A zinc-labeled porphyrin molecular cage and its preparation method and application

Abstract

The present invention discloses a zinc-labeled porphyrin molecular cage and a preparation method and application thereof. The zinc-labeled porphyrin molecular cage comprises a cationic cage and a counter anion. The cationic cage can be formed by the reaction of pyridylporphyrin and p-dibenzyl bromide to generate a porphyrin molecular cage and then coordinated with Zn(II). The zinc-labeled porphyrin molecular cage can be used to prepare X-ray irradiation dynamic therapy reagents and tumor treatment drugs. The zinc-labeled porphyrin molecular cage of the present invention is used to improve the energy loss during X-PDT treatment and the complex synthesis process of its photosensitizer, and is used as a triplet exciton generated by direct X-ray excitation for anti-tumor, aiming to promote its clinical transformation.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical drug molecules, and in particular to a zinc-labeled porphyrin molecular cage, a preparation method thereof, and an application thereof in anti-tumor. Background Art

[0002] In recent years, innovative dynamic treatment strategies have been developed using nanosensitizers with unique physicochemical properties to respond to high-penetrating stimuli for the treatment of various deep malignant tumors. They have the advantages of effectiveness, repeatability, flexibility, minimal invasiveness, and the ability to eliminate hidden cancer lesions, and have attracted increasing attention. Among them, photodynamic therapy has been widely studied due to its excellent therapeutic efficacy and minimal toxic side effects. However, in the treatment of solid tumors, it is still limited by many factors, such as: poor tissue penetration of excitation light (<1 cm), low photoconversion efficiency of photosensitizers, and complex synthesis of photosensitizers. Therefore, in order to overcome this limitation, people gradually use X-rays (X-rays) with strong tissue penetration and high photoconversion efficiency instead of excitation light sources to achieve X-ray-induced photodynamic therapy (X-PDT) for cancer.

[0003] The principle of X-PDT is usually to use a physical transducer to absorb X-ray energy and transfer it to a photosensitizer to produce cytotoxic singlet oxygen (OS) necessary for tumor destruction. 1 O2), thereby achieving photodynamic therapy. In this process, the transducer (commonly known as the scintillator) can exhibit X-ray stimulated optical luminescence, converting the absorbed X-ray energy into photons of appropriate wavelengths, which can be efficiently absorbed by the photosensitizer.

[0004] Generally speaking, the scintillation systems commonly used in XPDT currently include anchoring organic photosensitizers on the surface of the scintillator or constructing a metal-organic framework (MOF) structure with heavy metal units and photosensitive ligands. Among them, the research on organic scintillators has a long history. It is mainly composed of low atomic number elements such as hydrogen, carbon, nitrogen, and oxygen. It only produces fluorescence by ionizing and exciting single excitons, but at the same time faces a strong aggregation quenching (ACQ) effect. Inorganic scintillators constructed with heavy metals usually have large toxic side effects and their metabolic pathways in the body are still unclear. In addition, the combination of inorganic scintillators and organic scintillators not only complicates the synthesis of materials, but also undoubtedly leads to huge energy losses in the inefficient two-step energy transfer process.

[0005] The X-PDT strategy has the advantages of deep penetration, low-dose irradiation, and combined therapy, but there are still great challenges in the construction of the strategy, such as: (1) The complex two-step energy transfer inevitably leads to energy loss in X-ray irradiation, which in turn reduces the performance of X-PDT. (2) There is a lack of suitable photosensitizers or scintillators. Therefore, in order to overcome the problems caused by scintillators and the energy loss during energy conversion, it is urgent to develop innovative ionization energy-inducing organic molecules that can be directly activated by X-rays to effectively generate triplet excitons, ultimately achieving efficient X-ray dynamic therapy (X-DT).

[0006] Biomedical photosensitizers have made a lot of significant progress in the basic research of tumor treatment. However, there are still few research results that have been applied in clinic or are in clinical trials. Summary of the invention

[0007] In view of the above technical problems and the shortcomings in the art, the present invention provides a zinc-labeled porphyrin molecular cage and its preparation method and application. The zinc-labeled porphyrin molecular cage of the present invention can be prepared from the most commonly used photosensitizer in clinical practice, and can be prepared by substitution condensation reaction and metal coordination, and is used to improve the energy loss during X-PDT treatment and the complex synthesis process of its photosensitizer, and is used as a triplet exciton generated by direct X-ray excitation for anti-tumor, aiming to promote its clinical transformation.

[0008] Compared with free pyridylporphyrin molecules, the zinc-labeled porphyrin molecular cage (ZPC) obtained by the stepwise reaction of the present invention exhibits excellent X-ray irradiation kinetic characteristics and can efficiently generate hydroxyl radicals (·OH) and 1 Cytotoxic reactive oxygen species such as O2 show better oxidative damage to tumor cells and radiodynamic therapy (X-DT) efficacy.

[0009] [1] A zinc-labeled porphyrin molecular cage, comprising:

[0010] A cationic cage having the structure shown below:

[0011] ;

[0012] and, a counter anion.

[0013] In some embodiments, the zinc-labeled porphyrin molecular cage, the counter anion is PF6 - The counter anion is PF6 - When the zinc-labeled porphyrin molecular cage has good water solubility, it is beneficial for pharmaceutical and therapeutic applications.

[0014] The zinc-labeled porphyrin molecular cage, the cationic cage can be formed by reacting a porphyrin derivative with p-dibenzyl bromide to generate a porphyrin molecular cage and then coordinating with Zn(II). Specifically, the nitrogen atom in the porphyrin molecular ring of the porphyrin derivative coordinates with Zn(II), so that Zn(II) is doped therein.

[0015] The porphyrin derivative has a molecular structure as shown below:

[0016] .

[0017] [2] The method for preparing the zinc-labeled porphyrin molecular cage according to [1] comprises the steps of:

[0018] 1) reacting a porphyrin derivative and p-dibenzyl bromide in a first solvent at 95-105°C for 12-24 hours, generating insoluble matter after the reaction, first concentrating the solution, then adding dichloromethane and distilled water to remove the insoluble matter, separating and collecting the aqueous phase, washing the aqueous phase with dichloromethane, then adding an excess of a counter anion salt, precipitating a solid, collecting it, and drying it to obtain a porphyrin molecular cage;

[0019] The porphyrin derivative has a molecular structure as shown below:

[0020] ;

[0021] 2) reacting the porphyrin molecular cage obtained in step 1) with Zn(II) salt in a second solvent at 75-85° C. for 12-24 hours, spinning dry after the reaction, dissolving with acetonitrile, collecting the aqueous phase after washing with water, adding ether to the aqueous phase to generate a precipitate, collecting it, and drying it to obtain the zinc-labeled porphyrin molecular cage.

[0022] In step 1), the molar ratio of the porphyrin derivative to p-dibenzyl bromide may be 1:16-24.

[0023] In step 1), the first solvent may be dioxane.

[0024] In step 1), the ratio of the mass of the porphyrin derivative to the volume of the first solvent may be 0.3-0.4 g:150 mL.

[0025] In step 1), the counter anion salt may be an ammonium salt of the counter anion.

[0026] In step 2), the mass ratio of the porphyrin molecular cage obtained in step 1) to the Zn(II) salt can be 1:1.8-2.0.

[0027] In step 2), the Zn(II) salt may be zinc acetate.

[0028] In step 2), the second solvent may be acetonitrile and methanol. Furthermore, in the second solvent, the volume ratio of acetonitrile to methanol may be 3:1.

[0029] In step 2), the ratio of the mass of the porphyrin molecular cage obtained in step 1) to the volume of the second solvent can be 0.9-1.1 mg:1 mL.

[0030] [3] Use of the zinc-labeled porphyrin molecular cage described in [1] in the preparation of X-ray irradiation dynamic therapy agents.

[0031] [4] Use of the zinc-labeled porphyrin molecular cage described in [1] in the preparation of a tumor treatment drug. The tumor treatment method may be X-ray irradiation dynamic therapy.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. The present invention selects clinical photosensitizer porphyrin, constructs a porphyrin molecular cage through two simple steps, and then coordinates with zinc to obtain a zinc-labeled porphyrin molecular cage (ZPC).

[0034] 2. Compared with the uncaged porphyrin molecules, the ZPC synthesized in the present invention exhibits better X-ray radiation dynamics performance, showing better active oxygen generation performance and radiation dynamics therapeutic efficacy.

[0035] 3. The introduction of Zn(II) in the present invention brings about good cancer cell uptake performance of the drug ZPC, and ZPC can be effectively enriched in tumor tissues. At the same time, it absorbs X-ray irradiation to produce highly toxic reactive oxygen, showing excellent anti-tumor performance, and has no long-term toxic side effects on the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the chemical characterization hydrogen spectrum of pyridyl porphyrin.

[0037] Figure 2 This is the carbon spectrum of the chemical characterization of pyridyl porphyrin.

[0038] Figure 3 The chemical characterization hydrogen spectrum of ZPC.

[0039] Figure 4 UV spectra of ZPC and unlabeled porphyrin molecular cage (PC).

[0040] Figure 5Schematic diagram of the extracellular X-ray-induced reactive oxygen species production in ZPC, zinc-labeled pyridylporphyrin (ZPTT) and phosphate buffer (PBS), where: a) is a schematic diagram of the determination of hydroxyl radicals (·OH) using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a probe, and b) is a schematic diagram of the determination of hydroxyl radicals (·OH) using tetramethylpiperidone (TEMP) as a probe. 1 O2 schematic diagram.

[0041] Figure 6 The toxicity evaluation diagram of ZPC and PBS on different tumor cells, among which: a) is the survival rate of 4T1 tumor cells after incubation with different concentrations of ZPC for 24 hours without X-ray irradiation, b) is the survival rate of 4T1 tumor cells after incubation with the same concentration of ZPC for 24 hours under different doses of X-ray irradiation, and PBS is used as the control, c) is the survival rate of HepG2 tumor cells after incubation with the same concentration of ZPC for 24 hours under different doses of X-ray irradiation, and PBS is used as the control, d) is the survival rate of CT26 tumor cells after incubation with the same concentration of ZPC for 24 hours under different doses of X-ray irradiation, and PBS is used as the control.

[0042] Figure 7 The figure shows the evaluation of ZPC uptake efficiency by 4T1 tumor cells, where: a) is the localization of mitochondria in tumor cells, measured at different incubation time points using a laser confocal microscope, and b) is the evaluation using flow cytometry at different incubation time points.

[0043] Figure 8 Schematic diagram of the evaluation of ZPC uptake pathway in 4T1 tumor cells.

[0044] Fig. 9 Schematic diagram of the evaluation of the efficiency of reactive oxygen species produced by 4T1 tumor cells after uptake of ZPC and subsequent X-ray irradiation.

[0045] Fig.10 Schematic diagram of the changes in mitochondrial membrane potential of 4T1 tumor cells after treatment with different treatment methods, where: a) is a fluorescence staining image, and b) is a fluorescence ratio schematic diagram.

[0046] Fig.11 Fluorescent staining images of DNA damage in 4T1 tumor cells after different treatments.

[0047] Fig.12 These are co-stained fluorescence images of live cells (green fluorescence) / dead cells (red fluorescence) in 4T1 tumors after treatment with different treatments.

[0048] Fig.13 Schematic diagram of the apoptosis process of 4T1 tumor cells after different treatments.

[0049] Fig.14 Schematic diagram of the growth cycle of 4T1 tumor cells after different treatments.

[0050] Fig.15 Schematic diagram of the blood compatibility evaluation between 4T1 tumor cells and mice after treatment with different treatment methods.

[0051] Fig.16 This is the distribution of ZPC in major organs (heart, liver, spleen, lung, kidney) and tumors of mice 12 hours after ZPC was injected into the mouse body through the tail vein.

[0052] Fig.17 This is a blood cell count analysis chart of experimental mice on the 15th day after being treated with different treatments.

[0053] Fig.18 This is an analysis chart of serum biochemical indicators of experimental mice on the 15th day after treatment with different treatment methods.

[0054] Fig.19 The present invention shows a) a tumor growth curve of tumor-bearing experimental mice during treatment with different methods, b) a photo of the tumors of mice in each experimental group after the treatment, and c) a photo of the tumor-bearing experimental mice on the 15th day after the treatment.

[0055] Fig. 20 These are H&E staining, Ki67 staining and TUNEL staining images of tumor sections of mice in each experimental group after treatment.

[0056] Fig.21 This is an H&E staining analysis of sections of the main organs (brain, heart, liver, spleen, lung and kidney) of tumor-bearing mice after they were treated in different ways according to the present invention. Scale bar: 100 μm.

[0057] Fig. 22 The figure is a curve diagram of weight changes of tumor-bearing experimental mice during the treatment of tumor-bearing mice by different methods according to the present invention. DETAILED DESCRIPTION

[0058] The present invention will be further described below in conjunction with the accompanying drawings and specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The operating methods in the following examples that do not specify specific conditions are usually performed under conventional conditions or under conditions recommended by the manufacturer. All data in the following examples are expressed as mean ± standard deviation. Data comparisons were performed using Student's t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

[0059] Embodiment 1:

[0060] Step 1: Weigh 318.9 mg of porphyrin derivative (pyridylporphyrin) and 2.649 g of dibenzyl bromide, mix and add 150 mL of dioxane, then stir and react at 100 °C for 24 hours. After the reaction, insoluble matter is generated, which is first concentrated; then add appropriate amounts of dichloromethane and distilled water, filter out the insoluble matter, and collect the aqueous phase; finally, wash the aqueous phase three times with dichloromethane, collect the aqueous phase and add excess NH4PF6 to it, at this time a purple solid precipitates, filter and collect the solid and dry it to obtain a purple powder product, that is, the zinc-free porphyrin molecular cage PC.

[0061] Step 2: Weigh 20 mg of the product obtained in step 1, add 38.07 mg of zinc acetate, and finally add 15 mL of acetonitrile and 5 mL of methanol, and stir and react overnight at 80 °C. After the reaction is completed, spin dry, dissolve in acetonitrile, wash and collect the aqueous phase, and finally add ether to the aqueous phase. At this time, a precipitate is generated, which is filtered and dried to obtain a zinc-labeled porphyrin molecular cage, referred to as ZPC.

[0062] The structures of the porphyrin derivative in step 1 and ZPC in step 2 were characterized by H-NMR and C-NMR. The UV-visible absorption spectra of ZPC and PC were measured by UV-2100 spectrophotometer. The production of reactive oxygen species stimulated by extracellular X-rays was determined by electron paramagnetic resonance spectrometer. 5,5-dimethyl-1-pyrroline N-oxide (DMPO: 10 μL) or tetramethylpiperidone (TEMP: 10 μL) was mixed with ZPC solution (1 mL, 7 μM) or ZPTT solution (1 mL, 7 μM) or PBS and added to a 1 mL Eppendorf tube to capture reactive oxygen species (ROS). 4 Gy of X-ray irradiation was given during the process. The preparation method of ZPTT comprises: referring to the above step 2, replacing the product obtained in step 1 with the porphyrin derivative in step 1 to obtain zinc-labeled pyridylporphyrin ZPTT.

[0063] like Figure 1 and Figure 2 As shown in Figure 1, the precursor material of the porphyrin molecular cage, pyridyl porphyrin, has a suitable structure and no impurity molecules. Figure 3 As shown in Figure 2, ZPC was successfully synthesized and the zinc atom was successfully introduced. Figure 4 As shown in Figure 2, after ultraviolet spectrum analysis of ZPC, it is known that the molecular cage can be excited by an excitation wave of 520 nm and has a good fluorescence emission peak in the wavelength range of 550-570 nm. Figure 5 As shown in the figure, the results of the evaluation of the performance of X-ray induced reactive oxygen species production show that compared with uncaged porphyrin molecules, ZPC can efficiently produce reactive oxygen species under the action of X-rays, among which Figure 5In a), when DMPO is used as the active oxygen scavenger, ZPC shows obvious characteristic peaks of ·OH (peak intensity ratio: 1:2:2:1). Figure 5 In b), ZPC showed obvious 1 Characteristic peaks of O2 (peak intensity ratio: 1:1:1).

[0064] Example 2: Cell experiment.

[0065] Using mouse breast cancer 4T1 cells as a research model, the application of ZPC materials in tumor X-ray radiodynamic therapy was further explored.

[0066] (1) Cytotoxicity assay

[0067] The cell proliferation toxicity was detected using the CCK8 kit. CT26, HepG2 and 4T1 cells were seeded into 96-well plates and cultured overnight. Then, the cells were treated with different concentrations of ZPC (0, 2, 4, 6, 8, 10 μM) for 12 hours, and then the medium containing the drug was removed and replaced with fresh complete medium. Then, different doses of X-rays (0, 1, 2, 4 Gy) were irradiated. After the irradiation, the cells were cultured for 24 hours. Finally, CCK-8 reagent was added to each well and cultured at 37 °C for 1.5 hours. After the incubation, the absorbance (OD value) at 450 nm was measured using an enzyme reader. The cell survival rate of the blank control group was 100% as a control. The cell viability of each experimental group was calculated based on the absorbance ratio of each material experimental group to the blank control group. Figure 6 As shown in the figure, 4T1 cells still maintained about 80% survival when the ZPC dosage reached 10 μM, indicating that it has good safety in the absence of X-ray irradiation. Therefore, in subsequent experiments, we chose 7 μM ZPC as the optimal concentration. Then, the cytotoxicity of ZPC under X-ray irradiation was studied. The results showed that with the increase of X-ray dose intensity, the relative survival rate of 4T1 cells gradually decreased. When the X-ray irradiation dose reached 4 Gy, the survival rate of 4T1 cells was only about 20%, which shows that ZPC has good X-ray radiation dynamics performance, see Figure 6 b). Secondly, in order to better prove that ZPC has strong cell killing ability under X-ray irradiation, HepG2 cells and CT26 cells were selected. The results showed that the survival rate of cells after the same treatment was less than 60% and there was a dependence on the X-ray irradiation dose, see Figure 6 c) Figure 6 d) in.

[0068] (2) Cell uptake experiment

[0069] 4T1 cells were seeded into confocal culture dishes (1 × 105 Cells / dish) were incubated with ZPC (7 µM) for 1, 2, 4, and 8 hours. Cell nuclei were stained with Hoechst 33342. Finally, laser confocal microscopy and flow cytometry were used for analysis. Mitochondria were located to demonstrate the distribution of ZPC in cancer cells. The results showed that as the culture time of 4T1 cells increased, the fluorescence intensity of ZPC gradually increased and gradually overlapped with the green fluorescence of mitochondria, with a Pearson coefficient of 0.703. The maximum uptake was reached after 8 hours of incubation. Figure 7 At the same time, we further verified the above results using flow cytometry and quantified the fluorescence intensity of intracellular ZPC, and found that it had the same uptake results as the confocal test, see Figure 7 b in the figure). Finally, the uptake mechanism of cancer cells in the 4T1 cell model was analyzed. The cells were incubated with cytochalasin D (Cyt-D), chlorpromazine (CPZ), filipin, and wortm at 4 °C for 30 minutes, followed by the addition of CPZ and incubation for 8 hours before flow cytometry analysis. The results showed that temperature had the strongest ability to inhibit uptake, followed by chlorpromazine, cytochalasin D, and filipin, while the inhibitory effect of wortm was relatively low. This phenomenon is mainly attributed to the fact that temperature acts on energy regulation, while chlorpromazine and cytochalasin D are used to regulate clathrin- and actin-mediated endocytosis. Therefore, it is shown that the uptake of CPZ by 4T1 cells is mainly regulated by energy and cell surface proteins ( Figure 8 ).

[0070] (3) Evaluation of oxidative damage effects on 4T1 tumor cells

[0071] A. Generation of ROS in 4T1 tumor cells. Cells were first cultured with ZPC for 12 h, then the original culture medium was removed, 1 mL of DCFH-DA (7 μM) solution was added to each well and cultured for 20 min, while X-ray irradiation (4 Gy) was given. Finally, the cells were rinsed three times with PBS to remove excess DCFH-DA, and fluorescence images were captured using a confocal laser scanning microscope (CLSM). Fig. 9 As shown in the figure, compared with the PBS group and the X-ray group, the DCFH fluorescence signal of the ZPC group and the ZPC+X-ray group was enhanced, and the reactive oxygen species produced increased significantly. Quantitative analysis showed that the average fluorescence intensity of DCFH in the ZPC+X-ray group was 52.68%, which was 15 times that of the PBS group. At the same time, under X-ray irradiation, the average fluorescence intensity of DCFH in the ZPC+X-ray group was 1.3 times that of the ZPC group, indicating that X-ray effectively promoted ZPC to produce highly toxic reactive oxygen species in cells.

[0072] B. Evaluation of changes in mitochondrial membrane potential in 4T1 tumor cells. 4T1 cells were seeded into 96-well plates (7×10 3 After culturing for 24 hours, the original culture medium was removed and the culture medium containing ZPC was added to continue culturing for 12 hours, while irradiating with 4 Gy X-rays. After the culture was completed, the culture medium was removed and 100 μL of diluted JC-1 staining solution was added to each well for 30 minutes. Finally, the cells were washed three times with buffer and 100 μL of culture medium was added for detection. Fig.10 As shown in a), a stronger JC-1 monomer fluorescence signal was found in the ZPC+X-ray group, while the JC-1 dimer fluorescence signal was very weak, indicating that the mitochondria were damaged. On the other hand, the analysis of the fluorescence ratio (R / G) of JC-1 dimer (red) to JC-1 monomer (green) showed that the R / G ratio gradually decreased in different treatment groups, which means a decrease in mitochondrial membrane potential and an increase in oxidative stress in cancer cells, see Fig.10 b) in the above.

[0073] C. Evaluation of DNA oxidative damage in 4T1 tumor cells. 4T1 cells were inoculated in confocal culture dishes (1×10 5 The cells were then treated with drugs (ZPC, 7 µM) and X-ray irradiation (4 Gy) for 24 hours. After the incubation, the cells were stained according to the γ-H2AX immunofluorescence method. Finally, the staining solution was aspirated and the cells were washed three times with washing solution for confocal microscopy analysis. Fig.11 As shown, the green fluorescence of γ-H2AX represents DNA damage, and the DNA damage of cells is different after different treatments. In the blank control group, the DNA of the cells was not found to be damaged, but after single X-ray irradiation or ZPC treatment, the DNA was partially damaged. Finally, under the combined action of X-ray irradiation and ZPC, the DNA of tumor cells was severely damaged, and γ-H2AX showed a strong green fluorescence signal. This phenomenon is mainly attributed to the fact that ZPC produces more reactive oxygen after absorbing the energy of X-rays, further promoting DNA oxidative damage.

[0074] (4) Evaluation of 4T1 tumor cell killing effect

[0075] A. Live / dead staining analysis of 4T1 tumor cells: 4T1 cells were seeded into confocal culture dishes (1×10 5cells / dish), and then treated with drugs (ZPC, 7 µM) and X-ray irradiation (4 Gy) for 24 hours. After the culture, the culture medium was removed, and 1 mL of staining solution containing calcein-AM (2 μM) and iodide propidium (PI, 4 μM) was added to stain the cells at room temperature for 30 minutes. Finally, the staining solution was removed, the cells were rinsed three times with PBS, and analyzed using a laser confocal microscope. Fig.12 As shown in the figure, we observed significant changes in cell survival rate under different treatment conditions. Specifically, the cells in the control group grew well. On the contrary, after single X-ray or ZPC treatment, some cells died and the mortality rate was about 10%. It is worth noting that under the combined action of X-rays and ZPC, a large number of cells died, with a mortality rate of up to 90%.

[0076] B. Evaluation of 4T1 tumor cell apoptosis. Annexin V-FITC / PI staining was used for staining analysis. 4T1 cells were inoculated into 6-well culture plates (1×10 5 cells / well) for 24 hours, then ZPC (7 µM) was added and incubated for 12 hours, followed by 4 Gy of X-ray irradiation and incubation for 24 hours. Then, the cells were washed three times with PBS, stained with Annexin V-FITC / PI for 30 minutes, and then analyzed by fluorescence. For the apoptotic cycle, unlike apoptosis, the cells were first fixed with ethanol and then stained with PI for analysis. Fig.13 As shown, flow cytometry analysis of cell apoptosis showed that the cells gradually tended to the late apoptosis phase (Q2) after treatment, with a mortality rate gradually increasing from 0.382% to 16.66%. At the same time, the cell growth cycle showed that in the ZPC+X-ray group, cell division was blocked in the G1 phase, making it impossible for cells to divide and proliferate normally, ultimately inhibiting cell growth and promoting apoptosis ( Fig.14 ).

[0077] Example 3: Animal experiments.

[0078] Experimental animal model: Female BALB / c mice (weight 16-18 g) were purchased from the Experimental Animal Center of Zhejiang University.

[0079] Tumor model: 4T1 tumor cells were injected subcutaneously into the right hind hip of the experimental mice. When the tumor volume reached 80-100 mm 3 BALB / c mice bearing 4T1 subcutaneous tumors were used for animal experiments.

[0080] All animal experiments were in compliance with the animal protection regulations approved by the Experimental Animal Use Committee and the Experimental Animal Ethics Committee of Zhejiang University.

[0081] (1) Biological toxicity of material ZPC

[0082] 1 mL of red blood cell suspension (0.2%, v / v) was mixed with 1 mL of ZPC of different concentrations and incubated at 37 °C for 4 hours. After centrifugation at 3000 rpm for 5 minutes, the optical density (OD) of each solution at a wavelength of 540 nm was measured using a microplate reader. The hemolysis rate was calculated as follows:

[0083]

[0084] like Fig.15 As shown in the figure, compared with the positive control group (water), ZPC showed no hemolysis in the concentration range of 0-200 μM. The hemolysis rate at the corresponding concentration was then calculated, which was all below 5%, which was in line with the allowable limit of the hemolysis test (<5%), indicating that ZPC has good blood compatibility. Materials Evaluation of the distribution of ZPC in mice Female BALB / c mice (5-6 weeks old, about 18 g each) were inoculated with 4T1 cells (7.5×10 per mouse) 5 cells). On day 10, treatment was started by intravenous injection of ZPC, and the distribution of ZPC in mice was observed and photographed at 0, 1, 2, 4, 8, 12, 24, and 36 hours. Subsequently, major organs were harvested for in vitro imaging analysis, such as Fig.16 As shown in the figure, ZPC is mainly distributed in tumor tissue and liver in mice, which indicates that ZPC can be effectively enriched in tumor tissue and may eventually be metabolized by liver tissue. Finally, PBS or ZPC (7 μM, 100 μL) was injected into the experimental mice (n = 3) through the tail vein; serum and whole blood samples were collected on the 3rd and 7th days after injection for biochemical analysis and blood cell analysis. The concentration or content of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), serum albumin (ALB), and blood urea nitrogen (UREA) in the serum of experimental mice was measured using a fully automatic biochemical analyzer BS-220. The blood counts of white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets (PLT), mean platelet volume (MPV), platelet distribution width (PDW), platelet packed volume (PCT), and platelet ratio (P-LCR) in the blood of experimental mice before and after injection of nanomaterials were measured using a blood cell analyzer BC-2600. Fig.17 The concentration or content of each biological enzyme shown and Fig.18As shown in the blood cell count or concentration, there was no significant change in the biochemical indices and blood cell count of the experimental mice before and after administration, indicating that the ZPC material used did not cause obvious damage to the body functions and blood cells of the experimental mice, had low biological toxicity, and had a certain degree of biological application safety.

[0085] (2) Treatment effect of 4T1 tumors

[0086] Female BALB / c mice (5-6 weeks old, approximately 18 g each) were inoculated with 4T1 cells (7.5 × 10 5 cells). On the 7th day, mice bearing the 4T1 tumor model were randomly divided into 4 groups (PBS (blank control), X-ray, ZPC, ZPC+X-ray; n = 5 / group), and on the 8th day, intravenous injection of ZPC (7 μM, 100 μL) was started. 12 hours after injection, mice in the X-ray group and ZPC+X-ray group received X-ray irradiation treatment (1.5 Gy), and then the body weight and tumor size of the mice were collected every three days. After 15 days of treatment, tumors in all treatment groups were harvested and subjected to H&E staining, TUNEL staining, and Ki67 staining. In addition, the main organs of the mice (heart, liver, spleen, lung, and kidney) were harvested and subjected to H&E staining for histological analysis. Fig.19 As shown, during the entire treatment period, the ZPC+X-ray group showed the most significant inhibition of tumor growth and reduction of tumor weight, with a tumor inhibition rate of 92.13%. In contrast, the tumor growth trends of mice in the PBS, X-ray, and ZPC groups were similar, indicating a lower antitumor effect. In addition, hematoxylin and eosin (H&E) staining was used for histopathological analysis of the extracted tumor tissues. Fig. 20 As shown, compared with the control group, the ZPC+X-ray group had the most extensive damage in terms of tumor cell density, cell morphology regularity, and cell nuclear clarity. On the other hand, TUNEL staining and Ki67 staining further confirmed that cell proliferation in the ZPC+X-ray group was significantly reduced and apoptosis was significantly enhanced. The above results clearly show that ZPC can enhance cytotoxicity in tumor tissues after X-ray irradiation without the need for additional synergistic drugs, and can achieve significant anti-tumor effects.

[0087] After 15 days of treatment, all experimental mice were killed by cervical dislocation, and the main tissues and organs were removed by dissection: heart, liver, spleen, lung and kidney. The tissue sections were stained with H&E for histological analysis. Fig.21 As shown, no pathological changes occurred in the major organs of all experimental groups, indicating that these treatments did not cause significant damage to normal organs. In addition, during the entire treatment monitoring period, the experimental mice in all experimental groups did not show significant weight loss ( Fig. 22). This further confirms that the ZPC and treatment methods used in this example will not cause significant biological toxicity.

[0088] In summary, the present invention uses the clinically used photosensitizer porphyrin as a precursor, and prepares a Zn(II)-coordinated porphyrin molecular cage through a simple reaction. Compared with the precursor porphyrin molecule, the molecular cage ZPC has excellent X-ray radiation dynamics and can be better applied to cancer dynamics therapy. Using mouse breast cancer (4T1) as a research model, in in vitro cell experiments and in vivo tumor diagnosis and treatment experiments, the ZPC molecular cage showed high 4T1 cell uptake and 4T1 tumor inhibition ability, and efficiently mediated low-dose X-ray irradiation dynamics therapy of tumors, effectively inhibiting tumor growth and ultimately eliminating tumors. Because it uses clinical diagnostic and therapeutic agents as precursors, the molecular cage ZPC shows excellent biocompatibility and biological application safety, and has broad prospects for clinical transformation and application.

[0089] In addition, it should be understood that after reading the above description of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A zinc-labeled porphyrin molecular cage, characterized in that: It is composed of a cation cage and a counter anion; The cationic cage has the following structure: ； The counter anion is PF6 - .

2. The method for preparing the zinc-labeled porphyrin molecular cage according to claim 1, characterized in that: The cationic cage is formed by reacting a porphyrin derivative with p-dibenzyl bromide to generate a porphyrin molecular cage, which is then coordinated with Zn(II); The porphyrin derivative has a molecular structure as shown below: 。 3. The method for preparing the zinc-labeled porphyrin molecular cage according to claim 2, characterized in that: The method for preparing the zinc-labeled porphyrin molecular cage comprises the steps of: 1) reacting a porphyrin derivative and p-dibenzyl bromide in a first solvent at 95-105°C for 12-24 hours, generating insoluble matter after the reaction, first concentrating the solution, then adding dichloromethane and distilled water to remove the insoluble matter, separating and collecting the aqueous phase, washing the aqueous phase with dichloromethane, then adding an excess of a counter anion salt, precipitating a solid, collecting it, and drying it to obtain a porphyrin molecular cage; The porphyrin derivative has a molecular structure as shown below: ； 2) reacting the porphyrin molecular cage obtained in step 1) with Zn(II) salt in a second solvent at 75-85° C. for 12-24 hours, spinning dry after the reaction, dissolving with acetonitrile, collecting the aqueous phase after washing with water, adding ether to the aqueous phase to generate a precipitate, collecting it, and drying it to obtain the zinc-labeled porphyrin molecular cage.

4. The method for preparing the zinc-labeled porphyrin molecular cage according to claim 3, characterized in that: In step 1): The molar ratio of the porphyrin derivative to p-dibenzyl bromide is 1:16-24; The first solvent is dioxane; The ratio of the mass of the porphyrin derivative to the volume of the first solvent is 0.3-0.4 g:150 mL; The counter anion salt is an ammonium salt of the counter anion.

5. The method for preparing the zinc-labeled porphyrin molecular cage according to claim 3, characterized in that: In step 2): Step 1) The mass ratio of the obtained porphyrin molecular cage to the Zn(II) salt is 1:1.8~2.0; The Zn(II) salt is zinc acetate; The second solvent is acetonitrile and methanol; in the second solvent, the volume ratio of acetonitrile to methanol is 3:1; The ratio of the mass of the porphyrin molecular cage obtained in step 1) to the volume of the second solvent is 0.9-1.1 mg:1 mL.

6. Use of the zinc-labeled porphyrin molecular cage according to claim 1 in the preparation of an X-ray irradiation dynamic therapy agent.

7. Use of the zinc-labeled porphyrin molecular cage according to claim 1 in preparing tumor therapeutic drugs.

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