Iron-based photodynamic-chemodynamic synergistic immunonanopreparation for breast cancer treatment and preparation method thereof

Abstract

The application belongs to the field of tumor nano-immunotherapy, and particularly relates to an iron-based photodynamic-chemical kinetics synergistic immunonanopreparation for breast cancer treatment and a preparation method thereof. The effective component of the nanopreparation is a nanometer particle CyBT-Fe with good endoplasmic reticulum targeting stability, and through in-vitro and in-vivo experiments, the CyBT-Fe combined with sorafenib (SRF) shows great potential in breast cancer treatment. The nanometer particle CyBT-Fe is prepared from CyBT and Fe-Mil according to a mass ratio of 1:100, and the structure of the CyBT is as follows.

Description

Technical Field

[0001] This invention belongs to the field of tumor nanoimmunotherapy, specifically relating to an iron-based photodynamic-chemokinetic synergistic immunomodulatory nanoparticle for breast cancer treatment and its preparation method. Background Technology

[0002] Breast cancer, as one of the leading causes of cancer-related morbidity and mortality among women worldwide, faces challenges due to its complex tumor microenvironment (TME), including immunosuppression, treatment resistance, and heterogeneity. These factors severely limit the clinical efficacy of traditional radiotherapy, chemotherapy, targeted therapy, and immunotherapy, necessitating the development of multi-mechanism synergistic precision treatment strategies. Photodynamic therapy (PDT), with its advantages of spatiotemporally controllable cytotoxicity and low systemic toxicity, has become an important direction in precision cancer treatment. Its efficacy depends on the enrichment of photosensitizers (PS) in tumor tissue and the efficient generation of reactive oxygen species (ROS). Based on differences in their mechanisms of action, type I PDT generates free radicals such as superoxide anions through electron transfer, while type II PDT directly induces singlet oxygen (ROS). 1 Both O2 and PDT have their own advantages in regulating oxidative stress in the tumor microenvironment. However, single PDT is prone to incomplete treatment due to problems such as TME hypoxia and activation of the antioxidant system. Therefore, it is urgent to combine multimodal strategies to overcome the bottleneck.

[0003] Ferroplasmosis, as a novel regulatory cell death mechanism, induces tumor cell death by regulating the imbalance between intracellular iron ion metabolism and lipid peroxidation. It exhibits no cross-resistance with traditional apoptosis pathways, providing a new approach for breast cancer treatment. The tumor microenvironment (TME) is well-known as the "soil" for breast cancer development, possessing unique physiological characteristics such as low pH, high glutathione (GSH) concentration, and overexpression of specific enzymes. These characteristics provide an ideal "endogenous trigger" for the precise delivery of nanomedicines. TME-responsive smart nanoparticles, through rational design of carrier structure and function, can remain stable in the bloodstream to reduce damage to normal tissues. Upon entering the tumor region, they respond to TME stimulation, undergoing structural changes or degradation, achieving targeted release and activation of photosensitizers, significantly improving drug concentration and bioavailability at the tumor site. Iron-based nanoparticles, possessing both Fe... 2+ / Fe 3+ Cyclic-mediated Fenton reaction activity, high drug loading capacity, and TME responsiveness not only enable targeted delivery of PDT photosensitizers, but the released Fe can also amplify PDT-induced ROS generation by generating •OH through the Fenton reaction. 2+ / Fe 3+During circulation, O2 is generated to alleviate the hypoxic state of the tumor microenvironment. Sorafenib, as a multi-target tyrosine kinase inhibitor, not only inhibits tumor vascular endothelial growth factor receptor to block tumor angiogenesis and improve the hypoxic tumor microenvironment, regulates the immunosuppressive state in the TME, and enhances the anti-tumor activity of immune cells, but also promotes ferroptosis by inhibiting cysteine / glutamate transporters (SLC3A2 / SLC7A11) to reduce GSH, forming a highly efficient synergistic effect with PDT. Endoplasmic reticulum (ER) stress is a key adaptive mechanism for tumor cells to cope with oxidative stress and nutrient deprivation. The continuously activated unfolded protein response (UPR) causes the protein kinase RNA-like ER kinase (PERK) to dissociate from the ER molecular chaperone BiP (GRP78) and be activated. Activated PERK undergoes autophosphorylation, which in turn phosphorylates eukaryotic translation initiation factor 2α (elf2α). Phosphorylated elf2α inhibits the overall translation of proteins, but at the same time selectively promotes the translation of certain mRNAs, especially activating the translation of transcription factor 4 (ATF4). As a transcription factor, ATF4 upregulates the expression of glutathione-specific γ-glutamyl cyclotransferase 1 (CHAC1) through its translation. CHAC1 weakens the antioxidant capacity of glutathione peroxidase 4 (GPX4) by degrading glutathione, thereby significantly enhancing the sensitivity of tumor cells to ferroptosis by regulating lipid metabolism disorders. Meanwhile, the massive ROS generation induced by PDT can stimulate ER stress, providing an ideal target for combination therapy.

[0004] Based on this, the present invention designs a photosensitizer CyBT capable of generating type I / II ROS, loaded onto a TME-responsive iron-based nanocarrier, and combined with sorafenib to construct a synergistic therapeutic system of "PDT-ER stress-ferroptosis-immune activation". After the nanoparticles accumulate in tumor tissue, they trigger Fe under acidic conditions in the TME. 3+ Controlled drug release; ROS and Fe generated by PDT excitation 3 + The Fenton-mediated reaction creates a cascade amplification effect, causing ER stress, activating the PERK-elf2α-ATF4 pathway, upregulating CHAC1 expression, and significantly enhancing sorafenib-induced ferroptosis sensitivity. Simultaneously, sorafenib promotes ferroptosis by inhibiting the SLC3A2 / SLC7A11 transporter and reducing GSH. Ferroptosis and PDT-mediated tumor cell rupture release large amounts of damage-associated molecular patterns (DAMPs), activating dendritic cell (DC) maturation and promoting cytotoxic T lymphocyte (CTL) infiltration, reversing the TME immunosuppressive state, and ultimately achieving a synergistic effect of local tumor killing and systemic immune memory. This invention aims to develop a novel nanotherapy platform that combines targeting, responsiveness, and synergy, providing experimental evidence and theoretical support for solving the problems of drug resistance and immune escape in breast cancer treatment, and promoting the clinical translation of precision oncology technologies. Summary of the Invention

[0005] This invention provides a CyBT-Fe nanoparticle, characterized in that the preparation method of the CyBT-Fe nanoparticle includes the following steps:

[0006] CyBT was dissolved in ethanol, Fe-Mil was added, and after the reaction was complete, the precipitate was collected by centrifugation to obtain the CyBT-Fe nanoparticles.

[0007] The mass ratio of CyBT to Fe-Mil is 1:100;

[0008] The structure of CyBT is as follows:

[0009] .

[0010] The Fe-Mil is selected from MIL-88B(Fe), which can be purchased commercially or obtained by reacting H2BDC with Fe(NO3)3 or its hydrate (Fe(NO3)3·9H2O) in a molar ratio of 1:1; the reaction is preferably carried out under reflux in a mixed solvent of DMF / MeCN in a volume ratio of 1:1.

[0011] Another embodiment of the present invention provides the above-mentioned CyBT-Fe nanoparticles, characterized in that the average size of the CyBT-Fe nanoparticles is 147.05±1.92 nm.

[0012] Another embodiment of the present invention provides a method for preparing the above-mentioned nanoparticles, characterized by comprising the following steps:

[0013] CyBT was dissolved in ethanol, Fe-Mil was added, and after the reaction was complete, the precipitate was collected by centrifugation to obtain the CyBT-Fe nanoparticles.

[0014] The mass ratio of CyBT to Fe-Mil is 1:100;

[0015] The structure of CyBT is as follows:

[0016] ;

[0017] The Fe-Mil is selected from MIL-88B(Fe), which can be purchased commercially or obtained by reacting H2BDC with Fe(NO3)3 or its hydrate (Fe(NO3)3·9H2O) in a molar ratio of 1:1; the reaction is preferably carried out under reflux in a mixed solvent of DMF / MeCN in a volume ratio of 1:1.

[0018] Another embodiment of the present invention provides an intermediate CyBT, characterized in that the structure of the intermediate CyBT is as follows:

[0019] .

[0020] Another embodiment of the present invention provides the application of the above-mentioned intermediate CyBT in the preparation of CyBT-Fe nanoparticles.

[0021] Another embodiment of the present invention provides the application of the CyBT-Fe nanoparticles described herein in the preparation of nanoimmunotherapy agents for the treatment of breast cancer.

[0022] Another embodiment of the present invention provides a nanoimmunotherapy agent, characterized in that the nanoimmunotherapy agent uses CyBT-Fe nanoparticles as the active ingredient. The nanoimmunotherapy agent may also include pharmaceutically acceptable excipients. The nanoimmunotherapy agent may also include sorafenib (SRF).

[0023] Compared with existing technologies, the advantages of this invention are as follows: This invention provides CyBT-Fe nanoparticles with good endoplasmic reticulum targeting stability. Through in vitro and in vivo experiments, CyBT-Fe combined with SRF has shown great potential for treating breast cancer. In the TME, CyBT-Fe is dissociated to release Fe. 3+ CyBT, when irradiated with near-infrared (NIR) light at 660 nm, effectively generates reactive oxygen species (ROS) in tumor cells. The accumulation of ROS is amplified through the Fenton reaction of iron ions and H₂O₂, effectively inducing ER stress in tumor cells. This activates the PERK-elf2α-ATF4-CHAC1 signaling pathway, inducing ferroptosis. The consumption of GSH by iron ions and the reduction of GSH synthesis by SRF further promote ferroptosis. This series of reactions, leading to ferroptosis and oxidative stress, induces potent ICD, releasing a large number of damage-associated molecular patterns (DAMPs), activating anti-tumor immune responses, promoting dendritic cell maturation and T cell activation, increasing the frequency of effector memory T cells, and simultaneously reducing immunosuppressive cells, thus alleviating immunosuppression. This work establishes a multifunctional TME-activated nanoplatform that overcomes the key limitations of single-modality therapy. By integrating I / II photodynamic therapy, ER stress, ferroptosis, and immunotherapy, it provides a transformative strategy for overcoming the immunosuppressive tumor microenvironment in breast cancer. Attached Figure Description

[0024] Figure 1 Image a shows the HOMO and LUMO energy level diagrams of CyBT; image b shows the singlet and triplet energy level distributions of CyBT; images c, d, e, and f respectively depict the energy levels of CyBT+ABDA, CyBT+SOSG, CyBT+HPF, and CyBT+DHR123 at 660 nm (0.5 W / cm²). 2Ultraviolet absorption and fluorescence emission spectra of water under light irradiation over time.

[0025] Figure 2 In the image, a is the TEM image of CyBT-Fe (scale bar = 50 nm) and the corresponding elemental distribution; b is the particle size distribution of CyBT-Fe in ultrapure water; c is the Fe 2p spectrum of CyBT-Fe; d is the XPS measurement spectrum of CyBT-Fe; e and f are the UV absorption and fluorescence spectra of Fe-MIL, CyBT, and CyBT-Fe in water; g is the UV-Vis absorption spectrum of CyBT-Fe consuming GSH; h is the UV absorption spectrum of CyBT-Fe catalyzing the formation of ·OH from H2O2 using the MB method; i and j are Fe at different pH values. 3+ Release rate graph of CyBT.

[0026] Figure 3 Image a is a CLSM image of MCF-7 cells after 2 hours of treatment with 0.5 μM ER Tracker Green and 10 μg / mCyBT-Fe, representing spontaneous mitochondrial localization of CyBT-Fe (scale bar = 50 μm); image b is an image at 660 nm (20 mW / cm²). 2 Phototoxicity of CyBT and CyBT-Fe to MCF-7 cells under 10 min light irradiation; c is the result of irradiation with and without 660 nm (20 mW / cm²) light. 2 d is a graph showing the cytotoxicity of CyBT and CyBT-Fe to MCF-7 cells under 10 min light irradiation; d is a graph showing the co-localization experiment of MCF-7 cells co-incubated with ER green fluorescent probe and CyBT-Fe under different light intensities, scale bar = 50 μm; e is a graph showing the use of DCFH-DA probe to detect intracellular ROS production, scale bar = 50 μm; f is a graph evaluating the cytotoxicity of CyBT and CyBT-Fe at 660 nm (20 mW / cm²) using qualitative analysis of calcein AM and PI probes. 2 Image showing the killing effect of light on MCF-7 cells under 10 min light irradiation, scale bar = 200 μm; g is the killing effect of MCF-7 cells after different treatments analyzed by flow cytometry.

[0027] Figure 4 Image a is a schematic diagram of the immune model construction and processing flow; image b is a proximal tumor image in mice; image c is a proximal tumor volume image in mice; image d is a proximal tumor weight image in mice; image e is a distal tumor image in mice; image f is a distal tumor volume image in mice; image g is a distal tumor weight image in mice; image h is an immunofluorescence staining image of mouse tumor tissue, including CRT, HMGB1, SLC7A11 and GPX4, scale bar = 50 μm.

[0028] Figure 5 Figures a and b show the results of FCM detection and quantitative analysis of DC cells and Treg cells in proximal tumors; figures c and d show the results of FCM detection and quantitative analysis of DC cells and Treg cells in distal tumors; figure e shows the immunofluorescence staining of mouse tumor tissue, including CD4, CD8, and FOXP3, with a scale bar of 50 μm; figures fi show the concentrations of related cytokines IFN-γ, TNF-α, IL-12, and IL-6 in the serum of mice in the combined treatment group.

[0029] Figure 6 This is the mass spectrum of compound 1.

[0030] Figure 7 This is the mass spectrum of compound 2.

[0031] Figure 8 This is the mass spectrum of CyBT.

[0032] Figure 9 It's CyBT. 1 ¹H NMR (400 MHz, DMSO-d6) plot.

[0033] Figure 10 It's CyBT. 13 C NMR (100 MHz, DMSO-d6) plot. Detailed Implementation

[0034] The main analytical and testing methods of this invention are as follows:

[0035] 1. Determination of ROS in vitro

[0036] To evaluate the singlet oxygen of the photosensitizer ( 1 O2) generation efficiency was measured using singlet oxygen ( 1 O2 was detected indirectly by monitoring changes in the UV absorption of ABDA and the fluorescence intensity of SOSG using the specific trapping agent ABDA (9,10-anthracite dipropionic acid) and the singlet oxygen fluorescent probe SOSG. 1 O2 generation. CyBT-Fe was dissolved in 1 mL of DMSO, and a certain amount was dispersed in 4 mL of ultrapure water containing ABDA and SOSG, respectively. The resulting mixture was placed in a cuvette and irradiated with a 660 nm laser at a power density of 20 mW / cm² for different durations. The results were then evaluated by UV-Vis spectrophotometry and fluorescence emission spectroscopy. 1 The generation of O2 was compared with the UV and fluorescence spectra of ABDA and SOSG under separate irradiation.

[0037] For O2- The evaluation was performed using the fluorescent probe dihydrorhodamine 123 (DHR 123) to monitor the O2 in the solution. - The formation of the product was determined by dispersing CyBT-Fe dissolved in DMSO into 4 mL of ultrapure water containing DHR 123. The resulting mixture was placed in a cuvette and purified using a 20 mW / cm² solution. 2 The sample was irradiated with 660 nm near-infrared light for a certain period of time, and the fluorescence at 540 nm was measured immediately afterwards. The fluorescence spectrum of the sample irradiated with DHR 123 alone was used as a control.

[0038] The generation of ·OH was detected using hydroxyphenyl fluorescein (HPF) as an indicator. HPF emitted green fluorescence at 515 nm after reacting with CyBT-Fe. The resulting mixture was placed in a cuvette and purified using 20 mW / cm² water. 2 The sample was irradiated with 660 nm near-infrared light for a certain period of time, and the fluorescence at 515 nm was measured immediately afterwards. The fluorescence spectrum of the HPF irradiated alone was used as a control.

[0039] 2. Cell Culture

[0040] The cancer cells (4T1, MCF-7), human gastric mucosal cells GES-1, and monkey kidney cells COS7 were all cultured in DMEM medium containing 10% fetal bovine serum, penicillin, and streptomycin (100 U / mL) in a 37°C, 5% CO2 incubator.

[0041] 3. Cellular uptake

[0042] MCF-7 cells were seeded in 20 mm confocal culture dishes and cultured for 24 h. Subsequently, the cells were co-incubated with the drug for specific time periods. After washing away the drug-containing DMEM at the set time, the cells were fixed with fixative, and fluorescence images were collected under a laser confocal microscope.

[0043] 4. Intracellular ROS detection

[0044] MCF-7 cells were seeded in 20 mm confocal culture dishes and incubated for 24 h. MCF-7 cells were then incubated at 37°C for 4 h in DMEM containing or without CyBT and CyBT-Fe, according to their groupings, followed by washing three times with PBS buffer. The culture medium was then replaced with DMEM containing DCFHDA (2,7-dichlorodihydrofluorescein diacetate, 2.0 μM), and incubation continued for 30 min. The DMEM was then removed, and the cells were washed three times with PBS buffer. Subsequently, the cells were cultured at 660 nm, 20 mW / cm². 2Cells were irradiated with near-infrared light for 12 min. Subsequently, fluorescence imaging of the cells was performed using confocal microscopy. Confocal images were acquired under conditions of excitation wavelength of 488 nm and emission wavelength of 500–530 nm.

[0045] 5. Subcellular organelle colocalization experiment

[0046] MCF-7 cells were planted at a density of 1 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per 20 mm confocal culture dish and cultured for 24 h. Cells were first cultured in drug-containing DMEM for 4 h, then individually incubated with Hochest 33342 (500 nM), Golgi-Tracker Green (500 nM), ER-Tracker Green (1 µM), Lyso-Tracker Green (100 nM), or Mito-Tracker Green (200 nM) according to the manufacturer's instructions. All cells were washed three times with PBS and then imaged using a confocal laser scanning microscope (CLSM).

[0047] 6. Cytotoxicity (MTT) study

[0048] MCF-7 cells were planted at a density of 1 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 96-well plates and incubated for 24 hours. Subsequently, 100 μL of DMEM containing different drug concentrations was added to each well. After another 4 hours of incubation, the light group was exposed to an LED (660 nm, 20 mW / cm²). 2 Incubate for 15 minutes. After incubation for another 24 hours, add 20 µL of MTT (thiazolyl blue tetrazolium bromide, 5 mg / mL) to each well and incubate at 37°C and 5% CO2 for 4 hours. Then remove the solution from the 96-well plate and add 200 µL of DMSO to each well to dissolve the resulting purple formazan precipitate. Measure the absorbance of each well at 490 nm using a microplate reader. The cytotoxicity of CyBT and CyBT-Fe to other cell lines (COS7, GES-1) was evaluated using the same experimental method. All experiments were performed in parallel or in triplicate. IC50 was calculated as follows: 50 The value is derived from a curve plotted using the average OD value from three repeated trials against the drug concentration.

[0049] 7. Live / dead cell detection (live / dead cell staining)

[0050] MCF-7 cells were seeded in 20 mm confocal culture dishes and cultured for 24 h. Cells were then treated with CyBT and CyBT-Fe and cultured at 37 °C in a 5% CO2 incubator for 4 h. Subsequently, they were cultured with a 660 nm LED (20 mW / cm²).2 Irradiate for 0 or 15 min, then culture for 12 h. Light-treated cells serve as controls only. After light incubation, replace with fresh DMEM medium containing the green fluorescent precursor calceinam (5 μM) and propidium iodide (PI, 5 μM), and incubate for another 30 min. Remove the calceinam and PI solution, and wash cells three times with PBS buffer. Image using a confocal laser scanning microscope (CLSM).

[0051] 8. Flow cytometry detection

[0052] MCF-7 cells were seeded in 6-well plates and cultured for 24 h. Cells in specific wells were incubated with CyBT and CyBT-Fe for 4 h according to their respective groups, followed by irradiation for 0 or 15 min. The treated cells were then incubated for another 24 h and co-stained with Annexin V-FITC / PI; the irradiated group served as a control. Cells were then digested with trypsin, collected, and resuspended in buffer using a vortex mixer. All samples were then filtered through a 50 μm filter to remove air bubbles. Flow cytometry was performed using a 488 nm laser for excitation, and the data were analyzed using a FlowJo™ 10 instrument.

[0053] 9. Immunofluorescence assay (CRT, HMGB1, GRP78)

[0054] Cells at 1×10 6 Cells were re-seeded at a density of 1 mL in laser confocal culture dishes containing 1 mL of culture medium and incubated at 37°C for 24 h to ensure complete adhesion. Then, according to experimental requirements, cells were grouped, and the corresponding PBS, CyBT, and CyBT-Fe were dissolved in the culture medium and added to the culture dishes, incubated for 4 h. The cells were then exposed to NIR (660 nm, 20 mW / cm²). 2 Incubate for 10 minutes. After an additional 12 hours of incubation, remove the old culture medium and fix the cells overnight using immunostaining fixation solution. After cell fixation, incubate with primary antibody against GRP78 (1:100) for 4 hours. Subsequently, remove the primary antibody, wash the culture dish with PBS, and incubate with fluorescent secondary antibody (1:500) for 4 hours. After incubation, remove the secondary antibody, stain the cell nuclei with DAPI, and then wash with PBS. Then observe the samples under CLSM. HMGB1 and CRT were replaced with their corresponding specific antibodies, while all other steps remained unchanged.

[0055] 10. PDT experiment in mice

[0056] All experimental procedures followed the requirements of the "Guidelines for the Protection and Utilization of Laboratory Animal Resources" and were approved by the Laboratory Animal Ethics Committee of Shandong Second Medical University (Ethics Approval No.: 2022SDL381). Six- to eight-week-old female Balb / c mice (purchased from Beijing Viton Biotechnology Development Co., Ltd.) were used in the experiment, and the animals were allowed to acclimatize to the environment for one week after being introduced. 4T1 cells (5 × 10⁻⁶) were then used. 6 4T1 cells (100 μL PBS solution) were subcutaneously injected into the left axilla of each mouse. Three days later, 4T1 cells were injected into the right axilla to establish a mouse tumor model. Tumor volume was measured using calipers and calculated using the formula:

[0057]

[0058] V represents the tumor volume, and a and b represent the tumor's long and short diameters, respectively.

[0059] Subsequently, the mice were randomly divided into 6 different groups (n=5) for treatment: Group 1: PBS injection (100 μL); Group 2: PBS plus irradiation; Group 3: CyBT-Fe, no irradiation; Group 4: CyBT-Fe plus irradiation; Group 5: CyBT-Fe + SRF, no irradiation; Group 6: CyBT-Fe + SRF plus irradiation. Mice in groups 2, 4, and 6 were exposed to LED light (660 nm, 20 mW / cm²) 6 h after intratumoral injection. 2 Irradiation. Each group consisted of 5 mice and received three injections.

[0060] The efficacy of different treatment groups was monitored by measuring tumor size and mouse weight 14 days after PDT treatment. On day 14, mice were euthanized, tumors were dissected, and mice were weighed. For histological analysis, tumor tissue and major organs (heart, liver, spleen, lung, and kidney) were removed on day 14 post-treatment. Fluorescence images were acquired using an imaging system. The removed organs and tumors were fixed in 4% formaldehyde, embedded in paraffin, sectioned to a thickness of 5.0 μm, and stained with hematoxylin and eosin (H&E).

[0061] Example 1

[0062]

[0063] Preparation of Compound 1: 6-Bromo-2-methylbenzo[d]thiazole (10.0 mmol) and 4-(bromomethyl)acetophenone (10.0 mmol) were dissolved in 15 mL of acetonitrile. The mixture was heated to 80 °C and reacted for 24 hours. After the reaction was complete, the resulting pale green solid was collected by filtration and identified as Compound 1 (5.1 mmol, yield 51%, mass spectrometry shown). Figure 6 ).

[0064] Preparation of Compound 2: POCl3 (2 mL) was added dropwise to 9 mL of dimethylformamide (DMF) under ice bath conditions, and the reaction was allowed to proceed for 1 h. The reaction mixture was then stirred at room temperature for 3 h. Subsequently, phenylacetic acid (1.2 g) was added, and the mixture was heated to 90 °C, followed by vacuum distillation and reflux for 6 h. After the reaction was complete, the solution was cooled to room temperature, crushed ice was added, and 10 mL of saturated sodium perchlorate (NaClO4) solution was added with stirring, producing a large amount of solid. The solid was filtered and washed twice with saturated sodium perchlorate solution to remove impurities, yielding an intermediate product. Without further purification, the intermediate was directly transferred to 20 mL of sodium hydroxide solution (1.2 g) and heated to reflux at 90 °C until the precipitate was completely dissolved. The solution was then cooled to room temperature, and 10 mL of deionized water was added to dilute the mixture. Finally, the pH of the solution was adjusted to 3.0 with 10% hydrochloric acid (no further significant precipitation occurred), and the precipitate was separated by vacuum filtration to obtain Compound 2 as a pale yellow solid (0.96 g, 73.3%, mass spectrometry shown). Figure 7 ).

[0065] Preparation of CyBT: Compound 1 (585 mg) and Compound 2 (100 mg) were dissolved in 5 mL of anhydrous ethanol, and 3 drops of pyridine were added. The reaction mixture was then heated to 75 °C and reacted for 12 hours until the reaction was complete. The solvent was removed under reduced pressure, and the mixture was purified by column chromatography (eluent was a 20:1 mixture of dichloromethane and methanol) to obtain the target compound CyBT (263 mg, yield 46.7%, mass spectrometry and NMR spectra are shown in [reference needed]). Figure 8-10 ) 1 H NMR (400 MHz, DMSO-d6) δ 8.41 (s, 2H),7.92 (s, 1H), 7.88 (d, J = 6.3 Hz, 3H), 7.85 (s, 2H), 7.81 (s, 1H), 7.79 (s,1H), 7.73 (s, 1H), 7.71 (s, 1H), 7.44 (t, J = 7.4 Hz, 1H), 7.35 (s, 1H), 7.32 (d, J = 7.7 Hz, 1H), 7.11 (s, 2H), 7.09 (s, 2H), 6.79 (d, J = 7.5 Hz, 2H),5.83 – 5.68 (m, 2H), 5.48 (s, 4H), 2.57 (s, 6H). 13C NMR (100 MHz, DMSO-d6) δ197.89, 164.90, 150.88, 141.72, 139.98, 136.84, 132.19, 131.54, 129.72,129.39, 129.30, 128.20, 128.03, 127.42, 126.34, 117.85, 115.45, 100.42,49.63, 27.26. HRMS (ESI): m / z calcd for C 43 H 33 Br2N2O2S2 + [M] + 833.03, found833.03.

[0066] Mass spectrometry and NMR data confirmed the successful synthesis of CyBT. Next, we used density functional theory (DFT) to calculate the lowest vacant molecular orbital (LUMO) and highest occupied molecular orbital (HOMO) of CyBT, finding a band gap (ΔE) of 2.35 eV. Figure 1 a) A smaller bandgap can facilitate electron transfer. We then further calculated the singlet and triplet excited states of the CyBT molecule using time-dependent density functional theory (TD-DFT). For example... Figure 1 As shown in b, the values ​​of the lowest singlet excited state (S1), lowest triplet excited state (T1), and second lowest triplet excited state (T2) are 2.20, 1.09, and 2.38 eV, respectively. The band gaps between S1 and T1 and between S1 and T2 are 1.11 and 0.18 eV, respectively, indicating that CyBT can generate both type I and type II ROS. Next, to evaluate the ROS generation capacity of CyBT, using a blank as a control, its ability to generate singlet oxygen was measured using ABDA (9,10-anthrayl-bis(methylene)dimalonic acid) and the SOSG singlet oxygen sensor. The results show that CyBT has superior singlet oxygen generation capacity. 1 O2) production capacity ( Figure 1 c, d). In addition, we also used dihydrorhodamine 123 (DHR 123) and hydroxyphenylfluorescein (HPF) as detectors for O2. - The ability of CyBT to generate type I ROS was evaluated using indicators of ·OH. The results showed that CyBT exhibited strong ROS generation capabilities in ·O2. - It exhibits the best performance in terms of ·OH generation ( Figure 1 e, f). Consistent with theoretical calculations, CyBT produces type I and type II ROS after illumination.

[0067] Example 2: Preparation of CyBT-Fe nanoparticles

[0068] Fe-MIL (MIL-88B(Fe), 100 mg) was dissolved in ethanol (10 mL), CyBT (1 mg) was added, and the reaction was stirred at room temperature in the dark until it was completely reacted (about 24 h). The reaction mixture was centrifuged (10000 rpm, 10 min) to collect the precipitate, and dried to obtain the nanoparticles CyBT-Fe.

[0069] The Fe-MIL used in this invention is commercially available under the designation MIL-88B(Fe), and is obtained by reacting H2BDC with Fe(NO3)3 or its hydrate (Fe(NO3)3·9H2O) in a 1:1 molar ratio (preferably under reflux in a 1:1 DMF / MeCN mixed solvent). For example, it can be obtained by dissolving H2BDC (0.8 mmol, 132.9 mg) and Fe(NO3)3·9H2O (0.8 mmol, 323.2 mg) in a 1:1 DMF / MeCN (24 mL, 1:1 volume ratio) mixture, heating to 90°C until complete (approximately 5 h), centrifuging to collect the precipitate, washing with DMF and ethanol, and drying to obtain the Fe-MIL.

[0070] Example 3

[0071] The morphology of CyBT-Fe nanoparticles was observed using transmission electron microscopy (TEM). It was found that CyBT-Fe was prismatic in shape and approximately 150 nm in length. Elemental analysis was also performed to verify the elemental composition of the synthesized elongated nanomaterial. Figure 2 a). The presence of iron indicates successful loading of CyBT onto Fe-MIL. Next, a laser particle size analyzer was used to measure the particle size of the CyBT-Fe nanoparticles, confirming a size of 147.05 ± 1.92 nm. Figure 2 b). X-ray photoelectron spectroscopy (XPS) analysis showed that the valence state of iron in the compound was mainly Fe. 3+ Formal existence ( Figure 2 c), and XPS elemental analysis confirmed the presence of iron in CyBT-Fe ( Figure 2 d) This also proves the successful encapsulation of CyBT. In aqueous solution, CyBT-Fe exhibits a distinct absorption peak at approximately 661 nm and an emission peak at approximately 522 nm, showing a slight redshift compared to CyBT (λex = 603 nm, λem = 523 nm). Figure 2 e, f).

[0072] Subsequently, the ability of CyBT-Fe to consume GSH was evaluated using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). DTNB, as a GSH indicator, reacts with GSH to generate a colorimetric product. Experiments showed that the addition of CyBT-Fe to the GSH and DTNB mixture resulted in a decrease in UV-Vis absorption intensity at 412 nm. Figure 2 g). This confirmed that CyBT-Fe can effectively consume GSH. The catalytic activity of CyBT-Fe in consuming H2O2 was verified by methylene blue (MB) degradation experiments (the UV absorption peak of MB weakens after degradation by •OH). After mixing CyBT-Fe with 10 mM H2O2 and MB for 30 minutes, the UV absorption peak of MB at 665 nm gradually decreased, confirming that CyBT-Fe can mediate the generation of •OH from H2O2. Figure 2 (h). To evaluate the release performance of CyBT-Fe in an acidic environment, we added CyBT-Fe to PBS buffer solutions at different pH values. The results showed that, within the same time period, Fe in solutions at pH 5.5 and pH 6.5 increased significantly. 3+ The release rate of CyBT was significantly higher at pH 7.4 than at pH 7.4, indicating that CyBT exhibits pH responsiveness. Figure 2 (i, j) The morphology of CyBT-Fe in solutions of different pH values ​​after 1 hour was observed under transmission electron microscopy, showing that CyBT-Fe dissolves in acidic environments. Monitoring the absorbance of CyBT and CyBT-Fe under 60 min of light irradiation revealed that CyBT-Fe exhibited relatively better photostability compared to CyBT. Furthermore, particle size monitoring of CyBT-Fe in water over 14 days also demonstrated its good stability. The experiments show that loading CyBT onto Fe-MIL not only imparts acidic pH responsiveness but also improves its relative stability. This also indicates that we have successfully prepared nanoparticles based on TME released in acidic environments, effectively consuming GSH and catalyzing the production of •OH from H₂O₂.

[0073] Example 4: In vitro tumor cell killing effect

[0074] First, confocal laser scanning microscopy (CLSM) was used to study the cellular uptake of CyBT and CyBT-Fe in MCF-7 cells. We observed negligible fluorescence in MCF-7 cells after 30 minutes of incubation with CyBT and CyBT-Fe, and a significant enhancement of red fluorescence after 120 minutes, indicating that CyBT and CyBT-Fe were effectively internalized into MCF-7 cells within two hours. Subsequently, confocal fluorescence imaging was used to study the subcellular distribution of CyBT and CyBT-Fe. The fluorescence images of MCF-7 cells incubated with CyBT and CyBT-Fe overlapped well with the fluorescence images of ER Tracker-stained cells (Pearson correlation coefficient, PCC: 0.92 for CyBT, 0.93 for CyBT-Fe), indicating strong co-localization of CyBT and CyBT-Fe with the ER Tracker probe. Figure 3 a). This also indicates that CyBT and CyBT-Fe successfully targeted the endoplasmic reticulum (ER) of MCF-7 cells, and that CyBT loading to form CyBT-Fe did not affect their ER targeting. They could still accurately locate the ER when the light intensity was changed. Figure 3 d). The co-localization coefficients of the Golgi apparatus and lysosomes are very small. Next, we evaluated the cytotoxicity of CyBT and CyBT-Fe using the MTT assay and MCF-7 cells under and without 660nm laser irradiation. Under laser irradiation, cell death increased with increasing concentrations of both drugs, as measured by testing the IC50 of CyBT. 50 At 4.46 μg, CyBT-Fe IC 50 2.57 μg ( Figure 3 (b) In contrast, cells without laser irradiation did not show obvious signs of death. Figure 3 c). This demonstrates CyBT-Fe's excellent phototoxicity and extremely low dark toxicity. Subsequently, we also performed cytotoxicity tests on normal cells (GES-1, COS7) at the same concentration range. The results showed that the drug had no significant toxicity to normal cells, exhibiting extremely high biocompatibility and biosafety. To verify the ROS generation capacity of CyBT and CyBT-Fe in cells, we used DCFH-DA to detect intracellular ROS generation in MCF-7 cells after incubation with the drug under light. Strong green fluorescence was observed in the CyBT and CyBT-Fe light-illuminated groups, indicating the generation of a large amount of intracellular ROS (…). Figure 3 e). Simultaneously, SOSG and HPF tests also showed that CyBT and CyBT-Fe produced a large amount of [unclear - possibly referring to a specific chemical process or product] after illumination. 1O2 and ·OH are also major causes of drug cytotoxicity. The stronger green fluorescence of CyBT-Fe compared to CyBT is because the released CyBT produces [something] within the cell upon irradiation. 1 O2, and the released Fe also reacts with endogenous H2O2 to generate hydroxyl radicals and consume GSH, thereby weakening intracellular antioxidant defense and increasing oxidative stress in cancer cells. To further verify the antitumor ability of CyBT-Fe at the cellular level, we used Calcein-AM and propidium iodide (PI) as indicators for live / dead cell co-staining assays. It was observed that the unirradiated CyBT-Fe group showed a certain amount of cell death, while the irradiated CyBT-Fe effectively killed tumor cells (…). Figure 3 f). The reason is that in the intracellular environment, CyBT-Fe consumes GSH and releases iron ions, triggering the Fenton reaction and inducing cell death. When exposed to light, CyBT released by CyBT-Fe acts as a photosensitizer, generating a large amount of ROS, thereby effectively killing tumor cells. Flow cytometry also verified the cell-killing effect of CyBT-Fe, with results consistent with live / dead cell staining (f). Figure 3 g).

[0075] Example 5: In vivo anti-tumor effects and immune activation

[0076] To assess the accumulation of CyBT-Fe in solid tumors, a mouse solid tumor model was established by injecting 4T1 cells into the left axilla of BALB / c mice for in vivo fluorescence imaging. In vivo fluorescence imaging showed that CyBT-Fe effectively accumulated in orthotopic breast tumors 6 hours after tail vein injection. To further investigate its metabolism in vivo, mice were euthanized 12 hours after intravenous injection, and vital organs (heart, liver, spleen, lung, and kidney) and tumor tissues were collected for biodistribution assessment. Fluorescence signals were mainly observed in tumor tissues, but also concentrated in the liver, indicating that the nanomedicine is primarily metabolized by the liver. To increase drug accumulation in tumors and improve drug utilization, we used orthotopic injection to evaluate the in vivo therapeutic effect and immune activation of CyBT-Fe in an established bilateral 4T1 tumor model. Mice were randomly divided into 6 groups: PBS group, PBS+NIR group, CyBT-Fe group, CyBT-Fe+NIR group, CyBT-Fe+SRF group, and CyBT-Fe+SRF+NIR group. Then, the medication was administered systematically via in situ injection (days 0, 4, and 9) and followed by phototherapy. Figure 4a). By recording tumor volume and mouse weight at different time intervals, we were able to monitor tumor growth over time and the effect of treatment on mouse weight. At the end of treatment, mice were euthanized and the tumors of each mouse were excised. As expected, by assessing tumor growth curves and collecting tumor weight, we found that the CyBT-Fe+SRF+NIR group had the lowest tumor size and weight among all groups ( Figure 4 Throughout the treatment period, the tumor volume in all treatment groups was smaller than that in the control group, indicating that all treatment groups exhibited varying degrees of tumor suppression. Importantly, the body weight of mice increased slightly after different treatments. Furthermore, histopathological examination of major organs and tumors by H&E staining revealed no abnormalities in the heart, liver, spleen, lungs, and kidneys, while the intercytoplasmic space of tumor cells in the CyBT-Fe+SRF+NIR group was significantly increased, indicating extensive necrosis in this group. Tumor growth curves, mouse body weight, and H&E staining in mice demonstrate that the combination of NIR+CyBT-Fe and SRF effectively treats tumors with low systemic toxicity. Interestingly, the same tumor-suppressing effect was also observed in the untreated tumor on the other side. Figure 4 (e.g.), this is attributed to the activation of immune mechanisms by endoplasmic reticulum stress and ferroptosis-induced ICD.

[0077] To verify the ability of CyBT-Fe combined with sorafenib (SRF) to induce in vivo ICD and elicit an in vivo immune response, we measured CRT and HMGB1 levels in tumor tissues. Consistent with in vitro results, the CyBT-Fe+NIR group showed significant CRT translocation and a significantly reduced HMGB1 level. The CRT translocation and HMGB1 extranuclear exocytosis were even more pronounced after combining with SRF. Figure 4 h). This indicates that CyBT-Fe phototherapy strongly induces ICD in vivo. Combined with SRF, GPX4 in tumor tissue decreases due to the inhibition of the SLC3A2 / SLC7A11 transporter, leading to a reduction in GSH and thus triggering stronger ferroptosis and a more intense ICD. Measurement of SLC7A11 in tumor tissue showed a significant decrease after combined SRF treatment, while the group without SRF did not show a significant decrease, indicating that combined SRF effectively inhibits the SLC3A2 / SLC7A11 transporter, consistent with expectations. GPX4 levels in tumor tissue were also measured, consistent with in vitro cell experiments. The CyBT-Fe treatment group showed a significant decrease in GPX4, while the level was even lower after combined SRF treatment, indicating a strong ferroptosis response in tumor tissue. SRF more effectively inhibits the SLC3A2 / SLC7A11 transporter, leading to GPX4 downregulation and a more severe ferroptosis response.

[0078] Analysis of proximal tumor samples showed that, compared with the control group, the proportion of mature DCs in the tumors of CyBT-Fe+SRF+NIR mice in the combined treatment group was significantly increased. The proportion of mature DCs increased from 23.06% to 53.75%. Figure 5 a). This indicates that CyBT-Fe combined with SRF treatment activated the immune response and enhanced the anti-tumor immune response. It also has an effect on regulatory T cells (Tregs, immunosuppressive cells, CD25+). + The results of the detection of FOXP3 (a key surface marker and core functional transcription factor of Treg cells) showed that its proportion decreased to 26.39%. Figure 5 (b) indicates that the combination therapy reduced immunosuppression and enhanced the anti-tumor immune response. The same analysis was performed on distant tumors, and the results were consistent with those for proximal tumors. Figure 5 c, d). Simultaneously, immunofluorescence staining showed that the tumor CD4... + T cells and CD8 + T cells also increased significantly, while Treg cells decreased significantly. Figure 5 e), the results still indicate that CyBT-Fe combined with SRF treatment can promote immune activation. Notably, the serum levels of cytokines interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), IL-6, and IL-12 in the combined treatment group were higher than those in other groups. Figure 5 (fi) indicates that the combination of CyBT-Fe nanoparticles and SRF acts as an immune adjuvant, stimulating T cell infiltration and triggering effective and durable immune memory, thereby promoting photodynamic therapy. In summary, the combined treatment of NIR+CyBT-Fe and SRF not only effectively inhibits the primary tumor but also enhances the strong anti-tumor immune capacity, effectively inhibiting the growth of distant tumors. Furthermore, upon re-exposure to the same pathogen, it provides timely immune protection by secreting various cytokines such as TNF-α and IFN-γ.

Claims

1. A type of CyBT-Fe nanoparticle, characterized in that, The preparation method of the nanoparticles includes the following steps: CyBT was dissolved in ethanol, Fe-Mil was added, and after the reaction was complete, the precipitate was collected by centrifugation to obtain the CyBT-Fe nanoparticles. The mass ratio of CyBT to Fe-Mil is 1:100; The Fe-Mil is selected from MIL-88B(Fe); The structure of CyBT is as follows: 。 2. The CyBT-Fe nanoparticles according to claim 1, characterized in that, The average size of the CyBT-Fe nanoparticles is 147.05 ± 1.92 nm.

3. The method for preparing CyBT-Fe nanoparticles according to any one of claims 1-2, characterized in that, Includes the following steps: CyBT was dissolved in ethanol, Fe-Mil was added, and after the reaction was complete, the precipitate was collected by centrifugation to obtain the CyBT-Fe nanoparticles. The mass ratio of CyBT to Fe-Mil is 1:

100.

4. The use of intermediate CyBT in the preparation of the CyBT-Fe nanoparticles according to any one of claims 1-2, wherein the structure of intermediate CyBT is as follows: 。 5. The use of the CyBT-Fe nanoparticles according to any one of claims 1-2 in the preparation of nanoimmunotherapy agents for the treatment of breast cancer.

6. A nano-immunotherapy agent, characterized in that, The nanoimmunotherapy agent uses the CyBT-Fe nanoparticles as described in any one of claims 1-2 as its active ingredient.

7. The nano-immunotherapy agent according to claim 6, characterized in that, This nanoimmunotherapy agent also includes pharmaceutically acceptable excipients.

8. The nano-immunotherapy agent according to any one of claims 6-7, characterized in that, This nanoimmunotherapy agent also includes sorafenib.

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