Nanocapsules smcpm and their use in the preparation of a medicament for the treatment or prevention of osteosarcoma and lung metastasis

Abstract

This invention relates to the field of biomedical technology, specifically disclosing a nanocapsule SMCPM and its use in drugs for the treatment or prevention of osteosarcoma and lung metastases. The SMCPM consists of mesoporous silica-manganese oxide particles carrying a photosensitizer Ce6, an NRF2 inhibitor ML385, and an OS-targeting peptide PT-7. Experiments have demonstrated that the SMCPM exhibits high penetration ability and strong cell-killing effects; it can effectively repair the biochemically inhibitory microenvironment by downregulating NRF2 expression and reversing its nuclear translocation, significantly reducing cellular tolerance to reactive oxygen species and epithelial-mesenchymal transition induced by NOTCH1 signaling axis activation, enhancing cellular oxidative stress levels, and promoting PIT-induced immunogenic cell death; the released Mn... 2+ In synergy with double-stranded DNA, it can regulate the immunosuppressive microenvironment by activating the intracellular STING pathway, thereby inhibiting osteosarcoma growth and preventing lung metastasis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a nanocapsule SMCPM and its use in the preparation of drugs for the treatment or prevention of osteosarcoma and lung metastases. Background Technology

[0002] Osteosarcoma (OS) is the most common malignant bone tumor, with lung metastasis being a key prognostic factor. Lung metastasis can lead to pleural effusion or respiratory failure, impacting patients' quality of life. Currently, clinical practice for OS patients with lung metastases primarily relies on surgery combined with chemotherapy, radiotherapy, or other methods to prolong survival. However, difficulties in surgical resection, chemotherapy resistance, and radiotherapy insensitivity significantly limit the treatment efficacy of OS. Photoimmunotherapy (PIT), as a novel non-invasive treatment method, offers advantages such as high targeting, few side effects, and strong reproducibility. Reports indicate that PIT can effectively prevent osteosarcoma metastasis and recurrence by regulating intracellular oxidative stress, promoting immune-mediated cell death, and enhancing immune cell aggregation. However, the tumor microenvironment of OS greatly limits the effectiveness of PIT. Typical biochemically suppressive microenvironments, such as antioxidative stress, enhance OS cells' tolerance to reactive oxygen species, which can reduce the efficacy of PIT. Furthermore, the complex immunosuppressive microenvironment caused by tumor-associated macrophage (TAM) infiltration further increases the difficulty of treating lung metastases. Therefore, modulating the tumor microenvironment of osteosarcoma is key to further improving the efficacy of PIT treatment and inhibiting lung metastases.

[0003] Existing technologies, such as disrupting intracellular redox balance and reshaping the biochemically suppressive microenvironment by delivering oxygen or ROS, and leveraging PIT-induced immunogenic cell death (ICD) enhancement to effectively regulate the OS immunosuppressive microenvironment through immune cell infiltration, pro-inflammatory cytokine secretion, and cytotoxic T lymphocyte recruitment, have very limited effectiveness in inhibiting lung metastases.

[0004] Therefore, combining multiple mechanisms for synergistic treatment to enhance the inhibitory effect on primary and metastatic osteosarcoma lesions is expected to become a powerful weapon in the treatment of osteosarcoma lung metastases. Summary of the Invention

[0005] The purpose of this invention is to address the aforementioned shortcomings of the prior art by providing a nanocapsule SMCPM and its use in the preparation of drugs for the treatment or prevention of osteosarcoma and lung metastases. The nanocapsule SMCPM can be used to reshape the biochemical inhibition and immunosuppression of primary osteosarcoma lesions and lung metastases. In vivo experiments have demonstrated that SMCPM can inhibit osteosarcoma growth and prevent lung metastases, which has great potential and clinical significance.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A first aspect of the present invention is to provide a method for preparing SMCPM nanocapsules, comprising the following steps:

[0008] S1. Preparation of mesoporous organosilicon nanoparticles

[0009] Mesoporous organosilicon nanoparticles, namely MONs, were prepared by the sol-gel method using organosilicon sources and template agents.

[0010] Preparation of S2, manganese dioxide-doped mesoporous organosilicon particles

[0011] Mesoporous organosilicon nanoparticles were dispersed in a polyethyleneimine solution, then potassium permanganate solution was added, and after stirring for a preset time, oleic acid was added dropwise. The manganese dioxide-doped mesoporous organosilicon particles, i.e., SMONs, were collected by centrifugation.

[0012] Preparation of S3 and SMCPM nanocapsules

[0013] SMONs were first dispersed in a polyethyleneimine solution, centrifuged, and then redispersed in a gibberellin solution to obtain a mixed solution. Then, bovine serum albumin solution was added and incubated to obtain BSA-modified SMONs. Subsequently, Ce6 and PT7 were grafted onto the surface of BSA-modified SMONs via click reaction to obtain SMCP nanoparticles. Finally, ML385 was loaded onto the surface of SMCP nanoparticles through hydrophobic interactions to obtain nanocapsules SMCPM.

[0014] Further, in step S1, the organosilicon source is a mixed solution of tetraethyl orthosilicate and tetraethylammonium tetrafluoroborate, and the template agent includes any one of hexadecyltrimethylammonium bromide, sodium dodecyl sulfate, and octadecyltrimethylammonium chloride.

[0015] Further, in step S2, the mass concentration of the polyethyleneimine solution is 8~10 mg / mL, and the mass ratio of the MONs to the potassium permanganate is (5~6):10.

[0016] Furthermore, in step S3, the mass ratio of Ce6, PT7 and ML385 is 1:1:(2.5~3).

[0017] A second objective of this invention is to provide a nanocapsule SMCPM obtained by the above preparation method.

[0018] A third object of the present invention is to provide a pharmaceutical composition comprising the above-described nanocapsule SMCPM and optionally one or more pharmaceutically acceptable carriers.

[0019] The term "pharmaceutically acceptable carrier" refers to one or more inactive ingredients in an approved drug product. Inactive ingredients listed in the "Inactive Ingredients in Approved Drug Products" database maintained and updated by the U.S. Food and Drug Administration (FDA) are also appropriate. In some cases, a pharmaceutically acceptable carrier may also be referred to as an excipient.

[0020] A fourth objective of this invention is to provide the use of the above-described nanocapsule SMCPM or pharmaceutical composition in the treatment or prevention of osteosarcoma and lung metastases.

[0021] Furthermore, the nanocapsule SMCPM can remodel the biochemical and immunosuppressive microenvironment of primary osteosarcoma lesions and lung metastases.

[0022] Furthermore, the nanocapsule SMCPM can effectively repair the biochemically inhibitory microenvironment by downregulating NRF2 expression and reversing its nuclear translocation, thereby significantly reducing the cell's tolerance to reactive oxygen species and the epithelial-mesenchymal transition induced by the activation of the NRF2-NOTCH1-EMT signaling axis, thereby enhancing the cell's oxidative stress level and promoting photothermal immunotherapy-induced immunogenic cell death.

[0023] Furthermore, the SMCPM nanocapsule utilizes photothermal immunotherapy-induced immunogenic cell death to enhance and promote the release of double-stranded DNA, in conjunction with Mn 2+ It synergistically activates the intracellular STING pathway to regulate the immunosuppressive microenvironment.

[0024] Compared with the prior art, the beneficial effects of the technical solution provided by the present invention are as follows:

[0025] (1) The nanocapsule SMCPM provided by this invention disperses mesoporous organosilicon nanoparticles in a PEI solution. PEI is anchored to the surface of MONs through electrostatic adsorption or hydrogen bonding to form a MONS-PEI complex. The amino groups on PEI give the surface a positive charge, enhancing the subsequent anion (MnO4) -The binding ability of manganese dioxide is enhanced by amino groups, which act as reducing agents to reduce KMnO4 to MnO2, promoting in-situ deposition of manganese dioxide. The addition of oleic acid reduces the surface energy of the nanoparticles and prevents aggregation. This yields manganese dioxide-doped mesoporous organosilicon particles, which are further loaded with the photosensitizer Ce6, the OS-targeting peptide PT-7, and the NRF2 inhibitor ML385 to obtain nanocapsules (SMCPM). The SMCPM prepared in this invention exhibits a uniform particle size distribution and sensitive stiffness transition characteristics. In vitro experiments show that the penetration ability of this variable stiffness SMCPM is increased by 2.99 times, and the cell-killing effect is significantly enhanced.

[0026] (2) This invention innovatively constructs a nanocapsule SMCPM, a variable stiffness photothermal immunotherapy (PIT) nanoplatform based on manganese (Mn), for reshaping the biochemical and immunosuppressive microenvironment of primary osteosarcoma lesions and lung metastases. SMCPM can effectively repair the biochemically suppressive microenvironment by downregulating NRF2 expression and reversing its nuclear translocation, thereby significantly reducing cellular tolerance to reactive oxygen species (ROS) and epithelial-mesenchymal transition (EMT) induced by NOTCH1 signaling axis activation, ultimately enhancing cellular oxidative stress levels and promoting PIT-induced immunogenic cell death (ICD). During PIT treatment, the released Mn... 2+ Synergistically with double-stranded DNA (dsDNA), SMCPM can regulate the immunosuppressive microenvironment by activating the intracellular STING pathway, thereby inducing potent anti-tumor immune responses (such as dendritic cell maturation and cytotoxic lymphocyte infiltration). In vivo experiments have demonstrated that SMCPM can inhibit osteosarcoma growth and prevent lung metastasis, representing a synergistic therapeutic strategy with great potential and clinical significance. Attached Figure Description

[0027] Figure 1 A flowchart illustrating the preparation process of SMCPM nanocapsules provided by this invention;

[0028] Figure 2 Characterization of SMCPM: AB represents transmission electron microscopy, C represents scanning electron microscopy, D represents elemental distribution, E represents energy dispersive spectroscopy, F represents hydrated particle size, G represents the Zeta potential of SMCPM, H represents the UV-Vis spectrum of each nanoparticle, IJ represents the atomic force microscopy images and Young's modulus of SMCPM before and after GSH incubation, and K represents the concentration of manganese ions in SMCPM under different conditions.

[0029] Figure 3For the cytotoxicity and in vitro permeability assessment of SMCPM, A and B are confocal microscopy images of 3D tumor spheroids incubated with SMCPM in the presence and absence of GSH, along with their corresponding fluorescence intensities (scale bar = 100 μm); C is the cell viability assessment of OSK7M2 cells after 24 hours of co-incubation with different concentrations of SMCPM and SMCP; D is the cell viability assessment of K7M2 cells after 2 hours of co-incubation with different concentrations of SMCPM and SMCP, with or without exposure to 0.25 W / cm². 2 Cell viability after 3 minutes of 660nm laser irradiation. (E) Cell viability of K7M2 cells co-incubated with SMCPM at different irradiation times; F: Cell viability of K7M2 cells co-incubated with SMCPM at different laser power settings; GH: Quantitative analysis results and fluorescence images of live / dead staining experiments from different treatment groups (scale bar = 200 μm).

[0030] Figure 4 SMCPM inhibits NRF2, suppressing OS cell invasion and oxidative stress. A is a schematic diagram illustrating the mechanism by which ML385 inhibits EMT and metastasis. BC shows fluorescence images and quantitative analysis of reactive oxygen species (ROS) generation (scale bar = 100 μm). DE shows the Transwell assay results of the invasion region of K7M2 cells after different treatments. FG shows fluorescence images of NRF2 expression and nuclear translocation (scale bar = 8 μm). HK shows the results of Western blot analysis and quantitative analysis of NRF2 and its downstream protein expression in K7M2 cells after incubation with PBS, SMCP, SMCPM, and laser irradiation.

[0031] Figure 5 Immunofluorescence images of NRF2 nuclear translocation after laser irradiation in each group (scale bar = 25 μm);

[0032] Figure 6 Fluorescence images showing that SMCPM is mainly enriched in the lungs, liver, and kidneys (n=3);

[0033] Figure 7 The results of fluorescence intensity analysis of tumor regions at different time points after SMCPM injection are shown in A. Fluorescence image; B. Quantitative analysis results of tumor sites at different time points. The fluorescence intensity of the tumor region reaches its peak 6 hours after injection.

[0034] Figure 8 To demonstrate the activation of in vivo immune responses by SMCPM-based photodynamic therapy, A shows a schematic diagram of the animal experiment design; B and D show Western blotting images and quantitative analysis results indicating that the STING pathway is activated; C and E show immunofluorescence and quantitative results indicating CD8 in OS tissue. +T cells (scale bar = 100 μm); FI represents the results of flow cytometry analysis, showing mature DCs (CD80) in the tumor. + CD86 + ), activated CD8 + T cells (CD69) + CD8 + ) and Memory CD8 + T cells (CD44) + CD8 + The proportion of );

[0035] Figure 9 The results of flow cytometry analysis of lymph node and spleen immune cells after treatment in each group are shown. AC represents mature dendritic cells and activated CD8+ cells in the lymph nodes, respectively. + Flow cytometry analysis results of T cells and memory T cells; DF represents mature dendritic cells and activated CD8+ cells in the spleen, respectively. + T cells and memory CD8 + Flow cytometry analysis results of T cells;

[0036] Figure 10 To illustrate the antitumor effect of SMCPM-mediated photodynamic therapy on primary osteosarcoma and lung metastases, A shows images of primary osteosarcoma and lung metastatic nodules two weeks after treatment; B shows changes in mouse body weight after the above treatment; C shows changes in tumor volume in mice after the above treatment; DE shows the immunofluorescence results for each group, along with quantitative analysis of Ki67 expression (scale bar = 100 μm); F shows histological images of lungs stained with HE in different groups; G shows images of lung metastatic nodules two weeks after treatment; H shows statistical analysis of the number of lung nodules; IJ shows WB images and quantitative analysis of NRF2 expression levels; and KL shows WB images and quantitative analysis of NOTCH1 and EMT marker expression levels.

[0037] Figure 11 For the long-term observation results of SMCPM efficacy, A is the Kaplan-Meier survival curve assessment, and B is the number of lung metastatic nodules in each group after 60 days of treatment.

[0038] Figure 12 To assess the in vivo biocompatibility of SMCPM, serum biochemical analysis was performed on AF after two weeks of treatment in the control group, SCP group, SMCP group, and SMCPM group; one week after in vivo treatment, H&E staining was performed on pathological sections of the heart, liver, spleen, lung, and kidney to assess the tissue compatibility of the above groups (scale bar = 100 μm); ns: no significant difference, n = 5. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the specific embodiments and accompanying drawings are described in further detail below. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0040] The English abbreviations used in this invention are explained as follows:

[0041] OS: Osteosarcoma; PIT (Photoimmunotherapy); NRF2: Nuclear factor erythrocyte 2-related factor 2; EMT: The NOTCH1 / EMT signaling axis regulates epithelial-mesenchymal transition in OS; STING: Interferon gene stimulator; ICD: Immunogenic cell death; TME: Tumor microenvironment; Ce6: Photosensitizer chloroce6; ML385: Nuclear factor erythrocyte 2-related factor 2 inhibitor; PT-7: OS-targeting peptide (amino acid sequence: PPSHTPT); TEOS: Tetraethyl tetrafluoroborate; TETS: Tetraethyl tetrafluoroborate; BSA: Bovine serum albumin; CTAB: Cetyltrimethylammonium bromide.

[0042] This invention designs and synthesizes a flexible, variable nanocapsule (SMCPM) that enhances PIT (penetrating intracellular inflammatory response). The capsule consists of mesoporous silica-manganese oxide particles carrying a photosensitizer Ce6, an NRF2 inhibitor (ML385), and an OS-targeting peptide PT-7 (amino acid sequence: PPSHTPT). The prepared SMCPM exhibits a uniform size distribution of 264 nm and demonstrates good biocompatibility and cellular absorption and penetration. Figure 1 As shown, SMCPMs were prepared using a hard template method. Specifically, firstly, manganese oxide (MnO2) was mineralized in situ on mesoporous organosilica nanoparticles (MONs) to construct MONs-MnO2 nanoparticles (SMs) with variable stiffness. Subsequently, a carboxyl-rich photosensitizer Ce6 and a tumor-targeting peptide PT-7 were covalently linked to the amino groups inside the SM modified with bovine serum albumin (BSA). Finally, the NRF2 inhibitor ML385 was loaded onto SM-Ce6-PT-7 via electrostatic adsorption, thus obtaining SM-Ce6-PT7-ML385 (i.e., SMCPMs). The prepared SMCPMs can precisely target tumor cells and efficiently penetrate deep into tissues. Under laser irradiation, they can also enhance immunogenic cell death (ICD), reverse the immunosuppressive tumor microenvironment (TME), and block epithelial-mesenchymal transition (EMT) under inhibitor intervention.

[0043] Characterization of SMCPM: The morphology of SMCPM was characterized by transmission electron microscopy (TEM, HT7700 microscope, Tokyo, Japan) and scanning electron microscopy (SEM, S4800 microscope, Tokyo, Japan). The elemental distribution and Young's modulus of SMCPM were measured using a FEI Talos F200X electron microscope (Oxford Instruments, Asylum Research, Santa Barbara, California, USA). The hydrodynamic dimensions and Zeta potential of SMCPM were measured using a Brookhaven ZetaPALS analyzer.

[0044] Cells and Animals: The osteosarcoma cell line (K7M2 cells) was cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C and 5% CO2. Six-week-old male Balb / c mice were purchased from Wuhan Wanqianjiaxing Biotechnology Co., Ltd. (Wuhan, China) and housed in a standard environment at the Experimental Animal Center of Wuhan University. All experiments were conducted in accordance with the experimental animal guidelines established by the Experimental Animal Center of Wuhan University. Animal Ethics (AUP) Number: ZN2023199.

[0045] Cell penetration assay: 100 μL of warm agarose solution (16 mg / mL, dissolved in DMEM medium) was added to a 96-well plate to form an agarose gel. Then, 100 μL of K7M2 cells (1 × 10⁶ cells per well) were added. 5 (Number of cells) were seeded onto a gel and cultured for two days at 37°C and 5% CO2 to promote the formation of multicellular spheroids. The multicellular spheroids were then incubated with glutathione (GSH, 10 mM, GSH+) for 30 minutes, followed by co-incubation with 30 μL of SMCPM for 1 hour. Multicellular spheroids without added GSH (labeled GSH-) served as a negative control. Fluorescence images were observed using a Leica TCS SP8 confocal microscope.

[0046] Cell viability assay: K7M2 cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay kit (Biosharp Ltd., China). In short, 100 μL of K7M2 cells were seeded into 96-well plates (5 × 10⁶ cells per well). 3 Cells were cultured overnight. Then, the cells were incubated with different samples for 6 hours. After incubation, the culture medium was discarded, and fresh culture medium containing 10 μL of CCK-8 reagent was added, followed by incubation for another 2 hours. Finally, absorbance was measured using a Multiskan FC microplate reader (Thermo Fisher Scientific, USA). Cell viability was calculated using the following formula: Cell viability = (Sample OD value - Blank OD value) / (Control OD value - Blank OD value) × 100%.

[0047] Calcein-AM / PI staining: Calcein-AM / PI staining was performed using a live / dead cell detection kit (Beyotime, China) to distinguish between live and dead cells after different treatments. K7M2 cells in 12-well plates were treated, then the culture medium was removed, the cells were gently washed with PBS, and incubated in the dark with calcein-AM and PI fluorescent dyes for 30 minutes. Finally, images were observed and captured using a fluorescence microscope, and the ratio of live to dead cells was calculated.

[0048] Intracellular reactive oxygen species (ROS) measurement: ROS levels in K7M2 cells were assessed using fluorescence microscopy using 10 μM 2′,7′-dichlorodihydrofluorescein diethyl ester (DCFH) (Yeason, China) as the ROS detection probe. First, K7M2 cells were cultured in wells at 5 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 cells / well in 24-well plates and cultured overnight. After removing the culture medium, the cells were treated with different samples for 6 hours. Cells in the laser group were irradiated with a 660 nm laser (0.25 W, 1 min). Then, DCFH-DA was loaded into the cells and incubated for another 1 hour. Subsequently, the cells were washed twice with PBS and measured by fluorescence microscopy under 480 nm laser irradiation.

[0049] Matrigel invasion assay: Cell invasion was performed using 12-well transwell plates covered with Matrigel membranes and containing 8 μm pores. K7M2 cells were collected from different treatment groups, resuspended in 500 μL of serum-free medium (1 × 10⁵ / well), and then loaded into the upper layer of the transwell. The lower layer contained 500 μL of DMEM medium and 10% FBS. After incubation for 24 hours, cells that had crossed the Matrigel membrane were stained with crystal violet and counted.

[0050] Immunofluorescence: For cellular immunofluorescence, cells were fixed with 4% paraformaldehyde for 15 minutes and washed three times with PBS. Cells were then permeabilized with 0.5% Triton X-100 and washed again. Finally, cells were blocked with 5% BSA and incubated with antibodies. For tissue immunofluorescence, paraffin sections were dewaxed, hydrated, and subjected to heat-induced epitope repair (HIER) before incubation with antibodies. Rabbit anti-mouse anti-NRF2 (12721S, CST), anti-Ki67 (GB121141, Servicebio), and anti-CD8 (ab217344, Abcam) were used as primary antibodies. Goat anti-rabbit Alexa Fluor488 antibody (GB25303, Abclonal) was used as a secondary antibody. Nuclear counterstaining was performed using DAPI (C1005, Beyotime).

[0051] Western Blot: Proteins in cells and tissues were assessed by Western blot (WB). After total protein extraction, protein concentration was quantified using a BCA protein assay kit (G2026-1000T, Servicebio). Proteins were then separated by SDS-PAGE (PG112, Epizyme). The obtained proteins were transferred to a polyvinylidene fluoride membrane (WJ002S, Epizyme) in Tris-glycine buffer, blocked with 5% skim milk powder for 1 hour at room temperature, and then incubated overnight with primary antibody at 4°C. The membrane was subsequently washed and incubated with secondary antibody at room temperature for 2 hours. Finally, the membrane was washed with TBST solution and imaged in the dark using a gel imaging system (Tanon 1600, China). Primary antibodies were anti-NRF2 (12721S, CST), STING (19581-1-AP, Proteintech), p-STING (AF7416, Affinity), p-TBK1 (AF8190, Affinity), p-IRF3 (AF2436, Affinity), NOTCH1 (A7636, Abclonal), E-cadherin (A20798, Abclonal), N-cadherin (A19083, Abclonal), Snail (A5243, Abclonal), and Vimentin (A19607, Abclonal). Internal controls were Lamin B (ab133741, Abcam), GAPDH (60004-1-lg, Proteintech), and β-actin (bs-0061R, Bioss).

[0052] BMDC Differentiation: Mouse bone marrow-derived dendritic cells (BMDCs) were induced as described above. Bone marrow cells isolated from BALB / c mice were seeded in six-well plates with differentiation medium containing mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) (20 ng / ml, 315–03-20UG, PeproTech) and mouse IL-4 (20 ng / ml, RP01161, ABclonal). The medium was changed every 2 days. Finally, on day 6, non-adherent and loosely adherent cells were collected as BMDCs.

[0053] Co-culture model: The transwell co-culture model was used to assess BMDC maturation, STING signaling pathway activation, and cytokine production. In short, K7M2 cells and BMDCs were cultured at 1.0 × 10⁶ cells per well. 6 and 1.0×10 6Cells were seeded at a density of [number] cells per chamber in both upper and lower compartments. After overnight incubation, the cells were treated with PBS, LPS, SMCPM, and SMCPM + laser (0.25 W / cm²). 2 Processed (3 minutes). After 24 hours of incubation, BMDCs were collected, washed three times with PBS, and analyzed by flow cytometry with antibody staining. The supernatant was collected for ELISA. Total cellular protein was extracted for Western blotting.

[0054] Enzyme-linked immunosorbent assay (ELISA): After co-culturing K7M2 and BMDC, the supernatant was collected for ELISA. TNF-α and IL-6 levels were measured using the ELISA kit (MM-0132M2 / MM-0163M2, MEIMIAN). ELISA procedures were performed according to the manufacturer's recommendations.

[0055] Establishment of orthotopic osteosarcoma and spontaneous lung metastasis models: Orthotopic osteosarcoma and spontaneous lung metastasis models were established using 6-week-old male Balb / c mice. K7M2 cells (2×10⁻⁶) were used... 7 (50 μL, cells / mL) was injected into the bone marrow cavity of the right tibial plateau in each mouse. When the average tumor size reached approximately 50 mm... 3 Mice were randomly divided into four groups (n = 5): intravenously injected with 100 μL of saline, SCP (equivalent to 1 mg / kg Ce6), SMCP (equivalent to 1 mg / kg Ce6), and SMCPM (equivalent to 1 mg / kg Ce6). Four hours after injection, the tumor area was irradiated with a 660 nm laser (0.25 W, 2 min). After four treatments, all mice were sacrificed and tissues were collected for further analysis. The vertical diameter of the tumor was measured using calipers to monitor tumor growth. The tumor volume was calculated using the following formula: V = 4π / 3 × a / 2 × (b / 2) 2 (a: long axis of tumor; b: short axis of tumor.) Serum biochemical parameters, including ALT, AST, UREA, CR, CK, and CK-MB, were measured using an automated biochemical analyzer (RaytoLife and Analytical Sciences Co., Ltd., China) to assess the systemic toxicity of SMCPM. Major organs and tumors were fixed in 4% paraformaldehyde, embedded in paraffin, and stained with H&E. Images of all stained samples were captured using a fluorescence microscope (Olympus IX73, Japan).

[0056] Flow cytometry: To obtain a single-cell suspension, tissue was minced and digested at 37°C for 2 hours with collagenase IV (C8160, Solarbio), DNase I (D8071, Solarbio), and hyaluronidase (H8030, Solarbio). The suspension was then filtered through a 70 μm sieve (352350, Corning). For in vitro flow cytometry, BMDCs were collected using a cell scraper. Before staining, cells were blocked with purified CD16 / 32 (553141, BD). The detection antibodies and dyes used in the embodiments of this invention include: Fixed Viability Dye eFluor 450 (65-0863-18, eBioscience), PE-CD11c (N418, Biolegend), FITC-CD80 (16-10A1, Biolegend), PE-CY7-CD86 (105115, Biolegend), BV510-IA / IE (107635, Biolegend), PE-CD3 (100205, Biolegend), BV510-CD4 (100553, Biolegend), FITC-CD8 (100705, Biolegend), PE-CY7-CD44 (103029, Biolegend), PE-CY7-CD25 (101915, Biolegend), and PE-CY7-CD69 (104511, Biolegend). Flow cytometry was performed using a Beckman Cytoflex flow cytometer (USA). Data were analyzed using Flowjo v10.8.1.

[0057] To ensure data comparability, the dosage of the nanocapsule SMCPM in the pharmacological activity study was calculated as equivalent to 1 mg / kg Ce6.

[0058] Example 1

[0059] This embodiment provides the synthesis of SMCPM nanocapsules, specifically including the following steps:

[0060] 1.1 Synthesis of mesoporous organosilica nanoparticles (MONs)

[0061] Prepared via a CTAB-mediated sol-gel method. First, CTAB (0.16 g) and NH3·H2O (1 mL) were dissolved in a mixture of water (75 mL) and ethanol (30 mL) at 35 °C. After vigorous stirring for 1 h, a mixture of TEOS (0.25 mL) and TETS (0.1 mL) was added to the mixture. After stirring for 24 h, the product was collected by centrifugation (10,000 rpm, 10 min) and washed three times with ethanol. Finally, MONs in CTAB were extracted with a mixture of ethanol (200 mL) and hydrochloric acid (40 μL, 60 °C, 3 h), repeated three times.

[0062] 1.2 Synthesis of manganese dioxide-doped mesoporous organosilicon nanoparticles (SMONs).

[0063] 5.8 mg of MONs were dispersed in 5 mL of PEI solution (10 mg / mL), and the mixture was shaken for 4 hours. The MONS-PEI was collected by centrifugation and washed three times with water. The obtained MONS-PEI and 10 mg of KMnO4 were added to 5 mL of water, and the mixture was gently stirred at 35 °C for 24 h. A brown product formed; this was centrifuged and redispersed in 5 mL of water. Then, 100 μL of OA was added dropwise to the above system. After stirring at 35 °C for 24 h, the SMONs were collected, washed three times with ethanol, and dispersed in 10 mL of water for further use.

[0064] 1.3 Modify SMONs with BSA.

[0065] The specific procedure was as follows: 5.8 mg of SMONs were dispersed in PEI solution for 12 hours, then centrifuged and redispersed in 5 mL of GA solution (0.025% by mass). After 12 hours, BSA solution (5 mL, 4 mg / mL) was added to the above GA / PEI-SMONs solution, and incubated for 12 hours. BSA-modified SMONs were finally obtained.

[0066] 1.4 Modify SMONs with PT-7, Ce6 and ML385.

[0067] Ce6 and PT7 were grafted onto the surface of BSA-modified SMONs via a click reaction. The procedure was as follows: 0.4 mg of Ce6, 0.4 mg of PT7, 2 mg of NHS, and 2 mg of EDC were dissolved in 400 μL of DMF for 3 hours to prepare activated carboxylic acid esters Ce6 (NHS-Ce6) and PT7 (NHS-PT7). 50 μL of NHS-Ce6 and 50 μL of NHS-PT7 were simultaneously added to the BSA-modified SMONs, and the mixture was stirred for 12 hours to obtain SMONs-Ce6-PT7 (SMCP) nanoparticles. Finally, ML385 was loaded onto the surface of SMONs-Ce6-PT7 via hydrophobic interactions. Specifically, 1 mL of 1 mg / mL ML385 was mixed with SMONs-Ce6-PT7 for 12 hours to obtain the product SMONs-Ce6-PT7-ML385 (SMCPM).

[0068] The control group SCP consists of nanocapsules without MnO2 and ML385, which are obtained by grafting Ce6 and PT7 onto the surface of BSA-modified MONs via click reaction.

[0069] like Figure 2 As shown, the morphology and surface properties of SMCPM were observed. Low-magnification and high-magnification transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images show that SMCPM is quasi-spherical with a wrinkled surface structure. Figure 2 AC). This unique morphological feature is attributed to the reconstruction effect of KMnO4 on the MONs framework. Elemental distribution maps and energy-dispersive X-ray spectroscopy (EDS) analysis show that Si, O, S, N, and Mn elements are symmetrically distributed in the reconstructed framework. Figure 2 DE). These Mn particles embedded in SMCPMs not only regulate the transformation of particles into soft, hollow MONs, but also activate the STING signaling pathway, playing a key role in enhancing tissue penetration and reversing immunosuppressive TME. Furthermore, the average hydrodynamic diameter of the SMCPMs was measured to be 263.9 nm. Figure 2 F), which is basically consistent with the particle size calculated from TEM images (approximately 245 nm). When various drugs were grafted onto the surface of SMCPM, its Zeta potential increased from -36 mV to -18 mV ( Figure 2 G).

[0070] Suitable size and negative surface charge help reduce protein crown formation and prolong blood circulation time. Ultraviolet-visible spectroscopy (UV-vis) shows a distinct absorption peak at 667 nm. Figure 2 H), indicating that Ce6 (dihydroporphyrin e6, a photosensitizer) has been effectively covalently crosslinked on the surface of SMCPM (a multifunctional nanomaterial).

[0071] To verify the stiffness transformation characteristics of SMCPM, this invention investigated its Young's modulus changes and Mn (manganese) ion release in a simulated tumor microenvironment (TME). Specifically, atomic force microscopy (AFM) images showed that SMCPM in solution maintained a hemispherical morphology similar to that in transmission electron microscopy (TEM) images, with a measured Young's modulus of 4.43 GPa. However, when SMCPM was placed in a glutathione (GSH) solution, it exhibited an unexpanded spherical morphology; and after incubation in 20 mM GSH solution for 10 minutes, its Young's modulus decreased to 3.57 GPa. Figure 2 (IJ). These phenomena fully demonstrate that the SMCPM has a sensitive stiffness transition capability in response to environmental stimuli. Along with the stiffness transition, the internal Mn of the SMCPM was also monitored. 2+ The release of Mn was observed. Results showed that after incubation in PBS buffer (pH = 7.4) for 1 hour, Mn... 2+ The concentration was only 0.97 mg / L; when the incubation time was extended to 12 hours, Mn 2+ The concentration remained almost unchanged, indicating that SMCPM hardly changed during this period, and there was almost no change in Mn. 2+ Release. However, when SMCPM is dispersed in H2O2 solution (100 μM, pH = 6.5), Mn... 2+ The concentration increased to 2.11 mg / L, indicating the presence of a small amount of Mn under acidic H2O2 stimulation. 2+ Upon release, some SMCPM underwent transformation. During incubation in 20 mM GSH solution, Mn... 2+ The concentration was as high as 8.68 mg / L, which is 8.9 times and 4.1 times the concentration in PBS or H2O2 solution, respectively. Figure 2 The rapid increase in manganese ion concentration corresponds to a large release of manganese ions in SMCPM, indicating the disintegration of the particle skeleton and the transformation of particle rigidity.

[0072] Example 2

[0073] The in vitro permeation properties of SMCPM nanocapsules were investigated.

[0074] The changes in permeability induced by stiffness transition were evaluated in three-dimensional tumor cell spheroids. Confocal laser scanning microscopy (CLSM) images showed that in a physiological environment without glutathione treatment (GSH-free, i.e., GSH⁻), SMCPM-treated tumor spheroids exhibited weak fluorescence signals in the depth range of 0–90 μm, indicating poor permeability of SMCPM under these conditions. However, in the SMCPM / GSH group treated with 20 mM GSH, the fluorescence signal remained clearly visible as the depth of the three-dimensional tumor spheroids increased to 90 μm, demonstrating a significant penetration effect. These results indicate that in a simulated tumor microenvironment (containing GSH, i.e., GSH⁻), the fluorescence signal remains clearly visible even with increasing depth of the three-dimensional tumor spheroids. + In this study, the tumor penetration ability of SMCPM was enhanced.

[0075] The enhanced tumor penetration ability of deformable SMCPMs, due to the decrease in Young's modulus within a specified time, is likely attributed to their higher efficiency in traversing intercellular spaces and superior internalization ability compared to rigid particles, resulting in faster particle penetration and higher retention rates within spheroids. Furthermore, quantitative statistical results showed that, within the depth range of 0 to 90 micrometers, the fluorescence signal intensity of SMCPM / GSH-treated spheroids was enhanced by 1.92-fold, 2.30-fold, 2.22-fold, 2.91-fold, 2.99-fold, 2.28-fold, and 2.13-fold, respectively. Figure 3 (AB). The above results reveal that the SMCPM's tumor penetration capability in the simulated tumor microenvironment was significantly enhanced due to the change in stiffness.

[0076] Example 3

[0077] The in vitro antitumor properties of SMCPM nanocapsules were investigated.

[0078] This invention evaluated the cytotoxicity and antitumor effects of SMCPM on K7M2 cells. Figure 3 As shown in Figure C, after 24 hours of co-incubation with SMCP or SMCPM, the survival rate of K7M2 cells remained above 90%, indicating their good biocompatibility. Furthermore, the phototherapy effect of SMCPM on K7M2 cells was evaluated. The results showed that under laser irradiation, a concentration of SMCPM as low as 0.25 µg / ml could produce a significant anti-osteosarcoma effect (…). Figure 3 D). Furthermore, after irradiation with a 660 nm laser for 1, 2, 3, and 5 minutes, the survival rates of K7M2 cells treated with SMCPM were 19.64%, 14.48%, 8.20%, and 3.31%, respectively. Figure 3 E). Furthermore, when the laser power was reduced from 1 W to 0.25 W, the cell viability remained below 20% (E). Figure 3 F). These results indicate that SMCPM has excellent phototherapy effects.

[0079] To investigate its killing mechanism, K7M2 cells were co-incubated with SMCP and SMCPM at an equivalent Ce6 concentration of 0.25 µg / ml, and then irradiated with laser. The results showed that the survival rate of K7M2 cells treated with SMCPM was 32.70%, which was 0.49 times lower than that of cells treated with SMCP. This result indicates that ML385 in SMCPM plays an important role in regulating intracellular oxidative stress and inducing apoptosis. Live / dead cell staining results also showed that SMCPM achieved the most effective tumor killing under laser irradiation, indicating its significant phototherapy effect. Figure 3 GH).

[0080] Example 4

[0081] We investigated whether ML385 encapsulated in SMCPM could reverse phototherapy resistance and modulate epithelial-mesenchymal transition (EMT).

[0082] like Figure 4 A demonstrates that reactive oxygen species (ROS) can induce upregulation of NRF2 expression. Elevated NRF2 then undergoes nuclear translocation, further promoting the upregulation of protein expression along the NOTCH1-EMT signaling axis. This cascade reaction leads to downregulation of E-cadherin expression in tumor cells, while simultaneously upregulating N-cadherin and vimentin expression, ultimately triggering epithelial-mesenchymal transition (EMT) and promoting lung metastasis. This example investigates whether ML385 encapsulated in SMCPM has the ability to reverse this process.

[0083] To elucidate the phototherapy mechanism enhanced by SMCPM, this example assessed the generation of intracellular ROS levels. Confocal laser scanning microscopy (CLSM) images showed that almost no ROS signaling was observed in cells treated with SMCP or SMCPM alone. Figure 4 B). Conversely, significant ROS signals were observed in cells treated with both SMCP and SMCPM under 660 nm laser irradiation. Quantitative statistical results showed that the ROS intensity of the SMCPM-treated group was 185.8, which was 1.96 times that of the SMCP-treated group (94.89). Figure 4 C). The increased ROS levels in cells treated with SMCPM are likely attributable to the inhibitory effect of ML385 on the cellular antioxidant defense system.

[0084] Furthermore, consistent results from the matrix gel invasion assays indicated that the invasive ability of K7M2 cells was significantly reduced in the SMCPM + laser group compared to the SMCP + laser group. Figure 4D, E). Further Western blot (WB) and CLSM were used to clarify the intracellular expression and distribution of NRF2. WB grayscale images showed that, compared to the PBS control group, the NRF2 and nuclear NRF2 bands were thicker in the SMCP+ laser treatment group, indicating increased NRF2 expression. Figure 4 H). However, these bands in the SMCPM + laser treatment group are finer than those in the SMCP + laser treatment group ( Figure 4 I). These findings confirm the ML385-induced decrease in NRF2 expression. Furthermore, the distribution of NRF2 was also observed in this invention. CLSM images showed that only a weak fluorescence signal was observed around the nucleus in cells treated with SMCPM+ laser (I). Figure 4 FG, Figure 5 In contrast, the corresponding fluorescence signal in cells treated with SMCP + laser was very significant. This difference indicates a reduction in the nuclear distribution of NRF2. Overall, the restriction of NRF2 expression and the reduction in its nuclear distribution together suggest that SMCPM can effectively attenuate the NRF2-mediated defense system.

[0085] Therefore, the restricted NRF2 expression and reduced nuclear distribution in the SMCPM+ laser treatment group showed a significant advantage in regulating EMT. Further analysis of NRF2 downstream gene expression, including NOTCH1 and EMT-related markers (E-cadherin, N-cadherin, Snail, and vimentin), revealed that compared to the control group, SMCP-treated K7M2 cells showed increased gray values ​​for NOTCH1 and EMT-related markers (N-cadherin, Snail, and vimentin), while the gray value for E-cadherin decreased, indicating enhanced NOTCH1 expression and EMT activation. Figure 4 J). However, in the SMCPM+ laser treatment group, compared with the SMCP+ laser treatment group, the gray values ​​of NOTCH1, N-cadherin, Snail, and vimentin decreased, while the gray value of E-cadherin increased, indicating that NOTCH1 expression was suppressed and EMT process was inhibited in SMCPM+ laser-treated K7M2 cells. Figure 4 These results indicate that the ML385 in the SMCPM can further modulate the NRF2-NOTCH1-EMT signal axis and reverse the EMT process.

[0086] Example 5

[0087] SMCPM activates the STING pathway and enhances the body's immune response.

[0088] First, the distribution of SMCPM was evaluated in K7M2 tumor-bearing mice. For example... Figure 6 As shown in the fluorescence images, SMCPM fluorescence is observed in the lungs, liver, kidneys, and tumors; Figure 7 As shown, quantitative fluorescence results indicated that tumor fluorescence intensity reached its maximum 6 hours after injection, demonstrating effective and rapid tumor accumulation. Subsequently, the in vivo immune response mediated by SMCPM was assessed. Figure 8 A). Lymph nodes, spleen, and tumors were collected from K7M2 tumor-bearing mice treated with SMCPM+laser. For example... Figure 8 As shown in C and 8E, immunofluorescence images revealed that SMCPM significantly increased CD8+ in tumor tissue. + The number of T cells was increased, and this effect was significantly enhanced under laser irradiation. Compared with the PBS-treated group, Western blotting grayscale images of tumors in the SMCPM-treated group showed enhanced grayscale of p-STING, p-TBK1, and p-IRF3 labeled bands, indicating that SMCPM activated the STING pathway in tumor tissue. Furthermore, the grayscale value of the p-STING band in the SMCPM+laser-treated group was significantly higher than that in the SMCPM group, indicating that the double-stranded DNA released by PIT further activated the STING pathway. These results confirm a good activation effect of the STING signaling pathway. Figure 8 B and 8D). Strong STING activation favors DC maturation and T cell infiltration. Therefore, the distribution of these immune cells was evaluated. Flow cytometry analysis showed that the proportion of mature DCs in tumors treated with SMCPM+ laser was 30.53%, which was 3.83-fold and 1.59-fold higher than that in tumors treated with PBS- and SMCPM-, respectively, demonstrating the strongest immune activation (B and 8D). Figure 8 F and 8I). Simultaneously record activated CD8. + T cells (CD69) + CD8 + ) and memory CD8 + T cells (CD44) + CD8 + () Figure 8 Flow cytometry analysis showed that the proportion of activated T cells in tumors treated with SMCPM+laser increased to 27.83%, higher than that in tumors treated with PBS- (8.23%) or SMCPM- (18.80%). Similarly, the proportion of memory T cells gradually increased in PBS- (9.50%), SMCPM- (13.13%), and SMCPM+Laser (16.30%).

[0089] In addition, mature dendritic cells and activated CD8+ cells in lymph nodes and spleen were evaluated. + T cells and memory CD8 + The proportion of T cells. Flow cytometry results showed that, compared with PBS-treated lymphocytes, SMCPM+laser-treated lymphocytes had higher levels of mature dendritic cells and activated CD8+. + T cells and memory CD8+ The proportions of T cells increased to 33.43%, 22.37%, and 22.63%, respectively. Figure 9 AC). The proportion of these cells in the spleen treated with SMCPM+laser increased from 24.80%, 8.90%, and 6.70% to 48.13%, 19.23%, and 17.77% (AC). Figure 9 (DF). The increased proportion of unique immune cell subtypes in the lymph nodes and spleen confirms the super-strong in vivo immune response induced by SMCPM.

[0090] Example 6

[0091] To evaluate the inhibitory effect of SMCPM on osteosarcoma (OS) and lung metastases in vivo.

[0092] Mice bearing K7M2 osteosarcoma were injected with PBS, SCP, SMCP, and SMCPM, respectively. After laser irradiation, tumors and lung tissue were harvested. The tumor volume in the PBS-treated group (control group) continued to increase, reaching 286.6 mm² at the end of the observation period. 3 This indicates that it failed to effectively inhibit tumor growth. Although the tumor volume in the SCP-treated group continued to increase, its final tumor volume (167.7 mm) was lower. 3 The tumor volume in the SMCP group was smaller than that in the PBS group, indicating a weaker antitumor effect. In contrast, tumors in both the SMCP and SMCPM treatment groups were significantly inhibited. Corresponding tumor volume growth curves showed that the tumor volume in the SMCP treatment group remained at 61.40 mm. 3 The tumor size was similar to that before treatment; more surprisingly, the tumor volume in the SMCPM treatment group decreased from 50.22 mm. 3 Reduced to 9.02 mm 3 ( Figure 10 C). Tumor images also showed that the residual tumor in the bone area was the smallest ( Figure 10 A). Furthermore, all mice showed stable weight gain during treatment, indicating that SMCPM has minimal systemic toxicity. Figure 10 B). Ki67 immunofluorescence analysis further showed that SMCPM had a significant inhibitory effect on the proliferation of the primary lesion (B). Figure 10 These results indicate that SMCPM exhibits excellent tumor growth inhibition at the primary tumor site in mice.

[0093] Furthermore, lung imaging and whole-lung HE staining results showed that the number of metastatic nodules in the SMCP treatment group was 7.0, significantly lower than that in the PBS group (36.0) and the SCP group (17.0), indicating that it has a certain inhibitory effect on lung metastasis. However, the average number of lung metastatic nodules in the SMCPM treatment group was only 0.4, showing that it performed best in inhibiting lung metastasis. Figure 10FH, Figure 11 Researchers also investigated the mechanisms of lung metastasis. Western blot (WB) results showed that, compared with the SMCP treatment group, the gray intensity of NRF2 and intranuclear NRF2 was reduced in the SMCPM treatment group. Figure 10 These results indicate that NRF2 levels were suppressed in both the nucleus and cytoplasm. Furthermore, downstream markers associated with NRF (such as NOTCH1, E-cadherin, N-cadherin, Snail, and vimentin) were monitored. Western blotting showed reduced gray levels of these markers, suggesting well-suppressed epithelial-mesenchymal transition (EMT). Figure 10 SMCPM not only effectively inhibits EMT, but also has an immune-activating effect, which greatly improves its anti-tumor efficacy and inhibits lung metastasis of osteosarcoma.

[0094] like Figure 11 As shown, the Kaplan-Meier survival curves (A) show that SMCPM combined with laser therapy improved the survival rate of osteosarcoma (OS) mice (p<0.01), which is consistent with the reduction in the number of lung nodules observed on day 60 (B).

[0095] Example 7

[0096] To assess the biosafety of SMCPM nanocapsules.

[0097] After mice were injected with SMCPM, there were no significant differences in serum biochemical indicators or signs of major organ damage, indicating that SMCPM has good biocompatibility in vivo. Figure 12 AF). Furthermore, the effect of administering double the treatment dose within one week on normal tissues was assessed. Hematoxylin and eosin (H&E) staining of major organs revealed no signs of damage. Figure 12 G). These results indicate the biocompatibility of SMCPM in vivo.

[0098] Where there is no conflict, the above embodiments and features described herein can be combined with each other.

[0099] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of nanocapsules SMCPM for the preparation of a medicament for the treatment of osteosarcoma lung metastasis, characterized in that, The nanocapsule SMCPM is prepared by dispersing mesoporous organosilicon nanoparticles (MONs) in a polyethyleneimine (PEI) solution. PEI is anchored to the surface of MONs through electrostatic adsorption or hydrogen bonding, forming a MONS-PEI complex. The amino groups on PEI give the surface a positive charge. The amino groups act as reducing agents, reducing KMnO4 to MnO2 and promoting in-situ deposition of manganese dioxide. Oleic acid is added to reduce the surface energy of the nanoparticles and inhibit particle aggregation, resulting in manganese dioxide-doped mesoporous organosilicon particles (SMs). Subsequently, a carboxyl-rich photosensitizer Ce6 and a tumor-targeting peptide PT-7 are covalently linked to the amino groups of SM modified with bovine serum albumin, resulting in SM-Ce6-PT-7. Finally, the NRF2 inhibitor ML385 is loaded onto SM-Ce6-PT-7 by electrostatic adsorption, thus obtaining SMCPM. The amino acid sequence of PT-7 is PPSHTPT.

2. Use according to claim 1, characterized in that, The mass concentration of the polyethyleneimine solution is 8~10 mg / mL, and the mass ratio of the MONs to the potassium permanganate is (5~6):

10.

3. Use according to claim 1, characterized in that, The mass ratio of Ce6, PT-7 and ML385 is 1:1:(2.5~3).

4. Use according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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