Exosome-coated acid-responsive dendrimer nanogel as well as preparation and application thereof

Abstract

The invention relates to an exosome-coated acid-responsive dendrimer nanogel as well as preparation and application thereof, the exosome-coated acid-responsive dendrimer nanogel comprises an exosome and a polyamide-amine dendrimer nanogel, and the exosome is coated on the surface of the polyamide-amine dendrimer nanogel; wherein the polyamide-amine dendrimer nanogel is loaded with a drug, nanogold and an immunologic adjuvant. The nanogel is used as a nano vaccine and has CT imaging performance, and precise imaging and treatment monitoring of tumors can be realized.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to an exosome-coated acid-responsive dendrimer nanogel and its preparation and application. Background Art

[0002] Pancreatic cancer (PDAC) is one of the most aggressive and lethal malignant tumors, and its treatment faces huge challenges. Although conventional treatments such as chemotherapy have been widely used, the overall survival rate of pancreatic cancer patients has not been significantly improved. In recent years, immunotherapy, as an emerging treatment strategy, has attracted much attention in the treatment of pancreatic cancer. However, due to the unique tumor microenvironment (TME) and low tumor immunogenicity of pancreatic cancer, the efficacy of immunotherapy is limited. Therefore, how to effectively induce the infiltration of effector immune cells in the tumor and reverse the immunosuppressive tumor microenvironment has become a core issue in the treatment of pancreatic cancer (Ho et al. Nat. Rev. Clin. Oncol., 2020, 17 (9), 527-540).

[0003] Tumor-associated macrophages (TAMs) are the most abundant immune cell type in the tumor microenvironment. Under the complex conditions of the tumor microenvironment, TAMs mainly exhibit the M2 phenotype, which has the functions of immunosuppression and promoting tumor progression (DeNardo et al. Nat, Rev, Immunol., 2019, 19(6), 369-382). Studies have shown that the immunosuppressive tumor microenvironment has plasticity and can reverse the immunosuppressive state by activating infiltrating immune cells and regulating the repolarization of TAMs from M2 to anti-tumor M1. In addition, pancreatic cancer cells have significant immune escape characteristics, which further promotes tumor metastasis and recurrence. Nanovaccines, as a strategy that can reduce tumor cell immune escape and enhance the effect of immunotherapy, are usually composed of tumor-associated antigens (TAAs), immune adjuvants and nanocarriers (Zhang et al. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol., 2019, 11(5), 1559). However, the inhibitory effect of most nanovaccines on tumors is not ideal. This may be due to the degradation of TAAs in nanovaccines in the acidic lysosomes of antigen-presenting cells, resulting in reduced antigen presentation efficiency and the limited number of TAAs carried by nanovaccines.

[0004] Gold nanoparticles (Au NPs) exhibit unique advantages in tumor therapy. Studies have shown that Au NPs can increase the lysosomal pH of M2 TAMs and inhibit lysosomal-mediated antigen degradation, thereby improving the efficiency of antigen cross-presentation. Furthermore, Au NPs can promote the repolarization of M2 TAMs to M1, further enhancing the therapeutic effect of tumor vaccines. Furthermore, Au NPs can also be used as CT imaging contrast agents for tumor imaging. However, conventional gold nanoparticles lack tumor targeting, resulting in suboptimal tumor CT imaging capabilities.

[0005] Toyocamycin (Toy) is a small molecule chemotherapy drug that acts on the endoplasmic reticulum stress IRE1α-XBP1 pathway, preventing IRE1α from cleaving XBP1, amplifying the degree of endoplasmic reticulum stress in cells, and thus inducing tumor cell apoptosis (Wang et al. Adv. Mater., 2022, 34(7), 2107009). However, Toy's anti-tumor effect is limited due to its low bioavailability and poor targeting in vivo.

[0006] Dendrimer nanogels (DNGs) have been used in anti-tumor therapy research because they combine the properties of dendrimers and nanogels. However, existing studies have shown that traditional DNGs have limited specific responsiveness to the tumor microenvironment (TME), significantly hindering further improvement of their therapeutic efficacy.

[0007] A search of relevant domestic and international literature and patents has yet to reveal reports on the preparation and application of novel nanovaccines using dendrimer nanogels as carriers and integrating exosomes. Therefore, this invention possesses significant innovation and potential for application in the treatment of pancreatic cancer. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide an exosome-coated acid-responsive dendrimer nanogel and its preparation and application.

[0009] The present invention provides a composite nanogel, which comprises exosomes and polyamide-amine dendrimer nanogel, wherein the exosomes are coated on the surface of the polyamide-amine dendrimer nanogel; wherein the polyamide-amine dendrimer nanogel is loaded with drugs, nanogold and immune adjuvants.

[0010] Furthermore, the polyamidoamine dendrimer includes the third-generation polyamidoamine dendrimer G3.NH2PAMAM; the drug includes a chemotherapy drug; the immune adjuvant includes CpG; and the nanogold is gold nanoparticles.

[0011] Furthermore, the chemotherapy drug is Toy; and the exosomes are exosomes derived from pancreatic cancer cells.

[0012] Furthermore, the composite nanogel is prepared by modifying the surface of the third-generation polyamide-amine dendrimer with 4-bromomethylphenylboronic acid, and then loading Toy on the surface of the dendrimer through a boronate bond to obtain G3-Toy, and cross-linking it with a crosslinker FBA-PEG-FBA to prepare the dendrimer nanogel G3-Toy / DNGs, followed by in situ reduction loading of gold nanoparticles, further electrostatic adsorption of CpG, and finally integration with exosomes Exo derived from pancreatic cancer cells to prepare a composite nanogel, which can be used as a biomimetic nanogel vaccine.

[0013] The present invention provides a method for preparing a composite nanogel, comprising:

[0014] (1) 4-bromomethylphenylboronic acid PBA, G3.NH2 PAMAM, and a solvent are mixed, reacted, and purified to obtain G3.NH2-PBA;

[0015] (2) mixing G3.NH2-PBA and an aqueous solution of a chemotherapy drug, reacting, and purifying to obtain a pH-responsive G3-chemotherapeutic drug;

[0016] (3) G3-chemotherapeutic drug monomer, crosslinker, and water are mixed to form an aqueous phase, and then the aqueous phase is added to the organic phase to form a W / O mixture, which is ultrasonically dispersed to form a milky white polymer, and then a catalyst is added for catalysis, stirred, and purified to obtain a dendrimer nanogel loaded with chemotherapeutic drugs;

[0017] (4) dissolving the nanogel loaded with chemotherapy drugs in water, adding chloroauric acid under ice-bath stirring, continuing ice-bath stirring, then adding a reducing agent, stirring for reaction, and purifying to obtain a dendrimer nanogel loaded with chemotherapy drugs / nano-gold (i.e., a polyamide-amine dendrimer nanogel in situ encapsulating gold nanoparticles);

[0018] (5) mixing the dendrimer nanogel loaded with chemotherapy drugs / nano-gold, water, and immune adjuvant, and incubating at room temperature to obtain the dendrimer nanogel loaded with chemotherapy drugs / nano-gold / immune adjuvant;

[0019] (6) The dendrimer nanogel loaded with chemotherapy drugs / nanogold / immune adjuvants and exosomes are mixed to obtain a mixture, which is then extruded and centrifuged to obtain a composite nanogel.

[0020] Preferably, in step (1), the molar ratio of G3.NH2 PAMAM to PBA is 1:8 to 1:9; and the solvent comprises dimethyl sulfoxide.

[0021] Preferably, in step (1), the reaction is carried out at 65-70° C. with stirring for 24-26 hours.

[0022] Preferably, the chemotherapy drug in step (2) includes Toyocamycin Toy;

[0023] Preferably, in step (2), the molar ratio of PBA to chemotherapy drug in G3.NH2-PBA is 1:3 to 1:5;

[0024] Preferably, the reaction in step (2) is carried out at 55-60° C. with stirring for 24-26 hours, and the generated boronate bond has dual sensitivity to pH and active oxygen.

[0025] The purification in steps (1) and (2) is performed by dialysis; and the purification in steps (1) and (2) is followed by freeze-drying.

[0026] Preferably, in step (3), the cross-linking agent includes FBA-PEG-FBA; the oil phase is a mixture of Span 80, Tween 80 and an organic solvent; the organic solvent includes cyclohexane; and the catalyst includes triethylamine.

[0027] The structural formula of FBA-PEG-FBA is

[0028] Preferably, in step (3), the molar ratio of G3-chemotherapeutic drug, cross-linking agent, Span 80 and Tween 80 is 1:3:190:21 to 1:4:190:21; FBA-PEG-FBA is a pH-sensitive cross-linking agent.

[0029] The water in step (3) is pure water; wherein the volume ratio of water to the organic solvent in the oil phase is 1:10 to 1:11.

[0030] Preferably, the ultrasound in step (3) is probe ultrasound, wherein the power of the probe ultrasound is 70% to 75%, the ultrasound time is 5 to 8 minutes, and the ultrasound is performed for 2 to 3 seconds and then stopped for 3 to 4 seconds.

[0031] Preferably, the stirring time in step (3) is 12 to 14 hours.

[0032] The purification in step (3) is performed by centrifugation and dialysis, wherein the rotation speed used for centrifugation is 10000-12000 rpm.

[0033] After purification in step (3), freeze-drying is performed.

[0034] Preferably, in step (4), the mass ratio of the nanogel loaded with chemotherapy drugs, the reducing agent, and chloroauric acid is 5:2:1 to 3:1.5:1; and the reducing agent is sodium borohydride.

[0035] Preferably, in step (4), chloroauric acid is added under stirring in an ice bath, and stirring in the ice bath is continued for 30 to 40 minutes, and then a reducing agent is added and the reaction is stirred for 3 to 4 hours.

[0036] Preferably, the immune adjuvant in step (5) includes CpG.

[0037] In step (5), the immune adjuvant CpG is adsorbed onto the surface of the dendrimer nanogel loaded with chemotherapy drugs / nanogold by electrostatic adsorption.

[0038] Preferably, in step (5), the nitrogen-phosphorus ratio of the dendrimer nanogel loaded with chemotherapy drugs / nano-gold to the immune adjuvant is 1:3-5; and the incubation at room temperature is 30-40 minutes.

[0039] Preferably, in step (6), the mass ratio of the dendrimer nanogel loaded with chemotherapy drugs / nanogold / immune adjuvant to the exosomes is 1:3 to 1:5.

[0040] The exosomes in step (6) are exosomes derived from pancreatic cancer cells.

[0041] The method for extracting pancreatic cancer-derived exosomes is differential centrifugation.

[0042] Preferably, in the extrusion in step (6), the mixture is extruded 10 to 12 times on an Avanti micro-extruder; and the centrifugal speed is 10,000 to 12,000 rpm.

[0043] Furthermore, in step (6), the extrusion is performed 10 to 12 times through an Avanti extruder using a 220 nm polycarbonate film.

[0044] The invention provides a bionic tumor vaccine, which contains the composite nanogel.

[0045] Furthermore, the biomimetic tumor vaccine includes an exosome-integrated acid-responsive third-generation polyamidoamine dendrimer nanogel vaccine CpG@G3-Toy-Au / DNGs / Exo loaded with toyocamycin / gold nanoparticles / immune adjuvant CpG.

[0046] The present invention provides a use of any composite hydrogel or any composite hydrogel prepared by the method or the bionic tumor vaccine in preparing CT imaging drugs and chemotherapy / immunotherapy drugs for tumors, such as pancreatic cancer.

[0047] The present invention utilizes ultrasonic co-incubation of a dendrimer nanogel loaded with toyocamycin / gold nanoparticles / immune adjuvants with pancreatic cancer-derived exosomes to produce a dendrimer nanogel vaccine. This preparation method features simple reaction conditions, and the resulting product is easily purified and isolated with a high yield. The prepared dendrimer nanogel vaccine not only enables CT imaging of tumors but also targets pancreatic cancer cells through exosome-mediated homologous targeting, inducing immunogenic cell death in these cells. It also directly activates antigen-presenting cells and induces the transition of macrophages to an M1 phenotype, reversing the immunosuppressive tumor microenvironment and triggering a specific anti-tumor immune response, enabling combined chemotherapy / immunotherapy for pancreatic cancer.

[0048] This invention provides an acid-responsive third-generation polyamidoamine dendrimer nanogel coated with exosomes, loaded with toyocamycin, gold nanoparticles, and the immune adjuvant CpG. This gel can be used as a vaccine, laying the foundation for the research of novel nanovaccines. This invention utilizes a third-generation polyamidoamine dendrimer nanogel loaded with chemotherapy drugs, gold nanoparticles, and immune adjuvants, and then encapsulates exosomes acting as tumor antigens, thereby constructing a novel nanovaccine. This nanovaccine can achieve drug release responsive to the acidic tumor microenvironment, activate antigen-presenting cells, reverse the immunosuppressive tumor microenvironment, and further induce an immune response, enabling combined chemotherapy and immunotherapy for tumors.

[0049] The present invention discloses an exosome-encapsulated biomimetic nanogel vaccine, which comprises a third-generation polyamide-amine dendrimer nanogel as a carrier, loaded with chemotherapy drugs, gold nanoparticles, and immune adjuvants, and utilizes cancer cell-derived exosomes as tumor antigens.

[0050] This invention proposes a novel exosome-integrated biomimetic dendrimer nanogel vaccine. This vaccine grafts the chemotherapy drug Toy onto a third-generation polyamide-amine dendrimer (G3-PAMAM) via a boronate bond. Acid-responsive DNGs are then prepared using a reverse microemulsion method with a crosslinker. AuNPs are then in situ encapsulated, followed by electrostatic adsorption of CpG, and finally, pancreatic cancer cell-derived exosomes (Exo). This nanovaccine not only enables CT-guided precision therapy but also, through a combined chemotherapy / immunotherapy strategy, simultaneously kills tumor cells while activating the immune system, potentially significantly enhancing its anti-pancreatic cancer efficacy.

[0051] The nano vaccine is loaded with toyocamycin (Toy) / gold nanoparticles (AuNPs) / immune adjuvant CpG and has pH responsiveness. By injecting the nano vaccine through the tail vein, the exosomes precisely target tumor cells by virtue of homologous targeting, promote Toy to induce immunogenic cell death (ICD) of tumor cells, and stimulate the maturation of dendritic cells (DCs). CpG further promotes DCs maturation through TLR9, and Au NPs can induce M2 macrophages to repolarize to M1, reversing the immunosuppressive microenvironment. The vaccine has both CT imaging performance and can achieve precise tumor imaging and treatment monitoring. Through the synergistic action of multiple mechanisms, including promoting tumor cell apoptosis, inducing tumor cell immunogenic death, reversing the immunosuppressive microenvironment, and activating the immune response, it significantly inhibits tumor growth and recurrence, showing a powerful anti-tumor effect. The present invention provides a new chemotherapy / immunotherapy combination therapy strategy for clinical cancer treatment, which has broad clinical application potential.

[0052] The present invention uses nuclear magnetic resonance hydrogen spectroscopy ( 1 The physicochemical properties of the dendrimer nanogel vaccine were characterized by H NMR, ultraviolet-visible absorption spectroscopy (UV-vis), dynamic light scattering analysis (DLS), surface potential measurement, transmission electron microscopy (TEM), inductively coupled plasma atomic emission spectroscopy (ICP-OES), gel electrophoresis, and Western Blot. The cytotoxicity of CpG@G3-Toy-Au / DNGs / Exo was evaluated by CCK-8 assay. The phagocytic ability of tumor cells towards CpG@G3-Toy-Au / DNGs / Exo was investigated by flow cytometry. The externalization of calreticulin in tumor cells induced by CpG@G3-Toy-Au / DNGs / Exo was investigated by laser confocal microscopy. The effects of CpG@G3-Toy-Au / DNGs / Exo on tumor cell apoptosis, macrophage repolarization, and dendritic cell maturation were evaluated by flow cytometry. The release of high mobility group protein (HMGB-1) and adenosine triphosphate (ATP) in cancer cells treated with CpG@G3-Toy-Au / DNGs / Exo was detected by ELISA. Finally, a subcutaneous tumor-bearing mouse model was established to evaluate the tumor prevention efficacy and in vivo anti-tumor effect of the exosome-coated dendritic macromolecular nanogel vaccine.

[0053] Beneficial effects

[0054] (1) The preparation process of the exosome-encapsulated nanogel vaccine in the present invention is simple and easy to operate, and the product is easy to purify and separate, and has potential practical application value.

[0055] (2) The pancreatic cancer cell-derived Exo extracted in the present invention not only improves immunogenicity but also possesses homologous targeting, giving the nanogel vaccine the ability to actively target and enhance aggregation at the tumor site, providing a new approach for the construction of new nanovaccines. (3) The dendrimer nanogel vaccine prepared in the present invention has good stability, biocompatibility, and pH sensitivity, and can responsively release drugs in the tumor microenvironment, thereby improving drug utilization.

[0056] (4) The nanovaccine prepared by the present invention can effectively prevent tumor growth after being injected into mice through the tail vein for vaccination; after being injected into subcutaneous tumor-bearing mice through the tail vein, it can not only realize CT imaging of the tumor, but also induce immunogenic cell death of pancreatic cancer cells under the action of chemotherapy, directly activate antigen-presenting cells and induce the transformation of M2 macrophages to M1, reverse the immunosuppressive tumor microenvironment, trigger a specific anti-tumor immune response, and realize chemotherapy / immunotherapy combined with pancreatic cancer, providing important reference value for the development of clinical cancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Schematic diagram of the synthesis and application of CpG@G3-Toy-Au / DNGs / Exo prepared in the present invention;

[0058] Figure 2 The G3-PBA (A), Toy (B), and G3-Toy (C) prepared by the present invention are 1 H NMR spectrum;

[0059] Figure 3 TEM image of G3-Toy-Au / DNGs prepared in Example 1;

[0060] Figure 4 Agarose gel electrophoresis images of CpG@G3-Toy-Au / DNGs prepared in Example 1 under different nitrogen-phosphorus ratios;

[0061] Figure 5 Flow cytometric histograms (A) and fluorescence quantitative data (B) of cell phagocytosis of CpG@G3-Toy-Au / DNGs prepared under different nitrogen-phosphorus ratios after co-incubation with Panc-02 cells;

[0062] Figure 6 This is a TEM image of exosomes from Panc-02 cells extracted by ultracentrifugation in Example 1;

[0063] Figure 7 TEM image of CpG@G3-Toy-Au / DNGs / Exo prepared in Example 1;

[0064] Figure 8 This is a Western-blot image of the marker proteins of exosomes in CpG@G3-Toy-Au / DNGs / Exo prepared in Example 1;

[0065] Figure 9 In vitro CT imaging images of CpG@G3-Toy-Au / DNGs / Exo prepared in Example 1 at different gold concentrations (A) and the linear relationship between HU value and gold concentration (B);

[0066] Figure 10 The hydrated particle size changes of CpG@G3-Toy-Au / DNGs / Exo prepared in Example 1 at different pH values ​​(A) and the cumulative drug release curve of Toy (B);

[0067] Figure 11 Flow cytometry results (A, C) and fluorescence quantitative analysis (B, D) of free CpG and CpG@G3-Toy-Au / DNGs and CpG@G3-Toy-Au / DNGs / Exo prepared by the present invention after incubation with Panc-02 cells and L929 cells for 4 hours, respectively;

[0068] Figure 12 Figure 1 is a flow cytometric analysis of apoptosis after the nanovaccine prepared in the present invention was co-incubated with Panc-02 cells for 24 hours (A) and its quantitative analysis results (B);

[0069] Figure 13 Flow cytometric image (A) and quantitative analysis results (B) of the nanovaccine prepared in the present invention after co-incubation with interleukin-4 (IL-4)-induced RAW264.7 cells for 24 hours;

[0070] Figure 14 Immunofluorescence images (A) of calreticulin externalization caused by co-incubation of the nanovaccine prepared by the present invention with Panc-02 cells, as well as the detection results of the relative release of ATP (B) and HMGB-1 (C);

[0071] Figure 15 Figure 2 is a flow cytometric analysis of Panc-02 cells treated with the nanovaccine prepared in the present invention and co-incubated with DC cells for 24 hours (A) and quantitative analysis results of the proportion of mature DC cells (B);

[0072] Figure 16 Figure 2 shows the tumor growth curve (A) and body weight change curve (B) at different time points in Panc-02 subcutaneous tumor-bearing mice after treatment with different preparations.

[0073] Figure 17After Panc-02 subcutaneous tumor-bearing mice were treated with different preparations, CD11c in the lymph nodes was detected. + CD86 + (A) and CD11c + CD80 + (C) Flow cytometric analysis of dendritic cell expression and its quantitative results (B, D);

[0074] Figure 18 The CD4 + CD8 + Flow cytometry of T cells (A) and quantitative analysis results (B, C);

[0075] Figure 19 The expression of Foxp3 in tumor tissues of Panc-02 subcutaneous tumor-bearing mice was detected after treatment with different preparations. + CD25 + Flow cytometric images of Treg cells (A) and their quantitative analysis results (B);

[0076] Figure 20 This is a graph showing the tumor growth curve at different time points after mice were immunized with the nano vaccine prepared by the present invention and then subcutaneously inoculated with tumor cells;

[0077] Figure 21 In the preventive nanovaccine mouse model, CD11c + CD86 + (A) and CD11c + CD80 + (C) Flow cytometric analysis of dendritic cell expression and its quantitative results (B, D);

[0078] Figure 22 To investigate the expression of Foxp3 in tumor tissues in a preventive nanovaccine mouse model. + CD25 + Flow cytometric images of Treg (A) and quantitative analysis results (B). DETAILED DESCRIPTION

[0079] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0080] Unless otherwise specified, all chemical reagents were used directly without further purification. G3.NH2 was purchased from Dendritech, USA. 4-Bromomethylphenylboronic acid (PBA) was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd. Toyocaproin (Toy) was purchased from GLPBIO Shanghai Hongye Biotechnology Co., Ltd. FBA-PEG-FBA crosslinker was purchased from Shanghai Yayi Biotechnology Co., Ltd. Pan02 cells (mouse pancreatic cancer cell line) were purchased from Wuhan Punosai Life Science Co., Ltd. DC cells (mouse bone marrow-derived immature dendritic cells) were from the Affiliated Cancer Hospital of Fudan University. DMEM medium, 1640 medium, trypsin, and penicillin-streptomycin double antibody were purchased from Hangzhou Jinuo Biotechnology Co., Ltd. Fetal bovine serum was purchased from Nanjing Senbeijia Biotechnology Co., Ltd. IL-4 was purchased from Wuhan Yacoin Biotechnology Co., Ltd. Cell apoptosis kit was purchased from Jiangsu Keyi Biotechnology Co., Ltd. ATP detection kit and calreticulin (Anti-CRT) antibody were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. HMGB-1 was purchased from Wuhan Sewell Biotechnology Co., Ltd. Anti-CD80-PE, Anti-CD86-FITC, Anti-CD86-PI, Anti-CD206-FITC, Anti-CD11c-PE, Anti-CD3-PE, Anti-CD8-PE, Anti-CD4-FITC, Anti-CD25-FITC, and Anti-Foxp3-APC were purchased from Thermo Fisher Scientific (Shanghai). C57BL / 6 female mice were purchased from Hangzhou Regen Biotechnology Co., Ltd.

[0081] Example 1

[0082] (1) 80 mg of G3.NH2 PAMAM and 19.9 mg of PBA were dissolved in 5 mL of DMSO, mixed, and stirred in a 70°C water bath for 24 h. The resulting solution was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed against pure water for 3 days to remove DMSO and excess PBA. The dialyzed solution was lyophilized to obtain G3.NH2-PBA.

[0083] (2) Dissolve 10 mg of Toy in 8 mL of pure water and 14.7 mg of G3.NH2-PBA in 2 mL of pure water. Mix the two and stir in a 60°C water bath for 24 h. Transfer the resulting solution to a dialysis bag with a molecular weight cutoff of 1000 Da. Dilute the unloaded Toy with pure water for 3 days and lyophilize to obtain G3-Toy.

[0084] (3) 21 mg of G3-Toy and 15.84 mg of FBA-PEG-FBA were dissolved in 0.5 mL of pure water, respectively. The aqueous solution of G3-Toy and the aqueous solution of FBA-PEG-FBA were thoroughly mixed as the aqueous phase. 234 mg of Span 80 and 46 mg of Tween-80 were thoroughly dissolved in 20 mL of cyclohexane as the organic phase. The aqueous phase was quickly added to the organic phase and sonicated in a probe ultrasonic disruptor at a power of 75% with a 2-second on / 3-second off cycle for 7 min. Subsequently, 500 μL of triethylamine was added dropwise to the reaction system for catalysis while stirring at room temperature. The reaction was stirred at room temperature for 13 h. The reaction system was then centrifuged at 12,000 rpm for 10 min to remove cyclohexane, Tween-80, and Span 80. The G3-Toy / DNG obtained by the reaction was collected and resuspended in methanol. After the reaction, the product was dialyzed in water using a dialysis bag with a molecular weight cutoff of 8000-14000 Da for 3 days to remove the organic solvent, Tween-80 and Span 80, and then lyophilized to obtain purified G3-Toy / DNGs.

[0085] (4) Dissolve 40 mg of G3-Toy / DNGs in 3 mL of pre-cooled pure water, and slowly add 533 μL of pre-cooled HAuCl4·4H2O (30 mg / mL) and 100 μL of pre-cooled NaBH4 (80 mg / mL) to the G3-Toy / DNGs aqueous solution while stirring in an ice bath. Stir for 3 h in an ice bath. The reacted solution is placed in a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzed against pure water for 3 days, and lyophilized to obtain G3-Toy-Au / DNGs.

[0086] (5) The amino content of 2 mg / mL G3-Toy-Au / DNGs was determined using a nitrogen determination kit. G3-Toy-Au / DNGs and CpG were thoroughly mixed according to nitrogen-to-phosphorus ratios of 0.5, 1, 1.5, 2, 2.5, and 3, respectively. The mixture was incubated at room temperature for 30 min. The optimal nitrogen-to-phosphorus ratio for complete compression of CpG by G3-Toy-Au / DNGs was determined using a gel retardation experiment.

[0087] (6) Panc-02 cell-derived exosomes (Exo) were extracted by gradient centrifugation. First, Panc-02 cells in the logarithmic growth phase were inoculated into T75 cell culture flasks and cultured in a 37°C, 5% CO2 incubator for 24 h. The cell supernatant was discarded, washed once with phosphate buffered saline (PBS), and exosome-free culture medium was added for a further 48 h. The cell supernatant was collected and centrifuged at 300 g for 10 min. The supernatant was collected and centrifuged at 3000 g for 10 min. The cell supernatant was carefully collected and filtered with a 450 nm filter membrane to remove impurities. The supernatant was centrifuged at 10,000 g for 30 min. The supernatant was carefully collected and then centrifuged at 100,000 g for 75 min. After centrifugation, the supernatant was removed and the precipitate was resuspended in PBS to obtain purified Exo.

[0088] (7) 400 μg of CpG@G3-Toy-Au / DNGs was thoroughly mixed with Exo at a mass ratio of 1:1, 1:3, and 1:5, respectively. The mixture was placed in an ice bath and repeatedly extruded 10 times through a 220 nm polycarbonate mold on an Avanti microextruder. The mixed solution was centrifuged at 12,000 rpm for 10 min, and the precipitate was collected to obtain purified CpG@G3-Toy-Au / DNGs.

[0089] Example 2

[0090] 5 mg of each of G3.NH2-PBA, Toy, and G3-Toy prepared in Example 1 were dissolved in 650 μL of deuterated water and characterized by hydrogen nuclear magnetic resonance spectroscopy. Figure 2 As shown in (A), the proton peak with a chemical shift of 7.2-7.8 ppm is the characteristic proton peak on the benzene ring of PBA, indicating that PBA is successfully modified on the G3.NH2 surface. In addition, by integrating the peak areas of the characteristic peaks of G3.NH2 and PBA, it is calculated that 6 PBAs are modified on each G3.NH2 surface. Figure 2 As shown in (BC), the characteristic proton peak of Toy is 6.1 ppm, and a new proton peak appears at 6.1 ppm for G3-Toy, indicating that Toy is successfully connected to G3.NH2-PBA by forming a borate ester bond with PBA. By integrating the peak area, it is calculated that 4 Toys are connected to each G3.NH2.

[0091] Example 3

[0092] The G3-Toy-Au / DNGs prepared in Example 1 was dissolved in ultrapure water to prepare a 1 mg / mL G3-Toy-Au / DNGs solution. The solution was dropped onto a copper mesh and allowed to stand for 5 minutes. The excess liquid was then absorbed with filter paper. The morphology of the material was characterized using a JEM-2010F transmission electron microscope at a voltage of 200 kV. The TEM image of G3-Toy-Au / DNGs is shown in Figure 1. Figure 3 As shown, G3-Toy-Au / DNGs exhibited a uniformly distributed morphology, and gold nanoparticles aggregated in the nanogel.

[0093] Example 4

[0094] The CpG@G3-Toy-Au / DNGs prepared in Example 1 at different nitrogen-phosphorus ratios (N / P) were subjected to gel retardation experiments. Agarose gel electrophoresis was performed at a voltage of 100 V for 30 min, and gel imaging of the experimental results was performed. Figure 4 As shown in the figure, when N / P is 1.5, G3-Toy-Au / DNGs can completely compress CpG.

[0095] Example 5

[0096] The CpG@G3-Toy-Au / DNGs prepared at different nitrogen-phosphorus ratios (N / P) in Example 1 were co-incubated with Panc-02 cells for 4 h to evaluate the phagocytic ability of Panc-02 cells towards CpG@G3-Toy-Au / DNGs with different N / P ratios, and the optimal nitrogen-phosphorus ratio for the complexation of G3-Toy-Au / DNGs and CpG was determined. Panc-02 cells were cultured at a density of 2×10 6 Cells were seeded in a 6-well plate and incubated overnight in a 37°C, 5% CO2 cell culture incubator. The culture medium was discarded, and 1 mL of fresh culture medium containing CpG@G3-Toy-Au / DNGs with different N / P ratios was added and incubated in a 37°C, 5% CO2 cell culture incubator for 4 h. The culture medium was discarded, the cells were trypsinized, centrifuged, washed 3 times with PBS, and resuspended in 400 μL PBS. The fluorescence in the cells was detected by flow cytometry. Figure 5 As shown in (AB), when N / P=5, the fluorescence intensity in Panc-02 cells was the highest, indicating the best cell phagocytosis effect. Therefore, G3-Toy-Au / DNGs and CpG were complexed at a ratio of N / P=5 for subsequent experiments.

[0097] Example 6

[0098] The exosomes and CpG@G3-Toy-Au / DNGs / Exo extracted in Example 1 were dropped onto a copper grid and allowed to stand for 5 minutes. The excess liquid was absorbed with filter paper. The exosomes were negatively stained with 2% phosphotungstic acid for 5 minutes. The morphology of Exo and CpG@G3-Toy-Au / DNGs / Exo was photographed using a JEM-2010F transmission electron microscope (voltage 200kV). The results are shown in Figure 2. Figure 6 As shown in Figure 2, the edges of negatively stained exosomes are bright and appear disc-shaped or cup-shaped. The periphery of CpG@G3-Toy-Au / DNGs / Exo appears as a bright ring ( Figure 7 ), indicating that exosomes were successfully coated on the surface of CpG@G3-Toy-Au / DNGs.

[0099] Example 7

[0100] The Exo and CpG@G3-Toy-Au / DNGs / Exo cells and Panc-02 cells in Example 1 were lysed with lysis buffer, and the marker proteins (TSG101 and CD63) on the surface of exosomes were detected by Western blot. Figure 8 As shown in the figure, the characteristic proteins on the surface of Exo were retained in CpG@G3-Toy-Au / DNGs / Exo, further indicating that Exo was successfully coated on the surface of CpG@G3-Toy-Au / DNGs.

[0101] Example 8

[0102] Take the CpG@G3-Toy-Au / DNGs / Exo in Example 1, and use a CT imaging system to detect the X-ray attenuation performance of CpG@G3-Toy-Au / DNGs / Exo at different gold concentrations. The gold concentration in CpG@G3-Toy-Au / DNGs / Exo was diluted to 10, 20, 30, 40, and 50 mM, respectively, and the CpG@G3-Toy-Au / DNGs / Exo with different gold concentrations was imaged using a CT imaging system. Figure 9 As shown in the figure, with the increase of gold concentration, the CT value of CpG@G3-Toy-Au / DNGs / Exo gradually increased and exhibited a good X-ray attenuation coefficient, indicating that the exosome-coated dendrimer nanogel vaccine has good X-ray attenuation performance and has the potential to be used for in vivo CT imaging.

[0103] Example 9

[0104] PBS buffer solutions of pH = 7.4 and pH = 5.5 were prepared to analyze the pH responsiveness of CpG@G3-Toy-Au / DNGs / Exo. 1 mg / mL CpG@G3-Toy-Au / DNGs / Exo was prepared using PBS buffer solutions of pH = 7.4 and pH = 5.5, and the hydration kinetic diameter of CpG@G3-Toy-Au / DNGs / Exo in different pH buffers was measured using a Malvern particle size analyzer. Figure 10 As shown in A, the particle size of CpG@G3-Toy-Au / DNGs / Exo is concentrated at pH = 7.4, while two peaks appear at pH = 5.5, indicating that the size of CpG@G3-Toy-Au / DNGs / Exo changes in an acidic environment and the particle size is not uniform. CpG@G3-Toy-Au / DNGs / Exo was dissolved in 1 mL of PBS buffer solution at pH = 7.4 and pH = 5.5, respectively, and the concentration was 3 mg / mL. They were loaded into dialysis bags with a molecular weight of 3500 Da, and the dialysis bags were placed in 50 mL centrifuge tubes containing 9 mL of PBS buffer solution at pH = 7.4 and pH = 5.5, respectively, and shaken on a thermostatic shaker with a speed of 180 rpm and a temperature of 37 ° C. At different time points, 1 mL of the solution in the tube was taken each time, and 1 mL of the corresponding buffer was added to calculate the cumulative drug release of Toy at different time points. Figure 10 As shown in Figure B, the cumulative Toy release of CpG@G3-Toy-Au / DNGs / Exo at pH = 5.5 (51.9%) was significantly higher than that at pH = 7.4 (12.8%). These results indicate that CpG@G3-Toy-Au / DNGs / Exo is acid-responsive and can rapidly release drugs in the acidic tumor microenvironment.

[0105] Example 10

[0106] Panc-02 cells and L929 cells were used as cell models to evaluate the phagocytic ability of Free Toy, CpG@G3-Toy-Au / DNGs, and CpG@G3-Toy-Au / DNGs / Exo. Panc-02 cells and L929 cells in the logarithmic growth phase were taken and 1.5×10 5 Cells were seeded in a 6-well plate and cultured in a 37°C, 5% CO2 cell culture incubator for 16 h. The culture medium was discarded and serum-free culture medium containing different materials was added. The cells were incubated in the incubator for 4 h. The supernatant was discarded and the cells were washed twice with PBS buffer. After trypsin digestion and centrifugation, the cells were washed three times with PBS and resuspended in 350 μL PBS. The fluorescence intensity of the cells was detected by flow cytometry. Figure 11As shown in the results, free CpG was hardly taken up by Panc-02 and L929, while CpG@G3-Toy-Au / DNGs could be significantly taken up by Panc-02 and L929. In L929 cells, there was no significant difference between the uptake of CpG@G3-Toy-Au / DNGs / Exo and that of CpG@G3-Toy-Au / DNGs. In Panc-02 cells, the intracellular fluorescence intensity after treatment with CpG@G3-Toy-Au / DNGs / Exo was significantly higher than that of the CpG@G3-Toy-Au / DNGs group. This is because exosomes derived from pancreatic cancer cells can mediate homologous targeting, thereby increasing the phagocytic effect of Panc-02 on CpG@G3-Toy-Au / DNGs / Exo.

[0107] Example 11

[0108] Panc-02 cells were used as a cell model to investigate the apoptotic effects of Free Toy, G3-Toy / DNGs, G3-Toy-Au / DNGs, CpG@G3-Toy-Au / DNGs, and CpG@G3-Toy-Au / DNGs / Exo on Panc-02 cells. Panc-02 cells in the logarithmic growth phase were taken and 1.5×10 5 Cells were seeded in a 6-well plate and cultured in a cell culture incubator at 37°C and 5% CO2 for 16 hours. The culture medium was discarded, the cells were washed with PBS, and complete culture medium containing Free Toy, G3-Toy / DNGs, G3-Toy-Au / DNGs, CpG@G3-Toy-Au / DNGs, and CpG@G3-Toy-Au / DNGs / Exo (Toy concentration was 5 μg / mL) was added. The cells were incubated in a cell culture incubator for 24 hours. The apoptotic or necrotic cells in the supernatant were collected and the cells in the wells were digested with trypsin. After centrifugation, the cells were washed 3 times with PBS. The cells were gently blown into a single cell suspension with 500 μL Binding Buffer, 5 μL Annexin V-FITC was added and mixed, and then 5 μL PI was added and mixed. After incubation at room temperature in the dark for 10 minutes, the apoptosis of Panc-02 cells treated with different material groups was detected by flow cytometry. Figure 12 As shown in the figure, compared with the free and other material groups, the apoptosis rate of cells in the CpG@G3-Toy-Au / DNGs / Exo group was the highest. This is because exosomes have homologous targeting, which can increase the amount of drugs phagocytized by cancer cells and promote cell apoptosis.

[0109] Example 12

[0110] In order to verify the effect of CpG@G3-Toy-Au / DNGs / Exo on the repolarization process of mouse macrophage RAW 264.7, 1.5×10 5 RAW 264.7 cells were seeded into 6-well plates and cultured in a 37°C, 5% CO2 cell culture incubator for 16 h. The culture medium was discarded and 50 ng / mL IL-4 was added to induce the cells for 24 h to polarize the RAW 264.7 cells to M2 type. The culture medium was discarded and replaced with 50 ng / mL IL-4 containing lipopolysaccharide (LPS), Free Toy, G3-Toy / DNGs, G3-Toy-Au / DNGs, CpG@G3-Toy-Au / DNGs, CpG@G3-Toy-Au / DNGs / Exo (Toy concentration is 5μg / mL, LPS concentration is 2μg / mL) fresh culture medium and cells were co-incubated in the incubator for 24h, the supernatant was discarded, the cells were blown off with PBS and centrifuged, the supernatant was discarded, PBS containing Anti-CD206-FITC and Anti-CD86-PI antibodies was added, the cells were stained on ice in the dark for 30min, washed 3 times with PBS, and the cells were resuspended in 300μL PBS. Flow cytometry was used to detect the promotion of repolarization of M2 type RAW 264.7 cells by different materials. Figure 13 As shown in the data, compared with the IL-4 group, the expression of CD206 in the G3-Toy-Au / DNGs, CpG@G3-Toy-Au / DNGs, and CpG@G3-Toy-Au / DNGs / Exo groups was significantly decreased, and the expression of CD86 was significantly increased. In particular, the M1 / M2 ratio of the CpG@G3-Toy-Au / DNGs / Exo group was significantly higher than that of the other groups. This may be because Au and CpG can promote the repolarization of M2 macrophages, and the coating of exosomes may increase the phagocytosis of dendrimer nanogels by macrophages, indicating that CpG@G3-Toy-Au / DNGs / Exo can effectively promote the repolarization of M2 macrophages and contribute to the activation of anti-tumor immunity.

[0111] Example 13

[0112] In order to investigate whether the prepared nanovaccine can induce immunogenic cell death (ICD) in Panc-02 cells, the externalization of calreticulin (CRT) and the release of high mobility group protein (HMGB-1) and ATP in Panc-02 cells treated with different materials were investigated by confocal microscopy and ELISA. Panc-02 cells in the logarithmic growth phase were taken and plated at 1.5×10 5The cells were seeded at a density of 100 cells / mL in a confocal dish and cultured in a cell culture incubator at 37°C and 5% CO2 for 16 h. The supernatant was discarded and fresh culture medium containing Free Toy, G3-Toy / DNGs, G3-Toy-Au / DNGs, CpG@G3-Toy-Au / DNGs, and CpG@G3-Toy-Au / DNGs / Exo was added (the concentration of Toy was 5 μg / mL) and incubated with the cells for 24 h. The supernatant was discarded and CRT immunofluorescence staining was performed on cells treated with different materials. Figure 14 As shown in A, the green fluorescence intensity of cells in the G3-Toy / DNGs group was higher than that in the Free Toy group. Compared with the other material groups, the green fluorescence intensity of cells in the CpG@G3-Toy-Au / DNGs / Exo group was the strongest, indicating that compared with free drugs, the drug-loaded nanogels can be better phagocytosed by cancer cells. The homologous targeting performance mediated by exosomes further increased the phagocytic ability of cancer cells to the nanovaccine, thereby leading to more CRT externalization in cancer cells. In addition, Panc-02 cells were cultured at 1.5×10 5 Cells were seeded in 6-well plates and cultured in a 37°C, 5% CO2 cell culture incubator for 16 h. The supernatant was discarded and fresh culture medium containing Free Toy, G3-Toy / DNGs, G3-Toy-Au / DNGs, CpG@G3-Toy-Au / DNGs, and CpG@G3-Toy-Au / DNGs / Exo (Toy concentration was 5 μg / mL) was added, respectively. The cells were incubated in a cell culture incubator for 24 h, and the cell supernatant was collected. The release of HMGB-1 and ATP in the cell supernatant was detected by ELISA kit. Figure 14 As shown in Figures BC, the CpG@G3-Toy-Au / DNGs / Exo group had the highest relative release of extracellular ATP and HMGB-1 compared with the other formulation groups. These results indicate that CpG@G3-Toy-Au / DNGs / Exo can effectively promote ICD in Panc-02 cells and effectively activate anti-tumor immune responses.

[0113] Example 14

[0114] In order to verify the DC maturation induced by the prepared nanovaccine on tumor cell ICD, 1.0×10 5 DC cells were seeded in a 12-well plate and placed in the upper chamber of a 3 μm Transwell chamber at a rate of 1.0×10 cells per chamber. 5Panc-02 cells were inoculated, a Transwell system was established, and the cells were cultured in a 37°C, 5% CO2 cell culture incubator for 16 h. The supernatant in the chamber and well plate was discarded, and Free Toy, G3-Toy / DNGs, G3-Toy-Au / DNGs, CpG@G3-Toy-Au / DNGs, CpG@G3-Toy-Au / DNGs / Exo (Toy concentration is 5μg / mL) fresh culture medium, only fresh culture medium was added to the well plate, DC cells and Panc-02 cells were co-incubated in a cell culture incubator for 24h, the supernatant in the well plate was discarded, DC cells were trypsinized, the cells were washed 3 times with PBS, PBS containing Anti-CD86-FITC and Anti-CD80-PE antibodies was added, DC cells were stained on ice in the dark for 30min, washed 3 times with PBS, DC cells were resuspended in 300μL PBS, and the maturation degree of DC cells after co-incubation of Panc-02 cells treated with different materials with DC cells was detected by flow cytometry. Figure 15 As shown in the figure, compared with other groups, after Panc-02 cells treated with CpG@G3-Toy-Au / DNGs / Exo were co-cultured with DC cells, CD80 + CD86 + The proportion of DC cells is the highest. This is because CpG@G3-Toy-Au / DNGs / Exo can induce more Panc-02 cells to undergo ICD, release more damage-associated molecular patterns, and then promote DC cell maturation, which can effectively promote anti-tumor immune response in vivo. In addition, the CpG carried by the nanovaccine and the tumor antigens carried by exosomes can also directly activate DCs to cause anti-tumor immune activation.

[0115] Example 15

[0116] All animal experiments were conducted in strict accordance with the standards of the Animal Care Association. 4-5 week old C57BL / 6 female mice were purchased from Shanghai Slake Laboratory Animal Co., Ltd. To investigate the in vivo anti-tumor efficacy of exosome-encapsulated dendrimer nanogel vaccine as a therapeutic nanovaccine, a Panc-02 subcutaneous tumor model was established in C57BL / 6 female mice. Each mouse was treated with 2 × 10 7 The mice were subcutaneously injected with 100 μL of Panc-02 cells at a density of 100 μL / mL of Panc-02 cells on the right back of each mouse. 3The mice were randomly divided into 6 groups (5 mice in each group), specifically divided into PBS, Free Toy, G3-Toy / DNGs, G3-Toy-Au / DNGs, CpG@G3-Toy-Au / DNGs, CpG@G3-Toy-Au / DNGs / Exo (Toy concentration was 2 mg / kg), and the mice were administered via tail vein once every 3 days for a total of 5 times. The body weight and tumor volume of the mice were recorded every other day. Tumor volume (V) = L × W 2 / 2 (L is the length of the tumor, W is the width of the tumor). Figure 16 As shown in A, the tumor volume of the CpG@G3-Toy-Au / DNGs / Exo group was the smallest compared with the other groups, indicating that the exosome-encapsulated dendrimer nanogel vaccine can effectively inhibit tumor growth as a therapeutic nanovaccine. Figure 16 As shown in B, there was no significant change in the body weight of mice in different groups during tail vein administration, which indicates that the exosome-coated dendrimer nanogel vaccine has good biosafety.

[0117] To investigate the anti-tumor immune response in mice treated with different materials, lymph nodes, spleen, and tumor tissues were aseptically collected from mice in each treatment group, minced, ground, and passed through a 400-mesh cell sieve to obtain a cell suspension. The extracted lymph node cell suspension was washed with PBS, centrifuged, and then mixed with Anti-CD11c-FTIC / Anti-CD80-PE or Anti-CD11c-FTIC / Anti-CD86-PE. The cells were stained in an ice bath in the dark for 30 minutes, washed three times with PBS, and resuspended in 400 μL of PBS. Flow cytometry was used to analyze the degree of DC maturation in the lymph nodes treated with the different materials. In addition, the extracted spleen cell suspension was purified by nylon wool column to obtain T lymphocytes, the cells were washed with PBS and centrifuged, the purified T lymphocytes were incubated with Anti-CD4-FTIC and Anti-CD8-PE in an ice bath in the dark for 30 minutes, washed with PBS three times, resuspended in 500 μL PBS and transferred to flow cytometry tube, and flow cytometry was used to examine the CD4 in the spleen of mice treated with different materials. + T cells and CD8 + At the same time, the extracted tumor tissue cell suspension was washed with PBS and centrifuged, and the extracted tumor tissue cells were stained with Tregs cell markers (Anti-CD4-PE, Anti-CD25-FITC, Anti-Foxp3-APC) on ice for 30 minutes, washed with PBS three times, and resuspended in 500 μL PBS and transferred to a flow tube. Flow cytometry was used to examine the CD4 + CD25 + Foxp3 +The proportion of Tregs cells.

[0118] like Figure 17 As shown in AB, the maturation ratio of DC cells in lymphocytes of CpG@G3-Toy-Au / DNGs group (CD11c + CD86 + 、CD11c + CD86 + ) was significantly higher than in the other treatment groups. This may be because Toy induces ICD in cancer cells and releases more DAMPs, which can induce DC cell maturation, while gold nanoparticles and CpG can further stimulate DC maturation. In addition, the DC cell maturation rate in the CpG@G3-Toy-Au / DNGs / Exo group was higher than that in the CpG@G3-Toy-Au / DNGs group. This is because Exo, as a tumor antigen, can further promote DC cell maturation.

[0119] CD4 in spleen of tumor-bearing mice after different treatments + and CD8 + The proportion of T cells Figure 18 As shown, CD8 + T cells (33.1%) and CD4 + The proportion of T cells (40.6%) was significantly higher than that of the other treatment groups, which was consistent with the trend of DC maturation, indicating that CpG@G3-Toy-Au / DNGs / Exo stimulated DC maturation and presented antigens to T cells, thereby activating anti-tumor immunity and improving the effect of tumor treatment.

[0120] The flow cytometry results of Treg cells in the tumor sites of each treatment group showed that the level of Treg was significantly downregulated after treatment with nanomedicine, and the Treg level in the tumor site of the CpG@G3-Toy-Au / DNGs / Exo treatment group was the lowest. This result proves that the nanovaccine prepared by the present invention can effectively reverse the immunosuppressive microenvironment and help reactivate the anti-tumor immune response ( Figure 19 ).

[0121] Example 16

[0122] In order to investigate the tumor prevention effect of the nanovaccine prepared in Example 1, PBS, CpG@G3-Toy-Au / DNGs, and CpG@G3-Toy-Au / DNGs / Exo (Toy concentration was 2 mg / kg) were first injected into the tail vein of C57BL / 6 healthy female mice. The tail vein was administered once every 6 days for a total of 4 times. On the 6th day after the last administration, 2×10 7Panc-02 cells were inoculated 4 days later and the changes in tumor volume were monitored over 16 days. Figure 20 As shown, compared with the PBS group and the CpG@G3-Toy-Au / DNGs group, the tumor volume of the CpG@G3-Toy-Au / DNGs / Exo group was the smallest and the tumor growth was the slowest, indicating that after prevention by the exosome-coated dendrimer nanogel vaccine, the mice were able to inhibit tumor occurrence.

[0123] In order to investigate the immune effect of exosome-coated dendrimer nanogel vaccine as a preventive nanovaccine in mice. Lymph nodes, spleen and tumor tissues of mice in each prevention group were collected under sterile conditions, minced and ground, and then passed through a 400-mesh cell sieve to obtain a cell suspension. The extracted lymph node cells were labeled with Anti-CD11c-FTIC / Anti-CD80-PE or Anti-CD11c-FTIC / Anti-CD86-PE, and the extracted spleen cell suspension was purified through a nylon wool column to obtain T lymphocytes. T lymphocytes were labeled with Anti-CD4-FTIC and Anti-CD8-PE, and Tregs cells in the extracted tumor tissue cells were labeled with Anti-CD4-PE / Anti-CD25-FITC / Anti-Foxp3-APC. The above-stained and labeled cells were transferred to flow tubes, and flow cytometry was used to analyze the degree of DC maturation in lymph nodes and CD4 in spleen treated with different materials. + T cells and CD8 + The proportion of T cells and the proportion of Tregs cells in tumor tissues.

[0124] The DC cell maturation ratio in the lymph nodes of mice treated with preventive nanovaccines was Figure 21 As shown in the figure, compared with the PBS group, the proportion of DC cell maturation in the lymph nodes of the CpG@G3-Toy-Au / DNGs group and the CpG@G3-Toy-Au / DNGs / Exo group was increased, but the proportion in the CpG@G3-Toy-Au / DNGs / Exo group was even higher. This is because CpG as an immune adjuvant can promote the maturation of DC cells, while Exo as a tumor antigen can further stimulate the maturation of DC cells. + T cells and CD8 + The proportion of T cells, for example Figure 22 As shown in the figure, compared with the PBS group, both the CpG@G3-Toy-Au / DNGs prevention group and the CpG@G3-Toy-Au / DNGs / Exo prevention group could induce CD8 + T cells and CD4 +The proportion of T cells increased, and the increase was more obvious in the CpG@G3-Toy-Au / DNGs / Exo prevention group. This was because the dual effects of CpG and the antigens carried by Exo caused more DC cells to mature. The mature DC cells presented tumor antigens to T cells, causing an enhanced immune response.

[0125] In general, the CpG@G3-Toy-Au / DNGs / Exo constructed in the present invention can be used as both a therapeutic nanovaccine and a preventive nanovaccine. It can effectively promote DC maturation by inducing ICD in cancer cells and the CpG and tumor antigens it carries. At the same time, gold nanoparticles can also cause M1-type repolarization of macrophages, reverse the immunosuppressive tumor microenvironment, induce tumor-specific immune responses, and thus effectively inhibit tumor growth.

Claims

1. A composite nanogel, characterized in that: The composite nanogel comprises exosomes and polyamide-amine dendrimer nanogel, wherein the exosomes are coated on the surface of the polyamide-amine dendrimer nanogel; and the polyamide-amine dendrimer nanogel is loaded with drugs, nanogold and immune adjuvants.

2. A method for preparing a composite nanogel, comprising: (1) 4-bromomethylphenylboronic acid PBA, G3.NH2 PAMAM, and a solvent are mixed, reacted, and purified to obtain G3.NH2-PBA; (2) mixing G3.NH2-PBA and an aqueous solution of a chemotherapy drug, reacting, and purifying to obtain G3-chemotherapeutic drug; (3) mixing G3-chemotherapeutic drug, crosslinker, and water to form an aqueous phase, then adding the aqueous phase to the organic phase, sonicating, adding a catalyst, stirring, and purifying to obtain a dendrimer nanogel loaded with chemotherapeutic drug; (4) dissolving the nanogel loaded with chemotherapy drugs in water, adding chloroauric acid under ice-bath stirring, continuing ice-bath stirring, then adding a reducing agent, stirring for reaction, and purifying to obtain a dendrimer nanogel loaded with chemotherapy drugs / nano-gold; (5) mixing the dendrimer nanogel loaded with chemotherapy drugs / nano-gold, water, and immune adjuvant, and incubating at room temperature to obtain the dendrimer nanogel loaded with chemotherapy drugs / nano-gold / immune adjuvant; (6) The dendrimer nanogel loaded with chemotherapy drugs / nanogold / immune adjuvants and exosomes are mixed to obtain a mixture, which is then extruded and centrifuged to obtain a composite nanogel.

3. The preparation method according to claim 2, characterized in that: In step (1), the molar ratio of G3.NH2 PAMAM to PBA is 1:8 to 1:9; the solvent comprises dimethyl sulfoxide; In the step (1), the reaction is stirred at 65-70° C. for 24-26 hours.

4. The preparation method according to claim 2, characterized in that The chemotherapy drug in step (2) includes Toyocamycin Toy; the molar ratio of PBA to the chemotherapy drug in G3.NH2-PBA in step (2) is 1:3 to 1:5; The reaction in step (2) is carried out at 55-60° C. with stirring for 24-26 hours.

5. The preparation method according to claim 2, characterized in that: In step (3), the cross-linking agent includes FBA-PEG-FBA; the oil phase is a mixture of Span 80, Tween 80 and an organic solvent; and the catalyst includes triethylamine; In step (3), the molar ratio of G3-chemotherapeutic drug, cross-linking agent, Span 80 and Tween 80 is 1:3:190:21 to 1:4:190:21; The ultrasound in step (3) is probe ultrasound, wherein the power of the probe ultrasound is 70% to 75%, the ultrasound time is 5 to 8 minutes, and the ultrasound is continued for 2 to 3 seconds and then stopped for 3 to 4 seconds; The stirring time in step (3) is 12 to 14 hours.

6. The preparation method according to claim 2, characterized in that: In step (4), the mass ratio of the nanogel loaded with chemotherapy drugs, the reducing agent, and chloroauric acid is 5:2:1 to 3:1.5:1; the reducing agent is sodium borohydride; In the step (4), chloroauric acid is added under stirring in an ice bath, and stirring in the ice bath is continued for 30 to 40 minutes, and then a reducing agent is added, and the reaction is stirred for 3 to 4 hours.

7. The preparation method according to claim 2, characterized in that: In step (5), the immune adjuvant includes CpG; In the step (5), the nitrogen-phosphorus ratio of the dendrimer nanogel loaded with chemotherapy drugs / nano-gold to the immune adjuvant is 1:3-5; and the incubation at room temperature is 30-40 minutes.

8. The preparation method according to claim 2, characterized in that: In step (6), the mass ratio of the dendrimer nanogel loaded with chemotherapy drugs / nanogold / immune adjuvant to the exosomes is 1:3 to 1:5; In the extrusion step (6), the mixture is extruded 10 to 12 times on an Avanti micro-extruder; the centrifugal speed is 10,000 to 12,000 rpm.

9. A biomimetic tumor vaccine, characterized in that: The bionic tumor vaccine contains the composite nanogel according to claim 1.

10. Use of the composite hydrogel according to claim 1 or the composite hydrogel prepared by the method according to any one of claims 2 to 9, or the biomimetic tumor vaccine according to claim 9 in the preparation of CT imaging drugs or chemotherapy / immunotherapy combined treatment drugs for tumors.

Images