Anti-tumor milk exosome drug delivery system with double-cell targeting property as well as preparation and application of anti-tumor milk exosome drug delivery system

Abstract

The invention discloses an anti-tumor milk exosome drug delivery system with double-cell targeting as well as preparation and application of the anti-tumor milk exosome drug delivery system. According to the double-cell targeted drug-loading exosome system designed by the invention, an inhibitor of a key molecule miR-21-5p capable of generating a cancer promoting effect in both tumor cells and tumor-related macrophages is simultaneously delivered to the tumor cells and the tumor-related macrophages by modifying a targeted EGFR (Epidermal Growth Factor Receptor) and PD-L1 nano antibody. Compared with the conventional clinical experiment aiming at the miR-21-5p, the miR-21-5p has the advantage that the low targeting property and the strong toxic and side effects are avoided. After double-targeting modification, the effect of treating two cells is more effective than that of single-targeting treatment. In addition, the used carrier is the milk exosome extracted from fresh milk daily drunk by people, and the milk exosome has low immunogenicity and toxicity. The pharmaceutical composition can be applied to treatment of EGFR < + > and / or PD-L1 < + > colorectal cancer, gastric cancer, melanoma and other malignant tumors, and can be combined with radiotherapy and anti-PD-1 curative effect to improve the curative effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to an anti-tumor milk exosome drug delivery system with dual cell targeting, and a preparation and application thereof. Background Art

[0002] The tumor microenvironment is a key factor leading to the occurrence and development of tumors. Its importance in dynamically regulating cancer progression and affecting treatment outcomes has been widely recognized. In recent years, therapeutic strategies targeting the TME have become a promising cancer treatment method. Due to the complex interactive regulation between tumor cells and other cells in the tumor microenvironment, treatments targeting a single type of cell often fail to achieve satisfactory results. miR-21-5p shows strong pan-cancer promoting activity in both tumor cells and tumor-associated macrophages. However, miRNA inhibitors are easily degraded in vivo and lack targeting.

[0003] Exosomes (a type of double-layer membrane vesicles secreted by cells, with a diameter of 30 to 150 nm. It can transport biological molecules such as proteins, nucleic acids and lipids to receptor cells and is an important tool for intercellular communication. Due to its advantages such as strong tissue penetration, high biocompatibility and low immunogenicity, exosomes have become an ideal candidate for drug and gene delivery carriers in cancer treatment. Milk exosomes (mExo) use economically available milk as a source of exosomes, with the advantages of low immunogenicity, easy access and low cost. It is an excellent drug delivery carrier, but its clinical application is limited by its lack of cancer targeting. Nanobodies are a type of single-domain antibodies that can be easily obtained through expression systems and are low in cost. Compared with other tumor-targeting molecules (such as conventional antibodies, peptides or aptamers), they have higher tissue penetration, structural stability and target affinity. The modification of nanoantibodies can significantly improve the anti-tumor activity of killer cells and drug carriers against tumor cells and immune cells.

[0004] Nanobodies, also known as single-domain antibodies, are derived from the variable region of heavy-chain antibodies of camelids. Nanobodies are the smallest antibodies known to bind to antigens. They can easily pass through various physiological barriers and solid tissues, have the characteristics of strong specificity and good stability, and are more likely to recognize and bind to cleft-like and groove-like hidden epitopes that traditional antibodies cannot access. Therefore, if nanobodies are modified on milk exosomes, it is possible to achieve tumor-specific targeted drug delivery of mExo by utilizing their ability to recognize highly expressed antigens on tumor cells. EGFR is an important pan-cancer driver that is highly expressed on the surface of various types of tumor cells, while PD-L1 is not only expressed on the surface of cancer cells, but also on the surface of tumor-associated macrophages (TAMs) of various solid tumors. Therefore, milk exosomes can be targeted for modification of PD-L1 and EGFR to simultaneously target and deliver miR-21-5p inhibitors into tumor cells and TAMs to exert a synergistic anti-cancer effect.

[0005] Based on this, it is of great significance to develop an anti-tumor milk exosome drug delivery system with dual cell targeting. Summary of the invention

[0006] Purpose of the invention: In order to overcome the deficiencies in the prior art, the present invention provides an anti-tumor milk exosome drug delivery system with dual cell targeting, and its preparation and application. The present invention extracts mExo, encapsulates miRNA inhibitors (such as miR-21-5p inhibitors) in mExo by electroporation, and then performs surface modification of the drug-loaded mExo with dual nano antibodies (EGFR nano antibodies such as 7D12 and PD-L1 nano antibodies such as KN035), so that the treatment system has the ability to simultaneously target tumor cells and tumor-associated macrophages, avoiding the ineffectiveness of single cell therapy. In addition, the anti-tumor activity of the anti-tumor milk exosome drug delivery system modified with dual nano antibodies (7D12 and KN035) is significantly better than that of the corresponding anti-tumor milk exosome drug delivery system modified with single nano antibodies (7D12 or KN035).

[0007] Another object of the present invention is to provide a preparation method and application of the anti-tumor milk exosome drug delivery system with dual cell targeting.

[0008] Technical solution: In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is:

[0009] The first objective of the present invention is to provide an anti-tumor milk exosome drug delivery system with dual cell targeting, which is a milk exosome encapsulated with a microRNA inhibitor and surface-modified with two nanoantibodies.

[0010] Optionally, in some embodiments of the present invention, the nanobody includes EGFR nanobody and PD-L1 nanobody.

[0011] Optionally, in some embodiments of the present invention, the microRNA inhibitor comprises a miR-21-5p inhibitor.

[0012] Furthermore, the RNA nucleic acid sequence of the miR-21-5p inhibitor is: UCAACAUCAGUCUGAUAAGCUA.

[0013] Optionally, in some embodiments of the present invention, the nanobody is modified onto milk exosomes encapsulating RNA therapeutic drugs via Sortase A enzyme (abbreviated as SrtA enzyme) and a PEG linker.

[0014] Optionally, in some embodiments of the present invention, the EGFR nanobody includes nanobody 7D12.

[0015] Optionally, in some embodiments of the present invention, the PD-L1 nanobody includes nanobody KN035.

[0016] Optionally, in some embodiments of the present invention, the dual-cell targeted anti-tumor milk exosome drug delivery system comprises nanobody 7D12, nanobody KN035 and SrtA enzyme that mediates enzyme-linked reaction.

[0017] Further optionally, in some embodiments of the present invention, the specific overall structure of the Nanobody 7D12, Nanobody KN035 and the SrtA enzyme that mediates the enzyme-linked reaction is:

[0018]

[0019] Further optionally, in some embodiments of the present invention, the amino acid sequence of 7D12 is shown in Seq ID No.1, the amino acid sequence of KN035 is shown in Seq ID No.2, and the amino acid sequence of SrtA enzyme is shown in Seq ID No.3.

[0020] Optionally, in some embodiments of the present invention, the dual cell-targeted anti-tumor milk exosome drug delivery system comprises an intermediate NH2-GG-PEG that is connected to the nanoantibody and connects the nanoantibody to the drug-loaded milk exosomes. 2000 -DSPE.

[0021] Furthermore, the NH2-GG-PEG 2000 -DSPE structure is:

[0022]

[0023] The second object of the present invention is to provide a method for preparing an anti-tumor milk exosome drug delivery system with dual cell targeting, wherein the anti-tumor milk exosome drug delivery system with dual cell targeting is obtained by modifying milk exosomes encapsulating microRNA inhibitors with two nanoantibodies.

[0024] Optionally, in some embodiments of the present invention, the anti-tumor milk exosome drug delivery system with dual cell targeting is obtained by modifying milk exosomes encapsulating microRNA inhibitors with EGFR nanoantibodies and PD-L1 nanoantibodies.

[0025] Optionally, in some embodiments of the present invention, the anti-tumor milk exosome drug delivery system with dual cell targeting is obtained by modifying milk exosomes encapsulating miR-21-5p inhibitors with nanoantibodies 7D12 and KN035.

[0026] Optionally, in some embodiments of the present invention, the method for preparing the dual-targeting anti-tumor milk exosome drug delivery system comprises the following steps:

[0027] S1, electrotransferring microRNA inhibitors into milk exosomes to form drug-loaded milk exosomes;

[0028] S2. The two nano-antibodies are simultaneously modified on the surface of the drug-loaded milk exosomes through enzyme-linked reaction.

[0029] Optionally, in some embodiments of the present invention, the nanobody includes EGFR nanobody and PD-L1 nanobody.

[0030] Optionally, in some embodiments of the present invention, the microRNA inhibitor comprises a miR-21-5p inhibitor.

[0031] Optionally, in some embodiments of the present invention, the nanobody is synthesized by SrtA enzyme and NH2-GG-PEG 2000 -DSPE was modified onto milk exosomes encapsulating miR-21-5p inhibitor.

[0032] Optionally, in some embodiments of the present invention, in step S1, the electroporation method includes: transferring 400uL of electroporation buffer containing 400nM microRNA inhibitor and 640ug milk exosomes to a pre-cooled electroporation cup, and using an electroporator to perform electroporation at 350V with a pulse time of 10ms; thereafter, placing the solution at 37°C for 40min to restore the exosome structure and form drug-loaded milk exosomes.

[0033] Optionally, in some embodiments of the present invention, in step S2, the enzyme linkage reaction includes: inserting two nanoantibodies into the surface of the drug-loaded milk exosomes through SrtA enzyme and PEG connector to obtain anti-tumor drug-loaded milk exosomes with dual cell targeting.

[0034] Optionally, in some embodiments of the present invention, the PEG linker is NH2-GG-PEG 2000 -DSPE.

[0035] Optionally, in some embodiments of the present invention, the inserting of two nanoantibodies into the surface of the drug-loaded milk exosomes through SrtA enzyme and PEG connector specifically includes: adding PEG connector to 640ug drug-loaded milk exosomes at a final concentration of 100uM, and incubating at 37°C overnight; then adding SrtA reaction solution, and then adding two nanoantibodies, reacting at 4°C for 4h to form dual-targeted drug-loaded milk exosomes.

[0036] Optionally, in some embodiments of the present invention, the molar ratio of the drug-loaded milk exosomes, nanoantibodies, and SrtA is 10:1:1.

[0037] Optionally, in some embodiments of the present invention, the SrtA reaction solution is 1 / 10 of the final volume.

[0038] Optionally, in some embodiments of the present invention, the molar ratio of the two Nanobodies in the Nanobody is 1:1.

[0039] Optionally, in some embodiments of the present invention, the nanobody includes EGFR nanobody and PD-L1 nanobody, and the molar ratio of EGFR nanobody to PD-L1 nanobody in the nanobody is 1:1.

[0040] Further optionally, in some embodiments of the present invention, the nanobody is synthesized by SrtA enzyme and NH2-GG-PEG 2000 The specific reaction process of -DSPE modification to milk exosomes encapsulating miR-21-5p inhibitors includes: 2000 -DSPE was added to 640ug of drug-loaded exosomes at a final concentration of 100uM and incubated at 37°C for 12h; then SrtA reaction solution, nanoantibodies and SrtA enzyme were added and reacted at 4°C for 4h.

[0041] Optionally, in some embodiments of the present invention, the method for preparing milk exosomes encapsulating miRNA inhibitors is as follows: the miRNA inhibitor (such as miR-21-5p inhibitor) is electroporated into milk exosomes in an electroporation buffer.

[0042] Optionally, in some embodiments of the present invention, the method for preparing the nanobody (such as nanobody 7D12, KN035) and SrtA enzyme comprises: constructing an expression strain expressing the nanobody (such as nanobody 7D12, KN035) and SrtA enzyme by genetic engineering, and obtaining the corresponding protein by induction and purification.

[0043] Further optionally, in some embodiments of the present invention, a method for preparing the above-mentioned anti-tumor milk exosome drug delivery system with dual cell targeting comprises the following steps:

[0044] 1) obtaining two nanobodies (such as nanobody 7D12 and nanobody KN035) and SrtA enzyme through protein expression and affinity chromatography purification;

[0045] 2) obtaining milk exosomes by removing casein and ultracentrifugation;

[0046] 3) Electrotransferring miRNA inhibitors (such as miR-21-5p inhibitors) into milk exosomes;

[0047] 4) Removal of miRNA inhibitors not encapsulated into exosomes by ultrafiltration;

[0048] 5) NH2-GG-PEG 2000 -DSPE is incubated with drug-loaded milk exosomes, so that DSPE is inserted into the milk exosome membrane, and the NH2-GG end is displayed on the outside of the milk exosomes;

[0049] 6) Adding nanoantibodies, SrtA reaction solution and SrtA enzyme to the above reaction solution to form dual-targeted engineered drug-loaded milk exosomes;

[0050] 7) The reaction solution is ultrafiltered to remove unreacted and unmembrane-inserted substances outside the dual-targeted engineered drug-loaded milk exosomes to obtain specific dual-targeted engineered drug-loaded milk exosomes.

[0051] Furthermore, the expression host of the present invention preferably uses the prokaryotic host Escherichia coli.

[0052] The third object of the present invention is to provide the application of the dual-cell targeted anti-tumor milk exosome drug delivery system in the field of preparing tumor therapeutic drugs, including drugs for treating malignant tumors such as colorectal cancer, gastric cancer, melanoma, etc. with different EGFR / PD-L1 expression states, and the combined application of the treatment system with radiotherapy and Anti-PD-1 treatment.

[0053] The fourth object of the present invention is to provide a tumor therapeutic drug, comprising an anti-tumor milk exosome drug delivery system with dual targeting as described above.

[0054] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0055] 1) Since milk exosomes are natural substances extracted from nutrients that humans commonly drink, they avoid immunogenicity and potential toxic side effects.

[0056] 2) Since miRNA inhibitors such as miR-21-5p exert strong pro-oncogenic effects in both tumor cells and tumor-associated macrophages, the use of their inhibitors can mediate anti-cancer effects in both cell types, avoiding the potential toxic side effects caused by the combination of multiple drugs targeting different targets.

[0057] 3) It utilizes the body’s own natural antibodies, thus avoiding potential toxicity and immunogenicity.

[0058] 4) The present invention forms a dual-targeted treatment model for two types of cells that strongly express EGFR and PD-L1 in the tumor microenvironment, and can also produce anti-cancer effects on tumor cells with other EGFR and PD-L1 expression conditions, with good pan-cancer inhibition.

[0059] 5) The dual-targeting drug-loaded exosomes modified with 7D12 and KN035 of the present invention have more significant tumor tissue targeting and anti-cancer activity than the single-targeting drug-loaded exosomes modified with 7D12 or KN035. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 : It is a schematic diagram of the preparation method of the present invention.

[0061] Figure 2 : It is a schematic diagram of the principle of the present invention.

[0062] Figure 3 :Analysis of human colorectal cancer database was performed to detect the cell types in which EGFR and PD-L1 were mainly expressed in tumor tissues. Further verification was performed by in vitro experiments. A survival analysis, B tumor single cell analysis, CD tumor cell relative to normal intestinal epithelial cell EGFR and PD-L1 expression, EF tumor-associated macrophage relative to normal intestinal epithelial cell induced macrophage EGFR and PD-L1 expression, GH comparison of tumor cell and tumor-associated macrophage EGFR and PD-L1 expression.

[0063] Figure 4:Verify the effects of miR-21-5p inhibitors on tumor cells and tumor-associated macrophages. A Expression of miR-21-5p in tumor cells relative to normal intestinal epithelial cells, B Effect of miR-21-5p inhibitor on tumor cell proliferation, C Effect of miR-21-5p inhibitor on tumor cell apoptosis, D E Expression of macrophage miR-21-5p induced by tumor-associated macrophages relative to normal intestinal epithelial cells, and Effect of miR-21-5p inhibitor on polarization of tumor-associated macrophages.

[0064] Figure 5 :Verification of the extraction of milk exosomes and the purification of nanoantibodies 7D12, KN035 and SrtA enzyme. A Milk exosome particle size, B Exosome marker detection, C Exosome electron microscopy, D Acquisition of nanoantibodies 7D12, 7KN035 and SrtA enzyme, E Detection of the binding of nanoantibodies to tumor cells.

[0065] Figure 6 :Construction and characterization of dual-targeted drug-loaded milk exosomes. A Verification of exosome nanoantibody modification, B Detection of exosome markers, C Detection of exosome-encapsulated miRNA inhibitors, D Exosome nanosight and electron microscopy characterization, E Exosome zeta potential, F Exosome water and kinetic diameter detection, G Blood solubility detection.

[0066] Figure 7 :In vitro targeting and pan-cancer inhibition of dual-targeted engineered drug-loaded milk exosomes. A Detection of single-nanoantibody and dual-nanoantibody modified drug-loaded mExo on EGFR + PD-L1 + Cell targeting, B. Engineering exosomes to tumor-associated macrophages and EGFR + and / or PD-L1 + Targeting of tumor cells, pan-cancer inhibitory effects of C-engineered exosomes.

[0067] Figure 8 :The effect of dual-targeted engineered drug-loaded milk exosomes on tumor cells. A. Clone formation, B. Apoptosis detection, C. Schematic diagram of engineered exosomes acting on the co-culture system of tumor cells and T cells, D. Flow cytometry detection of CD8 + T cell ratio, E ELISA detection of cytokines IFN-γ, Gzms-B and IL-2.

[0068] Fig. 9:Effects of dual-targeted engineered drug-loaded milk exosomes on tumor-associated macrophages. A Western blot to detect macrophage markers, B flow cytometry to detect macrophage polarization, C flow cytometry to detect macrophage phagocytosis of tumor cells, D qPCR to detect the expression of macrophage iNOS and TNFα, E schematic diagram of engineered exosomes acting on the co-culture system of tumor-associated macrophages and T cells, F flow cytometry to detect CD8 + T cell ratio, G ELISA detection of cytokines IFN-γ, Gzms-B and IL-2.

[0069] Fig.10 :Biological distribution of dual-targeted engineered drug-loaded milk exosomes in tumor-bearing mice. AB in vivo imaging detected the enrichment of engineered exosomes in tumor sites, C in vivo imaging detected the enrichment of engineered exosomes in tissues and organs, D statistical analysis of fluorescence in tumor tissues after dissociation, and E immunofluorescence observation of co-localization of engineered exosomes with tumor cells and tumor-associated macrophages.

[0070] Fig.11 :In vivo antitumor effect of dual-targeted engineered drug-loaded milk exosomes. A Flow chart of exosome treatment of tumor-bearing mice, B tumor image, C tumor weight, D tumor volume change, E tumor growth inhibition rate, F expression of miR-21-5p in tumor tissue, G mouse weight change, HK expression of miR-21-5p in tumor tissue, H flow cytometry detection of M1 macrophages, M2 macrophages, and CD8 + T cells and granzyme B + CD8 + The proportion of T cells, the expression of Ki67 in tumor tissues was detected by immunohistochemistry, and the TUNEL signal in tumor tissues was detected by immunofluorescence.

[0071] Fig.12 :Verification of the non-toxic side effects of dual-targeted engineered drug-loaded milk exosomes. A Hematoxylin-eosin (HE) staining of major tissues and organs, B detection of biochemical indicators related to liver function and kidney function in mouse serum.

[0072] Fig.13 :Combined anti-tumor effects of dual-targeted drug-loaded milk exosomes with radiotherapy and Anti-PD-1. A Flow chart of radiotherapy combined with exosome treatment in tumor-bearing mice, B tumor image, C tumor weight, D tumor growth inhibition rate; E Flow chart of Anti-PD-1 combined with exosome treatment in tumor-bearing mice, F tumor image, G tumor weight, H tumor growth inhibition rate. DETAILED DESCRIPTION

[0073] The present invention is based on a certain research background to construct a dual-cell targeted anti-tumor milk exosome drug delivery system, including:

[0074] 1) Clinical data analysis shows that EGFR + PD-L1 + Colorectal cancer patients have lower survival rates than EGFR - PD-L1 - , EGFR + PD-L1 - EGFR - PD-L1 + Patients, demonstrating that cells within the tumor that primarily express EGFR and PD-L1 have a strong cancer-promoting effect.

[0075] 2) Single-cell analysis of colorectal cancer tissues revealed that EGFR and PD-L1 were mainly localized in tumor cells and TAMs, respectively, and the same conclusion was verified at the cellular level.

[0076] 3) Tumor cells and TAMs highly express miR-21-5p, and miR-21-5p inhibitors can exert tumor suppressor functions in both types of cells.

[0077] Based on this, the present invention proposes an anti-tumor milk exosome drug delivery system with dual targeting, as well as its preparation and application. Based on the fact that tumor cells in various common cancers mainly express EGFR and PD-L1 is mainly expressed by tumor-associated macrophages, the inhibitor of miR-21-5p, a key molecule that can promote cancer in both cells, is delivered to tumor cells and tumor-associated macrophages by modifying the surface of the drug-loaded exosome system with nanoantibodies targeting EGFR and PD-L1.

[0078] In the embodiment of the present invention, the microRNA inhibitor is selected as the inhibitor of miR-21-5p, and the PEG linker is NH2-GG-PEG 2000 -DSPE, the EGFR nanobody is nanobody 7D12, and the PD-L1 nanobody is nanobody KN035. Taking this as an example, the dual-targeted anti-tumor milk exosome drug delivery system of the present invention and its preparation and application are described in detail. Figure 1 , 2 As shown, the miR-21-5p inhibitor was encapsulated into milk exosomes, and NH2-GG-PEG 2000-DSPE is inserted into the membrane surface of drug-loaded milk exosomes, and under the action of SrtA enzyme, nanoantibodies 7D12 and KN035 are connected to form a new targeted treatment system integrating "microRNA inhibitor + milk exosomes + targeted nanoantibodies" - dual-targeted engineered drug-loaded milk exosomes. After injection through the tail vein, the exosomes can strongly target the tumor site, enter the tumor microenvironment, and bind to tumor cells and tumor-associated macrophages at the same time, promote the apoptosis of tumor cells, reverse the phenotype of tumor-associated macrophages, and mediate the corresponding anti-tumor effect, producing a synergistic anti-tumor effect.

[0079] Compared with previous clinical trials targeting miR-21-5p, the dual-targeted anti-tumor milk exosome drug delivery system of the present invention avoids low targeting and strong toxic side effects. After dual-targeted modification, the treatment of two cells shows a more effective effect than single targeted therapy. In addition, the carrier used is a natural product milk exosome extracted from daily human drinks, which has low immunogenicity and toxicity. The present invention can be applied to EGFR + and / or PD-L1 + It can be used in the treatment of malignant tumors such as colorectal cancer, gastric cancer, melanoma, etc., and can be used in combination with radiotherapy and anti-PD-1 therapy to improve the efficacy.

[0080] It should be noted that in the embodiments of the present invention, the RNA therapeutic drugs encapsulated by exosomes are not limited to miR-21-5p inhibitors, but are also applicable to other similar miRNA inhibitors that promote cancer in tumor cells and tumor-associated macrophages in tumor tissues. In addition, the targeting ligand is not limited to 7D12 nanoantibodies and KN035 nanoantibodies, but is also applicable to dual-cell targeted modification of drug-loaded milk exosomes by other dual nanoantibodies.

[0081] The present invention aims to provide an anti-tumor milk exosome drug delivery system with dual cell targeting and its preparation method and application. The treatment system is an exosome drug system loaded with miR-21-5p inhibitor and surface modified with EGFR nanoantibody 7D12 and PD-L1 nanoantibody KN035. Figure 1 As shown, the preparation steps of the anti-tumor milk exosome drug delivery system with dual cell targeting of the present invention are as follows:

[0082] 1) obtaining nanobody 7D12, nanobody KN035 and SrtA enzyme through protein expression and affinity chromatography purification;

[0083] 2) obtaining milk exosomes, and electroporating the miR-21-5p inhibitor into the milk exosomes to form drug-loaded milk exosomes;

[0084] 3) Through SrtA enzyme and NH2-GG-PEG 2000-DSPE inserted nanoantibodies into the surface of drug-loaded milk exosomes to obtain anti-tumor drug-loaded milk exosomes with dual cell targeting.

[0085] Further verification of the anti-tumor effect of the present invention:

[0086] 1) Verify the targeting of dual-targeted engineered drug-loaded milk exosomes to tumor cells and TAMs through in vivo and in vitro experiments;

[0087] 2) In vitro verification of the anti-tumor effects of dual-targeted engineered drug-loaded milk exosomes on tumor cells and tumor-associated macrophages;

[0088] 3) In vivo verification of the anti-cancer effect of dual-targeted engineered drug-loaded milk exosomes and their combined anti-tumor effect with radiotherapy and anti-PD-1 treatment.

[0089] The present invention is further described below in conjunction with examples. The specific examples described herein are only used to explain the present invention and are not intended to limit the present invention. All simple improvements to the preparation method of the present invention under the premise of the concept of the present invention belong to the protection scope of the present invention. The experimental methods of the following examples without specifying specific conditions are usually in accordance with the known methods in the art. According to the following examples, the present invention can be better understood. However, it is easy for those skilled in the art to understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0090] Unless otherwise specified, the test materials and cell lines used in the following examples were purchased from conventional reagent companies.

[0091] Example 1: Clinical Data Analysis of EGFR + and / or PD-L1 + Five-year survival rate of colorectal cancer patients, and single-cell analysis of cells that mainly express EGFR and PD-L1 in tumors; further, verification of the expression of EGFR and PD-L1 in tumor cells and TAMs at the cellular level

[0092] The specific method is as follows:

[0093] Survival analysis was performed on GSE39582 data, and single-cell analysis was performed on GSE132465 data to further analyze the cellular expression of EGFR and PD-L1. qPCR and Western blot were used to detect the expression of EGFR and PD-L1 in tumor cells and TAMs, and the expression of the two molecules in these two types of cells was compared.

[0094] The above results are Figure 3 As shown: Figure 3 A demonstrated that EGFR+ PD-L1 + Colorectal cancer patients have lower survival rates than EGFR - PD-L1 - , EGFR + PD-L1 - EGFR - PD-L1 + This suggests that cells expressing EGFR and PD-L1 have a strong tumor-promoting effect.

[0095] Figure 3 B demonstrates that EGFR in the tumor is mainly expressed in tumor cells, while PD-L1 is mainly expressed in TAMs.

[0096] Figure 3 CH demonstrated that human and mouse tumor cells and TAMs highly expressed EGFR and PDL1, and EGFR was more highly expressed in tumor cells, while PD-L1 was mainly expressed in TAMs.

[0097] Example 2: Expression of miR-21-5p in tumor cells and TAMs, and the anti-cancer effect of miR-21-5p inhibitor on tumor cells and TAMs

[0098] The specific method is as follows:

[0099] qPCR was used to detect the expression of miR-21-5p in tumor cells and TAMs. CCK8 and flow cytometry were used to detect the effects of miR-21-5p inhibitors on tumor cell proliferation and apoptosis. qPCR was used to detect the effects of miR-21-5p inhibitors on macrophage polarization and PD-L1 expression.

[0100] The above results are Figure 4 As shown: Figure 4 A demonstrated that human and mouse tumor cells highly expressed miR-21-5p relative to normal epithelial cells.

[0101] Figure 4 B demonstrated that miR-21-5p inhibitors can inhibit the growth of tumor cells.

[0102] Figure 4 C demonstrated that miR-21-5p inhibitor promoted apoptosis of tumor cells.

[0103] Figure 4 DE demonstrated that miR-21-5p inhibitor could reverse the M2 polarization of TAMs and reduce their PD-L1 expression.

[0104] Example 3: Extraction of milk exosomes and purification of nanobody 7D12, nanobody KN035 and SrtA enzyme

[0105] Milk exosomes were obtained by ultracentrifugation after removing casein; nanoantibodies and SrtA enzyme were obtained by prokaryotic expression and then purified by affinity chromatography.

[0106] The specific method is as follows:

[0107] Obtaining milk exosomes: Fresh milk was centrifuged at 13000×g for 30 minutes for defatting, and then the supernatant was separated and hydrochloric acid was added to make the pH reach 4.6 for isoelectric precipitation of casein. Centrifugation at 13000×g for 60 minutes removed casein to obtain a clear whey solution containing exosomes. Ultracentrifugation at 135000 for 60 minutes separated the exosomes and resuspended in PBS. The exosomes were then verified and characterized by Western blot, particle size, and electron microscopy.

[0108] Nanobodies and SrtA enzymes were expressed by prokaryotes and purified by affinity chromatography: Nanobodies and SrtA enzymes were genetically engineered into expression vectors and transformed into BL21 DE3 Escherichia coli. Take 20 μL of bacteria to 3 mL of ampicillin-resistant LB medium, culture overnight at 37°C and 200 rpm, and transfer to TB fermentation medium at a 4% inoculation volume the next day. After the bacterial concentration reaches OD600 of 0.6, add an inducer IPTG with a final concentration of 0.2 mmol / L for induction. The conditions for protein expression are 16°C, 200 rpm, and the expression time is 12 hours. After fermentation, centrifuge at 8000 rpm for 5 minutes to collect the bacteria and discard the supernatant. After the bacteria are resuspended with a wall-breaking buffer, they are broken with an ultrasonic disruptor. After the breaking is complete, centrifuge at 9000 rpm for 10 minutes, and repeat three times to completely remove cell debris and obtain a clear wall-breaking supernatant. After the cell wall supernatant was completely incubated with the nickel column, the nickel column was washed with a cell wall buffer containing 20 mmol / L imidazole to remove impurities. Finally, the protein was washed off with a cell wall buffer containing high concentration imidazole. The purified protein was desalted and placed at -20°C for standby use. The nanoantibody and SrtA enzyme were prepared and verified by SDS-PAGE and Western blot. + PD-L1 + 7D12 and KN035 were added to CT26 cells, and the unbound nanoantibodies were washed away with PBS after 3 hours. Primary antibodies: Anti-Myc and Anti-HA were further added, and the unbound primary antibodies were washed away with PBS. The corresponding fluorescent secondary antibodies were further incubated. After washing with PBS, the binding of nanoantibodies to tumor cells was finally analyzed by flow cytometry.

[0109] The above results are Figure 5 As shown: Figure 5 AC demonstrated the acquisition of milk exosomes.

[0110] Figure 5D confirms that the nanobody and SrtA enzyme are specifically captured.

[0111] Figure 5 E confirmed that nanoantibodies 7D12 and KN035 can bind to EGFR and PD-L1 on the surface of tumor cells, respectively.

[0112] Example 4: Construction and characterization of dual-targeted drug-loaded milk exosomes

[0113] After the miR-21-5p inhibitor was electroporated into the milk exosomes, its surface was modified with nano-antibodies to obtain dual-targeted engineered drug-loaded milk exosomes.

[0114] The specific method is as follows:

[0115] Obtain drug-loaded exosomes: Transfer the electrotransfer buffer (400uL in total volume) containing 400nM miRNA inhibitors (miR-21-5p inhibitor and miR-NC inhibitor, respectively) and 640ug mExos to a pre-cooled electroporation cup, and use an electroporator to electroporate at 350V with a pulse time of 10ms. Afterwards, place the solution at 37°C for 40min for exosome structure recovery to form iExo (mExo loaded with miR-21-5p inhibitor) and ncExo (mExo loaded with miR-NC inhibitor), respectively. In addition, remove the unbound miRNA inhibitors by centrifugation at 3000×g for 10 minutes through a 10Kd ultrafiltration tube, and then store iExo and ncExo for later use.

[0116] Nanoantibody modification to obtain dual-targeted drug-loaded milk exosomes: NH2-GG-PEG 2000 -DSPE was added to 640ug iExo at a final concentration of 100uM and incubated at 37°C overnight. SrtA reaction solution (1 / 10 of the final volume) was then added, followed by NH2-GG-PEG 2000 -DSPE: nanobody: SrtA was 10:1:1 (Mol ratio), and nanobodies (respectively: 7D12, KN035 and 7D12 / KN035 double nanobodies, where the molar ratio of KN035:7D12 in the double nanobody modification was 1:1) and SrtA were added. The reaction was carried out at 4°C for 4 hours to form single-targeted drug-loaded milk exosomes 7D12-iExo and KN035-iExo, and dual-targeted drug-loaded milk exosomes 7D12 / KN035-iExo.

[0117] To verify the success of the construction, we performed exosome marker detection and miRNA inhibitor encapsulation verification on all the engineered exosomes constructed, and also performed particle size, zeta potential, electron microscopy, and hemolysis safety analysis.

[0118] The above results are Figure 6 As shown: Figure 6 A proves that the nanoantibody is successfully modified on the surface of drug-loaded exosomes. Figure 6 B. Exosome markers were detected for all constructed engineered exosomes.

[0119] Figure 6 C verified that the inhibitors were successfully encapsulated by the corresponding exosomes.

[0120] Figure 6 DF shows that although the particle size becomes larger after modification of the nanoantibody, it is still within the normal exosome particle size range, which also shows that the relevant characteristics of the exosomes will not be changed; the increase in ZETA potential confirms the success of the nanoantibody modification and also characterizes the increased homogeneity of the nanoantibody in the liquid environment after modification.

[0121] Figure 6 G proves that the constructed engineered exosomes are non-hemolytic.

[0122] Example 5: In vitro targeting and pan-cancer inhibition of dual-targeted engineered drug-loaded milk exosomes

[0123] Verify the effect of dual-targeted exosomes on EGFR compared with single-targeted exosomes + PD-L1 + Improved cell targeting. At the same time, the dual-targeted drug-loaded milk exosomes were verified to target tumor-associated macrophages and surface EGFR + PD-L1 + HCT116 cells, surface EGFR - PD-L1 + SKMEL2 cells, surface EGFR + PD-L1 - Targeting and inhibition of MKN45 cells.

[0124] The specific method is as follows:

[0125] In vitro targeting validation: Exosomes were stained with DiO membrane dye and unbound DiO dye was removed by 100Kd ultrafiltration tube. 5 cells) added EGFR + PD-L1 +In HCT116, flow cytometry was used to detect the targeting of engineered exosomes to cells after 12 hours. In addition, DiO-labeled engineered exosomes were added to M0 macrophages, M1 macrophages, tumor-associated macrophages, HCT116 cells, MKN45 cells, and SKMEL2 cells and incubated for 4 hours. After washing with PBS, the cells were fixed with 4% paraformaldehyde, and the uptake of exosomes by cells was evaluated by confocal microscopy after DAPI staining and washing with PBS. Pan-cancer inhibitory verification: CCK8 verified the inhibitory effect of engineered exosomes on the growth of tumor cells.

[0126] The above results are Figure 7 As shown: Figure 7 A demonstrated that 7D12 and KN035 modified drug-loaded exosomes can inhibit EGFR + PD-L1 + The cell targeting is stronger than that of single 7D12 or KN035 modification.

[0127] Figure 7 B proves that dual-targeted engineered drug-loaded milk exosomes have strong targeting to tumor-associated macrophages in vitro, while M0 macrophages and M1 macrophages do not. It also shows that dual-targeted engineered drug-loaded milk exosomes have strong targeting to surface EGFR + PD-L1 + HCT116 cells have strong targeting, and the surface EGFR - PD-L1 + SKMEL2 cells, surface EGFR + PD-L1 - MKN45 cells were also well targeted.

[0128] Figure 7 C shows that dual-targeted drug-loaded milk exosomes can produce an anti-cancer effect on tumor cells of multiple cancer types.

[0129] Example 6: Effects of dual-targeted engineered drug-loaded milk exosomes on tumor cells

[0130] The effects of dual-targeted engineered drug-loaded milk exosomes on tumor cell proliferation and apoptosis were verified by CCK8 and apoptosis. After dual-targeted engineered drug-loaded milk exosomes were applied to tumor cells co-cultured with human peripheral blood T cells, the CD8 + T cell proportion and released anti-tumor related factors.

[0131] The specific method is as follows:

[0132] Colony formation: HCT116 cells were cultured at 2 × 10 3The cells were plated in six-well plates at a certain density, and the corresponding exosomes were added after the cells adhered to the plate. The culture medium was changed regularly, and cell clumps were visible on day 12. Subsequently, the cell colonies were detected and recorded.

[0133] Cell apoptosis: 2×10 cells per well 5 The cells were plated in a six-well plate at a density of 1:1, and the corresponding engineered exosomes were added after overnight culture. The cells were collected after 72 hours. The cells were stained with Annexin V-PE / 7-AAD apoptosis detection reagent and further detected by flow cytometry (FCM).

[0134] T cell immune activity detection: Peripheral blood lymphocytes were obtained using human peripheral blood lymphocyte separation fluid, and lymphocytes were isolated at 5×10 5 / well of a six-well plate, and human CD3 / CD28 activation magnetic beads were used to activate T cells. In addition, HCT116 cells treated with engineered exosomes for 48 hours were co-cultured with lymphocytes (cell number 4:1) for 72 hours, and FCM was used to detect CD8 + In addition, the T cell culture medium was collected and the contents of IFN-γ, Gzms-B, and IL-2 in the lymphocyte culture medium were detected using an ELISA kit.

[0135] The above results are Figure 8 As shown: Figure 8 AB proved that iExo can cause inhibition of tumor cell proliferation and increase of apoptosis. The anti-cancer effect of iExo was enhanced after modification with 7D12 or KN035, and the most significant tumor suppressor effect was 7D12 / KN035-iExo.

[0136] Figure 8 CE demonstrated that after 7D12 / KN035-iExo acted on tumor cells co-cultured with T cells, CD8 + The proportion of T cells increased significantly, and the levels of anti-cancer related factors IFN-γ, GzmB, and IL-2 released by T cells increased.

[0137] Example 7: Effects of dual-targeted drug-loaded milk exosomes on tumor-associated macrophages

[0138] The effects of dual-targeted drug-loaded milk exosomes on the polarization of tumor-associated macrophages were detected by qPCR, Western blot, and FCM. The effect of exosomes on tumor-associated macrophages after acting on tumor-associated macrophages was detected by FCM. After dual-targeted drug-loaded milk exosomes were applied to tumor-associated macrophages co-cultured with human peripheral blood T cells, the CD8 + T cell proportion and released anti-tumor related factors.

[0139] The specific method is as follows:

[0140] Macrophage polarization and phagocytosis detection: Engineered exosomes were added to tumor-associated macrophages, and macrophage polarization-related indicators were detected by Western blot and FCM 48 hours later; Engineered exosomes were added to tumor-associated macrophages, and GFP was expressed 48 hours later. + HCT116 cells were added to macrophages and the cells were harvested after four hours of co-culture. After incubation with anti-CD11b, FCM was used to detect the phagocytic effect of macrophages on tumor cells. + GFP + Macrophages that engulf tumor cells.

[0141] T cell immune activity detection: Peripheral blood lymphocytes were obtained using human peripheral blood lymphocyte separation fluid, and lymphocytes were isolated at 5×10 5 / well of a six-well plate was plated, and human CD3 / CD28 T cell activation magnetic beads were used to activate T cells. In addition, tumor-associated macrophages treated with exosomes were co-cultured with lymphocytes (cell number 4:1) for 48 hours, and FCM was used to detect CD8 + In addition, the T cell culture medium was collected and the contents of IFN-γ, GzmB, and IL-2 in the lymphocyte culture medium were detected using an ELISA kit.

[0142] The above results are Fig. 9 As shown: Fig. 9 AB proved that iExo can reverse the M2 polarization of tumor-associated macrophages. The reversal effect of iExo was enhanced after modification with 7D12 or KN035, and the most significant effect was 7D12 / KN035-iExo. 8C-D showed that 7D12 / KN035-iExo enhanced the phagocytic effect of tumor cells on tumor-associated macrophages, and the macrophage killing-related factors iNOS and TNF-α increased significantly.

[0143] Fig. 9 EG demonstrated that after 7D12 / KN035-iExo acted on tumor-associated macrophages co-cultured with T cells, CD8 + The proportion of T cells increased significantly, and the levels of IFN-γ, Gzms-B, and IL-2 released by T cells increased.

[0144] Example 8: Biological distribution of dual-targeted engineered drug-loaded milk exosomes in tumor-bearing mice

[0145] The engineered exosomes carrying DiD fluorescent dye were injected into tumor-bearing mice, and the biological distribution of the dual-targeted engineered drug-loaded milk exosomes in vivo was determined by in vivo imaging. Immunofluorescence (IF) was further used to detect the cellular targeting of the dual-targeted engineered drug-loaded milk exosomes in tumor tissues.

[0146] The specific method is as follows:

[0147] CT26 cells (5×10 5 / 100uL) for tumor-bearing mice. When the tumor size is about 200mm 3 At 30 hours after injection, the main organs and tumors of mice were separated for fluorescence capture. Furthermore, in order to verify the cell targeting of exosomes at the tumor site, the tumor tissue was made into frozen sections. Immunofluorescence staining was performed for CD206 or CK20, and the co-localization of CD206 or CK20 and DiD was captured using a confocal microscope.

[0148] The above results are Fig.10 As shown: Fig.10 AD showed that mExo could infiltrate less into the tumor site, but the infiltration amount decreased rapidly over time. Nanoantibody-modified mExo showed strong tumor tissue targeting and less normal tissue infiltration. The most significant tumor tissue targeting was mExo modified with 7D12 and KN035 at the same time.

[0149] Fig.10 E verified that 7D12-mExo mainly targets tumor cells in tumor tissue, KN035-mExo mainly targets tumor-associated macrophages in tumor tissue, and 7D12 / KN035-mExo can strongly target both types of cells at the same time in tumor tissue.

[0150] Example 9: In vivo anti-tumor effect of dual-targeted engineered drug-loaded milk exosomes

[0151] Tumor-bearing mice were treated with engineered exosomes, and the tumor size, weight of tumor-bearing mice, expression of miR-21-5p in tumor tissues, and immune cells in tumor tissues were analyzed. In order to detect tumor proliferation, tumor tissues were stained with TUNEL and Ki67.

[0152] The specific method is as follows:

[0153] CT26 cells (5×10 5The engineered exosomes (100ug / 100uL) were injected subcutaneously into BABL / c mice to form a CT26 colorectal cancer xenograft tumor model. The engineered exosome treatment was started on the 10th day after the injection of tumor cells. The engineered exosomes (100ug / 100uL) were injected once every two days through the tail vein for a total of five injections. The weight and tumor size of the mice were measured every two days during the treatment. The tumor volume was calculated using the formula: volume (mm 3 ) = length × width 2 / 2. The mice were killed 6 days after the last administration. Tumors, peripheral blood serum and major organs (heart, liver, spleen, lungs, and kidneys) were collected. The tumor weight was weighed. The tumor tissue was digested using a digestion solution containing type I collagenase (200μg / mL), hyaluronidase (100μg / mL) and DNase I (100μg / mL) to further form a tumor tissue cell suspension. Mononuclear cells were isolated using a mouse tumor infiltrating tissue mononuclear cell isolation kit. After filtering through a 0.22um filter membrane, the cells were incubated with the corresponding fluorescein-conjugated antibody, and finally CD8 was analyzed using FCM. + T cells, granzyme B + CD8 + T cell and macrophage subtypes. After the tumor tissue was sliced, TUNEL staining and Ki67 histochemistry were performed.

[0154] The above results are Fig.11 As shown: Fig.11 AE proved that iExo could inhibit tumor growth, and the anti-tumor effect in vivo was enhanced after nano-antibody modification, with the strongest effect being the dual-targeted engineered drug-loaded milk exosome treatment group.

[0155] Fig.11 F shows that dual-targeted engineered drug-loaded milk exosomes treatment significantly reduced the expression of miR-21-5p in tumor tissues.

[0156] Fig.11 G shows that exosome treatment does not cause cachexia-induced weight loss in mice.

[0157] Fig.11 HK showed that dual-targeted engineered drug-loaded milk exosomes significantly reduced the proportion of M2 macrophages and increased the proportion of M1 macrophages and CD8 macrophages compared with other engineered exosomes. + T cells and granzyme B + CD8 + The proportion of T cells.

[0158] Fig.11 L demonstrated that engineered exosome treatment reduced the expression of Ki67 in tumor tissues and increased the TUNEL signal representing apoptosis.

[0159] Example 10: Verification of the non-toxicity of dual-targeted engineered drug-loaded milk exosomes in vivo

[0160] HE staining was performed on the main tissues of mice in the in vivo treatment experiment: heart, liver, spleen, lung, and kidney to determine whether the tissue morphology was normal; biochemical tests were performed on the liver function and kidney function indicators of the serum of mice in the in vivo treatment experiment;

[0161] The specific method is as follows:

[0162] The sections of major organs (heart, liver, spleen, lung, and kidney) were stained with hematoxylin and eosin. Furthermore, the liver function indexes (ALT, AST) and kidney function indexes (BUN, Scr) of mouse serum samples were detected using a biochemical analyzer.

[0163] The above results are Fig.12 As shown: Fig.12 A proves that engineered exosome therapy does not cause substantial lesions in normal tissues.

[0164] Fig.12 B shows that the liver function indexes and kidney function indexes were within the normal range after external treatment, indicating that the treatment had no effect on the main metabolic organs, the liver and kidneys.

[0165] Example 11: Dual-targeted drug-loaded milk exosomes produce combined anti-tumor effects with radiotherapy and anti-PD-1 respectively.

[0166] The tumor-bearing mice were treated with dual-targeted engineered drug-loaded milk exosomes and radiotherapy. After radiotherapy caused changes in the tumor microenvironment, the tumor was further treated with dual-targeted engineered drug-loaded milk exosomes. The tumor-bearing mice were treated with dual-targeted engineered drug-loaded milk exosomes and anti-PD-1.

[0167] The specific method is as follows:

[0168] CT26 cells (5×10 5 The double-targeted drug-loaded milk exosomes combined with radiotherapy: When the tumor reached 50 mm 3 The tumor site was radiotherapyed with a dose of 8Gy. Exosome treatment was started the next day after radiotherapy. Exosomes (100ug / 100uL) were injected once every two days through the tail vein for a total of five injections. Dual-targeted drug-loaded milk exosomes combined with anti-PD-1 therapy: The exosome treatment method was consistent with the dual-targeted drug-loaded milk exosomes combined with radiotherapy. The anti-PD-1 treatment was 200ug / 100uL / mouse. The treatment method was intraperitoneal injection, and the time and frequency were consistent with exosome treatment. At the end of treatment, the tumor tissue was separated and weighed.

[0169] The above results are Fig.13 As shown: Fig.13 Both AD radiotherapy and dual-targeted drug-loaded milk exosomes can inhibit tumor growth. The combined treatment with dual-targeted drug-loaded milk exosomes on the basis of radiotherapy has a more obvious anti-cancer effect.

[0170] Fig.13 Anti-PD-1 treatment alone in EH has a small therapeutic effect, but combined treatment with dual-targeted drug-loaded milk exosomes can achieve a synergistic anti-tumor effect.

[0171] In addition, this experiment also demonstrated that the two nano-antibody-modified exosomes loaded with miR-NC inhibitors blocking EGFR and PD-L1 could produce a 38% tumor inhibition rate, while the tumor inhibition rate of the same modified exosome loaded with miR-21-5p inhibitors could be significantly improved.

[0172] The present invention discloses a dual-targeted anti-tumor milk exosome drug delivery system and its preparation and application. Compared with the existing targeted drug delivery technology and anti-tumor effect, the system simultaneously modifies ligands targeting different cells, has stronger tumor tissue targeting and target cell targeting, and exhibits stronger anti-tumor effect. The results also confirm the potential of the dual-targeted anti-tumor milk exosome drug delivery system provided by the present invention in the field of preparing tumor therapeutic drugs.

[0173] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An anti-tumor milk exosome drug delivery system with dual cell targeting, characterized in that: The drug delivery system is milk exosomes that encapsulate microRNA inhibitors and are modified with two nanoantibodies on the surface.

2. The anti-tumor milk exosome drug delivery system with dual cell targeting according to claim 1, characterized in that: The nanobodies include EGFR nanobodies and PD-L1 nanobodies; Preferably, the microRNA inhibitor comprises a miR-21-5p inhibitor.

3. The anti-tumor milk exosome drug delivery system with dual cell targeting according to claim 1, characterized in that: The nanobody is modified with Sortase A enzyme and PEG linker onto milk exosomes encapsulating RNA therapeutic drugs.

4. The method for preparing the anti-tumor milk exosome drug delivery system with dual cell targeting according to any one of claims 1-3, characterized in that: The following steps are involved: S1, electrotransferring microRNA inhibitors into milk exosomes to form drug-loaded milk exosomes; S2. The two nano-antibodies are simultaneously modified on the surface of the drug-loaded milk exosomes through enzyme-linked reaction.

5. The method according to claim 4, characterized in that The nanobodies include EGFR nanobodies and PD-L1 nanobodies; Preferably, the microRNA inhibitor comprises a miR-21-5p inhibitor.

6. The method according to claim 4, characterized in that In step S1, the electroporation method includes: transferring 400uL of electroporation buffer containing 400nM microRNA inhibitor and 640ug milk exosomes into a pre-cooled electroporation cup, and electroporating at 350V using an electroporator with a pulse time of 10ms; thereafter, placing the solution at 37°C for 40min to restore the exosome structure and form drug-loaded milk exosomes.

7. The method according to claim 4, characterized in that In step S2, the enzyme-linked reaction comprises: inserting two nano-antibodies into the surface of the drug-loaded milk exosomes through SrtA enzyme and PEG linker to obtain anti-tumor drug-loaded milk exosomes with dual cell targeting; Preferably, the PEG linker is NH2-GG-PEG 2000 -DSPE.

8. The method according to claim 7, characterized in that The method of inserting two nanobodies into the surface of the drug-loaded milk exosomes through the SrtA enzyme and the PEG connector specifically includes: adding the PEG connector to 640ug of the drug-loaded milk exosomes at a final concentration of 100uM, and incubating at 37°C overnight; then adding the SrtA reaction solution, and then adding the two nanobodies, reacting at 4°C for 4h to form dual-targeted drug-loaded milk exosomes; Preferably, the molar ratio of the drug-loaded milk exosomes, nanoantibodies, and SrtA is 10:1:1; Preferably, the SrtA reaction solution is 1 / 10 of the final volume; Preferably, the molar ratio of the two Nanobodies in the Nanobody is 1:

1.

9. Use of the anti-tumor milk exosome drug delivery system with dual cell targeting according to any one of claims 1-3 in the preparation of tumor therapeutic drugs.

10. A tumor treatment drug, characterized in that: It comprises an anti-tumor milk exosome drug delivery system with dual cell targeting as described in any one of claims 1-3.

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