Preparation method and application of carnosic acid carbon quantum dots

Abstract

The invention provides a preparation method of carnosic acid carbon quantum dots. The method has the advantages that a green synthesis process is adopted, the safety is high, and the biocompatibility is good; the carbon quantum dots of the carnosic acid carbon quantum dots prepared by the preparation method disclosed by the invention are used as an efficient ROS scavenger, have remarkable antioxidant and anti-inflammatory effects, can remarkably reduce oxidative injury of pulmonary epithelial cells, and can effectively promote lung injury repair of acute lung injury; compared with carnosic acid, the carnosic acid carbon quantum dot prepared by the invention has higher anti-inflammatory activity and better intracellular delivery efficiency, and has a better repair effect on acute lung injury.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular, it relates to a carboxylic acid carbon quantum dots and the application in preparation of the drug for treating acute lung injury. BACKGROUND

[0002] Acute lung injury is a severe lung disease caused by infection, trauma, toxin exposure and other factors, its main characteristics are alveolar epithelial cells and capillary endothelial cell damage, leading to alveolar-capillary barrier permeability increase, causing diffuse pulmonary edema and acute hypoxic respiratory failure. Oxidative stress and inflammation play a key role in the occurrence and progression of acute lung injury, excessive accumulation of reactive oxygen species can trigger inflammatory cascade, make alveolar macrophages and epithelial cells release pro-inflammatory factors (such as TNF-α, IL-1β, IL-6), exacerbate neutrophil infiltration, further damage lung tissue. In addition, ROS can also act on vascular endothelial cells, increase lung vascular permeability, exacerbate pulmonary edema, and ultimately lead to gas exchange dysfunction. The treatment strategies of acute lung injury include treatment of primary disease, respiratory support and drug intervention, etc. In recent years, new treatment methods are constantly explored in clinic, some of which are to alleviate inflammatory response by inhibiting inflammatory factors, some of which are to improve hypoxia by improving alveolar oxygenation, or to reduce pulmonary edema by promoting lung fluid clearance. These drugs include glucocorticoids, N-acetylcysteine, theophylline drugs, antioxidants, cytokine modulators, lung surfactants, fluid ventilation, nitric oxide inhalation and recombinant protein C, etc. However, these treatment methods still have problems such as limited efficacy, poor targeting or obvious side effects, and further research and large-scale clinical trials are still needed to verify their effectiveness and safety.

[0003] Therefore, it is necessary to develop another drug for treating acute lung injury. SUMMARY

[0004] The present application provides a preparation method of carboxylic acid carbon quantum dots, which can effectively alleviate oxidative stress response of lung epithelial cells and reduce the secretion of pro-inflammatory factors, and is helpful for the repair of lung injury.

[0005] The present application provides a preparation method of carboxylic acid carbon quantum dots, which can effectively alleviate oxidative stress response of lung epithelial cells and reduce the secretion of pro-inflammatory factors, and is helpful for the repair of lung injury.

[0006] S1, dispersing carboxylic acid in water, and then adding potassium hydroxide solution;

[0007] S2, the solution obtained in S1 is placed in an ultrasonic cleaner and ultrasonic treated for 30 min;

[0008] S3, the solution obtained in S2 is transferred to an autoclave, and then the closed autoclave is placed in a muffle furnace for heating, and after the reaction is completed, it is naturally cooled to room temperature;

[0009] S4, insoluble substances are removed, then centrifuged, and the crude product carbon quantum dot solution is obtained after dialysis, and the solution is freeze-dried after dialysis to obtain the product carboxylic acid carbon quantum dots.

[0010] Further, in S1, the mass ratio of carboxylic acid and potassium hydroxide is 1:1-1:3.

[0011] Further, the specific steps of S3 are as follows: the solution obtained in S2 is transferred to an autoclave, and then the closed autoclave is placed in a muffle furnace for heating, the temperature is 180-200℃, and the heating time is 6-8h, and after the reaction is completed, it is naturally cooled to room temperature.

[0012] The application also provides carboxylic acid carbon quantum dots prepared by the above preparation method.

[0013] Further, the carbon element content of the above-mentioned carbon quantum dots is 56.19%, the nitrogen element content is 11.74%, and the oxygen element content is 32.06%.

[0014] Further, the excitation wavelength of the above-mentioned carbon quantum dots is 379 nm, and the emission wavelength is 453 nm.

[0015] Further, the particle size of the above-mentioned carboxylic acid carbon quantum dots is 2-10 nm.

[0016] The application also provides the application of the above-mentioned carboxylic acid carbon quantum dots in preparing antioxidant anti-inflammatory drugs or drugs for treating acute lung injury.

[0017] The application has the following advantages:

[0018] The application provides a preparation method of carboxylic acid carbon quantum dots, which has the following advantages: green synthesis process, high safety, and good biocompatibility.

[0019] The carboxylic acid carbon quantum dots as high-efficiency ROS scavengers have significant antioxidant and anti-inflammatory effects, can significantly reduce oxidative damage of lung epithelial cells, and can effectively promote the repair of lung injury in acute lung injury.

[0020] The carboxylic acid carbon quantum dots prepared by the application have higher anti-inflammatory activity and better intracellular delivery efficiency than ordinary carboxylic acid, and have better repair effect on acute lung injury. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is a transmission electron micrograph based on carboxylic acid carbon quantum dots in Example 1.

[0022] Figure 2 Particle size distribution of the camphoric acid carbon quantum dots in Example 1;

[0023] Figure 3 Fluorescence emission spectra of the camphoric acid carbon quantum dots in Example 2 under different reaction conditions;

[0024] Figure 4 Cell toxicity test results of the camphoric acid carbon quantum dots in Example 3;

[0025] Figure 5 Detection of lung epithelial cell inflammatory factor indicators based on camphoric acid carbon quantum dots in Example 4;

[0026] Figure 6 Quantitative analysis of lung epithelial cell ROS fluorescence intensity based on camphoric acid carbon quantum dots in Example 5;

[0027] Figure 7 Detection of lung epithelial cell inflammatory factor indicators based on camphoric acid carbon quantum dots in Example 5 under different concentrations;

[0028] Figure 8 Fluorescence excitation spectrum and fluorescence emission spectrum of the camphoric acid carbon quantum dots in Example 6;

[0029] Figure 9 Fourier transform infrared spectrum of the camphoric acid carbon quantum dots in Example 6;

[0030] Figure 10 X-ray diffraction analysis of the camphoric acid carbon quantum dots in Example 6;

[0031] Figure 11 X-ray photoelectron spectroscopy analysis of the camphoric acid carbon quantum dots in Example 6;

[0032] Figure 12 Detection of lung epithelial cell ROS indicators based on camphoric acid carbon quantum dots in Example 7.

[0033] Figure 13 Animal tissue CAT content determination results based on camphoric acid carbon quantum dots in Example 8;

[0034] Figure 14 Animal tissue GSH-PX activity detection results based on camphoric acid carbon quantum dots in Example 8;

[0035] Figure 15 Animal tissue MDA activity detection results based on camphoric acid carbon quantum dots in Example 8;

[0036] Figure 16 The results of SOD activity detection in animal tissues based on sarsaparilla carbon quantum dots in Example 8;

[0037] Figure 17 The results of LDH content determination in animal tissues based on sarsaparilla carbon quantum dots in Example 8;

[0038] Figure 18 The ROS index of animal tissue lung slices based on sarsaparilla carbon quantum dots was detected in Example 8. Detailed Implementation

[0039] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to examples. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained in the art without creative effort should fall within the scope of protection of the present invention.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0041] Example 1: Preparation and characterization of carbon quantum dots based on oxalic acid under different NaOH mass ratios

[0042] 1. Experimental Methods

[0043] To determine the optimal precursor mass ratio for the alkaline hydrothermal synthesis of oxalic acid carbon quantum dots (CA-CQDs), four ratios of oxalic acid to potassium hydroxide (1:1, 1:1.5, 1:2, and 1:3) were systematically investigated. The specific procedures were as follows: The precursors at different mass ratios were dissolved in 20 mL of ultrapure water and ultrasonically dispersed for 30 min to form a homogeneous solution. This solution was then transferred to a 25 mL polytetrafluoroethylene-lined high-pressure reactor and reacted at 190 ℃ for 6 h. After natural cooling, the reaction solution was purified sequentially by centrifugation (12,000 rpm, 15 min) and dialysis (molecular weight cutoff 1 kDa, 24 h). The supernatant was collected for subsequent characterization and performance testing.

[0044] 2. Experimental Results

[0045] from Figure 1 and Figure 2Transmission electron microscopy (TEM) revealed that when the mass ratio was 1:1–1:2, the resulting CA-CQDs exhibited regular spherical shapes with a particle size distribution in the range of 1–10 nm (PDI < 0.1), demonstrating excellent monodispersity. CA-CQDs synthesized at this ratio showed higher emission intensity and quantum yield, and their surface contained abundant oxygen-containing functional groups, which is beneficial for binding to biological targets. This study focuses on the preparation and characterization of carbon quantum dots based on sagebrush acid.

[0046] Example 2: Study on the Influence of Temperature and Time Parameters on the Hydrothermal Synthesis of Carbon Quantum Dots Based on Scutellaria barbata

[0047] 1. Experimental Methods

[0048] To investigate the synergistic effect of temperature and time parameters on the synthesis performance of caryopsisic acid carbon quantum dots (CA-CQDs), this study employed a single-factor variable method to design an orthogonal experiment. Temperature gradient experiments: with a fixed precursor mass ratio (1:1) and reaction time (6 h), hydrothermal reactions were conducted at four temperature conditions: 180°C, 190°C, and 200°C. Time gradient experiments: after selecting the optimal reaction temperature, the effects of reaction times of 6 h, 7 h, and 8 h on product characteristics were further investigated. All reactions were carried out in a sealed autoclave and then naturally cooled to room temperature after completion.

[0049] 2. Experimental Results

[0050] from Figure 3 It can be seen that the CQDs obtained by heating at 190°C for 7 h have the strongest fluorescence emission intensity and stability, which are significantly better than other combinations.

[0051] Example 3: Comparison of the therapeutic effects of different concentrations on an acute lung injury model

[0052] 1. Experimental Methods

[0053] To systematically evaluate the dose-effect and biocompatibility of sarsaparilla carbon quantum dots (CA-CQDs) in inflammatory intervention, six concentration gradients (15.625, 31.25, 62.5, 125, 250, and 500 μg / mL) were set up. An in vitro inflammatory model was constructed by inducing human alveolar epithelial cells (such as the A549 cell line) with lipopolysaccharide (LPS, 10 μg / mL). After 6 h of LPS stimulation, the cells were treated with different concentrations of CA-CQDs for 24 h. Cell viability was quantified by the CCK-8 assay and morphological changes were observed by inverted microscopy to comprehensively analyze its anti-inflammatory efficacy and safety threshold.

[0054] 2. Experimental Results

[0055] from Figure 4It can be seen that CA-CQDs can effectively downregulate the expression of inflammatory factors and improve cell morphology within the concentration range of 15.625–500 μg / mL, while maintaining good cell viability. No obvious cytotoxicity was observed. Even at high concentrations (500 μg / mL), although the anti-inflammatory effect was not further enhanced, the cells still showed high survival rate and intact morphology, suggesting that CA-CQDs have good cell biocompatibility over a wide concentration range.

[0056] Example 4: Evaluation of the therapeutic effect of carbon quantum dots of different particle sizes in an acute lung injury model.

[0057] 1. Experimental Methods

[0058] By optimizing the hydrothermal synthesis process parameters (controlling precursor concentration and reaction time), carbon quantum dots (CA-CQDs) with precisely controllable particle size were successfully prepared, and their particle size-dependent anti-inflammatory effects were evaluated. In a lipopolysaccharide (LPS)-induced human alveolar epithelial cell inflammation model, ELISA detection showed that CA-CQDs of different particle sizes (50 μg / mL, treated for 24 h) had significantly different inhibitory effects on IL-6 (p<0.05). Cell morphology observation (phase contrast microscopy) showed that they could effectively alleviate the pseudopodia retraction and cell membrane wrinkling phenomena caused by LPS, suggesting that particle size control can significantly affect the biological effects of nanomaterials.

[0059] 2. Experimental Results

[0060] from Figure 5 The results showed that CA-CQDs with a particle size between 2–10 nm (medium particle size) significantly reduced the levels of inflammatory factors, maintained cell morphology, and significantly alleviated inflammatory damage. In contrast, CQDs with a particle size of 1 nm (small particle size) could rapidly enter cells, but their intracellular aggregation was weak, their effect duration was short, and their anti-inflammatory effect was limited. CQDs with a particle size greater than 10 nm (large particle size) showed relatively low intervention effects due to decreased endocytosis efficiency and cellular uptake barriers. In summary, CA-CQDs with a particle size between 2–10 nm exhibited the best anti-inflammatory capacity and cell compatibility in the cell model, suggesting that this particle size range may be the ideal size parameter for CA-CQDs in the intervention of lung diseases.

[0061] Example 5: Comparative Study of the Therapeutic Effects of Ordinary Succinate and Carbon Quantum Dot Formulations in an Acute Lung Injury Model

[0062] 1. Experimental Methods

[0063] To verify the synergistic mechanism of carbon quantum dot delivery system on the bioavailability of oxalic acid, a pharmacodynamic comparison experiment was conducted based on equimolar amounts of free oxalic acid (12.5-50 μg / mL) and CA-CQDs (calculated as equivalent oxalic acid). The control group consisted of free oxalic acid solution dissolved and diluted in complete culture medium using DMSO, while the experimental group consisted of CA-CQDs nanoformulations synthesized from the precursor. After 24 h of treatment with an LPS-induced human lung epithelial cell model (e.g., A549), the secretion levels of TNF-α and IL-6 in the supernatant were detected by ELISA, and cell morphology and apoptosis were observed.

[0064] 2. Experimental Results

[0065] from Figure 6 , Figure 7 The results showed that the CA-CQDs group was significantly superior to the ordinary kaempferol group in reducing the release of inflammatory factors, with more normal cell morphology and a significantly reduced apoptosis rate. The average reduction in inflammatory factors reached 42.3%. Furthermore, the CA-CQDs group exhibited stronger cytoplasmic distribution signals in intracellular fluorescence imaging, suggesting better intracellular delivery efficiency. These results demonstrate that CA-CQDs exhibit higher anti-inflammatory activity and drug bioavailability in cell models, validating its potential application as a kaempferol carrier in the treatment of lung diseases.

[0066] Example 6: Preparation and characterization of carbon quantum dots based on oxalic acid

[0067] 1. Experimental Methods

[0068] 0.5 g of oxalic acid and 0.5 g of sodium hydroxide were dissolved in 20 mL of deionized water. After complete dissolution with ultrasonic assistance (40 kHz, 100 W, 25 °C, 30 min), the solution was transferred to a 25 mL polytetrafluoroethylene high-pressure reactor and reacted at 190 °C for 6 h. The reaction solution was filtered through a 0.22 μm microporous membrane and centrifuged at 12000 rpm for 10 min to obtain the crude product. The crude product was then purified using a 500 Da molecular weight cutoff dialysis bag (1 L of deionized water, liquid change every 3 / 6 / 10 h). Finally, the product was freeze-dried for 24 h to obtain the target product. The optical properties, surface functional groups, crystal structure, and elemental composition of the product were systematically characterized using fluorescence spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and X-ray photoelectron spectroscopy.

[0069] 2. Experimental Results

[0070] from Figure 8 The results show that the optimal excitation wavelength of CA-CQD is 377 nm and the optimal emission wavelength is 454 nm.

[0071] fromFigure 9 The wavenumber is observed at 3136.46 cm⁻¹. -1 The absorption peak is due to the stretching vibration of CH, at 1675.10 cm⁻¹. -1 The absorption peak is due to the stretching vibration of the carbonyl group (C=O), at 1484.12 cm⁻¹. -1 The absorption peak is due to the stretching vibration of C=C, at 1399.98 cm⁻¹. -1 The absorption peak is due to the stretching vibration of CO.

[0072] from Figure 10 It can be seen that the carbon dots of sage oxalate may have a graphite-like crystal structure and exhibit sp. 2 The ordered stacking of hybrid carbons, with their graphitization characteristics, provides the structural basis for the properties of carbon dots.

[0073] from Figure 11 The data shows that the carbon point contains 61.92% carbon and 32.35% oxygen.

[0074] Example 7 In vitro experiment

[0075] 1. Experimental Methods

[0076] A549 human lung epithelial cell line was cultured in vitro, and the cell density was adjusted to 1×10⁻⁶. 6 Cells were seeded per well in 6-well plates. Five treatment groups (n=3) were set up: (1) normal control group; (2) LPS stimulation group (10 μg / mL); (3) LPS + 12.5 μg / mL CA-CQDs; (4) LPS + 25 μg / mL CA-CQDs; (5) LPS + 50 μg / mL CA-CQDs. After culturing at 37℃ and 5% CO2 for 24 hours, D-dihydroethidium (DHE) and Hoechst fluorescent probes were added, and the cells were incubated in the dark for 60 minutes (gently mixed every 10 minutes). Images were observed and acquired under a fluorescence microscope, and the intracellular ROS fluorescence intensity of each group was quantitatively analyzed using ImageJ software.

[0077] 2. Experimental Results

[0078] DHE fluorescence detection results showed that ( Figure 12 Compared with the normal control group, LPS stimulation significantly increased intracellular ROS levels. Treatment with different concentrations of sagebrush carbon quantum dots dose-dependently reduced LPS-induced ROS generation, with the 50 μg / mL treatment group showing the most significant ROS scavenging effect. These results indicate that sagebrush carbon quantum dots possess significant in vitro antioxidant activity.

[0079] Example 8 In vivo experiment

[0080] 1. Experimental Methods

[0081] Male C57BL6 mice aged 6-8 weeks were randomly divided into 5 groups (n=6): (1) control group (PBS treatment); (2) LPS model group (10 mg / kg); (3) LPS + 12.5 mg / kg CA-CQDs; (4) LPS + 25 mg / kg CA-CQDs; (5) LPS + 50 mg / kg CA-CQDs. After anesthesia with 5% chloral hydrate (6 μL / g) via intraperitoneal injection, an LPS-induced acute lung injury model was established by tracheal infusion. Animals were euthanized 24 hours later, and lung tissue was collected. A portion of the tissue was used to prepare a 10% homogenate, which was centrifuged at 4°C (3000 rpm, 15 min), and the supernatant was collected. Superoxide dismutase (SOD) activity (xanthine oxidase method), catalase (CAT) activity (H2O2 decomposition rate method), glutathione peroxidase (GSH-Px) activity (DTNB colorimetric method), malondialdehyde (MDA) content (TBA method), and lactate dehydrogenase (LDH) activity (colorimetric method) were measured using commercial kits. At the same time, another portion of lung tissue was used to prepare 5 μm thick frozen sections, which were incubated with 10 μM DCFH-DA in the dark for 30 min to detect reactive oxygen species levels. The cell nuclei were counterstained with DAPI for 5 min to label them. Finally, the cells were observed under a confocal microscope (excitation / emission wavelengths: 488 / 525 nm for DCF, 405 / 461 nm for DAPI), and the fluorescence intensity was quantitatively analyzed using ImageJ software.

[0082] 2. Experimental Results

[0083] from Figure 13 , 14 Figures 15, 16, 17, and 18 show that compared with the control group, the LPS model group exhibited significantly reduced SOD, CAT, and GSH-Px activities in lung tissue, and significantly increased MDA content and LDH release. The treatment group with sagebate carbon quantum dots showed a dose-dependent improvement in these indicators, with the 50 mg / kg group showing the most significant effect. DCF fluorescence detection revealed a significant increase in ROS levels in lung tissue of the LPS group, while the ROS fluorescence intensity in the treatment group decreased with increasing dose. These results indicate that sagebate carbon quantum dots can effectively alleviate LPS-induced acute lung injury by regulating redox balance.

Claims

1. A method for preparing carbon quantum dots of sagebrush acid, characterized in that: Includes the following steps: S1. Disperse oxalic acid in water, then add potassium hydroxide solution; S2. Place the solution obtained in S1 into an ultrasonic cleaner and sonicate for 30 minutes. S3. Transfer the solution obtained in S2 to an autoclave, then place the sealed autoclave in a muffle furnace for heating, and allow it to cool naturally to room temperature after the reaction is complete. S4. Remove insoluble substances, then centrifuge to obtain a crude carbon quantum dot solution, then dialyze it. After dialysis, freeze-dry the solution to obtain the product, sage carbon quantum dots.

2. The preparation method according to claim 1, characterized in that: In S1, the mass ratio of oxalic acid to potassium hydroxide is 1:1 to 1:

3.

3. The preparation method according to claim 2, characterized in that: The specific steps of S3 are as follows: the solution obtained in S2 is transferred to an autoclave, and then the sealed autoclave is placed in a muffle furnace for heating to 180 ℃-200 ℃ for 6-8 hours. After the reaction is completed, it is naturally cooled to room temperature.

4. Carbon quantum dots of sagebrush acid prepared by any one of the preparation methods of claims 1-3.

5. The carbon quantum dots of sagebrush as described in claim 4, characterized in that: The carbon quantum dots contain 56.19% carbon, 11.74% nitrogen, and 32.06% oxygen.

6. The sagebrush carbon quantum dots as described in claim 4, characterized in that: The carbon quantum dots have an excitation wavelength of 379 nm and an emission wavelength of 453 nm.

7. The sagebrush carbon quantum dots as described in claim 4, characterized in that: The particle size of the carbon quantum dots of the sagebrush acid is 2–10 nm.

8. The use of the sarsaparilla carbon quantum dots according to any one of claims 4-7 in the preparation of antioxidant and anti-inflammatory drugs or drugs for treating acute lung injury.

Images