Preparation method and application of silicon dioxide nano vaccine for mediating iNKT cells

Abstract

The invention discloses a preparation method of a silicon dioxide nano vaccine for mediating constant natural killer T (iNKT) cells and application of the silicon dioxide nano vaccine in the field of tumor immunotherapy. The iNKT cell is a special T cell subset which can be activated by a glycolipid antigen presented by CD1d, and can directly kill tumor cells and activate other immune cells. Clinically, the classical agonist alpha GC of the iNKT cell can lead to function depletion of the iNKT cell after multiple intravenous administration, and finally evolves into an inactive state. Although the CD1d / alpha GC protein complex relieves the iNKT cell anenergetic state to a certain extent, the CD1d / alpha GC protein complex is easily degraded and cleared in a complex physiological environment, and the treatment effect is weakened. The characteristics of large aperture and high specific surface area of dendritic mesoporous silica are utilized, CD1d / alpha GC is loaded into a pore channel of a mesoporous nano-material, and the design can protect protein drugs from being degraded in blood circulation, so that a nano-vaccine capable of effectively mediating iNKT cells and successfully relieving the inability of the iNKT cells is constructed; and the effectiveness of the compound in tumor treatment is verified. The patent is supported by the national natural science fund (82001958).

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a method for preparing and applying a silica nanovaccine that mediates iNKT cells. Background Technology

[0002] Immunotherapy represents a major revolution in cancer treatment. Immunotherapy based on inertial natural killer (iNKT) cells offers unique advantages: it is independent of specific tumor antigens, can synergize with multiple immune cells, and exhibits broad anti-cancer potential. iNKT cells are a special subset of T cells whose surface-expressed inertial T cell receptors are highly sensitive and rapidly responsive to non-peptide CD1d-restricted antigens (such as α-galactosylceramide). Activated iNKT cells can trigger a cascade of responses between innate and adaptive immune cells, acting as a bridge between the two. These activated iNKT cells exert their anti-tumor effects through multiple mechanisms, including direct tumor killing, recruitment and activation of various immune cells, improvement of the tumor immunosuppressive microenvironment, and promotion of immune memory. Because iNKT cell-induced anti-tumor immunity is independent of tumor antigens, iNKT cell-based immunotherapy holds promise as a novel strategy for pan-cancer treatment.

[0003] α-GalCer (αGC) is a glycosphingolipid derived from marine sponges and a classic iNKT cell activator. It was initially discovered during screening for antitumor compounds to activate iNKT cells. However, clinical studies have shown that repeated intravenous injection of free αGC leads to iNKT cell dysfunction, manifested as a loss of responsiveness to reactivation. iNKT cells are unable to effectively proliferate and release large amounts of cytokines, including interferon-γ (IFN-γ) and interleukin-4 (IL-4), thus losing their effective ability to attack pathogens or tumor cells. This phenomenon is called iNKT cell anergy and may be related to the exhaustion of iNKT cell function or alterations in regulatory mechanisms caused by repeated activation. Treatment should aim to avoid anergy and ensure that iNKT cells maintain effective immune function during treatment.

[0004]

[0005] CD1d is a conserved, non-classical MHC class I molecule, primarily expressed on the surface of antigen-presenting cells (including dendritic cells, monocytes, and B cells). Glycolipid antigens such as αGC can only bind to the TCR of iNKT cells and initiate an iNKT cell immune response by forming glycolipid antigen complexes with CD1d molecules expressed on the surface of antigen-presenting cells. Previous studies have shown that repeated injections of CD1d fusion proteins carrying αGC have induced sustained iNKT cell responses in mice. This discovery reveals an effective alternative to directly activating iNKT cells using CD1d / αGC complexes, providing a new pathway for iNKT cell activation. However, CD1d / αGC complexes are rapidly degraded and eliminated in vivo, making it difficult to maintain effective concentrations and thus weakening the therapeutic effect.

[0006] Nanoparticle delivery systems can protect protein drugs from degradation, improving their bioavailability and therapeutic efficiency. Currently, protein drug delivery systems include liposomes, polymer microspheres / nanoparticles, and viral vectors. However, these vectors suffer from low delivery efficiency, poor stability, and potential immunogenicity. New mesoporous materials such as mesoporous silica (MSNs), metal-organic frameworks (MOFs), and mesoporous carbon are being researched for protein delivery. Among these, MSNs exhibit significant advantages in protein delivery due to their high specific surface area, tunable pore size, excellent biocompatibility, and ease of surface functionalization. Traditional mesoporous silica has limited in vivo applications due to its small pore size and poor degradability. Dendritic mesoporous silica (DMSN), with its large pore size and easy degradation in physiological environments, can overcome the shortcomings of traditional mesoporous silica materials.

[0007] In summary, this invention improves upon DMSN by introducing thiol functional groups to obtain the modified material D-SH. Compared to the original DMSN, D-SH exhibits superior stability and can be further coated with polyethylene glycol (PEG) to meet specific functionalization requirements. The D-SH nanocarrier achieves an encapsulation efficiency of up to 93±5% for the CD1d / αGC complex, effectively protects the CD1d / αGC complex in complex physiological environments, alleviates iNKT cell anergy, and demonstrates good anti-tumor effects. Summary of the Invention

[0008] This invention aims to construct a silica-based nanovaccine that mediates iNKT cells by utilizing dendritic mesoporous silica nanomaterials as nanocarriers to deliver the large molecular protein complex CD1d / αGC. In this invention, DMSN was prepared in a heterogeneous oil-water two-phase layered reaction system. Thiol groups were modified with a silane coupling agent (3-mercaptopropyl)triethoxysilane (MPTES). The CD1d / αGC large molecular protein complex was then loaded into the pores using ultrasound to effectively protect the complex and prevent its degradation and clearance in vivo. After multiple intravenous administrations, the constructed mesoporous silica nanovaccine not only alleviated iNKT cell anergy but also exhibited good immune activation and significant anti-tumor effects. This vaccine opens a new avenue for iNKT cell-based immunotherapy, confirms the important value of iNKT cell immunotherapy, and provides a material and theoretical basis for further clinical applications and research.

[0009] The technical solution of the present invention is: a thiol-modified dendritic mesoporous silica, wherein the thiol-modified dendritic mesoporous silica nanoparticles are modified with thiol groups by (3-mercaptopropyl)triethoxysilane to obtain the thiol-modified dendritic mesoporous silica.

[0010] The silica, wherein the mass ratio of dendritic mesoporous silica to (3-mercaptopropyl)triethoxysilane is 1:1 to 5.

[0011] In some specific technical solutions, the mass ratio of dendritic mesoporous silica to (3-mercaptopropyl)triethoxysilane is 1:1 to 3.

[0012] The diameter of the dendritic mesoporous silica is 100–150 nm.

[0013] In some specific technical solutions, the diameter of the dendritic mesoporous silica is 100-120 nm.

[0014] The silica, specifically the dendritic mesoporous silica nanoparticles, is obtained by hydrolysis and condensation of triethanolamine, hexadecyltrimethylammonium chloride, and tetraethyl orthosilicate at the oil-water interface.

[0015] Specifically, the mass ratio of ethanolamine, hexadecyltrimethylammonium chloride, and tetraethyl orthosilicate is 0.1–0.3:6:1–8.

[0016] In some specific technical solutions, the mass ratio of ethanolamine, hexadecyltrimethylammonium chloride, and tetraethyl orthosilicate is 0.15–0.2:6:1–8.

[0017] The silica has a rich porous structure, which can be used to load macromolecular drugs.

[0018] The macromolecular drug is selected from at least one of proteins, peptides, and nucleic acids.

[0019] The thiol-modified dendritic mesoporous silica can be linked to functional compound groups via thiol groups.

[0020] Specifically, the thiol-modified dendritic mesoporous silica can be combined with mPEG. 2k -Mal reaction yields mPEG-linked 2k -Mal's dendritic mesoporous silica:

[0021]

[0022] A nanovaccine that mediates iNKT cells, the nanovaccine comprising a nanocarrier and a macromolecular protein complex loaded into the pores of the nanocarrier.

[0023] The nanocarrier is thiol-modified and PEG-coated dendritic mesoporous silica.

[0024] Specifically, the nanocarrier is the aforementioned thiol-modified dendritic mesoporous silica.

[0025] The macromolecular protein complex is CD1d / αGC.

[0026] The large protein complex CD1d / αGC was obtained by loading CD1d onto αGC. A stock solution of 1–2 mg / mL αGC was diluted to a concentration of 200–400 μg / mL with PBS solution containing 0.5–0.8% Tween 20 at pH 7.4. CD1d protein solution was added, and PBS was added to bring the Tween 20 concentration in the reaction solution to 0.05–0.1%. The reaction was carried out at 37°C for 3–4 h, and then incubated overnight (12–24 h) at 4°C. Unloaded free αGC was removed by ultrafiltration through a 10–30 kD ultrafiltration tube.

[0027] Specifically, the molar ratio of CD1d to α-galactosylceramide (αGC) is 1:6 to 12.

[0028] The novel nano-vaccine carrier D-SH has a specific surface area of ​​400–1000 m². 2 The average pore size is approximately 4-8 nm, with a density of approximately 0.5 g / g.

[0029] In some specific technical solutions, the specific surface area of ​​the novel nano-vaccine carrier D-SH is between 800 and 1000 m². 2 / g.

[0030] The hydrated particle size of the nanovaccine is approximately 100–150 nm, and the loading of the macromolecular protein complex on the dendritic mesoporous silica nanomaterial is 0.17–0.20 mg / mg.

[0031] In some specific technical solutions, the hydrated particle size of the nanovaccine is approximately 100–120 nm.

[0032] The novel nano-vaccine, wherein the thiol groups on the surface of the silica interact with... The following structures are modified by functionalizing groups via reaction connection:

[0033]

[0034] The functional group R can be methyl, carboxyl, amino, biotinylate, azide, glucose, mannose, or short peptide and protein molecules with targeting properties.

[0035] Polyethylene glycol (molecular weight 500-5000kD) is used as a linker, with maleimide attached to one end and a functional group R attached to the other end.

[0036] The method for preparing the novel nano-vaccine includes the following steps:

[0037] ① A dendritic mesoporous silica nanomaterial was synthesized by hydrolysis and condensation at the oil-water interface using triethanolamine, hexadecyltrimethylammonium chloride, and water as the aqueous phase and cyclohexane and tetraethyl orthosilicate as the oil phase.

[0038] ②The dendritic mesoporous silica nanomaterials obtained in step ① are heated and refluxed in a solvent to remove the template agent hexadecyltrimethylammonium chloride.

[0039] ③ React the dendritic mesoporous silica obtained in step ② with MPTES to obtain thiol-modified dendritic mesoporous silica.

[0040] ④ Add the macromolecular protein complex CD1d / αGC to the thiol-modified dendritic mesoporous silica dispersion obtained in step ③, and under ultrasonication, load the macromolecular protein complex CD1d / αGC into the pores of the silica. Remove the unloaded macromolecular protein complex to obtain the novel nanovaccine.

[0041] The preparation method described above involves combining the novel nanovaccine prepared in step ④ with mPEG. 2k -Mal room temperature reaction yields nano-vaccines with functionalized PEG groups.

[0042] Specifically, the present invention provides a method for preparing a nanovaccine mediated by iNKT cells, comprising the following steps:

[0043] Triethanolamine, hexadecyltrimethylammonium chloride and water were added to a round-bottom flask at a mass ratio of 0.1-0.3:6:60. The reaction solution was heated to 55-65°C in an oil bath at a speed of 100-150 rpm.

[0044] After reacting the reaction solution from step ① for 1 to 1.5 hours, add a mixture of tetraethyl orthosilicate and cyclohexane in a volume ratio of 1:2 to 19 to the reaction system.

[0045] The reaction solution from step ② was kept under magnetic stirring for 6–12 h. The upper organic phase was discarded, and the aqueous phase was centrifuged (15,000–20,000 rpm, 10–20 min) to collect the product.

[0046] The product collected after centrifugation in step ③ was heated and refluxed at 70-90°C for 24-48 h with a hydrochloric acid / ethanol mixture (volume ratio of 1:5-10) to completely remove the template agent hexadecyltrimethylammonium chloride and obtain DMSN.

[0047] In step ④, 10–20 mg of DMSN and 20–40 μL of MPTES were reacted overnight (24–48 h) in 20–40 mL of ethanol solution at 70–90 °C and 400–600 rpm to obtain thiol-modified DMSN (D-SH).

[0048] Take 1–1.5 mg of D-SH nanocarrier from step ⑤ and disperse it in an ice bath using an ultrasonic cell disruptor (50–150 W, 5 on, 5 s off) for 5–10 min. Then, add 0.1–0.3 mg of CD1d / αGC macromolecular protein complex to the dispersed D-SH aqueous solution and sonicate again in an ice bath for 5–10 min (50–150 W, 5 s on, 5 s off). Afterward, centrifuge (15,000–20,000 rpm, 10–20 min) and wash to remove free protein.

[0049] The free protein removed in step ⑥ was collected, and the concentration of unloaded free protein was determined using the Bradford Protein Detection Kit. The amount of protein loaded on D-SH was then determined using the differential method.

[0050] The D-SH&CD1d / αGC prepared in step ⑥ was mixed with 1–3 mg mPEG. 2k -Mal is dissolved in 1-2 mL of water and shaken at room temperature for 10-20 min, then centrifuged (15000-20000 rpm, 10-20 min) and washed to remove unmodified mPEG. 2k -Mal, thereby preparing nano-vaccines with the functionalized PEG group.

[0051] The application of the aforementioned silica or the novel nanovaccine in the preparation of antitumor drugs.

[0052] The tumors are selected from lymphoma, lung cancer, kidney cancer, melanoma with liver metastasis, colon cancer with liver metastasis, breast cancer with lung metastasis, liver cancer, and pancreatic cancer.

[0053] Specifically, the target tumor is a deep malignant metastatic tumor, preferably a melanocytic liver metastasis.

[0054] The beneficial effects of this invention are as follows: A high-encapsulation-efficiency, degradable, and functionalized three-dimensional dendritic mesoporous silica nanoparticle is successfully prepared. It exhibits uniform particle size and good dispersibility, effectively reducing the rapid clearance and degradation of large molecular drugs such as proteins in vivo. A single dose of the nanovaccine D-SH&CD1d / αGC of this invention can induce iNKT (TCRβ) in mice. + CD1d Tetramer + Effective activation of cells was achieved, and even after three repeated administrations, the activated iNKT cell function remained significantly higher than that of the small molecule free drug αGC and the free protein complex CD1d / αGC. In a mouse model of melanoma liver metastasis, the D-SH&CD1d / αGC nanovaccine exhibited enhanced immune activation and significant tumor suppression effects. After treatment, the proportions of iNKT cells and NK cells in the mouse liver (including the tumor area) increased, as did the proportions of DCs (CD80) cells. + CD86 + in CD11c + Increased maturity indicates that the efficiency of tumor antigen presentation has been effectively improved, thereby promoting CD8. + T cell infiltration in tumor areas. Furthermore, this vaccine can reduce immunosuppressive myeloid-derived suppressor cells (MDSCs, CD11b) in tumor tissue. + Gr-1 + The quantity of the nanovaccine is [not specified]. Meanwhile, the nanovaccine of this invention is applicable to various tumors, such as lymphoma, lung cancer, colon cancer (liver metastasis), breast cancer (lung metastasis), and liver cancer, and has the potential to achieve pan-cancer therapy. The nanovaccine of this invention is simple to prepare, low in cost, and can promote the construction of an immune interaction network in the body by activating iNKT cells, thus having broad prospects for clinical translation. Attached Figure Description

[0055] Figure 1 Figure I shows the characterization of dendritic mesoporous silica nanocarriers and the performance study of nanovaccines.

[0056] Figure (A) is a nitrogen adsorption-desorption diagram of DMSN and D-SH;

[0057] Figure (B) shows the average pore size distribution of DMSN and D-SH;

[0058] Figure (C) shows the small-angle X-ray diffraction (SAXS) patterns of CTAC@DMSN, DMSN, D-SH, and D-SH-PEG.

[0059] Figure (D) shows the Fourier transform infrared spectra of CTAC@DMSN, DMSN, D-SH, and D-SH-PEG.

[0060] Figure (E) shows the non-denaturing gel electrophoresis results of CD1d, D-SH & CD1d-647, and D-SH & CD1d-647-PEG.

[0061] Figure (F) shows the particle size distribution of CD1d protein before and after loading CD1d protein onto the D-SH nanocarrier;

[0062] Figure (G) shows the particle size distribution of CD1d protein before and after loading onto the D-SH-PEG nanocarrier;

[0063] Figure (H) shows the particle size changes of D-SH, D-SH-PEG, D-SH&CD1d, and D-SH&CD1d-PEG in aqueous solution over seven days.

[0064] (I) The figure shows the particle size changes of D-SH&CD1d in solutions with serum concentrations of 10%, 20%, 30%, and 40% over five days and the particle size changes of D-SH&CD1d-PEG in solutions with serum concentrations of 10% and 20% over five days.

[0065] Figure 2 Figure II shows the characterization of dendritic mesoporous silica nanocarriers and the performance study of nanovaccines.

[0066] Figure (G) is a transmission electron microscope image of the DMSN, with a scale bar of 100 nm.

[0067] Figure (H) is a transmission electron microscope image of D-SH, with a scale bar of 100 nm;

[0068] (I) The figure is a transmission electron microscope image of D-SH & CD1d, with a scale bar of 100 nm;

[0069] Figure (J) is a transmission electron microscope image of D-SH & CD1d / αGC, with a scale bar of 100 nm;

[0070] (K) is a transmission electron microscope (TEM) image of D-SH after 12 h of degradation in Krebs buffer solution, with a scale bar of 100 nm; (L) is a TEM image of D-SH after 24 h of degradation in Krebs buffer solution, with a scale bar of 100 nm; (M) is a graph showing the particle size changes of D-SH, D-SH & CD1d in aqueous solution over seven days.

[0071] Figure (N) shows the particle size changes of D-SH & CD1d over five days in solutions with serum concentrations of 10%, 20%, 30%, and 40%.

[0072] Figure 3 This is a diagram illustrating the in vitro performance of the nanovaccine.

[0073] (A) The figure shows the toxicity of different concentrations of D-SH nanocarriers to three cell types: RAW264.7, DC2.4, and AML-12.

[0074] (B) Figure is a flow cytometry plot of CD1d-647 and D-SH & CD1d-647 in BMDM cells;

[0075] (C) Figure is a flow cytometry plot of CD1d-647 and D-SH & CD1d-647 in BMDCs cells;

[0076] Figure (D) shows the uptake fluorescence of CD1d-647 and D-SH & CD1d-647 in BMDM cells;

[0077] Figure (E) shows the percentage change in M1 polarization of BMDM cells due to different concentrations of D-SH nanocarriers;

[0078] Figure (F) shows the percentage change in activation and maturation of BMDCs by different concentrations of D-SH nanocarriers; Figure (G) shows the polarization of BMDM cells by αGC, CD1d / αGC, and D-SH & CD1d / αGC.

[0079] Figure (H) shows the percentage changes in polarization of BMDM cells caused by αGC, CD1d / αGC, and D-SH & CD1d / αGC.

[0080] (I) The figure shows the percentage changes in activation and maturation of BMDCs by αGC, CD1d / αGC, and D-SH & CD1d / αGC.

[0081] (J) The figure shows the percentage changes in the activation and maturation of BMDCs by αGC, CD1d / αGC, and D-SH & CD1d / αGC.

[0082] Figure 4 This is a diagram illustrating the activation of iNKT cells in mice by D-SH & CD1d / αGC.

[0083] Figure (A) shows the flow cytometry method for detecting iNKT cells;

[0084] Figure (B) shows the proportion and absolute number of iNKT cells in mouse liver;

[0085] Figure (C) shows the proportion and absolute number of iNKT cells in the spleen of a mouse.

[0086] Figure (D) shows the proportion of iNKT cells in mouse peripheral blood.

[0087] Figure 5 Figure I shows the evaluation results of the antitumor effect of D-SH & CD1d / αGC in a mouse melanoma liver metastasis model and the results of immunological analysis.

[0088] (A) is a schematic diagram of the in vivo anti-tumor experiment design in tumor-bearing mice;

[0089] (B) The image shows the liver (containing tumor tissue) isolated from the mouse after treatment;

[0090] Figure (C) shows the H&E and Ki-67 immunohistochemical staining of the tumor area after treatment;

[0091] Figure (D) shows the changes in mouse body weight during treatment;

[0092] Figure (E) shows the weight of the mouse liver (including tumor tissue) after treatment;

[0093] Figure (F) shows the change in the percentage of NK cells in the liver (including tumor tissue) after treatment.

[0094] Figure (G) is an analysis of iNKT cells in the liver (including tumor tissue) after treatment;

[0095] Figure (H) shows the change in the percentage of iNKT cells in the liver (including tumor tissue) after treatment;

[0096] (I) The figure shows the change in the percentage of MDSC cells in the liver (including tumor tissue) after the treatment.

[0097] (J) Figure shows the change in the percentage of DCs in the liver (including tumor tissue) after treatment;

[0098] (K) diagram shows the CD8+ levels in the liver (including tumor tissue) after treatment. + Graph showing changes in the percentage of T cells;

[0099] (L) The image shows the CD8+ nucleotide sequence in the liver (including tumor tissue) after treatment. + OVA Tetramer + Graph showing changes in the percentage of T cells.

[0100] Figure 6 Figure II shows the evaluation results of the antitumor effect of D-SH & CD1d / αGC in a mouse melanoma liver metastasis model and the results of immunological analysis.

[0101] Figure (M) is an analysis of spleen iNKT cells after treatment;

[0102] Figure (N) shows the change in the percentage of iNKT cells in the spleen after treatment.

[0103] (O) The figure shows the CD8 count of the spleen after treatment. + T cell analysis diagram;

[0104] (P) The image shows the spleen's CD8 count after treatment. + Graph showing changes in the percentage of T cells;

[0105] (Q) The figure shows the T levels in the spleen after treatment. EM and T CM Cell analysis diagram;

[0106] (R) Figure is a TEM-type CD8 + graph showing changes in the percentage of T cells

[0107] (S) The figure shows the spleen CD8 after treatment. + OVA Tetramer + T cell analysis diagram;

[0108] The (T) graph shows the spleen's CD8+ levels after treatment. + OVA Tetramer + Graph showing changes in the percentage of T cells;

[0109] Figure (U) shows the peripheral blood iNKT cell analysis after treatment.

[0110] Figure (V) shows the change in the percentage of peripheral blood iNKT cells after treatment.

[0111] (W) Figure shows the peripheral blood CD8 count after treatment. + T cell analysis diagram;

[0112] (X) is the peripheral blood CD8+ figure after treatment. + Graph showing changes in the percentage of T cells. Detailed Implementation

[0113] The present invention will now be described in detail with reference to specific embodiments. The scope of the present invention is not limited to the specific embodiments, but is defined by the scope of the claims.

[0114] Unless otherwise specified, all raw materials and reagents used in the following examples are commercially available analytical grade products. Among them, αGC, CD1d, and mPEG are... 2k -Mal was purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd., MCE Company, and Shanghai Pengshuo Biotechnology Co., Ltd.

[0115] Example 1

[0116] Preparation of dendritic mesoporous silica nanocarriers (DMSN):

[0117] 0.18 g of triethanolamine, 6 g of hexadecyltrimethylammonium chloride, and 60 mL of water were added to a round-bottom flask. The reaction mixture was heated to 60 °C in an oil bath at 150 rpm for 1 h. After reacting, 20 mL of a mixture of cyclohexane and tetraethyl orthosilicate (volume ratio 1:19) was added to the reaction system, and the reaction was continued for 12 h under magnetic stirring. The upper organic phase was then discarded, and the aqueous phase was centrifuged at 15000 rpm for 20 min to collect the white solid product at the bottom, which was CTAC@DMSN. The collected product was extracted by reflux extraction with a hydrochloric acid / ethanol mixture (volume ratio 1:10) at 80 °C for 36 h to completely remove the template agent hexadecyltrimethylammonium chloride, yielding dendritic mesoporous silica DMSN.

[0118] Example 2

[0119] Preparation of dendritic mesoporous silica nanocarrier DMSN-1:

[0120] 0.1 g triethanolamine, 6 g hexadecyltrimethylammonium chloride, and 60 mL water were added to a round-bottom flask. The reaction mixture was heated to 55 °C in an oil bath at 100 rpm for 1.5 h. Then, 20 mL of a mixture of cyclohexane and tetraethyl orthosilicate (volume ratio 1:2) was added to the reaction system, and the reaction was continued for 6 h under magnetic stirring. The upper organic phase was then discarded, and the aqueous phase was centrifuged at 15000 rpm for 20 min to collect the white solid product at the bottom, which was CTAC@DMSN-1. The collected product was extracted by reflux extraction with a hydrochloric acid / ethanol mixture (volume ratio 1:7) at 70 °C for 24 h to completely remove the template agent hexadecyltrimethylammonium chloride, yielding dendritic mesoporous silica DMSN-1.

[0121] Example 3

[0122] Preparation of dendritic mesoporous silica nanocarrier DMSN-2:

[0123] 0.3 g of triethanolamine, 6 g of hexadecyltrimethylammonium chloride, and 60 mL of water were added to a round-bottom flask. The reaction mixture was heated to 65 °C in an oil bath at 120 rpm for 1.2 h. Then, 20 mL of a mixture of cyclohexane and tetraethyl orthosilicate (volume ratio 1:9) was added to the reaction system, and the reaction was continued for 10 h under magnetic stirring. The upper organic phase was then discarded, and the aqueous phase was centrifuged at 15000 rpm for 20 min to collect the white solid product at the bottom, which was CTAC@DMSN-2. The collected product was extracted by reflux extraction with a hydrochloric acid / ethanol mixture (volume ratio 1:5) at 90 °C for 48 h to completely remove the template agent hexadecyltrimethylammonium chloride, yielding dendritic mesoporous silica DMSN-2.

[0124] Example 4

[0125] Preparation of thiol-modified dendritic mesoporous silica nanocarriers (D-SH, D-SH-PEG):

[0126] The DMSN prepared in Example 1 was used, and then 10 mg of DMSN was reacted with 20 μL of (3-mercaptopropyl)triethoxysilane in 10 mL of ethanol at 80 °C and 500 rpm overnight. The reaction was then centrifuged and dried to obtain thiol-modified DMSN (D-SH). 1 mg of D-SH and 2 mg of mPEG were then... 2k -Mal was shaken in 1 mL of water at room temperature for 15 min, then centrifuged (15000 rpm, 10 min) and washed to remove unmodified mPEG. 2k -Mal, thereby preparing D-SH-PEG nanocarriers with the functionalized PEG group.

[0127] Example 5

[0128] Preparation of thiol-modified dendritic mesoporous silica nanocarriers (D-SH-1, D-SH-PEG-1):

[0129] The DMSN prepared in Example 1 was used, and then 10 μL of (3-mercaptopropyl)triethoxysilane and 10 mg of DMSN were reacted overnight in 10 mL of ethanol at 70 °C and 400 rpm. The reaction was then centrifuged and dried to obtain the thiol-modified DMSN (D-SH-1). 1 mg of D-SH-1 and 2 mg of mPEG were then... 2k -Mal was shaken in 1 mL of water at room temperature for 15 min, then centrifuged (20000 rpm, 10 min) and washed to remove unmodified mPEG. 2k -Mal, thereby preparing the nanocarrier D-SH-PEG-1 with the functionalized PEG group.

[0130] Example 6

[0131] Preparation of thiol-modified dendritic mesoporous silica nanocarriers (D-SH-2, D-SH-PEG-2):

[0132] DMSN from the vegetation in Example 1 was selected, and then 10 mg of DMSN was reacted with 50 μL of (3-mercaptopropyl)triethoxysilane in 10 mL of ethanol at 90 °C and 600 rpm overnight. After centrifugation and drying, thiol-modified DMSN (D-SH-2) was obtained. 1 mg of D-SH-2 and 2 mg of mPEG were then... 2k -Mal was shaken in 1 mL of water at room temperature for 15 min, then centrifuged (18000 rpm, 10 min) and washed to remove unmodified mPEG. 2k -Mal, thereby preparing the nanocarrier D-SH-PEG-2 with the functionalized PEG group.

[0133] Example 7

[0134] Preparation of the macromolecular protein complex CD1d / αGC:

[0135] αGC powder was dissolved in DMSO to prepare a 1 mg / mL stock solution. The solution was heated at 80°C for 5 min to ensure complete dissolution. The αGC stock solution was diluted with PBS (pH 7.4) containing 0.5% Tween 20 to a final concentration of 200 μg / mL. CD1d protein solution (αGC to CD1d molar ratio of 12:1) was added, and PBS containing 0.5% Tween 20 was added to bring the Tween 20 concentration in the reaction solution to 0.05%. The reaction was carried out at 37°C for 3 h, and then incubated overnight at 4°C. Unloaded free αGC was removed by ultrafiltration through a 30 kDa ultrafiltration tube. The concentration of the CD1d / αGC complex was determined using a Bradford protein assay kit.

[0136] Example 8

[0137] Preparation of nanovaccines D-SH&CD1d and D-SH&CD1d / αGC:

[0138] 1.5 mg of D-SH nanocarrier prepared in Example 4 (dispersed in 0.8 mL of deionized water) was first ultrasonically dispersed for 5 min in an ice bath using an ultrasonic cell disruptor (100 W, 5 s on, 5 s off). Then, 0.3 mg of CD1d or 0.2 mL of the CD1d / αGC protein complex prepared in Example 7 was added to the dispersed D-SH aqueous solution, and the mixture was ultrasonically dispersed again for 5 min in an ice bath. Afterwards, the mixture was centrifuged at 15000 rpm for 10 min to remove unloaded CD1d or CD1d / αGC complex, thus obtaining the nanovaccines D-SH&CD1d and D-SH&CD1d / αGC. The amount of CD1d or CD1d / αGC loaded on 1 mg of D-SH was 0.20 mg.

[0139] Example 9

[0140] Preparation of nanovaccines D-SH&CD1d-1 and D-SH&CD1d / αGC-1:

[0141] 1.5 mg of D-SH nanocarrier prepared in Example 4 (dispersed in 0.8 mL of deionized water) was first ultrasonically dispersed for 5 min in an ice bath using an ultrasonic cell disruptor (150 W, 5 s on, 5 s off). Then, 0.3 mg of CD1d or the CD1d / αGC protein complex prepared in Example 7 (0.2 mL) was added to the dispersed D-SH aqueous solution, and the mixture was ultrasonically dispersed again for 5 min in an ice bath. Afterwards, the mixture was centrifuged at 20,000 rpm for 10 min to remove unloaded CD1d or CD1d / αGC complex, thus obtaining the nanovaccines D-SH&CD1d-1 and D-SH&CD1d / αGC-1.

[0142] Example 10

[0143] Preparation of nanovaccines D-SH&CD1d-2 and D-SH&CD1d / αGC-2:

[0144] 1.5 mg of D-SH nanocarrier prepared in Example 4 (dispersed in 0.8 mL of deionized water) was first ultrasonically dispersed for 5 min in an ice bath using an ultrasonic cell disruptor (50 W, 5 s on, 5 s off). Then, 0.3 mg of CD1d or the CD1d / αGC protein complex prepared in Example 7 (0.2 mL) was added to the dispersed D-SH aqueous solution, and the mixture was ultrasonically dispersed again for 5 min in an ice bath. Afterwards, the mixture was centrifuged at 18000 rpm for 10 min to remove unloaded CD1d or CD1d / αGC complex, thus obtaining the nanovaccines D-SH&CD1d-2 and D-SH&CD1d / αGC-2.

[0145] Example 11

[0146] Preparation of nano-vaccines D-SH&CD1d-PEG and D-SH&CD1d / αGC-PEG:

[0147] Resuspend the D-SH&CD1d or D-SH&CD1d / αGC prepared in Example 8 in 1 mL of water, add 2 mg of mPEG 2k -MAL, react at room temperature for 15 min, centrifuge at 15000 rpm for 10 min, discard the supernatant, and collect the D-SH&CD1d-PEG or D-SH&CD1d / αGC-PEG nano-vaccine.

[0148] Example 12

[0149] Perform basic characterization on the nano-vaccines prepared in Example 1, Example 4, Example 8 and Example 11:

[0150] (1) Characterization test experiment 1: Nitrogen adsorption-desorption and determination of average pore size of dendritic mesoporous silica nanocarriers, SAXS characterization, Fourier transform infrared spectroscopy characterization

[0151] Prepare 100 mg of DMSN and D-SH prepared in Example 1 and Example 4, with the degassing temperature at 350 °C, and use a physical adsorption instrument to measure nitrogen adsorption-desorption and average pore size. As Figure 1 (A) shown by the nitrogen adsorption-desorption isotherm, obvious capillary condensation and hysteresis curves were observed near the relative pressure of 0.2 < P / P0 < 0.8, indicating a wide pore size distribution; from Figure 1 (B) the pore size distribution diagram, DMSN and D-SH have pore size distributions concentrated around 7.8 nm and 6 nm, and their specific surface areas are about 959 m 2 / g and 876 m 2 / g, respectively, which provides the possibility for loading macromolecular drugs such as proteins. Perform SAXS characterization on the powder samples of CTAC@DMSN, DMSN, D-SH, and D-SH-PEG prepared in Example 1 and Example 4. As Figure 1 (C) shown, a scattering peak was observed at 1.23 nm -1 , indicating a uniform mesoscopic structure. After drying the CTAC@DMSN, DMSN, D-SH, and D-SH-PEG samples in Example 1 and Example 4 into powders, take an appropriate amount and mix it with potassium bromide crystal powder, grind it thoroughly with an agate mortar and then press it into a tablet, and test it with a Fourier transform infrared spectrometer. As Figure 1 (D) shown, the peaks near 1076 cm -1 and 810 cm -1 are the characteristic infrared absorption peaks of silica. Among them, compared with CTAC@DMSN, DMSN and D-SH have peaks at 2927 cm -1The disappearance of the peak at 2927 cm⁻¹ confirms the successful removal of the template agent CTAC. The presence of a small peak in D-SH-PEG confirms the successful modification of PEG. The successful modification of PEG confirms the presence of SH. The MPTES compound used to modify SH contains CH bonds, hence the peak at 2927 cm⁻¹ in D-SH. -1 A small peak nearby proved SH's successful modification.

[0152] (2) Characterization Test Experiment 2: Characterization of whether the protein loaded in the dendritic mesoporous silica nanovaccine is located within the pores

[0153] Following the instructions for Alexa Fluor 647NHS fluorescent dye, the dye was labeled onto CD1d, the CD1d protein of the nanovaccine D-SH&CD1d prepared in Example 8, and D-SH&CD1d / αGC. Unlabeled dye was removed using ultrafiltration to prepare CD1d-647, D-SH&CD1d-647, and D-SH&CD1d-647-PEG. Non-denaturing polyacrylamide gel electrophoresis was performed at 120 mV. After electrophoresis, the gels were imaged using an Invitrogen gel imaging system. Following imaging, the gels were stained with Coomassie Brilliant Blue and then destained. The destained gels were then scanned for analysis. Figure 1 As shown in (E), lane 1 contains free protein, lane 2 contains D-SH & CD1d-647, and lane 3 contains D-SH & CD1d(647)-PEG. No protein bands were observed in lanes 2 and 3. Furthermore, after loading the fluorescently modified protein onto the nanocarrier, obvious fluorescent bands were visible at the inlets of lanes 2 and 3, demonstrating the successful loading of CD1d protein with D-SH and D-SH-PEG without interference from free CD1d protein. The nanocarriers D-SH, D-SH & CD1d, D-SH-PEG, and D-SH & CD1d-PEG from Examples 4, 8, and 11, before and after loading CD1d protein, were measured using a Malvern nanoparticle size and Zeta potential analyzer (model ZS90). Figure 1 As shown in (F), the particle size of D-SH and D-SH&CD1d remains almost unchanged, such as Figure 1 (G) shows that the particle size of D-SH-PEG and D-SH & CD1d-PEG also hardly changed, which further confirms that CD1d protein is loaded into the pores.

[0154] (3) Characterization Test Experiment 3: Stability Characterization of Dendritic Mesoporous Silica Nanoparticle Vaccine

[0155] The particle size changes of D-SH, D-SH-PEG, D-SH&CD1d, and D-SH&CD1d-PEG from Examples 4, 8, and 11 were monitored over seven days using a Malvern nanoparticle size and Zeta potential analyzer (model ZS90). Figure 1 As shown in (H), there was no significant difference in hydrated particle size within 7 days, indicating good stability.

[0156] The stability of D-SH&CD1d and D-SH&CD1d-PEG from Examples 8 and 11 in solutions containing different concentrations of fetal bovine serum (FBS) was investigated using a Malvern nanoparticle size and Zeta potential analyzer (model ZS90). FBS was prepared at different concentrations using deionized water. D-SH&CD1d prepared in Example 8 was resuspended in solutions containing 10%, 20%, 30%, and 40% FBS, respectively, while D-SH&CD1d-PEG prepared in Example 8 was resuspended in solutions containing 10% and 20% FBS, respectively. Changes in hydrated particle size were monitored using the nanoparticle size and Zeta potential analyzer for five consecutive days. Figure 1 As shown in (I), the particle size of D-SH&CD1d in solutions containing 10%, 20%, 30%, and 40% fetal bovine serum did not differ significantly over five days, and the particle size of D-SH&CD1d-PEG in solutions containing 10% and 20% fetal bovine serum did not differ significantly over five days.

[0157] (4) Characterization Test Experiment 4: Morphology and Degradability Characterization of Dendritic Mesoporous Silica Nanoparticles

[0158] 2.5 μL of DMSN, D-SH, D-SH&CD1d, and D-SH&CD1d / αGC samples prepared in Examples 1, 4, 8, and 11 (with a concentration of 1 mg / mL) were dropped onto a copper grid of a transmission electron microscope (TEM), dried under an infrared lamp, and then imaged using a 300 kV TEM (Tecnai G2 F30S-Twin). Figure 2 The dendritic morphology is clearly visible. A 300kV electron beam is too energetic for proteins and can damage them. Figure 2 TEM images of (L) and (M) after protein loading show that the edges of the nanoparticles did not become blurred, indirectly proving that the protein was loaded into the pores, and that the dendritic silica nanocarrier played a protective role for the protein. Furthermore, from... Figure 2 Based on the TEM images of (L) and (M), there is no difference between D-SH&CD1d and D-SH&CD1d / αGC.

[0159] Krebs buffer solution is commonly used to simulate physiological environments in vitro. The D-SH prepared in Example 4 was diluted to 2 mg / mL with Krebs buffer solution and incubated in a shaker at 37°C for 12 h and 24 h, respectively. Images were then taken using a 300 kV transmission electron microscope (Tecnai G2 F30 S-Twin). At 12 h, although D-SH retained some of its original morphology, the edges were noticeably blurred and the structure was disrupted, indicating that D-SH had begun to degrade. Figure 2 N). After 24 hours, the D-SH nanoparticle structure completely disintegrated, exhibiting fragmented and sand-like structures. Figure 2 These results indicate that dendritic mesoporous silica carriers degrade over time within 24 hours, suggesting their biodegradability and easier clearance by the body during in vivo application.

[0160] Example 13

[0161] The MTT assay was used to evaluate the cytotoxicity of D-SH prepared in Example 4 against AML-12, DC2.4, and RAW264.7. Inverted fluorescence microscopy and flow cytometry were used to compare the uptake efficiency of CD1d-647 and D-SH&CD1d-647 groups prepared in Example 12 in bone marrow-derived macrophages and dendritic cells (BMDM, BMDCs). The regulatory effects of D-SH and D-SH&CD1d / αGC prepared in Examples 4 and 8 on BMDM and BMDCs cells were investigated.

[0162] (1) The in vitro cytotoxicity of the D-SH nanocarriers prepared in Example 4 was evaluated by the MTT assay.

[0163] The MTT assay was used to evaluate the cytotoxicity of D-SH against three types of cells: mouse dendritic cells (DC2.4), mouse mononuclear macrophage leukemia cells (RAW264.7), and normal mouse hepatocytes (AML-12). Cells were seeded in 96-well plates at 8000 cells per well and cultured overnight at 37°C with 5% CO2. D-SH at concentrations of 0, 3.125, 6.25, 12.5, 25, and 50 μg / mL were prepared using complete culture medium and co-incubated with the cells for 24 h. The supernatant was then aspirated, and 100 μL of 0.5 mg / mL MTT solution was added. The cells were incubated at 37°C with 5% CO2 for 4 h. Excess MTT solution was aspirated, and 150 μL of DMSO was added. The absorbance at 570 nm was measured using a multi-sensor microplate reader. Cell viability was calculated using the following formula:

[0164] Cell viability (%) = OD hye / OD blank ×100%

[0165] like Figure 3 As shown in (A), when the concentration of D-SH was 0, 3.125, 6.25, 12.5, 25, and 50 μg / mL, the survival rate of these three cell types was higher than 75%. This result proves that D-SH has no significant cytotoxicity to DC2.4, RAW264.7, and AML-12 cell types in the concentration range of 50 μg / mL, and has good in vitro biosafety.

[0166] (2) Extraction of BMDCs and BMDM cells

[0167] Extraction of BMDCs: Eight-week-old C57BL / 6 mice were euthanized by cervical dislocation. Muscle tissue attached to the tibia was removed, sterilized by soaking in 75% ethanol, and quickly placed into culture medium. Both ends of the tibia were cut to expose the medullary cavity, and the bone marrow was expelled into the culture medium. After repeated rinsing several times, the cells were filtered through a sterile membrane. The cells were centrifuged at 1800 rpm for 5 min, the supernatant was discarded, and 2 mL of erythrocyte lysis buffer was added for lysis at room temperature for 2 min. Lysis was terminated by adding 3 mL of complete culture medium, and the cells were centrifuged at 1800 rpm for 5 min, and the supernatant was discarded. The collected cells were cultured in sterile Petri dishes in medium containing 20 ng / mL GM-CSF and 10 ng / mL LCM. Culture medium was added on the third day, and the cells were used on the sixth day.

[0168] BMDM cell extraction: Eight-week-old C57BL / 6 mice were euthanized by cervical dislocation. Muscle tissue attached to the tibia was removed, sterilized by soaking in 75% ethanol, and quickly placed in culture medium. Both ends of the tibia were cut to expose the medullary cavity, and the bone marrow was blown out into the culture medium. After repeated rinsing several times, the cells were filtered through a sterile membrane. The cells were centrifuged at 1800 rpm for 5 min, the supernatant was discarded, and 2 mL of erythrocyte lysis buffer was added for lysis at room temperature for 2 min. Lysis was terminated by adding 3 mL of complete culture medium, centrifuged at 1800 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL of M-SCF LCM medium containing 20 ng / mL for counting. Cells were seeded into wells according to different requirements and cultured. Culture medium was added on the third day, and the cells were used on the sixth day.

[0169] (3) Uptake efficiency of CD1d-647 and D-SH & CD1d-647 prepared in Example 12 in BMDM and BMDCs

[0170] Fluorescence uptake assay of BMDM cells was performed using an inverted fluorescence microscope: 3 × 10⁶ cells were seeded in 24-well plates. 5One BMDM cell was added to 500 μL of medium containing CD1d-647 and D-SH & CD1d-647 (Alexa Fluor 647NHS fluorescent dye concentration of 300 ng / mL) prepared in Example 12. After incubation for 1, 2, and 4 hours, the supernatant was aspirated, and the cells were washed three times with PBS. 4% paraformaldehyde fixative was added for fixation at 4°C in the dark for 30 minutes. After removing the paraformaldehyde fixative, DAPI (1 μg / mL) was added and incubated at 4°C in the dark for 20 minutes to label the cell nuclei. Finally, unlabeled DAPI dye was washed away with PBS, and fluorescence images of the cells were acquired using an inverted fluorescence microscope. The uptake results are as follows: Figure 3 As shown in (D), BMDM cells have a higher uptake efficiency of D-SH & CD1d-647 at any time point.

[0171] Flow cytometry was used to perform uptake experiments on BMDM and BMDCs cells: 5 × 10⁶ cells were seeded in 48-well plates. 5 BMDM and BMDC cells were added to 300 μL of culture medium containing CD1d-647, D-SH, and CD1d-647 (with an Alexa Fluor 647NHS fluorescent dye concentration of 100 ng / mL in CD1d-647 and D-SH & CD1d-647) prepared in Example 12. BMDM cells were incubated for 1, 2, and 4 h, while BMDC cells were incubated for 0.5, 1, and 2 h. After incubation, cells at the bottom of the plate were collected into 2 mL centrifuge tubes, centrifuged at 1800 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in FACS solution. The fluorescence intensity of the APC channel was analyzed using a flow cytometry analyzer. The results are as follows: Figure 3 As shown in (B) and (C), the uptake efficiency of D-SH & CD1d-647 is higher than that of CD1d-647.

[0172] (4) Regulation of BMDM and BMDCs cells by D-SH prepared in Example 4

[0173] Transmission electron microscopy (TEM) images revealed a dendritic structure in the nanocarrier. To further investigate the differences between the D-SH nanocarrier and conventional carriers, D-SH was prepared at concentrations of 50 and 100 μg / mL using LCM medium and co-incubated with BMDM and BMDCs. Flow cytometry was used to detect the expression of specific molecules F4 / 80 and CD86 on the surface of BMDM cells to assess the condition of the BMDM cells. Figure 3 As shown in (E), it has the effect of stimulating M0 type BMDM cells to activate and differentiate into M1 type BMDM cells, and the proportion of them differentiating into M1 type BMDM cells increases with increasing concentration.

[0174] By detecting the expression of specific molecules CD80 and CD86 on the surface of BMDCs, such as Figure 3 As shown in (F), 50 μg / mL and 100 μg / mL D-SH nanocarriers did not significantly activate BMDCs.

[0175] (5) Regulation of BMDM and BMDCs cells by D-SH & CD1d / αGC prepared in Example 8

[0176] 1×10⁻⁶ seeds per well of a 24-well plate 6 Cells were cultured in various media: Control group, LPS+INF-γ (positive control: LPS 25 ng / mL, INF-γ 100 ng / mL), αGC, CD1d / αGC, and D-SH&CD1d / αGC (αGC concentration 300 ng / mL for each group). After incubation at 37°C for 24 h, cells were collected and stained with anti-F4 / 80-PE, anti-CD80-PE / Cy7, and anti-CD86-APC antibodies at room temperature for 60 min in the dark. After centrifugation to remove unbound antibodies, the cells were analyzed by flow cytometry. Figure 3 (G)(H) αGC showed no significant change compared to the Control group and did not cause BMDM to polarize to the M1 type. CD1d / αGC, D-SH & CD1d / αGC could cause BMDM to polarize to the M1 type and were no different from the positive control group.

[0177] Inoculate 8×10⁸ cells into 96-well plates 4 BMDCs were cultured in various media: Control group, LPS (positive control: LPS 25 ng / mL), αGC, CD1d / αGC, and D-SH & CD1d / αGC (αGC concentration 300 ng / mL for each group). After incubation at 37°C for 24 h, cells were collected and stained with anti-CD11c-FITC, anti-CD80-PE, and anti-CD86-APC antibodies for 1 h at room temperature in the dark. After centrifugation to remove unbound antibodies, the cells were analyzed by flow cytometry. Figure 3 As shown in (I) and (J), αGC, CD1d / αGC, and D-SH & CD1d / αGC can all further promote the maturation of BMDCs.

[0178] Example 14

[0179] The iNKT cell analysis of the nanovaccine D-SH&CD1d / αGC prepared in Example 8 was performed in mice. Six- to eight-week-old female C57BL / 6 mice were randomly divided into seven groups: (1) Control group, (2) αGC single-dose group (αGC×1), (3) CD1d / αGC single-dose group (CD1d / αGC×1), (4) D-SH&CD1d / αGC single-dose group (D-SH&CD1d / αGC×1), (5) αGC triple-dose group (αGC×3), (6) CD1d / αGC triple-dose group (CD1d / αGC×3), and (7) D-SH&CD1d / αGC triple-dose group (D-SH&CD1d / αGC×3), with six mice in each group. For the single-dose and triple-dose groups, each mouse received αGC... The drug was administered at a dose of 300 ng / dose. In the single-dose group, liver, spleen, and peripheral blood were collected for immunoassay 72 hours after the first dose. In the triple-dose group, the interval between each dose was 7 days. In the triple-dose group, liver, spleen, and peripheral blood were collected for immunoassay 72 hours after the last dose.

[0180] (1) Treatment methods for different organs of mice

[0181] Liver: Mouse liver was minced in a centrifuge tube, ground, and filtered through a 70 μm filter to obtain a single-cell suspension. The single-cell suspension was brought to a final volume of 10 mL, and then 200 μL of heparin sodium solution (10 mg / mL) and 5 mL of Percoll cell separation medium were added. After gradient centrifugation, the supernatant was discarded. 5 mL of erythrocyte lysis buffer was added to the cell pellet for 5 min of lysis, followed by the addition of 5 mL of PBS to terminate erythrocyte lysis. After centrifugation, the supernatant was discarded to obtain a single-cell suspension of the spleen.

[0182] Spleen: Mouse spleens were collected and ground with a ground glass slide. The mixture was then passed through a 300-mesh filter to obtain a single-cell suspension. The suspension was centrifuged at 1800 rpm for 5 min, and the supernatant was discarded. 5 mL of red blood cell lysis buffer was added to lyse the cells at room temperature for 5 min. 5 mL of PBS was added to terminate the lysis. The suspension was centrifuged at 1800 rpm for 5 min, and the supernatant was discarded to obtain a single-cell suspension.

[0183] Peripheral blood: Blood from mice was collected in anticoagulated centrifuge tubes by orbital blood collection. The upper serum layer was discarded after centrifugation. Red blood cell lysis buffer was added and lysed at room temperature for 5 min. PBS was added to stop the lysis. The mixture was centrifuged at 1800 rpm for 5 min and the supernatant was discarded to obtain a single-cell suspension.

[0184] (2) Proportion of iNKT cells in different organs

[0185] The processed single-cell suspension was counted and monitored using flow cytometry. For example... Figure 4(A) shows iNKT cells labeled using TCRβ and CD1d Tetramer staining. Figure 4 (B) and (C) represent the percentage and absolute number of iNKT cells in the liver and spleen, respectively. In the liver, a single dose of D-SH & CD1d / αGC induced the activation of iNKT cells (35.9%), which was 3.9 times that of the Control group (8.99%), 1.5 times that of a single dose of αGC (22.65%), and 1.4 times that of a single dose of CD1d / αGC (24.8%). Compared with a single dose of αGC (0.88%) and three doses of CD1d / αGC (1.86%), three doses of D-SH & CD1d / αGC (5.81%) still showed a strong iNKT cell activation effect. Whether after a single dose or three repeated doses, the absolute number of iNKT cells in the D-SH & CD1d / αGC group in the liver was significantly higher than that in other groups. A single dose of D-SH&CD1d / αGC induced effective activation (7.46%) and a significant increase in the absolute number of iNKT cells in the spleen. The proportion of iNKT cells in the spleen did not differ significantly among the groups after three doses, as did the absolute number of iNKT cells in the spleen. Finally, the effects of the D-SH&CD1d / αGC nanovaccine on iNKT cells in peripheral blood were monitored. Figure 4 As shown in (D), the proportion of iNKT cells after a single dose of D-SH&CD1d / αGC was 1.25%, which was significantly higher than that of the other three groups.

[0186] Example 15

[0187] The D-SH&CD1d / αGC prepared in Example 8 was evaluated for its anti-melanoma liver metastasis in mice. Five days after injection of B16-F10-OVA-LUC cells, female C57BL / 6 mice (6-8 weeks) were randomly divided into four groups of five mice each: (1) Control group; (2) Free small molecule group (αGC); (3) Free protein complex group (CD1d / αGC); (4) Nanovaccine group (D-SH&CD1d / αGC). Figure 5 As shown in (A), each group received three doses via tail vein injection, with each dose administered 7 days apart. Each mouse in each group was given 300 ng of αGC per dose. 72 hours after the last administration, liver, spleen, and peripheral blood were collected for immunostaining analysis.

[0188] (1) Establishment of a mouse melanoma liver metastasis model

[0189] 1) Cell preparation: Select B16-F10-OVA-LUC cells in good growth condition, digest them with trypsin to obtain a single-cell suspension, and adjust the cell density to 4×10⁶ cells / cells.6 cells / mL, cell suspension kept on ice for later use;

[0190] 2) Anesthetize mice: Intraperitoneal injection of afodin was administered to anesthetize mice (C57BL / 6) at a dose of 240 mg / kg. After successful anesthesia, the mice were placed on their sides with their spleens facing upwards on a 37°C warming pad.

[0191] 3) Treatment of mice: Remove hair from the abdomen of the mice and disinfect with povidone-iodine;

[0192] 4) Make a 1 cm incision in the peritoneum in the mid-abdomen below the sternum, remove the spleen with flat-tipped forceps, clamp it with two titanium screws, and cut it in half. Put one half of the spleen back into the body, and inject 3×10 ppm of the spleen into the other half. 5 (75μL) B16-F10-OVA-LUC cells; leave the needle in place for 5 minutes, slowly remove the syringe, tie the blood vessels connecting the spleen to the liver with sterile sutures, cut off the spleen injected with tumor cells, suture the abdomen with a 4-0 suture needle in a double layer, disinfect with iodine, and feed as usual.

[0193] (2) Evaluation of the therapeutic effect of D-SH&CD1d / αGC on tumors prepared in Example 8

[0194] To evaluate the tumor-suppressive effect of D-SH & CD1d / αGC, mouse body weight was monitored from day 5 after tumor implantation until the end of the treatment cycle. Figure 5 As shown in (D), the mice maintained stable body weight during treatment, indicating that D-SH & CD1d / αGC has good biocompatibility. Tumor-bearing liver tissue was collected from the mice after treatment and photographed. Figure 5 As shown in (B), it is clearly evident that D-SH&CD1d / αGC exhibits the most significant tumor-suppressing effect, with a marked reduction in tumor nodules. The weight of the tumor-bearing liver in mice was also recorded. Figure 5 As shown in (E), compared with the Control group, the αGC and CD1d / αGC treatment groups can effectively delay tumor progression, but cannot completely inhibit tumor growth. D-SH & CD1d / αGC have a stronger tumor-inhibiting effect.

[0195] To further evaluate the therapeutic effect of D-SH & CD1d / αGC, liver tissue (including tumor tissue) was stained with hematoxylin and eosin (H&E). Figure 5 As shown in (C), significant tumor tissue was observed in all three groups: Control, αGC, and CD1d&αGC. The tumor tissue structure was dense and intact, while the tumor tissue was significantly reduced in the D-SH&CD1d / αGC group. Ki-67 immunohistochemical staining of tumor tissue sections showed a positive correlation between the Ki-67 positive ratio and tumor proliferation capacity. Figure 5As shown in (C), the positive rate of Control was the highest, while the positive rate of D-SH&CD1d / αGC was the lowest compared to other groups, indicating that D-SH&CD1d / αGC effectively killed tumor cells and inhibited tumor proliferation and metastasis.

[0196] (3) Flow cytometry analysis of immune markers in mouse liver (including tumor tissue)

[0197] Following the same treatment method for mouse livers as in Example 14, flow cytometry was then used to assess iNKT cells, NK cells, DCs, and CD8+ cells in the tumor microenvironment. + T cell expression, such as Figure 5 As shown in (G) and (H), the proportion of iNKT cells in the D-SH & CD1d / αGC group reached 5.18%, which was approximately 5 times higher than that in the Control group (0.94%) and the αGC group (1.06%), and 2.3 times higher than that in the CD1d / αGC group (2.22%). Figure 5 As shown in (F), the proportion of NK cells in the D-SH&CD1d / αGC group (6.51%) was significantly increased compared to the Control group (3.51%). The proportion of mature DCs after D-SH&CD1d / αGC treatment was significantly higher than that in the Control, αGC, and CD1d / αGC groups. Figure 5 J), which improved antigen presentation ability and thus produced effective CD8. + T cell response ( Figure 5 K).

[0198] MDSCs are a key inhibitor of TME, possessing a significant ability to impair immune responses. In our results, we observed that treatment with D-SH & CD1d / αGC reduced the proportion of MDSCs. Figure 5 I) This, to some extent, reversed the tumor microenvironment. Finally, to assess specific T-cell immune responses, we monitored CD8... + OVATetramer + In CD3 + The percentage of cells revealed specific CD8 in the D-SH & CD1d / αGC group. + The proportion of T cells was significantly higher in the group than in other groups. Figure 5 L) indicates that D-SH&CD1d / αGC increases T cell invasion of tumors and induces a specific T cell immune response in vivo.

[0199] (4) Flow cytometry analysis of immune markers in mouse spleen

[0200] Referring to the treatment method of mouse spleen in Example 14, flow cytometry was used to analyze mouse spleen iNKT cells and CD8+ cells. + T cells, TEM cells, specific CD8 + The expression of T cells was assessed. A higher proportion of iNKT cells was observed in the spleen after treatment with D-SH & CD1d / αGC. Figure 6 As shown in (M) and (N), the nanovaccine group (1.54%) was approximately 5 times higher than the control group (0.3%) and approximately 2 times higher than the αGC group (0.84%); CD8 + The proportion of T cells in the spleen Figure 6 As shown in (O) and (P), the D-SH&CD1d / αGC group (39.7%) was higher than the CD1d / αGC group (33.8%). Figure 6 As shown in (S) and (T), the D-SH&CD1d / αGC group (0.22%) showed specific CD8+. + The proportion of T cells was significantly higher than in other groups (0.02%). The development of immune memory is crucial for a durable anti-tumor immune response and prevention of tumor recurrence. When the tumor re-invades, central memory T cells (T cells) are activated. CM CD44 + CD62L + CD8 + CD3 + It can proliferate and produce a large number of effector T cells (T cells). EM CD44 + CD62L - CD8 + CD3 + Furthermore, effector memory T cells can be recruited to the site of infection for effective immune protection. For example... Figure 6 As shown in (Q) and (R), the D-SH&CD1d / αGC group (28.7%) T EM The proportion of cells was 2.7 times that of the Control group (10.4%), 2 times that of the αGC group (14.2%), and 1.1 times that of the CD1d / αGC group (24.9%). These results indicate that anti-tumor immunotherapy mediated by D-SH & CD1d / αGC nanovaccines can induce durable immune memory, which is beneficial for inhibiting tumor metastasis and recurrence.

[0201] (5) Flow cytometry was used to analyze iNKT cells and CD8 cells in mouse peripheral blood. + T cell analysis

[0202] Next, we analyzed iNKT cells and CD8 cells in mouse peripheral blood. +T cells were analyzed. Following the mouse peripheral blood processing method described in Example 14, flow cytometry was used to analyze iNKT cells and CD8+ cells in mouse peripheral blood. + The expression of T cells was analyzed. For example... Figure 6 As shown in (U) and (V), the proportion of activated iNKT cells in the D-SH&CD1d / αGC group (0.22%) was significantly higher than that in the αGC group (0.097%). Figure 6 (W) and (X) peripheral blood D-SH&CD1d / αGC group (38.3%) CD8 + The proportion of T cells was significantly higher than that in the control group (30.3%) and the αGC group (28.7%). These results indicate that anti-tumor immunotherapy mediated by D-SH&CD1d / αGC nanovaccines and iNKT cells can induce a systemic immune response.

Claims

1. A mercapto-modified dendritic mesoporous silica, characterized in that, The thiol-modified dendritic mesoporous silica nanoparticles were modified with thiol groups by (3-mercaptopropyl)triethoxysilane to obtain the thiol-modified dendritic mesoporous silica.

2. The silicon dioxide according to claim 1, characterized in that, The mass ratio of dendritic mesoporous silica to (3-mercaptopropyl)triethoxysilane is 1:1 to 5, and the diameter of the dendritic mesoporous silica is 100 to 150 nm.

3. The silicon dioxide according to claim 1, characterized in that, The dendritic mesoporous silica nanoparticles were obtained by hydrolysis and condensation of triethanolamine, hexadecyltrimethylammonium chloride, and tetraethyl orthosilicate at the oil-water interface.

4. The silicon dioxide according to claim 3, characterized in that, The mass ratio of triethanolamine, hexadecyltrimethylammonium chloride, and tetraethyl orthosilicate is 0.1–0.3:6:1–8.

5. The application of the silica-supported macromolecular drug according to any one of claims 1-4.

6. The application according to claim 5, characterized in that, The macromolecular drug is selected from at least one of proteins, peptides, and nucleic acids.

7. A novel nano-vaccine, characterized in that, The novel nanovaccine comprises silica as described in any one of claims 1-4 and a macromolecular protein complex CD1d / αGC loaded on the silica; the macromolecular protein complex CD1d / αGC is obtained by loading αGC onto CD1d.

8. The novel nanovaccine according to claim 7, characterized in that, The thiol groups on the surface of the silica interact with... The following structures are modified by functionalizing groups via reaction connection: The molecular weight of the polyethylene glycol linker is 500–5000 kDa; The functional group R is selected from methyl, carboxyl, amino, biotinylate, azide, glucose, mannose, short peptide groups with tumor-targeting properties, and protein molecule groups.

9. The novel nanovaccine according to claim 7, characterized in that: The hydrated particle size of the novel nanovaccine is 100–150 nm.

10. The novel nanovaccine according to claim 7, characterized in that: The specific surface area of ​​the silica is 500–1000 m². 2 / g, with an average pore size of 4–8 nm.

11. The novel nanovaccine according to claim 7, characterized in that: The loading amount of the macromolecular protein complex CD1d / αGC on silica is 0.17–0.20 mg / mg; The molar ratio of CD1d to α-galactosylceramide αGC is 1:6 to 12.

12. The method for preparing the novel nanovaccine according to claim 7, characterized in that, Includes the following steps: ① A dendritic mesoporous silica nanomaterial was synthesized by hydrolysis and condensation at the oil-water interface using triethanolamine, hexadecyltrimethylammonium chloride, and water as the aqueous phase and cyclohexane and tetraethyl orthosilicate as the oil phase. ②The dendritic mesoporous silica nanomaterials obtained in step ① are heated and refluxed in a solvent to remove the template agent hexadecyltrimethylammonium chloride. ③ The dendritic mesoporous silica obtained in step ② is reacted with (3-mercaptopropyl)triethoxysilane to obtain mercapto-modified dendritic mesoporous silica. ④ Add the macromolecular protein complex CD1d / αGC to the thiol-modified dendritic mesoporous silica dispersion obtained in step ③, and under ultrasonication, load the macromolecular protein complex CD1d / αGC into the pores of the silica. Remove the unloaded macromolecular protein complex to obtain the novel nanovaccine.

13. The preparation method according to claim 12, characterized in that, The novel nanovaccine prepared in step ④ was combined with mPEG. 2k -Mal room temperature reaction yields nano-vaccines with functionalized PEG groups.

14. The preparation method according to claim 12, characterized in that, In step ①, the mass ratio of triethanolamine, hexadecyltrimethylammonium chloride, and water is 0.10–0.3:6:60, and the volume ratio of tetraethyl orthosilicate and cyclohexane is 1:2–19. The mass ratio of dendritic mesoporous silica to (3-mercaptopropyl)triethoxysilane is 1:1 to 5; The loading amount of the macromolecular protein complex CD1d / αGC on silica is 0.17–0.20 mg / mg.

15. The preparation method according to claim 14, characterized in that, In step ①, the aqueous phase is heated to 55-65℃ and reacted for 1-1.5 hours, followed by the slow addition of the oil phase and a reaction time of 6-12 hours; the reaction speed is 100-150 rpm. The conditions for removing the template agent in step ② are as follows: using a hydrochloric acid / ethanol solution with a product ratio of 1:5 to 10, heating temperature of 70 to 90°C, reaction speed of 400 to 600 rpm, and reaction time of 24 to 48 h; The reaction conditions for modifying the thiol group in step ③ are as follows: the reaction solvent is ethanol, the reaction temperature is 70-90℃, the reaction speed is 400-600 rpm, and the reaction time is 10-12 h. In step ④, the ultrasound conditions are 50–150W, 5s on, 5s off.

16. The application of the silica according to claim 1 or the novel nanovaccine according to claim 7 in the preparation of antitumor drugs.

17. The application according to claim 16, characterized in that, The tumors are selected from lymphoma, lung cancer, kidney cancer, melanoma with liver metastasis, colon cancer with liver metastasis, breast cancer with lung metastasis, liver cancer, and pancreatic cancer.

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