A synergistic anti-tumor porphyrin-based covalent organic framework heterojunction material and a preparation method thereof

Abstract

The application discloses a kind of synergistic antitumor porphyrin-based covalent organic framework heterojunction materials and preparation method thereof, belong to biological medicine technical field. 5,10,15,20-tetra (4-aminobenzene) -21H,23H-porphyrin and 2,5-divinyl benzene formaldehyde are added to organic solvent to carry out solvothermal reaction, and porphyrin-based covalent organic framework (pCOF) is obtained;pCOF is mixed with 1,2-ethanedithiol and heated to obtain thiolated modified pCOF;Thiolated modified pCOF is stirred with glutathione aqueous solution ultrasonically, then tetra-chloroauric acid is added to carry out reaction, and synergistic antitumor porphyrin-based covalent organic framework confined gold nanocluster heterojunction material, recorded as Au@pCOF, is obtained.The Au@pCOF prepared in the application can be used as a kind of biocompatible antitumor therapeutic agent, under the irradiation of 808 nm near-infrared light, active oxygen is efficiently generated and heat is released, photothermal and photodynamic synergistic enhancement is realized, tumor cells are significantly killed, and is used for space-time integration synergistic antitumor therapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a porphyrin-based covalent organic framework heterojunction material with synergistic anti-tumor effects and its preparation. Background Technology

[0002] Photodynamic therapy (PDT) and photothermal therapy (PTT) have emerged as novel anti-tumor treatments due to their advantages such as spatiotemporal controllability and minimal invasiveness. However, they are often limited by issues such as limited efficacy of single-modality therapy and insufficient photosensitizer performance. Porphyrin-based photosensitizers have strong light-trapping capabilities due to their macrocyclic conjugated structure, but they face problems such as easy aggregation, poor water solubility, and low reactive oxygen species generation efficiency. Covalent organic frameworks (COFs), as porous crystalline materials with tunable structure, high specific surface area, and high stability, are ideal platforms for constructing photosensitive systems. However, their low dielectric constant makes it difficult for photogenerated electron-hole pairs to dissociate, resulting in significant exciton effects that severely restrict phototherapy performance.

[0003] Constructing heterojunctions is a mainstream optimization strategy to solve the problem of difficult separation of photogenerated electron-hole pairs in materials. The heterojunction interface forms a built-in electric field due to band differences, enabling efficient separation and transport of charge carriers, overcoming the performance limitations of single materials. Simultaneously, heterojunctions combine the functional advantages of each component, making them widely used in photocatalysis and a significant development direction for band structure engineering and advanced functional materials. Patent application CN201811037014.5 discloses a covalent organic framework heterojunction material that can achieve anti-tumor effects through the synergistic effect of photodynamics and photothermolysis. However, this material still has the following technical limitations: First, the irregular morphology and uneven size of the COF severely restrict its stable delivery and application in vivo; more importantly, the wide band gap of this donor-acceptor heterojunction limits its excitation wavelength to visible light at 635 nm, resulting in limited tissue penetration depth and difficulty in effectively targeting deep tumors; in addition, the required laser power also increases the risk of damage to normal tissues. Therefore, it is crucial to develop a material that not only possesses highly efficient photogenerated charge separation and photothermal conversion capabilities, but also extends the photoresponse range to the near-infrared region. Such materials can leverage their broad-spectrum absorption characteristics and low-power excitation advantages to achieve precise tumor treatment while minimizing damage to normal tissues.

[0004] Gold nanoclusters (AuNCs) possess excellent light absorption and utilization efficiency and phototherapy properties, showing potential in tumor treatment applications. Combining carbon nanofibers (COFs) with gold nanoclusters to construct heterojunction materials is expected to broaden the spectral absorption range, improve light absorption and utilization efficiency, enhance photodynamic and photothermal effects, and improve tumor cell killing efficacy. Patent application CN202211571456.4 discloses a COF-gold nanocluster composite material with both electrochemiluminescence and peroxidase-like activity. However, this material uses gold nanoclusters to fill the COF channels, with COF merely acting as a carrier rather than constructing a heterojunction material. Furthermore, it primarily focuses on electrochemiluminescence and does not address the photodynamic and photothermal synergistic effects in tumor treatment. Therefore, it is necessary to develop structurally controllable, multifunctional, synergistically synergistic COF-coupled gold nanocluster heterojunction materials through reasonable structural design and fabrication processes. This would significantly improve photogenerated charge separation efficiency and photothermal conversion capabilities, enhance photodynamic and photothermal synergistic effects, and ultimately improve tumor cell killing efficiency, which is of great significance for promoting the development of tumor diagnosis and treatment technologies. Summary of the Invention

[0005] To address the aforementioned limitations of existing technologies, the present invention aims to provide a porphyrin-based covalent organic framework heterojunction material with synergistic antitumor activity and its preparation method. The invention first prepares a porphyrin-based covalent organic framework (pCOF), then modifies the pCOF with thiol, and finally reduces tetrachloroauric acid in situ within the pores of the thiol-modified pCOF to prepare a porphyrin-based covalent organic framework-confined gold nanocluster heterojunction material with good photothermal and photodynamic activity. This material can serve as a biocompatible antitumor therapeutic agent, efficiently generating reactive oxygen species (ROS) and releasing heat under near-infrared light irradiation, achieving synergistic enhancement of photothermal and photodynamic activity, significantly killing tumor cells, and can be used for spatiotemporally integrated synergistic antitumor therapy.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for preparing a synergistic antitumor porphyrin-based covalent organic framework heterojunction material, comprising the following steps:

[0008] (1) 5,10,15,20-tetra(4-aminophenyl)-21H,23H-porphyrin (TAPP) and 2,5-divinyl terephthalaldehyde (Dva) were added to an organic solvent and ultrasonically dispersed. Acetic acid was then added for a solvothermal reaction. The mixture was centrifuged, washed, and vacuum dried to obtain pCOF.

[0009] (2) Under an argon atmosphere, pCOF was mixed with 1,2-ethylenedithiol, and azobisisobutyronitrile was added for stirring and heating reaction. After the reaction, the mixture was centrifuged and washed to obtain thiol-modified pCOF.

[0010] (3) The thiol-modified pCOF was ultrasonically stirred and mixed with glutathione aqueous solution, and then tetrachloroauric acid was added to react to obtain a synergistic antitumor porphyrin-based covalent organic framework confined gold nanocluster heterojunction material, denoted as Au@pCOF.

[0011] Preferably, in step (1), the structural formula of the 5,10,15,20-tetra(4-aminophenyl)-21H,23H-porphyrin is:

[0012]

[0013] The structural formula of 2,5-divinyl terephthalaldehyde is:

[0014]

[0015] Preferably, in step (1), the molar ratio of 5,10,15,20-tetra(4-aminobenzene)-21H,23H-porphyrin and 2,5-divinyl terephthalaldehyde is 1:1.2~1.8, preferably 1:1.5.

[0016] Preferably, in step (1), the organic solvent is a mixture of n-butanol and water; the volume ratio of n-butanol to water is 10:0~5, preferably 10:1.

[0017] Preferably, in step (1), the temperature of the solvothermal reaction is 20~120℃, preferably 80℃, and the time of the solvothermal reaction is 6~72h, preferably 24h.

[0018] Preferably, in step (2), the heating reaction temperature is 60~100℃, preferably 90℃, and the heating reaction time is 12~72h, preferably 48h.

[0019] Preferably, in step (3), the concentration of tetrachloroauric acid is 0.01~0.3mol / L, and more preferably 0.1mol / L.

[0020] Preferably, in step (3), the ultrasonic stirring speed is 300~800 rpm, preferably 600 rpm, the reaction temperature is 50~90℃, preferably 70℃, and the reaction time is 9~48h, preferably 24h.

[0021] In a second aspect, the present invention provides a porphyrin-based covalent organic framework heterojunction material with synergistic antitumor activity obtained by the above preparation method.

[0022] A third aspect of the present invention provides the application of porphyrin-based covalent organic framework heterojunction materials with synergistic antitumor effects in the preparation of antitumor drugs.

[0023] The beneficial effects of this invention are:

[0024] (1) This invention develops a novel porphyrin-based covalent organic framework confined gold nanoclusters heterojunction material (Au@pCOF) with negligible biotoxicity for spatiotemporal integrated photothermal and photodynamic synergistic antitumor therapy. Using an in-situ confined synthesis strategy, thiolization and metallization are sequentially performed on a porphyrin-based porous polymer. A novel covalent organic framework heterojunction is prepared by in-situ confined reduction of tetrachloroauric acid within the pCOF pores. The Au-S electron bridge at the pCOF-gold nanoclusters interface effectively mediates electron transfer, promotes photogenerated charge separation, and suppresses exciton effects. Under near-infrared light irradiation, the photogenerated electrons generated by Au@pCOF can react with oxygen to generate superoxide anions (•O2). - Photogenerated holes can react with water molecules to generate hydroxyl radicals (•OH), and simultaneously produce singlet oxygen through energy transfer. 1 O2). This process can significantly increase the amount of ROS generated, thereby promoting tumor cell apoptosis or necrosis. In addition, the pore confinement effect of the covalent organic framework can effectively enrich oxygen and water molecules, shorten the diffusion distance between reactants and photogenerated charge carriers, increase the contact probability and reaction efficiency between small molecules and photogenerated electrons and holes, enhance interfacial reaction kinetics, and thus further enhance the ROS generation efficiency.

[0025] (2) The porphyrin-based covalent organic framework-confined gold nanocluster heterojunction material provided by this invention can release heat after photoexcitation, which can be used for photothermal therapy of tumors, thereby achieving synergistic treatment of photodynamic and photothermal effects. Due to the coupling between the gold nanoclusters and pCOF, the absorption and utilization efficiency of near-infrared light of the heterojunction is also significantly improved. This allows it to efficiently generate ROS and produce a photothermal effect under low-power light irradiation, effectively killing tumor cells while reducing the impact on normal tissues. This opens up a potential avenue for the clinical application of COF-based materials in the field of anti-tumor therapy. Attached Figure Description

[0026] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0027] Figure 1 (a) Scanning electron microscope image of pCOF at a scale of 200 nm; (b) Transmission electron microscope image of pCOF at a scale of 100 nm; (c) Transmission electron microscope image of Au@pCOF at a scale of 100 nm; (d) Transmission electron microscope image of Au@pCOF at a scale of 10 nm.

[0028] Figure 2(a) Particle size distribution of pCOF; (b) Particle size distribution of AuNCs in Au@pCOF;

[0029] Figure 3 : Elemental distribution of the energy spectrum of Au@pCOF;

[0030] Figure 4 (a) FTIR spectra of TAPP, Dva and pCOF; (b) X-ray diffraction (XRD) spectra of pCOF and Au@pCOF; (c) X-ray photoelectron spectroscopy (XPS) spectra of pCOF and Au@pCOF; (d) Fine XPS spectrum of Au 4f;

[0031] Figure 5 (a) Photothermal curves of Au@pCOF under different laser power densities; (b) Photothermal curves of Au@pCOF with different concentrations; (c) Heating-cooling temperature change curves of Au@pCOF and water; (d) Relationship curve between cooling time and the negative natural logarithm of temperature for Au@pCOF.

[0032] Figure 6 (a) 1 (a) Electron paramagnetic resonance (EPR) spectrum of O2; (b) EPR spectrum of •OH; (c) •O2 - (d) EPR spectrum; Singlet oxygen generation capacity test;

[0033] Figure 7 (a) Statistical graph of cell survival rate of 4T1 cells after culture with different concentrations of Au@pCOF under light / dark conditions; (b) Half-maximal inhibitory concentration (IC50) curve of 4T1 cells; (c) Statistical graph of cell survival rate of A549 cells after culture with different concentrations of Au@pCOF under light conditions; (d) IC50 curve of A549 cells;

[0034] Figure 8 : Intracellular ROS assay in 4T1 cells;

[0035] Figure 9 :4T1 cell live / dead staining;

[0036] Figure 10 Thermographic images of 4T1 tumor-bearing mice in different treatment groups;

[0037] Figure 11 (a) Graph showing changes in mouse tumor volume; (b) Graph showing tumor inhibition rate.

[0038] Figure 12 (a) Hematoxylin and eosin (H&E) staining of tumor tissue sections; (b) Ki-67 staining of tumor tissue sections;

[0039] Figure 13 : Hemolysis rate of erythrocytes after treatment with different concentrations of Au@pCOF;

[0040] Figure 14 H&E staining of major organs of mice: heart, liver, spleen, lungs and kidneys; Detailed Implementation

[0041] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0042] As introduced in the background section, cancer is a global health threat, and traditional treatment methods (surgery, chemotherapy, and radiotherapy) have limitations such as poor targeting, significant side effects, and easy development of drug resistance. Phototherapy has become a novel minimally invasive treatment technology due to its advantages of spatial and temporal control and minimal trauma; however, its clinical application is still limited by issues such as insufficient efficacy of single treatment modalities and defects in photosensitizer performance. Therefore, the development of high-performance phototherapy materials to improve the efficacy of tumor phototherapy is of great significance for promoting the development of tumor diagnosis and treatment technologies.

[0043] Based on this, the purpose of this invention is to provide a porphyrin-based covalent organic framework heterojunction material with synergistic antitumor effects and its preparation method. This invention proposes a strategy for regulating exciton effects through heterojunctions, aiming to specifically prepare high-performance antitumor phototherapy agents. First, a thiol-modified porphyrin-based covalent organic framework is prepared. Then, tetrachloroauric acid is reduced in situ within the pCOF pores to prepare a porphyrin-based covalent organic framework-confined gold nanocluster heterojunction material (Au@pCOF) with good photothermal and photodynamic activity. The Au-S electron bridge at the pCOF and gold nanocluster interface can effectively mediate electron transfer, promote photogenerated charge separation, and suppress exciton effects. Under near-infrared light irradiation, the photogenerated electrons generated by Au@pCOF can react with oxygen to generate superoxide anions (•O2). - Photogenerated holes can react with water molecules to generate hydroxyl radicals (•OH), and simultaneously produce singlet oxygen through energy transfer. 1 O2). This process can significantly increase the generation of ROS, thereby promoting tumor cell apoptosis or necrosis. Furthermore, the pore confinement effect of the covalent organic framework can effectively enrich oxygen and water molecules, shorten the diffusion distance between reactants and photogenerated carriers, increase the contact probability and reaction efficiency between small molecules and photogenerated electrons and holes, and enhance interfacial reaction kinetics, thus further strengthening the ROS generation efficiency. Therefore, Au@pCOF can serve as a biocompatible antitumor therapeutic agent. Under near-infrared light irradiation, it can efficiently generate ROS and release heat, achieving synergistic enhancement of photothermal and photodynamic effects, significantly killing tumor cells, and can be used for spatiotemporally integrated synergistic antitumor therapy.

[0044] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0045] Note: Unless otherwise stated, the pH of the phosphate-buffered saline (PBS) used in this invention is 7.4. Unless otherwise stated, the control group (control group) in this invention refers to the group without any added drugs (such as pCOF, AuNCs, Au@pCOF), with PBS added and without laser treatment; all other treatments are the same as other groups. The laser group refers to the control group with laser treatment. In the ROS generation test experiment, the laser group refers to the group with laser treatment using water or methanol instead of the material solution.

[0046] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels.

[0047] Example 1: Preparation of Au@pCOF

[0048] (1) Preparation of porphyrin-based covalent organic frameworks:

[0049] 2,5-Divinyl-terephthalaldehyde (4.5 mg) and 5,10,15,20-tetra(4-aminophenyl)-21H,23H-porphyrin (10.8 mg) were dispersed in a mixed solution of deionized water (0.6 mL), n-butanol (6 mL), and acetic acid (6 mol / L), and ultrasonically dispersed for 15 min. After three cycles of vacuum evacuation followed by argon circulation to remove oxygen, the mixture was sealed and placed in an 80 °C oven for 24 h. After the reaction was complete, the solid precipitate was collected by centrifugation at 8000 rpm for 10 min and washed three times repeatedly with ethanol or acetone to remove unreacted monomers. The resulting brown product was vacuum dried at 70 °C for 12 h to obtain pCOF.

[0050] (2) Preparation of thiol-modified pCOF:

[0051] Under an argon (Ar) atmosphere, pCOF (20 mg) and azobisisobutyronitrile (2 mg) were added to a 25 mL round-bottom flask, followed by the addition of 1,2-ethylenedithiol (4 mL) and mixing thoroughly. The mixture was heated and stirred in a 90 °C oil bath for 36 h to complete the thiol functionalization reaction. After the reaction was completed, the mixture was centrifuged at 8000 rpm for 10 min and washed three times with ethanol or acetone to obtain the thiol-modified pCOF.

[0052] (3) Preparation of Au@pCOF:

[0053] Thiol-modified pCOF (10 mg) and glutathione (2 mg) were dispersed in 5 mL of ultrapure water and stirred at room temperature for 2 h to form a homogeneous mixture. Tetrachloroauric acid (0.1 mol / L) was then added to this mixture, and the mixture was magnetically stirred at room temperature for 2 h. The reaction was then heated in an oil bath at 70 °C for 24 h to complete the in-situ reduction of the gold precursor. After the reaction, the product was separated by centrifugation at 10,000 rpm for 10 min and washed three times with water. The product was then freeze-dried under vacuum to obtain a porphyrin-based covalent organic framework-confined gold nanocluster heterostructure material.

[0054] Example 2: Characterization of Au@pCOF

[0055] (1) The surface morphology of the synthesized pCOF and Au@pCOF was studied using field emission scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Figure 1 (a) and Figure 1 As shown in (b), the pCOF exhibits a uniform spherical morphology. TEM results show that the rough-surfaced spherical Au@pCOF is formed by stacking multiple layers of two-dimensional sheet-like structures. Figure 1 (c) is a TEM image of Au@pCOF. It can be seen that the confined growth of AuNCs in the pCOF channels did not change the morphology of pCOF, which also indicates that AuNCs are mainly anchored inside the pCOF channels rather than on the material surface. Figure 1 (d) is a TEM image of the edge of Au@pCOF, which clearly shows a large number of AuNCs uniformly anchored within pCOF.

[0056] (2) Figure 2 (a) shows that the particle size of the pCOF nanospheres is 117.5 ± 12.6 nm. Figure 2 (b) indicates that the ultrasmall AuNCs particle size in the pCOF channels is 1.7 ± 0.2 nm.

[0057] (3) From Figure 3 High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) also allows for direct observation of a large number of AuNCs ultrafine nanoparticles anchored within pCOF. Meanwhile, EDS elemental distribution maps further reveal that Au@pCOF is composed of C, N, S, and Au elements, with Au and S elements coexisting and being uniformly distributed within the Au@pCOF nanoparticles.

[0058] (4) Fourier transform infrared spectroscopy (FTIR) confirmed the successful synthesis of pCOF, such as Figure 4 As shown in (a), the carbonyl group (1683 cm) in the Dva monomer -1 ) and amino groups in TAPP monomers (3369cm) -1The characteristic peaks almost disappear in the FTIR of pCOF, while at 1640 cm⁻¹... -1 A C=N stretching vibration peak appears at this location.

[0059] Powder X-ray diffraction (PXRD) was used to characterize the periodic crystal structures of the prepared pCOF and Au@pCOF. For example... Figure 4 As shown in (b), pCOF exhibits a strong diffraction peak at a low angle of 2θ = 3.6°, corresponding to the 100 crystal plane of the tetragonal system. The PXRD pattern of Au@pCOF is similar to that of pCOF, indicating that the anchoring of AuNCs did not significantly alter the crystal structure of pCOF. The change in its broad peak in the range of 15° to 30° can be attributed to the distortion of the long-range ordered structure of pCOF and the alteration of the interlayer π-π stacking effect caused by the anchoring of AuNCs. Meanwhile, the new diffraction peak appearing at 38° corresponds to the Au(111) crystal plane of AuNCs in Au@pCOF.

[0060] The elemental composition and surface chemical valence states of the pCOF and Au@pCOF heterojunctions were analyzed using X-ray photoelectron spectroscopy (XPS). Figure 4 As shown in (c), the characteristic peak of Au element was detected only in the Au@pCOF heterojunction, confirming that AuNCs were successfully anchored in pCOF. Figure 4 (d) The fine spectrum of Au 4f shows two main peaks, corresponding to Au 4f and Au 4f respectively. 5 / 2 (88.20 eV) and Au4f 7 / 2 (84.45 eV). Au(0) 4f at 84.13 eV. 7 / 2 The peak originates from the metallic core of AuNCs, Au(I)4f at 84.63 eV. 7 / 2 The peaks are attributed to the Au-S bonds formed between AuNCs and thiol-functionalized pCOF. Simultaneously, the Au-S bonds also help anchor AuNCs firmly within the pCOF channels. XPS quantitative analysis showed that the proportions of Au(0) and Au(I) in Au@pCOF were 40.6% and 59.4%, respectively. This valence distribution provides an ideal electronic structural basis for synergistic photothermal and photodynamic therapy. The dominant Au(I) (59.4%) ensures a dense Au-S electronic bridge, which can both stably anchor AuNCs and promote efficient charge separation to improve ROS generation efficiency. Meanwhile, sufficient Au(0) (40.6%) maintains a stable metal core, providing a structural basis for enhancing the photothermal effect.

[0061] (5) Photothermal conversion performance test

[0062] Prepare a 100 μg / mL Au@pCOF aqueous solution and add it to a 1.5 mL centrifuge tube. Centrifuge tubes are then filled with solutions containing power densities of 0.25, 0.5, 0.75, 1, and 1.5 W / cm³. 2 The sample was irradiated with an 808nm laser for 5 minutes, with data collected every 30 seconds for plotting. Additionally, different concentrations of Au@pCOF aqueous solutions (0, 12.5, 25, 50, and 100 μg / mL) were prepared and added to 1.5mL centrifuge tubes, with a total volume of 1mL in each tube. Each centrifuge tube was placed in a centrifuge chamber at a wavelength of 808nm and a power density of 0.75W / cm². 2 The Au@pCOF was irradiated with a near-infrared laser for 5 minutes. Temperature values ​​were displayed on a thermal imaging device, and the temperature rise within 5 minutes was plotted every 30 seconds. Subsequently, heating / cooling experiments were conducted by switching the laser on and off to evaluate the photothermal conversion efficiency of Au@pCOF. A prepared Au@pCOF aqueous solution (50 μg / mL) and pure water were irradiated with a near-infrared laser (power density 0.75 W / cm²). 2 After irradiating for 5 minutes, the laser was turned off, and Au@pCOF was allowed to cool naturally to ambient temperature.

[0063] like Figure 5 As shown in (a), the temperature increase of Au@pCOF is positively correlated with the laser power density. When the concentration of Au@pCOF is 100 μg / mL, the laser power is 0.25, 0.5, 0.75, 1, and 1.5 W / cm². 2 At those times, the temperature increases were 13.9, 24.7, 34.9, 37.4, and 55.4℃, respectively. From Figure 5 (b) It can be seen that the heating behavior of Au@pCOF is related to its concentration. At 0.75 W / cm² 2 Under laser irradiation, as the concentration of Au@pCOF aqueous solution changed from 12.5 μg / mL to 100 μg / mL, the temperature increment of Au@pCOF changed from 15.3℃ to 34.9℃. In contrast, the temperature of pure water remained almost constant regardless of whether laser irradiation was present.

[0064] The results of the heating / cooling experiment are as follows Figure 5 As shown in (c), the photothermal conversion efficiency is calculated based on the temperature change during the cooling process. Figure 5 As shown in (d), the photothermal conversion efficiency of Au@pCOF is η(%) = 64.0%. The photothermal conversion efficiency was calculated using the formula published in application CN202310175497.X, entitled "A Photothermal-Fenton-like Artificial Nanoenzyme and Its Preparation Method and Application". Figure 5 (d) shows the values ​​of τ and θ in the formula for calculating photothermal conversion efficiency.

[0065] (6) ROS generation capacity test

[0066] Electron paramagnetic resonance (EPR) technology was used, with 2,2,6,6-tetramethylpiperidine (TEMP) or 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO) as spin trapping agents, to detect the generation of singlet oxygen (O2) in Au@pCOF under laser irradiation. 1 O2), hydroxyl radicals (•OH) and superoxide anions (•O2) - The ability of ) to. Mix 100 μL of DMPO or TEMP solution (200 mM) with 100 μL of 100 μg / mL pCOF and Au@pCOF solution, and then use an 808 nm laser (0.75 W / cm) to. 2 Irradiate for 5 min. After irradiation, transfer the mixture to a capillary for EPR testing. Use 9,10-anthratridimyl-bis(methylene)dimalonic acid (ABDA) as a probe to detect singlet oxygen in the solution. 1 O2) generation. 50 μg / mL pCOF and Au@pCOF were dispersed in 50 μM ABDA solution and analyzed using an 808 nm laser (0.75 W / cm²). 2 Irradiate the solution for 5 minutes and record the change in absorbance.

[0067] like Figure 6 (a) and Figure 6 As shown in (b), obvious TEMP / E2 could be observed in pCOF and Au@pCOF under laser irradiation. 1 The EPR signal peaks of the O2 and DMPO / •OH adducts indicate that pCOF and Au@pCOF can react with oxygen and water molecules to form... 1 O2 and •OH, and the corresponding EPR signal peak in Au@pCOF is significantly enhanced. Furthermore, Figure 6 (c) The Au@pCOF shows a clear superoxide anion radical signal, which is almost negligible in pure pCOF, indicating that the Au@pCOF heterojunction has increased •O2. - Its generation capability. Figure 6 (d) shows that under 808 nm laser irradiation, the absorbance of ABDA in the Au@pCOF group decreased significantly, and its degradation rate increased by nearly 3 times compared with pure pCOF, while the absorbance of the control group irradiated only by laser did not change significantly. These results confirm that Au@pCOF heterojunctions can efficiently generate ROS under mild light conditions.

[0068] Experimental Example 1: Cytotoxicity Experiment

[0069] In 96-well plates, mouse 4T1 breast cancer cells and A549 human non-small cell lung cancer cells (from the Chinese Academy of Sciences Cell Bank) were cultured at a density of 1 × 10⁶ cells per well. 4Cells were seeded at a density of 100 μL per well, and the edges of the plate were sealed with 100 μL of PBS to prevent excessive evaporation. After 12 h of culture, different concentrations of Au@pCOF (0, 5, 10, 25, 50, and 75 μg / mL) were added, and incubation continued for another 24 h. In the laser irradiation group, after adding different concentrations of Au@pCOF to the cells, incubation continued for 12 h, followed by irradiation with an 808 nm laser (0.75 W / cm²). 2 Irradiate for 5 minutes, then culture for 12 hours. Cell viability is detected by CCK-8 assay, and each experiment is repeated 3 times.

[0070] Test results are as follows Figure 7 As shown in (a), even with a Au@pCOF concentration as high as 75 μg / mL, the survival rate of 4T1 cells in the dark (no laser irradiation) group remained as high as 95%, effectively demonstrating that the synthesized Au@pCOF had virtually no cytotoxicity. However, in the 808nm laser irradiation group, the survival rate of 4T1 cells continuously decreased with increasing Au@pCOF concentration, and the cell killing rate reached over 80% at a concentration of 75 μg / mL. Figure 7 (b) The half-maximal inhibitory concentration (IC50) fitting results showed that its IC50 value was as low as 12.01 μg / mL. Figure 7 (c) For A549 cells, the cell killing rate also reached over 80% at a concentration of 75 μg / mL. Figure 7 (d) shows that its IC50 value is as low as 9.17 μg / mL. This indicates that this heterojunction material can enter cells well and exert a synergistic therapeutic effect of photodynamic and photothermal therapy, with significant anti-tumor effects and excellent biocompatibility, providing a novel and efficient nano-formulation for precision tumor treatment.

[0071] Experimental Example 2: Intracellular ROS Generation Assay

[0072] 4T1 cells were fed at a rate of 1×10 5 Cells were seeded at a density of 1 cell / well in 35 mm confocal culture dishes, and 2 mL of RPMI 1640 cell culture medium was added. After 12 h of incubation, PBS and 50 μg / mL of Au@pCOF, pCOF, pCOF+AuNCs, and Au@pCOF were added, respectively, and incubated for another 6 h. Then, 10 μmol / L 2',7'-dichlorofluorescein diacetate (DCFH-DA) reactive oxygen species fluorescent probe was added to the culture dishes, and incubation was continued for 20 min. The cells in the latter three groups were then treated with an 808 nm laser (0.75 W / cm²). 2 Irradiate for 5 minutes. Then wash the cells 3 times with PBS, replace with preheated phenol red-free RPMI 1640 medium, and immediately perform imaging observation using a laser scanning confocal microscope.

[0073] like Figure 8The observation results show that the intracellular fluorescence intensity of the Au@pCOF+ laser group was significantly higher than that of other treatment groups, indicating that the cell culture group with Au@pCOF added produced more ROS in the cells after laser irradiation.

[0074] Experimental Example 3: Cell Viability / Deadness Staining

[0075] 4T1 cells were fed at a rate of 1×10 5 Cells were seeded at a density of 1 cell / well in confocal culture dishes and cultured for 12 h. Subsequently, they were treated for 12 h with PBS, Au@pCOF, pCOF plus laser illumination, pCOF and AuNCs plus laser illumination, and Au@pCOF plus laser illumination (material concentration: 50 μg / mL). Following this, the cells were co-stained with calcein-AM / propidium iodide (Calcein-AM / PI) at 37 °C for 30 min. Fluorescence images were acquired using a laser scanning confocal microscope to assess cell viability and death.

[0076] like Figure 9 As shown, pCOF, pCOF, AuNCs, and Au@pCOF exhibited significant red fluorescence after laser irradiation. Furthermore, cells treated with Au@pCOF showed the strongest red fluorescence, indicating a significant increase in tumor cell death under the enhanced photodynamic and photothermal synergistic effects of Au@pCOF heterostructure.

[0077] Experiment 4: Photothermal Imaging of Tumors in Tumor-Bearing Mice

[0078] PBS, 2 mg / kg pCOF, and Au@pCOF were injected into mice via the tail vein. Twelve hours after injection, mice were subjected to treatment using an 808 nm laser (0.75 W / cm²). 2 The tumor area was irradiated for 5 minutes, and the temperature changes in the tumor area of ​​the mouse were recorded using an infrared thermal imager.

[0079] like Figure 10 As shown, the temperature change in the tumor area was not significant in the laser control group, but it was significant in the pCOF irradiation group. The Au@pCOF irradiation group showed the greatest increase in tumor temperature, reaching 50.7℃, and the precise location of the tumor could be clearly seen. This indicates that Au@pCOF has an enhanced in vivo photothermal effect, which can effectively induce tumor cell apoptosis and necrosis, making it suitable for photothermal therapy of tumors.

[0080] Experimental Example 5: Evaluation of in vivo tumor suppression effect

[0081] A 4T1 breast cancer tumor model was established using female Balb / c mice (6-8 weeks old, weighing approximately 20g) purchased from Shanghai Silex Laboratory Animal Co., Ltd. Mice were randomly divided into four groups: I: control (PBS); II: Au@pCOF; III: pCOF + laser; IV: Au@pCOF + laser, with five mice in each group. The drug was administered via tail vein injection at a dose of 2 mg / kg. Twelve hours after injection, the laser irradiation group received 808nm laser treatment (0.75W / cm²). 2 Irradiate for 5 minutes, and measure the tumor volume of mice at fixed times every two days. The tumor volume is calculated using the formula: V (mm²) 3 ) = (major axis × minor axis) 2 ) / 2. After 14 days of treatment, the mice were sacrificed, and tumor tissue was collected for hematoxylin and eosin (H&E) staining and Ki-67 immunohistochemical staining.

[0082] like Figure 11 As shown in (a), the tumor volume in the Au@pCOF+ laser group gradually decreased over time. Figure 11 (b) The results showed that the tumor inhibition rate of the Au@pCOF+laser group was as high as 97%, indicating that Au@pCOF produced a significant anti-tumor effect after laser irradiation.

[0083] Figure 12 (a) H&E staining results of tumor tissue showed that the tumor tissue in the Au@pCOF+ laser group showed large-area obvious necrosis. Figure 12 (b) Immunohistochemical staining of Ki-67 showed a significant decrease in Ki-67 expression, indicating a marked reduction in tumor cell proliferation activity. These results confirm that Au@pCOF heterojunctions have excellent anti-tumor therapeutic effects in vivo.

[0084] Experimental Example 6: In vivo and in vitro biocompatibility assessment

[0085] (1) Cell hemolysis experiment

[0086] Blood was collected from 6-8 week old female Balb / c mice (purchased from Shanghai Silex Laboratory Animal Co., Ltd.). Fresh anticoagulated blood was centrifuged at 2000 rpm for 15 min, and red blood cells were collected. The cells were then washed with PBS buffer (PBS buffer to red blood cells volume ratio approximately 1:1). After centrifugation, the supernatant was removed, and this process was repeated four times. The concentrated red blood cell suspension was then mixed with PBS buffer to prepare a 4% red blood cell suspension, which was then mixed with Au@pCOF (concentrations of 10, 20, 50, 100, and 150 μg / mL) at a 1:1 volume ratio and incubated at 37°C for 4 h. After centrifugation, 100 μL of the supernatant from each tube was added to a 96-well plate, and the absorbance was measured using a microplate reader. Distilled water served as a positive control, and the PBS group served as a negative control. The calculation formula is as follows:

[0087] Hemolysis rate (%) = (A-An) / (Ap-An) × 100%;

[0088] A: Absorbance of the supernatant after co-incubation of red blood cells with Au@pCOF;

[0089] An: Absorbance of the supernatant after red blood cells were co-incubated with PBS (negative control).

[0090] Ap: Absorbance of the supernatant after red blood cells are co-incubated with water (positive control).

[0091] like Figure 13 As shown, after co-incubation with blood red blood cells, Au@pCOF did not cause obvious hemolysis, and the hemolysis rate remained below 1.0%, indicating that Au@pCOF has good blood compatibility.

[0092] (2) H&E staining of tissues and organs

[0093] Heart, liver, spleen, lung, and kidney tissues of mice euthanized 14 days after treatment were collected and stained with hematoxylin and eosin (H&E).

[0094] from Figure 14 As can be seen, no significant damage or abnormalities were observed in the mouse organs and tissues after different treatments. All these results confirm that Au@pCOF heterojunctions have good biocompatibility and can be used as a safe and effective antitumor agent.

[0095] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a synergistic antitumor porphyrin-based covalent organic framework heterojunction material, characterized in that, Includes the following steps: (1) 5,10,15,20-tetra(4-aminophenyl)-21H,23H-porphyrin and 2,5-divinyl terephthalaldehyde were added to an organic solvent and ultrasonically dispersed. Acetic acid was then added for a solvothermal reaction. The mixture was centrifuged, washed, and vacuum dried to obtain pCOF. (2) Under an argon atmosphere, pCOF was mixed with 1,2-ethylenedithiol, and azobisisobutyronitrile was added for stirring and heating reaction. After the reaction, the mixture was centrifuged and washed to obtain thiol-modified pCOF. (3) The thiol-modified pCOF was ultrasonically stirred and mixed with glutathione aqueous solution, and then tetrachloroauric acid was added to react to obtain a synergistic antitumor porphyrin-based covalent organic framework confined gold nanocluster heterojunction material, denoted as Au@pCOF.

2. The preparation method according to claim 1, characterized in that, In step (1), the structural formula of the 5,10,15,20-tetra(4-aminophenyl)-21H,23H-porphyrin is: ， The structural formula of 2,5-divinyl terephthalaldehyde is: 。 3. The preparation method according to claim 1, characterized in that, In step (1), the molar ratio of 5,10,15,20-tetra(4-aminobenzene)-21H,23H-porphyrin and 2,5-divinyl terephthalaldehyde is 1:1.2~1.8, preferably 1:1.

5.

4. The preparation method according to claim 1, characterized in that, In step (1), the organic solvent is a mixture of n-butanol and water; the volume ratio of n-butanol to water is 10:0~5, preferably 10:

1.

5. The preparation method according to claim 1, characterized in that, In step (1), the temperature of the solvothermal reaction is 20~120℃, preferably 80℃, and the time of the solvothermal reaction is 6~72h, preferably 24h.

6. The preparation method according to claim 1, characterized in that, In step (2), the heating reaction temperature is 60~100℃, preferably 90℃, and the heating reaction time is 12~72h, preferably 48h.

7. The preparation method according to claim 1, characterized in that, In step (3), the concentration of tetrachloroauric acid is 0.01~0.3mol / L, preferably 0.1mol / L.

8. The preparation method according to claim 1, characterized in that, In step (3), the ultrasonic stirring speed is 300~800 rpm, preferably 600 rpm, the reaction temperature is 50~90℃, preferably 70℃, and the reaction time is 9~48h, preferably 24h.

9. The porphyrin-based covalent organic framework heterojunction material with synergistic antitumor activity obtained by the preparation method according to any one of claims 1 to 12.

10. The application of the porphyrin-based covalent organic framework heterojunction material with synergistic antitumor activity as described in claim 13 in the preparation of antitumor drugs.

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