Research method of breast cancer diagnosis and treatment nano material based on metal phenolic network

Abstract

The invention discloses a research method of a breast cancer diagnosis and treatment nano material based on a metal phenolic network, and relates to the technical field of nano materials. The nano material is prepared by the following steps: carrying out coordination reaction on tannic acid and ferric trichloride to generate photosensitive TAF nano particles, and loading mitoxantrone through pi-pi interaction to construct an MFTA nano platform. The preparation method of the nano material further comprises the following steps: dissolving mitoxantrone hydrochloride in methanol, neutralizing with trimethylamine to obtain hydrophobic MTO (Methanol To Olefins), then dissolving TA (Tetraacetic Acid) in absolute ethyl alcohol, and mixing with an MTO solution. According to the breast cancer diagnosis and treatment nano material based on the metal phenolic network provided by the invention, efficient and accurate treatment of TNBC is realized by integrating PTT, chemotherapy and ferroptosis strategies; intramolecular fluorescence of MTO is partially quenched under near-infrared excitation through pi-pi interaction between TAF and MTO, the photothermal effect of MFTA is enhanced, and a cascade photothermal enhancement mode is established.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials, and in particular to a research method for a nanomaterial for diagnosing and treating breast cancer based on a metal phenolic network. Background Art

[0002] Breast cancer is the most common tumor in women worldwide and poses a serious threat to women's health. Triple-negative breast cancer (TNBC), as the most aggressive molecular subtype, is characterized by a high tendency for distant metastasis and a high mortality rate. The lack of effective therapeutic targets and strategies is one of the main factors leading to its poor prognosis. To date, chemotherapy is the main treatment for TNBC, but traditional chemotherapy drugs such as anthracyclines have significant cardiotoxicity, which limits the dosage and course of treatment in clinical applications, thereby reducing the treatment effect and increasing the risk of heart disease in patients.

[0003] In addition, existing treatments have limitations in accurately locating tumor cells, reducing damage to normal tissues, and activating the body's own immune response, and new materials and methods are urgently needed to improve the current situation.

[0004] Therefore, it is of great significance to develop a new nanomaterial that can effectively treat TNBC and reduce side effects. Summary of the Invention

[0005] The purpose of the present invention is to solve the shortcomings of the existing technology and propose a research method for breast cancer diagnosis and treatment nanomaterials based on metal phenolic network.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A research method for a nanomaterial for the diagnosis and treatment of breast cancer based on a metal phenolic network, wherein the nanomaterial is prepared by the coordination reaction of tannic acid and ferric chloride to generate photosensitive TAF nanoparticles, and mitoxantrone is loaded onto the nanoplatform MFTA via π-π interaction;

[0008] The preparation method of the nanomaterial also includes dissolving mitoxantrone hydrochloride (MTO·2HCl) in methanol and neutralizing it with trimethylamine to obtain hydrophobic MTO, then dissolving TA in anhydrous ethanol and mixing it with the MTO solution, then adding FeCl3·6H2O aqueous solution and stirring overnight to evaporate the solvent, and finally collecting the precipitate by centrifugation to obtain MFTA.

[0009] Preferably, the particle size of the nanomaterial is in the range of 50-200 nm.

[0010] Preferably: the MTO, TA and Fe 3+ The molar ratio is 1:1:1.

[0011] Preferably: the use of the nanomaterial in drugs for the diagnosis and treatment of breast cancer.

[0012] Preferably, the research method includes the application of nanomaterials in in vitro experiments and in vivo experiments, wherein the in vitro experiments include:

[0013] Cellular uptake assay: 4T1 cells were seeded in confocal culture dishes and incubated for 24 hours. When the cell density reached 80%, the old culture medium was removed. DiI-labeled MFTA nanosuspension was added to the confocal culture dish in the dark and incubated with the tumor cells for 0.5, 1, 2, 4, and 6 hours. The dish was rinsed with PBS to remove unabsorbed MFTA. The cell nuclei were stained with 500 μL of DAPI staining solution for 10 minutes. After rinsing the excess dye three times with PBS, the cells were fixed with 4% paraformaldehyde fixative for 15 minutes and observed by CLSM.

[0014] Cytotoxicity assay: 4T1 cells were seeded in 96-well plates and cultured for 24 hours. The old culture medium was discarded, and gradient concentrations of MFTA nanosuspension were added to the plates and incubated for 12, 24, and 48 hours. Duplicate wells containing no-cell blanks and untreated controls were set up to detect 4T1 cell viability and perform statistical analysis.

[0015] Evaluation of synergistic treatment effects.

[0016] Preferably: in the cytotoxicity test: 10 4 4T1 cells were seeded into 96-well plates at a density of 1 / well and cultured for 24 h;

[0017] The gradient concentration of MFTA nanosuspension was controlled at 0.05, 0.1, 0.2, 0.5, 1, and 2 mg / mL and prepared in RPMI1640 medium.

[0018] The viability of 4T1 cells was detected according to the standard CCK-8 method.

[0019] Preferably, the synergistic treatment effect evaluation includes:

[0020] 4T1 cells were divided into 6 groups: blank control group, laser irradiation group, mitoxantrone treatment group, MFTA treatment group, TAF+laser group, and MFTA+laser group. The concentration of TAF in all groups was 0.5 mg / mL.

[0021] All laser irradiation groups were treated with 808 nm laser irradiation, and the irradiation parameters were controlled at: 1.5 W / cm 2 ,5min.

[0022] Preferably, the in vivo experiment is specifically as follows:

[0023] Animal model establishment;

[0024] Evaluation of treatment efficacy;

[0025] Verification of immune response induction effect;

[0026] The establishment of the animal model is as follows:

[0027] The 4T1 cells that were pre-cultured at a growth density of about 80% were digested and resuspended in sterile PBS and diluted to 1×10 6 / mL density cell suspension; use a 1mL syringe to slowly inject 200μL of cell suspension into the fourth pair of mammary fat pads of female Balb / c mice to establish a tumor-bearing mouse model; observe and monitor the survival status of the mice and the growth of the tumor every day after inoculation. 3 The tumor size was observed every 1-2 days and the tumor was kept at about 80 mm in volume. 3 For subsequent animal experiments.

[0028] Preferably: the treatment effect evaluation is specifically:

[0029] Mice were randomly divided into 6 groups: PBS, laser only, MTO, MFTA, TAF+laser, and MFTA+laser. When the tumor expanded to about 1 cm 3 At the same time, mice were divided into pre-set groups and received different treatments; all mice in the laser irradiation group were exposed to 808 nm laser (1 W / cm 2 , 5min); the body weight and tumor volume of each group of mice were recorded every 2 days during the entire 2-week treatment cycle; at the end of the treatment, the mice were killed and the tumors were weighed; the MFTA group alone showed some effect due to spontaneous drug release, while the laser irradiation group alone had a weak effect due to the lack of photothermal conversion material; the MFTA+laser group had the smallest tumor weight, indicating the best treatment effect; in addition, Ki-67, TUNEL and H&E staining results confirmed that the MFTA+laser group showed the highest tumor tissue apoptosis / necrosis rate among all groups.

[0030] Preferably, the immune response induction effect verification is specifically as follows:

[0031] Fresh tumor tissue was obtained to prepare 4-5 μm paraffin sections, which were baked at 60°C for 2 h, dewaxed in xylene, hydrated with graded ethanol, and washed three times with PBS;

[0032] Antigen retrieval: Immerse the sections in 0.01 M sodium citrate buffer, pH 6.0, microwave for 10 min, cool naturally, and wash with PBS.

[0033] Blocking: Add 5% goat serum for 30 min at room temperature, shake dry, add mouse anti-human HMGB-1 monoclonal antibody (1:200 dilution) and rabbit anti-human CD8 polyclonal antibody (1:150 dilution), and incubate at 4°C overnight;

[0034] Fluorescence staining: After washing three times with PBST, AlexaFluor488-labeled goat anti-mouse IgG (1:500) and Cy3-labeled goat anti-rabbit IgG (1:300) were added in the dark and incubated at room temperature for 1 h; DAPI was used to counterstain the nuclei for 5 min;

[0035] Sealing observation: The slides were sealed with anti-fluorescence fading mounting medium, and images of 405, 488, and 555 nm channels were collected under a confocal microscope for fluorescence intensity analysis;

[0036] Flow cytometry analysis of tumor immune cell infiltration: Mouse spleen tissue was minced and added to RPMI-1640 medium containing 0.5 mg / mL collagenase IV and 0.1 mg / mL DNase I. The tissue was digested at 37°C for 30 min, filtered through a 70 μm filter, and centrifuged to discard the supernatant. After treatment with red blood cell lysis buffer, the tissue was resuspended in PBS and stained with trypan blue to count live cells.

[0037] Antibody staining: 1×10 6 Cells were washed with CD3-PacificBlue (1:200), CD4-PE (1:150), and CD8-APC (1:200) antibodies and incubated at 4°C in the dark for 30 min. 200 μL of flow cytometry buffer (containing 2% FBS) was added to each tube and the cells were washed and centrifuged.

[0038] Fixation and storage: Fix with 2% paraformaldehyde for 10 min, resuspend in PBS and store at 4°C in the dark. Detect within 24 h.

[0039] Flow cytometry: FSC / SSC gate was set on the flow cytometer to exclude debris, CD3 + Analysis of CD4 / CD8 double positive cells within the gate: CD3 + CD4 + CD8 - For helper T cells, CD3 + CD4 - CD8 + Cytotoxic T cells, CD3 + CD4 + CD8 + For DC activation analysis, cell suspensions of tumors and lymph nodes were stained with another panel of fluorescently labeled antibodies (anti-CD11c-FITC, anti-CD80-PE, and anti-CD86-APC), and the proportion of activated DCs in tumor and lymph node tissues was assessed using FCM.

[0040] The beneficial effects of the present invention are:

[0041] 1. The breast cancer diagnosis and treatment nanomaterial based on the metal phenolic network provided by the present invention achieves efficient and precise treatment of TNBC by integrating PTT, chemotherapy and ferroptosis strategies; the π-π interaction between TAF and MTO partially quenches the intramolecular fluorescence of MTO under near-infrared excitation, enhances the photothermal effect of MFTA, and establishes a cascade photothermal enhancement mode.

[0042] 2. The nanomaterials of the present invention can enhance anti-tumor immune response and activate the immune system by inducing ICD. The combination of FeCl3 and TA can partially 3+ Reduction to Fe 2+ Under laser irradiation, the PTT of MFTA can generate a large amount of reactive oxygen species in TNBC cells, and the rich Fe 2+ Synergistically promote ferroptosis.

[0043] 3. The introduction of TA in the MFTA of the present invention reduces MTO-related cardiotoxicity and improves its safety by inhibiting oxidative stress and cell apoptosis.

[0044] 4. The nanomaterials of the present invention have real-time imaging capabilities and can be used to monitor the distribution of nanomaterials in tumor sites and the therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Schematic diagram of the particle size distribution (A) and zeta potential (B) of nanoparticles of different compositions of the present invention;

[0046] Figure 2 Schematic diagram of the encapsulation efficiency (A) and drug loading efficiency (B) of MFTA synthesized by using mitoxantrone and iron at different input ratios according to the present invention;

[0047] Figure 3 This is a schematic diagram of the temperature curve of 6 cycles of repeated irradiation according to the present invention;

[0048] Figure 4 Schematic diagram of the drug release rate of MFTA under different pH environments of the present invention;

[0049] Figure 5 This is a schematic diagram showing the change in the release rate of the MFTA drug after laser irradiation according to the present invention;

[0050] Figure 6 Schematic diagram of the effects of different treatments on cell viability determined by CCK-8 assay of the present invention;

[0051] Figure 7 Schematic diagram of the detection of cellular ATP levels of the present invention. DETAILED DESCRIPTION

[0052] The technical solution of the present invention will be further described in detail below in conjunction with specific implementation methods.

[0053] Example 1: Preparation of nanomaterials

[0054] Reagents and instruments

[0055] Reagents: tannic acid (TA, Sigma, USA), mitoxantrone (MTO, Macklin), trimethylamine (Chongqing Chuandong Chemical Group), methanol (Chongqing Chuandong Chemical Group), ferric chloride (FeCl3·6H2O, Chongqing Chuandong Chemical Group), PBS (Wuhan Boster Biotechnology Co., Ltd.), DiI cell membrane red fluorescent probe (Wuhan Boster Biotechnology Co., Ltd.), 4',6-diamidino-2-phenylindole (DAPI, China Beyotime Biotechnology Co., Ltd.), anhydrous ethanol (Chongqing Huierli Biotechnology Co., Ltd.), etc.

[0056] Instruments: electronic analytical balance (Shanghai Jingtian Electronic Instrument Co., Ltd.), ultrapure water system (Shanghai Rephile Company), low-temperature high-speed refrigerated centrifuge (Eppendorf Company, Germany), Malvern particle size and zeta potential instrument (Malvern Company, UK), X-ray diffractometer (Bruker Company, Germany), 808nm near-infrared laser (Beijing Hitech Optoelectronics Co., Ltd.), infrared thermal imager (Shanghai Thermal Imaging Technology Co., Ltd.), etc.

[0057] Preparation steps

[0058] MTO-loaded TA / Fe 3+ Preparation of nanocomposite materials (MFTA)

[0059] Mitoxantrone hydrochloride (MTO·2HCl) (5.17 mg, MR=517.4) was dissolved in 100 μL of methanol and neutralized with 3 μL of trimethylamine to obtain hydrophobic MTO.

[0060] Then, TA (17.01 mg, MR = 1701.2) was dissolved in anhydrous ethanol (100 μL) and mixed with the MTO solution at a molar ratio of 1:1 while stirring.

[0061] An aqueous solution of FeCl 3 ·6H 2 O (MR=270.2) (1 mg / mL, 27 mL) was added to the mixture, and the mixture was stirred overnight to evaporate the methanol and anhydrous ethanol solvents.

[0062] The mixture was then centrifuged at 13,000 rpm for 20 min at 4° C., and the precipitate after centrifugation was collected as MFTA.

[0063] TA / Fe without mitoxantrone loading 3+Preparation of nanocomposite materials (TAF)

[0064] TA (17.01 mg, MR=1701.2) was dissolved in 100 μL of anhydrous ethanol.

[0065] Then, FeCl3·6H2O (MR=270.2) aqueous solution (1 mg / mL, 27 mL) was added to the above TA solution and stirred at the desired molar ratio (the same as that of TA and Fe in the preparation of MFTA). 3+ The mixture is mixed with water (with the same molar ratio).

[0066] Stir overnight to evaporate the anhydrous ethanol solvent.

[0067] The mixture was then centrifuged at 13,000 rpm for 20 min at 4°C, and the precipitate was collected as TAF.

[0068] Example 2: Characterization and performance evaluation of nanomaterials

[0069] Particle size and Zeta potential detection

[0070] like Figure 1 As shown, the particle size distribution and zeta potential distribution of MFTA and TAF were measured by dynamic light scattering (DLS) using a Malvern particle size and zeta potential instrument. All samples were dissolved in water (5 mg / mL) before testing. MFTA was dissolved in different solutions (PBS, FBS, RPMI-1640, and saline) for 7 days to determine its hydrodynamic size changes.

[0071] Microstructure and morphology detection

[0072] The MFTA aqueous solution was dripped onto a copper grid and dried overnight. The nanoparticles' microstructure and morphology were then examined using TEM. Elemental mapping images and energy-dispersive X-ray spectroscopy (EDS) data were also obtained using TEM.

[0073] like Figure 2 As shown, the content of ingredients, encapsulation efficiency and loading capacity were tested

[0074] The concentration of MTO in the supernatant was calculated using the concentration-absorbance standard curve, and the encapsulation efficiency and loading capacity of MTO were then calculated. The calculation formula is as follows:

[0075] Encapsulation efficiency (% w / w) = (MTO addition amount - MTO remaining amount / specific ingredient addition amount) × 100%

[0076] Loading amount (μg / mL) = (MTO added amount - MTO remaining amount / MFTA total mass) × 100%

[0077] Spectral detection

[0078] The UV-visible absorption spectra of the nanoparticles loaded with MFTA, MTO and TAF were detected using a UV-visible spectrophotometer.

[0079] Infrared (IR) spectroscopy

[0080] Aqueous solutions of MFTA, MTO, and TAF were freeze-dried, and their IR absorption spectra were measured using a FOURIER transform infrared spectrometer to infer their composition. Sample preparation steps are as follows: Ground potassium bromide powder was placed in an infrared drying oven and dried for 10 minutes. An appropriate amount of potassium bromide was mixed with each dried sample (the ratio of potassium bromide to test sample was 100:1) and ground uniformly. The resulting mixture was compressed using a tableting device and then tested on an instrument.

[0081] Basic performance testing of nanoparticles

[0082] Temperature change experiment of MFTA with different concentrations under 808nm laser irradiation

[0083] Experimental Preparation: Prepare MFTA solutions of varying concentrations (0, 0.625, 1.25, 2.5, and 5 mg / mL) in a 96-well plate, with three replicates per sample. Ensure the plate is placed firmly to prevent sloshing of the solution that could affect the results.

[0084] Experimental operation: Using 808nm NIR laser (1.5W / cm 2 ) irradiate each sample well for 10 minutes, and use a thermal imager to observe and record data in real time. The data must be recorded with accuracy to the second and one decimal place. Analyze the temperature changes of different MFTA and TAF concentrations under this laser irradiation.

[0085] Experiment on the temperature effect of MFTA with different powers of 808nm laser

[0086] Experimental preparation: Place MFTA solutions of the same concentration (2 mg / mL) in 96-well plates, with n = 3 parallel experiments per group. Before the experiment, check the calibration of the infrared thermal imager to ensure measurement accuracy.

[0087] Experimental operation: The solution was exposed to different power intensities (0, 0.5, 1, 1.5 and 2 W / cm 2 ) laser, the temperature changes were continuously recorded with a thermal imager. Time-temperature curves of the MFTA at different powers were plotted.

[0088] Photothermal stability experiment of MFTA

[0089] Experimental Preparation: Place 2 mg / mL MFTA solution in a 96-well plate. Prepare a room temperature control device to ensure that the solution can effectively return to room temperature between each irradiation.

[0090] Experimental operation: Use 808nm laser (1.5W / cm 2 ) The solution was irradiated for 5 minutes and then stopped. After the solution temperature returned to room temperature, it was irradiated again, and the "On-Off" cycle was repeated for 6 cycles. During the process, the temperature change was continuously recorded with an infrared thermal imager, and the data was recorded every 15 seconds. The time-temperature change curve was plotted to evaluate the photothermal stability of MFTA. After the experiment, the photothermal stability was quantitatively evaluated; Figure 3 shown.

[0091] XPS valence state distribution test and intramolecular fluorescence quenching detection of iron element in MFTA

[0092] Dry MFTA powder was prepared by freeze-drying method, and the valence distribution characteristics of iron element in it were determined by XPS.

[0093] A 1 mmol / mL aqueous solution of FeCl3, TA, MTO, TAF and MFTA was prepared, and the fluorescence intensity was measured at 685 nm using a fluorescence microplate reader.

[0094] Drug release experiments of nanoparticles

[0095] like Figure 4 、 Figure 5 As shown;

[0096] Experimental preparation: First, prepare an MTO aqueous solution with a gradient concentration, measure the absorbance, and obtain the absorbance-concentration standard curve of MTO. Accurately weigh 5 mg of MFTA, disperse it in 1 mL of PBS, and then transfer it to a separate dialysis bag (molecular weight cutoff 8 kDa). Soak the dialysis bag containing the sample in flasks containing 100 mL of simulated body fluid (pH = 7.4), simulated tumor acidic environment (pH = 6.5) and weak alkaline (pH = 8.5), respectively. Place the flask in a constant temperature environment of 37°C and oscillate at a speed of 120 rpm. At the same time, verify the effect of laser irradiation on the release of MFTA drugs. During laser irradiation (808 nm, 1.5 W / cm 2 Before irradiation (5 min), the dialysis bag containing the loaded MFTA nanoparticles was thoroughly shaken. After irradiation, the shaking was continued for 2 days.

[0097] Experimental Procedure: At different time points (0, 0.5, 1, 2, 4, 6, 12, 24, and 48 hours), 1 mL of liquid was collected from each of three parallel flasks and 1 mL of buffer was added to each flask. The amount of MTO released in the supernatant was determined by UV-visible absorption peaks using a standard curve for MTO.

[0098] Example 3: Evaluation of the effect of nanoparticle photothermal synergistic therapy and exploration of in vitro therapeutic mechanism Figure 7 As shown;

[0099] Instruments, equipment, materials and reagents

[0100] Instruments and equipment: 808 nm near-infrared laser (LOS-BLD-0808-2W-C / P, Beijing Hi-Tech Optoelectronics Co., Ltd.), CO2 constant temperature incubator (Thermo Corporation, USA), upright optical microscope (IX53, Olympus Corporation, Japan), A1R laser scanning confocal microscope (Nikon Corporation, Japan), Annexin V-FITC cell apoptosis detection kit, Cell Counting Kit-8 (CCK-8 kit), DAPI staining solution (Shanghai Biyuntian Biotechnology Co., Ltd.), DiI staining solution (MedChemExpress, USA), CytoFLEX flow cytometer (Beckman Coulter, USA), etc.

[0101] Materials and reagents: breast cancer cell line 4T1 (Third Military Medical University), experimental nanoformulations (prepared as described above), sterile PBS (Hitachi), 96-well cell culture plates (Costar, USA), ATP detection kit (Biyuntian), anti-mouse CRT antibody (Wuhan Sewell Company), etc.

[0102] Murine TNBC cell line culture

[0103] The breast cancer mouse-derived cell line 4T1 was cultured in RPMI-1640 medium containing 10% by volume fetal bovine serum and 1% by volume penicillin / streptomycin at 37° C. and a 5% CO 2 cell culture incubator.

[0104] Detection of nanoparticle uptake effects in breast cancer cells

[0105] Laser confocal microscopy was used to observe cellular nanoparticle uptake: 4T1 cells were seeded in 35 mm confocal culture dishes and incubated for 24 hours. When the cell density reached 80%, the old culture medium was removed. Under dark conditions, DiI-labeled MFTA nanosuspension was added to the confocal culture dish and incubated with the tumor cells for 0.5, 1, 2, 4, and 6 hours. The dish was then rinsed with PBS to remove unabsorbed MFTA. Cell nuclei were stained with 500 μL of DAPI staining solution for 10 minutes. After rinsing the cells with PBS three times to remove excess dye, they were fixed with 4% paraformaldehyde for 15 minutes and observed by CLSM.

[0106] FCM observation of MFTA uptake by cells: 4T1 tumor cells were seeded in 6-well plates (5×10 5 After the cells reached a density of approximately 80%, the MFTA nanoparticle suspension was added to the 6-well plate and incubated with the tumor cells in the dark for 0.5, 1, 2, 4, and 6 hours. The culture dish was rinsed with PBS to remove any uningested nanoparticles. The cells were then digested and resuspended, and the fluorescence intensity of the incorporated MFTA was analyzed using a single-channel FCM.

[0107] CCK-8 assay to detect cytotoxicity of nanoparticles

[0108] like Figure 6 As shown;

[0109] Experimental grouping and treatment: 10 4 4T1 cells were seeded into 96-well plates at a density of 100 μg / well and cultured for 24 hours. The old culture medium was discarded, and gradient concentrations of MFTA nanosuspensions (prepared in RPMI1640 medium) were added to the plates and incubated for 12, 24, and 48 hours. Duplicate wells containing no cells and untreated controls were set up. 4T1 cell viability was measured using the standard CCK-8 assay, and statistical analysis was performed using GraphPad Prism software.

[0110] Laser irradiation treatment: 10 4 4T1 cells were seeded into 96-well plates at a density of 100 cells / well and cultured for 24 hours. The old culture medium was discarded, and then different concentrations of MFTA nanosuspensions were added to the wells for co-incubation. After 4 hours, the wells with different concentrations of nanosuspensions were irradiated with lasers of different powers (0.5, 1.0, 1.5 W / cm 2 ) and continued incubation for 4 hours. Duplicate wells were set up as a cell-free blank group and an untreated control group. After treatment, 100 μL of CCK-8 reagent was added to each well. 4T1 cell viability was measured according to the standard CCK-8 assay, and statistical analysis was performed using GraphPad Prism software.

[0111] Flow cytometry detection of breast cancer cell apoptosis

[0112] Experimental groups: There were 6 experimental groups in this part: blank control group (untreated group), simple laser irradiation group, mitoxantrone treatment group, MFTA treatment group, TAF+laser group, and MFTA+laser group.

[0113] Experimental procedure: 4T1 cells were seeded in a 6-well plate and incubated for 24 hours. When the cell density increased to about 80%, the old culture medium was discarded and different treatments were performed according to the group. The power density of the 808nm laser was 1.5W / cm 2 The plate was washed three times with PBS, and the cells were digested and collected into flow cytometry tubes. 15 μL of Annexin-VFITC and 10 μL of PI were added and stained for 20 minutes in the dark. Finally, the cells were washed three more times with PBS, and apoptotic cell populations were observed on a flow cytometer.

[0114] Detecting the live and dead state of breast cancer cells using confocal microscopy

[0115] Experimental groups: There were 6 experimental groups in this part: blank control group (untreated group), simple laser irradiation group, MTO treatment group, MFTA treatment group, TAF+laser group, and MFTA+laser group.

[0116] Experimental procedure: cells were seeded in a 35mm glass-bottom confocal culture dish and incubated for 24 hours. When the cell density reached about 80%, the old culture medium was discarded and different treatments were performed according to the group. The power density of the 808nm laser was 1.5W / cm 2 The cells were irradiated for 5 minutes. Then, 15 μL of Cal-AM and 10 μL of PI were added and the cells were stained for 20 minutes. Finally, the cells were washed three times with PBS and observed using a CLSM.

[0117] Statistical analysis: The experimental data were compared using analysis of variance (ANOVA) and t-test, with *p<0.05 and **p<0.001 as the significance test standards.

[0118] Example 4: Evaluation of the effect of nanoparticle synergistic therapy in inducing cell ferroptosis

[0119] Instruments, equipment, materials and reagents

[0120] Instruments and equipment: lipid peroxide (LPO) ELISA kit, glutathione peroxidase 4 (GPX4) ELISA kit, GSH and GSSG detection kits, mitochondrial membrane potential detection kit (JC-1), lipid peroxidation detection kit (BODIPY581 / 591C11), reactive oxygen species detection kit (Guangzhou Yibei Biotechnology Co., Ltd., Shanghai Biyuntian Biotechnology Co., Ltd., etc.).

[0121] Materials and reagents: breast cancer cell line 4T1 (Third Military Medical University), experimental nanoparticles (prepared as described above), sterile PBS (Wuhan Sewell Company), etc.

[0122] Detection of the effects of nanoparticle synergistic therapy on intracellular redox system-related factors (GSH / GSSG, GPX4, LPO)

[0123] Sample preparation: 4T1 cells were cultured at a volume of 2 × 10 5 The cells were cultured in a 6-well plate at a density of 10 cells / well and incubated for 24 hours. Subsequently, the cells were divided into 6 groups for different treatments: blank control group (untreated group), simple laser irradiation group, MTO treatment group, MFTA treatment group, TAF+laser group, MFTA+laser group. After treatment, continue incubation for 12 hours. Collect the cells, wash the cells with pre-cooled PBS 2-3 times, and collect the cell pellet by centrifugation. 6 100 μl of cells were added to the cell lysis buffer, lysed on ice for 15-30 min, and then centrifuged to obtain the supernatant as the sample.

[0124] GSH / GSSG Assay: GSH and GSSG were quantitatively determined using a GSH / GSSG assay kit. First, remove all reagents from the refrigerator, equilibrate to room temperature, and dilute as directed in the kit instructions to prepare the working solution. Next, construct a standard curve using the GSH or GSSG standard provided in the kit at six different concentrations (0, 5, 10, 20, 40, and 80 μM) and plate each well in a 96-well plate. Set up three replicates for each concentration. Next, add the prepared sample to a 96-well plate in duplicate, along with a blank control containing only lysis buffer or buffer. Then, add the working solution containing the specific enzyme or chromogen to the standard and sample wells, mix thoroughly, and incubate the 96-well plate in a 37°C incubator or water bath for 30-60 minutes to allow for full reaction. Finally, at the end of the incubation period, measure absorbance at 540 nm using a microplate reader according to the kit's assay principles. The GSH level in 4T1 cells treated with PBS was set to 100%.

[0125] GPX4 content detection: ELISA is used to detect GPX4 content. Coat the plate with the coated antibody, then add the diluted sample and GPX4 standard. Incubate at 37°C for 15 minutes to allow the GPX4 in the sample to fully bind to the coated antibody. Wash the plate to remove any unbound material, then add the enzyme-labeled antibody and continue incubating for 15 minutes. Wash again, then add the substrate for color development for 15 minutes. Finally, measure the absorbance at 540 nm using a microplate reader. Calculate the GPX4 content in the sample based on the standard curve drawn with the standard.

[0126] LPO content detection: ELISA is used to detect LPO content. Prepare the coating antibody, enzyme-labeled antibody, substrate, and other reagents in the ELISA kit. Fix the coating antibody to the ELISA plate. Then, add the diluted sample and LPO standard to the plate wells. Incubate at 37°C for 15 minutes to allow the LPO in the sample to specifically bind to the coating antibody. After incubation, wash to remove unbound substances, then add the enzyme-labeled antibody and continue incubation for 15 minutes. After washing again, add the substrate to induce a color reaction for 15 minutes. Finally, use a microplate reader to measure the absorbance at 540nm. Use the standard curve drawn with the standard to calculate the LPO content in the sample.

[0127] Determination of intracellular reactive oxygen species (ROS) levels

[0128] Experimental operation: 4T1 cells were detected by DCFH-DA staining. 4T1 cells were plated at 2×10 5 The cells were seeded at a density of 100 μg / dish in 35 mm confocal glass-bottom culture dishes. The culture dishes were randomly divided into 6 groups: a blank control group (untreated group), a laser irradiation group alone, a mitoxantrone treatment group, a MFTA treatment group, a TAF+laser group, and a MFTA+laser group. After incubation for 12 hours, the cells were stained with the fluorescent probe DCFH-DA for 20 minutes. After staining, the cells were imaged using a confocal laser scanning microscope (CLSM) system and the fluorescence intensity of the staining was quantitatively analyzed by FCM to accurately assess the changes in ROS levels in each group of cells.

[0129] Measurement of changes in mitochondrial inner membrane potential

[0130] CLSM observation: 4T1 cells were plated at 2×10 5 Cells were seeded at a density of 100 cells / mL in confocal microscopy (CMS) culture dishes. The dishes were randomly divided into six groups: a blank control group (untreated), a laser irradiation group alone, a MTO-treated group, a MFTA-treated group, a TAF+laser group, and a MFTA+laser group. The cells were stained with JC-1 and incubated in a cell culture incubator for 20 minutes. Changes in mitochondrial membrane potential were observed using CLSM.

[0131] FCM quantitative analysis: 4T1 cells were plated at 2×10 5 Cells were cultured in 6-well plates at a density of 10 cells / well and incubated overnight with 2 mL of culture medium. The cells were then divided into six different groups and incubated for 6 hours. Following this, the cells were washed three times with PBS, adherent cells were digested, collected into flow cytometers, and stained with JC-1 for 20 minutes. Changes in mitochondrial membrane potential were quantitatively analyzed using flow cytometry.

[0132] Observation of intracellular lipid peroxide accumulation

[0133] CLSM observation: LPO levels in 4T1 cells treated with different methods were measured using C11-BODIPY fluorescent probe. 4T1 cells were plated at 2×10 5 Cells were cultured in confocal culture dishes at a density of 10 cells / dish.

[0134] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A research method for nanomaterials for diagnosis and treatment of breast cancer based on metal phenolic network, comprising, characterized in that: The nanomaterial is obtained by the coordination reaction of tannic acid and ferric chloride to generate photosensitive TAF nanoparticles, and mitoxantrone is loaded through π-π interaction to construct an MFTA nano-platform. The preparation method of the nanomaterial also includes dissolving mitoxantrone hydrochloride in methanol and neutralizing it with trimethylamine to obtain hydrophobic MTO, then dissolving TA in anhydrous ethanol and mixing it with the MTO solution, then adding FeCl3·6H2O aqueous solution and stirring overnight to evaporate the solvent, and finally collecting the precipitate by centrifugation to obtain MFTA.

2. The research method of a nanomaterial for diagnosis and treatment of breast cancer based on a metal phenolic network according to claim 1, characterized in that: The particle size of the nanomaterial is in the range of 50-200 nm.

3. The research method of a nanomaterial for diagnosis and treatment of breast cancer based on a metal phenolic network according to claim 1, characterized in that: The MTO, TA and Fe 3+ The molar ratio is 1:1:

1.

4. The research method of a nanomaterial for diagnosis and treatment of breast cancer based on a metal phenolic network according to claim 1, characterized in that: Application of the nanomaterial in breast cancer diagnosis and treatment drugs.

5. The research method of a nanomaterial for diagnosis and treatment of breast cancer based on a metal phenolic network according to claim 1, characterized in that: The research method includes the application of nanomaterials in in vitro experiments and in vivo experiments, wherein the in vitro experiments include: Cellular uptake assay: 4T1 cells were seeded in confocal culture dishes and incubated for 24 hours. When the cell density reached 80%, the old culture medium was removed. DiI-labeled MFTA nanosuspension was added to the confocal culture dish in the dark and incubated with the tumor cells for 0.5, 1, 2, 4, and 6 hours. The dish was rinsed with PBS to remove unabsorbed MFTA. The cell nuclei were stained with 500 μL of DAPI staining solution for 10 minutes. After rinsing the excess dye three times with PBS, the cells were fixed with 4% paraformaldehyde fixative for 15 minutes and observed by CLSM. Cytotoxicity assay: 4T1 cells were seeded in 96-well plates and cultured for 24 hours. The old culture medium was discarded, and gradient concentrations of MFTA nanosuspension were added to the plates and incubated for 12, 24, and 48 hours. Duplicate wells containing no-cell blanks and untreated controls were set up to detect 4T1 cell viability and perform statistical analysis. Evaluation of synergistic treatment effects.

6. The method for researching nanomaterials for diagnosis and treatment of breast cancer based on metal phenolic network according to claim 5, characterized in that: In the cytotoxicity assay: 10 4 4T1 cells were seeded into 96-well plates at a density of 1 / well and cultured for 24 h; The gradient concentration of MFTA nanosuspension was controlled at 0.05, 0.1, 0.2, 0.5, 1, and 2 mg / mL and prepared in RPMI1640 medium. The viability of 4T1 cells was detected according to the standard CCK-8 method.

7. The method for researching nanomaterials for diagnosis and treatment of breast cancer based on metal phenolic network according to claim 6, characterized in that: The synergistic treatment effect evaluation includes: 4T1 cells were divided into 6 groups: blank control group, laser irradiation group, mitoxantrone treatment group, MFTA treatment group, TAF+laser group, and MFTA+laser group. The concentration of TAF in all groups was 0.5 mg / mL. All laser irradiation groups were treated with 808 nm laser irradiation, and the irradiation parameters were controlled at: 1.5 W / cm 2 ,5min.

8. The method for researching nanomaterials for diagnosis and treatment of breast cancer based on metal phenolic network according to claim 5, characterized in that: The in vivo experiments are specifically as follows: Animal model establishment; Evaluation of treatment efficacy; Verification of immune response induction effect; The establishment of the animal model is as follows: The 4T1 cells that were pre-cultured at a growth density of about 80% were digested and resuspended in sterile PBS and diluted to 1×10 6 / mL density cell suspension; 200μL of cell suspension was slowly injected into the fourth pair of mammary fat pads of female Balb / c mice using a 1mL syringe to establish a tumor-bearing mouse model; after inoculation, the survival status of the mice and the growth of the tumor were observed and monitored every day. 3 The tumor size was observed every 1-2 days and the tumor was used for subsequent experiments when it grew to a volume of 80 mm. 3 For subsequent animal experiments.

9. The method for researching nanomaterials for diagnosis and treatment of breast cancer based on metal phenolic network according to claim 8, characterized in that: The treatment effect evaluation is specifically as follows: Mice were randomly divided into 6 groups: PBS, laser only, MTO, MFTA, TAF+laser, and MFTA+laser. 3 At the same time, mice were divided into preset groups to receive different treatments; all mice in the laser irradiation group were exposed to 808nm laser; during the entire 2-week treatment cycle, the body weight and tumor volume of each group of mice were recorded every 2 days; at the end of the treatment, the mice were killed and the tumors were weighed.

10. The method for researching nanomaterials for diagnosis and treatment of breast cancer based on metal phenolic network according to claim 9, characterized in that: The immune response induction effect verification is specifically as follows: Fresh tumor tissue was obtained to prepare 4-5 μm paraffin sections, which were baked at 60°C for 2 h, dewaxed in xylene, hydrated with graded ethanol, and washed three times with PBS; Antigen retrieval: Immerse the sections in 0.01 M sodium citrate buffer, pH 6.0, microwave for 10 min, cool naturally, and wash with PBS. Blocking: Add 5% goat serum at room temperature for 30 minutes, shake dry, add mouse anti-human HMGB-1 monoclonal antibody and rabbit anti-human CD8 polyclonal antibody, and incubate at 4°C overnight; Fluorescence staining: After washing three times with PBST, AlexaFluor488-labeled goat anti-mouse IgG and Cy3-labeled goat anti-rabbit IgG were added in the dark and incubated at room temperature for 1 hour; DAPI counterstained the nucleus for 5 minutes; Sealing observation: The slides were sealed with anti-fluorescence fading mounting medium, and images of 405, 488, and 555 nm channels were collected under a confocal microscope for fluorescence intensity analysis; Flow cytometry analysis of tumor immune cell infiltration: Mouse spleen tissue was minced and added to RPMI-1640 medium containing 0.5 mg / mL collagenase IV and 0.1 mg / mL DNase I. The tissue was digested at 37°C for 30 min, filtered through a 70 μm filter, and centrifuged to discard the supernatant. After treatment with red blood cell lysis buffer, the tissue was resuspended in PBS and stained with trypan blue to count live cells. Antibody staining: 1×10 6 Cells were washed with a combination of CD3-PacificBlue, CD4-PE, and CD8-APC antibodies and incubated at 4°C in the dark for 30 min. 200 μL of flow cytometry buffer was added to each tube and the cells were centrifuged. Fixation and storage: Fix with 2% paraformaldehyde for 10 min, resuspend in PBS and store at 4°C in the dark. Detect within 24 h. Flow cytometry: FSC / SSC gate was set on the flow cytometer to exclude debris, CD3 + Analysis of CD4 / CD8 double positive cells within the gate: CD3 + CD4 + CD8 - For helper T cells, CD3 + CD4 - CD8 + Cytotoxic T cells, CD3 + CD4 + CD8 + For DC activation analysis, cell suspensions of tumors and lymph nodes were stained with another panel of fluorescently labeled antibodies, and the proportion of activated DCs in tumor and lymph node tissues was assessed using FCM.

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