Manganese dioxide nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to manganese dioxide nanoparticles as well as a preparation method and application thereof. The preparation method of the manganese dioxide nano-particles comprises the following steps: mixing tetraethoxysilane, stronger ammonia water and absolute ethyl alcohol, carrying out stirring reaction, after the reaction is finished, centrifugally collecting precipitates, and washing to obtain SiO2 nano-seeds; the SiO2 microspheres are prepared and obtained; the preparation method comprises the following steps: ultrasonically mixing a manganese acetate solution and SiO2 microspheres, dropwise adding an oxidizing agent, and stirring and reacting at 70-95 DEG C for 2-4 hours to obtain MnO2 coated SiO2 core-shell particles; and dialyzing the MnO2 coated SiO2 core shell to obtain the manganese dioxide nanoparticles. According to the invention, SiO2 microspheres with a particle size of 9-11 [mu] m are obtained by a step-by-step / seed growth method, and then a compact MnO2 shell layer with uniform thickness is formed by an adsorption-in-situ oxidation method. And then the manganese dioxide nanoparticles H-MnO2 with extremely low collapse rate and stable structure are obtained through a mild etching method.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, and specifically relates to a manganese dioxide nanoparticle, its preparation method, and its application. Background Technology

[0002] Glioblastoma (GBM), as the most aggressive and deadliest primary brain tumor, is characterized by diffuse infiltration, high proliferative capacity, and resistance to traditional therapies. [1,2] Currently, GBM treatment primarily involves surgical resection combined with radiotherapy and temozolomide (TMZ) chemotherapy. [3] However, under this treatment model, the prognosis for patients is poor, with a median survival of only 14-18 months. [4] The blood-brain barrier (BBB) ​​is a key bottleneck in the treatment of GBM, as its selective permeability severely hinders drug delivery to tumor tissue. [5] Furthermore, GBM typically induces resistance to TMZ through metabolic reprogramming and overexpression of efflux transporters. [6,7] Therefore, improving treatment outcomes has always been a major challenge for those skilled in the art.

[0003] Prior art CN 115054685 A discloses an ultrasound-assisted manganese dioxide vaccine delivery system, its preparation method, and its application. The preparation method of this vaccine delivery system includes the following steps: reacting tetraethyl orthosilicate, concentrated ammonia, and ethanol to obtain SiO2 nanoparticles; then reacting them with potassium permanganate to obtain mesoporous MnO2-coated SiO2; further reacting the mesoporous MnO2-coated SiO2 with Na2CO3 to obtain hollow mesoporous MnO2 nanoparticles; then reacting them with chitosan oligosaccharide solution to obtain MnO2@COS suspension; finally, mixing and incubating MnO2@COS with ovalbumin solution to obtain the vaccine delivery system. However, the vaccine delivery system prepared by this invention requires ultrasound assistance to promote mucosal penetration and cell entry efficiency, thus achieving mucosal vaccine therapy. This limits its practicality.

[0004] Existing technology CN 112370534 A relates to a tumor microenvironment-responsive degradation-type therapeutic nanoprobe and its preparation method. This nanoprobe uses novel mesoporous silica nanoparticles as a template. These nanoparticles are synthesized from tetraethyl orthosilicate, bis-[3-(triethoxysilyl)propyl]-tetrasulfide, surfactant, albumin, sodium hydroxide, ammonium nitrate, and anhydrous ethanol. MnO2 is loaded onto the nanoparticles using potassium permanganate, followed by electrostatic adsorption coating of bovine serum albumin. The fluorescent small molecule CQ4T is then coupled onto the nanoparticles, constructing a nanoprobe capable of multimodal imaging and therapy. However, the rod-shaped nanoprobe used in this invention has a low drug loading capacity, raising concerns about its practicality.

[0005] Existing technology CN 110293232 A discloses a method for preparing silicon-core gold-shell composite nanoparticles using an ultrasound-assisted hydroxylamine hydrochloride seed growth method. This method combines the hydroxylamine hydrochloride seed growth method with sonochemistry. The prepared silicon-core gold-shell composite nanoparticles use silica nanoparticles as the core. Under ultrasonic conditions, polyethyleneimine (PEI) is modified on the surface of the silica particles, causing it to self-assemble into cationic polymers on the silica nanoparticle surface. The strong positive charge of the PEI layer is used to electrostatically adsorb negatively charged small-diameter metal nanoparticles as seed structures. Finally, an ultrasound-assisted "seed growth method" is used to form a continuous and complete gold shell on the surface of the silica particles. However, the drug loading capacity of these gold-shell nanoparticles is relatively small, raising concerns about their practicality.

[0006] References:

[0007] 1. Wu, W., et al., Glioblastoma multiforme (GBM): An overview of current therapeutics and mechanisms of resistance. Pharmacol Res, 2021.171: p.105780.

[0008] 2.Lah, TT, M. Novak, and B. Breznik, Brain malignancies: Glioblastoma and brain metastases. Semin Cancer Biol, 2020.60: p.262-273.

[0009] 3.Khosla, D., Concurrent therapy to enhance radiotherapeutic outcomes in glioblastoma. Ann Transl Med, 2016.4(3):p.54.

[0010] 4. McNamara, MG, et al., Conditional probability of survival and post-progression survival in patients with glioblastoma in the temozolomidetreatment era. J Neurooncol, 2014.117(1):p.153-60.

[0011] 5. Ter Linden, E., et al., Overcoming Barriers in Glioblastoma-Advances in Drug Delivery Strategies. Cells, 2024.13(12).

[0012] 6. Immanuel, SRC, et al., Integrated genetic and metabolic landscapes predict vulnerabilities of temozolomide resistant glioblastoma cells. NPJ SystBiol Appl, 2021.7(1):p.2.

[0013] 7. Robey, RW, et al., Revisiting the role of ABC transporters in multidrug-resistant cancer. Nat Rev Cancer, 2018.18(7):p.452-464. Summary of the Invention

[0014] The purpose of this invention is to provide manganese dioxide nanoparticles that can achieve drug delivery without the assistance of ultrasound, as well as their preparation method and application.

[0015] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0016] A method for preparing manganese dioxide nanoparticles includes the following steps:

[0017] S1. Tetraethyl orthosilicate, concentrated ammonia and anhydrous ethanol are mixed and stirred to react. After the reaction is completed, the precipitate is collected by centrifugation, washed and SiO2 nanoseeds are obtained.

[0018] S2. Disperse SiO2 nanoseeds in anhydrous ethanol to form a seed suspension; mix the seed suspension with concentrated ammonia and anhydrous ethanol to obtain reaction system 1; add a mixed solution of tetraethyl orthosilicate and anhydrous ethanol dropwise to it, stir the reaction, and collect the precipitate by centrifugation after the reaction is completed.

[0019] S3. Mix the precipitate with concentrated ammonia and anhydrous ethanol to obtain reaction system 2; add a mixed solution of tetraethyl orthosilicate and anhydrous ethanol dropwise to it, stir the reaction, and collect the precipitate by centrifugation after the reaction is completed; wash and dry to obtain SiO2 microspheres;

[0020] After ultrasonically mixing S4, manganese acetate solution and SiO2 microspheres for 20-40 min, an oxidant is added dropwise, and the mixture is stirred at 70-95℃ for 2-4 hours to obtain MnO2@SiO2 core-shell particles.

[0021] S5. Suspend MnO2@SiO2 core-shell particles in deionized water and place them in a dialysis bag; then immerse the dialysis bag in Na2CO3 or NaOH solution and react in a water bath at 70-80℃. After the reaction is complete, transfer the dialysis bag to deionized water for dialysis for 24-48 hours to obtain manganese dioxide nanoparticles.

[0022] First, this invention obtains SiO2 microspheres with a controllable particle size range of approximately 9μm-11μm by adjusting the parameters of each component and using a stepwise / seed growth method. This step is crucial, as it yields a large, uniform, and more evenly dispersed SiO2 template, which is fundamental for preparing high-quality final nanoparticles and directly affects the repeatability of subsequent etching and dialysis steps and the uniformity of the final product. Second, existing technologies generally use rapid reduction reactions with strong oxidants, which easily form free MnO2 particles in solution, and the shell growth rate is fast, making it difficult to control uniformity. In contrast, the large-sized and more evenly dispersed SiO2 microsphere template obtained in the previous step more easily adsorbs MnO2 shells. This invention employs an "adsorption-in-situ oxidation" strategy, where MnO2... 2+First, the particles are adsorbed and then slowly and layer by layer transformed into MnO2 under the action of a mild oxidant, depositing on the SiO2 surface. This makes the adsorption and coating on the template surface more precise and uniform, ultimately forming a uniform and dense MnO2 shell that can better withstand the osmotic pressure impact and physical damage (such as high-speed stirring) caused by etching. Furthermore, existing SiO2 template etching techniques are generally "impact" etching methods, where particles are directly exposed to high-concentration alkaline solutions and mechanical stirring, causing physical damage and osmotic pressure impact to the fragile MnO2 shell, easily leading to breakage or collapse. In contrast, this invention uses a "dialysis bag-mediated mild etching method," which uses the diffusion principle driven by concentration gradients. The etchant (OH-) slowly penetrates into the dialysis bag, and the product (silicate) slowly effluxes. The process is gentle, avoiding mechanical shear force and osmotic pressure impact, which can significantly improve the integrity of the hollow sphere morphology and obtain hollow mesoporous manganese dioxide nanoparticles H-MnO2 with extremely low collapse rate, stable structure, and higher purity. Existing technologies for preparing similar hollow MnO2 nanoparticles often result in collapsed particles, inhomogeneous shells, and unstable final product structures, which directly affect drug delivery. A prerequisite for achieving direct and efficient "penetration" and "entry" into cells is a nanoparticle with a complete and stable structure, a uniform and dense shell, and consistent size and morphology. The H-MnO2 nanoparticles prepared in this invention are structurally highly complete, stable, and uniform, enabling them to act as a highly efficient and robust "nanomotor" for physical penetration. By reacting with hydrogen peroxide in the tumor or inflammatory microenvironment, they generate a powerful thrust, allowing the material to directly penetrate the cell membrane layer. In the weakly acidic tumor microenvironment, the manganese dioxide nanoparticles prepared in this invention are reduced to soluble manganese ions (MnO2). 2+ This process causes the entire nanoparticle to disintegrate and dissolve, releasing a high concentration of the drug within onto the cell membrane. At this point, the small-molecule drug can easily enter the cell through diffusion or transport proteins on the cell membrane. Therefore, when using the material of this invention to load drugs into tumor tissues or cells, the drugs can directly penetrate the cell membrane and enter the cell without the need for sonication.

[0023] According to embodiments of the present invention, the present invention can be further optimized, and the optimized technical solution is as follows:

[0024] In one preferred embodiment, the SiO2 microspheres have a particle size of 9-11 μm.

[0025] The particle size of SiO2 microspheres within this range can maximize drug loading; microspheres that are too large will be unstable, while microspheres that are too small will have too low a drug loading rate, affecting the final therapeutic effect.

[0026] In one preferred embodiment, in step S1, the volume ratio of tetraethyl orthosilicate, concentrated ammonia, and anhydrous ethanol is 3-8:8-13:100-200.

[0027] In one preferred embodiment, the reaction is stirred for 6-8 hours in step S1.

[0028] In one preferred embodiment, in step S1, the average particle size of the SiO2 nanoseeds is 300-500 nm.

[0029] In one preferred embodiment, in step S2, SiO2 nanoseeds are dispersed in 50-100 mL of anhydrous ethanol to form a seed suspension.

[0030] In one preferred embodiment, in step S2, the volume ratio of the seed suspension to concentrated ammonia and anhydrous ethanol is 20-100:10-20:200-400.

[0031] In one preferred embodiment, in step S2, the mixed solution of tetraethyl orthosilicate and anhydrous ethanol contains 15-25 mL of tetraethyl orthosilicate and 75-85 mL of anhydrous ethanol.

[0032] In one preferred embodiment, the reaction is stirred for 12-14 hours in step S2.

[0033] In one preferred embodiment, in step S2, the average particle size of the precipitate is 1-2 μm.

[0034] In one preferred embodiment, in step S3, reaction system 2 contains 250-350 mL of anhydrous ethanol and 10-20 mL of concentrated ammonia.

[0035] In one preferred embodiment, in step S3, the mixed solution of tetraethyl orthosilicate and anhydrous ethanol contains 30-40 mL of tetraethyl orthosilicate and 160-180 mL of anhydrous ethanol.

[0036] In one preferred embodiment, in step S3, the reaction is stirred for 24-28 hours.

[0037] In one preferred embodiment, in step S4, the mass ratio of manganese acetate to ultrapure water in the manganese acetate solution is 0.5-1:0.5.

[0038] In one preferred embodiment, in step S4, the mass ratio of manganese acetate solution to SiO2 microspheres is 1:1.0-1.5.

[0039] In one preferred embodiment, in step S4, the oxidant is ammonium persulfate ((NH4)2S2O8).

[0040] In one preferred embodiment, in step S4, the mass ratio of the oxidant to manganese acetate is 1.0-1.5:1.0.

[0041] In one preferred embodiment, in step S4, the oxidant is present in the form of an aqueous solution, wherein the mass ratio of oxidant to water in the oxidant solution is 1.0-1.5:0.5.

[0042] In one preferred embodiment, in step S4, the stirring speed of the reaction is 200-250 rpm.

[0043] Ammonium persulfate is used as a mild oxidant. In this invention, manganese acetate (Mn(OAc)2) solution is slowly oxidized under low stirring, and a controlled oxidation deposition method is used to adsorb a MnO2 shell on the template surface.

[0044] In one preferred embodiment, in step S5, the dialysis bag is permeable to molecular weights of 12,000-15,000 Da.

[0045] In one preferred embodiment, in step S5, the concentration of the Na2CO3 or NaOH solution is 0.5-1M.

[0046] In one preferred embodiment, in step S5, the reaction is carried out in a water bath for 18-24 hours.

[0047] In step S5, a mild etching method mediated by a dialysis bag is used to remove residual alkali and silicates, thereby obtaining H-MnO2 with a stable structure and higher purity.

[0048] Based on the same inventive concept, the present invention also claims protection for manganese dioxide nanoparticles prepared by the preparation method.

[0049] Based on the same inventive concept, the present invention also claims a delivery nanoparticle, the raw materials of which, by weight, include: 8-12 parts of the manganese dioxide nanoparticles, 8-15 parts of polyallylamine hydrochloride, 8-12 parts of N-hydroxysuccinimide-polyethylene glycol-maleimide, and 3-10 parts of tLyP-1 peptide.

[0050] In one preferred embodiment, in N-hydroxysuccinimide-polyethylene glycol-maleimide, the molecular weight of polyethylene glycol is 2000-4000.

[0051] NHS-PEG-MA1, N-hydroxysuccinimide-polyethylene glycol-maleimide, is a bifunctional polyethylene glycol derivative. It combines the high reactivity of N-hydroxysuccinimide ester (reacting with amino groups) and the specific reactivity of maleimide (reacting with thiol groups). The polyethylene glycol moiety provides good water solubility and biocompatibility, connecting these two active functional groups and enhancing the stability and dispersibility of the entire molecule in aqueous environments.

[0052] tLyp-1 (truncated LyP-1) is a tumor-homing transmembrane peptide. The truncated Lyp-1 (similar to iRGD, a cyclic tumor-homing non-peptide) was obtained through phage screening technology and has a C-endRule (CendR) fragment CGNKRTR. It can specifically target and bind to NRP proteins that are highly expressed in tumors, and then induce extravasation and tissue penetration through the mechanism of cell internalization.

[0053] Based on the same inventive concept, the present invention also claims protection for a method for preparing the delivered nanoparticles, comprising the following steps:

[0054] The manganese dioxide nanoparticles were ultrasonically mixed with polyallylamine hydrochloride, and then coated with N-hydroxysuccinimide-polyethylene glycol-maleimide. Finally, tLyP-1 peptide was added to the nanoparticles for coupling reaction. After the reaction was completed, the nanoparticles were washed and resuspended to obtain the final product, the delivery nanoparticles.

[0055] Based on the same inventive concept, the present invention also claims protection for the use of the delivery nanoparticles in the delivery of temozolomide (TMZ) and / or si-SLC16A1.

[0056] Based on the same inventive concept, the present invention also claims protection for the use of the delivered nanoparticles in the preparation of drugs for treating glioblastoma.

[0057] Therefore, this invention constructs a multifunctional delivery system for tLyP-1 modified polyethylene glycol (PEG)-coated manganese dioxide (MnO2) nanozymes. tLyP-1 is a targeting peptide with tumor-penetrating capabilities, which can enhance blood-brain barrier penetration and glioma-targeted accumulation through neuropilin-1 (NRP-1) receptor-mediated endocytosis. PEG modification improves the biostability and circulating half-life of nanoparticles while effectively reducing premature clearance rates. This invention synthesizes and characterizes multifunctional nanoparticles, evaluates their in vitro cellular uptake and gene silencing efficiency, and examines their in vivo therapeutic effects in an orthotopic glioma model. Extensive experimental data demonstrate that the engineered nanozyme system of this invention can achieve synergistic targeted delivery of temozolomide (TMZ) and si-SLC16A1, thereby integrating multiple functions such as chemotherapy, gene silencing, hypoxia regulation, and MRI-guided drug tracking. Attached Figure Description

[0058] Figure 1 This is a scanning electron microscope (SEM) image of MnO2-SiO2 nanoparticles.

[0059] Figure 2 Transmission electron microscopy (TEM) images of H-MnO2 and HM@si-SLC16A1 / TMZ-tLyP-1 nanoparticles; Figure 2 A is a TEM image of H-MnO2; where Figure 2 B is a TEM image of HM@si-SLC16A1 / TMZ-tLyP-1 nanoparticles.

[0060] Figure 3 A bar chart of zeta potential measurements.

[0061] Figure 4 This is an X-ray photoelectron spectroscopy (XPS) image.

[0062] Figure 5 Line graph showing the degradation of MnO2-based nanoparticles, drug release, and oxygen generation.

[0063] Figure 6 A bar graph for detecting cell viability in the CCK-8 assay.

[0064] Figure 7 Fluorescence pattern for assessing cytotoxicity in live / dead cell staining experiments.

[0065] Figure 8 Fluorescence image for assessing blood-brain barrier penetration in focused laser scanning microscopy (CLSM) images.

[0066] Figure 9 A bar graph for flow cytometry detection of cell apoptosis.

[0067] Figure 10 Fluorescence images of HIF-1α expression levels were obtained by confocal microscopy (CLSM).

[0068] Figure 11 A bar graph showing the changes in mitochondrial membrane potential detected by flow cytometry using the JC-10 fluorescent probe.

[0069] Figure 12 The in vivo MRI treatment follow-up results of a rat model of glioma after administration of HM@si-SLC16A1 / TMZ and HM@si-SLC16A1 / TMZ-tLyP-1 nanoparticles; among which Figure 12 A is an MRI scan of nanoparticles under T1 signal; Figure 12 B is the MRI scan image of the nanoparticles under T2 signal; Figure 12 C is a line graph of the T1 signal intensity of the nanoparticles in MRI scans; Figure 12 D is a line graph of the T2 signal intensity of the nanoparticles in an MRI scan.

[0070] Figure 13 In vitro fluorescence imaging of Cy5.5 labeled Cy5.5.

[0071] Figure 14 For monitoring tumor progression: T2-weighted MRI scans on days 1, 5, and 10 after injection.

[0072] Figure 15 Survival analysis curves performed using the Kaplan-Meier method.

[0073] Figure 16 Hematoxylin-eosin (H&E) staining images for histological analysis of major organs.

[0074] Figure 17 Image of hematoxylin-eosin (H&E) staining on a glioma tissue section.

[0075] Figure 18 The histological and immunohistochemical evaluation results of glioma tissues after treatment with different nanoparticles are shown; among them, Figure 18 A shows the TUNEL staining images of apoptotic cells in each group after treatment. Figure 18 B shows the IHC staining of Caspase-3, Ki67, and HIF-1α in apoptotic cells after treatment in each group; Figure 18 C is a bar chart of TUNEL staining of apoptotic cells in each group after treatment; Figure 18 D is a bar chart showing the expression levels of Caspase-3, Ki67, and HIF-1α in apoptotic cells after treatment in each group.

[0076] Figure 19 The bar chart shows the changes in the levels of liver function markers (ALT, AST) and kidney function markers (BUN, Cr) after 10 days of treatment. Detailed Implementation

[0077] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0078] The test reagents used in this invention include:

[0079] Potassium permanganate aqueous solution (KMnO4, catalog number #223468, Sigma-Aldrich, St. Louis, USA); silica nanoparticle suspension (catalog number #791342, Sigma-Aldrich); sodium carbonate solution (Na2CO3, catalog number #223484, Sigma-Aldrich); polyallylamine hydrochloride (PAH, molecular weight ≈15,000, Sigma-Aldrich); NHS-PEG2000-MAI (catalog number #PG2-MLNS-2k, Nanocs, New York, USA); maleimide-thiol reaction to tLyP-1 peptide (catalog number #HY-P10787, Med Chem Express, New Jersey, USA); SYBR Green nucleic acid dye (catalog number #A25743, Thermo Fisher). Scientific (Scientific Company); Fetal bovine serum (FBS, Gibco, Grand Island, USA); Temozolomide (TMZ, catalog number HY-17364, mce); si-SLC16A1 (purchased from Hunan Aorui Biotechnology Co., Ltd.); scrambled (si-SLC16A1 control, purchased from Hunan Aorui Biotechnology Co., Ltd.); Penicillin / Streptomycin (Sigma-Aldrich, St. Louis, Missouri, USA); Endothelial cell growth additive (ECGS, catalog number 1052, ScienCell); Endothelial cell culture medium (catalog number 1001, ScienCell, Carlsbad, California, USA); TRIzol reagent (catalog number 15596018, Thermo Fisher Scientific); Lipo 2000 (catalog number 12566014, Thermo Fisher Scientific); cDNA synthesis kit (catalog number K1622, Thermo Fisher Scientific); SYBR Green PCR premix (Catalog No. A25742, Thermo Fisher Scientific); RIPA lysis buffer (P0013C, Beyotime, Shanghai, China); protease inhibitor (P1046, Beyotime); PVDF membrane (Millipore, Burlington, Massachusetts, USA); SLC16A1 primary antibody (1:1000, Catalog No. 20139-1-AP, Proteintech, Wuhan, China); GAPDH primary antibody (1:50000, Catalog No. 60004-1-Ig, Proteintech); ECL chemiluminescence kit (P0018AS, Beyotime); CCK-8 kit (Dojindo, Japan); Calcein-AM / PI live / dead cell double staining kit (Catalog No. L3224, Thermo Fisher Scientific); Transwell chamber (0.4μm pore size (Corning, USA); Annexin V-FITC / propidium iodide (PI) apoptosis detection kit (BD Biosciences, San Jose, USA); anti-HIF-1α primary antibody (Abcam, catalog number ab51608); JC-10 mitochondrial membrane potential detection kit (Thermo Fisher Scientific, catalog number T3168); UNEL apoptosis detection kit (C1098, Beyotime); DAB chromogenic solution (Dako, Copenhagen, Denmark); alanine aminotransferase (ALT, catalog number E-BC-K235-M, Wuhan Elairite); aspartate aminotransferase (AST, catalog number E-BC-K236-M, Wuhan Elairite); blood urea nitrogen (BUN, catalog number C011-2-1, Nanjing Jiancheng Biotechnology Institute); creatinine (Cr, catalog number C013-1-1, Nanjing Jiancheng Biotechnology Institute).

[0080] The experimental equipment used in this invention includes:

[0081] Field emission scanning electron microscope (TALOSF200S, Thermo Fisher Scientific, Waltham, USA); transmission electron microscope (Verios 5XHR SEM, Thermo Fisher Scientific); dynamic light scattering system (Zetasizer Ultra, Malvern Panalytical, Malvern, UK); Zetasizer Ultra analyzer (Malvern Panalytical); X-ray photoelectron spectroscopy (XPS, Thermo Fisher Scientific, Waltham, USA); ultraviolet transmission electron microscope (Bio-Rad, Hercules, USA); dynamic light scattering system (Zetasizer Ultra, Malvern Panalytical); high performance liquid chromatography (HPLC, Agilent Technologies, Santa Clara, California, USA); NanoDrop spectrophotometry (Thermo Fisher Scientific); dissolved oxygen analyzer (Thermo Fisher Scientific). Scientific; Quantitative Real-Time PCR System (Applied Biosystems, Waltham, Massachusetts, USA); SDS-PAGE Electrophoresis System (Bio-Rad, Herlack, California, USA); Microplate Reader (Model VLBLATGD2, Thermo Fisher Scientific); Fluorescence Microscope (Leica Microsystems, Wetzlar, Germany); Flow Cytometer (BD FACSAria, BD Biosciences); Stereotype Analyzer (Reward Life Sciences, Shenzhen, China); Miniature Electric Skull Drill (Stoelting, Wooddale, Illinois, USA); Hamilton Microsyringe (Hamilton Company, Reno, Nevada, USA); 3.0T MRI Scanner (Bruker, Ettlingen, Germany); IVIS Spectrum In vivo Imaging System (PerkinElmer, Waltham, Massachusetts, USA); Optical Microscope (Olympus, Tokyo, Japan); Automated Biochemistry Analyzer (Beckman Coulter, Brea, California, USA); Fully Automated Blood Biochemistry Analyzer (Roche).

[0082] The experimental cells used in this invention include:

[0083] Brain microvascular endothelial cells (BMECs) were purchased from Bio-Innovation Technology Co., Ltd. (catalog number ABC-TC3524, Beijing, China). C6 glioma cells were purchased from the American Type Culture Collection (ATCC catalog number CCL-107, Manassas, Virginia, USA). Both cell lines were used to evaluate gene silencing efficiency and cellular responses to MnO2 nanoparticles.

[0084] Description of the experimental animals used in this invention:

[0085] All animal experiments were conducted in strict accordance with the guidelines of the Institute of College Animal Care and Use (IACUC) and the standards of the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals. The experimental procedures were approved by the Animal Ethics Committee. The Wistar rats used in the experiments (male, 6-8 weeks old, weighing 220-250g) were purchased from Hunan Slack Jingda Laboratory Animal Co., Ltd. (Changsha, China) and housed in a specific pathogen-free (SPF) environment with constant environmental conditions: temperature 22±2℃, 12-hour light-dark cycle, humidity 50±10%, and free access to food and water.

[0086] The data analysis principles of this invention are as follows:

[0087] All experiments were repeated three times (unless otherwise specified), and data are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using GraphPad Prism 9.0 software (GraphPad Software, San Diego, California, USA). Normality of the data was first assessed using the Shapiro-Wilk test and the Kolmogorov-Smirnov test: for normally distributed data, unpaired two-tailed Student's t-test was used for comparisons between two groups, and one-way or two-way ANOVA combined with Tukey's post-hoc test was used for comparisons among multiple groups; for non-normally distributed data, the Mann-Whitney U test (for two groups) or the Kruskal-Wallis test combined with Dunn's post-hoc test (for multiple groups) was used.

[0088] Survival analysis was performed using the Kaplan-Meier method, and differences in survival curves between groups were assessed using the Log-rank test (also known as the Mantel-Cox test). A p-value < 0.05 was considered statistically significant.

[0089] Example 1

[0090] The synthesis of HM@si-SLC16A1 / TMZ-tLyP-1 nanoparticles includes three steps, as follows:

[0091] Improve Method: In a reaction vessel containing 150 mL of anhydrous ethanol (product number Y263010, Beyotime) and 10 mL of concentrated ammonia (product number FF362, Beyotime) (25 wt%), 5 mL of tetraethyl orthosilicate (TEOS, product number T110595, Aladdin) was rapidly added under constant temperature of 30°C and mechanical stirring at 200 rpm. After the reaction continued for 6 hours, the product was collected by centrifugation (8000 rpm, 10 min) and washed three times with anhydrous ethanol. Finally, the obtained SiO2 nanoseeds (average particle size of approximately 400 nm) were redispersed in 60 mL of anhydrous ethanol to form a seed suspension. The above 60 mL seed suspension was added to a new reaction system containing 300 mL of anhydrous ethanol and 15 mL of concentrated ammonia. Under constant temperature of 30°C and mechanical stirring at 150 rpm, a mixed solution consisting of 20 mL of TEOS and 80 mL of anhydrous ethanol was added dropwise over 8 hours using a syringe pump. After the addition was complete, the reaction continued for 12 hours. The product was collected by centrifugation (3000 rpm, 5 min), at which point the average particle size was approximately 2 μm. All the product obtained in the previous round was redispersed in a new reaction system containing 300 mL of anhydrous ethanol and 15 mL of concentrated ammonia. Under constant temperature of 30 °C and mechanical stirring at 100 rpm, a mixed solution consisting of 35 mL of TEOS and 180 mL of anhydrous ethanol was added dropwise over 10 hours using a syringe pump. After the addition was complete, the reaction continued for 24 hours to ensure complete growth. Finally, the product was collected by low-speed centrifugation (1000 rpm, 5 min), washed three times sequentially with anhydrous ethanol and ultrapure water, and dried in a 60 °C oven for 12 hours to obtain SiO2 microsphere templates with an average particle size of 11 μm.

[0092] Mn(II) precursor controlled oxidation deposition method: Template dispersion: The prepared SiO2 nanotemplate was ultrasonically dispersed in water. Precursor addition: Manganese acetate (Mn(OAc)2) solution was added and stirred for 30 minutes to allow it to be fully adsorbed on the negatively charged SiO2 surface. Controlled oxidation: The reaction system was heated to 85℃, and then ammonium persulfate ((NH4)2S2O8) solution was slowly added dropwise as an oxidant. Rate-controlled reaction: The reaction was carried out in a constant temperature oil bath at 80℃ with low-speed stirring (200 rpm) for 2 hours.

[0093] Manganese acetate (Mn(OAc)2) solution: The solution was prepared with SiO2 template mass: manganese acetate (item number M131577, Aladdin): ultrapure water in a ratio of 1:1:0.5.

[0094] Ammonium sulfate ((NH4)2S2O8) solution: Based on the amount of manganese acetate to be used, calculate and weigh the required amount of ammonium persulfate (item number A112449, Aladdin) at a molar ratio of approximately 1:1.5, and quickly dissolve it in ultrapure water.

[0095] Dialysis bag-mediated mild etching: Sample loading: MnO2@SiO2 core-shell particles are suspended in a small amount of deionized water and placed in a dialysis bag (e.g., MWCO 14,000 Da). Mild etching: The sealed dialysis bag is completely immersed in a beaker containing 1M Na2CO3 or 1.5M NaOH. Diffusion control: The entire system is placed in an 80°C water bath and reacted for 20 hours under static or very low stirring (50 rpm). Purification: After the reaction, the dialysis bag is transferred to a large amount of deionized water for dialyzing for 36 hours to remove residual alkali and silicates, yielding H-MnO2 nanoshells.

[0096] The surface modification experimental steps are as follows: 10 mg of H-MnO2 nanoshells were ultrasonically mixed with 12 mg of polyallylamine hydrochloride for 2 h. Subsequently, the nanoparticles were coated with 11 mg of NHS-PEG2000-MA1 under ultrasonic conditions for 2 h. Finally, 6 mg of tLyP-1 peptide was coupled to the nanoparticles for 24 h via a maleimide-thiol reaction. The prepared tLyp-1-MnO2-PEG solution was collected by centrifugation, washed three times with water to remove impurities, and vacuum dried at 40 °C to obtain tLyp-1-MnO2-PEG solid, denoted as HM@tLyP-1.

[0097] The steps for sequentially loading siRNA and TMZ are as follows:

[0098] 10 mg of tLyp-1-MnO2-PEG was added to 5 ml of ultrapure water to prepare a nanoparticle dispersion. Then, 10 mg of TMZ was dissolved in 220 mg of dimethyl sulfoxide (DMSO), and this solution was slowly added dropwise to the nanoparticle dispersion. Simultaneously, 0.266 mg of si-SLC16A1 was dissolved in water and added to the mixture. The mixture was stirred at room temperature in the dark for 12 hours to ensure adequate loading. After the reaction was complete, the solution was centrifuged at 12000 rpm for 10 minutes, and the supernatant was discarded. The precipitate was washed twice with ultrapure water to remove unloaded drug, and finally dried under vacuum at 40 °C to obtain the final product, HM@si-SLC16A1 / TMZ-tLyP-1 nanoparticles.

[0099] Other methods for preparing nanoparticles are as follows:

[0100] 1. Preparation of HM@si-SLC16A1 / TMZ nanoparticles

[0101] 10 mg of H-MnO2 nanoshells were added to 5 ml of ultrapure water to prepare a nanoparticle dispersion. Then, 10 mg of TMZ was dissolved in 220 mg of dimethyl sulfoxide (DMSO), and this solution was slowly added dropwise to the nanoparticle dispersion. Simultaneously, 0.266 mg of Si-SLC16A1 was dissolved in water and added to the mixture. The mixture was stirred at room temperature in the dark for 12 hours to ensure adequate loading. After the reaction was complete, the solution was centrifuged at 12000 rpm for 10 minutes, and the supernatant was discarded. The precipitate was washed twice with ultrapure water to remove unloaded drug, and finally dried under vacuum at 40 °C to obtain the final product, HM@si-SLC16A1 / TMZ nanoparticles.

[0102] 2. Preparation of HM@TMZ-tLyP-1 nanoparticles

[0103] 10 mg of tLyp-1-MnO2-PEG was added to 5 ml of ultrapure water to prepare a nanoparticle dispersion. Subsequently, 10 mg of TMZ was dissolved in 220 mg of dimethyl sulfoxide (DMSO), and this solution was slowly added dropwise to the above nanoparticle dispersion. The mixture was stirred at room temperature in the dark for 12 hours to ensure sufficient loading. After the reaction was completed, the solution was centrifuged at 12000 rpm for 10 minutes, and the supernatant was discarded. The precipitate was washed twice with ultrapure water to remove unloaded drug, and finally dried under vacuum at 40 °C to obtain HM@TMZ-tLyP-1 nanoparticles.

[0104] 3. HM@si-SLC16A1-tLyP-1 nanoparticles

[0105] 10 mg of tLyp-1-MnO2-PEG was added to 5 ml of ultrapure water to prepare a nanoparticle dispersion. Then, 0.266 mg of si-SLC16A1 was dissolved in water, and this solution was slowly added dropwise to the nanoparticle dispersion. The mixture was stirred at room temperature in the dark for 12 hours to ensure adequate loading. After the reaction was complete, the solution was centrifuged at 12000 rpm for 10 minutes, and the supernatant was discarded. The precipitate was washed twice with ultrapure water to remove unloaded drug, and finally dried under vacuum at 40 °C to obtain HM@si-SLC16A1-tLyP-1 nanoparticles.

[0106] The prepared nanoparticles were characterized as follows:

[0107] 1. Scanning electron microscopy (SEM) analysis

[0108] The morphology of the nanoparticles was observed using field emission scanning electron microscopy. The specific sample preparation method was as follows: a suspension of nanoparticles was dropped onto a silicon wafer substrate, dried at room temperature, and then platinum was sputtered to enhance conductivity. Finally, the sample was imaged at an accelerating voltage of 5 kV.

[0109] SEM scans showed that the MnO2-SiO2 nanoparticles were spherical with a rough surface, confirming that Mn had been successfully deposited on the surface of the SiO2 spheres. Figure 1 ).

[0110] 2. Transmission electron microscopy (TEM) analysis

[0111] The specific sample preparation method is as follows: The nanoparticle suspension is dropped onto a carbon film copper mesh (200 mesh), dried at room temperature, and then the internal structure of the nanoparticles is analyzed by imaging with a transmission electron microscope at an accelerating voltage of 200 kV.

[0112] TEM observation showed that the H-MnO2 nanoparticles have a typical hollow mesoporous structure, indicating that the silica core has been successfully removed. Figure 2 A). TEM images of HM@si-SLC16A1 / TMZ-tLyP-1 nanoparticles after sequential loading of siRNA and TMZ show that they still retain the complete hollow mesoporous structure characteristics. Figure 2 B).

[0113] 3. Measurement of Zeta potential value

[0114] The surface charge of nanoparticles was determined using a Zetasizer Ultra analyzer. The specific method was as follows: a nanoparticle suspension (1 mg / mL, prepared with deionized water) was injected into a disposable folded capillary sample cell, and the sample was detected under constant temperature conditions of 25°C. The Zeta potential was calculated using the Smoluchowski equation.

[0115] Zeta potential analysis showed that with siRNA loading and tLyP-1 modification, the surface potential of the nanoparticles gradually decreased from -0.417 mV to -23.3 mV (HM@si-SLC16A1 / TMZ-tLyP-1). Figure 3 ).

[0116] 4. X-ray photoelectron spectroscopy (XPS) analysis

[0117] X-ray photoelectron spectroscopy was used to analyze the elemental composition of the nanoparticle surface. Specific detection conditions were as follows: After the sample was fixed on a dedicated sample stage, it was subjected to vacuum conditions (≤5×10⁻⁶). -9The mbar was excited by a monochromatic Al Kα source (1486.6 eV), and the characteristic binding energy spectra of Mn 2p, O 1s and C 1s were acquired and analyzed using CasaXPS software.

[0118] XPS detected characteristic peaks for Mn and O elements, with 641.8 eV and 653.5 eV corresponding to the Mn 2p3 / 2 and Mn 2p1 / 2 orbitals, respectively. The O 1s peaks at 529.8 eV and 532.9 eV were attributed to Mn-O-Mn and Mn-OH bonds, respectively. Characteristic peaks of 284.6 eV (C–C / C=C), 286.4 eV (C–O), and 287.0 eV (C=O) appeared in the C 1s spectrum. Figure 4 ).

[0119] These results confirm that the nanoparticles can maintain structural integrity under physiological conditions and have good long-term cycling potential.

[0120] Example 2

[0121] Drug performance evaluation of manganese dioxide (MnO2) based nanoparticles

[0122] 1. Degradation and drug release behavior of manganese dioxide (MnO2) based nanoparticles

[0123] To evaluate the degradation and drug release behavior of manganese dioxide (MnO2)-based nanoparticles under simulated tumor microenvironment (TME) conditions, HM@si-SLC16A1 / TMZ-tLyP-1 nanoparticles were placed in phosphate-buffered saline (PBS, pH 7.4 and pH 6.5) with different concentrations of H2O2 (50, 100, 150, and 200 μM) and incubated at 37 °C with shaking. The cumulative release of temozolomide (TMZ) and small interfering RNA (si-SLC16A1) was quantitatively detected by high-performance liquid chromatography (HPLC) and NanoDrop spectrophotometry at 0, 12, 24, 36, and 48 h.

[0124] 2. Oxygen release capacity analysis

[0125] To evaluate the ability of manganese dioxide (MnO2)-based nanoparticles to release O2 under tumor-like hypoxic conditions, the nanoparticles were incubated with phosphate-buffered saline (PBS, pH 7.4 and pH 6.5) containing 50, 100, 150, and 200 μM H2O2. Dissolved oxygen levels were monitored over 30 min using a dissolved oxygen meter, with measurements taken every 5 min.

[0126] The results are as follows Figure 5As shown in the figure. The results showed that TMZ release gradually increased over time, and after 48 hours, the cumulative release in the acidic environment (pH 6.5) reached approximately 60%, significantly higher than the 45% release under physiological conditions (pH 7.4). Figure 5 A) indicates that an acidic tumor microenvironment can effectively promote drug release. Similarly, siRNA release showed a similar trend, with approximately 55% released after 48 hours at pH 6.5, compared to 40% at pH 7.4. Figure 5 B) This further confirms that the acidic tumor microenvironment is conducive to siRNA release. To evaluate the oxygen release capacity of MnO2 nanoparticles, they were incubated with different concentrations of H2O2 (50-200 μM) at pH 6.5 and pH 7.4, respectively. The results showed that the oxygen generation significantly increased with increasing H2O2 concentration and reaction time. At pH 6.5, the oxygen concentration reached a peak of 0.18 μM after 30 min of treatment with 200 μM H2O2; while under the same conditions, the maximum oxygen release in the pH 7.4 group was only 0.03 μM. Figure 5 C), this result confirms that MnO2 has significant tumor microenvironment-responsive catalytic activity.

[0127] 3. Cell Culture

[0128] C6 glioma cells were cultured in DMEM containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. BMECs were cultured in endothelial cell-specific medium containing 5% fetal bovine serum, 1% endothelial cell growth additive, 100 U / mL penicillin, and 100 μg / mL streptomycin. Both cell lines were cultured at 37°C and 5% CO2. After reaching 80–90% confluence, the cells were passaged using 0.25% trypsin-EDTA. Pre-experimental testing confirmed the cells were free of mycoplasma contamination.

[0129] 4. Cell viability assay

[0130] The cytotoxicity of HM@si-SLC16A1 / TMZ-tLyP-1 nanoparticles was detected using a CCK-8 assay kit, with procedures strictly following the manufacturer's instructions. C6 glioma cells were cultured at 5 × 10⁶ cells per well. 3Cells were seeded at a density of 1000 μM in 96-well plates and cultured overnight at 37°C and 5% CO2. Cells were then treated with 200 μM PBS and TMZ, 0.5 mg of HM@TMZ-tLyP-1, HM@si-SLC16A1-tLyP-1, HM@si-SLC16A1 / TMZ, or HM@si-SLC16A1 / TMZ-tLyP-1 for 24 hours. After treatment, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance at 450 nm was measured using a microplate reader. To assess cell viability under normoxic (21% O2) and hypoxic (1% O2) conditions, C6 glioma cells were first cultured in the corresponding oxygen concentrations for 24 hours before the CCK-8 assay was performed.

[0131] The results are as follows Figure 6 As shown, the results indicated that, compared with the PBS group, cell viability was significantly reduced in all treatment groups except for the HM@si-SLC16A1-tLyP-1 nanoparticle group. Among them, the HM@si-SLC16A1 / TMZ-tLyP-1 group exhibited the strongest cytotoxicity due to the synergistic effect of TMZ and si-SLC16A1. Figure 6 ).

[0132] 5. Cell live / dead staining detection

[0133] Cell viability was further assessed using the Calcein-AM / PI live / dead cell double staining kit. C6 glioma cells were cultured at 2 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 cells / well in 24-well plates and treated with the same experimental group for 24 h. After washing with PBS, the cells were stained at 37 °C for 30 min in the dark with 2 μM Calcein-AM and 4 μM propidium iodide. Images were acquired using a fluorescence microscope. Live cells showed green fluorescence (Calcein-AM positive), while dead cells showed red fluorescence (PI positive).

[0134] The HM@si-SLC16A1 / TMZ and HM@si-SLC16A1 / TMZ-tLyP-1 nanoparticle treatment groups showed the highest proportion of dead cells (PI-positive, red fluorescence), while the PBS and TMZ treatment groups mainly showed green fluorescence (a marker of viable cells). Figure 7 Quantitative analysis of fluorescence intensity further showed that, compared with the other groups, the cell viability of the HM@si-SLC16A1 / TMZ and HM@si-SLC16A1 / TMZ-tLyP-1 treatment groups was significantly reduced, with HM@si-SLC16A1 / TMZ-tLyP-1 exhibiting the strongest cytotoxic effect. Figure 7 ).

[0135] 6. Blood-brain barrier penetration test

[0136] This invention establishes a Transwell chamber for assessing the blood-brain barrier penetration ability of a BMEC-C6 glioma cell co-culture model. Specifically, brain microvascular endothelial cells (BMECs) were seeded in the upper chamber, and C6 glioma cells were seeded in the lower chamber. After co-culturing for 48 hours, Cy5-labeled HM@si-SLC16A1 / TMZ-tLyP-1 nanoparticles were added to the upper chamber. After incubation for 6 hours, the fluorescence intensity in the lower chamber was detected using a microplate reader. Furthermore, the distribution of the nanoparticles in the co-culture model was observed using a laser confocal microscope.

[0137] Cellular uptake of nanoparticles was observed using confocal laser scanning microscopy (CLSM). Results showed that both siSLC16A1-FAM (green fluorescence) and Cy5-labeled nanoparticles (red fluorescence) signals were detected in both BMEC and C6 cells. CLSM imaging confirmed that Cy5-labeled nanoparticles could effectively cross the BMEC monolayer barrier and be internalized by C6 cells, indicating their highly efficient blood-brain barrier penetration capability. Figure 8 ).

[0138] 7. Flow cytometry

[0139] To detect apoptosis levels, C6 glioma cells were treated under normoxic and hypoxic conditions for 24 h, respectively, and then stained using the Annexin V-FITC / propidium iodide (PI) apoptosis detection kit. The proportions of early and late apoptotic cells were determined by flow cytometry, and the data were analyzed using FlowJo software.

[0140] The results showed that HM@si-SLC16A1 / TMZ-tLyP-1 treatment induced the highest proportion of apoptotic cells under both normoxic and hypoxic conditions, with the pro-apoptotic effect being more significant under normoxic conditions. Figure 9 Quantitative analysis showed that the apoptosis rate of cells treated with HM@si-SLC16A1 / TMZ-tLyP-1 reached approximately 60% under normoxic conditions and 50% under hypoxic conditions, fully demonstrating that the nanoparticles have a significant pro-apoptotic effect. Figure 9 ).

[0141] 8. Immunofluorescence staining

[0142] To assess the hypoxia-regulated effect, C6 glioma cells were treated under normoxic (21% O2) or hypoxic (1% O2) conditions for 24 h, and then fixed with 4% paraformaldehyde. They were then incubated overnight at 4°C with anti-HIF-1α primary antibody, followed by incubation with FITC-labeled secondary antibody in the dark. The cell nuclei were counterstained with DAPI, and images were finally acquired using a laser confocal microscope.

[0143] The results showed that under hypoxic conditions, cells treated with PBS and TMZ exhibited strong HIF-1α positive signals, while treatment with HM@si-SLC16A1 / TMZ and HM@si-SLC16A1 / TMZ-tLyP-1 significantly reduced HIF-1α expression. Figure 10 This indicates that nanoparticles can effectively alleviate the hypoxic state of glioma cells.

[0144] 9. Mitochondrial membrane potential detection (JC-10 staining method)

[0145] Mitochondrial dysfunction was assessed using the JC-10 mitochondrial membrane potential assay kit. C6 glioma cells were treated under normoxic (21% O2) or hypoxic (1% O2) conditions for 24 hours, respectively, and then stained with JC-10. Fluorescence intensity changes were detected by flow cytometry, and the red (aggregated) / green (monomerized) fluorescence ratio was calculated to analyze the degree of mitochondrial depolarization.

[0146] The results showed that compared with the PBS group, TMZ treatment induced moderate mitochondrial damage, while HM@si-SLC16A1 / TMZ and HM@si-SLC16A1 / TMZ-tLyP-1 treatments caused significant mitochondrial depolarization, manifested by a significantly increased red / green fluorescence ratio. Figure 11 Notably, HM@si-SLC16A1 / TMZ-tLyP-1 had a similar effect on mitochondrial membrane potential under normoxic and hypoxic conditions, indicating that the Mn released by the nanoparticles... 2+ The damage of ions to mitochondrial function is not affected by oxygen concentration. In summary, HM@si-SLC16A1 / TMZ-tLyP-1 nanoparticles exert a potent anti-tumor effect through a synergistic mechanism of significantly reducing cell viability, promoting apoptosis, alleviating the hypoxic tumor microenvironment, and disrupting mitochondrial function.

[0147] 10. Construction of an in situ glioma model

[0148] To establish an in situ glioma model, Wistar rats were anesthetized with a 2% isoflurane-oxygen mixture and fixed in a stereotaxic apparatus. After incising the scalp along the midline, a miniature electric craniotomy drill was used to drill a hole in the skull (location coordinates: 2.5 mm lateral to the anterior fontanelle, depth 3 mm). A suspension of C6 glioma cells (5 × 10⁻⁶ cells / mL) was injected using a Hamilton microsyringe. 5 A 5 μL PBS solution was slowly injected into the striatum over a period of 5 minutes to ensure uniform cell distribution. The needle was left in place for 5 minutes before being slowly withdrawn to prevent reflux. The incision was sutured, and the rats were monitored until they regained consciousness. Tumor formation was confirmed by T2-weighted MRI scan 5 days after inoculation. Only rats with tumor formation were randomly assigned to the experimental groups.

[0149] 11. In vivo MRI monitoring for glioma treatment

[0150] To evaluate the in vivo biodistribution and tumor-targeting ability of HM@si-SLC16A1 / TMZ-tLyP-1 nanoparticles, the nanoparticles were injected into a rat model of orthotopic glioma via tail vein injection. Imaging was performed using a 3.0T MRI scanner before injection and at 2, 6, 12, 24, and 72 hours post-injection. To monitor tumor progression in the rats with orthotopic glioma, MRI scans were performed on days 1, 3, 5, 7, and 10 post-injection. Tumor volume was quantified using T2-weighted images, and the trend over time was analyzed using ImageJ software.

[0151] T1-weighted imaging was used to evaluate the contrast enhancement effect on tumors after intravenous injection of HM@si-SLC16A1 / TMZ or HM@si-SLC16A1 / TMZ-tLyP-1 nanoparticles. The tumor / normal tissue (T / N) signal intensity ratio was calculated by measuring the signal intensity of the tumor region and the contralateral normal brain tissue. Simultaneously, T2-weighted imaging was used to monitor changes in tumor structure after nanoparticle administration. The T2 signal intensity of the tumor region was measured before injection (Pre), and at 6, 24, and 72 hours after injection. ImageJ software was used to analyze the T1 and T2 signal intensities, and signal intensity curves over time were plotted to compare the tumor enhancement efficiency of the two nanoparticles (HM@si-SLC16A1 / TMZ and HM@si-SLC16A1 / TMZ-tLyP-1).

[0152] The results showed that after injection of HM@si-SLC16A1 / TMZ-tLyP-1 nanoparticles, the tumor signal intensity increased significantly, reaching a peak at 24 hours post-injection, indicating that the nanoparticles possess effective tumor targeting capabilities. Figure 12 A and Figure 12 C). In contrast, the HM@si-SLC16A1 / TMZ group showed relatively low T1 signal enhancement, indicating less accumulation in tumors compared to tLyP-1 modified nanoparticles. Figure 12 A and Figure 12 C). T2-weighted imaging (T2WI) showed that the tumor signal intensity gradually decreased over time, reflecting possible structural changes in the tumor during treatment. Figure 12 B and Figure 12 D). Compared with the HM@si-SLC16A1 / TMZ group, the T2 signal intensity of the HM@si-SLC16A1 / TMZ-tLyP-1 group decreased more significantly, indicating that the accumulation of nanoparticles in the tumor was increased and the therapeutic effect was better. Figure 12 B and Figure 12D). Semi-quantitative analysis showed that at 6h, 24h, and 72h post-injection, the T1 signal intensity of the HM@si-SLC16A1 / TMZ-tLyP-1 group was significantly higher than that of the HM@si-SLC16A1 / TMZ group (p<0.01). Figure 12 C (left). Similarly, the T2 signal intensity of the HM@si-SLC16A1 / TMZ-tLyP-1 group decreased more rapidly, indicating that this group of nanoparticles has stronger tumor penetration ability and therapeutic effect. Figure 12 (D left). The T1 and T2 contrast ratio (the ratio of tumor to normal tissue, T / N) further confirms that HM@si-SLC16A1 / TMZ-tLyP-1 nanoparticles have superior tumor targeting ability compared to HM@si-SLC16A1 / TMZ. Figure 12 C right and Figure 12 D (right).

[0153] 12. In vivo biodistribution fluorescence imaging of nanoparticles

[0154] To detect the biodistribution of HM@si-SLC16A1 / TMZ-tLyP-1 nanoparticles, tumor-bearing Wistar rats were injected with Cy5.5-labeled nanoparticles via the tail vein. The animals were sacrificed 24 hours later, and liver, kidney, spleen, lung, heart, and brain tissues were collected. In vitro fluorescence imaging was performed using the IVIS Spectrum in vivo imaging system, and the fluorescence intensity of each organ was quantified using Living Image software to assess the accumulation of nanoparticles.

[0155] Fluorescence intensity analysis showed that the nanoparticles accumulated most significantly in the liver and kidneys, followed by moderate accumulation in the spleen and lungs, while almost none entered the heart. The fluorescence intensity in the brain was comparable to that in the kidneys, indicating that the nanoparticles successfully crossed the blood-brain barrier. Figure 13 These results demonstrate that tLyP-1 modification enhances the tumor-targeting efficiency of HM@si-SLC16A1 / TMZ-tLyP-1 nanoparticles, thereby achieving superior MRI contrast enhancement and therapeutic potential in glioma treatment.

[0156] Tumor progression was monitored on days 1, 5, and 10 post-injection using T2-weighted magnetic resonance imaging (MRI). The significant reduction in tumor volume in the TMZ treatment group compared to the PBS group, where tumor growth continued, confirmed its chemotherapeutic effect. Compared to free TMZ, the HM@TMZ-tLyP-1 treatment group further inhibited tumor growth, indicating that nanoparticle encapsulation improved drug delivery efficiency. Compared to the PBS group, the HM@si-SLC16A1-tLyP-1 treatment group showed moderate tumor growth inhibition, confirming the therapeutic relevance of silencing SLC16A1. Notably, the combination therapy of HM@si-SLC16A1 / TMZ and HM@si-SLC16A1 / TMZ-tLyP-1 showed superior tumor suppression compared to single-treatment groups, with the HM@si-SLC16A1 / TMZ-tLyP-1 treatment group showing a significant reduction in tumor volume, and tumor growth almost stopping by day 10. Figure 14 In summary, compared with free TMZ, TMZ-loaded nanoparticles can more effectively inhibit glioma growth, and silencing the SLC16A1 gene can further enhance the tumor suppression effect, thereby significantly prolonging survival.

[0157] 13. Kaplan-Meier Survival Analysis

[0158] To assess the impact of nanoparticle therapy on survival, tumor-bearing rats were monitored for 30 days post-injection. Survival curves were plotted using the Kaplan-Meier method, and statistical differences were analyzed using the log-rank test.

[0159] Kaplan-Meier survival curve analysis revealed that rats treated with HM@si-SLC16A1 / TMZ-tLyP-1 had the longest survival time, significantly better than other treatment groups (p<0.01, log-rank test). Figure 15 Compared with the PBS group, TMZ and HM@TMZ-tLyP-1 significantly prolonged the survival of rats, and HM@si-SLC16A1-tLyP-1 also prolonged survival, further confirming the therapeutic effect of silencing SLC16A1. Furthermore, the survival of the HM@si-SLC16A1 / TMZ and HM@si-SLC16A1 / TMZ-tLyP-1 combination therapy groups was significantly longer than that of the single therapy groups, with the HM@si-SLC16A1 / TMZ-tLyP-1 group exhibiting the longest survival. Figure 15 ).

[0160] 14. Histological analysis

[0161] Following standard procedures, tissue sections were dewaxed, hydrated, and stained with hematoxylin and eosin (HE) solution (Sigma-Aldrich). Histological changes, such as tumor cell density and necrotic areas, were observed using an optical microscope to assess the morphological and structural changes of the tumor after treatment. Ten days after intravenous injection, major organs (heart, liver, spleen, lung, and kidney) were extracted from a rat model of glioma for analysis to evaluate the systemic toxicity of the nanoparticles.

[0162] Histological analysis showed no significant pathological abnormalities in any of the treatment groups, indicating that nanoparticle administration did not cause detectable organ damage. Figure 16 ).

[0163] 15. TUNEL staining for apoptosis detection

[0164] The TUNEL cell apoptosis detection kit was used to detect cell apoptosis in tumor tissues. Tissue sections were processed according to the manufacturer's experimental procedures, TUNEL-positive cells were visualized by DAB staining, and counterstained with hematoxylin. The apoptosis index was quantified by calculating the percentage of TUNEL-positive cells in the tumor tissue sections.

[0165] Hematoxylin and eosin (H&E) staining showed that tumor cells in the PBS group were densely packed with only a few necrotic areas; the TMZ and HM@TMZ-tLyP-1 treatment groups showed decreased tumor cell density and moderate necrosis; the HM@si-SLC16A1-tLyP-1 treatment group showed mild tissue destruction. Notably, both the HM@si-SLC16A1 / TMZ and HM@si-SLC16A1 / TMZ-tLyP-1 combined treatment groups showed extensive tumor necrosis and cell disintegration, indicating a significantly enhanced therapeutic effect. Figure 17 ).

[0166] 16. Immunohistochemical (IHC) staining

[0167] Immunohistochemical staining was used to detect the expression levels of Caspase-3 (apoptosis marker), Ki67 (proliferation marker), and HIF-1α (hypoxia marker). The specific steps were as follows: tissue sections were dewaxed, hydrated, and then subjected to antigen retrieval. After blocking, they were incubated overnight at 4°C with primary antibodies against Caspase-3, Ki67, and HIF-1α, respectively. After washing, the sections were incubated with HRP-labeled secondary antibodies, developed using DAB chromogenic solution, and counterstained with hematoxylin. Images were acquired using an optical microscope, and quantitative analysis was performed using ImageJ software.

[0168] TUNEL staining showed that the proportion of apoptotic cells was low in the PBS group, while the proportion of apoptotic cells increased in the TMZ and HM@TMZ-tLyP-1 treatment groups. The apoptosis index in the HM@si-SLC16A1-tLyP-1 group was at a moderate level, while the proportion of apoptotic cells was highest in the HM@si-SLC16A1 / TMZ and HM@si-SLC16A1 / TMZ-tLyP-1 treatment groups, indicating a synergistic effect between TMZ and si-SLC16A1. Figure 18 A and Figure 18 C). IHC analysis showed that the PBS group had low Caspase-3 levels, while the TMZ and HM@TMZ-tLyP-1 groups showed gradually increasing Caspase-3 levels. The HM@si-SLC16A1-tLyP-1 group had intermediate Caspase-3 levels, while the HM@si-SLC16A1 / TMZ and HM@si-SLC16A1 / TMZ-tLyP-1 groups had the highest Caspase-3 levels, which confirms that the nanoparticles can enhance apoptosis activity. Figure 18 B and Figure 18 D). Ki67 staining showed higher proliferation activity in the PBS group, which decreased after treatment with TMZ and HM@TMZ-tLyP-1. HM@si-SLC16A1-tLyP-1 showed moderate proliferation inhibition, while the Ki67-positive cells were most prominent in the HM@si-SLC16A1 / TMZ and HM@si-SLC16A1 / TMZ-tLyP-1 groups, indicating that it can significantly inhibit cell proliferation. Figure 18 B and Figure 18 D). HIF-1α staining showed that tumors in the PBS group exhibited severe hypoxia, while the hypoxia in the TMZ and HM@TMZ-tLyP-1 groups was moderately reduced. HIF-1α was mildly downregulated in the HM@si-SLC16A1-tLyP-1 group, while HIF-1α levels were significantly downregulated in the HM@si-SLC16A1 / TMZ and HM@si-SLC16A1 / TMZ-tLyP-1 treatment groups, indicating a highly efficient regulatory effect on the tumor microenvironment. Figure 18 B and Figure 18 D). In summary, HM@si-SLC16A1 / TMZ-tLyP-1 nanoparticles demonstrated superior tumor inhibition, apoptosis induction, proliferation inhibition, and hypoxia relief effects in the treatment of glioma rats.

[0169] 20. Serum biochemical analysis

[0170] On the 10th day after treatment, blood samples were collected via cardiac puncture. Liver and kidney function were assessed by measuring serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr) levels. ALT and AST levels were measured using an automated biochemical analyzer, while BUN and Cr levels were measured using a fully automated blood biochemistry analyzer.

[0171] Serum biochemical analysis further confirmed the biocompatibility of the nanoparticles. ALT and AST levels, indicators of liver function, showed no significant differences among the groups, indicating that nanoparticle administration did not cause hepatotoxicity. Similarly, BUN and Cr levels remained stable in all treatment groups, indicating that the nanoparticles had no adverse effects on renal function. Figure 19 In summary, HM@si-SLC16A1 / TMZ and HM@si-SLC16A1 / TMZ-tLyP-1 nanoparticles exhibit excellent biocompatibility and do not cause systemic toxicity or organ damage, which strongly supports the safety of their potential therapeutic applications.

[0172] This invention designs a MnO2-based HM@si-SLC16A1 / TMZ-tLyP-1 nanoparticle system. By integrating siRNA-mediated metabolic inhibition and TMZ chemotherapy, it enhances anti-tumor effects while improving tumor oxygenation and increasing BBB penetration. These nanoparticles can responsively degrade in the tumor microenvironment (TME), achieve controlled drug release, silence the SLC16A1 gene via siRNA, enhance MRI imaging contrast, and effectively inhibit glioma growth in vivo. Notably, compared to HM@si-SLC16A1 / TMZ, HM@si-SLC16A1 / TMZ-tLyP-1 exhibits superior tumor-targeting properties and potent therapeutic effects, highlighting the crucial role of tLyP-1 modification in improving BBB penetration and glioma inhibition. The results show that SLC16A1 gene silencing and TMZ chemotherapy have a significant synergistic effect. This discovery provides a new approach to overcome glioblastoma drug resistance through multiple mechanisms, such as enhancing drug delivery efficiency, improving the tumor hypoxic microenvironment, and promoting tumor cell apoptosis.

[0173] Example 3

[0174] Based on the improvement of process parameters in the preparation of hollow mesoporous manganese dioxide nanoparticles (H-MnO2), the experiment was divided into 5 groups, namely: Comparison file 1 (H-MnO2-NC): Method + KMnO4 reduction method + batch alkaline etching method; Invention 1 (H-MnO2-1): Improved Method + Mn(II) controlled oxidation method + batch alkaline etching method; Invention 2 (H-MnO2-2): Improved Method + Mn(II) controlled oxidation method + batch alkaline etching method; Invention 3 (H-MnO2-3): Improved Method + KMnO4 reduction method + dialysis bag gentle etching method; the optimal preparation method of this invention 4 (H-MnO2-4): improved The preparation method is the same as that in Example 1, which is the method of controlled oxidation with Mn(II) and gentle etching with dialysis bags.

[0175] Comparison file 1 (H-MnO2-NC): The following methods are used: KMnO4 reduction method + batch alkaline etching method:

[0176] Method: Mix 4.5 mL of tetraethyl orthosilicate, 9 mL of 28% (w / w) concentrated ammonia, 62 mL of anhydrous ethanol, and 24.5 mL of deionized water, and stir at room temperature for 2 h (500 rpm). Centrifuge at 8000 rpm for 10 min, collect the precipitate, wash twice each with anhydrous ethanol and deionized water, and freeze-dry to obtain SiO2 nanoparticles.

[0177] KMnO4 Reduction Method: Under ultrasonic treatment (ultrasonic frequency 1MHz, power 6W, ultrasonic time 5 minutes: 5 seconds of operation followed by 5 seconds of rest), an aqueous solution of potassium permanganate containing 300mg of potassium permanganate (prepared by dissolving 4.5g of potassium permanganate in 90mL) was added to a SiO2 suspension containing 40mg of SiO2 nanoparticles (prepared by adding 600mg of SiO2 nanoparticles to 10mL). The mixture was stirred at room temperature for 6 hours (500rpm). After centrifugation at 10000rpm for 10min, the precipitate was collected, washed three times with deionized water, and then added to 10mL of deionized water to obtain a mesoporous MnO2-coated SiO2 suspension.

[0178] Batch alkaline etching method: The mesoporous MnO2-coated SiO2 suspension obtained in step (2) was added to a 2M sodium carbonate aqueous solution (containing 10.6g sodium carbonate, the reaction system used in the experiment was 50mL), and stirred at 60℃ for 12h (100rpm). Centrifuged at 10000rpm for 10min, the precipitate was collected, washed 3 times with deionized water, and freeze-dried to obtain hollow mesoporous MnO2 nanoparticles (referred to as MnO2).

[0179] The stability of the five types of nanoparticles prepared was tested as follows: Each group of nanoparticles was diluted with 10 times its volume of 1640 complete culture medium containing 10% FBS, and placed in a shaker at 37℃. The nanoparticles were collected at different time points (12h, 24h, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, and 8 days) to detect changes in particle size. The results are presented as the particle size on day 8. The results are shown in the table below.

[0180] Table 2 Results of stability tests for each nanoparticle

[0181] Nanoparticle grouping Initial particle size Repeat 1 Repeat 2 Repeat 3 mean Standard deviation <![CDATA[H-MnO2-NC]]> 350 310 312 317 313 3.61 <![CDATA[H-MnO2-1]]> 11000 10476 10467 10559 10500.67 50.72 <![CDATA[H-MnO2-2]]> 11000 10688 10674 10763 10708.33 47.86 <![CDATA[H-MnO2-3]]> 11000 10621 10797 10786 10734.67 98.59 <![CDATA[H-MnO2-4]]> 11000 10877 10897 10890 10888 10.15

[0182] The results showed that the initial particle size of H-MnO2-NC was significantly smaller than that of other groups. Secondly, from the nanoparticle size results after 8 days, the 8-day stability evaluation of each group in 10% fetal bovine serum (pH 7.4) showed that the nanoparticles in each group underwent a certain degree of degradation, and the degradation rate of each group was H-MnO2-4 (1%) < H-MnO2-1 (2.4%) < H-MnO2-2 (2.7%) < H-MnO2-3 (4.5%) < H-MnO2-NC (10%). The H-MnO2-4 of the present invention had the strongest stability.

[0183] Example 4

[0184] Five prepared nanoparticles were selected for the drug loading experiment of temozolomide (TMZ). That is, 10 mg of the five prepared nanoparticles were weighed and ultrasonically dispersed in 10 mL of PBS buffer (pH 7.3) respectively. Then, 0.5 mL of TMZ with a concentration of 0.5 mg / mL was added to each system, so that the total initial dosing mass of TMZ was 0.5 mg. The mixture was incubated at low speed on a shaker for 24 hours at room temperature and in the dark. After incubation, the mixture was centrifuged at 13,000 rpm for 20 minutes in a high-speed centrifuge to completely separate the precipitated nanoparticles after drug loading from the supernatant containing unencapsulated free TMZ. The supernatant was carefully aspirated, filtered through a 0.2 μm filter membrane, and then injected into a high-performance liquid chromatograph (HPLC) for analysis. The drug loading rate and encapsulation rate of TMZ were indirectly measured by measuring the content of TMZ in the supernatant with HPLC. The HPLC determination of TMZ used a Hedera ODS-C18 chromatographic column (250 mm × 4.6 mm, 5 μm), the mobile phase was acetonitrile-water (v∶v = 60∶40), the volume flow rate was 1 mL / min, the detection wavelength was 210 nm, the column temperature was room temperature, and the injection volume was 15 μL.

[0185] The results are shown in the following table.

[0186] Table 3 Results of encapsulation rate tests for each nanoparticle

[0187] Nanoparticle grouping Repeat 1 Repeat 2 Repeat 3 mean Standard deviation <![CDATA[H-MnO2-NC]]> 61.3％ 63.6％ 65.1％ 63.33％ 0.02 <![CDATA[H-MnO2-1]]> 63.4％ 73.2％ 70.3％ 68.97％ 0.05 <![CDATA[H-MnO2-2]]> 66.9％ 73.6％ 77.3％ 72.6％ 0.05 <![CDATA[H-MnO2-3]]> 65.3％ 66.8％ 70.4％ 67.5 0.03 <![CDATA[H-MnO2-4]]> 93.6％ 95.8％ 98.1％ 95.83％ 0.02

[0188] Table 4 Results of drug loading rate for each nanoparticle

[0189]

[0190]

[0191] The results showed that the drug loading rates of each group were H-MnO2-4 (88.33%) > H-MnO2-2 (61.1%) > H-MnO2-3 (57.8%) > H-MnO2-NC (55.1%) > H-MnO2-1 (53.07%), and the drug loading rate of H-MnO2-4 in this invention was higher.

[0192] Example 5

[0193] Preparation of PEI-modified silica: PEI-modified silica was prepared according to Example 1 of patent CN110293232A. The specific steps are as follows: Take a 200ml wide-mouth bottle, add 100ml anhydrous ethanol, 6ml deionized water, and 4ml 28% ammonia water sequentially, stir at room temperature for 20min, then add 4ml TEOS and continue stirring for 6h. After the reaction, centrifuge and concentrate the white SiO2 precipitate, wash twice with anhydrous ethanol, and finally centrifuge and concentrate in 40ml ethanol for later use. Under ultrasonic conditions, cationic polymer PEI was modified on the surface of 150nm SiO2 particles to form a uniform PEI interlayer with aminated structure, exhibiting strong positive charge. The specific steps are as follows: Take 1ml of the SiO2 nanospheres prepared in the first step, redisperse in 100ml deionized water, sonicate for 2-5min, add 12ml of 5mg / ml PEI solution, and sonicate for 40min. Centrifuge, wash twice with deionized water to remove excess PEI, and finally concentrate the PEI-modified silica to 3 ml with deionized water.

[0194] Mn(II) precursor controlled oxidation deposition method: Dispersion template: The prepared PEI-modified silica was ultrasonically dispersed in water. Precursor addition: Manganese acetate (Mn(OAc)2) solution was added, and the mixture was stirred for 30 minutes to allow it to be fully adsorbed onto the negatively charged SiO2 surface. Controlled oxidation: The reaction system was heated to 85℃, and then ammonium persulfate ((NH4)2S2O8) solution was slowly added dropwise as an oxidant. Rate-controlled reaction: The reaction was carried out in a constant temperature oil bath at 80℃ with low-speed stirring (200 rpm) for 2 hours.

[0195] Manganese acetate (Mn(OAc)2) solution: The solution was prepared with SiO2 template mass: manganese acetate (item number M131577, Aladdin): ultrapure water in a ratio of 1:1:0.5.

[0196] Ammonium sulfate ((NH4)2S2O8) solution: Based on the amount of manganese acetate to be used, calculate and weigh the required amount of ammonium persulfate (item number A112449, Aladdin) at a molar ratio of approximately 1:1.5, and quickly dissolve it in ultrapure water.

[0197] Dialysis bag-mediated mild etching: Sample loading: MnO2@SiO2 core-shell particles were suspended in a small amount of deionized water and placed in a dialysis bag (e.g., MWCO 14,000 Da). Mild etching: The sealed dialysis bag was completely immersed in a beaker containing 1M Na2CO3 or 1.5M NaOH. Diffusion control: The entire system was placed in an 80°C water bath and reacted for 20 hours under static or very low stirring (50 rpm). Purification: After the reaction, the dialysis bag was transferred to a large amount of deionized water and dialyzed for 36 hours to remove residual alkali and silicates, yielding H-MnO2 nanoshells.

[0198] It was compared with H-MnO2-4 prepared in Example 3, and the detection method was the same as in Example 4.

[0199] The results are shown in the table below.

[0200] Table 5. Encapsulation efficiency test results for each nanoparticle.

[0201] Nanoparticle grouping Repeat 1 Repeat 2 Repeat 3 mean Standard deviation <![CDATA[H-MnO2 nanoshell 5]]> 78.1％ 76.9％ 83.4％ 79.47％ 0.03 <![CDATA[H-MnO2-4]]> 93.6％ 95.8％ 98.1％ 95.83％ 0.02

[0202] Table 6. Drug loading rate results for each nanoparticle.

[0203] Nanoparticle grouping Repeat 1 Repeat 2 Repeat 3 mean Standard deviation <![CDATA[H-MnO2 nanoshell 5]]> 58.6％ 64.7％ 66.4％ 63.23％ 0.04 <![CDATA[H-MnO2-4]]> 85.3％ 89.6％ 90.1％ 88.33％ 0.03

[0204] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A method for preparing manganese dioxide nanoparticles, characterized in that, Includes the following steps: S1. Tetraethyl orthosilicate, concentrated ammonia and anhydrous ethanol are mixed and stirred to react. After the reaction is completed, the precipitate is collected by centrifugation, washed and SiO2 nanoseeds are obtained. S2. Disperse SiO2 nanoseeds in anhydrous ethanol to form a seed suspension; The seed suspension was mixed with concentrated ammonia and anhydrous ethanol to obtain reaction system 1; a mixed solution of tetraethyl orthosilicate and anhydrous ethanol was added dropwise to it, the mixture was stirred and reacted, and the precipitate was collected by centrifugation after the reaction was completed. S3. Mix the precipitate with concentrated ammonia and anhydrous ethanol to obtain reaction system 2; add a mixed solution of tetraethyl orthosilicate and anhydrous ethanol dropwise to it, stir the reaction, and collect the precipitate by centrifugation after the reaction is completed; wash and dry to obtain SiO2 microspheres; After ultrasonically mixing S4, manganese acetate solution and SiO2 microspheres for 20-40 min, an oxidant is added dropwise, and the mixture is stirred at 70-95℃ for 2-4 hours to obtain MnO2@SiO2 core-shell particles. S5. Suspend MnO2@SiO2 core-shell particles in deionized water and place them in a dialysis bag; then immerse the dialysis bag in Na2CO3 or NaOH solution and react in a water bath at 70-80℃. After the reaction is complete, transfer the dialysis bag to deionized water for dialysis for 24-48 hours to obtain manganese dioxide nanoparticles.

2. The preparation method according to claim 1, characterized in that, The particle size of SiO2 microspheres is 9-11 μm.

3. The preparation method according to claim 1, characterized in that, In step S1, the volume ratio of tetraethyl orthosilicate, concentrated ammonia, and anhydrous ethanol is 3-8:8-13:100-200; in step S2, the volume ratio of seed suspension, concentrated ammonia, and anhydrous ethanol is 20-100:10-20:200-400; in step S3, the mixed solution of tetraethyl orthosilicate and anhydrous ethanol contains 30-40 mL of tetraethyl orthosilicate and 160-180 mL of anhydrous ethanol.

4. The preparation method according to claim 1, characterized in that, In step S4, the mass ratio of manganese acetate solution to SiO2 microspheres is 1:1.0-1.5; the oxidant is ammonium persulfate ((NH4)2S2O8); and the mass ratio of the oxidant to manganese acetate is 1.0-1.5:1.

0.

5. The preparation method according to claim 1, characterized in that, In step S5, the dialysis bag is permeable to molecules with a molecular weight of 12000-15000 Da; the concentration of the Na2CO3 or NaOH solution is 0.5-1M.

6. Manganese dioxide nanoparticles prepared by the preparation method according to any one of claims 1-5.

7. A method for delivering nanoparticles, characterized in that, The raw materials, by weight, include: 8-12 parts of manganese dioxide nanoparticles according to claim 6, 8-15 parts of polyallylamine hydrochloride, 8-12 parts of N-hydroxysuccinimide-polyethylene glycol-maleimide, and 3-10 parts of tLyP-1 peptide.

8. The method for preparing delivery nanoparticles according to claim 7, characterized in that, Includes the following steps: The manganese dioxide nanoparticles were ultrasonically mixed with polyallylamine hydrochloride, and then coated with N-hydroxysuccinimide-polyethylene glycol-maleimide. Finally, tLyP-1 peptide was added to the nanoparticles for coupling reaction. After the reaction was completed, the nanoparticles were washed and resuspended to obtain the final product, the delivery nanoparticles.

9. The application of the delivery nanoparticles according to claim 7 in the delivery of temozolomide (TMZ) and / or si-SLC16A1.

10. The use of the delivery nanoparticles according to claim 7 in the preparation of a drug for treating glioblastoma.

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