Antibacterial carbon dots and their preparation method and application

Abstract

The present invention provides an antibacterial carbon dot and its preparation method and application, which belongs to the technical field of biomedicine and is used to solve the technical problem of poor antibacterial properties of carbon quantum dots. The antibacterial carbon dot preparation method of the present invention comprises the following steps: dissolving indocyanine green, curcumin and a carbon source in a solvent to prepare a mixed solution, and then performing a solvothermal reaction to obtain the antibacterial carbon dot. The antibacterial carbon dot of the present invention can effectively convert O 2 Converted into a large amount of singlet oxygen ( 1 O 2), the heat generated further enhances 1 The generation and release of O₂ further increases cell membrane permeability, effectively killing bacteria. Curcumin, with its anti-inflammatory and antioxidant properties, prevents bacterial aggregation and biofilm formation. Citric acid provides an acidic environment, which is unfavorable for bacterial growth. By combining these three ingredients through solvothermal synthesis, a near-infrared carbon dot material was created, creating a novel antibacterial agent that combines imaging and therapeutic properties, offering new insights into the clinical treatment of antibiotic resistance.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to antibacterial carbon dots and a preparation method and application thereof. Background Art

[0002] Antibiotic resistance has become one of the major public health threats of the 21st century. To combat this problem, research into new antimicrobial agents is underway, but this requires significant investment in both human and material resources. Research and development of new antibiotics can take as long as 10-15 years, costing hundreds of millions of yuan. Currently, there are approximately 400 anti-infective drug research projects worldwide, but none have yet reached Phase IV clinical trials. The pace of new drug development lags far behind the development of bacterial resistance. Therefore, developing new treatment options is a pressing global challenge that demands resolution.

[0003] Carbon dots, as a new type of fluorescent nanomaterial, have been applied in biomedicine and other fields due to their unique optical properties, excellent biocompatibility, and low or non-toxic properties. They show great potential in antibacterial therapy. Patent Publication No. CN 109021971 A discloses fluorescent carbon dots for nuclear staining, their application in nuclear imaging, and methods. The carbon dots are prepared by the following steps: 1) dissolving folic acid and m-phenylenediamine in water and heating for reaction; 2) after the reaction, centrifuging and subjecting the mixture to silica gel column chromatography to collect the yellow-green fluorescent fraction, which is then transferred to water after rotary evaporation and freeze-dried. Patent Publication No. CN113549448A discloses carbon dots with intrinsic antibacterial activity and photodynamically enhanced bactericidal effects, their preparation method, and applications. The technical solution is as follows: a bactericide and an organic solvent are uniformly mixed to form a mixed reaction solution. The mixed reaction solution is heated to 140-260°C and reacted for 30-24 hours. The reaction product is cooled and purified to obtain carbon dots with red fluorescence emission. These carbon dots can be used for cell and bacterial imaging, and have good biocompatibility, low toxicity, broad-spectrum bactericidal properties, and do not cause bacterial resistance. However, directly using bactericides as the carbon source for preparing carbon quantum dots reduces their antibacterial properties. Summary of the Invention

[0004] In response to the technical problem of poor antibacterial properties of carbon quantum dots, the present invention proposes an antibacterial carbon dot and its preparation method and application. The prepared antibacterial carbon dot integrates imaging and treatment in one, and has high antibacterial properties.

[0005] In order to achieve the above object, the technical solution of the present invention is achieved as follows:

[0006] A method for preparing antibacterial carbon dots comprises the following steps: dissolving a fluorescent dye, a phenolic compound and a carbon source in a solvent to prepare a mixed solution, and then performing a solvent thermal reaction to prepare the antibacterial carbon dots.

[0007] The fluorescent dye is indocyanine green and its derivatives, such as dihydroindocyanine green sodium salt, new indocyanine green (IR-820), pigment green 7, acid green 1, aluminum phthalocyanine chloride, titanium phthalocyanine, iron phthalocyanine, acid green 28 / 50, methylene green hemichloride zinc salt, solvent green 28, methyl green, 3,3,3',3'-tetramethyl-1,1'-bis(4-sulfobutyl)benzindole dicarbocyanine sodium, 1,1'-diethyl-3,3,3',3'-tetramethylindocyanine iodide, etc.

[0008] The polyphenol compounds are curcumin and its derivatives, such as demethoxycurcumin, demethoxycurcumin, dideoxycurcumin, octahydrocurcumin, dimethylcurcumin, dimethoxycurcumin, tetrahydrocurcumin, 3,4-difluorobenzocurcumin, tetrahydrocurcumin-d6, curcumin β-D-glucuronic acid, o-tert-butyldimethylsilylcurcumin, curcumin-d6, etc.

[0009] The carbon source is citric acid and its derivatives, such as citrate, citrate ester, citric anhydride, amine citrate, ethyl citrate, ammonium ferric citrate, etc.

[0010] The solvent is one or more of water, ethanol, methanol, propylene glycol, chloroform, N,N-dimethylformamide, ethyl acetate, dimethyl sulfoxide, carbon tetrachloride, benzene and acetone.

[0011] The mass ratio of the fluorescent dye, the phenolic compound and the carbon source is (1-500): (1-1000): (1-1500).

[0012] The concentration of the carbon source in the mixed solution is 0.1-2 mg / ml.

[0013] The temperature of the solvent thermal reaction is 50-1000° C., and the time is 30 min-24 h.

[0014] Application of antibacterial carbon dots as a drug for severe pneumonia caused by bacterial infection in children.

[0015] The beneficial effects of the present invention are: Due to the indocyanine green (ICG) component, O 2 Converted into a large amount of singlet oxygen ( 1 O2), the heat generated further enhances 1 The generation and release of O2 further increases cell membrane permeability, effectively killing bacteria. Curcumin, with its anti-inflammatory and antioxidant properties, prevents bacterial aggregation and biofilm formation. Citric acid provides an acidic environment, which is not conducive to bacterial growth. By combining these three ingredients through solvothermal synthesis, a near-infrared carbon dot material has been created, creating a novel antibacterial agent that combines imaging and therapeutic properties, offering new insights into the clinical treatment of antibiotic resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 This is the absorbance spectrum of the antibacterial carbon dots of the present invention.

[0018] Figure 2 : is the fluorescence spectrum of the antibacterial carbon dots of the present invention.

[0019] Figure 3 TEM image of the antibacterial carbon dots of the present invention.

[0020] Figure 4 This is the XPS graph of the antibacterial carbon dots of the present invention.

[0021] Figure 5 This is the FTIR graph of the antibacterial carbon dots of the present invention.

[0022] Figure 6 The antibacterial effect diagram of antibacterial carbon dots with different concentrations.

[0023] Figure 7 These are the results of in vivo imaging of bacteria in mice with severe pneumonia. DETAILED DESCRIPTION

[0024] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

[0025] Example 1

[0026] A preparation method of antibacterial carbon dots comprises the following steps:

[0027] (1) 3.1 mg of indocyanine green, 6.6 mg of curcumin, and 8.5 mg of citric acid were dissolved in 5 mL of 95% ethanol and ultrasonicated for 10 min. The mixture was then reacted by solvothermal method at 140°C for 10 h.

[0028] (2) After the reaction, the product is purified by centrifugal ultrafiltration, and then the solvent is removed by spin drying to prepare a solid product, which is dried and stored.

[0029] Sensing method: Disperse the product in a buffer solution and mix well, then test the fluorescence spectrum. Figure 1 and 2 As shown, the excitation wavelength is 788 nm and the emission is collected at 828 nm.

[0030] The prepared antibacterial carbon dots were then characterized. Figure 3 Transmission electron microscopy (TEM) can prove that the synthesized material is carbon dots. Figure 4 X-ray photoelectron spectroscopy (XPS) is performed by Figure 4 It can be seen that the antibacterial carbon dots are mainly composed of C, N, O, and S, accounting for C%: 78.63, N%: 1.15, O%, 18.96, and S%: 0.76. Figure 5 For Fourier transform infrared (FTIR) spectroscopy: 3000-3500cm -1 The enhanced OH stretching vibration peak at 2935 cm -1 (NH stretch), 1594cm -1 (C=O stretch), 1513cm -1 The peaks at 1278 cm-1 (C=N stretching), 1278 cm-1 (C=C stretching) and 1037 cm-1 (C-O-C stretching) indicate the synthesis of new substances.

[0031] Example 2

[0032] A preparation method of antibacterial carbon dots comprises the following steps:

[0033] (1) 3.1 mg of indocyanine green, 6.6 mg of curcumin, and 8.5 mg of citric acid were dissolved in 5 mL of 95% ethanol and sonicated for 10 min. The mixture was then reacted by solvothermal method at 160°C for 6 h.

[0034] (2) After the reaction, the product is purified by centrifugal ultrafiltration and then stored in a refrigerator.

[0035] Example 3

[0036] A preparation method of antibacterial carbon dots comprises the following steps:

[0037] (1) 5 mg of indocyanine green, 5 mg of curcumin, and 5 mg of citric acid were dissolved in 5 mL of 95% ethanol and ultrasonicated for 10 min. The mixture was then reacted by solvothermal method at 140°C for 10 h.

[0038] (2) After the reaction, the product is purified by centrifugal ultrafiltration and then stored in a refrigerator.

[0039] Example 4

[0040] A preparation method of antibacterial carbon dots comprises the following steps:

[0041] (1) 5 mg of indocyanine green, 5 mg of curcumin, and 5 mg of citric acid were dissolved in 5 mL of 95% ethanol and sonicated for 10 min. The mixture was then reacted by solvothermal method at 160°C for 6 h.

[0042] (2) After the reaction, the product is purified by centrifugal ultrafiltration and then stored in a refrigerator.

[0043] Example 5

[0044] A preparation method of antibacterial carbon dots comprises the following steps:

[0045] (1) 3.8 mg of indocyanine green, 1.8 mg of curcumin, and 1.4 mg of citric acid were dissolved in 5 mL of 95% ethanol and sonicated for 10 min. The mixture was then reacted by solvothermal method at 140°C for 10 h.

[0046] (2) After the reaction, the product is purified by centrifugal ultrafiltration and then stored in a refrigerator.

[0047] Example 6

[0048] A preparation method of antibacterial carbon dots comprises the following steps:

[0049] (1) 3.8 mg of indocyanine green, 1.8 mg of curcumin, and 1.4 mg of citric acid were dissolved in 5 mL of 95% ethanol and ultrasonicated for 10 min. The mixture was then reacted by solvothermal method at 160°C for 6 h.

[0050] (2) After the reaction, the product is purified by centrifugal ultrafiltration and then stored in a refrigerator.

[0051] Example 7

[0052] A preparation method of antibacterial carbon dots comprises the following steps:

[0053] (1) 3.1 mg of indocyanine green, 6.6 mg of curcumin, and 8.5 mg of citric acid were dissolved in 5 mL of 95% ethanol at a mass ratio of 3.1:6.6:8.5, and ultrasonicated for 10 min. The mixture was then reacted at 140°C for 14 h by a solvothermal method.

[0054] (2) After the reaction, the product is purified by centrifugal ultrafiltration, and then the solvent is removed by spin drying to prepare a solid product, which is dried and stored.

[0055] Sensing method: Disperse the product in a buffer solution and mix well. Test the fluorescence spectrum with an excitation wavelength of 786 nm and an emission collection wavelength of 821 nm.

[0056] Example 8

[0057] A preparation method of antibacterial carbon dots comprises the following steps:

[0058] (1) 5 mg of Acid Green 1, 5 mg of o-tert-butyldimethylsilylcurcumin, and 5 mg of triethyl citrate were dissolved in 10 mL of 95% ethanol, ultrasonicated for 10 min, and the mixture was reacted by solvothermal method at 160°C for 6 h;

[0059] (2) After the reaction, the product is purified by centrifugal ultrafiltration and then stored in a refrigerator.

[0060] Example 9

[0061] A preparation method of antibacterial carbon dots comprises the following steps:

[0062] (1) 5 mg of aluminum phthalocyanine chloride, 5 mg of demethoxycurcumin, and 5 mg of tributyl citrate were dissolved in 10 mL of 95% ethanol, ultrasonicated for 10 min, and the mixture was reacted by solvothermal method at 160°C for 6 h;

[0063] (2) After the reaction, the product is purified by centrifugal ultrafiltration and then stored in a refrigerator.

[0064] Example 10

[0065] A preparation method of antibacterial carbon dots comprises the following steps:

[0066] (1) 5 mg of 3,3,3',3'-tetramethyl-1,1'-bis(4-sulfobutyl)benzidinolyl dicarbocyanine sodium, 5 mg of tetrahydrocurcumin, and 5 mg of ammonium citrate were dissolved in 10 mL of 95% ethanol and ultrasonicated for 10 min. The mixture was then reacted by solvothermal method at 160°C for 6 h to synthesize the product.

[0067] (2) After the reaction, the product is purified by centrifugal ultrafiltration and then stored in a refrigerator.

[0068] Example 11

[0069] A preparation method of antibacterial carbon dots comprises the following steps:

[0070] (1) 5 mg of methylene green hemichloride zinc salt, 5 mg of bis-deoxycurcumin, and 5 mg of sodium citrate were dissolved in 10 mL of 95% ethanol, ultrasonicated for 10 min, and the mixture was reacted by solvothermal method at 160°C for 6 hours;

[0071] (2) After the reaction, the product is purified by centrifugal ultrafiltration and then stored in a refrigerator.

[0072] Example 12

[0073] A preparation method of antibacterial carbon dots comprises the following steps:

[0074] (1) 5 mg of iron phthalocyanine, 5 mg of dimethylcurcumin, and 5 mg of sodium citrate were dissolved in 10 mL of propylene glycol, ultrasonicated for 10 min, and the mixture was reacted by solvothermal method at 160 ° C for 6 hours;

[0075] (2) After the reaction, the product is purified by centrifugal ultrafiltration and then stored in a refrigerator.

[0076] Example 13

[0077] A preparation method of antibacterial carbon dots comprises the following steps:

[0078] (1) 1 mg of methyl green, 10 mg of dimethylcurcumin, and 5 mg of sodium citrate were dissolved in 10 mL of N,N-dimethylformamide, ultrasonicated for 10 min, and the mixture was reacted by solvothermal method at 500 °C for 30 min;

[0079] (2) After the reaction, the product is purified by centrifugal ultrafiltration and then stored in a refrigerator.

[0080] Example 14

[0081] A preparation method of antibacterial carbon dots comprises the following steps:

[0082] (1) 0.1 mg of methyl green, 10 mg of dimethylcurcumin, and 5 mg of sodium citrate were dissolved in 10 mL of carbon tetrachloride, ultrasonicated for 10 min, and the mixture was reacted by solvothermal method at 300 °C for 6 h;

[0083] (2) After the reaction, the product is purified by centrifugal ultrafiltration and then stored in a refrigerator.

[0084] Example 14

[0085] A preparation method of antibacterial carbon dots comprises the following steps:

[0086] (1) 10 mg of methyl green, 0.1 mg of dimethyl curcumin, and 10 mg of sodium citrate were dissolved in 10 mL of water, ultrasonicated for 10 min, and the mixture was reacted by solvothermal method at 50 °C for 24 h;

[0087] (2) After the reaction, the product is purified by centrifugal ultrafiltration and then stored in a refrigerator.

[0088] Example 15

[0089] A preparation method of antibacterial carbon dots comprises the following steps:

[0090] (1) 0.1 mg of methyl green, 0.1 mg of dimethyl curcumin, and 20 mg of sodium citrate were dissolved in 10 mL of propylene glycol, ultrasonicated for 10 min, and the mixture was reacted by solvothermal method at 200 °C for 12 h;

[0091] (2) After the reaction, the product is purified by centrifugal ultrafiltration and then stored in a refrigerator.

[0092] Example 16

[0093] A preparation method of antibacterial carbon dots comprises the following steps:

[0094] (1) 10 mg of titanium phthalocyanine, 10 mg of 3,4-difluorobenzocurcumin, and 1 mg of sodium citrate were dissolved in 10 mL of methanol, ultrasonicated for 10 min, and the mixture was reacted by solvothermal method at 200 °C for 12 h;

[0095] (2) After the reaction, the product is purified by centrifugal ultrafiltration and then stored in a refrigerator.

[0096] Test: The carbon dots prepared in Example 1 were subjected to minimum inhibitory test:

[0097] (1) The steps are as follows:

[0098] 1. Glycerol bacteria activation: Staphylococcus aureus at -80℃ was successfully revived by two plate streaking activations.

[0099] 2. Minimum inhibitory concentration determination: Prepare to OD = 0.20 (1*10^8 cfu / ml), dilute 200 times, add to 2 ml MH broth to prepare different gradient carbon dot concentrations of 1024μg / ml, 512μg / ml, 256μg / ml, 128μg / ml, 64μg / ml, 32μg / ml, 16μg / ml, 8μg / ml, 4μg / ml, 2μg / ml and in positive and negative control groups, make the system bacterial solution concentration 5*10^5 cfu / ml, observe the colony growth after 24 hours of incubation, spread the plate and incubate at 37℃ constant temperature incubator for 18-24 hours, record the colony growth (such as Figure 6 ).

[0100] 3. Figure 6 The concentration of carbon dot materials in tubes 1-10 was diluted two-fold (1024 μg / ml-2 μg / ml), and tubes 11 and 12 were positive control and negative control, respectively. Figure 6 The results on the left show that the minimum inhibitory concentration is 128μg / ml, Figure 6 The results on the right show that the minimum bactericidal concentration is 512μg / ml.

[0101] Test 2: In vivo imaging of drug administration via tracheal intubation in mice with severe pneumonia: The product was administered via tracheal intubation into both lungs of mice with severe pneumonia, and then observed and photographed using an in vivo imaging device.

[0102] (1) The steps are as follows:

[0103] 1. Construction of severe pneumonia model: Mice were anesthetized by intraperitoneal injection of 0.2ml / 10g tribromoethanol. The anesthetized mice were placed in a supine position on a fixed table to keep the trachea open. A rubber band was hung on the mouse's incisors. The mouse's mouth was opened with a light-emitting laryngoscope. The glottic fissure in the larynx could be clearly observed through the light source. A handheld liquid aerosol lung delivery device was then gently inserted into the airway. The piston was then pushed to aerosolize 50μL of bacterial solution (1×10^10 cfu / ml) into the lungs. After the lung delivery was completed, the mice were placed upright for 30s, keeping their heads high and feet low. The control group received an equal amount of PBS buffer in the lungs as described above.

[0104] 2. IVIS imaging of drug administration through endotracheal intubation: The method is the same as above, 50 μL of carbon dot material is administered to the lungs, and the samples are observed and photographed using an in vivo imaging device (e.g. Figure 7 ). Figure 7 The chest skin of mice was prepared, 50 μL of drug was administered through endotracheal intubation, and the in vivo imaging instrument was used for 808 nm excitation, which allowed the drug administration site and dynamic metabolic process to be clearly observed.

[0105] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing antibacterial carbon dots, characterized in that: The following steps are involved: A fluorescent dye, a phenolic compound, and a carbon source are dissolved in a solvent to prepare a mixed solution, which is then subjected to a solvothermal reaction to prepare antibacterial carbon dots. The fluorescent dye is indocyanine green, dihydroindocyanine green sodium salt, new indocyanine green, 3,3,3',3'-tetramethyl-1,1'-bis(4-sulfobutyl)benzindole dicarbocyanine sodium or 1,1'-diethyl-3,3,3',3'-tetramethylindocyanine iodide; The phenolic compound is curcumin, demethoxycurcumin, demethoxycurcumin, dideoxycurcumin, octahydrocurcumin, dimethylcurcumin, dimethoxycurcumin, tetrahydrocurcumin, 3,4-difluorobenzocurcumin, curcumin β-D-glucuronic acid or o-tert-butyldimethylsilylcurcumin; The carbon source is citric acid, citrate, citrate ester or citric anhydride.

2. The method for preparing antibacterial carbon dots according to claim 1, characterized in that: The solvent is one or more of water, ethanol, methanol, propylene glycol, chloroform, N,N-dimethylformamide, ethyl acetate, dimethyl sulfoxide, carbon tetrachloride, benzene and acetone.

3. The method for preparing antibacterial carbon dots according to claim 2, characterized in that: The mass ratio of the fluorescent dye, the phenolic compound and the carbon source is (1-500): (1-1000): (1-1500).

4. The method for preparing antibacterial carbon dots according to claim 1, wherein: The concentration of the carbon source in the mixed solution is 0.1-2 mg / ml.

5. The method for preparing antibacterial carbon dots according to claim 1, characterized in that: The temperature of the solvent thermal reaction is 50-1000° C., and the time is 30 min-24 h.

6. Antibacterial carbon dots prepared by the method according to any one of claims 1 to 5.

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