Drug-loaded nano-cluster as well as preparation method and application thereof

Abstract

The invention relates to the field of biological pharmacy, in particular to a drug-loaded nano-cluster, a preparation method and application thereof. The invention provides a drug-loaded nano-cluster. The drug-loaded nano-cluster comprises a nano-cluster and an anti-tumor drug, the mass ratio of the nanocluster to the anti-tumor drug is (92.00 to 92.32) to (7.68 to 8.00). According to the cobalt-based drug-loaded nano-cluster for delivering the lenvatinib, the drug-loaded characteristic and immunogenicity of ovalbumin and cobalt ion-mediated metal immunity are utilized, so that the immunosuppression microenvironment of hepatocellular carcinoma is improved, the treatment effect of the lenvatinib is improved, and the drug resistance is reduced; a novel immune activation scheme is formed by combining with a PD-1 monoclonal antibody and is used for systemic drug therapy, and the immunotherapy curative effect of hepatocellular carcinoma is enhanced.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular to drug-loaded nanoclusters, preparation methods and applications thereof. Background Art

[0002] As a highly lethal tumor disease, the incidence of primary liver cancer is increasing worldwide, causing serious disease and economic burden. Hepatocellular carcinoma (HCC) is the main type of primary liver cancer. Systemic drug therapy is the first choice for advanced HCC. Tyrosine kinase inhibitors represented by sorafenib and lenvatinib are the first-line treatment drugs for HCC as molecular targeted drugs. After the introduction of ICIs, new systemic treatment strategies have developed rapidly. ICIs represented by PD-1 / PD-L1 antibodies have affected the treatment of HCC, especially the molecular targeted drug atezolizumab combined with bevacizumab combined with immune checkpoint inhibitor therapy, which can significantly improve the prognosis of patients with liver cancer. Encouraged by this, more new combination regimens have entered the clinical trial stage of HCC, such as lenvatinib combined with pembrolizumab, durvalumab combined with tremelimumab, and nivolumab combined with ipilimumab. However, in the LEAP-002 clinical trial, the combination of lenvatinib and pembrolizumab did not show greater benefits, and neither OS nor PFS was better than lenvatinib monotherapy, suggesting that drug resistance caused by the immunosuppressive microenvironment still limits the potential application of immunotherapy. Researchers have begun to explore new immune activation schemes in order to overcome the tumor's immunosuppressive microenvironment and reduce drug resistance, and to combine molecular targeted drugs with immune checkpoint inhibitors to improve the therapeutic effect while reducing the dose required for optimal treatment. Many new immune activation schemes, including metal immunity and immune adjuvants, have received increasing attention.

[0003] At present, the research hotspot of metal immunity focuses on utilizing the immune activation properties of metal elements or using them as drug co-delivery platforms to enhance the efficacy of existing immunotherapy. Cobalt ions can be used as coordination sites of organic metal compounds to form coordination polymers, and can play a physiological role as a cofactor of vitamin B12 (cobalamin) in the body. The metal valence activity of cobalt elements makes it easy to synthesize cobalt-based nanoscale drug delivery systems. At the same time, due to their superior magnetic properties and proton relaxation effects, cobalt-based magnetic fluids have been designed for use in medical testing and imaging-related fields. Cobalt elements also play a positive role in enhancing anti-tumor immunity and reversing immunosuppressive TME, by enhancing the maturation and function of APCs or promoting the activation of the cGAS-STING pathway, thereby increasing the local infiltration and anti-tumor activity of T cells, NK cells, etc., and enhancing the killing ability of immune cells against tumor cells. In addition, cobalt ions can also interact with pathways related to tumor growth, angiogenesis, and energy metabolism by inducing ROS generation, regulating the tumor microenvironment, and thus affecting tumor growth and development. Therefore, cobalt can not only enhance the effect of immunotherapy by transforming the immunosuppressive TME, but also serve as one of the components of the nanocarrier system to exert therapeutic effects together with the drugs it carries.

[0004] Ovalbumin (OVA) is a monomeric phosphoglycoglobulin composed of 365 amino acids with a molecular weight between 42 and 47 kDa. It is a common model antigen. OVA is widely used to study tumor immune responses due to its significant immunogenicity. It can be captured by APCs and presented to T cells to induce specific immune responses. In recent years, the use of OVA as a carrier to construct drug-loaded nanoclusters in tumor treatment has also emerged. Its excellent biocompatibility and stability, as well as its specific amino acid structure, enable it to achieve self-assembly through chemical regulation, and then encapsulate a large number of drug molecules to form nanoclusters, achieving stable drug delivery and long-term sustained release. Through the strong antigenicity and drug-loading properties of OVA, OVA-based drug-loaded nanoclusters can serve as a bridge connecting metal immunity and liver cancer drug treatment, and is a platform for activating the immune microenvironment of liver cancer.

[0005] Numerous studies have found that activation of the cGAS-STING pathway in preclinical models can effectively improve the therapeutic effect of ICIs. The possible mechanisms include inducing the production of IFN I and related inflammatory factors after the STING pathway is activated, improving the antigen recognition ability of APCs, CD4+ T cells and CD8+ T cells, and enhancing the cytotoxicity of macrophages, DCs, NK cells, B cells and T cells against tumor cells, so as to improve the immunosuppressive microenvironment and provide good conditions for the implementation of immunotherapy such as ICIs. The cGAS-STING pathway is an evolutionarily conserved mechanism for the body to initiate an immune response against pathogens, and plays an important role in the initiation and enhancement of innate immunity and adaptive immunity. After cGAS recognizes dsDNA in the cytoplasm, it uses ATP and GTP as substrates to promote the production of cGAMP and bind it to STING on the endoplasmic reticulum membrane, thereby activating STING-related pathways. The role of metal ions in the cGAS-STING pathway is becoming increasingly clear, such as Mn 2+ The sensitivity of cGas to dsDNA can be increased by stabilizing the active center conformation of cGas or promoting the interaction between substrate and enzyme to form cGAMP, or Co 2+ 、Zn 2+ It can bind to certain key amino acid residues of the STING protein, changing its conformation and making it easier to bind to cGAMP. In the context of the combination of molecular targeted drugs and immune checkpoint inhibitors for the treatment of hepatocellular carcinoma, introducing metal immunity and constructing engineered nanocarriers to deliver drugs to activate the cGAS-STING pathway to activate the liver cancer immune microenvironment to improve the efficacy of immunotherapy is a promising strategy. Summary of the invention

[0006] In view of this, the present invention provides drug-loaded nanoclusters, preparation methods and applications thereof. The present invention develops a cobalt-based drug-loaded nanocluster for delivering lenvatinib, which uses cobalt ion-mediated metal immunity to improve the immunosuppressive microenvironment, improve the therapeutic effect of lenvatinib and reduce drug resistance, and is combined with PD-1 monoclonal antibody to form a new immune activation scheme for systemic drug treatment, thereby enhancing the efficacy of immunotherapy for hepatocellular carcinoma.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a drug-loaded nanocluster, comprising: a nanocluster and an anti-tumor drug;

[0009] The mass ratio of the nanoclusters to the drug is: (92.00-92.32): (7.68-8.00).

[0010] In some embodiments of the present invention, in the above-mentioned drug-loaded nanoclusters, the drug-loaded nanoclusters further include: a matrix; the matrix includes: protein; the protein includes: one or more of ovalbumin, human albumin, human hemoglobin, bovine serum albumin and bovine hemoglobin.

[0011] In some embodiments of the present invention, in the above-mentioned drug-loaded nanoclusters, the matrix of the drug-loaded nanoclusters is composed of protein allosteric self-assembly.

[0012] In some embodiments of the present invention, in the above-mentioned drug-loaded nanoclusters, the protein is ovalbumin.

[0013] In some embodiments of the present invention, in the above-mentioned drug-loaded nanoclusters, the anti-tumor drugs include: one or more of lenvatinib, sorafenib and regorafenib.

[0014] In some embodiments of the present invention, in the above-mentioned drug-loaded nanoclusters, the anti-tumor drug is: lenvatinib.

[0015] In some embodiments of the present invention, in the above-mentioned drug-loaded nanoclusters, the nanoclusters are obtained after being protected by metal ligands.

[0016] In some embodiments of the present invention, in the above-mentioned drug-loaded nanoclusters, the metal ligands include: one or more of cobalt ligands, manganese ligands, iron ligands, gold ligands and copper ligands.

[0017] In some embodiments of the present invention, in the above-mentioned drug-loaded nanoclusters, the metal ligand is a cobalt ligand.

[0018] The present invention also provides a method for preparing the drug-loaded nanoclusters, comprising the following steps:

[0019] S1: mixing the solution of the anti-tumor drug and the solution of the nanoclusters protected by the metal ligand, adding the mixture dropwise to the solution of the matrix, adjusting the pH value, and obtaining a mixed solution;

[0020] S2: Stirring the mixed solution, adjusting the pH value again, dialyzing, filtering, and freeze-drying to obtain the drug-loaded nanoclusters.

[0021] In some embodiments of the present invention, the pH value is adjusted to 2-3 in the above preparation method S1.

[0022] In some embodiments of the present invention, the pH value is adjusted to 7-8 in the above preparation method S2.

[0023] In some embodiments of the present invention, in the above preparation method, the concentration of the solution of the metal ligand-protected nanoclusters is 30 mg / mL; the concentration of the solution of the anti-tumor drug is 250 μg / mL; and the concentration of the matrix solution is 2 mg / mL.

[0024] In some embodiments of the present invention, in the above preparation method, the solution of the anti-tumor drug is obtained by mixing the anti-tumor drug with 5% acetic acid.

[0025] In some embodiments of the present invention, in the above preparation method, the solution of the metal ligand-protected nanoclusters is obtained by mixing the metal ligand-protected nanoclusters with 5% acetic acid.

[0026] In some embodiments of the present invention, the above-mentioned preparation method includes a pH-regulated self-assembly process: Different from traditional physical mixing or chemical synthesis methods, the present invention adopts pH-regulated self-assembly technology, and achieves efficient and stable loading of lenvatinib and cobalt ions on ovalbumin (OVA) to form drug-loaded nanoclusters by precisely adjusting the pH value. This method improves the encapsulation efficiency and stability of the drug.

[0027] In some embodiments of the present invention, in the above-mentioned preparation method, fine control of solution titration is adopted: during the preparation process, the mixed liquid (c) of the solution of the anti-tumor drug and the metal ligand solution is gradually added to the protein matrix solution (b) by titration, thereby ensuring the uniformity and controllability of the reaction and avoiding premature precipitation or inactivation of the drug components.

[0028] In some embodiments of the present invention, in the above-mentioned preparation method, post-treatment of dialysis and freeze-drying is adopted: dialysis is performed using a dialysis bag to effectively remove unreacted raw materials and low molecular weight impurities, thereby ensuring the purity and stability of the nanoclusters, and the subsequent freeze-drying step further ensures the long-term storage of the nanoclusters.

[0029] The present invention also provides the use of the drug-loaded nanoclusters and / or the drug-loaded nanoclusters obtained by the above preparation method in the preparation of any of the following products:

[0030] (I) downregulating the expression and / or translation efficiency of PD-1 in tumor cells; and / or

[0031] (II), inhibiting the growth of tumor cells; and / or

[0032] (III) inhibiting the proliferation of tumor cells; and / or

[0033] (IV), inhibiting the metastasis of tumor cells; and / or

[0034] (V), triggering immunogenic death of the tumor; and / or

[0035] (VI), activating the cGAS-STING pathway in tumor cells; and / or

[0036] (VII) promoting the maturation of dendritic cells; and / or

[0037] (VIII) enhancing the antigen presentation function of dendritic cells; and / or

[0038] (IX) Prevention and / or treatment of tumors;

[0039] The tumors include: hepatocellular carcinoma;

[0040] The product includes, but is not limited to, one or more of a drug, a formulation and / or a drug combination.

[0041] The present invention also provides a medicine and / or a preparation, comprising: the above-mentioned drug-loaded nanoclusters and / or the drug-loaded nanoclusters obtained by the above-mentioned preparation method.

[0042] The present invention also provides a drug combination, comprising: the above-mentioned drug and / or preparation and any other effective ingredients.

[0043] In some embodiments of the present invention, in the above-mentioned drug combination, the other arbitrary active ingredients include: PD-1 monoclonal antibody.

[0044] The present invention provides a drug-loaded nanocluster, comprising: a nanocluster and an anti-tumor drug;

[0045] The mass ratio of the nanoclusters to the anti-tumor drug is: (92.00-92.32): (7.68-8.00).

[0046] The beneficial effects of the present invention include:

[0047] (1) The present invention uses lenvatinib, a first-line treatment drug for advanced hepatocellular carcinoma, in combination with cobalt ions to achieve a synergistic anti-tumor effect.

[0048] (2) The synergistic effect of lenvatinib and cobalt ions provided by the present invention means that lenvatinib, as a first-line treatment for hepatocellular carcinoma, achieves anti-tumor effects by inhibiting multi-target tyrosine kinases. Cobalt ions, as a new type of metal immunomodulator, can activate immune cells and enhance the body's immune response to tumors. The present invention combines the two to achieve a synergistic anti-tumor effect of the drug.

[0049] (3) The present invention uses OVA as a model antigen and drug carrier. The present invention achieves the co-loading of lenvatinib and cobalt ions on OVA, which not only improves the passive targeted transport of drugs, but also enhances the body's immune response through the immunogenicity of OVA, achieving a therapeutic effect of "1+1>2".

[0050] (4) The Co+Len@OVA nanoclusters of the present invention enhance the activity of immune cells in the tumor microenvironment by activating tumor immunogenic death (ICD) and cGAS-STING pathway, promote the infiltration of immune cells into tumors, and thus improve the efficacy of immunotherapy.

[0051] (5) The combined use of the Co+Len@OVA nanoclusters of the present invention and PD-1 antibodies can induce the normalization of tumor blood vessels, increase the delivery efficiency of anti-tumor drugs, and improve the infiltration and survival of immune cells, thereby enhancing the overall effect of immunotherapy.

[0052] (6) The nanocarrier preparation process of the present invention is simpler, has lower cost, and is easy to mass produce. At the same time, the biocompatibility and passive targeting of the nanoclusters are improved through the pH-controlled self-assembly method. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0054] Figure 1 The database is combined with CADD molecular docking to screen eFT508-like drugs that act on PD-L1 expression; A shows that the small molecule compounds with similar structures to eFT-508 are scored by Ledock in the FDA database, and the top 200 compounds are screened out (the compounds are represented by the corresponding CAS numbers); B shows that the compounds screened out in A are sorted according to the quantitative estimation of drug similarity (QED), and compounds with QED>0.50 are screened out; C shows that the compounds screened out in B are sorted by Ledock scoring again, and manually inspected to select the top compounds. The 11th is Lenvatinib (cas: 417716-92-8); D is a schematic diagram of the sphere-stick (sphere-MNK1, stick-Lenvatinib) structure; E is a schematic diagram of the cartoon-stick (cartoon-MNK1, stick-Lenvatinib) structure; F is a schematic diagram of the receptor-ligand binding mode details of MNK1 and Lenvatinib; G is a comparison between the binding site of eFT508 and MNK1 and the binding site of Lenvatinib and MNK1 simulated by CADD;

[0055] Figure 2 The results show that lenvatinib combined with cobalt ions indirectly acts on the uORF of PD-L1 mRNA 5′-UTR to downregulate the translation of PD-L1; A shows that three sites of the uORF of PD-L1 WT 5′-UTR were mutated simultaneously (CU′G′ to CU′C′) to obtain PD-L1 all MUT 5′-UTR; B shows the luciferase activity of PD-L1 after SNU-449 was transfected with PD-L1 WT 5′-UTR luciferase reporter plasmid and PD-L1 all MUT 5′-UTR luciferase reporter plasmid respectively; C shows the luciferase reporter activity of PD-L1 after SNU-449 transfected with PD-L1 WT 5′-UTR plasmid was treated with different drugs; D shows the luciferase reporter activity of PD-L1 after SNU-449 transfected with PD-L1 all MUT 5′-UTR plasmid was treated with different drugs; E shows the luciferase reporter activity of PD-L1 after SNU-449 transfected with HBB plasmid was treated with different drugs. Luciferase reporter activity of β-globin after SNU-449 with WT 5′-UTR plasmid; F shows Western-Blotting detection of phosphorylated eIF4E (p- eIF4E) and PD-L1 expression in SNU-449 cells treated with different drug combinations, and quantification of the corresponding relative expression of p- eIF4E and PD-L1 proteins (G, H); data are expressed as mean ± standard deviation; (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001);

[0056] Figure 3 The wild-type and mutant sequences of the uORF of the 5′-UTR of human CD274 (PD-L1) mRNA are shown;

[0057] Figure 4 Synergistic effect of lenvatinib and cobalt ions on the inhibition of hepatocellular carcinoma; A and B represent cobalt ions (Co 2+ , blue circle) and lenvatinib (Len, red triangle) at different concentrations for 24 hours after treatment of liver cancer cell PLC / PRF / 5 cells and Hepa1-6 cells with single drug activity and dose-effect curves Fixed concentration of lenvatinib and concentration gradient of cobalt ions for combined treatment of liver cancer cells; C and D represent the cell activity and dose-effect curves of PLC / PRF / 5 cells and Hepa1-6 cells after 24 hours, respectively; E and F represent the contour maps of lenvatinib combined with cobalt ions on liver cancer cells PLC / PRF / 5 and Hepa1-6, respectively, generated according to the IC50 data in Table 2; data points of single drugs are represented by black squares, and data points of combined drugs are represented by red circles;

[0058] Figure 5UPLC detection of lenvatinib content is shown; wherein: A shows the chromatographic peak diagram; B shows the standard curve diagram;

[0059] Figure 6 Figure 1 shows the transmission electron microscope image (TEM) and element distribution of drug-loaded nanoclusters; A shows the transmission electron microscope (TEM) image of Co+Len@OVA; B shows the dark-field TEM image of Co+Len@OVA taken by TEM; C shows the distribution of visible particle sizes of drug-loaded nanoclusters of different components; D shows the distribution of Zeta potential of drug-loaded nanoclusters of different components; E shows the EDS element mapping of C, N, O, Cl, and Co in Co+Len@OVA; F shows the EDS energy spectrum of C, N, O, Cl, and Co in Co+Len@OVA;

[0060] Figure 7 The results show that drug-loaded nanoclusters inhibit the proliferation, migration, and invasion of liver cancer cells; A and B respectively represent the CCK-8 assay of cell activity of PLC / PRF / 5 and Hepa1-6 cells treated with different components of drug-loaded nanoclusters for 48 hours and the fitting curves and IC50 values ​​of cell activity of PLC / PRF / 5 and Hepa1-6 cells treated with different concentrations of Co+Len@OVA for 24 hours; C, D and G respectively represent the scratch test of PLC / PRF / 5 and Hepa1-6 cells treated with different components of drug-loaded nanoclusters, scale: 400 μm, and the corresponding quantification; E, F and H respectively represent the Transwell test of PLC / PRF / 5 and Hepa1-6 cells treated with different components of drug-loaded nanoclusters, scale: 200 μm, and the corresponding quantification; data are expressed as mean ± standard deviation, (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001);

[0061] Figure 8The results show that drug-loaded nanoclusters damage the nucleus and mitochondria of liver cancer cells and release dsDNA to activate the cGAS-STING pathway; A shows the immunofluorescence detection of nuclear damage in PLC / PRF / 5 and Hepa1-6 cells after 48 hours of treatment with drug-loaded nanoclusters of different components (γ-H2AX method; blue: nucleus, green: γ-H2AX), scale: 50 μm; B shows the detection of mitochondrial membrane potential in PLC / PRF / 5 and Hepa1-6 cells after 48 hours of treatment with drug-loaded nanoclusters of different components (blue: nucleus, green: JC-1 monomer, red: JC-1 polymer), scale: 20 μm and 50 μm; C shows the detection of dsDNA in PLC / PRF / 5 cells after 48 hours of treatment with drug-loaded nanoclusters of different components, the white arrow indicates the dsDNA released into the cytoplasm (blue: nucleus, red: TOMM20, green: dsDNA probe), scale: 20 μm; D and F respectively represent the expression of non-phosphorylated and phosphorylated forms of cGAS-STING related proteins (TBK1, IRF3, STING, NF-κB) and the corresponding quantification after the drug-loaded nanoclusters of different components acted on PLC / CRF / 5 cells by Western-Blotting; E and G respectively represent the expression of non-phosphorylated and phosphorylated forms of cGAS-STING related proteins (TBK1, IRF3, STING, NF-κB) and the corresponding quantification after the drug-loaded nanoclusters of different components acted on Hepa1-6 cells by Western-Blotting; ELISA detection of IFN-β (H) and TNF-α (I) secreted by PLC / CRF / 5 cells by drug-loaded nanoclusters of different components; data are expressed as mean ± standard deviation, (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001);

[0062] Fig. 9Figure 2 shows the flow cytometric detection of the culture supernatant of Hepa1-6 cells treated with drug-loaded nanoclusters acting on spleen lymphocytes; A shows the antigen presentation function of DC cells (FITC anti-mouse CD11c, APC / Cyanine7 anti-mouse MHC-II); B shows the immune co-stimulatory function of DC cells (FITC anti-mouse CD11c, PE anti-mouseCD40); C shows CD4+T cells (APC anti-mouse CD3, FITC anti-mouse CD4); D shows SIINFEKL / H-2Kb+ cells (PE anti-mouse CD45, APC anti-mouse SIINFEKL / H-2Kb); E shows CD8+T cells (APC anti-mouse CD3, PE anti-mouse CD8); F shows NK cells (PE anti-mouse CD45, APC / Cyanine7 anti-mouse NK1.1); Data are expressed as mean ± standard deviation, (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001); G from left to right are MFI quantification of MHC-II, MFI quantification of CD40, MFI quantification of CD4, MFI quantification of SIINFEKL / H-2Kb, MFI quantification of CD8, and MFI quantification of NK1.1;

[0063] Fig.10 The figure shows the anti-tumor effect of drug-loaded nanoclusters combined with PD-1 monoclonal antibody in subcutaneous tumor model; wherein: A shows a schematic diagram of the experimental process, including the establishment of Hepa1-6 tumor mouse model, the tail vein administration of drug-loaded nanoclusters and anti-PD1, and the time point of specimen collection; B shows a statistical graph of the average tumor volume of mice treated with different drug-loaded nanoclusters combined with anti-PD1 as the treatment time increases (n=4); C shows a statistical graph of the tumor volume of individual mice in different groups as the treatment time increases; D shows the tumor inhibition rate of each group of mice calculated based on the weight of the tumor specimens; E shows the change of the average weight of mice in each group as the treatment time changes; F shows the actual pictures of tumors of different groups obtained after the treatment; the data are expressed as mean ± standard deviation, (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001);

[0064] Fig.11The anti-tumor effect of drug-loaded nanoclusters combined with PD-1 monoclonal antibody in an orthotopic tumor model is shown; A shows the in vivo imaging of representative mice treated with different drug-loaded nanoclusters combined with anti-PD1 at different time points; B shows the statistics of the average radioactivity of liver tumors in each group of mice at different time points (n=3); data are expressed as mean ± standard deviation, (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001);

[0065] Fig.12 Figure 3 shows the organ distribution and therapeutic effect of drug-loaded nanoclusters in vivo; A shows near-infrared fluorescence (NIRF) imaging of DIR-Co+Len@OVA at 1, 4, 8, 12, 24, and 48 hours after tail vein injection; B shows NIRF imaging of the main organs and tumors of mice 48 hours after drug injection; C shows the bioluminescence image of the tumor 48 hours after drug injection; D shows the quantification of the relative average fluorescence intensity of the main organs in B in vitro NIRF imaging; E shows a schematic diagram of the experimental process, including the establishment of a mouse Hepa1-6 tumor model, tail vein injection, and Intravenous injection of drug-loaded nanoclusters and time points for sample collection; F shows a statistical graph showing the growth of the average tumor volume of each group of mice treated with different drug-loaded nanoclusters over time (n=3); G shows a statistical graph showing the growth of the tumor volume of individual mice in different groups over time; H shows the tumor inhibition rate of each group of mice calculated based on the tumor weight; I shows the change in the relative average body weight of each group of mice over time; data are expressed as mean ± standard deviation, (*, p<0.05; **, p<0.01; **c*, p<0.001; ****, p<0.0001). DETAILED DESCRIPTION

[0066] The invention discloses a drug-carrying nanocluster, a preparation method and an application thereof.

[0067] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0068] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0069] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0070] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0071] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0072] The present invention relates to the following English annotations:

[0073] Hepatocellular carcinoma (HCC)

[0074] Immune checkpoint inhibitors (ICIs)

[0075] Programmed cell death protein 1 (PD-1)

[0076] Programmed cell death protein 1 ligand (PD-L1)

[0077] Overall survival (OS)

[0078] Progression-free survival (PFS)

[0079] Antigen presenting cells (APCs)

[0080] Ovalbumin (OVA)

[0081] Cyclic GMP-AMP synthetase (cGAS)

[0082] Double-stranded DNA (dsDNA)

[0083] Cyclic GMP-AMP (cGAMP)

[0084] Stimulator of Interferon Genes (STING)

[0085] In Examples 1 to 9 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0086] The present invention will be further described below in conjunction with embodiments:

[0087] Example 1 Computer Aided Drug Design (CADD) and Screening Based on PD-L1 Intracellular Translation

[0088] First, we determined that the small molecule drugs and their targets that inhibit the PD-L1 expression pathway in tumor cells are the following three:

[0089] (1) Fraxinellone, which targets HIF-1α and STAT3, both of which are transcription factors of PD-L1. Fraxinellone downregulates the expression of PD-L1 by reducing the expression of HIF-1α and STAT3;

[0090] (2) Tomivosertib (eFT508), which has entered Phase II clinical trials, targets MNK1 / 2. eFT508 is an inhibitor of MNK1 / 2, thereby inhibiting the phosphorylation of eIF4E and ultimately downregulating the translation of PD-L1;

[0091] (3) Aloperine derivatives (SA-49), etc., target the melanogenesis-related transcription factor (MITF). SA-49 induces MITF nuclear translocation, thereby triggering lysosome-based PD-L1 degradation.

[0092] After comparing these three small molecule inhibitors in the database, only the complex structure of eFT508 binding to the target protein MNK1 / 2 was found in the PDB database - numbered 6CK6 in PDB ( Figure 1 D). Therefore, the present invention seeks clinical drugs for hepatocellular carcinoma with a mechanism of action similar to eFT508. In the FDA database, we used RDKit to process the molecules, removed some impurities and ions, used the ledock annealing algorithm to explore the binding space, and used the knowledge-based scoring function to sort the molecules. The Top 200 ( Figure 1 A).

[0093] The screened compounds were then ranked according to the quantitative drug similarity estimation (QED), and the compounds with QED>0.50 were screened out ( Figure 1 B). Finally, the samples were inspected manually and the ledock scores were sorted again ( Figure 1 C).

[0094] Among them, we noticed the small molecule drug lenvatinib, which ranked high in the score. It is the first-line drug for unresectable hepatocellular carcinoma. Lenvatinib is a multi-target tyrosine kinase inhibitor that can downregulate the expression of PD-L1 in liver cancer cells by blocking FGFR4.

[0095] The sphere-stick and cartoon-stick models in Pymol software were used to simulate the molecular docking of lenvatinib and MNK1 ( Figure 1 E and F), it was found that the binding mode and binding site of lenvatinib and MNK1 are in the same binding pocket as eFT508 ( Figure 1 G). The binding of eFT508 to the receptor depends on hydrogen bonds, with the sites being lysine 113, methionine 162, and aspartic acid 226 of the receptor. The binding of lenvatinib to the receptor also depends on hydrogen bonds, with the sites being leucine 90, alanine 94, and histidine 95 of the receptor. In summary, CADD simulation molecular docking predicts that lenvatinib may bind to MNK1 in a similar way to eFT508.

[0096] Example 2 Lenvatinib indirectly acts on the uORF of 5′-UTR of PD-L1 mRNA in a manner similar to eFT508 to downregulate the translation of PD-L1

[0097] (1) Dual luciferase reporter gene experiment to downregulate PD-L1 expression in liver cancer cells using cobalt ions combined with lenvatinib

[0098] The expression of PD-L1 in tumor cells is regulated by multiple factors, one of which is the mTOR signaling pathway. mTOR can promote the translation of PD-L1 mRNA by activating the MNK1 / eIF4E pathway. eIF4E is an important translation initiation factor. After binding to MNK1, eFT508 inhibits eIF4E phosphorylation and prevents eIF4E from bypassing the uORF of the 5′-UTR of PD-L1 mRNA, thereby reducing the translation efficiency of PD-L1 mRNA.

[0099] In the study of Example 1, CADD simulated that lenvatinib could competitively bind to the ATP binding pocket of MNK1 in a manner similar to eFT508. We then further explored whether lenvatinib, like eFT508, inhibited eIF4E phosphorylation, making it impossible for the translation of PD-L1 mRNA to bypass the uORF in the 5′-UTR, thereby downregulating the translation of PD-L1.

[0100] Metal elements play an important role in the expression of PD-L1 in tumor cells. Cobalt ions, as important trace elements in the body, play a role in tumor immunotherapy. So we explored whether cobalt ions can enhance the eFT508-like effect of lenvatinib. Studies have reported that liver cancer cell SNU-449 expresses a small amount of PD-L1, and the expression of PD-L1 is significantly different after transfection of PD-L1 plasmid, so SNU-449 was selected for research. In order to simulate the state of high expression of PD-L1 in liver cancer cells in cases where lenvatinib combined with PD-1 monoclonal antibody treatment fails, we mutated the uORF of the human PD-L1 5′-UTR ( Figure 3 ), a dual-luciferase reporter plasmid was further designed that was linked to PD-L1 WT 5′-UTR (wild type) and PD-L1 all MUT 5′-UTR (CU′G′ to CU′C′) (mutant type) ( Figure 2 A).

[0101] When the above two 5′-UTRs are connected to the luciferase reporter gene, the translation efficiency of luciferase will be regulated by different 5′-UTR sequences. Different treatments have different effects on the translation efficiency of the wild-type 5′-UTR and mutant 5′-UTR of PD-L1, so the activity of the corresponding connected luciferase reporter gene will also be different. We transfected SNU-449 with luciferase reporter gene plasmids connected with PD-L1 WT 5′-UTR and PD-L1 all MUT 5′-UTR (CU′G′ to CU′C′), and we found that the luciferase activity of cells transfected with PD-L1 all MUT 5′-UTR luciferase reporter plasmid was significantly increased compared with cells transfected with PD-L1WT 5′-UTR luciferase reporter plasmid ( Figure 2 B). SNU-449 cells transfected with PD-L1 WT 5′-UTR and PD-L1 all MUT 5′-UTR luciferase reporter plasmids were treated with different drug combinations: Control, eFT508, Lenvatinib, Cobalt, and Lenvatinib + Cobalt (Cobalt refers to cobalt ion (Co 2 +), which was introduced by adding CoCl2•6H2O and dissolving it in water under experimental conditions, the same applies below), and further detected and calculated the PD-L1 luciferase activity of each group (luciferase activity = firefly luciferase activity reading / sea renilla luciferase activity reading). The results showed that in SNU-449 cells transfected with PD-L1 WT 5′-UTR luciferase reporter plasmid, lenvatinib, like eFT508, could downregulate PD-L1 luciferase activity. The PD-L1 luciferase activity of the lenvatinib combined with cobalt ion group was the lowest, indicating that lenvatinib can downregulate the translation of PD-L1 by acting on the 5′-UTR of PD-L1 mRNA, and cobalt ions can enhance this effect ( Figure 2 C).

[0102] In SNU-449 cells transfected with PD-L1 all MUT 5′-UTR luciferase reporter plasmid, the degree of inhibition of PD-L1 luciferase activity by each drug combination was higher than that by SNU-449 cells transfected with PD-L1 WT 5′-UTR luciferase reporter plasmid. Moreover, in cells transfected with PD-L1 all MUT 5′-UTR plasmid, the down-regulated PD-L1 luciferase activity by Lenvatinib+ Cobalt group was stronger than that by eFT508 and the difference was significant (P<0.05) ( Figure 2 D), which indicates that lenvatinib combined with cobalt ions can significantly inhibit the high expression of PD-L1 caused by PD-L1 5′-UTR mutation. After SNU-449 cells were treated with different drug combinations for 48 hours, the expression of phosphorylated eIF4E (p-eIF4E) and PD-L1 in SNU-449 cells was detected by Western-Blotting ( Figure 2 F), the results verified the drug effects shown by the dual luciferase reporter gene experiment. The quantification results of the Western-Blotting bands also showed that the ability of lenvatinib to downregulate eIF4E phosphorylation and PD-L1 expression in SNU-449 cells was similar to that of eFT508. The combination of cobalt ions can further enhance the effect of lenvatinib, making it superior to eFT508 in the above effects ( Figure 2 G, H).

[0103] We also treated SNU-449 cells transfected with β-globin WT 5′-UTR luciferase plasmid with different drugs and found that the β-globin luciferase activity after different drug treatment was no different from that of the control group, indicating that lenvatinib acts on PD-L1 5′-UTR without affecting the expression of housekeeping proteins ( Figure 2E). In summary, lenvatinib combined with cobalt ions may bind to MNK1 and then affect the uORF of PD-L1 mRNA 5′-UTR, thereby downregulating the translation efficiency of PD-L1 in liver cancer cells.

[0104] (2) Sequence design and cloning

[0105] The uORF wild-type 5′-UTR sequence of human CD274 (PD-L1) (PD-L1 WT 5′-UTR), uORF mutant 5′-UTR sequence (PD-L1 MUT 5′-UTR), and human β-globin wild-type 5′-UTR sequence (PD-L1 WT 5′-HBB) were designed ( Figure 3 ), the sequences were cloned into the pSV40-MCS-firefly_Luciferase vector plasmid, and Shanghai GeneCare Gene Medicine Technology Co., Ltd. completed the plasmid cloning and verification and provided the Renilla luciferase plasmid.

[0106] (3) Drug treatment and transfection

[0107] First, SNU-449 cells were treated with drugs for 48 hours according to different groups including Control (containing the same amount of DMSO as Lenvatinib group), eFT508 (0.2 μg / mL), Lenvatinib (2 μg / mL), Cobalt (CoCl2•6H2O 20 μg / mL), and Lenvatinib+ Cobalt (Lenvatinib2 μg / mL, CoCl2•6H2O 20 μg / mL). Then, CD274 wild-type (PD-L1 WT 5′-UTR) or mutant (PD-L1 MUT 5′-UTR) firefly luciferase plasmid and Renilla luciferase plasmid were co-transfected into the target cells at a ratio of 50:1 using ViaFect™ transfection reagent (E4981, Promega).

[0108] (4) Dual luciferase activity detection

[0109] According to the instructions of the Dual-Luciferase® Reporter Assay System kit (E1910, Promega), 1× Passive Lysis Buffer (PLB), Luciferase Assay Reagent II (LAR II) and Stop & Glo® reagent were pre-prepared. 48 hours after transfection, the supernatant was aspirated, washed once with 1xPBS, and 65 μL of 1xPLB reagent was added to each well of the 48-well plate and lysed at room temperature for 15 minutes. 100 μL of LAR II reagent was added to the 96-well opaque white ELISA plate, and then 20 μL of lysate was added and mixed. Then, the firefly luciferase activity was detected using the Spark 10 M multi-function microplate reader (TECAN), and then 100ul of Stop & Glo® reagent was added, and the Renilla luciferase activity was continued on the machine:

[0110] PD-L1 luciferase activity = Firefly luciferase activity reading / Renilla luciferase activity reading

[0111] The luciferase reporter activity of β-globin was detected in the same manner as above.

[0112] Example 3 Lenvatinib combined with cobalt ions has a synergistic effect in inhibiting the growth of liver cancer cells

[0113] (1) Inhibitory effect of a single drug on the growth of liver cancer cells

[0114] The results of Example 2 have verified that lenvatinib combined with cobalt ions can downregulate the translation of PD-L1 in liver cancer cells. Studies have shown that downregulating PD-L1 expression in tumor cells can also directly inhibit the growth of tumor cells. Lenvatinib can also directly inhibit the growth of liver cancer cells by inhibiting pathways such as EGFR, FGFR, and IGF1R. Cobalt ions can destroy the redox homeostasis in tumor cells by generating ROS and directly kill tumor cells. Therefore, we further explored whether lenvatinib combined with cobalt ions has a synergistic effect in inhibiting the growth of liver cancer cells, thereby improving the efficacy of immunotherapy for hepatocellular carcinoma.

[0115] We first explored the inhibitory effect of a single drug on the growth of liver cancer cells. We treated liver cancer cell lines PLC / PRF / 5 and Hepa1-6 with lenvatinib or cobalt ions alone, and detected cell activity using the CCK-8 kit to draw a dose-effect curve. The results showed that both lenvatinib and cobalt ions alone can reduce liver cancer cell activity. Lenvatinib has a wider range of effective concentrations, but the cell activity decreases more slowly. Cobalt ions have a narrow range of effective concentrations, but the cell activity decreases rapidly ( Figure 4A and B). By analyzing the dose-effect curves of lenvatinib or cobalt ions on liver cancer cells PLC / PRF / 5 and Hepa1-6, the IC50 of single drug activity on PLC / PRF / 5 and Hepa1-6 cells can be further obtained (Table 1). The IC50 of lenvatinib on PLC / PRF / 5 and Hepa1-6 cells were 199.60 and 96.01 μg / mL, respectively, and the IC50 of cobalt ions on PLC / PRF / 5 and Hepa1-6 cells were 126.1 and 103.3 μg / mL, respectively.

[0116] Table 1 Concentration required to inhibit cell viability by 50% (IC50 ± standard deviation)

[0117]

[0118] (2) Calculation of the synergistic index and contour map of lenvatinib combined with cobalt ions in inhibiting the growth of liver cancer cells

[0119] Synergy occurs when the total effect of a combination of drugs is greater than the sum of the effects of the individual drugs. Synergistic drug combinations can improve efficacy while reducing drug doses, thereby reducing side effects. To determine the effect of lenvatinib combined with cobalt ions, we conducted a drug synergy study. We added a fixed concentration of lenvatinib to a range of cobalt ions to determine a new dose-effect curve ( Figure 4 C and D).

[0120] New IC50 values ​​were calculated based on the new dose-effect curves, and the Chou-Talalay synergy equation was used to generate the drug synergy index (CI) (Table 2). The data are intuitively presented in contour plots for each cell line and drug combination concentration ( Figure 4 E and F), where the X-axis represents the concentration of lenvatinib and the Y-axis represents the concentration of CoCl2•6H2O. The data points of a single drug are represented by black squares located on the X-axis and Y-axis, connected by a line. The data points of the combination are represented by red circles, and the combined data points that fall below the line indicate synergy, those above the line indicate additive effects, and those above the line indicate antagonism. The farther off the line, the stronger the effect. The results showed that lenvatinib combined with cobalt ions exhibited moderate synergistic or at least additive effects in PLC / PRF / 5 and Hepa1-6 cell lines ( Figure 4E and F). According to Table 2, for the PLC / PRF / 5 cell line, regardless of whether the concentration ratio of lenvatinib to cobalt ions is large or small, a moderate synergistic effect is observed. For the Hepa1-6 cell line, a higher concentration ratio of lenvatinib to cobalt ions corresponds to a moderate synergistic effect, while a lower concentration ratio corresponds to an additive effect. The reason for the synergistic effect of lenvatinib combined with cobalt ions in inhibiting the activity of liver cancer cells may be achieved through the mTOR signaling pathway. The specific mechanism may involve the activation of the mTOR signaling pathway by cobalt ions and the inhibition of the FGFR signaling pathway by lenvatinib, thereby regulating the metabolism and angiogenesis of tumor cells.

[0121] Table 2 Synergy index (CI) of lenvatinib and cobalt ions

[0122]

[0123] Note: The drug combination concentrations required for the PLC / PRF / 5 cells and Hepa1-6 cell lines to reach IC50 are listed. CI > 1 indicates antagonism, CI = 1 indicates additivity, 0.7 < CI < 1 indicates mild synergy, 0.3 < CI < 0.7 indicates moderate synergy, and CI < 0.3 indicates strong synergy. The degree of synergy is represented by grading symbols, where "±" indicates additivity, "+" indicates mild synergy, "++" indicates moderate synergy, and "+++" indicates strong synergy.

[0124] Example 4 Synthesis and characterization of cobalt-based drug-loaded nanoclusters

[0125] (1) Synthesis of cobalt-based drug-loaded nanoclusters

[0126] 1) Prepare 50 mL of a pure aqueous solution of ovalbumin (2 mg / mL), stir until clear to obtain solution a.

[0127] 2) Titrate solution a with hydrochloric acid solution (100 mM) to pH = 3.0 (any value between 2 - 3 is acceptable). The titration can be terminated when the phenomenon changes from cloudy to flocculent and then to clear to obtain solution b.

[0128] 3) Prepare 50 mL each of lenvatinib (250 μg / mL) and CoCl2•6H2O (30 mg / mL) solutions using acetic acid (5%), mix and stir until clear to obtain solution c.

[0129] 4) Gradually add solution c dropwise to solution b using a burette while continuously stirring to obtain solution d.

[0130] 5) Titrate solution d with sodium hydroxide solution (1 M) to pH = 7 - 8. The titration can be terminated when the phenomenon changes from clear to flocculent and then to cloudy to obtain solution e.

[0131] 6) Solution e was placed into a dialysis bag (8000-14000D) for dialysis. After dialysis, the solution was collected through a 0.22 μm filter and freeze-dried to obtain drug-loaded nanoclusters Co+Len@OVA loaded with lenvatinib and cobalt ions.

[0132] 7) Replace solution c in 3) with 100 mL of acetic acid (5%) solution containing only CoCl2•6H2O (30 mg / mL) (solution c') or 100 mL of acetic acid (5%) solution containing only lenvatinib (250 μg / mL) (solution c''), and follow the remaining steps 1) to 6) in the same manner to obtain Co@OVA or Len@OVA.

[0133] (2) Measurement of microscopic morphology, element distribution, and structural properties of cobalt-based drug-loaded nanoclusters

[0134] The microscopic morphology and structure of the prepared drug-loaded nanoclusters were photographed and analyzed using a transmission electron microscope (TEM). After the Co+Len@OVA sample was prepared on a copper grid, TEM images and EDS elemental maps were taken at a working voltage of 300 KV. A visual nanoparticle analyzer (NanoSight NS300, Malvern, UK) was used to perform nanoparticle tracking analysis (NTA) on OVA, Len@OVA, and Co+Len@OVA to measure the visible particle size of the drug-loaded nanoclusters. The Zeta potential of OVA, Len@OVA, and Co+Len@OVA was measured using a nanoparticle analyzer (Litesizer 500, Anton Paar, Austrial).

[0135] (3) Measurement of drug loading and encapsulation efficiency of lenvatinib in cobalt-based drug-loaded nanoclusters

[0136] "Drug loading" refers to the mass of drug that can be loaded per unit mass of nanoparticles, and "encapsulation efficiency" refers to the ratio between the actual mass of drug loaded in the nanoparticles and the maximum amount of drug that the particles can theoretically load. In order to detect the drug loading and encapsulation efficiency of lenvatinib loaded in drug-loaded nanoclusters, a certain amount of Co+Len@OVA was weighed and mixed in a mobile phase of 50 mM sodium dodecyl sulfate (pH 2.5) / acetonitrile (55.5 / 45, v / v). After sufficient ultrasonic disruption, it was centrifuged at 400 rpm for 4 minutes, the supernatant was obtained and the lenvatinib content therein was detected using an ultra-high performance liquid chromatograph (UPLC, Agilent 1290 InfinityI, Agilent Technologies). The chromatographic peak diagram and standard curve of lenvatinib calibration are shown in Figure 5 .

[0137] Table 3 Drug loading and encapsulation efficiency of lenvatinib loaded with drug-loaded nanoclusters (± standard deviation)

[0138]

[0139] "Drug loading" refers to the mass of drug that can be loaded per unit mass of nanoparticles, and "encapsulation efficiency" refers to the ratio between the actual mass of drug loaded in the nanoparticles and the maximum amount of drug that the particles can theoretically load. To determine the measured drug loading and encapsulation efficiency of lenvatinib in the drug-loaded nanoclusters, a certain amount of Co+Len@OVA was weighed and mixed in a mobile phase of sodium dodecyl sulfate (pH 2.5) / acetonitrile (55.5 / 45, v / v), and fully ultrasonically broken to allow lenvatinib loaded on the drug-loaded nanoclusters to be fully released into the system. The mixture was centrifuged at 400 rpm for 4 minutes, and the supernatant was used to detect the content of lenvatinib by ultra-high performance liquid chromatography (UPLC, Agilent 1290 Infinity I, Agilent Technologies). The drug loading and encapsulation efficiency of Co+Len@OVA were calculated based on the mass of the drug-loaded nanoclusters used and the corresponding mass of the feed amount (Table 3). The drug loading capacity of Co+Len@OVA loaded with lenvatinib was 7.84±0.16%, and the encapsulation efficiency was 62.7±1.82%.

[0140] Example 5 Antitumor effect of cobalt-based drug-loaded nanoclusters on liver cancer cells

[0141] (1) Toxicity of cobalt-based drug-loaded nanoclusters to liver cancer cells

[0142] PLC / PRF / 5 cells were seeded in 96-well plates (2×10 4After culturing for 12 hours, OVA, Co@OVA, Len@OVA, Co+Len@OVA and different drug-loaded nanoclusters (2 mg / mL) or free lenvatinib (Raw Len, the concentration is equal to the lenvatinib content in the drug-loaded nanoclusters) were co-cultured for 48 hours, and the cell activity was detected by CCK-8 method. PLC / PRF / 5 cells were co-cultured with different concentrations of Co+Len@OVA (0, 0.01, 0.02, 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 8, 10 mg / mL) for 24 hours, and the cell activity was detected by CCK-8 method, and the drug concentration required to reduce cell activity by 50% (IC50) was determined by the best fit trend line. The above steps were repeated on Hepa1-6.

[0143] (2) Hepatocellular carcinoma cell growth scratch experiment

[0144] The cell scratch test was used to investigate the effects of different drug-loaded nanoclusters on the migration of PLC / PRF / 5 cells. PLC / PRF / 5 cells were seeded in 24-well plates (5x10 5 cells / well), and after the cells were observed to be evenly grown on the well plate under a microscope, they were starved with serum-free DMEM for 24 hours; then a 200 μL sterile pipette was used to scratch the cells at the bottom of the well plate, and the cells were gently washed twice with 1×PBS to remove suspended cells and cell debris, and then replaced with DMEM containing 10% fetal bovine serum for continued culture. Different components of drug-loaded nanoclusters (2 mg / mL) or free lenvatinib (concentration equal to the lenvatinib content in the drug-loaded nanoclusters) were added and co-cultured for 24 hours. Photos were taken at 0 hours and 24 hours to record the changes in the scratch area. The changes in the scratch area were measured and statistically analyzed using Fiji software, and the following formula was used to express the effects of different components of drug-loaded nanoclusters on the migration of PLC / PRF / 5 cells:

[0145] Cell migration rate (%) = ((S0-St) / S0) × 100%

[0146] Where S0 is the initial scratch area at 0 hours, St is the scratch area at 24 hours, and 4 fields of view were randomly selected for calculation in each treatment group. The above steps were repeated on Hepa1-6.

[0147] (3) Transwell invasion assay of liver cancer cells.

[0148] At 4°C, Matrigel was diluted with serum-free cell culture medium and then evenly added to the upper chamber surface of the bottom membrane of the Transwell chamber. It was kept in a 37°C incubator for 3 hours to allow the matrix gel to polymerize into a thin film. The lower chamber of the 12-well plate was filled with complete culture medium (containing 10% FBS) and the Transwell chamber was then placed in it. Next, the PLC / PRF / 5 cell suspension was inoculated in the upper chamber (5x10 cells per well). 5 Cells were pre-cultured for 6 hours. Different components of drug-loaded nanoclusters (2 mg / mL) or free lenvatinib (concentration equal to the lenvatinib content in the drug-loaded nanoclusters) were added into the upper chamber for co-culture for 24 hours. After the upper chamber medium was dried, the chamber was removed and the cells and Matrigel in the upper chamber were wiped with a cotton swab. The cells were then fixed with paraformaldehyde (4%) and then stained with crystal violet. Finally, the cells were observed and photographed under an upright microscope, and 4 fields of view were randomly selected to count the positive cells (purple) and the results were statistically analyzed. Repeat the above steps on Hepa1-6.

[0149] Example 6 Cobalt-based drug-loaded nanoclusters damage liver cancer cell nuclei and mitochondria and activate cGAS-STING pathway

[0150] (1) Analysis of γ-H2AX aggregation foci in the nuclear damage of liver cancer cells induced by drug-loaded nanoclusters

[0151] PLC / PRF / 5 cells were seeded in confocal microplates (5 × 10 4 Cells were pre-cultured for 12 hours with different components of drug-loaded nanoclusters (2 mg / mL) or free lenvatinib (concentration equal to the lenvatinib content in drug-loaded nanoclusters) for 24 hours. Then, they were washed three times with PBS and fixed with 4% paraformaldehyde (PFA) for 15 minutes. After rinsing with PBS to remove PFA, the cells were permeabilized with 0.2% Triton X-100 for 30 minutes at room temperature. Then, after blocking with 5% bovine serum albumin (BSA) for 1 hour, the cells were incubated with anti-phosphorylated histone (γ-H2AX) monoclonal antibody at 4°C overnight. After washing with PBS three times, the cells were sealed with anti-fluorescence quenching blocking solution containing DAPI, and then observed and photographed under an inverted fluorescence microscope. The above steps were repeated on Hepa1-6.

[0152] (2) Drug-loaded nanoclusters induce mitochondrial damage in liver cancer cells

[0153] 1) JC-1 probe detection of drug-loaded nanoclusters induced mitochondrial membrane potential depolarization: PLC / PRF / 5 cells were seeded in confocal microplates (5×10 4Cells were pre-cultured with different components of drug-loaded nanoclusters (2 mg / mL) or free lenvatinib (concentration equal to the lenvatinib content in drug-loaded nanoclusters) for 24 hours. After washing with PBS, the changes in mitochondrial membrane potential were detected using the JC-1 kit (Biyuntian, Shanghai). When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix to form red fluorescent polymers; when the membrane potential decreases, JC-1 disperses into green fluorescent monomers. The ratio of green mean fluorescence intensity to red mean fluorescence intensity was calculated using Fiji software as an indicator to evaluate the degree of mitochondrial depolarization. The above steps were repeated on Hepa1-6.

[0154] 2) Drug-loaded nanoclusters induce dsDNA release into the cytoplasm of liver cancer cells: PLC / PRF / 5 cells were seeded in a confocal microplate (5×10 4 Cells were pre-cultured for 12 hours with different components of drug-loaded nanoclusters (2 mg / mL) or free lenvatinib (concentration equal to the lenvatinib content in drug-loaded nanoclusters) for 24 hours. The cells were then fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton-X-100, and blocked with 5% BSA. The cells were incubated overnight with anti-TOMM20 antibody-mitochondrial marker (1:100, Abcam) and dsDNA marker (1:100, Abcam), then incubated with Goat Anti-Mouse IgG H&L (Alexa Fluor® 568) for 1 hour and sealed with blocking solution containing DAPI anti-fluorescence quencher. The images were taken by inverted fluorescence microscopy. The above steps were repeated on Hepa1-6.

[0155] (3) Immunoblotting analysis of proteins related to cGAS-STING pathway activation induced by cobalt-based drug-loaded nanoclusters in liver cancer cells

[0156] PLC / PRF / 5 cells were seeded into 6-well plates (4x10 5Cells were pre-cultured for 12 hours with different components of drug-loaded nanoclusters (2 mg / mL) or free lenvatinib (concentration equal to the lenvatinib content in the drug-loaded nanoclusters) for 24 hours. After lysing the cells, the total protein supernatant was extracted by centrifugation, denatured, and its content was determined using a BCA kit. After determining the protein content, each cell lysate was diluted with the loading buffer at a volume ratio of 1:4. Subsequently, the mixture was heated at 95°C for 20 minutes to denature the protein. 40 μg of protein was taken from each sample and subjected to sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis. The proteins in the gel were next transferred to a nitrocellulose (NC) membrane. At room temperature, the NC membrane was soaked in 5% (w / v) skim milk containing an antimicrobial agent to block nonspecific protein binding sites. The blocking process lasted for 2 hours to enhance the specificity of subsequent Western Blotting analysis, and then washed with TBST. The NC membrane was incubated with the cGAS-STING related rabbit primary antibodies diluted and incubated overnight at 4°C, including anti-GAPDH antibody (Abcam, Cambridge, MA), anti-TBK1 antibody (CST, USA), anti-IRF3 antibody (CST, USA), anti-TBK1 antibody (CST, USA), anti-Phospho-TBK1 antibody (CST, USA), anti-STING antibody (CST, USA), anti-Phospho-STING antibody (CST, USA), and anti-NF-κB antibody (CST, USA). After washing the NC membrane, the NC membrane was incubated with GoatAnti-Rabbit IgG H&L (HRP) at room temperature for 1 hour. After washing, the proteins were visualized using an automatic chemiluminescence image analysis system (TANON, Shanghai) and an ECL immunoblotting detection kit (Advansta, CA, USA). The harvested bands were analyzed using Fiji software. The above steps were repeated on Hepa1-6.

[0157] Example 7 Cobalt-based drug-loaded nanoclusters indirectly activate spleen lymphocyte anti-tumor immunity through liver cancer cells

[0158] (1) Isolation and culture of spleen lymphocytes from C57BL / 6 mice

[0159] 6-8 week old mice were killed by cervical dislocation, placed in 75% alcohol for disinfection for 5 minutes, and then placed on a sterile table with the left ventral side facing up. Then, the skin and muscles were cut in the middle of the left ventral side of the mouse, the red long spleen was located, the lower peritoneum was gently lifted and cut and turned up to expose the spleen. Then, the spleen was gently clamped with forceps, the underlying connective tissue was separated with ophthalmic scissors, and the spleen was removed after being freed. The connective tissue around the spleen was stripped and rinsed with RPMI 1640. The spleen was placed on a 70 μm cell strainer and gently pressed with a syringe needle to obtain a cell suspension. Lymphocyte separation solution (Da Kewei, Shanghai) was added to another sterile test tube. The test tube was tilted to slowly add the cell suspension to the upper layer of the separation solution (separation solution volume: cell suspension volume = 1:2), and then centrifuged for 30 minutes (700-800 g). After centrifugation, the test tube was removed. The bottom of the test tube was red blood cells, the middle was separation solution, and the upper layer was plasma / tissue homogenate. The thin and dense white film between the plasma and the separation solution is the mononuclear cell layer. Remove the upper homogenate, take out the mononuclear cell layer, centrifuge for 10 minutes (speed 250g), and wash twice with PBS.

[0160] (2) Cobalt-based drug-loaded nanoclusters indirectly activate anti-tumor immunity of spleen lymphocytes through liver cancer cells

[0161] Hepa1-6 cells were treated with drug-loaded nanoclusters (2 mg / mL) of different components or free lenvatinib (concentration equal to the lenvatinib content in the drug-loaded nanoclusters) for 24 hours, and the cell culture supernatant was collected. The spleen lymphocytes extracted in Example 5 were incubated with the culture supernatant for 24 hours. After the cells were collected, FITC anti-mouse CD11c (Biolegend, California, USA), PE anti-mouse CD40 (Biolegend, California, USA), APC / Cyanine7anti-mouse MHC-II (Biolegend, California, USA), APC anti-mouse CD3 (Biolegend, California, USA), FITC anti-mouse CD4 (Biolegend, California, USA), PE anti-mouse CD8 (Biolegend, California, USA), PE anti-mouse CD45 (Biolegend, California, USA), APC / Cyanine7 anti-mouse NK1.1 (Biolegend, California, USA) were added. The cells were incubated with anti-mouse SIINFEKL / H-2Kb (Biolegend, California, USA) and APC anti-mouse SIINFEKL / H-2Kb (Biolegend, California, USA) at 4°C for 30 min, washed with PBS, resuspended and detected using CytoFLEX flow cytometry.

[0162] Example 8 In vivo therapeutic effect of drug-loaded nanoclusters combined with PD-1 monoclonal antibody on hepatocellular carcinoma

[0163] (1) Therapeutic effects of drug-loaded nanoclusters

[0164] Mice with subcutaneous hepatocellular carcinoma model were randomly divided into ①Control group; ②OVA group; ③Co@OVA group; ④Len@OVA group; ⑤Co+Len@OVA group; ⑥Raw Len group, 4 mice in each group. Mice were injected with various preparations (different components of drug-loaded nanoclusters (2 mg / mL) or free lenvatinib (concentration equal to the lenvatinib content in the drug-loaded nanoclusters) through the tail vein. Tail vein injection was performed once every 3 days, and the subcutaneous tumor volume and mouse body weight were monitored every 2 days. Mice were killed on the 20th day, and organs and tumor specimens were collected after perfusion and exsanguination, and tumor specimens were photographed. The heart, liver, spleen, lung, kidney, and tumor specimens obtained were sliced ​​and subjected to corresponding H&E, TUNEL, and various immunofluorescence staining.

[0165] (2) Therapeutic effect of drug-loaded nanoclusters combined with PD-1 monoclonal antibody

[0166] Mice with subcutaneous hepatocellular carcinoma model were randomly divided into ①Control group; ②OVA+anti-PD1 group; ③Lenvatinib+anti-PD1 group; ④Len@OVA+anti-PD1 group; ⑤Co+Len@OVA+anti-PD1 group; ⑥Co@OVA+anti-PD1 group, 4 mice in each group. Mice were injected with various preparations through the tail vein, including different components of drug-loaded nanoclusters (2 mg / mL) or free lenvatinib (concentration equal to the lenvatinib content in the drug-loaded nanoclusters), and the anti-PD1 dosage was 50 μg / mouse. The tail vein injection of drug-loaded nanoclusters and free lenvatinib was performed once every 3 days, and the injection of anti-PD1 was performed once every 4 days. The size of the primary tumor and the weight of the mice were monitored every 2 days. The mice were killed on the 14th day, and the tumors were collected and photographed, the draining lymph nodes (tdLNs), and the spleen lymphocytes were extracted for flow cytometry.

[0167] Mice with orthotopic hepatocellular carcinoma model were randomly divided into ①Control group; ②OVA+anti-PD1 group; ③Lenvatinib+anti-PD1 group; ④Len@OVA+anti-PD1 group; ⑤Co+Len@OVA+anti-PD1 group; ⑥Co@OVA+anti-PD1 group, 3 mice in each group. Mice were injected with various preparations via tail vein, including different components of drug-loaded nanoclusters (2 mg / mL) or free lenvatinib (concentration equal to the lenvatinib content in the drug-loaded nanoclusters), and the anti-PD1 dosage was 50 μg / mouse. The drug-loaded nanoclusters and free lenvatinib were injected into the tail vein once every 3 days, and anti-PD1 was injected once every 4 days. D–Luciferin (GlpBio, California, USA) was injected on the 1st, 6th, and 14th days of treatment for small animal live imaging and the mice were weighed.

[0168] Example 9 In vivo imaging of mice to observe the organ distribution of drug-loaded nanoclusters and PD-1 monoclonal antibodies

[0169] Since the injected tumor-forming Hepa1-6-luc can convert D-Luciferin into firefly luciferin. Three days after the in situ tumor surgery, the mice were anesthetized with isoflurane, the hair on the abdomen and chest of the mice was removed, and the sides were shaved to the posterior abdominal line to fully expose the liver surface area. The IVIS® Lumina III (PerkinElmer, Massachusetts, USA) was turned on to preheat the instrument. After the mice were anesthetized to a satisfactory degree, they were placed flat in the shooting area. After adjusting the bioluminescence and fluorescence parameters, the mice were imaged in vivo, and the fluorescence intensity of the liver was calculated and analyzed. After the in vivo imaging, the heart, lungs, liver, kidneys and spleen of the mice were removed, and they were placed in order on a special shooting blackboard, and fluorescence shooting was performed again. After the shooting was completed, some liver specimens were fixed in 4% paraformaldehyde, and the rest were quickly frozen in liquid nitrogen.

[0170] Orthotopic hepatocellular carcinoma model mice were randomly divided into 3 groups, 3 mice in each group: ①FITC-anti-PD1 group; ②Lenvatinib+FITC-anti-PD1 group; ③DIR-Co+Len@OVA+FITC-anti-PD1. Different preparations, including different components of drug-loaded nanoclusters (2 mg / mL) or free lenvatinib (concentration equal to the lenvatinib content in the drug-loaded nanoclusters), were injected into the tail vein, and the anti-PD1 dosage was 50 μg / mouse. Under isoflurane anesthesia, whole-body fluorescence images were recorded at 1, 8, 16, 24 and 48 hours using IVIS Lumina III IVIS®LuminaIII (PerkinElmer, Massachusetts, USA). The bioluminescence channel of the tumor observation channel was (Em / Ex) 488nm / 520nm for FITC-anti-PD1 and (Em / Ex) 754nm / 778nm for DIR-Co+Len@OVA. Mice were sacrificed 48 hours after administration, and organs (heart, liver, spleen, lung, and kidney) and tumors were collected for in vitro bioluminescence imaging and quantification using Living Image 4.4 software.

[0171] The obtained tumors were fixed with paraformaldehyde (4%) for 24 hours and transferred to melted paraffin for embedding. Tissue sections (5 μm thickness) were made using a Leica microtome (RM 2235, Germany). Paraffin-embedded tissue sections were deparaffinized in xylene and hydrated. The above tissue sections were blocked with 5% goat or donkey serum for 1 hour. The sections were then incubated with anti-CD31 antibody (CST, USA) at 4°C overnight. The washed sections were then incubated with Goat Anti-Mouse IgG H&L (Sulfo-Cyanine3). Finally, the washed sections were mounted with blocking solution containing DAPI anti-fluorescence quencher. Fluorescence images of the stained sections were taken using an inverted fluorescence microscope, and quantification of each stained section was performed using Fiji. In order to study the effect of drug-loaded nanoclusters on the diffusion of PD-1 monoclonal antibodies in tumors, two channels, FITC (Em / Ex) 488nm / 520nm and DIR (Em / Ex) 754nm / 778nm, were added when taking fluorescence photos.

[0172] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Drug-loaded nanoclusters, characterized in that: include: Nanoclusters and anti-tumor drugs; The mass ratio of the nanoclusters to the anti-tumor drug is: (92.00-92.32): (7.68-8.00).

2. The drug-loaded nanocluster according to claim 1, characterized in that: The drug-loaded nanocluster further comprises: a matrix; the matrix comprises: a protein; the protein comprises: one or more of ovalbumin, human serum albumin, human hemoglobin, bovine serum albumin and bovine hemoglobin.

3. The drug-loaded nanocluster according to claim 1 or 2, characterized in that: The anti-tumor drugs include: one or more of lenvatinib, sorafenib and regorafenib.

4. The drug-loaded nanocluster according to any one of claims 1 to 3, characterized in that: The nano cluster is obtained after being protected by a metal ligand.

5. The drug-loaded nanocluster according to claim 4, characterized in that: The metal ligands include: one or more of cobalt ligands, manganese ligands, iron ligands, gold ligands and copper ligands.

6. The method for preparing drug-loaded nanoclusters according to claim 4 or 5, characterized in that: The steps include: S1: mixing the solution of the anti-tumor drug and the solution of the nanoclusters protected by the metal ligand, adding the mixture dropwise to the solution containing the protein matrix, adjusting the pH value, and obtaining a mixed solution; S2: Stirring the mixed solution, adjusting the pH value again, dialyzing, filtering, and freeze-drying to obtain the drug-loaded nanoclusters.

7. The preparation method according to claim 6, characterized in that: Adjust the pH to 2-3 as described in S1.

8. The preparation method according to claim 7, characterized in that: Adjust pH to 7-8 as described in S2.

9. Use of the drug-loaded nanocluster according to any one of claims 1 to 5 and / or the drug-loaded nanocluster obtained by the preparation method according to any one of claims 6 to 8 in the preparation of any of the following products; (I) downregulating the expression and / or translation efficiency of PD-1 in tumor cells; and / or (II), inhibiting the growth of tumor cells; and / or (III) inhibiting the proliferation of tumor cells; and / or (IV), inhibiting the metastasis of tumor cells; and / or (V), triggering immunogenic death of the tumor; and / or (VI), activating the cGAS-STING pathway in tumor cells; and / or (VII) promoting the maturation of dendritic cells; and / or (VIII) enhancing the antigen presentation function of dendritic cells; and / or (IX) Prevention and / or treatment of tumors; The tumors include: Hepatocellular carcinoma; The product includes, but is not limited to, one or more of a drug, a formulation and / or a drug combination.

10. A drug and / or preparation, characterized in that include: The drug-loaded nanocluster according to any one of claims 1 to 5 and / or the drug-loaded nanocluster obtained by the preparation method according to any one of claims 6 to 8.

11. A drug combination, characterized in that include: The medicine and / or preparation as claimed in claim 10 and any other active ingredients.

12. The pharmaceutical combination according to claim 11, characterized in that The other arbitrary active ingredients include: PD-1 monoclonal antibody.

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