WO2023197901A1 - 荷载琥珀酸的肿瘤细胞来源微颗粒及其制备方法和应用 - Google Patents

荷载琥珀酸的肿瘤细胞来源微颗粒及其制备方法和应用 Download PDF

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WO2023197901A1
WO2023197901A1 PCT/CN2023/085972 CN2023085972W WO2023197901A1 WO 2023197901 A1 WO2023197901 A1 WO 2023197901A1 CN 2023085972 W CN2023085972 W CN 2023085972W WO 2023197901 A1 WO2023197901 A1 WO 2023197901A1
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cancer
tumor cell
succinic acid
tumor
loaded
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French (fr)
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唐科
黄波
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Huazhong University of Science and Technology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/194—Carboxylic acids, e.g. valproic acid having two or more carboxyl groups, e.g. succinic, maleic or phthalic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5068—Cell membranes or bacterial membranes enclosing drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5176—Compounds of unknown constitution, e.g. material from plants or animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C55/00—Saturated compounds having more than one carboxyl group bound to acyclic carbon atoms
    • C07C55/02—Dicarboxylic acids
    • C07C55/10—Succinic acid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the invention relates to the technical field of biomedicine, to the preparation of organic compound drug-loaded microparticles and their application, and specifically to tumor cell-derived microparticles loaded with succinic acid and their preparation methods and applications.
  • the immune system can maintain body health by identifying and eliminating tumor cells in the tumor microenvironment. In order to survive in the body, tumor cells use different strategies to suppress the body's immune system and thus cannot kill tumor cells normally, allowing tumor cells to survive various stages of the anti-tumor immune response.
  • Tumor immunotherapy is a treatment method that controls and eliminates tumors by restarting and maintaining the tumor-immune cycle and restoring the body's normal anti-tumor immune response. In recent years, tumor immunotherapy has demonstrated strong anti-tumor activity in the treatment of various tumors such as melanoma, non-small cell lung cancer, renal cancer, prostate cancer and other solid tumors.
  • Current tumor immunotherapy products include monoclonal antibody immune checkpoint inhibitors, therapeutic antibodies, cancer vaccines, cell therapy and small molecule inhibitors.
  • tumor cell-derived microparticles can induce tumor immune responses.
  • Microparticles derived from tumor cells are secreted by the tumors themselves and can be recognized and engulfed by tumor cells.
  • Reference 3 discloses integrating functional inorganic nanoparticles with cell-derived microparticles and encapsulating chemotherapy drugs to achieve the effect of treating cancer.
  • the tumor microenvironment is a complex environment in which tumor cells rely on to survive, and is mainly composed of a variety of different extracellular matrices and stromal cells.
  • Tumor-associated macrophages are macrophages infiltrating in tumor tissues and are the most abundant immune cells in the tumor microenvironment. Studies have shown that tumor-associated macrophages can promote the growth and metastasis of tumor cells through multiple pathways, and they promote tumor growth by regulating the metabolism of tumor cells. Studies have found that succinic acid, as a key metabolite of the tricarboxylic acid cycle, can promote the release of inflammatory factors. However, because it is a water-soluble organic compound, it cannot freely enter cells to play its role. According to the report cited in Reference 4, cancer cells can polarize macrophages into tumor-associated macrophages by releasing succinic acid into the tumor microenvironment, thereby promoting tumor growth and metastasis.
  • Cited literature 1 Zhang Huafeng. Research on the mechanism of anti-tumor immune response mediated by microparticles derived from tumor cells [D]. Huazhong University of Science and Technology, 2012.
  • Cited literature 2 Zhang H, Tang K, Zhang Y, Ma R, Ma J, Li Y, Luo S, Liang X, Ji T, Gu Z, Lu J, He W, Cao X, Wan Y, Huang B.Cell -free tumor microparticle vaccines stimulate dendritic cells via cGAS/STING signaling[J].
  • Cancer Immunol Res.2015,3(2):196-205 Zhang H, Tang K, Zhang Y, Ma R, Ma J, Li Y, Luo S, Liang X, Ji T, Gu Z, Lu J, He W, Cao X, Wan Y, Huang B.Cell -free tumor microparticle vaccines stimulate dendritic cells via cGAS/STING signaling[J].
  • Cancer Immunol Res.2015,3(2):196-205 Zhang H, Tang K, Zhang Y, Ma R, Ma J, Li Y, Luo S, Liang X, Ji T, Gu Z, Lu J, He W, Cao X, Wan
  • Cited document 3 CN109771376A
  • Cited literature 4 Wu JY, Huang TW, Hsieh YT, Wang YF, Yen CC, Lee GL, Yeh CC, Peng YJ, Kuo YY, Wen HT, Lin HC, Hsiao CW, Wu KK, Kung HJ, Hsu YJ, Kuo CC.Cancer-Derived Succinate Promotes Macrophage Polarization and Cancer Metastasis via Succinate Receptor[J].Mol Cell.2020 Jan 16;77(2):213-227.e5.
  • the present invention intends to provide tumor cell-derived microparticles loaded with succinic acid, as well as its preparation method and application. It provides a safer and more effective method reference for tumor treatment, while expanding the application scope of tumor cell-derived microparticles and succinic acid.
  • a kind of tumor cell-derived microparticles loaded with succinic acid which includes tumor cell-derived microparticles and succinic acid loaded thereon.
  • a method for preparing succinic acid-loaded tumor cell-derived microparticles which includes the following steps:
  • the cancer includes liver cancer, bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, endometrial cancer, cervical cancer, esophageal cancer, and eye cancer , head and neck cancer, kidney cancer, lung cancer, gallbladder cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, thyroid cancer, leukemia, lymphoma, myeloma.
  • a method for preventing and/or treating cancer comprising administering an effective amount of the succinic acid-loaded tumor cell source according to any one of [1] to [3] to an individual in need thereof.
  • the cancer includes liver cancer, bladder cancer, bone cancer, Brain cancer, breast cancer, colorectal cancer, endometrial cancer, cervical cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, gallbladder cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer , thyroid cancer, leukemia, lymphoma, myeloma.
  • Tumor cell-derived microparticles are endogenous substances in the body and can be better recognized and absorbed by tumor cells. On this basis, they can be used as drug carriers and are safer than antigen activation alone or other immune drugs. High, more abundant sources, easy to obtain, and it can target most tumors and infections, and has good universal applicability.
  • the succinic acid-loaded tumor cell-derived microparticles provided by the present invention are used as drug-loaded microparticles.
  • the preparation method of the tumor cell-derived microparticles provides a higher drug-loading capacity. The contained drugs further expand the type and scope of disease treatment, reduce costs and risks, and make the operation more convenient. More concise.
  • Figure 1A and Figure 1B show the particle size and morphology of tumor cell-derived microparticles respectively.
  • Figure 2 shows the concentration of succinic acid loaded on tumor cell microparticles.
  • Figures 3A, 3B, and 3C respectively show the tumor morphology, tumor volume, and mouse survival rate after treatment in the mouse breast cancer subcutaneous tumor model.
  • Figures 4A and 4B show the number of abdominal tumor nodules after treatment in the mouse colorectal cancer model
  • Figures 4C and 4D respectively show the tumor mass and mouse survival rate after treatment in the mouse colorectal cancer model.
  • Figures 5A, 5B, 5C, and 5D respectively show the tumor morphology, tumor mass, tumor volume, and mouse weight after treatment in the mouse liver cancer subcutaneous tumor model.
  • Figures 6A and 6B show the uptake of tumor cell microparticles by different immune cells.
  • Figure 7 shows the co-localization of macrophages and tumor cell microparticles.
  • the meaning expressed by "can” includes both the meaning of performing certain processing and not performing certain processing.
  • the terms “comprising”, “having”, “includes” or “containing” may mean inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Meanwhile, “comprises,” “having,” “includes” or “containing” can also mean closed terms, excluding additional, unrecited elements or method steps.
  • the numerical range represented by "numeric value A to numerical value B" or "numeric value A - numerical value B” means a range including the endpoint values A and B.
  • tumor cell microparticles In the present invention, “tumor cell microparticles”, “tumor cell-derived microparticles”, “tumor cell microparticles” “Microparticles derived from tumor cells” and “microparticles derived from tumor cells” are both interchangeable and refer to cell vesicles produced by tumor cell apoptosis, which do not contain any drug components.
  • succinic acid refers to succinic acid or a pharmaceutically acceptable salt thereof.
  • mammals include, but are not limited to, domestic animals (such as cattle, sheep, cats, dogs or horses, etc.), primates (such as humans, non-human primates such as monkeys or orangutans, etc.), rabbits, and rodents (e.g. mice, rats or guinea pigs, etc.).
  • domestic animals such as cattle, sheep, cats, dogs or horses, etc.
  • primates such as humans, non-human primates such as monkeys or orangutans, etc.
  • rabbits e.g. mice, rats or guinea pigs, etc.
  • tumor cells may be cells in solid tumors (including cells with the potential or ability to form solid tumors) or cells that are not solid tumors.
  • the term "about” may mean that a value includes the standard deviation of the error of the device or method used to determine the value. Unless otherwise expressly stated, it should be understood that all ranges, quantities, values and percentages used in the present invention are modified by "about”.
  • the invention provides tumor cell-derived microparticles loaded with succinic acid, which include tumor cell-derived microparticles and succinic acid loaded thereon.
  • the tumor cells are solid tumor cells or non-solid tumor cells, and accordingly, the tumor cell-derived microparticles are solid tumor cell-derived microparticles or non-solid tumor cell-derived microparticles.
  • the tumor cells include but are not limited to liver cancer, bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, endometrial cancer, cervical cancer, esophageal cancer, eye cancer , head and neck cancer, kidney cancer, lung cancer, gallbladder cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, leukemia, lymphoma, myeloma, etc.
  • the tumor cells are melanoma cells, lung cancer cells, leukemia cells or liver cancer cells, preferably liver cancer cells.
  • the particle size of the tumor cell-derived microparticles is 50-500 nm, preferably 100-300 nm, and more preferably 120-250 nm.
  • the content ratio of the tumor cell-derived microparticles to the succinic acid is 1 ⁇ 10 9 -1 ⁇ 10 11 pieces: 25-60 ng, preferably 1 ⁇ 10 10 pieces: 25-60 ng, preferably is 1 ⁇ 10 10 Each: 30-60ng, more preferably 1 ⁇ 10 10 : 35-60ng, further preferably 1 ⁇ 10 10 : 40-60ng, even more preferably 1 ⁇ 10 10 : 55ng.
  • the present invention provides a method for preparing the above-mentioned succinic acid-loaded tumor cell-derived microparticles, including an electroconversion method and a co-incubation method.
  • the electroconversion method includes the following steps: inducing tumor cell apoptosis to release tumor cell-derived microparticles, mixing the tumor cell-derived microparticles with succinic acid to obtain mixture I, and subjecting the mixture I to electroporation After transformation and centrifugation, the tumor cell-derived microparticles loaded with succinic acid are obtained.
  • the tumor cells are solid tumor cells or non-solid tumor cells.
  • the tumor cells include, but are not limited to, liver cancer, bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, endometrial cancer, cervical cancer, esophageal cancer, eye cancer, head and neck cancer Tumor cells in breast cancer, kidney cancer, lung cancer, gallbladder cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, leukemia, lymphoma, myeloma, etc.
  • the tumor cells are melanoma cells, lung cancer cells, leukemia cells or liver cancer cells, preferably liver cancer cells.
  • the method for inducing apoptosis of tumor cells is starvation treatment, preferably using physiological saline to starve the tumor cells.
  • the specific conditions are: suspending the tumor cells with physiological saline, and placing them at 37°C for 20-30 hours. It is best to leave it for 24 hours.
  • the microparticles are collected.
  • the specific collection method can be: collecting cell suspension, centrifuging at 500-1000g for 3-7 minutes, preferably at 800g. 5min, collect the supernatant, and centrifuge the supernatant at a gradient of 1000-50000g for 1-1.5h. It can be seen that the precipitate is the microparticles derived from tumor cells; the preferred specific step of gradient centrifugation is to centrifuge the supernatant at 15000rpm for 6 minutes to collect the supernatant.
  • the number of tumor cells inducing apoptosis is 1 ⁇ 10 7 -1 ⁇ 10 8
  • 1 ⁇ 10 10 -1 ⁇ 10 11 can be obtained The tumor cell-derived microparticles.
  • the number of tumor cell-derived microparticles is 1 ⁇ 10 10 -1 ⁇ 10 11 , preferably 1 ⁇ 10 10 ; the concentration of succinic acid in the mixture I is 0.5-2mM, preferably 0.5-1.5mM, more preferably 1mM.
  • the mass of succinic acid in the mixture I can be 100-500 ⁇ g, such as 100-472 ⁇ g, 118-500 ⁇ g, 118-472 ⁇ g, 100-354 ⁇ g, 118- 354 ⁇ g, 118 ⁇ g, 236 ⁇ g, 354 ⁇ g, 472 ⁇ g, etc.
  • the conditions for the above-mentioned electrical conversion are: voltage 150-300V, preferably voltage 270V; capacitance 100-200 ⁇ F, preferably capacitance 150 ⁇ F; pulse time 3-5ms, preferably pulse time 4ms; electric shock 1-6 times, It is preferred to shock 3 times.
  • the centrifugation conditions are: centrifugation at 500-50000g for 1-1.5h, preferably 16000g for 1h, preferably in a low temperature environment (4°C).
  • the co-incubation method includes the following steps: mixing tumor cells with succinic acid to obtain a mixture II, co-incubating the mixture II to induce apoptosis of the tumor cells in the mixture II and release succinic acid-loaded tumors. Cell-derived microparticles are centrifuged to obtain the tumor cell-derived microparticles loaded with succinic acid.
  • the tumor cells are solid tumor cells or non-solid tumor cells.
  • the tumor cells include, but are not limited to, liver cancer, bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, endometrial cancer, cervical cancer, esophageal cancer, eye cancer, head and neck cancer Tumor cells in breast cancer, kidney cancer, lung cancer, gallbladder cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, leukemia, lymphoma, myeloma, etc.
  • the tumor cells are melanoma cells, lung cancer cells, leukemia cells or liver cancer cells, preferably liver cancer cells.
  • the number of tumor cells is 1 ⁇ 10 7 -1 ⁇ 10 8 , preferably 1 ⁇ 10 7 ; the concentration of succinic acid in the mixture II is 0.5-2mM, preferably 0.5-1.5mM, more preferably 1mM.
  • the mass of succinic acid in the mixture II can be 100-500 ⁇ g, such as 100-472 ⁇ g, 118-500 ⁇ g, 118-472 ⁇ g, 100-354 ⁇ g, 118- 354 ⁇ g, 118 ⁇ g, 236 ⁇ g, 354 ⁇ g, 472 ⁇ g, etc.
  • the co-incubation conditions are: placing at 37°C for 20-30 hours, preferably 24 hours.
  • the method for inducing tumor cell apoptosis is the same as described in the above electroconversion method.
  • the microparticles are collected in the same manner as described in the above electroconversion method.
  • the number of tumor cells that induce apoptosis is 1 ⁇ 10 7 -1 ⁇ 10 8
  • 1 ⁇ 10 10 -1 ⁇ 10 11 of the succinic acid-loaded tumor cell-derived microorganisms can be obtained. Particles.
  • the succinic acid-loaded tumor cell-derived microparticles provided by the present invention can be used to prevent and/or treat cancer; preferably, the cancer includes liver cancer, bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, intrauterine cancer, etc. membrane cancer, cervical cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, gallbladder cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, thyroid cancer, leukemia, lymphoma, myeloma; More preferably, the cancer is liver cancer, breast cancer and colorectal cancer.
  • the succinic acid-loaded tumor cell-derived microparticles prepared by the method for preparing succinic acid-loaded tumor cell-derived microparticles provided by the present invention can be used to prevent and/or treat cancer; preferably, the cancer includes liver cancer, bladder cancer, and liver cancer. Cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, endometrial cancer, cervical cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, gallbladder cancer, melanoma, ovarian cancer, pancreatic cancer , prostate cancer, gastric cancer, thyroid cancer, leukemia, lymphoma, myeloma; more preferably, the cancer is liver cancer, breast cancer and colorectal cancer.
  • the present invention provides the use of the above-mentioned succinic acid-loaded tumor cell-derived microparticles in preparing drugs for preventing and/or treating cancer; preferably, the cancer includes liver cancer, bladder cancer, bone cancer, brain cancer, and breast cancer. , colorectal cancer, endometrial cancer, cervical cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, gallbladder cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, Thyroid cancer, leukemia, lymphoma, myeloma; more preferably, the cancer is liver cancer, breast cancer and colorectal cancer.
  • the succinic acid-loaded tumor cell-derived microparticles prepared by the method for preparing succinic acid-loaded tumor cell-derived microparticles provided by the present invention in the preparation of drugs for preventing and/or treating cancer; preferably, the
  • the above-mentioned cancers include liver cancer, bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, endometrial cancer, cervical cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, gallbladder cancer, and melanoma , ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, leukemia, lymphoma, myeloma; more preferably, the cancer is liver cancer, breast cancer and colorectal cancer.
  • the present invention provides a method for preventing and/or treating cancer, which includes administering an effective amount of the succinic acid-loaded tumor cell-derived microparticles to an individual in need thereof; preferably, the cancer includes liver cancer, bladder cancer, and liver cancer.
  • the present invention provides a method for preventing and/or treating cancer, which includes administering to an individual in need thereof an effective amount of a load prepared by the method for preparing succinic acid-loaded tumor cell-derived microparticles provided by the present invention.
  • Tumor cell-derived microparticles of succinic acid preferably, the cancers include liver cancer, bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, endometrial cancer, cervical cancer, esophageal cancer, eye cancer, head and neck cancer breast cancer, kidney cancer, lung cancer, gallbladder cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, leukemia, lymphoma, myeloma; more preferably, the cancer is liver cancer, breast cancer and colon cancer Rectal cancer.
  • Example 1 Preparation and particle size and electron microscope detection of tumor cell-derived microparticles
  • the H22 mouse liver cancer cells in the logarithmic growth phase were collected and counted, and then inoculated into the abdominal cavity of BALB/c mice at 1 ⁇ 10 5 cells/mouse.
  • One week later take the cells from the mouse ascites fluid, count them, resuspend them in physiological saline, spread them into a 15cm cell culture dish at 1 ⁇ 10 8 cells/dish, place them in a 37°C cell culture incubator for 24 hours, collect the cells, centrifuge them at 800g for 5 minutes, and collect the cells.
  • Figure 1A- Figure 1B showing the particle size and morphology of tumor cell-derived microparticles.
  • Figure 1A shows the particle size of tumor cell-derived microparticles detected by a nanoparticle tracker. It can be seen that the particle size distribution of tumor cell-derived microparticles is relatively uniform and concentrated at around 150nm.
  • Figure 1B is a morphological display of microparticles derived from tumor cells observed under a scanning electron microscope. It can be seen that the microparticles derived from tumor cells have a uniform shape and the particle size distribution is consistent with the detection results of the nanoparticle tracker.
  • succinic acid was added to 1 ⁇ 10 10 tumor cell microparticles at different concentrations (0.5-2mM).
  • the preferred succinic acid concentration is 1mM.
  • Example 3 Effect of succinic acid-loaded tumor cell microparticles on the treatment of mouse breast cancer subcutaneous tumor model, mouse colorectal cancer model, and mouse liver cancer subcutaneous tumor model
  • BALB/c mice were inoculated subcutaneously with 3 ⁇ 10 4T1 mouse breast cancer cells to construct a mouse breast cancer subcutaneous tumor model, and BALB/c mice were intraperitoneally injected with 3 ⁇ 10 CT26 mouse colorectal cancer cells to construct a small
  • BALB/c mice were subcutaneously inoculated with 3 ⁇ 10 5 H22 mouse liver cancer cells to construct a mouse liver cancer subcutaneous tumor model.
  • each model mouse was given intraperitoneal injection of microparticles and drug treatment.
  • Each model was divided into four groups (5-6 mice in each group): blank control group Con, microparticles alone group MP (1 ⁇ 10 11 pieces/bird), the succinic acid-only group SUCC (550ng/bird), and the succinic acid microparticle-loaded group SUCC-MP (1 ⁇ 10 11 pieces/bird, corresponding to 550ng succinic acid/bird). Administration was given every two days for two weeks. The subcutaneous tumors of mice were observed and their volumes were measured.
  • Figures 3A-5D show the therapeutic effect of succinic acid-loaded tumor cell microparticles on mouse tumor models.
  • Figures 3A and 3B show that the tumor volume of the mouse breast cancer subcutaneous tumor model became smaller after treatment.
  • the tumors shown in Figure 3A were obtained on the same day after two weeks of treatment.
  • Figure 3C shows that mouse survival was prolonged after treatment.
  • Figures 4A and 4B show that the abdominal tumor nodules of the mouse colorectal cancer model were reduced after two weeks of treatment.
  • Figure 4C shows that the weight of the mouse colorectal cancer tumors was reduced after two weeks of treatment.
  • Figure 4D shows that the survival period of the mice was prolonged after treatment.
  • Figures 5A, 5B, and 5C show that the tumor volume of the mouse liver cancer subcutaneous tumor model became smaller and the tumor weight decreased after treatment.
  • the tumors shown in Figure 5A were obtained on the same day after two weeks of treatment, and Figure 5B shows the statistics after two weeks of treatment.
  • Figure 5D shows that the survival time of mice was prolonged after treatment.
  • the results showed that the succinic acid-loaded tumor microparticles achieved therapeutic effects on mouse breast cancer models, mouse colorectal cancer models, and mouse liver cancer models.
  • Figures 6A-6B show that macrophages have the strongest ability to absorb tumor cell microparticles.
  • Figure 6B shows that macrophages have the highest ability to absorb tumor cell microparticles at 2 hours. The results indicate that macrophages can take up tumor cell microparticles.
  • the mouse liver cancer subcutaneous tumor model was constructed according to Example 3. After the treatment, the mouse subcutaneous tumor was peeled off and frozen sectioned. Circle the tissue to be stained with a histochemical pen and permeate it with 0.5% TritonX-100 (prepared in PBS) for 20 minutes at room temperature; block the sections with PBS containing 10% BSA for 1 hour at room temperature. Add flow cytometry antibody F4/80 to label macrophages and PKH-26 to label tumor cell microparticles (1:100), and incubate at 4°C in the dark for 1 hour. DAPI labeled cell nuclei (1:1000) and washed 3 times with PBS, 5 min/time. The slides were mounted with anti-fluorescence quenching mounting medium and detected by laser confocal microscopy.

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Abstract

涉及生物医药技术领域，具体涉及荷载琥珀酸的肿瘤细胞来源微颗粒及其制备方法和应用。提供了一种荷载琥珀酸的肿瘤细胞来源微颗粒，其包括肿瘤细胞来源微颗粒及其荷载的琥珀酸。实验数据显示，相比肿瘤细胞来源微颗粒或琥珀酸本身，提供的荷载琥珀酸的肿瘤细胞来源微颗粒对多种小鼠皮下肿瘤有明显的治疗作用，提高了小鼠的生存期。以肿瘤细胞来源微颗粒作为药物载体，安全性更高，来源更丰富，方便实际生产操作。此外，提供了荷载琥珀酸的肿瘤细胞来源微颗粒的制备方法，大大提高了微颗粒的载药量。

Description

荷载琥珀酸的肿瘤细胞来源微颗粒及其制备方法和应用
优先权和相关申请
本申请要求于2022年4月11日提交中国专利局、申请号为202210375748.4、发明名称为“荷载琥珀酸的肿瘤细胞来源微颗粒及其制备方法和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物医药技术领域，涉及一种有机化合物载药微颗粒的制备及其应用，具体涉及荷载琥珀酸的肿瘤细胞来源微颗粒及其制备方法和应用。
背景技术
近年来，肿瘤的发病率逐年升高，并且呈现显著的低龄化，肿瘤治疗也越来越成为人们关注的重点。当前，临床肿瘤治疗以放化疗为主，但该方法在杀伤肿瘤细胞的同时对患者自身也造成了不可逆的损伤，随着越来越多化疗药物的使用，肿瘤细胞的耐药性也成为了不可忽视的问题。寻找新的肿瘤治疗手段迫在眉睫。
免疫系统可以通过识别并清除肿瘤微环境中的肿瘤细胞，维持机体健康。肿瘤细胞为了能够在机体内存活，采用不同策略，使人体的免疫系统受到抑制，从而不能正常的杀伤肿瘤细胞，使得肿瘤细胞在抗肿瘤免疫应答的各阶段得以幸存。肿瘤免疫治疗就是通过重新启动并维持肿瘤-免疫循环，恢复机体正常的抗肿瘤免疫反应，从而控制与清除肿瘤的一种治疗方法。近几年，肿瘤免疫治疗在多种肿瘤如黑色素瘤，非小细胞肺癌、肾癌和前列腺癌等实体瘤的治疗中展示出了强大的抗肿瘤活性。目前肿瘤免疫治疗的产品包括单克隆抗体类免疫检查点抑制剂、治疗性抗体、癌症疫苗、细胞治疗和小分子抑制剂等。
根据引用文献1和引用文献2的报道，肿瘤细胞来源的微颗粒可以诱导肿瘤免疫应答。肿瘤细胞来源的微颗粒由肿瘤自身所分泌，其能够被肿瘤细胞识别并吞噬。目前已有部分对于肿瘤细胞来源的微颗粒的研究，例如引用文献3公开将功能性无机纳米颗粒与细胞来源的微颗粒进行整合，并将化疗药物包裹其中，从而达到治疗癌症的效果。
另一方面，肿瘤微环境是肿瘤细胞赖以生存的复杂环境，主要由多种不同的细胞外基质和基质细胞组成。肿瘤相关巨噬细胞是浸润在肿瘤组织中的巨噬细胞，是肿瘤微环境中最多的免疫细胞。研究表明肿瘤相关巨噬细胞可以通过多种途径促进肿瘤细胞的生长及转移，且其通过调控肿瘤细胞的代谢来促进肿瘤的生长。研究发现，琥珀酸作为三羧酸循环的关键代谢产物，其可以促进炎性因子的释放。但因其是水溶性的有机化合物，无法自由进入细胞内发挥作用。根据引用文献4的报道，癌细胞通过向肿瘤微环境中释放琥珀酸，可以将巨噬细胞极化为肿瘤相关巨噬细胞，从而促进肿瘤生长与转移。
引用文献：
引用文献1：张华锋.肿瘤细胞来源的微颗粒介导抗肿瘤免疫应答的机制研究[D].华中科技大学,2012.
引用文献2：Zhang H,Tang K,Zhang Y,Ma R,Ma J,Li Y,Luo S,Liang X,Ji T,Gu Z,Lu J,He W,Cao X,Wan Y,Huang B.Cell-free tumor microparticle vaccines stimulate dendritic cells via cGAS/STING signaling[J].Cancer Immunol Res.2015,3(2):196-205.
引用文献3：CN109771376A
引用文献4：Wu JY,Huang TW,Hsieh YT,Wang YF,Yen CC,Lee GL,Yeh CC,Peng YJ,Kuo YY,Wen HT,Lin HC,Hsiao CW,Wu KK,Kung HJ,Hsu YJ,Kuo CC.Cancer-Derived Succinate Promotes Macrophage Polarization and Cancer Metastasis via Succinate Receptor[J].Mol Cell.2020 Jan 16；77(2):213-227.e5.
发明内容
发明要解决的问题
针对上述现有技术中的问题，本发明拟提供荷载琥珀酸的肿瘤细胞来源微颗粒，并提供其制备方法及应用。为肿瘤治疗手段提供更加安全有效的方法参考，同时扩展肿瘤细胞来源微颗粒及琥珀酸的应用范围。
用于解决问题的方案
[1]、一种荷载琥珀酸的肿瘤细胞来源微颗粒，其包括肿瘤细胞来源微颗粒及其荷载的琥珀酸。
[2]、根据[1]所述的荷载琥珀酸的肿瘤细胞来源微颗粒，其中，所述肿瘤细胞为实体瘤细胞或非实体瘤细胞；优选地，所述肿瘤细胞来源微颗粒的粒径为50-500nm。
[3]、根据[1]或[2]所述的荷载琥珀酸的肿瘤细胞来源微颗粒，其中，所述肿瘤细胞来源微颗粒的数量与所述琥珀酸的含量比为1×109-1×1011个：25-60ng。
[4]、一种荷载琥珀酸的肿瘤细胞来源微颗粒的制备方法，其包括如下步骤：
(i)诱导肿瘤细胞凋亡释放肿瘤细胞来源微颗粒，将所述肿瘤细胞来源微颗粒与琥珀酸混合得到混合物I，将所述混合物I进行电转化，离心后，得到所述荷载琥珀酸的肿瘤细胞来源微颗粒；或，
(ii)将肿瘤细胞与琥珀酸混合得到混合物II，将所述混合物II进行共孵育，诱导混合物II中的肿瘤细胞凋亡释放荷载琥珀酸的肿瘤细胞来源微颗粒，离心后，得到所述荷载琥珀酸的肿瘤细胞来源微颗粒。
[5]、根据[4]所述的方法，其中，所述混合物II中肿瘤细胞的数量为1×107-1×108个；所述混合物I中肿瘤细胞来源微颗粒的数量为1×1010-1×1011个；所述混合物I或混合物II中琥珀酸的浓度为0.5-2mM。
[6]、根据[4]或[5]所述的方法，其中，所述诱导肿瘤细胞凋亡的条件为：利用生理盐水悬浮所述肿瘤细胞，37℃放置20-30h；所述离心的条件为： 500-50000g离心1-1.5h。
[7]、根据[4]或[5]所述的方法，其中，所述电转化的条件为：电压150-300V；电容100-200μF；脉冲时间3-5ms；电击1-6次。
[8]、根据[4]或[5]所述的方法，其中，所述共孵育的条件为：37℃放置20-30h。
[9]、根据[1]至[3]中任一项所述的荷载琥珀酸的肿瘤细胞来源微颗粒和/或根据权利要求[4]至[8]中任一项所述的方法制备的荷载琥珀酸的肿瘤细胞来源微颗粒在制备用于预防和/或治疗癌症的药物中的用途。
[10]、根据[9]所述的用途，其中，所述癌症包括肝癌、膀胱癌、骨癌、脑癌、乳腺癌、结肠直肠癌、子宫内膜癌、宫颈癌、食管癌、眼癌、头颈部癌、肾癌、肺癌、胆囊癌、黑色素瘤、卵巢癌、胰腺癌、前列腺癌、胃癌、甲状腺癌、白血病、淋巴瘤、骨髓瘤。
[11]、一种预防和/或治疗癌症的方法，其包括向对其有需要的个体施用有效量的根据[1]至[3]中任一项所述的荷载琥珀酸的肿瘤细胞来源微颗粒和/或根据权利要求[4]至[8]中任一项所述的方法制备的荷载琥珀酸的肿瘤细胞来源微颗粒；优选地，所述癌症包括肝癌、膀胱癌、骨癌、脑癌、乳腺癌、结肠直肠癌、子宫内膜癌、宫颈癌、食管癌、眼癌、头颈部癌、肾癌、肺癌、胆囊癌、黑色素瘤、卵巢癌、胰腺癌、前列腺癌、胃癌、甲状腺癌、白血病、淋巴瘤、骨髓瘤。
发明的效果
肿瘤细胞来源微颗粒为机体内源性物质，其本身能够更好的被肿瘤细胞识别并吞入，在此基础上将其作为药物载体，与单独抗原激活或其他免疫药物相比，其安全性更高，来源更丰富，易于获取，且其可以针对绝大部分肿瘤和感染，具有良好的普适性。本发明提供的荷载琥珀酸的肿瘤细胞来源微颗粒作为载药微颗粒，其制备方法使载药量更高，所载药物进一步扩大了疾病的治疗类型和范围，降低了成本和风险，使得操作更简洁。实验数据显示，相比肿瘤细胞来源微颗粒或琥珀酸本身，本发明提供的荷载琥珀酸的肿瘤细 胞来源微颗粒对多种小鼠皮下肿瘤有明显的治疗作用，提高了小鼠的生存期。
附图说明
图1A和图1B分别为肿瘤细胞来源微颗粒的粒径和形态。
图2为肿瘤细胞微颗粒荷载琥珀酸浓度。
图3A、3B、3C分别为小鼠乳腺癌皮下瘤模型治疗后肿瘤形态、肿瘤体积、小鼠存活率。
图4A和4B为小鼠结直肠癌模型治疗后腹腔肿瘤结节数目；图4C、4D分别为小鼠结直肠癌模型治疗后肿瘤质量、小鼠存活率。
图5A、5B、5C、5D分别为小鼠肝癌皮下瘤模型治疗后肿瘤形态、肿瘤质量、肿瘤体积、小鼠体重。
图6A和6B为不同免疫细胞摄取肿瘤细胞微颗粒的情况。
图7为巨噬细胞与肿瘤细胞微颗粒共定位的情况。
具体实施方式
以下对本发明的实施方式进行说明，但本发明不限定于此。
在本发明中，使用“可以”表示的含义包括了进行某种处理以及不进行某种处理两方面的含义。
在本发明中，“任选的”或“任选地”是指接下来描述的事件或情况可发生或可不发生，并且该描述包括该事件发生的情况和该事件不发生的情况。
在本发明中，术语“包含”、“具有”、“包括”或“含有”可以指包括在内的或开放式的，并不排除额外的、未引述的元件或方法步骤。与此同时，“包含”、“具有”、“包括”或“含有”也可以表示封闭式的，排除额外的、未引述的元件或方法步骤。
在本发明中，使用“数值A～数值B”或“数值A-数值B”表示的数值范围是指包含端点数值A、B的范围。
在本发明中，“肿瘤细胞微颗粒”、“肿瘤细胞来源微颗粒”、“肿瘤细胞 来源的微颗粒”和“肿瘤细胞源微颗粒”通用，是指肿瘤细胞凋亡产生的细胞囊泡，其没有荷载任何药物成分。
在本发明中，“琥珀酸”是指琥珀酸或其药学上可接受的盐。
在本发明中，“个体”、“对象”、“患者”或“受试者”包括哺乳动物。哺乳动物包括但不限于，家养动物(例如牛、羊、猫、狗或马等)，灵长类动物(例如人、非人灵长类动物如猴或猩猩等)，兔，以及啮齿类动物(例如小鼠、大鼠或豚鼠等)。
在本发明中，“肿瘤细胞”可以是实体瘤中的细胞(包括具有形成实体瘤的潜力或能力的细胞)或非实体瘤的细胞。
在本发明中，术语“约”可以表示：一个值包括测定该值所使用的装置或方法的误差的标准偏差。除非另有明确的说明，应当理解本发明所用的所有范围、数量、数值与百分比均经过“约”的修饰。
<荷载琥珀酸的肿瘤细胞来源微颗粒>
本发明提供了一种荷载琥珀酸的肿瘤细胞来源微颗粒，其包括肿瘤细胞来源微颗粒及其荷载的琥珀酸。
在一些实施方式中，所述肿瘤细胞为实体瘤细胞或非实体瘤细胞，相应的，所述肿瘤细胞来源微颗粒为实体瘤细胞来源的微颗粒或非实体瘤细胞来源的微颗粒。进一步地，在一些优选的实施方式中，所述肿瘤细胞包括但不限于肝癌、膀胱癌、骨癌、脑癌、乳腺癌、结肠直肠癌、子宫内膜癌、宫颈癌、食管癌、眼癌、头颈部癌、肾癌、肺癌、胆囊癌、黑色素瘤、卵巢癌、胰腺癌、前列腺癌、胃癌、甲状腺癌、白血病、淋巴瘤、骨髓瘤等中的肿瘤细胞。在一些具体的实施方式中，所述肿瘤细胞为黑色素瘤细胞、肺癌细胞、白血病细胞或肝癌细胞，优选为肝癌细胞。
在一些实施方式中，所述肿瘤细胞来源微颗粒的粒径为50-500nm，优选为100-300nm，更优选为120-250nm。
在一些实施方式中，所述肿瘤细胞来源微颗粒与所述琥珀酸的含量比为1×109-1×1011个：25-60ng，优选为1×1010个：25-60ng，优选为1×1010 个：30-60ng，更优选为1×1010个：35-60ng，进一步优选为1×1010个：40-60ng，甚至更优选为1×1010个：55ng。
<荷载琥珀酸的肿瘤细胞来源微颗粒的制备方法>
本发明提供了上述荷载琥珀酸的肿瘤细胞来源微颗粒的制备方法，包括电转化法和共孵育法。
(电转化法)
在一些实施方式中，所述电转化法包括如下步骤：诱导肿瘤细胞凋亡释放肿瘤细胞来源微颗粒，将所述肿瘤细胞来源微颗粒与琥珀酸混合得到混合物I，将所述混合物I进行电转化，离心后，得到所述荷载琥珀酸的肿瘤细胞来源微颗粒。
在一些实施方式中，所述肿瘤细胞为实体瘤细胞或非实体瘤细胞。在一些优选的实施方式中，所述肿瘤细胞包括但不限于肝癌、膀胱癌、骨癌、脑癌、乳腺癌、结肠直肠癌、子宫内膜癌、宫颈癌、食管癌、眼癌、头颈部癌、肾癌、肺癌、胆囊癌、黑色素瘤、卵巢癌、胰腺癌、前列腺癌、胃癌、甲状腺癌、白血病、淋巴瘤、骨髓瘤等中的肿瘤细胞。在一些具体的实施方式中，所述肿瘤细胞为黑色素瘤细胞、肺癌细胞、白血病细胞或肝癌细胞，优选为肝癌细胞。
在一些实施方式中，所述诱导肿瘤细胞凋亡的方法为饥饿处理，优选使用生理盐水饥饿处理所述肿瘤细胞，具体条件为：利用生理盐水悬浮所述肿瘤细胞，37℃放置20-30h，优选放置24h。
在一些实施方式中，在诱导肿瘤细胞凋亡释放肿瘤细胞来源微颗粒后，对所述微颗粒进行收集，具体收集方式可以为：收集细胞悬液，500-1000g离心3-7min，优选800g离心5min，收集上清液，对上清液1000-50000g梯度离心1-1.5h，可见沉淀即为肿瘤细胞来源微颗粒；其中梯度离心的优选的具体步骤为将上清15000rpm离心6min取上清，5000rpm离心10-15min取上清，14000g离心5min取上清，4℃，16000g离心1h。在一些实施方式中，诱导凋亡的肿瘤细胞的数量为1×107-1×108个时，即可获得1×1010-1×1011 个所述肿瘤细胞来源微颗粒。
为了提高肿瘤细胞来源微颗粒荷载琥珀酸的效率，在一些实施方式中，在上述混合物I中，所述肿瘤细胞来源微颗粒的数量为1×1010-1×1011个，优选为1×1010个；所述混合物I中琥珀酸的浓度为0.5-2mM，优选为0.5-1.5mM，更优选为1mM。在一些实施方式中，对应上述混合物I中琥珀酸的浓度，所述混合物I中琥珀酸的质量可以为100-500μg，例如100-472μg、118-500μg、118-472μg、100-354μg、118-354μg、118μg、236μg、354μg、472μg等。
在一些实施方式中，上述电转化的条件为：电压150-300V，优选电压为270V；电容100-200μF，优选电容为150μF；脉冲时间3-5ms，优选脉冲时间4ms；电击1-6次，优选电击3次。
在一些实施方式中，所述离心的条件为：500-50000g离心1-1.5h，优选16000g离心1h，优选在低温环境(4℃)中离心。
(共孵育法)
在一些实施方式中，所述共孵育法包括如下步骤：将肿瘤细胞与琥珀酸混合得到混合物II，将所述混合物II进行共孵育，诱导混合物II中的肿瘤细胞凋亡释放荷载琥珀酸的肿瘤细胞来源微颗粒，离心后，得到所述荷载琥珀酸的肿瘤细胞来源微颗粒。
在一些实施方式中，所述肿瘤细胞为实体瘤细胞或非实体瘤细胞。在一些优选的实施方式中，所述肿瘤细胞包括但不限于肝癌、膀胱癌、骨癌、脑癌、乳腺癌、结肠直肠癌、子宫内膜癌、宫颈癌、食管癌、眼癌、头颈部癌、肾癌、肺癌、胆囊癌、黑色素瘤、卵巢癌、胰腺癌、前列腺癌、胃癌、甲状腺癌、白血病、淋巴瘤、骨髓瘤等中的肿瘤细胞。在一些具体的实施方式中，所述肿瘤细胞为黑色素瘤细胞、肺癌细胞、白血病细胞或肝癌细胞，优选为肝癌细胞。
为了提高肿瘤细胞来源微颗粒荷载琥珀酸的效率，在一些实施方式中，在上述混合物II中，所述肿瘤细胞的数量为1×107-1×108个，优选为1× 107个；所述混合物II中琥珀酸的浓度为0.5-2mM，优选为0.5-1.5mM，更优选为1mM。在一些实施方式中，对应上述混合物II中琥珀酸的浓度，所述混合物II中琥珀酸的质量可以为100-500μg，例如100-472μg、118-500μg、118-472μg、100-354μg、118-354μg、118μg、236μg、354μg、472μg等。
在一些实施方式中，所述共孵育的条件为：37℃放置20-30h，优选放置24h。
在一些实施方式中，所述诱导肿瘤细胞凋亡的方法同上述电转化法中所述。在一些实施方式中，在诱导混合物II中的肿瘤细胞凋亡释放微颗粒后，对所述微颗粒的收集方式同上述电转化法中所述。在一些实施方式中，诱导凋亡的肿瘤细胞的数量为1×107-1×108个时，即可获得1×1010-1×1011个所述荷载琥珀酸的肿瘤细胞来源微颗粒。
<医药用途>
本发明提供的荷载琥珀酸的肿瘤细胞来源微颗粒可以用于预防和/或治疗癌症；优选地，所述癌症包括肝癌、膀胱癌、骨癌、脑癌、乳腺癌、结肠直肠癌、子宫内膜癌、宫颈癌、食管癌、眼癌、头颈部癌、肾癌、肺癌、胆囊癌、黑色素瘤、卵巢癌、胰腺癌、前列腺癌、胃癌、甲状腺癌、白血病、淋巴瘤、骨髓瘤；更优选地，所述癌症为肝癌、乳腺癌和结肠直肠癌。
通过本发明提供的荷载琥珀酸的肿瘤细胞来源微颗粒的制备方法所制备得到的荷载琥珀酸的肿瘤细胞来源微颗粒可以用于预防和/或治疗癌症；优选地，所述癌症包括肝癌、膀胱癌、骨癌、脑癌、乳腺癌、结肠直肠癌、子宫内膜癌、宫颈癌、食管癌、眼癌、头颈部癌、肾癌、肺癌、胆囊癌、黑色素瘤、卵巢癌、胰腺癌、前列腺癌、胃癌、甲状腺癌、白血病、淋巴瘤、骨髓瘤；更优选地，所述癌症为肝癌、乳腺癌和结肠直肠癌。
本发明提供了上述荷载琥珀酸的肿瘤细胞来源微颗粒在制备用于预防和/或治疗癌症的药物中的用途；优选地，所述癌症包括肝癌、膀胱癌、骨癌、脑癌、乳腺癌、结肠直肠癌、子宫内膜癌、宫颈癌、食管癌、眼癌、头颈部癌、肾癌、肺癌、胆囊癌、黑色素瘤、卵巢癌、胰腺癌、前列腺癌、胃癌、 甲状腺癌、白血病、淋巴瘤、骨髓瘤；更优选地，所述癌症为肝癌、乳腺癌和结肠直肠癌。
通过本发明提供的荷载琥珀酸的肿瘤细胞来源微颗粒的制备方法所制备得到的荷载琥珀酸的肿瘤细胞来源微颗粒在制备用于预防和/或治疗癌症的药物中的用途；优选地，所述癌症包括肝癌、膀胱癌、骨癌、脑癌、乳腺癌、结肠直肠癌、子宫内膜癌、宫颈癌、食管癌、眼癌、头颈部癌、肾癌、肺癌、胆囊癌、黑色素瘤、卵巢癌、胰腺癌、前列腺癌、胃癌、甲状腺癌、白血病、淋巴瘤、骨髓瘤；更优选地，所述癌症为肝癌、乳腺癌和结肠直肠癌。
本发明提供了一种预防和/或治疗癌症的方法，其包括向对其有需要的个体施用有效量的所述荷载琥珀酸的肿瘤细胞来源微颗粒；优选地，所述癌症包括肝癌、膀胱癌、骨癌、脑癌、乳腺癌、结肠直肠癌、子宫内膜癌、宫颈癌、食管癌、眼癌、头颈部癌、肾癌、肺癌、胆囊癌、黑色素瘤、卵巢癌、胰腺癌、前列腺癌、胃癌、甲状腺癌、白血病、淋巴瘤、骨髓瘤；更优选地，所述癌症为肝癌、乳腺癌和结肠直肠癌。
本发明提供了一种预防和/或治疗癌症的方法，其包括向对其有需要的个体施用有效量的通过本发明提供的荷载琥珀酸的肿瘤细胞来源微颗粒的制备方法所制备得到的荷载琥珀酸的肿瘤细胞来源微颗粒；优选地，所述癌症包括肝癌、膀胱癌、骨癌、脑癌、乳腺癌、结肠直肠癌、子宫内膜癌、宫颈癌、食管癌、眼癌、头颈部癌、肾癌、肺癌、胆囊癌、黑色素瘤、卵巢癌、胰腺癌、前列腺癌、胃癌、甲状腺癌、白血病、淋巴瘤、骨髓瘤；更优选地，所述癌症为肝癌、乳腺癌和结肠直肠癌。
实施例
本发明通过下述实施例进一步阐明，但任何实施例或其组合不应当理解为对本发明的范围或实施方式的限制。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件进行。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。除非另有定 义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。所有试剂、材料或仪器如无特殊说明，均为可以通过市购的常规产品。
实施例1：肿瘤细胞来源微颗粒的制备及粒径、电镜检测
将处于对数生长期的小鼠肝癌细胞H22收集计数，接种于BALB/c小鼠腹腔，1×105个/只。一周后，取小鼠腹水细胞，计数，用生理盐水重悬，以1×108个/皿铺入15cm细胞培养皿中，37℃细胞培养箱放置24h后收集细胞，800g离心5min，收集上清，将上清1000-50000g梯度离心1-1.5h(梯度离心具体步骤为：将上清15000rpm离心6min取上清，5000rpm离心10-15min取上清，14000g离心5min取上清，4℃，16000g离心1h所得沉淀即为细胞微颗粒)，收集肿瘤细胞微颗粒，弃去上清，将微颗粒(可见沉淀)用PBS重悬，4℃保存。1×107个肿瘤细胞可产生约1×1010个肿瘤细胞来源微颗粒。纳米颗粒跟踪分析(Nanoparticle Tracking Analysis，NTA)检测微颗粒粒径，电镜检测微颗粒形态。
结果如图1A-图1B所示，显示了肿瘤细胞来源微颗粒的粒径和形态。图1A是通过纳米颗粒追踪仪检测的肿瘤细胞来源微颗粒的粒径，可见，肿瘤细胞来源微颗粒的粒径分布较均匀，集中在150nm左右。图1B是扫描电镜观察肿瘤细胞来源微颗粒的形态展示，可见，肿瘤细胞来源微颗粒的形态均一，粒径分布与纳米颗粒追踪仪检测结果一致。
实施例2：肿瘤细胞微颗粒荷载琥珀酸浓度
根据实施例1中所得肿瘤细胞微颗粒，将琥珀酸以不同浓度(0.5-2mM)加入1×1010个肿瘤细胞微颗粒中。优选的琥珀酸浓度为1mM。将混合液(约2mL)加入电击杯中，设置电转仪参数(指数波270V，150μF，4ms)进行电转。电转三次，迅速放入超净台，将混合液转移至1.5mL EP管中，16000g，4℃离心1h，弃去上清，将载入琥珀酸的肿瘤细胞微颗粒用PBS重悬，4℃保存待用。
结果如图2所示，与直接加入琥珀酸与肿瘤细胞共孵育相比(共孵育具 体步骤为：将1×107个肿瘤细胞与1mM琥珀酸混合，37℃孵育24h后，收集细胞，800g离心5min，收集上清，将上清1000-50000g梯度离心1-1.5h，梯度离心具体步骤同实施例1，所得沉淀即为荷载琥珀酸的肿瘤细胞微颗粒)，通过电转方式获得的肿瘤细胞微颗粒中琥珀酸浓度更高。可见，电转的方式可以更好的提高肿瘤细胞微颗粒的琥珀酸荷载量。
实施例3：荷载琥珀酸肿瘤细胞微颗粒治疗小鼠乳腺癌皮下瘤模型、小鼠结直肠癌模型以及小鼠肝癌皮下瘤模型效果
在BALB/c小鼠皮下接种3×105个4T1小鼠乳腺癌细胞构建小鼠乳腺癌皮下瘤模型，在BALB/c小鼠腹腔注射3×105个CT26小鼠结直肠癌细胞构建小鼠结直肠癌模型，在BALB/c小鼠皮下接种3×105个H22小鼠肝癌细胞构建小鼠肝癌皮下瘤模型。一周后，开始对各模型小鼠进行腹腔注射微颗粒及药物治疗，分别将各模型分为四组(每组5-6只小鼠)：空白对照组Con，单加微颗粒组MP(1×1011个/只)，单加琥珀酸组SUCC(550ng/只)，荷载琥珀酸微颗粒组SUCC-MP(1×1011个/只，对应琥珀酸550ng/只)。每两天给药一次，连续治疗两周。观察小鼠皮下瘤，并测量体积。
结果如图3A-图5D所示，其显示了荷载琥珀酸肿瘤细胞微颗粒对小鼠肿瘤模型的治疗效果。图3A、3B显示治疗后小鼠乳腺癌皮下瘤模型肿瘤体积变小，其中图3A所展示的肿瘤为治疗两周后于同一天所得。图3C显示治疗后小鼠生存期延长。图4A、4B显示治疗两周后小鼠结直肠癌模型腹腔肿瘤结节减少，图4C显示治疗两周后小鼠结直肠癌肿瘤重量减轻，图4D显示治疗后小鼠生存期延长。图5A、5B、5C显示治疗后小鼠肝癌皮下瘤模型肿瘤体积变小，肿瘤重量减轻，其中图5A所展示的肿瘤为治疗两周后于同一天所得，图5B为治疗两周后的统计结果，图5D显示治疗后小鼠生存期延长。结果说明荷载琥珀酸肿瘤微颗粒对小鼠乳腺癌模型、小鼠结直肠癌模型以及小鼠肝癌模型达到了治疗效果。
实施例4：不同免疫细胞摄取肿瘤细胞微颗粒的情况
取C57/bl小鼠脾脏，充分研磨，裂解红细胞，收集细胞沉淀，将细胞以 1×106个/mL铺于6孔板中，每孔加入1×105个肿瘤细胞微颗粒。置入37℃细胞培养箱培养0.5h，2h。收集细胞，微颗粒以PKH-26染色，流式检测不同免疫细胞摄取微颗粒情况。
结果如图6A-图6B所示，图6A显示了巨噬细胞摄取肿瘤细胞微颗粒能力最强，图6B显示了在2h时巨噬细胞摄取肿瘤细胞微颗粒最多。结果说明巨噬细胞可以摄取肿瘤细胞微颗粒。
实施例5：巨噬细胞与肿瘤细胞微颗粒共定位的情况
根据实施例3所构建小鼠肝癌皮下瘤模型，治疗结束后将小鼠皮下瘤剥离，进行冰冻切片。用组化油笔将待染组织圈好，0.5％TritonX-100(PBS配制)室温通透20min；用含10％BSA的PBS室温封闭切片1小时。加入流式抗体F4/80标记巨噬细胞，PKH-26标记肿瘤细胞微颗粒(1:100)，4℃避光孵育1h。DAPI标记细胞核(1:1000)，PBS洗3次，5min/次。用抗荧光淬灭封片剂封片，激光共聚焦显微镜检测。
结果如图7所示，巨噬细胞与肿瘤细胞微颗粒发生了共定位。结果说明荷载琥珀酸的肿瘤微颗粒可能是因为被巨噬细胞摄取来发挥的抗肿瘤作用。

Claims (10)

  1. 一种荷载琥珀酸的肿瘤细胞来源微颗粒，其包括肿瘤细胞来源微颗粒及其荷载的琥珀酸。
  2. 根据权利要求1所述的荷载琥珀酸的肿瘤细胞来源微颗粒，其特征在于，所述肿瘤细胞为实体瘤细胞或非实体瘤细胞；优选地，所述肿瘤细胞来源微颗粒的粒径为50-500nm。
  3. 根据权利要求1或2所述的荷载琥珀酸的肿瘤细胞来源微颗粒，其特征在于，所述肿瘤细胞来源微颗粒的数量与所述琥珀酸的含量比为1×109-1×1011个：25-60ng。
  4. 一种荷载琥珀酸的肿瘤细胞来源微颗粒的制备方法，其包括如下步骤：
    (i)诱导肿瘤细胞凋亡释放肿瘤细胞来源微颗粒，将所述肿瘤细胞来源微颗粒与琥珀酸混合得到混合物I，将所述混合物I进行电转化，离心后，得到所述荷载琥珀酸的肿瘤细胞来源微颗粒；或，
    (ii)将肿瘤细胞与琥珀酸混合得到混合物II，将所述混合物II进行共孵育，诱导混合物II中的肿瘤细胞凋亡释放荷载琥珀酸的肿瘤细胞来源微颗粒，离心后，得到所述荷载琥珀酸的肿瘤细胞来源微颗粒。
  5. 根据权利要求4所述的方法，其特征在于，所述混合物II中肿瘤细胞的数量为1×107-1×108个；所述混合物I中肿瘤细胞来源微颗粒的数量为1×1010-1×1011个；所述混合物I或混合物II中琥珀酸的浓度为0.5-2mM。
  6. 根据权利要求4或5所述的方法，其特征在于，所述诱导肿瘤细胞凋亡的条件为：利用生理盐水悬浮所述肿瘤细胞，37℃放置20-30h；所述离心的条件为：500-50000g离心1-1.5h。
  7. 根据权利要求4或5所述的方法，其特征在于，所述电转化的条件为：电压150-300V；电容100-200μF；脉冲时间3-5ms；电击1-6次。
  8. 根据权利要求4或5所述的方法，其特征在于，所述共孵育的条件为：37℃放置20-30h。
  9. 根据权利要求1至3中任一项所述的荷载琥珀酸的肿瘤细胞来源微颗 粒和/或根据权利要求4至8中任一项所述的方法制备的荷载琥珀酸的肿瘤细胞来源微颗粒在制备用于预防和/或治疗癌症的药物中的用途。
  10. 根据权利要求9所述的用途，其特征在于，所述癌症包括肝癌、膀胱癌、骨癌、脑癌、乳腺癌、结肠直肠癌、子宫内膜癌、宫颈癌、食管癌、眼癌、头颈部癌、肾癌、肺癌、胆囊癌、黑色素瘤、卵巢癌、胰腺癌、前列腺癌、胃癌、甲状腺癌、白血病、淋巴瘤、骨髓瘤。
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