Manganese-doped mesoporous silica nanomaterial, and preparation method therefor and use thereof

Abstract

A manganese-doped mesoporous silica nanomaterial, and a preparation method therefor and the use thereof. The nanomaterial comprises a manganese-doped mesoporous silica nanosphere, an anti-angiogenic drug and a surface delivery system. The anti-angiogenic drug is adsorbed in the pores of the manganese-doped mesoporous silica nanosphere, and the surface delivery system is coated on the surface of the manganese-doped mesoporous silica nanosphere, wherein metal manganese ions are doped on the surface and in the framework of the mesoporous silica nanosphere. The anti-angiogenic drug is an HIF-2α inhibitor. After targeted degradation at a tumor site, the nanomaterial releases the manganese ions and the anti-angiogenic drug to realize the dual anti-tumor effects of activating local anti-tumor immunity and inhibiting angiogenesis within the tumor at a targeted tumor site; and the manganese ions and the anti-angiogenic drug have a synergistic effect. In addition, the nanomaterial has no potential toxic effect, has good biosafety, and provides a new choice for the treatment of tumors, especially pVHL-deleted tumors.

Description

A manganese-doped mesoporous silicon nanomaterial and its preparation method and application Technical Field

[0001] The present invention belongs to the field of medical nanomaterials, and in particular relates to a manganese-doped mesoporous silicon nanomaterial, a preparation method thereof, and an application thereof. Background Art

[0002] According to data from the National Cancer Center, there were 19.29 million new cases of cancer worldwide in 2020, of which 4.57 million were in China, accounting for 23.7% of the global total. At the same time, there were 9.96 million cancer deaths worldwide, 30.1% (3 million) of which occurred in China. Due to its high incidence and mortality rates, cancer has become the leading threat to human health worldwide. A major challenge in cancer treatment is the inherent and acquired resistance of tumors to chemotherapy drugs. The inhibitory effect of a single drug on a single mechanism may not achieve efficient tumor suppression. In combination chemotherapy, two or more therapeutic agents are used simultaneously for cancer treatment. These agents can synergistically act on different pathogenic mechanism targets, thereby achieving more effective cancer treatment effects compared to single drug treatment.

[0003] Research has shown that tumor growth and progression are critically dependent on an adequate blood supply of oxygen and nutrients. Consequently, tumors harbor a large number of abnormal vascular structures with increased permeability, a phenomenon particularly pronounced in tumors lacking the von Hippel-Lindau tumor suppressor protein (pVHL). Consequently, targeting the tumor vasculature has been a focus of intense research over the past 20 years. However, systemic administration of antiangiogenic drugs has been ineffective, potentially due to the development of tumor adaptive resistance. Therefore, combining antiangiogenic drugs with chemotherapy or immunotherapy is an effective approach to further expand the application of antiangiogenic therapy.

[0004] Currently, immunotherapies, including adoptive T cell therapy, immune checkpoint blockade (ICB), and therapeutic cancer vaccines, have demonstrated promising efficacy in clinical trials for treating various tumor types. However, current immunotherapies suffer from low efficacy, significant systemic side effects, and significant inter-individual variability. There is an urgent need to identify immunostimulatory agents that are broadly applicable to various tumor types, possess potent immune activation potential, and exhibit minimal toxic side effects. Manganese ions, an essential trace element for the human body, have recently been found to significantly activate innate and innate immunity by promoting the cyclic guanosine monophosphate-adenylate synthetase (cGAS)-stimulator of interferon genes (STING) signaling pathway, achieving significant tumor killing and inhibiting tumor recurrence. Manganese ions are expected to become a powerful innate immune stimulator. Summary of the Invention

[0005] To solve the above technical problems, the present invention discloses a manganese-doped mesoporous silicon nanomaterial, a preparation method and application thereof. The nanomaterial prepared by the present invention releases manganese ions and anti-angiogenic drugs after targeted degradation at the tumor site, thereby achieving the dual anti-tumor effects of activating local anti-tumor immunity at the tumor site and preventing angiogenesis inside the tumor. The two have a synergistic anti-tumor effect, providing a new option for the treatment of tumors, especially pVHL-deficient tumors.

[0006] To achieve the above objectives, the present invention first provides a manganese-doped mesoporous silicon nanomaterial, which comprises manganese-doped mesoporous silicon nanospheres, an anti-angiogenesis drug, and a surface delivery system. The anti-angiogenesis drug is adsorbed in the pores of the manganese-doped mesoporous silicon nanospheres, and the surface delivery system is coated on the surface of the manganese-doped mesoporous silicon nanospheres.

[0007] Wherein, metal manganese ions are doped on the surface and skeleton of the mesoporous silicon nanospheres; and the anti-angiogenesis drug is a HIF-2α inhibitor.

[0008] Preferably, the HIF-2α inhibitor comprises PT2385.

[0009] Preferably, the topical delivery system is folic acid polyethylene glycol phospholipid.

[0010] Another aspect of the present invention provides a method for preparing a manganese-doped mesoporous silicon nanomaterial, comprising the following steps:

[0011] S1, cetyltrimethylammonium toluenesulfonate (CTAT), water, and triethanolamine (TEA), after stirring evenly, quickly adding manganese chloride tetrahydrate, then dropwise adding tetraethyl orthosilicate (TEOS) and a silane coupling agent, continuously stirring, centrifuging, washing, and refluxing to obtain mesoporous silica nanospheres;

[0012] S2, mixing the mesoporous silicon nanospheres with disodium maleate and manganese sulfate aqueous solution, and preparing manganese-doped mesoporous silicon nanospheres MMSN by a hydrothermal method;

[0013] S3, mixing the MMSN obtained in step S2 with an anti-angiogenic drug, and continuously stirring to obtain drug-loaded MMSN;

[0014] S4, mixing the drug-loaded MMSN obtained in step S3 with the surface delivery system, performing water bath sonication and then centrifugation to obtain targeted drug-loaded MMSN;

[0015] Wherein, the anti-angiogenic drug is a HIF-2α inhibitor.

[0016] Preferably, the silane coupling agent includes TESPT, the HIF-2α inhibitor includes PT2385, and the surface delivery system is folic acid polyethylene glycol phospholipid.

[0017] Preferably, in step S1, the mass ratio of TEOS to TESPT is 5:4;

[0018] In step S2, the mass ratio of disodium maleate to manganese sulfate is 5:4;

[0019] In step S3, the mass ratio of MMSN to PT2385 is 5:1 to 50:1;

[0020] In step S4, the mass ratio of the drug-loaded MMSN to folic acid polyethylene glycol phospholipid is 5:1 to 50:1.

[0021] Preferably, in step S1, the reflux solvent is a mixed solution of methanol and hydrochloric acid.

[0022] Another aspect of the present invention further provides the use of the aforementioned manganese-doped mesoporous silicon nanomaterial, or the manganese-doped mesoporous silicon nanomaterial prepared by the aforementioned preparation method, in the preparation of anti-tumor drugs.

[0023] Preferably, the tumor is a tumor with pVHL deletion or low expression.

[0024] Furthermore, the tumor includes any one of melanoma and renal cancer.

[0025] Compared with the prior art, the beneficial effects of the present invention include at least:

[0026] 1. The present invention provides a manganese-doped mesoporous silicon nanomaterial, which includes manganese-doped mesoporous silicon nanospheres, an anti-angiogenic drug PT2385 loaded in the pores of the nanospheres, and a surface delivery system DSPE-PEG-FA that wraps the nanospheres. Folic acid (FA) improves the targeting of the nanomaterial to the tumor site; the nanosphere skeleton contains a reducing tetrasulfide bond that can react with hydrogen peroxide or glutathione, which is abundant in the tumor microenvironment, to undergo responsive degradation and then release manganese ions (Mn 2+ ) and the anti-angiogenic drug PT2385, achieving dual anti-tumor effects by targeting the tumor site to activate local anti-tumor immunity and inhibiting angiogenesis within the tumor, with a synergistic effect. Furthermore, the nanomaterials presented here have no potential toxic effects and exhibit excellent biosafety, providing a new treatment option for tumors, particularly those lacking pVHL.

[0027] 2. In the nanomaterial provided by the present invention, manganese ions are innovatively added to the nano-silicon skeleton for the first time, and manganese ions are also doped on the surface of the mesoporous silicon nanospheres during the subsequent etching process. The manganese ions first incorporated into the silicon skeleton will be lost during the elution process, thereby increasing the pores on the surface of the mesoporous silicon and occupying the void positions on the surface of the mesoporous silicon in advance. On the one hand, it is beneficial to the subsequent etching reaction and ensures the stability of the mesoporous silicon material and the uniformity of the pore size. On the other hand, it provides more space for the subsequent surface doping reaction of manganese ions, which is more conducive to increasing the manganese ion loading capacity, and ultimately helps to improve the efficiency of activating tumor immunity.

[0028] 3. The nanomaterial preparation method provided by the present invention is simple, does not require complex and expensive equipment, and is easy to achieve industrial production. Therefore, it has good application prospects in the field of tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG1 shows a transmission electron microscope (TEM) image of the nanomaterial prepared in Example 1 of the present invention, wherein:

[0030] A is the image of MMSN under transmission electron microscope;

[0031] B is the transmission electron microscopy image of MMSN@DSPE-PEG-FA.

[0032] FIG2 is the safety assessment result of PT / MMSN@DSPE-PEG-FA prepared in Example 1 of the present invention, wherein:

[0033] A is the content of alanine aminotransferase (ALT) in mouse serum;

[0034] B is the content of aspartate aminotransferase (AST) in mouse serum;

[0035] C is the total bilirubin (TBil) content in mouse serum;

[0036] D is the uric acid (UA) content in mouse serum;

[0037] E is the creatinine (CREA) content in mouse serum;

[0038] In the figure, PT / Mn@MSN-DSPE-PEG-FA represents PT / MMSN@DSPE-PEG-FA.

[0039] FIG3 shows the drug release results of PT / MMSN@DSPE-PEG-FA prepared in Example 1 of the present invention under different pH and different GSH conditions, wherein:

[0040] A is the pH response result;

[0041] B is the pH+GSH response result.

[0042] Figure 4 shows inverted fluorescence micrographs of MMSN@DSPE-PEG-FA loaded with red fluorescent probe Cy3 being taken up by cells for 2 hours and 4 hours.

[0043] Figure 5 shows the results of measuring the fluorescence content of the tumor site 48 hours after MMSN@DSPE-PEG-FA loaded with red fluorescent probe Cy5.5 was injected into mice, where:

[0044] A is a scan of a section obtained by dissecting the tumor site of a mouse 48 hours after tail vein injection of MMSN loaded with the fluorescent probe Cy5.5. The blue represents the cell nucleus and the red represents the fluorescent probe Cy5.5.

[0045] B is a scan of a section obtained by dissecting the tumor site of a mouse 48 hours after tail vein injection of MMSN@DSPE-PEG-FA loaded with the fluorescent probe Cy5.5. The blue represents the cell nucleus and the red represents the fluorescent probe Cy5.5.

[0046] C is a quantitative statistical graph of the average fluorescence intensity of the fluorescent probe Cy5.5 in A and B.

[0047] Figure 6 shows the cell death and viability staining results of B16-scr cells and B16-sh4 cells after different treatments, where PT represents the PT2385 group, Mn represents the MMSN@DSPE-PEG-FA group, and Mn+PT represents the PT / MMSN@DSPE-PEG-FA group.

[0048] FIG7 shows the effect of pVHL deletion on tumor proliferation and treatment, wherein:

[0049] AB represents the proliferation of cancer cells with or without pVHL deletion;

[0050] CD is the therapeutic effect of the nanomaterial of the present invention on cancer cells with or without pVHL deletion;

[0051] In the figure, scr represents the group inoculated with Renca-scr cells, sh1 represents the group inoculated with Renca-sh1 cells, sh4 represents the group inoculated with Renca-sh4 cells, untreated represents the group not treated with MMSN@DSPE-PEG-FA, and treated represents the group treated with MMSN@DSPE-PEG-FA.

[0052] FIG8 shows the evaluation of the anti-tumor activity of the nanomaterials of the present invention in vivo, wherein:

[0053] AB is a comparison of the in vivo tumor treatment effects of Renca-sh4 renal cancer model mice after treatment with PT2385, MMSN@DSPE-PEG-FA, and PT / MMSN@DSPE-PEG-FA, respectively;

[0054] CD is a comparison of the in vivo tumor treatment effects in B16-sh4 model melanoma mice after treatment with MMSN@DSPE-PEG-FA and PT / MMSN@DSPE-PEG-FA. DETAILED DESCRIPTION

[0055] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0056] As previously mentioned, studies have shown that angiogenesis plays a crucial role in tumor development and metastasis, and anti-angiogenesis is a potential target for tumor therapy. However, systemic anti-angiogenic drugs have been ineffective. Combining anti-angiogenic drugs with chemotherapy or immunotherapy can lead to better therapeutic effects, and this strategy is applicable to the treatment of most tumors. Manganese ions, an essential trace element for the human body, have recently been found to significantly activate innate and innate immunity by promoting the cyclic guanosine monophosphate-adenylate synthetase (cGAS)-stimulator of interferon genes (STING) signaling pathway, achieving significant tumor killing and inhibiting tumor recurrence, and are expected to become a powerful innate immune agonist.

[0057] Based on the above, the present invention has finally provided a manganese-doped mesoporous silicon nanomaterial after extensive research and experiments. The nanomaterial includes manganese-doped mesoporous silicon nanospheres, an anti-angiogenic drug and a surface delivery system. The anti-angiogenic drug is adsorbed in the pores of the manganese-doped mesoporous silicon nanospheres, and the surface delivery system is coated on the surface of the manganese-doped mesoporous silicon nanospheres; wherein metallic manganese ions are doped on the surface and skeleton of the mesoporous silicon nanospheres; and the anti-angiogenic drug is a HIF-2α inhibitor.

[0058] It should be noted that during the research and development process, the present invention first designed a mesoporous silicon nanosphere with a silicon skeleton doped with metallic manganese ions. However, during subsequent material characterization, it was found that the manganese ions were easily lost during the elution process (used to remove impurities and control material purity and particle size uniformity), making it difficult to provide sufficient manganese ions for immunotherapy in subsequent applications. To address the aforementioned problem, the present invention also designed a mesoporous silicon nanosphere with a surface doped with metallic manganese ions only after the elution process. However, the nanospheres obtained with this design suffered from problems such as poor stability and dispersibility. Ultimately, after extensive research, the present invention innovatively added manganese ions to the nanosilicon skeleton for the first time, while also doping the surface of the mesoporous silicon nanospheres with manganese ions. This resulted in the simultaneous doping of the surface and skeleton of the mesoporous silicon nanospheres with metallic manganese ions. This not only greatly increased the manganese ion loading capacity of the mesoporous silicon nanospheres, but also improved the stability and dispersibility of the material, which is conducive to improving the efficiency of activating tumor immunity.

[0059] Furthermore, experiments have shown that the manganese-doped mesoporous silica nanospheres (MMSNs) designed and prepared by the present invention are more sensitive to pVHL-deficient tumors and exhibit superior therapeutic efficacy. pVHL deficiency can lead to the accumulation of HIF-2α (hypoxia-inducible factor-2α), promoting tumor development and progression. Therefore, the present invention incorporates anti-angiogenic drugs, such as HIF-2α inhibitors, into the pores of the manganese-doped mesoporous silica nanospheres (MMSNs) through hydrophobic interactions. This nanomaterial simultaneously exerts both immunotherapy and anti-angiogenic effects, creating a synergistic effect that enhances the material's anti-tumor efficacy. As an example, the HIF-2α inhibitor includes PT2385.

[0060] Furthermore, the present invention also coats a surface delivery system on the surface of the above-mentioned drug-loaded manganese-doped mesoporous silica nanospheres. In some embodiments, the surface delivery system is folic acid polyethylene glycol phospholipid (DSPE-PEG-FA), wherein folic acid serves as a targeting ligand to improve the tumor targeting of the nanomaterial, polyethylene glycol phospholipid improves the stability and solubility of the nanomaterial, and the thiol group enables these components to combine, thereby producing a functional drug carrier with targeting ability.

[0061] Another aspect of the present invention provides a method for preparing a manganese-doped mesoporous silicon nanomaterial, comprising the following steps:

[0062] S1, cetyltrimethylammonium toluenesulfonate (CTAT), water, and triethanolamine (TEA) are mixed and stirred evenly, and then manganese chloride tetrahydrate is quickly added, followed by dropwise addition of tetraethyl orthosilicate (TEOS) and a silane coupling agent. The mixture is continuously stirred, centrifuged, washed, and refluxed to obtain mesoporous silica nanospheres.

[0063] In some embodiments, the reflux solvent is a mixed solution of methanol and hydrochloric acid. Using hydrochloric acid reflux in this step not only etches to form mesoporous silicon nanospheres, but also removes impurities, reduces and uniformizes the particle size, and controls the size of the mesoporous silicon nanospheres to the nanometer scale, thus preemptively achieving quality control. Without preemptive reflux etching, excessive impurities (i.e., large-sized nanoparticles) can prevent ultrasonically obtaining a uniform nanosolvent. Consequently, subsequent surface doping with manganese ions becomes impossible, resulting in insufficient manganese ion content in the nanomaterial, making it difficult to achieve a satisfactory immune-activating therapeutic effect.

[0064] S2, mixing the mesoporous silicon nanospheres, disodium maleate and manganese sulfate aqueous solution, and preparing manganese-doped mesoporous silicon nanospheres MMSN by a hydrothermal method.

[0065] Using disodium maleate and an aqueous solution of manganese sulfate as etchants further etches the mesoporous silicon nanospheres obtained in step S1, increasing the pore size and surface area, thereby boosting subsequent drug adsorption. Furthermore, during this secondary etching process, manganese ions are doped onto the surface of the mesoporous silicon nanospheres, increasing the manganese ion loading of the nanomaterial. It is important to note that this step is completed after the reflux elution process, eliminating the need for subsequent elution of the nanomaterial, thus preventing the loss of the doped manganese ions during the elution process.

[0066] S3, mixing the MMSN obtained in step S2 with the anti-angiogenic drug, and continuously stirring to obtain the drug-loaded MMSN.

[0067] S4, mixing the drug-loaded MMSN obtained in step S3 with the surface delivery system, performing water bath sonication and then centrifugation to obtain the drug-loaded MMSN modified with the surface delivery system; wherein the anti-angiogenic drug is a HIF-2α inhibitor.

[0068] In some embodiments, the silane coupling agent comprises TESPT, the HIF-2α inhibitor comprises PT2385, and the surface delivery system comprises folate-polyethylene glycol phospholipid. The addition of bis-[3-(triethoxysilyl)propyl]-tetrasulfide (TESPT) allows for the formation of tetrasulfide bonds within the mesoporous silica nanospheres, enabling the nanomaterials produced by the present invention to achieve dual responsiveness under both acidic and high glutathione (GSH) conditions within the tumor microenvironment.

[0069] In some embodiments, in step S1, the mass ratio of TEOS to TESPT is 5:4;

[0070] In step S2, the mass ratio of disodium maleate to manganese sulfate is 5:4;

[0071] In step S3, the mass ratio of MMSN to PT2385 is 5:1 to 50:1;

[0072] In step S4, the mass ratio of the drug-loaded MMSN to folic acid polyethylene glycol phospholipid is 5:1 to 50:1.

[0073] Another aspect of the present invention further provides the use of the aforementioned manganese-doped mesoporous silicon nanomaterial, or the manganese-doped mesoporous silicon nanomaterial prepared by the aforementioned preparation method, in the preparation of anti-tumor drugs.

[0074] In some embodiments, the tumor is a tumor with pVHL deletion or low expression; further, the tumor includes melanoma and renal cancer.

[0075] The experimental process and experimental results of the present invention are described in detail below. Unless otherwise specified, the reagents or materials used in the embodiments of the present invention are purchased from commercial products. The main reagents used include: hexadecyltrimethylammonium toluenesulfonate CTAT (purchased from Bid Pharmaceuticals, article number: BD120541), tetraethyl orthosilicate TEOS (purchased from Anaiji Chemicals, article number: A020302), bis-[3-(triethoxysilyl)propyl]-tetrasulfide TESPT (purchased from Wokai, article number: 40372-72-3), hypoxia-inducible factor 2 (HIF-2a) inhibitor PT2385 (purchased from S elleckchem, product number: S8352), folic acid polyethylene glycol phospholipid DSPE-PEG-FA (purchased from Aladdin, product number: B2224303), manganese chloride tetrahydrate MnCl2·4H2O (purchased from Aladdin, product number: G2227956), manganese sulfate MnSO4 (purchased from Aladdin, product number: 10034-96-5), disodium maleate (purchased from Aladdin, product number: K22181051).

[0076] Example 1. Preparation and performance characterization of nanomaterial PT / MMSN@DSPE-PEG-FA

[0077] (1) Preparation process

[0078] S1, add 480 mg of cetyltrimethylammonium toluenesulfonate (CTAT) to 25 mL of sterile water (ddH2O), heat to 80°C in a water bath, add 62 μL of 10% triethylamine, and stir at 80°C for 1 hour; add 1 mL of 50 mg / mL MnCl2•4H2O to the above solution as quickly as possible, and then add a mixture of 2 mL of TEOS and 1.6 mL of TESPT at a rate of 30 drops / min. Stir at 80°C for 16 hours to stop the reaction, cool to room temperature, and centrifuge at high speed. The obtained nanoparticles are washed alternately with ethanol and purified water, and added to a methanol / hydrochloric acid mixture and refluxed for 16 hours to obtain mesoporous silica nanospheres;

[0079] S2, an aqueous solution containing 30 mg of disodium maleate and 24 mg of manganese sulfate (MnSO4) was added dropwise to ddH2O containing 90 mg of mesoporous silicon nanospheres, mixed and stirred to a volume of 30 mL, and after high-temperature reaction, high-speed centrifugation was performed to obtain manganese-doped mesoporous silicon nanospheres MMSN;

[0080] S3, manganese-doped mesoporous silica nanospheres MMSN and anti-angiogenic drug PT2385 were mixed at a mass ratio of 20:1 and stirred for 24 h to obtain drug-loaded manganese-doped mesoporous silica nanospheres PT / MMSN;

[0081] S4, the PT / MMSN obtained in step S3 was mixed with folic acid polyethylene glycol phospholipid (DSPE-PEG-FA) at a mass ratio of 10:1, and the mixture was sonicated in a 37°C water bath for 30 minutes, followed by high-speed centrifugation. The supernatant was discarded, and the mixture was resuspended in 5% glucose solution to obtain targeted drug-loaded manganese-doped mesoporous silica nanospheres PT / MMSN@DSPE-PEG-FA.

[0082] (2) Material characterization

[0083] 1. Transmission electron microscopy characterization

[0084] The MMSN and MMSN@DSPE-PEG-FA nanomaterials prepared in this example were characterized by TEM. The results are shown in FIG1 . MMSN and MMSN@DSPE-PEG-FA are mesoporous solid silicon nanospheres with uniform morphology.

[0085] 2. Safety Assessment

[0086] Referring to Figure 2, the MMSN@DSPE-PEG-FA nanohydrate material prepared in this example was injected into mice, and the changes in the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBil) in the mouse serum were measured to evaluate the effect of the drug on the liver function of the mice. The changes in the levels of uric acid (UA) and creatinine (CREA) in the serum were measured to evaluate the effect of the drug on the renal function of the mice. The results show that the nanomaterial prepared by the present invention has no potential toxic effects and has good biosafety.

[0087] 3. Drug release detection under different pH and GSH conditions

[0088] Referring to Figure 3, as the environmental pH decreases, the nanomaterial of the present invention releases more PT2385 over time (Figure 3A); further, when GSH is also present in the environment, the drug release amount is even greater (Figure 3B), indicating that the nanomaterial of the present invention can react with the abundant hydrogen peroxide or glutathione in the tumor microenvironment to achieve responsive degradation.

[0089] 4. Cellular uptake and targeting performance testing of nanomaterials

[0090] The nanomaterial prepared in this example was loaded with the red fluorescent probe Cy3 and then cultured with Renca cells. Laser confocal microscopy was used to observe the cells after 2 and 4 hours of culture. As shown in Figure 4, the fluorescence intensity within the cancer cells increased over time, indicating that the cancer cells were able to absorb more MMSN@DSPE-PEG-FA nanomaterials over time.

[0091] The nanomaterial prepared in this example was loaded with the red fluorescent probe Cy5.5 and injected into BALB / c mice via the tail vein. 48 hours later, the mice were sacrificed, and tumor tissue was removed and sectioned to observe the fluorescence intensity of the tumor site after MMSN@DSPE-PEG-FA injection. As shown in Figure 5, compared to the MMSN control group, MMSN@DSPE-PEG-FA showed strong selectivity and targeting for the tumor site and a longer retention time within the tumor. MMSN@DSPE-PEG-FA is expected to provide more precise drug delivery to cancer cells with high FA receptor expression.

[0092] 5. Effects of pVHL loss on tumor proliferation and treatment

[0093] PT2385, MMSN@DSPE-PEG-FA, or PT / MMSN@DSPE-PEG-FA were added to B16-scr (wild-type) and B16-sh4 (pVHL knockdown: pVHL plasmid purchased, stable cell line established by lentiviral transfection) cells. After 48 hours, cells were stained for viability and death and photographed (red: dead cells; green: live cells). As shown in Figure 6, pVHL-deficient cells were more sensitive to treatment with MMSN@DSPE-PEG-FA and PT / MMSN@DSPE-PEG-FA, with a significant increase in the number of dead cells.

[0094] To further verify in vivo, Renca-scr cells expressing normal pVHL gene, Renca-sh1 cells and Renca-sh4 cells with knockdown of pVHL gene were cultured at 5×10 6BALB / c mice were subcutaneously inoculated with an inoculation density of 100 / mouse. After 30 days, the tumor volume and weight of the pVHL knockdown group (sh1, sh4) were significantly larger than those of the control group (scr) (Figure 7A and B). On the 10th, 12th, and 14th days after subcutaneous inoculation of BALB / c mice, MMSN@DSPE-PEG-FA was injected through the tail vein. The mice were killed on the 20th day, and the in vitro tumors were photographed and weighed. The results showed that the tumor volume and tumor size of mice with pVHL deficiency were significantly reduced after treatment, while there was no obvious therapeutic effect after treatment in mice with normal pVHL expression (Figure 7C and D).

[0095] 6. In vivo anti-tumor activity evaluation

[0096] The nanomaterial prepared in this example was prepared at 5 mg / mL for in vivo anticancer activity testing.

[0097] BALB / c mice were subcutaneously inoculated with 5×10 6 On the 7th, 9th, and 11th days after pVHL gene knockdown, Renca-sh4 cells were injected with PT2385, MSN@DSPE-PEG-FA, and PT / MMSN@DSPE-PEG-FA via the tail vein. The length and width of the tumor cells were measured starting from the 7th day. V (volume) = 1 / 2 × L (length) × W (width) 2 The proliferation curve of tumor volume was obtained, and the mice were killed on day 20, and the in vitro tumor photos were obtained by dissection.

[0098] The results are shown in Figure 8 AB. Compared with the PBS group, PT2385 alone did not exert a significant anti-tumor effect on renal cancer, while MMSN@DSPE-PEG-FA without drug loading showed a significant anti-tumor effect after treatment alone. By comparing the MMSN@DSPE-PEG-FA and PT / MMSN@DSPE-PEG-FA groups, it can be seen that PT2385 and MMSN@DSPE-PEG-FA exhibited a synergistic anti-tumor effect after co-stimulation at the target site.

[0099] C57 / BL6 mice were subcutaneously inoculated with 1×10 6 On the 7th, 9th, and 11th days after the inoculation of B16-sh4 cells, PT2385, MSN@DSPE-PEG-FA, and PT / MMSN@DSPE-PEG-FA were injected into the tail vein, respectively. The length and width of the tumor cells were measured starting from the 7th day. V (volume) = 1 / 2 × L (length) × W (width) 2 The proliferation curve of tumor volume was obtained, and the mice were killed on day 20, and the in vitro tumor photos were obtained by dissection.

[0100] The results showed that compared with the PBS group, the difference in tumor volume after PT2385 treatment alone was not statistically significant (ns), and it did not exert a significant anti-tumor effect on melanoma; while MMSN@DSPE-PEG-FA without drug loading showed a significant anti-tumor effect after treatment alone; and the tumor volume of mice treated with drug-loaded PT / MMSN@DSPE-PEG-FA was the smallest, indicating that after PT2385 and MMSN@DSPE-PEG-FA jointly stimulated the target site, the two exerted a synergistic anti-tumor effect (C and D in Figure 8).

[0101] In summary, the present invention provides a manganese-doped mesoporous silicon nanomaterial, which includes manganese-doped mesoporous silicon nanospheres, an anti-angiogenic drug PT2385 loaded in the pores of the nanospheres, and a surface delivery system DSPE-PEG-FA that wraps the nanospheres. Among them, folic acid (FA) improves the targeting of the nanomaterial to the tumor site; the nanosphere skeleton contains a reducing tetrasulfide bond, which can react with hydrogen peroxide or glutathione abundant in the tumor microenvironment to responsively degrade, and then release manganese ions (Mn 2+ ) and the anti-angiogenic drug PT2385, achieving dual anti-tumor effects by targeting the tumor site to activate local anti-tumor immunity and inhibiting angiogenesis within the tumor, with a synergistic effect. Furthermore, the nanomaterials presented here have no potential toxic effects and exhibit excellent biosafety, providing a new treatment option for tumors, particularly those lacking pVHL.

[0102] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A manganese-doped mesoporous silicon nanomaterial, characterized in that, The nanomaterials include manganese-doped mesoporous silica nanospheres, anti-angiogenic drugs, and a surface delivery system. The anti-angiogenic drugs are adsorbed in the pores of the manganese-doped mesoporous silica nanospheres, and the surface delivery system coats the surface of the manganese-doped mesoporous silica nanospheres; Among them, metal manganese ions are doped in the surface and framework of the mesoporous silica nanospheres; the anti-angiogenic drug is an HIF-2α inhibitor.

2. The manganese-doped mesoporous silicon nanomaterial according to claim 1, characterized in that, The HIF-2α inhibitor includes PT2385.

3. The manganese-doped mesoporous silica nanomaterial according to claim 1, wherein The surface delivery system is folic acid polyethylene glycol phospholipid.

4. A method for preparing a manganese-doped mesoporous silicon nanomaterial, characterized in that, It includes the following steps: S1. Mix cetyltrimethylammonium toluenesulfonate (CTAT), water, and triethanolamine (TEA), stir evenly, quickly add manganese chloride tetrahydrate, then dropwise add tetraethyl orthosilicate (TEOS) and a silane coupling agent, continuously stir, and after centrifugation, washing, and reflux, obtain mesoporous silica nanospheres; S2. Mix the mesoporous silica nanospheres with disodium maleate and an aqueous solution of manganese sulfate, and prepare manganese-doped mesoporous silica nanospheres (MMSN) by hydrothermal method; S3. Mix the MMSN obtained in step S2 with an anti-angiogenic drug, and continuously stir to obtain drug-loaded MMSN; S4. Mix the drug-loaded MMSN obtained in step S3 with a surface delivery system, perform water bath ultrasonic treatment and then centrifugation to obtain targeted drug-loaded MMSN; Among them, the anti-angiogenic drug is an HIF-2α inhibitor.

5. The preparation method of the manganese-doped mesoporous silicon nanomaterial according to claim 4, characterized in that, The silane coupling agent includes TESPT, the HIF-2α inhibitor includes PT2385, and the surface delivery system is folic acid polyethylene glycol phospholipid.

6. The preparation method of the manganese-doped mesoporous silicon nanomaterial according to claim 5, characterized in that, In step S1, the mass ratio of TEOS to TESPT is 5:4; In step S2, the mass ratio of disodium maleate to manganese sulfate is 5:4; In step S3, the mass ratio of MMSN to PT2385 is 5:1 to 50:1; In step S4, the mass ratio of the drug-loaded MMSN to folic acid polyethylene glycol phospholipid is 5:1 to 50:

1.

7. The preparation method of the manganese-doped mesoporous silicon nanomaterial according to claim 4, wherein, In step S1, the solvent for reflux is a mixed solution of methanol and hydrochloric acid.

8. The application of the manganese-doped mesoporous silica nanomaterial according to any one of claims 1-3, or the manganese-doped mesoporous silica nanomaterial prepared by the preparation method according to any one of claims 4-7 in the preparation of anti-tumor drugs.

9. The application according to claim 8, characterized in that, The tumor is a tumor with pVHL deletion or low expression.

10. The application according to claim 9, characterized in that, The tumor includes any one of melanoma and renal cancer.

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