Dendrimer-based polypeptide nano vaccine as well as preparation method and application thereof

Abstract

The invention relates to a dendrimer-based polypeptide nano vaccine as well as a preparation method and application thereof. The polypeptide nano vaccine is obtained by loading MnO2 nano particles on a functionalized dendrimer and wrapping a polypeptide antigen. After the polypeptide nano vaccine disclosed by the invention is injected into a mouse body through peritumoral injection, MnO2 plays a chemical kinetic treatment role through a Fenton-like reaction, and tumor cells are induced to generate immunogenic cell death, so that dendritic cell maturation is promoted; meanwhile, the polypeptide nano vaccine can activate an immune system and initiate potent immune response by activating a cGAS-STING pathway and being cooperated and combined with presentation of a polypeptide antigen OVA257-264, a new chemical kinetics / immune combined treatment strategy is provided, and the polypeptide nano vaccine has a remarkable inhibition effect on tumor growth and recurrence and has potential application value in the field of biological medicine.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of functional nanomaterials, and particularly relates to a dendrimer-based polypeptide nanovaccine as well as a preparation method and application thereof. BACKGROUND

[0002] The complexity and mutability of cancer occurrence mechanisms have brought great challenges to effective treatment. Traditional treatment methods (such as chemotherapy, radiotherapy and surgery) are still not the optimal solution for cancer treatment due to problems such as systemic toxicity, easy drug resistance and incomplete removal of cancer cells. In recent years, tumor vaccines, immune checkpoint therapy, cell therapy and other immunotherapies have become a research hotspot. Such therapies stimulate the host immune system to accurately recognize and eliminate tumors. Compared with traditional therapies, cancer immunotherapy has the advantages of higher specificity and smaller side effects. However, the low immunogenicity of tumors, insufficient T cell infiltration, low antigen presentation efficiency and immune tolerance seriously limit its clinical application. In order to overcome the above challenges and optimize tumor immunotherapy, nanomaterials have been widely used to regulate tumor microenvironment, increase antigen display, cross complex physical barriers and enhance T cell infiltration (Riley RS. et al. Nat. Rev. Drug Discovery. 2019, 18(3), 175-196).

[0003] Tumor polypeptide vaccines use epitope peptides derived from tumor-associated antigens or tumor-specific antigens as antigens, and are attracting attention due to their advantages such as high specificity, easy manufacturing, low cost and good safety. However, peptide antigens have weak immunogenicity, are easily degraded by enzymes, and have low accumulation in lymph nodes, so a high-efficiency delivery system is needed to enhance the immunogenicity and lymph node accumulation of antigens, thereby triggering a strong immune response (Zheng W. et al. J. Nanobiotechnol. 2024, 22(1), 562). The fifth-generation polyamide-amine dendrimer G5.NH2 PAMAM has the characteristics of clear composition, rich terminal functional groups, highly branched internal cavity and easy functionalization, and has been widely used in the field of nanomedicine. It is an ideal protein delivery carrier (Song C. et al. Coord. Chem. Rev. 2020, 421, 213463). Ovalbumin (OVA) is a widely used model antigen in cancer vaccine research, and its MHC-I antigen epitope OVA 257-264peptide antigen for high expression of OVA cancer cell line (Adv. Mater. 2025, DOI: 10.1002 / adma.202506174). In addition to suitable antigens and efficient delivery systems, suitable vaccine adjuvants and combination therapy strategies are also key factors for the successful development of cancer vaccines. Manganese-based nanomaterials show unique advantages in tumor immunotherapy and have been widely used in related research in the field of nanomedicine (Zhang K. et al. Adv. Mater. 2023, 35(19), e2205409). Manganese dioxide MnO2 not only consumes excess glutathione in the tumor microenvironment to generate divalent manganese ions, but also catalyzes the decomposition of hydrogen peroxide to generate toxic hydroxyl radicals through a Fenton-like reaction, achieving chemical kinetic therapy and inducing immunogenic cell death (ICD, Lin LS. et al. Angew. Chem., Int. Ed. 2018, 57(18), 4902-4906); at the same time, it can act as an adjuvant to regulate the tumor immunosuppressive microenvironment and activate the cGAS-STING pathway to trigger tumor immunotherapy (Luo Z. et al. ACS Nano 2024, 18(44), 30701-30715). SUMMARY

[0004] The technical problem to be solved by the present application is to provide a dendrimer-based polypeptide nanovaccine, a preparation method and application thereof. The dendrimer-based polypeptide nanovaccine is obtained by loading MnO2 nanoparticles on functionalized dendrimers and wrapping polypeptide antigens, and can achieve combined chemotherapy and immunotherapy, thereby effectively killing tumors.

[0005] The present application provides a dendrimer-based polypeptide nanovaccine, which is obtained by loading MnO2 nanoparticles on functionalized dendrimers and wrapping polypeptide antigens. 257-264 .

[0006] Preferably, the functionalized dendrimer is obtained by surface modification of a fifth-generation polyamide-amine dendrimer with polyethylene glycol and heptafluorobutyric acid.

[0007] The present application also provides a preparation method of a dendrimer-based polypeptide nanovaccine, comprising the following steps:

[0008] (1) Mix methoxy-polyethylene glycol-maleimide mPEG-Mal and fifth-generation polyamide-amine dendrimer G5.NH2 PAMAM to prepare solutions, stir and react, dialyze, and freeze-dry to obtain G5.NH2-PEG;

[0009] (2) The G5.NH2-PEG is configured into a solution, then triethylamine TEA and heptafluorobutyric anhydride HFAA are added in sequence, and stirred and reacted, and then dialyzed, freeze-dried to obtain a functional dendrimer GPF;

[0010] (3) The GPF is configured into a solution, then mixed with a potassium permanganate KMnO4 solution, stirred and reacted, dialyzed, and freeze-dried to obtain a nanoparticle MGPF;

[0011] (4) The MGPF and OVA 257-264 are respectively configured into a solution and mixed for incubation, and then ultrafiltration centrifugation is performed to obtain the polypeptide nano-vaccine, denoted as MGPF / OVA 257-264 .

[0012] Preferably, the molar ratio of G5.NH2 PAMAM to mPEG-Mal in the step (1) is 1:12.5-1:15; and the dispersion solvent of the solution is water.

[0013] Preferably, the stirring reaction temperature in the step (1) is room temperature, and the reaction time is 23-25 h.

[0014] Preferably, the molar ratio of G5.NH2-PEG to HFAA in the step (2) is 1:30-1:50; the volume ratio of HFAA to TEA is 1:2-1:3; and the dispersion solvent of the G5.NH2-PEG solution is methanol.

[0015] Preferably, in the step (2), TEA is first added into the G5.NH2-PEG solution and stirred for 20-40 min, then HFAA is added dropwise and stirred for 23-25 h, and the stirring reaction temperature is room temperature.

[0016] Preferably, the molar ratio of GPF to KMnO4 in the step (3) is 1:30-1:50; the dispersion solvent of the solution is water; and the mixing is that the KMnO4 solution is added dropwise into the GPF.

[0017] Preferably, the stirring reaction temperature in the step (3) is room temperature, and the reaction time is 1-3 h.

[0018] Preferably, the mass ratio of MGPF to OVA 257-264 in the step (4) is 1:1-2:1; and the dispersion solvent of the solution is water.

[0019] Preferably, the incubation time in the step (4) is 30 min-1 h, and the incubation temperature is room temperature.

[0020] Preferably, the centrifugation parameter in the step (4) is 4000-5000 g for 30 min-1 h.

[0021] The present invention also provides the application of a dendritic macromolecule-based polypeptide nanovaccine in the preparation of antitumor drugs.

[0022] This invention utilizes transmission electron microscopy (TEM), inductively coupled plasma atomic emission spectroscopy (ICP-OES), and nuclear magnetic resonance hydrogen spectroscopy (NMR spectroscopy). 1 The physicochemical properties of the peptide nanovaccine were characterized using methods such as ¹H NMR, dynamic light scattering analysis (DLS), surface potential measurement, X-ray photoelectron spectroscopy (XPS), and ultraviolet-visible absorption spectroscopy (UV-vis). The cytotoxicity of MGPF was evaluated using the CCK-8 assay; the effect of the peptide nanovaccine on glutathione (GSH) levels in cancer cells was detected using a GSH assay kit; the phagocytic capacity of dendritic cells (DCs) for the peptide nanovaccine was investigated using flow cytometry; the effects of the peptide nanovaccine on reactive oxygen species (ROS), lipid peroxidation (LPO), and calreticulin (CRT) levels in cancer cells were examined using laser confocal microscopy (CLSM); the release of high-mobility protein (HMGB-1) in cancer cells treated with the peptide nanovaccine was detected using ELISA; the release of adenosine triphosphate (ATP) in cancer cells treated with the peptide nanovaccine was detected using an ATP assay kit; the effect of the peptide nanovaccine on DC maturation by inducing intracellular dysplasia (ICD) in cancer cells was investigated using flow cytometry; finally, a subcutaneous colorectal cancer-bearing mouse model was established to evaluate the inhibitory effect of the peptide nanovaccine on tumor growth and recurrence.

[0023] Beneficial effects

[0024] (1) By Figure 1 As shown, the MnO2 contained in the polypeptide nanovaccine prepared by this invention can consume glutathione to generate MnO2 under the high concentration of glutathione in the tumor microenvironment. 2+ Bioactive Mn 2+ By catalyzing the decomposition of H₂O₂ into toxic hydroxyl radicals through a Fenton-like reaction, chemokinetics can kill tumor cells and achieve chemokinetic therapy. Furthermore, chemokinetic therapy can induce ICD in cancer cells. Simultaneously, Mn 2+ It can also activate the cGAS-STING pathway and, in conjunction with peptide antigen presentation, stimulate the immune system, triggering a powerful anti-tumor immune response.

[0025] (2) The polypeptide nanovaccine prepared in this invention can induce a strong immune response after being injected into tumor-bearing mice via peritumoral administration, effectively inhibiting tumor growth and recurrence, and achieving combined chemokinetic / immunotherapy, which has potential clinical application value.

[0026] (3) The synthesis method of the polypeptide nanovaccine prepared by the present invention is simple and easy to operate, and the product is easy to purify and separate, and has good development prospects. Attached Figure Description

[0027] Figure 1 MGPF / OVA prepared for this invention 257-264 Synthesis (a) and application diagram (b).

[0028] Figure 2 The G5.NH2-PEG prepared in this invention 1 1H NMR spectra (a) and G5-PEG.NHAc, GPF.NHAc 1 HNMR spectrum (b).

[0029] Figure 3 XPS total spectrum (a) and Mn2p spectrum (b) of MGPF prepared for this invention.

[0030] Figure 4 MGPF / OVA prepared for this invention 257-264 TEM image (a) and particle size distribution histogram (b).

[0031] Figure 5 The degradation of methylene blue (MB) by MGPF prepared in this invention under different conditions.

[0032] Figure 6 The cumulative release curve of manganese ions from MGPF prepared in this invention is shown.

[0033] Figure 7 Cell viability graphs of MC38-OVA cells after co-incubation with different concentrations of MGPF prepared in this invention for 24 h.

[0034] Figure 8 For OVA 257-264 and MGPF / OVA 257-264 The phagocytic results of cells after co-incubation with DCs for 6 h are shown in Figure (a) and the quantitative analysis figure (b).

[0035] Figure 9 For OVA 257-264 MGPF and MGPF / OVA 257-264 Intracellular GSH levels after co-incubation with MC38-OVA cells for 6 h.

[0036] Figure 10 For OVA 257-264 MGPF and MGPF / OVA 257-264 Laser confocal microscopy images showing changes in intracellular ROS levels after co-incubation with MC38-OVA cells for 6 h.

[0037] Figure 11 For OVA 257-264 MGPF and MGPF / OVA 257-264 Laser confocal microscopy images showing changes in intracellular LPO levels after co-incubation with MC38-OVA cells for 6 h.

[0038] Figure 12 For OVA 257-264 MGPF and MGPF / OVA 257-264 Laser confocal microscopy image of intracellular CRT eversion after co-incubation with MC38-OVA cells for 24 h.

[0039] Figure 13 For OVA 257-264 MGPF and MGPF / OVA 257-264 The results of cellular ATP (a) and HMGB-1 (b) release induced by co-incubation with MC38-OVA cells for 24 h.

[0040] Figure 14 For OVA 257-264 MGPF and MGPF / OVA 257-264 Flow cytometry analysis of treated MC38-OVA cells after co-incubation with DCs for 24 h (a) and quantitative analysis results of the proportion of mature DCs (b).

[0041] Figure 15 Tumor growth curves (a) and body weight change curves (b) at different time points in MC38-OVA subcutaneous tumor-bearing mice after treatment with different formulations.

[0042] Figure 16 After treatment with different formulations, CD3 levels in the tumor tissue of MC38-OVA subcutaneous tumor-bearing mice were measured. + CD8 + Flow cytometry plot of T cells (a) and its quantitative results (b).

[0043] Figure 17 After treatment with different formulations, CD3 levels in the tumor tissue of MC38-OVA subcutaneous tumor-bearing mice were measured. + CD4 + Flow cytometry plot of T cells (a) and its quantitative results (b).

[0044] Figure 18 After treatment with different formulations, CD80 levels in lymph nodes of MC38-OVA subcutaneous tumor-bearing mice were measured. + CD86 + Flow cytometry analysis of DC expression (a) and its quantitative results (b).

[0045] Figure 19 To illustrate the changes in tumor volume over 20 days and tumor weight after 20 days, the results were recorded in Figure (a) and Figure (b) after 20 days, following subcutaneous re-inoculation of MC38-OVA tumor cells into mice using the polypeptide nanovaccine prepared in this invention.

[0046] Figure 20 In a mouse model of a preventative peptide nanovaccine, CD80 in the lymph node region + CD86 + Flow cytometry analysis of DC expression (a) and its quantitative results (b).

[0047] Figure 21 In a mouse model of a prophylactic peptide nanovaccine, CD8 in the spleen + T cells and CD4 + Flow cytometry analysis of T cell expression (a) and its quantitative results (bc). Detailed Implementation

[0048] 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.

[0049] Unless otherwise specified, all chemical reagents are commercially available and can be used directly without further purification. G5.NH2PAMAM was purchased from Dendritech, USA. mPEG-MAL was purchased from Shanghai Yanyi Biotechnology Co., Ltd. Heptafluorobutyric anhydride and triethylamine were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Potassium permanganate was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. OVA 257-264 FITC-OVA 257-264All reagents were purchased from Shanghai Genetech Biotechnology Co., Ltd. MC38-OVA cells (mouse colorectal cancer cells) were obtained from the School of Pharmacy, Shanghai Jiao Tong University. DCs (immature dendritic cell lines derived from mouse bone marrow) were obtained from Fudan University Cancer Hospital. DMEM culture medium, penicillin-streptomycin antibiotics, and trypsin were purchased from Hangzhou Gino Biomedical Technology Co., Ltd. (Hangzhou). Fetal bovine serum was purchased from Gibco Life Sciences, Inc. (Carlsbad, CA). CellCounting Kit-8 (CCK-8) was purchased from Shanghai Qihai Biotechnology Co., Ltd. GSH assay kit was purchased from Nanjing Jiancheng Bioengineering Institute. ATP assay kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd. HMGB-1 ELISA kit was purchased from Beijing Solarbio Technology Co., Ltd. ROS assay kit was purchased from MedChemExpress. C11-BODIPY assay kit was purchased from Glpbio, Inc. Anti-CD80-PE, Anti-CD86-FITC, Anti-CD86-PE, Anti-CD80-FITC, Anti-CD11c-APC, Anti-CD3-PE, Anti-CD8-FITC, Anti-CD4-FITC, and Anti-CD8-PE were purchased from Thermo Fisher Scientific. Female C57BL / 6 mice were purchased from Shanghai JessJ Laboratory Animal Co., Ltd.

[0050] Example 1

[0051] (1) Weigh 50 mg of G5.NH2PAMAM and dissolve it in 5 mL of pure water to obtain a G5.NH2PAMAM solution. Weigh 48.1 mg of methoxy-polyethylene glycol-maleimide mPEG-Mal and dissolve it in 5 mL of pure water to obtain an mPEG-Mal solution. Add the above mPEG-Mal solution to the vigorously stirred G5.NH2 PAMAM solution and continue stirring at room temperature for 24 h. After the reaction is complete, transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 8000-14000 Da and dialyze it in pure water for 3 days. Finally, freeze-dry the product G5.NH2-PEG and store it in a sealed container at -20℃.

[0052] (2) Weigh 50 mg of G5.NH2-PEG and dissolve it in 10 mL of methanol to obtain a G5.NH2-PEG solution. Then, under vigorous stirring, add 28 μL of triethylamine (TEA) dropwise to the solution to make the reaction system weakly alkaline. After stirring at room temperature for 30 min, add 14 μL of heptafluorobutyric anhydride (HFAA) dropwise to the reaction system and continue stirring at room temperature for 24 h. After the reaction is completed, dialyze the product three times in PBS using a dialysis bag with a molecular weight cutoff of 8000-14000 Da, and then dialyze it six times in pure water. Freeze-dry the product to obtain the functionalized dendritic macromolecule GPF, and store it in a sealed container at -20℃.

[0053] (3) Weigh 50 mg of GPF and dissolve it in 10 mL of pure water to obtain a GPF solution; weigh 7.2 mg of KMnO4 and dissolve it in pure water to prepare a 0.25 mg / mL solution. Under vigorous stirring, add 28.8 mL of KMnO4 solution dropwise to the GPF solution using a syringe pump, and continue stirring at room temperature for 1 h. After the reaction is complete, dialyze the solution in pure water for 3 days using a dialysis bag with a molecular weight cutoff of 8000-14000 Da, freeze-dry to obtain the product MGPF nanoparticles, and store them in a sealed container at -20 ℃.

[0054] (4) Mix 1 mg MGPF and 1 mg OVA 257-264 Dissolve each OVA in 1 mL of pure water and obtain OVA 257-264 The solution and MGPF solution were mixed and incubated at room temperature for 40 min. Subsequently, the peptide nanovaccine, denoted as MGPF / OVA, was purified by centrifugation at 4500 g for 30 min three times with the addition of water using an ultrafiltration centrifuge tube with a molecular weight of 10000 Da. 257-264 .

[0055] Example 2

[0056] 8 mg of G5.NH2-PEG from Example 1 was weighed and dissolved in 2 mL of methanol. Then, under vigorous stirring, 50 μL of triethylamine was added dropwise to the solution to make the reaction system weakly alkaline. After stirring at room temperature for 30 min, excess 29 μL of acetic anhydride was added dropwise to the reaction system, and stirring was continued for 24 h. After the reaction was completed, the mixture was dialyzed against pure water for 3 days using a dialysis bag with a molecular weight cutoff of 8000-14000 Da. The product G5-PEG.NHAc was obtained by freeze-drying and stored in a sealed container at -20 °C. 8 mg of GPF was weighed and dissolved in 2 mL of methanol. Then, under vigorous stirring, 50 μL of triethylamine was added dropwise to the solution to make the reaction system weakly alkaline. After stirring at room temperature for 30 min, excess 29 μL of acetic anhydride was added dropwise to the reaction system, and stirring was continued for 24 h. After the reaction, the mixture was dialyzed against pure water for 3 days using a dialysis bag with a molecular weight cutoff of 8000-14000 Da. The product GPF.NHAc was then freeze-dried and stored in a sealed container at -20 °C. Six mg each of G5.NH2-PEG, G5-PEG.NHAc, and GPF.NHAc were weighed and dissolved in 600 μL of deuterated water, respectively. The 1H NMR spectra of the materials were then measured using a nuclear magnetic resonance spectroscopy (NMR) spectrometer. The NMR results for G5.NH2-PEG are shown below. Figure 2 As shown in Figure a, the proton peaks at chemical shifts of 2.2–3.5 ppm represent the methylene proton peaks of G5.NH2PAMAM, while the characteristic proton peak at 3.6 ppm is that of mPEG, indicating that mPEG has been successfully modified onto the surface of G5.NH2PAMAM. By integrating the proton peak regions of G5.NH2PAMAM and mPEG, it was calculated that each G5.NH2PAMAM surface was modified with 10 mPEG molecules. Figure 2 As shown in b, after acetylation, both G5.NH2-PEG and GPF.NHAc exhibited a new characteristic peak at 1.95 ppm, which is the proton peak of the -CH3 group in the acetylation group, indicating that the amino groups on the surface of the dendritic macromolecules were successfully acetylated. Calculations using integral and difference methods showed that each G5.NH2 PAMAM surface was modified with 40 heptafluorobutyric acid molecules.

[0057] Example 3

[0058] To determine the effect of MGPF on OVA 257-264 Encapsulation efficiency, according to MGPF and OVA 257-264 Different masses of MGPF were weighed at mass ratios of 1:1, 2:1, 5:1, and 10:1 and were respectively mixed with 50 μg of FITC-labeled OVA. 257-264Mix and incubate for 40 min. After incubation, use an ultrafiltration centrifuge tube with a molecular weight cutoff of 10000 Da to centrifuge / disperse in water three times at 4500 g for 30 min to separate the unencapsulated OVA. 257-264 Remove and collect the solution in the centrifuge tube, and prepare FITC-OVA at different concentrations. 257-264 Used to prepare a standard curve. Finally, the fluorescence intensity values ​​of the standard curve solution and the solution in the centrifuge tube were measured using a multi-functional microplate reader. The peptide encapsulation efficiency and drug loading were calculated according to the formulas: Encapsulation efficiency = (Total amount of peptide added - Amount of unloaded peptide) / Total amount of peptide added × 100%, and Drug loading rate = Amount of loaded peptide / Total mass of complex × 100%. As shown in Table 1, when MGPF and OVA... 257-264 When the mass ratio is 1:1, MGPF has a positive effect on OVA. 257-264 The encapsulation efficiency reached 93.8%. When the mass ratio increased, the encapsulation efficiency only increased slightly. Therefore, a mass ratio of 1:1 MGPF / OVA was selected. 257-264 The complex was used in subsequent experiments.

[0059] Table 1 MGPF and OVA 257-264 MGPF at different mass ratios for OVA 257-264 Encapsulation efficiency and drug loading rate

[0060]

[0061] Example 4

[0062] Take the GPF, MGPF, and MGPF / OVA from Example 1. 257-264 1 mg of each was dissolved in 1 mL of ultrapure water to determine surface potential, hydration kinetic diameter, and polydispersity index. As shown in Table 2, the hydration kinetic diameter of MGPF was 54.9 nm, and the surface potential was 9.2 mV, while the hydration kinetic diameter of GPF without MnO2 loading was 289.7 nm, and the surface potential was 23.8 mV, both higher than MGPF. This is because the MnO2 loading of GPF nanoparticles allows for more amino groups on the G5.NH2 PAMAM surface to stabilize the nanoparticles, thus lowering their potential; and the MnO2 encapsulation reduces the loose structure of the dendritic macromolecules, thereby decreasing the overall hydration kinetic diameter of the nanocomposite. MGPF encapsulates the peptide antigen to form MGPF / OVA. 257-264 The nanoparticles have a hydration kinetic diameter of 163.0 nm, a surface potential of 8.2 mV, and a polydispersity index of 0.17.

[0063] Table 2 GPF, MGPF, and MGPF / OVA 257-264 Surface potential, hydration dynamic diameter and polydispersity index

[0064]

[0065] Example 5

[0066] The MGPF powder prepared in Example 1 was dried and evenly spread on a conductive adhesive to obtain a sample. The sample was then analyzed using an Escalab 250Xi electron spectrometer. Figure 3 As shown in figure a, the XPS total spectrum of MGPF is in the range of 660-630 eV, confirming the presence of manganese. The XPS spectrum of Mn2p (…) Figure 3 b) Analyze the two characteristic peaks Mn2p 1 / 2 and Mn2p 3 / 2 The binding energies at 654 eV and 642.2 eV, respectively, with a binding energy of 11.8 eV, demonstrate the successful loading of MnO2.

[0067] Example 6

[0068] Take the MGPF / OVA prepared in Example 1 257-264 Dissolved in ultrapure water to prepare a 1 mg / mL solution, this solution was dropped onto the surface of a copper mesh with a carbon film and dried at room temperature to obtain the sample. The sample was then examined using a JEM-2100F transmission electron microscope at 200 kV. (MGPF / OVA) 257-264 TEM images and particle size distribution histograms are as follows: Figure 4 As shown in figures ab, the results indicate that the MnO2-containing nanoparticles are spherical with uniform size, averaging 2.6 ± 0.4 nm. Furthermore, the observation of nanoparticle aggregation confirms the presence of OVA. 257-264 Successful load.

[0069] Example 7

[0070] To test the Mn production of MGPF under different conditions 2+ The ability, and containing HCO 3- Under the condition of Mn 2+ The ability of glutathione (GSH) to generate toxic hydroxyl radicals (·OH) through a Fenton-like reaction with H₂O₂ was investigated, using methylene blue (MB) as an indicator to reflect the generation of ·OH. 1 mL of NaHCO₃ / CO₂ buffer ([NaHCO₃ / CO₂] = 25 mM) containing different concentrations of glutathione (GSH) (0, 1, 2, 5, 10 mM), MGPF (Mn = 0.25 mM), and MB (10 μg / mL) was prepared. Each solution was incubated at 37 ℃ for 30 min, and the UV absorption curves from 400–800 nm were measured afterward to calculate the MB degradation rate. Figure 5As shown, the characteristic absorption peaks of MB exhibit different absorbance values ​​under different conditions, indicating the formation of different concentrations of ·OH. Furthermore, when the GSH concentration is (0-1 mM), the MB degradation efficiency continuously increases, indicating that in the absence of GSH, MGPF can react with small amounts of H₂ in the solution. + It reacts with H2O2 to produce a small amount of Mn 2+ This leads to the formation of trace amounts of ·OH, causing a slight decrease in the MB absorption curve. When the solution contains 1 mM GSH, MGPF further reacts with GSH to form Mn. 2+ The increased concentration of ·OH in the solution enhances MB degradation efficiency. However, as the concentration of GSH in the solution increases, the MB degradation efficiency gradually decreases because excess GSH consumes the generated ·OH, hindering the reaction between ·OH and MB. These results indicate that MGPF can generate Mn in the tumor microenvironment. 2+ It further promotes the generation of ·OH, thereby enabling chemokinetic therapy.

[0071] Example 8

[0072] Phosphate buffer solutions with pH values ​​of 7.4, 6.5, 6.5 + 100 μM H₂O₂, 6.5 + 1 mM GSH, and 6.5 + 100 μM H₂O₂ + 1 mM GSH were prepared to analyze the responsive release of Mn from MGPF. 2+ The performance was assessed. 3 mg of MGPF was dissolved in 1 mL of PBS and placed in a dialysis bag with a molecular weight cutoff of 10000 Da. The dialysis bag was then placed into centrifuge tubes containing 9 mL of the corresponding buffer solution, and the mixture was shaken at 37 °C. At 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h, and 48 h, 1 mL of the extravasated dialysis fluid was collected, and 1 mL of the corresponding buffer solution was added to the centrifuge tubes. The Mn content in the collected fluid was then measured. 2+ Element content, calculate Mn 2+ Calculate the cumulative release amount and plot the cumulative release curve. For example... Figure 6 Mn 2+ As shown in the cumulative release curve, Mn in phosphate buffer at pH = 7.4 2+ Slow release, at pH = 6.5, Mn 2+ Release increased slightly. After adding H₂O₂ or GSH to phosphate buffer at pH = 6.5, Mn 2+ The release is significantly increased because MnO2 can react with H2O2 or GSH to generate Mn. 2+ Furthermore, when Mn contains both H2O2 and GSH, 2+The cumulative release reached 90.5% at 48 h. These results indicate that MGPF can effectively release Mn in the tumor microenvironment. 2+ This leads to chemokinetic therapy and immune activation.

[0073] Example 9

[0074] The in vitro antitumor activity of MGPF prepared in Example 1 was studied using MC38-OVA cells as a model cell. MC38-OVA cells in logarithmic growth phase were collected and cultured at 1 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 g / mL in 96-well plates using DMEM complete medium supplemented with 2 μg / mL MGPF and 10% fetal bovine serum (FBS). The cells were incubated at 37°C for 24 h at 5% CO2. The original medium was discarded, and each well was incubated with complete medium containing different concentrations of MGPF (Mn concentrations of 0, 0.5, 1, 2, 4, 8, and 10 μg / mL). The plates were then incubated at 37°C for another 24 h at 5% CO2. The original medium was then discarded, and serum-free medium containing 10% CCK-8 was added, followed by further incubation for 2–4 h. Finally, the absorbance of each well was measured at 450 nm using a microplate reader. Cells treated with PBS served as the control group (cell viability recorded as 100%), and cells without any drug treatment but containing only medium served as the blank group. Cytotoxicity was calculated. Results are shown below. Figure 7 As shown, within the experimental concentration range, cytotoxicity gradually increased with increasing Mn concentration. This indicates that MGPF induced the CDT effect, resulting in MGPF exhibiting manganese concentration-dependent cytotoxicity against cancer cells.

[0075] Example 10

[0076] DCs were used as a cell model to evaluate their phagocytic effect on different materials. DCs in the logarithmic growth phase were collected and cultured at a density of 2 × 10⁶ cells / well. 5 Cells were seeded at a density of 10% FBS and 1% penicillin-streptomycin in 6-well plates using DMEM complete medium. The cells were incubated in 5% CO2 at 37°C for 24 h. The original medium was then discarded, and serum-free medium or FITC-OVA was added to each well. 257-264 MGPF / FITC-OVA 257-264 Cells were incubated in serum-free medium (corresponding to a Mn concentration of 5 μg / mL) at 37 °C for 6 h at 5% CO2. The original medium was discarded, cells were washed three times with PBS, digested with trypsin, collected, and centrifuged. After washing three times with PBS, cells were resuspended in 300 μL PBS, and fluorescence intensity was detected by flow cytometry. Figure 8As shown, MGPF / OVA 257-264 The fluorescence value of the group is OVA 257-264 The fluorescence value of the group was 1.27 times that of the group, indicating that the MGPF / OVA vector... 257-264 This can improve DCs' response to OVA 257-264 Its ability to devour.

[0077] Example 11

[0078] The ability of different materials to deplete GSH in cancer cells was evaluated using MC38-OVA cells as a model. MC38-OVA cells in logarithmic growth phase were collected and cultured at 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 cells / well in 6-well plates and incubated at 37 °C with 5% CO2 for 24 h. The original culture medium was discarded, and each well was replenished with either culture medium or OVA-containing medium. 257-264 MGPF and MGPF / OVA 257-264 The culture medium (containing Mn at a concentration of 5 μg / mL) and cells were incubated for 6 h at 37 °C with 5% CO2. The original culture medium was discarded, and the cells were washed three times with PBS, trypsinized, collected, and centrifuged. After washing three more times, the cells were resuspended in 300 μL PBS, sonicated on ice for 40 min, and the supernatant was collected to determine GSH content. Figure 9 As shown, MGPF and MGPF / OVA 257-264 The intracellular GSH content in the treatment group was significantly higher than that in PBS and OVA. 257-264 The treatment group showed a significant reduction, and MGPF and MGPF / OVA were also significantly reduced. 257-264 There was no significant difference in intracellular GSH levels among the groups, indicating that OVA 257-264 It does not affect intracellular GSH levels, and this also demonstrates that MGPF / OVA 257-264 MnO2 in the cell can generate Mn by consuming intracellular GSH. 2+ It is used for subsequent tumor chemokinetic therapy.

[0079] Example 12

[0080] The effect of peptide nanovaccines on intracellular ROS levels in cancer cells was evaluated using MC38-OVA cells as a model. Cells were cultured at 1 × 10⁶ cells per dish. 5 Cells were seeded at a density of [number] cells per confocal dish and incubated at 37 °C with 5% CO2 for 24 h. The original culture medium was discarded, and each dish was replenished with culture medium or OVA-containing medium. 257-264 MGPF and MGPF / OVA 257-264The culture medium (containing Mn at a concentration of 5 μg / mL) and cells were incubated for 6 h at 37 ℃ with 5% CO2. After 6 h, the original culture medium was discarded, and the DCFH-DA probe diluted with serum-free medium was added. The mixture was then incubated at 37 ℃ for 30 min in the dark. The culture medium was discarded, and the cells were washed three times with PBS, fixed with 4% paraformaldehyde solution for 10 min, washed three times with PBS, stained with the nuclear dye DAPI for 10 min, and then washed three times with PBS. The experimental results were observed using a laser confocal microscope. DCFH-DA was initially non-fluorescent but could penetrate the cell membrane and enter the cell, where it was hydrolyzed by intracellular esterases to generate cell membrane-impermeable DCFH. Intracellular reactive oxygen species could oxidize the non-fluorescent DCFH to generate green fluorescent DCF. The results are as follows: Figure 10 As shown, OVA 257-264 Compared to the PBS group, there was no significant change in green fluorescence in the MGPF and MGPF / OVA groups. 257-264 The significantly increased green fluorescence intensity in the group indicates that MGPF / OVA 257-264 MnO2 is effectively converted into bioactive Mn 2+ It promotes the generation of intracellular ROS, which can be used for chemokinetic therapy of cancer cells.

[0081] Example 13

[0082] The effect of peptide nanovaccines on intracellular LPO levels in cancer cells was evaluated using MC38-OVA cells as a model. Cells were cultured at 1 × 10⁶ cells per dish. 5 Cells were seeded at a density of [number] cells per confocal dish and incubated at 37 °C with 5% CO2 for 24 h. The original culture medium was discarded, and each dish was replenished with culture medium or OVA-containing medium. 257-264 MGPF and MGPF / OVA 257-264 The culture medium (containing Mn at a concentration of 5 μg / mL) and cells were incubated for 6 h at 37 ℃ with 5% CO2. After 6 h, the original culture medium was discarded, and C11-BODIPY 581 / 591 probe diluted with serum-free medium was added. The mixture was then incubated at 37 ℃ for 30 min in the dark. The culture medium was discarded, the cells were washed three times with PBS, fixed with 4% paraformaldehyde solution for 10 min, washed three times with PBS, stained with the nuclear dye DAPI for 10 min, washed three times with PBS, and finally washed three times with PBS. The experimental results were observed using a laser confocal microscope. Lipid peroxides are peroxyl-containing lipids formed by ROS oxidation of biological membranes. The C11-BODIPY 581 / 591 probe is an oxidation-sensitive fluorescent fatty acid analog that is easily incorporated into the membrane. In its non-oxidized state, it exhibits red fluorescence, which turns green when induced by free radicals. Figure 11As shown, the red fluorescence nonoxidized intensity was the highest in the normal, unstimulated PBS group, and almost no green fluorescence oxidized signal was observed; after free OVA 257-264 No obvious green fluorescence was observed after stimulation. However, after stimulation with MGPF and MGPF / OVA... 257-264 After stimulation, intracellular red fluorescence was significantly reduced, while green fluorescence was significantly enhanced. This may be due to the MGPF / OVA ratio. 257-264 MnO2 is converted to Mn 2+ Mn 2+ The Fenton-like reaction increases intracellular ROS levels, leading to the accumulation of intracellular LPO.

[0083] Example 14

[0084] To investigate whether the prepared peptide nanovaccine could induce ICD in MC38-OVA cells, the eversion of the CRT and the release of HMGB-1 and ATP in MC38-OVA cells treated with different materials were examined. MC38-OVA cells were cultured at 1 × 10⁶ cells per dish. 5 Cells were seeded at a density of 1000 mcg / mL in confocal dishes and cultured at 37 °C in a 5% CO2 incubator for 24 h. The original culture medium was discarded, and culture medium or OVA was added to each well. 257-264 MGPF and MGPF / OVA 257-264 The culture medium (containing Mn at a concentration of 5 μg / mL) was used to incubate the cells with the cells at 5% CO2 and 37 ℃ for 24 h, followed by CRT immunofluorescence staining. Figure 12 As shown in the laser confocal image results, OVA 257-264 No obvious green signal was observed in the group, MGPF and MGPF / OVA 257-264 The significantly enhanced green fluorescence intensity of the group indicates that MGPF and MGPF / OVA 257-264 It can induce Mn-based chemokinetic killing in cancer cells, leading to ICD and CRT eversion. Furthermore, cells were spaced at 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 cells / well in 6-well plates and incubated at 37°C with 5% CO2 for 24 h. The original culture medium was then discarded, and the cells were incubated with OVA-containing medium. 257-264 MGPF and MGPF / OVA 257-264 The culture medium (containing Mn at a concentration of 5 μg / mL) was incubated for 24 h at 37 °C with 5% CO2. Afterward, the cell supernatant was collected, and the extracellular ATP content was detected using an ATP assay kit. The HMGB-1 content was detected using an HMGB-1 ELISA kit. Figure 13As shown in ab, compared with the PBS group, OVA 257-264 The relative release of extracellular ATP and HMGB-1 in the treatment groups showed no significant change, but after treatment with MGPF and MGPF / OVA... 257-264 The significant increase after treatment indicates that MGPF / OVA 257-264 ICD can be induced in cancer cells through the chemokinetics induced by Mn.

[0085] Example 15

[0086] To verify whether the prepared peptide nanovaccine could induce ICD in tumor cells and thus cause DCs to mature, DCs were prepared at a density of 1.0 × 10⁶ cells per well. 5 Cells were seeded in 12-well plates and simultaneously seeded in 3 μm Transwell chambers at a concentration of 1.0 × 10⁶ cells per chamber. 5 MC38-OVA cells were seeded to establish a Transwell system and cultured in a 37°C, 5% CO2 incubator for 16 h. The supernatant in the chambers and wells was discarded, and OVA-containing solutions were added to the chambers. 257-264 MGPF and MGPF / OVA 257-264 (All materials used were prepared with culture medium containing 5 μg / mL Mn.) Only fresh culture medium was added to each well of the cell culture plate. DCs and MC38-OVA cells were co-incubated in a cell culture incubator for 24 h. The supernatant was discarded, DCs were digested with trypsin, washed three times with PBS, and Anti-CD86-PE and Anti-CD80-FITC antibodies were added. The cells were stained on ice in the dark for 30 min, washed three times with PBS, and the DCs were resuspended in 300 μL of PBS. Flow cytometry was used to detect the maturation degree of the DCs. Figure 14 As shown, MGPF and MGPF / OVA 257-264 The ripening rate of DCs in the treatment group was significantly increased, which was due to MGPF and MGPF / OVA. 257-264 Inducing cancer cells to undergo ICD and release damage-associated molecular patterns (DAMPs), along with Mn 2+ Activation of the STING pathway releases related cytokines, which in turn promotes DC maturation and activates anti-tumor immunity.

[0087] Example 16

[0088] All animal experiments were conducted in strict accordance with relevant standards of animal protection associations. 4-5 week old C57BL / 6 female mice were purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd. To investigate the in vivo antitumor efficacy of the prepared peptide nanovaccine, a subcutaneous tumor-bearing model of MC38-OVA was established in C57BL / 6 female mice. Each mouse was subcutaneously injected with 2×10⁻⁶ dextrose nanoparticles in the axilla. 7MC38-OVA cells were used until the tumor volume reached 50-100 mm. 3 Mice were randomly divided into 4 groups (n=6 per group), as follows: PBS, OVA, etc. 257-264 MGPF, MGPF / OVA 257-264 (Mn = 2.5 mg / kg, OVA) 257-264 = 21 mg / kg), administered via peritumoral injection. The treatment diary begins on day 1, with peritumoral injections performed on days 1, 3, 5, and 7 for each group. Tumor volume (V) = (L × W) was recorded every two days during treatment. 2 (L is the length of the tumor, W is the width of the tumor) / 2 and the mouse's weight. For example... Figure 15 As shown in figure a, after 14 days of treatment, the tumor volume of mice in each group showed varying degrees of increase. Compared with the other groups, MGPF / OVA... 257-264 The mice exhibited the smallest tumor volume after treatment, indicating that Mn-based chemokinetics and peptide antigen presentation can effectively activate the immune system in mice, thereby effectively inhibiting tumor growth. Figure 15 b shows the weight change curve of mice during the treatment period. The results indicate that the weight of mice in each group increased slightly, indicating that the materials in each group had no obvious toxicity to mice and had good biocompatibility.

[0089] In addition, on day 14 after treatment, the anti-tumor immune effects in mouse tumor tissues and lymph nodes treated with different materials were analyzed by flow cytometry. Under aseptic conditions, tumor tissues and lymph nodes from each group of mice were collected, and immune cells were isolated for flow cytometry analysis. Tumor tissues were minced and ground, filtered through a 400-mesh filter, and erythrocytes were lysed to obtain cell suspensions. These cell suspensions were then stained with Anti-CD3-PE / Anti-CD8-FITC and Anti-CD3-PE / Anti-CD4-FITC antibodies, respectively, in an ice bath in the dark for 30 min. After washing with PBS and centrifugation, the cell pellet was resuspended in 500 μL of PBS. Lymph node tissues were minced and ground, filtered through a 400-mesh filter, and stained with Anti-CD11c-APC / Anti-CD80-PE / Anti-CD86-FITC antibodies, in an ice bath in the dark for 30 min. After washing with PBS and centrifugation, the cell pellet was resuspended in 500 μL of PBS. After staining, CD8+ in tumor tissues of mice in each group was analyzed by flow cytometry. ＋ T cells and CD4 ＋ The proportion of T cells and the degree of maturation of dendritic cells (DCs) in lymph nodes. For example... Figures 16-18 As shown, after 14 days of treatment, MGPF / OVA 257-264 CD8 in tumor tissue of mice +T cells and CD4 + T cell counts were significantly higher in the MGPF / OVA group than in the other groups, and the degree of dendritic cell maturation in lymph nodes was also significantly higher in the MGPF / OVA group. This indicates that the MGPF / OVA group has significantly higher T cell counts than in the other groups. 257-264 It can induce ICD in cancer cells, activate the cGAS-STING pathway, and promote peptide antigen presentation, thereby activating antigen-presenting cells and triggering a specific anti-tumor immune response.

[0090] Example 17

[0091] To investigate the in vivo tumor prevention effect of the peptide nanovaccine prepared in Example 1, healthy female C57BL / 6 mice aged 4-5 weeks were randomly divided into two groups (n=6 per group): PBS group and MGPF / OVA group. 257-264 Group (Mn = 2.5 mg / kg, OVA) 257-264 = 21 mg / kg), administered via subcutaneous injection in the armpit, once every 6 days, for a total of 4 doses. Six days after the last administration, mice were subcutaneously inoculated with 2 × 10⁻⁶ mg / kg in the armpit. 7 MC38-OVA cells were inoculated, with the day of inoculation recorded as day 0. Tumor volume changes in mice were assessed 7 days after inoculation, over a 14-day period. For example... Figure 19 As shown in figure a, compared with the PBS group, after 4 cycles of MGPF / OVA 257-264 Following immunization with the nano-vaccine, tumor growth was slower. Furthermore, 20 days after tumor cell inoculation, compared to the PBS group, the MGPF / OVA group showed significantly reduced tumor growth. 257-264 The tumors in this group were smaller in weight ( Figure 19 b). These results demonstrate that MGPF / OVA 257-264 It has the potential to be used as a preventive nanovaccine, which can effectively activate DCs in mice and mediate antigen presentation, thereby initiating T cell adaptive immune response and ultimately playing a role in preventing tumor growth.

[0092] To further investigate the immune response of peptide nanovaccines as prophylactic nanovaccines in mice, lymph nodes and spleens of mice from each group were removed under aseptic conditions, minced, ground, and filtered through a 400-mesh filter to obtain cell suspensions. The lymph node-derived cell suspensions were labeled with Anti-CD11c-APC / Anti-CD86-FITC / Anti-CD80-PE antibodies, while the spleen-derived cell suspensions were separated using nylon villous column chromatography to obtain T lymphocyte suspensions. These T lymphocytes were labeled with Anti-CD4-FTIC / Anti-CD8-PE antibodies. Flow cytometry was used to analyze the maturation degree of dendritic cells (DCs) in the lymph nodes and the CD4+ in the spleen. ＋ T cells and CD8 ＋ The proportion of T cells was analyzed. For example... Figure 20 As shown, compared with the PBS group, MGPF / OVA257-264 The proportion of mature DCs in the lymph nodes of the group of mice was significantly increased, indicating that MGPF / OVA 257-264 Mn 2+ Activation of the cGAS-STING pathway, along with peptide antigen presentation, jointly promotes DC maturation. For example... Figure 21 As shown, MGPF / OVA after immunization 257-264 The proportion of CD8⁺ T cells in the spleen of mice in the control group was significantly increased, indicating that it can effectively activate CD8⁺ T cells. + T-cell immune responses play a role in tumor prevention and recurrence suppression.

[0093] In summary, the MGPF / OVA constructed in this invention 257-264 Peptide nanovaccines possess both therapeutic and preventative vaccine functions. Through chemokinetics, they can induce ICD in cancer cells, activate the cGAS-STING pathway, and promote peptide antigen presentation. This synergistically promotes the maturation of antigen-presenting cells, alleviates the immunosuppressive tumor microenvironment, and achieves combined chemokinetics / immunotherapy, effectively inhibiting tumor growth and preventing tumor recurrence.

Claims

1. A polypeptide nanovaccine based on dendritic macromolecules, characterized in that, The peptide nanovaccine is obtained by loading MnO2 nanoparticles with functionalized dendritic macromolecules and encapsulating peptide antigens; the peptide antigen is OVA. 257-264 .

2. The polypeptide nanovaccine according to claim 1, characterized in that, The functionalized dendritic macromolecule is obtained by modifying the surface of a fifth-generation polyamide-amine dendritic macromolecule with polyethylene glycol and heptafluorobutyric acid.

3. A method for preparing a peptide nanovaccine based on dendritic macromolecules, characterized in that, Includes the following steps: (1) Methoxy-polyethylene glycol-maleimide mPEG-Mal and fifth-generation polyamide-amine dendritic macromolecule G5.NH2PAMAM were respectively prepared into solutions and mixed, stirred and reacted, dialyzed and lyophilized to obtain G5.NH2-PEG; (2) The above G5.NH2-PEG was prepared into a solution, and then triethylamine TEA and heptafluorobutyric anhydride HFAA were added in sequence. The mixture was stirred and reacted, dialyzed, and lyophilized to obtain functionalized dendritic macromolecule GPF. (3) The above GPF was prepared into a solution, then mixed with potassium permanganate KMnO4 solution, stirred and reacted, dialyzed, and freeze-dried to obtain nanoparticles MGPF; (4) Combine the above MGPF with OVA 257-264 The solutions were prepared separately, mixed and incubated, and then subjected to ultrafiltration and centrifugation to obtain the polypeptide nanovaccine.

4. The preparation method according to claim 3, characterized in that, In step (1), the molar ratio of G5.NH2PAMAM to mPEG-Mal is 1:12.5 to 1:15; the dispersing solvent of the solution is water; the stirring reaction temperature is room temperature, and the reaction time is 23-25 ​​h.

5. The preparation method according to claim 3, characterized in that, In step (2), the molar ratio of G5.NH2-PEG to HFAA is 1:30 to 1:50; the volume ratio of HFAA to TEA is 1:2 to 1:3; and the dispersing solvent of the G5.NH2-PEG solution is methanol.

6. The preparation method according to claim 3, characterized in that, In step (2), TEA is first added to the G5.NH2-PEG solution and stirred for 20-40 min, then HFAA is added dropwise and stirred for 23-25 ​​h. The stirring reaction temperature is room temperature.

7. The preparation method according to claim 3, characterized in that, In step (3), the molar ratio of GPF to KMnO4 is 1:30 to 1:50; the dispersing solvent of the solution is water; the mixing is the addition of KMnO4 solution dropwise to GPF; the stirring reaction temperature is room temperature, and the reaction time is 1 to 3 h.

8. The preparation method according to claim 3, characterized in that, In step (4), MGPF and OVA 257-264 The mass ratio is 1:1 to 2:1; the dispersing solvent of the solution is water.

9. The preparation method according to claim 3, characterized in that, The incubation time in step (4) is 30 min to 1 h, the incubation temperature is room temperature, and the centrifugation parameters are 4000 to 5000 g for 30 min to 1 h.

10. The application of a dendritic macromolecule-based polypeptide nanovaccine as described in any one of claims 1 to 2 in the preparation of antitumor drugs.

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