Preparation method of oxygen deficit and H2O2 dual-response COF nano-enzyme and application of oxygen deficit and H2O2 dual-response COF nano-enzyme in treatment of pancreatic cancer

Abstract

The invention discloses a preparation method of oxygen deficit and H2O2 dual-response COF nano-enzyme and application of the oxygen deficit and H2O2 dual-response COF nano-enzyme in treatment of pancreatic cancer, and belongs to the technical field of application materials. The COF is prepared from 1, 3, 6, 8-tetra (4-formylphenyl) pyrene (Py-CHO), p-diaminoazobenzene (Azo-NH2), ferroferric oxide (Fe3O4), dopamine hydrochloride (DA) and hyaluronic acid (HA), and the COF is prepared from the following raw materials in parts by weight: 1, 3, 6, 8-tetra (4-formylphenyl) pyrene (Py-CHO), p-diaminoazobenzene (Azo-NH2), ferroferric oxide ( Under a tumor microenvironment (TME), the CFPH can be split due to reductase existing in an anoxic environment, released Fe3O4 can react with H2O2 in TME to generate ROS, and pancreatic cancer cells are effectively killed. The COF prepared by the invention is low in cost, simple and convenient in preparation method and good in biocompatibility, and has a wide application prospect in the aspect of pancreatic cancer treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and specifically to a preparation method of an oxygen-deficient and H2O2 dual-responsive COF nanozyme and its application in the treatment of pancreatic cancer. Background Art

[0002] Pancreatic cancer is a highly malignant solid tumor in the digestive system and is known as the "king of cancers". In the past few decades, the global burden of pancreatic cancer has increased sharply. Research reports show that the number of global pancreatic cancer patients has increased sharply from 196,000 cases in 1990 to 441,000 cases in 2017. Currently, the late treatment methods mainly include radiotherapy, chemotherapy, etc. However, these treatments will have strong toxic and side effects on the human body. In addition, the dense extracellular matrix (ECM) in the pancreatic tumor microenvironment (TME) will compress the tumor blood vessels, reducing or blocking the blood flow to the tumor area, thereby reducing the oxygen supply and drug delivery of the tumor tissue. At the same time, the abnormally hard matrix in the tumor can also act as a physical barrier to hinder the diffusion of oxygen and drugs, further restricting tumor treatment. In order to effectively treat pancreatic cancer, currently, in addition to radiotherapy and chemotherapy, two or more methods are often used for combined treatment, such as photodynamic therapy (PDT), photothermal therapy (PTT), immunotherapy and other therapies to treat. Although progress has been made in the treatment of pancreatic cancer, the low patient response rate and potential immune-related adverse events are still the main clinical challenges. Therefore, there is an urgent need to seek a more precise and efficient treatment method for pancreatic cancer to reduce the mortality rate of pancreatic cancer. Therefore, it is of great significance to develop a highly efficient and convenient new pancreatic cancer treatment strategy.

[0003] In recent years, covalent organic frameworks (COFs), as a class of emerging organic crystalline polymer materials, have received extensive attention due to their advantages such as low density, high porosity, excellent biocompatibility and biodegradability. Because it does not introduce metal ions to bring additional toxicity, highly adjustable pore size, good drug loading capacity and excellent drug release ability, it has extremely high potential in the application in the biomedical field. Among them, using COF as an anti-tumor drug has gradually become a research hotspot in the field of tumor treatment, and there have been reports on the use of COF for tumor treatment. Guan et al. designed a nanoagent CaCO3@COF-BODIPY-2I @GAG based on a nanoscale covalent organic framework, and the glycosaminoglycan (GAG) targeting agent targets the CD44 receptor on colon cancer cells. Under light triggering 1 O2 directly kills tumor cells and also causes mitochondrial dysfunction and Ca 2+Overloading. The treatment rates for HCT-116 tumors and MCF-7 tumors were 90.6% and 58.1%, respectively, showing significantly enhanced and selective anti-tumor effects on colon tumors (Guan Q, Zhou L-L, Lv F-H, et al. A Glycosylated Covalent Organic Framework Equipped with BODIPY and CaCO3 for Synergistic Tumor Therapy[J]. Angewandte Chemie International Edition, 2020, 59(41):18042-18047.). Li et al. synthesized a redox-responsive disulfide-bonded porphyrin covalent organic framework for nanocrystallization through glutathione (GSH)-triggered biodegradation. The resulting nanoscale COF-based multifunctional nanomedicine after loading 5-fluorouracil (5-Fu) can be further effectively dissociated by endogenous GSH in tumor cells to efficiently release 5-Fu for selective chemotherapy of tumor cells. Together with GSH depletion-enhanced photodynamic therapy, ideal synergistic tumor therapy of MCF-7 breast cancer was achieved through ferroptosis (Li W-Y, Wan J-J, Kan J-L, et al. A biodegradable covalent organic framework for synergistic tumor therapy[J]. Chemical Science, 2023, 14(6): 1453-1460.). Currently, the research on COF in tumor diagnosis and treatment is mainly in its infancy, and there are still many challenges and problems to be solved. Based on the previous research progress of COF in the biomedical field, designing a multifunctional covalent organic framework with hypoxia responsiveness is of great significance in the treatment of pancreatic cancer. Summary of the Invention

[0004] In view of the existing technical problems, based on the characteristics of TME and nanozymes, the present invention designs a multifunctional covalent organic framework with dual responses to hypoxia and H2O2. The multifunctional covalent organic framework is composed of 1,3,6,8-tetra(4-formylphenyl)pyrene (Py-CHO), p-diaminoazobenzene (Azo-NH2), iron oxide (Fe3O4), dopamine hydrochloride (DA) and hyaluronic acid (HA). The obtained pyrene-based COF is COF-Fe3O4-PDA-HA (CFPH). When CFPH is used for pancreatic cancer treatment, after reaching the TME, CFPH spontaneously cleaves under the action of reductase to release Fe3O4 nanozymes. Due to the excessive H2O2 in the tumor microenvironment, CFPH can further react to generate ·OH, which can reduce the content of H2O2 and kill tumor cells to a certain extent. The inhibition rate of pancreatic cancer cells during in vitro cell treatment reaches 29.52%. This multi-component synergistic treatment COF nanozyme provides a simple and efficient treatment strategy and has broad application prospects in the field of pancreatic cancer treatment.

[0005] To achieve the above object, the present invention adopts the following technical solutions: A preparation method of a hypoxia and H2O2 dual-responsive COF nanozyme, comprising the following steps: (1) Weigh 1,3,6,8-tetra(4-formylphenyl)pyrene and p-diaminoazobenzene in a glass tube, then add n-butanol and acetic acid, ultrasonically treat for 20 minutes at room temperature until the powder is evenly dispersed in the solution. After three cycles of freezing-vacuum pumping-thawing, seal the glass tube at high temperature, place it in an oven at 120 °C and react for 3 d, wash with THF until the filtrate is clear, and finally dry in vacuo to obtain Azo-COF; (2) Take 14.2 mg of the Azo-COF obtained in step (1) and disperse it in 10 mL of ultrapure water, then add Fe3O4 nanoparticles, ultrasonically disperse and react under magnetic stirring for 6 h, centrifuge at high speed and wash three times with ultrapure water to obtain COF-Fe3O4; (3) Disperse the COF-Fe3O4 obtained in step (2) in 5 mL of Tris-HCl buffer solution, then add dopamine hydrochloride, stir for 6 h, wash three times with ultrapure water and dry in vacuo to obtain COF-Fe3O4-PDA; (4) Dissolve 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and hyaluronic acid in 5 mL of water, stand and activate in the dark for 30 min, then add COF-Fe3O4-PDA, stir in the dark for 12 h, and then wash 3 times with ultrapure water to obtain COF-Fe3O4-PDA-HA.

[0006] Furthermore, in step (1), the dosage ratio of 1,3,6,8-tetra(4-formylphenyl)pyrene, p-diaminoazobenzene, n-butanol and acetic acid is 12.4 mg: 8.5 mg: 1 mL: 30 μL.

[0007] Furthermore, in step (2), the dosage ratio of Azo-COF to Fe3O4 is 14.2 mg: 5 mg.

[0008] Furthermore, in step (3), the dosage ratio of COF-Fe3O4 to dopamine hydrochloride is 1:1, and the concentration of COF-Fe3O4 is 1 mg / mL.

[0009] Furthermore, in step (4), the dosage ratio of COF-Fe3O4-PDA, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and hyaluronic acid is 1:5:5:5.

[0010] Application of the hypoxia and H2O2 dual-responsive COF nanozyme prepared by the above preparation method in the preparation of products for treating pancreatic cancer.

[0011] The remarkable advantages of the present invention are as follows: The CFPH has hypoxia and H2O2 dual responsiveness. In the tumor hypoxic microenvironment, CFPH will undergo cleavage to release Fe3O4 nanozyme. Due to the excessive presence of H2O2 in the tumor microenvironment, Fe3O4 in CFPH can further react with H2O2 to generate ·OH, which can promote tumor cell apoptosis while reducing the content of H2O2, and has excellent anti-tumor effects. Description of the Drawings

[0012] Figure 1 are the XRD spectra of (a) Azo-COF, (b) CF and CFPH.

[0013] Figure 2 are the FTIR spectra of (a) Azo-COF and (b) CFPH.

[0014] Figure 3 are the particle size and Zeta potential diagrams of CFPH; where (a) particle size, (b) Zeta potential.

[0015] Figure 4 is the hypoxia response test of CFPH; where (a) the fluorescence spectra of CFPH with and without Na2S2O4 solution, (b) the change of fluorescence recovery of COF with time under the Na2S2O4-simulated tumor hypoxic microenvironment.

[0016] Figure 5ROS production performance of CFPH, where (a) ROS production performance of Fe3O4 nanozyme, (b) ROS production performance of CFPH at different pH values.

[0017] Figure 6 Cell viability assessment experiment of CFPH on pancreatic cancer cells, where PBS is used as the control group and gemcitabine is used as the commercial group.

[0018] Figure 7 Live and dead cell staining experiment of CFPH on pancreatic cancer cells, where PBS is used as the control group and gemcitabine is used as the commercial group. Detailed implementation manners

[0019] To make the content of the present invention easier to understand, the technical solutions of the present invention are further described below in conjunction with specific implementation manners, but the present invention is not limited thereto.

[0020] Example 1 (1) Place 12.4 mg of 1,3,6,8-tetra(4-formylphenyl)pyrene and 8.5 mg of p-diaminoazobenzene in a glass tube. Then add 1 mL of 1-butanol and 30 μL of acetic acid (AcOH) to the glass tube. Ultrasonically treat at room temperature for 20 minutes until the powder is evenly dispersed in the solution. After three cycles of freezing-vacuuming-thawing, seal the glass tube at high temperature. Place the glass tube in an oven at 120 °C and react for 3 days. Wash with THF until the filtrate is clear. Finally, dry in vacuo to obtain Azo-COF.

[0021] (2) Mix ferric chloride hexahydrate solution (0.27 g / mL) and ferrous chloride tetrahydrate solution (0.099 g / mL) in a molar ratio of 2:1. Stir in a 40 °C water bath for 10 min (500 rpm); add NaOH aqueous solution (2 mol / L) dropwise until pH = 9-10. Stir in a nitrogen atmosphere for 30 min (40 °C, 500 rpm) until black precipitate appears and stop the reaction. Wash with deionized water 3 times and dry in a vacuum drying oven at 50 °C to obtain Fe3O4 nanoparticles.

[0022] (3) Take 14.2 mg of the Azo-COF obtained in step (1) and disperse it in 10 mL of ultrapure water. Add 5 mg of Fe3O4 nanoparticles to the solution. After ultrasonic dispersion, react under magnetic stirring for 6 h. Centrifuge and wash to obtain COF-Fe3O4 (CF).

[0023] (4) Disperse 5 mg of COF-Fe3O4 in 5 mL of Tris-HCl buffer solution (pH = 8.5), then add 5 mg of dopamine hydrochloride, stir for 6 h, wash three times with ultrapure water, and dry in vacuum to obtain COF-Fe3O4-PDA (CFP).

[0024] (5) Dissolve 25 mg of EDC, 25 mg of NHS, and 25 mg of HA in 5 mL of ultrapure water, let it stand and activate for 30 min in a dark environment. After activation is completed, add 5 mg of COF-Fe3O4-PDA, continue to stir for 12 h in a dark environment, and then wash 3 times with ultrapure water to obtain COF-Fe3O4-PDA-HA (CFPH). Figure 1 are the XRD patterns of Azo-COF, CF, and CFPH. As can be seen from the figure, the XRD patterns of Azo-COF, CF, and CFPH all have obvious small-angle diffraction peaks at 3.05° and 6.12°, indicating that the synthesized COF has excellent crystallinity, which is basically consistent with the simulated peak shape on MS. At 30.01°, 35.4°, and 62.84° corresponding to the (220), (331), and (440) crystal planes of Fe3O4, which is basically consistent with the standard PDF card of Fe3O4.

[0025] Figure 2 are the FTIR spectra of Azo-COF and CFPH. From Figure 2 a, it can be observed that the characteristic absorption peak of -NH2 of p-diaminoazobenzene is at 3467 cm -1 , and the absorption peak of -CHO of 4-formylphenylpyrene is at 1695 cm -1 . Azo-COF shows a characteristic absorption peak of C=N at 1629 cm -1 , and the peaks of p-diaminoazobenzene and 4-formylphenylpyrene both show obvious decreases, indicating the successful synthesis of Azo-COF. And in Figure 2 b, it is observed that the peak at 1500 cm -1 is the characteristic absorption peak (C-N) of PDA, and the peak at 1620 cm -1 is the stretching vibration peak of the carboxyl group in HA, indicating that PDA and HA are successfully synthesized onto COF.

[0026] Figure 3 are the particle size and Zeta potential diagrams of CFPH. It can be known from the figure that the particle size of CFPH is 285.5 nm, meeting the conditions for tumor treatment of nanoparticles ( Figure 3 a). The surface charge changes from 0.29 mV to 7.84 mV, confirming the encapsulation of Fe3O4. After further modification with negatively charged PDA and HA, the Zeta potential drops to -16.43 mV (Figure 3 b). This proves that the negative charge on the surface can greatly weaken the interaction with negatively charged proteins in the blood and the aggregation effect of nanoparticles, thereby increasing the stability of the nano-delivery system.

[0027] Figure 4 For the hypoxia response test of CFPH. 1 mg of CFPH was added to 2 mL of PBS (10 mM, pH = 7.4), and sodium dithionite (Na2S2O4, 2 mM) was used to simulate azoreductase, and the fluorescence signal of Py-CHO monomer appeared ( Figure 4 a). The results showed that CFPH was triggered by the biomimetic azoreductase Na2S2O4 and could be degraded in the tumor hypoxia microenvironment. Due to the ACQ effect, the fluorescence of CFPH was initially quenched. After adding Na2S2O4 and mixing well, CFPH would undergo responsive cleavage, causing the fluorescence of Py-CHO to gradually recover over time ( Figure 4 b).

[0028] Figure 5 For the ROS generation performance of CFPH under different conditions. 10 mL of H2O and 20 mL of HAc / NaAc buffer solution (1:1, 0.1 M) were mixed and then 1 mL of 10 mM H2O2 was added. 50 mg of 3,3',5,5'-tetramethylbenzidine was dispersed in the above solution. 6 mL of the solution was taken and 2 mg of CFPH was added, and the reaction was carried out for 60 min, and the absorbance was measured by ultraviolet. As shown in the figure, in the absence of H2O2, Fe3O4 did not generate ROS. When H2O2 was added, Fe3O4, as a peroxidase (POD), could react with H2O2 to generate ROS ( Figure 5 a). Then the ROS generation effect of CFPH under the conditions of pH = 4.5 and pH = 7.4 was tested. It can be seen that CFPH has no effect under neutral conditions, while CFPH has a significant ROS generation effect under acidic conditions ( Figure 5 b). This means that CFPH can react with H2O2 to generate ROS under acidic conditions, which can not only reduce the H2O2 level in the tumor microenvironment but also effectively eliminate tumor cells.

[0029] Example 2 The cytotoxicity of CFPH was evaluated using CCK-8 and pancreatic cancer (Panc-1) cells. The specific steps are as follows. Panc-1 cells were cultured in DMEM medium (90% DMEM, 10% FBS, 1% penicillin-streptomycin) and incubated in a 37 °C, 5% CO2 cell culture incubator for 24 h to allow the cells to adhere. Then, Panc-1 cells were seeded onto 96-well plates, and PBS group (pH = 7.4, control group), Gemcitabine group, CF group, CF + H2O2 group, and CFPH + H2O2 group were set up. The above Gemcitabine, CF, and CFPH were dissolved in 10 mL of DMEM medium to make 300 μg / mL, and 1.3 mL of H2O2 was added to the CF + H2O2 group and CFPH + H2O2 group. 20 μL of the materials from the above different groups was added to each well. After further culturing the cells for 24 h, 10 μL of CCK-8 solution was added, and after incubating at 37 °C for 4 h, the absorbance of each well at 450 nm was measured using a microplate reader.

[0030] Figure 6 This was an experiment for evaluating the cell viability of COF against pancreatic cancer cells. In the absence of H2O2, the cell viability of pancreatic cancer cells with CF was 74.77%, which was higher than 51.58% of the commercial group (gemcitabine). Subsequently, in the presence of H2O2, CF showed an obvious cytotoxic effect, and the cell viability of pancreatic cancer cells decreased from 74.77% to 50.48%. After modification with PDA and HA, CFPH further reduced the cell viability to as low as 29.52%, showing excellent anti-pancreatic cancer cell effects.

[0031] Example 3 The cell live / dead state of COF was evaluated using AM / PI and pancreatic cancer (Panc-1) cells. The specific steps are as follows. Panc-1 cells were cultured in DMEM medium (90% DMEM, 10% FBS, 1% penicillin-streptomycin) and incubated in a 37 °C, 5% CO2 cell culture incubator for 24 h to allow the cells to adhere. Then, Panc-1 cells were seeded onto well plates, and PBS group (pH = 7.4, control group), Gemcitabine group, CF group, CFPH group, and CFPH + H2O2 group were set up. The above Gemcitabine, CF, and CFPH were dissolved in 10 mL of DMEM medium to make 300 μg / mL, and 1.3 mL of H2O2 was added to the CF + H2O2 group and CFPH + H2O2 group. 500 μL of the materials from the above different groups was added to each well. After further culturing the cells for 24 h, 100 μL of AM / PI solution was added, and after incubating at 37 °C for 0.5 h, the live / dead degree of the cells in each well was observed using an inverted fluorescence microscope.

[0032] Figure 7 This is the live / dead cell staining experiment of COF for pancreatic cancer cells. As shown in the figure, in the absence of H2O2, it can be seen that it is mainly green fluorescence, while the number of red fluorescence is scarce, indicating that CFPH has almost no effect on pancreatic cancer cells without H2O2. Under the condition of the presence of H2O2, the number of red fluorescence in the field of view surges, indicating that CFPH can effectively kill pancreatic cancer cells.

[0033] The above are only the preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims

1. A preparation method of an oxygen-deficient and H2O2 dual-responsive COF nanozyme, characterized in that: It includes the following steps: (1) Weigh 1,3,6,8-tetrakis(4-formylphenyl)pyrene and p-diaminoazobenzene in a glass tube. Then add n-butanol and acetic acid, and ultrasonically treat for 20 minutes at room temperature until the powder is evenly dispersed in the solution. After three cycles of freezing-vacuum pumping-thawing, seal the glass tube at high temperature, place it in an oven at 120 °C and react for 3 d. Wash with THF until the filtrate is clear, and finally dry in vacuum to obtain Azo-COF; (2) Disperse the Azo-COF obtained in step (1) in 10 mL of ultrapure water, add Fe3O4 nanoparticles, ultrasonically disperse and react under magnetic stirring for 6 h, then centrifuge and wash three times with ultrapure water to obtain COF-Fe3O4; (3) Disperse the COF-Fe3O4 obtained in step (2) in 5 mL of Tris-HCl buffer solution, add dopamine hydrochloride, stir for 6 h, wash three times with ultrapure water and dry in vacuum to obtain COF-Fe3O4-PDA; (4) Dissolve 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and hyaluronic acid in 5 mL of water, let it stand and activate in the dark for 30 min, then add COF-Fe3O4-PDA, stir in the dark for 12 h, and then wash 3 times with ultrapure water to obtain COF-Fe3O4-PDA-HA.

2. The preparation method according to claim 1, characterized in that: In step (1), the dosage ratio of 1,3,6,8-tetrakis(4-formylphenyl)pyrene, p-diaminoazobenzene, n-butanol and acetic acid is 12.4 mg: 8.5 mg: 1 mL: 30 μL.

3. The preparation method according to claim 1, characterized in that: In step (2), the dosage ratio of Azo-COF to Fe3O4 is 14.2 mg: 5 mg.

4. The preparation method according to claim 1, characterized in that: In step (3), the dosage ratio of COF-Fe3O4 to dopamine hydrochloride is 1:1, and the concentration of COF-Fe3O4 is 1 mg / mL.

5. The preparation method according to claim 1, characterized in that: In step (4), the dosage ratio of COF-Fe3O4-PDA, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and hyaluronic acid is 1:5:5:

5.

6. The hypoxia and H2O2 dual-responsive COF nanozyme prepared by the preparation method according to any one of claims 1-5.

7. Use of a hypoxia and H2O2 dual-responsive COF nanozyme as described in claim 6 in the preparation of a product for treating pancreatic cancer.

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