Load succinic acid tumor cell-derived microparticles, and preparation method and application thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to tumor cell-derived micro-particles loaded with succinic acid and a preparation method and application thereof. The present application provides tumor cell-derived micro-particles loaded with succinic acid, which comprises tumor cell-derived micro-particles and succinic acid loaded thereon. Experimental data show that, compared with tumor cell-derived micro-particles or succinic acid itself, the tumor cell-derived micro-particles loaded with succinic acid provided by the present application have obvious therapeutic effect on various mouse subcutaneous tumors, and improve the survival period of mice. The tumor cell-derived micro-particles are used as drug carriers, which are safer and more abundant in source, and are convenient for actual production operation. In addition, the present application provides a preparation method of tumor cell-derived micro-particles loaded with succinic acid, which greatly improves the drug loading capacity of the micro-particles.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the preparation and application of drug-loaded microparticles of organic compounds, and more specifically to tumor cell-derived microparticles loaded with succinic acid, their preparation methods, and applications. Background Technology

[0002] In recent years, the incidence of cancer has been increasing year by year, and it is showing a significant trend towards affecting younger people, making cancer treatment an increasingly important focus. Currently, clinical cancer treatment mainly relies on radiotherapy and chemotherapy. However, while this method kills tumor cells, it also causes irreversible damage to the patient. With the increasing use of chemotherapy drugs, drug resistance in tumor cells has become a problem that cannot be ignored. Finding new cancer treatments is urgently needed.

[0003] The immune system maintains bodily health by recognizing and eliminating tumor cells in the tumor microenvironment. Tumor cells employ various strategies to survive within the body, suppressing the immune system and preventing it from effectively killing them, thus allowing tumor cells to survive at various stages of the anti-tumor immune response. Tumor immunotherapy aims to control and eliminate tumors by restarting and maintaining the tumor-immune cycle, restoring the body's normal anti-tumor immune response. In recent years, tumor immunotherapy has demonstrated potent anti-tumor activity in the treatment of various tumors, such as melanoma, non-small cell lung cancer, kidney cancer, and prostate cancer. Current tumor immunotherapy products include monoclonal antibody immune checkpoint inhibitors, therapeutic antibodies, cancer vaccines, cell therapy, and small molecule inhibitors.

[0004] According to reports in cited references 1 and 2, tumor cell-derived microparticles can induce tumor immune responses. These microparticles are secreted by the tumor itself and can be recognized and phagocytosed by tumor cells. Some research on tumor cell-derived microparticles has been conducted; for example, cited reference 3 discloses the integration of functional inorganic nanoparticles with cell-derived microparticles, encapsulating chemotherapy drugs within them to achieve therapeutic effects for cancer.

[0005] On the other hand, the tumor microenvironment is a complex environment in which tumor cells depend for survival, mainly composed of various extracellular matrix and stromal cells. Tumor-associated macrophages (TAMs) are macrophages infiltrating tumor tissue and are the most abundant immune cells in the tumor microenvironment. Studies have shown that TAMs can promote tumor cell growth and metastasis through multiple pathways, and they promote tumor growth by regulating tumor cell metabolism. Research has 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 exert its effects. According to the report in reference 4, cancer cells can polarize macrophages into TAMs by releasing succinic acid into the tumor microenvironment, thereby promoting tumor growth and metastasis.

[0006] References:

[0007] Reference 1: Zhang, Huafeng. Study on the mechanism of anti-tumor immune response mediated by tumor cell-derived microparticles [D]. Huazhong University of Science and Technology, 2012.

[0008] 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 ImmunolRes. 2015, 3(2): 196-205.

[0009] Reference 3: CN109771376A

[0010] Cited literature 4: Wu JY, Huang TW, Hsieh YT, Wang YF, Yen CC, Lee GL, Yeh CC, PengYJ, Kuo YY, Wen HT, Lin HC, Hsiao CW, Wu KK, Kung HJ, Hsu YJ, Kuo CC. Cancer-DerivedSuccinate Promotes Macrophage Polarization and Cancer Metastasis via Succinate Receptor[J].Mol Cell.2020Jan 16;77(2):213-227.e5. Summary of the Invention

[0011] The problem the invention aims to solve

[0012] To address the problems in the existing technology, this invention aims to provide tumor cell-derived microparticles loaded with succinic acid, along with their preparation method and applications. This provides a safer and more effective methodological reference for tumor treatment, while expanding the application scope of tumor cell-derived microparticles and succinic acid.

[0013] Solution for solving the problem

[0014] [1] A tumor cell-derived microparticle loaded with succinic acid, comprising the tumor cell-derived microparticle and the succinic acid loaded thereon.

[0015] [2] The tumor cell-derived microparticles loaded with succinic acid according to [1], wherein the tumor cells are solid tumor cells or non-solid tumor cells; preferably, the particle size of the tumor cell-derived microparticles is 50-500 nm.

[0016] [3] Tumor cell-derived microparticles loaded with succinic acid as described in [1] or [2], wherein the ratio of the number of tumor cell-derived microparticles to the content of succinic acid is 1 × 10⁻⁶. 9 -1×10 11 Individual doses: 25-60 ng.

[0017] [4] A method for preparing tumor cell-derived microparticles loaded with succinic acid, comprising the following steps:

[0018] (i) Inducing tumor cell apoptosis to release tumor cell-derived microparticles, mixing the tumor cell-derived microparticles with succinic acid to obtain mixture I, subjecting mixture I to electroporation, and centrifuging to obtain the tumor cell-derived microparticles loaded with succinic acid; or,

[0019] (ii) Mixing tumor cells with succinic acid to obtain mixture II, co-incubating mixture II to induce apoptosis of tumor cells in mixture II and release tumor cell-derived microparticles loaded with succinic acid, and centrifuging to obtain the tumor cell-derived microparticles loaded with succinic acid.

[0020] [5] According to the method described in [4], the number of tumor cells in the mixture II is 1 × 10⁻⁶. 7 -1×10 8 The number of tumor cell-derived microparticles in mixture I is 1 × 10⁻⁶. 10 -1×10 11 The concentration of succinic acid in mixture I or mixture II is 0.5-2 mM.

[0021] [6] According to the method described in [4] or [5], the conditions for inducing tumor cell apoptosis are: suspending the tumor cells in physiological saline and placing them at 37°C for 20-30 h; and the conditions for centrifugation are: centrifuging at 500-50000g for 1-1.5 h.

[0022] [7] The method according to [4] or [5], wherein the conditions for the electrical conversion are: voltage 150-300V; capacitance 100-200μF; pulse time 3-5ms; electric shock 1-6 times.

[0023] [8] The method according to [4] or [5], wherein the co-incubation conditions are: 37°C for 20-30 hours.

[0024] [9] Use of tumor cell-derived microparticles loaded with succinic acid according to any one of [1] to [3] and / or tumor cell-derived microparticles loaded with succinic acid prepared by any one of [4] to [8] in the preparation of medicaments for the prevention and / or treatment of cancer.

[0025]

[10] According to the use described in [9], 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, kidney cancer, lung cancer, gallbladder cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, thyroid cancer, leukemia, lymphoma, and myeloma.

[0026]

[11] A method for preventing and / or treating cancer, comprising administering to an individual in need an effective amount of tumor cell-derived microparticles loaded with succinic acid according to any one of [1] to [3] and / or tumor cell-derived microparticles loaded with succinic acid prepared according to any one of [4] to [8]; preferably, 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, gastric cancer, thyroid cancer, leukemia, lymphoma, and myeloma.

[0027] The effects of the invention

[0028] Tumor cell-derived microparticles are endogenous substances that are more readily recognized and engulfed by tumor cells. Using them as drug carriers offers greater safety, more abundant sources, and easier access compared to antigen activation agents or other immunotherapies alone. Furthermore, they are effective against the vast majority of tumors and infections, demonstrating good versatility. The succinic acid-loaded tumor cell-derived microparticles provided in this invention, as drug-loaded microparticles, achieve higher drug loading through a specific preparation method. This expands the range and types of diseases that can be treated, reduces costs and risks, and simplifies the process. Experimental data show that, compared to tumor cell-derived microparticles or succinic acid itself, the succinic acid-loaded tumor cell-derived microparticles provided in this invention have a significant therapeutic effect on various subcutaneous tumors in mice and improve their survival. Attached Figure Description

[0029] Figure 1A and Figure 1B These represent the particle size and morphology of microparticles derived from tumor cells.

[0030] Figure 2 The concentration of succinic acid loaded onto tumor cell microparticles.

[0031] Figure 3A , 3B 3C represents the tumor morphology, tumor volume, and mouse survival rate after treatment in a mouse subcutaneous breast cancer tumor model, respectively.

[0032] Figure 4A and 4B The number of abdominal tumor nodules after treatment in a mouse colorectal cancer model; Figure 4C , 4D The values ​​represent tumor mass and mouse survival rate after treatment in a mouse colorectal cancer model, respectively.

[0033] Figure 5A , 5B 5C and 5D represent the tumor morphology, tumor mass, tumor volume, and mouse body weight after treatment in a mouse subcutaneous hepatocellular carcinoma model, respectively.

[0034] Figure 6A and 6B The situation of different immune cells taking up tumor cell microparticles.

[0035] Figure 7 This describes the co-localization of macrophages and tumor cell microparticles. Detailed Implementation

[0036] The following describes embodiments of the present invention, but the present invention is not limited thereto.

[0037] In this invention, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0038] In this invention, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both cases in which the event occurs and cases in which the event does not occur.

[0039] In this invention, the terms "comprising," "having," "including," or "containing" can mean included or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising," "having," "including," or "containing" can also mean closed-ended, excluding additional, uncited elements or method steps.

[0040] In this invention, the range of values ​​represented by “value A ~ value B” or “value A - value B” refers to the range that includes the endpoint values ​​A and B.

[0041] In this invention, the terms "tumor cell microparticles", "tumor cell-derived microparticles", "microparticles derived from tumor cells" and "tumor cell-sourced microparticles" are used interchangeably and refer to cell vesicles produced by tumor cell apoptosis that do not carry any drug components.

[0042] In this invention, "succinic acid" refers to succinic acid or its pharmaceutically acceptable salt.

[0043] In this invention, "individual," "object," "patient," or "subject" includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, or horses), primates (e.g., humans, non-human primates such as monkeys or chimpanzees), rabbits, and rodents (e.g., mice, rats, or guinea pigs).

[0044] In this invention, "tumor cells" can be cells in solid tumors (including cells with the potential or ability to form solid tumors) or cells in non-solid tumors.

[0045] In this invention, the term "about" can mean that a value includes the standard deviation of the error of the apparatus or method used to determine that value. Unless otherwise expressly stated, it should be understood that all ranges, quantities, numerical values, and percentages used in this invention are modified by "about".

[0046] <Tumor cell-derived microparticles loaded with succinic acid>

[0047] The present invention provides tumor cell-derived microparticles loaded with succinic acid, comprising tumor cell-derived microparticles and succinic acid loaded thereon.

[0048] In some embodiments, the tumor cells are solid tumor cells or non-solid tumor cells, and correspondingly, the tumor cell-derived microparticles are microparticles derived from solid tumor cells or non-solid tumor cells. Further, in some preferred embodiments, the tumor cells include, but are not limited to, tumor cells from 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. In some specific embodiments, the tumor cells are melanoma cells, lung cancer cells, leukemia cells, or liver cancer cells, preferably liver cancer cells.

[0049] In some embodiments, the particle size of the tumor cell-derived microparticles is 50-500 nm, preferably 100-300 nm, and more preferably 120-250 nm.

[0050] In some embodiments, the ratio of the tumor cell-derived microparticles to the succinic acid is 1 × 10⁻⁶. 9 -1×10 11 Quantity: 25-60 ng, preferably 1×10 10 Quantity: 25-60 ng, preferably 1×10 10 30-60 ng, more preferably 1×10 ng / ml 10 35-60 ng, more preferably 1×10 ng / ml 10 40-60 ng, or even more preferably 1×10 ng / ml 10 55ng.

[0051] <Preparation method of tumor cell-derived microparticles loaded with succinic acid>

[0052] This invention provides a method for preparing the above-mentioned tumor cell-derived microparticles loaded with succinic acid, including an electroconversion method and a co-incubation method.

[0053] (Electroconversion method)

[0054] In some embodiments, 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, electroconverting mixture I, and centrifuging to obtain the tumor cell-derived microparticles loaded with succinic acid.

[0055] In some embodiments, the tumor cells are solid tumor cells or non-solid tumor cells. In some preferred embodiments, the tumor cells include, but are not limited to, tumor cells from 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, etc. In some specific embodiments, the tumor cells are melanoma cells, lung cancer cells, leukemia cells, or liver cancer cells, preferably liver cancer cells.

[0056] In some embodiments, the method for inducing tumor cell apoptosis is starvation treatment, preferably using physiological saline to starve the tumor cells. The specific conditions are: suspending the tumor cells in physiological saline and placing them at 37°C for 20-30 hours, preferably for 24 hours.

[0057] In some embodiments, after inducing tumor cell apoptosis and releasing tumor cell-derived microparticles, the microparticles are collected. Specifically, the collection method may be as follows: collect the cell suspension, centrifuge at 500-1000g for 3-7 min, preferably at 800g for 5 min, collect the supernatant, and then centrifuge the supernatant at a gradient of 1000-50000g for 1-1.5 h. The precipitate is the tumor cell-derived microparticles. A preferred step for the gradient centrifugation is to centrifuge the supernatant at 15000rpm for 6 min, centrifuge at 5000rpm for 10-15 min, centrifuge at 14000g for 5 min, and then centrifuge at 16000g for 1 h at 4°C. In some embodiments, the number of tumor cells induced to undergo apoptosis is 1×10⁻⁶. 7 -1×10 8 At this time, you can get 1×10 10 -1×10 11 The tumor cell-derived microparticles.

[0058] To improve the efficiency of loading succinic acid onto tumor cell-derived microparticles, in some embodiments, the number of tumor cell-derived microparticles in the above mixture I is 1 × 10⁻⁶. 10 -1×10 11 One, preferably 1×10 10The concentration of succinic acid in mixture I is 0.5-2 mM, preferably 0.5-1.5 mM, and more preferably 1 mM. In some embodiments, corresponding to the concentration of succinic acid in mixture I, the mass of succinic acid in mixture I can be 100-500 μg, for example 100-472 μg, 118-500 μg, 118-472 μg, 100-354 μg, 118-354 μg, 118 μg, 236 μg, 354 μg, 472 μg, etc.

[0059] In some embodiments, the conditions for the above-mentioned electroconversion are: voltage 150-300V, preferably 270V; capacitance 100-200μF, preferably 150μF; pulse time 3-5ms, preferably 4ms; and 1-6 electric shocks, preferably 3 electric shocks.

[0060] In some embodiments, the centrifugation conditions are: centrifugation at 500-50000g for 1-1.5h, preferably at 16000g for 1h, and preferably at a low temperature environment (4°C).

[0061] (Co-incubation method)

[0062] In some embodiments, the co-incubation method includes the following steps: mixing tumor cells with succinic acid to obtain mixture II, co-incubating mixture II to induce apoptosis of tumor cells in mixture II to release tumor cell-derived microparticles loaded with succinic acid, and centrifuging to obtain the tumor cell-derived microparticles loaded with succinic acid.

[0063] In some embodiments, the tumor cells are solid tumor cells or non-solid tumor cells. In some preferred embodiments, the tumor cells include, but are not limited to, tumor cells from 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, etc. In some specific embodiments, the tumor cells are melanoma cells, lung cancer cells, leukemia cells, or liver cancer cells, preferably liver cancer cells.

[0064] To improve the efficiency of loading succinic acid onto tumor cell-derived microparticles, in some embodiments, the number of tumor cells in the above mixture II is 1 × 10⁻⁶. 7 -1×10 8 One, preferably 1 × 10 7The concentration of succinic acid in mixture II is 0.5-2 mM, preferably 0.5-1.5 mM, and more preferably 1 mM. In some embodiments, corresponding to the concentration of succinic acid in mixture II, the mass of succinic acid in mixture II can be 100-500 μg, for example 100-472 μg, 118-500 μg, 118-472 μg, 100-354 μg, 118-354 μg, 118 μg, 236 μg, 354 μg, 472 μg, etc.

[0065] In some embodiments, the co-incubation conditions are: 37°C for 20-30 hours, preferably 24 hours.

[0066] In some embodiments, the method for inducing tumor cell apoptosis is the same as that described in the electroporation method above. In some embodiments, after the tumor cells in mixture II release microparticles through apoptosis, the microparticles are collected in the same manner as described in the electroporation method above. In some embodiments, the number of tumor cells induced to undergo apoptosis is 1 × 10⁻⁶. 7 -1×10 8 At this time, you can get 1×10 10 -1×10 11 The tumor cell-derived microparticles loaded with succinic acid.

[0067] <Medical Uses>

[0068] The tumor cell-derived microparticles loaded with succinic acid provided by this invention can be used for the prevention and / or treatment of cancer; 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, kidney cancer, lung cancer, gallbladder cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, thyroid cancer, leukemia, lymphoma, and myeloma; more preferably, the cancers are liver cancer, breast cancer, and colorectal cancer.

[0069] The succinic acid-loaded tumor cell-derived microparticles prepared by the method provided in this invention can be used for the prevention and / or treatment of cancer; 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, kidney cancer, lung cancer, gallbladder cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, thyroid cancer, leukemia, lymphoma, and myeloma; more preferably, the cancers are liver cancer, breast cancer, and colorectal cancer.

[0070] This invention provides the use of the above-mentioned tumor cell-derived microparticles loaded with succinic acid in the preparation of medicaments for the prevention and / or treatment of cancer; 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, kidney cancer, lung cancer, gallbladder cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, leukemia, lymphoma, and myeloma; more preferably, the cancers are liver cancer, breast cancer, and colorectal cancer.

[0071] The use of succinic acid-loaded tumor cell-derived microparticles prepared by the method provided by the present invention in the preparation of medicaments for the prevention and / or treatment of cancer; 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, kidney cancer, lung cancer, gallbladder cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, leukemia, lymphoma, and myeloma; more preferably, the cancers are liver cancer, breast cancer, and colorectal cancer.

[0072] This invention provides a method for preventing and / or treating cancer, comprising administering an effective amount of the tumor cell-derived microparticles loaded with succinic acid to an individual in need; preferably, 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, and myeloma; more preferably, the cancer is liver cancer, breast cancer, and colorectal cancer.

[0073] This invention provides a method for preventing and / or treating cancer, comprising administering to an individual in need an effective amount of tumor cell-derived microparticles loaded with succinic acid prepared by the method provided by this invention; preferably, 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, gastric cancer, thyroid cancer, leukemia, lymphoma, and myeloma; more preferably, the cancer is liver cancer, breast cancer, and colorectal cancer.

[0074] Example

[0075] The present invention is further illustrated by the following embodiments, but any embodiment or combination thereof should not be construed as limiting the scope or implementation of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All reagents, materials, or instruments, unless otherwise specified, are commercially available conventional products.

[0076] Example 1: Preparation and particle size and electron microscopy detection of tumor cell-derived microparticles

[0077] H22 mouse liver cancer cells in the logarithmic growth phase were collected, counted, and inoculated into the peritoneal cavity of BALB / c mice at a dose of 1×10⁻⁶. 5 One week later, ascites cells were collected from mice, counted, resuspended in physiological saline, and analyzed at 1×10⁻⁶ cells / mouse. 8 Cells were cultured in 15cm dishes at 37°C for 24 hours. Cells were then collected and centrifuged at 800g for 5 minutes. The supernatant was collected and then serially centrifuged at 1000-50000g for 1-1.5 hours (specific steps: centrifuge at 15000rpm for 6 minutes, centrifuge at 5000rpm for 10-15 minutes, centrifuge at 14000g for 5 minutes, centrifuge at 16000g for 1 hour at 4°C; the resulting precipitate is the cell microparticles). Tumor cell microparticles were collected, the supernatant was discarded, and the microparticles (visible precipitate) were resuspended in PBS and stored at 4°C. 1×10⁻⁶ cells / dish 7 Each tumor cell can produce approximately 1 × 10⁻⁶ 10 Microparticles derived from tumor cells were analyzed. Particle size was determined using nanoparticle tracking analysis (NTA), and morphology was examined using electron microscopy.

[0078] The results are as follows Figures 1A-1B As shown, the particle size and morphology of microparticles derived from tumor cells are displayed. Figure 1A The particle size of tumor cell-derived microparticles was detected by a nanoparticle tracker. It can be seen that the particle size distribution of tumor cell-derived microparticles is relatively uniform, concentrated at around 150 nm. Figure 1B This is a morphological display of tumor cell-derived microparticles observed by scanning electron microscopy. It can be seen that the tumor cell-derived microparticles have a uniform morphology and the particle size distribution is consistent with the detection results of the nanoparticle tracker.

[0079] Example 2: Concentration of succinic acid loaded onto tumor cell microparticles

[0080] Based on the tumor cell microparticles obtained in Example 1, succinic acid was added at different concentrations (0.5-2 mM) to 1×10⁻⁶ cells / mL. 10 The succinic acid concentration is 1 mM. Approximately 2 mL of the mixture is added to an electroporation vessel, and the electroporation parameters are set (exponential wave 270 V, 150 μF, 4 ms). Electroporation is performed three times. The vessel is then quickly placed in a clean bench, and the mixture is transferred to a 1.5 mL EP tube. The tube is centrifuged at 16000 g, 4°C for 1 h. The supernatant is discarded, and the succinic acid-loaded tumor cell microparticles are resuspended in PBS and stored at 4°C for later use.

[0081] The results are as follows Figure 2 As shown, compared with direct addition of succinic acid and co-incubation with tumor cells (the specific steps of co-incubation are: adding 1×10... 7 Tumor cells were mixed with 1 mM succinic acid and incubated at 37°C for 24 h. The cells were then collected, centrifuged at 800 g for 5 min, and the supernatant was collected. The supernatant was then serially centrifuged at 1000-50000 g for 1-1.5 h (the specific steps of which are the same as in Example 1). The resulting precipitate is the succinic acid-loaded tumor cell microparticle. Tumor cell microparticles obtained by electroporation have a higher concentration of succinic acid. Therefore, electroporation can better improve the succinic acid loading of tumor cell microparticles.

[0082] Example 3: Effects of succinate-loaded tumor cell microparticles on the treatment of mouse subcutaneous breast cancer tumor models, mouse colorectal cancer models, and mouse subcutaneous liver cancer tumor models.

[0083] BALB / c mice were subcutaneously inoculated with 3×10 5 A mouse subcutaneous breast cancer tumor model was constructed using 4T1 mouse breast cancer cells. BALB / c mice were intraperitoneally injected with 3×10⁴ cells. 5 A mouse colorectal cancer model was established using CT26 mouse colorectal cancer cells. BALB / c mice were subcutaneously inoculated with 3 × 10⁶ CT26 mouse colorectal cancer cells. 5 A mouse subcutaneous hepatocellular carcinoma model was constructed using H22 mouse liver cancer cells. One week later, the mice were treated with intraperitoneal injections of microparticles and drugs. Each model was divided into four groups (5-6 mice per group): a blank control group (Con), a microparticle-only group (MP, 1×10⁻⁶), and a microparticle-only group. 11 (550 ng / animal), succinic acid-added group SUCC (550 ng / animal), succinic acid-loaded microparticle group SUCC-MP (1×10 11 Each mouse (550 ng succinic acid per mouse) was administered the medication every two days for two weeks. Subcutaneous tumors were observed and their volume was measured.

[0084] The results are as follows Figures 3A-5D As shown, it demonstrates the therapeutic effect of succinate-loaded tumor cell microparticles on a mouse tumor model. Figure 3A , 3B The study showed that the tumor volume decreased in a mouse subcutaneous breast cancer tumor model after treatment. Figure 3A The tumor shown was obtained on the same day, two weeks after treatment. Figure 3C The treatment showed that the survival time of mice was prolonged. Figure 4A , 4B The study showed a reduction in abdominal tumor nodules in a mouse model of colorectal cancer after two weeks of treatment. Figure 4C The study showed that the weight of colorectal cancer tumors in mice decreased after two weeks of treatment. Figure 4D The treatment showed that the survival time of mice was prolonged. Figure 5A , 5B 5C showed that after treatment, the tumor volume and weight of a mouse subcutaneous liver cancer tumor model decreased, among which... Figure 5A The tumor shown was obtained on the same day, two weeks after treatment. Figure 5B The results are statistical findings two weeks after treatment. Figure 5D The results showed that the survival time of mice was prolonged after treatment. These results indicate that the succinic acid-loaded tumor microparticles achieved a therapeutic effect on mouse models of breast cancer, colorectal cancer, and liver cancer.

[0085] Example 4: The uptake of tumor cell microparticles by different immune cells

[0086] Spleens from C57 / bl mice were thoroughly ground, erythrocytes were lysed, and the cell pellet was collected. The cells were then processed at a concentration of 1×10⁻⁶. 6 Spread 1×10⁻⁶ cells / mL into a 6-well plate, adding 1×10⁻⁶ cells / mL to each well. 5 Tumor cell microparticles were collected and incubated at 37°C for 0.5 h and 2 h. Cells were then collected, and the microparticles were stained with PKH-26. Flow cytometry was used to detect the uptake of microparticles by different immune cells.

[0087] The results are as follows Figures 6A-6B As shown, Figure 6A This showed that macrophages had the strongest ability to take up tumor cell microparticles. Figure 6B The results showed that macrophages took up the most tumor cell microparticles at 2 hours. This demonstrates that macrophages can take up tumor cell microparticles.

[0088] Example 5: Co-localization of macrophage and tumor cell microparticles

[0089] Based on the mouse subcutaneous hepatocellular carcinoma model constructed in Example 3, the subcutaneous tumors were dissected and frozen sectioned after treatment. The tissue to be stained was circled with a histochemical ink pen and permeated with 0.5% Triton X-100 (prepared in PBS) at room temperature for 20 min; the sections were then blocked with PBS containing 10% BSA at room temperature for 1 hour. Macrophages were labeled with flow cytometry antibody F4 / 80, and tumor cell microparticles were labeled with PKH-26 (1:100), and incubated at 4°C in the dark for 1 hour. Cell nuclei were labeled with DAPI (1:1000), and the sections were washed three times with PBS, 5 min each time. The sections were mounted with anti-fluorescence quenching mounting medium and examined using a laser confocal microscope.

[0090] The results are as follows Figure 7 As shown, macrophages and tumor cell microparticles co-localized. These results suggest that the antitumor effect of succinic acid-loaded tumor microparticles may be due to uptake by macrophages.

Claims

1. A tumor cell-derived microparticle loaded with succinic acid, comprising the tumor cell-derived microparticle and the succinic acid loaded thereon; The ratio of the number of tumor cell-derived microparticles to the content of succinic acid is 1 × 10⁻⁶. 9 -1×10 11 Quantity: 25-60ng; The tumor cells were liver cancer cells.

2. The tumor cell-derived microparticles loaded with succinic acid according to claim 1, characterized in that, The particle size of the tumor cell-derived microparticles is 50-500 nm.

3. A method for preparing tumor cell-derived microparticles loaded with succinic acid, comprising the following steps: (i) Inducing tumor cell apoptosis to release tumor cell-derived microparticles, mixing the tumor cell-derived microparticles with succinic acid to obtain mixture I, subjecting mixture I to electroporation, centrifuging, and obtaining the tumor cell-derived microparticles loaded with succinic acid; or, (ii) Mixing tumor cells with succinic acid to obtain mixture II, co-incubating mixture II to induce apoptosis of tumor cells in mixture II and release tumor cell-derived microparticles loaded with succinic acid, and centrifuging to obtain the tumor cell-derived microparticles loaded with succinic acid. The conditions for the electrical conversion are: voltage 150-300V; capacitance 100-200μF; pulse time 3-5ms; electric shock 1-6 times; The co-incubation conditions are: 37℃ for 20-30 hours; The tumor cells were liver cancer cells.

4. The method according to claim 3, characterized in that, The number of tumor cells in mixture II is 1 × 10⁻⁶ 7 -1×10 8 The number of tumor cell-derived microparticles in mixture I is 1 × 10⁻⁶. 10 -1×10 11 The concentration of succinic acid in mixture I or mixture II is 0.5-2 mM.

5. The method according to claim 3 or 4, characterized in that, The conditions for inducing tumor cell apoptosis are as follows: the tumor cells are suspended in physiological saline and placed at 37°C for 20-30 hours; the centrifugation conditions are as follows: centrifugation at 500-50000g for 1-1.5 hours.

6. Use of the tumor cell-derived microparticles loaded with succinic acid according to claim 1 or 2 and / or the tumor cell-derived microparticles loaded with succinic acid prepared by any one of claims 3 to 5 in the preparation of a medicament for the prevention and / or treatment of cancer; wherein the cancer is liver cancer, breast cancer or colorectal cancer.

Images