MIC) of CDs Synthesized from Different Precursors In order to screen CDs with the best antibacterial performance, this example measured the MIC values ​​of 4 representative bacteria, including Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli, and two drug-resistant bacteria MRSA and ESBL-E. The specific determination method is as follows: (1) Bacterial culture Bacterial strains obtained from the American Type Culture Collection (ATCC) included Escherichia coli (E. coli, ATCC 35218), extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E, ATCC 25922), Staphylococcus aureus (S. aureus, ATCC 6538), and methicillin-resistant Staphylococcus aureus (MRSA, ATCC 43300). The bacterial suspension was plated on sterile tryptic soy broth (TSB) agar plates and incubated at 37 °C for 18 h. Subsequently, bacterial colonies were picked from the plates and cultured overnight in liquid TSB to obtain bacteria growing in the logarithmic phase for further use.

[0034] (2) Minimum inhibitory concentration (MIC) determination The density is 1 × 10 5 Bacteria with colony forming units (CFU) / mL were inoculated into 96-well plates (100 μL / well). Then 100 μL of the above hydrazide-based CDs were mixed with different concentrations of bacteria in the wells. Bacteria incubated with CDs at a concentration of 0 μg / mL were used as negative controls. After the bacterial suspension was incubated at 37 °C for 18 h, the absorbance at 600 nm (OD 600) in each well was detected using a microplate reader (DNM-9602, China). The experiment was performed 5 times. The inhibition rate at each concentration was calculated using formula (1). The minimum inhibitory concentration (MIC) is the lowest concentration that shows an inhibition rate of more than 90%.

[0035] The measurement results are shown in Table 1: Table 1 MICs of CDs synthesized from different precursors (μg / mL) As can be seen from Table 1, all ten hydrazide derivative-based carbon dots have antibacterial activity, the MIC of methylhydrazide carbon dots is 50 or 100 μg / mL, the MIC of acetylhydrazide carbon dots is 100 or 200 μg / mL, the MIC of 3-pyridinecarboxyhydrazide carbon dots and p-carboxyphenylhydrazide carbon dots is 200 μg / mL, the MIC of thiodihydrazide carbon dots is 200 or 400 μg / mL, the antibacterial activity of benzoylhydrazide carbon dots and phenylhydrazide carbon dots against different bacteria is not exactly the same, with MICs of 100, 200, 400 or 800 μg / mL, the MIC of isophthalic acid dihydrazide carbon dots is 3200 μg / mL, and the MIC of salicylic acid hydrazide carbon dots is greater than 3200 μg / mL.

[0036] Furthermore, compared with other CDs using hydrazide derivatives as precursors, carbohydrazide-based CDs (CDH-CDs) showed the lowest MIC of 25 or 50 μg / mL ( Table 1 ), indicating that the antibacterial activity of CDH-CDs was superior to that of other hydrazide-based CDs.

[0037] 3. Material Characterization Taking carbohydrazide carbon dots (CDH-CDs) as an example, the CDH-CDs prepared above were characterized by the following method: Zeta potential was recorded by Zetasizer Nano ZS (Malvern Panalytical, UK). Ultraviolet–visible (UV–Vis) absorption spectra were measured by N4SUV-VIS spectrophotometer (INESA Analytical Instrument Co., Ltd, China). X-ray powder diffraction (XRD) analysis was performed on a D8 Advance (Bruker, Germany). Transmission electron microscopy (TEM) images were obtained using a JEM-2100 (JEOL, Japan). Fourier transform infrared (FT-IR) spectra were measured using a Nicolet iS50 FT-IR spectrometer (Thermo Fisher Scientific, USA). X-ray photoelectron spectroscopy (XPS) was performed by an ESCALAB 250Xi (Thermo Fisher Scientific, USA).

[0038] The characterization results are as follows Figures 2~4 shown.

[0039] exist Figure 2In the experiment, CDH-CDs were negatively charged, with Zeta potentials of -9.03 ± 1.58 mV or -6.06 ± 0.62 mV in neutral (pH 7.4) or acidic (pH 5.6) buffers, respectively. Compared with the precursor CDH, the absorption at 215.3 nm in the UV-visible absorption spectrum of CDH-CDs changed to 206.3 nm (Figure 4), which was attributed to the n→π* transition of -CONH-. Subsequently, we used TEM to examine the morphology of CDH-CDs. As shown in Figures 2B and C, CDH-CDs were quasi-spherical with diameters ranging from 1.5 to 5.0 nm and interplanar spacing of 0.21 nm. The XRD pattern of CDH-CDs shown in Figure 2D showed a broad peak near 25.5°, indicating the formation of disordered carbon atoms. Subsequently, we used TEM to examine the morphology of CDH-CDs. As shown in Figures 2B and C, CDH-CDs are quasi-spherical with diameters ranging from 1.5 to 5.0 nm and interplanar spacing of 0.21 nm. The XRD pattern of CDH-CDs shown in Figure 2D shows a broad peak near 25.5°, indicating the formation of disordered carbon atoms. In addition, the retention of surface groups on CDH-CDs was analyzed using FT-IR spectroscopy (Figure 2E). FT-IR analysis of CDH-CDs and its precursor CDH showed that the peak corresponding to -NH2 at 3389.50 cm - ¹, C=O at 1708.89 cm-1, CN at 1554.22 cm - ¹ and NH stretching vibration at 1196.28 cm-1. In addition, compared with CDH, the FT-IR spectrum of CDH-CDs shows a - ¹、952.61 cm - ¹ and 704.94 cm -New peaks appeared at ¹, which were attributed to the bond stretching of -NO2, CO, and CC, respectively. These results indicate that new chemical bonds were formed during the synthesis of CDH-CDs. In addition, the chemical bonds and basic components were examined by XPS, which was consistent with the FT-IR results. The XPS overall curve (Figure 2F) showed that CDH-CDs contained carbon, nitrogen, and oxygen, with elemental ratios of 36.08%, 43.68%, and 20.24%, respectively. The high-resolution C1s map (Figure 2G) showed three fitting peaks at 283.58 eV, 284.98 eV, and 287.41 eV, which were located as CC, CN, and C=O bonds, respectively. By examining the high-resolution N1s map (Figure 2H), three peaks were found, at 397.47 eV, 399.23 eV, and 400.77 eV, representing -NH2, NN, and -NO2, respectively. The two peaks at 530.19 eV and 531.44 eV in the high-resolution O1s map (Fig. 2I) were assigned to CO and C=O bonds.

[0040] Example 2 In vitro antibacterial properties of CDH-CDs 1. Taking carbohydrazide carbon dots (CDH-CDs) as an example, in order to further study the antibacterial activity of CDH-CDs in vitro, we first explored the antibacterial effects of different doses of CDH-CDs on different bacteria. The test method is as follows: (1) Inhibition zone detection MRSA or ESBL-E (1 × 10 5 CFU / mL) were spread on TSB or MacConkey (MAC) agar plates. A sterilized Oxford cup was then placed on the plate and filled with CDH or CDH-CD (2 mg / mL, 100 μL). The agar plates were incubated at 37 °C for 18 hours. The experiment was performed three times.

[0041] (2) Bacterial growth inhibition monitoring MRSA or ESBL-E (1 × 10 5 CFU / mL), and OD600 was measured every 2 hours until 24 hours to monitor bacterial growth inhibition. The experiment was performed 5 times.

[0042] (3) Live / dead bacterial staining MRSA or ESBL-E cells growing in the logarithmic phase were collected by centrifugation and resuspended in 0.85% NaCl solution. 8 CFU / mL) were further incubated with 800 μg / mL CDH-CDs at 37 °C for 1 h. Then, live or dead bacteria were stained using a bacterial viability assay kit (C2030S, Beyotime) according to the manufacturer's instructions and imaged by confocal laser scanning microscopy (CLSM, TCS SP8, Leica, Germany). Green channel, N,N-dimethylaniline N-oxide (DMAO), live and dead bacteria, λex = 488 nm, λem = 500-550 nm. Red channel, propidium iodide (PI), dead bacteria, λex = 552 nm, λem = 560-620 nm.

[0043] (4) Scanning electron microscopy (SEM) characterization of bacterial morphology MRSA or ESBL-E were incubated with 800 μg / mL CDH-CDs at 37 °C for 4 h and then collected by centrifugation. The collected bacteria were fixed and dehydrated. After drying in a freeze dryer and treated with spray gold, the samples were imaged with a scanning electron microscope (SEM, JSM-6390LV, JEOL, Japan).

[0044] The test results are as follows: As shown in Figures 5A and B, when incubated with 37 °C bacteria for 18 h at 37 °C, the MIC value of CDH-CDs against ESBL-E or MRSA was 25 μg / mL, which was much lower than that of CDH. In addition, CDH-CDs also had better antibacterial ability against Staphylococcus aureus and Escherichia coli (Figures 2C and D), indicating that CDH-CDs had excellent antibacterial activity. The excellent antibacterial effect of CDH-CDs was further verified by the disk diffusion test. The inhibition zone (ZOI) diameter of CDH-CDs against MRSA (Figures 2E and G) or ESBL-E (Figures 2F and H) was 2.7 or 2.5 times that of the precursor CDH, respectively. Bacterial growth inhibition monitoring showed that developing CDs at a concentration of 25.0 (MIC) or 50.0 (2 × MIC) μg / mL could effectively inhibit the growth of MRSA or ESBL-E within 24 h. When the concentration of CDH-CDs was lower than the MIC (e.g., 12.5 μg / mL, 1 / 2 MIC), bacterial growth was restricted to a certain extent, but eventually continued (Figure 6A and B). Meanwhile, DMAO / PI staining of live / dead bacteria further demonstrated that incubation with 800 μg / mL CDH-CDs at 37 °C for 1 h could cause severe damage to MRSA and ESBL-E, thereby inducing death (Figure 6C). SEM was then used to examine the morphological changes on the bacterial surface. After treatment with 800 μg / mL CDH-CDs at 37 °C for 4 h, obvious surface irregularities and collapse of MRSA and ESBL-E skeletons were observed ( Figure 6 D), confirming that CDH-CDs destroyed the integrity of bacterial membranes. All these results indicate that CDH-CDs have excellent antibacterial activity against various bacteria.

[0045] 2. Drug resistance test The development of drug resistance is a major concern, which will lead to a decrease in the antimicrobial efficacy of antibiotics. Therefore, we evaluated the ability of MRSA and ESBL-E to develop resistance to CDH-CDs. Two typical antibiotics, rifampicin and kanamycin, were used as controls. Serial passages of bacteria were cultured in the presence of subinhibitory concentrations of drugs to estimate resistance.

[0046] Drug resistance test: MRSA or ESBL-E were incubated with different concentrations of CDH-CDs, rifampicin, or kanamycin at 37 °C for 24 h to measure the MIC of each drug. Thereafter, bacteria in a 0.5-fold MIC suspension were added to fresh medium at a density of 1 × 105 CFU / mL and further treated with different concentrations of CDH-CDs, rifampicin, or kanamycin. This was repeated for 14 sessions.

[0047] The test results are shown in Figures 6E and F, and MRSA and ESBL-E developed significant resistance to rifampicin and kanamycin. Exposure to rifampicin resulted in a 256-fold or 128-fold increase in the MIC of MRSA or ESBL-E, respectively, after 14 passages. In addition, the MIC of kanamycin for MRSA and ESBL-E increased 64-fold after 14 passages. When cultured in the presence of CDH-CDs for 14 passages, no significant changes in the MIC of MRSA or ESBL-E were observed (MIC was 25 μg / mL at the first passage and 50 μg / mL at the last passage), indicating that neither MRSA nor ESBL-E developed resistance to CDH-CDs.

[0048] Example 3 In vitro antibiofilm activity of CDH-CDs Taking carbohydrazide carbon dots (CDH-CDs) as an example, this example evaluates the efficacy of CDH-CDs on biofilms. The evaluation method is as follows: (1) Biofilm formation inhibition assay MRSA or ESBL-E (1 × 10 5 CFU / mL) were inoculated with different concentrations (0, 25.0, 50.0, 100, 200, 400, 800 μg / mL) of CDH-CDs and cultured at 28 °C for 72 h without agitation. Bacteria incubated with CDH-CDs at a concentration of 0 μg / mL were used as negative controls. After rinsing five times with phosphate-buffered saline (PBS, pH 7.4) to remove planktonic bacteria, the formed biofilms were fixed, washed, and dried. Afterwards, the fixed biofilms were stained with 1 wt% crystal violet for 5 min and further rinsed with PBS three times. Finally, the stained biofilms were dissolved with 33.3% acetic acid (200 μL / well), photographed with a smartphone, and measured for absorbance at 570 nm (OD570) using a microplate reader. The experiment was performed five times. The biofilm inhibition rate at each concentration was calculated using formula (2).

[0049] (2) Biofilm removal assay Biofilm formation is a key factor in the development of antibiotic resistance, which has encouraged researchers to accelerate the development of effective strategies to eradicate biofilms. 5 CFU / mL) were incubated in TSB at 28 °C for 48 h without stirring to promote biofilm formation. After rinsing with PBS three times, the biofilms were incubated with different concentrations of CDH-CDs (0, 25.0, 50.0, 100, 200, 400, 800 μg / mL) for 24 h, and then fixed, stained, dissolved, and OD570 was measured. Biofilms incubated with CDH-CDs at a concentration of 0 μg / mL were used as negative controls. The experiment was performed five times. The biofilm removal rate at each concentration was determined using formula (3). The lowest concentration of antimicrobial agent that can kill all bacteria in the biofilm is defined as the minimum biofilm eradication concentration (MBEC).

[0050] MRSA or ESBL-E were incubated in TSB containing circular coverslips at 28 °C for 48 h to form biofilms, and then treated with different concentrations (0, 25.0, 50.0 μg / mL) of CDH-CDs for 24 h and then stained with DMAO and PI. The circular coverslips were imaged by CLSM to estimate the biofilm removal efficiency of CDH-CDs.

[0051] The test results are as follows Figure 7As shown, MRSA or ESBL-E were incubated with CDH-CDs at 28 °C for 72 h without agitation and then fixed and stained with crystal violet to quantify the biomass of the formed biofilm. As shown in Figures 7A and B, when MRSA or ESBL-E were incubated with CDH-CDs at concentrations higher than 100 μg / mL, the inhibition rate of biofilm formation exceeded 90%. In addition, CDH-CDs were also able to disrupt preformed biofilms, with an MBEC of 400 μg / mL for MRSA and 50 μg / mL for ESBL-E (Figures 7C and D). Staining of biofilms also indicated bacterial death in MRSA and ESBL-E biofilms treated with 25.0 μg / mL CDH-CDs (Figure 7E). However, when the biofilms were incubated with 50.0 μg / mL CDH-CDs, fewer live or dead bacteria were stained, which may be attributed to the removal of most of the biofilm on the round coverslips. Based on the above results, we can conclude that CDH-CDs can effectively eradicate MRSA and ESBL-E biofilms.

[0052] Example 4 Study on the antibacterial mechanism of CDH-CDs Taking carbohydrazide-based carbon dots (CDH-CDs) as an example, the antibacterial mechanism of CDH-CDs was further explored. The exploration method is as follows: (1) Generation of reactive oxygen species (ROS) in bacteria MRSA or ESBL-E were incubated with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) for 20 minutes. Subsequently, the bacteria were incubated with 200 μg / mL CDH-CD, 100 mM H2O2, reactive oxygen species (ROS) inducer Rosup (125 μg / mL) or sterile water (control) for 45 minutes. The generated ROS can oxidize the deacetylated DCFH-DA in the bacteria to produce fluorescent 2',7'-dichlorodihydrofluorescein (DCF). The fluorescence intensity of DCF was observed using an F-7000 fluorescence spectrophotometer (Hitachi, Japan) with an excitation wavelength of 488 nm. The content of ROS is proportional to the fluorescence intensity of DCF. (2) Production of hydroxyl radicals (•OH) in bacteria The generation of hydroxyl radicals (•OH) in MRSA was monitored by the change in the fluorescence intensity of terephthalic acid (TA). MRSA growing in the logarithmic phase was resuspended in PBS and then incubated with TA (1 mM), CDH-CDs (200 μg / mL), or a mixture of TA (1 mM) and CDH-CDs. After 1 hour of incubation, the fluorescence intensity of each sample was recorded using a fluorescence spectrophotometer (λex = 305 nm). The amount of •OH is proportional to the fluorescence intensity at 410 nm.

[0053] (3) Production of singlet oxygen (1O2) in bacteria The generation of singlet oxygen (1O2) in MRSA was monitored by the absorbance change of 1,3-diphenylisobenzofuran (DPBF). 8 CFU / mL) were incubated with 0 (control), 500 or 1000 μg / mL of DPBF (100 μg / mL) and CDH-CDs for 45 min. The UV-visible absorption spectrum of each sample was recorded, and the content of 1O2 was inversely proportional to the absorbance of DPBF.

[0054] (4) Superoxide anion (O2• - ) Monitoring of superoxide anion (O2• - ) production. MRSA (1× 10 8 CFU / mL) were treated with cytochrome c (0.5 mg / mL) and CDH-CDs (0, 50.0, 200, or 500 μg / mL) for 45 min. The absorbance at 550 nm (OD 550) was recorded and correlated with O 2 • - is proportional to the content.

[0055] (5) DNA gel electrophoresis Bacteria were incubated with CDH-CDs at a concentration of 0, 50.0, 100, 200, or 400 μg / mL at 37 °C for 4 h and collected by centrifugation. DNA was extracted from the treated bacteria using the Ezup Column Bacterial Genomic DNA Purification Kit (B518255, Sangon Biotech) for agarose gel electrophoresis (voltage: 140 V, 45 min). The gel was imaged under UV light (254 nm). Bacteria were incubated with CDH-CDs at a concentration of 0, 50.0, 100, 200, or 400 μg / mL at 37 °C for 4 h and collected by centrifugation. DNA was extracted from the treated bacteria using the Ezup Column Bacterial Genomic DNA Purification Kit (B518255, Sangon Biotech) for agarose gel electrophoresis (voltage: 140 V, 45 min). The gel was imaged under UV light (254 nm). (6) Fluorescence quantitative PCR (qPCR) Bacteria were incubated with 0 (control) or 50.0 μg / mL CDH-CDs at 37 °C for 18 h. Total RNA from treated bacteria was extracted using TRIzol reagent. Then, cDNA was synthesized by using RevertAid First-Strand cDNA Synthesis Kit (K1622, Thermo Fisher Scientific). The obtained cDNA was analyzed by real-time quantitative polymerase chain reaction (qPCR) using KiCqStart™ SYBR® GreenqPCR ReadyMix™ (KCQS0ri0, Sigma-Aldrich). After searching through the National Center for Biotechnology Information (NCBI), primer sequences for target genes were designed using Beacon Designer8 and the specificity was verified by Blast. The primer sequences are shown in Table 2.

[0056] Table 2 Primer sequences of qPCR target genes.

[0057] The results of the study are as follows: The prior art summarizes the basic mechanisms responsible for the antibacterial activity of CDs, including the generation of a large amount of ROS, the destruction of DNA structure, and the destruction of bacterial membrane structure. In order to explore the generation of ROS in bacteria treated with CDH-CDs, the present invention uses the ROS indicator DCFH-DA, which is first deacetylated and then oxidized by ROS to produce DCF with fluorescent properties in the cell. As shown in Figures 8A and Fig. 9As shown in Figure 8, the ROS levels in MRSA or ESBL-E treated with 200 μg / mL CDH-CDs increased significantly, even higher than those in bacteria incubated with 100 mM H2O2 or the ROS inducer Rosup (125 μg / mL). Further examination showed that the generated ROS mainly included hydroxyl radicals (•OH) (Figure 8B), singlet oxygen (1O2) (Figure 8C), and superoxide anions (O2• - ) (Figure 8D). We then performed DNA gel electrophoresis to confirm the damage to the DNA structure. Fig.10 In Figure 6D, it is clear that the DNA of bacteria incubated with CDH-CDs at concentrations up to 400 μg / mL maintained integrity, indicating that CDH-CDs do not interrupt the DNA structure to kill bacteria. We then investigated the damage to the bacterial membrane structure. The SEM in Figure 6D demonstrated the morphological changes on the bacterial surface after CDH-CDs treatment. The expression of genes related to MRSA membrane synthesis and bacterial division was then explored by qPCR. After incubation with CDH-CDs, the expression of FabI, FemA, FtsA, FtsL, FtsZ, MraY, MurB, and MurC increased (Figure 8E). When bacteria are treated with drugs, these genes may be upregulated to maintain the stability of the cell wall, repair damaged cell walls, or accelerate bacterial division to fight threats. At the same time, downregulation of MurA was also observed (Figure 8E), which inhibits the synthesis of peptidoglycan, an important component of the cell wall, by downregulating the expression of UDP-GlcNAc transferase. Most importantly, we concluded that CDH-CDs can induce oxidative stress to produce endogenous ROS, including •OH, 1O2, and O2• - To destroy the bacterial membrane structure. At the same time, gene expression in bacteria may be affected by CDH-CDs, thereby hindering the normal reproduction and growth of bacteria ( Figure 8 F). These aspects lead to the excellent antibacterial properties of CDH-CDs.

[0058] Example 5 In vivo antibacterial activity of CDH-CDs Taking carbohydrazide carbon dots (CDH-CDs) as an example, the in vivo antibacterial activity of CDH-CDs was further explored. Bacterial infection is an important cause of chronic wound progression and seriously hinders wound healing. Considering the excellent in vitro antibacterial activity of CDH-CDs, we developed an experimental model by inducing artificial wounds infected with MRSA on the back of mice to evaluate the in vivo antibacterial properties of CDH-CDs. The specific methods are as follows: (1) Antibacterial activity in vivo Normal male KM mice (6-8 weeks, Hubei Provincial Laboratory Animal Center) were used as experimental mice to evaluate the in vivo antimicrobial properties of CDH-CDs. The mice were gently cared for following the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. The procedures were approved by the Animal Care and Use Committee of Hubei University of Technology with the Animal Ethics and Welfare Approval Affidavit No. HBUT20240014. Briefly, infected mice were inoculated by making a full-thickness wound (circle with a diameter of 10 mm) on the dorsal skin. Then, MRSA suspension (10 μL, 1 × 10 6 CFU / mL) was added to the wound to establish a mouse wound infection model. One day later, the infected mice were randomly divided into 3 groups (5 mice in the first group), and the samples were dripped on the wound with 10 μL PBS (control), 500 μg / mL CDH-CDs or CDH. The wound area and body weight of each mouse were recorded every 2 days. The area and overlay of the wound surface were analyzed and obtained by ImageJ.

[0059] (2) Animal tissue observation At the end of treatment, mice were euthanized. Infected wounds were then collected for hematoxylin and eosin (H&E) staining and Masson's trichrome staining. In addition, the heart, liver, spleen, lung, and kidney of each mouse were harvested and stained with H&E for comparison with the heart, liver, spleen, lung, and kidney of healthy control mice.

[0060] 2.20. Hemolytic test First, the anticoagulant-stabilized rabbit whole blood was centrifuged and washed with 0.9% NaCl solution to obtain pure erythrocytes. Then, erythrocytes were treated with CDH-CDs (12.5, 25.0, 50.0, 100, 200, 400 μg / mL) at 37 °C for 1 h. Erythrocytes in 0.9% NaCl solution were used as negative controls, while 0.5 wt% Triton-X100 was used as positive controls. After centrifugation, each sample was photographed, and the absorbance at 405 nm (OD 450) was measured with the supernatant. The hemolysis rate of each concentration was calculated using formula (4).

[0061] The test results are as follows: like Figures 11 to 14As shown, 1 day after the establishment of the mouse model, the wounds were treated with PBS (control), developed CDS, CDH-CDs, or pro-CDH drops, respectively (Figure 13A). Figure 13B-D illustrates representative images and statistical analysis of wound areas at different time points. Excitingly, CDH-CDs treatment significantly accelerated the progress of wound healing compared with wounds treated with PBS or CDH. After 9 days of treatment, the relative wound area of ​​mice in the CDH-CDs-treated group decreased to 14.2%, which was significantly smaller than that of the PBS- (23.9%) and CDH-treated groups (36.0%). At the end of treatment, the mice were euthanized, and the infected wounds were collected for H&E staining and Masson's trichrome staining. As shown in Figure 13E, mild inflammation, as well as collagen deposition and follicle regeneration were observed in the stained sections of the wounds treated with CDH-CDs, while the wounds treated with PBS or CDH showed considerable inflammatory cell infiltration. These results describe the powerful antibacterial activity of CDH-CDs against bacterial infections. Subsequently, we evaluated the biocompatibility of CDH-CDs. The body weight of mice in each group was measured during the treatment regimen, and its changes were not obvious (Figure 11). Meanwhile, histological examination of the major organs of mice receiving different treatments showed no obvious abnormalities compared with healthy mice (Figure 14). Minimal hemolytic activity was re-detected at concentrations up to 400 μg / mL (Figure 12), indicating that CDH-CDs have good blood compatibility. In summary, our results indicate that the developed CDs have excellent in vivo antibacterial effects and high biocompatibility.

[0062] In summary, the present invention developed 10 hydrazide-based carbon dots by a simple one-step hydrothermal method, and then measured the MIC of the 10 hydrazide-based carbon dots respectively. It was found that the carbon hydrazide-based carbon dots had the best antibacterial activity, followed by methyl hydrazide-based carbon dots, acetyl hydrazide-based carbon dots and benzoyl hydrazide-based carbon dots, followed by thiodihydrazide-based carbon dots, 3-pyridinecarboxyhydrazide-based carbon dots, p-carboxyphenylhydrazide-based carbon dots, then phenylhydrazide-based carbon dots, and finally isophthaloyl hydrazide-based carbon dots and salicylic hydrazide-based carbon dots. In addition, the present invention took the carbon hydrazide-based carbon dots with the best antibacterial effect as an example to further explore the in vitro antibacterial activity, in vitro anti-biofilm activity, antibacterial mechanism, in vivo antibacterial activity, etc. of the hydrazide-based carbon dots, and found that CDH-CDs showed excellent antibacterial activity against Gram-positive bacteria Staphylococcus aureus, Gram-negative bacteria Escherichia coli, and two drug-resistant bacteria MRSA and ESBL-E. The MIC of CDH-CDs against these bacteria was 25.0 or 50.0 μg / mL, which was much lower than that of the precursor CDH. Compared with the typical antibiotics rifampicin and kanamycin, MRSA and ESBL-E found it more difficult to develop resistance to CDH-CDs. In addition, CDH-CDs also showed excellent anti-biofilm efficacy. Notably, CDH-CDs effectively killed bacteria by inducing oxidative stress to produce endogenous ROS to destroy the bacterial membrane structure and affect gene expression to hinder the normal reproduction and growth of bacteria. In vivo antibacterial studies further demonstrated the good antibacterial properties and biocompatibility of CDH-CDs, providing a simple method to overcome bacterial infections.

[0063] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing hydrazide derivative-based carbon dots, characterized in that: The following steps are involved: Hydrazide derivative-based carbon dots were prepared using hydrazide derivatives as raw materials through a one-step hydrothermal method.

2. The preparation method according to claim 1, characterized in that: The hydrazide derivatives include at least one of phenylhydrazine, methylhydrazine, isophthaloylhydrazine, thiodihydrazide, salicylic hydrazide, acetylhydrazine, 3-pyridinecarboxyhydrazide, benzoylhydrazide, p-carboxyphenylhydrazine and carbohydrazide.

3. The preparation method according to claim 1, characterized in that: The preparation method comprises the following specific steps: dissolving the hydrazide derivative in water and subjecting it to ultrasonic treatment to obtain a mixture; The mixture is heated to react to obtain hydrazide derivative-based carbon dots.

4. The preparation method according to claim 1, characterized in that: The addition ratio of the hydrazide derivative to water is 100 mg~140 mg: 10 mL~14 mL.

5. The preparation method according to claim 3, characterized in that: The ultrasonic treatment time is 20 min to 40 min.

6. The preparation method according to claim 3, characterized in that: The temperature of the heating reaction is 160°C to 200°C.

7. The preparation method according to claim 3, characterized in that: The heating reaction time is 10h~14h.

8. A hydrazide derivative-based carbon dot obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the hydrazide derivative-based carbon dots obtained by the preparation method according to any one of claims 1 to 7 or the hydrazide derivative-based carbon dots according to claim 8 in the preparation of antibacterial materials.

10. Use of the hydrazide derivative-based carbon dots obtained by the preparation method according to any one of claims 1 to 7 or the hydrazide derivative-based carbon dots according to claim 8 in the preparation of antibacterial resistant materials.

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