Preparation method and application of photodynamic-chemotherapy nano platform targeting tumor endogenous YAP1

Abstract

The invention relates to the technical field of medicine, in particular to a preparation method and application of a photodynamic-chemotherapy nano platform targeting tumor endogenous YAP1. The nano platform is hollow mesoporous silica nanoparticles (M (at) O-VNPs) loaded with oxaliplatin and VP of a macrophage membrane coating. The preparation method comprises the following steps: S1, synthesizing hollow mesoporous silica nanoparticles; s2, preparing the organic silicon nanoparticles loaded with the Verteporfin, and preparing the organic silicon nanoparticles loaded with the Verteporfin; s3, loading of the oxaliplatin; and S4, extraction and coating of a macrophage membrane. According to the invention, the macrophage membrane wrapped on the surface of the nano platform is utilized to provide good tumor targeting for the M (at) O-VNPs, and by camouflaging the macrophage membrane and the M (at) O-VNPs, not only are recognition and removal of an organism immune system successfully avoided, but also Verteporfin (VP) and oxaliplatin (OXA) can be delivered to a tumor site in a targeting manner, so that the tumor targeting of the drug is remarkably enhanced, and the drug has a good application prospect. The non-specific distribution in healthy tissues is reduced, and the systemic toxicity is reduced.

Description

Technical Field

[0001] The patent of this invention relates to the field of medical technology, and specifically, to a preparation method and application of a photodynamic-chemotherapy nanoplatform targeting endogenous YAP1 in tumors. Background Art

[0002] The treatment of gastric cancer (GC) still faces many challenges, especially in immune checkpoint inhibition (ICB) therapy, which has limited efficacy compared with other solid tumors. In the prior art, ICB therapy aims to restore the body's immune surveillance function against tumors by blocking the interaction between cancer cells and the immunosuppressive environment. However, due to the complex and suppressive tumor microenvironment (TME) of gastric cancer, many patients respond poorly to ICB therapy. Immunosuppressive cells such as regulatory T cells (Tregs), tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment further exacerbate this problem, leading to a weakened immune response. In addition, cancer cells enhance their ability to escape the immune system by changing their immunogenic characteristics, limiting the efficacy of ICB therapy.

[0003] YAP1 (Yes-Associated Protein 1), as a key regulator of the Hippo signaling pathway, has an important impact on tumor progression and immune escape. In tumors, overexpression of YAP1 is closely related to cell proliferation, inhibition of apoptosis, and promotion of metastasis, which directly promotes the occurrence and malignant progression of tumors. In addition, YAP1 also regulates the tumor microenvironment, promotes the recruitment of immunosuppressive cells such as SPP1+ macrophages and Tregs, and inhibits the function of CD8+ effector T cells, thereby weakening the effect of ICB therapy. Therefore, YAP1 is not only a driver of tumor growth, but also plays a core role in the immune escape process of tumors. Targeted inhibition of YAP1 has become an important strategy to improve the efficacy of ICB therapy.

[0004] Verteporfin (VP) is an effective YAP1 inhibitor that inhibits YAP1 activity by blocking the binding of YAP1 to TEAD. However, it is difficult to induce tumor regression significantly when using Verteporfin alone, probably because it only inhibits YAP1-related signaling pathways and fails to fully address the complex driving mechanism of tumors. Therefore, Verteporfin often needs to be used in combination with other anti-tumor drugs to enhance its efficacy. Oxaliplatin (OXA) is a classic platinum chemotherapy drug that is widely used in the treatment of various solid tumors including gastric cancer. Its main mechanism is to bind to tumor cell DNA to form platinum-DNA adducts, hinder DNA replication and transcription, and ultimately lead to tumor cell apoptosis. In addition, oxaliplatin also promotes the release of damage-associated molecular patterns (DAMPs) such as HMGB1, ATP, and calreticulin (CRT) by tumor cells by inducing immunogenic cell death (ICD), activating the immune system, and enhancing anti-tumor immune responses. Therefore, oxaliplatin can not only directly kill tumor cells, but also eliminate residual tumor cells through immune activation.

[0005] The combined use of verteporfin and oxaliplatin demonstrates many significant synergistic advantages. Verteporfin generates reactive oxygen species (ROS) through photodynamic action, which enhances the cytotoxicity of oxaliplatin and makes tumor cells more vulnerable under oxidative stress. At the same time, oxaliplatin further activates the patient's immune system by inducing ICD and enhances immune surveillance of tumors. This synergistic mechanism can not only accelerate the apoptosis of tumor cells, but also achieve a more lasting anti-tumor effect by enhancing the immune response. In addition, YAP1 inhibition can increase the sensitivity of tumors to chemotherapeutic drugs and further improve the efficacy of oxaliplatin. The combined use of the two not only enhances the direct killing effect of tumor cells, but also further enhances the anti-cancer effect by activating the immune system.

[0006] However, both Verteporfin and Oxaliplatin have significant side effects. Verteporfin is prone to skin phototoxicity under light conditions, and systemic application may cause liver toxicity, pulmonary fibrosis and renal impairment. Oxaliplatin mainly causes neurotoxicity and gastrointestinal reactions, such as numbness of the hands and feet, nausea and vomiting, which limits its dosage. These side effects not only affect the quality of life of patients, but also limit the therapeutic effects of the two drugs. Therefore, when Verteporfin and Oxaliplatin are used in combination, how to improve the targeting of the two is particularly critical. Drugs that lack targeting will not only affect healthy tissues during systemic distribution, but also increase the risk of toxicity and weaken the effectiveness of the drug. Therefore, the development of a drug delivery system that can be specifically delivered to the tumor microenvironment is an important way to improve the therapeutic effect and reduce toxicity. By accurately delivering drugs to the tumor site, it can not only enhance the lethality of drugs to cancer cells, but also reduce the accumulation of drugs in normal organs and reduce adverse reactions.

[0007] To this end, the present application provides a preparation method and application of a photodynamic-chemotherapy nanoplatform targeting endogenous YAP1 in tumors to solve the above problems. Summary of the invention

[0008] The purpose of the present invention is to solve the technical problems raised in the above-mentioned background technology and to provide a preparation method and application of a photodynamic-chemotherapy nanoplatform targeting endogenous YAP1 in tumors.

[0009] The above-mentioned purpose of the present invention is achieved like this:

[0010] One aspect of the present invention provides a method for preparing a photodynamic-chemotherapy nanoplatform targeting endogenous YAP1 in tumors, wherein the photodynamic-chemotherapy nanoplatform targeting endogenous YAP1 in tumors is hollow mesoporous silica nanoparticles (M@O-VNPs) loaded with oxaliplatin and VP and coated with macrophage membranes, and the preparation method comprises the following steps:

[0011] S1. Synthesis of hollow mesoporous silica nanoparticles:

[0012] First, 20 g of hexadecyltrimethylammonium chloride aqueous solution was mixed with 3.5 g of triethanolamine and stirred at 80° C. for 15 minutes;

[0013] Subsequently, 1 mL of tetraethoxysilane (TEOS) was added dropwise and reacted for 1 h;

[0014] Next, a mixture of 0.5 mL of TEOS and 1 mL of bis(triethoxysilyl)vinyl was added and the reaction was continued for 4 h;

[0015] After the reaction is completed, the product is washed with ethanol several times and then dispersed in 30 mL of methanol containing 1% sodium chloride to remove the template agent; the extraction step is repeated at least three times, each time for 12 hours, to ensure that the template agent is completely removed; the finally obtained mesoporous silica nanoparticles are selectively etched by ammonia water, reacted at 95°C for 3 hours, and washed with ethanol several times to obtain hollow mesoporous silica nanoparticles;

[0016] S2. Preparation of Verteporfin-loaded organosilicon nanoparticles:

[0017] OPSS-PEG-NH2 and carbodiimide were stirred in 2 mL of dimethyl sulfoxide at a molar ratio of 1:1 for 3 h, and 0.2 mL of triethylamine was added to activate the carboxyl groups;

[0018] Next, Verteporfin dissolved in 3 mL of DMSO and an additional 0.2 mL of TEA were added and stirred in the dark for 24 h to form OPSS-PEG-VP; 15 mg of hollow mesoporous silica nanoparticles were dispersed in 25 mL of ethanol, 0.15 mL of 3-mercaptopropyltriethoxysilane (MPTMS) and 0.2 mL of ammonia were added, and stirred overnight to generate thiolated hollow mesoporous silica nanoparticles (HMSN-SH);

[0019] OPSS-PEG-VP and 15 mg of HMSN-SH were stirred in 25 mL of ethanol in the dark overnight to finally obtain Verteporfin-loaded hollow mesoporous silica nanoparticles (VHMSNs), which were collected by centrifugation and vacuum drying;

[0020] S3. Oxaliplatin loading:

[0021] First, oxaliplatin was activated to ensure that it was positively charged; 10 mg of oxaliplatin and 10 mg of silver nitrate (AgNO3) were added to 10 ml of distilled water;

[0022] Subsequently, 100 μL of 5% HNO3 solution was added to the mixture to make the pH value <2; then the mixture was wrapped with aluminum foil and stirred at 80°C overnight to ensure that the silver ions reacted with the chloride ions in oxaliplatin;

[0023] Then, the AgCl precipitate was removed by centrifugation at 4000 rpm for 15 min; 4 mg of activated oxaliplatin was then mixed with 10 mg of VHMSN loaded with VP and subjected to ultrasonic treatment under alkaline conditions, followed by stirring in the dark for 24 h to form hollow mesoporous silica nanoparticles (O-VNPs) loaded with oxaliplatin and VP;

[0024] Then, the nanoparticles were subsequently passed through 0.45 μm and 0.22 μm filters, washed by centrifugation, and redispersed in phosphate buffer for later use;

[0025] S4. Extraction and coating of macrophage membranes:

[0026] Macrophage membranes were extracted from pretreated THP-1 cells or RAW 264.7 cells using a membrane protein extraction kit, according to the manufacturer's instructions; the extracted membrane proteins were sonicated on ice for 30 min, then filtered through 0.45 μm and 0.22 μm filters in sequence and quantified by the BCA method;

[0027] O-VNPs were mixed with macrophage membranes in a ratio of 1:2, sonicated in a water bath for 30 min, and then incubated with stirring at 4°C for 4 h; the resulting macrophage membrane-coated hollow mesoporous silica nanoparticles (M@O-VNPs) loaded with oxaliplatin and VP were collected by centrifugation.

[0028] Furthermore, the pH under the alkaline condition in step S3 is 8.5.

[0029] Furthermore, the surface protein retention of the macrophage membrane-coated hollow mesoporous silica nanoparticles (M@O-VNPs) loaded with oxaliplatin and VP obtained in step S4 was verified by SDS-PAGE gel electrophoresis and Coomassiebrilliant blue staining; and the presence of CD68 and CD86 macrophage membrane markers was detected by Western blot to demonstrate the successful coating of the macrophage membrane.

[0030] Another aspect of the scheme of the present invention also provides a method for preparing a photodynamic-chemotherapy nanoplatform targeting endogenous YAP1 in tumors, and uses the prepared hollow mesoporous silica nanoparticles (O-VNPs) loaded with oxaliplatin and VP in the preparation of anti-tumor drugs.

[0031] Another aspect of the present invention also provides a method for preparing a photodynamic-chemotherapeutic nanoplatform targeting endogenous YAP1 in tumors, and use of the prepared photodynamic-chemotherapeutic nanoplatform targeting endogenous YAP1 in tumors in the preparation of anti-tumor drugs.

[0032] The difficulty and significance of the technical problem solved by the present invention are:

[0033] Both Verteporfin and Oxaliplatin have significant side effects, which not only affect the quality of life of patients, but also limit the therapeutic effects of the two drugs. The key point is to improve the targeting of Verteporfin and Oxaliplatin in combination. At present, hollow mesoporous silica nanoparticles (HMSN) disguised by macrophage membranes show great advantages. Macrophages naturally have tumor tropism and can actively migrate to the immunosuppressive area in the tumor microenvironment. By disguising the macrophage membrane, nanoparticles can avoid being cleared by the immune system and target the tumor site. The structure of hollow mesoporous silica provides good drug loading capacity and drug release control, and can accurately release drugs under the stimulation conditions of the tumor microenvironment (such as high concentrations of glutathione), thereby ensuring that the effective concentration of drugs acts on tumor cells and reduces systemic side effects. Through research, the present invention aims to provide a drug loading system that can be specifically delivered to the tumor microenvironment, thereby improving the therapeutic effect and reducing toxicity, and providing an innovative strategy for anti-tumor treatment.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1. In the scheme of the present invention, the macrophage membrane wrapped on the surface of the nanoplatform provides M@O-VNPs with good tumor targeting, which is attributed to the natural tumor tropism of macrophages, which can recognize and migrate to the tumor microenvironment; by disguising the macrophage membrane, M@O-VNPs not only successfully avoid the recognition and clearance of the body's immune system, but also can target the delivery of Verteporfin (VP) and oxaliplatin (OXA) to the tumor site, significantly enhancing the tumor targeting of the drug, reducing the nonspecific distribution in healthy tissues, and reducing systemic toxicity.

[0036] 2. In addition, the disulfide bond connecting VP is broken in tumor cells with high glutathione, which can accurately release and effectively inhibit the YAP1 signaling pathway in tumor cells, limit tumor cell proliferation, and reshape the tumor microenvironment by organizing immunosuppressive cells, enhance the infiltration of effector T cells, and promote anti-tumor immune response.

[0037] 3. Moreover, VP's inhibitory effect on YAP1 also enhances the efficacy of oxaliplatin and reduces tumor resistance to oxaliplatin. At the same time, the combined use of VP and oxaliplatin further enhances the anti-tumor efficacy through the dual immunogenic cell death (ICD) effect. VP produces reactive oxygen species (ROS) through photodynamic therapy (PDT), inducing tumor cell death and releasing immune activation signals, while oxaliplatin binds to DNA to induce ICD reactions, further activating the immune system. The dual ICD effect and inhibition of YAP1 not only enhance anti-tumor immunity, but also significantly improve the effect of immune checkpoint inhibitor therapy by reshaping the tumor microenvironment, reducing immunosuppressive cells, and increasing the activity of effector T cells. Therefore, the M@O-VNPs in the present invention combine targeted delivery, YAP1 inhibition, dual ICD effects, and tumor microenvironment remodeling, showing strong potential in tumor treatment, especially in promoting immunotherapy, and provide innovative strategies for future anti-tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 :A is the potential of various nanoparticles and macrophage membrane; B is the nitrogen adsorption curve of hollow mesoporous silica nanoparticles; C is the average pore size of hollow mesoporous silica nanoparticles; D is the ultraviolet absorption spectrum (VHMSN) of hollow mesoporous silica nanoparticles (HMSN), Verteporfin (VP) and hollow mesoporous silica nanoparticles loaded with VP; E is the transmission electron microscopy image and energy dispersive X-ray spectroscopy image of hollow mesoporous silica nanoparticles (O-VNPs) loaded with oxaliplatin and VP; F is the transmission electron microscopy image of hollow mesoporous silica nanoparticles (M@O-VNPs) loaded with oxaliplatin and VP wrapped in macrophage membrane; G is the SDS-PAGE gel after Coomassie Brilliant Blue staining; H is the marker of macrophage membrane in M@O-VNPs detected by Western-Blot;

[0039] Figure 2 is the cumulative release profile of Verteporfin (VP) and oxaliplatin in M@O-VNPs;

[0040] Figure 3 Middle: A is the detection of the inhibitory ability of M@O-VNPs on YAP1 in human gastric cancer cells HGC-27; B is the flow cytometry detection of the killing ability of M@O-VNPs on HGC-27 cells;

[0041] Figure 4 The induction of (A) immunogenic cell death (ICD) and (B) infiltration of effector T cells (CD8+GZMB+) in different treatment groups were detected in a spontaneous gastric cancer mouse model.

[0042] Figure 5 It is to detect tumor regression in different treatment groups in a spontaneous gastric cancer mouse model;

[0043] Figure 6 The organ damage in different treatment groups was examined in a spontaneous gastric cancer mouse model. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0045] The implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0046] With reference to the drawings, the following are preferred embodiments of the present invention.

[0047] Example 1: Preparation of a photodynamic-chemotherapy nanoplatform targeting endogenous YAP1 in tumors

[0048] Step 1: 20 g of hexadecyltrimethylammonium chloride (CTAC) aqueous solution was mixed with 3.5 g of triethanolamine (TEA) and stirred at 80 ° C for 15 minutes. Subsequently, 1 mL of tetraethoxysilane (TEOS) was added dropwise and reacted for 1 hour, and then a mixture of 0.5 mL of TEOS and 1 mL of bis(triethoxysilyl)vinyl (BTES) was added and the reaction continued for 4 hours. After the reaction, 30 ml of anhydrous ethanol was used to wash and centrifuged at 13000 rpm for 15 minutes, repeated 3 times, and then dispersed in 30 mL of methanol containing 1% sodium chloride, stirred for 12 hours, and then centrifuged at 13000 rpm for 15 minutes, repeated 3 times. Next, the product was transferred to a 95 ° C water bath, hot water etched for 3 hours with the assistance of ammonia to corrode the internal core of the nanoparticles, washed 3 times and freeze-dried to obtain hollow mesoporous silica nanoparticles (HMSN).

[0049] The particle size and potential were measured by a particle size and surface potential meter. The particle size of HMSN was 40.32±1.22nm and the potential was -27.25±0.93mV (as shown in Figure 2). Figure 1 The specific surface area and pore size of HMSN were measured by Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) methods, which were 364.89 m 2 / g and 3.78nm (such as Figure 1 B and C).

[0050] Step 2: OPSS-PEG-NH2 and carbodiimide (CDI) were dissolved in 2 mL of DMSO at a molar ratio of 1:1 and stirred at room temperature for 3 hours, while 0.2 mL of triethylamine (TEA) was added to activate the carboxyl group. Next, Verteporfin (VP) dissolved in 3 mL of DMSO and an additional 0.2 mL of TEA were added and stirred at room temperature for 24 hours in the dark to form OPSS-PEG-VP. 15 mg of hollow mesoporous silica nanoparticles (HMSN) were dispersed in 25 mL of anhydrous ethanol, 0.15 mL of 3-mercaptopropyltriethoxysilane (MPTMS) and 0.2 mL of ammonia were added, and stirred at room temperature overnight to generate thiolated hollow mesoporous silica nanoparticles (HMSN-SH). OPSS-PEG-VP and 15 mg of HMSN-SH were stirred in 25 mL of ethanol in the dark overnight to finally obtain hollow mesoporous silica nanoparticles loaded with VP (VHMSN), which were collected by centrifugation and vacuum drying.

[0051] The particle size and potential were measured by a particle size and surface potential meter. The particle size of VHMSN was 54.36±1.58nm and the potential was -23.66±0.64mV (e.g. Figure 1 A). The UV spectrophotometer measured that the VHMSN spectrum showed a significant absorption peak at 687nm, proving the successful connection of VP ( Figure 1 D) The loading rate of VP in VHMSN was determined to be 9.37% using HPLC.

[0052] Step 3: 10 mg of oxaliplatin (OXA) and 10 mg of silver nitrate (AgNO3) were added to 10 ml of ddH2O, and 100 μL of 5% HNO3 solution was added to the mixture. Then it was wrapped with aluminum foil and stirred overnight at 80°C. Then it was centrifuged at 4000 rpm for 15 minutes to remove the AgCl precipitate. Subsequently, 4 mg of activated oxaliplatin was mixed with 10 mg of VHMSN, and ultrasonically treated in HEPES buffer (pH 8.5), and then stirred in the dark for 24 hours to obtain hollow mesoporous silica nanoparticles (O-VNPs) loaded with oxaliplatin and VP. The nanoparticles were then sequentially passed through 0.45 μm and 0.22 μm filters, centrifuged and washed, and redispersed in PBS solution for use.

[0053] The particle size and potential were measured by a particle size and surface potential meter. The particle size of VHMSN was 56.95±0.61nm and the potential was 33.5±0.46mV (e.g. Figure 1 Energy dispersive X-ray spectroscopy and transmission electron microscopy were used to verify the elemental distribution (as shown in Figure 1). Figure 1E), the Pt element is enriched inside the nanoparticles, proving that oxaliplatin is successfully encapsulated into the VHMSN. In addition, the encapsulation rate of oxaliplatin is measured to be 16.73% using an inductively coupled plasma mass spectrometer (ICP-MS).

[0054] Step 4: Use a membrane protein extraction kit to extract macrophage membranes from pretreated THP-1 cells. First, after THP-1 cells were cultured in RIPA-1640 medium with phorbol ester for 48 hours, approximately 4×10 7 Cells were washed with PBS and scraped with a cell scraper. The cells were centrifuged at 1000 rpm for 5 minutes at 4°C and the supernatant was removed to collect the cells. Repeat the centrifugation to remove the residual liquid as much as possible. Then 1 ml of membrane protein extraction reagent with PMSF added was added to the cells and resuspended, and ice bathed for 15 minutes. The cells were then repeatedly frozen and thawed in liquid nitrogen and room temperature for 6 times, centrifuged at 1000 rpm for 5 minutes at 4°C, and the supernatant was collected. Then centrifuged at 12000 rpm for 30 minutes at 4°C to completely precipitate the cell membrane. The extracted cell membrane was sonicated on ice for 30 minutes, then filtered through 0.45μm and 0.22μm filters in turn and quantified by BCA method. O-VNPs were mixed with macrophage membranes in a ratio of 1:2, sonicated in an ice bath for 30 minutes, and then incubated with stirring at 4°C for 4 hours. The macrophage membrane-coated hollow mesoporous silica nanoparticles (M@O-VNPs) loaded with oxaliplatin and VP were collected by centrifugation.

[0055] The potential of M@O-VNPs was measured by a potentiometer to be -12.93±0.47 mV ( Figure 1 Transmission electron microscopy results showed that M@O-VNPs were spherical in shape and had a particle size of approximately 61 nm (as shown in Figure 2A). Figure 1 The membrane proteins on the surface of M@O-VNPs were verified by SDS-PAGE gel electrophoresis and Coomassie Brilliant Blue staining (as shown in Figure 1 G). In addition, the presence of macrophage membrane markers such as CD68 and CD86 was detected by Western blot to confirm the successful coating of macrophage membranes (as shown in Figure 1 H).

[0056] Example 2: Preparation of a photodynamic-chemotherapy nanoplatform targeting endogenous YAP1 in tumors

[0057] Step 1: 20 g of hexadecyltrimethylammonium chloride (CTAC) aqueous solution was mixed with 3.5 g of triethanolamine (TEA) and stirred at 80 ° C for 15 minutes. Subsequently, 1 mL of tetraethoxysilane (TEOS) was added dropwise and reacted for 1 hour, and then a mixture of 0.5 mL of TEOS and 1 mL of bis(triethoxysilyl)vinyl (BTES) was added and the reaction continued for 4 hours. After the reaction, 30 ml of anhydrous ethanol was used to wash and centrifuged at 13000 rpm for 15 minutes, repeated 3 times, and then dispersed in 30 mL of methanol containing 1% sodium chloride, stirred for 12 hours, and then centrifuged at 13000 rpm for 15 minutes, repeated 3 times. Next, the product was transferred to a 95 ° C water bath, hot water etched for 3 hours with the assistance of ammonia to corrode the internal core of the nanoparticles, washed 3 times and freeze-dried to obtain hollow mesoporous silica nanoparticles (HMSN).

[0058] The particle size and potential were measured using a particle size and surface potential meter, and the particle size of HMSN was 42.17±0.95nm and the potential was -28.33±1.39mV. The specific surface area and pore size of HMSN were measured using the Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) methods, which were 355.71m 2 / g and 4.03nm.

[0059] Step 2: OPSS-PEG-NH2 and carbodiimide (CDI) were dissolved in 2 mL of DMSO at a molar ratio of 1:1 and stirred at room temperature for 3 hours, while 0.2 mL of triethylamine (TEA) was added to activate the carboxyl group. Next, Verteporfin (VP) dissolved in 3 mL of DMSO and an additional 0.2 mL of TEA were added and stirred at room temperature for 24 hours in the dark to form OPSS-PEG-VP. 15 mg of hollow mesoporous silica nanoparticles (HMSN) were dispersed in 25 mL of anhydrous ethanol, 0.15 mL of 3-mercaptopropyltriethoxysilane (MPTMS) and 0.2 mL of ammonia were added, and stirred at room temperature overnight to generate thiolated hollow mesoporous silica nanoparticles (HMSN-SH). OPSS-PEG-VP and 15 mg of HMSN-SH were stirred in 25 mL of ethanol in the dark overnight to finally obtain hollow mesoporous silica nanoparticles loaded with VP (VHMSN), which were collected by centrifugation and vacuum drying.

[0060] The particle size and potential were measured by a microparticle size and surface potential meter, and the particle size of VHMSN was 52.50±0.78nm and the potential was -24.19±0.82mV. The VHMSN spectrum measured by UV spectrophotometer showed a significant absorption peak at 687nm, proving the successful connection of VP. The loading rate of VP in VHMSN was 8.85% by high performance liquid chromatography.

[0061] Step 3: 10 mg of oxaliplatin (OXA) and 10 mg of silver nitrate (AgNO3) were added to 10 ml of ddH2O, and 100 μL of 5% HNO3 solution was added to the mixture. Then it was wrapped with aluminum foil and stirred overnight at 80°C. Then it was centrifuged at 4000 rpm for 15 minutes to remove the AgCl precipitate. Subsequently, 4 mg of activated oxaliplatin was mixed with 10 mg of VHMSN, and ultrasonically treated in HEPES buffer (pH 8.5), and then stirred in the dark for 24 hours to obtain hollow mesoporous silica nanoparticles (O-VNPs) loaded with oxaliplatin and VP. The nanoparticles were then sequentially passed through 0.45 μm and 0.22 μm filters, centrifuged and washed, and redispersed in PBS solution for use.

[0062] The particle size and potential were measured by a particle size and surface potential measuring instrument, and the particle size of VHMSN was measured to be 56.95±0.61 nm and the potential was 34.5±0.91 mV. The encapsulation rate of oxaliplatin was measured by an inductively coupled plasma mass spectrometer (ICP-MS) to be 15.41%.

[0063] Step 4: Use a membrane protein extraction kit to extract macrophage membranes from pretreated THP-1 cells. First, after THP-1 cells were cultured in RIPA-1640 medium with phorbol ester for 48 hours, approximately 4×10 7Cells were washed with PBS and scraped with a cell scraper. The cells were centrifuged at 1000 rpm for 5 minutes at 4°C and the supernatant was removed to collect the cells. Repeat the centrifugation to remove the residual liquid as much as possible. Then 1 ml of membrane protein extraction reagent with PMSF added was added to the cells and resuspended, and ice bathed for 15 minutes. The cells were then repeatedly frozen and thawed in liquid nitrogen and room temperature for 6 times, centrifuged at 1000 rpm for 5 minutes at 4°C, and the supernatant was collected. Then centrifuged at 12000 rpm for 30 minutes at 4°C to completely precipitate the cell membrane. The extracted cell membrane was sonicated on ice for 30 minutes, then filtered through 0.45μm and 0.22μm filters in turn and quantified by BCA method. O-VNPs were mixed with macrophage membranes in a ratio of 1:2, sonicated in an ice bath for 30 minutes, and then incubated with stirring at 4°C for 4 hours. The macrophage membrane-coated hollow mesoporous silica nanoparticles (M@O-VNPs) loaded with oxaliplatin and VP were collected by centrifugation.

[0064] The potential of VHMSN was measured by a potentiometer to be -15.92±2.03mV. The membrane proteins on the surface of M@O-VNPs were verified by SDS-PAGE gel electrophoresis and Coomassie Brilliant Blue staining. In addition, the presence of macrophage membrane markers such as CD68 and CD86 was detected by Western blot, proving the successful coating of macrophage membranes.

[0065] Example 3: Preparation of a photodynamic-chemotherapy nanoplatform targeting endogenous YAP1 in tumors

[0066] Step 1: 20 g of hexadecyltrimethylammonium chloride (CTAC) aqueous solution was mixed with 3.5 g of triethanolamine (TEA) and stirred at 80 ° C for 15 minutes. Subsequently, 1 mL of tetraethoxysilane (TEOS) was added dropwise and reacted for 1 hour, and then a mixture of 0.5 mL of TEOS and 1 mL of bis(triethoxysilyl)vinyl (BTES) was added and the reaction continued for 4 hours. After the reaction, 30 ml of anhydrous ethanol was used to wash and centrifuged at 13000 rpm for 15 minutes, repeated 3 times, and then dispersed in 30 mL of methanol containing 1% sodium chloride, stirred for 12 hours, and then centrifuged at 13000 rpm for 15 minutes, repeated 3 times. Next, the product was transferred to a 95 ° C water bath, hot water etched for 3 hours with the assistance of ammonia to corrode the internal core of the nanoparticles, washed 3 times and freeze-dried to obtain hollow mesoporous silica nanoparticles (HMSN).

[0067] The particle size and potential were measured using a particle size and surface potential meter, and the particle size of HMSN was measured to be 44.69±0.75nm and the potential was -25.26±1.88mV. The specific surface area and pore size of HMSN were measured using the Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) methods, which were 366.94m 2 / g and 4.85nm.

[0068] Step 2: OPSS-PEG-NH2 and carbodiimide (CDI) were dissolved in 2 mL of DMSO at a molar ratio of 1:1 and stirred at room temperature for 3 hours, while 0.2 mL of triethylamine (TEA) was added to activate the carboxyl group. Next, Verteporfin (VP) dissolved in 3 mL of DMSO and an additional 0.2 mL of TEA were added and stirred at room temperature for 24 hours in the dark to form OPSS-PEG-VP. 15 mg of hollow mesoporous silica nanoparticles (HMSN) were dispersed in 25 mL of anhydrous ethanol, 0.15 mL of 3-mercaptopropyltriethoxysilane (MPTMS) and 0.2 mL of ammonia were added, and stirred at room temperature overnight to generate thiolated hollow mesoporous silica nanoparticles (HMSN-SH). OPSS-PEG-VP and 15 mg of HMSN-SH were stirred in 25 mL of ethanol in the dark overnight to finally obtain hollow mesoporous silica nanoparticles loaded with VP (VHMSN), which were collected by centrifugation and vacuum drying.

[0069] The particle size and potential were measured by a particle size and surface potential meter, and the particle size of VHMSN was 54.39±1.03nm and the potential was -26.21±0.55mV. The VHMSN spectrum measured by UV spectrophotometer showed a significant absorption peak at 687nm, proving the successful connection of VP. The loading rate of VP in VHMSN was 9.71% determined by high performance liquid chromatography.

[0070] Step 3: 10 mg of oxaliplatin (OXA) and 10 mg of silver nitrate (AgNO3) were added to 10 ml of ddH2O, and 100 μL of 5% HNO3 solution was added to the mixture. Then it was wrapped with aluminum foil and stirred overnight at 80°C. Then it was centrifuged at 4000 rpm for 15 minutes to remove the AgCl precipitate. Subsequently, 4 mg of activated oxaliplatin was mixed with 10 mg of VHMSN, and ultrasonically treated in HEPES buffer (pH 8.5), and then stirred in the dark for 24 hours to obtain hollow mesoporous silica nanoparticles (O-VNPs) loaded with oxaliplatin and VP. The nanoparticles were then sequentially passed through 0.45 μm and 0.22 μm filters, centrifuged and washed, and redispersed in PBS solution for use.

[0071] The particle size and potential were measured by using a particle size and surface potential measuring instrument, and the particle size of VHMSN was measured to be 55.12±0.74 nm and the potential was 30.38±1.15 mV. The encapsulation rate of oxaliplatin was measured by using an inductively coupled plasma mass spectrometer (ICP-MS) to be 14.35%.

[0072] Step 4: Use a membrane protein extraction kit to extract macrophage membranes from pretreated THP-1 cells. First, after THP-1 cells were cultured in RIPA-1640 medium with phorbol ester for 48 hours, approximately 4×10 7 Cells were washed with PBS and scraped with a cell scraper. The cells were centrifuged at 1000 rpm for 5 minutes at 4°C and the supernatant was removed to collect the cells. Repeat the centrifugation to remove the residual liquid as much as possible. Then 1 ml of membrane protein extraction reagent with PMSF added was added to the cells and resuspended, and ice bathed for 15 minutes. The cells were then repeatedly frozen and thawed in liquid nitrogen and room temperature for 6 times, centrifuged at 1000 rpm for 5 minutes at 4°C, and the supernatant was collected. Then centrifuged at 12000 rpm for 30 minutes at 4°C to completely precipitate the cell membrane. The extracted cell membrane was sonicated on ice for 30 minutes, then filtered through 0.45μm and 0.22μm filters in turn and quantified by BCA method. O-VNPs were mixed with macrophage membranes in a ratio of 1:2, sonicated in an ice bath for 30 minutes, and then incubated with stirring at 4°C for 4 hours. The macrophage membrane-coated hollow mesoporous silica nanoparticles (M@O-VNPs) loaded with oxaliplatin and VP were collected by centrifugation.

[0073] The potential of VHMSN was measured by a potentiometer to be -13.81±1.75mV. The membrane proteins on the surface of M@O-VNPs were verified by SDS-PAGE gel electrophoresis and Coomassie Brilliant Blue staining. In addition, the presence of macrophage membrane markers such as CD68 and CD86 was detected by Western blot, proving the successful coating of macrophage membranes.

[0074] Example 4: Application of the photodynamic-chemotherapy nanoplatform targeting endogenous YAP1 in an in vitro gastric cancer model

[0075] 1. In vitro drug release behavior of the photodynamic-chemotherapeutic nanoplatform targeting endogenous YAP1 in tumors

[0076] To simulate the physiological conditions in the tumor microenvironment, a phosphate buffer solution (PBS) of pH 6.5 was prepared. 10 mg of M@O-VNPs (Example 1) was dispersed in 10 ml of PBS (pH 6.5), 5 mM glutathione buffer solution (pH 6.5) and 10 mM glutathione buffer solution (pH 6.5), and 2 mL of the solution was placed in a dialysis bag (MWCO: 5.0 kDa), and then the dialysis bag was placed in a release tube containing an appropriate amount of release medium and oscillated at room temperature at a rate of 60 rpm. Samples were taken at predetermined time points (0, 1, 2, 4, 6, 9, 12, 24, 36, 48 hours), and the sampled medium was replaced with a fresh release medium. The Verteporfin and Oxaliplatin contents in the samples were determined by high performance liquid chromatography (HPLC) and inductively coupled plasma mass spectrometry (ICP-MS), and the cumulative release percentage of the drug was calculated.

[0077] The test results showed that the in vitro release behavior of Verteporfin in M@O-VNPs was as follows: Figure 2 As shown, the nanomedicine exhibited obvious glutathione response characteristics. After 48 hours, the cumulative release percentage of Verteporfin in pH 6.5 buffer was 20.93±2.10%, the cumulative release percentage in 5mM glutathione buffer was 68.12±2.36%, and the cumulative release percentage in 10mM glutathione buffer was 76.64±4.751%. The release rate of oxaliplatin was as follows: the cumulative release percentage in pH 6.5 PBS buffer was 42.39±2.879%, the cumulative release percentage in 5mM glutathione buffer was 60.93±3.550%, and the cumulative release percentage in 10mM glutathione buffer was 66.83±1.986%.

[0078] 2. The inhibitory behavior of the photodynamic-chemotherapy nanoplatform targeting endogenous YAP1 in tumors on YAP1 in vitro

[0079] First, the human gastric cancer cell line HGC-27 was inoculated in a 35 mm confocal culture dish, and PBS, HMSN, VP, photoactivated VP, M@O-VNPs, and photoactivated M@O-VNPs were added and incubated for 24 hours. Subsequently, the cells were rinsed three times with PBS buffer, and then fixed with 4% paraformaldehyde for 15 minutes, and then rinsed with PBS three times for 5 minutes each time. After that, the blocking solution containing 5% BSA and 0.4% Triton was incubated at room temperature for 1 hour, and then a 1:500 dilution of YAP1 primary antibody (ab205270, Abcam) was added and incubated at 4°C overnight. The next day, after rinsing with PBS three times for 5 minutes each time, donkey anti-rabbit Alexa Fluor488 fluorescent secondary antibody was added, incubated for 1 hour at room temperature in the dark, and then rinsed with PBS three times for 5 minutes each time. To label F-actin, 555 fluorescein-labeled phalloidin was further incubated for 1 hour. After the final three washes, DAPI-containing mounting medium was added for sealing, and laser scanning confocal microscopy was used for imaging and quantitative analysis.

[0080] Fluorescence images taken by laser confocal microscopy showed that VP, photoactivated VP, M@O-VNPs, and photoactivated M@O-VNPs all significantly inhibited the nuclear expression of YAP1 in HGC-27 cells ( Figure 3 A).

[0081] 3. Study on the apoptosis of gastric cancer cells in vitro using a photodynamic-chemotherapy nanoplatform targeting endogenous YAP1 in tumors

[0082] Iron-blocking magnetic nanoparticles responsive to tumor microenvironment were prepared according to the method of Example 2, and PBS was used as the control group. Flow cytometry was used to study the effects of oxaliplatin, VP combined with oxaliplatin, and M@O-VNPs on photoactivation (690 nm, 5 min, 100 mW / cm 2) and the killing effect of gastric cancer cells under non-photoactivated state. In all experimental groups, the concentration of VP was 5μM and that of oxaliplatin was 10μM. In a 6-well culture plate with a density of 2×105 HGC-27 gastric cancer cells / well, when the cell density reached about 70%, the above drugs were added for co-incubation for 24 hours. After the incubation, the culture fluid of each well was collected and stored in a centrifuge tube, the cells were washed once with PBS solution, then trypsin was added for digestion, and the digestion was terminated with the culture fluid collected previously. The collected cells were centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, the cells were resuspended with PBS and counted, 50,000 to 100,000 cells were centrifuged again, the supernatant was discarded, 195μL AnnexinV-FITC binding solution was added for resuspending, AnnexinV-FITC (5μL) and PI (10μL) were added in sequence, mixed thoroughly, incubated at room temperature in the dark for 20 minutes, and then detected by flow cytometry.

[0083] The results showed that the apoptosis rate of tumor cells in vitro in each group was as follows Figure 3 As shown in B. There was no significant difference in the apoptosis rate of cells treated with oxaliplatin alone with or without laser irradiation, which were 23.5% and 26.9%, respectively. After the addition of VP, laser irradiation significantly increased the apoptosis rate from 40.6% to 66.0%, indicating the synergistic tumor killing effect of oxaliplatin and VP under light-activated conditions. The same synergistic effect was also observed in the M@O-VNPs group, and the effect was more significant. The apoptosis rate without light irradiation was 45.9%, while it reached 83.5% after light irradiation. These results show that M@O-VNPs can kill gastric cancer cells more effectively in the light-activated state.

[0084] Example 5: Application of the photodynamic-chemotherapy nanoplatform targeting endogenous YAP1 in a spontaneous gastric cancer model in vivo

[0085] 1. Photodynamic-chemotherapy nanoplatform targeting endogenous YAP1 in tumors induces immunogenic cell death (ICD) of tumor cells in vivo

[0086] In this study, a conditional knockout C57BL / 6 mouse model Tff1-CreERT2; Apc was used. fl / fl ;p53 fl / fl(TcPP). At 8 weeks of age, mice were orally given 100 mg / kg of tamoxifen for a week-long induction treatment. After 3 months, gastric cancer formed in the stomach of the mice. Then, the mice were injected with PBS, HMSN, oxaliplatin combined with VP and M@O-VNPs through the tail vein, once every two days for 3 weeks. The injection doses were: oxaliplatin 5 mg / kg, VP 1 mg / kg, M@O-VNPs 10 mg / kg. At the same time, the oxaliplatin combined with VP group and the M@O-VNPs group were respectively exposed to 690 nm wavelength laser (10 minutes, 100 mW / cm 2 ) or no laser irradiation. After 3 weeks of treatment, mice were euthanized, gastric tumors were removed, paraffin-embedded formalin-fixed specimens were prepared, and the specimens were cut into 4 μm thick serial sections. After dewaxing, rehydration and antigen retrieval of the sections, they were repaired with citrate buffer (pH 6.0) at high pressure and high temperature for 5 minutes. After cooling, the sections were blocked with blocking solution containing 5% BSA at room temperature for 2 hours to prevent nonspecific binding. Subsequently, the sections were incubated with Calreticulin antibody (ab92516, Abcam) diluted 1:250 in a humidified chamber at 4°C overnight. After washing away the primary antibody, the sections were incubated with donkey anti-rabbit Alexa Fluor 647 fluorescent secondary antibody for 1 hour at room temperature. After that, the sections were treated with blocking solution containing 0.4% TritonX-100 for 1 hour, and then incubated with HMGB1 antibody (MA5-17278, Invitrogen) diluted 1:200 in a humidified chamber at 4°C overnight. After washing away the primary antibody, the sections were incubated with donkey anti-mouse Alexa Fluor 555 fluorescent secondary antibody for 1 hour at room temperature. Finally, Alexa Fluor 488-conjugated panCK antibody (1:200, 53-9003-82, Invitrogen) was used to label gastric cancer cells. After staining, DAPI-containing mounting medium was added to the sections, and the sections were imaged and analyzed using a laser confocal microscope.

[0087] result( Figure 4A) shows that the M@O-VNPs group after laser irradiation induced 71.3% of gastric cancer cells to undergo immunogenic cell death (ICD) effect, which was significantly higher than the 27.7% of the M@O-VNPs group without laser irradiation and the 24.9% of the laser irradiated VP combined with oxaliplatin group. For non-tumor cells, the M@O-VNPs group after laser irradiation only induced 16.0% of cells to undergo ICD effect, which was much lower than the 31.9% of the laser irradiated VP combined with oxaliplatin group. These results show that M@O-VNPs can target and induce the immunogenic cell death effect of tumor cells, thereby activating antigen presenting cells and triggering a strong anti-tumor immune response. Compared with the treatment regimen of VP combined with oxaliplatin, M@O-VNPs has less effect on non-tumor cells and shows better safety.

[0088] 2. The photodynamic-chemotherapy nanoplatform targeting endogenous YAP1 in tumors reshapes the tumor microenvironment in vivo

[0089] Using the mouse gastric cancer tissue sections obtained above, after dewaxing, rehydration and antigen retrieval, the sections were repaired with citric acid buffer (pH 6.0) at high pressure and high temperature for 5 minutes. After cooling, the sections were blocked with a blocking solution containing 5% BSA and 0.4% TritonX-100 at room temperature for 2 hours to prevent nonspecific binding. Subsequently, the sections were incubated overnight in a humidified box at 4°C with a 1:250 dilution of Granzyme B antibody (ab255598, Abcam). After washing away the primary antibody, the sections were incubated with donkey anti-rabbit AlexaFluor 647 fluorescent secondary antibody at room temperature for 1 hour. After washing away the secondary antibody, the sections were incubated overnight in a 4°C humidified box with a 1:200 dilution of AlexaFluor488-conjugated panCK antibody (53-9003-82, Invitrogen) and a 1:100 dilution of AlexaFluor 647-conjugated CD8 antibody. After washing away the antibody, DAPI-containing mounting medium was added to mount the sections, and the sections were imaged and analyzed using a laser confocal microscope.

[0090] Figure 4 The results of B showed that the M@O-VNPs group after laser irradiation significantly promoted CD8 + GZM + The infiltration of T cells increased by 12.02 times compared with the PBS group and by 2.10 times compared with the laser-irradiated VP combined with oxaliplatin group, showing strong immune activation characteristics.

[0091] Example 6: Photodynamic-chemotherapy nanoplatform targeting endogenous YAP1 in tumors promotes the efficacy of PD-1 inhibitors in an in vivo spontaneous gastric cancer model

[0092] TcPP mice were used as a research model. Three months after tamoxifen induction, PBS, HMSN, oxaliplatin combined with VP, PD-1 inhibitor, M@O-VNPs, and M@O-VNPs combined with PD-1 inhibitor were injected into the tail vein once every two days for 4 weeks. The injection doses were: oxaliplatin 5mg / kg, VP 1mg / kg, PD-1 inhibitor 5mg / kg, and M@O-VNPs 10mg / kg. In addition, the oxaliplatin combined with VP group, M@O-VNPs group, and M@O-VNPs combined with PD-1 inhibitor group were all treated with 690nm wavelength laser (10 minutes, 100mW / cm 2 ) treatment. After 4 weeks of treatment, the mice were euthanized, and the tumors and organ specimens of each mouse were obtained to prepare paraffin-embedded formalin-fixed specimens, which were cut into 4μm thick continuous sections. The sections were stained with hematoxylin and eosin (H&E) to observe the inhibition of tumors and the damage to various organs after each group of treatment.

[0093] Figure 5 The results showed that the tumor area of ​​M@O-VNPs after laser irradiation was reduced by 5.27 times and 3.53 times compared with the PBS group and the oxaliplatin combined with VP group, respectively. In addition, compared with the use of PD-1 inhibitors alone, the tumor area was reduced by 10.66 times after the addition of M@O-VNPs after laser irradiation. This shows that M@O-VNPs not only has a strong killing effect on gastric cancer, but also can significantly enhance the effect of PD-1 inhibitors. The H&E staining results of the heart, liver, spleen, lung, kidney, and intestine showed that M@O-VNPs significantly reduced the damage to the liver, spleen, and intestines when combined with VP and oxaliplatin, proving that M@O-VNPs have good safety.

[0094] In summary, through the above examples and experiments of the present invention, the photodynamic-chemotherapy nanoplatform (M@O-VNPs) targeting endogenous YAP1 in tumors constructed by the embodiments of the present invention has the following advantages:

[0095] 1. Good targeting: In the spontaneous gastric cancer mouse model, after 24 hours of injection, the accumulation of M@O-VNPs in the tumor site after wrapping the macrophage membrane was 4.07 times higher than that of the unwrapped macrophage membrane, while the accumulation in the liver, spleen, and kidney decreased by 1.76 times, 1.51 times, and 1.46 times, respectively. At the cellular level, after incubating M@O-VNPs for 4 hours, the uptake rate of HGC-27 gastric cancer cells was 4.98 times higher than that of the unwrapped macrophage membrane.

[0096] 2. Significant anti-tumor effect: At the cellular level, the apoptosis rate of tumor cells treated with photoactivated M@O-VNPs increased by 3.1 times and 2.06 times compared with free oxaliplatin and free oxaliplatin combined with VP, respectively. In a spontaneous gastric cancer mouse model, the tumor size after M@O-VNPs treatment was reduced by 3.53 times compared with free Verteporfin (VP) combined with oxaliplatin (OXA). In terms of promoting the efficacy of immunotherapy, compared with the use of PD-1 alone, the addition of M@O-VNPs reduced the tumor size by 10.66 times and increased the tumor cell apoptosis rate by 6.1 times.

[0097] 3. Tumor-specific dual immunogenic cell death effect (ICD): In the spontaneous gastric cancer mouse model, compared with the free VP combined with OXA group, the proportion of tumor cells induced by photoactivated M@O-VNPs to produce ICD effect increased by 5.5 times, while the proportion of non-tumor cells that triggered ICD decreased by 2 times. This shows that M@O-VNPs not only triggers ICD efficiently, but also has high tumor selectivity.

[0098] 4. Effectively reshape the tumor microenvironment: In the spontaneous gastric cancer mouse model, compared with the control group and the free VP combined with OXA group, CD8 + The infiltration of effector T cells increased by 12.02-fold and 2.10-fold, respectively, and CD4 + The ratios of helper T cells increased by 4.93-fold and 1.44-fold.

[0099] 5. Better safety: No obvious signs of organ damage were found in the heart, liver, spleen, lung, kidney and intestine after M@O-VNPs treatment; while mice treated with free VP combined with oxaliplatin showed significant liver, spleen and intestinal damage.

[0100] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a photodynamic-chemotherapy nanoplatform targeting endogenous YAP1 in tumors, characterized in that: The photodynamic-chemotherapy nanoplatform targeting endogenous YAP1 in tumors is hollow mesoporous silica nanoparticles (M@O-VNPs) loaded with oxaliplatin and VP and coated with macrophage membranes, and the preparation method thereof comprises the following steps: S1. Synthesis of hollow mesoporous silica nanoparticles: First, 20 g of hexadecyltrimethylammonium chloride aqueous solution was mixed with 3.5 g of triethanolamine and stirred at 80° C. for 15 minutes; Subsequently, 1 mL of tetraethoxysilane (TEOS) was added dropwise and reacted for 1 h; Next, a mixture of 0.5 mL of TEOS and 1 mL of bis(triethoxysilyl)vinyl was added and the reaction was continued for 4 h; After the reaction is completed, the product is washed with ethanol several times and then dispersed in 30 mL of methanol containing 1% sodium chloride to remove the template agent; the extraction step is repeated at least three times, each time for 12 hours, to ensure that the template agent is completely removed; the finally obtained mesoporous silica nanoparticles are selectively etched by ammonia water, reacted at 95°C for 3 hours, and washed with ethanol several times to obtain hollow mesoporous silica nanoparticles; S2. Preparation of Verteporfin-loaded organosilicon nanoparticles: OPSS-PEG-NH2 and carbodiimide were stirred in 2 mL of dimethyl sulfoxide at a molar ratio of 1:1 for 3 h, and 0.2 mL of triethylamine was added to activate the carboxyl groups; Next, Verteporfin dissolved in 3 mL of DMSO and an additional 0.2 mL of TEA were added and stirred in the dark for 24 h to form OPSS-PEG-VP; 15 mg of hollow mesoporous silica nanoparticles were dispersed in 25 mL of ethanol, 0.15 mL of 3-mercaptopropyltriethoxysilane (MPTMS) and 0.2 mL of ammonia were added, and stirred overnight to generate thiolated hollow mesoporous silica nanoparticles (HMSN-SH); OPSS-PEG-VP and 15 mg of HMSN-SH were stirred in 25 mL of ethanol in the dark overnight to finally obtain Verteporfin-loaded hollow mesoporous silica nanoparticles (VHMSNs), which were collected by centrifugation and vacuum drying; S3. Oxaliplatin loading: First, oxaliplatin was activated to ensure that it was positively charged; 10 mg of oxaliplatin and 10 mg of silver nitrate (AgNO3) were added to 10 ml of distilled water; Subsequently, 100 μL of 5% HNO3 solution was added to the mixture to make the pH value <2; then the mixture was wrapped with aluminum foil and stirred at 80°C overnight to ensure that the silver ions reacted with the chloride ions in oxaliplatin; Then, the AgCl precipitate was removed by centrifugation at 4000 rpm for 15 min; 4 mg of activated oxaliplatin was then mixed with 10 mg of VHMSN loaded with VP and subjected to ultrasonic treatment under alkaline conditions, followed by stirring in the dark for 24 h to form hollow mesoporous silica nanoparticles (O-VNPs) loaded with oxaliplatin and VP; Then, the nanoparticles were subsequently passed through 0.45 μm and 0.22 μm filters, washed by centrifugation, and redispersed in phosphate buffer for later use; S4. Extraction and coating of macrophage membranes: Macrophage membranes were extracted from pretreated THP-1 cells or RAW 264.7 cells using a membrane protein extraction kit; the extracted membrane proteins were sonicated on ice for 30 min, then filtered through 0.45 μm and 0.22 μm filters in sequence and quantified by the BCA method; O-VNPs were mixed with macrophage membranes in a ratio of 1:2, sonicated in a water bath for 30 min, and then incubated with stirring at 4°C for 4 h; the resulting macrophage membrane-coated hollow mesoporous silica nanoparticles (M@O-VNPs) loaded with oxaliplatin and VP were collected by centrifugation.

2. The method for preparing a photodynamic-chemotherapy nanoplatform targeting endogenous YAP1 in tumors according to claim 1, characterized in that: The pH under the alkaline condition described in step S3 is 8.

5.

3. The method for preparing a photodynamic-chemotherapy nanoplatform targeting endogenous YAP1 in tumors according to claim 1, characterized in that: The surface protein retention of the macrophage membrane-coated hollow mesoporous silica nanoparticles (M@O-VNPs) loaded with oxaliplatin and VP obtained in step S4 was verified by SDS-PAGE gel electrophoresis and Coomassiebrilliant blue staining; and the presence of CD68 and CD86 macrophage membrane markers was detected by Western blot to demonstrate the successful coating of the macrophage membrane.

4. Use of hollow mesoporous silica nanoparticles (O-VNPs) loaded with oxaliplatin and VP prepared by the method for preparing a photodynamic-chemotherapy nanoplatform targeting endogenous YAP1 in tumors according to claim 1 in the preparation of anti-tumor drugs.

5. Use of the photodynamic-chemotherapy nanoplatform targeting endogenous YAP1 in tumors prepared according to the method for preparing the photodynamic-chemotherapy nanoplatform targeting endogenous YAP1 in tumors according to claim 1 in the preparation of anti-tumor drugs.

Images