A hydrazine derivative-based carbon dot and a preparation method and application thereof

Abstract

The application discloses a hydrazide derivative-based carbon dot and a preparation method and application thereof, relates to the technical field of antibacterial materials, and discloses a preparation method, which comprises the following steps: taking a hydrazide derivative as raw material, and preparing a hydrazide derivative-based carbon dot through a one-step hydrothermal method; the hydrazide derivative is at least one of phenylhydrazine, methylhydrazine, isophthalic dihydrazide, thio-diacid hydrazide, salicylic hydrazide, acetic hydrazide, 3-pyridine carbohydrazide, benzhydrazine, p-carboxyphenylhydrazine and carbon hydrazide; the application also protects the hydrazide derivative-based carbon dot prepared through the above method and the application of the hydrazide derivative-based carbon dot in the preparation of antibacterial materials and antibacterial drug resistance materials. The application has the beneficial effect that 10 kinds of hydrazide derivative-based carbon dots are prepared through a simple one-step hydrothermal method with 10 kinds of hydrazide derivatives as precursors; the 10 kinds of hydrazide derivative-based carbon dots all have antibacterial activity, and the carbon hydrazide-based carbon dot shows excellent antibacterial activity and can effectively eradicate biofilms.

Description

Technical Field

[0001] This invention relates to the field of antibacterial materials technology, specifically to an acylhydrazine derivative-based carbon dot, its preparation method, and its application. Background Technology

[0002] Antibiotics are widely used to treat bacterial infections, which can lead to the emergence and rapid spread of antibiotic resistance. Typical antibiotics are effective at eradicating common bacteria, but their effectiveness against resistant bacteria is significantly reduced. However, the development of alternative treatments has not kept pace with bacterial evolution. In particular, with the advent of biofilms, bacteria aggregate in extracellular polymers they secrete, further increasing their resistance to various antimicrobial agents. Many bacteria and chronic infections have been reported to be associated with bacterial biofilms. Faced with the serious problem of current bacterial infections and drug resistance, effective plans should be developed to regulate antibiotic use, slow the development of bacterial resistance, and maintain the effectiveness of critical medically available antibiotics. Many countries have enacted laws and regulations regarding the rational use of antibiotics. The discovery of novel antimicrobial agents is of great significance for treating bacterial infections and mitigating or altering the emergencies caused by antimicrobial resistance.

[0003] With the development of nanotechnology, nanomaterials with antibacterial activity have been extensively developed. Nanomaterials exhibit unique functions due to their extremely small particle size and large specific surface area, which has been widely explored in the field of antibacterial research. To date, various antibacterial nanoparticles have been reported, including metal-based nanoparticles, graphene oxide, dendritic macromolecules, and organic nanoparticles. Among them, carbon dots (CDs) are multifunctional carbon-based nanomaterials that show great potential in bioimaging, drug delivery, biosensing, tumor therapy, and antibacterial applications. 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 diverse structures, tunable structural defects, and enhanced optoelectronic properties. However, few existing technologies have reported on the antibacterial and anti-biofilm properties of CDs derived from such precursors. Summary of the Invention

[0004] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide an acylhydrazine derivative-based carbon dot, its preparation method and application.

[0005] This invention uses various acylhydrazine derivatives as precursors. Acylhydrazine derivatives are common raw materials in organic synthesis containing carbon and nitrogen elements, and are ideal precursors for preparing N-doped CDs derived from single precursors. This invention employs a simple hydrothermal synthesis method to prepare novel N-doped CDs. All 10 acylhydrazine derivative-based carbon dots synthesized in this invention exhibit certain antibacterial properties. Among them, CDs derived from inexpensive, safe, and environmentally friendly carbonylhydrazine (CDH-CDs) stand out due to their superior antibacterial performance. CDH-CDs exhibit the best antibacterial activity compared to other acylhydrazine derivative-based CDs, and also possess highly efficient anti-biofilm properties, low induction of bacterial resistance, and good biocompatibility. Furthermore, CDH-CDs can induce oxidative stress to produce endogenous ROS, thereby disrupting bacterial membrane structure and affecting gene expression, thus hindering normal bacterial reproduction and growth.

[0006] The technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for preparing hydrazide derivative-based carbon dots, comprising the following steps:

[0008] Acylhydrazine derivative-based carbon dots were prepared by a one-step hydrothermal method using acylhydrazine derivatives as raw materials.

[0009] This invention uses 10 acylhydrazine derivatives as precursors and prepares 10 acylhydrazine derivative-based carbon dots through a simple one-step hydrothermal method. All 10 acylhydrazine derivative-based carbon dots have antibacterial activity.

[0010] In a preferred embodiment of the present invention, the acylhydrazine derivative is at least one selected from phenylhydrazine, methylhydrazine, isophthalic acid hydrazine, thiodihydrazine, salicylhydrazine, acetylhydrazine, 3-pyridinecarboxylhydrazine, benzoylhydrazine, p-carboxyphenylhydrazine, and carbazine.

[0011] In a preferred embodiment of the present invention, the preparation method includes the following specific steps:

[0012] The acylhydrazine derivative was dissolved in water and subjected to ultrasonic treatment to obtain a mixture;

[0013] The mixture was heated to obtain hydrazide derivative-based carbon dots.

[0014] In a preferred embodiment of the present invention, the ratio of the acylhydrazine derivative to water is 100 mg~140 mg: 10 mL~14 mL. More preferably, it is 110 mg~130 mg: 11 mL~13 mL. More preferably, it is 115 mg~125 mg: 11.5 mL~12.5 mL.

[0015] In a preferred embodiment of the present invention, the ultrasonic treatment time is 20 min to 40 min. More preferably, it is 25 min to 35 min. More preferably, it is 28 min to 32 min.

[0016] 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.

[0017] In a preferred embodiment of the present invention, the heating reaction time is 10h to 14h. Preferably, the heating reaction time is 11h to 13h. More preferably, the heating reaction time is 11.5h to 12.5h.

[0018] Secondly, the present invention provides hydrazide derivative-based carbon dots obtained by the above-described preparation method.

[0019] Thirdly, the present invention provides the application of hydrazide derivative-based carbon dots obtained by the above preparation method or the above hydrazide derivative-based carbon dots in the preparation of antibacterial materials.

[0020] Fourthly, the present invention provides the application of hydrazine derivative-based carbon dots obtained by the above preparation method or the above hydrazine derivative-based carbon dots in the preparation of antibacterial drug-resistant materials.

[0021] This invention has at least one of the following beneficial effects:

[0022] This invention uses 10 acylhydrazine derivatives as precursors to prepare 10 acylhydrazine derivative-based carbon dots via a simple one-step hydrothermal method. All 10 acylhydrazine derivative-based carbon dots exhibit antibacterial activity. The carbon dot with the strongest antibacterial activity is the carbazide-based carbon dot, followed by methylhydrazine-based, acetylhydrazine-based, and benzoylhydrazine-based carbon dots, then thiodihydrazine-based, 3-pyridinecarboxylhydrazine-based, and p-carboxyphenylhydrazine-based carbon dots, then phenylhydrazine-based carbon dots, and finally isophthalic acid hydrazine and salicylhydrazine-based carbon dots. Among these, compared to other acylhydrazine derivative-based CDs, the carbazide-based carbon dots (CDH-CDs) exhibit superior antibacterial activity and can effectively eradicate biofilms. Compared to two typical antibiotics, rifampin and kanamycin, the two resistant bacteria did not show significant resistance to CDH-CDs. Mechanistic studies indicate that CDH-CDs kill bacteria by generating endogenous ROS and influencing gene expression. In vivo experiments further demonstrated the excellent antibacterial properties and biocompatibility of CDH-CDs. This invention proposes a simple method to overcome bacterial infections and reveals new insights into the potential applications of CDs. Attached Figure Description

[0023] Figure 1 shows the structural formulas of 10 acylhydrazide derivatives.

[0024] 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. Total XPS curves (F), C1s (G), N1s (H), and O1s (I) of CDH-CDs: High-resolution XPS curves.

[0025] Figure 3 shows the Zeta potential of CDH-CD in PBS (pH 7.4 or 5.6). Results are expressed as mean ± SD, n = 3.

[0026] Figure 4 shows the UV-Vis absorption spectra of the precursors CDH and CDH-CD.

[0027] Figure 5 The in vitro antibacterial 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. Photographs and corresponding bar graphs (indicated by black circles) of the ZOI developed during the paper disc dispersion test of CDH and CDH-CDs against MRSA (E and G) and ESBL-E (F and H), respectively. Results are expressed as mean ± SD, n = 3.

[0028] Figure 6In vitro antibacterial efficacy and bacterial resistance of CDH-CDs. Growth inhibition curves of different concentrations of CDH-CDs against MRSA (A) and ESBL-E (B). Results are expressed as mean ± SD, n = 5. MRSA (C) and ESBL-E (D) showed resistance to CDH-CDs, rifampin, and kanamycin, respectively. (E) Live / dead staining of MRSA or ESBL-E 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 with or without CDH-CDs. Surface shrinkage and fusion with pores are indicated by blue arrows. Scale bar, 1 μm.

[0029] Figure 7 In vitro anti-biofilm performance of CDH-CDs. Crystal violet staining photographs (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 photographs (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.

[0030] Figure 8The antibacterial mechanism of CDH-CDs. (A) ROS generated after culturing MRSA with sterile water (control), CDH-CDs, H2O2, or Rosup using DCFH-DA as a probe. Excitation: 488 nm. (B) Fluorescence spectra of MRSA after culturing with TA, CDH-CDs, or CDH-CDs and TA. The increased fluorescence intensity of TA at 410 nm indicates the generation of -OH. Excitation: 305 nm. (C) 1O2 generated by culturing different concentrations of MRSA with CDH-CDs using DPBF as a probe. (D) O2 generated by culturing different concentrations of MRSA with CDH-CDs using cytochrome c as a probe. Results are expressed as mean ± SD, n = 3. ***P < 0.001, Student's t-test was used. (E) Relative expression levels of mRNA in MRSA cultured with CDH-CDs. Results are expressed as mean ± SD, n = 3. (F) Proposed antimicrobial mechanism of CDH-CDs.

[0031] Figure 9 ROS generated in ESBL-E cultured using DCFH-DA as a probe with sterile water (control), CDH-CDs, H2O2, or Rosup. Excitation: 488 nm.

[0032] Figure 10 Agarose gel electrophoresis images of bacterial DNA cultured with different concentrations of CDH-CD.

[0033] Figure 11 The changes in body weight of MRSA-infected mice after different treatments are shown. Results are expressed as mean ± SD, n = 5.

[0034] Figure 12 Hemolysis assays were performed using different concentrations of CDH-CD. Results are expressed as mean ± SD, n = 3.

[0035] Figure 13 (A) Schematic diagram of treatment regimens for MRSA-infected mice. (B) Photographs of wounds in MRSA-infected mice after different treatments. (C) Relative wound areas in MRSA-infected mice after different treatments. Results are expressed as mean ± SD, n = 5. (D) B. (E) Schematic diagram of wounds after H&E and Masson trichrome staining at the end of treatment. Scale bar, 200 μm.

[0036] Figure 14H&E staining of major organs (heart, liver, spleen, lung, and kidney) in healthy mice or MRSA-infected mice after different treatments. Scale bar, 200 μm. Detailed Implementation

[0037] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0038] Example 1

[0039] 1. CD Synthesis

[0040] This embodiment uses a structural formula such as Figure 1 Using 10 acylhydrazine derivatives (phenylhydrazine, methylhydrazine, isophthalic acid hydrazine, thiodihydrazine, salicylhydrazine, acetylhydrazine, 3-pyridinecarboxylhydrazine, benzoylhydrazine, p-carboxyphenylhydrazine, and carbazine) as precursors, CD based on acylhydrazine derivatives was synthesized via a one-pot hydrothermal method. Specifically, these included:

[0041] 120 mg of each of the 10 acylhydrazine derivatives was dissolved in 12 mL of double-distilled water. After sonication for 30 minutes, the mixture was kept in an oven at 180 °C in a polytetrafluoroethylene-lined autoclave for 12 hours, and then cooled to room temperature. The resulting mixture was lyophilized, and the CDs were dispersed in ddwater at a concentration of 10 mg / mL for further use.

[0042] 2. Determine the minimum inhibitory concentration (MIC) of CDs synthesized from different precursors.

[0043] To screen for CDs with the best antibacterial properties, the MIC values ​​of four representative bacteria were determined in this embodiment, including Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli, and two drug-resistant bacteria, MRSA and ESBL-E. The specific determination methods are as follows:

[0044] (1) Bacterial culture

[0045] Bacterial strains obtained from the American Type Culture Collection (ATCC) included *Escherichia coli* (ATCC 35218), extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E, ATCC 25922), *Staphylococcus aureus* (ATCC 6538), and methicillin-resistant *Staphylococcus aureus* (MRSA, ATCC 43300). Bacterial suspensions were placed on sterile trypsin-soy broth (TSB) agar plates and incubated at 37 °C for 18 hours. Subsequently, bacterial colonies were picked from the plates and incubated overnight in liquid TSB to obtain bacteria in the logarithmic growth phase for further use.

[0046] (2) Determination of minimum inhibitory concentration (MIC)

[0047] With a density of 1 × 10 5 Bacteria at colony-forming units (CFU) / mL were inoculated into 96-well plates (100 μL / well). Then, 100 μL of the aforementioned hydrazide-based CD was mixed with different concentrations of bacteria in the wells. Bacteria incubated with CDs at a concentration of 0 μg / mL served as a negative control. After incubating the bacterial suspensions at 37 °C for 18 hours, the absorbance (OD 600) at 600 nm in each well was measured using a microplate reader (DNM-9602, China). The experiment was performed five times. The inhibition rate at each concentration was calculated using formula (1). The minimum inhibitory concentration (MIC) was the lowest concentration that showed an inhibition rate exceeding 90%.

[0048]

[0049] The measurement results are shown in Table 1:

[0050] Table 1. MIC (μg / mL) of CDs synthesized from different precursors

[0051]

[0052] As shown in Table 1, all 10 acylhydrazine derivative carbon dots exhibit antibacterial activity. The MIC of the methylhydrazine carbon dot is 50 or 100 μg / mL, the MIC of the acetylhydrazine carbon dot is 100 or 200 μg / mL, the MIC of the 3-pyridinecarboxylhydrazine carbon dot and the p-carboxyphenylhydrazine carbon dot is 200 μg / mL, the MIC of the thiodihydrazine carbon dot is 200 or 400 μg / mL, the antibacterial activity of the benzoylhydrazine carbon dot and the phenylhydrazine carbon dot is not completely the same against different bacteria, with MICs of 100, 200, 400 or 800 μg / mL, the MIC of the isophthalic acid hydrazine carbon dot is 3200 μg / mL, and the MIC of the salicylhydrazine carbon dot is greater than 3200 μg / mL.

[0053] Furthermore, compared with other CDs that use hydrazide derivatives as precursors, the carbazide-based CDs (CDH-CD) showed the lowest MICs, at 25 or 50 μg / mL (Table 1), indicating that CDH-CDs have superior antibacterial activity compared with other hydrazide-based CDs.

[0054] 3. Material Characterization

[0055] Taking carbon hydrazine-based carbon dots (CDH-CDs) as an example, the CDH-CDs prepared above were characterized using the following methods:

[0056] Zeta potentials were recorded using a Zetasizer Nano ZS (Malvern Panalytical, UK). UV-Vis absorption spectra were measured using an N4SUV-VIS spectrophotometer (INESA Analytical Instrument Co., Ltd, China). X-ray powder diffraction (XRD) analysis was performed on a D8 Advance (Bruck, 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 using an ESCALAB 250Xi (Thermo Fisher Scientific, USA).

[0057] Characterization results as follows Figures 2-4 As shown.

[0058] exist Figure 2In the buffer solutions, CDH-CDs are negatively charged, with Zeta potentials of -9.03 ± 1.58 mV and -6.06 ± 0.62 mV in neutral (pH 7.4) or acidic (pH 5.6) buffers, respectively. Compared to the precursor CDH, the absorption at 215.3 nm in the UV-Vis absorption spectrum of CDH-CDs changes to 206.3 nm (Figure 4), which is 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 2C, CDH-CDs are quasi-spherical with a diameter ranging from 1.5 to 5.0 nm and an interplanar spacing of 0.21 nm. The XRD pattern of CDH-CDs shown in Figure 2D exhibits a broad peak near 25.5°, indicating the formation of disordered carbon atoms. We then used TEM to examine the morphology of CDH-CDs. As shown in Figures 2B and 2C, CDH-CDs are quasi-spherical with a diameter ranging from 1.5 to 5.0 nm and an interplanar spacing of 0.21 nm. The XRD pattern of CDH-CDs shown in Figure 2D exhibits a broad peak near 25.5°, indicating the formation of disordered carbon atoms. Furthermore, 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 a peak corresponding to -NH2 at 3389.50 cm⁻¹. - At point ¹, C=O is at 1708.89 cm⁻¹, and CN is at 1554.22 cm⁻¹. - ¹ and the stretching vibration of NH at 1196.28 cm⁻¹. Furthermore, compared to CDH, the FT-IR spectra of CDH-CDs show a greater stretching vibration at 1323.67 cm⁻¹. - ¹、952.61 cm - ¹ and 704.94 cm -New peaks appeared at ¹, attributed to bond stretching of -NO2, CO, and CC, respectively. These results indicate the formation of new chemical bonds during the synthesis of CDH-CDs. Furthermore, the chemical bonds and basic composition were examined by XPS, consistent with FT-IR results. The overall XPS curve (Fig. 2F) shows that CDH-CDs contain carbon, nitrogen, and oxygen, with elemental proportions of 36.08%, 43.68%, and 20.24%, respectively. The high-resolution C1s plot (Fig. 2G) shows three fitted peaks at 283.58 eV, 284.98 eV, and 287.41 eV, located as CC, CN, and C=O bonds, respectively. Examination of the high-resolution N1s plot (Fig. 2H) revealed three peaks at 397.47 eV, 399.23 eV, and 400.77 eV, representing -NH2, NN, and -NO2, respectively. In the high-resolution O1s plot (Figure 2I), the two peaks at 530.19 eV and 531.44 eV are assigned to CO and C=O bonds.

[0059] Example 2: In vitro antibacterial properties of CDH-CDs

[0060] I. Taking carbon hydrazine-based 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 methods are as follows:

[0061] (1) Detection of inhibition zone

[0062] MRSA or ESBL-E (1 × 10) 5 CFU / mL were spread onto TSB or MacConkey (MAC) agar plates. Sterilized Oxford cups were then placed on the plates 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.

[0063] (2) Monitoring of bacterial growth inhibition

[0064] At 37 ℃, MRSA or ESBL-E were inoculated with different concentrations (0, 12.5, 25.0, 50.0 μg / mL) of CDH-CDs (1 × 10⁻⁶ μg / mL). 5 The concentration of CFU / mL was measured every 2 hours until 24 hours to monitor bacterial growth inhibition. The experiment was performed 5 times.

[0065] (3) Staining of live / dead bacteria

[0066] MRSA or ESBL-E grown in the logarithmic phase were collected by centrifugation and resuspended in 0.85% NaCl solution. Bacteria (1 × 10⁻⁶) were then... 8 CFU / mL) was 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.

[0067] (4) Scanning electron microscopy (SEM) characterization of bacterial morphology

[0068] MRSA or ESBL-E was incubated with 800 μg / mL CDH-CDs at 37 °C for 4 h, followed by centrifugation for collection. The collected bacteria were fixed and dehydrated. After being freeze-dried and treated with gold spray, the samples were imaged using a scanning electron microscope (SEM, JSM-6390LV, JEOL, Japan).

[0069] The test results are as follows:

[0070] As shown in Figures 5A and 5B, when incubated with bacteria at 37 ℃ for 18 h, the MIC value of CDH-CDs against ESBL-E or MRSA was 25 μg / mL, significantly lower than that of CDH. Furthermore, CDH-CDs also exhibited better antibacterial activity against Staphylococcus aureus and Escherichia coli (Figures 2C and 2D), indicating that CDH-CDs possess superior antibacterial activity. The disk diffusion assay further validated the excellent antibacterial effect of CDH-CDs. The diameter of the zone of inhibition (ZOI) of CDH-CDs against MRSA (Figures 2E and 2G) or ESBL-E (Figures 2F and 2H) was 2.7 times and 2.5 times that of the precursor CDH, respectively. Bacterial growth inhibition monitoring showed that contrast-enhanced CDs at concentrations of 25.0 (MIC) or 50.0 (2 × MIC) μg / mL effectively inhibited the growth of MRSA or ESBL-E within 24 h. When the concentration of CDH-CDs was below the MIC (e.g., 12.5 μg / mL, 1 / 2 MIC), bacterial growth was somewhat limited but eventually continued (Figs. 6A and 6B). Simultaneously, 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, inducing death (Fig. 6C). Morphological changes on the bacterial surface were then examined using SEM. After treatment with 800 μg / mL CDH-CDs at 37 °C for 4 h, significant surface irregularities and collapses were observed in the skeletons of MRSA and ESBL-E ( ). Figure 6 (D) confirms that CDH-CDs disrupt the integrity of bacterial membranes. All these results indicate that CDH-CDs possess excellent antibacterial activity against a variety of bacteria.

[0071] II. Drug Resistance Testing

[0072] The development of drug resistance is a major problem, leading to reduced antibacterial efficacy of antibiotics. Therefore, we evaluated the ability of MRSA and ESBL-E to induce resistance in CDH-CDs. Two typical antibiotics, rifampin and kanamycin, were used as controls. Successive passages of bacteria were cultured in the presence of subinhibitory concentrations of the drug to estimate resistance.

[0073] Drug resistance passage experiments:

[0074] MRSA or ESBL-E was incubated with different concentrations of CDH-CDs, rifampin, or kanamycin at 37 °C for 24 h to measure the MIC of each drug. Subsequently, bacteria in a 0.5-fold MIC suspension were added to fresh culture medium at a density of 1 × 10⁵ CFU / mL and further treated with different concentrations of CDH-CDs, rifampin, or kanamycin. This was repeated 14 times.

[0075] The test results are shown in Figures 6E and F. MRSA and ESBL-E developed significant resistance to rifampin and kanamycin. Exposure to rifampin led to a 256-fold and 128-fold increase in the MIC of MRSA or ESBL-E, respectively, after 14 passages. Furthermore, the kanamycin MIC of both MRSA and ESBL-E increased 64-fold after 14 passages. No significant changes in the MIC of MRSA or ESBL-E were observed after 14 passages in the presence of CDH-CDs (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.

[0076] Example 3: In vitro anti-biofilm activity of CDH-CDs

[0077] Taking carbon hydrazine-based carbon dots (CDH-CDs) as an example, this embodiment evaluates the therapeutic effect of CDH-CDs on biomembranes. The evaluation method is as follows:

[0078] (1) Determination of inhibition of biofilm formation

[0079] MRSA or ESBL-E (1 × 10) 5 Different concentrations (0, 25.0, 50.0, 100, 200, 400, 800 μg / mL) of CDH-CDs were inoculated and cultured at 28 °C for 72 h without stirring. Bacteria incubated with CDH-CDs at a concentration of 0 μg / mL served as a negative control. After rinsing five times with phosphate-buffered saline (PBS, pH 7.4) to remove planktonic bacteria, the biofilms were fixed, washed, and dried. Subsequently, the fixed biofilms were stained with 1 wt% crystal violet for 5 minutes and then rinsed three more times with PBS. Finally, the stained biofilms were dissolved in 33.3% acetic acid (200 μL / well), photographed with a smartphone, and the absorbance (OD570) at 570 nm was measured using a microplate reader. The experiment was performed five times. The biofilm inhibition rate at each concentration was calculated using formula (2).

[0080]

[0081] (2) Biofilm clearance assay

[0082] Biofilm formation is a key factor in antibiotic resistance, encouraging researchers to accelerate the development of effective strategies for biofilm eradication. MRSA or ESBL-E (1 × 10⁻⁶) 5 The biofilm (CFU / mL) was incubated in TSB at 28 °C for 48 h without stirring to promote biofilm formation. After rinsing three times with PBS, the biofilm was incubated with CDH-CDs at different concentrations (0, 25.0, 50.0, 100, 200, 400, 800 μg / mL) for 24 h, then fixed, stained, dissolved, and the 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).

[0083]

[0084] MRSA or ESBL-E were incubated in TSB containing circular coverslips at 28 °C for 48 h to form a biofilm. The biofilm was then treated with different concentrations (0, 25.0, 50.0 μg / mL) of CDH-CDs for 24 h, followed by DMAO and PI staining. The circular coverslips were imaged using CLSM to estimate the biofilm removal efficiency of CDH-CDs.

[0085] Test results are as follows Figure 7As shown, MRSA or ESBL-E were cultured with CDH-CDs at 28 °C for 72 hours without stirring, then fixed and stained with crystal violet to quantify the biomass of the formed biofilm. As shown in Figures 7A and 7B, the inhibition rate of biofilm formation exceeded 90% when MRSA or ESBL-E was incubated with CDH-CDs at concentrations higher than 100 μg / mL. Furthermore, CDH-CDs were also able to disrupt pre-formed biofilms, with an MBEC of 400 μg / mL for MRSA and 50 μg / mL for ESBL-E (Figures 7C and 7D). The staining of the 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 from the circular coverslip. Based on the above results, we can conclude that CDH-CDs can effectively eradicate MRSA and ESBL-E biofilms.

[0086] Example 4: Investigation into the antibacterial mechanism of CDH-CDs

[0087] Taking carbon hydrazine-based carbon dots (CDH-CDs) as an example, the antibacterial mechanism of CDH-CDs was further investigated. The investigation method is as follows:

[0088] (1) Generation of reactive oxygen species (ROS) in bacteria

[0089] MRSA or ESBL-E was incubated with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) for 20 minutes. Subsequently, the bacteria were incubated for 45 minutes with 200 μg / mL CDH-CD, 100 mM H2O2, the reactive oxygen species (ROS) inducer Rosup (125 μg / mL), or sterile water (control). The generated ROS oxidized 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 ROS content was directly proportional to the fluorescence intensity of DCF.

[0090] (2) Production of hydroxyl radicals (•OH) in bacteria

[0091] The generation of hydroxyl radicals (•OH) in MRSA was monitored by changes in the fluorescence intensity of terephthalic acid (TA). MRSA grown 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, respectively. After 1 hour of incubation, the fluorescence intensity of each sample was recorded using a fluorescence spectrophotometer (λex = 305 nm). The •OH content was proportional to the fluorescence intensity at 410 nm.

[0092] (3) Production of singlet oxygen (1O2) in bacteria

[0093] The generation of singlet oxygen (1O2) in MRSA was monitored by changes in the absorption of 1,3-diphenylisobenzofuran (DPBF). MRSA (1 × 10⁻⁶) was used as the absorbance of the MRSA. 8 The sample was incubated for 45 minutes with DPBF (100 μg / mL) at concentrations of 0 (control), 500, or 1000 μg / mL, and CDH-CDs. The UV-Vis absorption spectra of each sample were recorded, with the 1O2 content inversely proportional to the absorbance of DPBF.

[0094] (4) Superoxide anions (O2•) in bacteria - The generation of )

[0095] Monitoring superoxide anion (O2•) in MRSA by changes in cytochrome c uptake - The generation of MRSA (1 × 10⁻⁶). 8 Treatment with cytochrome c (0.5 mg / mL) and CDH-CDs (0, 50.0, 200, or 500 μg / mL) for 45 minutes was performed. The absorbance at 550 nm (OD 550) was recorded, and its relationship with O2·200 was analyzed. - The content is directly proportional to the content of.

[0096] (5) DNA gel electrophoresis

[0097] Bacteria were incubated with CDH-CDs at concentrations 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 (140 V, 45 min). The gel was imaged under UV light (254 nm).

[0098] (6) Quantitative real-time PCR (qPCR)

[0099] Bacteria were incubated with 0 (control) or 50.0 μg / mL CDH-CDs at 37 °C for 18 h. Total RNA was extracted from the treated bacteria using TRIzol reagent. cDNA was then synthesized using the RevertAid First-Strand cDNA Synthesis Kit (K1622, Thermo Fisher Scientific). The obtained cDNA was analyzed by real-time quantitative polymerase chain reaction (qPCR) using a KiCqStart™ SYBR® GreenqPCR ReadyMix™ (KCQS0ri0, Sigma-Aldrich). Primer sequences for the target genes were designed using Beacon Designer8 after searching the National Center for Biotechnology Information (NCBI), and specificity was verified by BLAST. The primer sequences are shown in Table 2.

[0100] Table 2 Primer sequences for qPCR target genes.

[0101]

[0102] The results of the investigation are as follows:

[0103] Existing technologies summarize the basic mechanisms responsible for the antibacterial activity of CDs, including the generation of large amounts of ROS, DNA structure disruption, and bacterial membrane structure disruption. To explore ROS generation in bacteria treated with CDH-CDs, this invention uses the ROS indicator DCFH-DA, which is first deacetylated and then oxidized by ROS to produce fluorescent DCF within the cell. (See Figure 8A and...) Figure 9 As shown, the ROS levels in MRSA or ESBL-E treated with 200 μg / mL CDH-CDs were significantly increased, even exceeding the ROS levels of bacteria incubated with 100 mM H2O2 or the ROS inducer Rosup (125 μg / mL). Further examination revealed that the generated ROS mainly consisted of hydroxyl radicals (•OH) (Figure 8B), singlet oxygen (1O2) (Figure 8C), and superoxide anions (O2•OH). - (Figure 8D). We then performed DNA gel electrophoresis to determine the disruption of the DNA structure. Figure 10 In the study, it was evident that the DNA of bacteria incubated with CDH-CDs at concentrations up to 400 μg / mL maintained its integrity, indicating that CDH-CDs do not disrupt DNA structure to kill bacteria. We then investigated damage to the bacterial membrane structure. SEM images in Figure 6D demonstrate the morphological changes on the bacterial surface after CDH-CDs treatment. Subsequently, qPCR was used to explore gene expression associated with MRSA membrane synthesis and bacterial division. Incubation with CDH-CDs resulted in increased expression of Fabi, FemA, FtsA, FtsL, FtsZ, MrY, MurB, and MurC (Figure 8E). These genes may be upregulated when bacteria are treated with drugs to maintain cell wall stability, repair damaged cell walls, or accelerate bacterial division to combat threats. Simultaneously, downregulation of MurA was observed (Figure 8E), which inhibits peptidoglycan synthesis by downregulating the expression of UDP-GlcNAc transferase, a crucial component of the cell wall. Most importantly, we conclude that CDH-CDs can induce oxidative stress to produce endogenous ROS, including •OH, 1O2, and O2•. - This disrupts the bacterial membrane structure. Simultaneously, gene expression in bacteria may be affected by CDH-CDs, thereby hindering normal bacterial reproduction and growth. Figure 8 F). These aspects contribute to the excellent antibacterial properties of CDH-CDs.

[0104] Example 5: In vivo antibacterial activity of CDH-CDs

[0105] Taking carbon hydrazine-based carbon dots (CDH-CDs) as an example, we further explored the in vivo antibacterial activity of CDH-CDs. Bacterial infection is a significant cause of chronic wound progression and severely hinders wound healing. Considering the excellent in vitro antibacterial activity of CDH-CDs, we developed an experimental model by inducing MRSA infection in the back of mice to evaluate the in vivo antibacterial performance of CDH-CDs. The specific methods are as follows:

[0106] (1) In vivo antibacterial activity

[0107] Normal male KM mice (6-8 weeks old, Hubei Provincial Experimental Animal Center) were used as experimental mice to evaluate the in vivo antimicrobial properties of CDH-CDs. Mice were cared for gently according to the guidelines outlined in the *Guidelines for the Care and Use of Laboratory Animals*. These procedures were approved by the Animal Care and Use Committee of Hubei University of Technology, with affidavit of approval number HBUT20240014. In short, infected mice were created by making a full-thickness incision (a circle with a diameter of 10 mm) on the dorsal side of the skin. Then, MRSA suspension (10 μL, 1 × 10⁻⁶) was applied. 6 A mouse wound infection model was established by adding CFU / mL of PBS to the wound. One day later, the infected mice were randomly divided into three groups (n=5 mice in the first group). Samples were applied to the wound with 10 μL PBS (control), 500 μg / mL CDH-CDs, or CDH, respectively. The wound area and body weight of each mouse were recorded every two days. The wound surface area and overlay images were obtained using ImageJ analysis.

[0108] (2) Animal tissue observation

[0109] At the end of treatment, the mice were euthanized. Infected wounds were then collected for hematoxylin and eosin (H&E) staining and Masson's trichrome staining. Additionally, the heart, liver, spleen, lung, and kidney of each mouse were harvested and H&E stained for comparison with those of healthy control mice.

[0110] 2.20. Hemolytic test

[0111] First, rabbit whole blood stabilized with anticoagulant was centrifuged and washed with 0.9% NaCl solution to obtain pure red blood cells. Then, the red blood cells were treated with CDH-CDs (12.5, 25.0, 50.0, 100, 200, 400 μg / mL) at 37 °C for 1 h. Red blood cells in 0.9% NaCl solution were used as a negative control, while 0.5 wt% Triton-X100 was used as a positive control. After centrifugation, each sample was photographed, and the absorbance (OD 450) at 405 nm was measured using the supernatant. The hemolysis rate for each concentration was calculated using formula (4).

[0112]

[0113] The test results are as follows:

[0114] like Figures 11-14 As shown in Figure 13A, one day after establishing the mouse model, wounds were treated with PBS (control), developed CDS, CDH-CDs, or the precursor CDH, respectively (Figure 13A). Figures 13B-D illustrate representative images and statistical analyses of the wound area at different time points. Excitingly, CDH-CDs treatment significantly accelerated wound healing compared to wounds treated with PBS or CDH. After 9 days of treatment, the relative wound area in the CDH-CDs-treated group decreased to 14.2%, significantly smaller than that in the PBS-treated group (23.9%) and the CDH-treated group (36.0%). At the end of treatment, the mice were euthanized, and infected wounds were collected for H&E staining and Masson trichrome staining. As shown in Figure 13E, mild inflammation, collagen deposition, and follicular regeneration were observed in stained sections of wounds treated with CDH-CDs, while wounds treated with PBS or CDH showed considerable inflammatory cell infiltration. These results describe the potent antibacterial activity of CDH-CDs against bacterial infection. Subsequently, we evaluated the biocompatibility of CDH-CDs. Body weight was measured in each group of mice during the treatment regimen, and the changes were not significant (Figure 11). Furthermore, histological examination of major organs in mice receiving different treatments showed no significant abnormalities compared to healthy mice (Figure 14). Minimal hemolytic activity was re-detected at concentrations up to 400 μg / mL (Figure 12), indicating good blood compatibility of CDH-CDs. In summary, our results demonstrate that the developed CDs possess excellent in vivo antibacterial efficacy and high biocompatibility.

[0115] In summary, this invention developed 10 acylhydrazine derivative-based carbon dots using a simple one-step hydrothermal method. The MICs of these 10 acylhydrazine derivative-based carbon dots were then determined. The results showed that the carbon dot with the best antibacterial activity was the carbazine-based carbon dot, followed by the methylhydrazine-based, acetylhydrazine-based, and benzoylhydrazine-based carbon dots, then the thiodihydrazine-based, 3-pyridinecarboxylhydrazine-based, and p-carboxyphenylhydrazine-based carbon dots, then the phenylhydrazine-based carbon dots, and finally the isophthalic acid hydrazine and salicylhydrazine-based carbon dots. Furthermore, taking the carbazine-based carbon dot with the best antibacterial effect as an example, this invention further investigated the in vitro antibacterial activity, in vitro anti-biofilm activity, antibacterial mechanism, and in vivo antibacterial activity of the acylhydrazine derivative-based carbon dots. The results showed that CDH-CDs exhibited excellent antibacterial activity against Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli, and two drug-resistant bacteria, MRSA and ESBL-E. The MICs of CDH-CDs against these bacteria were 25.0 or 50.0 μg / mL, significantly lower than the MIC of the precursor CDH. Compared to typical antibiotics rifampin and kanamycin, MRSA and ESBL-E showed greater difficulty in inducing resistance to CDH-CDs. Furthermore, CDH-CDs exhibited excellent anti-biofilm efficacy. Notably, CDH-CDs effectively killed bacteria by inducing oxidative stress to generate endogenous ROS, thereby disrupting bacterial membrane structure and affecting gene expression to inhibit normal bacterial reproduction and growth. In vivo antibacterial studies further demonstrated the good antibacterial properties and biocompatibility of CDH-CDs, providing a simple method for overcoming bacterial infections.

[0116] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. Use of a hydrazide derivative-based carbon dot in the preparation of an antibacterial material, characterized in that, The preparation method of the hydrazide derivative-based carbon dots comprises the following steps: The hydrazide derivative-based carbon dots are prepared by using carbon hydrazide as raw material through one-step hydrothermal method.

2. Use according to claim 1, characterized in that, The preparation method comprises the following specific steps: The carbon hydrazide is dissolved in water and ultrasonic treated to obtain a mixture; The mixture is heated to obtain the hydrazide derivative-based carbon dots.

3. Use according to claim 2, characterized in that, The adding ratio of the carbon hydrazide to water is 100 mg-140 mg:10 mL-14 mL.

4. Use according to claim 2, characterized in that, The ultrasonic treatment time is 20 min-40 min.

5. Use according to claim 2, characterized in that, The heating reaction temperature is 160 DEG C-200 DEG C.

6. Use according to claim 2, characterized in that, The heating reaction time is 10 h-14 h.

7. Use of a hydrazide derivative-based carbon dot in the preparation of an antibacterial resistance material, characterized by, The preparation method of the hydrazide derivative-based carbon dots comprises the following steps: The hydrazide derivative-based carbon dots are prepared by using carbon hydrazide as raw material through one-step hydrothermal method.

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