Curcumin carbon dot nano-enzyme and application thereof in preparation of medicine for preventing and treating lung injury

Abstract

The invention provides curcumin carbon dot nano-enzyme and application thereof in preparation of medicine for preventing and treating lung injury, a preparation method of the curcumin carbon dot nano-enzyme comprises the following steps: 1) adding tobacco powder, reduced glutathione and curcumin into formamide, stirring, performing ultrasonic treatment, and performing high-temperature reaction; and 2) after the reaction is finished, cooling to room temperature, filtering, collecting filtrate, transferring into a dialysis bag, enabling the dialyzed solution to pass through a water film, and freeze-drying to obtain the curcumin carbon dot nano-enzyme. The invention belongs to the technical field of nano-enzymes, and the curcumin carbon dot nano-enzyme provided by the invention has a dual-wavelength fluorescence emission characteristic, realizes effective utilization of tobacco leaf wastes, and is expected to play an important role in preparation of medicines for preventing and treating lung injury.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanozymes, and in particular relates to curcumin carbon dot nanozymes and applications thereof in the preparation of drugs for preventing and treating lung injury. Background Art

[0002] During tobacco planting and processing, a large amount of waste tobacco byproducts (such as tobacco leaves and tobacco stems) are generated, accounting for about 25% of the total tobacco production, resulting in serious waste of resources and environmental pollution. If waste tobacco byproducts can be effectively utilized, it can not only greatly increase the added value, but also be beneficial to environmental protection. How to achieve high-value recycling of tobacco waste is a hot issue that has long been concerned about in the industry.

[0003] Carbon dots (CDs) are a new type of nanocarbon material composed of dispersed spherical carbon particles. They are extremely small (below 10 nm), have fluorescent properties, and have the advantages of simple preparation, low toxicity, biocompatibility, and easy functionalization. They have shown great application potential in agriculture and materials. The paper entitled "Inhibition of oxidative stress in vivo through enzyme-like activity of carbon dots" published by Dong et al in 2021 confirmed that antioxidant CDs nanozymes have good biocompatibility and in vivo biosafety, and have broad prospects for biomedical applications.

[0004] Smoking has long been considered one of the major threats to severe respiratory diseases such as acute lung injury (ALI). Currently, the treatment of ALI mainly relies on the use of glucocorticoids or nicotinamide adenine dinucleotide phosphate oxidase inhibitors, which can downregulate inflammatory factors or increase blood flow in the ventilation area by inhaling vasodilator gases. However, these treatments have the disadvantages of high cost, poor prognosis, and inability to fundamentally neutralize excessive ROS around the inflammatory area.

[0005] Although curcumin has antioxidant, antibacterial, and anti-tumor effects, it is insoluble in water and has unstable chemical properties. It is easily degraded in neutral and alkaline environments and has low bioavailability, which limits its application in the fields of medicine. Chinese patent application CN 115501336 A discloses a water-soluble curcumin complex with antibacterial ability, and its preparation method includes the following steps: step 1, using citric acid as a carbon source and thiourea as a nitrogen source and a sulfur source, a carbon dot is prepared by a one-step hydrothermal method; step 2, the prepared carbon dot, EDC, NHS and dimethyl sulfoxide are stirred, allowed to stand, cholesterol dissolved in dimethyl sulfoxide is added, stirring is continued, and then a curcumin solution is added, stirring is continued, dialyzed, and freeze-dried to obtain a curcumin complex; by combining the prepared carbon dot with cholesterol and curcumin, using a variety of reagents for surface modification, etc., a composite drug with good water solubility is obtained, and the bioavailability of curcumin is improved. With the rapid development of nanomaterial technology, there have been many reports on antioxidant carbon dot nanozymes, but there have been no reports on the use of carbon dot nanozymes in preventing and treating lung injury.

[0006] Therefore, it is of great significance to provide a curcumin carbon dot nanozyme and its application in the preparation of drugs for preventing and treating lung injury. Summary of the invention

[0007] To solve the problems existing in the prior art, the present invention provides a curcumin carbon dot nanozyme (Cur@TB CDs) prepared from waste tobacco leaves as raw materials, and realizes the organic combination and synergistic enhancement of carbon dots and curcumin through chemical reactions, etc. The curcumin carbon dot nanozyme has dual-wavelength fluorescence emission characteristics, and the surface contains rich functional groups such as carbonyl, hydroxyl, carboxyl, and amino groups, and also contains sulfur oxide after combining curcumin, which improves the water solubility of the carbon dot nanozyme, enhances the superoxide dismutase (SOD) activity of the carbon dot nanozyme, has high reactive oxygen species (ROS) scavenging ability and anti-inflammatory activity, has good SOD activity and hydroxyl radical scavenging ability, and can improve acute lung injury (ALI) in vitro and in vivo. In addition, the curcumin carbon dot nanozyme provided by the present invention is a biomass-derived product with good biosafety.

[0008] The purpose of the present invention will be further illustrated by the following detailed description.

[0009] The present invention provides a curcumin carbon dot nanozyme, and the preparation method thereof comprises the following steps:

[0010] 1) Add tobacco powder, reduced glutathione and curcumin into formamide, stir, ultrasonicate and perform high temperature reaction;

[0011] 2) After the reaction is completed, the mixture is cooled to room temperature, the filtrate is collected by filtration, and the filtrate is transferred to a dialysis bag. The dialyzed solution is passed through a water membrane and then freeze-dried to obtain curcumin carbon dot nanozyme.

[0012] Preferably, in step 1), the mass ratio of tobacco powder, reduced glutathione and curcumin is (0.8-1.2):(2.5-3.5):(0.04-0.06). The amount of curcumin added should not be too high or too low, otherwise it will easily lead to residue or insufficient reaction, which will have an adverse effect on the stability and SOD activity of curcumin carbon dot nanozyme.

[0013] More preferably, in the step 1), the mass ratio of tobacco powder, reduced glutathione and curcumin is (0.9-1.1):(2.8-3.2):0.05.

[0014] Preferably, the conditions of the high temperature reaction include: temperature 160-190° C., time 7-10 h.

[0015] More preferably, the conditions of the high temperature reaction include: temperature 180° C., time 8 h.

[0016] Preferably, the molecular weight cutoff of the dialysis bag is 450-550 Dalton. More preferably, the molecular weight cutoff of the dialysis bag is 480-520 Dalton.

[0017] Preferably, the dialysis duration is 6-9 days.

[0018] Preferably, the tobacco powder is obtained by removing stems, cutting, crushing and sieving tobacco leaf samples. The source of tobacco leaf samples includes waste tobacco leaves.

[0019] Preferably, the particle size of the water film is 0.22 μm.

[0020] In addition, the present invention also provides the use of the curcumin carbon dot nanozyme in the preparation of a drug for preventing and treating lung injury. Preferably, the lung injury is acute lung injury.

[0021] Compared with the prior art, the beneficial effects of the present invention include:

[0022] (1) The present invention provides a novel curcumin carbon dot nanozyme (Cur@TB CDs), which is prepared by a one-pot hydrothermal method using tobacco powder, reduced glutathione and curcumin as raw materials and formamide as solvent; the organic combination and synergistic enhancement of carbon dots and curcumin are achieved through chemical reactions, etc. The curcumin carbon dot nanozyme has dual-wavelength fluorescence emission characteristics, which realizes the effective utilization of tobacco waste. The surface of the curcumin carbon dot nanozyme contains abundant functional groups such as carbonyl, hydroxyl, carboxyl and amino groups, and also contains sulfur oxide after combining with curcumin, which improves the water solubility of the carbon dot nanozyme and enhances the superoxide dismutase (SOD) activity of the carbon dot nanozyme.

[0023] (2) The present invention also provides the use of curcumin carbon dot nanozyme in the preparation of drugs for preventing and treating lung injury. Experiments have confirmed that the curcumin carbon dot nanozyme provided by the present invention has high reactive oxygen species (ROS) scavenging ability and anti-inflammatory activity, good SOD activity and hydroxyl free radical scavenging ability, can improve acute lung injury (ALI) in vitro and in vivo, and has good biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A schematic diagram of the preparation process of curcumin carbon dot nanozyme provided by the present invention.

[0025] Figure 2 The curcumin carbon dot nanozyme prepared in Example 1 was subjected to structural characterization and fluorescence spectrum analysis.

[0026] Figure 3 X-ray photoelectron spectroscopy analysis of the curcumin carbon dot nanozyme prepared in Example 1.

[0027] Figure 4 Figure 1 shows the stability test results of the curcumin carbon dot nanozyme prepared in Example 1 in deionized water.

[0028] Figure 5 Figure 1 shows the stability test results of the curcumin carbon dot nanozyme prepared in Example 1 in PBS.

[0029] Figure 6 In vivo biosafety analysis results of the curcumin carbon dot nanozyme prepared in Example 1.

[0030] Figure 7 The anti-inflammatory effect results of the curcumin carbon dot nanozyme prepared in Example 1 on LPS-induced ALI mice.

[0031] Figure 8 The dry-to-wet ratio results of lung tissue in different treatment groups. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0033] In the present invention, the experimental materials and equipment involved are conventional commercial products or can be obtained by conventional technical means in the art, and the detection method used is carried out according to the general method or kit instructions in the art. For example: SOD detection kit-WST (WST-1) was purchased from Japan Tongren Chemical Research Institute, and SOD detection kit-WST-8 (WST-8) was purchased from Suzhou Grace Biotechnology Co., Ltd. The male C57BL / 6 mice involved in this experiment were purchased from the Experimental Animal Center of Anhui Medical University, and all animal experiments were reviewed and approved by the Animal Care and Use Committee of Anhui Medical University (No.: LLSC20220731). A field emission transmission electron microscope (FEI Tal os F200X, USA) was used to obtain transmission electron microscopy (TEM) images of Cur@TB CDs. The zeta potential and hydrodynamic size of Cur@TB CDs were analyzed using a zetasizer (Nano-Z, Malvern, UK). All absorption spectra were recorded using a carry 50 UV spectrometer (Varian, USA). F-7000 fluorescence spectrometer (Hitachi, Japan) was used to collect fluorescence spectra. Infrared spectrophotometer (Perkin Elmer, USA) was used to detect the Fourier transform infrared spectrum (FT-IR) of Cur@TB CDs. ESCALAB 250Xi X-ray photoelectron spectrometer (XPS, Thermo Electron, USA) was used to perform elemental analysis on Cur@TB CDs. Bruker D8Advance (Germany) was used to obtain X-ray diffraction (XRD) data of Cur@TB CDs. Renishaw Invia instrument (Renishaw, UK) was used to collect Raman data of Cur@TBCDs. Electron paramagnetic resonance (EPR) spectrum of ROS was measured using Bruker A300-10 / 12 instrument (Bruker, Germany).

[0034] % content, if not explicitly stated, is understood as commonly used in the art. Unless otherwise specified, the results of this study are expressed as mean ± SD. The statistical comparison between the two groups was performed using the Student's t test. P < 0.05 was considered statistically significant. Statistical tests were performed using origin 2022.

[0035] Example 1 Preparation of Curcumin Carbon Dot Nanozyme

[0036] A curcumin carbon dot nanozyme, the process diagram of its preparation method is as follows Figure 1 As shown, the following steps are included:

[0037] 1) Add 0.1 g of waste tobacco powder, 0.3 g of reduced glutathione, and 0.005 g of curcumin into 10 mL of formamide, stir and ultrasonicate for 30 min, and perform high temperature reaction at 180 °C for 8 h;

[0038] 2) After the reaction is completed, the mixture is cooled to room temperature, the filtrate is collected by filtration, and transferred to a dialysis bag with a molecular weight cutoff of 500 Dalton. The dialyzed solution is passed through a 0.22 μm water membrane and then freeze-dried to obtain curcumin carbon dot nanozyme.

[0039] Example 2 Preparation of Curcumin Carbon Dot Nanozyme

[0040] A curcumin carbon dot nanozyme, the preparation method of which comprises the following steps:

[0041] 1) Add 0.12 g of waste tobacco powder, 0.32 g of reduced glutathione, and 0.006 g of curcumin into 10 mL of formamide, stir and ultrasonicate for 30 min, and perform high temperature reaction at 170 °C for 9 h;

[0042] 2) After the reaction is completed, the mixture is cooled to room temperature, the filtrate is collected by filtration, and transferred to a dialysis bag with a molecular weight cutoff of 500 Dalton. The dialyzed solution is passed through a 0.22 μm water membrane and then freeze-dried to obtain curcumin carbon dot nanozyme.

[0043] Example 3 Characterization and performance investigation of curcumin carbon dot nanozyme

[0044] In order to evaluate the effect of Cur@TB CDs prepared in Example 1 on 2 - The scavenging capacity of xanthine was firstly generated by 2.5 mM xanthine and 0.25 U / mL xanthine oxidase at room temperature. 2 - In order to evaluate the scavenging ability of Cur@TB CDs for ·OH, Fe 2+ and H 2 O 2 The samples were incubated with 0.0 (control group), 20.0, 50.0, 100.0, and 200.0 μg / mL of Cur@TB CDs and DMPO, respectively. Afterwards, EPR measurements were performed on all samples under the same conditions.

[0045] The curcumin carbon dot nanozyme (Cur@TB CDs) prepared in Example 1 was subjected to structural characterization and fluorescence spectroscopy analysis. Figure 2 As shown in Figure 2. Among them, the TEM image of Cur@TB CDs is shown in Figure 2. Figure 2 -A. The hydrodynamic size of Cur@TB CDs in deionized water is shown in Figure 2 -B, the main particle size distribution is 5-7nm. The XRD spectrum of Cur@TB CDs is shown in Figure 2 -C. The Raman, Fourier transform infrared and ultraviolet absorption spectra of Cur@TB CDs are shown in Figure 2 -D. Figure 2 -E, Figure 2 -F. The fluorescence spectra of Cur@TB CDs at different excitation wavelengths from 370 to 420 nm are shown in Figure 2 -G, where the inset is a photo of Cur@TB CDs before (left green EP tube) and after (right pink EP tube) excitation by 420nm UV light in deionized water. 2 - The EPR spectrum of DMPO (generated by xanthine and xanthine oxidase) is as follows: Figure 2 -H. ·OH (by Fe 2+ and H 2 O 2 The EPR spectra of the incubated Cur@TB CDs with DMPO and different concentrations (0.0 control group, 20.0, 50.0, 100.0 and 200.0 g / mL) are shown in Figure 2. Figure 2 -I. From Figure 2 It can be seen that the Cur@TB CDs provided by the present invention exhibit typical dual-wavelength fluorescence emission under a single excitation wavelength, which has the characteristics of being independent of excitation, which means that sp 2 The uniform surface state of the clusters leads to highly uniform sizes of Cur@TB CDs, and in the range of 0-200.0 g / mL, the higher the concentration of Cur@TB CDs, the better the free radical scavenging effect.

[0046] The curcumin carbon dot nanozyme prepared in Example 1 was subjected to X-ray photoelectron spectroscopy (XPS) analysis. The overall analysis of the curcumin carbon dot nanozyme is shown in FIG. Figure 3 -A, the high-resolution binding energy spectra of C1s, N 1s, O 1s and S2p in curcumin carbon dot nanozymes are shown as Figure 3 -B to 3-E. Figure 3 It can be seen that Cur@TB CDs mainly contain C, O, N and S elements. Through deep analysis, the high-resolution binding energy spectrum of C 1s showed two peaks at 286.04eV and 284.50eV, which were attributed to OC=O and C=C / CC, respectively, indicating that the sp 2Orbital hybridization is representative. In addition, in the high-resolution binding energy spectrum of O1s, a typical peak belonging to CO appeared at 532.28eV. These findings also prove the reason why the mass fractions of C (59.44%) and O (37.1%) elements in Cur@TB CDs are relatively high, while the mass fraction of N (3.11%) element is relatively low, which is consistent with the fact that the main component of tobacco is carbohydrate. In addition, there are two peaks at 400.10eV and 398.27eV, corresponding to CN=C and N=N, respectively, and two peaks at 167.65eV and 163.99eV, corresponding to S2p3 / 2 and sulfur oxide, respectively. It can be seen that the surface of Cur@TB CDs may have abundant functional groups such as carbonyl, amino, and hydroxyl groups, and also contains sulfur oxide after binding to curcumin, which helps to increase the water solubility of Cur@TB CDs.

[0047] Next, the present invention investigated the stability of Cur@TB CDs. 100.0 g / mL Cur@TB CDs were cultured in deionized water and PBS for 20.0 days, and the changes in the normalized fluorescence intensity at a wavelength of 650 nm were investigated. The results are as follows: Figure 4 and Figure 5 As shown, it can be seen that Cur@TB CDs exhibit good stability.

[0048] The kinetics of 5.0-60.0 g / mL Cur@TB CDs for water-soluble tetrazolium salts and O were tested using a commercial kit (WST-1). 2 - The SOD-like activity of Cur@TB CDs was quantitatively detected by using a SOD detection kit (WST-8). The principle of WST-1 and WST-8 to detect the SOD-like activity of Cur@TB CDs is as follows: The principle of the WST-1 kit to evaluate the SOD-like activity depends on the reaction of Cur@TB CDs with water-soluble tetrazolium salts (WST-1) and O 2 - ·Inhibition of the reaction (generated by xanthine and xanthine oxidase). Generally speaking, water-soluble tetrazolium salts react with O 2 - The product of the reaction is a chromogen of WST-1 methyl dye, whose absorption peak is at 450nm. In this case, the SOD-like activity of Cur@TB CDs can competitively scavenge O 2 -·, inhibiting the generation of WST-1 methyl dye, resulting in a decrease in absorbance at a wavelength of 450nm. Therefore, the inhibition rate of Cur@TB CDs on the generation of WST-1 methyl dye can be evaluated by measuring the change in the OD value at a wavelength of 450nm to evaluate the SOD-like activity of Cur@TB CDs. The higher the inhibition rate, the higher the SOD-like activity of Cur@TB CDs. Here, the calculation formula of "inhibition rate (%)" of the WST-1 kit is [(ODblank-1-ODblank-3)-(ODsample-ODblank-2)] / (ODblank-1-ODblank-3)×100%. Among them, ODblank-1 refers to the OD value at a wavelength of 450nm, that is, the blank control without Cur@TB CDs; ODblank-3 refers to the reaction system without Cur@TB CDs and xanthine oxidase; ODsample refers to the reaction system containing Cur@TB CDs; ODblank-2 refers to the reaction system containing Cur@TB CDs but without xanthine oxidase. The results showed that Cur@TB CDs had a 2 - The inhibitory effect of Cur@TB CDs on O 2 - The inhibition rate of · is almost 100%. Similarly, the SOD enzyme activity of Cur@TBCDs was calculated to be 614.70U / mg according to the commercial kit WST-8. The calculation formula of SOD enzyme activity (U / mg) is: =10×inhibition rate / (1-inhibition rate)×D. It can be seen that the curcumin carbon dot nanozyme provided by the present invention has good SOD enzyme activity and also has a significant elimination ability for ·OH.

[0049] Example 4 In vivo biosafety of curcumin carbon dot nanozyme and its efficacy against ALI

[0050] The safety of therapeutic nanomedicines in vivo is of vital importance. Therefore, the present invention evaluated the biosafety of Cur@TB CDs prepared in Example 1 from aspects such as in vivo toxicity analysis, hemolytic analysis, and serum biochemical analysis of the liver and kidney.

[0051] In vivo toxicity analysis: Nine healthy male C57BL / 6 mice aged 8.0-10.0 weeks and weighing 20.0-25.0 g were used in this study. The mice were housed under standard laboratory conditions, including a 12.0 h light / dark cycle, controlled temperature and humidity, and free access to food and water. After one week of acclimatization, the mice were randomly assigned to three groups (3 mice in each group). The first group received a single intratracheal injection of Cur@TB CDs in PBS at a dose of 20.0 mg / kg and was observed for 7.0 days; the second group received a single intratracheal injection of Cur@TB CDs in PBS at a dose of 20.0 mg / kg and was observed for 30.0 days; the third group received an intratracheal instillation of an equal volume of PBS and was observed for 30.0 days. All instillations were performed under light anesthesia to ensure accurate dosing and minimize suffering. At the end of the monitoring period in each group, the mice were euthanized by an overdose of isoflurane. The major organs, including liver, spleen, kidney, heart, and lung, were carefully removed, rinsed with PBS, and fixed in 10% neutral buffered formalin for 24.0 h. After fixation, the tissues were processed, embedded in paraffin, and sliced ​​at a thickness of 4.0 μm. The sections were stained with hematoxylin and eosin (H&E) to examine histopathological changes, and the sections were evaluated under light microscopy for potential lesions or morphological changes caused by Cur@TB CDs exposure.

[0052] Hemolytic analysis: In this study, erythrocytes were collected from healthy male C57BL / 6 mice aged 8.0-10.0 weeks and weighing 20.0-25.0 g and diluted to 2.0% with PBS. Different concentrations of Cur@TB CDs (50.0-300.0 μg / mL) were mixed with the erythrocyte suspension and incubated on a shaker at 37.0°C for 3.0 hours. Then, centrifugation was performed at 2000 rpm for 10.0 minutes. The positive and negative controls were treated similarly to the diluted blood and directly mixed with saline and deionized water. The absorbance of the supernatant at a wavelength of 540 nm was measured using an ELISA reader, and the hemolysis rate (%) was calculated as (ODsample-ODneg) / (ODpos-ODneg)×100%, where ODsample is the absorbance of the different concentration groups, ODneg is the absorbance of the negative control, and ODpos is the absorbance of the positive control.

[0053] Serum biochemical analysis of liver and kidney: Nine healthy male C57BL / 6 mice, aged 8.0-10.0 weeks and weighing 20.0-25.0 g, were used in this study. The mice were housed under standard laboratory conditions, including a 12.0-h light / dark cycle, controlled temperature and humidity, and free access to food and water. After one week of acclimatization, the mice were randomly assigned to three groups (3 mice in each group). The first group received a single intratracheal instillation of Cur@TB CDs in PBS at a dose of 20.0 mg / kg and was observed for 7.0 days. The second group received a single intratracheal instillation of Cur@TB CDs in PBS at a dose of 20.0 mg / kg and was observed for 30.0 days. At the end of each treatment period, blood samples were collected from mice in each group. The blood samples were centrifuged at 4000 rpm for 15 minutes at 4°C to collect serum, which was used to analyze liver function (aspartate aminotransferase AST, alkaline phosphatase ALP, alanine aminotransferase ALT) and renal function (blood urea nitrogen BUN and creatinine CR).

[0054] The results of in vivo biosafety analysis of Cur@TB CDs are shown in Figure 6 Among them, the H&E staining images of the liver, spleen, kidney, heart and lung of healthy mice after a single intratracheal instillation of 20.0 mg / kg Cur@TB CDs at 0.0 days (control group), 7.0 days and 30.0 days are shown in Figure 2. Figure 6 -A. The hemolysis rate of erythrocytes after incubation with 1: saline (positive), 2: deionized water (negative), 3-8: 50.0, 100.0, 150.0, 200.0, 250.0 and 300.0 μg / mL Cur@TB CDs is shown in Figure 2. Figure 6 -B, wherein the inset is the corresponding photograph of the above-treated red blood cells. The levels of liver and kidney function indicators (i.e., ALT, AST, ALP, BUN and CR) in the serum of the above-mentioned healthy mice after a single intratracheal infusion of 20.0 mg / kg Cur@TB CDs at 0.0 day (control group), 7.0 day and 30.0 day, as shown in FIG. Figure 6 -C to Figure 6 -G. Data are presented as mean ± SD (n = 3). Figure 6 It can be seen that even after a single intratracheal injection of Cur@TB CDs for 30.0 days, these major organs were not significantly adversely affected. At the same time, after incubating different concentrations of Cur@TB CDs with red blood cells for 3.0 hours, even 300.0 μg / mL of Cur@TB CDs had no significant effect on the state and hemolysis of red blood cells. The results of the hemolysis test showed that Cur@TBCDs had good blood compatibility. The results of serum biochemical analysis were consistent with the results of H&E staining, indicating that Cur@TB CDs did not damage liver and kidney function in vivo.

[0055] Sixteen healthy male C57BL / 6 mice aged 9-10 weeks and weighing 21.0-25.0 g were randomly assigned to four experimental groups (n=4 in each group), namely the control group (healthy mice), ALI model group, LPS+CurcuminNanozyme-Mediated Reactive Oxygen Species Scavenging and Anti-Inflammationfor Acute Lung Injury (Yuan, et al., 2022). ALI was induced by intratracheal instillation of LPS (5.0 mg / kg sterile saline) under light anesthesia in the experimental groups (excluding the control group) according to the reported paper "Fe-CurcuminNanozyme-Mediated Reactive Oxygen Species Scavenging and Anti-Inflammationfor Acute Lung Injury" (Yuan, et al., 2022). 5.0 minutes after administration, mice in the LPS+Curcumin@TB CDs group were intratracheally instilled with Cur@TB CDs (20.0 mg / kg), while mice in the LPS+DXMS group were intratracheally instilled with DXMS (5.0 mg / kg). After 24.0 h of treatment, all mice were euthanized by an overdose of isoflurane, and their lung tissues were collected for further analysis. In addition, the levels of proinflammatory cytokines (including TNF-α, IL-6, and IL-1β) in lung tissues were quantified by ELISA. Other lung tissue sections were histologically analyzed by H&E staining, DAPI staining (excitation wavelength: 358 nm, emission wavelength: 450-470 nm), and DHE staining (excitation wavelength: 518 nm, emission wavelength: 590-620 nm) to evaluate cellular inflammation and ROS generation, respectively. In addition to excessive ROS, the three proinflammatory factors TNF-α, IL-6, and IL-1 are often associated with the development of inflammation and tissue damage, and reducing these inflammatory factors is associated with the successful treatment of ALI.

[0056] Dexamethasone (DXMS) is commonly used in clinical treatment of ALI. Therefore, the present invention selects DXMS as a positive control to evaluate the efficacy of Cur@TB CDs on ALI. The results are shown in Figure 7. Figure 7 -A shows that the present invention successfully constructs the ALI model; from Figure 7 -B shows that after LPS treatment, the lung tissue emits obvious red fluorescence, indicating that O 2 -The content was high, while after treatment with Cur@TB CDs and DXMS, the red fluorescence weakened, indicating that Cur@TB CDs can significantly inhibit the production of excessive ROS in lung tissue. After LPS treatment, the levels of TNF-α, IL-6and IL-1β in bronchoalveolar lavage fluid (BALF) and lung tissue increased significantly, while after treatment with Cur@TB CDs and DXMS, the levels of inflammatory factors decreased significantly, such as Figure 7 -C and Figure 7 -D. From Figure 7 -C and Figure 7 -D shows that Cur@TB CDs can significantly inhibit the concentrations of TNF-α, IL-6 and IL-1β in the lung tissues and BALF of the above-treated mice, and the effect is comparable to that of the positive control. **** indicates P<0.0001. In addition, the dry-to-wet ratio (W / D) of the lungs after lung injury is an important indicator for evaluating the water content of the lungs. The lung tissues of mice treated as above (Control, LPS, LPS+Cur@TB CDs and LPS+DXMS) were taken, the surface moisture was gently wiped off, and then the wet weight was weighed. After recording the wet weight, the container containing the lung sample was placed in an oven and dried at 65.0°C for 48.0 hours, and then the dry weight was weighed. The ratio of the two lung tissue weights, that is, the dry-to-wet ratio (W / D) of the lung tissue, was calculated to reflect the degree of pulmonary edema. The results are shown in the figure. Figure 8 *** indicates P<0.001. Data are presented as mean ± SD (n = 3). Figure 8 It can be seen that the dry-wet ratio of the mouse lungs after LPS treatment was much higher than the normal value, while pulmonary edema was significantly improved after treatment with Cur@TB CDs and the clinical drug DXMS.

[0057] The above results indicate that Cur@TB CDs has a good therapeutic effect on ALI model mice and achieved a therapeutic effect comparable to that of the clinical drug DXMS, showing great potential in the treatment of inflammation.

[0058] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. A curcumin carbon dot nanozyme, characterized in that: The preparation method thereof comprises the following steps: 1) Add tobacco powder, reduced glutathione and curcumin into formamide, stir, ultrasonicate and perform high temperature reaction; 2) After the reaction is completed, the mixture is cooled to room temperature, the filtrate is collected by filtration, and the filtrate is transferred to a dialysis bag. The dialyzed solution is passed through a water membrane and then freeze-dried to obtain curcumin carbon dot nanozyme.

2. The curcumin carbon dot nanozyme according to claim 1, characterized in that: In the step 1), the mass ratio of tobacco powder, reduced glutathione and curcumin is (0.8-1.2):(2.5-3.5):(0.04-0.06).

3. The curcumin carbon dot nanozyme according to claim 1 or 2, characterized in that: In the step 1), the mass ratio of tobacco powder, reduced glutathione and curcumin is (0.9-1.1):(2.8-3.2):0.

05.

4. The curcumin carbon dot nanozyme according to claim 1 or 2, characterized in that: The conditions of the high temperature reaction include: temperature 160-190° C., time 7-10 h.

5. The curcumin carbon dot nanozyme according to claim 4, characterized in that: The conditions of the high temperature reaction include: temperature 180° C., time 8 h.

6. The curcumin carbon dot nanozyme according to claim 1 or 2, characterized in that: The molecular weight cut-off of the dialysis bag is 450-550 Dalton.

7. The curcumin carbon dot nanozyme according to claim 1 or 2, characterized in that: The dialysis time is 6-9 days.

8. The curcumin carbon dot nanozyme according to claim 1 or 2, characterized in that: The tobacco powder is obtained by removing stems, cutting into shreds, crushing and then sieving a tobacco leaf sample.

9. The curcumin carbon dot nanozyme according to claim 1 or 2, characterized in that: The particle size of the water film is 0.22 μm.

10. Use of the curcumin carbon dot nanozyme according to any one of claims 1 to 9 in the preparation of drugs for preventing and treating lung injury.

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