A nanocomposite material based on a porphyrin hydrogen-bonded organic framework, its preparation method and anti-tumor application.

Abstract

This invention relates to a nanocomposite material based on a porphyrin hydrogen-bonded organic framework, its preparation method, and its antitumor application. This application discloses a small-sized hydrogen-bonded organic framework prepared from 1,3,6,8-tetra(4-carboxyphenyl)pyrene and 5,10,15,20-tetra(4-aminophenyl)-21H,23H-porphyrin, and based on this framework, constructs a novel nanocomposite material that is small in size, possesses excellent photothermal properties, photodynamic properties, and antitumor activity, and efficiently triggers ferroptosis in tumor cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of material chemistry and biology technology, and particularly relates to a kind of nano-composite material based on porphyrin hydrogen bond organic framework and its preparation method and antitumor application. BACKGROUND

[0002] Inducing cell death is an important way of cancer treatment, and traditional cell death induction methods are often limited by treatment resistance, so exploring new cell death pathways has become a research hotspot in oncology.

[0003] Ferroptosis is an iron-dependent cell programmed death method characterized by accumulation of lipid peroxides. In recent years, studies have shown that ferroptosis is related to drug resistance in cancer treatment, and activation of tumor cell ferroptosis is an effective strategy to reverse tumor drug resistance. Due to the complexity of tumor microenvironment (TME), the interaction between TME and ferroptosis affects a large number of studies showing 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 immunosuppressive microenvironment of tumors. Considering that drugs for immunotherapy need to have high biocompatibility, and nano-drug delivery systems have the advantages of high biocompatibility and good targeting, the development of nano-immunological drug delivery systems based on ferroptosis has become a research hotspot in the field of biological medicine in recent years.

[0004] CN201910937168.8 discloses a metal-organic nanocomposite for triggering tumor cell ferroptosis, which has biocompatibility by wrapping hyaluronic acid on its surface through electrostatic adsorption and ionic cross-linking. The preparation process is relatively complicated. Hydrogen bond organic framework (HOFs) is a new type of porous crystalline material constructed by hydrogen bonds between organic components. Compared with covalent organic frameworks and metal-organic frameworks, HOFs are composed of hydrogen bonds, which 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 tumor sites. Therefore, it is urgent to explore suitable organic monomers and synthesis conditions to prepare smaller size HOFs as a hydrogen bond organic framework platform and construct nano-materials with targeting for triggering tumor cell ferroptosis based on it. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a kind of nano-composite material based on porphyrin hydrogen bond organic framework and its preparation method and antitumor application.

[0007] To solve the above problems, the technical solution adopted by the present application is:

[0008] Technical subject one

[0009] A porphyrin hydrogen-bonded organic framework is prepared from 1,3,6,8-tetra(4-carboxyphenyl)pyrene and 5,10,15,20-tetra(4-aminophenyl)-21H,23H-porphyrin.

[0010] As a further improvement of the present invention, the preparation steps of the porphyrin hydrogen-bonded organic framework are as follows:

[0011] S1: Add 1,3,6,8-tetra(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-tetra(4-aminophenyl)-21H,23H-porphyrin in water to form solution B;

[0012] S2: Mix solutions A and B, stir at 500-1000 rpm for 1-3 h at room temperature in the dark, centrifuge and wash with water to obtain hydrogen-bonded organic framework HOF-HT.

[0013] 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 mixture of S2 is 3.5-4.5:1;

[0014] As a further improvement of the present invention, the molar ratio of 1,3,6,8-tetra(4-carboxyphenyl)pyrene to 5,10,15,20-tetra(4-aminophenyl)-21H,23H-porphyrin is 4:1.

[0015] Technical Theme Two

[0016] A method for preparing a nanocomposite material based on the porphyrin hydrogen-bonded organic framework described in Technical Topic 1, comprising the following steps:

[0017] S1: Add 1,3,6,8-tetra(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-tetra(4-aminophenyl)-21H,23H-porphyrin in water to form solution B;

[0018] S2: Mix glucose oxidase with solution B and stir at room temperature for 10-30 min. Then add solution A to this solution and 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 that encapsulates the natural enzyme.

[0019] S3: Add the GOx@HOF obtained in S2 to water, add FeCl3 solution, sonicate and disperse, stir at 500-1000 rpm for 20-40 min, then add tannic acid solution and folic acid solution, sonicate and disperse, stir at 500-1000 rpm for 20-40 min, centrifuge and wash with water to obtain the nanocomposite material GOx@HOF@Fe-TF.

[0020] As a further improvement of the present invention, the amounts of glucose oxidase, 1,3,6,8-tetra(4-carboxyphenyl)pyrene and 5,10,15,20-tetra(4-aminophenyl)-21H,23H-porphyrin in S2 are in the following ratio: 0.3-0.45 g: 3.5-4.5 mmol: 1 mmol.

[0021] As a further improvement of the present invention, the amounts of glucose oxidase, 1,3,6,8-tetra(4-carboxyphenyl)pyrene and 5,10,15,20-tetra(4-aminophenyl)-21H,23H-porphyrin in S2 are in the following ratio: 0.37 g: 4 mmol: 1 mmol.

[0022] As a further improvement of the present invention, 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.

[0023] As a further improvement of the present invention, the mass ratio of GOx@HOF, FeCl3, tannic acid and folic acid in S3 is 1:0.37:0.7:0.07.

[0024] Technical Theme 3

[0025] A nanocomposite material obtained by the preparation method described in Technical Subject 2.

[0026] Technical Theme 4

[0027] The application of a porphyrin hydrogen-bonded organic framework as described in Technical Subject 1 and a nanocomposite material as described in Technical Subject 3 in the preparation of antitumor drugs.

[0028] As a further improvement of the present invention, the antitumor drug is a drug that triggers ferroptosis in tumor cells.

[0029] The beneficial effects of adopting the above technical solution are as follows:

[0030] 1. The HOFs prepared in this application have small size and good cell permeability. Based on their construction of composite materials for triggering tumor cell ferroptosis, a new approach is provided for the preparation of drugs that trigger tumor cell ferroptosis, and the biological application pathways of HOFs can be broadened.

[0031] 2. This application constructs a composite material based on HOFs, and introduces glucose oxidase GOx into smaller HOFs to form GOx@HOF, giving it glucose oxidase activity, without significantly changing its shape and size.

[0032] 3. The Fe of GOx@HOF@Fe-TF disclosed in this application 3+ - The TF layer exhibits excellent photothermal therapy (PTT) and chemokinetic therapy (CDT) capabilities. PTT promotes CDT, stimulating the generation of more hydroxyl radicals and further increasing reactive oxygen species (ROS) levels. GOx@HOF possesses glucose oxidase activity and photodynamic therapy (PDT), which can consume intratumoral glucose while lowering pH and increasing hydrogen peroxide content in the tumor microenvironment, providing raw materials for the subsequent Fenton reaction. 3+ -TF and GOx@HOF work synergistically to further amplify oxidative stress and enhance ferroptosis. Attached Figure Description

[0033] Figure 1 This is a flowchart illustrating the preparation process of GOx@HOF@Fe-TF in the examples;

[0034] Figure 2 These are transmission electron microscope (TEM) images of the materials prepared in this application, where A is a TEM image of HOF-H prepared in Comparative 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.

[0035] Figure 3 This is the infrared spectrum of the material prepared in this application, wherein HOF is HOF-HT prepared by the method described in Example 1 of this application, GOx@HOF is prepared by the method described in Example 4 of this application, GOx@HOF@Fe-TF is prepared by the method described in Example 4 of this application, TAPP is the raw material 5,10,15,20-tetra(4-aminophenyl)-21H,23H-porphyrin used in the examples, and H4TBAPy is the raw material 1,3,6,8-tetra(4-carboxyphenyl)pyrene used in the examples;

[0036] Figure 4 This is a powder X-ray diffraction (PXRD) pattern of the material prepared in this application, wherein HOF is HOF-HT prepared by the method described in Example 1 of this application, GOx@HOF is prepared by the method described in Example 4 of this application, and GOx@HOF@Fe-TF is prepared by the method described in Example 4 of this application;

[0037] Figure 5This is a graph showing the photothermal and photodynamic properties of GOx@HOF@Fe-TF, where A represents the photothermal and photodynamic properties of GOx@HOF@Fe-TF suspensions at different concentrations after treatment with 1 W / cm². 2 A) Temperature change after 808 nm laser irradiation for 8 min; B) Temperature change of 200 μg / mL GOx@HOF@Fe-TF suspension after irradiation with 808 nm lasers of different powers; C) Temperature change of 200 μg / mL GOx@HOF@Fe-TF suspension after irradiation with 1.5 W / cm² lasers. 2 Temperature changes after five light-cooling cycles following 808 nm laser irradiation for 5 min and cooling to room temperature; D is the ESR diagram of samples treated with different methods. Wherein, 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+660 nm group.

[0038] Figure 6 These are the enzyme activity graphs of GOx@HOF@Fe-TF. A is a comparison graph of pH changes over time for GOx@HOF@Fe-TF suspension and GOx@HOF@Fe-TF suspension containing glucose; B is the UV-Vis absorption spectrum of each solution containing titanium sulfate; C is the UV-Vis absorption spectrum of each solution at 652 nm, where laser is the wavelength of light emitted by a laser with a wavelength of 1 W / cm². 2 Irradiation with an 808 nm laser for 5 minutes; Glu represents glucose.

[0039] Figure 7 This is a graph showing the changes in breast cancer cell survival rate with varying concentrations of GOx@HOF@Fe-TF, the presence or absence of 808 nm laser irradiation, and the presence or absence of 660 nm laser irradiation.

[0040] Figure 8 This is a graph showing the change in survival rate of normal human hepatocytes (LO2 cells) with the concentration of GOx@HOF@Fe-TF;

[0041] Figure 9 This is a graph 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;

[0042] Figure 10These are ROS generation maps of each group detected using a DHE (dihydroethidium) probe under a fluorescence microscope. Groups 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.

[0043] Figure 11 This is a graph showing the intracellular malondialdehyde (MDA) content in breast cancer cells after different treatments. Groups 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.

[0044] Figure 12 This is a graph showing the glutathione peroxidase 4 (GPX4) content in breast cancer cells after different treatments. Groups 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.

[0045] Figure 13 This is a graph showing the tumor inhibition effect of GOx@HOF@Fe-TF on 4T1 tumor-bearing mice. A shows the change in tumor volume over days in mice in Example 16; B shows photographs of tumors in each group; C shows a comparison of tumor mass in each group; D shows the change in body weight of mice in each group over 14 days; and E shows H&E staining images of tumor tissue sections in each group. In A and E, 1-6 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+808 nm group, 5 is the GOx@HOF@Fe-TF+660 nm group, and 6 is the GOx@HOF@Fe-TF+808 nm+660 nm group.

[0046] Figure 14 This is a graph showing the content of immune cells in a single-cell suspension of mouse spleen from Example 16, where A represents CD4 immune cells. + B is an immune cell CD8. + C is the immune cell CD86. + D is the CD80 of immune cells. +In AD, 1-6 are as described in Example 16, where 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+808 nm group, 5 is the GOx@HOF@Fe-TF+660 nm group, and 6 is the GOx@HOF@Fe-TF+808 nm+660 nm group. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described clearly and completely below in conjunction with specific embodiments.

[0048] The concentration of PBS buffer used in the examples was 0.01 M.

[0049] Preparation Example 1

[0050] Add 8.6 mg of 1,3,6,8-tetra(4-carboxyphenyl)pyrene (H4TBAPy) to 0.8 mL of redistilled water, adjust the pH to 7-8 with ammonia, and add redistilled water to bring the solution to 1 mL to form solution A.

[0051] Preparation Example 2

[0052] Dissolve 17 mg of 5,10,15,20-tetra(4-aminophenyl)-21H,23H-porphyrin (TAPP) in 1 mL of redistilled water to form solution B.

[0053] Example 1

[0054] Take 555 μL of solution A (obtained by the preparation method in Preparation Example 1), add 70 μL of solution B (obtained by the preparation method in Preparation Example 2) and 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.

[0055] Example 2

[0056] Take 633 μL of solution A (obtained by the preparation method in Preparation Example 1), add 70 μL of solution B (obtained by the preparation method in Preparation Example 2) and 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.

[0057] Example 3

[0058] Take 492 μL of solution A (obtained by the preparation method in Preparation Example 1), add 70 μL of solution B (obtained by the preparation method in Preparation Example 2) and 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.

[0059] Example 4

[0060] The flowchart for the preparation of GOx@HOF@Fe-TF is attached. Figure 1 As shown.

[0061] 0.67 mg of glucose oxidase (GOx) was mixed with 70 μL of solution B (obtained by the method in Preparation Example 2) and stirred at room temperature for 20 min to obtain a suspension. Then, solution A (obtained by the method in Preparation Example 1, 12.6 mM, 557 μL) and 255 μL of redistilled water were added to the suspension, and the mixture was stirred at 800 rpm at room temperature in the dark for 2 h. After centrifugation and washing with water three times, GOx@HOF was obtained.

[0062] 0.7 mg of GOx@HOF prepared as described in Example 4 was dispersed in 1 mL of deionized water, and 0.2 mL of FeCl3 solution (1.3 mg / mL) was added. The mixture was sonicated for 10 s and stirred at 750 rpm for 30 min. Subsequently, 0.1 mL of tannic acid solution (5 mg / mL) and 0.1 mL of folic acid solution (0.5 mg / mL) were added, and the mixture was sonicated for 10 s and stirred at 750 rpm for 30 min. The mixture was then centrifuged and washed three times with water to obtain GOx@HOF@Fe-TF.

[0063] Example 5

[0064] 0.53 mg of glucose oxidase (GOx) was mixed with 70 μL of solution B (obtained by the method in Preparation Example 2) and stirred at room temperature for 20 min to obtain a suspension. Then, solution A (obtained by the method in Preparation Example 1, 12.6 mM, 492 μL) and 255 μL of redistilled water were added to the suspension, and the mixture was stirred at 500 rpm at room temperature in the dark for 1 h. After centrifugation and washing with water three times, GOx@HOF was obtained.

[0065] 0.7 mg of GOx@HOF prepared as described in Example 5 was dispersed in 1 mL of deionized water, and 0.16 mL of FeCl3 solution (1.3 mg / mL) was added. The mixture was sonicated for 10 s and stirred at 500 rpm for 30 min. Subsequently, 0.08 mL of tannic acid solution (5 mg / mL) and 0.08 mL of folic acid solution (0.5 mg / mL) were added, and the mixture was sonicated for 10 s and stirred at 500 rpm for 30 min. The mixture was then centrifuged and washed three times with water to obtain GOx@HOF@Fe-TF.

[0066] Example 6

[0067] 0.79 mg of glucose oxidase (GOx) was mixed with 70 μL of solution B (obtained by the method in Preparation Example 2) and stirred at room temperature for 20 min to obtain a suspension. Then, solution A (obtained by the method in Preparation Example 1, 12.6 mM, 633 μL) and 255 μL of redistilled water were added to the suspension, and the mixture was stirred at 1000 rpm at room temperature in the dark for 3 h. After centrifugation and washing with water three times, GOx@HOF was obtained.

[0068] Disperse 0.7 mg of GOx@HOF prepared as described in Example 6 in 1 mL of deionized water, add 0.24 mL of FeCl3 solution (1.3 mg / mL), sonicate for 10 s, and stir at 1000 rpm for 30 min. Then add 0.11 mL of tannic acid solution (5 mg / mL) and 0.11 mL of folic acid solution (0.5 mg / mL), sonicate for 10 s, and stir at 1000 rpm for 30 min. Centrifuge and wash three times with water to obtain GOx@HOF@Fe-TF.

[0069] Comparative Example 1

[0070] Dissolve 8.6 mg of 1,3,6,8-tetra(4-carboxyphenyl)pyrene (H4TBAPy) in 0.8 mL of redistilled water, adjust the pH to 7-8 with ammonia, and add redistilled water to bring the solution to 1 mL to form solution A. Take 555 μL of solution A, add 325 μL of redistilled water, stir at 800 rpm at room temperature in the dark for 2 h, centrifuge, and wash three times with water to obtain HOF-H.

[0071] Example 7

[0072] The HOF-H in Comparative Example 1 (attached) was examined using a transmission electron microscope. 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) Observations showed that, compared with HOF-H in Comparative Example 1, the HOF-HT disclosed in this application has a smaller size of only about 300-500 nm, providing a new approach for preparing drugs that trigger ferroptosis in tumor cells and broadening the biological application pathways of HOFs. (See attached...) Figure 2 As shown in Figure C, introducing glucose oxidase GOx into smaller HOFs did not significantly alter their shape or size. (See attached figure.) Figure 2 As shown in Figure D, Fe is introduced through the coordination of metal ions. 3+ Tannic acid and folic acid form a metal-polyphenol network on the surface of GOx@HOF.

[0073] The HOF-HT in Example 1, GOx@HOF in Example 4, and GOx@HOF@Fe-TF in Example 4 were detected by infrared spectroscopy, and the results are shown in the appendix. Figure 3 As shown, it can be seen that compared with TAPP and H4TBAPy, the 1691 cm⁻¹ of the Fourier transform infrared (FTIR) spectrum of HOF-HT is significantly higher. -1 C=O tensile vibration and at 1609 cm -1 The offset of C=N tensile vibration indicates that the tensile motion of C=O and C=N is restricted, suggesting that a rigid structure connected by hydrogen bonds is formed in HOF-HT.

[0074] 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 appendix. Figure 4 As shown, the analysis confirms that encapsulating GOx in HOF-HT has little impact on the crystal structure of HOF-HT.

[0075] Example 8

[0076] 1. The 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.

[0077] At 1W / cm 2 Temperature changes were detected and recorded within 0–8 min by irradiating redistilled water with an 808 nm laser and by using GOx@HOF@Fe-TF suspensions of different concentrations (100 μg / mL, 200 μg / mL, and 400 μg / mL). The results are shown in the attached figure. Figure 5 As shown in Figure A.

[0078] 2. Irradiation with different 808 nm laser powers (0.5 W / cm²) 2 1 W / cm 2 1.5 W / cm 2 The results of irradiating 200 μg / mL GOx@HOF@Fe-TF suspension with temperature changes recorded within 0-8 min are shown in the attached figure. Figure 5 As shown in B.

[0079] 3. At 1.5 W / cm 2 Irradiation of a 200 μg / mL GOx@HOF@Fe-TF suspension with an 808 nm laser for 5 min, followed by natural cooling to room temperature and cycling 5 times, is shown in the attached figure. Figure 5 As shown in C.

[0080] Appendix Figure 5As shown in Figures A and 5B, the thermometer readings demonstrate that GOx@HOF@Fe-TF possesses photothermal capabilities, and the photothermal effect is positively correlated with the concentration of GOx@HOF@Fe-TF and the laser irradiation power. Furthermore, after five light-cooling cycles, the temperature of GOx@HOF@Fe-TF remained stable, exhibiting good photothermal stability. Figure 5 C).

[0081] Example 9: Detection of Photodynamic Properties

[0082] The following groups of samples were analyzed using ESR (electron spin resonance):

[0083] Sample 1: 500 μL of redistilled water with 10 μL of 2,2,6,6-tetramethylpiperidine (TEMP trapping agent) added, and heated at a power of 0.6 W / cm². 2 Irradiate with 660 nm laser for 5 minutes.

[0084] Sample 2: 500 μL of a HOF-HT suspension with a concentration of 200 μg / mL prepared according to the method described in Example 1, and tested with a power of 0.6 W / cm². 2 Irradiate with 660 nm laser for 5 minutes.

[0085] Sample 3: 500 μL of a GOx@HOF@Fe-TF suspension prepared by the method described in Example 4 with 10 μL of TEMP capture agent and incubated for 5 min in the dark.

[0086] Sample 4: 500 μL of HOF-HT suspension prepared according to the method described in Example 1, with 10 μL of TEMP scavenging agent added, and controlled at a power of 0.6 W / cm². 2 Irradiate with 660 nm laser for 5 minutes.

[0087] Sample 5: A GOx@HOF@Fe-TF suspension was prepared by adding 10 μL of TEMP capture agent to a concentration of 200 μg / mL using the method described in Example 4, and the suspension was controlled at a power of 0.6 W / cm². 2 Irradiate with 660 nm laser for 5 minutes.

[0088] The results are attached. Figure 5As shown in Figure D, the ESR spectra of HOF-HT and GOx@HOF@Fe-TF suspensions containing TEMP trapping agent after irradiation at 660 nm show obvious 1:1:1 peaks, indicating that GOx@HOF@Fe-TF and HOF-HT have the ability to generate singlet oxygen under laser irradiation, and HOF-HT and GOx@HOF@Fe-TF have good photodynamic properties.

[0089] Example 10

[0090] 1. The generation of gluconic acid was detected by measuring the pH value of the mixture of GOx@HOF@Fe-TF prepared by the method described in Example 4 and glucose.

[0091] The pH values ​​of the GOx@HOF@Fe-TF suspension (200 μg / mL prepared according to the method described in Example 4) and the GOx@HOF@Fe-TF suspension (200 μg / mL containing 1 mg / mL glucose prepared according to the method described in Example 4, with a final glucose concentration of 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 Figure A, it is demonstrated that GOx@HOF@Fe-TF possesses glucose oxidase activity.

[0092] Using the same method, it was demonstrated that HOF-HT prepared by the method described in Example 1 does not possess glucose oxidase activity; GOx@HOF prepared by the method described in Example 4 does possess glucose oxidase activity, indicating that HOF-HT successfully encapsulates GOx.

[0093] 2. The formation of H2O2 was detected by detecting the characteristic absorption peak at 412 nm of the yellow titanium peroxide complex formed by the reaction of H2O2 and titanium sulfate.

[0094] 5 wt% titanium sulfate solution (final titanium sulfate concentration of 50 mM) was added to a suspension of GOx@HOF@Fe-TF prepared by the method described in Example 4 at a concentration of 200 μg / mL, a glucose solution at a concentration of 1 mg / mL, and a mixture containing a suspension of GOx@HOF@Fe-TF prepared by the method described in Example 4 and 1 mg / mL glucose, respectively. The mixtures were incubated for 10 min, and the characteristic absorption peak at 412 nm was detected. The results are shown in the attached figure. Figure 6 As shown in B.

[0095] 3. Prepare the sample as described below, incubate for 4 hours, centrifuge, take the supernatant, and use UV-Vis spectroscopy to detect the absorbance of the obtained supernatant at 652 nm.

[0096] Sample 1: A GOx@HOF@Fe-TF suspension (prepared by the method described in Example 4) containing 1 mg / mL glucose at a concentration of 200 μg / mL.

[0097] Sample 2: A GOx@HOF@Fe-TF suspension (prepared by the method described in Example 4) containing 0.42 mM 3,3',5,5'-tetramethylbenzidine (TMB) at a concentration of 200 μg / mL.

[0098] Sample 3: A GOx@HOF@Fe-TF suspension (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.

[0099] Sample 4: A suspension of GOx@HOF@Fe-TF (prepared as 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, and this sample was subjected to light treatment at 1 W / cm². 2 Irradiate with an 808 nm laser for 5 minutes.

[0100] The results are attached. Figure 6 As shown in C.

[0101] 4. Explanation of Experimental Results

[0102] like Figure 6 A-6B, GOx@HOF@Fe-TF possesses both glucose oxidase and peroxidase activities, and can self-supply hydrogen peroxide and gluconic acid, thereby catalyzing Fe... 3+ / Fe 2+ The Fenton reaction occurs, producing ROS. For example... Figure 6 As shown in Figure C, the generation of oxTMB was determined using UV-Vis spectroscopy, indicating that GOx@HOF@Fe-TF can self-supply H2O2, compensating for the insufficient hydrogen peroxide concentration in the tumor microenvironment. GOx@HOF@Fe-TF possesses photothermal properties, and PTT can enhance oxTMB generation, indicating a synergistic amplification of ROS by PTT and enzyme activity. Figure 6 C).

[0103] Example 11

[0104] Breast cancer cells (4T1 cells) were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. 5000 cells were added to each well of a 96-well plate. After 24 h, 5 μL of GOx@HOF@Fe-TF suspensions prepared according to the method described in Example 4 were added to bring the final concentrations of GOx@HOF@Fe-TF in the wells to 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 further cultured under the following conditions, in five parallel groups.

[0105] 1: Continue culturing at 37 ℃.

[0106] 2: At 1.0 W / cm 2 Irradiate with an 808 nm laser for 5 min, then continue culturing at 37 ℃.

[0107] 3: at 0.6 W / cm 2 Irradiate with 660 nm laser for 5 min, then continue culturing at 37 ℃.

[0108] 4: At 1.0 W / cm 2 808 nm, 0.6 W / cm 2 Irradiate with 660 nm laser for 5 min, then continue culturing at 37 ℃.

[0109] Cell viability = ×100%.

[0110] Cell viability was detected using the MTT assay. The absorbance at 570 nm was measured using a microplate reader, and the cell viability was calculated. The results are shown in the attached figure. Figure 7 As shown, the inhibition rate of GOx@HOF@Fe-TF on 4T1 cell viability increased with concentration. When the GOx@HOF@Fe-TF dose reached 75 μg / mL, the viability of 4T1 cells decreased to 20.2%, indicating that GOx@HOF@Fe-TF has a good inhibitory effect on 4T1 cells. Further laser irradiation (660 nm, 0.6 W / cm²) further enhanced the inhibition. 2 808 nm, 1.0 W / cm 2 The viability of 4T1 cells decreased to 7.1%, indicating that light exposure can promote the tumor-suppressive effect of GOx@HOF@Fe-TF.

[0111] Example 12

[0112] Human normal hepatocytes (LO2 cells) were cultured in 1640 medium containing 10% fetal bovine serum at 37 °C and 5% CO2. LO2 cells were cultured in 96-well plates, with 8000 cells added to each well. After 24 h, 5 μL of GOx@HOF@Fe-TF suspensions prepared according to the method described in Example 4 were added to bring the final concentrations of GOx@HOF@Fe-TF in the wells to 0, 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL, respectively. After another 24 h of culture, MTT reagent was added, and cell viability was calculated by measuring the absorbance at 570 nm using a microplate reader. The results are shown in the attached figure. Figure 8 As shown, GOx@HOF@Fe-TF had little effect on the cell viability of LO2 cells. This indicates that GOx@HOF@Fe-TF has virtually no toxic side effects on normal cells and can specifically kill tumor cells, indirectly verifying the targeting ability of GOx@HOF@Fe-TF.

[0113] Example 13

[0114] Breast cancer cells (4T1 cells) were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. 5000 cells were added to each well of a 96-well plate. After 22 h, 5 μL of PBS buffer containing 100 μM ferroptosis inhibitor Fer-1 or an equal volume of PBS buffer was added, and the cells were cultured for another 2 h. Then, 5 μL of a suspension of GOx@HOF@Fe-TF prepared as described in Example 4 (final concentration in the plate was 50 μg / mL) or an equal volume of PBS buffer was added, as shown in Table 1. After 24 h of further culture, MTT reagent was added, and the absorbance at 570 nm was measured using a microplate reader. Cell viability was calculated, and the results are shown in the attached table. Figure 9 As shown, the viability of 4T1 cells after treatment with GOx@HOF@Fe-TF reached 44.1%, while the viability of 4T1 cells increased to 62.0% after the addition of ferroptosis inhibitors, indicating that ferroptosis is one of the pathways by which GOx@HOF@Fe-TF induces cell death.

[0115] Table 1

[0116]

[0117] Example 14

[0118] ROS assay: 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% CO2. Cells were cultured for 24 h until adherence. 5 μL of a suspension of GOx@HOF@Fe-TF prepared as described in Example 4 (final concentration in the plate was 25 μg / mL) or an equal volume of PBS buffer was added for treatment. After 6 h, the light-treated group was exposed to light (808 nm, 1.0 W / cm²). 2 ; 5 min; 660 nm, 0.6 W / cm 2 The cells were incubated for 30 minutes (5 min) with the drug addition method shown in Table 2. The original culture medium was discarded, and the cells were incubated for another 30 minutes in serum-free DMEM medium containing 5 μM DHE probe. The cells were then washed three times with PBS, and intracellular ROS production was measured using a fluorescence microscope. The results are shown in the attached table. Figure 10 As shown, this demonstrates that GOx@HOF@Fe-TF can generate ROS in cells, and that both photothermal and photodynamic effects promote ROS generation.

[0119] Table 2

[0120]

[0121] Example 15

[0122] The study aimed to verify whether GOx@HOF@Fe-TF could induce ferroptosis by measuring changes in the levels of ferroptosis markers MDA (malondialdehyde) and GPX4 (glutathione peroxidase 4) in cells.

[0123] 4T1 cells were cultured in DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin solution) and stored in 5% carbon dioxide at 37 °C. 4T1 cells were then cultured at a rate of 2 × 10⁻⁶ cells / year. 4 After being implanted into 24-well plates and cultured for 24 hours, the cells were divided into two groups. One group was incubated for 2 hours with 5 μL of PBS buffer containing 100 μM ferroptosis inhibitor Fer-1 or an equal volume of PBS buffer for 2 hours to detect MDA. The other group was incubated for 2 hours without any drugs. For both groups, 5 μL of GOx@HOF@Fe-TF suspension (final concentration in the well plate was 50 μg / mL) prepared according to the method described in Example 4 or an equal volume of PBS was added to the culture medium as shown in Table 3. For the light-treated group, cells were exposed to light (808 nm, 1.0 W / cm²) 6 hours after drug administration. 2 5 min; 660 nm, 0.6 W / cm 2(5 min). Continue incubation for 12 h, then remove the 24-well plate and process it according to the corresponding ELISA kit to determine the content change. The results are as follows (see attached). Figure 11 Appendix Figure 12 As shown, GOx@HOF@Fe-TF has the ability to reduce GPX4 and increase MDA content, and light exposure can exacerbate lipid peroxidation, further indicating that GOx@HOF@Fe-TF induces cell death by activating the ferroptosis pathway.

[0124] Table 3

[0125]

[0126] Example 16

[0127] 4T1 tumor-bearing mice (6 weeks old, female) were randomly divided into 6 groups, with 6 mice in each group. The groups were: 1: PBS group; 2: PBS + 808 nm + 660 nm group; 3: GOx@HOF@Fe-TF group; 4: GOx@HOF@Fe-TF + 808 nm group; 5: GOx@HOF@Fe-TF + 660 nm group; and 6: GOx@HOF@Fe-TF + 808 nm + 660 nm group. On days 0 and 4, mice were administered the drug via tail vein injection, receiving either 100 μL of PBS buffer or 100 μL of PBS buffer containing 1 mg / mL GOx@HOF@Fe-TF, according to their respective groups. Six hours after administration, the drugs were injected at a concentration of 1.0 W / cm². 2 808 nm laser, 0.6 W / cm 2 Mice labeled with the corresponding laser group were irradiated with a 660 nm laser for five minutes. GOx@HOF@Fe-TF was prepared using the method described in Example 4.

[0128] Fourteen days later, the mice were euthanized, their tumors were photographed and weighed, and their spleens were collected for immune cell analysis.

[0129] Figure 13 As shown in A-13C, GOx@HOF@Fe-TF significantly inhibited tumor growth rate. The final group (GOx@HOF@Fe-TF + 660 nm + 808 nm) achieved a 98.3% inhibition rate against tumors, indicating that GOx@HOF@Fe-TF exhibits superior anti-tumor effects under laser irradiation. Figure 13 As shown in Figure D, the mouse body weight did not fluctuate significantly during treatment, indicating that the mice tolerated GOx@HOF@Fe-TF well. H&E staining results demonstrated that the GOx@HOF@Fe-TF plus light irradiation group significantly increased tumor necrosis and strongly inhibited tumor cell proliferation. Figure 13 E), which is consistent with the results of the tumor growth curve.

[0130] Tumors in different groups of mice were surgically removed. Cells were digested at 37 °C for 30 min using a digestion solution containing 1 mg / mL collagenase IV, 0.1 mg / mL hyaluronidase, and 1640 medium. After centrifugation, the cells were split into red blood cells and collected as single-cell suspensions through a 70 μm filter. A portion of the collected cells was incubated with antibodies against CD3, CD4, and CD8, while the remaining cells were stained with antibodies against CD11, CD80, and CD86. After staining, the cells were washed with PBS, filtered through a 40 μm filter, and collected as single-cell suspensions for flow cytometry analysis. Assays were performed according to standard protocols, using flow cytometry to detect DC cell maturity and CD4+. + and CD8 + Changes in cell content.

[0131] like Figure 14 As shown in A-14D, the number of DC cells and T cells in the treatment group mice was increased compared with that in the PBS group, and the final group (GOx@HOF@Fe-TF plus light irradiation group) significantly promoted DC cell maturation, stimulated T cell differentiation, and thus activated tumor immunity.

[0132] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features; 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 method for preparing a nanocomposite based on a porphyrin hydrogen-bonded organic framework, characterized in that, It comprises the following steps: S1: 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene is added to water, the pH is adjusted to 7-8 with a basic solution to form solution A, and 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphyrin is dissolved in water to form solution B; S2: glucose oxidase is mixed with solution B, stirred at room temperature for 10-30 min, then solution A is added to the solution to obtain a mixed solution, stirred at 500-1000 rpm at room temperature in the dark for 1-3 h, centrifuged and washed with water to obtain a natural enzyme-encapsulated hydrogen-bonded organic framework GOx@HOF; S3: the GOx@HOF obtained in S2 is added to water, an FeCl3 solution is added, ultrasonic dispersion is performed, stirred at 500-1000 rpm for 20-40 min, then a tannic acid solution and a folic acid solution are added, ultrasonic dispersion is performed, stirred at 500-1000 rpm for 20-40 min, centrifuged and washed with water to obtain a novel nanocomposite GOx@HOF@Fe-TF; 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.

2. The method of claim 1, wherein the nanocomposite is prepared by a method comprising: The amount ratio 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 of S2 is 0.3-0.45 g:3.5-4.5 mmol:1 mmol.

3. The method of claim 1, wherein the nanocomposite is prepared by a method comprising: The amount ratio 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 of S2 is 0.37 g:4 mmol:1 mmol.

4. The method of claim 1, wherein the nanocomposite is prepared by a method comprising: 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.

5. The method of claim 1, wherein the nanocomposite is prepared by a process comprising: The mass ratio of GOx@HOF, FeCl3, tannic acid and folic acid in S3 is 1:0.37:0.7:0.

07.

6. A nanocomposite obtained by the preparation method of any one of claims 1-5.

7. Use of the nanocomposite of claim 6 in the preparation of an anti-tumor drug.

8. Use according to claim 7, characterized in that, The anti-tumor drug is a drug that triggers ferroptosis of tumor cells.

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