Biomimetic nano preparation for mitochondrial targeted delivery of medicine based on biological orthogonal reaction as well as preparation method and application of biomimetic nano preparation

Abstract

The invention belongs to the technical field of drug delivery, and relates to a biomimetic nano preparation for mitochondrial targeted drug delivery based on biological orthogonal reaction, and a preparation method and application thereof. The bionic nano preparation comprises drug-loaded particles and a mitochondrial targeting bionic cell membrane coating the drug-loaded particles, the drug-loaded particles comprise a drug and a metal organic framework material loaded with the drug; the outer surface of the mitochondrial targeting bionic cell membrane is marked with a mitochondrial targeting group. The mitochondrial targeting group is marked to the surface of the bionic cell membrane, and the bionic nano preparation can be delivered to a focus (such as a tumor) and cell mitochondria of the focus in a cascade manner by utilizing the combined targeting property of the bionic cell membrane and the mitochondrial targeting group (such as TPP), so that a drug can be accurately delivered to the focus, and the drug delivery efficiency is improved. Meanwhile, the in-vivo residence time of the bionic nano preparation is also prolonged, the biological distribution of the medicine is improved, and the medication safety is improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of drug delivery, and relates to a biomimetic nano-preparation for mitochondria-targeted delivery of drugs based on a biological orthogonal reaction, a preparation method therefor and an application thereof. BACKGROUND

[0002] Hepatocellular carcinoma (HCC) is a primary liver cancer with high mortality. Globally, the incidence and mortality of HCC rank sixth and third respectively, and the incidence is increasing year by year, which seriously threatens human health. In China, the incidence of liver cancer is high, and it has become one of the important causes of death from malignant tumors in China.

[0003] Surgery, radiotherapy and chemotherapy are still the commonly used treatment methods for liver cancer in clinical practice at present, but these treatment methods have problems such as large side effects and low quality of life of patients. Data shows that the 5-year recurrence rate of HCC patients is as high as 50%-70%, and the treatment is more difficult after recurrence, and the prognosis is poorer. Therefore, new modes such as targeted therapy, immunotherapy, local combined systemic therapy are actively explored in clinical practice at present, in order to reduce side effects and reduce the risk of recurrence and metastasis.

[0004] Immunotherapy has been proven to effectively control tumor growth, prolong patient survival and improve quality of life, bringing new treatment hope to liver cancer patients. Among them, the immune checkpoint inhibitor represented by programmed death ligand 1 (PD-L1) / programmed death receptor 1 (PD-1) antibody is the main drug for liver cancer immunotherapy at present, and due to the existence of a large number of immunosuppressive cells in the microenvironment of HCC, the immunogenicity of liver tumor is low, and the natural immune tolerance characteristics of the liver as the largest metabolic organ of the human body further amplify the low immunogenicity of HCC, resulting in a lower response rate of HCC patients to immunotherapy and easy drug resistance. Therefore, it is urgent to develop a treatment strategy that can effectively improve the immunogenicity of liver tumor to achieve precise and efficient anti-liver cancer immunotherapy.

[0005] The cyclic guanosine monophosphate-adenosine monophosphate synthetase-interferon gene stimulator (cGAS-STING) pathway is an important immune response pathway in the cytoplasm, which regulates innate and adaptive immune responses by recognizing cytosolic DNA. When the cGAS protein detects DNA in the cytoplasm, it catalyzes the synthesis of the second messenger 2',3'-cGAMP, directly activates STING on the endoplasmic reticulum to transport to the Golgi apparatus, activates TANK binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3), promotes the secretion of type I interferons, induces the maturation of dendritic cells (DCs), promotes antigen presentation and activates T cell immune response. It is suggested that activation of the cGAS-STING pathway in tumor cells can enhance their immunogenicity, thereby effectively initiating an anti-tumor immune response.

[0006] Traditional Chinese medicine (TCM) is a traditional treasure of the Chinese nation, carrying thousands of years of wisdom and cultural accumulation. The monomer components of TCM with multi-target and multi-pathway treatment advantages have great potential in tumor treatment. For example, ginsenoside Rg3 can inhibit tumor progression by inhibiting tumor angiogenesis and enhancing T cell activity; oridonin can inhibit mitochondrial function of tumor cells and activate apoptosis-related genes, and can also reverse the drug resistance of tumor cells to chemotherapy drugs; curcumin can target the NF-κB inflammatory pathway and down-regulate the expression of PD-L1, and has direct tumor inhibition and immune regulation effects; artemisinin derivatives can induce immunogenic death of tumor cells and activate the killing function of NK cells, providing diversified natural drug options for tumor treatment.

[0007] Polyphyllin II (PPⅡ) is an important class of steroidal saponins in the traditional Chinese medicine Paris polyphylla, which has been proven to have significant anti-hepatoma effects. Its mechanism involves inducing endogenous substance disorder in hepatoma HepG2 cells, triggering cell S phase arrest, and inducing tumor cell apoptosis by interfering with intracellular sugar metabolism and tricarboxylic acid cycle. In addition, by regulating the p53 / SLC7A11 / GPX4 signaling axis, it can cause imbalance of the antioxidant system of cells and accumulation of lipid peroxides, and then induce ferroptosis of hepatoma HepG2 cells, which may also be one of the mechanisms of PPⅡ against hepatoma. The mitochondrial damage caused by cell ferroptosis and the massive release of mitochondrial DNA (mtDNA) are expected to serve as a “biological detonator” to “ignite” the cGAS-STING pathway and its downstream cascade reaction. However, PPⅡ has hepatorenal toxicity and poor mitochondrial targeting when used in large amounts for a long time, which may lead to insufficient activation of the cGAS-STING pathway. Therefore, there is an urgent need to develop new treatment strategies to achieve site-specific and controllable release of active ingredients of traditional Chinese medicines such as PPⅡ.

[0008] The rapid development of metal-organic frameworks (MOFs) provides a valuable idea for the effective delivery of active ingredients of traditional Chinese medicines such as PPⅡ. ZIF-8 is a metal-organic framework formed by zinc ions and 2-methylimidazole, which is widely used in nanodrug delivery systems. However, the nanomaterials synthesized by MOFs have the risk of being recognized and removed by the body's immune system, resulting in short circulation time in the body and ineffective targeting to tumor sites, which limits their biomedical applications. SUMMARY

[0009] Problem to be solved by the invention

[0010] Cell membrane biomimetic technology can effectively solve the problems of poor biocompatibility and insufficient targeting of traditional carriers by coating nanometer inner core with natural cell membrane. For example, red blood cell membrane-coated nanoparticles can evade immune clearance and prolong circulation time; tumor cell membrane-coated carriers can achieve homologous targeting. Mesenchymal stem cells (MSCs) are a kind of adult stem cells derived from mesoderm, which have the characteristics of directional migration to tumor microenvironment, and can achieve targeted delivery from "passive enrichment" to "active colonization". Moreover, MSCs themselves have low immunogenicity, and their membrane structure can evade phagocytic clearance by macrophages, significantly prolonging the circulation time in vivo.

[0011] If cell membrane biomimetic technology is combined with mitochondrial targeting strategy, the precision delivery efficiency of nanometer preparations can be further improved. Commonly used mitochondrial targeting heads include triphenylphosphine (TPP), TPO4 peptide, TAT peptide, and other mitochondrial penetrating peptides (MPPs), a-tocopherol succinate (a-TOS), diquafosine (DQA), and mitochondrial targeting coenzyme Q10 derivatives (MitoQ). As the most classic mitochondrial targeting ligand, the positive charge structure of TPP can be driven by the mitochondrial membrane potential to accumulate in mitochondria at high concentration. Therefore, if mitochondrial targeting heads (such as TPP) are designed in combination with cell membrane (such as MSC membrane) biomimetic carriers, it is expected to achieve multi-level targeting from tumor microenvironment colonization to mitochondrial subcellular level delivery, providing a new paradigm for tumor treatment.

[0012] Bioorthogonal reaction refers to a type of chemical reaction that can be efficiently carried out in a biological system without interfering with endogenous biochemical reactions. The core principle is to use highly specific reactions between special chemical groups that do not exist in the body, which occur rapidly under physiological conditions (such as room temperature, neutral pH, and aqueous environment) without cross-reactions with natural molecules in the body (such as proteins, nucleic acids, and carbohydrates). This allows for precise labeling, modification, or regulation of biological molecules or processes. Common chemical groups involved in the reaction include azido, alkyne, tetrazine, and trans-cyclooctene. These groups do not naturally occur in the body, so they only react with each other and do not react with common molecules such as amino acids, nucleotides, and sugars in the body, ensuring the specificity of the reaction.

[0013] Copper-free click chemistry has unique advantages in target head modification because it avoids the cytotoxicity of traditional copper-catalyzed click reactions and does not require extreme temperature, pH or organic solvents, and has become an important tool for precise chemical modification. The SPAAC reaction is one of the typical copper-free click chemistry reactions, which is based on the high reactivity between cyclooctyne (such as DBCO) with a ring strain and azido group. Without metal catalysts, the two can quickly undergo a cycloaddition reaction to form a stable triazole ring structure. The high efficiency, strong specificity, and high biocompatibility of copper-free click chemistry provide a feasible strategy for modifying mitochondrial targeting groups on the surface of biomimetic cell membranes and targeted delivery of drugs to mitochondria.

[0014] In summary, the present application aims to provide a mitochondrially targeted biomimetic nanofomulation, a preparation method and application thereof. By introducing mitochondrial targeting groups through biological orthogonal reactions, and combining biomimetic cell membrane technology and mitochondrial targeting strategies, the lesion (such as tumor) targeting ability and mitochondrial targeting of the biomimetic nanofomulation are significantly improved, the in vivo circulation time of the biomimetic nanofomulation is significantly prolonged, the biological safety is improved, and the drug can be efficiently delivered to the lesion (such as tumor) site to exert its excellent pharmacodynamic (such as antitumor) effect.

[0015] Solution for solving the problem

[0016] To achieve the above object, the technical scheme adopted by the present application is as follows:

[0017] In a first aspect, the present application provides a mitochondrially targeted biomimetic nanofomulation, wherein the biomimetic nanofomulation comprises drug-loaded particles and a mitochondrially targeted biomimetic cell membrane covering the drug-loaded particles.

[0018] The drug-loaded particles comprise a drug and a metal-organic framework material loaded with the drug.

[0019] The outer surface of the mitochondrially targeted biomimetic cell membrane is labeled with a mitochondrial targeting group.

[0020] Preferably, the mitochondrial targeting group is labeled to the outer surface of the mitochondrially targeted biomimetic cell membrane through a biological orthogonal reaction.

[0021] More preferably, the biological orthogonal reaction is carried out between a biomimetic cell membrane comprising a biological orthogonal group and a donor compound comprising a biological orthogonal ligand group, the donor compound comprising a biological orthogonal ligand group.

[0022] Further preferably, the biological orthogonal group comprises an azido group or a tetrazine group, and the biological orthogonal ligand group comprises an alkyne group or an alkenyl group.

[0023] Preferably, the drug comprises one or more of antitumor drugs, anti-inflammatory drugs, antibacterial drugs, osteoporosis treatment drugs, photosensitizers and protein hormone drugs.

[0024] More preferably, the antitumor drug comprises one or more of active ingredients of traditional Chinese medicine, chemically synthesized antitumor drugs, targeted therapy drugs, immunotherapy drugs, autophagy inhibitors and small nucleic acid antitumor drugs.

[0025] Further preferably, the active ingredient of traditional Chinese medicine comprises one or more of Chonglou saponin II, Chonglou saponin I, Xihingcao A, cucurbitacin B, astilbin, ginsenoside Rg3 and curcumin, preferably Chonglou saponin II.

[0026] Preferably, the metal organic framework material is ZIF-8, ZIF-67 or IRMOF-8; further, the metal organic framework material is etched by tannic acid.

[0027] Preferably, the biomimetic cell membrane comprises one or more of red blood cell membrane, macrophage membrane, platelet membrane, white blood cell membrane, tumor cell membrane, bone marrow mesenchymal stem cell membrane, adipose tissue mesenchymal stem cell membrane and embryonic mesenchymal stem cell membrane.

[0028] More preferably, the biomimetic cell membrane comprises one or more of bone marrow mesenchymal stem cell membrane, adipose tissue mesenchymal stem cell membrane and embryonic mesenchymal stem cell membrane.

[0029] Preferably, the mitochondrial targeting group is one or more of triphenylphosphine, TP04 peptide, TAT peptide, α-tocopherol succinate, dequalinium chloride and mitochondrial targeting coenzyme Q10 derivative.

[0030] More preferably, the mitochondrial targeting group is a group formed by triphenylphosphine.

[0031] Preferably, a hydrophilic group is further connected between the biological ortho coordination group and the mitochondrial targeting group; further, the hydrophilic group is a group formed by polyethylene glycol.

[0032] Preferably, the particle size of the biomimetic nano-preparation is 100-500 nm, preferably 150-300 nm.

[0033] In a second aspect, the present application provides a preparation method of the biomimetic nano-preparation, comprising the following steps:

[0034] (1) mixing the drug with the metal organic framework material and stirring to obtain drug-loaded particles;

[0035] (2) co-incubate the compound containing the bio-orthogonal group with the cell or cell fragment containing the biomimetic cell membrane, and extract the biomimetic cell membrane labeled by the bio-orthogonal group;

[0036] (3) mix the drug-loaded particles obtained in step (1) with the biomimetic cell membrane obtained in step (2), and perform ultrasonic treatment to obtain the drug-loaded particles coated by the biomimetic cell membrane;

[0037] (4) mix the drug-loaded particles coated by the biomimetic cell membrane obtained in step (3) with the donor compound containing the mitochondria-targeting group, and stir to obtain the biomimetic nano-preparation.

[0038] Preferably, in step (1), the metal organic framework material is tannic acid-etched ZIF-8, ZIF-67 or IRMOF-8, and the preparation method of the metal organic framework material comprises: mixing tannic acid and ZIF-8, ZIF-67 or IRMOF-8, and stirring to obtain the metal organic framework material.

[0039] Preferably, in step (2), the compound containing the bio-orthogonal group is one or more of tetraacetylated N-azidoacetylmannosamine, tetraacetylated N-azidoacetylglucosamine, tetraacetylated N-azidoacetylgalactosamine and tetrazine.

[0040] Preferably, the cell or cell fragment containing the biomimetic cell membrane is one or more of red blood cells, macrophages, platelets, white blood cells, tumor cells, bone marrow mesenchymal stem cells, adipose tissue mesenchymal stem cells and embryonic mesenchymal stem cells.

[0041] Preferably, in step (4), the donor compound containing the mitochondria-targeting group further contains a bio-orthogonal coordination group.

[0042] Preferably, the donor compound containing the mitochondria-targeting group further contains a hydrophilic group.

[0043] More preferably, the donor compound containing the mitochondria-targeting group is DBCO-PEG-TPP.

[0044] In a third aspect, the present application provides a pharmaceutical preparation comprising the biomimetic nano-preparation of the present application.

[0045] In a fourth aspect, the present application provides a pharmaceutical combination form comprising the biomimetic nano-preparation of the present application or the pharmaceutical preparation of the present application and at least one additional anti-tumor drug.

[0046] Preferably, the additional anti-tumor drug is a PD-L1 inhibitor or a PD-1 inhibitor.

[0047] Fifthly, the present invention provides the use of the biomimetic nanoformulations, pharmaceutical formulations, or drug combinations described herein in the preparation of a medicament for treating cancer.

[0048] Preferably, the cancer is liver cancer, and more preferably hepatocellular carcinoma.

[0049] Effects of the invention

[0050] This invention labels mitochondrial targeting groups onto the surface of a biomimetic cell membrane. Utilizing the combined targeting properties of the biomimetic cell membrane and the mitochondrial targeting groups (such as TPP), biomimetic nanoparticles can be delivered in a cascade to lesions (such as tumors) and the mitochondria within the lesion site. This not only achieves precise drug delivery to the lesion but also prolongs the in vivo retention time of the biomimetic nanoparticles and improves drug biodistribution. In particular, it can also reduce the damage to the liver and kidneys caused by antitumor drugs (such as active ingredients of traditional Chinese medicine), improving medication safety.

[0051] The biomimetic nano-formulation provided by this invention can be used for the efficient delivery of anti-tumor drugs (such as active ingredients of traditional Chinese medicine). This biomimetic nano-formulation has high biocompatibility, precise targeting, and high loading capacity, while also possessing the potential for multimodal synergistic therapy, which can better meet the needs of clinical oncology treatment. This invention provides a new research direction for the application of multifunctional biomimetic delivery platforms in oncology and has broad clinical translational potential.

[0052] Furthermore, the present invention utilizes copper-free click chemistry-based bioorthogonal modification to significantly improve the reaction efficiency between bioorthogonal groups and bioorthogonal ligand groups. Since the bioorthogonal ligand groups are linked to mitochondrial targeting groups, the present invention can precisely and efficiently modify mitochondrial targeting groups (such as TPP) onto the surface of biomimetic nanoparticles.

[0053] Furthermore, this invention utilizes hollow ZIF-8 cells etched with tannic acid as a carrier material for active ingredients in traditional Chinese medicine. This etching method is simple to operate, low in cost, and provides ample space for the drug (such as active ingredients in traditional Chinese medicine), achieving efficient loading of the drug (such as active ingredients in traditional Chinese medicine). In addition, the potential of the ZIF-8 cells after tannic acid etching changes from positive to negative, which is highly beneficial for the binding of active ingredients in traditional Chinese medicine. This method also provides a reference for loading drugs onto other metal-based organic materials, combining the advantages of large specific surface area of ​​MOFs with ample space for drugs, and providing a feasible approach and method for the efficient loading of drugs with large molecular weights.

[0054] Furthermore, this invention fully utilizes the inherent synergistic chemotherapy and immunotherapy properties of the active ingredients in traditional Chinese medicine. For example, Paris saponin II not only exerts its excellent chemotherapy effects by promoting the generation of reactive oxygen species, ferrous ion accumulation, excessive accumulation of lipid peroxides, mitochondrial functional and structural disorders, and inducing ferroptosis in tumor cells, but also releases a large amount of mtDNA during ferroptosis, which acts as a "bio-detonator" for activating the cGAS-STING immune pathway, thereby triggering this pathway and downstream cascade reactions, restarting the immune response, and exerting its immunotherapy effect. Therefore, the biomimetic nano-formulation of this invention does not require co-loading with other drugs, making the preparation process simple and easy, and avoiding the potential toxic side effects of other drugs. Attached Figure Description

[0055]

[0056]

[0057]

[0058] Detailed Implementation

[0059] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0060] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0061] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0062] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0063] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0064] In this invention, the range of values ​​represented by "value A to value B" refers to the range that includes the endpoint values ​​A and B.

[0065] In this invention, when "room temperature" or "room temperature" is used, the temperature can be 15~30℃, or more specifically 15~25℃, for example 20℃.

[0066] In this invention, "DBCO-PEG-TPP" represents dibenzocyclooctylene-polyethylene glycol-triphenylphosphine, a functional chemical molecule composed of three parts: DBCO, PEG, and TPP. DBCO is dibenzocyclooctylene, capable of undergoing copper-free click chemistry with azide groups; PEG is polyethylene glycol, acting as a bridge between the DBCO and TPP groups, providing hydrophilicity and biocompatibility, reducing immune recognition, and prolonging the molecule's in vivo circulation time; TPP is triphenylphosphine, possessing positive charge characteristics and mitochondrial membrane potential difference binding ability, enabling specific targeting of mitochondria. In this invention, DBCO-PEG-TPP is commercially available, and its chemical structure is as follows:

[0067]

[0068] In this invention, "Ac4ManNAz" represents tetraacetylated N-azidoacetylmannosamine, a functionalized sugar derivative with mannose as its backbone. The tetraacetyl modification increases the molecule's lipid solubility, allowing it to more easily penetrate cell membranes. The azide group can react with the alkyne group to generate a highly efficient copper-free click chemistry. Once Ac4ManNAz enters the cell, intracellular carboxylesterases remove the acetyl group, enabling the mannose portion to participate in normal intracellular sugar metabolism pathways. It is then integrated into glycoproteins, glycolipids, and other biomolecules on the cell surface, thus completing the N3 labeling of the cell membrane. The chemical structure of Ac4ManNAz is as follows:

[0069]

[0070] <Mitochondrial-targeted biomimetic nanoparticle formulations>

[0071] First, the present invention provides a mitochondrial-targeted biomimetic nanoformulation, wherein the biomimetic nanoformulation comprises drug-loaded particles and a mitochondrial-targeted biomimetic cell membrane coating the drug-loaded particles.

[0072] The drug-loaded particles comprise a drug and a metal-organic framework material on which the drug is loaded;

[0073] The outer surface of the mitochondrial-targeting biomimetic cell membrane is labeled with mitochondrial-targeting groups.

[0074] In some embodiments, the mitochondrial targeting group is labeled onto the outer surface of the mitochondrial-targeting biomimetic cell membrane via a bioorthogonal reaction.

[0075] In some embodiments, the bioorthogonal reaction is carried out between a biomimetic cell membrane and a donor compound containing the mitochondrial targeting group, the biomimetic cell membrane containing the bioorthogonal group and the donor compound containing the bioorthogonal coordination group.

[0076] In some preferred embodiments, the bioorthogonal group includes an azide group or a tetrazine group.

[0077] In some preferred embodiments, the bioorthogonal group is an azide group.

[0078] In some preferred embodiments, the bioorthogonal coordination group includes an alkynyl or alkenyl group.

[0079] In some preferred embodiments, the bioorthogonal coordination group is an alkynyl group.

[0080] In some specific implementations, the bioorthogonal group is an azide group, and the bioorthogonal coordination group is an alkynyl group.

[0081] In some implementations, the drug includes one or more of the following: antitumor drugs, anti-inflammatory drugs, antibacterial drugs, osteoporosis treatment drugs, photosensitizers, and protein-based hormone drugs.

[0082] In some implementations, the antitumor drug includes one or more of the following: active ingredients of traditional Chinese medicine for antitumor treatment, chemically synthesized antitumor drugs, targeted therapy drugs, immunotherapy drugs, autophagy inhibitors, and small nucleic acid antitumor drugs.

[0083] In some embodiments, the active ingredients of the antitumor traditional Chinese medicine include one or more of the following: Paris polyphylla saponin II, Paris polyphylla saponin I, oridonin A, cucurbitacin B, astilbene, ginsenoside Rg3, and curcumin.

[0084] In some specific implementation schemes, the active ingredient of the anti-tumor traditional Chinese medicine includes Paris saponin II.

[0085] In some specific implementation schemes, the active ingredient of the anti-tumor traditional Chinese medicine is Paris saponin II.

[0086] In this invention, the active ingredients of the anti-tumor traditional Chinese medicine can induce ferroptosis by promoting the generation of reactive oxygen species, the accumulation of ferrous ions, the excessive accumulation of lipid peroxides, and the dysfunction and structural disorders of mitochondria. They can also significantly activate the cGAS-STING pathway by promoting the release of mtDNA, thereby inducing dendritic cell maturation, promoting the transformation of M2 macrophages into M1 macrophages, and promoting T cell proliferation, thus exerting a significant immune effect and killing tumor cells.

[0087] In some embodiments, the chemically synthesized antitumor drugs include alkylating agents (such as cyclophosphamide, carmustine, etc.), antimetabolites (such as methotrexate, 6-mercaptopurine, 5-fluorouracil, cytarabine, etc.), platinum-based drugs (such as cisplatin, carboplatin, oxaliplatin, etc.), and antitumor antibiotics (such as doxorubicin).

[0088] In some implementations, the targeted therapeutic agents include small molecule inhibitors (such as imatinib, gefitinib, osimertinib, crizotinib, etc.), monoclonal antibodies (such as rituximab, trastuzumab, bevacizumab, etc.), etc.

[0089] In some implementations, the immunotherapeutic agents include PD-1 / PD-L1 inhibitors (such as atezolizumab, durvalumab, sugemalimab, pembrolizumab, nivolumab, etc.), CTLA-4 inhibitors (such as ipilimumab, etc.), hypoxia-activating prodrugs (terazamine), etc.

[0090] In some embodiments, the autophagy inhibitor includes 3-methyladenine, etc.

[0091] In some implementations, the small nucleic acid antitumor drugs include BCL-2 inhibitors (such as Venetoclax), siRNA drugs (such as Patisiran), etc.

[0092] In some implementations, the anti-inflammatory drug includes ibuprofen, etc.

[0093] In some implementations, the antibacterial drug includes gentamicin, vancomycin, etc.

[0094] In some implementations, the osteoporosis treatment drug includes risedronate sodium.

[0095] In some implementations, the photosensitizer includes zinc phthalocyanine, dihydroporphyrin 6, etc.

[0096] In some implementations, the protein-based hormone drug includes insulin, etc.

[0097] In some embodiments, the metal-organic framework material is ZIF-8, ZIF-67, or IRMof-8.

[0098] In some embodiments, the metal-organic framework material is ZIF-8, i.e., zeolite imidazole ester framework-8.

[0099] In some specific embodiments, the metal-organic framework material is etched with tannic acid (TA) (preferably chemical etching), for example, the metal-organic framework material can be obtained by etching and coordinating ZIF-8 with tannic acid. Etching in metal-organic framework materials refers to the process of selectively removing specific parts of the material, such as metal nodes, organic ligands, or carbon skeletons, through chemical or physical means to control its pore structure, expose active sites, or introduce defects. This technique can introduce new ligands by breaking the coordination bonds or chemical bonds of the metal-organic framework material.

[0100] In some embodiments, the biomimetic cell membrane is one or more of the following: erythrocyte membrane, macrophage membrane, platelet membrane, leukocyte membrane, tumor cell membrane, bone marrow mesenchymal stem cell membrane, adipose mesenchymal stem cell membrane, and embryonic mesenchymal stem cell membrane.

[0101] In some preferred embodiments, the biomimetic cell membrane is one or more of bone marrow mesenchymal stem cell membranes, adipose mesenchymal stem cell membranes, and embryonic mesenchymal stem cell membranes.

[0102] In some embodiments, the mitochondrial targeting group is one or more of the following: triphenylphosphine, TPO4 peptide, TAT peptide, α-tocopherol succinate (α-TOS), desquinoline chloride (DQA), and mitochondrial-targeting coenzyme Q10 derivative (MitoQ).

[0103] In some preferred embodiments, the mitochondrial targeting group is a triphenylphosphine-based group.

[0104] In some embodiments, a hydrophilic group is also attached between the bioorthogonal coordination group and the mitochondrial targeting group.

[0105] In some embodiments, the hydrophilic group is a group formed from polyethylene glycol.

[0106] In some specific embodiments, the average molecular weight of the polyethylene glycol of the present invention is 400-4000. For example, the polyethylene glycol of the present invention can be polyethylene glycol 1000, polyethylene glycol 2000, polyethylene glycol 4000, etc.

[0107] In some embodiments, the biomimetic nanoparticles of the present invention have a particle size of 100-500 nm, such as 150 nm, 200 nm, 250 nm, 300 nm, 400 nm, etc., preferably 150-300 nm.

[0108] In some embodiments, the mass ratio of the antitumor drug to the metal-organic framework material is 1:1 to 10:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 8:1, etc., preferably 2:1 to 6:1.

[0109] In some embodiments, the mass ratio of the drug-loaded particles to the mitochondrial-targeting biomimetic cell membrane is 1:1 to 1:5, such as 1:1.5, 1:2, 1:3, 1:4, etc., preferably 1:1 to 1:3.

[0110] <Preparation Methods of Bionic Nanoparticles>

[0111] This invention provides a method for preparing the biomimetic nanoparticle formulation described herein, comprising the following steps:

[0112] (1) Mix the drug with the metal-organic framework material and stir to obtain drug-loaded particles;

[0113] (2) Incubate a compound containing a bioorthogonal group with cells or cell fragments containing a biomimetic cell membrane, and extract the biomimetic cell membrane labeled with the bioorthogonal group.

[0114] (3) Mix the drug-loaded particles obtained in step (1) with the biomimetic cell membrane obtained in step (2), and sonicate to obtain drug-loaded particles coated with biomimetic cell membrane.

[0115] (4) The drug-loaded particles coated with biomimetic cell membranes obtained in step (3) are mixed with donor compounds containing mitochondrial targeting groups and stirred to obtain the biomimetic nano-formulation.

[0116] In some implementations, in step (1), the mass ratio of the drug to the metal-organic framework material is 1:1-10:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 8:1, etc., preferably 2:1-6:1.

[0117] In some embodiments, in step (1), the drug and the metal-organic framework material are dissolved in a solvent; further, the solvent is an alcohol solvent, such as methanol, ethanol, isopropanol, tert-butanol, etc.

[0118] In some embodiments, in step (1), the drug is first dissolved in a solvent, then the metal-organic framework material is added, stirred, centrifuged, and a precipitate is obtained. The precipitate is washed with the solvent and dried to obtain the drug-loaded particles. Further, the solvent is an alcohol solvent, such as methanol, ethanol, isopropanol, tert-butanol, etc. Further, the concentration of the drug after dissolving in the solvent is 1-15 mg / mL, such as 2 mg / mL, 3 mg / mL, 5 mg / mL, 8 mg / mL, 10 mg / mL, etc.

[0119] In some implementations, the stirring time in step (1) is 24-120 h, for example 48 h, 72 h, 96 h, etc.

[0120] In some implementations, in step (1), the metal-organic framework material is ZIF-8, ZIF-67, or IRMOF-8.

[0121] In some embodiments, the preparation method of ZIF-8 includes: mixing a zinc salt and 2-methylimidazole, stirring, to obtain ZIF-8. Further, the zinc salt is zinc nitrate, zinc chloride, or its hydrate, etc. Further, the molar ratio of the zinc salt to the 2-methylimidazole is 1:10-1:50, for example 1:15, 1:20, 1:30, 1:32, 1:40, etc., preferably 1:20-1:40. Further, the zinc salt and 2-methylimidazole are dissolved in a solvent; the solvent is an alcohol solvent, such as methanol, ethanol, isopropanol, tert-butanol, etc.; further, the concentration of the zinc salt after dissolving in the solvent is 0.05-1 mmol / mL, for example 0.08 mmol / mL, 0.1 mmol / mL, 0.15 mmol / mL, 0.2 mmol / mL, 0.5 mmol / mL, etc. Further, the preparation method of ZIF-8 includes: dissolving zinc salt and 2-methylimidazole in a solvent, stirring, centrifuging to obtain a precipitate, washing the precipitate with the solvent, and drying to obtain ZIF-8. Further, the stirring time is 1-5 h, for example, 2 h, 3 h, 4 h, etc.

[0122] In some implementations, in step (1), the metal-organic framework material is tannic acid-etched ZIF-8, ZIF-67, or IRMOF-8.

[0123] In some embodiments, the method for preparing tannic acid-etched ZIF-8 includes: mixing tannic acid and ZIF-8, stirring, to obtain tannic acid-etched ZIF-8. Further, the mass ratio of the tannic acid to the ZIF-8 is 1:1-10:1, for example, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 5:1, etc., preferably 1:1-5:1. Further, the tannic acid and the ZIF-8 are dissolved in a solvent; the solvent is water, for example, deionized water; further, the concentration of the tannic acid after dissolving in the solvent is 1-20 mg / mL, for example, 2 mg / mL, 5 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, etc. Further, the preparation method of tannic acid-etched ZIF-8 includes: dissolving tannic acid and ZIF-8 separately in a solvent, mixing, stirring, allowing to stand, centrifuging to obtain a precipitate, washing the precipitate with the solvent, and drying to obtain tannic acid-etched ZIF-8. Further, the stirring time is 10-30 min, for example, 15 min, 20 min, 25 min, etc. Further, the standing time is 0.5-5 h, for example, 1 h, 2 h, 3 h, 4 h, etc.

[0124] In some embodiments, in step (2), the compound containing the bioorthogonal group is one or more of tetraacetylated N-azidoacetylmannosamine, tetraacetylated N-azidoacetylglucosamine, tetraacetylated N-azidoacetylgalactosamine, and tetraazine.

[0125] In some preferred embodiments, in step (2), the compound containing the bioorthogonal group is tetraacetylated N-azidoacetylmannosamine.

[0126] In some implementations, in step (2), the cell or cell fragment containing the biomimetic cell membrane is one or more of the following: red blood cells, macrophages, platelets, white blood cells, tumor cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, and embryonic mesenchymal stem cells.

[0127] In some preferred embodiments, in step (2), the cells containing the biomimetic cell membrane are one or more of bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, and embryonic mesenchymal stem cells.

[0128] In some embodiments, in step (2), the cells or cell fragments containing the biomimetic cell membrane and the compound containing the bioorthogonal group are co-incubated in a complete cell culture medium (e.g., DMEM medium); further, the concentration of the compound containing the bioorthogonal group in the culture medium is 10-60 μg / mL, for example 15 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, etc.

[0129] In some implementations, the incubation time in step (2) is 24-120 h, such as 48 h, 72 h, 96 h, etc.

[0130] In some implementations, in step (2), the cells or cell fragments are broken up using a freeze-thaw method.

[0131] In some implementations, in step (3), the mass ratio of the drug-loaded particles to the biomimetic cell membrane is 1:1 to 1:5, such as 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 4:1, etc., preferably 1:1 to 3:1.

[0132] In some implementations, in step (3), the drug-loaded particles and the biomimetic cell membrane are dissolved in a solvent; further, the solvent is water, such as deionized water.

[0133] In some implementations, in step (3), the biomimetic cell membrane is coated onto the surface of the drug-loaded particles using a cell sonic disruptor.

[0134] In some implementations, in step (3), the power of the ultrasound is 100-300W, the ultrasound program is ultrasound for 1-3 seconds, stop for 1-3 seconds, and the total duration is 3-5 minutes.

[0135] In some implementations, in step (3), after sonication, centrifugation is performed to obtain a precipitate, which is then washed and freeze-dried to obtain drug-loaded particles coated with a biomimetic cell membrane.

[0136] In some implementations, in step (4), the donor compound containing the mitochondrial targeting group further contains a bioorthogonal coordination group.

[0137] In some embodiments, in step (4), the bioorthogonal coordination group is one or more of the groups formed from dibenzocyclooctylene, bicyclo[6,1,0]nonyne, 2-cyclooctyl-acetic acid, and trans-cyclooctene.

[0138] In some preferred embodiments, in step (4), the bioorthogonal coordination group is a group formed from dibenzocyclooctylene.

[0139] In some embodiments, in step (4), the donor compound containing the mitochondrial targeting group also contains a hydrophilic group; further, the hydrophilic group is polyethylene glycol, such as polyethylene glycol 1000, polyethylene glycol 2000, polyethylene glycol 4000, etc.

[0140] In some preferred embodiments, in step (4), the donor compound containing the mitochondrial targeting group is DBCO-PEG-TPP.

[0141] In some implementations, in step (4), the mass ratio of the drug-loaded particles coated by the biomimetic cell membrane to the donor compound containing the mitochondrial targeting group is 1:(50-500), for example 1:100, 1:200, 1:250, 1:300, 1:400, etc.

[0142] In some embodiments, in step (4), the drug-loaded particles coated with the biomimetic cell membrane are mixed with the donor compound containing the mitochondrial targeting group in a solvent. Further, the solvent is water, such as ultrapure water.

[0143] In some implementations, in step (4), the drug-loaded particles coated with the biomimetic cell membrane are dissolved in a solvent and mixed with the donor compound containing the mitochondrial targeting group, stirred, centrifuged, and a precipitate is obtained. The precipitate is washed and freeze-dried to obtain the biomimetic nano-formulation.

[0144] In some implementations, the stirring time in step (4) is 1-5 h, for example 2 h, 3 h, 4 h, etc.

[0145] <Pharmaceutical Preparations>

[0146] This invention provides a pharmaceutical formulation comprising the biomimetic nanoformulation described herein.

[0147] <Drug Combination Therapy>

[0148] This invention provides a combination of drugs, comprising the biomimetic nanoformulation or the pharmaceutical formulation described in this invention, and at least one additional antitumor drug.

[0149] In some embodiments, the drug combination of the present invention includes the biomimetic nanoformulations or the drug formulations of the present invention and an additional antitumor drug.

[0150] In some specific implementations, the additional antitumor drug is a PD-L1 inhibitor or a PD-1 inhibitor.

[0151] In some specific implementations, the PD-L1 inhibitor includes one or more drugs such as atezolizumab, durvalumab, and sugemalimab.

[0152] <Medical Uses>

[0153] This invention provides the use of the biomimetic nanoformulation, the pharmaceutical formulation, or the combination of drugs described herein in the preparation of a drug for treating cancer.

[0154] Furthermore, the biomimetic nano-formulation, the pharmaceutical formulation, or the combination of drugs described in this invention are used to treat cancer.

[0155] In addition, the present invention also provides a method for treating cancer, comprising: administering a therapeutically effective amount of the biomimetic nano-formulation, the pharmaceutical formulation, or the combination of drugs described herein to an individual in need.

[0156] In some embodiments, the cancer described in this invention is liver cancer, such as hepatocellular carcinoma.

[0157] <Abbreviations and Full Names>

[0158] The English abbreviations and full Chinese names involved in this invention are shown in the table below:

[0159]

[0160] Example

[0161] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. The MSC cells involved in the examples are bone marrow mesenchymal stem cells.

[0162] Example 1

[0163] Step 1: Preparation of metal-organic framework material TA-ZIF-8

[0164] ZIF-8 support was synthesized using magnetic stirring:

[0165] Accurately weigh 0.5 mmol Zn(NO3)2·6H2O and 16 mmol 2-MI and dissolve them in 5 mL of methanol. After sonication, transfer the solutions to 20 mL vials and stir with a magnetic stirrer (800 rpm, RT) for 2 h. Centrifuge (12000 rpm, 10 min) to remove the supernatant. Wash the precipitate three times with methanol and dry it in a vacuum drying oven at 60 °C for 24 h to obtain the ZIF-8 carrier.

[0166] Synthesis of TA-ZIF-8 nanoparticles using a chemically selective etching coordination method:

[0167] Disperse 0.1 g ZIF-8 in 2 mL of deionized water by sonication. Dissolve 0.25 g TA in 40 mL of deionized water by sonication, then introduce the ZIF-8 solution into the solution. Stir with a magnetic stirrer (800 rpm, RT) for 15 min and allow to stand at room temperature for aging for 1 h. Centrifuge (800 rpm, RT), remove the supernatant, wash the precipitate three times with deionized water, and dry in a vacuum drying oven at 60℃ for 24 h to obtain TA-ZIF-8.

[0168] Step 2: Preparation of drug-loaded particles PZ

[0169] PPⅡ was loaded into TA-ZIF-8 using an ex-situ encapsulation method:

[0170] Accurately weigh 40 mg of PPⅡ and place it in 8 mL of methanol solution. After sonication to dissolve, add 10 mg of TA-ZIF-8 and stir with a magnetic stirrer (800 rpm, RT) for 72 h. Centrifuge (12000 rpm, 30 min), wash the precipitate twice with methanol, combine all supernatants, and determine the drug loading by HPLC (drug loading was 34.95%). Dry the precipitate in a vacuum drying oven at 60 °C for 24 h to obtain PPⅡ / TA-ZIF-8 (PZ).

[0171] Step 3: Preparation of N3-labeled mesenchymal stem cell membranes

[0172] After co-incubating MSC cells with Ac4ManNAz (at a concentration of 30 μg / mL in DMEM complete cell culture medium) for 72 h, cells were carefully scraped off using a cell scraper and collected by centrifugation at 3000 rpm for 15 min. Following the kit instructions, cells were collected at a rate of 3 × 10⁶ cells per cell line. 7 Add 1 mL of membrane protein extraction reagent A (containing 1 mM PMSF) to each cell and incubate on ice for 15 min. Use a freeze-thaw method to lyse the cells: place the cells in liquid nitrogen for 1 min to rapidly freeze, then immediately transfer to a 37°C water bath for 5 min. Repeat this cycle 3 times. Centrifuge the cells at 4°C, 700 g for 10 min, carefully collecting the supernatant, taking care not to touch the precipitate. Centrifuge the supernatant at 4°C, 14000 g for 30 min. The precipitate is the cell membrane fragment. Wash the precipitate three times with PBS and then freeze-dry to obtain N3-labeled MSCs.

[0173] Step 4: Preparation of biomimetic nano-formulation PZ@M

[0174] N3-labeled MSCs were coated onto the surface of PPⅡ / TA-ZIF-8 (PZ) using a cell sonication disruptor:

[0175] PPⅡ / TA-ZIF-8 (PZ) and N3-labeled MSCm were ultrasonically dispersed in deionized water at concentrations of 1 mg / mL and 2 mg / mL, respectively. Equal volumes of the two were mixed, and the ultrasonic disruptor was set to 200 W with a 2-second sonication cycle and a 2-second pause, for a total duration of 4 min. The mixture was centrifuged at 12000 rpm and 4℃ for 30 min to obtain the precipitate. The precipitate was washed twice with deionized water, centrifuged again at 12000 rpm and 4℃ for 30 min, and then freeze-dried to obtain PPⅡ / TA-ZIF-8@M (PZ@M).

[0176] Step 5: Preparation of mitochondrial-targeting biomimetic nanoformulation PZ@MT

[0177] PZ@M (0.1 mg / mL, 10 mL, ultrapure water) was mixed with DBCO-PEG-TPP (molecular weight approximately 2650, 0.1 mmol) and stirred at 1200 rpm for 2 h. The mixture was centrifuged at 12000 rpm and 4 °C for 30 min to obtain a precipitate. The precipitate was washed twice with deionized water and freeze-dried to obtain the mitochondrial-targeting biomimetic nanoformulation PZ@MT.

[0178] Characterization of the mitochondrial-targeting biomimetic nanoformulation PZ@MT in experimental cases

[0179] Experimental Example 1: Observation of PZ@MT morphology using transmission electron microscopy (TEM)

[0180] Take an appropriate amount of PZ@MT in a methanol solution, disperse it evenly by ultrasonication, drop the sample onto a copper grid, dry the copper grid, and record the TEM image. The results are shown in [Figure number missing]. Figure 1 .

[0181] The results showed that PZ@MT retained the dodecahedral structure of TA-ZIF-8, with a clear film covering the surface, and the particle size of PZ@MT was around 200 nm.

[0182] Experimental Example 2: Dynamic Light Scattering Measurement (DLS)

[0183] Take an appropriate amount of PZ@MT and dissolve it evenly in methanol solution using ultrasonication. Then add it to a Malvern cuvette until the liquid level reaches 10-15 mm. Keep the outer surface of the cuvette dry, place it in the instrument's measuring chamber, select the refractive index and viscosity of the dispersion medium, set the measuring temperature to 25℃, and measure the particle size distribution of the sample. Results are shown below. Figure 2 .

[0184] The results of the Malvern particle size analyzer showed that the average particle size of PZ@MT prepared in Example 1 was 222.8 nm, which was consistent with the results of transmission electron microscopy.

[0185] Experimental Example 3: Measurement of Zeta Potential

[0186] Take an appropriate amount of PZ@MT in a methanol solution, disperse it evenly by ultrasonication, and then add it to a zeta potential measuring dish. Keep the outer surface of the dish dry, place it in the instrument's measuring chamber, select the refractive index and viscosity of the dispersion medium, set the measuring temperature to 25℃, and measure the zeta potential of the sample. Results are shown below. Figure 3 .

[0187] The Zeta potential results showed that the potential of PZ@MT was negative, with an average value of -7.54 mV.

[0188] Experimental Example 4: X-ray Diffraction Pattern Analysis (XRD)

[0189] Weigh approximately 20 mg of PZ@MT powder, pass it through a 300-mesh sieve, and place it in the sample holder. Mount the sample onto the sample stage with the sample surface facing upwards, aligning the sample center with the center line of the stage. Set the instrument parameters as follows: Cu-Kα (λ = 1.541 nm), high voltage 40 kV, tube current 40 mA, start angle 5°, end angle 45°, and scan speed 2° / min. The results are as follows. Figure 4 As shown.

[0190] The results showed that the overall peak shape of PZ@MT was similar to that of TA-ZIF-8, and a large broad peak appeared in the range of 15-30°, indicating that PZ@MT has an amorphous structure, which may be related to the encapsulation of mesenchymal stem cell membrane.

[0191] Experimental Example 5: Thermogravimetric Analysis (TGA)

[0192] Weigh an appropriate amount of PZ@MT powder into a thermogravimetric analyzer. The instrument is heated from 30℃ to 600℃ at a rate of 10℃ / min under a nitrogen atmosphere. Record the relationship between the mass of PZ@MT and temperature to obtain the thermogravimetric curve. (See figure) Figure 5 .

[0193] The thermogravimetric curves show that PZ@MT remains relatively stable at 0-240℃ with a weight loss of less than 10%, but exhibits significant weight loss at 260-380℃, with a mass loss exceeding 40%.

[0194] Experimental Example 6: Fourier Transmission Infrared Spectroscopy (FT-IR)

[0195] Take appropriate amounts of ZIF-8, TA-ZIF-8, PZ, and PZ@MT sample powders, disperse them evenly in a sample well under dry conditions, press them into thin sheets using the handle, and heat at 4000-400 cm⁻¹. -1 The Fourier transform infrared spectrum was measured within the specified range, and the characteristic vibrational peaks in the spectrum were observed. Results are shown below. Figure 6 .

[0196] As can be seen from the figure, 760 cm -1 and 420 cm -1 The peaks at these locations are attributed to the in-plane or out-of-plane bending of the imidazole ring and the stretching vibration of Zn-N in ZIF-8, respectively. After TA etching, characteristic C=O and CO peaks were detected in the TA-ZIF-8 spectrum, at 1705 cm⁻¹. -1 and 1065 cm -1 The aforementioned absorption peaks were also detected in the spectra of PZ and PZ@MT. Furthermore, in the 1435-1477 cm⁻¹ range... -1 Within the range, the bending vibration absorption peak of CH attributable to TPP was observed in the spectrum of PZ@MT, indicating the successful modification of TPP.

[0197] Experiment 7: PZ@MT protein retention

[0198] The retention of PZ@MT membrane proteins was detected using SDS-PAGE. First, the protein concentrations of MSC cell lysate, N3-labeled MSCm, PZ@M, and PZ@MT were determined using the BCA method. The protein samples were then mixed with 5× loading buffer and boiled at 100℃ for 5 min to denature the proteins. The protein loading volume was adjusted to 40 μg. After electrophoresis, the gel was incubated overnight with Coomassie Brilliant Blue staining solution. After washing off the staining solution, the protein bands were observed and photographed. The results are as follows: Figure 7 Repeat the above steps, blocking and trimming the membrane after transfer, and incubating it overnight at 4°C with primary antibodies of CD44 (1:1000), CD90 (1:1000), and CD105 (1:1000). Wash three times with TBST, add the corresponding secondary antibody, incubate at room temperature for 1 h, wash four times with TBST, develop in chromogenic reagent for 30 s, and develop using an ECL chemical imaging system. Results are as follows: Figure 8 .

[0199] Figure 7 SDS-PAGE analysis of protein expression in different nanoparticles and whole-cell lysates showed that after Coomassie brilliant blue staining, obvious protein bands appeared in each group, and the protein bands of PZ@M and PZ@MT were consistent with the protein bands of MSCm.

[0200] Figure 8 To visualize the expression of cell membrane markers in different nanoparticle formulations using immunoblotting, the results showed that both PZ@M and PZ@MT retained MSCm-specific proteins CD44, CD90, and CD105. These results indicate that the membrane coating process did not significantly affect the biological characteristics of MSCm.

[0201] Experimental Example 8: X-ray photoelectron spectroscopy (XPS) analysis

[0202] Take an appropriate amount of PZ@MT, grind it thoroughly in a mortar and pestle, compress it into a tablet, and then analyze the elemental composition of PZ@MT using an XPS analyzer. The results are shown below. Figure 9 .

[0203] The results showed that PZ@MT is composed of C, O, N, P and Zn, which indirectly proves the existence of mesenchymal stem cell membrane and TPP.

[0204] Experiment Example 9: Transmission Electron Microscopy-Energy Dispersive X-ray Element Mapping (TEM-EDS)

[0205] An appropriate amount of PZ@MT was dissolved in a methanol solution and ultrasonically dispersed until homogeneous. The sample was then dropped onto a copper grid, the grid was dried, and the TEM-EDS spectrum was recorded. The results are shown below. Figure 10 .

[0206] The results showed that, consistent with the XPS spectra, PZ@MT is composed of C, O, N, P and Zn, with C and O having high content and wide distribution, Zn mainly distributed in the framework structure, and N and P having relatively low content and distributed on the surface.

[0207] Evaluation of the in vitro targeting ability and in vitro pharmacodynamics of the mitochondrial-targeting biomimetic nanoformulation PZ@MT

[0208] Example 1: Cell targeting study

[0209] HepG2 cells were seeded in laser confocal microscopy (CLSM) culture dishes, and Cou-6-labeled nanoparticles were added to each group. After incubation for 4 h, the drug solution was removed, and the cells were washed twice with PBS. Then, a 200 nm Mito-Tracker Red CMXRos fluorescent probe was added and incubated for 30 min. After washing twice with PBS, Hoechst 33342 staining was added for 10 min, followed by two more washes with PBS. The fluorescence intensity was observed and photographed under a laser confocal scanning microscope, and the colocalization coefficient was evaluated using the Pearson coefficient (PCC). Results are shown below. Figure 11 .

[0210] Figure 11 CLSM images of HepG2 cells treated with Cou-6-labeled nanoparticles showed that the fluorescence intensity of Cou-6 in HepG2 cells treated with PZ@MT was significantly higher than that in the PZ@M group and the PZ group, and the PCC of this group was 0.81, which was significantly higher than that of other groups, indicating that TPP modification promoted the accumulation of PZ@MT in the mitochondria of HepG2 cells.

[0211] Example 2: Evaluation of cytotoxicity using the CCK8 assay

[0212] Harvesting vigorously growing HepG2 cells at a concentration of 8 × 10⁸ cells / cell, the cells were subjected to further processing. 3 Cells were seeded at a density of [number] cells / well in 96-well plates. After cell adhesion, PPⅡ, PZ, PZ@M, and PZ@MT were accurately weighed to prepare stock solutions of specific concentrations. Each stock solution was diluted to a predetermined concentration gradient (1, 2.5, 5, and 7.5 μg / mL for PPⅡ) using complete culture medium. After incubation for 24 h, 10 μL of CCK8 solution was added to each well, and the plates were incubated for another 2 h. The absorbance was then measured at 450 nm using a microplate reader to calculate cell viability. Results are shown below. Figure 12 .

[0213] The results showed that the cytotoxic effects of PPⅡ, PZ, PZ@M, and PZ@MT on HepG2 cells were dose-dependent, with PPⅡ having an IC50 value of approximately 5 μg / mL. PZ@MT exhibited the strongest cytotoxic effect at all concentrations, with PZ showing significantly stronger cytotoxicity than PPⅡ, and PZ@M showing even stronger cytotoxicity than PZ. These results indicate that loading PPⅡ with TA-ZIF-8, coating with MSCm, and modifying with TPP can all enhance the cytotoxicity of PPⅡ.

[0214] Example 3: PZ@MT on intracellular Fe in HepG2 cells 2+ Effect of content

[0215] HepG2 cells were harvested at a rate of 4 × 10⁻⁶. 5 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured overnight. The cells were then treated with PBS, TA-ZIF-8, PPⅡ, PZ, PZ@M, and PZ@MT (PPⅡ equivalent concentration: 5 μg / mL) for 24 h, respectively. After washing twice with PBS, 2 mL of 5 μM FeRhoNox-1 fluorescent probe was added to each well, and the cells were incubated for 30 min. After washing twice with PBS, Hoechst 33342 staining was added for 10 min, followed by two more washes with PBS. The fluorescence intensity in the cells was observed and photographed using an inverted fluorescence microscope. The results are shown below. Figure 13 Intracellular Fe was analyzed using flow cytometry. 2+ The intensity was quantitatively tested, and the results are shown below. Figure 14 .

[0216] Depend on Figure 13 It can be seen that, compared with the control group and the blank vector group, the fluorescence intensity of FeRhoNox-1 in cells was significantly enhanced after treatment with different formulations, with the PZ@MT group showing the strongest fluorescence, indicating that the FeRhoNox-1 fluorescence intensity in this group was significantly enhanced. 2+ It has the highest content, and cell ferroptosis is most significant.

[0217] Depend on Figure 14 It can be seen that the quantitative results of flow cytometry are consistent with the qualitative results. After processing with PZ@MT, Fe2+ The proportion of positive cells increased significantly, significantly higher than that of other groups, proving that the mitochondrial-targeted biomimetic nanoparticle formulation PZ@MT of the present invention can effectively induce intracellular ferroptosis.

[0218] Example 4: Effect of PZ@MT on intracellular lipid peroxide (LPO) content in HepG2 cells

[0219] HepG2 cells were harvested at a rate of 4 × 10⁻⁶. 5 Seeds were planted at a density of [number] cells / well in 6-well plates and cultured overnight. The plates were then treated with PBS, TA-ZIF-8, PPⅡ, PZ, PZ@M, and PZ@MT (PPⅡ equivalent concentration: 5 μg / mL) for 24 h, respectively. After washing twice with PBS, 2 μM of BODIPY 581 / 591 C11 fluorescent probe was added, and the plates were incubated at 37°C for 30 min. Fluorescence and quantification results were detected using an inverted fluorescence microscope and flow cytometry, respectively. Results are shown below. Figure 15 (Ⅰ. Control; Ⅱ. TA-ZIF-8; Ⅲ. PPⅡ; Ⅳ.PZ; Ⅴ. PZ@M; Ⅵ. PZ@MT).

[0220] The results showed that, compared with the control group and the blank vector group, the intracellular LPO level was significantly enhanced after treatment with PPⅡ, PZ, PZ@M and PZ@MT, indicating an imbalance of intracellular antioxidant activity. Among them, PZ@MT had the most significant effect and the difference was statistically significant.

[0221] Example 5: Effect of PZ@MT on intracellular reactive oxygen species (ROS) levels in HepG2 cells

[0222] HepG2 cells were harvested at a rate of 4 × 10⁻⁶. 5 Seeds were planted at a density of cells / well in 6-well plates and cultured overnight. The cells were then treated with PBS, TA-ZIF-8, PPⅡ, PZ, PZ@M, and PZ@MT (PPⅡ equivalent concentration: 5 μg / mL) for 24 h, respectively. After washing twice with PBS, 2 mL of 10 μM DCFH-DA fluorescent probe was added to each well, and the cells were incubated for 30 min. After washing twice with PBS, the ROS fluorescence intensity in the cells was observed and photographed using a fluorescence inverted microscope. The results are shown below. Figure 16 Intracellular ROS were quantitatively detected using flow cytometry. Results are shown in [Figure number missing]. Figure 17 .

[0223] Figure 16 The results showed that, compared with the control group and the blank vector group, the green fluorescence intensity in HepG2 cells treated with PZ@MT was significantly enhanced, indicating that the reactive oxygen species level in this group was the highest and the cells showed oxidative stress response.

[0224] Figure 17 The results showed that the quantitative results were consistent with the qualitative results. After treatment with PZ@MT, the proportion of ROS-positive cells increased significantly, which was significantly higher than that of other groups, proving that the mitochondrial-targeted biomimetic nano-formulation PZ@MT of the present invention can effectively promote oxidative stress response.

[0225] Example 6: Effects of mitochondrial-targeted biomimetic nanoparticles on the ultrastructure of mitochondria in HepG2 cells

[0226] HepG2 cells were harvested at a rate of 4 × 10⁻⁶. 5 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured overnight. They were then treated for 24 h with PBS, TA-ZIF-8, PPⅡ, PZ, PZ@M, and PZ@MT (PPⅡ equivalent concentration: 5 μg / mL), respectively. Cells were collected, fixed with 2.5% glutaraldehyde for 12 h, and 1% osmium tetroxide for 2 h. The cells were dehydrated using a series of gradient ethanol solutions, embedded in epoxy resin, sectioned, and double-stained with uranium oxide and lead citrate. Mitochondrial structure was observed and images were acquired using transmission electron microscopy. Results are shown below. Figure 18 (Ⅰ.Control; Ⅱ. TA-ZIF-8; Ⅲ. PPⅡ; Ⅳ. PZ; Ⅴ. PZ@M; Ⅵ. PZ@MT).

[0227] The results showed that, compared with the control group and the blank vector group, PZ@MT caused significant changes in mitochondrial structure, including the transformation of mitochondria from strip-shaped to round, the reduction or disappearance of mitochondrial cristae, and the appearance of more vacuolar structures in the cells. This may be related to the large accumulation of PZ@MT in mitochondria.

[0228] Example 7: Effect of mitochondrial-targeted biomimetic nanoformulation PZ@MT on ferroptosis protein

[0229] HepG2 cells were harvested at a rate of 4 × 10⁻⁶. 5 Cells were seeded at a density of cells / well in 6-well plates and treated with PBS, TA-ZIF-8, PPⅡ, PZ, PZ@M, and PZ@MT (PPⅡ equivalent concentration: 5 μg / mL) for 24 h, respectively. Cells were collected for total protein quantification. Protein samples were boiled for 5 min to denature, followed by SDS-PAGE (10%) gel electrophoresis, transferred to PVDF membranes, and blocked with Blotto at room temperature with shaking for 1.5 h. ACSL4, TFR1, SLC7A11, and GPX4 antibodies (1:750) were added, and the cells were incubated overnight, followed by 4 washes with TBST. Cells were then incubated with the corresponding secondary antibody (1:5000) for 2 h, followed by 4 washes with TBST. The cells were developed in chromogenic reagent for 30 s, and then visualized and photographed using an ECL chemical imaging system. Results are shown below. Figure 19 .

[0230] The results showed that, compared with the control group and the TA-ZIF-8 carrier material group, the expression of ACSL4 and TFR1 proteins in cells was significantly upregulated and the expression of SLC7A11 and GPX4 proteins was significantly downregulated after administration of PPⅡ, PZ, PZ@M and PZ@MT. The effect of PZ@MT was the most obvious, indicating that the mitochondrial-targeted biomimetic nano-formulation PZ@MT of the present invention can induce ferroptosis in HepG2 cells and significantly regulate the expression of related proteins.

[0231] Example 8: Effect of mitochondrial-targeted biomimetic nanoformulation PZ@MT on the cGAS-STING pathway

[0232] HepG2 cells were harvested at a rate of 4 × 10⁻⁶. 5 Cells were seeded at a density of cells / well into 6-well plates and treated with PBS, TA-ZIF-8, PPⅡ, PZ, PZ@M, and PZ@MT (PPⅡ equivalent concentration: 5 μg / mL) for 24 h, respectively. Cells were collected for total protein quantification. Protein samples were boiled for 5 min to denature, then subjected to SDS-PAGE (10%) gel electrophoresis, transferred to PVDF membranes, and blocked with Blotto at room temperature with shaking for 1.5 h. cGAS, p-STING, STING, p-TBK1, TBK1, p-IRF3, IRF3, and IFN-β antibody (1:700) were added, and the cells were incubated overnight, followed by 4 washes with TBST. Cells were then incubated with the corresponding secondary antibody (1:2500) for 2 h, followed by 4 washes with TBST. The cells were developed in chromogenic reagent for 30 s, and then visualized and photographed using an ECL chemical imaging system. Results are shown below. Figure 20 .

[0233] The results showed that, compared with the control group and the TA-ZIF-8 carrier material group, PZ@MT significantly increased the expression of cGAS and IFN-β proteins, and significantly increased the ratios of p-STING / STING, p-TBK1 / TBK1, and p-IRF3 / IRF3, indicating that the phosphorylation levels of STING, TBK1, and IRF3 proteins were significantly increased. This indicates that the mitochondrial-targeted biomimetic nanoformulation PZ@MT of the present invention can effectively activate the cGAS-STING pathway and has an immune-activating effect.

[0234] Example 9: In vivo animal targeting evaluation of the mitochondrial-targeting biomimetic nanoformulation PZ@MT

[0235] Establishment of a liver cancer-bearing mouse model: 100 µL of Hepa1-6 cells (7.5 × 10⁻⁶) were subcutaneously injected into the right axilla of male C57BL / 6 mice (20 ± 2 g). 6 A mouse xenograft tumor model was established using a caliper. Tumor size was monitored using calipers until the tumor reached 50 mm. 3It can be used for experiments when it is around 100 degrees Celsius.

[0236] Cou-6-labeled nanoparticles were injected into tumor-bearing mice via the tail vein. Fluorescence imaging was performed at 0, 6, 12, and 24 h using a BioLight GelView 6000M system. After 24 h, major organs and tumors were harvested for biodistribution studies. Results are shown below. Figure 21 . Figure 21 These are the results of the in vivo targeting study of the biomimetic nano-formulation of the present invention, wherein... Figure 21 A represents in vivo real-time fluorescence imaging; Figure 21 B represents the fluorescence image of isolated tissues and organs; Figure 21 C represents the fluorescence intensity of the tumor tissue; Figure 21 D represents the fluorescence intensity of tissues such as the heart, liver, spleen, lungs, and kidneys.

[0237] The results showed that 6 hours after injection of Cou-6-labeled nanoparticles, significant fluorescence was observed at the tumor sites of mice in all groups. The fluorescence intensity was highest at 12 hours. While the fluorescence decreased slightly at 24 hours, a significant fluorescence signal was still observed, indicating that the nanoparticles could reach the tumor site within 6 hours, accumulate continuously within 12 hours, and be gradually cleared between 12 and 24 hours. Compared with the control group, the fluorescence intensity of tumor tissues in the PZ@M and PZ@MT groups showed significant fluorescence signals at all time points, indicating that PZ@M and PZ@MT achieved high enrichment at the tumor site. Furthermore, the 24-hour fluorescence images also demonstrated that PZ@M and PZ@MT prolonged the in vivo time to some extent, achieving long-term in vivo retention.

[0238] In the fluorescence images of ex vivo tumor tissue, strong fluorescence signals were still observed in the PZ@M and PZ@MT groups, with PZ@MT showing the strongest fluorescence intensity, and the difference was statistically significant. This further demonstrates that the coating of MSCm and the modification of triphenylphosphine enhance the tumor-targeting ability of the nanoparticles and prolong their in vivo retention time. Furthermore, significant fluorescence signals were also observed in the ex vivo heart, liver, spleen, lung, and kidney, but these were significantly weaker than those in the tumor tissue, presumably related to the metabolic and excretory properties of the liver and kidney.

[0239] Example 10: In vivo pharmacodynamic evaluation of mitochondrial-targeted biomimetic nanoformulation PZ@MT

[0240] Establishment of a liver cancer-bearing mouse model: 100 µL of Hepa1-6 cells (7.5 × 10⁻⁶) were subcutaneously injected into the right axilla of male C57BL / 6 mice (20 ± 2 g). 6A mouse xenograft tumor model was established using a caliper. Tumor size was monitored using calipers until the tumor reached 50 mm. 3 It can be used for experiments when it is around 100 degrees Celsius.

[0241] Animal grouping and administration: Tumor volume was approximately 50 mm. 3 Forty-eight tumor-bearing mice were randomly divided into eight groups as follows: (I) Saline group; (II) TA-ZIF-8 group; (III) anti-PD-L1 group; (IV) PPII group; (V) PZ group; (VI) PZ@M group; (VII) PZ@MT group; (VIII) PZ@M-T+anti-PD-L1 group; among which, the anti-PD-L1 drug was Ultra-LEAF. TM Purified anti-mouse CD274 (B7-H1, PD-L1) Antibody. All treatment groups received 5.0 mg / kg (PPII) via tail vein injection, administered every other day for 5 doses. The anti-PD-L1 dose was 5.0 mg / kg via intraperitoneal injection, administered the day after the nano-formulation injection, for a total of 4 doses. After administration, whole blood was collected from mice in each group using ocular sampling. Mice were euthanized, and tumors and major organs (heart, liver, spleen, lungs, and kidneys) were dissected and preserved for later use.

[0242] In vivo tumor inhibition rate and hepatocellular carcinoma histopathological examination: Tumor volume of tumor-bearing mice was recorded daily. Dissected tumor tissue was photographed and weighed, and the tumor inhibition rate of each group was calculated. Liver tumor tissue from each group was fixed in 4% paraformaldehyde and then sequentially embedded in paraffin, sectioned, and... Staining and other procedures were performed, and the pathological structure of each group of tumor tissues was observed and images were acquired using an inverted fluorescence microscope. Results are shown below. Figures 22-23 . Figure 22 The tumor volume, tumor weight, and tumor inhibition rate of tumor-bearing mice were measured. Figure 23 tumor tissue Staining results.

[0243] The results showed that, compared with the control group and the blank carrier group, all treatment groups inhibited tumor growth, manifested as a slower tumor growth rate and significantly smaller tumor weight and volume compared to the control group. The tumor inhibition rate of the PZ@MT group was 76.7%, significantly higher than that of the control, TA-ZIF-8 (12.8%), PPⅡ (30.2%), PZ (43.5%), and PZ@M (58.0%), indicating that the mitochondrial-targeted biomimetic nanoparticle formulation PZ@MT of this invention can significantly inhibit tumor proliferation. Furthermore, the anti-PD-L1 group alone showed a tumor inhibition rate of 66.67%, also exhibiting good tumor inhibition. The combination of PZ@MT and anti-PD-L1 showed the best therapeutic effect, with a tumor inhibition rate of 86.60%, indicating that the combined application of anti-PD-L1 further enhanced the efficacy of tumor immunotherapy. Tumor HE staining results showed that after combined treatment with PZ@MT or PZ@MT combined with anti-PD-L1, tumor cell nuclei ruptured, cytoplasmic staining lightened, and tumor tissue necrosis occurred.

[0244] Example 11: In vivo immune activation effect of mitochondrial-targeted biomimetic nanoparticle formulation PZ@MT

[0245] Mouse tumor tissue from Effect Example 10 was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Immunofluorescence staining with HMGB-1 and CRT antibodies was performed for 12 h. After incubation with fluorescently labeled secondary antibody solution for 1 h in the dark, residual secondary antibody solution was washed away, and DAPI staining solution was added for 15 min. Observation was then performed using a laser confocal microscope. Tumor tissue was collected and ground into a cell suspension using a syringe plunger. A single-cell suspension was then prepared by passing the suspension through a cell filter. PE-anti-CD11c, FITC-anti-CD80, and APC-anti-CD86 staining were added, and the maturation ratio of dendritic cells (DCs) in lymph nodes was analyzed by flow cytometry. FITC-anti-F4 / 80 and PE-anti-CD206 staining were used to detect M2 type (F4 / 80) cells by flow cytometry. + CD206 + Macrophage ratio; FITC-anti-F4 / 80, APC-anti-CD86 staining, and flow cytometry were used to detect M1 type (F4 / 80). + CD86 + Macrophage ratio; PE-anti-CD3 and FITC-anti-CD4 staining were added, and CD4 count was detected by flow cytometry. + T cell segmentation ratio; PE-anti-CD3 and APC-anti-CD8 staining were performed, and CD8 counts were detected by flow cytometry. +The proportion of T cell clusters was determined by incubating the cells with FITC-anti-CD4, PE-Cy7-CD25, and PE-Foxp3 antibodies for 30 min, and the proportion of regulatory T cells (Tregs) was also measured. Liver tumor tissue was placed in homogenization tubes, PBS was added, magnetic beads were added, and the tissue cells were lysed using a homogenizer. After centrifugation, the supernatant was collected, and the levels of IFN-β, IFN-γ, IL-6, and TNF-α were detected using an ELISA kit. Results are shown below. Figures 24-29 .

[0246] Figure 24 The results of CRT and HMGB1 staining of tumor tissue. Figure 25 The results show the proportion of mature dendritic cells in tumor tissue. Figure 26 M2 macrophages (F4 / 80) in tumor tissue + CD206 + ) and M1 macrophages (F4 / 80 + CD86 + (Proportional results) Figure 27 CD4 in tumor tissue + T cells and CD8 + Results of T cell proportions. Figure 28 The results show the proportion of Tregs in tumor tissue. Figure 29 Results of pro-inflammatory factor content in tumor tissue.

[0247] The results showed that, compared with the control group and the blank vector group, the CRT level in each treatment group was significantly increased and the HMGB1 level was significantly decreased, indicating sufficient exposure of CRT and large release of HMGB1, demonstrating the significant activation of immunogenic cell death in mice, which is beneficial for the immunotherapy of liver cancer. Furthermore, PZ@MT significantly increased the proportion of mature dendritic cells, decreased the proportion of M2 macrophages, and increased the proportion of M1 macrophages, indicating that PZ@MT has a macrophage polarization-promoting effect; PZ@MT can also promote CD4+. + T cells and CD8 + The proliferation of T cells effectively increased the ratio of T cells to tumor cells, while the inhibition of immunosuppressive Tregs resulted in a beneficial adjustment of the T cell population ratio, manifesting as an overall state of immune activation and effectively killing tumor cells. The levels of pro-inflammatory factors in the PZ@MT group were significantly increased, further demonstrating the significant activation of the cGAS-STING pathway. These results demonstrate that the immune response in mice was fully activated after PZ@MT administration, and the tumor microenvironment was significantly improved. Furthermore, the combination of anti-PD-L1 further enhanced the immune activation state, which is one of the reasons why this group exhibited the best tumor-suppressive effect.

[0248] Example 12: In vivo safety study of mitochondrial-targeted biomimetic nanoformulation PZ@MT

[0249] In effect example 10, the following observations were also made:

[0250] Mouse weight change: During the administration period, the status and weight of each group of tumor-bearing mice were observed and recorded daily. At the end of the treatment, the weight change curve of the mice was plotted.

[0251] Organ and tissue morphology examination: The main organ tissues (heart, liver, spleen, lung and kidney) of each group were collected, fixed with 4% paraformaldehyde, and then paraffin embedded, sectioned and H&E stained. The morphological changes of the tissue sections of each group were observed and photographed for analysis using an inverted fluorescence microscope.

[0252] Complete blood count, liver and kidney function tests: Blood samples collected from the mouse eyeballs were used for complete blood count, liver and kidney function tests. The levels of white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), and platelets (PLT) in the blood were measured using a blood cell analyzer. Whole blood was centrifuged, and serum was collected for the determination of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels to investigate the effects of each group on mouse liver function. The levels of creatinine (CREA), urea (UREA), and uric acid (UA) in the serum were measured to investigate the effects of each group on mouse kidney function.

[0253] See results Figures 30-33 . Figure 30 The curves showing the changes in body weight of mice in each group are shown. Figure 31 HE staining results of mouse heart, liver, spleen, lung, and kidney tissues. Figure 32 The results of blood analysis for each group of mice are shown. Figure 33 The results of liver and kidney function analysis for each group of mice are shown.

[0254] The results showed that compared with the control group and the blank vector TA-ZIF-8 group, there were no significant changes in the body weight of mice in each group, and no obvious abnormalities were observed in tissue sections of the heart, liver, spleen, lung, and kidney, indicating that the mitochondrial-targeted biomimetic nanoparticle formulation PZ@MT and other drug administration groups had good safety. Blood routine tests on mice revealed no significant differences in WBC, RBC, HGB, and PLT levels among the groups. In liver and kidney function tests, when PPII was administered alone, liver function indicators such as ALP, ALT, and AST were significantly abnormal, and the levels of CREA and UREA were significantly increased, indicating that PPII monomer has certain hepatotoxicity and nephrotoxicity. However, no obvious abnormalities in liver and kidney indicators were observed in the mitochondrial-targeted biomimetic nanoparticle formulation PZ@MT group, indicating that the mitochondrial-targeted biomimetic nanoparticle formulation PZ@MT of the present invention has good safety and can alleviate liver and kidney damage caused by monomer injection.

[0255] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0256] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A mitochondrially targeted, biomimetic nanoformulation, wherein, The bionic nano-preparation comprises drug-loaded particles and a mitochondrion-targeting bionic cell membrane covering the drug-loaded particles; The drug-loaded particles comprise a drug and a metal-organic framework material loading the drug; The outer surface of the mitochondrion-targeting bionic cell membrane is marked with a mitochondrion-targeting group.

2. The bionanofactured formulation according to claim 1, wherein, The mitochondrion-targeting group is marked to the outer surface of the mitochondrion-targeting bionic cell membrane through a bio-orthogonal reaction; Preferably, the bio-orthogonal reaction is performed between a bionic cell membrane comprising a bio-orthogonal group and a donor compound comprising a bio-orthogonal coordination group, the donor compound comprising a mitochondrion-targeting group; More preferably, the bio-orthogonal group comprises an azide group or a tetrazine group, and the bio-orthogonal coordination group comprises an alkyne group or an alkenyl group.

3. The bionanofactured formulation as claimed in claim 1 or 2, wherein, The drug comprises one or more of an anti-tumor drug, an anti-inflammatory drug, an antibacterial drug, an osteoporosis treatment drug, a photosensitizer and a protein hormone drug; Preferably, the anti-tumor drug comprises one or more of an anti-tumor traditional Chinese medicine active ingredient, a chemically synthesized anti-tumor drug, a targeted therapy drug, an immunotherapy drug, an autophagy inhibitor and a small nucleic acid anti-tumor drug; More preferably, the anti-tumor traditional Chinese medicine active ingredient comprises one or more of Chonglou Saponin II, Chonglou Saponin I, Xingbeilv A, Cucurbitacin B, Shikikui, Ginsenoside Rg3 and Curcumin, preferably Chonglou Saponin II.

4. The bionanofarmaceutical formulation according to any one of claims 1 to 3, characterized in that, The metal-organic framework material is ZIF-8, ZIF-67 or IRMOF-8; further, the metal-organic framework material is etched by tannic acid.

5. The bionanofarmaceutical formulation according to any one of claims 2-4, wherein, The bionic cell membrane comprises one or more of a red blood cell membrane, a macrophage cell membrane, a platelet membrane, a white blood cell membrane, a tumor cell membrane, a bone marrow mesenchymal stem cell membrane, an adipose tissue mesenchymal stem cell membrane and an embryonic mesenchymal stem cell membrane, preferably one or more of a bone marrow mesenchymal stem cell membrane, an adipose tissue mesenchymal stem cell membrane and an embryonic mesenchymal stem cell membrane.

6. The bionanofarmaceutical formulation according to any one of claims 1-5, wherein, The mitochondrion-targeting group is one or more of a triphenylphosphine group, a TP04 peptide group, a TAT peptide group, an α-tocopherol succinate group, a dequalinium chloride group and a mitochondrion-targeting coenzyme Q10 derivative group; preferably, the mitochondrion-targeting group is a triphenylphosphine group.

7. The bionanofarmaceutical formulation according to any one of claims 2-6, wherein, A hydrophilic group is further connected between the bio-orthogonal coordination group and the mitochondrion-targeting group; further, the hydrophilic group is a polyethylene glycol group.

8. The bionanofactured formulation according to any one of claims 1-7, wherein, The bionic nano-preparation has a particle size of 100-500 nm, preferably 150-300 nm.

9. A preparation method of the bionic nano-preparation according to any one of claims 1-8, comprising the following steps: (1) mixing a drug with a metal-organic framework material, and stirring to obtain drug-loaded particles; (2) co-incubating a compound containing a bio-orthogonal group with cells or cell fragments containing a bionic cell membrane, and extracting the bionic cell membrane marked with the bio-orthogonal group; (3) mixing the drug-loaded particles obtained in step (1) with the bionic cell membrane obtained in step (2), and ultrasonically treating to obtain drug-loaded particles covered with the bionic cell membrane. (4) mixing the drug-loaded particles coated with the biomimetic cell membrane obtained in step (3) with a donor compound comprising a mitochondria-targeting group, and stirring to obtain the biomimetic nanoscale preparation.

10. The preparation method of claim 9, wherein, In step (1), the metal-organic framework material is tannic acid-etched ZIF-8, ZIF-67 or IRMOF-8, and the preparation method of the metal-organic framework material comprises mixing tannic acid and ZIF-8, ZIF-67 or IRMOF-8, and stirring to obtain the metal-organic framework material.

11. The preparation method of claim 9 or 10, wherein, In step (2), the compound containing a bio-orthogonal group is one or more of tetraacetylated N-azidoacetylmannosamine, tetraacetylated N-azidoacetylglucosamine, tetraacetylated N-azidoacetylgalactosamine and tetrazine; and / or The cell or cell fragment containing a biomimetic cell membrane is one or more of red blood cells, macrophages, platelets, white blood cells, tumor cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells and embryonic mesenchymal stem cells.

12. The preparation method of any one of claims 9-11, wherein, In step (4), the donor compound comprising a mitochondria-targeting group further comprises a bio-orthogonal coordination group; and / or The donor compound comprising a mitochondria-targeting group further comprises a hydrophilic group; Preferably, the donor compound comprising a mitochondria-targeting group is DBCO-PEG-TPP.

13. A pharmaceutical preparation comprising the biomimetic nanoscale preparation of any one of claims 1-8.

14. A pharmaceutical combination comprising the biomimetic nanoscale preparation of any one of claims 1-8 or the pharmaceutical preparation of claim 13 and at least one additional anti-tumor drug; preferably, the additional anti-tumor drug is a PD-L1 inhibitor or a PD-1 inhibitor.

15. Use of the biomimetic nanoscale preparation of any one of claims 1-8 or the pharmaceutical preparation of claim 13 or the pharmaceutical combination of claim 14 in the preparation of a medicament for treating cancer; preferably, the cancer is liver cancer, preferably hepatocellular carcinoma.

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