Application of traditional Chinese medicine composition and preparation thereof in synergistic anti-tumor with pd-1 inhibitor

Abstract

The present application relates to the application of traditional Chinese medicine active ingredient composition and its preparation in synergistic anti-tumor of PD-1 inhibitor. The traditional Chinese medicine active ingredient composition is a composition of oxymatrine and astragaloside B. The combination of the two can improve the anti-tumor effect of PD-1 inhibitor. In addition, it can also be used in the form of preparation to realize in vivo drug delivery in cooperation with its pharmaceutically acceptable carrier. The carrier can be selected from iron-based MOF and liposome, etc. to realize the co-loading of oxymatrine and astragaloside B with different polarity and efficient drug delivery. The iron-based MOF carrier has the advantages of high drug loading capacity and Fe ion which can cooperate with astragaloside B to improve the activity of tumor infiltrating T cells. The ferromagnetism and platelet membrane modification can also precisely target tumor tissue. The preparation technology of liposome is mature, which is conducive to industrialized production and has broad prospects for clinical transformation. The combination of the two with PD-1 inhibitor can significantly improve the anti-tumor effect of PD-1 inhibitor.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to application of a traditional Chinese medicine active ingredient composition and a preparation thereof in synergistic anti-tumor enhancement of a PD-1 inhibitor. BACKGROUND

[0002] Liver cancer and breast cancer are both common malignant tumors, and have the characteristics of high malignancy and easy recurrence. Among the treatment methods for malignant tumors in clinical practice, chemotherapy and radiotherapy often have strong side effects, and surgical resection has a high recurrence rate, and it is difficult to achieve ideal therapeutic effects. In recent years, the immune checkpoint blockade therapy represented by PD-1 inhibitors is considered to be one of the most promising methods for treating malignant tumors. However, for some solid tumors such as liver cancer and breast cancer, the lack of TILs quantity and activity caused by tumor-related fibroblasts (CAFs) and mitochondrial abnormalities of tumor-infiltrating T lymphocytes (TILs) in the tumor microenvironment, leads to the low objective response rate of PD-1 inhibitors in clinical practice, which seriously limits the anti-tumor effect and further application of the PD-1 inhibitors.

[0003] Oxymatrine and Astragaloside IV are the main effective components in Sophora flavescens and Astragalus membranaceus. Literature and our previous studies show that both of them have good anti-tumor and immunomodulatory effects. Among them, oxymatrine can effectively inhibit mouse liver fibrosis and reduce the expression of the CAF activation marker α-SMA, and has the effect of inhibiting CAF activation; Astragaloside IV can promote mitochondrial biogenesis and oxidative phosphorylation by up-regulating the expression of PGC-1α, and can be used to promote TILs mitochondrial normalization.

[0004] Through retrieval, there are existing technologies related to the anti-tumor or auxiliary anti-tumor effects of oxymatrine or Astragaloside IV.

[0005] Chinese patent applications related to oxymatrine include: Application of Traditional Chinese Medicine Monomer Oxymatrine in Preparation of Drugs for Treating or Preventing Non-small Cell Lung Cancer (CN201911068153.9), Drug Combination for Treating Hepatitis, Liver Fibrosis and Liver Cancer (CN201610255001.X), A Pharmaceutical Composition for Treating Colorectal Cancer (CN201210155469.3), Application of Oxymatrine in Preparation of Anti-tumor Drugs (CN201110379693.6), An Anti-cancer Drug Composition for Slow Release in vivo (CN200410075842.X), An Anti-tumor Composition, Its Preparation Method and Use (CN200110030181.5), Application of Oxymatrine in Preparation of Anti-tumor Drug Sensitizer (CN201610000529.2), An Anti-tumor Drug Composition and Its Preparation Method (CN201510367188.8), A Pharmaceutical Composition for Treating Colorectal Cancer (CN201210155469.3), A Drug Containing Oxymatrine (CN200810132186.0), etc.

[0006] Chinese patent applications related to astragaloside include: A Human Immunity Enhancer for Cancer Prevention and Treatment and Its Preparation Method (CN201911095186.2), A Resuscitation Method of CAR-T Cells (CN202011058265.9), Use of Combination of Astragaloside and Oxaliplatin in Preparation of Anti-tumor Drugs (CN201910632861.4), Preparation Method of Controlled-release Hydrogel with Photothermal Therapy and Wound Repair Function (CN201910618151.6), A Natural Product Combination and Its Medical Use (CN201810076809.0), A Detection Method of Anti-tumor Injection Preparation (CN201711436366.3), A New Traditional Chinese Medicine for Treating Leukopenia Caused by Cancer Radiotherapy and Chemotherapy and Its Preparation Method (CN201210023511.6), A New Compound Adjuvant Therapy Drug for Liver Cancer (CN201410434469.6), A Pharmaceutical Composition for Treating Cancer and Its Preparation Method and Use (CN201310698698.4), etc.

[0007] There are also reports in existing literature on the anti-liver cancer or adjuvant anti-liver cancer effects of oxymatrine and astragaloside respectively. However, neither the existing patents nor the existing literature reports the use of oxymatrine and astragaloside in a specific ratio to enhance the anti-tumor effect of PD-1 inhibitors, especially for liver cancer or breast cancer. There is also no report on the co-loading of the two in the same nano-preparation, or the use of metal-organic framework (MOF) and liposome drug delivery system to co-deliver the two to tumor tissues. SUMMARY

[0008] The first object of the present application is to provide an application of a traditional Chinese medicine active ingredient composition in synergistic tumor resistance enhancement of a PD-1 inhibitor.

[0009] The traditional Chinese medicine active ingredient composition is a composition of oxymatrine and astragaloside.

[0010] As a preferred embodiment, the mass ratio of oxymatrine to astragaloside is 1-8:1, preferably 1-2:1.

[0011] As a preferred embodiment, the tumor is liver cancer or breast cancer.

[0012] As a preferred embodiment, the composition further comprises a pharmaceutically acceptable carrier of the oxymatrine and astragaloside combination composition. That is, the composition can be prepared into a preparation for drug delivery.

[0013] As a preferred embodiment, the carrier is an iron-based MOF (Fe·MOF) or a liposome.

[0014] As a preferred embodiment, the tumor is liver cancer, and an iron-based MOF is used as the composition carrier.

[0015] As a preferred embodiment, the tumor is breast cancer, and a liposome is used as the composition carrier.

[0016] The present application combines water-soluble drug oxymatrine and lipid-soluble drug astragaloside in a specific ratio and prepares a nano-preparation. The two drugs of different polarity can be delivered to tumor tissues together, which can significantly improve the anti-tumor effect of the PD-1 inhibitor.

[0017] Another object of the present application is to provide a nano-preparation for synergistic tumor resistance enhancement of a PD-1 inhibitor, which comprises the above-mentioned traditional Chinese medicine active ingredient composition and a pharmaceutically acceptable carrier thereof.

[0018] As a preferred embodiment, the carrier is an iron-based MOF. The nano-preparation co-loading water-soluble drug oxymatrine and lipid-soluble drug astragaloside uses oxymatrine and astragaloside as raw drugs, an MOF system constructed by metal iron and organic ligand as a drug carrier, and uses surface modification to improve the targeting ability to liver cancer tissues.

[0019] The preparation method of the nano-preparation comprises: connecting metal nodes with organic ligands to form an iron-based MOF, activating the iron-based MOF, and placing it in a solution with the traditional Chinese medicine active ingredient composition as the solute to load the drug, obtaining a drug-loaded nano-preparation, and modifying the drug-loaded nano-preparation with a surface modification material to obtain a finished nano-preparation.

[0020] In the above preparation method, the raw materials include the following components by mass:

[0021] The Chinese medicine active ingredient composition is 198-792 parts, the metal node is 4-101 parts, the organic ligand is 32-64 parts, and the surface modification material is 10-160 parts; in the solution with the Chinese medicine active ingredient composition as the solute, the mass ratio concentration of the solute and the solvent is 0.15-2.4% (w / w).

[0022] As a preferred embodiment, the metal node adopts one or two of Fe3O4 nanoparticles, FeCl3·H2O, and Fe(NO3)3·9H2O; preferably Fe3O4 nanoparticles.

[0023] The organic ligand adopts one of trimesic acid, terephthalic acid, and 2-amino terephthalic acid, preferably trimesic acid.

[0024] The surface modification material adopts one of platelet membranes, red blood cell membranes, liver cancer cell membranes, and hyaluronic acid, preferably platelet membranes.

[0025] As a preferred embodiment, the iron-based MOF is prepared by a hydrothermal synthesis method.

[0026] As a preferred embodiment, the carrier is a liposome. The lipophilic drug astragaloside is loaded in the phospholipid bilayer of the liposome, the hydrophilic drug oxymatrine is encapsulated in the hydrophilic inner cavity, in vivo long circulation is achieved through PEGylation, and passive targeting of breast cancer tissue is achieved through the EPR effect.

[0027] The nano-preparation includes the following components in mass parts:

[0028] The Chinese medicine active ingredient composition is 15-120 parts, the natural phospholipid is 100-500 parts, the synthetic phospholipid is 60-200 parts, the cholesterol is 30-75 parts, and the DSPE-PEG2000 is 14-20 parts.

[0029] As a preferred embodiment, the liposome preparation method adopts the film dispersion method-pH gradient method and the ethanol injection method-pH gradient method, preferably the ethanol injection method-pH gradient method.

[0030] The natural phospholipid is preferably soybean lecithin; the synthetic phospholipid is preferably hydrogenated soybean lecithin. The mixing ratio of the soybean lecithin: hydrogenated soybean lecithin is 0.5-5:1, preferably 4:1.

[0031] The present research group found through long-term and large-scale experimental research that the combination of oxymatrine and astragaloside B can simultaneously increase the number and antitumor activity of TILs by inhibiting the activation of CAFs and promoting the normalization of TILs mitochondria. The improvement of antitumor efficacy is significantly better than that of single drug when combined with PD-1 inhibitor, showing a strong synergistic antitumor effect. The oxymatrine and astragaloside B optimized by prescription have application prospects for developing drugs for improving the antitumor efficacy of PD-1 inhibitors when formulated in a specific ratio.

[0032] However, oxymatrine and astragaloside B have problems such as fast metabolism, low bioavailability, and poor in vivo tissue distribution targeting. Oxymatrine has good water solubility, while astragaloside B has poor water solubility. Therefore, it is extremely challenging to efficiently co-deliver oxymatrine and astragaloside B with large polarity span into tumor tissues.

[0033] Iron-based MOF is a new type of crystalline porous material self-assembled from Fe ions or Fe clusters and organic ligands through coordination bonds. It has characteristics such as large drug polarity span and high drug loading capacity, and can be used for co-delivery of oxymatrine and astragaloside B. In addition, it contains Fe elements, which can be used to synergistically enhance the activity of TILs with astragaloside B. At the same time, MOF systems can achieve drug release through the breakage of coordination bonds under the acidic and reducing microenvironment of tumors. Magnetic iron-based MOF with magnetic Fe3O4 nanoparticles as the core can target liver cancer tissues under the action of an external magnetic field, and the surface modification of active targeting ligands can achieve double targeting while avoiding the clearance of the immune system, thus efficiently and accurately co-delivering oxymatrine and astragaloside B into liver cancer tissues.

[0034] Liposomes are microencapsulated vesicles composed of phospholipids and cholesterol with a lipid bilayer structure. Compared with traditional drug delivery systems, liposomes have unique drug loading advantages. The aqueous phase inside the liposome can encapsulate water-soluble drugs, and the lipid bilayer can encapsulate lipid-soluble drugs. Therefore, liposomes can simultaneously encapsulate water-soluble oxymatrine and lipid-soluble astragaloside B, making them ideal carriers for intratumoral co-delivery of the two drugs. Due to the EPR effect of tumor tissues and the cell-like membrane structure of liposomes, liposomes with a particle size less than 200 nm can easily accumulate in tumor sites and penetrate into cells by fusing with cell membranes, thereby promoting intracellular delivery of encapsulated drugs. Tumor-targeted delivery of drugs by liposomes also reduces drug metabolism and excretion in the systemic circulation, thereby enhancing the antitumor efficacy of drugs. At the same time, liposomes as drug carriers have good biocompatibility, and their preparation process is mature with high clinical translation degree.

[0035] Therefore, the application co-carries and delivers oxymatrine and astragaloside B in tumor tissues by using an iron-based MOF or a liposome, so as to improve the anti-tumor effect of the PD-1 inhibitor in a synergistic manner.

[0036] The active ingredient combination composition, preparation and application of the traditional Chinese medicine have the following beneficial effects:

[0037] 1. Under the guidance of the theory of traditional Chinese medicine, the oxymatrine in Sophora flavescens and the astragaloside B in Astragalus membranaceus are combined in a specific ratio according to the compatibility theory of traditional Chinese medicine. The oxymatrine plays a role in inhibiting the activation of tumor-related fibroblasts and increasing the number of tumor-infiltrating T lymphocytes, the astragaloside B plays a role in regulating the mitochondria of tumor-infiltrating T lymphocytes and increasing the anti-tumor activity thereof, and the combination with the PD-1 inhibitor plays a synergistic anti-tumor effect.

[0038] 2. The iron-based MOF nanodrug delivery system constructed by using metal nodes and organic ligands realizes the co-loading of oxymatrine and astragaloside B with large differences in chemical structure, molecular weight and solubility, and can also improve the drug loading capacity, with a drug loading capacity of 30-40wt%.

[0039] 3. The application uses magnetic iron-based MOF as a drug carrier, and modifies the surface with a surface modifier such as platelet membrane, so as to obtain double active targeting, avoid the clearance of the preparation by the immune system in the body, realize long circulation effect, realize precise and efficient targeting of tumors, and overcome the problems of low bioavailability and poor in vivo tissue distribution targeting of oxymatrine and astragaloside B.

[0040] 4. The application uses the MOF system as a drug carrier, which can avoid the metabolism and destruction of drugs in normal tissues and body fluids, and release the drugs in the tumor tissue by the instability of the MOF coordination bond to the acidic and reducing tumor microenvironment, so as to protect the stability of the drugs and ensure that they can be effectively released in the tumor to exert the drug effect.

[0041] 5. The application uses a liposome nanodrug delivery system, in which the water phase and the lipid outer layer can respectively load the water-soluble drug oxymatrine and the fat-soluble drug astragaloside B, and the surface is modified with PEG to realize long circulation effect in the body and improve the intratumoral accumulation of drugs.

[0042] 6、The application adopts phospholipid and cholesterol with good biocompatibility as a drug carrier, mixes natural phospholipid with synthetic phospholipid, successfully co-loads oxymatrine and astragaloside B with large solubility difference, and improves the drug loading capacity of the two drugs compared with single phospholipid. The liposome has a cell-like membrane structure, the surface is modified with PEG, and after reaching the tumor tissue by the EPR effect, membrane fusion with target cells is carried out, promoting intracellular drug delivery.

[0043] 7、The application adopts ethanol injection method combined with pH gradient method to construct a liposome nanodrug delivery system, adopts active drug loading technology such as pH gradient method to load water-soluble drug oxymatrine, and ethanol injection method to load fat-soluble drug astragaloside B. This liposome preparation technology is mature, is conducive to industrialized production, and has broad clinical conversion prospects. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 Chemical structural formula of oxymatrine and astragaloside B.

[0045] Figure 2 Effect of different matching proportions and concentrations of oxymatrine and astragaloside B in Example 1 on the activity of CTLL-2 mouse T cells (n=6, X±SD).

[0046] Figure 3 Effect of different matching proportions and concentrations of oxymatrine and astragaloside B in Example 2 on the activity of CAFs cells (n=6, X±SD).

[0047] Figure 4 Relative cell activity of CTLL-2 cells after 4T1 cells and CTLL-2 cells were co-cultured for 24h under the condition of oxymatrine:astragaloside B=2:1 (*P<0.05, **P<0.01).

[0048] Figure 5 Particle size distribution and zeta potential diagram of the iron-based MOF obtained in Example 4.

[0049] Figure 6 SEM diagram of the iron-based MOF obtained in Example 4.

[0050] Figure 7 Hepatocarcinoma C57BL / 6 mice were treated with the composition nanomedicine of the application combined with PD-1 inhibitor in Example 5. 22 Tumor weight, tumor volume and tumor index of each group of mice after orthotopic hepatocarcinoma C57BL / 6 mice were treated (n=3, X±SD); note: compared with the model group, ***P<0.001, **P<0.01, *P<0.05;

[0051] Figure 8To treat H22 hepatocarcinoma C57BL / 6 mice with the nano-preparation of the composition of the present application combined with PD-1 inhibitor in Example 5 22 The CD4 T cell immunofluorescence staining results of the tumor tissues of the mice in each group after the orthotopic hepatocarcinoma C57BL / 6 mice + The CD4 T cell immunofluorescence staining results of the tumor tissues of the mice in each group after the orthotopic hepatocarcinoma C57BL / 6 mice

[0052] Figure 9 To treat H22 hepatocarcinoma C57BL / 6 mice with the nano-preparation of the composition of the present application combined with PD-1 inhibitor in Example 5 22 The CD8 T cell immunofluorescence staining results of the tumor tissues of the mice in each group after the orthotopic hepatocarcinoma C57BL / 6 mice + The CD8 T cell immunofluorescence staining results of the tumor tissues of the mice in each group after the orthotopic hepatocarcinoma C57BL / 6 mice

[0053] Figure 10 The tumor weight and volume of the mice in each group after the 4T1 orthotopic breast cancer BALB / c mice were treated with the composition and nano-preparation of the present application combined with PD-1 inhibitor in Example 6 (n=6, X±SD); Note: compared with the model group, ***P<0.001, **P<0.01, *P<0.05; compared with the PD-1 group, ▲▲▲P<0.001, ▲▲P<0.01, ▲P<0.05.

[0054] Figure 11 The HE and Ki 67 staining results of the tumor tissues of the 4T1 orthotopic breast cancer BALB / c mice treated with the composition and nano-preparation of the present application combined with PD-1 inhibitor in Example 6 (the scale in the figure is 200 μm).

[0055] Figure 12 The TUNEL staining results of the tumor tissues of the 4T1 orthotopic breast cancer BALB / c mice treated with the composition and nano-preparation of the present application combined with PD-1 inhibitor in Example 6 (the scale in the figure is 40 μm).

[0056] Figure 13 The α-SMA staining results of the tumor tissues of the 4T1 orthotopic breast cancer BALB / c mice treated with the composition and nano-preparation of the present application combined with PD-1 inhibitor in Example 6 (the scale in the figure is 40 μm).

[0057] Figure 14 The particle size distribution and zeta potential of the co-loaded oxymatrine and astragaloside A liposomes obtained in Example 8.

[0058] Figure 15 The effect of different concentrations of the co-loaded oxymatrine and astragaloside A liposomes on the activity of CTLL-2 mouse T cells in Example 10 (n=6, X±SD).

[0059] Figure 16Effect of different concentrations of co-loaded oxymatrine and astragaloside IV liposomes on the activity of CAFs cells in mice (n = 6, X ± SD) in Example 10. DETAILED DESCRIPTION

[0060] The sources of cells, animals, drugs, reagents, and instruments involved in the examples are as follows:

[0061] Experimental cells: CTLL-2 mouse T cells, NIH3T3 mouse embryonic fibroblasts, H 22 -Luc mouse hepatocarcinoma cells, 4T1-Luc mouse breast cancer cells, all purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0062] Experimental animals: SPF C57BL / 6 male mice, SPF BALB / c female mice, all purchased from Jiangsu Jucu Pharmaceutical Biotechnology Co., Ltd. Experimental animal license number (SCXK (Su) 2018-0008), light / dark (12h / 12h), temperature (23±3)℃, and free diet and drinking water, experimental animal use license (SYXK (Su) 2016-0018).

[0063] Drugs and reagents: oxymatrine (HPLC≥98%, Chunqiu Biological); astragaloside IV (HPLC≥98%, Yuanye Biological); Tween 80 (Biofroxx); mouse PD-1 antibody for injection (Bioxcell); FeCl3·6H2O, Fe(NO3)3·9H2O (Araldite Reagent); anhydrous sodium acetate, nitric acid, hydrofluoric acid (Nanjing Chemical Reagent); diethylene glycol (Sigma); sodium acrylate, trimesic acid, terephthalic acid, 2-amino terephthalic acid, fluorescein potassium salt, cholesterol (Roen Reagent); methanol, ethanol (Xilong Scientific); protease inhibitors (CSNpharma); RPMI1640 medium (BasalMedia); DMEM medium (Gibco); fetal bovine serum (Deary Tech); CCK8 (Tongren Chemical); recombinant mouse TGF-β1 protein (Novoprotein); Matrigel, 24-well 0.4μm Transwell chamber (Corning), soybean lecithin (Lipoid), hydrogenated soybean lecithin (A.V.T).

[0064] Instruments: Mutiskan Go microplate reader, ST16R centrifuge, 311 CO2 incubator (Thermo Fisher); clean bench (Su'antai Air Technology Co., Ltd.); cell counter (IC1000, Countstar); TriStar II 3020 specific surface area and porosity analyzer (Micromeritics); MS205DU electronic balance (Mettler Toledo); XMTD-8222 high-temperature oven (Jinghong Instruments); OS20-Pro mechanical stirrer (SCILogex); ABM small animal gas inhalation anesthesia machine (Yu Yan Instruments); ChemStudio PLUS small animal live imaging instrument (Analytik Jena AG).

[0065] Other, unless otherwise specified, are commercially available.

[0066] Example 1: Effect of different compatibility ratios and concentrations of oxymatrine and astragaloside combination on T cell activity in mice

[0067] The CTLL-2 cells in the logarithmic growth phase were inoculated in a 96-well plate at 1×10 4 Each group had 6 parallel holes. The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum in a 5% CO2, 37°C incubator for 24 h, then different concentrations of oxymatrine (0.5, 1, 2, 4, 8 μg / mL), astragaloside (0.5, 1, 2, 4, 8 μg / mL) and oxymatrine and astragaloside combination (mass ratio 1:1, 2:1, 4:1 and 8:1, with astragaloside concentration 0.5, 1, 2, 4, 8 μg / mL) were added, and incubated at 37°C, 5% CO2 for 24 h.

[0068] After incubation, 10 μL of CCK8 reagent was added to each well, and incubation was continued for 1.5 h. The absorbance at 450 nm was measured by a microplate reader. The relative cell activity was calculated (relative cell activity = (absorbance of drug-treated wells - absorbance of medium) / (absorbance of blank group - absorbance of medium)).

[0069] Results: The combination of oxymatrine and astragaloside in the ratio of 1:1 to 8:1 can improve the activity of mouse T cells, and when the ratio of the two is 1:1, 2:1 and 8:1, it can significantly improve the activity of mouse T cells (P<0.05). The use of oxymatrine or astragaloside alone did not show a significant effect on improving the activity of mouse T cells (e.g. Figure 2

[0070] The results show that the combination of oxymatrine and astragaloside has a synergistic effect on the activity of T cells.​

[0071] Example 2: Effect of different proportions and concentrations of oxymatrine and astragaloside composition on the activity of CAFs cells in mice

[0072] NIH3T3 cells in the logarithmic growth phase were inoculated in a 96-well plate at 1 x 10 4 ng / mL of recombinant mouse TGF-β1 protein to induce activation of CAFs, and 6 parallel holes were set in each group. After the cells were cultured in a 37°C incubator with 5% CO2 and DMEM medium containing 10% fetal bovine serum for 24 h, different concentrations of oxymatrine (0.5, 1, 2, 4, 8 μg / mL), astragaloside (0.5, 1, 2, 4, 8 μg / mL), and oxymatrine and astragaloside composition (mass ratio 1:1, 1:2, 4:1, and 8:1, with the concentration of astragaloside being 0.5, 1, 2, 4, 8 μg / mL) were added, and incubation was carried out at 37°C and 5% CO2 for 24 h.

[0073] After the incubation, 10 μL of CCK8 reagent was added to each well, and incubation was continued for 1.5 h. The absorbance at 450 nm was determined by a microplate reader. The relative cell activity was calculated (relative cell activity = (absorbance of the drug-treated hole - absorbance of the culture medium) / (absorbance of the blank group - absorbance of the culture medium)).

[0074] Results: The oxymatrine and astragaloside composition in the proportion of 1:1 to 8:1 can inhibit the activity of mouse CAFs cells, and when the proportion of the two is 1:1, 2:1, and 8:1, it can significantly inhibit the activity of mouse CAFs cells (P < 0.05). However, the use of oxymatrine or astragaloside alone does not show the effect of inhibiting the activity of mouse CAFs cells (as shown in Figure 3 ).

[0075] The results show that the oxymatrine and astragaloside composition has a synergistic inhibitory effect on the activity of CAFs cells.

[0076] According to the results of Example 1, the oxymatrine and astragaloside composition can enhance the activity of T cells while inhibiting the activity of CAFs cells, and the effect is best when the proportion of the two is 2:1, which can be used to synergistically enhance the anti-tumor effect of PD-1 inhibitors.

[0077] Example 3: Effect of different concentrations of oxymatrine-astragaloside composition on the activity of T lymphocytes in a 4T1 cell and CTLL-2 cell co-culture model

[0078] Logarithmic growth phase 4T1 cells were inoculated in a 96-well plate at 5 x 10 4Cells were seeded at 1 x 10 5 After 24h incubation in 5% CO2, 37℃ incubator, different concentrations of the combination of oxymatrine and astragaloside B (concentration of astragaloside B was 0.25, 0.5, 1, 2, 4 μg / mL) were added. After 24h incubation in 5% CO2, 37℃ incubator, 10 μL CCK8 reagent was added to each well, and incubated for another 1.5h. The absorbance at 450nm was measured by microplate reader. The relative cell viability was calculated (relative cell viability = (absorbance of drug group - absorbance of culture medium) / (absorbance of blank group - absorbance of culture medium)).

[0079] Results: The combination of oxymatrine and astragaloside B with a mass ratio of 2:1 could improve the activity of T cells co-cultured with 4T1 tumor cells, especially in the range of 0.25-4 μg / mL (calculated by the concentration of astragaloside B), which could significantly improve the activity of T cells co-cultured with 4T1 tumor cells (as shown in Figure 4 ).

[0080] The results showed that the combination of oxymatrine and astragaloside B could improve the activity of tumor-infiltrating T lymphocytes, and could be used to synergize with PD-1 inhibitors to enhance the anti-tumor effect.

[0081] Example 4 Preparation of oxymatrine-astragaloside B iron-based MOF system and determination of its physical and chemical properties

[0082] Take 1 part of FeCl3·6H2O, 2 parts of anhydrous sodium acetate, 2 parts of sodium acrylate, and 66 parts of diethylene glycol. Dissolve FeCl3·6H2O, anhydrous sodium acetate and sodium acrylate in diethylene glycol, and react in a reaction kettle at 190℃ for 10 hours. Centrifuge the reaction at 5500 rpm for 1h, wash with ethanol for 2 times, centrifuge at 5500 rpm for 1h, collect the precipitate, vacuum dry at 40℃ for 12h, and get Fe3O4 nanoparticles.

[0083] Take oxymatrine 264 parts, astragaloside B 132 parts, Fe3O4 nanoparticles 8 parts, trimesic acid 32 parts, platelet membrane 20 parts, 0.1 mmol / mL nitric acid and 0.003 mmol / mL hydrofluoric acid aqueous solution 20000 parts, methanol 86000 parts. Oxymatrine and astragaloside B are dissolved in 66000 parts of methanol, ready for use; take the platelet membrane and disperse it in 10000 parts of methanol, ultrasonic 200w for 10 min, ready for use; take Fe3O4 nanoparticles and trimesic acid, disperse in 0.1 mmol / L nitric acid and 0.003 mmol / L hydrofluoric acid aqueous solution, place in a reaction kettle lined with polytetrafluoroethylene, react at 150°C for 4 hours, then centrifuge the reaction at 5500 rpm for 10 min, wash with 60°C hot water and ethanol each for 2 times, centrifuge at 5500 rpm for 10 min, collect the precipitate, get the iron-based MOF blank carrier (Fe·MOF), Fe·MOF is activated at 150°C for 8h, placed in oxymatrine-astragaloside B methanol solution, 300 rpm mechanical stirring in the dark for 24h, 5500 rpm centrifugation for 1h, collect the precipitate part, disperse in 10000 parts of methanol, add the platelet membrane methanol dispersion drop by drop under ultrasonic state, ultrasonic 10 min, 12000 rpm centrifugation for 1h, the precipitate part is washed with PBS for 2 times and centrifuged at 5500 rpm for 1h, collect the precipitate, which is platelet membrane modified oxymatrine-astragaloside B Fe·MOF nanometer preparation, namely Pm-Om-As-Fe·MOF, HPLC determination of its loading oxymatrine drug loading is 21.83wt%, loading astragaloside B drug loading is 10.97wt%, the total drug loading is 32.80wt%.

[0084] The particle size distribution, zeta potential and SEM of the Fe·MOF nanometer preparation are shown in Figure 5 、 6 .

[0085] Example 5 Platelet membrane modified oxymatrine-astragaloside B iron-based MOF combined with PD-1 inhibitor synergistic drug pharmacodynamic experiment on liver cancer in situ

[0086] Take H 22 -Luc cells in the logarithmic growth phase, centrifuge at 1500 rpm for 4 min, resuspend the precipitate with matrigel to 1×10 8 / mL cell suspension, store in crushed ice, ready for use.

[0087] After the mice were pre-depilated with depilatory cream to remove hair in an oval area of 3 cm long and 2 cm wide centered on the chest, the residual depilatory cream was removed with cotton balls dipped in water, and the mice were fasted for 12 h without water.

[0088] The mice were anesthetized with isoflurane and placed on the operating table on their backs. The abdominal area of the mice was disinfected with 75% ethanol. A 1 cm long opening was cut along the abdominal white line below the chest to expose the abdominal cavity, and the liver was gently squeezed out of the incision using a cotton swab soaked in normal saline. The liver lobes were turned over to expose the inner side, and 10 μL of H 22 -Luc cell suspension, cotton swab compression for 30 s.

[0089] The skin on both sides of the incision was gently lifted with tweezers, the liver was returned to the abdominal cavity, and the wound was sutured and disinfected with iodophor, and 0.02 mL of gentamicin injection was injected intramuscularly. Ten days after the operation, the mice that failed to model were removed by small animal live imaging, and the remaining mice were randomly divided into a model control group, an Om-As group, a Pm-Om-As-Fe·MOF group (here, the Pm-Om-As-Fe·MOF nano-preparation of Example 4 was used), a Pm-Om-As-Fe·MOF combined with PD-1 group, and a PD-1 single drug group.

[0090] Pm-Om-As-Fe·MOF was dispersed in PBS to 3 mg / mL, Om-As was prepared into a solution containing oxymatrine 0.96 mg / mL and astragaloside 0.48 mg / mL (mass ratio 2:1) using 1% Tween 80, and PD-1 antibody was prepared into a 0.5 mg / mL solution using standby PBS.

[0091] The mice were administered 0.1 mL per mouse once a day by tail vein according to the grouping, and on the 3rd and 7th days, 0.2 mL of PD-1 inhibitor was administered intraperitoneally. After administration, the Pm-Om-As-Fe·MOF group and the Pm-Om-As-Fe·MOF combined with PD-1 group bound a magnet to the liver site, and the magnetic targeting was 2 h.

[0092] After 10 days of administration, the mice were sacrificed by cervical dislocation, and their in situ liver cancer was dissected, and the tumor weight, tumor volume, and tumor index were measured and analyzed, and the tumor tissue was immunofluorescently sectioned to observe the number and distribution of CD4 + , CD8 + T cells in the tissue.

[0093] The statistical results of the tumor weight, tumor volume, and tumor index of the mice in each group are shown in Table 1. Figure 7 The results show that the combination of Pm-Om-As-Fe·MOF and PD-1 inhibitor can significantly reduce the tumor weight, tumor volume, and tumor index of in situ liver cancer. The immunofluorescent sections of the tumor tissue of the mice in each group are shown in Figures 1-3. Figure 8 9 The results show that the combination of Pm-Om-As-Fe·MOF and PD-1 inhibitor can effectively increase the number and distribution of CD4 + ​CD8 + The level of T cells. It is shown that Pm-Om-As-Fe·MOF can synergistically improve the anti-liver cancer effect of PD-1 inhibitors.

[0094] Example 6: Synergistic pharmacodynamic experiment of oxymatrine and astragaloside B composition and liposome preparation combined with PD-1 inhibitor against antigen site breast cancer

[0095] Take 4T1-Luc cells in the logarithmic growth phase, centrifuge at 1500 rpm for 4 min, resuspend the precipitate in 1640 complete culture medium to 1×10 7 6 / mL of cell suspension, and store in crushed ice for standby.

[0096] After the mice were pre-depilated with depilatory cream to remove the residual depilatory cream using a cotton ball dipped in water, and the mice were fasted for 12 h without water.

[0097] After the mice were anesthetized with isoflurane, they were placed on the operating table in a supine position. The abdominal area of the mouse was disinfected with 75% ethanol, a small incision was made between the fourth nipple and the midline, the fat pad was found, and the fat pad was squeezed from the bottom with forceps to completely expose it. A 100 μL 4T1-Luc cell suspension was injected into the fat pad using a syringe, and the leakage was checked. The wound was sutured and disinfected with iodophor, and 0.02 mL of gentamicin injection was intramuscularly injected. Two days after the operation, the mice were randomly divided into the blank control group, the model control group, the Om-As low-dose group, the Om-As high-dose group, the PD-1 single-drug group, the Om-As low-dose combined with PD-1 group, and the Om-As-Lip combined with PD-1 group by observing whether the breast part was swollen.

[0098] The Om-As low-dose group used 1% Tween 80 to prepare a solution containing oxymatrine 0.250 mg / mL and astragaloside B 0.125 mg / mL (mass ratio 2:1); the Om-As high-dose group used 1% Tween 80 to prepare a solution containing oxymatrine 0.320 mg / mL and astragaloside B 0.160 mg / mL (mass ratio 2:1); the PD-1 antibody was prepared into a 0.5 mg / mL solution with standby PBS, and the Om-As-Lip was prepared into a nanodrug delivery system according to Example 8.

[0099] The mice were administered by tail vein according to the grouping, 0.1 mL per mouse, once a day, and on the 3rd and 7th days, 0.2 mL of PD-1 inhibitor was administered intraperitoneally.

[0100] After 9 days of administration, the mice were decapitated by cervical dislocation, and their orthotopic breast cancer was dissected, and the tumor weight, tumor volume and tumor index were measured and analyzed, and the tumor tissue was stained by HE, Ki 67, TUNEL and α-SMA to observe the anti-tumor effect.

[0101] The tumor weight, tumor volume and tumor index of mice in each group were as shown in Figure 10 The results showed that Om-As-Lip combined with PD-1 could significantly reduce the tumor weight, tumor volume and tumor index of orthotopic breast cancer, and the effect of low-dose naked drug combined with PD-1 was better than that of single low-dose naked drug or PD-1. H&E and Ki67 staining as shown in Figure 11 The nuclear area of tumor cells in the Om-As low-dose combined with PD-1 group was significantly reduced, and a large area of tissue necrosis area was observed. At the same time, the Ki67 positive cell area in the Om-As low-dose combined with PD-1 group was significantly reduced, and the proliferation ability of cancer cells was weak. The TUNEL staining results as shown in Figure 12 The apoptosis of tumor tissue was labeled, and the apoptosis level in the Om-As low-dose combined with PD-1 group was significantly increased, indicating that the cancer cells appeared obvious apoptosis after combined drug use. The α-SMA staining results as shown in Figure 13 Each group could reduce the expression of α-SMA protein, and the combination group was the most significant, and α-SMA was the activated characteristic protein of CAFs, indicating that the combination of oxymatrine and astragaloside could effectively inhibit the activation of CAFs combined with PD-1.

[0102] Example 7: Preparation of Fe·MOF co-loaded with oxymatrine and astragaloside

[0103] This example tested the influence of different prescriptions on Fe·MOF nanoforumulation

[0104] Prescription 1: 8 parts of Fe3O4 nanoparticles and 32 parts of trimesic acid are dispersed in 20000 parts of an aqueous solution containing 0.01 mmol / mL of nitric acid and 0.003 mmol / mL of hydrofluoric acid, in a reaction kettle, reacted at 150°C for 4h, centrifuged at 5500 rpm for 10 min, washed with hot water and ethanol at 60°C for 2 times, centrifuged at 5500 rpm for 10 min, and the precipitate is collected to obtain Fe·MOF. Take 264 parts of oxymatrine and 132 parts of astragaloside, dissolve in 66000 parts of methanol, and prepare for use; Fe·MOF is activated at 150°C for 8h, placed in the methanol solution of oxymatrine and astragaloside, mechanically stirred at 300 rpm in the dark for 24h, centrifuged at 5500 rpm for 1h, and the precipitate part is collected as the drug-loaded nano-preparation. Take 20 parts of platelet membrane, disperse in 10000 parts of methanol, and prepare for use; the drug-loaded nano-preparation is dispersed in 10000 parts of methanol, and added dropwise into the platelet membrane dispersion liquid under the condition of 200w ultrasonic, ultrasonic for 10 min, centrifuged at 12000 rpm for 1h, the precipitate part is washed with PBS for 2 times and centrifuged at 5500 rpm for 1h, and the precipitate is collected, which is the Fe·MOF nano-preparation co-loaded with oxymatrine and astragaloside.

[0105] Prescription 2: 8 parts of Fe3O4 nanoparticles and 32 parts of trimesic acid are dispersed in 20000 parts of an aqueous solution containing 0.01 mmol / mL of nitric acid and 0.003 mmol / mL of hydrofluoric acid, in a reaction kettle, reacted at 150°C for 4h, centrifuged at 5500 rpm for 10 min, washed with hot water and ethanol at 60°C for 2 times, centrifuged at 5500 rpm for 10 min, and the precipitate is collected to obtain Fe·MOF. Take 528 parts of oxymatrine and 264 parts of astragaloside, dissolve in 66000 parts of methanol, and prepare for use; Fe·MOF is activated at 150°C for 8h, placed in the methanol solution of oxymatrine and astragaloside, mechanically stirred at 300 rpm in the dark for 24h, centrifuged at 5500 rpm for 1h, and the precipitate part is collected as the drug-loaded nano-preparation. Take 20 parts of platelet membrane, disperse in 10000 parts of methanol, and prepare for use; the drug-loaded nano-preparation is dispersed in 10000 parts of methanol, and added dropwise into the platelet membrane dispersion liquid under the condition of 200w ultrasonic, ultrasonic for 10 min, centrifuged at 12000 rpm for 1h, the precipitate part is washed with PBS for 2 times and centrifuged at 5500 rpm for 1h, and the precipitate is collected, which is the Fe·MOF nano-preparation co-loaded with oxymatrine and astragaloside.

[0106] Prescription 3: 8 parts of Fe3O4 nanoparticles and 32 parts of trimesic acid were dispersed in 20000 parts of an aqueous solution containing 0.01 mmol / mL of nitric acid and 0.003 mmol / mL of hydrofluoric acid, and reacted in a reaction kettle at 150°C for 4h, centrifuged at 5500 rpm for 10 min, washed with hot water and ethanol at 60°C for 2 times, centrifuged at 5500 rpm for 10 min, and the precipitate was collected to obtain Fe·MOF. 396 parts of oxymatrine and 198 parts of astragaloside were dissolved in 66000 parts of methanol for standby; Fe·MOF was activated at 150°C for 8h, and then placed in the methanol solution of oxymatrine and astragaloside, and mechanically stirred at 300 rpm in the dark for 24h, and then centrifuged at 5500 rpm for 1h, and the precipitate was collected as the drug-loaded nano-preparation. 20 parts of platelet membranes were dispersed in 10000 parts of methanol for standby; the drug-loaded nano-preparation was dispersed in 10000 parts of methanol, and then added dropwise into the platelet membrane dispersion liquid under the condition of 200w ultrasonic, and ultrasonic was performed for 10 min, and then centrifuged at 12000 rpm for 1h, and the precipitate was washed with PBS for 2 times and centrifuged at 5500 rpm for 1h, and then the precipitate was collected, which was the Fe·MOF nano-preparation co-loaded with oxymatrine and astragaloside.

[0107] Prescription 4: 8 parts of Fe3O4 nanoparticles and 32 parts of trimesic acid were dispersed in 20000 parts of an aqueous solution containing 0.01 mmol / mL of nitric acid and 0.003 mmol / mL of hydrofluoric acid, and reacted in a reaction kettle at 150°C for 4h, centrifuged at 5500 rpm for 10 min, washed with hot water and ethanol at 60°C for 2 times, centrifuged at 5500 rpm for 10 min, and the precipitate was collected to obtain Fe·MOF. 132 parts of oxymatrine and 66 parts of astragaloside were dissolved in 66000 parts of methanol for standby; Fe·MOF was activated at 150°C for 8h, and then placed in the methanol solution of oxymatrine and astragaloside, and mechanically stirred at 300 rpm in the dark for 24h, and then centrifuged at 5500 rpm for 1h, and the precipitate was collected as the drug-loaded nano-preparation. 20 parts of platelet membranes were dispersed in 10000 parts of methanol for standby; the drug-loaded nano-preparation was dispersed in 10000 parts of methanol, and then added dropwise into the platelet membrane dispersion liquid under the condition of 200w ultrasonic, and ultrasonic was performed for 10 min, and then centrifuged at 12000 rpm for 1h, and the precipitate was washed with PBS for 2 times and centrifuged at 5500 rpm for 1h, and then the precipitate was collected, which was the Fe·MOF nano-preparation co-loaded with oxymatrine and astragaloside.

[0108] Prescription 5: 8 parts of Fe3O4 nanoparticles and 32 parts of trimesic acid were dispersed in 20000 parts of an aqueous solution containing 0.01 mmol / mL of nitric acid and 0.003 mmol / mL of hydrofluoric acid, and reacted in a reaction kettle at 150°C for 4h, centrifuged at 5500 rpm for 10 min, washed with hot water and ethanol at 60°C for 2 times, centrifuged at 5500 rpm for 10 min, and the precipitate was collected to obtain Fe·MOF. 264 parts of oxymatrine and 132 parts of astragaloside were dissolved in 66000 parts of methanol for standby use; Fe·MOF was activated at 150°C for 8h, and then placed in the methanol solution of oxymatrine and astragaloside, and mechanically stirred at 300 rpm in the dark for 24h, and then centrifuged at 5500 rpm for 1h, and the precipitate was collected as a drug-loaded nano-preparation. 20 parts of liver cancer cell membrane was dispersed in 10000 parts of methanol for standby use; the drug-loaded nano-preparation was dispersed in 10000 parts of methanol, and then added dropwise into the liver cancer cell membrane dispersion liquid under the condition of 200w ultrasonic state, and ultrasonic treatment was performed for 10 min, and then centrifuged at 12000 rpm for 1h, and then the precipitate was washed with PBS for 2 times and centrifuged at 5500 rpm for 1h, and then the precipitate was collected, which was a Fe·MOF nano-preparation co-loading oxymatrine and astragaloside.

[0109] Prescription 6: 8 parts of Fe3O4 nanoparticles and 32 parts of trimesic acid were dispersed in 20000 parts of an aqueous solution containing 0.01 mmol / mL of nitric acid and 0.003 mmol / mL of hydrofluoric acid, and reacted in a reaction kettle at 150°C for 4h, centrifuged at 5500 rpm for 10 min, washed with hot water and ethanol at 60°C for 2 times, centrifuged at 5500 rpm for 10 min, and the precipitate was collected to obtain Fe·MOF. 264 parts of oxymatrine and 132 parts of astragaloside were dissolved in 66000 parts of methanol for standby use; Fe·MOF was activated at 150°C for 8h, and then placed in the methanol solution of oxymatrine and astragaloside, and mechanically stirred at 300 rpm in the dark for 24h, and then centrifuged at 5500 rpm for 1h, and the precipitate was collected as a drug-loaded nano-preparation. 20 parts of red blood cell membrane was dispersed in 10000 parts of methanol for standby use; the drug-loaded nano-preparation was dispersed in 10000 parts of methanol, and then added dropwise into the red blood cell membrane dispersion liquid under the condition of 200w ultrasonic state, and ultrasonic treatment was performed for 10 min, and then centrifuged at 12000 rpm for 1h, and then the precipitate was washed with PBS for 2 times and centrifuged at 5500 rpm for 1h, and then the precipitate was collected, which was a Fe·MOF nano-preparation co-loading oxymatrine and astragaloside.

[0110] Prescription 7: 8 parts of Fe3O4 nanoparticles and 32 parts of trimesic acid were dispersed in 20000 parts of an aqueous solution containing 0.01 mmol / mL nitric acid and 0.003 mmol / mL hydrofluoric acid, and reacted in a reaction kettle at 150°C for 4h, centrifuged at 5500 rpm for 10 min, washed with hot water and ethanol at 60°C for 2 times, centrifuged at 5500 rpm for 10 min, and the precipitate was collected to obtain Fe·MOF. 264 parts of oxymatrine and 132 parts of astragaloside were dissolved in 66000 parts of methanol for standby use; Fe·MOF was activated at 150°C for 8h, and then placed in the methanol solution of oxymatrine and astragaloside, and mechanically stirred at 300 rpm in the dark for 24h, and then centrifuged at 5500 rpm for 1h, and the precipitate was collected as a drug-loaded nano-preparation. 10 parts of platelet membranes were dispersed in 10000 parts of methanol for standby use; the drug-loaded nano-preparation was dispersed in 10000 parts of methanol, and then added dropwise into the platelet membrane dispersion liquid under the condition of 200w ultrasonic, and ultrasonic was performed for 10 min, and then centrifuged at 12000 rpm for 1h, and the precipitate was washed with PBS for 2 times and centrifuged at 5500 rpm for 1h, and then the precipitate was collected, which was the Fe·MOF nano-preparation co-loaded with oxymatrine and astragaloside.

[0111] Prescription 8: 8 parts of Fe3O4 nanoparticles and 32 parts of trimesic acid were dispersed in 20000 parts of an aqueous solution containing 0.01 mmol / mL nitric acid and 0.003 mmol / mL hydrofluoric acid, and reacted in a reaction kettle at 150°C for 4h, centrifuged at 5500 rpm for 10 min, washed with hot water and ethanol at 60°C for 2 times, centrifuged at 5500 rpm for 10 min, and the precipitate was collected to obtain Fe·MOF. 264 parts of oxymatrine and 132 parts of astragaloside were dissolved in 66000 parts of methanol for standby use; Fe·MOF was activated at 150°C for 8h, and then placed in the methanol solution of oxymatrine and astragaloside, and mechanically stirred at 300 rpm in the dark for 24h, and then centrifuged at 5500 rpm for 1h, and the precipitate was collected as a drug-loaded nano-preparation. 90 parts of platelet membranes were dispersed in 10000 parts of methanol for standby use; the drug-loaded nano-preparation was dispersed in 10000 parts of methanol, and then added dropwise into the platelet membrane dispersion liquid under the condition of 200w ultrasonic, and ultrasonic was performed for 10 min, and then centrifuged at 12000 rpm for 1h, and the precipitate was washed with PBS for 2 times and centrifuged at 5500 rpm for 1h, and then the precipitate was collected, which was the Fe·MOF nano-preparation co-loaded with oxymatrine and astragaloside.

[0112] Prescription 9: 8 parts of Fe3O4 nanoparticles and 32 parts of trimesic acid were dispersed in 20000 parts of an aqueous solution containing 0.01 mmol / mL of nitric acid and 0.003 mmol / mL of hydrofluoric acid, in a reaction kettle, reacted at 150°C for 4h, centrifuged at 5500 rpm for 10 min, washed with 60°C hot water and ethanol for 2 times respectively, centrifuged at 5500 rpm for 10 min, collected the precipitate to obtain Fe·MOF. Take 264 parts of oxymatrine and 132 parts of astragaloside, dissolved in 66000 parts of methanol, for standby use; Fe·MOF was activated at 150°C for 8h, placed in the methanol solution of oxymatrine and astragaloside, mechanically stirred at 300 rpm in the dark for 24h, centrifuged at 5500 rpm for 1h, collected the precipitate part as drug-loaded nano-preparation. Take 160 parts of platelet membrane, dispersed in 10000 parts of methanol, for standby use; the drug-loaded nano-preparation was dispersed in 10000 parts of methanol, added dropwise into the platelet membrane dispersion liquid under the condition of 200w ultrasonic, ultrasonic for 10 min, centrifuged at 12000 rpm for 1h, the precipitate part was washed with PBS for 2 times and centrifuged at 5500 rpm for 1h, collected the precipitate, which was the Fe·MOF nano-preparation co-loaded with oxymatrine and astragaloside.

[0113] Prescription 10: 8 parts of Fe3O4 nanoparticles and 64 parts of trimesic acid were dispersed in 20000 parts of an aqueous solution containing 0.01 mmol / mL of nitric acid and 0.003 mmol / mL of hydrofluoric acid, in a reaction kettle, reacted at 150°C for 4h, centrifuged at 5500 rpm for 10 min, washed with 60°C hot water and ethanol for 2 times respectively, centrifuged at 5500 rpm for 10 min, collected the precipitate to obtain Fe·MOF. Take 264 parts of oxymatrine and 132 parts of astragaloside, dissolved in 66000 parts of methanol, for standby use; Fe·MOF was activated at 150°C for 8h, placed in the methanol solution of oxymatrine and astragaloside, mechanically stirred at 300 rpm in the dark for 24h, centrifuged at 5500 rpm for 1h, collected the precipitate part as drug-loaded nano-preparation. Take 20 parts of platelet membrane, dispersed in 10000 parts of methanol, for standby use; the drug-loaded nano-preparation was dispersed in 10000 parts of methanol, added dropwise into the platelet membrane dispersion liquid under the condition of 200w ultrasonic, ultrasonic for 10 min, centrifuged at 12000 rpm for 1h, the precipitate part was washed with PBS for 2 times and centrifuged at 5500 rpm for 1h, collected the precipitate, which was the Fe·MOF nano-preparation co-loaded with oxymatrine and astragaloside.

[0114] Prescription 11: 4 parts of Fe3O4 nanoparticles and 32 parts of trimesic acid are dispersed in 20000 parts of an aqueous solution containing 0.01 mmol / mL of nitric acid and 0.003 mmol / mL of hydrofluoric acid, in a reaction kettle, reacted at 150°C for 4h, centrifuged at 5500 rpm for 10 min, washed with hot water and ethanol at 60°C for 2 times, centrifuged at 5500 rpm for 10 min, and the precipitate is collected to obtain Fe·MOF. Take 264 parts of oxymatrine and 132 parts of astragaloside, dissolve in 66000 parts of methanol, and prepare for use; Fe·MOF is activated at 150°C for 8h, placed in the methanol solution of oxymatrine and astragaloside, mechanically stirred at 300 rpm in the dark for 24h, centrifuged at 5500 rpm for 1h, and the precipitate part is collected as the drug-loaded nano-preparation. Take 20 parts of platelet membrane, disperse in 10000 parts of methanol, and prepare for use; the drug-loaded nano-preparation is dispersed in 10000 parts of methanol, and added dropwise into the platelet membrane dispersion liquid under the condition of 200w ultrasonic, ultrasonic for 10 min, centrifuged at 12000 rpm for 1h, the precipitate part is washed with PBS for 2 times and centrifuged at 5500 rpm for 1h, and the precipitate is collected, which is the Fe·MOF nano-preparation co-loaded with oxymatrine and astragaloside.

[0115] Prescription 12: 8 parts of Fe3O4 nanoparticles and 32 parts of trimesic acid are dispersed in 20000 parts of an aqueous solution containing 0.01 mmol / mL of nitric acid and 0.003 mmol / mL of hydrofluoric acid, in a reaction kettle, reacted at 150°C for 4h, centrifuged at 5500 rpm for 10 min, washed with hot water and ethanol at 60°C for 2 times, centrifuged at 5500 rpm for 10 min, and the precipitate is collected to obtain Fe·MOF. Take 264 parts of oxymatrine and 264 parts of astragaloside, dissolve in 66000 parts of methanol, and prepare for use; Fe·MOF is activated at 150°C for 8h, placed in the methanol solution of oxymatrine and astragaloside, mechanically stirred at 300 rpm in the dark for 24h, centrifuged at 5500 rpm for 1h, and the precipitate part is collected as the drug-loaded nano-preparation. Take 20 parts of platelet membrane, disperse in 10000 parts of methanol, and prepare for use; the drug-loaded nano-preparation is dispersed in 10000 parts of methanol, and added dropwise into the platelet membrane dispersion liquid under the condition of 200w ultrasonic, ultrasonic for 10 min, centrifuged at 12000 rpm for 1h, the precipitate part is washed with PBS for 2 times and centrifuged at 5500 rpm for 1h, and the precipitate is collected, which is the Fe·MOF nano-preparation co-loaded with oxymatrine and astragaloside.

[0116] Prescription 13: 8 parts of Fe3O4 nanoparticles and 32 parts of trimesic acid were dispersed in 20000 parts of an aqueous solution containing 0.01 mmol / mL of nitric acid and 0.003 mmol / mL of hydrofluoric acid, and reacted in a reaction kettle at 150°C for 4h, centrifuged at 5500 rpm for 10 min, washed with hot water and ethanol at 60°C for 2 times, centrifuged at 5500 rpm for 10 min, and the precipitate was collected to obtain Fe·MOF. 528 parts of oxymatrine and 66 parts of astragaloside were dissolved in 66000 parts of methanol for standby; Fe·MOF was activated at 150°C for 8h, and then placed in the methanol solution of oxymatrine and astragaloside, and mechanically stirred at 300 rpm in the dark for 24h, centrifuged at 5500 rpm for 1h, and the precipitate was collected as the drug-loaded nano-preparation. 20 parts of platelet membranes were dispersed in 10000 parts of methanol for standby; the drug-loaded nano-preparation was dispersed in 10000 parts of methanol, and then added dropwise into the platelet membrane dispersion liquid under the condition of 200w ultrasonic, ultrasonic for 10 min, centrifuged at 12000 rpm for 1h, the precipitate was washed with PBS for 2 times and centrifuged at 5500 rpm for 1h, and the precipitate was collected as the Fe·MOF nano-preparation co-loaded with oxymatrine and astragaloside.

[0117] Prescription 14: 8 parts of Fe3O4 nanoparticles and 32 parts of trimesic acid were dispersed in 20000 parts of an aqueous solution containing 0.01 mmol / mL of nitric acid and 0.003 mmol / mL of hydrofluoric acid, and reacted in a reaction kettle at 150°C for 4h, centrifuged at 5500 rpm for 10 min, washed with hot water and ethanol at 60°C for 2 times, centrifuged at 5500 rpm for 10 min, and the precipitate was collected to obtain Fe·MOF. 264 parts of oxymatrine and 132 parts of astragaloside were dissolved in 66000 parts of methanol for standby; Fe·MOF was activated at 150°C for 8h, and then placed in the methanol solution of oxymatrine and astragaloside, and mechanically stirred at 300 rpm in the dark for 24h, centrifuged at 5500 rpm for 1h, and the precipitate was collected as the drug-loaded nano-preparation. 40 parts of hyaluronic acid was dissolved in 10000 parts of water, and then the drug-loaded nano-preparation was added, mechanically stirred at 300 rpm in the dark for 1h, centrifuged at 5500 rpm for 1h, and the precipitate was collected as the Fe·MOF nano-preparation co-loaded with oxymatrine and astragaloside.

[0118] Prescription 15: 4 parts of Fe3O4 nanoparticles were dispersed in 20000 parts of water, 65 parts of FeCl3·6H2O and 55 parts of trimesic acid were added, and the reaction kettle was reacted at 150°C for 4h, centrifuged at 5500 rpm for 10 min, washed with 60°C hot water and ethanol for 2 times, centrifuged at 5500 rpm for 10 min, and the precipitate was collected to obtain Fe·MOF. 264 parts of oxymatrine and 132 parts of astragaloside were dissolved in 66000 parts of methanol for standby; Fe·MOF was activated at 150°C for 8h, and was placed in the oxymatrine and astragaloside methanol solution, and was mechanically stirred at 300 rpm in the dark for 24h, and was centrifuged at 5500 rpm for 1h, and the precipitate part was collected as the drug-loaded nano-preparation. 20 parts of platelet membrane were dispersed in 10000 parts of methanol for standby; the drug-loaded nano-preparation was dispersed in 10000 parts of methanol, and was added dropwise into the platelet membrane dispersion liquid under the condition of 200w ultrasonic, and was ultrasonically treated for 10 min, and was centrifuged at 12000 rpm for 1h, and the precipitate part was washed with PBS for 2 times and centrifuged at 5500 rpm for 1h, and the precipitate was collected, which was the Fe·MOF nano-preparation co-loaded with oxymatrine and astragaloside.

[0119] Prescription 16: 4 parts of Fe3O4 nanoparticles were dispersed in 20000 parts of water, 97 parts of Fe(NO3)3·9H2O and 55 parts of trimesic acid were added, and the reaction kettle was reacted at 150°C for 4h, centrifuged at 5500 rpm for 10 min, washed with 60°C hot water and ethanol for 2 times, centrifuged at 5500 rpm for 10 min, and the precipitate was collected to obtain Fe·MOF. 264 parts of oxymatrine and 132 parts of astragaloside were dissolved in 66000 parts of methanol for standby; Fe·MOF was activated at 150°C for 8h, and was placed in the oxymatrine and astragaloside methanol solution, and was mechanically stirred at 300 rpm in the dark for 24h, and was centrifuged at 5500 rpm for 1h, and the precipitate part was collected as the drug-loaded nano-preparation. 20 parts of platelet membrane were dispersed in 10000 parts of methanol for standby; the drug-loaded nano-preparation was dispersed in 10000 parts of methanol, and was added dropwise into the platelet membrane dispersion liquid under the condition of 200w ultrasonic, and was ultrasonically treated for 10 min, and was centrifuged at 12000 rpm for 1h, and the precipitate part was washed with PBS for 2 times and centrifuged at 5500 rpm for 1h, and the precipitate was collected, which was the Fe·MOF nano-preparation co-loaded with oxymatrine and astragaloside.

[0120] Prescription 17: 16 parts of Fe3O4 nanoparticles and 46 parts of terephthalic acid were dispersed in 20000 parts of water, reacted in a reaction kettle at 150℃ for 4h, centrifuged at 5500 rpm for 10 min, washed with hot water and ethanol at 60℃ for 2 times, centrifuged at 5500 rpm for 10 min, and the precipitate was collected to obtain Fe·MOF. 264 parts of oxymatrine and 132 parts of astragaloside were dissolved in 66000 parts of methanol for standby; Fe·MOF was activated at 150℃ for 8h, placed in the oxymatrine and astragaloside methanol solution, mechanically stirred at 300 rpm in the dark for 24h, centrifuged at 5500 rpm for 1h, and the precipitate part was collected as the drug-loaded nano-preparation. 31 parts of platelet membrane was dispersed in 10000 parts of methanol for standby; the drug-loaded nano-preparation was dispersed in 10000 parts of methanol, and the platelet membrane dispersion liquid was added dropwise under the condition of 200w ultrasonic, ultrasonic for 10 min, centrifuged at 12000 rpm for 1h, the precipitate part was washed with PBS for 2 times and centrifuged at 5500 rpm for 1h, and the precipitate was collected as the Fe·MOF nano-preparation co-loaded with oxymatrine and astragaloside.

[0121] Prescription 18: 8 parts of Fe3O4 nanoparticles and 46 parts of 2-amino terephthalic acid were dispersed in 20000 parts of water, reacted in a reaction kettle at 150℃ for 4h, centrifuged at 5500 rpm for 10 min, washed with hot water and ethanol at 60℃ for 2 times, centrifuged at 5500 rpm for 10 min, and the precipitate was collected to obtain Fe·MOF. 264 parts of oxymatrine and 132 parts of astragaloside were dissolved in 66000 parts of methanol for standby; Fe·MOF was activated at 150℃ for 8h, placed in the oxymatrine and astragaloside methanol solution, mechanically stirred at 300 rpm in the dark for 24h, centrifuged at 5500 rpm for 1h, and the precipitate part was collected as the drug-loaded nano-preparation. 31 parts of platelet membrane was dispersed in 10000 parts of methanol for standby; the drug-loaded nano-preparation was dispersed in 10000 parts of methanol, and the platelet membrane dispersion liquid was added dropwise under the condition of 200w ultrasonic, ultrasonic for 10 min, centrifuged at 12000 rpm for 1h, the precipitate part was washed with PBS for 2 times and centrifuged at 5500 rpm for 1h, and the precipitate was collected as the Fe·MOF nano-preparation co-loaded with oxymatrine and astragaloside.

[0122] Prescription 19: 8 parts of Fe3O4 nanoparticles and 32 parts of trimesic acid are dispersed in 20000 parts of an aqueous solution containing 0.01 mmol / mL of nitric acid and 0.003 mmol / mL of hydrofluoric acid, in a reaction kettle, and reacted at 150°C for 4h, centrifuged at 5500 rpm for 10 min, washed with hot water and ethanol at 60°C for 2 times, centrifuged at 5500 rpm for 10 min, and the precipitate is collected to obtain Fe·MOF. 528 parts of oxymatrine and 264 parts of astragaloside are dissolved in 33000 parts of methanol for standby use; Fe·MOF is activated at 150°C for 8h, and placed in the methanol solution of oxymatrine and astragaloside, and mechanically stirred at 300 rpm in the dark for 24h, centrifuged at 5500 rpm for 1h, and the precipitate part is collected as a drug-loaded nano-preparation. 20 parts of platelet membranes are dispersed in 10000 parts of methanol for standby use; the drug-loaded nano-preparation is dispersed in 10000 parts of methanol, and added dropwise into the platelet membrane dispersion liquid under the condition of 200w ultrasonic, ultrasonic for 10 min, centrifuged at 12000 rpm for 1h, the precipitate part is washed with PBS for 2 times and centrifuged at 5500 rpm for 1h, and the precipitate is collected as a Fe·MOF nano-preparation co-loaded with oxymatrine and astragaloside.

[0123] Prescription 20: 8 parts of Fe3O4 nanoparticles and 32 parts of trimesic acid are dispersed in 20000 parts of an aqueous solution containing 0.01 mmol / mL of nitric acid and 0.003 mmol / mL of hydrofluoric acid, in a reaction kettle, and reacted at 150°C for 4h, centrifuged at 5500 rpm for 10 min, washed with hot water and ethanol at 60°C for 2 times, centrifuged at 5500 rpm for 10 min, and the precipitate is collected to obtain Fe·MOF. 132 parts of oxymatrine and 66 parts of astragaloside are dissolved in 132000 parts of methanol for standby use; Fe·MOF is activated at 150°C for 8h, and placed in the methanol solution of oxymatrine and astragaloside, and mechanically stirred at 300 rpm in the dark for 24h, centrifuged at 5500 rpm for 1h, and the precipitate part is collected as a drug-loaded nano-preparation. 20 parts of platelet membranes are dispersed in 10000 parts of methanol for standby use; the drug-loaded nano-preparation is dispersed in 10000 parts of methanol, and added dropwise into the platelet membrane dispersion liquid under the condition of 200w ultrasonic, ultrasonic for 10 min, centrifuged at 12000 rpm for 1h, the precipitate part is washed with PBS for 2 times and centrifuged at 5500 rpm for 1h, and the precipitate is collected as a Fe·MOF nano-preparation co-loaded with oxymatrine and astragaloside.

[0124] The results show that in the traditional Chinese medicine active ingredient composition 198-792 parts, metal nodes 4-101 parts, organic ligand 32-64 parts, surface modification material 10-160 parts; under the condition that the mass ratio concentration of solute and solvent in the solution with the traditional Chinese medicine active ingredient composition as the solute is 0.15-2.4% (w / w), the Fe·MOF nano system co-loaded with oxymatrine and astragaloside can be prepared.

[0125] Example 8: Preparation of liposome nanoscale preparation co-loading oxymatrine and astragaloside and determination of physicochemical properties

[0126] Take 120 parts of soybean lecithin, 180 parts of hydrogenated soybean lecithin, 50 parts of cholesterol, 14 parts of DSPE-PEG2000, and 20 parts of astragaloside, and place them in a 5 mL test tube. Add 20 parts of anhydrous ethanol to dissolve to a clear and transparent state, and use as the oil phase. Take 50 parts of 150 mmol / L citric acid solution and place it in a 20 mL test tube, and use as the water phase. Under magnetic stirring at 800 rpm and 50°C, use a 1 mL syringe to slowly and uniformly inject the oil phase into the water phase, and stir for 30 min. Under vacuum rotary evaporation at 50°C and 30 rpm, remove the anhydrous ethanol, and probe ice bath ultrasonic for 10 min, which is the astragaloside liposome. Add 10 parts of 50 mmol / L HEPES buffer solution and 40 parts of 300 mmol / L Na2CO3 solution (pre-dissolved with 20 parts of oxymatrine), and incubate for 30 min in a 50°C water bath, which is the co-loading liposome of oxymatrine and astragaloside. Determine the particle size and potential by DLS. The oxymatrine encapsulation rate is 60.34, the drug loading is 1.96%, the astragaloside encapsulation rate is 95.60%, the drug loading is 5.38%, and the total drug loading is 7.34%.

[0127] Results: The particle size distribution and potential of the co-loading liposome of oxymatrine and astragaloside are shown in Table 1. Figure 14

[0128] Example 9: Preparation of liposome co-loading oxymatrine and astragaloside (Om-As-Lip)

[0129] This example tests the influence of different formulations on the liposome nanoscale preparation. Preparation method:

[0130] Formulation 1 (membrane dispersion method-pH gradient method): Take 300 parts of egg yolk lecithin, 75 parts of cholesterol, 14 parts of DSPE-PEG2000, and 10 parts of astragaloside, and place them in a 100 mL test tube. Add 20 parts of anhydrous ethanol to dissolve to a clear and transparent state, and remove the anhydrous ethanol under vacuum rotary evaporation at 50°C and 60 rpm to form a uniform lipid film layer. Add 50 parts of 150 mmol / L citric acid solution, and mechanically stir at 100 rpm and 50°C. Hydrate for 1 h, and ice bath probe ultrasonic for 10 min to form the astragaloside liposome. Add 10 parts of 50 mmol / L HEPES buffer solution and 40 parts of 300 mmol / L Na2CO3 solution (pre-dissolved with 10 parts of oxymatrine), and incubate for 30 min in a 50°C water bath, which is the co-loading liposome of oxymatrine and astragaloside.

[0131] ​Prescription 2 (ethanol injection method-pH gradient method): Take 300 parts of egg yolk lecithin, 75 parts of cholesterol, 14 parts of DSPE-PEG2000, and 20 parts of astragaloside A in a 5 mL vial, add 20 parts of anhydrous ethanol to dissolve to a clear and transparent state, and use as an oil phase. Take 50 parts of 150 mmol / L citric acid solution in a 20 mL vial as an aqueous phase. Under magnetic stirring at 800 rpm at 50°C, use a 1 mL syringe to slowly and uniformly inject the oil phase into the aqueous phase, and stir for 30 min. Remove the anhydrous ethanol under vacuum rotary evaporation at 50°C and 30 rpm, and sonicate for 10 min in an ice bath, to obtain astragaloside A liposomes. Add 10 parts of 50 mmol / L HEPES buffer solution and 40 parts of 300 mmol / L Na2CO3 solution (pre-dissolved with 10 parts of oxymatrine) to the astragaloside A liposomes, and incubate at 50°C in a water bath for 30 min, to obtain oxymatrine and astragaloside A co-loaded liposomes.

[0132] Prescription 3: Take 150 parts of soybean lecithin, 150 parts of hydrogenated soybean lecithin, 50 parts of cholesterol, 14 parts of DSPE-PEG2000, and 50 parts of astragaloside A in a 5 mL vial, add 20 parts of anhydrous ethanol to dissolve to a clear and transparent state, and use as an oil phase. Take 50 parts of 150 mmol / L citric acid solution in a 20 mL vial as an aqueous phase. Under magnetic stirring at 800 rpm at 50°C, use a 1 mL syringe to slowly and uniformly inject the oil phase into the aqueous phase, and stir for 30 min. Remove the anhydrous ethanol under vacuum rotary evaporation at 50°C and 30 rpm, and sonicate for 10 min in an ice bath, to obtain astragaloside A liposomes. Add 10 parts of 50 mmol / L HEPES buffer solution and 40 parts of 300 mmol / L Na2CO3 solution (pre-dissolved with 50 parts of oxymatrine) to the astragaloside A liposomes, and incubate at 50°C in a water bath for 30 min, to obtain oxymatrine and astragaloside A co-loaded liposomes.

[0133] Prescription 4: Take 150 parts of soybean lecithin, 150 parts of hydrogenated soybean lecithin, 50 parts of cholesterol, 14 parts of DSPE-PEG2000, and 10 parts of astragaloside A in a 5 mL vial, add 20 parts of anhydrous ethanol to dissolve to a clear and transparent state, and use as an oil phase. Take 50 parts of 150 mmol / L citric acid solution in a 20 mL vial as an aqueous phase. Under magnetic stirring at 800 rpm at 50°C, use a 1 mL syringe to slowly and uniformly inject the oil phase into the aqueous phase, and stir for 30 min. Remove the anhydrous ethanol under vacuum rotary evaporation at 50°C and 30 rpm, and sonicate for 10 min in an ice bath, to obtain astragaloside A liposomes. Add 10 parts of 50 mmol / L HEPES buffer solution and 40 parts of 300 mmol / L Na2CO3 solution (pre-dissolved with 10 parts of oxymatrine) to the astragaloside A liposomes, and incubate at 50°C in a water bath for 30 min, to obtain oxymatrine and astragaloside A co-loaded liposomes.

[0134] Prescription 5: Soybean lecithin 150 parts, hydrogenated soybean lecithin 150 parts, cholesterol 50 parts, DSPE-PEG2000 14 parts, astragaloside 30 parts are placed in a 5 mL vial, add 20 parts of anhydrous ethanol to dissolve to clear and transparent, as the oil phase for standby. Take 150 mmol / L citric acid solution 50 parts in a 20 mL vial, as the water phase for standby. Under 50℃, 800 rpm magnetic stirring, using 1 mL syringe, the oil phase is slowly and evenly injected into the water phase, stirring for 30 min. 50℃, 30 rpm under vacuum rotary evaporation to remove anhydrous ethanol, probe ice bath ultrasonic 10 min, which is astragaloside liposome. Add 50 mmol / L HEPES buffer solution 10 parts, 300 mmol / L Na2CO3 solution (pre-dissolved with 30 parts of oxymatrine) 40 parts, 50℃ water bath incubation for 30 min, which is oxymatrine and astragaloside co-loaded liposome.

[0135] Prescription 6: Soybean lecithin 150 parts, hydrogenated soybean lecithin 150 parts, cholesterol 50 parts, DSPE-PEG2000 14 parts, astragaloside 40 parts are placed in a 5 mL vial, add 20 parts of anhydrous ethanol to dissolve to clear and transparent, as the oil phase for standby. Take 150 mmol / L citric acid solution 50 parts in a 20 mL vial, as the water phase for standby. Under 50℃, 800 rpm magnetic stirring, using 1 mL syringe, the oil phase is slowly and evenly injected into the water phase, stirring for 30 min. 50℃, 30 rpm under vacuum rotary evaporation to remove anhydrous ethanol, probe ice bath ultrasonic 10 min, which is astragaloside liposome. Add 50 mmol / L HEPES buffer solution 10 parts, 300 mmol / L Na2CO3 solution (pre-dissolved with 40 parts of oxymatrine) 40 parts, 50℃ water bath incubation for 30 min, which is oxymatrine and astragaloside co-loaded liposome.

[0136] Prescription 7: Soybean lecithin 100 parts, hydrogenated soybean lecithin 200 parts, cholesterol 50 parts, DSPE-PEG2000 14 parts, astragaloside 20 parts are placed in a 5 mL vial, add 20 parts of anhydrous ethanol to dissolve to clear and transparent, as the oil phase for standby. Take 150 mmol / L citric acid solution 50 parts in a 20 mL vial, as the water phase for standby. Under 50℃, 800 rpm magnetic stirring, using 1 mL syringe, the oil phase is slowly and evenly injected into the water phase, stirring for 30 min. 50℃, 30 rpm under vacuum rotary evaporation to remove anhydrous ethanol, probe ice bath ultrasonic 10 min, which is astragaloside liposome. Add 50 mmol / L HEPES buffer solution 10 parts, 300 mmol / L Na2CO3 solution (pre-dissolved with 20 parts of oxymatrine) 40 parts, 50℃ water bath incubation for 30 min, which is oxymatrine and astragaloside co-loaded liposome.

[0137] Prescription 8: Soybean lecithin 150 parts, hydrogenated soybean lecithin 150 parts, cholesterol 50 parts, DSPE-PEG2000 14 parts, astragaloside A 20 parts were placed in a 5 mL vial, 20 parts of anhydrous ethanol was added to dissolve to clear and transparent, as the oil phase for standby. Take 50 parts of 150 mmol / L citric acid solution in a 20 mL vial, as the water phase for standby. Under 50℃, 800 rpm magnetic stirring, using a 1 mL syringe, the oil phase was slowly and evenly injected into the water phase, stirring for 30 min. 50℃, 30 rpm under vacuum rotary evaporation to remove anhydrous ethanol, probe ice bath ultrasonic 10 min, which is astragaloside A liposome. Add 50 mmol / L HEPES buffer solution 10 parts, 300 mmol / L Na2CO3 solution (pre-dissolved with 20 parts of oxymatrine) 40 parts, 50℃ water bath incubation for 30 min, which is oxymatrine and astragaloside A co-loaded liposome.

[0138] Prescription 9: Soybean lecithin 180 parts, hydrogenated soybean lecithin 120 parts, cholesterol 50 parts, DSPE-PEG2000 14 parts, astragaloside A 20 parts were placed in a 5 mL vial, 20 parts of anhydrous ethanol was added to dissolve to clear and transparent, as the oil phase for standby. Take 50 parts of 150 mmol / L citric acid solution in a 20 mL vial, as the water phase for standby. Under 50℃, 800 rpm magnetic stirring, using a 1 mL syringe, the oil phase was slowly and evenly injected into the water phase, stirring for 30 min. 50℃, 30 rpm under vacuum rotary evaporation to remove anhydrous ethanol, probe ice bath ultrasonic 10 min, which is astragaloside A liposome. Add 50 mmol / L HEPES buffer solution 10 parts, 300 mmol / L Na2CO3 solution (pre-dissolved with 20 parts of oxymatrine) 40 parts, 50℃ water bath incubation for 30 min, which is oxymatrine and astragaloside A co-loaded liposome.

[0139] Prescription 10: Soybean lecithin 200 parts, hydrogenated soybean lecithin 100 parts, cholesterol 50 parts, DSPE-PEG2000 14 parts, astragaloside A 20 parts were placed in a 5 mL vial, 20 parts of anhydrous ethanol was added to dissolve to clear and transparent, as the oil phase for standby. Take 50 parts of 150 mmol / L citric acid solution in a 20 mL vial, as the water phase for standby. Under 50℃, 800 rpm magnetic stirring, using a 1 mL syringe, the oil phase was slowly and evenly injected into the water phase, stirring for 30 min. 50℃, 30 rpm under vacuum rotary evaporation to remove anhydrous ethanol, probe ice bath ultrasonic 10 min, which is astragaloside A liposome. Add 50 mmol / L HEPES buffer solution 10 parts, 300 mmol / L Na2CO3 solution (pre-dissolved with 40 parts of oxymatrine) 40 parts, 50℃ water bath incubation for 30 min, which is oxymatrine and astragaloside A co-loaded liposome.

[0140] Prescription 11: Soybean lecithin 150 parts, hydrogenated soybean lecithin 150 parts, cholesterol 50 parts, DSPE-PEG2000 14 parts, astragaloside 5 parts are placed in a 5 mL vial, add 20 parts of anhydrous ethanol to dissolve to clear and transparent, as the oil phase for standby. Take 50 parts of 150 mmol / L citric acid solution in a 20 mL vial, as the water phase for standby. Under 50℃, 800 rpm magnetic stirring, using a 1 mL syringe, the oil phase is slowly and evenly injected into the water phase, stirring for 30 min. 50℃, 30 rpm under vacuum rotary evaporation to remove anhydrous ethanol, probe ice bath ultrasonic 10 min, which is astragaloside liposome. Add 50 mmol / L HEPES buffer solution 10 parts, 300 mmol / L Na2CO3 solution (pre-dissolved with 10 parts of oxymatrine) 40 parts, 50℃ water bath incubation for 30 min, which is oxymatrine and astragaloside co-loaded liposome.

[0141] Prescription 12: Soybean lecithin 150 parts, hydrogenated soybean lecithin 150 parts, cholesterol 50 parts, DSPE-PEG2000 14 parts, astragaloside 10 parts are placed in a 5 mL vial, add 20 parts of anhydrous ethanol to dissolve to clear and transparent, as the oil phase for standby. Take 50 parts of 150 mmol / L citric acid solution in a 20 mL vial, as the water phase for standby. Under 50℃, 800 rpm magnetic stirring, using a 1 mL syringe, the oil phase is slowly and evenly injected into the water phase, stirring for 30 min. 50℃, 30 rpm under vacuum rotary evaporation to remove anhydrous ethanol, probe ice bath ultrasonic 10 min, which is astragaloside liposome. Add 50 mmol / L HEPES buffer solution 10 parts, 300 mmol / L Na2CO3 solution (pre-dissolved with 10 parts of oxymatrine) 40 parts, 50℃ water bath incubation for 30 min, which is oxymatrine and astragaloside co-loaded liposome.

[0142] Prescription 13: Soybean lecithin 500 parts, hydrogenated soybean lecithin 100 parts, cholesterol 50 parts, DSPE-PEG2000 14 parts, astragaloside 20 parts are placed in a 5 mL vial, add 20 parts of anhydrous ethanol to dissolve to clear and transparent, as the oil phase for standby. Take 50 parts of 150 mmol / L citric acid solution in a 20 mL vial, as the water phase for standby. Under 50℃, 800 rpm magnetic stirring, using a 1 mL syringe, the oil phase is slowly and evenly injected into the water phase, stirring for 30 min. 50℃, 30 rpm under vacuum rotary evaporation to remove anhydrous ethanol, probe ice bath ultrasonic 10 min, which is astragaloside liposome. Add 50 mmol / L HEPES buffer solution 10 parts, 300 mmol / L Na2CO3 solution (pre-dissolved with 20 parts of oxymatrine) 40 parts, 50℃ water bath incubation for 30 min, which is oxymatrine and astragaloside co-loaded liposome.

[0143] Formulation 14: Soybean phosphatidylcholine 450 parts, hydrogenated soybean phosphatidylcholine 150 parts, cholesterol 50 parts, DSPE-PEG2000 14 parts, astragaloside 20 parts were placed in a 5 mL vial, 20 parts of anhydrous ethanol was added to dissolve to clear and transparent, as the oil phase ready for use. Take 50 parts of 150 mmol / L citric acid solution was placed in a 20 mL vial, as the water phase ready for use. Under 50℃, 800 rpm magnetic stirring, using a 1 mL syringe oil phase uniform slow injection into the water phase, stirring 30 min. 50℃, 30 rpm under vacuum rotary evaporation to remove anhydrous ethanol, probe ice bath ultrasonic 10 min, namely astragaloside liposome. Add 50 mmol / L HEPES buffer solution 10 parts, 300 mmol / L Na2CO3 solution (pre dissolved with 20 parts of oxymatrine) 40 parts, 50℃ water bath incubation for 30 min, namely oxymatrine and astragaloside co loaded liposome.

[0144] Formulation 15: Soybean phosphatidylcholine 400 parts, hydrogenated soybean phosphatidylcholine 200 parts, cholesterol 50 parts, DSPE-PEG2000 14 parts, astragaloside 20 parts were placed in a 5 mL vial, 20 parts of anhydrous ethanol was added to dissolve to clear and transparent, as the oil phase ready for use. Take 50 parts of 150 mmol / L citric acid solution was placed in a 20 mL vial, as the water phase ready for use. Under 50℃, 800 rpm magnetic stirring, using a 1 mL syringe oil phase uniform slow injection into the water phase, stirring 30 min. 50℃, 30 rpm under vacuum rotary evaporation to remove anhydrous ethanol, probe ice bath ultrasonic 10 min, namely astragaloside liposome. Add 50 mmol / L HEPES buffer solution 10 parts, 300 mmol / L Na2CO3 solution (pre dissolved with 20 parts of oxymatrine) 40 parts, 50℃ water bath incubation for 30 min, namely oxymatrine and astragaloside co loaded liposome.

[0145] Formulation 16: Soybean phosphatidylcholine 300 parts, cholesterol 30 parts, DSPE-PEG2000 20 parts, astragaloside 60 parts were placed in a 5 mL vial, 20 parts of anhydrous ethanol was added to dissolve to clear and transparent, as the oil phase ready for use. Take 50 parts of 50 mmol / L citric acid solution was placed in a 20 mL vial, as the water phase ready for use. Under 50℃, 800 rpm magnetic stirring, using a 1 mL syringe oil phase uniform slow injection into the water phase, stirring 30 min. 50℃, 30 rpm under vacuum rotary evaporation to remove anhydrous ethanol, probe ice bath ultrasonic 10 min, namely astragaloside liposome. Add 50 mmol / L HEPES buffer solution 10 parts, 300 mmol / L Na2CO3 solution (pre dissolved with 60 parts of oxymatrine) 40 parts, 50℃ water bath incubation for 30 min, namely oxymatrine and astragaloside co loaded liposome.

[0146] Results show that: thin film dispersion method-pH gradient method, ethanol injection method-pH gradient method, the active ingredient composition of traditional Chinese medicine 15-120 parts, natural phospholipid 100-500 parts, synthetic phospholipid 100-240 parts, cholesterol (membrane fluidity regulator) 30-75 parts, DSPE-PEG 2000 ( surface modifier) 14-20 parts, the oxymatrine and astragaloside B co-loaded liposome drug delivery system can be prepared in the range.

[0147] Example 10 Effect of co-loaded oxymatrine and astragaloside B liposome nanomedicine on CTLL-2 mouse T cell activity

[0148] Take the well-grown CTLL-2 cells and inoculate them in 96-well plates (1×10 4 / well), and incubate them at 37℃, 5% CO2 for 24h. Om-As-Lip (here, the Om-As-Lip of Example 8 is used) is filtered through a 0.22μm filter, the supernatant in the plate is discarded, and the drug solution is added by doubling dilution. Five holes are prepared in parallel, and blank holes and control holes are reserved. Incubate at 37℃, 5% CO2 for 24h.

[0149] After incubation, add 10μL CCK8 reagent to each well, continue to incubate for 2.5h, and then measure the absorbance at 450nm by enzyme label instrument. Calculate the relative cell activity.

[0150] [Relative cell activity = (absorbance of dosing hole - absorbance of culture medium) / (absorbance of blank group - absorbance of culture medium)]

[0151] Results: The co-loaded oxymatrine and astragaloside B liposome can significantly improve the activity of CTLL-2 mouse T cells in the concentration range of 0.25-7.5μg / mL (calculated by the concentration of astragaloside B). Figure 15

[0152] Example 11 Investigation on the inhibition of CAF activation by co-loaded oxymatrine and astragaloside B liposome nanomedicine

[0153] Take the well-grown NIH3T3 cells and inoculate them in 96-well plates (2.5×10 3 / well), and incubate them at 37℃, 5% CO2 for 24h. As-Om-Lip (here, the As-Om-Lip of Example 9- prescription 9 is used) is filtered through a 0.22μm filter, the supernatant in the plate is discarded, and the drug solution is added by doubling dilution. Five holes are prepared in parallel, and blank holes and control holes are reserved. Mix well and incubate for 1h, then add TGF-β1 (final concentration 5ng / mL) and incubate at 37℃, 5% CO2 for 24h.

[0154] ​After incubation, 10 μL CCK8 reagent was added to each well, and the absorbance at 450 nm was measured by a microplate reader after 1.5 h of further incubation. The relative cell activity was calculated. [Relative cell activity = (absorbance of drug-treated wells - absorbance of culture medium) / (absorbance of blank group - absorbance of culture medium)]

[0155] Results: The liposome co-loaded with oxymatrine and astragaloside B could significantly inhibit the activity of CAFs in the concentration range of 0.07-34.4 μg / mL (calculated based on the concentration of astragaloside B). Figure 16 According to the results of Example 10, the liposome co-loaded with oxymatrine and astragaloside B can effectively improve the anti-tumor ability of the PD-1 inhibitor.

Claims

1. Use of a traditional Chinese medicine active ingredient composition in the preparation of a synergistic PD-1 inhibitor antitumor synergistic drug, wherein the traditional Chinese medicine active ingredient composition is a combination of oxymatrine and astragaloside IV. The mass ratio of oxymatrine to astragaloside IV is 1-8:

1.

2. Use according to claim 1, characterized in that, The mass ratio of oxymatrine to astragaloside IV is 1-2:

1.

3. Use according to claim 1, characterized in that, The tumor is liver cancer or breast cancer.

4. Use according to claim 1, characterized in that, It also includes a pharmaceutically acceptable carrier.

5. Use according to claim 4, characterized in that, The carrier is an iron-based MOF or a liposome.

6. Use according to claim 4, characterized in that, The traditional Chinese medicine active ingredient composition is loaded into the carrier to form a nano preparation.

7. Use according to claim 6, characterized in that, The carrier is an iron-based MOF, and the preparation method of the nano preparation comprises the following steps: connecting the metal nodes with organic ligands to form an iron-based MOF, activating the iron-based MOF, and then placing it in a solution with the traditional Chinese medicine active ingredient composition as the solute to load the drug, obtaining the drug-loaded nano preparation, and modifying the drug-loaded nano preparation with a surface modification material to obtain the finished nano preparation. The raw materials for the preparation method include the following components in the following amounts: The traditional Chinese medicine active ingredient composition is 198-792 parts, the metal node is 4-101 parts, the organic ligand is 32-64 parts, and the surface modification material is 10-160 parts.

8. Use according to claim 7, characterized in that, The mass ratio of the solute to the solvent in the solution with the traditional Chinese medicine active ingredient composition as the solute is 0.15-2.4%. The metal node is selected from one or two of Fe3O4 nanoparticles, FeCl3·H2O, and Fe(NO3)3·9H2O. The organic ligand is selected from any one of trimesic acid, terephthalic acid, and 2-amino terephthalic acid.

9. Use according to claim 8, characterized in that, The surface modification material is selected from any one of platelet membrane, red blood cell membrane, tumor cell membrane, and hyaluronic acid.

10. Use according to claim 6, characterized in that, The surface modification material is selected from platelet membrane. Traditional Chinese medicine active ingredient composition 15-120 parts, natural phospholipid 100-500 parts, synthetic phospholipid 60-200 parts, cholesterol 30-75 parts, DSPE-PEG 2000 14-20 parts.

11. Use according to claim 10, characterized in that, The carrier is a liposome, and the nano preparation includes the following components in the following amounts: The natural phospholipid is selected from soy lecithin, and the synthetic phospholipid is selected from hydrogenated soy lecithin.

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