A composite and its preparation method and application

Abstract

The present invention relates to a complex comprising a polysaccharide conjugate, an anti-PD-L1 monoclonal antibody, and a PKM2 inhibitor. The complex of the present invention can accumulate and remain in tumor tissue, effectively stimulate dendritic cell maturation, alleviate the inhibitory immune microenvironment, and trigger a 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 patients with advanced HCC by reprogramming iRFA-induced tumor immunosuppression, and has great potential in improving the prognosis of patients with advanced HCC treated with RFA.

Description

Technical Field

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

[0002] Hepatocellular carcinoma (HCC) is a malignant tumor that develops in liver cells and is a common malignancy that affects a large number of patients. To alleviate the suffering of HCC patients and improve their quality of life, precise and effective treatments are now available. Radiofrequency ablation (RFA), approved by the US Food and Drug Administration, has emerged as a key thermal ablation technique. By directly intervening at the tumor site, it can induce a significant and immediate anti-tumor effect (primarily coagulative necrosis) in the clinical treatment of HCC and some other cancers with well-defined tumor boundaries.

[0003] However, because the tumor tissue in these advanced HCC patients is large and / or irregularly shaped, most often harbor multiple tumor lesions, making tumor boundaries difficult to define. This poses a significant challenge to determining RFA treatment parameters (such as duration and extent), leading to 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 a poor prognosis for patients with advanced HCC. Detailed mechanistic studies have demonstrated that iRFA treatment can worsen the tumor's 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. Consequently, the T cell-based anti-tumor immune response within the residual tumor mass is significantly suppressed, promoting local tumor recurrence and acquiring resistance 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 patient treatment 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 exploited to meet the high energy demands of iRFA-promoted tumor progression. In this context, large amounts of immunosuppressive lactate are 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 in 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] Problems to be solved by the invention

[0006] In view of the above problems existing in the prior art, the object of the present invention is to provide a nanomedicine that can reverse the tumor immunosuppression and tumor treatment failure exacerbated by iRFA.

[0007] The present invention provides a complex comprising a polysaccharide conjugate, an anti-PD-L1 monoclonal antibody, and a PKM2 inhibitor.

[0008] Preferably, the polysaccharide conjugate is a conjugate of a T cell promoter and a polysaccharide.

[0009] Preferably, the T cell promoter is spermidine, the polysaccharide is dextran, and the PKM2 inhibitor is shikonin.

[0010] Preferably, the complex further comprises PEG lipids, structural lipids and phospholipids;

[0011] 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;

[0012] The structural 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;

[0013] The phospholipid is selected from one or more of EPC, DAPC, DBPC, DLPC, DMPC, DOPC, DOPE, DOPG, DPPC, DSPC, POPC, and POPE.

[0014] Preferably, the particle size of the complex is 80-150 nm.

[0015] The present invention also provides a method for preparing the composite, comprising the following steps:

[0016] (1) conjugating the T cell promoter to the polysaccharide to prepare the polysaccharide conjugate;

[0017] (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;

[0018] (3) Using a closed impinging jet (CIJ) mixer, the PKM2 inhibitor is coated on the monodisperse nanocomplex prepared in step (2) to prepare the complex.

[0019] 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.

[0020] Preferably, step (3) is to dissolve the PKM2 inhibitor in a lipid solution, and then inject it and the monodisperse nanocomplex prepared in step (2) into a CIJ mixer at a constant flow rate of 1-20 mL / min.

[0021] 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.

[0022] Preferably, the T cell promoter in step (1) is spermidine, the polysaccharide is dextran, and the PKM2 inhibitor in step (3) is shikonin.

[0023] The present invention also provides a pharmaceutical composition comprising the complex and a pharmaceutically acceptable excipient.

[0024] 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.

[0025] Preferably, the tumor-related disease is selected from one or more of Wilms' tumor, Ewing sarcoma, neuroendocrine tumor, 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.

[0026] Preferably, the use comprises reversing tumor immunosuppression caused by iRFA.

[0027] The complex of the present invention can accumulate and remain in tumor tissue, effectively stimulating dendritic cell maturation, alleviating the suppressive immune microenvironment, and triggering a specific anti-tumor immune response. It can effectively inhibit the expression of PKM2, thereby suppressing glycolysis in tumor tissue and providing a more favorable environment for T lymphocyte infiltration of tumor tissue. The complex of the present invention is expected to enhance the therapeutic effect of RFA in patients with advanced HCC by reprogramming iRFA-induced tumor immunosuppression, and has great potential to improve the prognosis of patients with advanced HCC treated with RFA. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the H NMR spectrum of SPM-DEX.

[0029] Figure 2 Schematic diagram of the preparation of SPS-NPs and SPS-NP-mediated reversal of tumor immunosuppression induced by iRFA therapy to enhance HCC treatment.

[0030] Figure 3 Particle size distribution of SP-NP and SPS-NP analyzed by Zetasizer.

[0031] Figure 4 is the zeta potential of SP-NP and SPS-NP.

[0032] Figure 5 The morphology of SPS-NPs was photographed by TEM.

[0033] Figure 6 FT-IR spectrum of SPS-NP.

[0034] Figure 7 Particle size distribution of SPS-NPs prepared from different batches.

[0035] Figure 8 Figure 3 is the effect of flow rate on the particle size and polydispersity index (PDI) of SP-NPs.

[0036] Figure 9 In vitro release kinetics of SHK (left) and anti-PDL1 (right) at different pH values.

[0037] Figure 10 Figure 2 shows the biodistribution of SPS-NP in H22 tumor-bearing mice 24 hours after intravenous injection. All data are shown as SD ± mean (n = 3).

[0038] Figure 11 This is a graph showing the relationship between time and mouse tumor volume.

[0039] Figure 12 The weight of the excised lungs from mice in each group.

[0040] Figure 13 The biosafety test results of SPS-NP.

[0041] Figure 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).

[0042] Figure 15 Semi-quantitative analysis of VEGF and MMP2 expression in lung sections of each group.

[0043] Figure 16Immunofluorescence analysis of PKM2, CRT, and HMGB1 expressions in tumor tissues of mice in each group. Scale bar = 50 μm.

[0044] Figure 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).

[0045] Figure 18 Representative flow cytometric analysis of CD3+CD8+ cells and corresponding quantitative data.

[0046] Figure 19 Function of CD8+ lymphocytes after gating on Ki67+, TNF-α+, IFN-γ+, and GZMB+ was analyzed by flow cytometry.

[0047] Figure 20 Representative flow cytometric analysis of CD3+CD49b+ cells and corresponding quantitative data.

[0048] Figure 21 Representative flow cytometric analysis of Gr1+ CD11b+ cells and corresponding quantitative data.

[0049] Figure 22 Representative flow cytometric analysis of CD25+Foxp3+ cells and corresponding quantitative data.

[0050] Figure 23 Representative flow cytometric analysis and corresponding quantitative data of F4 / 80+CD86+ and F4 / 80+CD206+ cell populations.

[0051] Figure 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).

[0052] Figure 25 Western blot analysis of PKM2 expression in tumor tissues of mice in each group.

[0053] Description of reference numerals:

[0054] * represents p < 0.05, indicating statistically significant difference;

[0055] ** represents p < 0.01, indicating a significant statistical difference;

[0056] *** represents p < 0.001, indicating extremely significant statistical differences. DETAILED DESCRIPTION

[0057] To make the technical solutions and beneficial effects of the present invention more clearly understood, the following detailed description is given by way of specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly illustrate 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.

[0058] 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-amine. Such lipids are also referred to as PEGylated lipids. In certain embodiments, 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).

[0059] 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, alginosterol, 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.

[0060] As used herein, the term "phospholipid" refers to 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. Generally, a phospholipid comprises a phospholipid moiety and one or more fatty acid moieties. A phospholipid may comprise one or more multiple (e.g., double or triple) bonds (e.g., one or more degrees of unsaturation). A phospholipid or its analog or derivative may comprise choline. A phospholipid or its analog or derivative may not comprise choline. Certain phospholipids can promote fusion with membranes. In certain embodiments, a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cell or intracellular membrane). Fusion of a phospholipid with a membrane can allow one or more elements of a lipid-containing composition to pass through the membrane, thereby allowing, for example, delivery of the one or more elements 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.

[0061] The present invention provides a complex comprising a polysaccharide conjugate, an anti-PD-L1 monoclonal antibody, and a PKM2 inhibitor.

[0062] In certain embodiments, the polysaccharide conjugate is a conjugate of a T cell promoter and a polysaccharide.

[0063] In certain embodiments, the T cell promoter is spermidine, the polysaccharide is dextran, and the PKM2 inhibitor is shikonin.

[0064] In certain embodiments, the polysaccharide conjugate is a conjugate of spermidine and dextran.

[0065] In certain embodiments, the complex further comprises a PEG lipid, a structural lipid, and a phospholipid.

[0066] 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.

[0067] In certain embodiments, the PEG lipid is DMG-PEG.

[0068] 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.

[0069] In certain embodiments, the structured lipid is cholesterol.

[0070] 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.

[0071] In certain embodiments, the phospholipid is DPPC.

[0072] In certain embodiments, the complex comprises a conjugate of spermidine and dextran, an anti-PD-L1 monoclonal antibody, shikonin, DMG-PEG, cholesterol, and DPPC.

[0073] 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.

[0074] In certain embodiments, the particle size of the complex is 80-150 nm.

[0075] In certain embodiments, the particle size of the complex is 100-140 nm.

[0076] In certain embodiments, the complex has a particle size of about 130 nm.

[0077] The present invention also provides a method for preparing the composite, comprising the following steps:

[0078] (1) conjugating the T cell promoter to the polysaccharide to prepare the polysaccharide conjugate;

[0079] (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;

[0080] (3) Using a closed impinging jet (CIJ) mixer, the PKM2 inhibitor is coated on the monodisperse nanocomplex prepared in step (2) to prepare the complex.

[0081] In certain embodiments, step (2) is injecting the polysaccharide conjugate and the anti-PD-L1 monoclonal antibody into the CIJ mixer at a constant flow rate of 1-20 mL / min.

[0082] 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.

[0083] In certain embodiments, the flow rate in step (2) is 20 mL / min.

[0084] 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.

[0085] 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.

[0086] In certain embodiments, the flow rate in step (3) is 20 mL / min.

[0087] 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.

[0088] In certain embodiments, the concentration of the PEG lipid in the lipid solution is 1-10 mg / mL.

[0089] 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.

[0090] In certain embodiments, the concentration of the PEG lipid in the lipid solution is 3 mg / mL.

[0091] 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.

[0092] In certain embodiments, the PEG lipid is DMG-PEG.

[0093] In certain embodiments, the concentration of the structured lipid in the lipid solution is 0.5-3 mg / mL.

[0094] 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.

[0095] In certain embodiments, the concentration of the structured lipid in the lipid solution is 1.3 mg / mL.

[0096] 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.

[0097] In certain embodiments, the structured lipid is cholesterol.

[0098] In certain embodiments, the concentration of the phospholipid in the lipid solution is 0.5-3 mg / mL.

[0099] 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.

[0100] In certain embodiments, the concentration of the phospholipid in the lipid solution is 1 mg / mL.

[0101] In certain embodiments, the phospholipid is selected from one or more of DLPC, DMPC, DOPC, DPPC, DSPC, DUPC, and POPC.

[0102] In certain embodiments, the phospholipid is DPPC.

[0103] 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.

[0104] In certain embodiments, the concentration of the PKM2 inhibitor in the lipid solution is 5 mg / mL.

[0105] 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.

[0106] The present invention also provides a pharmaceutical composition comprising the complex and a pharmaceutically acceptable excipient.

[0107] 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.

[0108] In certain embodiments, the tumor-related disease is selected from one or more of Wilms' tumor, Ewing 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.

[0109] In certain embodiments, the tumor-related disease is hepatocellular carcinoma.

[0110] In certain embodiments, the use comprises reversing iRFA-induced tumor immunosuppression.

[0111] 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:

[0112] Material:

[0113] Spermidine (SPM, Macklin), dextran (DEX, Mw: 40 kDa, J&K), shikonin (SHK, MedChemExpress), 1,2-dipalmitoylphosphatidylcholine (DPPC, Aladdin), cholesterol (Chol, Aladin), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k, Sigma-Aldrich), 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.

[0114] Cell lines:

[0115] The H22 cell line used in this 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 in 5% CO2.

[0116] Antibody:

[0117] The anti-PD-L1 monoclonal antibody used in the present invention was commercially available, with clone number 10F.9G2.

[0118] animal:

[0119] The BALB / c mice used in this 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.

[0120] Unless otherwise stated, the reagents or instruments used in the following examples without manufacturer indication are all conventional products that can be purchased commercially. Experimental methods without specific conditions are usually performed under conventional conditions or those recommended by the manufacturers.

[0121] Example 1: Synthesis of SPM-DEX

[0122] 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 using a dialysis tube (14K) at 4°C for 2 days, 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.

[0123] Example 2: Preparation of SPS-NPs

[0124] (1) Based on the electrostatic interaction between SPM-DEX and anti-PD-L1, SP-NPs (such as Figure 2 shown).

[0125] 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 the SPM-DEX and anti-PD-L1 solutions through the corresponding inlets of a dual-inlet closed impinging jet (CIJ) mixer at a constant flow rate (1-20 mL / min) using a NE-4000 programmable multichannel syringe pump.

[0126] (2) The lipid layer was further coated on the SP-NPs to prepare SPS-NPs via a 3-inlet CIJ mixer.

[0127] 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 this solution 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.

[0128] The size distribution and surface charge of the prepared SP-NPs and SPS-NPs were evaluated using a nanoparticle size and high-sensitivity zeta potential analyzer (NanoBrook Omni, USA). The morphology and structure of the SP-NPs and SPS-NPs were observed and confirmed using transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR).

[0129] 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 6As shown, the stretching vibration peaks of amine and hydroxyl groups in SPM-DEX (~3276 cm -1 ) redshifted to ~3000 cm -1 , indicating that there is an electrostatic interaction between SPM-DEX and anti-PD-L1.

[0130] 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.

[0131] Example 3: Effect of flow rate on SP-NP particle size distribution

[0132] 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 the SPM-DEX and anti-PD-L1 solutions through the corresponding inlets of a dual-inlet closed impinging jet (CIJ) mixer at a constant flow rate (1-20 mL / min) using a NE-4000 programmable multichannel syringe pump.

[0133] The results are as follows Figure 8 As shown in the figure, the size of SP-NPs decreases with increasing flow rate (1-20 mL / min). When the flow rate reaches 20 mL / min, the size and PDI of SP-NPs are 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.

[0134] Example 4: In vitro release kinetics of SPS-NPs

[0135] 1 mL of the SPS-NP sample prepared in Example 2 was added to a dialysis tube and submerged in 9 mL of PBS at different pH values. The plate was then incubated on a shaker (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 added to maintain a constant volume. The concentration of SHK was measured at 520 nm using a Genesis 6 spectrophotometer, and the concentration of anti-PDL1 was measured using an ELISA kit. The results are shown in Figure 2. Figure 9 shown.

[0136] Example 5: Biodistribution of SPS-NPs

[0137] SPS-NPs were labeled with a DMG-PEG-Cy5.5 probe. Cy5.5@SPS-NPs were prepared using a similar procedure, replacing DMG-PEG-PEG with DMG-PEG-Cy5.5 in the lipid solution. The biodistribution of SPS-NPs following intravenous injection was studied in an H22 tumor-bearing mouse model.

[0138] H22 tumor-bearing mice were intravenously injected with Cy5.5@SPS-NPs (equivalent to 1 mg / kg Cy5.5). Fluorescence signals were then observed using an 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 tumor, heart, liver, spleen, lung, and kidney were collected for ex vivo imaging. The results are shown in Figure 10.

[0139] Example 6: In vivo anti-tumor efficacy of SPS-NPs

[0140] The antitumor efficacy of SPS-NP was evaluated using an H22 tumor-bearing mouse model, and iRFA was first applied on day 10 before SPS-NP treatment.

[0141] After anesthesia, the radiofrequency 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 5W 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. Figure 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.

[0142] Following the above-mentioned steps, an 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 by using 100 μL of H22 cells (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 the module 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 Figure 2. Figure 12 As shown in the figure (** represents p < 0.01), the lung weight of the control group was ~66% greater than that of the SPS-NP group due to abnormal tumor growth. This significant difference in lung weight between the two groups also partially confirms the anti-metastatic efficacy of SPS-NP.

[0143] Healthy BALB / C mice were used to evaluate the biosafety of SPS-NP.

[0144] 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 5 mg / 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 measured using an automatic biochemical analyzer. The results are shown in Figure 3. Figure 13 shown.

[0145] In addition, major organs including heart, liver, spleen, lung, and kidney were collected for H&E staining to further evaluate biological toxicity.

[0146] 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 minutes, anhydrous ethanol for 20 minutes, 75% alcohol for 5 minutes and PBS for 5 minutes. The treated sections were then stained with hematoxylin for 10 minutes and then rinsed with water. Subsequently, the sections were stained with eosin for 3 minutes. The obtained sections were then dehydrated and sealed with resin. The sections were then observed using the Vectra automated quantitative pathology imaging system (PerkinElmer), and the results were as follows. Figure 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.

[0147] 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 a light microscope. The results are shown in Figure 2. Figure 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 tumor cell metastasis was significantly inhibited. Considering that the anti-metastatic efficacy of SPS-NP stems from the overall anti-tumor immune response, cytokine levels in mouse serum were measured to verify the successful induction of the immune response.

[0148] Following the procedures described above, 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 treated with 3% hydrogen peroxide to inactivate endogenous peroxidases and washed three times with PBST. For immunofluorescence staining, sections were blocked with 10% goat serum for 20 minutes and then incubated with fluorescently labeled antibodies (conjugated to PKM2, Ki67, CRT, CD3, CD8, HIF-1α, HMGB1, Foxp3, and NKp46) overnight at 4°C.

[0149] The results are as follows Figure 16 As shown, anti-PD-L1 treatment increased CRT and HMGB1 levels in tumor tissue. Further enhancement was observed in the SHK and SPS-NP groups, with CRT levels elevated 2- and 6-fold, respectively. As CRT is a representative DAMP, its significant upregulation may indicate a reversal of the immunosuppressive tumor microenvironment.

[0150] TUNEL assay was used to assess the degree of cell apoptosis in the tumor site.

[0151] Collected tumor tissue sections were deparaffinized and immersed in PBST. The sections were mounted with a mixture of TDT enzyme, dUTP, and buffer (V / V / V = 1:5:50) and incubated at 37°C for 2 hours. The sections were mounted with glycerol and observed using a fluorescence microscope (ex: 450 nm, em: 515 nm).

[0152] The results are as follows Figure 17As shown, compared to the highly active cell division state (indicated by purple) in tumor sections from the control group, the purple color was significantly reduced in tumor sections from the anti-PD-L1 and SHK groups. Tumor sections from the SPS-NP group exhibited the least purple color among all groups, indicating that SPS-NPs significantly restricted tumor cell proliferation. Correspondingly, TUNEL signals from all these groups showed similar trends. Specifically, TUNEL signals were significantly elevated after treatment with anti-PD-L1 or SHK. The TUNEL signal was strongest in the SPS-NP group. Furthermore, SPS-NPs significantly inhibited Ki67 expression, indicating effective suppression of cancer cell proliferation.

[0153] Flow cytometry was used to investigate the anti-tumor mechanism induced by SPS-NPs. 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 minutes. The treated tissue was then ground on a cell strainer and washed simultaneously with RPMI 1640 medium (5% FBS). The cells were then centrifuged at 1000 rpm (5 minutes) and resuspended in RPMI1640 medium to obtain a single-cell suspension. The collected cells were then exposed to fluorescently labeled antibodies for 15 minutes, following a 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.

[0154] The results are as follows Figure 18-23As shown, the CD3+CD8+ cell populations in the anti-PD-L1 and SHK groups were significantly elevated by 3.5-fold and 1.6-fold, respectively, compared with the control group. Combined SHK and anti-PD-L1 treatment further increased the percentage of infiltrating T lymphocytes in the SPS-NP group (34.7%) by 0.46-fold and 1.5-fold, respectively, compared with the monotherapy groups, indicating that combined SHK and anti-PD-L1 treatment promoted a specific anti-tumor response in tumor tissue. The SPS-NP group exhibited higher proportions of CD8+Ki67+ cells (82.5%), CD8+IFN-γ+ cells (20.7%), and CD8+GZMB+ cells (36.8%); these percentages were 3-fold, 10-fold, and 3.4-fold higher than those in the control group, respectively. SPM-DEX also demonstrated beneficial effects 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.

[0155] A high proportion of NK cells was detected in tumor tissue, indicating that systemic administration of SPS-NPs also triggered an innate immune response. Furthermore, SPS-NP treatment effectively alleviated the inhibitory effects on the immune microenvironment in tumor tissue. The CD25+Foxp3+ subset in the SPS-NP group was ~0.3-, ~0.3-, ~0.5-, and ~0.6-fold higher than that in the control, SPM-DEX, anti-PDL1, and SHK groups, respectively. Furthermore, the number of F4 / 80+CD86+ cells in the SPS-NP group was 3.1-, 2.7-, 1.9-, and 1.4-fold higher than that in the control, SPM-DEX, anti-PDL1, and SHK groups, respectively.

[0156] Blood was collected from mice and centrifuged at 12,000 rpm for 20 minutes 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 6,000 rpm for 1 minute using a homogenizer. After incubation at 4°C for 30 minutes, 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 Figure 2. Figure 24 As shown in the data (*p < 0.05; **p < 0.01; ***p < 0.001), 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.

[0157] Resected tumors were homogenized in RIPA buffer and incubated at 4°C for 40 minutes to collect protein samples. After concentration was determined by BCA assay, samples containing 40 μg of protein were loaded onto Novex Tris-glycine mini-gels for electrophoresis and transferred to polyvinylidene difluoride membranes, which were then blocked with 5% nonfat milk in phosphate-buffered saline containing Tween-20 (PBST) buffer for 1 hour. The PVDF membranes were then incubated with primary antibodies (anti-PDL1 and anti-PKM2) at a 1:1000 dilution in 5% nonfat milk at 4°C overnight. Subsequently, the membranes were washed three times with PBST and incubated with secondary antibodies at a 1:2000 dilution (1 hour, room temperature). After washing three times with PBST, protein bands were visualized using a gel imaging system.

[0158] The results are as follows Figure 25 As shown in Figure 3, PKM2 expression was downregulated in the SHK group. However, this downregulation was less pronounced than in the SPS-NP group, as the nanoscale properties of the SPS-NP group resulted in faster distribution and longer retention in tumor tissue.

[0159] 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 to the above embodiments without departing from the scope of this 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 this invention.

Claims

1. A composite, characterized in that The complex includes a polysaccharide conjugate, an anti-PD-L1 monoclonal antibody, and a PKM2 inhibitor; the polysaccharide conjugate is a conjugate of a T cell promoter and a polysaccharide; the T cell promoter is spermidine, the polysaccharide is dextran, and the PKM2 inhibitor is shikonin; The preparation of the composite 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 nanocomplex prepared in step (2) to prepare the complex.

2. 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, alginosterol, tomatine, ursolic acid, and α-tocopherol; The phospholipid is selected from one or more of EPC, DAPC, DBPC, DLPC, DMPC, DOPC, DOPE, DOPG, DPPC, DSPC, POPC, and POPE.

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

4. The composite according to claim 1, 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.

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

6. The composite according to claim 5, characterized in that The lipid solution contains PEG lipids, structural lipids and phospholipids, and the concentration of the PKM2 inhibitor in the lipid solution is 1-10 mg / mL.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the complex according to any one of claims 1 to 6 and a pharmaceutically acceptable excipient.

8. Use of the complex according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating or preventing tumor-related diseases; The tumor-related disease is selected from one or more of Wilms' tumor, Ewing sarcoma, neuroendocrine tumor, 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.

9. The use according to claim 8, characterized in that The use includes reversing tumor immunosuppression caused by iRFA.

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