Application of carbon nanodots in the preparation of photocatalytic drugs

Abstract

The present invention discloses an application of carbon nanodots in the preparation of photocatalytic drugs. The surface of the carbon nanodots has electron-withdrawing groups, which can capture excited electrons generated by the carbon nanodots absorbing light and undergo a reduction reaction, thereby extending the life of the generated holes and achieving strong oxidation ability. Under light, the carbon nanodots can effectively kill cells and antibiotic-resistant bacteria in an aqueous solution by oxidizing hydroxide ions in water to generate hydroxyl radicals, and have good applications in tumor photocatalytic treatment and photocatalytic sterilization. In addition, the carbon nanodots have good biocompatibility, may be metal-free, and have high biosafety.

Description

Technical Field

[0001] The invention relates to the technical field of carbon nanomaterials, and in particular to an application of carbon nanodots in the preparation of photocatalytic drugs. Background Art

[0002] Reactive oxygen species are highly reactive chemical species that play an important role in antimicrobial and cancer therapy. Reactive oxygen species include singlet oxygen, peroxides, superoxide radicals, and hydroxyl radicals. Compared with other reactive oxygen species, hydroxyl radicals have the highest oxidative capacity and can cause more significant lipid peroxidation and oxidative DNA damage. Photodynamic therapy, chemodynamic therapy, and photocatalytic therapy can generate hydroxyl radicals. The generation of hydroxyl radicals in photodynamic therapy usually depends on the content of oxygen molecules in the aqueous medium, which limits its efficacy in the hypoxic microenvironment of tumors. For chemodynamic therapy, the generation of hydroxyl radicals depends not only on transition metal catalysts, but also on the content of hydrogen peroxide in the tumor microenvironment. The generation of hydroxyl radicals in photocatalytic therapy is based on the oxidation of hydroxide ions in water by holes generated by light, which can be independent of the content of oxygen and hydrogen peroxide in the tumor microenvironment.

[0003] So far, the photocatalysts reported for photocatalytic therapy are usually based on nanocomposites containing heavy metal elements, which have potential biosafety risks, which greatly hinders their application in the in vivo biomedical field. Therefore, biocompatible and metal-free photocatalysts that can generate hydroxyl radicals in aqueous solution under light induction have broad application prospects.

[0004] It can be seen that the existing photocatalysts used in photocatalytic therapy generally have the following problems: (1) they are usually nanocomposite materials with complex synthesis methods, large sizes, and poor biocompatibility; (2) they contain heavy metal elements and have potential biosafety risks.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide an application of carbon nanodots in the preparation of photocatalytic drugs to improve the above technical problems.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides an application of carbon nanodots in the preparation of photocatalytic drugs, wherein the carbon nanodots have electron-withdrawing groups on their surfaces.

[0009] Optionally, the atomic content of the electron-withdrawing groups is greater than 25%, preferably greater than 30%, of the atomic content of the carbon nanodots.

[0010] Optionally, the electron withdrawing group comprises at least one of a carbonyl group, a carboxyl group and a pyridinyl group.

[0011] Optionally, the carbon nanodots are mainly prepared by solvothermal reaction of citric acid and urea in formic acid.

[0012] Optionally, the size of the carbon nanodots is 2nm to 6nm.

[0013] Optionally, the main absorption band of the carbon nanodots in water is 400nm to 650nm.

[0014] Optionally, the mass ratio of citric acid to the urea is 1:1-5, preferably 1:2-4.

[0015] Optionally, the total concentration of citric acid and the urea in the formic acid is 0.1 g / mL to 0.4 g / mL.

[0016] Optionally, the reaction temperature of the solvent thermal reaction is 150° C. to 170° C., and the reaction time is 3 h to 5 h.

[0017] Optionally, the photocatalytic drug is a tumor photocatalytic drug or a photocatalytic antibacterial drug.

[0018] Optionally, the tumor treated by the tumor photocatalytic drug includes any one of non-small cell lung cancer, breast cancer, pancreatic cancer, liver cancer, colorectal cancer, prostate cancer, cervical cancer, ovarian cancer, malignant melanoma and leukemia, and preferably the tumor is breast cancer.

[0019] Optionally, the bacteria that the photocatalytic antibacterial drug fights are antibiotic-resistant bacteria, preferably tetracycline-resistant Escherichia coli.

[0020] In a second aspect, the present invention also provides an anti-tumor combination drug, which includes the above-mentioned carbon nanodots and other anti-tumor drugs.

[0021] The present invention has the following beneficial effects: the electron-withdrawing groups on the surface of the carbon nanodots can capture the excited electrons generated by the carbon nanodots absorbing light and undergo a reduction reaction, thereby extending the life of the generated holes and achieving strong oxidation ability. Under light, the carbon nanodots can effectively kill cells and antibiotic-resistant bacteria by oxidizing the hydroxide ions in the water to generate hydroxyl radicals, and have good applications in tumor photocatalytic therapy and photocatalytic sterilization. In addition, the carbon nanodots have good biocompatibility, may be metal-free, and have high biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 TEM image of carbon nanodots (CDs) in an embodiment of the present invention;

[0024] Figure 2 The electron paramagnetic resonance spectra of carbon nanodots (CDs) added with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) in the embodiment of the present invention under dark conditions and after visible light irradiation;

[0025] Figure 3 This is a fluorescence image of 4T1 cells in an embodiment of the present invention after being incubated with carbon nanodots (CDs) for 1 hour, with an excitation light wavelength of 560 nanometers;

[0026] Figure 4 The cell viability of 4T1 cells in the embodiment of the present invention incubated with different concentrations of carbon nanodots (CDs) for 24 hours in the dark and after irradiation with visible light for 5 minutes;

[0027] Figure 5 These are absorption spectra of aqueous solutions of photocatalytic carbon nanodots (a) without addition and (b) with addition of triethanolamine (TEA) in Example 1 of the present invention when irradiated under visible light for different time periods;

[0028] Figure 6 The excitation-emission spectra of carbon nanodots (CDs) added with triethanolamine in an aqueous solution (a) before visible light irradiation and (b) after visible light irradiation for 20 minutes;

[0029] Figure 7 The luminescence decay spectra of the carbon nanodots (CDs) in the embodiment of the present invention and the aqueous solution of the carbon nanodots (ir-CDs) after 20 minutes of visible light irradiation under 510 nm laser excitation and monitored at 638 nm (IRF is the instrument response function);

[0030] Figure 8 (a) XPS full spectrum and high-resolution (b) C 1s, (c) N 1s, (d) O 1s XPS spectra of carbon nanodots (CDs), acetic acid carbon nanodots (AcOH-CDs), acetone carbon nanodots (DMK-CDs) and dimethylformamide carbon nanodots (DMF-CDs) in the examples of the present invention;

[0031] Fig. 9 (a) Full XPS spectrum and high-resolution (b) C 1s, (c) N 1s, and (d) O 1s XPS spectra of the carbon nanodot CDs and ir-CDs in the embodiments of the present invention;

[0032] Fig.10 Schematic diagram and result diagram of carbon nanodots (CDs) used as antibacterial agents under visible light irradiation in an embodiment of the present invention;

[0033] Fig.11 Schematic diagram and result diagram of photocatalytic treatment of mice with 4T1 tumors by carbon nanodots (CDs) under visible light irradiation in an embodiment of the present invention;

[0034] Fig.12 The photocatalytic carbon nanodots of the present invention were used to treat mice with 4T1 tumors under visible light irradiation on day 12, and the tumor tissues were stained with hematoxylin-eosin (H&E), cleaved caspase3 (c-Cas-3), and Ki67. Scale bar: 100 μm. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0036] The application of the carbon nanodots proposed by the present invention in the preparation of photocatalytic drugs is described in detail below.

[0037] Some embodiments of the present invention provide a use of carbon nanodots in preparing photocatalytic drugs, wherein the carbon nanodots have electron-withdrawing groups on their surfaces.

[0038] After research and practice, the inventors found that the electron-withdrawing groups on the surface of carbon nanodots can capture excited electrons generated by light and undergo a reduction reaction, thereby extending the life of holes generated by illumination, making them have strong photo-oxidation ability. Under illumination, the carbon nanodots can effectively kill cells and antibiotic-resistant bacteria in an aqueous solution by oxidizing hydroxide ions in water to generate hydroxyl radicals, and have good applications in tumor photocatalytic therapy and photocatalytic sterilization. It is understandable that the more electron-withdrawing groups on the surface of carbon nanodots, the better their photocatalytic effect. In addition, the existing applications of carbon nanodots in the biological field have proved that they have the characteristics of simple preparation, wide absorption range, easy surface functionalization, good biocompatibility and low cytotoxicity. Therefore, they have better advantages in biocompatibility and biosafety.

[0039] In order to make the carbon nanodots have better application effects, in some embodiments, the atomic content of the electron-withdrawing groups is greater than 25% of the atomic content of the carbon nanodots, preferably greater than 30%.

[0040] Specifically, in some embodiments, the electron-withdrawing group includes at least one of a carbonyl group, a carboxyl group, and a pyridyl group. That is, the electron-withdrawing group may be multiple or a single one, as long as it can achieve a reduction reaction of the electron-withdrawing group after irradiation with light, and the generated holes oxidize the hydroxide ions in the water into hydroxyl radicals.

[0041] Furthermore, in some embodiments, the carbon nanodots are mainly prepared by solvothermal reaction of citric acid and urea in formic acid.

[0042] Specifically, the mass ratio of citric acid to urea is 1:1-5, for example 1:1, 1:2, 1:3, 1:4 or 1:5, preferably 1:2-4, more preferably 1:2.

[0043] In some embodiments, in order to achieve a better reaction effect, the total concentration of citric acid and urea in formic acid is 0.1 g / mL to 0.4 g / mL.

[0044] In some embodiments, the reaction temperature of the solvothermal reaction is 150°C to 170°C, for example 150°C, 155°C, 160°C, 165°C or 170°C, and the reaction time is 3h to 5h, for example 3h, 3.5h, 4h, 4.5h or 5h.

[0045] According to the test, the size of the carbon nanodots used for photocatalysis prepared by the solvent thermal reaction of citric acid and urea in formic acid is about 2nm to 6nm; after incubating with cells for one hour, they can enter cells and perform cell imaging; they can kill cells under visible light irradiation; the main absorption band of the carbon nanodots in water is 400nm-650nm, and under the excitation of the light source in the ultraviolet-visible light region, the red light emission peak is 600nm to 800nm, and the emission center changes after visible light irradiation and the fluorescence lifetime of the red light emission is shortened. In addition, the carbon nanodots have high water solubility, good biocompatibility, low cytotoxicity and can be excreted through the kidneys.

[0046] In some embodiments, the photocatalytic drugs prepared by the carbon nanodots are tumor photocatalytic drugs or photocatalytic antibacterial drugs.

[0047] Among them, the tumors treated by tumor photocatalytic drugs include but are not limited to any one of non-small cell lung cancer, breast cancer, pancreatic cancer, liver cancer, colorectal cancer, prostate cancer, cervical cancer, ovarian cancer, malignant melanoma and leukemia, and the preferred tumor to be treated is breast cancer.

[0048] The bacteria that the photocatalytic antibacterial drug fights are antibiotic-resistant bacteria, preferably tetracycline-resistant Escherichia coli.

[0049] Some embodiments of the present invention also provide an anti-tumor combination drug, which includes the above carbon nanodots and other anti-tumor drugs. That is, the carbon nanodots can be combined with some existing tumor treatment drugs, such as targeted antibody drugs, to treat tumors to achieve better treatment effects.

[0050] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0051] Carbon nanodots that can be used to prepare photocatalytic therapeutic drugs are prepared by the following steps:

[0052] 2g of citric acid and 4g of urea were dissolved in 20mL of formic acid, and the liquid was placed in a 50mL polytetrafluoroethylene high-pressure reactor for heating reaction at a temperature of 160°C for 4 hours in an oven. 60mL of ethanol was added to the solution after the reaction, and the reaction was centrifuged at a speed of 10,000 rpm to remove the upper solution, leaving a precipitate, which was centrifuged 3 times. The lower precipitate was freeze-dried to obtain a black solid powder, namely carbon nanodots (CDs).

[0053] According to the above steps, only the formic acid in the step was replaced by acetic acid, the solution after the reaction was put into a dialysis bag (cut-off molecular weight was 2000Da), and dialyzed with ultrapure water for two days to finally obtain acetic acid carbon nanodots (ACOH-CDs).

[0054] According to the above steps, only the formic acid in the step was replaced by acetone, the solution after the reaction was put into a dialysis bag (cut-off molecular weight was 2000Da), and dialyzed with ultrapure water for two days to finally obtain acetone carbon nanodots (DMK-CDs).

[0055] According to the above steps, only the formic acid in the step is replaced by dimethylformamide, and finally dimethylformamide carbon nanodots (DMF-CDs) are obtained.

[0056] Test Example 1

[0057] Carbon nanodots (CDs) were analyzed by transmission electron microscopy. The transmission electron microscopy TEM image is shown in Figure 1 shown.

[0058] Depend on Figure 1 It can be seen that the particle size of carbon nanodots (CDs) is around 2-6 nm.

[0059] Test Example 2

[0060] The electron paramagnetic resonance (EPR) spectra of carbon nanodots (CDs) were tested. Figure 2 shown.

[0061] Depend on Figure 2 It can be seen that after 10 minutes of visible light irradiation, characteristic signals with intensities of approximately 1:2:2:1 were detected in the aqueous solution of carbon nanodots. These signals are spin adducts of DMPO and hydroxyl groups.

[0062] Test Example 3

[0063] Carbon nanodots (CDs) were incubated with 4T1 cells for one hour, and the cell fluorescence imaging photos were shown in Figure 2. Figure 3 shown.

[0064] Depend on Figure 3 It can be seen that 4T1 cells incubated with carbon nanodots (CDs) exhibited red fluorescence under 560 nm excitation, indicating that carbon nanodots can enter the cells for fluorescence imaging.

[0065] Test Example 4

[0066] The cytotoxicity of carbon nanodots (CDs) on 4T1 cells under dark and visible light irradiation conditions was investigated by CCK-8 assay. Figure 4 shown.

[0067] Depend on Figure 4 It can be seen that after 24 hours of incubation in the dark, the control group cells and 250 μg mL -1 There was no significant difference between the cells treated with CNDs, indicating the inherent low cytotoxicity of CNDs. In contrast, as the concentration of CNDs increased to 250 μg mL -1 , high cytotoxicity was detected after 5 min of visible light irradiation and then incubation in the dark for 24 h, indicating that carbon nanodots (CDs) have strong visible light-induced cell killing ability.

[0068] Test Example 5

[0069] The absorption spectra of carbon nanodots (CDs) aqueous solution with or without hole sacrificial agent triethanolamine (TEA) under visible light irradiation for different time periods were investigated. Figure 5 As shown;

[0070] Depend on Figure 5 It can be seen that under visible light irradiation, the main absorption band of carbon nanodots (CDs) at 553nm gradually decreases with the increase of irradiation time. After adding TEA, the main absorption band of carbon nanodots decreases faster at the same irradiation time.

[0071] Test Example 6

[0072] Excitation-emission spectra of carbon nanodots (CDs) after adding triethanolamine in aqueous solution before visible light irradiation (CDs) and after visible light irradiation for 20 minutes (ir-CDs), as shown in Figure 6 As shown;

[0073] Depend on Figure 6 It can be seen that after 20 minutes of visible light irradiation, the red emission center of the carbon nanodots is no longer dominant.

[0074] Test Example 7

[0075] Under 510 nm excitation, the fluorescence decay curves of carbon nanodots (CDs) at 638 nm were monitored in aqueous solution before visible light irradiation (CDs) and after 20 min of visible light irradiation (ir-CDs). Figure 7 As shown;

[0076] Depend on Figure 7 It can be seen that after 20 min of visible light irradiation, the average fluorescence lifetime of the red emission of carbon nanodots was shortened, indicating that the red emission center of ir-CDs changed.

[0077] Test Example 8

[0078] X-ray photoelectron spectroscopy (XPS) analysis was performed on carbon nanodots (CDs), acetic acid carbon nanodots (ACOH-CDs), acetone carbon nanodots (DMK-CDs) and dimethylformamide carbon nanodots (DMF-CDs). Figure 8 As shown, Figure 8 (a) is the XPS full spectrum and high-resolution spectrum; (b) is the C1s spectrum, (c) is the N1s spectrum, and (d) is the O1s spectrum. The atomic contents of C, N, and O as well as the specific contents of N-related and O-related chemical bonds are shown in Table 1.

[0079] Table 1

[0080]

[0081] Depend on Figure 8 As shown in Table 1, the C=O content in CDs (17.43%) and DMF-CDs (16.26%) is much higher than that in AcOH-CDs (9.44%) and DMK-CDs (7.17%), while the NH and COH contents in DMF-CDs (10.48% and 7.70%, respectively) are higher than those in CDs (5.30% and 7.21%, respectively). In view of the electron-withdrawing properties of the C=O group and the proton-donating properties of the -NH and -OH groups, CDs exhibit the strongest surface electron-withdrawing structure among the four samples.

[0082] Test Example 9

[0083] X-ray photoelectron spectroscopy (XPS) analysis was performed on the carbon nanodots (CDs) and the carbon nanodots after visible light irradiation for 20 min (ir-CDs). Fig. 9 As shown, Fig. 9 (a) is the XPS full spectrum and high-resolution spectrum; (b) is the C1s spectrum, (c) is the N1s spectrum, and (d) is the O1s spectrum. The specific contents of different chemical bonds of C, N, and O are shown in Table 2.

[0084] Table 2

[0085]

[0086] Depend on Fig. 9 As shown in Table 2, the atomic content of carbon nanodots (CDs) has almost no change. After 20 minutes of visible light irradiation, the contents of pyridinic N (399.8 eV) and C=O (531.5 eV) in CDs decreased from 10.2% and 17.4% to 4.8% and 10.3%, respectively, while the contents of pyrrolic N (400.7 eV) and CO / OH (532.7 eV) in CDs increased from 5.3% and 7.2% to 8.8% and 15.0%, respectively. It can be inferred that the electron-withdrawing pyridinic N and C=O groups on the surface of CDs are reduced to pyrrolic N and C-OH after irradiation with visible light.

[0087] It can be seen that the surface of carbon nanodots (CDs) is rich in electron groups, such as C=O and pyridinic N. Under the excitation of visible light, the photogenerated electrons in the conduction band of carbon nanodots (CDs) are captured by the C=O and pyridinic N groups on the surface and photoinduced reduced to -OH and pyrrolic N groups, respectively. The photogenerated holes in the valence band of carbon nanodots (CDs) interact with water and oxidize the hydroxide ions in water to generate hydroxyl radicals. In the presence of triethanolamine, the photogenerated holes are consumed, the recombination of photoexcited electrons and holes is inhibited, and the photoinduced reduction of electron-withdrawing groups on the surface of carbon nanodots (CDs) is more favorable, resulting in a rapid decrease in the absorption band of carbon nanodots (CDs) at 553nm. The above results show that carbon nanodots (CDs) have the potential to be used in photocatalytic therapy.

[0088] Test Example 10

[0089] Carbon nanodots (CDs) were added to tetracycline (TC)-resistant Escherichia coli (E. coli), irradiated with visible light for 10 minutes, and then cultured on eosin methylene blue (EMB) agar plates for 48 hours. The colonies grown on the EMB agar plates are shown in the following figure. Fig.10 As shown in e, the colony images of the control group, CDs group, TC treatment group and visible light irradiation group are shown in Fig.10 As shown in the ad, Fig.10Figure f shows the normalized colony forming unit (CFU) statistics based on the control group. After 10 minutes of visible light irradiation, the CFU number of TC-resistant Escherichia coli treated with CDs was only 24% of that without irradiation. It can be seen that CDs exhibit good visible light-induced antibacterial properties.

[0090] Test Example 11

[0091] An aqueous solution of carbon nanodots (CDs) was intratumorally injected into mice bearing 4T1 tumors and irradiated with visible light for 10 minutes. The control groups included mice treated with CDs without irradiation (CDs group), mice irradiated with visible light (visible light group), and mice given phosphate buffered saline (PBS group). Photos of mice in each group are shown in Fig.11 After treatment, the tumor size of mice was measured every 2 days. Fig.11 As shown in d, the tumor size of the control group, i.e., CDs group, visible light irradiation group, and PBS group, grew rapidly, while the CDs+visible light group showed significant inhibition of tumor growth, confirming the effectiveness of photocatalytic tumor therapy based on carbon nanodots (CDs). To further evaluate the anti-tumor effect, histopathological and immunohistochemical (IHC) analyses were performed on the tumor tissues of mice on the 12th day after different treatments. Fig.12 It can be seen that there was no obvious tumor tissue damage in the control group. However, in the CDs+visible light group, hematoxylin and eosin (H&E), cleaved caspase 3 (c-Cas-3), and Ki67 staining images showed obvious cell damage and apoptosis, and decreased proliferation activity, confirming the effect of photocatalytic tumor therapy on tumors. In addition, after CDs-based photocatalytic tumor therapy, the 30-day survival rate of mice increased from 0 to 100% ( Fig.11 e) in.

[0092] from Figure 10-12 It can be seen that the carbon nanodots (CDs) provided in the embodiments of the present invention can be applied to photocatalytic antibacterial and photocatalytic tumor treatment in mice.

[0093] In summary, the carbon nanodots (CDs) in the embodiments of the present invention are characterized by having strong electron-withdrawing groups on the surface, which can capture excited electrons generated by the carbon nanodots absorbing light and undergo a reduction reaction, thereby extending the life of holes generated by light irradiation and achieving strong oxidation ability. The preparation method of the carbon nanodots is simple, low-cost, and easy to prepare in large quantities. It has good application prospects in the fields of biomedicine and the like, and can be specifically used in photocatalytic antibacterial and photocatalytic tumor treatment in mice.

[0094] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An application of carbon nanodots in the preparation of photocatalytic drugs, characterized in that: The carbon nanodots have electron-withdrawing groups on their surfaces; The carbon nanodots are mainly prepared by a solvothermal reaction of citric acid and urea in formic acid; the mass ratio of the citric acid to the urea is 1:1-5, and the total concentration of the citric acid and the urea in the formic acid is 0.1 g / mL-0.4 g / mL; the reaction temperature of the solvothermal reaction is 150°C-170°C, and the reaction time is 3h-5h; The photocatalytic drug is a tumor photocatalytic drug or a photocatalytic antibacterial drug. The tumor treated by the tumor photocatalytic drug is breast cancer, and the bacteria treated by the photocatalytic antibacterial drug is Escherichia coli.

2. The use according to claim 1, characterized in that: The atomic content of the electron-withdrawing groups is greater than 25% of the atomic content of the carbon nanodots.

3. The use according to claim 1, characterized in that: The atomic content of the electron-withdrawing groups is greater than 30% of the atomic content of the carbon nanodots.

4. The use according to claim 1, characterized in that: The electron withdrawing group includes at least one of a carbonyl group, a carboxyl group and a pyridyl group.

5. The use according to claim 1, characterized in that: The size of the carbon nanodots is 2nm~6nm.

6. The use according to claim 1, characterized in that: The main absorption band of the carbon nanodots in water is 400nm~650nm.

7. The use according to claim 1, characterized in that: The mass ratio of the citric acid to the urea is 1:2-4.

8. An anti-breast cancer combination drug, characterized in that: It comprises the carbon nanodots as described in any one of claims 1 to 7 and other anti-breast cancer drugs.

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