Pharmaceutical composition containing stem cell exosomes and preparation method thereof

Abstract

The present invention relates to a pharmaceutical composition containing stem cell exosomes and a preparation method thereof. After the exosomes are loaded with chemotherapeutic drugs, they can be effectively used for the treatment of ovarian cancer. When the exosomes loaded with chemotherapeutic drugs are used together with a monoclonal antibody against PI3K, the sensitivity of the chemotherapeutic drugs can be significantly improved, so that the two have a significant synergistic therapeutic advantage, and the application prospects show a good effect.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to a pharmaceutical composition comprising stem cell exosomes and a preparation method thereof. Background Art

[0002] Exosomes are extracellular vesicles (EVs) secreted by cells. They are 30-150nm in size and have a double-layer membrane structure. Exosomes carry important information such as various proteins, lipids, DNA and RNA of the mother cells and participate in the molecular transmission between cells. Exosomes are widely present in cell culture supernatants and various body fluids, including blood, lymph, saliva, urine, semen, breast milk, etc., and also in tissue samples, such as brain tissue, muscle tissue, adipose tissue, etc.

[0003] The contents of exosomes mainly depend on their cell origin. They are numerous and complex in composition, usually including some bioactive substances. It is known that exosomes from different tissues and cells contain 194 lipids, 4563 proteins, 1639 mRNAs and 764 microRNAs. Among them, the endosomal sorting transport complex and the members of the tetramolecular cross-linking body family (such as CD9, CD63, cD81, cD82, cDl51) are involved in the sorting and transport of exosome formation. In addition, studies have found that exosomes can express some MHcI molecules, HSP70, HSP90, HER2 / neu and Manl, and can also recruit a variety of cellular proteins from non-serosa, including MHc transmembrane tetraproteins, adhesion molecules and metalloproteinases. Exosomes mediate the transport of proteins, lipids and nucleic acids between cells and regulate various biological activities of receptor cells, such as affecting gene expression. Studies have found that exosomes from breast cancer can regulate the proinflammatory response of macrophages. In the immune system, on the one hand, exosomes from dendritic cells or tumor cells can induce anti-tumor activity; on the other hand, they can induce immune tolerance under physiological conditions, such as pregnancy, and immune tolerance in tumor progression. After the exosomes fuse with the target cell membrane, on the one hand, they can promote the transport of molecules and receptors on the cell surface from donor cells to recipient cells; on the other hand, the ligands expressed on the exosomes can interfere with the binding of cell surface receptors and ligands, and even reorganize the recipient cells, leading to the transformation of the recipient cells. In recent years, studies have found that exosomes play an important role in the occurrence and development of tumors, such as tumor formation, progression and metastasis, immune regulation, immune escape, angiogenesis, regulating the microenvironment of tumor metastasis, and promoting epithelial-mesenchymal transition (EMT).

[0004] Exosomes can also be used as a tool for disease treatment and drug delivery. RNA-based therapeutic drugs have great potential in treating various diseases by delivering small interfering RNA (siRNA) to silence pathological genes or by delivering exogenous mRNA to cells to express therapeutic proteins. In terms of mRNA / siRNA delivery, traditional carriers such as liposomes and nanoparticles have the advantages of non-infectivity, wide material sources, and easy mass preparation, which are irreplaceable advantages of viral vectors. However, they have low delivery efficiency, difficulty in passing through biological barriers to reach the lesion site, and are easily triggered by immune responses and are quickly cleared by the body. Unlike traditional carriers, on the one hand, exosomes contain transmembrane proteins and membrane-anchored proteins that can enhance endocytosis, thereby promoting the delivery of substances carried inside them; on the other hand, exosomes also contain some transmembrane proteins that inhibit phagocytosis and are not quickly cleared by the circulatory system, such as the exosomal protein CD47, which is an integrin-related protein widely expressed on the cell surface and can send a "don't eat me" signal to macrophages to protect cells from phagocytosis. Scholars have developed a biocompatible tumor cell-exosome sheathed PSiNPs as a drug carrier for targeted cancer chemotherapy. When tumor cells are incubated with PSiNPs (DOX@PSiNPs) loaded with doxorubicin, the tumor cells secrete exosomes loaded with DOX@PSiNPs (DOX@E-PSiNPs), which have a killing effect on a large number of cancer cells and cancer stem cells, thereby playing an anti-tumor role. Exosomes also have the ability to pass through the blood-brain barrier that many traditional drug carriers do not have. Studies have shown that exosomes produced by brain endothelial cells can be used as carriers for drugs to treat brain tumors. In the zebrafish primary brain cancer model, exosomes loaded with anticancer drugs can exert anti-tumor effects through the blood-brain barrier, significantly reducing the size of tumor lesions and reducing the fluorescence intensity and tumor growth marker content of xenograft cancer cells. Genetically modified exosomes were used to specifically deliver siRNA to prevent KRAS gene mutation, thereby alleviating the condition of mice with pancreatic cancer and improving the survival rate of mice. This confirmed that exosomes can act as a highly efficient RNA interference (RNAi) carrier, transporting siRNA and short hairpin RNA molecules that specifically target KRAS, which is more efficient than liposomes and has no obvious immune response.

[0005] Ovarian cancer is one of the common lethal gynecological malignancies and ranks fifth among the causes of cancer-related death in women. Due to the difficulty in early diagnosis, high recurrence rate and resistance to chemotherapy drugs, ovarian cancer patients have a poor prognosis, with a 5-year survival rate of only about 30%. Exosomes, as a carrier of bioactive substances, have great application prospects in the treatment of ovarian cancer. First of all, as a small vesicle outside the cell, exosomes can circulate freely in biological fluids, such as blood, urine, ascites, saliva and cerebrospinal fluid. Secondly, the membrane components of exosomes also have a property of cell membranes: optimal membrane fusion properties, and in some cases, unique cell tropism. And exosomes from ovarian cancer patients can better escape immune in polyethylene glycol liposomes in vitro. Through in vitro and in vivo experiments, nanotracking analysis and immunoblotting were used to analyze exosomes, and it was found that the extravasation kinetics of myocardial endothelial cells using exosomes as carriers of doxorubicin were relatively slow compared with doxorubicin alone; and in immunodeficient and immunocompetent mice, the tolerated dose of doxorubicin for exosomes as carriers was higher than that of doxorubicin alone. This study suggests that exosomes as carriers of doxorubicin can limit cardiac toxicity without affecting any other organs and increase the therapeutic index of doxorubicin in breast cancer and ovarian cancer mouse models. Ovarian cancer is the most lethal cause of gynecological malignancies, part of which is due to its chemotherapy resistance. Some related studies have found that ovarian cancer exosomes are involved in the regulation of tumor chemotherapy resistance. Studies have found that exosomes in platinum-resistant epithelial ovarian cancer cells can transfer some of their own chemotherapy-resistant phenotypes to platinum-sensitive cells, increase resistance-related EMT and sMAD4 mutations, which confirms that exosomes are regulators of platinum resistance in epithelial ovarian cancer. Moreover, studies have found that the exosomal miR21 of ovarian cancer can be transported to adjacent stromal cells, causing ovarian cancer cells to produce chemotherapy resistance and a more invasive phenotype. APAF1 is a direct target of miR21. Therefore, by upregulating APAF1 in ovarian cancer cells, the sensitivity of ovarian cancer cells to paclitaxel can be increased. Summary of the invention

[0006] In one aspect of the present invention, umbilical cord mesenchymal stem cell exosomes are prepared and obtained.

[0007] Furthermore, the chemotherapy drug cisplatin was introduced into the exosomes by electric shock, and drug-loaded exosomes were prepared and obtained.

[0008] Furthermore, the present invention provides use of drug-loaded exosomes in preparing a drug for treating ovarian cancer.

[0009] Furthermore, the present invention also provides a monoclonal antibody specific for PI3K, which can specifically inhibit the signaling pathway of PI3K, thereby inhibiting the proliferation of ovarian cancer cells and causing them to undergo apoptosis.

[0010] Furthermore, the present invention also provides the use of a monoclonal antibody of PI3K in preparing a drug for treating ovarian cancer.

[0011] Furthermore, the monoclonal antibody against PI3K of the present invention can inhibit the expression of p-AKT protein in ovarian cancer cells. p-AKT is the activated form of AKT. AKT has biological properties only when it is in the activated state. The monoclonal antibody against PI3K can inhibit the activity of PI3K, thereby reducing the activation of AKT and preventing its downstream molecules from having biological effects, leading to reduced cell proliferation and increased apoptosis, thereby treating ovarian cancer.

[0012] Furthermore, the monoclonal antibody of the present invention can enhance the synergistic therapeutic effect of the two by increasing the sensitivity of the chemotherapeutic drugs.

[0013] Therefore, further, the present invention also provides the use of a monoclonal antibody of PI3K in improving the drug sensitivity of cisplatin to ovarian cancer cells SK-OV-3.

[0014] The present invention also provides a pharmaceutical composition for treating ovarian cancer, which is composed of a monoclonal antibody against PI3K and exosomes loaded with cisplatin.

[0015] Furthermore, the pharmaceutical composition also contains a pharmaceutically acceptable carrier.

[0016] Furthermore, the carrier is one or more components selected from the group consisting of a binder, a filler, a lubricant and an effervescent couple, a wicking agent, a glidant, a disintegrant and a wetting agent.

[0017] In the context of the present invention, "for oral administration" means that the pharmaceutical composition is in a solid dosage form that is easily swallowed whole and is not substantially dissolved or suspended in water prior to administration.

[0018] Suitable disintegrants will be known to those skilled in the art, and non-limiting examples include crosslinked sodium carboxymethylcellulose, sodium starch glycolate, crosslinked polyvinyl pyrrolidone, povidone, starch (e.g., corn starch, pregelatinized starch), low-substituted hydroxypropyl cellulose, alginic acid, sodium alginate, trivalent calcium phosphate, calcium sulfate, carboxymethyl cellulose calcium, microcrystalline cellulose, powdered cellulose, colloidal silica, docusate sodium, guar gum, hydroxypropyl cellulose, magnesium aluminum silicate, methylcellulose, polycrilin potassium and polyvinyl pyrrolidone. Crosslinked sodium carboxymethyl cellulose is preferred. Disintegrants can be used alone or in combination with other substances.

[0019] Beneficial Effects

[0020] The present invention provides a stem cell exosome, which can be effectively used for the treatment of ovarian cancer after being loaded with chemotherapy drugs. When the exosome loaded with chemotherapy drugs is used together with a monoclonal antibody of PI3K, the sensitivity of the chemotherapy drugs can be significantly improved, so that the two have a significant synergistic therapeutic advantage, and the application prospects show a good effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Effects of different drug loadings of exosomes on cell survival

[0022] Figure 2 Effect of PI3K monoclonal antibody on the survival rate of ovarian cancer stem cells DETAILED DESCRIPTION

[0023] The present invention can be more easily understood by the subsequent description of some embodiments of the present invention and the detailed contents of the examples included therein. Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described, because these embodiments are necessarily diverse. It should also be understood that the terms used in this specification are only for the purpose of illustrating specific embodiments, rather than as limitations, because the scope of the present invention will be limited only in the appended claims.

[0024] Example 1 Preparation of stem cell exosomes

[0025] The umbilical cord of a full-term cesarean section fetus was sterilely removed and washed three times with PBS containing double antibodies in a clean bench to remove residual blood. The umbilical cord, vein and outer membrane were removed and cut into 1 mm pieces. 3 Size, evenly inoculated in disposable culture bottles, and cultured in a saturated humidity 37 ℃, 5% CO2 incubator with DMEM / F12 containing 10% FBS. The culture medium was replaced every 3 days. After about 10 days of culture, fibroblasts crawled out of the tissue. When the cells grew to 80% confluence, they were digested and passaged with 0.25% trypsin. The well-grown P4 generation MSCs were cultured in serum-free culture medium at 37 ℃ and 5% CO2 for 48 hours, and the supernatant was collected. At 4 ℃, centrifuged at 300×g for 5min, 2000×g for 20min, and 10000×g for 70min. The supernatant was then transferred to a high-speed centrifuge tube, centrifuged at 1000000×g for 120min at 4 ℃ to obtain a precipitate, filled the centrifuge tube with PBS, and centrifuged again at 1000000×g for 120min to obtain a purified exosome precipitate. The precipitate was resuspended in PBS, sterilized by filtration with a 0.22 μm filter membrane, and its protein concentration was detected by the BCA method. The protein concentration was adjusted to 1 g / L and stored at -80°C for future use.

[0026] The isolated and purified MSCs-derived exosomes were incubated with PE-labeled mouse anti-human CD73 and CD44 antibodies at room temperature in the dark for 30 minutes, washed once with PBS, resuspended with 1% paraformaldehyde, and analyzed by flow cytometry. The results showed that stem cell exosomes highly expressed CD44 (58.62%) and CD73 (34.52%), indicating that exosomes were isolated and prepared.

[0027] Take 20μL of exosome suspension and drop it on the smooth surface of the sample-loading copper grid. Let it stand at room temperature for 1 min. Use filter paper to absorb the liquid from one side. Add about 20μL of 20g / L phosphotungstic acid on the sample-loading copper grid. After negative staining at room temperature for 1 min, use filter paper to absorb the negative staining liquid. Bake it under an incandescent lamp for about 10 min. Observation under a transmission electron microscope showed that the exosomes were small round or oval membrane vesicles, and 80% of the diameters were distributed within 21.3nm.

[0028] Example 2 Stem cell exosome drug delivery method

[0029] The exosomes of Example 1 were evenly spread on a 10 cm culture dish, cultured conventionally, and cisplatin injection was added to a final mass concentration of 5 g / L, added to a cooled 4 mm electroporation cuvette, and the mixture was electroporated at 1000 kV for 5 ms using an Eppendorf Eporator, and incubated at 37 ° C for 30 minutes to restore the exosome membrane. Centrifuge at 400 × g for 10 min at 30 ° C, remove the precipitate, take the supernatant, centrifuge at 10000 × g for 2 min at 15 ° C, remove the precipitate again, take the supernatant, and centrifuge at 100000 × g for 60 min at 4 ° C to obtain the precipitate. Dissolve the precipitate in 0.5 mL PBS, centrifuge at 100000 × g for 60 min at 4 ° C, wash the precipitate repeatedly, and finally dissolve it in 0.5 mL PBS and store it at 4 ° C for use. The drug-loaded exosomes were diluted by multiples so that the mass concentration of cisplatin in the exosomes was 1, 5, 10, 20, 40, and 100 μmol / L, respectively.

[0030] Example 3 Ovarian cancer stem cell culture, passage, and identification

[0031] Human ovarian cancer SK-OV-3 cells were routinely cultured in DMEM / F12 medium containing 10% serum. Cells in the logarithmic growth phase were taken and perfused with 2.5 g / L trypsin to prepare a single cell suspension and count the cells. 7 / L were inoculated in SFM medium and cultured. The fifth generation suspended cell balls and normal adherent differentiated cells were digested with trypsin to obtain single cell suspension. The cells were washed twice with PBS buffer and the cell density was adjusted to 2×10 9 / L. Take 100μL of cell suspension and add 10μL of FITC-CD44 antibody to label the cells. Set isotype control for each fluorescent antibody, incubate at room temperature for 15min away from light, centrifuge at 1000r / / min for 5min, discard the supernatant, add 300μL of PBS, blow evenly and detect on flow cytometer. The fluorescence intensity of CD44 expressed in tumor cell spheres and adherent differentiated cells was 2.28±0.17 and 0.98±0.12, respectively, and the difference was significant (P<0.05). The present invention enriched and prepared ovarian cancer stem cells by serum-free suspension culture method.

[0032] Example 4 CCK-8 detection and screening of optimal exosome drug loading

[0033] Take the isolated and cultured ovarian cancer stem cells, add RPMI1640 medium containing 20 μg / L recombinant human epidermal growth factor and 20 μg / L basic fibroblast growth factor to prepare a single cell suspension, and use 1×10 7 / L of cells were inoculated in 96-well plates, and cell culture media containing cisplatin-loaded exosomes with concentrations of 1, 5, 10, 20, 40, and 100 μmol / L were added, respectively. Cells cultured in simple cell culture media were used as the control group. After 72 hours of culture, 10 μL of CCK-8 solution was added to each well, and the cells were cultured in an incubator at 37°C and 5% CO2 for 2 hours. Then, the absorbance A value was detected at a wavelength of 450 nm using an ELISA reader, and the cell survival rate was calculated. Cell survival rate = A value of the experimental group ÷ A value of the control group × 100%. The results are shown in Figure 1 shown.

[0034] from Figure 1 The results show that after 72 hours of culture, CCK-8 detection found that the proliferation activity of ovarian cancer stem cells was affected to varying degrees. With the increase of drug concentration, the cell survival rate gradually decreased. When the cisplatin drug concentration in the drug-loaded cell exosomes was 5 μmol / L, the cell survival rate decreased significantly. Therefore, the experiment selected exosomes with a cisplatin drug-loaded concentration of 5 μmol / L for subsequent experiments.

[0035] Example 5 Obtaining PI3K monoclonal antibody P-6F17

[0036] Recombinant human PI3K protein (ab125633, abcam) was used as an immunogen to immunize 8-week-old female BALB / c mice. The positive hybridoma cell lines were screened by conventional cell fusion methods in the art and cultured and screened using HAT and HT culture media. The limiting dilution method was used for subcloning. When the positive rate was 100%, the monoclonal antibody P-6F17 specific for PI3K was obtained. The antibody subtype is IgG1, which has good specificity and only reacts with PI3K protein. The dissociation constant was found to be 3.19×10 -9 M has good specificity. Its light chain variable region and heavy chain variable region are identified by sequence, as shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The hybridoma cell line of the antibody is subjected to ascites preparation and antibody purification for later use.

[0037] Example 6 Effect of PI3K monoclonal antibody P-6F17 on ovarian cancer stem cells

[0038] Take the isolated and cultured ovarian cancer stem cells, add RPMI1640 medium containing 20 μg / L recombinant human epidermal growth factor and 20 μg / L basic fibroblast growth factor to prepare a single cell suspension, and use 1×10 7 / L of cells were inoculated in a 96-well plate, and monoclonal antibodies containing monoclonal antibody P-6F17 at concentrations of 0, 1, 10, 50, 100, and 200 μg / mL were added respectively. Cells cultured in simple cell culture medium were used as the control group, and LY294002 with a concentration of 100 μg / mL was used as the positive control. After culturing for 72 h, 10 μL of CCK-8 solution was added to each well, and the cells were further cultured in an incubator at 37°C with a volume fraction of 5% CO2 for 2 h. The absorbance A value was then detected at a wavelength of 450 nm using a microplate reader, and the cell survival rate was calculated, cell survival rate = A value of the experimental group ÷ A value of the control group × 100%.

[0039] The results are as follows Figure 2 As shown in Figure 2, monoclonal antibody P-6F17 has a dose-dependent therapeutic effect on ovarian cancer stem cells. Under the condition of monoclonal antibody concentration of 200 μg / mL, the cell survival rate reached (34.39±

[0040] 2.93)%, which has a relatively good inhibitory effect compared with the positive control. Therefore, 200μg / mL monoclonal antibody P-6F17 was selected for subsequent experiments.

[0041] Example 7 Exosomes combined with monoclonal antibody P-6F17 sensitization experiment

[0042] Take the isolated and cultured ovarian cancer stem cells, add RPMI1640 medium containing 20 μg / L recombinant human epidermal growth factor and 20 μg / L basic fibroblast growth factor to prepare a single cell suspension, and use 1×10 7 The cells were inoculated in 96-well plates at a concentration of 100 / L and treated in the following groups:

[0043] Experimental group 1: 5 μmol / L cisplatin;

[0044] Experimental group 2: exosomes loaded with cisplatin at a final concentration of 5 μmol / L;

[0045] Experimental group 3: 200 μg / mL of monoclonal antibody P-6F17;

[0046] Experimental group 4: exosomes loaded with cisplatin at a concentration of 5 μmol / L + 200 μg / mL of monoclonal antibody P-6F17;

[0047] Blank control group: no drug was added, only culture medium was added;

[0048] After the cells were treated for 24 hours, a cell suspension was prepared, and the cells were washed with cold PBS after centrifugation, fixed with 75% ethanol at -20°C overnight, washed and centrifuged for 15 minutes, and then 500 μL of PBS containing 10 μg / mL PI and 0.1% RNaseA was added. After staining at room temperature in the dark for 30 minutes, the cell apoptosis rate was determined by flow cytometry. The results are shown in Table 1.

[0049] Table 1 Effects of each group on cell apoptosis rate

[0050] Each experimental group Apoptosis rate (%) Experimental Group 1 14.22±0.13* Experimental Group 2 18.39±0.24* Experimental Group 3 12.87±0.28* Experimental Group 4 34.28±0.31* Blank control group 1.32±0.12

[0051] (* indicates significant difference compared with blank, P<0.05)

[0052] From the results in Table 1, it can be seen that the same concentration of cisplatin, after being loaded with exosomes, has a certain improvement in the apoptosis rate of cells. However, when the monoclonal antibody is used in combination with exosomes loaded with cisplatin, the apoptosis rate is significantly improved.

[0053] The cells of each group treated with drugs were collected, the cells were lysed to extract total protein, 40 μg of protein was separated by 10% polyacrylamide gel (SDS-PAGE electrophoresis and then transferred to nitrocellulose membrane, blocked at room temperature for 2 hours, incubated with primary antibody p-AKT at 4°C overnight, rinsed 3 times with TBS buffer containing 0.05% Tween20, 10 minutes each time; added the corresponding alkaline phosphatase-labeled secondary antibody (1:500), incubated at room temperature for 1 hour, and developed with 5-hydroxy-4-chloro-3-indolyl phosphate / tetrazolyl nitro blue (BCIP / NBT / buffer) (1:1:50) kit, β-actin was used as internal reference, and each sample was repeated at least 3 times. The results are shown in Table 2.

[0054] Table 2 Effects of each group on cell p-AKT expression

[0055] Each experimental group Relative expression of p-AKT (p-AKT / β-actin) Experimental Group 1 0.51±0.05* Experimental Group 2 0.50±0.07* Experimental Group 3 0.11±0.02* Experimental Group 4 0.09±0.01* Blank control group 0.54±0.08

[0056] (* indicates significant difference compared with blank, P<0.05)

[0057] From the results in Table 2, it can be seen that monoclonal antibodies can inhibit the expression of p-AKT protein in ovarian cancer cells. p-AKT is the activated form of AKT, and AKT has biological characteristics only when it is activated. Therefore, monoclonal antibodies can inhibit PI3K, reduce the activation of AKT, and prevent its downstream molecules from having biological effects, resulting in reduced cell proliferation and increased apoptosis.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. P-6F17, a monoclonal antibody specific for PI3K, is characterized by The sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 1, and the sequence of the heavy chain variable region thereof is shown in SEQ ID NO:

2.

2. Use of the monoclonal antibody P-6F17 specifically targeting PI3K as claimed in claim 1 in the preparation of a drug for treating ovarian cancer by inhibiting the PI3K signaling pathway.

3. Use of umbilical cord mesenchymal stem cell exosomes loaded with the chemotherapy drug cisplatin and the monoclonal antibody P-6F17 specifically targeting PI3K in the preparation of a pharmaceutical composition for the treatment of ovarian cancer, wherein the light chain variable region sequence of the monoclonal antibody P-6F17 specifically targeting PI3K is shown in SEQ ID NO: 1, and its heavy chain variable region is shown in SEQ ID NO:

2.

4. The use according to any one of claims 2 to 3, wherein The ovarian cancer was caused by the cell line SK-OV-3.

5. The use according to claim 3, characterized in that The umbilical cord mesenchymal stem cell exosomes loaded with the chemotherapy drug cisplatin are prepared by mixing the exosomes with a cisplatin injection solution, adding the mixture to a cooled electroporation cuvette, introducing cisplatin into the exosomes by electroporation, and incubating at 37° C. for 30 minutes to restore the exosome membrane, thereby preparing the umbilical cord mesenchymal stem cell exosomes loaded with the chemotherapy drug cisplatin.

6. The use according to claim 5, characterized in that The pharmaceutical composition also contains a pharmaceutically acceptable carrier.

7. A pharmaceutical composition for treating ovarian cancer, the composition consisting of a monoclonal antibody P-6F17 specific for PI3K and umbilical cord mesenchymal stem cell exosomes loaded with cisplatin; wherein the light chain variable region sequence of the monoclonal antibody P-6F17 specific for PI3K is shown in SEQ ID NO: 1, and the heavy chain variable region sequence is shown in SEQ ID NO: 2.

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