Traditional Chinese medicine derived carbon dots for treating Parkinson's disease as well as preparation method and application of traditional Chinese medicine derived carbon dots

Abstract

The invention discloses traditional Chinese medicine derived carbon dots for treating Parkinson's disease as well as a preparation method and application thereof, belongs to the technical field of biomedical application, and aims to solve the technical problem that application of quercetin in treatment of Parkinson's disease is limited due to poor water solubility and low bioavailability of quercetin serving as a traditional Chinese medicine active component in the prior art. The preparation method comprises the following steps: dissolving quercetin and Congo red in water, carrying out hydrothermal reaction to prepare carbon dots, and drying reaction liquid to obtain the quercetin-derived carbon dots; the preparation method comprises the following steps: dissolving lecithin, cholesterol and DSPE-TK-PEG in an organic solvent to form a lipid membrane; and then adding a carbon dot PBS aqueous solution into the lipid membrane for hydration, and treating the obtained solution to obtain the traditional Chinese medicine derived carbon dots. The preparation method is simple and convenient in process and mild in condition, and the prepared product has good biocompatibility and oxidation resistance, can effectively remove active oxygen in a living body, and is used for preparing a medicine for treating Parkinson's disease to improve the neurological function and life quality of a patient.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical applications, and particularly relates to a traditional Chinese medicine derived carbon dot for treating Parkinson's disease and a preparation method and application thereof. BACKGROUND

[0002] Parkinson's disease (PD) is a second largest neurodegenerative disease caused by multiple causes, with the main pathological feature of progressive death of dopaminergic neurons. Its clinical manifestations include bradykinesia, rigidity and tremor, and affect millions of patients worldwide. The latest research results show that the disability and death caused by PD are growing at a faster rate than any other neurological disease worldwide. In China, with the deepening of population aging, the incidence of PD and other neurodegenerative diseases is increasing year by year. Due to the lack of effective treatment, the quality of life of PD patients is seriously decreased, and the labor loss brings a huge burden to the family and society. The medical expenses and nursing costs of PD patients in China are increasing year by year, which has become one of the important factors affecting the social and economic development. Therefore, it is crucial to develop safe, effective and easy-to-promote new methods and new drugs for PD treatment to improve the quality of life of patients and reduce the burden on families and society. Regarding the molecular mechanism of PD, it is currently believed to be closely related to alpha-synuclein aggregation, oxidative stress, neuroinflammation and mitochondrial dysfunction. Regarding the treatment of PD, the clinical mainly adopts left-handed dopamine replacement therapy, dopamine receptor agonists and other drugs, but these methods can only alleviate symptoms and cannot stop the progression of the disease, and long-term use can cause side effects such as motor fluctuations and dyskinesia. Therefore, developing new strategies to delay or stop the progression of PD is a research hotspot in the field of PD.

[0003] In recent years, the advantages of traditional Chinese medicine in the treatment of neurodegenerative diseases have attracted more and more attention. Traditional Chinese medicine has low toxicity and high safety, and is suitable for long-term use, which shows a good application prospect in the prevention and treatment of PD. Previous studies have reported that various active ingredients of traditional Chinese medicine such as curcumin, ginsenosides and quercetin have certain therapeutic effects on PD. Among them, quercetin, as a flavonoid compound widely existing in plants, has been confirmed to have multiple biological activities such as antioxidant, anti-inflammatory and neuroprotective effects. More attention is paid to the combination of quercetin and dasatinib, which is considered to be an effective senolytic agent at present. It has shown a good application prospect in the research of PD, Alzheimer's disease (AD) and other neurodegenerative diseases, and can improve the symptoms of the diseases through the elimination of senescent cells and the reduction of neuroinflammation. However, quercetin itself has problems such as poor water solubility and low bioavailability, which greatly limit its application and promotion in the clinic. How to modify quercetin to improve its therapeutic effect on PD has become a key problem to be solved in the current field. SUMMARY

[0004] It is an object of embodiments of the invention to address at least the above-mentioned problems and / or deficiencies and to provide at least the advantages stated below.

[0005] In view of the poor water solubility and low bioavailability of quercetin in the prior art, the present application provides a carbon dot-based nanomaterial treatment platform, a preparation method and application thereof, the prepared nanomaterial has good biocompatibility and can effectively remove active oxygen in the body.

[0006] The present application provides a traditional Chinese medicine derived carbon dot for treating Parkinson's disease model, a preparation method and application thereof, the carbon dot takes quercetin as a treatment unit, can well remove various active oxygen, has strong antioxidant capacity and good biocompatibility, and is successfully applied to the treatment of cell and mouse Parkinson's disease model. The present application also discloses a preparation method and application, the preparation method is simple to operate and has mild conditions.

[0007] To this end, the present application provides the following technical solutions: A traditional Chinese medicine derived carbon dot for treating Parkinson's disease, which is composed of quercetin derived carbon dots and active oxygen response liposome shell wrapping the quercetin derived carbon dots; The quercetin derived carbon dots are prepared by hydrothermal reaction with quercetin and Congo red as raw materials in a mass ratio of 1:0.8-1.2; The active oxygen response liposome is prepared from lecithin, cholesterol and DSPE-TK-PEG, and the mass ratio of lecithin, cholesterol and DSPE-TK-PEG is 7-9:0.8-1.2:0.5-1.5. DSPE-TK-PEG is short for distearoyl phosphatidyl ethanolamine-ketone thiol-polyethylene glycol.

[0008] A preparation method of the traditional Chinese medicine derived carbon dot, comprising the following steps: 1) Preparation of carbon dots: dissolve quercetin and Congo red in a mass ratio of 1:0.8-1.2 in water, react at 160-200 DEG C for 1-4 hours, and then purify and dry the reaction solution to obtain quercetin derived carbon dots; 2) Preparation of liposome: dissolve lecithin, cholesterol and DSPE-TK-PEG in an organic solvent in a mass ratio of 7-9:0.8-1.2:0.5-1.5, and spin to form a lipid film; 3) Preparation of traditional Chinese medicine derived carbon dots: add carbon dot PBS aqueous solution with a concentration of 0.5-2.0 mg / mL and pH of 7.2-7.4 to the lipid film obtained in step 2) for hydration, and then ultrasonic, filter membrane filtration and extrusion to obtain traditional Chinese medicine derived carbon dots CDs@LIP.

[0009] Preferably, in the preparation method, the temperature of the hydrothermal reaction in step 1) is 170-190 ℃, and the reaction time is 1.5-2.5 hours. The heating rate of the hydrothermal reaction is 3-5 ℃ / min, and after the reaction is completed, it is naturally cooled to room temperature. During the hydrothermal reaction process, the reaction temperature and pressure are monitored in real time to ensure that the temperature deviation is not more than ±2 ℃, and the pressure is maintained at 1-2 MPa.

[0010] Preferably, in the preparation method, the purification in step 1) includes centrifugation, filtration and dialysis, the centrifugation speed is 8000-12000 rpm, and the time is 10-20 minutes; the filtration uses a 0.22 μm microporous filter membrane; the dialysis uses a dialysis bag with a molecular weight cut-off of 1000 Da, and the dialysis time is 24-48 hours, and the temperature during dialysis is 2-8℃.

[0011] Preferably, in the preparation method, in step 2), the mass ratio of lecithin to cholesterol is 8:1, and the mass of DSPE-TK-PEG is 10% of the mass of lecithin. The organic solvent is a mixed solvent of dichloromethane and methanol in a volume ratio of 2:1; The temperature of rotary evaporation is 30-40 ℃, the pressure is 0.1-0.5 bar, and the rotary evaporation time is 20-40 minutes.

[0012] Preferably, in the preparation method, in step 3), the hydration temperature is 45-50 ℃, and the hydration time is 50-70 min; The ultrasonic conditions are: ice bath, power is 200-300 W, works for 5 seconds, intermittent for 5 seconds, and cycles for 25-35 times.

[0013] Preferably, in the preparation method, in step 3), the filter membrane filtration uses a 0.22 μm polycarbonate membrane; the extrusion uses a liposome extruder, and passes through a 100 nm polycarbonate membrane 3-5 times; The particle size of the traditional Chinese medicine derived carbon dots CDs@LIP is 80-120 nm, and the encapsulation efficiency is 85%-95%.

[0014] Preferably, in the preparation method, in step 1), the dialysis is carried out in a dialysis bag at 2-8 DEG C, and during the dialysis, samples are taken every 4-6 hours, the change of the carbon dot hydration particle size is monitored by dynamic light scattering method, and whether the Congo red characteristic absorption peak appears in the dialysate is detected by a UV-visible spectrophotometer at 390 nm; when the polydispersity index of the carbon dot hydration particle size distribution is less than 0.25 and the absorbance value of the dialysate at 390 nm is less than 0.05 for two consecutive times, the dialysis is terminated.

[0015] Preferably, in the preparation method, in step 1), in the purification, the dialysis step is carried out by using a tangential flow filtration system, the tangential flow filtration system is equipped with a filter membrane with a molecular weight cut-off of 1000 Da, and the operation conditions include that the transmembrane pressure is maintained at 0.5-1.5 bar, the feed liquid flow rate is controlled at 100-200 mL / min, and the UV absorbance of the permeate is continuously monitored during the dialysis; when the absorbance at 390 nm is continuously less than 0.02 for more than 10 minutes, the dialysis is terminated.

[0016] The application of the traditional Chinese medicine derived carbon dots in the preparation of a drug for treating Parkinson's disease.

[0017] The application at least has the following beneficial effects: the CDs@LIP nanomaterial obtained by the application takes traditional Chinese medicine carbon dots as a treatment unit, raw materials are easy to obtain, the synthesis steps are simple, and good biocompatibility is achieved; the synthesis steps of the liposome are simple, the synthesis conditions are mild, and after the material is combined with the carbon dots, the complex exhibits good biocompatibility, good treatment effect in the body, and great application value.

[0018] Other advantages, objects, and features of the application will be apparent from the following description, and will be appreciated by those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The transmission electron microscope and atomic force microscope images of the carbon dots CDs prepared in Example 1 of the application; Figure 2 The XRD pattern of the carbon dots CDs prepared in Example 1 of the application; Figure 3 The Fourier infrared spectrum of the carbon dots CDs prepared in Example 1 of the application; Figure 4 The XPS pattern of the carbon dots CDs prepared in Example 1 of the application; Figure 5 The UV spectrum of the carbon dots CDs prepared in Example 1 of the application for removing ABTS free radicals; Figure 6 UV spectrum of carbon dots CDs prepared in embodiment 1 of the present application for scavenging DPPH free radicals; Figure 7 UV spectrum of carbon dots CDs prepared in embodiment 1 of the present application for scavenging hydroxyl radicals; Figure 8 UV spectrum of carbon dots CDs prepared in embodiment 2 of the present application for scavenging superoxide anions; Figure 9 Transmission electron microscopy and dynamic light scattering diagram of CDs@LIP prepared in embodiment 3 of the present application; Figure 10 Cell toxicity test diagram of CDs@LIP prepared in embodiment 3 of the present application; Figure 11 Fluorescence imaging diagram of reactive oxygen species in a Parkinson's disease cell model of the material of the present application; Figure 12 Fluorescence imaging diagram of apoptosis in a Parkinson's disease cell model of the material of the present application; Figure 13 Biodistribution diagram of the material of the present application in a Parkinson's disease mouse model; Figure 14 Open field test result test diagram of the material of the present application in a Parkinson's disease mouse model; Figure 15 Rope climbing experiment result test diagram of the material of the present application in a Parkinson's disease mouse model; Figure 16 Rotarod experiment result test diagram of the material of the present application in a Parkinson's disease mouse model. DETAILED DESCRIPTION

[0021] The present application will be further described in detail below, so that those skilled in the art can implement it according to the description.

[0022] It should be understood that the terms such as "have", "contain" and "include" used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0023] It should be noted that the experimental methods in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0024] As a new type of carbon nanomaterial, carbon dots have many advantages such as small particle size, good water solubility, high biocompatibility, easy surface modification, excellent optical performance, etc., and show broad application prospects in the field of biomedicine. In recent years, the research on traditional Chinese medicine derived carbon dots for improving their performance has gradually increased, which provides a new idea for solving the problem of limited application of quercetin.

[0025] In the present application, first, quercetin-derived carbon dots are prepared by one-step hydrothermal method and their physicochemical properties are characterized; second, the effects of quercetin-derived carbon dots on treating PD are explored at the cell and in vivo levels; third, the mechanism of quercetin-derived carbon dots in PD models is studied in depth by using advanced technical means such as multi-omics analysis and transcriptome analysis; finally, the clinical application potential of quercetin-derived carbon dots is explored in PD patient-derived brain organoids. The development of this research not only provides new ideas for the treatment of PD, but also provides useful reference for the inheritance and innovation of traditional Chinese medicine.

[0026] A traditional Chinese medicine-derived carbon dot for treating Parkinson's disease, the material comprising quercetin-derived carbon dots and active oxygen-responsive liposomes, the liposomes encapsulating the quercetin-derived carbon dots.

[0027] A method for treating Parkinson's disease with traditional Chinese medicine-derived carbon dots, comprising the following steps: Step one, quercetin and congo red are mixed as raw materials to prepare carbon dots by hydrothermal reaction; the specific operation is as follows: quercetin and congo red are dissolved in ultrapure water, after ultrasonic dissolution, the mixed solution is transferred to a polytetrafluoroethylene reaction kettle, heated and reacted, the reaction conditions are as follows: reaction temperature 180 ℃, reaction time 2 h. The reacted solution is centrifuged, filtered with a filter membrane, dialyzed, and then freeze-dried to obtain carbon dots.

[0028] The mass ratio of quercetin to congo red is 1:1.

[0029] Step two, lecithin, cholesterol and DSPE-TK-PEG are dissolved in an organic solvent, the solvent is removed by rotary evaporation, and the carbon dot-containing PBS aqueous solution is added to hydrate to prepare CDs@LIP. The specific operation is as follows: appropriate amounts of lecithin, cholesterol and DSPE-TK-PEG are weighed into an EP tube, an organic solvent is added, and it is vortexed to dissolve thoroughly. Then it is transferred to a round-bottom flask, and the organic solvent is removed by rotary evaporation at 48 ℃ water bath under reduced pressure for 30 min to form a uniform lipid film at the bottom of the round-bottom flask. Add the carbon dot-containing PBS aqueous solution, place it in an oil bath, and stir magnetically at 48 ℃ for 60 min. The obtained solution is placed in an ultrasonic cell crusher, and ultrasonic treatment is carried out in an ice bath. Finally, it is filtered with a filter membrane, and then extruded through a liposome extruder.

[0030] The mass ratio of lecithin to cholesterol is 8:1, and the mass of DSPE-TK-PEG is 10% of the mass of lecithin.

[0031] The organic solvent is a mixed solvent composed of dichloromethane and methanol (v / v, 2:1).

[0032] The Chinese medicine derived carbon dots CDs@LIP for treating Parkinson's disease, and the application of the Chinese medicine derived carbon dots in treating Parkinson's disease model.

[0033] According to one of the embodiments of the present application, a Chinese medicine derived carbon dot for treating Parkinson's disease is composed of quercetin derived carbon dots and active oxygen response liposome shells wrapping the quercetin derived carbon dots. The quercetin derived carbon dots are prepared by hydrothermal reaction with quercetin and Congo red as raw materials in a mass ratio of 1:0.8-1.2. The active oxygen response liposome is prepared from lecithin, cholesterol and DSPE-TK-PEG, and the mass ratio of the lecithin, cholesterol and DSPE-TK-PEG is 7-9:0.8-1.2:0.5-1.5.

[0034] For example, the quercetin derived carbon dots are prepared by dissolving quercetin and Congo red in a mass ratio of 1:0.8 to 1.2 in water, performing hydrothermal reaction at 160 to 200°C for 1 to 4 hours, and drying the reaction solution after purification such as centrifugation, filtration and dialysis. The active oxygen response liposome is prepared by dissolving lecithin, cholesterol and DSPE-TK-PEG in an organic solvent in a mass ratio of 7-9:0.8-1.2:0.5-1.5, and forming a lipid membrane by rotary evaporation. Finally, the quercetin derived carbon dots are wrapped in the liposome by adding a carbon dot PBS aqueous solution with a concentration of 0.5 to 2.0 mg / mL to the lipid membrane for hydration, and then treated by ultrasonic, filtration and extrusion to obtain the Chinese medicine derived carbon dots.

[0035] The prior art directly uses quercetin as a therapeutic agent, which has the limitations of poor water solubility and low bioavailability, resulting in limited effect in the treatment of Parkinson's disease. The present application converts quercetin into carbon dots and wraps them in active oxygen response liposomes, which not only improves the solubility and bioavailability of quercetin, but also introduces the targeted antioxidant properties, thereby more effectively addressing the oxidative stress problem in Parkinson's disease. The Chinese medicine derived carbon dots have good biocompatibility and antioxidant capacity, can effectively scavenge active oxygen in the body, improve nerve function, and provide a safe and effective solution for the treatment of Parkinson's disease.

[0036] According to one of the embodiments of the present application, a preparation method of a Chinese medicine derived carbon dot for treating Parkinson's disease comprises the following steps: 1) Preparation of carbon dots: quercetin and congo red with a mass ratio of 1:0.8-1.2 are dissolved in water, and a hydrothermal reaction is carried out at 160-200°C for 1-4 hours; the reaction solution is purified, dried, and then quercetin derivative carbon dots are obtained; 2) Preparation of liposomes: lecithin, cholesterol and DSPE-TK-PEG are dissolved in an organic solvent at a mass ratio of 7-9:0.8-1.2:0.5-1.5, and a lipid film is formed by rotary evaporation; 3) Preparation of traditional Chinese medicine derivative carbon dots: the lipid film obtained in step 2) is hydrated with a carbon dot PBS aqueous solution with a concentration of 0.5-2.0 mg / mL and pH 7.2-7.4; the obtained solution is subjected to ultrasonic treatment, membrane filtration and extrusion, and then traditional Chinese medicine derivative carbon dots CDs@LIP are obtained.

[0037] For example, quercetin and congo red with a mass ratio of 1:0.8-1.2 are dissolved in water, and a hydrothermal reaction is carried out at 160-200°C for 1-4 hours to prepare carbon dots; the reaction solution is centrifuged, filtered and dialyzed, and then dried to obtain quercetin derivative carbon dots; lecithin, cholesterol and DSPE-TK-PEG are dissolved in an organic solvent at a mass ratio of 7:9:0.8:1.2:0.5:1.5, and a lipid film is formed by rotary evaporation; then a carbon dot PBS aqueous solution with a concentration of 0.5-2.0 mg / mL is added to the lipid film for hydration; the obtained solution is subjected to ultrasonic treatment, membrane filtration and extrusion treatment, and then traditional Chinese medicine derivative carbon dots are obtained. In the prior art, quercetin is directly used as a therapeutic agent, which has the limitations of poor water solubility and low bioavailability, resulting in limited effect in the treatment of Parkinson's disease; in the present application, quercetin is converted into carbon dots and encapsulated in active oxygen responsive liposomes, which not only improves the solubility and bioavailability of quercetin, but also introduces the targeting antioxidant property, thereby more effectively addressing the oxidative stress problem in Parkinson's disease. The preparation method is simple in operation and mild in conditions, and the prepared traditional Chinese medicine derivative carbon dots have good biocompatibility and antioxidant capacity, can effectively scavenge active oxygen in the body, and improve the nerve function.

[0038] According to one of the embodiments of the present application, as a preferred, the preparation method, the temperature of the hydrothermal reaction in step 1) is 170-190°C, and the reaction time is 1.5-2.5 hours; The heating rate of the hydrothermal reaction is 3-5°C / min, and the reaction is naturally cooled to room temperature after the reaction is completed; During the hydrothermal reaction, the reaction temperature and pressure are monitored in real time to ensure that the temperature deviation is not more than ±2°C, and the pressure is maintained at 1-2 MPa.

[0039] In the preparation of quercetin-derived carbon dots, the hydrothermal reaction is carried out at 170-190°C for 1.5-2.5 h, the heating rate is 3-5°C / min, after the reaction is completed, it is naturally cooled to room temperature, and the reaction temperature and pressure are monitored in real time, so as to ensure that the temperature deviation is not more than ±2°C, and the pressure is maintained at 1-2 MPa. The control of the hydrothermal reaction conditions in the prior art is not accurate, which may lead to inconsistent performance of the carbon dots; and the present application accurately controls the temperature, time and pressure parameters of the hydrothermal reaction, so as to ensure the repeatability and quality stability of the preparation of the carbon dots. By optimizing the hydrothermal reaction conditions, the uniformity and reaction efficiency of the carbon dots are improved, so as to enhance the therapeutic effect and reliability of the final product.

[0040] According to one of the embodiments of the present application, as preferred, the purification in step 1) of the preparation method comprises centrifugation, filtration and dialysis, the centrifugation is carried out at a speed of 8000-12000 rpm for 10-20 minutes; the filtration uses a 0.22 μm microporous filter membrane; the dialysis uses a dialysis bag with a molecular weight cut-off of 1000 Da, the dialysis time is 24-48 hours, and the temperature during dialysis is 2-8°C.

[0041] In the purification step after the preparation of quercetin-derived carbon dots, the centrifugation is carried out at a speed of 8000-12000 rpm for 10-20 min, the filtration uses a 0.22 μm microporous filter membrane, the dialysis uses a dialysis bag with a molecular weight cut-off of 1000 Da, the dialysis time is 24-48 h, and the temperature during dialysis is 2-8°C. The purification method in the prior art may be imperfect, resulting in impurities or unreacted raw materials in the carbon dots; and the present application effectively removes impurities through a systematic purification process, thereby improving the purity and biocompatibility of the carbon dots. Through careful purification treatment, the high purity and safety of the carbon dots are ensured, the potential side effects are reduced, and the applicability in biomedical applications is improved According to one of the embodiments of the present application, as preferred, in step 2) of the preparation method, the mass ratio of lecithin to cholesterol is 8:1, and the mass of DSPE-TK-PEG is 10% of the mass of lecithin. The organic solvent is a mixed solvent of dichloromethane and methanol in a volume ratio of 2:1; The temperature of rotary evaporation is 30-40°C, the pressure is 0.1-0.5 bar, and the rotary evaporation time is 20-40 minutes.

[0042] In the preparation of active oxygen response liposomes, the mass ratio of lecithin and cholesterol is controlled to be 8:1, the mass of DSPE-TK-PEG is 10% of lecithin, the organic solvent is a mixed solvent of dichloromethane and methanol with a volume ratio of 2:1, the rotary evaporation process is carried out at 30-40°C, the pressure is maintained at 0.1-0.5 bar, and the rotary evaporation time is 20-40 minutes. The existing liposome formula and film forming process have uncertainties, which may lead to poor stability of the liposomes or low drug loading efficiency; and the present application significantly improves the stability of the liposomes and the encapsulation capacity of the carbon dots by precisely controlling the proportion of each component and the rotary evaporation process parameters. Through optimization of the composition and preparation process of the liposomes, a structurally stable active oxygen response carrier is formed, which effectively protects the biological activity of the carbon dots and enhances the targeted delivery efficiency.

[0043] According to one of the embodiments of the present application, as a preference, the preparation method, the temperature of hydration in step 3) is 45-50°C, and the hydration time is 50-70 min; The ultrasonic conditions are: ice bath, power of 200-300 W, working for 5 seconds, intermittent for 5 seconds, and circulating for 25-35 times.

[0044] After adding the carbon dot PBS aqueous solution into the lipid film, hydration is carried out at 45-50°C for 50-70 min, followed by ultrasonic treatment, and the ultrasonic conditions are: ice bath, power of 200-300 W, working for 5 seconds, intermittent for 5 seconds, and circulating for 25-35 times. In the prior art, the hydration process may not be sufficient or the ultrasonic conditions may be too harsh, leading to destruction of the liposome structure or leakage of the carbon dots; and the present application realizes uniform combination of the liposomes and the carbon dots and maintains the structural integrity by controlling the hydration temperature and time and using intermittent ultrasonic treatment. By optimizing the hydration and ultrasonic parameters, it is ensured that the carbon dots are completely encapsulated in the liposomes, improving the uniformity and stability of the nanoparticles.

[0045] According to one of the embodiments of the present application, as a preference, the preparation method, the filter membrane filtration in step 3) uses a 0.22 μm polycarbonate membrane; and the extrusion uses a liposome extruder, and is passed through a 100 nm polycarbonate membrane for 3-5 times; The particle size of the traditional Chinese medicine derived carbon dots CDs@LIP is 80-120 nm, and the encapsulation efficiency is 85%-95%.

[0046] 0.22 pm polycarbonate membrane is used when filtering the solution after hydration, a liposome extruder is used for extrusion treatment and repeated 3-5 times through a 100 nm polycarbonate membrane, the particle size of the finally prepared traditional Chinese medicine derived carbon dots is controlled in 80-120 nm, and the encapsulation rate reaches 85%-95%. The particle size distribution of the nanoparticles in the prior art is wide and the encapsulation rate is not ideal, which affects the biological distribution and treatment effect; and the specific filtration and extrusion process of the present application obtains nanoparticles with uniform particle size and high encapsulation rate. By accurately controlling the particle size and encapsulation rate of the final product, the pharmacokinetic properties of the nanoparticles are optimized, so that they can more effectively target the lesion site and play a therapeutic role.

[0047] According to one of the embodiments of the present application, as preferred, in the preparation method, in step 1), dialysis is carried out in a dialysis bag at 2-8℃, sampling is carried out every 4-6 hours during dialysis, the change of the hydrated particle size of the carbon dots is monitored by dynamic light scattering method, and whether the characteristic absorption peak of Congo red appears in the dialysate is detected by ultraviolet-visible spectrophotometer at 390 nm wavelength; when the polydispersity index of the hydrated particle size distribution of the carbon dots is less than 0.25 and the absorbance value of the dialysate at 390 nm is less than 0.05 for two consecutive monitoring results, the dialysis is terminated.

[0048] The dialysis process in the prior art usually only relies on fixed time control and lacks real-time monitoring means, which may lead to insufficient purification or over-dialysis; and the present application realizes accurate judgment of the dialysis end point by introducing a multi-parameter real-time monitoring mechanism. By establishing a scientific dialysis end point determination standard, the consistency and reliability of the carbon dot purification quality are ensured, and the unreacted small molecule impurities are effectively removed.

[0049] The present application further relates to another embodiment of the purification step, in step 1), during purification, the dialysis step is carried out by using a tangential flow filtration system, the tangential flow filtration system is equipped with a filter membrane with a molecular weight cut-off of 1000 Da, the operating conditions include: the transmembrane pressure is maintained at 0.5-1.5 bar, the feed liquid flow rate is controlled at 100-200 mL / min, and the ultraviolet absorbance of the permeate is continuously monitored during dialysis, and when the absorbance at 390 nm wavelength is continuously less than 0.02 for more than 10 minutes, the dialysis is terminated.

[0050] The traditional dialysis method in the prior art has low efficiency and is easy to cause sample loss; and the present application significantly improves the purification efficiency by using tangential flow filtration technology. The automatic control system realizes efficient purification, greatly shortens the processing time while ensuring the purity of the carbon dots, and provides a technical basis for large-scale production.

[0051] According to one of the embodiments of the present application, the present application provides the application of the traditional Chinese medicine derived carbon dots in the preparation of drugs for treating Parkinson's disease. The traditional Chinese medicine derived carbon dots are used as active ingredients for the preparation of drugs for treating Parkinson's disease. The existing Parkinson's disease treatment drugs are mostly composed of chemical synthetic components, which have the limitations of large side effects and poor targeting; and the present application first applies the nano-preparation constructed by combining the quercetin derived carbon dots and the active oxygen responsive liposome to the treatment of Parkinson's disease. The application of the traditional Chinese medicine derived carbon dots in the field of treatment of neurodegenerative diseases is expanded, and a new nano-drug solution is provided for solving the problem of Parkinson's disease treatment.

[0052] According to one of the embodiments of the present application, the present application provides a preparation method of traditional Chinese medicine derived carbon dots for treating Parkinson's disease, comprising the following steps: step one, preparing carbon dots by mixing quercetin and congo red as raw materials through hydrothermal reaction; step two, dissolving lecithin, cholesterol and DSPE-TK-PEG in an organic solvent, removing the solvent by rotary evaporation, adding carbon dot-containing PBS aqueous solution to hydrate to prepare CDs@LIP.

[0053] The specific operation of step one is as follows: 0.100 g of quercetin and 0.100 g of congo red are dissolved in 30.0 mL of ultrapure water, and after complete dissolution, the mixed solution is transferred to a polytetrafluoroethylene reaction kettle, and then the reaction kettle is placed in a microwave hydrothermal instrument at 180 ℃ for reaction for 2 h. After the reaction is completed, the reaction kettle is transferred to room temperature for natural cooling, and the reaction mixture is centrifuged (12000 rpm, 10 min) to remove larger solid particles, and the supernatant is filtered through a 0.220 μm water-based microporous filter membrane. The obtained brown solution is dialyzed for 3 h (the molecular weight cut-off of the dialysis bag is 1000 Da). The liquid after dialysis is freeze-dried, and the obtained powder is the CDs.

[0054] The specific operation of step two is as follows: 16 mg of lecithin, 2 mg of cholesterol and 1.6 mg of DSPE-TK-PEG are weighed in an EP tube, 2 mL of a mixed solvent composed of dichloromethane and methanol (v / v, 2:1) is added as an organic phase, and the mixture is vortexed for 20 min to fully dissolve. Then it is transferred to a round-bottom flask, and the organic solvent is removed by rotary evaporation at 48 ℃ water bath under reduced pressure for 30 min, forming a uniform lipid film at the bottom of the round-bottom flask. 2 ml of PBS (pH=7.4) aqueous solution with a carbon dot concentration of 1 mg / mL is added to the flask, which is placed in an oil bath, and magnetically stirred at 48 ℃ for 60 min. The obtained solution is placed in an ultrasonic cell crusher, and ultrasonic treatment is carried out in an ice bath (working time 5 s, intermittent time 5 s, intensity 80%, 30 times). Finally, the solution is filtered through a 0.220 μm water-based microporous filter membrane, and then extruded 10 times through a liposome extruder.

[0055] Example 1 The synthesis method of fluorescent carbon dots includes the following steps: 1) Weigh 0.100 g of quercetin and 0.100 g of Congo red and dissolve them in 30.0 mL of ultrapure water by sonication. After complete dissolution, transfer the mixture to a polytetrafluoroethylene reactor and then place the reactor in an oven at 180 °C for 2 h.

[0056] 2) After the reaction was complete, the reactor was allowed to cool naturally to room temperature. Larger solid particles in the reaction solution were removed by centrifugation (12000 rpm, 10 min). The supernatant was collected and filtered through a 0.220 μm aqueous microporous membrane. The resulting brown solution was then dialyzed for 3 h (dialysis bag molecular weight cutoff of 1000 Da). After dialysis, the solution was freeze-dried to obtain CDs powder.

[0057] Figure 1 Image (A) is a transmission electron microscope characterization image of the carbon dot CDs prepared in Example 1. As can be seen from the image, the CDs prepared in this example are spherical, have good dispersibility, and do not have obvious agglomeration. Figure 1 (B) shows the atomic force microscopy characterization of the carbon dot CDs prepared in Example 1 and the corresponding thickness statistics. The results in the figure show that the CDs prepared in this example have good dispersibility in water and the thickness is mainly concentrated in 3-4 nm.

[0058] Figure 2 The image shows the XRD pattern of the carbon dot CDs obtained in Example 1. As can be seen from the results, the CDs obtained in this invention are mainly composed of amorphous carbon.

[0059] Figure 3 This is the Fourier transform infrared spectrum of the fluorescent carbon dots (CDs) prepared in this embodiment, where 3435 cm⁻¹ The absorption bands at 1593 cm⁻¹ belong to the stretching vibrations of OH or NH; the absorption bands at 1356 cm⁻¹ and 1112 cm⁻¹ belong to the stretching vibrations of CN and the bending vibrations of CO, respectively; and the absorption band at 619 cm⁻¹ belongs to the bending vibrations of NH. These results indicate that the fluorescent carbon dots (CDs) prepared in this example are rich in water-soluble groups such as carboxyl and amino groups, exhibiting good water solubility and high reactivity, and are easily functionalized.

[0060] Figure 4The XPS spectrum of the carbon dot CDs prepared in Example 1 is shown in Figure 4A. It can be seen from the result that the CDs prepared in this invention contain peaks of C1S, O1S, and N1S. High-resolution C1S (Figure 4B) shows four carbon signals, appearing at 284.8, 285.7, 286.0, and 288.9 eV, which can be attributed to C=C / CC, CN, CO, and C=O, respectively. High-resolution N1S (… Figure 4 Figure 4 (C) shows two nitrogen signals, 398.8 eV and 399.8 eV, corresponding to CNC and NH, respectively. The high-resolution O1S results (D in Figure 4) show two signals, 531.7 and 533.0 eV, which can be attributed to C=O and C-OH / COC, respectively. XPS results indicate that the carbon dots are mainly composed of carbon, nitrogen, and oxygen elements, and are rich in functional groups such as amino, hydroxyl, and carboxyl groups, consistent with the FTIR results.

[0061] Example 2 Study on the antioxidant properties of CDs: ABTS radical scavenging rate detection: First, 7.4 mM ABTS aqueous solution was mixed with 2.6 mM potassium persulfate and incubated for 12 hours to generate ABTS radical solution. The solution was diluted with PBS to obtain ABTS working solution. Different concentrations of CDs solution were added and mixed 1:1 with the ABTS working solution. The final concentrations of CDs were 0, 5, 10, 15, 20, and 25 μg / mL. The absorbance at 734 nm was measured using a UV-Vis spectrophotometer. The test time was 10 minutes, with absorbance measured every minute. The antioxidant capacity was evaluated by calculating the ABTS radical scavenging rate. ABTS radical scavenging rate = (A0 — A) / A0 × 100%. (A0 is the absorbance of the ABTS solution without sample; A is the absorbance of the sample after reaction with ABTS).

[0062] DPPH free radical scavenging rate determination: Different concentrations of CDs solutions were prepared by mixing them with 1 mL of 0.100 mM DPPH•ethanol solution. These CDs solutions were then mixed with an equal volume of 0.05 mM DPPH•ethanol solution, resulting in final CDs concentrations of 0, 10, 20, 30, 40, and 50 μg / mL. The absorbance at 519 nm was measured using a UV-Vis spectrophotometer. The testing period was 20 minutes, with absorbance measurements taken every minute. The antioxidant capacity was evaluated by calculating the DPPH free radical scavenging rate. DPPH free radical scavenging rate = (A0 — A) / A0 × 100%. (A0 is the absorbance of the DPPH solution without the sample; A is the absorbance of the sample after the reaction with DPPH).

[0063] Hydroxyl radical scavenging rate determination: The scavenging ability of CDs against hydroxyl radicals (·OH) was detected by the TMB colorimetric method. 0.0769 g of 3,3',5,5'-tetramethylbenzidine (TMB) was dissolved in 2 mL of dimethyl sulfoxide to prepare the TMB working solution. Then, the following were added sequentially: 1970 μL of CDs solutions of different concentrations, 10 μL of FeSO4 (supersaturated solution), 10 μL of hydrogen peroxide solution, and 10 μL of TMB working solution. The mixture was thoroughly mixed and reacted at 25°C in the dark for 5 minutes. Finally, the absorbance at 652 nm was measured using a UV-Vis spectrophotometer. The antioxidant capacity was evaluated by calculating the hydroxyl radical scavenging rate: Hydroxyl radical scavenging rate = (A0 — A) / A0 × 100%, where A and A0 are the absorbances with and without CDs, respectively.

[0064] Superoxide anion scavenging rate determination: Detection was performed using an SOD assay kit. Preparation of NBT working solution: Met buffer and NBT solution were mixed at a ratio of 23:3. Different concentrations of carbon dot solutions were mixed with 2.16 mL of NBT working solution and 0.240 mL of FD solution, and then transferred to 2 mL tubes. The centrifuge tubes were then exposed to natural light for 10 minutes. Finally, the absorbance at 560 nm was measured using a UV-Vis spectrophotometer. Dark treatment group: The same system was used for the reaction in the dark. Superoxide anion (O2) •− Clearance = (A0 — A) / A0 × 100%, where A and A0 are the absorbance with and without CDs, respectively.

[0065] like Figure 5 As shown, the scavenging effect of CDs on ABTS was evaluated by observing the change in absorbance at 734 nm. The results indicate that the scavenging effect of CDs on ABTS is concentration-dependent.

[0066] like Figure 6 As shown, the DPPH scavenging effect of CDs was evaluated by observing the change in absorbance at 519 nm. The results indicate that the DPPH scavenging effect of CDs is concentration-dependent.

[0067] like Figure 7 As shown, the scavenging effect of CDs on hydroxyl radicals was evaluated by observing the change in absorbance at 652 nm. The results indicate that the scavenging effect of CDs on hydroxyl radicals is concentration-dependent.

[0068] like Figure 8 As shown, the scavenging effect of CDs on superoxide anions was evaluated by observing the change in absorbance at 560 nm. The results indicate that the scavenging effect of CDs on superoxide anions is concentration-dependent.

[0069] Example 3 The synthesis method of the nanomaterial CDs@lip includes the following steps: 1) Weigh 16 mg of lecithin, 2 mg of cholesterol and 1.6 mg of DSPE-TK-PEG into an EP tube, add 2 mL of a mixed solvent of dichloromethane and methanol (v / v, 2:1) as the organic phase, and vortex for 20 min to dissolve it completely.

[0070] 2) Transfer the mixture to a round-bottom flask and remove the organic solvent by rotary evaporation under reduced pressure in a 48 °C water bath for 30 min, forming a uniform lipid film at the bottom of the flask. Add 2 mL of PBS (pH=7.4) aqueous solution with a carbon dot concentration of 1 mg / mL to the flask, place it in an oil bath, and hydrate with magnetic stirring at 48 °C for 60 min. Place the resulting solution in an ultrasonic cell disruptor and sonicate on ice (5 s working time, 5 s interval, 80% intensity, 30 cycles). Finally, filter through a 0.220 μm aqueous microporous membrane and then extrude 10 times using a liposome extruder.

[0071] Figure 9 Image (A) is a transmission electron microscope characterization image of the CDs@LIP prepared in Example 3. As can be seen from the image, the CDs@LIP prepared in this example is a regular sphere with a diameter of about 70 nm. Figure 9 (B) Dynamic light scattering (DLS) indicates that the hydrodynamic size of CDs@LIP is approximately 87.07 nm.

[0072] Example 4 CDs@LIP Cytotoxicity Test: 1) Cell culture: Rat adrenal pheochromocytoma cells (PC-12) were resuspended in DMEM medium (containing a mixture of 10% fetal bovine serum and 1% penicillin and streptomycin) and seeded into cell culture flasks. The flasks were then cultured in an incubator at 37 ℃ and 5% CO2 and passaged.

[0073] 2) MTT Test: After passaged three times, when the cell density reaches approximately 80%, the cells are digested, centrifuged, counted, and then seeded into 96-well plates (1×10⁶ cells / wells). 5 Cells were incubated in DMEM at different concentrations (0, 20, 40, 60, 80, and 100 μg / mL) for 24 h. After incubation for another 24 h, 100 μL of MTT solution was added to each well, and incubation continued for another 4 h. The MTT solution was discarded, and 150 μL of DMSO solution was added to each well. After shaking for 30 min, the absorbance at 490 nm was measured using a microplate reader. Cell viability was calculated. The test results are shown below. Figure 10 .

[0074] 3) Results of CDs@LIP cytotoxicity assay: like Figure 10 As shown, when the concentration of CDs@LIP is as high as 100 μg / mL, the cell viability is still above 80%, which proves that CDs@LIP has very low cytotoxicity and indicates that CDs@LIP has good biocompatibility and can be used in in vivo experiments.

[0075] Example 5 Changes in intracellular reactive oxygen species: PC-12 cells were seeded into 48-well plates. The experiment was divided into five groups: group 1 was the blank control group, group 2 was the rotenone-induced PD model group, group 3 was the quercetin treatment group, group 4 was the CDs treatment group, and group 5 was the CDs@LIP treatment group. After treatment, the cells were washed three times with PBS, and then 10 μM of DCFH-DA probe was added. After incubation for 30 min, the cells were washed three times again with PBS. The cells were observed and photographed using a microscope.

[0076] like Figure 11 As shown, fluorescence imaging results of cells in each treatment group were captured using a fluorescence microscope. It is clearly observed that the fluorescence intensity of the treatment group was significantly reduced compared to the rotenone-treated group, indicating that the material exhibits good antioxidant properties.

[0077] Example 6 CDs@LIP counteract rotenone-induced cell damage: PC-12 cells were seeded into 48-well plates, and the experiment was divided into 5 groups: blank control group, rotenone-induced PD model group, quercetin treatment group, CDs treatment group, and CDs@LIP treatment group. After treatment with different drugs, the cells were washed three times with PBS. AM-PI solution was prepared by adding 1 μL AM and 20 μL PI to each 1 mL PBS system. 200 μL of AM-PI solution was added to each well of cells, and the cells were incubated for 30 minutes. The cells were then washed again with PBS, and fluorescence imaging was performed using a fluorescence microscope.

[0078] like Figure 12 As shown, fluorescence imaging results of cells in each treatment group were captured using a fluorescence microscope. Calcein-AM stains live cells green, while PI stains dead cells red. The rotenone-induced group showed more red cells, indicating apoptosis, while the treatment group showed a decrease in the number of red cells, demonstrating the material's good therapeutic effect.

[0079] Example 7 Establishment of MPTP-induced PD model mice: A mouse model of Parkinson's disease was established using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP hydrochloride, MPTP). All mice were purchased from Beijing Huafukang Biotechnology Co., Ltd., and were 6 weeks old. After being housed at a constant temperature for one week, the model was established. The mice were then injected intraperitoneally with MPTP at a dose of 25 mg / kg for 7 consecutive days.

[0080] Example 8 Biodistribution of the material in mice: Rhodamine B-labeled CDs (administered at 20 mg / kg) were administered intranasally to PD model mice, and the distribution of nanomaterials in vivo was observed using a small animal in vivo imaging system.

[0081] like Figure 13 As shown in (A), the nanomaterials accumulated to varying degrees in the mouse brains 0.5 hours after administration. Four hours later, some of the nanomaterials in the brains had been metabolized. By 12 hours, the CDs@LIP group showed significant retention, with some residue remaining after 24 hours, while most of the nanomaterials in the other groups had been metabolized. Figure 13 (B) Analysis of the distribution of nanomaterials in the brain and major organs showed that the highest accumulation level was observed in the brain after 2 hours, and the material was completely metabolized after 48 hours. The results indicate that the material can effectively target the brain of PD mice.

[0082] Example 9 Application of materials in PD model mice Six-week-old C57BL / 6 black mice were used as a model and randomly divided into five groups: control group, MPTP group, quercetin treatment group, CDs treatment group, and CDs@LIP treatment group, with eight mice in each group. MPTP was administered intraperitoneally at a dose of 25 mg / kg for seven consecutive days. The administration of MPTP was repeated three days before, on the third and sixth day after injection, and on the second day after the completion of MPTP injection. Behavioral tests were performed one week after the end of treatment.

[0083] Before each behavioral test, place the mice in the behavioral laboratory half an hour in advance to allow them to acclimatize to the environment. The entire behavioral experiment must be conducted in a dark, quiet, and noise-free environment. Wipe the behavioral instruments clean with 75% alcohol beforehand.

[0084] Each time a test mouse is replaced, the instrument is wiped with 75% alcohol, the mouse feces are cleaned, and the next test is conducted after the 75% alcohol has completely evaporated.

[0085] Open field test: First, a square box measuring 40*40*40 cm was constructed, and the movement trajectory of the mouse was recorded using a video recording device. The experiment analyzed the total distance the mouse moved within 10 minutes, the number of times it entered the central area and the time it stayed there, as well as the mouse's movement speed, etc., as reference factors for measuring the mouse's motor coordination ability.

[0086] Pole climbing experiment: A solid wooden sphere with a diameter of 2.5 cm was fixed to a wooden pole with a diameter of 1 cm and a length of 50 cm. A layer of gauze was wrapped around the outside to prevent the mouse from slipping. The mouse was placed head up on the sphere at the top, and the time it took for the mouse to turn its head down and the time it took to turn its head to climb to the bottom of the pole were recorded.

[0087] Rotating bar experiment: Place the mouse on a rotating bar with gradually increasing speed. Slowly increase the speed to 30 rpm over 3 minutes. Record the time it takes for the mouse to fall.

[0088] like Figure 14 As shown, mice in the PD model group exhibited sluggish movement, lacked interest in exploring unknown areas, walked along the edges, spent less time in the central region, and covered a shorter total distance. Treatment with CDs and CDs@LIP improved the sluggishness caused by MPTP, increased the total distance covered, and slightly improved the movement speed. This indicates that the materials have a good therapeutic effect in the PD mouse model.

[0089] like Figure 15As shown, the mice in the PD model group had longer head-turning and crawling-down times. After treatment with CDs and CDs@LIP, both head-turning and crawling-down times decreased, and the mice's bradykinesia was significantly improved. This indicates that the materials have a good therapeutic effect in the PD mouse model.

[0090] like Figure 16 As shown, the PD model group mice had a shorter fall time, while the fall time was significantly prolonged after treatment with CDs and CDs@LIP, demonstrating a significant improvement in the mice's motor coordination. This indicates that the materials have a good therapeutic effect in the PD mouse model.

[0091] In this invention, firstly, quercetin-derived carbon dots were prepared using a one-step hydrothermal method, and their physicochemical properties were characterized. Secondly, the therapeutic effects of quercetin-derived carbon dots on Parkinson's disease (PD) were explored at the cellular and in vivo levels. This research not only provides new insights for the treatment of PD but also offers valuable lessons for the inheritance and innovation of traditional Chinese medicine.

[0092] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Other modifications can be readily made by those skilled in the art. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and examples shown and described herein.

Claims

1. A traditional Chinese medicine-derived carbon dot for treating Parkinson's disease, characterized in that, It consists of quercetin-derived carbon dots and a reactive oxygen species-responsive liposome shell encapsulating the quercetin-derived carbon dots; The quercetin-derived carbon dots are prepared by hydrothermal reaction using quercetin and Congo red in a mass ratio of 1:0.8-1.

2. The reactive oxygen species responsive liposomes are prepared from lecithin, cholesterol and DSPE-TK-PEG, wherein the mass ratio of lecithin, cholesterol and DSPE-TK-PEG is 7-9 : 0.8-1.2 : 0.5-1.

5.

2. A method for preparing traditional Chinese medicine-derived carbon dots as described in claim 1, characterized in that, Includes the following steps: 1) Preparation of carbon dots: Quercetin and Congo red were dissolved in water at a mass ratio of 1:0.8-1.2 and reacted at 160-200℃ for 1-4 hours. After purification and drying, quercetin-derived carbon dots were obtained. 2) Preparation of liposomes: Lecithin, cholesterol and DSPE-TK-PEG were dissolved in an organic solvent at a mass ratio of 7-9 : 0.8-1.2 : 0.5-1.5, and the mixture was rotary evaporated to form a lipid membrane; 3) Preparation of carbon dots derived from traditional Chinese medicine: Add a carbon dot PBS aqueous solution with a concentration of 0.5-2.0 mg / mL and pH 7.2-7.4 to the lipid membrane obtained in step 2) for hydration. After sonication, filtration through a filter membrane and extrusion, the resulting solution is used to obtain carbon dots CDs@LIP derived from traditional Chinese medicine.

3. The preparation method according to claim 2, characterized in that, In step 1), the hydrothermal reaction temperature is 170-190℃, and the reaction time is 1.5-2.5 hours. The heating rate of the hydrothermal reaction is 3-5 ℃ / min, and the reaction is allowed to cool naturally to room temperature after completion. During the hydrothermal reaction, the reaction temperature and pressure are monitored in real time to ensure that the temperature deviation does not exceed ±2 ℃ and the pressure is maintained at 1-2 MPa.

4. The preparation method according to claim 2, characterized in that, The purification in step 1) includes centrifugation, filtration and dialysis. The centrifugation speed is 8000-12000 rpm and the time is 10-20 minutes. The filtration uses a 0.22 μm microporous membrane. The dialysis uses a dialysis bag with a molecular weight cutoff of 1000 Da and the dialysis time is 24-48 hours. The dialysis temperature is 2-8℃.

5. The preparation method according to claim 2, characterized in that, In step 2), the mass ratio of lecithin to cholesterol is 8:1, and the mass of DSPE-TK-PEG is 10% of that of lecithin. The organic solvent is a mixture of dichloromethane and methanol in a volume ratio of 2:

1. The rotary evaporation temperature is 30-40 ℃, the pressure is 0.1-0.5 bar, and the evaporation time is 20-40 minutes.

6. The preparation method according to claim 2, characterized in that, In step 3), the hydration temperature is 45-50 ℃, and the hydration time is 50-70 min; The conditions for ultrasound are: ice bath, power of 200-300 W, working for 5 seconds, resting for 5 seconds, and repeating 25-35 times.

7. The preparation method according to claim 2, characterized in that, In step 3), a 0.22 μm polycarbonate membrane is used for filtration; the extrusion is performed using a liposome extruder, passing the material through a 100 nm polycarbonate membrane 3-5 times. The particle size of the traditional Chinese medicine-derived carbon dots CDs@LIP is 80-120 nm, and the encapsulation rate is 85%-95%.

8. The preparation method according to claim 4, characterized in that, In step 1), dialysis is performed in a dialysis bag at 2-8℃. During dialysis, samples are taken every 4-6 hours. The change in the hydrated particle size of carbon dots is monitored by dynamic light scattering, and the presence of Congo red characteristic absorption peaks in the dialysate is detected at a wavelength of 390 nm using a UV-Vis spectrophotometer. Dialysis can be terminated when two consecutive monitoring results show that the polydispersity index of the hydrated particle size distribution of carbon dots is less than 0.25 and the absorbance value of the dialysate at 390 nm is less than 0.

05.

9. The preparation method according to claim 4, characterized in that, In step 1), during purification, the dialysis step is performed using a tangential flow filtration system equipped with a filter membrane with a molecular weight cutoff of 1000 Da. The operating conditions include: maintaining the transmembrane pressure at 0.5-1.5 bar, controlling the feed flow rate at 100-200 mL / min, and continuously monitoring the UV absorbance of the permeate during dialysis. Dialysis is terminated when the absorbance at 390 nm wavelength remains below 0.02 for more than 10 minutes.

10. The use of the traditional Chinese medicine-derived carbon dots as described in claim 1 or the traditional Chinese medicine-derived carbon dots prepared as described in claims 2-9 in the preparation of a drug for treating Parkinson's disease.

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