An epigenetic regulatory in situ gel vaccine, its preparation method and application

Abstract

This invention belongs to the field of tumor immunotherapy and biomedical materials technology, specifically relating to an epigenetic regulatory in situ gel vaccine, its preparation method, and its application. The in situ gel vaccine comprises a thermosensitive hydrogel matrix composed of poloxamer F127 and poloxamer F68, and gold-silicon-based nanoparticles loaded therein. The surface of the gold-silicon-based nanoparticles is loaded with siRNA targeting YTHDF1. After intratumoral injection of the thermosensitive hydrogel, a drug reservoir can be formed locally in the tumor. Under near-infrared laser irradiation, the gold-silicon-based nanoparticles induce photothermal therapy, triggering immunogenic cell death and releasing tumor antigens. Simultaneously, by silencing YTHDF1 expression, it upregulates MHC-I molecule expression in tumor cells and enhances the ability of dendritic cells to cross-present tumor antigens. This invention can be used to prepare tumor immunotherapy drugs.

Description

Technical Field

[0001] This invention belongs to the field of tumor immunotherapy and biomedical materials technology, specifically relating to an epigenetic regulatory in situ gel vaccine and its preparation method and application. Background Technology

[0002] Tumor immunotherapy aims to mobilize the body's own immune system to recognize and eliminate tumor cells. Therapeutic tumor vaccines, as an active immunization strategy, activate tumor-specific T-cell responses by delivering tumor antigens, adjuvants, or inducing in situ antigen release, exhibiting durable and systemic anti-tumor potential. Compared to passive infusion of anti-tumor drugs, vaccines can mobilize the body's immune system to kill tumor cells and generate long-term immune memory, preventing recurrence and metastasis. In recent years, with a deeper understanding of the tumor immune microenvironment and antigen presentation mechanisms, in situ tumor vaccines have emerged—they induce immunogenic cell death (ICD) locally in the tumor, turning the tumor tissue itself into an "antigen factory," releasing tumor-specific antigens and damage-associated molecular patterns, thereby cross-activating dendritic cells (DCs) and CD8+. + The anti-tumor immune response of T cells. This strategy avoids the cumbersome process of in vitro antigen identification and personalized preparation, has broad applicability, and is considered an important direction for next-generation tumor immunotherapy.

[0003] However, many tumors still suffer from fundamental defects such as insufficient antigen presentation and immune escape, limiting the effectiveness of in situ vaccine therapy. Recent epigenetic studies have revealed a key mechanism: N6-methyladenosine (m6A) RNA methylation modification, particularly the recognition protein YTH N6-methyladenosine RNA-binding protein 1 (YTHDF1), plays a dual negative regulatory role in tumor immune escape. On the one hand, YTHDF1 promotes the expression of lysosomal proteases, impairing the cross-presentation capacity of dendritic cells (DCs) for tumor antigens, thereby inhibiting CD8. + On the other hand, YTHDF1 also directly promotes the degradation of major histocompatibility complex class I (MHC-I) molecules in tumor cells, reducing the antigen self-presentation capacity of tumor cells and further driving immune escape. Therefore, YTHDF1 is considered a potential new target for cancer immunotherapy.

[0004] Existing technologies have failed to address a core issue: how to simultaneously restore the cross-presentation function of dendritic cells (DCs) and enhance the expression of MHC-I in tumor cells within the same treatment system, thereby doubly enhancing antigen presentation and effectively reducing immune escape. More critically, existing in situ vaccine systems lack the ability to integrate ICD induction with localized and sustained silencing of YTHDF1, resulting in the in situ antigens generated by the tumor ICD effect not being effectively cross-presented, while tumor cells remain in a state of low MHC-I expression and CD8 expression.+ T cells are difficult to fully activate.

[0005] Therefore, there is an urgent need for an in situ vaccine system that can simultaneously achieve ICD induction and YTHDF1 silencing, thereby enhancing the anti-tumor immune response from the perspective of dual antigen presentation. Summary of the Invention

[0006] To address the aforementioned technological gaps and clinical needs, this invention creatively proposes an in-situ gel vaccine based on epigenetic regulation to enhance antigen cross-presentation and reduce tumor immune escape. This invention induces an ICD effect in tumor tissue through local photothermal therapy and integrates this effect with YTHDF1 silencing within a single injectable gel platform to construct an in-situ vaccine system. Specifically, gold-silicon nanoparticles (sAM) containing compressed YTHDF1 siRNA are loaded into a thermosensitive hydrogel composed of poloxamer F127 and poloxamer F68. On one hand, the photothermal heating capability of the gold-silicon nanoparticles induces ICD locally in the tumor, releasing antigens to produce an in-situ vaccine effect. On the other hand, it delivers YTHDF1 siRNA in a sustained manner, simultaneously reversing the cross-presentation barrier of dendritic cells (DCs) and upregulating MHC-I expression in tumor cells. This enhances both the antigen presentation capacity of DCs and the self-presentation efficiency of tumor cells, synergistically enhancing the body's anti-tumor immune response.

[0007] This design addresses the fundamental shortcomings of traditional in situ vaccines, such as insufficient tumor antigen presentation and immune escape. It employs a bidirectional synergistic epigenetic regulatory strategy to enhance the cross-presentation of tumor antigens by dendritic cells (DCs) and improve the self-presentation efficiency of tumor cells, thus providing a basis for reversing tumor immune escape and enhancing CD8 expression. + T-cell-mediated anti-tumor immune responses offer a novel approach.

[0008] The purpose of this invention is to provide an in situ gel vaccine that simultaneously enhances the antigen presentation capacity of DC cells and upregulates the expression of MHC-I molecules in tumor cells through epigenetic regulation, as well as its preparation method and application, in order to solve the problems of insufficient local drug retention in tumors, low antigen presentation efficiency, and difficulty in effectively reversing immune escape in the prior art.

[0009] The technical solution adopted in this invention is as follows: In a first aspect, the present invention provides an epigenetic regulatory in situ gel vaccine, comprising: A thermosensitive hydrogel matrix, wherein the thermosensitive hydrogel matrix is ​​composed of poloxamer F127 and poloxamer F68, and the thermosensitive hydrogel matrix has the property of undergoing sol-gel transition under body temperature conditions. Gold-silicon nanoparticles loaded in the thermosensitive hydrogel matrix, wherein the surface of the gold-silicon nanoparticles is loaded with siRNA targeting YTHDF1; The gold-silicon-based nanoparticles exhibit photothermal conversion capabilities under near-infrared laser irradiation.

[0010] Preferably, the mass ratio of poloxamer F127 to poloxamer F68 in the thermosensitive hydrogel matrix is ​​(5-12):(1-3).

[0011] More preferably, the mass ratio of poloxamer F127 to poloxamer F68 in the thermosensitive hydrogel matrix is ​​9:2.

[0012] Preferably, the gelation temperature of the thermosensitive hydrogel matrix is ​​32~40℃.

[0013] More preferably, the gelation temperature of the thermosensitive hydrogel matrix is ​​36.12°C.

[0014] Preferably, the gold-silicon-based nanoparticles are gold-core mesoporous silica nanoparticles with a particle size in the nanometer range.

[0015] More preferably, the gold-silicon-based nanoparticles are gold-core mesoporous silica nanoparticles with a particle size of 90±10 nm.

[0016] Preferably, the surface of the gold-silicon nanoparticles is modified with phosphate and coated with polyethyleneimine, and then loaded with the siRNA targeting YTHDF1 through electrostatic interaction, with an N / P ratio of 5:1 to 15:1.

[0017] Preferably, the gold-silicon-based nanoparticles can be heated to 40-50°C under near-infrared laser irradiation.

[0018] More preferably, the gold-silicon based nanoparticles are subjected to an 808 nm laser at 1.0 W / cm². 2 Under power density irradiation for 10 minutes, the local temperature of the tumor can rise to 44°C.

[0019] Preferably, the thermosensitive hydrogel matrix is ​​also loaded with granulocyte-macrophage colony-stimulating factor (GM-CSF) and / or anti-programmed death receptor 1 (PD-1) antibody.

[0020] Secondly, the present invention provides a method for preparing an epigenetic regulatory in situ gel vaccine, comprising the following steps: (1) Gold core mesoporous silica nanoparticles (AM) were ultrasonically dispersed in deionized water, and an ethanol solution of 3-(trihydroxysilyl)propylmethyl phosphate (THPMP) was added under stirring at 75-85℃. After the reaction, the product was collected, washed, and phosphate-modified AM-p was obtained. (2) AM-p was mixed with polyethyleneimine (PEI) in anhydrous ethanol and ultrasonically stirred to obtain PEI-coated AM-PEI; (3) AM-PEI was co-incubated with siRNA targeting YTHDF1 at an N / P ratio of 5:1 to 15:1 to obtain gold-silicon nanoparticles (sAM) loaded with YTHDF1 siRNA. (4) Disperse poloxamer F127 and poloxamer F68 in deionized water, hydrate them at low temperature to form a uniform precursor solution, add sAM and mix well to obtain drug-loaded hydrogel sAM-Gel.

[0021] Preferably, in step (1), the gold-core mesoporous silica nanoparticles are prepared by the following method: a one-pot method is used, in which a surfactant and an alkaline substance are mixed in water, a reducing agent and a gold source precursor are added for reduction reaction, and then a silicon source is added for hydrolysis and condensation reaction. After removing the template, the gold-core mesoporous silica nanoparticles are obtained; wherein, the surfactant is hexadecyltrimethylammonium chloride (CTAC), hexadecyltrimethylammonium bromide (CTAB) or hexadecyltrimethyltoluenesulfonate (CTAT); the alkaline substance is sodium hydroxide; and the reducing agent is formaldehyde. The silicon source is tetraethyl orthosilicate (TEOS), bis[3-(triethoxysilyl)propyl]-disulfide (BTESPD) or a combination thereof; and the gold source precursor is chloroauric acid trihydrate (HAuCl4·3H2O) or chloroauric acid tetrahydrate (HAuCl4·4H2O).

[0022] Preferably, in step (1), the mass-to-volume ratio of AM to THPMP is (300-800) mg:1 mL.

[0023] Preferably, in step (2), the mass ratio of AM-p to the cationic polymer is (1-5):1.

[0024] Preferably, in step (4), the mass ratio of poloxamer F127 to poloxamer F68 is (5-12):(1-3).

[0025] Preferably, the low temperature condition in step (4) is 2~8℃.

[0026] Thirdly, the present invention provides the application of the above-mentioned epigenetic regulatory in situ gel vaccine in the preparation of antitumor drugs.

[0027] Preferably, the application includes intratumoral injection of the in situ gel vaccine, followed by local tumor irradiation with near-infrared laser, and combined with granulocyte-macrophage colony-stimulating factor and / or anti-PD-1 antibody for anti-tumor immunotherapy.

[0028] Compared with the prior art, the present invention has the following beneficial effects: (1) The in situ gel vaccine constructed in this invention can be locally heated to a suitable temperature under near-infrared laser irradiation, killing tumor cells and inducing immunogenic cell death, releasing damage-related molecular patterns. Animal experiments have confirmed that after photothermal therapy, ROS expression in tumor tissue increases and CRT exposure level is enhanced.

[0029] (2) YTHDF1 siRNA in sAM-Gel can effectively reduce the expression of YTHDF1 in DC cells. Co-incubation with tumor antigen can stimulate DC maturation and enhance the ability of DC to cross-present tumor antigen.

[0030] (3) sAM-Gel can effectively silence YTHDF1 expression in tumor tissues and restore MHC-I expression in tumor cells, thereby enhancing the self-presentation of tumor cell antigens and potentially enhancing the body's immune surveillance to kill tumors.

[0031] (4) In vivo antitumor pharmacodynamic evaluation showed that sAM / G / P-Gel exhibited the best antitumor efficacy in mouse subcutaneous tumor model, with a tumor inhibition rate of 82.14 ± 1.78%.

[0032] (5) By integrating photothermal-induced ICD and YTHDF1 silencing into a single thermosensitive gel platform, this invention achieves the spatiotemporal synergy of "photothermal ICD-induced tumor antigen release", "epiggenetic regulation to enhance DC antigen presentation" and "epiggenetic regulation to enhance tumor self-presentation", forming a complete immune activation chain of "antigen release - DC recruitment - enhanced antigen presentation - continuous T cell activation - inhibition of immune escape". Attached Figure Description

[0033] The accompanying drawings, which form part of this specification, 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.

[0034] Figure 1 a: Electron micrographs of AM, AM-p, AM-PEI and sAM; b: Zeta potential changes of AM, AM-p, AM-PEI and sAM.

[0035] Figure 2 a: Scanning electron micrographs of blank hydrogel and drug-loaded hydrogel sAM-Gel; b: Rheological curve of sAM-Gel.

[0036] Figure 3 a: Evaluation of photothermal conversion capacity in vivo in the NS and sAM-Gel groups; b: ROS expression in tumor tissues of the NS and sAM-Gel groups; c: CRT expression in tumor tissues of the NS and sAM-Gel groups.

[0037] Figure 4 a: Expression of YTHDF1 mRNA in DC cells after treatment with different formulations; b: DC maturation rate after treatment with different formulations; c: MHC-I expression after treatment with different formulations. + DC ratio.

[0038] Figure 5 a: YTHDF1 expression in tumor tissues of the NS and sAM-Gel groups; b: MHC-Ⅰ expression in tumor tissues of the NS and sAM-Gel groups.

[0039] Figure 6 : Curves showing changes in tumor volume in mice after treatment with different formulations. Detailed Implementation

[0040] 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 herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0041] 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.

[0042] Example 1: Preparation and characterization of sAM (YTHDF1 siRNA-loaded gold-silicon nanoparticles) Gold-silicon based nanoparticles (Au@MSNs) (AM) possess excellent photothermal conversion capabilities and have abundant silanol groups on their surface, making them suitable for functionalization to load gene drugs. Specifically, phosphate groups are first introduced onto the AM surface to provide the necessary strong negative charge for subsequent assembly.

[0043] 1. Synthesis of AM Au@MSNs were synthesized using a one-pot sol-gel method. 0.5 g of CTAC was mixed with 0.5 mL of 10% (w / w) sodium hydroxide (NaOH) solution in 50 mL of deionized water and stirred at 80 °C for 30 min. Then, 0.5 mL of 3.7% (v / v) formaldehyde solution was added and mixed thoroughly. Subsequently, 3 mL of 1% chloroauric acid (HAuCl4) solution was added dropwise. After stirring for another 20 min, 0.5 mL of tetraethyl orthosilicate (TEOS) and 0.5 mL of bis[3-(triethoxysilyl)propyl]-disulfide (BTESPD) were mixed in 2 mL of anhydrous ethanol and added dropwise to the above mixture. The reaction solution was stirred for another 2 h to obtain a gold-core silica (Au@SiO2) solution. The product was centrifuged and washed three times. The CTAC surfactant was removed by reflux at 65°C for 6 h using NH4NO3 / ethanol solution (10 mg / mL). This process was repeated three times to obtain Au@MSNs (AM).

[0044] 2. Phosphate modification of AM 50 mg AM was ultrasonically dispersed in 10 mL of deionized water and stirred at 80 °C for 30 min. 0.1 mL of THPMP was dissolved in 1 mL of anhydrous ethanol and added dropwise to the reaction solution, and stirring was continued for 2 h. After the reaction was completed, the product was collected by centrifugation and washed three times each with PBS and anhydrous ethanol to obtain phosphate-modified AM (AM-p).

[0045] 3. PEI coating Subsequently, the AM-p surface was coated with the cationic polymer PEI to provide a strong positive charge for subsequent nucleic acid binding. Specifically, 50 mg of AM-p and 20 mg of PEI (branched type, Mw = 10 kDa) were mixed in 10 mL of anhydrous ethanol, sonicated for 20 s, and then stirred at room temperature for 30 min. After the reaction, the mixture was washed three times each with PBS and anhydrous ethanol to obtain PEI-coated AM nanoparticles (AM-PEI).

[0046] 4. Loading YTHDF1 siRNA Nucleic acid delivery is achieved through electrostatic interaction. AM-PEI and YTHDF1 siRNA are co-incubated in RNase-free water at an N / P ratio of 5:1 for 30 min to obtain AM (sAM) loaded with YTHDF1 siRNA (5′-GCACACAACCTCTATCTTTA-3′).

[0047] 5. Characterization Transmission electron microscopy images showed that AM, AM-p, AM-PEI, and sAM all exhibited spherical and uniformly shaped nanoparticles with a particle size of approximately 90 nm. Among them, AM and AM-p had smooth surfaces, while the modified AM-PEI and sAM had relatively rough surfaces. Figure 1 a). During the preparation process, the changes in the Zeta potential of the nanoparticles were monitored. The Zeta potentials of AM, AM-p, AM-PEI, and sAM were -8.63 ± 1.56 mV, -22.03 ± 2.00 mV, 21.03 ± 2.97 mV, and 15.53 ± 1.81 mV, respectively. Figure 1 (b) These successive changes are consistent with the introduction of strong negative phosphate groups, coating with positively charged PEI, and condensation with negatively charged siYTHDF1, indicating that the surface modification and siRNA loading were successful.

[0048] Example 2: Preparation and evaluation of sAM@Gel (drug-loaded thermosensitive hydrogel) This invention uses poloxamer F127 and F68 to prepare a thermosensitive hydrogel.

[0049] 1. Preparation of sAM@Gel 9 g of F127 and 2 g of F68 were dispersed in 50 mL of deionized water and allowed to fully hydrate at 4 °C until a homogeneous precursor solution was formed, thus obtaining a blank hydrogel. sAM (final concentration 100 μg / mL, based on AM) was added to the precursor solution and mixed thoroughly to form a drug-loaded hydrogel, sAM-Gel.

[0050] 2. Characterization The microstructure of the gel was observed using scanning electron microscopy (SEM). SEM results showed that the blank hydrogel had a highly porous and interconnected structure with pore sizes ranging from 2 to 20 μm. By adding sAM to an F127 / F68 solution, a drug-loaded hydrogel, sAM-Gel, was obtained. SEM results showed that its structural characteristics were similar to the blank hydrogel, and small granular protrusions were observed on the gel surface, indicating successful loading of sAM (…). Figure 2 a).

[0051] Thermal response behavior and sol-gel transition properties were evaluated using rheological analysis. Temperature dependence curves of the storage modulus (G') and loss modulus (G'') of the sAM-gel indicated a gelation temperature of 36.12°C, which is consistent with the conditions for in-situ gel formation after intratumoral injection. Figure 2 b).

[0052] Example 3: Validation of the mechanism of action of sAM@Gel in situ vaccine 1. Method BALB / c mice were injected subcutaneously into the right axilla with 1×10 6 A mouse model of 4T1 tumor-bearing cells was established using 4T1 cells. The tumor volume was increased to approximately 100 mm². 3 Mice were randomly divided into two groups (n=3 per group), and injected with an equal volume of saline (NS group) or sAM-Gel (50 μL, sAM-Gel group) into the tumor. Four hours after injection, the tumor was treated with an 808nm laser at 1.0W / cm². 2 The tumor was irradiated locally with a specific power for 10 min. Thermograms of mice were acquired at 1, 2, 4, 6, 8, and 10 min to monitor temperature changes. Tumor tissue was then collected within 12 h, fixed with 4% paraformaldehyde, and prepared into paraffin sections. The expression of reactive oxygen species (ROS) and calreticulin (CRT) in the tumor tissue was analyzed by histological and immunohistochemical staining.

[0053] 2. Results Thermal imaging results showed that under laser irradiation, the temperature of local tumor tissue in the sAM-Gel group was significantly higher than that in the NS group, reaching 44℃ within 10 minutes. Figure 3 a). This indicates its ability to implement a phototherapy-induced in situ vaccine strategy. Laser irradiation causes localized heating of tumor tissue; photothermal treatment generates reactive oxygen species (ROS) within tumor cells, inducing endoplasmic reticulum stress and immunogenic cell death, releasing damage-related molecular patterns, thereby forming an in situ vaccine. Tumor slice images show that, compared to the NS group, the sAM-Gel group exhibited a significantly increased amount of ROS generation in tumor tissue ( Figure 3 b), and accompanied by CRT eversion ( Figure 3 c), these results indicate that sAM-Gel forms an in situ tumor vaccine after intratumoral injection.

[0054] Example 4: Validation of the mechanism by which sAM promotes antigen cross-presentation 1. Method The effect of YTHDF1 siRNA on DC antigen presentation was investigated in vitro. Bone marrow cells were first isolated from the femur and tibia of BALB / c mice and induced and cultured for 7 days in a medium containing GM-CSF (20 ng / mL) and IL-4 (10 ng / mL) to obtain bone marrow-derived dendritic cells (BMDCs).

[0055] BMDCs were inoculated into the lower chamber of the Transwell chamber (5 × 10⁶). 5 4T1 cells were seeded in the upper chamber (1×10⁴ cells / well). 5(each hole), the upper chamber is provided with the following conditions: NS group: equal volume of PBS; TCL group: AM was added, and after incubation for 4 hours, 4T1 cells were irradiated for 5 minutes to establish tumor cell lysate (TCL) conditions; sLipo group: Liposomes loaded with YTHDF1 siRNA (final siRNA concentration 50 nM) were added and transfected using Lipofectamine 2000; sAM group: sAM (containing 50 nM siRNA) was added; TCL + sAM group: sAM (containing 50 nM siRNA) was added, and after incubation for 4 hours, 4T1 cells were irradiated for 5 minutes to establish tumor cell lysate (TCL) conditions; Each group was incubated at 37℃ in a 5% CO2 incubator for 24 hours. BMDC was collected, and the following indicators were measured: (1) YTHDF1 mRNA expression: The relative expression level of YTHDF1 mRNA in BMDC was detected by qRT-PCR ( Figure 4 a) (2) DC maturation rate: CD11c was detected by flow cytometry. + CD80 + CD86 + The proportion of mature DC ( Figure 4 b) (3) MHC-I cross-presentation: H-2Kb on the surface of BMDCs was detected by flow cytometry. + Cell ratio ( Figure 4 c).

[0056] 2. Results (1) YTHDF1 mRNA expression: qRT-PCR results showed that the expression level of YTHDF1 mRNA in BMDC of the sLipo group and sAM group was significantly reduced (compared with the NS group, p <0.001 indicates that sAM can effectively deliver siRNA into BMDC and silence YTHDF1 expression; there was no significant difference in YTHDF1 expression between the TCL group and the NS group ( p >0.05); the YTHDF1 expression level in the TCL+sAM group was comparable to that in the sAM group ( Figure 4 a).

[0057] (2) DC maturation rate: Flow cytometry analysis showed that the BMDC maturation rate in the NS group was 12.47±2.86%; the maturation rate in the TCL group was significantly increased to 31.67±3.89% (compared to the NS group, p<0.001 indicates that tumor antigen can stimulate DC maturation; the maturation rate in the sLipo group was 19.07±2.48%, and in the sAM group it was 21.43±3.27%, both of which were higher than those in the NS group ( p <0.05); the TCL+sAM group showed the highest DC maturity rate, reaching 47.27 ± 4.90% (compared to the TCL group, p <0.001)( Figure 4 b).

[0058] (3) MHC-I cross-presentation: Flow cytometry analysis showed that the proportion of MHC-I⁺ BMDCs in the NS group was 7.07±2.32%; the proportion of MHC-I⁺ in the TCL group was significantly increased to 30.26±3.91% (compared to the NS group, p <0.001), indicating that BMDCs can cross-present tumor antigens via the MHC-I pathway; the MHC-I⁺ proportion in the TCL+sAM group further increased to 45.43±3.02% (compared to the TCL group, p <0.001)( Figure 4 c).

[0059] The above results indicate that BMDCs can mature under TCL stimulation and cross-present tumor antigens via the MHC-I pathway; sAM can significantly enhance the maturation and MHC-I cross-presentation capacity of DCs by knocking down YTHDF1 expression in BMDCs through the delivery of YTHDF1 siRNA.

[0060] Example 5: Validation of the mechanism by which sAM@Gel upregulates tumor MHC-I 1. Method Establish a BALB / c mouse subcutaneous 4T1 breast cancer model, using 1×10 6 4T1 cells were inoculated into the right axilla of mice. The tumor volume was increased to approximately 100 mm². 3 Mice were randomly divided into two groups (n=3 per group), and injected intratumorally with either equal volume of physiological saline (NS group) or sAM-Gel (50 μL, containing 100 μg / mL of sAM, calculated as AM). Four hours after injection, the tumor was treated with an 808nm laser at 1.0W / cm². 2 The tumor was irradiated locally with a specific power for 10 minutes. Tumor tissue was then collected within 24 hours, fixed with 4% paraformaldehyde, and prepared into paraffin sections. The expression of YTHDF1 and MHC-I was analyzed by histological and immunohistochemical staining.

[0061] 2. Results The results showed that, compared with the NS group, the fluorescence signal of intracellular YTHDF1 protein in tumor tissue of the sAM-Gel group was significantly weakened, indicating that the expression of YTHDF1 protein was significantly reduced. Figure 5 a); Simultaneously, the fluorescence signal of MHC-I molecules on the surface of tumor cells in the sAM-Gel group was significantly enhanced, indicating that MHC-I expression was significantly upregulated ( Figure 5 b).

[0062] The above results indicate that sAM-Gel effectively silences YTHDF1 expression in tumor cells by delivering YTHDF1 siRNA, thereby relieving the negative regulation of MHC-I by YTHDF1, restoring the expression of MHC-I molecules on the surface of tumor cells, and enhancing the antigen self-presentation ability of tumor cells, thus potentially enhancing the body's immune surveillance and killing of tumors.

[0063] Example 6: Evaluation of in vivo antitumor effect 1. Method A mouse subcutaneous tumor model was established to evaluate the in vivo therapeutic potential of sAM-Gel in combination with GM-CSF (sAM / G-Gel) and its combination with anti-PD-1 antibody (sAM / G / P-Gel).

[0064] The preparation methods of sAM / G-Gel and sAM / G / P-Gel are the same as in Example 2. GM-CSF (final concentration 2 μg / mL) and anti-PD-1 antibody (final concentration 200 μg / mL) are added to the precursor solution respectively, and the mixture is stirred at 4°C to obtain the final product.

[0065] Specifically, this involves establishing a subcutaneous 4T1 triple-negative breast cancer model in BALB / c mice, using 1×10 6 4T1 tumor cells were inoculated into the right axilla of mice. The tumor volume was increased to approximately 100 mm². 3 Mice were randomly divided into 6 groups (n=6 per group) and received the following treatments: NS group: Intratumoral injection of an equal volume of normal saline (50 μL); Gel group: Intratumoral injection of blank hydrogel (50 μL); AM-Gel group: Intratumoral injection of AM-Gel (50 μL, containing AM 100 μg / mL) + 808 nm laser irradiation; sAM-Gel group: Intratumoral injection of sAM-Gel (50 μL, containing 100 μg / mL of sAM, calculated as AM) + 808 nm laser irradiation; sAM / G-Gel group: Intratumoral injection of sAM / G-Gel (50 μL, containing 100 μg / mL of sAM, calculated as AM, and 2 μg / mL of GM-CSF encapsulated in the gel) + 808 nm laser irradiation; sAM / G / P-Gel group: Intratumoral injection of sAM / G / P-Gel (50 μL, containing 100 μg / mL of sAM, calculated as AM, and 2 μg / mL of GM-CSF and 200 μg / mL of PD-1 antibody encapsulated in the gel) + irradiation with 808 nm laser.

[0066] Each group received a single intratumoral injection of the corresponding formulation. Laser irradiation groups (AM-Gel, sAM-Gel, sAM / G-Gel, sAM / G / P-Gel groups) received 808 nm laser light (1.0 W / cm²). 2 Irradiate the tumor site for 5 minutes, then repeat every 7 days for a total of three times. Measure the tumor's long diameter (L) and short diameter (W) every 2 days using electronic calipers, and calculate V = L × W. 2 / 2 Calculate tumor volume and plot tumor growth curve ( Figure 6 After treatment, the tumor inhibition rate (TIR) ​​was calculated using the formula: TIR (%) = (1 - mean tumor volume in the treatment group / mean tumor volume in the NS group) × 100%.

[0067] 2. Results Tumor volume monitoring results showed ( Figure 6 In both the NS and Gel groups, tumors showed progressive and rapid growth, with no significant difference between the two groups. Tumor growth in the AM-Gel group was inhibited to some extent, but the therapeutic effect was limited; the tumor volume in the sAM-Gel group was significantly smaller than that in the AM-Gel group. p < 0.05 indicates that YTHDF1 silencing-mediated upregulation of MHC-I expression and the resulting enhanced DC cross-presentation and tumor immune surveillance play a significant role in inhibiting tumor growth. After the addition of GM-CSF, the tumor volume in the sAM / G-Gel group was further reduced compared to the sAM-Gel group ( p < 0.05, attributed to GM-CSF enhancing the recruitment of dendritic cells (DCs) in the initial stage of antigen presentation. When used in combination with anti-PD-1 antibody, the tumor volume in the sAM / G / P-Gel group was the smallest among all groups ( pThe tumor inhibition rate reached 82.14±1.78% (< 0.05), indicating that relieving immune checkpoint inhibition can further enhance the T-cell immune response restored by epigenetic regulation. In summary, the synergistic enhancement of MHC-I expression and dendritic cell antigen presentation by epigenetic regulation is of great significance for improving the efficacy of in situ gel vaccines.

[0068] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An epigenetic regulatory in situ gel vaccine, characterized in that, include: A thermosensitive hydrogel matrix, wherein the thermosensitive hydrogel matrix is ​​composed of poloxamer F127 and poloxamer F68, and the thermosensitive hydrogel matrix has the property of undergoing sol-gel transition under body temperature conditions. Gold-silicon nanoparticles loaded in the thermosensitive hydrogel matrix, wherein the surface of the gold-silicon nanoparticles is loaded with siRNA targeting YTHDF1; The gold-silicon-based nanoparticles exhibit photothermal conversion capabilities under near-infrared laser irradiation.

2. The epigenetic regulatory in situ gel vaccine according to claim 1, characterized in that, The mass ratio of poloxamer F127 to poloxamer F68 in the thermosensitive hydrogel matrix is ​​(5-12):(1-3), and the gelation temperature of the thermosensitive hydrogel matrix is ​​32~40℃.

3. The epigenetic regulatory in situ gel vaccine according to claim 1, characterized in that, The gold-silicon-based nanoparticles are gold-core mesoporous silica nanoparticles with a particle size in the nanometer range. The surface of the gold-silicon-based nanoparticles is modified with phosphate and coated with polyethyleneimine, and then loaded with the siRNA targeting YTHDF1 through electrostatic interaction, with an N / P ratio of 5:1 to 15:

1.

4. The epigenetic regulatory in situ gel vaccine according to claim 1, characterized in that, The gold-silicon-based nanoparticles can be heated to 40-50°C under near-infrared laser irradiation, inducing immunogenic cell death in tumor cells and releasing tumor antigens; the siRNA targeting YTHDF1 silences YTHDF1 expression, upregulates MHC-I molecule expression in tumor cells, and enhances the cross-presentation ability of dendritic cells.

5. The epigenetic regulatory in situ gel vaccine according to claim 1, characterized in that, It also includes granulocyte-macrophage colony-stimulating factor and / or anti-PD-1 antibody.

6. A method for preparing an epigenetic regulatory in situ gel vaccine as described in any one of claims 1-5, characterized in that, Includes the following steps: (1) Gold core mesoporous silica nanoparticles were ultrasonically dispersed in deionized water, and an ethanol solution of 3-(trihydroxysilyl)propylmethyl phosphate was added under stirring at 75-85℃. After the reaction, the product was collected, washed, and phosphate-modified AM-p was obtained. (2) AM-p was mixed with polyethyleneimine in anhydrous ethanol and ultrasonically stirred to obtain PEI-coated AM-PEI; (3) AM-PEI was co-incubated with siRNA targeting YTHDF1 at an N / P ratio of 5:1 to 15:1 to obtain gold-silicon nanoparticles (sAM) loaded with YTHDF1 siRNA. (4) Disperse poloxamer F127 and poloxamer F68 in deionized water, hydrate them at low temperature to form a uniform precursor solution, add sAM and mix well to obtain drug-loaded hydrogel sAM-Gel.

7. The preparation method according to claim 6, characterized in that, In step (4), the mass ratio of poloxamer F127 to poloxamer F68 is (5-12):(1-3), and the low temperature condition is 2~8℃.

8. The use of an epigenetic regulatory in situ gel vaccine as described in any one of claims 1-5 in the preparation of antitumor drugs.

9. The application according to claim 8, characterized in that, The application includes injecting the in situ gel vaccine into the tumor and then irradiating the tumor site with near-infrared laser to induce immunogenic cell death in tumor cells and release tumor antigens; the siRNA targeting YTHDF1 silences YTHDF1 expression, enhances the ability of dendritic cells to cross-present tumor antigens, and upregulates the expression of MHC-I molecules in tumor cells.

10. The application according to claim 8, characterized in that, The application also includes combining granulocyte-macrophage colony-stimulating factor to promote immune cell infiltration, and / or combining anti-PD-1 antibody to relieve immunosuppression and carry out anti-tumor immunotherapy.

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