Bacteriostatic carbon dots as well as preparation method and application thereof

Abstract

The invention provides antibacterial carbon dots as well as a preparation method and application thereof, belongs to the technical field of biological medicines, and aims to solve the technical problem of poor antibacterial property of carbon quantum dots. The preparation method of the antibacterial carbon dots comprises the following steps: dissolving indocyanine green, curcumin and a carbon source in a solvent to prepare a mixed solution, and then performing solvothermal reaction to prepare the antibacterial carbon dots. Due to the indocyanine green component, O < 2 > can be effectively converted into a large amount of singlet oxygen (< 1 > O < 2 >), generation and release of < 1 > O < 2 > are further enhanced through generated heat, permeability of cell membranes is further improved, and therefore bacteria are effectively killed. Curcumin has the characteristics of inflammation resistance, oxidation resistance and the like, and can prevent bacteria from gathering to form a biological membrane. Citric acid provides an acidic environment which is not beneficial to growth of bacteria. The near-infrared carbon dot material is synthesized through solvothermal synthesis of the three components, the novel bacteriostatic agent integrates imaging and treatment, and a new thought is provided for clinical antibiotic resistance.

Description

Technical Field

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

[0002] Antibiotic resistance has become one of the major public health threats in the 21st century. In order to deal with the problem of drug resistance, research on new antibacterial drugs is carried out, but it requires a lot of manpower and material resources. Research statistics show that the research and development cycle of new antibiotics is as long as 10-15 years, and the research and development funds cost hundreds of millions of yuan. At present, there are about 400 anti-infective drug research projects in the world. So far, no drug has reached the standard for phase IV clinical use. The speed of new drug development lags far behind the speed of bacterial resistance. Therefore, the development of new treatment options is a global problem that needs to be solved urgently.

[0003] As a new type of fluorescent nanomaterial, carbon dots are applied to biomedicine and other fields due to their unique optical properties, good biocompatibility, low toxicity or non-toxic properties, and show great potential in antibacterial treatment. Patent Publication No. CN 109021971 A discloses a fluorescent carbon dot for nucleus staining and its application and method in nucleus imaging. The carbon dot is prepared by the following steps: 1) dissolving folic acid and m-phenylenediamine in water and heating for reaction; 2) after the reaction, centrifuging, passing through silica gel column chromatography, collecting the yellow-green fluorescent part, transferring it to water after rotary evaporation, and freeze-drying. Patent Publication No. CN113549448A discloses a carbon dot with intrinsic antibacterial activity and photodynamic enhanced bactericidal effect, its preparation method and application. The technical scheme is as follows: the bactericide and the organic solvent are mixed evenly to form a mixed reaction liquid, the mixed reaction liquid is heated to 140-260°C, and reacted for 30min-24h, the reaction product is cooled, and it is purified to obtain a carbon dot with red fluorescence emission. The carbon dots can be used for cell and bacterial imaging, have good biocompatibility, low toxicity, broad-spectrum bactericidal properties, and will not cause problems such as bacterial resistance. However, directly using bactericides as carbon sources for preparing carbon quantum dots results in reduced antibacterial properties. Summary of the invention

[0004] In view of the technical problem that carbon quantum dots have poor antibacterial properties, the present invention provides an antibacterial carbon dot and a preparation method and application thereof. The prepared antibacterial carbon dot integrates imaging and treatment 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 zinc hemichloride, 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, and the like.

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

[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 as follows: 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 the permeability of the cell membrane, thereby effectively killing bacteria. Curcumin has anti-inflammatory and antioxidant properties, which can prevent bacteria from aggregating to form biofilms. Citric acid provides an acidic environment, which is not conducive to bacterial growth. The three ingredients are combined to synthesize near-infrared carbon dot materials through solvent thermal synthesis, and a new antibacterial agent that integrates imaging and treatment is provided, providing new ideas for clinical 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 drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 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 be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Example 1

[0026] A method for preparing 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, ultrasonicated for 10 min, and the mixture was 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 obtain a solid product, which is then 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) was able to prove that the synthesized material was 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) suggest the synthesis of new substances.

[0031] Example 2

[0032] A method for preparing 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, ultrasonicated for 10 min, and the mixture was reacted at 160° C. for 6 hours by solvothermal method;

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

[0035] Example 3

[0036] A method for preparing 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, ultrasonicated for 10 min, and the mixture was 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 method for preparing 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, ultrasonicated for 10 min, and the mixture was reacted at 160° C. for 6 hours by solvothermal method;

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

[0043] Example 5

[0044] A method for preparing 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, ultrasonicated for 10 min, and the mixture was reacted at 140° C. for 10 h by solvothermal method;

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

[0047] Example 6

[0048] A method for preparing 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, ultrasonicated for 10 min, and the mixture was reacted at 160° C. for 6 hours by solvothermal method;

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

[0051] Example 7

[0052] A method for preparing 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 in a mass ratio of 3.1:6.6:8.5, and ultrasonicated for 10 min. The mixture was reacted at 140° C. for 14 hours 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 obtain a solid product, which is then 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 786nm and an emission collection wavelength of 821nm.

[0056] Example 8

[0057] A method for preparing 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 at 160° C. for 6 hours by a solvothermal method;

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

[0060] Example 9

[0061] A method for preparing antibacterial carbon dots comprises the following steps:

[0062] (1) 5 mg of phthalocyanine aluminum 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 at 160° C. for 6 hours by a solvothermal method;

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

[0064] Example 10

[0065] A method for preparing 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, ultrasonicated for 10 min, and the mixture was reacted at 160° C. for 6 hours by a solvothermal method to synthesize;

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

[0068] Embodiment 11

[0069] A method for preparing antibacterial carbon dots comprises the following steps:

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

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

[0072] Example 12

[0073] A method for preparing antibacterial carbon dots comprises the following steps:

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

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

[0076] Example 13

[0077] A method for preparing 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 at 500 °C for 30 min by solvothermal method;

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

[0080] Embodiment 14

[0081] A method for preparing antibacterial carbon dots comprises the following steps:

[0082] (1) 0.1 mg of methyl green, 10 mg of dimethyl curcumin 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 hours;

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

[0084] Embodiment 14

[0085] A method for preparing 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 at 50° C. for 24 hours by a solvothermal method;

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

[0088] Embodiment 15

[0089] A method for preparing 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 hours;

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

[0092] Example 16

[0093] A method for preparing 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 hours;

[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 operation steps are as follows:

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

[0099] 2. Determination of minimum inhibitory concentration: Prepare OD = about 0.20 (1*10^8cfu / ml), dilute 200 times, add 2ml 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 positive and negative control groups, make the system bacterial solution concentration 5*10^5cfu / ml, observe the colony growth after 24h of incubation, apply plates and incubate in a 37℃ constant temperature incubator for 18-24h to 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 in a gradient manner (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 right result shows that the minimum bactericidal concentration is 512μg / ml.

[0101] Test 2: In vivo imaging of drug administration via endotracheal intubation in mice with severe pneumonia: After the product was administered via endotracheal intubation into the lungs of mice with severe pneumonia, the products were observed and photographed using an in vivo imaging device.

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

[0103] 1. Construction of severe pneumonia model: Intraperitoneal injection of 0.2ml / 10g tribromoethanol anesthetized mice, placed the anesthetized mice in supine position on a fixed table, kept the trachea unobstructed, and the mouse incisors were hung with a rubber band. The mouse mouth was opened with a lighted laryngoscope, and the laryngeal glottic fissure could be clearly observed through the light source. Then, a handheld liquid aerosol lung delivery device was gently inserted into the airway, and then the piston was pushed to atomize and deliver 50μL of bacterial solution (1×10^10cfu / ml) to the lungs. After the lung delivery was completed, the mice were placed upright for 30s, keeping the head high and the 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 live imaging device is used for observation and photography (such as Figure 7 ). Figure 7 The chest skin of mice was prepared, 50 μL of drug was administered through endotracheal intubation, and the in vivo imager was excited at 808 nm to clearly observe the drug administration site and the dynamic metabolic process.

[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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing antibacterial carbon dots, characterized in that: The following steps are involved: The fluorescent dye, phenolic compound and carbon source are dissolved in a solvent to prepare a mixed solution, which is then subjected to a solvothermal reaction to obtain the antibacterial carbon dots.

2. The method for preparing antibacterial carbon dots according to claim 1, characterized in that: The fluorescent dye is indocyanine green and its derivatives.

3. The method for preparing antibacterial carbon dots according to claim 1, characterized in that: The polyphenol compound is curcumin and its derivatives.

4. The method for preparing antibacterial carbon dots according to claim 1, characterized in that: The carbon source is citric acid and its derivatives.

5. 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.

6. The method for preparing antibacterial carbon dots according to any one of claims 1 to 5, characterized in that: The mass ratio of the fluorescent dye, the phenolic compound and the carbon source is (1-500): (1-1000): (1-1500).

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

8. 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.

9. The antibacterial carbon dots prepared by the method according to any one of claims 1 to 8.

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

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