Modified exosomes and preparation methods thereof, and in-situ spray hydrogels of exosomes combined with PD-1 antibodies and their applications

Abstract

A modified exosome and its preparation method, and an in-situ spray hydrogel of exosomes combined with PD-1 antibody and its application, belong to the field of medical technology, CpG oligodeoxynucleotides and tumor-specific protein OVA are co-loaded on exosomes to obtain modified exosomes. And the in-situ spray hydrogel of exosomes combined with PD-1 antibody is prepared by loading the combined PD-1 antibody (aPD-1) and the above-mentioned modified exosomes into an in-situ spray hydrogel matrix. The in-situ spray hydrogel of the exosome combined with PD-1 antibody can release CpG to stimulate the maturation of dendritic cells at the tumor infiltration site, thereby activating tumor-infiltrating T cells. At the same time, the in-situ spray hydrogel of the exosome combined with PD-1 antibody can home to the tumor-draining lymph nodes, stimulate the maturation of dendritic cells in the lymph nodes, and increase the infiltration of T cells. And it can prolong the survival of mice and reduce the recurrence rate of melanoma after surgery.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and specifically relates to modified exosomes and a preparation method thereof, and an in-situ spray hydrogel of exosomes combined with PD-1 antibodies and applications thereof, and particularly relates to a modified exosome simultaneously loaded with CpG oligodeoxynucleotides and a tumor-specific protein OVA and a preparation method thereof, and relates to an application of exosomes combined with PD-1 antibodies loaded in an in-situ spray hydrogel in the treatment of postoperative recurrence of melanoma. Background Art

[0002] Blocking programmed cell death protein 1 using monoclonal anti-PD-1 antibodies (aPD-1) can relieve immunosuppression and has been shown to improve the efficacy of a range of cancers. However, the low response rate of immune checkpoint blockade always leads to postoperative tumor recurrence and metastasis. Tumor-infiltrating dendritic cells (TIDCs) and dendritic cells in tumor-draining lymph nodes (TDLNs) usually exhibit an immunosuppressive phenotype, usually due to their immature and inactive state leading to T cell immunosuppression. In addition, there are not enough activated tumor-infiltrating lymphocytes (TILs) in the resected tumor, and the treatment effect is not satisfactory. Therefore, effective dual activation of dendritic cells (TIDCs and TDLNs) at two sites and increasing tumor-infiltrating lymphocytes (TILs) to promote systemic immune responses are crucial for postoperative immunotherapy of melanoma.

[0003] CpG oligodeoxynucleotide (CpG ODN) is a synthetic oligodeoxynucleotide (ODN) containing unmethylated cytosine guanine dinucleotide (CpG), and is a Toll-like receptor 9 agonist. CpG oligodeoxynucleotide can directly promote the maturation and activation of dendritic cells, thereby inducing Th1 immune response, and can promote the secretion of cytokines and produce a strong immune response. However, it is difficult to effectively deliver CpG oligodeoxynucleotides to dendritic cells with existing methods, so the effect of treating melanoma is not good, and it cannot play a good anti-tumor effect. Summary of the invention

[0004] The present invention solves the problem that the treatment effect of postoperative recurrence of melanoma is not ideal, and provides a modified exosome and its preparation method, and an in situ spray hydrogel of exosomes combined with PD-1 antibodies and its application. The in situ spray hydrogel of exosomes combined with PD-1 antibodies is an in situ sprayable immunotherapy fibrin gel. The modified exosomes realize a dual response strategy, which can simultaneously activate dendritic cells at the tumor infiltration site and dendritic cells in the draining lymph nodes, thereby enhancing the effect of PD-1 antibodies. The prepared in situ spray hydrogel of exosomes combined with PD-1 antibodies forms a drug reservoir at the postoperative site, gradually and slowly releasing the modified exosomes and PD-1 antibodies, thereby increasing the infiltration of T lymphocytes in the tumor and improving the efficacy of the current PD-1 antibodies.

[0005] The present invention co-loads CpG oligodeoxynucleotides and tumor-specific protein OVA on exosomes to form modified exosomes, and then co-loads the modified exosomes with PD-1 antibodies in a spray hydrogel composed of fibrinogen and thrombin. The effects of delaying the growth of B16-F10-OVA melanoma, consolidating systemic T cell immunity, inhibiting tumor recurrence and metastasis, and prolonging the survival of mice are studied.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The modified exosomes of the present invention are exosomes carrying both CpG oligodeoxynucleotides and tumor-specific protein OVA.

[0008] The exosomes are dendritic cell-derived exosomes.

[0009] The number of dendritic cells producing dendritic cell-derived exosomes in proportion to the tumor-specific protein OVA is: 1×10 6 Piece: 200-400μg.

[0010] In terms of mass percentage, CpG oligodeoxynucleotides account for 3%-5%, and the remainder is tumor-specific protein OVA and exosomes.

[0011] The particle size of the modified exosomes is 120-150 nm.

[0012] A method for preparing modified exosomes of the present invention comprises the following steps:

[0013] S1: The serum-free culture medium of dendritic cells derived from mouse bone marrow and stimulated with OVA was collected, and the cells and cell debris were removed by an exosome extraction and separation kit. After resuspending with PBS, blank exosomes (DC-sEVs) were obtained;

[0014] S2: mixing the CpG ODN and the thiol reducing agent and stirring them thoroughly to obtain a mixed solution;

[0015] S3: Add the amine-thiol crosslinker to the mixed solution and continue stirring. After removing the excess thiol reducing agent and amine-thiol crosslinker, incubate with DC-sEVs in PBS solution at room temperature to obtain modified exosomes.

[0016] Furthermore, the dendritic cells are of mouse origin and are extracted from the femur and tibia of C57BL / 6 mice. The preparation process of the serum-free culture medium of the dendritic cells stimulated with OVA is as follows: 6 In the dendritic cell culture medium, 20ng / mL mouse granulocyte-macrophage colony stimulating factor (GM-CSF), 5ng / mL interleukin-4 (IL-4) and 50μM / L β-mercaptoethanol were added based on the volume of the culture medium. After culturing in a humidified incubator at 37°C for 3 days, the culture medium was replaced with fresh culture medium supplemented with the same volume of GM-CSF and IL-4. Dendritic cells were collected on the 6th day and incubated overnight in fresh culture medium containing 300μg / mL of OVA. 30ng / mL LPS was then added to stimulate dendritic cell maturation. Subsequently, dendritic cells expressing OVA were cultured in serum-free culture medium and their culture fluid was collected.

[0017] The thiol reducing agent is preferably tris(2-carboxyethyl)phosphine (TCEP) and / or dithiothreitol (DTT). In molar ratio, CpG ODN:thiol reducing agent=1:5000.

[0018] The amine-thiol crosslinking agent is preferably 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium salt (Sulfo-SMCC). In terms of molar ratio, CpG ODN: amine-thiol crosslinking agent is 1:1.5.

[0019] The method for removing excess thiol reducing agent and amine-thiol cross-linking agent is: using a 3kDa ultrafiltration tube to ultrafilter to remove excess thiol reducing agent and amine-thiol cross-linking agent.

[0020] The in situ spray hydrogel of exosomes combined with PD-1 antibody of the present invention comprises an in situ spray hydrogel matrix, PD-1 antibody (aPD-1) and the above-mentioned modified exosomes. The in situ spray hydrogel of exosomes combined with PD-1 antibody is prepared by loading the combined PD-1 antibody (aPD-1) and the above-mentioned modified exosomes into the in situ spray hydrogel matrix.

[0021] The in situ spray hydrogel matrix is ​​composed of fibrinogen and thrombin. When equal volumes of fibrinogen solution (10 mg / mL) are in contact with thrombin solution (100 U / mL), the matrix gels to obtain an in situ spray hydrogel matrix; by spraying and mixing equal volumes of thrombin solution containing modified exosomes and fibrinogen solution containing PD-1 antibodies, an in situ spray hydrogel of exosomes combined with PD-1 antibodies is formed.

[0022] According to the solid-liquid ratio, PD-1 antibody: CpG ODN contained in the modified exosomes: in situ spray hydrogel matrix = 6-8μg: 30-50μg: 100-300μL; among them, aPD-1 is uniformly dispersed in the fibrinogen solution of the in situ spray hydrogel matrix, and the modified exosomes are uniformly dispersed in the thrombin solution of the in situ spray hydrogel matrix.

[0023] The application of the in situ spray hydrogel of exosomes combined with PD-1 antibody is used as a drug for inhibiting and / or treating postoperative recurrence of melanoma.

[0024] The modified exosomes and exosomes combined with PD-1 antibody in situ spray hydrogel and preparation method and application thereof of the present invention have the following beneficial effects:

[0025] (1) An in situ spray hydrogel of exosomes combined with PD-1 antibodies was prepared to form a drug reservoir at the postoperative site, gradually and slowly releasing modified exosomes and PD-1 antibodies. An exosome preparation loaded with both CpG oligodeoxynucleotides and OVA was prepared, which can enhance the anti-tumor efficacy of PD-1 antibodies. The preparation method is simple and efficient, with good stability, and achieves efficient co-delivery of small molecule drugs.

[0026] (2) In order to solve the problem of postoperative tumor recurrence caused by the low response rate of PD-1 antibodies, an in situ spray hydrogel of combined modified exosomes and PD-1 antibodies was studied. The modified exosomes are loaded with CpG oligodeoxynucleotides and tumor-specific protein OVA. The CpG ODN carried by the modified exosomes can trigger the maturation and activation of tumor-infiltrating dendritic cells and dendritic cells in tumor-draining lymph nodes, thereby activating tumor-infiltrating T cells, which has a strong immunostimulatory effect. The modified exosomes can also deliver OVA to the same dendritic cells, triggering antigen-presenting cells to specifically express tumor antigens, thereby recognizing tumor cells carrying OVA antigens. At the same time, the exosomes can home to the tumor-draining lymph nodes, stimulate the maturation of dendritic cells in the lymph nodes, and increase the infiltration of T cells.

[0027] (3) The combination of PD-1 antibody and exosomes co-modified with immune adjuvants and tumor antigens in an in situ spray hydrogel can simultaneously enhance anti-tumor T cell immunity in the tumor microenvironment and lymph nodes, effectively reducing local tumor recurrence and metastasis. The dual activation strategy prolonged the survival of mice and reduced the recurrence rate of melanoma after surgery, providing a promising method for inhibiting tumor recurrence and metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a diagram of the preparation and characterization experiment of modified exosomes in Example 1 of the present invention.

[0029] A: Transmission electron microscopy images of DC-sEVs and DC-sEVs-CpG

[0030] B: Particle size and zeta potential diagram of DC-sEVs and DC-sEVs-CpG.

[0031] C: SDS-PAGE detection of DC, DC-sEVs and DC-sEVs-CpG.

[0032] D: Western blot detection of characteristic proteins of DC, DC-sEVs and DC-sEVs-CpG.

[0033] E: Confocal fluorescence image of DC-sEVs-CpG.

[0034] Figure 2 This is a diagram showing the preparation and property test of the in-situ spray hydrogel in Example 2 of the present invention.

[0035] A: Scanning electron microscopy image of the in situ sprayed hydrogel.

[0036] B: Dynamic rheological properties of in situ sprayed hydrogel.

[0037] C: Release kinetics of CpG ODN from gel at pH 6.5.

[0038] D: Release results of in situ spray hydrogel in mice.

[0039] Figure 3 This is the in vitro uptake diagram of DC-sEVs-CpG and the TLR9 localization diagram in Example 3 of the present invention.

[0040] A: Flow cytometry measurements of DCs treated with DC-sEVs-CpG and each control group.

[0041] B: Flow cytometry quantification of DCs treated with DC-sEVs-CpG and each control group, ***p<0.001, ****p<0.0001.

[0042] C: Confocal microscopy measurements of dendritic cells treated with DC-sEVs-CpG and each control group (0.5h and 2h).

[0043] Figure 4 This is a diagram of the in vitro activation experiment of DC-sEVs-CpG in Example 4 of the present invention.

[0044] A: Expression of CD80 on dendritic cells after treatment with DC-sEVs-CpG and each control group + CD86 + Figure 2. Flow cytometry measurements of .

[0045] B: Expression of CD80 on dendritic cells after treatment with DC-sEVs-CpG and each control group + CD86 + Flow cytometry quantification of , ***p<0.001.

[0046] C: Quantitative graphs of TNF-α and IL-2 after treatment with DC-sEVs-CpG and each control group, ***p<0.001, ****p<0.0001.

[0047] Figure 5 This is a diagram of the anti-tumor immune response experiment of aPD-1-DEV-CpG@Gel in Example 5 of the present invention.

[0048] A: Flow cytometry measurements of tumor-infiltrating M2 macrophages after treatment with Gel, DC-sEVs@Gel, CpG@Gel, DC-sEVs-CpG@Gel, aPD-1@Gel, and aPD-1-DEV-CpG@Gel, **p<0.01, ***p<0.001, ****p<0.0001.

[0049] B: Flow cytometry measurement of tumor-infiltrating M1 macrophages after treatment with each preparation, ***p<0.001, ****p<0.0001.

[0050] C: Tumor infiltrating CD8 after treatment with each preparation + Flow cytometry measurements of T cells, **p<0.01, ***p<0.001, ****p<0.0001.

[0051] D: Flow cytometry measurement of tumor-infiltrating regulatory T cells after treatment with each preparation, ***p<0.001, ****p<0.0001.

[0052] E: Flow cytometric measurement of the maturation of tumor-infiltrating dendritic cells after treatment with the three groups of formulations containing CpG ODN, ****p<0.0001.

[0053] F: Flow cytometry measurement of the maturation of dendritic cells in tumor-draining lymph nodes after treatment with the three groups of preparations containing CpG ODN, ****p<0.0001.

[0054] G: CD8 in the tumor-draining lymph nodes after treatment with the three groups of preparations containing CpG ODN + Flow cytometry measurement of T cells, ***p<0.001, ****p<0.0001.

[0055] Figure 6 This is a fluorescence intensity diagram of mouse lymph nodes in the targeted tumor-draining lymph node experiment in Example 6 of the present invention.

[0056] Figure 7 This is a diagram of the anti-tumor recurrence pharmacodynamics study of aPD-1-DEV-CpG@Gel in Example 7 of the present invention.

[0057] A: Changes in mouse tumor size after treatment with Gel, DC-sEVs@Gel, CpG@Gel, DC-sEVs-CpG@Gel, aPD-1@Gel, and aPD-1-DEV-CpG@Gel.

[0058] B: Changes in tumor volume after treatment with each preparation, ***p<0.001, ****p<0.0001.

[0059] C: Survival time of mice after treatment with each preparation.

[0060] D: Body weight changes of mice after treatment with each preparation.

[0061] Figure 8 This is a picture of the tumor and spleen in the pharmacodynamic investigation experiment in Example 7 of the present invention.

[0062] A: Photographs of dissected melanoma tumors after treatment with different types of agents.

[0063] B: The weight of dissected melanoma after treatment with different types of preparations, ***p<0.001, ****p<0.0001.

[0064] C: Photographs of dissected spleens after treatment with different types of preparations.

[0065] D: Spleen weight after dissection and treatment with different types of preparations, **p<0.01.

[0066] Fig. 9 This is a diagram showing the lung metastasis in the pharmacodynamic study experiment in Example 7 of the present invention.

[0067] Fig.10 This is an H&E staining image of the heart, liver, spleen, lung, kidney and tumor of the tumor-bearing mouse in Example 7 of the present invention.

[0068] Fig.11 This is a graph showing the anti-distant tumor pharmacodynamics experiment of aPD-1-DEV-CpG@Gel in Example 8 of the present invention.

[0069] A: Changes in tumor size in mice in the untreated group (Gel) and after aPD-1-DEV-CpG@Gel treatment.

[0070] B: Volume changes of primary tumor and distal tumor before and after treatment, ***p<0.001, ****p<0.0001.

[0071] Fig.12 This is a diagram showing the anti-distant tumor immune response of aPD-1-DEV-CpG@Gel in Example 8 of the present invention.

[0072] A: Flow cytometry measurement of M2 macrophages before and after treatment, ****p<0.0001.

[0073] B: Flow cytometry measurement of M1 macrophages before and after treatment, ****p<0.0001.

[0074] C: Untreated and treated CD8 + Flow cytometry measurement of T cells, ****p<0.0001.

[0075] D: Flow cytometry measurement of regulatory T cells before and after treatment, ****p<0.0001.

[0076] Fig.13 This is a diagram showing the tumor, spleen and major organs in the anti-distant tumor pharmacodynamics experiment of aPD-1-DEV-CpG@Gel in Example 8 of the present invention.

[0077] A: Photographs of anatomically isolated bilateral melanomas before and after treatment.

[0078] B: Photographs of dissected spleens after untreatment and treatment.

[0079] C: Dissected bilateral melanoma weights before and after treatment, ****p<0.0001.

[0080] D: Weights of dissected spleens after untreatment and treatment, ***p<0.001.

[0081] E: H&E staining of the heart, liver, spleen, lung, kidney, and tumor of untreated and treated tumor-bearing mice. DETAILED DESCRIPTION

[0082] The present invention is further described in detail below in conjunction with embodiments.

[0083] Example 1: Preparation and characterization of modified exosomes

[0084] The serum-free culture medium of dendritic cells extracted from the femur and tibia of C57BL / 6 mice and loaded with OVA was collected, the cell culture medium was centrifuged at 3000g for 15 minutes, and the supernatant was collected. The preparation process of the serum-free culture medium of dendritic cells loaded with OVA was as follows: in a solution containing 1×10 6 In the dendritic cell culture medium, 20ng / mL mouse granulocyte-macrophage colony-stimulating factor (GM-CSF), 5ng / mL interleukin-4 (IL-4) and 50μM / L β-mercaptoethanol were added based on the volume of the culture medium. After culturing in a 37°C humidified incubator for 3 days, the culture medium was replaced with fresh culture medium supplemented with the same volume of GM-CSF and IL-4. Dendritic cells were collected on the 6th day and incubated overnight in fresh culture medium containing 300μg / mL of OVA. Then 30ng / mL LPS was added to stimulate dendritic cell maturation. Subsequently, dendritic cells expressing OVA were cultured in serum-free culture medium, and their culture fluid was collected;

[0085] ExoQuick-TC TM The kit was added to the supernatant at a volume ratio of 1:5, mixed thoroughly, and placed at 4°C overnight. Next, the mixture was centrifuged at 1500g for 30min at 4°C, and the supernatant was removed and centrifuged at 1500g for 5min at 4°C. Finally, the exosomes obtained at the bottom of the tube were resuspended in PBS buffer. The thiolated CpG ODN was dissolved in PBS at a concentration of 100μM, combined with TCEP (1mol / L), and stirred at room temperature for 30min to reduce the disulfide bonds formed by the thiolated CpG ODN during transport. Sulfo-SMCC was then added to the solution and stirred for 30min. Each step was washed 3 times with PBS (molecular weight cutoff = 3kDa) to remove excess TCEP and Sulfo-SMCC. Finally, it was incubated with DC-sEVs at room temperature for 1h and purified in a centrifugal filter device (molecular weight cutoff = 100kDa) to obtain DC-sEVs-CpG.

[0086] The morphological characteristics of DC-sEVs and DC-sEVs-CpG were observed using a transmission electron microscope (TEM), and the particle size and potential of exosomes were determined using a Malvern laser particle size analyzer. Figure 1 A. Figure 1As shown in B. The protein integrity of exosomes was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting. Figure 1 C. Figure 1 D. To confirm that CpG ODN was successfully modified onto DC-sEVs through covalent bonds, Cy5-labeled CpG ODN and DiO-labeled DC-sEVs were observed under a confocal microscope, as shown in Figure 1 As shown in E, through Figure 1 E, it can be seen that CpG ODN was successfully modified onto DC-sEVs via covalent bonds.

[0087] Example 2: Preparation and property test of in-situ spray hydrogel

[0088] In situ spray hydrogels were prepared by spraying equal volumes of fibrinogen (10 mg / mL) and thrombin (100 U / mL) via a double-bottle sprayer, in which DC-sEVs-CpG was dissolved in the fibrinogen solution and PD-1 antibody was diluted in the thrombin solution.

[0089] The morphology of the in situ sprayed hydrogel was characterized using SEM. Figure 2 As shown in A, the dynamic rheological properties of the gel are Figure 2 Next, 1 mL of aPD-1-DEV-CpG@Gel was placed in 20 ml of PBS (pH = 6.5) for incubation, and in vitro release was simulated in a 37°C constant temperature water bath oscillator. The mixed release solution was taken out at the predetermined time points (0, 6, 12, 24, 48, 72, 96, 120, 144 h) and tested with a multifunctional microplate reader. The results are shown in Figure 2 As shown in Figure 3C, the in situ spray hydrogel can slowly and continuously release the drug. To measure the results of in vivo release, aPD-1, DC-sEVs-CpG, aPD-1@Gel, and DC-sEVs-CpG@Gel were administered to the postoperative site of mice, and the IVIS imaging system was used to observe the results on days 0, 2, 4, and 6. Figure 2 As shown in D, the in situ spray hydrogel (DC-sEVs-CpG@Gel) still contained the drug on the 6th day, while the amount of drug remaining in aPD-1 was very small on the 6th day. The amount of drug remaining in DC-sEVs-CpG was very small on the 6th day, while the amount of drug remaining in aPD-1@Gel was still large on the 6th day, indicating that the in situ spray hydrogel is a good drug reservoir.

[0090] Example 3: In vitro uptake of DC-sEVs-CpG and TLR9 localization experiments

[0091] To determine the in vitro uptake of DC-sEVs-CpG by DCs, Cy5-labeled CpG (6 μg), DC-sEVs+CpG (prepared by incubating DC-sEVs and CpG ODN directly in PBS solution), and DC-sEVs-CpG were incubated with 1×10 6 Mouse bone marrow derived dendritic cells were incubated in 12-well plates for 0.5 hours and 2 hours. First, dendritic cells were collected and tested by flow cytometry and analyzed using FlowJo software. The cells took up more DC-sEVs+CpG ( Figure 3 A. Figure 3 B). Secondly, the colocalization results were observed using confocal laser scanning microscopy ( Figure 3 C). Cy5-labeled CpG (6 μg), DC-sEVs+CpG and DC-sEVs-CpG were mixed with 1×10 6 The cells were incubated in confocal culture dishes for 0.5 h and 2 h, and the cells were stained with PE-conjugated TLR9 antibody at 4 °C, and then incubated with Hoechst33342 for 10 min at room temperature. The cells were washed 3 times with PBS between each process. It was found that DC-sEVs-CpG was more easily taken up by cells under confocal microscopy.

[0092] Example 4: In vitro activation experiment of DC-sEVs-CpG

[0093] To study the activation of dendritic cells in vitro, PBS, CpG (6 μg), DC-sEVs, DC-sEVs+CpG, and DC-sEVs-CpG were mixed with 1×10 6 Mouse bone marrow-derived dendritic cells were incubated overnight in a 12-well plate, and the cells were collected and stained with CD11c, CD80, and CD86 antibodies. The expression of these surface markers was tested by flow cytometry and analyzed by FlowJo software. DC-sEVs-CpG activated dendritic cells more significantly ( Figure 4 A. Figure 4 B). In addition, the cell culture supernatant was collected and measured by IL-2 mouse ELISA kit and TNF-α mouse ELISA kit. DC-sEVs-CpG treated cells produced more IL-2 and TNF-α ( Figure 4 C).

[0094] Example 5: Anti-tumor immune response experiment of aPD-1-DEV-CpG@Gel

[0095] B16-F10-OVA or Luc-B16-F10-OVA cells (5×10 per mouse) were inoculated 6The immunotherapy effect of the in situ spray hydrogel was investigated by subcutaneous injection of 100 cells into the right side of the abdomen of female C57BL / 6 mice (4-5 weeks old, average weight 18-22 g). Ten days after tumor cell inoculation, the tumor size reached 500 mm. 3 All mice were randomly divided into 6 groups, and the tumors were partially removed, leaving 1% of the tumor to simulate the postoperative residual tumor model. The process can be summarized as follows: First, the mice were anesthetized; then, the tumor tissue was removed using sterile surgical instruments, leaving about 1% of the tumor tissue; finally, various hydrogels (single gel Gel, modified exosome gel DC-sEVs@Gel, CPG gel CpG@Gel, in situ spray hydrogel DC-sEVs-CpG@Gel, antibody gel aPD-1@Gel, in situ spray hydrogel aPD-1-DEV-CpG@Gel of exosomes combined with PD-1 antibody) were immediately sprayed on the surgical wound, and the wound was closed with sutures or wound clips. In order to measure the immune effect of each spray gel, the tumors of the mice were collected on the 16th day and minced in a staining buffer containing digestive enzymes to obtain a single cell suspension. Staining with F4 / 80, CD80, CD206, CD3, CD4, CD8, Foxp3, CD11c, CD80 and CD86 antibodies. The stained cells were measured by flow cytometry and analyzed by FlowJo software ( Figure 5 A, 5B, 5C, 5D, 5E). The results showed that the number of M1 macrophages in the tumor increased, while the number of M2 macrophages decreased. + The proportion of T cells increased, while the proportion of regulatory T cells decreased. Tumor-draining lymph nodes (TDLNs) were transferred to a culture dish and crushed into single cells. CD3, CD4, CD8, Foxp3, CD11c, CD80 and CD86 antibodies were used for staining, and the stained cells were measured by flow cytometry and analyzed by FlowJo software ( Figure 5 F. Figure 5 G). The results showed that DCs in the tumor microenvironment and draining lymph nodes were activated to a higher degree after aPD-1-DEV-CpG@Gel treatment, leading to more effective CD8 + T cell immune effect.

[0096] Example 6: Targeted Tumor Draining Lymph Node Experiment

[0097] B16-F10-OVA or Luc-B16-F10-OVA cells (5×10 per mouse) were inoculated 6 10 cells) were injected subcutaneously into the right side of the abdomen of female C57BL / 6 mice (4-5 weeks old, average weight 18-22 g). Ten days after inoculation of tumor cells, the tumor size reached 500 mm 3All mice were randomly divided into 3 groups. The tumors were partially removed, leaving 1% of the tumor to simulate the residual tumor model after surgery. Then, CpG@Gel, DC-sEVs-CpG@Gel and aPD-1-DEV-CpG@Gel were given respectively. 24 hours after the administration, the tumor-draining lymph nodes of the mice were taken out and placed in the IVIS imaging system to observe the drug fluorescence intensity of each lymph node ( Figure 6 ). As can be seen in the figure, more CpG ODN accumulated in the draining lymph nodes after administration of aPD-1-DEV-CpG@Gel.

[0098] Example 7: Anti-tumor recurrence pharmacodynamics experiment of aPD-1-DEV-CpG@Gel

[0099] B16-F10-OVA or Luc-B16-F10-OVA cells (5×10 per mouse) were inoculated 6 The immunotherapy effect of the in situ spray hydrogel was investigated by subcutaneous injection of 100 cells into the right side of the abdomen of female C57BL / 6 mice (4-5 weeks old, average weight 18-22 g). Ten days after tumor cell inoculation, the tumor size reached 500 mm. 3 All mice were randomly divided into 6 groups, and the tumors were partially removed, leaving 1% of the tumor to simulate the postoperative residual tumor model. Gel, DC-sEVs@Gel, CpG@Gel, DC-sEVs-CpG@Gel, aPD-1@Gel, and aPD-1-DEV-CpG@Gel were respectively given to the postoperative wound site. D-luciferin (15 mg / mL) in DPBS solution was injected into the mouse peritoneum at a dose of 10 mg / mL, and the mice were imaged using an in vivo bioluminescence imaging system (IVIS) 10 minutes later ( Figure 7 A) According to the width 2 × length × 1 / 2 to measure and calculate the tumor size, and observe the survival of mice after 40 days of feeding (when the tumor volume exceeds 1.5cm 3 The mice were euthanized at 1 000 hrs and their body weights were recorded every other day ( Figure 7 B. Figure 7 C. Figure 7 D). The mice treated with aPD-1-DEV-CpG@Gel had significantly less tumor recurrence and a longer survival period. The other mice were killed on day 22, and the recurrent tumors and major organs were collected. The size and weight of the tumor and spleen are shown in the figure ( Figure 8 A. Figure 8 B. Figure 8 C. Figure 8 D) The tumors of mice treated with aPD-1-DEV-CpG@Gel were smaller and the size of the spleen was almost unchanged. Fig. 9As shown in Figure 2, lung metastasis was inhibited after aPD-1-DEV-CpG treatment. The heart, liver, spleen, lung, kidney and tumor were immersed in 4% paraformaldehyde for H&E staining. The images under the microscope are shown in Figure 2. Fig.10 shown.

[0100] Example 8: Anti-distant tumor pharmacodynamics experiment of aPD-1-DEV-CpG@Gel

[0101] For the distal tumor model, B16-F10-OVA or Luc-B16-F10-OVA cells (5 × 10 cells per mouse) were inoculated. 6 cells) were injected subcutaneously into the right side of the abdomen of female C57BL / 6 mice (4-5 weeks old, average weight 18-22 g) (primary tumor). After the primary tumor grew for 7 days, 1×10 6 B16-F10-OVA or Luc-B16-F10-OVA cells were injected into the left side of the mouse abdomen, and the second tumor obtained was used as the distal tumor. When the primary tumor grew for 10 days, the tumor on the right side was partially resected, and the resection site was given in situ spray hydrogel Gel and aPD-1-DEV-CpG@Gel. The mice were imaged and observed using the IVIS imaging system ( Fig.11 A) According to the width 2 × length × 1 / 2 to measure and calculate the size of the primary tumor recurrence and the size of the distant tumor ( Fig.11 B) The results showed that tumor recurrence and distal tumor size were suppressed in treated mice.

[0102] Tumors from mice were collected on day 16 and minced in a staining buffer containing digestive enzymes to obtain single-cell suspensions. They were stained with F4 / 80, CD80, CD206, CD3, CD4, CD8, and Foxp3 antibodies. The stained cells were measured by flow cytometry and analyzed by FlowJo software. Fig.12 A. Fig.12 B. Fig.12 C. Fig.12 D) After treatment, the number of M1 macrophages increased, while the number of M2 macrophages decreased. + The proportion of T cells increased, while the proportion of regulatory T cells decreased. Other mice were sacrificed on day 22, and the recurrent tumors, distal tumors, and major organs were collected. The size and weight of the tumor and spleen are shown in the figure ( Fig.13 A. Fig.13 B. Fig.13 C. Fig.13D) The recurrence of tumors and the size of distant tumors in treated mice were significantly inhibited, and the size of the spleen was almost unchanged. The heart, liver, spleen, lung, kidney and tumor were immersed in 4% paraformaldehyde for H&E staining. The images under the microscope are as follows Fig.13 As shown in E.

Claims

1. A modified exosome, characterized in that: The modified exosomes are exosomes that carry both CpG oligodeoxynucleotides and tumor-specific protein OVA. The exosomes are derived from dendritic cells, which are mouse-derived and extracted from the femur and tibia of mice. In terms of mass percentage, CpG oligodeoxynucleotides account for 3%-5%, and the remainder is tumor-specific protein OVA and exosomes. The modified exosomes simultaneously activate dendritic cells at the tumor infiltration site and dendritic cells in the draining lymph nodes, and are used in the preparation of a drug for inhibiting and / or treating postoperative recurrence of melanoma. In terms of ratio, the number of dendritic cells that produce exosomes derived from dendritic cells is: tumor-specific protein OVA is: 1×10 6 pcs: 200-400 μg.

2. The method for preparing modified exosomes according to claim 1, characterized in that: The following steps are involved: S1: Collect serum-free culture medium of dendritic cells derived from mouse bone marrow and stimulated with OVA, remove cells and cell debris using an exosome extraction and separation kit, and resuspend in PBS to obtain blank exosomes; The dendritic cells are mouse-derived and extracted from the femur and tibia of C57BL / 6 mice. The preparation process of the serum-free culture medium of dendritic cells stimulated with OVA is as follows: in a solution containing 1×10 6 To the dendritic cell culture medium, 20 ng / mL mouse granulocyte-macrophage colony-stimulating factor, 5 ng / mL interleukin-4 and 50 μM / L β-mercaptoethanol were added based on the volume of the culture medium; after culturing in a humidified incubator at 37°C for 3 days, the culture medium was replaced with fresh culture medium supplemented with the same volume of GM-CSF and IL-4; on the 6th day, the dendritic cells were collected and incubated overnight in fresh culture medium containing 300 μg / mL OVA; then 30 ng / mL LPS was added to stimulate the maturation of dendritic cells; subsequently, the dendritic cells expressing OVA were cultured in serum-free culture medium and their culture fluid was collected; S2: mixing CpG ODN and a thiol reducing agent at a molar ratio of CpG ODN:thiol reducing agent = 1:5000 and stirring thoroughly to obtain a mixed solution; the thiol reducing agent is tris(2-carboxyethyl)phosphine and / or dithiothreitol; S3: Add an amine-thiol crosslinker to the mixed solution and continue stirring. After removing excess thiol reducing agent and amine-thiol crosslinker, incubate with blank exosomes in PBS solution at room temperature to obtain modified exosomes; the amine-thiol crosslinker is 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt; in molar ratio, CpGODN: amine-thiol crosslinker is 1:1.

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3. An in situ spray hydrogel of exosomes combined with PD-1 antibody, characterized in that: The invention comprises an in-situ spray hydrogel matrix, a PD-1 antibody and the modified exosomes according to claim 1, wherein the in-situ spray hydrogel of the exosomes combined with the PD-1 antibody is prepared by loading the combined PD-1 antibody and the modified exosomes into the in-situ spray hydrogel matrix; The in situ spray hydrogel matrix is ​​composed of fibrinogen and thrombin. When equal volumes of a fibrinogen solution of 10 mg / mL are in contact with a thrombin solution of 100 U / mL, the matrix gels to obtain an in situ spray hydrogel matrix. By spraying and mixing equal volumes of a thrombin solution containing modified exosomes and a fibrinogen solution containing PD-1 antibodies, an in situ spray hydrogel of exosomes combined with PD-1 antibodies is formed. According to the solid-liquid ratio, PD-1 antibody: CpG ODN contained in the modified exosomes: in situ spray hydrogel matrix = 6-8 μg: 30-50 μg: 100-300 μL; among them, the PD-1 antibody is uniformly dispersed in the fibrinogen solution of the in situ spray hydrogel matrix, and the modified exosomes are uniformly dispersed in the thrombin solution of the in situ spray hydrogel matrix.

4. Use of the in situ spray hydrogel of exosomes combined with PD-1 antibody according to claim 3 for preparing a drug for inhibiting and / or treating postoperative recurrence of melanoma.

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