Exosome drug delivery system with anti-phagocytosis and tumor targeting ability and preparation and application thereof

Abstract

The application discloses an exosome drug delivery system with anti-phagocytosis and tumor targeting ability as well as a preparation method and application thereof. A tumor targeting group is flexibly connected to the outside of the cell membrane of a transmembrane protein with anti-phagocytosis ability through a linker of a fusion protein. A fusion gene sequence is designed and inserted into an expression vector to form a fusion expression vector, which is integrated into the chromosome of a cell to be modified to obtain an engineered cell stably expressing the fusion protein, which is used for secreting engineered exosomes. The cell to be modified includes a naked cell (an immortalized cell line, a primary cell), an engineered cell stably expressing a therapeutic nucleic acid molecule or a protein. The exosomes produced by the former cell can further package the therapeutic molecules to treat tumors, and the exosomes produced by the latter cell can be directly used for treating tumors after being separated, purified and collected. The half-life of the engineered exosomes produced by the application is significantly prolonged in the body, the treatment effect is improved, and the exosomes show strong tumor targeting and anti-tumor effects in vivo and in vitro.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine engineering, and particularly relates to an exosome drug delivery system with anti-phagocytosis and tumor targeting capabilities and preparation and application thereof. BACKGROUND

[0002] The incidence and mortality of cancer have been increasing year by year, which seriously threatens human health.

[0003] The tumor drug delivery treatment based on the exosome drug delivery system is the sixth cancer treatment mode after surgical treatment, radiotherapy, chemotherapy, targeted therapy and immunotherapy. Exosomes are vesicles with a double-membrane structure secreted by most cells, with a size of 30-150 nm. Exosomes can specifically enrich various biological molecules such as nucleic acids, proteins and lipids in cells, and then deliver them to recipient cells and regulate them. Because exosomes are derived from body cells, they have low immunogenicity, and in addition, they have small particle size and high bioavailability, can encapsulate various drugs (including hydrophobic drugs) and freely pass through physiological barriers to reach the deep part of tumors, and after being taken up by tumor cells, release the drugs to kill tumors. Through searching in the clinical trial official website clinicaltrials.gov using "exosomes" and "cancer" as keywords, only 3 clinical studies of tumor drug delivery by exosomes (NCT01294072, NCT01668849, NCT03608631) were found, and among them, 2 were based on plant-derived exosomes. It can be seen that the tumor drug delivery by exosomes as a new technology for tumor drug delivery treatment is still in the initial stage of clinical research, and has important research significance and broad application prospects.

[0004] In recent years, a large number of preclinical studies have reported the use of exosomes as a drug delivery system for tumor treatment. There are mainly two categories: 1. Because milk and serum are easy to obtain, the yield of exosomes is high, but exosomes do not contain drugs and lack targeting ability, so after obtaining milk exosomes or serum exosomes, they need to be targeted and loaded with drugs, and then anti-tumor research is carried out. 2. In order to simplify the subsequent processing process, researchers tend to use exosomes from normal cells. First, the cells are genetically engineered to allow the cells to stably express anti-tumor nucleic acid molecules (this scheme is mostly engineered), and after the cells are cultured, a large number of cell-derived exosomes are collected. These exosomes are rich in anti-tumor nucleic acid molecules, and then the exosomes are modified with tumor targeting groups for anti-tumor research; in addition, the exosome membrane protein can be modified to allow the cells to stably express targeting molecules, and the resulting exosomes carry tumor targeting groups, and then drug loading can also be obtained. Tumor-targeted drug delivery exosomes. However, the above scheme is relatively cumbersome in clinical application and is not easy to implement. In addition, with the deepening of research, researchers have also found that the exosome drug delivery system is easily phagocytosed and cleared by the endothelial reticular system (RES system) in the blood circulation. Matsumoto et al. found that when exosome drugs were injected into the tail vein of mice, 3h later, the content of exosomes in the blood was less than 5%, most of which accumulated in the liver and spleen, and very little reached the lesion. It can be seen that ordinary exosomes have a greatly shortened half-life during in vivo transport, thereby reducing the therapeutic effect.

[0005] In order to be applied in clinical, the exosome drug delivery system needs to establish a complete industrial large-scale production process. It is reported that the cell culture method for producing exosomes is more suitable for large-scale production, which has many advantages, such as: the culture process is easy to control, the quality control of the produced exosomes is good, etc. The preclinical research of exosomes mentioned in the foregoing is in full swing, but the clinical research is still in its infancy, the biggest reason is that the large-scale production process of exosomes has not been completely established, and it is still only in the stage of cell culture in the laboratory dish (bottle), the production is in short supply, and the production cost is high. SUMMARY

[0006] Invention purposes: In order to overcome the deficiencies in the prior art, the present application provides an exosome drug delivery system with anti-phagocytosis and tumor targeting ability, and its preparation and application, especially a method for producing anti-tumor exosome drugs conveniently. The method only needs to culture the engineered cells, collect the culture solution, and separate and purify the engineered exosomes from the culture solution. The surface of the exosomes is modified with a tumor targeting group, and when a stable strain is used as an engineered cell, the obtained exosomes carry anti-tumor effector molecules, which saves the subsequent processes of drug loading, targeting modification and re-purification of exosomes in the prior art. The clinical application of the exosome targeted drug delivery system can be quickly realized by using the technology provided by the present application.

[0007] Technical scheme: In order to achieve the above-mentioned purposes, the technical scheme adopted by the present application is:

[0008] The first purpose of the present application is to provide an exosome drug delivery system with anti-phagocytosis and tumor targeting ability, wherein the exosome drug delivery system is an engineered exosome modified with a tumor targeting group connected by an anti-phagocytosis protein on the surface, the engineered exosome is secreted by an engineered cell, and the engineered cell stably expresses a fusion protein.

[0009] The engineered cell includes a tool cell with a fusion expression vector integrated into the chromosome, the tool cell includes any one of a naked cell and a stable strain capable of expressing an anti-tumor effector molecule, the fusion expression vector is an expression vector with a fusion gene sequence inserted, and the fusion gene sequence is formed by flexibly connecting a tumor targeting group to the outside of the cell membrane of a transmembrane protein with anti-phagocytosis ability through a linker of a fusion protein. The naked cell and the tool cell capable of expressing an anti-tumor effector molecule are the cells to be modified.

[0010] Optionally, in an embodiment of the present application, the tumor targeting group is any one of a nano-antibody, a traditional antibody, a single-chain variable fragment (scFv), and a targeting peptide, wherein the traditional antibody includes an EGFR antibody (such as cetuximab), an HER2 antibody (such as trastuzumab), and the like.

[0011] Optionally, in an embodiment of the present application, the linker of the fusion protein is any one of a (GGGGS)n linker and a (G)n linker.

[0012] Optionally, in an embodiment of the present application, the transmembrane protein with anti-phagocytosis ability includes, but is not limited to, any one of a CD47 molecule, a CD24 molecule, an MHC-I molecule, and the like, which is a cell transmembrane protein inhibiting the phagocytosis of a reticuloendothelial system (RES system) and a DC cell.

[0013] Optionally, in an embodiment of the present application, the expression vector basic skeleton includes but is not limited to any one of a lentiviral expression vector, an adenoviral expression vector.

[0014] Optionally, in an embodiment of the present application, the engineered naked cells (immortal cell lines, primary cells) include normal cell lines or immune cells, wherein the normal cell lines include but are not limited to any one of a HEK293 cell line, a HEK293T cell line, an NK92 cell line, an NK92MI cell line, and the immune cells include but are not limited to any one of human primary NK cells, macrophages, and neutrophils.

[0015] Optionally, in an embodiment of the present application, the stable cell capable of stably expressing an anti-tumor effector molecule is an engineered cell stably expressing an anti-tumor miRNA, lncRNA, circRNA, or mRNA.

[0016] Optionally, in an embodiment of the present application, the stable cell capable of stably expressing an anti-tumor effector molecule includes but is not limited to any one of miR204-5p, miR139-5p, and CircMTO1.

[0017] A second object of the present application is to provide a preparation method of an exosome drug delivery system with anti-phagocytosis and tumor targeting capabilities, which utilizes the constructed fusion expression plasmid to engineer tool cells, and further utilizes the engineered cells to produce anti-tumor exosomes.

[0018] An exosome drug delivery system with anti-phagocytosis and tumor targeting capabilities requires the construction of a fusion expression plasmid, which has the following core part: the expressed protein uses an anti-phagocytosis transmembrane protein as a bridge to flexibly connect a tumor targeting group to the outside of the anti-phagocytosis transmembrane protein cell membrane through a fusion protein linker. Then the fusion expression plasmid is artificially integrated into the chromosome of a tool cell to obtain a stable expression cell line. The tool cell is a naked cell or a normal cell line or a primary cell stably expressing an anti-tumor nucleic acid drug. The stable expression cell line can separate and purify tumor-targeting drug-loaded exosomes from the culture solution after culture, and further prepare an anti-tumor nano-drug preparation, which can be directly applied to clinical use.

[0019] Optionally, in an embodiment of the present application, the preparation method of the exosome drug delivery system with anti-phagocytosis and tumor targeting capabilities includes the following steps:

[0020] S1, flexibly connecting a tumor targeting group to the outside of a cell membrane having an anti-phagocytosis transmembrane protein through a fusion protein linker to form a fusion gene sequence;

[0021] S2, the fusion gene sequence is inserted into an expression vector to form a fusion expression vector;

[0022] S3, the fusion expression vector is integrated into the chromosome of a tool cell to form an engineered cell that stably expresses a fusion protein, the engineered cell secretes a tumor-targeting drug-loaded engineered exosome, that is, an exosome drug delivery system, the surface of which is modified with a tumor-targeting group connected by an anti-phagocytic protein.

[0023] Optionally, in an embodiment of the present application, in step S3, the integration uses an engineered integration method, which includes but is not limited to viral transfection.

[0024] Optionally, in an embodiment of the present application, during the viral transfection, the fusion expression vector, the viral packaging plasmid, and the viral envelope plasmid are packaged in a mass ratio of 4-5:2-4:1-2.

[0025] Optionally, in an embodiment of the present application, the packaged virus is used to infect tool cells, and positive cells are screened using puromycin or a flow cytometer to obtain engineered cells with a tumor-targeting group connected by an anti-phagocytic transmembrane protein on the cell membrane.

[0026] Optionally, in an embodiment of the present application, the process of engineering the tool cell is as follows: using lentiviral infection, after the three-piece gene fusion is synthesized by a commercial company, it is cloned into a lentiviral expression vector (such as pLVX-AcGFP-N1), first, the fusion expression plasmid (pLVX-7hCD47-AcGFP-N1), the viral packaging plasmid (pSPAX2), and the viral envelope plasmid (pMDG2) are packaged in a mass ratio of 4-5:2-4:1-2; then when the tool cell is at a confluence of 20-30%, 5-8 μg / mL polybrene is added and cultured for 30-60 min, then the packaged virus is used to infect for 24-48 h, and then positive cells are screened using puromycin or a flow cytometer to produce engineered cells.

[0027] In order to realize the clinical transformation of the present application, a large-scale production method in the workshop is developed, including the treatment of engineered exosomes and tumor-targeting exosome drugs, and the two products are different according to the different tool cells:

[0028] When the tool cell is a naked cell, the engineered cell is an engineered naked cell, and the cell reactor is used to produce an engineered exosome modified with a tumor-targeting group and an anti-phagocytic protein on the surface, and the subsequent loading of an anti-tumor effect molecule can obtain an anti-tumor targeting exosome drug;

[0029] When the tool cell is a stable cell line that can stably express an anti-tumor effector molecule, the engineered cell is an engineered anti-tumor drug stable cell line, and only the cell culture needs to be replaced and the cell culture medium is collected, and then the subsequent exosome separation and purification are performed to produce exosomes carrying not only the anti-phagocytic transmembrane protein connected tumor targeting group on the membrane but also the anti-tumor effector molecule itself, which means that the obtained engineered exosomes are themselves a tumor-targeting exosome therapeutic drug (in the subsequent examples, when the tool cell is a stable cell line that can stably express an anti-tumor effector molecule, the tumor-targeting exosome therapeutic drug and the engineered exosome are synonymous, both refer to the product obtained by cell culture), which can continuously obtain tumor-targeting exosome therapeutic drugs without any subsequent modification and drug loading.

[0030] The two cases are as follows:

[0031] A third object of the present application is to provide a method for industrial large-scale production of engineered exosomes, which uses the above-mentioned exosome drug loading system or the exosome drug loading system prepared by the above-mentioned method, and is produced by microcarrier culture in a cell reactor, wherein the tool cell is a stable cell line that can express an anti-tumor effector molecule.

[0032] Optionally, in an embodiment of the present application, the method for industrial large-scale production of engineered exosomes is specifically culturing cells, replacing and collecting cell culture medium, and obtaining engineered exosomes by separation and purification.

[0033] Optionally, in an embodiment of the present application, the method for industrial large-scale production of engineered exosomes is specifically culturing engineered cells in a cell reactor, and for adherent cells, microcarriers are used for culture, and only the cell culture medium needs to be replaced and collected for exosome separation and purification.

[0034] Optionally, in an embodiment of the present application, a 10L cell reactor is selected, and the method for culturing in the cell reactor comprises: the cell reactor contains microcarriers cytodex3, the cells grow on the microcarriers, and the engineered cells are cultured using a culture medium containing 2%-5% exosome-free fetal bovine serum, the culture medium is collected, and then exosomes are separated and purified to prepare tumor-targeting drug-loaded exosomes.

[0035] Optionally, in an embodiment of the present application, the method for culturing in the cell reactor comprises culturing positive cells, replacing the complete culture medium containing 2%-5% exosome-free serum when the cell confluence is about 70%-80%, continuing to culture for 48h, and then collecting the culture medium, using ultracentrifugation to collect the precipitate, and the precipitate is the exosome.

[0036] Optionally, in an embodiment of the present application, the exosomes are resuspended in sterile PBS and stored in a -80°C refrigerator.

[0037] A fourth object of the present application is to provide an engineered exosome, which is secreted from the exosome drug delivery system described above or prepared by the method described above, or produced by the method for industrial large-scale production of engineered exosomes described above.

[0038] A fifth object of the present application is to provide a method for industrial large-scale production of tumor-targeting exosome therapeutic drugs, which uses the exosome drug delivery system described above or prepared by the method described above, and is produced by industrial large-scale production through microcarrier culture in a cell reactor, wherein the tool cells are stable cells that can express anti-tumor effector molecules.

[0039] Optionally, in an embodiment of the present application, the method for industrial large-scale production of tumor-targeting exosome therapeutic drugs is specifically culturing cells, replacing and collecting cell culture medium, and then isolating and purifying to obtain tumor-targeting exosome therapeutic drugs.

[0040] Optionally, in an embodiment of the present application, a 10L cell reactor is selected, and the method for culturing in the cell reactor includes that the cell reactor contains microcarriers cytodex3, the cells are grown on the microcarriers, the engineered cells are cultured using a culture medium containing 2% to 5% exosome-free fetal bovine serum, the culture medium is collected, and then the exosomes are isolated and purified to prepare tumor-targeting drug-loaded exosomes.

[0041] Optionally, in an embodiment of the present application, the method for culturing in the cell reactor includes culturing positive cells, replacing the complete culture medium containing 2% to 5% exosome-free serum when the cell confluence is about 70% to 80%, continuing to culture for 24h, then collecting the culture medium, collecting the precipitate by ultracentrifugation, and the precipitate is the exosomes.

[0042] Optionally, in an embodiment of the present application, the exosomes are resuspended in sterile PBS and stored in a -80°C refrigerator.

[0043] A sixth object of the present application is to provide a tumor-targeting exosome therapeutic drug, which is produced by the method for industrial large-scale production of tumor-targeting exosome therapeutic drugs described above.

[0044] A seventh object of the present application is to provide a medicine for treating cancer, which contains a target recognized by the tumor targeting group, including solid tumors, blood tumors and lymphomas, comprising the tumor targeting exosome therapeutic medicine as described above, or comprising the engineered exosome as described above for loading anti-tumor drugs, and obtaining the tumor targeting exosome therapeutic medicine after purification.

[0045] Optionally, in an embodiment of the present application, the drug loading method includes, but is not limited to, electric shock transformation, ultrasonic loading, liposome fusion loading, etc.

[0046] Optionally, in an embodiment of the present application, the anti-tumor drug includes, but is not limited to, chemotherapeutic drugs such as doxorubicin, and nucleic acid drugs such as small interfering RNA.

[0047] Advantages: Compared with the prior art, the present application has the following advantages:

[0048] 1. The exosome drug delivery system provided by the present application has anti-phagocytosis and tumor targeting ability. When a stable cell line stably expressing an anti-tumor effector molecule is used as a tool cell, the biggest advantage is that the obtained engineered exosome can directly enter the drug preparation production process, without the need for complicated targeting modification or drug loading process after exosome separation, which innovates the prior art, solves the problem of industrialization of exosome drug prepared by current technology, and makes the separated exosome carry tumor targeting groups and anti-tumor drugs (i.e. anti-tumor effector molecules) at the same time. Only large-scale culture of engineered cells to collect exosomes can directly enter clinical use, and the technology has good application prospect.

[0049] 2. The exosome drug delivery system provided by the present application has good universality. For different cancers, only the type of tumor targeting group (such as nanobody or targeting peptide) or anti-tumor drug needs to be changed, so that the engineered exosome can kill almost all tumor cells.

[0050] 3. The engineered exosome prepared by the present application can also mobilize the immune system of the body to play an anti-tumor function. Since it has an anti-phagocytosis transmembrane protein on the membrane, it can competitively bind to phagocytic cells such as macrophages, promote the phagocytosis of phagocytic cells such as macrophages to tumor cells, and further enhance the killing of tumor cells. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 It is a structural schematic diagram of the exosome drug delivery system of the present application.

[0052] Figure 2 It is a schematic diagram of the principle process of the present application.

[0053] Figure 3 The schematic diagram of the fusion expression plasmid of the application.

[0054] Figure 4 Verification of expression of the fusion expression plasmid in cells; A, Western Blot verification, B, immunofluorescence verification, C, flow cytometry analysis verification, D, cell enzyme-linked immunosorbent verification.

[0055] Figure 5 Characterization of engineered exosomes; A, detection of positive cells after flow cytometry sorting, B, Western Blot verification of engineered exosome fusion protein, C, nanosight characterization of engineered exosomes, D, zeta potential characterization of engineered exosomes, E, transmission electron microscope characterization of engineered exosomes, F, mode diagram of fusion protein on exosomes, G, pull down analysis of engineered exosomes, H, analysis of anti-tumor drug miR204 in engineered exosomes, I, blood compatibility analysis of engineered exosomes.

[0056] Figure 6 Anti-endothelial reticular system phagocytosis of engineered exosomes; A, anti-phagocytosis evaluation by fluorescence uptake, B, flow cytometry evaluation of anti-phagocytosis, C, anti-phagocytosis efficiency.

[0057] Figure 7 Engineered exosomes promote killing of tumor cells by macrophages; A, characterization of tumor cell CD47, B, evaluation of phagocytosis of tumor cells by macrophages by immunofluorescence, C, evaluation of phagocytosis of tumor cells by macrophages by flow cytometry.

[0058] Figure 8 Tumor targeting of engineered exosomes; A, observation of tumor targeting of engineered exosomes in EGFR-positive cells by immunofluorescence, B, observation of tumor targeting of engineered exosomes in EGFR-negative cells by immunofluorescence, C, analysis of tumor targeting of engineered exosomes in EGFR-positive cells by flow cytometry, D, analysis of tumor targeting of engineered exosomes in EGFR-negative cells by flow cytometry, E, detection of intracellular miR204 after uptake of engineered exosomes by EGFR-positive cells, F, detection of intracellular miR204 after uptake of engineered exosomes by EGFR-negative cells.

[0059] Figure 9 In vitro anti-tumor activity of nanobody-modified exosomes; A, CCK8 analysis of killing of EGFR-positive / negative tumor cells by engineered exosomes, B, observation of killing activity of engineered exosomes on tumors in 3D culture of tumor cells, C, analysis of apoptosis of tumor cells after treatment with engineered exosomes, D, detection of anti-tumor drug miR204 downstream targets after treatment of tumor cells with engineered exosomes, E, proliferation analysis of tumor cells after treatment with engineered exosomes.

[0060] Figure 10 Biodistribution of nanobody modified exosomes in tumor-bearing mice; A detection mode diagram, B in vivo bio-distribution of live imaging, C ex vivo fluorescence imaging of each major organ and tumor of mice, D fluorescence quantification in each major organ and tumor of mice.

[0061] Figure 11 In vivo anti-tumor activity of nanobody modified exosomes; A is the change of tumor volume during treatment, B is the tumor weight comparison after treatment, C is the change of mouse weight during treatment.

[0062] Figure 12 Anti-tumor activity of exosomes modified by targeting peptides; A CCK8 analysis of the killing of EGFR positive tumor cells by engineered exosomes, B is the change of tumor volume during in vivo treatment, C is the tumor weight comparison after in vivo treatment.

[0063] Figure 13 Exosomes produced after fusion expression vector engineering naked cells have anti-tumor activity; A CCK8 analysis of the killing of EGFR positive tumor cells by engineered exosomes encapsulating chemotherapeutic drug doxorubicin (DOX), B is the change of tumor volume during in vivo treatment, C is the tumor weight comparison after in vivo treatment.

[0064] Figure 14 In order to simulate the industrial production mode, the culture system is scaled up for production, and the exosomes are collected in a 10L cell reactor for verification; A comparison of exosome production before and after scaling up, B evaluation of exosome tumor killing activity before and after scaling up. DETAILED DESCRIPTION

[0065] In order to make the technical means, the purpose and effect of the present application easy to understand, the present application will be further illustrated by the following examples, but the present application is not limited by the examples.

[0066] EMBODIMENT

[0067] The present application can be better understood according to the following examples. However, those skilled in the art will readily understand that the specific material proportions, process conditions and results described in the examples are only for illustrating the present application, and should not and will not limit the present application described in detail in the claims.

[0068] The present application is intended to disclose a preparation method and application of an exosome drug delivery system with anti-phagocytosis and tumor targeting ability, such as Figure 1As shown, the exosome drug delivery system with both anti-phagocytosis and tumor targeting capabilities is an engineered exosome with a tumor targeting group connected to an anti-phagocytosis protein on the surface, which is secreted by an engineered cell stably expressing a fusion protein; the engineered cell includes a tool cell with a fusion expression vector integrated into the chromosome, the tool cell including a naked cell and a stable strain expressing an anti-tumor effector molecule, and the fusion expression vector is an expression vector with a fusion gene sequence connected to the outside of the cell membrane of the anti-phagocytosis transmembrane protein through a flexible linker of the fusion protein.

[0069] In the embodiment, 7D12 nanobody is selected as the tumor targeting group, CD47 is selected as the anti-phagocytosis transmembrane protein, and they are flexibly connected through (GGGGS)2. The fusion protein gene is cloned into a lentiviral vector pLVX-AcGFP-N1, and the tool cell is selected as a HEK293T cell line stably expressing anti-cancer miR204-5p. The exosomes produced after lentiviral infection and screening of positive cells can be directly used in clinical applications, and the subsequent cumbersome processes such as drug loading and tumor targeting group modification are omitted. This method is convenient, easy to mass-produce, and easy to transform. The following embodiments are not limited to the HEK293T cell line stably expressing anti-cancer miR204-5p, but also include other empty and normal cell lines stably expressing anti-cancer nucleic acid molecules and various primary cells; similarly, the targeting ligand is not limited to 7D12 nanobody, but also applies to other nanobodies, and also applies to other biological macromolecules such as scFv, targeting peptides, and other biological macromolecules with targeting and specific recognition functions, and the anti-phagocytosis transmembrane protein is not limited to CD47, but also applies to other anti-phagocytosis transmembrane proteins.

[0070] Embodiment 1: Construction of fusion expression plasmid

[0071] The fusion expression plasmid is a recombinant plasmid constructed by inserting a fusion protein composed of a tumor targeting group, a flexible linker, and an anti-phagocytosis transmembrane protein into a lentiviral vector, as shown in Figure 2 , and the specific form is as shown in Figure 3 . First, the fusion protein gene fragment is synthesized by a commercial company, and then cloned into the lentiviral expression vector pLVX-AcGFP-N1.

[0072] The specific method is as follows:

[0073] The company synthesized the fusion protein gene fragment containing the cloning vector was digested with Xho I and Bam HI, and then the fusion protein gene fragment was obtained by gel recovery. Then the lentiviral expression vector pLVX-AcGFP-N1 was also digested with Xho I and Bam HI, and the linearized vector skeleton was obtained by gel recovery. Then the fusion protein gene fragment and the linearized vector skeleton were mixed at a ratio of 2:1, and then reacted with T4 ligase at 16°C for 30 min. Then the DH5a was transformed, plated, cloned, plasmid extracted, and enzyme digested to obtain the successfully constructed fusion protein expression plasmid.

[0074] Example 2: Verification of fusion expression plasmid expression in cells

[0075] This example verifies whether the fusion protein expression plasmid is expressed in the tool cell. The fusion protein gene is integrated into the tool cell by lentiviral infection to obtain a stable transfection strain, and then WB, IF, FCM and cell ELISA are used for related verification from different angles.

[0076] The specific method is as follows:

[0077] Virus packaging and stable strain screening: HEK293T cells were plated in 10 cm culture dishes, and when the cell confluence was about 90%-95%, fresh DMEM complete medium was replaced for 1 h. Next, the transfection sample was prepared, the transfection reagent was diluted with DMEM to prepare premix A, and the fusion protein expression plasmid, virus packaging plasmid and virus envelope plasmid were mixed and diluted in DMEM to prepare premix B at a ratio of 4:3:1. Incubate for 5 min, then add premix A to premix B and mix well, incubate at room temperature for 15 min. After 1 h of cell culture, the transfection sample was added to the cell culture dish, and then the culture supernatant was collected at 24 h and 48 h, and the virus particles were collected by ultracentrifugation. Then the HEK293T(204) cell strain stably expressing miR204-5p was seeded in a 6-well plate, and when the confluence was 20%-30%, fresh DMEM medium containing 5 ug / mL polybrene was replaced for 30 min. Then the virus was added to the cell plate, and the infection was performed for 24-48 h. Because the recombinant expression plasmid contains a GFP tag, the GFP positive cells were sorted by flow cytometry, and the stable transfection strain (HEK293T-7hCD47(204)) stably expressing 7D12-hCD47(7hCD47) fusion protein and anti-cancer drug miR204-5p was obtained.

[0078] Stable strain detection: The stable strain protein was collected, and then WB detection was performed using Flag tag antibody, anti-nanobody antibody, and CD47 antibody, respectively. The results are as follows: Figure 4A shows that the stable strains have corresponding protein bands while the control cell strains have no bands; then IF and FCM are used to detect the fluorescence of the stable strains, and it is found that the Flag tag is outside the cell and the stable strain cells have obvious GFP fluorescence Figure 4 B and Figure 4 C); finally, the cell ELISA experiment further shows that the Flag tag, nanobody and CD47 functional domain are indeed on the outside of the membrane of the stable strain Figure 4 D), which is consistent with the preset structure, indicating that the fusion expression plasmid is successfully expressed in the cell.

[0079] Example 3: Collection and characterization of engineered exosomes

[0080] In this embodiment, the above-mentioned stable strains are cultured, and the exosomes in the culture solution are separated. Then, the collected exosomes are characterized by using WB, transmission electron microscopy, NTA, zeta potential and other technologies. Then, exosome pull down and anti-tumor miR204-5p in exosomes are verified, and then the blood compatibility of the collected engineered exosomes is analyzed.

[0081] The specific method is as follows:

[0082] As shown in Figure 5 A, the engineered cells are successfully constructed and effectively sorted, so 10L cell reactors are used to culture the positive cells. When the cell confluence is about 70% to 80%, the complete culture medium containing 2% exosome-free serum is replaced to continue culturing for 24h. Then, the culture solution is collected, centrifuged at 10000xg for 30min at 4°C, and the supernatant is taken and ultracentrifuged at 120000xg for 70min at 4°C. Then, the precipitate is washed once with ice-cold PBS and ultracentrifuged at 120000xg for 70min at 4°C. The precipitate is the exosomes, which are resuspended with sterile PBS and stored in a-80°C refrigerator.

[0083] Next, WB is used to characterize the exosome marker protein and fusion protein, and the results are shown in Figure 5 B, which shows that the exosome marker protein and fusion protein exist in the engineered exosome sample, indicating that the present technology can harvest engineered exosomes. Then, the transmission electron microscopy, NTA, zeta potential, exosome pull down and exosome miR204-5p analysis shown in Figure 5 C-5H further show that miR204-5p is highly expressed in the engineered exosomes, and the functional structure of the fusion protein, including the nanobody and CD47, is on the outside of the exosome membrane, which further proves that the collected engineered exosomes can play their intended functions. Then, the safety of the harvested engineered exosomes is analyzed, and the blood compatibility shown in Figure 5 I shows that the engineered exosomes do not cause hemolysis and have good blood safety.

[0084] Example 4: Anti-endothelial reticular system phagocytosis analysis of engineered exosomes

[0085] There are a large number of endothelial reticular systems in the body, and the presence of these sites can play a certain defense role to protect the body from foreign substances. The endothelial reticular system is mainly composed of endothelial cells, reticular cells, macrophages and the like, and only macrophages have phagocytosis. According to previous reports, injection of related nanomedicines into the human body will be partially removed by the endothelial reticular system, and only a small amount of drugs can reach the lesion to play a therapeutic function. Exosomes are no exception, although they are derived from the body itself, but will also be partially phagocytosed. Therefore, this embodiment focuses on the anti-phagocytic activity of the engineered exosomes obtained in Example 3. Since CD47 can inhibit the phagocytosis of macrophages through SIRPα on the macrophages, the engineered exosomes are co-cultured with macrophages to analyze their anti-phagocytic ability.

[0086] The specific method is as follows:

[0087] As shown in Figure 6 , the engineered exosomes labeled with DiO were co-cultured with macrophages labeled with DiI for 12h, and IF and FCM were used for analysis. The results showed that the anti-phagocytic ability of the engineered exosomes modified with CD47 was increased by 4 times.

[0088] Example 5: Engineered exosomes promote macrophages to kill tumor cells

[0089] Most tumor cells highly express CD47 and other immune checkpoints that inhibit macrophage phagocytosis, so that tumor cells can escape macrophage phagocytosis. In this embodiment, the engineered exosomes derived from Example 3 have high expression of CD47 on the surface, which can competitively bind SIRPα on macrophages, so that the inhibition of macrophages by tumor cells is significantly reduced, thereby inducing macrophages to kill tumor cells.

[0090] The specific method is as follows:

[0091] A three-component co-culture system was used, and the tumor cells highly expressing CD47 were labeled with GFP, and the macrophages were labeled with DiI. Then the two labeled cells were co-cultured with the engineered exosomes for 6h, and IF and FCM were used to detect the phagocytosis of macrophages to tumor cells, as shown in Figure 7 , the engineered exosomes can inhibit the anti-macrophage killing of tumor cells, and the phagocytosis of macrophages to tumor cells in the engineered exosome treatment group is about 40% higher than that in the control exosome treatment group, significantly improving the phagocytosis and killing activity of macrophages to tumor cells. This shows that the engineered exosomes obtained in the present application greatly prolong the half-life.

[0092] To verify the anti-tumor properties of the engineered exosomes obtained in Example 3 in vivo and in vitro, the present application carried out relevant experimental verification, specifically as in Examples 6-9.

[0093] Example 6: In vitro tumor targeting of nanobody-modified engineered exosomes

[0094] This example is an in vitro tumor targeting analysis of the engineered exosomes obtained in Example 3. The specific method is as follows: EGFR-positive tumor cells and negative tumor cells are selected as models for analysis, the exosomes and tumor cells are first labeled with two different colors of fluorescent dyes, respectively, then co-cultured, and then the ability of tumor cells to phagocytose exosomes is determined by IF and FCM, respectively.

[0095] The specific process is as follows:

[0096] The control exosomes and nanobody-modified engineered exosomes are first labeled with green fluorescent DiO, and the high EGFR-expressing HCT116 cells and low EGFR-expressing SW620 cells that are adherent overnight are labeled with red fluorescent DiR, then co-cultured with the labeled exosomes in the tumor cells for 12 h, washed with PBS, fixed, washed with PBS, and then imaged by laser confocal and detected by flow cytometry.

[0097] The results show that the uptake of nanobody-modified engineered exosomes by HCT116 is significantly higher than that of control exosomes, about 40% higher than that of control exosomes; while the uptake of nanobody-modified engineered exosomes by negative cells and control exosomes has no significant difference Figure 8 ). This indicates that the tumor targeting of exosomes is significantly enhanced after modification by nanobody 7D12.

[0098] Example 7: In vitro anti-tumor activity of nanobody-modified engineered exosomes

[0099] This example is an in vitro anti-tumor activity analysis of the engineered exosomes obtained in Example 3. The specific method is as follows: EGFR-positive tumor cells and negative tumor cells are selected as models for analysis, the control exosomes and nanobody-modified engineered exosomes are co-cultured with the tumor cells, and the killing of engineered exosomes on tumor cells is determined by CCK8, apoptosis, and tumor cell cluster formation.

[0100] The specific process is as follows:

[0101] The engineered exosomes modified by the nanobody have good tumor targeting property as described in Example 6, so the content of miR204-5p in the engineered exosomes modified by the nanobody is significantly higher than that in the exosomes without modification by the nanobody 7D12 and the control exosomes in the EGFR-positive HCT116 tumor cells by the analysis of fluorescent quantitative PCR, and there is no difference in the EGFR-negative SW620 tumor cells Figure 9 A); then each group of exosomes is co-cultured with the EGFR-positive HCT116, and the generation of tumor spheres of HCT116, the apoptosis of HCT116 and the staining of the proliferation marker ki67 of HCT116 are observed, and the results show that the engineered exosomes modified by the nanobody can significantly inhibit the growth of tumor cells Figure 9 B-9E).

[0102] Example 8: In vivo biodistribution of engineered exosomes modified by nanobody

[0103] This example is an in vivo biodistribution analysis of the engineered exosomes obtained in Example 3 to verify the in vivo targeting property. The specific method is as follows: EGFR-positive tumor cells are subcutaneously inoculated in nude mice, then DiR-labeled control exosomes and engineered exosomes modified by nanobody are injected into the tail vein, and then in vivo imaging analysis and ex vivo fluorescence imaging analysis of major organs are performed.

[0104] The specific process is as follows:

[0105] About 5-week-old nude mice are subcutaneously inoculated with tumor cells, and each mouse is injected with 3x10 6 HCT116 cells in the armpit, and when the tumor volume is about 100mm 3 , 5x10 11 DiR-labeled exosomes of each group are injected into the tail vein, and after feeding for 24h, imaging is performed using a small animal in vivo imaging system. The results are shown in Figure 10 , the enrichment of engineered exosomes modified by nanobody in tumor tissue is significantly higher than that of control exosomes, reaching 15 times; the fluorescence of engineered exosomes in the liver is significantly lower than that of control exosomes, reflecting the in vivo anti-macrophage phagocytosis ability.

[0106] Example 9: In vivo anti-tumor activity analysis of engineered exosomes modified by nanobody

[0107] This example is an in vivo anti-tumor activity analysis of the engineered exosomes obtained in Example 3. The specific method is as follows:

[0108] EGFR-positive HCT116 is subcutaneously inoculated in the armpit of nude mice, each with 3x10 6 , and when the tumor volume is about 50mm 3At approximately 10:00 AM, exosomes from each group were injected via the tail vein, once every 2 days for a total of 5 injections. Tumor size and mouse weight were measured at days 0, 3, 6, 9, 12, and 15. Results showed that after treatment with engineered exosomes modified with nanobodies, tumor volume decreased by 74%. Figure 11 A) The mouse weight did not change significantly. Figure 11 C); Fifteen days after treatment, the mice were euthanized and dissected to weigh the tumors. It was found that the tumor weight was reduced by 54% after treatment with engineered exosomes modified with nanobodies. Figure 11 B). This indicates that the engineered exosomes modified with nanobodies possess excellent in vivo antitumor activity and demonstrate therapeutic safety. Figure 11 ).

[0109] To better illustrate the universality of this invention and to enable those skilled in the art to further understand it, this invention replaces the tumor-targeting group with EGFR-targeting peptide-engineered exosomes, followed by analysis of related antitumor activity. Additionally, naked cells were engineered using the fusion protein expression plasmid from Example 1 above, and exosomes were then collected. Chemotherapy drugs were then loaded onto the exosomes, and the antitumor activity of the loaded cells was analyzed. Therefore, the following two examples further illustrate this.

[0110] Example 10: Analysis of the antitumor activity of engineered exosomes modified with EGFR-targeting peptides

[0111] This embodiment analyzes the antitumor activity of engineered exosomes modified with EGFR-targeting peptides. The specific method is as follows:

[0112] First, the EGFR-targeting nanobody sequence in Example 1 was replaced with an EGFR-targeting peptide sequence. A fusion protein expression plasmid was constructed using the same method. This plasmid was then transferred into a HEK293T cell line stably expressing miR204-5p. Positive cells were sorted, and exosomes were collected. The collected exosomes were then co-cultured with EGFR-positive HCT116 cells. The in vitro antitumor activity of the EGFR-targeting peptide-modified engineered exosomes was measured using CCK8 assay. Figure 12 A) The results showed that the engineered exosomes modified with EGFR-targeting peptides had strong antitumor activity, and the in vivo antitumor activity further confirmed the above results, with a tumor inhibition rate of 72%. Figure 12 (B and C).

[0113] Example 11: Analysis of the antitumor activity of exosomes generated from naked cells engineered with nanobody fusion protein expression plasmids

[0114] This embodiment analyzes the antitumor activity of exosomal cells generated from naked cells engineered with nanobody fusion protein expression plasmids. The specific method is as follows:

[0115] The fusion protein expression plasmid in Example 1 is used to engineer naked HEK293T cells, and then exosomes are collected according to the above method. Since the exosomes produced by naked HEK293T cells do not contain anti-tumor molecules, the engineered exosomes are loaded with the chemotherapeutic drug DOX by room temperature co-incubation for 30 min.

[0116] By Figure 13 The results show that CCK8 shows that the exosome drug prepared in this example has good tumor killing effect at the cell level in vitro, which is about 7 times higher than the control group Figure 13 A), the exosome drug delivery system obtained by this technology also has excellent anti-tumor activity in vivo, significantly increasing the targeted delivery effect of tumor chemotherapeutics, which is 67.5% higher than the control group Figure 13 B and C), further expanding the application range of the present application.

[0117] The biggest advantage of the present application is easy industrialization, so the present application also carries out a workshop pilot test in a 10L cell reactor, which is specifically described in the following examples.

[0118] Example 12: Verification of collecting exosomes in a 10L cell reactor

[0119] This example simulates the industrial production mode, and the culture system is scaled up for production. The verification of collecting exosomes in a 10L cell reactor is carried out.

[0120] The specific method is as follows:

[0121] The nanobody-modified engineered cells obtained in Example 2 are inoculated into a 10L cell reactor, which contains microcarriers cytodex3, so that the cells grow on the microcarriers. When the cell confluence is about 70%-80%, the engineered cells are cultured using a culture medium containing 2%-5% exosome-free fetal bovine serum. After 48h of culture, the culture medium is collected and subjected to ultracentrifugation for exosome separation and purification to prepare tumor-targeted drug-loaded exosomes.

[0122] As Figure 14 A shows, the yield of exosomes obtained by the 10L cell reactor is as high as 25.2ug, which is 5 times higher than the yield of exosomes obtained by the culture dish, greatly reducing the production cost. As Figure 14 B shows, the exosomes harvested by this method do not have significant changes in anti-tumor activity.

[0123] The application discloses an exosome drug delivery system with anti-phagocytosis and tumor targeting ability as well as a preparation and application thereof. A tumor targeting group is flexibly connected to the outside of the cell membrane of a transmembrane protein with anti-phagocytosis ability through a linker of a fusion protein. A fusion gene sequence is designed and inserted into an expression vector to form a fusion expression vector, and then integrated into the chromosome of a cell to be modified to obtain an engineered cell stably expressing the fusion protein, which is used for secreting engineered exosomes. The cell to be modified includes a naked cell, an engineered cell stably expressing a therapeutic nucleic acid molecule or protein. The exosomes produced by the former cell can further package the therapeutic molecules to treat tumors, and the exosomes produced by the latter cell can be directly used for treating tumors after being separated, purified and collected. The half-life of the engineered exosomes produced by the application is significantly prolonged in the body, the treatment effect is improved, and the exosomes show strong tumor targeting and anti-tumor effects in vivo and in vitro.

[0124] The above only describes the preferred embodiments of the application, and it should be pointed out that those skilled in the art can make several improvements and refinements without departing from the principles of the application, and these improvements and refinements should also be considered as the protection scope of the application.

Claims

1. An engineered exosome, characterized in that: The fusion protein is formed by the extracellular side of the anti-EGFR nanobody 7D12 connected to the transmembrane protein CD47 with anti-phagocytic ability by a (GGGGS)2 flexible linker, and is stably expressed in the engineered cells by a chromosomally integrated fusion expression vector, and the engineered exosomes are secreted by the engineered cells.

2. The engineered exosomes of claim 1, wherein the engineered cells are HEK293T cells stably expressing miR204-5p.

3. The engineered exosomes of claim 1 or 2, wherein the fusion expression vector is a lentiviral expression vector.

4. A method for preparing the engineered exosomes of any one of claims 1 to 3, comprising: S1: constructing a fusion gene sequence encoding the fusion protein, connecting 7D12 to the extracellular side of CD47 by a (GGGGS)2 flexible linker; S2: inserting the fusion gene sequence into an expression vector to form a fusion expression vector; S3: integrating the fusion expression vector into the chromosome of a tool cell to obtain engineered cells stably expressing the fusion protein; S4: collecting the engineered exosomes secreted by the engineered cells.

5. A method of industrial production of the engineered exosome of any one of claims 1 to 3, characterized in that: The positive engineered cells expressing the fusion protein are adherently cultured in a cell reactor containing Cytodex3 microcarriers, and when the cell confluence is 70-80%, the medium containing 2-5% exosome-free fetal bovine serum is replaced and the culture is continued for 24h or 48h, impurities are removed by centrifugation at 10000g, and exosomes are collected by ultracentrifugation at 120000g.

6. A drug-loaded engineered exosome, obtained by loading doxorubicin (DOX) into the engineered exosome of claim 1 via electroporation, sonication or liposome fusion. The engineered exosomes of claim 1 are secreted by naked engineered cells that do not express anti-tumor effector molecules.

7. The method of claim 4, wherein the positive cells expressing the fusion protein are obtained by puromycin screening and / or flow cytometer sorting.

8. The drug-loaded engineered exosomes of claim 6, which are suitable for intravenous injection administration.

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