A MEC nanoprobe, its preparation method and application

Abstract

This invention discloses a MEC nanoprobe, its preparation method, and its applications. The MEC nanoprobe is composed of Mn... 3+ The sample was prepared using TCPP, Erastin, and CD133mab, with CD133mab coupled to the surface. The preparation method involved adding Mn... 3+ Mn-TCPP-MOF was synthesized by stirring TCPP in an N,N-dimethylformamide solution containing acetic acid; Erastin was then added and stirred at room temperature, followed by centrifugation, washing, and redispersing in deionized water; the amide bonds were then activated via EDC / NHS and coupled to CD133mAb to obtain MEC nanoprobes. The combined application of this MEC nanoprobe with a TGFβ1R inhibitor can block both the classical ferroptosis resistance pathway and CAF-related pathways, achieving dual-channel blockade of ferroptosis resistance in pancreatic cancer. Furthermore, dual-modal imaging enables semi-quantitative in vivo visualization assessment of ferroptosis resistance, demonstrating promising clinical application prospects.

Description

Technical Field

[0001] This invention relates to the field of medicine, and more particularly to a MEC nanoprobe, its preparation method, and its uses. Background Technology

[0002] Pancreatic ductal adenocarcinoma is a malignant tumor of the digestive system with insidious onset, rapid progression, and extremely poor prognosis. Surgical resection is an effective treatment for pancreatic cancer, but due to the difficulty in early detection, it is often discovered at an intermediate or advanced stage, and the opportunity for radical surgery has been lost. Chemotherapy is an important treatment for intermediate and advanced pancreatic cancer, but it is prone to drug resistance and has poor efficacy. Current research shows that KRAS mutation in pancreatic cancer is the main cause of resistance to chemotherapy and immunotherapy, while ferroptosis therapy is sensitive to tumors with KRAS mutations and is a highly promising treatment strategy for pancreatic cancer. Although a large number of ferroptosis studies have been carried out on pancreatic cancer and some results have been achieved, there is still a large gap from clinical translation. The main reasons are: (1) The mechanism of ferroptosis resistance is unclear: In the past, ferroptosis resistance only considered pancreatic cancer cells themselves and ignored the influence of the complex tumor microenvironment of pancreatic cancer on them; (2) There is a lack of in vivo semi-quantitative visualization assessment methods for ferroptosis resistance: In the past, in vivo visualization strategies lacked assessment of ferroptosis resistance pathways, which will affect the selection of subsequent treatment plans.

[0003] Ferroplasmosis is a non-apoptotic form of cell death that depends on the accumulation of intracellular iron and leads to an increase in toxic lipid peroxides. A recent study published in *Science* showed that in pancreatic cancer, exogenous cystine is transported into the cell via the Xc-system to form cysteine ​​(Cys), providing an important raw material for GSH synthesis. This pathway is also known as the classical Xc-Cys-GSH metabolic axis (hereinafter referred to as the "classical pathway"). Previous research by our group indicated that, in addition to the classical pathway, pancreatic cancer cells resist ferroptosis through tumor microenvironmental ferroptosis regulation via a novel pathway involving tumor microenvironmental ferroptosis (CAFs). Specifically, CAFs mediate a series of metabolic remodeling processes through the TGF-β1 / SMAD3 / ATF4 signaling axis, significantly increasing the extracellular supply of Cys to support GSH synthesis, thereby enhancing tumor cell resistance to ferroptosis. In these two ferroptosis resistance pathways, GSH is a hallmark metabolite, and its metabolic level is closely related to ferroptosis resistance in pancreatic cancer.

[0004] Previous in vivo visual assessments of ferroptosis resistance have primarily relied on single imaging techniques, which often have significant limitations: Magnetic resonance imaging (MRI) offers advantages such as high tissue penetration depth and spatial resolution, but suffers from low sensitivity and cannot assess the degree of ferroptosis; fluorescence imaging, using nanoprobes, achieves high-sensitivity monitoring and specifically traces the ferroptosis biomarker GSH, but its tissue penetration depth is insufficient. The construction of Mn-TCPP-MOF nanoprobes provides a promising solution for the in vivo visual assessment of ferroptosis resistance. On one hand, Mn's T1-weighted MRI imaging can reflect the in vivo metabolism of the nanoprobes; on the other hand, the fluorescence imaging performance of the nanoprobes in response to GSH enables a semi-quantitative in vivo assessment of the degree of ferroptosis resistance. Furthermore, Mn-MOF nanoprobes can deplete GSH and generate ROS through sonodynamics and Fenton properties, ultimately inducing ferroptosis in tumor cells. Summary of the Invention

[0005] The purpose of this invention is to provide a GSH-responsive dual-modal imaging nanoprobe, Mn-TCPP-MOF-Era-CD133mab (MEC nanoprobe). This nanoprobe uses Mn-MOF as a carrier, loads the Xc-system inhibitor Erastin, and is externally coupled with CD133mab. The combination of this system with the TGFβR1 inhibitor Repsox achieves a dual-channel blocking strategy for ferroptosis in pancreatic cancer, inducing ferroptosis in pancreatic cancer cells by reducing GSH and increasing ROS. Furthermore, this nanosystem possesses dual-modal imaging capabilities, reflecting the metabolism of the nanoprobe at the tumor site using MRI T1 imaging and reflecting the degree of ferroptosis resistance using fluorescence imaging. This unique imaging mechanism enables in vivo detection of nanoprobe metabolism and the degree of ferroptosis resistance. In summary, this invention proposes a novel strategy for inducing ferroptosis in pancreatic cancer through "dual-channel blocking," and achieves in vivo visualization and assessment of nanoprobe metabolism and the degree of ferroptosis resistance through dual-modal imaging. This research contributes to deepening the understanding of the regulatory mechanism of ferroptosis in pancreatic cancer and lays a theoretical and practical foundation for targeted tumor microenvironment reversal of ferroptosis resistance in integrated diagnosis and treatment.

[0006] To achieve the above objectives, a MEC nanoprobe is characterized by being composed of Mn 3+ TCPP, Erastin, and CD133mab were used to prepare the sample, with CD133mab coupled to the surface.

[0007] The present invention also provides a method for preparing the MEC nanoprobe, characterized by comprising the following steps:

[0008] S1. With Mn 3+ Mn-TCPP-MOF was synthesized by stirring TCPP in an N,N-dimethylformamide solution containing acetic acid.

[0009] S2. Add Erastin to the Mn-TCPP-MOF obtained in S1, stir at room temperature, centrifuge, wash, and then disperse in deionized water to obtain Mn-TCPP-MOF-Era;

[0010] S3. The above Mn-TCPP-MOF-Era was coupled to CD133mAb via EDC and NHS-activated amide bonds to obtain MEC nanoprobes.

[0011] Furthermore, in step S1,

[0012] In the N,N-dimethylformamide solution containing acetic acid, the volume ratio of acetic acid to N,N-dimethylformamide is 1:4.

[0013] Optionally, the Mn 3+ TCPP: The ratio of N,N-dimethylformamide solution containing acetic acid to total volume is 10.4 * 10. - 2 mmol: 1.3 * 10 -2 mmol: 20ml;

[0014] Preferably, step S1 involves dissolving Mn 3+ Add the solution to N,N-dimethylformamide containing acetic acid and stir well; then slowly add TCPP to the above solution, stir at room temperature for 12±3 h, centrifuge, wash, and obtain Mn-TCPP-MOF.

[0015] Furthermore, in step S2,

[0016] Erastin and S1 Mn 3+ The dosage ratio is 3mg: 10.4*10 -2 mmol;

[0017] Preferably, step S2 involves adding Erastin to the Mn-TCPP-MOF DMSO solution obtained in step S1, stirring at room temperature for 24±5 h, centrifuging, washing, and then dispersing in deionized water to obtain Mn-TCPP-MOF-Era.

[0018] Further, step S3 involves adding an aqueous solution of EDC and NHS to the Mn-TCPP-MOF-Era prepared in step S2 and stirring until homogeneous. Then, CD133mAb is added and stirred until homogeneous. The MEC nanoprobe is obtained by centrifugation.

[0019] Preferably, the ratio of Mn-TCPP-MOF-Era, CD133mAb in EDC, NHS, water, and S2 is 15mg:20mg:10ml:40ml:100μg. The water is preferably deionized water.

[0020] The present invention also provides the use of the MEC nanoprobe for the preparation of drugs that induce ferroptosis in tumor cells.

[0021] The present invention also provides a drug for inducing ferroptosis in tumor cells, characterized in that it includes the MEC nanoprobe.

[0022] Furthermore, it also includes a TGFβR1 inhibitor; preferably, the TGFβR1 inhibitor is Repsox.

[0023] The present invention also provides a drug that simultaneously blocks the classical ferroptosis resistance pathway and CAFs-related pathways, characterized in that it comprises the MEC nanoprobe and a TGFβR1 inhibitor; preferably, the TGFβR1 inhibitor is Repsox.

[0024] This invention utilizes Mn-MOF to construct an integrated system for the diagnosis and treatment of ferroptosis. The mesoporous structure of MOF carries Erastin, an inhibitor of the classical ferroptosis resistance pathway, and is coupled externally with the pancreatic cancer stem cell targeting protein CD133mab, generating Mn-TCPP-MOF-Era-CD133mab (MEC). First, the nanoprobes penetrate the dense stromal barrier of pancreatic cancer through ultrasonic mechanical action and the targeting effect of CD133mab, increasing the accumulation of nanoprobes within the tumor. Second, the combined application of MEC and the TGFβR1 inhibitor Repsox simultaneously blocks both the classical ferroptosis resistance pathway and CAF-related pathways, achieving dual-channel blockade of pancreatic cancer ferroptosis resistance. Furthermore, this system is responsive to the tumor microenvironment, releasing Mn under high GSH conditions. 3+ and TCPP, Mn 3 + The Fenton-mediated effect and TCPP-mediated sonodynamic therapy jointly induce ferroptosis in tumor cells. Finally, the in vivo metabolic status of the nanoprobes and the degree of ferroptosis resistance are visualized and assessed using a dual-modal imaging strategy of magnetic resonance and fluorescence. Therefore, this integrated ferroptosis diagnosis and treatment system realizes a dual-channel blocking strategy for ferroptosis resistance and utilizes dual-modal imaging to achieve semi-quantitative in vivo visualization and assessment of ferroptosis resistance, showing promising clinical application prospects.

[0025] This invention uses some abbreviations, such as E which stands for Era, or Erastin.

[0026] C is CD133mab.

[0027] Mn-MOF is Mn-TCPP-MOF, and also Mn-TCPP-MOF nanoparticles.

[0028] M stands for Mn-TCPP-MOF, which is also Mn-TCPP-MOF NPS.

[0029] ME stands for Mn-TCPP-MOF-Era, which is also Mn-MOF-Era.

[0030] MC stands for Mn-TCPP-MOF-CD133mab.

[0031] MEC, which stands for Mn-TCPP-MOF-Era-CD133mab, is also a MEC nanoprobe and a GSH-responsive nanoprobe. Attached Figure Description

[0032] Figure 1 Figure 1 shows the synthesis and characterization results of MEC nanoprobes. Figure 2 shows the synthetic route and transmission electron microscopy (TEM) image of the nanoprobes (scale bar: 200 μm); Figure 3 shows the particle size distribution of the nanoprobes; Figure 4 shows the particle size and zeta potential of the nanoprobes; Figure 5 shows the FTIR spectrum of the nanoprobes; Figure 6 shows the UV-Vis absorption spectrum of the nanoprobes; Figure 7 shows the fluorescence intensity of MEC as a function of increasing GSH concentration; Figure 8 shows the Erastin release curve in 80 μm GSH and PBS buffer; Figure 9 shows the GSH consumption as detected by the DTNB method; Figure 10 shows the acoustic-dynamic properties of MEC; and Figure 11 shows the Fenton catalytic activity.

[0033] Figure 2 This is the XRD pattern of Mn-TCPP-MOF.

[0034] Figure 3 It is an Mn-TCPP-MOF element mapping image.

[0035] Figure 4 The graphs show the magnetic resonance imaging performance of the nanoprobes evaluated using a 0.5T MRI scanner. In the graphs, a is the fitting curve of the longitudinal relaxation rate (1 / T1) of the nanoprobes, and b is the fitting curve of the transverse relaxation rate (1 / T2) of the nanoprobes.

[0036] Figure 5 This is a TEM image showing the time-varying aqueous stability of MEC and GSH. Scale bar: 200 nm.

[0037] Figure 6 This is a graph showing the change in absorbance of DPBF in 10 μg / mL MEC under different power ultrasonic irradiation.

[0038] Figure 7This is a graph showing cellular uptake performance. Image a is a laser confocal microscopy image of Panc-02 cells co-incubated with CAFs, Mn-MOF, and MC for 0-6 h (scale bar: 20 μm); image b is a graph of Panc-02 cells co-incubated with CAFs, Mn-MOF, and MC for 4 h, along with ICP-OES quantitative analysis; image c is a soft X-ray 3D image of Panc-02 cells co-incubated with PBS, Mn-MOF, and MC for 4 h; image d is a laser confocal microscopy image showing the penetration of nanoprobes into a tumor spheroid model (scale bar: 100 μm).

[0039] Figure 8 These are images showing the cytotoxicity and cell therapy effects of the nanoprobes. a) shows the cytotoxicity of Mn-MOF and MC on Panc-02 cells (n=3). b) shows the cytotoxicity of Mn-MOF and MC on CAFs (n=3). c) shows the CCK-08 results of Panc-02 cells under different treatment conditions (n=3). d) shows the fluorescence images of reactive oxygen species (ROS) generation in Panc-02 cells under different treatment conditions. Scale bar: 200 μm. e) shows the live / dead cell staining of Panc-02 cells under different treatment conditions. Scale bar: 200 μm.

[0040] Figure 9 These are images of ferroptosis at the cellular level using nanoprobes. Image a shows lipid peroxidation induced in Panc-02 cells by different nanoprobes, as revealed by laser confocal microscopy. Scale bar: 20 μm. Image b shows GPX4 expression in Panc-02 cells treated with different nanoprobes, as shown by Western blot analysis. Image c shows GSH levels in Panc-02 cells treated with different nanoprobes, as revealed by a GSH assay kit. Image d shows TEM images of mitochondrial morphology induced by different nanoprobes. Scale bar: 1 μm.

[0041] Figure 10 This is a graph showing the biocompatibility assessment in vivo. (a) Weight change, (b) Blood biochemistry analysis of mice 14 days after intravenous injection of MEC, and (c) Routine blood analysis. (d) H&E staining of major organs. Scale bar: 200 micrometers. (e) Hemolysis results of different concentrations of MEC.

[0042] Figure 11This is an evaluation of the in vivo tumor treatment efficacy of nanoprobes. a) is a schematic diagram of in vivo nanoprobe treatment. b) is a tumor photograph 14 days after treatment. c) is the tumor volume 14 days after treatment. d) is an image of tumor tissue stained with H&E, Ki67, and TUNEL. Scale bar: 100 μm. e) is an immunofluorescence and immunohistochemical image showing changes in ROS, GPX4, and LPO levels after nanoprobe treatment. Scale bar: 100 μm. f) is a schematic diagram showing the changes in ferroptosis-related factors induced by MEC nanoprobe treatment.

[0043] Figure 12 This is a graph showing the changes in mouse body weight under different treatments over 14 days.

[0044] Figure 13 This is a diagram of Masson staining results of tumor tissue in an in vivo biosafety assessment experiment.

[0045] Figure 14 These are in vivo imaging images of the nanoprobes. Image a shows fluorescence images after in situ injection of the nanoprobes into the tumor and contralateral tissue; image b shows the quantitative statistics of mean fluorescence intensity (MFI); image c shows the changes in in vivo fluorescence intensity in mice from 0 to 72 h after different treatments; image d shows the in vitro fluorescence detection of major organs; image e shows T1-weighted magnetic resonance images of mouse tumors from 0 to 12 h after different treatments; and image fg shows the quantitative statistics of tumor MFI values ​​and the detection results of GSH content within the tumor at different time points. Detailed Implementation

[0046] The embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention. 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 the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0047] In the following embodiments:

[0048] In this invention, E refers to Era, which is also Erastin.

[0049] C is CD133mab.

[0050] Mn-MOF is Mn-TCPP-MOF, and also Mn-TCPP-MOF nanoparticles.

[0051] M stands for Mn-TCPP-MOF, which is also Mn-TCPP-MOF NPS.

[0052] ME stands for Mn-TCPP-MOF-Era, which is also Mn-MOF-Era.

[0053] MC stands for Mn-TCPP-MOF-CD133mab.

[0054] MEC, which stands for Mn-TCPP-MOF-Era-CD133mab, is also a MEC nanoprobe and a GSH-responsive nanoprobe.

[0055] Materials and Methods

[0056] 1) Materials

[0057] DMF was purchased from Aladdin (China). HAC, TCPP, dimethyl sulfoxide (DMSO), paraformaldehyde, and phosphate-buffered saline (PBS) were purchased from Macklin (China). Mn(OAc)3·2H2O was purchased from Sigma-Aldrich (Germany). Glutathione, Erastin, and lipid peroxidation fluorescent probes were purchased from APExBIO (USA). CD133 mAb and GPX4 mAb were purchased from Affinity (Canada). 1,3-Diphenylisobenzofuran (DPBF) was purchased from Bailingwei Technology (China). Reduced glutathione (GSH) assay kit was purchased from Addison (China). Cellulose dialysis bags (3500-5000) were purchased from Yuanye (China). Panc-02 cell culture medium was purchased from Pronosai (China). DMEM medium, penicillin-streptomycin, trypsin, and fetal bovine serum (FBS) were purchased from Gibco (USA). Cell counting kit (CCK-8) was purchased from Prile Biotechnology Co., Ltd. (China). The apoptosis kit, DCFH-DA, and Hoechst were purchased from Beyotime International (China). Repsox was purchased from Ron Chemical (China). Hydrogen peroxide was purchased from Sinopharm Chemical Reagent Co., Ltd. (China). The implementation of the following examples is not limited to these manufacturers; any manufacturer capable of achieving the technical solution of this invention may be used.

[0058] 2) Preparation of MEC nanoprobes

[0059] Preparation of Mn-TCPP-MOF nanoparticles: 10 ml of Mn(OAc)3·2H2O (DMF, 10.4 mM) was added to 20 ml of HAc / DMF (v / v=1:4) solution and stirred for 5 min. Then, 10 ml of TCPP (DMF, 1.3 mM) was slowly added to the above solution, and the mixture was stirred at room temperature for 12 h. After stirring, the mixture was centrifuged (12000 rpm, 10 min) and washed three times with DMF. Finally, the Mn-TCPP-MOF nanoparticles were dispersed in 5 ml of DMSO.

[0060] Synthesis of Mn-TCPP-MOF-Era (ME) and Calculation of Drug Loading and Encapsulation Efficiency: 5 ml of Erastin (0.6 mg / ml) was added to the above-mentioned Mn-TCPP-MOF DMSO solution, stirred at room temperature for 24 h, centrifuged (12000 rpm, 10 min), and washed three times with DMSO, then dispersed in 40 ml of deionized water. Next, the drug loading and encapsulation efficiency of Erastin were calculated. In summary, a supernatant containing Erastin was obtained after centrifugation, and the UV-Vis absorption of free Erastin (0-100 μg / ml) and the Erastin concentration-UV absorption curves were evaluated (UV-Vis Spectrophotometer, Cary300, USA). Finally, the loading rate and encapsulation efficiency of Erastin on the nanocarrier were calculated: Loading rate = ((total Erastin - Erastin in supernatant) / amount of nanocarrier) × 100%; Encapsulation efficiency = ((total Erastin - Erastin in supernatant) / total Erastin) × 100%.

[0061] Coupling of CD133mAb: ME is coupled to CD133mAb via an activated amide bond of EDC / NHS. In short, 15 mg EDC and 20 mg NHS are dissolved in 10 ml of deionized water and added to 40 ml of the above-mentioned deionized water solution of ME. The mixture is stirred for 20 min, then 100 μl of CD133mAb (1 μg / μl) is added and stirred for 12 h. The prepared MEC is collected by centrifugation and stored at 4 °C.

[0062] 3) Physicochemical property characterization

[0063] The microstructure of the nanoprobes was evaluated using biological transmission electron microscopy (TEM, HT7800, Japan), and the morphology and elemental distribution were further characterized by elemental mapping using high-resolution transmission electron microscopy (HRTEM, Talos F200X, USA). Next, the zeta potential and size distribution of the nanoprobes were detected using a particle size zeta potential analyzer (DLS, Litesizer500, Austria). The ultraviolet-visible absorption spectra were evaluated using a UV-Vis spectrophotometer (UV-Vis Spectrophotometer, Cary300, USA), and the functional groups in the nanoprobes were evaluated using Fourier transform infrared spectroscopy (FTIR, IS50, USA). The elemental composition was determined using inductively coupled plasma mass spectrometry (ICP-MS, Agilent, USA). The crystal structure and lattice parameters were determined by X-ray powder diffraction (XRD, D8 Advance, USA).

[0064] 4) Characterization of GSH response performance

[0065] The fluorescence changes of MEC at different GSH concentrations were evaluated using a fluorescence spectrophotometer (SPF, LS55, USA). 100 μl of MEC was dispersed in 1 ml of GSH solutions of different concentrations (0, 1, 2, 8, 16 μM). After incubation at 37 °C for 20 min, the fluorescence spectra of the solutions were detected (λex = 415 nm, λem = 630 nm). Subsequently, the erastin release performance of MEC under 80 μM GSH and PBS conditions was evaluated, and the drug release curves of MEC were detected using a UV-Vis spectrophotometer. Simultaneously, the morphological changes of MEC after co-incubation with 80 μM GSH for 0, 6, 12, and 24 h were evaluated using biological transmission electron microscopy.

[0066] 5) Characterization of GSH consumption and ROS generation

[0067] GSH depletion was detected using a reduced glutathione (GSH) content assay kit. Different concentrations of GSH (0 μM, 20 μM, 40 μM, 80 μM) were co-incubated with different concentrations of MEC (0 μM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM) for approximately 10 min. Then, DTNB was added according to the manufacturer's instructions, and the absorbance (A) was measured at 412 nm. x ), through A GSH The GSH concentration was calculated from the standard curve. Singlet oxygen (GSH) was then measured. 1 The generation of O2) and hydroxyl radicals (·OH) was detected using a UV-Vis spectrophotometer. (1) Different concentrations of MEC (2.5, 5.0, 10, 20 μg / ml) and different ultrasonic powers (0, 1.0, 1.5, 2.0 W / cm) were detected. 2 (1) Degradation kinetics of DPBF at 420 nm under the condition of UV-Vis spectrophotometer; (2) Degradation kinetics of 10 μg / mL MEC MB under 200 μM H2O2 environment were detected by UV-Vis spectrophotometer.

[0068] 6) Cell culture and cytotoxicity characterization

[0069] Mouse pancreatic cancer cells (Panc-02) were cultured in Panc-02 cell-specific medium, and tumor-associated fibroblasts (CAFs) were cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin at 37°C and 5% CO2. Cells were passaged regularly under these conditions. Panc-02 and CAF cells were seeded into 96-well plates and cultured for 24 h. After co-incubation with different concentrations of Mn-MOF and MC for 4 h, cell viability was assessed using the standard CCK-8 assay.

[0070] 7) Cellular uptake characterization

[0071] Confocal laser scanning microscopy imaging: Panc-02 cells and CAFs (4 × 10⁶ cells per well) were imaged. 5 Cells were seeded in confocal culture dishes and co-cultured with Mn-MOF and MC for different time periods, followed by Hoechst nuclear staining. (Hoechst: λex = 350 nm, λem = 460 nm; nanoprobes: λex = 415 nm, λem = 630 nm).

[0072] ICP-OES: To assess cell targeting ability, Panc-02 cells and CAFs were seeded in 6-well plates for 24 h and co-cultured with Mn-MOF and MC for 4 h. Cells were then digested with nitric acid, and the elemental content within the cells was determined by ICP-OES.

[0073] Cryosoft X-ray nano-CT imaging: In a confocal culture dish containing cell culture medium, use forceps to vertically immerse a nickel mesh into the medium, gently moving it back and forth 2-3 times to ensure wetting. Then release the forceps, allowing the nickel mesh to sink to the bottom of the culture dish with the front side facing up. Next, add a 4×10⁻⁶ nanoparticles of cell culture medium. 5 A suspension of Panc-02 cells / ml was seeded into culture dishes. The dishes were placed in a cell culture incubator to allow the cells to adhere to the surface for 12 h. Afterward, Mn-MOF and MC (150 μg / ml) were added to the culture medium, and the cells were incubated for 4 h. Following incubation, the cells were fixed with paraformaldehyde. This experiment was conducted at the National Synchrotron Radiation Laboratory of the University of Science and Technology of China.

[0074] Construction and Targeting Performance Characterization of 3D Multicellular Tumor Spheroid Model: Panc-02 cells (4000 cells per well) and CAFs (1000 cells per well) were added to ultra-low adhesion U-shaped 96-well plates. After 7 days of incubation, tumor spheroids formed. The tumor spheroids were transferred to confocal dishes, MC (150 μg / ml) was added to the culture medium, and the cells were subjected to sonication. Finally, Hoechst nuclear staining was performed, and the cells were observed using a confocal microscope.

[0075] 7) Intracellular reactive oxygen species assessment

[0076] Panc-02 cells were loaded at a rate of 4 × 10⁻⁶ 5 Cells were seeded at a density of [number] cells / ml into 12-well plates. After incubation in a cell culture incubator for 24 h, Mn-MOF, MC, ME, MEC at a concentration of 150 μg / ml and H2O2 at 200 μM were added to each group, respectively. After incubation for 4 h, reactive oxygen species in the cells were stained using DCFH-DA and incubated in the dark for 1 h. The sonication group was subjected to sonic stimulation (0.5 W / cm², 1 MHz, 50% duty cycle) for 5 min and observed using an inverted fluorescence microscope.

[0077] 8) Apoptosis analysis

[0078] Panc-02 cells were loaded at a rate of 4 × 10⁻⁶ 5 Cells were seeded at a density of [number] cells / ml into 12-well plates and incubated in a cell culture incubator for 24 h. After adding 150 μg / ml of Mn-MOF, MC, ME, MEC, and 200 μM H2O2 to each well, the cells were incubated for 4 h and then subjected to ultrasound therapy (0.5 W / cm²). 2 Cells were sonicated for 5 min at 1 MHz (50% duty cycle). After further culturing for 8 h in a cell culture incubator, AM / PI dye was added, and the cells were incubated together for 1 h. Observation was performed using an inverted fluorescence microscope. Live cells labeled with calcein AM emitted green fluorescence upon excitation at 490 nm, while dead cells labeled with PI emitted red fluorescence upon excitation at 545 nm. Simultaneously, Panc-02 cells were cultured at 4 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 100 cells / μl into 96-well plates and treated in the same manner as described above. Cell viability was then assessed using the standard CCK-8 assay.

[0079] 9) Characterization of ferroptosis at the cellular level

[0080] Characterization of cellular lipid peroxidation: The concentration of LPO in cells was detected using the C11-BODIPY 581 / 591 fluorescent lipid peroxidation probe. Panc-02 cells were treated with PBS, MC, MC+US, Erastin, MEC, and MEC+US. The concentration of the nanoprobe was 150 μg / mL, and the concentration of Erastin was 22 μg / mL (ultrasound power 0.5 W / cm²). 2 After incubation at 1 MHz, 50% duty cycle, for 5 min, 5 μM MC11-BODIPY reagent was added and incubated for 3 min. Images were then collected using a fluorescence confocal microscope. US here refers to sonication, and the same applies below.

[0081] Western blot experiment: To visually observe the expression level of GPX4 in Panc-02 cells treated with nanoprobes, we performed a Western blot experiment. The specific procedure was as follows: After complete cell adhesion, Panc-02 cells were treated with PBS, MC, MC+US, Erastin, MEC, and MEC+US. At the end of treatment, cell samples were collected and Western blot experiments were performed.

[0082] GSH detection: After cell treatment, cells in each group were lysed by sonication, and the GSH level in the supernatant was detected using a GSH detection kit.

[0083] Cellular TEM imaging: Panc-02 cells were treated with PBS, MC, MC+US, Erastin, MEC, and MEC+US. Cells were collected and fixed to prepare TEM samples and observe the morphology and number of mitochondria in the cells.

[0084] 10) In vivo imaging

[0085] All animal experimental procedures were conducted in accordance with the Ningbo University Guidelines for Laboratory Animal Care and Use, and were approved by the Ningbo University Animal Ethics Committee (License No.: SYXK Zhe 2024-0002). Female C57BL / 6 mice aged 4-6 weeks were used as experimental animals in this study. 5 × 10 6 One Panc-02 cell was injected into the subcutaneous tissue of the upper right thigh to establish a Panc-02 tumor model. Subsequent experiments began when the tumor diameter reached 5-7 mm.

[0086] In vivo MR imaging of mice was performed using three protocols (ME, MEC, and MEC+US). Tumor-bearing mice were injected intravenously with 100 μL of ME or MEC at a concentration of 1.5 mg / mL. Transverse MR imaging of the mice was performed before injection and at 4 h, 8 h, and 12 h post-injection using a 3.0T MR scanner (Vida, Siemens, Germany) and a small animal imaging coil. All images were acquired using spin-echo sequences with the following parameters: T1WI: TR = 450 ms, TE = 2 ms, field of view = 55 × 55 mm, matrix size = 256 × 256, number of slices = 8, slice thickness = 2 mm, flip angle = 70°.

[0087] In vivo fluorescence imaging of mice was performed using the same protocol. Tumor-bearing mice were injected intravenously with 100 μL of ME and MEC at a concentration of 1.50 mg / mL. Fluorescence imaging of the mice was performed using a small animal in vivo imaging system (IVIS Lumina XRMS Series III, Perkinelmer, USA) before injection and at 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h after injection. To further investigate the targeting and accumulation of the nanoprobes in various organs and tumor sites, mice were sacrificed 60 h after intravenous injection of the nanoprobes, and heart, liver, spleen, lung, kidney, and tumor tissues were collected for fluorescence imaging.

[0088] 11) Characterization of in vivo GSH response performance

[0089] 50 μL of MEC (1.50 mg / mL) was injected into the tumor and contralateral normal tissue of tumor-bearing mice. Fluorescence in the tumor and contralateral normal tissue was then observed at specific time points (0 h, 2 h, 4 h, and 6 h) using an IVIS imaging system. To further investigate the relationship between tumor fluorescence intensity and GSH content, 100 μL of MEC (150 μg / mL) was injected via the tail vein of tumor-bearing mice. The mean fluorescence intensity (MFI) of the tumor site was measured before injection and at 24 h, 36 h, 60 h, and 72 h after injection. Tumor tissue was removed at these time points, homogenized on ice, centrifuged, and the supernatant was analyzed using a GSH detection kit to determine the GSH level in the supernatant.

[0090] 12) In vivo anti-tumor research

[0091] Tumor-bearing mice were randomly divided into 11 groups: Control, Erastin, MC, MEC, Repsox, MEC / Repsox, Control / US, MC / US, MEC / US, Repsox / US, and MEC / Repsox / US (MOF: 1.5 mg / mL; Erastin: 220 μg / mL; Repsox: 2 mM in all groups; MC and MEC contained 1.5 mg / mL of Mn-TCPP-MOF). Each group contained 4 mice. Each mouse received a 100 μL injection via tail vein. Two hours after the injection, groups 7, 8, 9, 10, and 11 underwent 5 minutes of ultrasound therapy. In the group designation, US represents ultrasound (1.5 W / cm², 1 MHz, 50% duty cycle). Tumor length and diameter, as well as mouse weight, were measured daily. The results were calculated using the formula: V=W. 2 The tumor volume (V) was calculated as ×L / 2 (W and L are the shortest and longest diameters of the tumor). After 14 days of treatment, the excised tumor was weighed and photographed, and then tumor tissue was harvested for further histological analysis.

[0092] 13) In vivo safety testing

[0093] Female BALB / C mice aged 4-6 weeks were selected as experimental subjects. Blood was collected via orbital puncture to prepare erythrocyte suspensions. MEC nanoprobes were dissolved in PBS at concentrations of 0.25, 0.5, 0.75, 1, 1.25, and 1.5 mg / mL. Blood cells were incubated with these MEC nanoprobe solutions for 2 h, followed by photographic recording. Additionally, female BALB / C mice aged 4-6 weeks were injected intravenously via tail vein with 100 μL of PBS, MEC (7.5 mg / kg), and MEC (15 mg / kg), respectively. These mice were observed for 14 days, during which their weight was recorded. After 14 days, the mice were euthanized by cervical dislocation. Peripheral blood was collected for routine blood tests and biochemical analysis. Furthermore, the heart, liver, spleen, lungs, and kidneys were collected for hematoxylin-eosin (H&E) staining.

[0094] 14) Statistical Analysis

[0095] Quantitative results were based on at least three replicates. Data visualization and statistical analysis were performed using Origin 2021 and GraphPad Prism 10 software. Differences between two groups were assessed using unpaired t-tests. Statistical significance thresholds were defined as *P < 0.05, **P < 0.01, and ***P < 0.001.

[0096] Example 1: Synthesis and Characterization of MEC Nanoprobes

[0097] 1. Preparation of MEC nanoprobes

[0098] The preparation flowchart is as follows Figure 1 As shown in a.

[0099] 1.1 Figure 1 From “Mn” in a 3+ The step of "+TCPP to Mn-MOF":

[0100] Mn-TCPP-MOF nanoparticles were synthesized using Mn(OAc)3·2H2O and TCPP as raw materials in an N,N-dimethylformamide (DMF) solution containing acetic acid (HAc) with stirring for 12 h. Specifically:

[0101] 10 ml of Mn(OAc)3·2H2O (DMF, 10.4 mM) was added to 20 ml of HAc / DMF (v / v=1:4) solution and stirred for 5 min. Then, 10 ml of TCPP (DMF, 1.3 mM) was slowly added to the above solution, and the mixture was stirred at room temperature for 12 h. After centrifugation (12000 rpm, 10 min), the solution was washed three times with DMF. Finally, the solution was dispersed in 5 ml of DMSO to obtain a DMSO solution of Mn-TCPP-MOF, which is the Mn-TCPP-MOF nanoparticle.

[0102] X-ray diffraction (XRD) analysis showed a significant peak in the 0°-10° range, confirming that the synthesized nanoprobe is a MOF structure. Figure 2 In addition, elemental distribution analysis was performed, and the results showed that C, N, O, and Mn participated in the elemental composition of the nanoprobe. Figure 3 ).

[0103] 1.2 Figure 1 The step from "Mn-MOF to Mn-MOF-Era" in a:

[0104] Add 5 ml of Erastin (0.6 mg / ml) to the above Mn-TCPP-MOF DMSO solution, stir at room temperature for 24 h, centrifuge (12000 rpm, 10 min) and wash 3 times with DMSO, then disperse in 40 ml of deionized water to obtain Mn-MOF-Era (also called Mn-TCPP-MOF-Era, or ME).

[0105] Next, the drug loading and encapsulation efficiency of Erastin were calculated: the obtained supernatant containing Erastin was obtained by centrifugation, and the UV-Vis absorption of free Erastin (0-100 μg / ml) and the Erastin concentration-UV absorption curves were evaluated (UV-Vis Spectrophotometer, Cary300, USA). Finally, the loading rate and encapsulation efficiency of Erastin on the nanocarrier were calculated: Loading rate = ((total Erastin - Erastin in supernatant) / nanocarrier mass) × 100%; Encapsulation efficiency = ((total Erastin - Erastin in supernatant) / total Erastin) × 100%.

[0106] 1.3 Figure 1 The step from "Mn-MOF-Era to Mn-MOF-Era-CD133mAb" in a:

[0107] The coupling of CD133mAb occurs when ME couples with CD133mAb via an amide bond activated by EDC / NHS. The specific steps are as follows:

[0108] 15 mg EDC and 20 mg NHS were dissolved in 10 ml of deionized water and added to 40 ml of the above-mentioned deionized aqueous solution of Mn-MOF-Era. The mixture was stirred for 20 min, and then 100 μl of CD133mAb (1 μg / μl) was added and stirred for 12 h. The prepared MEC was collected by centrifugation and stored at 4 °C to obtain Mn-MOF-Era-CD133mAb (i.e., MEC nanoprobe).

[0109] Morphological observation using transmission electron microscopy (TEM) revealed that the MEC nanoprobes exhibited a spindle shape with a major axis of approximately 160 nm. Dynamic light scattering analysis using a particle size zeta potential analyzer (DLS, Litesizer 500, Austria) showed that the hydrodynamic diameter of the MEC nanoprobes was slightly increased, approximately 267 nm. Figure 1 b, where Mn-MOF is Mn-TCPP-MOF nanoparticles, ME is Mn-MOF-Era, and MEC is Mn-MOF-Era-CD133mab, which is also a MEC nanoprobe (the same applies below). Surface potential measurements show that after coupling with CD133mab, the surface potential changes from -8.59mV to -13.60mV ( Figure 1 (c), this change indicates that CD133mab has been successfully modified on the surface of the nanoprobe. Figure 1The d values ​​represent the FTIR spectra of Mn-MOF, ME, MEC, CD133mab, and Erastin. No significant differences were observed in the FTIR spectra of Mn-MOF and ME, possibly because the drug is inside the nanoprobe, and FTIR cannot detect signals within it. In the MEC nanoprobe, the 1732 cm⁻¹... -1 The presence of characteristic absorption peaks for CO-NH indicates that ME has been successfully linked to CD133mab NPs. The UV-Vis absorption spectra were evaluated using a UV-Vis spectrophotometer (Cary300, USA), and the UV spectra of various samples are shown below. Figure 1 As shown in Figure e, the absorption peak of the MEC nanoprobe exhibits a slight redshift compared to Mn-MOF and ME, which is mainly attributed to the coupling of CD133mab.

[0110] Example 2: Characterization of GSH response and ROS generation performance

[0111] The magnetic resonance imaging, GSH-responsive fluorescence imaging, and cleavage properties of the nanoprobe were evaluated.

[0112] The magnetic resonance imaging performance of the nanoprobe was evaluated using a 0.5 T MRI scanner. Figure 4 At the same Mn concentration, the longitudinal relaxation rate (r1) of MEC is approximately 9.70 Mm. -1 S -1 The transverse relaxation rate (r²) of MEC is approximately 27.81 Mm. -1 S -1 The r2 / r1 value of MEC is approximately 2.87, indicating that MEC is a suitable T1 contrast agent for magnetic resonance imaging, where r1 = 1 / T1 and r2 = 1 / T2. Figure 1 As shown in f, when the GSH concentration increased from 0 μM to 16 μM, the fluorescence intensity of Mn-MOF-EC increased by 2.72-fold, mainly due to the Förster resonance energy transfer (FRET) caused by the overlap between the broad-spectral absorption of Mn(III) and TCPP emission. Furthermore, MEC also exhibits GSH-responsive drug release properties. Based on the formulas: drug loading = encapsulated Erastin / total mass of Mn-MOF × 100%; encapsulation efficiency = encapsulated Erastin / added Erastin × 100%, the drug loading of the nanoprobe was calculated to be approximately 14.70%, and the encapsulation efficiency was approximately 22.05%. Figure 1 As shown in g, compared with the PBS group, the cumulative release of Erastin in the 80 μM GSH group within 24 h was as high as 46.10%, approximately 1.36 times that of the PBS group. Figure 1As shown in h, the consumption of GSH increases with increasing MOF and GSH concentration, indicating that GSH can be oxidized to GSSG in the presence of MOF. Furthermore, TEM results also show that MEC gradually decomposes in the GSH environment (…). Figure 5 ).

[0113] The acoustic dynamics and Fenton properties of nanoprobes were used to generate ROS (reactive oxygen species) to participate in the ferroptosis process. To verify the acoustic dynamics of MEC, singlet oxygen (ROS) generated by acoustic dynamics was generated using 1,3-diphenylisobenzofuran (DPBF). 1 O2) is detected. Figure 1 i, Figure 6 As shown, with increasing ultrasound irradiation duration, the absorption intensity of DPBF at 420 nm decreased significantly, indicating continuous ROS generation. When the MEC concentration was 10 μg / mL, ultrasound (1 MHz, 1.5 W / cm²) was used... 2 After irradiation with 50% duty cycle for 6 min, the DPBF decreased by approximately 70.47%. Subsequently, we evaluated the ability of 10 μg / mL MLMEC to generate hydroxyl radicals (··OH) via chemikinetic properties using the fluorescent indicator methylene blue (MB). Figure 1 As shown in Figure j, the UV-Vis absorbance of 10 μg / mL MEC at 660 nm exhibits a significant decreasing trend. These results indicate that MEC can generate a large amount of ROS through its acoustic and chemodynamic properties.

[0114] Example 3 Characterization of cellular uptake

[0115] CD133 is a key molecular biomarker for pancreatic cancer stem cells. To enhance the CD133 targeting ability of MEC nanoprobes, MEC nanoparticles were generated by coupling ME with CD133mab. The targeting ability of MEC NPs was evaluated using laser confocal microscopy, ICP-OES, and soft X-ray microscopy. Figure 7 The distribution of MEC nanoprobes in Panc-02 cells and CAFs is shown in Figure 1. Blue fluorescence indicates Dapi nuclear staining, and green fluorescence indicates TCPP in the MEC nanoprobes. In Panc-02 cells, the Mn-MOF-CD133mab (MC) group showed more aggregation in cells compared to the Mn-MOF group. However, in CAFs, there was no significant difference between the Mn-MOF group and the MC group. These results indicate that CD133mab can enhance the targeting ability of MEC nanoprobes to pancreatic cancer cells. Figure 7 (a). Figure 7The targeting ability of MEC nanoprobes to Panc-02 cells and CAFs was quantitatively detected by ICP-OES. The results showed the amount of intracellular Mn in Panc-02 cells or CAFs after co-incubation with Mn-MOF or MC for 4 h. Similarly, the amount of Mn in Panc-02 cells in the MC group was higher than that in the Mn-MOF group, while there was no significant difference in CAFs. 360° CT images of single cells were obtained by soft X-ray microscopy. Compared with the Control group and the Mn-MOF group, the lysosomes in the MC group showed higher contrast due to phagocytosis of MEC nanoprobes, indicating that CD133mab can enhance the targeting ability of MEC nanoprobes. Figure 7 (c). However, the targeting results of single cells cannot reflect the complex tumor microenvironment in vivo. In order to simulate the tumor microenvironment rich in fibrous matrix, such as pancreatic cancer, Panc-02 and CAFs were co-incubated to construct a tumor spheroid model. The uptake of MEC nanoprobes by the tumor spheroids was observed using laser confocal microscopy. Green fluorescence represents TCPP in the MEC nanoprobes, and blue fluorescence represents Hoechst 33342 nuclear staining. Compared with the Control group and the Mn-MOF-EC group, the MEC combined with ultrasound group showed more aggregation in the tumor spheroids, indicating that CD133 targeting combined with ultrasound can further improve the targeting ability of MEC nanoprobes (c). Figure 7 d).

[0116] Example 4: Cytotoxicity and Cell Therapy Characterization

[0117] First, the cytotoxicity of MEC nanoprobes was assessed to determine their intrinsic effects on Panc-02 cells and CAFs. After co-incubating Panc-02 cells and CAFs with nanoprobes (MC, Mn-MOF) for 4 hours, cell viability remained above 80% within a nanoprobe concentration range of 0-300 μg / mL, demonstrating low cytotoxicity of the nanoprobes. Figure 8 (a, b). To determine the in vitro therapeutic effect of the nanoprobes, different nanoprobes were co-incubated with Panc-02 cells, with or without ultrasound irradiation. CCK-8 results showed that under 200 μM H2O2 conditions, the cell viability of MEC nanoprobes combined with ultrasound treatment was significantly reduced to 32.93% (a, b). Figure 8 (c). Furthermore, live / dead cell staining assays were performed using AM and PI to demonstrate the killing effect of the nanoprobe on Panc-02. Figure 8(d). Similar to the CCK-8 results above, the MEC nanoprobe combined with ultrasound treatment resulted in the highest percentage of dead cells (red fluorescence). The ROS-generating ability of the nanoprobes was verified by DCFH-DA probe fluorescence, showing that the final treatment group produced significantly more ROS compared to the MEC / H2O2 and MEC / US groups. These findings confirm that combined sonodynamic and Fentonian effects lead to the generation of large amounts of ROS within cells, a result that also corresponds to the CCK-8 and live / dead cell staining results. Figure 8 (e).

[0118] Example 5: Characterization of ferroptosis at the cellular level

[0119] The effects of different nanoprobes and Erastin on lipid peroxidation in Panc-02 pancreatic cancer cells were evaluated using the BODIPY-C11 fluorescent probe. It was found that co-incubation of oxidized BODIPY-C11 with MEC / US resulted in significant fluorescence intensity in Panc-02 cells. Figure 9 (a) This may be due to Erastin blocking the classical ferroptosis pathway Xc-system, decreasing GSH, and increasing ROS production. In subsequent Western blotting experiments, we examined GPX4 expression. Consistent with the above results, GPX4 expression was significantly reduced in the MEC / US group, providing favorable validation for tumor cells entering the ferroptosis pathway. Figure 9 (b). Similarly, we also examined GSH levels within tumor cells, and the results showed that GSH levels significantly decreased after the addition of MEC and ultrasound therapy. Figure 9 (c). Finally, Bio-TEM imaging was used to understand the changes in subcellular organelles in Panc-02 pancreatic cancer cells. After different treatments, compared with the control group, different groups showed varying degrees of mitochondrial morphological changes, including shrinkage, cristae structure changes, and outer membrane rupture, which were most significant in the MEC / US group. These are generally considered to be characteristic manifestations of ferroptosis. Figure 9 (d). In summary, induced ferroptosis may be the main mechanism underlying the enhanced antitumor activity of MEC / US.

[0120] Example 6: Evaluation of biosafety and efficacy

[0121] PBS, 7.5 mg / kg MC, and 15 mg / kg MC were injected into C57 mice via the tail vein. Figure 10 Figure a shows the weight change in mice 14 days after MEC nanoprobe injection. Figure 10 Figures b and c show the changes in blood routine and blood biochemical indicators 14 days after MEC nanoprobe injection. Compared with the PBS group, no significant changes in indicators were observed in the experimental group. Figure 10Image d shows HE staining of tumor tissue 14 days after MEC nanoprobe injection. No obvious tissue damage or other changes were observed in the experimental group. The hemolysis experiment also indicated that the MEC nanoprobe has good biosafety and compatibility. Figure 10 (e). The above experiments all demonstrate that the MEC nanoprobes prepared in this invention have good biosafety within 14 days.

[0122] To evaluate antitumor efficacy, mice were divided into 11 groups: PBS, Erastin, MC, MEC, Repsox, MEC / R, PBS / US, MC / US, MEC / US, Repsox / US, and MEC / R / US (MOF: 1.5 mg / mL in all groups; Erastin: 220 μg / mL; Repsox: 2 mM; MC and MEC were administered at a concentration of 1.5 mg / mL of Mn-TCPP-MOF). On day 0, different nanoprobes were intravenously injected (100 μL in each group) into Panc-02 tumor-bearing mice (C57BL / 6, male), followed by ultrasound therapy (1 MHz, 1.5 W / cm²). 2 (50% duty cycle) Figure 11 (a), and measured changes in mouse body weight and tumor volume the following day. Figure 11 As shown in Figures b and c, MEC / R, MC / US, MEC / US, and MEC / R / US all exhibited significant tumor growth inhibition. Among them, MEC / R / US, through a dual-channel blocking strategy combined with ultrasound therapy, showed the most effective combined anti-tumor effect. During the 14-day treatment period, no significant change in mouse body weight was observed. Figure 12 H&E staining, Ki67, and TUNEL results of tumor tissue showed that the combination of the dual-channel blocking strategy and ultrasound therapy resulted in significant histological damage, reduced cell proliferation, and increased cell apoptosis. Figure 11 Masson staining of tumor tissue showed that the constructed pancreatic cancer model had abundant collagen fibers, and the tight junctions between tumor cells could be disrupted by the mechanical action of ultrasound. Figure 13 Further investigation was conducted to determine the degree of ferroptosis in pancreatic cancer tissue and to characterize key factors in the ferroptosis pathway, such as... Figure 11 As shown in Figure e, the MEC / R / US combined treatment produces more ROS, reduces GPX4 expression, and promotes LPO. These characterization results are consistent with the pattern. Figure 11 It matches f.

[0123] Example 7: Evaluation of in vivo imaging performance and characterization of GSH response performance

[0124] The metabolism of nanoprobes in vivo was explored through tail vein injection. For example... Figure 14As shown in Figure c, the fluorescence at the tumor site gradually brightened over time, with the maximum enrichment time observed at 60 hours post-injection. Compared to the ME and MEC groups, the MEC / US group exhibited higher fluorescence intensity at the tumor site, indicating that CD133 targeting combined with ultrasound can enhance the intratumoral enrichment effect of the nanoprobe. Simultaneously, we performed in vitro fluorescence detection on major organs in the ME, MEC, and MEC / US groups 60 hours after tail vein injection. Figure 14 d), results and Figure 14 The c-values ​​were consistent. Subsequently, the in vivo imaging effect of the nanoprobe was evaluated by MRI. The T1 signal at the tumor site gradually increased over time, and the maximum enrichment time within the tumor was observed at 8 hours. The T1 signal within the tumor decreased at 12 hours. Figure 14 (e). Notably, at 8 hours, the T1 signal at the tumor site in the MEC / US group became uniformly brighter, while the signal intensity at the MEC tumor site was uneven, showing only peritumoral enhancement. This indicates that the mechanical action of ultrasound helps to loosen the stromal barrier of the tumor, allowing the nanoprobe to successfully reach the tumor site. Based on the above experimental data, we found a significant difference between the maximum enrichment time of MRI (8 hours) and the maximum attachment time of fluorescence imaging (60 hours), mainly because MEC requires the response of intratumoral GSH to achieve a gradual increase in fluorescence intensity.

[0125] To further investigate the GSH-responsive imaging mechanism of MEC, MEC was injected into the tumor and contralateral normal tissue. A significant increase in fluorescence at the tumor site was observed from 0 h to 6 h, and the fluorescence at the tumor site was significantly higher than that in the contralateral normal tissue at each time point. This suggests that due to the high GSH level at the tumor site, MOF releases TCPP, leading to enhanced fluorescence at the tumor site. Figure 14 (ab). Then, the fluorescence intensity and GSH content at different time points in the tumor were detected. Figure 14 We found that the fluorescence intensity increased while the GSH content decreased in the tumor site from 0 to 60 hours, indicating that during the cleavage of MOF structures via GSH response, the remaining GSH content in the tumor was negatively correlated with the fluorescence intensity. This finding helps us to perform semi-quantitative detection of GSH content in tumors in vivo.

[0126] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A drug that simultaneously blocks both the classical ferroptosis resistance pathway and CAFs-related pathways, characterized in that, It includes MEC nanoprobes and a TGFβR1 inhibitor; the TGFβR1 inhibitor is Repsox. The preparation method of the MEC nanoprobe includes the following steps: S1. With Mn 3+ Mn-TCPP-MOF was synthesized by stirring TCPP in an N,N-dimethylformamide solution containing acetic acid. S2. Add Erastin to the Mn-TCPP-MOF obtained in S1, stir at room temperature, centrifuge, wash, and then disperse in deionized water to obtain Mn-TCPP-MOF-Era; S3. The above Mn-TCPP-MOF-Era was coupled to CD133mAb via EDC and NHS-activated amide bonds to obtain MEC nanoprobes.

2. The drug that simultaneously blocks the classical ferroptosis resistance pathway and CAF-related pathways as described in claim 1, characterized in that, In step S1 In the N,N-dimethylformamide solution containing acetic acid, the volume ratio of acetic acid to N,N-dimethylformamide is 1:

4. The Mn 3+ The ratio of TCPP to N,N-dimethylformamide solution containing acetic acid is 10.4 × 10⁻⁶. -2 mmol: 1.3×10 -2 mmol: 20ml.

3. The drug that simultaneously blocks the classical ferroptosis resistance pathway and CAF-related pathways as described in claim 1, characterized in that, In step S2, Erastin and Mn in S1 3+ The dosage ratio is 3mg: 10.4×10 -2 mmol.

4. The drug that simultaneously blocks the classical ferroptosis resistance pathway and CAF-related pathways as described in claim 1, characterized in that, In step S3, the aqueous solutions of EDC and NHS are added to the Mn-TCPP-MOF-Era prepared in step S2 and stirred until homogeneous. Then, CD133mAb is added and stirred until homogeneous. The MEC nanoprobe is obtained by centrifugation.

5. The drug that simultaneously blocks the classical ferroptosis resistance pathway and CAF-related pathways as described in claim 4, characterized in that, The dosage ratio of EDC, NHS, water, S2, and Mn-TCPP-MOF-Era, CD133mAb is 15mg:20mg:10ml:40ml:100μg.

6. The use of the drug according to claim 1, which simultaneously blocks the classical ferroptosis resistance pathway and CAFs-related pathways, in the preparation of a drug that induces ferroptosis in tumor cells; wherein the tumor is pancreatic cancer.

Images