Drug-loaded nanoclusters, preparation method and application thereof

Abstract

The present application relates to the field of biopharmaceuticals, in particular to drug-loaded nanoclusters, a preparation method and application thereof. The present application provides drug-loaded nanoclusters, comprising: nanoclusters and an antitumor drug; the mass ratio of the nanoclusters and the antitumor drug is (92.00~92.32):(7.68~8.00). The present application develops a cobalt-based drug-loaded nanocluster for delivering lenvatinib, which utilizes the drug-loading characteristics and immunogenicity of ovalbumin and metal immunity mediated by cobalt ions to improve the immunosuppressive microenvironment of hepatocellular carcinoma, improve the therapeutic effect of lenvatinib and reduce drug resistance, and combines with PD-1 monoclonal antibody to form a new immune activation scheme for systemic drug treatment, and enhances the immunotherapy effect of hepatocellular carcinoma.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceuticals, and more particularly to drug-loaded nanoclusters, their preparation methods, and their applications. Background Technology

[0002] Primary liver cancer, a highly lethal tumor, has an increasing incidence rate worldwide, causing severe disease and economic burden. Hepatocellular carcinoma (HCC) is the main type of primary liver cancer. Systemic drug therapy is the first-line treatment for advanced HCC, with tyrosine kinase inhibitors, such as sorafenib and lenvatinib, serving as molecularly targeted therapies and first-line treatments. Since the advent of immunotherapy inhibitors (ICIs), novel systemic treatment strategies have developed rapidly. ICIs, represented by PD-1 / PD-L1 antibodies, have influenced HCC treatment regimens, especially the combination of molecularly targeted therapies like atezolizumab and bevacizumab with immune checkpoint inhibitors, which has significantly improved the prognosis of liver cancer patients. Encouraged by this, more novel combination regimens have entered clinical trials for HCC, such as lenvatinib combined with pembrolizumab, durvalumab combined with trimemumab, and nivolumab combined with ipilimumab. However, in the LEAP-002 clinical trial, the combination of lenvatinib and pembrolizumab did not show greater benefit; neither overall survival (OS) nor progression-free survival (PFS) was superior to lenvatinib monotherapy, suggesting that drug resistance caused by the immunosuppressive microenvironment still limits the potential of immunotherapy. Researchers have begun exploring novel immune activation strategies to overcome the tumor's immunosuppressive microenvironment and thus reduce drug resistance. Combining molecularly targeted drugs and immune checkpoint inhibitors can improve therapeutic efficacy while reducing the optimal treatment dose. Many novel immune activation strategies, including metalloimmunization and immune adjuvants, have therefore received increasing attention.

[0003] Current research on metalloimmunology focuses on utilizing the immune-activating properties of metal elements or using them as drug co-delivery platforms to enhance the efficacy of existing immunotherapies. Cobalt ions can serve as coordination sites for organometallic compounds, forming coordination polymers, and in vivo, they can act as a cofactor for vitamin B12 (cobalamin). The metallic valence reactivity of cobalt facilitates the synthesis of cobalt-based nanoscale drug delivery systems. Furthermore, due to its superior magnetic properties and proton relaxation effect, cobalt-based magnetic fluids have been designed for use in medical testing and imaging. Cobalt also plays a positive role in enhancing anti-tumor immunity and reversing immunosuppressive tumor microenvironment (TME). By enhancing the maturation and function of APCs or promoting the activation of the cGAS-STING pathway, it increases the local infiltration and anti-tumor activity of T cells and NK cells, thereby 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 through mechanisms such as inducing ROS generation, regulating the tumor microenvironment, and thus influencing tumor growth and development. Therefore, cobalt can not only enhance the efficacy of immunotherapy by modifying immunosuppressive TMEs, but also serve as a component of nanocarrier systems to work synergistically with the drugs it carries to exert therapeutic effects.

[0004] Ovalbumin (OVA) is a monomeric phosphoglobulin composed of 365 amino acids with a molecular weight between 42 and 47 kDa, and is a common model antigen. Due to its significant immunogenicity, OVA is widely used in the study of tumor immune responses. It can be captured by APCs and presented to T cells, thereby triggering a specific immune response. In recent years, the application of OVA as a carrier for constructing drug-loaded nanoclusters in tumor therapy has also emerged. Its excellent biocompatibility and stability, as well as its specific amino acid structure, enable it to self-assemble through chemical regulation, thereby encapsulating 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 metalloimmunology and hepatocellular carcinoma drug therapy, acting as a platform for activating the immune microenvironment of hepatocellular carcinoma.

[0005] Numerous studies have found that activation of the cGAS-STING pathway in preclinical models can effectively improve the therapeutic efficacy of ICIs. Possible mechanisms include the induction of IFN-γ and related inflammatory cytokines after STING pathway activation, enhancing the antigen recognition capabilities of APCs, CD4+ T cells, and CD8+ T cells, and strengthening the cytotoxicity of macrophages, dendritic cells, NK cells, B cells, and T cells against tumor cells, thereby improving the immunosuppressive microenvironment and providing favorable conditions for the implementation of immunotherapies such as ICIs. The cGAS-STING pathway is an evolutionarily conserved mechanism for initiating an immune response against pathogens, playing a crucial role in the initiation and enhancement of innate and adaptive immunity. After recognizing dsDNA in the cytoplasm, cGAS uses ATP and GTP as substrates to promote the production of cGAMP, which then binds 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 site conformation of cGas or promoting substrate-enzyme interaction, thereby enabling the formation of cGAMP, or Co 2+ Zn 2+ It can bind to certain key amino acid residues of the STING protein, altering its conformation and making it easier for it to bind to cGAMP. In the context of drug combination therapy for hepatocellular carcinoma using molecularly targeted drugs and immune checkpoint inhibitors, activating the cGAS-STING pathway through methods such as introducing metal immunization and constructing engineered nanocarriers to deliver drugs, thereby enhancing 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. The present invention develops a cobalt-based drug-loaded nanocluster for delivering lenvatinib, utilizing cobalt ion-mediated metal-mediated immunity to improve the immunosuppressive microenvironment, enhance the therapeutic effect of lenvatinib, and reduce drug resistance. Combined with PD-1 monoclonal antibodies, it forms a novel immune activation regimen for systemic drug therapy, enhancing the efficacy of immunotherapy for hepatocellular carcinoma.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides drug-loaded nanoclusters, comprising: nanoclusters and antitumor drugs;

[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, the drug-loaded nanoclusters further include a matrix; the matrix includes a protein; the protein includes one or more of ovalbumin, human serum albumin, human serum globulin, bovine serum albumin, and bovine serum globulin.

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

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

[0013] In some embodiments of the present invention, the antitumor drug in the above-mentioned drug-loaded nanoclusters includes one or more of lenvatinib, sorafenib, and regorafenib.

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

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

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

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

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

[0019] S1: Mix the solution of the antitumor drug and the solution of the nanoclusters protected by the metal ligand, and then add the mixture dropwise to the solution of the matrix. Adjust the pH value to obtain a mixture.

[0020] S2: Stir the mixture, adjust the pH value again, dialyze, filter, and freeze dry to obtain the drug-loaded nanoclusters.

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

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

[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 antitumor 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 antitumor drug is obtained by mixing the antitumor 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 preparation method includes a pH-controlled self-assembly process: unlike traditional physical mixing or chemical synthesis methods, the present invention employs pH-controlled self-assembly technology, which achieves efficient and stable loading of lenvatinib and cobalt ions onto 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, the above preparation method employs precise control of solution titration: during the preparation process, the mixture (c) of the antitumor drug solution and the metal ligand solution is gradually added to the protein matrix solution (b) by titration, which ensures the uniformity and controllability of the reaction and avoids premature precipitation or inactivation of the drug components.

[0028] In some embodiments of the present invention, the above preparation method employs a post-treatment of dialysis and freeze-drying: dialysis is performed using dialysis bags, which effectively removes unreacted raw materials and low molecular weight impurities, ensuring the purity and stability of the nanoclusters. The subsequent freeze-drying step further ensures the long-term storage of the nanoclusters.

[0029] The present invention also provides the application of the above-described 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 PD-1 expression and / or translation efficiency in tumor cells; and / or

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

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

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

[0034] (V) Triggering immunogenic tumor cell death; and / or

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

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

[0037] (VIII) Enhance the antigen-presenting function of dendritic cells; and / or

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

[0039] The tumors include: hepatocellular carcinoma;

[0040] The products include, but are not limited to, one or more of drugs, formulations, and / or combinations of drugs.

[0041] The present invention also provides pharmaceuticals and / or formulations comprising: the drug-loaded nanoclusters described above and / or drug-loaded nanoclusters obtained by the preparation method described above.

[0042] The present invention also provides pharmaceutical combinations comprising: the aforementioned pharmaceuticals and / or formulations and other arbitrary active ingredients.

[0043] In some embodiments of the present invention, the other any active ingredient in the above-described drug combination includes: PD-1 monoclonal antibody.

[0044] This invention provides drug-loaded nanoclusters, comprising: nanoclusters and antitumor drugs;

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

[0046] The beneficial effects of this invention include:

[0047] (1) This invention uses lenvatinib, a first-line treatment for advanced hepatocellular carcinoma, and cobalt ions together to achieve a synergistic anti-tumor effect.

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

[0049] (3) In this invention, OVA is used as a model antigen and drug carrier. This invention achieves the co-loading of lenvatinib and cobalt ions on OVA, which not only improves the passive targeted delivery 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 the tumor immunogenic death (ICD) and cGAS-STING pathways, and promote the infiltration of immune cells into the tumor, thereby improving the efficacy of immunotherapy.

[0051] (5) The combined use of the Co+Len@OVA nanoclusters of the present invention with 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 preparation process of the nanocarrier of the present invention is simpler, lower in cost, and easier to scale up. At the same time, the biocompatibility and passive targeting of the nanoclusters are improved by pH-controlled self-assembly. Attached Figure Description

[0053] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0054] Figure 1 This study uses a database combined with CADD molecular docking to screen for eFT508-like drugs targeting PD-L1 expression. Specifically: A) Ledock scoring of small molecule compounds structurally similar to eFT-508 in the FDA database, selecting the top 200 compounds (compounds are represented by their corresponding CAS numbers); B) ranking the compounds selected in A according to quantitative drug similarity estimation (QED), selecting compounds with a QED > 0.50; C) re-ranking the compounds selected in B using Ledock scoring, followed by manual review to select the top-ranked compounds. The 11th lenvatinib (CAS: 417716-92-8); D shows a schematic diagram of the sphere-stick (sphere-MNK1, stick-Lenvatinib) structure; E shows a schematic diagram of the cartoon-stick (cartoon-MNK1, stick-Lenvatinib) structure; F shows a detailed schematic diagram of the receptor-ligand binding mode of MNK1 and lenvatinib; G shows a comparison between the binding site of eFT508 to MNK1 and the CADD-simulated binding site of lenvatinib to MNK1.

[0055] Figure 2 The study showed that lenvatinib, in combination with cobalt ions, indirectly acts on the uORF of the PD-L1 mRNA 5′-UTR, thereby downregulating PD-L1 translation. Specifically: A shows the simultaneous mutation of three sites (CU′G′ to CU′C′) of the uORF in the PD-L1 WT 5′-UTR to obtain the PD-L1 all MUT 5′-UTR; B shows the luciferase activity of PD-L1 after transfection with SNU-449 containing both the PD-L1 WT 5′-UTR and PD-L1 all MUT 5′-UTR luciferase reporter plasmids; C shows the luciferase reporter activity of PD-L1 after treatment with different drugs on SNU-449 transfected with the PD-L1 WT 5′-UTR plasmid; D shows the luciferase reporter activity of PD-L1 after treatment with different drugs on SNU-449 transfected with the PD-L1 all MUT 5′-UTR plasmid; E shows the luciferase reporter activity of PD-L1 after treatment with different drugs on HBB transfected with different drugs. β-globin luciferase reporter activity after SNU-449 cells were inoculated with the WT 5′-UTR plasmid; F shows Western-blotting analysis of phosphorylated eIF4E (p-eIF4E) and PD-L1 expression in SNU-449 cells treated with different drug combinations, and the corresponding quantification of the relative expression levels 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 Wild-type and mutant sequences of uORF in the 5′-UTR of human CD274 (PD-L1) mRNA;

[0057] Figure 4 The synergistic effect of sildenafil and cobalt ions in inhibiting hepatocellular carcinoma activity; where: A and B represent cobalt ions (Co... 2+ The table shows the cell viability and dose-response curves of PLC / PRF / 5 and Hepa1-6 liver cancer cells after 24 hours of single treatment with different concentrations of lenvatinib (blue circle) and lenvatinib (red triangle). It also shows the combined treatment of liver cancer cells with a fixed concentration of lenvatinib and a concentration gradient of cobalt ions. C and D represent the cell viability and dose-response curves of PLC / PRF / 5 and Hepa1-6 cells after 24 hours, respectively. E and F represent the contour plots of lenvatinib combined with cobalt ions on liver cancer cells PLC / PRF / 5 and Hepa1-6, generated based on the IC50 data in Table 2. Data points for single drugs are represented by black squares, and data points for combined drug treatment are represented by red circles.

[0058] Figure 5The diagram shows the UPLC determination of lenvatinib content; where: A shows the chromatogram; B shows the standard curve.

[0059] Figure 6 The images show transmission electron microscopy (TEM) images and elemental distributions of drug-loaded nanoclusters; where: A shows the TEM image of Co+Len@OVA; B shows the dark-field TEM image of Co+Len@OVA; C shows the visible particle size distribution of drug-loaded nanoclusters with different components; D shows the distribution of Zeta potentials of drug-loaded nanoclusters with different components; E shows the EDS elemental mapping of C, N, O, Cl, and Co in Co+Len@OVA; F shows the EDS energy dispersive spectroscopy of C, N, O, Cl, and Co in Co+Len@OVA.

[0060] Figure 7 This study demonstrates the inhibition of hepatocellular carcinoma cell proliferation, migration, and invasion by drug-loaded nanoclusters. Specifically: A and B represent the CCK-8 assay results of cell viability of PLC / PRF / 5 and Hepa1-6 cells treated with different drug-loaded nanoclusters for 48 hours, and the fitted curves and IC50 values ​​of cell viability of PLC / PRF / 5 and Hepa1-6 cells treated with different concentrations of Co+Len@OVA for 24 hours, respectively; C, D, and G represent the scratch assay results of PLC / PRF / 5 and Hepa1-6 cells treated with different drug-loaded nanoclusters (scale bar: 400 μm) and corresponding quantifications, respectively; E, F, and H represent the Transwell assay results of PLC / PRF / 5 and Hepa1-6 cells treated with different drug-loaded nanoclusters (scale bar: 200 μm) and corresponding quantifications, respectively. Data are expressed as mean ± standard deviation (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

[0061] Figure 8The study demonstrates how drug-loaded nanoclusters damage the nucleus and mitochondria of hepatocellular carcinoma cells, releasing dsDNA and activating the cGAS-STING pathway. Specifically: A shows immunofluorescence detection (γ-H2AX method; blue: nucleus, green: γ-H2AX) of PLC / PRF / 5 and Hepa1-6 cells after 48 hours of treatment with different drug-loaded nanoclusters, scale bar: 50 μm; B shows the detection of mitochondrial membrane potential (blue: nucleus, green: JC-1 monomer, red: JC-1 polymer) of PLC / PRF / 5 and Hepa1-6 cells after 48 hours of treatment with different drug-loaded nanoclusters, scale bars: 20 μm and 50 μm; C shows the detection of dsDNA in PLC / PRF / 5 cells after 48 hours of treatment with different drug-loaded nanoclusters, with white arrows indicating dsDNA released into the cytoplasm (blue: nucleus, red: TOMM20, green: dsDNA probe), scale bar: 20 μm. μm; D and F represent the expression and quantification of non-phosphorylation and phosphorylated forms of cGAS-STING-related proteins (TBK1, IRF3, STING, NF-κB) after Western blotting of different drug-loaded nanoclusters on PLC / CRF / 5 cells; E and G represent the expression and quantification of non-phosphorylation and phosphorylated forms of cGAS-STING-related proteins (TBK1, IRF3, STING, NF-κB) after Western blotting of different drug-loaded nanoclusters on Hepa1-6 cells; ELISA detection of IFN-β (H) and TNF-α (I) secreted by different drug-loaded nanoclusters on PLC / CRF / 5 cells; data are expressed as mean ± standard deviation (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

[0062] Figure 9Flow cytometry analysis of the effect of drug-loaded nanoclusters on the culture supernatant of Hepa1-6 cells on spleen lymphocytes; A shows the antigen-presenting function of dendritic cells (DCs) (FITC anti-mouse CD11c, APC / Cyanine7 anti-mouse MHC-II); B shows the immune co-stimulatory function of DCs (FITC anti-mouse CD11c, PE anti-mouse CD40); 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 represents the MFI quantization of MHC-II, MFI quantization of CD40, MFI quantization of CD4, MFI quantization of SIINFEKL / H-2Kb, MFI quantization of CD8, and MFI quantization of NK1.1;

[0063] Figure 10 This study demonstrates the antitumor effect of drug-loaded nanoclusters combined with PD-1 monoclonal antibody in a subcutaneous tumor model. Specifically: A shows a schematic diagram of the experimental process, including the establishment of the Hepa1-6 tumor mouse model, the tail vein administration of the drug-loaded nanoclusters and anti-PD1, and the time points for specimen collection; B shows a statistical graph of the increase in average tumor volume in mice treated with different drug-loaded nanoclusters combined with anti-PD1 over treatment time (n=4); C shows a statistical graph of the increase in tumor volume in individual mice in different groups over treatment time; D shows the tumor inhibition rate of each group of mice calculated based on the weight of the tumor specimens; E shows the change in average body weight of each group of mice over treatment time; F shows images of tumors obtained from different groups after treatment. Data are expressed as mean ± standard deviation (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001).

[0064] Figure 11This study demonstrates the antitumor effect of drug-loaded nanoclusters combined with PD-1 monoclonal antibody in an in situ tumor model. Specifically: A shows in vivo imaging of representative mice treated with different drug-loaded nanoclusters combined with anti-PD1 at different time points; B shows the statistical analysis of the mean radiation rate 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] Figure 12 This study illustrates the in vivo organ distribution and therapeutic effects of drug-loaded nanoclusters. Specifically: A shows near-infrared fluorescence (NIRF) images of DIR-Co+Len@OVA at 1, 4, 8, 12, 24, and 48 hours after tail vein injection; B shows NIRF images of major organs and tumors in mice 48 hours after drug injection; C shows bioluminescence images of tumors 48 hours after drug injection; D shows the quantification of the relative average fluorescence intensity of the major organs in in vitro NIRF imaging (as shown in B); and E shows a schematic diagram of the experimental process, including the establishment of a mouse Hepa1-6 tumor model and tail vein injection. The time points for intravenous injection of drug-loaded nanoclusters and sample collection; F shows the statistical graph of the increase in mean tumor volume of mice in each group treated with different drug-loaded nanoclusters over treatment time (n=3); G shows the statistical graph of the increase in tumor volume of individual mice in different groups over treatment time; H shows the tumor inhibition rate of mice in each group calculated based on tumor weight; I shows the change in relative mean body weight of mice in each group over treatment time; data are expressed as mean ± standard deviation (*, p<0.05; **, p<0.01; **c*, p<0.001; ****, p<0.0001). Detailed Implementation

[0066] This invention discloses drug-loaded nanoclusters, their preparation methods, and their applications.

[0067] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

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

[0069] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0070] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0071] Furthermore, 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 precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" 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 English annotations involved in this invention are:

[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 this invention, all raw materials and reagents used can be purchased from the market.

[0086] The present invention will be further illustrated below with reference to the embodiments:

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

[0088] First, the following three types of small molecule drugs and their targets were identified as inhibiting the PD-L1 expression pathway in tumor cells:

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

[0090] (2) Tomivosertib (eFT508) has entered phase II clinical trials and 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 melanogenesis-related transcription factor (MITF). SA-49 induces nuclear translocation of MITF, thereby triggering lysosomal-based PD-L1 degradation.

[0092] Subsequently, a database comparison of these three small molecule inhibitors was conducted. In the PDB database, only the structure of the complex formed by eFT508 binding to the target protein MNK1 / 2 was found - PDB number 6CK6. Figure 1 D). Therefore, this invention seeks hepatocellular carcinoma clinical drugs with a mechanism of action similar to eFT508. In the FDA database, we used RDKit to process molecules, removing some impurities and ions, used the ledock annealing algorithm to explore the binding space, and used a knowledge-based scoring function to rank the molecules, obtaining the Top 200 based on their ranking. Figure 1 A).

[0093] The selected compounds are then ranked based on quantitative drug similarity estimation (QED), and compounds with QED > 0.50 are selected. Figure 1 B). Finally, a manual visual inspection is performed, and the ledock scores are re-ranked. Figure 1 C).

[0094] Among the drugs we noted was lenvatinib, a small molecule drug that ranked highly in the scoring. Lenvatinib is a first-line treatment for unresectable hepatocellular carcinoma. It is a multi-target tyrosine kinase inhibitor that can downregulate PD-L1 expression in liver cancer cells by blocking FGFR4.

[0095] Molecular docking between lenvatinib and MNK1 was simulated using the sphere-stick and cartoon-stick models in Pymol software. Figure 1 E and F) revealed that lenvatinib and MNK1 binding patterns and binding sites were in the same binding pocket as eFT508 (E and F). Figure 1 (G). eFT508 binds to the receptor via hydrogen bonds at sites 113 (lysine), 162 (methionine), and 226 (aspartic acid) on the receptor. Lenvatinib also binds to the receptor via hydrogen bonds at sites 90 (leucine), 94 (alanine), and 95 (histidine). In summary, CADD molecular docking simulations predict that lenvatinib may bind to MNK1 via a similar mechanism to eFT508.

[0096] Example 2: Lenvatinib indirectly downregulates PD-L1 translation by acting on the uORF of the PD-L1 mRNA 5′-UTR in an eFT508-like manner.

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

[0098] PD-L1 expression 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 eFT508 binds to MNK1, it inhibits eIF4E phosphorylation, preventing eIF4E from bypassing the uORF of the PD-L1 mRNA 5′-UTR, thereby reducing the translation efficiency of PD-L1 mRNA.

[0099] In Example 1, CADD simulated that lenvatinib could competitively bind to the ATP-binding pocket of MNK1 in a manner similar to eFT508. Therefore, we further investigated whether lenvatinib, similar to eFT508, could inhibit eIF4E phosphorylation, preventing the translation of PD-L1 mRNA from bypassing the uORF in the 5′-UTR, thereby downregulating PD-L1 translation.

[0100] Metal elements play an important role in the expression of PD-L1 in tumor cells, and cobalt ions, as an important trace element in vivo, play a role in tumor immunotherapy. Therefore, we investigated whether cobalt ions could enhance the eFT508-like effect of lenvatinib. Previous studies reported that SNU-449 liver cancer cells expressed low levels of PD-L1, and that transfection with a PD-L1 plasmid significantly altered PD-L1 expression; therefore, SNU-449 was selected for this study. To simulate the state of high PD-L1 expression in liver cancer cells in cases where lenvatinib combined with PD-1 monoclonal antibody therapy failed, we mutated the uORF of the human PD-L1 5′-UTR (…). Figure 3 Furthermore, a dual-luciferase reporter plasmid was designed linking PD-L1 WT 5′-UTR (wild-type) and PD-L1 all MUT 5′-UTR (CU′G′ to CU′C′) (mutant). Figure 2 A).

[0101] When the two types of 5′-UTRs mentioned above are linked to luciferase reporter genes, the translation efficiency of luciferase will be regulated by different 5′-UTR sequences. Different treatments have different effects on the translation efficiency of wild-type and mutant 5′-UTRs of PD-L1, and therefore the activity of the corresponding linked luciferase reporter genes will also differ. We transfected luciferase reporter gene plasmids linked with PD-L1 WT 5′-UTR and PD-L1 all MUT 5′-UTR (CU′G′ to CU′C′) into SNU-449 cells, respectively. We found that cells transfected with the PD-L1 all MUT 5′-UTR luciferase reporter plasmid showed significantly higher luciferase activity compared to cells transfected with the PD-L1 WT 5′-UTR luciferase reporter plasmid. Figure 2 B). Different drug combinations, including Control, eFT508, Lenvatinib, Cobalt, and Lenvatinib + Cobalt, were used to treat SNU-449 cells transfected with PD-L1 WT 5′-UTR and PD-L1 all MUT 5′-UTR luciferase reporter plasmids (where Cobalt refers to cobalt ions). 2 +The PD-L1 luciferase activity was introduced by adding CoCl2•6H2O and dissolving it in water under experimental conditions (the same applies below). Further detection and calculation of PD-L1 luciferase activity in each group (luciferase activity = firefly luciferase activity reading / Renilla luciferase activity reading) showed that in SNU-449 cells transfected with the PD-L1 WT 5′-UTR luciferase reporter plasmid, lenvatinib, similar to eFT508, downregulated PD-L1 luciferase activity. The lenvatinib combined with cobalt ions group showed the lowest PD-L1 luciferase activity. This indicates that lenvatinib can downregulate PD-L1 translation 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 the PD-L1 all MUT 5′-UTR luciferase reporter plasmid, the inhibitory effect of each drug combination on PD-L1 luciferase activity was higher than that of SNU-449 cells transfected with the PD-L1 WT 5′-UTR luciferase reporter plasmid. Furthermore, in cells transfected with the PD-L1 all MUT 5′-UTR plasmid, the Lenvatinib + Cobalt group showed a stronger and more significant downregulation of PD-L1 luciferase activity than the eFT508 group (P<0.05). Figure 2 (D) This indicates that lenvatinib combined with cobalt ions can significantly inhibit PD-L1 overexpression caused by PD-L1 5′-UTR mutation. After treating SNU-449 cells 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 effect shown by the dual-luciferase reporter gene assay. The quantification results of Western blotting also showed that lenvatinib's ability to downregulate eIF4E phosphorylation and PD-L1 expression in SNU-449 cells was similar to that of eFT508. The combination with cobalt ions further enhanced the effect of lenvatinib, making its above effects superior to those of eFT508. Figure 2 G, H).

[0103] We treated SNU-449 cells transfected with the β-globin WT 5′-UTR luciferase plasmid with different drugs and found that the β-globin luciferase activity after different drug treatments was not different from that in the control group, indicating that the way lenvatinib acts on PD-L1 5′-UTR does not affect the expression of housekeeping proteins. Figure 2E). In summary, lenvatinib combined with cobalt ions may bind to MNK1 and thus affect the uORF of PD-L1 mRNA 5′-UTR, downregulating the translation efficiency of PD-L1 in liver cancer cells.

[0104] (2) Sequence design and cloning

[0105] The design included the uORF wild-type 5′-UTR sequence of human CD274 (PD-L1) (PD-L1 WT 5′-UTR), the uORF mutant 5′-UTR sequence (PD-L1 MUT 5′-UTR), and the human β-globin wild-type 5′-UTR sequence (PD-L1 WT 5′-HBB). Figure 3 The sequences were cloned into the pSV40-MCS-firefly_Luciferase vector plasmid, and the plasmid cloning and verification were completed by Shanghai Jikai Gene Medical Technology Co., Ltd., which also 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: Control (containing the same amount of DMSO as the Lenvatinib group), eFT508 (0.2 μg / mL), Lenvatinib (2 μg / mL), Cobalt (CoCl2•6H2O 20 μg / mL), and Lenvatinib+Cobalt (Lenvatinib 2 μg / mL, CoCl2•6H2O 20 μg / mL). Then, using ViaFect™ transfection reagent (E4981, Promega), firefly luciferase plasmids of wild-type (PD-L1 WT 5′-UTR) or mutant (PD-L1 MUT 5′-UTR) were co-transfected into the target cells with Renilla luciferase plasmids at a ratio of 50:1.

[0108] (4) Dual-luciferase activity detection

[0109] Following 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. Forty-eight hours after transfection, the supernatant was aspirated, and the plate was washed once with 1xPBS. 65 μL of 1xPLB reagent was added to each well of a 48-well plate, and lysis was performed at room temperature for 15 minutes. 100 μL of LAR II reagent was added to each well of a 96-well opaque white microplate, followed by 20 μL of the lysis product, and the mixture was stirred. Firefly luciferase activity was then detected using a Spark 10 M multi-well microplate reader (TECAN). 100 μL of Stop & Glo® reagent was then added, and the Renida luciferase activity was further detected.

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

[0111] The assay for β-globin luciferase reporter activity follows the same steps as above.

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

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

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

[0115] We first investigated the inhibitory effects of single drugs on the growth of hepatocellular carcinoma cells. We treated the hepatocellular carcinoma cell lines PLC / PRF / 5 and Hepa1-6 with lenvatinib or cobalt ions as single agents and plotted dose-response curves using a CCK-8 assay. The results showed that both lenvatinib and cobalt ions reduced the activity of hepatocellular carcinoma cells. Lenvatinib had a wider effective concentration range, but the decrease in cell activity was more gradual. Cobalt ions had a narrower effective concentration range, but the decrease in cell activity was rapid. Figure 4(A and B). By analyzing the dose-response curves of lenvatinib or cobalt ion monotherapy against PLC / PRF / 5 and Hepa1-6 liver cancer cells, the IC50 values ​​of the single drugs against PLC / PRF / 5 and Hepa1-6 cells can be further determined (Table 1). The IC50 values ​​of lenvatinib against PLC / PRF / 5 and Hepa1-6 cells were 199.60 and 96.01 μg / mL, respectively, while the IC50 values ​​of cobalt ion against PLC / PRF / 5 and Hepa1-6 cells were 126.1 and 103.3 μg / mL, respectively.

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

[0117]

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

[0119] Synergistic effects occur when the total effect of combined drug therapy exceeds the sum of the effects of individual drugs. Synergistic drug combinations can improve efficacy while reducing drug dosage, thereby mitigating side effects. To determine the efficacy of lenvatinib combined with cobalt ions, we conducted a synergistic drug effect study. We added fixed concentrations of lenvatinib to a range of cobalt ion concentrations to determine new dose-response curves (…). Figure 4 (C and D).

[0120] New IC50 values ​​were calculated based on the new dose-response curves, and the Combination Index (CI) was generated using the Chou-Talalay synergistic equation (Table 2). This data is visually presented in isopleth 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. Data points for individual drugs are represented by black squares on the X and Y axes, connected by a line. Data points for combination therapy are represented by red circles; data points falling below the line indicate synergistic effects, those on the line indicate additive effects, and those above the line indicate antagonistic effects. The further away from the line, the stronger the effect. Results showed that lenvatinib combined with cobalt ions exhibited moderate synergistic or at least additive effects in the 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 / cobalt ions is large or small, it has a moderate synergistic effect. For the Hepa1-6 cell line, a higher concentration ratio of lenvatinib / cobalt ions corresponds to a moderate synergistic effect, and a lower concentration ratio of lenvatinib / cobalt ions corresponds to an additive effect of the drugs. 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 Synergistic 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 additive effect, 0.7 < CI < 1 indicates mild synergistic effect, 0.3 < CI < 0.7 indicates moderate synergistic effect, and CI < 0.3 indicates strong synergistic effect. The degree of synergy is represented by grading symbols, where "±" indicates additive, "+" indicates mild synergistic effect, "++" indicates moderate synergistic effect, and "+++" indicates strong synergistic effect.

[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), and 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 with acetic acid (5%), mix and stir until clear to obtain solution c.

[0129] 4) Slowly add solution c drop by drop to solution b using a burette, and keep stirring during the titration process to obtain solution d.

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

[0131] 6) The solution e was placed in a dialysis bag (8000-14000D) for dialysis. After dialysis, the solution was collected through a 0.22 μm filter and lyophilized 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 the remaining steps 1)-6) are the same to obtain Co@OVA or Len@OVA.

[0133] (2) Measurement of the microstructure, elemental distribution, and structural properties of cobalt-based drug-loaded nanoclusters

[0134] The microstructure and structure of the prepared drug-loaded nanoclusters were photographed and analyzed using transmission electron microscopy (TEM). After preparing Co+Len@OVA samples on a copper mesh, TEM images and EDS elemental spectra were acquired at a working voltage of 300 kV. Nanoparticle tracking analysis (NTA) was performed on OVA, Len@OVA, and Co+Len@OVA to measure the visible particle size of the drug-loaded nanoclusters. The Zeta potentials of OVA, Len@OVA, and Co+Len@OVA were measured using a nanoparticle analyzer (Litesizer 500, Anton Paar, Austria).

[0135] (3) Measurement of the drug loading and encapsulation efficiency of lenvatinib loaded on 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 theoretical maximum amount of drug that the particle can load. To determine the drug loading and encapsulation efficiency of lenvatinib loaded onto 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 thorough ultrasonic disruption, the mixture was centrifuged at 400 rpm for 4 minutes, and the supernatant was collected. The lenvatinib content was then detected using ultra-high performance liquid chromatography (UPLC, Agilent 1290 InfinityI, Agilent Technologies). The chromatogram and standard curve for lenvatinib calibration are shown below. Figure 5 .

[0137] Table 3. Drug loading and encapsulation efficiency of lenvatinib loaded onto 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 theoretical maximum amount of drug that the particle can load. To determine the measured drug loading and encapsulation efficiency of the drug-loaded nanoclusters for lenvatinib, 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). The mixture was thoroughly ultrasonicated to release the lenvatinib loaded on the drug-loaded nanoclusters into the system. After centrifugation at 400 rpm for 4 minutes, the supernatant was analyzed for lenvatinib content using an ultra-high performance liquid chromatograph (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 feed amount (Table 3). The drug loading 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 (2×10⁶ cells / wells). 4After culturing cells / wells for 12 hours, they were co-cultured for 48 hours with drug-loaded nanoclusters containing different components of OVA, Co@OVA, Len@OVA, and Co+Len@OVA (2 mg / mL) or free lenvatinib (Raw Len, concentration equal to the lenvatinib content in the drug-loaded nanoclusters). Cell viability was assessed using the CCK-8 assay. PLC / PRF / 5 cells were co-cultured for 24 hours 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). Cell viability was assessed using the CCK-8 assay, and the optimal trend line was used to determine the drug concentration (IC50) required for a 50% reduction in cell viability. The above steps were repeated on Hepa1-6 cells.

[0143] (2) Scratch test for liver cancer cell growth

[0144] The effects of drug-loaded nanoclusters with different components on the migration of PLC / PRF / 5 cells were investigated using a cell scratch assay. PLC / PRF / 5 cells were seeded in 24-well plates (5 x 10⁻⁶ cells / wells). 5 After observing uniform cell growth in a well plate under a microscope, cells were starved in serum-free DMEM for 24 hours. Then, 200 μL sterile pipette tips were used to scratch the cells at the bottom of the wells. Cells were gently washed twice with 1×PBS to remove suspended cells and cell debris, and then cultured in DMEM containing 10% fetal bovine serum. Different 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. Changes in the scratched area were photographed at 0 and 24 hours. Changes in the scratched area were measured and statistically analyzed using Fiji software. The following formula represents the effect of different 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, and St is the scratch area at 24 hours. Four fields of view are randomly selected for calculation in each treatment group. The above steps are repeated on Hepa1-6.

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

[0148] Matrigel was diluted with serum-free cell culture medium at 4°C and then evenly added to the upper surface of the bottom membrane of a Transwell chamber. The plate was then incubated at 37°C for 3 hours to allow the Matrigel to polymerize into a thin film. The lower chamber of a 12-well plate was filled with complete culture medium (containing 10% FBS), and the Transwell chamber was then placed inside. Next, PLC / PRF / 5 cell suspensions were seeded into the upper chamber (5 x 10 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 within the drug-loaded nanoclusters) were added to the upper chamber for co-culturing for 24 hours. After aspirating the culture medium from the upper chamber, the chamber was removed, and the cells and Matrigel in the upper chamber were wiped clean with a cotton swab. Cells were then fixed with paraformaldehyde (4%) and stained with crystal violet. Finally, the cells were observed and photographed under an upright microscope. Four fields of view were randomly selected to count the positive cells (purple) and the results were statistically analyzed. The above steps were repeated on Hepa1-6.

[0149] Example 6: Cobalt-based drug-loaded nanoclusters damage the nucleus and mitochondria of liver cancer cells, thereby activating the cGAS-STING pathway.

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

[0151] PLC / PRF / 5 cells were seeded in confocal microplates (5×10⁶ cells / 10⁻ ... 4 Cells were pre-cultured (cells / well) for 12 hours. Cells were then cultured for 24 hours with drug-loaded nanoclusters of different components (2 mg / mL) or free lenvatinib (concentration equal to the lenvatinib content within the drug-loaded nanoclusters). Cells were then washed three times with PBS and fixed with 4% paraformaldehyde (PFA) for 15 minutes. After washing with PBS to remove PFA, cells were permeabilized with 0.2% Triton X-100 at room temperature for 30 minutes. Cells were then blocked with 5% bovine serum albumin (BSA) for 1 hour and incubated overnight at 4°C with an anti-phosphorylated histone (γ-H2AX) monoclonal antibody. After washing three times with PBS, cells were mounted with a DAPI-containing anti-fluorescence quenching blocking solution 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 dishes (5×10⁻⁶ cells / years). 4Cells were pre-cultured (cells / well) for 12 hours. Cells were then cultured for 24 hours with different components of drug-loaded nanoclusters (2 mg / mL) or free lenvatinib (concentration equal to the lenvatinib content within the drug-loaded nanoclusters). After washing with PBS, changes in mitochondrial membrane potential were detected using the JC-1 kit (Beyotime, Shanghai). When the mitochondrial membrane potential was high, JC-1 aggregated in the mitochondrial matrix to form a red fluorescent polymer; when the membrane potential decreased, JC-1 dispersed as green fluorescent monomers. The ratio of the average green fluorescence intensity to the average red fluorescence intensity was calculated using Fiji software as an indicator of mitochondrial depolarization. The above steps were repeated on Hepa1-6 cells.

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

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

[0156] PLC / PRF / 5 cells were seeded into 6-well plates (4 x 10⁻⁶ cells per well). 5Cells were pre-cultured (cells / well) for 12 hours, then co-cultured for 24 hours with drug-loaded nanoclusters of different components (2 mg / mL) or free lenvatinib (concentration equal to the lenvatinib content within the drug-loaded nanoclusters). After cell lysis, total protein supernatant was extracted by centrifugation, denatured, and its content was determined using a BCA kit. After protein content determination, each cell lysate was diluted with loading buffer at a 1:4 volume ratio. The mixture was then heated at 95°C for 20 minutes to denature the proteins. 40 μg of protein from each sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred from the gel to a nitrocellulose membrane (NC). The NC membrane was immersed in 5% (w / v) skim milk containing an antibacterial agent at room temperature to block non-specific protein binding sites for 2 hours to enhance the specificity of subsequent Western blotting analysis, followed by washing with TBST. Rabbit primary antibodies related to cGAS-STING were diluted and incubated overnight at 4°C on NC membranes. These antibodies included: 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, it was incubated with GoatAnti-Rabbit IgG H&L (HRP) at room temperature for 1 hour. After washing, the proteins were imaged using a fully automated chemiluminescence imaging system (TANON, Shanghai) and an ECL immunoblotting 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 lymphocytes for anti-tumor immunity via liver cancer cells.

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

[0159] Mice aged 6-8 weeks were euthanized by cervical dislocation and sterilized in 75% alcohol for 5 minutes before being placed on a sterile table with their left ventral side facing upwards. The skin and muscle were then cut open in the middle of the left ventral side of the mouse to locate the long, red spleen. The lower peritoneum was gently lifted and cut open to expose the spleen. The spleen was then gently grasped with forceps, and the underlying connective tissue was separated using ophthalmic scissors. The spleen was then freed and removed. The connective tissue surrounding the spleen was removed, and the spleen was rinsed with RPMI 1640. The spleen was placed on a 70 μm cell filter and gently crushed using a syringe needle core to obtain a cell suspension. Lymphocyte separation medium (Dakewei, 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 medium (separation medium volume: cell suspension volume = 1:2), followed by centrifugation for 30 minutes (700-800 g). After centrifugation, the test tube was removed; the bottom layer contained red blood cells, the middle layer contained the separation medium, and the upper layer contained plasma / tissue homogenate. The thin, dense white membrane between the plasma and the separation solution is the mononuclear cell layer. Remove the supernatant homogenate, extract the mononuclear cell layer, centrifuge for 10 minutes (250 g), and wash twice with PBS.

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

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

[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 a subcutaneous hepatocellular carcinoma model were randomly divided into four groups: ①Control group; ②OVA group; ③Co@OVA group; ④Len@OVA group; ⑤Co+Len@OVA group; and ⑥Raw Len group, with four mice in each group. Mice were injected via tail vein with various formulations (different components of drug-loaded nanoclusters (2 mg / mL) or free lenvatinib (concentration equal to the lenvatinib content within the drug-loaded nanoclusters). Tail vein injections were performed every three days, and subcutaneous tumor volume and mouse weight were monitored every two days. Mice were sacrificed on day 20, and organs and tumor specimens were collected after perfusion hemolysis. The tumor specimens were photographed. Sections of the obtained heart, liver, spleen, lung, kidney, and tumor specimens were subjected to appropriate H&E, TUNEL, and various immunofluorescence staining methods.

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

[0166] Mice with a subcutaneous hepatocellular carcinoma model were randomly divided into four groups: ① Control group; ② OVA + anti-PD1 group; ③ Lenvatinib + anti-PD1 group; ④ Len@OVA + anti-PD1 group; ⑤ Co + Len@OVA + anti-PD1 group; and ⑥ Co@OVA + anti-PD1 group, with four mice in each group. Mice were injected via tail vein with various formulations, including drug-loaded nanoclusters of different components (2 mg / mL) or free lenvatinib (concentration equal to the lenvatinib content within the drug-loaded nanoclusters), and anti-PD1 at a dose of 50 μg / mouse. Drug-loaded nanoclusters and free lenvatinib were injected via tail vein every 3 days, and anti-PD1 was injected every 4 days. Primary tumor size and mouse weight were monitored every 2 days. Mice were sacrificed on day 14, and tumors were collected, photographed, drained lymph nodes (tdLNs), and splenic lymphocytes were extracted for flow cytometry analysis.

[0167] Mice with an orthotopic hepatocellular carcinoma model were randomly divided into four groups: ① Control group; ② OVA + anti-PD1 group; ③ Lenvatinib + anti-PD1 group; ④ Len@OVA + anti-PD1 group; ⑤ Co + Len@OVA + anti-PD1 group; and ⑥ Co@OVA + anti-PD1 group, with three mice in each group. Mice were injected via tail vein with various formulations, including drug-loaded nanoclusters of different components (2 mg / mL) or free lenvatinib (concentration equal to the lenvatinib content within the drug-loaded nanoclusters), and anti-PD1 at a dose of 50 μg / mouse. Drug-loaded nanoclusters and free lenvatinib were injected via tail vein every 3 days, and anti-PD1 was injected every 4 days. D-Luciferin (GlpBio, California, USA) was injected on days 1, 6, and 14 of treatment for small animal in vivo imaging and mouse weight measurement.

[0168] Example 9: In vivo imaging observation of organ distribution of drug-loaded nanoclusters and PD-1 monoclonal antibody in mice.

[0169] Because injecting Hepa1-6-luc into the tumor can convert D-Luciferin into firefly luciferin, three days after the in situ tumorigenesis surgery, mice were anesthetized with isoflurane. Hair was removed from the abdomen and chest of the mice, and the sides were shaved to the posterior ventral line to fully expose the liver surface area. The IVIS® Lumina III (PerkinElmer, Massachusetts, USA) was preheated. After satisfactory anesthesia, the mice were placed supine in the imaging area. Bioluminescence and fluorescence parameters were adjusted before in vivo imaging was performed, and the fluorescence intensity of the liver was calculated and analyzed. After in vivo imaging, the mouse's heart, lungs, liver, kidneys, and spleen were removed and arranged sequentially on a dedicated imaging blackboard for further fluorescence imaging. After imaging, a portion of the liver specimen was fixed in 4% paraformaldehyde, and the remainder was rapidly cryopreserved in liquid nitrogen.

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

[0171] The acquired tumor tissue was fixed with paraformaldehyde (4%) for 24 hours and then embedded in molten paraffin. Tissue sections (5 μm thickness) were prepared using a Leica microtome (RM 2235, Germany). The paraffin-embedded sections were deparaffinized in xylene and hydrated. All sections were blocked with 5% goat or donkey serum for 1 hour. The sections were then incubated overnight at 4°C with anti-CD31 antibody (CST, USA). The washed sections were subsequently incubated with Goat Anti-Mouse IgG H&L (Sulfo-Cyanine3). Finally, the washed sections were mounted with a blocking solution containing DAPI anti-fluorescence quencher. Fluorescence images of the stained sections were captured using an inverted fluorescence microscope, and each stained section was quantified using Fiji. To investigate the effect of drug-loaded nanoclusters on the diffusion of PD-1 monoclonal antibody within tumors, two additional channels, FITC (Em / Ex) 488nm / 520nm and DIR (Em / Ex) 754nm / 778nm, were added during fluorescence imaging.

[0172] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Drug-loaded nanoclusters, characterized in that, Its preparation method is as follows: S1: Prepare 50 mL of pure aqueous solution of 2 mg / mL ovalbumin, stir until clear, and obtain solution a; S2: Titrate solution a with 100 mM hydrochloric acid solution until pH=3.

0. When the phenomenon of turbidity turning into flocculent and then clearing up is observed, the titration can be stopped to obtain solution b. S3: Prepare 50 mL each of 250 μg / mL lenvatinib and 30 mg / mL CoCl2•6H2O solutions using 5% acetic acid, mix them and stir until clear to obtain solution c; S4: Add solution c dropwise to solution b using a burette, stirring continuously during the titration process, to obtain solution d; S5: Titrate solution d with 1 M sodium hydroxide solution to pH=7-8. The titration can be stopped when the solution changes from clear to flocculent and then to turbid, and solution e is obtained. S6: Solution e was placed in an 8000-14000D dialysis bag for dialysis. After dialysis, the solution was collected through a 0.22μm filter and lyophilized to obtain drug-loaded nanoclusters Co+Len@OVA loaded with lenvatinib and cobalt ions.

2. The method for preparing drug-loaded nanoclusters as described in claim 1, characterized in that, The steps are as follows: S1: Prepare 50 mL of pure aqueous solution of 2 mg / mL ovalbumin, stir until clear, and obtain solution a; S2: Titrate solution a with 100 mM hydrochloric acid solution until pH=3.

0. When the phenomenon of turbidity turning into flocculent and then clearing up is observed, the titration can be stopped to obtain solution b. S3: Prepare 50 mL each of 250 μg / mL lenvatinib and 30 mg / mL CoCl2•6H2O solutions using 5% acetic acid, mix them and stir until clear to obtain solution c; S4: Add solution c dropwise to solution b using a burette, stirring continuously during the titration process, to obtain solution d; S5: Titrate solution d with 1 M sodium hydroxide solution to pH=7-8. The titration can be stopped when the solution changes from clear to flocculent and then to turbid, and solution e is obtained. S6: Solution e was placed in an 8000-14000D dialysis bag for dialysis. After dialysis, the solution was collected through a 0.22μm filter and lyophilized to obtain drug-loaded nanoclusters Co+Len@OVA loaded with lenvatinib and cobalt ions.

3. The use of the drug-loaded nanoclusters as described in claim 1 in the preparation of products for the prevention and / or treatment of hepatocellular carcinoma; The product is a drug and / or a combination of drugs.

4. A drug, characterized in that, include: The drug-loaded nanoclusters as described in claim 1.

5. A formulation, characterized in that, include: The drug-loaded nanoclusters as described in claim 1.

6. A drug combination, characterized in that, include: The drug as described in claim 4 and any other active ingredients.

7. The pharmaceutical combination as described in claim 6, characterized in that, The other possible active ingredients include: PD-1 monoclonal antibody.

Images