Zinc-labeled porphyrin molecular cage as well as preparation method and application thereof

Abstract

The invention discloses a zinc-labeled porphyrin molecular cage as well as a preparation method and application thereof. The zinc-labeled porphyrin molecular cage comprises a cation cage and a counter anion. And the cation cage can be formed by reacting pyridyl porphyrin with p-dibenzyl bromide to generate a porphyrin molecular cage and then coordinating with Zn (II). The zinc-labeled porphyrin molecular cage can be used for preparing X-ray irradiation dynamic therapeutic reagents and tumor therapeutic drugs. The zinc-labeled porphyrin molecular cage is used for improving energy loss in an X-PDT treatment process and a complex synthesis process of a photosensitive preparation of the zinc-labeled porphyrin molecular cage, and the zinc-labeled porphyrin molecular cage is used as an X-ray to directly excite to generate triplet excitons for resisting tumors and aims at promoting clinical transformation of the zinc-labeled porphyrin molecular cage.

Description

Technical Field

[0001] The present 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 treatment. Background Art

[0002] In recent years, the development of innovative dynamic therapeutic strategies utilizing nanosensitizers with unique physicochemical properties that respond to highly penetrating stimuli has garnered increasing attention for the treatment of various deep-seated malignancies. These strategies offer advantages such as effectiveness, reproducibility, flexibility, minimal invasiveness, and the potential to eliminate latent cancer lesions. Photodynamic therapy (PDT) has been extensively studied due to its exceptional therapeutic efficacy and minimal toxicity. However, its treatment of solid tumors remains limited by numerous factors, including poor tissue penetration of the excitation light (<1 cm), low photoconversion efficiency of the photosensitizer, and complex synthesis processes. To overcome these limitations, researchers have gradually replaced the excitation light source with X-rays (X-rays), which have strong tissue penetration and high photoconversion efficiency, resulting in 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 In this process, the transducer (often called a scintillator) can exhibit X-ray-stimulated optical luminescence, converting the absorbed X-ray energy into photons of appropriate wavelengths that can be efficiently absorbed by the photosensitizer.

[0004] Generally speaking, the scintillation systems commonly used in XPD involve anchoring organic photosensitizers on the scintillator surface or constructing metal-organic framework (MOF) structures with heavy metal units and photosensitizing ligands. Organic scintillators have a long history of research. They are primarily composed of low-atomic-number elements such as hydrogen, carbon, nitrogen, and oxygen, and generate fluorescence solely through ionization and excitation of single excitons, but they also suffer from strong aggregation quenching (ACQ) effects. Inorganic scintillators constructed from heavy metals often have significant toxic side effects and their metabolic pathways in the body are still unclear. Furthermore, combining inorganic and organic scintillators not only complicates material synthesis but also inevitably leads to significant 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 significant progress in basic research on tumor treatment. However, few of these research results have been applied clinically or are currently in clinical trials. Summary of the Invention

[0007] To address the aforementioned technical issues and shortcomings in the art, the present invention provides a zinc-labeled porphyrin molecular cage, its preparation method, and its application. The zinc-labeled porphyrin molecular cage of the present invention can be prepared from the most commonly used clinical photosensitizers, via substitution condensation reactions and metal coordination. It is used to mitigate energy loss during X-PDT treatment and the complex synthesis of its photosensitizers. It directly excites X-rays to generate triplet excitons for anti-tumor effects, aiming to promote its clinical translation.

[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 kinetics and can efficiently generate hydroxyl radicals (·OH) and 1 Cytotoxic reactive oxygen species such as O2 have shown better oxidative damage to tumor cells and radiodynamic therapy (X-DT) efficacy.

[0009] [1] A zinc-labeled porphyrin molecular cage, comprising: A cationic cage having the structure shown below: ; and, a counter anion.

[0010] In some embodiments, the zinc-labeled porphyrin molecular cage, the counter anion is PF6 - The counter anion is PF6 - At the same time, the zinc-labeled porphyrin molecular cage has good water solubility, which is beneficial for pharmaceutical and therapeutic applications.

[0011] The zinc-labeled porphyrin molecular cage can be formed by reacting a porphyrin derivative with p-dibenzyl bromide to form a porphyrin molecular cage, which is then coordinated with Zn(II). Specifically, the nitrogen atom in the porphyrin molecular ring of the porphyrin derivative coordinates with Zn(II), allowing Zn(II) to be doped therein.

[0012] The porphyrin derivative has the following molecular structure: .

[0013] [2] The method for preparing the zinc-labeled porphyrin molecular cage according to [1] 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. After the reaction, insoluble matter is generated. The reaction is first concentrated, and then dichloromethane and distilled water are added to remove the insoluble matter. The aqueous phase is separated and collected, and the aqueous phase is washed with dichloromethane. An excess of a counteranion salt is then added to precipitate a solid, which is then collected and dried to obtain a porphyrin molecular cage. The porphyrin derivative has the following molecular structure: ; 2) reacting the porphyrin molecular cage obtained in step 1) with a Zn(II) salt in a second solvent at 75-85° C. for 12-24 hours. After the reaction, the mixture is dried by rotary evaporation, dissolved in acetonitrile, washed with water, and the aqueous phase is collected. Ether is added to the aqueous phase to generate a precipitate, which is collected and dried to obtain the zinc-labeled porphyrin molecular cage.

[0014] In step 1), the molar ratio of the porphyrin derivative to p-dibenzyl bromide can be 1:16-24.

[0015] In step 1), the first solvent may be dioxane.

[0016] 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.

[0017] In step 1), the counter anion salt may be an ammonium salt of the counter anion.

[0018] 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.

[0019] In step 2), the Zn(II) salt may be zinc acetate.

[0020] In step 2), the second solvent may be acetonitrile and methanol. Furthermore, the volume ratio of acetonitrile and methanol in the second solvent may be 3:1.

[0021] 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.

[0022] [3] Application of the zinc-labeled porphyrin molecular cage described in [1] in the preparation of X-ray irradiation dynamic therapy agents.

[0023] [4] Use of the zinc-labeled porphyrin molecular cage according to [1] in the preparation of a tumor treatment drug. The tumor treatment method may be X-ray irradiation dynamic therapy.

[0024] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention selects clinical photosensitizer porphyrin and constructs a porphyrin molecular cage through a two-step simple reaction, which is then coordinated with zinc to obtain a zinc-labeled porphyrin molecular cage (ZPC).

[0025] 2. Compared with the non-caged 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.

[0026] 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 tissue. At the same time, it absorbs X-ray irradiation to produce highly toxic reactive oxygen species, showing excellent anti-tumor performance without causing long-term toxic side effects to the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the chemical characterization hydrogen spectrum of pyridyl porphyrin.

[0028] Figure 2 This is the chemical characterization carbon spectrum of pyridyl porphyrin.

[0029] Figure 3 This is the chemical characterization hydrogen spectrum of ZPC.

[0030] Figure 4 UV spectra of ZPC and unlabeled porphyrin molecular cage (PC).

[0031] Figure 5 Schematic 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.

[0032] Figure 6The figures are for evaluating the toxicity of ZPC and PBS on different tumor cells, where: 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, with PBS 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, with PBS 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, with PBS as the control.

[0033] Figure 7 The diagram below 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; b) is the evaluation using flow cytometry at different incubation time points.

[0034] Figure 8 Schematic diagram of the evaluation of ZPC uptake pathway in 4T1 tumor cells.

[0035] Figure 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.

[0036] Figure 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 diagram.

[0037] Figure 11 Fluorescent staining images of DNA damage in 4T1 tumor cells after treatment with different methods.

[0038] Figure 12 These are co-stained fluorescence images of live cells (green fluorescence) and dead cells (red fluorescence) in 4T1 tumors after treatment with different treatments.

[0039] Figure 13 Schematic diagram of the apoptosis process of 4T1 tumor cells after treatment with different treatment methods.

[0040] Figure 14 Schematic diagram of the growth cycle of 4T1 tumor cells after treatment with different treatment methods.

[0041] Figure 15 Schematic diagram of the blood compatibility evaluation between 4T1 tumor cells and mice after treatment with different treatment methods.

[0042] Figure 16This 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 via the tail vein.

[0043] Figure 17 This is the blood cell count analysis chart of experimental mice on the 15th day after treatment with different treatment methods.

[0044] Figure 18 This is an analysis chart of serum biochemical indicators of experimental mice on the 15th day after treatment with different treatment methods.

[0045] Figure 19 Figure 2 shows the tumor growth curve of the experimental mice during the treatment with different methods in the present invention, a) the tumor growth curve of the tumor-bearing experimental mice, b) the photos of the tumors of the mice in each experimental group after the end of treatment, and c) the photos of the tumor-bearing experimental mice after the 15th day of treatment.

[0046] Figure 20 These are H&E staining, Ki67 staining, and TUNEL staining images of tumor sections of mice in each experimental group after treatment.

[0047] Figure 21 These are H&E staining analysis images of sections of the main organs (brain, heart, liver, spleen, lung, and kidney) of tumor-bearing mice after different treatments in the present invention. Scale: 100 μm.

[0048] Figure 22 The figure is a curve diagram of weight changes of tumor-bearing experimental mice during the treatment with different methods according to the present invention. DETAILED DESCRIPTION

[0049] The present invention is further described below with reference to the accompanying drawings and specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or according to the conditions recommended by the manufacturer. All data in the following examples are expressed as mean ± standard deviation. Data were compared using the Student's t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

[0050] Example 1:

[0051] Step 1: Weigh 318.9 mg of a porphyrin derivative (pyridylporphyrin) and 2.649 g of p-dibenzyl bromide, mix them, add 150 mL of dioxane, and then stir and react at 100 °C for 24 hours. After the reaction, insoluble matter is generated, which is first concentrated; then add an appropriate amount 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 an excess of 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.

[0052] Step 2: Weigh 20 mg of the product from Step 1 and add 38.07 mg of zinc acetate. Finally, add 15 mL of acetonitrile and 5 mL of methanol. Stir and react at 80°C overnight. After the reaction, spin dry, dissolve in acetonitrile, wash with water, and collect the aqueous phase. Finally, add diethyl ether to the aqueous phase to form a precipitate. Filter and dry to obtain the zinc-labeled porphyrin molecular cage, referred to as ZPC.

[0053] The structures of the porphyrin derivative in step 1 and ZPC in step 2 were characterized by H-NMR and C-NMR spectroscopy. The UV-visible absorption spectra of ZPC and PC were measured using a UV-2100 spectrophotometer. Extracellular X-ray-induced reactive oxygen species (ROS) production was assayed using electron paramagnetic resonance spectroscopy. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO: 10 μL) or tetramethylpiperidone (TEMP: 10 μL) was mixed with a ZPC solution (1 mL, 7 μM) or a ZPTT solution (1 mL, 7 μM) or PBS and added to a 1 mL Eppendorf tube to capture reactive oxygen species (ROS). During this process, 4 Gy of X-ray irradiation was administered. The preparation method for ZPTT involved following the procedure described above for step 2, but substituting the porphyrin derivative from step 1 for the product, to obtain zinc-labeled pyridylporphyrin ZPTT.

[0054] like Figure 1 and Figure 2 As shown in Figure 2, 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 zinc atoms were successfully introduced. Figure 4 As shown in the figure, after ultraviolet spectrum analysis of ZPC, it is known that the molecular cage can be excited by 520 nm excitation wave 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 active oxygen production show that compared with the uncaged porphyrin molecules, ZPC can efficiently produce active oxygen under the action of X-rays. Figure 5In a), ZPC showed obvious characteristic peaks of ·OH (peak intensity ratio: 1:2:2:1) when DMPO was used as active oxygen scavenger. Figure 5 In b), ZPC showed obvious 1 Characteristic peaks of O2 (peak intensity ratio: 1:1:1).

[0055] Example 2: Cell experiment.

[0056] Using mouse breast cancer 4T1 cells as a research model, the application of ZPC materials in tumor X-ray radiodynamic therapy was further explored.

[0057] (1) Cytotoxicity assay 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 a microplate 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 Figure 2, 4T1 cells still maintained approximately 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 selected 7 μM ZPC as the optimal concentration. The cytotoxicity of ZPC under X-ray irradiation was then 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, to better demonstrate the strong cell-killing ability of ZPC under X-ray irradiation, HepG2 cells and CT26 cells were selected. The results showed that after the same treatment, the cell survival rate was less than 60% and was dependent on the X-ray irradiation dose. Figure 6 c) Figure 6 d) in the above example.

[0058] (2) Cell uptake experiment 4T1 cells were seeded into confocal culture dishes (1 × 10 5Cells were cultured in 4T1 cells (4T1 cells / dish) and incubated with ZPC (7 µM) for 1, 2, 4, and 8 hours. Cell nuclei were stained with Hoechst 33342. Finally, analysis was performed using laser confocal microscopy and flow cytometry. Mitochondria were located to demonstrate the distribution of ZPC within cancer cells. The results showed that with prolonged culture time, the fluorescence intensity of ZPC gradually increased and overlapped with the green fluorescence of mitochondria, with a Pearson coefficient of 0.703. 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 the uptake results were the same as those of the confocal microscopy test, see Figure 7 b in the figure). Finally, the uptake mechanism of cancer cells was analyzed using the 4T1 cell model. Cytochalasin D (Cyt-D), chlorpromazine (CPZ), filipin, and wortm were incubated at 4 °C for 30 minutes, followed by the addition of CPZ and incubation for 8 hours for 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 ).

[0059] (3) Evaluation of oxidative damage effects on 4T1 tumor cells A. To investigate ROS generation in 4T1 tumor cells, cells were first cultured with ZPC for 12 hours. The original culture medium was then removed, and 1 mL of DCFH-DA (7 μM) solution was added to each well and incubated for 20 minutes. The cells were then irradiated with X-rays (4 Gy). 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). Figure 9 As shown, compared with the PBS and X-ray groups, the DCFH fluorescence signal in the ZPC and ZPC+X-ray groups was enhanced, and the production of reactive oxygen species was significantly increased. Quantitative analysis showed that the average DCFH fluorescence intensity in the ZPC+X-ray group was 52.68%, 15 times that of the PBS group. Furthermore, under X-ray irradiation, the average DCFH fluorescence intensity in the ZPC+X-ray group was 1.3 times that of the ZPC group, indicating that X-ray effectively promotes the production of highly toxic reactive oxygen species in ZPC cells.

[0060] B. Evaluation of changes in mitochondrial membrane potential in 4T1 tumor cells. 4T1 cells were seeded into 96-well plates (7 × 103 After culturing for 24 hours, the original culture medium was removed and culture medium containing ZPC was added to continue culturing for 12 hours, while irradiating with 4 Gy X-rays. After the incubation period, the culture medium was removed and 100 μL of diluted JC-1 staining solution was added to each well and stained for 30 minutes. Finally, the cells were washed three times with buffer and 100 μL of culture medium was added for detection. Figure 10 As shown in (a), a stronger JC-1 monomer fluorescence signal was observed in the ZPC+X-ray group, while the JC-1 dimer fluorescence signal was very weak, indicating mitochondrial damage. Furthermore, 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 the different treatment groups, indicating a decrease in mitochondrial membrane potential and enhanced oxidative stress in cancer cells (see [1]). Figure 10 b) in the above example.

[0061] C. Evaluation of DNA oxidative damage in 4T1 tumor cells. 4T1 cells were seeded in confocal microplates (1×10 5 Cells were incubated with 400 μg of ZPC (100 μg / dish) for 24 hours. The cells were then treated with drugs (ZPC, 7 μM) and X-ray irradiation (4 Gy). After incubation, the cells were stained using the γ-H2AX immunofluorescence method. Finally, the staining solution was aspirated and the cells were washed three times with washing solution before analysis by confocal microscopy. Figure 11 As shown, the green fluorescence of γ-H2AX indicates DNA damage, and the extent of DNA damage varies after cells are treated differently. In the blank control group, no DNA damage was observed. However, after either X-ray irradiation or ZPC treatment alone, partial DNA damage was observed. Finally, under the combined effects of X-ray irradiation and ZPC, tumor cell DNA was severely damaged, with a strong green fluorescence signal from γ-H2AX. This phenomenon is primarily attributed to the fact that ZPC, after absorbing X-ray energy, generates more reactive oxygen species, further promoting oxidative DNA damage.

[0062] (4) Evaluation of 4T1 tumor cell killing effect A. Live / dead staining analysis of 4T1 tumor cells: 4T1 cells were seeded into confocal culture dishes (1×10 5 Cells were cultured for 24 hours and then treated with drugs (ZPC, 7 µM) and X-ray irradiation (4 Gy). After the culture was complete, the culture medium was removed and 1 mL of staining solution containing calcein-AM (2 µM) and propidium iodide (PI, 4 µM) was added. The cells were stained for 30 minutes at room temperature. Finally, the staining solution was removed, the cells were rinsed three times with PBS, and analyzed using a laser confocal microscope. Figure 12As 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. In contrast, after single X-ray or ZPC treatment, some cells died with a mortality rate of 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%.

[0063] B. Apoptosis evaluation of 4T1 tumor cells. Annexin V-FITC / PI staining was used for staining analysis. 4T1 cells were seeded into 6-well culture plates (1×10 5 Cells were cultured for 24 hours, and then ZPC (7 µM) was added and incubated for 12 hours. The cells were then irradiated with 4 Gy of X-rays and incubated for 24 hours. The cells were then rinsed three times with PBS and stained with Annexin V-FITC / PI for 30 minutes before fluorescence analysis. Unlike apoptosis, the cells were first fixed with ethanol and then stained with PI for analysis. Figure 13 As shown, flow cytometry analysis of cell apoptosis showed that after treatment, the cells gradually tended to the late apoptosis phase (Q2), 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 ( Figure 14 ).

[0064] Example 3: Animal experiment.

[0065] Experimental Animal Model: Female BALB / c mice (weighing 16-18 g) were purchased from the Experimental Animal Center of Zhejiang University.

[0066] Tumor model: 4T1 tumor cells were subcutaneously injected into the right hind hip of the experimental mice. When the tumor volume reached 80-100 mm, the mice were inoculated with subcutaneous 4T1 tumors. 3 BALB / c mice bearing 4T1 subcutaneous tumors were used for animal experiments.

[0067] All animal experiments complied with the animal protection regulations approved by the Laboratory Animal Use Committee and the Laboratory Animal Ethics Committee of Zhejiang University.

[0068] (1) Biological toxicity of material ZPC 1 mL of red blood cell suspension (0.2%, v / v) was mixed with 1 mL of various concentrations of ZPC and incubated at 37°C for 4 hours. After centrifugation at 3000 rpm for 5 minutes, the optical density (OD) of each solution at 540 nm was measured using a microplate reader. The hemolysis rate was calculated as follows:

[0069] like Figure 15 As shown, compared with the positive control (water), ZPC showed no hemolysis within the 0-200 μM concentration range. Subsequently, the hemolysis rates at the corresponding concentrations were calculated and were all below 5%, meeting the permissible limit for hemolysis testing (<5%), indicating that ZPC has good hemocompatibility. Materials To evaluate the distribution of ZPC in mice, female BALB / c mice (5-6 weeks old, approximately 18 g each) were inoculated with 4T1 cells (7.5×10 cells per mouse). 5 On day 10, ZPC was injected intravenously to start treatment. 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. Figure 16 As shown, ZPC was primarily distributed in tumor tissue and the liver in mice, suggesting that ZPC can be effectively accumulated in tumor tissue and ultimately metabolized in the liver. Finally, mice (n = 3) were injected intravenously with PBS or ZPC (7 μM, 100 μL). Serum and whole blood samples were collected on days 3 and 7 after injection for biochemical and hematological analyses. Serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), serum albumin (ALB), and blood urea nitrogen (UREA) were measured using a BS-220 automated biochemical analyzer. 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 were measured before and after the injection of nanomaterials. Figure 17 The concentration or content of each biological enzyme shown and Figure 18 As shown in the blood cell count or concentration, there was no significant change in the biochemical indicators 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.

[0070] (2) Treatment effect of 4T1 tumors Female BALB / c mice (5-6 weeks old, approximately 18 g each) were inoculated with 4T1 cells (7.5 × 10 cells per mouse). 5cells). 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. Figure 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 anti-tumor effect. In addition, the extracted tumor tissues were subjected to histopathological analysis using hematoxylin and eosin (H&E) staining. Figure 20 As shown, compared with the control group, the ZPC + X-ray group showed the most extensive damage in tumor cell density, cell morphology regularity, and nuclear clarity. Furthermore, TUNEL and Ki67 staining further confirmed that cell proliferation was significantly reduced and apoptosis was significantly enhanced in the ZPC + X-ray group. These results clearly demonstrate that ZPC can enhance cytotoxicity in tumor tissues after X-ray irradiation, achieving significant anti-tumor effects without the need for additional synergistic drugs.

[0071] 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 and subjected to histological analysis. Figure 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 ( Figure 22 ). This further confirms that the ZPC and treatment methods used in this example do not cause significant biological toxicity.

[0072] In summary, this invention uses a clinically used photosensitizer porphyrin as a precursor and prepares a Zn(II)-coordinated porphyrin molecular cage through a simple reaction. Compared to the precursor porphyrin molecule, the molecular cage ZPC exhibits superior X-ray radiation kinetics, making it more suitable for kinetic cancer therapy. Using murine breast cancer (4T1) as a research model, in vitro cell experiments and in vivo tumor therapy experiments demonstrated that the ZPC molecular cage exhibited high 4T1 cellular uptake and 4T1 tumor suppression. It also efficiently mediated low-dose X-ray kinetic therapy for tumors, effectively inhibiting tumor growth and ultimately eliminating them. Because it utilizes a clinical therapeutic agent as a precursor, the molecular cage ZPC exhibits excellent biocompatibility and biosafety, and has broad prospects for clinical translational applications.

[0073] 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: include: A cationic cage having the structure shown below: ； and, a counter anion.

2. The zinc-labeled porphyrin molecular cage according to claim 1, characterized in that: The counter anion is PF6 - .

3. 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: 。 4. The method for preparing a zinc-labeled porphyrin molecular cage according to any one of claims 1 to 3, characterized in that: Includes steps: 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.

5. The preparation method according to claim 4, 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.

6. The preparation method according to claim 4, 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.

7. Use of the zinc-labeled porphyrin molecular cage according to any one of claims 1 to 3 in the preparation of an X-ray irradiation dynamic therapy agent.

8. Use of the zinc-labeled porphyrin molecular cage according to any one of claims 1 to 3 in the preparation of tumor therapeutic drugs.

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