ART-MMF self-assembly capable of relieving allograft rejection and preparation method and application of ART-MMF self-assembly

Abstract

The invention discloses a nano self-assembly body capable of reducing allograft rejection. The nano self-assembly body is a nano particle obtained by self-assembling an artesunate-mycophenolic acid ester self-assembly body prodrug and DSPE-PEG2000, wherein the artesunate-mycophenolic acid ester self-assembly body prodrug and the DSPE-PEG2000 are of the following structure. The invention also discloses a preparation method and application of the nano self-assembly body. In addition, the invention also discloses a structure and a preparation method of the artesunate-mycophenolate self-assembly prodrug. In the preparation process of the nano self-assembly, artesunate and mycophenolic acid ester are synthesized into a prodrug through esterification reaction, and then the prodrug is combined with DSPE-PEG2000 to prepare the nano drug. The nano-drug has the characteristic of accurately targeting the spleen, and can effectively remodel the immune microenvironment after transplantation. Through research and development of the nano-drug, the problem of toxic and side effects caused by long-term use of a traditional immunosuppressor and the problem of poor treatment effect of a natural small-molecule immunosuppressor are successfully solved, and the nano-drug shows extremely wide development prospects and huge potential in the field of future clinical application.

Description

Technical Field

[0001] The present invention relates to the technical field of application of immunosuppressants after allogeneic organ transplant rejection, and mainly relates to a preparation method and application of an ART-MMF immunosuppressant self-assembly. Background Art

[0002] Organ transplantation, one of the most revolutionary breakthroughs in modern medicine, remains the core treatment for patients with end-stage organ failure to regain their lives. Whether it is solid organ transplantation such as kidney, liver, heart, or hematopoietic stem cell transplantation, it has significantly improved the quality of life and life expectancy of patients. In order to suppress the immune response, transplant patients need to use immunosuppressants for a long time. However, long-term use of traditional immunosuppressants such as sirolimus and mycophenolate mofetil may cause serious adverse reactions, such as bone marrow suppression, leukopenia, and risk of tumor recurrence. We combine natural small molecule compounds with traditional immunosuppressants and deliver them to immune organs through nano-modification, achieving immunosuppressive effects while reducing drug toxicity and side effects. This is a potential treatment option for future immunosuppressive therapy.

[0003] Traditional immunosuppressants commonly used in clinical practice, such as sirolimus and mycophenolate mofetil, can cause a range of serious adverse reactions with long-term use, significantly reducing the quality of life for transplant patients. In contrast, while natural small-molecule compounds have been reported to offer advantages such as lower toxicity and side effects, their immunosuppressive effects fall short of clinical needs. Furthermore, traditional immunosuppressants suffer from extremely low bioavailability and rapid metabolism. To ensure effective immunosuppressive therapy, clinical immunotherapy regimens require increased dosages and frequency, which not only further exacerbates the risk of adverse drug reactions but also imposes a greater financial burden and inconvenience on patients. Therefore, a novel approach has been proposed, combining natural small-molecule drugs with traditional immunosuppressants through rational structural modification and nano-modification technology for targeted delivery to immune organs. This innovative strategy offers multiple advantages, including cost-effectiveness, high bioavailability, and a favorable safety profile. It not only offers a new approach to alleviating transplant rejection but also opens up an effective approach to reducing the adverse effects associated with immunosuppressive therapy, demonstrating significant potential and promising prospects for clinical application. Summary of the Invention

[0004] This invention aims to provide an innovative nano-self-assembly composed of a traditional immunosuppressant and a natural drug, namely, an artesunate-mycophenolate mofetil self-assembly, as well as its preparation methods and application scope. This self-assembly can precisely target immune organs and allografts, optimizing and reconstructing the post-transplant immune microenvironment, significantly reducing the degree of graft rejection, and greatly prolonging graft survival, thereby significantly improving the prognosis of transplant patients, enhancing their quality of life and long-term survival rate.

[0005] The technical solution adopted by the present invention to solve its technical problem is:

[0006] A nanometer self-assembly capable of reducing allogeneic transplant rejection is a nanoparticle self-assembled from artesunate-mycophenolate mofetil self-assembly prodrug with the following structure and DSPE-PEG2000:

[0007]

[0008] Furthermore, the mass ratio of the artesunate-mycophenolate mofetil self-assembly prodrug to DSPE-PEG2000 is 1:0.1-0.9.

[0009] Furthermore, the mass ratio of the artesunate-mycophenolate mofetil self-assembly prodrug to DSPE-PEG2000 is 1:0.1.

[0010] An ART-MMF self-assembly capable of allograft rejection is shown below:

[0011]

[0012] Wherein, x represents the mass proportion of the artesunate-mycophenolate mofetil self-assembly prodrug in the polymer, and the mass proportion of the DSPE-PEG2000 modification accounts for 1 / 10 of the artesunate-mycophenolate mofetil self-assembly prodrug.

[0013] In the polymer system involved in the present invention, mycophenolate mofetil, as a traditional immunosuppressant widely used in clinical practice, has demonstrated remarkable results in the field of immunosuppressive therapy. Artesunate, on the other hand, is derived from natural substances and is a small molecule drug with excellent immunosuppressive potential. The synthesized artesunate-mycophenolate mofetil self-assembly prodrug is a light yellow oily liquid that is easily soluble in organic solvents such as dimethyl sulfoxide and acetone. After long-term systematic exploration and research by the inventors' team, it was found that after dissolving the artesunate-mycophenolate mofetil self-assembly prodrug in acetone, it was ultrasonically reacted with DSPE-PEG2000 in an aqueous phase system. Subsequently, the acetone solvent was effectively removed by rotary evaporation technology, and finally, a structurally stable nano-self-assembly was successfully prepared in the aqueous phase. In order to ensure that the structure of the self-assembly is in the most stable state, the value range of the variable x is set to 1-9; the value range of x / 10 is 0.1-0.9; under these conditions, the nano-self-assembly can maintain stable performance, so that it can better meet the needs of practical applications.

[0014] The method for preparing the artesunate-mycophenolate mofetil self-assembly comprises the following steps:

[0015] Step 1: Artesunate and mycophenolate mofetil undergo esterification reaction in the presence of a catalyst, and a self-assembled prodrug is obtained through a separation and purification process.

[0016] Step 2: After the esterification reaction, the self-assembled prodrug was dissolved in acetone. Under ultrasound assistance, the solution was slowly dripped into water in a certain proportion. The acetone was then removed by rotary evaporation to successfully prepare the artesunate-mycophenolate mofetil self-assembly.

[0017] Dichloromethane is selected as the solvent for the esterification reaction because it has good solubility and a low boiling point, which facilitates separation and purification after the reaction. In addition, it has relatively stable chemical properties and is not prone to side reactions with reactants or products.

[0018] The esterification reaction temperature is 40-50°C.

[0019] The catalyst is 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.

[0020] Furthermore, the esterification reaction is carried out in a reaction flask, the reaction temperature is maintained at 43° C., the reaction speed is 400 RPM, and the esterification reaction time is 8-12 hours.

[0021] The preliminary product after the esterification reaction is subjected to thin layer chromatography product identification, and the successful synthesis of the reaction product after the esterification reaction is confirmed by the thin layer chromatography result, and then the product is purified.

[0022] The product identified by thin layer chromatography was washed three times with 5% citric acid, saturated sodium bicarbonate and saturated brine in order to remove the catalyst residue and a small amount of unreacted raw material residue.

[0023] Subsequently, excess water of the product is removed with anhydrous sulfuric acid; the product is dissolved with dichloromethane and purified by thin layer chromatography; and the remaining water and dichloromethane are dried by rotary evaporation to obtain the final product.

[0024] The product was dissolved in the organic solvent acetone, mixed with DSPE-PEG2000 in a mass ratio of 1:10, and evenly dropped into water under ultrasound at a certain proportion. The acetone was then removed by rotary evaporation to obtain artesunate-mycophenolate mofetil self-assembly.

[0025] The present invention also provides a use of the nano self-assembly described in any one of the above technical solutions in preparing a drug for alleviating organ transplant rejection reaction after organ transplantation.

[0026] The present invention also provides a self-assembly prodrug having the following structure:

[0027]

[0028] The present invention also provides a method for preparing the self-assembly prodrug, which comprises: performing an esterification reaction on artesunate and mycophenolate mofetil in the presence of a catalyst, and performing post-treatment to obtain the self-assembly prodrug.

[0029] Preferably, 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide are used; the reaction temperature is 40-50° C. and the esterification reaction is carried out using dichloromethane.

[0030] The preparation process of this nano-self-assembly involves synthesizing artesunate and mycophenolate mofetil into a prodrug through an esterification reaction. This prodrug is then conjugated with DSPE-PEG2000 to form a nanodrug. This nanodrug precisely targets the spleen, effectively reshaping the immune microenvironment after transplantation. Furthermore, it significantly increases the drug's half-life and bioavailability, significantly enhancing the effectiveness of immunotherapy by reducing the dosage and frequency of administration.

[0031] The artesunate-mycophenolate mofetil self-assembly prepared by the present invention, as a new type of nanomedicine, not only has excellent water solubility and high biocompatibility, but can also accurately target spleen tissue. This self-assembly effectively prolongs the survival time of the transplant by reshaping the immune microenvironment after transplantation, which is of significant significance for improving the prognosis of organ transplant patients. This innovative achievement has successfully overcome the difficult problems of toxic side effects caused by long-term use of traditional immunosuppressants, as well as the dilemma of poor therapeutic effects of natural small molecule immunosuppressants. It is expected to bring new treatment hope to many organ transplant patients and show broad development prospects and huge application potential in the field of clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the synthesis of artesunate-mycophenolate mofetil prodrug;

[0033] Figure 2 To verify the structure of artesunate-mycophenolate mofetil self-assembly prodrug, H NMR spectrum;

[0034] Figure 3 To verify the high-resolution mass spectrometry structure of artesunate-mycophenolate mofetil self-assembly prodrug;

[0035] Figure 4 The electron microscopic structure, particle size, dispersion coefficient and potential diagram of artesunate-mycophenolate mofetil self-assembly;

[0036] Figure 5 To verify the in vitro stability and hydrolysis profile of artesunate-mycophenolate mofetil self-assembly;

[0037] Figure 6 The spleen targeting effect of artesunate-mycophenolate mofetil self-assembly in animals (scale bar: 400 μm);

[0038] Figure 7 The results are for the phagocytosis of artesunate-mycophenolate mofetil self-assembly by immune cells in spleen tissue of animals;

[0039] Figure 8 The blood drug concentration of artesunate-mycophenolate mofetil self-assembly in animals to achieve sustained release and the metabolite concentration in the transplant;

[0040] Figure 9 Toxicological evaluation of liver and kidney function in mice after 7 days of treatment with mycophenolate mofetil, artesunate, mycophenolate mofetil and artesunate combination, and artesunate-mycophenolate mofetil self-assembly;

[0041] Figure 10H&E staining of the main organs of mice after 7 days of treatment with mycophenolate mofetil, artesunate, mycophenolate mofetil and artesunate combination, and artesunate-mycophenolate mofetil self-assembly (Scale bar: 400μm), and evaluation of the long-term immune environment side effects of the animals after 28 days of treatment;

[0042] Figure 11 The graft phenotype, survival, and body weight changes of mice after 7 days of treatment with mycophenolate mofetil, artesunate, the combination of mycophenolate mofetil and artesunate, and artesunate-mycophenolate mofetil self-assembly.

[0043] Figure 12 H&E staining and immunohistochemistry images of transplants of mice treated with mycophenolate mofetil, artesunate, the combination of mycophenolate mofetil and artesunate, and artesunate-mycophenolate mofetil self-assembly for 7 days (scale bar 400 μm);

[0044] Figure 13 To investigate the effects of mycophenolate mofetil, artesunate, the combination of mycophenolate mofetil and artesunate, and artesunate-mycophenolate mofetil self-assembly on the immune phenotype of macrophages in the spleen of mice after 7 days of treatment.

[0045] Figure 14 The effect of artesunate-mycophenolate mofetil self-assembly on the polarization of bone marrow-derived macrophages toward M1 in vitro;

[0046] Figure 15 To evaluate the inhibitory effect of artesunate-mycophenolate mofetil self-assembly on T cell proliferation in mice;

[0047] Figure 16 Effects of mycophenolate mofetil, artesunate, mycophenolate mofetil and artesunate combination, and artesunate-mycophenolate mofetil self-assembly on T cell immunophenotype in the spleen of mice after 7 days of treatment

[0048] Figure 17 This is the co-culture result of macrophages and T cells treated with artesunate-mycophenolate mofetil self-assembly. DETAILED DESCRIPTION

[0049] The technical solution of the present invention is further described in detail below through specific embodiments.

[0050] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following examples, unless otherwise specified, are all conventional methods in the art.

[0051] Example 1 (Synthesis of Artesunate-Mycophenolate Mofetil Self-Assembly Prodrug):

[0052]

[0053] Artesunate-mycophenolate mofetil self-assembly prodrug

[0054] The artesunate-mycophenolate mofetil self-assembly prodrug was esterified using 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as catalysts. 100 mg of artesunate and 93.9 mg of mycophenolate mofetil were dissolved in 2 mL of anhydrous dichloromethane, and 38.1 mg of 4-dimethylaminopyridine and 48.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were added at a molar ratio of 1.2:1:1:1. The reaction was carried out in a 43°C reaction flask at 400 RPM for 12 hours. The reaction process is as follows: Figure 1 shown.

[0055] Place the reaction product in a suitable container and slowly add 50 mL of a 5% citric acid solution. Then, place the container at room temperature and shake it at an appropriate frequency for 3 minutes to allow the solution to fully mix and react. After 3 minutes, add saturated salt water to the container to stop any emulsification. This is the first pickling operation. To ensure the pickling effect, repeat the same pickling steps twice according to the above operation process, for a total of three pickling operations.

[0056] Transfer the product from the first acid wash to a reaction vessel and add saturated sodium bicarbonate solution. Shake the product at a constant speed for 3 minutes at room temperature to allow for sufficient contact and reaction between the product and the solution, completing the second alkaline wash. Repeat the above steps two more times, for a total of three alkaline washes.

[0057] Transfer the product from the second alkaline wash to a suitable reaction vessel and add a sufficient amount of saturated salt water. Shake the reaction system continuously at room temperature for 3 minutes to allow the product to fully react with the solution, completing the third salt wash. To ensure the effectiveness of the salt wash, repeat the above process twice, for a total of three salt washes.

[0058] The salt-washed product was dried over anhydrous sodium sulfate to remove water, and the solvent was removed by rotary evaporation. The product was then further separated and purified by thin-layer chromatography. After purification, a mixed solution of dichloromethane and methanol in a volume ratio of 10:1 was used for column chromatography elution. The eluate was again concentrated by rotary evaporation to finally obtain the target product. The structure was verified as follows: Figure 2-3 shown.

[0059] Example 2 (Synthesis of Artesunate-Mycophenolate Mofetil Self-Assembly):

[0060]

[0061] Artesunate-mycophenolate mofetil self-assembly

[0062] 5 mg of the artesunate-mycophenolate mofetil self-assembly prodrug obtained by esterification reaction was weighed by a precision balance, dissolved in 100 μL of acetone, and then 0.5 mg of DSPE-PEG2000 was added. Under the action of ultrasound, it was slowly dripped into 900 μL of double-distilled water and the acetone was removed by rotary evaporation. Stable artesunate-mycophenolate mofetil self-assembly (named AMNPs) was obtained. The shape, particle size and stability were as shown in FIG. Figure 4-5 As shown (particle size, PDI and zeta potential were performed in dd water; hydrolysis was performed in 40% acetonitrile solution).

[0063] The beneficial effects of the present invention are demonstrated below through specific test examples:

[0064] Test Example 1: Spleen targeting test of artesunate-mycophenolate mofetil self-assembly material:

[0065] 1. Artesunate-Mycophenolate Mofetil Spleen Targeted Test Method

[0066] Artesunate-mycophenolate mofetil self-assembly labeled with DIR dye was intraperitoneally injected into C57BL / 6 mice (35 mg / kg). A free DIR group served as a control group. Spleen tissue was harvested for fluorescence imaging at 6, 12, 24, 48, and 72 hours. Software was used to analyze the fluorescence intensity of the DIR dye in the spleens of both groups at different time points. Frozen sections of spleen tissue from both groups were prepared and stained for F4 / 80, CD11c, CD4, CD8, and B220, and the degree of overlap with the DIR dye was observed under an immunofluorescence microscope.

[0067] 2. Spleen-targeting effect of artesunate-mycophenolate mofetil self-assembly

[0068] like Figure 6 After intraperitoneal injection of artesunate-mycophenolate mofetil self-assembly labeled with DIR dye, the fluorescence intensity of the spleen at 12, 24 and 48 hours was significantly higher than that of the free DIR dye group, indicating that artesunate-mycophenolate mofetil self-assembly can have a significant accumulation effect in the immune organ spleen.

[0069] like Figure 7 These are the immunofluorescence imaging results of DIR dye-labeled artesunate-mycophenolate mofetil self-assemblies and free DIR dye groups with different immune cells. From the merge results analysis, artesunate-mycophenolate mofetil self-assemblies were mainly phagocytosed by F4 / 80-positive macrophages in the spleen, followed by CD11c-positive dendritic cells.

[0070] Test Example 2: Safety and efficacy test of artesunate-mycophenolate mofetil self-assembly material:

[0071] 1. Security testing methods

[0072] Healthy C57BL / 6 mice were selected and injected with artesunate-mycophenolate mofetil self-assembly through the tail vein at specific time points. One week later, organ tissues such as heart, liver, spleen, lung, and kidney were collected and stained with H&E, and the degree of organ damage was observed under a microscope. Blood samples were collected from mice and blood biochemical indicators were tested, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN), serum alkaline phosphatase (ALP), blood urea nitrogen (BUN), lactate dehydrogenase (LDH), and blood creatinine (CREA). In the fourth week, blood samples from mice were collected to detect inflammatory factor indicators, evaluate the degree of damage to various organs of the mice, and confirm the safety of the drug.

[0073] In the figure, Control is the normal saline group; ART is the artesunate group (dosage: ART 17.7 mg / kg); MMF is the mycophenolate mofetil group (dosage: 19.9 mg / kg); ART+MMF is a mixed group using artesunate / artesunate as two single drugs (dosage: ART (17.7 mg / kg) + MMF (19.9 mg / kg)); AMNPs is the artesunate-mycophenolate mofetil self-assembly group (the content of artesunate and mycophenolate mofetil is the same as the previous group).

[0074] 2. Safety test results

[0075] like Figure 13 Compared with the control group injected with normal saline, artesunate-mycophenolate mofetil self-assembly had no obvious damage to the organs in the animals, and artesunate-mycophenolate mofetil self-assembly had no long-term immune function toxicity and side effects on the animals. Figure 8-9 shown.

[0076] Test Example 3: Pharmacokinetics test of artesunate-mycophenolate mofetil self-assembly material:

[0077] 1. Pharmacokinetics of Artesunate-Mycophenolate Mofetil Self-Assembly

[0078] To investigate the pharmacokinetic characteristics of artesunate, mycophenolate mofetil, and artesunate-mycophenolate mofetil self-assemblies, Sprague-Dawley rats were randomly divided into two groups and treated as follows: one group received intraperitoneal injections of artesunate (35.4 mg / kg) and mycophenolate mofetil (39.8 mg / kg), while the other group received intraperitoneal injections of drug-loaded nanoprogenitor particles (containing the same artesunate and mycophenolate mofetil concentrations as the previous group). At 0.083, 0.166, 0.25, 0.5, 1, 2, 3, 12, 24, 31, 36, and 48 hours after drug administration, 500 μL of fresh blood was collected from the rats' orbital venous plexus into anticoagulant tubes. The blood samples were allowed to stand at room temperature for 1 hour, then centrifuged and the plasma layer collected. Grafts were harvested 0.5 and 2 hours after intraperitoneal injection of artesunate-mycophenolate mofetil self-assemblies, and the concentrations of mycophenolic acid (MPA) and dihydroartemisinin (DHA) in the blood and grafts were measured.

[0079] 2. Pharmacokinetic test results of artesunate-mycophenolate mofetil self-assembly

[0080] like Figure 10 The artesunate-mycophenolate mofetil self-assembly significantly improved the bioavailability of the drugs, increased the drug half-life of artesunate and mycophenolate mofetil, and had a higher drug concentration in the graft.

[0081] Test Example 4: Test on the immunosuppressive effect of artesunate-mycophenolate mofetil self-assembly:

[0082] 1. Test method for the immunosuppressive effect of artesunate-mycophenolate mofetil self-assembly

[0083] The skin of BALB / c mice was transplanted onto the back of C57BL / 6 mice, and the immunosuppressive effect was tested by treating with 35 mg / kg of artesunate-mycophenolate mofetil self-assembly, an equal amount of artesunate and mycophenolate mofetil combined, and artesunate and mycophenolate mofetil alone. The drugs were continuously injected intraperitoneally for 7 days, and then the graft survival was observed, the graft necrosis area was scored, and the weight changes of the mice during the administration period were detected.

[0084] 2. Test results of the immunosuppressive effect of artesunate-mycophenolate mofetil self-assembly

[0085] like Figure 11 The carvacrol-artesunate self-assembly treatment group significantly prolonged the graft survival, reduced the graft necrosis area, and had no significant effect on the mouse body weight.

[0086] Experimental Example 5: Test of Artesunate-Mycophenolate Mofetil Self-Assembly on Inflammatory Cell Infiltration in Transplants:

[0087] 1. Test method for the effect of artesunate-mycophenolate mofetil self-assembly on graft inflammatory cell infiltration

[0088] The skin of BALB / c mice was transplanted onto the back of C57BL / 6 mice, and the immunosuppressive effect was tested by treating with 35 mg / kg of artesunate-mycophenolate mofetil self-assembly, an equal amount of artesunate and mycophenolate mofetil combined, and artesunate and mycophenolate mofetil alone. The drugs were continuously injected intraperitoneally for 7 days, and then the mouse grafts were harvested for H&E staining and immunohistochemical staining.

[0089] 2. Test results of the effect of artesunate-mycophenolate mofetil self-assembly on graft inflammatory cell infiltration

[0090] like Figure 12 The carvacrol-artesunate self-assembly significantly improved the infiltration of inflammatory cells, especially CD4-positive T cells, CD8-positive T cells and macrophages, and improved the damage to the physiological structure of blood vessels caused by transplant rejection.

[0091] Experimental Example 6: Test on the immunomodulatory effect of artesunate-mycophenolate mofetil self-assembly on macrophages:

[0092] 1. Test method for the immunomodulatory effect of artesunate-mycophenolate mofetil self-assembly on macrophages

[0093] The immunosuppressive effects of BALB / c mouse skin transplants onto the backs of C57BL / 6 mice were tested by treating mice with 35 mg / kg of artesunate-mycophenolate mofetil self-assembly, an equal amount of artesunate and mycophenolate mofetil combined, or artesunate or mycophenolate mofetil alone. Treatment was intraperitoneally injected for 7 days. Spleens were then harvested to isolate monocytes, and immune markers of macrophages were assessed by flow cytometry. MHC-II expression was assessed in bone marrow-derived macrophages treated with artesunate-mycophenolate mofetil self-assemblies in vitro.

[0094] 2. Test results of the immunomodulatory effect of artesunate-mycophenolate mofetil self-assembly on macrophages

[0095] like Figure 13-14 Artesunate-mycophenolate mofetil self-assembly reduced the expression of inflammatory factors IL-6 and TNF-α in macrophages, as well as the expression of the chemokine receptor CXCR-2. Simultaneously, the proportion of pro-inflammatory M1 macrophages (M1 macrophages expressing MHC-II) decreased, while the proportion of anti-inflammatory M2 macrophages (M1 macrophages expressing CD206) increased. Furthermore, artesunate-mycophenolate mofetil self-assembly significantly inhibited the expression of MHC-II in bone marrow-derived macrophages stimulated by LPS in vitro.

[0096] Experimental Example 7: Test on the immunomodulatory effect of artesunate-mycophenolate mofetil self-assembly on T cells:

[0097] 1. Test method for the effect of artesunate-mycophenolate mofetil self-assembly on T cell immunomodulation

[0098] To further investigate the role of artesunate-mycophenolate mofetil self-assembly in the immune regulation mechanism, we labeled the spleen cell mononuclear cells of CD45.1-positive C57BL / 6 mice with CFSE dye and then reinfused them into CD45.2-positive transplanted C57BL / 6 mice through the tail vein. The mice were then treated with artesunate-mycophenolate mofetil self-assembly at 35 mg / kg intraperitoneal injection for 5 consecutive days, and the proliferation rate of CD45.1-positive T cells was detected by flow cytometry.

[0099] The immunosuppressive effects were tested by using 35 mg / kg of artesunate-mycophenolate mofetil self-assembly, an equal amount of artesunate and mycophenolate mofetil combined, and artesunate and mycophenolate mofetil alone. The drugs were continuously injected intraperitoneally for 7 days, and then the spleens of the mice were taken to isolate monocytes, and the immune indicators of T cells were detected by flow cytometry.

[0100] 2. Test results of the effect of artesunate-mycophenolate mofetil self-assembly on T cell immunomodulation

[0101] like Figure 15-16 Artesunate-mycophenolate mofetil self-assembly significantly inhibited the proliferation rate of T cells in the spleen and peripheral blood, and regulated the differentiation of T cells into the anti-inflammatory Th2 type and inhibited the differentiation of T cells into the pro-inflammatory Th1 type.

[0102] Experimental Example 7: Test of the effect of artesunate-mycophenolate mofetil self-assembly on in vitro immune cell interaction:

[0103] 1. Method for testing the interaction of artesunate-mycophenolate mofetil self-assembly with immune cells in vitro

[0104] Bone marrow-derived macrophages treated with artesunate-mycophenolate mofetil self-assembly were co-cultured with T cells, and T cells were stimulated to proliferate in vitro with IL-2 and CD3&CD28 antibodies. The proliferation rates of T cells in different groups were then analyzed.

[0105] 2. Results of in vitro immune cell interaction tests of artesunate-mycophenolate mofetil self-assembly

[0106] like Figure 17 As shown, after bone marrow-derived macrophages treated with artesunate-mycophenolate mofetil self-assembly were co-cultured with T cells, the proliferation rates of CD4-positive T cells and CD8-positive T cells in vitro were significantly reduced compared with the group not treated with artesunate-mycophenolate mofetil self-assembly.

[0107] In summary, the present invention provides a nano-self-assembly composed of a traditional immunosuppressant and a natural small molecule drug that significantly alleviates post-transplant rejection. This self-assembly is inexpensive, biocompatible, and effective. It could benefit organ transplant patients in my country and has excellent application prospects.

[0108] The embodiment described above is only a preferred solution of the present invention and does not limit the present invention in any form. Other variations and modifications are possible without exceeding the technical solution described in the claims.

Claims

1. A nano self-assembly capable of reducing allogeneic transplant rejection, characterized in that: Nanoparticles are self-assembled from artesunate-mycophenolate mofetil self-assembly prodrug and DSPE-PEG2000 with the following structure:

2. The nano self-assembly capable of reducing allograft rejection according to claim 1, characterized in that: The mass ratio of the artesunate-mycophenolate mofetil self-assembly prodrug to DSPE-PEG2000 is 1:0.1-0.

9.

3. The nano self-assembly capable of reducing allograft rejection according to claim 1, characterized in that: The mass ratio of the artesunate-mycophenolate mofetil self-assembly prodrug to DSPE-PEG2000 is 1:0.

1.

4. A method for preparing the nano self-assembly according to any one of claims 1 to 3, characterized in that: The steps include: Step 1: In the presence of a catalyst, artesunate and mycophenolate mofetil undergo an esterification reaction, followed by post-treatment to obtain the self-assembled prodrug; Step 2: dissolving the self-assembled prodrug in an organic solvent, adding DSPE-PEG2000, and slowly dripping it into water through ultrasonic treatment to remove the organic solvent, thereby finally obtaining artesunate-mycophenolate mofetil self-assembly.

5. Use of the nano self-assembly according to any one of claims 1 to 3 in preparing a drug for alleviating organ transplant rejection after organ transplantation.

6. A self-assembly prodrug, characterized in that: Has the following structure:

7. A method for preparing the self-assembly prodrug according to claim 6, characterized in that: include: In the presence of a catalyst, artesunate and mycophenolate mofetil undergo esterification reaction, and then undergo post-treatment to obtain the self-assembled prodrug.

8. The preparation method according to claim 7, characterized in that The reaction mixture is 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; the reaction temperature is 40-50°C.

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