Anti-tumor pharmaceutical composition and application thereof in improvement of tumor immunotherapy

Abstract

The invention relates to an anti-tumor pharmaceutical composition and an application of the anti-tumor pharmaceutical composition in improvement of tumor immunotherapy. The combined pharmaceutical composition comprises tripterine and DGAT1 siRNA. The combined application of the tripterine and the DGAT1 siRNA has stronger anti-tumor activity than a single component, and the combined pharmaceutical composition can significantly enhance the anti-tumor effect of an immune checkpoint inhibitor, can prolong the lifetime of tumor-bearing mice, shows good in-vivo safety, and has good clinical application prospects. And a new strategy is provided for realizing tumor treatment by enhancing the immune checkpoint inhibitor.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to an anti-tumor pharmaceutical composition and its application in improving tumor immunotherapy. Background Art

[0002] Currently, the main treatment methods for cancer are surgery, chemotherapy, radiotherapy, targeted therapy and immunotherapy, among which tumor immunotherapy has become a research hotspot in recent years. The emergence of immune checkpoint inhibitors represented by anti-PD-L1 monoclonal antibody has brought hope for the treatment of cancer. aPD-L1 blocks the binding of PD-1 and PD-L1, relieves the immune escape of tumor cells, and thus activates the immune system to attack tumor cells. Although studies have shown that immune checkpoint inhibitors can trigger anti-tumor immune responses, however, there are still a large number of cancer patients who are ineffective in the treatment with aPD-L1 alone, which may be closely related to insufficient lymphocyte infiltration in the tumor microenvironment, low immune response level and weak immunogenicity. Therefore, it is necessary to develop a combined strategy of immune checkpoint inhibitors with improved tumor immunogenicity and reconstituted TME to restore and enhance anti-tumor immunotherapy mediated by cytotoxic T lymphocytes.

[0003] Celastrol is a pentacyclic triterpenoid compound derived from the root bark of Tripterygium wilfordii Hook. f. Celastrol can inhibit the development of prostate cancer, liver cancer, breast cancer and gastric cancer through inducing apoptosis, autophagy, inhibiting angiogenesis and tumor metastasis; it also has great potential in tumor immunotherapy, and can enhance the anti-tumor immune response by inducing endoplasmic reticulum stress and autophagy in tumor cells, resulting in ICD; it has significant weight loss effect and lipid metabolism regulation function. Diacylglycerol acyltransferase 1 (DGAT1), as a key protein for the synthesis of triglyceride in lipid droplets, its siRNA can effectively inhibit the formation of cell lipid droplets and reduce lipid content. However, there are few detailed reports on the research of the combination of the two in tumor treatment and application. Summary of the Invention

[0004] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide an anti-tumor pharmaceutical composition and its application in improving tumor immunotherapy, which are specifically achieved through the following technical solutions:

[0005] An anti-tumor composition, the anti-tumor composition comprises active ingredients celastrol and DGAT1 siRNA, the mass of the celastrol is 1-4 mg, preferably 2 mg; the N / P ratio of the DGAT1 siRNA is 0-30 / 1, preferably 10 / 1.

[0006] Furthermore, celastrol is celastrol monomer; the DGAT1 siRNA sequence is Sense strand (5’-3’): CUGCAAGAUUCUUUGUUCATT; Antisense strand (5’-3’): UGAACAAAGAAUCUUGCAGTT.

[0007] Furthermore, the average particle size of the lipid polymer nanoparticles co-loaded with celastrol and DGAT1 siRNA is preferably 60 - 300 nm, more preferably 80 - 150 nm.

[0008] An anti-tumor drug, which is composed of the above anti-tumor composition and pharmaceutically acceptable excipients, and the anti-tumor drug is any pharmaceutically acceptable dosage form, preferably liposome, lipid polymer nanoparticle, micelle, solid dispersion, etc., more preferably lipid polymer nanoparticle.

[0009] Furthermore, the excipients include any one or a combination of more than one of soybean lecithin, egg yolk lecithin, cholesterol, DOTAP, DSPE-PEG2000, DSPE-PEG-RGD, high molecular polymer PGA or other anionic high molecular polymers, and high molecular polymer PEI or a high molecular polymer synthesized with PEI as a substrate or other cationic high molecular polymers.

[0010] The preparation method of the celastrol and DAGT1 siRNA lipid polymer nanoparticles includes the following steps:

[0011] Prepare the PEI / (PGA+siRNA) complex by using the "electrostatic adsorption" effect: Utilize the electrostatic adsorption effect of the cationic polymer (PEI) and the anionic polymer PGA+siRNA to form a negatively charged PEI / (PGA+siRNA) complex. Since siRNA has a low molecular weight and low charge density, it forms a loose and unstable complex when mixed with the polycationic polymer. Therefore, cationic liposomes are prepared by the thin film dispersion method to encapsulate the complex to protect siRNA from dissociation or clearance before reaching the tumor, and finally form the siD / CEL-NPs lipid polymer nanoparticles with a core-shell structure.

[0012] Furthermore, the administration form of the anti-tumor drug includes any one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration, preferably intravenous injection.

[0013] Furthermore, the application of the pharmaceutical composition in the preparation of anti-tumor drugs, wherein the tumors include liver cancer, lung cancer, pancreatic cancer, breast cancer, cervical cancer, endometrial cancer, colorectal cancer, gastric cancer, lung cancer, melanoma, prostate cancer, thyroid cancer, leukemia, lymphoma, esophageal cancer, oral cancer, intestinal cancer, nasal cancer, head and neck cancer, preferably breast cancer.

[0014] Furthermore, the application of the pharmaceutical composition in enhancing the anti-tumor effect of immune checkpoint inhibitors.

[0015] Furthermore, the immune checkpoint inhibitors include PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, TIM3 inhibitors, LAG3 inhibitors, preferably PD-L1 inhibitors.

[0016] The application of the composition provided by the present invention lies in cancer treatment, especially in enhancing tumor immune cell infiltration, improving the immunosuppressive microenvironment, and promoting the application of tumor immunotherapy; on the one hand, the pharmaceutical composition of the present invention effectively increases the solubility of celastrol and simultaneously effectively delivers DGAT1 siRNA, on the other hand, it realizes continuous and efficient ROS generation, targets and induces the ICD cascade amplification effect of tumor cells, thereby enhancing the effect of immune checkpoint inhibitors in treating TNBC. Description of the Drawings

[0017] Figure 1 It is the transmission electron micrograph of the siD / CEL-NPs lipid nanoparticles of the present invention;

[0018] Figure 2 It is the particle size distribution and potential map of the siD / CEL-NPs lipid nanoparticles of the present invention;

[0019] Figure 3 It is the stability detection result of the siD / CEL-NPs lipid nanoparticles of the present invention;

[0020] Figure 4 It is the investigation of the in vitro release rate of the siD / CEL-NPs lipid nanoparticles of the present invention;

[0021] Figure 5 It is the effect of the siD / CEL-NPs lipid nanoparticles of the present invention on the key protein DGAT1 of lipid droplet accumulation;

[0022] Figure 6This is the treatment effect diagram of siD / CEL-NPs lipid nanoparticles of the present invention on the animal model of breast cancer in situ (wherein, A is the schematic diagram of the administration plan of the lipid nanoparticles; B is the change in body weight of tumor-bearing mice during treatment; C is the change diagram of the tumor volume of tumor-bearing mice; D is the tumor weight of the dissected tumor of tumor-bearing mice on the 16th day after drug treatment; E is the dissected tumor diagram of tumor-bearing mice on the 16th day after different drug treatments; F is the change diagram of the tumor luminescence intensity of tumor-bearing mice; G is the BLIrel ratio curve of different drug treatment groups at different times; H is the TUNEL and H&E staining diagrams of tumor tissues).

[0023] Figure 7 It is for the investigation of the expression levels of CRT and HMGB1 in the tumor tissues of tumor-bearing mice after different drug treatments by immunofluorescence detection.

[0024] Figure 8 It is for the detection of the changes in mature DCs in the tumor-draining lymph nodes of tumor-bearing mice after different drug treatments by flow cytometry.

[0025] Figure 9 It is for the analysis of the changes in immune cells in the tumor microenvironment of tumor-bearing mice by flow cytometry and immunofluorescence.

[0026] Figure 10 It is for the H&E staining of the main organs of tumor-bearing mice (scale bar is 100 μm) and blood biochemical indexes ( 注 ： *P<0.05, **P<0.01, ***P<0.001; The t-test was used to evaluate the significant differences, and the results are expressed as mean±SD). Detailed implementation mode

[0027] The following further elaborates on the present invention in detail in combination with the specification drawings and specific embodiments for better understanding of the technical solution.

[0028] Example 1: Preparation of siD / CEL-NPs lipid nanoparticles

[0029] First, the PEI / (PGA+siRNA) complex was prepared by the "electrostatic adsorption" effect: by using the electrostatic adsorption effect between the cationic polymer (PEI) and the anionic polymer PGA+siRNA, a negatively charged PEI / (PGA+siRNA) complex was formed. Since siRNA has a low molecular weight and low charge density, a loose and unstable complex is formed when it is mixed with the polycationic polymer. Therefore, cationic liposomes were prepared by the thin film dispersion method to encapsulate the complex to protect siRNA from dissociation or clearance before reaching the tumor, and finally, the siD / CEL-NPs lipid nanoparticles with a core-shell structure were formed.

[0030] The preferred ratio of the siD / CEL-NPs lipid nanoparticles of the present invention (CEL = 1 mg) is shown in Table 1.

[0031] Table 1

[0032] DOTAP 20 mM Chol 20 mM N / P 10 / 1 CEL 1 mg

[0033] The preferred ratio of the siD / CEL-NPs lipid nanoparticles of the present invention (CEL = 2 mg) is shown in Table 2.

[0034] Table 2

[0035] DOTAP 20 mM Chol 20 mM N / P 10 / 1 CEL 2 mg

[0036] The preferred ratio of the siD / CEL-NPs lipid nanoparticles of the present invention (CEL = 4 mg) is shown in Table 3.

[0037] Table 3

[0038] DOTAP 20 mM Chol 20 mM N / P 10 / 1 CEL 4 mg

[0039] The particle size detection results of the siD / CEL-NPs lipid nanoparticles with different CEL dosages are similar. The average particle size of the lipid nanoparticles with CEL of 1 mg is (150.0 ± 2.9) nm, the average particle size of the lipid nanoparticles with CEL of 2 mg is (133.5 ± 0.7) nm, and the average particle size of the lipid nanoparticles with CEL of 4 mg is (214.6 ± 4.7) nm. It is preferred to use CEL of 2 mg for subsequent experiments.

[0040] The preferred siRNA ratio of the siD / CEL-NPs lipid nanoparticles of the present invention is (N / P = 0, 1, 5, 10, 20 and 30):

[0041] Specifically, PGA and siRNA are mixed at a molar ratio of 1:1, and the N / P ratio of the PEI / (PGA + siRNA) complex is adjusted. The encapsulation of siRNA by the nanoparticles is investigated by agarose gel electrophoresis. The principle is that free siRNA is negatively charged under neutral conditions and can migrate from the negative electrode to the positive electrode under the action of an external electric field. Due to the molecular sieve effect and charge effect of the agarose gel network structure, the siRNA stably encapsulated by the nanoparticles is blocked in its migration in the electric field. The non-covalent binding of GelGreen to RNA enables GelGreen to effectively interact with the charged groups in the RNA molecule, resulting in the fluorescence of RNA under ultraviolet light.

[0042] With the increase of N / P, the ability of nanoparticles to encapsulate siRNA gradually increases. When N / P is greater than 10, siDGAT1 still remains in the loading well, and no fluorescent band of siRNA migration appears in the gel electric field, indicating that the nanoparticles can completely compress siRNA at this time. When N / P increases from 1 to 10, the encapsulation efficiency of siDGAT1 increases significantly. When N / P is 10, the encapsulation efficiency reaches 89.8 ± 1.9%, and thereafter, even if N / P continues to increase, the change in the encapsulation efficiency of siDGAT1 is not obvious. Therefore, N / P of 10 was selected to prepare siD / CEL-NPs for subsequent experiments.

[0043] Example 2: Characterization of siD / CEL-NPs Lipid Nanoparticles

[0044] The particle size, distribution, and potential of siD / CEL-NPs diluted to an appropriate concentration were investigated using a laser particle size analyzer.

[0045] An appropriate amount of the prepared siD / CEL-NPs above was taken and diluted to an appropriate concentration. 20 μL was dropped on a copper grid, air-dried at room temperature, negatively stained with 2% phosphotungstic acid, and its morphology was observed by transmission electron microscopy.

[0046] The detection results are as Figure 1 and Figure 2 shown: It can be seen from the electron micrograph negatively stained with 2% phosphotungstic acid that the siD / CEL-NPs lipid nanoparticles ( Figure 1 ) have a complete surface, are spherical-like with an obvious core-shell structure, have a uniform particle size distribution, and the particle size is about 130 nm. It can be seen from the particle size distribution of the siD / CEL-NPs lipid nanoparticles ( Figure 2 ) measured by the Malvern laser particle size analyzer that the particle size distribution of the nanoemulsion is uniform, the average particle size is about (131.8 ± 2.8) nm, the polydispersity index (PDI) is 0.189 ± 0.027, and the Zeta potential is (+23.8 ± 0.8) mV.

[0047] Example 3: Stability Study of siD / CEL-NPs Lipid Nanoparticles

[0048] The serum stability of siD / CEL-NPs was investigated. siD / CEL-NPs were placed in PBS buffer containing 10% fetal bovine serum and incubated for different times, and the changes in particle size and PDI were detected. The results showed that there were no significant changes in the particle size and PDI of the siD / CEL-NPs nanoparticles, indicating that siD / CEL-NPs have good serum stability ( Figure 3 A).

[0049] Investigate the storage stability of siD / CEL-NPs. After storing siD / CEL-NPs at 4 °C for 7 days, the particle size and PDI slightly increased, but there were no significant changes, indicating good storage stability of siD / CEL-NPs( Figure 3 B).

[0050] Example 4: In vitro release study of siD / CEL-NPs lipid nanoparticles

[0051] Investigate the in vitro release of siD / CEL-NPs lipid nanoparticles. FAM-siRNA / CEL-NP was prepared by replacing siRNA with fluorescent FAM-siRNA, and free CEL was used as a control. PBS (pH 7.4, pH 6.8, and pH 5.5) containing 0.2% (w / v) Tween 80 was selected as the drug release medium to simulate three pH conditions: the human physiological environment, the tumor microenvironment, and the lysosome. Precisely pipette 1 mL of FAM-siRNA / CEL-NPs into a dialysis bag with a molecular weight cut-off of 3500 Da. Transfer the dialysis bag into a centrifuge tube containing 20 mL of the corresponding pH release medium and incubate (37 ± 0.5 °C, 100 rpm). At specific time points, pipette 2 mL of the drug-containing release medium and add an equal volume of blank medium, measure the contents of CEL and FAM-siRNA, calculate the cumulative release amount, and plot the in vitro drug release curve( Figure 4 ). The results showed that free CEL could reach complete release (>95%) within 8 h under the three different pH release conditions, indicating that the sink conditions required for CEL release were met. For siD / CEL-NPs, there was no obvious burst release of CEL from the lipid nanoparticles, and the release was slow. The cumulative release amount within 48 h was 46.53 ± 6.66%( Figure 4 ). As the pH gradually decreased, the cumulative release amount of CEL gradually increased to 65.53 ± 6.19% (pH 6.8) and 90.11 ± 0.36% (pH 5.5). The cumulative release amount of siRNA also increased significantly from 19.40 ± 1.08% to 61.28 ± 2.55%( Figure 4 ). The change in the release behavior of the drug and siRNA due to the change in pH may be attributed to the presence of PEI. PEI has the characteristic of "proton sponge effect" because it contains many tertiary amine groups itself, that is, it can be protonated in an acidic environment, resulting in a change in the structure of the nanoparticles and a decrease in stability, realizing the rapid release of CEL and siRNA.

[0052] Example 5: Effect of siD / CEL-NPs lipid nanoparticles on the key protein DGAT1 of lipid droplet accumulation

[0053] 4T1 cells were added to a 6-well plate at a density of 3×105 cells / well. After overnight adhesion, 2 mL of serum-free medium containing siNC-NPs (50 nM siNC), siD-NPs (25 - 100 nM siDGAT1), and siD / CEL-NPs (50 nM siDGAT1) was added to each group, and the cells were cultured for another 2 days. The 4T1 cells were digested with trypsin and collected. The DGAT1 mRNA level was measured by RT-qPCR, and the effect of each group on the DGAT1 protein expression level in 4T1 cells was determined by Western Blot. Meanwhile, the optimal in vitro administration concentration of siDGAT1 was selected.

[0054] As Figure 5 shown, compared with the Control group (siNC-NPs), lipid nanoparticles encapsulating siDGAT1 could significantly reduce the DGAT1 gene and protein expression levels. With the increase in the siDGAT1 concentration, the silencing efficiency also increased. When the concentration of siDGAT1 was 50 nM, the expression of DGAT1 had been significantly reduced (P < 0.001). The above results indicated that when the concentration of siDGAT1 was 50 nM, the constructed siD / CEL-NPs delivery system had effectively reduced the DGAT1 expression, laying a good foundation for subsequent experiments.

[0055] Example 6: Antitumor effect of siD / CEL-NPs lipid nanoparticles

[0056] Digest the mouse 4T1 breast cancer cells in the logarithmic growth phase with 0.25% trypsin, centrifuge at 1000 rpm for 3 minutes, adjust the cells to an appropriate concentration, and place the cells on ice for standby. Inoculate 4T1 cells (100 μL, 6×106 / mL) under the right second pair of mammary fat pads of BALB / c mice (8 weeks old). One week after inoculation, observe the tumor growth situation ( Figure 6 A).

[0057] The tumor-bearing mice were randomly divided into 6 groups, with 5 mice in each group. The experimental groups were PBS, aPD-L1, siD-NPs, CEL-NPs, siD / CEL-NPs, and siD / CEL-NPs + aPD-L1. Among them, the administration doses of CEL, siDGAT1, and aPD-L1 were 1 mg / kg, 500 μg / kg, and 5 mg / kg, respectively. aPD-L1 was intraperitoneally injected (100 μL), administered once every three days for three consecutive times; the remaining administration groups were intravenously injected through the tail vein (200 μL), administered once every three days for five times. One week after tumor inoculation, the tumor diameter was measured every two days, and the body weight change of the mice during the administration period was recorded ( Figure 6 B), the tumor volume was calculated ( Figure 6 C), and the change in tumor luminescence intensity was detected by in vivo fluorescence technology in miceFigure 6 F), calculate the BLIrel ratio curves of different drug treatment groups at different times ( Figure 6 G). On the 16th day after drug administration, the tumor-bearing mice were sacrificed, and the dissected tumors were obtained after dissection ( Figure 6 E), record the tumor weight of the dissected tumors of the tumor-bearing mice ( Figure 6 D), TUNEL and H&E stain the tumor tissues ( Figure 6 H).

[0058] The experimental results showed that siD / CEL-NPs could significantly inhibit tumor growth, and the tumor inhibition rate was 67.5 ± 2.5%. The combined use of aPD-L1 had a better therapeutic effect, reaching 86.3 ± 3.5%. The results of H&E and TUNEL staining showed that siD / CEL-NPs and the combined use of aPD-L1 group could effectively promote the apoptosis of tumor tissues.

[0059] Example 7: Investigation of the expression levels of CRT and HMGB1 in tumor tissues

[0060] CRT externalization and HMGB1 efflux are considered to be the hallmark features of ICD. These DAMPs can bind to pattern recognition receptors on the surface of innate immune cells, thereby recruiting antigen-presenting cells to the tumor site. Therefore, by effectively inducing the ICD effect, the effect of tumor immunotherapy can be improved.

[0061] The experimental results showed that the CEL-NPs group had obvious green fluorescence production, but the green fluorescence of the siD / CEL-NPs group was stronger ( Figure 7 ). The above results indicated that siD / CEL-NPs exhibited high-efficiency delivery ability of CEL in vivo to induce the ICD effect in tumor cells, and the combination of siDGAT1 could further enhance the "eat me" signals of CRT and HMGB1 in tumor cells.

[0062] Example 8: Determination of mature DCs in tumor-draining lymph nodes

[0063] The occurrence of the ICD effect in tumors will induce the exposure of CRT, promote the recognition and uptake of tumors by DCs, and activate the maturation of DCs. DCs play a key role in triggering the innate immune and adaptive immune response processes.

[0064] TDLNs of tumor-bearing mice after drug administration were collected, added with 1 mL of PBS, ground and passed through a 40-μm cell sieve, and the cells were collected. Centrifuged at 1500 rpm for 5 min at low temperature, and the supernatant was discarded. Resuspended at an appropriate density in 100 μL of PBS containing 2% BSA, and CD11c, CD80 and CD86 antibodies were added respectively, and incubated in the dark at room temperature for 30 min. After washing and resuspending with PBS containing 2% BSA, FCM was used to analyze the proportion of mature DCs in TDLNs. The results showed that compared with the siD-NPs group, after treatment with CEL-NPs, the number of mature DCs in TDLNs of mice increased significantly (~1.28-fold). The number of mature DCs in the TDLNs of the siD / CEL-NPs+aPD-L1 group was significantly higher than that of the siD-NPs group (~1.76-fold) and the CEL-NPs group (~1.37-fold), and there was no significant difference compared with the siD / CEL-NPs group( Figure 8 ). The above results indicate that CEL can effectively induce the maturation of DCs, and at the same time, the siD / CEL-NPs and the combined aPD-L1 group have a stronger ability to promote the maturation of DCs in TDLNs, which may be closely related to the ICD effect induced by the highly sustainable intracellular ROS generation mediated by them.

[0065] Example 9: Investigation of the infiltration of immune cells in tumor tissues

[0066] 80 mg of tumor tissue isolated from tumor-bearing mice after drug administration was ground and placed in a 5-mL centrifuge tube, and 2 mL of tissue dissociation solution (containing 5% FBS, DNase I, type I collagenase, type IV collagenase and RPMI 1640 medium) was added. Incubated on a shaker at 37 °C for 1 h. Ground through a 70-μm cell sieve to prepare a single-cell suspension. Centrifuged and resuspended with PBS containing 2% BSA, and the cell density was controlled at 1×106 cells / tube. Before staining, 0.2 μL of Zombie Aqua was added to each group in advance TMFixable Viability Kit (1:500), incubate on ice for 30 min, then wash, centrifuge, and resuspend with 100 μL of PBS containing 2% BSA for standby. For surface staining of protein markers, only add the corresponding antibody for staining; for intracellular protein staining, add Fixation / Permeabilization buffer for membrane permeabilization treatment; for nuclear protein staining, add Fixation buffer for nuclear permeabilization treatment and then perform antibody staining. Detect the proportions of infiltrated CD8+ T cells (CD3+CD4-CD8+), IFN-γ+CD8+ T cells (CD3+CD8+IFN-γ+), GZMB+CD8+ T cells (CD3+CD8+IFN-γ+GZMB+), Tregs (CD4+CD25+Foxp3+), and MDSCs (CD11b+Gr1+) in tumor tissues by FCM.

[0067] Embed the tumor tissues in paraffin, section, and dewax. Place the sections in antigen retrieval buffer for antigen retrieval, and add serum for blocking for 0.5 h. After serum blocking, label each type of cell with the corresponding antibody. For CD8+ T cells, add anti-CD3 and anti-CD8 primary antibodies; for MDSCs, add anti-CD11b and anti-Gr-1 primary antibodies. Incubate overnight at 4°C, wash 3 times with PBS, add fluorescently labeled secondary antibody, incubate for 1 h at room temperature in the dark, and wash 3 times with PBS.

[0068] The experimental results showed that the ICD effect induced by siD / CEL-NPs was significantly stronger than that of the single application group, with a stronger ability to promote the maturation of DCs in TDLNs and the infiltration of CD8+ T cells into tumor tissues, and effectively alleviated the immunosuppressive microenvironment by significantly reducing immunosuppressive Tregs and MDSCs, enhancing the immunotherapeutic effect of aPD-L1 ( Figure 9 ).

[0069] Example 10: H&E staining analysis of major organs and tumor tissues of tumor-bearing mice and blood biochemical index conditions

[0070] At the end of the experiment, sacrifice the mice by cervical dislocation, take out the major organs (heart, liver, spleen, lung, kidney) and tumor tissues of the mice, and fix them in 4% paraformaldehyde. After one week, perform tissue embedding, paraffin sectioning, and H&E staining, and observe whether there are tissue lesions in each organ under the microscope to evaluate the damage of each organ by the siD / CEL-NPs and aPD-L1 combination treatment group. The H&E staining results showed that there were no obvious pathological changes in the major organs of each treatment group ( Figure 10 B).

[0071] At the end of the experiment, the whole blood was obtained by removing the eyeballs of the tumor-bearing mice and placed in a centrifuge tube for natural coagulation for 1 h. Then, it was centrifuged (4 °C, 2000 rpm, 15 min), and the supernatant was collected to obtain serum. The serum samples were immediately sent to the blood biochemical testing room of the animal center to detect the concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine (CREA), and to evaluate the effects of the siD / CEL-NPs group and the combined aPD-L1 group on liver and kidney functions. The results showed that there were no significant differences in the liver function biochemical indexes (ALT and AST) and kidney function biochemical indexes (BUN and CREA) among the treatment groups compared with the PBS group, indicating that the hepatotoxic and nephrotoxic side effects were relatively small( Figure 10 A).

[0072] The above experiments showed that siD / CEL-NPs and the combined aPD-L1 had good anti-tumor effects.

Claims

1. An antitumor composition, characterized in that: The composition comprises an effective ingredient, tripterine, and diacylglycerol O-acyltransferase 1 (DGAT1) DGAT1 siRNA.

2. An antitumor composition according to claim 1, characterized in that: The tripterygium wilfordii is a tripterygium wilfordii monomer; the DGAT1 siRNA sequence is Sense strand (5'-3'): CUGCAAGAUUCUUUGUUCATT; Antisense strand (5'-3'): UGAACAAAGAAUCUUGCAGTT.

3. An antitumor composition according to claim 1, characterized in that: The dosage of tripterygium wilfordii is 1-4 mg, and the nitrogen-phosphorus ratio of DGAT1 siRNA is 0-30 / 1.

4. An anti-tumor drug, characterized in that: The anti-tumor drug is composed of the anti-tumor composition according to claim 1 and pharmaceutically acceptable excipients, and the anti-tumor drug is in any pharmaceutically acceptable dosage form.

5. An anti-tumor drug according to claim 4, characterized in that: The auxiliary materials include soybean lecithin, egg yolk lecithin, cholesterol, DOTAP, DSPE-PEG2000, DSPE-PEG-RGD, high molecular weight polymer sodium polyglutamate (PGA) or other anionic high molecular weight polymers and high molecular weight polymer polyethyleneimine (PEI) or high molecular weight polymers synthesized with PEI as a substrate or other cationic high molecular weight polymers, any one or a combination of more than one.

6. An anti-tumor drug according to claim 4, characterized in that: The anti-tumor drug can be administered in the form of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration.

7. Use of an antitumor composition according to any one of claims 1 to 3 in the preparation of antitumor drugs.

8. The use according to claim 7, characterized in that The tumor is any one of liver cancer, lung cancer, pancreatic cancer, breast cancer, cervical cancer, endometrial cancer, colorectal cancer, gastric cancer, lung cancer, melanoma, prostate cancer, thyroid cancer, leukemia, lymphoma, esophageal cancer, oral cancer, intestinal cancer, nasal cancer, and head and neck cancer.

9. The use according to claim 7, characterized in that Application of the anti-tumor composition in enhancing immune checkpoint inhibitors.

10. The use according to claim 9, characterized in that The immune checkpoint inhibitors include any one of CTLA-4 inhibitors, PD-L1 inhibitors, CD47 inhibitors, LAG3 inhibitors, and TIM3 inhibitors.

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