WO2021184449A1

WO2021184449A1 - Preparation method for and application of genetically engineered antitumor microparticle

Info

Publication number: WO2021184449A1
Application number: PCT/CN2020/083531
Authority: WO
Inventors: 郑俊武; 金红林; 万超; 黄浩; 郑成武; 卫路; 洪磊; 金秀妍; 刘群
Original assignee: 武汉圣润生物科技有限公司
Priority date: 2020-03-19
Filing date: 2020-04-07
Publication date: 2021-09-23
Also published as: CN111420031A

Abstract

The present invention provides a preparation method for a genetically engineered antitumor microparticle. The method comprises the following steps: S1, constructing recombinant plasmids of an over-expressed GM-CSF and a trans-membrane over-expressed GM-CSF; S2, obtaining ex-vivo tumor cells to cultivate primary tumor cells, and obtaining the primary tumor cells; S3, packaging lentiviral particles of the over-expressed GM-CSF and the trans-membrane over-expressed GM-CSF; S4, infecting the primary tumor cells, obtained in step S2, with the lentiviral particles to obtain primary tumor cells of the over-expressed GM-CSF and the trans-membrane over-expressed GM-CSF; S5, irradiating the primary tumor cells obtained in step S4 and a culture solution using an X-ray, and collecting a supernatant liquid after radiotherapy to obtain a mixture of a required microparticle and apoptotic tumor cell fragments; and S6, performing centrifugation on the mixture obtained in step S5. The present invention can solve the technical problems of poor effect, many side effects, proneness to drug resistance, and inhibition of organism immunity in conventional local chemotherapy.

Description

Preparation method and application of genetically engineered anti-tumor microparticles

Technical field

The invention relates to the field of anti-tumor technology, in particular to a preparation method and application of genetically engineered anti-tumor microparticles.

Background technique

Radiotherapy is an effective treatment method for routine clinical treatment of tumors. Tumor radiotherapy is a local treatment method that uses ionizing radiation generated by radiation to treat tumors. About 70% of cancer patients need radiotherapy during treatment, and about 40% of cancers can be cured with radiotherapy. The role and status of radiotherapy in tumor treatment have become increasingly prominent, and it has become one of the main methods for the treatment of malignant tumors, especially in the treatment of head and neck tumors, breast cancer, lung cancer and cervical cancer. However, some tumors do not receive radiotherapy routinely in clinical practice, such as gastric cancer, colon cancer, bladder cancer, kidney cancer, and malignant pleural effusion (MPE). Although the use of intratumoral irradiation methods such as seed implantation can expand the indications of radiotherapy, it requires higher surgical skills and has surgical risks. In addition, this method cannot be used to treat MPE.

Genetic engineering (genetic engineering), also known as gene splicing technology and DNA recombination technology, is based on molecular genetics and modern methods of molecular biology and microbiology as a means to combine genes from different sources according to pre-designed blueprints. Hybrid DNA molecules are constructed in vitro and then introduced into host cells to change the original genetic characteristics of organisms and obtain new features. The main tools of this technology include enzymes (restriction endonucleases and DNA ligases) and vectors (mainly plasmid vectors, phage vectors, Ti plasmids and artificial chromosomes, etc.). The main steps are to extract the target gene, target gene and carrier Combining and introducing the target gene into the recipient cell and the detection and expression of the target gene. At present, genetic engineering technology has a wide range of applications in various fields, especially in the field of medicine and health. Genetically engineered drugs (such as genetically engineered insulin, genetically engineered interferon, etc.) refers to the introduction of genes for biosynthesis of corresponding drug components into microbial cells, allowing them to produce corresponding drugs and then extracting drugs, which can greatly reduce production while solving the problem of yield cost. Gene therapy is the introduction of normal genes into the patient's body, so that the expression products of the gene can function, so as to achieve the purpose of treating diseases. This is the most effective means of treating genetic diseases. However, gene therapy technology is not yet mature, and some key issues have not been resolved, such as how to select effective therapeutic genes.

Summary of the invention

In order to solve the above problems, the present invention provides a preparation method, medicament and application of genetically engineered anti-tumor microparticles. The anti-tumor microparticles undergo indirect radiotherapy and chemoattract immune cells into the tumor microenvironment to achieve anti-tumor resistance. , To solve the technical problems of poor effect of conventional local chemotherapy, large side effects, easy to produce drug resistance, and suppression of the body's immunity.

The present invention is realized by the following technical means:

A method for preparing genetically engineered anti-tumor microparticles includes the following steps:

S1 constructs plasmids overexpressing GM-CSF and transmembrane overexpressing GM-CSF;

S2 Obtain isolated tumor cells for primary tumor cell culture, and obtain primary tumor cells;

S3 packages lentiviral particles overexpressing GM-CSF and transmembrane overexpressing GM-CSF;

S4 Infect the primary tumor cells obtained in step S2 with lentiviral particles to obtain primary tumor cells overexpressing GM-CSF and transmembrane overexpressing GM-CSF;

S5 subject the primary tumor cells and culture medium obtained in step S4 to X-ray irradiation, and collect the supernatant after radiotherapy to obtain a mixture of required microparticles and apoptotic tumor cell debris;

S6 Centrifuge the mixture obtained in step S5 to obtain the genetically engineered anti-tumor microparticles.

The centrifugation in step S6 includes the first centrifugation, the second centrifugation, and the third centrifugation in sequence. The first centrifugation has a speed of 1000 g and a time of 10 minutes to obtain the supernatant; the second centrifugation has a speed of 14000 g and the time is The supernatant is obtained in 2 minutes; the rotation speed of the third centrifugation is 14000g and the time is 60 minutes, and the precipitate is the fine particles.

The X-ray radiation dose in step S5 is 5-20 Gy, the X-ray energy is 6 MV, and the supernatant collection time is 2-7 days after radiotherapy.

The tumor cell line is a mouse lung cancer cell line Lewis cell, and the tumor cell in vitro is derived from a solid tumor.

An anti-tumor microparticle prepared by a method for preparing genetically engineered anti-tumor microparticles. The genetically engineered anti-tumor microparticles have a microvesicle structure that carries GM-CSF. The particle size of the structure is 100-1000nm.

A genetically engineered anti-tumor microparticle medicament is obtained from the genetically engineered anti-tumor microparticle through purification and concentration.

An application of genetically engineered anti-tumor microparticles prepared by a method for preparing genetically engineered anti-tumor microparticles in the preparation of anti-tumor drugs.

Beneficial effect

The invention provides a preparation method and application of genetically engineered anti-tumor microparticles. Has the following beneficial effects:

①For tumors that cannot be treated with radiotherapy, this technology can be used to prepare drugs to achieve indirect radiotherapy;

②Compared with radiotherapy, this technology can be administered repeatedly through intravenous injection of drugs, which can achieve an inhibitory effect on metastases;

③It can effectively chemoattract immune cells to the tumor microenvironment and promote the effect of anti-tumor immunity;

④The medicament made from microparticles of self-source has good biological safety and biocompatibility;

⑤The preparation process is simple, which is convenient for large-scale production;

⑥Extensible function: self-assembled peptide materials can be combined with small molecule targeted inhibitors, or directly loaded with chemotherapeutic drugs to directly kill tumors.

Description of the drawings

Figure 1 is an electron micrograph of microparticles, membrane GM-CSF microparticles and GM-CSF microparticles produced by radiotherapy of the present invention;

Figure 2 is a diagram of the particle size of the microparticles produced by radiotherapy of the present invention, membrane GM-CSF microparticles and GM-CSF microparticles;

Figure 3 is a map of plasmid vectors used in the present invention;

Figure 4 is the target gene sequence of the core plasmid 1 designed by the present invention;

Figure 5 is the target gene sequence of the core plasmid 2 designed by the present invention;

Figure 6 shows the detection of GM-CSF content on the surface of stable tumor cell membrane;

Figure 7 shows the detection of the overall GM-CSF content of microparticles, membrane GM-CSF microparticles and GM-CSF microparticles produced by radiotherapy of the present invention;

Figure 8 shows the detection of GM-CSF content on the surface of microparticles, membrane GM-CSF microparticles and GM-CSF microparticles produced by radiotherapy of the present invention;

Fig. 9 is a diagram showing the chemotactic effect of microparticles, membrane GM-CSF microparticles and GM-CSF microparticles produced by radiotherapy of the present invention;

Figure 10 is a statistical diagram of chemotaxis of microparticles, membrane GM-CSF microparticles and GM-CSF microparticles produced by radiotherapy of the present invention;

Figure 11 is a statistical diagram of the volume of the tumor body during the treatment of solid tumors with microparticles produced by radiotherapy, membrane GM-CSF microparticles, and GM-CSF microparticles;

Fig. 12 is a statistical diagram of the weight of mice during the treatment of solid tumors with microparticles produced by radiotherapy, membrane GM-CSF microparticles, and GM-CSF microparticles of the present invention;

Figure 13 is a statistical diagram of the survival of mice during the treatment of malignant pleural effusion with membrane GM-CSF microparticles of the present invention. .

Detailed ways

The principles and features of the present invention will be described below in conjunction with examples. The examples cited are only used to explain the present invention and are not used to limit the scope of the present invention.

The present invention relates to an anti-tumor technology based on genetic engineering that can replace radiotherapy for indirect radiotherapy. It mainly includes the development of an autologous source of genetically engineered anti-tumor microparticles. Engineered tumor cells are produced by radiation treatment. The genetically engineered anti-tumor microparticles have significant anti-tumor activity, can chemoattract immune cells to reach the tumor microenvironment, can improve anti-tumor immunity, and have a control effect on solid tumors. The genetically engineered anti-tumor microparticles are entirely derived from tumor cells themselves, and have good biological safety and biocompatibility. The genetically engineered anti-tumor microparticles can also be loaded with chemotherapeutic drugs and targeted drugs, or combined with small molecule targeted drugs to directly act on tumors.

The specific principles of the present invention are as follows:

A microparticle is a microvesicle structure that can be secreted by a variety of living cells with a diameter of 100-1000nm and is widely distributed in a variety of body fluids. In the past 10 years, research has found that microparticles are rich in biologically active molecules such as protein, DNA, RNA, and lipids, and participate in signal communication between cells. Microparticles can directly carry signal molecules and can also transfer Fas ligands from tumor cells. To T cells thereby mediate immune escape, and even mediate the transmission of genetic information. Microparticles are suitable for local treatment. Some studies have achieved certain effects in the treatment of MPE by encapsulating microparticles with chemotherapeutics, but their anti-tumor effects are limited, partly because the microparticle carrier itself has no anti-tumor effect.

Cytokines are small molecule polypeptides or glycoproteins synthesized and secreted by a variety of tissue cells (mainly immune cells). They can mediate the interaction between cells and have a variety of biological functions, such as regulating cell growth, differentiation and maturation, Maintain function, regulate immune response, participate in inflammatory response, wound healing and tumor growth and decline. Cytokines can be divided into interleukins, interferons, tumor necrosis factor superfamily, colony stimulating factors, chemokines, growth factors and other categories. Granulocyte-macrophage colony stimulating factor (GM-CSF) is a type of colony stimulating factor that stimulates the proliferation and differentiation of myeloid stem cells to mature granulocytes. The present invention uses genetic engineering technology to design a recombinant plasmid that can express GM-CSF on the surface of the host cell membrane, package it into lentiviral particles and then infect the host cell, so that GM-CSF is expressed on the surface of the host cell membrane. Microparticles are vesicle-like secretions peeled off from the cell membrane surface when eukaryotic cells are activated or apoptosis. They are considered a biological information carrier that can mediate the transfer and exchange of biological information materials between different types of cells. Radiotherapy can cause tumor cell apoptosis, which can also induce tumor cells to secrete microparticles. The microparticle membrane secreted by genetically engineered tumor cells contains GM-CSF, which can effectively chemoattract immune cells and therefore also has a good anti-tumor effect. Using the microparticle technology produced by in vitro radiotherapy, microparticles containing GM-CSF on the membrane surface can be obtained, and the infusion of these microparticles will provide more effective and safe local treatment methods for the treatment of tumors, thereby realizing the purpose of indirect radiotherapy .

S1. Construction of recombinant plasmids overexpressing GM-CSF and transmembrane overexpressing GM-CSF;

S2. Obtain isolated tumor cells for primary tumor cell culture, and obtain primary tumor cells;

S3. Packaging the lentiviral particles overexpressing GM-CSF and transmembrane overexpressing GM-CSF;

S4. Infect the primary tumor cells obtained in step S2 with lentiviral particles to obtain primary tumor cells overexpressing GM-CSF and transmembrane overexpressing GM-CSF;

S5. Perform X-ray irradiation on the primary tumor cells and culture medium obtained in step S4, and collect the supernatant after radiotherapy to obtain a mixture of required microparticles and apoptotic tumor cell debris;

S6. Centrifuge the mixture obtained in step S5 to obtain the anti-tumor microparticles.

The centrifugation in step S6 includes the first centrifugation, the second centrifugation, and the third centrifugation in sequence. The rotation speed of the first centrifugation is 1000 g and the time is 10 minutes to obtain the supernatant; the rotation speed of the second centrifugation is 14000 g and the time is 10 minutes. For 2 minutes, the supernatant is obtained; the rotation speed of the third centrifugation is 14000g, and the time is 60 minutes, and the precipitate is the fine particles.

The isolated tumor cells are tumor cells derived from autologous or tumor cells produced by tumor cell lines.

The isolated tumor cells are derived from solid tumors.

A genetically engineered anti-tumor microparticle prepared by a method for preparing genetically engineered anti-tumor microparticles, wherein the genetically engineered anti-tumor microparticles have a microvesicle structure carrying GM-CSF, The particle size of the microvesicle structure is 100-1000 nm.

After irradiating genetically engineered tumor cells with radiotherapy, the cells will secrete microparticles rich in GM-CSF on the surface. The obtained microparticles have anti-tumor ability and chemotactic immune cell function. In the present invention, the storage conditions for the genetically engineered microparticles to preserve the anti-tumor activity are 4° C. and within 7 days. The genetically engineered microparticle structure of the present invention is shown in FIG. 1, and the particle size is shown in FIG. 2.

The effect of the present invention will be further explained and verified below in conjunction with the examples.

Construction of recombinant plasmid and packaging into lentiviral particles to infect tumor cells

Determine the protein coding region sequence of GM-CSF on Pubmed website, add membrane signal peptide before this sequence, add transmembrane region and SFB tag sequence after the sequence, insert pCDH-CMV-MCS-EF1-copGFP-T2A-Puro In the multiple cloning site of the plasmid vector (shown in Figure 3), the core plasmid 1 (shown in Figure 4) that can overexpress GM-CSF across the membrane is constructed. On the basis of the core plasmid 2, the transmembrane region is removed to construct the core plasmid 2 overexpressing GM-CSF (as shown in Figure 5). Use 10% FBS (fetal bovine serum) medium 293T cells in a 10mm×10mm petri dish. When the cell density in the dish reaches about 50%, replace with 10 ml of fresh medium. Take 2 sterile, RNase-free EP tubes, add 1.5 mL of Opti-MEM optimized medium to one of them, add 30 μL of transfection reagent PEI, gently pipette the pipette tip to mix, and let stand at room temperature for 5 minutes. Add 1.5 mL of Opti-MEM optimized medium to another EP tube, add 6μg of core plasmid, 4.5μg of psPAX2 plasmid and 1.5μg of pMD2.G plasmid, and blow gently. Add the liquid in the EP tube with transfection reagent to the EP tube with plasmid, blow gently, and let it stand at room temperature for 20 minutes. Add the above mixed and static liquid to the 293T cells, gently shake it, and place it in an incubator for culture. After 24 hours, the 13mL medium was replaced. After culturing for 48 hours, the cell supernatant was filtered with 0.45μm. Use 10% FBS (fetal bovine serum) medium lewis cells in a 10mm×10mm culture dish. When the cell density in the dish is about 50%, replace the filtered supernatant 5mL and the fresh medium 5mL, and add 10μL gene transfer at the same time. Staining enhancer polybrene, continuous infection for two days. Screening was carried out with a medium containing puromycin (1:1000) to obtain stable tumor cells overexpressing GM-CSF and membrane overexpressing GM-CSF.

Verification of stable transgenic tumor cells overexpressing GM-CSF and transmembrane overexpressing GM-CSF

Digest the stabilized tumor cells into a single cell suspension by centrifugation and resuspend the cells with 1ml PBS (for current use) and count them, adjust the cells to an appropriate concentration, and transfer them to a flow tube. Add GM-CSF fluorescent antibody to the tube, incubate on ice for 30 minutes in the dark (mixing every 10 minutes), add 3ml PBS, centrifuge at 350g for 5 minutes, and repeat this step twice. Add an appropriate amount of 4% paraformaldehyde, fix at room temperature and avoid light for 30 minutes and then wash off. Resuspend the cells in PBS and wait for flow cytometric detection. As shown in Figure 6, stable tumor cells with membrane over-expressing GM-CSF have higher GM-CSF content.

Construction and BCA quantification of chemotactic radiotherapy microparticles and storage methods

Culture the tumor cells stably with 10% FBS (fetal bovine serum) medium in a 10mm×10mm petri dish. When the number of cells in the dish reaches about 5×106, perform radiotherapy at a dose of 20GY. The liquid was changed every day, and 20ml of medium containing 10% FBS was added. On the 3rd day, all the liquid in the petri dish was collected and the microparticles were extracted by gradient centrifugation. Take the cell culture medium after radiotherapy at 1000g and centrifuge for 10min, then take the supernatant, then centrifuge the supernatant at 14000g, centrifuge for 2min to remove debris and discard the precipitate. Finally, centrifuge the supernatant at 14000g at 4°C for 60min, discard the supernatant, the precipitate is the microparticles. Wash the precipitate twice with normal saline, resuspend in 1ml PBS (phosphate buffered saline) solution and store at 4°C. Centrifuge 100μl of liquid, add an appropriate amount of protein lysis solution, fully lyse on ice for 30 minutes, centrifuge at 12000g for 30 minutes, and take it up Add BCA quantitative solution to protein quantification.

Detection of the overall GM-CSF content of genetically engineered microparticles

Mix the quantitative microparticle lysate with a 5×SDS loading buffer of 1/4 the volume of the lysate, and heat at 100°C for 10 minutes. Prepare 12% separating gel and 5% concentrated gel according to the formula, add protein sample to the sample hole, and add an equal volume of 1×SDS sample buffer to the edge hole. During electrophoresis, the concentrated gel is at a constant voltage of 80V. When the protein markers are separated, the voltage is adjusted to a constant voltage of 120V. When the electrophoresis runs until the bromophenol blue is at the bottom of the separation gel, the electrophoresis is finished. Pour the film transfer liquid into the iron pan and put it into the transfer film clamp. Pry open the glass plate, use a rubber cutting board to cross-cut the desired target protein according to the position of the marker, place it on the black rubber filter paper, cover the PVDF membrane soaked with methanol on the rubber, and clamp the clamp. Put the clip into the film transfer tank, pour the film transfer liquid in the iron pan into the film transfer tank, the black of the clip faces the black side of the tank, put the film transfer tank into the ice foam box, select 200mA constant current transfer membrane. After 2 hours, the PVDF membrane was taken out and sealed with 5% skimmed milk blocking solution on a shaking table at room temperature for 1 hour. Wash with 1×TBST 3 times, 10 min each time, prepare the primary antibody solution with the primary antibody diluent according to a certain dilution ratio. The protein surface of the membrane is in contact with the antibody, and it is placed in a refrigerator at 4°C overnight. The membrane was washed 3 times with 1×TBST on a shaker for 10 minutes each time, and then incubated with HRP-labeled secondary antibody (diluted with 5% skim milk to 1:5000) on a shaker at room temperature for 1 h. Wash the film with 1×TBST on a shaker 3 times, 10 minutes each time, configure ECL developer solution (ECL A: ECL B = 1: S1, drop the ECL developer solution on the film, and expose with an exposure instrument. As shown in Figure 7 It shows that both GM-CSF radiotherapy microparticles and membrane GM-CSF radiotherapy microparticles contain GM-CSF.

Detection of GM-CSF content on the surface of genetically engineered microparticles

30μg each of radiotherapy microparticles, membrane GM-CSF radiotherapy microparticles and GM-CSF radiotherapy microparticles are combined with 10μl 4μm diameter aldehyde/sulfuric acid latex beads at room temperature for 15min, add 1ml PBS, gently shake at 37°C for 2h, add 10nmol/L glycine Block the reaction for 30 minutes, wash with PBS 3 times, resuspend in 500μl PBS, add GM-CSF fluorescent antibody, incubate for 1 hour in the dark, wash 3 times with PBS, and wait for flow-based detection. As shown in Figure 8, the surface of the film GM-CSF radiotherapy microparticles has a higher GM-CSF content.

Chemotaxis of genetically engineered microparticles

Add 1×105 macrophages to the upper chamber of the Transwell chamber, the cell suspension volume is 200μl, and add 500μl of medium containing PBS, radiotherapy microparticles, membrane GM-CSF radiotherapy microparticles and GM-CSF radiotherapy microparticles respectively in the lower chamber . Place the culture plate with the Transwell chamber in a 37°C 5% CO2 cell incubator, take out the chamber after 24 hours, fix the cells with 4% paraformaldehyde, stain them with crystal violet, wipe the inner cells carefully with a cotton swab, and observe under a microscope Number of migrating cells. As shown in Figure 9 and Figure 10, the number of cells migrated in the microparticle group of membrane GM-CSF radiotherapy was the most, which fully proved that GM-CSF on the surface of microparticles has a strong chemotaxis ability to immune cells.

Animal experiment of genetically engineered microparticles against solid tumors

Establish a subcutaneous transplantation tumor model in C57 mice: plant 5×105 Lewis cells per mouse subcutaneously in C57 mice, and when the tumor volume reaches about 50mm3 (about 8 days), on the first day and the third day, respectively 50 μL of 20GY microparticles (5 mg/kg) and PBS extracted on the third day after radiotherapy were injected intratumorally, and the tumor size was measured every two days from the day after drug injection. As shown in Figure 11, compared with the GM-CSF microparticle group, the tumor volume of the membrane GM-CSF microparticle group is significantly reduced, which fully proves that the presence of GM-CSF on the surface of the microparticle can significantly inhibit tumor growth. As shown in Figure 12, there was no significant difference in the body weight of the mice in each group, indicating that the various types of radiotherapy microparticles had no obvious toxic and side effects.

Animal experiment on the treatment of malignant pleural effusion with genetically engineered microparticles

Establish a model of malignant pleural effusion: inoculate 3×104 Lewis-LUC mouse lung cancer cells per mouse into the thoracic cavity of C57 mice, control the depth of needle penetration to 3.3 mm, and the injection volume to 50 μL. On the 8th day, small animal in vivo imaging was performed to prove that the model was successful, 50μL of stable transfected cells 20GY microparticles (5mg/kg) and PBS extracted on the 3rd day after radiotherapy were injected into the chest cavity for 7 consecutive days, and the survival of the mice was observed. time. As shown in Figure 13, membrane GM-CSF radiotherapy microparticles can significantly prolong the survival time of mice.

As shown in Figure 1, the electron microscope representative image of tumor cell microparticles after 5-20Gy, 6MV X-ray radiotherapy shows that it is a spherical vesicle-like structure with a particle size of about 500nm.

As shown in Figure 2, the particle size of the microparticles is analyzed by a Malvern particle size analyzer, and it is obtained that the particle size distribution of the microparticles is 100-1000nm.

As shown in Figure 3, it is the pCDH-CMV-MCS-EF1-copGFP-T2A-Puro vector plasmid map.

As shown in Figure 4, it is the target gene sequence of core plasmid 1 (membrane overexpression GM-CSF).

As shown in Figure 5, it is the target gene sequence of core plasmid 2 (overexpressing GM-CSF).

As shown in Figure 6, the use of flow cytometry to detect the construction of a Lewis stable line of mouse lung cancer cells proves that the membrane surface of the stable tumor cell membrane overexpressing GM-CSF has a higher GM-CSF content.

As shown in Figure 7, Western Blot was used to detect the content of SFB-tagged protein in different microparticles. The FLAG strip proved that both GM-CSF radiotherapy microparticles and membrane GM-CSF radiotherapy microparticles contained GM-CSF.

As shown in Figure 8, the use of flow cytometry to detect the content of GM-CSF on the surface of different microparticles proves that the surface of the membrane GM-CSF radiotherapy microparticles has a higher content of GM-CSF.

As shown in Figure 9, primary macrophages derived from mice were used to detect the chemotactic effects of different microparticles on macrophages, which proved that the number of cells migrated in the membrane GM-CSF radiotherapy microparticle group was the largest.

As shown in Figure 10, primary macrophages derived from mice were used to detect the chemotactic effects of different microparticles on macrophages, which proved that the number of cells migrated in the membrane GM-CSF radiotherapy microparticle group was the largest.

As shown in Figure 11, mouse lung cancer Lewis cells were used to make a mouse subcutaneous xenograft model. Common Lewis cells and stable Lewis cells were used for intratumoral injection of radiotherapy microparticles, which proved that the membrane GM-CSF radiotherapy microparticles are effective The tumor suppressor effect is the most significant.

As shown in Figure 12, mouse lung cancer Lewis cells were used to make a mouse subcutaneous xenograft model. The intratumoral injection and administration of radiotherapy microparticles released by ordinary Lewis cells and stable Lewis cells proved that various types of radiotherapy microparticles are effective against small tumors. The weight of the mouse has no effect.

As shown in Figure 13, mouse lung cancer Lewis-LUC cells were used to make a mouse pleural effusion model, and PBS and radiotherapy microparticles released by stable Lewis cells were used for pleural perfusion therapy, which proved that membrane GM-CSF radiotherapy microparticles can significantly prolong mice Survival time.

The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection of the present invention. Within range.

Claims

A method for preparing genetically engineered anti-tumor microparticles, which is characterized in that it comprises the following steps:

S1. Construction of plasmids overexpressing GM-CSF and transmembrane overexpressing GM-CSF;

S2. Obtain isolated tumor cells for primary tumor cell culture, and obtain primary tumor cells;

S3. Packaging the lentiviral particles overexpressing GM-CSF and transmembrane overexpressing GM-CSF;

S4. Infect the primary tumor cells obtained in step S2 with lentiviral particles to obtain primary tumor cells overexpressing GM-CSF and transmembrane overexpressing GM-CSF;

S5. Perform X-ray irradiation on the primary tumor cells and culture medium obtained in S4, and collect the supernatant after radiotherapy to obtain a mixture of required microparticles and apoptotic tumor cell debris;

S6. Centrifuge the mixture obtained in S5 to obtain the genetically engineered anti-tumor microparticles.
The method for preparing genetically engineered anti-tumor microparticles according to claim 1, wherein the centrifugation in S6 comprises sequentially performing a first centrifugation, a second centrifugation, and a third centrifugation, and the second centrifugation The speed of a centrifugation is 1000g and the time is 10 minutes to get the supernatant; the speed of the second centrifugation is 14000g and the time is 2 minutes to get the supernatant; the speed of the third centrifugation is 14000g and the time is 60 minutes, and the precipitate is obtained. For micro particles.
The method for preparing genetically engineered anti-tumor microparticles according to claim 1, wherein the X-ray radiation dose of the S5 is 5-20 Gy, the X-ray energy is 6 MV, and the supernatant collection time For the 2-7 days after radiotherapy.
The method for preparing genetically engineered anti-tumor microparticles according to claim 1, wherein the isolated tumor cells are tumor cells derived from autologous or tumor cell lines.
The method for preparing genetically engineered anti-tumor microparticles according to claim 1, wherein the isolated tumor cells are derived from solid tumors.
The method for preparing genetically engineered anti-tumor microparticles according to any one of claims 1 to 5, characterized in that the anti-tumor microparticles prepared therefrom are genetically engineered anti-tumor microparticles. The microvesicle structure carrying GM-CSF, the particle size of the microvesicle structure is 100-1000 nm.
A genetically engineered anti-tumor microparticle medicament, characterized in that it is obtained from the genetically engineered anti-tumor microparticle according to any one of claims 1-5 through purification and concentration.
Application of genetically engineered anti-tumor microparticles prepared by the method for preparing genetically engineered anti-tumor microparticles according to any one of claims 1 to 5 in the preparation of anti-tumor drugs.