Preparation method and application of nano platform for targeting tumor endogenous SOX9

Abstract

The invention relates to the technical field of medicine, in particular to a preparation method and application of a nano platform targeting tumor endogenous SOX9, and the technical key points are as follows: the preparation method comprises the following steps: S1, synthesizing hollow mesoporous silica nanoparticles; s2, preparing the organic silicon nanoparticles loaded by the Monensin; and S3, extraction of the macrophage membrane and preparation of the coating. The carrier prepared by the preparation method and the tumor endogenous SOX9 targeting nano platform are applied to the preparation of antitumor drugs. In the scheme of the invention, a novel nano delivery platform prepared based on Monensin is expected to become a powerful candidate drug for inhibiting SOX9 in gastric cancer treatment. According to the scheme, Monensin is delivered through a mesoporous silica platform camouflaged by a macrophage cell membrane, drug release can be accurately controlled, the targeting property and bioavailability of drugs can be effectively improved, and the scheme of the invention provides an innovative strategy with potential clinical application value for gastric cancer treatment.

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 nano-platform targeting endogenous SOX9 in tumors. Background Art

[0002] Gastric cancer is a malignant tumor with extremely high morbidity and mortality worldwide, especially in East Asia. Although existing surgical, radiotherapy and chemotherapy methods have extended the survival of some patients, the high recurrence rate, early metastasis and treatment resistance of gastric cancer still lead to poor prognosis. In recent years, studies have shown that cancer stem cells (CSCs) play a key role in the occurrence, development and recurrence of gastric cancer. These CSCs have the ability of self-renewal and multidirectional differentiation, can escape traditional treatment and promote tumor recurrence, and are closely related to the poor prognosis of patients. Therefore, targeted therapy for CSCs has become an important direction in the treatment of gastric cancer.

[0003] Among the many molecular mechanisms that regulate the characteristics of gastric cancer CSCs, SOX9, as a key transcription factor, can maintain the self-renewal, proliferation and drug resistance of CSCs and promote the malignant progression of tumors. Studies have found that high expression of SOX9 is closely related to the invasiveness and poor prognosis of gastric cancer. Therefore, inhibiting the function of SOX9 has become a potential therapeutic strategy to reduce the activity of CSCs and inhibit tumor growth.

[0004] However, inhibitors targeting SOX9 have many limitations in clinical applications, which significantly affect their therapeutic effects. First, as a transcription factor, SOX9 lacks a clear active site, which makes it difficult for traditional small molecule inhibitors to effectively target this protein. Therefore, the development of effective SOX9 inhibitors is full of challenges. Although the preparation method and application interference technology of a nano-platform targeting endogenous SOX9 in tumors in the prior art (such as SOX9-siRN, a preparation method and application of a nano-platform targeting endogenous SOX9 in tumors) can directly target the SOX9 gene and significantly reduce its expression, the application of this technology in vivo faces many challenges, including delivery difficulties, rapid degradation and immune response. In addition, SOX9 not only plays a role in tumors, but also has important physiological functions in normal tissues, especially stem cell-related tissues (such as bone marrow and intestinal epithelium). Nonspecific inhibition of SOX9 may cause damage to these normal tissues, thereby increasing the risk of side effects, further limiting the feasibility of its clinical application.

[0005] Therefore, the existing SOX9 inhibition strategies are greatly limited in clinical applications. Since traditional inhibition methods cannot take into account both specificity and safety, it is urgent to develop new delivery systems and more effective drugs to improve the targeting of SOX9 inhibition and reduce side effects.

[0006] To this end, the present application provides a method for preparing and applying a nanoplatform targeting endogenous SOX9 in tumors. Summary of the invention

[0007] 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 nano-platform targeting endogenous SOX9 in tumors.

[0008] The above-mentioned purpose of the present invention is achieved like this:

[0009] One aspect of the present invention provides a method for preparing a nano-platform targeting endogenous SOX9 in tumors, wherein the nano-platform is a macrophage membrane-coated hollow mesoporous silica nanoparticle (M@MoNPs) loaded with Monensin, and the preparation method comprises the following steps:

[0010] S1. Synthesis of hollow mesoporous silica nanoparticles:

[0011] First, 20 g of hexadecyltrimethylammonium chloride aqueous solution was mixed with 3.5 g of triethanolamine and stirred at 80° C. for 15 minutes;

[0012] Subsequently, 1 mL of tetraethoxysilane was added dropwise and reacted for 1 h;

[0013] Then, a mixture of 0.5 mL of TEOS and 1 mL of bis(triethoxysilyl)vinyl was added and the reaction was continued for 4 hours. After the reaction was completed, the product was 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 was repeated at least three times, each time for 12 hours, to ensure that the template agent was completely removed. The mesoporous silica nanoparticles finally obtained were selectively etched with ammonia water, reacted at 95° C. for 3 hours, and washed with ethanol several times to obtain hollow mesoporous silica nanoparticles.

[0014] S2. Preparation of Monensin-loaded organosilicon nanoparticles:

[0015] OPSS-PEG-COOH 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;

[0016] Next, Monensin 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-Monensin; 15 mg of hollow mesoporous silica nanoparticles were dispersed in 25 mL of ethanol, 0.15 mL of 3-mercaptopropyltriethoxysilane and 0.2 mL of ammonia were added, and stirred overnight to generate thiolated hollow mesoporous silica nanoparticles;

[0017] OPSS-PEG-Monensin 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 Monensin, which were collected by centrifugation and vacuum drying;

[0018] S3. Extraction and coating of macrophage membranes:

[0019] 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;

[0020] MoNPs 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 monensin-loaded hollow mesoporous silica nanoparticles (M@MoNPs) were collected by centrifugation.

[0021] Further, in step S3, the retention of proteins on the surface of hollow mesoporous silica nanoparticles (M@MoNPs) was verified by SDS-PAGE gel electrophoresis and Coomassie brilliantblue staining to demonstrate the successful coating of macrophage membranes.

[0022] Another aspect of the present invention provides a method for preparing a nano-platform targeting endogenous SOX9 in a tumor, and uses the hollow mesoporous silica nanoparticles loaded with Monensin prepared in the preparation of anti-tumor drugs.

[0023] Another aspect of the present invention provides a method for preparing a nano-platform targeting endogenous SOX9 in tumors, and uses the prepared macrophage membrane-coated monensin-loaded hollow mesoporous silica nanoparticles (M@MoNPs) in the preparation of anti-tumor drugs.

[0024] The difficulty and significance of the technical problem solved by the present invention are:

[0025] Through research, it was found that Monensin, as a known ion carrier antibiotic, can significantly inhibit the expression of SOX9 in gastric cancer cells. Monensin interferes with multiple signaling pathways by affecting the intracellular ion balance, especially in inhibiting tumor cell proliferation, inducing apoptosis and autophagy. Its wide range of mechanisms of action can not only inhibit the expression of SOX9, but also overcome the multidrug resistance of tumors, thereby enhancing the overall anti-tumor effect. Compared with the existing SOX9 inhibition strategy, Monensin has shown great therapeutic potential, but it still has problems such as poor water solubility, low bioavailability and non-specific toxicity in clinical applications. Therefore, in order to overcome these limitations, the present application designs a mesoporous silica nanoplatform disguised as a macrophage membrane to accurately deliver Monensin to the tumor site. The structure of hollow mesoporous silica provides excellent drug loading capacity and can accurately release drugs under specific stimulation conditions of the tumor microenvironment (such as high concentrations of glutathione), thereby ensuring that effective concentrations of Monensin act on tumor cells and reduce systemic side effects. In addition, the macrophage membrane camouflage technology gives nanoparticles the ability to escape immune system clearance, allowing them to evade the clearance of the immune system and maintain circulation stability for a long time. The macrophage membrane also has natural targeting and can actively migrate to the tumor microenvironment, further improving the accuracy and effectiveness of drug delivery. This nano-platform design significantly increases the cumulative concentration of Monensin at the tumor site and reduces toxicity to normal tissues. Both animal experiments and cell experiments have shown that this nano-delivery system can significantly inhibit tumor growth, delay metastasis, and significantly improve the therapeutic effect.

[0026] In summary, Monensin is expected to become a powerful candidate drug for inhibiting SOX9 in the treatment of gastric cancer through a new nano-delivery platform. Delivering Monensin through a mesoporous silica platform disguised as a macrophage membrane can not only accurately control drug release, but also effectively improve the targeting and bioavailability of the drug, providing an innovative strategy with potential clinical application value for the treatment of gastric cancer.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] In the scheme of the present invention, the macrophage membrane wrapped on the surface of the nanoplatform provides M@MoNPs 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@MoNPs not only successfully avoids the recognition and clearance of the body's immune system, but also can deliver Monensin in a targeted manner, significantly enhancing the tumor targeting of the drug, reducing the nonspecific distribution in healthy tissues, and reducing systemic toxicity. In addition, the disulfide bond connecting Monensin is broken in tumor cells with high glutathione, which can be accurately released in tumor cells and effectively inhibit the SOX9 signaling pathway, limiting tumor cell proliferation.

[0029] The method development of the present invention provides a novel delivery system and facilitates the development of more effective drugs to improve the targeting of SOX9 inhibition and reduce side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Middle: A is the nitrogen adsorption curve of hollow mesoporous silica nanoparticles; B is the average pore size of hollow mesoporous silica nanoparticles; C is the transmission electron microscopy image of hollow mesoporous silica nanoparticles (M@MoNPs) loaded with Monensin wrapped by macrophage membranes; D is the SDS-PAGE gel after Coomassie Brilliant Blue staining;

[0031] Figure 2 is the cumulative release curve of Monensin in M@MoNPs;

[0032] Figure 3 Middle: A is the expression of SOX9 in N87, KATO-III and AGS cells after being treated with different concentrations of M@MoNPs for 24 hours, detected by Western Blot; B is the immunofluorescence staining image of SOX2, SOX9 and Ki-67 in N87-derived gastric cancer organoids treated with different concentrations of M@MoNPs, the scale bar is 20μm; C is the proportion of SOX9+ cells in gastric cancer organoids treated with different concentrations of M@MoNPs (n=50 in each group);

[0033] Figure 4 Middle: A is a representative image of N87-derived gastric cancer organoids treated with different concentrations of M@MoNPs; B is a quantitative graph of the number and diameter of N87-derived gastric cancer organoids treated with different concentrations of M@MoNPs (n=3 for each group);

[0034] Figure 5Middle: A is an image of the inhibition of tumors in the nude mouse gastric cancer cell-derived xenograft model treated with PBS and M@MoNPs (10 mg / kg); B is the change in tumor size of mice during treatment; C is the change in body weight of mice during treatment; D is multiple immunofluorescence staining to detect the expression of SOX2 and SOX9 in tumors after PBS and M@MoNPs treatment, the scale bar is 50 μm. DETAILED DESCRIPTION

[0035] 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.

[0036] The implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0037] The embodiment of the present invention provides a method for preparing a nano-platform targeting endogenous SOX9 in tumors and its application in preparing anti-tumor drugs, referring to Figure 1-Figure 5 As shown, the following is a specific implementation of the scheme of the present invention.

[0038] Example 1: Preparation of a nanoplatform targeting endogenous SOX9 in tumors

[0039] 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).

[0040] 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 42.33±1.04nm and the potential was -26.25±1.22mV. 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 352.194m 2 / g and 3.66nm (such as Figure 1A and B in FIG.

[0041] 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, Monensin dissolved in 3 mL of DMSO and an additional 0.2 mL of TEA were added and stirred at room temperature in the dark for 24 hours to form OPSS-PEG-Monensin. 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 form thiolated hollow mesoporous silica nanoparticles (HMSN-SH). OPSS-PEG-Monensin and 15 mg of HMSN-SH were stirred in 25 mL of ethanol in the dark overnight to finally obtain hollow mesoporous silica nanoparticles (MoNPs) loaded with Monensin, which were collected by centrifugation and vacuum drying.

[0042] The particle size and potential were measured by a particle size and surface potential meter, and the particle size of MoNPs was 56.17±1.42nm and the potential was -19.21±1.38mV. The loading rate of Monensin in MoNPs was 12.14% by high performance liquid chromatography.

[0043] Step 3: 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. The centrifugation was repeated to remove the residual liquid as much as possible. Then 1 ml of membrane protein extraction reagent with PMSF 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. MoNPs 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 loaded with Monensin (M@MoNPs) were collected by centrifugation.

[0044] The potential of M@MoNPs was measured by a potential meter to be -31.45±1.62mV. Transmission electron microscopy results showed that M@MoNPs were spherical in shape and had a particle size of approximately 63.15nm (e.g. Figure 1 The membrane proteins on the surface of M@MoNPs were verified by SDS-PAGE gel electrophoresis, and the results confirmed the successful coating of macrophage membranes (as shown in D). Figure 1 ).

[0045] Example 2: Preparation of a nanoplatform targeting abnormally activated SOX9 in tumors

[0046] 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).

[0047] The particle size and potential were measured using a particle size and surface potential meter, and the particle size of HMSN was 42.33±1.04nm and the potential was -26.25±1.22mV. 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 361.05m 2 / g and 3.12nm.

[0048] 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, Monensin dissolved in 3 mL of DMSO and an additional 0.2 mL of TEA were added and stirred at room temperature in the dark for 24 hours to form OPSS-PEG-Monensin. 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 form thiolated hollow mesoporous silica nanoparticles (HMSN-SH). OPSS-PEG-Monensin and 15 mg of HMSN-SH were stirred in 25 mL of ethanol in the dark overnight to finally obtain hollow mesoporous silica nanoparticles (MoNPs) loaded with Monensin, which were collected by centrifugation and vacuum drying.

[0049] The particle size and potential were measured by a particle size and surface potential meter, and the particle size of MoNPs was 55.26±0.98nm and the potential was -22.34±0.95mV. The loading rate of Monensin in MoNPs was 11.04% by high performance liquid chromatography.

[0050] Step 3: 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. The centrifugation was repeated to remove the residual liquid as much as possible. Then 1 ml of membrane protein extraction reagent with PMSF 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. MoNPs 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 loaded with Monensin (M@MoNPs) were collected by centrifugation.

[0051] The potential of M@MoNPs was measured by a potential meter to be -34.62±2.35mV. Transmission electron microscopy results showed that M@MoNPs were spherical and had a particle size of about 59.24nm. The membrane proteins on the surface of M@MoNPs were verified by SDS-PAGE gel electrophoresis, and the results proved the successful coating of macrophage membranes.

[0052] Example 3: Preparation of a nanoplatform targeting SOX9 to inhibit tumor stemness

[0053] 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).

[0054] The particle size and potential were measured using a particle size and surface potential meter, and the particle size of HMSN was 45.17±2.32nm and the potential was -29.94±1.65mV. 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 369.13m 2 / g and 4.75nm.

[0055] 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, Monensin dissolved in 3 mL of DMSO and an additional 0.2 mL of TEA were added and stirred at room temperature in the dark for 24 hours to form OPSS-PEG-Monensin. 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 form thiolated hollow mesoporous silica nanoparticles (HMSN-SH). OPSS-PEG-Monensin and 15 mg of HMSN-SH were stirred in 25 mL of ethanol in the dark overnight to finally obtain hollow mesoporous silica nanoparticles (MoNPs) loaded with Monensin, which were collected by centrifugation and vacuum drying.

[0056] The particle size and potential were measured by a particle size and surface potential meter, and the particle size of MoNPs was 53.26±1.29nm and the potential was -25.09±1.34mV. The loading rate of Monensin in MoNPs was 9.74% as determined by high performance liquid chromatography.

[0057] Step 3: 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. The centrifugation was repeated to remove the residual liquid as much as possible. Then 1 ml of membrane protein extraction reagent with PMSF 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. MoNPs 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 loaded with Monensin (M@MoNPs) were collected by centrifugation.

[0058] The potential of M@MoNPs was measured by a potential meter to be -33.02±1.57mV. Transmission electron microscopy results showed that M@MoNPs were spherical and had a particle size of approximately 62.16nm. The membrane proteins on the surface of M@MoNPs were verified by SDS-PAGE gel electrophoresis, and the results proved the successful coating of macrophage membranes.

[0059] Example 4: Application of the nanoplatform targeting endogenous SOX9 in an in vitro gastric cancer model

[0060] 1. In vitro drug release behavior of the nanoplatform targeting endogenous SOX9 in tumors

[0061] To simulate the physiological conditions in the tumor microenvironment, a phosphate buffer solution (PBS) of pH 6.5 was prepared. 10 mg of M@MoNPs (in 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 each solution was placed in a dialysis bag (MWCO: 5.0 kDa). The dialysis bag was then placed in a release tube containing an appropriate amount of release medium and oscillated at 60 rpm at room temperature. 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 fresh release medium. Monensin in the sample was determined by high performance liquid chromatography (HPLC), and the cumulative release percentage of the drug was calculated.

[0062] The test results show that the in vitro release behavior of Monensin in M@MoNPs is as follows Figure 2 As shown in the figure, the nanomedicine exhibited obvious glutathione response characteristics. After 48 hours, the cumulative release percentage of monensin in pH 6.5 buffer was 24.47±1.42%, the cumulative release percentage in 5mM glutathione buffer was 68.83±1.72%, and the cumulative release percentage in 10mM glutathione buffer was 80.78±2.81%.

[0063] 2. The inhibitory behavior of the nanoplatform targeting endogenous SOX9 in tumors on SOX9 in vitro

[0064] Three human gastric cancer cell lines, N87, KATO-III and AGS, were cultured in RPMI 1640 medium containing 10% fetal bovine serum to 70-80% confluence. The experimental group treated the cells with different concentrations of M@MoNPs (1μg / ml, 10μg / ml, 50μg / ml) for 24 hours, and the PBS-treated group served as the control group. After the treatment, the culture medium was aspirated, washed twice with PBS at 4°C, and RIPA lysis buffer containing protease inhibitors was added. The lysate was collected after incubation on ice for 30 minutes. After the lysate was centrifuged at 12,000g for 15 minutes (4°C), the supernatant was taken and protein quantification was performed. The protein concentration was quantified using the BCA method, and the protein content of each group was calculated based on the standard curve. 30-50μg of protein sample was mixed with 5× protein loading buffer, heat denatured at 95°C for 5 minutes, and then electrophoresed in 10% SDS-PAGE gel, and then the protein was transferred to PVDF membrane (wet transfer, 300mA, 1 hour). After transfer, the membrane was blocked with 5% skim milk powder at room temperature for 1 hour, and then incubated with SOX9 and GAPDH primary antibodies at 4°C overnight. The next day, the membrane was washed 3 times with TBST solution (5 minutes each time), then incubated with HRP-labeled secondary antibodies at room temperature for 1 hour, and washed again with TBST 3 times. The bands were detected using ECL developer, and the expression of SOX9 and GAPDH was recorded by the chemiluminescence system. The results showed that with the increase in concentration, M@MoNPs significantly inhibited the expression of SOX9 in three gastric cancer cells ( Figure 3 A).

[0065] Next, N87, KATO-III and AGS gastric cancer cell lines were inoculated in Matrigel matrix to construct a three-dimensional gastric cancer organoid model, and cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) until organoids formed and grew stably. After organoid formation, they were divided into experimental and control groups. The experimental groups were treated with M@MoNPs at concentrations of 1μg / ml, 10μg / ml, and 50μg / ml for 24 hours, respectively, while the control group was treated with PBS. After treatment, the culture medium was aspirated and gently washed twice with PBS at 4°C, and then the organoids were fixed with 4% paraformaldehyde (PFA) for 30 minutes. After fixation, the organoids were washed with PBS and then embedded in paraffin. After dehydration and transparency, the organoids were embedded in paraffin, and serial sections with a thickness of 4-5μm were prepared for HE staining and multiple immunofluorescence staining, respectively. The steps of HE staining include: dewaxing of sections, gradient ethanol dehydration, hematoxylin staining of nuclei, 1% hydrochloric acid alcohol differentiation, eosin staining of cytoplasm, sealing after dehydration, and finally observing the histomorphological changes of organoids under an optical microscope. For multiple immunofluorescence staining, the sections were first antigen repaired, microwave heated in citrate buffer (pH 6.0) for 10-15 minutes, and washed with PBS three times for 5 minutes each after the sections cooled to room temperature. Then, they were blocked with 5% bovine serum albumin (BSA) at room temperature for 1 hour, and then primary antibodies of SOX2, SOX9 and Ki-67 of different species were added respectively, and incubated at 4°C overnight according to the recommended dilution ratio. On the second day, the sections were washed again with PBS for 3 times for 5 minutes each, and then the corresponding fluorescent-labeled secondary antibodies were added and incubated for 1 hour at room temperature in the dark. After that, they were washed with PBS three times, and then the nuclei were stained with DAPI, incubated for 5 minutes, and then rinsed with PBS, and finally sealed with anti-fluorescence quenching sealing agent. After staining, the fluorescence signals of DAPI (to mark the cell nucleus), SOX2, SOX9 and Ki-67 were observed in turn using a fluorescence microscope. Appropriate filters were used to observe different fluorescence channels, and fluorescence images were taken for recording. After the experiment, the fluorescence images were quantitatively analyzed using ImageJ software. The effects of M@MoNPs treatment on the expression of SOX2, SOX9 and Ki-67 in organoids were evaluated by measuring the changes in the fluorescence intensity of SOX2, SOX9 and Ki-67 in different concentration treatment groups. A bar graph was also drawn to show the effects of each concentration treatment on SOX9 expression. Figure 3 B and 3C show that with increasing concentration, M@MoNPs significantly inhibited the expression of SOX9 in all three gastric cancer organoids, while having no significant effect on SOX2 and Ki-67, indicating the targeted inhibition of SOX9 by M@MoNPs.

[0066] 3. Nanoplatform targeting endogenous SOX9 in tumors inhibits the stemness of gastric cancer organoids in vivo

[0067] First, the N87 cell line was cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) to 70-80% confluence. The cells were then collected and mixed with Matrigel matrix at an appropriate concentration of cell suspension to prepare cell-derived gastric cancer organoids. The cell-Matrigel mixture was inoculated into a 96-well plate, with a volume of 50-100 μL added to each well. The inoculated organoids were cultured in a 37°C, 5% CO2 incubator. After the organoids were formed and grew stably, the organoids were divided into experimental and control groups. The experimental groups were treated with M@MoNPs at concentrations of 1 μg / ml, 10 μg / ml, and 50 μg / ml, respectively, with 3 parallel wells in each group, and the control group was added with an equal amount of PBS as a negative control. After the treatment was completed, the organoids were returned to the incubator for continued incubation. The medium was changed every two days during the period. After 12 days, an inverted microscope was used to capture bright-field images of the organoids in each treatment group, ensuring that multiple fields of view were captured to comprehensively observe the growth status and morphological changes of the organoids in each group. Afterwards, the bright-field images were quantitatively analyzed using ImageJ analysis software, and the maximum diameter of the organoids in each treatment group was measured, the average diameter of the organoids in each group was calculated, and the total number of organoids in each group was also calculated. Finally, a comparative analysis was performed on the diameter and number of organoids in the M@MoNPs treatment groups with different concentrations and the PBS control group. Figure 4 A to B in the figure show that with increasing concentration, M@MoNPs significantly inhibited the number and diameter of gastric cancer organoids, indicating that M@MoNPs has a significant effect on the stemness of gastric cancer organoids.

[0068] Example 5: Application of the nanoplatform targeting endogenous SOX9 in tumors in an in vivo model

[0069] First, the N87 human gastric cancer cell line was cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) to 80-90% confluence, then trypsin was used to digest and collect the cell suspension, and the cell concentration was adjusted to 1×106 / 100μL PBS mixture. The cell suspension was inoculated into the left scapular region of female BALB / c nude mice aged 4-6 weeks using a sterile syringe, and 100μL was injected. After one week, the experimental treatment began. Each mouse in the experimental group was intraperitoneally injected with M@MoNPs at a dose of 10mg / kg, once every other day, for 24 days; the control group was injected with an equal amount of PBS in the same way. During the medication period, the weight changes and tumor size of the mice were recorded regularly, and their health status and tumor growth were observed. After 24 days of treatment, the mice were killed by cervical dislocation, the subcutaneous tumors were quickly removed, the tumors of each mouse were photographed, the tumor morphology was recorded, and the long diameter (L) and short diameter (W) of the tumor were measured using a vernier caliper to calculate the tumor volume. After the measurement, the tumor tissue was fixed in 4% paraformaldehyde and fixed at room temperature for 24 hours. After fixation, the tumor tissue was dehydrated, transparentized, and embedded in paraffin. The slicer was cut into continuous sections with a thickness of 4-5 μm for multiple immunofluorescence staining and DAPI staining, respectively. After the sections were dewaxed in xylene and dehydrated in gradient ethanol, antigen retrieval was performed using citric acid buffer (pH 6.0) and microwave heating for 10-15 minutes. After retrieval, the sections were washed with PBS three times for 5 minutes each time and blocked with 5% bovine serum albumin (BSA) for 1 hour. Subsequently, the primary antibodies for SOX2 and SOX9 were added and incubated at 4°C overnight at the recommended dilution ratio. The sections were washed with PBS three times the next day, and then fluorescently labeled secondary antibodies (such as AlexaFluor488 and AlexaFluor 555) were added and incubated at room temperature in the dark for 1 hour. After washing, the sections were incubated with DAPI staining solution for 5 minutes to mark the cell nuclei. After staining, the slides were sealed with anti-fluorescence quenching sealing agent, and the fluorescence signals of SOX2, SOX9 and DAPI were observed in turn under a laser confocal microscope, and images were taken.

[0070] The results showed that compared with the PBS group, the growth of tumors was significantly inhibited after treatment with M@MoNPs, indicating its excellent anti-tumor effect ( Figure 5 In addition, the body weight of mice treated with M@MoNPs did not change significantly compared with the PBS group, indicating its safety ( Figure 5 Finally, analysis of multiple immunofluorescence images showed that M@MoNPs inhibited the expression of SOX9 in tumors, but had no significant effect on SOX2. This shows the targeted inhibition of SOX9 by M@MoNPs ( Figure 5 D).

[0071] In summary, the above embodiments and experiments of the present invention prove that compared with the prior art, the novel nano-delivery platform prepared based on Monensin in the scheme of the present invention is expected to become a powerful candidate drug for inhibiting SOX9 in the treatment of gastric cancer. In the scheme of the present invention, Monensin is delivered through a mesoporous silica platform disguised as a macrophage membrane, which can not only accurately control the release of the drug, but also effectively improve the targeting and bioavailability of the drug. The scheme of the present invention provides an innovative strategy with potential clinical application value for the treatment of gastric cancer.

[0072] 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 nanoplatform targeting endogenous SOX9 in tumors, characterized in that: The nano-platform is a macrophage membrane-coated hollow mesoporous silica nanoparticle loaded with Monensin (M@MoNPs), 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 was added dropwise and reacted for 1 h; Then, a mixture of 0.5 mL of TEOS and 1 mL of bis(triethoxysilyl)vinyl was added and the reaction was continued for 4 hours. After the reaction was completed, the product was 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 was repeated at least three times, each time for 12 hours, to ensure that the template agent was completely removed. The mesoporous silica nanoparticles finally obtained were selectively etched with ammonia water, reacted at 95° C. for 3 hours, and washed with ethanol several times to obtain hollow mesoporous silica nanoparticles. S2. Preparation of Monensin-loaded organosilicon nanoparticles: OPSS-PEG-COOH 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, Monensin 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-Monensin; 15 mg of hollow mesoporous silica nanoparticles were dispersed in 25 mL of ethanol, 0.15 mL of 3-mercaptopropyltriethoxysilane and 0.2 mL of ammonia were added, and stirred overnight to generate thiolated hollow mesoporous silica nanoparticles; OPSS-PEG-Monensin 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 Monensin, which were collected by centrifugation and vacuum drying; S3. 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; MoNPs 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 monensin-loaded hollow mesoporous silica nanoparticles (M@MoNPs) were collected by centrifugation.

2. The method for preparing a nanoplatform targeting endogenous SOX9 in tumors according to claim 1, characterized in that: In step S3, the retention of proteins on the surface of hollow mesoporous silica nanoparticles (M@MoNPs) was verified by SDS-PAGE gel electrophoresis and Coomassie brilliantblue staining to demonstrate the successful coating of macrophage membranes.

3. The method for preparing a nanoplatform targeting endogenous SOX9 in tumors according to claim 1, characterized in that: The application of the hollow mesoporous silica nanoparticles loaded with Monensin in the preparation of anti-tumor drugs.

4. The method for preparing a nanoplatform targeting endogenous SOX9 in tumors according to claim 1, characterized in that: The invention discloses an application of the macrophage membrane-coated monensin-loaded hollow mesoporous silica nanoparticles (M@MoNPs) in the preparation of anti-tumor drugs.

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