Nanocomposite material based on porphyrin hydrogen bond organic framework as well as preparation method and anti-tumor application thereof

Abstract

The invention relates to a nano composite material based on a porphyrin hydrogen bond organic framework as well as a preparation method and anti-tumor application of the nano composite material. The invention discloses a hydrogen bond organic framework with a smaller size, which is prepared from 1, 3, 6, 8-tetra (4-carboxybenzene) pyrene and 5, 10, 15, 20-tetra (4-aminobenzene)-21H, 23H-porphyrin, and a hydrogen bond organic framework with a smaller size, a larger specific surface area and a larger specific surface area is constructed on the basis of the hydrogen bond organic framework. The novel nano composite material has good photo-thermal performance, photodynamic performance and anti-tumor activity, and can efficiently trigger ferroptosis of tumor cells.

Description

Technical Field

[0001] The invention relates to the technical field of material chemistry and biology, and in particular to a nanocomposite material based on a porphyrin hydrogen bond organic framework, a preparation method thereof and anti-tumor application thereof. Background Art

[0002] Inducing cell death is an important approach to cancer treatment. Traditional methods of inducing cell death are often limited in their anti-cancer effects due to treatment resistance. Therefore, exploring new cell death pathways has become a research hotspot in oncology.

[0003] Ferroptosis is an iron-dependent programmed cell death characterized by the accumulation of lipid peroxides. In recent years, studies have shown that ferroptosis is associated with resistance to cancer treatment, and activating ferroptosis in tumor cells is an effective strategy to reverse tumor resistance. Due to the complexity of the tumor microenvironment (TME), the TME interacts with ferroptosis, and a large number of studies have shown that ferroptosis in cancer cells can stimulate or regulate the immune response of tumors. Ferroptosis has become one of the feasible strategies to reverse the tumor immunosuppressive microenvironment. Considering that drugs used for immunotherapy need to have high biocompatibility, and nanodrug delivery systems have the advantages of high biocompatibility and good targeting, the development of nanoimmunotherapy drug delivery systems based on ferroptosis has become a research hotspot in the biomedical field in recent years.

[0004] CN201910937168.8 discloses a metal-organic nanocomposite for triggering ferroptosis of tumor cells, which wraps hyaluronic acid on its surface through electrostatic adsorption and ionic cross-linking to make the composite material biocompatible, and the preparation process is relatively cumbersome. Hydrogen-bonded organic frameworks (HOFs), as an emerging type of porous crystalline material, are constructed by organic components through hydrogen bond interactions. Compared with covalent organic frameworks and metal organic frameworks, HOFs are composed of hydrogen bonds, are easy to synthesize and have good biocompatibility, and are a potential platform for constructing functional materials.

[0005] However, most of the prepared HOFs are large in size and difficult to penetrate into the tumor site. Therefore, it is urgent to explore suitable organic monomers and synthesis conditions to prepare smaller HOFs as a hydrogen-bonded organic framework platform and to construct targeted nanomaterials based on them for triggering ferroptosis of tumor cells. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a nanocomposite material based on a porphyrin hydrogen bond organic framework, a preparation method thereof and anti-tumor application thereof.

[0007] To solve the above problems, the technical solution adopted by the present invention is: Technical Theme 1 A porphyrin hydrogen bond organic framework is prepared from 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene and 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphyrin.

[0008] As a further improvement of the present invention, the steps for preparing the porphyrin hydrogen bond organic framework are as follows: S1: Add 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene to water, adjust the pH to 7-8 with an alkaline solution to form solution A, and dissolve 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphyrin in water to form solution B; S2: Solution A and solution B were mixed, stirred at 500-1000 rpm for 1-3 h at room temperature and in the dark, centrifuged, and washed with water to obtain a hydrogen-bonded organic framework HOF-HT.

[0009] As a further improvement of the present invention, the molar ratio of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene to 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphyrin in the mixed solution of S2 is 3.5-4.5:1; As a further improvement of the present invention, the molar ratio of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene to 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphyrin is 4:1.

[0010] Technical Theme 2 A method for preparing a nanocomposite material of a porphyrin hydrogen-bonded organic framework based on the technical theme 1 comprises the following steps: S1: Add 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene to water, adjust the pH to 7-8 with an alkaline solution to form solution A, and dissolve 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphyrin in water to form solution B; S2: Mix glucose oxidase with solution B, stir at room temperature for 10-30 min, then add solution A to the solution, stir at 500-1000 rpm for 1-3 h at room temperature in the dark, centrifuge and wash with water to obtain the hydrogen-bonded organic framework GOx@HOF encapsulating the natural enzyme; S3: Add GOx@HOF obtained in S2 into water and add FeCl 3 solution, ultrasonically dispersed, stirred at 500-1000 rpm for 20-40 min, then added tannic acid solution and folic acid solution, ultrasonically dispersed, stirred at 500-1000 rpm for 20-40 min, centrifuged and washed with water to obtain the nanocomposite GOx@HOF@Fe-TF.

[0011] As a further improvement of the present invention, the usage ratio of glucose oxidase, 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene and 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphyrin in S2 is 0.3-0.45 g: 3.5-4.5 mmol: 1 mmol.

[0012] As a further improvement of the present invention, the dosage relationship of glucose oxidase, 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene and 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphyrin in S2 is 0.37 g:4 mmol:1 mmol.

[0013] As a further improvement of the present invention, GOx@HOF, FeCl 3 The mass ratio of tannic acid and folic acid is 1:0.3-0.45:0.6-0.8:0.06-0.08.

[0014] As a further improvement of the present invention, GOx@HOF, FeCl 3 The mass ratio of tannic acid and folic acid is 1:0.37:0.7:0.07.

[0015] Technical Theme 3 A nanocomposite material obtained by the preparation method described in technical subject two.

[0016] Technical Theme 4 An application of the porphyrin hydrogen-bonded organic framework as described in the first technical theme and the nanocomposite material as described in the third technical theme in the preparation of anti-tumor drugs.

[0017] As a further improvement of the present invention, the anti-tumor drug is a drug that triggers ferroptosis of tumor cells.

[0018] The beneficial effects of adopting the above technical solution are: 1. The HOFs prepared in this application have a small size and good cell permeability. The composite material for triggering ferroptosis of tumor cells based on the HOFs provides a new idea for the preparation of drugs that trigger ferroptosis of tumor cells and can broaden the biological application pathways of HOFs.

[0019] 2. This application constructs a composite material based on HOFs, and introduces glucose oxidase GOx into smaller HOFs to form GOx@HOF, which has glucose oxidase activity and its shape and size do not change significantly.

[0020] 3. Fe of GOx@HOF@Fe-TF disclosed in this application 3+-TF layer has excellent photothermal therapy (PTT) and chemodynamic therapy (CDT) capabilities, and PTT promotes CDT, which can stimulate the generation of more hydroxyl radicals and further increase the level of reactive oxygen species (ROS). GOx@HOF has glucose oxidase activity and photodynamic therapy (PDT), which can consume glucose in the tumor while lowering pH, increasing the hydrogen peroxide content in the tumor microenvironment, and providing raw materials for the subsequent Fenton reaction. 3+ -TF and GOx@HOF synergize with each other to further amplify oxidative stress and enhance the ferroptosis effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a flow chart of the preparation of GOx@HOF@Fe-TF in the embodiment; Figure 2 are transmission electron microscope (TEM) images of the materials prepared in the present application, wherein A is a TEM image of HOF-H prepared in Example 1, B is a TEM image of HOF-HT prepared in Example 1, C is a TEM image of GOx@HOF prepared in Example 4, and D is a TEM image of GOx@HOF@Fe-TF prepared in Example 4; Figure 3 is an infrared spectrum of the materials prepared in the present application, wherein HOF is HOF-HT prepared by the method described in Example 1 of the present application, GOx@HOF is prepared by the method described in Example 4 of the present application, GOx@HOF@Fe-TF is prepared by the method described in Example 4 of the present application, TAPP is the raw material 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphyrin used in the example, and H4TBAPy is the raw material 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene used in the example; Figure 4 is a powder x-ray diffraction pattern (PXRD) of the materials prepared in the present application, wherein HOF is HOF-HT prepared by the method described in Example 1 of the present application, GOx@HOF is prepared by the method described in Example 4 of the present application, and GOx@HOF@Fe-TF is prepared by the method described in Example 4 of the present application; Figure 5 is the photothermal and photodynamic properties of GOx@HOF@Fe-TF, where A is the GOx@HOF@Fe-TF suspension with different concentrations at 1 W / cm 2 A is the temperature change diagram of 200 μg / mL GOx@HOF@Fe-TF suspension irradiated with 808 nm laser for 8 min; B is the temperature change diagram of 200 μg / mL GOx@HOF@Fe-TF suspension irradiated with 808 nm laser of different powers; C is the temperature change diagram of 200 μg / mL GOx@HOF@Fe-TF suspension irradiated with 1.5 W / cm 2808 nm laser irradiation for 5 min, then cooled to room temperature, temperature change diagram after five irradiation-cooling cycles; D is the ESR diagram of samples treated differently. Among them, 1-5 correspond to samples 1-5 in the photodynamic property detection of Example 9, 1: TEMP+660 nm group; 2: HOF-HT+660 nm group; 3: TEMP+GOx@HOF@Fe-TF group; 4: TEMP+HOF-HT+660 nm group; 5: TEMP+GOx@HOF@Fe-TF+660nm group; Figure 6 Figure 1 is the effect diagram of the enzyme activity of GOx@HOF@Fe-TF. A is the comparison diagram of the pH change of GOx@HOF@Fe-TF suspension and GOx@HOF@Fe-TF suspension containing glucose over time. B is the UV-visible absorption spectrum of each solution containing titanium sulfate. C is the UV-visible absorption spectrum of each solution at 652 nm, where the laser is 1 W / cm 2 808 nm laser irradiation for 5 min, Glu represents glucose; Figure 7 This is a graph showing the changes in breast cancer cell survival rate with GOx@HOF@Fe-TF concentration, with or without 808 nm laser irradiation, and with or without 660 nm laser irradiation; Figure 8 This is a graph showing the change in survival rate of normal human liver cells (LO2 cells) with GOx@HOF@Fe-TF concentration; Fig. 9 1-4 are the graphs showing the changes in the survival rate of breast cancer cells after different treatments. 1-4 are as described in Example 13, 1: PBS group, 2: PBS+ferroptosis inhibitor (Fer-1) group, 3: GOx@HOF@Fe-TF group, 4: GOx@HOF@Fe-TF+Fer-1 group; Fig.10 It is a graph of ROS generation in each group detected by DHE (dihydroethidium) probe under a fluorescence microscope. Among them, 1-5 are as described in Example 14, 1: PBS group, 2: GOx@HOF@Fe-TF group, 3: GOx@HOF@Fe-TF+808 nm group, 4: GOx@HOF@Fe-TF+ 660 nm group, 5: GOx@HOF@Fe-TF+808 nm +660 nm group; Fig.11is a graph of intracellular malondialdehyde (MDA) content of breast cancer cells after different treatments. Among them, 1-5 are as described in Example 15, 1: PBS group, 2: GOx@HOF@Fe-TF group, 3: GOx@HOF@Fe-TF+808 nm group, 4: GOx@HOF@Fe-TF+660 nm group, 5: GOx@HOF@Fe-TF+808 nm +660 nm group; Fig.12 is a graph of intracellular glutathione peroxidase 4 (GPX4) content in breast cancer cells after different treatments. 1-5 are as described in Example 15, 1: PBS group, 2: GOx@HOF@Fe-TF group, 3: GOx@HOF@Fe-TF+808 nm group, 4: GOx@HOF@Fe-TF+ 660 nm group, 5: GOx@HOF@Fe-TF+808 nm+660 nm group; Fig.13 1 is a graph showing the tumor inhibition effect of GOx@HOF@Fe-TF on 4T1 tumor-bearing mice, wherein A is a graph showing the change of mouse tumor volume over days in Example 16, B is a photograph of the tumors of each group of mice, C is a comparison of the tumor mass of each group of mice, D is a graph showing the change of body weight of each group of mice within 14 days, and E is a H&E staining graph of tumor tissue sections of each group of mice. 1-6 in AE are as described in Example 16, 1 is the PBS group, 2 is the PBS+808 nm+660 nm group, 3 is the GOx@HOF@Fe-TF group, 4 is the GOx@HOF@Fe-TF+808nm group, 5 is the GOx@HOF@Fe-TF+660 nm group, and 6 is the GOx@HOF@Fe-TF+808nm+660 nm group; Fig.14 is a graph of immune cell content in mouse spleen single cell suspension in Example 16, wherein A is immune cell CD4 + , B is immune cell CD8 + , C is immune cell CD86 + , D is immune cell CD80 + , wherein 1-6 in AD are as described in Example 16, 1 is a PBS group, 2 is a PBS+808 nm+660 nm group, 3 is a GOx@HOF@Fe-TF group, 4 is a GOx@HOF@Fe-TF+808 nm group, 5 is a GOx@HOF@Fe-TF+660 nm group, and 6 is a GOx@HOF@Fe-TF+808 nm+660 nm group; DETAILED DESCRIPTION

[0022] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention is clearly and completely described below in conjunction with specific embodiments.

[0023] The concentration of PBS buffer used in the examples is 0.01 M.

[0024] Preparation Example 1 Add 8.6 mg of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene (H4TBAPy) to 0.8 mL of redistilled water, adjust the pH to 7-8 with aqueous ammonia, and add redistilled water to make up the solution to 1 mL to form solution A.

[0025] Preparation Example 2 Solution B was formed by dissolving 17 mg of 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphyrin (TAPP) in 1 mL of redistilled water.

[0026] Example 1 Take 555 μL of solution A (obtained by the preparation method of Preparation Example 1), add 70 μL of solution B (obtained by the preparation method of Preparation Example 2), mix, stir at 800 rpm at room temperature in the dark for 2 h, centrifuge, and wash with water 3 times to obtain HOF-HT.

[0027] Example 2 Take 633 μL of solution A (obtained by the preparation method of Preparation Example 1), add 70 μL of solution B (obtained by the preparation method of Preparation Example 2), mix, stir at 500 rpm at room temperature in the dark for 3 h, centrifuge, and wash with water 3 times to obtain HOF-HT.

[0028] Example 3 Take 492 μL of solution A (obtained by the preparation method of Preparation Example 1), add 70 μL of solution B (obtained by the preparation method of Preparation Example 2), mix, stir at 1000 rpm at room temperature in the dark for 1 h, centrifuge, and wash with water 3 times to obtain HOF-HT.

[0029] Example 4 The preparation flow chart of GOx@HOF@Fe-TF is as attached. Figure 1 shown.

[0030] 0.67 mg of glucose oxidase (GOx) was mixed with 70 μL of solution B (obtained by the preparation method of Preparation Example 2) and stirred at room temperature for 20 min to obtain a suspension. Then, solution A (obtained by the preparation method of Preparation Example 1, 12.6 mM, 557 μL) and 255 μL of redistilled water were added to the suspension, stirred at 800 rpm at room temperature in the dark for 2 h, centrifuged and washed with water 3 times to obtain GOx@HOF.

[0031] 0.7 mg of GOx@HOF prepared by the method described in Example 4 was dispersed in 1 mL of deionized water, and 0.2 mL of 1.3 mg / mL FeCl was added. 3The solution was ultrasonicated for 10 s and stirred at 750 rpm for 30 min. Then 0.1 mL of tannic acid solution (5 mg / mL) and 0.1 mL of folic acid solution (0.5 mg / mL) were added, ultrasonicated for 10 s, stirred at 750 rpm for 30 min, centrifuged and washed with water for 3 times to obtain GOx@HOF@Fe-TF.

[0032] Example 5 0.53 mg of glucose oxidase (GOx) was mixed with 70 μL of solution B (obtained by the preparation method of Preparation Example 2) and stirred at room temperature for 20 min to obtain a suspension. Then, solution A (obtained by the preparation method of Preparation Example 1, 12.6 mM, 492 μL) and 255 μL of redistilled water were added to the suspension, stirred at 500 rpm at room temperature in the dark for 1 h, centrifuged and washed with water 3 times to obtain GOx@HOF.

[0033] 0.7 mg of GOx@HOF prepared by the method described in Example 5 was dispersed in 1 mL of deionized water, and 0.16 mL of 1.3 mg / mL FeCl was added. 3 The solution was ultrasonicated for 10 s and stirred at 500 rpm for 30 min. Then 0.08 mL of tannic acid solution (5 mg / mL) and 0.08 mL of folic acid solution (0.5 mg / mL) were added, ultrasonicated for 10 s, stirred at 500 rpm for 30 min, centrifuged and washed with water for 3 times to obtain GOx@HOF@Fe-TF.

[0034] Example 6 0.79 mg of glucose oxidase (GOx) was mixed with 70 μL of solution B (obtained by the preparation method of Preparation Example 2) and stirred at room temperature for 20 min to obtain a suspension. Then, solution A (obtained by the preparation method of Preparation Example 1, 12.6 mM, 633 μL) and 255 μL of redistilled water were added to the suspension, stirred at 1000 rpm at room temperature in the dark for 3 h, centrifuged and washed with water 3 times to obtain GOx@HOF.

[0035] 0.7 mg of GOx@HOF prepared by the method described in Example 6 was dispersed in 1 mL of deionized water, and 0.24 mL of 1.3 mg / mL FeCl was added. 3 The solution was ultrasonicated for 10 s and stirred at 1000 rpm for 30 min. Then 0.11 mL of tannic acid solution (5 mg / mL) and 0.11 mL of folic acid solution (0.5 mg / mL) were added, ultrasonicated for 10 s, stirred at 1000 rpm for 30 min, centrifuged and washed with water for 3 times to obtain GOx@HOF@Fe-TF.

[0036] Comparative Example 1 8.6 mg of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene (H4TBAPy) was dissolved in 0.8 mL of redistilled water, the pH was adjusted to 7-8 with aqueous ammonia, and redistilled water was added to make up the solution to 1 mL to form solution A. 555 μL of solution A was taken, 325 μL of redistilled water was added, and the mixture was stirred at 800 rpm at room temperature in the dark for 2 h, centrifuged, and washed with water 3 times to obtain HOF-H.

[0037] Example 7 The HOF-H in Example 1 (attached) was examined by transmission electron microscopy. Figure 2 A) HOF-HT in Example 1 (attached Figure 2 B), GOx@HOF in Example 4 (attached Figure 2 C), GOx@HOF@Fe-TF in Example 4 (attached Figure 2 D) was observed. Compared with the HOF-H in Comparative Example 1, the HOF-HT disclosed in the present application is smaller in size, only about 300-500 nm, which provides a new idea for the preparation of drugs that trigger ferroptosis of tumor cells and can broaden the biological application of HOFs. Figure 2 As shown in Figure C, when glucose oxidase GOx was introduced into smaller HOFs, its shape and size did not change significantly. Figure 2 As shown in D, Fe is introduced through the coordination of metal ions. 3+ , tannic acid, and folic acid, forming a metal-polyphenol network on the surface of GOx@HOF.

[0038] The HOF-HT in Example 1, the GOx@HOF in Example 4, and the GOx@HOF@Fe-TF in Example 4 were detected by infrared spectroscopy, and the results are shown in the attached Figure 3 As shown in the figure, it can be seen that the Fourier transform infrared (FTIR) spectrum of HOF-HT has a higher peak at 1691 cm compared with TAPP and H4TBAPy. -1 C=O stretching vibration and 1609 cm -1 The shift of the C=N stretching vibration at indicates that the stretching motions of C=O and C=N are restricted, suggesting that a rigid structure connected by hydrogen bonds is formed in HOF-HT.

[0039] Powder X-ray diffraction was performed on HOF-HT in Example 1, GOx@HOF in Example 4, and GOx@HOF@Fe-TF in Example 4. The results are shown in the attached figure. Figure 4 As shown, the analysis confirmed that encapsulating GOx in HOF-HT had little effect on the crystal structure of HOF-HT.

[0040] Example 8 1. GOx@HOF@Fe-TF prepared by the method described in Example 4 was dispersed in redistilled water to prepare suspensions with concentrations of 100 μg / mL, 200 μg / mL, and 400 μg / mL, respectively.

[0041] At 1W / cm 2 808 nm laser irradiation was used to detect and record the temperature changes in 0-8 min for the redistilled water and the above GOx@HOF@Fe-TF suspensions of different concentrations (100 μg / mL, 200 μg / mL, 400 μg / mL). The results are shown in the attached figure. Figure 5 As shown in A.

[0042] 2. The irradiation power of 808 nm laser was different (0.5 W / cm 2 , 1 W / cm 2 , 1.5 W / cm 2 ) irradiated 200 μg / mL GOx@HOF@Fe-TF suspension and recorded the temperature change within 0-8 min. The results are shown in the attached figure. Figure 5 As shown in B.

[0043] 3. At 1.5 W / cm 2 808 nm laser irradiation of 200 μg / mL GOx@HOF@Fe-TF suspension for 5 min, followed by natural cooling to room temperature for 5 cycles. The results are shown in the attached figure. Figure 5 As shown in C.

[0044] Attached Figure 5 As shown in A and 5B, according to the change in the thermometer reading, it is proved that GOx@HOF@Fe-TF has photothermal capacity, and the photothermal effect is positively correlated with the GOx@HOF@Fe-TF concentration and the laser irradiation power. And after 5 cycles of illumination-cooling, the temperature change of GOx@HOF@Fe-TF is still stable, showing good photothermal stability ( Figure 5 C).

[0045] Example 9 Photodynamic property detection The following groups of samples were tested using ESR (electron spin resonance): Sample 1: 10 μL 2,2,6,6-tetramethylpiperidine (TEMP trap) in 500 μL redistilled water was added and the power was 0.6 W / cm 2 660 nm laser irradiation for 5 min.

[0046] Sample 2: 500 μL of a 200 μg / mL suspension of HOF-HT prepared by the method described in Example 1 and heated at a power of 0.6 W / cm 2660 nm laser irradiation for 5 min.

[0047] Sample 3: 10 μL of TEMP capture agent was added to 500 μL of a suspension of GOx@HOF@Fe-TF prepared by the method described in Example 4 at a concentration of 200 μg / mL and placed in the dark for 5 min.

[0048] Sample 4: 10 μL of TEMP trapping agent was added to 500 μL of HOF-HT suspension with a concentration of 200 μg / mL prepared by the method described in Example 1, and the power was 0.6 W / cm 2 660 nm laser irradiation for 5 min.

[0049] Sample 5: 10 μL of TEMP capture agent (500 μL, 200 μg / mL) was added to the suspension of GOx@HOF@Fe-TF prepared by the method described in Example 4, and the power was 0.6 W / cm 2 660 nm laser irradiation for 5 min.

[0050] The results are attached Figure 5 As shown in Figure D, the ESR spectra of HOF-HT and GOx@HOF@Fe-TF suspensions containing TEMP scavengers irradiated at 660 nm have obvious 1:1:1 peaks, indicating that GOx@HOF@Fe-TF and HOF-HT have the ability to produce singlet oxygen under laser irradiation, and HOF-HT and GOx@HOF@Fe-TF have good photodynamic properties.

[0051] Example 10 1. The generation of gluconic acid was detected by detecting the pH value of the mixture of GOx@HOF@Fe-TF prepared by the method described in Example 4 and glucose.

[0052] The pH values ​​of the 200 μg / mL suspension of GOx@HOF@Fe-TF prepared by the method described in Example 4 and the 200 μg / mL suspension of GOx@HOF@Fe-TF prepared by the method described in Example 4 containing 1 mg / mL glucose (final concentration of glucose was 1 mg / mL) were measured at different time points using a pH meter. The results are shown in the attached figure. Figure 6 As shown in A, it is proved that GOx@HOF@Fe-TF has glucose oxidase activity.

[0053] The same method was used to prove that the HOF-HT prepared by the method described in Example 1 did not have glucose oxidase activity; while the GOx@HOF prepared by the method described in Example 4 had glucose oxidase activity, indicating that HOF-HT successfully encapsulated GOx.

[0054] 2. By detecting H 2 O 2 The yellow titanium peroxide complex formed by reacting with titanium sulfate has a characteristic absorption peak at 412 nm to detect H 2 O 2 Generation of.

[0055] 5 wt% titanium sulfate solution (final concentration of titanium sulfate is 50 mM) was added to a suspension of GOx@HOF@Fe-TF prepared by the method described in Example 4 with a concentration of 200 μg / mL, a glucose solution with a concentration of 1 mg / mL, and a mixed solution containing a suspension of GOx@HOF@Fe-TF prepared by the method described in Example 4 with a concentration of 200 μg / mL and 1 mg / mL of glucose, respectively, and incubated for 10 min. The characteristic absorption peak at 412 nm was detected. The results are shown in the attached figure. Figure 6 As shown in B.

[0056] 3. Prepare the sample as follows, incubate for 4 h after preparation, centrifuge, take the supernatant, and detect the absorbance of the supernatant at 652 nm using UV-visible spectroscopy.

[0057] Sample 1: GOx@HOF@Fe-TF (prepared as described in Example 4) suspension with a concentration of 200 μg / mL and 1 mg / mL glucose.

[0058] Sample 2: GOx@HOF@Fe-TF (prepared as described in Example 4) suspension at a concentration of 200 μg / mL containing 0.42 mM 3,3',5,5'-tetramethylbenzidine (TMB).

[0059] Sample 3: GOx@HOF@Fe-TF (prepared by the method described in Example 4) suspension with a concentration of 200 μg / mL containing 1 mg / mL glucose and 0.42 mM 3,3',5,5'-tetramethylbenzidine.

[0060] Sample 4: A suspension of GOx@HOF@Fe-TF (prepared by the method described in Example 4) containing 1 mg / mL glucose and 0.42 mM 3,3',5,5'-tetramethylbenzidine at a concentration of 200 μg / mL was subjected to light treatment at a light intensity of 1 W / cm 2 The samples were irradiated with 808 nm laser for 5 min.

[0061] The results are as attached Figure 6 As shown in C.

[0062] 4. Experimental results like Figure 6A-6B, GOx@HOF@Fe-TF has glucose oxidase activity and peroxidase activity, and can self-supply hydrogen peroxide and gluconic acid, thereby catalyzing Fe 3+ / Fe 2+ Fenton reaction occurs to produce ROS. Figure 6 C, the generation of oxTMB was measured by UV-visible spectroscopy, indicating that GOx@HOF@Fe-TF can self-supply H 2 O 2 , which makes up for the lack of hydrogen peroxide concentration in the tumor microenvironment. GOx@HOF@Fe-TF has photothermal properties, and the PTT effect can enhance the generation of oxTMB, indicating that the synergistic amplification of ROS between PTT and enzyme activity ( Figure 6 C).

[0063] Embodiment 11 Breast cancer cells (4T1 cells) were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO 2 Breast cancer cells were cultured in a 96-well plate, with 5000 cells added to each well. After 24 h, 5 μL of the suspension of GOx@HOF@Fe-TF prepared by the method described in Example 4 was added to make the final concentrations of GOx@HOF@Fe-TF in the plate 0, 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, and 75 μg / mL, respectively. After 6 h of culture, the samples of each concentration were cultured under the following conditions in 5 parallel groups. 1: Continue to culture at 37°C.

[0064] 2: At 1.0 W / cm 2 The cells were irradiated with 808 nm laser for 5 min and then cultured at 37 °C.

[0065] 3: At 0.6 W / cm 2 The cells were irradiated with 660 nm laser for 5 min and then cultured at 37 °C.

[0066] 4: 1.0 W / cm 2 808 nm, 0.6 W / cm 2 The cells were irradiated with 660 nm laser for 5 min and then cultured at 37 °C.

[0067] Cell viability = ×100%.

[0068] The cell survival rate was detected by MTT reagent, and the absorbance at 570 nm was measured by an enzyme marker to calculate the cell survival rate. Figure 7As shown in the figure, the inhibition rate of GOx@HOF@Fe-TF on the cell activity of 4T1 cells increases with the concentration. When the dose of GOx@HOF@Fe-TF reaches 75 μg / mL, the viability of 4T1 cells decreases to 20.2%, indicating that GOx@HOF@Fe-TF has a good inhibitory effect on 4T1 cells. After further laser irradiation (660 nm, 0.6 W / cm 2 ; 808 nm, 1.0 W / cm 2 ), 4T1 cell viability decreased to 7.1%, indicating that light can promote the tumor inhibition effect of GOx@HOF@Fe-TF.

[0069] Example 12 Normal human hepatocytes (LO2 cells) were cultured in 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO. 2 LO2 cells were cultured in a 96-well plate, and 8000 cells were added to each well. After 24 h, 5 μL of different concentrations of the suspension of GOx@HOF@Fe-TF prepared by the method described in Example 4 was added to make the final concentrations of GOx@HOF@Fe-TF in the well plate 0, 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL, respectively. After continuing to culture for 24 h, MTT reagent was added, and the cell viability was calculated by measuring the absorbance value at 570 nm using an enzyme marker. The results are shown in the attached figure. Figure 8 As shown in the figure, GOx@HOF@Fe-TF has little effect on the cell viability of LO2 cells. This shows that GOx@HOF@Fe-TF has basically no toxic side effects on normal cells and can specifically kill tumor cells, which indirectly verifies the targeting of GOx@HOF@Fe-TF.

[0070] Embodiment 13 Breast cancer cells (4T1 cells) were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO 2 . Breast cancer cells were cultured in a 96-well plate, with 5000 cells added to each well. After 22 h, 5 μL of PBS buffer containing 100 μM of the ferroptosis inhibitor Fer-1 or an equal volume of PBS buffer was added, and the culture was continued for 2 h. Then, 5 μL of the suspension of GOx@HOF@Fe-TF prepared by the method described in Example 4 (the final concentration in the well plate was 50 μg / mL) or an equal volume of PBS buffer was added. The dosing method is shown in Table 1. After continuing to culture for 24 h, MTT reagent was added, and the absorbance value of the cells at 570 nm was measured using an enzyme marker to calculate the cell survival rate. The results are shown in the attached figure. Fig. 9As shown, after 4T1 cells were treated with GOx@HOF@Fe-TF, the cell viability reached 44.1%, while after the addition of ferroptosis inhibitors, the 4T1 cell viability increased to 62.0%, indicating that ferroptosis is one of the pathways of cell death induced by GOx@HOF@Fe-TF.

[0071] Table 1 Embodiment 14 ROS detection experiment: 4T1 cells (20,000 cells / well) were cultured in 24-well plates in DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin solution) at 37 °C and 5% CO 2 . Culture for 24 h until the cells adhered to the wall. Add 5 μL of the GOx@HOF@Fe-TF suspension prepared by the method described in Example 4 (final concentration in the well plate was 25 μg / mL) or an equal volume of PBS buffer solution, and irradiate the illumination group 6 h later (illumination conditions were 808 nm, 1.0 W / cm 2 ; 5 min; 660 nm, 0.6 W / cm 2 , 5 min), the dosing method is shown in Table 2, the original culture medium was discarded, and the serum-free DMEM medium containing 5 μM DHE probe was used for another 30 min. The cells were then washed three times with PBS, and the generation of intracellular ROS was measured using a fluorescence microscope. The results are shown in the attached figure. Fig.10 As shown, it is proved that GOx@HOF@Fe-TF can generate ROS in cells, and both photothermal effect and photodynamic effect can promote the generation of ROS.

[0072] Table 2 Embodiment 15 The changes in the content of ferroptosis markers MDA (malondialdehyde) and GPX4 (glutathione peroxidase 4) in cells were measured to verify whether GOx@HOF@Fe-TF could induce ferroptosis.

[0073] 4T1 cells were cultured in DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin solution) and kept in 5% carbon dioxide at 37°C. 4T1 cells were cultured at 2×10 4The cells were implanted in a 24-well plate at a density of 100 μM and cultured for 24 h. Then, the cells were divided into two groups. One group was added with 5 μL of PBS buffer containing 100 μM of ferroptosis inhibitor Fer-1 or an equal volume of PBS buffer and cultured for 2 h to detect MDA. The other group was cultured for 2 h without adding drugs. 5 μL of the suspension of GOx@HOF@Fe-TF prepared by the method described in Example 4 (the final concentration in the well plate was 50 μg / mL) or an equal volume of PBS was added to the culture medium of the two groups as shown in Table 3. For the illumination group, the cells were illuminated 6 h after administration (illumination conditions were 808 nm, 1.0 W / cm 2 , 5 min; 660nm, 0.6 W / cm 2 , 5 min). Continue to incubate for 12 h, take out the 24-well plate and process it according to the corresponding ELISA kit to determine the changes in its content. The results are as follows: Fig.11 , Attachment Fig.12 As shown, GOx@HOF@Fe-TF has the ability to reduce GPX4 and increase MDA content, and light can aggravate lipid peroxidation, further indicating that GOx@HOF@Fe-TF induces cell death by activating the ferroptosis pathway.

[0074] Table 3 Example 16 4T1 tumor-bearing mice (6 weeks, female) were randomly divided into 6 groups, 6 in each group. They were 1: PBS group, 2: PBS+808 nm+660 nm group, 3: GOx@HOF@Fe-TF group, 4: GOx@HOF@Fe-TF+808nm group, 5: GOx@HOF@Fe-TF+660 nm group, 6: GOx@HOF@Fe-TF+808nm+660 nm group. On day 0 and day 4, mice were injected with 100 μL PBS buffer or 100 μL PBS buffer containing 1 mg / mL GOx@HOF@Fe-TF by tail vein. After 6 h of administration, the mice were exposed to 1.0 W / cm 2 808 nm laser, 0.6 W / cm 2 The mice in the corresponding laser group were irradiated with 660 nm laser for five minutes. GOx@HOF@Fe-TF was prepared by the method described in Example 4.

[0075] After 14 days, the mice were euthanized, their tumors were photographed and weighed, and their spleens were collected for immune cell analysis.

[0076] Fig.13As shown in A-13C, GOx@HOF@Fe-TF has a significant inhibitory effect on tumor growth rate. The final group GOx@HOF@Fe-TF+660 nm+808 nm has an inhibition rate of 98.3% on tumors, indicating that GOx@HOF@Fe-TF has a more excellent anti-tumor effect under laser irradiation. Fig.13 As shown in D, there was no significant fluctuation in the weight of mice during the treatment period, indicating that the mice tolerated GOx@HOF@Fe-TF well. H&E staining results showed that the GOx@HOF@Fe-TF plus light group could significantly increase tumor necrosis and strongly inhibit the proliferation of tumor cells ( Fig.13 E), which is consistent with the results of the tumor growth curve.

[0077] The tumors of mice in different groups were surgically removed. The cells were digested with a digestion solution containing 1 mg / mL collagenase IV, 0.1 mg / mL hyaluronidase and 1640 culture medium at 37°C for 30 min, then centrifuged for red cell lysis, and the single cell suspension was collected by filtering with a 70 μm filter. Part of the collected cells were incubated with anti-CD3, anti-CD4 and anti-CD8 antibodies, and the other part of the collected cells were stained with anti-CD11, anti-CD80 and anti-CD86 antibodies. After antibody staining, the cells were washed with PBS, and the single cell suspension was collected by filtering with a 40 μm filter for cell analysis. The detection was carried out according to the standard protocol, and flow cytometry was used to detect DC cell maturity, CD4 + and CD8 + Changes in cell content.

[0078] like Fig.14 As shown in A-14D, the DC cell and T cell contents of mice in the treatment group increased compared with those in the PBS group, and the final group (GOx@HOF@Fe-TF plus light irradiation group) could significantly promote DC cell maturation, stimulate T cell differentiation, and thereby activate tumor immunity.

[0079] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A porphyrin hydrogen-bonded organic framework, characterized in that: The invention is prepared from 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene and 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphyrin.

2. A porphyrin hydrogen-bonded organic framework according to claim 1, characterized in that: The preparation steps are as follows: S1: Add 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene to water, adjust the pH to 7-8 with an alkaline solution to form solution A, and dissolve 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphyrin in water to form solution B; S2: Solution A and solution B were mixed, stirred at 500-1000 rpm for 1-3 h at room temperature in the dark, centrifuged, and washed with water to obtain a hydrogen-bonded organic framework HOF-HT.

3. A porphyrin hydrogen-bonded organic framework according to claim 2, characterized in that: The molar ratio of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene to 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphyrin in the mixed solution of S2 is 3.5-4.5:1, preferably 4:

1.

4. A method for preparing a nanocomposite material based on the porphyrin hydrogen-bonded organic framework according to claim 1, characterized in that: It includes the following steps: S1: Add 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene to water, adjust the pH to 7-8 with an alkaline solution to form solution A, and dissolve 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphyrin in water to form solution B; S2: Mix glucose oxidase with solution B, stir at room temperature for 10-30 min, then add solution A to the solution, stir at 500-1000 rpm for 1-3 h at room temperature in the dark, centrifuge and wash with water to obtain the hydrogen-bonded organic framework GOx@HOF encapsulating the natural enzyme; S3: Add the GOx@HOF obtained in S2 into water, add FeCl3 solution, ultrasonically disperse, and stir at 500-1000 rpm for 20-40 min. Then add tannic acid solution and folic acid solution, ultrasonically disperse, and stir at 500-1000 rpm for 20-40 min. Centrifuge and wash with water to obtain a new nanocomposite material GOx@HOF@Fe-TF.

5. The method for preparing the nanocomposite material according to claim 4, characterized in that: The dosage of glucose oxidase, 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene and 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphyrin in the mixed solution S2 is 0.3-0.45 g: 3.5-4.5 mmol: 1 mmol.

6. The method for preparing the nanocomposite material according to claim 4, characterized in that: The dosage relationship of glucose oxidase, 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene and 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphyrin in the mixed solution S2 is 0.37 g: 4 mmol: 1 mmol.

7. The method for preparing the nanocomposite material according to claim 4, characterized in that: The mass ratio of GOx@HOF, FeCl3, tannic acid and folic acid in S3 is 1:0.3-0.45:0.6-0.8:0.06-0.08, preferably 1:0.37:0.7:0.

07.

8. A nanocomposite material obtained by the preparation method according to any one of claims 4 to 7.

9. Use of the porphyrin hydrogen-bonded organic framework according to claim 1 or 2 and the nanocomposite material according to claim 8 in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that: The anti-tumor drug is a drug that triggers ferroptosis of tumor cells.

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