An aspirin derivative carbon dot-based targeted dual-drug nanoparticle and a preparation method and application thereof

Abstract

This invention belongs to the field of nanomedicine delivery technology and discloses an aspirin-derived carbon dot-based targeted dual-drug nanoparticle, its preparation method, and its application. The nanoparticle uses aspirin-derived carbon dots as the core carrier. The carbon dots are prepared through an alkaline carbon dotification reaction and then surface functionalized. The targeting molecule DSPE-PEG2K-lys-RVG29-FITC is coupled through amide bonds, and clopidogrel is loaded to construct a dual-drug synergistic delivery system. The nanoparticle has the characteristics of a multifunctional nanostructure with a carbon dot core-shell structure and a targeted modification layer. It has performance advantages such as enhanced brain-targeted delivery and blood-brain barrier penetration, synergistic effect of dual antiplatelet drugs, and significant reduction of inflammation and thrombosis-related indicators.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine delivery, specifically relating to an aspirin-derived carbon dot-based targeted dual-drug nanoparticle, its preparation method, and its application. Background Technology

[0002] Stroke is an acute focal neurological deficit caused by impaired blood circulation in a localized area of ​​the brain. Common symptoms include hemiparesis, dysarthria, sensory disturbances, aphasia, and visual impairment. Ischemic stroke has a high rate of disability, a high recurrence rate, and significant side effects from treatment.

[0003] Antiplatelet therapy is an effective means of reducing mortality, disability, and recurrence rates in ischemic stroke. Aspirin and clopidogrel are the first-line antiplatelet drugs. Numerous studies have shown that dual antiplatelet therapy with aspirin and clopidogrel can effectively improve clinical symptoms in patients with non-minor ischemic stroke, reduce stroke recurrence rates, restore neurological function, and without increasing the incidence of adverse events.

[0004] The blood-brain barrier (BBB) ​​is a major obstacle to the treatment of central nervous system diseases (such as stroke and neurodegenerative diseases), as its tightly connected endothelial cell structure prevents most drugs from effectively entering brain tissue. To overcome this limitation, nanocarrier technology has become a research hotspot, among which carbon dots (CDs) have been widely explored due to their advantages such as small particle size (<10 nm), good biocompatibility, and functionalizability. Zhang et al. (2022) proposed preparing carbon dots using aspirin as the sole precursor. Using aspirin as the carbon source, aspCDs were synthesized using a traditional hydrothermal method. aspCDs could effectively penetrate the blood-brain barrier in mice and zebrafish, and showed anti-inflammatory effects comparable to or even better than aspirin in in vitro and in vivo inflammation models. At the same time, aspCDs can be used as carriers to successfully load model drugs of different polarities (such as rhodamine B, doxorubicin, and emodin), demonstrating their great potential as a visualized, multifunctional nanomedicine delivery system. However, their targeting is insufficient and their efficacy is limited when used alone. Summary of the Invention

[0005] The purpose of this invention is to provide an aspirin-derived carbon dot-based targeted dual-drug nanoparticle and its preparation method. The nanoparticle can achieve synergistic delivery and brain-targeted enrichment of antiplatelet drugs, and improve the therapeutic effect of ischemic brain injury.

[0006] This invention provides the application of the above-mentioned aspirin-derived carbon dot-based targeted dual-drug nanoparticles in the preparation of drugs for treating ischemic stroke.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention provides an aspirin-derived carbon dot-based targeted dual-drug nanoparticle, comprising aspirin-derived carbon dots as a carrier core, wherein the carbon dot surface is connected to a DSPE-PEG2K-lys-RVG29-FITC targeted modification molecule via amide bonds, and clopidogrel is loaded to form the aspirin-derived carbon dot-based targeted dual-drug nanoparticle.

[0009] This invention provides a method for preparing the above-mentioned aspirin-derived carbon dot-based targeted dual-drug nanoparticles, the preparation method comprising the following steps:

[0010] 1) Aspirin and sodium hydroxide were dissolved in water and heated to react. After the reaction was completed, the product was cooled, dialyzed and dried to obtain aspirin carbon dots.

[0011] 2) Disperse aspirin carbon dots in water, add DSPE-PEG2K-lys-RVG29-FITC solution to carry out amidation reaction to obtain DSPE-PEG2K-lys-RVG29-FITC modified aspirin carbon dots;

[0012] 3) The aspirin carbon dots modified with DSPE-PEG2K-lys-RVG29-FITC were dissolved in PBS buffer solution and then added to clopidogrel solution for drug loading. After dialysis, the aspirin-derived carbon dot-based targeted dual-drug nanoparticles were obtained.

[0013] In step 1), the mass ratio of aspirin to sodium hydroxide is 1-5:1.

[0014] In step 1), the reaction temperature is 100-120℃ and the reaction time is 5-9h.

[0015] In step 1), the molecular cutoff of the dialysis bag used for dialysis is 500 Da, and the dialysis time is 36-60 h.

[0016] In step 2), the mass ratio of the aspirin carbon dots to DSPE-PEG2K-lys-RVG29-FITC is 1:1-5.

[0017] In step 2), the amidation reaction takes 12-48 hours, the reaction temperature is 25-40°C, and the reaction is carried out in the dark.

[0018] In step 3), the mass ratio of the DSPE-PEG2K-lys-RVG29-FITC modified aspirin carbon dots to clopidogrel is 1:1-2.

[0019] In step 3), the conditions for drug loading are: pH 7.35-7.45, temperature room temperature, and time 1-2 hours.

[0020] This invention provides the application of the above-mentioned aspirin-derived carbon dot-based targeted dual-drug nanoparticles in the treatment of ischemic stroke.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] 1. This scheme is based on the construction of a dual drug delivery system using aspirin-derived carbon dots, and introduces RVG29 brain-targeting peptide to achieve active targeted delivery. By enhancing drug enrichment in the brain and achieving synergistic effects of aspirin and clopidogrel, the antithrombotic and anti-inflammatory effects are improved.

[0023] 2. This scheme utilizes the BBB penetration capability of carbon dot nanostructures and combines it with the receptor-targeted transport mechanism mediated by RVG29 peptide to improve the ability to cross the blood-brain barrier and the efficiency of brain distribution.

[0024] 3. This approach reduces drug exposure in non-target organs and minimizes systemic stimulation through nanocarrier encapsulation and brain-targeted delivery, thereby improving safety;

[0025] 4. This regimen inhibits thrombus formation and reduces inflammatory response through synergistic delivery of two drugs, while nano-delivery enhances local drug concentration, achieving a simultaneous decrease in key indicators such as TXB2, P-selectin, and TNF-α. Attached Figure Description

[0026] Figure 1 This is a mechanism diagram of the aspirin-derived carbon dot-based targeted dual-drug nanoparticles described in this invention;

[0027] Figure 2A shows the UV-vis absorption spectra of the aspCD described in Example 1 in alkaline and aqueous solutions; B shows transmission electron microscopy images of the aspCD, with scale bars of 20 nm and 5 nm, respectively; C shows the UV-vis absorption spectra of aspirin-derived carbon dot-based targeted dual-drug nanoparticles. Absorption spectra: D represents the diameter of the aspirin-derived carbon dot-based targeted dual-drug nanoparticles measured by DLS; E represents the 24-hour cck8 toxicity test results of the aspirin-derived carbon dot-based targeted dual-drug nanoparticles described in Comparative Examples 1, 2, and 3 and Example 1 on mouse brain microvascular endothelial cells bEnd.3; F represents the 48-hour cck8 toxicity test results of the aspirin-derived carbon dot-based targeted dual-drug nanoparticles described in Comparative Examples 1, 2, and 3 and Example 1 on mouse brain microvascular endothelial cells bEnd.3; G represents the 24-hour cck8 toxicity test results of the aspirin-derived carbon dot-based targeted dual-drug nanoparticles described in Comparative Examples 1, 2, and 3 and Example 1 on mouse hippocampal neuron cells HT-22; H represents the 48-hour cck8 toxicity test results of the aspirin-derived carbon dot-based targeted dual-drug nanoparticles described in Comparative Examples 1, 2, and 3 and Example 1 on mouse hippocampal neuron cells HT-22.

[0028] Figure 3 A is a schematic diagram of constructing an in vitro blood-brain barrier model; B is a diagram of flow cytometry detection of drug entry into cells; C is the mean fluorescence intensity (MFI) detection result of the aspirin-derived carbon dot-based targeted dual-drug nanoparticles described in Example 1 in cells; D is a representative fluorescence imaging diagram (DAPI) of the aspirin-derived carbon dot-based targeted dual-drug nanoparticles described in Example 1 after 24 hours and 48 hours of drug treatment; E is a diagram of quantitative statistical results of DAPI; F is a diagram of the mean fluorescence irradiance detected in vivo at different time points after intravenous injection; G is a diagram of the quantitative statistical results of the mean fluorescence irradiance detected in vivo at different time points after tail vein injection.

[0029] Figure 4A shows the changes in cerebral blood flow in the MCAO model monitored by LSI laser speckle imaging, using the product described in Comparative Example 3 and Example 1; B shows the quantitative statistical results of blood flow detected by LSI; C shows the TTC results of the MCAO model using the product described in Comparative Example 3 and Example 1; D shows the quantitative statistical results of TTC in the treatment group using the MCAO model using the product described in Comparative Example 3 and Example 1; E shows the ELISA quantitative analysis of TXB2 expression levels in the control group, MCAO model, and treatment group using the product described in Comparative Example 3 and Example 1; F shows the ELISA quantitative analysis of P-selectin expression levels in the control group, MCAO model, and treatment group using the product described in Comparative Example 3 and Example 1; G shows the ELISA quantitative analysis of TNF-α expression levels in the control group, MCAO model, and treatment group using the product described in Comparative Example 3 and Example 1; H shows pathological sections of the heart, liver, spleen, lung, and kidney organs in the MCAO group and the treatment group using the product described in Example 1. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0032] Example 1

[0033] A method for preparing aspirin-derived carbon dot-based targeted dual-drug nanoparticles specifically includes the following steps:

[0034] 1) Weigh 800 mg of NaOH and dissolve it in 20 mL of ultrapure water. Weigh 2 g of aspirin into a 50 mL round-bottom flask and add the above NaOH solution. React at 110 °C with stirring on a constant temperature magnetic stirrer for 6 h. After the reaction is complete, let the solution cool to room temperature and dialyze it through a dialysis bag with a molecular weight of 500 D for 48 h. Then, rotary evaporate to obtain powder and obtain aspirin carbon dots, labeled as aspCD.

[0035] 2) Dissolve 5 mg of aspCD in 100 ml of ultrapure water and disperse it by ultrasonication to form a uniform suspension. Dissolve 5 mg of DSPE-PEG2K-lys-RVG29-FITC in 200 μL of DMSO solvent. Add the DMSO solution to the suspension and stir the reaction at 30 °C for 24 hours. During the reaction, the reaction was carried out in the dark to obtain DSPE-PEG2K-lys-RVG29-FITC modified aspirin carbon dots, which were labeled as aminated aspCD.

[0036] 3) Weigh 5 mg of aminated aspCD and dissolve it in 10 mL of PBS at pH 7.4. Weigh 10 mg of clopidogrel crystal type II and dissolve it in 3 mL of anhydrous ethanol and then add 5 mL of PBS to dissolve it. Mix the two and control the pH at 7.35. Stir at room temperature for 1.5 hours. Dialyze to remove free drug to obtain the aspirin-derived carbon dot-based targeted dual-drug nanoparticles, labeled as AspCD / RVG29-Clop.

[0037] Example 2

[0038] A method for preparing aspirin-derived carbon dot-based targeted dual-drug nanoparticles specifically includes the following steps:

[0039] The only difference between this embodiment and Embodiment 1 is that the dosage of DSPE-PEG2K-lys-RVG29-FITC in step 2) is 20 mg.

[0040] Example 3

[0041] A method for preparing aspirin-derived carbon dot-based targeted dual-drug nanoparticles specifically includes the following steps:

[0042] The only difference between this embodiment and Embodiment 1 is that the amount of NaOH used in step 1) is 2 g.

[0043] Comparative Example 1

[0044] The comparative example is aspirin, labeled Asp.

[0045] Comparative Example 2

[0046] This comparative example is clopidogrel crystal type II, labeled Clop.

[0047] Comparative Example 3

[0048] This comparative example is a mixture of aspirin and clopidogrel crystal type II, wherein the mass ratio of aspirin to clopidogrel crystal type II is 1:2, labeled Asp+Clop.

[0049] Test Example 1

[0050] TEM characterization:

[0051] The aspCD powder prepared in step 1) of Example 1 was dispersed in deionized water and sonicated for 10 minutes to form a uniform suspension. A drop of the suspension was placed on a copper mesh and allowed to dry naturally. The copper mesh was then placed in a transmission electron microscope, and the voltage was adjusted to 100 kV to observe the morphology and particle size distribution of the carbon dots. Figure 2 As shown in Figure B, the particle size of carbon dots was measured using image analysis software, and the particle size distribution was statistically analyzed. The particle size was approximately 2 nm.

[0052] Ultraviolet-Vis absorption spectroscopy (UV-Vis) characterization:

[0053] In step 1 of Example 1, the aspCD powder was dispersed in deionized water to form a uniform suspension with a concentration of 100 g / L. The absorption spectrum was scanned in the range of 200-800 nm using a UV-Vis spectrophotometer. The absorption peaks and optical band gaps of the carbon dots were determined. The obtained aspCD solution and an aspirin NaOH solution were subjected to UV absorption spectroscopy measurements. The mass ratio of aspirin to NaOH in the aspirin NaOH solution was 5:2. The test results are as follows: Figure 2 As shown in Figure A, the results show that the characteristic absorption peaks of aspCD are similar to those of aspirin in NaOH solution, with two characteristic absorption peaks at 230 nm and 295 nm, as shown in Figure A. Figure 2 As shown in Figure C, the results demonstrate that aspCD and clopidogrel were successfully assembled into a dual-drug delivery system, AspCD / RVG29-Clop.

[0054] DLS characterization:

[0055] The DLS test results of the AspCD / RVG29-Clop described in Example 1 are as follows: Figure 2 As shown in Figure D, the results show that the size of the AspCD / RVG29-Clop described in Example 1 is approximately 250 nm.

[0056] Test Example 2

[0057] In vitro efficacy and safety evaluation

[0058] Cytotoxicity assay (CCK-8):

[0059] bEnd.3 and HT22 were seeded in 96-well plates and cultured until adherent. Different dose gradients of Comparative Examples 1, 2, and 3, as well as AspCD / RVG29-Clop prepared in Example 1, were added. The different dose gradients refer to 0 μL, 5 μL, 10 μL, 50 μL, and 100 μL of aspirin at a concentration of 5 mg / mL. After incubation for 24 hours, CCK-8 reagent was added, and cell viability was calculated after 24 hours and 48 hours of incubation. The test results are shown below. Figure 2As shown in EH, the results showed that the survival rate of the AspCD / RVG29-Clop group in Example 1 was much higher than that of the other comparative treatment groups.

[0060] Construction of in vitro BBB model:

[0061] Transwell chambers with 0.4 μm pore size and 12 mm diameter were placed in 24-well plates. Mouse hippocampal neurons (HT22) were added to the lower chamber and seeded at the bottom of the plate. Mouse cerebral angioendothelioma cells (bEnd.3) were added to the upper chamber and seeded onto the Transwell membrane. The cells were cultured in an incubator at 37°C and 5% CO2, with the culture medium changed every 2-3 days. A complete monolayer cell barrier was formed, with the structure shown below. Figure 3 As shown in Figure A.

[0062] Fluorescence tracer method for evaluating penetration efficiency:

[0063] The ability of AspCD / RVG29-Clop prepared in Example 1 to penetrate the BBB was further evaluated by detecting the fluorescence intensity of cells in the lower chamber using flow cytometry. Fluorescently labeled AspCD / RVG29-Clop was dispersed in culture medium, adjusted to a concentration of 0.1 mg / mL, and then added to the upper chamber of a Transwell chamber. The chamber was incubated at 37°C and 5% CO2 for 6 hours. After incubation, the liquid was collected from the lower chamber. A control group was set up, consisting of parallel experimental groups without AspCD / RVG29-Clop, labeled "blank". The test results are as follows: Figure 3 As shown in Figures B and C, compared with the control group, the mean fluorescence intensity (MFI) increased to 55 (P<0.05), indicating a statistically significant difference, demonstrating that AspCD / RVG29-Clop can penetrate the BBB.

[0064] MCAO model construction:

[0065] Select healthy C57 mice, weighing 20-25 g. Anesthetize the mice and fix them on the operating table, ensuring appropriate depth of anesthesia. Disinfect the skin of the mouse's neck with alcohol, make an incision along the midline of the neck, separate the subcutaneous tissue and muscle, and expose the right common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA). Carefully separate the ECA with fine forceps and ligate the distal end of the ECA with sutures. Temporarily clamp the CCA and ICA to reduce bleeding. Make a small incision proximal to the ECA and insert a 0.22 mm diameter... A silicone-coated nylon suture was inserted into the ECA stump and slowly advanced to the ICA until resistance was encountered, at which point the suture had sealed the origin of the middle cerebral artery (MCA). The ECA stump was then secured with the suture to prevent movement. The neck incision was sutured with absorbable sutures, and the wound was disinfected. The mouse was placed in a warm environment to recover and returned to its cage after regaining consciousness. The success of the MCAO model was assessed using the Longa score, with the following scoring criteria: 0 points: no neurological deficit; 1 point: contralateral forelimb cannot be fully extended; 2 points: circling to the contralateral side; 3 points: tilting to the contralateral side; 4 points: unable to walk spontaneously, loss of consciousness. A score ≥ 1 indicates a successful model, and the result showed a score of 2.

[0066] DAPI staining:

[0067] The efficiency of cellular uptake of the complex over time was assessed by DAPI staining, such as... Figure 3 As shown in Figures D and E, after 24 hours of treatment, the proportion of positive staining cells was 40%; after 48 hours of treatment, the proportion of positive cells increased to 78%. This result confirms that the complex described in this invention can effectively enter the cell interior, and the cellular uptake shows a time-dependent increase.

[0068] In vivo tracer assay to detect BBB penetration:

[0069] The successful establishment of the MCAO model was marked as time point T0. The AspCD / RVG29-Clop solution prepared in Example 1 was injected into mice via tail vein at a dose of 5 mg / kg. The safe and effective dose range was determined through preliminary experiments, and the injection volume was controlled at 200 μL. Fluorescence signals were monitored in real time using an in vivo imaging system (IVIS), and imaging was performed at 0.5, 2, 4, and 8 h post-injection. Imaging software was used to analyze the fluorescence signal intensity, observe the distribution of carbon dots in the mouse brain, compare the fluorescence signals at different time points, and evaluate the BBB penetration ability and brain distribution dynamics of the carbon dots.

[0070] Tests such as Figure 3 As shown in F and G, the results indicate that the drug enters the mice via tail vein injection and targets the mouse brain, with an increasing amount of drug reaching the brain over time.

[0071] Efficacy assessment: Cerebral blood flow was monitored by LSI. The instrument was preheated for 30 minutes and calibrated. Mice were anesthetized and fixed on a three-dimensional imaging platform. Imaging was performed after MCAO and after treatment with the products of Example 1 and Comparative Example 3. Changes in cerebral blood flow were analyzed using ImageJ imaging software, and the blood flow recovery rate in the ischemic area was calculated. The test results are as follows: Figure 4 As shown in Figures A and B, the results indicate that the blood flow recovery rate in the Example 1 treatment group was higher than that in the Comparative Example 3 treatment group.

[0072] TTC staining:

[0073] TTC staining was used to detect infarct area. After sacrifice, mouse brain tissue was rapidly removed, washed with pre-cooled PBS, and cut into 2 mm thick coronal sections using a brain sectioning mold. The sections were incubated in 2% TTC solution at 37°C in the dark for 30 min, followed by fixation with 4 wt% paraformaldehyde for 24 h to prevent tissue shrinkage. Images were taken and the infarct area was quantified using ImageJ. The infarct volume of each brain section was calculated using the following formula:

[0074] Infarct volume (mm) 3 =Infarct area (mm) 2 ) × slice thickness (mm);

[0075] The total infarct volume is obtained by summing the infarct volumes of all slices.

[0076] Percentage of cerebral infarction volume (%) = (Total infarction volume / Total brain volume) × 100%.

[0077] Test results are as follows Figure 4 As shown in C and D, the results showed that the neuroprotective effect of the AspCD / RVG29-Clop treatment group was three times that of the combination therapy of aspirin and clopidogrel alone.

[0078] ELISA experiment:

[0079] The levels of plasma TXB2, P-selectin, and TNF-α were measured by ELISA to evaluate the ameliorative effect of AspCD / RVG29-Clop on inflammatory response and platelet activation in MCAO mice. After anesthesia, whole blood was collected from the orbital venous plexus of mice, mixed with EDTA anticoagulant, and centrifuged at 4 ℃ and 3000 rpm for 10 min. The supernatant plasma was collected, aliquoted, and stored at −80 ℃ for later use, avoiding repeated freeze-thaw cycles. Control group mice underwent only anesthesia and neck vessel exposure, without inserting sutures to occlude the middle cerebral artery; the remaining treatment procedures were consistent with the MCAO group, and these mice were labeled Sham. The treatment groups in Example 1 and Comparative Example 3 were treated according to the corresponding drug administration regimens after modeling. Subsequently, TXB2, P-selectin, and TNF-α were detected using an ELISA kit: 50 μL of the plasma sample to be tested was added to each well of the ELISA plate, followed immediately by 50 μL of biotinylated antibody working solution. After mixing, the ELISA plate was sealed with a sealing film and incubated at 37 ℃ for 45 minutes. After incubation, discard the liquid in the plate, add 300 μL of washing buffer to each well, let stand for 30 seconds and then discard. Repeat the washing 3 times. After the last wash, pat the microplate dry on absorbent paper.

[0080] Add 100 μL of HRP enzyme conjugate working solution to each well, seal the ELISA plate again with sealing film, and incubate at 37°C for 30 minutes. Discard the liquid in the plate, repeat the washing process 5 times, adding 300 μL of washing buffer to each well each time, letting it stand for 30 seconds, then discarding and patting dry. Add 90 μL of TMB substrate solution to each well, mix well, and incubate at 37°C in the dark for 15 minutes. After incubation, add 50 μL of stop solution to each well and mix well; within 15 minutes, measure the absorbance (OD value) of each well at 450 nm using an ELISA reader. Plot a standard curve based on the concentration of the standards and the corresponding OD values, substitute the OD values ​​of the test samples, and calculate the concentrations of TXB2, P-selectin, and TNF-α in plasma.

[0081] Test results are as follows Figure 4 As shown in Figures E, F, and G, the results indicate that the AspCD / RVG29-Clop treatment group prepared in Example 1 showed the greatest decrease in TXB2, P-selectin levels, and TNF-α, suggesting that the AspCD / RVG29-Clop prepared in Example 1 had the best anti-inflammatory effect.

[0082] The toxic side effects of AspCD / RVG29-Clop, such as Figure 4 As shown in the middle H, HE staining results showed that AspCD / RVG29-Clop had almost no toxicity to the heart, liver, spleen, lungs and kidneys of mice after administration.

[0083] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0084] The above description of the embodiments is intended to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. An aspirin derivative carbon dot based targeted dual drug nanoparticle, characterized in that, The aspirin-derived carbon dots serve as the core carrier, with the carbon dots surface linked to a DSPE-PEG2K-lys-RVG29-FITC targeting molecule via amide bonds, and loaded with clopidogrel to form the aspirin-derived carbon dot-based targeted dual-drug nanoparticles.

2. A method for preparing aspirin-derived carbon dot-based targeted dual-drug nanoparticles as described in claim 1, characterized in that, The preparation method includes the following steps: 1) Aspirin and sodium hydroxide are dissolved in water and heated to react. After the reaction is completed, the product is cooled, dialyzed and dried to obtain aspirin carbon dots. 2) Disperse aspirin carbon dots in water, add DSPE-PEG2K-lys-RVG29-FITC solution to carry out amidation reaction to obtain DSPE-PEG2K-lys-RVG29-FITC modified aspirin carbon dots; 3) The aspirin carbon dots modified with DSPE-PEG2K-lys-RVG29-FITC were dissolved in PBS buffer solution and then added to clopidogrel solution for drug loading. After dialysis, the aspirin-derived carbon dot-based targeted dual-drug nanoparticles were obtained.

3. The production method according to claim 2, characterized by, In step 1), the mass ratio of aspirin to sodium hydroxide is 1-5:

1.

4. The preparation method according to claim 2, characterized in that, In step 1), the reaction temperature is 100-120℃ and the reaction time is 5-9h.

5. The preparation method according to claim 2, characterized in that, In step 1), the molecular cutoff of the dialysis bag used for dialysis is 500 Da, and the dialysis time is 36-60 h.

6. The preparation method according to claim 2, characterized in that, In step 2), the mass ratio of the aspirin carbon dots to DSPE-PEG2K-lys-RVG29-FITC is 1:1-5.

7. The preparation method according to claim 2, characterized in that, In step 2), the amidation reaction takes 12-48 hours, the reaction temperature is 25-40°C, and the reaction is carried out in the dark.

8. The preparation method according to claim 2, characterized in that, In step 3), the mass ratio of the DSPE-PEG2K-lys-RVG29-FITC modified aspirin carbon dots to clopidogrel is 1:1-2.

9. The preparation method according to claim 2, characterized in that, In step 3), the conditions for drug loading are: pH 7.35-7.45, room temperature, and time 1-2 hours.

10. The application of the aspirin-derived carbon dot-based targeted dual-drug nanoparticles as described in claim 1 in the treatment of ischemic stroke.

Images