A nano-hydrogel precursor, a preparation method and application thereof

Abstract

The present application relates to the technical field of drug synthesis, and particularly relates to a nano-hydrogel precursor as well as a preparation method and application thereof. In the present application, the active ingredients GOx, Mn 2+ and HCQ loaded by the nano-hydrogel can strengthen cancer treatment by activating the STING pathway and regulating the autophagy pathway at the postoperative site. Among them, GOx and Mn 2+ act as a chemical kinetic therapy preparation, Mn 2+ acts as a STING agonist, and HCQ acts as an autophagy inhibitor; GOx and Mn 2+ can play the role of CDT, causing the release of cell membrane dsDNA and mitochondrial dsDNA, thereby further inducing the activation of the STING pathway, and HCQ can effectively inhibit the protective autophagy related to the activation of the STING pathway, further enhancing the anti-tumor immune response. The synergistic effect of the chemical kinetic therapy preparation, the STING agonist and the autophagy inhibitor can produce a specific immune response, significantly inhibit tumor recurrence, and thereby prolong the survival rate of the triple-negative breast cancer mice after surgery.

Description

Technical Field

[0001] This invention relates to the field of drug synthesis technology, and in particular to a nanohydrogel precursor, its preparation method, and its application. Background Technology

[0002] Triple-negative breast cancer is a special molecular subtype of breast cancer that is negative for estrogen receptor (ER), progesterone receptor (PR), and the proto-oncogene HER2. It accounts for approximately 15% of all breast cancers and is often poorly differentiated, exhibiting stem cell-like characteristics. It has a high recurrence rate, a short overall survival rate, and is considered a high-risk breast cancer with a poor prognosis. Postoperative recurrence and metastasis to other organs are common.

[0003] Studies have shown that tumor development and progression are closely related to the body's immune system. Anti-tumor immune response, following tumor ablation, is another powerful measure to activate the body's immune system, thereby eliminating tumor cells throughout the body and preventing metastasis. Among these measures, the interferon gene-stimulating factor (STING) pathway plays a crucial role in the initiation of anti-tumor immunity and the transformation of "cold" tumors into "hot" tumors. Chemokinetic therapy (CDT) converts hydrogen peroxide into reactive oxygen species (ROS) through Fenton / Fenton-like reactions. ROS are mostly hydroxyl radicals, which induce tumor cell apoptosis and necrosis. Glucose oxidase (GOx) reacts with intracellular glucose and oxygen to produce hydrogen peroxide (H2O2) and gluconic acid, which can cut off the nutrient supply to cancer cells, thereby inhibiting their proliferation. Research on surface manganese ions (Mn...) is also ongoing. 2+ This can enhance the sensitivity of cyclic GMP-AMP synthase (cGAS) to double-stranded DNA (dsDNA) and amplify STING activation. Furthermore, Mn... 2+ It can also undergo a Fenton-like reaction to decompose hydrogen peroxide, inducing the generation of reactive oxygen species. Meanwhile, studies have shown that H2O2 generated from glucose oxidation catalyzed by GOx promotes the production of Mn. 2+ The Fenton-like response mediated by the tumor leads to the death of more tumor cells. Moreover, the dsDNA produced from dead cell debris and the mitochondrial DNA released by excessive intracellular ROS damaging mitochondria can bidirectionally activate the cGAS-STING signaling pathway.

[0004] However, relying solely on the activation of the cGAS-STING signaling pathway for therapeutic effects remains unsatisfactory. This is because tumor cells, in response to cGAS-STING activation, also trigger their internal protective autophagy. Autophagy, as a conserved cellular protection mechanism, captures cytoplasmic components from autophagosomes and transfers them to lysosomes for degradation, converting them into metabolic substrates and thus maintaining homeostasis. Autophagy activation, in turn, leads to subsequent STING degradation, playing a negative feedback regulatory role in the STING pathway. Furthermore, autophagy can reduce stress responses, prevent the accumulation of ROS, especially ROS from damaged mitochondria, limit tumor cell-related damage, and promote tumor growth. Therefore, in the treatment of tumors, interrupting the autophagic degradation process and enhancing the immunotherapeutic effect induced by STING signaling pathway activation has become crucial for the development of tumor immunotherapy. Summary of the Invention

[0005] To overcome the above problems, this invention provides a nano-hydrogel precursor, its preparation method, and its application. The nano-hydrogel in this invention supports the active ingredients GOx and Mn. 2+ HCQ, along with other drugs, enhances cancer treatment at the postoperative site by activating the STING pathway and modulating the autophagy pathway. Among these, GOx and Mn... 2+ As a chemodynamic therapy agent, Mn 2+ As a STING agonist, HCQ acts as an autophagy inhibitor; GOx and Mn 2+ It can exert the effect of CDT, leading to the release of cell membrane dsDNA and mitochondrial dsDNA, thereby further inducing the activation of the STING pathway. HCQ can effectively inhibit protective autophagy associated with STING pathway activation, further enhancing the anti-tumor immune response. The synergistic effect of chemodynamic therapy agents, STING agonists, and autophagy inhibitors can generate a specific immune response, significantly inhibit tumor recurrence, and thus prolong the survival rate of triple-negative breast cancer mice after surgery.

[0006] A first aspect of the present invention provides a method for preparing a nano-hydrogel precursor, comprising the following steps:

[0007] (1) Disperse mesoporous silica (MSN) in anhydrous ethanol, add ammonia (NH3·H2O) and 3-aminopropyltrimethoxysilane (APTES) to the suspension, stir the reaction, and after the reaction is completed, aminated mesoporous silica MSN-NH2 is obtained.

[0008] (2) Disperse the MSN-NH2 prepared in step (1) in deionized water, add glucose oxidase (GOx), and incubate. After incubation, GOx-loaded nanoparticles G@MSN are obtained.

[0009] (3) Disperse the G@MSN prepared in step (2) in deionized water, and add tannic acid (TA), divalent manganese salt solution and 3-morpholinopropanesulfonic acid buffer (MOPS) in sequence to form an MPN coating. Then add bovine serum albumin solution (BSA) and vortex to form a BSA coating to form a loaded GOX and Mn. 2+ GM@MSN nanoparticles;

[0010] (4) After dialysis, the liposomes were incubated in a hydroxychloroquine (HCQ) solution. After incubation, liposomes loaded with hydroxychloroquine, LipHCQ, were obtained.

[0011] (5) Add sodium alginate to deionized water, stir, and then add the nanoparticles GM@MSN prepared in step (3) and LipHCQ prepared in step (4). Stir the reaction to obtain the nanohydrogel precursor solution H / GM@MSN.

[0012] The hydrogel precursor solution H / GM@MSN prepared in this invention reacts with endogenous calcium ions in the body to generate nano-hydrogel H / GM@Gel.

[0013] In a second aspect, the present invention provides a nanohydrogel precursor prepared by the above-described preparation method.

[0014] A third aspect of the present invention provides a pharmaceutical composition comprising the above-described nanohydrogel precursor.

[0015] A fourth aspect of the present invention provides a pharmaceutical formulation comprising the above-described nanohydrogel precursor.

[0016] A fifth aspect of the present invention provides the use of the above-described nanohydrogel precursor, the above-described pharmaceutical composition, or the above-described pharmaceutical preparation in the preparation of a drug for treating tumors.

[0017] The beneficial effects of this invention are as follows:

[0018] (1) The active ingredients GOx and Mn loaded on the nano-hydrogel in this invention 2+ HCQ, along with other drugs, enhances cancer treatment at the postoperative site by activating the STING pathway and modulating the autophagy pathway. Among these, GOx and Mn... 2+ As a chemodynamic therapy agent, Mn 2 + As a STING agonist, HCQ acts as an autophagy inhibitor; GOx and Mn 2+It can exert the effect of CDT, leading to the release of cell membrane dsDNA and mitochondrial dsDNA, thereby further inducing the activation of the STING pathway. HCQ can effectively inhibit protective autophagy associated with STING pathway activation, further enhancing the anti-tumor immune response. The synergistic effect of chemodynamic therapy agents, STING agonists, and autophagy inhibitors can generate a specific immune response, significantly inhibit tumor recurrence, and thus prolong the survival rate of triple-negative breast cancer mice after surgery.

[0019] (2) This invention achieves GOx loading based on electrostatic interactions, constructs the MPN coating based on coordination interactions, and prepares the BSA coating using hydrogen bonding interactions. Negatively charged MSN is aminated to reverse the surface charge of MSN, thereby achieving negatively charged GOx loading. Unreacted GOx is removed, and the MPN coating is constructed using the adhesive properties of TA and its strong coordination with MnCl2·4H2O. MOPS buffer is further added to enhance the stability of the MPN coating. Finally, the prepared nanoparticles are co-incubated with a BSA solution, utilizing the hydrogen bonds formed between TA and BSA to prepare the BSA coating, which improves the stability of the nanoparticles in the biological environment. To improve the sustained-release performance of HCQ in the gel system, it is encapsulated in liposomes, enabling the stepwise release of nanoparticles and drug molecules. Hydrogels, as carriers for drug formulations, offer injectability, good biocompatibility, and the ability to sustain drug release. Using hydrogels for sustained release of immunotherapy drugs can not only improve the efficacy of immunotherapy but also reduce side effects. Furthermore, alginate hydrogels can rapidly cross-link with endogenous calcium ions in the body at the tumor site, and the resulting hydrogels have a sustained therapeutic effect on residual tumor sites, providing the possibility of local inhibition of tumor recurrence and metastasis. Attached Figure Description

[0020] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0021] Figure 1 Transmission electron microscope (TEM) images of MSN, G@MSN in Example 1 and M@MSN in Comparative Example 1;

[0022] Figure 2 The images shown are transmission electron microscope (TEM) images and EDX spectra of GM@MSN in Example 1.

[0023] Figure 3 The images show the zeta potentials of MSN, MSN-NH2, and GM@MSN in Example 1 and M@MSN in Comparative Example 1.

[0024] Figure 4 In the middle, 'a' represents the precursor solution of the nano-hydrogel, H / GM@MSN, and the solution in Ca... 2+ Under the action of cross-linking, a hydrogel network is formed; b is a scanning electron microscope image of the hydrogel formed by cross-linking; c is a magnified scanning electron microscope image of the hydrogel; d is a CLSM image of the hydrogel.

[0025] Figure 5 The figure shows the in vitro cytotoxicity results of PBS, LipHCQ, M@MSN, GM@MSN and H / GM@MSN on 4T1 cells in Experiment Example 1;

[0026] Figure 6 Figure 1 shows the results of the live / dead cell staining assays using PBS, LipHCQ, M@MSN, GM@MSN, and H / GM@MSN in Experiment Example 1.

[0027] Figure 7 The expression levels of calreticulin (CRT) in patients treated with PBS, LipHCQ, M@MSN, GM@MSN, and H / GM@MSN;

[0028] Figure 8 Detection results for high-mobility group box 1 (HMGB1) treated with PBS, LipHCQ, M@MSN, GM@MSN and H / GM@MSN;

[0029] Figure 9 DC maturity analysis was performed on the PBS group, LipHCQ group, M@MSN group, GM@MSN group and H / GM@MSN group, where a is the quantitative analysis result and b is the flow cytometry.

[0030] Figure 10 Image a shows the Western blot analysis of STING pathway proteins in the PBS group, LipHCQ group, M@MSN group, GM@MSN group, and H / GM@MSN group; image b shows the results of quantitative analysis of IFN-β secretion by enzyme-linked immunosorbent assay; image c shows the LC3B-II / I ratio and p62 protein.

[0031] Figure 11 In Figure a, tumor volume changes are observed in the PBS, LipHCQ, M@MSN, GM@MSN, and H / GM@MSN groups; in Figure b, tumor mass changes are observed.

[0032] Figure 12 CD4 levels in recurrent tumors after treatment in the PBS group, LipHCQ group, M@MSN group, GM@MSN group, and H / GM@MSN group + and CD8 + Immunofluorescence images of T cells. Detailed Implementation

[0033] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0035] A first typical embodiment of the present invention provides a method for preparing a nano-hydrogel precursor, comprising the following steps:

[0036] (1) Disperse mesoporous silica (MSN) in anhydrous ethanol, add ammonia (NH3·H2O) and 3-aminopropyltrimethoxysilane (APTES) to the suspension, stir the reaction, and after the reaction is completed, aminated mesoporous silica MSN-NH2 is obtained.

[0037] (2) Disperse the MSN-NH2 prepared in step (1) in deionized water, add glucose oxidase (GOx), and incubate. After incubation, GOx-loaded nanoparticles G@MSN are obtained.

[0038] (3) Disperse the G@MSN prepared in step (2) in deionized water, and add tannic acid (TA), divalent manganese salt solution and 3-morpholinopropanesulfonic acid buffer (MOPS) in sequence to form an MPN coating. Then add bovine serum albumin solution (BSA) and vortex to form a BSA coating to form a loaded GOX and Mn. 2+ GM@MSN nanoparticles;

[0039] (4) After dialysis, the liposomes were incubated in a hydroxychloroquine (HCQ) solution. After incubation, liposomes loaded with hydroxychloroquine, LipHCQ, were obtained.

[0040] (5) Add sodium alginate to deionized water, stir, and then add the nanoparticles GM@MSN prepared in step (3) and LipHCQ prepared in step (4). Stir the reaction to obtain the nanohydrogel precursor solution H / GM@MSN.

[0041] The hydrogel precursor solution H / GM@MSN prepared in this invention reacts with endogenous calcium ions in the body to generate nano-hydrogel H / GM@Gel.

[0042] In one or more embodiments, in step (1), the mass ratio of MSN to the volume ratio of anhydrous ethanol is 120-140 mg: 3.5-4.5 mL, preferably 130 mg: 3.9 mL.

[0043] In one or more embodiments, in step (1), the mass ratio of MSN to the volume ratio of NH3·H2O and APTES is 120-140 mg: 200-230 μL: 120-150 μL, preferably 130 mg: 216 μL: 130 μL.

[0044] In one or more embodiments, in step (1), the stirring reaction time is 10 to 15 hours, preferably 12 hours.

[0045] In one or more embodiments, in step (1), after the reaction is completed, the sample is washed three times with ethanol and deionized water respectively.

[0046] In one or more embodiments, in step (2), the mass ratio of MSN-NH2 to GOx is 20:1 to 2, preferably 20:1.2.

[0047] In one or more embodiments, in step (2), the mass ratio of MSN-NH2 to deionized water is 1:800 to 1200, preferably 1:1000.

[0048] In one or more embodiments, in step (2), the incubation conditions are 3-5°C for 5-8 hours, preferably 4°C for 6 hours.

[0049] In one or more embodiments, in step (2), after incubation, centrifugation is performed to remove free GOx.

[0050] In one or more embodiments, in step (3), the divalent manganese salt is MnCl2, MnSO4, or Mn(CH3COO)2, preferably MnCl2.

[0051] In one or more embodiments, in step (3), the mass ratio of GOx-loaded nanoparticles to deionized water is 1:800 to 1200, preferably 1:1000.

[0052] In one or more embodiments, in step (3), the mass ratio of the GOx-loaded nanoparticles to TA and divalent manganese salt is 20:7 to 9:1.2 to 1.5, preferably 20:8:1.46.

[0053] In one or more embodiments, in step (3), the volume ratio of deionized water to MOPS is 1:0.8 to 1.2, preferably 1:1.

[0054] In one or more embodiments, in step (3), the concentration of the BSA solution is 0.8 to 1.2 mg / mL, preferably 1 mg / mL.

[0055] In one or more embodiments, in step (3), the mass ratio of the BSA solution to the GOx-loaded nanoparticles is 4 to 6:20, preferably 5:20.

[0056] In one or more embodiments, in step (3), the vortex conditions are vortexing at room temperature for 2 to 4 minutes, preferably 3 minutes; the rotation speed is 1800 to 2200 rpm, preferably 2000 rpm.

[0057] In one or more embodiments, in step (4), the preparation method of the liposomes after dialysis includes: dissolving hydrogenated soybean phospholipids (HSPC), cholesterol (Chol) and phospholipid-methoxy polyethylene glycol (DSPE-mPEG2000) in the organic solvent chloroform, removing the solvent by rotary evaporation to form liposomes; adding ammonium sulfate solution for hydration, using a high-pressure microfluidic device to squeeze the liposome suspension, and then using PBS buffer to dialyze the squeezed liposomes.

[0058] Furthermore, the mass ratio of HSPC, Chol, and DSPE-mPEG2000 is 3:0.9 to 1.1:0.9 to 1.1, preferably 3:1:1.

[0059] Furthermore, the concentration of the ammonium sulfate solution is 0.2–0.4 M, preferably 0.3 M.

[0060] Furthermore, the operating pressure for extrusion in the high-pressure microfluidizer is 13,000–18,000 psi, preferably 15,000 psi.

[0061] Furthermore, the dialysis time is 20–30 hours, preferably 24 hours.

[0062] In one or more embodiments, in step (4), the amount of HCQ administered is 5% to 25% of the liposome mass.

[0063] In one or more embodiments, in step (4), the incubation temperature is 50-70°C and the time is 20-40 min, preferably 60°C and 30 min.

[0064] In one or more embodiments, in step (4), after incubation, centrifugation is performed to remove free HCQ.

[0065] In one or more embodiments, in step (5), the mass ratio of sodium alginate to deionized water is 1:800 to 1200, preferably 1:1000.

[0066] A second typical embodiment of the present invention provides a nanohydrogel precursor prepared by the above preparation method.

[0067] A third typical embodiment of the present invention provides a pharmaceutical composition comprising the above-described nanohydrogel precursor.

[0068] In one or more embodiments, the pharmaceutical composition further includes tumor treatment-related drugs.

[0069] Preferably, the tumor treatment-related drugs are anti-tumor drugs, immune adjuvants, checkpoint inhibitors, or antigen proteins.

[0070] A fourth typical embodiment of the present invention provides a pharmaceutical formulation comprising the above-mentioned nanohydrogel precursor.

[0071] In one or more embodiments, the formulation is an injection.

[0072] A fifth typical embodiment of the present invention provides the use of the above-described nanohydrogel precursor or the above-described pharmaceutical composition or pharmaceutical formulation in the preparation of a drug for treating tumors.

[0073] In one or more embodiments, the tumor includes benign tumors and / or malignant tumors; the malignant tumors include solid tumors and hematomas, wherein solid tumors include breast cancer, preferably triple-negative breast cancer.

[0074] It should be noted that the term "tumor" is used in this invention as is known to those skilled in the art, and includes benign tumors and / or malignant tumors. A benign tumor is defined as the excessive proliferation of cells that cannot form an invasive, metastatic tumor in the body. Conversely, a malignant tumor is defined as cells with various cellular and biochemical abnormalities that can cause systemic disease (e.g., tumor metastasis in distant organs).

[0075] Solid tumors include those of the breast, bladder, bones, brain, central and peripheral nervous system, colon, endocrine glands (such as the thyroid and adrenal cortex), esophagus, endometrium, germ cells, head and neck, kidneys, liver, lungs, larynx and hypopharynx, mesothelioma, ovaries, pancreas, prostate, rectum, kidneys, small intestine, soft tissue, testes, stomach, skin (such as melanoma), ureters, vagina, and vulva. Malignant tumors include hereditary cancers such as retinoblastoma and nephroblastoma. Furthermore, malignant tumors include primary tumors in the aforementioned organs and corresponding secondary tumors in distant organs (tumor metastases). Hematologic malignancies include aggressive and painless forms of leukemia and lymphoma, namely non-Hodgkin's disease, chronic and acute myeloid leukemia (CML / AML), acute lymphoblastic leukemia (ALL), Hodgkin's disease, multiple myeloma, and T-cell lymphoma. It also includes myelodysplastic syndromes, plasmacytomas, tumor-like syndromes, cancers of unknown primary location, and AIDS-related malignancies.

[0076] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0077] Example 1

[0078] Preparation of nano-hydrogel precursors

[0079] (1) MSN (110nm, 130mg) was dispersed in 3.9mL of anhydrous ethanol. 216mL of NH3·H2O and 130μL of LAPTES were added to the suspension and the mixture was stirred for 12h. After the reaction was completed, the mixture was washed three times with ethanol and water respectively to obtain MSN-NH2.

[0080] (2) Disperse the MSN-NH2 (20mg) prepared in step (1) in deionized water (20mL), add GOx (1.2mg), and incubate at 4℃ for 6h. After incubation, GOx-loaded nanoparticles G@MSN are obtained.

[0081] (3) Disperse the G@MSN (20 mg) prepared in step (2) in deionized water (20 mL), then add 0.2 mL TA (40 mg / mL), 0.2 mL MnCl2·4H2O solution (7.3 mg / mL), and 20 mL MOPS (20 mM, pH = 7.4) sequentially to form an MPN coating. Then add 5 mL BSA solution (1 mg / mL) and vortex at 2000 rpm for 3 min at room temperature to form a BSA coating to load GOX and Mn. 2+ GM@MSN nanoparticles;

[0082] (4) Dissolve HSPC (75 mg), Chol (25 mg), and DSPE-mPEG2000 (25 mg) in 10 mL of chloroform. Remove the solvent using a rotary evaporator to form liposomes. Then, add 10 mL of ammonium sulfate solution (0.3 M) for hydration, and squeeze the liposome suspension four times under a high-pressure microfluidic apparatus at a working pressure of 15000 psi. Dialyze the obtained liposomes with PBS buffer (10 mM) for 24 h. Disperse 15 mg of HCQ in 20 mL of deionized water, mix the dialyzed liposomes with this solution, incubate at 60 °C for 30 min, and then centrifuge (50000 g, 40 min, 4 °C) to remove free HCQ.

[0083] (5) Add sodium alginate (50 mg) to deionized water (5 mL), stir vigorously for 2 h to dissolve completely; add the nanoparticles GM@MSN prepared in step (3) and LipHCQ prepared in step (4), mix to obtain the nanohydrogel precursor solution H / GM@MSN.

[0084] Comparative Example 1

[0085] (1) Disperse MSN (110nm, 130mg) in 3.9mL of anhydrous ethanol, add 216mL of NH3·H2O and 130μL of APTES to the suspension, stir for 12h, and wash three times with ethanol and water respectively to obtain MSN-NH2;

[0086] (2) The MSN-NH2 (20 mg) prepared in step (1) was dispersed in deionized water (20 mL), and 0.2 mL TA (40 mg / mL), 0.2 mL MnCl2·4H2O solution (7.3 mg / mL), and 20 mL MOPS (20 mM, pH = 7.4) were added sequentially to form an MPN coating. Then, 5 mL BSA solution (1 mg / mL) was added, and the mixture was vortexed at 2000 rpm for 3 min at room temperature to form a BSA coating, thus forming a loaded Mn 2+ The nanoparticles M@MSN.

[0087] Example 2

[0088] This embodiment characterizes MSN, MSN-NH2, and GM@MSN in Example 1 and M@MSN in Comparative Example 1.

[0089] Figure 1 The images are transmission electron microscopy (TEM) images of MSN, G@MSN in Example 1, and M@MSN in Comparative Example 1. Figure 1As can be seen, the morphology of MSN-NH2 and M@MSN did not change significantly, indicating that MSN has good monodispersity and the size of the nanoparticles is about 150 nm.

[0090] Figure 2 The images shown are transmission electron microscope (TEM) images and EDX spectra of GM@MSN from Example 1. Figure 2 As can be seen, the morphology of GM@MSN did not change significantly, and the EDX spectrum also proved the successful preparation of GM@MSN nanoparticles.

[0091] Figure 3 Zeta potential images of MSN, MSN-NH2, GM@MSN, and M@MSN in Comparative Example 1, from... Figure 3 It can be seen that the Zeta potential of MSN is around -16.83mV, the potential of M@MSN is around -9.38mV, and the potential rises to around 8.75mV after adsorption of GOx.

[0092] Example 3

[0093] The nanohydrogel precursor solution H / GM@MSN prepared in Example 1 was supplemented with Ca 2+ Afterwards, cross-linking can be used to form H / GM@Gel nanogels, such as... Figure 4 As shown in Figure a. Figure 4 Image b is a scanning electron microscope image of hydrogel-encapsulated nanoparticles, showing the porous structure of the hydrogel-encapsulated nanoparticles. Figure 4 In the middle, c is a magnified SEM image of the hydrogel-encapsulated nanoparticles. Figure 4 In the middle, d is a CLSM image of the nanoparticles encapsulated in the hydrogel, which shows that the nanoparticles are uniformly distributed in the hydrogel.

[0094] Experiment Example 1: Cytotoxicity Verification of Drug-Loaded Nanoparticles

[0095] The CCK-8 assay was used to study the in vitro cytotoxicity of different nanoparticles on 4T1 cells. 4T1 tumor cells (9 × 10⁶ cells per well) were used. 3 The cells were cultured in 96-well plates and incubated in a cell culture incubator for 24 h. The LipHCQ, GM@MSN, and H / GM@MSN prepared in Example 1 (equivalent GOx concentrations of 0.4, 0.8, 1.6, 3.1, and 6.3 μg / mL, respectively; equivalent Mn) were then added. 2+ M@MSNs prepared at concentrations of 1.2, 2.4, 4.8, 9.3 and 18.9 μg / mL, respectively, and Comparative Example 1 were cultured with cells for 24 h. CCK-8 solution was then added to each well to detect cell viability (absorbance at 450 nm).

[0096] Live / dead cell assay. 4T1 cells were cultured at 1 × 10⁻⁶ cells per well.5 The cells were seeded at a density of [number] cells per well in 24-well plates and incubated for 12 hours. After treatment with LipHCQ, M@MSN, GM@MSN, and H / GM@MSN (GOx, 0.6 μg / mL; Mn [missing information]), the cells were cultured in an incubator. 2+ Live and dead cells were stained with Calcein-AM (2 μM) and PI (4 μM) solutions, respectively, and the cells were imaged using a fluorescence microscope.

[0097] like Figure 5 As shown, due to the lack of GOx, the cytotoxicity of the M@MSN and LipHCQ groups was negligible, while GM@MSN and H / GM@MSN both exhibited dose-dependent cytotoxicity against 4T1 cells. The IC50 values ​​of GOx in the M@MSN and H / GM@MSN groups were 1.15 and 0.94 μg / mL, respectively. As expected, HCQ inhibited endoplasmosis and enhanced the STING pathway, thereby improving the killing effect of the H / GM@MSN group.

[0098] Live / dead cell staining assays also assessed the cytotoxicity of these nanoparticles; Calcein-AM staining positive (green signal) represented live cells, while propidium iodide staining positive (PI, red signal) represented dead cells. Figure 6 As shown, most cells survived after culture with M@MSN, indicating that M@MSN has low cytotoxicity. However, nearly 90% of cells died after culture with H / GM@MSN, consistent with the results for CCK-8. These results suggest that the synergistic effect of CDT and autophagy inhibition treatments enhances cytotoxicity.

[0099] Experimental Example 2: In Vitro ICD Detection

[0100] To evaluate the immunogenic cell death (ICD) effect induced by H / GM@MSN nanoparticles in 4T1 cells, calreticulin (CRT) and high-mobility group box 1 (HMGB1) proteins were analyzed in vitro. CRT exposure was detected by flow cytometry. 4T1 tumor cells (8 × 10⁶ cells per well) were used. 4 (Number of cells) were seeded into 24-well plates and cultured in a cell culture incubator for 24 h. Then, they were seeded with LipHCQ, M@MSN, GM@MSN and H / GM@MSN (GOx, 0.6 μg / mL; Mn) respectively. 2+ Cells were cultured with 1.8 μg / mL (HCQ, 10 μg / mL). After 24 h of culture, cells were collected and analyzed by flow cytometry using DyLight 488-labeled Affini pure goat anti-rabbit IgG. The supernatant from each well was collected, and the secretion of extracellular HMGB1 protein was detected using an HMGB1 enzyme-linked immunosorbent assay kit.

[0101] like Figure 7 As shown, the H / GM@MSN group had the highest CRT expression level compared with the PBS group, LipHCQ group, M@MSN group and GM@MSN group.

[0102] The released HMGB1 was quantitatively determined using an ELISA kit, such as... Figure 8 As shown, the HMGB1 release was also the highest in the H / GM@MSN group, at approximately 116 ng / mL, which was 1.28 times and 2.68 times that of the GM@MSN group and the M@MSN group, respectively.

[0103] The results above show that CDT can induce the release of more tumor antigens, thereby inducing ICD and stimulating DC maturation, laying the groundwork for subsequent immunotherapy.

[0104] Experimental Example 3: In vitro dendritic cell maturation analysis

[0105] Bone marrow-derived dendritic cells (BMDCs) were obtained from 6-week-old female C57BL / 6 mice and cultured in bacterial dishes at a concentration of 1×10⁻⁶. 6 Cells were cultured at a density of cells / mL in RPMI 1640 complete medium containing GM-CSF (20 ng / mL) and IL-4 (10 ng / mL). The cell culture medium was refreshed on days 2, 4, and 6. On day 7, immature BMDCs were harvested at a density of 3 × 10⁶ cells / well. 5 The cells were reseeded at a density of [number] cells per well in 24-well plates and incubated with the supernatant of 4T1 cells. The 4T1 cells were pre-treated with LipHCQ, M@MSN, GM@MSN, and H / GM@MSN (GOx, 0.6 μg / mL; Mn [missing information]). 2+ After incubation at 2.4 μg / mL, BMDCs were stained with APC-CD11c, FITC-CD80 and PE / Cyanine7-CD86, and then analyzed by flow cytometry.

[0106] like Figure 9 As shown, the DC maturation rates in the PBS group, LipHCQ group, M@MSN group, GM@MSN group, and H / GM@MSN group were 26.5%, 28.0%, 33.1%, 42.7%, and 49.8%, respectively. On the one hand, CDT can induce the release of more tumor antigens, thereby inducing ICD and stimulating DC maturation. On the other hand, based on Mn... 2+ CDT, along with the autophagy-inhibiting effect of LipHCQ, enhanced STING activation, thereby increasing IFN-β release. This further stimulated DC maturation, thereby activating anti-tumor immune responses and regulating TME. These results indicate that CDT and Mn... 2+The combination of release and autophagy inhibition can amplify STING signals, effectively promote DC maturation, and enhance the efficacy of intracellular anti-tumor immunotherapy.

[0107] Experiment 4: Detection of STING protein and autophagy protein

[0108] 4T1 cells were transferred to a density of 3×10⁻⁶ cells. 5 Cells were cultured in 6-well plates at 37°C for 24 h. Cells were then incubated with LipHCQ, M@MSN, GM@MSN, and H / GM@MSN (GOx, 0.8 μg / mL; Mn) respectively. 2+ Cells were treated with a 2.4 μg / mL solution for 24 h, then collected in 1.5 mL centrifuge tubes, and RIPA lysis buffer and PMSF were added. After incubation for 30 min, the cells were centrifuged at 13800 g for 15 min, and the supernatant was collected. Protein concentration was determined using a BCA protein assay kit. Equal volumes of protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane, which was then blocked in TBST solution containing 5% skim milk for 1 h. The membrane was incubated with primary antibodies of STING, P-STING (Ser366), LC3B, and SQSTM1 / p62 (p62) at 4 °C for 15 h on a shaker. Finally, Western blot results were obtained using a gel imaging system. β-actin was used as an internal control antibody.

[0109] Figure 10 As described in section a, Western blotting detected the expression of STING and phosphorylated STING (P-STING, Ser366), reflecting the activation effect of GM@MSN combined with LipHCQ on the STING pathway. The results showed that the expression levels of P-STING and [unspecified substance] in the M@MSN and H / GM@MSN groups were significantly higher than in other groups. Furthermore, to assess the effect of H / GM@MSN on the STING pathway, ELISA was used to evaluate the release of INF-β. Figure 10 As shown in Figure b, the H / GM@MSN group induced the largest release of IFN-β factor, confirming that CDT binds to Mn 2+ Release amplifies STING signaling, inducing 4T1 cells to release interferon. LipHCQ and Mn 2+ The combination can induce stronger STING activation.

[0110] To better understand STING activation, autophagy levels after treatment were also investigated. Western blotting was used to assess the expression of LC3B-II and SQSTM1 / p62 (p62). Compared with the PBS and GM@MSN groups, the LC3B-II / I ratio and p62 protein levels were significantly increased in LipHCQ-treated cells. Figure 10 c) indicates that autophagy inhibition leads to a large production of intracellular autophagosomes. Furthermore, p62 protein was downregulated in GM@MSN-treated cells, likely due to protective autophagy resulting from STING pathway activation. Importantly, the LC3B-II / I ratio and p62 expression in the H / GM@MSN group were higher than in other groups, suggesting that the internal protective autophagy process was largely suppressed. Blockage of this pathway should favor the exacerbation of oxidative damage and dsDNA accumulation, thereby further amplifying the STING pathway and enhancing immunotherapy.

[0111] Experimental Example 5: Verification of Antitumor Effect in Vivo

[0112] To verify the anti-recurrence effect of H / GM@Gel, a postoperative breast tumor model was established. In short, 4T1 cells (2×10⁻⁶) were used. 6 Injected into the right buttock and back of Balb / c mice. When the tumor volume reaches approximately 100–150 mm... 3 At that time, 90% of the primary tumor was removed and the incision was sutured. Post-surgery, mice were randomly divided into 5 groups, and 100 μL of hydrogel precursor (sodium alginate solution) loaded with different nanoparticles was injected into the surgical area, including PBS solution, LipHCQ, M@MSN, GM@MSN, and H / GM@MSN (GOx, 3 mg / kg; Mn...). 2+ 1-2 mg / kg; HCQ, 10 mg / kg). Measure body weight and recurrent tumor volume every 3 days, and calculate tumor size using the following formula:

[0113] Volume (mm) 3 = 0.5 × width 2 (mm) 2 ) × Length (mm).

[0114] On day 24, tumor samples were harvested for further analysis. Specifically, hematologic and epithelial studies (H&E) of tumor sections were used to investigate tumor cell necrosis. Flow cytometry was used for further analysis of CD4. + and CD8 + T cell expression.

[0115] A 4T1 tumor model was used to examine its inhibitory effect on postoperative tumor recurrence. Surgical resection was performed 9 days after inoculation with 4T1 tumor cells. Before suturing, the tumor resection cavity was injected with a hydrogel precursor (sodium alginate solution) loaded with different nanoparticles: PBS, LipHCQ, M@MSN, GM@MSN, and H / GM@MSN. Body weight was monitored every 3 days. Figure 11 As shown, 15 days after injection, the tumor volume in the PBS group, LipHCQ group, and M@MSN group increased by more than 1000 mm. 3 Tumors in the GM@MSN group tended to enlarge in later stages, which may be related to the immunosuppressive tumor microenvironment and lack of tumor-associated antigens. Conversely, mice treated with H / GM@MSN showed significant inhibition of tumor recurrence, indicating that gel-mediated GOx and Mn... 2+ It exhibits strong anti-tumor activity when combined with autophagy inhibitors. The weight of tumors harvested after different treatments confirms that H / GM@MSN has a superior anti-tumor effect compared to other groups.

[0116] In addition, immunofluorescence staining was performed on the tumor tissue to detect CD4. + T cells and CD8 + T cell expression. For example... Figure 12 As shown, the H / GM@MSN group exhibited more fluorescence signal, indicating that H / GM@MSN can enhance CD8+. + T and CD4 + T cell tumor infiltration level. In other words, CDT significantly enhanced Mn 2+ HCQ inhibits the protective autophagy induced by STING pathway activation, thereby leading to the secretion of more IFN-β, stimulating DC maturation, and subsequently recruiting more cytotoxic T lymphocytes (CTLs). These results confirm that the combination of enhanced STING pathway activation and autophagy inhibition can significantly activate anti-tumor immune responses.

[0117] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a nano-hydrogel precursor, characterized in that, Includes the following steps: (1) Disperse mesoporous silica in anhydrous ethanol, add ammonia and 3-aminopropyltrimethoxysilane to the suspension, stir the reaction, and after the reaction is completed, amino-modified mesoporous silica MSN-NH2 is obtained. (2) Disperse the MSN-NH2 prepared in step (1) in deionized water, add glucose oxidase, and incubate. After incubation, GOx-loaded nanoparticles G@MSN are obtained. (3) Disperse the G@MSN prepared in step (2) in deionized water, add tannic acid, divalent manganese salt solution and 3-morpholinopropanesulfonic acid buffer in sequence to form MPN coating, then add bovine serum albumin solution and vortex to form BSA coating, thus forming a loaded GOX and Mn. 2+ GM@MSN nanoparticles; (4) After dialysis, the liposomes were incubated in a hydroxychloroquine solution. After incubation, liposomes loaded with hydroxychloroquine, LipHCQ, were obtained. (5) Add sodium alginate to deionized water, stir, and then add the nanoparticles GM@MSN prepared in step (3) and LipHCQ prepared in step (4). Stir the reaction to obtain the nanohydrogel precursor solution H / GM@MSN.

2. The preparation method according to claim 1, characterized in that, The precursor solution of the nano-hydrogel, H / GM@MSN, reacts with endogenous calcium ions in the body to generate the nano-hydrogel, H / GM@Gel.

3. The preparation method according to claim 1, characterized in that, In step (2), the mass ratio of MSN-NH2 to GOx is 20:1~2; Alternatively, in step (3), the divalent manganese salt is MnCl2, MnSO4, or Mn(CH3COO)2; Alternatively, in step (3), the mass ratio of the GOx-loaded nanoparticles to TA and divalent manganese salt is 20:7~9:1.2~1.5; Alternatively, in step (4), the dosage of HCQ is 5% to 25% of the liposome mass.

4. The preparation method according to claim 3, characterized in that, In step (2), the mass ratio of MSN-NH2 to GOx is 20:1.2; Alternatively, in step (3), the divalent manganese salt is MnCl2; Alternatively, in step (3), the mass ratio of the GOx-loaded nanoparticles to TA and divalent manganese salt is 20:8:1.

46.

5. The nanohydrogel precursor solution H / GM@MSN prepared by the preparation method according to any one of claims 1 to 4.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the nanohydrogel precursor solution H / GM@MSN as described in claim 5.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition also includes tumor treatment-related drugs.

8. A pharmaceutical preparation, characterized in that, The pharmaceutical formulation comprises the nanohydrogel precursor solution H / GM@MSN as described in claim 5.

9. The pharmaceutical preparation according to claim 8, characterized in that, The preparation is an injectable formulation.

10. The use of the nanohydrogel precursor solution H / GM@MSN of claim 5 and / or the pharmaceutical composition of claim 6 and / or the pharmaceutical formulation of claim 9 in the preparation of a drug for treating tumors.

11. The application as described in claim 10, characterized in that, The tumors include benign tumors and / or malignant tumors; the malignant tumors include solid tumors and hematologic malignancies, wherein the solid tumors include breast cancer.

12. The application as described in claim 11, characterized in that, The breast cancer mentioned is triple-negative breast cancer.

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