CN111803519A

CN111803519A - Preparation method, medicament and application of anti-tumor microvesicle carrying chemotherapeutic drugs

Info

Publication number: CN111803519A
Application number: CN202010668225.XA
Authority: CN
Inventors: 金红林
Original assignee: Union Hospital Tongji Medical College Huazhong University of Science and Technology
Current assignee: Tongji Medical College of Huazhong University of Science and Technology; Union Hospital Tongji Medical College Huazhong University of Science and Technology
Priority date: 2020-07-13
Filing date: 2020-07-13
Publication date: 2020-10-23

Abstract

The invention relates to a preparation method, a medicament and application of an anti-tumor microbubble carrying chemotherapeutic drugs, which comprises the following steps: 1) culturing and subculturing primary tumor cells; 2) carrying out X-ray irradiation on the primary tumor cells cultured in the culture dish obtained in the step 1), and continuing culturing; 3) co-incubating the cells obtained in the step 2) with a chemotherapeutic drug, and collecting supernatant to obtain the microvesicles carrying the chemotherapeutic drug. The invention also provides an anti-tumor microbubble carrying chemotherapeutic drugs, which is obtained by purifying and concentrating the anti-tumor microbubble. The anti-tumor microvesicle prepared by the preparation method of the anti-tumor microvesicle carrying chemotherapeutic drugs can be used for preparing anti-tumor drugs, can realize more efficient killing effect on tumors, and expands the application range of radiotherapy and chemotherapy.

Description

Preparation method, medicament and application of anti-tumor microvesicle carrying chemotherapeutic drugs

The technical field is as follows:

the invention relates to the technical field of anti-tumor, in particular to a preparation method, a medicament and application of an anti-tumor microbubble carrying chemotherapeutic drugs.

Background art:

radiation-induced bystander effect (RIBE) refers to the death of irradiated cells and surrounding cells which are not irradiated after the cells are irradiated by radiation. The main principle of RIBE is that microvesicles produced by irradiated cells, which contain high levels of ROS and other DAMP-damaging molecules, can be efficiently taken up by surrounding tumor cells, resulting in the indirect death of the tumor cells. Early studies reveal that microvesicles generated after tumor cell radiotherapy is injected alone can effectively treat and delay the occurrence and development of tumors, and prove that tumor cell microvesicles from autologous sources have important clinical application value.

Chemotherapy is a common clinical means for treating middle and late stage tumors, but recent research reveals that part of tumors can generate multidrug resistance, the generation principle of the multidrug resistance is mainly related to high-expression multidrug resistance related proteins of tumor cells after stimulation, and the in vivo toxic effect of chemotherapeutic drugs on the tumor cells is greatly reduced. More research has focused on preventing the development of multidrug resistance in tumor cells in combination with different tumor treatment modalities, thereby achieving effective killing of in vivo tumors and reducing damage to normal organs. Patent No. ZL201910031298.5 is through using radiotherapy irradiation tumor cell after, the cell will secrete in a large number the microbubble that is rich in ROS, will be rich in cell microbubble of ROS and immunity activator physical mixing and make anti-tumor vaccine, can produce stronger external and lymph node DC activation effect, play the effect of preventing the tumor recurrence. However, the anti-tumor vaccine prepared by the method can only prevent the recurrence of the existing tumor, and has poor effect on preventing the tumor cells from generating multidrug resistance.

The invention content is as follows:

the technical problem to be solved is as follows:

the invention aims to provide a preparation method, a medicament and application of an anti-tumor microbubble carrying chemotherapeutic drugs, the microbubble obtained after radiotherapy of tumor cells carrying chemotherapeutic drugs is used for treating tumors, an effective treatment means of radiotherapy integration can be realized, and the problems of limitation in the prior art and multidrug resistance of the tumor cells after radiotherapy are solved.

The technical scheme is as follows:

in order to solve the technical problems, the invention adopts the following technical scheme: a preparation method of an anti-tumor microbubble carrying chemotherapeutic drugs comprises the following steps:

1) obtaining isolated tumor cells, culturing the primary tumor cells in a culture dish, and carrying out proliferation passage;

2) carrying out X-ray irradiation on the primary tumor cells obtained in the step 1) and placed in a culture dish, and continuing to culture;

3) incubating the cells obtained in the step 2) with chemotherapeutic drugs dissolved in a culture medium for a period of time, collecting supernatant, and performing ultrafiltration centrifugation to obtain microvesicles, namely the anti-tumor microvesicles containing the chemotherapeutic drugs;

4) identifying the anti-tumor microvesicle carrying the chemotherapeutic drug obtained in the step 3) by using an ultraviolet spectrophotometer or a high performance liquid chromatography, wherein the carried chemotherapeutic drug has a specific ultraviolet spectrum absorption characteristic or can observe chemotherapeutic drug components in the high performance liquid chromatography.

Further, the centrifugation in the step 3) comprises a first centrifugation, a second centrifugation and a third centrifugation which are sequentially carried out, wherein the rotation speed of the first centrifugation is 1000g, and the time is 10 minutes; continuously performing the second centrifugation at 14000g for 2min to obtain supernatant; the rotating speed of the third centrifugation is 14000g, the time is 60 minutes, and the obtained precipitate is the anti-tumor microvesicle carrying the chemotherapeutic drug.

Further, the X-ray radiation dose of the step 2) is 2-30Gy, and the X-ray energy is 6 MV; the collection time of the supernatant is 2-7 days after radiotherapy, and the chemotherapeutic drugs comprise alkylating agents, antimetabolites or antibiotics.

Further, the ex vivo tumor cell is a tumor cell produced from an autologous tumor or tumor cell line. The tumor cell lines comprise various human-derived and mouse-derived cell lines, human breast cancer cell lines MCF-1 and HBL-100, human lung cancer cell lines A549, H292, H1299, Calu-1, H460 and H522, human nasopharyngeal cancer cell lines 5-8F, CNE-2, SUNE-1 and HONE1, a mouse melanoma cell line B16 and mouse lung cancer cell line Lewis cells.

Further, the ex vivo tumor cell is from a malignant pleural effusion or a solid tumor.

Further, the alkylating agent is cyclophosphamide, the antimetabolite is methotrexate, 5-fluorouracil or gemcitabine, and the antibiotic is adriamycin or mitomycin.

Furthermore, the anti-tumor microvesicles are of a microvesicle structure carrying chemotherapeutic drugs, the particle size of the microvesicle structure is 100-1000nm, and the dose of the anti-tumor microvesicles carrying the chemotherapeutic drugs can be detected by an ultraviolet spectrophotometer or a high performance liquid chromatography.

An anti-tumor microvesicle medicament carrying a chemotherapeutic drug, obtained from said anti-tumor microvesicle by purification and concentration.

An application of the antineoplastic microvesicle prepared by the preparation method of the antineoplastic microvesicle carrying chemotherapeutic drugs in preparing antineoplastic drugs.

(III) the beneficial effects are as follows:

firstly, for tumors which can not be treated by radiation, the anti-tumor microbubble medicament prepared by the technology can be used for realizing indirect radiotherapy;

secondly, the tumor which is not sensitive to chemotherapy or has multidrug resistance can be prepared into the anti-tumor microvesicle medicament by adopting the technology to realize the high-efficiency killing of the chemotherapy medicament to the tumor;

compared with radiotherapy and chemotherapy, the technology can realize the inhibition effect on the metastatic tumor by repeatedly administering the medicament through intravenous injection for many times;

fourthly, the medicament prepared by carrying the chemotherapeutic drugs by the microvesicles from the self source has better biological safety and biocompatibility;

the preparation process is simple and is convenient for large-scale production.

Description of the drawings:

FIG. 1 is an electron microscope image of the anti-tumor microvesicles carrying chemotherapeutic agents of the invention;

FIG. 2 is a particle size chart of microbubbles carrying chemotherapeutic agents in accordance with the present invention;

FIG. 3 is a statistical graph of the tumor cell killing ability of the microbubbles of the invention carrying doxorubicin with doxorubicin radiation and doxorubicin alone;

FIG. 4 is a graph of an analysis of recruitment of higher numbers of CD3 positive T cells induced by doxorubicin-loaded radiotherapeutic microvesicles of the invention in a mouse malignant pleural effusion model prepared by treating Lewis cells;

FIG. 5 is a mouse survival analysis chart of a mouse malignant pleural effusion model prepared by the methotrexate-carrying radiotherapeutic microvesicle therapy Lewis cells of the invention;

FIG. 6 is a mouse survival analysis chart of a mouse malignant pleural effusion model prepared by treating Lewis cells with cisplatin-carrying radiation microvesicles of the present invention;

FIG. 7 is a mouse survival analysis chart of a mouse malignant pleural effusion model prepared by treating Lewis cells with radiotherapeutic microbubbles carrying doxorubicin according to the present invention;

FIG. 8 is a graph of statistical analysis of mouse body weight after tumor treatment with microbubbles of radiation therapy incorporating doxorubicin in accordance with the present invention;

the specific implementation mode is as follows:

the principles and features of this invention are described below in conjunction with examples, which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

The invention relates to a preparation method, a medicament and application of an anti-tumor microbubble carrying chemotherapeutic drugs, wherein the anti-tumor microbubble is generated by treating tumor cells with radioactive rays; the chemotherapy drugs are common clinical chemotherapy drugs. The anti-tumor microvesicles carrying chemotherapeutic drugs have broad-spectrum anti-tumor activity and can effectively kill tumor cells of autologous sources and non-autologous sources. Can be used for treating tumors insensitive to chemotherapy or tumors incapable of being directly treated by radiotherapy, and can be administered locally or systemically by intravenous injection. The anti-tumor principle mainly relates to the combined tumor killing effect of a large amount of active oxygen carried by the microvesicles and chemotherapeutic drugs, and the regulation and control of the tumor immune microenvironment, such as the promotion of the reprogramming of macrophages. The anti-tumor microvesicle carrying the chemotherapeutic drugs is completely derived from autologous tumor cells, and has good biological safety and biocompatibility.

A preparation method of an anti-tumor microbubble carrying chemotherapeutic drugs comprises the following steps:

The centrifugation in the step 3) comprises a first centrifugation, a second centrifugation and a third centrifugation which are sequentially carried out, wherein the rotation speed of the first centrifugation is 1000g, and the time is 10 minutes; continuously performing the second centrifugation at 14000g for 2min to obtain supernatant; the rotating speed of the third centrifugation is 14000g, the time is 60 minutes, and the obtained precipitate is the anti-tumor microvesicle carrying the chemotherapeutic drug.

The X-ray radiation dose of the step 2) is 2-30Gy, and the X-ray energy is 6 MV; the collection time of the supernatant is 2-7 days after radiotherapy, and the chemotherapeutic drugs comprise alkylating agents, antimetabolites or antibiotics.

The in vitro tumor cell is tumor cell produced by autologous tumor or tumor cell line. The tumor cell lines comprise various human-derived and mouse-derived cell lines, human breast cancer cell lines MCF-1 and HBL-100, human lung cancer cell lines A549, H292, H1299, Calu-1, H460 and H522, human nasopharyngeal cancer cell lines 5-8F, CNE-2, SUNE-1 and HONE1, a mouse melanoma cell line B16 and mouse lung cancer cell line Lewis cells.

The isolated tumor cells are from malignant pleural effusion or solid tumors.

The alkylating agent is cyclophosphamide, the antimetabolite is methotrexate, 5-fluorouracil or gemcitabine, and the antibiotic is adriamycin or mitomycin.

The anti-tumor microvesicle has a microvesicle structure carrying chemotherapeutic drugs, the particle size of the microvesicle structure is 100-1000nm, and the dose of the anti-tumor microvesicle carrying the chemotherapeutic drugs can be detected by an ultraviolet spectrophotometer or a high performance liquid chromatography.

The specific principle of the invention is as follows:

tumor microvesicles after radiation therapy have been confirmed to be the main cause of the production of RIBEs. The tumor cell microvesicle after radiotherapy can highly enrich ROS, and can be efficiently absorbed by adjacent tumor cells, DNA adjacent to the tumor cells is damaged through the ROS, and the tumor cell microvesicle has a good anti-tumor effect. In addition, the radiotherapy microvesicles can improve the tumor microenvironment by reprogramming macrophages and promote anti-tumor immune response. The microvesicles containing a large amount of ROS can be obtained by using a microvesicle technology generated by in vitro radiotherapy, and the microvesicles are proved to be capable of generating high-efficiency killing effect on solid tumors by administration of various ways, thereby realizing the aim of indirect radiotherapy.

Chemotherapy is a common clinical approach used to treat middle and late stage cancers, but the clinical response rate of chemotherapeutic drugs to the middle and late stage cancers is not high due to the appearance of multidrug resistance phenomenon. The combination of various therapeutic measures to break through the drug resistance property of tumor cells is an inevitable trend for the clinical development of chemotherapeutic measures. The microvesicles after radiotherapy have a strong clinical transformation characteristic as a microvesicle structure of an autologous source. By using the microvesicles after radiotherapy as a carrier, the microvesicles generated after carrying the chemotherapeutic drugs can realize the integrated radiotherapy and chemotherapy treatment of tumor cells, break the tolerance characteristics of the tumor cells to the chemotherapeutic drugs, greatly reduce the dosage of the chemotherapeutic drugs and ensure that the normal tissue area around is not damaged as much as possible while the chemotherapy is carried out. Meanwhile, the microbubbles carrying chemotherapeutic drugs after radiotherapy also contribute to the treatment of related cancers which cannot be directly treated by radiotherapy.

After irradiation of tumor cells with radiotherapy and incubation of the chemotherapeutic drug with the post-radiotherapy cells, the cells will secrete microvesicles carrying the chemotherapeutic drug. The obtained microvesicle carrying the chemotherapeutic drug has broad-spectrum anti-tumor capability, especially the tumor resistant to chemotherapy, and the microvesicle carrying the chemotherapeutic drug after the radiotherapy can kill donor tumor cells and other types of tumor cells. In the present invention, the storage conditions under which the antitumor activity of the antitumor microvesicles carrying chemotherapeutic drugs is preserved are 4 ℃ and within 7 days.

The experimental process tests that various chemotherapeutic drugs and tumor cells after radiotherapy are incubated to obtain the microvesicle carrying the chemotherapeutic drugs after radiotherapy, and the microvesicle has stronger killing effect than the microvesicle after simple radiotherapy or the microvesicle after simple chemotherapy drugs. Proves that the microvesicles derived from the tumor cells after radiotherapy and the carried chemotherapeutic drugs have the capability of killing the tumor cells in a synergistic way.

The structure of the anti-tumor microvesicle carrying the chemotherapeutic drug is shown in figure 1, wherein figure 1 is an electron microscope image of the extracted drug-loaded radiotherapy microvesicle which is a spherical vesicle-like structure with the particle size of about 400-500nm after tumor cells are subjected to radiotherapy by 6MV X-ray and then the chemotherapeutic drug adriamycin is added for continuous incubation for 72 hours; FIG. 2 is a graph of the particle size of the microbubbles obtained by analysis with a Malvern particle size analyzer, showing that the distribution of the particle size of the microbubbles is at 100-1000 nm.

The effects of the present invention will be further explained and verified by reference to examples.

In the first embodiment, the protein concentration of microvesicles after radiotherapy is constructed and quantified by BCA, the dose of the carried chemotherapeutic drug is quantified by high performance liquid chromatography, and the preservation method of the microvesicles after radiotherapy carrying the chemotherapeutic drug is also disclosed.

Culturing tumor cells in culture dish of 10mm × 10mm with culture medium of 10% FBS (fetal bovine serum), performing radiotherapy at a dose of 20GY when the cells in the culture dish reach about 5 × 106, changing the culture medium the first day after the radiotherapy, adding 20ml of culture medium containing 10% FBS and a certain concentration of chemotherapeutic drugs, collecting all the culture medium in the culture dish on the 3 rd day, and extracting microvesicles after the radiotherapy by gradient centrifugation. Centrifuging 1000g of cell culture medium after radiotherapy for 10min, taking supernatant, centrifuging 14000g of supernatant for 2min to remove fragments, discarding precipitate, centrifuging 14000g of supernatant at 4 ℃ for 60min, discarding supernatant, wherein the precipitate is microbubbles carrying chemotherapeutic drugs, washing the precipitate twice with physiological saline, resuspending 1ml of PBS (phosphate buffer) solution, storing at 4 ℃, centrifuging 100 mu l of liquid, adding a proper amount of protein lysate, fully lysing on ice for 30min, centrifuging 12000g for 30min, taking supernatant, and adding BCA quantitative liquid for protein quantification. At the same time, 200 μ l of liquid is centrifuged, and then organic solvent such as ethanol or acetone is added to dissolve the chemotherapeutic drug in the microbubbles, and then high performance liquid chromatography is carried out to separate and quantify the dose of the chemotherapeutic drug carried in the microbubbles.

Example two, killing effect of microvesicles against tumor cells after radiotherapy with chemotherapeutic drugs.

100. mu.l of cell suspension was prepared in a 96-well plate at a cell density of 5X 104 cells/ml. The 96-well plates were pre-incubated in an incubator for 24 hours (37 ℃, 5% CO 2). Different amounts of chemotherapy drug-bearing microvesicles (0.04, 0.08, 0.12, 0.16mg/ml) generated by radiotherapy were added to the culture plate, while microvesicles without chemotherapy drug alone were used as a control and chemotherapy drug alone was used as a control group, and the culture plate was incubated in an incubator for 48 hours. To each well was added 10. mu.l of CCK8(Cell counting kit-8) solution, and the plate was incubated in an incubator for 2 hours and the absorbance at 450nm was measured with a microplate reader. The absorbance value reflects the activity of the cell. As shown in fig. 3, with the increase of the number of anti-tumor microvesicles after the radiotherapy of the chemotherapy drugs, the killing of the microvesicles to the tumor cells is gradually enhanced and stronger than the killing ability of the pure chemotherapy drugs. The half-lethality concentration of the microvesicles carrying chemotherapeutic drugs after radiotherapy is obviously lower than that of the pure chemotherapeutic drug group.

Example three, animal experiments with microbubbles carrying chemotherapeutic drugs against malignant pleural effusion after radiation therapy.

Establishing a malignant pleural effusion model: 3X 104 Lewis-LUC mouse lung cancer cells per mouse were inoculated into the thoracic cavity of C57 mouse, the depth of the needle insertion was controlled to 3.3mm, and the injection volume was 50. mu.L. After successful model creation as evidenced by live imaging of small animals on

day

8, 50 μ L of post-radiotherapy microbubbles (5mg/kg) carrying chemotherapeutic drugs and PBS extracted 3 days after 20GY radiotherapy, and the same dose of chemotherapeutic drugs were injected into the thoracic cavity. The injection was continued for 7 days and the survival time of the mice was observed. As shown in FIG. 4, 7 days after the mouse malignant pleural effusion model prepared by using mouse Lewis cells and treated with adriamycin (3 mg/kg) or adriamycin-loaded radiotherapy microgranules (3 mg/kg of adriamycin), pleural lavage fluid was detected by flow-type assay, and CD3 was found therein⁺The proportion of T cells is obviously increased, and a good treatment effect is achieved; FIG. 5 is a statistical chart of mouse survival time for mice using Lewis lung carcinoma to make a pleural effusion model, and free methotrexate (2.4 mg/kg) or an equivalent amount of methotrexate-loaded radiotherapeutic microparticles administered by intrathoracic injection; FIG. 6 is a statistical chart of mouse survival time by using mouse Lewis lung cancer to make a mouse pleural effusion model, and free cisplatin (3.0 mg/kg) or radiotherapy microparticles loaded with the same amount of cisplatin to inject the drug into the thoracic cavity; FIG. 7 is a statistical chart of mouse survival time for mice using Lewis lung carcinoma to make a thoracic water model, and free adriamycin (4.0 mg/kg) or loaded with equal amount of adriamycin for administration by injection into thoracic cavity of radiotherapy microparticles; as shown in FIGS. 5-7, the mice survival time is significantly prolonged and the statistical significance is obtained by using the administration of the microvesicles carrying the chemotherapeutic drugs by injection compared with the group of the chemotherapeutic drugs alone.

Example four, toxicity verification of chemotherapeutic drug-loaded anti-tumor microvesicles.

The malignant pleural effusion model is established, and the weight of the mice in the adriamycin group is remarkably reduced by injecting the radiotherapy micro-particles into the pleural cavity of the mice as shown in figure 8, and the weight of the mice in the adriamycin radiotherapy micro-particle group and the weight of the negative control group are kept stable, which shows that the side effect of the adriamycin can be remarkably reduced after the adriamycin is loaded into the radiotherapy micro-particles.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A preparation method of an anti-tumor microbubble carrying chemotherapeutic drugs is characterized by comprising the following steps:

2. The method according to claim 1, wherein the centrifugation step 3) comprises a first centrifugation, a second centrifugation and a third centrifugation, wherein the first centrifugation is performed at 1000g for 10 min; continuously performing the second centrifugation at 14000g for 2min to obtain supernatant; the rotating speed of the third centrifugation is 14000g, the time is 60 minutes, and the obtained precipitate is the anti-tumor microvesicle carrying the chemotherapeutic drug.

3. The method for preparing chemotherapeutic drug-carrying anti-tumor microvesicles according to claim 1, wherein the X-ray radiation dose of step 2) is 2-30Gy, and the X-ray energy is 6 MV; the collection time of the supernatant is 2-7 days after radiotherapy, and the chemotherapeutic drugs comprise alkylating agents, antimetabolites or antibiotics.

4. The method of claim 1, wherein the ex vivo tumor cells are tumor cells derived from an autologous tumor or tumor cell line.

5. The method of claim 1, wherein the ex vivo tumor cells are from malignant pleural effusion or solid tumor.

6. The method of claim 3, wherein the alkylating agent is cyclophosphamide, the antimetabolite is methotrexate, 5-fluorouracil or gemcitabine, and the antibiotic is doxorubicin or mitomycin.

7. The method for preparing chemotherapeutic drug-carrying anti-tumor microvesicles according to any of claims 1 to 6, wherein the anti-tumor microvesicles have a microvesicle structure carrying chemotherapeutic drugs, the particle size of the microvesicle structure is 100-1000nm, and the dose of the anti-tumor microvesicles carrying chemotherapeutic drugs can be detected by UV spectrophotometer or high performance liquid chromatography.

8. An antitumor microbubble agent carrying a chemotherapeutic drug, which is obtained by purifying and concentrating the antitumor microbubble carrying a chemotherapeutic drug according to any one of claims 1 to 6.

9. Use of the anti-tumor microvesicles obtained by the method of any one of claims 1 to 6 for the preparation of an anti-tumor drug.