CN116036299B - Interval Pi Sute specific exosome carrier, medicine-carrying exosome containing same, preparation method and medical application of medicine-carrying exosome - Google Patents

Abstract

The present invention provides an exosome vector comprising a chimeric antigen receptor specific for Mesothelin (MSLN), wherein the chimeric antigen receptor comprises a transmembrane portion and an anti-MSLN single chain antibody displayed on the surface of an exosome membrane, the transmembrane portion being human CD63. The invention also provides a medicine carrying exosome containing the exosome carrier, a medicine composition and medical application thereof.

Description

Interval Pi Sute specific exosome carrier, medicine-carrying exosome containing same, preparation method and medical application of medicine-carrying exosome

Technical Field

The invention relates to the field of biological medicine, in particular to an mesothelin specific exosome carrier, a medicine-carrying exosome containing the mesothelin specific exosome carrier, a preparation method and medical application of the medicine-carrying exosome.

Background

In order to solve the characteristic of low utilization rate of the conventional drug administration, in recent years, the nanotechnology has been used for containing the research of the drug, which is quite hot, and has been better in the aspects of drug slow release and delivery. However, since the nanoparticles are exogenous substances and have immunogenicity, phagocytosis of macrophages in the body cannot be avoided, so that the stability of the circulation in the body is poor, the utilization rate is low, and even the rejection injury of the body can be caused, so that the final treatment effect is affected. Exosomes were originally found in sheep red blood cell supernatants cultured in vitro as small vesicles of uniform size, between 30 and 150nm in diameter, actively secreted by cells. Exosomes can carry a variety of proteins, mRNA, miRNA, involved in signal transduction and communication between cells. All human cells can secrete, can carry different proteins and nucleic acids, and play a role in intercellular communication. The exosomes are used as self cell secretions, have good biological stability and strong permeability, and are ideal drug delivery carriers. Therefore, the exosome can be used as a drug carrying system, and the therapeutic effect can be exerted to the greatest extent.

In fact, exosomes have been increasingly studied as carriers. Dai, S. reports that the exosome secreted by human colon cancer cells LS-174T contains a large amount of IL-18 in vivo by genetic engineering, which can effectively promote T cell proliferation and IFN-gamma release, thereby achieving the purpose of killing and inhibiting tumor cells. When miR-122, miR-134, miR146b or anti-miR-9 are highly expressed in exosomes, the anti-tumor effect can be achieved in the aspects of inhibiting the growth of tumor cells, promoting the apoptosis of the tumor cells or enhancing the drug sensitivity and the like. In 2015 Yang, T reports that after the exosomes secreted by brain endothelial cells are used for wrapping chemotherapeutic drugs doxorubicin and taxol, the drugs can pass through the blood brain barrier, the discovery has great significance for exosome-based treatment research, and the exosomes are suggested to have great application potential as drug transport carriers. In 2016, kim, M.S et al published nanomedicine shows that the application of exosomes secreted by the macrophage cell line Raw264.7 to encapsulate paclitaxel can effectively kill tumor cells with multidrug resistance, and this study demonstrates the high efficacy of exosomes as transport vehicles for chemotherapeutic drugs. In addition, toffoli studies demonstrate that doxorubicin cardiotoxicity can be effectively reduced when the doxorubicin is encapsulated with exosomes. However, the exosomes are used as drug delivery carriers simply, and have no tumor cell targeting effect, so that enrichment of drugs at tumor sites cannot be realized.

Ovarian cancer is the most common malignant tumor of gynecological cancers, and 80% of ovarian cancer patients already belong to advanced stages in diagnosis and the survival rate of advanced ovarian cancer is less than 30% in 5 years because of hidden onset, atypical clinical symptoms and signs and lack of effective early diagnosis methods. At present, the main treatment strategy of ovarian cancer is mainly combined with tumor reduction and chemotherapy, and is mainly combined with taxol and carboplatin for chemotherapy, but traditional chemotherapy drugs cannot directly target tumor cells and can only kill cells without selectivity, and normal cells can be killed while the tumor cells are killed, so that the immunity of the body is low. And most patients treated by chemotherapy have gastrointestinal reaction symptoms such as nausea, vomiting, constipation, diarrhea and the like. Therefore, it is important to find a treatment method capable of precisely killing tumor cells and reducing gastrointestinal reaction of patients and improving life quality of the patients.

Mesothelin (MSLN) is a cell surface expressed glycoprotein found when monoclonal antibody K1 (MabK) was isolated, which is expressed only in mesothelial cells of the body cavity, and which is highly expressed in malignant mesothelioma, ovarian malignancy, pancreatic cancer. The MSLN gene is one of the genes whose up-regulation of expression is most pronounced in ovarian cancer patients. Current studies show that MSLN can be used as a tumor marker for ovarian cancer and a prognostic predictor thereof.

Osthole, also known as methoxy Parsley phenol or Parsley phenol methyl ether, is a pure natural coumarin compound extracted and isolated from Umbelliferae plants. Studies show that osthole has various pharmacological activities such as anti-tumor, anti-osteoporosis, anti-hepatitis, anti-allergic reaction, platelet aggregation inhibition, antiviral, anti-mutagenesis and the like.

Disclosure of Invention

The present disclosure provides an exosome vector comprising an Mesothelin (MSLN) -specific chimeric antigen receptor, wherein the chimeric antigen receptor comprises a transmembrane portion and an anti-MSLN single chain antibody displayed on the surface of an exosome membrane, the transmembrane portion being human CD63.

In some embodiments, the amino acid sequence of human CD63 is as set forth in SEQ ID NO: 1.

MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEFNNNFRQQMENYPKNNHT

In some embodiments, the anti-MSLN single chain antibody has an amino acid sequence set forth in SEQ ID NO: 2.

QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGGGGSGGGGSSGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTYGAGTKLEIK

In some embodiments, the chimeric antigen receptor comprises a signal peptide and/or a linking peptide that links the anti-MSLN single chain antibody and the transmembrane portion.

In some embodiments, the chimeric antigen receptor has an amino acid sequence as set forth in SEQ ID NO:3.

MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEFNNNFRQQMENYPKNNHTQVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGGGGSGGGGSSGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTYGAGTKLEIK

In some embodiments, the chimeric antigen receptor consists of SEQ ID NO:4, and the nucleic acid sequence shown in FIG. 4.

atggcggtggaaggaggaatgaaatgtgtgaagttcttgctctacgtcctcctgctggccttttgcgcctgtgcagtgggactgattgccgtgggtgtcggggcacagcttgtcctgagtcagaccataatccagggggctacccctggctctctgttgccagtggtcatcatcgcagtgggtgtcttcctcttcctggtggcttttgtgggctgctgcggggcctgcaaggagaactattgtcttatgatcacgtttgccatctttctgtctcttatcatgttggtggaggtggccgcagccattgctggctatgtgtttagagataaggtgatgtcagagtttaataacaacttccggcagcagatggagaattacccgaaaaacaaccacactgccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgggatcccaggtacaactgcagcagtctgggcctgagctggagaagcctggcgcttcagtgaagatatcctgcaaggcttctggttactcattcactggctacaccatgaactgggtgaagcagagccatggaaagagccttgagtggattggacttattactccttacaatggtgcttctagctacaaccagaagttcaggggcaaggccacattaactgtagacaagtcatccagcacagcctacatggacctcctcagtctgacatctgaagactctgcagtctatttctgtgcaagggggggttacgacgggaggggttttgactactggggccaagggaccacggtcaccgtctcctcaggtggaggcggttcaggcggcggtggctctagcggtggcggatcggacatcgagctcactcagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagtgccagctcaagtgtaagttacatgcactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaactggcttctggagtcccaggtcgcttcagtggcagtgggtctggaaactcttactctctcacaatcagcagcgtggaggctgaagatgat

The exosome carrier disclosed by the invention is meta Pi Sute specific, has a targeting effect on ovarian cancer cells, and can be used for loading ovarian cancer therapeutic agents, such as bufogenin, osthole and the like, so that enrichment and accurate treatment of the drugs on ovarian cancer focus are realized.

The present disclosure also provides a drug-loaded exosome comprising the aforementioned exosome carrier and at least one drug loaded within the exosome carrier.

In some embodiments, the drug is an anti-tumor agent.

In some embodiments, the drug is an ovarian cancer therapeutic. Examples of ovarian cancer therapeutic agents include, but are not limited to: doxorubicin, paclitaxel, docetaxel, cisplatin, carboplatin, oxaliplatin, cyclophosphamide, ifosfamide, gemcitabine, olapari, lu Kapa, nilaparib, fluxapari, pamphle Mi Pali, valicari, tazopari, pazopani, ceridenib, nilamide, sunitinib, cabitinib, sorafenib, erlotinib, bupropion, temsirolimus, piriferin.

In some embodiments, the ovarian cancer therapeutic agent is a traditional Chinese medicine extract, such as bufogenin, osthole, and the like.

The present disclosure also provides an doxorubicin-loaded meta Pi Sute-specific exosome, which meta Pi Sute-specific exosome is as defined above for the exosome vector.

The present disclosure also provides a cinobufagin-loaded meta Pi Sute specific exosome, as defined above for the exosome vector.

The present disclosure also provides a pharmaceutical composition comprising the aforementioned drug-loaded exosomes, and a pharmaceutically acceptable carrier or diluent. Examples of carriers or diluents include, but are not limited to, water, saline, ringer's solution, dextrose solution, and 5% human serum albumin. The use of such media and compounds for pharmaceutically active substances is well known in the art. Unless any conventional medium or compound is incompatible with the exosomes described herein, its use in the composition is contemplated. The pharmaceutical compositions of the present disclosure may further comprise a second therapeutic agent. Generally, the pharmaceutical compositions are formulated to be compatible with their intended route of administration.

The present disclosure also provides the use of the above exosome carrier for loading or delivering a drug. The definition of drug is as described above.

The present disclosure also provides the use of the above exosome carrier, drug-loaded exosome or pharmaceutical composition in the manufacture of a medicament for treating a disease.

In some embodiments, the disease is a tumor or cancer. In some embodiments, the disease is a solid tumor.

In some embodiments, the tumor or cancer includes, but is not limited to: ovarian cancer, pancreatic cancer, lung cancer, mesothelioma, non-small cell lung cancer, thymoma, gastric cancer, cholangiocarcinoma, adenocarcinoma, fallopian tube cancer, peritoneal malignancy, pancreatic acinar cancer, ovarian epithelial cancer.

In some embodiments, the disease is ovarian cancer.

The present disclosure also provides a kit comprising any one of the above exosome carriers, drug-loaded exosomes and pharmaceutical compositions.

The present disclosure also provides a method of preparing the above exosome vector, comprising:

(1) Transfecting a producer cell with an expression plasmid comprising a nucleic acid encoding a Mesothelin (MSLN) -specific chimeric antigen receptor;

(2) After culturing, exosomes were collected.

The present disclosure also provides a method of preparing the drug-loaded exosomes described above, comprising:

(1) Transfecting a producer cell with an expression plasmid comprising a nucleic acid encoding a Mesothelin (MSLN) -specific chimeric antigen receptor;

(2) Separating and enriching exosomes after culturing;

(3) And (3) carrying out drug loading on the exosomes collected in the step (2) to obtain the drug-loaded exosomes.

In some embodiments, the producer cell comprises any type of cell capable of producing exosomes under suitable conditions (e.g., in suspension culture or pen-up culture or any other type of culture system).

In some embodiments, the producer cells may be selected from a wide range of cells and cell lines, such as stromal cells, fibroblasts, amniotic cells, myelosuppressive cells, adipocytes, endothelial cells, fibroblasts, human Embryonic Kidney (HEK) cells, chinese Hamster Ovary (CHO) cells, erythrocytes, erythroid progenitor cells, chondrocytes, stem cells, immune cells.

In some embodiments, the producer cell is a stem cell, such as an embryonic stem cell, a somatic stem cell, a Mesenchymal Stem Cell (MSC), an Induced Pluripotent Stem Cell (iPSC), and other stem cells derived by any method. For the patient to be treated, the cells may be allogeneic, autologous, or even xenogeneic in nature, i.e., the source cells may be from the patient himself or from unrelated, matched or unmatched donors.

In some embodiments, the producer cell is an immune cell, such as an antigen presenting cell, T cell, B cell, natural killer cell (NK cell), macrophage, M2 polarized macrophage, monocyte, dendritic cell, T helper cell, or regulatory T cell (Treg cell). In some embodiments, the producer cell is not an antigen presenting cell (e.g., a dendritic cell, a macrophage, a B cell, a mast cell, a neutrophil, a Kupffer-Browicz cell, or a cell derived from any such cell).

In some embodiments, the producer cell is not an immune cell.

In some embodiments, drug loading of the exosome vector is performed by: direct co-incubation, sonication, electroporation, freeze thawing, extrusion or permeabilization.

Exosomes can be isolated and enriched from the production cells by methods known in the art. In some embodiments, exosomes are released from the production cells into the cell culture medium. All known ways of isolating exosomes are contemplated as being suitable for use herein. For example, the physical properties of exosomes may be employed to separate them from a medium or other source material, including charge-based separations (e.g., electrophoretic separations), size-based separations (e.g., filtration, molecular sieves, etc.), density-based separations (e.g., conventional centrifugation or gradient centrifugation), svedberg constant-based separations (e.g., sedimentation with or without external forces, etc.). Alternatively or in addition, the separation may be based on one or more biological properties and include methods that may employ surface markers (e.g., for precipitation, reversible binding to a solid phase, FACS separation, specific ligand binding, non-specific ligand binding, affinity purification, etc.). Separation and enrichment can be performed in a general and non-selective manner, which typically includes continuous centrifugation. Alternatively, the isolation and enrichment may be performed in a more specific and selective manner, such as using exosomes or producing cell-specific surface markers. For example, specific surface markers can be used for immunoprecipitation, FACS sorting, affinity purification, and magnetic separation of bead-bound ligands. In some embodiments, size exclusion chromatography may be used to isolate exosomes. Size exclusion chromatography techniques are known in the art. Exemplary, non-limiting techniques are provided herein. In some aspects, the void volume fraction is isolated and comprises the target exosomes. Furthermore, in some aspects, the exosomes may be further isolated after chromatographic separation by centrifugation techniques (on one or more chromatographic fractions) as is well known in the art. In some aspects, for example, density gradient centrifugation may be utilized to further isolate extracellular vesicles. In certain aspects, it may be desirable to further isolate production cell-derived exosomes from exosomes of other origins. For example, production cell-derived exosomes can be separated from non-production cell-derived exosomes by immunoadsorption capture using antigen-antibody specific for the production cells. In some embodiments, the separation of exosomes may involve a combination of methods including, but not limited to, differential centrifugation, size-based membrane filtration, immunoprecipitation, FACS sorting, and magnetic separation.

In some embodiments, drug loading is performed by direct co-incubation of the exosomes collected in step (2) with the drug.

The interceptor Pi Sute foreign exosome carrier and the drug-loaded exosome have better targeting, lower toxic and side effects and more efficient anti-tumor effect, and are specifically:

(1) The exosome carrier of the present disclosure targets Mesothelin (MSLN), can specifically identify and bind MSLN on the surface of MSLN positive tumor cells, and then swallow the tumor cells, so that the drugs (such as bufogenin, osthole, etc.) are enriched in the tumor cells in a large amount, the effective utilization rate of the drugs can be greatly improved, and the effect of increasing the anti-tumor effect of the drugs is achieved;

(2) The side effects of serious injury of normal tissues, reduced immunity of patients, serious gastrointestinal reaction and the like caused by the nonspecific killing effect of the traditional chemotherapeutic drugs are obviously improved;

(3) The MSLN gene is one of the genes with the most significant up-regulation of expression in ovarian cancer patients, is an excellent target for ovarian cancer treatment, and therefore the MSLN specific exosome vector disclosed by the disclosure is particularly suitable for being used as a drug carrier in the treatment of ovarian cancer; for example, after carrying traditional Chinese medicine extracts (such as bufogenin, osthole and the like), the MSLN specific exosome carrier can obviously regulate the immune system of a patient, reduce intestinal tract reaction and improve the life quality of the patient while killing ovarian cancer cells; therefore, a novel strategy for treating ovarian cancer by combining traditional Chinese medicine and western medicine based on accurate intervention and system regulation is provided;

(4) The drug delivered by the exosome carrier has good slow release and long-acting effects, the membrane structure of the exosome has a protective effect on drug storage, and the targeted exosome loaded with the drug has better immunoregulation effect and better intestinal microecological regulation effect.

Drawings

The above and other aspects, features and advantages of the present invention will become apparent from the following detailed description of embodiments thereof, which is to be read in connection with the accompanying drawings.

FIG. 1 shows the targeting efficiency of anti-MSLN exosomes detected by fluorescence microscopy;

FIG. 2 shows the results of flow cytometry detection of anti-MSLN exosomes from HEK297T cells (A) and DC cell source (B);

FIG. 3 shows the results of the on-body drug efficacy of osthole-loaded anti-MSLN exosomes;

FIG. 4 shows the results of osthole-loaded anti-MSLN exosomes treatment SKOV3 cell proliferation assay-CCK 8;

FIG. 5 shows the microscopic examination of the anti-MSLN exosome treatment SKOV3 cell proliferation assay loaded with osthole.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The term "chimeric antigen receptor" or "CAR" as used herein refers to an engineered exosome that is engineered to express and specifically bind an antigen on the exosome. The CAR may also include an extracellular domain transmembrane domain and optionally an intracellular activation domain, the extracellular domain including a tumor-associated antigen binding region. In some aspects, the CAR comprises a fusion single chain variable fragment (scFv) -derived monoclonal antibody fused to an exosome-specific CD63 transmembrane domain. The specificity of CAR design may be derived from the ligand (e.g., peptide) of the receptor. In some embodiments, the CAR can target MSLN-positive cancer, for example, by redirecting the specificity of the exosomes expressing the CAR specific for the tumor-associated antigen.

The term "transfection" as used herein refers to the following process: exogenous nucleic acid is transferred or introduced into a host cell by this process. A "transfected" cell is a cell that has been transfected with an exogenous nucleic acid. Cells include primary subject cells and their progeny.

The process according to the invention is described in more detail below by way of a comparison of specific examples, in order to facilitate the understanding of the person skilled in the art. Unless otherwise indicated, reagents and equipment used in the present invention are commercially available products in the art, and experimental methods employed in the present invention are conventional in the art.

EXAMPLE 1 anti preparation and identification of MSLN-exosome vector

The anti-MSLN-exosome vector was prepared as follows:

1. Cell preparation: HEK293T cells were plated in six well plates of 8.5X10 ⁵ cells per well, cultured in a 5% CO ₂ cell incubator at 37℃and plasmid transfected the next day;

2. plasmid preparation: plasmid final concentration (MSLN-scFv plasmid: 210 ng/. Mu.l; empty vector: 206 ng/. Mu.l); the vector is pIRES2-EGFP, and the target gene sequence is as follows:

atggcggtggaaggaggaatgaaatgtgtgaagttcttgctctacgtcctcctgctggccttttgcgcctgtgcagtgggactgattgccgtgggtgtcggggcacagcttgtcctgagtcagaccataatccagggggctacccctggctctctgttgccagtggtcatcatcgcagtgggtgtcttcctcttcctggtggcttttgtgggctgctgcggggcctgcaaggagaactattgtcttatgatcacgtttgccatctttctgtctcttatcatgttggtggaggtggccgcagccattgctggctatgtgtttagagataaggtgatgtcagagtttaataacaacttccggcagcagatggagaattacccgaaaaacaaccacactgccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgggatcccaggtacaactgcagcagtctgggcctgagctggagaagcctggcgcttcagtgaagatatcctgcaaggcttctggttactcattcactggctacaccatgaactgggtgaagcagagccatggaaagagccttgagtggattggacttattactccttacaatggtgcttctagctacaaccagaagttcaggggcaaggccacattaactgtagacaagtcatccagcacagcctacatggacctcctcagtctgacatctgaagactctgcagtctatttctgtgcaagggggggttacgacgggaggggttttgactactggggccaagggaccacggtcaccgtctcctcaggtggaggcggttcaggcggcggtggctctagcggtggcggatcggacatcgagctcactcagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagtgccagctcaagtgtaagttacatgcactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaactggcttctggagtcccaggtcgcttcagtggcagtgggtctggaaactcttactctctcacaatcagcagcgtggaggctgaagatgat(SEQ ID NO:4)

3. Plasmid transfection:

1) Discarding the culture medium in the six-well plate, replacing the culture medium with Opti-Mem ^TM culture medium, 1.5ml of culture medium in each well, and then placing the culture medium in a 5% CO ₂ cell incubator at 37 ℃;

2) Preparing a solution B: MSLN-scFv plasmid (10. Mu.l Lipo 2000+250ml Opti-Mem ^TM) 3; empty vector (10 μl Lipo 2000+250ml Opti-Mem ^TM) 3 x 3

Gently and slowly blowing and sucking the mixture by using a gun head, and incubating the mixture for 5 minutes at room temperature;

3) Preparing solution A: MSLN-scFv plasmid (10. Mu.l MSLN-scFv plasmid+250 ml Opti-Mem ^TM) 3; empty vector (10 μl empty vector+250 ml Opti-Mem ^TM) 3 x 3

Respectively adding the incubated liquid B into the liquid A, slightly and slowly blowing and sucking the liquid A by using a gun head, and then incubating the liquid B for 20min at room temperature, wherein the mixed liquids are respectively marked as a and B;

4) Taking out a six-hole plate, adding 500 μl of the liquid a or b in the step 3), respectively, shaking the three holes slightly, and then placing the six holes in a 5% CO ₂ cell incubator at 37 ℃ for 5 hours;

5) After 5 hours, the medium in the six well plates was discarded, and replaced with DMEM (Gibco ^TM) medium containing 10% fbs, 1% streptavidin, 2ml per well;

4. the following day the medium was discarded and replaced with DMEM (Gibco ^TM) medium, 2ml per well;

5. Collecting supernatant after three days to extract exosomes;

6. Extracting exosomes: taking out a six-hole plate, collecting cell supernatants (about 5ml each), centrifuging (2000 g,4 ℃ C., 30 min), taking the supernatant (4.5 ml each), adding Total Exosome Isolation Reagent (Thermofisher/4478359) of 1/2 volume (2.25 ml) of the supernatant, vortex mixing, taking out overnight at4 ℃ C., shaking mixing, subpackaging into 1.5ml EP tubes, centrifuging (10000 g,4 ℃ C., 1 h), thoroughly discarding the supernatant, and finally re-suspending with 100 μl PBS to make the final concentration of MSLN-scFv exosomes 75.9 ng/. Mu.l, and the final concentration of empty exosomes 78.9 ng/. Mu.l;

7. Protein assay (BCA): diluting the exosome suspension obtained in step 610 times (MSLN-scFv exosome: 7.59 ng/. Mu.l, no-load exosome: 7.89 ng/. Mu.l);

8. Single chain antibody identification

1) Mu.l of DiL fluorescent dye (1 mM) was added to each of 100. Mu.l of MSLN-scFv exosomes and 100. Mu.l of empty vector exosomes, and the mixture was air-sucked and mixed;

2) Incubating in a water bath (37 ℃ C., shaking, light shielding) for 1 hour;

3) Volume was increased to 1ml with PBS;

4) Treating the liquid of step 3) as per desalting column instructions (thermofisher 89892);

5) Filtration with a filter (0.45 μm): operating with 1ml syringe, approximately 700 μl each remains;

6) Taking six-hole plates with 3X 10 ⁵ cells per hole and SKOV3 cells with cell density of about 80% on the previous day, changing the culture medium into 1ml per hole, adding 100 μl of the solution obtained in the step 5) into each hole, and mixing uniformly in three groups, namely PBS group, MSLN-scFv group and empty carrier group, wherein two holes are repeated in each group;

7) Placing the mixture into a cell incubator with 5% CO ₂ at 37 ℃ for incubation overnight;

8) Taking out the sample the next day, discarding the supernatant, and washing the sample three times with 1ml PBS;

9) Each well was further supplemented with 1mL Gibco RPMI 1640 medium containing 10% FBS, 1% streptavidin);

the results are shown in FIG. 1 when viewed under an inverted fluorescence microscope. anti-MSLN exosomes and non-specific exosomes were labeled with membrane dye and incubated with msln+ cell lines, and fluorescence microscopy examined the targeting efficiency, confirming that the targeting efficiency was due to specific binding of MSLN-scFv to MSLN antigen.

The same procedure as described above was also used to prepare Dendritic Cell (DC) -derived MSLN-specific exosome vectors.

Incubating the prepared two types of exosome carriers with corresponding exosome specific magnetic beads (Thermo Fisher: 106-06D), then adding MSLN antigen ((Sino Biological 13128-H08H-50)) for incubation for a period of time, then adding anti-MSLN-PE for incubation, and then detecting by using a flow cytometer to verify the specificity of the single-chain antibody on the exosome. Flow cytometry examination results showed that the above preparation method can effectively express single-chain antibodies on the surface of the exosomes as shown in a and B in fig. 2.

EXAMPLE 2 preparation of osthole-loaded anti-MSLN-exosomes

1) Drug loading: the mass of the anti-MSLN-exosome carrier and the non-specific exosome carrier is 7.5 mug (100 mug) respectively, 195.2 mug (5 mug) of osthole are blown and sucked evenly; the medicine carrying mode is as follows: co-incubating in a water bath at 37 ℃ for 4 hours; and (3) dialysis: sucking the liquid with the medicine, injecting into a dialysis card containing 1×PBS, and dialyzing at 4deg.C overnight; aspirate the dialysis card for approximately 800 μl each; filtered through a 0.45 μm filter, leaving approximately 650. Mu.l each.

2) HPLC (high Performance liquid chromatography) for measuring drug loading rate

Liquid phase conditions: chromatographic column XdbC, mobile phase acetonitrile: water = 7:3, detecting wavelength 303nm, flow rate 1ml/min, sample injection quantity 10 μl, and stopping time 5min;

Preparing a standard substance: osthole stock was 160mM in concentration and diluted with PBS to five concentrations of 0.390864 μg/ml,0.781728 μg/ml,1.95432 μg/ml,3.90864 μg/ml,7.81728 μg/ml, 1ml each, labeled a, b, c, d, e;

Sample preparation: the osthole-loaded exosomes after overnight dialysis at 4℃were aspirated from the dialysis card using a 5ml syringe, incubated for 2 hours (co-2), 4 hours (co-4) after total incubation, 2 hours (e-2) after electroporation, 4 hours (e-4) after electroporation, each liquid volume was 4.5ml, 1ml, 500 μl each was transferred to a new 1.5ml EP tube labeled co-2, co-4, e-2, e-4, centrifugation (4 ℃,7000 rpm) for 5min, 200 μl each was transferred to a new 1.5ml EP tube under four drug loading conditions;

Sample injection sequence: placing 50 μl of each sample into sample injection bottles, wherein the sample injection sequences are PBS, a, b, c, d, e, co-2, co-4, e-2 and e-4;

The osthole peak time is about 3 min.

The results of the drug loading rate measurement are shown in Table 1 and FIG. 3.

Table 1: drug loading efficacy under each drug loading condition

EXAMPLE 3 proliferation experiments of osthole-loaded anti-MSLN exosomes on SKOV3 cells

1. Cell preparation: SKOV3 was plated in six well plates of about 3 x10 ⁵ cells per well, cultured in complete medium (1640 medium containing 10% fbs and 1% penicillin), 2ml per well, and dosed the next day;

2. Adding the medicine: taking out a six-hole plate, discarding the supernatant, washing 3 times with 1ml of PBS, adding 1ml of complete culture medium into each hole, adding 300 μl of PBS, 300 μl of osthole-loaded anti-MSLN exosomes and 300 μl of osthole-loaded non-specific exosomes, repeating two holes, shaking lightly, and incubating in an incubator for 48 hours and 72 hours;

3. Adding CCK: after 48 hours, the supernatant was discarded, washed 2 times with 1ml PBS, then 1ml complete medium was added, then 100. Mu.l CCK solution was added to each well, mixed gently and placed into incubator for 2 hours;

4. After 2 hours incubation in incubator, the supernatant was transferred to 96-well plate, and 6-well plate was transferred to 6 wells of 96-well plate, 100 μl of each well, then the remaining supernatant was discarded, washed 2 times with 1ml PBS, and then 1ml complete medium was added to each well, and culturing was continued for 24 hours;

5. and (3) measuring by using an enzyme-labeled instrument: measuring the 96-well plate by using an enzyme-labeled instrument, wherein the single wavelength is 450nm;

6. Taking out the six-hole plate in the step 5, adding 100 mu l of CCK solution into each hole, mixing evenly with light shaking, and placing into an incubator for incubation for 2 hours;

7. Repeating the steps 4 and 5.

Detection result: the results of the 48-hour and 72-hour CCK assays are shown in FIG. 4, and the results of the 24-hour, 48-hour, and 72-hour fluorescence microscopy of the cell survival status are shown in FIG. 5, which both show that osthole-loaded anti-MSLN exosomes are toxic to SKOV3 cells (MSLN+) and that this effect increases with increasing incubation time. These results indicate that the osthole-loaded anti-MSLN exosomes should have low side effects (smaller doses of the same toxicity), slow release, long duration of action and progressively increased toxicity.

Claims (6)

1. A drug-loaded exosome comprising an exosome carrier and at least one drug loaded within the exosome carrier, wherein:

The exosome vector expresses a chimeric antigen receptor specific for Mesothelin (MSLN), wherein the chimeric antigen receptor consists of a transmembrane part and an anti-MSLN single-chain antibody displayed on the surface of an exosome membrane, the transmembrane part is human CD63, and the amino acid sequence of the human CD63 is shown as SEQ ID NO:1, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO:3 is shown in the figure; the exosome vector is produced by a cell selected from the group consisting of: stromal cells, fibroblasts, amniotic cells, myelosuppressive cells, adipocytes, endothelial cells, human Embryonic Kidney (HEK) cells, chinese Hamster Ovary (CHO) cells, erythrocytes, erythroid progenitor cells, chondrocytes, or stem cells;

The medicine is osthole.

2. A drug-loaded exosome comprising an exosome carrier and at least one drug loaded within the exosome carrier, wherein:

The exosome vector expresses a chimeric antigen receptor specific for Mesothelin (MSLN), wherein the chimeric antigen receptor consists of a transmembrane portion, an anti-MSLN single-chain antibody displayed on the surface of the exosome membrane, and a connecting peptide connecting the transmembrane portion and the anti-MSLN single-chain antibody, the transmembrane portion is human CD63, and the amino acid sequence of the human CD63 is shown as SEQ ID NO:1, wherein the amino acid sequence of the anti-MSLN single-chain antibody is shown as SEQ ID NO:2 is shown in the figure; the exosome vector is produced by a cell selected from the group consisting of: stromal cells, fibroblasts, amniotic cells, myelosuppressive cells, adipocytes, endothelial cells, human Embryonic Kidney (HEK) cells, chinese Hamster Ovary (CHO) cells, erythrocytes, erythroid progenitor cells, chondrocytes, or stem cells;

The medicine is osthole.

3. A pharmaceutical composition comprising the drug-loaded exosome according to claim 1 or 2, and a pharmaceutically acceptable carrier or diluent.

4. Use of a drug-loaded exosome according to claim 1 or 2 or a pharmaceutical composition according to claim 3 in the manufacture of a medicament for the treatment of a disease, said disease being ovarian cancer.

5. A method of preparing a drug-loaded exosome according to claim 1 or 2, comprising the steps of:

(1) Transfecting a producer cell with an expression plasmid comprising a nucleic acid encoding a Mesothelin (MSLN) -specific chimeric antigen receptor;

(2) Culturing the transfected production cells of step (1), and then separating and enriching exosomes;

(3) Loading osthole on the exosomes obtained in the step (2) to obtain the drug-loaded exosomes;

the production cells are selected from the group consisting of stromal cells, fibroblasts, amniotic cells, bone marrow-suppressive cells, adipocytes, endothelial cells, human Embryonic Kidney (HEK) cells, chinese Hamster Ovary (CHO) cells, erythrocytes, erythroid progenitor cells, chondrocytes, or stem cells.

6. The method of claim 5, wherein the producer cell is a HEK293T cell.