Glutathione-responsive exosome biomimetic dendrimer nanogel vaccine and preparation method and application thereof

Abstract

The present application relates to a kind of glutathione-responsive exosome biomimetic dendrimer nanogel vaccine and its preparation method and application.The vaccine is loaded with 5-Aza, coated with tumor cell Ex and modified R837 in turn to prepare dendrimer nanogel as carrier.The vaccine realizes precise drug delivery of tumor by virtue of the homologous targeting characteristics of Ex, and can target drug release in response to the high glutathione characteristics of tumor microenvironment.5-Aza exerts epigenetic therapy effect through DNA demethylation, while inducing pyroptosis type ICD of tumor cells.R837 immunoadjuvant can synergize with tumor-associated antigens carried by Ex to promote the maturation of dendritic cells, promote the polarization of M2 type tumor-associated macrophages to M1 type, and reverse the tumor immunosuppressive microenvironment.The present application combines epigenetic therapy and immunotherapy, providing a new strategy for the clinical treatment of pancreatic cancer, and has wide clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of functional nanomaterials and nanomedicines, and specifically relates to a glutathione-responsive exosome-inspired dendritic macromolecular nanogel vaccine, its preparation method, and its application. Background Technology

[0002] Malignant tumors pose a serious threat to human life and health. Commonly used clinical treatments for tumors include surgery, chemotherapy, and radiotherapy. However, these traditional therapies generally suffer from poor patient prognosis, high tumor recurrence rates, and difficulty in improving survival rates, resulting in limited overall treatment effectiveness (Yu et al.). Nat. Nanotechnol. , 2026, 21 (3), 443-454). In recent years, tumor immunotherapy has developed rapidly, showing excellent potential for anti-tumor application. Among them, tumor vaccines can induce the body to produce tumor-specific immune responses and form immune memory, which can effectively prevent tumor recurrence and are a highly advantageous immunotherapy method (Li et al.). Cancer Lett. (2025, 625, 217752). Currently, the clinical efficacy of tumor vaccines remains unsatisfactory, mainly due to multiple factors: strong tumor heterogeneity, weak antigenic immunogenicity, low in vivo vaccine delivery efficiency, and the immunosuppressive properties of the tumor microenvironment (TME) significantly weaken the body's anti-tumor immune response (Guo et al.). Bioact. Mater. (2024, 43, 129-144). Furthermore, tumor development and progression are closely related to epigenetic abnormalities, with DNA hypermethylation being the most prevalent type of epigenetic abnormality in tumors (Bhootra et al.). Med. Oncol. , 2023, 40 (2), 71). Therefore, improving the antigen delivery efficiency of tumor vaccines, improving immunosuppressive TME, and combining targeted epigenetic intervention are the core issues that urgently need to be addressed in the current field of tumor treatment.

[0003] In tumor cells, abnormal hypermethylation often occurs in the promoter regions of tumor suppressor genes. This process is catalyzed by DNA methyltransferase (DNMT), which transfers the methyl group of S-adenosylmethionine to a cytosine residue in DNA to generate 5-methylcytosine, causing the tumor suppressor gene to be silenced and inactivated, thereby driving tumor proliferation and metastasis (Liu et al.). Front. Pharmacol. ,2022, 13, 958146). 5-Azacytidine (5-Aza) is a highly specific DNMT inhibitor that can reverse abnormal tumor methylation, restart tumor suppressor genes, inhibit tumor growth and migration, and regulate the tumor cell cycle (Wong). Cell. Oncol., 2020,(5), 779-792). Pyroptosis is a programmed cell death process accompanied by an inflammatory response, characterized by cell swelling, cell membrane rupture, and release of immunogenic substances, and is mainly mediated by the GSDME protein (Wang et al.). Small , 2024,(17), 2307829). Studies have confirmed that 5-Aza can downregulate the methylation level of the GSDME gene, activate GSDME-mediated pyroptosis, and induce tumor immunogenic cell death (ICD) (Wang et al.). ACS Nano (2024, 18, 31, 20167–20180). In addition, 5-Aza can upregulate the expression of miR-7083-5p, and mediate the polarization of tumor-associated macrophages (TAMs) from the oncogenic M2 phenotype to the tumor suppressor M1 phenotype by inhibiting the expression of colony-stimulating factor 2 receptor α (CSF2RA) and CD43, providing an important basis for the activation of the body's anti-tumor immune system (Liu et al.). Cancer Lett. (2025, 632,217972). However, free 5-Aza has poor stability, lacks tumor targeting, and is prone to causing systemic toxicity, making it difficult to exert precise effects in the tumor microenvironment (TME), which greatly limits its anti-tumor efficacy. Therefore, developing intelligent nanodelivery systems to achieve tumor-specific release of 5-Aza is key to improving its therapeutic effect and reducing toxicity.

[0004] Simple demethylation therapy can only inhibit tumor proliferation and induce limited ICD effects, failing to generate a long-lasting immune response and thus making it difficult to completely eliminate tumor cells and prevent tumor recurrence. Compared to single-treatment regimens, nano-tumor vaccines possess stronger comprehensive therapeutic advantages and have become a research hotspot in cancer treatment. Nano-vaccines mainly consist of nanocarriers, tumor antigens, and immune adjuvants, enabling efficient co-delivery of multiple components and synergistically enhancing anti-tumor efficacy (Liu et al.). Sci. Adv. , 2024,10 (11), eadk2444).

[0005] Tumor cell-derived exosomes (Ex) are natural lipid bilayer vesicles with homologous tumor targeting capabilities (Zhou et al.). Biomaterials (2021, 268, 120546), and is rich in tumor-specific antigens, making it an ideal natural antigen for nanovaccines (Zou et al.). ACS Nano, 2025, 19 (18), 17309-17322). Imiquimod (R837), as a TLR7 / 8 agonist, is a highly effective immune adjuvant that can activate DC maturation, enhance antigen presentation efficiency, and promote the polarization of TAM in the TME from the immunosuppressive M2 type to the pro-inflammatory M1 type, effectively improving the tumor immunosuppressive microenvironment (Huang et al.). ACS Appl. Mater. Interfaces , 2024, 16 (21), 27187-27201).

[0006] Compared to other nanodelivery carriers, dendritic macromolecular nanogels (DNGs) combine the performance advantages of dendritic macromolecules and nanogels. They are not only highly branched and have flexible surface modification capabilities, but also possess excellent fluid properties and biocompatibility, enabling efficient loading of drugs, cytokines, and genes, and show promising prospects in anti-tumor research (Xiao et al.). Bioact. Mater. , 2025, 56, 3-14; Wang et al. Mater. Today Bio (2025, 35, 102614). This material can utilize its amplified solid tumor EPR effect to achieve efficient passive targeted enrichment of tumor tissue; at the same time, after structural design using functional cross-linking agents, DNGs can respond to glutathione (GSH), pH, and reactive oxygen species (ROS) within the TME, thereby achieving precise controlled release of drugs at the tumor site (Xu et al.). Biomacromolecules , 2023, 24 (2), 967-976;Zhang et al. Adv. Sci. , 2023, 10 (24), 2301759). With the above comprehensive advantages, DNGs are expected to improve the problems of insufficient vaccine antigen delivery efficiency and antagonism of tumor immunosuppressive microenvironment, while synergistically improving the efficacy of demethylation therapy. As an ideal multifunctional carrier platform, it can be applied to the epigenetic-immunotherapy system.

[0007] Based on domestic and international literature and patent searches, there are currently no reports on integrated dendritic macromolecular nanogel vaccines that combine GSH-responsive characteristics, exosome-inspired biomimetic targeting functions, and epigenetic regulation and immune activation. Summary of the Invention

[0008] The technical problem to be solved by this invention is to provide a method for preparing and applying a third-generation polyamide-amine dendritic macromolecular nanogel vaccine that is exosome-inspired and glutathione-responsive. This vaccine is loaded with 5-azacytidine and the immunoadjuvant R837, providing a research foundation for the development of novel nanovaccines. This invention uses a third-generation polyamide-amine dendritic macromolecular nanogel as a carrier, loads the epigenetic drug 5-azacytidine, encapsulates tumor cell-derived exosomes, and modifies the immunoadjuvant R837 to construct a novel nanovaccine. This vaccine can achieve targeted drug release in response to glutathione stimulation in the tumor microenvironment. Through tumor demethylation therapy and pyroptosis-mediated immunogenic cell death, it activates antigen-presenting cells, reverses the tumor immunosuppressive microenvironment, initiates an anti-tumor immune response, and ultimately achieves synergistic combined therapy of tumor epigenetics and immunity.

[0009] A method for preparing a glutathione-responsive exosome-inspired dendritic macromolecular nanogel vaccine includes the following steps: Step (1) The dendritic macromolecules and crosslinking agents are dispersed in pure water to form an aqueous phase; Sorbitan oleate (Span 80) and polyoxyethylene 20 sorbitan monooleate (Tween 80) are dissolved in an organic solvent to form an organic phase; The aqueous phase is rapidly added to the organic phase to form a W / O mixture, which is then ultrasonically dispersed by a probe to form a milky white polymer. A catalyst is added for catalysis, followed by stirring, centrifugation, dialysis, and freeze-drying to finally obtain dendritic macromolecular nanogel DNG. Step (2) The dendritic macromolecular nanogel DNG from step (1) is mixed with 5-azacytidine 5-Aza in an aqueous solution at different mass ratios, stirred at room temperature, and centrifuged using an ultrafiltration centrifuge tube to obtain nanogel Aza-DNG loaded with 5-Aza. Step (3) After mixing the Aza-DNG solution and Ex in step (2) at different mass ratios, the mixture is repeatedly extruded through a 220 nm polycarbonate porous membrane on an Avanti micro extruder 10-12 times, and then purified by centrifugation to obtain Aza-DNG@Ex; Step (4) 1,2-dococosyl-2-deoxy-3-phosphatidylglycerol-ethanolamine-N-[succinyl(polyethylene glycol-2000)]-terephthalaldehyde and imiquimod R837 were dispersed in an organic solvent, stirred and reacted under temperature control, dialyzed, and lyophilized to obtain DSPE-PEG-R837; Step (5) Aza-DNG@Ex from step (3) and DSPE-PEG-R837 from step (4) are mixed at different mass ratios and incubated to obtain Aza-DNG@Ex / R837.

[0010] The present invention also provides a nanogel vaccine, a glutathione-responsive nanovaccine, loaded with an epigenetic drug, encapsulated with tumor cell-derived exosomes and modified with a biomimetic immune adjuvant.

[0011] Preferably, the nanogel vaccine of the present invention comprises a dendritic macromolecule, wherein the dendritic macromolecule is a third-generation polyamide-amine dendritic macromolecule G3.NH2PAMAM; the epigenetic drug is 5-azacytidine 5-Aza; the immunoadjuvant is imiquimod R837; the exosomes are exosomes Ex derived from pancreatic cancer cells; and the cross-linking agent is N,N′-bis(acryloyl)cysteine ​​BAC; wherein the structural formula of BAC is: .

[0012] The present invention relates to the application of a biomimetic nanovaccine prepared by the method described in claim 1 in the combined epigenetic and immunotherapeutic treatment of tumors.

[0013] This invention provides a glutathione-responsive, exosome-inspired, dendritic macromolecular nanogel vaccine for pancreatic cancer. Using a third-generation polyamide-amine dendritic macromolecule as the matrix and N,N′-bis(acryloyl)cysteine ​​(BAC) as the cross-linking agent, GSH-responsive DNGs are prepared via a reverse microemulsion method and loaded with 5-Aza. Subsequently, pancreatic cancer cell-derived exosomes are coated onto the surface of the drug-loaded DNGs, endowing the formulation with homologous targeting ability and natural tumor antigens. Finally, R837 is modified onto the exosome surface via a phospholipid fusion method to construct an integrated biomimetic nanovaccine. This nanovaccine can mediate tumor demethylation through 5-Aza, activate GSDME-dependent pyroptosis, and trigger tumor ICD effects. At the same time, it can promote DC maturation, efficiently present tumor antigens, reshape the polarization phenotype of tumor macrophages, and reverse the immunosuppressive microenvironment of pancreatic cancer through the synergistic effect of epigenetic regulation and anti-tumor immunity.

[0014] This invention breaks through the bottleneck of existing single treatment technologies and constructs a novel synergistic anti-tumor nanovaccine system, which has outstanding innovation and good prospects for clinical translational application in the field of precise, low-toxicity and long-acting treatment of pancreatic cancer.

[0015] Compared with the prior art, the technical solution of this invention has the following advantages: 1. The exosome-inspired dendritic macromolecular nanogel vaccine of this invention has a simple and easy-to-operate preparation process, and the product purification and separation are easy, showing good prospects for practical transformation and application.

[0016] 2. This invention uses exosomes derived from pancreatic cancer cells, which can enhance the immunogenicity of the vaccine and endow the vaccine with homologous active targeting properties, effectively promoting the enrichment of the vaccine at the tumor site, and providing a new approach for the development of novel nanovaccines.

[0017] 3. The dendritic macromolecular nanogel vaccine prepared by this invention has both excellent stability and biocompatibility, and possesses glutathione-responsive properties, which can accurately respond to drug release in the tumor microenvironment and significantly improve drug utilization.

[0018] 4. The nanovaccine prepared by this invention can be adapted to various administration methods. Tail vein injection can effectively inhibit in situ tumor growth, while peritumoral injection can achieve tumor prevention. This vaccine can demethylate cancer cell DNA, induce pyroptosis and trigger immunogenic cell death, directly activate antigen-presenting cells, promote the polarization of M2 macrophages to M1, reverse the tumor immunosuppressive microenvironment, and initiate a specific anti-tumor immune response, achieving epigenetic and immunotherapeutic combined treatment of pancreatic cancer, providing important technical reference for the development of clinical anti-tumor drugs. Attached Figure Description

[0019] Figure 1 This diagram illustrates the synthesis and application of Aza-DNG@Ex / R837 prepared in this invention. Figure 2 The UV-vis spectrum of Aza-DNG prepared in Example 1; Figure 3 TEM image (A) and particle size distribution histogram (B) of Aza-DNG prepared in Example 1; Figure 4 TEM image and particle size distribution histogram (B) of Ex prepared in Example 1; Figure 5 The UV-vis spectra of DSPE-PEG-R837 and Aza-DNG@Ex / R837 prepared in Example 1 are shown. Figure 6 Western blot characterization of the Ex marker protein was performed on the Aza-DNG@Ex / R837 prepared in Example 1; Figure 7 The changes in hydrated particle size of Aza-DNG@Ex / R837 prepared in Example 1 with and without GSH (A) and the cumulative drug release curve of 5-Aza (B). Figure 8The flow cytometry results (A, C) and fluorescence quantitative analysis diagrams (B, D) of the Cy5.5-DNG, Cy5.5-Aza-DNG@Ex and Cy5.5-Aza-DNG@Ex / R837 prepared for this invention after co-incubation with Panc-02 cells and 4T1 cells for 4 h, respectively. Figure 9 Flow cytometry (A) and quantitative analysis results (B) of the nanovaccine prepared for this invention after co-incubation with interleukin-4 (IL-4) induced RAW264.7 cells for 24 h. Figure 10 Western blot results (A) and grayscale quantitative analysis results (B) of DNMT1 and GSDME-N protein expression in Panc-02 cells after treatment with different agents for 24 h. Figure 11 Confocal scanning microscopy images (A) and intracellular LDH activity detection results (B) of Panc-02 cells after treatment with different materials for 24 h. Figure 12 Immunofluorescence images (A) showing the outward rotation of calreticulin induced by co-incubation of different materials with Panc-02 cells for 24 h, and the results of detection of the relative release of ATP (B) and HMGB-1 (C). Figure 13 Flow cytometry analysis of Panc-02 cells treated with different materials after co-incubation with DCs for 24 h (A) and quantitative analysis results of the proportion of mature DCs (B). Figure 14 Flow cytometry analysis of Panc-02 cells after 24 h of treatment with different materials (A) and quantitative analysis results of the proportion of mature DCs (B). Figure 15 Tumor images (A) and tumor weight (B) of different formulations in Panc-02 orthotopic tumor-bearing mice after four tail vein administrations; Figure 16 After treatment with different formulations, CD11c in the lymph nodes of Panc-02 orthotopic tumor-bearing mice was measured. + CD86 + CD80 + Flow cytometry analysis of DC expression (A) and its quantitative results (B); Figure 17 After treatment with different formulations, CD4 counts in the spleen of Panc-02 orthotopic tumor-bearing mice were measured. + CD8 + Flow cytometry analysis of T cells (A) and quantitative analysis results (B, C); Figure 18 After treatment with different formulations, the CD4 content in the tumor tissue of Panc-02 orthotopic tumor-bearing mice was increased. + FoxP3 + CD25 + Flow cytometry images of Tregs cells (A) and their quantitative analysis results (B); Figure 19 The tumor growth curves (A) and tumor weight (B) at different time points after subcutaneous inoculation of tumor cells in mice immunized with the nano-vaccine prepared in this invention. Figure 20 To investigate the CD8+ levels in the spleen of a mouse model of a preventative nanovaccine. + CD44 + CD62L - and CD8 + CD44 + CD62L + Flow cytometry analysis of expression (A) and its quantitative results (B, C); Figure 21 To detect CD4 in tumor tissue in a mouse model of a preventative nanovaccine + FoxP3 + CD25 + Flow cytometry plot of Tregs cells (A) and quantitative analysis results (B). Detailed Implementation

[0020] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0021] This invention involves co-extruding 5-Aza-loaded dendritic macromolecular nanogels and pancreatic cancer cell-derived exosomes, followed by surface modification with the immunoadjuvant R837, to prepare an exosome-inspired dendritic macromolecular nanogel vaccine. The preparation process is simple, product separation and purification are easy, and the overall yield is excellent. The prepared exosome-inspired dendritic macromolecular nanogel vaccine can not only target pancreatic cancer cells through exosome-mediated homologous targeting, but also regulate pancreatic cancer DNA demethylation, induce pyroptosis-type immunogenic cell death, and directly activate antigen-presenting cells, promoting macrophage polarization to the M1 phenotype, improving the tumor immunosuppressive microenvironment, and stimulating a specific anti-tumor immune response, thereby achieving epigenetic / immunotherapy synergistic therapy for pancreatic cancer.

[0022] This invention uses ultraviolet-visible absorption spectroscopy (UV-vis), dynamic light scattering analysis (DLS), nanoparticle tracking analysis (NTA), surface potential measurement, transmission electron microscopy (TEM), gel electrophoresis, and Western blotting to characterize the physicochemical properties of dendritic macromolecular nanogel vaccines. The cytotoxicity of Aza-DNG@Ex / R837 was evaluated using the CCK-8 assay; the phagocytic capacity of tumor cells to Aza-DNG@Ex / R837 was examined using flow cytometry; the activity of lactate dehydrogenase (LDH) in tumor cells treated with Aza-DNG@Ex / R837 was detected using a lactate dehydrogenase assay kit; the calreticulin outward turning and pyroptosis induced by Aza-DNG@Ex / R837 in tumor cells were examined using laser confocal microscopy; the effects of Aza-DNG@Ex / R837 on macrophage repolarization and dendritic cell maturation were evaluated using flow cytometry; the release of high-migration protein (HMGB-1) and adenosine triphosphate (ATP) in cancer cells treated with Aza-DNG@Ex / R837 was detected using ELISA; finally, orthotopic and subcutaneous pancreatic cancer-bearing mouse models were established to evaluate the tumor therapeutic and preventive effects of the exosome-inspired dendritic macromolecular nanogel vaccine.

[0023] like Figure 1 As shown, a method for preparing a glutathione-responsive exosome-inspired dendritic macromolecular nanogel vaccine includes the following steps: Step (1) The dendritic macromolecules and crosslinking agents are dispersed in pure water to form an aqueous phase; Sorbitan oleate (Span 80) and polyoxyethylene 20 sorbitan monooleate (Tween 80) are dissolved in an organic solvent to form an organic phase; the aqueous phase is quickly added to the organic phase to form a W / O mixture, which is then ultrasonically dispersed by a probe to form a milky white polymer. A catalyst is added for catalysis, followed by stirring, centrifugation, dialysis, and freeze-drying to finally obtain dendritic macromolecular nanogel DNG.

[0024] Preferably, Tween 80 and Span 80 are dissolved in cyclohexane, and the crosslinking agent is N,N′ bis(acryloyl)cysteine ​​(BAC); the dendritic macromolecule is the third-generation polyamide-amine dendritic macromolecule G3.NH2PAMAM; the molar ratio of G3.NH2PAMAM, BAC, Tween 80, and Span 80 is 1:3:188:12 to 1:4:188:12; the ultrasonic power of the probe is 75% to 85%, the ultrasonic time is 8 to 10 min, with an ultrasonic interval of 2 to 3 s followed by a 3 to 4 s pause; the catalyst is triethylamine; the stirring reaction time is 12 to 14 h, and the centrifugation speed is 10,000 to 12,000 rpm.

[0025] Step (2) The dendritic macromolecular nanogel DNG from step (1) is mixed with 5-azacytidine 5-Aza in an aqueous solution at different mass ratios, stirred at room temperature, and centrifuged using an ultrafiltration centrifuge tube to obtain nanogel Aza-DNG loaded with 5-Aza.

[0026] Preferably, the mass ratio of DNG to Aza is 2:1 to 10:1, the stirring reaction time is 24 to 26 h, and the centrifugation speed is 3500 to 4000 rpm.

[0027] Step (3) After mixing the Aza-DNG solution and Ex in step (2) at different mass ratios, the mixture is repeatedly extruded through a 220 nm polycarbonate porous membrane on an Avanti micro extruder 10-12 times, and then purified by centrifugation to obtain Aza-DNG@Ex.

[0028] Preferably, the exosomes are derived from pancreatic cancer cells; the Avanti micro-extruder performs 10-12 extrusions; and the purification centrifugation speed is 10,000-12,000 rpm. The extraction method for the pancreatic cancer-derived exosomes is differential centrifugation.

[0029] Step (4) 1,2-dococosyl-2-deoxy-3-phosphatidylglycerol-ethanolamine-N-[succinyl(polyethylene glycol-2000)]-terephthalaldehyde and imiquimod R837 were dispersed in an organic solvent, stirred and reacted under temperature control, dialyzed, and lyophilized to obtain DSPE-PEG-R837.

[0030] Preferably, the mass ratio of Aza-DNG@Ex to DSPE-PEG-R837 is 2:1 to 3:1, the incubation temperature is 35 to 40 °C, and the incubation time is 4 to 6 h.

[0031] Step (5) Aza-DNG@Ex from step (3) and DSPE-PEG-R837 from step (4) are mixed at different mass ratios and incubated to obtain Aza-DNG@Ex / R837.

[0032] Unless otherwise specified, all chemical reagents can be used directly without further purification. G3.NH2PAMAM was purchased from Dendritech, USA; N,N′-bisacrylcysteine ​​(BAC) was purchased from Alfaesa (China) Co., Ltd.; 5-azacytidine (5-Aza) was purchased from Glpbio Biotechnology Co., Ltd. (Shanghai); Pan-02 cells (mouse pancreatic cancer cell line) and 4T1 cells (mouse breast cancer cell line) were purchased from Wuhan Pronosai Life Science Co., Ltd.; dendritic cells (immature dendritic cells derived from mouse bone marrow) were obtained from Fudan University Cancer Hospital; DMEM medium, RPMI-1640 medium, trypsin, and penicillin were used. Streptomycin bispecific antibody was purchased from Hangzhou Gino Biotechnology Co., Ltd.; fetal bovine serum was purchased from Nanjing Senbega Biotechnology Co., Ltd.; IL-4 was purchased from Wuhan Yacoin Biotechnology Co., Ltd.; ATP assay kit, LDH assay kit, and calreticulin (Anti-CRT) antibody were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; Anti-CD80-FITC, Anti-CD86-PE, Anti-CD86-FITC, Anti-CD206-PE, Anti-CD8-PE, and Anti-CD11c were also purchased. + Anti-APC, Anti-CD4-FITC, Anti-CD25-FITC, Anti-Foxp3-APC, Anti-CD44-FITC, and Anti-CD62L-APC were purchased from Thermo Fisher Scientific (Shanghai); female C57BL / 6 mice were purchased from Shanghai JessJet Laboratory Animal Co., Ltd.

[0033] Example 1: A method for preparing a glutathione-responsive exosome-inspired dendritic macromolecular nanogel vaccine, comprising the following steps: Step (1) Dissolve 20 mg of G3.NH2PAMAM and 2.26 mg of PBA-PEG-PBA in 0.5 mL of ultrapure water. Mix the above G3.NH2PAMAM aqueous solution and BAC aqueous solution thoroughly to obtain an aqueous phase; separately dissolve 234 mg of Span 80 and 46 mg of Tween 80 in 10 mL of cyclohexane to obtain an organic phase. Quickly add the aqueous phase to the organic phase and sonicate using a probe-type ultrasonic disruptor. Set the ultrasonic power to 80%, the ultrasonic mode to work for 2 s and then pause for 3 s, for a total ultrasonic duration of 8 min. Under stirring conditions at room temperature, add 500 μL of triethylamine dropwise to the reaction system to catalyze the reaction, and continue stirring at room temperature for 13 h. After the reaction is complete, place the mixture in a centrifuge and centrifuge at 12000 rpm for 10 min to remove cyclohexane, Tween-80, and Span 80. Collect the product DNG and resuspend it in 5 mL of methanol. Subsequently, a dialysis bag with a molecular weight cutoff of 8000~14000 Da was used to dialyze the sample in pure water for 3 days to completely remove residual organic solvents, Tween-80 and Span 80. Finally, the purified DNG was obtained by freeze drying.

[0034] Step (2) DNG and 5-Aza were mixed and dispersed in 1.5 mL of ultrapure water at different mass ratios of 2:1, 4:1, and 10:1, and stirred at room temperature for 24 h. The mixture was then centrifuged at 4000 rpm for 15 min using an ultrafiltration tube with a molecular weight cutoff of 10000 Da to remove free 5-Aza, thus obtaining Aza-DNG. The drug loading (DL%) and encapsulation efficiency (EE%) of DNG for 5-Aza at different mass ratios are shown in the table below:

[0035] By measuring the drug loading and encapsulation efficiency of 5-Aza under different mass ratios, the highest drug loading of 5-Aza (12.06%) was found when the mass ratio of DNG to 5-Aza was 4:1. Therefore, this ratio was selected for subsequent exosome encapsulation.

[0036] In step (3), exosomes (Ex) from Panc-02 cells were extracted using gradient centrifugation. Logarithmically growing Panc-02 cells were seeded into T75 cell culture flasks and cultured at 37 ℃ in a 5% CO2 incubator for 24 h. The cell culture supernatant was discarded, and the cells were washed once with phosphate-buffered saline (PBS), then cultured in exosome-free medium for another 48 h. The supernatant was then collected.

[0037] The supernatant was subjected to a gradient centrifugation process: first, centrifugation at 300 g for 10 min, collecting the supernatant; then, centrifugation at 3000 g for 10 min, collecting the supernatant; after filtering through a 450 nm filter to remove impurities, centrifugation at 10000 g for 30 min was continued, collecting the supernatant; finally, centrifugation at 100000 g for 75 min. After centrifugation, the supernatant was discarded, and the precipitate was resuspended in PBS to obtain purified Ex. The zeta potential and polydispersity index of Ex are shown in the table below:

[0038] Step (4) Aza-DNG and Ex were thoroughly mixed at mass ratios of 1:1, 1:3, and 1:5, respectively. The resulting mixtures were placed on an Avanti micro extruder and repeatedly extruded through a 220 nm polycarbonate membrane 10 times. The mixture was then centrifuged at 12000 rpm for 10 min, and the precipitate was collected to obtain purified Aza-DNG@Ex. The hydration kinetics, zeta potential, and polydispersity index of Aza-DNG@Ex prepared by co-extrusion of Aza-DNG and Ex at different mass ratios are shown in the table below:

[0039] By measuring the zeta potential of Aza-DNG@Ex prepared by Aza-DNG and Ex at different mass ratios, it can be observed that when the mass ratio is 1:5, the potential of Aza-DNG@Ex is -7.37 mV, which is close to the potential of Ex alone, indicating that Ex is completely coated on the surface of Aza-DNG.

[0040] Step (5) DSPE-PEG-terephthalaldehyde and R837 were dispersed in DMSO at a molar ratio of 2:1, stirred at 37 °C, dialyzed, and lyophilized to obtain DSPE-PEG-R837.

[0041] Step (6) Mix Aza-DNG@Ex and DSPE-PEG-R837 uniformly at a mass ratio of 2:1 and incubate at 37 ℃ for 4 h to obtain Aza-DNG@Ex / R837.

[0042] The Aza-DNG prepared in Example 1 was subjected to ultraviolet spectroscopy characterization and detection to verify the loading effect of DNG on 5-Aza. Figure 2 As shown, both 5-Aza and Aza-DNG exhibit the characteristic absorption peak of 5-Aza near a wavelength of 240 nm, confirming that 5-Aza has been successfully loaded into the DNG carrier.

[0043] Aza-DNG sample with a concentration of 1 mg / mL prepared in Example 1 was dropped onto a copper grid and allowed to stand for 5 min. After removing excess liquid from the surface of the copper grid using filter paper, the morphology of the sample was characterized using a JEM-2010F transmission electron microscope with an accelerating voltage of 200 kV. The transmission electron microscopy results of Aza-DNG are as follows: Figure 3 As shown in Figure A, the TEM image reveals that Aza-DNG exhibits a uniformly distributed spherical morphology with an average particle size of 100.1 nm. Figure 3 B).

[0044] Take Ex from Example 1, drop it onto a copper grid and let it stand for 5 min. After absorbing excess liquid from the surface with filter paper, negatively stain the Ex sample with 2% phosphotungstic acid solution for 5 min. Then, use a JEM-2010F transmission electron microscope with an accelerating voltage of 200 kV to image the morphology of Ex. Figure 4 As shown in Figure A, the negatively stained Ex particles have bright edges and exhibit a disc-like or cup-like shape, with an average particle size of approximately 115 nm. Figure 4 As shown in B.

[0045] The DSPE-PEG-R837 and Aza-DNG@Ex / R837 prepared in Example 1 were used, and the preparation effect of DSPE-PEG-R837 and the functionalization modification of Aza-DNG@Ex / R837 were verified by detecting the characteristic ultraviolet absorption peaks. Figure 5 As shown, R837, DSPE-PEG-R837 and Aza-DNG@Ex / R837 all exhibit the characteristic absorption peak of R837 at a wavelength of 330 nm, proving that DSPE-PEG-R837 was successfully prepared and that DSPE-PEG-R837 has been effectively modified on the surface of Aza-DNG@Ex.

[0046] Ex, Aza-DNG@Ex / R837, and Panc-02 cells from Example 1 were lysed with lysis buffer, and the exosome surface marker proteins TSG101 and CD81 were detected by Western blot. Figure 6 As shown, Aza-DNG@Ex / R837 still retains the characteristic proteins of Ex, including TSG101 and CD81, confirming that Ex has been effectively coated on the surface of Aza-DNG.

[0047] Prepare a 10 mM glutathione (GSH) buffer to evaluate the GSH response of Aza-DNG@Ex / R837. Measure the hydration kinetics of 1 mg / mL samples with and without 10 mM GSH using a Malvern particle size analyzer. Figure 7As shown in Figure A, Aza-DNG@Ex / R837 exhibits a concentrated particle size distribution in the absence of GSH. However, the addition of 10 mM GSH results in a bimodal particle size distribution and a decrease in particle size uniformity. One mL of Aza-DNG@Ex / R837 (2 mg / mL) was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and immersed in 9 mL of buffer solution with and without 10 mM GSH. The solution was then shaken at 37 ℃ and 180 rpm. Samples were taken periodically, and the buffer solution was replenished accordingly to determine the cumulative release of 5-Aza. Figure 7 As shown in Figure B, the cumulative release of 5-Aza in the 10 mM GSH group reached 71.27% at 48 h, significantly higher than the 32.8% in the group without 10 mM GSH. These results indicate that Aza-DNG@Ex / R837 is GSH-responsive and can release drugs in response to the high GSH environment of the tumor.

[0048] Using Panc-02 and 4T1 cells as models, the uptake capacity of three materials—Aza-DNG, Aza-DNG@Ex, and Aza-DNG@Ex / R837—was investigated. Panc-02 and 4T1 cells in logarithmic growth phase were harvested and added at doses of 1.5 × 10⁻⁶ cells per cell line. 5 Cells were seeded at a density of [number] cells / well in 6-well plates and incubated at 37 ℃ in a 5% CO2 incubator for 16 h. After incubation, the original culture medium was discarded, and serum-free medium containing Cy5.5 fluorescently labeled material (5-Aza concentration of 6 μg / mL) was replaced, and co-incubation continued for 4 h. After incubation, the supernatant was discarded, and the cells were washed once with PBS; after trypsin digestion and centrifugation, the cells were washed once more with PBS, and finally, 350 μL of PBS was added to resuspend the cells. The fluorescence intensity of each group of cells was detected by flow cytometry. Figure 8 As shown in the AD diagram, both Panc-02 and 4T1 cells effectively took up Aza-DNG. For Panc-02 cells, the mean fluorescence intensity of the Cy5.5-Aza-DNG@Ex group was significantly higher than that of the Cy5.5-Aza-DNG group, while there was no significant difference in fluorescence intensity between the Cy5.5-Aza-DNG@Ex group and the Cy5.5-Aza-DNG@Ex / R837 group. For 4T1 cells, except for the PBS group, there were no statistically significant differences in the mean fluorescence intensity of the other three groups. These results indicate that pancreatic cancer cell-derived Ex can imbue the nanogel with homologous targeting, significantly enhancing the uptake efficiency of the exosome-inspired nanogel by Panc-02 cells; R837 functionalization modification of the nanogel did not significantly alter the cell uptake effect.

[0049] To verify the regulatory effect of Aza-DNG@Ex / R837 on repolarization of mouse RAW 264.7 macrophages, cells were cultured at a rate of 1.5 × 10⁻⁶ cells / year.5 RAW264.7 cells were seeded at a density of 1 cell / well in 6-well cell culture plates and cultured at 37 °C in a 5% CO2 incubator for 16 h. After culture, the original culture medium was discarded, and medium containing 50 ng / mL IL-4 was added for continued induction culture for 24 h to polarize RAW264.7 cells into M2 macrophages. The induction culture medium was then discarded, and fresh medium containing lipopolysaccharide (LPS), DNG, 5-Aza, Aza-DNG, Aza-DNG@Ex, and Aza-DNG@Ex / R837 (5-Aza concentration of 6 μg / mL and LPS concentration of 2 μg / mL) was added for co-incubation. After continuous incubation in an incubator for 24 h, the culture supernatant was discarded, cells were aspirated with PBS and centrifuged, and the supernatant was discarded. PBS buffer containing Anti-CD206-PE and Anti-CD86-FITC antibodies was added to the cell system, and the cells were stained on ice in the dark for 30 min. After staining, the cells were washed twice with PBS, and finally, 300 μL of PBS was added to resuspend the cells. The regulatory effect of each material on the repolarization of M2 RAW 264.7 macrophages was detected by flow cytometry. Figure 9 China A Figure 9 As shown in Figure B, compared with the DNG group, the expression of CD206, a marker of M2 type macrophages, was significantly downregulated in the 5-Aza group and the Aza-DNG group, while the expression of CD86, a marker of M1 type macrophages, was significantly upregulated. Simultaneously, the M1 / M2 polarization ratio of the Aza-DNG@Ex / R837 group was significantly higher than that of the other nanoparticle formulations without R837 loading. These results indicate that this nanogel can co-deliver two functional components, 5-Aza and R837, effectively promoting the repolarization of M2 macrophages to M1 type, thereby reversing the immunosuppressive tumor microenvironment.

[0050] To verify whether Aza-DNG@Ex / R837 can induce demethylation and pyroptosis in Panc-02 cells, Western blot was used to detect the protein expression levels of DNA methyltransferase 1 (DNMT1) and pyrophorin E activated fragment (GSDME-N) in Panc-02 cells after different material treatments. Logarithmic growth phase Panc-02 cells were selected and stored at 1.5 × 10⁶ cells per well. 5Cells were seeded at a density of [number] cells / well in 6-well plates and incubated at 37 ℃ in a 5% CO2 incubator for 16 h. The original culture medium was removed, and cells were washed with PBS. Complete culture medium containing DNG, 5-Aza, Aza-DNG, Aza-DNG@Ex, and Aza-DNG@Ex / R837 (5-Aza final concentration 6 μg / mL) was added, and the cells were co-incubated for another 24 h. After incubation, the supernatant was discarded, and the cells were washed twice with PBS. Total cell protein was extracted, and the protein expression of DNMT1 and GSDME-N in each group of cells was detected by Western blot. Figure 10 China A Figure 10 As shown in Figure B, compared to the PBS control group, the expression of DNMT1 protein in the 5-Aza group was significantly downregulated, while the expression of GSDME-N protein was significantly upregulated. Compared to the experimental group without Ex, the DNMT1 protein expression level was lower and the GSDME-N protein expression level was higher in the nanogel group containing Ex, which may be because the Ex coating increased the uptake of the drug by cancer cells. The above results confirm that Aza-DNG@Ex / R837 can effectively induce DNA demethylation in Panc-02 cells and trigger GSDME-mediated pyroptosis.

[0051] To further verify the pyroptosis-inducing effect of Aza-DNG@Ex / R837 on Panc-02 cells, this study used laser confocal microscopy and enzyme-linked immunosorbent assay (ELISA) to observe the morphological changes and lactate dehydrogenase (LDH) release levels of cells after treatment with different materials. Logarithmic growth phase Panc-02 cells were selected and cultured at 1.5 × 10⁶ cells / plate. 5 Cells were seeded at a density of [number] cells / day in confocal culture dishes and incubated at 37 ℃ in a 5% CO2 incubator for 16 h. After removing the supernatant, complete culture medium containing DNG, 5-Aza, Aza-DNG, Aza-DNG@Ex, and Aza-DNG@Ex / R837 (5-Aza concentration 6 μg / mL) was added, and incubation continued for 36 h. The supernatant was then discarded, and cells were stained with Annexin V-FITC and propidium iodide (PI), where Annexin V-FITC labeled the cell membrane and PI labeled the cell nucleus. Figure 11 As shown in Figure A, all material groups containing 5-Aza exhibited typical pyroptosis morphological characteristics such as cell swelling and cell membrane bubbles. Figure 11 As shown in Figure B, the LDH release in the 5-Aza group was significantly higher than that in the DNG and PBS groups; the LDH level in the culture medium of the nanogel group coated with Ex was significantly higher than that in the material group without Ex coating, confirming that the cells experienced cell membrane damage and loss of cell integrity. These results indicate that Aza-DNG@Ex / R837 can effectively induce pyroptosis in Panc-02 cells.

[0052] To investigate whether the prepared biomimetic nanogel could induce pyroptosis-based intracellular drug reaction (ICD) in Panc-02 cells, laser confocal microscopy and ELISA were used to detect the outward movement of calreticulin (CRT) on the cell surface after treatment with different materials, as well as the release of extracellular high-mobility group box 1 (HMGB-1) and adenosine triphosphate (ATP). Logarithmic growth phase Panc-02 cells were collected and cultured at 1.5 × 10⁶ cells per dish. 5 Cells were seeded at a density of [number] cells / day in confocal culture dishes and incubated at 37 ℃ in a 5% CO2 incubator for 16 h. After removing the supernatant, complete culture medium containing DNG, 5-Aza, Aza-DNG, Aza-DNG@Ex, and Aza-DNG@Ex / R837 (5-Aza concentration 6 μg / mL) was added, and incubation continued for 24 h. The supernatant was discarded, and CRT immunofluorescence staining was performed on each group of cells. Figure 12 As shown in Figure A, the red fluorescence intensity of the Aza-DNG group was higher than that of the free 5-Aza group; compared with the group without Ex, the fluorescence intensity of the Ex-coated nanogel group was stronger. This indicates that the drug-loaded nanogel is more easily taken up by tumor cells than the free drug; the homologous targeting properties conferred by Ex can further enhance the uptake efficiency of the nano-formulation by tumor cells, ultimately leading to a significant increase in the outward turning level of the CRT on the cell surface. Panc-02 cells were also cultured at 1.5 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates and cultured for 16 h under the same conditions. The medium was then replaced with fresh medium containing the aforementioned materials (5-Aza concentration 6 μg / mL), and after incubation for 24 h, the cell supernatant was collected. The release of HMGB-1 and ATP in the supernatant was detected using an ELISA kit. Figure 12 B Figure 12 As shown in Figure C, the relative release of extracellular ATP and HMGB-1 in the Ex-coated nanogel group was higher than that in other experimental groups. These results confirm that Aza-DNG@Ex / R837 can effectively induce ICD in Panc-02 cells, thereby stimulating the body to produce an anti-tumor immune response.

[0053] To verify whether the biomimetic nanogel vaccine prepared in this study can promote the maturation of dendritic cells (DCs) by inducing intracellular dysplasia (ICD) in tumor cells, this experiment constructed a Transwell co-culture system for verification. DCs were cultured at a density of 1.0 × 10⁶ cells per well. 5 At a density of 1000 individuals, seeds were inoculated into the lower chamber of a 12-well plate, while simultaneously seeded into the upper chamber of a 3 μm Transwell chamber at a density of 1.0 × 10⁶ individuals per chamber. 5Panc-02 cells were seeded at a density of [number] cells / well, and the co-culture system was incubated at 37 ℃ in a 5% CO2 cell culture incubator for 16 h. After incubation, the supernatant in the Transwell chambers and 12-well plates was discarded. Fresh culture medium containing DNG, 5-Aza, Aza-DNG, Aza-DNG@Ex, and Aza-DNG@Ex / R837 (5-Aza concentration 6 μg / mL) was added to the Transwell chambers, and only fresh culture medium was added to the 12-well plate chambers. The system was then incubated in a cell culture incubator for another 24 h. After incubation, the supernatant in the lower chamber of the plate was discarded, and DCs were collected by trypsin digestion. After washing the cells twice with PBS, PBS buffer containing Anti-CD86-PE and Anti-CD80-FITC antibodies was added, and the DCs were stained on ice in the dark for 30 min. After staining, the cells were washed twice with PBS, resuspended in 300 μL PBS, and the maturation level of DCs co-cultured with Panc-02 cells treated with different materials in each group was detected by flow cytometry. Figure 13 China A Figure 13 As shown in Figure B, compared to the PBS and DNG groups, the 5-Aza group showed higher maturation (CD80). + CD86 + The significantly higher proportion of DCs confirmed that 5-Aza can induce pyroptosis-type ICD in Panc-02 cells, effectively promoting DC maturation. Simultaneously, the DC maturation rate in the Aza-DNG@Ex and Aza-DNG@Ex / R837 groups was significantly higher than in the other experimental groups, with no statistically significant difference between the two groups. This result may be because the Ex coating of the nanogel effectively enhances the uptake efficiency of 5-Aza by Panc-02 cells, inducing a stronger ICD effect and releasing a large amount of damage-associated molecular patterns (DAMPs), further promoting DC maturation; while the functionalization modification of the nanogel by R837 has no significant regulatory effect on the uptake of 5-Aza by Panc-02 cells, therefore the DC maturation rate between the Aza-DNG@Ex and Aza-DNG@Ex / R837 groups did not show a significant difference.

[0054] To verify whether the biomimetic nanogel vaccine prepared in this study can directly activate dendritic cells (DCs) in vitro, this study used flow cytometry to quantitatively detect the maturation level of DCs after treatment with different formulations. DCs were cultured at 2.0 × 10⁶ cells per well. 5DCs were seeded at a density of [number] cells / well in 6-well plates and cultured at 37 ℃ in a 5% CO2 cell culture incubator for 16 h. The supernatant was discarded, and fresh culture medium containing DNG, 5-Aza, Aza-DNG, Aza-DNG@Ex, and Aza-DNG@Ex / R837 (5-Aza concentration 6 μg / mL) was added, and the plates were incubated for a total of 24 h. The supernatant was discarded, and DCs were collected by trypsin digestion. After washing the cells twice with PBS, PBS buffer containing Anti-CD86-PE and Anti-CD80-FITC antibodies was added, and the DCs were stained on ice in the dark for 30 min. After staining, the cells were washed twice more with PBS, resuspended in 300 μL of PBS, and the maturation status of DCs in different material treatment groups was detected and analyzed by flow cytometry. Figure 14 China A Figure 14 As shown in Figure B, compared to the experimental group without Ex, the mature DCs (CD80) in the Aza-DNG@Ex group + CD86 + The significantly increased proportion of maturation dendritic cells (DCs) confirms that the abundant tumor-associated antigens carried by Ex can effectively induce and activate DC maturation. Meanwhile, the DC maturation rate in the Aza-DNG@Ex / R837 group was 32.50%, significantly higher than the 24.83% in the Aza-DNG@Ex group, further validating that the R837 immune adjuvant can synergistically enhance the maturation effect of DCs with the tumor-associated antigens carried by Ex.

[0055] All animal experiments in this study were conducted strictly in accordance with the experimental protocol approved by the Animal Care and Use Committee of Donghua University (Approval No.: DHUEC-STCSM-2023-10) and met relevant guidelines and management standards. Four- to five-week-old female C57BL / 6 mice were used in the experiments and purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd. To investigate the in vivo antitumor therapeutic effect of the exosome-inspired dendritic macromolecular nanogel vaccine prepared in Example 1, an orthotopic Pancreatic Panc-02 tumor-bearing model was constructed in C57BL / 6 mice. The concentration was 4 × 10⁻⁶. 7Panc-02 cell suspension at a concentration of 50 μL / mL was injected into the pancreas of C57BL / 6 mice via orthotopic inoculation. Ten days after modeling, tumor-bearing mice were randomly divided into six groups of five mice each: PBS group, DNG group, 5-Aza group, Aza-DNG group, Aza-DNG@Ex group, and Aza-DNG@Ex / R837 group (5-Aza dosage was 3 mg / kg). Administration was via tail vein, once every four days, for a total of four administrations. During the treatment period, mice were intraperitoneally injected with potassium fluorescein solution every seven days for tumor bioluminescence imaging, for a total of three imaging sessions. After the treatment cycle, mice were sacrificed 15 days after administration, and tumor tissue was dissected for photographing and weighing analysis.

[0056] like Figure 15 China A Figure 15 As shown in Figure B, compared to the other experimental groups, the Aza-DNG@Ex / R837 group of mice had the lowest tumor volume and tumor weight. This confirms that the exosome-inspired dendritic macromolecular nanogel vaccine, as a therapeutic nanovaccine, can effectively inhibit the growth of orthotopic pancreatic tumors in mice.

[0057] To investigate the antitumor immune response levels in tumor-bearing mice treated with different materials, lymph nodes, spleens, and tumor tissues from mice in each group were collected 15 days after drug administration for subsequent immunoassays. Under aseptic conditions, the tissue samples were minced, thoroughly ground, and filtered through a 400-mesh sieve to prepare single-cell suspensions. The lymph node single-cell suspensions were washed with PBS, centrifuged, and then a mixture of Anti-CD11c-APC, Anti-CD80-FITC, and Anti-CD86-PE antibodies was added. The cells were stained for 30 min in an ice bath in the dark. After staining, the cells were washed twice with PBS, resuspended in 400 μL of PBS, and the maturation level of dendritic cells (DCs) in the lymph nodes of each group of mice was detected by flow cytometry. Single-cell suspensions of spleen cells were purified using nylon puffing to obtain T lymphocytes. After washing with PBS and centrifugation, a mixture of Anti-CD4-FITC and Anti-CD8-PE antibodies was added, and the cells were incubated on ice in the dark for 30 min. After washing the cells twice with PBS, 400 μL of PBS was added to resuspend the cells, and the cells were transferred to flow cytometry tubes. Flow cytometry was used to detect CD4+ in the spleen of mice in each group. + T cells and CD8 +The percentage of T cells. Single-cell suspensions of tumor tissue were washed with PBS, centrifuged, and then stained on ice for 30 min in the dark using a combination of regulatory T cell (Treg) specific marker antibodies: Anti-CD4-PE, Anti-CD25-FITC, and Anti-Foxp3-APC. After staining, the cells were washed three times with PBS, resuspended in 500 μL of PBS, and transferred to flow cytometry tubes. Flow cytometry analysis was performed on CD4+ in tumor tissues of mice in each group. + CD25 + FoxP3 + The percentage of Treg cells.

[0058] like Figure 16 China A Figure 16 As shown in Figure B, mature DCs (CD11c) in the lymph nodes of mice in the Aza-DNG@Ex / R837 group + CD80 + CD86 + The proportion of 5-Aza was significantly higher than that of the other treatment groups. This result is attributed to the fact that 5-Aza can induce ICD in tumor cells and release a large number of DAMPs, while the combination of tumor-associated antigens carried by Ex and the immune adjuvant R837 can synergistically stimulate the efficient maturation and activation of DCs in lymph nodes.

[0059] CD4 in the spleen of tumor-bearing mice in each group + T cells and CD8 + The percentage of T cells is as follows: Figure 17 As shown in the middle AC, CD8 in the spleen of mice in the Aza-DNG@Ex / R837 group + T cells accounted for 26.93%, CD4 + The proportion of T cells was 39.53%, significantly higher than in other experimental groups, consistent with the trend of DC maturation. This indicates that Aza-DNG@Ex / R837 can enhance the in vivo therapeutic effect of pancreatic tumors by promoting DC maturation and mediating antigen presentation to activate the body's T cell-mediated anti-tumor immune response.

[0060] Flow cytometry analysis of Treg cells in tumor tissues showed that Treg cell levels were significantly downregulated in all nanomedicine treatment groups; the Aza-DNG@Ex / R837 group had the lowest proportion of Treg cells in mouse tumor tissues. This result confirms that the exosome-inspired biomimetic nanovaccine prepared in this invention can effectively reverse the tumor immunosuppressive microenvironment and reactivate the body's systemic anti-tumor immune response. Figure 18 China A Figure 18 (B)

[0061] To investigate the in vivo tumor prevention efficacy of the exosome-inspired biomimetic nanovaccine prepared in Example 1, healthy female C57BL / 6 mice were selected for a prophylactic immunization experiment. Mice were intramuscularly injected with PBS, Aza-DNG@Ex, or Aza-DNG@Ex / R837 (5-Aza dosage 3 mg / kg), respectively; the administration frequency was once every 6 days, for a total of 3 administrations. On day 6 after the last administration, 2 × 10⁻⁶ cells were subcutaneously injected into the right posterior back of each mouse. 7 Panc-02 tumor cells were inoculated. Five days after cell inoculation, the tumor volume of mice in each group was monitored for 15 consecutive days. After the monitoring period, tumor tissue was dissected and weighed for analysis.

[0062] like Figure 19 China A Figure 19 As shown in Figure B, compared to the PBS group and the Aza-DNG@Ex group, the Aza-DNG@Ex / R837 group of mice had the smallest tumor volume, the slowest tumor growth rate, and the lowest tumor weight. This confirms that the exosome-inspired dendritic macromolecular nanogel vaccine can effectively induce anti-tumor immune protection in mice and significantly inhibit tumor occurrence and growth.

[0063] To further investigate the immune response level activated by this exosome-inspired dendritic macromolecular nanogel vaccine as a prophylactic nanovaccine in mice, spleens and tumor tissues were harvested from mice in each prophylactic treatment group under aseptic conditions on day 20 after tumor inoculation. The tissue samples were minced, thoroughly ground, and filtered through a 400-mesh sieve to prepare single-cell suspensions. T lymphocytes were obtained from the spleen single-cell suspensions through nylon column chromatography and stained with a combination of Anti-CD8-PE, Anti-CD44-FITC, and Anti-CD62L-APC antibodies. Tregs were stained with a single-cell suspension from the tumor tissue using a combination of regulatory T cell-specific antibodies: Anti-CD4-PE, Anti-CD25-FITC, and Anti-Foxp3-APC. All stained cell samples were transferred to flow cytometry tubes, and the proportion of memory T cells in the spleen and the proportion of Tregs cells in the tumor tissue of each group of mice were detected by flow cytometry.

[0064] Flow cytometry results of mouse spleen memory T cells are as follows Figure 20 As shown in AC, compared to the other experimental groups, the Aza-DNG@Ex / R837 group mice had a higher concentration of central memory T cells (CD8+) in the spleen. + CD44 + CD62L + ) and effector memory T cells (CD8) + CD44 + CD62L -The proportions were significantly highest. This indicates that Aza-DNG@Ex / R837, as a preventative nanovaccine, can effectively activate the body to produce a strong and long-lasting immune memory effect.

[0065] Flow cytometry results of Tregs cells in mouse tumor tissue are as follows: Figure 21 China A Figure 21 As shown in Figure B, the proportion of Treg cells in the tumor tissue of mice in the Aza-DNG@Ex / R837 group was only 8.36%, the lowest among all experimental groups. This result further confirms that Aza-DNG@Ex / R837, as a prophylactic nanovaccine, can effectively enhance the body's anti-tumor immune response and improve the immunosuppressive state of the tumor microenvironment.

[0066] In summary, the Aza-DNG@Ex / R837 exosome-inspired biomimetic nanogel vaccine constructed in this invention can effectively promote DC maturation, mediate M2 macrophage repolarization, and reverse the tumor immunosuppressive microenvironment by inducing tumor cell DNA demethylation and triggering pyroptosis-mediated ICD, thereby significantly inhibiting tumor proliferation and growth. This novel biomimetic nanovaccine has both therapeutic and preventative effects, effectively inhibiting the in vivo growth of in situ pancreatic tumors and significantly blocking the occurrence and progression of subcutaneous pancreatic tumors, demonstrating excellent application prospects in the prevention and treatment of pancreatic tumors.

Claims

1. A method for preparing a glutathione-responsive exosome-inspired dendritic macromolecular nanogel vaccine, characterized in that... Includes the following steps: Step (1) The dendritic macromolecules and crosslinking agents are dispersed in pure water to form an aqueous phase; sorbitan oleate and polyoxyethylene 20 sorbitan monooleate are dissolved in an organic solvent to form an organic phase; the aqueous phase is quickly added to the organic phase to form a W / O mixture, which is then ultrasonically dispersed by a probe to form a milky white polymer. A catalyst is added for catalysis, followed by stirring, centrifugation, dialysis, and freeze-drying to finally obtain dendritic macromolecular nanogel DNG. Step (2) The dendritic macromolecular nanogel DNG from step (1) is mixed with 5-azacytidine 5-Aza in an aqueous solution at different mass ratios, stirred at room temperature, and centrifuged using an ultrafiltration centrifuge tube to obtain nanogel Aza-DNG loaded with 5-Aza. Step (3) After mixing the Aza-DNG solution and Ex in step (2) at different mass ratios, the mixture is repeatedly extruded through a 220 nm polycarbonate porous membrane on an Avanti micro extruder 10-12 times, and then purified by centrifugation to obtain Aza-DNG@Ex; Step (4) 1,2-dococosyl-2-deoxy-3-phosphatidylglycerol-ethanolamine-N-[succinyl(polyethylene glycol-2000)]-terephthalaldehyde and imiquimod R837 were dispersed in an organic solvent, stirred and reacted under temperature control, dialyzed, and lyophilized to obtain DSPE-PEG-R837; Step (5) Aza-DNG@Ex from step (3) and DSPE-PEG-R837 from step (4) are mixed at different mass ratios and incubated to obtain Aza-DNG@Ex / R837.

2. The preparation method according to claim 1, characterized in that, In step (1), sorbitan oleate and polyoxyethylene 20 sorbitan monooleate are dissolved in cyclohexane; the crosslinking agent is N,N′ bis(acryloyl)cysteine ​​(BAC); the dendritic macromolecule is the third-generation polyamide-amine dendritic macromolecule G3.NH2PAMAM; the molar ratio of G3.NH2PAMAM, BAC, sorbitan oleate and polyoxyethylene 20 sorbitan monooleate is 1:3:188:12~1:4:188:12; the ultrasonic power of the probe is 75%~85%, the ultrasonic time is 8~10 min, ultrasonic for 2~3 s, stop for 3~4 s; the catalyst is triethylamine; the stirring reaction time is 12~14 h, and the centrifugation speed is 10000~12000 rpm.

3. The preparation method according to claim 1, characterized in that, In step (2), the mass ratio of DNG to 5-Aza is 2:1 to 10:1, the stirring reaction time is 24 to 26 h, and the centrifugation speed is 3500 to 4000 rpm.

4. The preparation method according to claim 1, characterized in that, In step (3), the mass ratio of Aza-DNG to Ex is 1:1 to 1:5, the number of extrusions of the Avanti micro extruder is 10 to 12, and the rotation speed used for centrifugation is 10,000 to 12,000 rpm.

5. The preparation method according to claim 1, characterized in that, In step (4), the terminal aldehyde group in DSPE-PEG-terephthalaldehyde reacts with the primary amino group in R837 to form a Schiff base, and the molar ratio of the two is 1:1~2:

1. The organic solvent is dimethyl sulfoxide (DMSO), the reaction temperature is 35~40 °C, and the stirring time is 22~26 h.

6. The preparation method according to claim 1, characterized in that, In step (5), the mass ratio of Aza-DNG@Ex to DSPE-PEG-R837 is 2:1~3:1, the incubation temperature is 35~40 ℃, and the incubation time is 4~6 h.

7. A nanogel vaccine obtained by the preparation method according to any one of claims 1-6, characterized in that, A glutathione-responsive nanovaccine is loaded with an epigenetic drug, encapsulated with tumor cell-derived exosomes, and modified with a biomimetic immune adjuvant.

8. The biomimetic nanovaccine according to claim 7, characterized in that, The product comprises a dendritic macromolecule, wherein the dendritic macromolecule is a third-generation polyamide-amine dendritic macromolecule G3.NH2PAMAM; the epigenetic drug is 5-azacytidine 5-Aza; the immunoadjuvant is imiquimod R837; the exosomes are exosomes Ex derived from pancreatic cancer cells; and the cross-linking agent is N,N′-bis(acryloyl)cysteine ​​BAC; wherein the structural formula of BAC is: 。 9. The application of a biomimetic nanovaccine prepared by the method described in claim 1 in the combined epigenetic and immunotherapy of tumors.

Images