Antibacterial blue fluorescent carbon dots derived from fungi, preparation method and application thereof

Abstract

This invention discloses a fungal-derived antibacterial blue fluorescent carbon dot, its preparation method, and its application. The carbon dot is synthesized by heating an extract of *Metacarpus mesoderm*. Specifically, this invention uses a natural product extract of the endophytic bacterium YSP3 as a raw material and synthesizes blue fluorescent carbon dots (Du-CDs) using a one-step solvothermal method. The average particle size of these Du-CDs is 13.9 nm, and they are positively charged. The Du-CDs prepared by this invention can efficiently kill bacteria and fungi, exhibiting broad-spectrum antibacterial properties. This invention provides carbon dots with better bactericidal effects and wound-healing capabilities compared to natural fungal extracts, along with good biocompatibility, and develops novel antibacterial drugs with good fluorescence.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials and biotechnology, specifically relating to an antibacterial blue fluorescent carbon dot derived from fungi, its preparation method, and its application. Background Technology

[0002] Microbial infections have long posed a serious threat to global human health, and antibiotics are currently the most widely used antibacterial method. However, the overuse of antibiotics has led to increased microbial resistance, making the development of novel antibacterial therapies urgently needed. Research has shown that nanomaterials possess antibacterial and bactericidal properties. However, many antibacterial nanomaterials suffer from drawbacks such as poor water solubility and unsatisfactory biocompatibility, and most nanomaterials lack antifungal activity. Therefore, there is an urgent need to develop nanomaterials with broad-spectrum antibacterial properties and good biocompatibility to address these issues.

[0003] Carbon dots (CDs), as a novel type of zero-dimensional carbon-based fluorescent nanomaterial, have attracted increasing attention and become a research hotspot in recent years due to their outstanding properties. Compared with other nanomaterials, CDs are easy to synthesize and possess high water dispersibility, low toxicity, good biocompatibility, and excellent photoluminescence properties. Because their surfaces contain different types of functional groups, such as amino (-NH2), hydroxyl (-OH), and carboxyl (-COOH), CDs exhibit different electrical properties. Studies have shown that positively charged CDs can easily bind to and interact with negatively charged bacterial cell membranes through electrostatic interactions, killing bacteria by damaging the cell membrane.

[0004] Fungi are an important source of natural antimicrobial compounds. However, antimicrobial compounds extracted directly from fungi have drawbacks such as high toxicity, limited stability, difficulty in long-term storage, and general antimicrobial performance. Therefore, there is an urgent need to develop more efficient antimicrobial materials. Summary of the Invention

[0005] Purpose of the invention: To address the problems existing in the prior art, this invention proposes an antibacterial blue fluorescent carbon dot derived from fungi. This invention utilizes fungal extracts to prepare highly efficient antibacterial fluorescent carbon dots, providing a new and flexible strategy for developing novel antibacterial nanomaterials from natural sources. It also avoids the disadvantages encountered in the discovery and development of antibacterial drugs from natural products, such as high toxicity, limited stability, difficulty in long-term storage, and general antibacterial performance.

[0006] The present invention also provides a method for preparing and applying the antibacterial blue fluorescent carbon dots derived from the fungi.

[0007] Technical solution: In order to achieve the above objectives, the present invention provides an antibacterial blue fluorescent carbon dot derived from fungi, wherein the carbon dot is synthesized by heating an extract of *Metacarpa*.

[0008] The *Diaporthe unshiuensis* is YSP3.

[0009] The extract is an extract of ethyl acetate.

[0010] The method for preparing fungal-derived antibacterial blue fluorescent carbon dots according to the present invention includes the following steps:

[0011] (1) Culture the interstrain of *Syntheticus* to obtain mycelia;

[0012] (2) Transfer the mycelium to potato dextrose medium and continue culturing;

[0013] (3) The culture medium was filtered and then extracted with ethyl acetate to obtain the extract;

[0014] (4) The extract is heated to obtain a solvent-free substance;

[0015] (5) The solvent-free substance was dissolved in deionized water to form a light yellow solution. After high-speed centrifugation and filtration, an aqueous solution containing Du-CDs was obtained. After dialyzing, the solution was freeze-dried to obtain blue carbon dots.

[0016] In step (1), the culture conditions are 25-30℃ for 3-7 days; in step (2), the culture conditions are 20-30℃ for 1-5 days.

[0017] Preferably, in step (1), strain YSP3 is cultured on a PDA plate at 28°C for 5 days; in step (2), the mycelium is transferred from the plate to potato dextrose medium and cultured at 25°C and 150 rpm for 3 days.

[0018] In step (3), the culture medium is filtered through filter paper and then extracted 2-3 times with an equal volume of ethyl acetate.

[0019] As a preferred option, step (3) uses 185mm Whatman 1 filter paper, and 200mL of ethyl acetate is added in equal volume ratio during extraction, and the extraction is performed twice.

[0020] In step (4), the extract is transferred to a stainless steel autoclave and heated at 120-240°C for 6-18 hours to obtain a solvent-free substance.

[0021] Preferably, in step (4), the extract is transferred to a stainless steel autoclave and heated at 180°C for 12 hours to obtain a solvent-free substance.

[0022] Furthermore, the stainless steel autoclave is a stainless steel high-temperature autoclave with a polytetrafluoroethylene liner.

[0023] As a preferred method, (5) the solid substance is dissolved in deionized water to form a light yellow solution, centrifuged at high speed, and filtered with a 0.22 μm filter head to obtain an aqueous solution containing Du-CDs.

[0024] Further, in step (5), the centrifugation speed is 12000 rpm, the filtrate obtained by filtration is dialyzed with Milli-Q water for 6-18 h, and then freeze-dried to obtain fluorescent carbon dots Du-CDs.

[0025] The application of the fungal-derived antibacterial blue fluorescent carbon dots described in this invention in the preparation of in vivo and in vitro antibacterial drugs or reagents.

[0026] The antibacterial microorganisms include Gram-positive bacteria, Gram-negative bacteria, and fungi; the Gram-positive bacteria include Escherichia coli, Proteus, and Serratia marcescens; the Gram-negative bacteria include Staphylococcus aureus, Staphylococcus epidermidis, and Micrococcus luteus; and the fungi include Saccharomyces cerevisiae and Candida albicans.

[0027] The application of the fungal-derived antibacterial blue fluorescent carbon dots described in this invention in the preparation of drugs with wound-healing capabilities.

[0028] This invention synthesizes blue fluorescent carbon dots (Du-CDs) using *Diaporthe unshiuensis* YSP3 as raw material and natural product extracts of the endophytic fungus YSP3 as raw material via a one-step solvothermal method. The Du-CDs have an average particle size of 13.9 nm and are positively charged. Du-CDs can efficiently kill bacteria and fungi, exhibiting broad-spectrum antibacterial properties. This invention provides carbon dots with better bactericidal efficacy and wound-healing ability, as well as good biocompatibility, compared to natural fungal extracts, and develops novel antibacterial drugs with good fluorescence. The Du-CDs prepared by this invention can be stored for a longer period of time compared to fungal extracts and exhibit better resistance to traditional antibiotics.

[0029] In this invention, positively charged blue fluorescent carbon dots (Du-CDs) were successfully synthesized via a solvothermal method using the ethyl acetate extract of *Metacarpus mesoderm* YSP3 culture broth as a raw material. The antibacterial activity of Du-CDs against bacteria (Gram-negative and Gram-positive) and fungi was significantly enhanced compared to fungal extracts. Du-CDs exhibited enhanced antibacterial activity and wound-healing ability in mouse models, demonstrating good biocompatibility. The specific nanomaterials prepared in this invention possess unique properties due to their size, shape, charge, stability, and surface area, enabling them to penetrate pathogenic bacteria more quickly, interfere with cellular contents, and ultimately lead to bacterial death.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0031] The preparation process of the blue fluorescent carbon dots (Du-CDs) prepared by this invention is simple, rapid, economical, effective, and environmentally friendly. Compared with the extract of *Metacarpus mesoderm* YSP3, Du-CDs exhibit better antibacterial and antifungal effects and accelerate wound healing. Furthermore, Du-CDs also demonstrate good biocompatibility. This invention provides a new and flexible strategy for developing novel antibacterial nanomaterials from natural products, avoiding the drawbacks encountered in the discovery and development of antibacterial drugs from natural products. Attached Figure Description

[0032] Figure 1 Transmission electron microscopy (TEM) image of Du-CDs;

[0033] Figure 2 The Zeta potential of Du-CDs;

[0034] Figure 3 The UV-Vis absorption spectrum of Du-CDs;

[0035] Figure 4 The fluorescence spectrum of Du-CDs;

[0036] Figure 5 Antibacterial evaluation of Du-CDs;

[0037] Figure 6 Evaluation of the time-dependent antibacterial activity of Du-CDs;

[0038] Figure 7 To evaluate the in vivo antibacterial effect and wound healing of Du-CDs;

[0039] Figure 8 Evaluation of Du-CDs hemolytic activity.

[0040] In the attached figure, ** represents statistically significant p < 0.01, and *** represents statistically significant p < 0.001. Detailed Implementation

[0041] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0042] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer.

[0043] The *Diaporthe unshiuensis* YSP3 used in this invention is a known strain in the prior art; Chemical Investigation of Endophytic Diaporthe unshiuensis YSP3 Reveals New Antibacterial and Cytotoxic Agents, JOURNAL OF FUNGI, 2023, 9(2). Provided by Southeast University.

[0044] The other strains are all commonly used wild-type strains.

[0045] Example 1

[0046] Preparation of Du-CDs

[0047] Strain YSP3 was inoculated onto PDA plates and cultured at 28°C for 5 days. After 5 days of culture, the mycelium completely covered the plate. All strains on the plate were transferred to 200 mL of potato dextrose broth and cultured at 25°C and 150 rpm for 3 days. The culture broth was filtered through filter paper (185 mm Whatman 1 filter paper) and then extracted thoroughly with equal volumes of ethyl acetate (2 x 200 mL). The organic phases from the ethyl acetate extraction were combined, and all extracts were transferred to a stainless steel autoclave lined with polytetrafluoroethylene and heated at 180°C for 12 h to obtain a solvent-free solid. The solid was completely dissolved in deionized water and centrifuged at 12000 rpm for 10 min to obtain the supernatant. The supernatant was filtered through a 0.22 μm filter to obtain an aqueous solution containing Du-CDs. The obtained Du-CDs aqueous solution was further dialyzed at room temperature for 12 h using a 1000 Da dialysis bag containing ultrapure water. The dialysate was lyophilized to obtain Du-CDs blue fluorescent carbon dots, which were stored at 4°C for later use.

[0048] Example 2

[0049] 1. Morphological observation of Du-CDs

[0050] The Du-CDs fluorescent carbon dots prepared in Example 1 were redissolved and diluted with deionized water. 10 μL of each was dropped onto a copper grid and observed using a transmission electron microscope (TEM, JEM-2100, JEOL Ltd., Japan). Figure 1 As shown in the figure. Transmission electron microscopy observations revealed that the fluorescent carbon dots were graphite- or amorphous, with an average particle size of approximately 13.9 nm.

[0051] 2. Zeta potential of Du-CDs

[0052] The zeta potentials of the Du-CDs prepared in Example 1 were measured using a Zetasizer instrument (Malvern Instruments, Nano ZS, United Kingdom). Figure 2 As shown, the surface charge of Du-CDs is 9.2 ± 0.6 mV, indicating that Du-CDs have supercationic properties.

[0053] 3. Zeta potential of Du-CDs

[0054] The zeta potentials of the Du-CDs prepared in Example 1 were measured using a Zetasizer instrument (Malvern Instruments, Nano ZS, United Kingdom). Figure 2 As shown, the surface charge of Du-CDs is 9.2 ± 0.6 mV, indicating that Du-CDs have supercationic properties.

[0055] 4. Detection of Du-CDs by UV-Vis absorption spectroscopy

[0056] The UV-Vis absorption spectra of Du-CDs prepared in Example 1 in ultrapure water were measured using a UV-Vis spectrophotometer (UV-2600, Shimadzu, Japan). Figure 3 As shown, there is an absorption peak at around 280 nm, which is due to the absorption peak caused by the π-π* transition of C=C or C=O bonds.

[0057] 5. Fluorescence spectroscopy detection of Du-CDs

[0058] The fluorescence spectra of the Du-CDs prepared in Example 1 in water were detected using a fluorescence spectrophotometer (RF-5301PC, Shimadzu, Japan). Figure 4 As shown, the fluorescence emission spectrum of Du-CDs exhibits the strongest fluorescence at 390 nm, with an excitation wavelength of 316 nm. Figure 4 The mid-peak position corresponds to approximately 400 nm, proving that Du-CDs are blue. The above experiments effectively demonstrate the successful synthesis of blue fluorescent carbon dots for Du-CDs in this invention.

[0059] Example 3

[0060] Antibacterial evaluation of Du-CDs

[0061] Wild-type Staphylococcus aureus, Escherichia coli, and Candida albicans were used as model strains for Gram-positive bacteria, Gram-negative bacteria, and fungi, respectively. Each strain was cultured to the logarithmic growth phase, washed with 0.9% sodium chloride solution, and diluted to a final concentration of 1×10⁻⁶. 6CFU / mL. Diluted microbial cells were inoculated into 96-well plates, and either 50 μg / mL of the ethyl acetate fungal extract (raw material) prepared in Example 1 or 50 μg / mL Du-CDs were added. A bacterial culture without any added materials was used as a control. The plates were incubated at 37°C (bacteria) or 28°C (fungi) in a shaker at 180 rpm for 1 h to compare their antibacterial activity. Figure 5 As shown, the cell viability of Staphylococcus aureus, Escherichia coli, and Candida albicans treated with ethyl acetate fungal extracts was 34.5%, 37.2%, and 39.9%, respectively, while the cell viability of those treated with Du-CDs was significantly reduced to 7.2%, 8.1%, and 4.3%. This indicates that Du-CDs exhibit significantly enhanced antibacterial activity compared to the original extracts.

[0062] Example 4

[0063] Concentration-dependent antibacterial evaluation of Du-CDs

[0064] With a concentration of 1×10 6 CFU / mL suspensions of Staphylococcus aureus, Escherichia coli, and Candida albicans were inoculated into 96-well plates and treated with different concentrations of Du-CDs prepared in Example 1. Bacteria were cultured at 37°C and 160 rpm for 24 h, and fungi were cultured at 28°C and 160 rpm for 24 h. The OD values ​​of the bacterial suspensions were recorded. 600 The value changes over time. For example... Figure 6 As shown, when the concentrations of Du-CDs reached 10, 50, and 50 μg / mL, the cell growth of Staphylococcus aureus, Escherichia coli, and Candida albicans was completely inhibited.

[0065] Example 5

[0066] Evaluation of the broad-spectrum antibacterial properties of Du-CDs

[0067] 1×10 6 Bacteria / fungi at CFU / mL were cultured with different concentrations of Du-CDs at 37℃ (bacteria) or 28℃ (fungi) in a shaker at 180 rpm for 24 h without turbidity. The lowest CDs concentration that resulted in no turbidity was defined as the MIC value of CDs for that bacterium / fungus. As shown in Table 1, the MIC values ​​of Du-CDs for both Gram-positive and Gram-negative strains did not exceed 4 μg / mL, while the MIC values ​​for Candida albicans and Saccharomyces cerevisiae were relatively high, at 18 μg / mL and 24 μg / mL, respectively. These results indicate that Du-CDs possess broad-spectrum antibacterial activity.

[0068] Table 1 Evaluation of the broad-spectrum antibacterial activity of Du-CDs

[0069]

[0070] Example 6

[0071] Evaluation of the in vivo antibacterial effect and wound healing of Du-CDs

[0072] The dorsal hair of Balb / c mice aged 3-4 weeks was removed, and a full-thickness incision with a diameter of 10 mm was made with scissors. A wound containing 5×10⁻⁶ ppm of arbor tissue was then inserted. 8 A suspension of Staphylococcus aureus cells was inoculated onto the wound. Experimental mice were randomly divided into two groups of five, with each group treated with PBS (pH 7.4, control group) and Du-CDs prepared in Example 1 at 2 mg / mL, respectively. Starting 24 hours post-infection, 20 μL of the solution was added to the wound every 3 days for 21 days. Wound size and body weight were measured daily. Bacteria were collected from the wound tissue every 3 days using PBS swabs and diluted on LB agar; colony counts were performed to determine the severity of the infection. Figure 7 As shown, after day 3, the wound area reduction in the Du-CDs group was significantly greater than that in the control group. The wounds in the Du-CDs group healed completely by day 18, while those in the PBS group healed completely by day 21. The number of residual Staphylococcus aureus bacteria in the wounds treated with Du-CDs was significantly less than that in the wounds treated with PBS. Furthermore, the body weight of all tested mice gradually increased, with no significant difference between the Du-CDs and PBS groups, macroscopically demonstrating the good biocompatibility of Du-CDs. These results indicate that Du-CDs can significantly accelerate skin wound closure, enhance antibacterial activity, and exhibit good biocompatibility.

[0073] Example 7

[0074] Du-CDs hemolytic evaluation

[0075] Fresh red blood cells (RBCs) were extracted from whole blood of healthy mice by centrifugation at 5000 rpm for 5 min, washed three times, and resuspended in PBS. The separated RBCs were co-incubated at 37°C with Du-CDs prepared in Example 1 at final concentrations of 1, 2, 5, 10, and 15 μg / mL for 1 h, followed by centrifugation at 5000 rpm for 5 min. The supernatant was transferred to a 96-well plate, and the absorbance at 405 nm was measured to calculate the hemolysis rate. Red blood cells treated with PBS and deionized water were used as negative and positive controls, respectively. Figure 8 As shown, after incubation with 15 μg / mL Du-CDs for 1 h, only 13.14% of red blood cells were lysed, indicating that Du-CDs have good blood compatibility.

Claims

1. A fungal-derived antibacterial blue fluorescent carbon dot, characterized in that, The carbon dots are synthesized by heating an extract of *Metacarpa*.

2. The antibacterial blue fluorescent carbon dot derived from fungi according to claim 1, characterized in that, The *Diaporthe unshiuensis* is YSP3.

3. The antibacterial blue fluorescent carbon dot derived from fungi according to claim 1, characterized in that, The extract is an extract of ethyl acetate.

4. A method for preparing antibacterial blue fluorescent carbon dots derived from fungi, characterized in that, Includes the following steps: (1) Culture the interstrain of *Syntheticus* to obtain mycelia; (2) Transfer the mycelium to potato dextrose medium for further culture; (3) The culture medium was filtered and then extracted with ethyl acetate to obtain the extract; (4) The extract is heated to obtain a solvent-free substance; (5) Dissolve the solvent-free substance in deionized water, centrifuge and filter to obtain an aqueous solution containing Du-CDs, dialyze and freeze dry to obtain blue carbon dots.

5. The method for preparing fungal-derived antibacterial blue fluorescent carbon dots according to claim 4, characterized in that, The culture conditions in step (1) are 25-30℃ for 3-7 days; the culture conditions in step (2) are 20-30℃ for 1-5 days.

6. The method for preparing fungal-derived antibacterial blue fluorescent carbon dots according to claim 4, characterized in that, In step (3), the culture medium is filtered through filter paper and then extracted 2-3 times with an equal volume of ethyl acetate.

7. The method for preparing fungal-derived antibacterial blue fluorescent carbon dots according to claim 4, characterized in that, In step (4), the extract is transferred to a stainless steel autoclave and heated at 120-240℃ for 6-18 hours to obtain a solvent-free substance.

8. The use of the fungal-derived antibacterial blue fluorescent carbon dots of claim 1 in the preparation of in vivo and in vitro antibacterial drugs or reagents.

9. The application according to claim 8, characterized in that, The antibacterial microorganisms include Gram-positive bacteria, Gram-negative bacteria, and fungi; the Gram-positive bacteria include Escherichia coli, Proteus, and Serratia marcescens; the Gram-negative bacteria include Staphylococcus aureus, Staphylococcus epidermidis, and Micrococcus luteus; and the fungi include Saccharomyces cerevisiae and Candida albicans.

10. The use of the fungal-derived antibacterial blue fluorescent carbon dots of claim 1 in the preparation of a medicament with wound-healing capabilities.

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