Silicon-gold ratio screening method for tumor in situ vaccine and mild photothermal vaccine prepared therefrom and applications

Abstract

The present application belongs to the technical field of nanobiomedical materials and tumor in situ vaccine preparation, and particularly relates to a silicon-gold ratio screening method for a tumor in situ vaccine, a mild photothermal vaccine prepared from the silicon-gold ratio screening method and application. The present application prepares gold core mesoporous silica nanoparticles (Au@MSNs) with different volume ratios of silicon source to chloroauric acid (1:1, 1:2, 1:3 and 1:4), takes tumor cell calnexin (CRT) exocytosis as an immunogenic cell death marker, and comprehensively evaluates the structural stability and photothermal conversion efficiency to screen and determine that the Au@MSNs with a volume ratio of silicon source to chloroauric acid of 1:3 are the optimal carrier. Further, the HSP90 inhibitor is loaded into the mesoporous channels of the optimal Au@MSNs to obtain a mild photothermal in situ vaccine. The vaccine can induce immunogenic cell death of tumor cells and block HSP90-mediated heat repair escape under 808 nm laser irradiation, and is suitable for preparing an antitumor drug.

Description

Technical Field

[0001] This invention belongs to the technical fields of nanobiomedical materials and tumor in situ vaccine preparation, specifically involving a silicon-gold ratio screening method for tumor in situ vaccines and a mild photothermal vaccine prepared therefrom and its application. Background Technology

[0002] In situ tumor vaccines utilize the patient's own tumor tissue as an antigen library. By inducing tumor cells to release autologous tumor antigens in situ, they activate the body's specific anti-tumor immunity. They possess significant advantages such as high antigen matching, broad-spectrum activity, and the ability to inhibit tumor recurrence and metastasis, making them a core technological pathway for current personalized treatment. Mild photothermal therapy avoids the burns, inflammatory damage, and immunosuppression of normal tissues caused by high-intensity photothermal stimulation, making it a relatively safe in situ vaccine induction method. Gold nanoparticles possess excellent photothermal conversion performance and safety. Gold-core mesoporous silica nanomaterials combine excellent photothermal conversion performance, porous drug-loading properties, and good biocompatibility, making them a research hotspot for constructing photothermal in situ vaccine carriers. However, current research mostly uses core-shell nanocarriers with fixed silicon-gold ratios, lacking structural optimization designs guided by immune initiation. While inducing tumor cell damage and antigen release through single photothermal stimulation achieves preliminary in situ vaccine activation, the following problems severely restrict the immune activation efficiency and clinical therapeutic effect of in situ vaccines, leading to easy tumor recurrence. Specifically: ① The existing structural composition of silicon-gold nanocarriers has not been optimized with the ability to initiate immunity through tumor antigen release as the target, and there is a lack of vaccine-oriented silicon-gold structural optimization. If the silica shell is too thick, the degree of thermal damage to tumor cells is insufficient, and it is impossible to generate sufficient tumor antigens and immune stimulation signals; if the shell is too thin, the carrier structure is unstable and prone to pore collapse, making it difficult to continuously induce antigen release. Ultimately, this leads to insufficient antigen release, weak immunogenicity, and insufficient specific T cell response in in situ vaccines, making it difficult to form long-term anti-tumor immune memory.

[0003] ② Mild photothermal therapy relies solely on the passive release of antigens due to photothermal damage, without intervening in the stress protection mechanisms of tumor cells. Studies have shown that under mild photothermal stimulation, tumor cells significantly upregulate the expression of HSP90 heat shock protein, initiate self-repair of thermal damage, maintain tumor cell activity, inhibit antigen release and immune activation, directly leading to the failure of in situ vaccine anti-tumor immune response, local tumor recurrence, and distant metastasis.

[0004] Therefore, a comprehensive technical solution is urgently needed that can both optimize antigen release at the source and block tumor thermal repair escape. Currently, research on silicon-gold based photothermal in situ vaccines mainly focuses on material morphology improvement, enhancement of single photothermal properties, and construction of simple drug delivery systems. There is a lack of systematic screening studies on silicon-gold ratios optimized for tumor antigen release and immune initiation capabilities. Furthermore, regarding the problem of immune activation failure caused by HSP90 upregulation in tumor cells after mild photothermal therapy, existing technologies lack systematic solutions for synergistic treatment systems that can effectively block this thermal repair escape pathway in in situ vaccine applications. Summary of the Invention

[0005] Existing silicon-gold based photothermal in situ vaccine carriers suffer from the following problems: the silicon-gold ratio is not optimized to enhance the ability to initiate immunity through tumor antigen release, and there is a lack of structural optimization design to guide immune initiation, resulting in insufficient antigen release and weak immunogenicity. Furthermore, mild photothermal therapy relies solely on the passive release of antigens due to photothermal damage, without addressing the self-repair escape mechanism of tumor cells caused by the upregulation of HSP90 due to thermal damage, leading to limited efficacy and a high relapse rate. This invention provides a method for screening the silicon-gold ratio for tumor in situ vaccines, gold-core mesoporous silica nanoparticles obtained therefrom, and a mild photothermal in situ vaccine.

[0006] The purpose of this invention is: 1. A systematic method for screening and optimizing the silicon-gold ratio is provided. By precisely controlling the ratio parameters of the gold core and the mesoporous silica shell, the structural stability and photothermal responsiveness of the carrier are balanced, and a multifunctional nanodelivery carrier with optimal tumor antigen stimulation and release capability is obtained. This solves the problems of insufficient antigen release and weak immune activation capability of traditional carriers from the source, and improves the initiation efficiency of tumor in situ vaccines.

[0007] 2. Construct a synergistic treatment system combining mild photothermal therapy (≤45℃) and HSP90 inhibitors. By relying on mesoporous channels to stably load HSP90 inhibitors, the system can precisely block the self-repair pathway mediated by heat shock proteins in tumor cells, thus solving the problems of poor efficacy and easy recurrence in traditional mild photothermal therapy for tumor cell thermal repair.

[0008] 3. To develop an in-situ vaccine delivery platform that integrates photothermal therapy, drug delivery, antigen release, immune activation, and repair inhibition, thereby compensating for the shortcomings of single treatment modalities, improving the overall efficacy of tumor treatment, reducing the probability of tumor recurrence and metastasis, and promoting the clinical translation and application of high-performance tumor nanovaccines.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for screening the silicon-to-gold ratio for in situ tumor vaccines, comprising the following steps: Gold-core mesoporous silica nanoparticles with different silicon-to-gold ratios were prepared. Using immunogenic cell death markers of tumor cells as evaluation indicators, the immune initiation ability of gold-core mesoporous silica nanoparticles with different silicon-to-gold ratios was evaluated. Based on the results of the immune initiation capability evaluation, and combined with the structural stability and photothermal conversion efficiency of the gold-core mesoporous silica nanoparticles with different silicon-gold ratios, the optimal silicon-gold ratio was screened and determined.

[0010] Preferably, the preparation of gold-core mesoporous silica nanoparticles with different silicon-gold ratios includes: using a one-pot sol-gel method to react different volumes of chloroauric acid solution with a fixed amount of silicon source, that is, adjusting the volume of chloroauric acid solution while keeping the silicon source volume fixed, to obtain gold-core mesoporous silica nanoparticles with different silicon-gold ratios.

[0011] More preferably, the silicon source has a volume of 1 ml and a Si content of approximately 4.4-4.5 mmol, the chloroauric acid solution has a concentration of 1% (w / v, g / 100 mL), and the different volumes of chloroauric acid solution are 1 mL, 2 mL, 3 mL, and 4 mL, respectively, that is, the volume ratio of silicon source to chloroauric acid is 1:1, 1:2, 1:3, and 1:4, respectively, corresponding to a silicon-gold molar ratio of approximately 150:1, 75:1, 50:1, and 37.5:1.

[0012] Preferably, the immunogenic cell death marker is calreticulin (CRT); the evaluation of immune initiation capacity is achieved by in vivo CRT immunohistochemical staining of tumor tissue.

[0013] Preferably, the screening to determine the optimal silicon-gold ratio specifically includes: (1) The absorbance of gold core mesoporous silica nanoparticles with different silicon-gold ratios was measured at 808 nm by UV-Vis spectrophotometer, and the proportions with absorbance <0.2 were excluded. (2) The morphology and dispersion state of the gold core mesoporous silica nanoparticles with different silicon-gold ratios were observed by transmission electron microscopy, and the proportions with irregular morphology and severe adhesion were excluded. (3) The proportion of CRT with the strongest eversion was screened by evaluating the immune activation ability.

[0014] Preferably, the optimal silicon source to chloroauric acid volume ratio is 1:3.

[0015] More preferably, the gold core mesoporous silica nanoparticles with a silicon source to chloroauric acid volume ratio of 1:3 have a gold core particle size of 50 nm and an overall particle size of 90±10 nm, and are heated to 44.67±0.84℃ under irradiation with an 808 nm laser and a power density of 1.0 W / cm².

[0016] Secondly, the present invention provides a gold-core mesoporous silica nanoparticle, wherein the volume ratio of the silicon source to chloroauric acid in the gold-core mesoporous silica nanoparticle is 1:3.

[0017] Preferably, the gold core of the gold-core mesoporous silica nanoparticles has a gold core diameter of 50 nm and an overall particle size of 90±10 nm; it is heated to 44.67±0.84℃ under irradiation with an 808 nm laser and a power density of 1.0 W / cm².

[0018] Preferably, 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 a reduction reaction, and then a silicon source is added for a hydrolysis-condensation reaction. After removing the template, the gold-core mesoporous silica nanoparticles are obtained. 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 (HAuCl4).

[0019] Thirdly, the present invention provides a mild photothermal in situ vaccine, comprising: The above-mentioned gold-core mesoporous silica nanoparticles; And an HSP90 inhibitor loaded in the mesoporous channels of the gold-core mesoporous silica nanoparticles.

[0020] Preferably, the HSP90 inhibitor is galdromycin (GA), 17-allylaminogaldromycin (17-AAG), 17-dimethylaminoethylaminogaldromycin (17-DMAG), gambogeylic acid, or a combination thereof.

[0021] Preferably, the HSP90 inhibitor is loaded into the gold-core mesoporous silica nanoparticles by solvent evaporation: the HSP90 inhibitor and the gold-core mesoporous silica nanoparticles are dispersed in chloroform, stirred at room temperature until the solvent is completely evaporated, and then centrifuged and washed to obtain the final product.

[0022] Preferably, the gold-core mesoporous silica nanoparticles induce immunogenic cell death in tumor cells under 808 nm laser irradiation, releasing tumor antigens; the HSP90 inhibitor blocks the heat shock protein 90 (HSP90)-mediated thermal damage self-repair pathway in tumor cells.

[0023] Fourthly, the present invention provides the application of the above-mentioned mild photothermal in situ vaccine in the preparation of antitumor drugs.

[0024] Preferably, the application includes injecting the mild photothermal in situ vaccine into the tumor and then irradiating the tumor site with an 808 nm laser at a power density of 0.6-1.0 W / cm² for 5-10 minutes.

[0025] Compared with the prior art, the present invention has the following beneficial effects: 1. Achieve optimal tumor antigen stimulation and release effect, and improve the activation efficiency of tumor in situ vaccines.

[0026] This invention precisely screens the optimal volume ratio of silicon source to chloroauric acid (1:1, 1:2, 1:3, 1:4), using tumor cell CRT eversion as an evaluation index, and comprehensively assesses structural stability and photothermal conversion efficiency. A volume ratio of 1:3 for silicon source to chloroauric acid was determined to be the optimal ratio. This ratio achieves optimal matching of bulk structural stability, photothermal responsiveness, and mesoporous transport performance. The gold core particle size is 50 nm, and the overall particle size is 90 nm. Under 808 nm laser irradiation, it can be heated to 44.67℃, efficiently and continuously triggering the release of tumor in situ antigens, significantly increasing antigen abundance and immune stimulation intensity, thereby enhancing the activation efficiency of tumor in situ vaccines from the source and achieving a stronger specific anti-tumor immune response.

[0027] 2. It blocks the self-repair and escape of tumors by gentle photothermal therapy, solving the problems of poor efficacy and easy recurrence of traditional gentle photothermal therapy.

[0028] This invention constructs a G-Au@MSNs synergistic therapeutic system by loading the HSP90 inhibitor GA into the mesoporous channels of Au@MSNs with an optimal silicon-to-gold ratio (1:3). Western blot experiments confirmed that the HSP90 protein expression level in the G-Au@MSNs+L group was the lowest among all treatment groups, effectively blocking the HSP90-mediated self-repair pathway of tumor cells after thermal damage. While maintaining the advantages of mild photothermal therapy (≤45℃) with low toxicity and minimal invasiveness, this invention significantly improves the thoroughness of tumor killing, effectively solving the problems of poor tumor repair, low efficacy, and easy recurrence in traditional mild photothermal therapy.

[0029] 3. To achieve synergistic effects of multimodal tumor treatment and improve the overall effectiveness of tumor treatment.

[0030] This invention further constructs a multifunctional in-situ vaccine platform integrating photothermal physical killing, drug-targeted inhibition, and antigen-immune activation. The three mechanisms work synergistically: Au@MSNs (1:3) generate a mild photothermal effect under 808 nm laser irradiation, directly killing tumor cells and inducing immunogenic cell death to release tumor antigens; the HSP90 inhibitor loaded in the mesoporous channels precisely blocks the HSP90 self-repair pathway after thermal damage to tumor cells; the released tumor antigens further activate a specific anti-tumor immune response. Photothermal killing directly reduces tumor size, drug inhibition blocks repair, and immune activation clears residual and metastatic lesions. These three mechanisms work synergistically and complementarily, overcoming the limitations of single-treatment modalities such as limited efficacy, easy drug resistance, and high recurrence rates, significantly improving the overall efficacy of tumor treatment. Animal experimental results show that the tumor volume in the G-Au@MSNs+L group was significantly smaller than in other groups ( p The result of <0.01 verifies the synergistic effect of the integrated platform. Attached Figure Description

[0031] 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.

[0032] Figure 1 Morphology of Au@MSNs with different silicon source to chloroauric acid volume ratios.

[0033] Figure 2 a: UV-Vis absorbance of Au@MSNs at 808 nm with different silicon source to chloroauric acid volume ratios; b: Heating temperature of Au@MSNs after irradiation with 808 nm laser (1.0 W / cm², 5 min) with different silicon source to chloroauric acid volume ratios.

[0034] Figure 3 CRT immunohistochemical staining images of tumor tissues in vivo after administration of Au@MSNs in a B16F10 mouse model at different ratios; a: Au@MSNs (1:1); b: Au@MSNs (1:2); c: Au@MSNs (1:3); d: Au@MSNs (1:4).

[0035] Figure 4 a: Temperature rise curves of the optimal Au@MSNs at different powers; b: Temperature rise curves of the optimal Au@MSNs at different concentrations; c: Photothermal stability of the optimal Au@MSNs; d: Near-infrared thermal images of NS, MSNs and the optimal Au@MSNs after laser irradiation.

[0036] Figure 5 Expression of HSP90 protein in B16F10 cells after treatment with different formulations.

[0037] Figure 6 Changes in tumor volume in mice after treatment with different formulations. Detailed Implementation

[0038] 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.

[0039] 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.

[0040] Example 1: Preparation and characterization of Au@MSNs with different silicon-gold ratios 1. Preparation of Au@MSNs with different silicon-gold ratios Au@MSNs were synthesized via a one-pot sol-gel method. 0.5 g of CTAC was mixed with 0.5 mL of 10% (w / v, g / 100 mL) sodium hydroxide (NaOH) 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, different volumes (1 mL, 2 mL, 3 mL, 4 mL) of 1 w / v HAuCl4 solution were added dropwise, and stirring continued for 20 min. Then, 0.5 mL of TEOS and 0.5 mL of BTESPD were mixed in 2 mL of ethanol and added dropwise to the above mixture (the volume ratio of silicon source to chloroauric acid was 1:1, 1:2, 1:3, 1:4, corresponding to a silicon-to-gold molar ratio of approximately 150:1, 75:1, 50:1, and 37.5:1). The reaction solution was stirred for another 2 h to obtain gold-core silica (Au@SiO2) solutions with different silicon-to-gold ratios. The products were 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 with different silicon-to-gold ratios.

[0041] 2. Morphological characteristics The morphology of Au@MSNs with different silicon-gold ratios was observed using transmission electron microscopy (TEM). Figure 1The results showed that the gold nuclei gradually increased in size with increasing HAuCl4 content. At silicon-to-gold ratios of 1:1, 1:2, 1:3, and 1:4, the gold nuclei were 2 nm, 10 nm, 50 nm, and 80 nm, respectively. Notably, the Au@MSNs obtained at ratios of 1:1, 1:2, and 1:3 exhibited rounded morphology, uniform particle size, and an overall particle size of approximately 90 nm. In contrast, the Au@MSNs (1:4) showed irregular morphology and severe adhesion. Figure 1 ).

[0042] Example 2: Evaluation of the temperature rise capability of Au@MSNs with different silicon-to-gold ratios To screen the photothermal properties of Au@MSNs, the absorbance of Au@MSNs with different silicon-to-gold ratios at 808 nm was first measured using a UV-Vis spectrophotometer. The results are as follows: Figure 2 The results showed that, when evaluating the ultraviolet absorption at 808 nm, there was no statistically significant difference in the ultraviolet absorption of Au@MSNs(1:1) and Au@MSNs(1:2) at 808 nm compared to the MSNs group (both <0.2). p >0.05); while the UV absorption of Au@MSNs(1:3) and Au@MSNs(1:4) at 808 nm was significantly increased, at 0.59±0.05 and 0.71±0.04 respectively (compared to the MSNs group, p <0.001).

[0043] Furthermore, the Au@MSNs combinations were dispersed in PBS (100 μg / mL) and subjected to an 808 nm near-infrared (NIR) laser at a power of 1.0 W / cm². 2 Irradiated for 5 minutes under the specified conditions. Temperatures were recorded using a digital thermometer at predetermined time points. Results are as follows: Figure 2 b shows that the temperatures of the Au@MSNs(1:1) and Au@MSNs(1:2) groups after laser irradiation were not significantly higher than those of the MSNs group, while the temperatures of the Au@MSNs(1:3) and Au@MSNs(1:4) groups were significantly higher, reaching 44.67±0.84℃ and 52.40±0.92℃ respectively (compared to the MSNs group). p < 0.001).

[0044] Example 3: Evaluation of the immune initiation ability of Au@MSNs with different silicon-gold ratios Using melanoma as a model, a subcutaneous tumor model bearing B16F10 was constructed in mice. When the tumor volume reached approximately 100 mm³, mice were randomly divided into 5 groups (n=5 per group), receiving intratumoral injections of PBS (NS group), Au@MSNs (1:1 group), Au@MSNs (1:2 group), Au@MSNs (1:3 group), and Au@MSNs (1:4 group) (the Au@MSNs concentration in each group was 100 μg / mL, and the injection volume was 50 μL). Four hours after injection, the tumor was irradiated locally with an 808 nm laser (1.0 W / cm²) for 10 min. Twelve hours after irradiation, the mice were sacrificed, and tumor tissue was collected, fixed with 4% paraformaldehyde, and prepared into paraffin sections. Immunohistochemical staining was used to detect the outward expression of calreticulin (CRT) on the surface of tumor cells to evaluate the immune initiation capacity after in vivo administration of different proportions of Au@MSNs.

[0045] The results are as follows Figure 3 As shown, CRT expression in tumor tissues of the Au@MSNs (1:1) and Au@MSNs (1:2) groups was weak, with no significant difference compared to the NS group; however, CRT expression in tumor tissues of the Au@MSNs (1:3) and Au@MSNs (1:4) groups was significantly enhanced, indicating that these two groups could effectively induce immunogenic cell death (ICD) in tumor cells, promoting the translocation of CRT from the endoplasmic reticulum to the cell membrane surface. Further systematic evaluation was conducted based on morphological characteristics, dispersion stability, and photothermal conversion ability: Although Au@MSNs (1:4) exhibited the strongest photothermal conversion ability (52.40℃), TEM results showed that its morphology was irregular and heavily adhered, which was not conducive to stable in vivo delivery; Au@MSNs (1:3) had a rounded morphology, uniform particle size (90nm), good dispersibility, and suitable photothermal conversion performance (44.67℃). Therefore, Au@MSNs (1:3) was selected as the optimal silicon-gold ratio for subsequent experiments.

[0046] Example 4: Comprehensive evaluation of the photothermal performance of Au@MSNs (1:3) with the optimal silicon-to-gold ratio Based on the screening results of Example 3, Au@MSNs (1:3) was the optimal ratio. Further comprehensive evaluation of its photothermal performance was conducted, including the irradiation power of near-infrared laser, the concentration of Au@MSNs, and the photothermal cycling stability.

[0047] 1. Laser power dependence experiment Au@MSNs (1:3) were dispersed in PBS (100 μg / mL) and irradiated for 10 minutes with an 808 nm laser at 0.1, 0.6, 1.0, and 2.0 W / cm², respectively. Temperature changes were recorded using a digital thermometer, and thermal images were captured using an infrared thermal imager. The results are as follows: Figure 4As shown in Figure a, the temperature increases with increasing laser power: at 0.1, 0.6, 1.0, and 2.0 W / cm². 2 Under laser irradiation, the temperature increased from approximately 27°C to approximately 31.7°C, 39.0°C, 45.5°C, and 56.1°C, respectively.

[0048] 2. Concentration-dependent experiment Subsequently, the photothermal conversion efficiency of Au@MSNs at different concentrations was further determined. Au@MSNs at different concentrations (25, 50, 100, 200 μg / mL) (1:3) were dispersed in PBS and irradiated with an 808 nm laser at 1.0 W / cm² for 10 minutes. The results are as follows: Figure 4 As shown in b, the temperature increases with increasing Au@MSNs concentration: at concentrations of 25, 50, 100, and 200 μg / mL, the temperature rises from approximately 27 °C to approximately 32.4 °C, 37.6 °C, 45.3 °C, and 54.3 °C, respectively. These results indicate that the photothermal conversion efficiency is positively correlated with the power of the 808 nm laser and the concentration of Au@MSNs.

[0049] 3. Photothermal stability experiment At 1.0 W / cm 2 Au@MSNs (100 μg / mL) were subjected to three laser on / off cycles of irradiation (10 minutes each time, followed by natural cooling to initial temperature) under the same conditions. The results are as follows: Figure 4 As shown in Figure 4c, after three cycles, no significant change was observed in the photothermal conversion efficiency of Au@MSNs, indicating that it has good photothermal stability (Figure 4c).

[0050] 4. Infrared thermal imaging verification Mice were randomly divided into three groups: the NS group (intratumoral injection of saline), the MSNs group (intratumoral injection of blank mesoporous silica nanoparticles), and the Au@MSNs group (intratumoral injection of Au@MSNs (1:3)). All three groups were irradiated with an 808 nm laser (1.0 W / cm²) for 5 minutes, and temperature changes were visually detected using infrared thermography. The results are as follows: Figure 4 As shown in d, the NS group and MSNs group at 1.0 W / cm 2 After continuous irradiation with an 808nm laser for 5 minutes, the temperature did not change significantly, while the temperature of Au@MSNs (1:3) increased significantly under the same conditions, consistent with the results recorded by the digital thermometer.

[0051] The above results indicate that Au@MSNs with a silicon-to-gold ratio of 1:3 possess excellent photothermal conversion performance and photothermal stability. Their heating effect can be controlled by adjusting the laser power and material concentration, making them suitable as effective photosensitizers for photothermal therapy.

[0052] Example 5: Evaluation of the ability of G-Au@MSNs to inhibit HSP90 protein expression Upregulation of HSP90 heat shock protein is a major cause of failure in mild photothermal therapy. To block the self-repair pathway of tumor cells due to thermal damage, this invention further loaded the HSP90 inhibitor galdromycin (GA) into Au@MSNs (1:3) with an optimal silicon-gold ratio and evaluated its inhibitory effect on HSP90 protein expression in tumor cells.

[0053] 1. Preparation of G-Au@MSNs GA (10 mg) and Au@MSNs (1:3, 50 mg) were dissolved together in 10 mL of chloroform. The mixture was magnetically stirred at room temperature until the chloroform was completely evaporated, forming a GA-loaded nanocomposite film. 10 mL of deionized water was added, and the film was sonicated for 10 minutes to disperse the GA, yielding GA-loaded Au@MSNs (G-Au@MSNs), which were stored at 4 °C for later use. The concentration of free GA in the supernatant was determined by high-performance liquid chromatography (HPLC), and the encapsulation efficiency and drug loading of GA were calculated. The results showed that the encapsulation efficiency of GA in G-Au@MSNs was 87.6 ± 3.2%, and the drug loading was 8.9 ± 1.1% (w / w).

[0054] 2. Western blot detection of HSP90 protein expression B16F10 cells were seeded in 6-well plates (2 × 10⁻⁶ cells per well). 5 (1 well / well), after overnight incubation, divided into the following 6 groups (3 replicates per group): Control group: equal volume of PBS; Au@MSNs+L group: Au@MSNs (1:3, 100 μg / mL) + 808 nm laser irradiation (1.0 W / cm², 5 min) GA group: Free GA (final GA concentration 2 μg / mL); Au@MSNs+GA+L group: Au@MSNs (1:3, 100 μg / mL) + free GA (2 μg / mL) + 808 nm laser irradiation (1.0 W / cm², 5 min); G-Au@MSNs+L group: G-Au@MSNs (containing 100 μg / mL Au@MSNs and 2 μg / mL GA) + 808 nm laser irradiation (1.0 W / cm², 5 min); After treatment, each group was cultured for 24 hours at 37℃ in a 5% CO2 incubator. Cells were collected, and total protein was extracted using RIPA lysis buffer. Protein concentration was determined by the BCA method. An equal amount of protein (30 μg / well) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride (PVDF) membrane, blocked with 5% skim milk for 1 hour, and incubated overnight at 4℃ with anti-HSP90 primary antibody (1:1000) and anti-GAPDH internal control antibody (1:5000). The next day, horseradish peroxidase (HRP)-labeled secondary antibody (1:5000) was added, and the cells were incubated at room temperature for 1 hour. The cells were then developed using ECL chemiluminescence immunoassay, and images were acquired and analyzed using a gel imaging system.

[0055] 3. Results The results are as follows Figure 5 As shown: HSP90 expression was at a basal level in the Control group; HSP90 expression was significantly increased in the Au@MSNs+L group, indicating that mild photothermal stimulation induced tumor cells to initiate heat shock protein-mediated self-repair; HSP90 expression was suppressed in the GA group; HSP90 expression was significantly lower in the Au@MSNs+GA+L group than in the Au@MSNs+L group, indicating that free GA can partially block laser-induced HSP90 upregulation; the HSP90 expression level in the G-Au@MSNs+L group was the lowest among all treatment groups, indicating that G-Au@MSNs can efficiently deliver GA into cells and precisely block mild photothermal-induced HSP90 upregulation, and G-Au@MSNs has the best HSP90 protein inhibition ability.

[0056] Example 6: Evaluation of the in vivo antitumor efficacy of G-Au@MSNs in situ vaccine A C57BL / 6 mouse model of subcutaneous B16F10 tumor-bearing mice was established to evaluate the in vivo therapeutic effect of G-Au@MSNs (Figure 6). 1×10 6 One B16F10 tumor cell was subcutaneously inoculated into the right back of a mouse. When the tumor volume reached approximately 100 mm³, the mice were randomly divided into 3 groups (n=5 per group): NS: Inject an equal volume of normal saline into the tumor; Au@MSNs+L group: Au@MSNs (1:3, 100 μg / mL, 50 μL) were injected intratumorally, and irradiated with 808 nm laser (1.0 W / cm²) for 10 minutes 4 hours after injection; G-Au@MSNs+L group: Intratumoral injection of G-Au@MSNs (containing Au@MSNs 100 μg / mL and GA 2 μg / mL, 50 μL), followed by irradiation with 808 nm laser (1.0 W / cm²) for 10 minutes 4 hours after injection; The tumor's long diameter (L) and short diameter (W) are measured every two days using electronic calipers, according to the formula V = L × W. 2 / 2 Calculate tumor volume and plot tumor growth curve ( Figure 6 ) The results are as follows Figure 6 As shown, tumors in the NS group exhibited rapid and progressive growth; tumor growth in the Au@MSNs+L group was somewhat inhibited compared to the NS group, but the therapeutic effect was limited, and the tumor volume still showed an increasing trend. This is related to the upregulation of HSP90 expression in tumor cells and the initiation of self-repair after thermal damage following mild photothermal stimulation; the tumor volume in the G-Au@MSNs+L group was significantly smaller than that in the other groups. p < 0.01), demonstrating the optimal anti-tumor effect. These results indicate that Au@MSNs combined with mild photothermal therapy can induce tumor cell damage and antigen release; however, due to the HSP90-mediated self-repair mechanism, a single mild photothermal treatment is insufficient to achieve complete tumor killing. G-Au@MSNs, by delivering GA to block the HSP90 repair pathway, effectively overcome the tolerance deficiency of mild photothermal therapy, significantly enhancing the anti-tumor efficacy of the in situ vaccine, and validating the therapeutic potential of mild photothermal therapy combined with HSP90 inhibitors.

[0057] 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. A method for screening the silicon-to-gold ratio for in situ tumor vaccines, characterized in that, Includes the following steps: Gold-core mesoporous silica nanoparticles with different silicon-to-gold ratios were prepared. Using immunogenic cell death markers of tumor cells as evaluation indicators, the immune initiation ability of gold-core mesoporous silica nanoparticles with different silicon-to-gold ratios was evaluated. Based on the results of the immune initiation capability evaluation, and combined with the structural stability and photothermal conversion efficiency of the gold-core mesoporous silica nanoparticles with different silicon-gold ratios, the optimal silicon-gold ratio was screened and determined.

2. The silicon-gold ratio screening method according to claim 1, characterized in that, The preparation of gold-core mesoporous silica nanoparticles with different silicon-to-gold ratios includes: A one-pot sol-gel method was used to react a fixed amount of silicon source with different volumes of chloroauric acid solution, resulting in silicon source to chloroauric acid volume ratios of 1:1, 1:2, 1:3, and 1:4, respectively.

3. The silicon-gold ratio screening method according to claim 1, characterized in that, The immunogenic cell death marker is calreticulin; the evaluation of immune initiation capacity is achieved by immunohistochemical staining of calreticulin in in vivo tumor tissue.

4. The silicon-gold ratio screening method according to claim 1, characterized in that, The screening process to determine the optimal silicon-gold ratio specifically includes: (1) The absorbance of gold core mesoporous silica nanoparticles with different silicon-gold ratios was measured at 808 nm by UV-Vis spectrophotometer, and the proportions with absorbance <0.2 were excluded. (2) The morphology and dispersion state of the gold core mesoporous silica nanoparticles with different silicon-gold ratios were observed by transmission electron microscopy, and the proportions with irregular morphology and severe adhesion were excluded. (3) The proportion of calreticulin with the strongest outward folding was screened by evaluating the immune activation ability.

5. The silicon-gold ratio screening method according to claim 1, characterized in that, The optimal silicon-to-gold ratio is 1:

3.

6. The silicon-gold ratio screening method according to claim 5, characterized in that, The gold-core mesoporous silica nanoparticles with a silicon-to-gold ratio of 1:3 have a gold core particle size of 50 nm and an overall particle size of 90±10 nm. They are heated to 44.67±0.84℃ under irradiation with an 808 nm laser and a power density of 1.0 W / cm².

7. A gold-core mesoporous silica nanoparticle, characterized in that, The volume ratio of silicon source to chloroauric acid in the gold-core mesoporous silica nanoparticles is 1:

3.

8. A mild photothermal in situ vaccine, characterized in that, include: The gold-core mesoporous silica nanoparticles as described in claim 7; And an HSP90 inhibitor loaded in the mesoporous channels of the gold-core mesoporous silica nanoparticles.

9. The mild photothermal in situ vaccine according to claim 8, characterized in that, The HSP90 inhibitor is one or more of the following: galdromycin, 17-allylaminogaldromycin, 17-dimethylaminoethylaminogaldromycin, and gambogeylic acid.

10. The use of a mild photothermal in situ vaccine as described in claim 8 or 9 in the preparation of an antitumor drug.

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