Compound as well as preparation method and application thereof

Abstract

The invention relates to a compound. The compound comprises a polysaccharide conjugate, an anti-PD-L1 monoclonal antibody and a PKM2 inhibitor. The compound disclosed by the invention can be accumulated and retained in tumor tissues, so that dendritic cells are effectively stimulated to be mature, an inhibitory immune microenvironment is relieved, and a specific anti-tumor immune response is initiated. The expression of PKM2 can be effectively inhibited, so that glycolysis in tumor tissues is inhibited, and a more favorable environment is provided for infiltrating the tumor tissues with T lymphocytes. The compound disclosed by the invention is expected to enhance the treatment effect of RFA on advanced HCC patients by reprogramming iRFA-induced tumor immunosuppression, and has huge potential in the aspect of improving prognosis of the advanced HCC patients treated by the RFA.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a composite and a preparation method and application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) refers to a malignant tumor that occurs in liver cells. It is one of the most common malignant tumors and affects a large number of patients. In order to reduce the pain of HCC patients and improve their quality of life, precise and effective treatments are now available. Radiofrequency ablation (RFA), which has been approved by the US Food and Drug Administration, has now developed into a key thermal ablation technology that can induce significant and immediate anti-tumor effects (mainly coagulative necrosis) in the clinical treatment of HCC and some other cancers with clear tumor boundaries by directly intervening at the tumor site.

[0003] However, due to the large size and / or irregular shape of the tumor tissues in these advanced HCC patients, most of them carry multiple tumor lesions and the tumor boundaries are difficult to define, which poses a great challenge to determining the RFA treatment parameters (such as duration and range), resulting in a high frequency of incomplete radiofrequency ablation (iRFA). According to clinical studies, iRFA treatment often leads to rapid local recurrence and deterioration of health status, resulting in poor prognosis for patients with advanced HCC. Detailed mechanistic studies have shown that iRFA treatment can lead to worsening of the tumor immunosuppressive microenvironment by upregulating the number of myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), and other suppressive immune cells, as well as cytokines / chemokines in the residual tumor mass. As a result, the T cell-based anti-tumor immune response within the residual tumor mass is significantly suppressed, thereby promoting local tumor recurrence and acquiring tolerance to immune checkpoint blockade (ICB) and other immunotherapies.

[0004] With the rapid development of biomaterials and biomedical engineering, a series of powerful strategies have been proposed to improve the treatment of patients by inducing immunogenic ferroptosis of residual tumor cells and recruiting dendritic cells or natural killer cells, thereby reversing the tumor immunosuppression exacerbated by iRFA. Aerobic glycolysis, also known as the Warburg effect, through which cancer cells obtain energy, has been successfully used to meet the high energy demand of iRFA-promoted tumor progression. In this case, a large amount of immunosuppressive lactate is produced, which significantly promotes acidosis of residual tumors and impairs the infiltration and effector function of cytotoxic T cells. Studies have shown that iRFA can promote glycolysis of residual cancer cells by upregulating the expression of pyruvate kinase M2 (PKM2), thereby exacerbating tumor immunosuppression and leading to poor prognosis. Summary of the invention

[0005] Problem that the invention aims to solve In view of the above problems existing in the prior art, the object of the present invention is to provide a nano drug that can reverse the tumor immunosuppression and tumor treatment failure aggravated by iRFA.

[0006] The present invention provides a complex, which comprises a polysaccharide conjugate, an anti-PD-L1 monoclonal antibody, and a PKM2 inhibitor.

[0007] Preferably, the polysaccharide conjugate is a conjugate of a T cell promoter and a polysaccharide.

[0008] Preferably, the T cell promoter is spermidine, the polysaccharide is dextran, and the PKM2 inhibitor is shikonin.

[0009] Preferably, the complex further comprises PEG lipids, structural lipids and phospholipids; The PEG lipid is selected from one or more of DMG-PEG, DSG-PEG, DPG-PEG, DAG-PEG, DAA-PEG, DPPE-PEG, and DSPE-PEG; The structural lipid is selected from one or more of cholesterol, dihydrocholesterol, lanosterol, β-phytosterol, ergosterol, campesterol, stigmasterol, brassicasterol, algin sterol, tomatine, ursolic acid, α-tocopherol and derivatives thereof; The phospholipid is selected from one or more of EPC, DAPC, DBPC, DLPC, DMPC, DOPC, DOPE, DOPG, DPPC, DSPC, POPC, and POPE.

[0010] Preferably, the particle size of the complex is 80-150 nm.

[0011] The present invention also provides a method for preparing the composite, the method comprising the following steps: (1) conjugating the T cell promoter to the polysaccharide to prepare the polysaccharide conjugate; (2) using a closed impinging jet (CIJ) mixer, mixing the polysaccharide conjugate prepared in step (1) with the anti-PD-L1 monoclonal antibody to prepare a monodisperse nanocomplex; (3) Using a closed impinging jet (CIJ) mixer, the PKM2 inhibitor is coated on the monodisperse nanocomposite prepared in step (2) to prepare the complex.

[0012] Preferably, step (2) is to inject the polysaccharide conjugate and the anti-PD-L1 monoclonal antibody into the CIJ mixer at a constant flow rate of 1-20 mL / min.

[0013] Preferably, step (3) is to dissolve the PKM2 inhibitor in a lipid solution, and then inject the lipid solution and the monodisperse nanocomplex prepared in step (2) into a CIJ mixer at a constant flow rate of 1-20 mL / min.

[0014] Preferably, the lipid solution contains the PEG lipid, the structural lipid and the phospholipid, and the concentration of the PKM2 inhibitor in the lipid solution is 1-10 mg / mL.

[0015] Preferably, the T cell promoter in step (1) is spermidine, the polysaccharide is dextran, and the PKM2 inhibitor in step (3) is shikonin.

[0016] The present invention also provides a pharmaceutical composition, which comprises the complex and a pharmaceutically acceptable excipient.

[0017] The present invention also provides a use of the complex or the pharmaceutical composition in preparing a drug for treating or preventing tumor-related diseases.

[0018] Preferably, the tumor-related disease is selected from one or more of Wilms' tumor, Ewing's sarcoma, neuroendocrine tumors, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, bile duct cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, non-Hodgkin's lymphoma and bladder cancer.

[0019] Preferably, the use comprises reversing tumor immunosuppression caused by iRFA.

[0020] The complex of the present invention can accumulate and remain in tumor tissue, effectively stimulate dendritic cell maturation, alleviate the inhibitory immune microenvironment, and induce specific anti-tumor immune response. It can effectively inhibit the expression of PKM2, thereby inhibiting glycolysis in tumor tissue and providing a more favorable environment for T lymphocytes to infiltrate tumor tissue. The complex of the present invention is expected to enhance the therapeutic effect of RFA on advanced HCC patients by reprogramming iRFA-induced tumor immunosuppression, and has great potential in improving the prognosis of advanced HCC patients treated with RFA. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of SPM-DEX.

[0023] Figure 2Schematic diagram of the preparation of SPS-NPs and SPS-NP-mediated reversal of iRFA-induced tumor immunosuppression to enhance HCC treatment.

[0024] Figure 3 Particle size distribution of SP-NP and SPS-NP analyzed by Zetasizer.

[0025] Figure 4 is the zeta potential of SP-NP and SPS-NP.

[0026] Figure 5 The morphology of SPS-NPs was photographed by TEM.

[0027] Figure 6 FT-IR spectrum of SPS-NP.

[0028] Figure 7 Particle size distribution of SPS-NPs prepared from different batches.

[0029] Figure 8 The effect of flow rate on the particle size and polydispersity index (PDI) of SP-NPs.

[0030] Fig. 9 In vitro release kinetics of SHK (left) and anti-PDL1 (right) at different pH values.

[0031] Fig.10 The biodistribution of SPS-NP in H22 tumor-bearing mouse model 24 hours after intravenous injection. All data are shown as SD ± mean (n = 3).

[0032] Fig.11 This is a graph showing the relationship between time and mouse tumor volume.

[0033] Fig.12 The weights of the excised lungs from each group of mice.

[0034] Fig.13 The biosafety test results of SPS-NP.

[0035] Fig.14 Representative photographs of lungs excised from mice in each group and histological analysis of lung sections by H&E staining (scale bar = 200 μm) and immunohistochemical staining for VEGF and MMP2 (scale bar = 100 μm).

[0036] Fig.15 Semi-quantitative analysis of VEGF and MMP2 expression in lung sections of each group.

[0037] Fig.16Immunofluorescence analysis of PKM2, CRT and HMGB1 expression in tumor tissues of mice in each group. Scale bar = 50 μm.

[0038] Fig.17 Representative images of tumor sections and histological analysis by H&E staining, TUNEL assay, and Ki67 immunofluorescence staining (scale bar = 50 μm), all data are shown as SD ± mean (n = 3).

[0039] Fig.18 Representative flow cytometric analysis of CD3+CD8+ cells and corresponding quantitative data.

[0040] Fig.19 Function of CD8+ lymphocytes after gating on Ki67+, TNF-α+, IFN-γ+, and GZMB+ for flow cytometric analysis.

[0041] Fig. 20 Representative flow cytometric analysis of CD3+CD49b+ cells and corresponding quantitative data.

[0042] Fig.21 Representative flow cytometric analysis of Gr1+ CD11b+ cells and corresponding quantitative data.

[0043] Fig. 22 Representative flow cytometric analysis of CD25+Foxp3+ cells and corresponding quantitative data.

[0044] Fig.23 Representative flow cytometric analysis and corresponding quantitative data of F4 / 80+CD86+ and F4 / 80+CD206+ cell populations.

[0045] Fig.24 The levels of IL-2, IL-6, IL-10, and TGF-β in tumor tissues of mice in each group. All data are shown as SD±mean (n=3).

[0046] Fig.25 Figure 3 Western blot analysis of PKM2 expression in tumor tissues of mice in each group.

[0047] Description of reference numerals: * represents p < 0.05, indicating statistically significant difference; ** represents p < 0.01, indicating a significant statistical difference; *** represents p<0.001, indicating an extremely significant statistical difference. DETAILED DESCRIPTION

[0048] In order to make the technical solutions and beneficial effects of the present invention more clearly understandable, the following is a detailed description by listing specific embodiments. The drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0049] As used herein, the term "PEG lipid" refers to a lipid modified with polyethylene glycol (PEG). PEG-lipids include, but are not limited to, PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropane-3-amines. Such lipids are also referred to as PEGylated lipids. In certain embodiments, the PEG lipids include, but are not limited to, 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), polyethylene glycol diacrylate (PEG-DAA), PEG-dipalmitoylglycerol (DPG-PEG), PEG-disterylglycerol (PEG-DSG), PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-l,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA).

[0050] As used herein, the term "structured lipid" refers to sterols and lipids containing sterol moieties. Structured lipids include, but are not limited to, cholesterol, dihydrocholesterol, lanosterol, β-phytosterol, ergosterol, campesterol, stigmasterol, brassicasterol, algin sterol, tomatine, tomatin, ursolic acid, α-tocopherol, hopanes, plant sterols, steroids, and mixtures thereof. In certain embodiments, the structured lipid is a sterol. As used herein, the term "sterol" is a subgroup of steroids consisting of steroids.

[0051] As used herein, the term "phospholipid" is a lipid comprising a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. Phospholipids can be assembled into one or more lipid bilayers. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties. Phospholipids may comprise one or more multiple (e.g., double or triple) bonds (e.g., one or more degrees of unsaturation). Phospholipids or their analogs or derivatives may include choline. Phospholipids or their analogs or derivatives may not include choline. Specific phospholipids may promote fusion with membranes. In certain embodiments, cationic phospholipids may interact with one or more negatively charged phospholipids of a membrane (e.g., a cell or intracellular membrane). Fusion of phospholipids with a membrane may allow one or more elements of a lipid composition to pass through the membrane, thereby allowing, for example, one or more elements to be delivered to a cell. In certain embodiments, phospholipids include, but are not limited to, dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), di(lignoceryl)phosphatidylcholine (DLPC), dioleoylphosphatidylcholine (DOPC), sphingomyelin, ceramide, dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), phosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), and the like.

[0052] The present invention provides a complex, which comprises a polysaccharide conjugate, an anti-PD-L1 monoclonal antibody, and a PKM2 inhibitor.

[0053] In certain embodiments, the polysaccharide conjugate is a conjugate of a T cell promoter and a polysaccharide.

[0054] In certain embodiments, the T cell promoter is spermidine, the polysaccharide is dextran, and the PKM2 inhibitor is shikonin.

[0055] In certain embodiments, the polysaccharide conjugate is a conjugate of spermidine and dextran.

[0056] In certain embodiments, the complex further comprises a PEG lipid, a structural lipid, and a phospholipid.

[0057] In certain embodiments, the PEG lipid is selected from one or more of DMG-PEG, DSG-PEG, DPG-PEG, DAG-PEG, DAA-PEG, DPPE-PEG, and DSPE-PEG.

[0058] In certain embodiments, the PEG lipid is DMG-PEG.

[0059] In certain embodiments, the structured lipid is selected from one or more of cholesterol, dihydrocholesterol, lanosterol, β-phytosterol, ergosterol, campesterol, stigmasterol, brassicasterol, alginosterol, tomatine, ursolic acid, α-tocopherol and derivatives thereof.

[0060] In certain embodiments, the structured lipid is cholesterol.

[0061] In certain embodiments, the phospholipid is selected from one or more of EPC, DAPC, DBPC, DLPC, DMPC, DOPC, DOPE, DOPG, DPPC, DSPC, POPC, and POPE.

[0062] In certain embodiments, the phospholipid is DPPC.

[0063] In certain embodiments, the complex includes a conjugate of spermidine and dextran, an anti-PD-L1 monoclonal antibody, shikonin, DMG-PEG, cholesterol, and DPPC.

[0064] In certain embodiments, the complex consists of a conjugate of spermidine and dextran, an anti-PD-L1 monoclonal antibody, shikonin, DMG-PEG, cholesterol, and DPPC.

[0065] In certain embodiments, the particle size of the complex is 80-150 nm.

[0066] In certain embodiments, the particle size of the complex is 100-140 nm.

[0067] In certain embodiments, the complex has a particle size of about 130 nm.

[0068] The present invention also provides a method for preparing the composite, the method comprising the following steps: (1) conjugating the T cell promoter to the polysaccharide to prepare the polysaccharide conjugate; (2) using a closed impinging jet (CIJ) mixer, mixing the polysaccharide conjugate prepared in step (1) with the anti-PD-L1 monoclonal antibody to prepare a monodisperse nanocomplex; (3) Using a closed impinging jet (CIJ) mixer, the PKM2 inhibitor is coated on the monodisperse nanocomposite prepared in step (2) to prepare the complex.

[0069] In certain embodiments, step (2) is to inject the polysaccharide conjugate and the anti-PD-L1 monoclonal antibody into the CIJ mixer at a constant flow rate of 1-20 mL / min.

[0070] In certain embodiments, the flow rate in step (2) is 1 mL / min, or 2 mL / min, or 3 mL / min, or 4 mL / min, or 5 mL / min, or 6 mL / min, or 7 mL / min, or 8 mL / min, or 9 mL / min, or 10 mL / min, or 11 mL / min, or 12 mL / min, or 13 mL / min, or 14 mL / min, or 15 mL / min, or 16 mL / min, or 17 mL / min, or 18 mL / min, or 19 mL / min, or 20 mL / min.

[0071] In certain embodiments, the flow rate in step (2) is 20 mL / min.

[0072] In certain embodiments, step (3) is to dissolve the PKM2 inhibitor in a lipid solution, and then inject the lipid solution and the monodisperse nanocomplex prepared in step (2) into a CIJ mixer at a constant flow rate of 1-20 mL / min.

[0073] In certain embodiments, the flow rate in step (3) is 1 mL / min, or 2 mL / min, or 3 mL / min, or 4 mL / min, or 5 mL / min, or 6 mL / min, or 7 mL / min, or 8 mL / min, or 9 mL / min, or 10 mL / min, or 11 mL / min, or 12 mL / min, or 13 mL / min, or 14 mL / min, or 15 mL / min, or 16 mL / min, or 17 mL / min, or 18 mL / min, or 19 mL / min, or 20 mL / min.

[0074] In certain embodiments, the flow rate in step (3) is 20 mL / min.

[0075] In certain embodiments, the lipid solution contains the PEG lipid, the structural lipid, and the phospholipid, and the concentration of the PKM2 inhibitor in the lipid solution is 1-10 mg / mL.

[0076] In certain embodiments, the concentration of the PEG lipid in the lipid solution is 1-10 mg / mL.

[0077] In certain embodiments, the concentration of the PEG lipid in the lipid solution is 1 mg / mL, or 1.5 mg / mL, or 2 mg / mL, or 2.5 mg / mL, or 3 mg / mL, or 3.5 mg / mL, or 4 mg / mL, or 4.5 mg / mL, or 5 mg / mL, or 5.5 mg / mL, or 6 mg / mL, or 6.5 mg / mL, or 7 mg / mL, or 7.5 mg / mL, or 8 mg / mL, or 8.5 mg / mL, or 9 mg / mL, or 9.5 mg / mL, or 10 mg / mL.

[0078] In certain embodiments, the concentration of the PEG lipid in the lipid solution is 3 mg / mL.

[0079] In certain embodiments, the PEG lipid is selected from one or more of DMG-PEG, DSG-PEG, DAG-PEG, DLPE-PEG, DMPE-PEG, DPPC-PEG, DPPE-PEG, and DSPE-PEG.

[0080] In certain embodiments, the PEG lipid is DMG-PEG.

[0081] In certain embodiments, the concentration of the structured lipid in the lipid solution is 0.5-3 mg / mL.

[0082] In certain embodiments, the concentration of the structured lipid in the lipid solution is 0.5 mg / mL, or 0.6 mg / mL, or 0.7 mg / mL, or 0.8 mg / mL, or 0.9 mg / mL, or 1 mg / mL, or 1.1 mg / mL, or 1.2 mg / mL, or 1.3 mg / mL, or 1.4 mg / mL, or 1.5 mg / mL, or 1.6 mg / mL, or 1.7 mg / mL, or 1.8 mg / mL, or 1.9 mg / mL, or 2 mg / mL, or 2.1 mg / mL, or 2.2 mg / mL, or 2.3 mg / mL, or 2.4 mg / mL, or 2.5 mg / mL, or 21.6 mg / mL, or 2.7 mg / mL, or 2.8 mg / mL, or 2.9 mg / mL, or 3 mg / mL.

[0083] In certain embodiments, the concentration of the structured lipid in the lipid solution is 1.3 mg / mL.

[0084] In certain embodiments, the structured lipid is selected from one or more of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and derivatives thereof.

[0085] In certain embodiments, the structured lipid is cholesterol.

[0086] In certain embodiments, the concentration of the phospholipid in the lipid solution is 0.5-3 mg / mL.

[0087] In certain embodiments, the concentration of the phospholipid in the lipid solution is 0.5 mg / mL, or 0.6 mg / mL, or 0.7 mg / mL, or 0.8 mg / mL, or 0.9 mg / mL, or 1 mg / mL, or 1.1 mg / mL, or 1.2 mg / mL, or 1.3 mg / mL, or 1.4 mg / mL, or 1.5 mg / mL, or 1.6 mg / mL, or 1.7 mg / mL, or 1.8 mg / mL, or 1.9 mg / mL, or 2 mg / mL, or 2.1 mg / mL, or 2.2 mg / mL, or 2.3 mg / mL, or 2.4 mg / mL, or 2.5 mg / mL, or 21.6 mg / mL, or 2.7 mg / mL, or 2.8 mg / mL, or 2.9 mg / mL, or 3 mg / mL.

[0088] In certain embodiments, the concentration of the phospholipid in the lipid solution is 1 mg / mL.

[0089] In certain embodiments, the phospholipid is selected from one or more of DLPC, DMPC, DOPC, DPPC, DSPC, DUPC, and POPC.

[0090] In certain embodiments, the phospholipid is DPPC.

[0091] In certain embodiments, the concentration of the PKM2 inhibitor in the lipid solution is 1 mg / mL, or 1.5 mg / mL, or 2 mg / mL, or 2.5 mg / mL, or 3 mg / mL, or 3.5 mg / mL, or 4 mg / mL, or 4.5 mg / mL, or 5 mg / mL, or 5.5 mg / mL, or 6 mg / mL, or 6.5 mg / mL, or 7 mg / mL, or 7.5 mg / mL, or 8 mg / mL, or 8.5 mg / mL, or 9 mg / mL, or 9.5 mg / mL, or 10 mg / mL.

[0092] In certain embodiments, the concentration of the PKM2 inhibitor in the lipid solution is 5 mg / mL.

[0093] In certain embodiments, the T cell promoter in step (1) is spermidine, the polysaccharide is dextran, and the PKM2 inhibitor in step (3) is shikonin.

[0094] The present invention also provides a pharmaceutical composition, which comprises the complex and a pharmaceutically acceptable excipient.

[0095] The present invention also provides a use of the complex or the pharmaceutical composition in preparing a drug for treating or preventing tumor-related diseases.

[0096] In certain embodiments, the tumor-related disease is selected from one or more of Wilms' tumor, Ewing's sarcoma, neuroendocrine tumors, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, bile duct cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, non-Hodgkin's lymphoma and bladder cancer.

[0097] In certain embodiments, the tumor-related disease is hepatocellular carcinoma.

[0098] In certain embodiments, the use comprises reversing tumor immunosuppression caused by iRFA.

[0099] Unless otherwise specified, the materials and equipment used in the present invention can be obtained by conventional methods in the art, or through commercial channels, including but not limited to: Material: Spermidine (SPM, Macklin), dextran (DEX, Mw: 40 kDa, J&K), shikonin (SHK, MedChemExpress), 1,2-dipalmitoylphosphatidylcholine (DPPC, Aladin), cholesterol (Chol, Aladin), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k, Sigma\u2012Aldrich), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Macklin), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-Cy5.5 (DMG-PEG-Cy5.5, Thermo Fisher Scientific), and immunolabeling antibodies (Biolegend, USA) were used as needed.

[0100] Cell lines: The H22 cell line used in the present invention was purchased from the American Type Culture Collection (ATCC). H22 cells were cultured in RPIM-1640 medium supplemented with penicillin (100 units / mL), streptomycin (50 units / mL) and fetal bovine serum (10%) at 37°C and 5% CO2.

[0101] Antibody: The anti-PD-L1 monoclonal antibody used in the present invention was purchased from the market, with the clone number being 10F.9G2.

[0102] animal: The BALB / c mice used in the present invention were purchased from SPF Biotechnology, Inc. (Beijing, China). The H22 tumor-bearing mouse model was established to evaluate the antitumor efficacy of SPS-NP. Briefly, 2×10 6 H22 cells (dispersed in 100 μL PBS). Tumors were selected when the volume reached ~150 mm 3 mice for further experiments.

[0103] Unless otherwise specified, the reagents or instruments used in the following examples without indicating the manufacturer are conventional products that can be purchased commercially; experimental methods without indicating specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0104] Example 1: Synthesis of SPM-DEX Dextran (DEX) was dissolved in deionized water at a concentration of 50 mg / mL and then oxidized using NaIO4 at a molar ratio of 1:1 for 8 hours in the dark. Subsequently, the solution was collected, dialyzed at 4°C for 2 days using a dialysis tube (14K), and then freeze-dried. The oxidized DEX was dissolved in deionized water and added to spermidine (SPM) dissolved in borate buffer (0.1 M, pH=11.0) and reacted at a molar ratio of 1:1.3 for 24 hours. Afterwards, excess NaBH4 was added and stirred for 72 hours to reduce the reactants. After 5 days of dialysis against deionized water and complete freeze-drying, SPM-DEX was obtained in solid form. The structure of SPM-DEX was verified by hydrogen nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 1 As shown, SPM-DEX was successfully synthesized.

[0105] Example 2: Preparation of SPS-NPs (1) Based on the electrostatic interaction between SPM-DEX and anti-PD-L1, SP-NPs (e.g. Figure 2 as shown).

[0106] SPM-DEX (1 mg / mL) and anti-PD-L1 (B7-H1, clone 10F.9G2) (500 μg / mL) solutions were prepared using HEPES buffer (100 mM) at pH 3.2 and 7.5. SP-NPs were obtained by injecting SPM-DEX solution and anti-PD-L1 solution through the corresponding inlets at a constant flow rate (1-20 mL / min) using a dual-inlet closed impinging jet (CIJ) mixer using a NE-4000 programmable multichannel syringe pump.

[0107] (2) The lipid layer was further coated on the SP-NPs to prepare SPS-NPs via a 3-inlet CIJ mixer.

[0108] DPPC, CHOL, and DMG-PEG were dissolved in ethanol at concentrations of 3, 1.3, and 1 mg / mL to prepare lipid solutions. SHK was then dissolved in the above solutions to a concentration of 5 mg / mL. The prepared SP-NPs were injected through inlets 2 and 3, and the DPPC / Chol / DMG-PEG solution was injected through inlet 1 at a flow rate of 20 mL / min to prepare SPS-NPs.

[0109] The sizes of the prepared SP-NPs and SPS-NPs were tested by a nanoparticle size and high-sensitivity zeta potential analyzer (NanoBrook Omni, USA) to evaluate their size distribution and surface charge. The morphology and structure of the SP-NPs and SPS-NPs were observed and confirmed by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR).

[0110] The particle size distribution of SP-NP and SPS-NP analyzed by Zetasizer is shown in Figure 3 As shown in Figure 2, the size of SP-NP is 95.8 nm, the PDI is ~0.19; the size of SPS NP is ~134 nm, the PDI is ~0.075, and the state is highly uniform. Figure 4 As shown in Figure 2, the highly positively charged surface of SP-NP (+28.3 mV) becomes a neutrally charged surface (+2.3 mV) after being coated with the lipid layer, further confirming the successful coating of DPPC. Figure 5 As shown in Figure 2, the prepared SPS-NPs are highly uniform and spherical with a diameter of ~100 nm, which is consistent with the data provided by the particle analyzer. Figure 6 As shown in Figure 2, the stretching vibration peaks of amine and hydroxyl groups in SPM-DEX (~3276 cm -1 ) red-shifted to ~3000 cm -1 , indicating that there is an electrostatic interaction between SPM-DEX and anti-PD-L1.

[0111] SPS-NPs were prepared multiple times by the above method, and the size distribution curves of different batches were almost overlapping (e.g. Figure 7 ), the difference is negligible.

[0112] Example 3: Effect of flow rate on SP-NP particle size distribution SPM-DEX (1 mg / mL) and anti-PD-L1 (500 μg / mL) solutions were prepared using HEPES buffer (100 mM) at pH 3.2 and 7.5. SP-NPs were obtained by injecting SPM-DEX solution and anti-PD-L1 solution through the corresponding inlets at a constant flow rate (1-20 mL / min) using a dual-inlet closed impinging jet (CIJ) mixer via a NE-4000 programmable multichannel syringe pump.

[0113] The results are as follows Figure 8 As shown in Figure 2, the size of SP-NPs decreased with increasing flow rate (1-20 mL / min). When the flow rate reached 20 mL / min, the size and PDI of SP-NPs were the largest, indicating that the rapid and uniform mixing of the CIJ device can minimize the nucleation time and prepare nanoparticles in a more uniform manner.

[0114] Example 4: In vitro release kinetics of SPS-NPs 1 mL of the SPS-NP sample prepared in Example 2 was added to the dialysis tube and immersed in 9 mL of PBS at different pH values. The plate was then incubated on an oscillator (150 rpm) at 37°C. 1 mL of sample was collected at specific time intervals of 1, 2, 4, 8, 16, 24, 36, and 48 hours, and 1 mL of fresh PBS was supplemented to maintain a constant volume. The concentration of SHK was determined by Genesis 6 spectrophotometer at 520 nm, and the concentration of anti-PDL1 was determined using an ELISA kit, and the results are shown in Figure 2. Fig. 9 shown.

[0115] Example 5: Biodistribution of SPS-NPs SPS-NPs were labeled with DMG-PEG-Cy5.5 probes. Cy5.5@SPS-NPs were prepared by a similar procedure, only replacing DMG-PEG-PEG with DMG-PEG-Cy5.5 in the lipid solution. The biodistribution of SPS-NPs after intravenous injection was studied using an H22 tumor-bearing mouse model.

[0116] Cy5.5@SPS-NP (equivalent dose of 1 mg / kg Cy5.5) was injected intravenously into H22 tumor-bearing mice. Fluorescence signals were then observed by IVIS (PerkinElmer) at 0, 1, 2, 4, 8, 12, and 24 hours after injection. At the end of the experiment, the mice were euthanized, and the tumors, heart, liver, spleen, lungs, and kidneys were collected for ex vivo imaging. The results are shown in Figure 10.

[0117] Example 6: In vivo anti-tumor efficacy of SPS-NPs The antitumor efficacy of SPS-NPs was evaluated using the H22 tumor-bearing mouse model, and iRFA was first applied on day 10 before SPS-NP treatment.

[0118] After anesthesia, the RF electrode was inserted into the tumor xenograft under ultrasound guidance to a depth of one-third of the tumor length. Then iRFA was performed at an output power of 5 W for 30 seconds. After iRFA treatment, the tumor-bearing mice were randomly divided into 5 groups and treated with different preparations on days 11, 13, and 15: (1) control group: intravenous injection of 100 μL normal saline; (2) SPM-DEX group: iv injection of 100 μL SPM-DEX solution (10 mg / kg); (3) anti-PDL1 group: intravenous injection of 100 μL anti-PDL1 solution (5 mg / kg); (4) SHK group: iv injection of 100 μL SHK solution (5 mg / kg); (5) SPS-NP group: intravenous injection of 100 μL SPS-NP (equivalent dose of 10 mg / kg SPM-DEX, 5 mg / kg anti-PDL1 and 5 mg / kg SHK). Tumor volume was measured using a vernier caliper every 4 days after iRFA application, and the results are shown in Figure 2. Fig.11 Tumors were harvested on day 26 for further flow cytometric and histological analysis, and survival was monitored using multiple H22 tumor-bearing mice that received the same treatment.

[0119] Following the steps described above, the iRFA-treated H22 tumor-bearing mouse model was established and H22 tumors were inoculated on day 0. iRFA was used on day 10, and after drug injection on days 11, 13, and 15, BALB / C mice were injected with 1×10 6The lung metastatic cancer-bearing mouse model was established with 100 μL of H22 cells (100 μL, dispersed in cell culture medium). The mice were randomly divided into two groups: (1) the control group, which was intravenously injected with 100 μL of normal saline; (2) the SPS-NP group, which was intravenously injected with 100 μL of SPS-NP (equivalent doses of 10 mg / kg SPM-DEX, 5 mg / kg anti-PDL1 and 5 mg / kg SHK). It took about 14 days for modular scattering to occur in the lungs, and lung tissues were collected and analyzed on the 30th day. The lungs were weighed and histologically analyzed for further evaluation. The results are shown in Fig.12 As shown in Figure 2 (** represents p < 0.01), the lung weight of the control group was ~66% larger than that of the SPS-NP group due to abnormal tumor growth. The significant difference in lung weight between the two groups also partially confirmed the anti-metastatic efficacy of SPS-NP.

[0120] Healthy BALB / C mice were used to evaluate the biosafety of SPS-NPs.

[0121] The mice were randomly divided into two groups: (1) healthy mice, which received intravenous injection of 100 μL normal saline; (2) SPS-NP group: received intravenous injection of 100 μL SPS-NP (equivalent dose of 10 mg / kg SPM-DEX, 5 mg / kg anti-PDL1 and 5mg / kg SHK). After 30 days, the serum of mice in different groups was collected, and the levels of transaminase (AST), alanine aminotransferase (ALT), albumin (ALB), total protein (TP), creatinine clearance (CCr) and urea (Urea) were detected by automatic biochemical analyzer. The results are shown in Table 1. Fig.13 shown.

[0122] In addition, major organs including heart, liver, spleen, lung, and kidney were collected for H&E staining to further evaluate biological toxicity.

[0123] The collected tissues were fixed with 4% paraformaldehyde, embedded in paraffin and cut into thin sections (3-4 μm thick). The tissue sections were dewaxed and rehydrated by xylene dewaxing and rehydration for 40 min, anhydrous ethanol for 20 min, 75% alcohol for 5 min and PBS for 5 min. The treated sections were then stained with hematoxylin for 10 min and then rinsed with water. Subsequently, the sections were stained with eosin for 3 min. The obtained sections were then dehydrated and sealed with resin. These sections were then observed using the Vectra automated quantitative pathology imaging system (PerkinElmer), and the results were as follows Fig.14 As shown, a large number of metastatic lymph nodes were detected in the resected lung tissues of the control group, whereas almost no metastatic lymph nodes were observed in the SPS-NP group.

[0124] The collected lung tissue sections were rehydrated and immersed in PBS (0.05% Tween-20, PBST). After recovering the antigen and inactivating the endogenous peroxidase activity by heating, the sections were washed three times with PBST. The sections were then blocked with 10% goat serum for 20 minutes and then incubated with HRP-conjugated anti-mouse antibodies against MMP2 and VEGF for 1 hour. The treated sections were then stained with DAB substrate and hematoxylin for observation under an optical microscope. The results are shown in Figure 2. Fig.14 , 15 As shown, the expression of VEGF and MMP in the SPS-NP group was only ~0.5-fold and ~0.6-fold higher than that in the control group, respectively, indicating that the metastasis of tumor cells was significantly inhibited. Considering that the anti-metastatic efficacy of SPS-NPs originated from the overall anti-tumor immune response, the cytokine levels in the mouse serum were determined to verify the successful induction of the immune response.

[0125] Following the steps described above, the tumors were harvested and collected for fixation with paraformaldehyde. The collected tissues were embedded in paraffin and cut into thin sections (3-4 μm thick). These sections were then dewaxed and immersed in PBST. After 45 seconds, the samples were heated in a microwave oven for antigen retrieval. After storage at room temperature, the sections were then treated with 3% hydrogen peroxide to inactivate endogenous peroxidase and washed three times with PBST. For immunofluorescence staining, the sections were blocked with 10% goat serum for 20 minutes and then incubated with fluorescently labeled antibodies (coupled with PKM2, Ki67, CRT, CD3, CD8, HIF-1α, HMGB1, Foxp3, and NKp46) at 4°C overnight.

[0126] The results are as follows Fig.16 As shown in Figure 3, anti-PD-L1 treatment increased CRT and HMGB1 levels in tumor tissues. More enhancement could be observed in the SHK and SPS-NP groups, with CRT levels elevated by 2- and 6-fold, respectively. Since CRT is a representative DAMP, its significant upregulation may indicate a reversal of the immunosuppressive tumor microenvironment.

[0127] TUNEL assay was used to evaluate the degree of cell apoptosis in tumor sites.

[0128] The collected tumor tissue sections were dewaxed and immersed in PBST, and then covered with a mixed solution of TDT enzyme, dUTP, and buffer (V / V / V=1:5:50) and incubated at 37°C for 2 hours. The sections were covered with glycerol and observed using a fluorescence microscope (ex: 450nm, em: 515nm).

[0129] The results are as follows Fig.17As shown, the purple color in the tumor sections of the anti-PD-L1 group and the SHK group was significantly reduced compared with the highly active cell division state (indicated by purple) in the tumor sections of the control group. Among all the groups, the tumor sections of the SPS-NP group showed the least purple, indicating that SPS-NP can significantly limit the proliferation of tumor cells. Correspondingly, the TUNEL signals from all these groups showed similar trends. Specifically, after treatment with anti-PD-L1 or SHK, the TUNEL signal was significantly increased. In the SPS-NP group, the TUNEL signal was the strongest. In addition, SPS-NP had a significant inhibitory effect on the expression of Ki67, indicating that the proliferation of cancer cells was effectively inhibited.

[0130] Flow cytometry was used to investigate the anti-tumor mechanism induced by SPS-NPs. The resected tumor tissues and tumor-draining lymph nodes were collected, cut into small pieces, and then incubated with RPMI 1640 medium (containing 2 mg / mL collagenase and 0.5 mg / mL DNAse) on a shaker (37°C, 80 rpm) for 20 min. The treated tissues were then ground on a cell strainer and washed simultaneously with RPMI 1640 medium (5% FBS). The cells were then centrifuged at 1000 rpm (5 min) and resuspended in RPMI1640 medium to obtain a single-cell suspension. The collected cells were then exposed to fluorescently labeled antibodies for 15 min, following the method previously described by CytoFLEX (Beckman Coulter). The following protocols were tested: L / D-FITC, CD45-APC-CY7, CD3-percp-cy5.5, CD8-PE-cy7, MHCⅡ-APC, CD11c-BV421, CD80-PE, CD86-BV510 or L / D-FITC, CD45-APC-CY7, CD3-percp-cy5.5, CD8-BV510, GZMB-PE, TNF-α-APC, IFN-γ-PE-cy 7, Ki67-BV421 or L / D-FITC, CD45-APC-CY7, CD3-percp-cy5.5, CD4-BV510, CD49b-PE, Foxp3-APC, CD25-PE-cy7 or L / D-FITC, CD45-APC-CY7, CD11b-PE, Gr1-BV421, F4 / 80-percp-cy5.5, CD206-PE-cy7 and CD86-BV510.

[0131] The results are as follows Figure 18-23As shown, in the anti-PDL1 group and the SHK group, the CD3+CD8+ cell population was significantly increased by 3.5-fold and 1.6-fold compared with the control group, respectively. Under the combined treatment of SHK and anti-PD-L1, the percentage of infiltrating T lymphocytes in the SPS-NP group (34.7%) was further increased by 0.46-fold and 1.5-fold compared with the single treatment group, respectively, indicating that the combined treatment of SHK and anti-PD-L1 promoted a specific anti-tumor response in tumor tissues. The SPS-NP group had a higher proportion of CD8+Ki67+ cells (82.5%), CD8+IFN-γ+ cells (20.7%), and CD8+GZMB+ cells (36.8%); these percentages were 3, 10, and 3.4 times that of the control group, respectively. SPM-DEX also showed a beneficial effect on the function of infiltrating CD3+CD8+ cells. The percentages of CD8+Ki67+ cells, CD8+IFN-γ+ cells, and CD8+GZMB+ cells in the SPX-DEX group were also significantly increased by 53%, 82%, and 56%, respectively, compared with those in the control group.

[0132] A high proportion of NK cells can be detected in tumor tissues, which shows that systemic administration of SPS-NP also triggers innate immune responses. In addition, SPS-NP treatment also effectively alleviated the inhibitory effect on the immune microenvironment in tumor tissues. The CD25+Foxp3+ subpopulation in the SPS-NP group was ~0.3-, ~0.3-, ~0.5-, and ~0.6 times that of the control group, SPM-DEX group, anti-PDL1 group, and SHK group, respectively. In addition, the number of F4 / 80+CD86+ cells in the SPS-NP group was 3.1 times, 2.7 times, 1.9 times, and 1.4 times that of the control group, SPM-DEX group, anti-PDL1 group, and SHK group, respectively.

[0133] Blood was collected from mice and centrifuged at 12000 rpm for 20 min to obtain serum. Tumor tissue was homogenized using a mixture of RIPA buffer and PMSF (v:v=100:1), and then the solution was mixed with zirconium oxide grinding beads (diameter = 3 mm) and ground at 6000 rpm for 1 min using a homogenizer. After incubation at 4°C for 30 min, tumor lysis was performed. The levels of IL-2, IL-6, IL-10, TGF-β, TNF-α, IFN-γ, and IL-12 in the collected serum or tumor lysates were determined according to the corresponding procedures provided by NovusBiological. The results are shown in Fig.24 (* represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001) As shown, inflammatory cytokines (including IL-2 and IL-6) were significantly increased, and anti-inflammatory cytokines (including IL-10 and TGF-β) were correspondingly decreased in the SPS-NP group.

[0134] The resected tumors were homogenized in RIPA buffer and incubated at 4°C for 40 minutes to collect protein samples. After the concentration was determined by BCA, samples containing 40 μg of protein were loaded on Novex Tris-glycine mini gels for electrophoresis and transferred to polyvinylidene difluoride membranes, which were then blocked with 5% skim milk in phosphate-buffered saline containing Tween-20 (PBST) buffer for 1 hour. The PVDF membrane was then incubated with primary antibodies (anti-PDL1 and anti-PKM2) at a dilution of 1:1000 in 5% skim milk at 4°C overnight. Subsequently, the membranes were washed three times with PBST, and the PVDF membranes were incubated with secondary antibodies at a dilution of 1:2000 (1 hour, room temperature). After washing 3 times with PBST, protein bands were observed using a gel imaging system.

[0135] The results are as follows Fig.25 As shown in Figure 3, PKM2 expression was downregulated in the SHK group. However, this downregulation was not as obvious as that in the SPS-NP group, whose nanoscale properties enabled faster distribution and longer retention in tumor tissues.

[0136] It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. A composite, characterized in that The complex comprises a polysaccharide conjugate, an anti-PD-L1 monoclonal antibody, and a PKM2 inhibitor.

2. The composite according to claim 1, characterized in that The polysaccharide conjugate is a conjugate of a T cell promoter and a polysaccharide.

3. The composite according to claim 2, characterized in that The T cell promoter is spermidine, the polysaccharide is dextran, and the PKM2 inhibitor is shikonin.

4. The composite according to claim 1, characterized in that The complex also includes PEG lipids, structural lipids and phospholipids; The PEG lipid is selected from one or more of DMG-PEG, DSG-PEG, DPG-PEG, DAG-PEG, DAA-PEG, DPPE-PEG, and DSPE-PEG; The structural lipid is selected from one or more of cholesterol, dihydrocholesterol, lanosterol, β-phytosterol, ergosterol, campesterol, stigmasterol, brassicasterol, algin sterol, tomatine, ursolic acid, α-tocopherol and derivatives thereof; The phospholipid is selected from one or more of EPC, DAPC, DBPC, DLPC, DMPC, DOPC, DOPE, DOPG, DPPC, DSPC, POPC, and POPE.

5. The composite according to claim 1, characterized in that The particle size of the composite is 80-150 nm.

6. A method for preparing a composite according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: (1) conjugating the T cell promoter to the polysaccharide to prepare the polysaccharide conjugate; (2) using a closed impinging jet (CIJ) mixer, mixing the polysaccharide conjugate prepared in step (1) with the anti-PD-L1 monoclonal antibody to prepare a monodisperse nanocomplex; (3) Using a closed impinging jet (CIJ) mixer, the PKM2 inhibitor is coated on the monodisperse nanocomposite prepared in step (2) to prepare the complex.

7. The method according to claim 6, characterized in that Step (2) is to inject the polysaccharide conjugate and the anti-PD-L1 monoclonal antibody into the CIJ mixer at a constant flow rate of 1-20 mL / min.

8. The method according to claim 6, characterized in that Step (3) is to dissolve the PKM2 inhibitor in a lipid solution, and then inject the lipid solution and the monodisperse nanocomplex prepared in step (2) into a CIJ mixer at a constant flow rate of 1-20 mL / min.

9. The method according to claim 8, characterized in that The lipid solution contains the PEG lipid, the structural lipid and the phospholipid, and the concentration of the PKM2 inhibitor in the lipid solution is 1-10 mg / mL.

10. The method according to any one of claims 6 to 9, characterized in that: In step (1), the T cell promoter is spermidine, the polysaccharide is dextran, and in step (3), the PKM2 inhibitor is shikonin.

11. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the complex according to any one of claims 1 to 5 and a pharmaceutically acceptable excipient.

12. Use of the complex according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 11 in the preparation of a medicament for treating or preventing tumor-related diseases.

13. The use according to claim 12, characterized in that The tumor-related disease is selected from one or more of Wilms' tumor, Ewing's sarcoma, neuroendocrine tumors, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, bile duct cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, non-Hodgkin's lymphoma and bladder cancer.

14. The use according to claim 12, characterized in that The use includes reversing tumor immunosuppression caused by iRFA.

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